Discovery of β-carboline-(phenylsulfonyl)furoxan hybrids as potential anti-breast cancer agents

Article abstract of DOI:10.1016/j.bmcl.2021.127952

The cytotoxicity properties of the β-carboline alkaloids have been broadly investigated. However, the potential application of β-carbolines was hindered due to the moderate activity in cancer. In the present study, thirty β-carboline-(phenylsulfonyl)furoxan hybrids (11a–j, 12a–j and 13a–j) were designed and synthesized through esterification and amidation reaction strategy, and their inhibitory activities against the human breast cancer cell lines MCF-7 and MDA-MB-231 were evaluated by CCK-8 assay. Biological evaluation presented that the most promising amide derivative 13h, substituted with p-methoxyphenyl group at position 1, generated high concentration of NO and evidently depressed the MCF-7 (IC₅₀ = 0.89 μM) and MDA-MB-231 (IC₅₀ = 0.62 μM) cells proliferation. Particularly, the wound healing and transwell assays demonstrated that 13h significantly inhibited the migration and invasion of MDA-MB-231cells. Furthermore, the preliminary mechanisms studies indicated that 13h induced G2/M phase arrest and apoptosis possibly causing by ROS accumulation and ROS-mediated DNA damage. Based on these considerations, 13h may be a promising antimetastatic agent for breast cancer, which is noteworthy for further exploration.

Full text of DOI:10.1016/j.bmcl.2021.127952

Bioorg. Med. Chem. Lett. 40 (2021) 127952
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of β-carboline-(phenylsulfonyl)furoxan hybrids as potential
anti-breast cancer agents
,
,
,*
Xu Hua, Xiang Gaoa, Gang Gaoa, Yanbing Wangb, Hao Caob *, Dahong Lia *, Huiming Huaa
a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, and School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University, 103 Wenhua Road, Shenyang 110016, PR China
b School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The cytotoxicity properties of the β-carboline alkaloids have been broadly investigated. However, the potential
application of β-carbolines was hindered due to the moderate activity in cancer. In the present study, thirty
β-carboline-(phenylsulfonyl)furoxan hybrids (11a–j, 12a–j and 13a–j) were designed and synthesized through
esterification and amidation reaction strategy, and their inhibitory activities against the human breast cancer cell
lines MCF-7 and MDA-MB-231 were evaluated by CCK-8 assay. Biological evaluation presented that the most
promising amide derivative 13h, substituted with p-methoxyphenyl group at position 1, generated high con-
β-Carboline
NO donor
Breast cancer
Migration apoptosis
centration of NO and evidently depressed the MCF-7 (IC50 = 0.89 μM) and MDA-MB-231 (IC50 = 0.62 μM) cells
proliferation. Particularly, the wound healing and transwell assays demonstrated that 13h significantly inhibited
the migration and invasion of MDA-MB-231cells. Furthermore, the preliminary mechanisms studies indicated
that 13h induced G2/M phase arrest and apoptosis possibly causing by ROS accumulation and ROS-mediated
DNA damage. Based on these considerations, 13h may be a promising antimetastatic agent for breast cancer,
which is noteworthy for further exploration.
Although substantial advances in treatment have been achieved,
cancer remains the second leading cause of death globally in 2019,
which outnumber the human immunodeficiency syndrome, malaria,
and tuberculosis sum.1 Strikingly, breast cancer, the most common
malignant neoplasms affecting women, is the leading cause of cancer-
associated deaths.2,3 Currently, the treatment strategy for breast can-
cer mainly covers surgical mastectomy, hormone based therapies, ra-
diation therapy, chemotherapy, and anti-angiogenic therapies based on
tyrosine kinase receptors and hypoxia inducible factors (HIF) small
molecule inhibitors and so on.4 For estrogen receptor positive breast
cancer, these therapies exert a better prognosis, however, which fail to
produce prominent clinical benefits in the treatment of triple negative
breast cancers (TNBC).5,6 TNBC are short of human epidermal growth
factor receptor 2 (HER2), progesterone receptor (PR), and estrogen re-
ceptor (ER) expression, possessing highly metastatic and aggressive with
a worse prognosis.7 Moreover, some targeted therapeutics are also
barely satisfactory for TNBC.8–11
present, roughly 25% of drugs are arisen from natural resources, and the
others are synthetic derivatives based on prototype compounds isolated
from plants.14 β-carboline alkaloids, possessing the common tricyclic
pyrido-[3,4-b]indole ring, are mainly isolated from Zygophyllaceae,15
Simaroubaceae,16 Caryophyllaceae17 and Rubiaceae.18 Additionally,
this class of alkaloids has been extensively researched due to their broad-
spectrum of pharmacological activities, including anti-tumorigenic,19
anti-Alzheimer’s disease,20 anti-Huntington’s disease,21 antidepres-
sant,22
anti-diabetic
(inhibiting
dual
specificity
tyrosine
phosphorylation-regulated kinase 1A (DYRK1A)),23 antimicrobial,24
anti-inflammatory,25 and antiplasmodial26 effects and so on. Among
them, the antitumor activity of β-carbolines has become a hot research
topic. Accumulating evidences demonstrated that these alkaloids
restrained cancer cell proliferation and induced apoptosis through
diverse mechanisms, such as intercalating with DNA,27 blocking
angiogenesis,28 depressing I-Kappa-B kinase (IKK),29 topoisomerases I
and II (Topo I and II),30 mitogen activated protein kinase-activated
protein kinase 2 (MK-2),31 cyclin-dependent kinase (CDK),32 polo-like
kinase (PLK),33 kinesin-like protein Eg534 and histone deacetylases
(HDACs).35 Recent studies indicated that harmine (Fig. 1) inhibited
The alarming increase in cancer mortality has driven the search for
new anti-tumor drugs, and one of the feasible ways to look for effective
antineoplastic agents is still getting back to nature for answers.12,13 At
* Corresponding authors.
