Syntheses, biological activities and SAR studies of novel carboxamide compounds containing piperazine and arylsulfonyl moieties

Article abstract of DOI:10.1016/j.ejmech.2016.04.005

A series of novel carboxamide compounds 19a-19j, 20a-20j and 22a-22d containing piperazine and arylsulfonyl moieties have been synthesized. The bioassay results showed that some compounds exhibited favorable herbicidal activities against dicotyledonous plants and many of them possessed excellent antifungal activities. Among 24 novel compounds, some showed superiority over the commercial fungicides Chlorothalonil, Dimethomorph, Thiophanate-methyl, Iprodione, and Zhongshengmycin at 500 mg/L concentration. Some compounds also exhibited high KARI inhibitory activity at 100 γ1/4g/mL concentration and could be used as new KARI lead inhibitors for further studies. Moreover, SAR of these new compounds were comprehensively investigated using different computational methods in which 3D-QSAR model obtained provided useful information for further structural optimization for the discovery of new fungicides. The results of this research will contribute to explore comprehensive biological activities of piperazine-containing compounds in different areas of chemistry.

Full text of DOI:10.1016/j.ejmech.2016.04.005

Accepted Manuscript
Syntheses, biological activities and SAR studies of novel carboxamide compounds
containing piperazine and arylsulfonyl moieties
Bao-Lei Wang, Yan-Xia Shi, Shu-Jun Zhang, Yi Ma, Hong-Xue Wang, Li-Yuan Zhang,
Wei Wei, Xing-Hai Liu, Yong-Hong Li, Zheng-Ming Li, Bao-Ju Li
PII:
S0223-5234(16)30277-X
10.1016/j.ejmech.2016.04.005
EJMECH 8520
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 November 2015
Revised Date: 3 April 2016
Accepted Date: 4 April 2016
Please cite this article as: B.-L. Wang, Y.-X. Shi, S.-J. Zhang, Y. Ma, H.-X. Wang, L.-Y. Zhang, W.
Wei, X.-H. Liu, Y.-H. Li, Z.-M. Li, B.-J. Li, Syntheses, biological activities and SAR studies of novel
carboxamide compounds containing piperazine and arylsulfonyl moieties, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.04.005.
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ACCEPTED MANUSCRIPT
Graphical Abstract
24 novel carboxamide compounds containing piperazine and arylsulfonyl moieties were
synthesized. Several compounds exhibited excellent antifungal activities. 3D-QSAR study and
theoretical calculation were carried out.
R4
CH2
N
R3
N
R6
S
R6
H
R2
H
N
N
N
N
N
N
R5
20a-20j
S
O
O
O
19a-19j
O
O
O
NH
N
R6
S
H
N
Antifungal activities
... ...
O
O
O
22a-22d

ACCEPTED MANUSCRIPT
Syntheses, Biological Activities and SAR Studies of Novel
Carboxamide Compounds Containing Piperazine and Arylsulfonyl
Moieties
a
b
a
a
a
a
Bao-Lei Wang , Yan-Xia Shi , Shu-Jun Zhang , Yi Ma , Hong-Xue Wang , Li-Yuan Zhang ,
Wei Wei a, Xing-Hai Liu a, Yong-Hong Li a, Zheng-Ming Li a, *, Bao-Ju Li b,
*
a State-Key Laboratory of Elemento-Organic Chemistry, National Pesticide Engineering Research
Center (Tianjin), Collaborative Innovation Center of Chemical Science and Engineering, Nankai
University, Tianjin 300071, China
b Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081,
China
* Corresponding author. E-mail addresses: nkzml@vip.163.com (Z.-M. Li), libj@mail.caas.net.cn
(B.-J. Li).
ABSTRACT: A series of novel carboxamide compounds 19a-19j, 20a-20j and 22a-22d
containing piperazine and arylsulfonyl moieties have been synthesized. The bioassay results
showed that some compounds exhibited favorable herbicidal activities against dicotyledonous
plants and many of them possessed excellent antifungal activities. Among 24 novel compounds,
some showed superiority over the commercial fungicides Chlorothalonil, Dimethomorph,
Thiophanate-methyl, Iprodione, and Zhongshengmycin at 500 mg/L concentration. Some
compounds also exhibited high KARI inhibitory activity at 100 ꢀg/mL concentration and could be
used as new KARI lead inhibitors for further studies. Moreover, SAR of these new compounds
were comprehensively investigated using different computational methods in which 3D-QSAR
model obtained provided useful information for further structural optimization for the discovery of
new fungicides. The results of this research will contribute to explore comprehensive biological
activities of piperazine-containing compounds in different areas of chemistry.
Keywords: Piperazine derivatives; Arylsulfonyl group; Biological activities; 3D-QSAR;
1

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Theoretical calculation
1. Introduction
In recent two decades, the chemistry of piperazine derivatives with various structural moieties has
attracted much attention owing to their versatile properties [1-3]. As one of the important
six-membered heterocycles, piperazine ring has some drug-like advantages such as low toxicity,
easy formation of multiple hydrogen or ionic bonds, and functional effect that modulates drug
lipid water partition coefficient and acid-base equilibrium constant; all these make it often be used
as an essential pharmacophore for the drug design. It has been found that N-substituted piperazine
compounds possess a wide range of pharmaceutical activities, such as antimicrobial [4], anticancer
[5], antifungal [6], and tuberculostatic [7] activities. Furthermore, piperazine-containing
compounds play an important role in some agrochemicals, e.g. the systemic fungicide Triforine,
which has been found to be effective in controlling a number of diseases in ornamentals, cereal
grains, fruits, and vegetables [8]. Also, a few reports about their herbicidal and insecticidal
properties are available in the literature [9]. Despite having been widely applied in pharmaceutical
area, there are relatively few reports in the literature about the design and development of
agrochemicals containing piperazine motif.
It is known that arylsulfonyl is a multi-functional group that is present in many bioactive
compounds. Many arylsulfonamides and arylsulfonylureas possess a wide-range spectrum of
pharmacological, fungicidal, or herbicidal activities. For examples, Sulfanilamide is the first
synthetic antibacterial agent active against a wide range of infections [10]; Flusulfamide is a soil
fungicide used for the control of plasmodiophora brassicae on Chinese cabbage [11]; Pyroxsulam
and Chlorsulfuron are commercial sulfonamide and sulfonylurea herbicides, respectively [12,13].
Meanwhile , it has been found that some sulfonylureas also have antidiabetic [14], antituberculosis
[15] or antifungal [16] activities. In addition, the arylsulfone derivative ethyl
5-((2-chlorophenyl)sulfonyl)-1,2,3-thiadiazole-4-carboxylate was found to have potent in vitro
inhibitory activity against ketol-acid reductoisomerase (KARI; EC 1.1.1.86) which is a key
enzyme catalyzing the biosynthesis of branched-chain amino acids (mainly exist in plants and
microorganisms) and is a promising target for designing herbicides, fungicides or bactericides [17].
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It is worthy of note that some triazole Mannich bases containing piperazine moiety were also
found to have moderate KARI inhibitory activity in our earlier research [18, 19].
In view of the above-mentioned facts, and with the aim of exploring biological activities of
novel piperazine heterocyclic derivatives particularly in the pesticidal area, a series of new
carboxamide compounds containing piperazine and arylsulfonyl moieties combined via a urea
bridge have been synthesized. The herbicidal, antifungal and KARI inhibitory activities, and SAR
of these new compounds were comprehensively investigated in this paper.
