Optimization of novel oxidative DIMs as Nur77 modulators of the Nur77-Bcl-2 apoptotic pathway

Article abstract of DOI:10.1016/j.ejmech.2020.113020

Nur77, an orphan nuclear receptor, is a member of the nuclear receptor superfamily. Nur77 plays important roles in various biological processes. Previously we reported that BI1071(DIM-C-pPhCF₃⁺MeSO₃^-), an oxidiz

Full text of DOI:10.1016/j.ejmech.2020.113020

European Journal of Medicinal Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Optimization of novel oxidative DIMs as Nur77 modulators of the
Nur77-Bcl-2 apoptotic pathway
,
,
Xuhuang Tu a 1, Xiaohui Chen a 1, Dongliang Zhang a, Meichun Gao a, Jingmei Liang a,
Guoliang Bao a, Jie Zhang a, Shuangzhou Peng a, Xiaokun Zhang a, Zhiping Zeng a
,
,
*
, b, **
Ying Su a
a School of Pharmaceutical Science, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Fujian, 361002, China
b NucMito Pharmaceuticals, Xiamen 361000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nur77, an orphan nuclear receptor, is a member of the nuclear receptor superfamily. Nur77 plays
important roles in various biological processes. Previously we reported that BI1071(DIM-C-
pPhCFþ3 MeSO3- ), an oxidized form and methanesulfonate salt of (4-CF3-Ph-C-DIM), can modulate Nur77’s
non-genomic apoptotic pathway through that Nur77 translocated from the nucleus to mitochondria to
induce cytochrome c releasing and promote apoptosis of cancer cell. Here we report our efforts to further
optimize BI1071. A series of BI1071 analogs were designed, synthesized and their apoptosis potency was
systematically evaluated. Our preliminary structure-activity relationship study identified compound 10b
as a better modulator with strong binding to Nur77 and enhanced apoptotic activity. Binding studies
demonstrated that 10b could bind to its target Nur77 with an affinity value of 33 nM. Furthermore,
mechanism studies reveal that 10b acts as an anticancer agent by utilizing the Nur77-Bcl-2 apoptotic
pathway.
Received 21 July 2020
Received in revised form
10 November 2020
Accepted 11 November 2020
Available online xxx
Keywords:
Nur77
Nur77 modulator
Nur77/Bcl-2 apoptotic pathway
Oxidized DIM
BI1071
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
Paradoxically, Nur77 also exerts death effect, perhaps being the
most potent pro-apoptotic member in the nuclear receptor super-
Nur77 (also known as TR3 and NGFI-B), a unique orphan member
of the nuclear receptor superfamily and an immediate-early
response gene, regulates diverse biological processes in response
to various extracellular stimulations including growth factors, stress
and death signaling [1,2]. Nur77 was originally recognized for its pro-
active role in cell survival, proliferation and differentiation. It plays
an important role in cell proliferation, differentiation, apoptosis,
metabolism and immunity [3e11] of cancer cells. Overexpression of
Nur77 in precancerous or several cancer cells, including colon, lung,
and breast tumors [12e15], stimulates cell cycle progression and
proliferation to maintain their growth and survival [16]. In contrast,
low expression of Nur77 in cancer cells inhibits the growth, survival
and migration of tumor [8,17e19].
family [1,13,14,20e22]. Nur77 expression is rapidly induced during
apoptosis of immature thymocytes, T-cell hybridomas [23,24]. It is
also a key mediator of apoptosis of cancer cells induced by
chemotherapeutic agents [25e27]. The apoptotic effect of Nur77
appears to be clinically relevant, as gene expression profiles of
human tumor samples reveal that downregulation of Nur77 is
associated with metastasis of several primary solid tumors,
including lung cancer, breast cancer, and prostate cancer [28]. In
studying the molecular mechanism by which Nur77 triggers cell
death program, we found that it could translocate from the nucleus
to mitochondria, where it induces cytochrome c release and
apoptosis in response to some apoptotic stimuli [29]. Our later
studies demonstrated that the apoptotic effect of Nur77 is mediated
by its interaction with Bcl-2, an anti-apoptotic Bcl-2 family mem-
ber. The interaction between Nur77 and Bcl-2 converts Bcl-2 from
an anti-apoptotic to a pro-apoptotic protein to induce apoptosis of
cancer cells [30]. Overexpression of the anti-apoptotic protein Bcl-2
is commonly associated with various cancers including ER-positive
[31], triple negative breast cancer (TNBC) [32], and colon cancer
[33,34]. Thus, the ability of Nur77 to interact with Bcl-2 not only
* Corresponding author.
** Corresponding author. School of Pharmaceutical Science, Xiamen University,
NucMito Pharmaceuticals, Xiamen 361000, China.
E-mail addresses: zengzhiping@xmu.edu.cn (Z. Zeng), ying.su@nucmito.com
(Y. Su).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2020.113020
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
Please cite this article as: X. Tu, X. Chen, D. Zhang et al., Optimization of novel oxidative DIMs as Nur77 modulators of the Nur77-Bcl-2 apoptotic
pathway, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.113020

X. Tu, X. Chen, D. Zhang et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
suppresses its anti-apoptotic function but also converts Bcl-2 into a
pro-apoptotic molecule [29,30], providing an attractive strategy to
induce apoptosis of TNBC and colon cancer cells with elevated
levels of Nur77 and Bcl-2.
ortho/meta/para positions of the phenyl ring (compounds 8a - 8y).
In addition, we also studied the possibility of replacing the whole
phenyl group with other aromatic rings (9a - 9f).
We also found that the indolyl moiety 2 of BI1071 lack good
contacts with its surrounding hydrophobic region (sub-region 2,
Fig. 3A) formed by helices 5, 7 and 8. To take advantage of this
hydrophobic niche, several alkyl chains of different lengths were
introduced on the NeH position of indole ring 2 of BI1071(10b -
10f), aiming to enhance its binding affinity towards Nur77.
The PAINS (Pan analysis interfering compounds) evaluation was
performed to exclude any compounds with potential issues of
giving false positive results [40]. All of the designed compounds, 8a
e 8y, 9a - 9f, and 10a - 10f passed the tests carried out using
FAFDrugs4 (https://fafdrugs4.rpbs.univ-paris-diderot.fr/) or False
Positive Remover (https://www.cbligand.org/PAINS/).
However, agents that can bind directly to Nur77 to trigger its
mitochondrial localization and Bcl-2 interaction remain to be
explored. Our group previously reported that an oxidation form of
Ph-C-DIM (BI1071, DIM-C-pPhCFþ3 MeSO-3, Fig. 1B) binds Nur77 with
submicromolar affinity, and effectively induces apoptosis of cancer
cells in a Nur77-and Bcl-2-dependent manner [35]. As the first
identified small molecule modulator of the Nur77-Bcl-2 apoptotic
pathway, BI1071 directly binds to Nur77 to induce Nur77 trans-
location from nuclear to mitochondria where it interacts with Bcl-2,
resulting in the release of cytochrome c and apoptosis of cancer
cells, which warrants further optimization.
Here we optimized the hit compounds targeting Nur77 as
potent anticancer agent, BI1071, which belongs to oxidative DIMs.
We carried out the chemical optimization on the phenyl ring and
the NeH position of indolyl ring of BI1071 (Fig. 3B) by rational drug
design strategy (Figs. 2 and 3). Totally 36 novel oxidative DIM de-
rivatives were designed, synthesized and bio-evaluated for their
apoptosis activity in TNBC cell line MDA-MB-231 and colon cancer
cell line HCT116. Moreover, the primary biological mechanism
research, anticancer ability in vivo, and pharmacokinetic properties
of final optimized compounds were studied.
3. Chemistry
Preparation of the total 36 target compounds (8a - 8y, 9a - 9f,
and 10a - 10f) was described in Scheme 1. Commercially available
indole derivatives (1a, 1b) and aryl aldehyde derivatives (2a e 2y,
2a’ e 2f’) were treated with hydrochloric acid to yield Ph-C-DIM (3a
e 3y, 4a e 4f, 5a) [41]. For asymmetric Ph-C-DIM (7a - 7f), firstly,
indole (1a) and 4-(trifluoromethyl)benzaldehyde (2n) were treated
with tetramethylguanidine in H2O to yield secondary alcohol (6a)
[42]. The secondary alcohol is then treated with the indole de-
rivatives (1b - 1f) in trifluoroethanol at 50 ꢀC to obtain asymmetric
Ph-C-DIM (7a - 7f) with good yield [43]. The Ph-C-DIM (3a e 3y, 4a
e 4f, 5a, 7a - 7f) was oxidized by pyridinium dichromate in the
presence of methanesulfonic acid to yield Ph-C-DIMþMeSO3- (8a -
8i, 8l - 8n, and 10a - 10b), and hydrochloric acid to yield Ph-C-
DIMþClꢁ (8a-1, 8f-1, 8j - 8k, 8n-1, 8o - 8y, 9a - 9f, and 10c - 10f).
Reagents and conditions: (a) HCl, MeOH, r. t., (b) Pyridinium
dichromate, acid, MeOH, r. t., 1 h b1, MeSO3H, b2, HCl (37%). (c)
Tetramethylguanidine, H2O, r. t., 24 h. (d) 2,2,2-trifluoroethanol,
50 ꢀC, 6 h.
2. The rational design of novel BI1071 derivatives targeting
Nur77
Nur77 ligand binding pocket were occupied by bulk side chains
[36], and no endogenous ligands have yet been identified. However,
several grooves on the surface of the ligand binding domain (LBD)
of Nur77 were identified as small-molecule binding regions
[37e39]. Our previously work reported that BI1071 could bind to
the site B on Nur77-LBD, a surface groove formed by helices H1, H5,
H7, H8, loops H1eH2, and H5eB1 (Fig. 2A) [35]. In this binding
model, indole ring 1 interacts with His372 through hydrogen
bonds, and the other indole ring 2 forms pi-pi stacking with Try453.
Side chain of Lys456 interacts with phenyl ring of BI1071 through
pi-cation pattern. Furthermore, the phenyl ring and the indolyl
rings make van der Waals interactions with the sidechains of
Leu382, Leu373, Pro377, Ile463 and Val500 that form a hydrophobic
environment. (Fig. 2B).
The docking model also suggests that some binding regions of
the hit compound BI1071 could be optimized by chemistry efforts.
The phenyl group has only partial contacts with its surrounding
groove (sub-region 1, Fig. 3A) formed by Thr379, Leu382, Ile463,
and Lys456, implying that chemical substitutions on the phenyl
moiety of BI1071 could be explored. Thus, to study the spacious
tolerability of sub-region 1, a variety of substituents possessing
different electron properties and sizes were introduced to the
4. Result and discussion
4.1. Apoptosis assay and SAR analysis
First, all the compounds were evaluated by poly (ADP-ribose)
polymerase (PARP) cleavage assay for their apoptotic effects in
MDA-MB-231 and HCT116 cell lines. The compounds’ ability to
induce apoptosis was quantitatively analyzed using the intensity
ratio between the PARP cleavage band and the PARP band. Healthy,
normal cells displayed no PARP cleavage or little PARP cleavage,
while the apoptotic cells showed strong PARP cleavage. For easy
analyses, each compound’s apoptotic effect was compared to the
apoptotic effect of BI1071 (8n) of which the intensity ratio was set
to 1 and relative intensity ratios were calculated. Western blot re-
sults and the corresponding quantitative calculation of the relative
intensity ratio of PARP cleavage band/PARP band were shown in
Fig. 4 - 8, and Tables S1eS6. We then analyzed the structure and
apoptotic activity relationships (SAR) of the target compounds.
The organic-salt of Ph-DIM-CþMeSO-3 consists of the cationic
moiety of Ph-DIM-Cþ and the anion of MeSO-3, so, intuitively we
first investigated whether using different anions for the organic-
salt would have an impact on their apoptotic activities. Based on
the synthesis feasibility, inorganic acid HCl and organic acid
MeSO3H were compared. For 8n (BI1071), anions MeSO-3 (8n) and
Clꢁ (8n-1) displayed similar apoptotic effect in MDA-MB-231 and
HCT116 cell lines (Fig. 4). Similarly, for compound 8a and 8f (in
MeSO-3 form), when the anion was Clꢁ, not apparent differences
were observed for their apoptotic effects (Fig. 4). Impact of different
Fig. 1. (A) The structure of DIMs (4-CF3-Ph-C-DIM). (B) The oxidative DIMs (BI1071,
DIM-C-pPhCFþ3 MeSO3- ).
2

X. Tu, X. Chen, D. Zhang et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 2. The binding model of BI1071 targeting Nur77. A. The location of BI1071 on the surface of Nur77-LBD (3V3Q). The protein was represented as surface mode, and BI1071 was
shown as stick. The hydrophobic residues were colored in gray, and the polar residues were decorated with blue and red. B. the ligand-residue interaction of BI1071 with Nur77-
LBD. The H-bonds, pi-pi stacking and pi-cation interaction were shown in yellow dashed lines, cyan dashed lines, and magenta dashed lines, respectively. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of this article.)
Its activity wasn’t rescued when the strong electron-donating
group (-OH, 8f) was used to replace the hydrogen group (8a).
Increasing the steric effect at para-position by using a methoxy
group (8e) or the replacement of the eCF3 with methyl (8b), ethyl
(8c) or t-butyl (8d) group did not gain any improvement on the
apoptotic activity compared to 8n, though they are more active
than the hydrogen-substituted compound 8a (Fig. 5A). Substituents
with the electron-withdrawing natures, such as -Ph (8g), -Cl (8h),
eF (8i), eCOOCH3 (8j), eCOOH (8k) and eNO2 (8l) at the para-
position of the phenyl ring were explored. Unfortunately, all the
above analogs demonstrated weaker apoptotic effect than 8n
(Fig. 5B). Hence, we can conclude that the eCF3 group (8n) at the
para-position of the phenyl group is the best choice. It was reported
that fluorine substitution could enhance hydrophobic interactions
and the lipophilicity of drugs [44,45]. In the binding model of
BI1071 (Fig. 2), para-CF3 group is surrounded by hydrophobic side
chains of Leu 382, Ile463 and Lys456, which may explain why eCF3
group worked the best at the para-position of the phenyl group.
