Direct and two-step bioorthogonal probes for Bruton's tyrosine kinase based on ibrutinib: A comparative study

Article abstract of DOI:10.1039/c5ob00474h

Ibrutinib is a covalent and irreversible inhibitor of Bruton's tyrosine kinase (BTK) and has been approved for the treatment of haematological malignancies, such as chronic lymphocytic leukaemia, mantle cell lymphoma and Waldenstr?m's macroglobulinemia. The covalent and irreversible nature of its molecular mode of action allows identification and monitoring of its target in an activity-based protein profiling (ABPP) setting. Fluorescent and biotinylated ibrutinib derivatives have appeared in the literature in recent years to monitor BTK in vitro and in situ. The work described here complements this existing methodology and pertains a comparative study on the efficacy of direct and two-step bioorthogonal ABPP of BTK.

Full text of DOI:10.1039/c5ob00474h

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Journal Name
RSCPublishing
ARTICLE
Direct and two-step bioorthogonal probes for
Bruton’s tyrosine kinase based on ibrutinib: a
comparative study
Cite this: DOI: 10.1039/x0xx00000x
Nora Liua, Sascha Hoogendoorna, Bas van de Karb, Allard Kapteinb, Tjeerd
Barfb, Christoph Driessenc, Dmitri V. Filippova, Gijsbert A. van der Marela,
Received 00th January 2012,
Accepted 00th January 2012
Mario van der Stelta
*
and Herman S. Overkleefta
*
DOI: 10.1039/x0xx00000x
Ibrutinib is a covalent and irreversible inhibitor of Bruton’s tyrosine kinase (BTK) and has
been approved for the treatment of haematological malignancies, such as chronic lymphocytic
leukaemia, mantle cell lymphoma and Waldenström’s macroglobulinemia. The covalent and
irreversible nature of its molecular mode of action allows identification and monitoring of its
target in an activityꢀbased protein profiling (ABPP) setting. Fluorescent and biotinylated
ibrutinib derivatives have appeared in the literature in recent years to monitor BTK in vitro and
in situ. The work described here complements this existing methodology and pertains a
comparative study on the efficacy of direct and twoꢀstep bioorthogonal ABPP of BTK.
www.rsc.org/
probes (ABPs), are composed of a mechanismꢀbased enzyme
inhibitor modified to contain an identification tag, which can be
Introduction
Bruton’s tyrosine kinase (BTK), a nonreceptor tyrosine kinase
member of the Tec kinase family, is involved in B cell receptor
(BCR) signalling and governs Bꢀlymphocyte development,
differentiation, signalling and survival.1 Aberrant BTK activity
is a fundamental feature of the human Bꢀcell malignancies;
chronic lymphocytic leukaemia (CLL), mantle cell lymphoma
(MCL), follicular lymphoma (FL), diffuse large Bꢀcell
lymphoma (DLBCL), and Waldenström’s macroglobulinemia
(WM).2
a biotin (for visualisation and/or enrichment), a fluorophore (for
visualisation), or a bioorthogonal tag (to install a fluorophore or
affinity tag following enzyme labelling).
O
O
NH2
NH2
N
N
N
N
1 (Ibrutinib)
N
N
N
N
N
2
N
The firstꢀinꢀclass BTKꢀinhibitor ibrutinib (Imbruvica®, PCIꢀ
F
B
N
32765,
1, Figure 1) has recently been approved for the
O
N
O
HS
F
treatment of WM, MCL and CLL by the FDA.3 Apart from its
clinical efficacy, ibrutinib is of interest because of its
mechanism of action. Ibrutinib irreversibly blocks BTK activity
through covalent modification of Cys481 within the enzyme
ATPꢀbinding pocket following conjugate addition of the
N
O
N
N
H
Cys481
O
O
NH2
NH2
N
N
cysteine thiol to the acrylamide moiety in
1 (Figure 1), an event
N
N
N
N
N
N
N
N
3
that prevents phosphorylation of Tyr223, an essential step for
BTK activation. At present, numerous covalent kinase
inactivators are pursued for a range of human malignancies.4
Besides the clinical relevance of mechanismꢀbased inhibitors
(long residence time, lasting inhibitory effect which is only
dependent on de novo synthesis of the protein target), covalent
and irreversible enzyme inhibitors are highly useful starting
points for the development of activityꢀbased protein profiling
(ABPP) probes.5 Such probes, also termed activityꢀbased
S
O
O
Cys481
Figure 1. Mechanismꢀbased inactivation of Bruton’s tyrosine kinase
(BTK) by ibrutinib ( ) (left) and the direct ( ) and twoꢀstep ( ) BTK
activityꢀbased probes reported to date (right).
1
2
3
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In the recent literature both direct and twoꢀstep bioorthogonal findings we decided to use ibrutinib derivative 20 (Scheme 1)
ibrutinibꢀbased ABPs have been described. Honigberg and coꢀ as common core onto which the BODIPY fluorophores and
workers reported on the development of BODIPYꢀFLꢀibrutinib twoꢀstep bioorthogonal ligation handles as depicted in Figure 2
2
and its application in detection of BTK in BTKꢀpositive are grafted. Thus as the first research objective we set out to
tumour cells and mouse models of autoimmune disease.3c synthesise amine 20 (Scheme 1). Iodide 10 was prepared from
Cravatt and coꢀworkers in turn described ibrutinibꢀalkyne as commercially available aminopyrazolopyrimidine in an
part of a series of alkyneꢀmodified covalent kinase inhibitors in electrophilic aromatic substitution using ꢀiodosuccinimide as
3
9
N
a broadꢀspectrum Huisgen [2+3]ꢀcycloaddition ‘click’ꢀbased the iodinating agent. Ensuing Suzuki coupling with 4ꢀ
twoꢀstep ABPP study on kinase activities in a variety of tumour phenoxybenzene boronic acid according to the literature
tissues.6
procedure provided diphenyl ether 11, which was reacted with
enantiopure (S)ꢀ ꢀBocꢀ3ꢀhydroxypiperidine 12 under
Mitsunobu conditions to give intermediate 13 with full
inversion of stereochemistry.9, 10 Acid mediated removal of the
Boc protective group followed by condensation of the thus
liberated secondary amine with acryloyl chloride gave ibrutinib
N
O
F
N
B
F
O
N
4
NH2
N
1
, the analytical and spectral data of which were in full
O
agreement with those reported in the literature.10, 11
N
OMe
N
N
F
N
B
F
N
N
O
O
5
N
NH2
R
O
N
R
N
H
NH2
NH2
b)
N
N
N
N
N
H
N
H
N
N
c)
OH
O
O
O
N
N
N
N
N
N
N
N
H
N3
N
O
9
R = H
10 R = I
11
6
7
8
a)
NBoc
R
Figure 2. Direct (
4
and 5) and twoꢀstep bioorthogonal (6-8) BTK
12
13 R = Boc
14 R = H
e)
activityꢀbased probes that are subject of the here presented study.
d)
1
O
17
Br
O
N
Our ABPP research focuses amongst others on the headꢀtoꢀhead
comparison of direct and twoꢀstep bioorthogonal ABPP
methodologies.7 In these studies we include assessment of the
relative efficiency of established bioorthogonal chemistries
including Cu(I)ꢀcatalysed and strainꢀpromoted azideꢀalkyne
[2+3] cycloaddition, StaudingerꢀBertozzi ligation and inverseꢀ
electron demand DielsꢀAlder ligation. We have previously
shown that with the exception of strainꢀpromoted alkyneꢀazide
cycloadditions all of the above are effective bioorthogonal
chemistries in the twoꢀstep ABPP modification of the catalytic
activities of mammalian proteasomes and moreover that these
reactions can be executed consecutively in a multiplexing
fashion in a single biological sample.8 We here report on a
comparative study in which we probed for catalytically active
HO
R
HN
h)
g)
N
N
N
H
HO
N
NHBoc
18
15 R = H
16 R = Boc
f)
O
NH2
21
O
tag
19 R = Boc
20 R = H
i)
HO
4-8
N
N
N
N
j)
N
O
N
N
H
R
BTK levels in BTKꢀpositive Ramos cells with direct ABPs
4
and (Figure 2) as well as twoꢀstep ABPs and (Cu(I)ꢀ Scheme 1. Reagents and conditions: a)
5
6
7
Nꢀiodosuccinimide, 80 °C,
catalysed azideꢀalkyne [2+3] cycloaddition) and
electron demand DielsꢀAlder).
