Indian Journal of ChemistryVol. 45B, July 2006, pp. 1692-1698

':I:~'-J '~_' .r . ,

2,3-Diphenyltetrahydrofurans (DPTF) - A new class of stereogenic diarylheterocycles as potential COX-2 inhibitors - Computational evaluation of

COX-2-DPTF binding behaviour

Palwinder Singh*, Pervinder Kaur, Anu & Subodh KumarDepartment of Chemistry, Guru Nanak Dev University, Amritsar 143005 India

E-mail: palwinder_singh_2000@yahoo.com

Received 02 June 2005; accepted (revised) 05 September 2005

Based on the reported COX-2 selectivity of a,p-diarylheterocycles based NSAIDs' - a new class of 2,3-diphenyl-tetrahydrofurans (DPTF) possessing COOH, CH20H or CH2NH2 groups at C-5 and three chiral centers C-2, C-3 and C-5have been designed. In total 12 title compounds have been studied for their binding in the active sites of COX-2 and COX-Ienzymes. For comparison, the binding behavior of known COX-2 selective (rofecoxib, celecoxib, valdecoxib) and non-selective (aspirin, ibuprofen) drugs has also been studied. The trends of COX-2 selectivity of known NSAIDs' are parallelwith their reported results. In general, DPTF derivatives show binding energies in COX-2 active site better than rofecoxiband comparable to aspirin and poor binding energies in COX-I active sites pointing towards these molecules being selectiveCOX-2 inhibitors. The stereochemistries at C-2, C-3 and C-5 carbons of DPTF derivatives considerably affect their bindingsin the active sites of COX-I and COX-2 and the COX-2 selectivity. The ligands with configuration (2S, 3R, 5S) show higherselectivity for COX-2 over COX-I in comparison to other configurational isomers.

IPC: Int.CI.8C07D

Keywords: cyclooxygenase, NSAIDs, diphenyltetrahydrofurans, stereogenic molecules, doc kings

Cyclooxygenases (COXs) and lipoxygenases (LOXs)are the key enzymes in arachidonic acid metabolism'".One isoform of cyclooxygenase i.e., COX-I impart adesirable function in the formation of thromboxane,gastric acid secretions and induction of labor pains"while the second form, COX-2 is responsible for anundesirable role in the production of prostaglandinsduring its expression in inflammatory cells that leadsto pains". The traditional non-steroidal anti-inflam-matory drugs (NSAIDs) like aspirin, ibuprofen,diclofenac etc. block both the COX-I and COX-2enzymes and their non-selectivity leads to undesirableside effects'. Once the X-ray structures of COX-I andCOX-2 enzymes were established, a number ofselective inhibitors of COX-2 have been reported inwhich the basic skeleton involves the presence ofaryl/substituted aryl moieties at adjacent Sp2hybridized carbons of pyrazoles'", furanones'",cyclopentenes", imidazoles'", isoxazoles", pyrroles'j,oxazolones':', acyclic olefins", indoles'v'" etc., and afew like celecoxib ', rofecoxib", valdecoxib' are inclinical use. In a recent report", the theoretical studieshave been carried out for the inhibition mechanism of

COX-2 with various inhibitors like celecoxib,rofecoxib etc. In spite of the fact that COX-2 activesite being chiral and difference in activity of twoenantiomers of ibuprofen" is known, most of theCOX-2 selective inhibitors like celecoxib, rofecoxibare achiral in nature.

Bearing in mind the lack of flexibility of thereported COX-2 inhibitors at the two aryl rings due totheir presence on Sp2 hybridized atoms, reasonableflexibility in the COX-2 recognition site which canadapt itself to subtle changes in the conformations ofthe inhibitor molecule and due to chirality of theCOX-2 recognition site, the search of new moleculeshaving structural rationale similar to the knowninhibitors and having varied chiral centers for theeffective binding is necessitated. During theprogramme on the development of green approachesfor the synthesis of new organic molecules, a simplestrategy was developed for the synthesis of 1,2-diaryltetrahydrofuran (DPTF) derivatives". Here,DPTF derivatives 1-4 (Figure 1) have been designedin which the two aryl rings are placed at C-2 and C-3carbons on the opposite faces of the nearly planar

4

SINGH et a/: STEREOGENIC DIARYL HETEROCYCLES AS COX-2 INHIBITORS

R

la-c 2a-c

R

4a-c3a-c

Figure 1

tetrahydrofuran ring. Either of hydroxymethyl,aminomethyl or carboxylic acid group, are present onthe C-5 carbon.

