Chemical synthesis, in vitro acetohydroxyacid synthase (AHAS) inhibition, herbicidal activity, and computational studies of isatin derivatives

Article abstract of DOI:10.1021/jf2021607

Acetohydroxyacid synthase (AHAS) catalyzes the first common step in the biosynthesis of the branched-chain amino acids. As a result of its metabolic importance in plants, it is a target for many commercial herbicides. Virtual screening analysis inspired the evaluation of 19 commercially available isatin analogues and 13 newly synthesized isatin derivatives as novel AHAS inhibitors and for their herbicidal activity. The best compound demonstrated 95% inhibition of the activity of Arabidopsis thaliana AHAS at a concentration of 100 mg L^-1, whereas the herbicidal activities of three compounds reached 50% inhibition at a concentration of 10 mg L^-1 using the rape root growth test. CoMFA contour models were established to understand the structure-activity relationships for this class of AHAS inhibitor. The compounds were docked to the active site cavity of A. thaliana AHAS using FlexX, and the dominant binding mode was consistent with frontier molecular orbital from DFT calculations. This is the first comprehensive study of isatin derivatives as AHAS inhibitors and provides a valuable starting point for the design of new herbicides.

Full text of DOI:10.1021/jf2021607

ARTICLE
pubs.acs.org/JAFC
Chemical Synthesis, in Vitro Acetohydroxyacid Synthase (AHAS)
Inhibition, Herbicidal Activity, and Computational Studies of Isatin
Derivatives
Jianguo Wang,*,† Haizhong Tan,† Yonghong Li,† Yi Ma,† Zhengming Li,† and Luke W. Guddat‡
†State-Key Laboratory and Institute of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, China
‡School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia
S Supporting Information
b
ABSTRACT: Acetohydroxyacid synthase (AHAS) catalyzes the first common step in the biosynthesis of the branched-chain amino
acids. As a result of its metabolic importance in plants, it is a target for many commercial herbicides. Virtual screening analysis inspired
the evaluation of 19 commercially available isatin analogues and 13 newly synthesized isatin derivatives as novel AHAS inhibitors and
for their herbicidal activity. The best compound demonstrated 95% inhibition of the activity of Arabidopsis thaliana AHAS at a con-
centration of 100 mg Lꢀ1, whereas the herbicidal activities of three compounds reached 50% inhibition at a concentration of 10 mg Lꢀ1
using the rape root growth test. CoMFA contour models were established to understand the structureꢀactivity relationships for this
class of AHAS inhibitor. The compounds were docked to the active site cavity of A. thaliana AHAS using FlexX, and the dominant
binding mode was consistent with frontier molecular orbital from DFT calculations. This is the first comprehensive study of isatin
derivatives as AHAS inhibitors and provides a valuable starting point for the design of new herbicides.
KEYWORDS: AHAS, rational design, synthesis, isatin derivatives, biological activity, herbicide, QSAR, docking, DFT calculation,
molecular design
’ INTRODUCTION
monoamine oxidase B (MAO-B),18,19 where it triggers a dose-
and time-dependent switch from apoptosis to necrosis in human
neuroblastoma cells.20 Derivatives of isatin can also have anxio-
genic, sedative, antitubercular, and anticonvulsant activities and act
in vitro as potent antagonists on arterial natriuretic peptide
receptors.21 Herbicidal activities have also been reported for some
isatin derivatives.22ꢀ25 However, to date, isatins have not been
identified as inhibitors of AHAS. Although a large number of
publications and patents describing the synthesis and biological
activity of AHAS inhibitors have appeared in the literatures, very
few in recent years have identified new lead structures. This paper
describes the in vitro and in vivo activity of isatin derivatives as
AHASinhibitors. Inaddition, three-dimensional(3D) quantitative
structureꢀactivity relationship (3D-QSAR) analysis and density
functional theory (DFT) calculations have been undertaken to
determine the steric, electrostatic, and frontier molecular orbital
features that influence the inhibitory activity.
The branched-chain amino acids (BCAAs) can be biosynthe-
sized only by plants and micro-organisms. Animals do not have this
ability, obtaining these necessary nutrients from their diet to
maintain survival. Thus, enzymes involved in the biosynthesis of
the BCAAs are regarded as targets for the development of
herbicides or antimicrobial agents.1 Acetohydroxyacid synthase
(AHAS; EC 2.2.1.6, also referred to as acetolactate synthase, ALS)
has been a successful herbicidal target for nearly 30 years.2ꢀ4 Four
families of AHAS inhibitors that have been widely used as
herbicides (Figure 1) are sulfonylureas (SU), imidazolinones
(IMI), pyrimidinylthiobenzoates (PTB), and triazolopyrimidine
sulfonanilides (TP), and these have contributed enormously to
crop protection throughout the world.5,6 However, with the over-
use of these chemicals, resistant weed strains have become a major
problem in many areas.7ꢀ11 Thus, there is an urgent need to
discover AHAS inhibitors with new chemical structures as potential
replacements for those herbicides that are now no longer effective.