8
(inverseꢀ DMF, 87%; b) K3PO4, 4ꢀphenoxybenzene boronic acid, Pd(PPh3)4,
dioxane, 180 °C, 79%; c) 12, PPh3 polymerꢀbound, DIAD, THF, 56%;
d) 4.0 M HCl in dioxane, 100%; e) acryloyl chloride, TEA, DCM, 46%;
f) i) benzaldehyde, CbzꢀCl, toluene, 38%; ii) Boc2O, THF, 79%; iii)
Pd/C, H2, MeOH, 94%; g) 17, TEA, THF; h) 14, HATU, TEA, DMF,
Results and discussion
Synthesis of ibrutinib and ibrutinib-based ABPs
87%; i) 4.0 M HCl in dioxane, 100%; j) HATU, DiPEA, DMF, 21
, 4:
Perusal of the literature on structureꢀactivity relationship
studies on ibrutinib revealed that analogues featuring a
piperazinyl extension at the acrylamide side of the parent
compound are well tolerated by the target enzyme, BTK.
Weissleder and coꢀworkers used this finding in their design of
17%, : 10%, : 36%, : 22%, : 10%.
5
6
7
8
The synthesis of ABPs
4ꢀ8 requires access to piperazineꢀ
functionalised acrylate 18, for which synthesis we adapted the
patent literature.12 In the first instance, we attempted to prepare
direct BTK ABP
2
(Figure 1).9 Based on these literature
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the precursor, single Bocꢀprotected aminoethylpiperazine 16, as performed. C) Competition experiments of ibrutinib
described by treatment of aminoethylpiperazine with diꢀtert fluorescent ABP in Ramos cell lysates. Three independent
butyldicarbonate in the presence of benzyl aldehyde. However, experiments were performed. Ramos cell extracts were exposed to the
this oneꢀstep procedure, not unexpectedly,13 failed. Therefore, indicated concentrations of ibrutinib
for 1 hr at room temperature and
we resorted to the following threeꢀstep procedure as depicted.14 then incubated with ABP
(1 µM) for 1 hr at room temperature.
1 versus
ꢀ
4
1
4
Thus, in situ formation of the primary benzylimine and ensuing Proteins were analysed by SDSꢀPAGE using detection by inꢀgel
Cbz protection of the secondary amine and liberation of the fluorescent readout. See for complete gels the supplementary info. Lane
primary amine yielded monoꢀCbzꢀ aminoethylpiperazine, with 1: Dual Color protein standard.
the secondary amine temporarily protected as the
benzyloxycarbamate. Subsequent, introduction of the Boc As can be seen from Figure 3A, ibrutinib analogues
protective group at this stage followed by hydrogenolysis of the recombinant, purified BTK with IC50 values in the same range
Cbz group afforded compound 16, which was ꢀalkylated with as ibrutinib . The fluorescent analogues and are not
ꢀbromobutenoic acid 1715 to yield compound 18
compatible with the IMAP assay and thus we established
6ꢀ8 inhibit
N
1
4
5
E
.
Condensation of 14 and 18 under the agency of HATU and whether these compounds would be able to detect ibrutinib in
triethylamine followed by acidic removal of the Boc group an activityꢀbased protein profiling setting. As can be seen
afforded ibrutinib derivative 20. Condensation (HATU, DiPEA) (Figure 3B) both compounds label a single band with an
with the appropriately modified tags 21 (see for the used tags apparent molecular weight corresponding to that of BTK. Of
the experimental section) afforded target direct ABPs
4
and
) in moderate dependent manner by preꢀincubation with ibrutinib (Figure 3C).
We next set out to establish the ability of compounds to
target BTK in living cells. Figure 4A shows that both green (
Evaluation of the inhibitory potency and labelling efficiency and red ( ) fluorescent probes readily and selectively modify
We first established the potency of ABPs as BTK BTK in a concentrationꢀdependent manner and do so at
inhibitors. Nonꢀfluorescent derivatives were measured in an concentrations as low as 100 nM (for ABP ). Thus both direct
5 as note, labelling of this protein could be prevented in a doseꢀ
well as the twoꢀstep bioorthogonal ABPs (
yield but good purity after HPLC purification.
6ꢀ8
4ꢀ8
4
)
5
4ꢀ8
6
ꢀ
8
4
immobilized metal ion affinityꢀbased fluorescence polarization ABPs behave as expected, given the literature precedent.3c This
(IMAP) assay using recombinantly expressed human BTK with makes the probes also useful for the assessment of the
ibrutinib
1
included as benchmark. Fluorescent, direct ABPs
4
inhibitory potency and cell permeability of putative BTK
and were added to Ramos cell extract, which contains inhibitors in a competitive ABPP setting.
5
endogenously expressed BTK, after which the protein content
was denatured and resolved on SDSꢀPAGE.
Figure 4. A) In situ labelling of BTK in Ramos cells by
cells were exposed to the indicated concentrations of
4
and
5. Ramos
4
or
5
for 4 hrs at
37° C. Proteins were analysed by SDSꢀPAGE using detection by inꢀgel
fluorescent readout. Three independent experiments were performed.
Figure 3. A) BTK inhibitory potency (IC50) of ibrutinib
bioorthogonal ABPs . The mean IC50 values are calculated from two
independent experiments performed in duplicate. B) BTK labelling
efficiency of direct ABPs and in Ramos cell extract. Ramos cell
extracts were exposed for 1 hr at room temperature to the indicated
concentrations of ABP or . Three independent experiments were
1 and twoꢀstep
B) Competition experiments of BODIPYꢀFLꢀibrutinib
compounds in Ramos cells. Ramos cells were exposed to the
indicated concentrations of ABP , or for 3 hrs at 37° C and then
lysed. The cell lysates were exposed to 1 µM of ABP for 1 hr at room
4 versus
6ꢀ8
6ꢀ8
6,
7
8
4
5
4
temperature. Two independent experiments were performed. C)
4
5
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Competition experiments of ibrutinib
1 versus fluorescent ABP 4 in
Ramos cells. Two independent experiments were performed. Ramos
cells were exposed to the indicated concentrations of ibrutinib for 3
hrs at 37° C and then lysed. The cell lysates were exposed to 1 µM of
ABP for 1 hr at room temperature. Proteins were analysed by SDSꢀ
1
4
PAGE using detection by inꢀgel fluorescent readout. See for complete
gels the supplementary info. Lane 1: Dual Color protein standard.
To establish whether putative twoꢀstep ABPs 6ꢀ8 are able to
reach and modify BTK in live cells, we treated living Ramos
cells in culture with varying concentration of these compounds,
prior to treatment with ABP 4, cell lysis and SDSꢀPAGE. As is
evident from Figure 4B, the three compounds abolish labelling
at the lowest concentration (4 µM; approximately 10x IC50
value for all compounds, see Figure 3A) applied and we can
thus conclude that all 5 compounds – direct and twoꢀstep ABPs
– are able to modify BTK in live cells.
Figure 4C shows that BTK labelling in live cells could be
prevented in a doseꢀdependent manner by preꢀincubation with
ibrutinib, strongly suggesting BTK specificity of these probes
in live cells.
Figure 6. AꢀC) In vitro twoꢀstep bioorthogonal labelling of BTK in
Ramos cell extract using reagent pairs (6/22), (7/23) and (8/24). Ramos
cell lysates were exposed to ABP or
6
7 for 1 hr at room temperature
and then reacted with the indicated concentrations of BODIPYꢀazide
22 or BODIPYꢀalkyne 23 for 1 hr at room temperature. Copperꢀ
catalysed click reactions were performed in the presence of CuSO4 (6.5
mM), Tris(3ꢀhydroxypropyltriazolylmethyl) amine THPTA (6.5 mM)
The efficiency and selectivity of the twoꢀstep ABPs
modify BTK in living Ramos cells was assessed next.
6ꢀ8 to
and sodium Lꢀascorbate (6.5 mM). Alternatively, ABP
used for ligation with tetrazine 24. In control experiments different
ligation strategies were performed in the absence of ABP or after
. As a positive control, extracts
(1 µM). Three independent
8 (4 µM) was
6ꢀ8
competition by an excess of ibrutinib
were labelled with fluorescent ABP
1
4
experiments were performed. D) Twoꢀstep bioorthogonal profiling of
BTK activity by different ligation strategies. Ramos cells were exposed
Figure 5. Ligation reagents used in the here presented study.
to ABP
with their corresponding ligation reagent 22
room temperature. Alternatively, cells were consecutively exposed to
ABP and ligation reagent 24 in situ (lane 5). In control experiments
different ligation strategies were performed in the absence of ABP
and/or ligation reagent. As a positive control, cells were labelled with
fluorescent ABP (1 µM). Three independent experiments were
6
ꢀ
8
(4 µM) for 3 hrs at 37° C, washed, lysed, and then reacted
Figure 5 depicts the complementary reagents used in these
experiments, that is, BODIPYꢀFLꢀmodified bioorthogonal
reagent 22 (for copper(I)ꢀcatalysed alkyneꢀazide [2+3]
ꢀ24 (25 µM) for 1 hr at
8
cycloaddition (CuAAC) ligation to azideꢀmodified ibrutinib
BODIPYꢀgreenꢀalkyne 23 (for copper(I)ꢀcatalysed click
ligation to alkyneꢀmodified ibrutinib ) and BODIPYꢀFLꢀ
tetrazine 24 (for inverseꢀelectron demand DielsꢀAlder ligation
to norborneneꢀmodified ibrutinib ).
