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Design, Synthesis and in vitro Screening of Pyrazolines based compounds as Phytohaemagglutinin ( PHA) mimetic Raj Kumar 1, *, Gaurav Joshi 1 , and Sandeep Singh 2 1 Laboratory for Drug Design and Synthesis, Centre for Pharmaceutical Sciences and

  1. Design, Synthesis and in vitro Screening of Pyrazolines based compounds as Phytohaemagglutinin ( PHA) mimetic Raj Kumar 1, *, Gaurav Joshi 1 , and Sandeep Singh 2 1 Laboratory for Drug Design and Synthesis, Centre for Pharmaceutical Sciences and Natural Products, Central University of Punjab, 151 001, Bathinda, India ; 2 Centre for Genetics Diseases and Molecular Medicine, Central University of Punjab, 151 001, Bathinda, India * Corresponding author: raj.khungher@gmail.com; rajcps@cup.ac.in 1

  2. Design, Synthesis and in vitro Screening of Pyrazolines based compounds as Phytohaemagglutinin ( PHA) mimetic 2

  3. Abstract: Phytohaemagglutinin (PHA, or phytohemagglutinin) is a lectin found commonly in plants, especially legumes. It has some physiological effects on cell metabolism; it induces mitosis and affects the cell membrane regarding transport and permeability to proteins. It agglutinates most mammalian red blood cell types and have the mitogenic effect. This is the reason that PHA is extensively used in the laboratory as well as clinical set up for karyotyping analysis. The downside of PHA use is its cost and storage (-20 o C) resulting into increased cost. We have synthesised acetylated pyrazolines as anticancer agents and during their evaluation for anticancer potential in normal control cells, we were surprised by their cell proliferation activity. We thought of relating our compounds to PHA (PHA mimetic) and performed the basis karyotyping experiment keeping PHA as standard and found our compounds to be PHA mimics. The compounds are thus being evaluated for their further PHA mimetic potential using B/T cell specific cell cycle analysis and karyotyping experiment keeping PHA as standard and found our compounds outstanding PHA in every aspect. The compounds are thus being evaluated for their further PHA mimetic potential. Keywords: Phytohaemagglutinin; Mitogenic; PHA mimetic 3

  4. Introduction  Phytohaemagglutinin (PHA, or phytohemagglutinin) is a lectin found in plants, especially legumes.  The lectin has a number of effects on cell metabolism; it induces mitosis, and affects the cell membrane in regard to transport and permeability to proteins. It agglutinates most mammalian red blood cell types.  Lymphocytes cultured with phytohaemagglutinin can be used for karyotype analysis 4

  5. Hypothesis  While evaluating a series of newly synthesized compounds on human peripheral blood monocytic cells, the synthesized compounds did not exhibit any cytotoxic activity but they showed increased proliferation indicated by the increased intensity of formazan reduction.  It was, thus, hypothesized that the synthesized compounds may be increasing the cell similar to PHA. 5

  6. Synthetic Strategy 6

  7. Chemical structures of the synthetics 7

  8. Objectives  Determining the absolute cell count in different samples and their comparison with PHA as control.  Determining the Protein Concentration of the tested samples against PHA.  Arresting the dividing cells at metaphase and observing them under microscope at 10x and 40x. 8

  9. Result and Discussions 9

  10. No. of cells were counted on Automated Cell Counter 70,000 60,000 50,000 40,000 30,000 20,000 10,000 0 PHA Control JP-9 JP-11 JP-12 JP-14 JP-16 JP-17 10

  11. Evaluation of cytotoxic effect on PBC in response to treatment with synthesized compounds at concentrations of 1 µL, 2 µL and 5 µL for a time duration of 72 hrs. Data is expressed as mean values ± S.D. of three independent experiments. 1µL 2µL 5µL 0.12 0.1 0.08 0.06 0.04 0.02 0 Control Vehicle Control PHA Control JP-7 JP-11 JP-20 JP-14 JP-21 JP-24 JP-30 JP-31 11

  12. Evaluation of cytotoxic effect on PBC in response to treatment with synthesized compounds at concentrations of 1 µL, 2 µL and 5 µL for a time duration of 96 hrs. Data is expressed as mean values ± S.D. of three independent experiments. 1µL 2µL 5µL 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 Control Vehicle Control PHA Control JP-7 JP-11 JP-20 JP-14 JP-21 JP-24 JP-30 JP-31 12

  13. Determination of Protein Concentration 45 40 35 30 25 20 15 10 5 0 JP-16 JP-17 JP-14 JP-12 PHA JP-11 JP-9 Control 13

  14. Protocol for Metaphase Arrest PHA C1 (JP-14) C2 (JP-21) Media (RPMI) 2mL 2mL 2mL Blood 150 µL 150 µL 150 µL PHA 30 µL C1 30 µL C2 60 µL 14

  15. Day1: Treatment Strategy Incubation for 68h at 37˚C 15

  16. Day2: Metaphase arrest and Harvesting of Cells 16

  17. Day 3 Slides preparation For PHA For compound C1 17

  18. Conclusions  Absolute cell count showed that JP-12 and JP-14 had increased cell count as compared to the control and PHA.  Protein concentration was also found to be considerably higher in the synthesized compounds in comparison to PHA.  Similar to PHA, JP-12 was able to bring about the metaphase arrest at the 68 th hour.  From this, it can be concluded that, JP-12 and JP-14, the synthesized compounds show cell proliferation as PHA and have the potential to replace PHA. 18

  19. Future Prospective  It would be more helpful to determine the cell type that is more susceptible to cell proliferation  Studies should be undertaken to delve into the mechanism of how the synthesized compounds bring about this effect. 19

  20. References 1. Hamelryck, T. W., Dao-Thi, M. H., Poortmans, F., Chrispeels, M. J., Wyns, L., Loris, R. (1996). The crystallographic structure of phytohemagglutinin-L. The Journal of Biological Chemistry . 271 (34): 20479 – 20485. 2. Mire-Sluis,A. R., Wickremasinghe, R. G., Hoffbrand,A. V., Timms, A. M. Francis, G. E., (1987). Human T lymphocytes stimulated by phytohaemagglutinin undergo a single round of cell division without a requirement for interleukin-2 or accessory cells. Immunology. 60 (1), 7 – 12. 3. Serke,S., Serke,M., Brudler, O. (1987). Lymphocyte activation by phytohaemagglutinin and pokeweed mitogen: Identification of proliferating cells by monoclonal antibodies. 99 (2), 167-172. 20

  21. Acknowledgements R.K. and SS thank DST and UGC, New Delhi, India for the financial assistance (F.No. SR/FT/CS-71/2011) and F.30-13/2013(BSR), respectively. Encouragement and support from Prof. P. Ramarao, Dean, and Prof. R.K Kohli, Vice Chancellor Central University of Punjab, is gratefully acknowledged. Special thanks are also due to Miss. Jimi Marin Alex, who thoroughly helped in accomplishing this task. 21

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