N-Nitrourea Derivatives as Novel Potential Fungicides against Rhizoctonia solani: Synthesis, Antifungal Activities, and 3D-QSAR

Article abstract of DOI:10.1111/j.1747-0285.2012.01346.x

A series of N-nitrourea derivatives bearing various aryl substituents were conveniently obtained via three steps including nitration, carbamic chlorination, and aminolysis reactions. The structures of all newly synthesized compounds were characterized and confirmed by IR, ¹H-NMR, MS, and elemental analysis. The preliminary bioassays indicate that five compounds possess sufficient fungicidal activity against Rhizoctonia solani. Structure-activity relationship (SAR) is also discussed based on the experimental data, and the further quantitative structure-activity relationship (QSAR) was analyzed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA).

Full text of DOI:10.1111/j.1747-0285.2012.01346.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01346.x
Chem Biol Drug Des 2012; 80: 81–89
Research Article
N-Nitrourea Derivatives as Novel Potential
Fungicides against Rhizoctonia solani: Synthesis,
Antifungal Activities, and 3D-QSAR
Xiufang Cao1, Shengzhen Xu1, Xuegang
including herbicidal, antimicrobial, insecticidal activities, and so on.
Many studies have proved that modification of both sides of car-
bamide bridge's amines is an effective way to obtain new ana-
logues with higher activity. On the other hand, N-nitro-substituted
Li1, Xiaoxia Shen1, Qingye Zhang1,
Jianhong Li2 and Changshui Chen1,*
1College of Science, Huazhong Agricultural University, Wuhan
anilines displayed broad-spectrum biological activities including her-
bicidal properties (12), antifungal effects (13), and plant growth reg-
ulating activities (14). Keeping these considerations in mind, we
proposed that the urea derivatives bearing a new group nitro in
the NH-CO-NH bridge should display some interesting biological
activities.
430070, China
2Department of Plant Protection, Huazhong Agricultural University,
Wuhan 430070, China
*Corresponding author: Changshui Chen,
caoxiufang@mail.hzau.edu.cn
A series of N-nitrourea derivatives bearing various
aryl substituents were conveniently obtained via
three steps including nitration, carbamic chlorina-
tion, and aminolysis reactions. The structures of
all newly synthesized compounds were character-
ized and confirmed by IR, 1H-NMR, MS, and ele-
mental analysis. The preliminary bioassays
indicate that five compounds possess sufficient
fungicidal activity against Rhizoctonia solani.
Structure–activity relationship (SAR) is also dis-
cussed based on the experimental data, and the
further quantitative structure–activity relationship
(QSAR) was analyzed using comparative molecular
field analysis (CoMFA) and comparative molecular
similarity indices analysis (CoMSIA).
As a continuation of our ongoing project aimed, we report herein
the detailed synthetic procedures of series of N-nitrourea deriva-
tives and bioassay results, and the further quantitative structure–
activity relationships of these synthesized compounds were also
analyzed using comparative molecular field analysis (CoMFA) (15)
and comparative molecular similarity indices analysis (CoMSIA)
(16,17). Fortunately, some compounds with promising fungicidal
activities were identified.
Experimental Section
Instrumentation and chemicals
All reagents were commercially available, and all solvents and
liquid reagents were dried by standard methods and distilled before
use. Melting points were determined with a digital melting point
apparatus and are uncorrected. IR spectra were recorded on a
Key words: antifungal activities, N-Nitrourea, quantitative struc-
ture–activity relationship, Rhizoctonia solani
1
Thermo Nicolet FT-IR Avatar 330 instrument. H-NMR spectra were
Received 21 June 2011, revised 8 January 2012 and accepted for publi-
cation 8 January 2012
measured on a Bruker AM spectrometer (Bruker, Fallanden, Switzer-
land) with tetramethylsilane as internal standard and DMSO-d6 as
solvent. Elemental analysis was performed on a Vario EL III Elemen-
tal analysis instrument (Elementar Analysensysteme GmbH, Hanau,
Germany). The progress of the reactions was monitored by TLC on
silica gel plates visualized with UV light.
Rice sheath blight caused by Rhizoctonia solani is one of the most
destructive rice diseases worldwide and severely impairs both rice
yield and quality (1). Under conditions favorable for disease develop-
ment, rice grain yield losses, ranging from 4% to 50%, have been
attributed to sheath blight (2). In Japan, this disease causes a yield loss
of as high as 20% and affects about 120 000–190 000 ha. In the Uni-
ted States, a yield loss of 50% is reported when susceptible cultivars
are planted (3). In China, sheath blight disease affects about 15–
20 million hectares and causes a yield loss of 6 million tons of rice
grains per year (4). Therefore, the research on novel inhibitors control-
ling the Rhizoctonia solani becomes active, necessary, and meaningful.
General procedure for the preparation of target
compounds 4–41
Preparation of compound 4–41: Fuming nitric acid (16 mL) was
slowly added dropwise to the stirred acetic anhydride (30 mL) for
30 min at 10–12 ꢀC and kept stirring for 1 h. The formed crude
product of acetyl nitrate 1 would be added dropwise to a solution
of 2,4,6-trichloroaniline (50 g) in dry ethanoic acid (300 mL) and
acetic anhydride (10 mL) for 1 h at 14 ꢀC. The reaction mixture was
stirred for another 1 h and then poured into ice water (2 L).The
Nowadays, urea derivatives have occupied a pivotal position in
pesticide chemistry because of their significant activities (5–11),
81

Cao et al.
precipitate 2 was filtered and washed with water (1 L), which was
recrystallized from ethanol and dried in vacuo. Precipitate 2
(10 mmol) with 2 mL triethylamine was added dropwise to the BTC
(3.3 mmol) toluene solution. The mixture was stirred at )5 ꢀC for
1 h and then heated to 50 ꢀC and kept reacting for 2 h. The inter-
mediate 3 was formed and without isolated. After quenching the
unreacted phosgene with dry nitrogen, a series of substituted ani-
lines (The structure of various anilines are shown in Table 1) with
2 mL triethylamine were added dropwise to the unseparated prod-
uct 3, churned at 50–80 ꢀC for 1–10 h, and then cooled to room
temperature. The respective ureas 4–41 were precipitated, filtered,
and washed with toluene, excess water, and acetone. Crude prod-
ucts were further purified by recrystallization (DMF ⁄ acetone).
H), 3066 (Ar-H), 1646 (C=O), 1257 (N-NO2). 1H NMR (400 MHz,
DMSO-d6): d 8.98 (s, 1H, NH), 7.76 (s, 2H, ArH), 7.45 (d, J = 8.0 Hz,
2H, ArH), 7.29 (d, J = 7.6 Hz, 2H, ArH), 6.99 (t, J = 7.6, 6.8 Hz, 1H,
ArH). Anal. Calcd for C13H8Cl3N3O3: C, 43.30; H, 2.24; N, 11.65;
Found: C, 43.23; H, 2.12; N, 11.80.