2. Results and discussion
2.1. Chemistry
The synthetic procedures for the intermediates are shown in Scheme 1, Scheme 2, Scheme 3,
Scheme 4 and Scheme 5 and for the title compounds - in Scheme 6, Scheme 7 and Scheme 8. For
the synthesis of the title compounds 19 and 20, initially the intermediates arylsulfonylcarbamates
and 4-substitutedpiperazine were refluxed in toluene for several hours, however, only a little of
desired products was obtained. It indicates that lots of the starting material didn’t undergo the
reaction as shown from the TLC monitoring. It was postulated that such nucleophilic substitution
reaction may exist in the reversible equilibrium in which the generated product and the ethanol
may be reacted further to yield corresponding starting materials. Subsequently, the Dean-Stark
trap was adopted to continuously remove the ethanol produced; as a result, the title compounds
were efficiently synthesized with 40.7%-93.5% yields. Using this procedure, after the completion
of the reaction the product can easily precipitate from the reaction solution or directly crystallize
after cooling down, with satisfactory purity. Under the same condition, the attempts to obtain the
possible N1,N4-bis(arylsulfonyl)piperazine-1,4-dicarboxamide (21a-d) using anhydrous piperazine
as a nucleophile and arylsulfonylcarbamate with 1 : 2 molar ratio indicate that the product
obtained was N-(arylsulfonyl)piperazine-1-carboxamide (22a-d) (yields 76.4%-94.1%). This may
be attributed to the fact that after production of the insoluble mono-substituted piperazine
(compound 22), the nucleophilicity of the secondary amine part in the piperazine ring decreases
due to the electron-withdrawing effect of arylsulfamidocarbonyl group, which makes it difficult
for compound 22 to undergo further nucleophilic reaction to give di-substituted piperazine
compound 21.
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1
Compounds 19, 20 and 22 were identified by melting point, H NMR, 13C NMR, MS and IR
spectra. The measured elemental analyses were also consistent with the corresponding calculated
values. All the corresponding data can be found in the supplementary information.
2.2. Herbicidal activities
As shown in Table 1, most of the compounds exhibited obvious herbicidal activities based on
the rape (Brassica campestris) root test at a concentration of 100 ꢀg/mL, while their herbicidal
activities in barnyardgrass (Echinochloa crusgalli) cup test were weak. Based on the result for
compounds 19 and 20 in the rape root test, it was found that the overall sequence of R6 related to
their herbicidal activities is Cl > NO2 > CO2CH3 ≈ CO2C2H5; the overall sequence of the
substituent in piperazine ring with the same R6 is 2,4-dichlorobenzyl > 4-chlorobenzyl for
compounds 19, or 4,6-dimethylpyrimidin-2-yl ≈ 4-methylpyrimidin-2-yl > pyrimidin-2-yl for
compounds 20. As for the compounds 22 whose piperazine part didn’t bear substituent, the
herbicidal activities in general were unfavorable. These preliminary results indicate that both
substituents in benzene ring of arylsulfonyl part and benzyl or pyrimidyl group in piperazine part
make great contribution to their herbicidal activities, and these compounds may have good
herbicidal effect against dicotyledonous plants.
Partial compounds that displayed comparatively higher herbicidal activities in the preliminary
tests were selected for the further investigation in glasshouse on four herbs representing
monocotyledonous and dicotyledonous plants. As shown in Table 2, most of the tested compounds
exhibited apparent herbicidal activities against dicotyledonous Amaranthus retroflexus and
Brassica campestris at a dosage of 1500 g/ha; the most favorable activity was shown in
post-emergence treatment of Amaranthus retroflexus. Among them, compounds 19c, 19d, 19e,
19h, 20c, 20f, 20g and 20i possessed the inhibition rates of 68.6%, 78.1%, 67.6%, 70.4%, 79.2%,
73.2%, 79.8% and 71.3%, respectively.
2.3. Antifungal activities
As shown in Table 3 for the in vivo antifungal test results, most of the compounds displayed
fair or weak antifungal activities against Ascochyta citrullina Smith and Sclerotinia sclerotiorum;
compounds 19i, 20j and compounds 19h, 20e, 20h, 20i and 22a held efficacy rates of 66.53%,
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73.89% and 75.83%, 87.57%, 66.73%, 80.53%, 72.55% against Pseudomonas syringae pv.
lachrymans and Pseudoperonospora cubensis, respectively, which were comparable with all the
control fungicides; the antifungal activities agains Corynespora cassiicola with efficacy rates of
50% ~ 93%, were significant for almost all the compounds, especially 20f (90.43%), 20g (90.99%)
and 20i (92.77%) were more effective than controls Thiophanate-methyl, Iprodione and
Zhongshengmycin, and were comparable with Chlorothalonil and Dimethomorph. In order to well
illustrate the structure-activity relationship of such kind of compounds and guide future molecular
design of novel fungicides against Corynespora cassiicola, a subsequent 3D-QSAR study was
carried out.
2.4. KARI inhibitory activity
From Table 4, it was found that almost half of the compounds exhibited significant in vitro
inhibitory activity against rice KARI at the test concentration of 100 ꢀg/mL and compounds 19
and 22 were more effective than compounds 20. Particularly, 19c, 19e and 19j displayed inhibition
rates of 98.5%, 86.7% and 79.1%, respectively, which could be considered as new KARI inhibitor
lead structures used for further structural optimization.
2.5. 3D-QSAR analysis
The CoMFA is a widely used method for drug design, from which the predictive information of
QSAR can be rapidly generated based on the biological activity of synthesized compounds [20].
Starting from the structural alignment of Fig. 1, comprehensive CoMFA analyses were performed.
During the experiments, the data for compounds 19b, 19e, 20b and 22b were removed, since
along with other compounds they couldn’t give a satisfying computational result. The comparison
of experimental and predicted activities by CoMFA for the compounds in training set and test set
(marked with asterisk) in this study was presented in Table 5. The cross-validated results were
assessed by their q2 value, where the value above 0.3 indicates the probability of correlation less
than 5% and the value over 0.5 is significant. As a result, a predictive CoMFA model was
established with the cross-validated coefficient q2 = 0.687 and the conventional correlation
coefficient r2 = 0.813. The contributions of steric and electrostatic fields are 79.2% and 20.8% as
shown in panels A and B of Fig. 2, respectively.
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In Fig. 2, the isocontour diagrams of the steric and electrostatic field contributions obtained
from the CoMFA analysis are illustrated. The steric field contour map is plotted in Fig. 2A. The
green area displays three positions, where the introduction of a bulky group would favor the
higher antifungal activity. In contrast, the yellow area indicates the positions where the decrease in
bulkiness of the target molecules is favorable. As shown in Fig. 2A, the CoMFA steric contour
plots obviously indicates that the yellow region is located around the 2 and 4 positions of the
benzene and piperazine rings, respectively. This means that the bulky substituents at those
positions will decrease the antifungal activity. For example, compounds 19i and 19j bearing the
bulky -CO2C2H5 group at the 2 position of the benzene ring displayed lower antifungal activity.
The electrostatic contour plot is shown in Fig. 2B. The blue contour defines a region where the
increase of the positive charge will result in the increased activity, whereas the red contour defines
a region of space where the increase of electron density is favorable. As shown in Fig. 2B, the title
compounds bearing an electron-donating group at the 4 or 6 position of the pyrimidine ring or an
electron-withdrawing group at the 2 position of the benzene ring, such as 20f and 20i, displayed
higher activity. According to the 3D-QSAR model obtained, four compounds (19a, 19g, 20e and
22a) as test set compounds were subsequently analyzed. As shown in Table 5, their activities
were estimated fairly accurately from the analysis, indicating that the alignment of the molecules
was valid. These results provide useful information for the further optimization of the compounds.
