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Dr. Rasool Khan Department of chemistry Abdul Wali Khan University, Mardan

Identification of novel quinazolin-4(3 H )-ones as inhibitors of thermolysine , the prototype of M4 family of proteinase. Dr. Rasool Khan Department of chemistry Abdul Wali Khan University, Mardan. Introduction. Thermolysine (TLN, EC 3.4.24.27)

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Dr. Rasool Khan Department of chemistry Abdul Wali Khan University, Mardan

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  1. Identification of novel quinazolin-4(3H)-ones as inhibitors of thermolysine , the prototype of M4 family of proteinase Dr. Rasool Khan Department of chemistry Abdul Wali Khan University, Mardan AWKUM-Chemistry

  2. Introduction • Thermolysine (TLN, EC 3.4.24.27) zinc-containing eubacterial endoproteniase, from bacillus thermoproteolyticus Require one zinc ion for enzyme activity Four ca2+ ions for structural stability AWKUM-- Chemistry

  3. Importance of Thermolysine • Enzyme of M4 family suppress the innate immune system of infected host during pathogenesis • Therefore, inhibition of several M4 enzymes is believed to be a novel strategy in the development new generation of antibacterial drugs ( a step to drug discovery) AWKUM-Chemistry

  4. Function of thermolysine • Catalyze the hydrolysis of peptide bond of protein, containing hydrophobic amino acid • Also catalyze the formation of peptide bond i.e. reverse of hydrolysis • i.e. Formation and hydrolysis peptide bond both are catalyzed by this enzyme (Reversible Process) • Biochem Biophys Res Commun1969, 37, 333. • Eur J Biochem1970, 15, 374. • Nature2003, 421, 551 AWKUM-- Chemistry

  5. Quinazolin-4(3H)-ones • Heterocyclic alkaloid ring system frequently encountered in medicinal chemistry. have attracted much focus by synthetic and medicinal chemists and consequently a plethora of reports are available describing the synthesis and biological activity Rasool khan

  6. Biological activities • Quinazolin-4(3H)-one structure analogous have been found to have different biological properties including; • anticonvulsant, sedative, tranquilizer, analgesic, antimicrobial, anesthetic, anticancer, antiviral, antihypertensive, anti-inflammatory, diuretic and muscle relaxant • Bioorg Med Chem2003, 11, 5293 • J Med Chem2008, 51, 4359 • Chem Biol Drug Des2007, 70, 254 AWKUM -- Chemistry

  7. Present study • Synthesis of structurally diverse library of 2,3-disubstituted Quinazolin-4(3H)-ones • TLN inhibition using in vitro binding assays. • To study structure activity relationship, in search of better thermolysine inhibitors AWKUM -- Chemistry

  8. Part-I; Chemistry Synthesis • Three different methods were used for synthesis of 2, 3-disubstituted quinazolin-4(3H)-ones • The synthesis of 2-aryl 3-amino quinazolin-4 (3H)-ones was accomplished using a recent reported method via benzoxazinoines intermediate AWKUM -- Chemistry

  9. Part-I; Chemistry, Synthesis M. Arfan, Rasool Khan, J. Chem. Soc. Pak., 2008, 30, 299. Reagents and conditions: a) M.W, 1 min., b&c) Hydrazine hydrate, ethanol, reflux, 2 h., d) Hydrazine hydrate, Pyridine or benzene, reflux, 6-8 h.,e) NH2CH2CH2CH2NH2, reflux, dry benzene, 2h. AWKUM -- Chemistry

  10. Table : List of Compounds (2-16) Synthesized AWKUM-- Chemistry

  11. Synthesis of quinazolin-4(3H)-one Schiff bases a) acid halid, Pyridine or benzene (dry), stir, 2-5h., b) Urea, ethanol(dry), 5h., c) i. ethanol dry, H2SO4, reflux, ii. 5%-10%, NaHCO3., d) triethylorthoacetate, stir .,e) acetic acid, reflux, 6 h., f) KMnO4­, water, reflux. AWKUM-- Chemistry

