Synthesis and fungicidal activity of novel aminophenazine-1-carboxylate derivatives

Article abstract of DOI:10.1021/jf904408c

A series of novel 6-aminophenazine-1-, 7-aminophenazine-1- and 8-aminophenazine-1-carboxylate derivatives were synthesized by a facile method, and their structures were characterized by ¹H NMR, ¹³C NMR and high-resolution mass spectrometry. Some unexpected byproducts V-7b-V-8d were noticed and isolated, and their structures were identified by 2D NMR spectra including heteronuclear multiple-quantum coherence (HMQC), heteronuclear multiple-bond correlation (Hmbc) and H-H correlation spectrometry (H-H COSY) approach. Their fungicidal activities against five fungi were evaluated, which indicated that most of the title compounds showed low fungicidal activities in vitro against Alternaria solanl, Cercospora arachidicola, Fusarium omysporum, Gibherella zeae, and Physalospora piricola at a dosage of 50 μg mL ^-1, while compounds IV-6a and IV-6b exhibited excellent activities against P. piricola at that dosage. Compound IV-6a could be considered as a leading structure for further design of fungicides. 2010 American Chemical Society.

Full text of DOI:10.1021/jf904408c

J. Agric. Food Chem. 2010, 58, 3651–3660 3651
DOI:10.1021/jf904408c
Synthesis and Fungicidal Activity of Novel
Aminophenazine-1-carboxylate Derivatives
M
ING-ZHONG
W
ANG, HAN
X
U
, SHU-JING
Y
U
, Q
I
F
ENG, S -HUA WANG, and
U
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 6-aminophenazine-1-, 7-aminophenazine-1- and 8-aminophenazine-1-carboxylate
derivatives were synthesized by a facile method, and their structures were characterized by 1H
NMR, 13C NMR and high-resolution mass spectrometry. Some unexpected byproducts V-7b-V-8d
were noticed and isolated, and their structures were identified by 2D NMR spectra including
heteronuclear multiple-quantum coherence (HMQC), heteronuclear multiple-bond correlation
(Hmbc) and H-H correlation spectrometry (H-H COSY) approach. Their fungicidal activities
against five fungi were evaluated, which indicated that most of the title compounds showed low
fungicidal activities in vitro against Alternaria solani, Cercospora arachidicola, Fusarium omysporum,
Gibberella zeae, and Physalospora piricola at a dosage of 50 μg mL-1, while compounds IV-6a and
IV-6b exhibited excellent activities against P. piricola at that dosage. Compound IV-6a could be
considered as a leading structure for further design of fungicides.
KEYWORDS: Aminophenazine-1-carboxylate; derivatives; synthesized; fungicidal activities; leading
structure
INTRODUCTION
Those compounds’ fungicidal activities against five fungi were
evaluated, and their possible structure-activity relationships
(SAR) were discussed. In order to enhance the lipophilic proper-
ties, aminophenazine-1-carboxylic acid was derivatized to its ester
with corresponding CH3(CH2)nOH (n=0, 1, 2, 3); The -NH2
group was substituted at different positions of the phenazine ring;
Different carboxamide and sulfonamide derivatives were opti-
mized with acyl chloride or sulfonyl chloride; the phenazine rings
of some carboxamide derivatives were further nitrified. All the
new derivatives were designed to explore whether that might
improve or decrease the fungicidal activities.
In the past century, many potential antibiotics containing the
planar tricyclic heteroaromatic phenazine were isolated from the
marine microorganism Streptomyces antibioticus strain Tu
€
2706 (1). The derivatives included the diphenazine antibiotics
esmeraldin A and B as well as simpler monomeric structures
containing 6-(1-hydroxyethyl)-1-phenazine carboxylic acid (A,
Figure 1) have shown extensive antimicrobial activity toward a
broad range of bacteria (2). Phenazine-1-carboxylic acid (A,
Figure 1) isolated from Pseudomonas sp. had high antimicrobial
activity against nine bacterial strains, inhibiting settlement of
barnacle larvae, and reducing Ulva lactuca spore settlement and
percent cover of germlings (3). Recently, a series of substituted
(-Cl, -OCH3, -CH3) phenazine-1-carboxylic acids and the
corresponding carboxamides (B, Figure 1) were prepared as
antitumor drugs acting on electron-deficient DNA-intercalating
ligands and evaluated against L1210 leukemia invitro and against
P388 leukemia and Lewis lung carcinoma in vivo (4).
The total synthesis of aminophenazine-1-carboxylic acid was
completed in the 1960s (5-7), but few published studies reported
their biological activity. Considering the other potential biological
activity of phenazine structures, the derivatives of aminophena-
zine-1-carboxylic acid with the phenazine skeleton were synthe-
sized and tested against fungi in vitro. In this paper, forty-six
6-aminophenazine-1-, 7-aminophenazine-1- and 8-aminophena-
zine-1-carboxylate derivatives, including six known compounds
(C, Figure 1), were synthesized with a facile synthetic method.
MATERIALS AND METHODS
Instruments. 1H NMR, 13C NMR, HMQC, HMBC, H-H COSY
spectra were obtained at 300 MHz using a Bruker AV300 spectrometer or
at 400 MHz using a Bruker AV400 spectrometer in CDCl3 or d6-DMSO
solution with tetramethylsilane as the internal standard. Chemical shift
values (δ) are given in ppm. High-resolution mass spectrometry (HRMS)
data were obtained on a Varian QFT-ESI instrument. The melting points
were determined on an X-4 binocular microscope melting point apparatus
(Beijing Tech Instruments Co., Beijing, China) and are 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. Silica gel (200-300 mesh) was obtained
from Qingdao Marine Chemical Factory, Qingdao, P. R. China. Three
kinds of benzene-diamine, 2-bromo-3-nitrobenzoic acid and NaBH4 were
bought from the Alfa Aesar Company (Tianjin, China). Phenazine-1-
carboxylic acid was synthesized according to the literature (4).
General Synthetic Procedures for I-a, I-b and I-c. To a solution of
o-phenylenediamine (8.0 g, 74.0 mmol) in dry THF (50.0 mL) was added
dropwise a solution of Ac2O (8.0 mL) in dry THF (20.0 mL) under 15 °C
*To whom correspondence should be addressed. Tel: þ86-22-
23503732. Fax: þ86-22-23505948. E-mail: nkzml@vip.163.com.
© 2010 American Chemical Society
Published on Web 02/18/2010
pubs.acs.org/JAFC

3652 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Wang et al.
Figure 1. Chemical structure of compounds A-C.
Scheme 1. Synthetic Routes to Compounds III
within 2 h. The mixture was stirred for 5 h at 15 °C, and the solvent was
removed, followed by cooling to get the crude product and recrystalliza-
tion with ethyl acetate to give N-(2-aminophenyl) acetamide (I-a, 5.3 g,
47.7%).
cooled mixture was diluted with 0.2 N aqueous NaOH, clarified with
charcoal, and filtered through Celite. The resulting clear solution was
acidified with dilute HCl to give 2-(2-acetamidophenylamino)-3-nitroben-
zoic acid (II-a) (3.0 g, 35.8%) (9). 2-(3-acetamidophenylamino)-3-nitro-
benzoic acid (II-b) and 2-(4-acetamidophenylamino)-3- nitrobenzoic acid
(II-c) were given through the same process. All the substituents at the
aromatic rings were listed in the Scheme 1.
To a solution of m-phenylenediamine (12.0 g, 111.0 mmol) in dry THF
(30.0 mL) was added dropwise a solution of Ac2O (9.0 mL) in dry THF
(35.0 mL) under -10 °C within 2 h. The mixture was stirred for 3 h keeping
the temperature under -10 °C, and dilute hydrochloric acid (20.0 mL,
18.5%) was added to the mixture, followed by cooling for 30 min to get the
white salt. Then the salt was dissolved in dilute sodium hydroxide and
extracted with ethyl acetate to give N-(3-aminophenyl) acetamide (I-b, 5.7 g,
34.2%). N-(4-Aminophenyl) acetamide (I-c) was synthesized according to
the literature (8). All the substituents at the aromatic rings are listed in
Scheme 1.
Data for II-a. Yield: 35.8%; orange solid; mp, 202-204 °C (acetone/
petroleum ether). 1H NMR (400 MHz, d6-DMSO) δ: 13.56 (s, 1H,
COOH), 9.80 (s, 1H, NHAc), 9.63 (s, 1H, Ar-NH-Ar), 8.14 (d, 3JHH
=
7.2 Hz, 1H, Ar-H), 8.01 (d, 3JHH=7.6 Hz, 1H, Ar-H), 7.39 (d, 3JHH=6.8
Hz, 1H, Ar-H), 7.02 (t, 3JHH=8.4 Hz, 3H, Ar-H)), 6.82 (d, 3JHH=7.2
Hz, 1H, Ar-H), 2.07 (s, 3H, COCH3).
Data for II-b. Yield: 62.6%; orange solid; mp, 222-224 °C (acetone/
petroleum ether). 1H NMR (400 MHz, d6-DMSO) δ: 9.88 (s, 1H, NHAc),
9.84 (s, 1H, Ar-NH-Ar), 8.21 (d, 3JHH=7.6 Hz, 1H, Ar-H), 8.08 (d,
3JHH=8.0 Hz, 1H, Ar-H), 7.25 (s, 1H, Ar-H), 7.18 (d, 3JHH=7.6 Hz, 1H,
Ar-H), 7.15 (d, 3JHH=7.6 Hz, 1H, Ar-H), 7.11 (d, 3JHH=7.6 Hz, 1H,
Ar-H), 6.56 (d, 3JHH=7.6 Hz, 1H, Ar-H), 2.01 (s, 3H, COCH3).
Data for II-c. Yield: 50.6%; orange solid; mp, 218-220 °C (acetone/
petroleum ether). 1H NMR (400 MHz, d6-DMSO) δ: 9.93 (s, 1H,
Ar-NH-Ar), 9.87 (s, 1H, NHAc), 8.19 (d, 3JHH=7.6 Hz, 1H, Ar-H),
8.04 (d, 3JHH=8.0 Hz, 1H, Ar-H), 7.44 (d, 3JHH=8.4 Hz, 2H, Ar-H),
7.02 (t, 3JHH=8.0 Hz, 1H, Ar-H), 6.88 (d, 3JHH=8.4 Hz, 2H, Ar-H), 2.01
(s, 3H, COCH3).
General Synthetic Procedures for III-a, III-b and III-c. A solution
of 2-(3-acetamido phenylamino)-3-nitrobenzoic acid (II-b) (6.0 g, 19.0
mmol) and NaBH4 (4.8 g, 126.0 mmol) in 2 N NaOH (500.0 mL) was
refluxed for 4 h. Cooling gave the sodium salt of the phenazine acid, which
was acidified to give a mixture of 6-aminophenazine-1-carboxylic acid
(III-a) and 8-aminophenazine-1-carboxylic acid (III-c). The mixture con-
sisted of 50% III-a and 50% III-c, as determined by 1H NMR. A small
Data for I-a. Yield: 47.7%; white crystal; mp, 129-131 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, d6-DMSO) δ: 9.11 (s, 1H,
NHAc), 7.14 (d, 3JHH=7.6 Hz, 1H, Ar-H), 6.88 (t, 3JHH=7.6 Hz, 1H,
Ar-H), 6.70 (d, 3JHH=7.6 Hz, 1H, Ar-H), 6.52 (t, 3JHH=7.6 Hz, 1H,
Ar-H), 4.84 (s, 2H, NH2), 2.02 (s, 3H, COCH3).
