Biological evaluation of mitochondria targeting small molecules as potent anticancer drugs

Article abstract of DOI:10.1016/j.bioorg.2021.105055

Cancer therapy targets specific metabolic pathways or a single gene. This may result in low therapeutic effects due to drug selectivity and drug resistance. Recent studies revealed that the mitochondrial membrane potential and transmembrane permeability of cancerous mitochondria are differed from normal mitochondria. Thus, chemotherapy targeting cancerous mitochondria could be an innovative and competent strategy for cancer therapy. Previously, our work with a novel group of mitochondria targeting small molecules presented promising inhibitory capability toward various cancer cell lines and suppressed adenosine triphosphate (ATP) generation. Therefore, it is critical to understand the anticancer effect and targeting mechanism of these small molecules. This study investigated the inhibitory activity of mitochondria targeting small molecules with human cervical cancer cells – HeLa to further explore their therapeutic potential. HeLa cells were exposed to 10 μM of synthesized compounds and presented elevation in intracellular reactive oxygen species (ROS) level, impaired mitochondrial membrane potential and upregulation of apoptosis as well as necrosis. In vivo, HeLa cell tumor-bearing BALB/c nude mice were treated with mitochondria targeting small molecules for 12 days consecutively. Throughout this chemotherapy study, no deleterious side effects nor the appearance of toxicity was observed. Furthermore, mitochondria targeting small molecules treated groups exhibited significant down-regulation with both tumor volume and tumor weight compared to the Doxorubicin (DOX) treated group. Thus, inhibition of mitochondrial ATP synthesis, activation of intracellular ROS production, down-regulation of mitochondrial membrane potential and upregulation of apoptosis and necrosis rates are the indications of cancer therapy. In this work, we examined the anticancer capability of four mitochondria targeting small molecules in vitro and in vivo, and demonstrated a novel therapeutic approach in cancer therapy with tremendous potential.

Full text of DOI:10.1016/j.bioorg.2021.105055

Bioorganic Chemistry 114 (2021) 105055
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Short communication
Biological evaluation of mitochondria targeting small molecules as potent
anticancer drugs
Shuhua Luoa, Xin Danga, Juntao Wanga, Chang Yuana, Yixin Hua, Shuwen Leia, Yang Zhanga,
,b,*
Dan Lua, Faqin Jianga, Lei Fua
a Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, School of Pharmacy, Shanghai Jiao Tong University (SJTU), No. 800 Dongchuan Rd. Minhang
District, Shanghai, 200240, PR China
b SJTU-Agilent Technologies Joint Laboratory for Pharmaceutical Analysis, School of Pharmacy, Shanghai Jiao Tong University (SJTU), No. 800 Dongchuan Rd.
Minhang District, Shanghai, 200240, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cancer therapy targets specific metabolic pathways or a single gene. This may result in low therapeutic effects
due to drug selectivity and drug resistance. Recent studies revealed that the mitochondrial membrane potential
and transmembrane permeability of cancerous mitochondria are differed from normal mitochondria. Thus,
chemotherapy targeting cancerous mitochondria could be an innovative and competent strategy for cancer
therapy. Previously, our work with a novel group of mitochondria targeting small molecules presented promising
inhibitory capability toward various cancer cell lines and suppressed adenosine triphosphate (ATP) generation.
Therefore, it is critical to understand the anticancer effect and targeting mechanism of these small molecules.
This study investigated the inhibitory activity of mitochondria targeting small molecules with human cervical
cancer cells – HeLa to further explore their therapeutic potential. HeLa cells were exposed to 10 µM of syn-
thesized compounds and presented elevation in intracellular reactive oxygen species (ROS) level, impaired
mitochondrial membrane potential and upregulation of apoptosis as well as necrosis. In vivo, HeLa cell tumor-
bearing BALB/c nude mice were treated with mitochondria targeting small molecules for 12 days consecu-
tively. Throughout this chemotherapy study, no deleterious side effects nor the appearance of toxicity was
observed. Furthermore, mitochondria targeting small molecules treated groups exhibited significant down-
regulation with both tumor volume and tumor weight compared to the Doxorubicin (DOX) treated group.
Thus, inhibition of mitochondrial ATP synthesis, activation of intracellular ROS production, down-regulation of
mitochondrial membrane potential and upregulation of apoptosis and necrosis rates are the indications of cancer
therapy. In this work, we examined the anticancer capability of four mitochondria targeting small molecules in
vitro and in vivo, and demonstrated a novel therapeutic approach in cancer therapy with tremendous potential.
Mitochondria
Mitochondria-targeting compounds
ROS
Mitochondrial membrane potential
Apoptosis
Mitochondrial chemotherapy
1. Introduction
cytosolic calcium levels [6,7]. Until today, scientists have discovered
that the mitochondrial genome encompasses between one to two thou-
The mitochondrion is a highly evolved system for coordinating en-
ergy production and distribution based on available calories and oxy-
gen. The biochemical process, known as cellular respiration, is
descriptive of the metabolic reactions within the cell of organisms [1].
