The small molecule tool (S)-(-)-blebbistatin: Novel insights of relevance to myosin inhibitor design

Article abstract of DOI:10.1039/b801223g

The small molecule blebbistatin is now a front line tool in the study of myosin function. Chemical modification of the tricyclic core of blebbistatin could deliver the next generation of myosin inhibitors and to help address this we report here on the impact of structural changes in the methyl-substituted aromatic ring of blebbistatin on its biological activity. Chemical methods for the preparation of isomeric methyl-containing analogues are reported and a series of co-crystal structures are used to rationalise the observed variations in their biological activity. These studies further support the view that the previously identified binding mode of blebbistatin to Dictyostelium discoideum myosin II is of relevance to its mode of action. A discussion of the role that these observations have on planning the synthesis of focused libraries of blebbistatin analogues is also provided including an assessment of possibilities by computational methods. These studies are ultimately directed at the development of novel myosin inhibitors with improved affinity and different selectivity profiles from blebbistatin itself. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b801223g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
The small molecule tool (S)-(−)-blebbistatin: novel insights of relevance to
myosin inhibitor design†
b
Cristina Lucas-Lopez,‡a John S. Allingham,‡§ Tomas Lebl,a Christopher P. A. T. Lawson,a Ruth Brenk,c
James R. Sellers,d Ivan Rayment*b and Nicholas J. Westwood*a
Received 23rd January 2008, Accepted 25th March 2008
First published as an Advance Article on the web 21st April 2008
DOI: 10.1039/b801223g
The small molecule blebbistatin is now a front line tool in the study of myosin function. Chemical
modification of the tricyclic core of blebbistatin could deliver the next generation of myosin inhibitors
and to help address this we report here on the impact of structural changes in the methyl-substituted
aromatic ring of blebbistatin on its biological activity. Chemical methods for the preparation of
isomeric methyl-containing analogues are reported and a series of co-crystal structures are used to
rationalise the observed variations in their biological activity. These studies further support the view
that the previously identified binding mode of blebbistatin to Dictyostelium discoideum myosin II is of
relevance to its mode of action. A discussion of the role that these observations have on planning the
synthesis of focused libraries of blebbistatin analogues is also provided including an assessment of
possibilities by computational methods. These studies are ultimately directed at the development of
novel myosin inhibitors with improved affinity and different selectivity profiles from blebbistatin itself.
Introduction
The investigation of normal and aberrant cellular function is
greatly enhanced by chemical tools that precisely and rapidly
inhibit specific targets in a defined manner. These tools are
particularly valuable for dissecting complex cellular processes such
as cytokinesis.1 Thus the recent discovery that (S)-(−)-blebbistatin
(1) (Scheme 1) inhibits a limited number of myosin II isoforms has
allowed the role of these myosins to be assessed in a wide range of
cellular processes including cytokinesis1 and cellular migration2 in
a manner that was previously inaccessible. As such 1 represents
an exciting addition to the strategies available for studying the
cytoskeleton in vivo. Importantly, the introduction of 1 has allowed
the focus to shift from small molecule ligand interactions with
actin3 to the control of individual classes of motor proteins.
At present more than 18 classes of myosin have been recognised
by phylogenetic analyses4 and all are characterised by the presence
of a motor domain that interacts with actin, hydrolyzes ATP,
and mediates a nucleotide-dependent conformational change that
results in directed movement. For the most part they share the
Scheme 1 Numbering scheme and chemical structures of analogues 4–7
aSchool of Chemistry and the Centre for Biomolecular Sciences, University of
and their synthetic precursors. Reagents and conditions: for the synthesis
of 8–12, including yields, see Experimental section; (i) LiHMDS, THF,
13 (84%), 14 (90%)9, 15 (95%), 16 (95%), 17 (95%); (ii) LiHMDS, THF,
oxaziridine 18 or 19; for 18, 4 (87% yield), 1 (82%)9, 5 (83%), 6 (63%), 7
(78%); for yields using 19 see Experimental section. aGeneralised structures
used to represent a particular stage in the synthesis process. bObserved
enantiomeric excess (ee) as a function of substrate and structure of the
oxaziridine used. cSee ref. 9 for details.
St Andrews, North Haugh, St Andrews, Fife, UK KY16 9ST. E-mail: njw3@
st-andrews.ac.uk
bDepartment of Biochemistry, University of Wisconsin, Madison, WI 53706,
USA. E-mail: ivan_rayment@biochem.wisc.edu
cDivision of Biological Chemistry and Drug Discovery, College of Life
Sciences, University of Dundee, Dow Street, Dundee, UK DD1 5EH
dLaboratory of Molecular Physiology, National Heart, Lung and Blood
Institute, National Institutes of Health, Bethesda, MD 20892-1762, USA
† Electronic supplementary information (ESI) available: More detailed
discussion regarding cyclisation of 11; biological activity of (S)-(−)-
blebbistatin analogues; data collection and refinement statistics for the
co-crystal structures of S1dC with 4–7. See DOI: 10.1039/b801223g
‡ These authors contributed equally to this work.
§ Current Address: Department of Biochemistry, Queen’s University,
Kingston, ON, K7L 3N6, Canada.
same chemical mechanism and given this commonality would
not be expected to be good targets for individual chemical
regulation. However, a series of functional, kinetic and structural
investigations have shown that (S)-(−)-blebbistatin (1) achieves
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selectivity by binding in a highly hydrophobic cleft in the myosin
motor domain.5–8 Surprisingly, this is one of the most conserved
components of all myosin motor domains where a few small
sequence and structural differences lead to the ability of 1 to
discriminate between myosin isoforms.
Given our detailed knowledge of the molecular interactions of
(S)-(−)-blebbistatin (1) with non-muscle myosin II, there is every
reason to believe that additional biological tools can be synthesised
with improved potency and specificity. In particular it is ultimately
desirable to design, or select from libraries of analogues, ligands
that can discriminate between distinct classes of myosin. Our initial
approach has been to carry out chemical optimisation studies
on 1 coupled closely with biochemical, computational and X-ray
crystallographic analysis of the impact of any structural changes.
We have previously reported that a nitro-functional group can be
incorporated into the core structure of 1 without significant loss
of activity and that this leads to a dramatic improvement in the
light stability of 1.9
Results and discussion
Analysis of the co-crystal structure of 1 bound to S1dC
The recently reported co-crystal structure of (S)-(−)-blebbistatin
(1) bound to the metastable state of Dictyostelium discoideum
myosin II (S1dC) provided an explanation for its mechanism of
inhibition and specificity at a molecular level.8 It showed that 1
binds near the apex of the 50 kDa cleft of myosin (Fig. 1a). The
structure also revealed that, in part, binding of 1 is stabilised, and
perhaps its orientation controlled, by the formation of a hydrogen
bonding network between the main chain carboxylate oxygen
of Leu262, the main chain amide hydrogen of Gly240, and the
hydroxyl group of the (S)-(−)-enantiomer (Fig. 1b).8 However,
most of the binding strength seems to be controlled by the
‘hydrophobic effect’ as the blebbistatin binding pocket encloses the
largely hydrophobic compound in a complimentary hydrophobic
environment. For example, the N-phenyl ring–myosin II interface
is comprised of residues Leu262, Phe466, Glu467, Cys470, and
Val630, whilst the tricyclic core interacts with Tyr261, Ser456,
Ile471, Thr474, Tyr634, Gln637, Leu638, and Leu641 (Fig. 1b).