Data for I-b. Yield: 34.2%; white crystal; mp, 82-84 °C (ethyl acetate/
petroleum ether). 1H NMR (400 MHz, d6-DMSO) δ: 9.60 (s, 1H, NHAc),
6.92 (s, 1H, Ar-H), 6.89 (t, 3JHH=8.0 Hz, 1H, Ar-H), 6.65 (d, 3JHH=8.0
Hz, 1H, Ar-H), 6.23 (dd, 3JHH=8.0 Hz, 3JHH=1.2 Hz, 1H, Ar-H), 5.02
(s, 2H, NH2), 1.99 (s, 3H, COCH3).
Data for I-c. Yield: 72.0%; white crystal; mp, 162-164 °C (ethyl
acetate/petroleum ether). 1H NMR (300 MHz, d6-DMSO) δ: 9.46 (s, 1H,
NHAc), 7.18 (d, 3JHH=8.4 Hz, 2H, Ar-H), 6.48 (d, 3JHH=8.4 Hz, 2H,
Ar-H), 4.80 (s, 2H, NH2), 1.94 (s, 3H, COCH3).
General Synthetic Procedures for II-a, II-b and II-c. A mixture of
N-(2-aminophenyl) acetamide (I-a) (4.0 g, 26.6 mmol), 2-bromo-3-nitro-
benzoic acid (6.3 g, 26.6 mmol), CuI (1.3 g, 6.8 mmol) in triethylamine
(12.5 mL) and ethane-1,2-diol (50.0 mL) were stirred at 80 °C for 3 h. The

Article
J. Agric. Food Chem., Vol. 58, No. 6, 2010 3653
Scheme 2. Synthetic Routes to the Title Compounds IV, VI and VII
amount of the mixture was purified by flash chromatography on silica gel
[elution solvent: ethyl acetate/petroleum ether (60-90 °C), 1:4, v/v].
Similar procedures were used to prepare 7-aminophenazine-1-carboxylic
acid (III-b) by reductive cyclization of 2-(4-acetamido phenylamino)-3-
nitrobenzoic acid (II-c). The compound III-x was the only product by
reductive cyclization of 2-(2-acetamido phenylamino)-3-nitrobenzoic acid
(II-a). All the substituents at the aromatic rings are listed in Scheme 1.
Data for III-a. Yield: 59.0%; purple solid; mp, 306-308 °C (acetone/
petroleum ether) (5). 1H NMR (300 MHz, d6-DMSO) δ: 15.27 (s, 1H,
COOH), 8.62 (d, 3JHH=6.6 Hz, 1H, Ar-H), 8.48 (d, 3JHH=8.4 Hz, 1H,
Ar-H), 8.03 (t, 3JHH=7.5 Hz, 1H, Ar-H), 7.82 (t, 3JHH=7.8 Hz, 1H,
Ar-H), 7.40 (d, 3JHH=8.4 Hz, 1H, Ar-H), 6.98 (d, 3JHH=7.5 Hz, 1H,
Ar-H), 6.74 (s, 2H, NH2).
Data for III-b. Yield: 69.0%; red solid; mp, beyond 340 °C (acetone/
petroleum ether) (4). 1H NMR (400 MHz, d6-DMSO) δ: 8.33 (d, 3JHH=6.0
Hz, 1H, Ar-H), 8.23 (d, 3JHH=8.0 Hz, 1H, Ar-H), 8.05 (d, 3JHH=8.0 Hz,
1H, Ar-H), 7.91 (t, 3JHH=7.2 Hz, 1H, Ar-H), 7.58 (d, 3JHH=8.0 Hz, 1H,
Ar-H), 6.95 (s, 1H, Ar-H), 6.81 (s, 2H, NH2).
Data for III-c. Yield: 59.0%; red solid; mp, beyond 340 °C (acetone/
petroleum ether) (5). 1H NMR (300 MHz, d6-DMSO) δ: 8.41 (d, 3JHH=6.6
Hz, 1H, Ar-H), 8.34 (d, 3JHH=8.7 Hz, 1H, Ar-H), 7.98 (d, 3JHH=9.6 Hz,
1H, Ar-H), 7.78 (d, 3JHH=7.8 Hz, 1H, Ar-H), 7.57 (d, 3JHH=9.0 Hz, 1H,
Ar-H), 7.25 (s, 2H, NH2), 6.86 (s, 1H, Ar-H).
methanol was then removed by distillation, and the residue was diluted
with water. The black solution was made alkaline with NH3 H2O (5) and
3
extracted with dichloromethane (2 ꢀ 50.0 mL). The extraction was
evaporated and purified by flash chromatography on silica gel eluting
[elution solvent: ethyl acetate/petroleum ether (60-90 °C), 1:4, v/v] to
provide red solid IV-6a (0.52 g, Yield, 25.1%) and IV-8a (0.25 g, yield,
11.8%). Compounds IV-6b-IV-8d were prepared according to the same
process. V-7b, V-7c, V-7d, and V-8d were the byproducts of IV-7b, IV-7c,
IV-7d and IV-8d respectively. All the substituents at the phenazine rings
are listed in Scheme 2 and Table 1.
Data for IV-6a. Yield: 25.1%; red solid; mp, 142-144 °C (ethyl acetate/
petroleum ether) (6). 1H NMR (400 MHz, CDCl3) δ: 8.14 (d, 3JHH=8.8
Hz, 1H, Ar-H), 8.06 (d, 3JHH=6.8 Hz, 1H, Ar-H), 7.60 (t, 3JHH=8.0 Hz,
1H, Ar-H), 7.51 (d, 3JHH=8.4 Hz, 1H, Ar-H), 7.47 (d, 3JHH=8.4 Hz, 1H,
Ar-H), 6.74 (d, 3JHH=6.0 Hz, 1H, Ar-H), 5.16 (s, 2H, NH2), 3.98 (s, 3H,
OCH3). 13C NMR (100 MHz, CDCl3) δ: 167.3, 144.3, 143.9, 141.2, 140.4,
134.9, 133.4, 132.6, 131.9, 131.0, 127.8, 117.7, 108.0, 52.6.
Data for IV-6b. Yield: 41.7%; red solid; mp, 108-110 °C (ethyl acetate/
petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.20 (dd, 3JHH=8.8 Hz,
4
4JHH=1.2 Hz, 1H, Ar-H), 8.09 (dd, 3JHH=7.2 Hz, JHH=1.2 Hz,1H,
Ar-H), 7.65 (dd, 3JHH=8.8 Hz, 3JHH=7.2 Hz, 1H, Ar-H), 7.52 (d, 3JHH
=
4.4 Hz, 2H, Ar-H), 6.80 (d, 3JHH=4.4 Hz, 1H, Ar-H), 5.16 (s, 2H, NH2),
4.50 (q, 3JHH=7.2 Hz, 2H, OCH2), 1.42 (t, 3JHH=7.2 Hz, 3H, CH3). 13
C
NMR (100 MHz, CDCl3) δ: 165.8, 143.3, 142.8, 140.2, 139.4, 133.9, 132.1,
131.4, 130.5 (2C), 126.9, 116.9, 107.0, 60.5, 13.3. HRMS: m/z 268.1082.
Calcd for C15H13N3O2: 268.1081 [M þ H]þ.
Data for III-x. Yield: 20.0%; red solid; mp, 305-307 °C (acetone/
1
petroleum ether) (10). H NMR (400 MHz, d6-DMSO) δ: 10.41 (s, 1H,
NHCO), 8.79 (s, 1H, Ar-NH-Ar), 8.20 (d, 3JHH=7.2 Hz, 1H, Ar-H),
8.05 (d, 3JHH=6.8 Hz, 1H, Ar-H), 7.13 (t, 3JHH=8.0 Hz, 1H, Ar-H),
7.02-7.06 (m, 4H, Ar-H).
General Synthetic Procedures for Compounds IV-6a-V-8d. To a
solution of the mixture 6-aminophenazine-1-carboxylic acid (III-a) and 8-
aminophenazine-1-carboxylic acid (III-c) (2.0 g) in methanol (500.0 mL),
H2SO4 (0.5 mL) was added dropwise and refluxed for 7 h. Most of the
Data for IV-6c. Yield: 33.1%; red solid; mp, 98-100 °C (ethyl acetate/
petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.16 (d, 3JHH=8.4 Hz,
1H, Ar-H), 8.07 (d, 3JHH=6.4 Hz, 1H, Ar-H), 7.62 (t, 3JHH=7.6 Hz, 1H,
Ar-H), 7.50 (brd, 4JHH=1.2 Hz, 2H, Ar-H), 6.76 (brs, 1H, Ar-H), 5.15
(s, 2H, NH2), 4.41 (t, 3JHH=6.0 Hz, 2H, OCH2), 1.77-1.82 (m, 2H, CH2),
1.04 (t, 3JHH=7.2 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 167.2,

3654 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Wang et al.
Table 1. The R Group and the Value of n in Compounds IV-6a-VII-e
Ar-H), 7.79 (d, 3JHH=8.0 Hz, 1H, Ar-H), 7.49 (d, 3JHH=8.4 Hz, 1H,
Ar-H), 6.91 (s, 1H, Ar-H), 6.65 (s, 2H, NH2), 3.96 (s, 3H, OCH3). 13
C
NMR (100 MHz, d6-DMSO) δ: 167.4, 151.5, 145.8, 142.5, 139.2, 136.5,
132.0, 130.9, 130.4, 128.9, 127.4, 126.9, 100.7, 52.3.
Data for IV-7b. Yield: 59.7%; red solid; mp, 190-192 °C (ethyl acetate/
petroleum ether). 1H NMR (300 MHz, CDCl3) δ: 8.20 (dd, 3JHH=7.5 Hz,
4JHH=1.2 Hz, 1H, Ar-H), 8.07 (d, 3JHH=9.3 Hz, 1H, Ar-H), 8.03 (dd,
3JHH=7.2 Hz, 4JHH=1.2 Hz, 1H, Ar-H), 7.74 (dd, 3JHH=8.7 Hz, 3JHH
=
7.2 Hz, 1H, Ar-H), 7.32 (dd, 3JHH=9.3 Hz, 4JHH=2.4 Hz, 1H, Ar-H),
7.13 (s, 1H, Ar-H), 4.58 (q, 3JHH=6.9 Hz, 2H, OCH2), 4.53 (s, 2H, NH2),
1.49 (t, 3JHH =6.9 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 167.1,
148.8, 145.4, 143.1, 140.4, 138.7, 132.2, 131.7 (2C), 129.2, 128.8, 125.7,
104.5, 61.5, 14.4. HRMS: m/z 290.0899. Calcd for C15H13N3O2: 290.0900
[M þ Na]þ.
Data for IV-7c. Yield: 29.9%; red solid; mp, 170-172 °C (ethyl acetate/
petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.20 (d, 3JHH=8.4 Hz,
1H, Ar-H), 8.03 (brd, 3JHH=6.8 Hz, 2H, Ar-H), 7.73 (t, 3JHH=7.6 Hz,
1H, Ar-H), 7.29 (d, 3JHH=9.6 Hz, 1H, Ar-H), 7.10 (s, 1H, Ar-H), 4.61
(s, 2H, NH2), 4.47 (t, 3JHH=6.0 Hz, 2H, OCH2), 1.87-1.89 (m, 2H, CH2),
1.12 (t, 3JHH=6.8 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 167.3,
148.9, 145.4, 143.1, 140.3, 138.6, 132.1, 131.8 131.5, 129.1 128.8, 125.8,
104.3, 67.1, 22.1, 10.6. HRMS: m/z 304.1059. Calcd for C16H15N3O2:
304.1056 [M þ Na]þ.
