Design, synthesis, and fungicidal activity of novel carboxylic acid amides represented by N-benzhydryl valinamode carbamates

Article abstract of DOI:10.1039/c4ob00744a

Carboxylic acid amide (CAA) fungicides are an important class of agricultural fungicide with oomycete activity and low toxicity toward mammalian cells. To find CAA analogues with high activity against resistant pathogens, a series of substituted N-benzhydryl valinamide carbamate derivatives were designed and synthesized by introducing substituted aromatic rings into valinamide carbamate leads. Bioassays showed that some title compounds exhibited very good in vitro fungicidal activity against Phytophthora capsici and in vivo fungicidal activities against Pseudoperonospora cubensis. Topomer CoMFA was performed to explore the structure-activity relationship on the basis of the in vitro data. The dimethoxy substituted aromatic analogue 9e was found to display higher in vitro fungicidal activity against Phytophthora capsici than iprovalicarb but lower activity than mandipropamid, and higher in vivo fungicidal activity against Pseudoperonospora cubensis than dimethomorph at a dosage of 6.25 μg mL^-1. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00744a

Organic &
Biomolecular Chemistry
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Design, synthesis, and fungicidal activity of novel
carboxylic acid amides represented by
N-benzhydryl valinamode carbamates†
Cite this: Org. Biomol. Chem., 2014,
12, 5427
Xiu-Jiang Du,a Qiang Bian,*b Hong-Xue Wang,b Shu-Jing Yu,a Jun-Jie Kou,b
Zhi-Peng Wang,a Zheng-Ming Lia and Wei-Guang Zhao*a
Carboxylic acid amide (CAA) fungicides are an important class of agricultural fungicide with oomycete
activity and low toxicity toward mammalian cells. To find CAA analogues with high activity against resistant
pathogens, a series of substituted N-benzhydryl valinamide carbamate derivatives were designed and syn-
thesized by introducing substituted aromatic rings into valinamide carbamate leads. Bioassays showed
that some title compounds exhibited very good in vitro fungicidal activity against Phytophthora capsici
and in vivo fungicidal activities against Pseudoperonospora cubensis. Topomer CoMFA was performed to
explore the structure–activity relationship on the basis of the in vitro data. The dimethoxy substituted
aromatic analogue 9e was found to display higher in vitro fungicidal activity against Phytophthora capsici
than iprovalicarb but lower activity than mandipropamid, and higher in vivo fungicidal activity against
Received 9th April 2014,
Accepted 9th May 2014
DOI: 10.1039/c4ob00744a
www.rsc.org/obc
Pseudoperonospora cubensis than dimethomorph at a dosage of 6.25 µg mL−1
.
Introduction
Oomycetes are biologically and biochemically distinct from
ascomycetes,1 and exposure to the former can lead to the
occurrence of several destructive diseases in a range of impor-
tant crop plants, such as late blight on potatoes, blue mold on
tobacco, and downy grape mildew. Carboxylic acid amide
(CAA) fungicides exhibit high levels of activity against most
foliar oomycete plant pathogens, such as Plasmopara viticola in
grapes, Phytophthora infestans in potatoes and tomatoes, and
Pseudoperonospora cubensis in cucurbits. CAA fungicides were
officially announced by the Fungicide Resistance Action Com-
mittee (FRAC) in 20051 because they exhibited a common cross
resistance pattern to the vast majority of Plasmopara viticola.
CAA fungicides can be divided into three different sub-classes
based on differences in their structure, including (1) cinnamic
acid amides, such as dimethomorph,2 flumorph,3 and pyri-
morph;4 (2) valinamide carbamates, such as benthiavalicarb,5
benthiavancarb-isopropyl, iprovalicarb,6 and valiphenal;7 and
(3) mandelic acid amides, such as mandipropamid8 (Fig. 1).
Although the first CAA fungicides have been routinely used for
Fig. 1 Structures of commercial CCA fungicides.
the last 20 years, their mode of action was not elucidated until
2010. In 2010, Blum et al.9,10 reported that both of the known
mutations in the PiCesA3 gene of P. infestans, which is known
to be involved in the synthesis of cellulose, resulted in a
change to the same amino acid residue (glycine-1105) in the
protein, and that these mutations were responsible for the
mandipropamid-insensitive phenotype. Furthermore, these
aCollaborative Innovation Center of Chemical Science and Engineering (Tianjin),
State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin
300071, China. E-mail: zwg@nankai.edu.cn
bNational Pesticide Engineering Research Center (Tianjin), Nankai University,
Tianjin 300071, China. E-mail: bianqiang@nankai.edu.cn
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob00744a
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Fig. 2 The common structural fragments of CCA fungicides.
mutants showed reduced sensitivity to compounds belonging
to all CAA sub-classes. Several other mutant pathogen species
have recently been reported in the literature as being resistant
to CAA fungicides.9,11–13 Changes to G1105 V, G1105W or
G1105S have been reported to be responsible for CAA-resis-
tance in Ps. cubensis11 and P. viticola.14 The CAA compounds
exert their fungicidal activity by targeting cellulose synthases
in the oomycetes.
