Design, synthesis, and fungicidal activity of novel analogues of pyrrolnitrin

Article abstract of DOI:10.1021/jf902320e

A series of novel analogues of pyrrolnitrin containing a thiophene moiety were designed and synthesized by a facile method, and their structures were characterized by 1H nuclear magnetic resonance (NMR) and high-resolution mass spectrometry. The isomers IV-h and V-h were isolated, and their structures were identified by 2D NMR, including heteronuclear multiple-quantum coherence (HMQC), heteronuclear multiple-bond correlation (HMBC), and nuclear Overhauser effect spectrometry (NOESY) spectra. Their fungicidal activities against five fungi were evaluated, and the results indicated that some of the title compounds showed excellent fungicidal activities in vitro against Alternaria solani, Gibberella zeae, Physalospora piricola, Fusarium omysporum, and Cercospora arachidicola at the dosage of 50 μg mL^-1. Some compounds shown moderate activity at low dosage. Compound V-h could be considered as a leading structure for further design of agricultural fungicides.

Full text of DOI:10.1021/jf902320e

7912 J. Agric. Food Chem. 2009, 57, 7912–7918
DOI:10.1021/jf902320e
Design, Synthesis, and Fungicidal Activity of Novel Analogues
of Pyrrolnitrin
MING-ZHONG
WANG, HAN
X
U
, Q
I
F
ENG, L
I
-ZHONG
W
ANG, S
U
-HUA
W
ANG AND
,
Z
HENG-MING
LI*
State Key Laboratory of Elemento-organic Chemistry, Research Institute of Elemento-organic Chemistry,
Nankai University, Tianjin 300071, China
A series of novel analogues of pyrrolnitrin containing a thiophene moiety were designed and
synthesized by a facile method, and their structures were characterized by 1H nuclear magnetic
resonance (NMR) and high-resolution mass spectrometry. The isomers IV-h and V-h were isolated,
and their structures were identified by 2D NMR, including heteronuclear multiple-quantum coher-
ence (HMQC), heteronuclear multiple-bond correlation (HMBC), and nuclear Overhauser effect
spectrometry (NOESY) spectra. Their fungicidal activities against five fungi were evaluated, and the
results indicated that some of the title compounds showed excellent fungicidal activities in vitro
against Alternaria solani, Gibberella zeae, Physalospora piricola, Fusarium omysporum, and
Cercospora arachidicola at the dosage of 50 μg mL-1. Some compounds shown moderate activity
at low dosage. Compound V-h could be considered as a leading structure for further design of
agricultural fungicides.
KEYWORDS: Pyrrolnitrin; thiophene; fungicidal activities; leading structure; synthesized
INTRODUCTION
know whether that might improve or decrease the fungicide
activity (9). In addition, adding some halogen atoms to the aromatic
ring might enhance the lipophilic ability and fungicidal activity of
those compounds (4). A total of 24 analogues (D, Figure 1) were
synthesized with a facile synthetic method. The target compounds
were evaluated for fungicide activity in vitro against five fungi,
and some compounds showed moderate activity at low dosage.
Pyrrolnitrin (A, Figure 1) is an antibiotic first isolated from the
bacterial cells of Pseudomonas by Arima et al. (1), and the
structure was established by the same author (1). The antibiotic
was totally synthesized by Nakano et al. in 1965 (1). It was also
isolated from a non-obligate predator bacterial strain 679-2,
which has growth-inhibitory properties against bacteria, fungi,
and other predator bacteria and yet with an ability to thrive in the
absence of prey microorganisms (2). Pyrrolnitrin is one of the
most important natural products inhibiting Mycobacterium tu-
berculosis in early days (3), and this structure has been studied
in crop protection. Fenpiclonil (B, Figure 1) and fludioxonil
(C, Figure 1) were discovered as a novel class of agrochemical
fungicides on the basis of the synthetic optimization of the natural
structure (4). Both compounds have broad-spectrum activity
across many classes of fungi and are safe to mammals and the
environment (4).
In the design of new fungicides, when the pyrrole group in
pyrrolnitrin was replaced by furanone, pyrazole, and isoxazoli-
nol, respectively, all of the analogues reported were found to be
devoid of biological activity (5). The thiophene and pyrrole rings
are considered to be bioisosteric analogues, and there are many
fungicides that have the thiophene group, such as bethoxazin (6),
penthiopyrad (7), ethaboxam (8), etc. Therefore, in this paper,
the pyrrole ring was replaced by the thiophene ring. To study the
structure-activity relationship (SAR) of those analogues, the
thiophene and aromatic rings of some analogues were nitrified to
MATERIALS AND METHODS
Instruments. 1H nuclear magnetic resonance (NMR), 13C NMR, hete-
ronuclear multiple-quantum coherence (HMQC), heteronuclear multiple-
bond correlation (HMBC), and nuclear Overhauser effect spectrometry
(NOESY) spectra were obtained at 300 MHz using a Bruker AV300
spectrometer or at 400 MHz using a Varian Mercury Plus400 spectrometer
in CDCl3 solution, with tetramethylsilane as the internal standard.
Chemical-shift values (δ) were given in parts per million (ppm). High-
resolution mass spectrometry (HRMS) data were obtained on a VG ZAB-
HS instrument. The melting points were determined on an X-4 binocular
microscope melting point apparatus (Beijing Tech Instruments Co.,
Beijing, China) and were uncorrected. Yields were not optimized. The
reagents were all analytically or chemically pure. All solvents and liquid
reagents were dried by standard methods in advance and distilled before
use. 1-Bromo-2-nitro-benzene (II-a) and all halogen-benzene were
bought from the Alfa Aesar Company (Tianjin, China). Fenpiclonil (B)
::
was bought from the laboratory of Dr. Ehrenstorfer-Schafers (Augsburg,
Germany).
