Bioorganic Chemistry 99 (2020) 103863

Contents lists available at ScienceDirect

Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

Anti-cancer potential of sophoridine and its derivatives: Recent progress and T

future perspectives
Haroon ur Rashida,b, , Shagufta Rasoolb, Yousaf Alib, Kamin Khanb,
⁎

Marco Antonio Utrera Martinesa,

⁎

a
Institute of Chemistry, Federal University of Mato Grosso do Sul, Campo Grande, MS, Brazil
b
Department of Chemistry, Sarhad University of Science and Information Technology, Peshawar, Khyber Pakhtunkhwa, Pakistan

ARTICLE INFO ABSTRACT

Keywords: Cancer is the second leading cause of mortality and has resulted in about 9.6 million deaths around the world in
Sophoridine 2018. Cancer-caused deaths are expected to be 11.5 million by 2030 all over the world. Because of the fatal
Phytochemical nature of cancer, substantial efforts are made all over the world to combat it. Phytoconstituents such as certain
Alkaloid alkaloids, saponins, tannins, polyphenols, and terpenoids exhibit anticancer effects. Sophoridine is a tetracyclic
Cancer
quinolizidine alkaloid isolated from the stem and leaves of medicinal plants Sophora alopecuroides L., and
Topoisomerase I
Euchresta japonica Benth, and roots of Sophora alopecuroides Ait. Chinese Food and Drug Administration (CFDA)
approved sophoridine as an antitumor agent in 2005. This review covers the antitumor activities of sophoridine
and its derivatives. The efficacy of sophoridine analogs is expressed with respect to their half-maximal inhibitory
concentration (IC50 values). Structure-activity relationship (SAR) study for most of the sophoridine derivatives
has been explained. Moreover, the current market of anticancer drugs and its expected growth are discussed.
Prospects provide suggestions and clues for novel sophoridine-based anticancer agents with enhanced expected
efficacy and minimum toxicity.

1. Introduction
Cancer represents the uncontrolled division of abnormal body cells.
Cancer cells attack or reach to the adjacent healthy tissues of the body
via the bloodstream and lymphatic systems, causing malignant tumors.
On the contrary, cells in benign tumors neither invade the neighboring
tissues nor spread to other body parts. Nearly 90% of cancer-related
mortalities are triggered by tumor dispersion, a phenomenon termed as
metastasis. (Fig. 1) [1,2] DNA damage is reckoned to be the primary
cause of cancer growth. It produces mutations and epimutations, which
then lead to cancer progression through the process of natural selection.
(Fig. 2) [3] Cancer is the second-highest cause of deaths in the world,
only excelled by cardiovascular ailments [4].
According to a recent press release of the International Agency for
Research on Cancer (IARC), 18.1 million new cancer cases, and 9.6
million cancer- caused deaths were reported worldwide in 2018 [5].
According to a report from the World Health Organization (WHO), over
70% of the cancer-related mortalities happened in developing coun-
tries. Cancer-related deaths will increase and are expected to reach an
approximate number of 11.5 million by 2030 all over the world. Major
risk factors for cancer include alcohol consumption, smoking, dietary
aspects (comprising inadequate vegetables and fruits consumption),
obesity, lack of physical activity, exposure to ionizing and non-ionizing
radiations, and exposure to toxins, chronic infections from helicobacter
pylori, human papillomavirus (HPV), hepatitis B virus (HBV), and he-
patitis C virus (HCV) [6].
Approximately 100 kinds of cancer have been recognized, which are
characteristically named for the organs or tissues where they arise.
Statistics show that prostate and breast cancers are the two principal
kinds of cancer identified in males and females, respectively [1,7]. In
modern medicine, surgery, radiotherapy, and systemic therapy (such as
chemotherapy, immunotherapy, hormonal therapy, and targeted
therapy) represent the key modalities applied to treat different types of
cancer [8,9]. Owing to the deadly nature of cancer, considerable efforts
are made worldwide to combat it by the exploration of novel antitumor
agents, which has resulted in a noteworthy success. US Food and Drug
Administration (FDA) recommended approximately 150 antitumor
drugs from 1949 to 2014. Depending on their mode of action, these
drugs have been classified into two sets (a) 61 cytotoxic drugs and (b)
89 target-oriented drugs [10–13]. Selected FDA approved drugs are

⁎
Corresponding authors at: Institute of Chemistry, Federal University of Mato Grosso do Sul, Campo Grande, MS, Brazil (H. ur Rashid).
E-mail addresses: haroongold@gmail.com (H. ur Rashid), marcomartines@gmail.com (M.A.U. Martines).

https://doi.org/10.1016/j.bioorg.2020.103863
Received 17 January 2020; Received in revised form 1 March 2020; Accepted 16 April 2020
Available online 18 April 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.
H. ur Rashid, et al.
Fig. 1. Difference between the proliferation of normal and abnormal cells.
Abnormal cell division leads to a malignant tumor.
given in Table 1, whereas their chemical structures are given in Fig. 3.
However, the toxicity and high price of the anticancer drugs restrict
their scope. Owing to their toxic nature, they carry various aftereffects,
for instance, tiredness, headache, alopecia, weakness, vomiting, nausea,
diarrhea, mouth sores, abdominal cramps, memory impairment, dry
mouth, and dizziness of the patients [14]. Therefore, the exploration of
novel cost-effective antitumor agents with the least toxicity is vital in
the pharmacological research, and offers a demanding obligation to the
researchers [1].
2. Phytochemicals as anticancer agents
The toxicity linked to the existing anticancer drugs may be over-
whelmed by utilizing plant-derived compounds either alone or in
combination. An analysis of the plant kingdom can provide novel pre-
cursors for prompt development of new anticancer agents.
Phytochemicals represent a rich source of efficient and safer anticancer
agents. Phytoconstituents such as alkaloids, saponins, tannins, poly-
phenols, and terpenoids have been reported to possess anticancer ac-
tivities [15]. Phytochemicals and their resulting metabolites are found
in leaf, root, stem, bark, and flower of plants. Taxol, Camptothecin,
Vincristine, Paclitaxel, Irinotecan, Etoposide, Ellagic acid, Colchicine,
Curcumin, Camptothecin, Gingerol, and Matrine represent few plant-
derived anticancer agents. Chemical structures of selected anticancer
phytochemicals are given (Fig. 4) [16].
Among phytochemicals, alkaloids characterize minor metabolites
that are synthesized by plants for their safety. They are not related to
any biological function but represent a distinct class of compounds,
possessing a nitrogen-containing ring configuration. The heterocyclic
ring contains a nitrogen atom inside it [17,18]. Nearly, 21,120 alkaloids
are known to exist in various families of plant kingdom such as An-
nonaceae, Amaryllidaceae, Berberidaceae, Apocynaceae, Boraginaceae,
Asteraceae, Celastraceae, Fabaceae, Liliaceae, Buxaceae, Lauraceae,
Menispermaceae, Papaveraceae, Loganiaceae, Poaceae, Piperaceae,
Ranunculaceae, Rutaceae, Solanaceae, and Rubiaceae [19]. Plant-de-
rived alkaloids are the most important bioactive compounds which
exhibit potent activity against various cancer cell lines. Some of them
have already been approved as chemotherapeutic agents, for example,
camptothecin, a well-known agent that suppresses the activity of to-
poisomerase I, and vinblastine, which aims at tubulin [20]. Among
biologically active alkaloids, quinazolines represent a useful group of
nitrogen-containing heterocyclic compounds with promising anticancer
2
Bioorganic Chemistry 99 (2020) 103863
activities. To date, about 150 naturally occurring quinazoline alkaloids
have been isolated from numerous families of the plant kingdom as well
as microorganisms and animals. Plant family Rutaceae abundantly
contain quinazolines alkaloids [21,22]. Naturally-occurring quinazo-
lines and their synthetic analogs exhibit substantial antitumor activities
against numerous cancer cell lines [23]. Substantial studies on struc-
ture–activity-relationship (SAR) analysis, hemisynthesis, and total
synthesis of various alkaloid derivatives have resulted in their trans-
formation into antitumor agents of enhanced potency, solubility, se-
lectivity, bioavailability, stability, and lesser toxicity in humans
[24,25]. Studies on the mode of action revealed that alkaloids and their
analogs target protein synthesis or DNA replication on cancer cells,
leading to the death of tumor cells [26–29].
3. Sophoridine as an anticancer agent
Sophoridine (C15H24N2O, molecular weight 248.36) is a quinolizi-
dine-based alkaloid obtained from the stem and leaves of medicinal
plants Euchresta japonica Benth and Sophora alopecuroides L., and roots
of Sophora alopecuroides Ait. (Fig. 5a and 5b) It is known to exhibit
numerous therapeutic effects, comprising antiinflammation, anti-
anaphylaxis, antitumor, antiarrhythmia, antiviral (Fig. 5c). Sophoridine
also affects the central nervous and immune systems [30–33].
Several research groups tested sophoridine for its antitumor po-
tency. Chinese Food and Drug Administration (CFDA) in 2005 approved
sophoridine to treat various kinds of cancer such as gastric, lung, and
liver cancers with minimum toxicity [34]. The mechanism of action
indicates that sophoridine induces cancer cells apoptosis by suppressing
the activity of DNA topoisomerase I, and arresting the cell cycle at the
G0/G1 stage [35,36]. Apoptosis or programmed cell mortality is an
instinctive phenomenon that happens in all living things. Cell mortality
is a carefully regulated practice that does not cause even slight in-
flammation. The primary function of apoptosis is to control the me-
chanism of the cell cycle. Antitumor agents work by forcing the tumor
cells to undergo apoptosis, triggering death alerts by causing DNA im-
pairment or cellular stress. Numerous apoptosis-inducing agents are
under study, and some are currently in the phase of clinical tests
[37,38]. Contrary to other DNA type I topoisomerases (Topo I) in-
hibitors, for example, Irinotecan (CPT-11), topotecan and 10-hydro-
xycamptothecin (HCPT), sophoridine possesses several benefits, for
example, simple, small and flexible structure, enhanced water solubi-
lity, convenient chemical synthesis, prospects of preparation for oral
administration and low toxicity profile [39]. Nevertheless, the mild
anticancer effects of sophoridine restrict its application as a drug for
clinical purposes, signifying that it is a prime compound for novel
modification as well as improvement. Consequently, the synthesis and
isolation of new sophoridine analogs are needed to identify further
useful drugs. This review is principally devoted to cover the break-
throughs achieved in the research area of antitumor activities of so-
phoridine and its analogs.
3.1. Anticancer activity of sophoridine against gastric cancer
Gastric cancer is a common type of cancer and sophoridine has been
examined as anticancer agent against gastric cancer cell lines.
Sophoridine has been investigated for its apoptosis-inducing potential
gastric carcinoma MGC-803 cells. Apoptosis was measured by
Fluorescence microscopy, electron microscopy, flow cytometry (FCM),
and DNA gel electrophoresis. Studies confirmed that sophoridine pro-
motes the suppression of cell growth through apoptosis. The growth of
MGC-803 cells was substantially suppressed by 0.4–3.2 mg/mL of so-
phoridine, and structural features of apoptosis were noted along with
the shrinkage of the nucleus, fizzing of cytoplasm, the fragment of a
nucleus, and a ladder-resembling shape of DNA disintegration in
agarose gel electrophoresis. The underlying mechanism revealed that
hindering S period cells is involved in apoptosis. The half-maximal
H. ur Rashid, et al. Bioorganic Chemistry 99 (2020) 103863

