Novel homologated-apio adenosine derivatives as A₃ adenosine receptor agonists: design, synthesis and molecular docking studies

Abstract

Synthesis of a series of novel homologated-apio adenosine analogues including homologated-apio IB-MECA and Cl-IB-MECA has been accomplished from a commercially available, inexpensive starting material D-ribose. The synthetic route includes a controlled oxidative cleavage of vicinal diol and Mitsunobu condensation reactions as the key steps. The molecular docking studies of the synthesized compounds to the A₃ adenosine receptor model indicated them as agonists. Interestingly, some of the molecules showed good binding interactions at the active site as evident by docking scores and MM/GBSA binding affinity.

---

Introduction

The adenosine receptors (ARs) belong to the rhodopsin-like purinergic G protein-coupled receptors (GPCRs) and are comprised of four distinct subtypes A₁, A_2A, A_2B and A₃. ARs show a significant responsibility in the regulation of many physiological functions through binding with an endogenous adenosine.¹ They are considered as promising therapeutic targets for the treatment of many types of diseases related to signal transduction pathways because the binding of adenosine to the ARs alters the levels of the second messenger such as diacylglycerol (DAG) and cyclic 3′,5′-monophosphate (cAMP) for signal transduction.² In the past decades many efforts have been made to develop therapeutic agents based on selective interaction³ with one of the four adenosine receptor sub types. A₃AR, the most recently identified among the four has been modulated for the treatment of several diseases such as cancer, cardiac ischemia, asthma, glaucoma, and inflammation.⁴ On the basis of structure–activity relationship studies (SAR), several AR ligands have been synthesized and explored as agonist and/or antagonist to it.⁵ However, a few nucleoside agonists of the A₃ adenosine receptor (A₃AR) are currently in clinical trials such as N⁶-(3-iodobenzyl)-5′-N-methyl-carboxamidoadenosine (IB-MECA) 1 for the treatment of autoimmune inflammatory diseases, rheumatoid arthritis as well as plaque psoriasis and Cl-IB-MECA 2 for treatment of hepatocellular carcinoma and liver cancer.⁶ In addition, CP-532,903 3 exhibits an anti-inflammatory effect, involves in myocardial ischemia and reduces superoxide production in damaged tissues.⁷ These modified nucleoside drugs are especially very attractive as they can directly stifle or block transcription and the expression of proteins that cause the diseases.

With the recent emergence of the new frontier of drug discovery based on the GPCRs, design, synthesis and pharmacological evaluation of novel A₃AR agonists of high affinity and selectivity are urgently warranted. On this context, as an annexe to the SAR of AR agonists, we designed some homologated apio adenosine derivatives as potential A₃AR modulators (Fig. 1). Apionucleosides, one of the sugar ring modified nucleoside where the hydroxymethyl side chain is shifted from 4′ to the 3′ position, have concerned much attention ascribed to the stabilization of the glycosyl bond under acidic conditions, metabolic resistance and significance biological activity.⁸ On the other hand, introduction of a C–C single bond between the nucleobase and sugar ring augments the free rotation which permits the nucleoside to adopt conformations appropriate to induce good interactions at the binding site of A₃AR receptor.⁹ Herein, we wish to report the design, molecular docking studies and the synthesis of novel homologated apio adenosine analogues as A₃ adenosine receptors.

Fig. 1 The rationale for the design of the target nucleosides.

