Discovery of (S)-3′-hydroxyblebbistatin and (S)-3′-aminoblebbistatin: polar myosin II inhibitors with superior research tool properties

Article abstract of DOI:10.1039/c7ob00006e

In search of myosin II inhibitors with superior research tool properties, a chemical optimization campaign of the blebbistatin scaffold was conducted in this paper. (S)-Blebbistatin is the best known small-molecule inhibitor of myosin II ATPase activity. Unfortunately, as a research tool this compound has several deficiencies: it is photolabile and (photo)toxic, has low water solubility, and its (fluorescent) precipitates interfere in (fluorescence) readouts. In view of obtaining tool compounds with improved properties, both enantiomers of a series of D-ring modified polar analogs were prepared. We identified (S)-3′-hydroxyblebbistatin (S)-2 and (S)-3′-aminoblebbistatin (S)-3 as two myosin II inhibitors with a 30-fold higher water solubility than (S)-blebbistatin. These molecules furthermore do not cause interference in (fluorescence) readouts. (S)-2 and (S)-3 thus are superior alternatives to (S)-blebbistatin as research tools to study myosin II.

Full text of DOI:10.1039/c7ob00006e

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Discovery of (S)-3’-hydroxyblebbistatin and (S)-3’-aminoblebbistatin: polar
myosin II inhibitors with superior research tool properties
Sigrid Verhasselt,[a] Bart I. Roman,[a]* Olivier De Wever,[b] Kristof Van Hecke,[c] Rik Van
Deun,[c] Marc E. Bracke,[b] and Christian V. Stevens[a]*
[a] SynBioC Research Group, Department of Sustainable Organic Chemistry and Technology,
Ghent University, Coupure Links 653, 9000 Ghent (Belgium)
*E-mail: bart1.roman@ugent.be, chris.stevens@ugent.be
[b] Laboratory of Experimental Cancer Research, Department of Radiation Oncology and
Experimental Cancer Research, Ghent University, De Pintelaan 185, 9000 Ghent (Belgium)
[c] Department of Inorganic and Physical Chemistry, Ghent University, Krijgslaan 281 S3, 9000
Ghent (Belgium)
ABBREVIATIONS. ∆, reflux; 2-mTHF, 2-methyltetrahydrofuran; APCI, atmospheric pressure
chemical ionization; cps, counts per second; CV, column volume; DMEM, Dulbecco’s modified
Eagle medium; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; N.D.,
not determined; s.d., standard deviation.
1

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ABSTRACT. In search of myosin II inhibitors with superior research tool properties, a chemical
optimization campaign of the blebbistatin scaffold was conducted in this paper. (S)-blebbistatin
is the best known small-molecule inhibitor of myosin II ATPase activity. Unfortunately, as a
research tool this compound has several deficiencies: it is photolabile and (photo)toxic, has low
water solubility, and its (fluorescent) precipitates interfere in (fluorescence) readouts. In view of
obtaining tool compounds with improved properties, both enantiomers of a series of D-ring
modified polar analogs were prepared. We identified (S)-3’-hydroxyblebbistatin (S)-2 and (S)-3’-
aminoblebbistatin (S)-3 as two myosin II inhibitors with a 30-fold higher water solubility than
(S)-blebbistatin. These molecules furthermore do not cause interference in (fluorescence)
readouts. (S)-2 and (S)-3 thus are superior alternatives to (S)-blebbistatin as research tools to
study myosin II.
2

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INTRODUCTION
Myosins are actin-based ATP-driven molecular motor proteins.1 They play a key role in many
forms of eukaryotic motility, such as muscle contraction, cytokinesis, intracellular trafficking,
phagocytosis, and cell movement.2-4 (S)-blebbistatin (S)-1 (Figure 1) is a micromolar, cell-
permeable ATPase cycle inhibitor of myosin II. Its main virtue is specificity: it does not
modulate myosins I, V, and X.5
Figure 1. Overview of the state of the art and present work.
Since its discovery in 2001,6 (S)-blebbistatin (S)-1 has rapidly been embraced as a research
tool: it has played a significant role in the understanding of the function of myosin II in
biological processes,2-4 and has provided insight into the cellular and molecular events that drive
a range of myosin II-related diseases, such as invasion in malignant disease,7-10 viral
infections,11-13 bacterial infections,14 glaucoma,15 progressive renal disease,16 and
methamphetamine use relapse.17 However, as is the case for other ‘first-in-class’ tool
compounds, (S)-blebbistatin (S)-1 bears deficiencies that encumber or prohibit its use in
3

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sophisticated model systems: it is photolabile,18-23 (photo)toxic,18,21,22 has limited water
solubility,23-25 and its (fluorescent) precipitates interfere in (fluorescence) readouts.26-28 The main
objective in the present study was therefore to develop (S)-blebbistatin analogs with superior
research tool characteristics. We hypothesized that this would require the introduction of polar
functionalities on its scaffold (Figure 1): the ensuing increased aqueous solubility would
eliminate assay readout issues by preventing the formation of precipitates.
Following this rationale, the novel analogs (S)-3’-hydroxyblebbistatin (S)-2, (S)-3’-
aminoblebbistatin (S)-3, (S)-3’-cyanoblebbistatin (S)-13, (S)-3’-carbamoylblebbistatin (S)-16,
and (S)-3’-carboxyblebbistatin (S)-17, and their R-enantiomers (negative controls),29 were
synthesized. The myosin II inhibitory activity of these compounds was evaluated in a
biochemical assay (i.e. an ATPase assay). We furthermore discuss the physicochemical
properties of these molecules, including solubility and fluorescence data. During the final
preparation of this manuscript, a paper on a single polar blebbistatin derivative with improved
physicochemical properties was published by Varkúti et al.30 Our study provides broader context
by the exploration of an elaborate family of polar analogs. We furthermore provide detailed
synthetic protocols and analytical data, in order to facilitate the broad use of our molecules as
research tool compounds to study myosin II.
RESULTS AND DISCUSSION
1. Chemistry
Polar blebbistatin analogs (S)- and (R)-2,3,13,16,17 were prepared after select adaptation of a
route that has proven successful for (S)-blebbistatin (S)-1.20 The syntheses commenced with
arylations of 2-pyrrolidinone (5) in very good yields (Scheme 1).
4

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Scheme 1. Synthesis of quinolones 9a–d. Reagents and conditions: (a) CuI, 5, tris(3,5-dimethyl-
1H-pyrazol-1-yl)methane, K3PO4, 1,4-dioxane, reflux, 24–72 h; (b) 1) POCl3, CH2Cl2, rt, 24 h,
2) 7, CH2Cl2, 35 °C, 24 h; (c) LiHMDS, THF, 0 °C, 1 h.
The synthesis of amidine 8a, starting from pyrrolidinone 6a and amine 7, was performed in the
presence of phosphorus oxychloride via a modified procedure of Lucas-Lopez et al.20 The
amount of phosphorus oxychloride was doubled and the reaction time for the formation of the
chloroiminium intermediate was increased from 3 h to 24 h. In this way, a conversion of 95%
was obtained (Table 1). Isolation of the amidine out of the crude reaction mixture via acid-base
extraction, as reported in the procedure of Lucas-Lopez et al.,20 proved difficult and afforded
amidine 8a in 2% yield. This compound was isolated in 78% yield when the same crude reaction
mixture was purified by reversed phase automated flash chromatography. This procedure affords
amidine 8a was obtained in a much higher yield than the previously reported strategies. With
these conditions in hand, amidines 8b–d were synthesized in the same manner (Scheme 1). The
presence of (mild) electron withdrawing groups (e.g. protonated amino group (formed under the
acidic conditions used) in 6c, cyano group in 6d) in pyrrolidinones 6c–d hampered the
nucleophilic character of the aryl amide, leading to rather high amounts of unreacted starting
material. A similar observation was made for the electron donating allyloxy group in 6b.
