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  1. Medicinal chemistry An- Najah National University By Anwar Khanfa Taqwa Kabaha Sara Diab Instructor Mohyeddin Assali
  2. 1,4-Diaryl-substituted triazoles as cyclooxygenase-2 inhibitor : synthesis, biological evaluation and molecular modeling studies
  3. Introduction • Over the last 100 years, nonsteroidal anti-inflammatory drugs (NSAIDs) were among the most prominent and frequently used drugs in medicine for the treatment of pain, fever, inflammation and arthritis-associated disorders. • The main therapeutic mechanism of NSAIDs action involves inhibition of cyclooxygenase-mediated biosynthesis of prostaglandins (PGs). • Cyclooxygenase (prostaglandin endoperoxide synthase or COX) enzyme exists in at least two closely related isoforms1–4 viz. COX-1 and COX- 2. COX-1 is consecutively expressed in most tissues. • COX-1 is involved in homeostasis of cytoprotective PGs in the gastrointestinal tract, homeostasis of proaggregatory thromboxane A2 (TXA2) in blood platelets and in overall vascular homeostasis.
  4. • In contrast, inducible COX-2 isoform is absent in most organs and tissues, except for brain, kidney, pancreas, uterus, ovary, and macrophages. • However, upon inflammatory or carcinogenic stimulation, COX-2 expression levels are elevated, and COX-2 is responsible for the production of inflammatory PGs. • Moreover, high levels of COX-2 were also found in various human cancers like gastric, breast, lung, colon, oesophageal, prostate, and hepatocellular carcinomas. • Classical NSAIDs (aspirin, ibuprofen, meclofenamic acid and indomethacin) block the activity of both COX isoforms non-selectively to exhibit their anti-inflammatory effects, which leads to unwanted side effects by the inhibition of COX-1
  5. • Inhibition of consecutively expressed COX-1 isoform and/or downfall in the production of physiological PGs by classical NSAIDs is considered as the basis of life-threatening side effects such as gastrointestinal irritation, ulcer, hepatic and renal toxicity. • In this context, selective inhibition of COX-2 was proposed to be a safer therapeutical approach to reduce deleterious side effects of classical NSAIDs. • As a result, a variety of selective COX-2 inhibitors (COXIBs) such as celecoxib (1), rofecoxib (2), valdecoxib (3), etoricoxib (4) and lumiracoxib (5) have been developed (fig1)
  6. • Selective COX-2 inhibitors have widely been used for the treatment of inflammation and a variety of diseases such as rheumatoid arthritis, osteoarthritis, Alzheimer’s disease (AD) and Parkinson’s disease. • Besides these therapeutic applications, NSAIDs have also been found to play an important role in the prevention of several types of human cancer such as colon, lung, breast and prostate cancers. • Over the last two decades, numerous studies have been carried out to improve the COX-2 selectivity and safety profile.
  7. • However, various selective COX-2 inhibitors came under scrutiny due to an increased risk of adverse cardiovascular side effects. • This development led to the withdrawal of rofecoxib and valdecoxib from the market and stimulated the development of novel classes of selective and safe COX-2 inhibitors with reduced cardiovascular side effects. • In this line, several nitric oxide releasing NSAIDs and dual COX/LOX inhibitors have been developed recently.
  8. • Among the different classes of COXIBs, several vicinal diaryl heterocycles bearing a p-methylsulfonyl- or p-aminosulfonyl-aryl substituent rings represent the most important class of selective COX- 2 inhibitors. • Structure–activity relationship (SAR) data indicate that the nature and position of substituents on two vicinal aryl rings is an important determinant. • It has been observed that compounds containing RSO2 (R = NH2, CH3,) groups effectively interact with amino acid residues of the COX-2 secondary pocket resulting in a generally high COX-2 selectivity and potency profile. • Recently, our group has reported the synthesis of various 1,4 and 1,5- diaryl substituted 1,2,3-triazoles (6–11, Fig. 1) as COX-2 inhibitors. • .