Subsequently, we examined the impact of introducing sub-
stituents at the meta-position of the phenyl ring. Results of PARP
cleavage assay showed that the electron donating group such as 8o
(-CH3) or electron-withdrawing groups such as 8p (-OCH3), 8q
(-OH), 8r (-Cl), and 8s (-F) displayed sharp decrease in the apoptotic
activity (Fig. 6). However, 8t with meta-CF3 substitution exhibited
better apoptotic activity in MDA-MB-231 cell line (relative intensity
Fig. 3. Design strategy based on the BI1071 binding profile. A. Surface binding analysis
of BI1071 in Complex with Nur77-LBD (3V3Q). The protein was represented as surface
mode, and BI1071 was shown as green stick. The hydrophobic residues were colored in
gray, and the polar residues were decorated with blue and red, respectively. B. Design
strategies used in this present study. (For interpretation of the references to color in
this figure legend, the reader is referred to the Web version of this article.)
ratio ¼ 1.76, Table S4) compared to reference compound 8n.
Docking model showed that 8t shared similar binding pose with 8n
(Fig. S1). Notably, the meta-CF3 group of 8t was buried and may
make stronger interactions with the hydrophobic surroundings
formed by Pro377, Leu382 and Ile463.
Similarly, introducing eOH, -Cl, or eF substituent at the ortho-
position of the phenyl ring (8u (-OH), 8v (-Cl) and 8w (-F)) led to an
activity reduction (Fig. 6). However, it is surprising to find that the
ortho-CF3 group (8x, eCF3) or 2,4-di-CF3 substituent (8y, 2,4-di-CF3)
were not favorable changes, acting differently from the para-CF3
group or meta-CF3 group (Fig. 6). In binding model of BI1071, the
angle between phenyl ring and bis-indole rings 1 and 2 were 62.2ꢀ
and 73.4ꢀ, respectively. And the ring angel of the two bis-indole
rings is 80.6ꢀ (Fig. S2). However, in the minimized-energy confor-
mation of 8x, the ring angle were altered largely because of the
steric hindrance of ortho-CF3 group. The angle between phenyl ring
and each bis-indole rings 1 and 2 were 79.0ꢀ and 80.5ꢀ respectively,
and the ring angel of the two bis-indole rings was 57.0ꢀ (Fig. S2). It
is likely due to this conformation change, 8x could no longer dock
well into the Nur77-LBD binding site.
anions on the in vitro anticancer effect was also evaluated. The IC50
of compound 8n was measured as 0.22
231 cell line and 0.27 0.10 M in HCT116 cell line respectively. 8n-
1 displayed almost the same anticancer effect with an IC50 value of
0.22 0.09 M in MDA-MB-231 and 0.26 0.11 M in HCT116 cell
0.10 mM in MDA-MB-
m
m
m
lines, respectively (Table 1). Taken together, these results indicated
that the anion type MeSO-3 and Clꢁ have no obvious different effects
on the compounds’ bioactivities. Therefore, either MeSO-3 or Clꢁ
was used to make BI1071 analogs, based on the synthesis feasibility.
Next, we examined the SAR at the phenyl group (-R) of the
BI1071. A variety of substituents possessing different electron
property were introduced to the ortho-, meta-, and para-positions
of the phenyl ring of BI1071 for structural optimization. For phenyl
ring without eCF3 substitution, 8a (relative intensity ratio ¼ 0.01,
Table S2) showed significant decline in apoptotic activity in MDA-
MB-231 cancer cell compared to reference compound BI1071(8n).
Replacing the whole phenyl moiety with other aromatic het-
erocyclic groups, such as five-member ring (2-furyl 9a, 2-thienyl
3

X. Tu, X. Chen, D. Zhang et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Scheme 1. Synthetic route of target compounds.
Fig. 4. Apoptotic activity of BI1071 derivatives with anion type of MeSO-3 or Clꢁ. MDA-MB-231 and HCT116 cells were treated with compounds (0.5
cleaved PARP were analyzed by western blotting. The relative intensity ratio of PARP cleavage band/PARP band was counted and analyzed. Data are presented as mean SD.
mM) for 6 h, then PARP and
9b), six-member ring (3-pyridyl 9c), and aromatic fused ring (3-
Indolyl 9d, 3-benzothienyl 9e), resulted in a dramatical decrease
in the apoptosis activity (Fig. 7). These results are consistent with
8a (Fig. 5A). Given the importance of the para-CF3 group or the
meta-CF3 group, we introduced one eCF3 group on the 3-pyridyl
ring (9f). Consistently 9f exhibited comparable activity to B1071
(Fig. 7, Table S5). Together, the results indicated that a eCF3 group
that is properly positioned on a aryl ring is essential for the
apoptotic activity.
The binding model of BI1071 (Fig. 2B) showed that the NeH of one
indole ring (indolyl 1, Fig. 2B) could form a hydrogen bond with the
sidechain of His372. The importance of this hydrogen bond
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European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 1
IC50 values of selected compounds against TNBC and colorectal cancer cells.
Ar
R1
X-
IC50 (mM)
MDA-MB-231
BT549
MCF10A
HCT116
SW620
NCM460
8n
8n-1
8t
4-CF3-Ph
4-CF3-Ph
3-CF3-Ph
6-CF3-3-pyridyl
4-CF3-Ph
H
H
H
H
-Me
-Et
-Pr
MeSO3
Cl
Cl
Cl
MeSO3
Cl
0.22 0.10
0.22 0.09
0.29 0.10
0.23 0.11
0.06 0.04
0.08 0.06
0.17 0.10
0.17 0 .01
0.17 0.01
0.07 0.02
0.05 0.02
0.04 0.02
0.05 0.02
0.10 0.02
4.29 0.09
3.58 0.14
5.03 0.09
2.98 0.16
2.19 0.07
1.50 0.11
1.31 0.16
0.27 0.10
0.26 0.11
0.31 0.11
0.35 0.13
0.09 0.05
0.10 0.07
0.35 0.08
0.18 0.02
0.18 0.02
0.13 0.02
0.14 0.02
0.10 0.03
0.10 0.03
0.16 0.02
6.84 0.07
5.13 0.20
10.0 0.15
8.10 0.25
4.63 0.20
3.08 0.13
5.09 0.10
9f
10b
10c
10d
4-CF3-Ph
4-CF3-Ph
Cl
IC50: Concentration of the compound producing 50% cell growth inhibition after 24 h of drug exposure, as determined by the MTT assay. Data are presented as mean SD. Each
experiment was performed at least 6 times. -Me: methyl, -Et: ethyl, -Pr: n-propyl.
Fig. 5. Apoptotic activity of BI1071 derivatives with different groups at the para-position of phenyl ring. MDA-MB-231 and HCT116 cells were treated with compounds (0.5 mM) for
6 h, and then cleaved PARP were analyzed by western blotting. The relative intensity ratio of PARP cleavage band/PARP band were counted and analyzed. (A) electron-donating
groups as substitute at the para-position. (B) electron-withdrawing groups as substitute at the para-position. Data are presented as mean SD.
interactionwasvalidated by10a (Fig. 8), inwhichreplacing theH with
eCH3 on the NeH of both indole rings led to a sharp decrease in its
apoptotic activity. The binding model of BI1071 (Fig. 2B) also showed
that the other indole ring (indolyl 2, Fig. 2B) bound to an open groove
with limited contacts with the protein and lipophilic groups as sub-
stituents at the NeH of the indole ring could introduce additional
hydrophobic interaction with the protein. Indeed, compound 10b
with a eCH3 group at the NeH displayed much enhanced apoptotic
activity compared to 8n (Fig. 8, relative intensity ratio ¼ 8.93 in MDA-
MB-231 and 13.54 in HCT116, Table S6). Next, we explored the
apoptotic effect of alkyl chains with different lengths at the NeH
position of just one indole ring. Result exhibited that the apoptotic
activity was gradually reduced when the length of alkyl chain was
increased (10b, 10c, 10d, 10e and 10f) (Fig. 8). Modeling showed that
the alkyl chain substituents at the NeH of indole ring 2 could enhance
the interactions between the compound and the protein, however,
steric hindrance would occur when the size of the alkyl chain
increased, weakening the compound binding (Fig. 11).
PARP cleavage assay (Fig. 9A). 10b showed stronger apoptotic ac-
tivity than 8n. The effects of 8n and 10b on cell death were further
assessed using flow cytometry-based Annexin V/Propidium iodide
(PI) apoptosis assay. Fig. 9B showed that about 65.53% of MDA-MB-
231 cells were apoptotic when treated with 0.5 mM of 10b for 6 h,
while only 12.83% of cells were apoptotic in 8n-treated cells.
Similar results were observed in BT549 (8n-24.86%, 10b-84.72%),
HCT116 (8n-14.63%, 10b-64.42%) and SW620 (8n-28.15%, 10b-
92.54%) cancer cells (Fig. 9B and C). Collectively, these data sug-
gested that the pro-apoptotic effect of 10b was stronger than 8n in
cancer cells.
4.2. In vitro anti-proliferative activity of the potent compounds
The potent compounds 8n(BI1071), 8n-1, 8t, 9f, 10b, 10c and
10d were further evaluated for their anticancer effect in both TNBC
(MDA-MB-231 and BT 549) and colorectal (HCT116 and SW620)
cancer
cells
using
the
3-(4,5-dimethylthiazol-2-yl)-2,5-
The apoptotic effects of 8n(BI1071) and 10b were further tested
in TNBC cell line BT549 and colorectal cancer cell line SW620 using
diphenyltetrazolium bromide (MTT) assay, and the IC50 values
were listed in Table 1. Among these compounds, compounds 10b
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European Journal of Medicinal Chemistry xxx (xxxx) xxx
cells MCF10A was about 40-fold higher than in MDA-MB-231 and
BT549, while in normal colon cells NCM460 was about 50-fold
higher than in HCT116 and SW620.
4.3. Compounds’ binding affinities to Nur77 protein
Next we evaluated the binding affinities of 10b and 10c to Nur77
protein. Fluorescence quenching assay and surface plasmon reso-
nance (SPR) were utilized. The fluorescence emission spectra of
Nur77-LBD (1
mM) in the presence of serial compound concentra-
tions (0.01e1
mM) are shown in Fig. 10A. Interactions between the
protein and compounds induced fluorescence quenching. The
dissociation constant (Kd) of BI1071 (8n) was measured as 217 nM
(Fig. 10A). Compounds 10b and 10c bound to Nur77 with higher
affinity than 8n (Kd was 66 nM and 66 nM respectively, Fig. 10A).
SPR was performed on BIAcore T200 instrument. Nur77-LBD was
immobilized on a CM5 sensor chip, and gradient concentrations of
compounds (0.01e5
mM) were injected into the flow cells in
running buffer. The dissociation constant (Kd) of 8n, 10b and 10c
was measured as 110 nM, 33 nM and 36 nM, respectively (Fig. 10B).
Taken together, results from the fluorescence quenching assay and
the SPR methods consistently demonstrated that both 10b and 10c
bind better to Nur77 than 8n.
4.4. Molecular docking analysis
Fig. 6. Apoptotic activity of BI1071 derivatives with different groups at the meta- and
ortho-position of phenyl ring. MDA-MB-231 and HCT116 cells were treated with
Compounds 10b and 10c displayed higher binding affinity to
Nur77-LBD and improved anticancer activity, thus we perform
molecular docking to further understand their binding mechanism.
For 10b and 10c, the optimization strategy was to mimic the
binding model of BI1071 while introducing different alkyl groups at
the NeH group on one of the indole rings to enhance compound’s
interaction with Nur77. The docking results showed that the
binding models of 10b and 10c were almost identical to BI1071
(Fig. 11). Indole ring 1 without alkyl substitution could establish a
hydrogen bond with the polar residue of His372. Also, the phenyl
ring could form pi-cation with Lys456. It is worth noting that the
central carbonium of 10b and 10c other than the indolyl like BI1071
could form additional pi-cation interaction with Try453. The
NeCH3 of 10b and the NeCH2eCH3 of 10c positioned closely to
His494 to form good van der Waals interaction. The docking results
may explain why compounds 10b and 10c exhibit slightly enhanced
activity compared with BI1071.
compounds (0.5
mM) for 6 h, and then cleaved PARP were analyzed by western blot-
ting. The relative intensity ratio of PARP cleavage band/PARP band were counted and
analyzed. Data are presented as mean
SD. *p < 0.05 compared to the 8n treated
group (n ¼ 3).
and 10c displayed stronger anti-proliferative activity than 8n in
both of breast cancer cells (MDA-MB-231: 0.06 0.04 M for 10b
and 0.08 0.06 M for 10c, BT549: 0.04 0.02 M for 10b and
M for 10c) and colorectal cancer cells (HCT116:
M for 10b and 0.10 0.07 M for 10c, SW620:
M for 10c). We further used
m
m
m
0.05
0.09
0.02
0.05
m
m
m
0.10 0.03
mM for 10b and 0.10 0.03 m
the non-cancerous breast epithelial cell line MCF10A and the non-
transformed human colonic epithelial cell line NCM460 to test the
compounds’ selectivity against cancer cell lines. Compounds dis-
played good selectivity against cancer cells (Table 1). The most
potent compound 10b showed that the IC50 in normal mammary
Fig. 7. Apoptotic activity of BI1071 with its phenyl ring replaced by different aromatic rings. MDA-MB-231 and HCT116 cells were treated with compounds (0.5 mM) for 6 h, and then
cleaved PARP were analyzed by western blotting. The relative intensity ratio of PARP cleavage band/PARP band were counted and analyzed. Data are presented as mean SD.
6

X. Tu, X. Chen, D. Zhang et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 8. Apoptotic activity of B1071 derivatives with substituents at the NeH of one indole ring. MDA-MB-231 and HCT116 cells were treated with compounds (0.5 mM) for 6 h, and
then cleaved PARP were analyzed by western blotting. -Me: methyl, -Et: ethyl, -Pr: n-propyl, -Bu: n-butyl, -Pe: n-pentyl. The relative intensity ratio of PARP cleavage band/PARP
band was counted and analyzed. Data are presented as mean SD. ***p < 0.001 compared to 8n treated group (n ¼ 3).
4.5. 10b induces Nur77 mitochondrial targeting and its apoptotic
effect is Nur77-dependent
treated with 10b (Fig. 13B). Moreover, confocal microscopy analysis
revealed that 10b promoted extensive mitochondrial colocalization
of transfected GFP-Nur77 (Fig. 13C) or Myc-Nur77-LBD (Fig. 13D)
with Bcl-2 in cells. Thus, binding of compound 10b to Nur77 pro-
motes Nur77 interaction with Bcl-2 and their mitochondrial co-
localization.
We next determined whether the strong apoptotic effect of 10b
is Nur77-dependent by examining its apoptotic activity in mouse
embryonic fibroblast (MEF) and MEF lacking Nur77 (Nur77ꢁ/ꢁMEF).
