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. 2021 Jul 15;12(9):1604–1611. doi: 10.1039/d1md00095k

Small molecule-mediated induction of endoplasmic reticulum stress in cancer cells†

Shalini Pandey 1,2, Virender Kumar Sharma 3, Ankur Biswas 1, Mayurika Lahiri 3, Sudipta Basu 2,✉
PMCID: PMC8459384  PMID: 34671742

Abstract

The endoplasmic reticulum (ER) is one of the crucial sub-cellular organelles controlling myriads of functions including protein biosynthesis, folding, misfolding and unfolding. As a result, dysregulation of these pathways in the ER is implicated in cancer development and progression. Subsequently, targeting the ER in cancer cells emerged as an interesting unorthodox strategy in next-generation anticancer therapy. However, development of small molecules to selectively target the ER for cancer therapy remained elusive and unexplored. To address this, herein, we have developed a novel small molecule library of sulfonylhydrazide-hydrazones through a short and concise chemical synthetic strategy. We identified a fluorescent small molecule that localized into the endoplasmic reticulum (ER) of HeLa cells, induced ER stress followed by triggering autophagy which was subsequently inhibited by chloroquine (autophagy inhibitor) to initiate apoptosis. This small molecule showed remarkable cancer cell killing efficacy in different cancer cells as mono and combination therapy with chloroquine, thus opening a new direction to illuminate ER-biology towards the development of novel anticancer therapeutics.

Sulfononylhydrazide-hydrazone based small molecules as ER stress modulators for anti-cancer therapygraphic file with name d1md00095k-ga.jpg

Introduction

The endoplasmic reticulum (ER) is a vital organelle that is known to govern the synthesis, folding and processing of over a third of all the cellular proteins.1–4 Several ER associated chaperones assist in ensuring proper folding and modification of these proteins before they traffic out of the ER.5–7 Despite the robustness of this protein folding machinery, the success rate for optimum protein folding is low owing to the wide range of cellular disturbances that disrupt its efficiency.8 As a result, several unfolded and misfolded proteins start accumulating inside the ER lumen leading to a state known as ER stress.9,10 In response to this burden of unfolded proteins, a cytoprotective program known as the unfolded protein response (UPR) is launched by the cell.11–13 Together, the three mechanistically different branches of the UPR regulate the expression of numerous genes that resolve this ER stress and maintain homeostasis or induce apoptotic signals in case the stress remains unmitigated.14 Very recently, this deregulated ER homeostasis has been implicated in various pathological states and particularly with cancer.15,16 The baseline activity level of the ER stress response system is elevated in cancer cells as compared to normal cells. Thus, the ER stress signalling is involved in tumorigenesis and development leading it to be the new Achilles heel in the development of cancer therapeutics.17–19

Over the last couple of decades, small molecules have emerged as important tools to illuminate myriads of biological phenomena in cancer cells leading to the development of novel anti-cancer therapies.20,21 In this context, few small molecules have been explored that target various aspects of the unfolded protein response and ER physiology.22–30 Molecules like salubrinal, GSK2606414, and sunitinib are well reported to manipulate one or more arms of ER stress pathways.31 However, due to the scarcity of the targeting moieties, the development of ER targeted therapeutics is still in its infancy and therefore the presence of such ER stress inducers and UPR inhibitors is limited. Hence, there is a serious need to develop novel small molecules that can serve as ER stress modulators. Very recently, a few molecules were demonstrated to accumulate inside the ER in cancer cells to induce ER stress or as a tool to understand ER physiology.32–35 For instance, Rocchi et al. reported the development of HA15 that serves as an ER stress inducer and overcomes BRAF-inhibitor resistance in melanoma cells.36 On the other hand, Xiao et al. reported an ER-H2O2 probe to understand the generation of hydrogen peroxide at the ER during apoptosis.37 A remarkable highlight in these small molecules is the presence of sulfonamide moieties. Furthermore, it was reported that the sulphonamide moiety facilitates the internalization of small molecules in the intracellular ER owing to the presence of sulphonamide receptors on the ER surface.38,39 Moreover, sulphonamide as well as hydrazide-hydrazone containing small molecules demonstrated diverse biological activities making them privileged structures for natural and non-natural products (Scheme 1a).40–47

Scheme 1. (a) Structures of the biologically active sulphonamide natural products and sulfonylhydrazide-hydrazone focused library. (b) Synthetic scheme of the sulfonylhydrazide-hydrazone library. (c) Schematic representation of the mechanism of action of compound 1 in cancer cells to induce ER stress.

