Design, synthesis and evaluation of N-benzoylindazole derivatives and analogues as inhibitors of human neutrophil elastase

Article abstract of DOI:10.1016/j.bmc.2011.06.036

Human neutrophil elastase (HNE) plays an important role in tumour invasion and inflammation. A series of N-benzoylindazoles was synthesized and evaluated for their ability to inhibit HNE. We found that this scaffold is appropriate for HNE inhibitors and that the benzoyl fragment at position 1 is essential for activity. The most active compounds inhibited HNE activity with IC₅₀ values in the submicromolar range. Furthermore, docking studies indicated that the geometry of an inhibitor within the binding site and energetics of Michaelis complex formation were key factors influencing the inhibitor's biological activity. Thus, N-benzoylindazole derivatives and their analogs represent novel structural templates that can be utilized for further development of efficacious HNE inhibitors.

Full text of DOI:10.1016/j.bmc.2011.06.036

Bioorganic & Medicinal Chemistry 19 (2011) 4460–4472
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of N-benzoylindazole derivatives
and analogues as inhibitors of human neutrophil elastase
Letizia Crocetti a, Maria Paola Giovannoni a, , Igor A. Schepetkin b, Mark T. Quinn b, Andrei I. Khlebnikov c,
⇑
Agostino Cilibrizzi a, Vittorio Dal Piaz a, Alessia Graziano a, Claudia Vergelli a
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Italy
b Department of Immunology and Infectious Diseases, Montana State University, Bozeman, MT 59717, USA
c Department of Chemistry, Altai State Technical University, Barnaul 656038, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Human neutrophil elastase (HNE) plays an important role in tumour invasion and inflammation. A series
of N-benzoylindazoles was synthesized and evaluated for their ability to inhibit HNE. We found that this
scaffold is appropriate for HNE inhibitors and that the benzoyl fragment at position 1 is essential for
activity. The most active compounds inhibited HNE activity with IC50 values in the submicromolar range.
Furthermore, docking studies indicated that the geometry of an inhibitor within the binding site and
energetics of Michaelis complex formation were key factors influencing the inhibitor’s biological activity.
Thus, N-benzoylindazole derivatives and their analogs represent novel structural templates that can be
utilized for further development of efficacious HNE inhibitors.
Received 28 February 2011
Revised 10 June 2011
Accepted 13 June 2011
Available online 7 July 2011
Keywords:
Indazoles
1-Benzoylindazoles
Human neutrophil elastase
Inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
Structure–activity relationship
1. Introduction
agents able to modulate the proteolytic activity of HNE represent
promising therapeutics for pathological conditions involving
Human neutrophil elastase (HNE) is a member of the chymo-
trypsin superfamily of serine proteases and is expressed primarily
in neutrophils.1 It is a small, soluble protein of about 30 kDa, con-
taining 218 amino acid residues and is stabilized by four disulfide
bridges.2 HNE is a basic glycoprotein, with a catalytic triad consist-
ing of Ser195, His57, and Asp102.3 HNE efficiently degrades a vari-
ety of extracellular matrix proteins, such as elastin, fibronectin,
collagen, proteoglycans and laminin.4 HNE also activates some ma-
trix metalloproteinases (i.e., MMP-2, MMP-3 and MMP-9)5 and is
able to modulate cytokine expression and growth factors.6,7 Under
physiological conditions and during inflammation, endogenous
excessive HNE activity.17
Over the past two decades, intensive research has resulted in
the development of a number of HNE inhibitors, including peptidic
and non-peptidic compounds, some of which have been tested in
clinical trials.18–22 More recently, low molecular weight HNE inhib-
itors have appeared in literature,21–29 and examples of different
structural classes of these inhibitors are shown in Figure 1. Among
these, the 2-pyridin-3-yl-benzo[d]1,3-oxazin-4-ones (structure A)
exhibited a very good balance of potency (Ki = 28 nM) and stability
(t1/2 ꢀ 23.3 h at pH 9.2),28 the 3,3-diethyl-1-(4-tolyl)azetidine-2,4-
diones (structure B), had Ki values in the low micromolar range and
acted as an irreversible inhibitors of elastase,26 and the
N-benzoylpyrazoles (structure C) were potent, competitive,
pseudoirreversible inhibitors of HNE with Ki values in the nanomo-
lar range.23 However, despite the numerous inhibitors described in
the literature, the only small-molecule HNE inhibitor currently
available for clinical use is Sivelestat (ONO-5046), which is
marketed in Japan and Korea.30 ONO-5046 is a competitive and
selective inhibitor of HNE and is used for the treatment of acute
lung injury (ALI) associated with systemic inflammatory response
syndrome (SIRS).31
HNE inhibitors, such as
a1-antitrypsin and elafin, protect tissues
from damage by elastase.8,9
Several pathological conditions are known to result from imbal-
ance between HNE and its endogenous inhibitors. In fact, HNE is
considered to be the primary source of tissue damage associated
with some inflammatory diseases, such as adult respiratory dis-
tress syndrome (ARDS),10 chronic obstructive pulmonary disease
(COPD),11 cystic fibrosis (CF),12 and other inflammatory disor-
ders.13–15 Massive HNE release can also contribute to the move-
ment of neoplastic cells through tissues.16 Thus, it is clear that
Based on previous studies showing that N-benzoylpyrazoles
were potent HNE inhibitors,23 we synthesized a series of N-ben-
zoylindazoles (Fig. 1, structure D), which build upon the basic pyr-
azole feature (structure C) by linking the benzoyl fragment to the
⇑
Corresponding author. Tel.: +39 055 4573682; fax: +39 055 4573780.
E-mail address: mariapaola.giovannoni@unifi.it (M.P. Giovannoni).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.036

L. Crocetti et al. / Bioorg. Med. Chem. 19 (2011) 4460–4472
4461
When the benzoyl fragment at position 1 was substituted at the
meta position with an amino group, it was necessary to protect the
NH2 function of 9 with di-terbutyldicarbonate to obtain the inter-
mediate 1036 (Scheme 2). This compound was treated with ethyl
chloroformate in anhydrous tetrahydrofuran (THF) in the presence
of trietylamine, resulting in the mixed anhydride 11, which was re-
acted with the appropriate indazole to give compounds 12a–f. The
last step was removal of the protecting group by using triofluoro-
acetic acid and dichloromethane.
Scheme 3 depicts the synthetic route which, starting from the
precursors 2 and 4, allowed us to modify the amidic function at po-
sition 1 by inserting a sulphonamidic moiety (14a,b), replacing the
CO group with a CH2 (14c), eliminating the CO between N-1 and
phenyl ring (14d), inserting a methylenic spacer between N-1
and CO (14e), and finally introducing a CONH fragment (14f,g).
The sulphonamide derivatives 14a,b were obtained by treatment
with the desired sulphonyl chloride in dichloromethane and trieth-
ylamine; compounds 14c and 14e were synthesized using the
appropriate benzyl or phenacyl halides in anhydrous solvent and
K2CO3; synthesis of derivative 14d was performed through a
cross-coupling reaction with 4-chlorophenylboronic acid, using
copper acetate as catalyst and a weak base (triethylamine) in
dichloromethane; and treatment of 4 with substituted phenyl iso-
cyanate in anhydrous THF afforded the semicarbazone derivatives
14f,g.
O
N
O
N
O
O
N
O
N
N
N
R
A
B
HO
O
O
N
N
NH
R
1
O
R
2
C
NH
S
O
O
R
O
1
N
N
R
O
2
Scheme 4 depicts the synthetic routes followed to obtain 17a,b
and 19a,b, where the function at position 3 was replaced with a
methyl group (17a,b), a carboxylic group (19b), or a hydrogen
(19a). Treatment of the commercially available o-bromoacetophe-
none 15 with hydrazine hydrate at high temperature afforded the
3-methylindazole 16,37 which was transformed into the final com-
pounds 17a,b, as reported in Scheme 1. Following the same proce-
dure, final product 19a38 was synthesized. Finally, treatment of
compound 5c with 6 N NaOH at 60 °C afforded 19b.
O
O
D
Sivelestat
Figure 1. Structures of selected HNE inhibitors.
cyclic amide, and evaluated these compounds and various deriva-
tives for their ability to inhibit HNE. We also utilized molecular
modelling to evaluate binding of the compounds to the HNE active
site and obtained insight into the differences in inhibitory activity
of the various derivatives.
3. Biological results and discussion
3.1. Structure–activity relationship analysis
All compounds were evaluated for their ability to inhibit HNE,
and the results are presented in Tables 1–5. The first compounds
synthesized were the 3-phenylamido (3a–q, 13a) and 3-carbeth-
oxy (5a–s, 13b) derivatives (Table 1). Compounds 3a and 5a, bear-
ing an unsubstituted benzoyl group at position 1, exhibited
2. Chemistry
We followed a simple synthetic route that allowed us to obtain
a large number of 1-aroylindazole derivatives and to obtain preli-
minary information about the structural requirements for the
activity of this new scaffold.
submicromolar inhibitory activity (IC50 = 0.4 lM). This suggested
that the 1-benzoylindazole system could represent a suitable scaf-
fold for HNE inhibitors. Thus, as a primary approach, we modified
the benzoyl fragment. Introduction of a methyl (3b and 5b) or a
chlorine (3e and 5e) at the ortho position of the phenyl ring led
to decreased activity for both series of compounds. In particular,
compounds 3b and 5b had 3 orders of magnitude lower activity
The synthesis of all compounds bearing an aroyl fragment
[compounds 3a–q, 5a–s (5a,32 5b–k33), and 8a–p] or an acyl frag-
ment [compounds 6a–e (6a32)] at position 1 of the indazole nu-
cleus is depicted in Scheme 1, and the final products are grouped
into the 3, 5, 6 or 8 series in order to facilitate discussion of the
pharmacological results. Using previously described conditions,
we first transformed the commercially available indazole-3-car-
boxylic acid 1 into the corresponding phenylamide 234 and the
known esters 432 and 7a–c.35 The novel 7d was prepared from a
reaction of 1 with trifluoroethanol via the acid chloride. Introduc-
tion of the acyl function at N-1 of various indazoles was performed
following three different approaches, as follows: (1) treating the
appropriate aroyl or acyl chloride, where commercially available,
with dichloromethane and triethylamine; (2) preparing the appro-
priate aroyl chloride starting from the corresponding acid and then
carrying out the reaction in anhydrous toluene and triethylamine;
and (3) coupling with the appropriate acid in dimethylformamide
(DMF) by treatment with triethylamine and diethylcyanophospho-
nate (DCF). Compounds 3, 5, 6 and 8 are defined in Tables 1–3.
compared to 3a and 5a (IC50 = 34 and 52 lM, respectively),
whereas 3e and 5e were completely devoid of activity, suggesting
that the ortho position has to remain unsubstituted to permit free
rotation, which is in agreement with that reported for the pyra-
zoles (see Fig. 1, structure C).23
The introduction of substituents in the para position resulted in
variable effects on HNE inhibitory activity. For example, the p-
methyl (3d and 5d) and the 4-CN (3m and 5m) derivatives were
only weak inhibitors, both for the phenylamide and carbethoxy
series (IC50 = 29 and 36 lM, respectively). In contrast, compounds
3i and 5i, both bearing a 4-NO2 group, were completely inactive.
The 4-Cl (3g and 5g) and the 4-F-benzoyl (3k and 5k) derivatives
were the only products that retained inhibitory activity, although
still lower when compared to the unsubstituted reference