N¢-(4-methylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (5): Ammon-
olysis at 50 ꢀC for 1 h. Yield 78.7%, m.p. 232–233 ꢀC; IR (KBr) ⁄ cm:
1
3280 (N-H), 3079 (Ar-H), 2919 (C-H), 1654 (C=O), 1283 (N-NO2); H
NMR (400 MHz, DMSO-d6): d 8.85 (s, 1H, NH), 7.75 (s, 2H, ArH),
7.32 (d, J = 6.4 Hz, 2H, ArH), 7.07(d, J = 6.8 Hz, 2H, ArH), 2.24 (s,
3H, CH3). Anal. Calcd for C14H10Cl3N3O3: C, 44.89; H, 2.69; N,
11.22; Found: C, 45.01; H, 2.64; N, 11.35.
N-nitro-N¢-phenyl-N-(2,4,6-trichlorophenyl)urea (4): Ammonolysis at
50 ꢀC for 4 h. Yield 63.7%, m.p. 229–230 ꢀC; IR (KBr) ⁄ cm: 3267 (N-
N¢-(3-methylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (6): Ammon-
olysis at 50 ꢀC for 2 h. Yield 83.4%, m.p. 227–228 ꢀC; IR (KBr) ⁄ cm:
3280 (N-H), 3076 (Ar-H), 2916 (C-H), 1644 (C=O), 1284 (N-NO2); H
1
NMR (400 MHz, DMSO-d6): d 8.89 (s, 1H, NH), 7.75 (s, 2H, ArH),
7.29 (s, 1H, ArH), 7.22 (d, J = 8.4 Hz, 1H, ArH), 7.15 (t, J = 7.2,
6.0 Hz, 1H, ArH), 6.79 (d, J = 7.6 Hz, 1H, ArH), 2.26 (s, 3H, CH3).
Anal. Calcd for C14H10Cl3N3O3: C, 44.89; H, 2.69; N, 11.22; Found:
C, 44.99; H, 2.63; N, 11.34.
Table 1: Structures and biological activities of the compounds
used in training and test sets
IC50
Compounds
Ar
Yeild (%)
mp (ꢀC)
(lg ⁄ mL)
4a
5
Phenyl
4-CH3Ph
3-CH3Ph
2-OCH3Ph
4-NO2Ph
2-NO2Ph
4-OCH3Ph
2-CH3Ph
4-BrPh
4-FPh
1-Naphthyl
2,6-di-CH3Ph
4-OC2H5Ph
2-OC2H5Ph
3-BrPh
3-Cl-4-FPh
2,4-di-BrPh
2,4-di-FPh
3-F-4-CH3Ph
3-Cl-4-CH3Ph
3,4-di-CH3Ph
3-NO2-4-CH3Ph
3,4-di-OCH3Ph
2-FPh
63.7
78.7
83.4
70.6
48.4
46.1
77.3
86.8
59.3
69.7
54.9
73.7
57.1
68.2
53.6
50.1
50.4
62.6
76.6
72.5
76.8
59.1
50.2
69.4
70.1
82.4
51.7
72.6
65.6
72.5
78.1
75.5
74.2
66.7
58.5
47.8
52.6
54.5
229–230
232–233
227–228
235–237
286–288
273–274
245–246
241–243
244–245
223–225
247–248
252–253
267–269
238–239
258–260
242–245
241–242
238–239
245–246
241–242
242–244
250–251
237–238
233–234
231–232
242–244
260–261
259–261
236–238
246–248
249–251
236–238
245–246
243–244
278–279
239–241
249–250
283–285
448
220
193
567
106
149
219
323
121
170
227
978
239
287
119
133
93
N¢-(2-methoxyphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (7): Am-
monolysis at 50 ꢀC for 4 h. Yield 70.6%, m.p. 235–237 ꢀC; IR
(KBr) ⁄ cm: 3304 (N-H), 3079 (Ar-H), 2835 (C-H), 1660 (C=O), 1220 (N-
NO2). 1H NMR (400 MHz, DMSO-d6): d 8.93 (s, 1H, NH), 8.05 (d,
J = 8.4 Hz, 1H, ArH), 7.74 (s, 2H, ArH), 7.01 (d, J = 7.6 Hz, 1H,
ArH), 6.90–6.98 (m, 1H, ArH), 6.86 (d, J = 8.4 Hz, 1H, ArH), 3.88 (s,
3H, OCH3). Anal. Calcd for : C14H10Cl3N3O4 C, 43.05; H, 2.58; N,
10.76; Found: C, 43.14; H, 2.42; N, 10.88.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21a
22
23
24
25
26
27
28a
29a
30
31a
32
33
34a
35
36a
37
38
39
40a
41
N-nitro-N¢-(4-nitrophenyl)-N-(2,4,6-trichlorophenyl)urea (8): Ammonol-
ysis at 80 ꢀC for 10 h. Yield 48.4%, m.p. 286–288 ꢀC; IR (KBr) ⁄ cm:
3281 (N-H), 3080 (Ar-H), 1656 (C=O), 1373 (N-NO2). 1H NMR
(400 MHz, DMSO-d6): d 10.04 (s, 1H, NH), 8.02 (d, J = 8.4 Hz, 2H,
ArH), 7.69 (d, J = 8.4 Hz, 2H, ArH), 7.28 (s, 2H, ArH). Anal. Calcd
for C13H7Cl3N4O5: C, 38.50; H, 1.74; N, 13.81; Found: C, 38.43; H,
1.87; N, 13.62.
104
107
72
N-nitro-N¢-(2-nitrophenyl)-N-(2,4,6-trichlorophenyl)urea (9): Ammonol-
ysis at 80 ꢀC for 10 h. Yield 46.1%, m.p. 273–274 ꢀC; IR (KBr) ⁄ cm:
3295 (N-H), 3082 (Ar-H), 1658 (C=O), 1338 (N-NO2). 1H NMR
(400 MHz, DMSO-d6): d 9.42 (s, 1H, NH), 8.52 (t, J = 9.6, 2.4 Hz,
1H, ArH), 8.47 (d, J = 8.6 Hz, 1H, ArH), 8.35 (d, J = 3.2 Hz, 1H,
ArH), 8.18 (t, J = 8.8, 3.6 Hz, 1H, ArH), 7.77 (s, 2H, ArH). Anal.
Calcd for C13H7Cl3N4O5: C, 38.50; H, 1.74; N, 13.81; Found: C,
38.44; H, 1.89; N, 13.65.