In previous studies,15–18 we reported the synthesis and
evaluation of the fungicidal activities of a series of mandelic
acid amide derivatives. Our attempts to optimize the aromatic
substituents as well as the structural features of the core
scaffold did not lead to significant improvements in the fungi-
cidal activities of these derivatives. Although dimethomorph
was the first CAA fungicide to be introduced in 1988, followed
by iprovalicarb in 1998, to date only two other cinnamic acid
amide compounds (flumorph and pyrimorph) and two other
valinamide carbamate compounds (benthiavalicarb and vali-
phenal) have progressed through development to be marketed
as commercial products. The two pairs of compounds are very
similar to those of dimethomorph and iprovalicarb, respectively.
In spite of differences in their overall structures, com-
pounds belonging to all three sub-classes possess very similar
structural fragments, including amide, halobenzene (or
methylbenzene) and/or dialkoxy benzene moieties (Fig. 2).
Compounds belonging to the cinnamic acid amide and man-
delic acid amide sub-classes possess all three of these active
structural fragments, whereas compounds belonging to the
valinamide carbamate sub-class only contain two of these
active structural fragments. With this in mind, we became
interested in synthesizing and evaluating the fungicidal activi-
ties of valinamide carbamate analogues that contained all
three of the active structural fragments.
Scheme 1 The synthetic route to compounds 9a–v.
according to a previously described procedure.13 The substi-
tuted benzoyl chlorides were obtained by treating the corres-
ponding benzoic acids 1a–g with oxalyl chloride in the
presence of a catalytic amount of N,N-dimethylformamide.
The benzoyl chlorides were then subjected to a Friedel–Crafts
acylation reaction with 1,2-dimethoxybenzene in the presence
of AlCl3 to give the ketone products 4a–g. Several other com-
pounds 4h–v (where R2 or R3 were not Me) were obtained by
the oxidation of the corresponding diphenylmethanols 3h–v
with IBX in dimethylsulfoxide. Compounds 3h–v were
obtained by the reaction of the substituted vanillin com-
pounds 2h–v with the appropriate aryl Grignard reagent. The
condensation of 4a–v with hydroxylamine hydrochloride gave
the corresponding oximes 5a–v in excellent yield. The oximes
5a–5v were selectively reduced to the corresponding amines
6a–v using zinc dust and ammonium formate. These reduction
conditions were used instead of hydrogenation over Pd/C to
avoid the loss of chlorine from the phenyl ring. The addition
of isopropyl chloroformate to a solution of L-valine 7 in
aqueous sodium hydroxide gave isopropyloxycarbonyl-L-valine
8 in good yield. The carboxylic acid moiety in compound 8 was
then treated with ethyl chloroformate under basic conditions
in tetrahydrofuran to give the mixed anhydride, which was not
isolated. Subsequent treatment with triethylamine and amines
Herein, we describe the synthesis and evaluation of the fungi-
cidal activities of a series of novel substituted N-benzhydryl valin-
amide carbamate derivatives, and show that these compounds
display promising antioomycetic activity.19 Stiez and colleagues
have reported a series of monoalkoxy substituted derivatives.20
Results and discussion
Chemistry
Compounds 9a–v were synthesized as shown in Scheme 1. The
precursor compounds 4a–g (R2, R3 = Me) were synthesized
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6a–v in tetrahydrofuran gave the title compounds 9a–v in 234.1 µg mL−1), showed much lower levels of fungicidal
85–95% yield. The synthesized compounds were characterized activity.
by 1H and 13C NMR and HR-MS. All of the spectral and analyti-
cal data were consistent with the assigned structures.
Compounds with a large group at the para- or meta-position
of ring B showed lower levels of antioomycetic activity than
compounds with smaller groups at the same position, as
shown in Table 1. The dimethoxy phenyl ring (ring B) provided
the highest activity for the compounds tested in the N-benz-
hydryl valinamide carbamate sub-class. When R2 ≠ Me, the
optimum ring A was found to be an unsubstituted phenyl ring
(R1 = H). Compound 9h (R1 = H, R2 = Et) was the most active
derivative with EC50 = 0.53 µg mL−1. Compound 9i (R1 = F,
R2 = Et) was the next most active analogue (EC50 = 10.07 µg
mL−1). Substitution at the para-position with larger sub-
stituents again had a negative effect on the fungicidal activity,
indicating that the SAR at this position is particularly tight.
Further increasing the bulk of the substituent at the para-
position of ring B or ring A led to a significant decrease in
fungicidal activity (compounds 9j–p all gave EC50 values
>100 µg mL−1).