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L. Crocetti et al. / Bioorg. Med. Chem. 19 (2011) 4460–4472
COOH
N
N
COOR
N
H
1
b
a
b
CONHPh
N
N
H
COOC2H5
7a-d
7
a
b
c
d
R
N
N
CH3
n.C3H7
i.C3H7
CH2CF3
N
H
2
H
c
4
c
c
c
COOC2H5
N
CONHPh
COOC2H5
N
COOR
N
N
N
N
N
N
8a-p
R1
O
O
R1
5a-s
3a-q
O
O
6a-e
R1
(cyclo)alkyl
Scheme 1. Synthesis of indazoles 3a–q, 5a–s, 6a–e and 8a–p. Reagents and conditions: (a) SOCl2, Et3N, THF, PhNH2, 0 °C–rt; (b) for 4, 7a–c: ROH, H2SO4, reflux; for 7d: SOCl2,
Et3N, CF3CH2OH, dry toluene, rt; (c) for 3a–k, 3m and 3q, 5a–k, 5m, 5q and 5r, 6b–c and 6e, 8a–b, 8e–f, 8i–j and 8m–n: aroylchloride, Et3N, DCF, dry CH2Cl2, 0 °C–reflux; for 3l
and 3o–p, 5l and 5o–p: (hetero)aryl carboxylic acid, Et3N, DCF, dry DMF, rt; for 3n, 5n and 5s, 6d, 8g–h, 8k–l, 8o–p: (ethero)aryl carboxylic acid, SOCl2, Et3N, dry toluene,
reflux.
Table 1
Table 2
HNE inhibitory activity of indazole derivatives 3a–q, 5a–s, and, 13a,b
HNE inhibitory activity of indazole derivatives 8a–p and 13c–f
CONHPh
COOC H
COOR
2
5
1
N
N
N
N
N
N
R
R
R
O
O
O
IC50 (l
M)a
3a-q, 13a
5a-s, 13b
Compound
R
R1
8a
8b
8c
8d
13c
8e
Ph
CH3
CH3
CH3
CH3
CH3
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
iC3H7
iC3H7
iC3H7
iC3H7
iC3H7
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
0.089 0.031
0.13 0.051
0.55 0.21
0.31 0.12
NA
0.40 0.18
0.78 0.32
1.9 0.78
R
Compound
IC50
(
l
M)a
Compoundb
IC50 (l
M)a
3-CH3Ph
3-OCH3Ph
3-Thienyl
3-NH2Ph
Ph
3-CH3Ph
3-OCH3Ph
3-Thienyl
3-NH2Ph
Ph
3-CH3Ph
3-OCH3Ph
3-Thienyl
3-NH2Ph
Ph
3-CH3Ph
3-OCH3Ph
3-Thienyl
3-NH2Ph
Ph
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
—
0.42 0.21
34 5.2
0.93 0.30
29 4.8
NA
3.9 1.6
6.1 2.4
11 3.8
NA
5.9 2.1
8.1 3.2
1.8 0.71
38 5.3
NA
3.1 1.1
33 4.6
25 3.9
5a32
5b33
5c33
5d33
5e33
5f33
5g33
5h33
5i33
5j33
5k33
5l
5m
5n
5o
5p
5q
0.40 0.19
52 7.3
0.41 0.11
36 4.9
NA
1.7 0.61
19 4.1
NA
2-CH3Ph
3-CH3Ph
4-CH3Ph
2-ClPh
3-ClPh
4-ClPh
3-NO2Ph
4-NO2Ph
3-FPh
4-FPh
3-OCH3Ph
4-CNPh
4-Pyridyl
3-Thienyl
5-Benzodioxole
1-Naphthyl
3-CF3Ph
3,4,5-OCH3Ph
3-NH2Ph
8f
8g
8h
13d
8i
8j
8k
8l
13e
8m
8n
8o
8p
13f
1.9 0.72
0.70 0.26
0.24 0.088
0.65 0.28
0.76 0.34
0.69 0.31
0.51 0.24
0.26 0.11
0.46 0.21
0.89 0.37
0.51 0.23
0.34 0.13
NA
1.7 0.52
5.7 2.3
0.80 0.33
38 4.7
NA
0.93 0.37
28 3.4
NA
5r
5s
13b
NA
NA
1.6 0.58
—
13a
NA, no activity was observed at concentrations below 55
l
M.
The IC50 values are presented as the mean SD of three independent
experiments.
3.1 1.2
a
NA, no activity was observed at concentrations below 55
l
M.
The IC50 values are presented as the mean SD of three independent
experiments.
a
b
exhibited different activity in the two series, where 3g was about
3 times more potent than 5g.
References for previously reported compounds are indicated.
When substituents were introduced at the meta position of the
benzoyl group, the most active products were the 3-methyl and 3-
methoxy derivatives, which had IC50 values similar to those of the
compounds 3a and 5a (3g, IC50 = 6.1
lM; 5g, IC50 = 19 lM; 3k,
IC50 = 8.1 M; 5k, IC50 = 5.7 M). Moreover, the 4-Cl derivatives
l
l

L. Crocetti et al. / Bioorg. Med. Chem. 19 (2011) 4460–4472
COR
4463
Table 3
COR
N
HNE inhibitory activity of indazole derivatives 6a–e
a (for 14a,b)
b (for 14c)
COOC H
2
5
N
N
H
N
X
c (for 14d)
d (for 14e)
e (for 14f,g)
N
2, 4
N
14a-g
R1
R1
Compd.
R
R
O
2
4
NHPh
OC2H5
Compounda
R
IC50 (l
M)b
6a32
6b
6c
6d
6e
CH3
C2H5
4.8 1.9
0.40 0.18
0.18 0.068
0.48 0.19
NA
14
R
X
nC3H7
a
b
c
d
e
f
4-CH3-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
Ph
NHPh SO2
NHPh SO2
NHPh CH2
Cyclopentyl
Cyclohexyl
NA, no activity was observed at concentrations below 55
lM.
NHPh
-
a
References for previously reported compounds are indicated.
The IC50 values are presented as the mean SD of three independent
OC2H5 CH2CO
OC2H5 CONH
OC2H5 CONH
b
4-Cl-Ph
g
3-OCH3-Ph
experiments.
Scheme 3. Synthesis of indazoles 14a–g. Reagents and conditions: (a) Et3N,
appropriate sulfonylchloride, anhydrous CH2Cl2, rt; (b) 4-chlorfobenzylchloride,
K2CO3, anhydrous DMF, 80 °C; (c) 4-chlorobenzeneboronic acid, Cu(Ac)2, Et3N,
CH2Cl2, rt; (d) phenacylbromide, K2CO3, anhydrous acetone, rt; (e) appropriate
phenylisocyanide, anhydrous THF, rt.
unsubstituted compounds. In particular, compounds 5c and 5l had
IC50 values of 0.41 and 0.80 lM, respectively. Compounds bearing
an amino group (13a and 13b), a chlorine (3f and 5f), or a fluorine
(3j and 5j) at the same position retained HNE inhibitory activity,
but at lower levels (Table 1). In comparison, insertion of a nitro
(3h and 5h) or a trifluoromethyl group (5r) led to weak or inactive
compounds. These results indicate that the presence of an elec-
tron-withdrawing group in this position is detrimental. The in-
creased steric bulk due to the insertion of 3,4,5-OCH3Ph led to a
complete loss of activity (5s), as did replacement of the phenyl
linked to CO at position 1 of the indazole nucleus with a heterocy-
clic ring, such as 4-pyridyl (3n and 5n), 5-benzodioxole (3p and
5p), or 1 naphtyl (3p and 5p). The only exception was substitution
with a 3-thienyl ring, which produced active compounds (3o and
5o). In particular 5o, belonging to the carbethoxy series, had an
3-methyl-benzoyl, 3-methoxy-benzoyl, 3-amino-benzoyl, and 3-
thienyl) (see Table 2). Analysis of the biological activity of these
compounds (8a–p and 13c–f) demonstrated that HNE inhibitory
activity improved by changing –COOEt to –COOMe, –COOnPr,
–COOiPr, or –COOCH2CF3 (Table 2). In fact, for most compounds,
these modifications resulted in increased HNE inhibitory activity
(about one order of magnitude) with respect to the –COOEt deriv-
atives. In particular, the –COOMe derivative 8a was the most active
in this series (IC50 = 89 nM). On the other hand, not all such deriv-
atives were active, and the 3-amino derivative 13c was completely
inactive.
IC50 = 0.93 lM.
We also evaluated the effects of replacement of the ring with a
(cyclo)alkyl fragment (compounds 6a–e) (Table 3). The most active
compound was 6c, with R = nPr (IC50 = 0.18 lM). The activity of
We next performed modifications of residue R1 of the ester
function in position 4, while maintaining at position R2 the best
substituent features determined above for each series (i.e., benzoyl,
O
O
O
COOH
COOH
a
b
OC2H5
BOC
NH2
N
H
BOC
N
H
10
9
11
COR
N
+
c
N
COR
N
H
COR
N
2, 4, 7a-d
12,13
R
N
a
b
c
d
e
f
NHPh
N
OC2H5
OCH3
d
O
O
OC3H7n
OC3H7i
OCH2CF3
NH2
NHBoc
13a-f
12a-f
Scheme 2. Synthesis of indazoles 13a–f. Reagents and conditions: (a) (BOC)2O, Et3N, dioxane, H2O, rt; (b) (1) Et3N, dry THF-7 °C/-5 °C; (2) ClCOOEt, 0 °C; (c) rt–60 °C; (d)
CH2Cl2, CF3COOH, rt.

