1,5-Disubstituted indazol-3-ols with anti-inflammatory activity

Article abstract of DOI:10.1002/(SICI)1521-4184(199801)331:1<13::AID-ARDP13>3.0.CO;2-M

A series of new indazol-3-ol derivatives was synthesized. Some of these compounds exhibit interesting anti-inflammatory activities in various models of inflammation. 5-Methoxy-1-[quinoline-2-yl-methoxy)-benzyl]-1H-indazol-3- ol (27) strongly inhibits the oxidation of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid catalyzed by 5-lipoxpgenase (IC₅₀ = 44 nM). 27 also inhibits the contraction of sensitized guinea pig tracheal segments (IC₅₀ = 2.9 μM). In guinea pigs treated with 27 (1 mg/kg i.p.) 2 h before antigen provocation, there was a marked inhibition (47%) of the antigen-induced airway eosinophilia. After topical application of 1 μg/ear 27 inhibits the arachidonic acid induced mouse ear edema (41%).

Full text of DOI:10.1002/(SICI)1521-4184(199801)331:1<13::AID-ARDP13>3.0.CO;2-M

1,5-Disubstituted Indazol-3-ols
13
1,5-Disubstituted Indazol-3-ols with Anti-Inflammatory Activity ✩
a)
b)
a)
a)
a)
a)
Rudolf Schindler* , Ilona Fleischhauer , Norbert Höfgen , Wolfgang Sauer , Ute Egerland , Hildegard Poppe ,
a)
a)
b)
b)
Sabine Heer , Istvan Szelenyi , Bernhard Kutscher , and Jürgen Engel
a) Department of Chemical Research, Corporate R&D, ASTA Medica Group, Arzneimittelwerk Dresden GmbH, Meissner Str. 35, D-01445 Radebeul,
Germany
b) ASTA Medica AG, Weismuellerstr. 45, D-60314 Frankfurt a. M., Germany
Key Words: Indazole derivatives; inhibitors of 5-lipoxygenase; antiinflammatory activity; asthma
In the following we report about synthesis and biological
activities of indazol-3-ols – a new class of 5-LOX inhibitors.
Summary
A series of new indazol-3-ol derivatives was synthesized. Some of
these compounds exhibit interesting anti-inflammatory activities
in various models of inflammation. 5-Methoxy-1-[quinoline-2-yl-
methoxy)-benzyl]-1H-indazol-3-ol (27) strongly inhibits the oxi-
dation of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid
catalyzed by 5-lipoxygenase (IC50 = 44 nM). 27 also inhibits the
Results and Discussion
Chemistry
[5,6]
Indazol-3-ols are synthesized by common procedures
.
contraction of sensitized guinea pig tracheal segments (IC50
=
We used 5-substituted 2-halogenobenzoic acids (3) and hy-
drazine hydrate as starting materials which were heated to
give hydrazino benzoic acid derivatives. Acid catalyzed cy-
clization of these intermediates yielded 5-substituted indazol-
3-ols (4,5). 5-Methoxy-1H-indazol-3-ol (4) and 5-nitro-
1H-indazol-3-ol (5) were used for further derivatisation. We
focused on N-1 alkylation with halogenoalkylaryls or -het-
eroaryls. As potential side products N-2, O- and various
bisalkyl derivatives may occur. For the synthesis of 5-substi-
tuted 1-alkyl-indazol-3-ols (6–50) best selectivity was
achieved by using aqueous sodium hydroxide.
2.9 µM). In guinea pigs treated with 27 (1 mg/kg i.p.) 2 h before
antigen provocation, there was a marked inhibition (47%) of the
antigen-induced airway eosinophilia. After topical application of
1 µg/ear 27 inhibits the arachidonic acid induced mouse ear edema
(41%).
Introduction
Allergic diseases like asthma are characterized by inflam-
matory processes. Important mediators involved in these
processes are arachidonic acid metabolites, such as leuko-
trienes, prostaglandins, and thromboxanes. The oxidative
formation of leukotrienes is catalyzed by the enzyme 5-
lipoxygenase (5-LOX). This enzyme has been found in eosi-
nophils, neutrophils, monocytes, macrophages, mast cells,
basophils, and B lymphocytes. Therefore inhibition of this
enzyme is a promising approach in the treatment of asthma
and other allergic diseases. Recent studies have demonstrated
[1]
the therapeutic benefit of 5-LOX inhibitors . Zileuton (1) is
one of the first launched 5-LOX inhibitors for the treatment
of asthma (Scheme 1). In comparison to treatment with as
needed inhaled salbutamol the additional administration of
zileuton in case of mild to moderate bronchial asthma inhib-
Scheme 2
Starting with 4 we synthesized a wide range of substituted
1-benzyl derivatives varying the substitution pattern in the
benzyl moiety (Table 1). Many of them proved to be active
in the in vitro testing of 5-LOX inhibition.