Fig. 2. Cancer produced through DNA damage or mutation in the gene.

Table 1
Selected FDA-recommended antitumor agents.
Drug Year of approval Kind of cancer Target gene Transfer Type

Mechlorethamine 1949 Lung; Leukemia; Lymphoma DNA synthesis Single

Vinblastine sulfate 1965 Lymphoma; Testicular cancer; Choriocarcinoma TUBA1A; TUBB; TUBD1; TUBE1; TUBG1 Combination
Thioguanine 1966 Leukemia DNA synthesis Combination
Procarbazine Hydrochloride 1969 Lymphoma DNA synthesis Combination
Floxuridine 1970 Stomach DNA synthesis Single
Fluorouracil 1970 Breast; Colorectal; Stomach; Pancreatic DNA Synthesis Single
Lomustine 1976 Brain; Lymphoma DNA Synthesis Both
Cisplatin 1978 Testicular cancer; Ovarian cancer DNA Synthesis Both
Streptozocin 1982 Pancreatic cancer DNA synthesis; SLC2A2 Single
Carboplatin 1989 Ovarian DNA Synthesis Both
Thiotepa 1994 Breast; Ovarian; Bladder DNA Synthesis Single
Bexarotene 1999 Cutaneous T cell lymphoma RXRA Single
Mitomycin 2002 Stomach; Pancreatic DNA Synthesis Both
Vorinostat 2006 Lymphoma HDAC1; HDAC2; HDAC3; HDAC6 Single
Pertuzumab 2012 Breast cancer ERBB2 Both
Pomalidomide 2013 Multiple myelomas CRBN Single
Hydroxyurea 2010 Melanoma; Leukemia RRM1 Single
Idelalisib 2014 Leukemia; Lymphoma PIK3CD Both

inhibitory concentration (IC50) was measured by 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
For a sophoridine dose of 3.2 mg/mL, the suppression of tumor cells
was observed to be about 70% (Fig. 6), and the IC50 value was noted to
be 1.27 mg /mL [40].
Sophoridine also exhibited antiproliferative and apoptosis-inducing
effects against human gastric cancer MKN45 cells. The gastric cancer
cells were arbitrarily classified into control and sophoridine (including
6 subgroups) groups. MTT assay results showed that sophoridine caused
proliferation inhibition of human gastric cancer cells (IC50 = 2.51 mg/
mL) dose-dependently after 48 h treatment (Fig. 7). Western blot and
immunocytochemical staining analyses revealed that the sophoridine-
treated group possessed reduced high mobility group-box 3 (HMGB3)
protein expressions in comparison to the control group. Additionally,
flow cytometry confirmed an increased apoptotic rate in MKN45 cells.
The data suggest that sophoridine has the capability to prohibit the
progression of MKN45 cells and can promote their apoptosis, which
may be associated with the reduction of HMGB3 protein expression
[41].
Zhuang and coworkers examined the influence of sophoridine on
the polarity level of gastric tumor-related macrophages (TAMs). Co-
culture analysis was performed through key bone marrow-derived
macrophages (BMDMs) and vital CD8+ T cells. Sophoridine-treated
TAMs diverge to M1-TAMs and inhibited M2-TAMs polarity via TLR4/
IRF3 core. Sophoridine-exposed TAMs displayed robust pro-in-
flammatory activity via increasing the expression of INOS, IFN-β, and
IL-12α, and decreasing the expression of Arg-1, CD206, and IL-10.
Sophoridine-cultured TAMs also promoted the propagation and cyto-
toxic function of CD8+ T by increasing the expression of Granzyme-B,
TNF-α, and Perforin, and reduced the expression of CD8+ T cells
function exhaustion markers PD-1, Tim-3, and Lag-3. Moreover, so-
phoridine suppressed the migration capability of macrophage by re-
ducing the CCR2 expression. Therefore, sophoridine stimulated mac-
rophages and CD8+ T cells to redesign gastric cancer immune
microhabitat. These results delivered a preclinical base for the clinical
use of sophoridine [42].
3.2. Anticancer activity of sophoridine against colon cancer
Few research groups investigated the anticancer behavior of so-
phoridine against colon cancer cells. Lei et al. observed that sophor-
idine hindered the proliferation and encouraged apoptosis of human
colon cancer SW620 cells. MTT assay was carried out to calculate the
half-inhibitory concentration of sophoridine against SW620 cells,
whereas apoptosis was detected through fluorescence microscopy,
electron microscopy, DNA fragmentation analysis, and flow cytometry.
Results showed that sophoridine prevented the proliferation of SW620
cells cultured in vitro, and promoted apoptosis in a dose- and time-de-
pendent fashion. The IC50 value of sophoridine versus SW620 cells was
noted to be 2.8 mmol .L−1 after 48 h. The growth of SW620 cells was
considerably suppressed after their exposure to sophoridine (Fig. 8),
and morphological features of apoptosis such as shrinkage of the nu-
cleus, the bubble of cytoplasm and fragment of the nucleus were ob-
served. Moreover, a DNA ladder pattern of internucleosomal

3
H. ur Rashid, et al. Bioorganic Chemistry 99 (2020) 103863

Fig. 3. Chemical structures of selected FDA-recommended antitumor agents.

Fig. 4. Chemical structures of selected anticancer phytochemicals.