Further, since the tetrahydrofuran ring has threechiral centers and it is possible to have eight stereo-isomers, the fixation of trans-relative geometries ofthe two aryl rings at C-2 and C-3 carbons results infour stereo isomers. These constitute two pairs ofdiastereorners.

All the four stereo isomers of tetrahydrofuran(Figure 1) possessing hydroxymethyl, aminomethylor COOH group at C-5 have been considered forcomputational evaluation of their interactions withCOX-2 and COX-I enzymes. So, in total twelvetetrahydrofuran derivatives have been studied. Forcomparison and val idation of the procedures, theknown NSAlDs, both selective (rofecoxib, celecoxib,valdecoxib) and non-selective (ibuprofen, diclofenac)towards COX-2 have also been made part of dockingstudies. The results clearly demonstrate the role ofstereogenecity and the nature of the substituents onthe selective binding of OPTF derivatives withCOX-2.

Computational detailsMethodology

All molecular techniques used in this manuscriptwere performed on CAChe Worksystem Pro 6.1. Thecrystal structures of the enzymes COX-l and COX-2were downloaded from Protein Data Bank(http://www.rcsb.org/)_as POB files.

COX-2: The file containing the crystal structure ofCQX-? 'Jiit)l. its: selective inhibitor SC-558 in the

1693

active site (POB entry 6COX) was downloaded. It is adimeric structure with chains A and B each consistingof 550 residues. Each chain has SC-558, 3 moleculesof Nsacetyl-Dcglucosamine (NAG) and 1 heme(HEM) group. The chain A.with the residues, waterand the hetero groups (HEM, NAG) within a radius of5 A was refined and further cleaned by ascertainingthe hybridization and valence of each atom of SC-558and introducing H-atoms to the protein residues. Thecleaned structure of COX-2 carried a net charge of -3and 9047 atoms. The active site residues of COX-2are shown in Figure 2.

COX-I: The crystal structure of COX-l cornplexedwith ibuprofen (POB entry 1EQG)2o was downloaded.It was having a dimeric structure with two chains,each having 551 residues, 7 molecules of N-acetyl-O-glucosamine (NAG), 2 molecules of Bvocrylglucoside(BOG), one heme group and a molecule of ibuprofen.The monomer within the 5 A radius of''chain A 'wasrefined and cleaned by checking the: 'hYbd~!.izatjbli,valence of the ligand and introducing;;H-atoms.1(Hheprotein residues. COX-l carries a ~n€t,~:cgai-ge':df"+2and' 9680 atoms. The active site residues' or G:0)~Ylare shown in Figure 3. ' ,:i',' :';'Jfi1i ,',)"

All the tetrahydrofuran derivatives were built in theCAChe workspace and the structures were refined byperforming an optimized geometry calculation inMOPAC using PM5 parameters.·..",

Docking and binding evaluationsIn the automated module of CAChe Work system

]?ro 6.1, the ligands were docked into the active site ofCOX-2 and COX-J using a genetic algorithm with afast, simplified Potential of Mcan Force (PMF)2I, It is

Figure 2 - Active site residues of COX-2 with SC-558.Hydrogens are suppressed for clarity

1694 INDIAN 1. CHEM., SEC B, JULY 2006

Figure 3 - Active site residues of COX-I with ibuprofen.Hydrogens are suppressed for clarity

assumed that the protein and the ligand dock non-covalently. The standard PMF implementation usedAmber" van der Waal's potential for this purpose.The docking is carried with flexible ligand into a rigidprotein active site. The general procedure for dockingprocess starts with the addition of energy minimizedtarget ligand on the enzyme. The active site and theligand were specified in the programme. The differentstarting parameters were optimized by using15x15x15 A box located at the centre of the targetactive site using a united atom (explicit hydrogens arenot considered) potential of mean force (PMF) with adocking algorithm that has a population of 50chromosomes and runs for 6000 generations. Theprocess of docking is repeated until a constant valueof docking score is reached. This takes about 12000-18000 generations. The final results are parameterized.in terms of docking score in KcaIlmol. The dockedligand-COX-2 complex is interpreted by looking atthe H-bonding or hydrophobic interactions of theligand with the amino acid residues in the active site.The same procedure was followed for docking ofdifferent DPTFs' into the active site of COX-l andCOX-2.