General Synthetic Procedures for II-b-II-h. A mixture of 65%
nitric acid (1.5 mL) and 1-bromo-4-fluorobenzene (I-b, 3.4 mL, 30 mmol)
in 98% sulfuric acid (12.0 mL) was kept 15 min at 0 °C, then poured onto
crushed ice (20 g), and extracted with dichloromethane (2ꢀ 20 mL), and
the solvent was removed. The residue was purified by flash chromato-
graphy on silica gel eluting with petroleum ether (60-90 °C) to provide
*To whom correspondence should be addressed. Telephone: þ86-
22-23503732. E-mail: nkzml@vip.163.com.
pubs.acs.org/JAFC
Published on Web 08/14/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 17, 2009 7913
II-b as a light yellow crystal (10). Compounds II-c, II-d, II-e, II-f, II-g, and
II-h were prepared through the same process. In compounds II-e and II-f
preparation procedures, the mixture was kept 40 min at 0 °C and then
poured onto crushed ice. In compounds II-g preparation procedures, the
mixture was kept 60 min at 60 °C and then poured onto crushed ice.
Compound II-h was obtained from II-c, and the mixture was kept 30 min
at 60 °C and then poured onto crushed ice. All of the substituents at the
aromatic rings were listed in Scheme 1.
Synthesis of 3-Bromo-thiophene. 3-Bromo-thiophene was synthe-
sized through an improved procedure referenced in the literature (11, 12):
A solution of thiophene (93.0 g, 1.1 mol) in 130 mL of chloroform was
cooled to 0 °C. Bromine (150 mL, 2.9 mol) was added dropwise over 4.5 h
and then refluxed for 3.5 h, after which 200 mL of 2 M sodium hydroxide
was added and stirred for 0.5 h, then extracted with chloroform (150 mL),
washed with water, and dried, and then the solvent was removed to afford
a light reddish oil, which was used in the subsequent step without further
purification. In a 2 L flask equipped with a distillation apparatus, zinc
powder (220 g, 3.3 mol) was added to 500 mL of water; glacial acetic acid
was then added (210 mL, 3.64 mol). The crude product obtained above
was added. The mixture was stirred and refluxed for 8 h. The subsequent
steps of distillation are the same as in the literature. Distillation afforded
3-bromo-thiophene as a colorless liquid. Yield, 80.8%. 1H NMR (300 MHz,
Data for II-b. Yield, 61.1%. mp, 37-39 °C (acetone/petroleum
ether). 1H NMR (300 MHz, CDCl3) δ: 7.19-7.25 (m, 1H, Ar-H), 7.62
(dd, 3JHH=7.8 Hz, 4JHF =3.0 Hz, 1H, Ar-H), 7.74 (dd, 3JHF=9.0 Hz,
4JHH =5.1 Hz, 1H, Ar-H).
Data for II-c. Yield, 59.2%. mp, 39-41 °C (acetone/petroleum
ether). 1H NMR (300 MHz, CDCl3) δ: 7.15-7.19 (m, 1H, Ar-H), 7.39
(dd, 3JHF=8.1 Hz, 4JHH=2.4 Hz, 1H, Ar-H), 7.89 (dd, 4JHF=5.4 Hz,
3JHH =9.0 Hz, 1H, Ar-H).
CDCl3) δ: 6.90 (dd, 4JHH =2.1 Hz, 3JHH =4.2 Hz, 1H), 7.08 (d, 3JHH
=
4.2 Hz, 1H), 7.10 (s, 1H).
General Synthetic Procedures for Target Compounds III-a-III-h.
Compound III-a was synthesized by a facile method according to the
literature (13). Cuprous iodide (7.6 g, 39.8 mmol) and 3-bromo-thiophene
(8.2 g, 50.0 mmol) were mixed together and heated to 160 °C for 1 h. Then,
the bath temperature was raised and maintained at 200 °C. With stirring,
compound II-a (8.0 g, 39.8 mmol) was dissolved in 3-bromo-thiophene
(16.4 g, 100.0 mmol) before it was added dropwise into the above mixture.
The process of adding would need 50 min. During the above adding
process, copper powder (24.0 g, 375.0 mmol) was added in five portions.
After II-a was added up, the mixture reacted for 15 h while keeping the
bath temperature between 200 and 210 °C. Upon cooling, the products
were extracted with dichloromethane (2ꢀ50 mL) and the extraction was
filtered through celite and evaporated to give a brown oil, which was
purified by flash chromatography on silicagel eluting with petroleum ether
(60-90 °C) to provide an orange oil III-a (3.27 g; yield, 40.1%).
Compounds III-b, III-c, III-d, III-e, III-f, III-g, and III-h were prepared
according to the same process. All of the substituents at the aromatic rings
were listed in Scheme 1.
Data for II-d. Yield, 48.7%. mp, 65-67 °C (acetone/petroleum
ether). 1H NMR (300 MHz, CDCl3) δ:7.42(dd,3JHH=8.4Hz,4JHH=2.4Hz,
1H, Ar-H), 7.68 (d, 3JHH =8.7 Hz, 1H, Ar-H), 7.85 (s, 1H, Ar-H).
Data for II-e. Yield, 90.0%. mp, 63-65 °C (acetone/petroleum
ether). 1H NMR (300 MHz, CDCl3) δ: 7.61 (d, 4JHH=2.4 Hz, 1H, Ar-H),
7.66 (d, 4JHH =2.4 Hz, 1H, Ar-H).
