N-acylanilines, herbicide-CHA chimera, and amino acid analogues as novel chemical hybridizing agents for wheat (Triticum aestivum L.)

Article abstract of DOI:10.1021/jf051458t

In the absence of viable alternative technology of hybrid wheat development, chemical induction of male sterility mediated technology based on chemical hybridizing agents (CHAs) holds a great potential. N-Acylaniline derivatives, namely, ethyl 4′-fluoro oxanilate (1) and ethyl 4′-trifluoromethyl oxanilate (2) containing halogen atoms in the para position of the aryl ring and substituted amide linkage (-CO-NH-) in the acyl side chain induced >98% spikelet sterility on three genotypes of wheat, namely, PBW 343, HD 2046 and HD 2733, at 1500 ppm. The active moieties were incorporated in the form of herbicide-CHA chimera and amino acid analogues using glycine and alanine as templates. The target activity was made more selective by synthesizing chimeric structure of herbicide (2,4-dichlorophenoxyacetic acid and dalapon) and the most active CHA templates, namely, 4-fluoroanilinyl and β-ethoxycarbonyl moieties. Among herbicide-CHA chimera ethyl 2′,4′-dichlorophenyl oxalate (14) induced 79.11% male sterility, whereas benzyl methyl 2-oxo-3-azaadipate (20) was the best, inducing 73.87% male sterility in HD 2733, among amino acid analogues. The CHAs were found to modify the reproductive biology to ensure cross-pollination in the cleistogamous wheat flowers, increasing the probability for the development of hybrids.

Full text of DOI:10.1021/jf051458t

J. Agric. Food Chem. 2005, 53, 7899−7907 7899
N-Acylanilines, Herbicide−CHA Chimera, and Amino Acid
Analogues as Novel 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
In the absence of viable alternative technology of hybrid wheat development, chemical induction of
male sterility mediated technology based on chemical hybridizing agents (CHAs) holds a great
potential. N-Acylaniline derivatives, namely, ethyl 4′-fluoro oxanilate (1) and ethyl 4′-trifluoromethyl
oxanilate (2) containing halogen atoms in the para position of the aryl ring and substituted amide
linkage (-CO-NH-) in the acyl side chain induced >98% spikelet sterility on three genotypes of
wheat, namely, PBW 343, HD 2046 and HD 2733, at 1500 ppm. The active moieties were incorporated
in the form of herbicide-CHA chimera and amino acid analogues using glycine and alanine as
templates. The target activity was made more selective by synthesizing chimeric structure of herbicide
(2,4-dichlorophenoxyacetic acid and dalapon) and the most active CHA templates, namely, 4-
fluoroanilinyl and â-ethoxycarbonyl moieties. Among herbicide-CHA chimera ethyl 2′,4′-dichlorophenyl
oxalate (14) induced 79.11% male sterility, whereas benzyl methyl 2-oxo-3-azaadipate (20) was the
best, inducing 73.87% male sterility in HD 2733, among amino acid analogues. The CHAs were
found to modify the reproductive biology to ensure cross-pollination in the cleistogamous wheat flowers,
increasing the probability for the development of hybrids.
KEYWORDS: Chemical hybridizing agents (CHAs); wheat; N-acylanilines; herbicide-CHA chimera; amino
acid analogues
INTRODUCTION
by interrupting microsporogenesis to prevent self-pollination and
to promote fertilization (1, 2). Induction of male sterility by
CHAs may be a good alternative to hand emasculation in hybrid
breeding as well as in population improvement programs. Unlike
the CGMS system, the CHAs have the unique advantage of
saving time and labor because no restorer-maintainer lines are
required. Effective CHAs allow the sterilization of a large
number of potential parents of wheat and permit the production
of large numbers of hybrids. The lack of safe and selective
chemicals capable of the induction of male sterility without
adverse effects on plant development has been the main
constraint of this approach. The second generations of CHAs
were developed specifically for their pollen-suppressing activity.
They provide much improved safety with significantly reduced
phytotoxic effects. Earlier results with ethyl oxanilates on rice
(3), wheat (4-6), and chickpea (7) revealed their paramount
importance in heterosis breeding. Herbicides such as dalapon
(8), 2, 4-dichlorophenoxyacetic acid (2, 4-D) (9), maleic
hydrazide (10), and TIBA (11) were tested as possible CHAs
in various studies, but were found to induce strong phytotoxic
response on different agronomic parameters and female fertility.
In a program of design and development of potential CHAs we
have undertaken the synthesis of several N-acylanilines, amino
acid analogues, and herbicide-CHA chimera. The target activity
of the herbicides (2, 4-D and dalapon) was made more selective
by synthesizing the chimeric structure of the herbicide and the
The production of hybrid wheat (Triticum aestiVum L.) offers
an opportunity for overcoming the stagnating yield plateau of
wheat. There exists a yield gap of 2.0-2.3 t/ha over most of
the 9.5 million hectares of the North East Plain Zone and 9.0
million hectares area under wheat in the North Western Plain
Zone of India. There is a need to bridge this technology gap to
achieve a quantum jump in production. Heterosis breeding offers
a means to increase current yield levels in wheat. In self-
pollinated crops such as wheat with the male and female organs
in the same flower, selective sterilization of pollen is of
paramount importance for heterosis breeding. Exploitation of
heterosis at the commercial level depends on the availability of
stable male sterile lines. A viable and stable cytoplasmic-
genetic male sterile (CGMS) system along with perfect restorer
lines in wheat is not in place, although considerable research
efforts are underway. In view of this, the other option of
regulating pollen growth using chemical hybridizing agents
(CHAs) involving two-line hybrid breeding needs to be pursued
intensively. The CHAs facilitate cross-breeding in plant species
with perfect flowers by selectively sterilizing male sex cells or
* Address correspondence to this author at Division PNP, Central Marine
Fisheries Research Institute, Ernakulam North P.O., P.B. 1603, Cochin
682018, Kerala, India (telephone 91-484-2394867, ext. 272; fax 91-484-
2394909; e-mail kajal_iari@yahoo.com.
10.1021/jf051458t CCC: $30.25
© 2005 American Chemical Society
Published on Web 09/10/2005

7900 J. Agric. Food Chem., Vol. 53, No. 20, 2005
Chakraborty and Devakumar
was added over a period of 45 min to a solution of 4-fluoroaniline
(0.025 mol, 2.3 mL) and dry pyridine (0.05 mol, 3.96 mL) in toluene
(50 mL) kept at e5 °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 chilled hexane was added to get the
precipitate. Toluene was removed under vacuum. The precipitate was
filtered off to get a solid product, which was diluted with cold ethyl
acetate (100 mL) and treated with saturated ammonium chloride solution
(50 mL). The organic phase was washed with water, dried, and
evaporated to give a yellowish solid. Recrystallization of the residue
with a mixture of diethyl ether/hexane (1:1) yielded halogenated
acetanilides (9-12) as crystalline solids, which were homogeneous,
by TLC. The duration of reaction, yield, and physicochemical char-
acteristics of the products thus prepared were as follows.
most active agrophore CHA templates, namely, 4-fluoroanilinyl
and â-ethoxycarbonyl moieties. In this group the dalapon
analogue, namely, N-(2,2-dichloropropanoyl)-4-fluoroaniline
(13), and the 2,4-D analogues, 2,4-dichlorophenyl oxalate,
malonate and acetoacetanilide (14-16) were synthesized. The
amino acid profiles of both sterile and fertile pollens have shown
the deficiency of certain amino acids (12, 13). A novel way to
derive new analogues of glycine and alanine has been tried
because they are absent in sterile pollens.
