Synthesis and fluorescence properties of 4,5-, 4,6- and 5,6-disubstituted benzofurazan (2,1,3-benzoxadiazole) compounds

Article abstract of DOI:10.3987/com-01-s(m)4

New 4,5-, 4,6- and 5,6-disubstituted benzofurazan (2,1,3-benzoxadiazole) compounds having various substituent groups were synthesized. Their fluorescence properties were obtained and the discussions were made to develop new sensitive and selective fluores

Full text of DOI:10.3987/com-01-s(m)4

HETEROCYCLES, Vol. 58, 2002, pp. 165-173, Received, 13th December, 2001
SYNTHESIS AND FLUORESCENCE PROPERTIES OF 4,5-, 4,6- AND 5,6-
DISUBSTITUTED BENZOFURAZAN (2,1,3-BENZOXADIAZOLE) COMPOUNDS
Natsuko Okiyama, Seiichi Uchiyama, Maki Onoda, Kazuhiro Imai, and Tomofumi Santa*
Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
E-mail: santa@mol.f.u-tokyo.ac.jp
Abstract – New 4,5-, 4,6- and 5,6-disubstituted benzofurazan (2,1,3-benzoxadiazole)
compounds having various substituent groups were synthesized. Their fluorescence properties
were obtained and the discussions were made to develop new sensitive and selective
fluorescent reagents.
INTRODUCTION
Until now, over forty 4,7-disubstituted benzofurazan (2,1,3-benzoxadiazole) compounds (the
benzofurazan skeleton was shown in Figure 1) have been reported as fluorogenic and fluorescent labeling
4
N
5
O
6
N
7
Figure 1: Benzofurazan (2,1,3-benzoxadiazole) skeleton
example, 4-fluoro-7-nitro-2,1,3-benzoxadiazole
(NBD-F),2
7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F),3 4-mercapto-7-methylthio-2,1,3-benzoxadiazole
(MTBD-SH),4 (PSBD-NCO),5
7-phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isocyanate and
reagents.1
For
ammonium
7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MTBD-NCS)6 are now used as highly-sensitive
fluorogenic reagents for amines, thiols, carboxylic acids, alcohols, and peptides, respectively. Thus,
sensitive and selective determinations of biologically important compounds have been achieved with
these benzofurazan reagents.1
While the synthesis and fluorescence properties of various 4,7-disubstituted benzofurazan compounds
including the reagents mentioned above have been reported,7 there are few reports on the synthesis and
the fluorescence properties of 4,5-, 4,6- and 5,6-disubstituted benzofurazan compounds.8 In this paper, we

report a synthesis of new 4,5-, 4,6- and 5,6-disubstituted benzofurazan compounds having -NHAc, -NH2,
-Cl, -NAc2, -NMe2, -F, -OMe, -SMe and -NO2 groups and their fluorescence properties to develop new
useful fluorescent reagents.
RESULTS AND DISCUSSION
Synthesis
The new benzofurazan compounds synthesized in this study are summarized in Table 1. In this paper, the
disubstituted benzofurazan compounds are denoted using substituent groups and position numbers, e.g.
7-fluoro-4-nitro-2,1,3-benzoxadiazole (NBD-F) was denoted as 4-NO2/7-F.
