Bracke, Marc

Roman, Bart

Stevens, Christian

Van den broecke, Tom

Verhasselt, Sigrid

English

Sigrid Verhasselt

Christian V. Stevens

Tom Van den broecke

Marc E. Bracke

Bart I. Roman

Crossref

Bioorganic & Medicinal Chemistry Letters

Insights into the myosin II inhibitory potency of A-ring-modified ( S )-blebbistatin analogs

bracke, marc

Ghent University Academic Bibliography

1  Insights into the myosin II inhibitory potency of A-ring modified (S)-blebbistatin analogs Sigrid Verhasselt,a Christian V. Stevens,a,* Tom Van den broecke,a Marc E. Bracke,b Bart I. Romana,* a SynBioC Research Group, Department of Sustainable Organic Chemistry and Technology, Campus Coupure, Ghent University, Coupure Links 653, 9000 Ghent, Belgium. b Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium. *Corresponding author (bart1.roman@ugent.be or bart.roman@gmail.com; chris.stevens@ugent.be) Abstract Myosin II is an interesting target for therapeutic intervention, as it is involved in a large number of motility-based diseases. (S)-Blebbistatin is a known micromolar inhibitor of this protein. A new series of (S)-blebbistatin derivatives with a modified A-ring were synthesized and their myosin II inhibitory properties were evaluated in vitro. In this way, we gained insight into the influence of structural modifications in this part of the scaffold on myosin II inhibitory potency. Our results indicate there are few possibilities for potency enhancement via ring A modification of the blebbistatin scaffold. Keywords blebbistatin, A-ring modification, myosin II     2  (S)-Blebbistatin (S)-1 (Figure 1) is a well-known and widely used micromolar inhibitor of myosin II.1 Given the multiple roles of this protein in a diverse range of motility-based diseases,2−7 it is a promising target for therapeutic intervention. We have previously reported on the feasibility of improving (S)-blebbistatin’s myosin II inhibitory potency and physicochemical properties.8 We have also generated and analyzed SAR data on D-ring modified analogs.9 These studies have shown that D-ring modification enables fine-tuning of (S)-blebbistatin’s physicochemical properties, but potency enhancement cannot be pursued in this manner.8−12   Figure 1. State-of-the-art on SAR-information of the (S)-blebbistatin scaffold and present work.8−14 In the present report, our search for improved activity focused on the impact of structural changes in ring A. Analysis of the co-crystal structure of (S)-blebbistatin (S)-1 bound to the metastable state of Dictyostelium discoideum myosin II (PDB: 1YV3)15 shows that the residues in closest proximity to rings AB are Tyr261, Thr474, Tyr634, Gln637 and Leu641 (Figure 2). Extending the (aromatic) ring system in this part of the molecule therefore has potential to improve binding affinity through additional hydrophobic interactions and π-π stacking with Tyr261. Lucas-Lopez et al. previously reported that small-sized substituents are 3  of little influence at the C5, C6 and C7 positions, but are undesired at the C8 position.13,14 In this study, we incorporated larger cyclic substituents fused at positions C6 and C7. Analog (S)-2 was envisioned to accommodate π-π stacking with Tyr261. To overcome potential solubility issues associated with the latter compound, a more polar analog (S)-3 was also prepared. Indoline (S)-4 and N-allyl protected synthetic intermediate (S)-5 were included as aliphatic counterparts.  