Design, synthesis and structure-activity relationship of novel diphenylamine derivatives

Article abstract of DOI:10.1016/j.bmc.2015.09.032

Diphenylamine derivatives have been reported with good fungicidal, insecticidal, acaricidal, rodenticidal and/or herbicidal activities. To find new lead compound of this kind, a series of novel diphenylamine derivatives were designed and synthesized by the approach of Intermediate Derivatization Methods. All compounds were identified by ¹H NMR and elemental analysis. Bioassays demonstrated that some compounds substituted at 2,4,6-positions or 2,4,5-positions of phenyl ring B exhibited excellent fungicidal activities. The optimal compounds P30 and P33 showed 80% and 85% control respectively against cucumber downy mildew at 12.5 mg L^-1, both 100% control against rice blast at 0.3 mg L^-1 and both 100% control against cucumber gray mold at 0.9 mg L^-1. The relationship between structure and fungicidal activities was discussed as well.

Full text of DOI:10.1016/j.bmc.2015.09.032

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and structure–activity relationship of novel
diphenylamine derivatives
⇑
Huichao Li, Aiying Guan, Guang Huang, Changling Liu , Zhinian Li, Yong Xie, Jie Lan
State Key Laboratory of the Discovery and Development of Novel Pesticide, Shenyang Research Institute of Chemical Industry Co. Ltd, Shenyang 110021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Diphenylamine derivatives have been reported with good fungicidal, insecticidal, acaricidal, rodenticidal
and/or herbicidal activities. To find new lead compound of this kind, a series of novel diphenylamine
derivatives were designed and synthesized by the approach of Intermediate Derivatization Methods.
All compounds were identified by 1H NMR and elemental analysis. Bioassays demonstrated that some
compounds substituted at 2,4,6-positions or 2,4,5-positions of phenyl ring B exhibited excellent fungici-
dal activities. The optimal compounds P30 and P33 showed 80% and 85% control respectively against
cucumber downy mildew at 12.5 mg Lꢀ1, both 100% control against rice blast at 0.3 mg Lꢀ1 and both
100% control against cucumber gray mold at 0.9 mg Lꢀ1. The relationship between structure and fungici-
dal activities was discussed as well.
Received 29 June 2015
Revised 17 September 2015
Accepted 18 September 2015
Available online xxxx
Keywords:
Diphenylamine derivatives
Intermediate Derivatization Methods
Fungicidal activity
Ó 2015 Elsevier Ltd. All rights reserved.
Structure–activity relationship
1. Introduction
in agrochemical filed for many years mainly as a post-harvest
fungicidal protectant and scald inhibitor for pome fruit.5,6 Early
There are several approaches well known and widely used for
the discovery of new agrochemicals, including Random Synthesis
and Screening, Modification of Natural Compounds, ‘Me Too Chem-
istry’, Combinatorial Chemistry, and Rational Design. Meanwhile,
new methods that are more efficacious are continuously sought.
Recently, we have summarized an efficient method, Intermediate
Derivatization Methods (IDM), which has some significant advan-
tages over the traditional ones.1–4 IDM can be divided into three
types, namely Common Intermediate Method (CIM), Terminal
group Replacement Method (TRM) and Active compound Derivati-
zation Method (ADM). Among them, TRM focuses on novel key
intermediates synthesized through chemical reactions starting
from raw chemicals. Those unique intermediates are potential to
replace terminal moieties of known agrochemicals, pharmaceuti-
cals or natural products to obtained structurally new compounds,
which are further optimized to obtain new lead compounds and
develop novel products with excellent bioactivity. In this way,
the bioactivity of the original compounds may be maintained or
improved resulting in a high success rate of discovering patentable
structures.3
diphenylamine derivatives exhibited certain fungicidal activity as
well.7,8 However, great advancements were made when dipheny-
lamine derivatives substituted by multiple nitro and/or trifluo-
romethyl groups were discovered. Those compounds not only
showed good fungicidal activity9–12 but also provided excellent
insecticidal,13–16 acaricidal,17–20 rodenticidal21–23 or herbicidal
activity.24 Also, analogs in which one of the phenyl groups of
diphenylamine derivatives was replaced with a pyridinyl group
produced good activities.25–28 An important compound of this type
is fluazinam (Fig. 1), which is a broad-spectrum fungicide with pro-
tective action.29–31 Given this background, we viewed the dipheny-
lamine class as targets for the development of new fungicides.
In this paper, we present our results from the application of
TRM to generate new diphenylamine derivatives. 2,6-Dichloro-
toluene (1) was chosen as the starting raw material due to several
reasons: (1) it has a proven application as an important intermedi-
ate in several agrochemicals including bistrifluron (insecticide),32
methoxyfenozide (insecticide),33 tembotrione (herbicide),34 and
methoxyacrylate compound 2 with excellent acaricidal activity35,36
(Fig. 2); (2) the simple chemical structure and relatively low
molecular weight provided it more chances to be modified; and
(3) it can be readily functionalized, opening the door to a wide vari-
ety of new compounds. 2,6-Dichlorotoluene (1, Fig. 3) was nitrated
to obtain the key intermediate 2,6-dichloro-3,5-dinitrotoluene (3),
which has not been used as a building block of agrochemicals yet.
Using the chemical structure of fluazinam as a scaffold, we
Compounds of diphenylamine class are simple in structure, easy
to be synthesized, and have diverse bioactivities. The simplest
one of this class is diphenylamine (Fig. 1), which has been used
⇑
Corresponding author. Tel.: +86 24 85869078; fax: +86 24 85869137.
E-mail address: liuchangling@vip.163.com (C. L. Liu).
http://dx.doi.org/10.1016/j.bmc.2015.09.032
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Li, H. C. et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.032

2
H. C. Li et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Cl
NO2 Cl
aniline (6) in DMF under basic condition is a convenient method to
H
N
H
N
synthesize the title compounds, with which compounds P1–P21,
F3C
CF3
P25–P28 and P33 were prepared (Scheme 1).39
N
NO2
Since diphenylamine can be nitrated in ortho or para-position of
phenyl ring, nitration is another method to prepare the title com-
pounds with nitro-group(s). When nitration happened in phenyl
ring B, compounds P22–P24, P31, P32 and P34 were synthesized
as shown in Scheme 2. Compounds P6–P8 and P26 were nitrated
with mixture of fuming nitric acid and concentrated sulfuric acid
to generate compounds P22–P24 and P34 with one more nitro-
group in yields of 65–79%. Compounds P5 and P8 were nitrated
to generate compounds P32 and P31 with two more nitro-groups
in yields of 65% and 46%. When nitration happened in phenyl ring
A or both rings, the synthetic route is shown in Scheme 3.
3-Chloro-2-methylaniline (7) reacted with o/p-nitrochlorobenzene
in basic condition to afford intermediate 8, which could be nitrated
to generate compounds P29, P30, P35 and P36 in yields of 34–65%.
The target compounds P37–P40 were synthesized as shown in
Scheme 4. Compounds P8, P12 or P20 reacted with CH3I respec-
tively to give corresponding compounds P37–P39, compound
P37 was further nitrated to afford compound P40.
diphenylamine
fluazinam
Figure 1. Chemical structures of diphenylamine and fluazinam.
replaced the substituted phenyl ring of fluazinam with intermedi-
ate 3 resulting in compound 4, from which a class of N-pyridiny-
laniline analogues (5) with good fungicidal activity were
designed and synthesized.37 With the aim of discovering new com-
pounds with better bioactivities, we designed a series of dipheny-
lamine derivatives of the general formula P (Fig. 3) by replacing the
pyridinyl ring of compounds 5 with a diverse set of substituted
phenyl groups. The design strategy of the target compounds P1–
P40 is shown in Figure 4.
