5-Aryl-2-(naphtha-1-yl)sulfonamido-thiazol-4(5: H)-ones as clathrin inhibitors

Article abstract of DOI:10.1039/c6ob02308h

The development of a (Z)-5-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (2), rhodanine-based lead that led to the Pitstop 2 family of clathrin inhibitors is described herein. Head group substitution and bioisosteric replacemen

Full text of DOI:10.1039/c6ob02308h

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A. horatscheck, S. Sauer, L. von Kleist, J. R. Baker, W. Stahlschmidt, M. Nazare, A. Whiting, N. Chau, P. J.
Robinson, V. Haucke and A. McCluskey, Org. Biomol. Chem., 2016, DOI: 10.1039/C6OB02308H.
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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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5-Aryl-2-(naphtha-1-yl)sulfonamido-thiazol-4(5H)-ones as clathrin
inhibitors
Received
January 20xx,
00th
Mark J Robertson,a André Horatscheck,b Samantha Sauer,a Lisa von Kleist,b Jennifer R Baker,a Wiebke Stahlschmidt,b Marc
Nazaré,b Ainslie Whiting,c Ngoc Chau,c Phillip J Robinson,c Volker Hauckeb and Adam McCluskeya*
The development of a (Z)-5-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (2), rhodanine-
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
based lead that led to the Pitstop® 2 family of clathrin inhibitors is described herein. Head group substitution and
bioisosteric replacement of the rhodanine core with a 2-aminothiazol-4(5H)-one scaffold eliminated off target dynamin
activity. A series of N-substituents gave first phenylglycine (20, IC50~ 20 ꢀM) then phenyl (25, IC50 ~ 7.1 ꢀM) and 1-napthyl
sulfonamide (26, Pitstop® 2 compound, IC50 ~ 1.9 μM) analogues with good activity, validating this approach. A final
library exploring the head group resulted in three analogues displaying either slight improvements or comparable activity
(33, 38, and 29 with IC50 ~ 1.4, 1.6 and 1.8 ꢀM respectively) and nine others with IC50 < 10 ꢀM. These results were
rationalized using in silico docking studies. Docking studies predicted enhanced Pitstop® 2 family bindng, not a loss of
binding, within the Pistop® groove of the reported clathrin mutant clathrin, invalidating recent assumptions of poor
selectivity for this family of clathrin inhibitors.
together with a variety of membrane curvature effectors, i.e.
BAR domain proteins,8 drives further membrane invagination
Introduction
Clathrin is best known for its functions in endocytosis and
vesicle trafficking between the trans-Golgi network and
endosomes, however an additional role in mitosis is now well
established.1–4 Combined, these key roles in cell physiology
make the inhibition of clathrin an attractive medicinal
chemistry target for infectious disease and novel cancer anti-
mitotics.
of nascent cargo loaded coated pits, which eventually undergo
fission mediated by dynamin GTPase. The resulting clathrin-
coated vesicles (CCV) rapidly uncoat and fuse with endosomes
to deliver their internalized cargo into the cell interior. A
similar clathrin dependent vesicle formation process occurs at
the trans-Golgi network and at endosomes. Moreover, clathrin
has also been found to play an important role in mitosis that is
independent of its function in vesicular traffic. Here it
organises and stabilises the spindle microtubules allowing
proper formation of the mitotic spindle during anaphase.2,4,9,10
The clathrin terminal domain (CTD) is crucial to clathrin’s
function and its interaction within cells. The CTD is localised at
the end of each of the three legs of the clathrin triskelia and
consists of a seven bladed WD40-like propeller structure,11
which contains three defined binding sites.12,13 These sites
associate with so-called clathrin box peptide ‘motifs’ which
comprise short peptide sequences contained within
endogenous clathrin binding proteins. These include the
Endocytosis is
a cellular process that mediates the
internalisation of extracellular material including ligand-bound
receptors or pathogens into the cell interior, and can be
broadly classified as either clathrin-mediated endocytosis
(CME) or clathrin-independent endocytosis.5–7 In CME, a
complex and highly orchestrated protein-protein interaction
cascade results in the formation of polyhedral coats,
comprising self-assemblies of clathrin protein around plasma
membrane-derived vesicles. The formation of these vesicles
occurs through distinct, largely reversible stages.8 The initial
nucleation phase is defined by the adaptor protein-mediated
accumulation of transmembrane cargo proteins at
phosphatidylinositol (4,5)-bisphosphate containing plasma
membrane sites, which facilitates recruitment of clathrin and/
or adaptor-binding accessory proteins. Clathrin polymerization
endocytic accessory proteins amphiphysin, AP180,
β-arrestins,
CALM and PI3KC2
α
that bind to the CTD.14 Protein-protein
interactions (PPIs) between endocytic proteins bearing such
peptide motifs and the CTD play a central role in CME but
have proved difficult to dissect using current tools such as
dominant-negative proteins and peptides, monoclonal
antibodies and gene fusion approaches.15,16 A small molecule
inhibitor of these interactions could potentially abrogate these
limitations and provide a means to explore clathrin cell
physiology.17
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Currently there are three reported classes of clathrin identified two leads active at 100
inhibitor: bolinaquinone identified by a chemical proteomics have previously reported the development of lead
approach, but with no reported IC50 value,18 the Pitstop® 1 rise to the Pitstop® 1 , , family of compounds (Figure 1).19
family and the cell permeable Pitstop® 2 analogues.1,19–21 We now report on the process of discovery and development
These inhibitors have been used as molecular probes in order of the second lead (Z)-2-(5-((6,8-dichloro-4-oxo-4H-
ꢀ
M,
1
and
2
(Figure 1). We
1, that gave
3
2,
to understand the internalisation of various cargos,24–28 roles chromen-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl,
of endocytosis in other processes such as neuronal which gave rise to Pitstop® 2, 26, and the broader family of
excitability,29 and intracellular trafficking.30 They have also Pitstop® 2 compounds (Figure 2).1,20
been used to differentiate clathrin-dependent from clathrin-
independent endocytic pathways31,32 including the
identification of CME as viral entry routes for HIV1 and
Crimean Congo haemorrhagic fever.31 Targeting receptor
trafficking via clathrin inhibition has also been identified as a
potential therapeutic opportunity to block tumour
angiogenesis.33 Non-endocytic roles of clathrin have also been
explored and have been identified as a potential new class of
antimitotic drug targets.2–4
The specificity of Pitstop® 2 was questioned based on its
ability to block the uptake of the Major Histocompatibility
Complex I (MHCI),22 and based on an expectation that
mutations within the CTD (the C+-CTD has the following 4
point mutations: T87A, Q89A, K96E, K98E) that should block
Figure 2. The bioisosteric and fragment approaches used to develop new clathrin
clathrin-box motifs (this was not tested in the study due to
lack of access to functionally active clathrin mutants) would
also effect the binding of Pitstop® 2.23 We have demonstrated
that MHCI internalisation was clathrin dependent, not clathrin
independent.20 Neither Pitstop® 2 nor clathrin binding domain
peptide binding data was provided in this prior study to
support retention or loss of function of the C+-CTD mutants.
Our modelling approach was adopted to removal ambiguity
around ‘fold and functionality’ issues that may have resulted
as a result of point mutations.34
inhibitors.
Results and Discussion
Lead
2 was considered moderately attractive, possessing
three readily modifiable regions: the aromatic moiety, the
rhodanine core and the N-substituent; and thus amenable to
focused library development (Figure 1). Of some concern was
the presence of the PAINS (pan-assay interference
compounds) promiscuous rhodanine core.35,36
While
rhodanine cores are still reported as novel leads,37 our
strategy sought to end-stage modulate potential promiscuity
through bioisosteric replacement.38 The rhodanine core was
only retained in early, isolated protein based assay studies.
ELISA-based clathrin PPI screening of
rhodanine library identified two clathrin-active 6,8-dichloro-
4H-chromen-4-one analogues ( 10; Table 1; , IC50 = 4.5
and , IC50 ~ 10
M),35 but off-target effects against dynamin
a pre-existing
4-
7
ꢀM
8
ꢀ
GTPase (also critical to CME) were noted (unpublished
results). A 3,5-dichlorobenzylidene based analogue showed
retention of dynamin activity but a loss in clathrin activity
(Table 1; 9, IC50 ~ 70 ꢀM), but a 4-bromobenzylidene based
analogue displayed good clathrin activity (Table 1; 10, IC50 ~ 10
ꢀ
M) and was devoid of dynamin activity.