Fungicidal activity
The in vitro fungicidal activities of compounds 9a–v towards
Phytophthora capsici are shown in Table 1 (reported as EC50
values in µg mL−1). The EC50 values were obtained from petri
dish trials and represent the concentrations at which the
tested compounds showed 50% inhibitory activity. Some com-
pounds showed very good fungicidal activity against Phyto-
phthora capsici and structure–activity relationship analysis
indicated that the substituents on the benzene ring had a sig-
nificant effect on the fungicidal activity. It was clear from the
data that compounds containing small alkyl groups at the
para-position of ring A (e.g. 9e, R1 = Me), gave high levels of
fungicidal activity against Phytophthora capsici (EC50 = 0.15 µg
mL−1), when ring B was a dimethoxyphenyl. Furthermore,
compound 9e showed higher antioomycetic potency than ipro-
valicarb (EC50 = 0.27 µg mL−1) and a level of potency very
similar to that of dimethomorph (EC50 = 0.12 µg mL−1), but it
was less active than mandipropamid (EC50 = 0.04 µg mL−1).
The para-methoxy derivative 9g (EC50 = 0.52 µg mL−1), unsub-
stituted derivative 9a (EC50 = 0.89 µg mL−1) and para-fluoro
derivative 9b (EC50 = 1.19 µg mL−1) all possessed interesting
levels of fungicidal activity. It is noteworthy, however, that
compounds containing a large and sterically bulky halogen or
In contrast, the use of bulkier groups at the meta-position
of ring B appeared to be reasonably well tolerated, with only a
relatively small drop in activity. For example, compound 9q
(R1 = H, R2 = Me, R3 = Et) gave an EC50 value of 5.35 µg mL−1
,
although the activity of this compound was 10-fold weaker
than that of its para-ethoxy substituted counterpart 9h.
Surprisingly, the para-bromo compound 9t (R1 = Br; EC50
=
1.97 µg mL−1) showed the highest fungicidal activity of the
compounds containing a halogen at this position, with the
order of fungicidal activity being Br > Cl > F. This trend was in
alkyl group, e.g. 9d (EC50 = 139.4 µg mL−1) and 9f (EC50
=
Table 1 Fungicidal activity against phytophthora capsici (in vitro) and predicted activities of the title compounds 9
Substituents
pEC50/μg mL−1
EC50 (95% confidence
interval)/μg mL−1
No.
R1
R2
R3
y = a + bx
r2
Exp.
Calcdb
9a
9b
9c
9d
9ea
9f
9g
9h
9i
H
F
Cl
Br
Me
t-Bu
MeO
H
F
Cl
Me
Me
Me
Me
Me
Me
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
y = 5.0599 + 1.1453x
y = 4.8445 + 2.0197x
y = 4.2690 + 0.7323x
y = 2.5569 + 1.1393x
y = 5.6249 + 0.7524x
y = 1.1442 + 1.5007x
y = 5.9592 + 3.3480x
y = 5.5692 + 2.0599x
y = 3.9643 + 1.0324x
y = 2.3445 + 1.2400x
y = 2.1691 + 1.2624x
y = 1.9304 + 1.5246x
y = 2.6194 + 1.0414x
y = 1.8955 + 1.3721x
y = 2.3025 + 1.1610x
y = 3.8242 + 0.4946x
y = 4.4663 + 0.7331x
y = 1.5614 + 1.5529x
y = 3.6425 + 0.8646x
y = 4.6247 + 1.2712x
y = 2.3287 + 1.3014x
y = 2.1550 + 1.2509x
y = 6.5558 + 1.6550x
y = 6.2088 + 2.1245x
y = 8.4774 + 2.2311x
0.9662
0.9817
0.9729
0.9954
0.9692
0.9925
0.9719
0.9710
0.9871
0.9983
0.9918
0.9804
0.9988
0.9911
0.9976
0.9982
0.9568
0.9744
0.9930
0.9998
0.9912
0.9782
0.9930
0.9966
0.9685
0.89 (0.65–1.21)
1.19 (1.00–1.32)
9.14 (6.31–13.25)
139.4 (109.0–178.4)
0.15 (0.06–0.35)
234.1 (181.4–302.1)
0.52 (0.46–0.58)
6.05
5.92
5.04
3.86
6.83
3.63
6.29
6.28
4.50
3.83
3.76
3.99
3.71
3.73
3.67
3.54
5.27
3.79
4.43
5.71
3.95
3.72
5.94
5.48
4.82
4.79
5.95
3.27
5.97
5.60
5.14
4.48
3.96
3.91
4.33
3.68
3.65
3.16
5.68
5.22
4.56
4.55
4.05
3.43
0.53 (0.46–0.61)
Et
Et
Et
10.07 (7.76–13.08)
147.8 (116.7–187.1)
174.8 (136.3–224.1)
102.18 (86.2–123.3)
193.2 (141.7–263.3)
183.1 (144.4–232.1)
210.6 (156.7–282.9)
238.3 (184.9–306.6)
5.35 (3.33–8.57)
163.85 (133.8–200.5)
37.16 (23.9–57.7)
1.97 (1.39–2.80)
112.9 (91.8–138.9)
188.1 (144.9–244.0)
0.12 (0.10–0.24)
9j
9k
9l
Me
Me
F
Propargyl
Benzyl
Benzyl
Benzyl
Benzyl
Me
Me
Me
Me
Me
9m
9n
9o
9p
9q
9ra
9sa
9ta
9u
9v
Cl
Br
Me
H
F
Cl
Br
Me
Me
Et
Et
Et
Et
Me
Benzyl
Dimethomorph
Iprovalicarb
Mandipropamid
0.27 (0.22–0.33)
0.04 (0.03–0.06)
a Testing set. b Predicted were using topomer CoMFA.