Data for II-f. Yield, 49.5%. mp, 24-26 °C (acetone/petroleum
3
ether). 1H NMR (400 MHz, CDCl3) δ: 7.76 (d, JHF = 8.0 Hz, 1H,
Ar-H), 7.83 (d, 4JHF =6.8 Hz, 1H, Ar-H).
Data for II-g. Yield, 67.8%. mp, 100-102 °C (acetone/petroleum
ether). 1H NMR (300 MHz, CDCl3) δ: 7.73 (s, 1H, Ar-H).
Data for II-h. Yield, 55.0%. mp, 95-97 °C (acetone/petroleum
ether). 1H NMR (300 MHz, CDCl3) δ: 7.78 (d, 3JHF=9.6 Hz, 1H, Ar-H),
8.72 (d, 4JHF =6.9 Hz, 1H, Ar-H).
1
Data for III-a. Yield, 40.1%; oil. H NMR (300 MHz, CDCl3) δ:
6.96 (dd, 4JHH =1.2 Hz, 3JHH =5.1 Hz, 1H, thiophene), 7.21 (d, 4JHH
=
4
3
1.2 Hz, 1H, thiophene), 7.25 (dd, JHH = 3 Hz, JHH = 5.1 Hz, 1H,
thiophene), 7.32 (t, 3JHH=7.5 Hz, 1H, Ar-H), 7.36 (t, 3JHH=7.8 Hz, 1H,
3
3
Ar-H), 7.44 (dd, JHH = 7.5 Hz, JHH = 7.5 Hz, 1H, Ar-H), 7.66 (d,
3JHH=7.8 Hz, 1H, Ar-H). HRMS, m/z 205.0156. Calcd for C10H7NO2S,
205.0197. All of the data are the same as the known compound (13).
1
Data for III-b. Yield, 39.8%; oil. H NMR (400 MHz, CDCl3) δ:
3
4
7.06 (d, JHH = 4.8 Hz, 1H, thiophene), 7.31 (d, JHH = 2.4 Hz, 1H,
thiophene), 7.33 (dt, 4JHH=2.4 Hz, 3JHF =8.0 Hz, 1H, Ar-H), 7.40 (dd,
4JHH =3.2 Hz, 3JHH =4.8 Hz, 1H, thiophene), 7.49 (dd, 4JHF =5.6 Hz,
3JHH=8.4 Hz, 1H, Ar-H), 7.55 (dd, 4JHH=2.4 Hz, 3JHF=8.0 Hz, 1H,
Ar-H). HRMS, m/z 223.0103. Calcd for C10H6FNO2S, 223.0109.
Data for III-c. Yield, 42.2%; oil. 1H NMR (400 MHz, CDCl3)
3
4
Figure 1. Chemical structures of compounds A-D.
δ: 7.07 (dd, JHH = 5.2 Hz, JHH = 1.2 Hz, 1H, thiophene), 7.12-7.16
Scheme 1. Synthetic Routes to the Title Compounds III, IV, and V

7914 J. Agric. Food Chem., Vol. 57, No. 17, 2009
Wang et al.
4
3
(m, 1H, Ar-H), 7.19 (dd, JHH = 2.4 Hz, JHF = 8.8 Hz, 1H, Ar-H),
7.36-7.37 (m, 1H, thiophene), 7.41 (dd, 4JHH=2.8 Hz, 3JHH=5.2 Hz, 1H,
thiophene), 7.88 (dd, 4JHF =5.2 Hz, 3JHH=8.8 Hz, 1H, Ar-H). HRMS,
m/z 223.0103. Calcd for C10H6FNO2S, 223.0113.
7.41 (d, 4JHH=2.0 Hz, 1H, thiophene), 7.58 (dd, 3JHH=8.4 Hz, 4JHH=2.0
Hz, 1H, Ar-H), 7.83 (d, 4JHH=2.0 Hz, 1H, thiophene), 7.90 (d, 4JHH=2.0,
1H, Ar-H). HRMS, m/z 283.9651. Calcd for C10H5ClNO2S, 283.9658.
Data for IV-e. Yield, 41.7%. mp, 96-98 °C (acetone/petroleum
1
4
Data for III-d. Yield, 34.8%; oil. H NMR (400 MHz, CDCl3) δ:
ether). 1H NMR (400 MHz, CDCl3) δ: 7.45 (d, JHH = 1.6 Hz, 1H,
7.07 (dd, 4JHH=1.2 Hz, 3JHH=4.8 Hz, 1H, thiophene), 7.33-7.34 (m, 1H,
thiophene), 7.40 (dd, 4JHH =2.8 Hz, 3JHH=4.8 Hz, 1H, thiophene), 7.45
(d, 3JHH =8.0 Hz, 1H, Ar-H), 7.57 (dd, 4JHH =2.0 Hz, 3JHH =8.0 Hz,
1H, Ar-H), 7.81 (d, 4JHH =2.0 Hz, 1H, Ar-H). HRMS, m/z 238.9807.
Calcd for C10H6ClNO2S, 238.9806.
thiophene), 7.50 (s, 1H, Ar-H), 7.82 (d, 4JHH =2.0 Hz, 1H, thiophene),
8.20 (s, 1H, Ar-H). HRMS, m/z 317.9266. Calcd for C10H4Cl2NO2S,
317.9268.
