Discovery of Novel Benzimidazole and Indazole Analogues as Tubulin Polymerization Inhibitors with Potent Anticancer Activities

Article abstract of DOI:10.1021/acs.jmedchem.0c01837

Novel indazole and benzimidazole analogues were designed and synthesized as tubulin inhibitors with potent antiproliferative activities. Among them, compound 12b exhibited the strongest inhibitory effects on the growth of cancer cells with an average IC50 value of 50 nM, slightly better than colchicine. 12b exhibited nearly equal potency against both, a paclitaxel-resistant cancer cell line (A2780/T, IC50 = 9.7 nM) and the corresponding parental cell line (A2780S, IC50 = 6.2 nM), thus effectively overcoming paclitaxel resistance in vitro. The crystal structure of 12b in complex with tubulin was solved to 2.45 ? resolution by X-ray crystallography, and its direct binding was confirmed to the colchicine site. Furthermore, 12b displayed significant in vivo antitumor efficacy in a melanoma tumor model with tumor growth inhibition rates of 78.70% (15 mg/kg) and 84.32% (30 mg/kg). Collectively, this work shows that 12b is a promising lead compound deserving further investigation as a potential anticancer agent.

Full text of DOI:10.1021/acs.jmedchem.0c01837

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Article
Discovery of Novel Benzimidazole and Indazole Analogues as
Tubulin Polymerization Inhibitors with Potent Anticancer Activities
⊥
⊥
⊥
Yichang Ren, Yuxi Wang, Gang Li, Zherong Zhang, Lingling Ma, Binbin Cheng, and Jianjun Chen*
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sı Supporting Information
ABSTRACT: Novel indazole and benzimidazole analogues were designed and synthesized as tubulin inhibitors with potent
antiproliferative activities. Among them, compound 12b exhibited the strongest inhibitory effects on the growth of cancer cells with
an average IC value of 50 nM, slightly better than colchicine. 12b exhibited nearly equal potency against both, a paclitaxel-resistant
5
0
cancer cell line (A2780/T, IC50 = 9.7 nM) and the corresponding parental cell line (A2780S, IC50 = 6.2 nM), thus effectively
overcoming paclitaxel resistance in vitro. The crystal structure of 12b in complex with tubulin was solved to 2.45 Å resolution by X-
ray crystallography, and its direct binding was confirmed to the colchicine site. Furthermore, 12b displayed significant in vivo
antitumor efficacy in a melanoma tumor model with tumor growth inhibition rates of 78.70% (15 mg/kg) and 84.32% (30 mg/kg).
Collectively, this work shows that 12b is a promising lead compound deserving further investigation as a potential anticancer agent.
INTRODUCTION
Although no CBSIs have been approved for clinical uses so
far, several CBSIs (e.g., BNC-105p, CA-4P, AVE8062, and
VERU-111) are being actively investigated in clinical trials for
the treatment of various cancers such as colon cancer, lung
cancer, and prostate cancer (www.clnicaltrials.gov). One of
these investigational CBSIs, VERU-111 (in phase 2 clinical
■
Microtubules, composed of α-tubulin and β-tubulin hetero-
dimers, are the key components of the cytoskeleton and are of
1
great importance for many cellular functions such as mitosis,
cell motility, cell shape maintenance, intracellular transport,
2
and cell signaling. Owing to the polymerization dynamics of
2
8
tubulin, microtubules are considered to be an important target
for anticancer drugs, such as microtubule-targeting agents
trial, NCT03752099), was originally discovered by us and
was found to have benign toxicity profiles (www.verupharm.
3
,4
29
(
MTAs), which induce apoptosis by disrupting microtubule
com) when compared to known tubulin inhibitors (e.g.,
5
dynamics. MTAs bind to one of the six binding sites on
paclitaxel) targeting the taxane site. Thus, VERU-111
represents a promising CBSI with high therapeutic value in
cancer treatment.
6
,7
8,9
tubulin, the paclitaxel site, the vinca site, the laulimalide
1
0,11
12
13,14
site,
the pironetin site, the maytansine site,
and the
1
5,16
colchicine site.
All currently marketed MTAs (e.g.,
We have previously reported the discovery of several classes
1
7
18
taxanes and vinca alkaloids ) bind to the paclitaxel site or
vinca site and are effective against a variety of cancers. Their
clinical applications are often hindered by several drawbacks
of CBSIs such as 2-aryl-4-benzoyl-imidazoles (ABI, Figure
28
1
), as mentioned above (code name VERU-111), 4-
19
including low aqueous solubility, dose-limiting toxicity, and
multi-drug resistance (MDR).
shown that colchicine-binding site inhibitors (CBSIs) have the
potential to overcome the abovementioned disadvantages
associated with taxanes and vinca alkaloids, due to their
structural simplicity, non-P-gp (P-glycoprotein) substrate
20−23
Received: October 21, 2020
Published: March 31, 2021
Extensive research has
2
4−27
nature, and reasonable pharmacokinetic properties.
©
2021 American Chemical Society
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Figure 1. Design of target compounds by the ring-fusion strategy.
a
Scheme 1. Synthesis of Series 1 Compounds (6a−j)
a
Reagents and reaction conditions: (a) (Boc) O, TEA, DMAP, and ACN; (b) 3,4,5-trimethoxyphenylboronic acid, PdCl (dtbpf), Na CO ,
2
2
2
3
dioxane−H O, 80 °C; (c) Pd/C, H and MeOH-THF; (d) (i) NaNO , 37%HCl, 0 °C; (ii) KI, 0 °C to room temperature, reflux; (e)
2
2
2
PdCl (dtbpf), Na CO , dioxane−H O, 80 °C; and (f) NH OHHCl, NaHCO , EtOH, 65 °C.
2
2
3
2
2
3
3
0
substituted methoxybenzoyl-aryl-thiazoles (SMART), and
by replacing the five-membered imidazole B-ring of ABI with a
six-membered phenyl or pyridine ring; ABP compounds
exhibited high antiproliferative activities as tubulin inhibitors.
Inspired by this, we designed series 1 by cyclizing the carbonyl
moiety into a pyrazole ring to improve the metabolic stability.
Series 2 was designed by switching the positions of the C-ring
and A-ring of series 1. Series 3 (“bent” conformation) and
series 4 (“straight” conformation) were designed by shifting
the positions of the A-ring to explore the preferred
31
substituted-2-aryl imidazoles (SAI) analogues. These CBSIs
exhibited high in vitro antiproliferative activities and significant
in vivo antitumor efficacy and were able to overcome P-gp-
mediated MDR effectively. In spite of the excellent biological
activities, the ABI and SMART analogues share a common
structural feature, the carbonyl (ketone) group between the B-
ring and C-ring (Figure 1), which is a metabolic soft spot and
is susceptible to metabolic reduction by liver microsomes. In
order to improve the metabolic stability, we designed five
series of compounds by incorporating the carbonyl group into
a five-membered pyrazole ring (series 1 and 2: indazole
analogues), a six-membered phenyl ring (series 3: benzimida-
zole analogues), and a six-membered pyrazine ring (series 4:
pyrazine imidazole analogues) or a pyrimidine ring (series 5:
selenium compounds based on the pyrazolo[1,5-a]pyrimidine
scaffold). Previous co-crystal structures of an ABI compound in
complex with tubulin showed that the binding pocket near the
32
36
conformations by the binding pocket. Series 5 was designed
by replacing the oxygen atom of the 3′-methoxy moiety with a
selenium as selenium-containing compounds have been proven
37,38
to be potent tubulin inhibitors.
In summary, the main
strategy is to fuse the B-ring and cyclized ketone as the ring-
fusion strategy is a common approach used in medicinal
39,40
chemistry for drug design.
Here, we describe the design,
synthesis, and biological evaluation of novel benzimidazole and
indazole analogues as potential anticancer agents targeting
tubulin polymerization.
33,34
B-ring may accommodate a bulkier group,
and Li’s group
35
designed 6-aryl-2-benzoyl-pyridine/phenyl (ABP) analogues
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a
Scheme 2. Synthesis of Series 2 Compounds (10a−j)
a
Reagents and reaction conditions: (a) 3,4,5-trimethoxyphenylboronic acid, PdCl (dtbpf), Na CO , dioxane−H O, 80 °C; (b) NIS and Actone;
2
2
3
2
and (c) PdCl (dtbpf), Na CO , dioxane−H O, 80 °C.
2
2
3
2
a
Scheme 3. Synthesis of Series 3 Compounds (12a−h)
a
Reagents and reaction conditions: (a) TsOH, dioxane, 95 °C; (b) 3,4,5-trimethoxyphenylboronic acid, PdCl (dtbpf), Na CO , dioxane−H O, 80
2
2
3
2
°C.
a
Scheme 4. Synthesis of Series 4 Compounds (14a−d)
a
Reagents and reaction conditions: (a) PdCl (dtbpf), Na CO , dioxane−H O, 80 °C.
2
2
3
2
RESULTS AND DISCUSSION
compounds 6g and 6h with hydroxylamine hydrochloride
provided compounds 6i and 6j.
■
Chemistry. The synthesis of series 1 compounds (6a−j) is
illustrated in Scheme 1. 3-Bromo-5-nitro-1H-indazole (1) was
Series 2 compounds (10a−j) were synthesized and are
outlined in Scheme 2. 5-Bromo-1H-indazole (7) was coupled
with 3,4,5-trimethoxyphenylboronic acid to give the indazole
compound 8 followed by an iodination reaction to produce 3-
iodo-indazole compound 9. Compound 9 underwent the
Suzuki coupling reactions with various arylboronic acids to
generate compounds 10a−h. Treating compounds 10g and
10h with hydroxylamine hydrochloride provided compounds
10i and 10j.
protected by (Boc) O to give intermediate 2, which underwent
2
Suzuki reaction to yield intermediate 3. Intermediate 3 was
reduced with hydrogen and Pd/C (10%) to generate
intermediate 4, which was then converted to the 5-iodo-
indazole intermediate 5 under reported conditions. Sub-
sequently, intermediate 5 was coupled with a variety of
arylboronic acid to produce compounds 6a−h. Treating
41
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a
Scheme 5. Synthesis of Series 5 Selenium Compounds (23a−h)
a
Reagents and reaction conditions: (a) HNO , AcOH, 3 h, rt; (b) dimethylsulfate, K CO , acetone, 48 h, 80 °C; (c) Pd/C, H , CH OH/THF, rt,
3
2
3
2
3
overnight; (d) (i) HCl, H O, NaNO , 40 min, −5 °C; (ii) NaOAc, 30 min, 0 °C; (iii) KSeCN, 3 h, 0 °C, rt; (e) NaBH , CH I, EtOH, 10 min, rt;
2
2
4
3
(
f) N,N-dimethylformamide dimethyl acetal, DMF, 120 °C, 6 h; (g) 3-bromo-1H-pyrazol-5-amine, AcOH, 80 °C, 8 h; and (h) appropriate aryl
boronic acid, Pd(dppf)Cl , Na CO aq., DMF, 95 °C, 12 h.
2
2
3
Series 3 compounds (12a−h) were prepared and are
illustrated in Scheme 3. 3-Bromo-1,2-benzenediamine was
cyclized with different aldehydes to produce the benzimidazole
intermediates 11a−f. Compounds 11a−f underwent Suzuki
reaction with 3,4,5-trimethoxyphenylboronic acid to give
compounds 12a−f. Treating compounds 12e and 12f with
hydroxylamine hydrochloride provided compounds 12g and
methyl-indole substituted benzimidazole analogues showed
higher activities (average IC s of 1.39, 0.05, and 0.70 μM for
5
0
12a, 12b, and 12c, respectively) than the corresponding 1-
butyl-indole analogues (IC50 > 10 μM for 12d). These results
suggest that small substituents (e.g., methyl) are preferred over
bulky functional groups (e.g., butyl) at N-1 of the indole ring
(12a−c vs 12d). Compound 12b exhibited the highest activity
with an average IC of 50 nM which is slightly better than that
12h.