Retaining the 4-BrPh moiety, we next explored the routes
shown in Scheme 1 for rhodanine core to 2-aminothiazol-
4(5H)-one replacement. Neither pathway was broadly
applicable to library synthesis. Knoevenagel condensation of
11 (and 12) with 4-bromobenzaldehyde, either via microwave
or thermal approaches using sodium acetate or piperidine,
Figure 1. An ELISA-based HTS identified (1) and (2) as inhibitors of the clathrin TD-
amphiphysin interaction. Lead (1) was previously used to develop the Pitstop® 1 series
(3) of clathrin inhibitors.19
gave 13 in 64 and 83% yield respectively (12 gave rise to 14
)
In an effort to identify potential clathrin inhibitors we
previously conducted high-throughput screening (HTS) of a
17,000 small molecule library (ChemBioNet) from which we
(Scheme 1). However subsequent N-alkylation of 13 was only
successful with 1-bromohexane to give 16 isolated in 16%
yield. Direct N-alkylation of 11 proved similarly
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unsuccessful.39,40 Treatment of 14 with octylamine under
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DOI: 10.1039/C6OB02308H
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microwave conditions gave 17 in a 41% yield; and 1-
naphthylamine 18 was accessed in a 16% yield only via a
mercuric chloride mediated pathway from 14 (Scheme 1).40,41
A similar yield, 10%, of the 2,2-diphenyl 19 was obtained in a
one-pot reaction sequence.42 An alternative route, a base-
catalysed nucleophilic displacement with the required amine,
however, gave analogues 16
89%.21 In all cases, NMR analysis revealed the presence of
tautomers in this series (ESI
).43
, 17 and 19 in yields from 44-
†
Scheme 1. Reagents and conditions: (i) 4-bromobenzaldehyde, sodium acetate, glacial
Table 1. Inhibition of dynamin I GTPase activity and the clathrin TD – amphiphysin
interaction by rhodanine analogues 2, 4-10 (Library 1).
acetic acid or piperidine (cat), reflux 18
h or MW, 150 °C, 150 W, 30 min; (ii)
hexylamine, DMF, triethylamine, RT, 18 h; (iii) octylamine, ethanol, MW, 200 W, 120
oC, 30 min; (iv) 1-napthylamine, HgCl2, 25 oC, 18 h; (v) 2,2-diphenylethylamine, 4-
bromobenzaldehyde, glacial acetic acid (cat), MW, 200W, 130 oC, 45 min; (vi) CH3I,
DIPEA, CH3CN, RT, 18 h; (vii) glycine, 2-phenylglycine or benzylamine, DIPEA, 25 oC, 3 h;
(viii) hexylamine, octylamine, 2,2-diphenylethylamine or glycine, DIPEA, CH3CN, reflux
18 h.
Dyn I
R1
R2
CTD – Amph IC50 (ꢀM)
IC50 (ꢀM)
To increase library diversity (and improve clathrin
inhibition) we developed a more robust synthetic approach via
H
24.2 ± 9.1
~100
a
thiomethyl intermediate 15, which was accessed by
treatment of 14 with methyl iodide in acetonitrile in excellent
yield (90%) using a simple filtration workup.21 Coupling of 15
with the requisite glycine afforded an analogue equivalent to
2
4
5
6
7
8
7
. This approach also facilitated the synthesis of alanine 20
phenylalanine 21 and benzylamine 22 in 56-90% yields. While
analogues 12 19 were devoid of clathrin inhibition, the
phenylalanine (21, IC50 = 20.2 M) was a modest clathrin
,
5.1 ± 0.8
-
-
ꢀ
inhibitor (Table 2). Removal of either the phenyl (20) or
a
-
~ 135
carboxylate (22) moieties resulted in a significant reduction in
clathrin inhibition with IC50 values > 50 ꢀM (Table 2). This
suggested a dual requirement of a polar moiety with an
adjacent hydrophobic moiety for clathrin inhibition.
~ 7.4
-
Installation of a sulfonamide isostere permitted removal of
the chiral centre associated with 21 (Scheme 2). After
considerable sequence optimization,21 treatment of 11 with 1-
naphthylsulfonyl chloride in DMF with 2.2 equivalents of NaH
afforded
direct
precipitation
of
the
desired
3.4 ± 0.4
10.2 ± 6.6
4.5
~10
1-naphthylsulfonamide (24) on careful addition of water in an
85% yield. The phenyl analogue 23 required extractive
workup. With 23 and 24 in hand, coupling with 4-
bromobenzaldehyde at ethanol reflux (or microwave heating
at 120 °C) gave the phenyl 25 and naphthyl 26 (Pitstop® 2)
sulfonamide analogues in yields of 63 and 73% respectively.
The use of a piperidine/benzoic acid co-catalyst negated any
issues associated with the formation of piperidine salts. Both
25 and 26 were potent clathrin inhibitors returning IC50 values
5.5 ± 0.8
≥100
~70
~10
9
of 7.1 and 1.9 ꢀM respectively (Table 2). Importantly, neither
analogue inhibited dynamin (at 100 µM), with 25 displaying
similar, and 26 superior, clathrin activity than the previously
reported Pitstop® 1 compound (IC50 ~6.9 µM).19
10
a (-) Not Active at 100 ꢀM compound concentration
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withdrawing group, -NO2
(
36), or sterically bulky moiety,
Table 2. The inhibition of the clathrin TD–amphiphysin interaction by (5Z)-5-(4-
brombenzylidene)-2-(amino)thiazol-4(5H)-one analogues 16-26 (Library 2).
benzylpiperidine (37), essentially removed clathrin activity.
These are presumably electronic and steric driven effects
respectively. The introduction of 3,4-dibromo (38, IC50 = 1.6
µM), 3,5-dichloro (39, IC50 = 4.7 µM; Pitstop® 2-100),20 and
2,5-difluoro (40, IC50 = 5.4 µM) moieties had limited effect in
activity.
CTD – Amph
Compound
R
IC50 (ꢀM)
Table 3. The inhibition of the clathrin TD–amphiphysin PPI by (5Z)-5-(2-benzylidene)-2-
(naphth-1-ylsulfonamido)thiazol-4(5H)-one analogues 26-40 (Library 3).
16
17
-
-
18
-
19
20
-
CTD – Amph
IC50 (µM)
Compound
26
R
>50
1.9 ± 0. 4
21
20.2
27
28
2.6 ± 0.5
3.1 ± 0.6
22
25
>50
7.1
29
30
31
32
33
34
35
1.9 ± 0.6
4.2 ± 0.9
3.6 ± 0.6
4.0 ± 0.8
1.41 ± 0.1
5.7
26
1.9 ± 0.4
a (-) Not Active at 100 μM compound concentration; b Pitstop®-2 compound15
7.2 ± 1.9
36
37
>50
>50
Scheme 2. Reagents and conditions: (i) NaH, DMF, R1SO2Cl, RT 2-18h (ii) R2CHO,
piperidine (cat), benzoic acid (cat), ethanol, MW, 120 °C, 200W, 20 min (iii) R2CHO,
piperidine (cat), benzoic acid (cat), ethanol, reflux, 18 h.
With 26 as the new lead we developed a focused library
exploring the 4-bromophenyl moiety using the chemistry
described in Scheme 2 (Table 3). The 4-bromo-substitution
was found to be preferred with 3- (27, IC50 = 2.6 µM) and 2-
38
39
1.6 ± 0.03
4.7 ± 0.8
(
28, IC50 = 3.1 µM) substitution showing a minor reduction in
activity. This trend was also observed on Br to Cl interchange
29 - 31). Overall, 4-substituted halogens were well tolerated
with activities I (33, IC50 = 1.4 µM) >Br (26, IC50 = 1.9 µM) ~Cl
29, IC50 = 1.9 µM) >F (32, IC50 = 4.0 µM) (Table 3). These small
(
40
5.4 ± 2.67
(
differences in halogen-dependent activity suggested only very
minor interactions between these moieties and the CTD.
These minor effects were also reflected in results for clathrin
activity obtained upon halogen removal (34, IC50 = 5.7 µM), or
We previously reported the co-crystallization of 26 with
the CTD revealed key hydrogen bond (HB) interactions
between the thiazol-4(5H)-one ring with R64,1 and
hydrophobic interactions between the sulfonamide
naphthalene ring and a hydrophobic pocket comprising: I52,
I66, I80, I93, L82, V50 and F91 (yellow coloured structure,
the use of a nitrile group as a halogen bioisostere (35, IC50
=
7.2 µM).43 The introduction of either a powerful electron
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Figure 3a). We emulated these studies using molecular clathrin PPI inhibition data, especially with respect to the
docking approaches using PyRx,44 running Autodock Vina.45 benzylidine moieties.
Our initial efforts saw the docking of compound 26 into the
Prior to our modelling analysis, Royle et al postulated that
binding pocket identified in the co-crystal structure to allow CTD clathrin box mutations T87A, Q89A, K96E, K98E should
direct comparison of predicted and known binding poses. The negate the Pitstop® 2 inhibition of CME.23 Intriguingly these
results highlighted the importance of the hydrophobic pocket, assumptions pertaining to Pitstop® 2 and the C+ mutants were
with either the naphthyl or 4-bromobenzlidene moiety founded on Royle’s assertion that it “is particularly
docking into this position. The remainder of the molecule noteworthy as these mutants are predicted to be unable to
clustered into the shallow groove to the left or right bind peptides bearing clathrin-box motifs”, in the absence of
(exemplified by an example of each binding motif, coloured ligand-mutant binding data. From this, Royle et al suggested
red and green respectively, Figure 3a). In both binding that these mutations would prevent Pitstop® 2 binding, and
clusters, the naphthalene moiety was shifted slightly to better concluded that the Pitstops® were non-specific.
align for a potential F91
predicted binding affinity pose saw R64 HB with the mutations adversely affecting protein folding and
sulfonamide S=O and a thiazol-4(5 )-one ring and K96 π- consequently function, a plausible reason for the Royle
π
-stacking interaction. The highest
To avoid potential complications arising from point
H
cation interaction (Figure 3a, red structure). In the second observations, we adopted a modelling approach to evaluate
binding cluster, the 4-bromobenzylidine moiety ‘flips’ to the the potential effect of the C+-CTD mutations. Our data is
other side of the hydrophobic pocket resulting in the loss of unequivocal, and contrary to Royle et al’s findings and clearly
the R64 HB, but is compensated by an additional benzyl and demonstrates no critical Pitstop® 2 contacts are affected by
thiazol-4(5H)-one ring K98 π-cation interaction (Figure 3a, T87A or Q89A mutations. The K96E and K98E mutants present
green structure). A single binding pose (Figure 3a coloured by a more profound change in basic amino acid structure, but
element) was identified that docked into a similar pose to that retain key HB interactions, consistent with our initial
found in the co-crystal structure with 26. Critically, this modelling that demonstrated flexibility in the thiazol-4(5H)-
analysis highlights the importance of the hydrophobic binding one moiety to R64, K96 and K98 HB interaction across the
pocket and a degree of flexibility in potential HB interactions observed binding.
