Optimization of N-benzoylindazole derivatives as inhibitors of human neutrophil elastase

Article abstract of DOI:10.1021/jm400742j

Human neutrophil elastase (HNE) is an important therapeutic target for treatment of pulmonary diseases. Previously, we identified novel N-benzoylindazole derivatives as potent, competitive, and pseudoirreversible HNE inhibitors. Here, we report further development of these inhibitors with improved potency, protease selectivity, and stability compared to our previous leads. Introduction of a variety of substituents at position 5 of the indazole resulted in the potent inhibitor 20f (IC₅₀ ～10 nM) and modifications at position 3 resulted the most potent compound in this series, the 3-CN derivative 5b (IC₅₀ = 7 nM); both derivatives demonstrated good stability and specificity for HNE versus other serine proteases. Molecular docking of selected N-benzoylindazoles into the HNE binding domain suggested that inhibitory activity depended on geometry of the ligand-enzyme complexes. Indeed, the ability of a ligand to form a Michaelis complex and favorable conditions for proton transfer between Hys57, Asp102, and Ser195 both affected activity.

Full text of DOI:10.1021/jm400742j

Article
pubs.acs.org/jmc
Optimization of N‑Benzoylindazole Derivatives as Inhibitors of
Human Neutrophil Elastase
Letizia Crocetti,† Igor A. Schepetkin,‡ Agostino Cilibrizzi,† Alessia Graziano,† Claudia Vergelli,†
Donatella Giomi,§ Andrei I. Khlebnikov,∥ Mark T. Quinn,‡ and Maria Paola Giovannoni*,†
†NEUROFARBA, Sezione di Farmaceutica e Nutraceutica, Universita
Italy
̀
degli Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino,
‡Department of Immunology and Infectious Diseases, Montana State University, Bozeman, Montana 59717, United States
§Dipartimento di Chimica, Universita
̀
degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
∥Department of Chemistry, Altai State Technical University, Barnaul, Russia
S
* Supporting Information
ABSTRACT: Human neutrophil elastase (HNE) is an important therapeutic
target for treatment of pulmonary diseases. Previously, we identified novel N-
benzoylindazole derivatives as potent, competitive, and pseudoirreversible HNE
inhibitors. Here, we report further development of these inhibitors with improved
potency, protease selectivity, and stability compared to our previous leads.
Introduction of a variety of substituents at position 5 of the indazole resulted in the
potent inhibitor 20f (IC50 ∼10 nM) and modifications at position 3 resulted the
most potent compound in this series, the 3-CN derivative 5b (IC50 = 7 nM); both
derivatives demonstrated good stability and specificity for HNE versus other serine
proteases. Molecular docking of selected N-benzoylindazoles into the HNE binding domain suggested that inhibitory activity
depended on geometry of the ligand−enzyme complexes. Indeed, the ability of a ligand to form a Michaelis complex and
favorable conditions for proton transfer between Hys57, Asp102, and Ser195 both affected activity.
the large number of HNE inhibitors described in the literature,
Sivelestat is the only nonpeptidic inhibitor marketed, and it is
limited to use in Japan and Korea for the treatment of acute
lung injury, since its development in the USA was terminated in
2003.21,22 On the other hand, the neutrophil elastase inhibitor
AZD9668 is currently being evaluated in clinical trials for
patients with bronchiectasis and COPD.23,24
Recently, we discovered that N-benzoylpyrazoles and N-
benzoylindazoles are potent HNE inhibitors.25−27 In the
present studies, we further optimize HNE inhibitors with an
N-benzoylindazole scaffold and evaluate their biological activity.
Introduction of a variety of substituents at the phenyl ring of
the indazole nucleus and elaboration of the ester function at
position 3 demonstrated that the cyclic amides, as well as the
benzoyl fragment at position 1, are essential for activity. Studies
on the most active derivatives, which were active in the low
nanomolar range, showed that these compounds are com-
petitive, pseudoirreversible inhibitors of HNE, with an
appreciable selectivity toward HNE versus other kinases tested.
We also utilized molecular modeling to evaluate binding of the
compounds to the HNE active site and determined that both
the ability of an inhibitor to form a Michaelis complex and
favorable conditions for proton transfer between His57,
Asp102, and Ser195 affected inhibitory activity. Thus, the N-
INTRODUCTION
■
Human neutrophil elastase (HNE) is a member of the
chymotrypsin superfamily of serine proteases involved in the
response to inflammatory stimuli.1,2 HNE is stored in
azurophilic granules of neutrophils and is very aggressive and
cytotoxic, as its substrates include many components of the
extracellular matrix.3 Because of the destructive potency of
HNE, its extracellular activity is regulated under physiological
conditions by endogenous inhibitors such as α1-proteinase
inhibitor (α1-PI) and α2-macroglobulin.4,5 When the appro-
priate balance between HNE and its inhibitors fails in favor of
the protease, the excess HNE activity may lead to tissue damage
and the consequent development of a variety of diseases.
Among the pathologies associated with increased HNE activity
are acute respiratory distress syndrome (ARDS),6 chronic
obstructive pulmonary disease (COPD),7,8 cystic fibrosis
(CF),9 and other disorders with an inflammatory component
such as atherosclerosis, psoriasis, and dermatitis.10−14 Recently
HNE has also been implicated in the progression of nonsmall
cell lung cancer progression.15 Thus, a number of clinical
observations indicate that HNE represents a good therapeutic
target for the treatment of inflammatory diseases and might be
of value as therapeutic agents in lung cancer.15−20
The design of new HNE inhibitors has focused primarily on
the development of different inhibitor types, including
mechanism-based inhibitors, acylating-enzyme inhibitors, tran-
sition-state analogues, and noncovalent inhibitors.16−20 Despite
Received: May 20, 2013
Published: July 12, 2013
© 2013 American Chemical Society
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Journal of Medicinal Chemistry
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benzoylindazole scaffold has great potential for the develop-
ment of additional potent HNE inhibitors.
Scheme 2 shows the synthetic routes followed to obtain
nitro-derivatives 14a−h (14e32), 15, and 16. Treatment of
a
CHEMISTRY
Scheme 2
■
All final compounds were synthesized as reported in Schemes
1−6, and the structures were confirmed on the basis of
analytical and spectral data. The synthetic pathway leading to
the final benzoyl-1H-indazoles 5a−e and 8, which are
substituted at position 3 of the indazole, is depicted in Scheme
1. Compounds 5a−d were obtained starting from the
a
Scheme 1
a
Reagents and conditions: (a) conc HNO3, conc H2SO4, 0 °C, 10 min.
(b) 14a,b,e,f, 15, 16: Ar-COCl, NEt3, anhydrous CH2Cl2, 0 °C, 2 h
and then rt, 2 h. 14c,d,g,h: step 1, Ar-COOH, SOCl2, reflux, 1 h; step
2, NEt3, anhydrous toluene, reflux, 6 h.
compounds 932 and 1031 with H2SO4/HNO3 1:1 v/v at 0 °C
resulted in a mixture of the intermediates 11a,b,32 12a,b
(12a33), and 1334 in different yields (60% for 11a,b, 10% for
12a,b, and 5% for 13), which were separated by column
chromatography and subsequently transformed into the final
compounds 14a−h, 15, and 16 following the same procedures,
as described previously.
a
Reagents and conditions: (a) 5a−c,e: Ar-COCl, NEt3, anhydrous
CH2Cl2, 0 °C, 2 h and then rt, 2−12 h. 5d: step 1, m-toluic acid, NEt3,
anhydrous THF, −7/−5 °C, 30 min; step 2, ClCOOEt, 0 °C, 1 h and
then rt, 24 h; (b) conc CH3COOH, conc H2SO4, 100 °C, 2 h; (c)
(NH4)2Ce(NO3)6, anhydrous CH3CN, 3,4-dihydro-2H-pyrano, rt, 24
h; (d) CH2Cl2/CF3COOH 6:1, rt, 3 h.
The 5-amino and 5-alkyl(aryl)amido derivatives 18, 19, and
20a−h were obtained starting from a common precursor, the 5-
NO2 derivative 14f described above, which was transformed
into the corresponding 5-amino 17 through catalytic reduction
with a Parr instrument. Treatment of 17 with iodomethane,
(cyclo)alkylcarbonyl chloride, or phenylboronic acid resulted in
the final compounds 18, 19, 20a−g, and 20h, respectively.
In Scheme 4, the synthetic pathways leading to the 7-
sulfamoyl (24a,b) and 5-bromo (26a−h, 26e32) indazole
derivatives are shown. Introduction of the sulfamoyl moiety was
carried out by treatment of the commercially available 21 with
chlorosulfonic acid and 33% aqueous ammonia (22).
Esterification of 22 and further insertion of the benzoyl
fragment resulted in the final compounds 24a,b. The
previously described precursors 3 and 4,28,29 following two
different procedures: treatment with the appropriate benzoyl-
chloride and Et3N in anhydrous CH2Cl2 (5a−c) or treatment
with m-toluic acid, Et3N, and ethyl cloroformate in anhydrous
THF (5d). For synthesis of 5e, the (1H-indazol-3-yl)-methanol
130 was transformed into the ester derivative 2, which was
treated with m-toluoyl chloride and Et3N in CH2Cl2 to obtain
the final compound. The same precursor 130 was also used for
the synthesis of compound 8. First, it was necessary to protect
the alcohol function at position 3 with 3,4-dihydro-2H-pyran
(compound 6), followed by insertion of the benzoyl fragment
at N-1, and finally removal of the protecting group with
trifluoroacetic acid.