4464
L. Crocetti et al. / Bioorg. Med. Chem. 19 (2011) 4460–4472
CH3
N
CH3
COCH3
Br
a
b
N
N
N
17a,b
R1
H
16
15
O
17
R1
a
b
3-CH3-Ph
3-OCH3-Ph
R1
N
COOC2H5
H
N
b
c
N
N
N
N
H
O
18
O
19a,b
5c
CH3
CH3
19
R1
a
b
H
COOH
Scheme 4. Synthesis of indazoles 17a,b and 19a,b. Reagents and conditions: (a) NH2NH2ÁH2O, 120 °C; (b) for 17a and 19a: m-toluoyl chloride, Et3N, anhydrous CH2Cl2, 0 °C–rt ;
for 17b: 3-methoxybenzoic acid, SOCl2, Et3N, anhydrous toluene, 100 °C; (c) 6 N NaOH, 60 °C.
Table 5
Table 4
HNE inhibitory activity of indazole derivatives 17a,b and 19a,b
HNE inhibitory activity of indazole derivatives 14a–g
R
1
COOR
1
N
N
N
N
R
R
O
Compound
R
R1
IC50 (l
M)a
Compounda
R
R1
IC50 (l
M)b
14a
14b
14c
14d
14e
14f
4-CH3-PhSO2
4-Cl-PhSO2
4-Cl-PhCH2
4-Cl-Ph
PhCOCH2
4-Cl-PhNHCO
3-OCH3-PhNHCO
NHPh
NHPh
NHPh
NHPh
OC2H5
OC2H5
OC2H5
NA
NA
NA
NA
NA
NA
NA
17a
3-CH3-Ph
3-OCH3-Ph
3-CH3-Ph
3-CH3-Ph
CH3
CH3
H
22 6.8
NA
10 2.2
NA
17b
19a38
19b
COOH
NA, no activity was observed at concentrations below 55
lM.
14g
a
References for previously reported compounds are indicated.
The IC50 values are presented as the mean SD of three independent
a
b
No activity was observed at concentrations below 55 lM.
experiments.
compounds in this series decreased both by shortening the alkylic
chain (e.g., 6a with R = Me was 10-fold less active than 6c), and by
increasing the bulk, as demonstrated by the complete inactivity of
6e bearing a cyclohexyl group. These results suggest that a specific
steric requirement is necessary at this position.
To consider the effects of modifications at the level of the
amidic group, we synthesized a number of 3-phenylamide and 3-
carbomethoxy derivatives. These modifications included replace-
ment of the amidic CO with SO2 (14a,b) or a methylenic linker
(14c), bonding of the phenyl ring directly to the indazole by
deletion of the amidic CO (compound 14d), insertion of a methy-
lenic spacer between the amidic nitrogen and the amidic CO
(14e), and insertion of a CONH group (14f,g) (Table 4). In all cases,
these modifications led to a complete loss of activity. These data
demonstrate that the cyclic amidic function is essential for HNE
inhibitory activity, suggesting that interaction with the enzyme is
mediated by the nucleophilic attack at the amide carbonyl group.
We also analyzed the importance of the ester function at position
3 of the indazole scaffold by hydrolyzing, removing, or replacing
this feature with a methyl group. As shown in Table 5, these elab-
orations significantly affected inhibitory activity, resulting in inac-
tive compounds (17b and 19b) or weak inhibitors (17a and 19a).
The most active N-benzoylindazole derivatives identified here
(IC50 ꢀ 90–100 nM) are similar in HNE inhibitory activity to other
currently reported small-molecule HNE inhibitors, such as Sivele-
stat (ONO-5046; IC50 = 44 nM),30 Midesteine (MR889; IC50
1–10
M),39 and 2-pyridin-3-yl-benzoxazinone (IC50 = 61 nM).40
=
l

L. Crocetti et al. / Bioorg. Med. Chem. 19 (2011) 4460–4472
4465
Table 6
and several known HNE inhibitors are also relatively potent chy-
motrypsin inhibitors.41,42
Analysis of N-benzoylindazole inhibitory specificity
Compound
IC50 (lM)
3.3. Kinetic features and stability
Thrombin
Urokinase
Chymotrypsin
6c
8a
8b
8d
8i
5.2 1.9
14 4.2
5.1 1.8
NA
7.8 2.7
6.7 2.4
14 3.5
0.43 0.13
0.081 0.018
0.030 0.013
0.26 0.087
0.047 0.011
0.0029 0.0012
To better understand the mechanism of action of these com-
pounds, we performed kinetic experiments with two of the most
active compounds (8a and 8b). As shown in Fig. 2, the representa-
tive double-reciprocal Lineweaver–Burk plot of fluorogenic sub-
strate hydrolysis by HNE in the absence and presence of
compound 8a indicates that this compound is a competitive HNA
inhibitor. Similar results were observed with kinetic analysis of
compound 8b (data not shown).
1.2 0.028
0.87 0.33
12 4.2
6.3 2.1
10 3.1
8m
3.2. Specificity
Compound 8a was further evaluated for chemical stability in
aqueous buffer using spectrophotometry to detect compound
hydrolysis.23 Fig. 3 shows that the absorbance maxima at 246
and 314 nm decreased over time, indicating that the compound
was hydrolyzed almost completely after 10 h in aqueous buffer.
This relatively rapid rate of spontaneous hydrolysis allowed us to
evaluate reversibility of HNE inhibition over time. As shown in
Fig. 4A, HNE was rapidly inhibited, with no lag period after addi-
To evaluate inhibitor specificity, we analyzed effects of the six
most potent N-benzoylindazole derivatives on three other serine
proteases, including human pancreatic chymotrypsin (EC
3.4.21.1), human thrombin (EC 3.4.21.5), and human urokinase
(urokinase-type plasminogen activator precursor, EC 3.4.21.73).
As shown in Table 6, most of selected compounds inhibited throm-
bin and urokinase at micromolar concentrations, whereas the
derivatives were potent chymotrypsin inhibitors. This is not sur-
prising, as HNE and chymotrypsin belong to same enzyme family,
A
80
vehicle
0.55
70
Compound 8a
[I] = 160 nM
0.45
0.35
0.25
0.15
0.05
-0.05
60
50
[I] = 80 nM
[I] = 40 nM
[I] = 0 nM
6c
40
30
-0.025 0.000 0.025 0.050 0.075 0.100
1/[S]
8a
20
8d
Figure 2. Kinetics of HNE inhibition by compound 8a. Representative double-
reciprocal Lineweaver–Burk plot from three independent experiments.
10
0
8m
8b, 8i
0
30
60
90
120
150
180
Time (min)
0.45
0
S
25
0.40
65
B
100
80
60
40
20
0
120
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
175
vehicle
240
325
395
8a
8i
465
8d, 8m
8b
6c
630 min
0
100
200
300
Time (min)
400
500
600
230
255
280
305
330
Figure 4. Evaluation of HNE inhibition by representative N-benzoylindazoles over
extended periods of time. HNE was incubated with the indicated compounds
(600 nM), and kinetic curves monitoring substrate cleavage catalyzed by HNE
during the first 180 min (Panel A) and from 0 to 10 h (Panel B) are shown. The arrow
indicates addition of additional fluorogenic substrate (S). Representative curves are
from two independent experiments.
Wavelength (nm)
Figure 3. Analysis of spontaneous hydrolysis of N-benzoylindazole derivative 8a.
The change in absorbance spectrum of compound 8a (20 M in 0.05 M phosphate
buffer, pH 7.3, 25 °C) was monitored over time in solution. Representative scans are
l
from two independent experiments.

4466
L. Crocetti et al. / Bioorg. Med. Chem. 19 (2011) 4460–4472
tion of compound, and inhibition was maximal during the first
90 min with compounds 6c and 120 min with the other tested
compounds (8a, 8b, 8d, 8m, and 8i). However, inhibition by com-
pound 6c was soon reversed, and full recovery of HNE activity
was observed by ꢀ5 h after treatment with up to 600 nM of the
compound (Fig. 4B). In comparison, reversal of inhibition by 8a,
8d, 8m, and 8i was much slower, and HNE was still inhibited to
50% of control level by 600 nM of these compounds at 5 h after
treatment (Fig. 4B). Furthermore, subsequent addition of more
fluorogenic substrate showed that the enzyme was still active after
the 10 h incubation period (indicated by arrow in Fig. 4B). Thus,
these results suggest that the active N-benzoylindazoles may be
pseudoirreversible inhibitors of HNE, which covalently attack the
enzyme active site but can be reversed by hydrolysis of the acyl-
enzyme complex, as discussed previously for structure-related
N-benzoylpyrazole-derived HNE inhibitors.23
3.4. Molecular modelling
To investigate binding of compounds to the HNE active site and
obtain insight into the differences in inhibitory activity of the var-
ious derivatives, we selected active and inactive compounds and
performed molecular modelling studies using a 23-residue model
of the HNE binding site, as described previously.23 The binding site
was derived from the 1.84 Å resolution crystal structure of HNE
complexed with a peptide chloromethyl ketone inhibitor.43 We im-
ported this structure of the HNE binding site into ArgusLab and
performed docking studies to identify binding modes of the flexible
ligands that were favourable for the interaction between the hy-
droxyl group of Ser195 and the carbonyl group of the compounds
under investigation. This approach is based on a number of reports
showing that the mechanism of action of serine protease inhibitors
containing an N–C@O fragment involves interaction between the
inhibitor’s carbonyl group and Ser19544–46 and/or other targets in
the active site.42,47 This mechanism of interaction has been shown
to involve formation of an intermediate Michaelis complex, where
the inhibitor carbonyl carbon atom is located 1.8–2.6 Å from the
Ser195 hydroxyl oxygen (distance d1), while the angle of Ser195
Figure 6. Molecular docking of active (8a) and inactive (14d) compounds into the
binding site of HNE. The Ser195 hydroxyl oxygen is yellow. For the ligand
structures, carbon is gray, hydrogen is white, nitrogen is violet, oxygen is red, and
chlorine is green.
the carbonyl carbon atom is more accessible to the Ser195 hydro-
xyl oxygen in active compounds, as compared to inactive com-
pounds (d1 = 3.49 0.47 versus 5.91 1.01 Å, respectively)
(Table 7). Docking poses for inactive compounds are characterized
by remarkably obtuse angle
a (148.4 9.4), whereas this angle is
67.2 4.1° for active indazole derivatives. Obviously, these geo-
metric parameters strongly influence the ability of a molecule to
form Michaelis complex and define energy of molecular distortion
on the formation of such complex (see below).
Oc
Á Á ÁC@O, denoted as angle
a
, is limited to 80–120°8,45,48 (Fig. 5).
The most favourable binding modes for six active (5c, 6c, 8a, 8b,
Three residues, Ser195, His57, and Asp102, form an active site
triad that catalyzes synchronous proton transfer from Ser195 to
Asp102, with His57 acting as a general base to enhance nucleophi-
licity of the Ser195 hydroxyl oxygen and activate the hydrolytic
H2O23,43,49,50 (Fig. 2). While rotational lability of the catalytic triad
residues could result in a more favourable environment for sub-
strate binding and cleavage, certain rotational freedom could also
destroy the conformationally sensitive ‘channel’ of proton migra-
tion during formation of the Michaelis complex. To explore this is-
sue, five torsion angles (r1–r5) in Ser195, His57, and Asp102 (Fig. 2)
were varied during optimization with the MM+ force field, while
8d, and 13f) and five inactive (14b, 14c, 14d, 17b, and 19b) com-
pounds were chosen from ligand docking poses lying within a
2 kcal/mol energy gap above the lowest-energy binding mode for
each compound. Representative binding modes for active 8a and
inactive 14d are shown in Fig. 6. The d1 and
a values show that
concurrently applying harmonic restraints to d1 and a, as described
previously.23 This approach allowed us to evaluate if rotational
flexibility in Ser195, His57, and Asp102 could lead to a favourable
orientation for Michaelis complex formation with an optimized li-
gand. Differences in the conformational energies (DE) of a mole-
cule after and before MM+ optimization (Table 6) within the
partially flexible binding site showed that active compounds may
form a Michaelis complex with suitable d1 and
values) without strong distortion of their optimal molecular geom-
etry, as indicated by relatively low average value
a (see optimized
D
E
(7.59 4.49 kcal/mol). In addition, geometric rearrangement for
these compounds did not lead to steric collisions between
nonbonded atoms. Alternatively, geometry suitable for Michaelis
complex formation with inactive molecules was not attainable
starting from their docking poses because their penetration to
Figure 5. Geometric parameters important for formation of a Michaelis complex in
the HNE active site. The important distances (d) and relevant angle ( ), as specified
a
in the text, are indicated. Based on the model of synchronous proton transfer from
the oxyanion hole in HNE. Reproduced from Ref.22