The methyl-containing aromatic ring of (S)-(−)-blebbistatin
(1) interacts with the deepest point of the 50 kDa cleft (Fig. 1
and 2). Based on the position of this ring and the surrounding
hydrophobic residues, the location of the attached methyl group
is predicted to have a significant influence on the molecular
complementarity of the ligand–protein interface, and thus the
affinity for its binding site in S1dC myosin II. In order to probe
the effects of structural changes in this portion of 1 on myosin
inhibition, a series of analogues of 1 have been synthesised that
differ in the position (or existence) of the methyl group.
Fig. 1 Structure of the Dictyostelium discoideum myosin II S1dC–
MgADP–vanadate–(S)-(−)-blebbistatin (1) complex. (a) Myosin is shown
as a ribbon diagram (grey) with nucleotide (blue carbon and nitrogen
atoms) and (S)-(−)-blebbistatin (1) (yellow carbon atoms) bound at their
respective sites (PDB accession: 1YV3). The binding pocket of 1 is formed
by the U50 linker (green) from the upper face of the 50 kDa cleft and the
HP (blue) and HW (pink) helices, which form part of the lower face of
the 50 kDa cleft.10 (b) Stereoview of the blebbistatin binding site showing
the hydrogen bonding interactions and largely hydrophobic nature of the
interface residues (side chains of these residues are shown with blue carbon
atoms and red oxygen atoms).
formation of the amidines 8–12 varied with the position of the
methyl-substituent with the synthesis of the 5-substituted amidine
9 (R2 = Me, Scheme 1) being the most efficient, presumably
reflecting the increased nucleophilicity of the aniline nitrogen in
the starting anthranilate. In contrast, the sterically encumbered
3-methyl-substituted amidine 11 (R4 = Me) was formed in low
yield under reaction conditions analogous to those used in the
synthesis of 9. Sufficient quantities of 11 could be prepared
following a significant increase in the reaction time. Second, the
cyclisation conditions required to convert the amidines 8–12 to the
corresponding quinolones 13–17 (Scheme 1) varied as a function
of structure with yields of 84–95% being obtained in 3 h at 0 ◦C for
quinolones 13–15 and 17. However, it was necessary to increase the
temperature and the reaction time to prepare quinolone 16 in 95%
yield probably reflecting a strong preference for a conformation
of the lithium anion of 11 that minimizes the interaction between
the 3-methyl and N–Li groups (Scheme 1).†
(S)-(−)-Blebbistatin (1) analogue synthesis
We have recently established a synthetic route to analogues of 1
using Davis’ asymmetric hydroxylation methodology to convert
quinolones of the general type 2 to the corresponding a-hydroxy
ketones 3 (Scheme 1).9 Whilst the route was sufficiently robust
to enable the synthesis of analogues 4–7 of 1, several interesting
differences were observed across the series. First, the ease of
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Fig. 2 Overlays of the Dictyostelium discoideum myosin II S1dC–MgADP–vanadate binding pocket structures for (S)-(−)-blebbistatin (1) and each of
the blebbistatin analogues (PDB accession: 3BZ7, 3BZ8, 3BZ9). The X-ray crystal structures of the myosin’s interaction with (a) analogue 7 (pink carbon
atoms), (b) analogue 4 (light blue carbon atoms), (c) analogue 5 (green carbon atoms), and (d) the electron density observed on incubation with analogue
6 are shown superimposed upon the original ternary structure of 1 (yellow carbon atoms in all cases). All alignments were performed by superimposing
the a-carbon atoms for the analogue-bound form of myosin (blue) onto the (S)-(−)-blebbistatin (1)-bound form (white). Selected residues that form
the inhibitor–myosin interface are shown. Electron density maps contoured at 3r are shown for all analogue complexes (green mesh). The maps were
calculated with coefficients of the form Fo−Fc where the inhibitor was omitted from the refinement and phase calculation. For two of the complexes
[myosin-4 and -5, (b) and (c) respectively], an additional area of electron density was observed in the region of the 8-position of the aromatic ring. All
attempts using a range of chemical analytical techniques to identify any impurity in the samples of 4 and 5 proved unsuccessful. In addition, HPLC
analysis of incubations of 5 with the components of the co-crystallisations either on their own or in combination, including the presence of the protein,
indicated no apparent degradation of 5. This electron density remains unassigned.
An interesting variation in the degree of asymmetric induction in
the quinolone hydroxylation reaction was also observed as a func-
tion of structure, although in all cases the absolute configuration of
the resulting analogue was the same [(S)-(−)]. Quinolones 15–17
all reacted with (−)-[(8,8-dichlorocamphoryl)sulfonyl]oxaziridine
(18)11 under optimised conditions to give the desired analogues
5–7 respectively in good yield and high enantiomeric excess (ee
86–90%) in line with our report9 on the synthesis of 1 (Scheme 1,
column 8). As expected,9 the ee of the asymmetric hydroxylation
reaction for these substrates was reduced significantly when
the dihydro-oxaziridine 19 was used instead of 18 (Scheme 1,
column 9). Bach et al.12 have reported a detailed computational
analysis of the transition state involved in lithium enolate hydrox-
ylation reactions using an oxaziridine. The steric bulk associated
with the oxygen-bound lithium counter ion was identified as a
major factor in determining the observed absolute stereochemical
outcome of these reactions. Interestingly in our hands, 7Li NMR
analysis of the species formed on addition of 1.2 equivalents of
LiHMDS to a dry THF solution of quinolones 15, 16 and 17 at
0.68 ppm respectively (Fig. 3b–d). LiHMDS in dry THF gave a
single peak at d 0.33 (Fig. 3e).