It is essential to develop selective and highly potent CHAs
for the development of hybrid wheat. With this view in mind,
we tried to get an insight into the synthesis, spectroscopic
analysis, and structure-activity relationship governing N-
acylanilines, herbicide-CHA chimera, and amino acid ana-
logues as CHAs, and in this paper, we report our results.
Ethyl 4′-Fluoro Oxanilate (1). To a solution of diethyl oxalate (0.03
mol, 4.06 mL) in toluene (50 mL) were added 4 -fluoroaniline (0.025
mol, 2.3 mL) and boric acid (0.1 g) under reflux for 0.5 h following
Figure 1 to obtain a white crystalline solid: yield, 4.96 g (94%); mp
MATERIALS AND METHODS
1
118-119 °C; TLC Rf 0.50; GC tR ) 7.82 min; H NMR (CDCl3) δ
1.65 (t, J ) 6 Hz, 3H, -CH3), 4.70 (q, J ) 6 Hz, 2H, -OCH2), 7.40
(t, J ) 6 Hz, 2H, Hb,Hb′-aromatic), 7.95 (d, J ) 6 Hz, 1H, Ha-aromatic),
8.10 (d, J ) 6 Hz, 1H, Ha′-aromatic), 9.44 (s, 1H, NH); EI-MS, m/z
(rel intensity %) 211 (M+, 100), 139 (18), 138 (93), 137 (72), 110
(92), 75 (12), 83 (32), 63 (5).
Substituted anilines, diethyl malonate, ethyl acetoacetate, ethyl
pyruvate, and amino acids were procured from Aldrich Chemical Co.
Inc. Other esters were prepared in the laboratory. Chloroacetyl chloride,
dichloroacetyl chloride, and trichloroacetyl chloride were procured from
E. Merck. The structures of synthesized compounds were confirmed
by 1H NMR and mass spectroscopy. Melting points (mp) were
determined by using a sulfuric acid bath and are uncorrected. Anhydrous
reactions were performed under an inert atmosphere, the setup
assembled and cooled under dry nitrogen. Unless otherwise noted,
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) or sodium sulfate
(Na2SO4), evaporated on a rotatory evaporator, and under reduced
pressure. 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 developing medium. All reactions were
monitored by UV fluorescence or staining with iodine. Preparative TLC
was performed on 1.0 mm silica gel, 60 Å, 20 × 20 plates. All solvents
used in chromatography had been distilled. All test compounds gave
correct elemental analyses using a Euro Vector elemental analyzer
(model EA3011). Mass spectral assays were obtained using a Fisons
Trio 1000 (HRGC Mega-2 coupled with an 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 was 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 internal
standard (TMS, δ 0.0 for 1H NMR). Chemical shifts (δ) are expressed
in parts per million (ppm), and the coupling constants (J) are expressed
in hertz (Hz). Multiplicities are described by the following abbrevia-
tions: s for singlet, d for doublet, dd for doublet of doublets, t for
triplet, q for quartet, and m for multiplet.
General Procedure of Synthesis of N-Acylanilines (1-12). The
following method illustrates the general scheme of synthesis of the
title compounds using different substituted anilines and esters.
To a solution of para-substituted anilines (0.025 mol) in toluene (50
mL) was added alkyl alkanoates (diethyl oxalate, dimethyl oxalate,
diisopropyl oxalate, ethyl pyruvate, 2-methoxyethanol, diethyl malonate,
and ethyl acetoacetate) (0.03 mol) in toluene (50 mL). The aliquot was
added with boric acid (0.1 g) as catalyst to a round-bottom flask fitted
with a Dean-Stark apparatus, over an oil bath, and refluxed for 0.5-4
h; the azeotrope was collected in the receiver tube. The product was
cooled to 90 °C to let the product solidify, triturated with boiling
ethanol, and refrigerated to allow for recrystallization of 1-8 (Figure
1), which were homogeneous, by TLC [hexane/ethyl acetate (4:1) as
developing medium]. Acetanilides (including halogenated acetanilides)
(9-12) were synthesized by condensation of acid chlorides with aniline.
A solution of acid chloride (chloroacetyl chloride, dichloracetyl chloride,
and trichloracetyl chloride) (0.03 mol) suspended in anhydrous toluene
Ethyl 4′-Trifluoromethyl Oxanilate (2). A white crystalline solid of
the title compound was obtained in a reaction between 4-trifluoro-
methylaniline (0.025 mol, 3.96 mL) and diethyl oxalate (0.03 mol, 4.06
mL) in toluene (50 mL) for 2 h: yield 5.81 g (89%); mp 139-141 °C;
TLC Rf 0.66; GC tR 10.78 min; 1H NMR (CDCl3) δ 1.35 (t, J ) 6 Hz,
3H, CH3), 4.35 (q, J ) 6 Hz, 2H, OCH2), 7.35 (m, 2H, Hb,Hb′-aromatic),
7.85 (m, 2H, Ha,H′a-aromatic), 8.85 (s, 1H, NH); EI-MS, m/z (rel
intensity %) 261 (M+, 5), 207 (4), 188 (9), 145 (7), 59 (12), 58 (100).
Methyl 4′-Fluoro oxanilate (3). A stirred solution of 4-fluoroaniline
(0.025 mol, 2.3 mL), dimethyl oxalate (0.03 mol, 3.5 g), and boric
acid (0.1 g) suspended in toluene (50 mL) was heated to reflux for 1
h following the method of Figure 1. The reaction was followed by
TLC [hexane/ethyl acetate (4:1) as developing medium] until comple-
tion. The resulting suspension was filtered with diethyl ether (70 mL),
washed with water (100 mL), and dried in a desiccator for 1 day.
Recrystallization of the crude product with ethyl alcohol furnished the
title compound (3) in the form of white crystals: yield 4.24 g (86%);
mp 136-137 °C; TLC Rf ) 0.37; GC tR ) 10.26 min; 1H NMR (CDCl3)
δ 3.80 (s, 3H, OCH3), 7.10 (dd, J ) 6 Hz, 2H, Hb,Hb′-aromatic), 7.60
(dd, J ) 6 Hz, 2H, Ha,Ha′-aromatic), 8.80 (s, 1H, NH); EI-MS, m/z
(rel intensity %) 197 (M+, 15), 138 (100), 139 (12), 137 (28), 112 (9),
110 (73), 77 (11), 75 (9), 65 (11).
