Linear propargylic alcohol functionality attached to the indazole-7-carboxamide as a JAK1-specific linear probe group

Article abstract of DOI:10.1016/j.bmc.2013.12.033

Selective inhibition of JAK1 has recently been proposed as an appropriate therapeutic rationale for the treatment of inflammatory diseases such as rheumatoid arthritis (RA) In this study, through pairwise comparison and 3D alignment of the JAK isozyme structures bound to the same inhibitor molecule, we reasoned that an alkynol functionality would serve as an isozyme-specific probe group, which would enable the resulting inhibitor to differentiate the ATP-binding site of JAK1 from those of other isozymes The 3-alkynolyl-5- (4′-indazolyl)indazole-7-carboxamide derivatives were thus prepared, and in vitro evaluation of their inhibitory activity against the JAK isozymes revealed that the propargyl alcohol functionality endowed the 5-(4′-indazolyl)indazole-7-carboxamide scaffold with JAK1 selectivity over other JAK isozymes, particularly JAK2

Full text of DOI:10.1016/j.bmc.2013.12.033

Bioorganic & Medicinal Chemistry 22 (2014) 1156–1162
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Linear propargylic alcohol functionality attached to the
indazole-7-carboxamide as a JAK1-specific linear probe group
⇑
Mi Kyoung Kim , Heerim Shin , Seo Young Cho, Youhoon Chong
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Selective inhibition of JAK1 has recently been proposed as an appropriate therapeutic rationale for the
treatment of inflammatory diseases such as rheumatoid arthritis (RA). In this study, through pairwise
comparison and 3D alignment of the JAK isozyme structures bound to the same inhibitor molecule, we
reasoned that an alkynol functionality would serve as an isozyme-specific probe group, which would
enable the resulting inhibitor to differentiate the ATP-binding site of JAK1 from those of other isozymes.
The 3-alkynolyl-5-(40-indazolyl)indazole-7-carboxamide derivatives were thus prepared, and in vitro
evaluation of their inhibitory activity against the JAK isozymes revealed that the propargyl alcohol func-
tionality endowed the 5-(40-indazolyl)indazole-7-carboxamide scaffold with JAK1 selectivity over other
JAK isozymes, particularly JAK2.
Received 13 November 2013
Revised 4 December 2013
Accepted 7 December 2013
Available online 22 December 2013
Keywords:
Janus kinase (JAK)
Isozyme-specificity
Propargylic alcohol
Probe group
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
RA. In this regard, accumulating evidence suggests that, in signal
transduction through cc-containing cytokine receptors, functional
The JAK/STAT pathway is the major signaling cascade in re-
sponse to inflammatory and proliferative signals such as cytokine,
chemokine, and growth factors. This signaling pathway consists of
the Janus protein tyrosine kinase family (JAK3, JAK2, JAK1, and
TYK2) and the STAT (signal transduction and transcription) family
of transcription factors. Being engaged with the cytoplasmic do-
mains of the transmembrane receptors, the JAKs become activated
upon ligand-receptor binding to result in phosphorylation, dimer-
ization, and nuclear translocation of downstream STAT proteins,
which regulate transcription of STAT-dependent genes. The JAKs
are coupled with specific cytokines that play essential roles in im-
mune function,1 inflammation,2 and hematopoiesis.3 For instance,
activity of JAK1 is dominant while JAK3 plays a secondary role to
merely enhance the effect of JAK1.7–11 Thus, JAK1 has become
an appropriate target for immune modulation, and several
JAK1-selective inhibitors have recently been identified.12,13 In
addition, ideal therapeutic agent for RA is also anticipated to have
high JAK1 versus JAK2 selectivity because concurrent inhibition of
JAK2 would result in significant anemia particularly dangerous for
patients under immunomodulating therapy.14
Structure-based rational drug design, a well-known process to
accelerate inhibitor lead design and optimization, has been ham-
pered in the field of the isozyme-selective JAK kinase inhibitors be-
cause the JAK kinases share similar structure which is
characterized by the presence of seven JAK homology (JH)
domains. In this study, we reasoned that pairwise comparison
and 3D alignment of the JAK isozyme structures bound to the same
inhibitor molecule would delineate the active site variants respon-
sible for isozyme specificity of the JAK inhibitors, which culmi-
nated in identification of the relative position of the glycine-rich
loop as the key structural variants among the JAK isozymes. Herein,
we report structure-based design of the JAK1-selective inhibitor,
synthesis of a series of (indazolyl)indazole derivatives, and evalua-
tion of their inhibitory activity against all JAK isozymes.
signaling by the gamma common (cc) family of cytokines (interleu-
kins IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) is transmitted exclusively
by JAK1 and JAK3.4 As loss of function mutation in the cc chain or
JAK3 results in severe combined immunodeficiency (SCID), abroga-
tion of signaling by
cc-cytokines and thereby inhibition of the
cc-linked JAK3 has long been regarded as an attractive target for
the treatment of immunologic disorders such as rheumatoid
arthritis (RA).2,5 Among the synthetic JAK inhibitors identified to
date, Pfizer’s Xelianz (Tofacitinib, CP-690-550), a pan-JAK inhibitor
initially reported as a selective JAK3 inhibitor,5b has recently been
approved by FDA for the treatment of RA.6 However, in spite of the
clinical success of Xelianz, it is unclear whether pan-JAK or iso-
zyme-selective inhibition is required for clinical intervention of
2. Results and discussion
2.1. Structure-based design of the title compounds
⇑
Corresponding author. Tel.: +82 220496100.