In contrast, oxidation of quinolone 13 with 18 (Scheme 1) gave
4 in good yield but with a significantly lower ee (65%) than for
the other analogues as judged by chiral HPLC analysis of the
crude reaction mixture. When 13 was treated with LiHMDS and
the reaction analysed by 7Li NMR, one major peak was observed
with a chemical shift of 0.18 ppm (Fig. 3a). A second minor peak
was also observed. It is tempting to speculate that the observed
difference in ee for the hydroxylation of the enolate of 13 (cf .
the enolates of 15, 16, 17 and 149) results from the formation
of an alternative transition state in which the lithium atom is
coordinated to the nitrogen atom rather than the oxygen. The
formation of the O–Li enolate could be disfavored due to the
potential steric clash with the C5-methyl-substituent in 13 (R1 =
Me, Scheme 1). Differences in the aggregation states of lithium
enolates may also account for the observed differences in both the
7Li NMR spectra and the differential reactivity.13 To the best of
our knowledge, these studies in the blebbistatin system are the first
time that the enolate of a quinolone system, which can potentially
◦
−78 C showed the presence of a single peak at d 0.69, 0.69 and
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Co-crystal structures with S1dC
In order to correlate the observed biological activities of the
blebbistatin analogues against S1dC with their interactions with
this myosin at the molecular level, we have performed co-
crystallisation experiments with each of the analogues 4–7 and the
MgADP–vanadate complex of Dictyostelium discoideum myosin
II. The complexes with 4, 5, 6 and 7 were solved by molecular
˚
˚
˚
˚
replacement to resolutions of 2.0 A, 2.2 A, 2.15 A and 2.1 A
respectively, using the (S)-(−)blebbistatin (1)–myosin structure as
the starting model.† With the exception of analogue 6, the electron
density observed for each compound allowed all atoms to be
modelled into the 50 kDa cleft where their position and orientation
is nearly identical to that of 1 (Fig. 2). Alignment of the myosin
complexes formed with 4, 5 and 7 onto the model of 1 shows
that these analogues do not significantly alter the conformation
of residues comprising the binding pocket relative to those of
the blebbistatin-bound form. In addition, their interaction did
not produce any significant conformational changes in the overall
protein structure as the a-carbons of all complexes superimpose
on the original (S)-(−)-blebbistatin (1) model with a root mean
˚
square deviation of less than 0.2 A.† Furthermore, their hydrogen
bonding interactions with myosin and the surrounding water
activity within the 50 kDa cleft were identical to the structure of 1.
Unlike the other analogues, the electron density observed in
the co-crystallisation experiment using 6 was not sufficiently
unambiguous to allow this analogue to be modelled into the
blebbistatin binding pocket. The absence of 6 in the model is
further justified by the characteristics of Leu262 and Tyr634. In
the (S)-(−)-blebbistatin (1)-bound form, the side chains of these
residues move considerably in order to accommodate the N-phenyl
ring and tricyclic core. Such movements are not observed for the
co-crystallisation experiment with 6. The lack of occupancy for
6 can be rationalised based on the observation that the methyl-
substituent at position 8 (Scheme 1 for numbering) would create
Fig. 3 7Li NMR spectra for the different enolate anions. Conditions:
−78 ◦C, THF, LiHMDS. A capillary containing acetone-d6 was used as a
lock signal in each case. 7Li chemical shifts are given with respect to 0.1 M
solution of Li2SO4 as an external standard, with a d = 0 ppm in H2O.
(a) 13, (b) 15, (c) 16, (d) 17, (e) LiHMDS.
have the lithium atom coordinated to the oxygen or the nitrogen in
the transition state for hydroxylation, has been used in the Davis
reaction. Our results suggest that this may prove an interesting
system for more detailed study including computational analysis.
Highly optically enriched samples of the analogues 4–7 (ee
>99%) were obtained by a single recrystallisation from acetonitrile
of the product from the Davis hydroxylation reaction in line with
our studies on 1.9 With the analogues of 1 in hand, assays against
two myosin fragments (skeletal S1 and S1dC) were used to identify
structure–activity relationships.
˚
an unacceptably close hydrophobic contact (< 2.5 A) with Tyr634,
˚
whose side chain has already been shown to move ∼3.6 A to
accommodate 1 (even in the absence of the 8-methyl-substituent,
Fig. 2d). Therefore, it is apparent that 6 does not have suitable
structural characteristics for formation of a stable interaction with
Dictyostelium discoideum myosin II.
When taken together with the measured inhibitory effects
of the analogues against both rabbit skeletal myosin S1 and
Dictyostelium myosin II S1dC fragments, the crystallographic
data strongly support the view that the inhibitor binding mode
that is observed by X-ray crystallography is of relevance to the
mechanism of myosin II inhibition by 1 and its analogues. Indeed,
a methyl group at the C5, C6 or C7-position of the tricyclic core
(in 4, 1 or 5 respectively), or the absence of this group altogether as
in 7, allows the compound to remain structurally compatible with
the 50 kDa cleft of Dictyostelium discoideum myosin II and able
to trap the protein in the metastable state of the contractile cycle,
inhibiting myosin activity. Methyl incorporation at the 8-position,
on the other hand, is incompatible with adoption of a functional
binding mode and thus does not have a robust inhibitory effect on
Dictyostelium discoideum myosin II. Moreover, the conservation
of residue identity or similarity in the equivalent pocket of skeletal
myosin S1 fragment provides the basis for the parallels observed
in the inhibitory effect of the analogues on both proteins.8
Biological activity against skeletal S1 and S1dC
myosin fragments
A previous report has described the inhibition of a class II myosin
(S1dC) obtained from the slime mould Dictyostelium discoideum
by (S)-(−)-blebbistatin (1).5 Using an analogous approach to that
described previously,5 1 (as expected) and 3 of the 4 analogues
were shown to inhibit completely the ATPase activity of S1dC
at a final concentration of 50 lM (ESI, Table S1†). However, a
clear outlier was also identified with analogue 6 (for structure see
Fig. 1) being significantly less potent (65% residual S1dC ATPase
activity remaining under analogous assay conditions†). This trend
for reduced potency of 6 compared to the other analogues was
mirrored in studies carried out using rabbit skeletal myosin S1
fragment at a final inhibitor concentration of both 5 lM and
50 lM, although at the higher concentration 6 was observed to
reduce the ATPase activity by >70%.†
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The design of focused libraries based on (S)-(−)blebbistatin (1)
Conclusion
The synthetic approach currently adopted for preparing analogues
of 1 (Scheme 1) requires that the methyl-containing aromatic ring
is incorporated from the outset of the synthesis. Our analysis of
analogues with variations in this ring in complex with S1dC has
shown that small substituents on the C5, C6 and C7-positions
are well tolerated (Fig. 2 and Scheme 1 for numbering system).
By varying these substituents to create more diverse analogues it
may prove possible to prepare analogues that have either improved
affinity for the subset of myosin II isoforms that 1 inhibits or that
are specific for different myosin subtypes. We have further analysed
the co-crystal structures reported here with the aim of designing
inhibitors with increased affinity. Tyr261 forms a p–p stacking
interaction with the aromatic ring of the core fragment of 1 which
appears to contribute significantly to the binding affinity (Fig. 2
and 4). Interactive modelling of potential analogues of 1 using
the MAB force field as implemented in Moloc14 revealed that
this interaction could be improved by extending the aromatic ring
system, for example, by incorporating an additional thiophene
ring to give analogue 20 (Fig. 4). Based on the modelling, both
a ring joining the C6 and C7 positions and a ring joining the
C5 and C6 positions could be accommodated in the binding
pocket. However, the fact that the selectivity of the asymmetric
hydroxylation reaction was shown here to decrease in the presence
of a C5-substituent suggests that future studies should focus on
the preparation of analogues such as 20 with substitutents at the
C6 and C7 positions only.