E-mail addresses: caohao2008@163.com (H. Cao), lidahong0203@163.com (D. Li), huimhua@163.com (H. Hua).
https://doi.org/10.1016/j.bmcl.2021.127952
Received 29 January 2021; Received in revised form 26 February 2021; Accepted 7 March 2021
Available online 17 March 2021
0960-894X/© 2021 Published by Elsevier Ltd.

X. Hu et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127952
NONOate (A) (Fig. 2), a long-acting NO donor, induced the mitochon-
drial apoptosis apoptosis in African American by enhancing Bax and
reactive oxygen species (ROS) levels, activating caspase-3, and depola-
rizing mitochondrial membrane potential in TNBC.49 Additionally, JS-K
(B) (Fig. 2), a NO-releasing prodrug, induced autophagy by up-
regulating microtubule light chain 3-II expression in breast cancer
cells, while sparing normal mammary epithelial cells.50 Subsequently,
Huang et al. found that O2-3-aminopropyl diazeniumdiolate (C) (Fig. 2)
inhibited highly metastatic TNBC cells proliferation, and decreased
adhesion, invasion and migration in vitro. Moreover, C also suppressed
implanted TNBC growth and metastasis in vivo via blocking microvescile
formation through NO-based epigenetic regulation of miR-203/RAB22A
expression.51 Considerable evidences suggest that NO donors may be
potential benefits for the treatment of breast cancer.
Fig. 1. Natural β-carboline alkaloids with anti-breast cancer properties.
Particularly interesting is the role of furoxans as NO donors in cancer
cells, there was report that furoxan derivative (D) (Fig. 2) inhibited
MCF-7 cells colony formation, and restrained MDA-MB-231 cells
migration by down-regulating pNF-κB-p65 expression.52 Our group has
developed several (phenylsulfonyl)furoxan-based NO-donor derivatives
possessing potent selective antitumor effects.53–59 Given that both
β-carboline and NO exert antiproliferative activity against breast cancer
cells, we envisioned that novel furoxan/β-carboline hybrids could
release high concentrations of NO, leading to superactive cytotoxicity
against breast cancer cells. In the present study, a series of NO-releasing
β-carboline derivatives (11a–j, 12a–j and 13a–j) were designed and
synthesized by conjugating the carboxyl group of 5a–c to
phenylsulfonyl-substituted furoxan via different chemical linkers.
Whereafter, their antiproliferative activity against MCF-7 and MDA-MB-
231 cells, NO-releasing ability, cell cycle analysis, induction of
apoptosis, comet assay, and the inhibitory effects of migration and in-
vasion were evaluated.
Fig. 2. NO donating derivatives with anti-breast cancer effects.
breast cancer resistance protein (BCRP) to decrease resistance to the
anticancer drugs mitoxantrone and camptothecin in MDA-MB-231
cells.36 Wink et al. showed that harmine suppressed telomerase activ-
ity of MCF-7 cells by down-regulating the expression of human telo-
merase reverse transcriptase (hTERT) mRNA, and inducing the
accelerated senescence phenotype through the p53/p21 pathway.37
Moreover, harmine restrained breast cancer cells growth and migration,
induced apoptosis in vitro (MDA-MB-231 and MCF-7 cells) and in vivo
(MCF-7 xenograft mice model), the underlying molecular mechanism
identified that harmine exerted these properties through reducing the
expression of transcriptional co-activator with PDZ-binding motif (TAZ),
phosphorylation of extracellular regulated protein kinases (p-ERK),
phosphorylation of v-Akt murine thymoma viral oncogene (p-Akt) and
Bcl-2, and increasing Bax expression.38 Picrasidine G (Fig. 1), a natural
dimeric β-carboline, caused apoptosis and inhibited proliferation
through aggrandizing chromatin condensation, sub-G1 population,
cleavage of caspase 3 and poly(ADP-ribose) polymerase (PARP), sup-
pressing the epidermal growth factor receptor (EGFR)-induced signal
transducer and activator of transcription 3 (STAT3) phosphorylation
and survivin transcription in the EGFR-overexpressed MDA-MB 468
cells.39 In addition, Sheu et al. found that flavopereirine (Fig. 1) induced
apoptosis and S phase cell cycle arrest via the Akt/p38/mitogen-
activated protein kinases (MAPK)/ERK1/2 signaling pathway in MDA-
MB-231 cells.40
The synthesis of target hybrids 11a–j, 12a–j and 13a–j is illustrated
in Scheme 1. The β-carboline intermediates 5a–c were obtained by a
four-step sequence. First, L-tryptophan 1 was treated with differently
substituted aldehydes in the presence of sodium hydroxide solution or
trifluoroacetic acid (TFA) and 1,2-dichloroethane (DCE) to offer 2a–c by
Pictet–Spengler reaction.60 Then 3a–c were prepared by esterification of
2a–c through using SOCl2. Meanwhile, 3a–c were oxidized by KMnO4 in
anhydrous N,N-dimethylformamide (DMF) to afford 4a–c. Finally, 4a–c
were hydrolyzed by sodium hydroxide to generate intermediates 5a–c.