E-mail address: chongy@konkuk.ac.kr (Y. Chong).
Three-dimensional crystal structures of the JAK isozymes bound
to C-2 methyl imidazopyrrolopyridines (CMP) were retrieved from
These two authors contributed equally on this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.033

M. K. Kim et al. / Bioorg. Med. Chem. 22 (2014) 1156–1162
1157
the Protein Data Bank (PDB) [PDB ID: 3EYH (JAK1),15 2B7A
(JAK2),16 3LXL (JAK3),17 3LXP (Tyk2)17], and aligned by using the
‘protein structure alignment’ module incorporated in the molecu-
lar modeling software Maestro. Except 3LXP, which showed signif-
icant structural difference compared with other isozymes,
structures of JAK1, JAK2, and JAK3 superposed well to show only
minimal deviation particularly around the ATP-binding site
(Fig. 1a). Nevertheless, subtle but significant difference was ob-
served at the so-called ‘glycine loop’. Thus, due to formation of a
hydrogen bond between Asp1003 and His885, the ATP-binding site
of JAK1 is markedly narrow compared with JAK2 and JAK3. More-
over, Phe860 in JAK2, which corresponds to His885 in JAK1, locates
its aromatic side chain away from Asp976 in the N-terminal lobe to
result in relatively open ATP-binding site. Due to this interesting
feature, JAK1 forms a small cavity right below the CMP-binding
site, which seems to be able to accommodate short linear alkyl
chain (lined with the block arrows in Fig. 1b). In addition, two polar
residues, Asp1003 and His885, at the bottom of the cavity are capa-
ble of forming hydrogen bonds with the incoming inhibitor
molecule.
Based on these observations, we reasoned that positioning a po-
lar group attached to a linear tether around the His885 of JAK1
(Phe860 of JAK2) would enable the resulting inhibitor to differen-
tiate the ATP-binding sites of JAK1 from those of other isozymes.
For this purpose, propargyl alcohol (n = 1, Fig. 2) and but-3-yn-1-
ol (n = 2, Fig. 2) were chosen as the isozyme-specific linear probe
group, which were attached to the 3-position of the indazole-7-
carbamate, a hydrogen-bonding scaffold to the hinge motif of the
kinase. In order to increase binding interaction with the enzyme,
additional indazole functionality was installed at the 5-position
to result in the title compound, 3-alkynolyl-5-(40-indazolyl)inda-
zole-7-carboxamide (1, Fig. 2).
H
N
Hinge Backbone
O
H
H
N
N
5
6'
R2
Isozyme-specific
probe
3
4'
3'
NH
N
n
HO
1
Hydrophobic Contact
Figure 2. Structure of the designed title compound.
lan-2-yl)-1H-indazole derivatives (3a–3b) were prepared starting
from methyl 2-amino-3-methylbenzoate (Scheme 2) and
4
2,4-dinitrotoluene 8 (Scheme 3), respectively. Thus, treatment of
4 with NBS proceeded exclusively at the para position of the amino
functionality to give 5 in 62% yield.19 Construction of the indazole
core structure was then accomplished by acetylation followed by
diazotization of 5 to give a separable mixture of 6a (46% yield)
and 6b (35% yield).20 The acetyl group of 6a was removed by treat-
ment with 6 N HCl in MeOH to give 6b in quantitative yield. The
methyl ester 6b, thus obtained, was converted into the correspond-
ing Boc-protected amide 2a via hydrolysis followed by amidation
and Boc-protection in 95% combined yield. Treatment of 2a with
NIS provided 3-iodoindazole 7 in 54% yield, which gave the desired
indazolyl-3-alkynols 2b and 2c by Sonogashira coupling reac-
tions21 with propargyl alcohol (36% yield) and but-3-yn-1-ol
(30% yield), respectively.
Similar strategy was applied to the synthesis of the 4-(4,4,5,5-
2.2. Syntheses of the title compounds
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole
derivatives
(3a–3b) (Scheme 3). Bromination of 2,4-dinitrotoluene (8) with
NBS22 followed by reduction of the nitro functionality with iron
powder23 provided an inseparable mixture of nitroanilines, 3-bro-
mo-4-methyl-5-nitroaniline and 3-bromo-2-methyl-5-nitroani-
line, in 3:1 ratio (74% combined yield). Boc-protection of the
resulting amino functionality and reduction of the remaining nitro
functionality gave a mixture of 9a and 9b, which were readily sep-
arated by silica gel column chromatography. The desired regio-
isomer 9a underwent cyclization to the indazole core structure
Syntheses of the 5-(40-indazolyl)indazole-7-carboxamide
derivatives 1a–1f were performed by Suzuki coupling18 of the
3-alkynolyl-5-bromo-indazole-7-carboxamides (2a–2c) and 4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole deriva-
tives (3a–3b) followed by removal of the Boc-protecting group in
35–68% combined yield (Scheme 1).
The key intermediates 3-alkynolyl-5-bromo-indazole-7-car-
boxamides (2a–2c) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
Figure 1. (a) Superposition of the ATP-binding sites of the 3D-aligned JAK isozymes bound to the same inhibitor molecule, CMP. Color code: atom type (JAK1), green (JAK2),
pink (JAK3). Tyk2 structure, which showed significant structural difference compared with others, was omitted for the sake of clarity. (b) Molecular surface of JAK1 ATP-
binding site. The putative inhibitor binding site is indicated by block arrows.

