Bioorg. Med. Chem.

64 (2022) 116759

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Small molecule NSAID derivatives for impairing powerhouse in cancer cells

Aman Bajpai a, c, Deepshikha b, Dimple Chhabria a, Tripti Mishra a, Sivapriya Kirubakaran a,
Sudipta Basu a, *
a
Discipline of Chemistry, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar, Gujarat 382355, India
b
Department of Chemistry, Indian Institute of Science Education and Research (IISER), Pune, Dr. Homi Bhabha Road, Pashan, Pune, Maharashtra 411008, India
c
Chemistry Department, Bharat Institute of Engineering and Technology, Mangalpally Village, Ibrahimpatnam Mandal, Telangana 501510, India

A R T I C L E I N F O A B S T R A C T

Keywords: Mitochondrion emerged as an important therapeutic target for anti-cancer strategy due to its involvement in
Mitochondria cancer progression and development. However, progress of novel small molecules for selective targeting of
Indomethacin mitochondria in cancer cells remained a major challenge. To address this, herein, through a concise synthetic
Ibuprofen
strategy, we have synthesized a small molecule library of indomethacin and ibuprofen (non-steroidal anti-
Cancer
inflammatory drugs, NSAIDs) derivatives having triarylphosphonium moiety for mitochondria localization.
Two of the library members were identified to induce mitochondrial damage through outer membrane per­
meabilization (MOMP) followed by generation of reactive oxygen species (ROS) leading to the remarkable MCF7
breast cancer cell death through apoptosis. These novel mitochondria targeted NSAID derivatives could open a
new direction in understanding mitochondrial biology towards anti-cancer therapeutics in future.

1. Introduction small molecule based mitochondrial impairment induced generation of

In last couple of decades, mitochondrion (powerhouse of the cells)
emerged as an interesting organelle due to its contribution in numerous
biological phenomena including bioenergetics, metabolism, biosyn­
thesis, signalling and programmed cell death.1–4 Consequently, mito­
chondrial functions have been highjacked by cancer cells for cancer
progression and development.5–9 As a result, small molecule anticancer
drugs are detoured in mitochondria for better therapeutic efficacy and
overcoming drug resistance.10–14.
In this context, clinically approved non-steroidal anti-inflammatory
drugs (NSAIDs) showed promise in anti-cancer therapy in different types
of cancer.15–20 Moreover, growing evidence showed that NSAIDs can
perturb mitochondria in different types of cells.21–25 However, selective
routing of NSAIDs into the mitochondria of cancer cells to improve their
therapeutic efficacy remained elusive and unexplored until now.
To address this, herein, we have synthesized non-steroidal anti-in­
flammatory drugs (NSAIDs), indomethacin V and ibuprofen derivatives
(8 compounds) having triarylphosphonium moieties for mitochondria
targeting. Two of the small molecule derivatives demonstrated
remarkable breast cancer cell killing by triggering mitochondrial outer
membrane permeabilization (MOMP) followed by mitochondrial
morphology damage compared to indomethacin and ibuprofen. This
reactive oxygen species (ROS) leading to the programmed cell death
(apoptosis). To the best of our knowledge, this is the first example of
mitochondria targeted NSAID derivatives showing highly improved
anti-cancer efficacy by impairing mitochondria.
2. Results and discussion
2.1. Synthesis of the Mito-NSAID library
The synthetic scheme of the mitochondria targeted NSAID de­
rivatives is outlined in Fig. 1a. In short, parallelly we treated indo­
methacin V (1) and ibuprofen (6) with 5-bromopentanol (2) in presence
of EDC and catalytic amount of DMAP for 24 h to obtain indomethacin
V-bromopentanol ester (3) and ibuprofen-bromopentanol ester (7) in
87% and 84% yield respectively. Both compound 3 and 7 were further
treated with different commercially available triarylphosphines (4a-4d)
in presence of sodium iodide in refluxing condition in acetonitrile for 24
h to afford indomethacin V-triarylphosphonium derivatives (5a-5d) and
ibuprofen-triarylphosphonium derivatives (8a-8d) in 74–87% yields.
The intermediates and the final products were characterized by 1H, 13C,
19
F, 31P NMR and HR-MS spectroscopy (Fig. S1-S40). We hypothesize
that these mito-NSAID derivatives will home into mitochondrial of the

* Corresponding author.
E-mail address: Sudipta.basu@iitgn.ac.in (S. Basu).

https://doi.org/10.1016/j.bmc.2022.116759
Received 29 January 2022; Received in revised form 5 April 2022; Accepted 16 April 2022
Available online 20 April 2022
0968-0896/© 2022 Elsevier Ltd. All rights reserved.
A. Bajpai et al. Bioorganic & Medicinal Chemistry 64 (2022) 116759

Fig. 1. (a) Synthetic scheme of Mito-NSAID library. (b) Schematic representation of the mitochondrial damage by compound 5a and 8a.