2.6 Theoretical calculation
According to the frontier molecular orbital theory, HOMO has the priority to provide electrons,
while LUMO accept electrons in the first place [21]; these two frontier orbitals are the most
important factors that affect the bioactivity of compounds [22]. Therefore, by means of
DFT/B3LYP, we calculated the frontier molecular orbitals of representative compounds 19c, 19e,
compounds 20c, 20e and compounds 22a, 22b that have good, weak and moderate KARI
inhibition, respectively. From Fig. 3, we observed that the LUMOs of those compounds are
mainly locatated on the benzene ring and the sulfonylamino group, espcially in case of 19c and
19e, while the HOMOs of latter ones are mainly located on the piperazine ring, carbonyl group,
methylene group and a small part of the phenyl group adjacent to methylene group. Besides the
piperazine and carbonyl groups, the HOMOs of 20c and 20e are also located on the whole
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pyrimidine ring. There is no substituent on the N4 atom of piperazine ring, so the HOMOs of 22a
and 22b are mainly located on the piperazine ring and carbonyl group. It was found there are
striking differences between the frontier molecular obitals of those compounds. So the fact is that
the arylsulfonyl, piperazine ring and carbonyl are key functionalities for the biological activity. It
is interesting to note that for compounds 20c and 20e, a large portion of the HOMO is located on
the pyrimidine ring at the N4-position of piperzine ring, but the inhibitory activities of those
compounds against KARI enzyme are rather weak. This may be due to the fact that electrons of
these compounds can not be easily transferred from HOMO to the receptor (KARI) since there is a
strong conjugative effect of N4 atom with pyrimidine (stablize the molecule). This conjugative
effect can also be inferred from the bond length of 0.1367 nm and 0.1366 nm between N4 of
piperazine ring and C2 of pyrimidine ring for 20c and 20e, respectively, as both of them are in
between the normal C-N (0.1471 nm) and C=N (0.1273 nm) bond length values [23](see Table
S2). Moreover, DFT calculations indicate that there is a huge difference between the dihedrals of
piperazine N4 part and its adjacent ArCH2 or Pyrimidyl part. (Table S2). Also they show that the
atomic net charges (Table S3) for piperazine N4 of those compounds correlate with their KARI
inhibitory activities -- the lower the negative charge on N4 of piperazine ring, the higher the
activity of the compound. In addition, electrostatic potential (ESP) of the most active 19c has also
been calculated. According to Fig. 4, the charges for both oxygen and nitrogen atoms in ESP
mapping were found to be negative, especially for oxygen atoms, which indicates the oxygen and
nitrogen atoms may have some interactions with the receptor.
Overall, from all these theoretical calculations it can be inferred that the arylsulfonyl groups and
piperazine moieties in the title compounds make a major contribution to the inhibitory activity
against KARI. These are largely through π-π stacking or hydrophobic interactions and are obvious
in the HOMO and LUMO maps. While substitutents in the N4-position of piperazine ring may
play a supplementary role, the conjugative substituent like pyrimidyl linked directly to piperazine
would not be a favorable factor to improve the activity for this type of structures.
3. Conclusion
In summary, we have synthesized a series of novel carboxamide compounds containing
piperazine and arylsulfonyl moieties. The bioassay results showed that some compounds exhibited
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favorable herbicidal activities against dicotyledonous plants. The antifungal tests indicated that
several compounds possessed excellent fungicidal activities. Among 24 novel compounds, some
showed superiority over the commercial fungicides Chlorothalonil, Dimethomorph,
Thiophanate-methyl, Iprodione, and Zhongshengmycin in the present studies. Some compounds
also exhibited favorable KARI inhibitory activity and could be used as new KARI inhibitors for
the further structural optimization. In addition, significant 3D-QSAR and theoretical calculation
results were obtained through the SAR studies, in which 3D-QSAR results will serve as the useful
guide to accelerate the discovery of compounds with high antifungal activity. These work will be a
significant contribution towards the exploration of comprehensive biological activities of
piperazine-containing compounds.
4. Experimental
4.1. Materials and methods
All reagents were analytical grade. Melting points were determined using an X-4 melting
1
apparatus (uncorrected). H NMR spectra were measured on a Bruker AVANCE instrument
(300MHz or 400MHz) using tetramethylsilane as an internal standard and CDCl3 or DMSO-d6 as
solvent. Infrared spectra were recorded on a BrukerEquinox55 spectrophotometer as potassium
bromide tablets. Mass spectra were recorded on a Thermo Finnigan LCQ Advantage instrument or
a VG ZAB-HS instrument. Elemental analyses were performed on a Yanaco CHN CORDER
MT-3 elemental analyzer. HRMS data were obtained on a Varian QFT-ESI instrument.
4.2. Synthesis
4.2.1. Synthetic procedure of intermediates arylsulfonylcarbamates (3a-b, 8, 12)
As shown in Schemes 1, 2, and 3, ethyl 2-(N-(methoxycarbonyl)sulfamoyl)benzoate (3a) and
ethyl 2-(N-(ethoxycarbonyl)sulfamoyl)benzoate (3b) were prepared from saccharin (1) according
to the procedure of literature [24, 25], ethyl ((2-nitrophenyl)sulfonyl)carbamate (8) was prepared
from 2-chloronitrobenzene (4) according to the procedure of literature [26-28, 25], and ethyl
((2-chlorophenyl)sulfonyl)carbamate (12) was prepared from 2-chloroaniline (9) according to the
procedure of literature [29-31, 25].
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4.2.2. Synthetic procedure of intermediates 4-(substituted)benzylpiperazine (15a-c)
The procedures are similar to that described previously (32). To a solution of anhydrous
piperazine (50 mmol) in 20 mL 95% of ethanol was added concentrated hydrochloric acid (25
mmol). The mixture was stirred under reflux, and substituted benzyl chlorine (25 mmol) was
added dropwise for over 5 min. The mixture was refluxed for 4-8 h with TLC monitoring, then left
to stand overnight at room temperature. The solid precipitated was filtered and washed with
ethanol, the filtrate was evaporated in vacuum, and the residue was dissolved in 30 mL of
saturated K2CO3 aq., extracted with chloroform (8 mL × 5). The chlroform solution was dried
with anhydrous Na2SO4 and evaporated in vacuum. The residue was then distilled under reduced
pressure to give compound as a colorless liquid.
15a: yield 58%, bp 131-134 oC⁄10 mmHg (Lit. bp 154-160 oC⁄18 mmHg [33]); 15b: yield 41%,
o
o
o
bp 138-141 C⁄10 mmHg (Lit. bp 92-96 C⁄0.1 mmHg [34]); 15c: yield 36%, bp 147-150 C⁄6
mmHg (Lit. bp 106-108 oC⁄0.05 mmHg [34]).
4.2.3. Synthetic procedure of intermediates 4-(4,6-disubstituted-pyrimidin-2-yl)piperazine (18a-c)
2-Chloro-4,6-disubstituted-pyrimidines 17 were prepared by the reaction of the diazoniumsalts
of 4,6-disubstituted-pyrimidin-2-amines (16) with concentrated hydrochloric acid and ZnCl2 [35].
Compound 18 was prepared according to literature [32], and the method was improved. To a
stirred solution of piperazine (45 mmol) and K2CO3 (16.5 mmol) in water (20 mL) was added
o
chloropyrimidine 17 (18 mmol) in small portions at 50-65 C. The mixture was stirred for 1 h at
o
o
60-65 C and cooled to 35 C. The yellow solid, 1,4-bispyrimidylpiperazine byproduct, was
filtered off, and the filtrate was then extracted three times with chloroform, dried over Na2SO4,
and evaporated in vacuum to give compound 18, which was used for the following reactions
without further purification.