1158
M. K. Kim et al. / Bioorg. Med. Chem. 22 (2014) 1156–1162
O
NHBoc
Br
H
N
O
NH2
R2
H
N
H
N
N
a, b
N
+
N
R2
B
O
O
R1
2a R1 = H
2b R1 = CCCH2OH
2c R1 = CC(CH2)2OH
R1
NH
N
1a R1 = H, R2 = H
1b
3a R2 = NHBoc
3b R2 = H
R1 = H, R2 = NH2
1c R1 = CCCH2OH,
1d
R
2 = H
1 = CCCH2OH, R2 = NH2
1e R1 = CC(CH2)2OH, 2 = H
1f R1 = CC(CH2)2OH, R2 = NH2
R
R
Scheme 1. Synthesis of the title compounds 1a–1f. Reagents and conditions: (a) ArB(OR)2, K2CO3, Pd(PPh3)2, toluene, 90 °C; (b) 2 N HCl, THF, rt.
O
OMe
O
OMe
Br
O
OMe
Br
O
NHBoc
Br
R
H
N
H2N
H3C
H2N
H3C
N
a
b
d ~ f
N
N
R1
2a R1 = H
R1 = I
4
5
6a R = Ac
6b
c
g
h
7
R = H
2b R1 = CCCH2OH
2c R1 = CC(CH2)2OH
Scheme 2. Preparation of the 3-alkynolyl-5-bromo-indazole-7-carboxamides 2a–2c. Reagents and conditions: (a) NBS, DMF, rt; (b) KOAc, Ac2O, CHCl3, 0 °C to rt; iso-amyl
nitrite, 18-crow-6; (c) 6 N HCl, MeOH, rt; (d) LiOH, H2O/THF, 50 °C; (e) (COCl)2, CH2Cl2; NH3, MeOh; (f) Boc2O, DMAP, CH2Cl2, rt; (g) NIS, DMF, rt; (h) propargyl alcohol or
3-butynyl alcohol, Pd(PPh3)2, Cul, TEA, CH2Cl2, rt.
R
H
O2N
NO2
CH3
R2
R
R2
R2
N
N
e
a ~ d
g
N
N
CH3
Br
Br
11a R2 = NBoc2, R = Ac
11b 2 = NBoc2, R = H
B
O
O
8
9a R2 = NBoc2, R = NH2
9b R2 = NH2, R = NBoc2
f
f
R
10 R2 = H, R = NH2
12a R2 = H, R = Ac
12b R2 = H, R = H
3a R2 = NHBoc
3b R2 = H
Scheme 3. Preparation of the 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole derivatives 3a–3b. Reagents and conditions: (a) NBS, DMF, H2SO4, 80 °C; (b) iron
power, concd HCl, EtOH; (c) Boc2O, BMAP, CH2Cl2, rt; (d) H2, Pd/C, MeOH; (e) KOAc, Ac2O, CHCl3, 0 ° to rt; 18-crow-6, iso-amyl nitrite, rt to 100 °C; (f) 6 N HCl, MeOH, rt; (g)
bis(pinacolato)diboron, PdCl2(dppf)2, KOAc, DMSO, 80 °C.
under the same reaction conditions described above to give a
mixture of the cyclized products 11a and 11b in 81% yield. After
deacetylation, 11a was converted into 11b in quantitative yield.
Finally, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-inda-
zole derivative 3a was prepared by treatment of 11b with bis(pina-
coloato)diboron and PdCl2(dppf)2 in the presence of KOAc in 36%
yield.24 On the other hand, 3b was prepared in an analogous
manner starting from the commercially available 3-bromo-2-
methylaniline 10 in 44% overall yield.
As anticipated, it is clearly shown that the title compounds lack
the inhibitory activity against JAK2, and their JAK1 selectivity over
JAK2 (1.9 to >67.0) is much higher than that of the reported
JAK1-selective inhibitors (6.4 fold).12 Among the series, the unsub-
stituted 5-(40-indazolyl)indazole-7-carboxamide derivatives (1a
and 1b) showed lowest inhibitory activity against JAK1 while the
4-alkynol-substituents significantly enhanced the JAK1-inhibitory
activity of the corresponding derivatives (1c–1f). In particular,
propargyl alcohol contributed more to the JAK1-inhibitory activity
than butynol, which resulted in higher inhibitory activity of 1c and
1d compared with their but-3-yn-1-ol counterparts, 1e and 1f,
respectively. The possible binding role of the 60-NH2 functionality
is also noteworthy because those with 60-NH2 substituents (1d
and 1f) showed higher inhibitory activity compared with the
unsubstituted derivatives (1c and 1e). The title compounds exhib-
ited similar level of inhibition against JAK3 with the exception of
the propargyl alcohol-substituted derivatives 1c and 1d, which
2.3. Biological activity of the title compounds
In vitro inhibitory activity of the indazole derivatives on the JAK
isozymes was determined by Z0-LYTE™ Kinase Assay Kit-Tyr 6 Pep-
tide (JAK1–JAK3) and Tyr 3 Peptide (Tyk2) (Invitrogen) according to
the manufacturer’s instruction, and%-inhibition of each JAK isozyme
by 10 lM of the title compounds are summarized in Table 1.