Table 1: Synthesis of 4,5-, 4,6-, and 5,6- benzofurazan compounds
Product
From
Yield (%)
Product
From
Yield (%)
Cyclization to benzofurazan structure
Reduction of nitro group
4-Cl/6-Cl
4-Cl/6-NO2
5-F/6-NO2
2,4-dichloro-6-nitroaniline
6-chloro-2,4-dinitroaniline
2,4-dinitro-5-fluoroaniline
37
68
41
4-NH2/5-NMe2
4-NH2/5-Cl
4-NH2/5-OMe
4-NH2/5-SMe
4-SMe/5-NH2
5-NH2/6-F
4-NO2/5-NMe2
24
67
45
13
85
4.7
4-NO2/5-Cl
4-NO2/5-OMe
4-NO2/5-SMe
4-SMe/5-NO2
5-NO2/6-F
Replacement of halogen group with nucleophile
4-Cl/6-NMe2
4-NO2/5-NMe2 4-NO2/5-Cl
4-NO2/5-SMe
4-SMe/5-NO2
5-SMe/6-NH2
4-NO2/5-OMe
4-Cl/6-Cl
9.6
76
35
93
57
63
4-NO2/5-Cl
4-Cl/5-NO2
5-NH2/6-F
4-NO2/5-Cl
Acetylation of amino group
4-NHAc/5-NMe2 4-NH2/5-NMe2
4-NAc2/5-Cl
4-NAc2/5-SMe
83
45
12
4-NH2/5-Cl
4-NH2/5-SMe
Construction of a benzofurazan ring. In general, the benzofurazan ring is synthesized from
o-nitroaniline by four steps: (1) diazotization, (2) reaction of diazonium salt with sodium azide, (3)
cyclization for the construction of a benzofuroxan (2,1,3-benzoxadiazole N-oxide) ring, and (4) reduction
to a benzofurazan ring with triphenylphosphine (TPP).9 4-Cl/6-Cl, 4-Cl/6-NO2, and 5-F/6-NO2 were
obtained by these procedures. On the other hand, benzofurazan compounds having a nitro group at the
4-position could not be synthesized in a similar way. The reaction of benzofuroxan compounds having a
nitro group at the 4-position with TPP did not produce the corresponding benzofurazan compounds
because of the strong electron attractive nitro group.
Nucleophilic substitution. A halogeno group of benzofurazan compounds can be replaced by various
nucleophilic groups such as –NR1R2, -SR, and -OR.1 The fluoro group is more reactive to nucleophiles
than the chloro group and the 5-(or 6-)position of the benzofurazan skeleton is also more reactive than the
4-(7-) position.10 Therefore, 4-Cl/6-NMe2 and 4-NO2/5-NMe2 were synthesized from the corresponding
halogenobenzofurazans using dimethylamine solution. 4-NO2/5-SMe, 4-SMe/5-NO2, and 5-SMe/6-NH2

were obtained with methyl mercaptan sodium salt, while 4-NO2/5-OMe was with methanol. The results
indicated that the 4,5-, 4,6-, and 5,6-disubstituted benzofurazan compounds with the halogeno group
could be used as derivatization reagents for amines, thiols, and alcohols as same as 4,7-disubstituted
benzofurazan reagents such as NBD-F and SBD-F.
Reduction of a nitro group to an amino group. An amino group can be inserted into the benzofurazan
compounds with changing a nitro group using iron and hydrochloric acid.11 Thus, 4-NH2/5-NMe2,
4-NH2/5-Cl, 4-NH2/5-OMe, 4-NH2/5-SMe, 4-SMe/5-NH2, and 5-NH2/6-F were obtained. The amino
group can be easily converted to reactive isocyano and isothiocyano groups for the useful derivatization
reagents.
Acetylation of an amino group. 4-NHAc/5-NMe2, 4-NAc2/5-Cl, and 4-NAc2/5-SMe were synthesized
from the corresponding amines with an excess of acetic anhydride. 4-NHAc/5-Cl and 4-NHAc/5-SMe
were not obtained from 4-NH2/5-Cl under this condition, while the reason was unknown.
Table 2: Absorption and fluorescence properties of the disubstituted benzofurazan compounds
in cyclohexane
in acetonitrile
103
/nm*
*
/nm
103
/nm*
*
Φ
-1
-1
-1
-1
/nm
λab.
378
401
323
339
395
394
379
353
401
398
230
409
399
391
334
325
321
316
294
285
ε /
M cm λem.
Φ
λab.
394
438
332
354
403
398
392
362
448
451
244
420
408
400
347
250
321
321
307
291
ε /
M cm λem.