Cellular respiration converts biochemical energy from nutrients into
ATP [2]. In addition, other vital cellular parameters are controlled by
the mitochondria, including but not limited to modulation of the oxi-
dation–reduction status [3], generation of ROS [4,5] and control of
sand nuclear DNA (nDNA) genes, plus thousands of mitochondrial DNA
(mtDNA) copies that reside within the mitochondrion [8,9]. The mu-
tation of mtDNA could lead to various cancers, which most commonly
include astrocytic tumors, colon cancer, thyroid tumors, breast tumors,
head and neck tumors, and ovarian tumors, prostate and bladder cancer,
neuroblastomas, and oncocytomas [8,10–14]. Moreover, mutations
with mtDNA encoded for the mitochondrial enzymes have been found to
associate with specific cancers [15,16]. Thus, targeting cancerous
* Corresponding author at: Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, School of Pharmacy, Shanghai Jiao Tong University (SJTU), No.
800 Dongchuan Rd. Minhang District, Shanghai, 200240, PR China.
E-mail address: leifu@sjtu.edu.cn (L. Fu).
https://doi.org/10.1016/j.bioorg.2021.105055
Received 11 April 2021; Received in revised form 17 May 2021; Accepted 1 June 2021
Available online 3 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Luo et al.
Bioorganic Chemistry 114 (2021) 105055
mitochondria could be an efficient approach in cancer therapy. Mean-
while, one of the most challenging obstacles we confront in chemo-
therapy discovery today is the optimization of drug selectivity for
cancerous mitochondria. Consequently, treatment with high selectivity
in targeting the cancerous mitochondria is urgent [17].
2. Result and discussion
2.1. Chemistry
Recently within our group, 40 mitochondria targeting small mole-
cules were synthesized and evaluated the cytotoxicity against three
different cancerous cell lines – HeLa, MCF-7 and PC-3; and a non-
cancerous cell line – CHO (See Appendix A). The structural design of
these compounds contained three parts: the warhead, the linker and the
tail group, as shown in Fig. 1. Briefly, the synthesis of these mitochon-
dria targeting compounds shared a common tail-group - TPP, a group
known to target the mitochondrion [24,30]. The warhead contained a
modified thiazole derivative based on our previous findings [28,29].
Among all 40 mitochondria targeting compounds, four compounds -
R12, R13, D15 and N2, demonstrated high potential in cancerous-
proliferation inhibition. Subsequent in vitro analysis confirmed that
these four compounds inhibited ATP production of HeLa cells at a low
concentration (See Appendix A). Therefore, further examinations were
designed and performed to evaluate the anticancer capability and
explore possible mechanisms of these compounds. The synthetic routes
for these four compounds, R12, R13, D15 and N2 were illustrated in
Scheme 1-3. All structures were verified by 1HNMR, 13CNMR and HRMS.
Mitochondrial targeting antioxidant and anticancer compounds were
first discovered during the 1990’s, and these mitochondrial targeting
moieties included triphenylphosphonium (TPP), rhodamine 123 and
dequalinium [18–22]. The concept of mitochondria targeting was based
on the membrane potential difference between cellular plasma and
mitochondria [23]. Generally, plasma membrane potential ranges from
ꢀ 30 to ꢀ 60 mV, while typical mitochondrial membrane potential rea-
ches up to ꢀ 180 mV [24]. This phenomenon facilitates positively
charged molecules to have a much higher accumulation in mitochondria
than cytosol. Additionally, studies have shown that cancerous mito-
chondrial membrane exhibited higher accessibility and transmembrane
potential than normal mitochondrial membrane [19,22,25]. In the
studies with rhodamine 123 (florescent lipophilic cation dye) targeting
the mitochondrial matrix, rhodamine 123 was found to have markedly
higher accumulation and longer retention period with cancerous mito-
chondria than normal mitochondria in vitro, and similar results were also
observed with malignant cells implanted with mice models [19,25].
Additional studies with delocalized lipophilic cations, including thio-
pyrylium and TPP, revealed comparable results as well [26]. Together,
these phenomena were used as the basis to develop mitochondria tar-
geting antioxidant or anticancer compounds for cancer cells. Recent
studies with mitochondria targeting antioxidant or anticancer com-
pounds focused on enhancing mitochondrial accumulation, thereby
improving therapeutic effects and overcoming drug resistance [23].
Meanwhile, mitochondria targeting compounds would selectively
accumulate in cancerous mitochondria, constantly induce intracellular
ROS generation, impair mitochondrial membrane potential and even-
tually lead to apoptotic cell death [27]. Thus, it is crucial to understand
the signs related to mitochondria targeting compounds and their cellular
effects.
2.2. ROS generation analysis
Intracellular ROS elevation has been reported as a sign of early-stage
apoptosis [31]. The production of intracellular ROS mainly relies upon
the mitochondrion. During OXPHOS, ROS are constantly produced and
released from complexes I and III on the electron transport chain in
order to afford ATP production [32]. Excessive intracellular ROS can
overwhelm the cellular antioxidant defense system and lead to apoptosis
[27]. To determine whether R12, R13, D15 and N2 are capable of
inducing ROS generation, 10 µM of the respective compounds were
incubated with HeLa cell for 24 h and stained with fluorogenic probe ꢀ
2′,7′-Dichlorofluorescin diacetate (DCFH-DA). Intracellular ROS eleva-
tion was observed and evaluated by fluorescence microscope and flow
cytometry respectively. As illustrated in Fig. 2-a, all 4 treated groups
(R12, R13, D15 and N2) and the positive control group (Ros-up) pre-
sented vigorous green fluorescence intensity, while the control group
exhibited no obvious green fluorescence. By evaluating the DCFH-DA
intensity (Fig. 2-b), intracellular ROS production of HeLa cells was
increased by 1.65 (R12), 1.87 (R13), 1.44 (D15) and 1.95 (N2) fold in
comparison to the control group. This phenomenon implied that these
mitochondria targeting compounds might take a significant role in the
early stage of HeLa cell apoptosis.