(R64, K96 and K98). This is consistent with our observed
Figure 3: Examination of the binding interactions of 26 (Pitstop® 2, coloured yellow) within the co-crystal structure of CTD (PDB code 2X2E) and C+ mutant; a) Selected binding
poses within the groove to the left (red structure) and right (green structure) for 2X2E; b) Identification of the C+ mutant sites (yellow colored surface). No major binding
interactions are predicted in these regions with 26 (Pitstop® 2); c) Selected binding poses within the groove to the left (red structure) and right (green structure) for C+ mutant.
Binding was more pronounced towards the left region of the groove with only the single pose (shown) to the right.
Docking simulations with the C+-CTD quad-mutant reveal interaction with F91. The second highest scoring pose had
these mutations lie in close proximity to the 4-BrPh moiety of similar naphthyl moiety hydrophobic interactions, with the 4-
Piotstop® 2 (Figure 3b, yellow structure).23 Our SAR herein BrPh moiety gaining a HB interaction with R64 via the
demonstrates that minimal interaction occur in this region, sulfonamide (S=O) as well as hydrophobic interactions with
and we further note only superficial differences docking I62 and I93 (Figure 3c, red structure). Of the C+-CTD quad-
modes between wild type and the C+ quad mutant CTDs mutant, T87A and Q89A are remote from the Pitstop® 2
(Figure 3c versus Figure 3a). A slight bias towards binding in pocket, with negligible predicted interaction with these
the left side of the groove with only a single pose shifted residues. Neither K96E nor K98E, external to the ‘Pitstop
towards the right (Figure 3c, green structure). In this pose the groove’, interacted with Pitstop® 2. Docking analysis with wild
naphthyl and benzylidene portions have swapped positions type CTD supported potential π -cation interactions, however,
within the hydrophobic pocket, consistent with our findings the C+-mutant supported enhanced engagement via π-cation
with the wild type clathrin. The highest scoring pose interactions. Despite marginal predicted increase in
a
positioned the naphthyl moiety in a pocket with hydrophobic potential engagement with the C+-mutant, no calculated
interactions with V50, I52 and I80 (Figure 3c, coloured by binding affinity differences between wild type and C+ CTD
element). The 4-BrPh moiety gains an additional
π
-stacking were observed. This binding site flexibility has been noted
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previously through the simultaneous binding of clathrin-box
motif peptides to three binding sites in the CTD.12
The authors acknowledge the financial support of the
Deutsche Forschungsgemeinschaft (SFB765/B04 and
The Pitstop® analogues reported herein have SFB958/A01 to VH), the Australian Research Council, the
demonstrated functional activity in cell based models National Health and Medical Research Council (Australia,
consistent with the inhibition of clathrin as suggested by our in GNT1069493, GNT 1047070 and GNT 1022218), the
vitro screening data. Analogue 26 blocks the internalisation of Ramaciotti Foundation the Australian Cancer Research
HIV through inhibition of clathrin mediated endocytosis and in Foundation and the Cancer Institute NSW.
other studies treatment of HeLa cells with 26 and 39 resulted
in specific inhibition of the internalisation of the major and AM; MJR, AH, JRB and SS conducted synthesis; MN
histocompatibility complex.1,20
In this latter case, the contributed to compound design and supervision of AH; LvK
Author Contribution: Manuscript written by MJR, VH, PJR
observed phenotype matched that observed by clathrin or AP- and WS conducted clathrin screening supervised by VH; AW
2 depletion, confirming that MCHI endocytosis is clathrin and NC conducted off-target screening supervised by PJR.
dependent. Combined these data demonstrate the on target Original project concept developed by VH, PJR and AM.
and functional activity of the Pitstop® compound series.
Author Statement
The authors declare the following competing financial
Conclusions
interest(s): We have entered into a commercial agreement
with Abcam Biochemicals (Bristol, UK) for the supply of our
dynamin and clathrin inhibitors. This includes some of the
compounds listed in this paper.
Initial HTS screening of a ChemBioNet 17,000 molecule
library identified the rhodanine based
2 as a 100 ꢀM potent
inhibitor of clathrin. Evaluation of an in-house rhodanine
library identified two additional potent clathrin inhibitors, the
#Pitstop
® is a registered trademark of Children’s Medical
Research Institute and Newcastle Innovation Ltd. Compounds
26 (Pitstop®-2), 39 (Pitstop®-2-100), 18 (Pitstop Inactive
control 2) and 19 (Pitstop Inactive control 2B) described in this
paper are available from Abcam Biochemicals (Bristol, UK).
6,8-dichlorochrom-4-one based
analogues showed off-target effects against another key
endocytic pathway protein, dynamin GTPase, activity.
7 and 8. However these
I
Introduction of a 4-bromobenzylidine moiety gave 10, devoid
of dynamin I GTPase activity. This lack of dynamin activity was
maintained throughout the other compounds in this study.
Experimental Section
Simple rhodanine bioisosteres were inactive (13, 16-20, 22),
however introduction of phenyl alanine moiety (21) and ELISA-based clathrin inhibitor binding assay
subsequent installation of a sulfonamide moiety combined The clathrin inhibitor assay was based on that of our
with the thiazole-4(5
)-one rhodanine bioisostere afforded a previous report.13 Bacterially expressed recombinant purified
wide range of highly active clathrin inhibitors (25 40). Within His6-tagged amphiphysin 1 (amino acids 250-578) in screening
this series the 4-nitro 36 and sterically bulky buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 1 mM DTT, 1 mM
H
-
(
)
N-
benzylpiperidine (37) were inactive. Larger halogens show PMSF) was added to a 384 well ELISA plate (high-binding PS
marginally better activity and di-halogenated analogues were Microplate, Greiner Bio-One) and bound to the plastic for 1 hr
also well tolerated, although
a
5-substituent (with at room temperature. Non-specific binding was reduced by
L blocking buffer (20 mM
disubstituted aromatic analogues) saw an activity reduction. overnight incubation with 50
ꢀ
The analogues reported herein currently represent the most HEPES, pH 7.4, 50 mM NaCl, 1 mM PMSF, 2% BSA, 2.5% skim
potent small molecule inhibitors of clathrin, with 26 (Pitstop® milk) at 4 °C. Following extensive washes (with 20 mM HEPES,
2)1,21 and 39 (Pitstop® 2-100)20,21 have been widely used to pH 7.4, 50 mM NaCl, 0.05% Tween 20), chemical compounds
probe clathrin mediated biochemical pathways. The Pitstop® diluted in DMSO (10 µL) were added and incubated together
2 compound series are CME inhibitors in cells.1,20
with bacterially expressed recombinant GST-tagged clathrin
To date the Pitstops have proven to be selective clathrin heavy chain TD (amino acids 1–364) for 1 hr at room
inhibitors, despite some claims to the contrary.20,23 Our temperature in screening buffer. After 3 washes, horseradish
molecular docking studies highlighted the importance of the peroxidase-coupled anti-GST antibodies were added in
naphthyl moiety interaction with the hydrophobic pocket and screening buffer and the plate incubated for 15 min at room
the facile movement of key HB across R64, K96 and K98 with temperature. Following additional washes, 50 ꢀL TMB
the thiazol-4(5 )-one and sulfonamide moieties. Subsequent (3,3′,5,5′-tetramethylbenzidine) (Pierce Biotechnology)
H
evaluation of the Royle C+-CTD quad-mutant (T87A, Q89A, chromogenic substrate of horseradish peroxidase was added
K96E and K98E) revealed, at best, superficial impact on and the plate was incubated for 20 min before the reaction
Pitstop® 2 binding in the CTD.34 Modelling predicts that the was terminated by adding 50
ꢀ
L 1 N sulfuric acid to produce a
K96E and K98E mutations would result in enhanced yellow color. The amount of bound protein was determined
engagement with the surface-binding groove and, thus would by spectrophotometric measurement in plate reader.
a
be expected to show enhanced clathrin inhibition.
Relative binding was calculated as a percentage of DMSO
control.