2. Results and discussion
2.1. Chemistry
2.2. Biological activity and the structure–activity relationship
The synthetic routes for the key intermediate 3 and all target
compounds were illustrated in Schemes 1–4. Starting from 2,6-
dichlorotoluene (1), the key intermediate 2,6-dichloro-3,5-dinitro-
toluene (3) was prepared by nitration with fuming nitric acid in the
presence of concentrated sulfuric acid according to known
method.38 The reaction of key intermediate (3) with corresponding
Each compound was bioassayed against cucumber downy mil-
dew (CDM), rice blast (RB) and cucumber gray mold (CGM). Based
on the bioassay results, a relationship between chemical structures
and fungicidal activities was gradually developed.
CF3
Cl
F
O
F
O
CN
F
O
Cl
Cl
N
H
N
H
CF3
CH3
NH2
F
bistrifluron
CH3
CH3
O
CH3
O
NC
OCH3
OCH3
N
O
OCH3
HO
H3C
N
H
CH3
methoxyfenozide
Cl
Cl
O
O
Cl
O
Cl
O
Cl
CH3
SCH3
H3C
1
O
CF3
H3C
O
Br
O
SO2CH3
SO2CH3
tembotrione
Cl
CH3
CH3
H
N
H3C
Cl
N
O
Cl
Cl
NH2
Cl
Cl
NO2
N
O
O
CH3
CF3
2
O
H3C
Figure 2. Agrochemicals derived from 2,6-dichlortoluene.
CH3
Cl
CH3 Cl
Cl
CH3
Cl
CH3
Cl
NO2
Cl
Cl
H
N
H
N
O2N
CF3
O2N
O2N
Cl
(R)n
N
N
NO2
4
NO2
5
1
3
CH3
R1
Cl
NO2 Cl
(R)n
H
N
O2N
N
F3C
CF3
N
NO2
P
NO2
fluazinam
Figure 3. Derivation of compounds P.
Please cite this article in press as: Li, H. C. et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.032

H. C. Li et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
CH3
3
Cl
Cl
CH3
Cl
CH3 O2N
n=0 or 1
R1=H
R
optimization
H
H
H
N
COCH3
or NO2
O2N
N
O2N
R
N
O2N
NO2
NO2
NO2
Compounds P5-P8 (R=electron-withdrawing
group) displayed better activity against RB
than P9 and P10 (R=electron-donating group).
Compounds P2-P4 (R=Cl)
displayed better activity
against RB than P1 (R=H)
Compounds P22 and P24
showed enhanced effect
against CDM and RB.
screening
Cl
CH3 Cl
Cl
CH3 R2
Cl
CH3
n=2
screening
optimization
Cl
Cl
H
N
H
N
H
O2N
Cl
O2N
R3
O2N
N
R1=H
NO2
NO2
Compounds P16-P24
NO2
Compounds P11-P15
Compound P12
Cl
CH3 Cl
Cl
CH3 R2
optimization
Cl
H
N
H
N
O2N
O2N
R3
CH3
Cl
2'
2
3
Cl
CH3
R1
Cl
Cl
(R)n
4
NO2
Cl
screening
Cl
NO2
R4
1
n=3
O2N
N
A
B
H
Compound P26
Compounds P29, P30
O2N
N
R1=H
6
5
5'
NO2
P1-P40
screening
Cl
CH3 Cl
NO2
H
N
Compounds P25-P28
O2N
Cl
Cl
CH3 Cl
H
N
NO2 Cl
O2N
NO2
Cl
CH3 R2
Compound P28
n=4
H
N
NO2
Cl
O2N
R3
optimization
CH3 R2
Compound P30 showed 80% control
against CDM at 12.5 mg L-1, 100%
control against RB at 0.3 mg L-1 and
CGM at 0.9 mg L-1
R1=H
NO2 R5
R4
Cl
Compounds P34-P36 were
either phytotoxic or inferior to
the corresponding compounds
trisubstituted
H
N
O2N
R3
Lead 1
NO2 R4
Compounds P31-P33
screening
Cl
CH3
CH3
N
n=1-3
(R)n
O2N
R1=CH3
Compound P33 showed 85% control
against CDM at 12.5 mg L-1, 100%
control against RB at 0.3 mg L-1 and
CGM at 0.9 mg L-1
Cl
CH3 Cl
NO2
H
N
Compounds P37-P40 for the most
part were less active than their N-H
counterparts
O2N
CF3
NO2 Cl
Lead 2
Figure 4. Design strategy of the target compounds.
Cl
CH3
Cl
NO2
2.2.1. Preliminary screening of position and type of substituent
To begin with, the unsubstituted compound P1 was synthesized
as a measuring stick, which showed no activity at all (Table 1).
Then, the effect of substituent position (2, 3 or 4-position) was
tested by using the chlorine atom as a probe. As a result, com-
pounds P2–P4 (2-Cl, 3-Cl or 4-Cl, respectively) showed 0%, 50%
and 50% control against RB at 12.5 mg Lꢀ1. Finally, to screen the
effect of different substituents, compounds P5–P10 with elec-
tron-withdrawing or electron-donating substituents at 4-position
of ring B were synthesized. Bioassays indicated that compounds
P5–P8 with electron-withdrawing substituents showed better
control against RB than others, especially compound P5 with CF3
at 4-positon. All compounds synthesized in this section exhibited
very weak activities against CDM and CGM. Therefore, at the first
stage, no compound with excellent fungicidal activities was found,
but some useful information for further studies was obtained. (1) 3
CH3
i
Cl
Cl
O2N
1
3
Cl
CH3
Cl
NO2
Cl
CH3
(R)n
(R)n
ii
H
H2N
O2N
O2N
N
NO2
P1-P21, P25-P28, P33
6
3
Scheme 1. Synthesis of compounds P1–P21, P25–P28 and P33. Reagents and
conditions: (i) fuming nitric acid, concentrated sulfuric acid, 0–5 °C to 45 °C; (ii)
NaH, THF.
Cl
O2N
CH3
(R)n
NO2
H
N
Cl
CH3
NO2
P22-P24, P34
(R)n
i
H
O2N
N
B
Cl
CH3
NH2
Cl
CH3
Cl
CH3
Cl
O2N
CH3
NO2
(R)n
NO2
(R)n
i
ii
H
H
NO2
P5-P8, P26
(R)n
A
A
O2N
N
B
N
B
H
N
(NO2)2
NO2
P29, P30, P35, P36
7
8
NO2
P31, P32
Scheme 3. Synthesis of compounds P29, P30, P35 and P36. Reagents and
conditions: (i) corresponding o/p-nitrochlorobenzene, NaH, THF; (ii) fuming nitric
acid, concentrated sulfuric acid, 0 °C.
Scheme 2. Synthesis of compounds P22–P24, P31, P32 and P34. Reagents and
conditions: (i) fuming nitric acid, concentrated sulfuric acid, 0 °C.