Previously, our research group discovered a novel batch of mito-
chondria targeting small molecules that contained the TPP moiety based
on Collman’s work [28]. This novel group of mitochondria targeting
compounds, also called the Mito-Fu family, was adapted to the thiazole
warhead. Previous studies with the Mito-Fu have shown promising
capability in anti-aging and anti-obesity in vivo [29]. In concern with the
indispensable relationship between oxidative phosphorylation
(OXPHOS) and tumor metabolism, further derivations were adapted to
the Mito-Fu compounds focusing on the anticancer activity. Most
recently, our discovery with four mitochondrial targeting small mole-
cules presented high cytotoxicity toward cancerous cell lines (unpub-
lished). Moreover, substantial ATP inhibition was observed with treated
HeLa cells (unpublished). Thus, it is reasonable to further study the
anticancer effects of these mitochondria targeting compounds both in
vitro and in vivo, uncovering the underlying mechanisms.
2.3. Mitochondrial membrane potential analysis
Mitochondrial membrane potential is one of the most essential fac-
tors during oxidative phosphorylation for ATP production. Typically,
the mitochondrial membrane potential of cancer cells is displayed in the
hyperpolarized state [33]. The reduced mitochondrial membrane
Fig. 1. Structural Design of the Mito-Fu Family compounds. The Mito-Fu Family compounds’ basal structure contained three parts: the warhead, the linker, and the
tail group.
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Bioorganic Chemistry 114 (2021) 105055
Scheme 1. Synthetic route for R12-13 from 4-hydroxybenzothioamide and ethyl 3-bromo-2-chloro-3-oxopropanoate. Reagents and conditions: a: EtOH, 80 ℃
reflux, overnight; b1: (bromomethyl)benzene/1-(bromomethyl)-4-(trifluoromethyl)benzene, DMF, 80 ℃ reflux, overnight; c: LiAlH4, THF, 0 ℃, 3 h; d: 2-bromoacetyl
bromide, DCM, 0 ℃ to room temperature, overnight; e: PPh3, toluene, room temperature, overnight.
Scheme 2. Synthetic route for D15 from 4-hydroxybenzothioamide and ethyl 3-bromo-2-oxopropanoate. Reagents and conditions: a: EtOH, 80 ℃ reflux, overnight;
b2: 1-(bromomethyl)-4-(trifluoromethyl)benzene, K2CO3, CH3CN, 80 ℃ reflux, overnight; c: LiAlH4, THF, 0 ℃, 3 h; d: 2-bromoacetyl bromide, DCM, 0 ℃ to room
temperature, overnight; e: PPh3, toluene, room temperature, overnight.
Scheme 3. Synthetic route for N2 from 4-hydroxybenzothioamide and ethyl 2-chloro-4-methyl-3-oxopentanoate. Reagents and conditions: a: EtOH, 80 ℃ reflux,
overnight; c: LiAlH4, THF, 0 ℃, 3 h; d: 2-bromoacetyl bromide, DCM, 0 ℃ to room temperature, overnight; e: PPh3, toluene, room temperature, overnight.
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Bioorganic Chemistry 114 (2021) 105055
Fig. 2. Effects of ROS production with HeLa cells treated with mitochondria targeting compounds. Intracellular production of ROS in HeLa cells incubated with 10
µM of the indicated compound for 24 h was (a) observed with fluorescence microscope and (b) measured with flow cytometry; Ros-up was used as positive control
and provided by the test kit. Three independent experiments were performed, and data was shown as mean ± SD. Asterisks indicated statistical significance compare
to control using the t-test, (*p < 0.01; **p < 0.001; ***p < 0.0001).
potential is seen as a sign of early-stage apoptosis [34]. Furthermore,
excessive production of ROS could collapse the mitochondrial mem-
brane potential and lead to cytochrome c release and apoptosis ma-
chinery activation. In order to determine whether R12, R13, D15, and
N2 sufficiently modifies mitochondrial membrane potential, 10 µM of
the respective compounds were incubated with HeLa cells for 24 h.
Subsequently, HeLa cells were stained with JC-1 fluorescent probe to
determine and evaluate the changes with mitochondrial membrane
potential via fluorescence microscope and flow cytometry respectively.
As illustrated in Fig. 3-a, all four treated groups (R12, R13, D15 and N2)
and the positive control group (CCCP) displayed substantial enhance-
ment of green fluorescence (JC-1 monomer) intensity in comparison to
the control group. This phenomenon proposed depolarization of mito-
chondrial membrane potential of treated HeLa cells. The fluorescence
intensity of JC-1 monomers was increased by 2.55 (R12), 2.73 (R13),
1.64 (D15) and 2.62 (N2) fold in comparison to the control group, as
shown in Fig. 3-b. In summary, these data suggested that all compounds
sufficiently collapsed the mitochondrial membrane potential, and
eventually led to cell death.
2.4. Cellular apoptosis and necrosis analysis
Apoptosis and necrosis are not only vital signs for cell survival, but
they also play significant roles in cancer therapy. During apoptosis, cells
undergo a programmed cell death process triggered by various chemical,
physical or biological factors [35]. In contrast, necrosis is an unpro-
grammed cell death induced by cell injury [35]. Thus, it is crucial to
examine the capability of our compounds R12, R13, D15 and N2 to
induce cancer cell death. In order to study which type of cell death our
compounds initiate, the Hoechst 33342/PI dual staining assay was
performed. 10 µM of each compound was incubated with HeLa cells for
24 h, and 0.35 µM doxorubicin (DOX) was used as the positive control.