Acknowledgements
Dynamin GTPase assay
6 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
Page 7 of 12
Journal Name
Native dynamin I was purified from sheep brain by MHz respectively, or
View Article Online
DOI: 10.1039/C6OB02308H
ARTICLE
a
Bruker Avance III 400 MHz
extraction from the peripheral membrane fraction of whole spectrometer, where H-NMR and 13C-NMR were acquired at
1
brain and affinity purification on GST-Amph2-SH3-sepharose 400 and 100 MHz respectively. All spectra were recorded in
as previously described.43 The GTPase assay was based on deuterated dimethyl sulfoxide (DMSO-d6
) or deuterated
colorimetric detection of phosphate release from GTP using acetone (acetone-d6), obtained from Sigma Aldrich or
the Malachite Green method as previously described.43,44 Cambridge Isotope Laboratories Inc., unless otherwise stated,
except that except that the GTPase assay buffer contained 5 with the residual solvent peaks used as the internal reference
mM Tris-HCl, 10 mM NaCl, 2 mM Mg2+, pH 7.4, 1
1
ꢀ
g/mL (δ 2.49 (quintet) and δ 39.7 (septet) for H-NMR and 13C-NMR
leupeptin, 0.1 mM PMSF and 0.3 mM GTP. Dynamin (20 nM) respectively for DMSO-d6 and δ 2.05 (quintet) and δ 29.8
activity was stimulated by
g/mL sonicated (septet) for 1H-NMR and 13C-NMR respectively for acetone-d6).
phosphatidylserine in the presence of test compounds for 30 Chemical shifts (δ) were measured in parts per million (ppm)
4
ꢀ
min at 37 °C.
and referenced against the internal reference peaks. Coupling
constants ( ) were measured in Hertz (Hz). Multiplicities are
denoted as singlet (s), broad singlet (br s), doublet (d), doublet
J
Molecular Modelling & Docking Studies
Molecular modeling was conducted using the PyRx of doublets (dd), doublet of doublet of doublets (ddd), triplet
platform45 running the autodock Vina molecular modelling (t), triplet of doublets (td), doublet of triplets (dt), quartet (q),
system.46 The structure comprising the compound 26
-
quintet (quin) and multiplet (m). Peaks are listed in increasing
clathrin-TD co-crystal was used as the modeling base (PDB chemical shift in the following format: chemical shift
Code: 2Z2E). The Discovery Studio 3.5 Visualiser (DSS) (integration (1H), multiplicity (1H), coupling constant (1H)).
software was used for manipulation of the compound data
General
Procedure
1
:
To
a
suspension
of
set, visualizing and creation of the molecular graphics images. pseudothiohydantoin 11 (3 eq.) in DMF (30 mL) was added,
The compounds used for docking were created in DSS, saved slowly in portions, solid sodium hydride (60% dispersion in
in pdb form and imported into the PyRx software to be saved mineral oil) (3 eq.). After stirring at room temperature (RT) for
as pdbqt files using the default settings. The binding site was 30 mins the desired sulfonyl chloride (1 eq.) was then slowly
defined by the sulfur of compound 26 (coordinates -50.042, - added. The mixture was stirred for the requisite time before
9.024, 26.138), which was then removed, and the protein also being acidified by the addition of 10% HCl(aq) solution (10 mL)
imported into the PyRx software using the default settings. and then water (50 mL). The solid formed was filtered off via
Docking was carried out using default settings using a 25 Å vacuum filtration, washed with water, EtOH and finally ether
grid around the previously defined binding site. A binding to afford the desired product. This material was usually used
affinity was then calculated for each compound, and for each as is, but can be recrystallized from EtOH or CH3CN if
ligand the score for up to nine different conformations were necessary.
produced. Both the docking poses and scores were exported
in sdf format for subsequent manipulation using DSS.
General Procedure 2: To a 10 mL reaction vessel was added
sulfonamide precursor (1 eq.), aldehyde (1.1 eq.),
piperidine/benzoic acid mix (0.5M of each, 0.1 mL) and
ethanol (5 mL). The resulting suspension was heated using
microwave irradiation (200 W, 120 °C) for 15 minutes. After
cooling overnight, the resulting precipitate was filtered,
washed with small portions of cold ethanol (2 x 10 mL) and
cold diethyl ether (2 x 10 mL) and air dried.
Chemistry General Procedures
All reactions were performed using standard laboratory
equipment and standard laboratory glassware. Solvents and
reagents were purchased from Sigma Aldrich, Lancaster
International or TCI and used as received. Compounds 3-10, 15
have been described previously35 and were used directly from
our in-house store. Organic solvents were bulk quality, and
were distilled from glass prior to use. Flash chromatography
was carried out using silica gel 200-400 mesh (60Å). Organic
solvent extracts were dried with magnesium sulfate (MgSO4),
and dried under reduced pressure with either Büchi or
Heidolph rotary evaporators. Melting points were recorded in
open capillaries on a Stuart SMP11 Melting Point Apparatus.
Temperatures are expressed in degrees Celsius (°C) and are
uncorrected. Where available, literature values are provided
and appropriately referenced. Electrospray mass spectra were
recorded using 10% DMSO/H2O or HPLC-grade methanol or
General Procedure 3:
A suspension of sulfonamide
precursor (1 eq.) in ethanol (30 mL), aldehyde (1.5 eq.) and
piperidine/benzoic acid mix (0.5M of each, 0.1 mL) was heated
at reflux for 18 h. After cooling overnight, the resulting
precipitate was filtered, washed with small portions of cold
ethanol (2 x 10 mL) and cold diethyl ether (2 x 10 mL) and air
dried. If no precipitate was formed the solvent was removed
and the resulting residue was purified using column
chromatography using
CH3OH/CH2Cl2.
a
gradient from CH2Cl2-10%
General Procedure 4: A suspension of rhodanine precursor
(1 eq.), amine (1.2 eq.) and DIPEA (1.5 eq.) in MeCN (15 mL)
was heated at reflux for 24 hours. After cooling overnight, the
resulting precipitate was filtered, washed with water (2 x 10
mL) and cold diethyl ether (2 x 10 mL) and air dried.
acetonitrile as carrier solvents on
spectrometer. All compounds were
determined by HPLC and LCMS analysis.
a
Shimadzu LC-MS
95% purity as
≥
NMR spectroscopy was performed on a Bruker Avance 300
MHz spectrometer, where proton NMR (1H-NMR) spectra and
carbon NMR (13C-NMR) spectra were acquired at 300 and 75
(Z)-5-(4-Bromobenzylidene)-2-iminothiazolidin-4(5H)-one
(13). This compound was synthesized via two methods:
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Page 8 of 12
View Article Online
DOI: 10.1039/C6OB02308H
ARTICLE
Journal Name
Method A: A mixture of pseudothiohydantoin 11 (2.0 g, 17.2
(Z)-5-(4-Bromobenzylidene)-2-(octylamino)thiazol-4(5H)-
mmol), 4-bromobenzaldehyde (3.5 g, 18.9 mmol, 1.1 equiv.) one (17). Was synthesised using General Procedure 4 with 13
and sodium acetate (1.41 g, 17.2 mol) in glacial acetic acid (30 (150 mg, 0.50 mmol), -octylamine (100 µL, 0.60 mmol) and
n
mL) was prepared, and the mixture was heated at reflux DIPEA (131 µL, 0.75 mmol) in CH3CN (15 mL) to afford 17 as a
overnight before being allowed to cool. The yellow solid white solid (86.2 mg, 44%). M.P: 173-176 °C.
produced was collected by filtration, washed with water (3 x
30 mL) and finally ether (3 x 30 mL) and air dried (4.02 g, 83%).
1H NMR (400 MHz, DMSO-d6): δ 9.73 (bs, NH), 7.72 (d,
J
=
8.4 Hz, 2H), 7.59-7.56 (m, 1H), 7.51 (d,
J
= 8.5 Hz, 2H), 3.50 (t,
J
Method B: A mixture of pseudothiohydantoin 11 (500 mg, = 7.0 Hz, 2H), 1.58 (quin,
J = 6.8 Hz, 2H), 1.29-1.26 (m, 10H),
4.30 mmol), 4-bromobenzaldehyde (796 mg, 4.30 mmol) and 0.87-0.84 (m, 3H).13C NMR (101 MHz, DMSO-d6): δ 179.4,
sodium acetate (353 mg, 4.30 mmol) were placed in 3 mL MW 172.9, 133.4, 132.2 (2C), 131.2 (2C), 129.7, 127.5, 122.7, 44.6,
reactor and acetic acid (3 mL) added. It was then heated by 31.2, 28.6, 28.5, 28.4, 26.2, 22.1, 13.9s. IR νmax (cm-1): 2950,
microwave irradiation (150 W, 150 oC, 30 min) before being 2849, 1683, 1601, 1249, 819, 525. Mass Spectral Analysis:
allowed to cool. The yellow/orange solid produced was LRMS: m/z (ES-) 395/397 [M+H]+ (100%). 393/395 [M-H]-
collected by filtration, washed with water and finally ether and (100%); HRMS: Exact mass calculated for C18H2279BrN2OS [M-
air dried (778 mg, 64%). M.P: 182 °C (dec)
1H NMR (400 MHz, DMSO-d6): δ 9.48 (s, NH), 9.21 (s, NH), C18H2281BrN2OS, 395.0621. Found 395.0678 (M-H)-.
H]-, 393.0642. Found 393.0704. Exact mass calculated for
7.72 (s, 2H), 7.54 (d,
J
= 14.5 Hz, 3H). 13C NMR (101 MHz,
(Z)-5-(4-Bromobenzylidene)-2-(naphthalen-1-
DMSO-d6): δ 180.3, 175.4, 133.4, 132.2 (2C), 131.3 (2C), 130.4,
127.8, 122.8. IR νmax (cm-1): 3061, 2848, 1700, 1594, 1577, 824,
514. Mass Spectral Analysis: LRMS: m/z (ES-) 281/283 [M-H]-
(100%); HRMS: Exact mass calculated for C10H7BrN2OS [M+H]+,
281.9462. Found 281.9534. HRMS: Exact mass calculated for
C10H6Br79N2OS [M-H]-, 280.9389. Found 280.9453. Exact mass
calculated for C10H681BrN2OS [M-H]-, 282.9369. Found
282.9453.
ylamino)thiazol-4(5H)-one (Pitstop Inactive Control 2) (18).