84
132
105
44
98
59
88
100
57
3-FPh
3,5-di-CH3Ph
3,5-di-ClPh
3-CH3-5-ClPh
2,4-di-ClPh
2-CH3-3-ClPh
2,4-di-CH3Ph
2,5-di-CH3Ph
2,3-di-CH3Ph
2,6-di-FPh
4-ClPh
66
N¢-(4-methoxyphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (10): Am-
monolysis at 50 ꢀC for 4 h. Yield 77.3%, m.p. 245–246 ꢀC; IR
(KBr) ⁄ cm: 3281 (N-H), 3078 (Ar-H), 2834 (C-H), 1645 (C=O), 1250 (N-
NO2). 1H NMR (400 MHz, DMSO-d6): d 8.75 (s, 1H, NH), 7.72 (s,
2H, ArH), 7.33 (d, J = 8.8 Hz, 2H, ArH), 6.84 (d, J = 8.8 Hz, 2H,
ArH), 3.69 (s, 3H, OCH3). Anal. Calcd for C14H10Cl3N3O4: C, 43.05; H,
2.58; N, 10.76; Found: C, 43.16; H, 2.44; N, 10.90.
189
125
75
29
30
69
55
32
3-ClPh
2-ClPh
2-NO2-4-ClPh
aTest set compounds mp (melting point, ꢀC).
82
Chem Biol Drug Des 2012; 80: 81–89

N-Nitrourea Derivatives as Novel Potential Fungicides
N¢-(2-methylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (11): Am-
monolysis at 50 ꢀC for 2 h. Yield 86.8%, m.p. 241–243 ꢀC; IR
(KBr) ⁄ cm: 3267 (N-H), 3067 (Ar-H), 2972 (C-H), 1647 (C=O), 1286 (N-
NO2); 1H NMR (400 MHz, DMSO-d6): d 8.87 (s, 1H, NH), 7.75 (s,
2H, ArH), 7.21 (d, J = 6.4 Hz, 1H, ArH), 7.13–7.20 (m, 1H, ArH),
6.96–7.08 (m, 1H, ArH), 6.81 (d, J = 8.0 Hz, 1H, ArH), 2.23 (s, 3H,
CH3). Anal. Calcd for C14H10Cl3N3O3: C, 44.89; H, 2.69; N, 11.22;
Found: C, 45.01; H, 2.57; N, 11.32.
7.2 Hz, 3H, CH3). Anal. Calcd for C15H12Cl3N3O4: C, 44.52; H, 2.99;
N, 10.38; Found: C, 44.64; H, 3.11; N, 10.49.
N¢-(3-bromophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (18): Am-
monolysis at 60 ꢀC for 5 h. Yield 53.6%, m.p. 258–260 ꢀC; IR (KBr)
1
cm-1: 3277 (N-H), 3075 (Ar-H), 1641 (C=O), 1274 (N-NO2); H NMR
(400 MHz, DMSO-d6): d 9.19 (s, 1H, NH), 8.35 (s, 1H, ArH), 7.78 (s,
2H, ArH), 7.34 (d, J = 7.6 Hz, 1H, ArH), 7.23 (t, J = 8.0 Hz, 1H,
ArH), 7.15 (d, J = 7.2 Hz, 1H, ArH). Anal. Calcd for C13H7BrCl3N3O3:
C, 35.53; H, 1.61; N, 9.56; Found: C, 35.62; H, 1.70; N, 9.81.
N¢-(4-bromophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (12): Am-
monolysis at 60 ꢀC for 5 h. Yield 59.3%, m.p. 244–245 ꢀC; IR
(KBr) ⁄ cm: 3275 (N-H), 3078 (Ar-H), 1653 (C=O), 1278 (N-NO2); 1H
NMR(400 MHz, DMSO-d6): d 9.13 (s, 1H, NH), 7.86 (d, J = 7.6 Hz,
2H, ArH), 7.75 (d, J = 6.4 Hz,2H, ArH), 7.43 (s, 2H, ArH). Anal. Calcd
for C13H7BrCl3N3O3: C, 35.53; H, 1.61; N, 9.56; Found: C, 35.64; H,
1.73; N, 9.82.
N¢-(3-chloro-4-fluorophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea(19):
Ammonolysis at 70 ꢀC for 7 h. Yield 50.1%, m.p. 242–245 ꢀC; IR
(KBr) ⁄ cm: 3309 (N-H), 3084 (Ar-H), 1650 (C=O), 1263 (N-NO2); 1H
NMR (400 MHz, DMSO-d6): d 9.20 (s, 1H, NH), 8.38 (s, 1H, ArH),
7.77 (s, 2H, ArH), 7.33 (d, J = 7.2 Hz, 2H, ArH). Anal. Calcd for
C13H6Cl4FN3O3: C, 37.80; H, 1.46; N, 10.17; Found: C, 37.89; H,
1.63; N, 10.31.
N¢-(4-fluorophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (13): Am-
monolysis at 60 ꢀC for 4 h. Yield 69.7%, m.p. 223–225 ꢀC; IR
(KBr) ⁄ cm: 3314 (N-H), 3081 (Ar-H), 1669 (C=O), 1270 (N-NO2). 1H
NMR (400 MHz, DMSO-d6): d 8.30 (s, 1H, NH), 7.73 (s, 2H, ArH),
7.30–7.47 (m, 2H, ArH), 7.08 (t, J = 8.8 Hz, 2H, ArH). Anal. Calcd
for C13H7Cl3FN3O3: C, 41.24; H, 1.86; N, 11.10; Found: C, 41.33; H,
1.94; N, 11.25.
N¢-(2,4-dibromophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(20):
Ammonolysis at 80 ꢀC for 6 h. Yield 50.4%, m.p. 241–242 ꢀC; IR
(KBr) ⁄ cm: 3288 (N-H), 3081 (Ar-H), 1650 (C=O), 1284 (N-NO2); 1H
NMR (400 MHz, DMSO-d6): d 9.19 (s, 1H, NH), 8.01 (s, 1H, ArH),
7.90 (d, J = 6.0 Hz, 1H, ArH), 7.87 (s, 2H, ArH), 7.54 (d, J = 8.8 Hz,
1H, ArH). Anal. Calcd for C13H6Br2Cl3N3O3: C, 30.12; H, 1.17; N,
8.11; Found: C, 30.23; H, 1.09; N, 8.30.
N¢-1-naphthyl-N-nitro-N-(2,4,6-trichlorophenyl)urea (14): Ammonolysis
at 70 ꢀC for 5 h. Yield 54.9%, m.p. 247–248 ꢀC; IR (KBr) ⁄ cm: 3269
(N-H), 3049 (Ar-H), 1646 (C=O), 1267 (N-NO2); 1H NMR (400 MHz,
DMSO-d6): d 9.04 (s, 1H, NH), 8.15 (d, J = 8.0 Hz, 1H, ArH), 7.93 (t,
J = 6.4, 7.6 Hz, 2H, ArH), 7.67 (s, 2H, ArH), 7.50–7.66 (m, 3H, ArH),
7.47 (t, J = 7.6, 8.0 Hz, 1H, ArH). Anal. Calcd for C17H10Cl3N3O3: C,
49.72; H, 2.45; N, 10.23; Found: C, 49.66; H, 2.38; N, 10.33.