6),
6ꢀ8
7
4
performed. Proteins were analysed by SDSꢀPAGE using detection by
inꢀgel fluorescent readout. Lane 1: Dual Color protein standard.
8
Ramos cell lysates were treated with ibrutinibꢀalkyne
6,
ibrutinibꢀazide or ibrutinibꢀnorbonene at 4 µM final
7
8
concentrations for one hour at room temperature. Next the
samples were treated with the complementary bioorthogonal
ligation handles 22ꢀ24 at various final concentrations for one
hour. The proteins were resolved on SDSꢀPAGE and the wet
gel slabs analysed by fluorescence readꢀout. As can be seen
(Figure 6A) both copper(I)ꢀcatalysed click reactions give BTK
labelling in a concentrationꢀdependent manner, as does the
inverseꢀelectron demand DielsꢀAlder (IEDDA) ligation.
Though the two click ligation steps appear about equally
effective in terms of activity, differences are apparent when
considering aspecific reaction of the (click/tetrazine) ligation
handles, with alkyneꢀazide click ligation giving the optimal
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result. The IEDDA ligation appears to proceed equally selective spectrometer. Chemical shifts are given in ppm (δ) relative to
though the labelling intensity appears somewhat lower.
tetramethylsilane (1HꢀNMR) or CDCl3 (13CꢀNMR) as internal
As a final set of experiments we explored the ability of our twoꢀ standard. Mass spectra were recorded on a PE/Sciex API 165
step BTK ABPs to modify BTK in situ in living Ramos cells instrument equipped with an Electrospray Interface (ESI) (Perkinꢀ
(Figure 6D). For this purpose, we treated Ramos cells with 4 Elmer). Highꢀresolution MS (HRMS) spectra were recorded with a
µM
click ligations the cells were lysed, whereas IEDDA ligation Shimadzu FTIRꢀ8300 and absorptions are given in cmꢀ1. Optical
(addition of norbornene 24) was performed in situ and in vitro
rotations [α]D23 were recorded on a Propol automatic polarimeter at
6ꢀ8 for 1 hour at 37 °C. For the purpose of the two CuAAC Finnigan LTQꢀFT (Thermo Electron). IR spectra were recorded on a
.
As can be seen (Figure 6D) also these partial or complete in situ room temperature. LCꢀMS analysis was performed on a Jasco HPLC
ligations proved successful, with here the two CuAAC ligations system with a Phenomenex Gemini 3 µm C18 50 x 4.6 mm column
more selective compared to the IEDDA ligation.
In conclusion, we have expanded the BTK activityꢀbased probe API 165 mass spectrometer with ESI. HPLC gradients were 10 →
set by the development of two direct ABPs and equipped 90%, 0 → 50% or 10 → 50% ACN in 0.1% TFA/H2O. Chiral HPLC
(detection simultaneously at 214 and 254 nm), coupled to a PE Sciex
4
5
with BODIPY fluorophores that emit at two distinct analysis was performed on a Spectroflow 757 system (ABI
wavelengths, as well as three bioorthogonal twoꢀstep ABPs that Analytical Kratos Division, detection at 254 nm) equipped with a
can be addressed through either CuAAC or IEDDA Chiralcel OD column (150 x 4.6 mm). The compounds were purified
bioorthogonal chemistry. As such our BTK imaging toolset on a Gilson HPLC system coupled to a Phenomenex Gemini 5 µm
adds to the existing BTK probes – both direct and twoꢀstep –for 250 x 10 mm column and a GX281 fraction collector. The used
measuring catalytically active BTK in various settings. Though gradients were either 0 → 30% or 10 → 40% ACN in 0.1%
direct imaging with BODIPYꢀibrutinib derivatives
4
and
5
TFA/water, depending on the lipophilicity of the product.
appears most effective in live cells, the situation may differ in Appropriate fractions were pooled, and concentrated in a Christ
case live animals are to be subject of study and for this purpose rotary vacuum concentrator overnight at room temperature at 0.1
the IEDDA bioorthogonal ABP pair 8/24 may be most effective mbar.
– even though labelling efficiency appears the lowest. In an
alternative setting, BTK occupancy may be monitored in
conjunction with other enzymatic activities in a multiplexing
setting, using either the direct probes in conjunction with ABPs
targeting other enzymes and equipped with complementary
fluorophores, or by making use of a number of mutually
exclusive bioorthogonal ligations.
3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10)
To a solution of 4ꢀaminopyrazolo[3,4ꢀd]pyrimidine (9, 2.50 g, 18.5
mmol) in DMF (43 mL) was added Nꢀiodosuccinimide (1.5 eq., 6.24
g, 27.7 mmol) and the mixture was heated to 80 °C overnight, before
being cooled to 0 °C. H2O (110 mL) was added and the mixture was
allowed to stand at 0 °C for 30 min. followed by filtration of the
solid. The residue was washed with iceꢀcold EtOH, Et2O and EtOAc
and dried in vacuo. The title compound was obtained without further
purification as a pale yellow solid (yield: 4.22 g, 16.17 mmol, 87%).
Experimental
General: Tetrahydrofuran (THF) was distilled over LiAlH4 before
use. Acetonitrile (ACN), dichloromethane (DCM), N,Nꢀ
dimethylformamide (DMF), methanol (MeOH) and trifluoroacetic
acid (TFA) were of peptide synthesis grade, purchased at Biosolve,
and used as received. All general chemicals (Fluka, Acros, Merck,
Aldrich, Sigma) were used as received. Traces of water were
removed from reagents used in reactions that require anhydrous
conditions by coevaporation with toluene. Solvents that were used in
reactions were stored over 4Å molecular sieves, except methanol and
acetonitrile, which were stored over 3Å molecular sieves. Molecular
sieves were flame dried before use. Unless noted otherwise all
reactions were performed under an argon atmosphere. Column
chromatography was performed on Silicycle SiliaꢀP Flash Silica Gel,
with a particle size of 40 – 63 µm. The eluents toluene and ethyl
acetate were distilled prior to use. TLC analysis was conducted on
Merck aluminium sheets (Silica gel 60 F254). Compounds were
visualized by UV absorption (254 nm), by spraying with a solution
of (NH4)6Mo7O24 4 H2O (25 g/L) and (NH4)4Ce(SO4)4·2H2O (10
g/L) in 10% sulphuric acid, a solution of KMnO4 (20 g/L) and
K2CO3 (10 g/L) in water, or ninhydrin (0.75 g/L) and acetic acid
(12.5 mL/L) in ethanol, where appropriate, followed by charring at
1
RF = 0.30 (10% MeOH/DCM). H NMR (400 MHz, DMSOꢀd6) δ
13.84 (s, 1H, NH), 8.17 (s, 1H, CH). 13C NMR (101 MHz, DMSOꢀ
d6) δ 157.50, 155.90, 155.0, 102.51, 89.88.
3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (11)
Compound 10 (0.2 g, 0.77 mmol), K3PO4 (3 eq., 0.49 g, 2.30 mmol),
4ꢀphenoxybenzene boronic acid (3 eq., 0.49 g, 2.29 mmol) and
Pd(PPh3)4 (0.14 eq., 0.12 g, 0.11 mmol) were dissolved in sonicated
dioxane (2.5 mL) in a microwave vial. The resulting mixture was
heated to 180 °C for 10 min. under microwave irradiation. EtOAc
(10 mL) was added and the mixture was washed with H2O and brine
before being dried over MgSO4, filtered and concentrated under
reduced pressure. The title compound was obtained after purification
by column chromatography (100% DCM ꢀ 4% MeOH/DCM) as a
white solid (yield: 0.18 g, 0.61 mmol, 79%). RF = 0.19 (5%
MeOH/DCM). 1H NMR (400 MHz, DMSOꢀd6) δ 13.58 (s, 1H, NH),
8.22 (s, 1H, CH), 7.67 (d, J = 8.8 Hz, 2H, 2xCH), 7.43 (t, J = 8.0 Hz,
2H, 2xCH), 7.20 – 7.11 (m, 5H, 5xCH). 13C NMR (101 MHz,
DMSOꢀd6) δ 158.11, 157.04, 156.33, 155.82, 143.99, 130.17,
128.48, 123.82, 119.05, 96.95. LCꢀMS analysis: Rt 5.63 min (linear
1
ca. 150°C. Hꢀ and 13CꢀNMR spectra were recorded on a Bruker
DMXꢀ400 (400 MHz) or
a Bruker DMXꢀ600 (600 MHz)
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DOI: 10.1039/C5OB00474H
gradient 10ꢀ90% acetonitrile in H2O, 0.1% TFA, 15 min). ESIꢀMS 3.71 (t, J = 10.8Hz, 0.5H), 3.26 – 3.19 (m, 1H), 3.04 (t, J = 12.0 Hz,
(m/z): 304.07 [M+H+].
