Novel Indazole-based MKK7–TIPRL Interaction Inhibitors as TRAIL Sensitizers

Article abstract of DOI:10.1002/bkcs.11561

This work describes the process by which a metabolically unstable TRT-0002 compound exhibiting Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitizing activity for Huh7 cells at a high working concentration (40 μM) is converted to more potent and metabolically improved analogues by modifying the 5-amino group and the 1-aryl moiety in the 1H-indazole skeleton. The efforts enabled us to identify 5-sulfonamido derivatives, TRT-0029 and TRT-0173 compounds, working at lower concentrations (10 and 20 μM, respectively) and with improved metabolic stabilities. As reported previously by us, co-treating cultured Huh7 cells with either TRT-0029 or TRT-0173 and TRAIL resulted in TRAIL-induced apoptosis due to the inhibition of the MKK7–TOR signaling pathway regulator-like (TIPRL) interaction and subsequent phosphorylation of MKK7 and JNK. In addition, the injection of TRT-0029 or TRT-0173 compound suppressed tumor growth in combination with TRAIL in an in vivo hepatocellular carcinoma (HCC) mouse xenograft model. TRT-0029 and TRT-0173 compounds and the relevant structure–activity relationship can provide an insight into further study on optimization of potency and metabolic stability.

Full text of DOI:10.1002/bkcs.11561

Article
DOI: 10.1002/bkcs.11561
BULLETIN OF THE
S. Gu et al.
KOREAN CHEMICAL SOCIETY
Novel Indazole-based MKK7–TIPRL Interaction Inhibitors as TRAIL
Sensitizers
Sujin Gu,† Myoung Eun Jung,† Ji-Yong Yoon,‡ Sang-Eun Yoon,† Jeong-Ju Lee,‡ Kwangho Lee,†,§
‡,¶,
†,
Gildon Choi,†,§, Nam-Soon Kim,
*
*
*
and Moon-Kook Jeon
†Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, Daejeon
34114, Republic of Korea. *E-mail: moteta@krict.re.kr
‡Genome Research Center, Korea Research Institute of Biosciences and Biotechnology, Daejeon
305-333, Republic of Korea
§Department of Medicinal Chemistry and Pharmacology, Korea University of Science and Technology,
Daejeon 34113, Republic of Korea. *E-mail: gchoi@krict.re.kr
¶Department of Functional Genomics, Korea University of Science and Technology, Daejeon 34113,
Republic of Korea. *E-mail: nskim37@kribb.re.kr
Received May 31, 2018, Accepted July 26, 2018
This work describes the process by which a metabolically unstable TRT-0002 compound exhibiting
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitizing activity for Huh7 cells at a
high working concentration (40 μM) is converted to more potent and metabolically improved analogues
by modifying the 5-amino group and the 1-aryl moiety in the 1H-indazole skeleton. The efforts enabled
us to identify 5-sulfonamido derivatives, TRT-0029 and TRT-0173 compounds, working at lower concen-
trations (10 and 20 μM, respectively) and with improved metabolic stabilities. As reported previously by
us, co-treating cultured Huh7 cells with either TRT-0029 or TRT-0173 and TRAIL resulted in TRAIL-
induced apoptosis due to the inhibition of the MKK7–TOR signaling pathway regulator-like (TIPRL)
interaction and subsequent phosphorylation of MKK7 and JNK. In addition, the injection of TRT-0029 or
TRT-0173 compound suppressed tumor growth in combination with TRAIL in an in vivo hepatocellular
carcinoma (HCC) mouse xenograft model. TRT-0029 and TRT-0173 compounds and the relevant
structure–activity relationship can provide an insight into further study on optimization of potency and
metabolic stability.
Keywords: Tumor necrosis factor-related apoptosis-inducing ligand, Apoptosis, Sensitizer, TOR signal-
ing pathway regulator-like, Indazole
Introduction
and compounds inhibiting the function of these factors.
Natural/small compounds, such as doxorubicin, cisplatin,
curcumin, and wogonin, have been reported as TRAIL
sensitizers,6–9 but the mode of action (MOA) of these com-
pounds is not well known.
Tumor necrosis factor (TNF)-related apoptosis-inducing
ligand (TRAIL), a member of the TNF superfamily, is con-
sidered a promising anticancer agent owing to its anticancer
activity and its ability to selectively induce apoptosis in
cancer cells, but not in normal cells.1 TRAIL is well known
for binding to five receptors: the death receptor (DR) 4/5,
decoy receptor (DcR) 1/2, and osteoprotegerin. Among
them, DR 4/5 contain a death domain (DD) that initiates
TRAIL-induced apoptosis. The binding of TRAIL to DR
4/5 recruits the FAS-associated DD (FADD) and DD. This
leads to apoptosis owing to poly ADP ribose polymerase
(PARP) and caspase.2–4 However, many cancer cells have
been demonstrated to exhibit TRAIL resistance owing to
several factors such as: (1) overexpression of antagonistic
receptors (DcR 1/2), (2) overpexression of anti-apoptotic
proteins (Bcl-2 family), (3) defective functional FADD.5
One strategy that has been considered for sensitizing
TRAIL-induced apoptosis is the combination of TRAIL
Recently, we reported that TOR signaling pathway
regulator-like (TIPRL) protein plays an important role in
TRAIL resistance by enabling the interaction of the
mitogen-activated protein kinase kinase 7 (MKK7) and pro-
tein phosphatase type 2A (PP2Ac). The MKK7–TIPRL–
PP2Ac complex prevents the phosphorylation of MKK7
and c-Jun N-terminal kinase (JNK), inhibiting the apoptosis
cascade caused by TRAIL. In addition, TIPRL depletion
induces TRAIL-induced apoptosis via the phosphorylation
of MKK7 and activation of JNK.10 To identify TRAIL sen-
sitizers based on these mechanisms, we established an
in vitro enzyme-linked immunosorbent assay (ELISA)
screening system for detecting MKK7–TIPRL interaction.
Using this screening system, we identified natural com-
pounds such as Tussilago farfara L. (commonly known as
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KOREAN CHEMICAL SOCIETY
coltsfoot)11 and Taraxacum officinale F. H. Wigg (com-
monly known as dandelion)12 that inhibit the
MKK7-TIPRL interaction, which we reported as TRAIL
sensitizers.
chromatography on a silica gel column (n-hexane: dichloro-
methane: ethyl acetate = 1: 5: 1) to give the intermediate
1
1 (1.00 g, 6%). H NMR (500 MHz, CDCl3): δ 8.11 (s,
1H), 8.05 (s, 1H), 7.94 (s, 1H), 7.09 (d, J = 8.5 Hz, 1H),
6.88 (d, J = 8.5 Hz, 1H), 6.86 (s, 1H), 5.02 (br s, 2H), 2.63
(s, 3H); MS (ESI): m/z for C15H11Cl2N3O, found
320 [M + H+].
We also identified small chemical compounds that act as
TRAIL sensitizers.13 In the study, we screened over 6000
small molecule compounds using the high-throughput
ELISA system and found TRT-0002 (1, Figure 1) inhibit-
ing the MKK7–TIPRL interaction as a hit compound. After
intensive hit-to-lead optimization, two lead compounds,
TRT-0029 (2, Figure 1) and TRT-0173 (3, Figure 1), were
selected from the 280 synthesized analogues of TRT-0002.
These two lead compounds were observed to initiate
TRAIL-induced apoptosis in Huh7 cells by inhibiting the
MKK7-TIPRL interactions and activating MKK7/JNK. The
TRAIL-sensitizing activity of the two lead compounds was
also confirmed using an in vivo xenograft animal model.13
These results indicate that the pharmacological inhibition of
the MKK7–TIPRL interaction is a promising strategy to
overcome TRAIL resistance in hepatocellular carci-
noma (HCC).
Representative Synthetic Procedure for 1-(5-Acetyl-2,-
4-Dichlorophenyl)-5-Alkylamino-1H-Indazoles (5). To a
solution of the precursor 1 (30 mg, 0.094 mmol) in DMF
(2 mL) at room temperature was added 1-bromo-
2-methylpropane (26 μL, 0.23 mmol) and potassium car-
bonate (39 mg, 0.28 mmol). The mixture was heated at
80ꢀC overnight and partitioned between dichloromethane
and water after cooling to room temperature. The organic
layer was washed several times with water and dried over
magnesium sulfate. The solvent was evaporated in vacuo
and the residue was separated by chromatography on a sil-
ica gel column (n-hexane: ethyl acetate = 5: 1) to give
1-(5-acetyl-2,4-dichlorophenyl)-5-isobutylamino-1H-indazo
1
le (5a, 4 mg, 10%). H NMR (300 MHz, CDCl3): δ 8.06
(s, 1H), 7.74 (s, 1H), 7.68 (s, 1H), 7.06 (d, J = 9.5 Hz,
1H), 6.88 (m, 2H), 2.99 (d, J = 6.8 Hz, 2H), 2.66 (s, 3H),
1.97 (m, 1H), 1.03 (d, J = 6.6 Hz, 6H); MS (ESI): m/z for
C19H19Cl2N3O, found 376 [M + H+].
However, in the previous report, the optimization process
by which the lead compounds TRT-0029 (2) and TRT-
0173 (3) were obtained from an initial hit compound TRT-
0002 (1) was only schematically described. Herein, we
explain the process in more detail from the medicinal
chemistry perspective. During the hit-to-lead stage, we con-
centrated on variations of substituents around the 5-amino-
1-aryl-1H indazole core as represented by the general struc-
ture 4 in Figure 1. The TRAIL-sensitizing activity of the
derivatives was evaluated based on a cell viability assay.
Other in vitro properties, such as liver microsomal stability
and solubility, will be reported for representative
compounds.