Fig. 2. Dose dependent viability of MCF7 cells in presence of Mito-NSAID library at 24 h post-incubation by MTT assay.

cancer cells and impair them for improved therapeutic efficacy (Fig. 1b).
2.2. Cytotoxicity of the mito-NSAID derivatives
To evaluate the efficacy of the mito-NSAID derivatives in cancer
cells, we incubated the MCF7 breast cancer cells with compounds 5a-5d
and 8a-8d in a dose dependent manner for 24 h followed by the quan­
tification of cell viability by MTT assay. We used indomethacin V and
ibuprofen as controls. Compound 5a-5d demonstrated highly improved
MCF7 cell killing ability compared to indomethacin V with compound
5a having the lowest IC50 = 1 μM (Fig. 2a, Table S1). On the other hand,
compound 8a-8c also showed much better cell killing ability compared

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A. Bajpai et al. Bioorganic & Medicinal Chemistry 64 (2022) 116759

Fig. 3. Confocal microscopy images of MCF7 cells treated with compound 5a, 8a, indomethacin V and ibuprofen followed by JC1 staining to observe mitochondrial
outer membrane permeabilization (MOMP). Scale bar = 10 μm.

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A. Bajpai et al.
Fig. 4. Confocal microscopy images of MCF7 cells after treatment with indomethacin V, ibuprofen, compound 5a and compound 8a for 24 h followed by staining
with MitoTracker Red dye to visualize the mitochondrial morphology. Scale bar = 10 μm.
to ibuprofen and compound 8d, except compound 8a showing lowest
IC50 = 10 μM (Fig. 2b, Table S2). It is interesting to observe that the
electron withdrawing p-fluoro-phenyl-phosphonium derivatives (5d and
8d) are least effective in cell killing. On the other hand, triphenylphos­
phonium derivatives (5a and 8a) showed highest effectivity with mod­
erate activity for the electron donating p-methyl and p-methoxy-phenyl-
phosphonium derivatives (5b, 5c, 8b and 8d). To evaluate the effect of
the mito-NSAIDs on non-cancerous cells, we incubated RPE-1 human
retinal epithelial cells with the library members in a dose dependent
manner for 24 h and performed MTT assay to assess cell viability. It was
observed that all the library members showed high IC50 values with less
cell killing (Fig. S41, Table S3 and Table S4). We have chosen compound
5a and 8a for further biological assays as they are the most potent
compounds in their respective series. We also evaluated the purity of
compound 5a and 8a through high performance liquid chromatography
(HPLC), which showed that 5a and 8a were 94.0 and 97.9 % pure
respectively (Fig. S42 and S43). These MTT assays clearly demonstrated
that compound 5a and 8a induced increased cancer cell killing
compared to non-cancerous cells.
2.3. Mitochondrial outer membrane permeabilization (MOMP)
We hypothesized that the mito-NSAIDs would show improved cancer
cell killing efficacy by impairing the mitochondria. To validate our hy­
pothesis, we assessed the mitochondrial membrane depolarization in
presence of mito-NSAIDs by JC1 assay.26 JC1 remains in monomeric and
aggregated states showing green and red fluorescence emission at λmax
= 525 nm and 590 nm respectively. Inside the cells having healthy
mitochondria, JC1 shows both green fluorescence signals (monomer) in
cytosol and red fluorescence signals (aggregated) in mitochondria. We
treated MCF7 cells with compound 5a and 8a for 24 h followed by in­
cubation with JC1 dye. We used indomethacin V and ibuprofen as
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Bioorganic & Medicinal Chemistry 64 (2022) 116759
controls. The cells were visualized by confocal microscopy. The non-
treated control cells showed both green and red fluorescence signals in
nearly equal intensity (J monomer: J-aggregate = 1.5) leading to the
generation of the merged yellow fluorescence signals, which clearly
confirmed the presence of healthy mitochondria (Fig. 3). Interestingly,
both indomethacin V and ibuprofen showed slight increase in green
fluorescence signals compared to the red fluorescence signals (J-mono­
mer: J-aggregate = 1.9 and 1.7 respectively) which indicated that both
the NSAIDs induced marginal mitochondrial depolarization (Fig. 3).
However, confocal microscopy images of the MCF7 cells treated with
compound 5a and 8a demonstrated remarkably high intensity of green
fluorescence signals compared to the red fluorescence signals (J-mono­
mer: J-aggregate = 2.7 and 2 respectively) (Fig. 3). This significant in­
crease in green fluorescence signals compared to the red fluorescence
signals clearly confirmed that compound 5a and 8a induced mitochon­
drial depolarization leading to the sequestration of the aggregated red
fluorescent JC1 from mitochondria to the cytosol triggering the forma­
tion of monomeric green fluorescent JC1 dye.
To further validate the mitochondrial impairment, we visualized the
morphology of the mitochondria in presence of mito-NSAIDs through
confocal microscopy. We treated MCF7 cells with compound 5a and 8a
for 24 h followed by staining the mitochondria by MitoTracker Red dye.