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Table 2 Fungicidal activity against Phytophthora capsici and Pseudoperonospora cubensi (in vivo)
P. capsici (% control at given concentration mg L−1
)
P. cubensi (% control at given concentration mg L−1
)
No.
50
25
12.5
6.25
3.125
100
50
25
12.5
6.25
c log P
9a
9b
9e
9g
9h
9t
87
100
96
100
100
83
100
100
77
83
63
96
83
66
100
100
59
75
46
91
61
53
100
100
55
33
38
87
35
36
87
71
37
17
33
83
30
17
63
46
79
85
88
77
73
77
82
—
59
83
87
68
58
72
74
—
56
79
77
65
51
59
69
—
42
51
72
56
47
50
61
—
39
23
71
49
31
30
47
—
3.94
4.09
4.44
3.86
4.47
5.33
2.73
3.2
Dimethomorph
Iprovalicarb
complete contrast to what had been observed above. The (0.2 mg L−1) and 9g (1.3 mg L−1) were poorly soluble in water.
results of the current study appear to suggest that when R2 is The aqueous solubilities of the compounds provided some
methyl, the introduction of an ethyl group at the meta-position understanding as to why they exhibited low in vivo fungicidal
of ring B (i.e., R3 = Et) could have a significant steric effect that activities at high assay doses.
could lead to a change in the binding mode of the substrates
to the target enzyme.
It was envisaged that the more lipophilic compounds would
be tightly bound to the leaf’s waxy layer. This would allow
A further in vivo assay was conducted in a greenhouse to these compounds to provide a highly effective, weatherproof
estimate the fungicidal activities of the most active compounds and long-lasting barrier to disease by foliar sprays. Most of the
against Phytophthora capsici and Pseudoperonospora cubensi. As compounds in Table 2 exhibited very good inhibition against
shown in Table 2, the compounds showed lower levels of Pseudoperonospora cubensi. A comparison of all of the test com-
in vivo fungicidal activity against Phytophthora capsici than the pounds revealed a strong positive correlation between the EC50
control compounds dimethomorph and iprovalicarb. Under values estimated for in vitro fungicidal activity against Phyto-
equivalent dosage conditions of 50 mg L−1, compounds 9b, 9g phthora capsici and in vivo fungicidal activity against Pseudo-
and 9h show 100% inhibition against Phytophthora capsici, peronospora cubensi. Compounds 9e and 9g displayed
whereas compound 9e, which was identified as the most significant levels of control of 71 and 49% against Pseudo-
potent compound from the in vitro fungicidal assay, showed a peronospora cubensi at 6.25 µg mL−1, respectively, and showed
slightly lower level of inhibition (96%). The activities of these higher levels of antioomycetic activity than dimethomorph
compounds against Phytophthora capsici were therefore (47%) at the same concentration.
measured using dose reduction with serial two-fold dilution.
Tomoper CoMFA analysis
The fungicidal activities of all of the test compounds became
progressively lower against Phytophthora capsici at concen-
trations of 25 and 12.5 µg mL−1, whereas the control com-
pounds still showed 100% inhibition at these concentrations.
The fungicidal activity of the most potent synthesized com-
pound 9g decreased significantly with decreasing concen-
tration, although its potency (83%) was greater than that of the
reference dimethomorph (63%) and iprovalicarb (46%) at the
3.125 µg mL−1 concentration.
The introduction of a second phenyl ring into the lead com-
pounds led to a significant increase in their c log P values. The
high log P values represent a major stumbling block for drug
absorption through the root. With this in mind, we calculated
the log P values (octanol–water) of the test compounds using
Sybyl, and the results are shown in Table 2. The results show
that the in vivo fungicidal activities are related to their c log P
values. For example, compound 9g (c log P = 3.86) exhibited
83% inhibition down to a dose of 3.125 mg L−1, whereas com-
pounds 9e (c log P = 4.44) and 9h (c log P = 4.47) exhibited
inhibition values of 33% and 30% at the same dose levels,
respectively.
To further explore the influence of the different substituents
on the benzhydryl group on the activities of the compounds,
we used a topomer CoMFA method to develop a deeper under-
standing of the quantitative structure–activity relationships.
The topomer CoMFA method provides the means for an align-
ment-independent 3D-QSAR approach,21 which is a more
rational way to explain the hypothesis as it does not involve
alignment, and it provides a means for automated activity
searches in fragment libraries. In this topomer CoMFA study,
we designated a training set of 18 compounds by fragmenting
the molecules into three parts: valinamide carbamates, halo-
benzene (or methylbenzene) and dialkoxy benzene moieties,
topologically aligned R1 and R2 fragments as shown in Fig. 3.