Representative Synthetic Procedure for 1-(5-Acetyl-2,-
4-Dichlorophenyl)-5-Dialkylamino-1H-Indazoles (6). To a
solution of the precursor 1 (50 mg, 0.16 mmol) in DMF
(2 mL) at room temperature was added iodomethane
(24 μL, 0.39 mmol) and potassium carbonate (65 mg,
0.45 mmol). The mixture was heated at 60ꢀC overnight and
partitioned between dichloromethane and water after cool-
ing to room temperature. The organic layer was washed
several times with water and dried over magnesium sulfate.
The solvent was evaporated in vacuo and the residue was
separated by chromatography on a silica gel column (n-hex-
ane: ethyl acetate = 1: 1) to give 1-(5-acetyl-2,-
Experimental
4-dichlorophenyl)-5-dimethylamino-1H-indazole
(6a,
Synthesis of Compounds 5–10
1
11 mg, 20%). H NMR (300 MHz, DMSO‑d6): δ 8.22 (s,
1H), 8.06 (s, 1H), 7.96 (s, 1H), 7.23 (d, J = 8.6 Hz, 1H),
7.16 (dd, J = 8.6, 1.9 Hz, 1H), 7.01 (d, J = 1.9 Hz, 1H),
2.91 (s, 6H), 2.62 (s, 3H); MS (ESI): m/z for
C17H15Cl2N3O, found 348 [M + H+].
Synthetic Procedure for 1-(5-Acetyl-2,4-Dichlorophenyl)-
5-Amino-1H-Indazole (1)13. A mixture of 5-amino-1H-
indazole (6.40 g, 48.1 mmol), 1-acetyl-2,4-dichloro-
5-fluorobenene (10.0 g, 48.3 mmol) and potassium carbon-
ate (66.8 g, 483 mmol) in DMF (200 mL) was heated at
110ꢀC for 20 h. After cooling to room temperature, the
reaction mixture was partitioned between dichloromethane
and water, and the organic layer was washed several times
with water and dried over magnesium sulfate. The solvent
was evaporated in vacuo and the residue was separated by
Representative Synthetic Procedure for 1-(5-Acetyl-2,-
4-Dichlorophenyl)-5-Amido-1H-Indazoles (7). To a solu-
tion of the precursor 1 (30 mg, 0.094 mmol) in DCM
(2 mL) at room temperature was added 3-(1H-indol-3-yl)
propanoic acid (21 mg, 0.11 mmol) and EDC (22 mg,
0.14 mmol). The mixture was stirred at the same tempera-
ture overnight and the solvent was evaporated in vacuo.
The residue was separated by chromatography on a silica
gel column (n-hexane: ethyl acetate = 3: 2) to give
1-(5-acetyl-2,4-dichlorophenyl)-5-(3-(1H-indol-3-yl)propa-
noyl)amino-1H-indazole (7a, 36 mg, 78%). 1H NMR
(300 MHz, DMSO‑d6): δ 10.77 (br s, 1H), 10.04 (br s,
1H), 8.39 (s, 1H), 8.28 (s, 1H), 8.08 (s, 1H), 8.01 (s, 1H),
Figure 1. Representation of indazole-based compounds.
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MKK7–TIPRL Interaction Inhibitors as TRAIL Sensitizers
KOREAN CHEMICAL SOCIETY
7.59 (d, J = 7.7 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H),
7.34–7.30 (m, 2H), 7.14 (s, 1H), 7.07 (t, J = 7.5 Hz, 1H),
6.98 (t, J = 7.5 Hz, 1H), 3.05 (t, J = 7.6 Hz, 2H), 2.71 (t,
J = 7.6 Hz, 2H), 2.62 (s, 3H); MS (ESI): m/z for
C26H20Cl2N4O2, found 491 [M + H+].
6.60 (s, 1H), 3.95 (s, 3H), 3.86 (s, 3H); MS (ESI): m/z for
C22H17Cl2N3O4S, found 490 [M + H+].
1-(5-Acetyl-2,4-dichlorophenyl)-5-phenylmethanesulfo-
namido-1H-indazole (10b). 1H NMR (300 MHz,
DMSO‑d6): δ 9.87 (br s, 1H), 8.42 (s, 1H), 8.10 (s, 1H),
8.02 (s, 1H), 7.72 (s, 1H), 7.39–7.29 (m, 7H), 4.45 (s, 2H),
2.63 (s, 3H); MS (ESI): m/z for C22H17Cl2N3O3S, found
474 [M + H+].
Representative Synthetic Procedure for 1-(5-Acetyl-2,-
4-Dichlorophenyl)-5-Oxycarbonylamino-1H-Indazoles (8).
To a solution of the precursor 1 (50 mg, 0.16 mmol) in
DCM (2 mL) at room temperature was added methyl chlor-
oformate (14 μL, 0.18 mmol) and pyridine (19 μL,
0.24 mmol). The mixture was stirred at the same tempera-
ture overnight and the solvent was evaporated in vacuo.
The residue was separated by chromatography on a silica
gel column (n-hexane: ethyl acetate = 3: 1) to give
1-(5-acetyl-2,4-dichlorophenyl)-5-methoxycarbonylamino-
1H-indazole (8a, 52 mg, 86%). 1H NMR (300 MHz,
DMSO‑d6): δ 9.73 (br s, 1H), 8.38 (s, 1H), 8.08 (s, 1H),
8.03–8.00 (m, 2H), 7.47 (dd, J = 9.0, 1.7 Hz, 1H), 7.32 (d,
J = 9.0 Hz, 1H), 3.69 (s, 3H), 2.62 (s, 3H); MS (ESI): m/z
for C17H13Cl2N3O3, found 378 [M + H+].
Representative Synthetic Procedure for 1-(5-Acetyl-2,-
4-Dichlorophenyl)-5-Ureido-1H-Indazoles (9). To a solu-
tion of the precursor 1 (50 mg, 0.16 mmol) in DCM (2 mL)
at room temperature was added ethyl isocyanate (15 μL,
0.19 mmol) and pyridine (19 μL, 0.24 mmol). The mixture
was stirred at the same temperature overnight and the solvent
was evaporated in vacuo. The residue was separated by chro-
matography on a silica gel column (n-hexane: ethyl acetate =
1: 1) to give 1-(5-acetyl-2,4-dichlorophenyl)-5-ethylamino-
carbonylamino-1H-indazole (9a, 35 mg, 56%). 1H NMR
(300 MHz, DMSO‑d6): δ 8.51 (br s, 1H), 8.31 (s, 1H), 8.07
(s, 1H), 7.99 (s, 2H), 7.36 (dd, J = 9.0, 1.8 Hz, 1H), 7.25 (d,
J = 9.0 Hz, 1H), 6.10 (br t, J = 5.4 Hz, 1H), 3.12 (m, 2H),
2.62 (s, 3H), 1.07 (t, J = 7.1 Hz, 3H); MS (ESI): m/z for
C18H16Cl2N4O2, found 391 [M + H+].
Synthesis of Compounds 15
Synthetic Procedure for 5-Amino-1-(2,4-Dichloro-5-Meth-
oxycarbonylphenyl)-1H-Indazole (11a).
A mixture of
5-amino-1H-indazole (3.46 g, 26.0 mmol), methyl
2,4-dichloro-5-fluorobenzoate (4.00 mL, 26.0 mmol), and
potassium carbonate (35.9 g, 260 mmol) in DMF (40 mL)
was heated at 100ꢀC for 3 days. After cooling to room tem-
perature, the reaction mixture was partitioned between
dichloromethane and water, and the organic layer was
washed several times with water and dried over magnesium
sulfate. The solvent was evaporated in vacuo and the resi-
due was separated by chromatography on a silica gel col-
umn (n-hexane:dichloromethane:ethyl acetate = 1:1:1) to
give the intermediate 11a (430 mg, 5%). 1H NMR
(300 MHz, CDCl3): δ 8.05 (s, 1H), 8.04 (s, 1H), 7.72 (s,
1H), 7.07 (d, J = 8.8 Hz, 1H), 7.00 (d, J = 1.4 Hz, 1H),
6.89 (dd, J = 8.8, 1.4 Hz, 1H), 3.92 (s, 3H), 3.70 (br s,
2H); MS (ESI): m/z for C15H11Cl2N3O2, found
336 [M + H+].
Synthetic Procedure for 5-Amino-1-(3-Methoxycarbonyl-
phenyl)-1H-Indazole (11b). A mixture of 5-amino-1H-
indazole (1.00 g, 7.51 mmol), methyl 3-iodobenzoate
(1.97 g, 7.51 mmol), cuprous iodide (140 mg, 0.75 mmol),
trans-cyclohexane-1,2-diamine (450 μL, 3.76 mmol), and
tripotassium phosphate (2.90 g, 1.35 mmol) in 1,4-dioxane
(40 mL) was heated at 100ꢀC overnight. After cooling to
room temperature, the reaction mixture was partitioned
between dichloromethane and water, and the organic layer
was dried over magnesium sulfate. The solvent was evapo-
rated in vacuo and the residue was separated by chromatog-
raphy on a silica gel column (n-hexane: ethyl acetate = 1:
Representative Synthetic Procedure for 1-(5-Acetyl-2,-
4-Dichlorophenyl)-5-Sulfonamido-1H-Indazoles
(10)13.
Benzenesulfonyl chloride (14 μL, 0.11 mmol) was added to
a solution of compound 1 (30 mg, 0.094 mmol) and pyridine
(11 μL, 0.14 mmol) in dichloromethane (2 mL) at room
temperature and the resulting mixture was stirred at the same
temperature for 15 h. The reaction mixture was partitioned
between dichloromethane and water, and the organic layer
was dried over magnesium sulfate. The solvent was evapo-
rated in vacuo, and the residue was separated by chromatog-
raphy on a silica gel column (n-hexane: ethyl acetate = 4: 1)
to give 1-(5-acetyl-2,4-dichlorophenyl)-5-benzenesulfona-
1
1) to give the intermediate 11b (1.25 g, 63%). H NMR
(500 MHz, DMSO‑d6): δ 8.28 (s, 1H), 8.12 (s, 1H), 8.06
(dd, J = 7.9, 1.4 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.71 (t,
J = 7.9 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 6.95 (dd,
J = 8.9, 2.1 Hz, 1H), 6.89 (s, 1H), 5.08 (s, 2H), 3.92 (s,
3H); MS (ESI): m/z for C15H13N3O2, found 268 [M + H+].