The small molecule (S)-(−)-blebbistatin (1) has been quickly
adopted by the myosin research community as a means of moving
forward our understanding of the role of myosins in complex bio-
logical processes. Through modification of the chemical structure
of 1 there is considerable potential to develop a second generation
of tools that are either more potent than 1 or exhibit a modified
selectivity across the myosin superfamily. Here, a combination
of studies in chemistry and biology have enabled us to develop
an initial view of what structural modifications can and cannot
be accommodated in the methyl-substituted aromatic ring of 1.
In general, this region of 1 is relatively tolerant to changes in
the position of the methyl-substituent with the exception of its
incorporation at the 8-position. X-Ray crystallographic studies
using Dictyostelium discoideum myosin II (S1dC) have led to a clear
rationalisation of the observed variations in biological activity
with a steric interaction between the 8-methyl-substituent and
Tyr634 being responsible for the observed significant reduction
in activity associated with analogue 6. Our observed correlation
between the ATPase activity and ability to adopt the same binding
mode as 1 across a series of analogues further strengthens the view
that this is the biologically relevant binding mode.
During the course of our synthesis of these analogues of 1,
it became clear that whilst acceptable from a biological activity
perspective, the incorporation of a substituent in the 5-position
(as in 4) results in a reduction in the efficiency of the key asym-
metric hydroxylation reaction. We therefore concluded that the
incorporation of substituents at the 6- and 7-positions should be
focused on in future attempts to optimise the potency of 1 against
this particular myosin subclass. A preliminary computational
assessment of this has been included that helps to define future
synthetic targets.
Experimental
Unless otherwise noted, starting materials and reagents were
obtained from commercial suppliers and were used without
further purification. PE 40–60 refers to the fraction of light
petroleum ether boiling in the range 40–60 ◦C. Melting points were
recorded using an Electrothermal 9100 capillary melting point
apparatus. Values are quoted to the nearest 0.5 ◦C. IR spectra were
measured on a Perkin-Elmer Paragon 1000 FT-IR spectrometer.
1H and 13C NMR spectra were measured on a Bru¨ker Advance
300/500 instrument. J values are quoted in Hz. Chemical shifts
are calibrated with reference to the residual proton and carbon
resonances of the solvent (CDCl3: dH = 7.26, dC = 77.0 ppm).
Low and high-resolution mass spectral analyses were recorded
using ES operating in positive or negative ion mode.
General procedure for the synthesis of amidines 8, 10–12
Phosphorus oxychloride (1.0 equiv) was added dropwise to
a solution of 1-phenyl-2-pyrrolidinone (1.1 equiv.) in dry
dichloromethane (5.0–10 mL) and the reaction mixture was stirred
for 3 h at room temperature. A solution of the corresponding
anthranilate (1.0 equiv) in dry dichloromethane (15–25 mL)
was then added and the mixture refluxed for 16–72 h, cooled
and concentrated in vacuo. The resulting solid was dissolved in
Fig. 4 Modeled binding mode of an analogue of 1 with an extended
aromatic ring system (carbon atoms coloured in green) superimposed
onto the crystallographically determined binding mode of 1 (carbon atoms
coloured in yellow). The extended ring system is designed to increase the
p–p stacking interaction with Tyr261.
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aqueous hydrochloric acid (0.30 M, 100 mL) and extracted with
dichloromethane (3 × 100 mL). The aqueous phase was basified
with aqueous sodium hydroxide solution (2.0 M, pH adjusted to
8) and extracted with ethyl acetate (3 × 100 mL). The first organic
extracts were concentrated in vacuo and the resulting solid was
carried through the above procedure three more times. All ethyl
acetate extracts were combined, dried (MgSO4) and concentrated
in vacuo to give the desired amidine.
d = 1.99–2.11 (m, 2 H, 4ꢀ-H), 2.48 (t, 3J = 7.9, 2 H, 3ꢀ-H), 3.83 (s,
3 H, OCH3), 3.88 (t, 3J = 6.9, 2 H, 5ꢀ-H), 6.82 (dd, 3J = 8.0, 4J =
1.0, 1 H, 3-H), 6.98–7.10 (m, 2 H, 4ꢀꢀ, 5-H), 7.31–7.41 (m, 3 H,
3ꢀꢀ, 4-H), 7.77–7.89 ppm (m, 3 H, 2ꢀꢀ, 6-H). 13C NMR (75.5 MHz,
CDCl3): d = 19.6 (CH2), 29.1 (CH2), 50.5 (CH2), 51.6 (OCH3),
120.2 (CH), 121.5 (CH), 122.1 (C), 122.9 (CH), 123.1 (CH), 128.4
(CH), 130.7 (CH), 132.5 (CH), 141.2 (C), 153.0 (C), 159.4 (C),
167.5 ppm (C). LRMS (ES+) m/z (%): 295 (100) [M + H]+, 263
(22) [M-OMe]+. HRMS (ES+): m/z calcd for C18H19N2O2 [M +
H]+: 295.1447; found: 295.1449.
Methyl 6-methyl-2-(1ꢀ -phenylpyrrolidin-2ꢀ -ylideneamino)benzo-
◦
ate (8). White solid (0.32 g, 1.0 mmol, 28%), mp 97–98 C. IR
=
=
(Nujol): mmax = 1718 (s) (C O), 1647 (s) (C N), 1587 (m), 761 (m)
(Ar-H), 722 (m), 692 cm−1 (m). 1H NMR (300 MHz, CDCl3): d =
1.96–2.08 (m, 2 H, 4ꢀ-H), 2.34 (s, 3 H, CH3), 2.59 (t, 3J = 7.8, 2 H,
3ꢀ-H), 3.79–3.86 (m, 5 H, 5ꢀ-H, OCH3), 6.62–6.68 (m, 1 H, 5-H),
6.82–6.88 (m, 1 H, 3-H), 7.03–7.10 (m, 1 H, 4ꢀꢀ-H), 7.14–7.21 (m, 1
H, 4-H), 7.31–7.39 (m, 2 H, 3ꢀꢀ-H), 7.76–7.83 ppm (m, 2 H, 2ꢀꢀ-H).
13C NMR (75.5 MHz, CDCl3): d = 19.4 (CH3), 19.8 (CH2), 29.3
(CH2), 50.5 (CH2), 51.8 (OCH3), 118.7 (CH), 120.0 (C), 123.0 (C),
123.7 (CH), 127.5 (C), 128.5 (CH), 129.7 (CH), 135.3 (C), 141.2
(C), 149.4 (C), 161.0 (C), 170.2 ppm (C). LRMS (ES+) m/z (%):
309 (100) [M + H]+. HRMS (ES+): m/z calcd for C19H21N2O2 [M +
H]+: 309.1603; found: 309.1610.