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a
Scheme 3
BIOLOGICAL RESULTS AND DISCUSSION
■
Structure−Activity Relationship Analysis. All com-
pounds were evaluated for their ability to inhibit HNE, and
the results are reported in Tables 1−3, together with
representative reference compounds from the previous series
of N-benzoylindazole-derived HNE inhibitors (designated as
compounds A through H here).25 Keeping at positions 1 and 3
those substituents that produced the best results in the previous
series,25 we first introduced a variety of groups at position 5 of
the indazole nucleus, such as nitro, bromine, chlorine,
(substituted) amino, etc. (Table 1). The introduction of
substituents was generally favorable for HNE inhibitory activity,
and most of the newly synthesized compounds showed 1 order
of magnitude higher or a similar activity compared to the
unsubstituted reference compounds A−H. In particular,
introduction of a nitro group led to the most active
compounds, which had IC50 values of 15−50 nM, irrespective
of substituents Ar and R1 at positions 1 and 3, respectively
(compounds 14a−h). Likewise, the presence of an amide
(compounds 20a−c, 20f, and 20g) was beneficial for activity,
and these derivatives had similar activity as the 5-nitro
derivatives (IC50 = 12−50 nM) (Table 1). Results for these
5-amidic derivatives (20a−g) suggested the importance of
steric hindrance by the group linked to the amide CO because
the most bulky phenyl (20d) and cyclohexyl (20e) derivatives
were less active by about 1 order of magnitude (IC50 = 0.21 and
0.10 μM, respectively). Introduction of bromine resulted in
increased potency for all compounds of this series (26a−h)
compared to reference compounds A−H (Table 1). Com-
pounds containing methyl (31a), chlorine (31b), fluorine
(31c), methoxy (31d,e), or trifluoromethoxy (31g) at the same
position increased HNE inhibitory activity by 2−3-fold
compared to the reference compounds A and F, with the
exception of 31f, which had an IC50 = 60 nM. However, the 5-
(substituted)amino derivatives 17−19 and 20h retained HNE
inhibitory activity in the same activity range as reference
compound A (Table 1). On the other hand, 5-OH (31h) and
5-SO2NH2 (31i) had low or no activity, respectively. Thus, the
introduction of substituents at position 5 of the indazole
nucleus is clearly beneficial for activity; however, there does not
appear to be a generalizable correlation between activity and
nature of the substituents. On the other hand, it is clear that
acidic groups, such as OH and SONH2, are not tolerated at this
position because compounds 31h and 31i had low or no
activity. Regarding the variety of other substituents and taking
into account their different electronic and/or steric properties,
we hypothesize that an electron withdrawing group within a
given size is necessary to improve the potency. Aside from this
characteristic, and with the exception of the limitations of an
acid group mentioned above, compounds with other
substituents retained similar levels of activity as their
unsubstituted analogues.
a
Reagents and conditions: (a) H2, Pd/C, EtOH, rt 2 h. (b) CH3I,
K2CO3, anhydrous DMF, 60 °C, 3 h. (c) 20a−f: RCOCl, NEt3,
anhydrous CH2Cl2, 0 °C, 2 h, rt, 2 h. 20g: step 1, cyclo-
pentanecarboxylic acid, SOCl2, reflux, 1 h; step 2, NEt3, anhydrous
toluene, reflux, 5−6 h. 20h: phenylboronic acid, anhydrous CH2Cl2,
Cu(Ac)2, NEt3, rt, 2−4 h.
substitution at position 7 (rather than at position 4) for these
sulfamoyl derivatives was confirmed by performing spectral
analyses on intermediate 23. For this compound, the
1
substitution pattern was established on the basis of the H
NMR spectrum showing a pseudo triplet for H-5 at δ 7.50, as
the result of the 3JH,H couplings with H-4 and H-6 protons and
definitively proved on the basis of heteronuclear correlation
experiments. The g-HMBC spectrum clearly showed two cross-
3
peaks for the JC,H couplings of C-7a at δ 135.2 with H-4 and
H-6 protons and a cross-peak for the 3JC,H coupling of C-3a at δ
124.0 with H-5. The 5-bromo derivatives 26a−h shown in the
same scheme were obtained from 25a,b28 following the two
procedures described for 5a−c,e and for 14c,d,g,h.
Many substituents at position 5 have been introduced
following methods previously reported in the literature,35 which
allowed us to obtain the indazole nucleus starting from the
corresponding isatine. The synthetic route is shown in Scheme
5 using precursor isatines 27a−f (27a−e are commercially
available; 27f36), which resulted in synthesis of compounds
28a−f (28a−c,37 28d,38 28e39). Intermediates 28a−f were then
subjected to esterification, forming 29a−f (29a−c,37 29d38),
which were finally converted into the desired compounds 31a−
i. To obtain 31h, it was necessary to convert the OCH3 group
of 29d into OH by treatment with BBr3 in CH2Cl2 under
nitrogen at −78 °C, followed by treatment with m-toluic acid,
Et3N, and diethylcyanophosphonate (DCF) in dimethylforma-
mide (31h).
Evaluation of C-6 and C-7 substitutions (Table 2) showed
that the 6-substituted nitroderivative 15 (IC50 = 20 nM) was as
active as its 5-isomer 14b, while introduction of group at
position 7 gave rise to inactive compounds 16, 24a, and 24b.
Thus the C-6 position seems to be modifiable, while the total
inactivity of 7-substituted derivatives confirms that the position
neighboring the amidic nitrogen must be unsubstituted to allow
free rotation of N−CO bond, which is consistent with our
previous observations with other N-benzoylindazole deriva-
tives.25
Lastly, the synthesis of 33 (Scheme 6) was performed,
starting from precursor 3240 and using the same conditions as
described for compounds 5a−c,e (see Scheme 1).
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a
Scheme 4
a
Reagents and conditions: (a) Step 1, H3SO2Cl, 0 °C−reflux, 4−5 h; step 2, 33% NH3, 0 °C. (b) Anhydrous EtOH, conc H2SO4, reflux, 5 h. (c) Ar-
COCl, NEt3, anhydrous CH2Cl2, 0 °C, 2 h, rt, 2 h. (d) Anhydrous EtOH or MeOH, conc H2SO4, reflux, 5 h; (e) Br2, anhydrous DMF, 0 °C, 1 h, rt,
3 h. (f) 26a,b,e,f: Ar−COCl, NEt3, anhydrous CH2Cl2, 0 °C, 2 h and then rt, 2 h. 26c,d,g,h: step 1, Ar-COOH, SOCl2, reflux, 1 h; step 2, NEt3,
anhydrous toluene, reflux, 6 h.
The next modifications were performed at position 3 (Table
3). The introduction of an hydroxymethyl (8) or an inverse
ester (5e) was detrimental for activity, while the replacement of
the ester function with a primary amide gave different results
depending on the group at position 1 (5c and 5d). Conversely,
a remarkable increase in potency was obtained by introducing a
CN group, which resulted in the most active derivative of this
series (5b) with an IC50 of 7 nM. To verify if an additive effect
was possible to achieve by inserting in the same molecule both
of the groups that separately led to increased activity (i.e., 5-
nitro derivative 14c; 3-CN derivative 5b), we synthesized
compound 33. However, no additive effects were observed, as
33 had similar inhibitory activity (IC50 = 31 nM) as the singly
substituted derivatives 14c and 5b.
Inhibitor Specificity. To evaluate inhibitor specificity, we
analyzed effects of the 10 most potent N-benzoylindazoles on
four other serine proteases, including human pancreatic
chymotrypsin (EC 3.4.21.1), human thrombin (EC 3.4.21.5),
human plasma kallikrein (EC 3.4.21.34), and human urokinase
(EC 3.4.21.73), and an aspartic protease, cathepsin D (EC
3.4.23.5). As shown in Table 4, none of the tested derivatives
inhibited cathepsin D and only compound 14a inhibited
kallikrein. Compound 5b inhibited thrombin and urokinase at
micromolar concentrations. Although all tested compounds
inhibited chymotrypsin at nanomolar concentrations, com-
pound 20f had the lowest activity for this enzyme. Moreover,
only compound 20f had no inhibitory activity for urokinase
(Table 4). Thus, compounds 5b and 20f appear to be the most
specific HNE inhibitors.
Stability and Kinetic Features. The most potent and
specific N-benzoylindazoles were further evaluated for chemical
stability in aqueous buffer using spectrophotometry to detect
compound hydrolysis. As shown in Figure 1, the absorbance
maxima at 242 and 322 nm for compound 5b decreased over
time, indicating that this compound was hydrolyzed almost
completely after 35 min in aqueous buffer with a t1/2 of 21.7
min. The other nine compounds had t1/2 values from 27.2 to
117.5 min (Table 5), indicating that the tested N-
benzoylindazoles were more stable than our previously
described HNE inhibitors with the N-benzoylpyrazole scaf-
fold.25
The relatively rapid rate of spontaneous hydrolysis allowed
us to evaluate inhibitor reversibility over time. As shown in
Figure 2, inhibition was maximal during the first 5 h for
compounds 20b and 20f and during the first 4 h for
compounds 5b, 14b, 14f, 15, and 20a at 5 μM concentrations.
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a
Scheme 5
Table 1. HNE Inhibitory Activity of Indazole Derivatives
14a−h, 17−19, 20a−h, 26a−h, and 31a−i
a
compd
Ar
R1
R2
IC50 (μM)
14a
14b
14c
14d
14e32
14f
Ph
CH3
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NH2
0.03 0.011
0.015 0.0018
0.05 0.021
0.05 0.019
0.02 0.018
0.02 0.028
0.025 0.015
0.03 0.031
0.33 0.042
0.31 0.11
0.14 0.23
0.03 0.037
0.018 0.021
0.05 0.012
0.21 0.13
0.10 0.11
0.012 0.030
0.05 0.029
0.33 0.052
0.08 0.37
0.08 0.11
0.21 0.23
0.16 0.21
0.05 0.042
0.08 0.018
0.27 0.088
0.26 0.12
0.22 0.052
0.15 0.033
0.10 0.019
0.08 0.021
0.14 0.027
0.06 0.018
0.11 0.028
6.4 1.1
m-CH3-Ph
m-OCH3-Ph
3-thienyl
Ph
CH3
CH3
CH3
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
CH3
m-CH3-Ph
m-OCH3-Ph
3-thienyl
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
Ph
14g
14h
17
18
NHCH3
19
N(CH3)2
20a
20b
20c
20d
20e
20f
NHCOCH3
NHCOC2H5
NHCOnC3H7
NHCOPh
a
Reagents and conditions: (a) step 1, 0.5 N NaOH, H2O, 30 min, 50
NHCOcC6H11
°C; step 2, NaNO2 sol, H2SO4, 1 h, 0 °C; step 3, SnCl2·2H2O, conc
HCl, 0 °C, 2 h and then rt, 12 h. (b) anhydrous EtOH, conc H2SO4,
100 °C, 5 h. (c) for 29d: BBr3/CH2Cl2 (1M), N2, −78 °C, 15 min and
rt, 12 h. (d) 31a−g,i, Ar-COCl, NEt3, anhydrous CH2Cl2, 0 °C, 2 h
and then rt, 2 h; for 31h, m-toluic acid, DCF, NEt3, anhydrous DMF, 0
°C, 30 min and then rt, 12 h.