Treated HeLa cells were collected and stained with Hoechst 33342/ PI
stains and analyzed with flow cytometry. As shown in Fig. 4, the per-
centage of apoptotic cells (lower right quadrant) was increased by 6.09
(R12), 8.85 (R13), 9.50 (D15) and 7.88 (N2) fold after incubation with
the respective compound. In addition, compounds R12 and R13 induced
a 7.5-fold and 39.5-fold increase with necrotic cells (upper right quad-
rant) compared to the control group. These results proved that all four
compounds effectively induced HeLa cell apoptosis, while compounds
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Bioorganic Chemistry 114 (2021) 105055
Fig. 3. Changes in mitochondrial membrane potential with HeLa cell treated with mitochondria targeting compounds. Changes in mitochondrial membrane po-
tential in HeLa cells treated with 10 µM of the indicated compound for 24 h were (a) observed with fluorescence microscope and (b) evaluated with flow cytometry;
10 µM CCCP was used as positive control and incubated with HeLa cells for 30 min. Three independent experiments were performed, and data was illustrated as
mean ± SD. Asterisks indicated statistical significance compared to the control using the t-test, (*p < 0.02; **p < 0.002).
R12 and R13 triggered cell necrosis.
10 mg/kg), 63% (R13-20 mg/kg), 61% (N2-20 mg/kg) and 79% (N2-50
mg/kg). Fig. 6 presented the histological slices of tumor from treated
BALB/C nude mice. In comparison with the control group, both R13-20
mg/kg and N2-50 mg/kg treated group manifested the increasing tumor
necrosis (black arrows) and down-regulation of the number of tumor
nodes (lower row). This implied that the compounds effectively pre-
vented tumor migration. Furthermore, R13-20 mg/kg shown dimin-
ished cancerous nucleolus (red arrows), indicating the restraint of
cancer cell growth. These data together supported that R13 and N2
could repress tumor growth and transition in vivo.
2.5. Chemotherapy study
HeLa tumor cells-bearing BALB/C nude mice by subcutaneous in-
jection of indicated compounds with different concentrations consecu-
tively. During this in vivo study, tumor volume, body weight, as well as
the physiological function of each animal was monitored and recorded
daily. Subsequently, the final tumor was isolated and weighed for his-
tological study. As shown in Fig. 5-a, treated groups demonstrated
convincing inhibition in tumor growth compared to the control group
during 12 days. Additionally, the body weight of all mice remained the
average weight of 21 g (Fig. 5-b), and no death case was caused by the
compounds. As tumors reached over 15 mm in diameter, nude mice
were sacrificed. All collected tumors were categorized, weighed and
preserved in 4% paraformaldehyde. Besides down-regulation of tumor
volume, the average tumor weight was decreased in treated groups as
compared to the control group. As illustrated in Fig. 4-c, the tumor
weight of treated group demonstrated down-regulation by 40% (R13-
2.6. Chemico-genetic Analysis: R13 and N2 induced DEGs and functional
analysis
Chemico-genetic analysis of R13 and N2 induced differentially
expressed genes (DEG) were examined and analyzed. As shown in
Fig. 7a-b, volcano plots illustrated the DEG amount between R13, N2
groups and their corresponding control groups. In total, 153 DEGs dis-
played differential expression after treatment with compound R13,
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Bioorganic Chemistry 114 (2021) 105055
Fig. 4. Mitochondria targeting compounds induced cell apoptosis and necrosis. (a) Cell apoptosis and necrosis were analyzed using the Apoptosis and Necrosis
Analysis Kit with 10 µM of the indicated compound for 24 h by flow cytometry; 0.35 µM Dox was used as positive control and incubated with HeLa cells for 24 h.
Changes in (b) apoptosis rate and (c) necrosis rate of treated HeLa cells were evaluated with flow cytometry and shown in column graphs. Three independent
experiments were performed, and data was shown as mean ± SD. Asterisks indicated statistical significance compared to the control using the t-test, (*p < 0.01; **p
< 0.001).
including 86 upregulated and 67 downregulated DEGs. In N2 treated
tumor, 459 DEGs were displayed in total, in which 258 DEGs were
upregulated and 201 were downregulated. The overall alteration in gene
expression of DEG of R13 and N2 among the gene samples was visual-
ized in Fig. 7c-d through Heatmap clustering analysis. These data
confirmed gene expression changes after treatment with R13 and N2.
Subsequently, the Gene Ontology (GO) pathway (Fig. 7e-f) and the
Kyoto encyclopedia of genes and genomes (KEGG) pathway (Fig. 7g-h)
analysis was analyzed using the Database for Annotation, Visualization,
and Integrated Discovery (DAVID). Both Go and KEGG pathways pro-
vided clues about the targeting mechanisms of compounds R13 and N2.