Next, 9 was synthesized by a three-step strategy.61 Briefly, benzenethiol
6 was reacted with chloroacetic acid to gain compound 7. 7 was then
oxidized by 30% H2O2 to give 8. Diphenylsulfonylfuroxan 9 was syn-
thesized from 8 accordingly with fuming HNO3, which was next con-
verted to diverse furoxan intermediates 10a–j by reaction with various
amino-substituted alcohol and diol. Finally, the treatment of β-carbo-
line intermediates 5a–c with furoxan derivatives 10a–f in the presence
of 4-dimethylaminopyridine (DMAP) and 1-(3-(dimethylamino)-pro-
pyl)-3-ethylcarbodiimide hydrochloride (EDCI) produced eighteen
novel β-carboline/phenylsulfonylfuroxan derivatives 11a–f, 12a–f and
13a–f. Simultaneously, twelve novel hybrids 11g–j, 12g–j and 13g–j
were synthesized by acylation of 5a–c with 10g–j under the condition of
EDCI and 1-hydroxybenzotriazole (HOBT). The target compounds
11a–j, 12a–j and 13a–j were purified by silica gel column chromatog-
raphy, and all structures were identified by 1H NMR, 13C NMR, and high
resolution mass spectrometry (HR-MS). (data in Supplementary
Materials.)
Besides participating in a majority of physiological and pathophys-
iological processes, nitric oxide (NO) was found to be an effective
anticancer candidate via different mechanisms, including ERK, Akt,
mammalian target of rapamycin (mTOR), Ras, cyclin D1/retinoblas-
toma (Rb), nuclear factor kappa B (NF-κB) signaling pathways and so
on.41–43 Nevertheless, the role of NO is quite intricate in tumor biology,
since it inhibits or promotes tumor proliferation mainly depending on
concentration and cell sensitivity to NO.44 It is worth mentioning that
high concentration of NO released by NO donors not only induces tumor
cells apoptosis through reducing the cell cycle proteins translation, but
also sensitizes cells to radiation, immunotherapy, and chemotherapy in
vitro and in vivo.45–47 Engagingly, a recent study showed that NO-
targeted therapy inhibited cancer stem cells and increased anti-
hormonal therapy efficacy avoiding drug resistance in estrogen
receptor-positive breast cancer cells.48 Pervin et al. reported that DETA-
To estimate the antineoplastic activity of the target derivatives, the
inhibitory action on the proliferation of human mammary gland tumor
cell line MCF-7 and TNBC cell line MDA-MB-231 was primarily tested by
CCK-8 assay.62 The IC50 values of all test compounds against two solid
tumor cells are emerged in Table 1. The results displayed that most of
the synthesized derivatives were remarkably more potent than the
natural harmine. Eleven out of 30 hybrids even stronger than doxoru-
bicin or 9. The structure–activity relationships (SARs) analysis showed
that 11a–j, 12a–j and 13a–j with different linkers or a variety of 1-
2

X. Hu et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127952
Scheme 1. Synthesis of compounds 11a–j, 12a–j and
13a–j. Reagents and conditions: (a) 0.4 N NaOH so-
lution, 37% formaldehyde solution, 37 ℃, 3 d; or
TFA, DCE, acetaldehyde/p-methoxybenzaldehyde,
110 ℃, 0.5 h; (b) SOCl2, MeOH, 0 ◦C, 1 h, and then
reflux, 6 h; (c) KMnO4, anhydrous DMF, 0 ◦C, 1 h, and
then rt, 24 h; (d) NaOH/MeOH, reflux, 6 h; (e)
ClCH2COOH, NaOH solution, reflux, 2 h; (f) 30%
H2O2, AcOH, rt, 3 h; (g) Fuming HNO3, 90 ◦C, 4 h; (h)
Diol or alkanolamine, THF, NaH, 0 ◦C, 4 h; (i) 10a–f,
EDCI, DMAP, anhydrous DCM, rt, 8–12 h; or 10g–j,
HOBT, EDCI, anhydrous DCM, rt, 8–12 h.
3

X. Hu et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127952
presented more potent inhibitory activity than the compounds (11a–f,
12a–f and 13a–f) linked with diol, which might be due to the fact that
the ester-linked hybrids were usually easily metabolized into acids and
alcohols in the body to cause weakened activity. As a matter of fact, the
ester derivatives (11a, 11c, 11d and 11e) showed the lowest activity
against breast cancer cells.
Table 1
The antiproliferative activities of 11a–j, 12a–j and 13a–j against MCF-7 and
MDA-MB-231 cells.
Compounds
IC50
(μ
M)a
MCF-7
MDA-MB-231
11a
11b
11c
11d
11e
11f
11.47 ± 0.39
4.64 ± 0.28
9.57 ± 0.23
13.73 ± 0.70
19.04 ± 0.65
5.27 ± 0.40
7.53 ± 0.38
4.64 ± 0.44
6.94 ± 0.36
8.28 ± 0.47
4.58 ± 0.35
4.88 ± 0.19
4.28 ± 0.15
6.85 ± 0.30
5.89 ± 0.41
8.86 ± 0.48
4.96 ± 0.35
2.97 ± 0.14
3.80 ± 0.17
3.29 ± 0.27
4.80 ± 0.16
3.49 ± 0.16
2.92 ± 0.27
5.36 ± 0.32
3.92 ± 0.21
2.35 ± 0.27
1.45 ± 0.12
0.89 ± 0.05
1.56 ± 0.10
1.33 ± 0.11
> 30
9.56 ± 0.43
7.96 ± 0.45
12.54 ± 0.68
8.87 ± 0.76
15.85 ± 0.52
5.89 ± 0.31
6.75 ± 0.47
7.50 ± 0.42
5.97 ± 0.27
6.83 ± 0.35
7.46 ± 0.37
2.24 ± 0.20
4.54 ± 0.22
5.75 ± 0.24
7.67 ± 0.52
6.91 ± 0.55
3.84 ± 0.16
1.62 ± 0.10
2.20 ± 0.15
3.47 ± 0.19
3.59 ± 0.29
2.86 ± 0.22
4.26 ± 0.29
2.98 ± 0.44
2.53 ± 0.26
1.16 ± 0.08
0.87 ± 0.05
0.62 ± 0.05
2.27 ± 0.16
0.92 ± 0.04
> 30
Among the amide hybrids of β-carbolines, compounds 13h with an
aminopropanol linker possessed evidently inhibition on the prolifera-
tion of breast cancer cells, compared with aminoalcohol linkers, such as
13g, 13i and 13j. As previously noticed, the compounds 13g–j indicated
relatively low IC50 values (1.45, 0.89, 1.56 and 1.33 μM, respectively)
11g
11h
11i
against MCF-7 cells, compared with the IC50 values of 5c, harmine,
positive control doxorubicin (14.3, 16.94, and 3.09 M, respectively).