General procedure for the synthesis of quinolones 13, 15–17
A solution of the corresponding amidine (1.0 equiv.) in anhydrous
THF (50 mL) was cooled to −78 ◦C and stirred for 15 min. Lithium
bis(trimethylsilyl)amide (1.0 M in THF, 3.0 equiv.) was added
dropwise to the reaction mixture, which was warmed to 0 ◦C over
3 h or room temperature over 12 h and quenched with saturated
aqueous ammonium chloride (5.0 mL). Further saturated aqueous
ammonium chloride (150 mL) was added. The aqueous phase
was extracted with dichloromethane (3 × 100 mL) and the
combined organic extracts dried (MgSO4) and concentrated in
vacuo. Purification by flash column chromatography on silica gel
eluting with (50–100% ethyl acetate–PE 40–60) gave the desired
compound.
Methyl 4-methyl-2-(1ꢀ -phenylpyrrolidin-2ꢀ -ylideneamino)benzo-
ate (10). Waxy solid (0.66 g, 2.1 mmol, 24%). IR (Nujol): mmax
=
=
=
1690 (s) (C O), 1602 (s) (C N), 1529 (m), 779 (m), 750 (m) (Ar-
5-Methyl-1-phenyl-2,3,4,9-tetrahydro-1H-pyrrolo[2,3-b]quinolin-
4-one (13). Off-white solid (0.044 g, 0.16 mmol, 84%), mp
H), 693 cm−1 (m). H NMR (300 MHz, CDCl3): d = 1.97–2.08
1
[m, 2 H, 4ꢀ-H], 2.32 (br s, 3 H, CH3), 2.47 (t, J = 7.8, 2 H,
3
◦
240–241 C. IR (Nujol): mmax = 1614 (m), 1570 (s), 1310 (s), 754
3ꢀ-H), 3.80 (s, 3 H, OCH3), 3.86 (t, J = 6.9, 2 H, 5ꢀ-H), 6.66
3
1
(m) (Ar-H), 722 cm−1(m). H NMR (300 MHz, d8-THF): d =
3
4
4
2.85 (s, 3 H, CH3), 3.16 (t, 3J = 8.3, 2 H, 3-H), 4.02 (t, 3J = 8.3,
2 H, 2-H), 6.85–6.90 (m, 1 H, 6-H), 6.91–6.97 (m, 1 H, 4ꢀ-H),
7.21 (dd, 3J = 8.2, 3J = 7.2, 1 H, 7-H), 7.26–7.34 (m, 2 H, 3ꢀ-H),
7.41–7.46 (m, 1 H, 8-H), 7.93–8.00 ppm (m, 2 H, 2ꢀ-H). 13C NMR
(75.5 MHz, d8-THF)¶: d = 23.0 (CH2), 24.4 (CH3), 49.9 (CH2),
106.6 (C), 118.7 (CH), 118.9 (C), 121.9 (CH), 124.5 (CH), 125.6
(CH), 128.4 (CH), 129.0 (CH), 135.7 (C), 143.3 (C), 149.0 (C),
156.1 (C), 158.0 ppm (C). IR (Nujol): mmax = 1614 (m), 1570 (s),
1310 (s), 754 (m) (Ar-H), 722 cm−1(m). LRMS (ES+), m/z (%):
277 (100) [M + H]+. HRMS (ES+): m/z calcd for C18H17N2O [M +
H]+: 277.1341; found: 277.1336.
(br s, 1 H, 3-H), 6.83 (ddq, J = 8.0, J = 1.6, J = 0.5, 1 H,
5-H), 7.02–7.09 (m, 1 H, 4ꢀꢀ-H), 7.31–7.40 (m, 2 H, 3ꢀꢀ-H), 7.80
(d, 3J = 8.0, 1 H, 6-H), 7.82–7.87 ppm (m, 2 H, 2ꢀꢀ-H). 13C NMR
(75.5 MHz, CDCl3): d = 19.5 (CH2), 21.3 (CH3), 29.0 (CH2), 50.3
(CH2), 51.4 (OCH3), 118.9 (C), 120.0 (CH), 122.4 (CH), 122.7
(CH), 123.5 (CH), 128.3 (CH), 130.9 (CH), 141.2 (C), 143.2 (C),
153.1 (C), 159.2 (C), 167.2 ppm (C). LRMS (ES+) m/z (%): 309
(100) [M + H]+, 277 (24) [M-MeOH]+. HRMS (ES+): m/z calcd
for C19H21N2O2 [M + H]+: 309.1603; found: 309.1597.
Methyl 3-methyl-2-(1-phenylpyrrolidin-2-ylideneamino)benzoate
(11). White crystalline solid after 72 h reflux (0.65 g, 2.1 mmol,
52%), mp 95–96 ◦C. IR (Nujol): mmax = 1720 (s) (C O), 1654
=
7-Methyl-1-phenyl-2,3,4,9-tetrahydro-1H-pyrrolo[2,3-b]quinolin-
4-on◦e (15). Off-white solid (0.51 g, 1.8 mmol, 95%), mp 186–
187 C. IR (Nujol): mmax = 1630 (m), 1578 (s), 1308 (w), 750 (m)
(Ar-H), 722 (m), 688 cm−1(w). 1H NMR (500 MHz, d8-THF): d =
2.41 (s, 3 H, CH3), 3.16 (t, 3J = 8.2, 2 H, 3-H), 4.01 (t, 3J = 8.2, 2
H, 2-H), 6.90–6.97 (m, 1 H, 4ꢀ-H), 7.00 (m, 1 H, 6-H), 7.25–7.34
(m, 2 H, 3ꢀ-H), 7.41 (br s, 1 H, 8-H), 7.92 (d, 3J = 8.2, 1 H, 5-H),
7.96–8.05 ppm (m, 2 H, 2ꢀ-H). 13C NMR (75.5 MHz, d8-THF)¶:
d = 21.5 (CH3), 22.8 (CH2), 49.8 (CH2), 105.5 (C), 117.7 (C), 118.8
(CH), 121.8 (CH), 122.2 (CH), 124.0 (CH), 128.0 (CH), 129.0
(CH), 139.0 (C), 143.3 (C), 144.0 (C), 149.2 (C), 160.6 ppm (C).
LRMS (CI+) m/z (%): 305 (11), 277 (99) [M + H]+. HRMS (CI+):
m/z calcd for C18H17N2O [M + H]+: 277.1341; found: 277.1342.
−1
1
=
(s) (C N), 1587 (m), 747 (m) (Ar-H), 722 cm (m). H NMR
(300 MHz, CDCl3): d = 1.98–2.10 (m, 2 H, 4ꢀ-H), 2.10–2.25 (m,
4 H, 3ꢀ-H, CH3), 2.52–2.67 (m, 1 H, 3ꢀ-H), 3.78 (s, 3 H, OCH3),
3.89 (t, J = 6.8, 2 H, 5ꢀ-H), 6.91–6.97 (m, 1 H, 5-H), 7.07 (m,
3
1 H, 4ꢀꢀ-H), 7.29–7.34 (m, 1 H, 4-H), 7.34–7.41 (m, 2 H, 3ꢀꢀ-H),
7.70 (m, 1 H, 6-H), 7.83–7.90 ppm (m, 2 H, 2ꢀꢀ-H). 13C NMR
(75.5 MHz, CDCl3): d = 18.2 (CH3), 19.3 (CH2), 29.4 (CH2), 50.2
(CH2), 51.3 (OCH3), 119.8 (CH), 120.9 (CH), 121.2 (C), 122.4
(CH), 128.1 (CH), 128.2 (CH), 130.0 (C), 133.4 (CH), 141.3 (C),
151.3 (C), 158.9 (C), 167.5 ppm (C). IR (Nujol): mmax = 1720 (s)
−1
=
=
(C O), 1654 (s) (C N), 1587 (m), 747 (m) (Ar-H), 722 cm (m).