The effect of modification of the substituents in the aro-
matic ring of the benzyl moiety is summarized as follows:
ited asthma exacerbations, improved the lung function, and
[2,3]
reduced the use of salbutamol
. BAY × 1005 (2) has been
reported as leukotriene synthesisinhibitor(Scheme 1). Added
to inhaled corticosteroids it has a significant effect on the
[4]
pulmonary function . Several other 5-LOX inhibitors are
under development.
mono subst. halogen:
disubst. halogen:
alkyl:
4-Cl > 3-Cl > 2-Cl
4-Cl > 4-Br > 4-F > 4-CF
3
2,4-Cl > 3,4-Cl > 2,6-Cl >
2
2
2
2-Cl, 6-F
4-tBu > 3-Me, 4-Me >
2-MeO, 4-MeO > 3-MeO,
4-MeO, 5-MeO
(hetero)aryl:
4-O-CH -quinolin-2-yl >
2
4-O-Bzl > Ph
Scheme 1
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0365-6233/98/0101/0013 $17.50 +.50/0

14
Schindler and co-workers
Table 1. 5-LOX inhibition of 1-benzyl-5-methoxy-1H-indazol-3-ols.
O
H
CH3
O
N
N
R
——————————————————————————————————————————————————
No. Mp (°C) MW
Analysis C, H, Na IC50 [nM]b
——————————————————————————————————————————————————
R
6
4-Br
187–189
194–196
167–168
190–191
202–204
214–215
194–195
214–215
189
333.19
288.74
288.74
288.74
272.28
323.18
323.18
323.18
306.73
306.73
333.73
322.29
315.29
314.34
344.37
282.35
310.40
298.30
312.33
330.39
360.42
C15H13BrN2O2
C15H13ClN2O2
C15H13ClN2O2
C15H13ClN2O2
C15H13FN2O2
C15H12Cl2N2O2
C15H12Cl2N2O2
C15H12Cl2N2O2
C15H12ClFN2O2
C15H12ClFN2O2
C15H12ClN3O4
C16H13F3N2O2
C15H13N3O5
765 ± 132
400 ± 88
470 ± 76
710 ± 120
1100 ± 146
100 ± 32
410 ± 33
750 ± 156
>1000
7
4-Cl
8
3-Cl
9
2-Cl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4-F
2,4-Cl2
3,4-Cl2
2,6-Cl2
2-Cl, 6-F
3-Cl, 2-F
4-Cl, 2-NO2
4-CF3
194–195
212–215
165–167
224–227
151–154
180
829 ± 153
>1000
>1000
2-OH, 4-NO2
2,4-(OMe)2
3,4,5-(OMe)3
3,4-(Me)2
4-tBu
>1000
C17H18N2O4
>1000
C18H20N2O5
>1000
209
C17H18N2O2
977 ± 207
696 ± 103
>1000
202–205
220–223
182–186
214–218
177.5–179
C19H22N2O2
4-COOH
4-CH2-COOH
4-Ph
C16H14N2O4
C17H16N2O4
>1000
C21H18N2O2
257 ± 57
171 ± 31
4-O-Bzl
C22H20N2O3
CH2
181
484.39
C25H23Cl2N3O3
27
44 ± 8
N
O
2 HCl
—————————————————————————————————————————————
Zileuton (1)
2160 ± 291
18.9 ± 2.9
Bay x 1005 (2)
—————————————————————————————————————————————
a: Analysis C, H, N within ± 0.4% unless otherwise indicated. b: Mean of 4 independent experiments.
Arch. Pharm. Pharm. Med. Chem. 331, 13–21 (1998)

1,5-Disubstituted Indazol-3-ols
15
Table 2. 5-LOX inhibition of 1-substituted 5-methoxy-1H-indazol-3-ols (further CH2 spacer linked derivatives).
O
H
CH3
O
N
N
R
————————————————————————————————————————————————————
No. Mp (°C) MW
Analysis C, H, Na IC50 [nM]b
R
————————————————————————————————————————————————————
CH2
187
304.35
C19H16N2O2
28
265 ± 56
N
CH2
180–184
166–167
305.34
255.28
C18H15N3O2
C14H13N3O2
> 1000
> 1000
29
30
CH2
N
CH2
153
255.28
C14H13N3O2
> 1000
31
N
CH2
N
150
186
255.28
260.32
C14H13N3O2
> 1000
32
33
CH2
C13H12N2O2S
500 ± 110
S
CH2
N
191–195
210–214
273.29
294.32
C14H15N3O3
> 1000
> 1000
34
35
O
N
N
CH2
C16H14N4O2
H
CH2
Cl
O
O
227–229
332.75
C16H13ClN2O4
36
874 ± 179
————————————————————————————————————————————————————
a: Analysis C, H, N within ± 0.4% unless otherwise indicated. b: Mean of 4 independent experiments.
Arch. Pharm. Pharm. Med. Chem. 331, 13–21 (1998)

16
Schindler and co-workers
Table 3. 5-LOX inhibition of 1-substituted 5-methoxy-1H-indazol-3-ols (more than one atom between the indazole skeleton and the phenyl ring).