Representative Synthetic Procedure for 5-((tert-Butoxy-
carbonyl)amino)-1-(2,4-Dichloro-5-Methoxycarbonylphe-
nyl)-1H-Indazole (12a) and 5-((tert-Butoxycarbonyl)
amino)-1-(3-Methoxycarbonylphenyl)-1H-Indazole (12b).
To a solution of compound 11a (100 mg, 0.300 mmol) in
DMF (1 mL) at 0 ꢀC was added DIPEA (260 μL,
1.49 mmol) and Boc2O (190 mg, 0.892 mmol). The mix-
ture was stirred at 0 ꢀC for 2 h and the reaction temperature
was elevated to room temperature. In addition, the mixture
was stirred at room temperature overnight. The reaction
mixture was partitioned between dichloromethane and
1
mido-1H-indazole (TRT-0029, 2, 25 mg, 58%). H NMR
(500 MHz, CDCl3): δ 8.18 (s, 1H), 7.76 (t, J = 7.5 Hz, 3H),
7.72 (s, 1H), 7.57–7.54 (m, 2H), 7.46 (t, J = 8.00 Hz, 2H),
7.13 (d, J = 1.5 Hz, 2H), 6.69 (s, 1H), 2.70 (s, 3H); MS
(ESI): m/z for C21H15Cl2N3O3S, found 460 [M + H+].
1-(5-Acetyl-2,4-dichlorophenyl)-5-(4-methoxybenzenesulfo-
1
namido)-1H-indazole (10a). H NMR (500 MHz, CDCl3): δ
8.19 (s, 1H), 8.05 (s, 1H), 7.75 (s, 1H), 7.69 (d, J = 8.9 Hz,
2H), 7.53 (s, 1H), 7.15 (m, 2H), 6.91 (d, J = 8.9 Hz, 2H),
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MKK7–TIPRL Interaction Inhibitors as TRAIL Sensitizers
KOREAN CHEMICAL SOCIETY
water, and the organic layer was washed several times with
water and dried over magnesium sulfate. The solvent was
evaporated in vacuo and the residue was separated by chro-
matography on a silica gel column (n-hexane: ethyl ace-
5-((tert-Butoxycarbonyl)amino)-1-(3-(benzylcarbamoyl)-
4,6-dichlorophenyl)-1H-indazole (14b). NMR
1H
(300 MHz, CDCl3): δ 8.15 (s, 1H), 7.94 (s, 1H), 7.85
(s, 1H), 7.67 (s, 1H), 7.35 (m, 5H), 7.31 (m, 1H), 7.15
(d, J = 9.1 Hz, 1H), 6.55 (s, 1H), 6.47 (s, 1H), 4.65 (d, J =
5.6 Hz, 2H), 1.54 (s, 9H); MS (ESI): m/z for
C26H24Cl2N4O3, found 511 [M + H+].
1
tate = 4: 1) to give the intermediate 12a (93 mg, 72%). H
NMR (300 MHz, CDCl3): δ 8.17 (s, 1H), 8.05 (s, 1H),
7.94 (s, 1H), 7.73 (s, 1H), 7.31 (d, J = 8.9 Hz, 1H), 7.13
(d, J = 8.9 Hz, 1H), 6.55 (br s, 1H), 3.92 (s, 3H), 1.54
(s, 9H); MS (ESI): m/z for C20H19Cl2N3O4, found
436 [M + H+].
5-((tert-Butoxycarbonyl)amino)-1-(2,4-dichloro-5-(phe-
nethylcarbamoyl)phenyl)-1H-indazole (14c). 1H NMR
(300 MHz, CDCl3): δ 8.14 (s, 1H), 7.94 (br s, 1H), 7.75 (s,
1H), 7.63 (s, 1H), 7.33–7.21 (m, 6H), 7.14 (d, J = 8.9 Hz,
1H), 6.56 (br s, 1H), 6.22 (br t, J = 6.8 Hz, 1H), 3.74 (q,
J = 6.8 Hz, 2H), 2.95 (t, J = 6.8 Hz, 2H), 1.54 (s, 9H); MS
(ESI): m/z for C27H26Cl2N4O3, found 525 [M + H+].
Representative Synthetic Procedure for 5-Amino-
1-(3-Carbamoylphenyl)-1H-Indazoles (15). A solution of
compound 14a (4.4 mg, 0.0088 mmol) in DCM/TFA
(0.5 mL/0.5 mL) at room temperature was stirred for 2 h
and the solvent was evaporated in vacuo to give 5-amino-
1-(2,4-dichloro-5-(phenylcarbamoyl)phenyl)-1H-indazole
trifluoroacetic acid salt (15a, 3.5 mg, 78%). 1H NMR
(300 MHz, DMSO‑d6): δ 10.65 (s, 1H), 8.43 (s, 1H), 8.14
(s, 1H), 7.91 (s, 1H), 7.69 (d, J = 7.7 Hz, 2H), 7.64 (s,
1H), 7.42–7.25 (m, 4H), 7.12 (m, 1H); MS (ESI): m/z for
C20H14Cl2N4O, found 397 [M + H+].
5-Amino-1-(3-(benzylcarbamoyl)-4,6-dichlorophenyl)-
1H-indazole trifluoroacetic acid salt (15b). 1H NMR
(300 MHz, DMSO‑d6): δ 9.15 (br t, J = 5.7 Hz, 1H), 8.40
(s, 1H), 8.06 (s, 1H), 7.73 (s, 1H), 7.58 (s, 1H), 7.34 (m,
5H), 7.25 (m, 2H), 4.45 (d, J = 5.7 Hz, 2H); MS (ESI): m/z
for C21H16Cl2N4O, found 411 [M + H+].
5-Amino-1-(2,4-dichloro-5-(phenethylcarbamoyl)phe-
nyl)-1H-indazole trifluoroacetic acid salt (15c). 1H NMR
(500 MHz, DMSO‑d6): δ 8.71 (t, J = 6.8 Hz, 1H), 8.41 (s,
1H), 8.04 (s, 1H), 7.58–7.55 (m, 1H), 7.56 (s, 1H), 7.35
(m, 1H), 7.25 (m, 5H), 7.17 (m, 1H), 3.44 (q, J = 6.8 Hz,
2H), 2.82 (t, J = 6.8 Hz, 2H); MS (ESI): m/z for
C22H18Cl2N4O, found 425 [M + H+].
5-((tert-Butoxycarbonyl)amino)-1-(3-methoxycarbonyl-
1
phenyl)-1H-indazole (12b). H NMR (300 MHz, CDCl3):
δ 8.40 (s, 1H), 8.14 (s, 1H), 8.01 (d, J = 7.8 Hz, 1H),
7.96–7.92 (m, 2H), 7.69 (d, J = 9.0 Hz, 1H), 7.61 (t, J =
7.9 Hz, 1H), 7.32 (dd, J = 9.0, 2.0 Hz, 1H), 6.59 (br s,
1H), 3.96 (s, 3H), 1.55 (s, 9H); MS (ESI): m/z for
C20H21N3O4, found 368 [M + H+].
Representative Synthetic Procedure for 5-((tert-Butoxy-
carbonyl)amino)-1-(3-Carboxy-4,6-Dichlorophenyl)-1H-
Indazole (13a) and 5-((tert-Butoxycarbonyl)amino)-
1-(3-Carboxyphenyl)-1H-Indazole (13b). A mixture of
compound 12a (160 mg, 0.380 mmol) and lithium hydrox-
ide monohydrate (24 mg, 0.57 mmol) in THF/MeOH
(2 mL/1 mL) was stirred at 50ꢀC for 2 h. After completion
of the reaction, the reaction mixture was cooled to room
temperature and acidified to pH 4 by addition of 1 N aque-
ous HCl solution. The precipitated solid was filtered,
washed with water, and dried to give the intermediate 13a
1
(140 mg, 90%). H NMR (300 MHz, DMSO‑d6): δ 9.43
(s, 1H), 8.34 (s, 1H), 8.06 (s, 1H), 8.02 (s, 1H), 7.94 (s,
1H), 7.47 (d, J = 8.7 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H),
1.50 (s, 9H); MS (ESI): m/z for C19H17Cl2N3O4, found
422 [M + H+].
5-((tert-Butoxycarbonyl)amino)-1-(3-carboxyphenyl)-
1H-indazole (13b). 1H NMR (500 MHz, DMSO‑d6): δ
9.48 (br s, 1H), 8.34 (s, 1H), 8.26 (s, 1H), 8.06–8.03 (m,
2H), 7.93 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 9.1 Hz, 1H),
7.72 (t, J = 7.9 Hz, 1H), 7.53 (dd, J = 9.1, 1.5 Hz, 1H),
1.50 (s, 9H); MS (ESI): m/z for C19H19N3O4, found
354 [M + H+].
