Synthesis and chemiluminescent activity of pyridazino[4,5-b]indole-1,4(2H,3H)-diones

Article abstract of DOI:10.3987/com-00-9049

Substituted and non-substituted anilines were transformed to the corresponding triazole derivatives, which were converted to dimethyl indole-2,3-dicarboxylates by photocyclization. The reaction of diester with hydrazine hydrate gave 1,2,3,4-tetrahydro-5H-

Full text of DOI:10.3987/com-00-9049

HETEROCYCLES, Vol. 53, No. 12, 2000, pp. 2809 - 2819, Received, 30th August, 2000
SYNTHESIS AND CHEMILUMINESCENT ACTIVITY OF
PYRIDAZINO[4,5-b]INDOLE-1,4(2H,3H)-DIONES
Masateru Kurumi, Kenji Sasaki,* Hiroko Takata, and Taiji Nakayama
Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka,
Okayama, 700-8530, Japan
Abstract - Substituted and non-substituted anilines were transformed to the corresponding
triazole derivatives, which were converted to dimethyl indole-2,3-dicarboxylates by photo-
cyclization. The reaction of diester with hydrazine hydrate gave 1,2,3,4-tetrahydro-5H-
pyridazino[4,5-b]indole-1,4(2H,3H)-diones (5). Some of compounds (5) were found to have
chemiluminescent activity in a similarly to luminol in the presence of hydrogen peroxide and
peroxidase in a basic solution.
Since the discovery of the chemiluminescence of luminol by Albrecht,1 its analytical usefulness has been
extensively studied.2 The chemiluminescent assay is an attractive analytical method because of its high
sensitivity, rapid reaction and wide dynamic range.3 Polycyclic heterocyclic compounds containing the
pyridazinedione moiety are well known to have chemiluminescent activity as luminol analogue.4 And the
development of luminol analogs has been performed actively to meet the requests of clinical analyses for
something more useful than luminol. We describe here the synthesis of pyridazino[4,5-b]indole-
1,4(2H,3H)-diones (5) as luminol analogue and evaluation of their chemiluminescent activity.
The synthetic route of pyridazinoindoles (5) from aniline derivatives (1) is shown in Scheme 1. Among
final compounds (5), 5a,5 5c,6 5d6 and 5'd6 have been already known. All these compounds have been
prepared by the reaction of the corresponding dimethyl (or diethyl) indole-2,3-dicarboxylates (4) with
hydrazine hydrate. This synthetic method for preparing the pyridazine-1,4-dione moiety from diester is
well known, so that we also employed this procedure in this study. As for preparing 4, Diels et al.7 have
reported the synthesis of 4a by the reaction of 1,2-diphenylhydrazine with DMAD. All other groups
described above also employed this strategy. Especially Bare et al.6 used this procedure for preparing

NH2
R1
N3
N
N
CO2Me
CO2Me
a
R1
R2
N
c
R2
R3
R1
R2
1a-d
R3
b
3a-e
NH2
2a-e
R3
d
R1 R2 R3
OMe
1e
a
H
H
H
OH
H
H
CO2Me
CO2Me
CO2Me
CO2Me
R2
HN
b
c
d
e
HN
R1
R2
OMe H
H
+
OMe
H
H
H
OMe
H
R3
4a-e
4'b,d
e
e
O
O
HN
NH
NH
HN
NH
NH
R1
R2
O
O
R2
R3
5'b,d
5a-e
a: i) N aNO2 /AcOH, conc.H2SO4. ii) NaN 3. b: i) NaNO2 /10% HCl. ii) NaN3.
c: DMAD/dioxane. d: hν/MeOH. e: NH2NH2•H2O/EtOH.
Scheme 1
diethyl analogue of 4c, 4d and 4'd, however, the yields of them were very low. Huntress et al.8 also
reported the synthesis of 4a by the reaction of phenylhydroxylamine with DMAD followed by heating in
xylene. But their method is also not so efficient. On the other hand, Nagawa et al.9 has reported the good
yield synthesis of diethyl 1H-benz[g]indole-2,3-dicarboxylate from naphthylamine in a few steps via
photocyclization of diethyl 1-(1-naphthyl)-1H-1,2,3-triazole-4,5-dicarboxylate. We applied this
procedure for preparing 4 in this work.
