awkum-chemistry identification of novel quinazolin-4(3h)- ones as inhibitors of thermolysine, the...

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Identification of novel quinazolin-4(3Identification of novel quinazolin-4(3HH)-ones as )-ones as inhibitors of thermolysine , the prototype of M4 inhibitors of thermolysine , the prototype of M4

family of proteinase family of proteinase

Dr. Rasool Khan

Department of chemistry

Abdul Wali Khan University, Mardan

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Introduction Introduction • Thermolysine (TLN, EC 3.4.24.27)

zinc-containing eubacterial endoproteniase,

from bacillus thermoproteolyticusRequire one zinc ion for enzyme activityFour ca2+ ions for structural stability

Importance of Importance of Thermolysine 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)

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

•

Rasool khanRasool khan

Quinazolin-4(3Quinazolin-4(3HH)-ones )-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

Biological activitiesBiological 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

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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


Part-I; Chemistry Part-I; Chemistry Synthesis 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


Part-I; Chemistry, Part-I; Chemistry, Synthesis Synthesis


COOMe

NH2

F3C COOH+

COOMe

NH

CF3O

N

N

O

Ar2/R2

Ar1/R1

COOMe

NH2N

O

O

Ar1

N

N

O

C6H5

NH O

NHO

2-16

171

a

b

c d

e

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.

M. Arfan, Rasool Khan, J. Chem. Soc. Pak., 2008, 30, 299.


Compound # Ar1/R1 Ar2/R2

2 CF3 NH2

3 CF3 Phenyl

4 CH3 NH2

5 NHNH2 NH2

6 PHENYL NH2

7 P-tolyl 2-thiazolo

8 -p-nitrophenyl NH2

9 Phenyl P-iodophenyl

10 P-tolyl -CH2CH2NH2

11 P-tolyl -CH2CH2CH2NH2

12 Phenyl 2-thiazolo

13 P-chlorophenyl NH2

14 Phenyl 2,3,5, trimethyl phenyl

15 H -CH2CH2OH

16 CH3 -CH2CH2OH

Table : List of Compounds (2-16) Synthesized

Synthesis of quinazolin-4(3Synthesis of quinazolin-4(3HH)-one Schiff )-one Schiff basesbases


N

N

O

Ar/R

NH2

N

N

O

CH3

N NH2

NH2

N

N

O

Ar/R

N

Ar

N

N

O

Ar/R

NH

O R

N

N

O

C6H5

N

CH3

OCH3 N

N

O

C6H5

N

CH3

NH

2 -1 61 8 -2 2

2 3 -2 7

2 8

2 93 0

a

b

c

d

eNH

N

O

Ar

31 ,32

f

A r = 3 1 = p -ch loroph en yl 3 2 = p -n itroph en yl

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.

Synthesis Of Quinazolin-4(3Synthesis Of Quinazolin-4(3HH)-)-onesones


N

NH

O

R1

N

R2

R1

R2

NH

NH2

O

NH2OCH3

NH2

O

NH

N

O

R

33 ,34 35 ,36

a bc

R =3 3 = -C H 2 C H 3

3 4 = -C H 2 C O 2 C 2 H 5

3 5 . R 1, R 2= CH 3

3 6 . R2= C 6H 5, R=H

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

Biological Studies of the Biological Studies of the CompoundsCompounds

• 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)



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.

Comp. IC50 values

(μM)

Ki

(μM)

Comp. IC50 values

(μM)

Ki

(μM)

1 LA a - 20 LAa -

2 37.86 37.9 21 LAa -

3 0.0115 0.0115 22 LAa -

4 54.89 54.9 23 42.03 42.00

5 LAa - 24 1832 1830

6 Inactive b - 25 4002 4000

7 76.85 59.3 26 LAa -

8 3118 3120 27 LAa -

9 LAa - 28 LAa -


10 LAa - 29 122637 106000

11 Laa - 30 LAa -

12 Laa - 31 1.25 1.25

13 12743 10100 32 LAa -

14 Inactive b - 33 LAa -

15 Laa - 34 LAa -

16 Inactive b - 35 0.2477 0.243

17 Laa - 36 LAa -

18 Laa - 37 Inactive b -

19 Laa -

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

The structure-activity The structure-activity

relationship (SAR)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


(SAR) cont. (SAR) cont. • Replacing the position-3 amino

group of compound 2 with phenyl increased affinity more then 3000 time


N

N

O

NH2

CF3

Com p.2; IC 50 (37.9)

N

N

O

CF3

Com p.2; IC 50 (0.0115)

(SAR) cont.(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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Com p.29; IC 50 (122,637)

N

N

O

N

CH3

O C2H5

Com p.4; IC 50 (54.89)

N

N

O

N

Cl

N

N

O

N

N

CH3

CH3

Com p.23; IC 50 (42.03)

N

N

O

N

Cl

Com p.25; IC 50 (4002)

Substituents on aromatic Substituents on aromatic groupgroup

• 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


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N

N

O

N

Cl

comp. 4; (54.89)

N

N

O

N

Cl

comp. 25; (4002)

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)



N

N

NH2

O

CF3

N

N

O

CF3

NH

N

O

CH3

N

NH

O

CH3

N

CH3

CH3

(comp.2) 54.89(comp.2) 0 .0115 (comp.2) 1 .24 (comp.2) 0 .2477

N

N

NH2

O

CF3

(comp.2) 54.89

N

N

NH2

O

N

CH3

CH3

N

N

NH2

O

N

CH3

CH3(comp.8) 3118(comp.13) 12743

Docking 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


Schematic illustration of the interactions of compound 3 at the active site of TLN as 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 theindentified by LigPlot. (B) Corresponding 3D view of compound 3 at the

active site of TLN. The binding pocket is in grey transparent modeactive site of TLN. The binding pocket is in grey transparent mode


Conclusion 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)


Most potent inhibitorsMost potent inhibitors


NH

N

O

N

N

N

O

F

F

F

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

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

Acknowledgments 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



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awkum-chemistry identification of novel quinazolin-4(3h)- ones as inhibitors of thermolysine, the...

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