To a solution of rhodanine 14 (0.5 g, 3.73 mmol), 1-
naphthylamine (0.54 g, 3.77 mmol) and DIPEA (218 mg, 1.69
mmol) in dry CH2Cl2 (15 mL) was added mercuric chloride
(1.02 g, 3.76 mmol). The resulting mixture was allowed to stir
overnight before being filtered through Celite. The filtrate was
then washed with water (2 × 15 mL), dried over MgSO4 and
evaporated to dryness. This crude mixture was suspended in
ethanol (50 mL) and 4-bromobenzaldehyde (0.22 g, 1.19
mmol) and finally piperidine (4 drops) added. The resultant
suspension was heated at reflux overnight before being
evaporated to dryness. The product was purified using column
chromatography (gradient CH2Cl2 - 3% CH3OH/CH2Cl2) to yield
a yellow solid (80 mg, 16%). M.P: 254–256 °C.
(Z)-5-(4-Bromobenzylidene)-2-thioxothiazolidin-4-one (14).
This compound was synthesized as previously reported35 to
form a yellow solid (85%); M.P: 232 °C (dec).
1H NMR (400 MHz, DMSO-d6): δ 13.89 (bs, NH), 7.75 (d,
J =
8.5 Hz, 2H), 7.63 (s, 1H), 7.55 (d,
J
= 8.6 Hz, 2H). 13C NMR (101
MHz, DMSO-d6): δ 195.5, 169.5, 132.5 (2C), 132.2 (2C), 130.2,
1H NMR (400 MHz, DMSO-d6): δ 12.69 (bs, NH), 7.96 – 7.91
126.5, 124.3.
(m, 2H), 7.77 (d,
J
= 8.0 Hz, 1H), 7.62 – 7.50 (m, 6H), 7.38 (d, J =
13C NMR (101 MHz, DMSO-
(Z)-5-(4-Bromobenzylidene)-2-(methylthio)thiazol-4(5H)-
one (15). This compound was prepared as previously
reported21 to afford 15 as a pale yellow solid (90%). M.P: 171–
174 °C.
8.1 Hz, 2H), 7.13 (d,
J
= 6.7 Hz, 1H)
.
d6): δ 167.7*, 144.2, 133.9, 132.5, 132.2 (2C), 131.5 (2C),
128.3, 128.0, 127.0, 126.6 (2C), 126.1 (2C), 124.9, 124.1,
123.2, 123.0, 116.0. *Confirmed by HMBC correlations. IR νmax
(cm-1): 3128, 2980, 1701, 1598, 1340, 806, 562. Mass Spectral
Analysis: LRMS: m/z (ESI+) 408/410 [M+H]+ (100%). 406/408
[M-H]- (100%); HRMS: Exact mass calculated for
C20H1279BrN2OS [M-H]-, 406.9859. Found 406.9894. Exact mass
calculated for C20H1281BrN2OS [M-H]-, 408.9838. Found
408.9865.
1H NMR (400 MHz, DMSO-d6): δ 7.79 (s, 1H), 7.60 (d,
8.5 Hz, 2H), 7.38 (d,
= 8.5 Hz, 2H), 2.84 (s, 3H); 13C NMR (101
J =
J
MHz, DMSO-d6): δ 192.9, 179.9, 134.5, 132.67 (2C), 132.65,
131.8 (2C), 127.3, 125.4, 16.3.
(Z)-5-(4-Bromobenzylidene)-2-(hexylamino)thiazol-4(5H)-
one (16). Was synthesised using General Procedure 4 with 13
(150 mg, 0.50 mmol) and n-hexylamine (80 µL, 0.60 mmol) in
MeCN (15 mL) to afford 16 as a white solid (87.2 mg, 48%).
(Z)-5-(4-Bromobenzylidene)-2-(2,2-diphenylethylamino)
thiazol-4(5H)-one (Pitstop Inactive Control 2B) (19). Was
synthesised using General Procedure 4 with 15 (317 mg, 1.00
mmol), 2,2-diphenylethylamine (220 mg, 1.10 mmol) and
DIPEA (261 µL, 1.50 mmol) in CH3CN (15 mL) to afford 19 as an
off-white solid (391 mg, 85%). M.P: 240 °C (dec).
M.P: 124-126 °C.
1H NMR (400 MHz, DMSO-d6): δ 9.74 (bs, NH), 7.72 (d,
J
=
J
8.4 Hz, 2H), 7.59-7.56 (m, 1H), 7.51 (d,
J = 8.4 Hz, 2H), 3.51 (t,
= 7.0 Hz, 2H), 1.58 (quin, = 6.8 Hz, 2H), 1.29 (s, 6H), 0.86 (t,
J
J
= 6.4 Hz, 3H). 13C NMR (101 MHz, DMSO-d6): δ 179.4, 172.9,
133.4, 132.2 (2C), 131.2 (2C), 129.7, 127.5, 122.7, 44.6, 30.8,
28.4, 25.9, 22.0, 13.9. IR νmax (cm-1): 2970, 2855, 1686, 1605,
1249, 823, 517. Mass Spectral Analysis: LRMS: m/z (APCI-)
365/367 [M-H]- (100%); HRMS: Exact mass calculated for
C16H2079BrN2OS [M+H]+, 367.0475. Found 367.0472. Exact
mass calculated for C16H2081BrN2OS, 369.0454. Found
369.0449.
1H NMR (400 MHz, DMSO-d6): δ 9.76 (bs, NH), 7.70 (d,
8.3 Hz, 2H), 7.57 (s, 1H), 7.48 (d, = 8.3 Hz, 2H), 7.36-7.30 (m,
8H), 7.22 (t, = 6.5 Hz, 2H), 4.42 (t, = 7.6 Hz, 1H), 4.19* (d,
8.0 Hz, 1.7H), 4.03* (d,
= 8.4 Hz, 0.3H). 13C NMR (101 MHz,
J =
J
J
J
J =
J
DMSO-d6): δ 179.4, 173.4, 142.0 (2C), 133.4, 132.2 (2C), 131.2
(2C), 129.6, 128.6 (4C), 128.0, 127.9 (4C), 126.7 (2C), 122.8,
49.6, 48.6. *Tautomeric signals. IR νmax (cm-1): 3172, 2966,
8 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
Page 9 of 12
Journal Name
1680, 1585, 1276, 1183, 744, 522. Mass Spectral Analysis:
View Article Online
DOI: 10.1039/C6OB02308H
ARTICLE
2-(Phenylsulfonamido)thiazol-4(5H)-one (23).
Was
LRMS: m/z (APCI-) 463 [M+H]+ (100%). 461 [M-H]- (100%); synthesized using an adaption of General Procedure 1 with
HRMS: Exact mass calculated for C24H2079BrN2OS [M+H]+, pseudothiohydantoin 11 (205 mg, 1.70 mmol) and
463.0475. Found 463.0477. Exact mass calculated for phenylsulfonyl chloride (100 mg, 0.570 mmol). No
C24H2081BrN2OS [M+H]+, 465.0454. Found 465.0464.
precipitation was evident post aqueous quench and so the
mixture was evaporated to near dryness and the resultant
residue partitioned between EtOAc (25 mL) and water (15 mL).
After separation the organic phase was washed with water (3
x 10 mL), dried (MgSO4), and evaporated to produce a brown
oil. After precipitation by the addition of CH2Cl2/Hexane, the
solid was collected by filtration and dried in vacuo to afford 23
as a pale brown solid. (146 mg, 59%). M.P: 165-169 °C. (Trace
amounts of DMF remained; however, the product was taken
directly through to the next step without further purification).
1H-NMR (400 MHz, DMSO-d6) δ 12.52 (bs, NH), 7.87-7.85
(5Z)-5-(4-Bromobenzylidene)-2-(carboxymethylene
amino)thiazol-4(5H)-one (20). Was synthesised using General
Procedure 4 with 15 (150 mg, 0.5 mmol), glycine (56 mg, 0.75
mmol) and DIPEA (131 µL, 0.75 mmol) in CH3CN (15 mL) to
afford 20 as a white solid (26.5 mg, 16%). M.P: 174 °C (dec).
(Trace amounts of DIPEA remained; this was calculated to be
less than 1.5% by weight.) M.P: 174 °C (dec).
1H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 7.72 (d,
Hz, 2H), 7.57-7.50 (m, 3H), 4.02* (s, 1.8H), 3.76* (s, 0.2H), 3.33
(s, 2H), 2.82 (s, 2H), 1.12 (d, = 5.5 Hz, 15H)
13C NMR (151
J = 8.2
J
.