Results

Validation of PMF method: To ensure thevalidity of the programme, before docking the testcompounds, the docking of SC-558 into the active siteof COX-2 was performed. This selective inhibitor ofCOX-2 binds in the active site with a binding score of-85 KcaIlmol and nns deviation of 0.45 is observed.The docked structure of SC-558 in the active site of

Figure 4 - Figure showing the close overlapping of dockedstructure of Sc-558 with its crystal structure (rms error is 0.45).

Native ligand is labeled, other is docked ligand.

Table I - Comparison of ICso (IlM) of various COX-2 andCOX-I inhibitors in Human Blood with their docking scores

(Kcal/mol)

Entry Ligand IC50 (IlM) Dock score(Kcal/mol)

COX-I COX-2 COX-1 COX-2

I Celecoxib 14 1.2 1713 -87.2

2 Rofecoxib 40 0.3 833 -54.4

3 Valdecoxib 25 0.89 479 -55.4

4 Etoricoxib 12 I.l 1042 -58

5 Ibuprofen Non-selective -21 -47.4

6 Diclofenac Non-selective -41 -27.4

COX-2 is shown in Figure 4. The close overlappingof the docked structure with the native ligand (X-raycrystal structure) ensures the validity of theprogramme.

Docking of known selective and non-selectivecyclooxygenase inhibitors into the active sites ofCOX-l and COX-2: The non-selective behaviour ofibuprofen, diclofenac and selective behaviour ofcelecoxib, rofecoxib, valdecoxib towards COX-2 isquite in agreement with their binding energies(docking score) with COX-1 and COX-2 as calculatedfrom the docking of these compounds in COX-l andCOX-2 active site (Table I). In case of COX-2selective NSAIDs viz. celecoxib, rofecoxib,valdecoxib, etoricoxib, the negative binding energiesfor COX-2 and positive binding energies with COX-l

-------_. __._...._--._----- -----

SINGH et al: STEREOGENIC D1ARYL HETEROCYCLES AS COX-2 INHIBITORS

are in agreement with their COX-2 selectivity asreported in terms of ICso values (entries 1-4, Table I).The non-selective compounds ibuprofen anddiclofenac show negative binding score with bothCOX-l and COX-2 enzymes in agreement with theirreported non-selectivity. During binding of theseknown NSAIDs in the binding pocket of COX-2, theconformational placement of amino acid residues inthe active site is observed.

Celecoxib binds in the active site of COX-2 mainlythrough hydrophobic interactions. Nr-phenyl ring issurrounded by Y355 and V523 and the sulphonamidegroup present on this ring approaches A516, H90 andG192 where NHcelecoxib---O=CG192distance is in therange of weak H-bonding (2.31 A). C-2 phenyl ring isenveloped by F518, W387, M522, G526, Y385, S353.The CF3 group present at C-4, through H-bondingbetween CF---HNRI20 (1.73 A) has blocked R120, theprominent residue involved in arachidonic acidmetabolism. Therefore, the aromatic rings present oncelecoxib through 1t-1t interactions with amino acidresidues keep it placed in the active site of COX-2and CF3 group blocks R120 via H-bonding.

Docking of DPTF Derivatives 1-4 into COX-1and COX-2 active sites: The dockings of 12 DPTFderivatives (Figure 1) in COX-2 and COX-l activesites has been studied (Table II). In these compounds,all the carbon atoms of the central core(tetrahydrofuran ring) are Sp3hybridized. The C-2 andC-3 carbons carry aryl moieties and the groups chosento be present at C, are H-donors (CH20H) as well asH-acceptors/H-donors (COOH, CH2NH2). The pairs