N¢-(3,4-difluorophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (21): Am-
monolysis at 50 ꢀC for 4 h. Yield 62.6%, m.p. 238–239 ꢀC; IR
(KBr) ⁄ cm: 3271 (N-H), 3084 (Ar-H), 1650 (C=O), 1250 (N-NO2); 1H
NMR (400 MHz, DMSO-d6): d 9.20 (s, 1H, NH), 8.34 (s, 2H, ArH),
7.74 (s, 1H, ArH), 7.60 (d, J = 6.8 Hz,1H, ArH), 7.27 (d,
J = 6.4 Hz,1H, ArH). Anal. Calcd for C13H6Cl3F2N3O3: C, 39.37; H,
1.53; N, 10.60; Found: C, 39.53; H, 1.62; N, 10.81.
N¢-(2,6-dimethylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(15):
Ammonolysis at 50 ꢀC for 1 h. Yield 73.7%, m.p. 252–253 ꢀC; IR
(KBr) ⁄ cm: 3274 (N-H), 3070 (Ar-H), 2974 (C-H), 2921 (C-H), 1646
(C=O), 1264 (N-NO2); H NMR (400 MHz, DMSO-d6): d 8.33 (s, 1H,
NH), 7.72 (s, 2H, ArH), 7.06 (d, J = 4.0 Hz, 2H, ArH), 6.85 (t,
J = 6.8, 7.2 Hz, 1H, ArH), 2.23 (s, 6H, -CH3). Anal. Calcd for
C15H12Cl3N3O3: C, 46.36; H, 3.11; N, 10.81; Found: C, 46.44; H, 3.22;
N, 10.95.
N¢-(3-fluoro-4-methylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(22): Ammonolysis at 50 ꢀC for 2 h. Yield 76.6%, m.p. 245–246 ꢀC;
IR (KBr) ⁄ cm: 3280 (N-H), 3076 (Ar-H), 2921 (C-H), 1655 (C=O), 1274
1
1
(N-NO2); H NMR (400 MHz, DMSO-d6): d 9.10 (s, 1H, NH), 8.28 (s,
2H, ArH), 7.76 (s, 1H, ArH), 7.73 (d, J = 6.0 Hz,1H, ArH), 7.04 (d,
J = 6.4 Hz, 1H, ArH), 2.16 (s, 3H, CH3). Anal. Calcd for
C14H9Cl3FN3O3: C, 42.83; H, 2.31; N, 10.70; Found: C, 42.93; H,
2.18; N, 10.84.
N¢-(4-ethoxyphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (16): Am-
monolysis at 60 ꢀC for 2 h. Yield 57.1%, m.p. 267–269 ꢀC; IR
(KBr) ⁄ cm: 3273 (N-H), 3076 (Ar-H), 2977 (C-H), 1642 (C=O), 1246 (N-
NO2); 1H NMR (400 MHz, DMSO-d6): d 8.75 (s, 1H, NH), 7.74 (s,
2H, ArH), 7.33 (d, J = 9.2 Hz, 2H, ArH), 6.84 (d, J = 8.8 Hz, 2H,
ArH), 3.96 (m, 2H, -CH2), 1.30 (t, J = 7.2, 6.8 Hz, 3H, -CH3). Anal.
Calcd for C15H12Cl3N3O4: C, 44.52; H, 2.99; N, 10.38; Found: C,
44.61; H, 3.10; N, 10.48.
N¢-(3-chloro-4-methylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(23): Ammonolysis at 70 ꢀC for 3 h. Yield 72.5%, m.p. 241–242 ꢀC;
IR (KBr) ⁄ cm: 3279 (N-H), 3080 (Ar-H), 2914 (C-H), 1652 (C=O), 1283
1
(N-NO2); H NMR (400 MHz, DMSO-d6): d 9.32 (s, 1H, NH), 8.55 (s,
2H, ArH), 8.00 (s, 1H, ArH), 7.91 (d, J = 6.8 Hz,1H, ArH), 7.46 (d,
J = 6.0 Hz, 1H, ArH), 2.49 (s, 3H, CH3). Anal. Calcd for
C14H9Cl4N3O3: C, 41.11; H, 2.22; N, 10.27; Found: C, 41.24; H, 2.12;
N, 10.42.
N¢-(2-ethoxyphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (17): Am-
monolysis at 60 ꢀC for 2 h. Yield 68.2%, m.p. 238–239 ꢀC; IR
(KBr) ⁄ cm: 3291 (N-H), 3072 (Ar-H), 2979 (C-H), 1645 (C=O), 1263 (N-
NO2); 1H NMR (400 MHz, DMSO-d6): d 9.08 (s, 1H, NH), 8.06 (d,
J = 7.6 Hz, 1H, ArH), 7.76 (s, 2H, ArH), 7.01 (d, J = 8.0 Hz, 1H,
ArH), 6.92 (q, J = 1.2, 6.4, 8.0 Hz, 1H, ArH), 6.85 (t, J = 8.0, 7.2 Hz,
1H, ArH), 4.14 (q, J = 6.8, 7.2, 6.8 Hz, 2H, CH2), 1.42 (t, J = 6.8,
N¢-(3,4-dimethylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(24):
Ammonolysis at 50 ꢀC for 2 h. Yield 76.8%, m.p. 242–244 ꢀC; IR
(KBr) ⁄ cm: 3280 (N-H), 3074 (Ar-H), 2921 (C-H), 1654 (C=O), 1267 (N-
NO2); 1H NMR (400 MHz, DMSO-d6): d 8.75 (s, 1H, NH), 7.74 (s,
2H, ArH), 7.22 (s, 1H, ArH), 7.14 (d, J = 5.6 Hz,1H, ArH), 7.00 (d,
Chem Biol Drug Des 2012; 80: 81–89
83

Cao et al.
J = 4.8 Hz, 1H, ArH), 2.17 (s, 3H, CH3), 2.15 (s, 3H, CH3). Anal.
Calcd for C15H12Cl3N3O3: C, 46.36; H, 3.11; N, 10.81; Found: C,
46.27; H, 3.19; N, 10.98.
6H, CH3). Anal. Calcd for C15H12Cl3N3O3: C, 46.36; H, 3.11; N,
10.81; Found: C, 46.27; H, 3.19; N, 10.98.
N¢-(3,5-dichlorophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(30):
N¢-(4-methyl-3-nitrophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (25):
Ammonolysis at 80 ꢀC for 5 h. Yield 59.1%, m.p. 250–251 ꢀC; IR
(KBr) ⁄ cm: 3313 (N-H), 3076 (Ar-H), 2989 (C-H), 1654 (C=O), 1310 (N-
NO2); 1H NMR (400 MHz, DMSO-d6): d 9.40 (s, 1H, NH), 8.44 (s,
2H, ArH), 7.77 (s, 1H, ArH), 7.56 (d, J = 7.2 Hz,1H, ArH), 7.41 (d,
J = 6.8 Hz, 1H, ArH), 2.45 (s, 3H, CH3). Anal. Calcd for
C14H9Cl3N4O5: C, 40.07; H, 2.16; N, 13.35; Found: C, 40.16; H,2.10;
N, 13.46.