The binding modes of the sulfonylureas and one imidazoli-
none (imazaquin) to AHAS were previously determined by X-ray
crystallography.12ꢀ16 These studies have opened the way to the
rational design of novel AHAS inhibitors. In 2007, we reported
the identification of several novel AHAS inhibitors using virtual
screening.17 Among these was an indigo-based compound, and
we hypothesized that the structure of isatin, a half moiety of
indigo, might also be active toward AHAS.17 Thus, we built a
library of isatin derivatives to test their inhibition of AHAS and to
see if these possess herbicidal activity.
’ MATERIALS AND METHODS
General Synthesis and Instruments. Compounds 1ꢀ19 were
purchased from Alfa-Aesar or Sigma-Aldrich. Compounds 20ꢀ32 were
synthesized in our laboratory, and the commercial sources of the starting
materials were purchased from the Tianjin Guangfu Fine Chemical
Research Institute, Alfa-Aesar, or Sigma-Aldrich. The crystal structure of
Received: June 1, 2011
Revised:
August 5, 2011
Isatin, a marine natural product, has previously been reported to
display significant antifungal activity due to its inhibition of
Accepted: August 12, 2011
Published: August 13, 2011
r
2011 American Chemical Society
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Journal of Agricultural and Food Chemistry
ARTICLE
57%; mp, 273ꢀ274 °C; 1H NMR (DMSO-d6, 300 MHz), δ 13.82 (s,
1H, NNH), 11.38 (s, 1H, NH), 7.77 (d, 1H, J = 7.8 Hz, Ar1ꢀH4),
7.74ꢀ7.54 (m, 5H, 4Ar2ꢀH and Ar1ꢀH6), 6.93 (t, 1H, J = 7.8 Hz,
Ar1ꢀH5); ESI-MS, m/z 408 [M ꢀ H]ꢀ. Anal. Calcd for C15H9FIN3O2:
C, 44.03; H, 2.22;N, 10.27. Found: C, 43.68;H, 2.47; N, 10.43. The same
method was used to synthesize and purify compounds 20, 21, and 23.
Analytical data for compound 20: yellow solid; yield, 65%; mp,
284ꢀ286 °C; 1H NMR (DMSO-d6, 300 MHz), δ 13.76 (s, 1H, NNH),
11.61 (s, 1H, NH), 8.02ꢀ7.42 (m, 6H, 4Ar2ꢀH and Ar1ꢀH4,
Ar1ꢀH6), 7.06 (t, 1H, J = 8.1 Hz, Ar1ꢀH5); ESI-MS, m/z 360 [M ꢀ
H]ꢀ. Anal. Calcd for C15H9BrFN3O2: C, 49.75; H, 2.50; N, 11.60.
Found: C, 49.49; H, 2.68; N, 11.82.
Analytical data for compound 21: brownꢀyellow solid; yield, 66%;
1
mp, 287ꢀ289 °C; H NMR (DMSO-d6, 300 MHz), δ 13.81 (s, 1H,
NNH), 11.68 (s, 1H, NH), 7.73ꢀ7.53 (m, 6H, 4Ar2ꢀH and Ar1ꢀH4,
Ar1ꢀH6), 7.08 (t, 1H, J = 7.8 Hz, Ar1ꢀH5); ESI-MS, m/z 360 [M ꢀ
H]ꢀ. Anal. Calcd for C15H9BrFN3O2: C, 49.75; H, 2.50; N, 11.60.
Found: C, 49.56; H, 2.78; N, 11.71.
Figure 1. Representative examples of four AHAS inhibitors.
compound 24 has previously been published by our group.22 All solvents
were distilled before use. Melting points were determined using an X-4
melting apparatus and were uncorrected. 1H NMR spectra were obtained
using a 300 MHz Bruker AC-P300 spectrometer or a 400 MHz Varian
Mercury Plus 400 spectrometer in DMSO-d6 with TMS as an internal
standard. Elemental analyses were determined on a Yanaca CHN Corder
MT-3 elemental analyzer. Mass spectra were recorded on a Thermo
Finnigan LCQ Advantage LC/mass detector instrument. The control
herbicide, monosulfuron, was synthesized in our laboratory as described
previously.26 The structures of all compounds purchased or synthesized
are presented in Figure 2. Molecular modeling studies were computed
using an SGI 350 server and an SGI Fuel workstation.