Data for IV-7d. Yield: 31.7%; red solid; mp, 202-204 °C (ethyl acetate/
petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.20 (d, 3JHH=8.4 Hz,
1H, Ar-H), 8.07 (d, 3JHH=9.6 Hz, 1H, Ar-H), 8.03 (t, 3JHH=6.8 Hz, 1H,
Ar-H), 7.75 (t, 3JHH=8.0 Hz, 3JHH=6.8 Hz, 1H, Ar-H), 7.33 (dd, 3JHH
=
9.6 Hz, 4JHH=2.0 Hz, 1H, Ar-H), 7.14 (d, 4JHH=2.4 Hz, 1H, Ar-H),
4.51 (t, 3JHH=6.4 Hz, 2H, OCH2), 4.46 (s, 2H, NH2), 1.81-1.88 (m, 2H,
CH2), 1.57-1.62 (m, 2H, CH2), 1.02 (t, JHH=7.2 Hz, 3H, CH3). 13C
3
NMR (100 MHz, CDCl3) δ: 167.3, 148.7, 145.4, 143.1, 140.4, 138.8, 132.2,
131.9, 131.7, 129.2, 128.8, 125.7, 104.6, 65.4, 30.8, 19.3, 13.8. HRMS: m/z
296.1391. Calcd for C17H17N3O2: 296.1393 [M þ H]þ.
Data for IV-8a. Yield: 11.8%; red solid; mp, 185-187 °C (ethyl acetate/
petroleum ether) (6). 1H NMR (400 MHz, CDCl3) δ: 8.27 (d, 3JHH=8.4
Hz, 1H, Ar-H), 8.16 (d, 3JHH=6.8 Hz, 1H, Ar-H), 7.99 (d, 3JHH=9.2 Hz,
1H, Ar-H), 7.67 (dt, 3JHH=8.4 Hz, 3JHH=7.2 Hz, 1H, Ar-H), 7.34 (d,
3JHH=9.2 Hz, 1H, Ar-H), 7.25 (s, 1H, Ar-H), 4.58 (s, 2H, NH2), 4.08 (s,
3H, OCH3).
Data for IV-8b. Yield: 19.0%; red solid; mp, 136-138 °C (ethyl acetate/
petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.18 (d, 3JHH=8.0 Hz,
1H, Ar-H), 8.06 (d, 3JHH=7.2 Hz, 1H, Ar-H), 7.93 (d, 3JHH=9.2 Hz, 1H,
Ar-H), 7.60 (dd, 3JHH=8.4 Hz, 3JHH=7.2 Hz, 1H, Ar-H), 7.25 (dd, 3JHH
=9.3 Hz, 4JHH=2.4 Hz, 1H, Ar-H), 7.17 (d, 4JHH=2.4 Hz, 1H, Ar-H),
4.49 (q, 3JHH=7.2 Hz, 2H, OCH2), 4.37 (s, 2H, NH2), 1.42 (t, 3JHH =7.2
Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 166.0, 147.5, 144.7, 140.5,
139.6, 138.9, 132.2, 130.5, 129.7 (2C), 125.5, 124.9, 104.7, 60.4, 13.3.
HRMS: m/z 268.1082. Calcd for C15H13N3O2: 268.1081 [M þ H]þ.
Data for IV-8c. Yield: 21.2%; red solid; mp, 144-146 °C (ethyl acetate/
petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.16 (d, 3JHH=8.4 Hz,
1H, Ar-H), 8.05 (d, 3JHH=6.8 Hz, 1H, Ar-H), 7.88 (d, 3JHH=9.2 Hz, 1H,
Ar-H), 7.58 (dd, 3JHH=8.0 Hz, 3JHH=7.2 Hz, 1H, Ar-H), 7.21 (dd, 3JHH
3
=9.2 Hz, JHH=8.0 Hz, 1H, Ar-H), 7.13 (s, 1H, Ar-H), 4.50 (s, 2H,
NH2), 4.39 (t, 3JHH=6.0 Hz, 2H, OCH2), 1.77-1.82 (m, 2H, CH2), 1.04 (t,
3JHH=6.8 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 166.3, 147.7,
144.7, 140.5, 139.5, 138.8, 132.2, 130.5 129.7 129.6 125.4, 125.0, 104.4, 66.0,
21.1, 9.6. HRMS: m/z 282.1243. Calcd for C16H15N3O2: 282.1237 [M þ
H]þ.
144.3, 143.8, 141.2, 140.4, 134.9, 133.2, 132.5, 131.6 (2C), 127.9, 117.8,
108.0, 67.2, 22.2, 10.7. HRMS: m/z 282.1241. Calcd for C16H15N3O2:
282.1237 [M þ H]þ.
Data for IV-6d. Yield: 60.1%; brown solid; mp, 110-112 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.30 (dd, 3JHH
=
8.8 Hz, 4JHH=1.2 Hz, 1H, Ar-H), 8.17 (dd, 3JHH=6.8 Hz, 4JHH=1.2 Hz,
1H, Ar-H), 7.75 (dd, 3JHH=8.8 Hz, 4JHH=2.8 Hz, 1H, Ar-H), 7.63 (dt,
Data for IV-8d. Yield: 50.5%; red solid; mp, 141-143 °C (ethyl acetate/
petroleum ether).1H NMR (400 MHz, CDCl3) δ: 8.10 (d, 3JHH=8.4 Hz,
1H, Ar-H), 7.99 (d, 3JHH=6.8 Hz, 1H, Ar-H), 7.74 (d, 3JHH=9.2 Hz, 1H,
Ar-H), 7.51 (t, 3JHH=7.6 Hz, 1H, Ar-H), 7.13 (d, 3JHH=9.2 Hz, 1H,
Ar-H), 7.06 (s, 1H, Ar-H), 4.78 (s, 2H, NH2), 4.37 (t, 3JHH=6.4 Hz, 2H,
3JHH=8.8 Hz, 3JHH=6.8 Hz, 1H, Ar-H), 7.61 (dt, 3JHH=8.8 Hz, 3JHH
6.8 Hz, 1H, Ar-H), 6.91 (dd, 3JHH=6.8 Hz, 3JHH=1.6 Hz, 1H, Ar-H),
5.24 (s, 2H, NH2), 4.53 (t, 3JHH=6.4 Hz, 2H, OCH2), 1.83-1.87 (m, 2H,
=
OCH2), 1.67-1.70 (m, 2H, CH2), 1.39-1.44 (m, 2H, CH2), 0.85 (t, 3JHH
=
CH2), 1.58-1.64 (m, 2H, CH2), 1.03 (t, JHH=7.2 Hz, 3H, CH3). 13C
3
7.2 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 166.2, 148.2, 144.8,
140.3, 139.3, 138.7, 132.1, 130.4, 129.5, 129.4, 125.2 (2C), 103.7, 64.2, 29.7,
18.2, 12.7. HRMS: m/z 296.1392. Calcd for C17H17N3O2: 296.1393 [M þ
H]þ.
NMR (100 MHz, CDCl3) δ: 167.1, 144.3, 143.8, 141.3, 140.5, 135.0, 133.2,
132.5, 131.7, 131.6, 128.0, 117.9, 108.1, 65.5, 30.8, 19.3, 13.8. HRMS: m/z
296.1394. Calcd for C17H17N3O2: 296.1393 [M þ H]þ.
Data for IV-7a. Yield: 47.6%; red solid; mp, 201-203 °C (acetone/
petroleum ether) (5). 1H NMR (400 MHz, d6-DMSO) δ: 8.11 (d, 3JHH=8.4
Hz, 1H, Ar-H), 7.90 (d, 3JHH=8.8 Hz, 1H, Ar-H), 7.86-7.87 (brd, 1H,
Data for V-7b. Yield: 5.1%; red solid; mp, 159-161 °C (ethyl acetate/
petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.22 (d, 3JHH=8.4 Hz,

Article
J. Agric. Food Chem., Vol. 58, No. 6, 2010 3655
1H, H-4), 8.04 (d, 3JHH=6.8 Hz, 1H, H-2), 7.66-7.72 (m, 1H, H-3), 7.35
(s, 1H, H-9), 7.13 (s, 1H, H-6), 4.89 (s, 2H, NH2), 4.56 (t, 3JHH=7.2 Hz,
2H, CH2-20), 4.29 (t, 3JHH=7.2 Hz, 2H, CH2-100), 1.55 (q, 3JHH=7.2 Hz,
3H, CH3-200), 1.48 (q, 3JHH=7.2 Hz, 3H, CH3-30). 13C NMR (100 MHz,
CDCl3) δ: 167.0 (s, C-10), 152.8 (s, C-8), 143.6 (s, C-7), 142.2 (s, C-9a),
142.2 (s, C-5a), 141.2 (s, C-4a), 138.6 (s, C-10a), 131.9 (d, C-4), 130.5 (s, C-
1), 129.1 (d, C-2), 127.3 (d, C-3), 105.7 (d, C-9), 103.4 (d, C-6), 64.9 (t, C-
100), 61.3 (t, C-20), 14.5 (q, C-200), 14.4 (q, C-30). HRMS: m/z 334.1163.
Calcd for C17H17N3O3: 334.1162 [M þ Na]þ.
NHCO), 8.81 (d, 3JHH=7.6 Hz, 1H, Ar-H), 8.33 (dd, 3JHH=8.8 Hz, 4JHH
=1.2 Hz, 1H, Ar-H), 8.23 (dd, 3JHH=6.8 Hz, 4JHH=1.2 Hz, 1H, Ar-H),
7.95 (d, 3JHH=8.0 Hz, 1H, Ar-H), 7.81 (dt, 3JHH=8.8 Hz, 4JHH=1.6 Hz,
2H, Ar-H), 4.11 (s, 3H, OCH3), 2.43 (s, 3H, COCH3). 13C NMR (100
MHz, CDCl3) δ: 168.8, 166.9, 143.3, 141.2, 140.3, 134.3, 133.9, 132.7,
132.2, 132.1, 131.4, 129.1, 123.7, 116.4, 52.7, 25.1. HRMS: m/z 296.1031.
Calcd for C16H13N3O3: 296.1030 [M þ H]þ.