L. Crocetti et al. / Bioorg. Med. Chem. 19 (2011) 4460–4472
4467
Table 7
Results of docking and subsequent molecular optimization for favourable poses in the binding site of HNE for selected active (5c, 6c, 8a, 8b, 8d and 13f) and inactive (14b-d, 17b
and 19b) compounds
No.
a
(deg)
Optimization
Distances (Å)
Optimization
Molecular deformation,
E (kcal/mol)
D
Docking
Docking
d1
d1
d2
d3
L = d2,min + d3
Active
5c
6c
8a
8b
71
70
69
60
68
65
86
81
90
82
90
87
4.09
3.27
3.03
3.11
3.34
4.07
2.39
2.32
2.37
2.40
2.36
2.39
2.17/2.45
1.95/2.52
3.38/3.56
2.21/2.47
3.72/3.97
2.17/2.45
2.99
2.77
3.88
3.00
4.15
3.01
5.16
4.72
7.26
5.21
7.87
5.18
12.89
0.32
4.65
10.40
7.92
9.38
8d
13f
Inactive
14b
14c
14d
17b
160
139
147
140
156
86
90
85
69
88
5.55
6.74
4.55
7.08
5.65
2.32
2.40
2.36
2.49
2.39
2.16/2.52
2.88/3.76
3.21/3.34
3.76/3.94
3.05/4.62
2.86
3.31
3.58
4.94
4.72
5.02
6.19
6.79
8.70
7.77
18.07
18.36
18.70
32.90
26.50
19b
Ser195 under the forces of harmonic restraints led to dramatic
molecular distortion characterized by E = 22.91 6.61 kcal/mol.
5. Experimental section
5.1. Chemistry
D
The sum of d3 distance and shorter d2 value, denoted here as L
(Table 6), represents the length of the channel for synchronous
proton transfer from Ser195 through His57 to Asp102. It should
be noted that there were no significant differences in distance L be-
tween active and inactive compounds investigated, which is in
contrast to HNE inhibitors with a benzoylpyrazole scaffold.23
MM+ optimization of docked structures with the applied harmonic
restraints led to noticeable rotation of His57, both for active inda-
zoles 8a, 8d and inactive compounds 14c, 14d, 17b, 19b. These
geometric changes can be explained by the bulkiness of the mole-
cules. Unlike benzoylpyrazoles,23 the indazole derivatives investi-
gated here contain a fused benzene ring, and their stiff scaffolds
cause conformational changes in the receptor residues during
Michaelis complex formation. Thus, it can be concluded that effects
All melting points were determined on a Büchi apparatus
and are uncorrected. 1H NMR spectra were recorded with an
Avance 400 instruments (Bruker Biospin Version 002 with
SGU). Chemical shifts are reported in ppm, using the solvent
as an internal standard. Extracts were dried over Na2SO4, and
the solvents were removed under reduced pressure. Merck F-
254 commercial plates were used for analytical TLC to follow
reactions. Silica gel 60 (Merck 70-230 mesh) was used for col-
umn chromatography. Microanalyses were performed with a
Perkin-Elmer 260 elemental analyzer for C, H, and N, and the
results were within 0.4% of the theoretical values, unless
otherwise stated. Reagents and starting materials were commer-
cially available.
of distortion energy
DE of a docked molecule on subsequent for-
mation of the Michaelis complex is the main factor influencing bio-
logical activity of the compounds.
5.1.1. General procedures for 3a–k, 3m, and 3q
To a cooled (0 °C) suspension of 1H-indazole-3-carboxylic acid
phenylamide 234 (0.42 mmol) and a catalytic amount of Et3N
(0.05 mL) in anhydrous CH2Cl2 (1–2 mL), the appropriate aroyl
chloride (1.25 mmol) was added. The mixture was stirred for 1–
2 h at 0 °C and then for 1–3 h at room temperature. The precipitate
was filtered off, and the solvent was evaporated in vacuo. Cold
water (20 mL) was added, the mixture was neutralized with
0.5 N NaOH, and the precipitate was recovered by vacuum filtra-
tion. For compounds 3c, 3e–f, 3k, and 3q, the reaction mixture
was extracted with CH2Cl2 (3 Â 15 mL) after dilution. The solvent
was dried over sodium sulphate to obtain the desired final com-
pounds. Compound 3c was purified by column chromatography
using toluene/ethyl acetate 8:2 as eluent.
4. Conclusions
Our data demonstrate that the N-benzoylindazole scaffold is an
appropriate system for development of HNE inhibitors and that a
benzoyl substituent to the cyclic amide at N-1 was fundamental
for activity, in agreement with the potent N-benzoylpyrazoles pre-
viously described. Our results also showed that the inclusion of a
methyl or methoxy substituent at the meta position of this benzoyl
group was favorable for activity. Likewise, modification of the ester
function at position 4 of the indazole demonstrated that com-
pounds with a methyl ester at this position were the most active.
The most active derivatives (8a and 8b) were found to be compet-
itive, pseudoirreversible inhibitors of HNE and were relatively spe-
cific for HNE and chymotrypsin, as compared to other serine
proteases, including thrombin and urokinase. Docking studies with
partial flexibility of His57, Asp102, and Ser195 residues showed
that orientation of active compounds in the HNE binding site is
suitable for the interaction between the hydroxyl group of
Ser195 and the carbonyl group of the inhibitor molecule. More-
over, changes in conformational energy during Michaelis complex
formation for inactive compounds were significantly higher than
those for the active inhibitors investigated. Further studies are in
progress to improve our knowledge of structure–activity relation-
ships of HNE inhibitors with an N-benzoylindazole scaffold.
5.1.1.1. 1-Benzoyl-1H-indazole-3-carboxylic acid phenylamide,
3a.
Yield = 29%; mp = 142–143 °C (EtOH); 1H NMR (CDCl3) d
7.20 (t, 1H, Ar, J = 7.6 Hz), 7.40 (t, 2H, Ar, J = 7.6 Hz), 7.55–7.65
(m, 3H, Ar), 7.70 (m, 4H, Ar), 8.10 (m, 2H, Ar), 8.55 (m, 2H, Ar),
8.70 (exch br s, 1H, NH). Anal. (C21H15N3O2) C, H, N.
5.1.1.2. 1-(2-Methylbenzoyl)-1H-indazole-3-carboxylic acid
phenylamide, 3b.
Yield = 20%; mp = 129–130 °C (EtOH); 1H
NMR (CDCl3) d 2.45 (s, 3H, CH3), 7.20 (m, 1H, Ar), 7.40 (m, 4H,
Ar), 7.50–7.75 (m, 6H, Ar), 8.55 (s, 2H, Ar), 8.60 (exch br s, 1H,
NH). Anal. (C22H17N3O2) C, H, N.