Data for IV-f. Yield, 46.1%. mp, 107-109 °C (acetone/petroleum
4
ether). 1H NMR (400 MHz, CDCl3) δ: 7.01 (d, JHH = 4.0 Hz, 1H,
thiophene), 7.63 (d, 4JHF=7.2 Hz, 1H, Ar-H), 8.20 (d, 3JHF=7.6 Hz, 1H,
Data for III-e. Yield, 45.3%. mp, 70-72 °C (acetone/petroleum
4
ether). 1H NMR (400 MHz, CDCl3) δ: 7.07 (dd, 4JHH =1.2 Hz, 3JHH
=
Ar-H), 7.89 (d, JHH = 4.0 Hz, 1H, thiophene). HRMS, m/z 301.9569.
4
4
Calcd for C10H4ClFNO2S, 301.9564.
4.8 Hz, 1H, thiophene), 7.28 (dd, JHH = 1.2 Hz, JHH = 2.8 Hz, 1H,
thiophene), 7.45 (dd, 4JHH =2.8 Hz, 3JHH=5.1 Hz, 1H, thiophene), 7.69
(d, JHH = 2.0 Hz, 1H, Ar-H), 7.71 (d, JHH = 2.4 Hz, 1H, Ar-H).
HRMS, m/z 272.9418. Calcd for C10H5Cl2NO2S, 272.9414.
Data for IV-g. Yield, 42.1%. mp, 105-107 °C (acetone/petroleum
4
4
4
ether). 1H NMR (400 MHz, CDCl3) δ: 7.54 (d, JHH = 1.6 Hz, 1H,
thiophene), 7.79 (s, 1H, Ar-H), 7.91 (d, 4JHH =2.0 Hz, 1H, thiophene).
HRMS, m/z 351.8868. Calcd for C10H3Cl3NO2S, 351.8879.
1
Data for III-f. Yield, 35.0%; oil. H NMR (400 MHz, CDCl3) δ:
7.06 (dd, 4JHH=1.2 Hz, 3JHH=4.8 Hz, 1H, thiophene), 7.34 (dd, 4JHH
1.2 Hz, 4JHH =2.8 Hz, 1H, thiophene), 7.41 (dd, 4JHH =2.8 Hz, 3JHH
4.8 Hz, 1H, thiophene), 7.57 (d, 4JHF=7.2 Hz, 1H, Ar-H), 7.71 (d, 3JHF
=
=
=
Data for IV-h. Yield, 34.2%. mp, 113-115 °C (acetone/petroleum
ether). 1H NMR (400 MHz, CDCl3) δ: 7.48 (d, 3JHF=10.0 Hz, H-60), 7.64
4
4
4
(d, JHH = 1.6 Hz, H-2), 7.93 (d, JHH = 1.6 Hz, H-4), 8.79 (d, JHF
=
13
6.8 Hz, H-30). C NMR (400 MHz, CDCl3) δ: 120.9, 121.2 (dd, C-60),
122.8 (d, C-30), 126.5 (d, C-2), 129.8 (d, C-4), 132.6 (s, C-3), 135.4 (s, C-10),
142.6 (s, C-50), 152.2 (s, C-5), 154.2 (s, C-20), 156.9 (s, C-40). HRMS,
m/z 312.9802. Calcd for C10H4FN3O6S, 312.9804.
8.0 Hz, 1H, Ar-H). HRMS, m/z 256.9713. Calcd for C10H5ClFNO2S,
256.9747.
Data for III-g. Yield, 45.8%; mp, 66-68 °C (acetone/petroleum
ether). 1H NMR (400 MHz, CDCl3) δ: 7.10 (d, JHH = 4.8 Hz, 1H,
3
thiophene), 7.37 (s, 1H, thiophene), 7.43 (dd, 4JHH=2.8 Hz, 3JHH=5.1 Hz,
1H, thiophene), 7.74 (s, 1H, Ar-H). HRMS, m/z 306.9028. Calcd for
C10H4Cl3NO2S, 306.9027.
Data for V-a. Yield, 29.4%. mp, 108-110 °C (acetone/petroleum
3
ether). 1H NMR (400 MHz, CDCl3) δ: 7.01 (d, JHH = 5.2 Hz, 1H,
thiophene), 7.38 (dd, 3JHH=7.6 Hz, 4JHH=1.2 Hz, 1H, Ar-H), 7.59 (d,
3JHH =5.2 Hz, 1H, thiophene), 7.64 (dt, 3JHH = 8.0 Hz, 4JHH = 1.6 Hz,
1H, Ar-H), 7.72 (dt, 3JHH=7.6 Hz, 4JHH=1.2 Hz, 1H, Ar-H), 8.25 (dt,
3JHH=8.4 Hz, 4JHH=1.2 Hz, 1H, Ar-H). HRMS, m/z 249.0057 [M-1]þ.
Calcd for C10H6NO2S, 250.0048. All of the data are the same as the known
compound (15).
Data for III-h. Yield, 35.8%; viscosity. 1H NMR (400 MHz, CDCl3)
3
3
δ: 6.98 (d, JHH = 5.2 Hz, 1H, thiophene), 7.34 (t, JHH = 5.2 Hz, 1H,
thiophene), 7.37 (s, 1H, thiophene), 7.38 (s, 1H, Ar-H), 8.47 (d, 3JHF
=
6.8 Hz, 1H, Ar-H). HRMS, m/z 267.9955. Calcd for C10H5FN2O4S,
267.9954.
Data for V-b. Yield, 24.0%. mp, 132-134 °C (acetone/petroleum
General Synthetic Procedures for Target Compounds IV-a-V-h.