LRMS (ES+) m/z (%): 309 (100) [M + H]+. HRMS (ES+): m/z
calcd for C19H21N2O2 [M + H]+: 309.1603; found: 309.1610.
8-Methyl-1-phenyl-2,3,4,9-tetrahydro-1H-pyrrolo[2,3-b]quinolin-
4-one (16). Off-white solid (0.42 g, 1.5 mmol, 95%), mp
Methyl 2-(1-phenylpyrrolidin-2-ylideneamino)benzoate (12).
White crystalline solid, (0.40 g, 1.4 mmol, 26%), mp 127–
128 ◦C. IR (Nujol): mmax = 1721 (s) (C O), 1654 (s) (C N), 1591
=
=
¶ The assignment of 13C signals was made by using Pendant, HMBC and
(m), 756 (m) (Ar-H), 692 cm−1 (m). 1H NMR (300 MHz, CDCl3):
HSQC NMR analysis.
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231–232 ◦C. IR (Nujol): mmax = 1566 (s), 1539 (m), 1307 (s), 1075
(w), 750 (m) (Ar-H), 722 (m), 693 cm−1(m). 1H NMR (500 MHz,
d8-THF): d = 2.65 (s, 3 H, CH3), 3.16 (t, 3J = 8.1, 2 H, 3-H), 4.07
(m, 1 H, 8-H), 7.30–7.38 (m, 2 H, 3ꢀ-H), 8.08–8.14 ppm (m, 2
H, 2ꢀ-H). 13C NMR (75.5 MHz, d8-THF): d = 21.5 (CH3), 29.6
(CH2), 48.4 (CH2), 75.0 (C), 120.1 (CH), 121.1 (C), 123.9 (CH),
125.2 (CH), 127.0 (CH), 129.04 (CH), 134.3 (CH), 141.4 (C),
142.0 (C), 153.3 (C), 166.0 (C), 197.0 ppm (C). LRMS (ES+): m/z
(%): 293 (100) [M + H]+. HRMS (ES+): m/z calcd for C18H17N2O2
[M + H]+: 293.1290; found: 293.1288. Anal. calcd for C18H16N2O2:
C, 73.95; H, 5.52; N, 9.58; found: C, 73.75; H, 5.54; N, 9.67. An
analytical sample of 4 was prepared by recrystallisation from
acetonitrile; [a]2D5 = −450 (c = 0.07 in dichloromethane). Chiral
HPLC analysis showed that after a single recrystallisation 4 was
prepared with an ee of 98.9%. When this reaction was carried out
using 19 instead of 18, the ee was 17%.
3
3
(t, J = 8.1, 2 H, 2-H), 6.89–6.96 (m, 1 H, 4ꢀ-H), 7.05 (dd, J =
3
8.1, J = 7.0, 1 H, 6-H), 7.27–7.36 (m, 3 H, 3ꢀ-H,7-H), 7.84 (br
dd, 3J = 8.1, 4J = 0.8, 1 H, 5-H), 8.12–8.19 ppm (m, 2 H, 2ꢀ-H).
13C NMR (125.7 MHz, d8-THF)¶: d = 18.8 (CH3), 22.4 (CH2),
48.8 (CH2), 106.1 (C), 117.7 (CH), 119.2 (C), 119.6 (CH), 121.1
(CH), 121.6 (CH), 128.8 (CH), 129.4 (CH), 134.3 (C), 143.5 (C),
148.7 (C), 155.2 (C), 160.0 ppm (C). LRMS (ES+) m/z (%): 277
(100) [M + H]+. HRMS (ES+): m/z calcd for C18H17N2O [M +
H]+: 277.1341; found: 277.1336.
1-Phenyl-2,3,4,9-tetrahydro-1H-pyrrolo[2,3-b]quinolin-4-one (17).
Off-white solid (0.42 g, 1.6 mmol, 95%), mp 189–190 ◦C. IR
(Nujol): mmax = 1630 (s), 1575 (s), 1297 (w), 756 (m) (Ar-H), 722
S-3a-Hydroxy-7-methyl-1-phenyl-2,3,3a,4-tetrahydro-1H-pyr-
rolo[2,3-b]quinolin-4-one (5). Bright yellow solid (0.045 g,
0.15 mmol, 83%), ee 90% as determined by chiral-phase HPLC
analysis (Daicel Chiralpak AD, acetonitrile–water 50 : 50, flow
1
(m), 695 cm−1 (m). H NMR (300 MHz, d8-THF): d = 3.19 (t,
3
3J = 8.2, 2 H, 3-H), 4.07 (t, J = 8.2, 2 H, 2-H), 6.91–6.98 (m,
rate 1.0 mL min−1, k = 254 nm, major enantiomer: tR = 9.39 min,
1 H, 4ꢀ-H), 7.11–7.18 (m, 1 H, 6-H), 7.27–7.34 (m, 2 H, 3ꢀ-H),
7.36–7.43 (m, 1 H, 7-H), 7.58–7.64 (m, 1 H, 8-H), 8.00 (dd, 3J =
8.2, 4J = 1.3, 1 H, 5-H), 8.02–8.08 ppm (m, 2 H, 2ꢀ-H). 13C NMR
(75.5 MHz, d8-THF)¶: d = 22.6 (CH2), 49.5 (CH2), 106.4 (C),
118.6 (CH), 119.6 (C), 121.8 (CH), 122.0 (CH × 2), 126.6 (CH),
128.9 (CH × 2), 143.3 (C), 148.4 (C), 157.7 (C), 159.5 ppm (C).
LRMS (ES+) m/z (%): 263 (100) [M + H]+. HRMS (ES+): m/z
calcd for C17H15N2O [M + H]+: 263.1184; found: 263.1180.