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L. Crocetti et al. / Bioorg. Med. Chem. 19 (2011) 4460–4472
5.1.2. General procedures for 3l, 3o, and 3p
Ar, J = 8.0 Hz), 8.60 (d, 1H, Ar, J = 8.4 Hz), 8.95 (s, 1H, Ar). Anal.
(C15H12N2O3S) C, H, N.
To a cooled and stirred solution of the appropriate acid (3-
methoxybenzoic acid, thiophene 3-carboxylic acid and piperonylic
acid) (0.42 mmol) and a catalytic amount of Et3N (0.05 mL) in
anhydrous DMF (1–2 mL), diethylcyanophosphonate (DCF)
(1.68 mmol) and 1H-indazole-3-carboxylic acid phenylamide 234
(0.42 mmol) were added. The mixture was stirred at room temper-
ature for 12 h. After dilution with cold water, the suspension was
extracted with CH2Cl2 (3 Â 15 mL), and the solvent was evaporated
in vacuo, resulting in a residue oil, which was purified by column
chromatography using toluene/ethyl acetate (8:2) as eluent.
5.1.5. General procedures for 5m, 5q, and 5r
Compounds 5m, 5q and 5r were obtained from compound 4,32
following the general procedure described for 3a–k, 3m and 3q.
For compound 5m, cold water (15 mL) was added after evapora-
tion of the solvent, and the mixture was neutralized with 0.5 N
NaOH. The precipitate was recovered by suction.
5.1.5.1. 1-(4-Cyanobenzoyl)-1H-indazole-3-carboxylic acid ethyl
ester, 5m.
Yield = 38%; mp = 140–142 °C (EtOH); 1H NMR
5.1.2.1. 1-(3-Methoxybenzoyl)-1H-indazole-3-carboxylic acid
(CDCl3) d 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 4.55 (q, 2H, CH2CH3,
J = 7.2 Hz), 7.55 (t, 1H, Ar, J = 8.0 Hz), 7.70 (t, 1H, Ar, J = 8.4 Hz),
7.85 (d, 2H, Ar, J = 8.4 Hz), 8.25 (d, 2H, Ar, J = 8.4 Hz), 8.30 (d, 1H,
Ar, J = 8.0 Hz), 8.60 (d, 1H, Ar, J = 8.4 Hz). Anal. (C18H13N3O3) C, H, N.
phenylamide, 3l.
Yield = 15%; oil (purified by column chro-
matography using toluene/ethyl acetate 8:2 as eluent); 1H NMR
(CDCl3) d 3.95 (s, 3H, OCH3), 7.15–7.25 (m, 2H, Ar), 7.40 (t, 2H,
Ar, J = 8.0 Hz), 7.50 (t, 1H, Ar, J = 8.0 Hz), 7.55 (t, 1H, Ar,
J = 7.6 Hz), 7.60 (s, 1H, Ar), 7.65–7.75 (m, 4H, Ar), 8.55 (d, 2H, Ar,
J = 8.4 Hz), 8.70 (exch br s, 1H, NH). Anal. (C22H17N3O3) C, H, N.
5.1.5.2. 1-(Naphthalene-1-carbonyl)-1H-indazole-3-carboxylic
acid ethyl ester, 5q.
Yield = 28%; mp = 150–152 °C (EtOH);
1H NMR (CDCl3) d 1.45 (t, 3H, CH2CH3, J = 7.2 Hz), 4.50 (q, 2H,
CH2CH3, J = 7.2 Hz), 7.55–7.65 (m, 4H, Ar), 7.75 (m, 1H, Ar), 7.85
(d, 1H, Ar, J = 7.2 Hz), 7.95 (m, 1H, Ar), 8.05–8.15 (m, 2H, Ar),
8.30 (d, 1H, Ar, J = 8.0 Hz), 8.70 (d, 1H, Ar, J = 8.4 Hz). Anal.
(C21H16N2O3) C, H, N.
5.1.2.2.
1-(Thiophene-3-carbonyl)-1H-indazole-3-carboxylic
acid phenylamide, 3o.
Yield = 21%; mp = 186–187 °C (EtOH);
1H NMR (CDCl3) d 7.20 (t, 1H, Ar, J = 7.6 Hz), 7.40–7.50 (m, 3H, Ar),
7.55 (t, 1H, Ar, J = 8.0 Hz), 7.70 (t, 1H, Ar, J = 7.6 Hz), 7.75 (d, 2H, Ar,
J = 7.6 Hz), 7.95 (d, 1H, Ar, J = 5.2 Hz), 8.55 (d, 1H, Ar, J = 8.0 Hz),
8.60 (d, 1H, Ar, J = 8.4 Hz), 8.65 (s, 1H, Ar), 8.75 (exch br s, 1H,
NH). Anal. (C19H13N3O2S) C, H, N.
5.1.6. General procedures for 5n and 5s
Compounds 5n and 5s were obtained from 432 and the appro-
priate carboxylic acid, following the same procedure as described
for 3n. Compound 5n was purified by column chromatography
using CH2Cl2/MeOH 9.5:0.5 as eluent, and compound 5q was
recrystallized with ethanol.
5.1.3. 1-(Pyridine-4-carbonyl)-1H-indazole-3-carboxylic acid
phenylamide, 3n
Isonicotinic acid (0.42 mmol) was dissolved in SOCl2 (0.7 mL)
and refluxed for 1 h. After cooling, the excess of SOCl2 was removed
in vacuo, and the residue oil was dissolved in cold anhydrous tolu-
ene (3 mL). To this solution, a mixture of H-indazole-3-carboxylic
acid phenylamide 234 (0.42 mmol) and Et3N (0.46 mmol) in
2.8 mL of anhydrous toluene was added. The mixture was stirred
at 100 °C for 6 h. After cooling, the precipitate was recovered by
vacuum filtration and washed with water, followed by 0.5 N NaOH,
resulting in 3n, which was recrystallized with ethanol. Yield = 21%;
mp = 180 °C dec. (EtOH); 1H NMR (CDCl3) d 7.20 (t, 1H, Ar,
J = 7.6 Hz), 7.40 (t, 2H, Ar, J = 7.60 Hz), 7.60 (t, 1H, Ar, J = 8.0 Hz),
7.70 (d, 2H, Ar, J = 8.0 Hz), 7.75 (t, 1H, Ar, J = 8.4 Hz), 7.95 (d, 2H,
Ar, J = 4.8 Hz), 8.55 (exch br s, 1H, NH), 8.60 (d, 2H, Ar, J = 8.4 Hz),
8.95 (d, 2H, Ar, J = 4.8 Hz). Anal. (C20H14N4O2) C, H, N.
5.1.6.1. 1-(Pyridine-4-carbonyl)-1H-indazole-3-carboxylic acid
ethyl ester, 5n.
Yield = 13%; mp = 119 °C dec. (EtOH); 1H NMR
(CDCl3) d 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 4.55 (q, 2H, CH2CH3,
J = 7.2 Hz), 7.60 (t, 1H, Ar, J = 8.0 Hz), 7.75 (t, 1H, Ar, J = 7.6 Hz),
8.00 (d, 2H, Ar, J = 6 Hz), 8.30 (d, 1H, Ar, J = 8.0 Hz), 8.60 (d, 1H,
Ar, J = 8.4 Hz), 8.90 (d, 2H, Ar, J = 6.0 Hz). Anal. (C16H13N3O3) C, H, N.
5.1.6.2. 1-(3,4,5-Trimethoxybenzoyl)-1H-indazole-3-carboxylic
acid ethyl ester, 5s.
Yield = 50%; mp = 160–163 °C (EtOH);
1H NMR (CDCl3) d 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 3.95 (s, 6H, 2
OCH3), 4.00 (s, 3H, OCH3), 4.55 (q, 2H, CH2CH3, J = 7.2 Hz), 7.55 (t,
1H, Ar, J = 8.0 Hz), 7.60 (s, 2H, Ar), 7.70 (t, 1H, Ar, J = 8.4 Hz), 8.30
(d, 1H, Ar, J = 8.0 Hz), 8.60 (d, 1H, Ar, J = 8.4 Hz). Anal.
(C20H20N2O6) C, H, N.
5.1.4. General procedures for 5l, 5o, and 5p
Compounds 5l, 5o, and 5p were obtained from 1H-indazole-3-
carboxylic acid ethyl ester 432 and the appropriate acid, following
the same procedure described for 3l, 3o and 3p. Compounds 5l
and 5o were purified by column chromatography using toluene/
ethyl acetate (8:2) as eluent, while compound 5p was purified by
crystallization from ethanol.
5.1.7. General procedures for 6b, 6c, and 6e
Compounds 6b, 6c, and 6e were obtained from 432 and the
appropriate (cyclo)alkylchloride, following the same general pro-
cedure described for 3a–k, 3m, and 3q. For these compounds, the
mixtures were stirred at 0 °C for 2 h, and the solvent was evapo-
rated in vacuo. Cold water was then added, the resulting suspen-
sions were alkalinized with 0.5 N NaOH, and the precipitates
were recovered by vacuum filtration.
5.1.4.1. 1-(3-Methoxybenzoyl)-1H-indazole-3-carboxylic acid
ethyl ester, 5l.
Yield = 20%; mp = 81–83 °C (EtOH); 1H NMR
(CDCl3) d 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 3.90 (s, 3H, OCH3), 4.55
(q, 2H, CH2CH3, J = 7.2 Hz), 7.20 (d, 1H, Ar, J = 8.8 Hz), 7.45 (t, 1H,
Ar, J = 8.4 Hz), 7.55 (t, 1H, Ar, J = 7.2 Hz), 7.65–7.80 (m, 3H, Ar),
8.30 (d, 1H, Ar, J = 8.0 Hz), 8.60 (d, 1H, Ar, J = 8.8 Hz). Anal.
(C18H16N2O4) C, H, N.
5.1.7.1. 1-Propionyl-1H-indazole-3-carboxylic acid ethyl ester,
6b.
Yield = 55%; mp = 101–103 °C (EtOH); 1H NMR (CDCl3) d
1.40 (t, 3H, COCH2CH3, J = 7.2 Hz), 1.55 (t, 3H, OCH2CH3,
J = 7.2 Hz), 3.40 (q, 2H, COCH2CH3, J = 7.2 Hz), 4.55 (q, 2H, OCH2CH3,
J = 7.2 Hz), 7.50 (t, 1H, Ar, J = 8.0 Hz), 7.60 (t, 1H, Ar, J = 8.6 Hz), 8.25
(d, 1H, Ar, J = 8.0 Hz), 8.50 (d, 1H, Ar, J = 8.6 Hz). Anal. (C13H14N2O3)
C, H, N.
5.1.4.2.
1-(Thiophene-3-carbonyl)-1H-indazole-3-carboxylic
Yield = 14%; mp = 57–58 °C (EtOH); 1H
acid ethyl ester, 5o.
NMR (CDCl3) d 1.55 (t, 3H, CH2CH3, J = 7.2 Hz), 4.60 (q, 2H, CH2CH3,
J = 7.2 Hz), 7.40 (d, 1H, Ar, J = 5.2 Hz), 7.50 (t, 1H, Ar, J = 7.6 Hz),
7.65 (t, 1H, Ar, J = 7.2 Hz), 8.00 (d, 1H, Ar, J = 5.2 Hz), 8.30 (d, 1H,
5.1.7.2. 1-Butyryl-1H-indazole-3-carboxylic acid ethyl ester,
6c.
Yield = 37%; mp = 57–59 °C (EtOH); 1H NMR (CDCl3) d