Ammonolysis at 80 ꢀC for 5 h. Yield 51.7%, m.p. 260–261 ꢀC; IR
(KBr) ⁄ cm: 3275 (N-H), 3079 (Ar-H), 1649 (C=O), 1268 (N-NO2); 1H
NMR (400 MHz, DMSO-d6): d 9.39 (s, 1H, NH), 8.53 (s, 2H, ArH),
7.78 (s, 1H, ArH), 7.56 (s, 2H, ArH). Anal. Calcd for C13H6Cl5N3O3: C,
36.36; H, 1.41; N, 9.78; Found: C, 36.44; H, 1.52; N, 9.95.
N¢-(3-chloro-5-methylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(31): Ammonolysis at 70 ꢀC for 3 h. Yield 72.6%, m.p. 259–261 ꢀC;
IR (KBr) ⁄ cm: 3274 (N-H), 3079 (Ar-H), 1644 (C=O), 1267 (N-NO2); H
1
N¢-(3,4-dimethoxyphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (26):
Ammonolysis at 80 ꢀC for 5 h. Yield 50.2%, m.p. 237–238 ꢀC; IR
(KBr) ⁄ cm: 3283 (N-H), 3077 (Ar-H), 2993 (C-H), 1644 (C=O), 1238 (N-
NO2); 1H NMR (400 MHz, DMSO-d6): d 9.24 (s, 1H, NH), 8.19 (s,
2H, ArH), 7.48 (s, 1H, ArH), 7.25 (d, J = 7.6 Hz,1H, ArH), 7.06 (d,
J = 7.2 Hz, 1H, ArH), 4.05 (s, 3H, CH3), 4.03 (s, 3H, CH3). Anal.
Calcd for C15H12Cl3N3O5: C, 42.83; H, 2.88; N, 9.99; Found: C, 42.94;
H,2.98; N, 10.10.
NMR (400 MHz, DMSO-d6): d 8.59 (s, 1H, NH), 7.75 (s, 2H, ArH),
7.64 (s, 1H, ArH), 7.18 (s, 2H, ArH), 2.30 (s, 3H, CH3). Anal. Calcd
for C14H9Cl4N3O3: C, 41.11; H, 2.22; N, 10.27; Found: C, 41.20; H,
2.14; N, 10.42.
N¢-(2,4-dichlorophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(32):
Ammonolysis at 80 ꢀC for 8 h. Yield 65.6%, m.p. 236–238 ꢀC; IR
(KBr) ⁄ cm: 3283 (N-H), 3080 (Ar-H), 1658 (C=O), 1279 (N-NO2); 1H
NMR (400 MHz, DMSO-d6): d 8.55 (s, 1H, NH), 7.74 (s, 2H, ArH),
7.56 (s, 2H, ArH), 7.05 (d, J = 6.4 Hz, 1H, ArH), 6.78 (d, J = 8.0 Hz,
1H, ArH); GC-MS (DMSO) m ⁄ z(%): 427(1), 282(50). Anal. Calcd for
C13H6Cl5N3O3: C, 36.36; H, 1.41; N, 9.78; Found: C, 36.42; H, 1.52;
N, 9.96.
N¢-(2-fluorophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (27): Am-
monolysis at 50 ꢀC for 3 h. Yield 69.4%, m.p. 233–234 ꢀC; IR
(KBr) ⁄ cm: 3270 (N-H), 3076 (Ar-H), 1652 (C=O), 1273 (N-NO2); 1H
NMR (400 MHz, DMSO-d6): d 8.85 (s, 1H, NH), 8.63 (s, 2H, ArH),
8.05 (d, J = 7.2 Hz, 1H, ArH), 7.14–7.29 (m, 1H, ArH), 7.0–7.14 (m,
1H, ArH), 6.98 (d, J = 6.0 Hz, 1H, ArH). Anal. Calcd for
C13H7Cl3FN3O3: C, 41.24; H, 1.86; N, 11.10; Found: C, 41.31; H,
1.80; N, 11.22.
N¢-(3-chloro-2-methylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(33): Ammonolysis at 60 ꢀC for 4 h. Yield 72.5%, m.p. 246–248 ꢀC;
1
IR (KBr) ⁄ cm: 3279 (N-H), 3078 (Ar-H), 1650 (C=O), 1271 (N-NO2); H
NMR (400 MHz, DMSO-d6): d 8.59 (s, 1H, NH), 7.75 (s, 2H, ArH),
7.56–7.69 (m, 1H, ArH), 7.10–7.19 (m, 2H, ArH), 2.30 (s, 3H, CH3);
GC-MS (DMSO) m ⁄ z(%): 407 (1), 281(57), 91(72). Anal. Calcd for
C14H9Cl4N3O3: C, 41.11; H, 2.22; N, 10.27; Found: C, 41.21; H, 2.33;
N, 10.43.
N¢-(3-fluorophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (28): Am-
monolysis at 50 ꢀC for 3 h. Yield 70.1%, m.p. 231–232 ꢀC; IR
(KBr) ⁄ cm: 3277 (N-H), 3083 (Ar-H), 1655 (C=O), 1277 (N-NO2); 1H
NMR (400 MHz, DMSO-d6): d 9.24 (s, 1H, NH), 8.33 (s, 2H, ArH),
7.76 (s, 1H, ArH), 7.43 (d, J = 6.0 Hz, 1H, ArH), 7.28 (t, J = 7.6,
6.4 Hz, 1H, ArH), 7.14 (d, J = 7.2 Hz, 1H, ArH). Anal. Calcd for
C13H7Cl3FN3O3: C, 41.24; H, 1.86; N, 11.10; Found: C, 41.32; H,
1.82; N, 11.24.
N¢-(2,4-dimethylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(34):
Ammonolysis at 50 ꢀC for 4 h. Yield 78.1%, m.p. 249–251 ꢀC; IR
(KBr) ⁄ cm: 3280 (N-H), 3078 (Ar-H), 2919 (C-H), 1651 (C=O), 1269 (N-
NO2); 1H NMR (400 MHz, DMSO-d6): d 8.46 (s, 1H, NH), 7.73 (s,
2H, ArH), 7.53 (d, J = 6.8 Hz, 1H, ArH), 6.99 (s, 1H, ArH), 6.93 (d,
J = 6.0 Hz, 1H, ArH), 2.22 (s, 3H, CH3), 2.21 (s, 3H, CH3); GC-MS
(DMSO) m ⁄ z(%): 387(2), 359(4), 282(63), 91(28). Anal. Calcd for
C15H12Cl3N3O3: C, 46.36; H, 3.11; N, 10.81; Found: C, 46.26; H, 3.22;
N, 10.97.