By contrast, the compounds 13g, 13h and 13j showed the minimum IC50
values (0.87, 0.62, and 0.92 M, respectively) against MDA-MB-231
cells, compared with the IC50 values of 5c, harmine, doxorubicin
μ
11j
12a
12b
12c
12d
12e
12f
μ
(12.63, 21.91, and 2.86 μM, respectively). To take these elements into
consideration, compound 13h was the most promising for in depth
exploration into the action mechanism against TNBC MDA-MB-231
cells.
12g
12h
12i
Compound 13h was consisted of the β-carboline moiety (compound
5c) and furoxan (9) through an aminopropanol linker. As shown in
12j
13a
13b
13c
13d
13e
13f
Table 1, the IC50 values of 13h against MCF-7 (0.89 μM) and MDA-MB-
231 cells (0.62
μ
M) were obviously lower than that of 5c (14.3 and
12.63 M) and 9 (3.92 and 4.84
μ
μ
M), respectively. These data showed
that the potent anti-breast cancer activity of 13h might be attributed to
the synergistic effects of β-carboline and NO donor. Therefore, in order
to affirm the contribution of NO to the antiproliferative activity, the NO
released concentrations by several selected target derivatives were
tested using the Griess assay.63 As outlined in Fig. 3, hybrids (13a, 13d
and 13g–j) substituted with p-methoxyphenyl at position 1 possessed
stronger NO release ability than that of analogues substituted with
methyl group (12d, 12df, 12h and 12i) or H (11d, 11e, 11f and 11h).
Among compounds 13a, 13d, and 13g–j, the amide hybrids (13g–j)
released higher NO levels than that of the ester derivatives (13a and
13d). Intriguingly, the most promising compound 13h produced the
highest concentration of NO. These results supported that the NO levels
released by several selected target derivatives were positively correlated
with their antiproliferative activity against MDA-MB-231 cells.
13g
13h
13i
13j
5a
5b
19.24 ± 0.67
14.30 ± 0.32
3.92 ± 0.10
16.94 ± 0.47
3.09 ± 0.16
22.64 ± 0.86
12.63 ± 0.28
4.84 ± 0.22
21.91 ± 1.25
2.86 ± 0.18
5c
9
harmine
doxorubicin
a
IC50: Half inhibitory concentrations were measured by the CCK-8 assay, cells
were treated with indicated compounds for 72 h, and the values are expressed as
averages ± standard deviations of three independent experiments.
Numerous compounds suppressed cancer cell growth mainly by
decreasing cell division to arrest cell cycle.64 To verify whether 13h
affects cell cycle progression, propidium iodide (PI) staining-based cell
cycle analysis was conducted by flow cytometry. As shown in Fig. 4a,
treatment with 13h hindered the cell cycle at the G2/M stage. Compared
to the control cells, the culture of MDA-MB-231 cells with different
concentrations of 13h (0.15, 0.3 and 0.6 μM) enhanced the cells per-
centage in the G2/M phase from 10.90% to 23.54%, simultaneously the
cells percentage of G1 phase reduced, and that of S phase remained
essentially unchanged (Fig. 4b). The above results affirmed that 13h
inhibited MDA-MB-231 cells proliferation via arresting cell cycle at G2/
M phase.
Since various anticancer drugs cause the tumor cells death through
inducing apoptosis,65 additionally, cell cycle arrest induced by multiple
factors may also lead to apoptosis. So whether compound 13h induced
cancerous cell apoptosis was also investigated. Whereafter, vehicle- or
13h-incubated MDA-MB-231 cells were stained with Annexin V-FITC
and PI. As illustrated in Fig. 5a and b, treatment with 13h caused a
Fig. 3. NO-releasing ability of several selected target compounds. The values
are expressed as averages of three independent experiments.
concentration-dependent (0.15, 0.3, and 0.6 μM) increase in both the
substituted β-carbolines indicated discrepant antiproliferative activity
against breast cancer cells. For instance, the antitumor activity of
β-carbolines derivatives (13a–j) substituted with p-methoxyphenyl
group at position 1 was significantly stronger than that of analogues
substituted with methyl (12a–j) or H (11a–j) at position 1, which re-
flected that the steric hindrance and electric effect of substituted groups
were of great importance in the biological activities. Furthermore, the
compounds (11g–j, 12g–j and 13g–j) linked with alkanolamine
early- and late-stage apoptosis of MDA-MB-231 cells (12.67%, 23.99%
and 54.18%, respectively), which demonstrated that 13h inhibited
MDA-MB-231 cells proliferation by inducing apoptosis.