5
0
Series 4 compounds (14a−d) were synthesized through a
of colchicine (IC = 65 nM), indicating that the 4-substituted
indolyl moiety is optimal for activity (12b vs 12a and 12c) in
comparison to the 3- and 5-substituted indolyl groups. Series 4
50
one-pot Suzuki reaction and are shown in Scheme 4.
Series 5 selenium compounds (23a−h) were prepared and
are illustrated in Scheme 5. The intermediates 16−20 were
compounds (14a−d) showed low activity (IC5 > 10 μM)
0
38
prepared according to a reported procedure. The selenium
compound 20 was condensed with N,N-dimethylformamide
dimethyl acetal at 120 °C to give compound 21, which was
then stirred with 3-bromo-1H-pyrazol-5-amine at 80 °C to
provide the key intermediate 22. Compound 22 underwent the
Suzuki coupling reactions with various arylboronic acids to
generate selenium compounds 23a−h.
probably due to their “straight” conformation which is not
preferred for binding to the colchicine-binding site in tubulin
as compared to the “bent” conformation of series 3
compounds; similar results were also observed by Zhang’s
36
group. Series 5 compounds (23a−h) demonstrated high
antiproliferative activities with IC values in the low
5
0
nanomolar to low micromolar range. In this series, compounds
bearing an indolyl A-ring (e.g., 23a, 23e, and 23g) showed
significantly better antiproliferative activities (IC50 ranging
from 91 to 856 nM) than those with a phenyl A-ring (e.g., 23d,
23f, and 23h, IC50 ranging from 3 to 10 μM), consistent with
our previous observations that the indolyl A-ring is optimal for
Biological Evaluation. In Vitro Antiproliferative Activ-
ities. The newly synthesized compounds 6a−j, 10a−j, 12a−h,
1
4a−d, and 23a−h were evaluated for their in vitro cytotoxicity
against four cancer cell lines by the MTT assay with colchicine
as the positive control. As shown in Table 1, series 1
compounds, the indazole derivatives with a trimethoxyphenyl
28
activity.
(
TMP) moiety connected to the 3-position of indazole,
Compound 12b Inhibited the Viability and Colony
Formation of Cancer Cells. To further validate the in vitro
activities, the most potent compound 12b was chosen for the
MTT and colony formation assay. On the MTT assay,
compound 12b significantly decreased the viability of MCF-7,
A549, Hela, and B16-F10 cancer cells in a dose- and time-
dependent manner (Figure 2A). In the colony formation assay
using MCF-7 cells, compound 12b was able to suppress the
formation of colonies in a dose-dependent manner, with the
colony-forming ability of MCF-7 cells being totally suppressed
after 24 h treatment with 500 nM of compound 12b (Figure
2B).
exhibited moderate to high antiproliferative activities in general
with IC50 values in the low nanomolar to low micromolar
range (e.g., 6a−j). Compound 6a with a 1-methyl-1H-indolyl
moiety at the 5-position of indazole displayed the highest
activity in this series with an average IC of 81 nM. Replacing
the 1-methyl-1H-indolyl moiety with an indazole (6b), a
substituted phenyl (6c−h), or an N-hydroxycarbamimidoyl-
phenyl (6i,j) led to diminished activities with average IC
values ranging from 0.22 μM (6c) to 6.6 μM (6j). Series 2
compounds (10a−j) lost their activities (IC50 ∼ 10 μM) due
to the shift of the TMP group to the 5-position of indazole
when compared with series 1 compounds (e.g., 10a vs 6a, 10b
vs 6c, and 6d−j vs 10d−j). For series 3 compounds, the 1-
50
5
0
Effects of Compound 12b on Paclitaxel-Resistant Cell
Line. Resistance to tubulin inhibitors such as paclitaxel and
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a
Table 1. In Vitro Antiproliferative Activities of Target Compounds against Four Cancer Cell Lines
a
Cells were exposed to different concentrations of compounds continuously for 48 h prior to determination of cell viability by the MTT assay. IC50
values are presented as the mean ± SD (standard error) of at least three independent experiments.
colchicine is a major obstacle impeding the clinical use of these
agents. In our previous work, the ABI analogues demonstrated
the ability to circumvent paclitaxel resistance effectively. We
hypothesized that the cyclized ketone analogues designed on
the basis of the ABI scaffold would maintain the ability to
overcome paclitaxel resistance. To verify this hypothesis, we
examined the antiproliferative activity of compound 12b in a
paclitaxel-resistant cancer cell line (A2780/T) and the
corresponding parental cells (A2780S), with paclitaxel and
colchicine as positive controls. In the A2780/T cell line, P-gp
is overexpressed, which may represent the primary mechanism
42
of resistance to paclitaxel. As shown in Table 2, compound
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Figure 2. Growth inhibitory effects of compound 12b on cancer cells. (A) Four cancer cell lines, MCF-7, A549, Hela, and B16-F10 were treated
with the indicated concentration of compound 12b for 48 h (left panel) or compound 12b (100 nM) for the indicated time (right panel). Cell
viability was determined by MTT assays. (B) Number of colonies was counted after treating MCF-7 cells with the indicated concentration of
compound 12b for 24 h (n = 3, **P < 0.05 vs DMSO control).
Table 2. Drug Tolerances of Compound 12b against
A2780/T
nM) in the A2780/T cell line with RIs of 421.8 and 46.9,
respectively.
Because a colony formation assay has been generally used to
predict the therapeutic efficacy of a drug to solid tumors, we
therefore performed it to examine the effect of compound 12b
on paclitaxel-resistant cells (A2780/T). As shown in Figure S1
(Supporting Information), compound 12b suppressed the
formation of A2780/T colonies in a dose-dependent manner,
and even at the lowest tested concentration of 10 nM, 12b was
able to effectively inhibit A2780/T colony formation.
However, paclitaxel could not inhibit the formation of
A2780/T colonies even at a high concentration of 100 nM.
These results suggest that compound 12b is probably not a
substrate for P-gp and thus could overcome P-gp-mediated
MDR in vitro in A2780/T cells.
IC50 (nM)
a
drug
2b
paclitaxel
colchicine
A2780/T
A2780S
RI
1
9.7 ± 4.1
3164.0 ± 147.1
783.8 ± 62.7
6.2 ± 2.1
7.5 ± 3.4
16.7 ± 6.6
1.6
421.8
46.9
a
Resistant index: (IC of drug-resistant cancer cell)/(IC of parental
cancer cell).
1
(
2b was nearly equally potent against both the parental
A2780S) and its paclitaxel-resistant cell line (A2780/T) with
IC values of 6.2 and 9.7 nM, respectively, and a resistance
index (RI) of 1.6. While paclitaxel and colchicine showed low
nanomolar potency (7.5 and 16.7 nM) in the parental A2780S
cells, the activity was significantly lower (3164.0 and 783.8
In Vitro Tubulin Polymerization Assay and Binding
Kinetics to Tubulin. Based on the high in vitro antiproliferative
activities, we further evaluated the inhibitory effects of
Figure 3. Inhibition of tubulin polymerization in vitro. (A) Inhibition of tubulin polymerization by compound 12b. (B) Inhibition of tubulin
polymerization by colchicine. (C) Compound 12b exhibited a dose-dependent inhibition of tubulin polymerization. Data are expressed as mean ±
standard deviation from three independent experiments.
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Figure 4. Effects of compound 12b on microtubules. B16-F10 cells were treated with vehicle control 0.1% DMSO (A), compound 12b (10 nM)
B), compound 12b (20 nM) (C), and compound 12b (40 nM) (D) for 6 h. Microtubules were visualized with an anti-β-tubulin antibody (green),
(
and the cell nucleus was visualized with DAPI (blue). Fluorescence images were collected by a LSM 880 laser confocal microscope (Carl Zeiss,
Germany).
Figure 5. Cell cycle arrest induced by compound 12b. (A) Compound 12b induced G2/M arrest in MCF-7 cells. MCF-7 cells were incubated with
varying concentrations of compound 12b (5, 10, and 20 nM) for 48 h. The percentages of cells in different phases of the cell cycle were analyzed by
FlowJo 7.6.1. (B) Histograms display the percentage of cell cycle distribution after treatment with compound 12b. Data are expressed as mean ±
standard deviation from three independent experiments.
compound 12b on tubulin polymerization with colchicine as a
positive control. As shown in Figure 3, compound 12b
potently inhibited tubulin polymerization with an IC50 of 2.52
compound 12b dose dependently binds to tubulin with a K of
D
12.7 μM, similar to that of colchicine (K = 25.4 μM). These
D
results indicate that 12b could bind to tubulin at a similar
magnitude to that of colchicine.
±
0.63 μM (Figure 3A), which is better than that of colchicine
(
1
IC = 7.30 ± 0.44 μM, Figure 3B). Furthermore, compound
2b inhibited tubulin polymerization in a dose-dependent
Antimicrotubule Effects in B16-F10 Cells. The inhibitory
effect of compound 12b on microtubule organization was
further studied in B16-F10 cells by immunofluorescence
staining. As illustrated in Figure 4, in the control experiment
[dimethyl sulfoxide (DMSO)], the microtubule networks
appeared in a normal arrangement with slim and fibrous
5
0
manner (Figure 3C). In addition, we also performed an surface
plasmon resonance (SPR) experiment to explore the binding
affinity of our compounds to tubulin with colchicine as a
control. As shown in Figure S2 (Supporting Information),
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Figure 6. Effects of compound 12b on cancer cell apoptosis. (A) Compound 12b induced apoptosis in MCF-7 cells. MCF-7 cells were exposed to
increasing concentrations of compound 12b (5, 10, and 20 nM) for 48 h. The percentages of cells in each stage of cell apoptosis were quantified by
flow cytometry: necrosis cells (upper left quadrant); late apoptotic cells (upper right quadrant); live cells (bottom left quadrant); and early
apoptotic cells (bottom right quadrant). (B) Histograms displayed the percentage of cell cycle distribution after treatment with compound 12b.
Data are expressed as mean ± standard deviation from three independent experiments.
Figure 7. Effects on the B16-F10 cell migration. (A) Representative images of wound area in a wound healing assay. Images were obtained at 0 and
24 h after treatment with 0, 5, 10, and 20 nM of compound 12b. (B) Histograms display the length of the scratches at 0 and 24 h after treatments
with 0, 5, 10, and 20 nM of compound 12b (n = 3, **P < 0.05 vs DMSO control).
microtubules (green) wrapped around the cell nucleus (blue).
However, the microtubule networks in cytosol were disrupted
when cells were exposed to compound 12b (10, 20, and 40
nM) for 6 h. These results suggest that compound 12b may
induce a dose-dependent collapse of the microtubule networks.
Cell Cycle Analysis. Tubulin polymerization inhibitors have
been previously reported to disrupt cell mitosis and induce cell
in Figure 6A,B, after treating MCF-7 cells with increasing
concentrations of compound 12b (5, 10, and 20 nM) for 48 h,
the total numbers of early (annexin-V+/PI−) and late
(
±
annexin-V+/PI+) apoptotic cells were 13.71 ± 1.49, 33.28
3.12, and 40.66 ± 1.61%, respectively. These results
confirmed that 12b could induce efficient apoptosis of MCF-
7
cells.
4
3
cycle arrest. Thus, flow cytometry analysis was performed to
examine the effects of compound 12b on the cell cycle of
MCF-7 cells. As shown in Figure 5A,B, treatment of cells with
compound 12b for 48 h caused a significant accumulation of
MCF-7 cells in the G2/M phase with the percentage of cells
distributed in the G2/M phase of around 62% at 10 and 20
nM, as compared to the control group (17%).