NHCOcC3H5
20g
20h
26a
26b
26c
26d
26e32
26f
NHCOcC5H9
NH-Ph
Br
m-CH3-Ph
m-OCH3-Ph
3-thienyl
Ph
CH3
Br
CH3
Br
a
CH3
Br
Scheme 6
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
CH3
Br
m-CH3-Ph
m-OCH3-Ph
3-thienyl
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
Ph
Br
26g
26h
31a
31b
31c
31d
31e
31f
Br
Br
CH3
Cl
F
a
OCH3
OCH3
OCF3
OCF3
OH
SO2NH2
H
Reagents and conditions: (a) m-toluoyl chloride, NEt3, anhydrous
CH2Cl2, 0 °C, 2 h, and then rt, 2 h.
m-CH3-Ph
Ph
31g
31h
31i
A25
B25
C25
D25
E25
F25
G25
H25
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-OCH3-Ph
m-OCH3-Ph
Ph
However, inhibition by compounds 14a, 14g, and 14h was
soon reversed, and recovery of HNE activity was observed by
∼1−2 h after treatment with up to 5 μM of these compounds
(Figure 2). Although the rate of reversibility for the last three
compounds (14a, 14g, and 14h) correlated with the rate of
spontaneous hydrolysis, this was not the case for the other N-
benzoylindazole derivatives tested. For example, hydrolysis of
the acyl−enzyme complex for compound 5b was much slower
than spontaneous hydrolysis of this nitrile derivative, and HNE
was still inhibited to 80% of control level at 7 h after treatment
(compare Figures 1 and 2). Thus, these results suggest that the
active N-benzoylindazoles may be pseudoirreversible HNE
inhibitors, which covalently attack the enzyme active site but
can be reversed by hydrolysis of the acyl−enzyme complex,
which is similar to the structurally related N-benzoylpyrazole-
derived HNE inhibitors.25
b
NA
0.41 0.11
0.13 0.051
0.55 0.21
0.8 0.33
H
CH3
H
C2H5
CH3
H
H
0.089 0.031
0.40 0.19
0.31 0.12
0.93 0.37
Ph
C2H5
CH3
H
3-thienyl
3-thienyl
H
C2H5
H
a
The IC50 values are presented as the mean
SD of three
b
independent experiments. NA: no inhibitory activity was found at
the highest concentration of compound tested (50 μM).
favorable specificity profiles (5b and 20f). As shown in Figure
3, the representative double-reciprocal Lineweaver−Burk plot
of fluorogenic substrate hydrolysis by HNE in the absence and
presence of compound 5b indicates that this compound is a
To better understand the mechanism of action of these N-
benzoylindazole HNE inhibitors, we performed kinetic experi-
ments with two of the most active compounds that had
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Table 2. HNE Inhibitory Activity of Indazole Derivatives 15,
16, and 24a,b
a
compd
Ar
R1
R2
R3
IC50 (μM)
15
m-CH3-Ph
m-CH3-Ph
Ph
CH3
NO2
H
H
0.02 0.018
b
16
CH3
NO2
NA
24a
24b
A25
B25
F25
C2H5
C2H5
C2H5
CH3
H
SO2NH2
NA
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
Ph
H
SO2NH2
NA
H
H
H
H
0.41 0.11
0.13 0.051
0.40 0.19
H
C2H5
H
a
The IC50 values are presented as the mean
SD of three
b
Figure 1. Analysis of changes in compound absorbance resulting from
spontaneous hydrolysis. The changes in absorbance spectra of
compound 5b (20 μM) were monitored over time with 2 min
intervals in 0.05 M phosphate buffer (pH 7.5, 25 °C). Representative
scans are from three independent experiments.
independent experiments. NA: no inhibitory activity was found at
the highest concentration of compound tested (50 μM).
Table 3. HNE Inhibitory Activity of Indazole Derivatives
5a−e, 8, and 33
Table 5. Half-Life (t1/2) for the Spontaneous Hydrolysis of
Selected Indazole Derivatives
a
compd
max (nm)
t1/2 (min)
5b
242
274
272
262
272
268
270
268
266
266
21.7
117.5
27.2
78.8
32.8
37.9
47.8
41.5
51.0
51.7
14f
14g
20a
14h
14a
14b
15
a
compd
Ar
R1
R2
IC50 (μM)
5a
5b
5c
5d
5e
8
Ph
CN
CN
H
0.04 0.011
0.007 0.0015
0.26 0.042
12.5 2.7
m-CH3-Ph
Ph
H
CONH2
CONH2
CH2OCOCH3
CH2OH
CN
H
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
m-CH3-Ph
H
H
6.4 1.2
20b
20f
b
H
NA
33
A25
NO2
H
0.031 0.018
0.41 0.11
a
Absorption maximum used for monitoring spontaneous hydrolysis.
COOC2H5
a
The IC50 values are presented as the mean
SD of three
b
competitive HNE inhibitor. Similar results were observed for
the kinetic analysis of compound 20f (data not shown).
Molecular Modeling. To evaluate complementarity of the
inhibitors to the HNE binding site, we performed molecular
docking studies of selected compounds (5b, 5d, 8, 14f, 26b,
independent experiments. NA: no inhibitory activity was found at
the highest concentration of compound tested (50 μM).
Table 4. Analysis of Inhibitory Specificity for Selected Indazole Derivatives
a
IC50 (μM)
compd
thrombin
chymotrypsin
kallikrein
urokinase
cathepsin D
b
5b
1.9 0.62
0.48 0.12
0.082 0.033
0.31 0.059
0.37 0.12
0.83 0.14
1.1 0.35
0.066 0.019
0.040 0.012
0.021 0.057
0.17 0.049
0.046 0.003
0.031 0.008
0.015 0.004
0.038 0.012
0.14 0.026
0.37 0.045
NA
6.6 2.7
1.5 0.65
0.48 0.11
13.7 3.6
25.3 6.2
0.62 0.17
0.44 0.13
0.31 0.018
14.8 4.6
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
14f
14g
20a
14h
14a
14b
15
NA
NA
NA
NA
11.2 2.6
NA
0.64 0.19
0.039 0.011
0.16 0.035
NA
20b
20f
NA
NA
a
b
The IC50 values are presented as the mean
concentration of compound tested (50 μM).
SD of three independent experiments. NA: no inhibition activity was found at the highest
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Figure 4. Superimposed docking poses of compound 5b (dark-green)
and peptide chloromethyl ketone inhibitor. The peptide inhibitor is
shown with a ball−stick rendering. The surface corresponds to the
initial enzyme structure from the PDB file.
Figure 2. Evaluation of HNE inhibition by selected indazole
derivatives over time. HNE was incubated with the indicated
compounds at 5 μM concentrations, and kinetic curves monitoring
substrate cleavage catalyzed by HNE from 0 to 7 h are shown.
Representative curves are from three independent experiments.
obtained by docking are presented in Table 6. Although the
docking poses are not Michaelis complexes themselves, it is
Table 6. Biological Activities of HNE Inhibitors and
Geometric Parameters of the Enzyme−Inhibitor Complexes
Predicted by Docking
a
b
compd IC50 (μM)
α
d1
d2
d3
L
5b
0.007
12.5
NA
119.3
105.5
169.4
129.8
75.5
3.448
4.348
4.590
4.079
3.874
4.222
2.181; 3.755
5.702; 5.844
1.796; 3.357
2.158; 3.749
1.730; 3.312
2.362; 3.959
3.142
2.449
3.272
3.119
2.753
3.303
5.323
8.151
5.068
5.277
4.483
5.665
5d
8
14f
26b
31h
0.02
0.08
6.4
Figure 3. Kinetics of HNE inhibition by compound 5b. Representative
double-reciprocal Lineweaver−Burk plot from three independent
experiments.
96.4
a
b
HNE inhibitory activity. Length of the channel for proton transfer
calculated as d3 + min(d2).
and 31h) into the HNE binding site using the HNE structure
from the Protein Data Bank (1HNE entry) where the enzyme
is complexed with a peptide chloromethyl ketone inhibitor.41
Previously, we used an approach where the HNE conformation
was adopted to be rigid, and a limited refinement of docking
poses was undertaken with flexibility of only three residues
(His57, Asp102, and Ser195) representing the catalytic triad of
serine proteases.25,27 In the present study, we applied a more
sophisticated methodology and performed docking with 42
flexible residues in the vicinity of the HNE binding site (see
Experimental Section) using Molegro software.
The search area for docking was defined as a sphere 10 Å in
radius centered at the nitrogen atom of the 5-membered ring of
the complexed inhibitor from the PDB file. This sphere
embraced almost the entire peptide chloromethyl ketone
inhibitor molecule and the nearest HNE residues, including
the catalytic triad of His57, Asp102, and Ser195. The best
docking poses obtained are located near the tail of the
cocrystallized peptide chloromethyl ketone inhibitor (see in
Figure 4 where the peptide and the pose of compound 5b are
shown superimposed).
reasonable to regard their structures as approximations to the
geometries of these complexes, keeping in mind that many
residues were treated as flexible in the docking procedure.
According to previous reports,42,43 it is optimal when the
Michaelis complex formed with participation of an inhibitor’s
amido moiety has the ligand carbonyl carbon atom located
1.8−2.6 Å from the Ser195 hydroxyl oxygen (distance d1),
while the angle α is within 80−120°. As shown in Table 6, the
highly active compounds 5b, 14f, and 26b in their best docking
poses had shorter d1 values than the less active inhibitors 5d
and 31h or inactive compound 8. Hence, the distance optimal
for Michaelis complex is more attainable from these poses.
Moreover, all active inhibitors were characterized by angle α in
the optimal range (5b, 5d, and 31h) or close to this range (14f
and 26b). On the other hand, angle α was too obtuse in the
docking pose of inactive compound 8 (Table 6). A visual
inspection of this pose (Supporting Information Figure S2)
indicated that an optimal value of α is hardly reachable from the
calculated binding mode of 8 because the molecule is anchored
by strong H-bonds formed between the oxymethyl group and
backbone heteroatoms in Cys191 and Ser195. Thus, such an
anchoring causes a very high energy barrier for reorienting 8 to
form the Michaelis complex within the binding site.
Analysis of the docking poses was performed to determine
the distances d1−d3 and angle O(Ser195)···CO (α,
Supporting Information Figure 1S), important for the
consideration of Michaelis complex formation and for easy
proton transfer within the oxyanione hole.42−44 The geometric
parameters of the lowest-energy enzyme−inhibitor complexes
The three residues, His57, Asp102, and Ser195, form a
catalytic triad, which acts as general base and enhances
nucleophilicity of Ser195 by the synchronous proton transfer
from the OH group of Ser195 to Asp102 through the His57
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imidazole moiety.41,45,46 Conformational changes of the
enzyme in the process of Michaelis complex formation can
destroy the channel of proton transfer because of possible
unfavorable orientation of the residues in the catalytic triad. To
evaluate conditions for synchronous proton transfer, we have
calculated an effective length (L) of the channel as a sum of
distances d2 and d3 (Supporting Information Figure S1). The
most active compounds (5b, 14f, and 26b) had shorter L values
than the moderately active inhibitors 5d and 31h (Table 6).
Although a relatively low L was obtained for inactive compound
8, the main reason of its inactivity is the unfavorable positioning
of the ligand due to H-bond formation, as described above.
The results of this investigation, as well as those of our
previous studies,25,27 show that HNE inhibitory activity
strongly depends on the geometric characteristics of ligand−
enzyme complexes. The ability of a ligand to form a Michaelis
complex and favorable conditions for proton transfer within the
binding site affected inhibitor activity. Hence, both an inhibitor
orientation with respect to Ser195 and the relative positioning
of residues in the catalytic triad are important.
General Procedure for 5a−c and 5e. To a cooled (0 °C)
suspension of the appropriate substrate 2, 3,28 or 429 (0.42 mmol) in
anhydrous CH2Cl2 (1−2 mL), a catalytic amount of Et3N (0.05 mL)
and the (substituted)-benzoyl chloride (1.26 mmol) were added. The
solution was stirred at 0 °C for 1−2 h and then for 1−3 h at room
temperature. The precipitate was removed by suction, and the organic
solvent was evaporated under vacuum. The residue was mixed in with
ice-cold water (20 mL) and neutralized with 0.5 N NaOH, and the
suspension was extracted with CH2Cl2 (3 × 15 mL). Evaporation of
the solvent resulted in the final compounds 5a−c and 5d, which were
purified by crystallization from ethanol (compounds 5a,b) or by
column chromatography using cycloexane/ethyl acetate 2:1 (for 5c) or
toluene/ethyl acetate 9.5:0.5 (for 5e) as eluent.
