Development of inositol-based antagonists for the D-myo-inositol 1,4,5-trisphosphate receptor

Abstract

The syntheses of four D-myo-inositol 1,4,5-trisphosphate (InsP₃) derivatives, incorporating phosphate bioisosteres at the 5-position, are reported. The methyl phosphate ester and sulfate derivatives retain InsP₃receptor (InsP₃R) agonist activity; the compounds that possess a methylphosphonate or a carboxymethyl moiety are InsP₃R antagonists.

---

D-myo-Inositol 1,4,5-trisphosphate (InsP₃, 1, Fig. 1) is a ubiquitous intracellular second messenger that mediates a plethora of cellular functions via interaction with defined receptors (InsP₃Rs).^1,2 These receptors are ligand-gated Ca²⁺-releasing channels that are located predominantly on the endo- or sarcoplasmic reticulum (ER/SR). The central signalling role of InsP₃ has promoted many chemical syntheses of the natural compound and numerous unnatural derivatives.^3,4 With only a few exceptions,^5–8 these compounds have all displayed full agonist activity at InsP₃Rs. However, to enable chemical dissection of the InsP₃signalling pathway, the development of selective InsP₃R antagonists is an essential aim. Although some antagonists of this receptor do exist, these compounds are not selective for the InsP₃Rs over other Ca²⁺ channels and pumps. For example, 2-aminoethoxydiphenyl borate (2-APB), the most commonly used InsP₃R antagonist,⁹ displays a number of biological actions, including inhibition of SERCA pumps¹⁰ and inhibition of store operated Ca²⁺ entry.^11,12

Fig. 1 The structures of InsP₃ (1) and its four 5-position derivatives, 2–5.

Most existing InsP₃R antagonists are not structurally related to InsP₃; indeed, only one InsP₃ derivative (3) has been reported to act as an InsP₃R antagonist and no biological data were provided for this compound.^7,8 However, it seems probable that InsP₃-based antagonists are most likely to be selective for InsP₃Rs over other cellular targets. The elucidation of the X-ray crystal structure of the mouse type 1 InsP₃R in complex with InsP₃ enables the rational design of InsP₃R ligands.¹³ This structure shows that InsP₃ binds between two flexible lobes, the α-domain and the β-domain, leading to proposals of a “clam-shell-like” mode of receptor activation.¹⁴ The 1- and 5-position phosphates of InsP₃ bind to the α-domain, whereas the 4-position phosphate binds predominantly to the β-domain. The structure confirms the findings of previous structure–activity studies that the 4- and 5-position phosphate groups are the most important for InsP₃-receptor interactions.³ We previously hypothesised that reduction of charge at the 4-position, by introducing a phosphate isostere, would lead to compounds that can bind to the InsP₃Rs. The reduced charge of these compounds would lead to them being unable to evoke the conformational change required for receptor activation, hence they would acts as competitive antagonists.¹⁵ None of the 4-position-modified compounds synthesised exhibited agonist or antagonist activity at InsP₃Rs, leading to the conclusion that the 4-position phosphate group is essential for InsP₃R binding.¹⁵ Furthermore, we hypothesised that the conformational change undergone by InsP₃Rs upon InsP₃ binding is modest, and mediated by interaction of the 5-position phosphate of InsP₃ with its receptor. To test this hypothesis, our attention turned to the development of InsP₃ derivatives that contain phosphate bioisosteres at the 5-position (Fig. 1). Again, phosphate isosteres that possess reduced charge, compared to a phosphate group, were utilised. However, more polar isosteres than used previously have been employed, to maximise the likelihood of these compounds binding to the polar InsP₃ binding pocket of the InsP₃Rs.

Herein, we report the synthesis of four 5-position-modified InsP₃ derivatives (Fig. 1). It is shown that, while compounds with a methyl phosphate ester (2) or a sulfate group (5) at the 5-position retain InsP₃R agonist activity, compounds with a methylphosphonate (3) and a carboxymethyl (4) derivative act as InsP₃R antagonists.