(m, 2H), 7.70 (t, J = 7.4 Hz, 1H), 7.62 (t, J = 7.5 Hz, 2H), 4.05 (s,
MHz, DMSO-d6) δ 179.4, 172.9, 169.3, 133.5, 132.2, 131.2,
130.1, 127.5, 122.7, 47.6, 18.6. *Tautomeric signals. IR νmax
(cm-1): 3130, 1704, 1553, 1326, 1154, 825, 731. HRMS: Exact
mass calculated for C12H1079BrN2O3S [M+H]+, 340.9591. Found
340.9599. Exact mass calculated for C12H1081BrN2O3S [M+H]+,
342.9570. Found 342.9585.
2H). 13C NMR (101 MHz, DMSO) δ 173.8, 172.9, 140.6, 133.2,
129.3 (2C), 126.4 (2C), 35.0. IR νmax (cm-1): 3149, 2943, 1713,
1549, 1147, 728, 557. HRMS: Exact mass calculated for
C9H9N2O3S2[M+H]+, 257.0049. Found 257.0040.
2-(Naphth-1-ylsulfonamido)thiazol-4(5H)-one (24). Was
synthesized
using
General
Procedure
1
with
(5Z)-2-((5-(4-Bromobenzylidene)-4-oxo-4,5-
pseudothiohydantoin 11 (4.0 g, 34.3 mmol) and 1-
naphthalenesulfonyl chloride (2.96 g, 13.1 mmol) with an 18
hr reaction time to afford 24 as a pale yellow solid (3.41 g,
85%). M.P: 218–220 °C. (Trace amounts of DMF remained;
however, the product was taken directly through to the next
step without further purification).
dihydrothiazol-2-yl)amino)-2-phenylacetic
acid
N-
diisopropyl-N-ethylammonium salt (21). To a solution of 15
(150 mg, 0.50mmol), in 15 mL CH3CN was added DIPEA (87 µL,
0.50 mmol) and 2-phenylglycine (76 mg, 0.50 mmol). After
stirring for 18 h at room temperature the reaction mixture
was isolated via filtration to afford 21 as a yellow solid (45 mg,
22% yield. 95% pure by HPLC analysis). This solid was
subjected to reverse-phase chromatography (1:1 CH3CN: H2O)
to afford 21 as a white solid (6.5 mg, 3%). M.P: 166-169 ºC
1H NMR (400 MHz, DMSO-d6): δ 12.53 (bs, NH), 8.58 (d,
J =
8.4 Hz, 1H), 8.29–8.24 (m, 2H), 8.11 (d, = 7.9 Hz, 1H), 7.76–
J
7.66 (m, 3H), 4.04 (s, 2H). 13C NMR (101 MHz, DMSO-d6): δ
173.7, 173.2, 135.5, 134.4, 133.8, 128.9, 128.11, 128.08,
127.7, 127.0, 125.0, 124.5, 35.2. IR νmax (cm-1): 3141, 1710,
1553, 1124, 764, 514. Mass Spectral Analysis: LRMS: m/z
(APCI-) 307 [M+H]+ (100%). 305 [M-H]- (100%); HRMS: Exact
mass calculated for C13H10N2O3S2, 306.0133. Found 306.0187
[M+H]+.
1H NMR (400 MHz, acetone-d6) δ 7.68 (d,
J = 8.5 Hz, 2H),
7.60 (s, 1H), 7.56-7.52 (m 4H), 7.44-7.36 (m, 3H), 5.88 (s, 1H),
1.37-1.35 (m, 2H). 13C NMR (151 MHz, acetone-d6) δ 180.2,
174.4, 171.1, 137.5, 134.7, 133.1, 132.1, 130.9, 129.7, 129.5, 129.4,
128.6, 123.9, 115.1, 62.4, 54.5, 4.8, 12.6; HRMS: Exact mass
calculated for C18H1379BrN2NaO3S [M+Na]+, 438.9722. Found
438.9712. Exact mass calculated for C18H1381BrN2NaO3S
[M+Na]+, 440.9702. Found 440.9695.
(5Z)-5-(4-Bromobenzylidene)-2-
(phenylsulfonamido)thiazol-4(5H)-one (25). Was synthesized
using General Procedure 3 with 23 (113 mg, 0.44 mmol) and 4-
bromobenzaldehyde (122 mg, 0.66 mmol) to afford 25 as an
orange-beige solid (187 mg, 63%). M.P: 265 °C (dec).
(5Z)-5-(4-Bromobenzylidene)-2-(benzylamino)thiazol-
4(5H)-one (22). To a solution of 15 (20 mg, 63 µmol), in
1.7 mL CH3OH/CH2Cl2 (2:1 v/v) was added DIPEA (11 µL,
1H NMR (400 MHz, DMSO-d6) δ 13.23 (bs, NH), 7.92 (d,
J =
0.13 mmol) and benzylamine (7 ꢀL, 63 µmol). After stirring for
7.4 Hz, 2H), 7.80 (d, = 8.5 Hz, 2H), 7.76 (s, 1H), 7.74 – 7.70 (m,
J
2 h at room temperature the precipitate formed was collected
by filtration, washed with CH3OH/CH2Cl2 (2:1 v/v; 2 x 10 mL) to
yield a yellow solid (13 mg, 56%). M.P: 252-254 °C.
1H), 7.65-7.61 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ
166.7, 165.4, 140.2, 133.5, 132.5 (2C), 132.3, 132.14, 132.07
(2C), 129.5 (2C), 126.5 (2C), 124.4, 122.8. IR νmax (cm-1): 3130,
1704, 1553, 1326, 1154, 825, 731. HRMS: Exact mass
calculated for C16H1279BrN2O3S2 [M+H]+, 422.9467. Found
422.9481. Exact mass calculated for C16H1281BrN2O3S2 [M+H]+,
424.9447. Found 424.9471.
1H-NMR (400 MHz, DMSO-d6) δ 10.13 (bs, NH), 7.72 (d,
J =
7.7 Hz, 2H), 7.60 (s, 1H), 7.52 (d, = 7.8 Hz, 2H),7.39 – 7.32 (m,
J
5H), 4.75* (s, 1.8H), 4.61* (s, 0.2H). 13C-NMR (101 MHz,
DMSO-d6) δ 179.5, 173.5, 137.1, 133.3, 132.2 (2C), 131.2 (2C),
129.6, 128.6 (2C), 128.0, 127.8 (2C), 127.6,122.8, 47.9.
*Tautomeric signals. IR νmax (cm-1): 2676, 1680, 1616, 1256,
(5Z)-5-(4-Bromobenzylidene)-2-(naphth-1-
1188, 739, 521. HRMS: Exact mass calculated for ylsulfonamido)thiazol-4(5H)-one (Pitstop® 2) (26).
Was
C17H1479BrN2OS [M+H]+, 373.0005. Found 373.0021. Exact synthesized using General Procedure 3 with 24 (306 mg, 1.00
mass calculated for C17H1481BrN2OS [M+H]+, 374.9985. Found mmol) and 4-bromobenzaldehyde (203 mg, 1.1 mmol) to
375.0009.
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afford 25 as a pale yellow solid (345 mg, 73%). M.P: 298 °C
(5Z)-5-(3-Chlorobenzylidene)-2-(naphth-1-
(dec).
ylsulfonamido)thiazol-4(5H)-one (30). Was synthesized using
General Procedure 3 with 24 (37 mg, 0.12 mmol) and 3-
1H NMR (400 MHz, DMSO-d6): δ 13.04 (bs, NH), 8.60 (d,
J =
8.6 Hz, 1H), 8.31–8.29 (m, 2H), 8.12 (d,
7.67 (m, 6H), 7.62 (d,
= 8.5 Hz, 2H). 13C NMR (101 MHz, beige solid (33 mg, 66%). M.P: 229-231 °C.
DMSO-d6): δ 166.7, 165.6, 135.3, 134.9, 133.9, 132.6 (2C),
1H NMR (400 MHz, DMSO-d6) δ 13.26 (bs, NH), 8.60 (d,
132.5, 132.2 (2C), 129.2, 128.5, 128.3, 127.7, 127.3, 124.9, 8.5 Hz, 1H), 8.30 (d, = 7.5 Hz, 2H), 8.12 (d, = 8.1 Hz, 1H),
J = 8.2 Hz, 1H), 7.82– chlorobenzaldehyde (18 mg, 0.13 mmol) to afford 30 as a
J
J
=
J
J
124.8, 124.6, 122.8. IR νmax (cm-1): 3054, 2953, 1699, 1555, 7.79-7.58 (m, 8H). 13C NMR (101 MHz, DMSO-d6) δ 166.5,
1328, 1129, 828, 711. Mass Spectral Analysis: LRMS: m/z 165.3, 135.2, 135.1, 134.8, 134.0, 133.8, 131.9, 131.3, 130.4,
(APCI-) 471/473 [M-H]- (100%). HRMS: Exact mass calculated 130.2, 129.0, 128.4, 128.2, 127.9, 127.6, 127.1, 124.8, 124.7,
for C20H1479BrN2O3S2 [M+H]+, 472.9624. Found 472.9624. Exact 123.8. IR νmax (cm-1): 3130, 2962, 1710, 1564, 1309, 1129, 854,
mass calculated for C20H1481BrN2O3S2 [M+H]+, 474.9604. Found 725. HRMS: Exact mass calculated for C20H14ClN2O3S2 [M+H]+,
474.9621.
429.0129. Found 429.0139.
(5Z)-5-(2-Chlorobenzylidene)-2-(naphth-1-
(5Z)-5-(3-Bromobenzylidene)-2-(naphth-1-
ylsulfonamido)thiazol-4(5H)-one (31). Was synthesized using
General Procedure 3 with 24 (111 mg, 0.36 mmol) and 2-
chlorobenzaldehyde (56 mg, 0.40 mmol) to afford 31 as a
beige solid (94 mg, 60%). M.P: 232-234 °C.
ylsulfonamido)thiazol-4(5H)-one (27). Was synthesized using
General Procedure 3 with 24 (37 mg, 0.12 mmol) and 3-
bromobenzaldehyde (24 mg, 0.13 mmol) to afford 27 as an off
white solid (29 mg, 59%). M.P: 230-231 °C.