We found significant enrichment with GO analysis, including regulation
of cell growth, regulation with membrane potential, negative regulation
of nuclear division, regulation of intracellular transport, regulation of
mRNA catabolic and metabolic process, regulation of apoptotic
signaling pathway, epithelial cell migration and tissue migration applied
to both R13 and N2 treated tumors. In the KEGG pathways, DEGs were
enriched in cell cycle, apoptosis, RNA degradation, Wnt signaling
pathway, MAPK signaling pathway and other pathways in cancer when
applied to both R13 and N2 treated models. To be noted, the Wnt
signaling pathway was marked as the sixth most enhanced pathway
among 30 KEGG analytical pathways in both R13 and N2 treated
samples.
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Bioorganic Chemistry 114 (2021) 105055
Fig. 5. Treatment analysis of compounds R13 and
N2 on the animal subcutaneous model. (a). The
tumor volumes of HeLa tumor-bearing nude mice
treated with indicated compounds over time; tumor
volumes were calculated using the ellipsoid for-
mula = 2 × (length × width2); (b). the body weight
of each group of treated nude mice was recorded
after each subcutaneous injection; (c) the isolated
tumor-weight of each treated group after 12 days of
treatment. Data was shown as mean ± SD; n = 5 for
each group. Asterisks indicated statistical signifi-
cance compare to control using the t-test, (*p < 0.1;
**p < 0.01; n.s. = not significant).
2.7. Chemico-genetic Analysis: The Co-expression network of R13 and N2
induced DEGs
induced DEGs co-expression network, ADCY10 demonstrated the high-
est correlation among all DEGs. ADCY10 is a protein-coding gene for
soluble adenylyl cyclase (sAC), and sAC is localized in the mitochondria
responsible for mitochondrial cAMP production [36]. A recent study
presented the role of sAC in controlling mitochondrial-dependent
apoptosis, and suppression of sAC activity with selective inhibitor or
knockdown of sAC with small interfering RNA simultaneously abolished
mitochondrial-dependent apoptosis [37]. Therefore, we hypothesized
that mitochondrial-dependent apoptosis was modulated by upregulation
of ADCY10 in this study. Within the N2-induced DEGs co-expression
All DEGs from the KEGG analysis were utilized as genes of interest to
study the possible targeting mechanisms. Moreover, co-expression net-
works were built based upon the selected DEGs. The established co-
expression network contained 15 nodes in both R13 and N2 induced
DGEs models (Fig. 7k-l). Heatmap clustering analysis of respective DEGs
was illustrated in Fig. 7i-j, and the expression magnitudes of DEGs were
indicated with various intensities of red and blue. As shown in the R13-
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Fig. 6. H&E Staining of HeLa Tumors of BALB/c Mice. Tumors were presented in histological slides. Upper row: Red arrows indicate diminished tumor nucleolus
and black arrows indicate tumor necrosis. All histology images share the same magnification of 200X. Lower row: The decrease in the number of tumor nodes
indicated the prevention of tumor migration with treated groups compared to the control group. Tumor sizes were indicated with scale bars. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
network, two genes, CCNA1 and NKD2, manifested the highest corre-
lation. CCND1 is a well-known oncogene in many diseases, and this gene
encodes protein Cyclin D1 which is abundant throughout the cell cycle.
Accumulating studies have proven that the knockdown of CCND1
induced glioblastoma cell apoptosis, suppressed liver cancer stem cell
differentiation, regulated ovarian cancer proliferation and more
[38–40]. Thus, down-regulation with CCND1 in this study strongly
confirmed the anticancer activity of N2. Besides CCND1, NDK2 is the
other most correlated DEGs in this model, and this gene encodes a
protein that acts as a negative regulator of the Wnt signaling pathway.
Another study has proven that overexpression of NKD2 deregulates
tumor proliferation, invasion and migration ability within the osteo-
sarcoma tumor [41]. Therefore, it is reasonable to investigate the role of
NKD2 in our study model relating to the mitochondrial targeting
mechanisms in the future.
ADCY10, NKD2 and a few others. Further investigations are underway to
evaluate their roles not only in anticancer activity but also in
mitochondria-targeting mechanisms. The discovery of anticancer capa-
bility both in vitro and in vivo supported the hypothesis of mitochondria-
targeting chemotherapy in cancer. Subsequently, our research group
shall focus on the underlying mechanisms in hope of moving us a step
closer toward understanding the dynamic and perhaps multi-regulation
of the Mito-Fu family compounds.
4. Experimental section
4.1. General information
Chemicals. All chemical (reagent grade) and solvents were purchased
from commercial sources and purified using standard methods.
3. Conclusion
4.2. Experimental section
Previously, our group discovered 40 mitochondria-targeting com-
pounds. Selected compounds displayed promising capability toward
4.2.1. General procedure for preparation of compounds R12-R13
Commercially available 1 and 2 were dissolved in anhydrous
ethanol. Mixture was stirred and refluxed at 80 ℃ overnight. Upon
completion, anhydrous ethanol was distilled off and extracted with
minimum amount of ethyl acetate. Then intermediate 3 was extracted
via recrystallization in an ice bath as a white solid. A mixture of inter-
mediate 3 and commercially available (bromomethyl)benzene/1-(bro-
cancerous cell line toxicity (R13, IC50 = 5.52 μM), and up to 64% down-
regulation with ATP generation when HeLa cells are exposed to
mitochondria-targeting compound for 24 h. These results have guided us
to further investigate the mechanisms and anticancer activity of the
Mito-Fu family.