Data for VI-6b. Yield: 35.7%; yellow solid; mp, 165-167 °C (ethyl
1
acetate/petroleum ether). H NMR (400 MHz, CDCl3) δ: 10.03 (s, 1H,
NHCO), 8.77 (d, 3JHH=7.6 Hz, 1H, Ar-H), 8.22 (d, 3JHH=6.8 Hz, 1H,
Ar-H), 8.18 (d, 3JHH=8.4 Hz, 1H, Ar-H), 7.99 (d, 3JHH=8.8 Hz, 1H,
Ar-H), 7.82 (dt, 3JHH=7.2 Hz, 4JHH=2.4 Hz, 2H, Ar-H),4.89 (s, 2H,
OCH2), 4.11 (s, 3H, OCH3), 2.42 (s, 3H, COCH3). 13C NMR (100 MHz,
CDCl3) δ: 169.3, 166.8, 165.4, 143.2, 141.2, 140.3, 134.3, 132.8, 132.6,
132.2, 132.0, 131.5, 129.3, 124.5, 116.8, 63.3, 52.7, 20.8. HRMS: m/z
376.0899. Calcd for C18H15N3O5: 376.0904 [M þ Na]þ.
Data for V-7c. Yield: 10.6%; red solid; mp, 140-142 °C (ethyl acetate/
petroleum ether). 1H NMR (300 MHz, CDCl3) δ: 8.14 (dd, 3JHH=7.2 Hz,
4JHH=1.2 Hz, 1H, Ar-H), 7.97 (dd, 3JHH=6.9 Hz, 3JHH=1.2 Hz, 1H,
Ar-H), 7.61 (dd, 3JHH =7.2 Hz, 4JHH=1.2 Hz, 1H, Ar-H), 7.28 (s, 1H,
Ar-H), 7.04 (s, 1H, Ar-H), 4.72 (s, 2H, NH2), 4.39 (t, 3JHH=6.6 Hz, 2H,
3
OCH2), 4.13 (t, JHH =6.6 Hz, 2H, OCH2), 1.85-1.92 (m, 2H, CH2),
1.77-1.85 (m, 2H, CH2), 1.04-1.06 (m, 6H, 2CH3). 13C NMR (100 MHz,
CDCl3) δ: 166.2, 151.8, 142.3, 141.6, 141.0, 140.6, 137.7, 131.2, 129.6,
128.1, 126.1, 104.6, 102.7, 69.6, 65.9, 21.2 (2C), 9.6, 9.5. HRMS: m/z
340.1653. Calcd for C19H21N3O3: 340.1656 [M þ H]þ.
Data for VI-6c. Yield: 80.3%; orange solid; mp, 158-160 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 9.70 (s, 1H,
NHCO), 8.78 (d, 3JHH=7.6 Hz, 1H, Ar-H), 8.32 (d, 3JHH=8.4 Hz, 1H,
Ar-H), 8.22 (d, 3JHH=6.8 Hz, 1H, Ar-H), 7.96 (d, 3JHH=8.8 Hz, 1H,
Ar-H), 7.82 (t, 3JHH=8.0 Hz, 2H, Ar-H), 4.11 (s, 3H, OCH3), 3.75 (t,
3JHH=6.4 Hz, 2H, CH2Cl), 2.85 (t, 3JHH=6.8 Hz, 2H, CH2CO), 2.29-2.35
(m, 2H, CH2). 13C NMR (100 MHz, CDCl3) δ: 170.4, 166.9, 143.3, 141.2,
140.3, 134.3, 133.7, 132.8, 132.1 (2C), 131.4, 129.1, 123.9, 116.4, 52.7, 44.4,
34.5, 27.8. HRMS: m/z 380.0774. Calcd for C18H16ClN3O3: 380.0772 [M þ
Na]þ.
Data for V-7d. Yield: 30.2%; red solid; mp, 134-136 °C (ethyl acetate/
petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.20 (d, 3JHH=8.0 Hz,
1H, Ar-H), 8.04 (d, 3JHH=7.2 Hz, 1H, Ar-H), 7.68 (dd, 3JHH =8.4 Hz,
3JHH=7.2 Hz, 1H, Ar-H), 7.35 (s, 1H, Ar-H), 7.11 (s, 1H, Ar-H), 4.83
(s, 2H, NH2), 4.51 (t, 3JHH=6.8 Hz, 2H, OCH2), 4.23 (t, 3JHH=6.4 Hz, 2H,
OCH2), 1.82-1.92 (m, 4H, 2CH2), 1.54-1.60 (m, 4H, 2CH2), 1.00-1.03
(m, 6H, 2CH3). 13C NMR (100 MHz, CDCl3) δ: 167.3, 152.9 143.4, 142.5,
142.0, 141.6, 138.7, 132.2, 130.7, 129.1, 127.2, 105.6, 103.7, 68.9, 65.2, 30.8
(2C), 19.3 (2C), 13.8, 13.7. HRMS: m/z 390.1786. Calcd for C21H25N3O3:
390.1788 [M þ Na]þ.
Data for VI-6d. Yield: 90.1%; yellow solid; mp, 191-193 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 9.60 (s, 1H,
NHCO), 8.82 (d, 3JHH=6.8 Hz, 1H, Ar-H), 8.25 (d, 3JHH=8.4 Hz, 1H,
Ar-H), 8.20 (d, 3JHH=6.0 Hz, 1H, Ar-H), 7.96 (d, 3JHH=8.4 Hz, 1H,
Ar-H), 7.82 (t, 3JHH=8.0 Hz, 2H, Ar-H), 7.31 (brs, 4H, Ar-H), 7.19
(brs, 1H, Ar-H), 4.10 (s, 3H, OCH3), 3.17 (t, 3JHH=7.2 Hz, 2H, CH2CO),
Data for V-8d. Yield: 14.6%; red solid; mp, 198-200 °C (ethyl acetate/
petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.13 (d, 3JHH=8.4 Hz,
1H, Ar-H), 7.99 (d, 3JHH=6.8 Hz, 1H, Ar-H), 7.57 (t, 3JHH=7.2 Hz, 1H,
Ar-H), 7.18 (s, 1H, Ar-H), 7.13 (s, 1H, Ar-H), 4.70 (s, 2H, NH2), 4.43 (t,
3JHH=6.4 Hz, 2H, OCH2), 4.17 (t, 3JHH=6.4 Hz, 2H, OCH2), 1.83-1.86
(m, 2H, CH2), 1.75-1.78 (m, 2H, CH2), 1.48-1.54 (m, 4H, 2CH2),
0.94-0.99 (m, 6H, 2CH3). 13C NMR (100 MHz, CDCl3) δ: 166.4, 152.0
142.1, 142.0, 140.4, 139.4, 138.9, 131.3, 129.6, 128.9, 125.2, 103.9, 103.5,
67.8, 64.2, 29.8 (2C), 18.3 (2C), 12.8 (2C). HRMS: m/z 368.1970. Calcd for
C21H25N3O3: 368.1969 [M þ H]þ.
3
2.96 (t, JHH=7.2 Hz, 2H, Ar-CH2). 13C NMR (100 MHz, CDCl3) δ:
170.8, 166.9, 143.3, 141.1, 140.6, 140.3, 134.3, 133.8, 132.8, 132.3, 132.2,
131.3, 129.1, 128.6 (2C), 128.4 (2C), 126.4, 123.7, 116.4, 52.8, 39.7, 31.3.
HRMS: m/z 386.1501. Calcd for C23H19N3O3: 386.1499 [M þ H]þ.
Data for VI-6e. Yield: 92.0%; yellow solid; mp, 226-228 °C (ethyl
1
acetate/petroleum ether). H NMR (400 MHz, CDCl3) δ: 10.63 (s, 1H,
NHCO), 8.98 (d, 3JHH=7.2 Hz, 1H, Ar-H), 8.33 (d, 3JHH=8.4 Hz, 1H,
Ar-H), 8.25 (d, 3JHH=6.4 Hz, 1H, Ar-H), 8.06 (d, 3JHH=8.8 Hz, 1H,
Ar-H), 7.92 (t, 3JHH=7.2 Hz, 2H, Ar-H), 7.85 (d, 3JHH=8.0 Hz, 1H,
Ar-H), 7.56 (s, 1H, Ar-H), 7.43 (d, 3JHH=8.0 Hz, 1H, Ar-H), 4.11 (s,
3H, OCH3). 13C NMR (100 MHz, CDCl3) δ: 166.9, 163.6, 143.3, 141.4,
140.5, 137.6, 134.6, 133.8, 133.2, 132.9, 132.3, 132.1, 131.9 (2C), 131.4,
130.5, 129.3, 127.8, 124.6, 117.1, 52.7. HRMS: m/z 426.0408. Calcd for
C21H13Cl2N3O3: 426.0407 [M þ H]þ.
General Synthetic Procedures for Target Compounds VI-6a-VI-
7n. Methyl-6-aminophenazine-1-carboxylate (IV-6a, 50.0 mg, 0.2 mmol)
was dissolved in the solution of dry CH2Cl2 (4.0 mL) and anhydrous
pyridine (0.2 mL). Acetyl chloride (0.2 mL) diluted in CH2Cl2 (6.0 mL)
was added dropwise to the above solution. The mixture was stirred under
15 °C for 0.5 h, and then 20 mL of CH2Cl2 was added, followed by washing
with dilute 2 N hydrochloric acid, saturated sodium bicarbonate and water
respectively. Separating the organic layer, the solvent was removed by
distillation. The residue was recrystallized with ethyl acetate/petroleum
ether (60-90 °C) to give methyl-6-acetamidophenazine-1-carboxylate (VI-
6a, 23.0 mg, 40.0%). Compounds VI-6b-VI-6e, VI-6h and VI-7h were
prepared with corresponding acyl chloride through the same process.
Compounds VI-6f and VI-6g were prepared as follows: Methyl-6- ami-
nophenazine-1-carboxylate (IV-6a, 50.0 mg, 0.2 mmol) and 4-nitroben-
zene-1-sulfonyl chloride (88 mg, 0.4 mmol) were dissolved in a solution of
CH2Cl2 (20.0 mL) and pyridine (0.2 mL). The mixture was refluxed for 1 h,
and then 20 mL of CH2Cl2 was added, followed by washing with dilute 2 N
hydrochloric acid, saturated sodium bicarbonate and water respectively.
Leaving the organic layer, the solvent was removed by distillation. The
residue was recrystallized with ethyl acetate/petroleum ether (60-90 °C) to
give methyl 6-(4-nitrophenylsulfonamido) phenazine-1-carboxylate (VI-
6f, 52.0 mg, 60%). To be pointed out, in the process of preparing
compounds VI-7a-VI-7e and VI-7j-VI-7n, methyl-7-aminophenazine-
1-carboxylate (IV-7a) was dissolved only in the anhydrous pyridine. The
rest of the procedures were the same as preparing for compound VI-6a. In
the process of preparing compounds VI-7f, VI-7g and VI-7i, compound
IV-7a and the corresponding sulfonyl chloride were refluxed in anhydrous
pyridine (11). The other procedures were the same as preparing for
compound VI-6f. All the substituents at the phenazine rings are listed in
Scheme 2 and Table 1.
Data for VI-6f. Yield: 54.2%; yellow solid; mp, 226-228 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, d6-DMSO) δ: 11.12 (s, 1H,
NHSO2), 8.29-8.31 (brd, 3JHH=8.4 Hz, 3H, Ar-H), 8.25 (d, 3JHH=6.8
Hz, 1H, Ar-H), 8.20 (d, 3JHH=8.8 Hz, 2H, Ar-H), 8.00-8.04 (m, 2H,
Ar-H), 7.96 (t, 3JHH=7.2 Hz, 1H, Ar-H), 7.87 (d, 3JHH=6.8 Hz, 1H,
Ar-H), 3.99 (s, 3H, OCH3). 13C NMR (100 MHz, d6-DMSO) δ: 166.5,
149.7, 145.2, 142.6, 140.6, 139.9, 136.4, 133.2, 132.1, 131.8, 131.6 (2C),
130.2, 128.5 (2C), 125.9, 124.3 (2C), 121.5, 52.5. HRMS: m/z 439.0708.
Calcd for C20H14N4O6S: 439.0707 [M þ H]þ.
Data for VI-6g. Yield: 83.3%; brown solid; mp, 192-194 °C (ethyl
1
acetate/petroleum ether). H NMR (400 MHz, CDCl3) δ: 10.00 (s, 1H,
NHSO2), 8.37 (d, 3JHH=8.8 Hz, 1H, Ar-H), 8.23 (d, 3JHH=9.2 Hz, 1H,
Ar-H), 8.10-8.13 (m, 1H, Ar-H), 8.08 (d, 3JHH=7.2 Hz, 1H, Ar-H),
8.01 (d, 3JHH=9.2 Hz, 1H, Ar-H), 7.85 (d, 3JHH=8.0 Hz, 1H, Ar-H),
7.82 (d, 3JHH=8.0 Hz, 2H, Ar-H), 7.59-7.61 (m, 2H, Ar-H), 4.07 (s, 3H,
OCH3). 13C NMR (100 MHz, CDCl3) δ: 166.7, 147.9, 143.3, 141.5, 140.9,
134.8, 134.1, 133.3, 132.9, 132.7, 132.6, 132.5, 131.3, 131.2, 131.1, 129.6,
125.7, 125.4, 116.3, 52.7. HRMS: m/z 439.0705. Calcd for C20H14N4O6S:
439.0707 [M þ H]þ.