Results and discussion

As part of our ongoing research interest in development of potent modified nucleoside templates, in our previous work we have reported the synthesis of apio and homologated apio pyrimidine nucleosides by lead tetraacetate mediated one pot oxidative cleavage and acetylation strategy.¹⁰ Here the synthetic strategy to the desired homologated apio adenosine derivatives involved a controlled oxidative cleavage of vicinal diol and condensation of the homologated glycosyl donor with purine base under Mitsunobu condition as the key steps. The apio homologated glycosyl donor 15 was first synthesized from D-ribose as shown in Scheme 1. The lactol 8 was readily prepared from D-ribose by the reported procedure.¹¹ The stereoselective hydroxymethylation on lactol 8 using 37% aqueous formaldehyde in methanol gave the hydoxymethylated compound 9 which constructed the apio architecture. The selective primary hydroxyl group protection of the hydroxymethylated compound 9 was perpetrated by TBDPSCl to provide tert-butyldiphenylsilyl ether 10 in very good yield. The reduction of compound 10 using sodium borohydride in methanol formed the diol 11 and subsequent exposure of the diol 11 to p-tolunesulfonylchloride in dry pyridine at room temperature furnished the desired olefin 12 by primary alcohol tosylation and in situ tetrahydrofuran formation.^10,12 The dihydroxylation of olefin derivative 12 was performed by OsO₄ and NMO to exhibit the diol 13 in good yield. The controlled oxidative cleavage of the diol 13 by lead tetraacetate at 0 °C presented the aldehyde 14, which upon reduction with sodium borohydride produced the pivotal alcohol 15.

Scheme 1 Synthesis of the key glycosyl donor 15.

The condensation of 6-chloropurine with the glycosyl donor 15 was successfully achieved under Mitsunobu condition¹³ to afford the purine derivative 16, which underwent desilylation by tetrabutylammonium fluoride (TBAF) to give the compound 17. The removal of the isopropylidine group of 17 using 3 N HCl rendered the homologated apio purine 18. Treatment of 6-chloropurine derivative 18 with 3-halobenzyl amines yielded the N⁶-(3-halobenzyl)amine derivatives 6a–d (Scheme 2).

Scheme 2 Synthesis of 6-Cl purine derivatives.

Similarly the glycosyl donor 15 was condensed to 2,6-dichloro purine under Mitsunobu condition to produce the 2,6-dichloro purine derivative 19. However, unlike the previous instance the TBDPS deprotection of the compound 19 by TBAF dispensed the desired desilylated product in a poor yield. As a consequence, to avoid the pitfall we swapped the sequence of the reactions as described in Scheme 3.

Scheme 3 Synthesis of 2,6-dichloro purine derivatives.

The 2,6-dichloro purine derivative 19 was first treated with 3-halobenzyl amines to deliver the corresponding substituted products 20a–d regioselectively. Then the N⁶-(3-halobenzyl)amine derivatives 20a–d were sequentially deprotected, initially desilylation using TBAF without any difficulty followed by acetonide hydrolysis by 3 N HCl to give the final nucleosides 7a–d.

The synthesis of homologated apio analogues of IBMECA 4 and Cl-IBMECA 5 were also been achieved successfully as illustrated in Scheme 4. The 6-chloro purine derivative 16 and 2,6-dichloro purine derivative 19 were treated with 3-iodobenzyl amines individually to furnish the corresponding N⁶-(3-iodobenzyl)amino purine derivatives 22 and 20d. The TBDPS group of the N⁶-(3-iodobenzyl)amino purine derivatives 22 and 20d were deprotected by TBAF and the resulting desilylated compounds 23 and 21d were treated with PDC in DMF to afford the respective acids 24 and 25. The coupling of methyl amine with the acid derivatives 24 and 25 in presence of HOBt and EDC yielded the methylamido adducts 26 and 27. The deprotection of the acetonide groups by 3 N HCl in 26 and 27 afforded the final IB-MECA and Cl-IB-MECA analogues 4 and 5 respectively. All the synthesized compounds were thoroughly characterized by ¹H NMR, ¹³C NMR, ¹⁹F NMR, HRMS and UV spectroscopic techniques.

Scheme 4 Synthesis of homologated-apio IB-MECA and Cl-IB-MECA.