Figure 2. Co-crystal structure of (S)-blebbistatin (S)-1 bound to the metastable state of Dictyostelium discoideum myosin II (PDB: 1YV3)15 indicates possible additional π-π stacking interactions with Tyr261 by extending the aromatic system in ring A of the scaffold. (S)-Benzo[h]blebbistatin (S)-2 was prepared via a route that was optimized previously by us.8,9 The synthesis started from commercially available 3-amino-2-naphthoic acid (6) (Scheme 1), which was converted to methyl ester 7 upon treatment with sulfuric acid in MeOH (step (a), 96%). Reaction of pyrrolidinone 8 with POCl3 and amine 7 resulted in a 75% conversion to amidine 9 and isolated yield of 65% (step (b)). Intramolecular ring closure of the latter compound was induced after deprotonation by LiHMDS (step (c)). Asymmetric α-hydroxylation of intermediate 10 using Davis’ oxaziridine methodology yielded analog (S)-2 (step (d), 76%, ee 72%). A single recrystallization from CH3CN afforded enantiopure (S)-benzo[h]blebbistatin (S)-2 (ee >99%).  The synthesis of (S)-(N-allyl-2,3-dihydro-1H)-pyrrolo[3,2-h]blebbistatin (S)-5,  (S)-(2,3-dihydro-1H)-pyrrolo[3,2-h]blebbistatin (S)-4 and (S)-(1H)-pyrrolo[3,2-h]blebbistatin (S)-3 required the preparation of precursor 16, which was synthesized in an analogous way as described by Showalter et al. (Scheme 2).16 In short, nitration of methyl 3-methyl-4-nitrobenzoate (12) resulted in a 85:15 mixture of methyl 5-methyl-2,4-dinitrobenzoate (13) and its isomer methyl 3-methyl-2,4-dinitrobenzoate. Isolation of compound 13 out of this 4  mixture of isomers proved difficult. Efforts to purify it via normal phase and reversed phase automated flash chromatography both failed and initial recrystallization attempts in 2-propanol were not successful either. Recrystallization parameters (solvent volume, timing) appeared to be crucial and eventually we were able to isolate methyl 5-methyl-2,4-dinitrobenzoate (13) in 55% yield (step (a)). Condensation with reagent 14 gave enamine 15 (step (b)), which was reductively cyclized to precursor 16 (step (c), 81%) without intermediate isolation. Subsequent protection with allyl bromide yielded a crude mixture of unreacted starting material and mono- and diallylated regioisomers, out of which indole 17 was purified via reversed phase automated flash chromatography (step (d), 71%). Reduction with NaCNBH3 in glacial acetic acid yielded indoline 18 (step (e), 92%). The synthesis of amidine 19 proved difficult due to the rather low nucleophilic propensity of aniline 18 (step (f), 48%). One-pot intramolecular cyclization (step (g)) and enantioselective hydroxylation (step (h), 73%, ee 92%) afforded highly optically enriched (S)-(N-allyl-2,3-dihydro-1H)-pyrrolo[3,2-h]blebbistatin (S)-5 upon recrystallization from CH3CN (ee 96%). (S)-(2,3-Dihydro-1H)-pyrrolo[3,2-h]blebbistatin (S)-4 was obtained after successful allyl deprotection (step (i), 95%, ee 98%). Finally, oxidation with MnO2 yielded (S)-(1H)-pyrrolo[3,2-h]blebbistatin (S)-3 (step (j), 96%). not isolatedaSScheme 1. Synthesis of (S)-benzo[h]blebbistatin (S)-2. Reagents and conditions: (a) H2SO4, MeOH, reflux, 48 h; (b) (1) 8, POCl3, CH2Cl2, rt, 24 h, (2) 7, CH2Cl2, 35 °C, 48 h; (c) LiHMDS, THF, 0 °C, 1.5 h; (d) 11, THF, −15 °C, 16 h. a The reaction mixture initially 5  consisted of 25 mol% of 8 and 75 mol% of 9. b Determination of ee via chiral HPLC analysis. c After recrystallization from CH3CN. Scheme 2. Synthesis of (S)-(N-allyl-2,3-dihydro-1H)-pyrrolo[3,2-h]blebbistatin (S)-5, (S)-(2,3-dihydro-1H)-pyrrolo[3,2-h]blebbistatin (S)-4 and (S)-(1H)-pyrrolo[3,2-h]blebbistatin (S)-3. Reagents and conditions: (a) HNO3, H2SO4, −20 °C, 20 h; (b) 14, 1,4-dioxane, reflux, 20 h; (c) Pd/C, H2 (1 bar), 1,4-dioxane/MeOH (5:2), rt, 16 h; (d) (1) NaH, DMF, 0 °C, 30 min, (2) allyl bromide, DMF, 0 °C, 30 min; (e) NaCNBH3, glacial acetic acid, rt, 4h; (f) (1) 8, POCl3, CH2Cl2, rt, 24 h, (2) 18, CH2Cl2, 35 °C, 3 days; (g) LiHMDS, THF, 0 °C, 1.5 h; (h) 11, THF, −15 °C, 16 h; (i) N,N’-dimethylbarbituric acid, Pd(PPh3)4, CH2Cl2, reflux, 6 h; (j) MnO2, DMF, rt, 25 min. a The reaction mixture initially consisted of 44 mol% of 8 and 56 mol% of 19. b Determination of ee via chiral HPLC analysis by comparison of (R)-enantiomer enriched fractions. c After recrystallization from CH3CN. (S)-Blebbistatin derivatives (S)-2–5 were evaluated for their myosin II inhibitory properties in a steady-state ATPase assay using rabbit skeletal muscle myosin II and (S)-blebbistatin (S)-1 was used as a benchmark. Dose-response curves are presented in Figure 3 and half-maximum 6  inhibitory concentrations (IC50) are summarized in Table 1. Extending the aromatic system of ring A of the blebbistatin scaffold ((S)-2 and (S)-3) did not result in improved binding affinity. The aliphatic ring systems in analogs (S)-4 and (S)-5 too had a negative impact on myosin II inhibitory activity. Moreover, Table 1 shows there is no correlation between compound solubility and potency. Our data indicate there is little tolerance toward linear extension of the blebbistatin scaffold at the side of ring A. In addition, Lucas-Lopez et al. observed that moving the small methyl group to C8 resulted in a reduced potency and in silico analysis shows no room for fused substituents at C5 and C6.14 Thus, other modifications of ring A are also unlikely to result in improved potency. This SAR-investigation, focused on ring A, complements earlier data on ring D.8−12 Taken together, little room for potency enhancement remains except for ring C.  Figure 3. Overview of the myosin II inhibitory properties of compounds (S)-2–5, evaluated in a steady-state ATPase assay with rabbit skeletal muscle myosin II. Examples of dose-response curves and 4-parameter logistic curve fitting obtained for (A) (S)-benzo[h]blebbistatin (S)-2 and (S)-(1H)-pyrrolo[3,2-h]blebbistatin (S)-3 and (B) (S)-(2,3-dihydro-1H)-pyrrolo[3,2-h]blebbistatin (S)-4 and (S)-(N-allyl-2,3-dihydro-1H)-pyrrolo[3,2-h]blebbistatin (S)-5. Data points represent mean ± s.d. of at least three samples (N = 1). (S)-blebbistatin (S)-1 is shown 7  as a benchmark. Concentrations exceeding 10 µM, 40 µM and 20 µM caused compounds (S)-2, (S)-4 and (S)-5, respectively, to precipitate in the assay buffer. As an approximation, the relative ATPase activity obtained for (S)-blebbistatin (S)-1 at a concentration of 32.5 µM was used to set the lower asymptote of the fitted curve for these compounds. Table 1. Evaluation of the myosin II inhibitory properties of compounds (S)-1–5: half-maximum inhibitory concentrations (IC50) for the steady-state ATPase activity of rabbit skeletal muscle myosin II and solubility in the latter assay.  Compound IC50a (µM) Solubility in ATPase assay (µM)b (S)-1 1.02 ± 0.05 <100 (S)-2 7.97 ± 0.02 <15 (S)-3 >20c <40 (S)-4 14.5 ± 2.2 <50 (S)-5 8.46 ± 1.22 <40 a Data represent mean ± s.d. of two independent experiments. b Concentrations resulting in compound precipitation. c Highest compound concentration used was 20 µM, as concentrations exceeding 20 µM resulted in compound precipitation in the assay buffer. In conclusion, a new series of (S)-blebbistatin derivatives with a modified A-ring was developed in this study. The myosin II inhibitory properties of (S)-benzo[h]blebbistatin (S)-2, (S)-(1H)-pyrrolo[3,2-h]blebbistatin (S)-3, (S)-(2,3-dihydro-1H)-pyrrolo[3,2-h]blebbistatin (S)-4 and (S)-(N-allyl-2,3-dihydro-1H)-pyrrolo[3,2-h]blebbistatin (S)-5 were evaluated in a steady-state ATPase assay with rabbit skeletal muscle myosin II and proved less potent than the parent compound. Therefore, potency enhancement via modification of ring A of the blebbistatin scaffold seems unattainable. Future attempts to improve (S)-blebbistatin’s potency will focus on ring C. Acknowledgement This work was supported by the Ghent University (BOF).  