Synthesis of Compounds 19
Representative Synthetic Procedure for 1-Aryl-
2-(2-Fluoro-5-Nitrobenzylidene)hydrazines (16)13. To a
Representative Synthetic Procedure for 5-((tert-Butoxy-
carbonyl)amino)-1-(3-Carbamoylphenyl)-1H-Indazoles
(14). To a solution of compound 13a (13 mg, 0.030 mmol)
and aniline (3 μL, 0.03 mmol) in DCM (1 mL) at room tem-
perature was added EDC (7 mg, 0.05 mmol). The mixture
was stirred at the same temperature overnight and the sol-
vent was evaporated in vacuo. The residue was separated by
chromatography on a silica gel column (n-hexane: ethyl ace-
tate = 3: 1) to give 5-((tert-butoxycarbonyl)amino)-1-(2,-
4-dichloro-5-(phenylcarbamoyl)phenyl)-1H-indazole (14a,
4.4 mg, 29%). 1H NMR (300 MHz, CDCl3): δ 8.15 (s, 1H),
7.99–7.85 (m, 3H), 7.72 (s, 1H), 7.62 (d, J = 7.7 Hz, 2H),
7.38 (m, 2H), 7.28 (s, 1H), 7.18 (m, 2H), 6.58 (s, 1H), 1.54
(s, 9H); MS (ESI): m/z for C25H22Cl2N4O3, found
497 [M + H+].
solution
of
2-fluoro-5-nitrobenzaldehyde
(50 mg,
0.27 mmol) in ethanol was added 4-fluorophenylhydrazine
(37 mg, 0.29 mmol) and toluenesulfonic acid monohydrate
(3.0 mg, 0.02 mmol), and the mixture was stirred at reflux
for 1 h. After cooling to room temperature, the precipitated
solid was collected by filtration to give 1-(2-fluoro-5-nitro-
benzylidene)-2-(4-fluorophenyl)hydrazine (16a, 65 mg,
1
87%). H NMR (300 MHz, CDCl3): δ 8.84 (dd, J = 6.2,
2.9 Hz, 1H), 8.12 (m, 1H), 7.86 (s, 1H), 7.23 (t,
J = 9.3 Hz, 1H), 7.08 (m, 4H); MS (ESI): m/z for
C13H9F2N3O2, found 278 [M + H+].
1-(4-Chlorophenyl)-2-(2-fluoro-5-nitrobenzylidene)hydra
zine (16b). 1H NMR (500 MHz, CDCl3):
δ 8.84
(dd, J = 6.2, 2.8 Hz, 1H), 8.17 (m, 1H), 7.90 (s, 1H), 7.31
(d, J = 8.7 Hz, 2H), 7.24 (t, J = 9.2 Hz, 1H), 7.12 (d, J =
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MKK7–TIPRL Interaction Inhibitors as TRAIL Sensitizers
KOREAN CHEMICAL SOCIETY
8.7 Hz, 2H); MS (ESI): m/z for C13H9ClFN3O2, found
294 [M + H+].
Synthetic Procedure for 1-(4-Fluoro-2-Methylphenyl)-
5-Nitro-1H-Indazole (17f). The mixture of 5-nitro-1H-
indazole (50 mg, 0.31 mmol), 4-fluoro-2-methylphenylboronic
acid (94 mg, 0.61 mmol), and cupric acetate (50 mg,
0.28 mmol) in pyridine/dichloromethane (0.1 mL/3 mL) was
stirred at room temperature overnight. The solvent was evapo-
rated in vacuo and the residue was chromatographed on a sil-
ica gel column (n-hexane: ethyl acetate = 4: 1) to give
1-(4-fluoro-2-methylphenyl)-5-nitro-1H-indazole (17f, 40 mg,
1-(2-Chlorophenyl)-2-(2-fluoro-5-nitrobenzylidene)hydra
zine (16c). 1H NMR (500 MHz, CDCl3): δ 8.91 (dd, J = 6.2,
2.9 Hz, 1H), 8.46 (br s, 1H), 8.19 (m, 1H), 8.05 (s, 1H),
7.70 (dd, J = 8.6, 1.4 Hz, 1H), 7.33 (m, 2H), 7.23 (t,
J = 9.2 Hz, 1H), 6.91 (dt, J = 7.8, 1.4 Hz, 1H); MS (ESI):
m/z for C13H9ClFN3O2, found 294 [M + H+].
1-(2,4-Difluorophenyl)-2-(2-fluoro-5-nitrobenzylidene)hy
drazine (16d). 1H NMR (300 MHz, CDCl3): δ 8.85
(dd, J = 6.2, 2.9 Hz, 1H), 8.16 (m, 1H), 7.97 (s, 1H), 7.59
(m, 1H), 7.22 (m, 1H), 6.96–6.82 (m, 2H); MS (ESI): m/z
for C13H8F3N3O2, found 296 [M + H+].
1
48%). H NMR (500 MHz, CDCl3): δ 8.84 (s, 1H), 8.44
(s, 1H), 8.30 (dd, J = 9.2, 2.1 Hz, 1H), 7.38 (m, 1H), 7.26
(m, 1H), 7.17 (m, 1H), 7.11 (m, 1H), 2.11 (s, 3H); MS (ESI):
m/z for C14H10FN3O2, found 272 [M + H+].
1-(2-Fluoro-5-nitrobenzylidene)-2-(2-methylphenyl)hydr
Representative Synthetic Procedure for 5-Amino-1-Aryl-
1H-Indazoles (18a–f)13. To a solution of compound 17a
(17 mg, 0.066 mmol) in dichloromethane (5 mL) was
added 10% palladium on carbon (10 mg) at room tempera-
ture and the mixture was shaken under hydrogen atmo-
sphere (60~70 psi) at the same temperature for 5 h. The
reaction mixture was filtered through Celite and the filtrate
was evaporated in vacuo to afford 5-amino-1-(4-fluorophe-
azine (16e). 1H NMR (300 MHz, CDCl3):
δ 8.90
(dd, J = 6.2, 2.9 Hz, 1H), 8.13 (m, 1H), 7.97 (s, 1H), 7.60
(d, J = 7.9 Hz, 1H), 7.26 (m, 1H), 7.19 (d, J = 9.2 Hz,
1H), 7.12 (d, J = 7.5 Hz, 1H), 6.90 (t, J = 7.5 Hz, 1H),
2.28 (s, 3H); MS (ESI): m/z for C14H12FN3O2, found
274 [M + H+].
1
Representative Synthetic Procedure for 1-Aryl-5-Nitro-
1H-Indazoles (17a–e)13. The mixture of compound 16a
(61 mg, 0.22 mmol) and potassium carbonate (134 mg,
0.97 mmol) in N,N-dimethylformamide (2 mL) was heated
at 100ꢀC for 15 h. After cooling to room temperature, the
reaction mixture was partitioned between ethyl acetate and
water. The organic layer was washed with water several
times and dried over magnesium sulfate. The solvent was
evaporated in vacuo and the residue was chromatographed
on a silica gel column (n-hexane: ethyl acetate = 1: 1) to
give 1-(4-fluorophenyl)-5-nitro-1H-indazole (17a, 17 mg,
nyl)-1H-indazole (18a, 14 mg, 93%). H NMR (300 MHz,
CDCl3): δ 8.03 (s, 1H), 7.74 (dd, J = 8.1, 5.2 Hz, 2H),
7.55 (d, J = 8.9 Hz, 1H), 7.38 (t, J = 8.7 Hz, 2H), 6.88
(t, J = 8.2 Hz, 2H), 5.00 (br s, 2H); MS (ESI): m/z for
C13H10FN3, found 228 [M + H+].
5-Amino-1-(4-chlorophenyl)-1H-indazole (18b). 1H
NMR (500 MHz, DMSO‑d6): δ 8.08 (s, 1H), 7.74 (dd, J =
8.8 Hz, 2H), 7.63 (d, J = 8.9 Hz, 1H), 7.60 (d, J = 8.8 Hz,
2H), 6.92 (dd, J = 8.9, 2.1 Hz, 1H), 6.87 (d, J = 2.1 Hz,
1H), 5.06 (br s, 2H); MS (ESI): m/z for C13H10ClN3, found
244 [M + H+].
1
30%). H NMR (300 MHz, CDCl3): δ 8.80 (d, J = 2.0 Hz,
5-Amino-1-(2-chlorophenyl)-1H-indazole
(18c). 1H
1H), 8.40 (s, 1H), 8.32 (dd, J = 9.2, 2.1 Hz, 1H), 7.68 (m,
3H), 7.30 (t, J = 6.1 Hz, 2H); MS (ESI): m/z for
C13H8FN3O2, found 258 [M + H+].
NMR (300 MHz, DMSO‑d6): δ 8.04 (s, 1H), 7.72 (m, 1H),
7.54 (m, 3H), 6.97 (d, J = 9.7 Hz, 1H), 6.83 (m, 2H), 4.95
(br s, 2H); MS (ESI): m/z for C13H10ClN3, found
244 [M + H+].
1
1-(4-Chlorophenyl)-5-nitro-1H-indazole (17b). H NMR
(300 MHz, CDCl3): δ 8.80 (d, J = 2.0 Hz, 1H), 8.41
(s, 1H), 8.33 (dd, J = 9.2, 2.0 Hz, 1H), 7.74 (d, J = 9.2 Hz,
1H), 7.67 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H);
MS (ESI): m/z for C13H8ClN3O2, found 274 [M + H+].
5-Amino-1-(2,4-difluorophenyl)-1H-indazole (18d). 1H
NMR (500 MHz, DMSO‑d6): δ 8.08 (s, 1H), 7.68 (m, 1H),
7.59 (m, 1H), 7.32 (m, 1H), 7.12 (m, 1H), 6.89–6.85 (m,
2H), 5.00 (br s, 2H); MS (ESI): m/z for C13H9F2N3, found
246 [M + H+].
1
1-(2-Chlorophenyl)-5-nitro-1H-indazole (17c). H NMR
(300 MHz, CDCl3): δ 8.81 (d, J = 1.5 Hz, 1H), 8.44
(s, 1H), 8.29 (dd, J = 9.2, 1.9 Hz, 1H), 7.65 (dd, J = 7.7,
2.2 Hz, 1H), 7.57–7.42 (m, 3H), 7.30 (d, J = 9.3 Hz, 1H);
MS (ESI): m/z for C13H8ClN3O2, found 274 [M + H+].