4
H. ur Rashid, et al.
Fig. 5. (a) Chemical structure of sophoridine (C15H24N20, MW = 248.36) (b) an image of Sophora alopecuroides L, and (c) Various pharmacological activities of
sophoridine.
fragmentation was detected. Sophoridine treatment caused an increase
in the proportion of the G0/G1 stage and the S stage cells against the
control group. Sophoridine might promote the suppression of cell
growth by apoptosis in a concentration- and time-dependent manner.
The underlying mechanism indicated that blocking of G0/G1 phase of
cells was responsible for apoptosis [43].
Liang et al. reported that sophoridine exhibited an anticancer effect
against SW480 human colon cancer cells in vivo and in vitro. MTT assay
was performed to measure the antiproliferative influence of sophor-
idine against colon cancer cells. Whereas western blot technique was
executed to estimate the apoptosis of cancer cells by the detection of
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Bioorganic Chemistry 99 (2020) 103863
apoptosis-associated proteins viz. poly-ADP-ribose-poly-merase
(PARP), and three types of cysteine-aspartic proteases (caspase-3, cas-
pase-7, and caspase-9). For in vivo studies, xenograft tumors were re-
cognized by subcutaneous injection of SW480 cells in the armpit of
nude mice. Research findings showed that sophoridine substantially
blocked the multiplication of SW480 cells in a concentration- and time-
dependent mode (Fig. 9) with no obvious toxicity. The underlying
mechanism confirmed that apoptosis induction in SW480 human colon
cancer cells is responsible for the antitumor effect of sophoridine [44].
The antineoplastic effect of sophoridine against human colorectal
cancer cells has been evaluated by PharmMapper and KEGG databXase
H. ur Rashid, et al.
Fig. 6. Growth suppression of MG C-803 cells after their treatment with various
doses of sophoridine. Reproduced with permission from Z. Binggang, et al.,
Tumor, 23 (2003) 197–199. Copyright © 2003 China National Knowledge
Infrastructure (CNKI) [Ref. [40]].
Fig. 7. Proliferation suppression of MKN45 cells by sophoridine identified via
MTT assay. Reproduced with permission from Dong et al. Acta Physiol. Sin. 70
(2018) 391–396. Copyright © 2020, Editorial office Acta Physiol. Sin [Ref.
[41]].
Fig. 8. The growth of SW620 cells was substantially suppressed by sophoridine.
The IC50 value of sophoridine after 48 h is 2.8 mmol· L−1. Reproduced with
permission from Lei et al. Chinese Pharmacol. Bull. 24 (2008) 782–787.
Copyright © 2008 Chinese Pharmacological Society [Ref. [43]].
6
Bioorganic Chemistry 99 (2020) 103863
Fig. 9. MTT assay showing (%) inhibition to the growth of SW480 cells after
treatment with sophoridine. The progression curve of the sophoridine group is
considerably lower compared to that of the control group (vs. Control
**P < 0.001) (Reproduced with permission from L. Liang et al. Life Sci. 2012,
91, 1295–303, © 2012 Elsevier) [Ref. [44]].
analysis. Results showed that sophoridine blocked colorectal cancer
development by aiming at phospho-MAPKAPK2 (Thr222). (Fig. 10)
Molecular docking studies revealed that sophoridine selectively deac-
tivates MAPKAPK2 and directly attaches to the ATP site of MAPKAPK2.
Moreover, a direct binding between sophoridine and MAPKAPK2 was
confirmed by drug affinity responsive targets stability assay and cellular
thermal shift assay. Western blot analysis indicated that sophoridine
considerably downregulated MAPKAPK2 time-dependently. However,
no obvious change in MAPKAPK2 expression of colorectal cancer cells
was noted. Overall, the results revealed that MAPKAPK2 plays a vital
role in sophoridine-inhibited growth and invasion in colorectal cancers
[45].
3.3. Anticancer activity of sophoridine against lung cancer
Lung cancer is one of the most common types of cancer all over the
world. Xiong et al. reported that sophoridine suppressed lung cancer
cell propagation via stimulating hippo signaling and P53 pathway.
Anticancer effects of sophoridine against numerous human lung cancer
cells were examined via colony formation, CCK-8, transwell invasion,
and migration assays. The fundamental mechanism for the prohibitory
activity of sophoridine against the propagation of lung cancer cells was
investigated by quantitative real-time PCR and western-blot analyses.
Experimental outcomes revealed that sophoridine considerably sup-
pressed lung cancer cells proliferation, invasion, and relocation.
Quantitative real-time PCR indicated that sophoridine could intensely
inhibit the downstream targets of the Hippo-YAP1 passageway, such as
FOXM1, CYR61, CDX2, VEGF, and c-Myc. Overall the data re-
commended that sophoridine behaves as a new candidate to treat lung
cancer [46]. Sophoridine-loaded poly(lactide-co-glycolide) (PLGA) mi-
crospheres were reported to enhance the treatment efficiency of so-
phoridine against lung malignancy. Such microspheres were synthe-
sized by oil-in-oil emulsion-solvent vaporization technique. The results
revealed that the synthesized microspheres had appropriate physico-
chemical characteristics and particle size distribution. Moreover, they
also exhibited a combination of lung-targeting and efficient drug re-
lease properties [47].
3.4. Anticancer activity of sophoridine against brain cancer
Studies have shown that sophoridine also exhibits anticancer
H. ur Rashid, et al. Bioorganic Chemistry 99 (2020) 103863

Fig. 10. Sophoridine suppressed colorectal cancer by aiming at phospho-MAPKAPK2. (A–C) Cells were treated with or without 160 mmol/L of sophoridine for 48 h
and then heated at different temperature for 3 min. After freeze-thaw cycles for cell rupturing, the soluble MAPKAPK2 protein extents bound to a drug were observed
by western blot analysis. Right, comparative band strength of MAPKAPK2 (Reproduced by permission from the American Association for Cancer Research. R. Wang,
H. Liu, Y. Shao, K. Wang, S. Yin, Y. Qiu, H. Wu, E. Liu, T. Wang, X. Gao, H. Yu, Sophoridine Inhibits Human Colorectal Cancer Progression via Targeting MAPKAPK2,
Mol. Cancer Res. 17 (2019) 2469–2479. https://doi.org//10.1158/1541-7786.mcr-19-0553) [45].

Fig. 12. Sophoridine showing antiproliferative effect against human medullo-

Fig. 11. MTT assay results showing the inhibitory effect of sophoridine on
U87MG cell proliferation. U87MG cells exposed to several concentrations (0.
0.1, 0.2, 0.5, 1.0, 2.0, 5.0 & 10.0 mg/mL) of sophoridine for 24 h. (Reproduced
with permission from F.W. W. Wan et al. Int. J. Clin. Exp. Med. 8 (2015)
464–471, © 2015 e-Century Publishing Corporation) [Ref. [48]].
activity against cancer cells originating in the brain. Wang et al. studied
the antineoplastic action of sophoridine against the in vitro progression
of human glioma U87MG cells. MTT analysis indicated that sophoridine
caused noticeable inhibition to the growth of U87MG cells (Fig. 11),
and the mean IC50 value was reported to be 5.3 mg/mL for 24 h.
Moreover, sophoridine caused G2/M phase arrest, cell apoptosis-in-
duction, reactive oxygen species (ROS) production, and a decrease in
GSH content. Sophoridine substantially reduced the expression of p27,
Survivin, E2F1, CDK2, Bcl-2, Livin, and the transcriptional activity of
FoxM1, NF-κb, and AP-1, in the meantime, enhanced the expression of
caspase-3/8, Smac, p53, p38-MAPK, and c-JNK. Ubiquitin-Proteasome
Pathway (UPP) in tumor cells was substantially inhibited by sophor-
idine. It was concluded that sophoridine exhibits noticeable anticancer
activity against glioma cells by inducing cell apoptosis, promoting ROS
deposition, and stimulating mitochondrial signal pathways [48].
Yue and coworkers studied the influence of sophoridine on the
growth of human medulloblastoma (brain tumor). Human medullo-
blastoma D283-Med cells were exposed to numerous doses of sophor-
idine (0, 0.5, 1, or 2 mg/mL) for 24, 48, or 72 h. MTT and lactate
dehydrogenase assays were carried out to analyze the cell proliferation
and cytotoxicity respectively. Flow cytometry or spectrophotometry
analyses were performed to cell apoptosis and caspase-3/8 activity. The
variation in FoxM1, BDNF, TrkB, AP-1, and NF-κB expression was
blastoma cells. **P < 0.01 vs. D283-Med cells treated with 0 µM sophoridine.
Reproduced with permission from Z. Yue, et al. Oncol. Lett. 14 (2017)
7941–7946, © 2017 Spandidos publications [49].
investigated through western blot analysis. The research findings re-
vealed that sophoridine substantially inhibited the proliferation dose-
and time-dependently (Fig. 12) and promoted apoptosis of human
medulloblastoma cells. Moreover, the caspase‑3/8 action and cyto-
toxicity in human medulloblastoma were enhanced after sophoridine
treatment. Protein expression of FoxM1, BDNF, TrkB, AP-1, and NF-κB
in human medulloblastoma cells was suppressed by sophoridine [49].
3.5. Anticancer activity of sophoridine against pancreatic cancer
Sophoridine has also been evaluated for its anticancer effect against
human pancreatic cancer cells (PANC-1) in vivo. Xenografts in nude
mice were employed to assess the impact of sophoridine on the pan-
creatic cancer cells. Cell growth was examined by CCK-8 and colony
formation analysis. Flow cytometry was performed to measure cell
apoptosis and cell cycle dissemination (Fig. 13). The intracellular ROS
levels were measured by peroxide-sensitive fluorescent probe DCFH-
DA, whereas the extents of cell cycle and apoptosis-linked proteins were
determined by the western blot technique. Experimental outcomes
suggested that sophoridine inhibited the propagation of PANC-1 cells
(IC50 value = 19.23 μM) dose and time-dependently, and also en-
couraged cell cycle arrest at S stage and mitochondrial-associated
apoptosis. Additionally, sophoridine promoted a constant stimulation of
the phosphorylation of JNK and ERK, and triggered the production of
reactive oxygen species (ROS) in pancreatic cancer cells. In vivo