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4
H. C. Li et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Cl
CH3
Cl
Cl
CH3
Table 2
CH3
N
(R)n
i
(R)n
H
Chemical structures and fungicidal activities of compounds P11 to P24
O2N
Cl
N
O2N
O2N
Compound (R)n
R1
Control (%) at given concentration
(mg Lꢀ1
NO2
P8, P12, P20
NO2
P37-P39
)
CDM
100 50
RB
CGM
CH3 O2N
CH3
N
CH3
CH3
N
ii
12.5 25
2.8 0.3 25
NO2
O2N
NO2
P11
P12
P13
P14
P15
P16
P17
P18
P19
P20
P21
P22
P23
P24
Fluazinam
2,3-2Cl
2,4-2Cl
2,6-2Cl
3,4-2Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0
50
0
0
0
0
20
0
/
40
0
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
0
/
100 60
100 80
0
0
0
/
/
/
0
0
0
0
80
0
0
50
0
0
NO2 O2N
NO2
P37
P40
0
0
/
/
80
50
80
Scheme 4. Synthesis of compounds P37–P40. Reagents and conditions: (i) CH3I,
NaH, DMF; (ii) fuming nitric acid, concentrated sulfuric acid, 0 °C.
3,5-2Cl
4-Cl-2-CH3
4-Cl-2-NO2
4-COCH3-2-Cl
2-Cl-4-NO2
2-Cl-4-CF3
2,4-2F
4-COCH3-2-NO2
4-CN-2-NO2
2,4-2NO2
/
100 80
100 100 50
100 100 50
/
70
/
/
or 4-position seems a little superior to 2-position. (2) Compounds
with one substituent are more active than that unsubstituted in
ring B. (3) Electron-withdrawing substituents (especially CF3) dis-
play better effect than electron-donating ones.
100 100
0
/
0
/
0
0
0
50
100 98
20
100 100 50
100 100 95
100 100 50
100 100
100 100 50
/
0
100 100 100 100
2.2.2. Further screening of position and type of substituent
Further effort was carried out on disubstituted compounds
(Table 2). To evaluate the effect of substituent positions on fungi-
cidal activities, compounds P11–P15 with two chlorines at differ-
/ stands for no data.
ent positions of ring
B were synthesized. Fungicidal assays
2.2.3. Discovery of the lead compounds
indicated that compound P12 with 2,4-2Cl exhibited better activity
against RB than others, but all compounds substituted by two chlo-
rines showed no or very weak activities against CDM and CGM.
Then, an effort aiming at modifying compound P12 was made
and compounds P16–P24 were synthesized. When 4-Cl was hold
constant and 2-Cl was replaced by 2-CH3 or 2-NO2, we synthesized
compounds P16 (4-Cl-2-CH3) and P17 (4-Cl-2-NO2), which respec-
tively showed inferior and comparable activities against RB to P12;
When 2-Cl was hold unchanged and 4-Cl was replaced by electron-
withdrawing groups (COCH3, NO2, CF3), all compounds generated
(P18–P20) exhibited superior activity against RB than P12; Consid-
ering the unique property of fluorine atom, compound P21 (2,4-2F)
was synthesized but did not show any activity at all; When both Cl
were changed to other electron-withdrawing groups, compounds
P22–P24 displayed enhanced activity against RB than P12. More-
over, the activity against CDM of P22 (4-COCH3-2-NO2, 98% control
at 50 mg Lꢀ1) and P24 (2,4-2NO2, 100% control at 50 mg Lꢀ1) was
improved greatly compared to P12 (50% control at 100 mg Lꢀ1).
Based on the results above, the following conclusions were
obtained: (1) 2,4-positions may play an important role in activity
improvement. (2) Strong electron-withdrawing groups at both 2
and 4 positions of phenyl ring B may enhance activities against
CDM and RB.
According to studies above, we noticed that compounds disub-
stituted showed better activities than those monosubstituted in
general. This result encouraged us to synthesis compounds with
more substituents. First of all, compounds P25–P28 with three
chlorines in ring B were synthesized to screen the effect of differ-
ent substituent positions on activities (Table 3). Among the four
compounds, P26 (2,4,5-3Cl) and P28 (2,4,6-3Cl) showed better
activities against both CDM and RB than others. This result was
consistent with that obtained above, namely 2,4-positions may
play an important role in activity improvement. Then optimization
around P26 and P28 were carried out respectively. Changing Cl at
2-positon or 4-position of P26 to NO2, we got compounds P29
(4,5-2Cl-2-NO2) and P30 (2,5-2Cl-4-NO2), whose fungicidal effect
increased considerably. Particularly, compound P30 showed
approximated activities to fluazinam against CDM (80% vs 95% at
12.5 mg Lꢀ1), RB (both 100% at 0.3 mg Lꢀ1) and CGM (both 100%
at 0.9 mg Lꢀ1) at the tested concentration. At the same time, mod-
ification of compound P28 was in progress. According to the
obtained idea above that strong electron-withdrawing groups
may enhance compound’s fungicidal activities, compounds P31
and P32 whose 2, 4 and 6 positions were occupied by NO2 or CF3
were synthesized. As a result, compound P31 with 2,4,6-3NO2
did not show better activities than P28, while P32 with 2,6-
2NO2-4-CF3 did. This result proved it again that CF3 at 4-posion
is important for activity improvement. So, compound P33 (2,6-
2Cl-4-CF3) was synthesized by changing Cl in 4-position of P28
into CF3. To our satisfaction, P33 showed comparable activities to
fluazinam at the tested concentration (85% vs 95% control against
CDM at 12.5 mg Lꢀ1, both 100% control against RB at 0.3 mg Lꢀ1
and both 100% control against CGM at 0.9 mg Lꢀ1). It seems that
introducing electron-withdrawing groups to 2, 4 or 6 position of
phenyl ring B can indeed improve the fungicidal activities. But
when 2, 4 and 6 positions of ring B were all occupied by strong
electron-withdrawing groups such as NO2 and/or CF3, the com-
pounds obtained were not the most active ones. Then, three com-
pounds (P26, P30 and P32) were chosen as examples for further
modification by adding one substituent to each of them. Com-
pound P34 with one more NO2 than P26, showed better activity,
but was phytotoxic. P35 and P36 generated by introducing a NO2
and Cl to P30 and P32, respectively did not show any enhanced
activity.
Table 1
Chemical structures and fungicidal activities of compounds P1 to P10
Compound
(R)n
R1
Control (%) at given concentration (mg Lꢀ1
)
CDM
RB
CGM
100
50
25
2.8
0.3
25
P1
P2
P3
P4
P5
P6
P7
P8
H
2-Cl
3-Cl
4-Cl
4-CF3
4-COCH3
4-CN
4-NO2
4-OCH3
4-C(CH3)3
H
H
H
H
H
H
H
H
H
H
/
0
0
0
0
0
/
20
10
0
0
/
/
/
/
/
0
0
0
/
0
/
0
0
0
50
0
0
0
0
0
0
0
0
50
0
0
0
50
0
100
100
100
100
100
100
100
80
50
50
100
80
50
80
0
P9
P10
Fluazinam
0
/
100
0
100
0
100
0
100
100
100
/ stands for no data.