The synthesis of compounds 3–5 commenced from myo-inositol, and employed a well-established sequence of transformations to afford the chiral, non-racemic, camphor derivative 7 (Scheme 1).^16,17 Removal of the chiral auxiliary under acidic conditions followed by treatment with dibutyltin oxide and benzyl bromide furnished the desired 3-O-benzyl regioisomer (8) in high yield. Phosphitylation and oxidation of the 1- and 4-position hydroxyl groups gave the protected bisphosphate. The allyl group was removed by treatment with PdCl₂ in methanol, yielding the key intermediate 9. The methyl phosphonate was installed using conditions reported by Dreef et al. (Scheme 2).^7,8 In our hands, this reaction was capricious and the greatest success was achieved when using freshly prepared reagents and rigorously dry conditions. Hydrogenolysis in the presence of sodium bicarbonate afforded the methyl phosphonate 3 as its presumed pentakis sodium salt. The carboxymethyl derivative 4 was synthesised by cleavage and oxidation of the allyl group,^18,19 followed by hydrogenolysis as before. The sulfate 5 was afforded by reaction of 9 with sulfur trioxide in pyridine, followed by hydrogenolysis.

Scheme 1 The synthesis of the key intermediate 9. Reagents and conditions: a. i. AcCl, MeOH, CH₂Cl₂, 83%; ii. Bu₂SnO, BnBr, TBABr, 3 Å molecular sieves, MeCN, 79%; b. i. 1. (BnO)₂PNⁱPr₂, 1H-tetrazole, CH₂Cl₂; 2. mCPBA, CH₂Cl₂, 81%; ii. PdCl₂, MeOH, 80–84%.

Scheme 2 The synthesis of the 5-position-modified InsP₃ derivatives 3–5. Reagents and conditions: a. i. 1. Bis[6-(trifluoromethyl)benzotriazol-1-yl] methylphosphonate, pyridine; 2. BnOH, 36–60%; ii. Pd black, H₂, ^tBuOH, H₂O, NaHCO₃, 93%. b. i. RuCl₃·H₂O, NaIO₄, CCl₄, MeCN, H₂O, 44%; ii. Pd black, H₂, ^tBuOH, H₂O, NaHCO₃, 98%. c. i. SO₃, pyridine, 52%; ii. ^tBuOH, H₂O, NaHCO₃, 94%.

The methyl phosphate ester 2 was synthesised from intermediate 11, which was prepared as described previously.²⁰ Deprotection of the TIPS group, using TBAF, furnished a 1,4-diol. This compound was phosphitylated and oxidised to give the protected bisphosphate 12 (Scheme 3). Oxidative cleavage of the PMB ether, followed by phosphitylation and oxidation, gave the fully protected methyl phosphate ester 13. Hydrogenolysis, as above, furnished 2 as the presumed pentakis sodium salt.

Scheme 3 The synthesis of the 5-position-modified InsP₃ derivative 2. Reagents and conditions: a. i. TBAF, THF, 89%; ii. 1. (BnO)₂PNⁱPr₂, 1H-tetrazole, CH₂Cl₂; 2. mCPBA, CH₂Cl₂, 89%; b. i. CAN, MeCN, H₂O, 75%; ii. 1. (BnO)(OCH₃)PNⁱPr₂, 1H-tetrazole, CH₂Cl₂; 2. mCPBA, CH₂Cl₂, 92%; c. Pd black, H₂, ^tBuOH, H₂O, NaHCO₃, 51%.

The ability of 2–5 to mobilise Ca²⁺ or inhibit InsP₃-induced Ca²⁺ release (IICR) was investigated using either a unidirectional ⁴⁵Ca²⁺ flux assay in permeabilised cells or a Ca²⁺ fluorescence assay in sea urchin egg homogenate. In the ⁴⁵Ca²⁺ assay, either MEF cells or L15 cells were employed.¶ Using the ⁴⁵Ca²⁺ assay in MEF cells, the methyl phosphate ester (2) acts as an InsP₃R agonist, with an EC₅₀ = 9.7 μM (InsP₃EC₅₀ = 5.2 μM in the same system, see ESI‡). The sulfate 5 also acts as a weak InsP₃R agonist, evoking InsP₃R-mediated Ca²⁺ release when applied at 100 and 300 μM in permeabilised L15 cells (see ESI‡). The methylphosphonate 3 showed little activity when applied to permeabilised L15 cells alone. However, application of 3 (300 μM) before and during application of InsP₃ (0.25 μM) caused approximately 40% reduction in the amount of IICR (Fig. 2).

Fig. 2 Fractional loss plots of ⁴⁵Ca²⁺ in L15 cells when treated with the methylphosphonate analogue 3. The cells were permeabilised, leading to an initial loss of ⁴⁵Ca²⁺ (0–3 min). Application of InsP₃ evokes a large loss of ⁴⁵Ca²⁺ as a result of InsP₃R activation. This loss of ⁴⁵Ca²⁺ is reduced by ∼40% in the presence of the methylphosphonate 3 (300 μM), indicating that this compound is behaving as an InsP₃R antagonist.