1-(2,4-Difluorophenyl)-5-nitro-1H-indazole (17d). 1H
NMR (300 MHz, CDCl3): δ 8.80 (d, J = 2.0 Hz, 1H), 8.44
(s, 1H), 8.33 (dd, J = 9.2, 2.1 Hz, 1H), 7.68–7.59 (m, 1H),
7.40 (dd, J = 9.2, 3.1 Hz, 1H), 7.16–7.09 (m, 2H); MS
(ESI): m/z for C13H7F2N3O2, found 276 [M + H+].
5-Amino-1-(2-methylphenyl)-1H-indazole (18e). 1H
NMR (300 MHz, DMSO‑d6): δ 7.99 (s, 1H), 7.45–7.34
(m, 4H), 6.96 (d, J = 8.5 Hz, 1H), 6.84 (s, 1H), 6.82 (m,
1H), 4.93 (br s, 1H), 2.06 (s, 3H); MS (ESI): m/z for
C14H13N3, found 224 [M + H+].
5-Amino-1-(4-fluoro-2-methylphenyl)-1H-indazole (18f).
1H NMR (500 MHz, DMSO‑d6): δ 8.00 (s, 1H), 7.40 (dd,
J = 8.7, 5,6 Hz, 1H), 7.33 (dd, J = 9.7, 2.8 Hz, 1H), 7.21
(m, 1H), 6.95 (d, J = 8.6 Hz, 1H), 6.85–6.82 (m, 2H), 4.94
(br s, 2H), 2.04 (s, 3H); MS (ESI): m/z for C14H12FN3,
found 242 [M + H+].
1
1-(2-Methylphenyl)-5-nitro-1H-indazole (17e). H NMR
(300 MHz, CDCl3): δ 8.81 (s, 1H), 8.41 (s, 1H), 8.26 (d,
J = 9.2 Hz, 1H), 7.44 (m, 2H), 7.38 (m, 2H), 7.27 (m, 1H),
2.11 (s, 3H); MS (ESI): m/z for C14H11N3O2, found
254 [M + H+].
Representative Synthetic Procedure for 5-Amino-1-Aryl-
1H-Indazoles (18 g–i). A mixture of 5-amino-1H-indazole
(50 mg, 0.38 mmol), 3-chloro-1-fluoro-4-iodobenzene
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MKK7–TIPRL Interaction Inhibitors as TRAIL Sensitizers
KOREAN CHEMICAL SOCIETY
(100 μL, 0.376 mmol), cuprous iodide (7 mg, 0.04 mmol),
trans-cyclohexane-1,2-diamine (25 μL, 0.19 mmol), and
tripotassium phosphate (140 mg, 0.676 mmol) in
1,4-dioxane (2 mL) was heated at 100ꢀC for 3 days. After
cooling to room temperature, the reaction mixture was par-
titioned between dichloromethane and water, and the
organic layer was dried over magnesium sulfate. The sol-
vent was evaporated in vacuo and the residue was separated
by chromatography on a silica gel column (n-hexane: ethyl
acetate = 1: 1) to give 5-amino-1-(2-chloro-4-fluorophe-
5-(2-Chlorobenzenesulfonamido)-1-(2,4-dichlorophenyl)-
1H-indazole (19c). H NMR (300 MHz, CDCl3): δ 8.13 (s,
1H), 7.94 (dd, J = 7.9, 1.4 Hz, 1H), 7.60–7.53 (m, 3H),
7.47 (m, 1H), 7.38 (s, 2H), 7.31 (m, 1H), 7.20 (m, 1H),
7.15 (s, 1H), 7.08 (d, J = 8.9 Hz, 1H); MS (ESI): m/z for
C19H12Cl3N3O2S, found 451 [M + H+].
1
1-(2,4-Dichlorophenyl)-5-(3-methylbenzenesulfonamido)
1
-1H-indazole (19d). H NMR (500 MHz, CDCl3): δ 8.18
(s, 1H), 7.63 (s, 1H), 7.58 (s, 1H), 7.53–7.48 (m, 2H),
7.46–7.43 (m, 2H), 7.37–7.33 (m, 2H), 7.13 (s, 2H), 6.61
(br s, 1H), 2.36 (s, 3H); MS (ESI): m/z for
C20H15Cl2N3O2S, found 432 [M + H+].
1
nyl)-1H-indazole (18 g, 10 mg, 10%). H NMR (500 MHz,
DMSO‑d6): δ 8.05 (s, 1H), 7.76 (dd, J = 8.7, 2.8 Hz, 1H),
7.63 (dd, J = 8.8, 5.7 Hz, 1H), 7.44 (m, 1H), 6.97 (d,
J = 9.6 Hz, 1H), 6.86–6.83 (m, 2H), 4.97 (br s, 2H); MS
(ESI): m/z for C13H9ClFN3, found 262 [M + H+].
1-(2,4-Dichlorophenyl)-5-(2-methylbenzenesulfonamido)
1
-1H-indazole (19e). H NMR (500 MHz, CDCl3): δ 8.14
(s, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.62 (s, 1H), 7.47 (m,
2H), 7.42 (s, 2H), 7.33 (m, 1H), 7.28 (m, 1H), 7.11 (s, 2H),
6.80 (s, 1H), 2.70 (s, 3H); MS (ESI): m/z for
C20H15Cl2N3O2S, found 432 [M + H+].
5-Amino-1-(2,4-dichlorophenyl)-1H-indazole (18h). 1H
NMR (300 MHz, DMSO‑d6): δ 8.06 (s, 1H), 7.92 (d,
J = 2.0 Hz, 1H), 7.61 (m, 2H), 7.01 (d, J = 9.3 Hz, 1H),
6.86 (m, 2H), 4.97 (br s, 2H); MS (ESI): m/z for
C13H9Cl2N3, found 278 [M + H+].
1-(2,4-Dichlorophenyl)-5-(2-trifluoromethylbenzenesul-
fonamido)-1H-indazole (19f). 1H NMR (500 MHz, CDCl3):
δ 8.16 (s, 1H), 7.96–7.92 (m, 2H), 7.68 (m, 1H), 7.63 (s,
1 h), 7.55 (m, 1H), 7.53 (s, 1H), 7.43 (s, 2H), 7.13–7.11
(m, 2H), 6.75 (br s, 1H); MS (ESI): m/z for
C20H12Cl2F3N3O2S, found 486 [M + H+].
5-Amino-1-phenyl-1H-indazole
(18i).
1H
NMR
(300 MHz, DMSO‑d6): δ 8.04 (s, 1H), 7.72 (d, J = 7.8 Hz,
2H), 7.63–7.54 (m, 3H), 7.33 (m, 1H), 6.90 (d, J = 9.1 Hz,
1H), 6.86 (s, 1H), 5.00 (br s, 2H); MS (ESI): m/z for
C13H11N3, found 210 [M + H+].
1-(2,4-Dichlorophenyl)-5-(2-thiophenesulfonamido)-1H-
1
indazole (19g). H NMR (500 MHz, CDCl3): δ 8.20 (s,
Representative Synthetic Procedure for 1-Aryl-5-Sulfona-
mido-1H-Indazoles (19)13. To a solution of compound 18a
(7 mg, 0.03 mmol) and pyridine (4 μL, 0.05 mmol) in
dichloromethane (2 mL) at room temperature was added
4-methoxybenzenesulfonyl chloride (8 mg, 0.04 mmol),
and the mixture was stirred at the same temperature for
15 h. The reaction mixture was partitioned between
dichloromethane and water. The organic layer was dried
over magnesium sulfate. The solvent was evaporated in
vacuo and the residue was chromatographed on a silica gel
column (n-hexane: ethyl acetate = 4: 1) to give
1-(4-fluorophenyl)-5-(4-methoxybenzenesulfonamido)-1H-i
1H), 7.64 (s, 1H), 7.60–7.56 (m, 2H), 7.48–7.44 (m, 3H),
7.20–7.17 (m, 2H), 7.04–7.02 (m, 1H), 6.77 (br s, 1H); MS
(ESI): m/z for C17H11Cl2N3O2S2, found 424 [M + H+].
5-Benzenesulfonamido-1-(2,4-difluorophenyl)-1H-
1
indazole (19h). H NMR (300 MHz, CDCl3): δ 8.15 (s,
1H), 7.73 (m, 2H), 7.58–7.41 (m, 5H), 7.20–7.15 (m, 1H),
7.15–7.03 (m, 3H), 6.61 (br s, 1H); MS (ESI): m/z for
C19H13F2N3O2S, found 386 [M + H+].
1-(2,4-Difluorophenyl)-5-(4-methoxybenzenesulfona-
1
mido)-1H-indazole (19i). H NMR (300 MHz, CDCl3): δ
8.15 (s, 1H), 7.66 (d, J = 8.9 Hz, 2H), 7.55 (m, 1H), 7.49
(s, 1H), 7.26–7.19 (m, 1H), 7.15–7.03 (m, 3H), 6.88 (d, J =
8.9 Hz, 2H), 6.49 (br s, 1H), 3.82 (s, 3H); MS (ESI): m/z
for C20H15F2N3O3S, found 416 [M + H+].
1
ndazole (TRT-0173, 3, 8 mg, 70%). H NMR (500 MHz,
CDCl3): δ 8.13 (s, 1H), 7.69–7.64 (m, 4H), 7.61 (d,
J = 9.0 Hz, 1H), 7.50 (d, J = 1.8 Hz, 1H), 7.25 (t,
J = 8.6 Hz, 2H), 7.17 (dd, J = 9.0, 2.0 Hz, 1H), 6.91 (dd,
J = 7.2, 1.7 Hz, 2H), 6.55 (s, 1H), 3.85 (s, 1H); MS (ESI):
m/z for C20H16FN3O3S, found 398 [M + H+].