Data for VI-6h. Yield: 37.0%; yellow solid; mp, 101-103 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 9.76 (s, 1H,
NHCO), 8.81 (d, 3JHH=7.2 Hz, 1H, Ar-H), 8.36 (d, 3JHH=8.8 Hz, 1H,
Ar-H), 8.22 (d, 3JHH=6.8 Hz, 1H, Ar-H), 7.96 (d, 3JHH=8.8 Hz, 1H,
Ar-H), 7.87 (d, 3JHH=6.8 Hz, 1H, Ar-H), 7.83 (d, 3JHH=7.6 Hz, 1H,
Data for VI-6a. Yield: 79.3%; yellow solid; mp, 212-214 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 9.66 (s, 1H,

3656 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Wang et al.
3
3
Ar-H), 4.54 (t, JHH=6.8 Hz, 2H, OCH2), 3.75 (t, JHH=6.4 Hz, 2H,
CH2Cl), 2.84-2.88 (m, 2H, CH2CO), 2.31-2.34 (m, 2H, CH2), 1.84-1.88
(m, 2H, CH2), 1.58-1.64 (m, 2H, CH2), 1.04 (t, 3JHH=7.2 Hz, 3H, CH3).
13C NMR (100 MHz, CDCl3) δ: 170.4, 166.8, 143.3, 141.3, 140.4, 134.3,
133.8, 132.6, 132.1 (2C), 131.8, 129.3, 123.8, 116.4, 65.6, 44.4, 34.5, 30.8,
27.8. 19.3, 13.7. HRMS: m/z 400.1419. Calcd for C21H22ClN3O3: 400.1422
[M þ H]þ.
118.1, 52.8. HRMS: m/z 461.0531. Calcd for C20H14N4O6S: 461.0526 [M
þ Na]þ.
Data for VI-7h. Yield: 40.1%; yellow solid; mp, 200-202 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.44 (s, 1H,
NHCO), 8.29 (d, 3JHH=8.8 Hz, 1H, Ar-H), 8.15 (d, 3JHH=7.2 Hz, 1H,
Ar-H), 8.13 (d, 3JHH=9.2 Hz, 1H, Ar-H), 8.11 (d, 3JHH=6.0 Hz, 1H,
Ar-H), 7.94 (dd, 3JHH=8.0 Hz, 4JHH=1.2 Hz, 1H, Ar-H), 7.82 (dd, 3JHH
=8.0 Hz, 3JHH=7.2 Hz, 1H, Ar-H), 4.53 (t, 3JHH=6.8 Hz, 2H, OCH2),
3.67 (t, 3JHH=6.0 Hz, 2H, CH2Cl), 2.65-2.68 (m, 2H, CH2CO), 2.22-2.25
(m, 2H, CH2), 1.83-1.87 (m, 2H, CH2), 1.57-1.62 (m, 2H, CH3), 1.03 (t,
3JHH=7.2 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 170.6, 167.1,
143.9, 143.1, 141.3, 140.2, 139.8, 132.9, 131.8, 131.0, 130.9, 129.2, 126.2,
115.2, 65.6, 44.3, 34.2, 30.8, 27.7. 19.3, 13.8. HRMS: m/z 400.1423. Calcd
for C21H22ClN3O3: 400.1422 [M þ H]þ.
Data for VI-7a. Yield: 34.5%; yellow solid; mp, 234-236 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.40 (s, 1H,
NHCO), 8.25 (d, 3JHH=8.8 Hz, 1H, Ar-H), 8.12-8.14 (brd, 3JHH=8.4
Hz, 2H, Ar-H), 7.95 (s, 1H, Ar-H), 7.87 (d, 3JHH=8.8 Hz, 1H, Ar-H),
3
7.75 (t, JHH = 7.2 Hz, 1H, Ar-H), 4.03 (s, 3H, OCH3), 2.22 (s, 3H,
COCH3). 13C NMR (100 MHz, CDCl3) δ: 167.8, 166.2, 143.0, 142.1,
140.3, 139.1 (2C), 132.0, 130.3, 130.2, 130.0, 128.1, 125.2, 113.9, 51.7, 23.8.
HRMS: m/z 318.0847. Calcd for C16H13N3O3: 318.0849 [M þ Na]þ.
Data for VI-7b. Yield: 50.1%; yellow solid; mp, 206-208 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.56 (s, 1H,
NHCO), 8.28 (brs, 4H, Ar-H), 7.85-7.96 (brd, 2H, Ar-H), 4.80 (s, 2H,
OCH2), 4.11 (s, 3H, OCH3), 2.28 (s, 3H, COCH3). 13C NMR (100 MHz,
CDCl3) δ: 169.3, 167.1, 165.5, 150.9, 143.9, 143.2, 141.5, 140.4, 138.7,
133.2, 131.6, 131.4, 129.3, 126.0, 116.1, 63.4, 52.7, 20.8. HRMS: m/z
376.0902. Calcd for C18H15N3O5: 376.0904 [M þ Na]þ.
Data for VI-7i. Yield: 35.5%; yellow solid; mp, 183-185 °C (ethyl
acetate/petroleum ether). 1H NMR (300 MHz, CDCl3) δ: 8.22 (d, 3JHH
=
8.7 Hz, 1H, Ar-H), 8.09 (d, 3JHH=6.3 Hz, 1H, Ar-H), 8.07 (d, 3JHH=6.9
Hz, 1H, Ar-H), 7.75-7.78 (brd, 4H, Ar-H), 7.62 (d, 3JHH=8.7 Hz, 1H,
Ar-H), 7.13 (d, 3JHH=8.1 Hz, 2H, Ar-H), 4.01 (s, 3H, OCH3), 2.23 (s,
3H, Ar-CH3). 13C NMR (100 MHz, CDCl3) δ: 166.0, 143.5, 141.7, 141.0,
139.1, 134.5, 131.2, 130.8, 130.5, 130.3, 128.9 (2C), 126.4 (2C), 125.0,
123.4, 122.9, 118.0, 112.4, 51.7, 20.5. HRMS: m/z 430.0839. Calcd for
C21H17N 3O4S: 430.0832 [M þ Na]þ.
Data for VI-7c. Yield: 47.5%; brown solid; mp, 198-200 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.49 (s, 1H,
NHCO), 8.45 (s, 1H, Ar-H), 8.31 (d, 3JHH=8.4 Hz, 1H, Ar-H), 8.12 (d,
3JHH=7.2 Hz, 1H, Ar-H), 8.10 (d, 3JHH=9.2 Hz, 1H, Ar-H), 8.00 (d,
3JHH=9.2 Hz, 1H, Ar-H), 7.79 (dd, 3JHH=8.4 Hz, 3JHH=7.2 Hz, 1H,
Ar-H), 4.03 (s, 3H, OCH3), 3.58 (t, 3JHH=6.0 Hz, 2H, CH2Cl), 2.62 (t,
3JHH=6.8 Hz, 2H, CH2CO), 2.12-2.15 (m, 2H, CH2). 13C NMR (100
MHz, CDCl3) δ: 171.0, 166.9, 142.3, 141.7, 141.4, 141.1, 140.0, 131.6,
131.4, 131.3, 131.1, 130.1, 126.7, 113.3, 52.8, 44.3, 34.3, 27.6. HRMS: m/z
380.0777. Calcd for C18H16ClN3O3: 380.0772 [M þ Na]þ.
Data for VI-7j. Yield: 45.7%; yellow solid; mp, 202-204 °C (ethyl
1
acetate/petroleum ether). H NMR (400 MHz, CDCl3) δ: 10.22 (s, 1H,
Ar-H), 8.57 (s, 1H, Ar-H), 8.21-8.26 (m, 3H, Ar-H), 8.10 (d, 3JHH=6.8
Hz, 1H, Ar-H), 8.07 (d, 3JHH=8.8 Hz, 1H, Ar-H), 7.76 (t, 3JHH=7.2 Hz,
1H, Ar-H), 7.41 (brs, 1H, Ar-H), 7.07 (brs, 1H, 1H, Ar-H), 6.95 (d, 3JHH
=7.2 Hz, 1H, Ar-H), 4.09 (s, 3H, OCH3), 4.05 (s, 3H, OCH3). 13C NMR
(100 MHz, CDCl3) δ: 167.1, 163.5, 157.1, 144.2, 143.1, 141.4, 140.5, 140.0,
133.7, 133.1, 132.5, 131.3, 131.1, 130.9, 128.9, 127.0, 121.6, 120.9, 115.3,
111.4, 56.2, 52.6. HRMS: m/z 410.1111. Calcd for C22H17N3O4: 410.1111
[M þ Na]þ.
Data for VI-7d. Yield: 65.7%; yellow solid; mp, 215-217 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.44 (s, 1H,
Data for VI-7k. Yield: 20.1%; yellow solid; mp, 238-240 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, d6-DMSO) δ: 10.88 (s, 1H,
NHCO), 8.95 (s, 1H, Ar-H), 8.43 (d, 3JHH=8.4 Hz, 1H, Ar-H), 8.37 (d,
3JHH=8.0 Hz, 1H, Ar-H), 8.29 (d, 3JHH=8.4 Hz, 1H, Ar-H), 8.21 (d,
3
NHCO), 8.33 (d, JHH = 8.0 Hz, 1H, Ar-H), 8.28 (s, 1H, Ar-H),
3
8.12-8.17 (m, 2H, Ar-H), 7.95 (d, JHH=8.0 Hz, 1H, Ar-H), 7.81 (t,
3JHH = 7.2 Hz, 1H, Ar-H), 7.20-7.26 (m, 5H, Ar-H), 4.08 (s, 3H,
OCH3), 3.06 (t, 3JHH=6.4 Hz, 2H, CH2CO), 2.80 (t, 3JHH=6.4 Hz, 2H,
Ar-CH2). 13C NMR (100 MHz, CDCl3) δ: 171.2, 167.1, 143.3, 142.2,
141.5, 140.7, 140.3, 140.0, 132.5, 131.4, 131.2, 131.0, 129.5, 128.6 (2C),
128.4 (2C), 126.7, 126.4, 114.4, 52.8, 39.6, 31.3. HRMS: m/z 408.1322.
Calcd for C23H19N3O3: 408.1319 [M þ Na]þ.
3JHH=6.0 Hz, 1H, Ar-H), 8.02-8.04 (brd, 3H, Ar-H), 7.47 (d, 3JHH
=
6.4 Hz, 2H, Ar-H), 4.06 (s, 3H, OCH3), 2.48 (s, 3H, CH3). 13C NMR (100
MHz, d6-DMSO) δ: 166.9, 166.4, 155.0, 143.8, 142.4, 142.2, 141.5, 140.4,
138.8, 132.0, 131.5, 130.1, 129.8, 129.7, 129.0 (2C), 127.9 (2C), 127.8,
114.4, 52.5, 21.0. HRMS: m/z 394.1159. Calcd for C22H17N3O3: 394.1162
[M þ Na]þ.
Data for VI-7e. Yield: 30.1%; yellow solid; mp, 246-248 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 11.27 (s, 1H,
NHCO), 8.83 (s, 1H, Ar-H), 8.38 (d, 3JHH=8.4 Hz, 1H, Ar-H), 8.25 (d,
3JHH=9.6 Hz, 1H, Ar-H), 8.16 (d, 3JHH=6.0 Hz, 1H, Ar-H), 8.12 (d,
3JHH=8.8 Hz, 1H, Ar-H), 7.99 (dd, 3JHH=7.6 Hz, 3JHH=6.8 Hz, 1H,
Ar-H), 7.85 (s, 1H, Ar-H), 7.79 (d, 3JHH=8.0 Hz, 1H, Ar-H), 7.64 (d,
3JHH=7.2 Hz, 1H, Ar-H), 4.01 (s, 3H, OCH3). 13C NMR (100 MHz,
CDCl3) δ: 166.8, 165.0, 143.7, 142.5, 140.8, 140.5, 139.0, 135.3, 135.1,
132.0 (2C), 131.2, 130.5, 130.3 (2C), 129.9, 129.3, 127.5, 126.9, 114.4, 52.5.