1H NMR (400 MHz, DMSO-d6) δ 13.30 (bs, NH), 8.59 (d,
J =
1H NMR (400 MHz; DMSO-d6): δ 8.54 (d,
8.27 (d = 7.4 Hz, 2H), 8.08 (d, = 7.6 Hz, 1H), 7.71-7.68 (m,
J = 8.3 Hz, 1H),
8.4 Hz, 1H), 8.29 (t, = 8.7 Hz, 2H), 8.12 (d, = 8.0 Hz, 1H), 7.88
J
J
J
J
(s, 1H), 7.77 – 7.56 (m, 7H). 13C NMR (101 MHz, DMSO-d6) δ
166.4, 165.5, 135.2, 134.8, 134.5, 133.8, 132.3, 130.9, 130.5,
129.3, 129.0, 128.4, 128.36, 128.33, 128.2, 127.6, 127.1,
125.8, 124.8, 124.7; IR νmax (cm-1): 3134, 2970, 1707, 1557,
1307, 1125, 839, 759. HRMS: Exact mass calculated for
C20H14ClN2O3S2 [M+H]+, 429.0129. Found 429.0144.
6H), 7.60-7.59 (m, 1H), 7.53-7.51 (m, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 166.5, 165.3, 135.3, 135.2, 134.8, 133.8, 133.3,
133.1, 131.8, 131.5, 129.0, 128.4, 128.24, 128.16, 127.6,
127.1, 124.8, 124.7, 123.7, 122.5. IR νmax (cm-1): 3130, 2958,
1712, 1564, 1309, 1128, 848, 706. HRMS: Exact mass
calculated for C20H1479BrN2O3S2 [M+H]+, 472.9624. Found
472.9642. Exact mass calculated for C20H1481BrN2O3S2 [M+H]+,
474.9604. Found 474.9630.
(5Z)-5-(4-Fluorobenzylidene)-2-(naphth-1-
ylsulfonamido)thiazol-4(5H)-one (32). Was synthesized using
General Procedure 3 with 24 (100 mg, 0.33 mmol) and 4-
fluorobenzaldehyde (61 mg, 0.49 mmol). Upon cooling, the
solid was collected by filtration and washed with acetone and
cold ether and dried in vacuo to afford 32 as a beige solid
(136 mg, 53%). M.P: 261-263 °C.
(5Z)-5-(2-Bromobenzylidene)-2-(naphth-1-
ylsulfonamido)thiazol-4(5H)-one (28). Was synthesized using
General Procedure 3 with 24 (37 mg, 0.12 mmol) and 2-
bromobenzaldehyde (24 mg, 0.13 mmol) to afford 28 as a
beige solid (31 mg, 62%). M.P: 236-238 °C.
1H NMR (400 MHz, DMSO-d6) δ 13.14 (bs, NH), 8.61 (d,
8.6 Hz, 1H), 8.30 (d, = 7.7 Hz, 2H), 8.12 (d, = 8.1 Hz, 1H),
7.79 – 7.69 (m, 6H), 7.45 (t,
= 8.8 Hz, 2H). 13C NMR (101 MHz,
DMSO-d6) δ 166.5, 165.5, 163.1 (d, = 251 Hz), 135.2, 134.8,
133.8, 132.8 (d, = 37.3 Hz, 2C), 132.6, 129.5 (d, J = 3.0Hz),
J =
1H NMR (400 MHz, DMSO-d6): δ 8.59 (d,
J = 8.4 Hz, 1H),
J
J
8.31-8.27 (m, 2H), 8.12 (d, = 7.8 Hz, 1H), 7.83-7.67 (m, 8H),
J
7.48-7.44 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 166.2,
165.5, 135.1, 134.8, 133.8, 133.7, 132.6, 132.4, 131.2, 129.4,
129.0, 128.8, 128.4, 128.1, 127.6, 127.1, 125.7, 125.3, 124.8,
124.7. IR νmax (cm-1): 3137, 2966, 1708, 1568, 1306, 1122, 837,
712. HRMS: Exact mass calculated for C20H1479BrN2O3S2
[M+H]+, 472.9624. Found 472.9648. Exact mass calculated for
C20H1481BrN2O3S2 [M+H]+, 474.9604. Found 474.9621.
J
J
J
129.0, 128.3, 128.1, 127.6, 127.1, 124.8, 124.7, 121.5 (d, J =
2.0 Hz), 116.7 (d, J = 22.2Hz, 2C); IR νmax (cm-1): 3107, 2977,
1710, 1548, 1156, 1129, 804, 768. HRMS: Exact mass
calculated for C20H14FN2O3S2 [M+H]+, 413.0424. Found
413.0436.
(5Z)-5-(4-Chlorobenzylidene)-2-(naphth-1-
(5Z)-5-(4-Iodobenzylidene)-2-(naphth-1-
ylsulfonamido)thiazol-4(5H)-one (29). Was synthesized using
General Procedure 3 with 24 (37 mg, 0.12 mmol) and 4-
chlorobenzaldehyde (18 mg, 0.13 mmol) to afford 29 as a
white solid (23 mg, 46%). M.P: 290-292 °C.
ylsulfonamido)thiazol-4(5H)-one (33). Was synthesized using
General Procedure 3 with 24 (37 mg, 0.12 mmol) and 4-
iodobenzaldehyde (30 mg, 0.13 mmol) to afford 33 as a yellow
solid (42 mg, 67%). M.P: 292 °C (dec).
1H NMR (400 MHz, DMSO-d6) δ 13.24 (bs, NH), 8.61 (d,
8.5 Hz, 1H), 8.30 (d, = 7.7 Hz, 2H), 8.12 (d, = 8.0 Hz, 1H),
7.77 (t,
= 7.2 Hz, 2H), 7.73 – 7.65 (m, 6H). 13C NMR (101 MHz,
J =
1H NMR (400 MHz, DMSO-d6): δ 13.22 (bs, NH), 8.60 (d,
8.5 Hz, 1H), 8.30 (d, = 7.6 Hz, 2H), 8.12 (d, = 8.0 Hz, 1H),
7.97 (d, = 8.3 Hz, 2H), 7.77-7.67 (m, 4H), 7.45 (d, = 8.3 Hz,
J =
J
J
J
J
J
J
J
DMSO-d6) δ 166.6, 165.4, 135.4, 135.2, 134.8, 133.8, 132.2,
131.9 (2C), 131.8, 129.6 (2C), 129.0, 128.3, 128.1, 127.6,
127.1, 124.8, 124.7, 122.7; IR νmax (cm-1): 3050, 2958, 1699,
1569, 1326, 1129, 829, 711. HRMS: Exact mass calculated for
C20H14ClN2O3S2 [M+H]+, 429.0129. Found 429.0138.
2H). 13C NMR (101 MHz, DMSO-d6) δ 166.5, 165.3, 138.4 (2C),
135.2, 134.8, 133.8, 132.7, 132.3, 131.8 (2C), 129.0, 128.3,
128.1, 127.6, 127.1, 124.8, 124.7, 122.6, 98.4; IR νmax (cm-1):
3130, 2950, 1703, 1563, 1315, 1126, 806, 767. HRMS: Exact
10 | J. Name., 2012, 00, 1-3
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mass calculated for C20H14IN2O3S2 [M+H]+, 520.9485. Found 2H), 2.90 (s, 2H), 2.33 (s, 1H), 1.83 – 1.80 (m, 2H), 1.63 (s, 2H).
520.9494.
(5Z)-5-(Benzylidene)-2-(naphth-1-ylsulfonamido)thiazol-
13C NMR (101 MHz, DMSO-d6): δ 177.9, 173.6, 138.5, 133.7,
133.1, 132.8, 132.4, 130.9 (2C), 129.2, 128.8 (2C), 128.5,
128.0, 127.2, 127.1, 126.5, 125.9, 124.4, 59.4, 50.6, 36.8,
27.6. IR νmax (cm-1): 3435, 2950, 1670, 1446, 1115, 883, 741.
Mass Spectral Analysis: LRMS: m/z (APCI-) 490 [M-H]- (100%);
HRMS: Exact mass calculated for C26H24N3O3S2 [M-H]-,
490.1264. Found 490.1278.
4(5H)-one (34). Was synthesized using General Procedure 3
with 24 (216 mg, 0.71 mmol) and benzaldehyde (90 mg, 0.85
mmol) to afford 34 as a yellow solid (126 mg, 45%). M.P: 227-
229 °C.
1H NMR (400 MHz, DMSO-d6): δ 13.15 (bs, NH), 8.61 (d,
J =
8.8 Hz, 1H), 8.30 (d, = 6.8 Hz, 2H), 8.12 (d, = 8.0 Hz, 1H),
J
J
(5Z)-5-(3,4-Dibromobenzylidene)-2-(naphth-1-
7.78-7.66 (m, 6H), 7.61 - 7.58 (m, 2H), 7.54 – 7.51 (m, 1H). 13C ylsulfonamido)thiazol-4(5H)-one (38). Was synthesized using
NMR (101 MHz, DMSO-d6) δ 166.4, 165.6, 135.2, 134.8, 133.8, General Procedure 3 with 24 (37 mg, 0.12 mmol) and 3,4-
132.8, 130.9, 130.3 (2C), 129.5 (2C), 129.0, 128.3, 128.1, dibromobenzaldehyde (34 mg, 0.13 mmol) to afford 38 as a
127.6, 127.1, 124.8, 124.7, 121.7; IR νmax (cm-1): 3061, 1706, pale yellow solid (33 mg, 66%). M.P: 290 °C (dec).