As shown in Scheme 1, at first, aniline derivatives (1) was converted to azidobenzenes (2) by the
method of Forster et al.10 In the case of 1e, using hydrochloric acid instead of H2SO4 improved the yield
of 2e. These azides (2) were used as a starting material for the next step without further purification
because of their sufficient purity. Compounds (2) were transformed to 4 via photocyclization reaction of
triazole (3) accompanied by denitrogenation by the method of Nagawa et al.9 In the case of the

photocyclization of 3b, two isomers (4b and 4'b) were obtained. Similarly, two isomers (4d and 4'd)
were obtained from 3d. All structures of 4 were well consisted with their instrumental and analytical
data. As described above compound (4a)7,8 has been already reported, however, its instrumental data
1
(including the H-NMR assignment) have not been described in detail. So, we report its detail data in
EXPERIMENTAL in this paper. Finally compounds (4) were derived to the corresponding desired
pyridazinoindoles (5) in 92-40% yields by the reaction of hydrazine hydrate. This reaction promptly
proceeded by using large excess of hydrazine hydrate and employing external heating at high
temperature (120 °C), which is higher than the refluxing temperature of EtOH. Compounds (5a,5 5c,6
1
5d6 and 5'd6) are already reported. But their spectral data (especially assignments of their H-NMR
spectra) have not been reported as in the case of 4. We now describe their detail data in
EXPERIMENTAL in this paper. All these instrumental data consisted their structures well.
The evaluation of the chemiluminescent activity of compounds (5) was performed in the presence of
Triton X-100, hydrogen peroxide, and Arthromyces ramosus peroxidase (ARP) in a phosphate buffer
solution at pH 8.0 and 10. The chemiluminescent intensity of these pyridazinoindoles is shown in Table
1. All of methoxy derivatives had chemiluminescent activity, however, these activities were 1/20-1/40 of
that of luminol at pH 10. These compounds showed increasing light intensity with rise in pH and this
phenomenon is the same as that of luminol. It seems that the presence of methoxy group is important for
chemiluminescent activity of this series.
Table 1 Chemiluminescent Production of Pyridazino[4,5-b]indole-
1,4(2H, 3H)dione Drivatives at pH 8 and 10
Compound
pH 8
pH 10
5a
5b
5'b
5c
NPa
NPa
NPa
NPa
NPa
NPa
2.3 x 10-3
2.7 x 10-3
2.9 x 10-3
2.5 x 10-3
3.7 x 10-1
2.5 x 10-2
5.7 x 10-2
2.0 x 10-2
3.1 x 10-2
5d
5'd
5e
Luminol
1.0b
Arthromyces ramosus peroxidase (ARP) was used as peroxydase. Amount of ARP was 55 Unit and
hydrogen peroxide was 0.22 mmol per 3 mL of test solution containing 0.3 mg of each compound.
a NP means no production of chmiluminescence. b Value of the total chmiluminescence intensity of
luminol at pH 10 is presented as 1.0 for standard.

EXPERIMENTAL
All melting points were determined on a Yanagimoto micro-melting point apparatus, and are
uncorrected. Elemental analyses were performed on a Yanagimoto MT-5 CHN Corder elemental
analyzer. The FAB-MS spectra were measured on a VG 70 mass spectrometer and glycerol or m-
nitrobenzyl alcohol was used as a matrix agent. The IR spectra were recorded on a Japan Spectroscopic
1
IRA-102 diffraction grating infrared spectrophotometer and frequencies are expressed in cm-1. H-NMR
spectra were recorded on a Varian VXR-200 instrument working at 200 MHz in CDCl3 with TMS as an
internal standard. Chemical shifts are given in ppm (δ) and J values in Hz. Photocyclization reaction was
performed using an Eikohsha High-Pressure Mercury Arcs EHB-W1 (500 watt). The chemiluminescence
intensity was measured with a Shimadzu Multiconvertible Spectrophotometer Double-40.
General Procedure of Preparation of Azide (2)
To a mixture of aniline derivative (107 mmol) in AcOH (92 mL) and conc. H2SO4 (43 mL) was added
sodium nitrite (7.70 g, 111 mmol) in water (50 mL) dropwise under vigorous stirring at 0-5 °C. After 10
min aqueous urea was added to the reaction mixture to remove excess sodium nitrite. Then sodium azide
(7.70 g, 118 mmol) in water (50 mL) was added to the reaction mixture at 0-5 °C for 3 h. The reaction
mixture was poured into ice water and the resulting mixture was basified with sodium hydroxide and
extracted with ethyl acetate. The organic layer was washed with water, dried over anhydrous sodium
sulfate and evaporated to give corresponding azide (2), which was employed as a starting material for
the next step without purification, because it gave almost single spot on TLC using several solvent
systems.