Please cite this article in press as: Li, H. C. et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.032

H. C. Li et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Table 3
Chemical structures and fungicidal activities of compounds P25 to P36
Compound
(R)n
R1
Control (%) at given concentration (mg Lꢀ1
RB
)
CDM
50
CGM
2.8
100
12.5
25
2.8
0.3
25
0.9
P25
P26
P27
P28
P29
P30
P31
P32
P33
P34
P35
P36
2,3,4-3Cl
2,4,5-3Cl
3,4,5-3Cl
2,4,6-3Cl
4,5-2Cl-2-NO2
2,5-2Cl-4-NO2
2,4,6-3NO2
2,6-2NO2-4-CF3
2,6-2Cl-4-CF3
2,4,5-3Cl-6-NO2
2,5-2Cl-4,6-2NO2
2,6-2NO2-3-Cl-4-CF3
H
H
H
H
H
H
H
H
H
H
H
H
0
/
/
/
40
/
/
/
/
/
/
80
/
70
85
/
100
100
80
50
80
/
0
0
/
0
0
0
0
0
100
100
0
100
100
100
100
100
100
/
/
/
/
/
/
/
/
40
⁄
100
60
98
100
100
100
/
100
100
100
100
100
100
100
100
100
100
100
100
100
0
100
100
100
100
100
100
/
0
100
/
100
100
100
80
100
100
/
95
30
100
100
⁄
100
/
/
100
100
0
50
100
100
/
80
100
100
0
98
100
100
30
45
100
/
/
95
50
100
Fluazinam
/ stands for no data.
⁄ means no data because of phytotoxicity.
3,4,6-positions > 2,4-positions > other position(s) > no substituent.
Generally speaking, electron-withdrawing substituents were
superior to electron-donating ones. Our assessment of the effect
of amount, position and type of substituent(s) on fungicidal activ-
ity suggests that the best fungicidal activity was observed in those
compounds where the phenyl ring B is substituted at 2,4,5-posi-
tions or 2,4,6-positions with chlorines and strong electron-with-
drawing groups such as CF3 and NO2. The optimal compounds
P30 and P33 were identified as the lead compounds for further
study.
Table 4
Chemical structures and fungicidal activities of compounds P37 to P40 and their N–H
counterparts
Compound
(R)n
R1
Control (%) at given concentration
(mg Lꢀ1
)
CDM
100
RB
CGM
25
50
25
2.8
0.3
P8
4-NO2
4-NO2
2,4-2Cl
2,4-2Cl
2-Cl-4-CF3
2-Cl-4-CF3
2,4,6-3NO2
2,4,6-3NO2
H
CH3
H
CH3
H
CH3
H
20
0
0
/
/
/
/
/
30
/
100
80
100
50
100
80
100
100
80
/
80
/
100
/
0
0
/
0
/
0
/
/
0
0
0
0
0
0
0
0
P37
P12
P38
P20
P39
P31
P40
50
30
40
60
100
0
3. Conclusion
A series of novel diphenylamine derivatives were designed and
synthesized from key intermediate 3 by using IDM. Two lead com-
pounds (P30 and P33) were found, their fungicidal activities are
comparable to that of fluazinam at the tested concentration (80%
and 85% control vs 95% control against CDM at 12.5 mg Lꢀ1, all
100% control against RB at 0.3 mg Lꢀ1 and all 100% control against
CGM at 0.9 mg Lꢀ1). Some diphenylamine fungicides and fluazinam
are uncouplers of oxidative phosphorylation which are less suscep-
tible to the onset of resistance than many other fungicides.40 Based
on the similarity of chemical structure, we inferred that the com-
pounds synthesized in this study are uncouplers as well. Further
detailed studies on structural modification and mechanism are in
progress.
CH3
50
/
/ stands for no data.
2.2.4. Modification of the nitrogen bridge
To check the effect of an N-methyl substituent on fungicidal
activity, we synthesized four N-methyl diphenylamine compounds
(P37–P40), which for the most part were less active than their N–H
counterparts (Table 4).
Based on the data listed in Tables 1–4 and the discussions
above, a clear-cut, well-defined relation between chemical struc-
tures and biological activities was gradually developed. The effect
of amount, position and type of substituent(s) in phenyl ring B
on fungicidal activities were studied. When substituent amount
was considered independently, the trend was 3 substituents > 2
substituents > 1 substituent > no substituent. The role of sub-
stituent position in imparting fungicidal activity has been summa-
rized as follows (all in decreasing order of activity):
4. Experimental methods
4.1. General
All starting materials and reagents were commercially available
and used without further purification except as indicated. Melting
points were determined on a Büchi melting point apparatus M-565
and were uncorrected. 1H NMR spectra were recorded with
Mercury 300 (Varian, 300 MHz) spectrometer with CDCl3 or
DMSO-d6 as the solvent and tetramethylsilane as the internal stan-
dard. Elemental analyses were determined on a Yanaco MT-3CHN
elemental analyzer. Petroleum ether used for column chromatog-
raphy had a boiling range of 60–90 °C. Chemical names were
generated using ChemDraw Ultra (Cambridge Soft, version 14.0).
All plant and bacteria materials were obtained from the Agrochem-
ical Discovery Department in Shenyang Research Institute of
Chemical Industry.
(1) For compounds monosubstituted
3-position, 4-position > 2-position
(2) For compounds disubstituted
2,4-positions > 2,3-positions > 3,4-positions >
3,5-positions > 2,6-positions
(3) For compounds trisubstituted
2,4,6-positions > 2,4,5-positions > 2,3,4-positions > 3,4,
5-positions
When amount and position of substituent were considered
combined, the sequence was 2,4,6-positions > 2,4,5-positions > 2,
Please cite this article in press as: Li, H. C. et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.032

6
H. C. Li et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
4.2. Chemistry
4.2.1.8. 3-Chloro-2-methyl-4,6-dinitro-N-(4-nitrophenyl)aniline
(P8). Yellow solid; mp 172–174 °C; yield 72%. 1H NMR (300 MHz,
CDCl3): d 9.49 (s, 1H, NH), 8.64 (s, 1H, Ph-50-H), 8.06 (d, 2H, Ph-3,5-
2H, J = 9.0 Hz), 6.83 (d, 2H, Ph-2,6-2H, J = 9.0 Hz), 2.34 (s, 3H, Ph-20-
CH3). Anal. Calcd for C13H9ClN4O6: C, 44.27; H, 2.57; N, 15.89.
Found: C, 44.28; H, 2.54; N, 15.83.
4.2.1. General procedure for the synthesis of compounds P1–21,
P25–P28 and P33
Substituted/unsubstituted aniline 6 (1 equiv) was added in por-
tions to a suspension of NaH (60%, 2 equiv) and tetrahydrofuran
(THF) at room temperature, the mixture was stirred for 30 min.
2,6-Dichloro-3,5-dinitrotulune (3, 1.2 equiv) in THF was added
within 30 min, then stirred for another 5 h. After the reaction
was over by thin-layer chromatography (TLC) monitoring, the reac-
tion mixture was filtered. The filtrate was concentrated under
reduced pressure, and the residue was purified by silica gel column
chromatography (ethyl acetate/petroleum ether = 1:20) to give the
target compounds.
4.2.1.9. 3-Chloro-N-(4-methoxyphenyl)-2-methyl-4,6-dinitroani-
line (P9). Brown solid; mp 107–109 °C; yield 65%. 1H NMR
(300 MHz, CDCl3): d 9.42 (s, 1H, NH), 8.74 (s, 1H, Ph-50-H), 6.89
(m, 4H, Ph-2,3,5,6-4H), 3.82 (s, 3H, Ph-4-OCH3), 2.06 (s, 3H,
Ph-20-CH3). Anal. Calcd forC14H12ClN3O5: C, 49.79; H, 3.58; N,
12.44. Found: C, 49.77; H, 3.50; N, 12.47.
4.2.1.10. N-(4-tert-butylphenyl)-3-chloro-2-methyl-4,6-dini-
troaniline (P10). Yellow solid; mp 183–185 °C; yield 68%. 1H
NMR (300 MHz, CDCl3): d 9.37 (s, 1H, NH), 8.75 (s, 1H, Ph-50-
H), 7.35 (d, 2H, Ph-3,5-2H, J = 8.7 Hz), 6.85 (d, 2H, Ph-2,6-2H,
J = 8.4 Hz), 2.10 (s, 3H, Ph-20-CH3), 1.32 (s, 9H, Ph-4-(CH3)3). Anal.