—————————————————————————————————————————————————————————————
No.
R
Mp (°C)
MW
Analysis C, H, Na
IC50 [nM]b
—————————————————————————————————————————————————————————————
37
38
39
40
41
42
43
44
45
46
(CH2)3Ph
160
282.34
280.33
284.32
298.34
296.33
313.32
329.32
328.35
357.37
353.21
C17H18N2O2
C17H16N2O2
C16H16N2O3
C17H18N2O3
C17H16N2O3
C16H15N3O4
C16H15N3O5
C18H17FN2O3
C18H19N3O5
C16H14Cl2N2O3
150 ± 36
299 ± 47
745 ± 130
655 ± 123
> 1000
CH2CH=CHPh
183–187
165
(CH2)2OPh
(CH2)3OPh
138–142
171–173
238–240
221–224
163–165
225
(CH2)2COPh
(CH2)2 C6H4NO2(4)
(CH2)2OC6H4NO2(4)
(CH2)3COC6H4F(4)
CH2CONHC6H3(OMe)2(3,4)
CH2CHOHC6H3Cl2(2,4)
> 1000
> 1000
679 ± 118
> 1000
232–235
> 1000
—————————————————————————————————————————————————————————————
a: Analysis C, H, N within ± 0.4% unless otherwise indicated. b: Mean of 4 independent experiments.
The most active compounds of this series are 11 and 27.
By reduction of the 5-nitro group we obtained 5-amino-in-
Compounds with a heteroaryl- or naphthyl moiety linked dazol-3-ols (51–53) (Scheme 3).
51–53 were precursors of 5-urea derivatives (54–57) ob-
by a methylene group to the indazole skeleton were also
prepared (Table 2).
Only the 1-naphthalen-2-ylmethyl derivative 28 and the
tained by reaction with isocyanates. In addition, 51–53 were
used to prepare indazol-3,5-diols 58–60 via diazotisation and
elimination of nitrogen. Alternatively these compounds are
accessible by ether cleavage of 7, 11, or 12. Selected com-
pounds with 5-amino-, 5-urea, and 5-hydroxy-substituent are
summarized in Table 4. With the exception of 54, 59, 60 none
of these compounds showed any activity.
1-thiophen-2-ylmethyl substituted indazole 33 inhibited the
enzyme 5-LOX in concentrations smaller than 1 µM. The
heterocylic substituted compounds show no or only little
activity.
Extending the linkage between the indazole skeleton and
the aryl ring resulted in some derivatives with moderate in
vitro activity (Table 3).
Structure-Activity Discussion
The propyl linked (37) and the allyl linked (38) indazoles
have the highest activity in the series of more than one atom
spacer linked compounds.
According to the (preliminary) results of the in-vitro assay
indazole derivatives with the following substitution pattern
did not show any 5-LO activity:
The synthesis of 1-substituted 5-nitro-indazol-3-ols was
carried out in analogy to the corresponding 5-methoxy de-
rivatives. In all cases 5-nitro-substitution led to a loss of in
vitro activity (Table 4).
1-benzyl (subst.)
1-aryl (subst.)
1-benzoyl (subst.) 3-OH
1-benzyl (subst.)
3-OH
3-OH
5-H
5-OMe
5-OMe
3-Hal, 3-SH, 3-OR 5-OMe
On the contrary, the combination of the following structural
features seemed to be relevant for 5-LO activity:
Scheme 3
1-benzyl (subst.)
3-OH
5-OMe, 5-OH, ...
Arch. Pharm. Pharm. Med. Chem. 331, 13–21 (1998)

1,5-Disubstituted Indazol-3-ols
17
Table 4. 5-LOX inhibition of 5-nitro-, 5-amino-, 5-urea-, and 5-hydroxy-1H-indazol-3-ols.
___________________________________________________________________________________________________
No.
___________________________________________________________________________________________________
R1
R2
Mp (°C)
MW
Analysis C, H, Na
IC50 [nM]b
47
48
3,4-Cl2
2,4-Cl2
NO2
NO2
247
338.15
338.15
C14H9Cl2N3O3
C14H9Cl2N3O3
> 1000
> 1000
236–240
O
NO2
264–266
218–221
375.39
C21H17N3O4
> 1000
49
4 -
N
O
NO2
426.44
C24H18N4O4
> 1000
50
4 -
51
52
53
4-Cl
NH2
NH2
NH2
200
273.72
308.17
308.17
C14H12ClN3O
C14H11Cl2N3O
C14H11Cl2N3O
> 1000
> 1000
> 1000
3,4-Cl2
2,4-Cl2
216–220
199–203
54
55
56
57
4-Cl
NHCONHC6H3Cl2(3,4)
282 dec.