Azidobenzene (2a)
Aniline (1a) (10.0 g, 107 mmol) was used as a starting material and 2a (12.4 g, 104 mmol) was obtained
as pale yellow viscous oil (97%), IR (neat) cm-1: 2100 (N3).
3-Azidophenol (2b)
3-Aminophenol (1b) (6.00 g, 55.0 mmol) was used as a starting material and 2b (6.80 g, 50.0 mmol)
was obtained as brown viscous oil (91%), IR (neat) cm-1: 2110 (N3).
2-Azidoanisole (2c)
2-Aminoanisole (1c) (10.0 g, 81.3 mmol) was used as a starting material and 2c (12.0 g, 80.5 mmol)
was obtained as brown viscous oil (99%), IR (neat) cm-1: 2110 (N3).
3-Azidoanisole (2d)

3-Aminoanisole (1d) (1.00 g, 8.10 mmol) was used as a starting material and 2d (1.14 g, 7.65 mmol)
was obtained as brown viscous oil (94%), IR (neat) cm-1: 2145 (N3).
4-Azidoanisole (2e)
4-aminoanisole (1e) (233 mg, 1.90 mmol) was used as a starting material and 10% hydrochloric acid (10
mL) was used instead of AcOH and conc. H2SO4. Compound (2e) (167 mg, 1.12 mmol) was obtained as
pale brown crystals (59%), IR (neat) cm-1: 2105 (N3).
General Procedure of Preparation of Triazole (3)
To a solution of 2 (100 mmol) in dioxane (200 mL) was added dimethyl acetylenedicarboxylate
(DMAD, 56.8 g, 400 mmol) and the resulting mixture was stirred at rt for 10 d in a dark. After
evaporation of the solvent the viscous oily residue was triturated with n-hexane. Precipitated solid was
collected, washed with ether and recrystallized from appropriate solvent to give corresponding 3.
Dimethyl 1-Phenyl-1H-1,2,3-triazole-4,5-dicarboxylate (3a)
Compound (2a) was employed as a starting material and 3a was obtained as colorless needles (from
MeOH), yield 60%, mp 124-125 °C. FAB-MS m/z: 262 (MH+). IR (KBr) cm-1: 1725 (CO). NMR
(CDCl3) δ: 3.92 (3H, s, 5-ester Me), 4.01 (3H, s, 4-ester Me), 7.56 (5H, s, Ph-H). Anal. Calcd for
C12H11N3O4: C, 55.17; H, 4.24; N, 16.09. Found: C, 54.77; H, 4.25; N, 16.05.
Dimethyl 1-(3-Hydroxyphenyl)-1H-1,2,3-triazole-4,5-dicarboxylate (3b)
Compound (2b) was employed as a starting material and 3b was obtained as colorless needles (from
benzene), yield 58%, mp 158-160 °C. FAB-MS m/z: 555 (2MH+), 278 (MH+). IR (KBr) cm-1: 3240
(OH), 1732 (CO). NMR (DMSO-d6) δ: 3.85 (3H, s, 5-ester Me), 3.90 (3H, s, 4-ester Me), 6.95-7.06 (3H,
m, 2'-, 4'- and 6'-H), 7.43 (1H, t, J = 7.9, 5'-H), 10.26 (1H, br s, OH). Anal. Calcd for C12H11N3O5: C,
51.99; H, 4.00; N, 15.16. Found: C, 51.92; H, 4.21; N, 15.34.
Dimethyl 1-(2-Methoxyphenyl)-1H-1,2,3-triazole-4,5-dicarboxylate (3c)
Compound (2c) was employed as a starting material. In this case solid did not precipitate. So the residue
was column-chromatographed on silica gel and eluate by n-hexane-ethyl acetate (3:2, v/v) gave 3c as
yellow granules (from benzene-n-hexane), yield quantitatively, mp 68-69 °C. FAB-MS m/z: 292 (MH+).
IR (KBr) cm-1: 1740 (CO). NMR (CDCl3) δ: 3.78, 3.86, 4.01 (each 3H, each s, Me x 3), 7.05 (1H, dd, J =
8.0, J = 1.1, 3'-H), 7.14 (1H, td, J = 8.0, J = 1.1, 5'-H), 7.52 (1H, td, J = 7.9, J = 1.5, 4'-H), 7.57 (1H, dd,
J = 8.0, J = 1.5, 6'-H). Anal. Calcd for C13H13N3O5: C, 53.61; H, 4.50; N, 14.43. Found: C, 53.37; H,
4.85; N, 14.58.