Molecular modelling and docking studies

Preliminary protein models were built using Modeller v9.13 software utilizing the ligand supported homology modelling mode.¹⁴ The generated models were validated for stereochemical accuracy using Ramachandran plot analysis. It was found that majority of the modelled residues were well within the allowed regions of the plot with only a few (<1%) in the non-allowed regions. Additionally, a visual inspection of residues lining the cavity of A₃AR binding pocket especially Ser271, Asn250 and Phe168 were checked for proper orientation and interactions with adenosine molecule. The generated model was quite similar structurally, as indicated by calculated root mean square deviation (RMSD) of 0.261 and well superimposed modelled active site residues with the template (PDB ID: 2YDO) residues (Fig. 2). The modelled A₃AR resembled the structure of template helical bundle and ECL2, which is the loop near adenosine binding site and has a defined secondary structure (antiparallel beta-sheet). This loop appears to be constrained in its position by means of the disulfide bridge (Cys83-Cys166) that linked it to the upper part of TM3. This equipped structure has been employed for extensive docking studies of A₃AR agonists and the results were compared with SARs data to identify key ligand–receptor interactions involved in the molecular recognition process so as to provide a basis for rational design of new A₃AR agonists. The receptor grid box was generated by employing the modelled structure and keeping the adenosine molecule in the complex as grid centre with applying default settings. The molecules were then docked in the A₃AR binding site using standard precision (SP) dock in Glide by treating a single HB constraint of Ser271.¹⁵ This constraint was chosen because it is known that the Ser271 plays a critical role in molecular recognition process for agonists of this receptor.^3c The docking solutions were manually checked and the poses were selected based on interactions with important residues and docking score.

Fig. 2 The cartoon representation of active sites of A₃AR modelled protein based on A₂AR template protein (PDB ID: 2YD0). Where each coloured helix represents the individual transmembrane domain.

As stated earlier the molecules were docked in the modelled A₃AR structure using SP dock protocol and binding energy estimation was carried out by MM/GBSA tool in Maestro.¹⁶ During the binding energy estimation, water molecules were excluded and partial flexibility was applied to the residues within 5 Å of the docked pose. All other default settings were used for MM/GBSA calculation.¹⁷ The reported agonists and synthesized molecules were checked for the docking orientation and interactions with in the active site residues (Table 1). All molecules were docked into the active sites where each molecule exposed its possible binding position to fit inside the pocket. From the docking score and MM/GBSA score it could be easily understood that all the synthesized molecules showed good binding affinity towards A₃AR receptor pocket. All of them have shown docking score greater than −5.8 kcal mol⁻¹ and MM/GBSA score greater than −30 kcal mol⁻¹. Moreover, from the detailed analysis, we could also observe that the molecules have shown good relative binding interactions, comparing with reported molecules.^3c,5b,6

Table 1 The docking and (MM/GBSA) scores of the docked molecules^a

Reported compounds					Synthesized compounds
Sl No.	Compound name	Docking score (kcal mol^−1)	MM/GBSA score (kcal mol^−1)	Interactions	Sl No.	Compound name	Docking score (kcal mol^−1)	MM/GBSA score (kcal mol^−1)	Interactions
a S = Ser, F = Phe, N = Asn, T = Thr, L = Leu.
1	IBMECA	−8.1	−47.9	S271,N250,F168,S73,T94	1	6a	−7.0	−30.3	S271,T94
2	Cl-IBMECA	−8.7	−76.0	S271,N250,F168,S73,T94	2	6b	−5.8	−45.4	N250
3	Thio-Cl-IB-MECA^5b,f	−9.3	−101.3	S271,N250,F168,S73,T94	3	6c	−6.3	−70.7	S271,N250
4	N^6-(3-Iodobenzyl)apioadenosine^3c	−7.2	−43.7	S271,N250,F168,S73,T94	4	6d	−6.5	−88.7	N250,L90
					5	7a	−7.1	−82.7	S271,N250,F168
					6	7b	−7.3	−98.3	S271,N250,F168
					7	7c	−7.3	−91.3	S271,N250,F168
					8	7d	−7.0	−101.1	S271,N250,F168
					9	4	−7.4	−82.1	F168,S181
					10	5	−6.8	−82.0	S271,F168,S181