Nitration of compound III-a (2.0 g, 9.8 mmol) in 10 mL of acetic
3
ether). 1H NMR (400 MHz, CDCl3) δ: 6.92 (d, JHH = 5.2 Hz, 1H,
thiophene), 7.30 (dt, 3JHH=8.4 Hz, 4JHF=5.6 Hz, 1H, Ar-H), 7.37 (dt,
anhydride was added dropwise to a solution of Cu(NO3)2 3H2O (2.4 g,
3
3JHH =8.4 Hz, 3JHF =7.2 Hz, 1H, Ar-H), 7.53 (d, 3JHH =5.2 Hz, 1H,
9.9 mmol) in 10 mL of acetic anhydride. The solutionwas held at 10-12 °C
for 2 h. Then, the copper salts were removed by filtration, and the residue
was poured into ice water. Continuous extraction with dichloromethane
(2ꢀ 20 mL) gave a thick oil, which was evaporated and purified by flash
chromatography on silica gel eluting with petroleum ether (60-90 °C) to
provide a brown mixture, which consisted of 60% IV-a (4JHH = 2.0 Hz,
thiophene) and 40% V-a (3JHH=5.2 Hz, thiophene), as determined by 1H
NMR (14, 15). Compounds IV-a and V-a were isolated with preparative
thin-layer chromatography [elution solvent: ethyl acetate/petroleum ether
(60-90 °C), 1:9 (v/v)]. Compounds IV-b-V-h were prepared according to
the same process, except compounds IV-g, V-g, IV-h, and V-h, which were
isolated with preparative thin-layer chromatography [elution solvent:
chloroform/petroleum ether (60-90 °C), 1:3 (v/v)]. All of the substituents
at the aromatic rings were listed in Scheme 1.
3
thiophene), 7.88 (d, JHF = 8.0 Hz, 1H, Ar-H). HRMS, m/z 267.9947.
Calcd for C10H5FNO2S, 267.9954.
Data for V-c. Yield, 20.9%. mp, 150-152 °C (acetone/petroleum
3
ether). 1H NMR (400 MHz, CDCl3) δ: 6.92 (d, JHH = 5.6 Hz, 1H,
thiophene), 7.01 (dd, 3JHF=8.4 Hz, 4JHH=2.4 Hz, 1H, Ar-H), 7.37 (ddt,
3JHF=9.2 Hz, 3JHH=7.2 Hz, 4JHH=2.4 Hz, 1H, Ar-H), 7.54 (d, 3JHH
=
5.2 Hz, 1H, thiophene), 8.24 (dd, 3JHH=8.4 Hz, 4JHF =4.8, 1H, Ar-H).
HRMS, m/z 267.9966. Calcd for C10H5FNO2S, 267.9954.
Data for V-d. Yield, 26.0%. mp, 108-110 °C (acetone/petroleum
3
ether). 1H NMR (400 MHz, CDCl3) δ: 6.91 (d, JHH = 5.2 Hz, 1H,
thiophene), 7.25 (d, 3JHH =8.0 Hz, 1H, Ar-H), 7.54 (d, 3JHH =5.6 Hz,
1H, thiophene), 7.62 (dd, 3JHH=8.0 Hz, 4JHH=2.0 Hz, 1H, Ar-H), 8.16
4
(d, JHH = 2.0, 1H, Ar-H). HRMS, m/z 283.9639. Calcd for
C10H5ClNO2S, 283.9658. All of the data are the same as the known
compound (6).
Data for IV-a. Yield, 44.6%. mp, 118-120 °C (acetone/petroleum
ether). 1H NMR (400 MHz, CDCl3) δ: 7.48 (d, 3JHH=7.6 Hz, 1H, Ar-H),
7.49 (d, 4JHH=2.0 Hz, 1H, thiophene), 7.59 (t, 3JHH=7.2 Hz, 1H, Ar-H),
7.69 (t, 3JHH=7.6 Hz, 1H, Ar-H), 7.93 (d, 4JHH=1.6 Hz, 1H, thiophene),
7.98 (d, JHH = 8.0 Hz, 1H, Ar-H). HRMS, m/z 250.0047. Calcd for
C10H6NO2S, 250.0048. All of the data are the same as the known
Data for V-e. Yield, 24.5%. mp, 118-120 °C (acetone/petroleum
3
ether). 1H NMR (400 MHz, CDCl3) δ: 7.01 (d, JHH = 5.2 Hz, 1H,
3
thiophene), 7.47 (s, 1H, Ar-H), 7.63 (d, 3JHH =5.2 Hz, 1H, thiophene),
8.52 (s, 1H, Ar-H). HRMS, m/z 317.9265. Calcd for C10H4Cl2NO2S,
317.9268.
compound (15).
Data for IV-b. Yield, 49.3%. mp, 97-99 °C (acetone/petroleum
Data for V-f. Yield, 20.2%. mp, 125-127 °C (acetone/petroleum
4
ether). 1H NMR (400 MHz, CDCl3) δ: 6.99 (d, JHH = 4.0 Hz, 1H,
3
ether).1H NMR (400 MHz, CDCl3) δ: 6.99 (d, JHH = 5.6 Hz, 1H,
3
4
thiophene), 7.47 (d, 4JHF = 7.2 Hz, 1H, Ar-H), 7.63 (d, 3JHH = 5.6 Hz,
1H, thiophene), 8.08 (d, 3JHF=8.0 Hz, 1H, Ar-H). HRMS, m/z 301.9569.
Calcd for C10H4ClFNO2S, 301.9564.
thiophene), 7.43 (dt, JHF = 7.6 Hz, JHH = 2.4 Hz, 1H, Ar-H), 7.56
(dd, 3JHH=8.4 Hz, 4JHF=5.6 Hz,1H, Ar-H), 7.71 (dd, 3JHF=7.6, 4JHH
2.4 Hz, 1H, Ar-H), 7.89 (d, JHH = 4.0 Hz, 1H, thiophene). HRMS,
m/z 267.9936. Calcd for C10H5FNO2S, 267.9954.