Calcd for C17H18ClN3O4: C, 56.13; H, 4.99; N, 11.55. Found: C,
56.20; H, 4.97; N, 11.58.
4.2.1.1. 3-Chloro-2-methyl-4,6-dinitro-N-phenylaniline (P1)
Yellow solid; mp 132–134 °C; yield 75%. 1H NMR (300 MHz,
CDCl3): d 9.34 (s, 1H, NH), 8.74 (s, 1H, Ph-50-H), 7.35 (t, 2H, Ph-
3,5-2H, J = 8.1 Hz), 7.18 (t, 1H, Ph-4-H, J = 8.1 Hz), 6.90 (d, 2H, Ph-
2,6-2H), 2.11 (s, 3H, Ph-20-CH3). Anal. Calcd for C13H10ClN3O4: C,
50.75; H, 3.28; N, 13.66. Found: C, 50.70; H, 3.30; N, 13.69.
4.2.1.2. 3-Chloro-N-(2-chlorophenyl)-2-methyl-4,6-dinitroani-
line (P2). Orange solid; mp 150–151 °C; yield 79%. 1H NMR
(300 MHz, CDCl3): d 9.02 (s, 1H, NH), 8.69 (s, 1H, Ph-50-H), 7.48
(dd, 1H, Ph-3-H, 3J = 7.8 Hz, 4J = 1.5 Hz), 7.14–7.19 (m, 1H, Ph-4-
H), 7.04–7.10 (m, 1H, Ph-5-H), 6.61 (d, 1H, Ph-6-H, J = 8.1 Hz),
2.13 (s, 3H, Ph-20-CH3). Anal. Calcd for C13H9Cl2N3O4: C, 45.64;
H, 2.65; N, 12.28. Found: C, 45.73; H, 2.69; N, 12.24.
4.2.1.11. 3-Chloro-N-(2,3-dichlorophenyl)-2-methyl-4,6-dini-
troaniline (P11). Yellow solid; mp 158–160 °C; yield 75%. 1H
NMR (300 MHz, CDCl3): d 8.90 (s, 1H, NH), 8.66 (s, 1H, Ph-50-
H), 7.22 (d, 1H, Ph-4-H, J = 8.1 Hz), 7.11 (t, 1H, Ph-5-H,
J = 7.8 Hz), 2.16 (s, 3H, Ph-20-CH3). Anal. Calcd for C13H8Cl3N3O4:
C, 41.46; H, 2.14; N, 11.16. Found: C, 41.50; H, 2.12; N, 11.19.
4.2.1.12. 3-Chloro-N-(2,4-dichlorophenyl)-2-methyl-4,6-dini-
troaniline (P12). Orange solid; mp 136–137 °C; yield 73%. 1H
NMR (300 MHz, CDCl3): d 8.93 (s, 1H, NH), 8.68 (s, 1H, Ph-50-H),
7.49 (s, 1H, Ph-3-H), 7.17 (d, 1H, Ph-5-H, J = 9.0 Hz), 6.53 (d, 1H,
Ph-6-H, J = 9.0 Hz), 2.14 (s, 3H, Ph-20-CH3). Anal. Calcd for C13H8Cl3-
N3O4: C, 41.46; H, 2.14; N, 11.16. Found: C, 41.53; H, 2.23; N, 11.08.
4.2.1.3. 3-Chloro-N-(3-chlorophenyl)-2-methyl-4,6-dinitroani-
line (P3). Orange solid; mp 120–122 °C; yield 75%. 1H NMR
(300 MHz, CDCl3): d 9.09 (s, 1H, NH), 8.68 (s, 1H, Ph-50-H), 7.24–
7.29 (m, 1H, Ph-5-H), 7.12 (d, 1H, Ph-4-H, J = 7.5 Hz), 6.88 (s,
1H, Ph-2-H), 6.76 (d, 1H, Ph-6-H, J = 8.1 Hz), 2.16 (s, 3H, Ph-20-
CH3). Anal. Calcd for C13H9Cl2N3O4: C, 45.64; H, 2.65; N, 12.28.
Found: C, 45.68; H, 2.61; N, 12.30.
4.2.1.13. 3-Chloro-N-(2,6-dichlorophenyl)-2-methyl-4,6-dini-
troaniline (P13). Brown solid; mp 166–168 °C; yield 70%. 1H
NMR (300 MHz, CDCl3): d 9.29 (s, 1H, NH), 8.73 (s, 1H, Ph-50-H),
7.40 (d, 2H, Ph-3,5-2H, J = 8.1 Hz), 7.19 (t, 1H, Ph-4-H,
J = 8.1 Hz), 1.94 (s, 3H, Ph-20-CH3). Anal. Calcd for C13H8Cl3N3O4:
C, 41.46; H, 2.14; N, 11.16. Found: C, 41.43; H, 2.17; N, 11.24.
4.2.1.4. 3-Chloro-N-(4-chlorophenyl)-2-methyl-4,6-dinitroani-
line (P4). Brown solid; mp 154–156 °C; yield 81%. 1H NMR
(300 MHz, CDCl3): d 9.17 (s, 1H, NH), 8.70 (s, 1H, Ph-50-H), 7.31
(d, 2H, Ph-3,5-2H, J = 9.0 Hz), 6.83 (d, 2H, Ph-2,6-2H, J = 9.0 Hz),
2.13 (s, 3H, Ph-20-CH3). Anal. Calcd for C13H9Cl2N3O4: C, 45.64; H,
2.65; N, 12.28. Found: C, 45.73; H, 2.70; N, 12.33.
4.2.1.14. 3-Chloro-N-(3,4-dichlorophenyl)-2-methyl-4,6-dini-
troaniline (P14). Red solid; mp 106–108 °C; yield 78%. 1H
NMR (300 MHz, CDCl3): d 9.04 (s, 1H, NH), 8.69 (s, 1H, Ph-50-H),
7.39 (d, 1H, Ph-5-H, J = 7.8 Hz), 6.98 (s, 1H, Ph-2-H), 6.72 (dd,
4.2.1.5. 3-Chloro-2-methyl-4,6-dinitro-N-(4-(trifluoromethyl)
phenyl)aniline (P5). Red solid; mp 91–94 °C; yield 73%. 1H
NMR (300 MHz, CDCl3): d 9.20 (s, 1H, NH), 8.71 (s, 1H, Ph-50-H),
7.21 (d, 2H, Ph-3,5-2H, J = 8.4 Hz), 6.91 (d, 2H, Ph-2,6-2H,
J = 8.4 Hz), 2.14 (s, 3H, Ph-20-CH3). Anal. Calcd for C14H9ClF3N3O4:
C, 44.76; H, 2.41; N, 11.19. Found: C, 44.65; H, 2.37; N, 11.23.
4
1H, Ph-6-H, 3J = 8.4 Hz, J = 2.4 Hz), 2.17 (s, 3H, Ph-20-CH3). Anal.
Calcd for C13H8Cl3N3O4: C, 41.46; H, 2.14; N, 11.16. Found: C,
41.39; H, 2.11; N, 11.25.