---

An analysis of crystal structure of A₂AR complexed with adenosine (PDB ID: 2YDO) revealed that adenosine interacts with residues namely Phe168, Glu169, Asn253, Ser277 and His278. In case of reported molecules, the docking in A₃AR showed that Phe168, Ser271, and Asn250 were commonly involved in agonistic binding of molecule. These results were in-line with earlier findings where it was known that interactions with Phe168, and Asn250 were required for binding and interaction with Ser271 was required for agonistic activity.^3c The docking of synthesized molecules in A₃AR indicated that 2,6-dichloro purine derived molecules 7a–d (Fig. 3) settled nicely in the cavity, making strong hydrogen bond and π–π interaction with the active site. The side chain amino group of Asn250 (TM6) was directly involved in hydrogen bonding with 2′-hydroxyl group of ribose moiety. The molecules remained anchored inside the pocket by π–π stacking interaction between rigid aromatic ring of Phe168 (TM5) and adenine ring. Additionally, the 3′-hydroxyl group of sugar moiety and Ser271 (TM7) forms a hydrogen bonding interaction that is required for agonist binding. On the other hand, rest of the molecules showed interaction with only two of the three residues while a few were unsuccessful to interact with these residues. Since all molecules were sharing a common core, MM/GBSA binding energies were applied to rank them as per their binding affinity. It is also important to note that MM/GBSA procedure includes minimization of the binding site residues that mimic the flexibility of the binding site. The 2,6-dichloro purine derived molecules 7a–d showed better binding in term of docking and (MM/GBSA) scores as compared with rest of the molecules. The binding interaction between ligand and residues is shown in Fig. 3.

Fig. 3 Summary of the binding interactions of the modelled A₃AR protein active site residues with the synthesized molecules. (A) Native adenosine, (B) 7c, (C) 7a, (D) 7b, (E) 7d, (F) superimposition of all the ligands (adenosine and 7a–d).

Conclusion

In summary, a series of N⁶-substituted homologated apio adenosine derivatives along with homologated apio IB-MECA and Cl-IB-MECA have been designed, synthesized and their molecular docking studies to A₃AR have also been depicted. An effective and proficient synthetic protocol towards homologated apio adenosines has been delineated using Pb(OAc)₄ mediated controlled oxidative cleavage of vicinal diol and Mitsunobu condensation reactions as the key steps. Most of the compounds have shown to be potent A₃AR agonists by molecular docking studies. Interestingly, some of the molecules (7a–d) showed interactions with critical active site residues that are required for binding and agonist activity. It is evident from the docking scores and MM/GBSA binding affinity that the molecules possess selective binding interactions at the active sites of the receptor. This study may provide good insight towards the rational design of new molecules with strong binding affinity to A₃ARs. The detailed pharmacological evaluation of the compounds is under process and will be published in due course.

Acknowledgements

This work was partially supported by Department of Biotechnology (DBT), Govt. of India (No. BT/PR5430/MED/29/566/2012). The authors thank CIF, IIT Bhubaneswar for characterization.