1557, 1336, 1129, 833, 764, 521. Mass Spectral Analysis:
LRMS: m/z (ESI+) 395 [M+H]+ (100%); (ESI-) 393 (M-H)-. HRMS: 8.5 Hz, 1H), 8.32 – 8.29 (m, 2H), 8.12 (d,
Exact mass calculated for C20H15N2O3S2 [M+H]+, 395.0519. 1H), 8.00 (d,
= 8.3 Hz, 1H), 7.79 - 7.67 (m, 4H), 7.56 (d,
1H NMR (400 MHz, DMSO-d6): δ 13.07 (bs, NH), 8.60 (d,
= 8.1 Hz, 1H), 8.07 (s,
= 8.3
J =
J
J
J
Found 395.0508.
Hz, 1H). 13C NMR (101 MHz, DMSO) δ 166.5, 165.1, 135.5,
135.2 134.8, 134.7, 134.2, 133.8, 130.9, 129.4, 129.0, 128.4,
128.2, 127.6, 127.1, 126.3, 125.0, 124.8, 124.7, 124.3.; IR νmax
(cm-1): 3168, 3011, 1707, 1568, 1312, 1129, 844, 714. HRMS:
Exact mass calculated for C20H1379Br2N2O3S2 [M+H]+,
(5Z)-5-(4-Cyanobenzylidene)-2-(naphth-1-
ylsulfonamido)thiazol-4(5H)-one (35). Was synthesized using
General Procedure 3 with 24 (102 mg, 0.33 mmol) and 4-
cyanobenzaldehyde (66 mg, 0.49 mmol) to afford 35 as a
yellow solid (80 mg, 57%). M.P: 301-304 °C.
550.8729.
Found 550.8802. Exact mass calculated for
C20H1379Br81BrN2O3S2 [M+H]+, 552.8709. Found 552.8797.
Exact mass calculated for C20H1381Br2N2O3S2 [M+H]+, 554.8688.
Found 554.8791.
1H NMR (400 MHz, DMSO-d6) δ 13.22 (bs, NH), 8.60 (d,
8.6 Hz, 1H), 8.32 - 8.29 (m, 2H), 8.12 (d, = 8.1 Hz, 1H), 8.05 (d,
= 8.3 Hz, 2H), 7.85 (s, 1H), 7.83 (d, = 4.3 Hz, 2H), 7.79 - 7.68
(m, 3H). 13C NMR (101 MHz, DMSO-d6) δ 166.5, 165.2, 137.4,
J =
J
J
J
(5Z)-5-(3,5-Dichlorobenzylidene)-2-(naphth-1-
135.1, 134.8, 133.8, 133.2 (2C), 131.3, 130.6 (2C), 129.0, ylsulfonamido)thiazol-4(5H)-one (Pitstop® 2-100) (39). Was
128.4, 128.2, 127.6, 127.1, 125.6, 124.8, 124.7, 118.4, 112.3; synthesized using General Procedure 3 with 24 (106 mg, 0.34
IR νmax (cm-1): 3134, 3053, 2226, 1709, 1554, 1347, 1130, 807, mmol) and 3,5-dichlorobenzaldehyde (65 mg, 0.37 mmol) to
768. Mass Spectral Analysis: LRMS: m/z (ESI-) 418 [M-H]- afford 39 as a cream solid (44 mg, 28%). M.P. 220 °C (dec).
(100%); HRMS: Exact mass calculated for C21H14N3O3S2 [M+H]+,
420.0471. Found 420.0462.
1H NMR (400 MHz, DMSO-d6): δ 8.59 (d,
8.30 (t, = 7.0 Hz, 2H), 8.13 (d, = 8.0 Hz, 1H), 7.80-7.68 (m,
J = 8.5 Hz, 1H),
J
J
7H). 13C NMR (101 MHz, DMSO-d6) δ 166.3*, 164.8, 136.4,
134.9, 134.8 (2C), 134.6, 133.6, 130.1, 129.5, 128.8, 128.2,
128.00, 127.97 (2C), 127.4, 126.9, 125.4, 124.6, 124.5.
*Confirmed by HMBC correlations. Mass Spectral Analysis:
LRMS: m/z (APCI-) 461 [M-H]- (100%); IR νmax (cm-1): 2934,
1713, 1579, 1312, 1126, 851, 713. HRMS: Exact mass
calculated for C20H11Cl2N2O3S2 [M-H]-, 460.9588. Found
460.9654.
(5Z)-5-(4-Nitrobenzylidene)-2-(naphth-1-
ylsulfonamido)thiazol-4(5H)-one (36). Was synthesized using
General Procedure 3 with 24 (100 mg, 0.33 mmol) and 4-
nitrobenzaldehyde (74 mg, 0.49 mmol) to afford 36 as a pale
yellow solid (88 mg, 60%). M.P: 278 °C (dec).
1H NMR (400 MHz, DMSO-d6) δ 13.06 (bs, NH), 8.61 (d,
8.6 Hz, 1H), 8.40 (d, = 8.2 Hz, 2H), 8.30 (d, = 7.6 Hz, 2H),
8.12 (d, = 7.7 Hz, 1H), 7.92 (d, = 8.2 Hz, 2H), 7.86 (s, 1H),
7.77 – 7.69 (m, 3H). 13C NMR (101 MHz, DMSO-d6) δ 166.8*,
J =
J
J
J
J
(5Z)-5-(2,5-Difluorobenzylidene)-2-(naphth-1-
165.4, 147.7, 139.2, 135.2, 134.8, 133.8, 131.1 (2C), 130.6, ylsulfonamido)thiazol-4(5H)-one (40). Was synthesized using
129.0, 128.3, 128.2, 127.6, 127.1, 126.6, 124.8, 124.7, 124.4 General Procedure 2 with 24 (150 mg, 0.50 mmol) and 2,5-
(2C); *Confirmed by HMBC correlations. IR νmax (cm-1): 3138, difluorobenzaldehyde (78 mg, 0.55 mmol) to afford 40 as an
2943, 1712, 1557, 1516, 1350, 1130, 809, 771. HRMS: Exact off white solid after chromatography (80 mg, 37%). M.P: 190
mass calculated for C20H14N3O5S2 [M+H]+, 440.0369. Found °C (dec).
440.0369.
1H NMR (400 MHz, DMSO-d6): δ 8.60 (d,
J = 8.6 Hz, 1H),
8.32 – 8.29 (m, 2H), 8.12 (d, J = 8.0 Hz, 1H), 7.79-7.75 (m, 1H),
7.72 – 7.66 (m, 3H), 7.51 – 7.42 (m, 3H). 13C NMR (101 MHz,
(Z)-N-(5-((1-Benzylpiperidin-4-yl)methylene)-4-oxo-4,5-
dihydrothiazol-2-yl)naphthalene-1-sulfonamide (37). Was
synthesized using General Procedure 2 with 24 (306 mg, 1.0
DMSO-d6): δ 166.4, 165.2, 158.6 (dd, J = 158, 2.0 Hz), 156.1
(dd, J = 164, 2.0 Hz), 135.1, 134.8, 133.8, 129.0, 128.3, 128.2,
mmol) and
N-benzylpiperidine-4-carboxaldehyde (240 mg,
127.6, 127.1, 126.6, 124.8, 124.7, 123.8, 122.2 (dd, J = 15.3,
8.7 Hz), 119.5 (dd, J = 24.5, 9.2 Hz), 118.2 (dd, J = 24.8, 9.0 Hz),
115.5 (dd, J = 25.5, 2.3 Hz). IR νmax (cm-1): 3077, 2089, 1710,
1567, 1308, 1127, 796, 522. Mass Spectral Analysis: LRMS:
1.18 mmol) to afford 37 as a light brown solid (300 mg, 61%).
M.P: 172 °C (dec).
1H NMR (400 MHz, DMSO-d6): δ 9.71 (bs, NH), 8.67 (d,
J =
8.4 Hz, 1H), 8.15 – 8.12 (m, 2H), 8.02 (d, J = 7.6 Hz, 1H), 7.62-
7.60 (m, 3H), 7.45 (s, 5H), 6.32 (bs, NH), 4.16 (s, 2H), 3.23 (s,
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m/z (APCI-) 429 [M-H]- (100%); HRMS: Exact mass calculated
for C20H11Cl2N2O3S2 [M-H]-, 429.0179. Found 429.0206.
25 E. Tkachenko, D. Tse, O. Sideleva, S. J. Deharvengt, M. R.
Luciano, Y. Xu, C. L. McGarry, J. Chidlow, P. F. Pilch, W. C.
Sessa, D. K. Toomre and R. V. Stan, PLoS One 2012,
e32655.
7,
26 G. R. Ares and P. A. Ortiz, J. Biol. Chem. 2012, 287, 37824–
37834.
27 S. K. Mishra, L. Funair, A. Cressley, G. K. Gittes and R.
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