Evaluation of N-arylhydrazone derivatives of orotic acid as stimulators of human mesenchymal stem cell ( hMSCs ) differentiation

Saeed Ali Syed, Ahmed BariAmer Mahmood, Sarah Abuelreich, Eric C. Hosten, Richard Betz, Abdulrahman M. Al-Obaid, Abdulrahman Ghadeer Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box: 2457, Riyadh, 11451, Saudi Arabia Research Center, College of Pharmacy, King Saud University, P.O. Box: 2457, Riyadh, 11451, Saudi Arabia Stem Cell Unit, Department of Anatomy, King Saud University, P.O. Box: 2457, Riyadh, 11451, Saudi Arabia Nelson Mandela Metropolitan University, Department of Chemistry, PO Box 77000, Port Elizabeth 6031, South Africa. *abari@ksu.edu.sa

their evaluation as stimulators of human mesenchymal stem cells.Some of the analogs show good to moderate proliferation rate.

Introduction:
Heterocycles and heterocyclic derivatives are continuously serves as a versatile tool to synthesize variety of natural product due to the presence of various chromophores (1)(2)(3)(4)(5).Pyrimidine carboxylic acid which is commonly called as orotic acid is a well known compound and found in many naturally occurring products like, milk whey and serves as an intermediate in the biosynthesis of pyrimidine which is an essential component in DNA and RNA synthesis.
Moreover, orotic acid is also reported to enhance cardiac output and aid in recovery from heart failure.It also has growth stimulant effects in mammals and may assist in absorption of calcium, magnesium and other essential nutrients.It has also been reported that orotic acid is used to reduce the level of bilirubin in infants and is also useful for the treatment of gout.Many orotic acid analogues have shown promising results against antitumor and antimicrobial activities.Some of them also serve as enzyme inhibitors and gain the attention of chemists and molecular biologists (6)(7)(8)(9).Hydrazones constitutes an important class of compounds in organic synthesis due to azomethine group being part of the molecule.In addition, hydrazones and hydrazides are considered as one of the most useful synthetic intermediate to synthesize variety of molecules and possible drug candidates (10).Since many years, hydrazones derivatives have been the focus of interest for many synthetic chemists and biologists because of the synthetic importance and the biological activity associated with them.The pharmacological profile includes their antimicrobial, antiviral, anticancer and anti inflammatory activities.The bioactivity of the hydrazide-hydrazone analogues is apparently not limited to the core moiety but also depends on the molecules attached to the terminal nitrogen.It is long been known that introduction of aromatic molecules to the heterocyclic system results in more potent molecules (11)(12)(13).types.Synthetic design of heterocyclic compounds have been aging in substantial numbers of molecular platform including substituted purines pyrimidines, quinazolines, pyrazines, pyrrolopyrimidine, pyrazolopyrimidine, pyridazines, and hydrazones which lend appropriate chemical concern to look into modulate complex cellular mechanism (14)(15)(16)(17)(18).However, to the best of our knowledge, an application with pyrimidine carboxylic acid has not yet been reported.
Keeping in mind the biological potential of orotic acid and in continuation to our interest in hydrazone-hydrazide chemistry, the present works describe the preparation of N-arylhydrazones starting from orotic acid.Current research work also presents the stem cell proliferation potential of hydrazone derivatives with human stromal stem cells which was never studied before.

Results:
Proliferation Method: In this study we used the already established hTERT-MSC-CL1 (hMSC) cell lines, the cells were used in between passage 24-28.The cells were cultured in T75 culture flask (BD FalconTM, NJ, USA).Cells were monitored with inverted light microscope (Observer A1, Zeiss®, Gottingen, Germany).hMSC were grown in media composed of D-MEM (Gibco, Cat No. 41966052) upplemented with 10% FBS (Gibco, Cat No. 26140087), 1% pen/strep (10,000 units of penicillin and 10,000 g of streptomycin/ml; Gibco, Cat No. 15140122) and 1% NEAA (X100; Gibco, Cat No. 11140035).After the cells reached 80-90% confluences in culture flasks, the cells were trypsinized and they were transferred in falcon tubes.Cells were counted using the Neubauer hemocytometer counting chamber (PAUL MARIENFELD GMBH & CO.KG.), the cells were seeded at a density of 0.01×106 cells per well of a 96-well tissue culture plate.Following day the diluted compounds were added to the cells at the desired concentration in triplicate, after additional 2 days the media is changed to normal growth media.The next day is designated as day 1 of proliferation.

Alamar Blue Cell Viability Assay:
Cell viability was measured using alamar blue assay according to the manufacturer's recommendations (AbD Serotec, Raleigh, NC, USA).In brief, we cultured cells 96-well plates in 100μl of the appropriate medium and at the indicated time point, and 10μl of Alamar Blue substrate was added and plates were incubated in the dark at 37°C for 1h.Reading was subsequently taken using fluorescent mode (Ex 530nm/Em 590nm) using BioTek Synergy II microplate reader (BioTek Inc., Winooski, VT, US.

Chemistry:
All solvents and reagents were purchased from Aldrich Chemical Co and were used as obtained.
IR spectra were recorded with a Perkin-Elmer spectrum BX FT-IR spectrometer using KBr pellets. 1 H and 13 C NMR spectra were recorded on a Bruker instrument (500 and 125 MHz, respectively) in DMSO-d6, Mass spectra were obtained on a JEOL JMS-700 mass spectrometer, ionization method was EI (70 eV).Melting points were measured with a Thermo Scientific 9100 apparatus and are uncorrected.Thin-layer chromatography (TLC) was performed with fluorescent silica gel HF254 plates (Merck) and visualized under UV 254 and charring with the EtOH-H2SO4 (5:1) system.Merck silica gel 60 (230-400 mesh) was used for column chromatography.
General procedure for the synthesis of compound 2 and 3 (19).
To a solution of orotic acid 1 (2 mmol) in ethanol/butanol (50 mL) was added catalytic amount of HCl.The resulting mixture was refluxed for 10 h with stirring followed by the evaporation of solvent in vacuo.The resulting solid obtained was washed with cold water several times, recrystallized with ethanol-water mixture afforded compounds 2 and 3.