References

1. (a) J. F. Chen, H. K. Eltzschig and B. B. Fredholm, Nat. Rev. Drug Discovery, 2013, 12, 265; (b) B. B. Fredholm, A. P. IJzerman, K. A. Jacobson, J. Linden and C. E. Müller, Pharmacol. Rev., 2011, 63, 1; (c) S. A. Hutchinson and P. J. Scammells, Curr. Pharm. Des., 2004, 10, 2021; (d) C. E. Müller, Curr. Top. Med. Chem., 2003, 3, 445; (e) P. Fishman and S. Bar-Yehuda, Curr. Top. Med. Chem., 2003, 3, 463; (f) B. B. Fredholm, A. P. IJzerman, K. A. Jacobson, K. N. Klotz and J. Linden, Pharmacol. Rev., 2001, 53, 527; (g) M. E. Olah and G. L. Stiles, Pharmacol. Ther., 2000, 85, 55; (h) K. A. Jacobson, P. J. M. van Galen and M. Williams, J. Med. Chem., 1992, 35, 407.
2. (a) K. A. Jacobson and Z. G. Gao, Nat. Rev. Drug Discovery, 2006, 5, 247; (b) K. N. Klotz, Naunyn-Schmiedeberg's Arch. Pharmacol., 2000, 362, 382.
3. (a) A. Rodríguez, A. Guerrero, H. Gutierrez-de-Terán, D. Rodríguez, J. Brea, M. I. Loza, G. Roselle and M. P. Bosch, Med. Chem. Commun., 2015, 6, 1178; (b) S. M. Devine, L. T. May and P. J. Scammells, Med. Chem. Commun., 2014, 5, 192; (c) K. S. Toti, S. M. Moss, S. Paoletta, Z. G. Gao, K. A. Jacobson and S. Van Calenbergh, Bioorg. Med. Chem., 2014, 22, 4257; (d) L. S. Jeong, S. A. Choe, P. Gunaga, H. O. Kim, H. W. Lee, S. K. Lee, D. K. Tosh, A. Patel, K. K. Palaniappan, Z.-G. Gao, K. A. Jacobson and H. R. Moon, J. Med. Chem., 2007, 50, 3159; (e) S. K. Kim, Z. G. Gao, P. V. Rompaey, A. S. Gross, A. Chen, S. V. Calenbergh and K. A. Jacobson, J. Med. Chem., 2003, 46, 4847.
4. (a) S. L. Cheong, S. Federico, G. Venkatesan, A. L. Mandel, Y. M. Shao, S. Moro, G. Spalluto and G. Pastorin, Med. Res. Rev., 2011, 33, 235; (b) G. J. Chen, B. K. Harvey, H. Shen, J. Chou, A. Victor and Y. Wang, J. Neurosci. Res., 2006, 84, 1848; (c) N. J. Press, T. H. Keller, P. Tranter, D. Beer, K. Jones, A. Faessler, R. Heng, C. Lewis, T. Howe, P. Gedeck, L. Mazzoni and J. R. Fozard, Curr. Top. Med. Chem., 2004, 4, 863; (d) L. Madi, S. Bar-Yehuda, F. Barer, E. Ardon, A. Ochaion and P. Fishman, J. Biol. Chem., 2003, 278, 42121–42130; (e) P. G. Baraldi, B. Cacciari, R. Romagnoli, S. Merighi, K. Varani, P. A. Borea and G. Spalluto, Med. Res. Rev., 2000, 20, 103.
5. (a) D. K. Tosh, S. Paoletta, Z. Chen, S. Crane, J. Lloyd, Z.-G. Gao, E. T. Gizewski, J. A. Auchampach, D. Salveminib and K. A. Jacobson, Med. Chem. Commun., 2015, 6, 555; (b) L. S. Jeong, H. W. Lee, K. A. Jacobson, H. O. Kim, D. H. Shin, J. A. Lee, Z.-G. Gao, C. Lu, H. T. Duong, P. Gunaga, S. K. Lee, D. Z. Jin, M. W. Chun and H. R. Moon, J. Med. Chem., 2006, 49, 273; (c) Y. Chen, R. Corriden, Y. Inoue, L. Yip, N. Hashiguchi, A. Zinkernagel, V. Nizet, P. A. Insel and W. G. Junger, Science, 2006, 314, 1792; (d) S. Merighi, A. Benini, P. Mirandola, S. Gessi, K. Varani, E. Leung, S. Maclennan and P. A. Borea, Biochem. Pharmacol., 2006, 72, 19; (e) H. Yang, M. Y. Avila, K. Peterson-Yantorno, M. Coca-Prados, R. A. Stone, K. A. Jacobson and M. M. Civan, Curr. Eye Res., 2005, 30, 747; (f) L. S. Jeong, D. Z. Jin, H. O. Kim, D. H. Shin, H. R. Moon, P. Gunaga, M. W. Chun, Y.-C. Kim, N. Melman, Z.-G. Gao and K. A. Jacobson, J. Med. Chem., 2003, 46, 3775; (g) Z. G. Gao, S.-K. Kim, T. Biadatti, W. Chen, K. Lee, D. Barak, S. G. Kim, C. R. Johnson and K. A. Jacobson, J. Med. Chem., 2002, 45, 4471; (h) R. Volpini, S. Costanzi, C. Lambertucci, S. Vittori, K.-N. Klotz, A. Lorenzen and G. Cristalli, Bioorg. Med. Chem. Lett., 2001, 11, 1931; (i) K. A. Jacobson, S. M. Siddiqi, M. E. Olah, X. d. Ji, N. Melman, K. Bellamkonda, Y. Meshulam, G. L. Stiles and H. O. Kim, J. Med. Chem., 1995, 38, 1720.