1-Benzoyl-1H-Indazole-3-carbonitrile (5a). Yield = 35%; mp =
1
156−157 °C (EtOH). H NMR (CDCl3) δ 7.63−7.74 (m, 4H, Ar),
7.88 (t, 1H, Ar, J = 8.0 Hz), 8.00−8.03 (m, 2H, Ar), 8.10 (d, 1H, Ar, J
= 8.0 Hz), 8.52 (d, 1H, Ar, J = 8.0 Hz).
1-(3-Methylbenzoyl)-1H-indazole-3-carbonitrile (5b). Yield =
1
22%; mp = 135−136 °C (EtOH). H NMR (CDCl3) δ 2.43 (s, 3H,
CH3), 7.48−7.57 (m, 2H, Ar), 7.68 (t, 1H, Ar, J = 8.0 Hz), 7.81 (s, 2H,
Ar), 7.87 (t, 1H, Ar, J = 8.4 Hz), 8.09 (d, 1H, Ar, J = 8.0 Hz), 8.51 (d,
1H, Ar, J = 8.4 Hz).
1-Benzoyl-1H-indazole-3-carboxylic Acid Amide (5c). Yield =
1
10%; mp = 204−206 °C (EtOH). H NMR (DMSO-d6) 7.53−7.62
CONCLUSIONS
■
(m, 3H, Ar), 7.69−7.76 (m, 2H, Ar), 7.84 (exch br s, 1H, NH2), 7.94
(exch br s, 1H, NH2), 8.14 (d, 2H, Ar, J = 7.2 Hz), 8.29 (d, 1H, Ar, J =
8.0 Hz), 8.47 (d, 1H, Ar, J = 8.0 Hz).
These results confirm that the N-benzoylindazole scaffold is an
appropriate structure for development of potent HNE
inhibitors and that, by maintaining a benzoyl substituent at
N-1, which is essential for activity, it is possible to increase the
potency by inserting substituents at the phenyl ring of the
indazole. In particular, the best results were obtained
introducing nitro, bromine, or acylamino groups at position
5, which resulted in corresponding HNE inhibitors with IC50
values of 12−50 nM. Furthermore, modifications at position 3
led to the most potent HNE inhibitor, 5b, with IC50 = 7 nM.
Analysis of specificity showed that compounds 5b and 20f were
relatively selective for HNE versus other proteases evaluated.
Finally, molecular docking studies strongly suggested that the
geometry of ligand−enzyme complexes was the main factor
influencing interaction of inhibitors with the HNE binding site.
Besides the ability of a compound to form a Michaelis complex,
a suitable orientation of His57, Asp102, and Ser195 was also
important for effective proton transfer from the oxyanione hole.
Thus, the novel HNE inhibitors reported here represent
potential leads for future optimization and in vivo studies.
Acetic Acid 1-(3-Methylbenzoyl)-1H-indazol-3-yl Methyl Ester
1
(5e). Yield = 22%; mp = 135−136 °C (EtOH). H NMR (CDCl3) δ
2.16 (s, 3H, Ph-CH3), 2.48 (s, 3H, COCH3), 5.50 (s, 2H, CH2), 7.42−
7.47 (m, 3H, Ar), 7.66 (t, 1H, Ar, J = 8.4 Hz), 7.85 (d, 1H, Ar, J = 8.0
Hz), 7.89 (s, 2H, Ar), 8.58 (d, 1H, Ar, J = 8.4 Hz).
1-(3-Methylbenzoyl)-1H-indazole-3-carboxylic Acid Amide
(5d). To a cooled (−5 to −7 °C) suspension of m-toluic acid (0.62
mmol) in anhydrous THF (5 mL), 2.17 mmol of Et3N was added. The
suspension was stirred for 30 min, and after warming up to 0 °C, ethyl
chloroformate was added (0.68 mmol). The mixture was stirred for 1
h, and then 1.24 mmol of 328 was added. The reaction was carried out
at room temperature for 12 h, and evaporation of the solvent resulted
in the desired final compound, which was purified by column
chromatography (cycloexane/ethyl acetate gradient 4:1 to 3:1). Yield
1
= 10%; mp = 220 °C dec (EtOH). H NMR (CDCl3) δ 2.33 (s, 3H,
CH3), 6.98 (t, 1H, Ar, J = 7.6 Hz), 7.22 (d, 1H, Ar, J = 8.0 Hz), 7.60 (t,
1H, Ar, J = 8.0 Hz), 7.73 (t, 1H, Ar, J = 8.4 Hz), 7.83 (d, 1H, Ar, J =
8.0 Hz), 7.93 (s, 1H, Ar), 8.55 (d, 2H, Ar, J = 8.4 Hz), 10.81 (exch br
s, 2H, NH2).
3-(Tetrahydro-2H-pyran-2-yloxymethyl)-1H-indazole (6). To
a mixture of a catalytic amount of ammonium cerium(IV) nitrate
(CAN) in anhydrous CH3CN (2.5 mL), 0.53 mmol of 1,30 and 0.53
mmol of 3,4 dihydro-2H-pyran (commercially available) were added,
and the suspension was stirred at room temperature for 24 h. After
evaporation of the solvent, water was added (20 mL), the mixture was
extracted with CH2Cl2, and the organic layer was evaporated in vacuo,
resulting in crude 6, which was purified by column chromatography
EXPERIMENTAL SECTION
Chemistry. All melting points were determined on a Buchi
apparatus and are uncorrected. H NMR spectra were recorded with
■
̈
1
an Avance 400 instrument (Bruker Biospin Version 002 with SGU).
Chemical shifts are reported in ppm, using the solvent as 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 the course of reaction. Silica gel
60 (Merck 70−230 mesh) was used for column 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 commercially available.
1
using CH2Cl2/CH3OH 9.5:0.5 as eluent. Yield = 33%; oil. H NMR
(CDCl3) δ 1.59−1.86 (m, 6H, cC5H9O), 3.60−3.78 (m, 1H,
cC5H9O), 3.90−4.05 (m, 1H, cC5H9O), 4.83 (s, 1H, O−CH−O),
4.90−5.00 (m, 1H, C−CH2−O), 5.15−5.22 (m, 1H, C−CH2−O)
7.15−7.22 (m, 1H, Ar), 7.37−7.51 (m, 2H, Ar), 7.87−7.93 (m, 1H,
Ar), 10.62 (exch br s, 1H, NH).
3-(Tetrahydro-2H-pyran-2-yloxymethyl)-indazol-1-yl]-m-
tolyl-methanone (7). Compound 7 was obtained starting from
intermediate 6 by reaction with m-toluoyl chloride, following the
general procedure described for 5a−c and 5e. After evaporation of the
solvent, the final compound was purified by column chromatography
Acetic Acid 1H-Indazol-3-yl Methyl Ester (2). A mixture of 130
(0.2 mmol), 1 mL of acetic acid, and 0.12 mL of conc H2SO4 was
heated at 100 °C for 2 h. After cooling, cold water was added and the
mixture was extracted with ethyl acetate (3 × 10 mL). Evaporation of
the solvent resulted in compound 2. Yield = 98%; oil. 1H NMR
(CDCl3) δ 2.17 (s, 3H, CH3), 5.60 (s, 2H, CH2), 7.30 (t, 1H, Ar, J =
8.4 Hz), 7.54 (t, 1H, Ar, J = 8.4 Hz), 7.64 (d, 1H, Ar, J = 8.4 Hz), 7.88
(d, 1H, Ar, J = 8.4 Hz).
1
using toluene/ethyl acetate 9.5:0.5 as eluent. Yield = 50%; oil. H
NMR (CDCl3) δ 1.60−1.88 (m, 6H, cC5H9O), 2.47 (s, 3H, CH3),
3.55−3.65 (m, 1H, cC5H9O), 3.90−4.06 (m, 1H, cC5H9O), 4.84 (s,
1H, O−CH−O), 4.90−4.98 (m, 1H, C−CH2−O), 5.10−5.20 (m, 1H,
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C−CH2−O), 7.42 (s, 3H, Ar), 7.57−7.68 (m, 1H, Ar), 7.89 (s, 2H,
Ar), 7.90−8.00 (m, 1H, Ar), 8.52−8.61 (m, 1H, Ar).
Ar), 8.54 (d, 1H, Ar, J = 9.2 Hz), 8.77 (d, 1H, Ar, J = 9.2 Hz), 9.00 (s,
1H, Ar), 9,21 (s, 1H, Ar).
1-(3-Methoxybenzoyl)-5-nitro-1H-indazole-3-carboxylic Acid
Ethyl Ester (14g). Yield = 54%; mp = 193−194 °C (EtOH). 1H
NMR (CDCl3) δ 1.53 (t, 3H, CH2CH3, J = 7.2 Hz), 3.93 (s, 3H,
OCH3), 4.61 (q, 2H, CH2, J = 7.2 Hz), 7.25 (d, 1H, Ar, J = 5.6 Hz),
7.50 (t, 1H, Ar, J = 8.0 Hz), 7.74 (s, 1H, Ar), 7.80 (d, 1H, Ar, J = 8.0
Hz), 8.55 (d, 1H, Ar, J = 7.2 Hz), 8.72 (d, 1H, Ar, J = 9.2 Hz), 9,22 (s,
1H, Ar).
5-Nitro-1-(thiophene-3-carbonyl)-1H-indazole-3-carboxylic Acid
Ethyl Ester (14h). Yield = 48%; mp = 156−159 °C (EtOH). 1H NMR
(CDCl3) δ 1.53 (t, 3H, CH3, J = 7.2 Hz), 4.65 (q, 2H, CH2, J = 7.2
Hz), 7.43−7.49 (m, 1H, Ar), 8.02 (d, 1H, Ar, J = 5.2 Hz), 8.53 (d, 1H,
Ar, J = 7.2 Hz), 8.76 (d, 1H, Ar, J = 9.2 Hz), 9.01 (d, 1H, Ar, J = 1.6
Hz), 9.2 (m, 1H, Ar).
General Procedures for 15 and 16. Compounds 15 and 16 were
obtained starting from compounds 12a33 and 1334 following the same
procedure described for 5a−c and 5e. After dilution with cold water
and neutralization with 0.5 N NaOH, the suspension was extracted
with CH2Cl2 (3 × 15 mL). Evaporation of the solvent resulted in the
final compounds, which were purified by column chromatography
using cyclohexane/ethyl acetate 4:1 as eluent.
1-(3-Methylbenzoyl)-6-nitro-1H-indazole-3-carboxylic Acid
Methyl Ester (15). Yield = 5%; mp = 180−181 °C (EtOH). 1H
NMR (CDCl3) δ 2.50 (s, 3H, CH3-Ph), 4.13 (s, 3H, CH3), 7.47−7.54
(m, 2H, Ar), 7.94−7.99 (m, 2H, Ar), 8.55 (d, 1H, Ar, J = 9.2 Hz), 8.71
(d, 1H, Ar, J = 7.2 Hz), 9.22 (m, 1H, Ar).
(3-Hydroxymethyl-indazol-1-yl)-m-tolylmethanone (8). A
mixture of 7 (0.09 mmol), trifluoroacetic acid (0.28 mL), and
CH2Cl2 (1.72 mL) was stirred at room temperature for 3 h.