Scheme 1

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Inspired by the scarcity of novel ER stress modulators, we develop a focussed library of sulfonohydrazide-hydrazone based small molecules (4′, Scheme 1a) to identify novel ER stress inducers in cancer cells. To address this, herein, we report a short and concise synthesis of a library of sulfonohydrazide-hydrazone based small molecules comprising 66 compounds (Scheme 1b). Upon screening these compounds in the HeLa cell line, we identified four molecules as potential ER stress inducers. One of the four lead molecules was found to be fluorescent in nature and localized inside the subcellular ER to induce ER stress through CHOP and IRE-1α pathways (Scheme 1c). Interestingly, the ER stress induced by the lead molecule triggered autophagy followed by apoptosis in HeLa cells. Further inhibition of autophagy by chloroquine (CQ), a classic inhibitor that decreases the autophagic flux by inhibiting the autophagosome–lysosome fusion,48 resulted in improved cell killing in lung and breast cancer cells including drug resistant-breast cancer cells.

Results and discussion

Synthesis of the small molecule library

The synthesis of sulfonohydrazide-hydrazone molecules is shown in Scheme 1b. Briefly, commercially available substituted aromatic sulfonyl chlorides (1′) were reacted with hydrazine monohydrate at 0 °C to obtain substituted aromatic sulfonyl hydrazides (2′) in 60–70% yield (Fig. S1, ESI†). Further, these aromatic sulfonyl hydrazides were reacted with different aromatic aldehydes (3′) in the presence of p-toluenesulfonic acid as a catalyst to afford 66 different sulfonylhydrazide-hydrazones (4′) in 50–80% yield (Fig. S2, ESI†). The final products were characterized by 1H NMR, 13C NMR and HR-MS (Fig. S3–S200, ESI†).

To evaluate their cancer cell killing activity, we first incubated these library members in HeLa cervical cancer cells at a concentration of 30 μM for 24 hours. The cell viability was quantified by the MTT assay. Interestingly, only four compounds (compounds 1, 10, 17 and 55) were found to induce more than 60% HeLa cell killing even at 30 μM concentration (Fig. S201, ESI†). After finding out the potential lead compounds, we evaluated their cancer cell killing ability in a dose dependent manner. HeLa cells were incubated with compounds 1, 10, 17 and 55 for 24 h in different concentrations and the cell viability was evaluated by the MTT assay. It was observed that at 24 h post incubation, compounds 1, 10, 17 and 55 induced HeLa cell killing with IC50 values of 9 μM, 16 μM, 18 μM and 19 μM respectively (Fig. 1). From this cell viability assay, it was evident that compound 1 showed the best cell killing efficacy with the lowest IC50 value. Interestingly, we observed that the electron donating groups (Cl, Br, dansyl) on the benzene ring attached to the sulfonyl moiety increased the cytotoxicity effect, whereas, the electron withdrawing group (NO2) reduced the cytotoxicity. Moreover, we also observed that the 6-bromoindole moiety also increased the cytotoxicity of the compounds.47 However, a detailed structure–activity relationship (SAR) analysis needs to be performed to understand the effect of the functional groups in the sulfonylhydrazide-hydrazone library. Moreover, compound 1 was found to be fluorescent with λmax (emission) at 538 nm (Fig. S202, ESI†). This inherent fluorescence property of compound 1 will aid in its cellular tracking into the endoplasmic reticulum (ER), and hence we chose to carry out further studies using compound 1.

Fig. 1. Cell viability assay of compounds 1, 10, 17 and 55 in HeLa cells at 24 h post-incubation in a dose dependent manner. ***p < 0.0001, the p value is calculated using two-way ANOVA.