Attempts to improve this transformation, using alternative reagents or starting materials, and/or
by the addition of base, were unsuccessful (Table 1).
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Table 1. Optimization of the synthesis of amidines 8a–d.
Amidine
Starting
material
Reaction conditions
Conversion[a]
Yield
(%)
(%)
1) 2 equiv POCl3, CH2Cl2, 24 h, rt
95
2[b,c]
78[b,d]
50[d]
8a
8a
8b
8c
8d
8d
8d
8d
8d
8d
8d
6a
6a
2) 1.05 equiv 7, CH2Cl2, 24 h, 35 °C
1) 2 equiv POCl3, CH2Cl2, 24 h, rt
95
62[e]
53[e]
37[e]
0
2) 1.05 equiv 7, CH2Cl2, 24 h, 35 °C
1) 2 equiv POCl3, CH2Cl2, 24 h, rt
6b
2) 1.05 equiv 7, CH2Cl2, 24 h, 35 °C
1) 2 equiv POCl3, CH2Cl2, 24 h, rt
29[d,f]
30[d]
6c
2) 1.05 equiv 7, CH2Cl2, 24 h, 35 °C
1) 2 equiv POCl3, CH2Cl2, 24 h, rt
6d
2) 1.05 equiv 7, CH2Cl2, 24 h, 35 °C
1) 2 equiv Et3N, 1.2 equiv Mukaiyama’s reagent, CH2Cl2, 5 min, rt
2) 1.2 equiv 7, CH2Cl2, 45 h, r.t.
[g]
–
6d
1) 2 equiv Et3N, 1.2 equiv Mukaiyama’s reagent, CH2Cl2, 5 min, rt
2) 1.2 equiv 7, CH2Cl2, 24 h, 35 °C
[g]
10[h]
10[h]
10[h]
10[h]
10[h]
10[h]
13
25
3
–
1) 2 equiv Et3N, 2 equiv Mukaiyama’s reagent, CH2Cl2, 5 min, rt
2) 1.2 equiv 7, CH2Cl2, 24 h, 35 °C
[g]
–
1) 2 equiv Et3N, 2 equiv POCl3, CH2Cl2, 5 min, rt
[g]
–
2) 1.05 equiv 7, CH2Cl2, 24 h, 35 °C
1) 2 equiv Et3N, 2 equiv POCl3, CH2Cl2, 24 h, rt
[g]
3
–
2) 1.05 equiv 7, CH2Cl2, 24 h, 35 °C
1) 2 equiv POCl3, CH2Cl2, 5 min, rt
[g]
14
15
–
2) 1.05 equiv 7, CH2Cl2, 24 h, 35 °C
1) 2 equiv POCl3, CH2Cl2, 24 h, rt
[g]
–
8d
2) 1.05 equiv 7, CH2Cl2, 24 h, 35 °C
1
[a] H NMR. [b] Previously prepared by Lucas-Lopez et al. in 41% yield: 1) 1.1 equiv 6a,
1 equiv POCl3, CH2Cl2, 3 h, rt; 2) 1 equiv 7, CH2Cl2, 16 h, 35 °C.20 [c] Purified via acid-base
extraction. [d] Purified via reversed phase automated flash chromatography. [e] Unreacted
starting material was fully recovered. [f] 85% purity. [g] Not isolated. [h] Pyrrolidinethione 10
was prepared from pyrrolidinone 6d and Lawesson’s reagent.31 When excess Lawesson’s reagent
(more than 0.5 equivalents) and/or prolonged reaction times (more than 30 minutes) were used at
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elevated temperatures (70 °C), thionation of the nitrile functionality in pyrrolidinethione 10 also
occurred.
Intramolecular cyclization gave quinolones 9a–d in good to excellent yields (Scheme 1). The
ensuing asymmetric α-hydroxylation (Scheme 2) was carried out using Davis’ oxaziridine
methodology. In all cases, quinolones 9a–d were completely converted to α-hydroxy ketones
(S)- and (R)-1,11–13, but isolated yields varied. A single recrystallization from CH3CN or EtOH
resulted in highly optically enriched (S)- and (R)-1,11 and (S)- and (R)-12, respectively.
Intermediates 11 and 12 were deprotected in high yields (Scheme 3) without affecting the
blebbistatin scaffold. Nitriles (S)- and (R)-13 were selectively hydrolyzed to amides (S)- and (R)-
16, which were successfully optically enriched by recrystallization from EtOH. Amides (S)- and
(R)-16 were further converted into carboxylic acids (S)- and (R)-17. Isolation of these acids from
the crude reaction mixture proved tedious, due to their polar and zwitterionic nature. (S)-17 was
only recovered in low yield by preparative TLC. Conversely, a quick work-up of the crude
mixture afforded (R)-17 in good yield and similar purity as obtained for (S)-17 after preparative
TLC. Finally, the absolute configuration of (S)-2 and (R)-2 was determined by X-ray analysis
(see the Supporting Information), and this confirms the formation of (S)- and (R)-α-hydroxy
ketones using Davis’ oxaziridines 14 and 15, respectively (Scheme 2).
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Scheme 2. Asymmetric α-hydroxylation of quinolones 9a–d. Reagents and conditions: 1)
LiHMDS, THF, −78 °C, 30 min, 2) 14 (for (S)-1,11–13) or 15 (for (R)-1,11–13), THF, −15 °C,
16 h. [a] Determination of ee via chiral HPLC analysis. [b] After recrystallization from CH3CN.
[c] After recrystallization from EtOH.
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Scheme 3. Deprotection of (S)- and (R)-11–13: formation of polar blebbistatin analogs (S)- and
(R)-2,3,16,17. Reagents and conditions: (a) Pd(PPh3)4, K2CO3, MeOH, 50 °C, 1 h; (b) N,N’-
dimethylbarbituric acid, Pd(PPh3)4, CH2Cl2, reflux, 1 h; (c) Et2NOH, CH2Cl2, reflux, 48 h; (d)
H2SO4, H2O, 70 °C, 14 h. [a] Determination of ee via chiral HPLC analysis. [b] After
recrystallization from EtOH. [c] Determination of ee via chiral HPLC analysis of corresponding
methyl ester.
The key steps in the synthesis of blebbistatin derivatives 2, 3, 13, 16 and 17 were the
successful introduction and removal of a suitable masked polar group. Of the evaluated
alternatives (OMEM, OAlloc, OAllyl, NHMEM, NHAlloc, N(Allyl)2, CN), only the allyl
protecting group and cyano function proved appropriate for the preparation of blebbistatin
analogs 2,3 and 13,16,17, respectively. Finally, enantiopurity of both enantiomers of all newly
synthesized blebbistatin derivatives 2,3,11–13,16,17 was determined via chiral HPLC analysis.
Unfortunately, this feature is often neglected in literature.24,30 Knowledge of the enantiopurity of
(S)-blebbistatin or its derivatives is highly important when using these molecules to modulate
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myosin II function in biochemical or biological experiments, as the (R)-enantiomers display no
affinity for myosin II (vide infra).
2. Biochemical evaluation
The myosin II inhibitory properties of both enantiomers of compounds 1–3, 13, 16 and 17
were evaluated in vitro in a steady state ATPase assay using rabbit skeletal muscle myosin II.
The half-maximum inhibitory concentrations (IC50) of (S)-3’-hydroxyblebbistatin (S)-2 and (S)-
3’-aminoblebbistatin (S)-3 in the ATPase assay (Figure 2, Table 2) lie somewhat higher, but still
in the same order of magnitude as that of parent compound (S)-1. (S)-3’-cyanoblebbistatin (S)-13
proved less potent. The maximal extent of inhibition is the same for all four compounds at high
concentrations (50–100 µM). (S)-3’-carbamoylblebbistatin (S)-16 and (S)-3’-carboxyblebbistatin
(S)-17, on the other hand, were ineffective at concentrations up to 100 µM. As expected,29 all
(R)-enantiomers lacked efficacy.