259–263
302–306
308–310
461.74
496.18
457.32
433.34
C21H15Cl3N4O2
C21H14Cl4N4O2
C22H18Cl2N4O3
C21H22Cl2N4O2
278 ± 60
> 1000
> 1000
> 1000
3,4-Cl2 NHCONHC6H3Cl2(3,4)
2,4-Cl2 NHCONHC6H4OMe(4)
2,4-Cl2
HNCOHN
58
59
60
4-Cl
OH
OH
OH
n.d. (resin)
239–243
216
274.71
309.15
309.15
C14H11ClN2O2
C14H10Cl2N2O2
C14H10Cl2N2O2
> 1000
3,4-Cl2
2,4-Cl2
748 ± 119
258 ± 44
___________________________________________________________________________________________________
a: Analysis C, H, N within ± 0.4% unless otherwise indicated. b: Mean of 4 independent experiments.
Therefore, we first concentrated our synthetic efforts on the
– Halogen compounds are more active than those with
variation of the substituents in the 1-benzyl moiety of the
3-hydroxy-5-methoxy-indazole skeleton.
The resulting structure-activity relationships can be sum-
marized as follows:
(branched) alkyl, alkyloxy, nitro, OH or CF substituents
3
in the benzyl group.
– Bulky lipophilic substituents in 4-position of the benzyl
ring are superior to small substituents.
– If there is only one substituent in the benzyl moiety, the
4-position seems to be the most active one.
– Naphthylmethyl or 6-chloropiperonylmethyl moieties in
1-position of the indazole ring reveal similar activity as the
benzyl group.
– In the series of halogen derivatives the chloro substituents
are superior to bromo or fluoro substituents.
– Heterocyclic analogues of the benzyl group show (almost)
no activity.
– Among the compounds with dihalosubstituted benzyl
groups those with 2,4- or 3,4-substitution prove to be the
most active ones.
The distance between the indazole skeleton and the aryl ring
system significantly influences 5-LO activity. Linking by
Arch. Pharm. Pharm. Med. Chem. 331, 13–21 (1998)

18
Schindler and co-workers
propyl-or allyl-chains results in best activity; compounds as well as on the arachidonic acid (AA) induced ear edema in
with ethyloxy or propyloxy chains were slightly less active. mice.
Carbonyl, amido, or hydroxy functions in this part of the
molecule lead to loss of activity.
In vitro experiments for investigation of bronchodilating
activity were carried out in the presence of the H -receptor
1
When modifying the 5-position in 3-hydroxy indazoles antagonist mepyramine and of the cyclooxygenase inhibitor
with selected 1-benzyl substitution we found that 5-nitro and indomethacin to exclude the reactions of histamine and pro-
[7]
5-amino derivatives did not show any 5-LO activity. In the staglandins . Compounds were tested in the concentration-
series of 5-urea derivatives only for 54 an IC value < 1 µM range from 0.3 to 10 µM (Table 5). While compounds 12 and
50
was found. However , introduction of the 5-hydroxy group
results in compounds almost equipotent to the analogues with
5-methoxy substituent.
26 exhibited maximum inhibition at 3 µM (40%) and at 1 µM
(32%) respectively, compound 27 (IC = 2.9 µM) showed a
50
strong inhibitory effect. This effect was comparable with 2
(BAY × 1005, IC = 2.1 µM).
50
Table 5. Effect of the 5-LOX inhibitors on allergen-induced contraction of
actively sensitized guinea pig tracheal segments.
Late phase asthmatic reaction is characterized by a signifi-
cant increase in the number of eosinophils in the bron-
choalveolar fluid resulting from pronounced migration of
——————————————————————————————
[8]
these cells into the lung . The corresponding model com-
Compound
IC50 µmol/l
(with 95% confidence intervals in parentheses)
prises the application of the test compounds to sensitized and
ovalbumin (OVA) challenged guinea pigs
[9]
.
——————————————————————————————
In contrast to vehicle only 12, 26, and 27 significantly
reduced the number of eosinophils in the bronchoalveolar
fluid (Table 6). A similar effect could be demonstrated for 2
(BAY × 1005).
12
26
27
3 µmol/l = maximum inhibition: 40%
1 µmol/l = maximum inhibition: 32%
2.9 (1.2–6.9)
Topical application of AA produces an acute transient
inflammatory reaction characterized by vasodilation, tissue
edema and elevated tissue concentration of prostaglandins
and leukotrienes. Neutrophils migrate into the dermis of the
——————————————————————————————
2 (BAY × 1005) 2.1 (1.4–2.9)
——————————————————————————————
Table 6. Effect of 5-LOX inhibitors on inflammatory cell influx into BAL fluid in the actively sensitized guinea-pig by prophylactic
administration 2 hours prior to antigen challenge.
———————————————————————————————————————————————————————
n
Eosinophil
%
numbers
inhibition of
increase
(×106 cells/animal)
———————————————————————————————————————————————————————
Naive
9
58
56
0.83 ± 0.31
0.84 ± 0.45
6.44 ± 2.49###
Saline challenged + vehicle
OVA challenged + vehicle
0
OVA challenged + 12 (1.0 mg/kg i.p.)