Analytical data for compound 23: yellow solid; yield, 60%; mp,
291ꢀ293 °C; 1H NMR (DMSO-d6, 400 MHz), δ 11.76 (s, 1H, NNH),
11.12 (s, 1H, NH), 8.00 (d, 1H, J = 8 Hz, Ar2ꢀH3), 7.72 (d, 1H, J = 8 Hz,
Ar1ꢀH4), 7.59ꢀ6.97 (m, 5H, Ar2ꢀH4, Ar2ꢀH5, Ar2ꢀH6 and
Ar1ꢀH6, Ar2ꢀOH), 6.90 (t, 1H, J = 7.6 Hz, Ar1ꢀH5); ESI-MS m/z
406 [M ꢀ H]ꢀ. Anal. Calcd for C15H10IN3O3: C, 44.25; H, 2.48; N,
10.32. Found: C, 44.52; H, 2.39; N, 10.17.
Preparation of Compounds 24ꢀ26, Example Compound 24. A
mixture of 3-chloro-2-hydrazinylpyridine (e) (1.15 g, 8 mmol), isatin
(1.18 g, 8 mmol), and acetic acid (2.5 mL) was refluxed in a water/
ethanol solution (1:5, 40 mL) for 6 h. The mixture was then cooled to
room temperature and an orange solid precipitated out. The precipitate
was filtered, washed with water, dried, and recrystallized from ethanol to
give yellow crystals. Analytical data for compound 24 have been
previously reported.22 The intermediates e, f, and g were synthesized
by using a previously described method.27ꢀ29 The same method was
used to synthesize and purify compounds 25 and 26.22
Analytical data for compound 25: yellow solid; yield, 67%; mp,
256ꢀ258 °C; 1H NMR (DMSO-d6, 300 MHz), δ 12.75 (s, 1H, NNH),
11.27 (s, 1H, NH), 7.57 (br s, 1H, ArꢀH4), 7.39 (t, 1H, J = 7.5 Hz,
ArꢀH6), 7.10 (t, 1H, J = 7.2 Hz, ArꢀH5), 6.94 (d, 1H, J = 7.2 Hz,
ArꢀH7), 5.87 (s, 1H, HetꢀCH), 5.36 (s, 1H, COCH2), 5.01 (s, 1H,
COCH2), 2.19 (s, 3H, HetꢀCH3), 2.10 (s, 3H, HetꢀCH3); ESI-MS m/
z 296 [M ꢀ H]ꢀ. Anal. Calcd for C15H15N5O2: C, 60.60; H, 5.09; N,
23.56. Found: C, 60.33; H, 5.12; N, 23.27.
Analytical data for compound 26: yellow solid; yield, 67%; mp,
265ꢀ266 °C; 1H NMR (DMSO-d6, 400 MHz), δ 12.95 (s, 1H, NNH),
11.31 (s, 1H, NH), 7.61 (d, 1H, J = 7.6 Hz, ArꢀH4), 7.39 (t, 1H, J = 7.6
Hz, ArꢀH6), 7.12 (t, 1H, J = 7.6 Hz, ArꢀH5), 6.94 (d, 1H, J = 7.6 Hz,
ArꢀH7), 2.69ꢀ2.67 (br s, 6H, Hetꢀ2CH3); ESI-MS, m/z 299
[MꢀH]ꢀ. Anal. Calcd for C14H12N4O2S: C, 55.99; H, 4.03; N, 18.65.
Found: C, 55.89; H, 3.91; N, 18.29.
Preparation of Compounds 27ꢀ32, Example Compound 28. To a
solution of ethyl benzoate (22.53 g, 150 mmol) in anhydrous ethanol
(65 mL) was added 85% hydrazine hydrate (35 mL). The resulting
mixture was refluxed for 4 h and then cooled to room temperature. The
product precipitated out of solution, white crystals were collected by
filtration, and these were washed with cold water and dried to give
benzhydrazide (h) (18.5 g, 90%). A mixture of isatin (0.59 g, 4 mmol)
and potassium carbonate (2.5 g, 18 mmol) was heated at 65 °C in DMF
(30 mL). After 1 h, 1-(chloromethyl)-3-fluorobenzene (0.58 g, 4 mmol)
was added in portions, and the stirring was continued at 65 °C until no
isatin was detectable (TLC). The mixture was then cooled to room
temperature and poured into water (60 mL) with continual stirring.
After 40 min, 1-(3-fluorobenzyl)indoline-2,3-dione (i) was filtered,
washed with cold water, and then dried to give a crude red product,
Procedures for the Synthesis of Compounds 20ꢀ32
(Scheme 1). Preparation of Compounds 20ꢀ23, Example Com-
pound 22. 3-Fluorobenzoic acid (8.4 g, 60 mmol) was dissolved in
anhydrous ethanol (150 mL) in an iceꢀwater bath, and then SOCl2
(7.5 mL, 100 mmol) was added dropwise before stirring for 4 h at 0 °C.
Water (50 mL) was then added after the organic solvent, and SOCl2 was
removed under vacuum. The pH of the solution was adjusted to 8 by the
addition of 1 M NaOH and then extracted with EtOAc. The organic
solvent was removed to give the crude product ethyl 3-fluorobenzoate
(a) (8.24 g, 83%) as a colorless liquid. Hydrazine hydrate (85%, 10 mL)
was added to liquid a (8.24 g, 50 mmol), and the mixture was refluxed for
6 h before being cooled to room temperature. The product precipitated
out of solution after the resulting ethanol was removed. The white
crystals were collected by filtration, washed thoroughly with cold EtOAc,
and dried to give 3-fluorobenzoylhydrazine (b) (5.86 g, 76%). A mixture
of chloral hydrate (8.0 mL, 80 mmol) and Na2SO4 (85 g, 600 mmol) was
warmed to 40 °C in aqueous solution (180 mL), and then 2-iodoaniline
(2.19 g, 10 mmol) in 10% HCl (85 mL) was added dropwise within 0.4 h,
and the mixture was stirred for 0.5 h at that temperature. Hydroxylamine
hydrochloride (14 g, 200 mmol) was added quickly. The mixture was
stirred and heated to 75 °C for 4 h and then cooled to room temperature.