M. K. Kim et al. / Bioorg. Med. Chem. 22 (2014) 1156–1162
1159
showed almost two-fold decreased inhibitory activity against JAK3
compared with JAK1.
acetonitrile in water containing 0.1% formic acid (50–54 min) and
20% acetonitrile in water containing 0.1% formic acid (54–
60 min). Flow rate was 1 mL/min. UV was detected at 340 nm.
3. Conclusion
4.2. Syntheses of the title compounds
Taken together, the propargyl alcohol functionality endowed
the 5-(40-indazolyl)indazole-7-carboxamide scaffold with JAK1
selectivity over other JAK isozymes, particularly JAK2, and this
observation supports our hypothesis that a polar group attached
to a linear tether would serve as an isozyme-specific linear probe
group, which would enable the resulting inhibitor to differentiate
the ATP-binding sites of JAK1 from those of other isozymes. Further
optimization study of the aklynol-substituted 5-(40-indazolyl)inda-
zole-7-carboxamide derivatives as JAK1-selective inhibitors are
now under way and will be published elsewhere.
4.2.1. 1-(N,N-Di-Boc-amino)-5-bromo-4-methyl-3-nitrobenzene
(9a)
To
a
solution of 1-methyl-2,4-dinitrobenzene (1.00 g,
5.50 mmol) in sulfuric acid (10 ml) was added NBS (1.17 g,
6.60 mmol) in three portions over 45 min while keeping the reac-
tion temperature at 80 °C. Once the reaction was complete, the
reaction mixture was cooled to rt. Ice-cold water and CH2Cl2 were
added to the reaction mixture and, after phase separation, the or-
ganic layer was washed with saturated aqueous Na2S2O3 solution.
Concentration of the organic layer under reduced pressure pro-
vided 1-bromo-2-methyl-3,5-dinitrobenzene as
a white solid
4. Experimental
(1.40 g, 98% yield): 1H NMR (400 MHz, CD3COCD3) d 8.72 (d,
J = 2.3 Hz, 1H), 8.68 (d, J = 8.3 Hz, 1H), 2.66 (s, 3H).
4.1. Materials and general methods
1-Bromo-2-methyl-3,5-dinitrobenzene (1.00 g, 3.83 mmol) ob-
tained above was dissolved in a mixture of EtOH (10 ml) and THF
(2 ml), and iron powder was added at 0 °C. After stirring at 80 °C
for 2 h, the reaction mixture was cooled to rt, diluted with a mix-
ture of CH2Cl2 and water, and filtered through Celite washing with
CH2Cl2. Organic phase of the filtrate was mixed with water and
then treated with K2CO3 until the aqueous phase became basic.
After phase separation, the organic layer was dried over MgSO4, fil-
tered, and concentrated under reduced pressure to give a yellow
residue. Purification by column chromatography on silica gel pro-
vided an inseparable mixture of 3-bromo-4-methyl-5-nitroaniline
and 3-bromo-2-methyl-5-nitroaniline, in 3:1 ratio (0.65 g, 74%
combined yield).
The nitroaniline derivatives thus obtained were dissolved in
CH2Cl2 (12 ml) and treated with DMAP (1.04 g, 8.49 mmol) and
Boc2O (1.85 g, 8.49 mmol). After stirring for 3 h at rt, the reaction
mixture was concentrated under reduced pressure and the residue
was purified by column chromatography on silica gel (hexanes/
CH2Cl2/Et2O = 100:100:1) to give an inseparable mixture of
di-tert-butyl (5-bromo-4-methyl-3-nitrophenyl)carbamate and
Unless otherwise indicated, all chemicals and solvents were
purchased from standard sources and used without further purifi-
cation. Nuclear magnetic resonance spectra were recorded on a
Bruker 400 AMX spectrometer (Karlsruhe, Germany) at 400 MHz
for 1H NMR and 100 MHz for 13C NMR with tetramethylsilane as
the internal standard. Chemical shifts (d) are given in parts per mil-
lion (ppm) relative to the remaining protons of the deuterated sol-
vents used as internal standard. Coupling constants J are reported
in Hertz (Hz) and spin multiplicities are reported as s (singlet), d
(doublet), dd (doublet of doublets), t (triplet), q (quartet), m (mul-
tiplet), or br s (broad singlet). TLC was performed on silica gel 60
F254 purchased from Merck. Column chromatography was per-
formed using silica gel-60 (220–440 mesh) for flash chromatogra-
phy. Mass spectrometric data (MS) were obtained by electronspray
ionization (ESI). High resolution fast atom bombardment (FAB)
mass spectra were recorded using a JEOL JMS-700 mass spectrom-
eter at the Daegu center of KBSI, Korea. All tested compounds were
P95% purity, as determined by reverse phase HPLC. HPLC was per-
formed on Agilent 1050 (Hewlett-Packard) equipment with vari-
able wavelength (VW) UV detector using Polaris 5, C18-A
250 ꢀ 4.6 mm (Varian) column. Analytical conditions were as fol-
lows: gradient used was 20–25% acetonitrile in water containing
0.1% formic acid (0–8 min), 25–35% acetonitrile in water contain-
ing 0.1% formic acid (8–18 min), 35% acetonitrile in water contain-
ing 0.1% formic acid (18–25 min), 35–80% acetonitrile in water
containing 0.1% formic acid (25–40 min), 80–100% acetonitrile in
water containing 0.1% formic acid (40–45 min), 100% acetonitrile
in water containing 0.1% formic acid (45–50 min), 100–20%
di-tert-butyl (3-bromo-2-methyl-5-nitrophenyl)carbamate as
a
yellow solid (0.99 g, 81% yield).