18a: yellow oil, yield 88%. 18b: yellow solid, yield 81%, mp 45-48 oC. 18c: yellow solid, yield
79%, mp 82-84 oC.
4.2.4.
General
synthetic
procedure
for
the
title
compounds
4-(substitutedbenzyl)-N-(arylsulfonyl)piperazine-1-carboxamide (19a-j)
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To a 100 mL three-neck round bottom flask equipped with magnetic stirrer, Dean-Stark trap
and reflux condenser, arylsulfonylcarbamate 3, 8, or 12 (2 mmol), 4-(substituted)benzylpiperazine
15 (2 mmol) and dry toluene (30 mL) were added and stirred under reflux for 1.5 h; a clear
solution was formed. The mixture was further refluxed while the solvent (toluene) and the
produced ethanol were collected in the Dean-Stark trap. They (toluene and ethanol) were released
from the side tube of the Dean-Stark trap when it was full. Meanwhile, fresh dry toluene was
added frequently into the reaction flask. This procedure was repeated several times until the
reaction was complete as confirmed by TLC monitoring. During the course of reaction, the
solution color changed from light yellow to pale brown. At the last stage the volume of the
reaction mixture was maintained at approximately 15 mL. After the reaction mixture was cooled
down to room temperature, the solid precipitate was filtered off, washed with toluene (3-4 times)
and petroleum ether (2 times) to give the title compounds 19a-j.
4.2.4.1. 4-Benzyl-N-((2-chlorophenyl)sulfonyl)piperazine-1-carboxamide (19a)
o
1
White solid, yield 55.9%, m.p. 222-224 C. H NMR (400MHz, DMSO-d6) δ (ppm): 7.93 (d, J =
8.0 Hz, 1H, Ph-H), 7.40-7.45 (m, 8H, Ph-H), 3.98 (s, 2H, CH2), 3.38 (bs, 4H, Piperazine-H), 2.74
(bs, 4H, Piperazine-H). ESI-MS (m/z): 392 ([M-H]-). HRMS (m/z): calcd. for C18H20ClN3O3S
[M+H]+: 394.0987; found: 394.0993.
4.2.4.2. 4-(4-Chlorobenzyl)-N-((2-chlorophenyl)sulfonyl)piperazine-1-carboxamide (19b)
o
1
White solid, yield 93.5%, m.p. 103-106 C. H NMR (300MHz, DMSO-d6) δ (ppm): 7.88 (d, J =
7.2 Hz, 1H, Ph-H), 7.29-7.42 (m, 7H, Ph-H), 3.52 (s, 2H, CH2), 3.05 (t, J = 4.5 Hz, 4H,
Piperazine-H), 2.23 (bs, 4H, Piperazine-H). ESI-MS (m/z): 426 ([M-H]-). Elemental analysis for
C18H19Cl2N3O3S, calcd: C 50.47, H 4.47, N 9.81; found: C 50.32, H 4.68, N 9.55.
4.2.4.3. N-((2-Chlorophenyl)sulfonyl)-4-(2,4-dichlorobenzyl)piperazine-1-carboxamide (19c)
White solid, yield 82.2%, m.p. 209-211 oC. 1H NMR (300MHz, DMSO-d6) δ (ppm): 8.53 (bs, 1H,
NH), 7.98 (d, J = 8.7 Hz, 1H, Ph-H), 7.41-7.63 (m, 6H, Ph-H), 3.61 (s, 2H, CH2), 3.35 (bs, 4H,
Piperazine-H), 2.40 (bs, 4H, Piperazine-H). ESI-MS (m/z): 460 ([M-H]-). Elemental analysis for
C18H18Cl3N3O3S, calcd: C 46.72, H 3.92, N 9.08; found: C 46.47, H 4.11, N 8.66.
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4.2.4.4. 4-Benzyl-N-((2-nitrophenyl)sulfonyl)piperazine-1-carboxamide (19d)
o
1
White solid, yield 87.5%, m.p. 251-252 C. H NMR (400MHz, DMSO-d6) δ (ppm): 7.96 (d, J =
7.6 Hz, 1H, Ph-H), 7.46-7.60 (m, 8H, Ph-H), 4.24 (s, 2H, CH2), 3.38 (bs, 4H, Piperazine-H), 2.92
(bs, 4H, Piperazine-H). ESI-MS (m/z): 403 ([M-H]-). HRMS (m/z): calcd. for C18H20N4O5S
[M+H]+: 405.1227; found: 405.1235.
4.2.4.5. 4-(4-Chlorobenzyl)-N-((2-nitrophenyl)sulfonyl)piperazine-1-carboxamide (19e)
White solid, yield 83.6%, m.p. 225-226 oC. 1H NMR (300MHz, DMSO-d6) δ (ppm): 9.57 (bs, 1H,
NH), 7.48-8.00 (m, 8H, Ph-H), 4.19 (s, 2H, CH2), 3.35 (bs, 4H, Piperazine-H), 2.88 (bs, 4H,
Piperazine-H). ESI-MS (m/z): 437 ([M-H]-). HRMS (m/z): calcd. for C18H19ClN4O5S [M+H]+:
439.0837; found: 439.0838.
4.2.4.6. Methyl 2-(N-(4-benzylpiperazine-1-carbonyl)sulfamoyl)benzoate (19f)
o
1
White solid, yield 68.7%, m.p. 158-161 C. H NMR (400MHz, DMSO-d6) δ (ppm): 7.93 (d, J =
8.4 Hz, 1H, Ph-H), 7.39-7.54 (m, 8H, Ph-H), 3.92 (s, 2H, CH2), 3.75 (s, 3H, CH3), 3.38 (bs, 4H,
Piperazine-H), 2.69 (bs, 4H, Piperazine-H). ESI-MS (m/z): 416 ([M-H]-). HRMS (m/z): calcd. for
C20H23N3O5S [M+H]+: 418.1431; found: 418.1438.
4.2.4.7. Methyl 2-(N-(4-(4-chlorobenzyl)piperazine-1-carbonyl)sulfamoyl)benzoate (19g)
White solid, yield 54.6%, m.p. 172-174 oC. 1H NMR (300MHz, DMSO-d6) δ (ppm): 8.49 (bs, 1H,
NH), 7.31-7.99 (m, 8H, Ph-H), 3.84 (s, 2H, CH2), 3.75 (s, 3H, CH3), 3.35 (bs, 4H, Piperazine-H),
2.45 (bs, 4H, Piperazine-H). 13C NMR (101 MHz, CDCl3) δ (ppm): 168.19, 151.58, 138.92,
135.58, 133.23, 132.86, 131.73, 131.41, 130.44, 130.36, 128.57, 61.83, 53.46, 52.30. IR (KBr),
v/cm-1: 3434 (N-H), 3008, 2950, 2856 (C-H), 1744, 1725 (C=O), 1580, 1557, 1451 (C=C), 1170,
1121, 1051 (O=S=O). ESI-MS (m/z): 450 ([M-H]-). HRMS (m/z): calcd. for C20H22ClN3O5S
[M+H]+: 452.1041; found: 452.1048.
4.2.4.8. Methyl 2-(N-(4-(2,4-dichlorobenzyl)piperazine-1-carbonyl)sulfamoyl)benzoate (19h)
White solid, yield 82.3%, m.p. 179-181 oC. 1H NMR (400MHz, DMSO-d6) δ (ppm): 7.42-8.00 (m,
11

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7H, Ph-H), 3.82 (s, 3H, CH3), 3.61 (s, 2H, CH2), 3.37 (bs, 4H, Piperazine-H), 2.42 (bs, 4H,
Piperazine-H). ESI-MS (m/z): 484 ([M-H]-). Elemental analysis for C20H21Cl2N3O5S, calcd: C
49.39, H 4.35, N 8.64; found: C 48.99, H 4.05, N 8.31.