9
8
2.92 ± 1.25***
3.32 ± 1.55***
3.82 ± 1.87**
3.82 ± 2.80***
62.9
55.7
46.8
45.5
OVA challenged + 26 (1.0 mg/kg i.p.)
OVA challenged + 27 (free base), (1.0 mg/kg i.p.)
OVA challenged + 27 (free base), (10 mg/kg i.p.)
10
20
———————————————————————————————————————————————————————
OVA challenged + 2 (BAY × 1005) (10 mg/kg i.p.) 3.23 ± 1.88*** 57.3
9
———————————————————————————————————————————————————————
### p < 0.001 versus saline challenged; ** p < 0.01, *** p < 0.001 versus OVA challenged plus vehicle. Data are presented as mean ± s.d.
ear 30 min after AA-application and reach the maximum
concentration 1 h when edema culminates. The quantity of
polymorphonuclear leukocytes (PMNL) migrating into the
Pharmacology
Based on the in vitro results the compounds 12, 26, and 27
were selected for further pharmacological characterization.
The antiinflammatory effect of 5-LOX inhibitors was tested
on the in vitro model of allergen-induced contraction of
tracheal segments and in vivo on the antigen-induced bron-
inflammatory sites corresponds to the degree of development
[10,11]
of edema
. 5-LOX inhibitors are known to reduce the
[12]
development of edema in mice
.
There were equipotent effects of 27 and 2 (BAY × 1005)
chopulmonary eosinophilia in actively sensitized guinea pigs after topical application of 0.5 and 1 µg/ear (Table 7).
Arch. Pharm. Pharm. Med. Chem. 331, 13–21 (1998)

1,5-Disubstituted Indazol-3-ols
19
Table 7. Inhibitory effect of 5- LOX inhibitors after topical administration
on arachidonic acid (AA)-induced edema in mice.
——————————————————————————————
Yield: 1.0 g ( 15%) 26, mp 177.5–179 °C.– Anal. (C22H20N2O3) C, H, N.–
1H-NMR ([D6]DMSO): δ = 3.59 (s, 3H, CH3), 4.88 (s, 2H, CH2), 5.07 (s,
2H, CH2), 6.75–7.31 (12H, aromatic), 10.30 (s, 1H, OH).– 13C-NMR
([D6]DMSO): δ = 50.53 CH2N, 54.99 CH3O, 68.75 CH2O, 99.41–153.73
18 C-aromatic, 157.22 C-OH.
Compound
Topical Dose
Mean % change
AA-edema
µg/ear
–—————————————————————————————
27 (free base)
0.5
1
–19 *
–41*
5-Methoxy-1-[quinoline-2-ylmethoxy)-benzyl]-1H-indazol-3-ol Dihydro-
chloride (27)
——————————————————————————————
2 (BAY × 1005)
0.5
1
20
–17
–48 *
–54 *
5-Methoxy-1H-indazol-3-ol (4) (3.6 g, 21.9 mmol), 2-(4-chloromethyl-
phenoxymethyl)-quinoline hydrochloride (6.4 g, 22.6 mmol) and sodium
hydroxide (2.4 g, 60.0 mmol) in DMSO (50 ml) were stirred for 6 h at
20–30°C. The mixture was extracted with CH2Cl2 (300 ml) and water (400
ml), the CH2Cl2 phase was washed successively with water (3 × 400 ml),
dried over Na2SO4 and concentrated. The crude product was recrystallized
twice from EtOAc in the presence of activated charcoal.
Yield: 0.6 g (7%) 27 (free base), mp 165–169 °C. Anal. (C25H21N3O3) C,
H, N.– 1H-NMR ([D6]DMSO): δ = 3.73 (s, 3H, CH3), 5.22 (s, 2H, CH2N),
5.34 (s, 2H, CH2O), 6.96–9.41 (21H, aromatic), 10.46 (s, 1H, OH).– 13C-
NMR ([D6]DMSO): δ = 50.83 CH2N, 55.32 CH3O, 70.80 CH2O, 99.75–
157.47 22 C-aromatic.
27 (free base) (1.5 g, 3.6 mmol) were dissolved in acetone (550 ml) under
reflux. After cooling isopropanolic HCl (1.71 ml of 7 N) were added and the
solution was stirred for 3 h at 20–25°C. The product was filtered off and
recrystallized from ethanol.
yield: 1.1 g (62%) 27, mp 181 °C. Anal. (C25H23Cl2N3O3) C, H, N.–
1H-NMR ([D6]DMSO): δ = 3.72 (s, 3H, CH3), 5.26 (s, 2H, CH2N), 5.69 (s,
2H, CH2O), 6.95–9.05 (21H, aromatic), 11.5 (s, 2H, OH, NH).– 13C-NMR
([D6]DMSO): δ = 50.74 CH2N, 55.41 CH3O, 66.55 CH2O, 100.16–156.71
22 C-aromatic.
1 (Zileuton)
——————————————————————————————
* p < 0.05 compared to the vehicle-treated group.