Evaporation of the solvent resulted in compound 8, which was
purified by column chromatography using CH2Cl2/CH3OH 9.9:0.1 as
1
eluent. Yield = 87%; oil. H NMR (CDCl3) δ 2.20 (exch br s, 1H,
OH), 2.47 (s, 3H, CH3), 5.08 (s, 2H, CH2OH) 7.44 (s, 3H, Ar), 7.62
(m, 1H, Ar), 7.80−7.91 (m, 3H, Ar), 8.50−8.56 (m, 1H, Ar).
6-Nitro-1H-indazole-3-carboxylic Acid Ethyl Ester (12b). To a
cooled and stirred solution of conc H2SO4 and conc HNO3 (1.5 mL,
1:1, v/v), 0.45 mmol of 10 was slowly added, and the mixture was kept
under stirring at 0 °C for 10 min. After dilution with ice-cold water,
the precipitate was filtered and washed with water (10−20 mL).
Finally, compound 12b was purified by column chromatography using
cycloexane/ethyl acetate 4:1 as eluent. Yield = 10%; mp = 120−121
°C (EtOH). 1H NMR (CDCl3) δ 1.54 (t, 3H, CH3, J = 7.2 Hz), 4.64
(q, 2H, CH2, J = 7.2 Hz), 8.46 (d, 1H, Ar, J = 9.6 Hz), 8.73 (d, 1H, Ar,
J = 9.2 Hz), 9.27 (s, 1H, Ar), 11,67 (exch br s, 1H, NH).
General Procedures for 14a,b, and 14f. Compounds 14a,b and
14f were obtained starting from 11a and 11b, respectively, following
the general procedure described for 5a−c and 5e. For compound 14a,
after dilution with cold water and neutralization with 0.5 N NaOH, the
precipitate was filtered off and purified by crystallization from ethanol.
For compound 14b and 14e, after dilution and neutralization with
NaOH, the suspension was extracted with CH2Cl2 (3 × 15 mL), and
evaporation of the solvent resulted in the final compounds, which were
recrystallized from ethanol.
1-(3-Methylbenzoyl)-7-nitro-1H-indazole-3-carboxylic Acid
1
Methyl Ester (16). Yield = 17%; mp = 118−119 °C (EtOH). H
NMR (CDCl3) δ 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 2.48 (s, 3H, Ph-
CH3), 4.56 (q, 2H, CH2, J = 7.2 Hz), 7.49−7.56 (m, 2H, Ar), 7.65 (t,
1H, Ar, J = 8.0 Hz), 7.99 (s, 2H, Ar), 8.23 (d, 1H, Ar, J = 8.0 Hz), 8.66
(d, 1H, Ar, J = 8.0 Hz).
1-Benzoyl-5-nitro-1H-indazole-3-carboxylic Acid Methyl Ester
1
(14a). Yield = 62%; mp = 156−157 °C (EtOH). H NMR (CDCl3)
δ 4.13 (s, 3H, CH3), 7.60 (t, 2H, Ar, J = 8.0 Hz), 7.72 (t, 1H, Ar, J =
8.0 Hz), 8.19 (d, 2H, Ar, J = 8.0 Hz), 8.56 (d, 1H, Ar, J = 7.2 Hz), 8.73
(d, 1H, Ar, J = 9.2 Hz), 9.23 (d, 1H, Ar, J = 2.0 Hz).
5-Amino-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic
Acid Ethyl Ester (17). Compound 14f (0.31 mmol) was subjected to
catalytic reduction in EtOH (6 mL) for 2 h with a Parr instrument
using 70 mg of 10% Pd/C as catalyst and the pressure kept constant at
30 psig. The catalyst was filtered off, and the solvent was evaporated
under vacuum, resulting in the final compound, which was purified by
crystallization from ethanol. Yield = 30%; mp = 131−133 °C (EtOH).
1H NMR (CDCl3) δ 1.48 (t, 3H, CH2CH3, J = 7.2 Hz), 2.47 (s, 3H,
Ph-CH3), 3.94 (exch br s, 2H, NH2), 4.52 (q, 2H, CH2, J = 7.2 Hz),
7.05 (dd, 1H, Ar, J = 2.4 Hz, J = 6.4 Hz), 7.40−7.46 (m, 2H, Ar), 7.47
(d, 1H, Ar, J = 2.4 Hz), 7.94 (s, 2H, Ar), 8.36 (d, 1H, Ar, J = 8.8 Hz).
General Procedures for 18 and 19. A mixture of 0.31 mmol of
17, 0.68 mmol of K2CO3, and 0.54 mmol of CH3I in 1 mL of
anhydrous DMF was stirred at 50 °C for 3 h. After cooling, ice-cold
water was added (10−15 mL), and the suspension was extracted with
CH2Cl2 (3 × 15 mL). The organic layer was evaporated in vacuo, and
the residue was purified by flash column chromatography using
cyclohexane/ethyl acetate 3:1 as eluent.
1-(3-Methylbenzoyl)-5-nitro-1H-indazole-3-carboxylic Acid
1
Methyl Ester (14b). Yield = 52%; mp = 180−181 °C (EtOH). H
NMR (CDCl3) δ 2.50 (s, 3H, CH3-Ph), 4.13 (s, 3H, OCH3), 7.47−
7.53 (m, 2H, Ar), 7.90 (d, 2H, Ar, J = 7.2 Hz), 8.55 (d, 1H, Ar, J = 5.2
Hz), 8.71 (d, 1H, Ar, J = 9.2 Hz), 9.22 (d, 1H, Ar, J = 2.0 Hz).
1-(3-Methylbenzoyl)-5-nitro-1H-indazole-3-carboxylic Acid Ethyl
Ester (14f). Yield = 76%; mp = 150−153 °C (EtOH). 1H NMR
(CDCl3) δ 1.54 (t, 3H, CH2CH3, J = 7.2 Hz), 2.50 (s, 3H, Ph-CH3),
4.62 (q, 2H, CH2CH3, J = 7.2 Hz), 7.47−7.54 (m, 2H, Ar), 7.98 (s,
2H, Ar), 8.54 (d, 1H, Ar, J = 7.2 Hz), 8.71 (d, 1H, Ar, J = 9.2 Hz), 9.22
(d, 1H, Ar, J = 2.0 Hz).
General Procedures for 14c,d, and 14g,h. The appropriate
(hetero)arylcarboxylic acids (0.90 mmol) were dissolved in 2 mL of
SOCl2 and heated at 80−90 °C for 1 h. After cooling, excess SOCl2
was removed under vacuum and the residue was dissolved in 3.5 mL of
anhydrous toluene. A solution of 11a32 or 11b32 (0.45 mmol) and
Et3N (0.50 mmol) in anhydrous toluene (3.5 mL) was added to this
mixture, and it was stirred at 110 °C for 3−6 h. After cooling, the
precipitate was removed by filtration, and the organic solvent was
evaporated under vacuum. Addition of cold water to the residue and
neutralization with 0.5 N NaOH resulted in the final compounds.
Compounds 14c, 14g, and 14h were recovered by suction and
recrystallized from ethanol, while the crude 14d was recovered by
extraction with ethyl acetate (3 × 15 mL) and evaporation of the
solvent. Compound 14d was finally crystallized from ethanol.
1-(3-Methoxybenzoyl)-5-nitro-1H-indazole-3-carboxylic Acid
5-Methylamino-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic
1
Acid Ethyl Ester (18). Yield = 34%; mp = 152−154 °C (EtOH). H
NMR (CDCl3) δ 1.49 (t, 3H, CH2CH3, J = 7.2 Hz), 2.47 (s, 3H, Ph-
CH3), 2.98 (s, 3H, NCH3), 4.02 (exch br s, 1H, NH), 4.53 (q, 2H,
CH2, J = 7.2 Hz), 6.99 (d, 1H, Ar, J = 6.4 Hz), 7.30 (m, 1H, Ar), 7.40−
7.47 (m, 2H, Ar), 7.96 (s, 2H, Ar), 8.35 (d, 1H, Ar, J = 9.2 Hz).
5-Dimethylamino-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic
1
Acid Ethyl Ester (19). Yield = 28%; mp = 114−116 °C (EtOH). H
NMR (CDCl3) δ 1.49 (t, 3H, CH2CH3, J = 7.2 Hz), 2.47 (s, 3H, Ph-
CH3), 3.09 (s, 6H, N(CH3)2), 4.53 (q, 2H, CH2, J = 7.2 Hz), 7.21 (d,
1H, J = 6.4 Hz), 7.40−7.48 (m, 3H, Ar), 7.97 (s, 2H, Ar), 8.41 (d, 1H,
Ar, J = 9.2 Hz).
1
Methyl Ester (14c). Yield = 56%; mp = 151−152 °C (EtOH). H
NMR (CDCl3) δ 3.93 (s, 3H, Ph-OCH3), 4.13 (s, 3H, COOCH3),
7.25 (dd, 1H, Ar, J = 2.4 Hz, J = 5.6 Hz), 7.50 (t, 1H, Ar, J = 8.0 Hz),
7.71 (s, 1H, Ar), 7.78 (d, 1H, Ar, J = 7,6 Hz), 8.56 (dd, 1H, Ar, J = 2.0
Hz, J = 7.2 Hz), 8.72 (d, 1H, Ar, J = 9.2 Hz), 9.22 (d, 1H, Ar, J = 2.0
Hz).
General Procedures for 20a−f. To a cooled (0 °C) suspension
of 17 (0.31 mmol) in anhydrous CH2Cl2 (2 mL), a catalytic amount of
Et3N and 0.93 mmol of the appropriate (cyclo)alkylcarbonyl or
benzoylchloride were added. The mixture was stirred at 0 °C for 2 h
and then at room temperature for 2 h. Finally, the precipitates were
recovered by vacuum filtration and recrystallized with ethanol.
5-Acetylamino-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic
5-Nitro-1-(thiophene-3-carbonyl)-1H-indazole-3-carboxylic Acid
1
Methyl Ester (14d). Yield = 54%; mp = 193−194 °C (EtOH). H
1
NMR (CDCl3) δ 4.17 (s, 3H, CH3), 7.47 (s, 1H, Ar), 8.02 (m, 1H,
Acid Ethyl Ester (20a). Yield = 27%; mp = 155−158 °C (EtOH). H
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NMR (DMSO-d6) δ 1.39 (t, 3H, CH2CH3, J = 7.2 Hz), 2.12 (s, 3H,
COCH3), 2.43 (s, 3H, Ph-CH3), 4.44 (q, 2H, CH2, J = 7.2 Hz), 7.49−
7.57 (m, 2H, Ar), 7.80−7.86 (m, 3H, Ar), 8.38 (d, 1H, Ar, J = 9.2 Hz),
8.65 (s, 1H, Ar), 10.32 (exch br s, 1H, NH).
chlorosulfonic acid was slowly added. The mixture was stirred at 80−
90 °C for 4−5 h. After cooling, ice-cold water (15 mL) and 33%
aqueous ammonia (5 mL) were added, resulting in a precipitate, which
was recovered by suction. Yield = 10%; mp = 257 °C dec (EtOH). 1H
NMR (DMSO-d6) δ 6.50 (exch br s, 1H, OH), 7.24 (t, 1H, Ar, J = 8.4
Hz), 7.73 (d, 1H, Ar, J = 6.8 Hz), 8.10 (exch br s, 2h, NH2), 8.45 (d,
1H, Ar, J = 7.6 Hz).