1-(2,4-Difluorophenyl)-5-(4-fluorobenzenesulfonamido)-
1
1H-indazole (19j). H NMR (300 MHz, CDCl3): δ 8.16
(s, 1H), 7.74 (m, 2H), 7.61–7.52 (m, 1H), 7.51 (s, 1H),
7.23–7.20 (m, 1H), 7.14–7.03 (m, 5H), 6.60 (br s, 1H);
MS (ESI): m/z for C19H12F3N3O2S, found 404 [M + H+].
1-(2,4-Difluorophenyl)-5-(3-fluorobenzenesulfonamido)-
1-(2,4-Dichlorophenyl)-5-(4-methoxybenzenesulfonamid
1
o)-1H-indazole (19a). H NMR (300 MHz, CDCl3): δ 8.15
1
(s, 1H), 7.67 (d, J = 8.9 Hz, 2H), 7.61 (s, 1H), 7.49 (s,
1H), 7.42 (s, 2H), 7.11 (s, 2H), 6.88 (d, J = 8.9 Hz, 2H),
6.62 (s, 1H), 3.83 (s, 3H); MS (ESI): m/z for
C20H15Cl2N3O3S, found 448 [M + H+].
1H-indazole (19k). H NMR (300 MHz, CDCl3): δ 8.17 (s,
1H), 7.62–7.38 (m, 5H), 7.30–7.20 (m, 2H), 7.13–7.03 (m,
3H), 6.61 (br s, 1H); MS (ESI): m/z for C19H12F3N3O2S,
found 404 [M + H+].
1-(2,4-Dichlorophenyl)-5-(2-fluorobenzenesulfonamido)-
1H-indazole (19b). H NMR (500 MHz, CDCl3): δ 8.16 (s,
1-(2,4-Difluorophenyl)-5-(2-fluorobenzenesulfona-
mido)-1H-indazole (19l). H NMR (300 MHz, CDCl3):
1
1
1H), 7.79 (m, 1H), 7.63–7.55 (m, 3H), 7.42 (s, 2H),
7.25–7.19 (m, 3H), 7.12 (d, J = 8.9 Hz, 1H), 6.90 (br s,
1H); MS (ESI): m/z for C19H12Cl2FN3O2S, found
436 [M + H+].
δ 8.13 (s, 1H), 7.77 (t, J = 7.7 Hz, 1H), 7.56–7.48
(m, 3H), 7.25–7.15 (m, 4H), 7.08–7.00 (m, 2H), 6.89
(br s, 1H); MS (ESI): m/z for C19H12F3N3O2S, found
404 [M + H+].
Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
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BULLETIN OF THE
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MKK7–TIPRL Interaction Inhibitors as TRAIL Sensitizers
KOREAN CHEMICAL SOCIETY
5-(4-Chlorobenzenesulfonamido)-1-(2,4-difluorophenyl)-
1H-indazole (19m). H NMR (300 MHz, CDCl3): δ 8.17
(s, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.66–7.51 (m, 1H), 7.51
(s, 1H), 7.41 (d, J = 8.6 Hz, 2H), 7.26–7.20 (m, 1H),
7.18–7.02 (m, 3H), 6.62 (br s, 1H); MS (ESI): m/z for
C19H12ClF2N3O2S, found 420 [M + H+].
8.15 (s, 1H), 8.03 (s, 1H), 7.85 (m, 2H), 7.65 (m, 2H),
7.61–7.57 (m, 2H), 7.53 (s, 1H), 7.28–7.23 (m, 2H), 7.14
(m, 1H), 6.63 (br s, 1H); MS (ESI): m/z for
C20H13F4N3O2S, found 436 [M + H+].
1
5-(4-Cyanobenzenesulfonamido)-1-(4-fluorophenyl)-1H-
1
indazole (19w). H NMR (500 MHz, CDCl3): δ 8.16 (s,
5-(2-Chlorobenzenesulfonamido)-1-(2,4-difluorophenyl)-
1H), 7.85 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H),
7.68–7.64 (m, 2H), 7.58 (d, J = 9.0 Hz, 1H), 7.55 (s, 1H),
7.28–7.24 (m, 2H), 7.12 (d, J = 8.9 Hz, 1H), 6.96 (br s,
1H); MS (ESI): m/z for C20H13FN4O2S, found
393 [M + H+].
1
1H-indazole (19n). H NMR (300 MHz, CDCl3): δ 8.13 (s,
1H), 7.93 (dd, J = 7.9, 1,4 Hz, 1H), 7.56–7.43 (m, 4H),
7.29–7.25 (m, 1H), 7.20 (m, 2H), 7.12 (br s, 1H),
7.08–7.00 (m, 2H); MS (ESI): m/z for C19H12ClF2N3O2S,
found 420 [M + H+].
1-(4-Fluorophenyl)-5-(2-thiophenesulfonamido)-1H-
1
1-(2,4-Difluorophenyl)-5-(4-methylbenzenesulfonamido)-
indazole (19x). H NMR (500 MHz, CDCl3): δ 8.16 (s,
1
1H-indazole (19o). H NMR (300 MHz, CDCl3): δ 8.14 (s,
1H), 7.69–7.65 (m, 2H), 7.61–7.56 (m, 3H), 7.46 (m, 1H),
7.28–7.20 (m, 3H), 7.02 (m, 1H), 6.63 (br s, 1H); MS
(ESI): m/z for C17H12FN3O2S2, found 374 [M + H+].
5-(4-Methoxybenzenesulfonamido)-1-(2-methylphenyl)-
1H), 7.62 (d, J = 8.3 Hz, 2H), 7.59–7.49 (m, 1H), 7.49 (s,
1H), 7.22 (d, J = 8.3 Hz, 2H), 7.21–7.18 (m, 1H),
7.18–7.02 (m, 3H), 6.64 (br s, 1H), 2.38 (s, 3H); MS (ESI):
m/z for C20H15F2N3O2S, found 400 [M + H+].
1
1H-indazole (19y). H NMR (300 MHz, CDCl3): δ 8.11 (s,
1-(2-Chloro-4-fluorophenyl)-5-(4-methoxybenzenesulfo-
1H), 7.67 (d, J = 8.9 Hz, 2H), 7.48 (s, 1H), 7.39–7.32 (m,
4H), 7.08 (m, 2H), 6.88 (d, J = 8.9 Hz, 2H), 6.66 (s, 1H),
3.83 (s, 3H), 2.09 (s, 3H); MS (ESI): m/z for C21H19N3O3S,
found 394 [M + H+].
1
namido)-1H-indazole (19p). H NMR (300 MHz, CDCl3):
δ 8.14 (s, 1H), 7.66 (d, J = 8.9 Hz, 2H), 7.50–7.44 (m,
2H), 7.34 (m, 1H), 7.16 (m, 1H), 7.11 (m, 2H), 6.88 (d,
J = 8.9 Hz, 2H), 6.58 (s, 1H), 3.83 (s, 3H); MS (ESI): m/z
for C20H15ClFN3O3S, found 432 [M + H+].
5-Benzenesulfonamido-1-phenyl-1H-indazole (19z). 1H
NMR (300 MHz, CDCl3): δ 8.11 (s, 1H), 7.74–7.60 (m,
5H), 7.57–7.34 (m, 7H), 7.12 (d, J = 8.9 Hz, 1H), 6.48
(br s, 1H); MS (ESI): m/z for C19H15N3O2S, found
350 [M + H+].
1-(4-Chlorophenyl)-5-(4-methoxybenzenesulfonamido)-
1
1H-indazole (19q). H NMR (500 MHz, CDCl3): δ 8.14 (s,
1H), 7.70–7.68 (m, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.59 (d,
J = 9.0 Hz, 1H), 7.53–7.50 (m, 3H), 7.19 (m, 1H), 6.90 (d,
J = 8.9 Hz, 2H), 3.84 (s, 3H); MS (ESI): m/z for
C20H16ClN3O3S, found 414 [M + H+].
5-(4-Methoxybenzenesulfonamido)-1-phenyl-1H-
1
indazole (19aa). H NMR (500 MHz, CDCl3): δ 8.14 (s,
1H), 7.72–7.63 (m, 5H), 7.52 (m, 1H), 7.51 (s, 1H), 7.40
(t, J = 7.6 Hz, 1H), 7.15 (dd, J = 9.0, 2.0 Hz, 1H), 6.91
(dd, J = 7.0, 1.9 Hz, 2H), 6.48 (s, 1H), 3.85 (s, 3H); MS
(ESI): m/z for C20H17N3O3S, found 380 [M + H+].
1-(2-Chlorophenyl)-5-(4-methoxybenzenesulfonamido)-
1
1H-indazole (19r). H NMR (300 MHz, CDCl3): δ 8.15 (s,
1H), 7.67 (d, J = 8.9 Hz, 2H), 7.61–7.57 (m, 1H),
7.51–7.46 (m, 2H), 7.46–7.41 (m, 2H), 7.11 (s, 2H), 6.88
(d, J = 8.9 Hz, 2H), 6.75 (br s, 1H), 3.82 (s, 3H); MS
(ESI): m/z for C20H16ClN3O3S, found 414 [M + H+].
5-(4-Fluorobenzenesulfonamido)-1-(4-fluorophenyl)-1H-
indazole (19s). 1H NMR (300 MHz, CDCl3): δ 8.12 (s,
1H), 7.76–7.70 (m, 2H), 7.66–7.60 (m, 2H), 7.55 (d,
J = 8.9 Hz, 1H), 7.50 (s, 1H), 7.23–7.19 (m, 2H),
7.14–7.07 (m, 3H), 6.51 (br s, 1H); MS (ESI): m/z for
C19H13F2N3O2S, found 386 [M + H+].
