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

Article abstract of DOI:10.1016/j.bmcl.2017.05.008

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

Full text of DOI:10.1016/j.bmcl.2017.05.008

Accepted Manuscript
Insights into the myosin II inhibitory potency of A-ring modified (S)-blebbis-
tatin analogs
Sigrid Verhasselt, Christian V. Stevens, Tom Van den broecke, Marc E. Bracke,
Bart I. Roman
PII:
S0960-894X(17)30489-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.008
BMCL 24953
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
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Revised Date:
Accepted Date:
1 April 2017
2 May 2017
3 May 2017
Please cite this article as: Verhasselt, S., Stevens, C.V., Van den broecke, T., Bracke, M.E., Roman, B.I., Insights
into the myosin II inhibitory potency of A-ring modified (S)-blebbistatin analogs, Bioorganic & Medicinal
Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.05.008
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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
1

(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
−7
diseases,2 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
−12
this manner.8
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
2

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)
3

and its isomer methyl 3-methyl-2,4-dinitrobenzoate. Isolation of compound 13 out of this
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%).
Scheme 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)
°C, 16 h. a The reaction mixture initially
−15
LiHMDS, THF, 0 °C, 1.5 h; (d) 11, THF,
4

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,
a
DMF, rt, 25 min. 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
5

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
6

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.
IC50a (µM) Solubility in ATPase assay (µM)b
Compound
(S)-1
(S)-2
(S)-3
(S)-4
(S)-5
1.02 0.05
7.97 0.02
>20c
<100
<15
<40
<50
<40
14.5 2.2
8.46 1.22
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).
7

A. Supplementary material
Materials and methods, 1H and 13C NMR spectra, chiral HPLC chromatograms.
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