1-(3-Methylbenzoyl)-5-propionylamino-1H-indazole-3-carboxylic
1
Acid Ethyl Ester (20b). Yield = 95%; mp = 116−119 °C (EtOH). H
NMR (CDCl3) δ 1.32 (t, 3H, COCH2CH3, J = 7.2 Hz), 1.51 (t, 3H,
OCH2CH3, J = 7.2 Hz), 2.47 (s, 3H, Ph-CH3), 2.51 (q, 2H,
COCH2CH3, J = 7.2 Hz), 4.55 (q, 2H, OCH2CH3, J = 7.2 Hz), 7.42−
7.48 (m, 3H, Ar), 7.83 (d, 1H, Ar, J = 9.2 Hz), 7.95 (s, 2H, Ar), 8.46
(exch br s, 1H, NH), 8.51 (d, 1H, Ar, J = 9.2 Hz).
7-Sulfamoyl-1H-indazole-3-carboxylic Acid Ethyl Ester (23).
A mixture of 22 (0.62 mmol) and a catalytic amount of conc H2SO4 in
anhydrous ethanol (7.5 mL) was heated at 100 °C for 5 h. After
cooling, ice-cold water was added and the precipitate was recovered by
suction and recrystallized from ethanol. Yield = 48%; mp = 244 °C dec
5-Butyrylamino-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic
1
1
Acid Ethyl Ester (20c). Yield = 72%; mp = 122−125 °C (EtOH). H
(EtOH). H NMR (DMSO-d6) δ 1.40 (t, 3H, CH3, J = 7.2 Hz), 4.43
NMR (CDCl3) δ 1.07 (t, 3H, CH2CH2CH3, J = 7.2 Hz), 1.51 (t, 3H,
OCH2CH3, J = 7.2 Hz), 1.84 (m, 2H, CH2CH2CH3, J = 7.2 Hz), 2.43
(t, 2H, COCH2, J = 7.2 Hz), 2.47 (s, 3H, Ph-CH3), 4.54 (q, 2H,
OCH2CH3, J = 7.2 Hz), 7.37 (s, 1H, Ar), 7.42−7.48 (m, 2H, Ar), 7.82
(d, 1H, Ar, J = 8.8 Hz), 7.96 (s, 2H, Ar), 8.46 (exch br s, 1H, NH),
8.51 (d, 1H, Ar, J = 8.8 Hz).
(q, 2H, CH2, J = 7.2 Hz), 7.50 (t, 1H, Ar, J = 8.0 Hz), 7.71 (exch br s,
2H, SO2NH2), 7.90 (d, 1H, Ar, J = 7.2 Hz), 8.34 (d, 1H, Ar, J = 8.0
Hz), 13.91 (exch br s, 1H, NH). 13C NMR (DMSO-d6) δ 14.4 (q),
60.9 (t), 122.7 (d), 124.0 (s), 125.7 (2d), 127.5 (s), 135.2 (s), 136.0
(s), 162.0 (s).
General Procedures for 24a,b. Compounds 24a,b were obtained
starting from 23 following the general procedure described for 5a−c,e.
After dilution with water and neutralization with 0.5 NaOH, the
mixture was extracted with ethyl acetate (3 × 15 mL). Evaporation of
the solvent resulted in the final compounds, which were purified by
flash chromatography using as eluents cyclohexane/ethyl acetate 2:1
for 24a and 1:2 for 24b.
5-Benzoylamino-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic
1
Acid Ethyl Ester (20d). Yield = 78%; mp = 196−198 °C (EtOH). H
NMR (CDCl3) δ 1.52 (t, 3H, CH2CH3, J = 7.2 Hz), 2.48 (s, 3H, Ph-
CH3), 4.55 (q, 2H, CH2, J = 7.2 Hz), 7.42−7.47 (m, 2H, Ar), 7.55−
7.60 (m, 3H, Ar), 7.94−8.00 (m, 5H, Ar), 8.04 (exch br s, 1H, NH),
8.58 (d, 2H, Ar, J = 9.2).
5-(Cyclohexanecarbonylamino)-1-(3-methylbenzoyl)-1H-inda-
zole-3-carboxylic Acid Ethyl Ester (20e). Yield = 64%; mp = 189−191
°C (EtOH). 1H NMR (CDCl3) δ 1.29−1.39 (m, 3H, cC6H11), 1.51 (t,
3H, CH2CH3, J = 7.2 Hz), 1.63 (t, 2H, cC6H11, J = 5.6 Hz), 1.70−1.80
(m, 1H, cC6H11), 1.85−1.95 (m, 2H, cC6H11), 2.00−2.2.05 (m, 2H,
cC6H11), 2.28−2.38 (m, 1H, cC6H11), 2.48 (s, 3H, Ph-CH3), 4.55 (q,
2H, CH2CH3, J = 7.2 Hz), 7.32−7.40 (m, 1H, Ar), 7.40−7.46 (m, 2H,
Ar), 7.84 (d, 1H, Ar, J = 9.6 Hz), 7.96 (s, 2H, Ar), 8.45 (exch br s, 1H,
NH), 8.51 (d, 1H, Ar, J = 8.4 Hz).
5-(Cyclopropanecarbonylamino)-1-(3-methylbenzoyl)-1H-inda-
zole-3-carboxylic Acid Ethyl Ester (20f). Yield = 82%; mp = 204−205
°C (EtOH). 1H NMR (CDCl3) δ 0.92 (m, 2H, cC3H11), 1.17 (m, 2H,
cC3H11), 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 1.58 (m, 1H, cC3H11),
2.47 (s, 3H, Ph-CH3), 4.53 (q, 2H, CH2CH3, J = 7.2 Hz), 7.42−7.47
(m, 2H, Ar), 7.60 (exch br s, 1H, NH), 7.,85 (d, 1H, Ar), 7.95 (s, 2H,
Ar), 8.44 (s, 1H, Ar), 8.50 (d, 1H, Ar, J = 9.2 Hz).
5-(Cyclopentanecarbonylamino)-1-(3-methylbenzoyl)-1H-
indazole-3-carboxylic Acid Ethyl Ester (20g). Compound 20g was
obtained starting from 17 following the same procedure described for
14c,d,g,h. After dilution with cold water, the mixture was neutralized
with 0.5 N NaOH and extracted with ethyl acetate (3 × 15 mL).
Evaporation of the solvent resulted in a residue, which was purified by
crystallization from ethanol. Yield = 20%; mp = 157−160 °C (EtOH).
1H NMR (CDCl3) δ 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 1.65−1.70 (m,
1-Benzoyl-7-sulfamoyl-1H-indazole-3-carboxylic Acid Ethyl Ester
1
(24a). Yield = 29%; mp = 139−140 °C (EtOH). H NMR (DMSO-
d6) δ 1.38−1.42 (m, 3H, CH3), 4.45−4.60 (m, 2H, CH2), 7.61−7.70
(m, 2H, Ar), 7.72−7.80 (m, 1H, Ar), 7.90−8.05 (m, 1H, Ar), 8.15 (d,
2H, Ar, J = 6.8 Hz), 8.45 (d, 1H, Ar, J = 7.2 Hz), 8.52 (d, 1H, Ar, J =
7.2 Hz).
1-(3-Methylbenzoyl)-7-sulfamoyl-1H-indazole-3-carboxylic Acid
Ethyl Ester (24b). Yield = 17%; mp = 176−178 °C (EtOH). 1H
NMR (CDCl3) δ 1.50−1.60 (m, 3H, CH3), 2.51 (s, 3H, Ph-CH3),
4.58−4.65 (m, 2H, CH2), 7.52 (s, 2H, Ar), 7.80−7.90 (m, 1H, Ar),
8.17 (s, 1H, Ar), 8.22−8.27 (m, 2H, Ar), 8.51 (d, 1H, Ar).
General Procedures for 26a,b and 26f. Compounds 26a,b and
26f were obtained starting from 25a,b32 following the same general
procedure described for 5a−c,e. For compound 26a, after dilution
with water and neutralization with NaOH, the crude precipitate was
recovered by suction and crystallized by ethanol. The suspension of
26b and 26f was extracted with CH2Cl2 (3 × 15 mL), and evaporation
of the solvent resulted in the final compounds, which were
recrystallized from ethanol.
1-Benzoyl-5-bromo-1H-indazole-3-carboxylic Acid Methyl Ester
(26a). Yield = 82%; mp = 143−146 °C (EtOH). 1H NMR (CDCl3) δ
4.08 (s, 3H, CH3), 7.55−7.60 (m, 2H, Ar), 7.65−7.71 (m, 1H, Ar),
7.77 (d, 1H, Ar, J = 7.2 Hz), 8.12−8.17 (m, 2H, Ar), 8.47 (d, 1H, Ar, J
= 6.4 Hz), 8.49 (s, 1H, Ar).
2H, cC5H9), 1.85−1.88 (m, 2H, cC5H9), 1.95−2.01 (m, 4H, cC5H9),
2.47 (s, 3H, Ph-CH3), 2.73−2.80 (m, 1H, cC5H9), 4.55 (q, 2H,
CH2CH3, J = 7.2 Hz), 7.38−7.47 (m, 3H, 1H NH e 2H Ar), 7.85 (d,
1H, Ar, J = 9.2 Hz), 7.95 (s, 2H, Ar), 8.46 (s, 1H, Ar), 8.50 (d, 1H, Ar,
J = 9.2 Hz).
5-Bromo-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic Acid
1
Methyl Ester (26b). Yield = 58%; mp = 121−123 °C (EtOH). H
NMR (CDCl3) δ 2.48 (s, 3H, Ph-CH3), 4.07 (s, 3H, OCH3), 7.43−
7.49 (m, 2H, Ar), 7.77 (d, 1H, Ar, J = 9.2 Hz), 7.93 (d, 2H, Ar, J = 7.2
Hz), 8.46 (d, 1H, Ar, J = 9.2 Hz), 8.48 (s, 1H, Ar).
1-(3-Methylbenzoyl)-5-phenylamino-1H-indazole-3-carbox-
ylic Acid Ethyl Ester (20h). A mixture of activated powdered 4A
molecular sieves (500 mg), 0.30 mmol of 17, 5 mL of anhydrous
CH2Cl2, 0.60 mmol of phenylboronic acid, 0.45 mol of Cu(Ac)2, and
0.60 mol of Et3N was stirred for 24 h at room temperature. Molecular
sieves were removed by suction filtration, and the organic layer was
washed with 33% aqueous ammonia (3 × 5 mL). Evaporation of the
solvent resulted in the final compound, which was purified by flash
chromatography using toluene/ethyl acetate 7:3 as eluent. Yield =
5-Bromo-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic Acid
Ethyl Ester (26f). Yield = 50%; mp = 116−117 °C (EtOH). 1H
NMR (CDCl3) δ 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 2.48 (s, 3H, Ph-
CH3), 4.55 (q, 2H, CH2, J = 7.2 Hz), 7.43−7.49 (m, 2H, Ar), 7.76 (d,
1H, Ar, J = 8.0 Hz), 7.95 (s, 2H, Ar), 8.45 (s, 2H, Ar).