Dimethyl 1-(3-Methoxyphenyl)-1H-1,2,3-triazole-4,5-dicarboxylate (3d)
Compound (2d) was employed as a starting material. In this case also solid did not precipitate. So the
residue was column-chromatographed on silica gel and eluate by n-hexane-ethyl acetate (7:3, v/v) gave
3d as colorless needles (from benzene-n-hexane), yield 85%, mp 94-95 °C. FAB-MS m/z: 292 (MH+).
IR (KBr) cm-1: 1725 (CO). NMR (CDCl3) δ: 3.87, 3.93, 4.01 (each 3H, each s, Me x 3), 7.05-7.14 (3H,
m, 2’-, 4’- and 6’-H), 7.44 (1H, td, J = 8, J = 1, 5’-H). Anal. Calcd for C13H13N3O5: C, 53.61; H, 4.50; N,
14.43. Found: C, 53.45; H, 4.50; N, 14.56.
Dimethyl 1-(4-Methoxyphenyl)-1H-1,2,3-triazole-4,5-dicarboxylate (3e)
Compound (2e) was employed as a starting material. In this case also solid did not precipitate. So the
residue was column-chromatographed on silica gel and eluate by n-hexane-ethyl acetate (13:7, v/v) gave
3e as colorless needles (from benzene-n-hexane), yield 94%, mp 96-97 °C. FAB-MS m/z: 292 (MH+). IR
(KBr) cm-1: 1745, 1720 (CO). NMR (CDCl3) δ: 3.88, 3.91, 4.00 (each 3H, each s, Me x 3), 7.02 (2H, br
d, J = 6.8, 3'- and 5'-H), 7.46 (2H, br d, J = 6.8, 2'- and 6'-H). Anal. Calcd for C13H13N3O5: C, 53.61; H,
4.50; N, 14.43. Found: C, 53.38; H, 4.48; N, 14.49.
General Procedure of Preparation of 1H-Indole-2,3-dicarboxylates (4)
A solution of 3 in MeOH was irradiated with a 500 watt high pressure mercury lamp under nitrogen
atmosphere at rt. After evaporation of the solvent the residue was column-chromatographed on silica gel
to isolate the desired 4.
Dimethyl 1H-Indole-2,3-dicarboxylate (4a)
Compound (3a) (300 mg, 1.15 mmol) in MeOH (500 mL) was employed as a starting material. The
reaction time was 2 h. The eluate by benzene-ether (9:1, v/v) gave 4a as colorless needles (from
benzene-n-hexane), yield 77%, mp 112-113 °C (lit., 114 °C7, 112-112.5 °C8). FAB-MS m/z: 202 (MH+ -
32), 234 (MH+). IR (KBr) cm-1: 3305 (NH), 1725, 1690 (CO). NMR (CDCl3) δ: 3.99 (6H, s, Me x 2),
7.22-7.42 (2H, m, 5- and 6-H), 7.45 (1H, br d, J = 7, 7-H), 8.06 (1H, br d, J = 8.2, 4-H), 9.36 (1H, br s,
NH). Anal. Calcd for C12H11NO4: C, 61.80; H, 4.75; N, 6.00. Found: C, 61.63; H, 4.75; N, 5.83.
Dimethyl 6-Hydroxy-1H-indole-2,3-dicarboxylate (4b) and Dimethyl 4-Hydroxy-
1H-indole-2,3-dicarboxylate (4’b)
Compound (3b) (300 mg, 1.08 mmol) in MeOH (600 mL) was employed as a starting material. The
reaction time was 3 h. The former eluate by n-hexane-ethyl acetate (3:1, v/v) gave 4b as yellow granules
(from benzene) and the latter eluate by the same eluent afforded a mixture of unreacted 3b and 4’b.

This mixture was re-chromatographed on silica gel and the eluate by chloroform-ethyl acetate (8:2, v/v)
gave 4’b as colorless needles (from benzene-ether).