Isopropyl 4′-Fluoro Oxanilate (4). 4-Fluoroaniline (0.025 mol, 2.3
mL) and diisopropyl oxalate (0.03 mol, 5.2 g) were refluxed in toluene
for 3 h following the general procedure to furnish a white crystalline
solid of the title compound: yield 4.27 g (76%); mp 107 °C; TLC Rf
) 0.42; GC tR ) 12.26 min; 1H NMR(CDCl3) δ 5.25 [m, 7H,
OCH(CH3)2], 7.10 (dd, J ) 6 Hz, 2H, Hb,Hb′-aromatic), 7.60 (dd, J )
6 Hz, 2H, Ha,Ha′-aromatic), 8.80 (s, 1H, NH); EI-MS, m/z (rel intensity
%) 225 (M+, 11), 166 (14), 138 (100), 120 (56), 110 (73), 107 (35),
94 (17), 93 (20), 77 (18), 75 (37), 65 (14), 59 (8).
4′-Fluoro PyruVanilide (5). Reaction of 4-fluoroaniline (0.025 mol,
2.3 mL) in toluene (50 mL) with ethyl pyruvate (0.03 mol, 3.48 mL)
following the method of Figure 1 furnished a white crystalline solid
of 5: yield 3.39 g (75%); mp 180 °C (dec); TLC Rf ) 0.39; GC tR )
10.43 min; 1H NMR (CDCl3) δ 1.30 (t, J ) 6 Hz, 3H, CH3), 7.00 (m,
2H, Hb,Hb′-aromatic), 7.20 (m, 2H, Ha,Ha′-aromatic), 10.35 (s, 1H, NH);
EI-MS, m/z (rel intensity %) 181 (M+, 27), 139 (7), 138 (69), 137
(13), 110 (100), 77 (13), 65 (13).
2-Methoxyethyl 4′- Fluoro Oxanilate (6). To a stirred solution of
2-methoxyethanol (0.03 mol) and dimethyl oxalate (0.03 mol) was
added 4-fluoroaniline (0.025 mol, 2.3 mL) dissolved in dry toluene
(50 mL), and the mixture was heated to reflux for 3 h following the
general procedure to furnish grayish white crystals of the title
compound: yield 3.78 g (63%); mp 113 °C; Rf ) 0.31; tR ) 9.59 min;
1H NMR (CDCl3) δ 5.13 (s, 3H, OCH3), 5.15 (s, 4H, C2H4), 6.90 (m,

N-Acylanilines, Herbicides, and Amino Acids as CHAs
J. Agric. Food Chem., Vol. 53, No. 20, 2005 7901
Figure 1. Synthesis of N-acylanilines and N-(2,2-dichloropropanoyl)-4-fluoroanilide.
2H, Hb,Hb′-aromatic), 7.55 (m, 2H, Ha,Ha′-aromatic), 9.60 (s, 1H, NH);
EI-MS, m/z (rel intensity %) 241 (M+, 15), 210 (9), 196 (12), 166
(72), 110 (100), 137 (12), 77 (9), 65 (13).
GC tR ) 6.11 min; 1H NMR (CDCl3) δ 2.45 (s, 3H, COCH3), 3.70 (s,
2H, CH2CO), 7.25 (dd, J ) 6 Hz, 2H, Hb,Hb′-aromatic), 7.70 (dd, J )
6 Hz, 2H, Ha,Ha′-aromatic), 9.50 (s, 1H, NH); El-MS m/z (rel intensity
%) 181 (M+, 1), 137 (11), 124 (3), 112 (8), 111 (100), 97 (4), 83 (12).
Ethyl 4′-Fluoro Malonanilate (7). A solution of 4-fluoroaniline (0.025
mol, 2.3 mL) suspended in anhydrous xylene was added to a solution
of diethyl malonate (0.03 mol, 4.8 mL) in xylene (50 mL), and the
aliquot was refluxed for 1.5 h following the method of Figure 1. 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 filtered, washed with water (100 mL), and
dried in a desiccator for 1 day. Recrystallization of the residue with
ethyl alcohol yielded the title compound as a white crystalline solid,
which was homogeneous by TLC: yield 4.65 g (83%); mp 72-73 °C;
4′-Fluoro Acetanilide (9). A stirred solution of acetyl chloride (0.03
mol, 2.3 mL) in toluene and 4-fluoroaniline (0.025 mol, 2.3 mL) in
dry pyridine (0.05 mol, 3.96 mL) kept at e5 °C were reacted, followed
by recrystallization of the residue in diethyl ether/hexane (1:1) following
the method of Figure 1 to obtain a white crystalline solid of the title
compound: yield 3.40 g (89%); mp 152 °C; TLC Rf 0.62; GC tR
)
1
9.03 min; H NMR (CDCl3-d6-DMSO) δ 2.12 (s, 3H, COCH3), 7.10
(t, 2H, Hb,Hb′-aromatic), 7.65 (dd, 2H, Ha,Ha′-aromatic), 10.00 (s, 1H,
NH); El-MS, m/z (rel intensity %) 153 (M+, 12), 138 (29), 111 (100),
110 (9), 78 (26), 77 (67), 65 (58).
1
TLC Rf 0.58; GC tR ) 6.12 min; H NMR (CDCl3) δ 1.23 (t, J ) 6
4-Fluoro Chloroacetanilide (10). The title compound (grayish
crystalline solid) was made as described for the synthesis of halogenated
acetanilides (Figure 1) by the condensation of 4-fluoroaniline (0.025
mol, 2.3 mL) and chloroacetyl chloride (0.03 mol, 3.35 mL) under an
inert atmosphere of nitrogen followed by crystallization: yield 4.00 g
Hz, 3H, CH3), 3.46 (s, 2H, CH2), 4.15 (q, J ) 6 Hz, 2H, OCH2), 7.11
(t, J ) 6 Hz, 2H, Hb,Hb′-aromatic), 7.64 (dd, J ) 6 Hz, 2H, Ha,Ha′-
aromatic), 9.70 (s, 1H, NH); EI-MS, m/z (rel intensity %) 225 (M+,
30), 180 (4), 111 (100), 110 (11), 109 (4), 95 (4), 83 (12), 75 (2), 69
(2).
1
(86%); mp 109-112 °C; TLC Rf 0.49; GC tR ) 9.02 min; H NMR
4′-Fluoro Acetoacetanilide (8). 4-Fluoroaniline (0.025 mol, 2.3 mL)
and ethyl acetoacetate (0.03 mol, 3.83 mL) were refluxed together using
toluene as solvent for 2 h following the method of Figure 1 to furnish
a white crystalline solid: yield 4.00 g (89%); mp 110 °C; TLC Rf 0.58;
(CDCl3-d6-DMSO) δ 4.05 (s, 2H, CH2Cl), 7.10 (m, 2H, Hb,Hb′-
aromatic), 7.50 (m, 2H, Ha,H′a-aromatic), 9.95 (s, 1H, NH); El-MS,
m/z (rel intensity %) 187 (M+, 2), 138 (30), 124 (12), 111 (100), 110
(69), 78 (11), 77 (75), 65 (81), 64 (15).