We also treated the MCF7 cells with indomethacin V and ibuprofen as
controls. The confocal images of the non-treated control cells demon­
strated elongated thread like mitochondrial morphology (Fig. 4). Like­
wise, the mitochondrial morphology was found to be elongated and
threadlike even after treatment with indomethacin V and ibuprofen. On
the other hand, the typical filamentous mitochondrial morphology was
completely damaged in the MCF7 cells by the treatment with compound
5a and 8a leading to the development of fragmented punctate structures,
confirming the mitochondrial morphology impairment.
To further confirm the mitochondrial damage, we treated MCF7 cells
A. Bajpai et al.
Fig. 5. Confocal microscopy images of MCF7 cells after treatment with indomethacin V, ibuprofen, compound 5a and compound 8a for 24 h followed by staining
with TMRM dye to visualize the mitochondrial damage. Scale bar = 10 μm.
with compound 5a and 8a for 24 h and incubated the cells with tetra­
methylrhodamine methyl ester (TMRM) dye followed by visualization
through confocal microscopy. We also treated MCF7 cells with indo­
methacin V and ibuprofen as controls. Red fluorescent TMRM dye stains
sub-cellular undamaged mitochondria. However, after mitochondrial
damage, TMRM dye is effluxed from the cells. The confocal microscopy
images of the non-treated control cells showed high intensity of the red
fluorescent TMRM dye inside the cells confirming the undamaged
mitochondria (Fig. 5). Interestingly, indomethacin V and ibuprofen also
showed significant amount of red fluorescence signal, which indicated
marginal mitochondrial damage. However, after treatment with com­
pound 5a and 8a, the sub-cellular red fluorescent intensity in MCF7 cells
remarkably reduced which evidently confirmed the presence of
damaged mitochondria. These JC1, morphology and TMRM assays
clearly confirmed that compound 5a and 8a induced mitochondrial
damage in MCF7 cells leading to the improved cell death.
2.4. Generation of reactive oxygen species (ROS)
Impairment of mitochondria in cancer cells would trigger the
increased generation of reactive oxygen species (ROS).27 Superoxide
species is one of the ROS generated after mitochondrial damage inside
the cells. To assess the generation of superoxide, we treated MCF7 cells
with compound 5a and 8a for 24 h, followed by staining the cells with
MitoSox dye, which generates red fluorescence signal in presence of sub-
cellular superoxides. The cells were visualized with confocal micro­
scopy. The control cells hardly showed any red fluorescence signals
confirming the absence of ROS. Interestingly, ibuprofen and indo­
methacin V also showed negligible increase in red fluorescence signals
(Fig. 6). However, compound 5a and 8a treated cells demonstrated
remarkable increase in red fluorescence signals compared to the control
cells (Fig. 6).
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Bioorganic & Medicinal Chemistry 64 (2022) 116759
To further evaluate the global increase in ROS after mitochondrial
damage, we treated the MCF7 cells with compound 5a and 8a for 24 h
followed by staining the cells with H2DCFDA dye which reacts with the
sub-cellular ROS to produce green fluorescent DCF dye. The cells were
visualized under confocal microscopy. As controls, we also treated the
cells with indomethacin V and ibuprofen for 24 h. The confocal images
exhibited that the non-treated control cells produced weak green signals
due to the presence of endogenous ROS (Fig. 7). Furthermore, indo­
methacin V and ibuprofen treated cells also showed considerable
amount of green fluorescence DCF generation (2.4 folds and 1.3 folds
compared to the non-treated control cells respectively) due to the for­
mation of moderate amount of ROS. However, treatment with com­
pound 5a and 8a produced remarkably increased green fluorescence
signals (2.4 folds and 3.2 folds compared to the control cells) confirming
the highly increased production of ROS. These H2DCFDA and MitoSox
assay confirmed the generation of ROS in MCF7 cells after mito-NSAID
mediated mitochondrial damage.
2.5. Induction of apoptosis
Mito-NSAID-based mitochondrial damage would lead to the pro­
grammed cell death (apoptosis) in cancer cells. To evaluate the
apoptosis induction, MCF7 cells were treated with compound 5a and 8a
for 24 h followed by incubation with the red fluorescent propidium io­
dide (PI) dye which binds with the sub-cellular DNA in apoptotic and
necrotic stage, keeping the healthy cell DNA intact. The confocal mi­
croscopy images of the non-treated cells demonstrated no red fluores­
cence signal indicating no induction of apoptosis (Fig. 8a). Interestingly,
indomethacin V and ibuprofen treated cells also showed marginal red
fluorescence signals representing negligible induction of apoptosis.
However, after treatment with compound 5a the MCF7 cells displayed
remarkable increase in the red fluorescence signals confirming the
A. Bajpai et al. Bioorganic & Medicinal Chemistry 64 (2022) 116759