=
4
Data for V-g. Yield, 20.4%; oil. 1H NMR (400 MHz, CDCl3) δ: 7.03
(d, 3JHH = 4.0 Hz, 1H, thiophene), 7.81 (s, 1H, Ar-H), 7.89 (d, 3JHH
=
Data for IV-c. Yield, 49.0%. mp, 99-101 °C (acetone/petroleum
4
ether). 1H NMR (400 MHz, CDCl3) δ: 7.02 (d, JHH = 4.0 Hz, 1H,
4.0 Hz, 1H, thiophene). HRMS, m/z 351.8876. Calcd for C10H3Cl3NO2S,
351.8879.
thiophene), 7.25 (dt, 3JHF=7.6 Hz, 4JHH=2.4 Hz, 1H, Ar-H), 7.33 (ddt,
3JHF=9.6 Hz, 3JHH=9.2 Hz, 4JHH=2.4 Hz, 1H, Ar-H), 7.89 (d, 4JHH
=
Data for V-h. Yield, 22.1%; viscosity. 1H NMR (400 MHz, CDCl3)
δ: 7.06 (d, 3JHH=4.0 Hz, H-4), 7.49 (d, 3JHF=10.0 Hz, 1H, H-60), 7.85 (d,
3JHH = 4.0 Hz, H-5), 8.68 (d, 4JHF = 6.8 Hz, 1H, H-30). 13C NMR (400
MHz, CDCl3) δ: 121.6, 121.8 (dd, C-60), 122.8 (d, C-30), 127.2 (d, C-4),
127.4 (d, C-5), 133.0 (s, C-3), 135.8 (s, C-10), 139.1 (s, C-2), 143.1 (s, C-50),
4.0 Hz, 1H, thiophene), 8.06 (dd, 3JHH=8.8 Hz, 4JHF=4.8, 1H, Ar-H).
HRMS, m/z 267.9955. Calcd for C10H5FNO2S, 267.9954.
Data for IV-d. Yield, 42.2%. mp, 136-138 °C (acetone/petroleum
ether). 1H NMR (400 MHz, CDCl3) δ: 7.35 (d, 3JHH=8.4 Hz, 1H, Ar-H),

Article
J. Agric. Food Chem., Vol. 57, No. 17, 2009 7915
Table 1. Fungicidal Activity of the Compounds III-a-V-ha
concentration
(μg mL-1
A. solani
inhibition (%)
G. zeae
inhibition (%)
P. piricola
inhibition (%)
F. omysporum
inhibition (%)
C. arachidicola
inhibition (%)
compound
)
III-a
50
20
10
50
20
10
50
20
10
50
20
10
50
20
10
50
20
10
50
50
50
20
10
50
20
10
50
20
10
50
20
10
50
20
10
50
20
10
50
20
10
50
50
20
10
50
20
10
50
20
10
50
20
10
50
20
10
50
20
10
50
20
10
50
50
75
30
0
65
43
13
39
30
8
88
68
20
80
20
0
68
31
12
75
50
0
18
0
0
III-b
III-c
III-d
III-e
III-f
55
35
10
75
25
5
0
0
0
65
43
21
39
21
13
39
26
8
72
36
0
87
50
18
75
43
25
87
81
56
87
43
18
36
100
0
25
12
0
55
20
0
80
38
14
82
72
42
80
74
64
90
100
20
9
25
6
0
60
55
20
60
35
0
25
18
0
47
30
0
25
18
0
III-g
III-h
IV-a
49
68
22
16
6
47
100
25
7
50
100
20
16
8
0
1
0
0
IV-b
IV-c
IV-d
IV-e
IV-f
41
25
6
12
0
46
10
4
25
17
7
44
27
11
33
22
0
0
35
16
0
16
0
27
8
28
10
0
0
0
19
0
4
2
17
0
2
0
0
0
0
0
0
0
0
41
32
25
25
16
0
24
16
8
25
17
10
14
2
39
32
21
10
7
27
16
5
0
16
11
0
0
0
0
0
IV-g
34
26
17
58
25
16
9
0
24
10
0
4
35
20
5
0
0
0
0
IV-h
V-a
54
23
14
6
100
30
25
0
60
22
13
0
100
25
16
8
V-b
V-c
V-d
V-e
V-f
48
25
9
0
14
4
17
7
16
0
0
0
0
0
0
29
16
0
0
10
0
21
0
11
0
0
0
0
0
0
9
0
25
10
0
17
0
0
0
0
0
0
0
0
0
38
32
16
61
54
48
34
26
0
0
27
17
2
28
14
10
39
28
14
22
0
27
11
0
0
0
20
4
42
36
12
40
13
0
18
5
0
0
V-g
0
40
25
5
0
0
0
V-h
fenpiclonil
100
100
100
100
100
100
100
18
85
100
a The data are the average of three duplicate results.

7916 J. Agric. Food Chem., Vol. 57, No. 17, 2009
Wang et al.