0.5H), 2.30 – 2.23 (m, 1H), 2.15 – 2.13 (m, 1H), 1.95 – 1.92 (m,
1H), 1.66 – 1.54 (m, 1H). 13C NMR (150 MHz, DMSOꢀd6): δ
164.64, 157.48, 156.15, 152.66, 144.73, 130.11, 128.28, 127.49,
127.21, 126.95, 126.12, 123.86, 119.02, 115.76, 97.06, 53.03, 52.41,
49.220, 45.65, 45.12, 41.54, 29.49, 29.27, 24.74, 23.13. IR film (cmꢀ
1): 2936.8, 1688.8, 1609.7, 1586.58, 1516.1, 1489.1, 1436.1, 1233.5,
1197.9, 1134.2, 856.9, 801.6, 759.6, 725.3, 698.6. HRMS: calcd. for
C25H24N6O2 [M+H+]: 441.20335; found: 441.20315.
tert-butyl(R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-
d]pyrimidin-1-yl)piperidine-1-carboxylate (13)
To a suspension of (S)ꢀ1ꢀBocꢀ3ꢀhydroxypiperidine 12 (2 eq., 1.24 g,
6.2 mmol) and polymerꢀbound triphenylphosphine (3 eq., 3.08 g, 9.2
mmol) in THF (20 mL) was added dropwise diisopropyl
diazodicarboxylate (2 eq., 1.21 mL, 6.1 mmol) and the reaction
mixture was stirred for 5 min. Compound 11 (0.93 g, 3.08 mmol)
was added and the resulting mixture was heated for 5 min. The
tert-butyl (2-(piperazin-1-yl)ethyl)carbamate (16)
suspension was stirred overnight before being filtered over Celite to 1ꢀ(2ꢀaminoethyl)piperazine (15, 32.8 mL, 250 mmol) and
remove the resins and the resins were washed with MeOH and benzaldehyde (1 eq., 25.5 mL, 250 mmol) were dissolved in toluene
DCM. The filtrate was concentrated and the target compound was (200 mL) and the reaction mixture was refluxed over a DeanꢀStark
obtained by column chromatography (40% ꢀ 55% EtOAc/Pentane) apparatus in 3 hrs, cooled to 0 °C, and treated with dropwise
as a yellow solid (yield: 0.84 g, 1.72 mmol, 56%). RF = 0.50 (90% addition of benzylchloroformate (1 eq., 38 mL, 250 mmol). The
1
EtOAc/Pentane). H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H, CH), resulting mixture was stirred overnight before being concentrated.
7.65 (d, J = 8.4 Hz, 2H, 2xCH), 7.35 (t, J = 8.0 Hz, 2H, 2xCH), 7.15 The residue was dissolved in MeOH (500 mL), cooled to 0 °C and
– 7.12 (m, 3H, 3xCH), 7.06 (d, J = 8.0 Hz, 2H, 2xCH), 4.90 – 4.81 treated with 2 N HCl (125 mL). The resulting mixture (pH 1ꢀ2) was
(m, 1H, CH), 4.36 – 4.19 (m, 1H), 4.12 – 4.06 (m, 1H), 3.50 – 3.34 allowed to warm up to RT and concentrated under reduced pressure.
(m, 1H), 2.86 (t, J = 10.8 Hz, 1H), 2.31 – 2.16 (m, 2H), 1.93 – 1.88 The aqueous layer was washed with DCM before being made basic
(m, 1H), 1.72 – 1.67 (m, 1H), 1.44 (s, 9H). 13C NMR (101 MHz, with NH4OH (pH = 10) and extracted with DCM (3x), washed with
CDCl3) δ 158.10, 157.97, 156.02, 154.87, 154.30, 143.63, 130.60, brine, dried, filtered and evaporated. The residue was applied to
128.50, 124.44, 119.37, 98.09, 79.51, 52.51, 48.20, 44.05, 29.90, silica column chromatography (4% ꢀ 6% MeOH/DCM + 1% TEA)
28.95, 24.51. LCꢀMS analysis: Rt 8.26 min (linear gradient 10ꢀ90% to afford benzyl 4ꢀ(2ꢀaminoethyl)piperazineꢀ1ꢀcarboxylate as a
acetonitrile in H2O, 0.1% TFA, 15 min). ESIꢀMS (m/z): 487.13 yellowish oil (yield: 25.15 g, 95.5 mmol, 38%). RF = 0.20 (20%
1
[M+H+].
MeOH/DCM). H NMR (400 MHz, CDCl3) δ 7.36 – 7.28 (m, 5H,
CHar), 5.12 (s, 2H, CH2), 3.51 (t, J = 4.8 Hz, 4H, 2xCH2), 2.79 (t, J =
6.0 Hz, 2H, CH2), 2.44 (t, J = 6.0 Hz, 2H, CH2), 2.40 (br s, 4H,
2xCH2), 2.30 (s, 2H, NH2). 13C NMR (101 MHz, CDCl3) δ 154.76,
136.32, 128.09, 127.59, 127.44, 67.10, 60.06, 52.48, 43.42, 38.03.
After the solution of the carboxylate product (12.58 g, 47.75 mmol)
in THF (200 mL) was cooled to 0 °C, Boc2O (1.2 eq., 12.51 g, 57.30
mmol) was added portion wise and the reaction mixture was allowed
to warm up to RT overnight before being concentrated. The residue
was further purified by silica column chromatography (20% ꢀ 30%
(R)-3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (14)
Compound 13 (0.05 g, 0.1 mmol) was stirred in 4.0 M HCl in
dioxane (1 mL) for 2 hrs. The reaction mixture was concentrated in
vacuo and the residue was suspended in EtOAc before being filtered.
The residue was washed with EtOAc and dried under reduced
pressure. The title compound was obtained without further
purification as a white solid (yield: 0.042 g, 0.1 mmol, 100%). RF =
0.05 (90% EtOAc/Pentane). LCꢀMS analysis: Rt 5.34 min (linear
gradient 10ꢀ90% acetonitrile in H2O, 0.1% TFA, 15 min). ESIꢀMS
(m/z): 387.01 [M+H+].
EtOAc/Pentane)
to
afford
benzyl
4ꢀ(2ꢀ((tertꢀ
butoxycarbonyl)amino)ethyl)piperazineꢀ1ꢀcarboxylate as a yellowish
oil (13.71 g, 37.72 mmol, 79%). RF = 0.30 (80% EtOAc/Pentane). 1H
NMR (400 MHz, CDCl3) δ 7.37 ꢀ 7.30 (m, 5H, CHar), 5.13 (s, 2H,
CH2), 4.98 (br s, 1H, NH), 3.51 (t, J = 5.2 Hz, 4H, 2xCH2), 3.25 –
3.21 (m, 2H, CH2), 2.46 (t, J = 6.0 Hz, 2H, CH2), 2.41 (br s, 4H,
2xCH2). 13C NMR (101 MHz, CDCl3) δ 155.85, 155.11, 136.59,
128.42, 127.96, 127.82, 79.17, 67.05, 57.14, 52.53, 43.67, 36.93,
28.35. HRMS: calcd. for C19H29N3O4 [M+H+]: 364.21581; found:
Ibrutinib (1)
To a solution of crude amine 14 (0.20 mmol) in DCM (1 mL) were
added TEA (3.0 eq., 84 uL, 0.6 mmol) and acryloyl chloride (1.3 eq.,
20 uL, 0.26 mmol). The resulting mixture was stirred overnight prior
to washing with aqueous solution of citric acid (5%, 5 mL) and
brine. The title compound was obtained after RPꢀHPLC purification
(linear gradient 40% ꢀ 60% ACN in H2O, 0.1% TFA, 15 min) as a
364.20315.