8  A. Supplementary material Materials and methods, 1H and 13C NMR spectra, chiral HPLC chromatograms. References 1. Straight AF, Cheung A, Limouze J, Chen I, Westwood NJ, Sellers JR, Mitchison TJ. Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor. Science. 2003;299:1743-1747. 2. Newell-Litwa KA, Horwitz R, Lamers ML. Non-muscle myosin II in disease: mechanisms and therapeutic opportunities. Dis Model Mech. 2015;8:1495-1515. 3. Heissler SM, Manstein DJ. Nonmuscle myosin-2: mix and match. Cell Mol Life Sci. 2013;70:1-21. 4. Young EJ, Blouin AM, Briggs SB, Sillivan SE, Lin L, Cameron MD, Rumbaugh G, Miller CA. Nonmuscle myosin IIB as a therapeutic target for the prevention of relapse to methamphetamine use. Mol Psychiatry. 2016;21:615-623. 5. Zhang M, Rao PV. Blebbistatin, a novel inhibitor of myosin II ATPase activity, increases aqueous humor outflow facility in perfused enucleated porcine eyes. Invest Ophthalmol Vis Sci. 2005;46:4130-4138. 6. Liu Z, van Grunsven LA, Van Rossen E, Schroyen B, Timmermans J-P, Geerts A, Reynaert H. Blebbistatin inhibits contraction and accelerates migration in mouse hepatic stellate cells. Br J Pharmacol. 2010;159:304-315. 7. Zhai K, Tang Y, Zhang Y, Li F, Wang Y, Cao Z, Yu J, Kou J, Yu B. NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3β-NF-κB signalling pathways in the endothelium. J Thromb Haemost. 2015;114:173-185. 8. Verhasselt S, Roman BI, De Wever O, Van Hecke K, Van Deun R, Bracke M, Stevens CV. Discovery of (S)-3'-hydroxyblebbistatin and (S)-3'-aminoblebbistatin: polar myosin II inhibitors with superior research tool properties. Org Biomol Chem. 2017;15:2104-2118. 9  9. Verhasselt S, Roman BI, Bracke ME, Stevens CV. Improved synthesis and comparative analysis of the tool properties of new and existing D-ring modified (S)-blebbistatin analogs. Eur J Med Chem. 2017; doi: 10.1016/j.ejmech.2017.04.072. 10. Képiró M, Várkuti BH, Végner L, Vörös G, Hegyi G, Varga M, Málnási-Csizmadia A. para-Nitroblebbistatin, the non-cytotoxic and photostable myosin II inhibitor. Angew Chem, Int Ed. 2014;53:8211-8215. 11. Várkuti BH, Képiró M, Horváth IÁ, Végner L, Ráti S, Zsigmond Á, Hegyi G, Lenkei Z, Varga M, Málnási-Csizmadia A. A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative. Sci Rep. 2016;6:26141. 12. Képiró M, Várkuti BH, Bodor A, Hegyi G, Drahos L, Kovács M, Málnási-Csizmadia A. Azidoblebbistatin, a photoreactive myosin inhibitor. Proc Natl Acad Sci USA. 2012;109:9402-9407. 13. Lucas-Lopez C, Patterson S, Blum T, Straight AF, Toth J, Slawin AMZ, Mitchison TJ, Sellers JR, Westwood NJ. Absolute stereochemical assignment and fluorescence tuning of the small molecule tool, (–)-blebbistatin. Eur J Org Chem. 2005;1736-1740. 14. Lucas-Lopez C, Allingham JS, Lebl T, Lawson CPAT, Brenk R, Sellers JR, Rayment I, Westwood NJ. The small molecule tool (S)-(–)-blebbistatin: novel insights of relevance to myosin inhibitor design. Org Biomol Chem. 2008;6:2076-2084. 15. Allingham JS, Smith R, Rayment I. The structural basis of blebbistatin inhibition and specificity for myosin II. Nat Struct Mol Biol. 2005;12:378-379. 16. Showalter HDH, Sun L, Sercel AD, Winters RT, Denny WA, Palmer BD. Concise syntheses of the novel 1H-pyrrolo[3,2-g]quinazoline ring system and its [2,3-f] angular isomer. J Org Chem. 1996;61:1155-1158. 

Insights into the myosin II inhibitory potency of A-ring-modified (S)-blebbistatin analogs

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Insights into the myosin II inhibitory potency of A-ring-modified (S)-blebbistatin analogs

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