N¢-(3,5-dimethylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(29):
Ammonolysis at 50 ꢀC for 1 h. Yield 82.4%, m.p. 242–244 ꢀC; IR
(KBr) ⁄ cm: 3276 (N-H), 3077 (Ar-H), 3014 (Ar-H), 2917 (C-H), 1648
1
(C=O), 1278 (N-NO2); H NMR (400 MHz, DMSO-d6): d 8.75 (s, 1H,
NH), 8.14 (s, 2H, ArH), 7.74 (s, 2H, ArH), 7.02 (s, 1H, ArH), 2.17 (s,
A
B
Figure 1: Structure of N-nitro urea derivatives: (A) General structure for title compounds; (B) 3D view of all the aligned molecules in train-
ing and test sets.
84
Chem Biol Drug Des 2012; 80: 81–89

N-Nitrourea Derivatives as Novel Potential Fungicides
N¢-(2,5-dimethylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(35):
C15H12Cl3N3O3: C, 46.36; H, 3.11; N, 10.81; Found: C, 46.25; H, 3.22;
N, 10.97.
Ammonolysis at 50 ꢀC for 4 h. Yield 75.5%, m.p. 236–238 ꢀC; IR
(KBr) ⁄ cm: 3284 (N-H), 3077 (Ar-H), 2921 (C-H), 1648 (C=O), 1283 (N-
NO2); 1H NMR (400 MHz, DMSO-d6): d 8.55 (s, 1H, NH), 7.74 (s,
2H, ArH), 7.57 (s, 1H, ArH), 7.05 (d, J = 6.4 Hz, 1H, ArH), 6.78 (d,
J = 6.0 Hz, 1H, ArH), 2.22 (s, 3H, CH3), 2.20 (s, 3H, CH3); GC-MS
(DMSO) m ⁄ z(%): 387(2), 281(57), 91(42). Anal. Calcd for
C15H12Cl3N3O3: C, 46.36; H, 3.11; N, 10.81; Found: C, 46.24; H, 3.21;
N, 10.99.
N¢-(2,6-difluorophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (37): Am-
monolysis at 60 ꢀC for 5 h. Yield 66.7%, m.p. 243–244 ꢀC; IR
(KBr) ⁄ cm: 3282 (N-H), 3071 (Ar-H), 1661 (C=O), 1243 (N-NO2); 1H
NMR (400 MHz, DMSO-d6): d 8.50 (s, 1H, NH), 7.74 (s, 2H, ArH),
7.22–7.35 (m, 1H, ArH), 7.04–7.20 (m, 2H, ArH); GC-MS (DMSO)
m ⁄ z(%): 395(1), 282(38), 45(100). Anal. Calcd for C13H6Cl3F2N3O3: C,
39.37; H, 1.53; N, 10.60; Found: C, 39.28; H, 1.59; N, 10.81.
N¢-(2,3-dimethylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea
(36):
Ammonolysis at 50 ꢀC for 4 h. Yield 74.2%, m.p. 245–246 ꢀC; IR
(KBr) ⁄ cm: 3288 (N-H), 3067 (Ar-H), 2956 (C-H), 1648 (C=O), 1273 (N-
NO2); 1H NMR (400 MHz, DMSO-d6): d 8.44 (s, 1H, NH), 7.73 (s,
2H, ArH), 7.43 (d, J = 7.2 Hz, 1H, ArH), 7.02 (t, J = 6.4, 6.8 Hz, 1H,
ArH), 6.92 (d, J = 6.4 Hz, 1H, ArH), 2.25 (s, 3H, CH3), 2.15 (s, 3H,
CH3); GC-MS (DMSO) m ⁄ z(%): 387(2), 282(85), 9(25). Anal. Calcd for
N¢-(4-chlorophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (38): Am-
monolysis at 80 ꢀC for 5 h. Yield 58.5%, m.p. 278–279 ꢀC; IR
(KBr) ⁄ cm: 3293 (N-H), 3075 (Ar-H), 1655 (C=O), 1279 (N-NO2). 1H
NMR (400 MHz, DMSO-d6): d 8.86 (s, 1H, NH), 7.77 (s, 2H, ArH),
7.47 (d, J = 8.0 Hz, 2H, ArH), 7.32 (d, J = 8.0 Hz, 2H, ArH). Anal.
Calcd for C13H7Cl4N3O3: C, 39.53; H, 1.79; N, 10.64; Found: C,
39.43; H, 1.88; N, 10.82.
N¢-(3-chlorophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (39): Am-
monolysis at 80 ꢀC for 5 h. Yield 47.6%, m.p. 239–241 ꢀC; IR
(KBr) ⁄ cm: 3273 (N-H), 3078 (Ar-H), 1641 (C=O), 1274 (N-NO2); 1H
NMR (400 MHz, DMSO-d6): d 9.31 (s, 1H, NH), 8.42 (s, 1H, ArH),
7.76 (s, 2H, ArH), 7.67 (d, J = 5.6 Hz, 1H, ArH), 7.27 (d, J = 7.2 Hz,
1H, ArH), 7.00–7.08 (m, 1H, ArH). Anal. Calcd for C13H7Cl4N3O3: C,
39.53; H, 1.79; N, 10.64; Found: C, 39.42; H, 1.89; N, 10.81.
Table 2: CoMFA and CoMSIA results
LOO
NV
Fields
q2
N
r2
SEE
F
CoMFA
Both steric and
electrostatic
Steric
0.773
4
0.959 0.077 146.833
0.729
0.480
3
2
0.902 0.117
0.762 0.179
79.975
43.160
N¢-(2-methylphenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (40): Am-
monolysis at 80 ꢀC for 5 h. Yield 52.6%, m.p. 249–250 ꢀC; IR
(KBr) ⁄ cm: 3273 (N-H), 3077 (Ar-H), 1653 (C=O), 1290 (N-NO2). 1H
NMR (400 MHz, DMSO-d6): d 8.86 (s, 1H, NH), 7.77 (t, J = 8.8,
9.6 Hz, 1H, ArH), 7.65 (s, 2H, ArH), 7.37 (d, J = 8.4 Hz, 1H, ArH),
7.22 (d, J = 8.8 Hz, 1H, ArH), 7.13 (d, J = 4.8, 5.2 Hz, 1H, ArH).
Anal. Calcd for C14H10Cl3N3O3: C, 44.89; H, 2.69; N, 11.22; Found:
C, 45.01; H, 2.80; N, 11.37.