HRMS: m/z 426.0408. Calcd for C21H13Cl2N3O3: 426.0407 [M þ H]þ.
Data for VI-7f. Yield: 38.0%; green solid; mp, 235-237 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, d6-DMSO) δ: 11.68 (s, 1H,
NHSO2), 8.38 (d, 3JHH=8.4 Hz, 2H, Ar-H), 8.29 (d, 3JHH=8.4 Hz, 1H,
Ar-H), 8.19 (d, 3JHH=8.4 Hz, 2H, Ar-H), 8.16 (d, 3JHH=6.8 Hz, 1H,
Ar-H), 8.12 (d, 3JHH=6.8 Hz, 1H, Ar-H), 7.94 (t, 3JHH=8.0 Hz, 1H,
Ar-H), 7.82 (s, 1H, Ar-H), 7.78 (d, 3JHH=8.8 Hz, 1H, Ar-H), 3.95 (s,
3H, OCH3). 13C NMR (100 MHz, d6-DMSO) δ: 166.7, 150.1, 144.2, 143.1,
142.3, 140.2, 139.7, 139.0, 131.9, 131.2, 130.5, 130.1, 128.3 (2C), 126.2,
124.9 (3C), 112.9, 52.5. HRMS: m/z 461.0528. Calcd for C20H14N4O6S:
461.0526 [M þ Na]þ.
Data for VI-7l. Yield: 41.9%; yellow solid; mp, 100-102 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.53 (s, 1H,
NHCO), 8.42 (s, 1H, Ar-H), 8.16 (d, 3JHH=8.8 Hz, 1H, Ar-H), 8.06 (d,
3JHH=6.8 Hz, 1H, Ar-H), 8.00 (d, 3JHH=8.8 Hz, 1H, Ar-H), 7.92 (d,
3JHH=8.8 Hz, 1H, Ar-H), 7.69 (t, 3JHH=8.0 Hz, 1H, Ar-H), 4.00 (s, 3H,
OCH3), 2.21-2.27 (m, 1H, cyclohexyl-CH), 1.81-1.87 (m, 2H, cyclohex-
yl-CH2), 1.65-1.67 (m, 2H, cyclohexyl-CH2), 1.41-1.52 (m, 3H, cyclo-
hexyl-CH2), 1.07-1.09 (m, 3H, cyclohexyl-CH2). 13C NMR (100 MHz,
CDCl3) δ: 174.5, 166.2, 142.8, 141.7, 140.2, 139.7, 138.8, 131.9, 130.2,
130.0, 129.5, 128.0, 125.9, 113.8, 51.7, 45.4, 28.4 (2C), 24.5 (3C). HRMS:
m/z 386.1482. Calcd for C21H21N3O3: 386.1475 [M þ Na]þ.
Data for VI-7m. Yield: 21.7%; orange solid; mp, 60-62 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.63 (s, 1H,
NHCO), 8.51 (d, 4JHH=2.0 Hz, 1H, Ar-H), 8.25 (d, 3JHH=8.8 Hz, 1H,
Ar-H), 8.18 (d, 3JHH=9.2 Hz, 1H, Ar-H), 8.10 (d, 3JHH=6.8 Hz, 1H,
Ar-H), 7.99 (dd, 3JHH=9.2 Hz, 4JHH=2.0 Hz, 1H, Ar-H), 7.79 (d, 3JHH
=
8.0 Hz, 1H, Ar-H), 7.75 (dd, 3JHH=7.2 Hz, 3JHH=6.8 Hz, 1H, Ar-H),
7.44 (d, 3JHH=8.0 Hz, 1H, Ar-H), 7.25 (t, 3JHH=8.0 Hz, 1H, Ar-H), 7.10
(d, 3JHH=8.8 Hz, 1H, Ar-H), 4.02 (s, 3H, OCH3), 2.28 (s, 3H, COCH3).
13C NMR (100 MHz, CDCl3) δ: 168.3, 166.1, 163.1, 146.9, 143.0, 142.1,
140.4, 139.2, 138.9, 132.1, 131.6, 130.4, 130.3, 129.9, 128.8, 128.2, 127.0,
125.5, 125.1, 122.4, 114.4, 51.7, 20.1. HRMS: m/z 416.1248. Calcd for
C23H17N3O5: 416.1241 [M þ H]þ.
Data for VI-7g. Yield: 37.1%; orange solid; mp, 199-201 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 8.30 (d, 3JHH
=
8.4 Hz, 1H, Ar-H), 8.25 (d, 3JHH=9.6 Hz, 1H, Ar-H), 8.20 (d, 3JHH=7.2
Hz, 1H, Ar-H), 7.99-8.01 (brd, 2H, Ar-H), 7.85-7.87 (brd, 2H,
3
3
Ar-H), 7.82 (d, JHH =10.0 Hz, 1H, Ar-H), 7.67 (t, JHH =7.6 Hz,
Data for VI-7n. Yield: 47.7%; yellow solid; mp, 246-248 °C (ethyl
1H, Ar-H), 7.56 (t, 3JHH=7.6 Hz, 1H, Ar-H), 4.09 (s, 3H, OCH3). 13
C
acetate/petroleum ether). 1H NMR (400 MHz, d6-DMSO) δ: 11.48 (s, 1H,
3
NMR (100 MHz, CDCl3) δ: 166.9, 148.1, 143.3, 143.1, 141.7, 140.6, 138.1,
134.5, 133.0, 132.8, 132.1, 131.9, 131.8, 131.7, 131.5, 129.7, 127.0, 125.6,
NHCO), 8.67 (s, 1H, Ar-H), 8.38 (d, JHH = 8.8 Hz, 1H, Ar-H),
8.23-8.29 (m, 2H, Ar-H), 8.18 (d, JHH = 6.4 Hz, 1H, Ar-H), 7.99
3

Article
J. Agric. Food Chem., Vol. 58, No. 6, 2010 3657
(dd, 3JHH=8.0 Hz, 3JHH=7.2 Hz, 1H, Ar-H), 4.00 (s, 3H, OCH3). 13
NMR (100 MHz, d6-DMSO) δ: 166.7, 160.2, 143.1, 142.4, 140.6, 139.6,
139.3, 132.1, 131.9, 130.8, 130.2, 130.0, 127.2, 116.8, 51.5. HRMS: m/z
419.9688. Calcd for C16H10Cl3N3O3: 419.9680 [M þ Na]þ.
C
Table 2. Fungicidal Activity of the Compounds III-a-VII-e at Dosage of
50 μg mL-1a
inhibition (%)
compd
A. solani C. arachidicola F. omysporum G. zeae P. piricola
General Synthetic Procedures for Target Compounds VII-a-
VII-e. At 0 °C, 65% nitric acid (0.2 mL) was dropped to the solution of
VI-6c (80 mg, 0.22 mmol) in 98% sulfuric acid (10.0 mL). The mixture was
stirred and kept at 0 °C for 2 h, then poured onto crushed ice (20 g),
and extracted with dichloromethane (2 ꢀ 20 mL), followed by washing
with saturated sodium bicarbonate and water respectively. The solvent of
the organic layer was removed (12). The residue was recrystallized with
ethyl acetate/petroleum ether (60-90 °C) to give methyl 6-amino-7,9-
dinitrophenazine-1-carboxylate (VII-b, 40.0 mg, 53.3%). Two recrystalli-
zations from ethyl acetate/petroleum ether (60-90 °C) gave methyl-6-(4-
chlorobutanamido)-7-nitrophenazine-1-carboxylate (VII-a, 5.0 mg,
5.5%). Compounds VII-c and VII-d were gained from VI-6h with the
same process as above. Compound VII-e was the only product from VI-7h.
The structures of VII-c, VII-d and VII-e were testified by the 2D-NMR
spectra. All the substituents at the phenazine rings are listed in Scheme 2
and Table 1.
III-a
III-b
III-c
0
7.7
11.1
11.5
26.9
32.0
30.0
8.3
13.8
2.7
12.9
1.6
19.2
4.9
5.1
5.9
6.9
33.9
36.3
41.8
43.6
2.7
9.6
IV-6a
IV-6b
IV-6c
IV-6d
IV-7a
IV-7b
IV-7c
IV-7d
IV-8a
IV-8b
IV-8c
IV-8d
V-7b
100
40.0
33.3
36.7
21.4
10.8
31.0
10.0
13.3
23.3
16.7
33.3
26.7
6.9
100
100
57.1
51.0
46.7
24.2
33.3
29.4
16.7
43.3
10.0
20.0
20.2
23.3
17.6
20.0
13.3
17.4
13.8
10.3
13.8
4.3
15.6
25.0
44.2
19.6
29.6
37.1
51.4
48.6
34.3
26.9
26.5
25.5
34.3
46.4
32.1
41.1
31.4
10.7
17.9
28.6
0
0
7.7
15.1
11.3
25.5
7.3
26.1
17.4
4.0
18.2
20.0
27.3
23.6
14.5
20.0
23.6
42.4
18.2
22.7
25.0
30.3
27.3
31.8
43.2
0
12.0
20.0
24.0
11.5
21.7
13.0
16.7
8.3
Data for VII-a. Yield: 5.5%; orange solid; mp, beyond 340 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 9.95 (s, 1H,
NHCO), 8.90 (d, 3JHH=8.4 Hz, 1H, Ar-H), 8.56 (d, 3JHH=8.4 Hz, 1H,
Ar-H), 8.44 (d, 3JHH=8.0 Hz, 1H, Ar-H), 8.40 (d, 3JHH=6.0 Hz, 1H,
Ar-H), 8.02 (dd, 3JHH=8.0 Hz, 3JHH=7.2 Hz, 1H, Ar-H), 4.16 (s, 3H,
OCH3), 3.77 (brs, 2H, CH2Cl), 2.93 (brs, 2H, CH2CO), 2.34 (brs, 2H,
CH2). 13C NMR (100 MHz, CDCl3) δ: 170.9, 166.9, 141.8, 140.4 (2C),
138.5, 135.6, 134.4, 133.9, 133.6, 132.5, 131.3, 130.0, 113.1, 53.0, 44.2, 34.5,
27.5. HRMS: m/z 426.0620. Calcd for C18H15ClN4O5: 425.0623 [M þ
Na]þ.
V-7c
V-7d
0
0
V-8d
20.0
14.3
21.4
0
VI-6a
VI-6b
VI-6c
VI-6d
VI-6e
VI-6f
0
0
0
28.0
7.1
3.4
0
13.8
3.4
25.0
0
17.9
0
Data for VII-b. Yield: 53.3%; orange solid; mp, 257-259 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, d6-DMSO) δ: 9.66-9.77
(brd, 2H, NH2) 9.14 (s, 1H, Ar-H), 8.44 (d, 3JHH=8.4 Hz, 1H, Ar-H),
8.37 (d, 3JHH=6.8 Hz, 1H, Ar-H), 8.11 (t, 3JHH=7.2 Hz, 1H, Ar-H), 3.97
(s, 3H, OCH3). 13C NMR (100 MHz, d6-DMSO) δ: 166.4, 148.7, 140.7,
138.9, 137.9, 135.3, 133.9, 132.3, 132.0, 131.7, 131.4, 127.8, 121.4, 52.4.
HRMS: m/z 366.0442. Calcd for C14H9N5O6: 366.0445 [M þ Na]þ.