7
H. ur Rashid, et al.
0 μM
investigations indicated that sophoridine blocked the neoplasm pro-
gression in mouse xenograft models [50].
3.6. Anticancer activity of sophoridine against liver cancer
The inhibitory effect of sophoridine against hepatocellular carci-
noma has been studied by in vitro and in vivo investigations. MTS assay
was performed to determine the cell proliferation after treatment of
HepG2 cells (human liver cancer cells) with different concentrations of
sophoridine. The results indicated that sophoridine inhibited HepG2
proliferation dose-dependently. The extent of proliferation in different
groups of HepG2 cells treated with varying concentrations (0 μg/mL,
5 μg/mL, 10 μg/mL, 20 μg/mL) of sophoridine was observed to be
134.62 ± 5.76%, 98.96 ± 4.88%, 80.26 ± 6.70% and
80.08 ± 5.65%, respectively, which is considerably lower than 0 mg/
mL group (134.62 ± 5.76%) (P < 0.05, respectively). (Fig. 14). Flow
cytometry results indicated that cell apoptosis rate in 5 mg/mL, 10 mg/
mL and 20 mg/mL sophoridine treated HepG2 cell groups was
14.80 ± 2.97%, 42.36 ± 6.72%, 59.55 ± 4.72% respectively, which
is substantially higher compared to 0 mg/mL group (4.97 ± 3.78%)
(P < 0.05). Overall, the results confirmed that sophoridine possessed
the anticancer potential to inhibit HepG2 activities by modulating
PTEN/PI3K/AKT, Caspase-3/-9, and MMP-2/-9 signaling passageway
[51].
Fig. 14. The cell proliferation rates (%) in various HepG2 cells exposed to
different doses of sophoridine Reproduced with permission from B.C. Wang
et al. Biomed. Pharmacother. (2017) 95, 324–330. Copyright © 2017 Elsevier
Masson SAS [Ref. [51]].
8
Bioorganic Chemistry 99 (2020) 103863
Fig. 13. Flow cytometry findings in-
20 μM
dicate the cell cycle dissemination of
pancreatic cancer cells (PANC-1) after
their exposure to 0 μM or 20 μM doses
of sophoridine for a specified time.
(Readapted from Z. Xu, F. Zhang, C.
Bai, C. Yao, H. Zhong, C. Zou, X, J.
Exp. Clin. Cancer Res. (2017) 36, 124.
The image is registered under the
Creative Commons Attribution 4.0
International Consent, which au-
thorizes unconditional usage, circula-
tion, modification, and reproduction
in any means, http://creative-
commons.org/licenses/by/4.0/ [Ref.
[50]].
3.7. Anticancer activity of sophoridine against miscellaneous cancer cells
Sophoridine activities against the grafted solid tumor SW480, as
well as the expressions of p53 and vascular endothelial growth factor
(VEGF) in the tumor of nude mice, have also been studied. Nude mouse
model containing grafted solid tumor SW480 (Fig. 15) was exposed to
sophoridine, and variations in the weight and volume of the tumors
were observed. The expressions of VEGF and p53 proteins were in-
vestigated by immunohistochemistry and western blotting respectively,
and mRNA expression in the tumor tissues of the two proteins was
detected by quantitative fluorescence PCR. The experimental findings
revealed that sophoridine treatment triggered a reduction in weight and
volume of the tumor xenograft compared to those in the control group.
Furthermore, reduced expressions of p53 and VEGF proteins and mRNA
in the tumor tissues (with a suppression rate of 34.07%) against the
control group in nude mice were detected. Overall, these results con-
firmed that sophoridine could suppress the progression of transplanted
SW480 human colon cancer cells by the inhibition of VEGF and p53
expressions [52].
Li and coworkers reported that sophoridine possesses noticeable
antitumor activity. The results indicated that small therapeutic dose
(10 mg/kg ig) of sophoridine suppressed the growth of transplanted
tumors of Lewis Lung Carcinoma (LLC), S 180, U 14 and esophageal
carcinoma (ECA) with rate inhibition of 30–60% (p < 0.05) and IC50
values greater than 100 µg. mL−1. The acute intraperitoneal injection
(ip) and intravenous injection (iv) at Lethal Dose (LD) in mice were
observed to be 60 + 0.1 and 50.4 + 0.4 mg/kg, respectively. The
proportions of RNA and DNA in tumor and spleen reduced somewhat in
normal mice or mice bearing S 180 and U 14 solid tumors after injecting
them with sophoridine doses (20 mg/kg qd × 10 d). Sophoridine
caused apoptosis of cancer cells and disturbed the G1 and G2 stages of
the proliferation cycle. Moreover, the activities of the TOPO 1 enzyme
were suppressed after the exposure of cancer cells to sophoridine. The
results also revealed that sophoridine is quickly absorbed, distributed,
and excreted unchanged by glomerular filtration. The mitochondria in S
180 sarcoma cells expanded, and melted vacuolations appeared within
the cytoplasm of the tumor cells, and the microvilli of the cell mem-
brane reduced (Fig. 16). Overall, these results verified the antitumor
activity of sophoridine [53,54].
4. Anticancer activities of sophoridine derivatives
To improve the moderate anticancer activities of sophoridine, sev-
eral research groups reported the synthesis of different sophoridine
derivatives and evaluated them for their potential application as an-
ticancer agents against various cancer cell lines.
Tan et al. synthesized eight novel sophoridine derivatives (2–7, 8,
H. ur Rashid, et al. Bioorganic Chemistry 99 (2020) 103863

Fig. 15. Human colon cancer transplanted compact cancer cells in bare mice SW480 VEGF Positive expression (Envision, ×200) A. classical control group; B. sputum
base group. Reproduced with permission from Wang et al. J. South. Med. Univ. 30 (2010) 1953–1956. Copyright © 2010 J. South. Med. Univ [Ref. [52]].

9) by modification at the 14-position (Scheme 1). Sulforhodamine B
(SRB) colourimetric assay was applied to assess the anticancer activity
of the synthesized compounds against five human cancer cell lines (KB,
A549, KB-VIN, MCF7, MDA-MB-231). Structure-activity relationship
(SAR) study indicated that neither a conformation modification at C-6,
or C-5, nor the existence of unsaturation in the D or C ring caused a
noteworthy increase in antiproliferative activity. However, the sub-
stitution of the phenylmethylene group on C-14 resulted in anticancer
activity enhancement. Concerning the substitution on the phenyl ring,
3-methoxy-4-methyl substituted 3 exhibited more potent activity than
2-methyl-4-methoxy substituted 4. Compound 8 which was obtained by
keeping the 3,4-substituted pattern with an ethoxy moiety at position-3
and a benzyloxy moiety at position-4, showed considerable inhibitory
activity, with its IC50 values about 20 µM against all five cancer cells,
four times greater than those of parent sophoridine. Remarkably, a
switch from a benzyloxy (8) to methoxymethoxy (9) moiety on the
same location of the phenyl ring did not cause an increase in the in-
hibitory effect. Thus, the benzyloxy group in sophoridine derivatives is
key to enhance the antiproliferative activity [55].
Xu et al. reported the synthesis of sophoridine imine derivatives
possessing conjugated planar structure. The obtained derivatives were
assessed for their in vitro antineoplastic activity against HepG-2 and
HeLa cells. Experimental findings showed that most of the target deri-
vatives showed strong in vitro antitumor activity. Derivative 10
(Fig. 17) displayed exceptional anti-proliferative activities with its IC50
values to be 5.7 µM and 8.5 µM versus HepG-2 and HeLa cell lines,
respectively. Additionally, SAR investigation showed that the
incorporation of conjugated frameworks on the 15-carbonyl site results
in imines generation which possesses better anticancer activity. The
activity can further be improved by the cyclization of the moiety.
Docking studies revealed that π-π stacking connection between the
conjugated structure and DNA was the primary binding mode. The
underlying mechanism indicated that the synthesized compounds sup-
pressed the activity of DNA Topo I succeeded by the inhibition of G0/
G1 stage.[56]
Li et al. reported the synthesis and antineoplastic activity of 58
sophoridine analogs (bearing indole or pyrrole scaffold) against the
HepG2 cancer cells. Amongst the 58 synthesized derivatives, 33 ex-
hibited strong anticancer activities with their IC50 values lower than
10 µM. Derivative 11 (Fig. 18) was observed to be the most potent
anticancer agent. Therefore, its inhibitory activity was evaluated
against six cancer cell lines (Hela, HepG2, SMMC-7721, MCF7, CNE1,
and CNE2). Research findings revealed that 11 exhibited antineoplastic
activity against all six cell lines with its IC50 values of 0.93–1.89 µM,
much smaller compared to the IC50 values of parent sophoridine. SAR
investigation indicated that the presence of N-benzyl indole fragment
on the 14-carbon in sophoridine skeleton could substantially increase
the anticancer effect. Molecular docking study and enzymatic assay
confirmed that 11 suppressed DNA Topo 1 activity. Additionally,
apoptosis assay indicated that 11 could substantially promote the
apoptosis of HepG2 cells dose-dependently by stimulating caspase-3,
up-regulating the expression of cleaved caspase-3, and down-regulating
the percentage of Bcl-2/Bax. Moreover, the investigations also in-
dicated that 11 inhibited the proliferation of HepG2 xenografts in nude