The precipitate corresponding to 20-hydroxyimino-2-iodoacetanilide (c)
was filtered and dried to give a light-brown solid (18.53 g, 98%).
Concentrated sulfuric acid (70 mL) was warmed to 75 °C in a
200 mL flask fitted with an efficient stirrer, and dry c (18.53 g, 68 mmol)
was added at a suitable speed to keep the temperature between 75 and
80 °C. After that, the solution was heated to 80 °C for 0.5 h to complete
the reaction. The reaction mixture was next cooled to room temperature
and poured into crushed ice (30 g) with continual stirring. After 1 h, the
product was filtered, washed with cold water, and then dried to give a
crude red product, 7-iodoindoline-2,3-dione (d) (16.12 g, 86%). A
mixture of d (2.19 g, 8 mmol), b (1.23 g, 8 mmol), and acetic acid
(2.5 mL) was refluxed in a water/ethanol solution (1:5, 50 mL) for 6 h.
The mixture was then cooled to room temperature and a yellow solid
precipitated out. The precipitate was filtered, washed with water, dried,
and then recrystallized from ethanol to give a pure yellow solid 22: yield,
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Figure 2. Structures of isatin (compound 1) and derivatives tested in this study as novel AHAS inhibitors.
purified by flash column chromatography on silica gel (0.82 g, 80%), the
eluting solvent for which is ethyl acetate/petroleum ether (1:3). A
mixture of h (0.51 g, 2 mmol), I, and acetic acid (1.0 mL) was refluxed in
a water/alcohol solution (1:5, 25 mL) for 5 h. The reaction mixture was
next cooled to room temperature and a yellow solid precipitated out.
This precipitate was filtered, washed with water, and then dried to give a
crude yellow product. Recrystallization from ethanol gave pure yellow
Ar2ꢀH6), 7.73ꢀ7.10 (m, 10H, 4 Ar3ꢀH, Ar2ꢀH3, Ar2ꢀH4, Ar2ꢀH5,
Ar1ꢀH4, Ar1ꢀH5, and Ar1ꢀH6), 7.07 (d, 1H, J = 7.6 Hz, Ar1ꢀH7),
5.05(s, 2H, Ar3ꢀCH2N); ESI-MS, m/z 372 [M ꢀ H]ꢀ. Anal. Calcd for
C22H16FN3O2: C, 70.77; H, 4.32; N, 11.25. Found: C, 70.42; H, 4.39; N,
11.11. The same method was used to synthesize and purify compounds
27 and 29ꢀ32.
Analytical data for compound 27: yellow solid; yield, 98%; mp,
242ꢀ243 °C; 1H NMR (DMSO-d6, 300 MHz), δ 13.81 (s, 1H, NNH),
7.94 (d, 2H, J = 7.8 Hz, Ar2ꢀH2 and Ar2ꢀH6), 7.83 (d, 2H, J = 8.4 Hz,
1
solid 28: yield, 71%; mp, 165ꢀ166 °C; H NMR (DMSO-d6, 400
MHz), δ 13.84 (s, 1H, NNH), 7.94 (d, 2H, J = 6.8 Hz, Ar2ꢀH2 and
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Scheme 1. Synthetic Routes for New Isatin Derivatives 20ꢀ32
Ar3ꢀH3 and Ar3ꢀH5), 7.71ꢀ7.60 (m, 6H, Ar3ꢀH2, Ar3ꢀH6, Ar2ꢀH3,
Ar2ꢀH4, Ar2ꢀH5, and Ar1ꢀH4), 7.42 (t, 1H, J = 7.8 Hz, Ar1ꢀH6), 7.19
(t,1H, J=7.5 Hz, Ar1ꢀH5), 7.06 (d, 1H,J=8.1Hz, Ar1ꢀH7), 5.13(s, 2H,
Ar3ꢀCH2N); ESI-MS, m/z 379 [M ꢀ H]ꢀ. Anal. Calcd forC23H16N4O2:
C, 72.62; H, 4.24; N, 14.73. Found: C, 72.88; H, 4.39; N, 14.76.
Ar2ꢀH3, Ar2ꢀH4, Ar2ꢀH5, Ar1ꢀH4, and Ar1ꢀH6),7.21(t, 1H,J=7.5Hz,
Ar1ꢀH5), 6.99 (d, 1H, J= 7.8 Hz, Ar1ꢀH7), 5.07 (s, 2H, Ar3ꢀCH2N); ESI-
MS, m/z422 [Mꢀ H]ꢀ. Anal. Calcd for C22H15Cl2N3O2:C, 62.28;H, 3.56;
N, 9.90. Found: C, 62.07; H, 3.66; N, 9.70.