4.2.4.9. Ethyl 2-(N-(4-(4-chlorobenzyl)piperazine-1-carbonyl)sulfamoyl)benzoate (19i)
White solid, yield 70.2%, m.p. 201-202 oC. 1H NMR (300MHz, DMSO-d6) δ (ppm): 8.53 (bs, 1H,
NH), 7.28-7.99 (m, 8H, Ph-H), 4.22 (q, J = 7.2 Hz, 2H, CH2CH3), 3.64 (s, 2H, CH2), 3.36 (bs, 4H,
Piperazine-H), 2.43 (bs, 4H, Piperazine-H), 1.26 (t, J = 7.2 Hz, 3H, CH2CH3). ESI-MS (m/z): 464
([M-H]-). Elemental analysis for C21H24ClN3O5S, calcd: C 54.13, H 5.19, N 9.02; found: C 54.60,
H 4.80, N 8.75.
4.2.4.10. Ethyl 2-(N-(4-(2,4-dichlorobenzyl)piperazine-1-carbonyl)sulfamoyl)benzoate (19j)
White solid, yield 66.7%, m.p. 158-160 oC. 1H NMR (300MHz, DMSO-d6) δ (ppm): 8.57 (bs, 1H,
NH), 7.28-8.00 (m, 7H, Ph-H), 4.24 (q, J = 7.2 Hz, 2H, CH2CH3), 3.61 (s, 2H, CH2), 3.35 (bs, 4H,
Piperazine-H), 2.36 (bs, 4H, Piperazine-H), 1.28 (t, J = 7.2 Hz, CH2CH3). ESI-MS (m/z): 498
([M-H]-). Elemental analysis for C21H23Cl2N3O5S, calcd: C 50.41, H 4.63, N 8.40; found: C 50.53,
H 4.84, N 8.07.
4.2.5.
General
synthetic
procedure
for
the
title
compounds
N-(arylsulfonyl)-4-(substitutedpyrimidin-2-yl)piperazine-1-carboxamide (20a-j)
The synthesis was similar to the synthesis of compounds 19a-j. Using arylsulfonylcarbamate (3,
or 8, or 12) and 4-(4,6-disubstituted-pyrimidin-2-yl)piperazine (18) as materials, title compounds
20a-j was obtained.
4.2.5.1. N-((2-Chlorophenyl)sulfonyl)-4-(pyrimidin-2-yl)piperazine-1-carboxamide (20a)
Yellow crystals, yield 69.9%, m.p. 198-200 oC. 1H NMR (400MHz, CDCl3) δ (ppm): 8.30 (d, J =
4.8 Hz, 3H, Pyrimidine-H + Ph-H), 7.48-7.59 (m, 3H, Ph-H), 6.53 (t, J = 4.8 Hz, 1H,
Pyrimidine-H), 3.83 (t, J = 5.2 Hz, 4H, Piperazine-H), 3.52 (t, J = 5.2 Hz, 4H, Piperazine-H).
ESI-MS (m/z): 380 ([M-H]-). HRMS (m/z): calcd. for C15H16ClN5O3S [M+Na]+: 404.0555; found:
404.0547.
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4.2.5.2. N-((2-Chlorophenyl)sulfonyl)-4-(4-methylpyrimidin-2-yl)piperazine-1-carboxamide (20b)
Yellow crystals, yield 67.4%, m.p. 175-176 oC. 1H NMR (400MHz, CDCl3) δ (ppm): 8.30 (d, J =
8.0 Hz, 1H, Ph-H), 8.15 (d, J = 5.2 Hz, 1H, Pyrimidine-H), 7.48-7.59 (m, 3H, Ph-H), 6.42 (d, J =
5.2 Hz, 1H, Pyrimidine-H), 3.83 (t, J = 5.2 Hz, 4H, Piperazine-H), 3.51 (t, J = 5.2 Hz, 4H,
Piperazine-H), 2.32 (s, 3H, CH3). ESI-MS (m/z): 396 ([M-H]-). Elemental analysis for
C16H18ClN5O3S, calcd: C 48.55, H 4.58, N 17.69; found: C 49.14, H 5.01, N 17.52.
4.2.5.3. N-((2-Chlorophenyl)sulfonyl)-4-(4,6-dimethylpyrimidin-2-yl)piperazine-1-carboxamide
(20c)
Yellow crystals, yield 79.4%, m.p. 181-183 oC. 1H NMR (400MHz, CDCl3) δ (ppm): 8.30 (d, J =
8.0 Hz, 1H, Ph-H), 7.47-7.58 (m, 3H, Ph-H), 6.31 (s, 1H, Pyrimidine-H), 3.83 (t, J = 4.8 Hz, 4H,
Piperazine-H), 3.49 (t, J = 4.8 Hz, 4H, Piperazine-H), 2.27 (s, 6H, CH3). 13C NMR (101 MHz,
CDCl3) δ (ppm): 167.22, 161.44, 150.69, 136.86, 134.52, 132.79, 131.60, 131.48, 127.35, 109.75,
43.18, 24.03. ESI-MS (m/z): 410 ([M-H]-). HRMS (m/z): calcd. for C17H20ClN5O3S [M+Na]+:
432.0868; found: 432.0862.
4.2.5.4. N-((2-Nitrophenyl)sulfonyl)-4-(pyrimidin-2-yl)piperazine-1-carboxamide (20d)
Yellow crystals, yield 44.0%, m.p. 191-193 oC. 1H NMR (400MHz, CDCl3) δ (ppm): 8.42 (d, J =
7.6 Hz, 1H, Ph-H), 8.32 (d, J = 4.8 Hz, 2H, Pyrimidine-H), 7.75-7.85 (m, 3H, Ph-H), 6.56 (t, J =
4.8 Hz, 1H, Pyrimidine-H), 3.88 (t, J = 5.2 Hz, 4H, Piperazine-H), 3.55 (t, J = 5.2 Hz, 4H,
Piperazine-H). ESI-MS (m/z): 391 ([M-H]-). Elemental analysis for C15H16N6O5S, calcd: C 45.91,
H 4.11, N 21.42; found: C 46.10, H 4.55, N 21.32.
4.2.5.5. 4-(4-Methylpyrimidin-2-yl)-N-((2-nitrophenyl)sulfonyl)piperazine-1-carboxamide (20e)
Yellow crystals, yield 61.6%, m.p. 170-172 oC. 1H NMR (400MHz, CDCl3) δ (ppm): 8.43 (d, J =
7.6 Hz, 1H, Ph-H), 8.17 (d, J = 5.2 Hz, 1H, Pyrimidine-H), 7.74-7.84 (m, 3H, Ph-H), 6.43 (d, J =
5.2 Hz, 1H, Pyrimidine-H), 3.88 (t, J = 5.2 Hz, 4H, Piperazine-H), 3.52 (t, J = 5.2 Hz, 4H,
Piperazine-H), 2.33 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ (ppm): 167.95, 161.23, 157.18,
150.98, 148.07, 134.32, 133.73, 132.87, 132.63, 124.82, 110.33, 43.12, 24.30. ESI-MS (m/z): 405
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([M-H]-). Elemental analysis for C16H18N6O5S, calcd: C 47.29, H 4.46, N 20.68; found: C 47.49,
H 4.80, N 20.51.
4.2.5.6.