As expected 10b, 10c, 10d, and 9f and could indeed induce the PARP
cleavage in MEFs. However, such an apoptotic effect was signifi-
cantly diminished in Nur77ꢁ/ꢁMEFs (Fig. 12A), implying that the
apoptotic activities of these BI1071 analogs are Nur77-dependent.
We showed previously that 8n exerted its Nur77-dependent
apoptotic effect by promoting Nur77 mitochondrial targeting
[35]. Thus, we compared the effect of 10b with 8n on Nur77
mitochondrial targeting. HEK293T cells were transfected with Myc-
4.7. Antitumor effect of compound 10b in MMTV-PyMT-transgenic
mouse models
10b showed stronger apoptotic effect than BI1071 in vitro.
Therefore, its anticancer therapeutic potential was further investi-
gated in vivo. MMTV-PyMT-transgenic mouse model was used for
evaluating the in vivo efficacy of 10b [35]. Administration of the
MMTV-PyMT mice with BI1071 (8n) and 10b (3 mg/kg) potently
inhibited the growth of PyMT mammary tumor (Fig.14A and B).10b
possessed stronger in vitro apoptotic effect than 8n in both of MDA-
MB-231 and HCT116 cancer cells (Fig. 8 and Table 1). However, this
differencewas notdirectly translated in invivomodel. We foundthat
the potency of 10b was only slightly superior to 8n (Fig. 14C), which
could be attributed to their differences in ADME properties [46]. It is
worthwhile noting that administration of 10b did not show any
apparent toxic effects such as loss of body weight (Fig. 14D).
Nur77 and subsequently treated with 0.5
mM 8n or 10b.
Mitochondria-specific tracker using confocal microscopy revealed
the localization of Myc-Nur77-LBD. The result showed that signif-
icantly more amount of transfected Myc-Nur77 accumulated in the
mitochondria fraction when cells were treated with 10b than with
8n (Fig. 12C). The result was further confirmed by Western blotting
(Fig. 12B). Taken together, these data demonstrated that compound
10b exerted its Nur77-dependent apoptotic effect by inducing
stronger Nur77 mitochondrial targeting than 8n.
4.6. Compound 10b promotes the interaction between Nur77 and
4.8. Pharmacokinetic (PK) study of compounds 10b and BI1071 in
Bcl-2 to induce Bcl-2 dependent apoptosis
rats
We have reported that BI1071 (8n) induces apoptosis of cancer
cells by activating the Nu77-Bcl-2 apoptotic pathway [35]. We
therefore asked if derivatives of BI1071 utilize the same pathway to
induce apoptosis by promoting the interactions between Nur77
and Bcl-2. The apoptotic effect of compounds 8n, 10b, 10c, 10d, and
9f was evaluated in MEFs and MEFs lacking Bcl-2 (Bcl-2ꢁ/ꢁMEF). At
To gain some insight into why 10b’s superiority to BI1071(8n)
in vitro did not translate in vivo, we carried out pharmacokinetic
(PK) study of compounds 10b and BI1071 (8n) in Sprague-Dawley
(SD) rats. Compounds were administered by oral absorption (PO,
5 mg/kg) or intravenous injection (IV, 0.5 mg/kg). It is worth noting
that 8n displays more favorable pharmacokinetic properties in fe-
male rat than male rat (Table S7). As shown in Table 2, 8n has a
better oral bioavailability in female rats (F ¼ 22.79%) than in male
rats (F ¼ 20.12%), and possesses a higher maximal plasma con-
0.5 mM, all test compounds effectively induced PARP cleavage in
MEFs, while they had no apparent effect on PARP cleavage in Bcl-
2ꢁ/ꢁMEFs (Fig. 13A). Then we determined if 10b can induce Nur77
and Bcl-2 interaction. Cell-based Co-IP showed that Nur77 trans-
fected in HEK293T cells interacted with Bcl-2 when cells were
centration as well in female rats (Cmax ¼ 203.25
17.15 mg/L in
female and Cmax ¼ 143.37 20.05 g/L in male). In addition, the
m
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X. Tu, X. Chen, D. Zhang et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
maximum plasma concentration. Tmax, the time of maximum
concentration. T1/2, elimination half-life. MRT, mean residence
time. CL, total plasma clearance. Vss, apparent volume of the plasma
compartment. F, oral bioavailability. PO, per oral. IV, intravenous. a,
the number of rats is 3. b, the number of rats is 4, 2 male and 2
female rats.
5. Conclusion
Compound BI1071 (8n) is a novel modulator of Nur77. In this
study we carried out some preliminary optimization work of
BI1071 using the structure-based design approach. 36 BI1071 de-
rivatives were synthesized and evaluated for their apoptosis ac-
tivities. Analyses of the structure activity relationship (SAR)
indicated that: a): A eCF3 group at the meta- or para-position of
phenyl ring is essential for maintaining the outstanding apoptosis
activity. b): The ortho-substituent on the phenyl ring goes against
its activity. c): The activity of the asymmetric mono-alkyl substi-
tution on the NeH of one indole moiety is better than the sym-
metric di-alkyl substitution, and the alkyl chain with 1e3 carbons is
the most favorable moiety. Among the 36 derivatives, compound
10b showed the most potent activity at molecular, cellular and
animal levels. 10b could bind to target protein Nur77 with higher
affinity and strongly activate the Nur77-Bcl-2 apoptotic pathway.
The PK profiling in rats reflected that 10b displayed a suitable drug
exposure (AUC) and half-life (T1/2), but a low bioavailability. Hence,
compound 10b has been selected as a lead candidate and is
currently undergoing further optimization in our follow-up studies.
6. Experimental section
6.1. Chemistry
All the materials were obtained from commercial suppliers and
used without purification, unless otherwise specified. 1H NMR
(600 MHz) and 13C NMR (150 MHz) spectra were performed on
Bruker spectrometers (Bruker in Asia Pacific, Beijing, China) in
DMSO‑d6 or CD3OD with TMS as the internal reference. The values
of chemical shifts are expressed in ppm. MS spectra were taken in
ESI mode on Q-Exactive apparatus (ThermoFisher, Shanghai,
China). Column chromatography was performed on Biotage
(Prime). The purity of all tested compounds was detected by HPLC
performed on waters e2695 instrument.
Fig. 9. Apoptotic activity of 8n(BI1071) and 10b in TNBC and colorectal cancer cells. (A)
BT549 and SW620 cells were treated with 0.5 mM 8n and 10b respectively for 6 h, and
then cleaved PARP were analyzed by western blotting. Data are presented as
mean SD. ***p < 0.001 compared to 8n treated group (n ¼ 3). (BeC) Annexin V/PI
staining of MDA-MB-231, BT549, HCT116 and SW620 cells treated with 0.5
mM 8n and
6.1.1. General procedure for the synthesis of symmetric Ph-C-DIM
(3a e 3y, 4a - 4f, 5a)
10b respectively for 6 h was analyzed by flow cytometry. Data are presented as
mean SD. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control group (n ¼ 3).
Indole (50.0 g, 0.427 mol,
2
equiv) and p-tri-
fluoromethylbenzaldehyde (37.1 g, 0.214 mol, 1 equiv) and CH3OH
(200 mL) were added to the round-bottom flask (500 mL). Then 37%
HCl(2.34g,0.0214mol, 0.1equiv)wasdilutedinwater(19mL,ten-fold
dilution), the aqueous was slowly added to round-bottom flask. The
mixture was stirred at room temperature, and monitored by TLC until
thereactionwascompleted. Thesolutionwasneutralizedby5%NaOH
to pH ¼ 7. The solvent methanol was evaporated under reduced
pressure to give oily liquid mixed with water. The resident was
extracted with ethyl acetate (50 mL * 3). The EA phase was combined
and dried over anhydrous Na2SO4. The organic solvent was evapo-
rated to obtain a crude product which was purified by recrystalliza-
tion (CH3OH/H2O), p-CF3-Ph-C-DIM yielded as a white powder
(80.2 g, 96.4%)
time of maximum concentration (Tmax ¼ 5.33 2.31 h) was the
same for both the male and female rats, but the elimination half-life
and the mean residence times in female rats (T1/2 ¼ 6.81 4.35 h,
MRT ¼ 14.50
0.78 h) are slight longer than male rats (T1/
¼ 5.28 2.91 h, MRT ¼ 12.32 1.06 h). It is interesting to note that
2
10b displayed less gender differences between male and female
rats (Table S8). However, compound 10b reveals poorer PK profile
in rats compared to compound 8n with a low AUC, short half-life,
and low bioavailability (Table 2). The low bioavailability may be
the reason why compound 10b did not display significantly better
antitumor activity in vivo than 8n, although it had a much stronger
apoptotic effect in vivo than 8n.
3a e 3y, 4a - 4f, and 5a (0.1e1 mmol) were synthesized ac-
cording this general method, the crude products were purified by
silica-gel column chromatography (Ethyl acetate/Hexane).
Detected by LC/MS/MS, liquid chromatography/tandem mass
spectrometry. AUC, area under the concentrationꢁtime curve. Cmax
,
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European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 10. Nur77 binding affinity of 10b and 10c. (A) fluorescence quenching assay, 1 mM Nur77-LBD was titrated by successive additions of a 0.01e1 mM compound solution. The
excitation wavelength was 280 nm, and the emission measurement range was 300e500 nm. The maximum fluorescence intensities at 330 nm with increasing concentrations of
compound were selected for curve fitting. (B) surface plasmon resonance (SPR) assay. Nur77-LBD was immobilized on a CM5 sensor chip, and gradient concentrations of compounds
(0.01e5
mM) were injected into the flow cells in running buffer at a flow rate of 30 mL/min for a 150-s association phase followed by a 420-s dissociation phase and a 30-s
regeneration phase.
Fig. 11. Proposed binding model for 10b (cyan stick, A) and 10c (yellow stick, B) in site B of Nur77-LBD (PDB: 3V3Q). The protein was represented as surface mode, the hydrophobic
residues were colored in gray, and the polar residues were decorated with blue and red, respectively. The H-bonds and pi-cation were shown in yellow dashed lines and magenta
dashed lines, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
6.1.2. General procedure for the synthesis of asymmetric Ph-C-DIM
(7a - 7f)
6.1.3. General procedure for the synthesis of Ph-C-DIMþ (8a - 8y, 9a
- 9f, and 10a - 10f)
Indole (1.17 g, 10 mmol, 3 equiv), 4-(trifluoromethyl)benzalde-
hyde (0.58 g, 3.3 mmol, 1 equiv), and tetramethyl guanidine (TMG,
76 mg, 0.66 mmol, 0.2 equiv) were added to a round bottom flask
(100 mL). Pure water (50 mL) was added to the flask, and the re-
action was stirred vigorously at room temperature for 24 h. The
reaction mixture was extracted with ethyl acetate (50 mL * 3). The
organic phase was collected, dried over anhydrous Na2SO4 and
purified by silica-gel column chromatography (Ethyl acetate/Hex-
ane), to give a white product (1H-3-indolyl)(4-CF3-phenyl)meth-
anol (6a, 623 mg, yield: 65%).
6a (1 mmol, 1 equiv) and alkyl-N-indole 1b - 1f (1.5 mmol, 1.5
equiv) were added to 25 mL sealed tube equipped with tri-
fluoroethanol (5 mL). The reaction was stirred at 50 ꢀC for 4 h, and
the solvent was evaporated under reduced pressure. The final crude
product was purified by silica-gel column chromatography (Ethyl
acetate/Hexane) to give white compound 7a - 7f.
The synthesis procedure of Ph-C-DIMþMeSO3- (8a - 8i, 8l - 8n
and 10a - 10b).
Ph-C-DIM (1 equiv), pyridinium dichromate (1.4 equiv) and
MeSO3H (10 equiv) were added to a flask containing methanol. The
mixture was stirred at room temperature for 1 h. Then, the meth-
anol was evaporated under reduced pressure to obtain a mixture.
The mixture was dissolved with 1-butanol and washed with water.
The organic layer was evaporated under reduced pressure to obtain
a crude product. Finally, the crude product was purified by silica-gel
column chromatography (CH2Cl2/MeOH/MeSO3H) to afford Ph-C-
DIMþMeSO-3.
The synthesis procedure of Ph-C-DIMþClꢁ (8a-1, 8f-1, 8j - 8k,
8n-1, 8o - 8y, 9a - 9f and 10c - 10f).
Ph-C-DIM (1 equiv), pyridinium dichromate (1.4 equiv) and HCl
(37%, 10 equiv) were added to a flask containing methanol. The
mixture was stirred at room temperature for 1 h. Then, the
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European Journal of Medicinal Chemistry xxx (xxxx) xxx
6.1.4. Di(1H-indol-3-yl)(phenyl)methylium methanesulfonate (8a)
Compound 8a was obtained according to general procedure
6.1.3. 0.310 mmol Ph-C-DIM was used. Compound 8a was obtained
as red solid (94 mg, yield ¼ 73%). HPLC purity: 99.85%. 1H NMR
(600 MHz, DMSO‑d6) 14.02 (br. s., 2H,1-indole-H), 8.68 (br. s., 2H, 2-
indole-H), 7.84e7.98 (m, 1H, 4-Ar-H), 7.63e7.79 (m, 6H, 4-indole-H,
2,3,5,6-Ar-H), 7.39 (t, J ¼ 7.61 Hz, 2H, 6-indole-H), 7.15 (t, J ¼ 7.52 Hz,
2H, 5-indole-H), 6.67 (br. s., 2H, 7-indole-H), 2.37 (s, 3H, eSO3CH3).
13C NMR (151 MHz, DMSO‑d6) 169.7(Cþ, 1C), 147.8 (2C), 140.3 (2C),
136.7 (1C), 133.9 (2C), 133.0 (1C),129.9 (2C), 127.2 (2C), 126.2 (2C),
124.7 (2C), 121.8 (2C), 121.5 (2C), 115.0 (2C). HRMS (ESI) calcd for
C
23H17Nþ2 [M]þ: 321.1386, found: 321.1385.
6.1.5. Di(1H-indol-3-yl)(phenyl)methylium chloride (8a-1)
Compound 8a-1 was obtained according to general procedure
6.1.3. 0.310 mmol Ph-C-DIM was used. Compound 8a-1 was ob-
tained as red solid (85 mg, yield ¼ 77%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.44 (br. s., 2H,1-indole-H), 8.68 (br. s., 2H, 2-
indole-H), 7.88 (td, J ¼ 7.29, 1.38 Hz, 1H, 4-Ar-H), 7.74 (d, J ¼ 8.07 Hz,
2H, 4-indole-H), 7.65e7.72 (m, 4H, 2,3,5,6-Ar-H), 7.38 (t, J ¼ 7.61 Hz,
2H, 6-indole-H), 7.12 (t, J ¼ 7.51 Hz, 2H, 5-indole-H), 6.66 (br. s., 2H,
7-indole-H). 13C NMR (151 MHz, DMSO‑d6) 169.3 (Cþ, 1C), 147.3
(2C), 139.8 (2C), 137.8 (1C), 133.5 (2C), 132.3 (1C), 129.4 (2C), 126.7
(2C), 125.7 (2C), 124.2 (2C), 121.3 (2C), 121.0 (2C), 114.5 (2C). HRMS
(ESI, m/z) calcd for C23H17N2þ[M]þ: 321.1386, found: 321.1385.