ROS plays a central role in modulating cell apoptosis by disturbing
the intracellular environment in various tumor cells, and accesses to the
nucleus to inflict DNA damage.66 Accordingly, to study whether com-
pound 13h promoted intracellular ROS generation, the 2′,7′-dichlor-
odihydrofluorescein diacetate (DCFH-DA) was used to survey the levels
4

X. Hu et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127952
Fig. 4. Compound 13 h induced G2/M arrest in MDA-MB-231 cancer cells. MDA-MB-231 cells were incubated with different concentrations of 13 h (0, 0.15, 0.3, and
0.6
μM) for 72 h. (a) Cells were harvested and stained with PI and then analyzed by flow cytometry. (b) Histograms display the cell cycle distribution percentages.
5

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Bioorganic & Medicinal Chemistry Letters 40 (2021) 127952
Fig. 5. Compound 13 h induced apoptosis of MDA-MB-231 cells. (a) MDA-MB-231 cells were incubated with varying concentrations of 13 h (0, 0.15, 0.3 and 0.6
μ
M). After 72 h of incubation, cells were collected and stained with Annexin V/PI, followed by flow cytometric analysis. (b) Histograms display the percentage of cell
distribution. Data are represented as mean ± SD of three independent experiments. *p < 0.05 vs control group.
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Bioorganic & Medicinal Chemistry Letters 40 (2021) 127952
Fig. 6. Accumulation of ROS production in MDA-MB-231 cells induced by 13 h (72 h). (a) Generation of ROS was measured using the ROS-detecting fluorescent dye
DCFH-DA in combination with FACScan flow cytometry. (b) Corresponding histograms of FACScan flow cytometry are shown. Data are represented as mean ± SD of
three independent experiments. *p < 0.05 compared to control cells.
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Bioorganic & Medicinal Chemistry Letters 40 (2021) 127952
Fig. 7. DNA damage induced by compound 13 h. (a) Cells were incubated for 72 h and DNA damage was evaluated by comet assay. Representative photomicro-
graphs are presented. (b) Head DNA%, tail DNA%, tail movement, and Olive tail movement were measured. Tail and Olive tail movements were presented as
estimated DNA damage in arbitrary units. Data are represented as mean ± SD of three independent experiments. *p < 0.05 compared to control cells.
8

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Bioorganic & Medicinal Chemistry Letters 40 (2021) 127952
Fig. 8. Effects of 13 h on metastasis, invasion, and adhesion of MDA-MB-231 cells. (a) Scratches of MDA-MB-231 cell colonies were generated with pipette tips (200
L). After 24 h of incubation with 0, 0.15, 0.3 and 0.6 M compound 13 h, representative images were captured using phase contrast microscopy. (b) Wound-healing
rate of MDA-MB-231 cells induced by 13 h. (c) MDA-MB-231 cells were seeded onto chambers and incubated with 13 h (0, 0.15, 0.3, and 0.6 M) for 24 h. Cells that
migrated through the chambers were stained with crystal violet, and representative images were captured. (d) Histograms display the number of cells. (e) MDA-MB-
μ
μ
μ
231 cells were incubated with 13 h (0, 0.15, 0.3 and 0.6 μM) in 96-well plates, and the nonadherent cells were washed away with phosphate-buffered saline, while
the adhesive cells were counted using phase contrast microscopy. All data are represented as the mean ± SD of three independent experiments. *p < 0.05 vs the
control group.
of ROS in the presence and absence of 13h. As listed in Fig. 6a and 6b,
13h increased the generation of intracellular ROS in a dose-dependent
manner. The DCFH-DA positive cells ratio increased from 3.04% in
cells incubated with dimethyl sulfoxide (DMSO) to 51.62% in cells
13h significantly increased the endogenous DSBs generation. Addi-
tionally, 13h remarkably raised tail DNA percentage with increasing
concentration, and by contrast, reduced the head DNA percentage.
Meanwhile, DNA migration of tail and Olive tail was perceived with
increasing concentration of 13h (Fig. 7b), announcing that 13h inhibi-
ted DNA replication and induced DNA damage. Taken together, these
results preliminarily proved that compound 13h induced MDA-MB-231
cells apoptosis by stimulating ROS generation, which in turn led to DNA
damage.
incubated with 0.6 μM 13h.
As is well known, DNA damage is deemed to a mark of apoptosis.
Moreover, it was well documented that excess accumulation of intra-
cellular ROS might led to DNA damage and lipid membrane peroxida-
tion, eventually causing cell death.67 Therefore, the DNA damage by
detecting double-strand DNA breaks (DSBs) adducts in MDA-MB-231
cells using neutral comet assays was evaluated. Fig. 7a revealed that
It is noteworthy that TNBC is the most invasive and metastatic sub-
type of breast cancer, the effects of compound 13h on migration and
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Bioorganic & Medicinal Chemistry Letters 40 (2021) 127952
Fig. 8. (continued).
invasion by wound healing and transwell assays in MDA-MB-231 cells
were further explored. As shown in Fig. 8a and b, after treating without
drugs for 24 h, the wound closure distance was significantly shortened,
yet the addition of 13h had remarkable inhibitory effect on wound
biologically evaluated. After incubating the tumor cells for 72 h, most
derivatives presented stronger antiproliferative activity than harmine
against MCF-7 and MDA-MB-231 cells. Notably, 13g, 13h and 13j dis-
played more potent inhibition on MDA-MB-231 cells compared to those
of doxorubicin. Particularly, 13h exhibited the most excellent anti-
closure. Moreover, the wound closure rate of cells exposed to 0.3 μM
13h was less than half compared to the control. With the increasing of
13h to 0.6 M, the wound closure rate decreased lower than that treated
with 0.3
proliferative activity, with an IC50 value of 0.62
231 cells. Interestingly, 13h also generated a continuous NO release in
vitro, and produced the NO concentration of above 65 M/L at 3 h.