Data for VII-c. Yield: 18.0%; yellow solid; mp, 141-143 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 9.85 (s, 1H,
NHCO), 8.80 (d, 3JHH=8.8 Hz, 1H, Ar-H), 8.41 (d, 3JHH=8.4 Hz, 1H,
Ar-H), 8.33 (d, 3JHH=8.8 Hz, 1H, Ar-H), 8.27 (d, 3JHH=6.8 Hz, 1H,
Ar-H), 7.92 (t, 3JHH=8.0 Hz, 1H, Ar-H), 4.48 (t, 3JHH=7.2 Hz, 2H,
OCH2), 3.69 (t, 3JHH=6.0 Hz, 2H, CH2Cl), 2.85 (t, 3JHH=7.2 Hz, 2H,
CH2CO), 2.25-2.28 (m, 2H, CH2), 1.79-1.82 (m, 2H, CH2), 1.41-1.46
VI-6g
VI-6h
VI-7a
VI-7b
VI-7c
VI-7d
VI-7e
VI-7f
6.1
3.3
8.3
10.7
0
0
4.0
12.7
4.8
3.9
1.8
6.5
5.3
0
37.7
19.1
3.0
7.7
2.6
36.8
2.4
0
11.1
7.1
0
0
8.0
0
0
6.5
15.8
6.7
0
18.2
2.0
VI-7g
VI-7h
VI-7i
13.3
6.1
10.9
8.3
12.7
0
3.6
0
8.0
33.3
6.5
22.2
24.0
0
13.5
2.6
17.5
13.1
14.6
9.8
VI-7j
23.6
0
VI-7k
VI-7l
19.1
23.4
17.2
0
0
12.8
4.2
11.1
10.7
5.3
7.4
3
(m, 2H, CH2), 0.91 (t, JHH=7.2 Hz, 3H, CH3). 13C NMR (100 MHz,
VI-7m
VI-7n
VII-a
36.4
4.8
33.9
21.8
34.4
50.0
37.1
42.9
16.1
CDCl3) δ: 169.8, 165.6, 140.8, 139.7, 139.4, 137.3, 134.6, 132.5, 132.0,
131.6, 131.2, 130.2, 128.4, 112.0, 65.3, 43.2, 33.5, 29.6, 26.5, 18.2, 12.7.
HRMS: m/z 467.1100. Calcd for C21H21ClN4O5: 467.1093 [M þ Na]þ.
Data for VII-d. Yield: 51.9%; orange solid; mp, 176-178 °C (ethyl
acetate/petroleum ether). 1H NMR (400 MHz, CDCl3) δ: 9.30 (s, 1H, H-
8), 9.01 (s, 1H, NH-R), 8.40 (brs, 1H, NH-β), 8.40 (brs, 1H, H-4), 8.40 (brs,
12.0
35.0
22.2
11.1
5.6
20.5
21.7
17.4
17.4
17.2
16.3
32.0
16.0
24.0
17.9
65.4
40.4
57.6
51.5
53.0
25.0
43.1
VII-b
VII-c
VII-d
VII-e
12.5
92.3
1H, H-2), 8.02 (dd, 3JHH=7.2 Hz, 3JHH=6.8 Hz, 1H, H-3), 4.54 (t, 3JHH
=
phenazine-
1-carboxylic
acid
100
100
5.6 Hz, 2H, CH2-20), 1.85-1.88 (m, 2H, CH2-30), 1.48-1.51 (m, 2H, CH2-
40), 0.99 (t, 3JHH=6.4 Hz, 3H, CH3-50). 13C NMR (100 MHz, CDCl3) δ:
166.2 (s, C-10), 147.2 (s, C-9), 142.5 (s, C-5a), 139.9 (s, C-4a), 138.1 (s, C-7),
135.0 (d, C-4), 134.1 (s, C-6), 133.7 (s, C-10a), 132.9 (s, C-1), 132.5 (d, C-2),
131.6 (d, C-3), 126.5 (d, C-8), 122.6 (s, C-9a), 66.4 (t, C-20), 30.6 (t, C-30),
19.2 (t, C-40), 13.7 (q, C-50). HRMS: m/z 386.1102. Calcd for C17H15N5O6:
386.1095 [M þ H]þ.
a The data is the average of three duplicate results.
relative inhibitory ratio (%) had been determined by using the mycelium
growth rate method (13). Phenazine-1-carboxylic acid was used as a
control. After the mycelia grew completely, the diameters of the mycelia
were measured and the inhibition rate was calculated according to the
formula
Data for VII-e. Yield: 50.8%; yellow solid; mp, 181-183 °C (ethyl
acetate/petroleum ether).1H NMR (400 MHz, CDCl3) δ: 8.33 (d, 3JHH=8.4
Hz, 1H, Ar-H), 8.16 (d, 3JHH=7.2 Hz, 1H, Ar-H), 8.08 (d, 3JHH=9.2 Hz,
1H, Ar-H), 7.84 (dd, 3JHH=8.4 Hz, 3JHH=7.2 Hz, 1H, Ar-H), 7.35 (d,
3JHH=9.2 Hz, 1H, Ar-H), 6.91 (s, 2H, NH2), 4.50 (t, 3JHH=6.4 Hz, 2H,
I ¼ ðD1 -D2Þ=D1ꢀ100%
OCH2), 1.82-1.85 (m, 2H, CH2), 1.56-1.58 (m, 2H, CH2), 1.02 (t, 3JHH
=
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 ratio of those compounds at
the dose of 50 μg mL-1 is summarized in Table 2. The EC50 of compounds
IV-6a, IV-6b and phenazine-1-carboxylic acid had been experimented and
calculated by the Scatchard method. The results are summarized in
Table 3.
7.2 Hz, 3H, CH3). 13C NMR (100MHz, CDCl3) δ: 166.6, 146.8, 142.7, 139.5,
138.7, 138.5, 136.3, 133.1, 131.2, 131.1, 130.1, 125.8, 123.8, 65.5, 30.7, 19.3,
13.7. HRMS: m/z 341.1236. Calcd for C17H16N 4O4,: 341.1244 [M þ H]þ.
Bioassays. The fungicidal activities of the compounds III-a-VII-e
were tested in vitro against Alternaria solani, Cercospora arachidicola,
Fusarium omysporum, Gibberella zeae, Physalospora piricola, and their

3658 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Wang et al.
RESULTS AND DISCUSSION
compounds V-7b-V-8d were testified by 1D NMR and 2D NMR
spectra. One typical compound (V-7b) was elucidated as follows:
The molecular formula of V-7b was revealed as C17H17N3O3 by
HRMS data [M þ Na]þ (found 334.1163, calcd 334.1162). The
1H and 13C NMR (Data for V-7b) spectra showed the signals of
eight quaternary, five CH, two CH2, two CH3 carbon atoms.
Considering the reagents, the HMQC spectra showed as follows:
8.22 (d, H-4), 8.04 (d, H-2), 7.66-7.72 (m, H-3), 7.35 (s, H-9) and
7.13 (s, H-6) belonged to the Ar-H, which was confirmed by 3JHH
Synthesis and Structural Elucidation. In the previous papers
(5, 6), compounds III-a-III-c and IV-6a-IV-8a were synthe-
sized as follows: methyl-3-nitro-2-(3-nitrophenylamino) benzoate
was hydrogenated over PtO2 to give methyl 3-amino-2-(3-
aminophenylamino) benzoate. The above product was refluxed
in nitrobenzene for 30 h to give a mixture of IV-6a and IV-8a
(1:11). Compound III-a was the hydrolysis product of IV-6a. III-c
was the product from 3-nitro-2-(3-nitrophenylamino) benzoic
acid (6). III-b and IV-7a were synthesized by the same author with
the same process in principle (5). The whole procedure was rather
tedious, and the total yield was low.
Compounds III-a-IV-8d were synthesized through an im-
proved procedure referencing to the literature (4-7) (14-16):
Compound II-b with NaBH4 in 2 N NaOH was refluxed for 4-
5 h, cooled, and acidified to give a mixture of III-a and III-c,
which consisted of 50% III-a and 50% III-c, as determined by
1H NMR. The mixture of III-a and III-c was refluxed in CH3-
(CH2)nOH (n=0, 1, 2, 3) with a catalytic amount of H2SO4 to give
IV-6a-IV-6d and IV-8a-IV-8d, which were easily purified by
flash chromatography on silica gel [elution solvent: ethyl acetate/
petroleum ether (60-90 °C)]. Similar procedures were used to
prepare III-b and IV-7a-IV-7d from II-c. Regrettably, com-
pound III-x was the only product from II-a. So the compound
9-aminophenazine-1-carboxylic acid did not come into being.
In the preparation of IV-7a-IV-7d, some unexpected bypro-
ducts (V-7b-V-7d) were isolated by flash chromatography on
silica gel, and the yields increased with increasing value of n in
CH3(CH2)nOH. However, no corresponding byproduct was
isolated when the n was equal to 0. V-8d was the only byproduct
from IV-8d. No corresponding byproduct was isolated when n
was equal to 0, 1, or 2 in the series of IV-8. The structures of
3
= 8.4 Hz, JHH = 6.8 Hz and HMBC spectra; 4.89 (s, 2H)
belonged to NH2; 4.56 (t, CH2-20), 4.29 (t, CH2-100), 1.55 (q,
CH3-200), 1.48 (q, CH3-30) belonged to two side chains, which was
confirmed bythe 3JHH=7.2 Hzand HMBC spectra. The chemical
shift values of C-9a and C-5a in 13C NMR spectra overlapped; the
chemical shift values of C-4a and C-1 in 13C NMR spectra were
not easily differentiated by HMBC spectra, which were men-
tioned by the reference data of V-7c, V-7d and known com-
pound (17).
Based on the HMBC spectra, the correlations between H-4 (δH
=8.22) and C-2, C-10a [δC=129.1 (d, C-2), 138.6 (s, C-10a)], the
correlations between H-2 (δH=8.04) and C-10, C-4, C-10a [δC=
167.0 (s, C-10), 131.9 (d, C-4), 138.6 (s, C-10a)], the correlations
between H-3 (δH=7.66-7.72) and C-1, C-4a [δC=130.5 (s, C-1),
141.2 (s, C-4a)], the correlations between H-9 (δH=7.35) and C-
5a, C-7, C-8, C-9a [δC=142.2 (s, C-5a), 143.6 (s, C-7), 152.8 (s, C-
8), 142.2 (s, C-9a)], the correlations between H-6 (δH=7.13) and
C-8, C-9a [δC=152.8 (s, C-8), 142.2 (s, C-9a)], the correlations
between H-20 (δH=4.56) and C-10, C-30 [δC=167.0 (s, C-10), 14.4
(q, C-30)], the correlations between H-30 (δH=1.48) and C-20 [δC=
61.3 (t, C-20)], the correlations between H-100 (δH=4.29) and C-8,
C-200 [δC =152.8 (s, C-8), 14.5 (q, C-200)], and the correlations
between H-200 (δH=1.55) and C-100 [δC=64.9 (t, C-100)] indicated
the structure of V-7b should be as shown in Figure 2.
In the H-H COSY spectra, the correlations between H-3 (δH=
7.66-7.72) and H-4 (δH=8.22), the correlations between H-3 (δH
=7.66-7.72) and H-2 (δH=8.04) indicated H-4, H-3 and H-2
were in nearby positions. The correlations between H-20 (δH =
4.56) and H-30 (δH=1.48) and the correlations between H-100 (δH
=4.29) and H-200 (δH=1.55) further verified the positions of two
side chains were right in the structure of V-7b. As the possible
synthesis mechanisms to those unexpected byproducts would be
further studied in the future.
Compounds VI-7a-VI-7g and VI-7i-VI-7n were given using
IV-7a with corresponding acyl chloride or sulfonyl chloride. As
the amount of IV-6a was limited, compounds VI-6a-VI-6g were
synthesized using IV-6a with acyl chloride or sulfonyl chloride
which were selected based on the SAR of the VI-7 series.