The topomer CoMFA model gives both statistical and
graphical results. Statistically, the conventional non-cross-
validated correlation coefficient, r2 of 0.82 using 2 PLS com-
ponents, while the cross-validation gave a q2 of 0.43, F = 34.70,
and a r2pred of 0.46. The predicted activities of the dataset,
along with R1 and R2 fragment contribution, are shown in
Table 1. Plots of the predicted vs. actual (experimental) activity
of the training set and the test set molecules are shown in
Fig. 4.
The solubilities of compounds 9e and 9g were also tested
in water at pH 7. The results revealed that compounds 9e
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Yellow contours (Fig. 5a) dominate the R1 group in the
steric field and red contours (Fig. 5b) occupy the electrostatic
field; this suggests that small bulky groups with electronega-
tive potential at the R1 site would be favorable for activity.
Regarding the contours of the R2 group, yellow contours
(Fig. 5c) are located near R2 and R3; the red contours are
located in the middle of the electrostatic field, and blue con-
tours are located far away from the phenyl ring (Fig. 5d); this
demonstrates that a small bulky group with electronegative
potential at the R2 site would improve the fungicidal activity.
Fig. 3 Diagram of split title compounds into three pieces for the
topomer CoMFA modeling.
Conclusions
We have designed and synthesized a series of substituted
N-benzhydryl valinamide carbamate derivatives as potential
cellulose synthase inhibitors. Subsequent evaluation of the
fungicidal activities of these compounds revealed that the
introduction of a dimethoxyphenyl group into the valinamide
carbamate lead compound gave very good levels of fungicidal
activity against Phytophthora capsici and Pseudoperonospora
cubensi. The overall analyses (topomer CoMFA), suggest that
the small bulky and electronegative nature of the halobenzene
(or methylbenzene) and dialkoxybenzene moieties are respon-
sible for the higher potency of the molecules. Compounds 9e
and 9g are particularly active against Phytophthora capsici
and Pseudoperonospora cubensi and are also more potent than
the control compound dimethomorph but less potent than
mandipropamid. Further studies are currently under way in
our laboratory to conduct field trials.
Fig. 4 Graph of experimental versus predicted pEC50 values from
topomer CoMFA.
Experimental
Material and methods
1H and 13C NMR spectra were measured on a Bruker AC-P500
instrument using TMS as the internal standard and CDCl3 as
the solvent. Melting points were determined on an X-4 bino-
cular microscope melting point apparatus (Beijing Tech Instru-
ments, Beijing, China) and were uncorrected. HRMS were
recorded on an Ionspec 7.0-T Fourier-transform ion-cyclotron
resonance (FTICR) mass spectrometer. All reagents are analyti-
cal grade.
Synthesis of (3,4-dimethoxyphenyl)(substituted phenyl)-
methanones (4a–4g).22 To a suspension of substituted benzoic
acid (54.28 mmol) in dry dichloromethane (60 mL), oxalyl
chloride (108.57 mmol) and dimethylformamide (three drops)
were added at room temperature. The crude mixture was
refluxed for 2 h. It was concentrated in vacuo, washed with
dichloromethane and concentrated again, twice. The substi-
tuted benzoyl chloride was isolated as a yellow oil in quantita-
tive yield. This was transferred into a round-bottomed flask and
diluted with dichloromethane (50 mL). Aluminium trichloride
(5.31 g, 39.81 mmol) was then added and the mixture was
cooled to 0 °C. 1,2-Dimethoxybenzene (5.0 g, 36.19 mmol) was
added dropwise and the mixture was refluxed for 2 h. The
Fig. 5 Contour maps of topomer CoMFA around R1 and R2. (a) Steric
contour map for R1, (b) Eelectrostatic contour map for R1, (c) steric
contour map for R2, (d) electrostatic contour map for R2.
The topomer CoMFA 3D contour maps around R1 and R2
(shown in Fig. 5) were generated by plotting the coefficients
from the model. Green polyhedra surrounded regions where
more bulk is ‘good’ for increasing potency, while yellow poly-
hedra surrounded regions where less bulk is ‘good’. Red and
blue contours show regions of desirable negative and positive
electrostatic interactions, respectively.
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temperature was cooled to room temperature and the organic chloride (2.07 g, 30 mmol) in anhydrous ethanol (50 mL), and
layer was washed with ice water (50 mL), 2 M HCl (20 mL), the resulting mixture was heated at reflux for 6 h. The reaction
saturated aqueous sodium hydrogen carbonate (50 mL), dried was then cooled to room temperature and concentrated
over anhydrous sodium sulfate, and concentrated in vacuo to in vacuo to give a residue, which was extracted with dichloro-
give compounds 4.
methane (3 × 50 mL), and the combined organic layers were
General synthetic procedures for (3,4-dialkoxylphenyl)(sub- washed with water (3 × 15 mL), dried over anhydrous sodium
stituted phenyl)methanols (3h–v).23 To a suspension of mag- sulfate, and concentrated in vacuo to give compounds 5.