General Procedures for 26c,d, and 26g,h. These compounds
were obtained starting from compounds 25a32 or 25b32 following the
general procedure described for 14c,d,g,h. For 26d and 26h, the
precipitate was recovered by suction and recrystallized from ethanol;
for compounds 26c and 26g, after dilution and neutralization with 0.5
N NaOH, the suspension was extracted with CH2Cl2 (3 × 15 mL),
and evaporation of the solvent resulted in the final compounds, which
were purified by crystallization from ethanol.
1
71%; mp = 123−125 °C (EtOH). H NMR (CDCl3) δ 1.45 (t, 3H,
CH2CH3, J = 7.2 Hz), 2.46 (s, 3H, Ph-CH3), 4.50 (q, 2H, CH2, J = 7.2
Hz), 7.03 (t, 1H, Ar, J = 7.2 Hz), 7.19 (d, 2H, Ar, J = 8.0 Hz), 7.34 (t,
2H, Ar, J = 8.0 Hz), 7.39−7.44 (m, 3H, Ar), 7.94 (s, 3H, Ar), 8.44 (d,
1H, Ar, J = 9.2 Hz), 8.95 (exch br s, 1H, NH).
5-Bromo-1-(3-methoxybenzoyl)-1H-indazole-3-carboxylic Acid
1
7-Sulfamoyl-1H-indazole-3-carboxylic Acid (22). To 0.43
mmol of 21 (commercially available) cooled at 0 °C, 1 mL of
Methyl Ester (26c). Yield = 26%; mp = 105−108 °C (EtOH). H
NMR (CDCl3) δ 3.91 (s, 3H, OCH3), 4.07 (s, 3H, COOCH3), 7.21
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(d, 1H, Ar, J = 6.4 Hz), 7.47 (t, 1H, Ar, J = 8.0 Hz), 7.68 (s, 1H, Ar),
7.76 (t, 2H, Ar, J = 7.2 Hz), 8.45−8.53 (m, 2H, Ar).
5-Chloro-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic Acid
1
Ethyl Ester (31b). Yield = 27%; mp = 108 °C (EtOH). H NMR
(CDCl3) δ 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 2.48 (s, 3H, Ph-CH3),
4.55 (q, 2H, CH2, J = 7.2 Hz), 7.43−7.49 (m, 2H, Ar), 7.63 (d, 1H, Ar,
J = 8.8 Hz), 7.95 (s, 2H, Ar), 8.28 (s, 1H, Ar), 8.52 (d, 1H, Ar, J = 9.2
Hz).
5-Bromo-1-(thiophene-3-carbonyl)-1H-indazole-3-carboxylic
Acid Methyl Ester (26d). Yield = 36%; mp = 136−138 °C (EtOH). 1H
NMR (CDCl3) δ 4.12 (s, 3H, CH3), 7.43 (t, 1H, Ar, J = 4.8 Hz), 7.75
(d, 1H, Ar, J = 8.8 Hz), 7.99 (d, 1H, Ar, J = 5.2 Hz), 8.46 (s, 1H, Ar),
8.50 (d, 1H, Ar, J = 9.2 Hz), 8.94 (d, 1H, Ar, J = 3.2 Hz).
5-Bromo-1-(3-methoxybenzoyl)-1H-indazole-3-carboxylic Acid
Ethyl Ester (26g). Yield = 54%; mp = 115−117 °C (EtOH). 1H
NMR (CDCl3) δ 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 3.91 (s, 3H,
OCH3), 4.55 (q, 2H, CH2CH3, J = 7.2 Hz), 7.21 (d, 1H, Ar, J = 8.0
Hz), 7.47 (t, 1H, Ar, J = 8.0 Hz), 7.72 (s, 1H, Ar), 7.77 (d, 2H, Ar, J =
8.0 Hz), 8.46 (d, 2H, Ar, J = 8.4 Hz).
5-Fluoro-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic Acid
1
Ethyl Ester (31c). Yield = 23%; mp = 105 °C (EtOH). H NMR
(CDCl3) δ 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 2.48 (s, 3H, Ph-CH3),
4.55 (q, 2H, CH2, J = 7.2 Hz), 7.40−7.49 (m, 3H, Ar), 7.92−7.96 (m,
3H, Ar), 8.56 (d, 1H, Ar, J = 5.2 Hz).
1-Benzoyl-5-methoxy-1H-indazole-3-carboxylic Acid Ethyl Ester
1
(31d). Yield = 21%; mp = 111−112 °C (EtOH). H NMR (DMSO-
d6) δ 1.38 (t, 3H, CH2CH3, J = 7.2 Hz), 3.90 (s, 3H, OCH3), 4.45 (q,
2H, CH2, J = 7.2 Hz), 7.39 (d, 1H, Ar, J = 9.2 Hz), 7.58−7.72 (m, 4H,
Ar), 7.78−7.82 (m, 2H, Ar), 8.37 (d, 1H, Ar, J = 8.8 Hz).
5-Bromo-1-(thiophene-3-carbonyl)-1H-indazole-3-carboxylic
1
Acid Ethyl Ester (26h). Yield = 35%; mp = 106−108 °C (EtOH). H
NMR (CDCl3) δ 1.53 (t, 3H, CH2CH3, J = 7.2 Hz), 4.60 (q, 2H,
CH2CH3, J = 7.2 Hz), 7.40−7.7.45 (m, 1H, Ar), 7.75 (d, 1H, Ar, J =
9.2 Hz), 8.00 (d, 1H, Ar, J = 5.2 Hz), 8.45 (s, 1H, Ar), 8.50 (d, 1H, Ar,
J = 9.2 Hz), 8.96 (s, 1H, Ar).
5-Methoxy-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic Acid
1
Ethyl Ester (31e). Yield = 20%; mp = 89−90 °C (EtOH). H NMR
(DMSO-d6) δ 1.37 (t, 3H, CH2CH3, J = 7.2 Hz), 2.42 (s, 3H, Ph-
CH3), 3.90 (s, 3H, OCH3), 4.44 (q, 2H, CH2, J = 7.2 Hz), 7.39 (d, 1H,
Ar, J = 9.2 Hz), 7.48−7.53 (m, 2H, Ar), 7.58 (s, 1H, Ar), 7.78−7.82
(m, 2H, Ar), 8.35 (d, 1H, Ar, J = 8.8 Hz).
5-Sulfamoyl-1H-indazole-3-carboxylic Acid (28f). A mixture of
27f36 (0.31 mmol) and 0.32 mmol of 0.5 N NaOH in 1 mL of water
was stirred at 50 °C for 30 min. After cooling, a solution of NaNO2
(0.31 mol) in water (0.1 mL) followed by a solution of conc H2SO4
(0.60 mmol) in 0.8 mL of cold water were added, maintaining the
mixture reaction under stirring at 0 °C for 1 h. Finally, a cold solution
of SnCl2 (0.74 mmol) in 0.5 mL of conc HCl was added, and the
mixture was stirred for further 2 h at 0 °C and 16 h at room
temperature. The precipitate was recovered by suction. Yield = 38%;
1-Benzoyl-5-trifluoromethoxy-1H-indazole-3-carboxylic Acid
1
Ethyl Ester (31f). Yield = 22%; mp = 91−92 °C (EtOH). H NMR
(CDCl3) δ 1.51 (t, 3H, CH2CH3, J = 7.2 Hz), 4.56 (q, 2H, CH3CH2, J
= 7.2 Hz), 7.54−7.60 (m, 3H, Ar), 7.68 (t, 1H, Ar, J = 7.6 Hz), 8.16−
8.19 (m, 3H, Ar), 8.62 (d, 1H, Ar, J = 9.2 Hz).
1-(3-Methylbenzoyl)-5-trifluoromethoxy-1H-indazole-3-carbox-
ylic Acid Ethyl Ester (31g). Yield = 28%; mp = 112−113 °C (EtOH).
1H NMR (CDCl3) δ 1.51 (t, 3H, CH2CH3, J = 7.2 Hz), 2.48 (s, 3H,
1
mp > 300 °C dec (EtOH). H NMR (DMSO-d6) δ 6.10 (exch br s,
1H, OH), 7.57 (d, 1H, Ar, J = 7.2 Hz), 7.68 (d, 1H, Ar, J = 8.4 Hz),
8.18 (s, 1H, Ar), 8.40 (exch br s, 2H, NH2), 13.95 (exch br s, 1H,
NH).
General Procedures for 29e,f. Compounds 29e and 29f were
obtained starting from 28e39 and 28f and following the same
procedure described for compound 23.
Ph-CH3), 4.56 (q, 2H, CH2, J = 7.2 Hz), 7.43−7.50 (m, 2H, Ar), 7.54
(d, 1H, Ar, J = 8.8 Hz), 7.96 (s, 2H, Ar), 8.16 (s, 1H, Ar), 8.61 (d, 1H,
Ar, J = 9.2 Hz).
1-(3-Methylbenzoyl)-5-sulfamoyl-1H-indazole-3-carboxylic Acid
1
Ethyl Ester (31i). Yield = 28%; mp = 52−54 °C (EtOH). H NMR
(CDCl3) δ 1.43 (t, 3H, CH2CH3, J = 7.2 Hz), 2.46 (s, 3H, Ph-CH3),
4.49 (q, 2H, CH2, J = 6.4 Hz), 7.41−7.47 (m, 2H, Ar), 7.83 (d, 1H, Ar,
J = 7.6 Hz), 7.91 (s, 2H, Ar), 8.46 (s, 1H, Ar), 8.53 (d, 1H, Ar, J = 8.0
Hz).
5-Trifluoromethoxy-1H-indazole-3-carboxylic Acid Ethyl Ester
1
(29e). Yield = 45%; mp = 179−181 °C (EtOH). H NMR (CDCl3)
δ 1.53 (t, 3H, CH3, J = 7.2 Hz), 4.57 (q, 2H, CH2, J = 7.2 Hz), 7.39 (d,
1H, Ar, J = 8.8 Hz), 7.70 (d, 1H, Ar, J = 8.8 Hz), 8.11 (s, 1H, Ar),
12.87 (exch br s, 1H, NH).
5-Hydroxy-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic
Acid Ethyl Ester (31h). To a cooled and stirred solution of m-toluic
acid (0.52 mmol) and Et3N (0.05 mL) in anhydrous DMF (1−2 mL),
diethylcyanophosphonate (DCF) (2.08 mmol) and 0.52 mmol of 30
were added. The mixture was stirred at room temperature for 16 h.
After dilution with ice-cold water (10 mL), the suspension was
extracted with CH2Cl2 (3 × 15 mL) and evaporation of the solvent
resulted in the final compound, which was purified by column
chromatography using toluene/ethyl acetate 8:2 as eluent. Yield =
5-Sulfamoyl-1H-indazole-3-carboxylic Acid Ethyl Ester (29f).