The data generated from the ATPase assay yield useful SAR information. They highlight that
the (S)-configuration is essential for activity, confirming a similar binding mode for (S)-3’-
hydroxyblebbistatin (S)-2, (S)-3’-aminoblebbistatin (S)-3 and (S)-3’-cyanoblebbistatin (S)-13 as
for (S)-1. The data also reveal that small hydrophilic groups in the meta-position of ring D can be
accommodated, but do not influence potency in an appreciable manner. Large polar groups are
not well tolerated. The kinetics of the chemo-mechanical cycle of myosin may play an important
role in the observed ligand discrimination. Preliminary in silico studies (data not shown) suggest
that steric and temporal restrictions imposed by the path(s) leading toward the binding site may
be a possible reason behind this observation. A full report of these investigations will be
disclosed in due course.
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Figure 2. Biochemical evaluation of the myosin II inhibitory properties of both enantiomers of
compounds 1–3, 13, 16 and 17, evaluated in an ATPase assay with rabbit skeletal muscle myosin
II. Examples of dose-response curves and 4-parameter logistic curve fitting obtained for (A) (S)-
blebbistatin (S)-1 and (R)-blebbistatin (R)-1, (B) (S)-3’-hydroxyblebbistatin (S)-2 and (R)-3’-
hydroxyblebbistatin (R)-2, (C) (S)-3’-aminoblebbistatin (S)-3 and (R)-3’-aminoblebbistatin (R)-
3, (D) (S)-3’-cyanoblebbistatin (S)-13 and (R)-3’-cyanoblebbistatin (R)-13, (E) (S)-3’-
carbamoylblebbistatin (S)-16 and (R)-3’-carbamoylblebbistatin (R)-16, and (F) (S)-3’-
carboxyblebbistatin (S)-17 and (R)-3’-carboxyblebbistatin (R)-17. Data points represent the
means ± s.d. of at least three samples (N = 1). Concentrations higher than 40 µM caused (S)-3’-
cyanoblebbistatin (S)-13 to precipitate in the assay buffer (Figure S72 in the Supporting
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Information), so the relative ATPase activity of (S)-blebbistatin (S)-1 at a concentration of
32.5 µM was used to set the lower asymptote of the fitted curve.
Table 2. Biochemical and physicochemical evaluation of compounds (S)- and (R)-1–3, 13, 16,
and 17: half-maximum inhibitory concentrations (IC50) for the steady state ATPase activity of
rabbit skeletal muscle myosin II, and solubility data in PBS pH 7.4 buffer.
[a]
[a]
Compound
IC50
(µM)
Solubility[b] Compound IC50
(µM)
(µM)
>100[c]
>300[d]
>300[d]
>40[f]
(S)-1
(S)-2
2.16 ± 0.14 6.18 ± 0.08
(R)-1
(R)-2
19.3 ± 0.5
14.1 ± 0.1
48.5 ± 0.1
>100[c]
193 ± 1
186 ± 9
N.D.[e]
(S)-3
(R)-3
(S)-13
(S)-16
(S)-17
(R)-13
(R)-16
(R)-17
86.3 ± 6.7
>200[g]
>100[c]
>100[c]
>100[c]
[a] Data represent mean ± s.d. of two independent experiments (1.6% DMSO). [b] Data represent
mean ± s.d. of two independent experiments (2% DMSO). [c,d,f,g] Highest compound
concentrations used were 100, 300, 40, and 200 µM, respectively. [e] Not determined. [f]
Concentrations >40 µM resulted in compound precipitation in the assay buffer (Figure S73 in the
Supporting Information).
3. Physicochemical evaluation
A major issue with (S)-blebbistatin (S)-1 is its limited solubility in aqueous media. Since high
(S)-1 concentrations (50–100 µM) are typically required to halt myosin II dependent cellular
processes, experiments are de facto carried out in oversaturated solutions.5 This creates
uncertainty about actual assay concentrations and, as will be shown below, can produce
interfering precipitates. We found that the solubility of (S)-blebbistatin (S)-1 in PBS pH 7.4
buffer (Table 2) is 6.18 ± 0.08 µM at 2% (v/v) DMSO. As anticipated, the solubility of the newly
synthesized alcohol, amine, amide and carboxylic acid derivatives under the same conditions
proved significantly higher. (S)-3’-cyanoblebbistatin (S)-13 was not evaluated, since
precipitation was noted above 40 µM in the ATPase assay. Most importantly, the solubility of
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the most potent derivatives, (S)-3’-hydroxyblebbistatin (S)-2 and (S)-3’-aminoblebbistatin (S)-3,
is over 30 times higher than that of (S)-blebbistatin (S)-1.
A second, even more severe limitation of (S)-blebbistatin (S)-1 is its interference when using
fluorescence as a readout. Two of the most important experiments in this context are cardiac
optical mapping,26,32 and the study of myosin II function in the cell. Both are routinely conducted
using (S)-blebbistatin (S)-1 as a tool to inhibit myosin II. In the former experiment, calcium
concentrations are measured using fluorescent Ca2+ indicators (Fluo-3, Fluo-4, Fluo-5F). When
studying myosin II function in the cell, fluorescence microscopy with GFP-labeled myosin II is
often used. In both experimental setups, the use of (S)-blebbistatin (S)-1 is accompanied with
fluorescence interference upon excitation in the 488 nm region. It is often assumed that (S)-
blebbistatin (S)-1 solutions are highly fluorescent and thereby cause this interference.20,23
However, during our own prior biological work in media containing high (S)-blebbistatin (S)-1
concentrations, we had realized that (S)-blebbistatin (S)-1 crystals, rather than (S)-blebbistatin
(S)-1 in solution, were the actual cause of the observed interference. We therefore hypothesized
that the use of more water soluble blebbistatin analogs would eliminate fluorescence
interference. To test this assumption, we exposed MCF-7/6 breast carcinoma cells, a commonly
used cell line in our laboratories,7 to low and high concentrations of (S)-blebbistatin (S)-1 and
imaged after 24 h of incubation (Figure 3). At low (S)-blebbistatin (S)-1 concentrations (Figures
3A and 3B), no fluorescence upon 488 nm excitation was observed. Conversely, at high (S)-
blebbistatin (S)-1 concentrations (Figures 3C and 3D), fluorescent precipitates were clearly
visible. When repeating the experiment with the most potent polar derivatives, (S)-3’-
hydroxyblebbistatin (S)-2 (Figure S75 in the Supporting Information) and (S)-3’-
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aminoblebbistatin (S)-3 (Figures 3E and 3F, and Figure S74 in the Supporting Information), no
fluorescence was detected.
Figure 3. Low solubility of (S)-blebbistatin (S)-1 results in precipitates that interfere during
fluorescence imaging when applied at high concentrations. Fluorescence imaging of MCF-7/6
breast carcinoma cells treated with (S)-blebbistatin (S)-1 and (S)-3’-aminoblebbistatin (S)-3. (A)
Brightfield and (B) fluorescence image (488 nm excitation) of treatment with 5 µM (S)-
blebbistatin (S)-1. (C) Brightfield and (D) fluorescence image (488 nm excitation) of treatment
with 50 µM (S)-blebbistatin (S)-1. (E) Brightfield and (F) fluorescence image (488 nm
excitation) of treatment with 50 µM (S)-3’-aminoblebbistatin (S)-3. Exposure time in B, D, and F
is equal. Scale bar, 50 µm.