To the solution of the intermediate obtained above in MeOH
(10 ml) was added Pd/C (0.05 g), and the reaction flask was
charged with H2 by using a balloon. The reaction mixture was stir-
red for 4 h at rt, and filtered through a Celite pad. The filtrate was
concentrated under reduced pressure and the residue was purified
by column chromatography on silica gel (hexanes/EtOAc = 5:1) to
give the desired compound 9a as a yellow powder (0.37 g, 40%
Table 1
%-Inhibition of the JAK isozymes by 10
l
M of the title compounds
%-Inhibition at 10
Compd
l
Ma
Selectivityb
JAK1
JAK2
JAK3
Tyk2
JAK1/JAK2
JAK1/JAK3
1a
1b
1c
22.2 1.2
17.4 1.6
46.7 2.4
67.0 3.2
27.3 2.5
59.8 4.0
4.3
1.2 0.3
9.1 1.6
2.2 0.6
ꢁ0.4 0.1
6.7 0.9
2.5 1.2
1.3
22.5 2.3
15.7 2.1
24.9 1.8
38.1 2.7
32.3 3.4
48.1 2.5
12.6
7.8 0.8
12.1 1.2
2.5 0.6
0.6 0.1
9.3 2.3
27.2 2.7
5.1
18.5
1.9
21.2
>67.0
4.1
1.0
1.1
1.9
1.8
0.8
1.2
2.9
1d
1e
1f
23.9
0.3
Inhc
(IC50, nM)
(3.3)d
(2.5)d
(10.9)d
(3.6)d
a
b
c
Each experiment was repeated at least three times.
Selectivity = % inhibition of JAK1 at 10 lM/% inhibition of JAK2 (or JAK3) at 10 lM.
25
Pan-JAK inhibitor, ‘pyridine 6’.
d
IC50 values reported in Ref. 25.

1160
M. K. Kim et al. / Bioorg. Med. Chem. 22 (2014) 1156–1162
yield): 1H NMR (400 MHz, CD3COCD3) d 6.70 (d, J = 2.0 Hz, 1H), 6.56
(d, J = 2.0 Hz, 1H), 4.93 (br s, 2H), 2.24 (s, 3H), 1.45 (s, 18H).
with 2 N NaOH. The combined base extracts were washed with
brine, dried over MgSO4, concentrated under reduced pressure
and purified by column chromatography on silica gel (hexanes/
Et2O = 4:3) to gain 3a as a white solid (0.046 g, 31% yield): 1H
NMR (400 MHz, CD3COCD3) d 12.33 (br s, 1H), 8.68 (br s, 1H),
7.98 (s, 1H), 7.90 (d, J = 0.9 Hz, 1H), 7.50 (d, J = 1.5 Hz, 1H), 2.84
(s, 12H), 1.51 (s, 9H).
4.2.2. Di-tert-butyl (4-bromo-1H-indazol-6-yl)carbamate (11b)
To a solution of di-tert-butyl (3-amino-5-bromo-4-methyl-
phenyl)carbamate (9a) (0.37 g, 0.91 mmol) in CHCl3 (3 ml) was
added KOAc (0.09 g, 0.96 mmol) at rt. After cooling to 0 °C, Ac2O
(0.17 ml, 1.82 mmol) was added and the reaction mixture was stir-
red at rt until white solid formed (ca. 15 min). Then, 18-crown-6
(0.05 g, 0.18 mmol) and isopentyl nitrite (0.27 ml, 2.00 mmol)
were added, and the reaction mixture was stirred at 65 °C for
18 h. After cooling to rt, the reaction mixture was diluted with
CHCl3 and washed sequentially with saturated aqueous NaHCO3
solution and brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The residue was purified
by column chromatography on silica gel (hexanes/EtOAc = 6:1) to
give di-tert-butyl (1-acetyl-4-bromo-1H-indazol-6-yl)carbamate
11a (0.19 g, 45% yield).
Compound 11a obtained above (0.19 g, 0.41 mmol) was dis-
solved in MeOH (5 mL) and treated with 6 N HCl (2 ml). The reaction
mixture was stirred at rt for 2 h and then concentrated under re-
duced pressure. The residue was taken with EtOAc and washed with
water. The organic layer was concentrated to give the desired di-
tert-butyl (4-bromo-1H-indazol-6-yl)carbamate (11b) as a bright
brown solid (0.15 g, 89% yield): 1H NMR (400 MHz, CD3COCD3) d
8.33 (d, J = 0.5 Hz, 1H), 8.23 (s, 1H), 7.56 (d, J = 1.5 Hz, 1H), 1.41 (s,
18H); 13C NMR (100 MHz, acetone-d6) d 171.7, 152.1, 142.2, 140.1,
139.3, 128.7, 125.8, 114.8, 113.7, 83.6, 28.0, 23.0.