1
Yield = 95%; mp = 290 °C (EtOH). H NMR (DMSO-d6) δ 1.15−
1.25 (m, 3H, CH3), 4.24 (q, 2H, CH2, J = 7.2 Hz) 7.62 (d, 1H, Ar, J =
7.2 Hz), 7.72 (d, 1H, Ar, J = 8.8 Hz), 8.08 (s, 1H, Ar), 9.05 (exch br s,
2H, NH2), 14.10 (exch br s, 1H, NH).
5-Hydroxy-1H-indazole-3-carboxylic Acid Ethyl Ester (30).
To a cooled (−78 °C) solution of 29d38 (1.08 mmol) in 1 mL of
anhydrous CH2Cl2, 4.15 mL of BBr3 (1 M in CH2Cl2) were added.
The reaction was carried out under nitrogen, and after 10 min the
mixture was allowed to warm to room temperature and stirred for 3 h.
After dilution with water, the mixture was extracted with ethyl acetate
(3 × 15 mL), and the organic layers were evaporated to obtain
compound 30, which was purified by flash column chromatography
1
51%; mp = 145−146 °C (EtOH). H NMR (CDCl3) δ 1.49 (t, 3H,
CH2CH3, J = 7.2 Hz), 2.47 (s, 3H, Ph-CH3), 4.53 (q, 2H, CH3CH2, J =
7.2 Hz), 7.23 (d, 1H, Ar, J = 8.8 Hz), 7.42−7.45 (m, 2H, Ar), 7.65 (s,
1H, Ar), 7.96 (s, 2H, Ar), 8.46 (d, 1H, Ar, J = 9.2 Hz).
1-(3-Methylbenzoyl)-5-nitro-1H-indazole-3-carbonitrile (33).
Compound 33 was obtained starting from compound 3240 and
following the general procedure described for 5a−c and 5e. The final
compound was purified by flash chromatography using toluene/ethyl
1
using cyclohexane/ethyl acetate 1:1 as eluent. Yield = 14%; oil. H
1
NMR (CDCl3) δ 1.51 (t, 3H, CH3, J = 7.2 Hz), 4.53 (q, 2H, CH2, J =
7.2 Hz), 7.11 (d, 1H, Ar, J = 8.8 Hz), 7.47 (d, 1H, Ar, J = 9.2 Hz), 7.61
(s, 1H, Ar).
General Procedures for 31a−g and 31i. Compounds 31a−g
and 31i were obtained following the general procedure described for
5a−c and 5e starting from compounds 29a−f (29a−c37 and 29d38).
Compounds 31a−g were recrystallized from ethanol, while compound
31i was purified by flash chromatography using toluene/ethyl acetate
99:1 as eluent.
5-Methyl-1-(3-methylbenzoyl)-1H-indazole-3-carboxylic Acid
Ethyl Ester (31a). Yield = 13%; mp = 88 °C (EtOH). 1H NMR
(CDCl3) δ 1.50 (t, 3H, CH2CH3, J = 7.2 Hz), 2.47 (s, 3H, Ph-CH3),
2.57 (s, 3H, 5-CH3), 4.54 (q, 2H, CH2, J = 7.2 Hz), 7.43−7.45 (m, 2H,
Ar), 7.50 (d, 1H, Ar, J = 8.4 Hz), 7.93−7.97 (m, 2H, Ar), 8.07 (s, 1H,
Ar), 8.45 (d, 1H, Ar, J = 8.4 Hz).
acetate 8:2 as eluent. Yield = 32%; mp = 140−143 °C (EtOH). H
NMR (CDCl3) δ 2.51 (s, 3H, CH3), 7.50 (t, 1H, Ar, J = 8.0 Hz), 7.55
(d, 1H, Ar, J = 7.6 Hz), 7.90 (s, 2H, Ar), 8.62 (dd, 1H, Ar, J = 2.0 Hz, J
= 9.6 Hz), 8.77 (d, 1H, Ar, J = 9.6 Hz), 8.89 (s, 1H, Ar).
HNE Inhibition Assay. Compounds were dissolved in 100%
DMSO at 5 mM stock concentrations. 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.
Reactions were initiated by addition of 25 μM elastase substrate (N-
methylsuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin, Calbio-
chem) in a final reaction volume of 100 μL/well. Kinetic measure-
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Journal of Medicinal Chemistry
Article
ments were obtained every 30 s for 10 min at 25 °C using a
Fluoroskan Ascent FL fluorescence microplate 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 % inhibition 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%.
Leu99B, Asp102, Trp141, Gly142, Leu143, Leu167, Arg177, Val190,
Cys191, Phe192, Gly193, Asp194, Ser195, Gly196, Ser197, Ala213,
Ser214, Phe215, Val216, Arg217A, Gly218, Gly219, Cys220, Ser222,
Leu223, Tyr224, Asp226, Ala227, Phe228). To simulate the receptor
flexibility, the standard technique built in the Molegro program was
employed, i.e., docking a ligand with softened potentials was followed
by optimization of flexible side chains with respect to the found pose.
Further simultaneous minimization of these side chains and the pose
using nonsoftened potentials was applied (Molegro Virtual Docker.
User Manual, 2010). Values of 0.9 and 0.7, respectively, were assigned
to the “Tolerance” and “Strength” parameters of the MVD “Side chain
Flexibility” wizard. Fifteen docking runs were performed for each
compound, with full flexibility of a ligand around all rotatable bonds
and side chain flexibility of the selected residues of the enzyme (see
above).
The docking poses corresponding to the lowest-energy binding
mode of each inhibitor were evaluated for the ability to form a
Michaelis complex between the hydroxyl group of Ser195 and the
carbonyl group in the amido moiety of an inhibitor. For this purpose,
values of d1 [distance O(Ser195)···C between the Ser195 hydroxyl
oxygen atom and the carbonyl carbon atom of the amido moiety] and
α [angle O(Ser195)···CO, where CO is the carbonyl group of an
inhibitor amido moiety] were determined for each docked
compound44 (Supporting Information Figure S1). In addition, we
estimated the possibility of proton transfer from Ser195 to Asp102
through His57 (the key catalytic triad of serine proteases41,45,46,47) by
calculating distances d2 between the NH hydrogen in His57 and
carboxyl oxygen atoms in Asp102. The distance between the hydroxyl
proton in Ser195, and the pyridine-type nitrogen in His57 is also
important for proton transfer. However, because of easy rotation of the
hydroxyl about the C−O bond in Ser195, we measured distance d3
between the oxygen in Ser195 and the basic nitrogen atom in His57.
The effective length L of the channel for proton transfer was calculated
as L = d3 + min(d2).
Analysis of Inhibitor Specificity. Selected compounds were
evaluated for their ability to inhibit a range of proteases in 100 μL
reaction volumes at 25 °C, as described previously.25 Briefly, analysis
of chymotrypsin inhibition was performed in reaction mixtures
containing 0.05 M Tris-HCl, pH 8.0, 30 nM human pancreas
chymotrypsin, test compounds, and 100 μM substrate (Suc-Ala-Ala-
Pro-Phe-7-amino-4-methylcoumarin). Thrombin inhibition was eval-
uated in reaction mixtures containing 0.25 M sodium phosphate, pH
7.0, 0.2 M NaCl, 0.1% PEG 8000, 1.7 U/mL human plasma thrombin,
test compounds, and 20 μM substrate (benzoyl-Phe-Val-Arg-7-amino-
4-methylcoumarin). Analysis of kallikrein inhibition was performed in
reaction mixtures containing 0.05 M Tris-HCl, pH 8.0, 0.1 M NaCl,
0.05% Tween-20, 2 nM human plasma kallikrein, test compounds, and
50 μM substrate (benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcou-
marin). Analysis of urokinase inhibition assay was performed in
reaction mixtures containing 0.1 M Tris-HCl, pH 8.0, 30 U/mL
human urine urokinase, test compounds, and 30 μM substrate
(benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin). Analy-
sis of cathepsin D inhibition was performed in reaction mixtures
containing 0.1 M sodium acetate, pH 5.0, 0.1 U/mL human spleen
cathepsin D, test compounds, and 5 μM substrate (MOCAc-Gly-Lys-
Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2). Cathepsin D
assays were monitored with a Fluoroskan Ascent FL microtiter plate
reader at excitation and emission wavelengths of 340 and 390 nm,
respectively. For all serine proteases (chymotrypsin, thrombin,
kallikrein, and urokinase), 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 vs 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%.
Analysis of Compound Stability. Spontaneous hydrolysis of
selected indazole derivatives was evaluated at 25 °C in 0.05 M
phosphate buffer, pH 7.3. Kinetics of hydrolysis were monitored by
measuring changes in absorbance spectra over time using a
SpectraMax Plus microplate spectrophotometer (Molecular Devices,
Sunnyvale, CA). Absorbance (At) at the characteristic absorption
maxima of each N-benzoylpyrazole was measured at the indicated
times until no further absorbance decreases occurred (A∞).43 Using
these measurements, we created semilogarithmic plots of log(At −
A∞) vs time and k′ values were determined from the slopes of these
plots. Half-conversion times were calculated using t1/2 = 0.693/k′, as
described previously.25
Molecular Modeling. For molecular modeling, we used Hyper-
Chem 7.0 (Hypercube Inc., Waterloo, ON, Canada) and Molegro
Virtual Docker (MVD), version 4.2.0 (CLC bio, Denmark) software.
Molecular structures of compounds 5b, 5d, 8, 14f, 26b, and 31h were
generated in HyperChem and optimized with the use of the
semiempirical PM3 method. These structures were then saved in the
Tripos Mol2 format and imported into the MVD program for docking
into the HNE binding site.
ASSOCIATED CONTENT
* Supporting Information
■
S
Chemical and physical characteristics and spectral data for all
the remaining new intermediates 5c, 5e, 14f−h, 20c, 20d−f,
26f−h, 31c−g, and 31i, elemental analyses for all final new
compounds, geometric parameters important for formation of a
Michaelis complex in the HNE active site, Docking pose of
inactive compound 8. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +39-055-4573682. Fax: +39 55 4573671. E-mail:
mariapaola.giovannoni@unifi.it.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by an Institutional
Development Award (IDeA) from the National Institute of
General Medical Sciences of the National Institutes of Health
under grant number GM103500, an equipment grant from the
M. J. Murdock Charitable Trust, and the Montana State
University Agricultural Experimental Station.
The structure of HNE complexed with a peptide chloromethyl
ketone inhibitor41 was downloaded from the Protein Data Bank
(1HNE entry of the database). The search area for docking poses was
defined as a sphere with 10 Å radius centered at the nitrogen atom in
the five-membered ring of the peptide chloromethyl ketone inhibitor.
After removal of this peptide and cocrystallized water molecules from
the program workspace, we set side chain flexibility for 42 residues
closest to the center of the search area (His40, Phe41, Cys42, Gly43,
Ala55, Ala56, His57, Cys58, Val59, Ala60, Tyr94, Pro98, Asn99A,
ABBREVIATION USED
■
HNE, human neutrophil elastase; ARDS, acute respiratory
distress syndrome; CF, cystic fibrosis; His, histidine; Asp,
aspartic acid; Ser, serine; Cys, cysteine; SD, standard deviation;
NA, not active
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