4b: yield 28%, mp 222-223 °C. FAB-MS m/z: 250 (MH+). IR (KBr) cm-1: 3320, 3260 (NH and OH),
1691, 1672 (CO). NMR (CDCl3) δ: 3.82 and 3.86 (each 3H, each s, ester Me x 2), 6.75 (1H, dd, J = 8.8,
J = 2.1, 5-H), 6.82 (1H, d, J = 2.1, 7-H), 7.66 (1H, d, J = 8.8, 4-H), 9.55 (1H, s, NH or OH), 12.14 (1H,
br s, NH or OH). Anal. Calcd for C12H11NO5: C, 57.83; H, 4.45; N, 5.62. Found: C, 58.01; H, 4.61; N,
5.62.
4’b: yield 18%, mp 143-144 °C (decomp). FAB-MS m/z: 250 (MH+). IR (KBr) cm-1: 3440, 3205 (NH
and OH), 1740 (CO). NMR (CDCl3) δ: 3.99 and 4.00 (each 3H, each s, ester Me x 2), 6.73 (1H, d, J = 8,
5-H), 6.92 (1H, d, J = 8, 7-H), 7.27 (1H, t, J = 8, 6-H), 9.25 (1H, br s, NH or OH), 10.73 (1H, s, NH or
OH). Anal. Calcd for C12H11NO5: C, 57.83; H, 4.45; N, 5.62. Found: C, 57.66; H, 4.44; N, 5.37.
Dimethyl 7-Methoxy-1H-indole-2,3-dicarboxylate (4c)
Compound (3c) (100 mg, 0.344 mmol) in MeOH (500 mL) was employed as a starting material. The
reaction time was 1.5 h. The eluate by n-hexane-ethyl acetate (7:3, v/v) gave 4c as colorless prisms
(from benzene-n-hexane), yield 43%, mp 108-109 °C. FAB-MS m/z: 264 (MH+). IR (KBr) cm-1: 3300
(NH), 1700 (CO). NMR (CDCl3) δ: 3.978, 3.982 and 3.99 (each 3H, each s, Me x 3), 6.76 (1H, d, J = 8,
6-H), 7.18 (1H, t, J = 8, 5-H), 7.62 (1H, d, J = 8, 4-H), 9.42 (1H, br s, NH). Anal. Calcd for C13H13NO5:
C, 59.31; H, 4.98; N, 5.32. Found: C, 59.24; H, 4.87; N, 5.07.
Dimethyl 6-Methoxy-1H-indole-2,3-dicarboxylate (4d) and Dimethyl 4-Methoxy-
1H-indole-2,3-dicarboxylate (4’d)
Compound (3d) (500 mg, 1.72 mmol) in MeOH (600 mL) was employed as a starting material. The
reaction time was 3.5 h. Unreacted 3d (75.0 mg) was recovered from the former eluate by n-hexane-
ethyl acetate (3:1, v/v). The latter eluate by the same eluent afforded 4d as colorless prisms (from
benzene) and then 4’d as colorless needles (from benzene) respectively.
4d: yield 48%, mp 138-140 °C (decomp). FAB-MS m/z: 232 (MH+ - 32), 264 (MH+). IR (KBr) cm-1:
3325 (NH), 1730, 1685 (CO). NMR (CDCl3) δ: 3.86, 3.976 and 3.982 (each 3H, each s, Me x 3), 6.83
(1H, d, J = 2.2, 7-H), 6.93 (1H, dd, J = 9.0, J = 2.2, 5-H), 7.91 (1H, d, J = 9.0, 4-H), 9.18 (1H, br s, NH).
Anal. Calcd for C13H13NO5: C, 59.31; H, 4.98; N, 5.32. Found: C, 59.03; H, 4.91; N, 5.06.
4’d: yield 33%, mp 150-152 °C. FAB-MS m/z: 232 (MH+ - 32), 264 (MH+). IR (KBr) cm-1: 3360 (NH),
1715 (CO). NMR (CDCl3) δ: 3.92, 3.93 and 3.99 (each 3H, each s, Me x 3), 6.54 (1H, br d, J = 8, 5-H),

7.00 (1H, dd, J = 8, J = 1, 7-H), 7.27 (1H, t, J = 8, 6-H), 8.95 (1H, br s, NH). Anal. Calcd for C13H13NO5:
C, 59.31; H, 4.98; N, 5.32. Found: C, 59.45; H, 4.86; N, 5.11.