Considering the high activity of the IV-6 series and the activity
contribution of butan-1-ol to IV-7d and 4-chlorobutanoyl chlo-
ride to VI-7c, compounds VI-6h and VI-7h were designed and
synthesized. In order to know whether the nitryl group on the
phenazine ring improved the fungicidal activity or not, some
nitration derivatives were given from VI-6c, VI-6h and VI-7h. In
the nitration process of VI-6c and VI-6h, both compounds had
Table 3. EC50 of the Compounds IV-6a, IV-6b, and Phenazine-1-carboxylic
Acid
Y = a þ bx
EC50
R
IV-6a
A. solani
Y = 3.913 þ 0.827x
20.53
0.96
C. arachidicola
F. omysporum
G. zeae
>50
>50
>50
P. piricola
Y = 4.329 þ 1.402x
Y = 3.250 þ 1.0908x
3.00
0.96
0.94
IV-6b
A. solani
40.13
>50
C. arachidicola
F. omysporum
G. zeae
>50
>50
P. piricola
phenazine-1-carboxylic acid
A. solani
Y = 4.149 þ 1.312x
4.44
0.98
Y = 4.400 þ 0.472x
Y = 3.185 þ 1.615x
Y = 4.108 þ 0.758x
18.5
0.99
0.96
0.98
C. arachidicola
F. omysporum
G. zeae
13.2
14.9
>50
3.58
P. piricola
Y = 3.445 þ 2.803x
0.99
Figure 2. Key HMBC correlations (left) and key H-H COSY (right) correlations of V-7b.

Article
J. Agric. Food Chem., Vol. 58, No. 6, 2010 3659
Figure 3. Key HMBC correlations (left) and key H-H COSY (right) correlations of VII-d.
two nitration derivatives at 0 °C. One had a single nitryl in the 7-
position, and the other had two nitryls in the 7,9-position with the
carboxamide bond hydrolyzed. Compound IIV-e was the only
nitration product from IV-7h whenever the reaction’s tempera-
ture was kept at 0 or 20 °C. The structures of compounds VII-
a-VII-d were proved by 1D NMR and 2D NMR spectra. One
typical compound (VII-d) was elucidated as follows: The molec-
ular formula of VII-d was revealed as C17H17N3O3 by HRMS
F. omysporum, G. zeae, and P. piricola of the title compounds III-
a-VII-e. Table 3 showed the EC50 of the high fungicidal activity
compounds IV-6a, IV-6b and phenazine-1-carboxylic acid.
Fungicidal Activity against A. solani. The screening data of
Table 2 indicated that, at a dosage of 50 μg mL-1, most
compounds of III-a-VII-e exhibited low activities against A.
solani except the series of compounds IV-6. The inhibition activity
of IV-6a was 100%, which was equal to that of the phenazine-1-
carboxylic acid. The inhibition activity of IV-6b was 51.0%; as the
side chain prolonged, the fungicidal activities of compounds IV-
6c and IV-6d evidently decreased. The series of compounds IV-7
and IV-8 did not show satisfactory fungicidal activity, and their
activity rules seemed to be contrary to the IV-6 series, such as the
typical compounds IV-7d and IV-8d only exhibited 43.4% and
23.3% inhibition respectively. That might be due to the side chain
extension. To be mentioned, compared with the biological
activities of III-a-III-c, the carboxyl group substituted by the
ester group in aminophenazine derivatives had a vital function for
improving the fungicidal activity.
1
data [M þ Na]þ (found 334.1163, calcd 334.1162). The H and
13C NMR (Data for VII-d) spectra showed the signals of nine
quaternary, four CH, three CH2, one CH3 carbon atoms. Con-
sidering the reagents (VI-6h), the HMQC spectra showed as
follows: 9.30 (s, H-8), 8.40 (brs, H-4), 8.40 (brs, H-2), 8.02 (dd,
H-3) belonged to the Ar-H, which was confirmed by 3JHH=7.2
Hz, 3JHH=6.8 Hz (H-3) and HMBC spectra; 9.01 (s, H-R), 8.40
(brs, H-β) belonged to NH2; 4.54 (t, CH2-20), 1.85-1.88 (m, CH2-
30), 1.48-1.51 (m, CH2-40), 0.99 (t, CH3-50) belonged to a side
3
chain, which was confirmed by the JHH value and HMBC
spectra. The chemical shift values of H-4, H-2 and NH-β in 1H
NMR spectra overlapped; the chemical shift values of C-4a and
C-1 in 13C NMR spectra were not easily differentiated by HMBC
spectra, which were mentioned by the reference data of VII-
a-VII-c and known compound (17).
Fungicidal Activity against C. arachidicola. At a dosage of
50 μg mL-1, only compounds IV-6a, IV-6b and IV-6c exhibited
moderate activity. The fungicidal activity of IV-6a, IV-6b and IV-
6c were 26.9%, 32.3% and 30.0% respectively, which might be
attributed to the -NH2 group in the 6-position of phenazine ring.
Most compounds of III-a-VII-e exhibited low activities against
C. arachidicola.
Based on the HMBC spectra, the correlations between H-8 (δH
=9.03) and C-6, C-7, C-9, C-9a [δC=134.1 (s, C-6), 138.1 (s, C-7),
147.2 (s, C-9), 122.6 (s, C-9a)], the correlations between NH-β (δH
=8.40) and C-6, C-5a [δC=134.1 (s, C-6), 142.5 (s, C-5a)], the
correlations between H-4 (δH=8.40) and C-2, C-10a [δC=132.5
(d, C-2), 133.7 (s, C-10a)], the correlations between H-2 (δH =
8.40) and C-10, C-4, C-10a [δC=166.2 (s, C-10), 135.0 (d, C-4),
133.7 (s, C-10a)], the correlations between H-3 (δH=8.02) and
C-1, C-4a [δC=132.9 (s, C-1), 139.9 (s, C-4a)], the correlations
between H-20 (δH=4.54) and C-10, C-30, C-40 [δC=166.2 (s, C-10),
30.6 (t, C-30), 19.2 (t, C-40)], the correlations between H-30 (δH=
1.85-1.88) and C-20, C-40, C-50 [δC=66.4 (t, C-20), 19.2 (t, C-40),
13.7 (q, C-50)], the correlations between H-40 (δH =1.48-1.51)
and C-20, C-30, C-50 [δC=66.4 (t, C-20), 30.6 (t, C-30), 13.7 (q, C-
50)], and the correlations between H-50 (δH=0.99) and C-30, C-40
[δC=30.6 (t, C-30), 19.2 (t, C-40)] indicated the structure of VII-d
should be as shown in Figure 3.
In the H-H COSY spectra, the correlations between H-3 (δH=
8.02) and H-4 (δH=8.40) and the correlations between H-3 (δH=
8.02) and H-2 (δH=8.40) indicatedH-4, H-3 and H-2 were nearby.
The correlations between H-R (δH =9.01) and H-β (δH=8.40)
further proved H-R and H-β belonged to NH2-6. The chemical
shift value of H-R in 1H NMR spectra might be influenced by the
nitryl in the 7-position. The correlations between H-20 (δH=4.54)
and H-30 (δH=1.85-1.88), the correlations between H-30 (δH=
1.85-1.88) and H-40 (δH =1.48-1.51), and the correlations
between H-40 (δH =1.48-1.51) and H-50 (δH = 0.99) indicated
H-20, H-30, H-40 and H-50 were adjacent.
Fungicidal Activity against F. omysporum. The screening
data of Table 2 indicated that, at a dosage of 50 μg mL-1, most
compounds of III-a-VII-e exhibited low activities against F.
omysporum except the compounds IV-6a, IV-6b and IV-6c. The
fungicidalactivitiesof IV-6a, IV-6b and IV-6c were 40.0%, 33.3%
and 36.7% respectively, which further explained that the -NH2
group in the 6-position of the phenazine ring was vital for
improving their biological activity. Regrettably, the fungicidal
activities of VI-6a-VI-7n against F. omysporum were as low as
those against C. arachidicola. It might be concluded that the
-NH2 group being substituted by -NHCO- or -NHSO2-
group would result in the fungicidal activity being decreased.
Fungicidal Activity against G. zeae. The biological activity
rules of III-a-VI-7n against G. zeae were generally the same as
the test against C. arachidicola and F. omysporum showed. To be
pointed out, compounds VII-a-VII-d were the nitration deriva-
tives of VI-6c and VI-6h. The fungicidal activities of VII-a-VII-d
were 40.4%, 57.6%, 51.5% and 53.0% respectively. The fungi-
cidal activities of VII-a-VII-d were higher than those of IV-
6a-IV-6d or VI-6c, VI-6h (Table 2). It might be explained by that
the nitration of the phenazine ring resulted in the activity against
G. zeae being increased.
Fungicidal Activity against P. piricola. The screening data of
Table 2 indicated that most target compounds exhibited low
activities against P. piricola except for IV-6a and IV-6b. At a
dosage of50μg mL-1, compounds IV-6a, IV-6b and phenazine-1-
carboxylic acid exhibited 100% inhibition. The EC50 values of
Biological Assay and Structure-Activity Relationship. Table 2
showed the fungicidal activities against A. solani, C. arachidicola,

3660 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Wang et al.
IV-6a, IV-6b and phenazine-1-carboxylic acid against F. omysporum
were 3.00 μg mL-1, 4.44 μg mL-1, 3.58 μg mL-1 (Table 3), which
fully explained the rules as follows: The side chain of C-1 should not
be too long. The -NH2 group should be in the 6-position of the
phenazine ring and not form the -NHCO- or -NHSO2- group.
The above terms were essential for high fungicidal activity of the
aminophenazine derivatives.
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Though the screening data of Table 2 and Table 3 indicated
that the fungicidalactivitiesof most targetcompounds werelower
than that of phenazine-1-carboxylic acid, compound IV-6a had
higher fungicidal activity than that of phenazine-1-carboxylic
acid against P. piricola. IV-6a also showed excellent activity
against A. solani at low dosage. So IV-6a could be developed as
a leading compound for further structural optimization. In
conclusion, a novel and facile procedure for preparation of
derivatives of aminophenazine-1-carboxylate was developed
from 2-bromo-3-nitrobenzoic acid with corresponding substi-
tuted benzene-diamine. Forty-six aminophenazine-1-carboxylate
derivatives were synthesized. The results of bioassay showed that
the fungicidal activities of most target compounds were lower
than that of phenazine-1-carboxylic acid; a few of the title
compounds exhibited moderate activities against A. solani, C.
arachidicola, F. omysporum, G. zeae, P. piricola at a dosage of
50 μg mL-1; IV-6a and IV-6b exhibited excellent activity against
A. solani and P. piricola at that dosage; the EC50 of IV-6a against
P. piricola was 3.00 μg mL-1, which was lower than that of
phenazine-1-carboxylic acid (Table 3). To our knowledge, IV-6a
was a known compound; however, no biological activity or
feasible synthesis method was reported in any literature. So IV-
6a could be developed as a leading compound for further
structural optimization. The possible SAR of aminophenazine-
1-carboxylate derivatives were as follows: The -NH2 group
should be in the 6-position of the phenazine ring and not be
substituted by the -NHCO- or -NHSO2- group. The amino-
phenazine-1-carboxylic acid should be esterified to aminophena-
zine-1-carboxylate, and the side chain of C-1 should not be too
long. The above terms were essential for high fungicidal activity
of the aminophenazine derivatives. The nitration of the phenazine
ring could increase the fungicidal activity against G. zeae but not
for A. solani or P. piricola. The byproducts V-7b-V-8d did not
show noticeable fungicidal activities.
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