Fig. 16. A. Sarocoma 180 cells. × 7150. Er = Endoplasmic reticulum, F = microvilli of cell membrane, f/ = microvilli of plasma membrane, M = mitochondria,
N = nucleus, V = virus, Ri = ribosome. B. After ip sophoridine 20 mg/kg. × 8370. (Reproduced with permission from X. M. Li et al. Acta Pharmacol. Sin., 1987, 8,
153–158, © 2017 Springer Nature) [Ref. [53]].

9
H. ur Rashid, et al.
Scheme 1. (1a) Synthesis of sophoridine derivations 2–7. (a) Aromatic aldehyde, THF, NaH, reflux for 6 h, and (1b) Synthesis of sophoridine derivatives 8 & 9. (b)
BnBr, 10% K2CO3, rt; (c) Chloromethyl methyl ether, N, N-Diisopropylethylamine, 0 °C to rt.
Fig. 17. Chemical Structure of compound 10.
mice without causing any apparent toxicity [57].
The tricyclic sophoridinic derivatives have been identified as a new
group of autophagy suppressors, which promote autophagy-dependent
apoptosis in tumor cells and specify their role as antitumor drug can-
didates. SAR study of 12N-substituted sophoridinamine analogs in-
dicates that an appropriate arylethyl or arylidene moiety at the N′-end
might increase antitumor potency. Among the synthesized derivatives,
the tricyclo sophoridinic derivative viz. N′-3,4,5-trimethoxylbenzyl-
12N-p-chlorobenzyl sophoridinamine 12 (Fig. 19) showed strong anti-
proliferative activity against three human cancer cells comprising leu-
kemia (K562), breast cancer (HMLE), and HepG2, with its IC50 values to
be 0.55–1.7 μM. The basic mechanism revealed that 12 suppresses
autophagic flow in cancer cells, inhibits lysosomal acidification, and
10
Bioorganic Chemistry 99 (2020) 103863
Fig. 18. Chemical structure of derivative 11.
thus suppresses cancer cell viability [58,59].
A new N-substituted sophoridinic acid analog, IMB-6G (13)
(Fig. 20) was examined for its anticancer effect against human naso-
pharyngeal carcinoma (NPC) cell lines. NPC cell line C666-1 was ex-
posed to numerous doses of IMB-6G (0, 1, 2, and 5 µM) for 24 h. Cell
survival was measured through a cell counting kit-8 assay, while
apoptosis was studied via flow cytometry. Western blot and reverse
transcription-quantitative polymerase chain reaction analyses were
applied to ascertain the expression levels of proteins and genes, re-
spectively. Experimental data showed that IMB-6G suppressed C666-1
cell progression and encouraged apoptosis in a dose-dependent manner.
The underlying mechanism indicated that IMB-6G promoted apoptosis
via triggering endoplasmic reticulum (ER) stress (by stimulating IRE1α
and PERK signaling passages). Overall, the results suggested that IMB-
6G is a possible antineoplastic agent that works by promoting ER stress-
H. ur Rashid, et al.
Fig. 19. Chemical structure of compound 12.
Fig. 20. Chemical structure of N-substituted sophoridine acid derivative, IMB-
6G, 13.
related apoptosis in human NPC cells. Moreover, targeting ER stress by
IMB-6G might be a useful technique to treat human NPC [60].
Xu and coworkers synthesized of thirty new indole-bearing so-
phoridine derivatives as Topo I inhibitors. The target derivatives were
evaluated for their antiproliferative and enzymatic suppressive effects.
Preliminary data showed that some of the target compounds had potent
Topo I inhibitory activity. Derivative 14 (Fig. 21) was the most effective
inhibitor against Topo I enzyme. MTT assay revealed that 14 caused
significant proliferation inhibition of HepG2, CNE-2 (nasopharyngeal
carcinoma) and A549 (adenocarcinoma human alveolar basal epithelial
cells) tumor cells with its IC50 values of 3.1 ± 0.8, 3.5 ± 1.3 and
22.1 ± 3.5 µM, respectively. SAR study indicated that the incorpora-
tion of the indole scaffold enhanced the anticancer activities of the
target derivatives. Moreover, the insertion of an aryl or alkyl moiety on
the nitrogen atom of indole ring resulted in lipophilicity enhancement,
which in turn improved the anticancer activity [61].
Synthesis and anticancer activities of 47 sophoridinic acid deriva-
tives have been reported. Among all the synthesized derivatives, com-
pound 15 (Fig. 22) showed potent anticancer effects versus six human
cancer cell lines (lung, breast, glioma, colon, nasopharyngeal & liver).
Its IC50 values against HepG2 (hepatoma), HCT116 (colon cancer)
H1299 (lung cancer) U87 (malignant glioma) MCF-7 (breast cancer)
and KB (nasopharyngeal epidermoid carcinoma) cell lines were re-
ported to be 9.37 ± 0.49, 10.64 ± 0.22, 5.55 ± 0.31,
Fig. 21. Chemical structure of compound 14.
11
Bioorganic Chemistry 99 (2020) 103863
Fig. 22. Chemical structure of compound 15.
4.52 ± 10.04, 5.73 ± 0.48, and 19.71 ± 2.02 µM, respectively. The
underlying mechanism indicated that 15 suppressed the action of DNA
Topo I, with the subsequent arrest of the G0/G1 phase. SAR study
showed that (a) R-configuration at the 5-position is key; (b) in-
corporation of a chlorobenzyl on the 12-nitrogen atom of the sophor-
idinol can improve the antineoplastic effect [62].
Li et al synthesized novel phosphoramide mustard containing so-
phoridine derivatives. The target derivatives showed potent antitumor
effect versus numerous cancer cell lines, and their IC50 values were
reported to be 1.01–3.65 µM against H22 and S180 cancer cells. They
were more cytotoxic against cancer cells in comparison to standard cell
L929. Furthermore, derivatives (16–18) (Fig. 23) were tested for their
in vivo antitumor activities against mice having H22 liver cancer. Re-
sults indicated that compounds (16–18) exhibited modest tumor in-
hibition with their IC50 values of 1.01 µM, 1.85 µM, and 2.17 µM re-
spectively, without evident organ toxicity in vivo. Additionally, they
caused tumor cell arrest in the G1 stage and promoted cancer cell
apoptosis [63].
Using sophoridine and chalcone as starting materials, Xu and cow-
orkers synthesized α, β-unsaturated sophoridinic derivatives and ex-
amined theirs in vitro cytotoxicity. Among the target derivatives, com-
pounds 19 and 20 (Fig. 24) displayed potential activities against CNE-2
and HepG-2 human cancer cell lines via MTT analysis. The IC50 values
of 19 were recorded to be 38 ± 2.1 µM and 35.1 ± 2.9 µM against
CNE-2 and HepG-2 cancer cell lines, respectively. For compound 20, the
IC50 values were observed to be 33.8 ± 2.3 µM and 25.5 ± 3.3 µM
against CNE-2 and HepG-2 cancer cells, respectively. The elementary
mechanism indicated that the course of action of α, β-unsaturated so-
phoridinic derivatives involves the suppression of DNA Topo I activity,
succeeded by the arrest of the G0/G1 stage. SAR study suggested that
Fig. 23. Chemical structures of compounds 16–18.
H. ur Rashid, et al.
Fig. 24. Chemical Structures of compounds 19 and 20.
the fabrication of α, β-unsaturated ketone, and the introduction of
heterocyclic moiety could improve the activity efficiently. Docking
study indicated that the induction of heterocyclic and aryl rings might
strengthen the bonding tendency of the target derivatives with the
purine ring of DNA in DNA-Topo I complex by π–π stacking synergy,