To
a solution of 4ꢀ(2ꢀ((tertꢀbutoxycarbonyl)amino)ethyl)piperazineꢀ1ꢀ
carboxylate (1.82 g, 5.0 mmol) in MeOH (20 mL) Pd/C (10% w/w,
150 mg) was added. Hydrogen gas was then bubbled through the
mixture overnight. The reaction mixture was filtered over Celite and
concentrated to obtain the target compound as a yellowish oil (yield:
1.08 g, 4.71 mmol, 94%). The target compound was used without
1
white solid (yield: 12.0 g, 92.0 µmol, 46%). H NMR (600 MHz,
DMSOꢀd6): δ 8.42 (s, 1H), 7.67 (d, J = 7.2 Hz, 2H), 7.44 (t, J = 7.2
Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 7.16 (d, J = 9.0 Hz, 2H), 7.13 (d, J
= 9.0 Hz, 2H), 6.85 (t, J= 13.2, 0.5 H), 6.69 (t, J = 15.6 Hz, 0.5 H),
6.13 (d, J = 16.2 Hz, 0.5H), 6.06 (d, J = 16.8 Hz, 0.5H), 5.70 (d, J =
10.2 Hz, 0.5H), 5.59 (d, J = 9.6 Hz, 0.5H), 4.79 – 4.71 (m, 1H), 4.56
(d, J = 10.8 Hz, 1H), 4.19 (br s, 1H), 4.06 (d, J = 13.2 Hz, 0.5H),
1
further purification. RF = 0.29 (1/1/1 v/v/v H2O/ACN/tBuOH). H
NMR (400 MHz, MeOD) δ 3.80 ꢀ 3.65 (m, 6H, 3xCH2), 3.57 – 3.51
(m, 4H, 2xCH2), 3.38 (t, J = 7.6 Hz, 2H, CH2), 1.46 (s, 9H, 3xCH3).
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View ArticAle ROnTliInCeLE
DOI: 10.1039/C5OB00474H
13C NMR (101 MHz, MeOD) δ 158.03, 80.43, 57.74, 54.59, 53.30, analysis: Rt 6.05 min (linear gradient 10ꢀ90% acetonitrile in H2O,
49.87, 41.93, 36.11, 35.09, 28.67. HRMS: calcd. for C11H23N3O2 0.1% TFA, 15 min). ESIꢀMS (m/z): 682.13 [M+H+].
[M+H+]: 230.17903; found: 230.17907.
(R,E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-
(E)-4-bromobut-2-enoic acid (17)
d]pyrimidin-1-yl)piperidin-1-yl)-4-(4-(2-aminoethyl)piperazin-1-
yl)but-2-en-1-one (20)
To a solution of commercially available crotonic acid (10 g, 116
mmol) in benzene (150 mL) was added Nꢀbromosuccinimide (1.1 Compound 19 (0.015 g, 0.022 mmol) was stirred in 4.0 M HCl in
eq., 22.74 g, 120 mmol) and benzoyl peroxide (0.01 eq, 0.45 g, 1.4 dioxane (2 mL) for 2 hrs. The reaction mixture was concentrated in
mmol) and the resulting mixture was refluxed for 4 hrs. The reaction vacuo and the residue was suspended in EtOAc before being filtered.
mixture was then allowed to cool to 0 °C, which resulted in The residue was washed with EtOAc and dried under reduced
precipitation of succinimide crystals. The crystals were filtered over pressure. The title compound was obtained without further
Celite and washed with toluene. The filtrate was concentrated and purification as a white solid (yield: 0.013 g, 0.022 mmol, 100%). RF
the residue was recrystallized from hexanes yielding the title = 0.05 (90% EtOAc/Pentane). LCꢀMS analysis: Rt 5.26 min (linear
compound as a pale yellow solid (yield: 9.55 g, 57.9 mmol, 50%). RF gradient 10ꢀ90% acetonitrile in H2O, 0.1% TFA, 15 min). ESIꢀMS
= 0.79 (1/1/1 v/v/v H2O/ACN/tBuOH). 1H NMR (400 MHz, CDCl3) (m/z): 587.27 [M+H+].
δ 9.04 (bs, 1H), 7.12 (m, 1H), 6.05 (d, J = 15.2 Hz, 1H), 4.04 (dd, J1
= 1.2 Hz, J2 = 7.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 170.41,
(R,E)-N-(2-(4-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-oxobut-2-en-1-
yl)piperazin-1-yl)ethyl)-3-(5,5-difluoro-1,3,7,9-tetramethyl-5H-
5l4,6l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-2-
yl)propanamide (4)
144.20, 123.79, 28.72.
(E)-4-(4-(2-((tert-butoxycarbonyl)amino)ethyl)piperazin-1-
yl)but-2-enoic acid (18)
Compound 17 (1 eq., 0.22 g, 1 mmol) was dissolved in THF (5 mL). Carboxaldehyde pyrrole16 (90 mg, 0.43 mmol, 1 eq) was dissolved
A solution of compound 16 (0.27 g, 1 mmol) and TEA (3 eq., 0.42
mL, 3.0 mmol) in THF (2 mL) was added and the mixture was
stirred overnight and concentrated under reduced pressure. The title
compound was used without further purification. RF = 0.29 (1/1/1
v/v/v H2O/ACN/tBuOH). LCꢀMS analysis: Rt 5.69 min (linear
gradient 0ꢀ50% acetonitrile in H2O, 0.1% TFA, 15 min). ESIꢀMS
(m/z): 313.93 [M+H+].
in MeOH (5 mL) and 2,4ꢀ
dimethylpyrrole (41 mg, 44 ꢁL,
0.43 mmol, 1 eq) was added. The
resulting mixture was cooled to
0°C, and hydrobromic acid, 48%
solution in water (0.072 mL, 0.64
mmol, 1.5 eq) was added. After 2 h of stirring a yellowish precipitate
formed and TLC analysis showed complete consumption of the
starting materials. The crude dipyrrole HBr salt was concentrated
and coevaporated with DCE (3x) and dissolved in DCE (10 mL)
under an argon atmosphere. Triethylamine (0.178 mL, 1.29 mmol, 3
tert-butyl (R,E)-(2-(4-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-oxobut-2-en-1-
yl)piperazin-1-yl)ethyl)carbamate (19)
.
eq) and BF3 Et2O (0.57 mL, 2.15 mmol, 5 eq) were added, and the
HATU (2.2 eq., 1.25 g, 3.3 mmol) was added to a solution of
compound 18 (4 eq., 1.88 g, 6.0 mmol) and TEA (7 eq., 1.46 mL,
10.5 mmol) in DMF (5 mL) and the reaction mixture was allowed to
stir for 1 min. A solution of amine 14 (0.63 g, 1.5 mmol) in DMF (2
mL) was added and the resulting mixture was stirred overnight.
EtOAc (25 mL) was added and the organic layer was washed with
sat. aq. NaHCO3 and brine, dried over MgSO4, filtered and
concentrated in vacuo. The title compound was obtained after
column chromatography (4% ꢀ 8% MeOH/DCM) as a brown solid
(yield: 0.89 g, 1.31 mmol, 87%). RF = 0.55 (10% MeOH/DCM). 1H
NMR (400 MHz, DMSOꢀd6) δ 8.26 (s, 1H), 7.68 (d, J = 8.8 Hz, 2H),
7.42 (t, J = 7.6Hz, 2H), 7.20 – 7.11 (m, 5H), 6.52 (t, J = 9.8 Hz, 1H),
6.48 – 6.42 (m, 2H), 5.88 (br s, 1H), 4.78 – 4.72 (m, 1H), 4.33 (d, J
= 12.0 Hz, 1H), 4.01 (dt, J = 16, 8 Hz, 1H), 3.60 – 3.57 (m, 1H),
3.51 (t, J = 12 Hz, 1H), 3.27 – 3.18 (m, 1H), 3.10 – 3.04 (m, 6H),
2.42 – 2.37 (m, 6H), 2.21 – 2.17 (m, 1H), 2.03 – 1.96 (m, 1H), 1.70
– 1.59 (m, 1H), 1.41 (s, 9H). 13C NMR (101 MHz, DMSOꢀd6) δ
164.36, 157.69, 156.91, 154.96, 153.82, 142.61, 139.95, 129.39,
127.66, 123.12, 122.37, 118.42, 97.38, 77.08, 57.99, 56.64, 52.32,
52.07, 47.51, 42.81, 37.39, 28.57, 27.73, 23.18. HRMS: calcd. for
C37H47N9O4 [M+H+]: 682.37510; found: 682.37513. LCꢀMS
reaction was subsequently stirred at room temperature until TLC
showed completion of the reaction. The solution was concentrated
and the product purified by silica gel column chromatography (0%
→ 2% EtOAc in toluene) which gave 4,4ꢀDifluoroꢀ1,3,7,9ꢀ
tetramethylꢀ2ꢀ(2ꢀ(ethoxycarbonylmethyl))ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀ
indacene (106 mg, 0.32 mmol, 74%). Rf = 0.4 (6:1 toluene:EtOAc).
1HꢀNMR (400 MHz, CDCl3) δ 6.99 (s, 1H), 6.00 (s, 1H), 3.66 (s,
3H), 2.70 (t, J = 7.8 Hz, 2H), 2.50 (s, 6H), 2.43 (t, J = 7.8, 2H), 2.18
(d, J = 15.4, 6H). 13CꢀNMR (101 MHz, CDCl3) δ 173.41, 156.51,
155.86, 141.20, 138.54, 133.59, 133.07, 128.61, 120.05, 119.08,
77.80, 77.48, 77.16, 52.04, 34.45, 19.86, 14.96, 13.03, 11.56, 9.87.