As (S)-blebbistatin (S)-1, (S)-3’-hydroxyblebbistatin (S)-2 and (S)-3’-aminoblebbistatin (S)-3
show similar, weak, fluorescence upon 488 nm excitation (Figure 4), these observations confirm
that the described fluorescence issues of (S)-blebbistatin (S)-1 are due to its low aqueous
solubility and the resulting formation of precipitates. (S)-3’-hydroxyblebbistatin (S)-2 and (S)-3’-
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aminoblebbistatin (S)-3 are therefore superior tools for cardiac optical mapping and myosin II
studies.
Figure 4. Fluorescent properties of (S)-blebbistatin (S)-1, (S)-3’-hydroxyblebbistatin (S)-2 and
(S)-3’-aminoblebbistatin (S)-3. Emission spectra upon excitation at a wavelength of (A) 488 nm
and (B) 365 nm.
CONCLUSIONS
Both enantiomers of a series of polar blebbistatin analogs (2, 3, 13, 16 and 17) were
synthesized in search of myosin II inhibitors with enhanced physicochemical properties and
superior research tool characteristics as compared to (S)-blebbistatin (S)-1. The most important
steps in their preparation consisted of (i) introduction of a suitable masked polar group in the
beginning of the sequence (i.e. an allyl protecting group and cyano function for 2,3 and 13,16,17,
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respectively), (ii) an optimized synthesis and purification of the amidine intermediates and (iii)
removal of the protecting group at the end of the pathway without affecting the blebbistatin
scaffold. The enantiopurity of both enantiomers of these newly synthesized blebbistatin
derivatives was confirmed. All (R)-enantiomers lacked myosin II inhibitory properties. This
highlights the importance of a correct assessment of the enantiopurity of (S)-blebbistatin
(derivatives) in terms of their use as myosin II inhibitors. Our biochemical and physicochemical
evaluation demonstrated that both (S)-3’-hydroxyblebbistatin (S)-2 and (S)-3’-aminoblebbistatin
(S)-3 combine good myosin II inhibitory properties with a 30 times higher aqueous solubility
than (S)-blebbistatin (S)-1. These polar analogs moreover do not show interference in
fluorescence readouts. Given these virtues, (S)-3’-hydroxyblebbistatin (S)-2 and (S)-3’-
aminoblebbistatin (S)-3 can be considered superior research tool molecules for the study of
myosin II.
MATERIALS AND METHODS
1. Chemistry
The synthesis of compounds 1–3, 11–13, 16 and 17 is provided below. The purity of all final
compounds was 95% or higher, as determined by LC-MS analysis. The synthesis of compounds
9a–d is detailed in the Supporting Information.
1.1. General
Dichloromethane was dried by heating under reflux over CaH2 and distilled under an
atmosphere of nitrogen. Tetrahydrofuran was dried by heating under reflux with
sodium/benzophenone under a nitrogen atmosphere and collected by distillation. Dry
1,4-dioxane and dry methanol were purchased from Sigma-Aldrich and Acros Organics,
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respectively. Reagents were purchased at the highest commercial quality and were used as
received without further purification.
Yields refer to chromatographically and spectroscopically (1H NMR) homogeneous material,
unless otherwise stated.
Reactions were monitored on an Agilent 1200 series HPLC system fitted with an Ascentis®
Express C18-column (2.7 µm particle size, 4.6 mm internal diameter), using acetonitrile/water
(5 mM NH4OAc) as eluent. Low-resolution mass spectra were recorded on an Agilent 1100
series VL mass spectrometer (ESI, 70 eV). High-resolution mass spectra (HRMS) were recorded
using an Agilent Technologies 6210 series time-of-flight (TOF) mass spectrometer equipped
with an ESI/APCI-multimode source. Thin layer chromatography (TLC) was carried out on
0.25 mm Merck silica plates (60 F254) and spots were visualized by UV light (254 nm). Flash
column chromatography was performed on an automated Reveleris® X2 flash chromatography
system, using Reveleris® C18 or Reveleris® silica cartridges. Melting points were determined
using a Wagner & Munz WME Heizbank Kofler bench.
NMR spectra were recorded on a Bruker Avance III instrument. 1H NMR (400 MHz) chemical
shifts are recorded in CDCl3 or DMSO-d6, reported in ppm and measured relative to
tetramethylsilane or the residual undeuterated solvent as the internal reference, respectively.
13C NMR (100.6 MHz) chemical shifts are reported in ppm and were measured relative to the
residual undeuterated solvent as the internal reference. The following abbreviations were used to
explain NMR peak multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m =
multiplet, br = broad.
The enantiomeric excess (ee) of chiral compounds was determined via chiral HPLC analysis
using a Daicel Chiralpak IA column (5 µm particle size, 150 mm length, 2.1 mm internal
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diameter). Detection wavelengths were set at 268, 234 and 296 nm. Analyses under reversed
phase and normal phase conditions were performed at 25 °C and 35 °C, respectively. Optical
rotations were obtained on a Jasco P-2000 polarimeter and are reported in deg mL g-1 dm-1;
concentrations are reported in grams per 100 mL.
1.2. General procedure for the asymmetric α-hydroxylation of quinolones
The asymmetric α-hydroxylation of quinolones 9a,b,d was adopted from Lucas-Lopez et al.20
An oven-dried bulb of 50 mL, evacuated and back-filled with argon (5×), was loaded with
5 mL of dry tetrahydrofuran and quinolone (1.00 mmol, 1 equiv). Then, the solution was cooled
to −78 °C and 1.20 mL of lithiumbis(trimethylsilyl)amide (1 M solution in tetrahydrofuran,
1.20 mmol, 1.2 equiv) was added. The reaction was stirred for 30 minutes at −78 °C and a
solution of Davis’ oxaziridine 14 or 15 (0.716 g, 2.40 mmol, 2.4 equiv) in dry tetrahydrofuran
(10 mL) was added via syringe. Subsequently, the mixture was stirred at −15 °C for 16 hours
before being quenched by the addition of 35 mL of saturated aqueous NH4Cl. Saturated aqueous
Na2S2O3 (100 mL) was then added and the reaction mixture was extracted with diethyl ether
(3× 150 mL). The combined organic extracts were concentrated in vacuo. The residue was
dissolved in 40 mL of dichloromethane and subsequently washed with 0.3 M aqueous HCl
(3× 60 mL). The combined aqueous layers were washed with 60 mL of diethyl ether, basified
with 3 M aqueous NaOH until pH 10 was reached, and extracted with ethyl acetate (2× 250 mL).
The combined organic extracts were dried over magnesium sulfate and evaporated in vacuo.
1.2.1. (S)-3a-hydroxy-6-methyl-1-phenyl-1,2,3,3a-tetrahydro-4H-pyrrolo[2,3-b]quinolin-4-one
((S)-1)20
(S)-blebbistatin (S)-1 was synthesized from quinolone 9a (0.276 g, 1.00 mmol, 1 equiv) using
Davis’ oxaziridine 14 (0.716 g, 2.40 mmol, 2.4 equiv). Evaporation in vacuo gave (S)-1 (0.250 g,
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85%) as bright yellow powder. The enantiomeric excess was 75% as determined by chiral HPLC
analysis. This powder was redissolved in 10 mL of boiling acetonitrile and left untouched
overnight at room temperature, which afforded (S)-blebbistatin (S)-1 (0.108 g, 37%) as bright
yellow crystals with an enantiomeric excess of >99%. Chiral HPLC: tR ((S)-1) = 7.32 min,
tR ((R)-1) = 9.77 min (Daicel Chiralpak IA column, acetonitrile/water (50:50), 1.0 mL min-1,
1
25 °C). H NMR (400 MHz, DMSO-d6): δ = 2.23–2.30 (m, 2H), 2.31 (br. s, 3H), 3.90–4.02 (m,
1H), 4.02–4.12 (m, 1H), 6.84 (s, 1H), 7.13 (d, J = 8.1 Hz, 1H), 7.15 (t, J = 7.3 Hz, 1H), 7.38 (dd,
J = 8.1, 1.8 Hz, 1H), 7.40–7.47 (m, 2H), 7.54 (d, J = 1.8 Hz, 1H), 8.09 (d, J = 7.9 Hz, 2H).