Fig. 12. 10b induced Nur77-dependent apoptosis and Nur77 mitochondrial targeting.
(A) PARP cleavage in MEFs or Nur77ꢁ/ꢁMEFs treated with 0.5
mM potent compounds as
showed for 6 h was determined by Western blotting. (B). HEK293T cells were trans-
fected with Myc-Nur77. Whole cell lysate (WCL) and mitochondria fractions (Mito)
were prepared from HEK293T cells treated with 0.5 mM 8n and 10b respectively for 2 h,
and analyzed by Western blotting. Expression of nuclear PARP and mitochondrial
Tom20 was shown to ensure the purity of mitochondrial fraction. (C). HEK293T cells
transfected with Myc-Nur77-LBD were treated with 8n and 10b (0.5 mM) respectively
for 2 h were immunestained with Mito-Tracker (Green) and visualized by confocal
microscopy. (For interpretation of the references to color in this figure legend, the
reader is referred to the Web version of this article.)
6.1.6. Di(1H-indol-3-yl)(p-tolyl)methylium methanesulfonate (8b)
Compound 8b was obtained according to general procedure
6.1.3. 0.15 mmol Ph-C-DIM was used. Compound 8b was obtained
as red solid (52 mg, yield ¼ 81%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 13.95 (br. s., 2H,1-indole-H), 8.60 (br. s., 2H, 2-
indole-H), 7.72 (d, J ¼ 0 8.07 Hz, 2H, 2,6-Ar-H), 7.59 (d, J ¼ 7.70 Hz,
2H, 4-indole-H), 7.52 (d, J ¼ 7.70 Hz, 2H, 3,5-Ar-H), 7.39 (t,
J ¼ 7.61 Hz, 2H, 6-indole-H), 7.16 (t, J ¼ 7.52 Hz, 2H, 5-indole-H),
6.77 (d, J ¼ 6.97 Hz, 2H, 7-indole-H), 2.55 (s, 3H, eCH3), 2.35 (s, 3H,
eSO3CH3). 13C NMR (151 MHz, DMSO‑d6) 169.3(Cþ, 1C), 147.2 (2C),
144.7 (2C), 139.7 (2C), 133.1 (1C), 130.0 (2C), 126.7 (2C), 125.7 (3C),
124.2 (2C), 121.4 (2C), 121.1 (2C), 114.5 (2C), 39.1 (1C), 21.4 (1C).
HRMS (ESI) calcd for C24H19N2þ[M]þ: 335.1543, found: 335.1541.
methanol was evaporated under reduced pressure to obtain a
mixture. The mixture was dissolved with 1-butanol and washed
with saturated NaCl solution. The organic layer was evaporated
under reduced pressure to obtain a crude product. Finally, the crude
product was purified by silica-gel column chromatography (CH2Cl2/
MeOH/HCl) to afford Ph-C-DIMþClꢁ.
Fig. 13. 10b induced Bcl-2 dependent apoptosis by promoting the interaction between Nur77 and Bcl-2. (A) PARP cleavage in MEFs or Bcl-2ꢁ/ꢁMEFs treated with 0.5
compounds as showed for 6 h was determined by Western blotting. (B) HEK293T transfected with Flag-Bcl-2 together with Myc-Nur77 were treated with or without 0.5
10b respectively for indicated time and analyzed by co-immunoprecipitation assays using anti-Flag antibody. (C-D) Co-localization of Nur77 with Bcl-2. HEK293T cells were
mM potent
mM 8n and
transfected with Flag-Bcl-2 together with GFP-Nur77 (C) or GFP-Nur77-LBD (D), treated with or without 0.5 mM 8n and 10b respectively for 2 h, stained with anti-flag antibody, and
visualized using confocal microscopy.
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European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 14. Antitumor effect of compound 10b in MMTV-PyMT-transgenic mouse models. (A-B) Representative images of MMTVePyMT mammary tumor model mice and tumors from
mice administered with or without 8n and 10b respectively. For MMTV-PyMT mammary tumor model, female wild-type MMTV-PyMT mice of 12 weeks old were randomly divided
into two groups (n ¼ 6), treated daily with an oral dose of 3 mg/kg 8n and 10b respectively for 14 days. (C) Inhibition of PyMT tumor growth by 8n and 10b. Mice treated with 8n
and 10b respectively, and tumors were weighted (n ¼ 6). Data are presented as mean
SD. **p < 0.01, ***p < 0.001 compared to control group (n ¼ 6) (D) 14 days after
administration of 8n and 10b respectively, MMTV-PyMT mice without tumor were weighted. Data are presented as mean SD, ns, no significant difference.
Table 2
Pharmacokinetic parameters of compound 10b and BI1071 in rats. Data are presented as mean SD.
Compound
Route
AUC0-t
(
m
g/L*h)
Cmax
(
m
g/L)
Tmax (h)
T 1/2 (h)
MRT0-t (h)
CL (L/h/kg)
Vss (L/kg)
F (%)
BI1071 (8n) IV(0.5 mg/kg) Malea
femalea
1116.00 136.33 879.60 123.02
1731.98 69.97 1636.90 191.04
2245.71 491.07 143.37 20.05
4.05 0.91 4.31 0.86
5.91 1.48 3.59 0.32
5.33 1.16 5.28 2.91 12.32 1.06
5.33 2.31 6.81 4.35 14.50 0.78
2.66 0.30 0.56 0.68
0.45 0.05 2.56 0.27
0.28 0.01 2.38 0.53
PO(5 mg/kg)
Malea
20.12
22.79
femalea
3947.28 97.00
203.25 17.15
560.79 91.56
57.93 9.15
10b
IV(0.5 mg/kg) Male/femaleb 501.35 72.71
PO(5 mg/kg)
1.02 0.17 3.69 0.63
Male/femaleb 733.56 138.08
3
0
4.49 1.93 9.59 1.44
14.63
6.1.7. (4-ethylphenyl)di(1H-indol-3-yl)methylium
methanesulfonate (8c)
indole-H), 7.73 (m, 4H, 2,6-Ar-H, 4-indole-H), 7.62 (d, J ¼ 8.25 Hz, 2H,
3,5-Ar-H), 7.40 (t, J ¼ 7.61 Hz, 2H, 6-indole-H), 7.16 (t, J ¼ 7.61 Hz, 2H,
5-indole-H), 6.74 (br. s., 2H, 7-indole-H), 2.37 (s, 3H, eSO3CH3), 1.43
(s, 9H, eCH3). 13C NMR (151 MHz, DMSO‑d6) 169.7 (1C, Cþ), 158.0
(2C), 147.6 (2C), 140.0 (2C), 133.8 (1C), 126.6 (4C), 126.2 (3C), 124.7
(2C), 121.8 (2C), 121.6 (2C), 115.0 (2C), 35.6 (1C), 31.4 (3C). HRMS(ESI)
calcd for C27H25Nþ2 [M]þ: 377.2012, found: 377.2013.
Compound 8c was obtained according to general procedure
6.1.3. 0.57 mmol Ph-C-DIM was used. Compound 8c was obtained
as red solid (172 mg, yield ¼ 68%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 13.93 (br. s., 2H,1-indole-H), 8.60 (br. s., 2H, 2-
indole-H), 7.72 (d, J ¼ 8.07 Hz, 2H, 2,6-Ar-H), 7.62 (d, J ¼ 7.89 Hz, 2H,
3,5-Ar-H), 7.55 (d, J ¼ 8.07 Hz, 2H, 4-indole-H), 7.40 (t, J ¼ 7.61 Hz,
2H, 6-indole-H), 7.16 (t, J ¼ 7.61 Hz, 2H, 5-indole-H), 6.76 (br. s., 2H,
7-indole-H), 2.85 (q, J ¼ 7.64 Hz, 2H, eCH2-), 2.35 (s, 3H, eSO3CH3),
1.33 (t, J ¼ 7.61 Hz, 3H, eCH3). 13C NMR (151 MHz, METHANOL-d4)
172.5 (1C, Cþ), 153.5 (2C), 147.5 (2C), 141.3 (2C), 135.6 (1C), 130.2
(2C), 128.2 (2C), 127.4 (3C), 125.8 (2C), 123.7 (2C), 123.1 (2C), 115.3
(2C), 39.6 (1C), 30.2 (1C), 15.8 (1C). HRMS (ESI) calcd for
6.1.9. Di(1H-indol-3-yl)(4-methoxyphenyl)methylium
methanesulfonate (8e)
Compound 8e was obtained according to general procedure
6.1.3. 0.16 mmol Ph-C-DIM was used. Compound 8e was obtained
as red solid (62 mg, yield ¼ 87%). HPLC purity >98%.1H NMR
(600 MHz, DMSO‑d6) 13.83 (br. s., 2H,1-indole-H), 8.56 (br. s., 2H, 2-
indole-H), 7.72 (d, J ¼ 8.07 Hz, 2H, 2,6-Ar-H), 7.69 (d, J ¼ 7.89 Hz, 2H,
3,5-Ar-H), 7.40 (t, J ¼ 7.61 Hz, 2H, 6-indole-H), 7.26 (d, J ¼ 8.62 Hz,
2H, 4-indole-H), 7.17 (t, J ¼ 7.61 Hz, 2H, 5-indole-H), 6.86 (br. s., 2H,
7-indole-H), 3.98 (s, 3H, eOCH3), 2.36 (s, 3H, eSO3CH3). 13C NMR
(151 MHz, DMSO‑d6) 168.9 (1C, Cþ), 164.7 (1C, eOCH3-C), 146.7
(2C), 139.5 (2C), 129.1 (1C), 126.2 (4C), 124.1 (2C), 121.1 (4C), 115.0
(2C), 114.3 (4C), 56.0 (1C, eOCH3). HRMS (ESI) calcd for
C
25H21Nþ2 [M]þ: 349.1699, found: 349.1701.
6.1.8. (4-(tert-butyl)phenyl)di(1H-indol-3-yl)methylium
methanesulfonate (8d)
Compound 8d was obtained according to general procedure
6.1.3. 0.53 mmol Ph-C-DIM was used. Compound 8d was obtained as
red solid (202 mg, yield ¼ 81%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 13.91 (br. s., 2H, 1-indole-H), 8.57 (br. s., 2H, 2-
C
24H19N2Oþ[M]þ: 351.1492, found: 351.1488.
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European Journal of Medicinal Chemistry xxx (xxxx) xxx
6.1.10. (4-hydroxyphenyl)di(1H-indol-3-yl)methylium
methanesulfonate (8f)
red solid (198 mg, yield ¼ 80%). HPLC purity: 97%. 1H NMR
(600 MHz, DMSO‑d6) 13.99 (br. s., 2H,1-indole-H), 8.65 (br. s., 2H, 2-
indole-H), 7.77 (d, J ¼ 8.34 Hz, 2H, 3,5-Ar-H), 7.72 (d, J ¼ 8.07 Hz, 2H,
2,6-Ar-H), 7.55 (t, J ¼ 8.71 Hz, 2H, 4-indole-H), 7.40 (t, J ¼ 7.61 Hz,
2H, 6-indole-H), 7.18 (t, J ¼ 7.61 Hz, 2H, 5-indole-H), 6.76 (br. s., 2H,
7-indole-H), 2.34 (s, 3H, eSO3CH3). 13C NMR (151 MHz, DMSO‑d6)
167.8 (1C, Cþ), 165.55 (d, J ¼ 257.49 Hz, 1C, -C-F), 147.7 (2C), 139.8
(2C), 135.5 (1C), 126.6 (2C), 125.9 (4C), 124.4 (2C), 121.5 (2C), 121.2
(2C),116.74 (d, J ¼ 20.91 Hz, 2C, -C-C-F),114.5 (2C). HRMS (ESI) calcd
for C23H16FNþ2 [M]þ: 339.1292, found: 339.1296.
Compound 8f was obtained according to general procedure
6.1.3. 0.30 mmol Ph-C-DIM was used. Compound 8f was obtained
as red solid (73 mg, yield ¼ 56%). HPLC purity: 97%.1H NMR
(600 MHz, DMSO‑d6) 13.73 (br. s., 2H, 1-indole-H), 11.12 (br. s., 1H,
eOH), 8.50 (br. s., 2H, 2-indole-H), 7.71 (d, J ¼ 8.07 Hz, 2H, 2,6-Ar-
H), 7.59 (d, J ¼ 7.34 Hz, 2H, 3,5-Ar-H), 7.39 (t, J ¼ 7.70 Hz, 2H, 6-
indole-H), 7.17 (t,
J
¼
7.43 Hz, 2H, 5-indole-H), 7.07 (d,
J ¼ 8.62 Hz, 2H, 4-indole-H), 6.90 (br. s., 2H, 7-indole-H), 2.35 (s, 3H,
eSO3CH). 13C NMR (151 MHz, DMSO‑d6) 169.5 (1C, Cþ), 164.4 (1C,
-C-OH), 146.2 (2C), 139.4 (2C), 127.3 (1C), 125.5 (2C), 123.9 (2C),
121.0 (4C), 116.6 (4C), 114.2 (4C). HRMS (ESI) calcd for
6.1.15. Di(1H-indol-3-yl)(4-(methoxycarbonyl)phenyl)methylium
chloride (8j)
C
23H17N2Oþ[M]þ: 337.1335, found: 337.1332.
Compound 8j was obtained according to general procedure
6.1.3. 0.30 mmol Ph-C-DIM was used. Compound 8j was obtained as
red solid (110 mg, yield ¼ 89%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.40 (br. s., 2H,1-indole-H), 8.72 (br. s., 2H, 2-
indole-H), 8.22 (d, J ¼ 7.34 Hz, 2H, 4-indole-H), 7.82 (d, J ¼ 7.15 Hz,
2H, 3,5-Ar-H), 7.72 (d, J ¼ 7.34 Hz, 2H, 2,6-Ar-H), 7.38 (br. s., 2H, 6-
indole-H), 7.19 (br. s., 2H, 5-indole-H), 6.62 (br. s., 2H, 7-indole-H),
3.96 (s, 3H, eCH3). 13C NMR (151 MHz, DMSO‑d6) 167.6 (1C, Cþ),
166.1 (1C, eC]O), 148.3 (2C), 140.4 (2C), 133.6 (2C), 133.0 (1C),
130.4 (2C), 126.9 (2C), 126.4 (3C), 125.0 (2C), 121.9 (2C), 121.6 (2C),
115.1 (2C), 53.2 (1C). HRMS (ESI) calcd for C25H19N2Oþ2 [M]þ:
379.1441, found: 379.1434.