Fig. 1

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ER homing

We hypothesized that the presence of the sulphonamide moiety in compound 1 will enable its localization specifically inside the subcellular ER.49,50 To validate this hypothesis, we incubated the HeLa cells with compound 1 (concentration = 5 μM) (green fluorescent) for two different time points of 3 h and 6 h followed by staining the ER with ER Tracker Red. We then visualised the cells using confocal laser scanning microscopy (CLSM). It was observed that compound 1 started accumulating in the ER within 3 h and the accumulation increased at 6 h (Fig. 2 and S203, ESI†) which was observed from the yellow fluorescence resulting from merging of red and green fluorescence. The ER localization of compound 1 was further quantified from the CLSM images through Pearson's coefficients of 0.84 and 0.86 and Mander's coefficients of 0.98 and 0.97 at 3 h and 6 h, respectively (Table S1, ESI†). These confocal images and quantification showed that indeed compound 1 homed into the subcellular ER in HeLa cancer cells over 6 h.

Fig. 2. Confocal microscopy images of HeLa cells at 3 h and 6 h post-incubation with compound 1 (green fluorescence). The cells were counter stained with ER-Tracker Red dye. Scale bar = 10 μm.

Fig. 2

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ER stress

Once internalized inside the ER, compound 1 is expected to induce ER stress in the HeLa cervical cancer cells. Elevated ER stress levels lead to an increment in the expression levels of protooncogene CHOP which is a marker for ER stress induced apoptosis.51,52 To demonstrate the onset of ER stress we evaluated the levels of CHOP in HeLa cells by an immunofluorescence assay. We treated HeLa cells with compound 1 for 24 h. We then incubated the cells with a primary antibody specific to CHOP for 4 h followed by incubation with an Alexa-Fluor 594 (red fluorescence) tagged secondary antibody. The nuclei of the cells were stained with DAPI (blue fluorescent). We then visualized the cells with confocal microscopy. The fluorescence microscopy images showed that the control cells expressed negligible CHOP which was evident from no red fluorescence signals (Fig. 3a). However, the cells treated with compound 1 showed remarkably increased red fluorescence signals indicating that compound 1 increased (1.7 fold) the expression of CHOP protein in HeLa cells. Furthermore, the overlap of red and blue fluorescence to give purple fluorescence indicated the accumulation of CHOP in the nucleus which is a marker of ER stress induction.52 Interestingly, the immunofluorescence staining of CHOP showed a characteristic punctate structure after treatment with compound 1 which is the hallmark of ER stress (Fig. S204, ESI†). We also evaluated the expression of CHOP by gel electrophoresis. We treated HeLa cells with compound 1 for 24 h, followed by lysis of the cells. We separated the sub-cellular proteins to perform western blot analysis. In accordance with the confocal microscopy study, the cells after treatment with compound 1 showed a significant increase (1.6 fold) in the expression of CHOP compared to the non-treated control cells (Fig. 3b and S205a, ESI†). To further estimate the ER stress, we evaluated the expression levels of another ER stress marker IRE1-α.53 We found that the cells treated with compound 1 exhibited an enhanced expression level of IRE1-α as compared to the control cells (1.8 fold) (Fig. 3b and S205b, ESI†). We also evaluated the expression of PERK and BiP as ER stress markers and activation of caspase-12 as an ER stress-mediated apoptosis marker by gel electrophoresis.10,36,54 We found that compound 1 induced enhanced expression of PERK (1.6 fold), BiP (1.6 fold) and caspase-12 (1.4 fold) compared to the non-treated control cells (Fig. 3c and S205c–e, ESI†). These immunofluorescence assays and gel electrophoresis revealed that compound 1 indeed induced ER stress in HeLa cells.

Fig. 3. (a) Confocal images of HeLa cells after treatment with compound 1 for 24 h followed by treatment with a CHOP primary antibody and an Alexa Fluor 594-tagged secondary antibody (red). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (b and c) The western blot images of CHOP, IRE1α, PERK, BiP and caspase-12 proteins as the markers of ER stress in HeLa cells after treatment with compound 1 for 24 h.

Fig. 3

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The ER is the major site of lipid metabolism as several enzymes involved in it are compartmentalized in the ER.55 As a result, excessive ER stress is known to increase lipid accumulation inside the cell.56 We anticipated that HeLa cells treated with compound 1 will also show an enhanced lipid content in them. To validate this hypothesis, we performed an Oil Red O staining experiment.57,58 We treated HeLa cells with compound 1 for 24 h followed by staining the cells with Oil-Red-O dye. Oil Red O is a lipid soluble lysochrome dye which stains the lipid droplets in cells or frozen tissues.59 We also stained the nucleus of the cells with blue fluorescent DAPI. We visualised the Oil-Red-O stained cells by confocal microscopy. The confocal microscopy images showed that the control cells contained traces of lipid molecules as expected (Fig. 4). However, compound 1 treatment remarkably increased the level of the lipids inside the cells, which was clear from the increased red fluorescence signals from the Oil-Red-O dye in Fig. 4.