LCꢀMS analysis (10% ꢀ 90% ACN) Rt: 9.18 min, ESIꢀMS (m/z):
[M+H]+: 335.0; [MꢀF]+: 315.2
4,4ꢀDifluoroꢀ1,3,7,9ꢀtetramethylꢀ2ꢀ
(2ꢀ(ethoxycarbonylmethyl))ꢀ4ꢀ
boraꢀ3a,4aꢀdiazaꢀsꢀindacene (106
mg, 0.32 mmol) was dissolved in
MeOH (20 mL) and aq. NaOH
(3.68 mL, 0.1 M, 1.15 eq, 0.37 mmol) was added. The mixture was
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heated to reflux for 1.5 h, after which byꢀproduct formation started ACN in H2O, 0.1% TFA, 15 min) as a purple solid (yield: 12.19 mg,
1
to occur. The reaction was quenched by the addition of aq. HCl (3.68 10.25 µmol, 10.3%). RF = 0.05 (90% EtOAc/Pentane). H NMR
mL, 0.1 M, 1.15 eq), followed by extraction with EtOAc (3x). The (600 MHz, DMSOꢀd6): δ 8.26 (s, 1H), 7.85 (d, J = 6.6 Hz, 2H), 7.66
organic layers were dried (MgSO4), concentrated and 4,4ꢀDifluoroꢀ (d, J = 7.8 Hz, 3H), 7.43 (t, J = 10.8 Hz, 2H), 7.19 (t, J = 7.2 Hz,
1,3,7,9ꢀtetramethylꢀ2ꢀ(2ꢀ(carboxyethyl))ꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀ
1H), 7.16 – 7.11 (m, 5H), 7.02 (d, J = 7.8 Hz, 2H), 6.69 (br s, 1H),
indacene was obtained by silica column chromatography (0 ꢀ1% 6.61 – 6.58 (m, 1H), 6.5 (br s, 1H), 4.73 – 4.67 (m, 1H), 4.54 (d, J =
EtOAc in toluene (starting material)ꢀ 1% EtOAc in toluene + 1% 11.4 Hz, 1H), 4.14 (d, J = 10.2 Hz, 1H), 4.03 (d, J = 11.4 Hz, 1H),
AcOH (product)) as a red powder (65 mg, 0.2 mmol, 64% (74% 3.82 (s, 3H), 3.76 – 3.72 (m, 1H), 3.24 – 3.19 (m, 3H), 3.09 (q, J =
based on recovered starting material)). Rf = 0.2 (6:1 toluene:EtOAc 7.8 Hz, J = 15.0 Hz, 4H), 2.66 – 2.61 (m, 3H), 2.55 ꢀ 2.54 (m, 2H),
1
+ AcOH). λabs 514 nm/λem 523 nm (MeOH). HꢀNMR (400 MHz, 2.49 (s, 3H), 2.26 (d, J = 6.6 Hz, 3H), 2.22 (s, 3H), 2.12 (d, J = 10,2
CDCl3) δ 7.01 (s, 1H), 6.02 (s, 1H), 2.72 (t, J = 7.7 Hz, 2H), 2.56 – Hz, 1H), 1.96 – 1.89 (m, 1H), 1.63 – 1.52 (m, 1H), 1.18 (t, J = 7.2
2.40 (m, 8H), 2.21 (d, J = 14.7 Hz, 6H). 13CꢀNMR (101 MHz, Hz, 4H). 13C NMR (150 MHz, DMSOꢀd6): δ 159.97, 158.18, 157.14,
CDCl3) δ 178.68, 156.57, 155.48, 141.10, 138.22, 133.42, 132.77, 156.28, 155.66, 153.96, 143.34, 143.20, 140.58, 134.68, 133.92,
127.97, 119.84, 118.95, 34.16, 19.37, 14.76, 12.81, 11.36, 9.69. LCꢀ 131.07, 130.39, 130.12, 128.30, 127.89, 124.90, 124.19, 123.80,
MS analysis (10% ꢀ 90% ACN) Rt: 7.94 min, ESIꢀMS (m/z): 118.97, 117.98, 113.78, 97.39, 55.71, 45.76, 40.06, 34.93, 19.80,
[M+H]+: 320.93; [MꢀF]+: 301.13.
12.90, 9.25, 8.59. IR film (cmꢀ1): 1672.3, 1604.8, 1523.8, 1464.0,
1234.4, 1201.7, 1180.4, 1139.9, 1045.4. HRMS: calcd. for
C53H58BF2N11O4 [M+2H2+]: 481.7444; found: 481,74402.
HATU (1.3 eq., 49 mg, 0.13 mmol) was added to a solution of
compound 4,4ꢀDifluoroꢀ1,3,7,9ꢀtetramethylꢀ2ꢀ(2ꢀ(carboxyethyl))ꢀ4ꢀ
boraꢀ3a,4aꢀdiazaꢀsꢀindacene (2 eq., 64 mg, 0.20 mmol) and TEA (5 Ibrutinib-alkyne (6)
eq., 70 µL, 0.5 mmol) in DMF (0.4 mL) and the reaction mixture
DiPEA (4.0 eq., 70 µL, 0.4 mmol) and 6ꢀheptynoicꢀOSu18 (2.2 eq.,
was allowed to stir for 1 min. A solution of amine 20 (42 mg, 0.1
mmol) in DMF (0.3 mL) was added and the resulting mixture was
stirred overnight. EtOAc (10 mL) was added and the organic layer
was washed with sat. aq. NaHCO3 and brine, dried over MgSO4,
filtered and concentrated under reduced pressure. The title
compound was obtained after RPꢀHPLC purification (linear gradient
40% ꢀ 60% ACN in H2O, 0.1% TFA, 15 min) as a red/brown solid
4.9 mg, 0.25 mmol) were added to a solution of crude amine 20 (42
mg, 0.1 mmol) in DMF (0.5 mL). The reaction mixture was stirred
overnight before being evaporated. The title compound was obtained
after RPꢀHPLC purification (linear gradient 40% ꢀ 60% ACN in
H2O, 0.1% TFA, 15 min) as a white solid (yield: 32.92 mg, 35.87
µmol, 35.9%). RF = 0.05 (90% EtOAc/Pentane). 1H NMR (600
MHz, DMSOꢀd6): δ 8.39 (s, 1H), 8.03 (br s, 1H), 7.67 ꢀ 7.66 (m,
2H), 7.44 (t, J = 7.2 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 7.16 (d, J =
9.0 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 15.0 Hz, 0.5H),
6.70 (d, J = 15.0 Hz, 0.5H), 6.63 ꢀ 6.61 (m, 0.5H), 6.56 – 6.54 (m,
0.5H), 4.77 – 4.71 (m, 2H), 4.57 (d, J = 12 Hz, 1H), 4.19 (d, J = 12.0
Hz, 2H), 4.06 (d, J = 12.6 Hz, 1H), 3.75 (t, J = 11.4 Hz, 1H), 3.58 (s,
1H), 3.44 (s, 1H), 3.35 (br s, 3H), 3.23 (q, J = 11.4 Hz, J = 22.2
Hz,3H), 3.07 – 2.93 (m, 4H), 2.73 (s, 1H), 2.28 – 2.24 (m, 1H), 2.20
(br s, 3H), 2.12 – 2.11 (m, 2H), 1.95 – 1.93 (m, 1H), 1.63 – 1.58 (m,
3H), 1.47 – 1.44 (m, 2H). 13C NMR (150 MHz, DMSOꢀd6): δ
174.26, 163.81, 157.40, 156.58, 156.18, 152.98, 144.44, 130.12,
127.16, 126.12, 123.63, 119.00, 115.78, 97.15, 85.35, 71.18, 62.04,
61.17, 52.95, 52.33, 50.06, 19.23, 45.70, 45.19, 41.60, 34.66, 34.35,
29.53, 27.51, 24.80, 24.51, 23.48, 17.43. IR film (cmꢀ1): 3290.7,
2940.0, 1663.7, 1614.5, 1520.9, 1490.1, 1455.4, 1235.5, 1198.8,
1131.3, 831.4, 711.2. HRMS: calcd. for C39H47N9O3 [M+2H2+]:
345.69737; found: 345.69732.