4.2.1.15. 3-Chloro-N-(3,5-dichlorophenyl)-2-methyl-4,6-dini-
troaniline (P15). Brown solid; mp 148–150 °C; yield 80%. 1H
NMR (300 MHz, CDCl3): d 8.91 (s, 1H, NH), 8.70 (s, 1H, Ph-50-H),
7.12 (s, 1H, Ph-4-H), 6.73 (s, 2H, Ph-2,6-2H), 2.21 (s, 3H, Ph-20-
CH3). Anal. Calcd for C13H8Cl3N3O4: C, 41.46; H, 2.14; N, 11.16.
Found: C, 41.55; H, 2.18; N, 11.19.
4.2.1.6. N-(4-acetylphenyl)-3-chloro-2-methyl-4,6-dinitroani-
line (P6). Brown solid; mp 206–208 °C; yield 70%. 1H NMR
(300 MHz, CDCl3): d 9.08 (s, 1H, NH), 8.69 (s, 1H, Ph-50-H), 7.95
(d, 2H, Ph-3,5-2H, J = 8.4 Hz), 6.87 (d, 2H, Ph-2,6-2H, J = 8.4 Hz),
2.59 (s, 3H, Ph-4-COCH3), 2.19 (s, 3H, Ph-20-CH3). Anal. Calcd for
C
15H12ClN3O5: C, 51.52; H, 3.46; N, 12.02. Found: C, 51.58; H,
3.43; N, 12.09.
4.2.1.16. 3-Chloro-N-(4-chloro-2-methylphenyl)-2-methyl-4,6-
dinitroaniline (P16). Red solid; mp 121–123 °C; yield 71%. 1H
NMR (300 MHz, CDCl3): d 9.03 (s, 1H, NH), 8.74 (s, 1H, Ph-50-H),
7.27 (s, 1H, Ph-3-H), 7.10 (dd, 1H, Ph-5-H, 3J = 8.4 Hz,
4J = 2.1 Hz), 6.53 (d, 1H, Ph-6-H, J = 8.4 Hz), 2.40 (s, 3H, Ph-2-
CH3), 2.02 (s, 3H, Ph-20-CH3). Anal. Calcd for C14H11Cl2N3O4: C,
47.21; H, 3.11; N, 11.80. Found: C, 47.18; H, 3.15; N, 11.88.
4.2.1.7. 3-Chloro-N-(4-cyanophenyl)-2-methyl-4,6-dinitroani-
line (P7). Reddish brown solid; mp 184–186 °C; yield 68%. 1H
NMR (300 MHz, CDCl3): d 8.93 (s, 1H, NH), 8.66 (s, 1H, Ph-50-H),
7.62 (d, 2H, Ph-3,5-2H, J = 8.7 Hz), 6.87 (d, 2H, Ph-2,6-2H,
J = 8.7 Hz), 2.22 (s, 3H, Ph-20-CH3). Anal. Calcd for C14H9ClN4O4:
C, 50.54; H, 2.73; N, 16.84. Found: C, 50.61; H, 2.76; N, 16.81.
Please cite this article in press as: Li, H. C. et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.032

H. C. Li et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
4.2.1.17.
3-Chloro-N-(4-chloro-2-nitrophenyl)-2-methyl-4,6-
4.2.1.26. 3-Chloro-N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-
2-methyl-4,6-dinitroaniline (P33). Yellow solid; mp 143–
144 °C; yield 70%. 1H NMR (300 MHz, CDCl3): d 9.10 (s, 1H, NH),
8.70 (s, 1H, Ph-50-H), 7.66 (s, 2H, Ph-3,5-2H), 1.98 (s, 3H, Ph-20-
CH3). Anal. Calcd for C14H7Cl3F3N3O4: C, 37.82; H, 1.59; N, 9.45.
Found: C, 37.88; H, 1.53; N, 9.46.
dinitroaniline (P17). Yellow solid; mp 204–205 °C; yield 76%. 1H
NMR (300 MHz, CDCl3): d 10.36 (s, 1H, NH), 8.54 (s, 1H, Ph-50-H),
8.26 (s, 1H, Ph-3-H), 7.43 (d, 1H, Ph-5-H, J = 8.4 Hz), 6.48 (d, 1H,
Ph-6-H, J = 8.4 Hz), 2.31 (s, 3H, Ph-20-CH3). Anal. Calcd for C13H8Cl2-
N4O6: C, 40.33; H, 2.08; N, 14.47. Found: C, 40.37; H, 2.02; N, 14.40.
4.2.2. General procedure for the synthesis of compounds P22–
P24 and P34
4.2.1.18. N-(4-acetyl-2-chlorophenyl)-3-chloro-2-methyl-4,6-di-
nitro aniline (P18). Yellow solid; mp 178–180 °C; yield 70%. 1H
NMR (300 MHz, CDCl3): d 8.88 (s, 1H, NH), 8.64 (s, 1H, Ph-50-H),
8.08 (s, 1H, Ph-3-H), 7.77 (d, 1H, Ph-5-H, J = 8.4 Hz), 6.51 (d, 1H,
Ph-6-H, J = 8.4 Hz), 2.58 (s, 3H, Ph-4-COCH3), 2.23 (s, 3H, Ph-20-
CH3). Anal. Calcd for C15H11Cl2N3O5: C, 46.90; H, 2.89; N, 10.94.
Found: C, 46.85; H, 2.93; N, 10.89.
Compound P6–P8 or P26 (1 equiv) was dissolved in concen-
trated sulfuric acid (96%) and cooled to 0 °C. Fuming nitric acid
(1.2 equiv) in concentrated sulfuric acid was added evenly, the
reaction mixture was stirred for 5 min. After the reaction was over
by TLC monitoring, the reaction mixture was poured into ice water,
extracted with ethyl acetate, washed with water and then brine.
The organic phase was dried over anhydrous MgSO4, evaporated,
and the residue was purified by silica gel column chromatography
(ethyl acetate/petroleum ether = 1:10).
4.2.1.19. 3-Chloro-N-(2-chloro-4-nitrophenyl)-2-methyl-4,6-di-
nitroaniline (P19). Yellow solid; mp 191–193 °C; yield 75%. 1H
NMR (300 MHz, CDCl3): d 8.79 (s, 1H, NH), 8.62 (s, 1H, Ph-50-H),
8.41 (s, 1H, Ph-3-H), 8.06 (d, 1H, Ph-5-H, J = 8.7 Hz), 6.48 (d, 1H,
Ph-6-H, J = 8.7 Hz), 2.29 (s, 3H, Ph-20-CH3). Anal. Calcd for C13H8Cl2-
N4O6: C, 40.33; H, 2.08; N, 14.47. Found: C, 40.40; H, 2.18; N, 14.42.
4.2.2.1. N-(4-acetyl-2-nitrophenyl)-3-chloro-2-methyl-4,6-dini-
troaniline (P22). Yellow solid; mp 171–173 °C; yield 79%. 1H
NMR (300 MHz, CDCl3): d 10.60 (s, 1H, NH), 8.87 (s, 1H, Ph-50-H),
8.53 (s, 1H, Ph-3-H), 8.07 (d, 1H, Ph-5-H, J = 9.0 Hz), 6.53 (d, 1H,
Ph-6-H, J = 9.0 Hz), 2.63 (s, 3H, Ph-4-COCH3), 2.37 (s, 3H, Ph-20-
CH3). Anal. Calcd for C15H11ClN4O7: C, 45.64; H, 2.81; N, 14.19.
Found: C, 45.60; H, 2.89; N, 14.25.