Analysis of Cancer Cell Apoptosis. Knowing that mitotic
arrest of tumor cells by tubulin polymerization inhibitors is
generally associated with cellular apoptosis, an annexin V-
FITC and propidium iodide (PI) assay of compound 12b was
performed with MCF-7 cells, in order to assess whether
compound 12b could induce cancer cell apoptosis. As shown
Inhibition of Cancer Cell Migration. It is implicated that
tubulin and microtubule played an important role in cell
migration and motility. Therefore, we performed a wound
healing assay in order to confirm the effects of compound 12b
in cell migration via microtubule destabilization. B16-F10 cells
were treated with 0, 5, 10, and 20 nM of compound 12b for 24
h. The result of the wound closure is shown in Figure 7. The
control cells were able to migrate into the wound channel,
recovering 51.92 ± 2.02% of the area. Treating with 5, 10, or
20 nM of compound 12b, B16-F10 cells only migrated into
29.42 ± 4.93, 15.68 ± 2.03, and 6.60 ± 2.09% of the wound
area, respectively. Obviously, displaying that the migration of
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Figure 8. Compound 12b inhibits melanoma tumor growth in vivo. After being administered with vehicle, PTX (10 mg/kg per days), compound
2b (15 mg/kg per day), and compound 12b (30 mg/kg per day) for 14 days, the mice were sacrificed, and the tumors were weighed. (A) Images
1
of tumors from mice at 14 days after initiation of treatment. (B) Body weight changes of mice during treatment. (C) Weight of the excised tumors
of each group. **P < 0.05 vs control group. (D) Tumor volume changes of mice during treatment.
Figure 9. Pathological sections of major tissues (liver and kidney) were obtained from mice bearing melanoma tumors. Organs were stained with
H&E, and the representative images are captured.
cancer cells was suppressed by compound 12b in a dose-
dependent manner.
[composed of two α/β tubulin dimers, the stathmin-like
protein RB3, and tubulin tyrosine ligase (TTL)] in complex
with compound 12b at a resolution of 2.45 Å (PDB code:
7DBA). The X-ray diffraction data and structure refinement
statistics are shown in Table 3.
As expected, compound 12b occupies the colchicine-binding
site at the interface of the α- and β-tubulin opposite to the
GTP molecule (Figure 10A). There is a hydrogen bond
formed between compound 12b and β-tubulin: the nitrogen
atom of the imidazole ring of compound 12b and the −OH in
β-Asn258 (Figure 10B−D). In addition, compound 12b is
wrapped firmly in the colchicine-binding site with a hydro-
phobic “sandwich” formed by the side-chains of β-Cys241, β-
Leu248, and β-Lys352 from one side and that of β-Lys254
from the other side. The observed hydrogen bond and the
strong hydrophobic interaction between the ligand and tubulin
may explain the high activity of 12b.
In vivo Antitumor Efficacy. The in vivo antitumor efficacy of
compound 12b was further evaluated in the mice melanoma
tumor model, established by subcutaneous inoculation of B16-
F10 cells into the mice. BALB/c mice bearing melanoma
tumors were treated with vehicle control, paclitaxel (PTX, 10
mg/kg), and compound 12b (15, 30 mg/kg) via i.p. injection
every once a day for 14 days. As shown in Figure 8, compound
1
2b demonstrated significant in vivo antitumor efficacy with
tumor growth inhibition (TGI) of 78.70 and 84.32% at 15 and
0 mg/kg, respectively, slightly less than that of PTX (88.66%)
3
at 10 mg/kg. In addition, compound 12b did not cause an
obvious body weight loss at 15 mg/kg, indicating that the
compound was well-tolerated at low doses (Figure 8B).
Furthermore, the hematoxylin and eosin (H&E) staining of
liver and kidney showed that compound 12b had no significant
toxic effects on the major organs of mice (Figure 9).
X-ray Structures of Compound 12b in Complex with
Tubulin. To reveal the binding interactions of compound 12b
with tubulin, we determined the crystal structures of T2R-TTL
CONCLUSIONS
In summary, we designed, synthesized, and bioevaluated novel
indazole and benzimidazole analogues as tubulin inhibitors,
■
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(
ppm). Multiplicities are reported as follows: singlet (s), doublet (d),
Table 3. X-ray Data Collection and Refinement Statistics
triplet (t), quartet (q), double doublet (dd), and multiplet (m).
Compound purities were estimated by reversed phase C18 high-
performance liquid chromatography, with UV detection at 254 nm.
The major peak area of each tested compound was ≥95% of the
combined total peak area. High-resolution mass spectrometry
T2R-TTL-12b (PDB code: 7DBA)
data collection
space group
P121221
cell dimensions
(
HRMS) was obtained on a Micromass Q-TOF mass spectrometer.
unit cell (Å)
unit cell (°)
wavelength (Å)
resolution (Å)
Rmerge
105.4 158.9 181.6
90.0, 90, 90
0.9793
Column chromatography was carried out using commercially available
silica gel (200−300 mesh) under pressure.
Synthesis of Intermediate 2. To a solution of 3-bromo-5-nitro-
31.3−2.45
1H-indazole (1) (1.0 g, 4.13 mmol) and (Boc) O (947 mg, 4.34
mmol) in 20 mL of acetonitrile (ACN) were added 4-
dimethylaminopyridine (DMAP) (202 mg, 1.65 mmol) and TEA
2
0.14.1 (0.80)
97.7 (74.1)
12.3 (1.5)
CC1/2
I/sigma
(
triethyl amine, 859 μL, 6.20 mmol). The mixture was stirred at room
temperature overnight. The solvent was removed in vacuo, and the
residue was diluted by H O and extracted with EtOAc (3 × 20 mL).
The combined organic layer was then washed with brine, dried over
anhydrous Na SO , and concentrated in vacuo to provide the crude
product, which was directly subjected to silica gel column
chromatography eluted with petroleum PE/EtOAc (9/1, v/v) to
give intermediate 2 as a white solid (1.1 g, 79.2%). H NMR (400
MHz, CDCl ): δ 8.57 (d, J = 2.1 Hz, 1H), 8.45 (dd, J = 9.2, 2.1 Hz,
completeness (%)
redundancy
99.9 (99.6)
6.5 (6.4)
2
structure refinement
2
4
number of measured reflections
number of unique reflections
717,379
110,366
18.92/22.82
18,135
17,520
433
1
Rwork/Rfree (%)
no. atoms
protein
3
1H), 8.31 (d, J = 9.2 Hz, 1H), 1.73 (s, 9H).
water
Synthesis of Intermediate 3. To a solution of intermediate 2
(
1.0 g, 2.92 mmol) in 18 mL of dioxane and 2 mL of H O were added
ligand
average B value (Å )
protein
others
182
53.35
53.67
43.18
2
2
(3,4,5-trimethoxyphenyl)boronic acid (682 mg, 3.21 mmol), Na CO
2 3
(620 mg, 5.85 mmol), and catalytic equivalent of PdCl (dtbpf). The
2
mixture was stirred at 80 °C under a nitrogen atmosphere for 4 h.
Then, the solvent was removed in vacuo, and the residue was diluted
by H O and extracted with EtOAc (3 × 20 mL). The combined
organic layer was then washed with brine, dried over anhydrous
r.m.s. deviations
2
bonds (Å)
angle (°)
0.01
0.83
Na SO , and concentrated in vacuo to provide the crude product,
2
4
Ramachandran plot statistics (%)
96.92
which was directly subjected to silica gel column chromatography
most favorable
allowed
disallowed
eluted with petroleum PE/EtOAc (4/1, v/v) to give intermediate 3 as
1
3.08
0
yellow oil (693 mg, 55.9%). H NMR (400 MHz, CDCl ): δ 8.87 (d,
3
J = 1.8 Hz, 1H), 8.45 (dd, J = 9.3, 2.1 Hz, 1H), 8.34 (d, J = 9.2 Hz,
1H), 7.16 (s, 2H), 3.98 (s, 6H), 3.94 (s, 3H), 1.77 (s, 9H).
Synthesis of Intermediate 4. To a stirred solution of
targeting the colchicine-binding site. These compounds
intermediate 3 (500 mg, 1.16 mmol) in 5 mL of THF and 5 mL of
MeOH was added Pd/C (10%) to hydrogenate for 24 h. The mixture
was then filtered, and the filtrate was concentrated to dryness in vacuo.
The residue was purified by silica gel column chromatography eluted
showed high in vitro antiproliferative activities with IC values
5
0
in the nanomolar range. Among them, compound 12b
displayed the highest activity against a panel of cancer cell
lines with an average IC value 50 nM, which is slightly better
50
with PE/EtOAc (1/1, v/v) to give intermediate 4 as yellow oil (440
1
than colchicine. Compound 12b exhibited nearly equal activity
against both, a paclitaxel-resistant cancer cell line (A2780/T)
and the corresponding parental cell line (A2780S). Thus,
compound 12b can effectively overcome paclitaxel resistance in
vitro. Mechanism studies revealed that compound 12b
inhibited tubulin polymerization, arrested the cell cycle in
the G2/M phase, and induced apoptosis in MCF-7 cells. The
crystal structure of 12b in complex with tubulin was solved to
mg, 94.6%). H NMR (400 MHz, CDCl ): δ 7.97 (d, J = 8.9 Hz, 1H),
3
7.18−7.13 (m, 3H), 6.97 (dd, J = 8.9, 2.1 Hz, 1H), 3.95 (s, 6H), 3.91
s, 3H), 1.73 (s, 9H).
Synthesis of Intermediate 5. Intermediate 4 (200 mg, 0.501
(
mmol) was suspended in 37% HCl (5 mL) at 0 °C. An ice-cold
solution of NaNO (52 mg, 0.751 mmol) in H O (2.5 mL) was added
2
2
dropwise, and the resulting solution was stirred at 0−5 °C for 10 min.
An ice-cold suspension of KI (104 mg, 0.626 mmol) in 37% HCl (2.5
mL) was added dropwise. The mixture was stirred at room
temperature for 10 min, then at reflux for 3 h, and at room
temperature for 16 h. NaOH solution was added to adjust the mixture
to neutral. Then, the mixture was filtered, and the filter residue was
2
.45 Å resolution by X-ray crystallography and confirmed its
direct binding to the colchicine site. In addition, compound
2b displayed significant in vivo antitumor efficacy in a
1
purified by silica gel column chromatography eluted with PE/EtOAc
melanoma tumor model with a significant TGI of 78.70% (15
mg/kg) and 84.32% (30 mg/kg) without apparent toxicity.
Collectively, this work shows that compound 12b is a
promising lead compound deserving further investigation as
a potential anticancer agent.
1
(
2/1, v/v) to give intermediate 5 as a white solid (85 mg, 41.4%). H
NMR (400 MHz, CDCl ): δ 7.95 (d, J = 1.2 Hz, 1H), 7.45 (d, J = 8.8
3
Hz, 1H), 7.39 (dd, J = 8.8, 1.8 Hz, 1H), 7.12 (s, 2H), 3.97 (s, 6H),
.93 (s, 3H).
3
General Procedures for the Preparation of Compounds 6a−
6
h. To a solution of intermediate 5 (20 mg, 0.039 mmol) in 4.5 mL
EXPERIMENTAL SECTION
■
of dioxane and 0.5 mL of H
mmol), Na CO (9 mg, 0.078 mmol), and catalytic equivalent of
PdCl
2
O were added arylboronic acid (0.043
General Methods. Unless otherwise specified, all chemicals and
reagents were obtained from commercial suppliers and were used
without further purification. The reference compounds (paclitaxel and
colchicine) were acquired from InvivoChem (Libertyville, IL 60048,
2
3
(dtbpf). The mixture was stirred at 80 °C under a nitrogen
2
atmosphere for 4 h. Then, the solvent was removed in vacuo, and the
residue was diluted by H O and extracted with EtOAc (3 × 10 mL).
2
1
13
USA). H and C NMR spectra were recorded on a Bruker Avance at
00 MHz, using CDCl or DMSO-d as solvent and tetramethylsilane
The combined organic layer was then washed with brine, dried over
4
anhydrous Na SO , and concentrated in vacuo to provide the crude
3
6
2
4
as internal standard. Chemical shifts are reported in parts per million
product, which was directly subjected to silica gel column
4
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Figure 10. T2R-TTL crystal in complex with compound 12b (PDB code: 7DBA). (A) Overall structure of the α/β-tubulin and 12b complex.