Dimethyl 5-Methoxy-1-H-indole-2,3-dicarboxylate (4e)
Compound (3e) (200 mg, 0.687 mmol) in MeOH (500 mL) was employed as a starting material. The
reaction time was 15 min. After evaporation of the solvent the crystalline residue was recrystallized from
benzene-n-hexane to give 4e as pale yellow granules and unchanged 3e (20.3 mg) was recovered from
its mother liquor.
4e: yield 69%, mp 152-154 °C (decomp). FAB-MS m/z: 232 (MH+ - 32), 264 (MH+). IR (KBr) cm-1:
3285 (NH), 1720, 1690 (CO). NMR (CDCl3) δ: 3.89 (3H, s, methoxy Me), 3.99 (6H, s, ester Me), 7.04
(1H, dd, J = 9.2, J = 2.5, 6-H), 7.32 (1H, d, J = 9.2, 7-H), 7.48 (1H, d, J = 2.5, 4-H), 9.16 (1H, br s, NH).
Anal. Calcd for C13H13NO5: C, 59.31; H, 4.98; N, 5.32. Found: C, 59.43; H, 4.90; N, 4.95.
General Procedure of Preparation of 1,2,3,4-Tetrahydro-5H-pyridazino [4,5-b]-
indole-1,4 (2H,3H)- diones (5)
A mixture of 4 and 100% hydrazine hydrate in EtOH (280 mL) was refluxed for appropriate period.
After evaporation of the reaction mixture the resulting crystalline residue was dissolved in DMSO. The
solution was acidified with diluted hydrochloric acid and precipitated crystalline solid was washed with
water and recrystallized from an appropriate solvent to afford 5.
1,2,3,4-Tetrahydro-5H-pyridazino[4,5-b]indole-1,4(2H,3H)-dione (5a)
A mixture of 4a (1.30 g, 5.58 mmol) and 100% hydrazine hydrate (55.0 g, 1.10 mol) in EtOH (280 mL)
was employed. The reflux time was 5 h. Recrystallization from MeOH gave 5a as pale yellow powder,
yield 92%, mp >300 °C (lit., >300 °C5a, >330 °C5b, >330 °C5c). FAB-MS m/z: 202 (MH+), 403 (2MH+).
IR (KBr) cm-1: 3420, 3135, 2980 (NH), 1638 (CO). NMR (DMSO-d6) δ: 7.30 and 7.47 (each 1H, each br
t, J = 7.3, 7- and 8-H), 7.61 (1H, br d, J = 7.3, 6-H), 8.11 (1H, br d, J = 7.3, 9-H), 11.55 (2H, s, NH x 2),
12.54 (1H, s, NH). Anal. Calcd for C10H7N3O2·1/5H2O: C, 58.65; H, 3.64; N; 20.52. Found: C, 58.98; H,
4.03; N, 20.54.
7-Hydroxy-1,2,3,4-tetrahydro-5H-pyridazino[4,5-b]indole-1,4(2H,3H)-dione (5b)
A mixture of 4b (670 mg, 2.70 mmol) and 100% hydrazine hydrate (13.5 g, 270 mmol) in MeOH (68
mL) was employed. The reflux time was 5 h. Recrystallization from acetone gave 5b as pale yellow
powder, yield 74%, mp >300 °C. FAB-MS m/z: 218 (MH+), 435 (2MH+). IR (KBr) cm-1: 3308, 3248,
3148 (NH and OH), 1650 (CO). NMR (DMSO-d6) δ: 6.77 (1H, dd, J = 8.5, J = 2.0, 8-H), 6.91 (1H, d, J

= 2.0, 6-H), 7.85 (1H, d, J = 8.5, 9-H), 9.65 (1H, br s, OH or NH), 11.40 (2H, br, OH or NH), 12.13 (1H,
s, OH or NH). Anal. Calcd for C10H7N3O3: C, 55.30; H, 3.25; N; 19.35. Found: C, 55.31; H, 3.56; N,
19.17.
9-Hydroxy-1,2,3,4-tetrahydro-5H-pyridazino[4,5-b]indole-1,4(2H,3H)-dione (5’b)
A mixture of 4’b (150 mg, 0.602 mmol) and 100% hydrazine hydrate (3.00 g, 60.0 mmol) in MeOH (15
mL) was employed. The reflux time was 5 h. Recrystallization from MeOH gave 5’b as white powder,
yield 40%, mp >300 °C. FAB-MS m/z: 218 (MH+). IR (KBr) cm-1: 3437, 3167, 3032 (NH and OH), 1620
(CO). NMR (DMSO-d6) δ: 6.55 and 6.99 (each 1H, each d, J = 8.0, 6- and 8-H), 7.30 (1H, t, J = 8.0, 7-
H), 11.95 (1H, br s, OH or NH), 12.48 (1H, s, OH or NH). Anal. Calcd for C10H7N3O3·1/3H2O: C, 53.82;
H, 3.46; N; 18.83. Found: C, 53.95; H, 3.66; N, 18.65.