μM against MDA-MB-
μ
μ
M, revealing that 13h significantly inhibited MDA-MB-231
μ
cells migration via a concentration-dependent manner. The results of
the transwell assay are presented in Fig. 8c and d. Treatment with 13h
distinctly restrained the invasion of MDA-MB-231 cells at micromolar
Accordingly, 13h was selected for further research to explore the action
mechanisms in MDA-MB-231 cell lines. The results demonstrated that
13h induced G2/M-phase arrest and cell apoptosis by the activation of
ROS-mediated DNA damage. Additionally, compound 13h suppressed
the MDA-MB-231 cells metastasis, invasion, and adhesion. Collectively,
the novel β-carboline-(phenylsulfonyl)furoxan hybrid 13h possessed
prominent antineoplastic activity and sustained NO release capacity in
vitro. Hence, compound 13h may be a promising antimetastatic agent
for TNBC, which is noteworthy for further investigation.
concentrations (0.3–0.6
μM). Subsequently, the adhesion assays
(Fig. 8e) verified that 13h dose-dependently suppressed the adhesion of
MDA-MB-231 cells. As outlined above, 13h possessed superactive ac-
tivity against the migration, invasion, and adhesion of MDA-MB-231
cells in vitro.
In conclusion, thirty hybrids of β-carbolines and (phenylsulfonyl)
furoxan (11a–j, 12a–j and 13a–j) were designed, synthesized and
10

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Bioorganic & Medicinal Chemistry Letters 40 (2021) 127952
Fig. 8. (continued).
breast cancer: pooled and subgroup analyses of data from 2447 patients. Ann Oncol.
2013;24:2773–2780.
Declaration of Competing Interest
11 Islam R, Lam KW. Recent progress in small molecule agents for the targeted therapy
of triple-negative breast cancer. Eur J Med Chem. 2020;207, 112812.
12 Harvey AL, Edrada-Ebel R, Quinn RJ. The re-emergence of natural products for drug
discovery in the genomics era. Nat Rev Drug Discov. 2015;14:111–129.
13 Rodrigues T, Reker D, Schneider P, Schneider G. Counting on natural products for
drug design. Nat Chem. 2016;8:531–541.
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.
14 Newman DJ, Cragg GM. Natural products as sources of new drugs over the nearly
four decades from 01/1981 to 09/2019. J Nat Prod. 2020;83:770–803.
15 Li SG, Wang YT, Zhang Q, et al. Pegaharmols A-B, axially chiral β-carboline-
quinazoline dimers from the roots of Peganum harmala. Org Let. 2020;22:7522–7525.
16 Ohishi K, Toume K, Arai MA, et al. 9-Hydroxycanthin-6-one, a β-carboline alkaloid
from Eurycoma longifolia, is the first wnt signal inhibitor through activation of
Acknowledgements
This paper was financially supported by the National Natural Science
Foundation of China (81773604) and Career Development Support Plan
for Young and Middle-aged Teachers in Shenyang Pharmaceutical
University.
glycogen synthase kinase 3β without depending on casein kinase 1
2015;78:1139–1146.
α. J Nat Prod.
17 Chen YF, Kuo PC, Chan HH, et al. β-carboline alkaloids from Stellaria dichotoma var.
lanceolata and their anti-inflammatory activity. J Nat Prod. 2010;73:1993–1998.
18 de Oliveira FP, Perdomo RT, Garcez FR, et al. Further constituents of Galianthe
Appendix A. Supplementary data
thalictroides (Rubiaceae) and inhibition of DNA topoisomerases I and IIα by its
cytotoxic β-carboline alkaloids. Bioorg Med Chem Lett. 2014;24:1358–1361.
19 Le Moigne R, Subra F, Karam M, Auclair C. The β-carboline harmine induces actin
dynamic remodeling and abrogates the malignant phenotype in tumorigenic cells.
Cells. 2020;9:1168.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127952.
20 He D, Wu H, Wei Y, et al. Effects of harmine, an acetylcholinesterase inhibitor, on
spatial learning and memory of APP/PS1 transgenic mice and scopolamine-induced
memory impairment mice. Eur J Pharmacol. 2015;768:96–107.
References
21 Jain S, Panuganti V, Jha S, Roy I. Harmine acts as an indirect inhibitor of
intracellular protein aggregation. ACS Omega. 2020;5:5620–5628.
22 Farzin D, Mansouri N. Antidepressant-like effect of harmane and other β-carbolines
in the mouse forced swim test. Eur Neuropsychopharmacol. 2006;16:324–328.
23 Kumar K, Wang P, Wilson J, et al. Synthesis and biological validation of a harmine-
based, central nervous system (CNS)-avoidant, selective, human β-cell regenerative
dual-specificity tyrosine phosphorylation-regulated kinase A (DYRK1A) inhibitor.
J Med Chem. 2020;63:2986–3003.
1 Cortes J, Perez-Garcia JM, Llombart-Cussac A, et al. Enhancing global access to
cancer medicines. CA Cancer J Clin. 2020;70:105–124.
2 Sparano JA, Gray RJ, Makower DF, et al. Adjuvant chemotherapy guided by a 21-
gene expression assay in breast cancer. New Engl J Med. 2018;379:111–121.
3 DeSantis C, Ma JM, Bryan L, Jemal A. Breast cancer statistics, 2013. Ca-Cancer J Clin.
2014;64:52–62.