Analytical data for compound 32: yellow solid; yield, 59%; mp,
171ꢀ172 °C; 1H NMR (DMSO-d6, 300 MHz), δ 13.87 (s, 1H, NNH),
7.94 (d, 2H, J = 7.2 Hz, Ar2ꢀH2 and Ar2ꢀH6), 7.71ꢀ7.17 (m, 10H,
4Ar3ꢀH, Ar2ꢀH3, Ar2ꢀH4, Ar2ꢀH5, Ar1ꢀH4, Ar1ꢀH5, and
Ar1ꢀH6), 7.14 (d, 1H, J = 7.5 Hz, Ar1ꢀH7), 4.98 (s, 2H, Ar3ꢀCH2N),
1.24 (s, 9H, Ar3ꢀC(CH3)3); ESI-MS, m/z 411 [M ꢀ H]ꢀ. Anal. Calcd
for C26H25N3O2: C, 75.89; H, 6.12; N, 10.21. Found: C, 75.66; H, 5.95;
N, 10.20.
Analytical data for compound 29: light-yellow solid; yield, 92%;
1
mp, 213ꢀ214 °C; H NMR (DMSO-d6, 400 MHz), δ 13.85 (s, 1H,
NNH), 7.94 (d, 2H, J = 7.6 Hz, Ar2ꢀH2 and Ar2ꢀH6), 7.73ꢀ7.16 (m,
10H, 4 Ar3ꢀH, Ar2ꢀH3, Ar2ꢀH4, Ar2ꢀH5, Ar1ꢀH4, Ar1ꢀH5, and
Ar1ꢀH6), 7.10 (d, 1H, J = 8.0 Hz, Ar1ꢀH7), 5.01 (s, 2H, Ar3ꢀCH2N);
ESI-MS, m/z 372 [M ꢀ H]ꢀ. Anal. Calcd for C22H16FN3O2: C, 70.77;
H, 4.32; N, 11.25. Found: C, 70.88; H, 4.48; N, 11.59.
Analytical data for compound 30: orange solid; yield, 88%; mp,
187ꢀ188 °C; 1H NMR (DMSO-d6, 300 MHz), δ 13.86 (s, 1H, NNH),
7.93 (d, 2H, J = 8.1 Hz, Ar2ꢀH2 and Ar2ꢀH6), 7.72ꢀ7.08 (m, 9H,
4Ar3ꢀH, Ar2ꢀH3, Ar2ꢀH4, Ar2ꢀH5, Ar1ꢀH4, and Ar1ꢀH6), 7.06 (t,
1H, J = 7.8 Hz, Ar1ꢀH5), 6.96 (d, 1H, J = 7.8 Hz, Ar1ꢀH7), 5.01 (s, 2H,
Ar3ꢀCH2N), 2.40 (s, 3H, Ar3ꢀCH3); ESI-MS, m/z 368 [M ꢀ H]ꢀ.
Anal. Calcd for C23H19N3O2: C, 74.78; H, 5.18; N, 11.37. Found: C,
74.51; H, 5.08; N, 11.29.
Analytical data for compound 31: yellow solid; yield, 85%; mp,
202ꢀ204 °C; 1H NMR (DMSO-d6, 300 MHz), δ 13.77 (s, 1H, NNH),
7.92 (d, 2H, J=6.9Hz,Ar2ꢀH2 and Ar2ꢀH6), 7.73ꢀ7.30 (m, 8H, 3Ar3ꢀH,
Biological Assays. Determination of Inhibition of Arabidopsis
thaliana AHAS (AtAHAS). The expression and purification of AtAHAS
has been described previously.30 AHAS activity was measured using the
colorimetric assay in a previous publication.17
In Vivo Inhibition of the Root Growth of Rape (Brassica campestris L).
The in vivo data were determined using our previously published
method.31
Molecular Modeling. CoMFA. Chemical structures of the com-
pounds were built within Sybyl 7.3 (Tripos Inc., St. Louis, MO). All of
the molecules were assigned MMFF94 charges and minimized by
the Tripos force field when convergence reached 0.001 kcal molꢀ1 Åꢀ1
.
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Table 1. In Vitro and in Vivo Biological Activity of the Isatins
2; HOMO, highest occupied molecular orbital; LUMO, lowest unoccu-
pied molecular orbital) was calculated precisely. Frequency analysis was
performed, and partial atomic charges were derived from a population
analysis based on the natural bond orbital (NBO) method.38
AtAHAS inhibition (%)
rape root length inhibition (%)
no.
100 mg Lꢀ1
D
100 mg Lꢀ1
10 mg Lꢀ1
Molecular Docking. The molecular docking of the isatins to AHAS
was performed by FlexX.39 The crystal structure of AtAHAS in complex
with chlorimuron ethyl (pdb code 1YBH) was retrieved from the pdb
databank.6 All amino acids in the protein were modified in the
“Biopolymer” module to correct side-chain rotomer conformations.