4.2.5. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
indazole (3b)
A solution of 4-bromo-1H-indazole (12b) (0.20 g, 1.02 mmol) in
Et2O (6 mL) was cooled to ꢁ78 °C and then treated with 1.7 M t-BuLi
(1.78 ml, 3.06 mmol) slowly. The resulting cream color mixture was
stirred for 1 h at -78 °C. Pinacol borane (0.44 ml, 3.06 mmol) was
added to this solution in a dropwise manner and then the mixture
was stirred for 1 h at ꢁ78 °C. The reaction mixture was stirred for
additional 2 h at rt, and the resulting sticky mixture was taken with
EtOAc. The organic layer was washed sequentially with saturated
aqueous ammonium chloride solution and brine. The combined or-
ganic layer was separated, dried over MgSO4 and concentrated un-
der reduced pressure. The crude mixture was purified by column
chromatography on silica gel (hexanes/Et2O = 4:3) to obtain 3b
(0.11 g, 44% yield) as white powder: 1H NMR (400 MHz, CD3COCD3)
d 8.36 (s, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.60 (d, J = 6.8 Hz, 1H), 7.38
(dd, J = 6.8, 8.3 Hz, 1H), 1.40 (s, 12H).
4.2.6. Methyl 2-amino-5-bromo-3-methylbenzoate (5)
To a solution of methyl 2-amino-3-methylbenzoate (4) (1.00 g,
6.10 mmol) in DMF (10 ml) was added NBS (1.10 g, 6.10 mmol),
and the reaction mixture was stirred at rt for 6 h. The reaction mix-
ture was diluted with EtOAc (10 ml) and washed with saturated
aqueous Na2CO3 solution (10 ml ꢀ 3). The organic layer was dried
over MgSO4, filtered, and concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel
(hexanes/EtOAc = 100:1) to give compound 5 as pale gray solid
(0.93 g, 62% yield): 1H NMR (400 MHz, CDCl3) d (ppm) 7.88 (d,
J = 2.3 Hz, 1H), 7.28 (d, J = 2.3 Hz, 1H), 5.82 (s, 2H), 3.86 (s, 3H),
2.15 (s, 3H).
4.2.3. 4-Bromo-1H-indazole (12b)
To
a solution of 3-bromo-2-methylaniline (10) (0.30 g,
1.61 mmol) in CHCl3 (5 ml) was added KOAc (0.17 g, 1.69 mmol)
at rt. After cooling to 0 °C, Ac2O (0.30 ml, 3.22 mmol) was added
and the reaction mixture was stirred at rt until white solid formed
(ca. 15 min). Then, 18-crown-6 (0.085 g, 0.32 mmol) and isopentyl
nitrite (0.48 mL, 3.54 mmol) were added, and the reaction mixture
was stirred at 65 °C for 18 h. After cooling to rt, the reaction mix-
ture was diluted with CHCl3 and washed sequentially with satu-
rated aqueous NaHCO3 solution and brine. The organic layer was
dried over MgSO4 and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel (hex-
anes/EtOAc = 5:1) to give 1-(4-bromo-1H-indazol-1-yl)ethanone
12a (0.19 g, 49% yield) and 4-bromo-1H-indazole 12b (0.13 g,
40% yield).
Compound 12a (0.19 g, 0.79 mmol) was dissolved in MeOH
(4 mL) and treated with 6 N HCl (2 ml). The reaction mixture was
stirred at rt for 2 h and then concentrated under reduced pressure.
The residue was taken with EtOAc and washed with water. The or-
ganic layer was concentrated to give the desired 4-bromo-1H-inda-
zole (12b) as a bright brown solid (0.14 g, 90% yield): 1H NMR
(400 MHz, CD3COCD3) d 1H NMR (400 MHz, acetone-d6) d 12.86
(br s, 1H), 8.05 (s, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.34 (d, J = 7.3 Hz,
1H), 7.29 (t, J = 7.8 Hz, 1H).
4.2.7. Methyl 5-bromo-1H-indazole-7-carboxylate (6b)
To a stirred solution of methyl 2-amino-5-bromo-3-meth-
ylbenzoate (5) (0.50 g, 2.20 mmol) in CHCl3 (5 ml) was added KOAc
(0.22 g, 2.30 mmol) at rt. After cooling to 0 °C, Ac2O (0.40 ml,
4.20 mmol) was added and the reaction mixture was stirred at rt
until white solid formed (ca. 15 min). Then, 18-crown-6 (0.12 g,
0.40 mmol) and isopentyl nitrite (0.60 ml, 4.80 mmol) were added,
and the reaction mixture was stirred at 65 °C for 18 h. After cooling
to rt, the reaction mixture was diluted with CHCl3 and washed
sequentially with saturated aqueous NaHCO3 solution and brine.
The organic layer was dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica gel (hexanes/EtOAc = 6:1) to give 6a (0.30 g, 46%
yield) and 6b (0.20 g, 35% yield).
Compound 6a (0.30 g, 1.00 mmol) was dissolved in MeOH (5 mL)
and treated with 6 N HCl (2 ml). The reaction mixture was stirred at
rt for 2 h and then concentrated under reduced pressure. The residue
was taken with EtOAc and washed with water. The organic layer was
concentrated to give the desired methyl 5-bromo-1H-indazole-7-
carboxylate (6b) as a bright brown solid (0.25 g, 98% yield): 1H
NMR (400 MHz, CDCl3) d (ppm) 12.85 (s, 1H), 8.16 (d, J = 1.5 Hz,
1H), 8.13 (t, J = 1.5 Hz, 1H), 8.09 (d, J = 1.5 Hz, 1H), 4.04 (s, 3 H).