(yield: 15.60 mg, 16.51 µmol, 16.5%). RF
= 0.05 (90%
1
EtOAc/Pentane). H NMR (600 MHz, DMSOꢀd6): δ 8.38 (s, 1H),
8.10 (br s, 1H), 7.66 (t, J = 7.8 Hz, 2H), 7.61 (s, 1H), 7.44 (t, J = 7.2
Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 7.13 (d, J
= 7.8 Hz, 2H), 6.84 (d, J = 15.0 Hz, 0.5H), 6.69 (d, J = 15.0 Hz),
6.62 – 6.58 (m, 0.5H), 6.55 – 6.52 (m, 0.5H), 6.15 (s, 1H), 4.76 –
4.66 (m, 1H), 4.57 (d, J = 11.4 Hz, 1H), 4.20 – 4.19 (m, 2H), 4.06
(d, J = 12.6 Hz, 1H), 3.77 (t, J = 12 Hz, 1H), 3.53 (br s, 1H), 3.40 (br
s, 1H), 3.37 – 3.32 (m, 4H), 3.24 – 3.21 (m, 2H), 3.04 (m, 1H), 2.98
– 2.88 (m, 4H), 2.61 – 2.60 (m, 2H), 2.42 (s, 3H), 2.40 (s, 1H), 2.25
(s, 6H), 2.22 (s, 3H), 2.14 ꢀ ꢀ2.12 (m, 1H), 1.95 – 1.93 (m, 1H), 1.63
– 1.56 (m, 1H). 13C NMR (150 MHz, DMSOꢀd6): δ 172.0, 163.79,
158.79, 158.56, 157.40, 156.76, 156.24, 155.70, 154.55, 153.38,
153.07, 144.43, 144.03, 140.94, 139.24, 132.43, 130.09, 129.22,
127.24, 126.87, 123.90, 121.47, 119.04, 118.45, 117.45, 97.22,
56.69, 55.02, 52.96, 52.32, 50.16, 49.32, 45.77, 45.21, 41.63, 35.16,
34.13, 29.47, 24.88, 23.25, 19.57, 14.21, 12.51, 10.96, 9.25. IR film
(cmꢀ1): 1670.4, 1603.9, 1517.1m 1471.8, 1436.1, 1227.7, 1199.8,
1131.3, 974.1, 832.3, 799.5, 720.4, 666.4. HRMS: calcd. for
C48H56BF2N11O3 [M+2H2+]: 442.73912; found: 442.73849.
Ibrutinib-N3 (7)
DiPEA (4.0 eq., 70 µL, 0.4 mmol) and azidoꢀPNP ester19 (2.5 eq., 63
mg, 0.25 mmol) were added to a solution of crude amine 20 (42 mg,
0.1 mmol) in DMF (0.5 mL). The reaction mixture was stirred
overnight before being evaporated. The title compound was obtained
after RPꢀHPLC purification (linear gradient 40% ꢀ 60% ACN in
H2O, 0.1% TFA, 15 min) as a white solid (yield: 20.39 mg, 22.14
Ibrutinib-BODIPY-TMR (5)
DiPEA (3.5 eq., 60 µL, 0.35 mmol) and BODIPYꢀTMRꢀOSu17 (2.2
eq., 0.11 g, 0.22 mmol) were added to a solution of crude amine 20
(42 mg, 0.1 mmol) in DMF (0.5 mL). The reaction mixture was
stirred overnight before being evaporated. The title compound was
obtained after RPꢀHPLC purification (linear gradient 40% ꢀ 60%
1
µmol, 22.1%). H NMR (600 MHz, DMSOꢀd6): δ 8.37 (s, 1H), 8.08
(br s, 1H), 7.66 (br s, 2H), 7.44 (t, J = 8.4 Hz, 2H), 7.20 (t, J = 7.2
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DOI: 10.1039/C5OB00474H
Hz, 1H), 7.16 (d, J = 9.0 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 6.84 (d, pH 7=0, 250 mM sucrose, 5 mM MgCl2, 1 mM DTT, 0.025%
J= 15.0 Hz, 0.5H), 6.70 (d, J = 14.4 Hz, 0.5H), 6.63 – 6.60 (m, digitonin; 3x pellet volume), incubation on ice for 30 min and
0.5H), 6.56 – 6.52 (m, 0.5H), 4.76 – 4.71 (m, 1H), 4.56 (d, J = 12.0 centrifugation for 15 min at 16, 000 g (4° C), after which the
Hz, 0.5H), 4.19 (d, J = 12.0 Hz, 0.5H), 4.06 (d, J = 12.6 Hz, 0.5H), supernatants containing the cytosolic fractions were collected and
3.75 (t, J = 10.2 Hz, 0.5H), 3.37 – 3.33 (m, 4H), 3.23 – 3.20 (m, 2H), the protein concentration was determined by Qubit® Protein Assay
3.07 – 2.97 (m, 4H), 2.31 – 2.24 (m, 2H), 2.19 – 2.13 (m, 6H), 1.95 kit. Precipitations of proteins was done using a chloroform/methanol
– 1.93 (m, 2H), 1.77 – 1.75 (m, 4H), 1.63 – 1.53 (m, 2H). 13C NMR (c/m) precipitation protocol.20 SDSꢀPAGE analysis: inꢀgel
(150 MHz, DMSOꢀd6): δ 171.95, 163.81, 157.37, 156.19, 153.07, fluorescence was measured on a ChemiDoc MP system (Cy2
152.79, 144.31, 143.91, 130.12, 127.25, 126.13, 123.85, 119.00, settings, 530/30 filter and Cy3 settings, 605/50 filter) and analysed
115.78, 97.17, 56.67, 55.04, 52.92, 52.30, 50.16, 49.30, 45.71, using Image Lab 4.1. As a loading control gels were stained with
45.19, 41.60, 34.07, 32.12, 29.54, 29.38, 24.81, 24.29, 23.18, 22.94. Coomassie Blue. Protein standard is Dual Color protein standard
IR film (cmꢀ1): 3317.7, 2097.7, 1671.4, 1587.5, 1520.9, 1490.1, (DC, BioꢀRad).
1437.0, 1236.4, 1201.7, 1131.3, 760.3, 613.4. HRMS: calcd. for
C36H44N12O3 [M+2H2+]: 347.19024; found: 347.19022.
IMAP Reaction Buffer (10 mM TrisꢀHCl, 10 mM MgCl2, 0.01%
Tweenꢀ20, 0.05% NaN3 pH 7.2 ) and the IMAP Progressive Binding
Ibrutinib-norbornene (8)
System (IMAP Progressive Binding Buffer A, IMAP Progressive
Binding Buffer B, and IMAP Progressive binding Reagent), were all
from Molecular Devices.
Azidoꢀibrutinib 7 (69 mg, 0.1 mmol) and Nꢀ(2ꢀpropynyl)ꢀ5ꢀ
norborneneꢀ2ꢀcarboxamide8 (1.5 eq., 26.3 mg, 0.15 mmol) were
dissolved in DMF (0.5 mL). The reaction mixture was stirred IMAP FP assay:
overnight after addition of CuSO4·5 H2O (0.2 eq., 20 uL 1 M in
Compounds 1, 6, 7 and 8 (5 µL 10x solution in DMSO/kinase
H2O, 20 umol) and sodium ascorbate (0.4 eq., 40 uL 1 M in H2O, 40
umol). The mixture was then concentrated and purified by RPꢀHPLC
(linear gradient 40% ꢀ 60% ACN in H2O, 0.1% TFA, 15 min) to
yield the title compound as a white solid (yield: 10.75 mg, 9.81
reaction (KR)ꢀbuffer, such that the final concentration of DMSO was
4%) and 100 mU/mL BTK (5 µL 100 mU/mL in KRꢀbuffer) were
incubated for 60 min at room temperature. Next, 50 nM of
fluoresceinated substrate (5 µL 200 nM in KRꢀbuffer) Fluorescein
labeled Blk/Lyntide substrate (5FAMꢀEFPIYDFLPAKKKꢀNH2)
and 5 µM ATP (5 µL 20 µM in KRꢀbuffer) were added to the
mixture and incubated for 120 min at room temperature. The
reaction was stopped after 120 min by a 40 µL addition of IMAP
binding reagent in binding buffer and read on the Envision 2102
Multilabel Reader, Dichroic mirror D505FP/D535, exitation filter:
480 nm cwl. Parallel and perpendicular filters 535 nm cwl. For every
measurement 18 wells were used as minimum wells (wells with
ATP, 0% effect), 18 wells were used as maximum wells (wells
without ATP, 100% effect). 16 wells were used to measure the
background signal, which contained no substrate. Enzyme, substrate,
and ATP were prepared in kinase reaction buffer containing 1 mM
DTT. The IMAP binding reagent was 2000x diluted in the binding
buffer.