L. Crocetti et al. / Bioorg. Med. Chem. 19 (2011) 4460–4472
4469
1.10 (t, 3H, CH2CH2CH3, J = 7.2 Hz), 1.55 (t, 3H, OCH2CH3, J = 7.2 Hz),
1.85–1.95 (m, 2H, CH2CH2CH3, J = 7.2 Hz), 3.35 (t, 2H, CH2CH2CH3,
J = 7.2 Hz), 4.60 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.50 (t, 1H, Ar,
J = 8.0 Hz), 7.60 (t, 1H, Ar, J = 8.4 Hz), 8.25 (d, 1H, Ar, J = 8.0 Hz),
8.50 (d, 1H, Ar, J = 8.4 Hz). Anal. (C14H16N2O3) C, H, N.
2H, Ar), 8.30 (d, 1H, Ar, J = 8.0 Hz), 8.60 (d, 1H, Ar, J = 8.4 Hz). Anal.
(C17H14N2O3) C, H, N.
5.1.11. General procedures for 8c and 8d
Compounds 8c and 8d were obtained starting from 1H-inda-
zole-3-carboxylic acid methyl ester 7a35 and the appropriate acid
(3-methoxybenzoic acid and thiophene 3-carboxylic acid) follow-
ing the same procedure described for 3l, 3o, and 3p. In this case,
the mixtures were stirred at room temperature for 24 h. After dilu-
tion with cold water, compound 8c suspension was extracted with
CH2Cl2 (3 Â 15 mL), the solvent was evaporated in vacuo, and the
residue oil was purified by column chromatography using tolu-
ene/ethyl acetate (8:2) as eluent. Compound 8d was filtered and
purified by crystallization from ethanol.
5.1.8. 1-Cyclopentanecarbonyl-1H-indazole-3-carboxylic acid
ethyl ester, 6d
Cyclopentanecarboxylic acid (0.525 mmol) dissolved in SOCl2
(1 mL) was stirred at 100 °C for 1 h. After cooling, the excess SOCl2
was removed in vacuo, and the residue was dissolved in cold anhy-
drous toluene (3–4 mL). To this solution, a mixture of 1H-indazole-
3-carboxylic acid ethyl ester 432 (0.525 mmol) and Et3N
(0.577 mmol) in toluene anhydrous (3 mL) was added, and the sus-
pension was stirred at 110 °C for 5 h. After cooling, the precipitate
was filtered off, the solvent was evaporated in vacuo, cold water
(15 mL) was added, and resulting mixture was neutralized with
0.5 N NaOH and extracted with CH2Cl2 (3 Â 15 mL). Evaporation
of the solvent resulted in the final compound 6d. Yield = 86%; oil;
1H NMR (CDCl3) d 1.55 (t, 3H, OCH2CH3, J = 7.2 Hz), 1.75–1.85 (m,
4H, cC5H9), 1.95–2.05 (m, 2H, cC5H9), 2.10–2.20 (m, 2H, cC5H9),
4.20–4.30 (m, 1H, cC5H9), 4.60 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.50
(t, 1H, Ar, J = 8.0 Hz), 7.60 (t, 1H, Ar, J = 8.4 Hz), 8.25 (d, 1H, Ar,
J = 8.0 Hz), 8.50 (d, 1H, Ar, J = 8.4 Hz). Anal. (C16H18N2O3) C, H, N.
5.1.11.1. 1-(3-Methoxybenzoyl)-1H-indazole-3-carboxylic acid
methyl ester, 8c.
Yield = 18%; mp = 112–116 °C (EtOH); 1H
NMR (CDCl3) d 3.90 (s, 3H, OCH3), 4.05 (s, 3H, COOCH3), 7.20 (d,
1H, Ar, J = 8.0 Hz), 7.45 (t, 1H, Ar, J = 8.0 Hz), 7.55 (t, 1H, Ar,
J = 7.6 Hz), 7.70 (m, 2H, Ar), 7.75 (d, 1H, Ar, J = 7.6 Hz), 8.30 (d,
1H, Ar, J = 8.0 Hz), 8.60 (d, 1H, Ar, J = 8.4 Hz). Anal. (C17H14N2O4)
C, H, N.
5.1.11.2.
1-(Thiophene-3-carbonyl)-1H-indazole-3-carboxylic
acid methyl ester, 8d.
Yield = 25%; mp = 113–114 °C (EtOH);
5.1.9. 1H-Indazole-3-carboxylic acid 2,2,2-trifluoroethyl ester,
7d
1H NMR (CDCl3) d 4.10 (s, 3H, OCH3), 7.45 (m, 1H, Ar), 7.55 (t,
1H, Ar, J = 7.6 Hz), 7.65 (t, 1H, Ar, J = 8.0 Hz), 8.00 (d, 1H, Ar,
J = 5.2 Hz), 8.30 (d, 1H, Ar, J = 8.0 Hz), 8.60 (d, 1H, Ar, J = 8.4 Hz),
8.95 (s, 1H, Ar). Anal. (C14H10N2O3S) C, H, N.
A solution of indazole-3-carboxylic acid (1.85 mmol) 1 in 6 mL
of SOCl2 was refluxed for 8 h. After cooling, the excess SOCl2 was
removed in vacuo, and the residue was dissolved in 6 mL of dry tol-
uene. To this mixture, a solution of trifluoroethanol (26.6 mmol)
and of Et3N (6.24 mmol) in anhydrous toluene (6 mL) was added,
and the mixture was stirred at room temperature. After 8 h, the
solvent was evaporated in vacuo, and cold water was added to
the residue oil to form a precipitate, which was recovered by
suction. Yield = 77%; mp = 183–185 °C (EtOH); 1H NMR (CDCl3) d
4.85 (q, 2H, OCH2CF3, J = 8.4 Hz), 7.40 (t, 1H, Ar, J = 8.0 Hz),
7.55 (t, 1H, Ar, J = 8.4 Hz), 7.65 (d, 1H, Ar, J = 8.0 Hz), 8.20 (d, 1H,
Ar, J = 8.4 Hz), 11.45 (exch br s, 1H, NH). Anal. (C10H7F3N2O2) C,
H, N.
5.1.12. General procedures for 8g, h, k, l, o, and p
Compounds 8g, h, k, l, o, and p were obtained from 7b,35 7d, and
the appropriate carboxylic acid (3-methoxybenzoic acid and thio-
phene 3-carboxylic acid), following the same procedure described
for 3n, with the exception of compound 8h, which was refluxed for
10 h. After cooling, the precipitates were filtered off, and the sol-
vent was evaporated under vacuum. After addition of cold water
(20 mL), the mixtures were extracted with CH2Cl2. Evaporation of
the solvent resulted in the final compounds, which were purified
by flash chromatography using cyclohexane/ethyl acetate (4:1) as
eluent for compounds 8k and toluene/ethyl acetate (8:2) for 8o.
Compounds 8g, 8h, 8l, and 8p were recrystallized from ethanol.
5.1.10. General procedures for 8a, b, e, f, i, j, m, and n
Compounds 8a, b, e, f, i, j, m, and n were obtained starting from
7a–d (7a–c35) and the appropriate ethero(aryl) chloride following
the general procedures described for 3a–k, 3m, and 3q. For com-
pounds 8a, 8b, and 8m, the mixtures were stirred for 1–2 h at
0 °C and then for 12 h at room temperature. For compounds 8e,
8f, 8i, and 8j, the reactions were carried out at 60 °C for 4–5 h.
The resulting precipitates were filtered off, the solvent was evapo-
rated, cold water (20 mL) was added, and the mixtures were neu-
tralized with 0.5 N NaOH. Compound 8a was recovered by vacuum
filtration, whereas compounds 8b, e, f, i, j, m, and n were extracted
with CH2Cl2 (3 Â 15 mL), and the solvent was evaporated in vacuo
to afford the desired final compounds, which were recrystallized
from ethanol.
5.1.12.1. 1-(3-Methoxybenzoyl)-1H-indazole-3-carboxylic acid
propyl ester, 8g.
Yield = 55%; mp = 93–94 °C (EtOH); 1H
NMR (CDCl3) d 1.10 (t, 3H, CH2CH2CH3, J = 7.2 Hz), 1.90 (m, 2H,
CH2CH2CH3), 3.90 (s, 3H, OCH3), 4.45 (t, 2H, CH2CH2CH3,
J = 6.8 Hz), 7.20 (d, 1H, Ar, J = 8.0 Hz), 7.45 (t, 1H, Ar, J = 8.0 Hz),
7.55 (t, 1H, Ar, J = 7.6 Hz), 7.70 (t, 1H, Ar, J = 8.0 Hz), 7.75 (s, 1H,
Ar), 7.80 (d, 1H, Ar, J = 8.0 Hz), 8.30 (d, 1H, Ar, J = 8.4 Hz), 8.60 (d,
1H, Ar, J = 8.0 Hz). Anal. (C19H18N2O4) C, H, N.
5.1.12.2.
1-(Thiophene-3-carbonyl)-1H-indazole-3-carboxylic
acid propyl ester, 8h.
Yield = 52%; mp = 61–62 °C (EtOH);
1H NMR (CDCl3) d 1.15 (t, 3H, CH2CH2CH3, J = 7.2 Hz), 1.95 (m,
2H, CH2CH2CH3), 4.50 (t, 2H, CH2CH2CH3, J = 6.8 Hz), 7.40 (m, 1H,
Ar), 7.50 (t, 1H, Ar, J = 8.0 Hz), 7.65 (t, 1H, Ar, J = 8.4 Hz), 8.00 (d,
1H, Ar, J = 5.2 Hz), 8.30 (d, 1H, Ar, J = 8.0 Hz), 8.60 (d, 1H, Ar,
J = 8.4 Hz), 8.95 (s, 1H, Ar). Anal. (C16H14N2O3S) C, H, N.
5.1.10.1. 1-Benzoyl-1H-indazole-3-carboxylic acid methyl ester,
8a.
Yield = 37%; mp = 115–117 °C (EtOH); 1H NMR (CDCl3) d
4.10 (s, 3H, OCH3), 7.55 (m, 3H, Ar), 7.65 (m, 2H, Ar), 8.15 (m,
2H, Ar), 8.30 (d, 1H, Ar, J = 8.0 Hz), 8.60 (d, 1H, Ar, J = 8.4 Hz). Anal.
(C16H12N2O3) C, H, N.
5.1.13. General procedures 12a–f
To a cooled (À5 °C) and stirred solution of 3-tert-butoxycarbon-
ylamino benzoic acid (0.26 mmol), 10 (prepared from precursor 9,
as reported in literature36) in anhydrous THF (2 mL) and 0.91 mmol
of Et3N were added. After 30 min, the mixture was allowed to
warm up to 0 °C, and ethyl chloroformate was added (0.29 mmol).
5.1.10.2. 1-(3-Methylbenzoyl)-1H-indazole-3-carboxylic acid
methyl ester, 8b.
NMR (CDCl3) d 2.50 (s, 3H, CH3), 4.05 (s, 3H, OCH3), 7.45 (m, 2H,
Ar), 7.55 (t, 1H, Ar, J = 8.0 Hz), 7.70 (t, 1H, Ar, J = 8.4 Hz), 7.95 (d,
Yield = 37%; mp = 109–110 °C (EtOH); 1H