1H NMR spectrum and chiral HPLC chromatograms are provided in Figures S29 and S48–S49,
respectively, in the Supporting Information.
1.2.2. (R)-3a-hydroxy-6-methyl-1-phenyl-1,2,3,3a-tetrahydro-4H-pyrrolo[2,3-b]quinolin-4-one
((R)-1)
(R)-blebbistatin (R)-1 was synthesized from quinolone 9a (0.138 g, 0.500 mmol, 1 equiv)
using Davis’ oxaziridine 15 (0.358 g, 1.20 mmol, 2.4 equiv). Evaporation in vacuo resulted in
(R)-1 (0.0918 g, 63%) as a yellow powder. The enantiomeric excess was 75% as determined by
chiral HPLC analysis. The powder was redissolved in 5 mL of boiling acetonitrile and left
untouched overnight at room temperature, which resulted in (R)-blebbistatin (R)-1 (0.0299 g,
20%) as bright yellow crystals with an enantiomeric excess of 99%. Chiral HPLC:
tR ((R)-1) = 9.77 min, tR ((S)-1) = 7.31 min (Daicel Chiralpak IA column, acetonitrile/water
1
(50:50), 1.0 mL min-1, 25 °C). H NMR (400 MHz, DMSO-d6): δ = 2.23–2.30 (m, 2H), 2.31
(br. s, 3H), 3.90–4.02 (m, 1H), 4.02–4.12 (m, 1H), 6.84 (s, 1H), 7.13 (d, J = 8.1 Hz, 1H), 7.15 (t,
J = 7.3 Hz, 1H), 7.38 (dd, J = 8.1, 1.8 Hz, 1H), 7.40–7.47 (m, 2H), 7.54 (d, J = 1.8 Hz, 1H), 8.09
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(d, J = 7.9 Hz, 2H). MW = 292.33. Chiral HPLC chromatogram is provided in Figure S50 in the
Supporting Information.
1.2.3.
(S)-1-(3-(allyloxy)phenyl)-3a-hydroxy-6-methyl-1,2,3,3a-tetrahydro-4H-pyrrolo[2,3-
b]quinolin-4-one ((S)-11)
(S)-3’-allyloxyblebbistatin (S)-11 was synthesized from quinolone 9b (11.5 g, 34.6 mmol,
1 equiv) using Davis’ oxaziridine 14 (24.8 g, 83.0 mmol, 2.4 equiv). Evaporation in vacuo
resulted in (S)-11 (8.19 g, 68 %) as an orange powder. The enantiomeric excess was 78% as
determined by chiral HPLC analysis. The powder was redissolved in 80 mL of boiling
acetonitrile and left untouched for 30 minutes at room temperature. After 20 minutes crystals had
formed. After an additional 10 minutes, the obtained crystals were filtered off. This resulted in
(S)-3’-allyloxyblebbistatin (S)-11 (2.29 g, 19%) as bright, gold leaf-like crystals with an
enantiomeric excess of >99%. mp 176 °C. Chiral HPLC: tR ((S)-11) = 11.2 min, tR ((R)-
11) = 14.2 min (Daicel Chiralpak IA column, acetonitrile/water (50:50), 1.0 mL min-1, 25 °C).
1
[α]25D = −327 ± 2 (c = 0.11 in tetrahydrofuran). H NMR (400 MHz, DMSO-d6): δ = 2.22–2.29
(m, 2H), 2.31 (s, 3H), 3.92–4.00 (m, 1H), 4.00–4.09 (m, 1H), 4.62 (dt, J = 5.2, 1.5 Hz, 2H), 5.29
(dq, J = 10.6, 1.5 Hz, 1H), 5.44 (dq, J = 17.3, 1.5 Hz, 1H), 6.09 (ddt, J = 17.3, 10.6, 5.2 Hz, 1H),
6.75 (dd, J = 8.2, 2.0 Hz, 1H), 6.83 (s, 1H), 7.14 (d, J = 8.1 Hz, 1H), 7.32 (t, J = 8.2 Hz, 1H),
7.39 (dd, J = 8.1, 1.8 Hz, 1H), 7.50 (dd, J = 8.2, 2.0 Hz, 1H), 7.54 (d, J = 1.8 Hz, 1H), 7.99 (t,
J = 2.0 Hz, 1H). 13C NMR (100.6 MHz, DMSO-d6): δ = 20.7, 28.7, 48.0, 68.7, 73.4, 106.9,
110.3, 112.2, 118.0, 121.5, 126.4, 126.8, 129.9, 132.9, 134.2, 137.0, 142.2, 149.4, 158.8, 165.8,
195.0. MS (ESI): m/z (%) = 348.6 ([M+H]+, 100). HRMS (ESI): calculated for C21H21N2O3
([M+H]+) 349.1547; found 349.1544. MW = 348.40. 1H NMR spectrum, 13C NMR spectrum and
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chiral HPLC chromatograms are provided in Figures S30–S31 and Figures S51–S52 in the
Supporting Information.
1.2.4.
(R)-1-(3-(allyloxy)phenyl)-3a-hydroxy-6-methyl-1,2,3,3a-tetrahydro-4H-pyrrolo[2,3-
b]quinolin-4-one ((R)-11)
(R)-3’-allyloxyblebbistatin (R)-11 was synthesized from quinolone 9b (2.66 g, 8.00 mmol,
1 equiv) using Davis’ oxaziridine 15 (5.73 g, 19.2 mmol, 2.4 equiv). Evaporation in vacuo
resulted in (R)-11 (2.15 g, 77%) as an orange powder. The enantiomeric excess was 79% as
determined by chiral HPLC analysis. The powder was redissolved in 20 mL of boiling
acetonitrile and left untouched for 30 minutes at room temperature. After 20 minutes crystals had
formed, which were filtered off after an additional 10 minutes. This resulted in (R)-3’-
allyloxyblebbistatin (R)-11 (1.00 g, 36%) as bright, gold leaf-like crystals with an enantiomeric
excess of >99%. mp 170 °C. Chiral HPLC: tR ((R)-11) = 14.2 min, tR ((S)-11) = 11.2 min (Daicel
Chiralpak IA column, acetonitrile/water (50:50), 1.0 mL min-1, 25 °C). [α]25D = +313 ± 3
1
(c = 0.12 in tetrahydrofuran). H NMR (400 MHz, DMSO-d6): δ = 2.22–2.29 (m, 2H), 2.31 (s,
3H), 3.92–4.00 (m, 1H), 4.00–4.09 (m, 1H), 4.62 (dt, J = 5.2, 1.5 Hz, 2H), 5.29 (dq, J = 10.6,
1.5 Hz, 1H), 5.44 (dq, J = 17.3, 1.5 Hz, 1H), 6.09 (ddt, J = 17.3, 10.6, 5.2 Hz, 1H), 6.75 (dd,
J = 8.2, 2.0 Hz, 1H), 6.83 (s, 1H), 7.14 (d, J = 8.1 Hz, 1H), 7.32 (t, J = 8.2 Hz, 1H), 7.39 (dd,
J = 8.1, 1.8 Hz, 1H), 7.50 (dd, J = 8.2, 2.0 Hz, 1H), 7.54 (d, J = 1.8 Hz, 1H), 7.99 (t, J = 2.0 Hz,
1H). 13C NMR (100.6 MHz, DMSO-d6): δ = 20.7, 28.7, 48.0, 68.7, 73.4, 106.9, 110.3, 112.2,
118.0, 121.5, 126.4, 126.8, 129.9, 132.9, 134.2, 137.0, 142.2, 149.4, 158.8, 165.8, 195.0. MS
(ESI): m/z (%) = 348.6 ([M+H]+, 100). HRMS (ESI): calculated for C21H21N2O3 ([M+H]+)
349.1547; found 349.1551. MW = 348.40. Chiral HPLC chromatogram is provided in
Figure S53 in the Supporting Information.