6-Methoxy-1,2,3,4-tetrahydro-5H-pyridazino[4,5-b]indole-1,4(2H,3H)-dione (5c)
A mixture of 4c (1.40 g, 5.32 mmol) and 100% hydrazine hydrate (42.0 g, 840 mmol) in EtOH (280 mL)
was employed. The reflux time was 2 h. Recrystallization from EtOH gave 5c as white powder, yield
73%, mp >300 °C (lit.,6 >260 °C). FAB-MS m/z: 232 (MH+). IR (KBr) cm-1: 3310, 3065, 2970, 2890
(NH), 1655 (CO). NMR (DMSO-d6) δ: 3.96 (3H, s, OMe), 6.99 and 7.68 (each 1H, each d, J = 7.3, 7-
and 9-H), 7.21 (1H, t, J = 7.3, 8-H). Anal. Calcd for C11H9N3O3·1/6H2O: C, 56.41; H, 4.02; N; 17.94.
Found: C, 56.69; H, 4.32; N, 17.94.
7-Methoxy-1,2,3,4-tetrahydro-5H-pyridazino[4,5-b]indole-1,4(2H,3H)-dione (5d)
A mixture of 4d (170 mg, 0.650 mmol) and 100% hydrazine hydrate (14.0 g, 280 mmol) in MeOH (30
mL) was employed. The reflux time was 8.5 h. Recrystallization from acetone-n-hexane gave 5d as pale
yellow powder, yield 82%, mp >300 °C (lit.,6 >260 °C). FAB-MS m/z: 232 (MH+), 463 (2MH+). IR
(KBr) cm-1: 3150, 3040, 2960 (NH), 1630 (CO). NMR (DMSO-d6) δ: 3.84 (3H, s, OMe), 6.93 (1H, dd, J
= 8.7, J = 2.1, 8-H), 7.01 (1H, d, J = 2.1, 6-H), 7.94 (1H, d, J = 8.7, 9-H), 11.45 (2H, br s, NH x 2), 12.40
(1H, br s, NH). Anal. Calcd for C11H9N3O3·1/4H2O: C, 56.05; H, 4.06; N; 17.83. Found: C, 56.23; H,
4.15; N, 17.91.
9-Methoxy-1,2,3,4-tetrahydro-5H-pyridazino[4,5-b]indole-1,4(2H,3H)-dione (5’d)
A mixture of 4’d (430 mg, 1.63 mmol) and 100% hydrazine hydrate (13.0 g, 260 mmol) in EtOH (86
mL) was employed. The reflux time was 2 h. Recrystallization from acetone-n-hexane gave 5’d as
colorless granules, yield 69%, mp >300 °C (lit.,6 >260 °C). FAB-MS m/z: 232 (MH+). IR (KBr) cm-1:
3285, 3035, 2910, 2750 (NH), 1650 (CO). NMR (DMSO-d6) δ: 4.00 (3H, s, OMe), 6.84 and 7.21 (each

1H, each d, J = 8.0, 6- and 8-H), 7.42 (1H, t, J = 8.0, 7-H), 10.30, 11.70 and 12.80 (each 1H, each br, NH
x 3). Anal. Calcd for C11H9N3O3·1/5H2O: C, 56.27; H, 4.04; N; 17.90. Found: C, 56.32; H, 4.27; N,
17.78.
8-Methoxy-1,2,3,4-tetrahydro-5H-pyridazino[4,5-b]indole-1,4(2H,3H)-dione (5e)
A mixture of 4e (400 mg, 1.50 mmol) and 100% hydrazine hydrate (12.0 g, 240 mmol) in MeOH (50
mL) was employed. The reflux time was 3 h. Recrystallization from acetone-n-hexane gave 5e as pale
brown powder, yield 63%, mp >300 °C. FAB-MS m/z: 232 (MH+). IR (KBr) cm-1: 3125, 3040, 2960
(NH), 1650 (CO). NMR (DMSO-d6) δ: 3.83 (3H, s, OMe), 7.10 (1H, dd, J = 9.0, J = 2.6, 7-H), 7.49 (1H,
d, J = 9.0, 6-H), 7.53 (1H, d, J = 2.6, 9-H), 11.49 (2H, br s, NH x 2), 12.40 (1H, br s, NH). Anal. Calcd
for C11H9N3O3·1/5H2O: C, 56.27; H, 4.04; N; 17.90. Found: C, 56.56; H, 4.26; N, 17.61.