Conclusion
The synthesized indazole derivatives enabled us to study
the influence of substituents on in vitro inhibition of 5-lipoxy-
genase. According to the in vivo results 27 proved to be the
most potent compound of the synthesized series. 27 inhibited
the contraction of tracheal segments of guinea pigs, reduced
the infiltration of eosinophils into the lung (guinea pigs), and
exhibited dose dependent antiinflammatory activity in the
mouse earedema model. Therefore27 is expected to be useful
in the treatment of a variety of leukotriene-mediated disorders
including asthma.
Experimental Part
1-(3,4-Dichlorobenzyl)-5-nitro-1H-indazol (47)
Chemistry
A solution of 5-nitro-1H-indazol-3-ol (5) (14.4 g, 80 mmol) and 3,4-di-
chlorobenzyl chloride (15.6 g, 80 mmol) in NaOH (80 ml of 1 N) was stirred
for 4 h at 70°C, additional 3,4-dichlorobenzyl chloride (5.5 g, 28 mmol) and
NaOH (20 ml of 1 N) were added and stirred for another 2.5 h at 70°C. After
cooling and filtration the solid was recrystallized from n-BuOH with acti-
vated charcoal.
Yield 22.1 g (81%) 47, mp 247 °C. Anal. (C14H9Cl2N3O3) C, H, N.–
1H-NMR ([D6]DMSO): δ = 5.34 (s, 2H, CH2N), 7.04–8.55 (6H, aromatic).–
13C-NMR ([D6]DMSO): δ = 50.60 CH2N, 110.27–142.69 12 C-aromatic,
157.62 C-3.
Melting points: melting point apparatus by Boetius, uncorrected.– IR
spectra: Perkin Elmer FT-IR 1725 X, KBr.– 1H and 13C NMR spectra (TMS
as internal standard): Bruker ARX 300 and Bruker AMX 500 spectrometers,
303 K, chemical shifts in δ units. All NH and OH were replaceable by D2O.–
Microanalyses were within ± 0.4% of the theoretical values for all elements
listed, unless otherwise stated.- Silica gel chromatography was performed
using Merck silica gel 60 (63–200 mesh). Reagents used were purchased
from the Aldrich Chemical Co., E. Merck KGaA and Lancaster Synthesis
GmbH.
All compounds were characterized by elemental analysis, 1H, 13C-NMR,
and IR spectroscopy, melting points and thin layer chromatography. In all
cases the IR and NMR spectra indicated the presence of the 3-hydroxy group
and not a carbonyl group (no carbonyl band at about ν ≈ 1700 cm–1 s ).
5-Amino-(2,4-dichlorobenzyl)-1H-indazol-3-ol (53)
1-(2,4-Dichlorobenzyl)-5-nitro-1H-indazol-3-ol (48) (16.2 g, 48 mmol)
were hydrogenated in dioxane (800 ml) with Ra-Ni (5.0 g, 20 bar, 100°C,
6 h). After filtration and concentration under reduced pressure the crude
product was recrystallized from n-BuOH with activated charcoal.
yield: 8.7 g (59%) 53, mp 199–203 °C. Anal. (C14H11Cl2N3O) C, H, N.–
1H-NMR ([D6]DMSO): δ = 4.75 (s, 2H, NH2), 5.24 (s, 2H, CH2N), 6.67–7.58
(6H, aromatic), 10.25 (s, 1H, OH).– 13C-NMR ([D6]DMSO): δ = 48.52
CH2N, 100.86–141.63 12 C-aromatic, 154.18 C-3.
Synthesis of 5-substituted 1H-indazol-3-ols
5-Methoxy-1H-indazol-3-ol (4) was prepared by the method of
Baiocchi [5]
.
5-Nitro-1H- indazol-3-ol (5) was prepared by the method of Pfannstiel et
al. [6]
.
Synthesis of 1-substituted indazol-3-ols 6–50
Compounds 6–50 were prepared by similar methods as reported pre-
viously [5]
.
1-[1-(3,4-Dichlorobenzyl)-3-hydroxy-1H-indazole-5-yl]-3-(4-methoxy-
phenyl)-urea (56)
Typical procedures are described in the following:
A solution of 5-amino-1-(3,4-dichlorobenzyl)-1H-indazol-3-ol (52)
(1.54 g, 5.0 mmol) and 4-methoxyphenylisocyanate (1.12 g, 7.5 mmol) in
THF (75 ml) was stirred for 5 h at room temp. The solution was concentrated
under reduced pressure to one third of its volume. After standing overnight
the solid was filtered and recrystallized from n-BuOH.
Yield: 1.55 g (68%) 56, mp 267 °C. Anal. (C22H18Cl2N4O3) C, H, N.–
1H-NMR ([D6]DMSO): δ = 3.75 (s, 3H, CH3), 5.29 (s, 2H, CH2N), 6.87–8.52
(18H, aromatic), 10.5 (s, 1H, OH).– 13C-NMR ([D6]DMSO): δ = 52.65
CH2N, 57.76 CH3O, 111.26–141.71 17 C-aromatic, 155.75, 156.99, 157.31
C-3, C-OCH3, C=O.