6.1.11. (4-hydroxyphenyl)di(1H-indol-3-yl)methylium chloride (8f-
1)
Compound 8f-1 was obtained according to general procedure
6.1.3. 1.48 mmol Ph-C-DIM was used. Compound 8f-1 was obtained
as red solid (314 mg, yield ¼ 57%). HPLC purity: 96%. 1H NMR
(600 MHz, DMSO‑d6) 13.86 (br. s., 2H, 1-indole-H), 11.20 (br. s., 1H,
eOH), 8.50 (br. s., 2H, 2-indole-H), 7.71 (d, J ¼ 8.07 Hz, 2H, 2,6-Ar-
H), 7.59 (d, J ¼ 7.34 Hz, 2H, 3,5-Ar-H), 7.39 (t, J ¼ 7.70 Hz, 2H, 6-
indole-H), 7.17 (t,
J
¼
7.43 Hz, 2H, 5-indole-H), 7.09 (d,
J ¼ 8.62 Hz, 2H, 4-indole-H), 6.91 (br. s., 2H, 7-indole-H). 13C NMR
(151 MHz, DMSO‑d6) 169.6 (1C, Cþ), 164.4 (1C, -C-OH), 146.0 (2C),
139.4 (2C), 127.3 (1C), 125.4 (2C), 123.9 (2C), 120.9 (4C), 116.6 (4C),
114.2 (4C). HRMS (ESI) calcd for C23H17N2Oþ[M]þ: 337.1335, found:
337.1331.
6.1.16. Bis(1H-indol-3-yl)(4-carboxyphenyl)methylium chloride
(8k)
Compound 8k was obtained according to general procedure
6.1.3 (acetone was used as solvent). 0.30 mmol Ph-C-DIM was used.
Compound 8k was obtained as red solid (67 mg, yield ¼ 56%). HPLC
purity >98%. 1H NMR (600 MHz, DMSO‑d6) 14.21 (br. s, 2H, 1-
indole-H), 13.56 (br. s., 1H, eCOOH), 8.67 (br. s., 2H, 2-indole-H),
8.20 (d, J ¼ 8.25 Hz, 2H, 4-indole-H), 7.79 (d, J ¼ 8.07 Hz, 2H, 3,5-Ar-
H), 7.71 (d, J ¼ 8.07 Hz, 2H, 2,6-Ar-H), 7.39 (t, J ¼ 7.61 Hz, 2H, 6-
indole-H), 7.15 (t, J ¼ 7.61 Hz, 2H, 5-indole-H), 6.66 (br. s., 2H, 7-
indole-H). 13C NMR (151 MHz, DMSO‑d6) 172.0 (1C, Cþ), 167.2 (1C,
eC]O), 148.2 (2C), 142.3 (1C), 140.8 (2C), 134.8 (2C), 132.8 (1C),
130.5 (2C), 127.0 (2C), 126.3 (2C), 124.8 (2C), 122.0 (2C), 121.6 (2C),
115.2 (2C). HRMS (ESI) calcd for C24H17N2Oþ2 [M]þ: 365.1285, found:
365.1272.
6.1.12. [1,10-biphenyl]-4-yldi(1H-indol-3-yl)methylium
methanesulfonate (8g)
Compound 8g was obtained according to general procedure
6.1.3. 0.50 mmol Ph-C-DIM was used. Compound 8g was obtained
as red solid (216 mg, yield ¼ 88%). HPLC purity >98%.1H NMR
(600 MHz, DMSO‑d6) 13.98 (br. s., 2H,1-indole-H), 8.67 (br. s., 2H, 2-
indole-H), 8.06 (d, J ¼ 8.25 Hz, 2H, 3,5-Ar-H), 7.93 (d, J ¼ 7.34 Hz, 2H,
2,6-Ar-H), 7.79 (d, J ¼ 7.89 Hz, 2H, 20,60-Ar-H), 7.74 (d, J ¼ 8.07 Hz,
2H, 4-indole-H), 7.58 (t, J ¼ 7.70 Hz, 2H, 30,50-Ar-H), 7.48e7.53 (m,
1H, 40-Ar-H), 7.41 (t, J ¼ 7.61 Hz, 2H, 6-indole-H), 7.18 (t, J ¼ 7.61 Hz,
2H, 5-indole-H), 6.83 (br. s., 2H, 7-indole-H), 2.35 (s, 3H, eSO3CH3).
13C NMR (151 MHz, DMSO‑d6) 168.21 (1C, Cþ), 147.9 (2C),145.5 (1C),
140.1 (2C), 138.8 (2C), 134.9 (1C), 129.8 (2C), 129.4 (2C), 127.8 (2C),
127.6 (4C), 126.3 (2C), 124.5 (2C), 121.9 (2C), 121.7 (2C), 115.0 (2C).
HRMS (ESI) calcd for C29H21N2þ[M]þ: 397.1699, found: 397.1703.
6.1.17. Di(1H-indol-3-yl)(4-nitrophenyl)methylium
methanesulfonate (8l)
Compound 8l was obtained according to general procedure
6.1.3. 0.54 mmol Ph-C-DIM was used. Compound 8l was obtained as
red solid (184 mg, yield ¼ 74%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.14 (br. s., 2H, 1-indole-H), 8.75 (br. s., 2H, 2-
indole-H), 8.50 (d, J ¼ 8.44 Hz, 2H, 4-indole-H), 7.97 (d, J ¼ 8.44 Hz,
2H, 3,5-Ar-H), 7.73 (d, J ¼ 8.07 Hz, 2H, 2,6-Ar-H), 7.42 (t, J ¼ 7.61 Hz,
2H, 6-indole-H), 7.17 (t, J ¼ 7.52 Hz, 2H, 5-indole-H), 6.70 (br. s., 2H,
7-indole-H), 2.35 (s, 3H, eSO3CH3). 13C NMR (151 MHz, DMSO‑d6)
165.6 (1C, Cþ), 150.0 (2C), 148.4 (2C), 145.1 (1C), 139.9 (2C), 133.4
(1C), 126.2 (4C), 124.7 (2C), 124.4 (2C), 121.6 (2C), 121.4 (2C), 114.7
(2C). HRMS (ESI) calcd for C23H16N3Oþ2 [M]þ: 366.1241, found:
366.1241.
6.1.13. (4-chlorophenyl)di(1H-indol-3-yl)methylium
methanesulfonate (8h)
Compound 8h was obtained according to general procedure
6.1.3. 0.75 mmol Ph-C-DIM was used. Compound 8h was obtained
as red solid (239 mg, yield ¼ 71%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 13.92 (br. s, 2H, 1-indole-H) 8.65 (br. s., 2H, 2-
indole-H) 7.78 (d, J ¼ 8.25 Hz, 2H, 3,5-Ar-H) 7.68e7.75 (m, 4H, 2,6-
Ar-H, 4-indole-H) 7.41 (t, J ¼ 7.61 Hz, 2H, 6-indole-H) 7.19 (t,
J ¼ 7.52 Hz, 2H, 5-indole-H) 6.76 (d, J ¼ 6.79 Hz, 2H, 7-indole-H)
2.35 (br. s, 3H, eSO3CH3). 13C NMR (151 MHz, DMSO‑d6) 167.47 (1C,
Cþ), 148.27 (2C), 140.38 (2C), 139.11 (2C), 134.97 (1C), 130.03 (2C),
126.88 (2C), 126.39 (2C),124.92 (3C),121.96 (2C), 121.66 (2C), 115.08
(2C). HRMS (ESI) calcd for C23H16ClNþ2 [M]þ: 355.0997, found:
355.0991.
6.1.18. Di(1H-indol-3-yl)(4-(trifluoromethyl)phenyl) methylium
methanesulfonate (8n)
Compound 8n was obtained according to general procedure
6.1.3. 0.26 mmol Ph-C-DIM was used. Compound 8n was obtained
as red solid (104 mg, yield ¼ 83%). HPLC purity >98%.1H NMR
(600 MHz, DMSO‑d6) 14.06 (br. s., 2H,1-indole-H), 8.71 (br. s., 2H, 2-
indole-H), 8.07 (d, J ¼ 8.07 Hz, 2H, 3,5-Ar-H), 7.91 (d, J ¼ 7.70 Hz, 2H,
2,6-Ar-H), 7.73 (d, J ¼ 8.07 Hz, 2H, 4-indole-H), 7.42 (t, J ¼ 7.61 Hz,
6.1.14. (4-fluorophenyl)di(1H-indol-3-yl)methylium
methanesulfonate (8i)
Compound 8i was obtained according to general procedure
6.1.3. 0.57 mmol Ph-C-DIM was used. Compound 8i was obtained as
12

X. Tu, X. Chen, D. Zhang et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
2H, 6-indole-H), 7.19 (t, J ¼ 7.52 Hz, 2H, 5-indole-H), 6.67 (br. s., 2H,
7-indole-H), 2.33 (br. s., 3H, eSO3CH3). 13C NMR (151 MHz,
DMSO‑d6) 166.6 (1C, Cþ), 148.3 (2C), 139.9 (2C), 133.2 (1C), 132.4 (q,
JC-F ¼ 29.7 Hz,1C, 4-Ar-C), 126.2 (6C),126.1 (2C),124.6 (2C), 124.0 (q,
JC-F ¼ 274.0 Hz, 1C, eCF3), 121.6 (2C), 121.3 (2C), 114.7 (2C). HRMS
(ESI) calcd for C24H16F3N2þ[M]þ: 389.1260, found: 389.1255.
6.1.23. (3-chlorophenyl)di(1H-indol-3-yl)methylium chloride (8r)
Compound 8r was obtained according to general procedure
6.1.3. 0.25 mmol Ph-C-DIM was used. Compound 8r was obtained
as red solid (90 mg, yield ¼ 92%). HPLC purity >98%. 1H NMR
(400 MHz, DMSO‑d6) 14.47 (br. s., 2H,1-indole-H), 8.93 (br. s., 2H, 2-
indole-H), 7.83 (br. s., 2H, 4-indole-H), 7.55e7.75 (m, 4H), 7.37 (br. s.,
2H, 6-indole-H), 7.14 (br. s., 2H, 5-indole-H), 6.45 (br. s., 2H, 7-
indole-H). 13C NMR (151 MHz, DMSO‑d6) 164.2 (1C, Cþ), 148.3
(2C), 141.6 (2C), 134.4 (1C), 132.9 (1C), 131.7 (2C), 131.2 (1C), 127.1
(1C), 126.2 (2C), 124.6 (2C), 124.6 (2C), 122.2 (2C), 121.5 (2C), 114.9
(2C). HRMS (ESI) calcd for C23H16ClN2 þ[M]þ: 355.0997, found:
355.0990.
6.1.19. Di(1H-indol-3-yl)(4-(trifluoromethyl)phenyl) methylium
chloride (8n-1)
Compound 8n-1 was obtained according to general procedure
6.1.3. 0.26 mmol Ph-C-DIM was used. Compound 8n-1 was ob-
tained as red solid (93 mg, yield ¼ 85%). HPLC purity >98%.1H NMR
(600 MHz, DMSO‑d6) 14.48 (br. s., 2H,1-indole-H), 8.69 (br. s., 2H, 2-
indole-H), 8.05 (br. s., 2H, 3,5-Ar-H), 7.89 (br. s., 2H, 2,6-Ar-H), 7.74
(br. s., 2H, 4-indole-H), 7.40 (br. s., 2H, 6-indole-H), 7.17 (br. s., 2H, 5-
indole-H), 6.64 (br. s., 2H, 7-indole-H). 13C NMR (151 MHz,
DMSO‑d6) 166.3 (1C, Cþ), 148.0 (2C), 140.0 (2C), 133.1 (1C), 132.4 (q,
JC-F ¼ 29.7 Hz,1C, 4-Ar-C),126.2 (6C),126.0 (2C),124.6 (2C),124.0 (q,
JC-F ¼ 274.0 Hz, 1C, eCF3), 121.6 (2C), 121.2 (2C), 114.7 (2C). HRMS
(ESI, m/z) calcd for C24H16F3Nþ2 [M]þ: 389.1260, found: 389.1255.
6.1.24. (3-fluorophenyl)di(1H-indol-3-yl)methylium chloride (8s)
Compound 8s was obtained according to general procedure
6.1.3. 0.21 mmol Ph-C-DIM was used. Compound 8s was obtained
as red solid (71 mg, yield ¼ 91%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.42 (br. s., 2H,1-indole-H), 8.72 (br. s., 2H, 2-
indole-H), 7.74 (m, 4H), 7.58 (d, J ¼ 8.80 Hz, 1H, 4-Ar-H), 7.52 (d,
J ¼ 5.69 Hz, 1H, 6-Ar-H), 7.40 (t, J ¼ 6.60 Hz, 2H, 6-indole-H), 7.18 (t,
J ¼ 6.60 Hz, 2H, 5-indole-H), 6.68 (br. s., 2H, 7-Ar-H). 13C NMR
(151 MHz, METHANOL-d4) 170.0 (1C, Cþ), 164.3 (d, JC-F ¼ 247.59 Hz,
1C, 3-F-Ar-C),148.1 (2C),141.2 (2C),132.6 (2C),127.8 (2C),127.6 (4C),
126.0 (2C), 123.4 (2C), 122.9 (2C), 122.5 (d, JC-F ¼ 20.9 Hz, 1C, AreC),
115.6 (2C). HRMS (ESI, m/z) calcd for C23H16FN2 þ[M]þ: 339.1292,
found: 339.1288.