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1.2.5. (S)-1-(3-(diallylamino)phenyl)-3a-hydroxy-6-methyl-1,2,3,3a-tetrahydro-4H-pyrrolo[2,3-
b]quinolin-4 one ((S)-12)
(S)-3’-diallylaminoblebbistatin (S)-12 was synthesized from the crude quinolone 9c (3.96 g,
10.7 mmol, 1 equiv) using Davis’ oxaziridine 14 (7.64 g, 25.6 mmol, 2.4 equiv). Evaporation in
vacuo resulted in (S)-12 (3.02 g, 73%) as an orange powder. The enantiomeric excess was 76%
as determined by chiral HPLC analysis. The powder was redissolved in 60 mL of boiling
absolute ethanol and left untouched for 1 hour at room temperature. After 40 minutes fibers had
formed, which were filtered off after an additional 20 minutes. This resulted in (S)-3’-
diallylaminoblebbistatin (S)-12 (1.03 g, 25%) as bright orange cotton-like fibers with an
enantiomeric excess of >99%. mp 167 °C. Chiral HPLC: tR ((S)-12) = 27.1 min,
tR ((R)-12) = 31.1 min (Daicel Chiralpak IA column, acetonitrile/water (45:55), 1.0 mL min-1,
25 °C). [α]25D = −248 ± 2 (c = 0.11 in tetrahydrofuran). 1H NMR (400 MHz, DMSO-d6):
δ = 2.19–2.27 (m, 2H), 2.30 (s, 3H), 3.87–3.95 (m, 1H), 3.94–4.07 (m, 5H), 3.12–5.24 (m, 4H),
5.92 (ddt, J = 17.1, 10.2, 5.1 Hz, 2H), 6.47 (dd, J = 8.1, 2.0 Hz, 1H), 6.79 (s, 1H), 6.98 (dd,
J = 8.1, 2.0 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 7.15 (t, J = 8.1 Hz, 1H), 7.38 (dd, J = 8.1, 1.8 Hz,
1H), 7.52 (d, J = 1.8 Hz, 1H), 7.94 (t, J = 2.0 Hz, 1H). 13C NMR (100.6 MHz, DMSO-d6):
δ = 20.7, 28.8, 47.9, 53.0, 73.5, 104.8, 107.4, 108.3, 116.4, 121.4, 126.2, 126.8, 129.4, 132.5,
134.8, 137.0, 141.9, 148.9, 149.8, 165.7, 195.1. MS (ESI): m/z (%) = 387.5 ([M+H]+, 100).
HRMS (ESI): calculated for C24H26N3O2 ([M+H]+) 388.2020; found 388.2035. MW = 387.47.
1H NMR spectrum, 13C NMR spectrum and chiral HPLC chromatograms are provided in
Figures S34–S35 and Figures S57–S58 in the Supporting Information.
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1.2.6.
(R)-1-(3-(diallyl-amino)phenyl)-3a-hydroxy-6-methyl-1,2,3,3a-tetrahydro-4H-
pyrrolo[2,3-b]quinolin-4 one ((R)-12)
(R)-3’-diallylaminoblebbistatin (R)-12 was synthesized from the crude quinolone 9c (0.967 g,
2.60 mmol, 1 equiv) using Davis’ oxaziridine 15 (1.86 g, 6.24 mmol, 2.4 equiv). Evaporation in
vacuo resulted in (R)-3’-diallylaminoblebbistatin (R)-12 (0.686 g, 68%) as an orange powder.
The enantiomeric excess was 74% as determined by chiral HPLC analysis. The powder was
redissolved in 15 mL of boiling absolute ethanol and left untouched for 50 minutes at room
temperature. After 30 minutes fibers had formed, which were filtered off after an additional
20 minutes. This resulted in (R)-3’-diallylaminoblebbistatin (R)-12 (0.160 g, 16%) as bright
orange cotton-like fibers with an enantiomeric excess of 98%. mp 167 °C. Chiral HPLC:
tR ((R)-12) = 31.1 min, tR ((S)-12) = 27.7 min (Daicel Chiralpak IA column, acetonitrile/water
(45:55), 1.0 mL min-1, 25 °C). [α]25D = +327 ± 3 (c = 0.18 in tetrahydrofuran). 1H NMR
(400 MHz, DMSO-d6): δ = 2.19–2.27 (m, 2H), 2.30 (s, 3H), 3.87–3.95 (m, 1H), 3.94–4.07 (m,
5H), 3.12–5.24 (m, 4H), 5.92 (ddt, J = 17.1, 10.2, 5.1 Hz, 2H), 6.47 (dd, J = 8.1, 2.0 Hz, 1H),
6.79 (s, 1H), 6.98 (dd, J = 8.1, 2.0 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 7.15 (t, J = 8.1 Hz, 1H),
7.38 (dd, J = 8.1, 1.8 Hz, 1H), 7.52 (d, J = 1.8 Hz, 1H), 7.94 (t, J = 2.0 Hz, 1H). 13C NMR
(100.6 MHz, DMSO-d6): δ = 20.7, 28.8, 47.9, 53.0, 73.5, 104.8, 107.4, 108.3, 116.4, 121.4,
126.2, 126.8, 129.4, 132.5, 134.8, 137.0, 141.9, 148.9, 149.8, 165.7, 195.1. MS (ESI): m/z (%) =
387.5 ([M+H]+, 100). HRMS (ESI): calculated for C24H26N3O2 ([M+H]+) 388.2020; found
388.2023. MW = 387.47. Chiral HPLC chromatogram is provided in Figure S60 in the
Supporting Information.
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1.2.7.
(S)-3-(3a-hydroxy-6-methyl-4-oxo-2,3,3a,4-tetrahydro-1H-pyrrolo[2,3-b]quinolin-1-
yl)benzonitrile ((S)-13)
(S)-3’-cyanoblebbistatin (S)-13 was synthesized from quinolone 9d (1.51 g, 5.00 mmol,
1 equiv) using Davis’ oxaziridine 14 (3.58 g, 12.0 mmol, 2.4 equiv). Evaporation in vacuo
resulted in (S)-13 (1.54 g, 97%) as a yellow powder. The enantiomeric excess was 83% as
determined by chiral HPLC analysis. mp 235 °C. Chiral HPLC: tR ((S)-13) = 7.68 min,
tR ((R)-13) = 10.1 min (Daicel Chiralpak IA column, acetonitrile/water (50:50), 1.0 mL min-1,
25 °C). [α]25D = −248 ± 2 (c = 0.11 in tetrahydrofuran). 1H NMR (400 MHz, DMSO-d6):
δ = 2.25–2.36 (m, 5H), 3.97–4.12 (m, 2H), 6.92 (s, 1H), 7.19 (d, J = 8.2 Hz, 1H), 7.42 (dd,
J = 8.2, 2.0 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.59 (dt, J = 8.0, 1.3 Hz, 1H), 7.65 (t, J = 8.0 Hz,
1H), 8.45 (ddd, J = 8.0, 2.2, 1.1 Hz, 1H), 8.61 (t, J = 1.7 Hz, 1H). 13C NMR (100.6 MHz,
DMSO-d6): δ = 20.7, 28.6, 47.8, 73.2, 112.1, 119.3, 121.6, 122.6, 124.2, 126.6, 126.9, 127.1,
130.6, 133.6, 137.0, 141.7, 148.7, 165.9, 194.7. MS (ESI): m/z (%) = 317.6 ([M+H]+, 100).
HRMS (ESI): calculated for C19H16N3O2 ([M+H]+) 318.1237; found 318.1247. MW = 317.35.
