Identification of novel quinazolin-4(3
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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

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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



  • Thermolysine (TLN, EC

    zinc-containing eubacterial endoproteniase,

    from bacillus thermoproteolyticus

    Require one zinc ion for enzyme activity

    Four ca2+ ions for structural stability

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Importance of thermolysine
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)


Function of thermolysine
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

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Quinazolin 4 3 h 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

Biological activities
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

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Present study
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

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Part i chemistry synthesis
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

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Part i chemistry synthesis1
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.

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Synthesis of quinazolin 4 3 h one schiff bases
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.

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Synthesis of quinazolin 4 3 h ones
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

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Biological studies of the compounds
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

  • (Khan, M. T.; Fuskevag, O. M.; Sylte, I. J Med Chem 2009, 52, 48)

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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.

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

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The structure activity relationship sar
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

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Sar cont
(SAR) cont.

  • Replacing the position-3 amino group of compound 2 with phenyl increased affinity more then 3000 time

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Sar cont1
(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

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Substituents on aromatic group
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

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Size of the substituent
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)

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  • 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

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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

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Conclusion the active site of TLN as indentified by

  • 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)

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Most potent inhibitors
Most potent inhibitors the active site of TLN as indentified by

3-Phenyl-2-(trifluoromethyl) quinazolin-4(3H)-one

3-(Isopropylideneamino)-2, 2-dimethyl-2, 3-dihydroquinazolin-4(1H)-one (35)

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Acknowledgments the active site of TLN as indentified by

  • 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

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THANK YOU the active site of TLN as indentified by

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