The overall objective of the project was to synthesis of coumarin based NIR probes (MGRK) with model trigger & leaving group and evaluate its photochemical properties, release kinetics in buffer. Upon validation, the selected MGRK probe will be conjugated with cancer specific trigger, which is Q3PA and a drug (Topotecan or monomethyl auristatin E (MMAE) or ROS generator JCHD) with the PSMA targeting ligand, DUPA. Finally, the obtained theranostic agent (MGRK) will be thoroughly profiled in vitro followed by in vivo assays. As proposed, NIR probe (10, without benzyl alcohol group at ortho position) was synthesized successfully and found to be more stable and soluble in buffer. Next in ordered to validate the stimuli responsive nature, as a proof of concept, NIR probe was protected with an allyl carbonate group and made carbon monoxide (CO) probe. It was known that, in the presence of palladium and CO, allyl carbonate group gets cleaved (Tsuji–Trost-type reaction) and produce free probe with an increase in the fluorescence signal to detect CO. As expected, the probe was found to be stable in buffer and increase in the fluorescence signal was seen only in the presence of Pd+CO in buffer pH 7.4 at 37ºC. Although, NIR-CO probe emission falls in the NIR region (Ex: 670 nm; Em: 710 nm; stokes shift: 40 nm; 633 nm laser beams can be used for confocal imaging) but unstable carbonate linker made it not suitable for in vivo imaging. Recently there are few allyl ethers-based CO probes have been reported and allyl ether linkage was found to be more stable than allyl carbonate in cellular conditions. Next, synthesis of allyl ether based NIR CO probe was optimized, which can be extend further to image CO in vivo. After optimizing all the conditions for NIR probe without leaving group, next try to introduce leaving group at ortho position of NIR Probe. Attempted different reaction conditions but unable get the NIR probe with leaving group and study further. Parallelly, synthesis of NIR probe (7) was started. Before making it, as a proof of concept, probe ((E)-2-(3-(4-hydroxy-3-(hydroxymethyl)styryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile) was synthesized and its fluorescence emission also falls in the NIR region (Ex: 419 nm; Em: 633 nm; stock shift = 244 nm) and this novel theranostic tool can be extended further for cancer targeted drug delivery and imaging applications. After optimizing all the reaction conditions, next coumarin based NIR Probe (7) was started. 2,4-dihydroxy benzaldehyde was reacted with diethyl glutaconate in the presence of piperidine to obtain coumarin derivative followed by formylation reaction to introduce aldehyde group at ortho position of coumarin. Corresponding coumarin aldehyde was reacted with propargyl bromide, a model protecting group and followed by NaBH4 reduction to give corresponding coumarin benzyl alcohol derivative. The product was purified and confirmed, however the yield of formylation and propargyl protection reactions were very low. Different reaction conditions were attempted, low reactivity, solubility, low yield made it unfeasible to obtain the NIR probes with model leaving group.
Next, compound Q3PA, 4, 5 and ROS generator JCHD were synthesized successfully. Before attaching of JCHD to NIR probe, test reaction was performed, where JCHD OH group was protected with an esterase sensitive protecting group and made POM-JCHD. POM-JCHD was found to be an efficient ROS generator in buffer as well as in cells. Cytotoxicity of POM-JCHD was evaluated previously, found to be more potent against different cancer cells (IC50 ~4-5 μM, 4h). Computational studies were performed in collaboration, IDO1 enzyme was found to be the top predicted target for JCHD, which can be explored further. Also, these two ROS generating molecules (JCHD, POM-JCHD) were tested against SARS-CoV-2 virus and was found to be more effective (IC50 0.31 μM), which is under progress, this can be extended further in the context of antiviral (COVID-19) drugs. Currently manuscript describing these results is under progress has been done by the researcher and when published he will appear as a co-corresponding author, which is a demonstration of his independence during the development of the research.