Fig. 4. The confocal microscopy images of HeLa cells after incubation with compound 1 for 24 h followed by staining with Oil Red O dye (red) and DAPI. Scale bar = 10 μm.

Fig. 4

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To confirm that the fluorescence signal from compound 1 does not interfere with the Oil-Red-O, we further treated the HeLa cells with non-fluorescent compound 10 for 24 h and performed the Oil-Red-O staining. From the confocal imaging it was evident that compound 10 also enhanced the red fluorescence signal significantly compared to the non-treated control cells (Fig. S206, ESI†). This confocal microscopy exhibited that indeed the lipidic content was increased inside the cells after treatment with compound 1 and compound 10 because of elevated ER stress.

Autophagy induction

ER stress induction acts as a potent trigger for autophagy which is primarily a cytoprotective secondary response to the excessive protein build up in the ER.60–62 To investigate if autophagy was launched as a survival mechanism by the HeLa cells in response to treatment with compound 1, we evaluated the cellular expression level of LC3B, a key autophagy marker, by an immunofluorescence assay.63 We treated HeLa cells with compound 1 for 24 h followed by fixing the cells with paraformaldehyde. These fixed cells were then permeabilized with a buffer containing 0.1% Tween-20 followed by incubation with a primary antibody specific to LC3B for 4 h. The cells were then incubated with a secondary antibody tagged with Alexa-Fluor-488 (green fluorescent). The nuclei of the cells were stained with blue fluorescent DAPI. The cells were visualised by confocal microscopy. The green fluorescence signals were hardly seen in the control cells (Fig. 5a). However, the cells treated with compound 1 exhibited highly enhanced green fluorescence (5 fold) compared to the control cells. More interestingly, we observed the formation of autophagosomes (green puncta in the images) which is regarded as the hallmark of autophagy.64 To confirm that the fluorescence signal of compound 1 does not interfere with the immunofluorescence assay, we treated the HeLa cells with non-fluorescent compound 10 for 24 h. The cells were then fixed, permeabilized and stained with a primary antibody specific to LC3B and a secondary antibody tagged with Alexa-Fluor-488. The control cells were treated in a similar manner without compound 10. The confocal imaging clearly showed that compound 10 also increased the expression of LC3B remarkably in the HeLa cells compared to the control cells (Fig. S207, ESI†).

Fig. 5. (a and b) Confocal images of HeLa cells after treatment with compound 1 for 24 h followed by LC3B and Beclin specific primary antibodies followed by an Alexa Fluor 488 tagged secondary antibody (green) and an Alexa Fluor 594 tagged secondary antibody (red), respectively. Scale bar = 10 μm. (c) Western blot images of the LC3-I, LC3-II and Beclin protein expressions in HeLa cells after treatment with compound 1 for 24 h.

Fig. 5

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We also evaluated the expression level of Beclin, another autophagy marker, by immunofluorescence.65 HeLa cells were treated with compound 1 for 24 h followed by incubation with a Beclin specific primary antibody. The cells were then incubated with a secondary antibody tagged with Alexa-Fluor-594 (red fluorescent). The nuclei of the cells were stained with DAPI. We visualised the cells by confocal microscopy and found that an increased red fluorescence signal was observed in the compound 1 treated cells compared to that in the non-treated control cells (8 fold) (Fig. 5b). The enhanced expression levels of LC3B and Beclin in the treated cells as evaluated by the immunofluorescence assay evidently demonstrated that autophagy was induced upon elevation of ER stress in the HeLa cells.