4 Waks AG, Winer EP. Breast cancer treatment: a review. JAMA-J Am Med Assoc. 2019;
321:288–300.
24 Nenaah G. Antibacterial and antifungal activities of (β)-carboline alkaloids of
Peganum harmala (L) seeds and their combination effects. Fitoterapia. 2010;81:
779–782.
5 Brufsky AM, Dickler MN. Estrogen receptor-positive breast cancer: exploiting
signaling pathways implicated in endocrine resistance. Oncologist. 2018;23:528–539.
6 Carey L, Winer E, Viale G, Cameron D, Gianni L. Triple-negative breast cancer:
disease entity or title of convenience? Nat Rev Clin Oncol. 2010;7:683–692.
7 Sharma S, Barry M, Gallagher DJ, Kell M, Sacchini V. An overview of triple negative
breast cancer for surgical oncologists. Surg Oncol. 2015;24:276–283.
8 Costa A, Kieffer Y, Scholer-Dahirel A, et al. Fibroblast heterogeneity and
immunosuppressive environment in human breast cancer. Cancer Cell. 2018;33:
463–479.
25 Nguyen HD, Choo YY, Nguyen TD, Nguyen HN, Chau VM, Lee JH. 7-Methoxy-(9H-
β-carbolin-1-il)-(E)-1-propenoic acid, a β-carboline alkaloid from Eurycoma longifolia,
exhibits anti-inflammatory effects by activating the Nrf2/heme oxygenase-1
pathway. J Cell Biochem. 2016;117:659–670.
26 Perkovic I, Raic-Malic S, Fontinha D, et al. Harmicines - harmine and cinnamic acid
hybrids as novel antiplasmodial hits. Eur J Med Chem. 2020;187, 111927.
27 Wu J, Zhao M, Qian K, Lee KH, Morris-Natschke S, Peng S. Novel N-(3-carboxyl-9-
benzyl-β-carboline-1-yl)ethylamino acids: synthesis, anti-tumor evaluation,
intercalating determination, 3D QSAR analysis and docking investigation. Eur J Med
Chem. 2009;44:4153–4161.
9 Audeh MW. Novel treatment strategies in triple-negative breast cancer: specific role
of poly(adenosine diphosphate-ribose) polymerase inhibition. Pharmgenomics Pers
Med. 2014;7:307–316.
´
´
10 Miles DW, Dieras V, Cortes J, Duenne AA, Yi J, O’Shaughnessy J. First-line
bevacizumab in combination with chemotherapy for HER2-negative metastatic
11

X. Hu et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127952
28 Liu Y, He Y, Yang F, et al. A novel synthetic small molecule YF-452 inhibits tumor
growth through antiangiogenesis by suppressing VEGF receptor 2 signaling. Sci China
Life Sci. 2017;60:202–214.
49 Martinez L, Thames E, Kim J, Chaudhuri G, Singh R, Pervin S. Increased sensitivity of
African American triple negative breast cancer cells to nitric oxide-induced
mitochondria-mediated apoptosis. BMC Cancer. 2016;16:559.
50 McMurtry V, Saavedra JE, Nieves-Alicea R, Simeone AM, Keefer LK, Tari AM. JS-K, a
nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal
mammary epithelial cells. Int J Oncol. 2011;38:963–971.
29 Castro AC, Dang LC, Soucy F, et al. Novel IKK inhibitors: β-carbolines. Bioorg Med
Chem Lett. 2003;13:2419–2422.
30 Funayama Y, Nishio K, Wakabayashi K, et al. Effects of β- and γ-carboline derivatives
of DNA topoisomerase activities. Mutat Res. 1996;349:183–191.
31 Trujillo JI, Meyers MJ, Anderson DR, et al. Novel tetrahydro-β-carboline-1-
carboxylic acids as inhibitors of mitogen activated protein kinase-activated protein
kinase 2 (MK-2). Bioorg Med Chem Lett. 2007;17:4657–4663.
51 Kang F, Zhu J, Wu J, et al. O2-3-Aminopropyl diazeniumdiolates suppress the
progression of highly metastatic triple-negative breast cancer by inhibition of
microvesicle formation via nitric oxide-based epigenetic regulation. Chem Sci. 2018;
9:6893–6898.
32 Song Y, Wang J, Teng SF, et al. β-carbolines as specific inhibitors of cyclin-dependent
kinases. Bioorg Med Chem Lett. 2002;12:1129–1132.
52 Gao Y, Sun TY, Bai WF, Bai CG. Design, synthesis and evaluation of novel
phenothiazine derivatives as inhibitors of breast cancer stem cells. Eur J Med Chem.
2019;183, 111692.
33 Han X, Zhang J, Guo L, et al. A series of β-carboline derivatives inhibit the kinase
activity of PLKs. PLoS ONE. 2012;7, e46546.
53 Zhao N, Tian KT, Cheng KG, et al. Antiproliferative activity and apoptosis inducing
effects of nitric oxide donating derivatives of evodiamine. Bioorg Med Chem. 2016;24:
2971–2978.
34 Barsanti PA, Wang W, Ni ZJ, et al. The discovery of tetrahydro-β-carbolines as
inhibitors of the kinesin Eg5. Bioorg Med Chem Lett. 2010;20:157–160.
35 Ling Y, Xu C, Luo L, et al. Novel β-carboline/hydroxamic acid hybrids targeting both
histone deacetylase and DNA display high anticancer activity via regulation of the
p53 signaling pathway. J Med Chem. 2015;58:9214–9227.
54 Li D, Han T, Tian K, et al. Novel nitric oxide-releasing spirolactone-type diterpenoid
derivatives with in vitro synergistic anticancer activity as apoptosis inducer. Bioorg
Med Chem Lett. 2016;26:4191–4196.