Compounds
4-SMe/5-NH2
4-Cl/6-NMe2
5-SMe/6-NH2
5-NH2/6-F
2.14
4.28
3.24
2.26
2.12
2.58
5.52
2.56
3.22
3.40
9.96
5.90
4.96
3.42
4.44
3.78
4.34
4.50
3.04
8.56
490
503
465
413
516
515
488
426
527
542
340
-
0.31
0.26
0.16
0.11
0.092
0.071
0.048
0.044
0.043
0.014
0.0040
0
3.26
4.88
7.76
4.08
2.42
4.98
2.92
4.60
2.96
3.06
8.26
9.18
5.02
6.38
5.30
14.5
2.92
3.42
4.12
7.38
528
0.048
561
0.036
509
0.077
454
0.077
4-NH2/5-SMe
4-NHAc/5-NMe2
4-NH2/5-Cl
582
0.0026
549
0.0014
537
0.017
4-NAc2/5-SMe
4-NH2/5-OMe
4-NH2/5-NMe2
5-F/6-NO2
468
0.028
-
-
-
-
-
-
-
-
-
-
-
-
0
0
0
0
0
0
0
0
0
0
0
0
4-NO2/5-NMe2
4-NH2/5-NO2
4-NO2/5-SMe
4-NO2/5-OMe
4-Cl/6-NO2
-
0
-
0
-
0
-
0
4-Cl/5-NO2
-
0
4-Cl/6-Cl
4-NAc2/5-Cl
4-NO2/5-Cl
-
-
0
0
-
0
* excited at maximum absorption wavelength (λab.)
Fluorescence properties. The absorption and fluorescence properties of the twenty 4,5-, 4,6-,
5,6-disubstituted benzofurazan compounds including 4-Cl/5-NO2,12 4-NH2/5-NO2,12 and 4-NO2/5-Cl13

synthesized previously were summarized in Table 2. The absorption and especially fluorescence
properties of these compounds were quite different from each other, and the difference was derived from
the different substituent groups and their positions. The results shown in Table 2 suggested that the
nucleophilic group such as -NH2, -SMe, and -OMe was necessary for the benzofurazan compound to be
fluorescent. Compared the absorption and fluorescence properties of fluorescent 4,5-, 4,6-, and
5,6-disubstituted benzofurazan compounds in Table 2 with those of 4,7-disubstituted benzofurazan
compounds,14 Φ values of the 4,5-, 4,6-, and 5,6-disubstituted benzofurazan compounds are greatly
influenced by the solvent polarity more than those of 4,7-disubstituted benzofurazan compounds. These
results indicated that the 4,5-, 4,6-, and 5,6-disubstituted benzofurazan structures such as 4-SMe/5-NH2
and 4-Cl/6-NMe2 would be polarity sensitive fluorophores. Moreover, the benzofurazan skeletons studied
in this paper are also suitable to fluorogenic reagents. For instance, non-fluorescent 4-Cl/6-Cl is
converted to fluorescent 4-Cl/6-NMe2 with dimethylamine, meaning that 4-Cl/6-Cl is the fluorogenic
reagent for amines.
CONCLUSION
Twenty 4,5-, 4,6-, and 5,6-disubstituted benzofurazan compounds were synthesized and their
fluorescence properties were obtained. The fluorescence properties of these compounds were quite
different from each other by the substituent groups and positions, suggesting us the new coming
fluorogenic 4,5-, 4,6-, or 5,6-disubstitued benzofurazan reagents. If the relationship between the chemical
structures and the fluorescence properties of these compounds is elucidated, new highly-sensitive
fluorogenic reagents would be developed as previously reported of 4,7-disubstituted benzofurazan
compounds.7
EXPERIMENTAL
Materials. Triphenyl phosphine (TPP), 2,4-dichloro-6-nitroaniline, and 2,4-dinitro-5-fluoroaniline
were purchased from Aldrich (Milwaukee, WI, USA). Hydrochloric acid and cyclohexane were obtained
from Wako Pure Chemicals (Osaka, Japan). Methyl mercaptan sodium salt solution (15 % in water) and
6-chloro-2,4-dinitroaniline were purchased from Tokyo Kasei Kogyo (Tokyo, Japan). Iron powder,
dimethylamine solution (50 % in water), acetic anhydride, acetic acid, dichloromethane, diethylene glycol,
n-hexane, ethyl acetate, chlorobenzene, toluene, acetonitrile, methanol, triethylamine, o-dichlorobenzene,
sulfuric acid, sodium nitrite, anhydrous sodium sulfate, and sodium hydrogen carbonate were obtained
from Kanto Chemicals (Tokyo, Japan). Sodium azide was purchased from Nacalai tesque (Kyoto, Japan).