All water molecules were removed, and hydrogen atoms were added
in the standard geometry. Any amino acid residue within 6.5 Å of the
location of the chlorimuron ethyl was considered to be in the binding
pocket. Cscore calculation was enabled and set to serial mode. Database
docking and subsequent scoring procedures were performed using the
default parameters of FlexX within Sybyl7.3. The figures were generated
by PYMOL (DeLano Scientific, South San Francisco, CA) and Chem-
Draw (ACD Laboratories, Toronto, Canada).
1
70 ( 3
55 ( 4
70 ( 3
65 ( 2
60 ( 2
65 ( 3
60 ( 3
45 ( 3
50 ( 4
60 ( 3
60 ( 3
60 ( 2
50 ( 3
75 ( 5
10 ( 2
60 ( 3
25 ( 3
70 ( 4
80 ( 4
95 ( 2
60 ( 3
75 ( 3
95 ( 1
70 ( 3
35 ( 2
30 ( 2
20 ( 1
95 ( 3
80 ( 3
50 ( 3
50 ( 2
60 ( 3
90 ( 1
6.14
6.69
6.94
6.39
6.57
6.60
6.68
6.72
6.62
6.70
7.21
6.59
6.61
6.26
8.02
6.83
7.42
6.41
6.27
6.40
7.50
7.35
6.55
6.94
7.69
7.75
8.34
6.44
7.11
7.71
7.89
7.67
52 ( 2
95 ( 1
92 ( 2
88 ( 2
30 ( 3
65 ( 2
82 ( 1
55 ( 2
24 ( 1
53 ( 1
27 ( 2
59 ( 1
80 ( 1
26 ( 1
18 ( 3
93 ( 1
33 ( 1
83 ( 2
59 ( 2
39 ( 1
16 ( 3
81 ( 1
25 ( 1
19 ( 1
27 ( 2
21 ( 3
48 ( 1
40 ( 1
15 ( 2
0
24 ( 1
28 ( 2
31 ( 3
52 ( 1
20 ( 1
10 ( 1
7 ( 1
0
2
3
4
5
6
7
8
9
7 ( 2
20 ( 1
13 ( 1
19 ( 2
13 ( 2
14 ( 2
4 ( 1
27 ( 2
2 ( 1
51 ( 2
31 ( 2
14 ( 1
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
C*a
’ RESULTS AND DISCUSSION
Synthetic Chemistry of the Title Compounds. Compounds
20ꢀ32 were synthesized by a mild condensation reaction from
indoline-2,3-dione (or substituted) and a substituted hydrazine
in the presence of acetic acid in ethanol solution under refluxing
conditions and gave favorable yields. The synthetic methods for
the intermediates in Scheme 1 are detailed under Materials and
Methods. It should be noted that it is theoretically possible for
both the E and Z isomers for every Schiff base to be synthesized.
However, for all of the title compounds only a single isomer was
obtained. From selected X-ray structures of isatin Schiff
bases,22,40,41 all of the molecules exist as Z isomers so that an
intramolecular hydrogen bond is formed between the isatin
unreacted carbonyl oxygen atom and the hydrogen atom bonded
to the nitrogen atom in the hydrazone group. This forms a large π
electron conjugate plane (formed by the three connected rings)
stabilizing the molecule. The identities and structures of all the
compounds synthesized were determined by ESI-MS, elemental
analysis, and 1H NMR.
51 ( 1
0
0
0
0
2
0
0
0
6 ( 1
0
Biological Activity. The in vitro and in vivo biological activities
of the 32 isatin derivatives are summarized in Table 1. The in vitro
activity was evaluated at a concentration of 100 mg Lꢀ1 against
AtAHAS, whereas the in vivo activity was measured using the rape
root inhibition method developed at Nankai University.42 The
sulfonylurea herbicide monosulfuron was used as the control.
These results show that most of the compounds exhibited con-
siderable in vitro and in vivo inhibitory activities. The most potent
of the compounds, 20, 23, and 28, showed 95% in vitro inhibition
against AtAHAS at a concentration of 100 mg Lꢀ1. At the same
concentration monosulfuron inhibited the activity of AtAHAS by
90%. In the rape root test these three compounds were not as
potent as expected, with no more than 40% inhibition of growth at
a concentration of 100 mg Lꢀ1, compared with 78% for mono-
sulfuron. On the other hand, compounds 2ꢀ4, 7, 13, 16, 18, and
22 displayed 80ꢀ95% inhibition of rape root growth at 100 mg
15 ( 1
78 ( 1
0
64 ( 2
a C*, control herbicide, monosulfuron.
The molecules were superimposed using isatin (1) as the template.