Cell Viability Assay. Cell viability was assessed using a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT, Sigma-Aldrich, St. Louis, MO, USA) assay.
The Huh7 cells were seeded at a density of 1 × 104 cells
per well and treated with the compounds and/or TRAIL.
After 24 or 48 h, MTT (2 mg/mL) was added to each well,
and the absorbance was measured using a microplate reader
(Bio-Rad, Hercules, CA, USA) at 570 nm. The cell viabil-
ity was calculated as the percentage of viable cells in the
drug-treated group versus the untreated control using the
following equation: cell viability (%) = (OD [drug] − OD
[blank])/(OD [control] − OD [blank]) × 100. The cell via-
bility difference between the compound/TRAIL combina-
tion treatment and the treatment of only the compound is
considered as the TRAIL sensitizing activity of the com-
pound. The term working concentration is defined as the
minimum concentration of a compound exhibiting the larg-
est TRAIL sensitizing activity.
1-(4-Fluorophenyl)-5-(4-methylbenzenesulfonamido)-
1
1H-indazole (19t). H NMR (500 MHz, CDCl3): δ 8.12 (s,
1H), 7.67–7.62 (m, 4H), 7.56 (d, J = 8.9 Hz, 1H), 7.51 (s,
1H), 7.27–7.23 (m, 4H), 7.19–7.15 (m, 1H), 6.59 (br s,
1H), 2.40 (s, 3H); MS (ESI): m/z for C20H16FN3O2S, found
382 [M + H+].
1-(4-Fluorophenyl)-5-(3-methylbenzenesulfonamido)-
1
1H-indazole (19u). H NMR (500 MHz, CDCl3): δ 8.13 (s,
1H), 7.65 (m, 2H), 7.61 (s, 1H), 7.58–7.51 (m, 3H),
7.37–7.32 (m, 2H), 7.25 (m, 2H), 7.17 (d, J = 9.0 Hz, 1H),
6.62 (br s, 1H), 2.38 (s, 1H); MS (ESI): m/z for
C20H16FN3O2S, found 382 [M + H+].
Liver Microsomal Stability Assay. Liver microsomal sta-
bility was determined in human, rat, and mouse liver micro-
somes. The compound (1 μM) was mixed with human, rat,
or mouse liver microsomes (0.5 mg/mL; BD Biosciences
Gentest, CA) in 100 mM potassium phosphate buffer
1-(4-Fluorophenyl)-5-(3-trifluoromethylbenzenesulfona-
1
mido)-1H-indazole (19v). H NMR (500 MHz, CDCl3): δ
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(pH 7.4) and incubated at 37ꢀC for 5 min. The reaction was
initiated by NADPH regeneration solution (BD Biosciences)
and terminated by three times volume of ice-cold acetonitrile
with imipramine (80 ng/mL) as internal standard at single-
time-point 30 min. After pre-treatment of biological samples
with vortex and centrifuge, the samples were analyzed by
LC/MS/MS system.
derivatization reactions of TRT-0002 (1) are shown in
Figure 2. The TRAIL-sensitizing activity of the derivatives
was assessed based on the cell viability using an MTT assay
in Huh7 cells. The results for compounds exhibiting a cell
viability difference greater than 25% between the compound/
TRAIL combination treatment and the treatment of only the
compound, together with the working concentration and treat-
ment time are shown in Table 1. The effective working con-
centration was determined based on the responses of Huh7
cells treated with various doses in the presence and absence
of TRAIL. The data of the TRT-0002 compound (1) are
listed for reference (a cell viability difference of 52% at a
40-μM working concentration).13 The synthetic procedures
for the compounds listed in Table 1 are described in Experi-
mental section. In addition, the representative procedures for
the synthesis of derivatives 8 and 9 are described in Experi-
mental section. Among the synthesized alkylamino (5) and
dialkylamino (6) derivatives, 5-isobutylamino (5a) and
5-dimethylamino (6a) analogues satisfied the criterion, with
cell viability differences of 26% and 39%, respectively. The
compound with only the 3-(1H-indol-3-yl)propanoylamino
group (7a) results in a cell viability difference of more than
25% in the case of 5-amido derivatives 7. The carbamate (8)
and ureido (9) analogues did not meet the criterion of the cell
viability difference. Noticeably, 5-benzenesulfonamido deriva-
tive 2 (TRT-0029) showed a 58% cell viability difference at
a lower working concentration of 10 μM.13 In addition, in
Results and Discussion
The hit compound TRT-0002 (1) exhibited low metabolic
stability in the liver microsomal stability test (0.54%, 0.54%,
and 26.8% remaining after 30 min for the mouse, rat, and
human
specimens,
respectively).
Firstly,
the
1,4-diaminobenzene moiety in the TRT-0002 compound was
expected to be vulnerable to oxidative metabolism. Hence, as
an initial trial for overcoming the metabolic instability and
exploring the chemical space beyond the 5-amino group, we
attempted to transform the amino group in the 5 position in
TRT-0002 into various amino-related functionalities such as
alkylamino, dialkylamino, amido, carbamate, ureido, and sul-
fonamido groups. The derivatives of TRT-0002 were synthe-
sized as shown in Scheme 1. The precursor TRT-0002 (1)
was prepared via the reaction of 5-amino-1H-indazole and
20,40-dichloro-50-fluoroacetophenone under basic conditions.13
The structure of TRT-0002 was discriminated from its 2-aryl-
2H-indazole regioisomer based on HMBC and NOE experi-
ments as described in the previous report.13 The alkylation of
TRT-0002 with alkyl halides resulted in the formation of
mono-alkyl (5) and/or bis-alkyl (6) derivatives depending on
the amount of alkyl halides and the reaction temperature. The
acylation of TRT-0002 with acid chlorides or acids afforded
5-amido derivatives 7. Carbamate (8), ureido (9), and sulfo-
namido (10)13 derivatives were obtained from the reactions
with chloroformates, isocyanates, and sulfonyl chlorides in an
analogous manner. The building blocks used in the
the
case
of
5-sulfonamido
derivatives
10,
5-(4-methoxybenzenesulfonamido)
(10a)
and
Figure 2. Building blocks used in derivatization reactions of
TRT-0002 (1).
Scheme 1. Derivatization reactions of TRT-0002 (1).
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Table 1. Results of cell viability assay for derivatives 5, 6, 7, 8, 9, and 10.
Compound
Working concentration (μM) Treatment time (h) Compound only (%)a Compound + TRAIL (%)a Difference (%)
40
48
81
29
52
TRT-0002 (1)
40
40
24
24
54
62
28
23
26
39
(5a)
(6a)
(7a)
40
10
24
24
61
69
25
11
36
58
TRT-0029 (2)
10
20
48
24
59
51
7
52
28
(10a)
23
(10b)
a
Cell viability assays were performed in duplicate or triplicate. Errors for the individual values were found to be 0.5 ~ 9.6%.
5-phenylmethanesulfonamido (10b) analogues displayed a
52% and 28% cell viability difference at 10 μM and 20 μM,
respectively. In particular, compound TRT-0029 (2) caused
TRAIL-induced apoptosis by activating MKK7/JNK via the
inhibition of the MKK7–TIPRL interaction, as described in
our previous report.13 Further, the TRT-0029 compound
exhibited a slightly improved metabolic stability in the liver
microsomal stability test (5.4%, 2.1%, and 42.6% remaining
after 30 min for the mouse, rat, and human specimens,
respectively) compared with that of the TRT-0002 com-
pound (1).
Next, we focused on replacing the acetyl group on the
1-aryl moiety in TRT-0002 (1) with a carbamoyl group, as
represented by the target compounds 15 in Scheme 2,
because we thought that the acetyl group is potentially lia-
ble to reductive metabolism and we intended to explore the
chemical space near it. The intermediate 5-amino-1-(2,-
indazole and methyl 2,4-dichloro-5-fluorobenzoate under
basic conditions. The dechlorinated analogue 11b (X = H)
was prepared by a copper-catalyzed reaction14 involving
5-amino-1H-indazole and methyl 3-iodobenzoate. Protec-
tion of the 5-amino functionality in compounds 11 with the
Boc group and subsequent hydrolysis of the ester group in
compounds 12 afforded the carboxylic acid intermediates
13. Amide coupling reactions of intermediates 13 with vari-
ous primary and secondary amine building blocks listed in
Figure 3 provided Boc-protected carbamoyl derivatives 14,
which were deprotected to give the final target products 15.
The TRAIL-sensitizing activities of the analogues are
shown in Table 2 for compounds exhibiting a cell viability
difference of more than 25% between the compound/
TRAIL combination treatment and the compound alone
treatment; the working concentrations and treatment times
are also shown. Unfortunately, only three compounds ful-
filled the cell viability difference requirement. Further they
exhibited less potent TRAIL-sensitizing activities (of 33%,
4-dichloro-5-methoxycarbonylphenyl)-1H-indazole
(11a,
X = Cl) was synthesized from the reaction of 5-amino-1H-
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25%, and 29% for 15a, 15b, and 15c, respectively, at a
working concentration of 40 μM) compared with those of
compounds TRT-0002 (1) and TRT-0029 (2) (52% and
58% at working concentrations of 40 and 10 μM, respec-
tively). The synthetic procedures for the compounds listed
in Table 2 are described in Experimental section.
