1. AWKUM-Chemistry 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

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

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. AWKUM-- Chemistry 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 Commun 1969, 37, 333. Eur J Biochem 1970, 15, 374. Nature 2003, 421, 551

5. Rasool khan 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

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 Chem 2003, 11, 5293 J Med Chem 2008, 51, 4359 Chem Biol Drug Des 2007, 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 AWKUM -- Chemistry 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) NH 2 CH 2 CH 2 CH 2 NH 2, reflux, dry benzene, 2h. M. Arfan, Rasool Khan, J. Chem. Soc. Pak., 2008, 30, 299.

10. AWKUM-- Chemistry Compound #Ar 1 /R 1 Ar 2 /R 2 2 CF 3 NH 2 3 CF 3 Phenyl 4 CH 3 NH 2 5 NHNH 2 NH 2 6 PHENYLNH 2 7 P-tolyl2-thiazolo 8 -p-nitrophenylNH 2 9 PhenylP-iodophenyl 10 P-tolyl-CH 2 CH 2 NH 2 11 P-tolyl-CH 2 CH 2 CH 2 NH 2 12 Phenyl2-thiazolo 13 P-chlorophenylNH 2 14 Phenyl2,3,5, trimethyl phenyl 15 H-CH 2 CH 2 OH 16 CH 3 -CH 2 CH 2 OH Table : List of Compounds (2-16) Synthesized

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

12. Synthesis Of Quinazolin-4(3H)-ones AWKUM-- Chemistry Conditions and reagents: a) NH 2 NH 2.H 2 O, 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

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 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 (IC 50 and K i values) of 2,3-disubstituted quainzolin-4(3H)-ones. The K i values were determined from IC 50 values using the Cheng-Prusoff relationship. Comp. IC 50 values (μM) K i (μM) Comp. IC 50 values (μM) K i (μM) 1LA a -20LA a - 237.8637.921LA a - 30.0115 22LA a - 454.89 54.9 2342.03 42.00 5LA a -241832 1830 6Inactive b -254002 4000 776.85 59.3 26LA a - 83118 3120 27LA a - 9 -28LA a -

15. AWKUM -- Chemistry 10LA a -29122637106000 11La a -30LA a - 12La a -311.25 13127431010032LA a - 14Inactive b -33LA a - 15La a -34LA a - 16Inactive b -350.24770.243 17La a -36LA a - 18La a -37Inactive b - 19La a - Notes: LA: low activity, a due to the low activity the IC 50 values were not possible to calculate; b completely inactive.

16. The structure-activity relationship (SAR) Position 2 and 3 are important for activity 12 compounds were found inhibitors out of all synthesized The IC 50 values for compound 3 (IC 50 = 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 of para 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

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 the active 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 AWKUM -- Chemistry 3-Phenyl-2-(trifluoromethyl) quinazolin-4(3H)-one 3-(Isopropylideneamino)-2, 2-dimethyl-2, 3-dihydroquinazolin-4(1H)-one (35)

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