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Background The endoplasmic reticulum (ER) is an essential organelle that is involved in multiple cellular processes that are required for cell survival and normal cellular functions 1 . ER is particularly important for the maintenance of protein homeostasis 1 . Different stimulus such as hypoxia, glucose depletion, and oxidative stress, may result in ER stress and the subsequent activation of unfolded protein response (UPR) as a defense mechanism 2 . UPR activate several pathways as shown in figure 1 2 . Piperine, a natural amide, is the major component of P. nigrum and biologically acts as an antioxidant, anti- inflammatory agent (Structure shown in figure 2a) 3 . Several studies have demonstrated the protective role of different piperine analogs in a variety of diseases 3 . To our knowledge, the impact of piperine derivatives on ER stress and oxidative stress in kidney cells has not been investigated yet. Synthesis and Characterization of Novel Piperine Derivatives Ayat Hammad, Shankar Munusamy and Ashraf Khalil College of Pharmacy, Qatar University, Doha, Qatar Experimental Methodology Literature search was conducted to ensure the novelty of the project and to develop the methodology. 2 M Ethanolic potassium hydroxide was prepared. Preparation of Piperic Acid (PA) Intermediate 1.0 g of Piperine was dissolved in 100 mL of 2 M ethanolic potassium hydroxide, and refluxed for 25 h. The reaction was monitored by Thin Layer Chromatography (TLC). After 25 h, the ethanolic solution was evaporated. The remaining solid potassium piperate was dissolved in 50 mL of hot water. The suspension was acidified with 6 M HCl (pH <1). Reaction mixture was filtered and the formed precipitate was washed with cold aqueous ethanol solution (1:5 ethanol/water). The product was purified by recrystallization from hot ethanol. A yellow precipitate was obtained. The chemical structure of the obtained product was identified by infrared (IR) spectra in comparison with the starting material, piperine. 5-(3,4-methylenedioxyphenyl)-2,4 pentadienoylcyclohexylamine (CHP) preparation 0.218 g of piperic acid and 0.227 g of N,N’- dicyclohexylcarbodiimide (DCC) were dissolved together in 15 mL of dichloromethane (DCM). 0.114 mL of cyclohexylamine was added to the previous mixture dropwise. Study Objective • The purpose of this study is to carry out synthesis and characterization of novel amide piperine derivatives to further assess their impact on different ER and oxidative stress markers using Normal Rat Kidney (NRK-52E) cells. Limitations Delay in the arrival of chemicals led to shortage of available time for completion of the project. Unavailability of some analytical devices at QU. Results and Discussion PA was obtained by alkaline hydrolysis of piperine and used as a precursor for preparation of the piperine derivatives. Figure 3 shows the DSC thermogram of purified PA. As can be seen, the melting point of PA is 218 o C and no other interfering peaks were detected as an indication of purity. Figure 4 shows the FT-IR spectrum of PA. The graph shows a band of carbonyl functional group at 1716 cm-1 and a broad OH stretch between 3500- 2500 cm-1, which are characteristic of PA. Figure 2b on the other hand illustrates the FT-IR of piperine, which lacks the characteristics of PA. Figure 5 a and b showing the MS spectra of PA and CHP confirms that the molecular weights of PA and CHP are 218 and 299 g/mol respectively. GC-MS was performed for CHP and resulted in the product decomposition as seen in figure 6. Experimental Methodology (Cont.) The solution was left at room temperature while stirring overnight. The reaction mixture was then filtered and washed with 20 mL of distilled water, 20 mL of HCl and 20 mL of distilled water sequentially. The solution was collected and mixed with anhydrous sodium sulfate. Next, the solution was evaporated, and column chromatography was performed using a mobile phase of DCM (70%) and ethyl acetate (30%). Scheme 1 illustrates the chemical scheme of the preparation. Characterization of the prepared compounds Differential Scanning Calorimetry (DSC) were performed using a Perkin Elmer® DSC-8000. Fourier transform infrared (FT-IR) spectra were obtained at room temperature using a Jasco® FT/IR 6000 spectrometer. Gas Chromatography-Mass Spectrometry (GC-MS), Liquid Chromatography- Mass Spectrometry (LC-MS) and elemental analysis were performed. Figure 1. ER Stress and UPR Pathways Figure 2a. Piperine Structure Scheme 1. Synthesis of Piperine Derivatives Figure 3. DSC of PA • This work was made possible by a grant from Qatar University Office of Academic Research (Student grant # QUST-CPH-SPR-12/13-5). • We would like to thank Qatar University Central Lab Unit staff and Taxas A & M Qatar for their great help and support Acknowledgments Conclusions A new amide piperine derivative was successfully synthesized from piperic acid. Additional work is still needed to optimize the reaction condition and prepare more amide piperine analogs. Further spectroscopic analyses are required for full structural elucidation of the synthesized compounds including 1 H and 13 C-NMR and FT-IR. References 1. Kim I, Xu W, Reed J. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic. Nature. 2008 Dec;7 (12):1013-30. 2. Araki E, Oyadomari S, Mori M. Impact of Endoplasmic Reticulum Stress Pathway on Pancreatic β-Cells and Diabetes Mellitus. Exp Biol Med. 2003, 228:1213-1217. 3. Ferreira C, Soares DC, Barreto-Junior CB, Nascimento MT, Freire-de-Lima L, Delorenzi JC, et al. Leishmanicidal effects of piperine, its derivatives, and analogues on Leishmania amazonensis. Phytochemistry. 2011 Dec;72(17):2155-64. Figure 2b. FT-IR of Piperine Figure 4. FT-IR of PA 50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 0 50 100 % 56 99 143 224 70 41 100 83 126 181 113 155 167 195 209 244 232 Figure 6. GC-MS of CHP Figure 5. LC-MS PA (a) and CHP (b) a b % T

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