The biological activity of 3 and 4 was also investigated using the sea urchin egg homogenate-based assay.^21–23|| In this assay both 3 and 4 behaved as InsP₃R antagonists (Fig. 3). The methylphosphonate (3) gave 37% inhibition of IICR when applied at a concentration of 1.67 mM. The carboxymethyl derivative 4 demonstrated 58% inhibition of IICR when applied at a concentration of 5.00 mM. In both cases, the compounds evoked no Ca²⁺ release when applied in the absence of InsP₃. Additionally, preliminary data (not shown) indicate that 4 has no significant effect on cyclic adenosine diphosphate ribose-induced Ca²⁺ release, which occurs viaryanodine receptors. These data indicate that 4 is acting at InsP₃Rs and not non-selectively at other Ca²⁺ channels or pumps. Taken together, our data show that 3 and 4 are acting as InsP₃R antagonists. These data represent the first conclusive proof that InsP₃ derivatives can act as antagonists at InsP₃Rs.

Fig. 3 Compounds 3 and 4 inhibit IICR. (A) A representative trace obtained from sea urchin egg homogenate loaded with the fluorescent Ca²⁺ dye Fluo-3. Application of InsP₃ (400 nM) evokes an increase in fluorescence due to Ca²⁺ release from InsP₃Rs (black trace). Application of compound 4 (5.00 mM) alone does not cause Ca²⁺ release, however, application of InsP₃ (400 nM) in the presence of 4 evokes significantly reduced Ca²⁺ release, demonstrating that 4 is an InsP₃R antagonist (grey trace). (B) A comparison of the Ca²⁺ release evoked by InsP₃ alone and in the presence of compound 3 or compound 4. The Ca²⁺ release caused by InsP₃ (400 nM) alone is taken as 100% (top bar). When InsP₃ (400 nM) and compound 3 (1.67 mM) were applied to the sea urchin egg homogenate, a smaller increase in fluorescence (63%, n = 4) was observed (middle bar), consistent with reduced Ca²⁺ release from InsP₃Rs. When InsP₃ and compound 4 (5.00 mM) were applied a similarly reduced increase in fluorescence was seen (42%, n = 4, bottom bar). These data show that both 3 and 4 are acting as InsP₃R antagonists. Error bars show SEM.

The structural similarity of the methyl phosphate ester 2 and the methylphosphonate 3, which differ by one oxygen atom yet possess opposing activity at InsP₃Rs, potentially provides unique insight into the mechanism of InsP₃R activation and inhibition. Our current hypothesis is that all compounds bind to the InsP₃R ligand-binding domain in a similar orientation to InsP₃. The methyl phosphate ester (2) and the sulfate (5) derivatives have the same number of atoms as a phosphate group (such as in InsP₃) from which to make polar contacts. However, the methylphosphonate (3) and the carboxylate (4) have one less polar atom at the 5-position. It is possible that this reduced interaction with the receptor renders the compounds unable to evoke the conformation change required for InsP₃R activation, and hence 3 and 4 act as competitive antagonists at InsP₃Rs.

In conclusion, four InsP₃ derivatives with phosphate bioisosteres at the 5-position have been synthesised. Biological evaluation of these compounds has revealed the methyl phosphate ester (2) and sulfate (5) derivatives retain InsP₃R agonist activity. However, the methylphosphonate (3) and carboxymethyl (4) derivatives behave as InsP₃R antagonists. These studies provide the first proof that InsP₃ derivatives can act as antagonists at InsP₃Rs. The compounds developed herein will likely prove useful tools for the study of InsP₃Rs and provide new insight into the mechanism of InsP₃R activation and inhibition.

The authors thank the University of St Andrews (DB) and the BBSRC (NSK, TA) for funding. SJC thanks St Hugh's College, Oxford, for research support.