1H NMR spectrum, 13C NMR spectrum and chiral HPLC chromatograms are provided in
Figures S32–S33 and Figures S54–S55 in the Supporting Information.
1.2.8.
(R)-3-(3a-hydroxy-6-methyl-4-oxo-2,3,3a,4-tetrahydro-1H-pyrrolo[2,3-b]quinolin-1-
yl)benzonitrile ((R)-13)
(R)-3’-cyanoblebbistatin (R)-13 was synthesized from quinolone 9d (1.51 g, 5.00 mmol,
1 equiv) using Davis’ oxaziridine 15 (3.58 g, 12.0 mmol, 2.4 equiv). Evaporation in vacuo
resulted in (R)-13 (1.50 g, 95%) as a yellow powder. The enantiomeric excess was 84% as
determined by chiral HPLC analysis. mp 232 °C. Chiral HPLC: tR ((R)-13) = 9.88 min,
tR ((S)-13) = 7.67 min (Daicel Chiralpak IA column, acetonitrile/water (50:50), 1.0 mL min-1,
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25 °C). [α]25D = +251 ± 2 (c = 0.14 in tetrahydrofuran). 1H NMR (400 MHz, DMSO-d6):
δ = 2.25–2.36 (m, 5H), 3.97–4.12 (m, 2H), 6.92 (s, 1H), 7.19 (d, J = 8.2 Hz, 1H), 7.42 (dd,
J = 8.2, 2.0 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.59 (dt, J = 8.0, 1.3 Hz, 1H), 7.65 (t, J = 8.0 Hz,
1H), 8.45 (ddd, J = 8.0, 2.2, 1.1 Hz, 1H), 8.61 (t, J = 1.7 Hz, 1H). 13C NMR (100.6 MHz,
DMSO-d6): δ = 20.7, 28.6, 47.8, 73.2, 112.1, 119.3, 121.6, 122.6, 124.2, 126.6, 126.9, 127.1,
130.6, 133.6, 137.0, 141.7, 148.7, 165.9, 194.7. MS (ESI): m/z (%) = 317.6 ([M+H]+, 100).
HRMS (ESI): calculated for C19H16N3O2 ([M+H]+) 318.1237; found 318.1236. MW = 317.35.
Chiral HPLC chromatogram is provided in Figure S56 in the Supporting Information.
1.3. General procedure for the allyl deprotection of allyl phenyl ethers
The allyl deprotection of allyl phenyl ethers (S)-11 and (R)-11 was adopted from a publication
of Vutukuri et al.33
In a flame-dried bulb of 250 mL containing 200 mL of dry methanol, allyl phenyl ether
(4.21 mmol, 1 equiv) and 0.243 g of tetrakis(triphenylphosphine)palladium(0) (0.211 mmol,
0.05 equiv) were brought together under a nitrogen atmosphere and stirred for 5 minutes at room
temperature. Then, 3.49 g of K2CO3 (25.3 mmol, 6 equiv) was added and the reaction mixture
was stirred for 1 hour at 50 °C. Afterwards, the mixture was concentrated to a volume of 25 mL,
diluted with 400 mL of ethyl acetate, washed with saturated aqueous NH4Cl (2× 75 mL). Next,
the organic layer was dried over magnesium sulfate and evaporated in vacuo.
1.3.1.
(S)-3a-hydroxy-1-(3-hydroxyphenyl)-6-methyl-1,2,3,3a-tetrahydro-4H-pyrrolo[2,3-
b]quinolin-4-one ((S)-2)
(S)-3’-hydroxyblebbistatin (S)-2 was synthesized from (S)-3’-allyloxyblebbistatin (S)-11
(1.47 g, 4.21 mmol, 1 equiv). Evaporation in vacuo afforded a bright red powder (1.53 g) which
was dissolved in tetrahydrofuran and coated under reduced pressure onto 4.60 g of silica.
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Subsequently, purification was performed via automated flash chromatography with
hexane/ethyl acetate as eluent on a Reveleris® 80 g silica cartridge (2% sample loading; flow
rate: 60 mL min-1; eluent: 2 CV of 15% ethyl acetate, followed by a gradient from 15% to 100%
ethyl acetate over 15 CV and finally 2 CV of 100% ethyl acetate; detection wavelengths: 220 nm
and 264 nm). This resulted in (S)-3’-hydroxyblebbistatin (S)-2 (1.03 g, 79%) as a bright orange
powder. The enantiomeric excess was 99% as determined by chiral HPLC analysis. mp 179 °C.
Rf = 0.40 (hexane/ethyl acetate, 2:3). Chiral HPLC: tR ((S)-2) = 3.87 min, tR ((R)-2) = 5.18 min
(Daicel Chiralpak IA column, acetonitrile/water (50:50), 1.0 mL min-1, 25 °C). [α]25D = −332 ± 3
1
(c = 0.11 in tetrahydrofuran). H NMR (400 MHz, DMSO-d6): δ = 2.20–2.27 (m, 2H), 2.31 (s,
3H), 3.87–3.95 (m, 1H), 3.96–4.06 (m, 1H), 6.56 (dd, J = 8.1, 2.1 Hz, 1H), 6.81 (s, 1H), 7.12 (d,
J = 8.1 Hz, 1H), 7.19 (t, J = 8.1 Hz, 1H), 7.35–7.41 (m, 2H), 7.53 (d, J = 1.6 Hz, 1H), 7.71 (t,
J = 2.1 Hz, 1H), 9.50 (s, 1H). 13C NMR (100.6 MHz, DMSO-d6): δ = 20.7, 28.7, 48.0, 73.4,
107.6, 110.8, 111.2, 121.4, 126.3, 126.8, 129.8, 132.7, 136.9, 142.1, 149.7, 158.0, 165.7, 195.1.
MS (ESI): m/z (%) = 308.7 ([M+H]+, 100). HRMS (ESI): calculated for C18H17N2O3 ([M+H]+)
1
309.1234; found 309.1233. MW = 308.33. H NMR spectrum, 13C NMR spectrum and chiral
HPLC chromatograms are provided in Figures S36–S37 and Figures S60–S61 in the Supporting
Information. X-ray data are provided in Figures S1–S2 in the Supporting Information.
1.3.2.
(R)-3a-hydroxy-1-(3-hydroxyphenyl)-6-methyl-1,2,3,3a-tetrahydro-4H-pyrrolo[2,3-
b]quinolin-4-one ((R)-2)
(R)-3’-hydroxyblebbistatin (R)-2 was synthesized from (R)-3’-allyloxyblebbistatin (R)-11
(0.182 g, 0.521 mmol, 1 equiv). Evaporation in vacuo afforded an orange oil (0.200 g) which
was dissolved in tetrahydrofuran and coated under reduced pressure onto 0.600 g of silica.
Subsequently, purification was performed via automated flash chromatography with
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hexane/ethyl acetate as eluent on a Reveleris® 12 g silica cartridge (2% sample loading; flow
rate: 36 mL min-1; eluent: 2 CV of 15% ethyl acetate, followed by a gradient from 15% to 100%
ethyl acetate over 15 CV and finally 2 CV of 100% ethyl acetate; detection wavelengths: 220 nm
and 264 nm). This resulted in (R)-3’-hydroxyblebbistatin (R)-2 (0.120 g, 75%) as a bright orange
powder. The enantiomeric excess was 98% as determined by chiral HPLC analysis. mp 202 °C.