1695

1a-c, 4a-c and 2a-c, 3a-c are enantiomers and la-c,2a-c; 1a-c, 3a-c; 3a-c, 4a-c; 2a-c, 4a-c havediastereomeric relation with each other. These DPTFderivatives having two phenyl rings on thetetrahydrofuran moiety have some structuralsimilarities with COX-2 selective drug rofecoxib-a3,4-diphenyl-5H-furan-2-one. The decrease in stericrepulsion between two phenyl rings due to theirpresence on the opposite faces of the tetrahydrofuranring provides a degree of freedom for free rotation ofthe two aryl rings. All the 12 diphenyltetrahydrofurans go into the active site of COX-2 andshow negative docking scores. These DPTFs showpositive docking score (except for 4b, 3c and 4c) forthe COX-l enzyme, resulting in reasonable to highbinding specificities for COX-2. Therefore thesemolecules have potential to act as COX-2 selectiveNSAIDs. The difference in binding of various DPTFmolecules with COX-2 and COX-l enzymes pointstowards the significant role of various stereo isomersand the C-5 substituents in their binding abilities.

DiscussionAll the 12 DPTF molecules show binding in the

COX-2 active site with binding scores between -53.0and -67.4 Kcal/mol. However, in case of dockings ofDPTF molecules in COX-l cavity, the docking scoresvary between -19.4 to 94.2 Kcal/mol. These resultsclearly point that the stereo chemistries at C-2, C-3and C-5 centers of DPTF molecules and the nature ofthe substituent at C-5 significantly affect their bindingin COX-lICOX-2 active sites.

Table II-Docking score (Kcal/mol) of various DPTF derivatives 1-4 with COX-I(pdb 10: 1EQG) and COX-2 (pdb 10: 6COX).

ligand Configuration at Docking score (Kcal/mol) COX-2 selectivityC2, C3, C5 COX-I (Ei) COX-2 (E2) (Ei-E2)

1a 2S,3R,5R 30.8 -61.0 91.8

2a 2S,3R,5S 47.2 -56.3 103.5

3a 2R,3S,5R 28.7 -54.6 83.34a 2R,3S,5S 8.6 -60.0 68.6

Ib 2S,3R,5R 52.6 -67.4 120

2b 2S,3R,5S 94.2 -62.7 156.9

3b 2R,3S,5R 68.0 -60.0 1284b 2R,3S,5S -19.4 -65.6 85

Ie 2S,3R,5R 16.9 -60.1 77

2e 2S,3R,5S 39.4 -55.7 95.1

3e 2R,3S,5R -1.9 -53.0 54.9

4e 2R,3S,5S -11.8 -60.8 72.6

1696 INDIAN J. CHEM., SEC B, JULY 2006

Amongst the four stereo isomers, irrespective ofthe substituent at C-5, the (2R,3S,5S)-stereoisomers(4a, 4b and 4e) show highest order of binding withCOX-I resulting in lowest binding specificities ineach category. The ligands with (2S,3R,5R)-configuration (la, Ib and Ic) which are enantiomersof respective ligands 4, exhibit highest binding withCOX-2 in each category with appreciable selectivityfor COX-2 over COX-I. The graph plotting thebinding energy of DPTF molecules with COX-2 vstheir energy difference for binding with COX-2 andCOX-l (selectivity) clearly shows that in case ofenantiomers la-c and 4a-c, the selectivity are directlyproportional to the binding energies. As the bindingsof enantiomers la-c and 4a-c in the COX-2 active siteincrease, their selectivity towards COX-2 alsoincrease (Figure 5). Among the enantiomers Ia-c and4a-e, 1b shows highest binding energy (dockingscore) and also the highest selectivity for COX-2 overCOX-I. The compounds 2 and 3 are enantiomers with

1b•

----.- .• -2-b _ ... ---- .. ---- -"--160 .~

!140 -i

.3b I120 -J

o.- I> ,•.::: Iul<1)1vVJ

-70

100 l80,

3c 60 ~. .

---- --- --- 40~

-65 -60 -55 -50Binding energy (KCallmoI)

.4b

Figure 5 - The plot of binding energies of DPTF derivativeswith COX-2 (E2) vs. difference in binding energies (E1-E2)

respect to each other and have diastereomericrelationship with I and 4. Here again, the selectivity isdirectly proportional to binding with COX-2 (Figure5). 2b shows the highest selectivity among the 12DPTFs. Based upon the substituent at C-5, in all the12 diphenyl tetrahydrofurans, the order of bindingwith COX-2 as well as the selectivity for COX-2 overCOX-l is COOH >NH2>OH.