Silica gel 60 was supplied by Merck (Darmstadt, Germany). Water was purified using a Milli-Q system
(Millipore, Bedford, MA, USA). All reagents were of analytical, HPLC or guaranteed grade and used

without further purification.
Apparatus. Melting points were measured on a Yanagimoto Micro Point Apparatus (Tokyo, Japan) and
are uncorrected. 1H-NMR spectra were obtained using a JEOL LA-500 spectrometer (Tokyo, Japan) with
tetramethylsilane as the internal standard in CDCl3. J values are given in Hz. MS spectra were measured
using a Hitachi M-1200H mass spectrometer (atmospheric pressure chemical ionization (APCI) system)
(Tokyo, Japan). UV-VIS absorption spectra (5 µM) were measured using a JASCO (Japan Spectroscopic
Co., Ltd.) Ubest-50 spectrophotometer (Tokyo, Japan). Fluorescence spectra (5 µM) were measured using
a Hitachi F-4010 fluorescence spectrophotometer (Tokyo, Japan). The fluorescence quantum yields (Φ)
were determined using quinine sulfate in 0.1 M sulfuric acid (Φ = 0.55; λex., 355 nm) as the standard.
Synthesis. 4-Chloro-5-nitro-2,1,3-benzoxadiazole (4-Cl/5-NO2),12 4-amino-5-nitro-2,1,3-benzoxadiazole
(4-NH2/5-NO2),12 and 5-chloro-4-nitro-2,1,3-benzoxadiazole (4-NO2/5-Cl)13 was synthesized and
purified as described previously.
4,6-Dichloro-2,1,3-benzoxadiazole (4-Cl/6-Cl). 2,4-Dichloro-6-nitroaniline (550 mg, 2.7 mmol) was
dissolved in a mixture of acetic acid (6 mL) and conc. sulfuric acid (2 mL). After the addition of sodium
nitrite (200 mg, 2.9 mmol) solution (5 mL) at 0 °C, sodium azide (190 mg, 2.9 mmol) solution (5 mL)
was added and the mixture was stirred at rt for 30 min. The mixture was concentrated in vacuo and the
residue was chromatographed on silica gel with CH2Cl2–n-hexane (2:5) to afford
2,4-dichloro-6-nitrophenyl azide. 2,4-Dichloro-6-nitrophenyl azide was dissolved in diethylene glycol (5
mL) and stirred for 1 h at 150 °C. The mixture was chromatographed on silica gel with CH2Cl2–n-hexane
(2:5) to afford 4,6-dichlorobenzofuroxan (300 mg, 55%) as yellow crystals. 4,6-Dichlorobenzofuroxan
(300 mg, 1.4 mmol) was dissolved in chlorobenzene (5 mL). TPP (416 mg, 1.6 mmol) was added and
refluxed for 1 h. The reaction mixture was chromatographed on silica gel with n-hexane to afford
4-Cl/6-Cl (185 mg, 68%) as white crystals: mp 55 – 56 °C. δH 7.78 (1H, s), 7.40 (1H, s). Anal. Calcd for
C6H2N2OCl2: C, 38.13; H, 1.07; N, 14.82. Found: C, 38.13; H, 1.32; N, 14.81.
4-Chloro-6-nitro-2,1,3-benzoxadiazole (4-Cl/6-NO2). 6-Chloro-2,4-dinitroaniline (129 mg, 0.59 mmol)
was dissolved in a mixture of acetic acid (8 mL) and conc. sulfuric acid (4 mL). After the addition of
sodium nitrite (50 mg, 0.72 mmol) solution (5 mL) at 0 °C, sodium azide (48 mg, 0.74 mmol) solution (5
mL) was added and the mixture was stirred at rt for 1 h. The mixture was concentrated in vacuo and the
residue was chromatographed on silica gel with n-hexane–ethyl acetate (3:1) to afford
2-chloro-4,6-dinitrophenyl azide as yellow oil. 2-Chloro-4,6-dinitrophenyl azide was dissolved in
o-dichlorobenzene (5 mL) and refluxed for 1. The mixture was chromatographed on silica gel with
n-hexane–ethyl acetate (3:1) to afford 4-chloro-6-nitrobenzofuroxan (100 mg, 78%) as yellow crystals.