Fig. 6. Confocal microscopy images of MCF7 cells after treatment with compound 5a, 8a, indomethacin V and ibuprofen for 24 h followed by staining with MitoSox
dye to visualize the generation of superoxides. Scale bar = 10 μm.

Fig. 7. Confocal microscopy images of MCF7 cells after treatment with indomethacin V, ibuprofen, compound 5a and compound 8a for 24 h followed by staining
with H2DCFDA dye to visualize the generation of ROS. Scale bar = 10 μm.

6
A. Bajpai et al.
Fig. 8. (a) Confocal microscopy images of MCF7 cells treated with compound 5a, 8a, indomethacin and ibuprofen for 24 h followed by the staining with propidium
iodide (PI) to observe the apoptotic and necrotic cells. Scale bar = 10 μm. (b) Western blot image to visualize the expression of anti-apoptotic Bcl-2 and Caspase-3 in
MCF7 cells.
triggering of the apoptosis (Fig. 8a). On the other hand, compound 8a
also showed considerable amount of red fluorescence signals to
demonstrate substantial apoptosis induction.
To further understand the apoptosis mechanism, we also evaluated
the expression of anti-apoptotic protein Bcl-2 and executioner caspase-3
as apoptosis markers by Western blot analysis. We treated MCF7 cells
with 5a and 8a for 24 h and the whole cell proteins were subjected to gel
electrophoresis. The Western blot images (Fig. 8b) showed that the
expression of Bcl-2 and caspase-3 were reduced as expected compared to
the control cells as well as indomethacin V treated cells. These confocal
images and gel electrophoresis evidently confirmed the significant
initiation of programmed cell death by the mito-NSAIDs compared to
their parent drugs, which is also in accordance with the cell viability
assays.
3. Conclusion
In conclusion, we have developed a concise synthetic strategy to
produce a small library of indomethacin V and ibuprofen derivatives
containing triarylphosphonium moieties. Two small molecules were
identified by the cell viability and confocal microscopy studies to induce
mitochondrial impairments followed by the generation of the reactive
oxygen species (ROS). This mito-NSAID derivative mediated mito­
chondrial damage lead to the induction of apoptosis and remarkable cell
killing ability in MCF7 breast cancer cells compared to the parent drugs.
We envision, these novel mitochondria targeted NSAID molecules can
open a direction to understand mitochondrial chemical biology as well
as next-generation cancer therapy.
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Bioorganic & Medicinal Chemistry 64 (2022) 116759
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.
Acknowledgement
S.B. sincerely thanks IIT Gandhinagar internal funding, Gujarat
Council on Science and Technology (GUJCOST/STI/R&D/2020-21/
1302) and Science and Engineering Research Board (CRG/2020/
001127) for financial support. A.B. sincerely thanks CSIR-UGC for
doctoral fellowship. We thank Dr. Bhaskar Datta, IIT Gandhinagar for
providing the lab facility for biological experiments. We also thank Dr.
Sharad Gupta, IIT Gandhinagar for providing HPLC analysis.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2022.116759.
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