7902 J. Agric. Food Chem., Vol. 53, No. 20, 2005
Chakraborty and Devakumar
Figure 2. Synthesis of 2,4-dichlorophenyl esters of oxalate, malonate, and acetoacetate.
4-Fluoro Dichloroacetanilide (11). 4-Fluoroaniline (0.025 mol, 2.3
mL) and dichloroacetyl chloride (0.03 mol, 4.4 mL) were reacted under
stirring at e5 °C to provide the title compound as a grayish white
crystalline solid: yield 4.70 g (85%); mp 119-120 °C; TLC Rf 0.32;
filtered, washed with water (100 mL), and dried in a desiccator for a
day. Recrystallization of the residue with boiling ethanol furnished the
title compounds (Figure 2) in the form of crystalline solids, which
were homogeneous in TLC [hexane/ethyl acetate (4:1) as developing
medium].
1
GC tR ) 9.09 min; H NMR (CDCl3) δ 5.80 (s, 1H, -CHCl2), 6.80
(m, 2H, Hb,Hb′-aromatic), 7.20 (m, 2H, Ha,Ha′-aromatic), 8.10 (s, 1H,
NH); El-MS, m/z (rel intensity %) 221 (M+, 5), 158 (5), 150 (7), 138
(28), 110 (799), 109 (8), 95 (19), 85 (33), 83 (100), 76 (28), 75 (15).
4-Fluoro Trichloroacetanilide (12). The title compound (grayish
white crystalline solid) was made as described for the synthesis of
halogenated acetanilides (Figure 1) by the condensation of 4-fluoro-
aniline (0.025 mol, 2.3 mL) and trichloroacetyl chloride (0.03 mol,
5.32 mL) under an inert atmosphere of nitrogen followed by crystal-
lization in diethyl ether/hexane (1:1): yield 4.10 g (69%); mp 128 °C;
Ethyl 2′,4′-Dichlorophenyl Oxalate (14). To a stirred solution of 2,4-
dichlorophenol (0.025 mol, 4.08 g) was added diethyl oxalate (0.03
mol, 4.06 mL) in DMF (5 mL), and the mixture was heated to reflux
for 4 h following the general procedure (Figure 2) to furnish a solid
residue, which was recrystallized from ethanol to furnish a creamy white
crystalline solid of the title compound: yield 3.49 g (53%); mp 189-
1
192 °C; TLC Rf 0.29; H NMR (CDCl3-d6-DMSO) δ 1.40 (t, J ) 6
Hz, 3H, OCH2CH3), 4.30 (q, J ) 6 Hz, 2H, OCH2), 7.45 (m, 1H, Hb′-
aromatic), 7.65 (m, 1H, Hb-aromatic), 7.90 (m, 1H, Ha′-aromatic); El-
MS, m/z (rel intensity %) 263 (M+, 18), 165 (52), 163 (100), 162 (25),
148 (12), 146 (39), 145 (7), 121 (41), 114 (8), 112 (21), 101 (8), 94
(55), 75 (12), 73 (40), 72 (8), 65 (51), 63 (86), 62 (49), 61 (40), 60
(25).
1
TLC Rf 0.29; GC tR ) 13.42 min; H NMR (CDCl3) δ 7.00 (m, 4H,
aromatic), 9.80 (s,1H, NH); El-MS, m/z (rel intensity %) 238 (M+, 7),
204 (15), 169 (22), 121 (10), 119 (37), 111 (100), 110 (17), 78 (13),
77 (8), 65 (10).
Synthesis of N-(2,2-Dichloropropanoyl)-4-Fluoro Anilide (13). To
a solution of 4-fluoroaniline (0.025 mol, 2.3 mL) in xylene (50 mL)
was added ethyl pyruvate (0.03 mol, 3.48 mL). The reaction mixture
was refluxed for 3 h at 160 °C to obtain a dark colored aliquot, which
after cooling gave a solid product. The reaction was followed by TLC
[hexane/ethyl acetate (4:1) as developing medium] until completion.
The resultant solid intermediate (N-2-oxopropanoyl-4-fluoroaniline;
0.025 mol, 4.5 g) was chlorinated using PCl5 in xylene (10 mL) to
furnish a solid residue. Chilled hexane was added to get a precipitate,
which was filtered off to get a solid product that was diluted with cold
ethyl acetate (50 mL) and treated with a saturated ammonium chloride
solution (50 mL). The organic phase was washed with water (5 mL),
dried with magnesium sulfate, filtered, and evaporated to give a solid.
The resulting crude solid product was 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 grayish white
crystals of the title compound [N-(2,2-dichloropropanoyl)-4-fluoro
anilide] (Figure 1), which was homogeneous by TLC: yield 3.4 g
(58%); mp 165 °C; TLC Rf 0.37; 1H NMR (CDCl3-d6-DMSO) δ 5.65
(s, 3H, CCl2CH3), 7.20 (m, 2H, Hb,Hb′-aromatic), 8.30 (m, 2H, Ha,Ha′-
aromatic), 9.40 (s, 1H, NH); El-MS, m/z (rel intensity %) 236 (M+,
12), 224 (8), 222 (19), 138 (100), 128 (10), 126 (26), 125 (10), 111
(52), 107 (39), 100 (51), 98 (63), 97 (36), 95 (28), 91 (86), 77 (24), 65
(20).
Ethyl 2′,4′-Dichlorophenyl Malonate (15). A stirred solution of 2,4-
dichlorophenol (0.025 mol, 4.08 g) in DMF (5 mL) and diethyl malonate
(0.03 mol, 4.8 mL) was heated to reflux for 5 h to furnish a suspension,
which was filtered, washed with water (100 mL), and dried in a
desiccator. Recrystallization of the residue with boiling ethanol
furnished the title compound in the form of a white crystalline solids:
1
yield 3.12 g (45%); mp 159-160 °C (dec); TLC Rf 0.32; H NMR
(CDCl3-d6-DMSO) δ 1.40 (t, J ) 6 Hz, 3H, OCH2CH3), 2.20 (s, 2H,
CH2CO), 4.40 (q, J ) 6 Hz, 2H, OCH2), 7.45 (m, 1H, Ha′-aromatic),
7.25 (m, 2H, Hb,Hb′-aromatic); El-MS, m/z (rel intensity %) 277 (M+,
10), 165 (34), 163 (100), 148 (16), 146 (47), 135 (42), 121 (28), 114
(14), 112 (38), 77 (11), 75 (31), 73 (52), 72 (20), 65 (39), 63 (55), 62
(35).