4-(4,6-Dimethylpyrimidin-2-yl)-N-((2-nitrophenyl)sulfonyl)piperazine-1-carboxamide
(20f)
Yellow crystals, yield 95.2%, m.p. 138-141 oC. 1H NMR (400MHz, CDCl3) δ (ppm): 8.43 (d, J =
7.6 Hz, 1H, Ph-H), 7.74-7.85 (m, 3H, Ph-H), 7.20 (bs, 1H, NH), 6.32 (s, 1H, Pyrimidine-H), 3.88
(t, J = 4.8 Hz, 4H, Piperazine-H), 3.52 (t, J = 4.8 Hz, 4H, Piperazine-H), 2.29 (s, 6H, CH3).
ESI-MS (m/z): 419 ([M-H]-). HRMS (m/z): calcd. for C17H20N6O5S [M+Na]+: 443.1108; found:
443.1114.
4.2.5.7. Methyl 2-(N-(4-(pyrimidin-2-yl)piperazine-1-carbonyl)sulfamoyl)benzoate (20g)
o
1
White crystals, yield 40.7%, m.p. 102-104 C. H NMR (400MHz, CDCl3) δ (ppm): 8.35 (d, J =
7.2 Hz, 1H, Ph-H), 8.32 (d, J = 4.8 Hz, 2H, Pyrimidine-H), 7.83 (d, J = 7.2 Hz, 1H, Ph-H),
7.64-7.72 (m, 2H, Ph-H), 6.54 (t, J = 4.8 Hz, 1H, Pyrimidine-H), 3.99 (s, 3H, CH3), 3.85 (t, J =
5.2 Hz, 4H, Piperazine-H), 3.55 (t, J = 5.2 Hz, 4H, Piperazine-H). ESI-MS (m/z): 404 ([M-H]-).
HRMS (m/z): calcd. for C17H19N5O5S [M+Na]+: 428.0999; found: 428.0997.
4.2.5.8. Methyl 2-(N-(4-(4-methylpyrimidin-2-yl)piperazine-1-carbonyl)sulfamoyl)benzoate (20h)
Light yellow crystals, yield 41.8%, m.p. 147-149 oC. 1H NMR (400MHz, CDCl3) δ (ppm): 8.34 (d,
J = 7.2 Hz, 1H, Ph-H), 8.16 (d, J = 4.8 Hz, 1H, Pyrimidine-H), 7.83 (d, J = 7.6 Hz, 1H, Ph-H),
7.64-7.72 (m, 2H, Ph-H), 6.42 (d, J = 4.8 Hz, 1H, Pyrimidine-H), 3.99 (s, 3H, CO2CH3), 3.85 (t, J
= 5.2 Hz, 4H, Piperazine-H), 3.53 (t, J = 5.2 Hz, 4H, Piperazine-H), 2.33 (s, 3H, Pyrimidine-CH3).
ESI-MS (m/z): 418 ([M-H]-). Elemental analysis for C18H21N5O5S, calcd: C 51.54, H 5.05, N
16.70; found: C 51.42, H 5.08, N 16.54.
4.2.5.9. Methyl 2-(N-(4-(4,6-dimethylpyrimidin-2-yl)piperazine-1-carbonyl)sulfamoyl)benzoate
(20i)
Yellow crystals, yield 70.4%, m.p. 175-176 oC. 1H NMR (400MHz, CDCl3) δ (ppm): 8.34 (d, J =
7.6 Hz, 1H, Ph-H), 7.83 (d, J = 7.6 Hz, 1H, Ph-H), 7.64-7.71 (m, 2H, Ph-H), 6.31 (s, 1H,
14

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Pyrimidine-H), 3.99 (s, 3H, CO2CH3), 3.85 (t, J = 4.8 Hz, 4H, Piperazine-H), 3.52 (t, J = 4.8 Hz,
4H, Piperazine-H), 2.28 (s, 6H, Pyrimidine-CH3). 13C NMR (101 MHz, CDCl3) δ (ppm): 168.05,
167.23, 161.48, 151.12, 138.61, 133.08, 131.85, 131.71, 130.58, 130.37, 109.74, 53.53, 43.20,
24.03. IR (KBr), v/cm-1: 3257 (N-H), 3000, 2957, 2865 (C-H), 1735, 1692 (C=O), 1578, 1500,
1440 (C=N + C=C), 1165, 1118, 1058 (O=S=O). EI-MS (m/z, 70eV): 433 (M+, 1.0%), 402 (3.0%),
241 (16.4%), 210 (100%), 150 (72.8%), 124 (67.3%). Elemental analysis for C19H23N5O5S, calcd:
C 52.65, H 5.35, N 16.16; found: C 52.54, H 5.13, N 15.79.
4.2.5.10. Ethyl 2-(N-(4-(pyrimidin-2-yl)piperazine-1-carbonyl)sulfamoyl)benzoate (20j)
Yellow crystals, yield 43.4%, m.p. 171-173 oC. 1H NMR (400MHz, CDCl3) δ (ppm): 8.31 (d, J =
4.8 Hz, 2H, Pyrimidine-H), 8.29 (d, J = 7.2 Hz, 1H, Ph-H), 7.78 (d, J = 7.2 Hz, 1H, Ph-H),
7.62-7.69 (m, 2H, Ph-H), 6.53 (t, J = 4.8 Hz, 1H, Pyrimidine-H), 4.44 (q, J = 7.2 Hz, 2H, CH2
CH3), 3.84 (t, J = 5.2 Hz, 4H, Piperazine-H), 3.55 (t, J = 5.2 Hz, 4H, Piperazine-H), 1.42 (t, J =
7.2 Hz, 3H, CH2 CH3). ESI-MS (m/z): 418 ([M-H]-). Elemental analysis for C18H21N5O5S, calcd:
C 51.54, H 5.05, N 16.70; found: C 51.88, H 5.22, N 16.87.
4.2.6.
General
synthetic
procedure
for
the
title
compounds
N-(arylsulfonyl)piperazine-1-carboxamide (22a-d)
The synthesis was similar to the synthesis of compounds 19a-j. Using arylsulfonylcarbamate (3,
or 8, or 12) and anhydrous piperazine (14) as materials, title compounds 22a-d was obtained.
4.2.6.1. N-((2-Chlorophenyl)sulfonyl)piperazine-1-carboxamide (22a)
White solid, yield 94.1%, m.p. 283-285 oC. 1H NMR (400MHz, DMSO-d6) δ (ppm): 8.48 (bs, 1H,
NH), 7.31-7.91 (m, 4H, Ph-H), 3.71 (s, 3H, CH3), 3.50 (bs, 4H, Piperazine-H), 2.94 (bs, 4H,
Piperazine-H). EI-MS (m/z, 70eV): 303 (M+, 1.5%), 219 (11.5%), 217 (34.8%), 177 (22.1%), 175
(66.5%), 113 (33.2%), 111 (100%). Elemental analysis for C11H14ClN3O3S, calcd: C 43.50, H 4.65,
N 13.83; found: C 43.30, H 4.67, N 13.76.
4.2.6.2. N-((2-Nitrophenyl)sulfonyl)piperazine-1-carboxamide (22b)
White solid, yield 76.4%, m.p. 277-279 oC. 1H NMR (400MHz, DMSO-d6) δ (ppm): 8.52 (bs, 1H,
15

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NH), 7.57-7.98 (m, 4H, Ph-H), 3.49 (bs, 4H, Piperazine-H), 2.96 (bs, 4H, Piperazine-H). 13C
NMR (101 MHz, DMSO-d6) δ (ppm): 159.55, 148.24, 138.88, 131.10, 130.88, 130.77, 122.89,
43.45. ESI-MS (m/z): 313 ([M-H]-). Elemental analysis for C11H14N4O5S, calcd: C 42.03, H 4.49,
N 17.83; found: C 41.69, H 4.80, N 17.67.