36 Ma Y, Wink M. The β-carboline alkaloid harmine inhibits BCRP and can reverse
resistance to the anticancer drugs mitoxantrone and camptothecin in breast cancer
cells. Phytother Res. 2010;24:146–149.
55 Jiao R, Xu F, Huang X, et al. Antiproliferative chromone derivatives induce K562 cell
death through endogenous and exogenous pathways. J Enzyme Inhib Med Chem.
2020;35:759–772.
37 Zhao L, Wink M. The β-carboline alkaloid harmine inhibits telomerase activity of
MCF-7 cells by down-regulating hTERT mRNA expression accompanied by an
accelerated senescent phenotype. PeerJ. 2013;1, e174.
56 Li D, Hu X, Han T, et al. Synthesis, Biological activity, and apoptotic properties of
NO-donor/enmein-type ent-kauranoid hybrids. Int J Mol Sci. 2016;17:747.
57 Han T, Li J, Xue J, et al. Scutellarin derivatives as apoptosis inducers: Design,
synthesis and biological evaluation. Eur J Med Chem. 2017;135:270–281.
58 Tian K, Xu F, Gao X, et al. Nitric oxide-releasing derivatives of brefeldin A as potent
and highly selective anticancer agents. Eur J Med Chem. 2017;136:131–143.
59 Huang X, Cheng K, Liu L, et al. Design, synthesis and apoptosis-related
antiproliferative activities of chelidonine derivatives. Bioorg Med Chem Lett. 2020;30,
126913.
38 Ding Y, He J, Huang J, et al. Harmine induces anticancer activity in breast cancer
cells via targeting TAZ. Int J Oncol. 2019;54:1995–2004.
39 Yamashita N, Kondo M, Zhao S, et al. Picrasidine G decreases viability of MDA-MB
468 EGFR-overexpressing triple-negative breast cancer cells through inhibition of
EGFR/STAT3 signaling pathway. Bioorg Med Chem Lett. 2017;27:2608–2612.
40 Yeh HT, Tsai YS, Chen MS, et al. Flavopereirine induces cell cycle arrest and
apoptosis via the AKT/p38 MAPK/ERK1/2 signaling pathway in human breast
cancer cells. Eur J Pharmacol. 2019;863, 172658.
60 Eagon S, Anderson MO. Microwave-assisted synthesis of tetrahydro-β-carbolines and
β-carbolines. European J Org Chem. 2014;2014:1653–1665.
41 Szabo C. Gasotransmitters in cancer: from pathophysiology to experimental therapy.
Nat Rev Drug Discov. 2016;15:185–203.
61 Ling Y, Ye X, Zhang Z, et al. Novel nitric oxide-releasing derivatives of
farnesylthiosalicylic acid: synthesis and evaluation of antihepatocellular carcinoma
activity. J Med Chem. 2011;54:3251–3259.
42 Fukumura D, Kashiwagi S, Jain RK. The role of nitric oxide in tumour progression.
Nat Rev Cancer. 2006;6:521–534.
62 Lakshmi Manasa K, Thatikonda S, Sigalapalli DK, et al. Design and synthesis of
β-carboline linked aryl sulfonyl piperazine derivatives: DNA topoisomerase II
inhibition with DNA binding and apoptosis inducing ability. Bioorg Chem. 2020;101,
103983.
43 Bonavida B, Garban H. Nitric oxide-mediated sensitization of resistant tumor cells to
apoptosis by chemo-immunotherapeutics. Redox Biol. 2015;6:486–494.
44 Chang CF, Diers AR, Hogg N. Cancer cell metabolism and the modulating effects of
nitric oxide. Free Radic Biol Med. 2015;79:324–336.
63 Kaur J, Bhardwaj A, Huang Z, et al. Synthesis and biological investigations of nitric
oxide releasing nateglinide and meglitinide type II antidiabetic prodrugs: in-vivo
antihyperglycemic activities and blood pressure lowering studies. J Med Chem. 2012;
55:7883–7891.
45 Bonavida B, Baritaki S, Huerta-Yepez S, Vega MI, Chatterjee D, Yeung KJNO. Novel
therapeutic applications of nitric oxide donors in cancer: roles in chemo-and
immunosensitization to apoptosis and inhibition of metastases. Nitric Oxide. 2008;19:
152–157.
64 Strzyz P. Cell signalling: Signalling to cell cycle arrest. Nat Rev Mol Cell Biol. 2016;17:
536.
46 Pervin S, Singh R, Chaudhuri G. Nitric oxide, Nω-hydroxy-L-arginine and breast
cancer. Nitric Oxide. 2008;19:103–106.
65 Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature. 2001;
411:342–348.
47 Huang Z, Fu J, Zhang Y. Nitric oxide donor-based cancer therapy: advances and
prospects. J Med Chem. 2017;60:7617–7635.
66 Zheng A, Li H, Wang X, et al. Anticancer effect of a curcumin derivative B63: ROS
production and mitochondrial dysfunction. Curr Cancer Drug Targets. 2014;14:
156–166.
48 Lopez-Sanchez LM, Mena R, Guil-Luna S, et al. Nitric oxide-targeted therapy inhibits
stemness and increases the efficacy of tamoxifen in estrogen receptor-positive breast
cancer cells. Lab Invest 2020; online ahead of print. DOI: 10.1038/s41374-020-
00507-z.
67 Wu CF, Efferth T. Miltirone induces G2/M cell cycle arrest and apoptosis in CCRF-
CEM acute lymphoblastic leukemia cells. J Nat Prod. 2015;78:1339–1347.
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