4.2.4. tert-Butyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-indazol-6-yl)carbamate (3a)
A solution of di-tert-butyl (4-bromo-1H-indazol-6-yl)carba-
mate (11b) in Et2O was treated with 1.7 M t-BuLi (0.72 ml,
1.23 mmol) at ꢁ78 °C in a dropwise manner and stirred for 1 h.
Pinacol borane (0.18 ml, 1.23 mmol) was slowly added and the
reaction mixture was stirred for 1 h at ꢁ78 °C before being allowed
to warm to rt. More anhydrous Et2O was added to facilitate stir-
ring. After stirring for 24 h, the resulting sticky mixture was diluted
with Et2O and transferred in portions with stirring to a precooled
solution of 2 N HCl. After stirring for 30 min, the acidic mixture
was extracted with Et2O and the combined extracts were washed
4.2.8. tert-Butyl (5-bromo-1H-indazole-7-carbonyl)carbamate
(2a)
To a stirred solution of tert-butyl (5-bromo-1H-indazole-7-car-
bonyl)carbamate (6b) (0.55 g, 2.20 mmol) in a mixture of THF

M. K. Kim et al. / Bioorg. Med. Chem. 22 (2014) 1156–1162
1161
(2 ml) and water (8 ml), LiOH (0.20 g, 8.60 mmol) was added, and
the reaction mixture was stirred at 50 °C for 4 h. 2 N HCl was added
to the reaction mixture to adjust pH to 2, and the resulting white
solid was filtered. The solid was dissolved in CH2Cl2 (5 ml) and
treated with (COCl)2 (0.20 ml, 2.20 mmol). After stirring for 1 h,
the reaction mixture was concentrated under reduced pressure.
The residue was taken with saturated methanolic ammonia
(5 ml), and stirred for 4 h at rt. The reaction mixture was concen-
trated under reduced pressure, and the residue was purified by col-
umn chromatography on silica gel (hexanes/acetone = 2:1) to give
the 5-bromo-1H-indazole-7-carboxamide (0.45 g, 85% yield) as a
pale brown solid: 1H NMR (400 MHz, MeOD) d (ppm) 11.24 (s,
1H), 10.63 (s, 2H), 8.16 (d, J = 1.5 Hz, 1H), 8.12 (t, J = 1.5 Hz, 1H),
8.09 (d, J = 1.5 Hz, 1H).
5-Bromo-1H-indazole-7-carboxamide (0.45 g, 1.90 mmol) ob-
tained above was dissolved in CH2Cl2 and treated with Boc2O
(0.42 g, 1.90 mmol) and DMAP (0.23 g, 1.90 mmol). After stirring
at rt for 6 h, the reaction mixture was concentrated under reduced
pressure, and the residue was purified by column chromatography
on silica gel (hexanes/EtOAc = 2:1) to give the desired tert-butyl (5-
bromo-1H-indazole-7-carbonyl)carbamate (2a) as a pale brown oil
(0.61 g, 95% yield): 1H NMR (400 MHz, CDCl3) d (ppm) 8.18 (d,
J = 1.5 Hz, 1H), 8.14 (t, J = 1.5 Hz, 1H), 8.10 (d, J = 1.5 Hz, 1H), 1.50
(s, 9H).
EtOAc (10 ml). The organic layer was washed with brine
(10 ml ꢀ 3), dried over MgSO4, filtered, and concentrated under re-
duced pressure. The residue was taken with THF (10 ml) and trea-
ted with 2 N HCl (5 ml). After stirring at rt for 4 h, the reaction
mixture was neutralized with 2 N NaOH and extracted with EtOAc
(10 ml ꢀ 3). The combined organic layer was dried over MgSO4, fil-
tered, and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (CH2Cl2/
MeOH = 20:1) to give the desired compound 1 in 37–68% yield.
Compound 1a (0.19 g, 68% yield): 1H NMR (400 MHz, MeOD) d
(ppm) 12.86 (br s, 2H), 8.14 (d, J = 1.5 Hz, 1H), 8.12 (t, J = 1.5 Hz,
1H), 8.08 (d, J = 1.5 Hz, 1H), 8.05 (s, 1H), 7.61 (d, J = 7.8 Hz, 1H),
7.52 (br s, 2H), 7.32 (d, J = 7.8 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H);
LC/MS (ESI) m/z found: 278.2 [M+H]+; calcd for C15H11N5O: 278.10.
Compound 1b (0.12 g, 41% yield): 1H NMR (400 MHz, MeOD) d
(ppm) 12.86 (br s, 2H), 8.14 (d, J = 1.5 Hz, 1H), 8.12 (t, J = 1.5 Hz,
1H), 8.08 (d, J = 1.5 Hz, 1H), 8.05 (s, 1H), 7.90 (d, J = 1.5 Hz, 1H),
7.52 (br s, 2H), 7.85 (d, J = 1.5 Hz, 1H), 4.95 (br s, 2H); LC/MS
(ESI) m/z found: 293.3 [M+H]+; calcd for C15H12N6O: 293.11.