We further validated the expression levels of LC3B and Beclin by western blot analysis. We treated HeLa cells with compound 1 for 24 h followed by cell lysis. The cellular proteins were then subjected to gel electrophoresis. The western blot analysis revealed that treatment with compound 1 resulted in increased levels of LC3-I (2.6 fold), LC3-II (10 fold) and Beclin (2 fold) indicating the onset of autophagy (Fig. 5c and S208a–c, ESI†). Moreover, the high LC3-I/LC3-II ratio (3.8 fold) clearly confirmed the induction of autophagy by compound 1. The immunofluorescence assay and western blot analysis thus confirmed that autophagy was triggered upon ER stress induction on treatment with compound 1 which is in agreement with our previous reports as well as other reports.36,49,50

Induction of apoptosis

Induction of ER stress is expected to induce apoptosis in HeLa cells. To estimate the apoptosis induced by compound 1 in HeLa cells, we performed a flow cytometry assay. We treated HeLa cells with compound 1 for 24 h followed by staining the apoptotic and necrotic cells with Annexin V-FITC and propidium iodide (PI), respectively. The cells were then analysed by flow cytometry. We found that treatment with compound 1 resulted in 64.6% cells in the early apoptotic stage and 17.8% cells in the late apoptotic stage compared to 1.26% and 0.08% in the control cells, respectively (Fig. 6a). We anticipate that compound 1 was not as effective to induce apoptosis due to induction of autophagy as a survival mechanism. We thus hypothesized that the concomitant inhibition of autophagy will improve the apoptotic outcome in HeLa cells. To validate this hypothesis, we cotreated HeLa cells with compound 1 and 50 μM chloroquine, an autophagy inhibitor, for 24 h and performed a flow cytometry assay.66 We observed that autophagy inhibition indeed improved the apoptotic outcome with 28.6% and 62% cells in the early and late apoptotic stages, respectively (Fig. 6a). The flow cytometry data thus confirmed that compound 1 induced apoptosis in HeLa cells which further improved upon inhibition of autophagy with chloroquine.

Fig. 6. (a) Flow cytometry analysis of compound 1 and CQ in HeLa cells to evaluate apoptosis. (b) Dose dependent cell viability assay of compound 1 and CQ in HeLa, MCF7, A549 and MDA-MB-231 cells at 24 h post incubation. ***p < 0.001 and **p < 0.01, the p value is calculated using two-way ANOVA.

Fig. 6

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To further investigate the effect of autophagy inhibition on the cell killing efficacy of compound 1, we performed a cell viability assay. We cotreated HeLa cells with compound 1 in a dose dependent manner along with 50 μM chloroquine for 24 h. The cell viability was measured by the MTT assay. We found that inhibiting autophagy improved the IC50 value of compound 1 from 9 μM to 4 μM in HeLa cells (Fig. 6b). We also evaluated the cell killing efficacy of compound 1 on three different cancer cell lines by the MTT assay. We treated MCF-7 (human breast cancer), A549 (human lung carcinoma) and MDA-MB-231 (drug resistant triple negative breast cancer) with compound 1 in a dose dependent manner for 24 h. We found that compound 1 exhibited IC50 values of 15 μM, 10 μM and 17 μM in MCF-7, A549 and MDA-MB-231, respectively (Fig. 6b). Furthermore, inhibition of autophagy by treatment with chloroquine resulted in enhanced cell killing and improved IC50 values of 10 μM, 5 μM and 13 μM in MCF-7, A549 and MDA-MB-231, respectively (Fig. 6b). Compound 1 thus efficiently induced cell killing in different cancer cell lines which further improved upon autophagy inhibition.

We also evaluated the inhibition of autophagy along with induction of ER stress with the other lead molecules (compounds 10, 17 and 55) on HeLa cells. We treated HeLa cells with CQ in combination with compounds 10, 17 and 55 in a dose dependent manner for 24 h and the cell viability was measured by the MTT assay. We observed that the combination treatment of CQ with compounds 10, 17 and 55 induced much improved HeLa cell killing with IC50 = 4.4 μM, 5.5 μM and 16.1 μM respectively (Fig. S209, ESI†).

To be successful in future cancer therapy, compound 1 should selectively induce cancer cell death without collateral damage to the non-cancerous healthy cells. To evaluate the effect of compound 1 on healthy cells, we incubated HEK293 human embryonic kidney cells with compound 1 in a dose dependent manner for 24 h and quantified the cell viability by the MTT assay. To our surprise, we found that compound 1 showed only 52.8% cell viability even in 30 μM concentration with IC50 = 31.7 μM, which was quite high compared to the IC50 value in different cancer cells (Fig. S210, ESI†). We further calculated the selectivity index (SI) of compound 1 in HeLa, MCF7, A549 and MDA-MB-231 cells to understand its effect on cancer cells compared to the healthy cells.67 It was found that the SI of compound 1 was 3.52, 2.11, 3.17 and 1.86 in HeLa, MCF7, A549 and MDA-MB-231 cells, respectively, which indicated that compound 1 is more selective for HeLa and A549 cells, compared to the other tested cancer cells.