  9. • From this study it was concluded that compounds containing a vicinal diaryl substitution pattern display higher COX-2 inhibition potency compared to their corresponding 1,4- diaryl-substituted counterparts. Both series of compounds showed moderate COX-2 selectivity profiles. • As an extension to this work, we now describe the synthesis of a novel class of 1,4 diaryl-substituted triazoles as potential selective COX-2 inhibitors. • Novel compounds were designed based on Cu(I)-mediated click chemistry between azides containing the SO2NH2 COX-2 pharmacophore attached to an aryl ring and various aryl acetylenes containing different substitutents (H, F, Cl, CH3 or OCH3) attached to the para position of the aryl ring
  10. • . Furthermore, the size of the molecule was increased through the introduction of an alkyl linker to enhance flexibility of the compound to facilitate COX-2 binding. The COX-1/COX-2 inhibitory activities of these compounds were determined in a COX binding assay, and plausible binding interactions within the COX-1/COX-2 active sites were explored using molecular docking studies
  11. Chemistry There is three categories of compounds which is 1,4 - elozairt detutitsbuslyraid : 1. Category 14 _ 18 2. Category 21 _ 25 3. Category 28 _ 32 They are all di substituted triazole but they have a linker (n= 0,1,2 HC,lC,F,H=R dna ( 3 HCO, 3 All contain triazole ring as the name implies and two aromatic rings.
  12. General structure
  13. Point of view To discuss the effect of chain length, size and flexibility of the molecule on the COX- 1 XOC/ - 2 inhibitory potency and selectivity (SI). The chemical approach used here is popular click chemistry Cu+ mediated [ 2+3 ediza citamora eerht fo noitcaer noitiddaolcyc ] ) erutcurts enelyteca lyra detutitsbus suoirav htiw 4 - , H 4 - ,F 4 - ,lC 4 - HC 3,4 - HCO 3 ekam ot ( 1,4 - elozairt detutitsbus lyraid . Equimolar amount are used in precence of ETOH/H2O ,Cu+and triethylamine at 60 ° rof C 14 _ 16 hour. Moderate to good chemical yeild 54 _ 89% after purification.
  14. Results and Discussion
  15. • The COX-1/2 inhibitory potency and selectivity profile of compounds 14 – 18 , 21 – 25 dna , 28 – 32 was determined in triplicate using a fluorescent COX- 1 XOC/ - 2 inhibitor screening assay kit. • In vitro study showed that all classes exhibit potency on COX- 2 over COX-1. • Range of IC50 of COX-2 = 0.17-28 Mm • Range of IC50 of COX-1 = 21 to >100 mM
  16. • Synthesis of compounds 14, 15, 17 and 18 as potential inhibitors of carbonic anhydrases VA and VB was reported recently.
  17. Category I (14-18) N=0 R=H,F,Cl,CH3,OCH3 Para-phenyl sulfonamide attached directly to N-1 of the triazole ring.
  18. • N= 0, R=H • Most potent and selective cox-2 inhibitor. • COX-1 IC50 = >100mM • COX-2 IC50 = 1.3mM • SI= >77
  19. • Has satisfactory cox-2 inhibitor potency but less selectivity. • COX-2 IC50 = 1.3mM. • SI= 16.1
  20. Category II 25 - 21 ( ) N=1, R=H,F,Cl,CH3,OCH3
  21. • MOST POTENT and SELECTIVE inhibitor of COX-2 . • COX-1 IC50 = > 100mM. • COX-2 IC50 = 1.2mM. • SI = 83
  22. • displayed comparable COX-2 inhibitory potency but COX-2 selectivity index of compound 22 was smaller than that of compound 24. • COX-2 IC50 = 1.5mM. • SI = 13.3
  23. Category III ) 32 - 28 ( • N=2, R=H,F,Cl,CH3,OCH3 Compound 30 the most potent cox-2 inhibitor. • COX-2 IC50 = 0.17mM. •SI = >588
  24. • COX-1 IC50 = >100mM. • COX-2 IC50 = 1.1mM. • SI = 91
  25. • COX-1 IC50 = >100mM. • COX-2 IC50 = 1mM. Compounds 28,29 have low potency toward cox- 2 but high cox-2 selectivity.
  26. What can be used as lead compound ? The most effective compound is 30 because it display high potency and selectivity. So it can be a lead compound.