4.2.1.20. 3-Chloro-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-
methyl-4,6-dinitroaniline (P20). Yellow solid; mp 106–
108 °C; yield 78%. 1H NMR (300 MHz, CDCl3): d 8.87 (s, 1H, NH),
8.65 (s, 1H, Ph-50-H), 7.75 (s, 1H, Ph-3-H), 7.43 (d, 1H, Ph-5-H,
J = 8.4 Hz), 6.55 (d, 1H, Ph-6-H, J = 8.4 Hz), 2.22 (s, 3H, Ph-20-
CH3). Anal. Calcd for C14H8Cl2F3N3O4: C, 41.00; H, 1.97; N, 10.25.
Found: C, 41.10; H, 1.93; N, 10.28.
4.2.2.2. 3-Chloro-N-(4-cyano-2-nitrophenyl)-2-methyl-4,6-dini-
troaniline (P23). Yellow solid; mp 259–261 °C; yield 71%. 1H
NMR (300 MHz, CDCl3): d 10.51 (s, 1H, NH), 8.62 (s, 1H, Ph-50-H),
8.50 (s, 1H, Ph-3-H), 7.70 (d, 1H, Ph-5-H, J = 8.4 Hz), 6.54 (d, 1H,
Ph-6-H, J = 8.4 Hz), 2.38 (s, 3H, Ph-20-CH3). Anal. Calcd for C14H8-
ClN5O6: C, 44.52; H, 2.14; N, 18.54. Found: C, 44.46; H, 2.17; N,
18.60.
4.2.1.21. 3-Chloro-N-(2,4-difluorophenyl)-2-methyl-4,6-dini-
troaniline (P21). Orange solid; mp 136–138 °C; yield 80%. 1H
NMR (300 MHz, CDCl3): d 9.00 (s, 1H, NH), 8.72 (s, 1H, Ph-50-H),
6.90–6.99 (m, 3H, Ph-3,5,6-3H), 2.12 (s, 3H, Ph-20-CH3). Anal.
Calcd for C13H8ClF2N3O4: C, 45.43; H, 2.35; N, 12.23. Found: C,
45.48; H, 2.31; N, 12.29.
4.2.2.3. 3-Chloro-N-(2,4-dinitrophenyl)-2-methyl-4,6-dinitroa-
niline (P24). Yellow solid; mp 185–186 °C; yield 70%. 1H NMR
(300 MHz, CDCl3): d 10.59 (s, 1H, NH), 9.23 (s, 1H, Ph-3-H), 8.52
(s, 1H, Ph-50-H), 8.31 (d, 1H, Ph-5-H, J = 9.3 Hz), 6.56 (d, 1H, Ph-
6-H, J = 9.3 Hz), 2.41 (s, 3H, Ph-20-CH3). Anal. Calcd for C13H8ClN5-
O8: C, 39.26; H, 2.03; N, 17.61. Found: C, 39.17; H, 2.06; N, 17.67.
4.2.1.22. 3-Chloro-2-methyl-4,6-dinitro-N-(2,3,4-trichlorophe-
nyl)aniline (P25). Brown solid; mp 122–123 °C; yield 78%. 1H
NMR (300 MHz, CDCl3): d 8.84 (s, 1H, NH), 8.65 (s, 1H, Ph-50-H),
7.28 (d, 1H, Ph-5-H, J = 8.7 Hz), 6.42 (d, 1H, Ph-6-H, J = 8.7 Hz),
2.17 (s, 3H, Ph-20-CH3). Anal. Calcd for C13H7Cl4N3O4: C, 37.99;
H, 1.72; N, 10.22. Found: C, 37.97; H, 1.78; N, 10.26.
4.2.2.4. 2,4,5-Trichloro-N-(3-chloro-2-methyl-4,6-dinitrophe-
nyl)-6-nitroaniline (P34). Reddish brown solid; mp 133–
135 °C; yield 65%. 1H NMR (300 MHz, CDCl3): d 9.22 (s, 1H, NH),
8.69 (s, 1H, Ph-50-H), 7.70 (s, 1H, Ph-5-H), 2.07 (s, 3H, Ph-20-
CH3). Anal. Calcd for C13H6Cl4N4O6: C, 34.24; H, 1.33; N, 12.29.
Found: C, 34.18; H, 1.36; N, 12.23.
4.2.1.23. 3-Chloro-2-methyl-4,6-dinitro-N-(2,4,5-trichlorophe-
nyl)aniline (P26). Red solid; mp 142–143 °C; yield 74%. 1H
NMR (300 MHz, CDCl3): d 8.80 (s, 1H, NH), 8.67 (s, 1H, Ph-50-H),
7.58 (s, 1H, Ph-2-H), 6.59 (s, 1H, Ph-6-H), 2.20 (s, 3H, Ph-20-CH3).
Anal. Calcd for C13H7Cl4N3O4: C, 37.99; H, 1.72; N, 10.22. Found:
C, 38.03; H, 1.69; N, 10.20.
4.2.3. General procedure for the synthesis of compounds P31
and P32
Compounds P31 and P32 were prepared from P8 and P5,
respectively by the same procedure of 4.2.2, except using 3 equiv
fuming nitric acid.
4.2.1.24. 3-Chloro-2-methyl-4,6-dinitro-N-(3,4,5-trichlorophe-
nyl)aniline (P27). Orange solid; mp 163–164 °C; yield 75%. 1H
NMR (300 MHz, CDCl3): d 8.94 (s, 1H, NH), 8.87 (s, 1H, Ph-50-H),
6.88 (s, 2H, Ph-2,6-2H), 2.22 (s, 3H, Ph-20-CH3). Anal. Calcd for
4.2.3.1. 3-Chloro-N-(2,4,6-trinitrophenyl)-2-methyl-4,6-dinitro-
aniline (P31). Red oil; yield 65%. 1H NMR (300 MHz, CDCl3): d
10.93 (s, 1H, NH), 9.09 (s, 2H, Ph-3,5-2H), 8.52 (s, 1H, Ph-50-H),
2.27 (s, 3H, Ph-20-CH3).
C
13H7Cl4N3O4: C, 37.99; H, 1.72; N, 10.22. Found: C, 38.10; H,
1.65; N, 10.27.
4.2.1.25. 3-Chloro-2-methyl-4,6-dinitro-N-(2,4,6-trichlorophe-
nyl)aniline (P28). Yellow solid; mp 160–162 °C; yield 79%. 1H
NMR (300 MHz, CDCl3): d 9.19 (s, 1H, NH), 8.72 (s, 1H, Ph-50-H),
7.41 (s, 2H, Ph-3,5-2H), 1.95 (s, 3H, Ph-20-CH3). Anal. Calcd for
4.2.3.2. 3-Chloro-N-(2,6-dinitro-4-(trifluoromethyl)phenyl)-2-
methyl-4,6-dinitroaniline (P32). Brown solid; mp 93–95 °C;
yield 46%. 1H NMR (300 MHz, CDCl3): d 10.42 (s, 1H, NH), 8.56
(s, 1H, Ph-50-H), 8.14 (s, 2H, Ph-3,5-2H), 2.19 (s, 3H, Ph-20-CH3).
Anal. Calcd for C14H7ClF3N5O8: C, 36.11; H, 1.52; N, 15.04. Found:
C, 36.17; H, 1.54; N, 14.96.
C
13H7Cl4N3O4: C, 37.99; H, 1.72; N, 10.22. Found: C, 38.03; H,
1.77; N, 10.19.
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8
H. C. Li et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
4.2.4. General procedure for the synthesis of compounds P29,
P30, P35 and P36
4.2.5.3.