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L. Crocetti et al. / Bioorg. Med. Chem. 19 (2011) 4460–4472
After 1 h, 0.52 mmol of the appropriate 1H-indazole (2, 4, 7a–d)
were added, and the reaction was carried out at room temperature
for 12 h. For compounds 12d and 12e, the mixtures were stirred at
50–60 °C for 5–10 h. The suspensions were concentrated in vacuo,
diluted with cold water, neutralized with 0.5 N NaOH, and ex-
tracted with CH2Cl2 (3 Â 15 mL). The solvent was evaporated to af-
ford the desired final compounds, which were purified by column
chromatography using toluene/ethyl acetate (8:2) as eluent.
5.1.15.2. 1-(4-Chlorobenzenesulfonyl)-1H-indazole-3-carbox-
ylic acid phenylamide, 14b.
Yield = 78%; mp = 190–193 °C
(EtOH); 1H NMR (CDCl3) d 7.20 (t, 1H, Ar, J = 7.6 Hz), 7.45 (m, 2H,
Ar), 7.50 (m, 3H, Ar), 7.65 (m, 1H, Ar), 7.80 (d, 2H, Ar, J = 8.0 Hz),
7.95 (d, 2H, Ar, J = 8.4 Hz), 8.25 (d, 1H, Ar, J = 8.0 Hz), 8.45 (d, 1H,
Ar, J = 8.4 Hz), 8.80 (exch br s, 1H, NH). Anal. (C20H14ClN3O3S) C,
H, N.
5.1.16. 1-(4-Chlorobenzyl)-1H-indazole-3-carboxylic acid
phenylamide, 14c
5.1.13.1. [3-(3-Phenylcarbamoyl-indazole-1-carbonyl)-phenyl]-
carbamic acid tert-butyl ester, 12a.
Yield = 15%; mp = 75–
A mixture of 1H-indazole-3-carboxylic acid phenylamide 234
(0.63 mmol), K2CO3 (1.97 mmol) and 4-chlorobenzyl chloride
(1.1 mmol) in 2 mL of anhydrous DMF was stirred at 80 °C for
1 h. After cooling, the mixture was diluted with cold water and
the precipitate recovered by vacuum filtration.
78 °C (EtOH); 1H NMR (CDCl3) d 1.45 (s, 9H, C(CH3)3), 6.65 (exch
br s, 1H, NHCOO), 7.15 (t, 1H, Ar, J = 7.6 Hz), 7.30–7.40 (m, 2H,
Ar), 7.50–7.60 (m, 3H, Ar), 7.70 (t, 1H, Ar, J = 7.6 Hz), 7.75 (m, 2H,
Ar), 7.85 (d, 1H, Ar, J = 7.6 Hz), 8.55–8.65 (m, 3H, Ar), 9.15 (exch
br s, 1H, NH). Anal. (C26H24N4O4) C, H, N.
Yield = 52%; mp = 127–130 °C (EtOH); 1H NMR (CDCl3) d 5.65 (s,
2H, CH2Ar), 7.20 (m, 3H, Ar), 7.30–7.45 (m, 7H, Ar), 7.80 (d, 2H, Ar,
J = 8.0 Hz), 8.50 (d, 1H, Ar, J = 8.0 Hz), 8.85 (exch br s, 1H, NH). Anal.
(C21H16ClN3O) C, H, N.
5.1.13.2. 1-(3-tert-Butoxycarbonylamino-benzoyl)-1H-indazole-
3-carboxylic acid ethyl ester, 12b.
Yield = 10%; oil; 1H NMR
(CDCl3) d 1.30 (t, 3H, CH2CH3, J = 7.2 Hz), 1.55 (s, 9H, C(CH3)3),
4.55 (q, 2H, CH2CH3, J = 7.2 Hz), 6.65 (exch br s, 1H, NHCO), 7.45–
7.55 (m, 2H, Ar), 7.65 (t, 1H, Ar, J = 7.6 Hz), 7.80–7.90 (m, 2H, Ar),
8.05 (s, 1H, Ar), 8.30 (d, 1H, Ar, J = 7.6 Hz), 8.55 (m, 1H, Ar). Anal.
(C22H23N3O5) C, H, N.
5.1.17. 1-(4-Chlorophenyl)-1H-indazole-3-carboxylic acid
phenylamide, 14d
A mixture of activated, powdered 4A molecular sieves (700 mg),
dry CH2Cl2 (8 mL), 234 (0.63 mmol), 4-chlorobenzenboronic acid
(1.26 mmol), Cu(Ac)2 (0.94 mmol), and Et3N (1.26 mmol) was stir-
red at room temperature for 3 h. After filtration of the molecular
sieves, the organic layer was washed with water (3 Â 20 mL), fol-
lowed by 33% aqueous ammonia (3 Â 5 mL). The organic phase
was dried over sodium sulfate, and the solvent was evaporated in
vacuo to afford the final compound 14d, which was purified by
column chromatography using toluene/ethyl acetate (9.9:0.1) as
eluent. Yield = 15%; mp = 130–132 °C (EtOH); 1H NMR (CDCl3) d
7.20 (t, 1H, Ar, J = 7.6 Hz), 7.40 (m, 3H, Ar), 7.55 (t, 1H, Ar,
J = 8.0 Hz), 7.60 (m, 2H, Ar), 7.70–7.80 (m, 5H, Ar), 8.60 (d, 1H,
Ar, J = 8.4 Hz), 8.95 (exch br s, 1H, NH). Anal. (C20H14ClN3O)
C, H, N.
5.1.14. General procedures for 13a–f
A mixture of 12a–f (0.043 mmol), trifluoroacetic acid (0.28 mL),
and CH2Cl2 (1.72 mL) was stirred at room temperature for 3 h.
After evaporation of the solvent, diethyl ether was added to the
residue, and the precipitate was recovered by vacuum filtration.
5.1.14.1. 1-(3-Aminobenzoyl)-1H-indazole-3-carboxylic acid
phenylamide, 13a.
Yield = 35%; mp = 85–88 °C (EtOH); 1H
NMR (DMSO-d6) d 6.90 (d, 1H, Ar, J = 8.4 Hz), 7.15 (t, 1H, Ar,
J = 7.2 Hz), 7.20–7.30 (m, 2H, Ar), 7.40 (m, 3H, Ar), 7.60 (t, 1H, Ar,
J = 8.0 Hz), 7.75 (t, 1H, Ar, J = 8.4 Hz), 7.80 (m, 2H, Ar), 8.25 (d,
1H, Ar, J = 8.0 Hz), 8.45 (d, 1H, Ar, J = 8.4 Hz), 9.05 (exch br s, 2H,
NH2), 10.45 (exch br s, 1H, NH). Anal. (C21H16N4O2) C, H, N.
5.1.18. 1-(2-Oxo-2-phenylethyl)-1H-indazole-3-carboxylic acid
ethyl ester, 14e
5.1.14.2. 1-(3-Aminobenzoyl)-1H-indazole-3-carboxylic acid
ethyl ester, 13b.
A mixture of 1H-indazole-3-carboxylic acid ethyl ester 432
(0.42 mmol), phenacyl bromide (0.42 mmol), and K2CO3
(1.26 mmol) in anhydrous acetone was stirred at room tempera-
ture for 6 h. After evaporation of the solvent, cold water was added,
and the precipitate was recovered by vacuum filtration.
Yield = 98%; mp = 92–95 °C (EtOH); 1H NMR (CDCl3) d 1.50 (t, 3H,
OCH2CH3, J = 7.2 Hz), 4.55 (q, 2H, OCH2CH3, J = 7.2 Hz), 6.00 (s,
2H, CH2COPh), 7.35 (m, 2H, Ar), 7.45 (t, 1H, Ar, J = 7.6 Hz), 7.55 (t,
2H, Ar, J = 8.0 Hz), 7.70 (t, 1H, Ar, J = 7.6 Hz), 8.05 (d, 2H, Ar,
J = 7.6 Hz), 8.30 (d, 1H, Ar, J = 8.0 Hz). Anal. (C18H16N2O3) C, H, N.
Yield = 80%; mp = 152–155 °C (EtOH); 1H
NMR (DMSO-d6) d 1.40 (t, 3H, CH2CH3, J = 7.2 Hz), 4.15 (q, 2H,
CH2CH3, J = 7.2 Hz), 6.95 (s, 1H, Ar), 7.20–7.30 (m, 3H, Ar), 7.60 (t,
1H, Ar, J = 8.0 Hz), 7.75 (t, 1H, Ar, J = 7.2 Hz), 8.25 (d, 1H, Ar,
J = 8.0 Hz), 8.40 (d, 1H, Ar, J = 8.4 Hz), 9.10 (exch br s, 2H, NH2).
Anal. (C17H15N3O3) C, H, N.
5.1.15. General procedures for 14a and 14b
To a cooled (0 °C) suspension of 1H-indazole-3-carboxylic acid
phenylamide 234 (0.25 mmol) and a catalytic amount of Et3N
(0.05 mL) in anhydrous CH2Cl2 (1–2 mL), 0.25 mmol of the appropri-
ate sulfonyl chloride (tosyl chloride and 4-chlorobenzenesulfonyl
chloride) were added. The mixture was stirred at room temperature
for 3–5 h. The precipitate was filtered off, the solvent was evapo-
rated under vacuum, and water was added to the residue. Finally,
the mixture was neutralized with 0.5 N NaOH, and the precipitate
obtained was filtered and recrystallized with ethanol.
5.1.19. General procedure for 14f and 14g
A mixture of 1H-indazole-3-carboxylic acid ethyl ester 432
(0.52 mmol) and the appropriate phenylisocyanide (4-chlorophe-
nylisocyanide and 3-methoxyphenylisocyanide) in 2 mL of anhy-
drous THF was stirred at room temperature for 12–20 h. After
evaporation of the solvent, the residue was purified by crystalliza-
tion from ethanol.
5.1.15.1. 1-(Toluene-4-sulfonyl)-1H-indazole-3-carboxylic acid
phenylamide, 14a.
5.1.19.1. 1-(4-Chlorophenylcarbamoyl)-1H-indazole-3-carbox-
Yield = 72%; mp = 185–188 °C (EtOH); 1H
ylic acid ethyl ester, 14f.
Yield = 73%; mp = 125–127 °C
NMR (CDCl3) d 2.45 (s, 3H, Ar), 7.20 (t, 1H, Ar, J = 7.6 Hz), 7.25
(m, 2H, Ar), 7.45 (m, 3H, Ar), 7.65 (t, 1H, Ar, J = 7.6 Hz), 7.75 (d,
2H, Ar, J = 8.0 Hz), 7.90 (d, 2H, Ar, J = 8.4 Hz), 8.25 (d, 1H, Ar,
J = 8.0 Hz), 8.45 (d, 1H, Ar, J = 8.4 Hz), 8.85 (exch br s, 1H, NH). Anal.
(C21H17N3O3S) C, H, N.
(EtOH); 1H NMR (CDCl3) d 1.55 (t, 3H, OCH2CH3, J = 7.2 Hz), 4.55
(q, 2H, OCH2CH3, J = 7.2 Hz), 7.40 (d, 2H, Ar, J = 8.4 Hz), 7.50 (t,
1H, Ar, J = 8.0 Hz), 7.65 (m, 3H, Ar), 8.25 (d, 1H, Ar, J = 8.0 Hz),
8.50 (d, 1H, Ar, J = 8.4 Hz), 9.20 (exch br s, 1H, NH). Anal.
(C17H14ClN3O3) C, H, N.