4-Chloro-6-nitrobenzofuroxan (28 mg, 0.13 mmol) was dissolved in toluene (3 mL). After the addition of
TPP (38 mg, 0.15 mmol), the mixture was refluxed for 1 h and concentrated in vacuo. The residue was

chromatographed on silica gel with CH2Cl2–n-hexane (1:2) to afford 4-Cl/6-NO2 (23 mg, 88%) as white
needles: mp 72 °C. δH 8.81 (1H, s), 8.24 (1H, s). Anal. Calcd for C6H2N3O3Cl: C, 36.11; H, 1.01; N,
21.06. Found: C, 36.33 ; H, 1.12 ; N, 21.30.
5-Fluoro-6-nitro-2,1,3-benzoxadiazole (5-F/6-NO2). 2,4-Dinitro-5-fluoroaniline (1.1 g, 5.2 mmol) was
dissolved in a mixture of acetic acid (40 mL) and conc. sulfuric acid (20 mL). After the addition of
sodium nitrite (400 mg, 5.8 mmol) solution (10 mL) at 0 °C, sodium azide (480 mg, 7.4 mmol) solution
(5 mL) was added and the mixture was stirred at rt for 1 h. After the extraction with ethyl acetate (3 × 50
mL), the organic layer was refluxed for 2.5 h. The reaction mixture was concentrated in vacuo and the
residue was chromatographed on silica gel with n-hexane–ethyl acetate (4:1) to afford 5-fluoro-6-
nitrobenzofuroxan (810 mg, 78%) as yellow crystals. 5-Fluoro-6-nitrobenzofuroxane (810 mg, 4.1 mmol)
was dissolved in ethyl acetate (10 mL). After the addition of TPP (1.3 g), the mixture was refluxed for 1 h.
The mixture was chromatographed on silica gel with CH2Cl2–n-hexane (1:2) to afford
5-fluoro-6-nitro-2,1,3-benzoxadiazole (390 mg, 52%) as yellow needles: mp 33 °C. δH 8.62 (1H, d, J =
6.7), 7.73 (1H, m). Anal. Calcd for C6H2N3O3F: C, 39.54; H, 1.10; N, 22.95. Found: C, 39.34; H, 1.35; N,
23.07.
4-Chloro-6-N,N-dimethylamino-2,1,3-benzoxadiazole (4-Cl/6-NMe2). 4-Cl/6-Cl (20 mg, 0.11 mmol)
was dissolved in acetonitrile (2 mL). After the addition of 50% dimethylamine solution (1.5 mL, 12
mmol), the mixture was refluxed for 4 h. The reaction mixture was chromatographed on silica gel with
CH2Cl2–n-hexane (1:2) to afford 4-Cl/6-NMe2 (2.0 mg, 9.6%) as yellow crystals: mp 60 – 61 °C. δH 7.02
(1H, s), 5.95 (1H, s), 3.34 (6H, s). Anal. Calcd for C8H8N3OCl: C, 48.62; H, 4.08; N, 21.26. Found: C,
48.87; H, 4.28; N, 21.41. APCI-MS: m/z 198 [M + H]+.
5-N,N-Dimethylamino-4-nitro-2,1,3-benzoxadiazole (4-NO2/5-NMe2). 4-NO2/5-Cl (29 mg, 0.15 mmol)
was dissolved in acetonitrile (2 mL). After the addition of 50% dimethylamine solution (200 µL, 1.6
mmol), the mixture was stirred at rt for 2 h. The reaction mixture was chromatographed on silica gel with
ethyl acetate–n-hexane (2:1) to afford 4-NO2/5-NMe2 (23 mg, 76%) as yellow crystals: mp 166 °C. δH
7.83 (1H, d, J = 10.7), 7.42 (1H, d, J =10.7), 3.24 (6H, s). Anal. Calcd for C8H8N4O3: C, 46.16; H, 3.87;
N, 26.91. Found: C, 46.17 ; H, 3.82 ; N, 26.96. APCI-MS: m/z 209 [M + H]+.