6.1.20. Di(1H-indol-3-yl)(m-tolyl)methylium chloride (8o)
Compound 8o was obtained according to general procedure
6.1.3. 0.60 mmol Ph-C-DIM was used. Compound 8o was obtained
as red solid (189 mg, yield ¼ 85%). HPLC purity: 97%.1H NMR
(600 MHz, DMSO‑d6) 14.11 (br. s, 2H, 1-indole-H) 8.60 (br. s., 2H, 2-
indole-H) 7.71 (d, J ¼ 7.70 Hz, 2H, 4-indole-H) 7.68 (d, J ¼ 6.97 Hz,
1H, 6-Ar-H) 7.58 (t, J ¼ 7.15 Hz, 1H, 5-Ar-H) 7.48 (s, 1H, 2-Ar-H) 7.44
(d, J ¼ 6.97 Hz, 1H,4-Ar-H) 7.37 (t, J ¼ 7.06 Hz, 2H, 6-indole-H) 7.13
(t, J ¼ 7.24 Hz, 2H, 5-indole-H) 6.69 (d, J ¼ 5.69 Hz, 2H, 7-indole-H)
2.39 (s, 3H, eCH3). 13C NMR (151 MHz, DMSO‑d6) 168.9 (1C, Cþ),
147.2 (2C), 140.0 (2C), 138.8 (1C), 134.1 (2C), 129.6 (1C), 129.3 (1C),
126.7 (1C), 125.7 (3C), 124.2 (3C), 121.5 (2C), 121.1 (2C), 114.6 (2C),
20.8 (1C). HRMS (ESI) calcd for C24H19Nþ2 [M]þ: 335.1543, found:
335.1540.
6.1.25. Di(1H-indol-3-yl)(3-(trifluoromethyl)phenyl)methylium
chloride (8t)
Compound 8t was obtained according to general procedure
6.1.3. 0.427 mmol Ph-C-DIM was used. Compound 8t was obtained
as red solid (161 mg, yield ¼ 89%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.62 (br. s., 2H,1-indole-H), 8.70 (br. s., 2H, 2-
indole-H), 8.22 (d, J ¼ 7.89 Hz, 1H, 4-Ar-H), 8.02 (s, 1H, 2-Ar-H),
7.96e8.00 (m, 1H, 6-Ar-H), 7.91e7.95 (m, 1H, 5-Ar-H), 7.76 (d,
J ¼ 8.25 Hz, 2H, 4-indole-H), 7.39 (t, J ¼ 7.61 Hz, 2H, 6-indole-H),
7.15 (t, J ¼ 7.61 Hz, 2H, 5-indole-H), 6.62 (d, J ¼ 5.69 Hz, 2H, 7-
indole-H). 13C NMR (151 MHz, DMSO‑d6) 166.2 (1C, Cþ), 148.4
(2C), 140.7 (2C), 139.3 (1C), 136.7 (1C), 131.1 (q, JC-F ¼ 3.3 Hz, 1C,
AreC), 130.5 (q, JC-F ¼ 31.9 Hz, 1C, 3-Ar-C), 129.9 (q, JC-F ¼ 3.3 Hz, 1C,
AreC), 129.2 (1C), 126.9 (2C), 126.3 (2C), 124.8 (2C), 124.3 (q, JC-
6.1.21. Di(1H-indol-3-yl)(3-methoxyphenyl)methylium chloride
(8p)
Compound 8p was obtained according to general procedure
6.1.3. 0.57 mmol Ph-C-DIM was used. Compound 8p was obtained
as red solid (167 mg, yield ¼ 76%). HPLC purity >98%.1H NMR
(600 MHz, DMSO‑d6) 14.08 (br. s., 2H,1-indole-H), 8.62 (br. s., 2H, 2-
indole-H), 7.71 (d, J ¼ 7.70 Hz, 2H, 4-indole-H), 7.60 (br. s., 1H, 5-Ar-
H), 7.43 (d, J ¼ 7.15 Hz, 1H, 6-Ar-H), 7.37 (t, J ¼ 7.24 Hz, 2H, 6-indole-
H), 7.21 (d, J ¼ 7.15 Hz, 1H, 4-Ar-H), 7.18 (br. s., 1H, 2-Ar-H), 7.14 (t,
J ¼ 7.34 Hz, 2H, 5-indole-H), 6.72 (d, J ¼ 7.15 Hz, 2H, 7-indole-H),
3.76 (s, 3H, eOCH3). 13C NMR (151 MHz, DMSO‑d6) 168.1 (1C, Cþ),
159.6 (1C, -C-OCH3), 147.4 (2C), 140.1 (2C), 139.3 (1C), 130.6 (2C),
126.7 (1C), 125.8 (2C), 124.3 (2C), 121.5 (2C), 121.2 (2C), 119.1 (2C),
117.1 (1C), 114.6 (2C), 55.6 (1C). HRMS (ESI) calcd for
¼ 272.9 Hz, 1C, eCF3), 122.0 (2C), 121.4 (2C), 115.3 (2C). HRMS (ESI,
F
m/z) calcd for C24H16F3N2þ[M]þ: 389.1260, found: 389.1264.
6.1.26. (2-hydroxyphenyl)di(1H-indol-3-yl)methylium chloride
(8u)
Compound 8u was obtained according to general procedure
6.1.3. 0.75 mmol Ph-C-DIM was used. Compound 8u was obtained
as red solid (156 mg, yield ¼ 56%). HPLC purity: 96%. 1H NMR
(600 MHz, DMSO‑d6) 14.10 (br. s., 2H, 1-indole-H), 10.36 (br. s., 1H,
eOH), 8.71 (br. s., 2H, 2-indole-H), 7.68 (d, J ¼ 8.07 Hz, 2H, 4-indole-
H), 7.60e7.66 (m, 1H, -H-Ar), 7.36 (t, J ¼ 7.61 Hz, 1H, 4-Ar-H),
7.26e7.30 (m, 1H,-H-Ar), 7.20 (d, J ¼ 8.25 Hz, 1H, 3-Ar-H), 7.15 (t,
J ¼ 7.70 Hz, 2H, 6-indole-H), 7.04 (t, J ¼ 7.52 Hz, 2H, 5-indole-H),
6.79 (d, J ¼ 6.05 Hz, 2H, 7-indole-H). 13C NMR (151 MHz, DMSO‑d6)
166.9 (1C, Cþ), 156.6 (1C, -C-OH), 146.5 (2C), 139.5 (2C), 134.5 (2C),
132.5 (1C), 127.0 (1C), 125.5 (2C), 124.6 (1C), 124.2 (2C), 122.0 (1C),
120.6 (2C), 119.9 (2C), 117.0 (1C), 114.2 (2C). HRMS (ESI) calcd for
C
24H19N2Oþ[M]þ: 351.1492, found: 351.1489.
6.1.22. (3-hydroxyphenyl)di(1H-indol-3-yl)methylium chloride (8q)
Compound 8q was obtained according to general procedure
6.1.3. 0.70 mmol Ph-C-DIM was used. Compound 8q was obtained
as red solid (170 mg, yield ¼ 65%). HPLC purity >98%. 1H NMR
(400 MHz, DMSO‑d6) 14.28 (br. s., 2H, 1-indole-H), 10.17 (br. s., 1H,
eOH), 8.65 (br. s., 2H, 2-indole-H), 7.73 (br. s., 2H, 4-indole-H), 7.50
(br. s., 1H, 5-Ar-H), 7.39 (br. s., 2H, 6-indole-H), 7.30 (br. s., 1H, 6-Ar-
H), 7.17 (br. s., 2H, 5-indole-H), 7.07 (br. s., 1H, 4-Ar-H), 7.04 (br. s.,
1H, 2-Ar-H), 6.77 (br. s., 2H, 7-indole-H). 13C NMR (151 MHz,
DMSO‑d6) 169.4 (1C, Cþ), 158.0 (1C, -C-OH), 147.2 (1C), 139.7 (2C),
139.1 (1C), 130.5 (2C), 126.7 (1C), 125.7 (2C), 124.3 (2C), 122.8 (1C),
121.3 (2C) 121.2 (2C), 120.5 (2C), 128.3 (1C), 114.3 (2C). HRMS (ESI)
calcd for C23H17N2O þ[M]þ: 337.1335, found: 337.1331.
C
23H17N2Oþ[M]þ: 337.1335, found: 337.1330.
6.1.27. (2-chlorophenyl)di(1H-indol-3-yl)methylium chloride (8v)
Compound 8v was obtained according to general procedure
6.1.3. 0.27 mmol Ph-C-DIM was used. Compound 8v was obtained
as red solid (84 mg, yield ¼ 79%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.59 (br. s., 2H,1-indole-H), 8.94 (br. s., 2H, 2-
13

X. Tu, X. Chen, D. Zhang et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
indole-H), 7.84 (br. s., 2H, 4-indole-H), 7.73 (m, 2H, AreH), 7.67 (br.
s., 1H, AreH), 7.61 (br. s., 1H, AreH), 7.37 (br. s., 2H, 6-indole-H), 7.14
(br. s., 2H, 5-indole-H), 6.46 (br. s., 2H, 7-indole-H). 13C NMR
(151 MHz, DMSO‑d6) 164.1 (1C, Cþ), 147.4 (2C), 139.6 (2C), 136.2
(1C), 133.3 (1C), 131.8 (1C), 131.5 (1C), 130.8 (1C), 128.5 (1C), 126.2
(2C), 125.8 (2C), 124.7 (2C), 121.0 (2C), 120.2 (2C), 114.5 (2C). HRMS
(ESI, m/z) calcd for C23H16ClNþ2 [M]þ: 355.0997, found: 355.0992.
6.1.32. Di(1H-indol-3-yl)(thiophen-2-yl)methylium chloride (9b)
Compound 9b was obtained according to general procedure
6.1.3. 1.00 mmol Ph-C-DIM was used. Compound 9b was obtained
as red solid (270 mg, yield ¼ 75%). HPLC purity >98%.1H NMR
(600 MHz, DMSO‑d6) 14.24 (br. s., 2H, 1-indole-H), 8.64 (d,
J ¼ 4.22 Hz, 1H, 4-Ar-H), 8.62 (s, 2H, 2-indole-H), 8.00 (d,
J ¼ 2.57 Hz, 1H, 2-Ar-H), 7.76 (d, J ¼ 8.07 Hz, 2H, 4-indole-H), 7.64 (t,
J ¼ 3.85 Hz, 1H, 3-Ar-H), 7.40 (t, J ¼ 7.61 Hz, 2H, 6-indole-H), 7.14 (t,
6.1.28. (2-fluorophenyl)di(1H-indol-3-yl)methylium chloride (8w)
Compound 8w was obtained according to general procedure
6.1.3. 0.27 mmol Ph-C-DIM was used. Compound 8w was obtained
as red solid (57 mg, yield ¼ 56%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.48 (br. s., 2H,1-indole-H), 8.82 (br. s., 2H, 2-
indole-H), 7.90 (br. s., 1H, -H-Ar), 7.72 (br. s., 2H, 4-indole-H), 7.62
(br. s.,1H, -H-Ar), 7.50e7.58 (m, 2H, -H-Ar), 7.38 (br. s., 2H, 6-indole-
H), 7.15 (br. s., 2H, 5-indole-H), 6.64 (br. s., 2H, 7-indole-H). 13C NMR
(151 MHz, DMSO‑d6) 160.6 (1C, Cþ), 159.43 (d, J ¼ 251.99 Hz, 1C, -C-
F), 147.5 (2C), 140.0 (2C), 135.25 (d, J ¼ 7.70 Hz, 1C, -C-C-C-F), 133.0
(1C), 126.5 (2C), 125.9 (3C), 125.2 (1C), 124.6 (2C), 121.6 (2C), 120.3
(2C),116.98 (d, J ¼ 22.01 Hz,1C, -C-C-F),114.7 (2C). HRMS (ESI) calcd
for C23H16FNþ2 [M]þ: 339.1292, found: 339.1292.
J ¼ 7.52 Hz, 2H, 5-indole-H), 6.85 (d, J ¼ 7.15 Hz, 2H, 7-indole-H). 13
C
NMR (151 MHz, METHANOL-d4) 159.4 (1C, Cþ), 145.1 (2C), 143.0
(1C), 141.4 (1C), 139.8 (1C), 139.3 (2C), 130.6 (1C), 125.9 (4C), 124.1
(2C), 121.8 (2C), 121.0 (2C), 113.8 (2C). HRMS (ESI, m/z) calcd for
C
21H15N2Sþ[M]þ: 327.0950, found: 327.0955.
6.1.33. Di(1H-indol-3-yl)(pyridin-3-yl)methylium chloride (9c)
Compound 9c was obtained according to general procedure
6.1.3. 0.62 mmol Ph-C-DIM was used. Compound 9c was obtained
as red solid (147 mg, yield ¼ 67%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.55 (br. s., 2H, 1-indole-H), 9.07 (br. s.,1H, 2-
Ar-H), 8.89 (br. s., 1H, 6-Ar-H), 8.69 (br. s., 2H, 2-indole-H), 8.23 (br.
s., 1H, 4-Ar-H), 7.84 (br. s., 1H, 5-Ar-H), 7.75 (br. s., 2H, 4-indole-H),
7.41 (br. s., 2H, 6-indole-H), 7.18 (br. s., 2H, 5-indole-H), 6.72 (br. s.,
2H, 7-indole-H). 13C NMR (151 MHz, METHANOL-d4) 160.6 (1C, Cþ),
151.6 (1C), 149.1 (2C), 148.4 (1C), 140.4 (2C), 127.3 (3C), 126.0 (4C),
125.8 (2C), 123.8 (2C), 123.3 (2C), 115.0 (2C). HRMS (ESI, m/z) calcd
for C22H16Nþ3 [M]þ: 322.1339, found: 322.1341.
6.1.29. Di(1H-indol-3-yl)(2-(trifluoromethyl)phenyl)methylium
chloride (8x)
Compound 8x was obtained according to general procedure
6.1.3. 0.26 mmol Ph-C-DIM was used. Compound 8x was obtained
as red solid (101 mg, yield ¼ 93%). HPLC purity >98%.1H NMR
(600 MHz, DMSO‑d6) 14.63 (br. s., 2H,1-indole-H), 8.94 (br. s., 2H, 2-
indole-H), 8.11 (m, 1H, 3-Ar-H), 7.99 (br. s., 2H, 4-indole-H), 7.68 (m,
3H, AreH), 7.31 (br. s., 2H, 6-indole-H), 7.08 (br. s., 2H, 5-indole-H),
6.22 (br. s., 2H, 7-indole-H). 13C NMR (151 MHz, DMSO‑d6) 164.4
(1C, Cþ), 147.0 (2C), 139.0 (2C), 136.0 (1C), 133.4 (q, JC-F ¼ 3.3 Hz, 1C,
AreC), 131.6 (1C), 130.2 (q, JC-F ¼ 3.3 Hz, 1C, AreC), 127.3 (1C), 126.3
(q, JC-F ¼ 33.0 Hz,1C, 2-Ar-C), 125.8 (2C),125.4 (2C),124.3 (2C),122.8
(q, JC-F ¼ 274.0 Hz, 1C, eCF3), 120.5 (2C),119.9 (2C),114.2 (2C). HRMS
(ESI, m/z) calcd for C24H16F3Nþ2 [M]þ: 389.1260, found: 389.1262.