Compound 12b and GTP are shown in violet and yellow spheres, respectively. (B) 3D detailed interactions between compound 12b and tubulin at
the colchicine-binding domain. (C) 2D detailed interactions between compound 12b and tubulin at the colchicine-binding domain. (D) 90°
clockwise rotation of corresponding structures along the x-axis. Oxygen and nitrogen atoms are colored read and blue, respectively. Hydrogen bond
is indicated in green dashed line.
chromatography eluted with petroleum PE/EtOAc (2/1, v/v) to give
corresponding pure compounds 6a−6h.
3-(3,4,5-Trimethoxyphenyl)-5-phenyl-1H-indazole (6d). The
compound was obtained as a white solid; yield: 59.7%. m.p. 161.2−
1
3
-(3,4,5-Trimethoxyphenyl)-5-(1-methyl-1H-indole-5-yl)-1H-in-
161.8 °C. H NMR (400 MHz, CDCl
3
): δ 8.16 (s, 1H), 7.69 (dd, J =
dazole (6a). The compound was obtained as a white solid; yield:
3
1
8.7, 1.5 Hz, 1H), 7.64 (d, J = 7.3 Hz, 2H), 7.54 (d, J = 8.7 Hz, 1H),
1
4.3%. m.p. 190.1−191.5 °C. H NMR (400 MHz, CDCl ): δ 8.21 (s,
7.47 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 7.22 (s, 2H), 3.96 (s,
3
+
H), 7.89 (s, 1H), 7.78 (d, J = 8.6 Hz, 1H), 7.58−7.52 (m, 2H), 7.44
6H), 3.94 (s, 3H). HRMS m/z: calcd for C22
H
20
N
2
O
3
[M + H] ,
(
d, J = 8.5 Hz, 1H), 7.28 (s, 1H), 7.27 (s, 1H), 7.13 (d, J = 2.9 Hz,
360.1474; found, 361.1599.
3-(3,4,5-Trimethoxyphenyl)-5-(4-fluorophenyl)-1H-indazole (6e).
1
3
H), 6.57 (d, J = 2.9 Hz, 1H), 5.32 (s, 1H), 3.97 (d, J = 5.3 Hz, 9H),
.86 (s, 3H). 13C NMR (101 MHz, CDCl ): δ 153.81, 146.31, 140.99,
The compound was obtained as a white solid; yield: 51.3%. m.p.
3
1
1
(
62.4−162.9 °C. H NMR (400 MHz, CDCl ): δ 8.09 (s, 1H), 7.64
136.91, 136.25, 133.32, 130.07, 130.04, 129.78, 129.36, 129.21,
3
dd, J = 8.7, 1.4 Hz, 1H), 7.60−7.55 (m, 3H), 7.20 (s, 2H), 7.16 (t, J
= 8.7 Hz, 2H), 3.96 (s, 6H), 3.94 (s, 3H). HRMS m/z: calcd for
C H FN O [M + H] , 378.1380; found, 379.1484.
1
28.04, 121.80, 119.78, 119.15, 110.23, 109.74, 105.11, 101.44, 61.15,
+
56.48, 33.12. HRMS m/z: calcd for C H N O [M + H] , 413.1739;
2
5
23
3
3
+
found, 414.1818.
22 19
2
3
3
-(3,4,5-Trimethoxyphenyl)-5-(4-methoxyphenyl)-1H-indazole
3
-(3,4,5-Trimethoxyphenyl)-5-(1-methyl-1H-indazole-5-yl)-1H-
(
6f). The compound was obtained as a white solid; yield: 61.5%. m.p.
indazole (6b). The compound was obtained as a white solid; yield:
1
1
158.1−158.8 °C. H NMR (400 MHz, CDCl
dd, J = 8.7, 1.4 Hz, 1H), 7.56 (d, J = 8.7 Hz, 2H), 7.49 (d, J = 8.7 Hz,
): δ 8.10 (s, 1H), 7.64
3
4
1
8
0.8%. m.p. 192.4−193.6 °C. H NMR (400 MHz, CDCl ): δ 8.19 (s,
3
(
1
H), 8.07 (s, 1H), 7.96 (s, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.72 (d, J =
.7 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.25
H), 7.22 (s, 2H), 7.01 (d, J = 8.7 Hz, 2H), 3.96 (s, 6H), 3.94 (s,
+
s, 2H), 4.15 (s, 3H), 3.99 (s, 6H), 3.96 (d, J = 0.7 Hz, 3H). 13
3H), 3.87 (s, 3H). HRMS m/z: calcd for C23
90.1580; found, 391.1690.
-(3,4,5-Trimethoxyphenyl)-5-(4-cyanophenyl)-1H-indazole
6g). The compound was obtained as a white solid; yield: 52.6%. m.p.
H N O [M + H] ,
22 2 4
(
C
3
NMR (101 MHz, CDCl ): δ 153.65, 146.17, 140.92, 139.22, 138.38,
3
3
1
1
35.54, 134.65, 134.37, 133.02, 128.97, 127.56, 126.67, 124.65,
(
1
21.61, 119.29, 119.18, 110.33, 109.32, 105.01, 60.96, 56.31, 35.61.
1
84.8−185.6 °C. H NMR (400 MHz, DMSO): δ 8.30 (s, 1H), 7.98
(d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.7 Hz, 1H),
.71 (d, J = 8.7 Hz, 1H), 7.23 (s, 2H), 3.89 (s, 6H), 3.73 (s, 3H).
+
HRMS m/z: calcd for C H N O [M + H] , 414.1692; found,
2
4
22
4
3
4
15.1779.
-(3,4,5-Trimethoxyphenyl)-5-(4-methylphenyl)-1H-indazole
6c). The compound was obtained as a white solid; yield: 45.7%. m.p.
7
3
+
HRMS m/z: calcd for C H N O [M + H] , 385.1426; found,
2
3
19
3
3
(
1
3
86.1519.
1
64.8−165.0 °C. H NMR (400 MHz, CDCl ): δ 8.16 (s, 1H), 7.72
3
3-(3,4,5-Trimethoxyphenyl)-5-(3-cyanophenyl)-1H-indazole
(dd, J = 8.7, 1.4 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 8.1 Hz,
(6h). The compound was obtained as a white solid; yield: 49.3%. m.p.
1
2
6
H), 7.31 (d, J = 7.9 Hz, 2H), 7.23 (s, 2H), 6.11 (s, 1H), 3.99 (s,
H), 3.96 (s, 3H), 2.44 (s, 3H). HRMS m/z: calcd for C H N O
181.1−181.8 °C. H NMR (400 MHz, DMSO): δ 8.29 (s, 1H), 8.25
2
3
22
2
3
(s, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.79 (t, J = 7.5 Hz, 2H), 7.71−7.65
(m, J = 15.5, 8.0 Hz, 2H), 7.23 (s, 2H), 3.90 (s, 6H), 3.74 (s, 3H).
+
[
M + H] , 374.1630; found, 375.1740.
4
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Article
+
HRMS m/z: calcd for C H N O [M + H] , 385.1426; found,
3
3-(4-Methylphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-indazole
2
3
19
3
3
86.1515.
General Procedures for the Preparation of Compounds 6i
and 6j. To a solution of compound 6g or 6h (27 mg, 0.070 mmol) in
mL of anhydrous EtOH were added hydroxylamine hydrochloride
8 mg, 0.105 mmol) and NaHCO3 (18 mg, 0.210 mmol). The
mixture was stirred at 65 °C under a nitrogen atmosphere for 6 h.
Then, the solvent was removed in vacuo, and the residue was
subjected to silica gel column chromatography eluted with petroleum
PE/EtOAc (1/2, v/v) to give corresponding pure compound 6i or 6j.
-(3,4,5-Trimethoxyphenyl)-5-[4-(N-hydroxycarbamimidoyl)]-
H-indazole (6i). The compound was obtained as a white solid; yield:
3.4%. m.p. 174.7−175.9 °C. H NMR (400 MHz, DMSO): δ 13.27
s, 1H), 9.65 (s, 1H), 8.23 (s, 1H), 7.77 (s, 4H), 7.74 (s, 1H), 7.68
(10b). The compound was obtained as a white solid; yield: 48.2%.
1
m.p. 176.0−176.7 °C. H NMR (400 MHz, CDCl
): δ 8.15 (d, J =
3
7.8 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.77−7.61 (m, 2H), 7.60−7.53
(m, 1H), 7.38 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 6.84 (s,
5
(
2H), 3.99 (s, 3H), 3.97 (s, 3H), 3.94 (s, 3H), 2.47 (s, 3H). HRMS m/
+
z: calcd for C23
H
N
22
O
2
[M + H] , 374.1630; found, 375.1743.
3
3-(3,4,5-Trimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-inda-
zole (10c). The compound was obtained as a white solid; yield:
1
5
1
0.6%. m.p. 183.5−184.8 °C. H NMR (400 MHz, CDCl ): δ 8.13 (s,
3
H), 7.67 (dd, J = 8.6, 1.2 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.25 (s,
3
1
2H), 6.84 (s, 2H), 4.04−3.95 (m, 15H), 3.94 (s, 3H). HRMS m/z:
1
+
4
calcd for C25H N O [M + H] , 450.1791; found, 451.1888.
26 2 6
(
(
3
3-Phenyl-5-(3,4,5-trimethoxyphenyl)-1H-indazole (10d). The
d, J = 8.7 Hz, 1H), 7.24 (s, 2H), 5.83 (s, 2H), 3.90 (s, 6H), 3.74 (s,
H). HRMS m/z: calcd for C H N O [M + H] , 418.1641; found,
compound was obtained as a white solid; yield: 66.7%. m.p. 162.1−
+
1
163.0 °C. H NMR (400 MHz, CDCl
): δ 8.13 (s, 1H), 8.02 (d, J =
2
3
22
4
4
3
4
19.1729.
3
7.2 Hz, 2H), 7.61 (dd, J = 8.6, 1.1 Hz, 1H), 7.56 (t, J = 7.5 Hz, 2H),
7.50−7.44 (m, 2H), 6.81 (s, 2H), 3.95 (s, 6H), 3.92 (s, 3H). HRMS
-(3,4,5-Trimethoxyphenyl)-5-[3-(N-hydroxycarbamimidoyl)]-
H-indazole (6j). The compound was obtained as a white solid; yield:
0.9%. m.p. 172.8−173.9 °C. H NMR (400 MHz, DMSO): δ 13.27
s, 1H), 9.64 (s, 1H), 8.28 (s, 1H), 8.04 (s, 1H), 7.77 (t, J = 6.5 Hz,
H), 7.71−7.66 (m, 2H), 7.47 (t, J = 7.7 Hz, 1H), 7.25 (s, 2H), 5.93
s, 2H), 3.91 (s, 6H), 3.74 (s, 3H). HRMS m/z: calcd for
C H N O [M + H] , 418.1641; found, 419.1733.
Synthesis of Intermediate 8. To a solution of 5-bromo-1H-
indazole (7) (1.0 g, 5.08 mmol) in 27 mL of dioxane and 3 mL of
H O were added (3,4,5-trimethoxyphenyl)boronic acid (1.18 g, 5.58
mmol), catalytic equivalent of PdCl (dtbpf), and Na CO (1.08 g,
0.15 mmol). The mixture was stirred at 80 °C under a nitrogen
atmosphere for 4 h. Then, the solvent was removed in vacuo, and the
residue was diluted by H O and extracted with EtOAc (3 × 20 mL).
The combined organic layer was then washed with brine, dried over
anhydrous Na SO , and concentrated in vacuo to provide the crude
product, which was directly subjected to silica gel column
chromatography eluted with petroleum PE/EtOAc (4/1, v/v) to
give intermediate 8 as a white solid (995 mg, 68.9%). H NMR (400
MHz, CDCl ): δ 8.15 (s, 1H), 7.91 (s, 1H), 7.63 (d, J = 8.7 Hz, 1H),
.56 (d, J = 8.6 Hz, 1H), 6.82 (s, 2H), 3.95 (s, 6H), 3.91 (s, 3H).
Synthesis of Intermediate 9. To a stirred solution of
intermediate 8 (800 mg, 2.81 mmol) in 20 mL of acetone was
added NIS (N-iodosuccinimide, 760 mg, 3.38 mmol). After stirring at
room temperature overnight, the solvent was removed in vacuo, and
+
1
m/z: calcd for C H N O [M + H] , 360.1474; found, 361.1591.