5-Methylthio-4-nitro-2,1,3-benzoxadiazole (4-NO2/5-SMe). 4-NO2/5-Cl (200 mg, 1.0 mmol) was
dissolved in the mixture of acetonitrile (10 mL) and 50 mM borate buffer (pH 9.3, 4 mL). After the
addition of 15 % methyl mercaptan sodium salt solution (800 µL, 1.9 mmol), the mixture was stirred at rt
for 1.5 h. The reaction mixture was concentrated in vacuo and the residue was chromatographed on silica
gel with CH2Cl2–n-hexane (2:1) to afford 4-NO2/5-SMe (75 mg, 35%) as orange crystals: mp 137 °C. δH
8.08 (1H, d, J = 9.8), 7.64 (1H, d, J = 9.8), 2.70 (3H, s). Anal. Calcd for C7H5N4O3S: C, 39.81; H, 2.39; N,
19.90. Found: C, 39.89; H, 2.66; N, 19.68.
5-Methylthio-6-amino-2,1,3-benzoxadiazole (5-SMe/6-NH2). 5-Amino-6-fluoro-2,1,3-benzoxadiazole

(5-NH2/6-F, 15 mg, 0.098 mmol) was dissolved in acetonitrile (12 mL). After the addition of 15 %
methyl mercaptan sodium salt solution (1.6 mL, 3.7 mmol), the mixture was stirred at rt for 10 h. The
reaction mixture was concentrated in vacuo and the residue was chromatographed on silica gel with
dichloromethane to afford 5-SMe/6-NH2 (10 mg, 57%) as yellow crystals: mp 183 – 184 °C. δH 7.42 (1H,
s), 6.69 (1H, s), 4.49 (2H, br), 2.58 (3H, s). Anal. Calcd for C7H7N3OS: C, 46.39; H, 3.89; N, 23.19.
Found: C, 46.50; H, 4.04; N, 22.94. APCI-MS: m/z 182 [M + H]+.
5-Methoxy-4-nitro-2,1,3-benzoxadiazole (4-NO2/5-OMe). 4-NO2/5-Cl (300 mg, 1.5 mmol) was
dissolved in methanol (15 mL). After the addition of triethylamine (0.5 mL, 3.6 mmol), the mixture was
stirred at rt for 4 h. The reaction mixture was concentrated in vacuo and the residue was chromatographed
on silica gel with CH2Cl2–n-hexane (1 : 1) to afford 4-NO2/5-OMe (185 mg, 63%) as yellow crystals: mp
120 °C. δH 8.15 (1H, d, J = 9.7), 7.54 (1H, d, J = 9.7), 4.21 (3H, s). Anal. Calcd for C7H5N3O4: C, 43.09;
H, 2.58; N, 21.53. Found: C, 43.06; H, 2.78; N, 21.48. APCI-MS: m/z 196 [M + H]+.
4-Amino-5-N,N-dimethylamino-2,1,3-benzoxadiazole (4-NH2/5-NMe2). 4-NO2/5-NMe2 (207 mg, 1.0
mmol) was dissolved in the mixture of dichloromethane (8 mL), conc. hydrochloric acid (4 mL) and
methanol (10 mL). After the addition of iron powder (800 mg, 14 mmol), the mixture was stirred at rt for
1 h. The reaction mixture was poured into water and extracted using dichloromethane. The organic layer
was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was chromatographed
on silica gel with CH2Cl2–n-hexane (2 : 1) to afford 4-NH2/5-NMe2 (42 mg, 24%) as red crystals: mp 75
°C. δH 7.31 (1H, d, J = 9.4), 7.12 (1H, d, J = 9.4), 4.69 (2H, br), 2.67 (6H, s). Anal. Calcd for C8H10N4O:
C, 53.92; H, 5.66; N, 31.44. Found: C, 54.13; H, 5.46; N, 31.48. APCI-MS: m/z 179 [M + H]+.