4.2.6.3. Methyl 2-(N-(piperazine-1-carbonyl)sulfamoyl)benzoate (22c)
White solid, yield 78.6%, m.p. 226-228 oC. 1H NMR (400MHz, DMSO-d6) δ (ppm): 8.43 (bs, 1H,
NH), 7.27-7.92 (m, 4H, Ph-H), 3.71 (s, 3H, CH3), 3.50 (bs, 4H, Piperazine-H), 2.94 (bs, 4H,
Piperazine-H).ESI-MS (m/z): 326 ([M-H]-). HRMS (m/z): calcd. for C13H17N3O5S [M+H]+:
328.0962; found: 328.0963.
4.2.6.4. Ethyl 2-(N-(piperazine-1-carbonyl)sulfamoyl)benzoate (22d)
White solid, yield 83.8%, m.p. 168-171 oC. 1H NMR (400MHz, DMSO-d6) δ (ppm): 8.50 (bs, 1H,
NH), 7.27-7.93 (m, 4H, Ph-H), 4.18 (q, J = 6.8 Hz, 2H, CH2CH3), 3.50 (bs, 4H, Piperazine-H),
2.94 (bs, 4H, Piperazine-H), 1.26 (t, J = 6.8 Hz, 3H, CH2CH3). ESI-MS (m/z): 340 ([M-H]-).
HRMS (m/z): calcd. for C14H19N3O5S [M+H]+: 342.1118; found: 342.1122.
4.3. The herbicidal activity tests
4.3.1. Inhibition of the Root-growth of Rape (Brassica campestris)
Herbicidal activity of the compounds were determined by rape root test according to the method
of Lit. [36]. The percentage inhibition was calculated relative to controls using distilled water.
4.3.2. Inhibition of the Seedling-growth of Barnyardgrass (Echinochloa crusgalli)
Herbicidal activity of the compounds were determined by barnyardgrass cup test according to
the method of Lit. [36]. The percentage inhibition was calculated relative to controls using
distilled water.
For both tests, commercial herbicide Chlorsulfuron was used as control at the same condition.
4.3.3. Glasshouse tests [36]
4.3.3.1. Pre-emergence. Sandy clay (100 g) in a plastic box (11 × 7.5 × 6 cm) was wetted with
water. Sprouting seeds (15) of the weed under test were planted in fine earth (0.6 cm depth) in the
16

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glasshouse and sprayed with the test compound dissolved in a suitable solvent at 1500 g ha−1.
4.3.3.2. Post-emergence. Seedlings (one leaf and one stem) of the weed were sprayed with the test
compounds at the same rate as used for the pre-emergence test.
For both methods, the fresh weights were determined 15 days later, and the percentage
inhibition relative to water-sprayed controls was calculated. Also commercial herbicides
Monosulfuron and Chlorsulfuron were used as controls at the same condition.
4.4. Antifungal activity
Antifungal activities of the title compounds 19a-j, 20a-j and 22a-d against Corynespora
cassiicola, Pseudomonas syringae
pv. lachrymans, Ascochyta citrullina Smith,
Pseudoperonospora cubensis and Sclerotinia sclerotiorum were evaluated according to Lit. [37],
and a potted plant test method was adopted. Five commercial fungicides Chlorothalonil,
Dimethomorph, Thiophanate methyl, Iprodione, and Zhongshengmycin as controls against above
mentioned five fungi were evaluated at the same condition. The antifungal activities were
evaluated when the non-treated cucumber plant (blank) fully developed symptoms. The area of
inoculated treated leaves covered by disease symptoms was assessed and compared to that of
non-treated ones to determine the average disease index [37]. The relative control efficacy of
compounds compared to the blank assay was calculated via the following equation:
Relative control efficacy (%) = [(CK-PT) / CK] × 100%
where CK is the average disease index during the blank assay and PT is the average disease
index after treatment during testing.
4.5 KARI activity test
The cloning of rice KARI has been described previously [38], and enzyme expression and
purification followed that protocol. Protein concentrations were estimated via the bicinchoninic
acid method and protein purity was assessed by SDS-PAGE [39, 40]. KARI activity was measured
with a continuous assay method [38], following the consumption of NADPH at 340 nm and 30 oC.
Assay solutions contained 0.2 mM NADPH, 1 mM MgCl2, 0.1 mM substrate (2-acetolactate), and
inhibitors [19a-j, 20a-j, 22a-d and CPD (a potent KARI inhibitor)], in 0.1 M phosphate buffer
(pH 8.0). Inhibitors were preincubated with the enzyme, NADPH, and MgCl2 in phosphate buffer
17

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o
at 30 C for 10 min. The reaction was then started by adding the substrate. The percentage of the
inhibition was calculated using the following equation:
Relative inhibition rate (%) = [(Absblank - Abssample) / Absblank] × 100%
where Absblank and Abssample are the absorption curve slopes of NADPH at 340 nm with blank
and inhibitor in testing solution, respectively.
4.6 Calculation methods
Molecular modeling was performed using SYBYL 6.91 software and the comparative
molecular field analysis (CoMFA) method for three-dimensional quantitative structure-activity
relationship (3D-QSAR) study has been done according to our previous work [41]. The antifungal
activity of the compounds against Corynespora cassiicola (% I) at 500 mg/L used to derive the
CoMFA analyses model were listed in Table 5. The activity was expressed in terms of D by the
formula D = log{[I/(100 - I)] × MW}, where I is percent control efficacy and MW is the molecular
weight of the tested compounds. The compound 20i, owing to its highest antifungal activity
against Corynespora cassiicola, was used as a template to build the other molecular structures.
Each structure was fully geometry optimized using a conjugate gradient procedure based on the
TRIPOS force field and Gasteiger and Hückel charges. Because these compounds share a common
skeleton, 18 atoms marked with an asterisk were used for RMS-fitting onto the corresponding
atoms of the template structure (Fig. 5 and Fig. 1). CoMFA steric and electrostatic interaction
fields were calculated at each lattice intersection on a regularly spaced grid of 2.0 Å. The grid
pattern was generated automatically by the SYBYLꢁCoMFA routine, and an sp3 carbon atom with
a van der Waals radius of 1.52 Å and a +1.0 charge was used as the probe to calculate the steric
(Lennard-Jones 6–12 potential) field energies and electrostatic (Coulombic potential) fields with a
distance-dependent dielectric at each lattice point. Values of the steric and electrostatic fields were
truncated at 30.0 kcalꢁmol. The CoMFA steric and electrostatic fields generated were scaled by the
CoMFA-STD method in SYBYL. The electrostatic fields were ignored at the lattice points with
maximal steric interactions. A partial least-squares approach was used to derive the 3D-QSAR, in
which the CoMFA descriptors were used as independent variables, and ED values were used as
dependent variables. The cross-validation with the leave-one-out option and the SAMPLS
program [42] were carried out to obtain the optimal number of components to be used in the final
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analysis. After the optimal number of components was determined, a non-cross-validated analysis
was performed without column filtering. The modeling capability (goodness of fit) was judged by
the correlation coefficient squared, r2, and the prediction capability (goodness of prediction) was
indicated by the cross-validated r2 (q2).
In addition, in view of the fact that several compounds possessed favorable inhibitory activity in
vitro at 100 ꢀg/mL against rice KARI, some theoretical calculations were also carried out. The
DFT-B3LYP/6-31G (d,p) [43] methods in Gaussian 03 package [44] were used to optimize the
structures of six representative compounds. Vibration analysis showed that the optimized
structures were in accordance with the minimum points on the potential energy surfaces. All the
convergent precisions were the system default values, and all the calculations were carried out on
the Nankai Stars supercomputer at Nankai University.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Nos. 21372133
and 21272129) and “111” Project of Ministry of Education of China (No. B06005).