2
2
20
2
3
1
4
3-(4-Fluorophenyl)-5-(3,4,5-trimethoxyphenyl)-1H-indazole
(
2
(10e). The compound was obtained as a white solid; yield: 51.4%.
1
m.p. 158.2−158.6 °C. H NMR (400 MHz, CDCl
): δ 8.06 (s, 1H),
3
8
.00−7.95 (m, 2H), 7.64 (dd, J = 8.7, 1.5 Hz, 1H), 7.54 (d, J = 8.7
Hz, 1H), 7.22 (t, J = 8.4 Hz, 2H), 6.80 (s, 2H), 3.95 (s, 6H), 3.91 (s,
H). HRMS m/z: calcd for C H FN O [M + H] , 378.1380;
(
+
23
22
4
4
+
3
22 19 2 3
found, 379.1499.
3
-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-indazole
2
(
10f). The compound was obtained as a white solid; yield: 59.1%.
2
2
3
1
m.p. 155.7−156.6 °C. H NMR (400 MHz, CDCl ): δ 8.09 (s, 1H),
.94 (d, J = 8.8 Hz, 2H), 7.61 (dd, J = 8.7, 1.4 Hz, 1H), 7.49 (d, J =
1
3
7
8
(
.7 Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 6.81 (s, 2H), 3.95 (s, 6H), 3.91
2
+
s, 3H), 3.90 (s, 3H). HRMS m/z: calcd for C H N O [M + H] ,
23 22
2
4
3
90.1580; found, 391.1716.
-(4-Cyanophenyl)-5-(3,4,5-trimethoxyphenyl)-1H-indazole
10g). The compound was obtained as a white solid; yield: 50.8%.
2
4
3
(
1
1
m.p. 192.3−194.0 °C. H NMR (400 MHz, CDCl
3
): δ 8.14 (d, J =
.5 Hz, 2H), 8.08 (s, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.68 (dd, J = 8.7,
.4 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 6.80 (s, 2H), 3.95 (s, 6H), 3.92
8
1
(
3
7
+
s, 3H). HRMS m/z: calcd for C H N O [M + H] , 385.1426;
23 19 3 3
found, 386.1513.
3
-(3-Cyanophenyl)-5-(3,4,5-trimethoxyphenyl)-1H-indazole
(
10h). The compound was obtained as a white solid; yield: 46.7%.
1
m.p. 190.6−192.3 °C. H NMR (400 MHz, CDCl ): δ 8.30 (s, 1H),
.25 (d, J = 7.8 Hz, 1H), 8.05 (s, 1H), 7.72−7.06 (m, 4H), 6.80 (s,
H), 3.96 (s, 6H), 3.92 (s, 3H). HRMS m/z: calcd for C H N O
3
3
the residue was diluted by H O and extracted with EtOAc (3 × 20
mL). The combined organic layer was then washed with brine, dried
over anhydrous Na SO , and concentrated in vacuo to provide the
2
8
2
[
2
3
19
3
2
4
+
M + H] , 385.1426; found, 386.1501.
crude product, which was directly subjected to silica gel column
chromatography eluted with petroleum PE/EtOAc (2/1, v/v) to give
General Procedures for the Preparation of Compounds 10i
and 10j. To a solution of compound 10g or 10h (27 mg, 0.070
mmol) in 5 mL of anhydrous EtOH were added hydroxylamine
1
intermediate 9 as a white solid (666 mg, 57.8%). H NMR (400 MHz,
CDCl ): δ 7.67 (dd, J = 8.7, 1.5 Hz, 1H), 7.62 (s, 1H), 7.55 (d, J = 8.7
Hz, 1H), 6.82 (s, 2H), 3.96 (s, 6H), 3.91 (s, 3H).
3
hydrochloride (8 mg, 0.105 mmol) and NaHCO (18 mg, 0.210
3
mmol). The mixture was stirred at 65 °C under a nitrogen
atmosphere for 6 h. Then, the solvent was removed in vacuo, and
the residue was subjected to silica gel column chromatography eluted
with petroleum PE/EtOAc (1/2, v/v) to give corresponding pure
compound 10i or 10j.
General Procedures for the Preparation of Compounds
1
0a−h. To a solution of intermediate 9 (30 mg, 0.073 mmol) in 4.5
mL of dioxane and 0.5 mL of H O were added arylboronic acid
2
(
0.080 mmol), Na CO (16 mg, 0.146 mmol), and catalytic
equivalent of PdCl (dtbpf). The mixture was stirred at 80 °C under
a nitrogen atmosphere for 4 h. Then, the solvent was removed in
vacuo, and the residue was diluted by H O and extracted with EtOAc
3 × 10 mL). The combined organic layer was then washed with
brine, dried over anhydrous Na SO , and concentrated in vacuo to
2 3
2
3-[4-(N-Hydroxycarbamimidoyl)]-5-(3,4,5-trimethoxyphenyl)-
1
H-indazole (10i). The compound was obtained as a white solid;
1
2
yield: 41.9%. m.p. 182.1−183.0 °C. H NMR (400 MHz, DMSO): δ
3.34 (s, 1H), 9.71 (s, 1H), 8.22 (s, 1H), 8.07 (d, J = 8.3 Hz, 2H),
7.84 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 8.6 Hz, 1H), 7.66 (d, J = 8.7 Hz,
H), 6.96 (s, 2H), 3.88 (s, 6H), 3.70 (s, 3H). HRMS m/z: calcd for
(
1
2
4
provide the crude product, which was directly subjected to silica gel
column chromatography eluted with petroleum PE/EtOAc (2/1, v/v)
to give corresponding pure compounds 10a−10h.
1
+
C H N O [M + H] , 418.1641; found, 419.1721.
2
3
22
4
4
3
-[3-(N-Hydroxycarbamimidoyl)]-5-(3,4,5-trimethoxyphenyl)-
3
-(1-Methyl-1H-indole-5-yl)-5-(3,4,5-trimethoxyphenyl)-1H-in-
1H-indazole (10j). The compound was obtained as a white solid;
1
dazole (10a). The compound was obtained as a white solid; yield:
yield: 55.6%. m.p. 179.9−180.6 °C. H NMR (400 MHz, CDCl ): δ
3
1
3
1
7
1
3
4
6.8%. m.p. 182.4−183.8 °C. H NMR (400 MHz, CDCl ): δ 8.23 (s,
8.26 (s, 1H), 7.99 (s, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 7.6
Hz, 1H), 7.53−7.41 (m, 3H), 6.72 (s, 2H), 5.24 (s, 2H), 3.87 (s, 9H).
3
H), 8.20 (s, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H),
.53 (d, J = 8.6 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 3.0 Hz,
H), 6.82 (s, 2H), 6.60 (d, J = 2.8 Hz, 1H), 3.94 (s, 6H), 3.91 (s,
+
HRMS m/z: calcd for C H N O [M + H] , 418.1641; found,
2
3
22
4
4
419.1741.
+
H), 3.86 (s, 3H). HRMS m/z: calcd for C H N O [M + H] ,
General Procedures for the Preparation of Intermediates
11a−11f. To a solution of 3-bromo-1,2-benzenediamine (100 mg,
2
5
23
3
3
13.1739; found, 414.1837.
4
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Article
0
.535 mmol) in 5 mL of dioxane were added aldehyde (0.641 mmol)
2-(1-Methyl-1H-indole-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-ben-
and TsOH (0.080 mmol). The mixture was stirred at 95 °C under a
nitrogen atmosphere for 48 h. Then, the solvent was removed in
vacuo, and the residue was diluted by H O and extracted with EtOAc
zimidazole (12c). The compound was obtained as a yellow solid;
1
yield: 40.7%. m.p. 182.8−183.0 °C. H NMR (400 MHz, DMSO): δ
8
.67 (d, J = 7.8 Hz, 1H), 8.11 (s, 1H), 7.71 (s, 2H), 7.58 (d, J = 8.2
2
Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.32−
(3 × 10 mL). The combined organic layer was then washed with
7
3
4
.29 (m, J = 7.3 Hz, 1H), 7.25−7.20 (m, 2H), 3.96 (s, 6H), 3.93 (s,
brine, dried over anhydrous Na SO , and concentrated in vacuo to
2
4
+
H), 3.77 (s, 3H). HRMS m/z: calcd for C H N O [M + H] ,
provide the crude product, which was directly subjected to silica gel
column chromatography eluted with petroleum PE/EtOAc (2/1, v/v)
to give corresponding pure intermediates 11a−f.
25 23
3
3
13.1739; found, 414.1884.
2
-(1-Butyl-1H-indole-4-yl)-4-(3,4,5-trimethoxyphenyl)-1H-benzi-
midazole (12d). The compound was obtained as a yellow solid; yield:
Intermediate 11a. The compound was obtained as a yellow solid;
1
1
33.6%. m.p. 179.6−180.2 °C. H NMR (400 MHz, DMSO): δ 7.82
d, J = 7.4 Hz, 1H), 7.65 (d, J = 6.1 Hz, 3H), 7.59 (d, J = 2.8 Hz, 1H),
yield: 68.8%. H NMR (400 MHz, DMSO): δ 8.43 (s, 1H), 8.04 (dd,
J = 8.7, 1.4 Hz, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.50 (d, J = 7.8 Hz,
(
7
(
1
.55 (d, J = 3.0 Hz, 1H), 7.51 (dd, J = 7.7, 1.8 Hz, 2H), 7.37−7.24
m, 2H), 4.24 (t, J = 6.9 Hz, 2H), 3.93 (s, 6H), 3.76 (s, 3H), 1.81−
.73 (m, 2H), 1.30−1.26 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H). HRMS
1H), 7.45 (d, J = 3.0 Hz, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.10 (d, J =
7.8 Hz, 1H), 6.61 (d, J = 2.9 Hz, 1H), 3.86 (s, 3H).
Intermediate 11b. The compound was obtained as a yellow solid;
+
1
m/z: calcd for C28
-(4-Cyanophenyl)-4-(3,4,5-trimethoxyphenyl)-1H-benzimida-
zole (12e). The compound was obtained as a yellow solid; yield:
H N O [M + H] , 455.2209; found, 456.2357.
29 3 3
yield: 49.5%. H NMR (400 MHz, DMSO): δ 7.82 (d, J = 7.3 Hz,
2
1
H), 7.64 (d, J = 8.2 Hz, 1H), 7.57−7.48 (m, 3H), 7.42 (d, J = 7.2
Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.16 (t, J = 7.8 Hz, 1H), 3.88 (s,
H).
Intermediate 11c. The compound was obtained as a yellow solid;
1
4
(
7
(
3.5%. m.p. 192.8−193.7 °C. H NMR (400 MHz, DMSO): δ 8.38
d, J = 8.3 Hz, 2H), 8.05 (d, J = 8.3 Hz, 2H), 7.62−7.55 (m, 3H),
.55 (d, J = 1.5 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 3.91 (s, 6H), 3.75
3
1
yield: 53.9%. H NMR (400 MHz, DMSO): δ 8.54 (d, J = 7.2 Hz,
+
s, 3H). HRMS m/z: calcd for C H N O [M + H] , 385.1426;
23 19 3 3
1
7
H), 8.13 (s, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H),
.35 (d, J = 7.7 Hz, 1H), 7.32−7.22 (m, 2H), 7.08 (dd, J = 14.3, 6.5
found, 386.1505.
2
-(3-Cyanophenyl)-4-(3,4,5-trimethoxyphenyl)-1H-benzimida-
Hz, 1H), 3.92 (s, 3H).
zole (12f). The compound was obtained as a yellow solid; yield:
1
Intermediate 11d. The compound was obtained as a yellow solid;
40.8%. m.p. 191.3−191.8 °C. H NMR (400 MHz, DMSO): δ 8.66−
8.49 (m, 2H), 8.01−7.66 (m, 3H), 7.60−7.55 (m, 3H), 7.34 (t, J =
7.5 Hz, 1H), 3.90 (s, 6H), 3.75 (s, 3H). HRMS m/z: calcd for
1
yield: 40.3%. H NMR (400 MHz, DMSO): δ 7.79 (d, J = 7.4 Hz,
1
Hz, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.31 (s, 1H), 7.15 (t, J = 7.8 Hz,
H), 7.67 (d, J = 8.1 Hz, 1H), 7.58−7.52 (m, 2H), 7.48 (d, J = 2.9
+
C H N O [M + H] , 385.1426; found, 386.1505.