On the basis of the above-mentioned results, we specu-
lated that the replacement of the 5-amino group in TRT-
0002 compound 1 by a 5-sulfonamido group and the intro-
duction of an aryl moiety without the acetyl group at the
1 position on the indazole ring system, as represented by the
general structure 19 in Scheme 3, would give compounds
with superior TRAIL-sensitizing activity and metabolic sta-
bility. The precursors 18 were prepared through three differ-
ent routes. In one approach, the condensation reaction of
respectively. In the case of the 1-(4-fluorophenyl)-
1H-indazole compounds 3 (TRT-0173) and 19s–x,
analogues with 4-methoxybenzenesulfonamido (3, TRT-
0173),
3-methylbenzenesulfonamido
(19u),
and
3-trifluoromethylbenzenesulfonamido (19v) groups at the
5 position on the indazole ring system displayed clearly
high cell viability differences of 80%,13 86%, and 83% at
a working concentration of 20 μM, respectively. These
compounds (3, 19u and 19v) also exhibited significant
TRAIL-sensitizing activities (cell viability differences of
31%, 41%, and 40%, respectively) even at 10 μM con-
centration of the compounds. In addition, the
5-(4-cyanobenzenesulfonamido) derivative (19w) showed
a 40% TRAIL-sensitizing activity at a working concentra-
tion of 20 μM. Finally, compounds 19y (5-(4-methoxy-
benzenesulfonamido)-1-(2-methylphenyl)-1H-indazole),
19z (5-benzenesulfonamido-1-phenyl-1H-indazole), and
19aa (5-(4-methoxybenzenesulfonamido)-1-phenyl-1H-
indazole) exhibited 44%, 29%, and 37% TRAIL-
sensitizing activities at a working concentration of
20 μM, respectively. Through MOA studies, it was real-
ized that TRT-0173 (3) enhances TRAIL-induced apopto-
sis in Huh7 cells by inhibiting MKK7–TIPRL interactions
and activating MKK7/JNK.13 Although the TRT-0173
compound operated at a higher working concentration
(20 μM) than that of TRT-0029 (10 μM), TRT-0173 dem-
onstrated an improved metabolic stability in liver micro-
somal stability tests (mouse, 32.1%; rat, 34.6%; and
human, 66.4% remaining after 30 min) compared with
those of TRT-0002 (1) and TRT-0029 (2). The metabolic
stability data for compounds 19u and 19v were 6.2% and
34.6% for mouse, <1% and 0.42% for rat, and 36.7% and
65.3% for human (remaining after 30 min), respectively.
2-fluoro-5-nitrobenzaldehyde
with
arylhydrazines
(Ar = 4-Cl-C6H4, 2-Cl-C6H4, 4-F-C6H4, 2,4-di-F-C6H3,
2-Me-C6H4) in the presence of toluenesulfonic acid resulted
in the formation of the hydrazones 16a–e, which were then
cyclized in the presence of potassium carbonate to give
1-aryl-5-nitro-1H-indazoles (17a–e).13 In another approach,
compound 17f (Ar = 4-F-2-Me-C6H3) was obtained from
the copper-catalyzed reaction15 of 5-nitro-1H-indazole with
4-fluoro-2-methylphenylboronic acid. The nitro reduction of
compounds 17a–f yielded the intermediates 18a–f.13 The
5-amino-1-aryl-1H-indazole compounds (18g–i) were also
synthesized directly from copper-catalyzed reactions14 of
5-amino-1H-indazole with aryl iodides (Ar = 2,4-di-Cl-
C6H3, 2-Cl-4-F-C6H3, C6H5). The precursors 18 were sulfo-
nylated under typical conditions to give 1-aryl-5-sulfona-
mido-1H-indazole compounds 19.13 The sulfonyl chloride
building blocks used in the derivatization reactions are shown
in Figure 4. The TRAIL-sensitizing activities of the analogues
19 are shown in Table 3 for compounds exhibiting a cell via-
bility difference of more than 25% between the compound/
TRAIL combination treatment and the compound alone treat-
ment, together with their corresponding working concentra-
tions and treatment times. The synthetic procedures for the
compounds listed in Table 3 are described in Experimental sec-
tion. In the case of 1-(2,4-dichlorophenyl) derivatives 19a–g,
Conclusion
We attempted to convert a metabolically unstable TRT-
0002 compound exhibiting TRAIL-sensitizing activity for
Huh7 cells at a high working concentration (40 μM) to
more potent and metabolically improved analogues by
modifying the 5-amino group and the 1-aryl moiety in the
1H-indazole skeleton. The first trial that varied the 5-amino
group to obtain amino-related functionalities such as alkyla-
mino, dialkylamino, amido, carbamate, ureido, and sulfona-
5-(2-fluorobenzenesulfonamido)
(19b)
and
5-(3-methylbenzenesulfonamido) (19d) analogues showed
relatively good TRAIL-sensitizing activities (of 51% and
40% at a working concentration of 20 μM, respectively).
Among the 1-(2,4-difluorophenyl)-1H-indazole compounds
19h–o, derivatives with 5-(4-methoxybenzenesulfonamido)
mido groups resulted in the identification of
a
5-sulfonamido derivative TRT-0029 compound working at
a lower concentration (10 μM) with a slightly improved
metabolic stability. Next, we replaced the acetyl group in
the 1-aryl group of the TRT-0002 compound with various
carbamoyl groups. The effort was fruitless and no ana-
logues with improved potency were obtained. Subse-
quently, we focused on the synthesis of 5-sulfonamido
derivatives having a 1-aryl moiety without an acetyl group.
The resulting TRT-0173 compound was found to exhibit
TRAIL-sensitizing activity at a working concentration
20 μM and improved metabolic stability compared with
(19i),
5-(2-fluorobenzenesulfonamido)
(19l),
and
5-(4-methylbenzenesulfonamido) (19o) groups exhibited
enhanced sensitivity for TRAIL (54%, 55%, and 50%
at a working concentration of 20 μM, respectively).
Compounds 19p (1-(2-chloro-4-fluorophenyl)-5-(4-meth-
oxybenzenesulfonamido)-1H-indazole), 19q (1-(4-chlorophe-
nyl)-5-(4-methoxybenzenesulfonamido)-1H-indazole), and 19r
(1-(2-chlorophenyl)-5-(4-methoxybenzenesulfonamido)-
1H-indazole) exhibited 35%, 43%, and 54% cell viabil-
ity differences at a working concentration of 20 μM,
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Scheme 3. Synthesis of derivatives 19.
Scheme 2. Synthesis of derivatives 15.
Figure 4. Building blocks used in the synthesis of derivatives 19.
that of the TRT-0029 analogue. As reported previously,13
co-treatment of cultured Huh7 cells with either TRT-0029
or TRT-0173 and TRAIL resulted in TRAIL-induced apo-
ptosis owing to the inhibition of the MKK7–TIPRL interac-
tion and subsequent phosphorylation of MKK7 and JNK.
In addition, the injection of these two compounds sup-
pressed tumor growth in combination with TRAIL in an
in vivo HCC mouse xenograft model. With regard to the
physicochemical aspect, the TRT-0029 and TRT-0173
compounds seemed to be somewhat lipophilic based on
Figure 3. Building blocks used in the synthesis of derivatives 15.
Table 2. Results of cell viability assay for derivatives 15.
Compound
Working concentration (μM) Treatment time (h) Compound only (%)a Compound + TRAIL (%)a Difference (%)
40
24
80
47
33
(15a)
(15b)
40
40
48
48
56
48
31
19
25
29
(15c)
a
Cell viability assays were performed in duplicate or triplicate. Errors for the individual values were found to be 0.6 ~ 7.0%.
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Table 3. Results of cell viability assay for derivatives 19.
Compound
Working concentration (μM) Treatment time (h) Compound only (%)a Compound + TRAIL (%)a Difference (%)
20
24
37
10
27
(19a)
20
24
62
11
51
(19b)
20
24
68
37
31
(19c)
(19d)
20
20
24
24
48
43
8
40
31
12
(19e)
(19f)
(19g)
20
20
20
24
24
24
48
34
61
15
7
33
27
37
24
(19h)
(19i)
20
20
24
24
66
57
12
31
54
26
(19j)
20
20
24
24
50
62
11
7
39
55
(19k)
(19l)
20
24
32
3
29
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Table 3 (continued)
Compound
Working concentration (μM) Treatment time (h) Compound only (%)a Compound + TRAIL (%)a Difference (%)
(19m)
20
20
24
24
45
62
13
12
32
50
(19n)
(19o)
(19p)
20
20
24
24
63
47
28
4
35
43
(19q)
(19r)
20
20
24
24
66
92
12
12
54
80
TRT-0173 (3)
20
20
20
24
24
24
46
94
92
7
69
6
39
25
86
(19s)
(19t)
(19u)
(19v)
20
20
24
24
87
53
4
83
40
13
(19w)
20
24
68
41
27
(continued overleaf )
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Article
MKK7–TIPRL Interaction Inhibitors as TRAIL Sensitizers
KOREAN CHEMICAL SOCIETY
Table 3 (continued)
Compound
Working concentration (μM) Treatment time (h) Compound only (%)a Compound + TRAIL (%)a Difference (%)
(19x)
(19y)
20
20
24
24
63
61
19
32
44
29
(19z)
20
24
69
32
37
(19aa)
a
Cell viability assays were performed in duplicate or triplicate. Errors for the individual values were found to be 0.4 ~ 14.0%.
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their CLogP values (5.07 and 4.60, respectively), and their
solubilities were too low to be detected in an equilibrium
solubility test in water. Despite such limitations, it is
assumed that two compounds and the relevant structure–
activity relationship would provide an insight to enable fur-
ther study on optimization of potency and metabolic
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Acknowledgments. This research was possible thanks to
generous financial support from the Bio & Medical Technol-
ogy Development Program of the National Research Foun-
dation (NRF) funded by the Korean government (NRF-
2012M3A9C) and the Korea Research Institute of Chemical
Technology (KK1803-B00). This work was also supported
by the Basic Science Research Program of the NRF funded
by the Ministry of Education, Science and Technology
(NRF-2014M3A9A5034157) and the KRIBB Research Ini-
tiative Program. The TIPRL and MKK7 clones were pro-
vided by the Korea Human Gene Bank, KRIBB, Korea.
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Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
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