causing double-strand cleavage and eventually apoptotic cell death
[64].
Compound 21 (Fig. 25), an analog of sophoridine, displays an im-
proved antitumor effect against various cancer cells (Table 2). It could
suppress Topo 1 action via strengthening the DNA-Topo I complex and
promote mitochondria-facilitated apoptosis through inducing DNA
single- and double-strand cracking (facilitated by Topo 1). Moreover,
21 promoted HCT116 cell arrest at G1 phase by deactivating CDK2/
CDK4–Rb–E2F and cyclinD1–CDK4–p21 check-point signal routes.
Compound 21 hindered the Ataxia Telangiectasia Mutated (ATM) and
Rad3-associated (ATR) stimulation. It also reduced 53BP level, which
supported DNA impairment repair. The results signified that 21 could
be useful to enhance the anticancer activity of DNA detrimental agent
by suppressing ATR and ATM stimulation and 53BP level. Additionally,
the importance of molecular features and druggability (for example, a
plain structure and fabrication for oral intake) also verify that 21 might
be a potential agent to target topoisomerase [65].
Peng et al. studied the relationship between novel sophoridine de-
rivatives (synthesized by structural modifications) and their antitumor
activity. Sophoridine derivatives were synthesized by carrying out
substitution at the 14-position (Scheme 2). The synthesized compounds
were assessed for their antitumor activity versus glioblastomas, mela-
noma, liver, lung, colon, gastric and cervical cancer cell lines by MTT
cell proliferation assay. Six out of total sophoridine derivatives pos-
sessed different configurations at 14-position. Data indicated that
14–chloro substituted compounds 22, 23, 24, and 29 had better an-
ticancer activity than sophoridine against most of the cancer cell lines.
Overall, the experimental data showed that substitution by chlorine at
Fig. 25. Chemical structure of compound 21.
12
Bioorganic Chemistry 99 (2020) 103863
Table 2
The IC50 values of 21 and sophoridine for numerous cancer cell lines.
Reproduced with permission from W. Zhao et al. Onco Targets Ter. 9 (2016)
2805–2817. Copyright © 2019 Dove Medical Press Ltd [Ref. [65]].
Tumor cell lines IC50 (µg/mL)
21 Sophoridine
human non-small-cell lung cancer A549 4.31 ± 0.21 > 40
Human fbrosarcoma HT1080 5.5 ± 0.11 > 40
human glioblastoma U87-MG 5.07 ± 0.86 > 40
human hepatoma HepG2 4.6 ± 0.25 > 40
human breast cancer MCF-7 2.8 ± 0.24 > 40
human colon carcinoma HCT116 5.6 ± 0.11 > 40
human breast cancer MDA-MB-231 10.4 ± 0.9 > 40
human colon carcinoma HCT116 p53KO 10.33 ± 0.6 > 40
human leukemia cells K562 10 ± 0.58 > 40
14-position of sophoridine increased the antineoplastic effect of the
synthesized derivatives [66].
Synthesis and antineoplastic activity of six nitrogen mustard so-
phoridine analogs (Scheme 3) have been reported. MTT assay indicated
that the synthesized derivatives had more potent antitumor activity in
vivo against human HepG2 cells than parent sophoridine (Table 3).
Moreover, the antitumor activity of 12-acid benzyl sophoridine 17-{4-
[N,N-bis(2-chloroethyl)}benzamide (35) was comparable to that of the
commercial drug Melphalan [67].
Dai et al. synthesized and evaluated the anticancer activity of so-
phoridine derivatives possessing an acyclic aryloxy phosphoramidate
mustard moiety (Scheme 4). The cytotoxic activities of the target de-
rivatives were assessed against H22, MCF-7, S180, K562, LoVo, and
SMMC-7721 cancer cells. All the derivatives were more susceptible to
H22 and S180 cell lines, with their IC50 values ranging from 2.10 to
7.21 μM. Moreover, all compounds selectively caused more cytotoxicity
to cancer cells in comparison to healthy cells (L929 cell line). Deriva-
tives (37b-37e) exhibited modest antineoplastic activity in vivo against
mice carrying H22 liver tumors without obvious side effects. Molecular
docking indicated that the binding mechanism of the derivatives (37b-
37e) could interact with DNA-Top1 binding sack through pi–pi (π–π)
stacking interaction [68].
Synthesis of a new family of phosphoramide mustard sophoridinic
acid derivatives has been described. The target derivatives were eval-
uated for their Topo I inhibitory effect and antiproliferative activity
versus six tumor cell lines (SMMC‐7721, MCF‐7, K562, H22, S180, and
LoVo). Five of the target compounds (38a-e) (Fig. 26) were observed to
be strong Topo I inhibitors, which prevented the linkage of Topo I to
DNA and suppressed the DNA rupture. Docking study indicated that the
binding energy of the derivatives was similar to that of the typical Topo
I inhibitors HCPT and CPT, signifying that these derivatives had in-
teracted with Topo I and DNA. Moreover, five derivatives displayed
strong cytotoxicity versus H22 and S180 cells with their IC50 values
ranging from 1 to 4 μM. SAR study indicated that the addition of
phosphoramide mustard and substituted phenyl ring moieties on the
12‐N of the three‐ring moiety was effective for maintaining a better
anticancer effect [69].
Li et al. also synthesized a number of nitrogen mustard sophoridinic
acid analogs for their prospective application as antineoplastic agents.
Among the target derivatives, 35 (Fig. 27) showed the most potent
antiproliferative activity in vitro and in vivo against HepG2 cells. The
IC50 value of 35 for HepG2 cells was reported to be 0.82 μM as com-
pared to that of a standard chemotherapeutic agent, melphalan
(IC50 = 1.34 μM). SAR study showed that the addition of a nitrogen
mustard group to the sophoridinic acid considerably improved the an-
titumor effect. Furthermore, docking investigation indicated that the
insertion of benzyl moiety at 12-N, and aryl nitrogen mustard moiety at
the 40-carboxyl position of 35 lead to considerable enhancement of
anticancer activity. These results provided a solid foundation for more
structural variations in nitrogen mustard sophoridinic acid analogs,
H. ur Rashid, et al. Bioorganic Chemistry 99 (2020) 103863

Scheme 2. Synthesis of sophoridine at substitution at the 14-position.

Scheme 3. Synthesis of nitrogen mustard sophoridine derivatives.

useful for the synthesis of novel, strong antitumor agents [70]. amines, and imines were synthesized. All the derivatives were tested for
Synthesis of 12-N-p-chlorobenzyl sophoridinol analogs for their their cytotoxic effect in human HepG2 hepatoma cells through MTT
possible use as anticancer agents has been described. Several new so- assay. Experimental data showed that majority of the synthesized
phoridinic derivatives, for example, sophoridinic ketones, alkenes, compounds had considerable antiproliferative effects, with their IC50

13
H. ur Rashid, et al. Bioorganic Chemistry 99 (2020) 103863

Table 3
The IC50 values of nitrogen mustard sophoridine derivatives against HepG2
cells. Reproduced with permission from C.X. YAN Guo-rui, ZHENG Xiaohui,
TAO Zun-wei, ZHAO Yong-an, Chem. Reagents. 36 (2014) 109–113. Copyright ©
2014 China National Knowledge Infrastructure (CNKI) [67].
−1 − 1
Compounds IC50 / (μg·mL ) Compounds IC50 / (μg·mL )