Electrostatic
CoMSIA
Steric
Electrostatic
Hydrophobic
H-bond donor
H-bond acceptor
Steric, electrostatic
0.615
0.522
0.260
0.492
0.350
0.720
6
6
4
2
5
6
5
0.869 0.143
0.889 0.132
0.669 0.219
0.629 0.223
0.659 0.226
25.488
30.758
12.649
22.907
9.293
0.936 0.100 56.051
0.694 0.215 10.803
H-bond donor, H-bond acceptor 0.499
N¢-(4-chloro-2-nitrophenyl)-N-nitro-N-(2,4,6-trichlorophenyl)urea (41):
Ammonolysis at 80 ꢀC for 10 h. Yield 54.5%, m.p. 283–285 ꢀC; IR
(KBr) ⁄ cm: 3314 (N-H), 3081 (Ar-H), 1669 (C=O), 1270 (N-NO2). 1H
NMR (400 MHz, DMSO-d6): d 9.65 (s, 1H, NH), 8.75 (s, 1H, ArH),
8.30 (d, J = 8.8 Hz, 1H, ArH), 8.13 (d, J = 2.4 Hz, 1H, ArH), 7.77 (s,
Note: N is the optimal number of components, NV is No-Validation, q2 is
the leave-one-out (LOO) cross-validation coefficient, r2 is the non-cross-vali-
dation coefficient, SEE is the standard error of estimation, and F is the F-
test value. The best models are marked in bold.
Scheme 1: Synthetic route for
target compounds 4–41 and the
structure of ArNH2 are shown in
Table 1.
Chem Biol Drug Des 2012; 80: 81–89
85

Cao et al.
2H, ArH). Anal. Calcd for C13H6Cl4N4O5: C, 35.48; H, 1.37; N, 12.73;
Found: C, 35.37; H, 1.54; N, 12.89.
4, 6-trichlorophenyl)amine 2 in yields of 75–86%. Then, at the pres-
ence of triethylamine as base (27), a subsequent reaction of BTC
and intermediate 2 produced nitro (2,4,6-trichlorophenyl) carbamic
chloride 3. Without further isolation, the key intermediate 3 reacted
with the corresponding amine to afford the compounds 4–41 in
the yields of 45–87%.
Antifungal activities
The in vivo fungicidal activities of N-nitrourea derivatives were
tested against Rhizoctonia solani by the modified agar cup method
according to the reported method in literature (18).
In vivo antifungal activities
Thirty-eight N-nitrourea derivatives 4–41 were synchronously
tested the in vivo fungicidal activities against Rhizoctonia solani by
agar cup method (18). As shown in Table 1, Compounds 20, 23,
Molecular modeling
The 38 target compounds were divided into a training set and a
testing set as shown in Table 1. The IC50 values were converted
into pIC50 (-log IC50) for use in 3D-QSAR analysis. CoMFA and CoM-
SIA study was performed using SYBYL 7.3 molecular modeling soft-
ware (19). The 3D structures of all compounds were sketched by
the Build ⁄ Edit module of SYBYL7.3. Partial atomic charges were
calculated by the Gasteiger–Huckel method, and energy minimiza-
tions were performed by using the Tripos force and the Powell con-
Table 3: Observed and predicted activities for training and test
sets' compounds by 3D-QSAR models
CoMFA model
CoMSIA model
Compounds Observed pIC50 Predicted Residual Predicted Residual
Training set
jugate gradient algorithm with
a convergence criterion of
0.05kcal ⁄ (mol ꢀ) (20,21). The potent compound 41 was chosen as
the template. The common substructure is displayed in Figure 1.
Each compound in the training and testing sets was aligned to the
template using the database alignment function because of its easy
implementation and effectiveness. The aligned compounds are
shown in Figure 1.
5
3.66
3.71
3.24
3.98
3.83
3.66
3.49
3.92
3.76
3.64
3.01
3.62
3.55
3.92
3.88
4.03
3.97
4.14
4.08
3.88
3.98
4.36
4.05
4.24
4.18
3.90
4.54
4.53
4.16
4.49
3.66
3.74
3.28
4.03
3.73
3.57
3.55
3.96
3.77
3.69
2.94
3.62
3.53
3.95
3.86
4.00
3.97
4.07
4.12
4.00
4.02
4.38
4.11
4.26
4.10
3.98
4.51
4.27
4.16
4.52
0.00
0.03
0.04
0.05
)0.10
)0.09
0.06
0.04
0.01
0.05
)0.07
0.00
)0.02
0.03
)0.02
)0.03
0.00
)0.07
0.04
0.12
0.04
0.02
3.61
3.65
3.23
3.91
3.86
3.55
3.56
3.89
3.89
3.59
2.98
3.69
3.60
3.98
3.99
3.99
4.05
4.06
4.17
3.91
4.02
4.38
4.13
4.27
3.98
4.02
4.48
4.28
4.23
4.44
)0.05
)0.06
)0.01
)0.07
0.03
)0.11
0.07
)0.03
0.13
)0.05
)0.03
0.07
0.05
0.06
0.11
)0.04
0.08
)0.08
0.09
0.03
0.04
0.02
0.08
0.03
6
7
8
9
10
11
12
A grid that extends 4 ꢀ units beyond the dimensions of aligned
molecules was established (22). The CoMFA steric and electrostatic
fields were calculated at grid points using Tripos Standard with a
default energy cutoff of 30 kcal ⁄ mol. For CoMSIA, steric, electro-
static, hydrophobic, hydrogen-bond donor, and acceptor fields were
evaluated using probe atom with +1 charge, radius of 1 ꢀ, and +1
hydrophobicity on the same lattice as the CoMFA used. Each single
and some possible combinations of fields were calculated. The rela-
tionship between activity data and fields of training set was ana-
lyzed by partial least squares (PLS) methods (23,24). The leave-one-
out (LOO) cross-validation was performed to determine optimum
number of components (N) and cross-validated coefficient q2, which
indicates the consistency and prediction of models for the training
set. Then, no validation was performed to derive the final PLS
regression models, and the results were shown in Table 2.
13
14
15
16
17
18
19
20
22
23
24
25
26
27
30
32
0.06
0.02
33
35
)0.08
0.08
)0.03
)0.26
0.00
)0.20
0.12
)0.06
)0.25
0.07
The external predictive ability of the models can be measured
by Rp2red
,
Rp2red was according to the formula: Rp2red
¼
37
38
ðSD ꢀ PRESSÞ=SD, where SD is the sum of the squared deviations
between the biological activities of the test set compounds and
mean activity of the training set compounds and PRESS is the sum
of squared deviations between experimental and predicted activities
of the test set compounds (25).
39
41
0.03
)0.05
Test set
4a
3.35
3.92
4.10
4.23
4.00
3.72
4.12
4.26
3.60
3.79
4.24
4.04
4.03
4.00
4.16
4.17
0.25
)0.13
0.14
)0.19
0.03
0.28
0.04
)0.09
0.14
3.58
4.00
4.35
4.41
4.21
4.01
4.06
4.25
0.23
0.08
0.25
0.18
0.21
0.29
)0.06
)0.01
0.16
21a
28a
29a
31a
34a
36a
40a
Average
Results and Discussion
Synthesis
The synthetic route for target compounds 4–41 is outlined in
Scheme 1. According to the reported procedures (26), 2,4,6-trichlo-
roaniline was nitrified by acetyl nitrate 1 to give the N-nitro-N-(2,
aThe selective compounds as test set compounds.