In this study, we confirmed the mitochondria targeting capability of
four small molecules (R12, R13, D15 and N2) and their ability to
upregulate ROS generation, impair mitochondrial membrane potential,
and induce cellular apoptosis and necrosis to various extents. Further in
vivo study confirmed the excellent anticancer capability of R13 and N2
toward HeLa tumors on mice models. Consequently, chemico-genetic
analysis was performed on treated tumors and disclosed the possible
targeting mechanisms of these chemicals. Additionally, the down-
regulation of cancer-hallmark genes such as CCND1 strongly sup-
ported the anticancer capability of our mitochondria-targeting com-
pounds. Beyond chemico-genetic analysis, numbers of highly correlated
DEGs were extensively studied during our current research, including
momethyl)-4-(trifluoromethyl)benzene
was
dissolved
in
dimethylformamide and refluxed at 80 ℃ overnight. The reaction was
quenched by H2O and extracted by ethyl acetate. The mixture of ethyl
acetate solutions was dried and evaporated, and intermediate 4a-b were
afforded by silica chromatography as white solids. Then intermediates
4a-b were dissolved in tetrahydrofuran, and LiAlH4 was added to solu-
tion dropwise under the ice bath. Reaction mixture was stirred at room
temperature for 3 h. Then reaction was quenched with H2O dropwise
under ice-bath and extracted by ethyl acetate. Collected ethyl acetate
solutions were dried and evaporated. Silica chromatography was per-
formed to afford intermediate 5a-b as yellow solids. A solution of 5a-b
dissolved in dichloromethane was added with 2-bromoacetyl bromide
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Bioorganic Chemistry 114 (2021) 105055
Fig. 7. R13 and N2 Induced Gene Expression Changes in HeLa Tumor Bearing Mice. The volcano plots of R13 (a) and N2 (b) induced gene expression changes based
on all genes detected by the microarray; The heatmap of R13 (c) and N2 (d) induced DEGs are illustrated; DEG fold change ≥ 2.0. GO pathway analysis of R13 (e)
and N2 (f) induced DEGs, 10 most enrich biological processes were selected in each model; KEGG pathway analysis of R13 (g) and N2 (h) induced DEGs, 10 most
enrich biological processes were selected in each model; The heatmaps of R13 (i) and N2 (j) induced DEGs from the respective KEGG pathways; R13-induced DEGs
co-expression network (k) and N2-induced DEGs co-expression network (l), red circles represented upregulation of gene expression and blue circles represented
down-regulation of gene expression determined by the log (fold change) with each model; the size of each circle corresponding to the correlation degree, and the
larger the circle represents a higher correlation degree and vice versa. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
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Bioorganic Chemistry 114 (2021) 105055
Fig. 7. (continued).
dropwise under an ice bath. Reaction mixture was stirred at room
temperature overnight and reaction was monitored via TLC. Then, re-
action mixture was washed with saturated NaHCO3, and organic layers
were collected, dried, and evaporate. Silica chromatography was per-
formed to afford intermediates 6a-b as white solids. Finally,
intermediates 6a-b dissolved in toluene were added with PPh3. Reaction
mixture was stirred at room temperature overnight and monitored with
TLC. Upon completion, the precipitate was collected and washed with
toluene to provide R12 & R13 as white solids.
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Bioorganic Chemistry 114 (2021) 105055
Fig. 7. (continued).
4.2.1.1. (2-((2-(4-(benzyloxy)phenyl)thiazol-4-yl)methoxy)-2-oxoethyl)
triphenylphosphonium bromide (R12). White solid, yield 75%. 1H NMR
(400 MHz, DMSO‑d6) δ 7.87 – 7.74 (m, 11H), 7.70 (td, J = 7.7, 3.7 Hz,
6H), 7.57 (s, 1H), 7.50 – 7.47 (m, 2H), 7.45 – 7.39 (m, 2H), 7.37 – 7.32
(m, 1H), 7.18 – 7.12 (m, 2H), 5.46 (d, J = 14.6 Hz, 2H), 5.20 (s, 4H).
13C NMR (101 MHz, Chloroform-d) δ 155.15, 141.85, 140.21,
138.92, 138.82, 135.24, 135.11, 133.66, 132.95, 120.64, 45.39, 45.18,
44.97, 44.76, 44.55, 44.34, 44.13. HRMS(ESI) m/z C37H31BrNO3PS
calcd. for [M]+ 600.1757, found: 600.1757
134.07, 130.49, 130.36, 128.52, 128.23, 126.36, 125.81, 125.77,
119.72, 118.92, 118.04, 115.94, 69.01, 63.33, 30.17. HRMS(ESI) m/z
C38H30BrF3NO3PS calcd for [M]+ 668.1631, found: 668.1611
4.2.2. General procedure for preparation of compounds D15
Commercially available 7 and 8 were dissolved in anhydrous
ethanol. Mixture was stirred and refluxed at 80 ℃ overnight. Upon
completion, anhydrous ethanol was distilled off and extracted with
minimum amount of ethyl acetate. Then intermediate 9 was extracted
via recrystallization in an ice bath as a white solid. A mixture of inter-
mediate
9
and commercially available 1-(bromomethyl)-4-(tri-
4.2.1.2. (2-oxo-2-((2-(4-((4-(trifluoromethyl)benzyl)oxy)phenyl)thiazol-
4-yl)methoxy)ethyl)triphenylphosphonium bromide (R13). White solid,
yield 93%. 1H NMR (400 MHz, DMSO‑d6) δ 7.90 – 7.76 (m, 13H), 7.70
(td, J = 7.8, 3.3 Hz, 8H), 7.60 (s, 1H), 7.18 (d, J = 8.5 Hz, 2H), 5.50 (d, J
= 14.6 Hz, 2H), 5.34 (s, 2H), 5.21 (s, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 167.83, 160.22, 150.43, 142.01, 135.47, 135.44, 134.18,
fluoromethyl)benzene was dissolved in acetonitrile with potassium
carbonate and refluxed at 80 ℃ overnight. The reaction was quenched
by H2O and extracted by ethyl acetate. The mixture of ethyl acetate
solutions was dried and evaporated, and intermediate 10 was afforded
by silica chromatography as white solids. Then intermediate 10 was
11