2′,4′-Dichlorophenyl Acetoacetate (16). 2,4-Dichlorophenol (0.025
mol, 4.08 g) and ethylacetoacetate in DMF (5 mL) were reacted together
for 4 h following the general procedure (Figure 2) followed by
recrystallization with boiling ethanol to furnish a grayish white
crystalline solid of the title compound: yield 2.97 g (48%); mp 173
1
°C (dec); TLC Rf 0.35; H NMR (CDCl3-d6-DMSO) δ 3.70 (s, 2H,
CH2CO), 3.40 (t, J ) 6 Hz, 3H, COCH3), 6.90 (m, 2H, Hb,Hb′-aromatic),
7.20 (m, 1H, Ha′-aromatic); El-MS, m/z (rel intensity %) 247 (M+, 18),
165 (35), 163 (100), 148 (11), 146 (37), 138 (38), 77 (9), 75 (22), 73
(14).
General Procedure of Synthesis of Amino Acid Analogues (17-
21). In this series five analogues, namely, benzyl glycine p-toluene-
sulfonic acid salt (17), benzyl glycine (18), benzyl â-alanine p-toluene-
sulfonic acid salt (19), benzyl methyl 2-oxo-3-aza-adipate (20), and
trityl glycine (21), were synthesized.
General Procedure of Synthesis of 2,4-Dichlorophenyl Esters
(14-16). In this series three analogues, namely, ethyl 2′,4′-dichloro-
phenyl oxalate (14), ethyl 2′,4′-dichlorophenyl malonate (15), and 2′,4′-
dichlorophenyl acetoacetate (16) were synthesized.
To a stirred solution of 2,4-dichlorophenol (0.025 mol, 4.08 g) in
DMF (5 mL) was added diethyl esters of oxalate, malonate, or
ethylacetoacetate (0.03 mol). The resulting mixture was refluxed in
the presence of anhydrous K2CO3 (0.025 mol, 3.5 g) for 3-5 h, and
ethanol was collected as an azeotrope. The dark colored aliquot so
produced was cooled below 90 °C to solidify. The resulting solid was
To a mixture of glycine (0.25 mol, 18.8 g) in dry benzene (50 mL)
were added fresh benzyl alcohol (100 mL), p-toluenesulfonic acid
(PTSA) (0.26 mol, 48.5 g) and dry benzene (50 mL), and the mixture
was distilled to reflux for 3 h with continuous stirring. The reaction
was followed by TLC [hexane/ethyl acetate (4:1) as developing
medium] until completion. The distillate was diluted with ether and

N-Acylanilines, Herbicides, and Amino Acids as CHAs
J. Agric. Food Chem., Vol. 53, No. 20, 2005 7903
Figure 3. General procedure of synthesis of benzyl amino acids and benzyl methyl 2-oxo-3-aza-adipate.
was cooled to obtain a solid residue, which was washed with water
and dried with Na2SO4. The product was recrystallized from methanol
to obtain grayish white crystals of the intermediate salt (17) (benzyl
glycine p-toluenesulfonic acid salt). To the suspension of the resultant
solid intermediate in benzene was added an aqueous solution of 5 N
NaOH, and the reaction mixture was stirred vigorously at reflux for
4.5 h until benzyl glycine (free base) (18) was oiled out. Chilled hexane
(20 mL × 2) was added to the mixture to remove benzene. The
complete removal of benzene was ensured by adding a few milliliters
of methanol, and reconcentration was done in a vacuum. The excess
hexane was evaporated off, and benzyl glycine was condensed with
dimethyl oxalate for 3 h in toluene to furnish a solid residue, which
was poured into ice-cold water followed by recrystallization in solvent
ether/MeOH (5:2) to obtain white crystalline solids of benzyl methyl
2-oxo-3-aza-adipate (20) (Figure 3), which was homogeneous, by TLC.
In another series, to a stirred solution of a â-amino acid (â-alanine)
(0.25 mol, 22.3 g) were added PTSA (0.26 mol, 48.5 g) and benzyl
alcohol (100 mL) suspended in dry benzene (50 mL) and heated to
reflux for 2 h. The resulting suspension was filtered with diethyl ether
(50 mL), washed with water, and dried in a desiccator for a day.
Recrystallization of the crude product with diethyl ether/hexane (1:1)
furnished benzyl â-alanine p-toluenesulfonic acid salt (19) in the form
of white crystals. The details regarding yield and physicochemical
characteristics of the products thus prepared are as follows.
Benzyl Methyl 2-Oxo-3-aza-adipate (20). Benzyl glycine (0.015 mol)
was condensed with dimethyl oxalate for 3 h in toluene followed by
recrystallization in solvent ether/MeOH (5:2) following the method of
Figure 3 to furnish white crystalline solids of the title compound: yield
1
1.58 g (42%); mp 68-71 °C; TLC Rf ) 0.49; H NMR (CDCl3) δ
2.10 (s, 2H, -OCH2), 2.30 (s, 3H, -OCH3), 4.50 (s, 2H, -COCH2),
5.20 (s, 1H, NH), 7.25 (m, 5H, aromatic); El-MS, m/z (rel intensity %)
251 (M+, 10), 179 (13), 163 (26), 148 (57), 136 (46), 107 (22), 103
(11), 91 (100), 77 (15), 65 (12).
Trityl Glycine (21). A solution of triethylamine (10 mL, 10% v/v)
suspended in dry CHCl3 was added dropwise over a period of 30 min
to a chilled solution of trityl chloride (3 g) in dry CHCl3 (30 mL) kept
at e5 °C with continuous stirring to make reagent 1. A solution of
glycine (0.75 g) in triethylamine (1 mL) suspended in dry CHCl3 (10
mL) was added to reagent 1 kept at e10 °C with continuous stirring
followed by the addition of diethylamine (10% v/v) in CHCl3 (7 mL).
The resulting solution was diluted with ammonium chloride solution
(25 mL, 10% w/v), and the organic phase was separated and dried over
anhydrous Na2SO4. Chilled hexane (50 mL) was added to the organic
phase to get the precipitate of trityl glycine. The reaction was followed
by TLC [hexane/ethyl acetate (4:1) as developing medium] until
completion. The precipitate was filtered off to get a solid product, which
was washed with water (50 mL) and dried with anhydrous Na2SO4.
The complete removal of CHCl3 was ensured by adding a few milliliters
of methanol, and reconcentration was done in a vacuum. The excess
hexane was evaporated off, and the mixture was poured into ice-cold
water followed by recrystallization of the residue in ethanol to obtain
a white crystalline solid of 9, which was homogeneous, by TLC; yield
Benzyl Glycine p-Toluenesulfonic Acid Salt (17). To a solution of
glycine (0.25 mol, 18.8 g) were added fresh benzyl alcohol (100 mL),
p-toluenesulfonic acid (PTSA) (0.26 mol, 48.5 g), and dry benzene
(50 mL), reacted following Figure 3 to obtain grayish white crystals
of the title compound: yield 26.98 g (32%); mp 132-134 °C; TLC Rf
) 0.21; 1H NMR (CDCl3) δ 4.95 (s, 2H, -OCH2), 3.70 (s, 2H,
-COCH2), 2.20 (s, 3H, -CH3), 7.60 (m, 2H, Ha,Ha′-aromatic), 7.20
(m, 1H, Hb-aromatic), 7.10 (m, 2H, Hb′,Hc-aromatic), 9.01 (s, 1H, NH).