4-Amino-5-chloro-2,1,3-benzoxadiazole (4-NH2/5-Cl). 4-NO2/5-Cl (200 mg, 1.0 mmol) was dissolved
in the mixture of dichloromethane (4 mL), conc. hydrochloric acid (3 mL) and methanol (10 mL). After
the addition of iron powder (500 mg, 9.0 mmol), the mixture was stirred at rt for 1 h. The reaction
mixture was poured into water and extracted using dichloromethane. The organic layer was dried over
anhydrous sodium sulfate and concentrated in vacuo. The residue was chromatographed on silica gel with
CH2Cl2–n-hexane (1 : 1) to afford 4-NH2/5-Cl (113 mg, 67%) as yellow crystals: mp 130 °C. δH 7.24 (1H,
d, J = 9.4), 7.11 (1H, d, J = 9.4), 4.91 (2H, br). Anal. Calcd for C6H4N3OCl: C, 42.50; H, 2.38; N, 24.78.
Found: C, 42.44; H, 2.51; N, 24.62. APCI-MS: m/z 168 [M – H]-.
4-Amino-5-methoxy-2,1,3-benzoxadiazole (4-NH2/5-OMe). 4-NO2/5-OMe (50 mg, 0.26 mmol) was
dissolved in the mixture of dichloromethane (4 mL), conc. hydrochloric acid (2 mL) and methanol (6 mL).
After the addition of iron powder (200 mg, 3.6 mmol), the mixture was stirred at rt for 1 h. The reaction
mixture was poured into water and extracted using dichloromethane. The organic layer was dried over
anhydrous sodium sulfate and concentrated in vacuo. The residue was chromatographed on silica gel with
dichloromethane to afford 4-NH2/5-OMe (19 mg, 45%) as red crystals: mp 88 °C. δH 7.28 (1H, d, J =

9.4), 7.17 (1H, d, J = 9.4), 4.40 (2H, br), 3.92 (3H, s). Anal. Calcd for C7H7N3O2: C, 50.91; H, 4.27; N,
25.44. Found: C, 50.77; H, 4.40; N, 25.17. APCI-MS: m/z 166 [M + H]+.
4-Amino-5-methylthio-2,1,3-benzoxadiazole (4-NH2/5-SMe). 4-NO2/5-SMe (200 mg, 0.95 mmol) was
dissolved in the mixture of dichloromethane (15 mL), conc. hydrochloric acid (9 mL) and methanol (35
mL). After the addition of iron powder (1.1 g, 20 mmol), the mixture was stirred at rt for 30 min. The
reaction mixture was poured into water and extracted using dichloromethane. The organic layer was dried
over anhydrous sodium sulfate and concentrated in vacuo. The residue was chromatographed on silica gel
with CH2Cl2–n-hexane (2 : 1) to afford 4-NH2/5-SMe (23 mg, 13%) as orange crystals: mp 66 °C. δH
7.36 (1H, d, J = 9.2), 7.07 (1H, d, J = 9.2), 5.27 (2H, br), 2.35 (3H, s). Anal. Calcd for C7H7N3OS: C,
46.39; H, 3.89; N, 23.19. Found: C, 46.37; H, 3.90; N, 23.13.
5-Amino-4-methylthio-2,1,3-benzoxadiazole (4-SMe/5-NH2). 4-Cl/5-NO2 (162 mg, 0.81 mmol) was
dissolved in the mixture of acetonitrile (12 mL) and saturated sodium hydrogen carbonate solution (3 mL).
After the addition of 15 % methyl mercaptan sodium salt solution (100 µL, 0.23 mmol), the mixture was
stirred at rt for 20 min. The reaction mixture was concentrated in vacuo and the residue was
chromatographed on silica gel with CH2Cl2–n-hexane (1:1) to afford 4-methylthio-5-nitro-2,1,3-
benzoxadiazole (4-SMe/5-NO2, 93%) as yellow crystals. δH 8.06 (1H, d, J = 5.8), 7.60 (1H, d, J = 5.8),
3.04 (3H, s). 4-SMe/5-NO2 (160 mg, 0.76 mmol) was dissolved in the mixture of dichloromethane (20
mL), conc. hydrochloric acid (1.1 mL) and methanol (8 mL). After the addition of iron powder (517 mg,
9.3 mmol), the mixture was stirred at rt for 20 min. The reaction mixture was poured into water and
extracted using dichloromethane. The organic layer was dried over anhydrous sodium sulfate and
concentrated in vacuo. The residue was chromatographed on silica gel with CH2Cl2–n-hexane (5 : 2) to
afford 4-SMe/5-NH2 (117 mg, 85%) as yellow needles: mp 102 °C. δH 7.60 (1H, d, J = 9.4), 6.96 (1H, d,
J = 9.4), 5.03 (2H, br), 2.41 (3H, s). Anal. Calcd for C7H7N3OS: C, 46.39; H, 3.89; N, 23.19. Found: C,
46.15; H, 3.83; N, 23.10. APCI-MS: m/z 182 [M + H]+.