S. Luo et al.
Bioorganic Chemistry 114 (2021) 105055
dissolved in tetrahydrofuran, and LiAlH4 was added to solution drop-
wise under ice-bath. Reaction mixture was stirred at room temperature
for 3 h. Then reaction was quenched with H2O dropwise under ice-bath
and extracted by ethyl acetate. Collected ethyl acetate solutions were
dried and evaporated. Silica chromatography was performed to afford
intermediate 11 as yellow solids. A solution of 11 dissolved in
dichloromethane was added with 2-bromoacetyl bromide dropwise
under ice-bath. Reaction mixture was stirred at room temperature
overnight and reaction was monitored via TLC. Then, reaction mixture
was washed with saturated NaHCO3, and organic layers were collected,
dried, and evaporate. Silica chromatography was performed to afford
intermediate 12 as white solid. Finally, intermediates 12 dissolved in
toluene were added with PPh3. Reaction mixture was stirred at room
temperature overnight and monitored with TLC. Upon completion,
precipitate was collected and washed with toluene to provide D15 as
white solids.
purchased from HyClone. Fetal Bovine Serum (FBS), Trypsin-EDTA
(2.5%) and Phenol red were purchased from UBI. HeLa cell line was
grown in the supplementation of 10% FBS and 1% 100X Penicillin/
Streptomycin Solution in a humidified atmosphere of 5% CO2 at 37 ◦C.
4.4. ROS assay
HeLa cells were grown at a density of 5 × 105 cells/mL in a 6-well
plate and treated with compounds R12, R13, D15 and N2 at a concen-
tration of 10 µM for 24 h with proper culture medium in 5% CO2 at
37 ◦C. For fluorescent microscope observation, treated HeLa cells were
washed twice with PBS and stained with 10 mM DCFH-Da dissolved in
DMEM and incubated at 37 ◦C in the dark for 20 min. Then, DCFH-Da
stained cells were washed twice with ice-cold PBS and fluorescent mi-
croscope observation was performed at the proper excitation wave-
length (488 nm) and emission wavelength (525 nm). For flow cytometry
evaluation, treated cells were washed twice with PBS and collected at
1500 rpm. Then, treated cells were resuspended in 10 mM DCFH-DA
that dissolved in DMEM and incubated at 37 ◦C in the dark for 20
min. Subsequently, DCFH-Da stained cells were collected, washed twice
with ice-cold PBS and resuspended in DMEM. Intracellular ROS con-
centration was evaluated with flow cytometry at the proper excitation
wavelength (488 nm) and emission wavelength (525 nm).
4.2.2.1. (2-((4-methyl-2-(4-((4-(trifluoromethyl)benzyl)oxy)phenyl)thia-
zol-5-yl)methoxy)-2-oxoethyl)triphenylphosphonium bromide (D15).
White solid, yield 71%.1H NMR (400 MHz, DMSO‑d6) δ 7.86 – 7.69 (m,
23H), 5.40 (d, J = 14.7 Hz, 2H), 5.32 (d, J = 3.9 Hz, 4H), 2.25 (s, 3H).
13C NMR (101 MHz, DMSO‑d6) δ166.63,164.90, 160.29,153.75, 135.57,
135.50, 135.47, 134.15, 134.04, 130.55, 130.42, 128.51, 128.07,
126.38, 126.01, 125.85, 125.81, 125.74, 123.78, 118.89, 118.00,
115.97, 68.98, 59.53, 40.41, 40.20, 29.99, 15.26. HRMS(ESI) m/z
4.5. Mitochondrial membrane potential assay
C
39H32F3NO3PS, calcd for [M + H]+ 683.1865, found: 683.1833
HeLa cells were grown at a density of 5 × 105 cells/ml in a 6-well
plate and treated with compounds R12, R13, D15 and N2 at a concen-
tration of 10 µM for 24 h with proper culture medium in 5% CO2 at
37 ◦C. For fluorescent microscope observation, treated HeLa cells were
washed with PBS and stained with JC-1 staining solution and incubated
at 37 ◦C in the dark for 20 min. Then, stained cells were washed with ice-
cold JC-1 Buffer. JC-1 monomers were observed by fluorescent micro-
scope at an excitation of 490 nm and emission of 530 nm. For flow
cytometry evaluation, treated cells were collected at 1500 rpm and
stained with JC-1 staining solution and incubated at 37 ◦C in the dark for
20 min. Subsequently, cells were rinsed with JC-1 buffer and resus-
pended in JC-1 Buffer. Loss of mitochondrial membrane potential was
determined with flow cytometry by evaluating the relative JC-
1monomer and aggregate ratio.