86
Chem Biol Drug Des 2012; 80: 81–89

N-Nitrourea Derivatives as Novel Potential Fungicides
24, 32, 33, and 39 display good activities, as the IC50 are less
than 100 lg ⁄ mL. Compounds 27, 37, 38, 40, and 41 exhibit good
antifungal activities with lower IC50 below 50 lg ⁄ mL.
values are all under 150 lg ⁄ mL except compound 15 (2,6-di-CH3,
978 lg ⁄ mL) and 34 (2,4-di-CH3, 189 lg ⁄ mL). In addition, the com-
pounds with optimal activity are 41 (2-NO2-4-Cl, 32 lg ⁄ mL) and
37 (2,6-di-F, 29 lg ⁄ mL).
For structure–activity relationship (SAR), the biological data
obtained the various substituents on N¢-benzene ring displayed dif-
ferent activities. For mono-substituted compounds, 27 (2-F) >40 (2-
Cl) > 9 (2-NO2) > 17 (2-OC2H5) > 11 (2-CH3) > 7 (2-OCH3); 39 (3-
Cl) > 28 (3-F) > 18 (3-Br) > 6 (3-CH3); 38 (4-Cl) > 8 (4-NO2) > 13
(4-F) > 10 (4-OCH3) > 5 (4-CH3) > 16 (4-OC2H5). Most of the disub-
stituted compounds displayed good antifungal activities. Their IC50
Structure–activity relationships
The statistical results of the 3D-QSAR models are summarized in
the Table 2, in which the best models are marked in bold. As seen,
both the CoMFA (q2 = 0.773, r2 = 0.959) and CoMSIA (q2 = 0.720,
r2 = 0.936) models show good prediction capability.
A
B
Figure 2: Plot of the predicted versus observed pIC50 values for all the molecules based on CoMFA (q2 = 0.773, r2 = 0.959) model (A) and
CoMSIA (q2 = 0.720, r2 = 0.936) model (B).
A
B
Figure 3: CoMFA contour maps: (A) steric contour map; (B) electrostatic contour map.
B
A
Figure 4: CoMSIA contour maps: (A) steric contour map; (B) electrostatic contour map.
Chem Biol Drug Des 2012; 80: 81–89
87

Cao et al.
The predicted and residual pIC50 values for the training and testing
set compounds are listed in Table 3. The relative plots of the pre-
dicted versus experimental pIC50 values for the two models are
shown in Figure 2. The average residual values for the test set of
two models were 0.14 and 0.16. The external testing set yields a
predictive Rp2red of 0.662 and 0.568 for CoMFA and CoMSIA models.
As seen, the values indicate that CoMFA and CoMSIA models pos-
sess a high predictive capacity, and the CoMFA model is considered
more predictability with higher q2, r2, and Rp2red than the CoMSIA
model. So we could predict that the hydrogen bond is not the key
element for the activity of these inhibitors.
pIC50 = 4.49). But near 2, 6-position suggests that substituents on
this region would be small. It can be proven by compound 15(2, 6-
di-CH3, pIC50 = 3.01) with lower activity.
Moreover, in the CoMSIA electrostatic contour map (Figure 4B),
there are two bulky red contours near 2, 3-position before and
behind the phenyl ring, which indicates that electronegative groups
here benefits the activity, the CoMSIA steric and electrostatic con-
tour maps indicate that linkers composed of small and electronega-
tive groups on 2-position, bulky and electronegative substituents on
3-position, bulky groups on 4-region, small groups on 6-position
may increase the activity.
The steric and electrostatic contribution contour maps of the best
models of CoMFA and CoMSIA are plotted in Figures 3 and 4. In
the steric contour maps, the green contours (80% contribution) rep-
resent regions that bulky substituents would increase the inhibitory
activity, while the yellow contours (20% contributions) represent
regions that steric bukly group would be unfavorable. While in the
electrostatic contour maps, the blue and red contours (80% and
20% contributions) signify the position where positively charged
groups and negatively charged groups would be favorable, respec-
tively.
Conclusions
Using a simple and convenient BTC one-pot synthetic method, we
have prepared 38 N-nitrourea derivatives, and compounds 27, 37,
38, 40, and 41 exhibited excellent antifungal activities. Based on
the experimental data, two best CoMFA and CoMSIA models with
the cross-validated (LOO) q2 values of 0.773 and 0.72 and no vali-
dated r2 values of 0.959 and 0.936 were obtained, respectively. The
testing set of compounds that gave a predictive Rp2red of 0.662 and
0.568 for CoMFA and CoMSIA models indicate that the two best
models could be effectively used to predict the activity of new
inhibitors and guide the further modification of these compounds,
just as small and electronegative groups on 2-position (2-F), bulky
and electronegative substituents on 3-position (3-CF3, 3-NO2), bulky
and electropositive groups on 4,5-region, and small and electroneg-
ative groups on 6-position may increase the activity.
As shown in CoMFA steric map (Figure 3A), a bulky group in the
region of the green contour on 4-position of N¢-phenyl is favor-
able for activity. By comparing the structures and activities of
4-substituted compounds 4 (4-H), 5 (4-CH3), 8 (4-NO2), and 12 (4-
Br), the activity order is: 8>12>5>4, which coincides with the
model prediction. The yellow contours near 2-position suggests
that the smaller substituents on this region would be advanta-
geous. It can be proven by the compounds 7, 11, and 17 with
lower activity.
Acknowledgements
In the CoMFA electrostatic contour map (Figure 3B), a red and blue
contour are, respectively, distributed before and behind the N¢-phe-
nyl ring. On the 2-position, there is red contour before the ring and
also blue contour behind it, which indicates that both electroposi-
tive and electronegative group here benefit activity, such as com-
pounds 9 (2-NO2, pIC50 = 3.83), 11 (2-CH3, pIC50 = 3.49), 27 (2-F,
pIC50 = 4.36), and 40 (2-Cl, pCI50 = 4.26) result better inhibitory
activity than compound 4 (2-H, pIC50 = 3.35). There is a bulky blue
contour near 4,5-position, which shows electropositive groups here
would benefit activity. For example, compounds 8 (4-NO2), 13 (4-F),
and 38 (4-Cl) possess significant inhibitory activity. And near 6-posi-
tion, a red contour indicates electronegative groups are needed
here. The CoMFA steric and electrostatic contour maps show that
the linkers composed of small substituents on 2-position, bulky and
electropositive groups on 4,5-region, electronegative groups on 6-
position may increase the activity.
We gratefully acknowledge the support of this work by the Funda-
mental Research Funds for the Central Universities (2010JC001) and
the National Natural Science Foundation of China (31101467).
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