32a 167. 32 32e 24. 98

32b 91. 21 35 0. 47
32c 44. 57 Sophoridine 1160. 00
32d 2. 39 Melphalan 0.41

values to be 3.8–5.4 μM. SAR studies revealed that the addition of an

appropriate methyleneamine fragment to the 3/-site might significantly
increase the anticancer activity. Compounds 39 and 40 (Fig. 28) de-
monstrated equally potent anticancer activity in both adriamycin
(AMD)-prone and resistive MCF-7 breast carcinoma cells, showing an
opposing mechanism from AMD. The underlying mechanism indicated
that 39 caused cell cycle arrest at the G0/G1 stage, indicating the si-
milarity of 39 in anticancer behavior to its precursor sophoridine.[71]
Synthesis and antitumor activity of 27 new sophoridinic acid/ester Fig. 26. Chemical structures of compounds 38a-e.
and sophoridinol analogs have been reported. The antiproliferative ef-
fect of the target derivatives was examined versus human HepG2 he-
patoma cells. Compound 41 (Fig. 29) exhibited antiproliferative effect
with its IC50 value of 3.1 μM. Moreover, it caused an equally potent
anticancer activity against both AMD-resistant MCF-7 (MCF-7/AMD)
and wild MCF-7 breast cancer cell lines. The underlying mechanism
indicated that 41 caused cell cycle arrest at the G0/G1 phase, showing
uniformity in antitumor effect to its forerunner sophoridine. SAR study
revealed that (a) two appropriate moieties on the 12-N and carboxyl
zone were useful for maintaining a strong antineoplastic activity. (ii)
The two moieties could be incorporated by fabricating several sub-
stituents to control the drug-like physicochemical characteristics of the
target derivatives. Overall, the experimental data confirmed the so-
phoridinol derivatives to be a new group of beneficial antineoplastic
agents with the potential to suppress drug-resistant cancer cells [72].
Synthesis of a new class of novel N-substituted sophoridinic acid Fig. 27. Chemical structure of compound 35.
derivatives has been reported. (Scheme 5). Using 10-Hydro-
xycamptothecin (HCPT) as a positive control, the synthesized products
were investigated for their antiproliferative activity against HepG2 caused S-phase arrest, followed by apoptosis, giving better antitumor
hepatoma cells by sulforhodamine B (SRB) analysis. Among the tested activity than its parent sophoridine. SAR study showed that the addi-
derivatives, 44b (with a bromoacetyl side-chain) (Fig. 30) exhibited tion of a substituent (e.g., an aliphatic acyl) at the 12-site could con-
moderate anticancer activity versus four kinds of human tumor cells siderably improve the antitumor effect of this class of derivatives.
(breast, lung, liver, and colon). The IC50 value of 44b in HepG2 hepa- However, bulkier side-chains at 12-position were not useful for the
toma cells was recorded to be 38.40 μM. The fundamental mechanism antitumor activity [73].
showed that 44b suppressed the activity of DNA topoisomerase I and The potency of a few vital sophoridine analogs is given in table 4

Scheme 4. Synthesis of sophoridine derivatives carrying an acyclic aryloxy phosphoramidate mustard moiety (37a-37e).

14
H. ur Rashid, et al. Bioorganic Chemistry 99 (2020) 103863

Fig. 28. Chemical structures of compounds 39 and 40.

Fig. 30. Chemical structure of compound 44b.

Fig. 29. Chemical structure of compound 41.
below.
5. Comprehensive SAR analysis of sophoridine derivatives
The complete SAR study of sophoridine and its derivatives is pre-
sented in Fig. 31. It can be summarized in the following points,
(a) The insertion of phenylmethylene group on C-14 position in parent
sophoridine causes anticancer activity enhancement [55].
(b) The addition of a conjugated structure to 15-carbonyl position via
imine generation leads to improvement in anticancer activity [56].
(c) The incorporation of an N-benzyl indole scaffold on the C-14 atom
of sophoridine leads to significant activity enhancement of the re-
sulting derivatives [57].
(d) The incorporation of an appropriate arylidene or arylethyl moiety
at the N′-end of 12N-substituted sophoridinamine analogs can lead
to significant activity enhancement [58].
(e) The introduction of indole moiety on the sophoridine scaffold en-
hances the anticancer activity of the target derivatives.
Furthermore, incorporation of an aryl or alkyl fragment on the ni-
trogen atom of the indole ring also boosts the antineoplastic prop-
erty by improving the lipophilicity [61].
(f) The insertion of chlorobenzyl on the 12-N atom of the sophoridinol
leads to significant activity enhancement [62].
(g) The fabrication of α, β-unsaturated ketone and the introduction of
heterocyclic moiety on sophoridine improve the activity [64].

Scheme 5. Synthesis of N-substituted sophoridinic acid derivatives (44a-44 m).

15
H. ur Rashid, et al. Bioorganic Chemistry 99 (2020) 103863

Table 4
Potency of selected sophoridine derivative with respect to their IC50 values.
Compound No. IC50/µM Target Cancer Cells Reference

8 ~ 20 KB, A549, KB-VIN, [55]

MCF7, MDAMB-231
9 ~ 20 KB, A549, KB-VIN, [55]
MCF7, MDAMB-231
10 5.7 HepG-2 [56]
10 8.5 HeLa [56]
11 0.93 –1.89 HepG2, SMMC-7721, [57]
Hela, CNE1, CNE2,
MCF7
12 0.55 –1.7 Leukemia (K562), Breast [58,59]
cancer (HMLE), HepG2 Fig. 32. Molecular docking models of sophoridine in the active site of
14 3.1 ± 0.8 HepG2 [61] DNA–Topo I complex (PDB ID. 1T8I). This is an open-access article circulated
14 3.5 ± 1.3 CNE-2 [61] under the Creative Commons Attribution License which allows unrestricted use,
14 22.1 ± 3.5 A549 [61] distribution, and reproduction in any medium provided the original work is
15 4.52 ± 10.04 HepG2, HCT116, H1299, [62] properly cited.
–19.71 ± 2.02 U87, MCF-7, KB
16 1.01 H22 [63]
17 1.85 H22 [63] (h) The substitution of chlorine at 14-site of sophoridine could improve
18 2.17 H22 [63] the anticancer effect of the synthesized sophoridine analogs [66].
19 38 ± 2.1 CNE-2 [64]
(i) The addition of substituted phenyl ring, phosphoramide mustard,
19 35.1 ± 2.9 HepG-2 [64]
20 33.8 ± 2.3 CNE-2 [64] and nitrogen mustard moieties on the 12‐N atom of the three‐ring
20 25.5 ± 3.3 HepG-2 [64] sophoridinic acid skeleton is suitable for keeping excellent antic-
35 0.82 HepG2 [70] ancer activity [69,70].
37a-37e 2.10 –7.21 S180, H22, K562, MCF- [68] (j) The introduction of a substituent (e.g., an aliphatic acyl) at the 12-
7, SMMC-7721, LoVo
position might considerably improve the antitumor activity of N-
38a-e 1– 4 H22, S180 [69]
39 4.9 ± 1.2 HepG2 [71] substituted sophoridinic acid derivatives. However, bulkier side-
40 3.8 ± 1.0 HepG2 [71] chains at the 12-position were not suitable for the anticancer ac-
41 3.1 HepG2 [72] tivity. Moreover, ring D of sophoridine might not be vital for an-
44b 38.40 HepG2 [73]
ticancer activity [73].

Fig. 31. Comprehensive SAR study of sophoridine and its derivatives.