Compound 1c (0.13 g, 39% yield): 1H NMR (400 MHz, MeOD) d
(ppm) 12.86 (br s, 2H), 8.38 (d, J = 1.5 Hz, 1H), 8.21 (d, J = 1.5 Hz,
1H), 8.04 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.52 (br s, 2H), 7.32 (d,
J = 7.8 Hz, 1H), 7.28 (t, J = 7.8 Hz), 4.95 (br s, 2H), 4.12–4.16 (m,
2H); LC/MS (ESI) m/z found: 332.2 [M+H]+; calcd for C18H13N5O2:
332.11.
4.2.9. tert-Butyl (5-bromo-3-iodo-1H-indazole-7-
carbonyl)carbamate (7)
Compound 1d (0.13 g, 37% yield): 1H NMR (400 MHz, MeOD) d
(ppm) 12.86 (br s, 2H), 8.38 (d, J = 1.5 Hz, 1H), 8.21 (d, J = 1.5 Hz,
1H), 8.04 (s, 1H), 7.85 (d, J = 1.5 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H),
7.52 (br s, 2H), 4.95 (br s, 2H), 4.12–4.16 (m, 2H); LC/MS (ESI) m/
z found: 347.3 [M+H]+; calcd for C18H14N6O2: 347.12
To a stirred solution of tert-butyl (5-bromo-1H-indazole-7-car-
bonyl)carbamate (2a) (0.61 g. 1.80 mmol) in DMF (5 ml) was added
NIS (0.24 g, 1.80 mmol). After stirring at rt for 6 h, the reaction
mixture was diluted with EtOAc (10 ml) and washed with satu-
rated aqueous Na2CO3 solution (10 ml ꢀ 3). The organic layer was
dried over MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by column chromatography on silica
gel (hexanes/EtOAc = 2:1) to give tert-butyl (5-bromo-3-iodo-1H-
indazole-7-carbonyl)carbamate (7) (0.45 g, 54% yield) as a pale
gray solid: 1H NMR (400 MHz, CDCl3) d (ppm) 12.86 (s, 1H),
10.58 (s, 1H), 8.44 (d, J = 1.5 Hz, 1H), 8.30 (d, J = 1.5 Hz, 1H), 1.50
(s, 9H).
Compound 1e (0.12 g, 38% yield): 1H NMR (400 MHz, MeOD) d
(ppm) 12.86 (br s, 2H), 8.38 (d, J = 1.5 Hz, 1H), 8.21 (d, J = 1.5 Hz,
1H), 8.04 (s, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.52 (br s, 2H), 7.31 (d,
J = 7.8 Hz, 1H), 7.26 (t, J = 7.8 Hz), 4.95 (br s, 2H), 4.12–4.16 (m,
2H), 2.26–2.30 (m, 2H); LC/MS (ESI) m/z found: 346.5 [M+H]+;
Calcd. for C19H15N5O2: 346.12
Compound 1f (0.13 g, 37% yield): 1H NMR (400 MHz, MeOD) d
(ppm) 12.86 (br s, 2H), 8.38 (d, J = 1.5 Hz, 1H), 8.21 (d, J = 1.5 Hz,
1H), 8.04 (s, 1H), 7.85 (d, J = 1.5 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H),
7.52 (br s, 2H), 4.95 (br s, 2H), 4.12–4.16 (m, 2H), 2.26–2.29 (m,
2H); LC/MS (ESI) m/z found: 362.4 [M+H]+; calcd for C19H16N6O2:
362.13.
4.2.10. tert-Butyl (5-bromo-3-(3-hydroxyprop-1-yn-1-yl)-1H-
indazole-7-carbonyl) carbamate (2b) and tert-butyl (5-bromo-
3-(4-hydroxybut-1-yn-1-yl)-1H-indazole-7-carbonyl)carbamate
(2c)
Acknowledgments
To a stirred solution of tert-butyl (5-bromo-3-iodo-1H-inda-
zole-7-carbonyl)carbamate (7) (0.45 g, 1.00 mmol) in CH2Cl2 were
added Pd(PPh3)4 (0.23 g, 0.20 mmol), CuI (0.095 g, 0.50 mmol),
propargyl alcohol or 3-butynyl alcohol (1.00 mmol), and TEA
(0.30 ml, 2.00 mmol). After stirring at rt for 24 h, the reaction mix-
ture was diluted with CH2Cl2 and washed with brine (10 ml ꢀ 3).
The organic layer was dried over MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by column chro-
matography on silica gel (hexanes/acetone = 2:1) to give com-
pound 2b or 2c.
This research was supported by a grant of the Korean Health
Technology R&D project, Ministry of Health & Welfare, Republic
of Korea (A111698), by Priority Research Centers Program through
the National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (2009-0093824),
and by a grant from Next Generation Bio-Green 21 PJ007982
(Korea Rural Development Administration).
References and notes
Compound 2b (0.14 g, 36% yield): 1H NMR (400 MHz, CDCl3) d
(ppm) 12.85 (s, 1H), 10.58 (s, 1H), 8.40 (d, J = 1.5 Hz, 1H), 8.28 (d,
J = 1.5 Hz, 1H), 3.62–3.64 (m, 2H), 2.28–2.30 (m, 2H), 1.50 (s, 9H).
Compound 2c (0.12 g, 30% yield): 1H NMR (400 MHz, CDCl3) d
(ppm) 12.85 (s, 1H), 10.58 (s, 1H), 8.40 (d, J = 1.5 Hz, 1H), 8.28 (d,
J = 1.5 Hz, 1H), 4.12–4.16 (m, 2H), 1.49 (s, 9H).
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