nesium shavings (0.59 g, 24.75 mmol) in anhydrous
Example data of 5k is shown as follows, whereas data for
tetrahydrofuran (30 mL, nitrogen atmosphere) was added a the other compounds can be found in the ESI.†
small crystal of iodine. Substituted bromobenzene
Data for 5k: white solid, yield = 81.7%. mp 87–89 °C. 1H
(22.5 mmol) was then added slowly (over 30 minutes), so as to NMR (400 MHz, CDCl3) δ 7.29 (ddd, J = 25.4, 22.1, 4.9 Hz, 4H,
maintain a gentle reflux. The mixture was heated to reflux for Ph-H), 7.14 (d, J = 8.0 Hz, 1H, Ph-H), 6.96 (dd, J = 12.0,
3 h. After cooling to room temperature, the mixture was trans- 10.3 Hz, 1H, Ph-H), 6.80 (dt, J = 21.7, 5.2 Hz, 1H, Ph-H), 4.13
ferred to a pressure-equalizing dropping funnel with a syringe. (dd, J = 22.3, 7.0 Hz, 2H, OCH2CH3), 3.84 (d, J = 5.6 Hz, 3H,
To a solution of 3,4-dialkoxyl benzaldehyde (15 mmol) in an- OCH3), 2.41 (s, 1H), 1.51–1.45 (m, 3H, OCH2CH3). HRMS calcd
hydrous tetrahydrofuran (25 mL, nitrogen atmosphere), was for C17H19NO3 ([M + H]): 285.1442; found: 285.1365.
slowly added the solution of substituted-phenylmagnesium
General procedure for the synthesis of the (3,4-dialkoxyl-
bromide. After stirring at room temperature for 4 h, saturated phenyl)(substituted phenyl)methanamines (6a–v). A solution
ammonium chloride (30 mL) was added and the reaction was of 28% (w/w) aqueous ammonia (70 mL) was added to a sus-
extracted with diethyl ether (3 × 20 mL). The combined pension of (3,4-dialkoxylphenyl)(substituted phenyl)metha-
organic layers were washed with brine (3 × 15 mL), dried over none oxime (23.11 mmol), zinc powder (7.51 g, 115.56 mmol)
anhydrous sodium sulfate, and concentrated in vacuo to give and ammonium acetate (1.78 g, 23.11 mmol) in ethanol
compounds 3.
Example data of 3i is shown as follows, whereas data for the 1 h. The mixture was then cooled to room temperature and fil-
other compounds can be found in the ESI.† tered to remove the zinc. The filtrate was collected and the
(140 mL) and the resulting mixture was heated at reflux for
Data for 3i: white solid, yield = 93.0%, mp 95–96 °C. 1H organic solvent removed in vacuo to give a residue, which was
NMR (300 MHz, CDCl3) δ 7.34 (dd, J = 8.4, 5.6 Hz, 2H, Ph-H), extracted with ethyl acetate (4 × 50 mL). The combined orga-
7.02 (t, J = 8.7 Hz, 2H, Ph-H), 6.89 (s, 1H, Ph-H), 6.83 (s, 2H, nics were then washed sequentially with saturated aqueous
Ph-H), 5.78 (s, 1H, Ph-H-Ph), 4.08 (q, J = 7.0 Hz, 2H, sodium hydrogen carbonate (50 mL) and brine (3 × 50 mL)
OCH2CH3), 3.84 (s, 3H, OCH3), 1.45 (t, J = 7.0 Hz, 3H, before being dried over anhydrous sodium sulfate and concen-
OCH2CH3). HRMS calcd for C16H17FO3 ([M + Na]): 276.1054; trated in vacuo to give compounds 6.
found: 276.1162.
Example data of 6i is shown as follows, whereas data for the
General procedure for the synthesis of the (3,4-dialkoxyl- other compounds can be found in the ESI.†
phenyl)(substituted phenyl)methanones (4h–v). 2-Iodoxy-
Data for 6i: yellow oil (4.63 g). Yield = 77.9%. 1H NMR
benzoic acid (IBX) (15 mmol) was added to a solution of (3,4- (400 MHz, CDCl3) δ 7.40–7.28 (m, 2H, Ph-H), 6.98 (t, J = 8.4 Hz,
dialkoxylphenyl)(substituted phenyl)methanol 3 (10 mmol) in 2H, Ph-H), 6.93–6.74 (m, 3H, Ph-H), 5.14 (s, 1H, CHNH), 4.06
dimethylsulfoxide (25 mL), and the resulting mixture was (dd, J = 13.6, 6.7 Hz, 2H, OCH2CH3), 3.83 (s, 3H, OCH3), 1.44
stirred at room temperature for 4 h. Water (100 mL) was then (t, J = 6.8 Hz, 3H, OCH2CH3). HRMS calcd for C16H18FNO2
added, and the resulting mixture was extracted with diethyl ([M + H]): 275.1137; found: 275.1322.