Comparative molecular field analysis (CoMFA) was used for the 3D-QSAR
analysis using default parameters.32 The “leave-one-out” (LOO) cross-
validation method was applied to determine the optimum number of
partial least-squares (PLS) components. The biological activities were
expressed in terms of D (Table 1) using the formula33ꢀ35
D ¼ log10½a=ð1 ꢀ aÞꢁ þ log10 Mw
where a is the percent inhibition of AHAS in vitro, Mw is the molecular
weight of the inhibitor, and D is the biological activity index.
DFT Calculation. Representative compounds 6, 20, and 29 were
chosen for DFT geometry optimization by the SCF method using the
B3LYP (Becke, three-parameter, LeeꢀYangꢀParr) function with a basis
set of 6-31 G(d) to describe their molecular properties.36,37 Gaussian03
was used to perform the calculations.38 The initial conformations were
chosen from the structural database for CoMFA. All computations were
conducted for the ground states of these molecules as singlet states. All
of the convergent precisions used were the system’s default values. The
frontier molecular orbital (HOMO-k, k = 0, 1, 2 and LUMO+k, k = 0, 1,
L
ꢀ1, which are values better than that observed for monosulfuron.
It is encouraging that compounds 4, 18, and 22 showed ∼50%
in vivo inhibition at 10 mg Lꢀ1, whereas at this level monosufuron
reduced the in vivo activity by 63.8%. Compounds 4, 18, and 22
were able to inhibit AtAHAS by 65, 70, and 75%, respectively.
Reports dating back to 1957 indicate that isatins can have
herbicidal activity, although the mechanism of action for these
compounds is unknown.23ꢀ25 It has previously been demonstrated
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Figure 4. LUMO, HOMO, and HOMO-1 maps for compounds 6, 20,
and 29 from DFT calculations. The green parts represent positive
molecular orbital, and the red parts represent negative molecular orbital.
components (CoMFA-2), with standard errors of prediction
(SEP) of 0.431 and 0.425, respectively. Both values are acceptable
considering the fairly large molecular diversity of the isatin
training set. The PLS statistics of all CoMFA models and the
figures for predicted activity versus experimental activity can be
found in the Supporting Information. The alignment of com-
pounds in the training set and 3D contour maps for CoMFA-1
and CoMFA-2 are shown in Figure 3. Compound 20 was used to
illustrate the structureꢀactivity relationships. The CoMFA-1 and
CoMFA-2 3D contour models are very similar, suggesting that for
this training set, using five components is acceptable. For the
steric contour map (Figure 3b,d), the green region displays a
position where a bulky group would be favorable for higher
AtAHAS inhibition; this region is located at the 5-position of the
isatin ring. The sterically unfavorable region is in yellow, and this
is mostly near the 1- and 7-positions of the isatin ring. For the
electrostatistic contour map (Figure 3c,e), the blue contour
defines a region where an increase in the positive charge will
result in an increase in affinity, whereas the red contour defines a
region of space where negative charge is favorable. From the
maps, it can be observed that the electrostatically favored region is
located near the 5-position of isatin and projects forward of the
3-position. An electrostatically disfavored region projects behind
the 3-position and is far from the 1- and 2-positions of isatin. On
the basisof these results it will be possibleto design andsynthesize
more potent isatin inhibitors of plant AHAS.
Figure 3. (a) Alignment of the isatins in the training set for CoMFA
analysis with the numbers of positions: (bꢀe) ball and stick model of
compound 20; (b, d) steric contour maps for the CoMFA models
(sterically favored and disfavored regions are shown in green and yellow,
respectively); (c, e) electrostatic contour maps for the CoMFA models
(positively charged favored regions are shown in blue, and negatively
charge favored regions are shown in red).
that inhibition of AHAS by the sulfonylureas (and all of the
compounds in Figure 1) is the basis for their herbicidal activity.31
The data in Table 1 show that isatin inhibitors of AtAHAS also
exhibit herbicidal activity, suggesting that their activity is due to their
inhibition of AHAS. Further studies are required to confirm the
linkage between the herbicidal activity of isatins and their inhibition
of AtAHAS. Such experiments may include control in vivo rape root
length studies in the presence of BCAA.
The current isatins have weaker in vivo activity than many of
the commercial sulfuonylurea herbicides, but the discovery of a
new family of AHAS inhibitors is of major significance, particu-
larly in view of the emergence of weeds resistant to many of the
currently available AHAS inhibitors. Further structural optimiza-
tion of the isatins should therefore be undertaken.
CoMFA Results and the 3D Contour Models. For computa-
tional analysis all of the molecules were built as the Z isomer.
Compounds 14, 15, 17, 30, and 32 were excluded from the
database because they were statistical outliers in the training set.
The inclusion of any of these molecules did not yield a satisfactory
leave-one-out q2. The best score that could be obtained was <0.3.