Table 2. EC50 Values of the Compounds III-g, III-h, IV-h, V-h, and Fenpiclonil
III-g
y = a þ bx
EC50
R
III-h
y = a þ bx
EC50
R
A. solani
G. zeae
>50
>50
36.67
A. solani
G. zeae
y = 2.392 þ 1.860x
y = 0.559 þ 3.084x
y = 4.044 þ 1.481x
y = 1.855 þ 2.628x
y = 3.127 þ 2.190x
25.20
27.53
4.41
0.99
0.99
0.99
0.99
0.99
P. piricola
F. omysporum
C. arachidicola
y = 3.399 þ 1.023x
0.96
P. piricola
F. omysporum
C. arachidicola
>50
>50
4.42
7.16
IV-h
V-h
A. solani
G. zeae
y = 2.868 þ 1.398x
y = 1.458 þ 2.795x
y = 4.048 þ 1.481x
y = 3.584 þ 0.893x
y = 3.443 þ 1.602x
33.49
0.99
0.97
0.99
0.94
0.96
A. solani
G. zeae
y = 3.461 þ 1.045x
y = 3.104 þ 2.059x
y = 3.854 þ 2.070x
y = 3.932 þ 1.434x
y = 3.798 þ 1.487x
29.62
8.33
3.57
5.55
6.42
0.94
0.95
0.99
0.99
0.99
18.50
4.39
P. piricola
F. omysporum
C. arachidicola
P. piricola
F. omysporum
C. arachidicola
38.44
9.36
fenpiclonil
A. solani
G. zeae
y = 5.340 þ 1.322x
y = 4.584 þ 1.780x
y = 5.226 þ 2.352x
0.55
1.71
0.80
0.96
0.97
0.99
P. piricola
F. omysporum
C. arachidicola
>50
0.38
y = 5.477 þ 1.153x
0.95
153.9 (s, C-20), 156.6 (s, C-40). HRMS, m/z 312.9817. Calcd for
C10H4FN3O6S, 312.9804.
The molecular formula of IV-h was revealed as C10H4FN3O6S
by HRMS data [M]þ (calcd., 312.9804; found, 312.9802). The 1H
and 13C NMR (Data for IV-h) spectra showed the signals of six
quaternary, four CH, carbon atoms. Considering the reagents,
the HMQC spectra showed thatδH=7.48 (d, H-60) and δH=8.79
Bioassays. The fungicidal activity of the compounds III-a-III-h and
IV-a-V-h were tested in vitro against Alternaria solani, Gibberella zeae,
Physalospora piricola, Fusarium omysporum, and Cercospora arachidicola,
and their relative inhibitory ratio (%) has been determined using the
mycelium growth rate method (16). Fenpiclonil was used as a control.
After the mycelia grew completely, the diameters of the mycelia were
measured, and the inhibition rate is calculated according to the formula:
(d, H-30) belong to the Ar-H, which was confirmed by 3JHF
=
10.0 Hz and 4JHF=6.8 Hz; δH=7.64 (d, H-2) and δH=7.93 (d,
H-4) belong to the thiophene ring, which was confirmed by the
4JHH=1.6 Hz. The chemical-shift value of C-60 in the 13C NMR
spectra was split by Ar-F into two signals δ = 120.9 and 121.2,
which was confirmed by the HMQC spectra.
I ¼ ðD1 - D2Þ=D1 ꢀ 100%
In the formula, I is the inhibition rate, D1 is the average diameter of
mycelia in the blank test, and D2 is the average diameter of mycelia in the
presence of those compounds. The inhibition ratios of those compounds
at the dose of 50, 20, and 10 μg mL-1 have been determined, and the
experimental results are summarized in Table 1. The EC50 values of the
high fungicidal activity compounds III-g, III-h, IV-h, and V-h and
fenpiclonil have been calculated by the Scatchard method. The results
are summarized in Table 2.
On the basis of the HMBC spectra, the correlations between H-
60 (δH=7.48) and C-10, C-50, C-20, and C-40 [δC=135.4 (s, C-10),
142.6 (s, C-50), 154.2 (s, C-20), and 156.9 (s, C-40)], the correlations
between H-30 (δH = 8.79) and C-10, C-50, C-20, and C-40 [δC =
135.4 (s, C-10), 142.6 (s, C-50), 154.2 (s, C-20), and 156.9 (s, C-40)],
the correlations between H-2 (δH =7.64) and C-3 and C-4 [δC =
132.6 (s, C-3) and 129.8 (d, C-4)], the correlations between H-4
(δH = 7.93) and C-10, C-2, C-3, and C-5 [δC = 135.4 (s, C-10),
126.5 (d, C-2), 132.6 (s, C-3), and 152.2 (s, C-5)] indicated that the
structure of IV-h should be as follows in Figure 2.
RESULTS AND DISCUSSION
Synthesis and Structure Elucidation. In the previous paper,
compound III-a was synthesized using 1-bromo-2-nitrobenzene
and 3-iodothiophene by the Ullmann coupling reaction but the
yield was not reported (13). Under the same conditions, III-a was
synthesized using 1-bromo-2-nitrobenzene and 3-bromo-thio-
phene, which reacted for 20 h with 20.0% yield and obtained
the byproduct 2,20-dinitrobiphenyl (yield, 80.0%). One molecular
equivalent of cuprous iodide was added, mixed with 3-bromo-
thiophene, and then heated to 160 °C for 1 h before 1-bromo-2-
nitrobenzene was added. Compound III-a (yield, 40.1%) and
2,20-dinitrobiphenyl (yield, 59.9%) were obtained after 15 h. The
other target compounds were all synthesized in this novel way.
Regrettably, 2-bromo-1-nitro-4-(trifluoromethyl)benzene, 1-bro-
mo-3-nitrobenzene, and 2-bromo-1-nitro-3-(trifluoromethyl) ben-
zene were not successful in obtaining the corresponding target
compounds. Additionally, in the process of the synthesis of
3-bromo-thiophene, the production of bromization of thiophene
in the first step was used in the subsequent step without further
purification and had a satisfactory yield (80.8%).
In the NOESY spectra, the correlations between H-60 (δH
=
7.48) and H-4 (δH=7.93) indicated that H-60 and H-4 are on the
same side.