Compounds Ib, 2b and 4b show the highestbinding with COX-2 in comparison to their respectiveisomers. The enantiomers 2b and 3b with COOHfunctional group at C-5 show higher specificity than2a, 2e and 3a, 3e respectively. Hence, out of the 12compounds lb and 2b show appreciable binding aswell as selectivity for COX-2 over COX-I.

The further rationalization of mode of binding ofthese DPTF molecules in active site of COX-2 hasbeen based upon the amino acid residues presentaround the ligands in the active site pocket of COX-2.On the basis of structural features essential forbinding in the cavity, the DPTF molecules could bedivided into three segments: 2-phenyl, 3-phenyl and5-carboxylic/amino, hydroxymethyl. The residuessurrounding each phenyl group and the C-5substituent have been determined (Table III). Basedupon the stereochemistry at the three chiral centers oftetrahydrofuran, three segments of DPTFs are placedinto different sub-pockets of COX-2 active site. Themost prominent bindings are observed in the case ofligands Ib and 2b. For ligand 2b, the C-2 phenyl ringis stabilized by hydrophobic interactions from Y385,W387, M522 and C-3 phenyl ring is surrounded byH90, S353 and V523. The C-5 substituent is havingvery characteristic orientation where it forms H-

ligand Configuration at C2,C), C,

Table III - Amino acid residues around each segment ofDPTF derivatives when docked into COX-2.

Ia

2a

3a4a

Ib

2b

3b

4b

Ie

2e

3e

4e

2S,3R,5R2S,3R,5S

2R,3S,5R2R,3S,5S

2S,3R,5R2S,3R,5S

2R,3S,5R2R,3S,5S

2S,3R,5R2S,3R,5S

2R,3S,5R

2R,3S,5S

2-phenyl

R 120, Y355, A527, L531,

L352, W387, Y385,

V116, RI20, Y355, A527, L531

R I20, S353, Y355, A527

L352,S353, F518, V523

Y385, W387, M522

L384, W387, S530

RI20, V349, Y355, L531

R120, A527, L531

V349, L352, Y385, W387

Y355, A527, L531

R120, Y355, A527

Amino acid residues surrounding3-phenyl

F381, Y385, W387, S530

H90, Y385, W387, F518

H90, L352, Y355, R513, V523,

L384, Y385, W387, M522

Y348, Y385, W387

H90,S353,S523

R I20, S353, Y385, A527

L352, Y385, W387, F518

Y385, W387, S530

H90, S353, F518

H90,L352, Y355,V523

L352, Y385, W387,F518

5-substituent

S353,L352

RI20,L352

W387

H90, S353, V523

RI20, Y355, V523,

RI20

H90

H90

L352, S353, V523

R120, Y355

F381, G526, S530

S353

SINGH et al: STEREOGENIC DIARYL HETEROCYCLES AS COX-2 INHIBITORS

I¥

Figure 6 - 2b docked in the active site of COX-2

~8.

Figure 7 - I b docked in the active site of COX-2

bonding with two NH groups of R120 (Figure 6).This functionality of R120 is responsible forarachidonic acid metabolism and blockage of thisguanidine moiety would render the COX-2 inactive. Itseems as 2b could be the potential COX-2 inhibitor.

In case of lb, the C-2 phenyl ring is surrounded byL352, S353, F518, V523 and C-3 phenyl ring isenveloped by Y348, Y385, W387. Its carboxylicgroup at C-5 has shifted away from the guanidine partof R 120 with distances 2.21 A and 2.85 A from twoNHs of RI20 (Figure 7) and does not show H-bonding with R120.

1697

Conclusions

The control of stereochemistries at C-2, C-3 and C-5 carbons of DPTF molecules significantly affects thebinding and selectivity in COX-l active site whichresults these molecules to be COX-2 selective withvaried selectivity orders. The presence of COOHmoiety at C-5 further enhances the binding capacityand selectivity and results in better discriminationbetween COX-l and COX-2 active sites.

Acknowledgements

The authors thank CSIR [project no.0I(l735)/02/EMR-II] and DST (project no.SR/FTP/CS-20/2001) for financial support. One of theauthors (Anu) thanks CSIR for SRF.

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;II