ether (4 × 20 mL). The combined organic layers were washed
with brine (6 × 20 mL), dried over anhydrous sodium sulfate, butanoic acid (8).24 L-Valine 7 (0.3 mol) and isopropyl chloro-
and concentrated in vacuo to give compounds 4. carbonate (0.36 mol) were added sequentially in a drop-wise
Example data of 4i is shown as follows, whereas data for the manner to a stirred solution of sodium hydroxide (0.6 mol) in
other compounds can be found in the ESI.† water (300 mL) at 0 °C, and the resulting mixture was stirred at
Synthesis of (S)-2-((isopropoxycarbonyl)amino)-3-methyl-
Data for 4i: white solid, yield = 94.5%, mp 92–94 °C. room temperature for 1 h. The reaction mixture was then
1H NMR (400 MHz, CDCl3) δ 7.80 (dd, J = 8.4, 5.6 Hz, 2H, washed with ethyl acetate (100 mL) before being acidified
Ph-H), 7.46 (d, J = 1.5 Hz, 1H, Ph-H), 7.32 (dd, J = 8.3, 1.6 Hz, with 2 M HCl to pH 2–3 and extracted with ethyl acetate (4 ×
1H, Ph-H), 7.15 (t, J = 8.5 Hz, 2H, Ph-H), 6.88 (d, J = 8.4 Hz, 50 mL). The combined extracts were dried over anhydrous
1H, Ph-H), 4.19 (q, J = 7.0 Hz, 2H, OCH2CH3), 3.93 (s, 3H, sodium sulfate and concentrated in vacuo to give compound 8
OCH3), 1.52 (t, J = 7.0 Hz, 3H, OCH2CH3). HRMS calcd for as a white solid (mp: 70–72 °C, yield = 91.0%).
C16H15FO3 ([M + H]): 274.1084; found: 274.1005.
General procedure for the synthesis of the (3,4-dialkoxyl- 1-(((3,4-dialkoxylphenyl)(substituted
phenyl)(substituted phenyl)methanone oximes (5a–v). 3-methyl-1-oxobutan-2-yl)carbamates
General procedure for the synthesis of the isopropyl ((2S)-
phenyl)methyl)amino)-
(9a–v). Triethylamine
Sodium hydroxide (1.20 g, 30 mmol) was added in a single (6 mmol) was added to a solution of (S)-2-((isopropoxycarbo-
portion to solution of (3,4-dialkoxylphenyl)(substituted nyl)amino)-3-methylbutanoic acid 8 (5 mmol) in anhydrous
phenyl)methanone 4 (20 mmol) and hydroxylamine hydro- tetrahydrofuran (20 mL) followed by ethyl chloroformate
a
5432 | Org. Biomol. Chem., 2014, 12, 5427–5434
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(5 mmol), and the resulting mixture was stirred at 0 °C for 1 h. of a solution of each chemical by making 1 cm long incisions
A solution of ((3,4-dialkoxylphenyl)(substituted phenyl)metha- in the pepper plant 1 day prior to the inoculation with
namine 6 (6 mmol) in anhydrous tetrahydrofuran (10 mL) was P. capsici. The zoospore inoculation of the pepper roots was
then added to the reaction in a drop-wise manner, and the performed by inoculating 1 mL of zoospore inoculum of
resulting mixture was stirred at room temperature for 2 h. The P. capsici according to the same procedure used for the chemi-
reaction mixture was then filtered and the filtrate concentrated cal treatment process. Six seedlings at the four-leaf stage were
under vacuum to give a residue, which was extracted with ethyl transplanted into a plastic pot (5 × 30 × 5 cm) containing the
acetate (3 × 20 mL). The combined organics were washed with soil mix described previously. Seedlings that had been
brine (2 × 15 mL), dried over anhydrous sodium sulfate, and drenched with sterile distilled water instead of the zoospore
concentrated in vacuo to give compounds 9.
Example data of 9h is shown as follows, whereas data for evaluated after 7 days using a 0–5 scale (0 = no visible disease
the other compounds can be found in the ESI.† symptoms; 1 = leaves slightly wilted with brownish lesions
inoculum were used as a negative control. Disease severity was
1
Data for 9h: white solid, yield = 94.1%. mp 142–144 °C. H beginning to appear on their stems; 2 = 30–50% of the entire
NMR (400 MHz, CDCl3) δ 7.36–7.24 (m, 3H, Ph-H), 7.21 (d, J = plant diseased; 3 = 50–70% of the entire plant diseased; 4 =
7.1 Hz, 2H, Ph-H), 6.77 (dd, J = 11.6, 8.6 Hz, 2H, Ph-H), 6.69 (d, 70–90% of the entire plant diseased; 5 = dead plant). The
J = 5.8 Hz, 1H, Ph-H), 6.17 (d, J = 7.6 Hz, 1H, CHNHCO), 5.17 results are listed in Table 2.
(d, J = 7.7 Hz, 1H, Ph-CHNH), 4.85 (d, J = 6.3 Hz, 1H, Ph-CH-
Fungicidal activity against Pseudoperonospora cubensi
Ph), 4.14–4.03 (m, 2H, CH3CH2O), 4.02–3.92 (m, 1H, (in vivo).30 Cucumber plants, which were cultivated by
COCHNH), 3.79 (s, 3H, OCH3), 3.21–2.92 (m, 1H, (CH3)2CHO), Shandong Mici with one fully expanded true leaf, were main-
2.15 (m, 1H, (CH3)2CHCH), 1.44 (t, J = 7.0 Hz, 3H, CH3CH2O), tained in a greenhouse. The commercial fungicide dimetho-
1.20 (dd, J = 12.7, 6.6 Hz, 6H, (CH3)2CHO), 0.93 (dd, J = 17.8, morph was used as a positive control and its antifungal
6.3 Hz, 6H, (CH3)2CHCH). HRMS calcd for C25H34N2O5 activity compared with those of the synthesized compounds 9.
([M + H]): 442.2540; found: 442.2468.