5-Amino-6-fluoro-2,1,3-benzoxadiazole (5-NH2/6-F). 5-NO2/6-F (391 mg, 2.1 mmol) was dissolved in
the mixture of dichloromethane (50 mL), conc. hydrochloric acid (3 mL) and methanol (20 mL). After the
addition of iron powder (1.1 g, 20 mmol), the mixture was stirred at rt for 15 min. The reaction mixture
was poured into water and extracted using dichloromethane. The organic layer was dried over anhydrous
sodium sulfate and concentrated in vacuo. The residue was chromatographed on silica gel with
dichloromethane to afford 5-NH2/6-F (15 mg, 4.7%) as white crystals: mp 186 °C. δH 7.34 (1H, d, J =
10.3), 6.74 (1H, d, J = 7.9), 4.47 (2H, br). Anal. Calcd for C6H4N3OF: C, 47.07; H, 2.63; N, 27.44.
Found: C, 46.94; H, 2.84; N, 27.28. APCI-MS: m/z 154 [M + H]+.
4-Acetylamino-5-N,N-dimethylamino-2,1,3-benzoxadiazole (4-NHAc/5-NMe2). 4-NH2/5-NMe2 (5.0
mg, 0.028 mmol) was dissolved in acetic anhydride (2 mL, 22 mmol). The mixture was stirred at rt for 8

h and chromatographed on silica gel with ethyl acetate to afford 4-NHAc/5-NMe2 (5.1 mg, 83%) as
yellow crystals: mp 210 – 211 °C. δH 7.57 (1H, d, J = 9.7), 7.29 (1H, d, J = 9.7), 2.99 (6H, s), 2.24 (3H, s).
Anal. Calcd for C10H12N4O2: C, 54.54; H, 5.49; N, 25.44. Found: C, 54.64; H, 5.36; N, 25.31. APCI-MS:
m/z 221 [M + H]+.
5-Chloro-4-diacetylamino-2,1,3-benzoxadiazole (4-NAc2/5-Cl). 4-NH2/5-Cl (30 mg, 0.18 mmol) was
dissolved in acetic anhydride (2 mL, 22 mmol). The mixture was stirred at 130 °C for 2 h and
chromatographed on silica gel with ethyl acetate–n-hexane (1 : 2) to afford 4-NAc2/5-Cl (20 mg, 45%) as
white crystals: mp 110 °C. δH 7.90 (1H, d, J = 9.4), 7.50 (1H, d, J = 9.7), 2.33 (6H, s). Anal. Calcd for
C10H8N3O3Cl: C, 47.35; H, 3.18; N, 16.57. Found: C, 47.47; H, 3.43; N, 16.38. APCI-MS: m/z 253 [M]–.
4-Diacetylamino-5-methylthio-2,1,3-benzoxadiazole (4-NAc2/5-SMe). 4-NH2/5-SMe (20 mg, 0.11
mmol) was dissolved in acetic anhydride (2 mL, 22 mmol). The mixture was heated at 100 °C for 2 h and
chromatographed on silica gel with ethyl acetate–n-hexane (2 : 1) to afford 4-NAc2/5-SMe (3.5 mg, 12%)
as yellow crystals: mp 142 – 143 °C. δH 7.84 (1H, d, J = 9.5), 7.41 (1H, d, J = 9.5), 2.53 (3H, s), 2.28 (6H,
s). Anal. Calcd for C11H11N3O3S: C, 49.80; H, 4.18; N, 15.84. Found: C, 49.92; H, 4.44; N, 15.57.
APCI-MS: m/z 266 [M + H]+.
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