6.1.34. Tri(1H-indol-3-yl)methylium chloride (9d)
Compound 9d was obtained according to general procedure
6.1.3. 0.55 mmol Ph-C-DIM was used. Compound 9d was obtained
as red solid (201 mg, yield ¼ 92%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 13.81 (br. s., 3H, 1-indole-H), 8.40 (s, 3H, 2-
indole-H), 7.75 (d,
J
¼
8.07 Hz, 3H, 4-indole-H), 7.33 (t,
J ¼ 7.89 Hz, 3H, 6-indole-H), 7.06 (t, J ¼ 6.88 Hz, 3H, 5-indole-H),
6.98 (br. s., 3H, 7-indole-H). 13C NMR (151 MHz, DMSO‑d6) 160.9
(1C, Cþ), 143.1 (3C), 139.5 (3C), 127.6 (3C), 125.3 (3C), 123.6 (3C),
121.7 (3C),120.8 (3C),114.3 (3C). HRMS (ESI, m/z) calcd for C25H18N3þ
[M]þ: 360.1495, found: 360.1495.
6.1.30. (2,4-bis(trifluoromethyl)phenyl)di(1H-indol-3-yl)methylium
chloride (8y)
Compound 8y was obtained according to general procedure
6.1.3. 0.25 mmol Ph-C-DIM was used. Compound 8y was obtained
as red solid (116 mg, yield ¼ 94%). HPLC purity: 98%. 1H NMR
(600 MHz, DMSO‑d6) 14.55 (br. s., 2H,1-indole-H), 8.91 (br. s., 2H, 2-
indole-H), 8.48 (br. s., 1H, 3-Ar-H), 8.40 (d, J ¼ 6.60 Hz, 1H, 5-Ar-H),
7.99 (d, J ¼ 6.60 Hz, 1H, 6-Ar-H), 7.71 (d, J ¼ 7.15 Hz, 2H, 4-indole-H),
7.38 (br. s., 2H, 6-indole-H), 7.16 (br. s., 2H, 5-indole-H), 6.38 (br. s.,
2H, 7-indole-H). 13C NMR (151 MHz, DMSO‑d6) 162.5 (1C, Cþ), 148.8
(2C), 141.2 (1C), 140.5 (2C), 133.1 (q, JC-F ¼ 3.3 Hz, 1C, AreC), 132.40
(q, JC-F ¼ 31.9 Hz, 1C, AreC), 131.2 (1C), 129.1 (q, JC-F ¼ 34.1 Hz, 1C,
AreC), 126.6 (2C), 126.5 (2C), 125.6 (2C), 125.4 (1C), 123.6 (q, JC-
6.1.35. Benzo[c]thiophen-1-yldi(1H-indol-3-yl)methylium chloride
(9e)
Compound 9e was obtained according to general procedure
6.1.3. 1 mmol Ph-C-DIM was used. Compound 9e was obtained as
red solid (206 mg, yield ¼ 50%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.16 (br. s., 2H, 1-indole-H), 8.76 (br. s., 2H, 2-
indole-H), 8.61 (s, 1H, 2-Ar-H), 8.27 (d, J ¼ 8.25 Hz, 1H, 4-Ar-H), 7.72
(d, J ¼ 8.07 Hz, 2H, 4-indole-H), 7.47 (t, J ¼ 8.16 Hz, 1H, 6-Ar-H), 7.37
(d, J ¼ 7.34 Hz, 2H, 6-indole-H), 7.20e7.27 (m, 2H), 7.09 (br. s., 2H),
6.63 (br. s, 2H, 7-indole-H). 13C NMR (151 MHz, DMSO‑d6) 160.41
(1C, Cþ), 147.28 (2C), 140.74 (2C), 140.14 (2C), 126.95 (1C), 126.21
(4C), 126.10 (2C), 124.81 (2C), 124.16 (2C), 123.27 (2C), 121.99 (1C),
121.30 (2C), 114.97 (2C). HRMS (ESI) calcd for C25H17N2Sþ[M]þ:
377.1107, found: 377.1109.
¼ 272.9 Hz, 1C, AreC), 123.2 (q, JC-F ¼ 275.1 Hz, 1C, AreC), 121.7
F
(2C), 121.2 (2C), 115.4 (2C). HRMS (ESI, m/z) calcd for C25H15F6N2þ
[M]þ: 457.1134, found: 457.1135.
6.1.31. Furan-2-yldi(1H-indol-3-yl)methylium chloride (9a)
6.1.36. Di(1H-indol-3-yl)(6-(trifluoromethyl)pyridin-3-yl)
Compound 9a was obtained according to general procedure
6.1.3. 0.64 mmol Ph-C-DIM was used. Compound 9a was obtained as
red solid (196 mg, yield ¼ 88%). HPLC purity: 98%.1H NMR (600 MHz,
DMSO‑d6) 14.19 (br. s., 2H, 1-indole-H), 8.66 (s, 2H, 2-indole-H), 8.60
(s, 1H, AreH), 7.71e7.81 (m, 3H), 7.40 (t, J ¼ 7.43 Hz, 2H, 6-indole-H),
7.15e7.23 (m, 3H), 6.94 (d, J ¼ 7.15 Hz, 2H, 7-indole-H). 13C NMR
(151 MHz, METHANOL-d4) 153.9 (1C, Cþ),152.5 (1C), 151.1 (1C), 144.7
(2C), 139.2 (2C), 129.7 (1C), 126.0 (2C), 125.7 (2C), 124.1 (2C), 121.7
(2C), 118.0 (2C), 115.7 (1C), 113.8 (2C). HRMS (ESI, m/z) calcd for
methylium chloride (9f)
Compound 9f was obtained according to general procedure
6.1.3. 0.38 mmol Ph-C-DIM was used. Compound 9f was obtained
as red solid (130 mg, yield ¼ 80%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.60 (br. s., 2H,1-indole-H), 8.98 (br. s.,1H, 2-
Ar-H), 8.70 (br. s., 2H, 2-indole-H), 8.40 (br. s., 1H, 6-Ar-H), 8.23 (br.
s., 1H, 5-Ar-H), 7.78 (d, J ¼ 4.40 Hz, 2H, 4-indole-H), 7.45 (br. s., 2H,
6-indole-H), 7.22 (br. s., 2H, 5-indole-H), 6.76 (br. s., 2H, 7-indole-
H). 13C NMR (151 MHz, DMSO‑d6) 162.3 (1C, Cþ),152.6 (1C), 149.1 (q,
JC-F ¼ 34.1 Hz, 1C, 4-Ar-C), 148.3 (2C), 142.9 (1C), 139.8 (2C), 126.1
C
21H15N2Oþ[M]þ: 311.1179, found: 311.1170.
14

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European Journal of Medicinal Chemistry xxx (xxxx) xxx
(2C), 125.6 (2C), 124.6 (2C), 121.7 (2C), 121.6 (1C), 121.4 (q, JC-
H), 7.02e7.31 (m, 2H), 6.46e6.87 (m, 2H), 4.06 (br. s., 2H, -N-CH2-),
1.12e1.36 (m, 3H, eCH3), 0.94 (br. s., 2H, eCH2-). 13C NMR (151 MHz,
DMSO‑d6) 165.7 (1C, Cþ), 149.5 (1C), 149.5 (1C), 147.8 (1C), 139.7
(1C), 139.6 (1C), 133.2 (1C), 132.8 (q, J ¼ 33.01 Hz, 1C, -C-CF3), 126.5
(1C), 126.1 (1C), 126.0 (3C), 125.9 (2C), 124.9 (1C), 124.5 (1C), 124.01
(q, J ¼ 275.10 Hz, 1C, eCF3), 121.3 (1C), 121.1 (1C), 120.4 (1C), 120.3
(1C), 114.6 (1C), 113.3 (1C), 47.5 (1C), 19.2 (1C), 13.4 (1C). HRMS (ESI)
calcd for C27H22F3Nþ2 [M]þ: 431.1730, found: 431.1735.
¼ 274.0 Hz, 1C, eCF3), 121.0 (2C), 120.9 (1C), 114.7 (2C). HRMS (ESI,
F
m/z) calcd for C23H15F3N3þ[M]þ: 390.1213, found: 390.1207.
6.1.37. Bis(1-methyl-1H-indol-3-yl)(4-(trifluoromethyl)phenyl)
methylium methanesulfonate (10a)
Compound 10a was obtained according to general procedure
6.1.3. 2.5 mmol Ph-C-DIM was used. Compound 10a was obtained
as red solid (909 mg, yield ¼ 71%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 8.74 (br. s., 2H, 2-indole-H), 8.07 (d,
J ¼ 8.07 Hz, 2H, 3,5-Ar-H), 7.90 (d, J ¼ 7.89 Hz, 2H, 2,6-Ar-H), 7.87 (d,
J ¼ 8.07 Hz, 2H, 4-indole-H), 7.51 (t, J ¼ 7.61 Hz, 2H, 6-indole-H),
7.26 (t, J ¼ 7.61 Hz, 2H, 5-indole-H), 6.71 (br. s., 2H, 7-indole-H), 4.13
(s, 6H, eCH3), 2.30 (s, 3H, eSO3-CH3). 13C NMR (151 MHz, DMSO‑d6)
164.6 (1C, Cþ), 150.2 (2C), 140.4 (2C), 133.8 (1C), 132.5 (q, JC-
6.1.41. (1-butyl-1H-indol-3-yl)(1H-indol-3-yl)(4-(trifluoromethyl)
phenyl)methylium chloride (10e)
Compound 10e was obtained according to general procedure
6.1.3. 0.24 mmol Ph-C-DIM was used. Compound 10e was obtained
as red solid (96 mg, yield ¼ 82%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.63 (br. s., 1H, 1-indole-H), 8.84 (br. s., 1H, 2-
indole-H), 8.71 (br. s., 1H, 20-indole-H), 8.07 (d, J ¼ 7.89 Hz, 2H, 3,5-
Ar-H), 7.93 (d, J ¼ 8.25 Hz, 1H, 4-indole-H), 7.91 (d, J ¼ 7.70 Hz, 2H,
2,6-Ar-H), 7.77 (d, J ¼ 8.07 Hz, 1H, 40-indole-H), 7.48 (t, J ¼ 7.52 Hz,
1H, 6-indole-H), 7.41 (t, J ¼ 7.34 Hz, 1H, 60-indole-H), 7.24 (t,
J ¼ 7.52 Hz, 1H, 5-indole-H), 7.18 (t, J ¼ 7.43 Hz, 1H, 50-indole-H),
6.66 (br. s., 2H, 7,70-indole-H), 4.51 (t, J ¼ 7.06 Hz, 2H, -N-CH2-), 1.94
(m, 2H, -N-C-CH2-), 1.39 (m, 2H, eCH2-CH3), 0.95 (t, J ¼ 7.34 Hz, 3H,
eCH3). 13C NMR (151 MHz, DMSO‑d6) 165.8 (1C, Cþ), 149.6 (1C),
148.0 (1C), 140.0 (1C), 139.7 (1C), 133.9 (1C), 132.5 (q, JC-F ¼ 33.0 Hz,
1C, 4-Ar-C), 126.7 (1C), 126.2 (5C), 126.1 (1C), 126.0 (1C), 125.9 (1C),
124.9 (1C), 123.9 (q, JC-F ¼ 274.2 Hz, 1C, eCF3), 121.5 (2C), 121.4 (1C),
120.3 (1C), 114.7 (1C), 113.5 (1C), 47.7 (1C), 31.0 (1C), 19.4 (1C), 13.5
(1C). HRMS (ESI, m/z) calcd for C28H24F3Nþ2 [M]þ: 445.1886, found:
445.1889.
¼ 29.7 Hz,1C, 4-Ar-C),126.3 (2C),126.1 (4C),125.1 (4C),124.0 (q, JC-
F
¼ 272.9 Hz, 1C, eCF3), 121.5 (2C), 120.2 (2C), 113.3 (2C), 35.0 (2C).
F
HRMS (ESI) calcd for C26H20F3Nþ2 [M]þ: 417.1573, found: 417.1570.
6.1.38. (1-methyl-1H-indol-3-yl)(1H-indol-3-yl)(4-
(trifluoromethyl)phenyl)methylium methanesulfonate (10b)
Compound 10b was obtained according to general procedure
6.1.3. 0.74 mmol Ph-C-DIM was used. Compound 10b was obtained
as red solid (287 mg, yield ¼ 78%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.07 (br. s., 1H, 1-indole-H), 8.78 (br. s., 1H, 2-
indole-H), 8.69 (br. s., 1H, 20-indole-H), 8.07 (d, J ¼ 8.07 Hz, 2H, 3,5-
Ar-H), 7.91 (d, J ¼ 8.07 Hz, 2H, 2,6-Ar-H), 7.87 (d, J ¼ 8.25 Hz, 1H, 4-
indole-H), 7.75 (d, J ¼ 8.07 Hz, 1H, 40-indole-H), 7.51 (t, J ¼ 7.61 Hz,
1H, 6-indole-H), 7.42 (t, J ¼ 7.61 Hz, 1H, 60-indole-H), 7.27 (t,
J ¼ 7.61 Hz, 1H, 5-indole-H), 7.18 (t, J ¼ 7.61 Hz, 1H, 50-indole-H),
6.67 (br. s., 2H, 7,70-indole-H), 4.13 (s, 3H, eCH3), 2.34 (s, 3H, eSO3-
CH3). 13C NMR (151 MHz, DMSO‑d6) 165.64 (1C, Cþ), 150.8 (1C),
147.6 (1C), 140.6 (1C), 140.0 (1C), 133.0 (1C), 132.6 (q, JC-F ¼ 33.0 Hz,
1C, 4-Ar-C), 127.1 (1C), 126.9 (1C), 126.7 (1C), 126.6 (3C), 126.5 (1C),
125.3 (1C), 124.5 (1C), 123.5 (q, JC-F ¼ 274.0 Hz, 1C, eCF3), 121.9 (2C),
121.2 (1C), 120.6 (1C), 114.6 (1C), 113.4 (1C), 39.1 (1C), 35.0 (1C).
HRMS (ESI) calcd for C25H18F3Nþ2 [M]þ: 403.1417, found: 403.1412.