3
2
19
3
3
1
2
H), 4.25 (t, J = 7.0 Hz, 2H), 1.80−1.74 (m, 2H), 1.25 (d, J = 3.8 Hz,
H), 0.91−0.87 (m, 3H).
General Procedures for the Preparation of Compounds 12g
and 12h. To a solution of compound 12e or 12f (30 mg, 0.078
mmol) in 5 mL of anhydrous EtOH were added hydroxylamine
Intermediate 11e. The compound was obtained as a yellow solid;
1
yield: 60.1%. H NMR (400 MHz, DMSO): δ 8.38 (d, J = 7.3 Hz,
hydrochloride (8 mg, 0.117 mmol) and NaHCO (20 mg, 0.156
3
2
7
H), 8.05 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 7.5 Hz, 1H), 7.48 (d, J =
.6 Hz, 1H), 7.21 (t, J = 7.9 Hz, 1H).
mmol). The mixture was stirred at 65 °C under a nitrogen
atmosphere for 6 h. Then, the solvent was removed in vacuo, and
the residue was subjected to silica gel column chromatography eluted
with petroleum PE/EtOAc (1/2, v/v) to give corresponding pure
compound 12g or 12h.
Intermediate 11f. The compound was obtained as a yellow solid;
1
yield: 55.7%. H NMR (400 MHz, DMSO): δ 8.60 (s, 1H), 8.52 (d, J
=
7.7 Hz, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.62
3
-[4-(N-Hydroxycarbamimidoyl)]-5-(3,4,5-trimethoxyphenyl)-
(d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.20 (t, J = 7.9 Hz, 1H).
1
H-indazole (12g). The compound was obtained as a yellow solid;
General Procedures for the Preparation of Compounds
1
yield: 51.6%. m.p. 186.7−187.6 °C. H NMR (400 MHz, DMSO): δ
3.08 (s, 1H), 9.79 (s, 1H), 8.20 (d, J = 8.2 Hz, 2H), 7.86 (d, J = 8.2
Hz, 2H), 7.60 (s, 2H), 7.51 (d, J = 7.8 Hz, 2H), 7.30 (t, J = 7.8 Hz,
H), 5.88 (s, 2H), 3.90 (s, 6H), 3.74 (s, 3H). HRMS m/z: calcd for
1
2a−f. To a solution of intermediate 11a (11b, 11c, 11d, 11e, or
1
1
1f) (0.092 mmol) in 4.5 mL of dioxane and 0.5 mL of H O were
2
added (3,4,5-trimethoxyphenyl)boronic acid (23 mg, 0.110 mmol),
1
Na CO3 (20 mg, 0.184 mmol), and catalytic equivalent of
PdCl (dtbpf). The mixture was stirred at 80 °C under a nitrogen
atmosphere for 4 h. Then, the solvent was removed in vacuo, and the
residue was diluted by H O and extracted with EtOAc (3 × 10 mL).
The combined organic layer was then washed with brine, dried over
anhydrous Na SO , and concentrated in vacuo to provide the crude
product, which was directly subjected to silica gel column
chromatography eluted with petroleum PE/EtOAc (2/1, v/v) to
give corresponding pure compounds 12a−f.
2
+
C H N O [M + H] , 418.1641; found, 419.1725.
23 22
4
4
2
3
-[3-(N-Hydroxycarbamimidoyl)]-5-(3,4,5-trimethoxyphenyl)-
1
H-indazole (12h). The compound was obtained as a yellow solid;
2
1
yield: 57.2%. m.p. 185.4−185.7 °C. H NMR (400 MHz, DMSO): δ
3.11 (s, 1H), 9.71 (s, 1H), 8.59 (s, 1H), 8.21 (d, J = 7.8 Hz, 1H),
.77 (d, J = 7.8 Hz, 1H), 7.67 (s, 2H), 7.58 (t, J = 7.8 Hz, 1H), 7.53
1
7
2
4
(
3
t, J = 7.5 Hz, 2H), 7.33−7.28 (m, 1H), 5.90 (s, 2H), 3.93 (s, 6H),
+
.76 (s, 3H). HRMS m/z: calcd for C H N O [M + H] , 418.1641;
23 22 4 4
found, 419.1730.
2
-(1-Methyl-1H-indole-5-yl)-4-(3,4,5-trimethoxyphenyl)-1H-ben-
General Procedures for the Preparation of Compounds
zimidazole (12a). The compound was obtained as a yellow solid;
1
4a−d. To a solution of intermediate 13 (0.860 mmol) in 9 mL of
1
yield: 34.8%. m.p. 182.1−182.7 °C. H NMR (400 MHz, CDCl ): δ
3
dioxane and 1 mL of H O were added (3,4,5-trimethoxyphenyl)-
2
8
6
.28 (d, J = 1.1 Hz, 1H), 7.98 (dd, J = 8.6, 1.6 Hz, 1H), 7.68 (dd, J =
.7, 2.3 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H), 7.33−7.28 (m, 2H), 7.11
boronic acid (200 mg, 0.946 mmol), 1-methy-1H-indole-5-boronic
acid (166 mg, 0.946 mmol), Na CO (182 mg, 1.72 mmol), and
2
3
(d, J = 3.1 Hz, 1H), 7.04 (s, 2H), 6.56 (d, J = 3.1 Hz, 1H), 3.94 (s,
catalytic equivalent of PdCl (dtbpf). The mixture was stirred at 80 °C
2
6H), 3.93 (s, 3H), 3.83 (s, 3H). HRMS m/z: calcd for C H N O
25 23 3 3
under a nitrogen atmosphere for 4 h. Then, the solvent was removed
+
[
M + H] , 413.1739; found, 414.1859.
in vacuo, and the residue was diluted by H O and extracted with
2
2
-(1-Methyl-1H-indole-4-yl)-4-(3,4,5-trimethoxyphenyl)-1H-ben-
EtOAc (3 × 10 mL). The combined organic layer was then washed
zimidazole (12b). The compound was obtained as a yellow solid;
with brine, dried over anhydrous Na SO , and concentrated in vacuo
1
2
4
yield: 41.2%. m.p. 181.1−181.5 °C. H NMR (400 MHz, DMSO): δ
to provide the crude product, which was subjected to silica gel column
chromatography eluted with petroleum PE/EtOAc (4/1, v/v) to give
corresponding pure compounds 14a−d.
7
.85 (d, J = 7.4 Hz, 1H), 7.66 (s, 2H), 7.62−7.59 (m, 2H), 7.53−7.50
(m, 3H), 7.38−7.27 (m, 2H), 3.93 (s, 6H), 3.87 (s, 3H), 3.76 (s, 3H).
13
C NMR (101 MHz, DMSO): δ 152.71, 152.00, 141.64, 137.27,
3
-(1-Methyl-1H-indol-5-yl)-8-(3,4,5-trimethoxyphenyl)imidazo-
136.99, 135.38, 134.01, 130.73, 130.04, 125.88, 122.67, 121.41,
[1,2-a]pyrazine (14a). The compound was obtained as a white solid;
1
120.92, 120.02, 118.14, 111.65, 110.27, 106.27, 102.19, 60.16, 55.90,
yield: 13.8%. m.p. 162.4−163.2 °C. H NMR (400 MHz, CDCl ): δ
8.27 (d, J = 4.6 Hz, 1H), 8.15 (s, 2H), 7.97 (d, J = 4.6 Hz, 1H), 7.92
(s, 1H), 7.86 (s, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 8.4 Hz,
3
+
32.66. HRMS m/z: calcd for C H N O [M + H] , 413.1739;
2
5
23
3
3
found, 414.1888.
4
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Article
1
H), 7.20 (d, J = 3.0 Hz, 1H), 6.62 (d, J = 3.0 Hz, 1H), 4.05 (s, 6H),
.98 (s, 3H), 3.91 (s, 3H). HRMS m/z: calcd for C H N O [M +
7-(3,4-Dimethoxy-5-(methylselanyl)phenyl)-2-(p-methylphenyl)-
3
pyrazolo[1,5-a]pyrimidine (23b). The compound was obtained as a
2
4
22
4
3
+
1
H] , 414.1692; found, 415.1816.
-(3,4,5-Trimethoxyphenyl)-8-(3,4,5-trimethoxyphenyl)imidazo-
1,2-a]pyrazine (14b). The compound was obtained as a white solid;
yellow solid; yield: 78.8%; m.p. 152.1−152.9 °C. H NMR (400
3
MHz, CDCl ): δ 8.49 (d, J = 4.4 Hz, 1H), 7.93 (d, J = 8.0 Hz, 2H),
3
[
7.76 (d, J = 1.6 Hz, 1H), 7.68 (d, J = 1.6 Hz, 1H), 7.29 (d, J = 8.0 Hz,
2H), 7.06 (s, 1H), 6.92 (d, J = 4.4 Hz, 1H), 4.01 (s, 3H), 3.99 (s,
1
yield: 11.2%. m.p. 165.8−166.8 °C. H NMR (400 MHz, CDCl ): δ
8
4
CDCl ): δ 154.04, 153.00, 150.02, 140.21, 139.66, 138.85, 133.74,
1
3
1
3
.21 (s, 1H), 8.13 (s, 2H), 8.01 (s, 1H), 7.88 (s, 1H), 6.79 (s, 2H),
3H), 2.43 (s, 3H), 2.37 (s, 2H). C NMR (101 MHz, CDCl ): δ
3
1
3
.04 (s, 6H), 3.96 (d, J = 3.5 Hz, 12H). C NMR (101 MHz,
155.8, 151.8, 151.2, 148.7, 148.5, 145.5, 138.9, 130.0, 129.4, 128.1,
127.3, 126.3, 121.3, 111.6, 106.6, 93.3, 60.1, 56.1, 21.3, 5.1. HRMS
3
+
31.29, 129.19, 127.11, 123.46, 115.05, 107.08, 105.73, 60.97, 60.87,
m/z: calcd for C H N O Se [M + H] , 439.0799; found, 440.0917.
2
2
21
3
2
+
5
6.38, 56.23. HRMS m/z: calcd for C H N O [M + H] , 451.1743;
7-(3,4-Dimethoxy-5-(methylselanyl)phenyl)-2-(1-methyl-1H-in-
2
4
25
3
6
found, 452.1835.
dazol-6-yl)pyrazolo [1,5-a]pyrimidine (23c). The compound was
1
3
-(3,4,5-Trimethoxyphenyl)-8-(1-methyl-1H-indol-5-yl)imidazo-
obtained as a yellow solid; yield: 81.6%; m.p. 158.2−159.0 °C. H
[1,2-a]pyrazine (14c). The compound was obtained as a white solid;
NMR (400 MHz, CDCl ): δ 8.50 (d, J = 4.4 Hz, 1H), 8.38 (s, 1H),
3
1
yield: 14.4%. m.p. 166.0−166.3 °C. H NMR (400 MHz, CDCl ): δ
8.12 (d, J = 8.8, 1H), 8.07 (s, 1H), 7.75 (d, J = 1.9 Hz, 1H), 7.70 (d, J
3
9
(
1
.17 (s, 1H), 8.59 (d, J = 8.6 Hz, 1H), 8.18 (s, 1H), 8.03 (s, 1H), 7.92
s, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.13 (s, 1H), 6.81 (s, 2H), 6.69 (s,
H), 3.96 (s, 9H), 3.88 (s, 3H). HRMS m/z: calcd for C H N O
= 1.9 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.12 (s, 1H), 6.92 (d, J = 4.4
1
3
Hz, 1H), 4.13 (s, 3H), 4.02 (s, 3H), 4.00 (s, 3H), 2.38 (s, 3H). C
2
4
22
4
3
NMR (101 MHz, CDCl ): δ 156.1, 151.8, 151.3, 148.8, 148.6, 145.5,
3
+
[
M + H] , 414.1692; found, 415.1778.