1-(4-Benzyloxybenzyl)-5-methoxy-1H-indazol-3-ol (26)
5-Methoxy-1H-indazol-3-ol (4) (3.0 g, 18.0 mmol) and 4-benzyloxy-ben-
zyl chloride (4.2 g, 18.0 mmol) were stirred in NaOH (20 ml of 5 N) for 2.5 h
at 70°C. After cooling the viscous solid was filtered, washed with water,
dissolved in DMF (60 ml), and purified by column chromatography on a
silica gel column eluating with dichloromethane-methanol (95:5, v/v). The
purified fraction was concentrated under reduced pressure and the residue
was crystallized with MeCN. The solid was filtered and recrystallized from
MeCN in the presence of activated charcoal.
Arch. Pharm. Pharm. Med. Chem. 331, 13–21 (1998)

20
Schindler and co-workers
1-(4-Chlorobenzyl)-1H-indazol-3,5-diol (58)
Inhibition of the allergen-induced contraction of tracheal segments in ac-
tively sensitized guinea pigs
To a solution of 5-amino-1-(4-chlorobenzyl)-1H-indazol-3-ol (51) (2.0 g,
7.3 mmol) in n-BuOH (180 ml) and NaOH (20 ml of 3.3 N) a solution of
NaNO2 (1.26 g, 18.2 mmol) in water (5 ml) was dropped at 0 °C. After
stirring for 1 h at 0–5 °C the solution was stirred for another hour at 80 °C.
After cooling the product was concentrated under reduced pressure to dry-
ness. The residue was extracted with water (100 ml) and tBu-O-Me (200 ml).
The ether phase was washed with water (100 ml), dried over Na2SO4, and
evaporated to dryness.
Actively sensitized guinea pigs were sacrificed and the trachea was re-
moved. The trachea was divided into 3 to 4 small segments and equilibrated
for 0.5 hours at 37°C under isotonic conditions (1 g pretension, isometric
transducers and amplifier, TSE in vitro apparatus, Bad Homburg). The
isolated preparations were suspended in 20 ml organ baths containing modi-
fied Krebs’ solution (4.7 mM KCl, 1.2 mM KH
2PO4, 1.62 mM MgSO4 ×
7 H2O, 118 mM NaCl, 25 mM NaHCO3, 6.1 glucose, 2.52 CaCl2 × 2 H2O),
which was gassed with a mixture of 95% O2 and 5% CO2. Indomethacin
(1 µM) and mepyramine (28 µM) were added to the organ bath to inhibit
bronchoactive prostanoid and histamine. After an equilibration period car-
bachol (0.5 µM) was applied as spasmogen, left in contact with the organs
up to the maximum contraction (about 3 – 5 min) and washed out. After
30 minutes 10 µg/ml ovalbumin was added as spasmogen, left in contact with
the organs for 1 hour. The contractile reaction of the sensitized tissues was
measured after a single challenge of ovalbumin. The test compounds were
applied as a suspension in Tween 80 (0.2 ml with a final concentration of
0.0013%) to the organ bath (30 ml), 30 minutes prior to allergen challenge.
There was no effect of vehicle (Tween 80) - treated control group at the final
concentration of 0.0013% on allergen-induced contraction of tracheal seg-
ments. The anaphylactic (contractile) responses were expressed as percent-
age of the maximum carbachol response obtained at the beginning of the
experiment.
Yield: 2.01 g ( 99%) 58. Anal. (C14H11ClN2O2) C, H, N.– 1H-NMR
([D6]DMSO): δ = 5.35 (s, 2H, CH2N), 7.13–7.78 (7H, aromatic), 10.44 (s,
2H, OH).– 13C-NMR ([D6]DMSO): δ = 50.96 CH2N, 110.98–140.46 12
C-aromatic, 154.89 C-3.
1-(3,4-Dichlorobenzyl)-1H-indazol-3,5-diol (59)
A solution of 1-(3,4-dichlorobenzyl)-5-methoxy-1H-indazol-3-ol (12)
(4.85 g, 15.0 mmol) in acetic acid (30 ml) and HBr (30 ml of 5.93 N) was
refluxed for 4h. After cooling water (250 ml) were added, pH 14 was adjusted
by adding conc. NaOH, and the aqueous solution was extracted twice with
tBu-O-Me. The aqueous phase was acidified with H2SO4, and extracted with
tBu-O-Me (3 × 200 ml). The combined ether phases were dried over Na2SO4
and concentrated under reduced pressure. The crude solid was crystallized
with tBu-O-Me.
