Hydroxyl groups of GCV (Table 1). These benefits recommend that the conjugation of lipid chain to GCV drastically enhances lipophilicity which may perhaps help in enhancing GCV bioavailability along with slow release of GCV. Mass spectrometry All the lengthy chain lipid GCV prodrugs and GCV have been subjected to molecular weight analysis in constructive mode with mass spectroscopy. Evaluation revealed GCV and all its derivatives as proton adduct [M+1]+except C13-mono-(O-acyl) derivative which wasNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptAdv Ophthalmol Vis Syst. Author manuscript; offered in PMC 2014 October 30.Cholkar et al.Pageidentified as a sodium adduct [M+Na]+ (Table 1). Figure 1 shows the broad mass spectra for C13-mono-(O-acyl) GCV derivative (100 ng mL-1) using a mass of 452.four Dalton and an intensity of 7.4 ?106 cps.Triethyl(ethynyl)silane custom synthesis All theoretically calculated masses for lengthy chain lipid derivatives were in agreement with mass spectroscopy results (Table 1), indicating that all compounds were steady. Nuclear magnetic resonance The proton NMR spectral data and assignments for mono- and di-(O-acyl) derivatives have been calculated in comparison to the parent compound, GCV (Table two) [23]. Figures 2a and 2b shows the proton NMR for GCV and GCV-C5 conjugated lipid prodrugs. As might be seen within the spectra for GCV prodrug (Figure 2a), we usually do not observe any alkyl resonance peaks downstream i.e. in between 0 ppm and 1.9 ppm. But, GCV-C5 exhibits the proton peaks corresponding to lipid chain conjugated to hydroxyl group of GCV (Figure 2b) which on integration determined the corresponded to proton quantity in the lipid chain. Related proton NMR spectra have been collected for other prodrugs (GCV-C10 and GCV-13). Chemical shifts within the resonance peaks for the lipid derivatives have been evident relative to GCV. Conjugation of aliphatic carbon chain (C5, C10 and C13) deshielded proton NMR chemical shifts of carbon (CH2OCO) to which these groups are conjugated plus the -situated (OCH) protons.Formula of C5 Lenalidomide All other resonance peaks remained very same to that of GCV.PMID:35116795 Spectra had been similar for mono- and di-(O-acyl) derivatives of GCV but the total number of protons calculated was double in number for di-(O-acyl) GCV lipid derivatives. 13C NMR information for all ready GCV lipid conjugates is presented in Table two. The ester conjugated carbon (RCOO) resonance peak was evident in all the lipid prodrugs except the parent GCV molecule (Table two). These final results indicate that the extended carbon chain is conjugated to hydroxyl group of GCV. Cell culture To evaluate the cytotoxic effects of lengthy chain lipid GCV prodrugs MTS assay have been performed on ARPE-19 cells for 24 h. Percent viable cells had been compared with of damaging manage (culture medium) (Figure three). DMSO (10 ) served as optimistic manage and lowered the % cell viable to 35 . ARPE-19 cell viability immediately after exposure to our novel long chain mono- and di-(O-acyl) GCV prodrugs was comparable to that of damaging control. The result indicates that exposure of prodrugs to ARPE-19 cells for prolonged period did not induce any toxicity and the compounds were well-tolerated. Benefits from this study clearly recommend that our novel long chain lipid prodrugs do not cause any cytotoxicity and are protected for ocular application.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptConclusionsIn summary, we have successfully synthesized lengthy chain lipid GCV prodrugs (mono- and di-(O-acyl)). These lipid prodrugs are effectively eluted and separat.