6.1.42. (1H-indol-3-yl)(1-pentyl-1H-indol-3-yl)(4-(trifluoromethyl)
phenyl)methylium chloride (10f)
Compound 10f was obtained according to general procedure
6.1.3. 0.24 mmol Ph-C-DIM was used. Compound 10f was obtained
as red solid (104 mg, yield ¼ 87%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.59 (br. s., 1H, 1-indole-H), 8.84 (br. s., 1H, 2-
indole-H), 8.71 (br. s., 1H, 20-indole-H), 8.07 (d, J ¼ 8.07 Hz, 2H, 3,5-
Ar-H), 7.94 (s, 1H, 4-indole-H), 7.91 (d, J ¼ 7.89 Hz, 2H, 2,6-Ar-H),
7.77 (d, J ¼ 7.15 Hz, 1H, 40-indole-H), 7.48 (t, J ¼ 7.61 Hz, 1H, 6-
6.1.39. (1-ethyl-1H-indol-3-yl)(1H-indol-3-yl)(4-(trifluoromethyl)
phenyl)methylium chloride (10c)
indole-H), 7.41 (t,
J
¼
7.61 Hz, 1H, 60-indole-H), 7.24 (t,
Compound 10c was obtained according to general procedure
6.1.3. 0.50 mmol Ph-C-DIM was used. Compound 10c was obtained
as red solid (206 mg, yield ¼ 91%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.51 (br. s., 1H, 1-indole-H), 8.83 (br. s., 1H, 2-
indole-H), 8.75 (br. s., 1H, 20-indole-H), 8.07 (d, J ¼ 7.89 Hz, 2H, 3,5-
Ar-H), 7.87e7.96 (m, 3H), 7.75 (d, J ¼ 7.70 Hz, 1H, 40-indole-H), 7.48
(t, J ¼ 7.52 Hz, 1H, 6-indole-H), 7.41 (t, J ¼ 7.34 Hz, 1H, 60-indole-H),
7.24 (t, J ¼ 7.43 Hz,1H, 5-indole-H), 7.18 (t, J ¼ 7.24 Hz,1H, 50-indole-
H), 6.65 (br. s., 2H, 7,70-indole-H), 4.54 (q, J ¼ 6.97 Hz, 2H, eCH2-),
1.56 (t, J ¼ 7.15 Hz, 3H, eCH3). 13C NMR (151 MHz, DMSO‑d6) 165.5
(1C, Cþ), 149.2 (1C), 148.1 (1C), 140.1 (1C), 139.6 (1C), 133.4 (1C),
132.5 (q, JC-F ¼ 33.0 Hz,1C, 4-Ar-C),126.8 (1C),126.7 (1C),126.2 (4C),
126.1 (1C), 126.0 (1C), 124.8 (1C), 124.6 (1C), 123.9 (q, JC-
J ¼ 7.52 Hz, 1H, 5-indole-H), 7.18 (t, J ¼ 7.34 Hz, 1H, 50-indole-H),
6.67 (br. s., 2H, 7,70-indole-H), 4.50 (t, J ¼ 7.06 Hz, 2H, -N-CH2-), 1.95
(m, 2H, -N-C-CH2-), 1.36 (m, 4H), 0.84e0.94 (m, 3H, eCH3). 13C NMR
(151 MHz, DMSO‑d6) 166.1 (1C, Cþ), 149.7 (1C), 148.0 (1C), 139.9
(2C),132.8 (q, JC-F ¼ 29.2 Hz,1C, 4-Ar-C),129.2 (1C),126.8 (1C),126.4
(2C), 126.3 (5C), 125.4 (1C), 125.0 (1C), 124.0 (q, JC-F ¼ 274.2 Hz, 1C,
eCF3), 121.6 (2C), 121.3 (1C), 120.8 (1C), 114.9 (1C), 113.7 (1C), 48.1
(1C), 28.8 (1C), 28.4 (1C), 21.9 (1C), 14.0 (1C). HRMS (ESI, m/z) calcd
for C29H26F3Nþ2 [M]þ: 459.2043, found: 459.2048.
6.2. Biological assay
6.2.1. Cell culture
¼ 274.0 Hz, 1C, eCF3), 121.5 (2C), 121.2 (1C), 120.5 (1C), 114.8 (1C),
Human TNBC breast cancer cell line MDA-MB-231 and BT549,
mouse embryonic fibroblast (MEF) cells and HEK293T were
cultured in Dulbecco’s Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS). Human colorectal carcinoma
cancer cell line HCT116 and SW620 were cultured in RPMI-1640
supplemented 10% FBS. Normal mammary cell line MCF10A and
normal colon cell line NCM460 were cultured in DMEM with 20%
FBS and 10 ng/ml Epidermal Growth Factor (EGF).
F
113.4 (1C), 43.1 (1C), 14.6 (1C). HRMS (ESI, m/z) calcd for
C
26H20F3Nþ2 [M]þ: 417.1573, found: 417.1577.
6.1.40. (1H-indol-3-yl)(1-propyl-1H-indol-3-yl)(4-
(trifluoromethyl)phenyl)methylium chloride (10d)
Compound 10d was obtained according to general procedure
6.1.3. 0.46 mmol Ph-C-DIM was used. Compound 10d was obtained
as red solid (159 mg, yield ¼ 74%). HPLC purity >98%. 1H NMR
(600 MHz, DMSO‑d6) 14.45 (br. s., 1H, 1-indole-H), 8.73 (br. s., 2H,
2,20-indole-H), 8.04 (br. s., 2H, 4,40-indole-H), 7.91e7.95 (m, 1H),
7.88 (br. s., 2H, 3,5-Ar-H), 7.73 (br. s., 1H), 7.34e7.55 (m, 2H, 2,6-Ar-
6.2.2. Plasmids construction and transfection
Plasmids pcmv-myc-Nur77, pcmv-Myc-Nur77-LBD, pFlag-cmv-
2-Bcl-2, pcmv-myc-Bcl-2, pEGFP-C1-Nur77, pEGFP-C1-Nur77-LBD
15

X. Tu, X. Chen, D. Zhang et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
were constructed with standard methods as described previously
[35]. Cell transfection was carried out by using lipofectamine 2000
according to the manufacture’s instruction.
2,5-diphenyltetrazolium bromide (MTT) for 4 h at 37 ꢀC. MTT su-
pernatant in each well was removed carefully, and 100 mL of DMSO
was added. Absorbance was measured at 490 nm. The IC50 of each
compound was calculated by GraphPad Prism 8.0 software.
6.2.3. Western blotting
Cell lysates were electrophoresed by SDS-PAGE gel and trans-
ferred to polyvinylidene difluoride (PVDF) membrane. The mem-
branes were blocked with 5% skimmed milk in TBST (50 mM Tris-
HCl (pH 7.4), 150 mM NaCl and 0.1% Tween20) for 1 h at room
temperature, then incubated with primary antibodies and sec-
ondary antibodies and detected using ECL system (Thermo). The
dilutions of the primary antibodies were all in 1:1000.
6.2.9. Protein expression and purification
The human Nur77-LBD (367e598) was cloned as an N-terminal
histidine-tagged fusion protein in pET15b expression vector and
overproduced in Escherichia coli BL21 DE3 strain. Cells were har-
vested and sonicated, and the extract was incubated with the His60
Ni Superflow resin.
6.2.10. Fluorescence quenching
6.2.4. Apoptosis assay
The binding of 8n, 10b and 10c to Nur77 was measured by
Varioskan scanning spectrofluorometer and spectrophotometer. In
Cells plated at a density of 1 ꢂ 106 per well on six-well plates
were treated with 0.5
m
M of 8n or 10b for 6 h, and then the sus-
each assay, 3.0 mL portion of Nur77-LBD PBS solution (1
added accurately into the quartz cell and it was titrated by suc-
cessive additions of a 0.01e1
M stock solution at 25 ꢀC. The exci-
mM/L) was
pension and the adherent cells were collected, stained with
Annexin V-FITC for 10 min and with propidium iodide for 5 min,
and analyzed immediately by cytoFLEX Flow Cytometry System
(Beckman-Coulter, Miami, FL, USA) using FITC and PC5.5.
m
tation wavelength was 280 nm, and the emission measurement
range was 300e500 nm. To estimate the dissociation constant (Kd),
the maximum fluorescence intensities at 330 nm with increasing
concentrations of compound were measured, and the values of Kd
were calculated according to the reported formula [47].
6.2.5. CoIP assay
Cells transfected with various plasmids were lysed in lysis buffer
(20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA,
30 mM NaF, 2 mM sodium pyrophosphate) with a cocktail of pro-
teinase inhibitors (Roche). 5% of cell lysates were taken out to
represent Input. Lysates were incubated with the appropriate
antibody at 4ꢀCfor 12 h and subsequently incubated with protein A-
Sepharose beads for 3 h. Then collect the immunoprecipitants at
3000 rpm and washed three times with PBS. The proteineantibody
complexes recovered on beads were subjected to Western blotting.
6.2.11. Surface plasmon resonance (SPR)
The binding kinetics between Nur77-LBD and 8n, 10b and 10c
were performed on a BIAcore T200 instrument (GE Healthcare) at
25 ꢀC. Nur77-LBD were diluted to 0.05 mg/mL in 10 mM NaAc (pH
5.0) and immobilized on a CM5 sensor chip (GE Healthcare) by
amine coupling at densities ~9000 RU according to the manufac-
turer’s instructions. Gradient concentrations of compounds were
injected into the flow cells in running buffer (PBS, 0.4% DMSO) at a
6.2.6. Immunostainning
flow rate of 30 mL/min for 150 s of association phase followed by a
Cells incubated on glass slides were fixed in 4% para-
formaldehyde overnight and permeabilized with PBS containing
0.1% Triton X-100 for 15 min on ice. Cells were blocked with PBS
containing 1% bovine serum albumin (BSA) for 30 min at room
temperature, and incubated with various primary antibodies at 4 ꢀC
for 12 h. Then wash the cells with PBS and incubated with sec-
ondary antibodies for another 3 h at room temperature. Mito-
chondria were marked by Mitotracker (Deep Red) (1: 20000
dilution) for additional 30 min before fixed in 4% para-
formaldehyde. Cells were co-stained with 4060-diamidino-2- phe-
nylindole (DAPI) (Sigma) for the visualization of nuclei. Fluorescent
images were collected and analyzed by using a fluorescence mi-
croscopy or MRC-1024 MP laser-scanning confocal microscope
(Bio-Rad, Hercules, CA).
420 s dissociation phase and a 30 s regeneration phase (10 mM
Glycin-HCl, pH 2.5). The data were analyzed using BIAcore T200
Evaluation Software 2.0 and referenced for blank injections and
reference Surface. The dissociation constant (Kd) was fitted to the
standard 1:1 interaction model and calculated using the global
fitting of the kinetic data from gradient concentrations.
6.2.12. MMTV-PyMT transgenic mouse study
12-week-old female mice of a transgenic mouse on the FVB/N
genetic background expressing the PyMT oncogene under the
control of MMTV LTR promoter40 were randomized into different
groups (n ¼ 6) and oral gavaged with 8n or 10b for 14 days. 8n and
10b were dissolved in DMSO and diluted with normal saline con-
taining 5.0% (V/V) Tween-80 to a final concentration 3 mg/mL. All
experimentations and animal usage were performed and approved
by the Animal Care and Use Committee of Xiamen University.
6.2.7. Mitochondrial separation
10% of the collected cells were taken out to represent the whole
cells. Resuspend other cells in solution Buffer D (10 mM Tris-HCl pH
7.5, 2 mM MgCl2, 10 mM KCl, 250 mM sucrose, 0.5 mM DTT) con-
taining a cocktail of protease inhibitors for 15 min on ice. Use a
homogenizer to stick the cells for 20 times. Centrifuge at 500 g for
5 min to get supernatant S1, then centrifuge S1 at 5000 g for 10 min
to get precipitate P1 as a mitochondrial component. The various
fractions were analyzed by SDS-PAGE. Anti-PARP antibody was used
to ensure the purity of nuclear. Anti-Tom20 antibody was used to
detect the purity of mitochondria.
6.2.13. Statistical analysis
Data were expressed as mean SD. Each assay was repeated in
triplicate in three independent experiments. The statistical signif-
icance of the differences among the means of several groups was
determined using Student’s t-test. Differences were considered
statistically significant with P < 0.05.
6.2.14. In vivo pharmacokinetic (PK) profile in rats
The pharmacokinetic analysis of compound BI1071(8n) and 10b
was performed on Sprague-Dawley rats weighing between 200 and
220 g. All experiments with animals were approved by the Animal
Care and Use Committee of Xiamen University, in accordance with
the animal care and use guidelines. The oral dose of compounds
was 5 mg/kg, and the intravenous dose was 0.5 mg/kg. Compounds
were dosed as a solution in DMSO/T-80/H2O (1 : 5: 94 by volume).
6.2.8. The cell viability assay
Cell viability was determined by the MTT assay. Cells were
seeded in 96-well plates at 104 cells/well and then treated with
various concentrations of potent compounds for 24 h. Then the
cells were incubated with 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-
16

X. Tu, X. Chen, D. Zhang et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
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5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 36 h
after intravenous administered, and 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h,
14 h, 24 h and 36 h after oral administered. For 10b, the rats were
randomly assigned into two groups consisting of four ones per
group. Blood samples were collected by tubes containing Na hep-
arin (10 mg/mL, 5 mL) at 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h,
8 h, 12 h, 24 h and 36 h after intravenous administered, and 0.5 h,
1 h, 3 h, 4 h, 6 h, 8 h,10 h,12 h, 24 h and 36 h after oral administered.
The bloods were centrifuged at 5000 r/min for 15 min at 4 ꢀC to
separate the plasma (50
of 10.0 g/mL internal standard (IS: Carbamazrpine), and vortex-
mixed for 15 s, then the 445 L methanol (2% HCl) were added
mL). Plasma samples were added with 5 mL
m
m
into the solution and vortex-mixed for 1 min to precipitate pro-
teins. The supernatant was collected after centrifuging at 12000 r/
min for 15 min at 4 ꢀC, filtered through a 0.22
mm filter membrane,
and injected with 5
mL into the liquid chromatography tandem
mass spectrometry (LC-MS/MS) system for the analysis (Waters
Acquity UPLC- XEVOTQSMIC). The plasma concentrationꢁtime data
of 8n and 10b were analyzed using the DAS 3.0 to obtain the
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Declaration of competing interest
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The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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7. Acknowledgement
This work was supported by the grants from the National Nature
Science Fund of China (NSFC-31271453, NSFC-31471318, NSFC-
91129302 and NSFC-81301888), the Fundamental Research Funds
for the Central Universities (2013121038, 20720180052), the Nat-
ural Science Foundation of Fujian Province of China (No.
2018J01133).
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Appendix A. Supplementary data
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