Notes and references

1. M. J. Berridge, M. D. Bootman and H. L. Roderick, Nat. Rev. Mol. Cell Biol., 2003, 4, 517.
2. M. J. Berridge, Biochim. Biophys. Acta, 2009, 1793, 933.
3. B. V. L. Potter and D. Lampe, Angew. Chem., Int. Ed. Engl., 1995, 34, 1933.
4. S. J. Conway and G. J. Miller, Nat. Prod. Rep., 2007, 24, 687.
5. A. M. Rossi, A. M. Riley, S. C. Tovey, T. Rahman, O. Dellis, E. J. A. Taylor, V. G. Veresov, B. V. L. Potter and C. W. Taylor, Nat. Chem. Biol., 2009, 5, 631.
6. S. T. Safrany, R. A. Wilcox, C. Liu, D. Dubreuil, B. V. L. Potter and S. R. Nahorski, Mol. Pharmacol., 1993, 43, 499.
7. C. E. Dreef, J. P. Jansze, C. J. J. Elie, G. A. van der Marel and J. H. van Boom, Carbohydr. Res., 1992, 234, 37.
8. C. E. Dreef, W. Schiebler, G. A. van der Marel and J. H. van Boom, Tetrahedron Lett., 1991, 32, 6021.
9. T. Maruyama, T. Kanaji, S. Nakade, T. Kanno and K. Mikoshiba, Jpn. J. Biochem., 1997, 122, 498.
10. J. G. Bilmen, L. L. Wootton, R. E. Godfrey, O. S. Smart and F. Michelangeli, Eur. J. Biochem., 2002, 269, 3678.
11. M. D. Bootman, T. J. Collins, L. Mackenzie, H. L. Roderick, M. J. Berridge and C. M. Peppiatt, FASEB J., 2002, 16, 1145.
12. C. M. Peppiatt, T. J. Collins, L. Mackenzie, S. J. Conway, A. B. Holmes, M. D. Bootman, M. J. Berridge, J. T. Seo and H. L. Roderick, Cell Calcium, 2003, 34, 97.
13. I. Bosanac, J. R. Alattia, T. K. Mal, J. Chan, S. Talarico, F. K. Tong, K. I. Tong, F. Yoshikawa, T. Furuichi, M. Iwai, T. Michikawa, K. Mikoshiba and M. Ikura, Nature, 2002, 420, 696.
14. C. W. Taylor, P. C. A. da Fonseca and E. P. Morris, Trends Biochem. Sci., 2004, 29, 210.
15. D. Bello, T. Aslam, G. Bultynck, A. M. Z. Slawin, H. L. Roderick, M. D. Bootman and S. J. Conway, J. Org. Chem., 2007, 72, 5647.
16. G. F. Painter, S. J. A. Grove, I. H. Gilbert, A. B. Holmes, P. R. Raithby, M. L. Hill, P. T. Hawkins and L. R. Stephens, J. Chem. Soc., Perkin Trans. 1, 1999, 923.
17. S. J. Conway, J. Gardiner, S. J. A. Grove, M. K. Johns, Z. Y. Lim, G. F. Painter, D. Robinson, C. Schieber, J. W. Thuring, L. S. M. Wong, M. X. Yin, A. W. Burgess, B. Catimel, P. T. Hawkins, N. T. Ktistakis, L. R. Stephens and A. B. Holmes, Org. Biomol. Chem., 2010, 8, 66.
18. P. H. J. Carlsen, T. Katsuki, V. S. Martin and K. B. Sharpless, J. Org. Chem., 1981, 46, 3936.
19. C. S. Liu and B. V. L. Potter, J. Org. Chem., 1997, 62, 8335.
20. N. S. Keddie, G. Bultynck, T. Luyten, A. M. Z. Slawin and S. J. Conway, Tetrahedron: Asymmetry, 2009, 20, 857.
21. D. L. Clapper, T. F. Walseth, P. J. Dargie and H. C. Lee, J. Biol. Chem., 1987, 262, 9561.
22. A. Galione, S. Patel and G. C. Churchill, Biol. Cell, 2000, 92, 197.
23. A. J. Morgan and A. Galione, Methods, 2008, 46, 194.

---

Footnotes

† This article is part of the ‘Emerging Investigators’ themed issue for ChemComm.

‡ Electronic supplementary information (ESI) available: Additional biological data, compound characterisation data and NMR spectra for compounds 2–5. See DOI: 10.1039/c0cc03003a

§ These authors contributed equally to this work.

¶ MEF cells are immortalised wild-type mouse embryonic fibroblast cells expressing normal levels of InsP₃R1 and InsP₃R3. L15 cells are mouse L-fibroblast cells stably over-expressing InsP₃R1 by ∼8–10 fold.

|| Sea urchin eggs possess only one subtype of InsP₃R.

---