-(1-Methyl-1H-indol-5-yl)-8-(1-methyl-1H-indol-5-yl)imidazo-
1,2-a]pyrazine (14d). The compound was obtained as a white solid;
140.1, 133.4, 128.2, 127.3, 125.6, 125.2, 124.3, 121.3, 119.2, 111.5,
3
109.2, 106.6, 93.2, 60.2, 56.1, 35.6, 5.1. HRMS m/z: calcd for
+
[
C H N O Se [M + H] , 479.0860; found, 480.0962.
2
3
21
5
2
1
yield: 16.9%. m.p. 163.9−164.9 °C. H NMR (400 MHz, CDCl ): δ
3
7-(3,4-Dimethoxy-5-(methylselanyl)phenyl)-2-(4-methoxy-3-
9
.20 (s, 1H), 8.62 (d, J = 8.5 Hz, 1H), 8.25 (d, J = 4.4 Hz, 1H), 8.00
nitrophenyl)pyrazolo [1,5-a]pyrimidine (23d). The compound was
1
(d, J = 4.5 Hz, 1H), 7.97 (s, 1H), 7.88 (s, 1H), 7.52 (t, J = 8.1 Hz,
obtained as a yellow solid; yield: 83.5%; m.p. 149.8−150.2 °C. H
2
H), 7.45 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 3.0 Hz, 1H), 7.13 (d, J =
NMR (400 MHz, CDCl ): δ 8.57 (s, 1H), 8.54 (d, J = 4.3 Hz, 1H),
3
3.0 Hz, 1H), 6.70 (d, J = 2.9 Hz, 1H), 6.62 (d, J = 2.9 Hz, 1H), 3.91
8.15 (d, J = 8.7 Hz, 1H), 7.75 (s, 1H), 7.65 (s, 1H), 7.20 (d, J = 8.7
+
(
3
s, 3H), 3.88 (s, 3H). HRMS m/z: calcd for C H N [M + H] ,
Hz, 1H), 7.05 (s, 1H), 6.98 (d, J = 4.3 Hz, 1H), 4.04 (s, 3H), 4.02 (s,
2
4
19
5
1
3
77.1640; found, 378.1730.
Synthesis of Intermediate 21. To a solution of intermediate 20
5.0 g, 25.7 mmol) in 15 mL of dimethylformamide (DMF) was
3H), 4.01 (s, 3H), 2.38 (s, 3H). C NMR (101 MHz, CDCl ): δ
3
153.3, 153.1, 151.9, 151.4, 149.2, 148.8, 145.6, 139.8, 131.9, 128.4,
126.9, 125.8, 123.7, 121.2, 113.7, 111.5, 107.1, 93.3, 60.2, 56.7, 56.1,
(
+
added N,N-dimethylformamide dimethyl acetal (9.2 g, 77.2 mmol).
The mixture was stirred at 120 °C for 6 h, then the reaction was
quenched by H O. After being extracted with CH Cl and
concentrated in vacuo, the crude product was purified by column
chromatography to give intermediate 21 as a white solid; yield:
5.2. HRMS m/z: calcd for C H N O Se [M + H] , 500.0599;
2
2
20
4
5
found, 501.0684.
2
2
2
7-(3,4-Dimethoxy-5-(methylselanyl)phenyl)-2-(1H-indol-3-yl)-
pyrazolo[1,5-a]pyrimidine (23e). The compound was obtained as a
1
yellow solid; yield: 88.7%; m.p. 160.1−161.2 °C. H NMR (400
1
85.4%; H NMR (400 MHz, CDCl ): δ 7.65 (d, J = 12.3 Hz, 1H),
MHz, CDCl ): δ 8.69 (s, 1H), 8.49 (d, J = 4.4 Hz, 1H), 8.44 (d, J =
3
3
7.25 (d, J = 1.7 Hz, 1H), 7.24 (d, J = 1.7 Hz, 1H), 5.53 (d, J = 12.3
8.5 Hz, 1H), 7.83 (d, J = 1.5 Hz, 1H), 7.74 (s, 1H), 7.68 (d, J = 1.6
Hz, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 2.98 (s, 3H), 2.78 (s, 3H), 2.19
Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.29−7.23 (m, 2H), 7.03 (s, 1H),
1
3
(s, 3H).
6.88 (d, J = 4.4 Hz, 1H), 4.03 (s, 3H), 4.00 (s, 3H), 2.36 (s, 3H). C
Synthesis of Intermediate 22. To a solution of intermediate 21
2.0 g, 8.0 mmol) in 10 mL of AcOH was added 3-bromo-1H-
NMR (101 MHz, CDCl ): δ 152.7, 151.9, 150.7, 148.7, 148.5, 145.4,
136.5, 128.0, 127.8, 125.4, 124.0, 122.7, 121.5, 121.4, 120.7, 111.6,
3
(
pyrazol-5-amine (1.6 g, 9.6 mmol). The mixture was stirred at 80 °C
for 8 h. After the completion of the reaction, the cooled mixture was
111.3, 110.4, 106.0, 60.2, 56.1, 5.2. HRMS m/z: calcd for
+
C H N O Se [M + H] , 464.0751; found, 465.0847.
2
3
20
4
2
deposited dropwise in H O, and the precipitated solid was filtered and
dried to obtain intermediate 22. White solid; yield: 89.5%; H NMR
7-(3,4-Dimethoxy-5-(methylselanyl)phenyl)-2-phenylpyrazolo-
2
1
[1,5-a]pyrimidine (23f). The compound was obtained as a yellow
1
(
1
3
400 MHz, CDCl ): δ 8.52 (d, J = 4.4 Hz, 1H), 7.54 (d, J = 1.7 Hz,
H), 7.48 (d, J = 1.7 Hz, 1H), 6.92 (d, J = 4.4 Hz, 1H), 6.84 (s, 1H),
.99 (s, 3H), 3.96 (s, 3H), 2.34 (s, 3H).
solid; yield: 82.9%; m.p. 158.6−159.2 °C. H NMR (400 MHz,
3
CDCl ): δ 8.52 (d, J = 4.2 Hz, 1H), 8.04 (d, J = 7.1 Hz, 2H), 7.76 (d,
3
J = 1.9 Hz, 1H), 7.68 (d, J = 1.9 Hz, 1H), 7.48 (t, J = 7.3 Hz, 2H),
7.42 (t, J = 7.3 Hz, 1H), 7.10 (s, 1H), 6.94 (d, J = 4.4 Hz, 1H), 4.02
General Procedures for the Preparation of Compounds
1
3
2
3a−h. To a solution of intermediate 22 (117.5 mg, 0.275 mmol)
(s, 3H), 4.00 (s, 3H), 2.38 (s, 3H). C NMR (101 MHz, CDCl ): δ
3
and corresponding boronic acid (0.360 mmol) in 5 mL of DMF and 1
mL of H O, Na CO (73 mg, 0.688 mmol) and Pd(dppf)Cl (10 mg,
155.7, 151.8, 151.2, 148.9, 148.6, 145.6, 132.8, 129.0, 128.7, 128.2,
2
2
3
2
127.3, 126.4, 121.3, 111.6, 106.8, 93.6, 60.1, 56.1, 5.1. HRMS m/z:
+
0
.014 mmol) were added. The mixture was stirred at 95 °C for 12 h,
calcd for C H N O Se [M + H] , 425.0642; found, 426.0746.
2
1
19
3
2
then the reaction was quenched by H O and extracted with CH Cl .
7-(3,4-Dimethoxy-5-(methylselanyl)phenyl)-2-(1-hydroxymeth-
2
2
2
The combined organic layers were washed with brine, dried over
yl-1H-indole-3-yl)pyrazolo [1,5-a]pyrimidine (23g). The compound
1
anhydrous Na SO , and concentrated in vacuo to provide the crude
was obtained as a yellow solid; yield: 80.6%; m.p. 150.3−151.6 °C. H
2
4
product, which was purified by column chromatography with DCM/
MeOH (100:1) to give pure compounds 23a−h.
NMR (400 MHz, methanol-d ): δ 8.41 (d, J = 4.5 Hz, 1H), 8.36 (d, J
4
= 7.8 Hz, 1H), 7.81 (s, 1H), 7.76 (d, J = 1.7 Hz, 1H), 7.67 (d, J = 1.7
Hz, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.28 (t, J = 7.3 Hz, 1H), 7.21 (t, J =
7
-(3,4-Dimethoxy-5-(methylselanyl)phenyl)-2-(1-methyl-1H-
indol-5-yl)pyrazolo[1,5-a]pyrimidine (23a). The compound was
7.4 Hz, 1H), 6.97 (s, 1H), 6.90 (d, J = 4.5 Hz, 1H), 5.63 (s, 2H), 3.98
1
13
obtained as a yellow solid; yield: 80.2%; m.p. 156.8−157.7 °C. H
NMR (400 MHz, CDCl ): δ 8.48 (d, J = 4.3 Hz, 1H), 8.31 (s, 1H),
(s, 3H), 3.97 (s, 3H), 2.33 (s, 3H). C NMR (101 MHz, CD OD): δ
3
3
152.9, 151.8, 150.2, 148.6, 148.4, 145.9, 136.4, 128.1, 127.6, 127.5,
126.4, 122.6, 121.5, 121.3, 120.8, 111.5, 109.9, 109.5, 106.0, 92.3,
7.96 (d, J = 8.5 Hz, 1H), 7.81 (s, 1H), 7.74 (s, 1H), 7.41 (d, J = 8.6
+
Hz, 1H), 7.12 (s, 1H), 7.10 (d, J = 3.0 Hz, 1H), 6.89 (d, J = 4.3 Hz,
69.3, 60.0, 55.9, 4.8. HRMS m/z: calcd for C H N O Se [M + H] ,
2
4
22
4
3
1
3
H), 6.58 (d, J = 2.8 Hz, 1H), 4.03 (s, 3H), 4.01 (s, 3H), 3.85 (s,
494.0857; found, 495.0949.
7-(3, 4-Dimethoxy-5-(methylselanyl)phenyl)-2-(4-
(methylsulfonyl)phenyl)pyrazolo [1,5-a]pyrimidine (23h). The
1
3
H), 2.40 (s, 3H). C NMR (101 MHz, CDCl ): δ 157.3, 151.8,
3
151.3, 148.5, 148.5, 145.3, 137.2, 129.6, 128.7, 128.1, 127.5, 124.2,
1
21.3, 120.4, 119.3, 111.6, 109.4, 106.2, 101.6, 93.0, 60.1, 56.1, 32.9,
compound was obtained as a yellow solid; yield: 85.6%; m.p.
+
1
5.2. HRMS m/z: calcd for C H N O Se [M + H] , 478.0908;
156.6−157.7 °C. H NMR (400 MHz, CDCl ): δ 8.56 (d, J = 4.1 Hz,
2
4
22
4
2
3
found, 479.0994.
1H), 8.21 (d, J = 8.2 Hz, 2H), 8.04 (d, J = 8.2 Hz, 2H), 7.69 (s, 1H),
4
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Article
.65 (s, 1H), 7.17 (s, 1H), 7.00 (d, J = 4.1 Hz, 1H), 4.02 (s, 3H), 3.98
incubated for 30 min in the dark. Then, samples were analyzed for the
DNA content with flow cytometry (BD FACSCanto II).
1
3
3
1
1
53.4, 151.9, 151.3, 149.4, 148.8, 145.9, 140.4, 138.2, 128.4, 127.9,
Analysis of Cancer Cell Apoptosis. MCF-7 cells were seeded into
6-well plates and cultured overnight. After treating with 12b (5, 10,
and 20 nM) for 48 h, cells were harvested and suspended in binding
buffer containing annexin V-FITC (0.5 mg/mL) and PI (0.5 mg/
mL). After that, samples were incubated for 30 min in the dark and
analyzed with a flow cytometer (BD FACSCanto II).