  12. Synthesis Of Quinazolin-4(3H)-ones Conditions and reagents: a) NH2NH2.H2O, ethanol, reflux, 2h., b) aldehyde/ketone, silica gell, stir., c) alkyl cyanide, dioxane(dry), HCl(dry), stirr M. Arfan, Rasool Khan, Chinese Chemical Letters 19 (2008) 161–165 AWKUM-- Chemistry

  13. Biological Studies of the Compounds • The steady-state enzyme assays were performed at 25 °C using the spectrophotometric method of Feder and Schuck (Feder, J.; Schuck, J. M. Biochemistry 1970, 9, 2784) • Molecular docking of compound 3 was performed as previously described using the Internal Coordinate Mechanics (ICM) program from Molsoft http://www.molsoft.com • (Khan, M. T.; Fuskevag, O. M.; Sylte, I. J Med Chem 2009, 52, 48) AWKUM -- Chemistry

  14. Table : TLN inhibitory activities (IC50 and Ki values) of 2,3-disubstituted quainzolin-4(3H)-ones. The Ki values were determined from IC50 values using the Cheng-Prusoff relationship. AWKUM -- Chemistry

  15. Notes: LA: low activity, a due to the low activity the IC50 values were not possible to calculate; b completely inactive. AWKUM -- Chemistry

  16. The structure-activity relationship (SAR) • Position 2 and 3 are important for activity • 12 compounds were found inhibitors out of all synthesized • The IC50 values for compound 3(IC50 = 0.0115 µM) most potent in the whole series AWKUM -- Chemistry

  17. (SAR) cont. • Replacing the position-3 amino group of compound 2 with phenyl increased affinity more then 3000 time AWKUM -- Chemistry

  18. (SAR) cont. • Affinity of compound 29 is lower then 4, 23 and 25 • Which suggest that aromatic substituent on position 3 is favorable for strong TLN affinity WWU -- Chemistry

  19. AWKUM -- Chemistry

  20. Substituents on aromatic group • Choloro group (comp. 4) is changed from meta to para position (comp. 25), affinity drops • Replacement ofpara chloro group with dimethyl amino group slightly increase the activity AWKUM -- Chemistry

  21. WWU -- Chemistry

  22. Size of the substituent • Compounds with small group on position 2 (comp. 2, 3, 31 and 35) showed high activity, compared to lager groups on same position • Trifluoromethyl group at position 2 (comp.2) binds strongly than the aromatic bulky group ( comp. 8 and 13) AWKUM -- Chemistry

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  24. Docking • Docking of compound 3 into the active site of TLN indicated that the trifluoromethyl group in position 2 interacted in the region of Asn112 (S1’-subsite) and Phe114 (S1-subsite) • phenyl ring in position 3 interacted Asn111, Asn112 and His231 in the S1’ and S2’ subsites AWKUM-- Chemistry

  25. Schematic illustration of the interactions of compound 3 at the active site of TLN as indentified by LigPlot. (B) Corresponding 3D view of compound 3 at theactive site of TLN. The binding pocket is in grey transparent mode AWKUM -- Chemistry

  26. Conclusion • Trifloromethyl group at position 2 • Aromatic substituent at position 3 enhance the activity In the present series of Quinazolin-4 (3H)-ones • Compound 3 and 35 were found most potent TLN inhibitors • (Rasool Khan et al, Bioorganic and medicinal Chemistry 18 (2010), 4317-4327) AWKUM -- Chemistry

  27. Most potent inhibitors 3-Phenyl-2-(trifluoromethyl) quinazolin-4(3H)-one 3-(Isopropylideneamino)-2, 2-dimethyl-2, 3-dihydroquinazolin-4(1H)-one (35) AWKUM -- Chemistry

  28. Acknowledgments • Prof. Dr. Muhammad Arfan, ICS, UOP, my Ph.D Supervisor • HEJ-RIC, University of KARACHI • M. Tariq and Y. Wuxiur, Department of medical biology, University of Tromso, Norway • Director, Dr. Bashir , Centre of Biotechnology and microbiology, for giving opportunity AWKUM-- Chemistry

  29. THANK YOU AWKUM -- Chemistry

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