16
H. ur Rashid, et al.
6. Molecular docking study of sophoridine
To understand better the effectiveness of sophoridine, Xu et al
performed its docking analysis with the human DNA-Topo I complex
(Fig. 32). Molecular docking studies of sophoridine were carried out to
determine its binding affinity into the binding site of DNA-Topo I
complex, which contributed to explaining its mechanism of action.
Moreover, molecular docking analysis facilitated numerous interactions
between the ligands and the receptor in detail. Human topoisomerase I
break a single DNA strand. Topo I poisons can attach to the covalent
Topo I-DNA complex by several interactions, causing double-strand
cleavage and eventually apoptosis. It can be seen in Fig. 32 that the
primary interactions of the sophoridine structure with DNA-Topo I
complex are through the creation of force like Van der Waals, and
hydrophobic bond with residues of the protein [64].
7. The market of anticancer drugs and its estimated growth
The extensive occurrence of cancer in the world has led to an in-
crease in demand for cancer drugs. However, the high price of anti-
tumor drugs is considered to be a global issue. The average cost of a
new anticancer drug in the USA normally surpasses US$100,000 per
year. Compared to other countries, the prices of anticancer drugs are
higher in the USA. However, anticancer drugs are the most expensive in
financially developing countries such as China and India. Oncology
drugs represent the major expenditure of any specialty [74]. Worldwide
oncology/cancer drugs market was estimated to be $97,401 million in
2017 and is expected to reach $176,509 million by 2025, recording a
CAGR (compound annual growth rate) of 7.6% from 2018 to 2025 [75].
Several aspects that steer the market progress include rising Patient
Assistance Programs (PAPs), growing government initiatives for cancer
concern, increasing the occurrence of cancer globally, and robust R&D
initiatives from major companies. Molecularly targeted therapy is pre-
dicted to display rapid progress (CAGR of 9.68%) in the forecast period.
In targeted therapy, drugs function by targeting cancer's specific genes,
proteins, or the tissue surroundings which assist in cancer propagation
and viability. This kind of therapy is getting attention owing to its
specialty toward cancer cells without harming the healthy cells. Breast
cancer is supposed to make up the maximum market proportion in the
predicted period of time. This is primarily linked to the greater and
growing occurrence of breast cancer around the globe. As per data of
the Breast Cancer Organization in 2018, it is predicted that more than
2,66,120 new cases of invasive breast cancer and about 63,960 new
cases of non-invasive breast cancer are likely to be detected among
females in the United States [76], North America controls the market
for cancer treatment and is expected to maintain its dominance for
several more years. This part of the world is predicted to enhance its
commercialization due to the greater use of anticancer drugs in the
coming years. The United States controls most of the market in the
North American continent, owing to the increasing occurrence of
cancer in the country. As reported by the US National Cancer Institute
(NCI), cancer was diagnosed in 1.6 million people, and around 0.5
million people perished from cancer in 2016. The data show that the
occurrence of cancer is growing in the United States [77].
8. Conclusions
Cancer is the second leading cause of fatality around the globe and
has, therefore, become a serious health threat. Significant efforts are
made around the globe to control cancer by the discovery of new an-
ticancer drugs [1,2]. Plant-derived compounds are investigated as safe
anticancer agents [15]. Sophoridine, an alkaloid obtained from Sophora
flavescens, has been reported to exhibit numerous therapeutic effects
comprising anticancer activity [33]. Extensive research on the anti-
neoplastic effects of sophoridine against a variety of cancer cells has
been performed. To improve the moderate anticancer activity of
17
Bioorganic Chemistry 99 (2020) 103863
sophoridine, several research groups synthesized a number of sophor-
idine derivatives and subsequently tested them against various cancer
cell lines. Moreover, SAR studies provided useful information for the
synthesis of new sophoridine derivatives of improved anticancer ac-
tivity.
9. Future research prospects
Regarding the research on the anticancer activities of sophoridine,
only superficial events have been investigated without accurate eva-
luation and clinical trials. The latest molecular procedures can be ap-
plied to perform research on the antitumor mechanism, and clinical
investigations to assess the efficiency and safety of sophoridine-based
drugs against several types of human cancer. Moreover, useful in-
formation can be derived from SAR analysis for the synthesis of novel
sophoridine derivatives of better potency, minimum toxicity, and
higher efficacy.
The anticancer effect of sophoridine and its derivatives can be en-
hanced through drug synergism. Drug Synergy refers to the interaction
between two or more drugs that causes the total therapeutic effect of
the drugs to be larger than the sum of the discrete effects of each drug.
Drug synergy works by targeting multiple components of complex
diseases such as cancer. Therefore, the application of sophoridine de-
rivatives, in combination with other anticancer drugs, can also lead to
the enhancement of anticancer activity in a synergistic manner of these
drugs. Moreover, lower doses of each drug could potentially be used
which could allow for lesser toxicity whereas still giving the anticipated
results, such as apoptosis.
Synthetic modifications in parent sophoridine such as the opening
of ring D or functional group transformation can prove handy for the
synthesis of novel analogs as robust and efficient antitumor agents [1].
Alkaloids-based drugs exhibit low bioavailability and poor water
solubility and cannot easily access the target cancer sites. Therefore,
altering the drug delivery system could be an alternative approach to
structural modification in sophoridine derivatives. The advancement of
nanotechnology can resolve these issues [78,79]. Nanoparticles possess
great potential to act as an efficient drug transport system. Nanosystems
with varying structures and biological features have been widely in-
vestigated for drug delivery applications. Nanoparticles are absorbed by
cells more efficiently than bulkier micromolecules and consequently,
could be applied as effective transport and delivery systems. Nano-
particles can be used in targeted drug delivery at the position of disease
to enhance the intake of sparingly soluble drugs, the targeting of drugs
to a particular location, and drug bioavailability. For this purpose, so-
phoridine derivatives can either be incorporated in the matrix of var-
ious types of nanoparticles or be attached to their surface through
chemical bonds or adsorption. In this regard, several types of nano-
materials such as polymeric nanoparticles, solid lipid nanoparticles,
nanocrystals & nanosuspensions, polymeric micelles, liposomes, den-
drimers, mesoporous silica nanoparticles, and gold nanoshells, etc. can
be chosen.
Numerous plant-isolated compounds, for example, piperine, sino-
menine, genistein, quercetin, naringin, nitrile glycoside, and glycyr-
rhizin have been reported to enhance the bioavailability of several
medicines. Consequently, such compounds can be applied as possible
bioavailability enhancing agents in the future to increase the bioa-
vailability of new and reported sophoridine-based anticancer agents as
well [80]. Alkaloids obtained from plants and their synthetic analogs
are not always safe. Drug doses, their delivery method, and therapeutic
techniques, amongst others, are extremely vital. Therefore, the mod-
ification of chemical structures and the use of a new mode of admin-
istration may decrease the side effects of sophoridine derivatives.
Coordination complexes of Platinum and Palladium have been re-
ported to be effective chemotherapeutic agents. They are known to be
more potent and helpful against a variety of drug-resistant cancers
[81,82]. Cisplatin (a platinum-based antineoplastic agent) is a well-
H. ur Rashid, et al.
known example of such complexes [83]. Therefore, a useful approach
might be the design and synthesis of Platinum and Palladium co-
ordination complexes with ligandable sophoridne derivatives. Such
complexes are likely to exhibit better antineoplastic properties com-
pared to sophoridine derivatives. Nevertheless, thermodynamic stabi-
lity and kinetic inertness of such complexes must be guaranteed for
their safe in vivo application as antitumor agents.
It is an acknowledged fact that the discovery or synthesis of novel
drugs is a complex, expensive skeptical and laborious job. The com-
putational chemistry methodology is an effective tool that can accel-
erate the proficiency of the classical approach used for drug discovery
and design. This technique utilizes mathematical algorithms and com-
puter simulation for chemical occurrences in addition to computing the
structures and chemical features of molecules [80,84]. Distinct com-
putational chemistry methodologies are applied to estimate and cal-
culate events, such as the drug binding to its target and the chemical
characteristics for designing possible novel drugs. Additionally, huge
databases are utilized to combine the chemical concept and modeling
with experimental elucidations. Thus, this key technique can be cost-
effective and time-saving in the discovery and design of novel sophor-
idine-based antitumor drugs [85]. Likewise, mathematical models
should be applied to elucidate the deterioration process of sophoridine-
based antitumor drugs and compute the results of manufacturing and
process refinement and scale-up.
Another useful approach to improve the antitumor activity of so-
phoridine derivatives is through their coupling with other chemother-
apeutics such as Etoposide [86], Frankincense [87], Taxol [88], and
Curcumin [89]. The resulting conjugates are likely to display better
antineoplastic activities than both of their parent precursors on account
of improvement in their cytotoxicities, attributed to synergism.
Furthermore, for future work on the anticancer activity of sophor-
idine (a) the precise anticancer mechanisms of sophoridine derivatives
should further be investigated by novel pharmacological tools (b) the
chemical structures of reported sophoridine derivatives may further be
modified by pharmaceutical chemistry (c) the current combinational
treatment approaches may be examined (d) efficient drug delivery
systems should be formulated (e) clinical antitumor tests for sophor-
idine derivatives need to be performed. Additionally, clinical in-
vestigations must seek substantial positive effects for patients in order
to attain real advantage from novel sophoridine-based antitumor drugs.
Such novel drugs should be more effective than existing antitumor
drugs or any other available therapy. If there is no enhancement in
terms of efficacy, they should nevertheless be less toxic, more tolerable,
convenient to use, or more affordable as compared to the available
anticancer drugs. Furthermore, the adverse effects of novel sophor-
idine-based anticancer drugs can be minimized by designing more po-
tent and selective cytotoxic agents. The selectivity of cytotoxic agents
can be enhanced either by safeguarding normal cells, increasing toxi-
city to cancer cells or by both. Overall, the design and synthesis of new
sophoridine derivatives are essential owing to their encouraging pro-
spects to behave as leading compounds against cancer.
Declaration of Competing Interest
The authors have no conflict of interest to declare.
Acknowledgment
The authors are grateful to the Universidade Federal de Mato Grosso
do Sul. This work was sponsored in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior—Brasil
(CAPES)—Finance Code 001. The authors are also thankful to the
Conselho Nacional de Desenvolvimento Científico e Tecnológico—
Brasil (CNPq)—Finance Code 420852/2018-2 and Fundação de Apoio
ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato
18
Bioorganic Chemistry 99 (2020) 103863
Grosso do Sul—Brasil (FUNDECT-MS)—grants 036/2017 (59/300.074/
2017) for providing funds to execute this project.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103863.
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