Supplementary data
The total energy, the frontier orbital energy, the equilibrium geometries optimized from
DFT/B3LYP and the main atomic net charges of the representative compounds 19c, 19e, 20c, 20e,
22a and 22b; 1H NMR and spectra for compounds 19, 20 and 22, 13C NMR, IR and EI-MS spectra
for partial compounds. These supplementary data associated with this article can be found in the
online version.
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Figure Captions
Scheme 1. The synthetic route of the intermediates 3a and 3b.
Scheme 2. The synthetic route of the intermediate 8.
Scheme 3. The synthetic route of the intermediate 12.
Scheme 4. The synthetic route of the intermediates 15a-15c.
Scheme 5. The synthetic route of the intermediates 18a-18c.
Scheme 6. The synthetic route of the title compounds 19a-19j.
Scheme 7. The synthetic route of the title compounds 20a-20j.
Scheme 8. The synthetic route of the title compounds 22a-22d.
Fig. 1. Superposition modes of compounds.
Fig. 2. (A) Steric map from the CoMFA model; (B) Electrostatic map from the CoMFA model.
Fig. 3. Frontier molecular orbitals of compound 19c: (A) HOMO; (B) LUMO, compound 20e: (C)
HOMO; (D) LUMO, and 22b: (E) HOMO; (F) LUMO.
Fig. 4. ESP mapping of compound 19c.
Fig. 5. The asterisk skeleton of title compounds.
24

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Table 1. Herbicidal activity data of compounds (% inhibition).
Rape root test
Barnyardgrass cup test
(Echinochloa crusgalli)
(Brassica campestris)
Compd.
19a
R2 or R4
R3 or R5
R6
10 ꢀg/mL
100 ꢀg/mL
79.9
12.3
69.8
48
10 ꢀg/mL
100 ꢀg/mL
H
H
Cl
18.9
0
0
20
0
H
Cl
Cl
H
Cl
0
0
19b
19c
Cl
H
Cl
0
10
15
14.6
25
10
15
5
NO2
0
0
19d
19e
H
Cl
H
NO2
0
42
8.1
0
H
CO2CH3
CO2CH3
CO2CH3
CO2C2H5
CO2C2H5
Cl
0
28.8
30.4
37.9
30.8
53.3
31
19f
H
Cl
Cl
Cl
Cl
H
0
5
19g
Cl
H
0
10
0
19h
19i
0
Cl
H
0
31.5
0
39.8
10
20
20
0
19j
0
20a
CH3
CH3
H
H
Cl
18.4
12.3
0
82.4
86.8
14.1
61
5
20b
20c
CH3
H
Cl
2
NO2
0
20d
20e
CH3
CH3
H
H
NO2
20.3
26.9
0
0
5
CH3
H
NO2
52.7
33.7
36.5
42.5
17.4
0
0
5
20f
CO2CH3
CO2CH3
CO2CH3
CO2C2H5
Cl
0
0
20g
CH3
CH3
H
H
0
0
3
20h
20i
CH3
H
0
0
5
0
0
5
20j
-
-
0
0
0
22a
-
-
NO2
0
22.8
36.2
5
0
0
22b
22c
-
-
CO2CH3
CO2C2H5
-
5
0
0
-
-
0
0
0
22d
Chlorsulfuron
-
-
76.0
80.4
8.2
29.9
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Table 2. Herbicidal activity data of compounds in glasshouse tests at 1500 g/ha (% inhibition).
Brassica campestris Amaranthus retroflexus
Echinochloa crusgalli
Digitaria adscendens
Compd.
Post *c
Pre *d
5.0
0
Post
0
Pre
13.2
0
Post
0
Pre
9.0
0
Post
0
Pre
0
0
19a
0
68.6
78.1
67.6
70.4
23.9
47.5
79.2
43.3
73.2
79.8
71.3
18.0
24.4
100
100
0
0
0
19c
0
0
21.8
30.4
0
0
0
0
0
19d
0
0
0.2
0
0
0
0
19e
0
15.0
0
0
0
0
19h
0
0
8.9
9.4
0
39.8
0
0
5.0
0
19j
0
24.8
20.9
8.3
29.1
10.0
19.9
0
0
0
20b
0
0
0
0
0
20c
0
0
0
14.0
0
0
0
20e
0
10.0
6.8
0
0.2
17.0
0
0
0
20f
0
17.3
0
0
0
20g
0
0
0
20i
0
0
0
0
0
0
22c
5.0
51.5
100
0
0
0
22.6
95.0
96.6
5.1
100
22.7
0
22d
Monosulfuron *a
Chlorsulfuron *b
39.7
100
100
100
100
73.1
100
100
* a: 30 g/ha; b: 30 g/ha; c: Post ---- post-emergence; d: Pre ---- pre-emergence.
Table 3. Antifungal activity data of compounds at 500 mg/L (% relative control efficacy).
Pseudomonas
syringae
Ascochyta
citrullina
Corynespora
cassiicola
Pseudoperonospora Sclerotinia
Compd.
cubensis
sclerotiorum
pvꢀlachrymans Smith
76.55
89.52
41.39
59.07
-1.20
47.76
31.70
47.47
-23.03
-12.72
19a
19b
26

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81.62
87.19
59.91
68.09
55.49
87.08
49.80
75.29
56.81
69.69
55.33
86.70
88.72
90.43
90.99
78.43
92.77
85.23
80.43
77.68
66.63
88.29
95.16
95.31
78.75
53.52
90.15
36.38
28.97
21.66
17.38
36.61
40.23
66.53
50.09
43.75
28.27
31.40
52.07
54.30
51.62
54.92
26.08
56.72
73.89
49.51
32.50
39.94
49.16
26.88
13.98
54.15
38.49
69.69
37.97
-25.37
39.81
-24.06
9.51
31.55
48.20
53.13
14.63
48.11
75.83
55.08
58.15
22.33
27.95
56.24
50.56
87.57
57.01
38.48
66.73
80.63
41.11
72.55
59.83
48.45
54.33
45.22
83.75
68.71
55.92
62.57
-4.12
-4.12
0.09
19c
19d
19e
-6.94
-23.48
27.44
31.15
28.54
5.00
19f
19g
24.59
20.05
62.26
40.45
1.99
19h
19i
19j
20a
-3.72
-1.26
64.82
12.79
-14.47
51.94
28.54
2.92
20b
45.32
20c
20d
-7.52
20e
-30.59
41.23
-0.90
20f
20g
20h
-36.47
20i
8.89
20j
26.02
30.62
48.88
56.78
42.69
6.56
22a
22b
22c
25.56
22d
74.89
98.35
83.39
97.84
84.43
Chlorothalonil
Dimethomorph
Thiophanate-methyl
Iprodione
Zhongshengmycin
98.35
100.00
98.35
100.00
27

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Table 4. Inhibitory activity data(%) of compounds against rice KARI at 100 ꢀg/mL in vitro.
Compd.
19a
inhibition rate, %
Compd.
19i
inhibition rate, %
Compd.
20g
inhibition rate, %
39.1
42.4
98.5
30.7
86.7
42.3
73.1
22.6
100
11.6
79.1
2.6
0
0
0
19b
19c
19j
20h
20i
0
20a
20b
20c
0
19d
19e
20j
0
64.4
49.0
39.0
31.1
22a
0
19f
20d
20e
22b
22c
0
19g
11.3
19h
CPD
20f
22d
28