27.1, 126.8, 121.2, 111.5, 107.6, 94.6, 60.2, 56.1, 44.5, 5.1. HRMS
+
m/z: calcd for C H N O SSe [M + H] , 503.0418; found, 504.0492.
22
21
3
4
Biological Assays. Cell Lines and Cell Culture. The cancer cell
lines were purchased from American Type Culture Collection
(ATCC). Human breast cancer cells (MCF-7) and human lung
cancer cells (A549) were grown in RPMI-1640 medium (Life
Technologies, USA). Human cervical cancer cells and murine
melanoma cells were grown in Dulbecco’s modified Eagle’s medium
Wound Healing Assay. B16-F10 cells were seeded and incubated
in a 6-well plates overnight. Scratches were made in confluent
monolayers by a 200 μL pipette tip. Then, wounds were washed twice
with media to remove debris and uprooted cells. Cells were treated
with different concentrations (0, 5, 10, and 20 nM) of compound 12b.
Images were obtained using phase contrast microscopy at 0 and 24 h.
The migration distance of cells in the wound area was measured
manually.
(
Life Technologies, USA). The medium for all cell lines was
supplemented with 10% fetal bovine serum (Life Technologies, USA)
and 1% penicillin−streptomycin (Life Technologies, USA) and
maintained at 37 °C in a humidified atmosphere with 5% CO .
2
Cytotoxicity Assay. The cytotoxicity of the test compounds was
evaluated against MCF-7, A549, Hela, B16-F10, A2780S, and A2780/
T using the MTT assay. Cells were seeded into 96-well plates at a
density of 5000 cells/well. After removing the medium, 100 μL of
medium with 0.1% DMSO containing our antitubulin compounds at
different concentrations was added to each well and incubated at 37
In Vivo Antitumor Evaluation. The animal experiments were
performed with the approval of the National Institutional Animal
Care and Use Committee (IACUC) of Southern Medical University.
Male BALB/c mice, 6−8 weeks old, purchased from SPF (Beijing)
Biotechnology Co. Ltd., were used to study the inhibition effect of
compound 12b on the subcutaneous transplanted model of melanoma
°C for another 48 h. The MTT (5 mg/mL in PBS) was added and
6
incubated for another 4 h, and then, the absorbance was detected with
a microplate reader at a wavelength of 570 nm. The IC50 values were
calculated by nonlinear regression analysis using GraphPad Prism. All
the experiments were repeated in at least three independent
experiments.
cells. Logarithmic growth phase B16-F10 cells (4 × 10 per mL) were
suspended in PBS before injecting into mice. Tumors were
established by inoculating subcutaneously with 200 μL containing 8
5
× 10 cells into each mouse. Mice were divided into control or
treatment groups (n = 8) randomly. Compound 12b or paclitaxel was
dissolved in a 1:1 ratio of PEG300/PBS solution to produce the
desired concentrations. The vehicle control solution was formulated
with equal parts PEG300 and PBS only. Dose of the drug treatment
(paclitaxel, 10 mg/kg; compound 12b, 15, 30 mg/kg) or vehicle
control was administered via i.p. injection every day once for 14 days.
Body weight was monitored during the entire experiment period to
assess acute toxicity. Tumor volume was measured with a caliper and
In Vitro Antiproliferative Assay. Cells were seeded into 96-well
plates at a density of 5000 cells/well. After being treated with
compound 12b at the indicated concentrations or time points, the
MTT (5 mg/mL in PBS) was added and incubated for another 4 h.
Cell viability was detected with a microplate reader at a wavelength of
5
70 nm.
Colony Formation Assay. MCF-7 cells (1000 units) were counted
2
and seeded in 6-well plates. After being cultured for 24 h at 37 °C, the
medium was replaced with medium added with compound 12b at the
indicated concentration. After 24 h treatment, the medium was
changed to normal. After being cultured for another 10−14 days, the
colonies were fixed with 4% paraformaldehyde for 30 min and stained
with 0.1% crystal violet for 15 min. Following washing with PBS, the
colonies were then photographed and quantified using Image J.
In Vitro Tubulin Polymerization Assay. Pig brain microtubule
protein was isolated via three cycles of temperature-dependent
assembly/disassembly in 100 mM PIPES (pH 6.5), 1 mM GTP, 1
calculated using the formula a × b × 0.5, where a and b represent the
larger and smaller diameters, respectively. Mice were sacrificed 14
days after the initiation of the experiment, and the tumors were
weighed. The TGI value was calculated using the formula: TGI (%) =
[1 − W /W ] × 100%, where W and W are the mean tumor weight
t
v
t
v
of the treatment group and vehicle control. The harvested organs
(liver and kidney) were fixed in 4% paraformaldehyde, processed into
paraffin routinely, stained with H&E, and captured by the microscope.
Protein Expression and Purification. TTL protein was expressed
44
and purified from Escherichia coli, as described by Prota et al. (2013)
45
mM MgSO , 2 mM EGTA, and 1 mM 2-mercaptoethanol. Glycerol
and Wang et al. (2016). The stathmin-like domain of RB3 (RB3-
SLD) and TTL recombinant plasmids were transformed into E. coli
strain BL21(DE3) (TransGen Biotech) for protein expression. The
bacterial cells were grown in Luria broth supplemented with antibiotic
at 37 °C. RB3-SLD protein was induced by the addition of 0.4 mM
isopropyl-β-D-thiogalactoside at 37 °C for 3 h. Cells were collected
and sonicated in lysis buffer (20 mM Tris pH 8.0, 200 mM NaCl, 1
mM EDTA, 2 mM DTT, and 1 mM PMSF). Then, the protein was
purified via anion-exchange chromatography and gel filtration
chromatography. TTL was concentrated to 20 mg/mL and stored
at −80 °C.
4
and phenylmethylsulfonyl fluoride were added in 4 M and 0.2 mM,
respectively, in the first cycle of polymerization. Tubulin was prepared
from microtubule protein by phosphocellulose (P11) chromatog-
raphy, stored at −70 °C. Tubulin was mixed with different
concentrations of compounds in PEM buffer (100 mM PIPES, 1
mM MgCl , and 1 mM EGTA) containing 1 mM GTP and 5%
2
glycerol. Microtubule polymerization was detected by a spectropho-
tometer at 340 nm at 37 °C. The plateau absorbance values were used
for calculations.
Immunofluorescence Staining. B16-F10 cells were seeded on
glass coverslips in 24-well plates and then treated with vehicle control
Crystallization, Data Collection, and Processing. Detailed process
44
0
4
.1% DMSO, 12b (10, 20, 40 nM) for 6 h. The cells were fixed with
% paraformaldehyde and then penetrated with PBS containing 0.2%
to obtain crystals of T2R-TTL was described by Prota et al. (2013)
46
and Wang et al. (2017). The T2R-TTL complex was formed by
mixing tubulin, RB3-SLD, and TTL at a 2:1.3:1.2 molar ratio. Then, 5
mM tyrosine, 10 mM DTT, and 1 mM AMPPCP were added, and the
complex was concentrated to 20 mg/mL at 4 °C. Protein
crystallization trials were performed using 1 μL of protein mixed
with 1 μL of reservoir solution at 16 °C. Crystals appeared after
incubation at 16 °C and formed rodlike crystals within 3−5 days. A
volume of 0.1 μL of compound 12b, dissolved in 10 mM
dimethylsulfoxide, was added to a sitting drop containing crystals at
16 °C for soaking for 4 h. For data collection, all crystals were cryo-
protected by briefly soaking in reservoir solution supplemented with
20% (v/v) glycerol before flash-cooling in liquid nitrogen. Structures
Triton X-100. After blocking for 30 min by adding 100 μL of goat
serum albumin at room temperature, cells were incubated with a
monoclonal antibody (anti-β-tubulin) at 37 °C for 1 h. Then, the cells
were washed three times by PBS following staining by the
fluorescence secondary antibody. Nuclei were labeled by 4,6-
diamidino-2-phenylindole (DAPI). Cells were finally visualized
using a fluorescence microscope.
Cell Cycle Analysis. MCF-7 cells were seeded into 6-well plates and
cultured overnight. Cells were treated with 12b (5, 10, and 20 nM)
for 48 h. The collected cells were fixed at 4 °C with 70% ethanol
overnight. The fixed cells were washed and resuspended using PBS
with 10 mg/mL RNaseA and 50 mg/mL PI. After that, samples were
47
were solved by molecular replacement with Phaser using the T2R-
4
512
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Article
TTL structure (PDB ID: 7DBA) as template. The initial models were
ACKNOWLEDGMENTS
48
■
refined alternating cycles of automatic refinement with Refmac5 in
49
We thank Dr. Benoît Gigant [Institute for Integrative Biology
of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université
Paris-Saclay, France] and Dr. Michel O. Steinmetz (Paul
Scherrer Institute, Switzerland) for kindly providing the
plasmids of RB3-SLD and TTL. We also thank Jianjian
Zhou of Beijing Loham Trading Co. Ltd. for SPR assistance.
This work was supported by the scientific research project of
high-level talents (no. G618319142) in Southern Medical
University of China; International Science and Technology
Cooperation Projects of Guangdong Province (no.
G819310411); Thousand Youth Talents Program (no.
C1080092) from the Organization Department of the CPC
Central Committee, China; GDAS Special Project of Science
and Technology Development (2016GDASRC-0104 and
CCP4 program suite and manual model building with Coot. The
model quality checked with the PROCHECK program shows a good
50
stereochemistry, according to the Ramachandran plot. PYMOL
(http://www.pymol.org) was used to generate the figures.
ASSOCIATED CONTENT
■
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01837.
Colony formation assay on A2780T cells, binding
kinetics of compound 12b to tubulin (T2R-TTL),
binding poses for colchicine and compound 12b in
tubulin (PDB code: 7DBA), in vitro metabolic stability
2
018GDASCX-0107); and Guangdong Basic Research Foun-
1
13
studies, HRMS, infrared, H NMR, C NMR, and
HPLC (PDF)
dation (no. 2019A1515110616). The X-ray work was
supported by the National Major Scientific and Technological
Special Project for “Significant New Drugs Development” (no.
Molecular formula strings (CSV)
2
018ZX09201018-021), National Natural Science Foundation
of China (no. 81703553), China Postdoctoral Science
Foundation (nos. 2017M610607 and 2018T110984), and
Sichuan Science and Technology Program (no.
AUTHOR INFORMATION
■
Corresponding Author
2019YFS0003).
Jianjun Chen − School of Pharmaceutical Sciences, Guangdong
Provincial Key Laboratory of New Drug Screening, Southern
Medical University, Guangzhou 510515, China;
orcid.org/0000-0001-5668-6572; Email: jchen21@
smu.edu.cn
ABBREVIATIONS
■
ABI, 2-aryl-4-benzoyl-imidazoles; ABP, 6-aryl-2-benzoyl-pyr-
idine/phenyl; CBSIs, colchicine-binding site inhibitors; H&E,
hematoxylin and eosin; HRMS, high-resolution mass spec-
trometry; MDR, multi-drug resistance; MTAs, microtubule-
targeting agents; P-gp, P-glycoprotein; RI, resistance index;
SAI, substituted-2-aryl imidazoles; SMART, 4-substituted
methoxybenzoyl-aryl-thiazoles; SPR, surface plasmon reso-
nance; TGI, tumor growth inhibition; TMP, trimethoxyphenyl
Authors
Yichang Ren − School of Pharmaceutical Sciences, Guangdong
Provincial Key Laboratory of New Drug Screening, Southern
Medical University, Guangzhou 510515, China
Yuxi Wang − Targeted Tracer Research and Development
Laboratory, Precision Medicine Research Center, Department
of Respiratory and Critical Care Medicine, West China
Hospital, Sichuan University, Chengdu 610041, China
Gang Li − Guangdong Key Laboratory of Animal
Conservation and Resource Utilization, Guangdong Public
Laboratory of Wild Animal Conservation and Utilization,
Institute of Zoology, Guangdong Academy of Sciences,
Guangzhou 510260, China; orcid.org/0000-0003-4191-
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