However, the leave-one-out q2 is 0.498 when using five optimum
components (CoMFA-1) and is 0.535 when using six optimum
DFT Calculation. Frontier molecular orbital theory should be
taken into account when structureꢀactivity relationships are
investigated because the HOMO and LUMO make significant
contributions to ligandꢀenzyme interactions.43ꢀ45 We therefore
calculated the frontier molecular orbital of representative com-
pounds 6, 20, and 29 that have good AtAHAS inhibition by
means of DFT/B3LYP. LUMO+1, LUMO+2, and HOMO-2
maps did not give useful information. However, meaningful
results were observed from LUMO, HOMO, and HOMO-1
maps (Figure 4). In all of the frontier molecular orbital maps, it
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Journal of Agricultural and Food Chemistry
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binding site of AtAHAS, demonstrating that the inhibitor fits
neatly into the binding cavity. A two-dimensional representation
shows which atoms of the inhibitor interact directly with
AtAHAS (Figure 5c). The diagram emphasizes that hydrophobic
contacts to the protein are through the indole moiety and
the carbonyl carbon atom in the bridge. This is in good
agreement with the observations from the frontier MO and
DFT results. From Figure 5c it can be observed that the inhibitor
makes hydrophobic contact with the G654, K256, and G121 of
AtAHAS. Hydrogen bonds are also observed between the
carbonyl oxygen of compound 20 and the side chains of S653
and R377.
The location of compound 20 when docked to the surface of
AtAHAS resembles that of the sulfonylureas in that it is bound to
an area referred to as the herbicide binding site, which is located
directly above the active site. With this site filled by an inhibitor it
is not possible for the substrate to gain access to the active site to
allow catalysis to occur. In common between the structures of the
sulfonylureas and compound 20 is that they both possess two
aromatic rings and a bridge that links them together. In both
cases, the rings are orthogonal to each other due to the bending
of the bridge within the herbicide binding site. Another similarity
is that two residues, R377 and S653, form hydrogen bonds to the
bridge sections of both inhibitors. However, there are also some
differences when the molecular modeling results of the docked
isatins into the crystal structure of AtAHAS are compared with
the crystal structures of the sulfonlyureas or imazaquin when in
complex with this enzyme.15 For example, in the crystal struc-
tures, W574 is crucial for the sulfonylurea to form πꢀπ stacking,
whereas compound 20 does not make such an interaction with
this residue. Additionally, D376 and M200 do not make any
contacts with compound 20, yet both play important roles when
binding with the sulfonylureas or imazaquin. The side chain of
R199 forms part of the surface where compound 20 is bound, but
does not participate directly in any noncovalent ligandꢀenzyme
interactions (Figure 5b,c). It will be interesting to compare the
binding affinities of the isatins to those of mutant AHAS
enzymes, whereby resistance to the sulfonylureas or imidizoli-
nones has already developed. In particular, the W574L mutation
is the most commonly observed resistant biotype found in field
isolates. Because the isatins do not bind directly to W574, we
would anticipate that the isatins are inhibitors of this variant.
In summary, 32 isatin derivatives were assessed as novel AHAS
inhibitors both in enzymatic assays and in the rape root growth
test. The combination of 3D-QSAR, DFT calculation, and
molecular docking provided meaningful clues as to the structural
features of this new family of AHAS inhibitors that are important
for binding and will help in the design of more potent com-
pounds in the future.
Figure 5. FlexX docking of isatins to AtAHAS: (a) overlay of docked
conformations for compounds 1, 3, 20ꢀ22, 25, 26, and 29; (b)
Connolly surface of AtAHAS with compound 20 shown as a stick model
(the two subunits of AtAHAS are in different shades of green); (c)
hydrogen bonds between the enzyme and inhibitor shown as red dashed
lines and distances in Å units, amino acid residues within van der Waals
contact of compound 20 shown as green arcs, an atom belonging to
compound 20 that participates in van der Waals attractions to the
enzyme identified by green star. Residues labeled with a prime are from
an alternate subunit that completes the active site.
can be seen that the isatin moiety in all of the molecules makes a
major contribution to the binding of AtAHAS. These are largely
through πꢀπ or hydrophobic interactions and are most obvious
in the HOMO maps. The frontier molecular orbital maps show
that the carbonyl positioned adjacent to the hydrazone nitrogen
atom also plays an important role in binding, especially for
compounds 20 and 29. It is interesting to note that for
compound 29, only a small portion of the frontier molecular
orbital spreads into the adjacent aromatic ring at the 1-postion of
isatin, indicating that the group at the 1-position plays a very
minor role. Thus, it can be concluded that the isatin moiety
makes a significant contribution to the binding to AtAHAS.
Molecular Docking. All of the isatins were docked to the
binding site of AtAHAS by using the program FlexX. Com-
pounds 1, 3, 20ꢀ22, 25, 26, and 29 all adopt a similar
conformation and location in the active site (Figure 5a), whereas
none of the other 24 compounds yielded consistent docking
poses. Figure 5b shows the docking of compound 20 in the
’ ASSOCIATED CONTENT
S
Supporting Information. Predicted activities versus ex-
b
perimental activities of isatins and PLS statistics of CoMFA
models for isatins. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +86-(0)22-23499414. Fax: +86-(0)22-23503627. E-mail:
nkwjg@nankai.edu.cn.
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