3-Chloro-N-(2-chloro-4-(trifluoromethyl)phenyl)-N,
(P39). Yellow solid; mp
2-dimethyl-4,6-dinitroaniline
3-Chloro-2-methylaniline (7) reacted with o/p-nitrochloroben-
zene according to the synthesis method of Section 4.2.1 to afford
intermediate 8. Compounds P29, P30, P35 and P36 were prepared
from intermediate 8 by the same procedure of Section 4.2.2, except
using 3 equiv fuming nitric acid.
138–140 °C; yield 45%. 1H NMR (300 MHz, CDCl3): d 8.08 (s, 1H,
Ph-50-H), 7.57 (d, 1H, Ph-5-H, J = 8.7 Hz), 7.48 (s, 1H, Ph-3-H),
7.23 (d, 1H, Ph-6-H, J = 8.7 Hz), 3.37 (s, 3H, NCH3), 2.58 (s, 3H,
Ph-20-CH3). Anal. Calcd for C15H10Cl2F3N3O4: C, 42.48; H, 2.38; N,
9.91. Found: C, 42.43; H, 2.41; N, 9.97.
4.2.5.4. 3-Chloro-N-(2,4,6-trinitrophenyl)-N,2-dimethyl-4,6-dini-
troaniline (P40). Brown solid; mp 140–142 °C; yield 43%. 1H NMR
(300 MHz, CDCl3): d 8.57 (s, 2H, Ph-3,5-2H), 8.38 (s, 1H, Ph-50-H),
3.30 (s, 3H, NCH3), 2.58 (s, 3H, Ph-20-CH3). Anal. Calcd for C14H9ClN6-
4.2.4.1. 3-Chloro-N-(2-nitro-4,5-dichlorophenyl)-2-methyl-4,6-
dinitroaniline (P29). Reddish-brown solid; mp 127–129 °C; yield
65%. 1H NMR (300 MHz, CDCl3): d 10.31 (s, 1H, NH), 8.54 (s, 1H, Ph-
50-H), 8.40 (s, 1H, Ph-3-H), 6.55 (s, 1H, Ph-6-H), 2.36 (s, 3H, Ph-20-
CH3). Anal. Calcd for C13H7Cl3N4O6: C, 37.04; H, 1.67; N, 13.29.
Found: C, 37.15; H, 1.69; N, 13.25.
O10: C, 36.82; H, 1.99; N, 18.40. Found: C, 36.75; H, 2.07; N, 18.51.
4.3. Fungicidal assay
4.2.4.2. 3-Chloro-N-(2,5-dichloro-4-nitrophenyl)-2-methyl-4,6-
dinitroaniline (P30). Brown solid; mp 169–171 °C; yield 42%.
1H NMR (300 MHz, CDCl3): d 8.62 (s, 1H, NH), 8.60 (s, 1H, Ph-50-
H), 8.20 (s, 1H, Ph-3-H), 6.42 (s, 1H, Ph-6-H), 2.32 (s, 3H, Ph-20-
CH3). Anal. Calcd for C13H7Cl3N4O6: C, 37.04; H, 1.67; N, 13.29.
Found: C, 37.08; H, 1.64; N, 13.34.
Each of the test compounds was first dissolved in proper sol-
vent, water containing 0.1% Tween 80 was then added to generate
the stock solution. Serial test solutions were prepared by diluting
the above solution. The untreated control was a solution of solvent
and water containing Tween 80. The fungicidal activity against
cucumber downy mildew (CDM) was tested in vivo, while rice
blast (RB) and cucumber gray mold (CGM) were tested in vitro.
Biological data were reported in the range 0 (indicates no control)
to 100% (complete control).
Evaluation of fungicidal activity in vivo: Cucumber seedling
with 2–3 leaves of greenhouse-grown was used as the host plant,
which was sprayed with the testing solution by a special plant
sprayer and then placed in a drying hood at room temperature.
Three replicates were used for each treatment. After 24 h, the
cucumber plants were inoculated with fungus and stored in a
humidity chamber and then transferred into greenhouse after
infection was finished. The activity of each tested compound was
based on a subjective estimate of percent disease control (leaf area
free of disease symptoms and sporulation). The first true leaf was
used for disease evaluation. After 7 days, the untreated plant was
considered ‘0% control’ and was the basis for rating all the
treatments.
4.2.4.3. 3-Chloro-N-(2,5-dichloro-4,6-dinitrophenyl)-2-methyl-
4,6-dinitroaniline (P35). Brown solid; mp 159–162 °C; yield
34%. 1H NMR (300 MHz, CDCl3): d 8.91 (s, 1H, NH), 8.63 (s, 1H,
Ph-50-H), 8.23 (s, 1H, Ph-5-H), 2.16 (s, 3H, Ph-20-CH3). Anal. Calcd
for C13H6Cl3N5O8: C, 33.47; H, 1.30; N, 15.01. Found: C, 33.38; H,
1.33; N, 15.06.
4.2.4.4. 3-Chloro-N-(3-chloro-2,6-dinitro-4-(trifluoromethyl)
phenyl)-2-methyl-4,6-dinitroaniline
(P36). Reddish-brown
solid; mp 136–138 °C; yield 59%. 1H NMR (300 MHz, CDCl3): d
10.10 (s, 1H, NH), 8.72 (s, 1H, Ph-50-H), 8.50 (s, 1H, Ph-5-H), 2.41
(s, 3H, Ph-20-CH3). Anal. Calcd for C14H6Cl2F3N5O8: C, 33.62; H,
1.21; N, 14.00. Found: C, 33.72; H, 1.25; N, 13.92.
4.2.5. General procedure for the synthesis of compounds P37–
P40
Evaluation of fungicidal activity in vitro: HTS method was used.
In a no animalcule condition, the testing solution and the patho-
gens suspension were added into the cells of 96 cells culture board,
and four replicates were used for each treatment. The board was
placed in the constant temperature box. After 24 h, the activity of
test compound was evaluated based on germination of pathogen
under the microscope.
Compound P12, P12 or P20 (1 equiv) was added to a suspension
of NaH (60%, 2.5 equiv) and DMF. The mixture was stirred for 1 h
and then added thereto CH3I (3 equiv), the resulting mixture was
allowed to react for 5 h. After the reaction was over by TLC moni-
toring, the reaction mixture was poured into saturated brine and
extracted with ethyl acetate. The extract were washed with brine,
dried with anhydrous MgSO4, and filtered. The filtrate was evapo-
rated and the crude product was purified by silica gel column chro-
matography (ethyl acetate/petroleum ether = 1:10) to give
compounds P37–P39. Compound P40 was prepared from com-
pound P37 by the same procedure of Section 4.2.2., except using
3 equiv fuming nitric acid.
Acknowledgements
We thank Dr. Xiaoping Yang in University of Colorado and Dr.
Mark Dekeyser (Canada) for assistance with manuscript
preparation.
References and notes
4.2.5.1.
3-Chloro-N,2-dimethyl-4,6-dinitro-N-(4-nitrophenyl)
aniline (P37). Yellow solid; mp 150–152 °C; yield 40%. 1H NMR
(300 MHz, CDCl3): d 8.29 (s, 1H, Ph-50-H), 8.15 (d, 2H, Ph-3,5-2H,
J = 7.2 Hz), 6.58 (d, 2H, Ph-2,6-2H, J = 7.2 Hz), 3.36 (s, 3H, NCH3),
2.34 (s, 3H, Ph-20-CH3). Anal. Calcd for C14H11ClN4O6: C, 45.85; H,
3.02; N, 15.28. Found: C, 45.74; H, 3.07; N, 15.36.
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