1
The H and 13C NMR (Data for V-h) spectra of V-h are the
same as those of IV-h on the whole. However, there are evident
differences in the 2D NMR as follows: In the HMBC spectra, the
correlations between H-60 (δH = 7.49) and C-10, C-50, C-20, and
C-40 [δC=135.8 (s, C-10), 143.1 (s, C-50), 153.9 (s, C-20), and 156.6
(s, C-40)], the correlations between H-30 (δH = 8.68) and C-10,
C-50, C-20, and C-40 [δC = 135.8 (s, C-10), 143.1 (s, C-50), 153.9
(s, C-20), and 156.6 (s, C-40)], the correlations between H-4 (δH =
7.06) and C-5, C-3, C-2, and C-10 [δC = 127.4 (d, C-5), 133.0 (s,
C-3), 139.1 (s, C-2), and 135.8 (s, C-10)], the correlations between
H-5 (δH=7.85) and C-4 and C-2 [δC=127.2 (d, C-4) and 139.1 (s,
C-2)] indicated that the structure of V-h should be as follows in
Figure 2.
In the NOESY spectra, the correlations between H-4 (δH
=
7.06) and H-5 (δH=7.85) indicated that H-4 and H-5 are nearby.
Biological Assay and SAR. The data in Table 1 show the
fungicidal activities against A. solani, G. zeae, P. piricola,
F. omysporum, and C. arachidicola of the title compounds
Because the compounds IV-a-V-h were not easily separated,
they were finally isolated with the preparative layer chromato-
graphy. The structures of two important compounds (IV-h and
V-h) were elucidated by 1D and 2D NMR.

Article
J. Agric. Food Chem., Vol. 57, No. 17, 2009 7917
Figure 2. Key HMBC correlations and key NOESY correlations of IV-h and V-h.
III-a-V-h. The data in Table 2 show the EC50 values of the high
fungicidal activity compounds III-g, III-h, IV-h, and V-h and the
commercial fungicide fenpiclonil.
clonil exhibited 100% inhibition against C. arachidicola and V-h
showed 85% inhibition. Owning to adding nitro to the aromatic
ring might be essential for high fungicidal activity.
The screening data of Tables 1 and 2 indicated that, athough the
fungicidal activities of most target compounds are lower than that
of fenpiclonil, compound V-h has higher fungicidal activity than
fenpiclonil against F. omysporum. Therefore, the V-h could be deve-
loped as a leading compound for further structural optimization.
In conclusion, a novel and facile procedure for preparation
analogues of pyrrolnitrin from 3-bromo-thiophene with corre-
sponding substituted nitrobenzene was described, and 24 3-(2-
nitrophenyl) thiophene derivatives were synthesized. The results
of the bioassay showed that some of these title compounds
exhibited favorable fungicidal activities against A. solani, G. zeae,
P. piricola, F. omysporum, and C. arachidicola at the dosage of
50 μg mL-1; III-h, IV-h, and V-h still exhibited moderate activity
against P. piricola at low dosage. Although the fungicidal
activities of most target compounds are lower than that of fenpi-
Fungicidal Activity against A. solani. The screening data of
Table 1 indicated that, at the dosage of 50 μg mL-1, some
compounds of III-a-V-h exhibited excellent activity against
A. solani. For instance, the inhibition activity of III-a-III-h were
more than 50%, and the inhibition activity of V-h was equal to the
commercialized fenpiclonil. At the dosage of 20 and 10 μg mL-1
,
the fungicidal activities evidently decreased and the fungicidal
activities of compounds IV-a-V-h are lower than those of III-
a-III-h. It may be owing to the nitration of the thiophene ring
that resulted in the activity to be decreased.
Fungicidal Activity against G. zeae. At the dosage of 50 μg
mL-1, compounds of III-a, III-c, and IV-h exhibited moderate
activity and IV-f, IV-g, V-b-V-e, and V-g exhibited no activity.
However, the fungicidal activity of III-h and V-h were equal to
the fenpiclonil at this dosage. As the concentration of compounds
III-a-V-h declining, its biological activity decreased.
Fungicidal Activity against P. piricola. At the dosageof50μg
mL-1, compounds of III-a-III-h exhibited excellent activity,
while IV-a-IV-g and V-a-V-g exhibited low activity against
P. piricola. Compound III-g showed 90% inhibition, which might
be owing to the biological activity of adding halogen atoms to the
aromatic ring. The fungicidal activities of III-h, IV-h, and V-h
were the same as fenpiclonil at this dosage. Although at the low
concentrations the fungicidal activities evidently declined, III-h,
IV-h, and V-h still exhibited moderate activity, which can be
shown from their EC50 values (Table 2). This might be owing to
the biological activity of adding nitro to the aromatic ring.
Fungicidal Activity against F. omysporum. The biological
activity rules of III-a-V-h against F. omysporum are generally the
same as the test against P. piricola. The EC50 value of V-h against
F. omysporum is 5.55 μg mL-1, while that of fenpiclonil is more
than 50 μg mL-1 (Table 2).
clonil, the EC50 value of V-h against F. omysporum is 5.55 μg mL-1
,
while that of fenpiclonil is more than 50 μg mL-1 (Table 2).
Therefore, the V-h could be developed as a leading compound for
further structural optimization. The possible SAR is as follows:
The nitration of the thiophene ring may result from the activity to
be decreased. The adding of halogen atoms and nitro to the
aromatic ring might be essential for high fungicidal activity.
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Received May 12, 2009. Accepted July 31, 2009. We gratefully
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National Natural Science Foundation of China for the financial
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