Finally, as a proof of concept, to confirm that ER stress induction resulted in apoptosis, we treated HeLa cells with compound 1 and 4-phenyl butyric acid (4-PBA), a chemical chaperone that alleviates both toxicity and proteomic alterations induced by an ER stress inducer, by aiding in protein folding in the ER.68–70 Although 4-PBA is also known as a potent HDAC inhibitor, it was shown to be non-toxic even at a higher concentration in HeLa cells.71 We co-treated HeLa cells with compound 1 along with 15 μM 4-PBA for 24 h and then performed the cell viability assay. We found that cotreatment with 4-PBA resulted in reduced cell killing (IC50 = 28 μM) in HeLa cells as compared to the treatment with compound 1 only (Fig. S211, ESI†). From these cell viability assays, it was evident that compound 1 triggered apoptosis after induction of autophagy. Furthermore, co-treatment of compound 1 and an autophagy inhibitor would induce improved cancer cell killing without inducing significant damage to the non-cancerous cells.

Conclusions

In conclusion, we synthesised a library comprising 66 small molecules based on sulfonohydrazide-hydrazones through short and concise synthetic steps. Upon screening, we identified four molecules as potential ER stress inducers. Further studies with the dansyl based sulfonohydrazide-hydrazone (compound 1) revealed its accumulation in the ER within 3 h in HeLa cervical cancer cells. The identified small molecule efficiently induced ER stress mediated apoptosis and autophagy in HeLa cells. It also induced ER stress associated apoptosis in lung, breast and drug resistant breast cancer cell lines. Finally, the treatment in combination with an autophagy inhibitor, chloroquine, remarkably improved the cell killing efficacy of the small molecule in the cancer cell lines. We envision that these small molecules can be employed in understanding the ER biology and functions in cancer and pave the way for the development of novel ER stress inducers thereby improving the cancer therapeutics.

Experimental section

Materials

All the chemicals were purchased from commercial suppliers unless otherwise noted. MCF-7 cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC) (Salisbury, UK). HeLa, HEK-293, MDA-MB-231 and A549 cells were obtained from the National Centre for Cell Science (NCCS) (Pune, India).

Synthesis of sulfonohydrazides

The sulfonohydrazides were prepared according to a previously reported procedure.72 Briefly, to a solution of sulfonyl chloride (1 equiv.) in THF at −30 °C, hydrazine monohydrate (5 equiv.) was added dropwise. The solution was then allowed to stir for 30 minutes. The progress of the reaction was monitored through TLC and after the reaction was over, ethyl acetate was added into the cold reaction mixture. The reaction mixture was washed multiple times with ice cold 10% brine solution. The organic layer was collected and dried over anhydrous Na2SO4 and the solvent was then evaporated under reduced pressure. The solid obtained was washed with pentane three times and the product was stored at 4 °C for further use.

Synthesis of sulfonohydrazide-hydrazones

To a solution of sulfonohydrazide (1 eq.) in ethanol, aldehyde was added (1 eq.) along with a catalytic amount of para-toluene sulfonic acid and the reaction was allowed to stir at room temperature overnight. The extent of reaction was monitored by TLC. On completion, the solvent ethanol was evaporated, and the residue obtained was dissolved in organic solvents (DCM/ethylacetate). The organic layer was washed with water, collected and evaporated. The residue obtained was then purified using column chromatography. The procedure was utilized to generate a library of 66 small molecules.

Author contributions

SP, VKS and AB performed all the experiments and collected the data. SP, ML and SB wrote the manuscript.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

MD-012-D1MD00095K-s001

Acknowledgments

SB acknowledges IIT Gandhinagar internal funding and DST [SB/NM/NB-1083/2017 (G)] for financial support. SP acknowledges IISER Pune for a doctoral fellowship. We sincerely thank Dr. Nirmalya Ballav from IISER Pune for constructive and helpful scientific discussion and support.

†

Electronic supplementary information (ESI) available: 1H NMR, 13C NMR, and HR-MS spectra, cell viability, fluorescence spectra, western blot quantification, and cell viability assay. See DOI: 10.1039/d1md00095k

Notes and references

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