◦
and minor enantiomer: tR = 12.42 min), mp 223–224 C, [a]D25
=
−354 (c = 0.1 in dichloromethane). IR (Nujol): mmax = 1664 (m),
1593 (m), 1300 (w), 1264 (w), 752 (m) (Ar-H), 722 cm−1(w). H
1
NMR (300 MHz, d8-THF): d = 2.16–2.32 (m, 2 H, 3-H), 2.33 (s,
3 H, CH3), 3.89–3.97 (m, 1 H, 2-H), 4.12 (dt, 2J = 9.7, 3J = 6.2, 1
H, 2-H), 6.84 (m, 1 H, 6-H), 7.05–7.10 (m, 2 H, 4ꢀ,8-H), 7.30–7.38
3
(m, 2 H, 3ꢀ-H), 7.66 (d, J = 7.8, 1 H, 5-H), 8.10–8.16 ppm (m,
2 H, Ar-2ꢀ-H). 13C NMR (75.5 MHz, d8-THF): d = 21.6 (CH3),
29.6 (CH2), 48.3 (CH2), 74.0 (C), 120.2 (CH), 120.5 (C), 123.9
(CH), 124.5 (CH), 127.2 (CH), 127.4 (CH), 129.0 (CH), 142.0 (C),
146.7 (C), 152.7 (C), 167.0 (C), 194.3 ppm (C). LRMS (ES+) m/z
(%): 293 (100) [M + H]+. HRMS (ES+): m/z calcd for C18H17N2O2
[M + H]+: 293.1290; found: 293.1296. Anal. calcd for C18H16N2O2:
C, 73.95; H, 5.52; N, 9.58; found: C, 73.90; H, 5.56; N, 9.69. An
analytical sample of 5 was prepared by recrystallisation from
acetonitrile; [a]2D5 = −450 (c = 0.1 in dichloromethane). Chiral
HPLC analysis showed that after a single recrystallisation 5 was
prepared with an ee of >99%. When this reaction was carried out
using 19 instead of 18, the ee was 28%.
General procedure for the synthesis of analogues 4–7
A
solution of the corresponding quinolone (1.0 equiv.)
in dry THF (10–28 mL) was added dropwise to lithium
bis(trimethylsilyl)amide (1.0 M in THF, 1.2 equiv.) in dry THF
(2.0–5.0 mL) at −78 ◦C under argon. The reaction was stirred for
30 min at −78 ◦C and a solution of 18 (2.4 equiv.) (or 19) in dry
THF (4–10 mL) was added via cannula. After 16 h at −10 ◦C
saturated aqueous ammonium iodide (5.0 mL, 10 equiv) and
diethyl ether (5.0 mL) were added and the reaction mixture grad-
ually warmed to room temperature. Saturated aqueous sodium
thiosulfate (5–15 mL) was then added and the reaction mixture
extracted with diethyl ether (2 × 10 mL). The combined organic
extracts were dried (MgSO4) and concentrated in vacuo. The solid
was partitioned between dichloromethane (100 mL) and aqueous
hydrochloric acid solution (0.30 M, 100 mL). The aqueous phase
was washed with dichloromethane (3 × 100 mL), basified with
aqueous sodium hydroxide solution (2.0 M, pH adjusted to 8) and
extracted with ethyl acetate (2 × 100 mL). The organic extracts
were dried (MgSO4) and concentrated in vacuo to give the desired
compound.
S-3a-Hydroxy-8-methyl-1-phenyl-2,3,3a,4-tetrahydro-1H-pyr-
rolo[2,3-b]quinolin-4-one (6). Bright yellow solid (0.13 g,
0.45 mmol, 63%), ee 86% as determined by chiral-phase HPLC
analysis (Daicel Chiralpak AD, acetonitrile–water 50 : 50, flow
rate 0.8 mL min−1, k = 254 nm, major enantiomer: tR = 16.67 min,
◦
and minor enantiomer: tR = 52.30 min), mp 214–215 C, [a]D25
=
−420 (c = 0.1 in dichloromethane). IR (Nujol): mmax = 3329 (s),
1670 (s), 1618 (s), 1590 (s), 1297 (m), 754 cm−1 (m) (Ar-H). H
1
NMR (500 MHz, d8-THF): d = 2.21–2.35 (m, 2 H, 3-H), 2.45
2
3
(s, 3 H, CH3), 3.94–3.99 (m, 1 H, 2-H), 4.13 (dt, J = 9.6, J =
3
S-3a-Hydroxy-5-methyl-1-phenyl-2,3,3a,4-tetrahydro-1H-pyr-
rolo[2,3-b]quinolin-4-one (4). Bright yellow solid (0.020 g,
0.070 mmol, 87%), ee 64% as determined by chiral-phase HPLC
analysis (Daicel Chiralpak AD, acetonitrile–water 50 : 50, flow
6.0, 1 H, 2-H), 6.90–6.94 (m, J = 7.5, 1 H, 6-H), 7.05–7.09 (m,
1 H, 4ꢀ-H), 7.33–7.39 (m, 3 H, 3ꢀ,7-H), 7.62–7.65 (m, 1 H, 5-H),
8.16–8.20 ppm (m, 2 H, 2ꢀ-H). 13C NMR (125.7 MHz, d8-THF):
d = 18.3 (CH3), 29.6 (CH2), 48.1 (CH2), 73.4 (C), 119.9 (CH),
122.0 (C), 123.1 (CH), 123.8 (CH), 124.9 (CH), 129.1 (CH), 134.3
(C), 136.9 (CH), 142.0 (C), 150.5 (C), 165.6 (C), 195.2 ppm (C).
LRMS (ES+) m/z (%): 293 (100) [M + H]+. HRMS (ES+): m/z
calcd for C18H17N2O2 [M + H]+: 293.1290; found: 293.1300. An
analytical sample of 6 was prepared by recrystallisation from
acetonitrile. Anal. calc’d for C18H16N2O2: C, 73.95; H, 5.52; N,
9.58; found: C, 73.62; H, 5.58; N, 9.60. Chiral HPLC analysis
rate 0.8 mL min−1, k = 254 nm, major enantiomer: tR = 6.05 min,
◦
and minor enantiomer: tR = 8.02 min), mp 227–228 C, [a]D25
=
−310 (c = 0.1 in dichloromethane). IR (Nujol): mmax = 1686 (m),
1618 (m), 1590 (m), 1313 (w), 1262 (w), 723 cm−1(m) (Ar-H). 1H
NMR (300 MHz, d8-THF): d = 2.19–2.37 (m, 2 H, 3-H), 2.53 (s,
3 H, CH3), 3.90–3.98 (m, 1 H, 2-H), 4.11 (dt, 2J = 9.6, 3J = 6.4, 1
H, 2-H), 6.82 (m, 1 H, 6-H), 7.02–7.11 (m, 2 H, 4ꢀ,7-H), 7.25–7.29
2082 | Org. Biomol. Chem., 2008, 6, 2076–2084
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showed that after a single recrystallisation, 6 was prepared with
an ee of >99%. When this reaction was carried out using 19
instead of 18, the ee was 40%.
with an equal volume of well solution containing 100 mM MOPS
(pH 7.0), 250 mM MgCl2, 12% PEG 8000, 1 mM TCEP, and
2 mM Thymol. The protein/precipitant solutions for all analogue
complexes were streak-seeded from preliminary crystals and grew
to maximum dimensions within 2 to 3 weeks.