Evaluation of Chemiluminescent Activity
The reaction solution contains 10 mM phosphate buffer (for pH 8) or 10 mM carbonate buffer (for pH
10), Triton X-100 (0.05%) and test compound (0.1 mg/mL). The concentration of hydrogen peroxide
solution was 3 mM. Arthromyces ramosus peroxidase (ARP) solution was prepared as 730 U/mL. The
reaction solution (3 mL) was transferred to a sample tube and immediately placed in a water bath (37 °C)
for 10 min. At the end of the incubation period, the sample tube to be counted was incorporated into a
luminometer. Photons were counted after addition of 75 µL of hydrogen peroxide solution (final
concentration is 0.22 mmol of hydrogen peroxide/3 mL of the reaction solution) and 75 µL of ARP
solution (final concentration is 55 Unit of ARP/3 mL of the reaction solution).
ACKNOWLEDGEMENTS
We are grateful to The SC-NMR Laboratory of Okayama University for 200 MHz 1H-NMR experiment.
REFERENCES
1.
2.
H. O. Albrecht, Z. Phys. Chem., 1928, 136, 321.
Bioluminescence and Chemiluminescence, ed. by A. K. Campbell, L. J. Kricka, and P. E. Stanley,
John Wiley & Sons, Chichester, 1994 and 1996, and references cited therein.
3.
a) D. C. Young, S. D. Ryan, and F. J. Dutko, Anal. Biochem., 1993, 215, 24; b) J. A. Matthews,
A. Batki, C. Hynds, and L. J. Kricka, Anal. Biochem., 1985, 151, 205; c) M. P. Wymann, V.
Tscharner, D. A. Deraulean, and M. Baggiolini, Anal. Biochem., 1987, 165, 371; d) R. Lock, A.

Johansson, K. Orselius, and C. Dahlgren, Anal. Biochem., 1988, 173, 450; e) H. P. Misra and P.
M. Squatrito, Arch Biochem. Biophys, 1982, 215, 59; f) J. Arnhold, S. Mueller, K. Arnold, and
K. Sonntag, J. Biolumin. Chemilumin., 1991, 6, 189; g) G. Merenyi, J. Lind, and T. E.
Eriksen, J. Biolumin. Chemilumin., 1990, 5, 53.
4.
a) T. G. Burdo and W. Rudolf Seitz, Anal. Chem., 1975, 47, 1639; b) R. B. Brundrett, D. F.
Roswell, and E. H. White, J. Am. Chem. Soc., 1972, 94, 7536; c) C. C. Wei and E. H. White,
Tetrahedron Lett., 1971, 3559; d) M. Ii, H. Yoshida, Y. Aramaki, H. Masuya, T. Hada, M.
Terada, M. Hatanaka, and Y. Ichimori, Biochem. Biophys. Res. Comm., 1993, 193, 540.
a) E. H. Huntress and W. M. Hearon, J. Am. Chem. Soc., 1941, 63, 2762; b) A. Monge and I.
Aldana, J. Heterocycl. Chem., 1981, 18, 1533; c) A. Güven and R. A. Jones, Tetrahedron, 1993,
49, 11145.
5.
6.
T. M. Bare, J. F. Resch, and P. F. Jackson, Eup. Pat. 512 817. (Chem. Abstr., 1993, 118,
101975g).
7.
8.
9.
O. Diels and J. Reese, Ann., 1934, 511, 168.
E. H. Huntress, T. E. Lesslie, and W. M. Hearon, J. Am. Chem. Soc., 1956, 78, 419.
a) Y. Nagawa, K. Honda, and H. Nakanishi, Bull. Chem. Soc. Jpn., 1987, 60, 2931; b) Y.
Nagawa, M. Goto, K. Honda, and H. Nakanishi, Bull. Chem. Soc. Jpn., 1989, 62, 3109; c) Y.
Nagawa, K. Honda, and H. Nakanishi, Heterocycles, 1999, 51, 1093.
10.
M. O. Forster and H. E. Fierz, J. Chem. Soc., 1907, 91, 1942.