L. Crocetti et al. / Bioorg. Med. Chem. 19 (2011) 4460–4472
4471
5.1.19.2.
boxylic acid ethyl ester, 14g.
1-(3-Methoxyphenylcarbamoyl)-1H-indazole-3-car-
Yield = 74%; mp = 102–105 °C
5.3. Analysis of compound stability
(EtOH); 1H NMR (CDCl3) d 1.55 (t, 3H, OCH2CH3, J = 7.2 Hz), 3.90
(s, 3H, OCH3), 4.55 (q, 2H, OCH2CH3, J = 7.2 Hz), 6.75 (d, 1H, Ar,
J = 8.0 Hz), 7.25 (d, 1H, Ar, J = 7.6 Hz), 7.35 (t, 1H, Ar, J = 8.0 Hz),
7.40 (s, 1H, Ar), 7.45 (t, 1H, Ar, J = 8.0 Hz), 7.65 (t, 1H, Ar,
J = 7.6 Hz), 8.25 (d, 1H, Ar, J = 8.0 Hz), 8.55 (d, 1H, Ar, J = 8.4 Hz),
9.20 (exch br s, 1H, NH). Anal. (C18H17N3O4) C, H, N.
Spontaneous hydrolysis of selected N-benzoylindazoles was
evaluated at 25 °C in 0.05 M phosphate buffer, pH 7.3. Kinetics of
N-benzoylindazole hydrolysis was monitored by measuring
changes in absorbance spectra over the incubation time using a
SpectraMax Plus spectrophotometer (Molecular Devices, Sunny-
vale, CA).
5.1.20. (3-Methylindazol-1-yl)-m-tolyl-methanone, 17a
5.4. Analysis of inhibitor specificity
Compound 17a was obtained from compound 1637 by reaction
with m-toluoyl chloride, following the procedure described for 3a–
k, 3m, and 3q. After evaporation of the solvent, cold water was
added to the residue, followed by neutralization with 0.5 N NaOH.
The mixture was then extracted with ethyl acetate, and the solvent
was evaporated in vacuo to afford the desired final compound 17a.
Yield = 20%; mp = 79–80 °C (EtOH); 1H NMR (CDCl3) d 2.45 (s, 3H,
Ph-CH3), 2.60 (s, 3H, CH3), 7.45 (m, 3H, Ar), 7.65 (t, 1H, Ar,
J = 7.6 Hz), 7.70 (d, 1H, Ar, J = 8.4 Hz), 7.90 (m, 2H, Ar), 8.55 (d,
1H, Ar, J = 8.4 Hz). Anal. (C16H14N2O) C, H, N.
Selected compounds were evaluated for their ability to inhibit
various proteases in 100 lL reaction volumes at 25 °C. Analysis of
chymotrypsin inhibition was performed in reaction mixtures con-
taining 0.05 M Tris–HCl, pH 8.0, 30 nM human pancreas chymo-
trypsin (Calbiochem, San Diego, CA), test compounds, and
100 lM substrate (Suc-Ala-Ala-Pro-Phe-7-amino-4-methylcouma-
rin). Thrombin inhibition was evaluated in reaction mixtures con-
taining 0.25 M sodium phosphate, pH 7.0, 0.2 M NaCl, 0.1% PEG
8000, 1.7 U/mL human plasma thrombin (Calbiochem), test com-
pounds, and 20 lM substrate (benzoyl-Phe-Val-Arg-7-amino-4-
5.1.21. (3-Methoxyphenyl)-(3-methylindazol-1-yl)-methanone,
17b
methylcoumarin). Analysis of urokinase inhibition assay was per-
formed in reaction mixtures containing 0.1 M Tris–HCl, pH 8.0,
30 U/mL human urine urokinase (Calbiochem), test compounds,
Compound 17b was obtained from 1637 by reaction with 3-
methoxybenzoic acid following the same procedure described for
3n. After cooling, the solvent was evaporated under vacuum, cold
water was added, and the mixture was neutralized with 0.5 N
NaOH and extracted with CH2Cl2. Evaporation of the solvent re-
sulted in 17b. Yield = 10%; mp = 62–65 °C (EtOH); 1H NMR (CDCl3)
d 2.60 (s, 3H, CH3), 3.90 (s, 3H, OCH3), 7.15 (d, 1H, Ar, J = 8.0 Hz),
7.45 (m, 2H, Ar), 7.65 (t, 2H, Ar, J = 8.0 Hz), 7.70 (t, 2H, Ar,
J = 8.4 Hz), 8.55 (d, 1H, Ar, J = 8.4 Hz). Anal. (C16H14N2O2) C, H, N.
and 30 lM substrate (benzyloxycarbonyl-Gly-Gly-Arg-7-amino-
4-methylcoumarin). Enzyme activity was monitored at excitation
and emission wavelengths of 355 and 460 nm, respectively. For
all compounds tested, the concentration of inhibitor that caused
50% inhibition of the enzymatic reaction (IC50) was calculated by
plotting % inhibition versus logarithm of inhibitor concentration
(at least six points), and the data are the mean values of at least
three experiments with relative standard deviations of <15%.
5.1.22. 1-(3-Methylbenzoyl)-1H-indazole-3-carboxylic acid, 19b
A mixture of 5c (0.357 mmol) and 6 N NaOH (8 mL) was stirred
at 50 °C for 5 h. After cooling, the mixture was acidified with 6 N
HCl, and the precipitate was recovered by vacuum filtration and
recrystallized with ethanol. Yield = 20%; mp = 267–269 °C dec.
(EtOH); 1H NMR (CDCl3) d 2.35 (s, 3H, CH3), 7.30 (t, 1H, Ar,
J = 7.6 Hz), 7.35–7.45 (m, 3H, Ar), 7.65 (d, 1H, Ar, J = 8.4 Hz), 7.75
(m, 2H, Ar), 8.10 (d, 1H, Ar, J = 8.0 Hz), 13.80 (exch br s, 1H, 0H).
Anal. (C16H12N2O3) C, H, N.
5.5. Molecular modeling
For computer-assisted molecular modeling, we employed
HyperChem, version 7.0 (Hypercube Inc., Waterloo, ON, Canada)
and ArgusLab 4.0.1 (Planaria Software LLC, Seattle, WA). Molecular
structures were first created and optimized by HyperChem with
the use of the molecular mechanics MM+ force field. These struc-
tures were then subjected to docking computations by ArgusLab
with AScore scoring functions51 to find the lowest-energy binding
modes. Flexibility of an inhibitor was accounted for around all
rotatable bonds automatically identified by ArgusLab. The binding
site of HNE was considered to be rigid in the docking procedure,
and its geometry was obtained by downloading a crystal structure
of HNE complexed with a peptide chloromethyl ketone inhibitor43
from the Protein Data Bank (code 1HNE). Amino acid residues
within 7 Å of any non-hydrogen atom of the inhibitor were re-
garded as belonging to the binding site, and 22 residues satisfied
this condition (Phe41, Cys42, Ala55, His57, Cys58, Leu99B,
Asp102, Val190, Cys191, Phe192, Gly193, Asp194, Ser195,
Gly196, Ser197, Ala213, Ser214, Phe215, Val216, Arg217A,
Gly218, and Tyr224). Although Ala56 is 8.3 Å away from the near-
est inhibitor atom, we included Ala56 in the binding site to main-
tain continuous sequence from Ala55 to Cys58. We then removed
the chloromethyl ketone inhibitor and co-crystallized water mole-
cules to obtain the 23-residue model of the HNE binding site that
was used for docking.
5.2. HNE inhibition assay
Compounds were dissolved in 100% DMSO at 5 mM stock con-
centrations. The final concentration of DMSO in the reactions was
1%, and this level of DMSO had no effect on enzyme activity. The
HNE inhibition assay was performed in black flat-bottom 96-well
microtiter plates. Briefly, a buffer solution containing 200 mM
Tris–HCl, pH 7.5, 0.01% bovine serum albumin, and 0.05%
Tween-20 and 20 mU/mL of HNE (Calbiochem) was added to
wells containing different concentrations of each compound.
The reaction was initiated by addition of 25
strate (N-methylsuccinyl-Ala-Ala-Pro-Val-7-amino-4-methyl-
coumarin, Calbiochem) in a final reaction volume of 100 L/
lM elastase sub-
l
well. Kinetic measurements were obtained every 30 s for
10 min at 25 °C using a Fluoroskan Ascent FL fluorescence micro-
plate reader (Thermo Electron, MA) with excitation and emission
wavelengths at 355 and 460 nm, respectively. For all compounds
tested, the concentration of inhibitor that caused 50% inhibition
of the enzymatic reaction (IC50) was calculated by plotting % inhi-
bition versus logarithm of inhibitor concentration (at least six
points). The data are presented as the mean values of at least
three independent experiments with relative standard deviations
of <15%.
Binding modes found for each inhibitor within a 2 kcal/mol gap
above the lowest-energy mode were examined for the potential to
form a Michaelis complex between the hydroxyl group of Ser195
and the inhibitor’s carbonyl group in amido moiety, and values
of d1 and
a (Fig. 2) were determined for each docked compound.
These modes were further optimized by molecular mechanics with
the MM+ force field using HyperChem. During this optimization,

4472
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Acknowledgements
This work was supported in part by National Institutes of
Health grant P20 RR-020185 and the Montana State University
Agricultural Experimental Station.
Supplementary data
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