Benzyl Glycine (18). The title compound (yellowish oil) was made
as described for the synthesis of benzyl glycine (Figure 3) by the
addition of an aqueous solution of 5 N NaOH to a stirred suspension
of PTSA-benzyl glycine salt (0.025 mol) in benzene: yield 1.6 mL
1
1.09 (23%); mp 178-179 °C; TLC Rf ) 0.33; H NMR (CDCl3) δ
1.60 (d , J ) 6 Hz, 2H, -CH2), 7.30 (m, 15H, aromatic), 10.20 (s, 1H,
NH); El-MS, m/z (rel intensity %) 315 (M+, 24), 259 (32), 243 (100),
167 (15), 107 (29), 91 (74), 77 (31), 65(14).
Field Evaluation of CHAs. Three high-yielding varieties of bread
wheat (Triticum aestiVum L.), namely, PBW 343, HW 2046, and HD
2733, recommended for the North Western Plain Zone of India, were
chosen for chemical induction of male sterility. The experiment was a
randomized block design (RBD) 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. Row to row
distance was kept at 30 cm. Five rows of 2 m length were taken as a
plot in which the outermost two rows were treated as the pollen parents
and the inner three rows as female parents. The incoming foreign pollens
were prevented from entering 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) containing Tween-80 (1 g) was prepared. From EC 5 stocks
appropriate dilutions with water furnished a spray emulsion of 1500
ppm for trials. The synthetic compounds were tested in the winter of
2001-2002, sprayed at premeiotic stage when the length of the spike
1
(39%); TLC Rf ) 0.49; H NMR (CDCl3) δ 1.05 (s, 2H, -COCH2),
1.70 (s, 2H, -OCH2), 5.00 (s, 1H, NH2), 7.20 (m, 5H, aromatic); El-
MS, m/z (rel intensity %) 165 (M+, 19), 164 (70), 148 (25), 107 (59),
58 (12), 107 (100), 91 (62), 77 (72), 65 (22).
Benzyl â-Alanine p-Toluenesulfonic Acid Salt (19). To a stirred
solution of â-alanine (0.25 mol, 22.28 g) were added PTSA (0.26 mol,
48.5 g) and benzyl alcohol (100 mL) in dry benzene (50 mL), and the
mixture was heated to reflux following the general procedure to furnish
a solid compound. The resulting solid was recrystallized from diethyl
ether/hexane (1:1) to furnish a white crystalline solid of the title
1
compound: yield 28%; TLC Rf ) 0.16; H NMR (CDCl3) δ 4.97 (s,
2H, -OCH2), 2.20(s, 3H, -CH3), 7.80 (m, 2H, H′a,Ha-aromatic), 7.20
(m, 3H, Hb,Hb′,Hc-aromatic).

7904 J. Agric. Food Chem., Vol. 53, No. 20, 2005
Chakraborty and Devakumar
Figure 4. Sketch model of herbicide−CHA chimera in dalapon and 2,4-D analogue.
emerging from the first node was 6-9 mm (60 days after sowing) (14).
Pollen from the pollinator was dusted on 10 spikes of female parents,
and bagging was done immediately to prevent any further cross-
pollination. 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
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 percentage. To
study the floret fertility, the numbers of fertile (filled) and sterile
(unfilled) grains per spike were counted, and percent male sterility was
computed using the following formula: % floret sterility ) (Sc - Sf)/
Sc × 100, where Sc ) seeds per spike in control plants and Sf ) seeds
per spike in bagged and treated plants. Data were subjected to statistical
analysis, and on the basis of the significance at 5% levels (p ) 0.05)
the results were compared.
glycine or alanine, thus competitively inhibiting the active site
of the enzyme required to synthesize the amino acids. Moreover,
N-acylaniline derivatives containing a substituted amide linkage
[-CO-NH- or -CO-(CH2)n-NH- moiety] in the acyl side
chain were found to be essential to impart CHA activity of the
compounds as obvious from the very high activity (>98%
spikelet sterility) of ethyl 4′-fluoro oxanilate (1). This idea
prompted us to utilize these templates in synthesizing various
amino acid analogues, namely, the benzyl ester of glycine (18)
and N-tritylglycine (21), by means of C- and N-protection
strategies. Benzyl methyl 2-oxo-3-aza-adipate (20) was synthe-
sized by condensation between dimethyl ester of oxalate and
benzyl glycine (Figure 3).
The compounds were purified using physical and chromato-
graphic separation methods. Their structures were confirmed
using 1H NMR and mass spectroscopy. The characteristic feature
RESULTS AND DISCUSSION
1
of the H NMR spectra of N-acylanilines was the presence of
a broad singlet due to the anilide NH proton around δ 8.68 (
2.58. The 4′-fluoro and trifluoromethyl substituents in the aryl
ring of N-acylanilines caused marked deshielding of the highly
exchangeable NH protons due to its electron-withdrawing -I
effect. The group -COC2H4OMe as in 6 caused deshielding of
Ha by about 0.15 ppm and of Hb and H′b aromatic protons by
about 0.70 and 0.50 ppm when compared to that with 4′-
fluoroacetanilide (δHa ) 7.65, δHb ) 7.10, δNH ) 10.00). The
deshielding effect was due to the strong electron-withdrawing
phenyl and carboxymethyl groups, which withdraw the electron
cloud from the aromatic ring, resulting in the deshielding of
the aromatic protons. The descending order of shielding of
different side chains was COOCH3 (-1.2 ppm) > COOiPr
(-1.0 ppm) > COC2H4OCH3 (-0.73 ppm) compared to the
base compound. The methylene and methine protons had
chemical shifts of δ 4.0-4.05 and δ 4.4-6.2, respectively, in
chloroacetanilides and dichloroacetanilides, corresponding to the
electronegativity effect of the chlorine atom. In N-(2,2-di-
chloropropanoyl)-4-fluoro anilide, the side chain -C(Cl)2CH3
caused substantial deshielding of the aromatic protons, due to
the electronegative chlorine atom. In benzyl glycine the aromatic
Synthesis and Spectral Analyses. Several N-acylanilines
containing variations at the acyl domain were prepared by
essentially keeping fluoro at the para-position of the aromatic
ring and screened as CHAs. The N-acylanilines (1-12) were
synthesized by condensation of substituted anilines with ap-
propriate diesters or acid chlorides or monoesters as the case
might be as detailed under the earlier section. Dalapon
(H3CCH2COO-Na+) was reported to have CHA activity (9),
albeit with adverse phytotoxic effects. It was of interest to
understand whether the target gametocidal activity could be
made more selective by using a chimeric structure drawn from
dalapon and 4-fluoro anilinyl moieties (Figure 4). This chimeric
compound was synthesized from the synthon N-2-oxopropanoyl-
4-fluoroaniline, which was chlorinated by PCl5 to obtain N-(2,2-
dichloropropanoyl)-4-fluoroanilide (Figure 1) (13). 2,4-Di-
chorophenol and diethyl esters of oxalate, malonate, or
ethylacetoacetate containing anhydrous K2CO3 were refluxed
to obtain ethyl 2′,4′-dichlorophenyl oxalate, malonate, and