yield: 2.40 g (52%) 59, mp 239–243 °C. Anal. (C14H10Cl2N2O2) C, H, N.–
1H-NMR ([D6]DMSO): δ = 5.33 (s, 2H, CH2N), 6.98–7.65 (6H, aromatic),
9.13 (s, 2H, OH).– 13C-NMR ([D6]DMSO): δ = 49.17 CH2N, 101.45–149.69
12 C-aromatic, 153.33 C-3.
Effect on late phase eosinophilia in actively sensitized guinea pigs
Male Dunkin-Hartley guinea pigs (200–250 g) were obtained from
Schoenwalde, FRG. Animals were actively sensitized with a suspension of
10 µg ovalbumin (OVA) + 1 mg Al(OH)3 subcutaneously and two weeks
later boosted. 7 days after boosting the animals were exposed for 30 s to an
aerosol of OVA (5 mg/ml)administeredviaanebulizerdrivenbycompressed
air at 19.6 kPa in an exposure chamber. To prevent anaphylactic death
mepyramine (10 mg/kg i.p.) was given 0.5 minutes before challenge. 24 h
later bronchoalveolar lavage (BAL) was performed with 2 × 5 ml saline in
animals sacrificed by an overdose of urethane. Lavage fluid was pooled,
centrifuged at 400g for 10 minutes and the cell pellet suspendedin1ml saline.
Eosinophils were counted in a Neubauer chamber after staining them using
the Becton Dickinson Test kit (No. 5877) for eosinophils. Mean and standard
deviation (sd) was calculated. Each group of animals treated with test
compounds was compared with the placebo treated challenged group (un-
paired t test). For drug administration all compounds were administered
intraperitoneally suspended in 10% polyethylene glycol 300 and 0.5% hy-
droxyethyl-cellulose with a volume of 0.1 ml per 100 g body weight. Animals
received test compounds prophylactically 2 hours before allergen challenge.
Each drug was tested in at least 8–20 animals. For each group mean and
standard deviation (sd) was calculated.
Supplementary Material
NMR data are available from the authors.
Pharmacological Methods
Inhibition of 5-lipoxygenase
Paraffin (2 ml per animal) was administered intraperitoneally to ten male
Wistar rats (360–400 g) to induce the production of macrophages. After
4 days the rats were sacrificed. The abdominal cavity was opened, the cavity
was washed with sodium-phosphate buffer (20 ml of 3 mM, pH 7, containing
5.5 mM glucose, 150 mM NaCl, 3.8 mM KCl) and the cell suspension was
transferred into plastic vials. The cells were centrifuged at 400 g for 10 min.
The supernatant was removed and the cells were washed twice with cold
sodium-phosphate buffer (50 ml). After addition of aq. CaCl2 (500 µl of
200 mM) and [3H]-arachidonic acid (15 µl, Bio Trend) the vials were closed,
shaken gently and centrifuged at 400 g for 10 min. The radioactive super-
natant was removed, the cell pellet resuspended and buffer (50 ml) was
added. The cells were washed twice.
Antiinflammatory activity in the AA induced mouse ear edema
Investigations into the effect of test substances on arachidonic acid (AA)
induced ear edema were carried out in male mice (25–30 g). The test
compounds or vehicle, dissolved in acetone, were administered topically
30 minutes prior to topical application of AA. Arachidonic acid was dis-
solved in acetone and applied to the inner side of the right ear in a volume of
15 µl (2 mg AA/ear) by an automatic pipette. The left ear received 15 µl
acetone alone. One hour after AA application, the thickness of both ears was
measured by means of an Oditest dial gauge calipers in units of 0.01 mm.
The development of the ear edema was calculated by subtracting the thick-
ness of the left ear (vehicle control) from that of the right ear (treated ear).
The incubation mixture (2 ml) contained sodium-phosphate buffer (3 mM,
pH 7), indomethacin (0.025 mM), glutathione (0.6 mM), Ca-ionophore
(0.01 mg/ml), the prepared cell preparation (500 µl) and the test compound
in different concentrations. Nonspecific enzyme activity was determined in
presence of E 6080 (20 µM). This mixture was preincubated for 30 min at
37 °C. After addition of aq. CaCl2 (25 µl of 200 mM) incubation was
continuedfor 10min at 37 °C. The reaction was stopped by addition of formic
acid (25 µl).
The reaction products of 5-lipoxygenase were separated by an extraction
procedure. Isopropanol/diethyl ether (5 ml of 1/4, v/v) was added, the vials
were shaken and centrifuged at 3000 g for 5 min at 4 °C. The upper organic
phase was transferred into new vials. After addition of aq. NH3 (25 µl of
25%), n-hexane (2.5 ml) was added, the vials were shaken and centrifuged
at 3000 g for 5 min at 4 °C. The vials were placed into a freezer at –70°C.
After removing the upper organic phase the lower aqueousphase wasthawed.
100 µl of the aqueous phase, which contained the [3H] labeled products
(mixture of the polar metabolic products of arachidonic acid), was transferred
into scintillation vials. After addition of scintillation fluid (3 ml) the vials
were counted for radioactivity in a liquid scintillation counter (Wallac 1409,
The percentage inhibition was calculated according to Kotyuk [11]
.
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✩
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Received: August 19, 1997 [FP248]
Arch. Pharm. Pharm. Med. Chem. 331, 13–21 (1998)