Rf = 0.40 (hexane/ethyl acetate, 2:3). Chiral HPLC: tR ((R)-2) = 5.11 min, tR ((S)-2) = 3.91 min
(Daicel Chiralpak IA column, acetonitrile/water (50:50), 1.0 mL min-1, 25 °C). [α]25D = +320 ± 3
1
(c = 0.12 in tetrahydrofuran). H NMR (400 MHz, DMSO-d6): δ = 2.20–2.27 (m, 2H), 2.31 (s,
3H), 3.87–3.95 (m, 1H), 3.96–4.06 (m, 1H), 6.56 (dd, J = 8.1, 2.1 Hz, 1H), 6.81 (s, 1H), 7.12 (d,
J = 8.1 Hz, 1H), 7.19 (t, J = 8.1 Hz, 1H), 7.35–7.41 (m, 2H), 7.53 (d, J = 1.6 Hz, 1H), 7.71 (t,
J = 2.1 Hz, 1H), 9.50 (s, 1H). 13C NMR (100.6 MHz, DMSO-d6): δ = 20.7, 28.7, 48.0, 73.4,
107.6, 110.8, 111.2, 121.4, 126.3, 126.8, 129.8, 132.7, 136.9, 142.1, 149.7, 158.0, 165.7, 195.1.
MS (ESI): m/z (%) = 308.6 ([M+H]+, 100). HRMS (ESI): calculated for C18H17N2O3 ([M+H]+)
309.1234; found 309.1239. MW = 308.33. Chiral HPLC chromatogram is provided in
Figure S62 in the Supporting Information. X-ray data are provided in Figures S3–S4 in the
Supporting Information.
1.4. General procedure for the allyl deprotection of diallyl phenyl amines
The allyl deprotection of diallyl phenyl amines (S)-12 and (R)-12 was based on a publication
of Belardi and Micalizio.34
In a flame-dried bulb of 25 mL containing 20 mL of dry dichloromethane, 0.358 g of diallyl
phenyl amine (0.924 mmol, 1 equiv), 0.865 g of N,N’-dimethylbarbituric acid (5.55 mmol,
6 equiv) and 0.106 g of tetrakis(triphenylphosphine)palladium(0) (0.0924 mmol, 0.1 equiv) were
brought together under a nitrogen atmosphere and stirred for 1 hour at reflux temperature.
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Afterwards, the reaction mixture cooled to room temperature, diluted with 150 mL of ethyl
acetate and washed with saturated aqueous NaHCO3 (2× 50 mL). The organic layer was dried
over magnesium sulfate and evaporated in vacuo.
1.4.1.
(S)-1-(3-aminophenyl)-3a-hydroxy-6-methyl-1,2,3,3a-tetrahydro-4H-pyrrolo[2,3-
b]quinolin-4-one ((S)-3)
(S)-3’-aminoblebbistatin (S)-3 was synthesized from (S)-3’-diallylaminoblebbistatin (S)-12
(0.358 g, 0.924 mmol, 1 equiv). Evaporation in vacuo afforded a brown oil (0.496 g) which was
taken up in tetrahydrofuran and coated under reduced pressure onto 1.49 g of silica.
Subsequently, purification was performed via automated flash chromatography with
hexane/ethyl acetate as eluent on a Reveleris® 40 g silica cartridge (1% sample loading; flow
rate: 40 mL min-1; eluent: 2 CV of 20% ethyl acetate, followed by a gradient from 20% to 100%
ethyl acetate over 20 CV and finally 2 CV of 100% ethyl acetate; detection wavelengths: 220 nm
and 264 nm). This resulted in (S)-3’-aminoblebbistatin (S)-3 (0.215 g, 76%) as a dark yellow-
orange powder. The enantiomeric excess was >99% as determined by chiral HPLC analysis.
mp 207 °C. Rf = 0.33 (hexane/ethyl acetate, 1:4). Chiral HPLC: tR ((S)-3) = 4.41 min,
tR ((R)-3) = 6.35 min (Daicel Chiralpak IA column, acetonitrile/water (50:50), 1.0 mL min-1,
25 °C). [α]25D = −383 ± 3 (c = 0.10 in tetrahydrofuran). 1H NMR (400 MHz, DMSO-d6):
δ = 2.18–2.26 (m, 2H), 2.30 (s, 3H), 3.82–3.91 (m, 1H), 3.93–4.05 (m, 1H), 5.14 (br. s, 2H), 6.37
(dd, J = 8.0, 1.8 Hz, 1H), 6.78 (s, 1H), 7.04 (t, J = 8.0 Hz, 1H), 7.11 (d, J = 8.1 Hz, 1H), 7.17
(dd, J = 8.0, 1.8 Hz, 1H), 7.34 (t, J = 1.8 Hz, 1H), 7.37 (dd, J = 8.1, 1.9 Hz, 1H), 7.51 (d,
J = 1.9 Hz, 1H). 13C NMR (100.6 MHz, DMSO-d6): δ = 20.7, 28.3, 48.0, 73.4, 106.1, 108.2,
110.3, 121.4, 126.3, 126.7, 129.4, 132.4, 136.9, 141.7, 149.4, 150.0, 165.6, 195.2. MS (ESI):
m/z (%) = 307.7 ([M+H]+, 100). HRMS (ESI): calculated for C18H18N3O2 ([M+H]+) 308.1394;
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1
found 308.1387. MW = 307.35. H NMR spectrum, 13C NMR spectrum and chiral HPLC
chromatograms are provided in Figures S38–S39 and Figures S63–S64 in the Supporting
Information.
1.4.2.
(R)-1-(3-aminophenyl)-3a-hydroxy-6-methyl-1,2,3,3a-tetrahydro-4H-pyrrolo[2,3-
b]quinolin-4-one ((R)-3)
(R)-3’-aminoblebbistatin (R)-3 was synthesized from (R)-3’-diallylaminoblebbistatin (R)-12
(0.0530 g, 0.137 mmol, 1 equiv). Evaporation in vacuo afforded a brown oil (0.0731 g) which
was taken up in tetrahydrofuran and coated under reduced pressure onto 0.219 g of silica.
Subsequently, purification was performed via automated flash chromatography with
hexane/ethyl acetate as eluent on a Reveleris® 12 g silica cartridge (1% sample loading; flow
rate: 36 mL min-1; eluent: 2 CV of 20% ethyl acetate, followed by a gradient from 20% to 100%
ethyl acetate over 20 CV and finally 2 CV of 100% ethyl acetate; detection wavelengths: 220 nm
and 264 nm). This resulted in (R)-3’-aminoblebbistatin (R)-3 (0.0301 g, 72%) as a dark yellow-
orange powder. The enantiomeric excess was 95% as determined by chiral HPLC analysis.
mp 174 °C. Rf = 0.33 (hexane/ethyl acetate, 1:4). Chiral HPLC: tR ((R)-3) = 6.35 min, tR ((S)-
3) = 4.44 min (Daicel Chiralpak IA column, acetonitrile/water (50:50), 1.0 mL min-1, 25 °C).
1
[α]25D = +358 ± 4 (c = 0.11 in tetrahydrofuran). H NMR (400 MHz, DMSO-d6): δ = 2.18–2.26
(m, 2H), 2.30 (s, 3H), 3.82–3.91 (m, 1H), 3.93–4.05 (m, 1H), 5.14 (br. s, 2H), 6.37 (dd, J = 8.0,
1.8 Hz, 1H), 6.78 (s, 1H), 7.04 (t, J = 8.0 Hz, 1H), 7.11 (d, J = 8.1 Hz, 1H), 7.17 (dd, J = 8.0,
1.8 Hz, 1H), 7.34 (t, J = 1.8 Hz, 1H), 7.37 (dd, J = 8.1, 1.9 Hz, 1H), 7.51 (d, J = 1.9 Hz, 1H).
13C NMR (100.6 MHz, DMSO-d6): δ = 20.7, 28.3, 48.0, 73.4, 106.1, 108.2, 110.3, 121.4, 126.3,
126.7, 129.4, 132.4, 136.9, 141.7, 149.4, 150.0, 165.6, 195.2. MS (ESI): m/z (%) = 307.7
([M+H]+, 100). HRMS (ESI): calculated for C18H18N3O2 ([M+H]+) 308.1394; found 308.1403.
29