acetoacetate (Figure 2) (14-16). The amino acid analogues
(17-20) synthesized appeared to act as mimics of two essential
precursors for sporopollenin synthesis in pollen grains, namely,

N-Acylanilines, Herbicides, and Amino Acids as CHAs
J. Agric. Food Chem., Vol. 53, No. 20, 2005 7905
Table 1. Effect of N-Acylanilines, Herbicide
Test Concentrations on Three Genotypes of Wheat in Winter 2001
−
CHA Chimera, and Amino Acid Analogues on the Percent Induction of Spikelet Sterility at 1500 ppm
−2002
spikelet sterility (%)
HW 2046
Compound Number
X
R
PBW 343
99.97
99.57
84.32
67.15
51.71
87.07
84.66
89.12
61.35
39.92
63.75
HD 2733
99.99
99.98
84.71
69.29
52.35
87.38
84.46
89.36
64.13
41.28
65.10
1
2
3
4
5
6
7
8
4-F
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
NHCOCOOEt
NHCOCOOEt
99.48
99.83
81.69
64.20
50.38
84.06
80.30
87.00
59.33
38.17
56.05
85.00
71.33 (68.45)
75.10 (65.71)
58.00
60.99
38.10
4-CF3
4-F
NHCOCOOCH3
NHCOCOOiPr
NHCOCOCH3
NHCOCOC2H4OMe
NHCOCH2COOEt
NHCO CH2COCH3
NHCOCH3
NHCOCH2Cl
NHCOCHCl2
NHCOCCl3
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
9
10
11
12
13
14
15
16
17
18
19
20
21
control
4-F
4-F
89.61
91.20
a
NHC(Cl)2CH3
75.28 (76.29)
77.77 (69.15)
61.79
63.01
41.15
51.13
37.22
71.53
28.16
76.83 (75.22)
79.11 (71.58)
65.02
67.87
42.75
53.86
38.93
73.87
30.24
2,4-di-Cl
2,4-di-Cl
2,4-di-Cl
H
OCOCOOEtb
OCOCH2COOEt
OCOCH2COCH3
R1c
R2c
R3c
R4c
R5c
H
H
H
H
44.16
30.83
68.07
26.49
0.33
0.33
0.49
CD (p
)
0.05)
6.96
3.82
5.22
a Corresponding values of dalapon are in parentheses. b Corresponding values of 2,4-D are in parentheses.
c
protons appeared at about δ 7.20 as a singlet and the aromatic
in the side chain of chloroacetanilides (10-12) led to an increase
in the activity. 4′-Fluoro trichloroacetanilide (12) showed the
highest induction of male sterility (88.6 ( 2.6%) in the three
genotypes of wheat. As may be seen from Table 2, dalapon
was phytotoxic to wheat at 1500 ppm, but the herbicide
analogue, namely, N-(2,2-dichloropropanoyl)-4-fluoro anilide
(13), was not phytotoxic. Moreover, it had shown profound
effect on male sterility on the three genotypes of wheat (74.5
( 2.3%). Among 2,4-dichlorophenyl ester analogues, maximum
male sterility was induced by ethyl 2′,4′-dichlorophenyl oxalate
(14) followed by 2′,4′-dichlorophenyl acetoacetate (16).
Benzyl glycine (18) as well as its p-toluenesulfonic acid salt
(17) exhibited higher induction of male sterility as compared
to benzyl â-alanine PTSA salt (19) and trityl glycine (21).
Maximum male sterility (73.87% in HD 2733) was induced by
benzyl methyl 2-oxo-3-azaadipate (20). This offers the op-
portunity of exogenous application of amino acid or their
derivatives containing substituted amide linkage in the side chain
to induce male sterility. It was of interest to note that amidate
showed induction of >70% male sterility, making this series
of compounds deserving of further investigations.
protons did not split up significantly. The order of deshielding
of aromatic protons in benzyl â-alanine PTSA salt appeared to
be the same as that of benzyl glycine PTSA salt, the order of
deshielding being Ha ) Hb > Hc > Ha′ ) Hb′. In benzyl methyl-
2-oxo-3-aza-adipate (20) the aromatic protons did not split up
much and appeared as a multiplet at δ 7.25.
The M+ ion was conspicuous in the mass spectra of alkyl
oxanilates; the base peak was found to be either a protonated
aryl isocyanate moiety (m/z 138) or an azatropylium ion,
whereas in malonanilates and chloroacetanilides the breakdown
to parent anilines (m/z 111) appeared to be the base peak. In
N-(2,2-dichloropropanoyl)-4-fluoroanilide (13), the aryl iso-
cyanate moiety was found to be the base peak, whereas in 2,4-
dichlorophenyl esters, 2,4-dichlorophenol (m/z 163) appeared
to be the base peak. Azatropylium (m/z 91) ion was determined
to be the base peak in benzyl methyl 2-oxo-3-aza-adipate (20).
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 are given
in Table 1. The para-substituted ethyl oxanilates (1 and 2)
containing F and CF3, respectively, were found to be the best
in that order. Among N-acylanilines, 2-methoxyethyl 4′-fluoro
oxanilate (6) was found to induce 86.2 ( 1.5% male sterility
in the wheat genotypes. An increasing number of chlorine atoms
Effect of CHAs on Pollen Sterility. An assessment of pollen
sterility using KI-I2 (2%) or acetocarmine (1%) staining showed
that pollen sterility was highly correlated (r ) 0.9872) with
spikelet sterility. Sterile pollen grains were transparent and

7906 J. Agric. Food Chem., Vol. 53, No. 20, 2005
Chakraborty and Devakumar
study provide for improved seed quality and are able to be used
on a wide array of genotypes. The most potent CHAs had less
impact on various agronomic features and female fertility. Ethyl
4′-trifluoromethyl oxanilate (2) appeared to be a highly selective
CHA in this study (Table 2). The guiding principles both for
activity and for selectivity of CHAs were designed by utilizing
different descriptor variables, namely, Swain-Lupton field
constant (Fp), hydrophobic parameter π, molar refractivity, bulk
descriptor parachor, etc. As far as activity was concerned, Fp
appeared to be most important. Ethyl 4′-fluoro (1) and trifluoro-
methyl oxanilate (2) were very active because F and CF3 have
very high Fp values (Fp ) 0.43 and 0.38, respectively). In a
competitive binding at the bioreceptor site, a negative Fp term
could mean unfavorable conformational changes in the enzyme-
inhibitor complex as compared to favorable conformational
changes caused by the enzyme-substrate complex. The lead
derived from the present study can be valuable in exploring
the primary site and mode of action of these CHAs. The guiding
principle for selectivity was the π value. For the more selective
trifluoromethyl analogue (2), the π value is positive [ethyl 4′-
trifluoromethyl oxanilate; π(CF3) ) 0.88]. It is apparent from
the study that increased hydropobicity (π) as in ethyl 4′-
trifluoromethyl oxanilate has an efficient macromolecular recep-
tor fit at the enzyme active site. The compounds in the
N-acylaniline series are lipophilic esters allowing a better
penetration inside wheat. The esters appeared to be transformed
in the leaves to give the free acids, which are very probably
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 stamen at this stage. However, the amino acid
analogues, namely, 17-21, seem to be highly hydrophilic, and
their uptake is likely to be difficult. They are also probably
submitted to hydrolysis in the apoplastic space. That might
explain why these compounds are poorly effective as CHA.