S-3a-Hydroxy-1-phenyl-2,3,3a,4-tetrahydro-1H-pyrrolo[2,3-b]-
quinolin-4-one (7). Bright yellow solid (0.040 g, 0.15 mmol, 78%),
ee 86% as determined by chiral-phase HPLC analysis (Daicel
Chiralpak AD, acetonitrile–water 50 : 50, flow rate 0.9 mL min−1,
k = 254 nm, major enantiomer: tR = 7.72 min, and minor
enantiomer: tR = 11.07 min), mp 203–204 ◦C, [a]D25 = −414 (c = 0.1
in dichloromethane). 1H NMR (300 MHz, CDCl3): d = 2.21–2.34
(m, 1 H, 3-H), 2.41–2.50 (m, 1 H, 3-H), 3.83–3.92 (m, 1 H, 2 H),
Data collection
Crystals were transferred from the original drop to a synthetic
mother liquor containing 100 mM MOPS (pH 7.0), 225 mM
MgCl2, 2 mM thymol, 12% PEG 8000, 1X MgADP.Vi trapping
solution, and 0.5 mM of each blebbistatin analogue. Prior to
freezing in a stream of nitrogen gas, the crystals were gradually
transferred from the synthetic mother liquor to a cryoprotectant
solution of 100 mM MOPS (pH 7.0), 300 mM MgCl2, 16% PEG
8000, 2 mM thymol, 1X MgADP.Vi trapping solution, 0.5 mM
blebbistatin, and 25% ethylene glycol. X-Ray data were collected
on a MAR-165 detector at the COM-CAT 32-ID beamline at
the Advanced Photon Source in Arg◦onne, Il with a wavelength
2
3
3
4
4.03 (dt, J = 9.8, J = 5.8, 1 H, 2-H), 7.05 (dt, J = 7.5, J =
1.1, 1 H, 6-H), 7.16–7.23 (m, 2 H, 4ꢀ,8-H), 7.37–7.47 (m, 3 H,
3ꢀ,5-H), 7.83–7.87 (m, 1 H, 7-H), 7.88–7.93 ppm (m, 2 H, 2ꢀ-H).
13C NMR (75.5 MHz, d8-THF): d = 29.6 (CH2), 48.3 (CH2), 74.0
(C), 120.3 (CH), 122.44 (C), 123.5 (CH), 124.0 (CH), 127.0 (CH),
127.1 (CH), 129.0 (CH), 135.7 (CH), 141.9 (C), 152.6 (C), 166.7
(C), 194.8 ppm (C. IR (Nujol): mmax = 1698 (m), 1618 (m), 1593 (m),
1294 (w), 723 cm−1(m) (Ar-H). LRMS (ES+) m/z (%): 279 (100)
[M + H]+. HRMS (ES+): m/z calcd for C17H15N2O2 [M + H]+:
279.1134; found: 279.1141. Anal. calc’d for C17H14N2O2: C, 73.37;
H, 5.07; N, 10.07; found: C, 73.26; H, 4.84; N, 10.28. An analytical
sample of 7 was prepared by recrystallisation from acetonitrile;
[a]2D5 = −460 (c = 0.1 in dichloromethane. Chiral HPLC analysis
showed that after a single recrystallisation, 7 was prepared with an
ee of >99%. When this reaction was carried out using 19 instead
of 18, the ee was 55%.
˚
of 1.0 A. An oscillation range of 0.5 was used for all datasets.
Diffraction data were integrated and scaled with the program
HKL2000.17
Structure determination and refinement
The structures of each analogue–myosin complex were solved by
molecular replacement using the Blebbistatin-MgADP.Vi-S1dC
structure (PDB accession code 1YV3) as the starting model.8 The
structure models were subjected to manual and automated refine-
ment using Coot and Refmac5, respectively.18,19 Water molecules
were added to the coordinate sets using Coot with subsequent
manual verification.18 Blebbistatin analogues were built into the
electron density using the original blebbistatin coordinates as a
starting model, followed by subsequent manual atom position
refinement in Coot. Relevant X-ray data collection and refinement
statistics are shown in the ESI, Table S1† in the supplementary
material. Ramachandran statistics for the myosin polypeptide
chain of each complex were determined using PROCHECK.20
For the 5–myosin complex 91.9% are in the most favored region,
7.7% additionally allowed, 0.3% generously allowed, and 0%
disallowed. For the 4–myosin complex 93.7% are in the most
favored region, 6% additionally allowed, 0.3% generously allowed,
and 0% disallowed. For the 7–myosin complex 92.7% are in the
most favored region, 7.1% additionally allowed, 0.2% generously
allowed, and 0% disallowed. For the 6–myosin complex 92.4%
are in the most favored region, 7.3% additionally allowed, 0.3%
generously allowed, and 0% disallowed.
General procedure for preparation and 7Li NMR analysis of
quinolone enolates
A solution of the quinolone (0.025 g, 0.090 mmol, 1.0 eq) in
anhydrous THF (10 mL) was cooled to −78 ◦C and stirred for
5 min. Lithium bis(trimethylsilyl)amide (1.0 M in THF, 0.11 mL,
0.11 mmol, 1.2 equiv.) was then added to the reaction mixture, at
−78 ◦C. An aliquot from the reaction mixture was transfer to an
NMR tube, under an inert atmosphere, and analysed by 7Li NMR
◦
(116.6 MHz), at −78 C. A capillary containing acetone-d6 was
used as a lock signal in each case. 7Li chemical shifts are given with
respect to 0.1 M solution of Li2SO4 (as external standard, with a
d = 0 ppm) in H2O.
Procedures for crystallisation studies
A truncated version of Dictyostelium myosin II (S1dC) containing
residues Asp2-Asn762 was used for all crystallisation experiments.
The S1dC construct was cloned into a recombinant version of the
pDXA vector (pDTEV2), which inserts a 6-His tag followed by
an rTEV protease recognition site at the N-terminus of myosin.
This construct was expressed and purified from Dictyostelium
discoideum as previously described.8,15 Co-crystallisation of this
myosin with each of the blebbistatin analogues was achieved by
addition of the analogue at a final concentration of 0.5 mM
followed by one tenth of the volume of a 10X MgADP.Vi stock
solution containing 10 mM MgCl2, 20 mM ADP, and 30 mM
sodium vanadate.16 This mixture was incubated on ice in the dark
for 1 h to allow the analogue and the nucleotide to form a stable
complex with ◦myosin. Crystals of these complexes were grown in
the dark at 4 C by vapor diffusion after mixing 5 ll of protein
Structure analysis and figure preparation
Structure alignments were performed with Superpose.21 Figures
were prepared using MacPymol.22
Acknowledgements
We would like to thank Dr Stephen Patterson for providing a
sample of 8 and Laura Walker for assistance with the blebbistatin
analogue–myosin co-crystallisations. We would also like to thank
the following for funding: The Royal Society (NJW fellow-
ship), EPSRC (CL-L PhD funding) and BBSRC (CPATL PhD
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funding), NIH grant AR35186 (IR) and a Canadian Institutes
of Health postdoctoral fellowship (64606 to JSA). The structural
coordinates for the blebbistatin analogue-myosin complexes have
been deposited in the RCSB under accession numbers 3BZ7 (4-
myosin complex), 3BZ8 (5-myosin complex), and 3BZ9 (7-myosin
complex).
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