4.2.3. General procedure for preparation of compounds N2
Commercially available 13 and 14 were dissolved in anhydrous
ethanol. Mixture was stirred and refluxed at 80 ℃ overnight. Upon
completion, anhydrous ethanol was distilled off and extracted with
minimum amount of ethyl acetate. Then intermediate 15 was extracted
via recrystallization in an ice bath as a white solid. Then intermediates
15 was dissolved in tetrahydrofuran, and LiAlH4 was added to solution
dropwise under ice-bath. Reaction mixture was stirred at room tem-
perature for 3 h. Then reaction was quenched with H2O dropwise under
ice-bath and extracted by ethyl acetate. Collected ethyl acetate solutions
were dried and evaporated. Silica chromatography was performed to
afford intermediate 16 as yellow solids. A solution of 16 dissolved in
dichloromethane was added with 2-bromoacetyl bromide dropwise
under ice-bath. Reaction mixture was stirred at room temperature
overnight and reaction was monitored via TLC. Then, reaction mixture
was washed with saturated NaHCO3, and organic layers were collected,
dried, and evaporate. Silica chromatography was performed to afford
intermediate 17 as white solid. Finally, intermediates 17 dissolved in
toluene were added with PPh3. Reaction mixture was stirred at room
temperature overnight and monitored with TLC. Upon completion, the
precipitate was collected and washed with toluene to provide N2 as
white solids.
4.6. Apoptosis and necrosis assay
Hoechst 33342 and PI dual staining were used for apoptosis and
necrosis analysis. HeLa cells were grown at a density of 5 × 105 cells/ml
in a 6-well plate and treated with compounds R12, R13, D15 and N2 at a
concentration of 10 µM for 24 h with proper culture medium in 5% CO2
at 37 ◦C. Then HeLa cells were collected at 1500 rpm and resuspended in
the staining buffer. Subsequently, Hoechst/PI probes were resuspended
with treated cells and incubated at 0 ◦C for 20 min. Samples were
analyzed with flow cytometry and data was analyzed with FlowJo
software.
4.2.3.1. (2-((4-isopropyl-2-(4-(trifluoromethyl)phenyl)thiazol-5-yl)
methoxy)-2-oxoethyl)triphenylphosphonium bromide (N2). White solid,
yield 66%. 1H NMR (400 MHz, DMSO‑d6) δ 7.91 – 7.69 (m, 19H), 5.45
(d, J = 14.6 Hz, 2H), 5.41 (s, 2H), 3.17 (dt, J = 13.6, 6.9 Hz, 1H), 1.18
(d, J = 6.8 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6) δ 165.35, 164.99,
163.60, 136.75, 135.51, 135.48, 134.17, 134.06,133.74, 130.60,
130.55, 130.47, 130.42, 127.15, 126.79, 126.75, 125.20, 118.89,
118.00, 58.88, 30.02, 27.98, 23.03. HRMS(ESI) m/z C34H30F3NO2PS,
calcd for [M]+ 604.1681, found: 604.1668
4.7. Chemotherapy study – Tumor model and treatment
All animal experimental procedures were approved by Shanghai Jiao
Tong University’s review board and administrative panel on laboratory
animal according to national guidelines (Ethic Number: 20201127–01).
Animals used for this research project include female BALB/c mice
(Shanghai SLAC Laboratory Animal Co. Ltd). A total of 45 female BALB/
c mice were group-housed in a pathogen-free facility with 21 ◦C and 12 h
light/12 h dark cycle, with full access to water and standard laboratory
food.
4.3. Cell culture and maintenance
All HeLa cells used in this study were purchased from China Life
Science College (Shanghai, PRC). DMEM culture medium, Phosphate
Buffered Saline (PBS) and 100X Penicillin/Streptomycin Solution were
In order to build the tumor model, HeLa cells with concentration of 1
× 106 cells /100 µL in serum-free DMEM were inoculated subcutane-
ously in the lower right thigh of each nude mouse. When the diameter of
12

S. Luo et al.
Bioorganic Chemistry 114 (2021) 105055
the tumor reached 4–5 mm, nude mice were randomly divided into six
groups and labeled. Group I – the control group and treatment with 100
µL saline solution; Group II – the positive control group and treatment
with 0.5 mg/kg DOX; Group III – treatment with 20 mg/kg N2 com-
pound; Group IV – treatment with 50 mg/kg N2 compound; Group V –
treatment with 10 mg/kg R13 compound; Group VI – treatment with 20
mg/kg R13 compound. All compounds were dissolved in saline (contain
0.1% DMSO) and 100 µL of each solution were administrated subcuta-
neously to nude mice. This experiment was carried out consecutively
from day 0 to day 11. Meanwhile, the width and length of ellipsoid tu-
mors, as well as weights, were measured and recorded after each sub-
cutaneous administration (from day 0 to 12). On day 12, nude mice were
sacrificed. Tumors, kidneys and livers were isolated and preserved for
future analysis.
Declaration of Competing Interest
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.
Acknowledgements
This research was supported by the Chinese government scholarship
(CSC for SH Luo), Medical-Engineering Joint Fund of Shanghai Jiao
Tong University (No. YG2017MS29, No. ZH2018ZDB07), and Agilent
research Award (Agilent Grant ID #4608). The authors would like to
also thank Tony Zhang for his careful language proofreading and
corrections.
Appendix A. Supplementary data
4.8. Hematoxylin-eosin (H&E) staining
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105055.
Tumor tissues collected from BALB/c nude mice were preserved in
4% paraformaldehyde and stained with H&E staining according to the
manual H&E Staining protocol [42]. Sectioned tissues were observed
and photographed under 200X and 400X microscope.
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