  27. Molecular modeling(docking) studies Structure of COX1 & COX2 enzyme :
  28. Molecular modeling (docking) studies were carried out for compounds (16, 23, 30) to gain more insights into the probable nature of their respective binding interactions within the active site of COX-1 and COX-2 isozymes. Docking experiments were performed using X-ray crystal structure data for COX-1 and COX-2 obtained from the protein data bank.
  29. Compound 16, when docked into the COX-2 binding site , comfortably enters into the COX-2 active site and places its para-Cl-phenyl substituent near S530, L531residues, while one of the nitrogen atom of the triazole ring indicates hydrogen bonding interactions with V523 residue of COX-2 active site (NO = 1.55 Å). The SO2NH2 group as prominent and effective COX-2 pharmacophore) is pointed near the COX-2 isozyme secondary pocket amino acid residues (A516, F518, R513, Q192 and H90).
  30. In a comparable manner, compound 23 displays a favorable orientation in the COX-2 active site and the SO2NH2 group is oriented at the entrance of the secondary pocket. This places the SO2NH2 group near the R513, L352 residues. The 4-chlorophenyl substituent was moved near to the L359 amino acid of the COX-2 binding site.
  31. A similar molecular docking simulation for compound 30 within the COX-2 active site, showed noticeable interactions between compound 30 and active binding site residues .The SO2NH2 group in compound 30 deeply enters into the secondary pocket region of the COX-2 active site, where it is oriented towards Q192, R513, H90, and S353 residues. The nitrogen atom of the SO2NH2 group indicates hydrogen bonding interactions with the oxygen of carbonyl group of Q192 ,and one of the oxygen atom of SO2NH2 group is hydrogen bonded to the nitrogen atom of H90 residue.
  32. However, in contrast to binding interactions of compound 16 and 23, the docking results of compound 30 indicate that upon increasing the size of the linker chain (n = 2), there is an increase in the flexibility of the molecule which facilitates the deeper insertion of the SO2NH2 group into the secondary binding pocket and more significant intermolecular binding interactions. This favorable orientation and electrostatic binding interactions of compound 30 with COX-2 active site residues are consistent with experimentally observed high COX-2 inhibitory potency (COX-2 IC50 = 0.17 lM). A comparison of the binding mode of compounds 16, 23 and 30 in the COX-2 active site using a molecular docking structure containing all three compounds (16, 23 and 30) docked in the same COX-2 active site is given in the Supplementary section.
  33. Alternatively, docking results for compound 16, 23 and 30 in COX-1 active site show that compound 16 comfortably enters into the COX-1 active site and its p-Cl-phenyl substituent has moved into the hydrophobic subpocket of COX-1isozyme constituted by W387, Y385 and F381 residues.
  34. However, during docking studies of compound 23 and 30 into COX-1, compounds were not able to enter into the COX-1 active site completely .This inability is likely due to the increase in their size or/and due to the steric conflict with side chains of amino acid residues of the active site. This body of data evidently supports the experimentally observed weak COX-1inhibitory activity of compound 23 and 30. 23 30
  35. Conclusions In conclusion, a new class of 1,4-diaryl-substituted triazoles was synthesized using high yielding Cu(I)-mediated click chemistry. In vitro COX-1/COX-2 inhibition studies showed that some compounds of this novel class of 1,4-diaryl-substituted triazoles are able to inhibit COX-2 with high otency and selectivity. Especially compounds (28–30) were identified as highly potent (IC50 range 0.17–1.1Mm) and selective novel COX-2 inhibitors (SI >91 to >588). Among all investigated compounds, a ‘lead compound’ 4-{2-[4-(4-Chloro-phenyl)-[1,2,3]triazol-1-yl]-ethyl}- benzenesulfon-amide was identified that exhibited appreciable COX-2 inhibitory potency and selectivity (COX-1 IC50 >100 lM, COX-2 IC50 = 0.17 lM; SI >588).
  36. Molecular docking analyses for compounds 16, 23 and 30 on the COX-1/COX-2 isozymes complement the experimental results. From both the experimental structure– activity data and the molecular docking results it can be concluded that an increase of the length of the linker chain is accompanied with an enhancement in molecular interactions between functional groups of the compounds and amino acid residues of the secondary pocket of the COX-2 active site.
  37. The End