6. (a) M. H. Silverman, V. Strand, D. Markovits, M. Nahir, T. Reitblat, Y. Molad, I. Rosner, M. Rozenbaum, R. Mader, M. Adawi, D. Caspi, M. Tishler, P. Langevitz, A. Rubinow, J. Friedman, L. Green, A. Tanay, A. Ochaion, S. Cohen, W. D. Kerns, I. Cohn, S. Fishman-Furman, M. Farbstein, S. B. Yehuda and P. Fishman, J. Rheumatol., 2008, 35, 41; (b) S. Bar-Yehuda, S. M. Stemmer, L. Madi, D. Castel, A. Ochaion, S. Cohen, F. Barer, A. Zabutti, G. Perez-Liz, L. Del Valle and P. Fishman, Int. J. Oncol., 2008, 33, 287; (c) Z. G. Gao and K. A. Jacobson, Expert Opin. Emerging Drugs, 2011, 16, 597.
7. (a) T. C. Wan, Z. D. Ge, A. Tampo, Y. Mio, M. W. Bienengraeber, W. R. Tracey, G. J. Gross, W. M. Kwok and J. A. Auchampach, J. Pharmacol. Exp. Ther., 2007, 324, 234; (b) D. van der Hoeven, T. C. Wan and J. A. Auchampach, Mol. Pharmacol., 2008, 74, 685.
8. (a) T. B. Sells and V. Nair, Tetrahedron Lett., 1993, 34, 3527; (b) V. Nair and Z. Nuesca, J. Am. Chem. Soc., 1992, 114, 7951; (c) Y. Terao, M. Akamatsu and K. Achiwa, Chem. Pharm. Bull., 1991, 39, 823.
9. (a) H. W. Lee, W. J. Choi, K. A. Jacobson and L. S. Jeong, Bull. Korean Chem. Soc., 2011, 32, 1620; (b) H. W. Lee, H. O. Kim, W. J. Choi, S. Choi, J. H. Lee, S.-G. Park, L. Yoo, K. A. Jacobson and L. S. Jeong, Bioorg. Med. Chem., 2010, 18, 7015.
10. A. Panda, S. Islam, M. K. Santra and S. Pal, RSC Adv., 2015, 5, 82450.
11. (a) A. Panda, S. Das and S. Pal, Carbohydr. Res., 2014, 398, 13; (b) H. R. Moon, H. O. Kim, K. M. Lee, M. W. Chun, J. H. Kim and L. S. Jeong, Org. Lett., 2002, 4, 3501.
12. R. J. B. H. N. van den Berg, T. J. Boltje, C. P. Verhagen, R. E. J. N. Litjens, G. A. van der Marel and H. S. Overkleeft, J. Org. Chem., 2006, 71, 836.
13. W. J. Choi, H. J. Chung, G. Chandra, V. Alexander, L. X. Zhao, H. W. Lee, A. Nayak, M. S. Majik, H. O. Kim, J. H. Kim, Y. B. Lee, C. H. Ahn, S. K. Lee and L. S. Jeong, J. Med. Chem., 2012, 55, 452.
14. N. Eswar, B. Webb, M. A. Marti-Renom, M. S. Madhusudhan, D. Eramian, M. Y. Shen, U. Pieper and A. Sali, Comparative Protein Structure Modeling Using Modeller, Current protocols in bioinformatics, ed. A. D. Baxevanis, et al., 2006, ch. 5, unit 5.6.
15. R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren, J. J. Klicic, D. T. Mainz, M. P. Repasky, E. H. Knoll, M. Shelley, J. K. Perry, D. E. Shaw, P. Francis and P. S. Shenkin, J. Med. Chem., 2004, 47, 1739.
16. Schrödinger Release 2015-4: Maestro, version 10.4, Schrödinger, LLC, New York, NY, 2015.
17. S. Sirin, R. Kumar, C. Martinez, M. J. Karmilowicz, P. Ghosh, Y. A. Abramov, V. Martin and W. Sherman, J. Chem. Inf. Model., 2014, 54, 2334.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra26416b

---