Table 2. Effect of CHAs on Different Agronomic Traits and Female
Fertility on PBW 343 at 1500 ppm Test Concentration
spike
length
(cm)
female
fertility
(%)
Compound
Number
plant
ht (cm)
1000-grain
wt (g)
germination
(%)
1
2
8
12
dalapon
2,4-D
13
14
15
16
17
18
19
20
21
66.22
69.25
74.44
63.07
45.97
48.62
62.55
63.81
66.70
60.25
56.32
64.01
58.25
66.11
64.29
69.89
6.48
10.30
10.92
10.73
8.53
7.26
7.53
9.34
9.59
10.42
10.90
8.55
9.64
8.02
10.83
9.73
10.99
1.26
38.70
38.65
36.83
29.73
24.14
25.83
38.83
38.24
36.81
31.88
28.73
33.11
28.57
34.12
37.91
39.82
0.94
96.52
96.81
93.99
79.96
47.21
44.08
85.61
82.93
84.25
89.57
66.92
89.43
67.88
85.00
87.71
99.88
2.51
97.93
98.89
97.13
81.66
57.73
52.59
92.58
88.21
95.60
84.72
70.91
82.55
68.39
88.60
91.15
99.80
0.72
emulsion control
CD (p
) 0.05)
a Compound names of the corresponding compound number are mentioned in
Table 1.
clearly distinguishable from fertile pollens, which stained
uniform deep red or blue in the acetocarmine or KI-I2 stain
test, respectively (Figure 5). The sterile pollen grains showed
with irregular shape and increasing disintegration. The absence
of any starch material in the sterile pollen grains as shown by
the KI-I2 stain test could be indicative of either of the processes
leading to starch depletion or blocking its synthesis. This could
provide an important lead in unraveling the mode of action of
the CHAs.
Effect of CHAs on Performance Evaluation. The chemical
induction of male sterility through the suppression of pollen
formation is a rapid and flexible method to prepare hybrids.
The first generation of chemical compounds to be tested as
CHAs generally caused a high degree of phytotoxicity at rates
required for effective sterility. This often resulted in poor female
receptivity or fertility or failed to produce adequate male sterility.
The risk of commercial production or their utility in developing
breeding populations is unacceptable with any of this class of
compounds. The second generations of CHAs developed by this
The acidic pH generated by the hydrolysis of the ester moiety
in N-acylaniline series of CHAs led to an imbalance of acid-
base equilibria in the pollen mother cells, resulting in dissolution
of the microsporocyte callose wall during the meiotic -I
prophase by premature callase secretion. The high hydrophilicity
of the acids (viz., oxanilic acid obtained from the hydrolysis of
N-acylanilines, 1-5) results in an easy water solubility and
phloem transport, which add to the efficiency of the active
Figure 5. Sterile pollens of wheat due to treatment of N-(2,2-dichloropropanoyl)-4-fluoroanilide (13) vis-a`-vis fertile pollens as revealed from KI−I2 stain
test: (A) fertile pollens with fully developed cytoplasm of T. aestivum in untreated plants; (B) sterile pollen grains of T. aestivum in CHA-treated plants;
(C) magnified view of sterile pollen grains showing sterile transparent cytoplasm.

N-Acylanilines, Herbicides, and Amino Acids as CHAs
J. Agric. Food Chem., Vol. 53, No. 20, 2005 7907
LITERATURE CITED
CHAs. Therefore, chemicals with high water solubility would
be preferred as active CHAs. The solubility and log(octanol/
water) value of ethyl 4′-fluoro oxanilate (1) were experimentally
recorded to be 0.5332 g/L and 1.506 at 25 °C, which indicate
the high hydrophilicity of the CHA. The chimeric synthon
derived from herbicides (dalapon and 2,4-D) and CHA agro-
phores (4-fluoro anilinyl and â-ethoxycarbonyl moieties) ap-
peared to be more selective than their parent herbicides. It is
apparent that unlike dalapon, the herbicide-CHA chimera,
N-(2,2-dichloropropanoyl)-4-fluoro anilide (13), did not show
any significant reduction in plant height (7.34 cm with respect
to the emulsion control). Among amino acid analogues, the
hydrophilic salts (e.g., benzyl glycine p-toluenesulfonic acid salt)
were found to be less selective in action as compared to their
neutral analogues (Table 2). The key to the use of a CHA in
the commercial production of hybrid wheat is to achieve a high
degree of selectivity in terms of male sterility versus female
fertility. Past attempts using herbicides and plant growth
regulators, as gametocides have not proved to be successful due
to less selectivity, namely, induction of very high female
sterility. The data pertaining to female fertility revealed that
N-(2,2-dichloropropanoyl)-4-fluoro anilide (13) and 2,4-dichloro-
phenyl ester analogues (14-16) showed a marginal reduction
in female fertility. Among amino acid derivatives, benzyl glycine
(18) and benzyl methyl 2-oxo-3-aza-adipate (20) exhibited a
moderate (10-15%) reduction in female fertility at 1500 ppm.
Dalapon and 2,4-D, in comparison, had marked detrimental
effect on female fertility, showing a reduction of 52-55% in
PBW 343. There is ample scope for the development of potent
CHA analogues based on the leads postulated and predicted in
the present study. The CHAs not only induce 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 increase the probability for the development
of hybrids. Second floret opening started after the lodicules had
collapsed; the carpels in the sterile floret continued to grow palea
and lemma apart. This second opening lasted for longer 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.
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ACKNOWLEDGMENT
We are thankful to the Director, IARI, New Delhi, for providing
necessary facilities to carry out the work. We also thank Dr. S.
M. S. Tomar, Principal Scientist, Division of Genetics, IARI,
New Delhi, for useful discussion and help in the execution of
the study.
Received for review June 21, 2005. Revised manuscript received August
10, 2005. Accepted August 11, 2005.
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