The synthesized compounds 9 and dimethomorph dissolved
separately in DMF, and the resulting solutions were diluted to
Fungicidal activities
give the concentrations of 12.5, 25, 50.0, 100 and 200 µg mL−1
.
Fungicidal activity against Phytophthora capsici (in vitro). The plants were then sprayed with these different fungicide
The in vitro fungicidal activities of the synthesized compounds solutions and allowed to dry in air for approximately 2 h. Cul-
against Phytophthora capsici were determined as previously tures of P. cubensis were maintained on the cucumber plants
described.25–27 The results are summarized in Table 1.
and the spores were collected by shaking the leaves in water.
Fungicidal activity against Phytophthora capsici (in vivo).28 The lower surfaces of the treated cucumber leaves were then
Plants infected with P. capsici were collected from pepper sprayed with a spore suspension of ca. 100 000 spores mL−1
.
fields in Tianjin, China, in 2012. The pathogenic fungal iso- The plants were transferred to a thermo-hygrostat and held at
lates of P. capsici were grown on V8 juice agar medium at room 20 °C and >90% humidity for 5 days to allow for infection. The
temperature for 7 days.29 To simulate the formation of sporan- plants were then held in the greenhouse for 5 more days
gium, the cultures were maintained under light at 24 °C for before being examined for their disease control score. The
seven days. When abundant sporangia had formed, the sterile results are listed in Table 2.
solution was replaced with sterile distilled water and the plates
were maintained at 4 °C for 30 min and then left at room
Topomer CoMFA and prediction of the inhibitory activity
temperature (20–24 °C) for 1 h to enhance the release of zoo- The Topomer CoMFA program in the SYBYL-X software
spores from the sporangia. The flooding water, which contained package was used to perform all calculations. A 3D-QSAR
zoospores and mycelium, was filtered using two layers of model was generated by splitting the molecules into frag-
cheesecloth, and the concentration of the zoospore suspension ments, topomerically aligning each fragment, and calculating
was subsequently adjusted to approximately 1 × 106 zoospores steric and electrostatic field descriptor values for the topomeri-
mL−1 using a hemocytometer. The inoculum was then used cally aligned fragments to create a CoMFA table with the field
immediately.
descriptor values. All molecules of the dataset were separated
The inoculation tests were conducted under greenhouse into two fragments shown as R1 and R2 groups as shown in
and growth chamber conditions. The pepper plants (Capsicum Fig. 3. Template molecules 9g were chosen for fragmentation.
annuum L.) were grown in plastic trays (50 × 30 × 5 cm) in a Each topomer fragment was applied with topomer alignment
greenhouse at 25 2 °C in a sterilized mixture of vermiculite to make a 3D invariant representation. Steric and electrostatic
and worm cast (5 : 1 – v/v). The commercial fungicide dimetho- interaction energies were calculated using the carbon sp3
morph was used as a positive control to compare its antifungal probe.
activity with those of the synthesized compounds 9. The title
All the models were investigated using the full cross-vali-
compounds 9 and dimethomorph were dissolved separately in dated r2 (q2) PLS leave-one-out (LOO) method with CoMFA
DMF, and the resulting solutions were diluted to give concen- standard options for scaling of the variables. A progressive
trations of 6.25, 12.5, 25, 50.0 and 100 µg mL−1. When the scrambling method was applied to determine the sensitivity of
plants had six leaves, each pepper root was treated with 1 mL QSAR models to chance correlations.
This journal is © The Royal Society of Chemistry 2014
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Paper
Organic & Biomolecular Chemistry
12 Y. Aoki, S. Furuya and S. Suzuki, Pest Manage. Sci., 2011,
67, 1557–1561.
Acknowledgements
We are grateful for financial support for this work from the 13 Z. Pang, J. Shao, L. Chen, X. Lu, J. Hu, Z. Qin and X. Liu,
National Natural Science Foundation of China (21172124), PLoS One, 2013, 8, e56513.
the National Basic Research Science Foundation of China 14 M. Blum, M. Waldner, G. Olaya, Y. Cohen, U. Gisi and
(2010CB126105), and the National Key Technologies R&D H. Sierotzki, Pest Manage. Sci., 2011, 67, 1211–1214.
Program (2011BAE06B05). We are also grateful for Professor 15 N. Su, Z. J. Wang, L. Z. Wang, X. Zhang, W. L. Dong,
Hong-Yu Zhang, Dr Dei-Xing Kong and Rong Wang from Huaz-
hong Agricultural University for their technical assistance in
performing the SYBYL.
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