1
µmol, 10.2%). H NMR (600 MHz, DMSOꢀd6): δ 8.34 (s, 1H), 8.09
(br s, 1H), 8.00 (br s, 1H), 7.08 (s, 1H), 7.67 – 7.66 (m, 2H), 7.45 (t,
J = 7.2 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 7.16 (d, J = 9.0 Hz, 2H),
7.13 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 14.4 Hz, 0.5H), 6.69 (d, J =
14.4 Hz, 0.5 H), 6.63 – 6.61 (m, 0.5H), 6.56 – 6.53 (m, 0.5H), 6.10 –
6.08 (m, 1H), 5.80 – 5.78 (m, 1H), 4.75 – 4.70 (m, 1H), 4.56 (br s,
1H), 4.33 (br s, 2H), 4.23 (br s, 2H), 4.08 – 4.05 (m, 1H), 3.78 (dd, J
= 2.4 Hz, J = 5.4 Hz, 2H), 3.34 (br s, 2H), 3.25 – 3.21 (m, 1H), 3.17
(br s, 1H), 3.14 (br s, 1H), 2.97 – 2.92 (br s, 2H), 2.82 – 2.79 (m,
2H), 2.28 – 2.23 (m, 1H), 2.16 9 2.14 (m, 1H), 2.12 – 2.09 (m, 4H),
2.07 (s, 1H), 2.11 (br s, 2H), 2.00 – 1.93 (m, 1H), 1.78 – 1.72 (m,
3H), 1.68 – 1.54 (m, 2H), 1.33 – 1.22 (m, 9H). 13C NMR (150 MHz,
DMSOꢀd6): δ 172.87, 172.66, 171.70, 163.80, 158.32, 158.10,
157.32, 156.19, 153.20, 147.73, 145.42, 136.83, 132.21, 132.02,
130.12127.73, 127.47, 126.59, 123.84, 122.51, 118.99, 97.23, 81.61,
72.37, 69.75, 55.08, 54.03, 49.34, 48.71, 47.37, 46.64, 45.48, 43.23,
42.07, 34.33, 31.85, 28.41, 27.83, 25.72. IR film (cmꢀ1): 3337.0,
2945.4, 1663.7, 1518.0, 1490.1, 1446.7, 1418.7, 1235.5, 1199.8,
1185.3, 839.1, 799.5, 720.4. HRMS: calcd. for C47H57N13O4
[M+2H2+]: 434.74010; found: 434.74012.
In vitro labeling of BTK in Ramos cells (ABPs 4 and 5)
Ramos cell lysates (30 µg total protein per experiment) in lysis
buffer (9 µL) were exposed to the indicated concentrations of the
ABP (1 µL 10x solution in DMSO) for 1 hr at room temperature.
The reaction mixtures were then boiled for 5 min at 95° C with 3.3
µL 4x Laemmli’s sample buffer containing 2ꢀmercaptoethanol and
resolved on 10% SDSꢀPAGE, followed by fluorescence scanning
(Cy2 or Cy3 settings) and CBB staining.
Experimental procedures: biochemistry
General
Ramos cells, a Burkitt’s lymphoma B lymphocyte cell line, were
cultured on Dulbecco's Modified Eagle Medium: Nutrient Mixture
Fꢀ12 (DMEM/Fꢀ12) supplemented with 10% Fetal Bovine Serum,
0.1 mg/mL penicillin, 0.1 mg/mL streptomycin in a 5% CO2
humidified incubator at 37° C. Cell lysates were prepared from cell
pellets by resuspension in cold digitonin lysis buffer (50 mM Tris
In situ labelling of BTK in Ramos cells (ABPs 4 and 5)
Ramos cells (±1x107 cells per experiment) were exposed to the
indicated concentrations of the ABP (1 µL 10000x solution in
DMSO) in 10 mL fresh medium for 4 hrs at 37° C, before being
centrifuged for 5 min at 1200 rpm, and washed with PBS (3x). The
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Journal Name
DOI: 10.1039/C5OB00474H
cell pellets were then flash frozen in liquid nitrogen and lysed in 30 for 5 min at 1200 rpm, and washed with PBS (3x), before flash
µL lysis buffer. The lysates (30 µg total protein per experiment) in freezing the cell pellets in liquid nitrogen and cell lysis in 15 µL
lysis buffer (10 µL) were boiled for 5 min at 95° C with 3.3 µL 4x lysis buffer. The lysates (50 µg total protein per experiment) in lysis
Laemmli’s sample buffer containing 2ꢀmercaptoethanol and resolved buffer (10 µL) were precipitated by c/m precipitation and taken up in
on 10% SDSꢀPAGE. Inꢀgel visualization of the fluorescent labeling 10 µL 8 M urea and 3.5 µL 4x Laemmli’s sample buffer containing
was performed in the wet gel slabs directly using Cy2 or Cy3 2ꢀmercaptoethanol, boiled for 5 min at 95° C. the proteins were
settings.
resolved on 10% SDSꢀPAGE. Fluorescence was measured in the wet
gel slabs using Cy2 settings.
Competition experiments of ABP 4 versus ABPs 6-8: in situ
modification of BTK by ABP 4 and in vitro competition by ABPs For postꢀlysis ligation experiments, cells were harvested directly
6-8 .
after treatment with ABP 8 and lysed. The lysates (50 µg total
protein per experiment) in lysis buffer (10 µL) were boiled for 5 min
at 95° C after addition of 1% SDS (1.11 µL 10% SDS in H2O) and
boiling for 5 min at 95° C. Next, the lysates were exposed for 1 hr at
room temperature to 25 µM of tetrazine 24 (1.23 µL 10x solution in
DMSO). After the ligation reaction proteins were precipitated by c/m
precipitation and taken up in 10 µL 8 M urea and 3.5 µL 4x
Laemmli’s sample buffer containing 2ꢀmercaptoethanol, boiled for 5
min at 95° C and resolved on 10% SDSꢀPAGE. Inꢀgel visualization
of the fluorescent labeling was performed in the wet gel slabs
directly using Cy2 settings.
Ramos cells (±5x106 cells per experiment) were seeded in 6 cm petri
dishes and grown 2 hrs at 37° C, before being exposed to the
indicated concentrations of ABP (2 µL 1000x solution in DMSO) in
2 mL fresh medium for 3 hrs at 37° C. Next, the cells were
centrifuged for 5 min at 1200 rpm, and washed with PBS (3x),
before flash freezing the cell pellets in liquid nitrogen and cell lysis
in 15 µL lysis buffer. The lysates (30 µg total protein per
experiment) in lysis buffer (10 µL) were exposed to 1 µM ABP 4
(1.11 µL 10x solution in DMSO) for 1 hr at room temperature,
boiled for 5 min at 95° C with 3.7 µL 4x Laemmli’s sample buffer
containing 2ꢀmercaptoethanol and resolved on 10% SDSꢀPAGE. Inꢀ Copper(I)-catalyzed click ligation in vitro
gel visualization of the fluorescent labeling was directly performed
in the wet gel slabs using Cy2 or Cy3 settings.
Ramos lysates (50 µg total protein per experiment) in lysis buffer (9
µL) were exposed to 4 µM ABP 6 or 7 (1 µL 40 µM in DMSO) for 1
hr at room temperature. The reaction mixtures were subsequently
diluted with an additional 9 µL buffer containing 6.5 mM CuSO4
(0.58 µL 100 mM in H2O), 6.5 mM THPTA (0.58 µL 100 mM in
H2O), 6.5 mM sodium Lꢀascorbate (0.58 µL 100 mM in H2O) and
exposed for 1 hr at room temperature to the indicated concentrations
of azide 22 (reaction with 6) or alkyne 23 (reaction with 7) (1 µL
20x solution in DMSO). In control experiments, lysates were treated
with ABP 4 (1 µM) (positive control) or subjected to azide or alkyne
labeling in the absence of an ABP (background control).
Alternatively, click ligation was performed on lysates pretreated
with 20 µM ibrutinib (1) (negative control). Next, proteins were
precipitated by c/m precipitation and taken up in 10 µL 8 M urea and
3.5 µL 4x Laemmli’s sample buffer containing 2ꢀmercaptoethanol,
boiled for 5 min at 95° C and resolved on 10% SDSꢀPAGE. Inꢀgel
visualization of the fluorescent labeling was performed in the wet gel
slabs directly using Cy2 settings.
In vitro tetrazine ligation
Ramos lysates (50 µg total protein per experiment) in lysis buffer (9
µL) were exposed to 4 µM ABP 8 (1 µL 40 µM in DMSO) for 1 hr
at room temperature, followed by addition of 1% SDS (1.11 µL 10%
SDS in H2O) and boiling for 5 min at 95° C. Hereafter, the lysates
were exposed for 1 hr at room temperature to the indicated
concentrations of tetrazine 24 (1.23 µL 10x solution in DMSO). In
control experiments, lysates were treated with ABP 4 (1 µM)
(positive control) or subjected to tetrazine labeling in the absence of
ABP 8 (background control). As a negative control, tetrazine ligation
was performed on lysates pretreated with 20 µM ibrutinib (1). After
the ligation reaction proteins were precipitated by c/m precipitation
and taken up in 10 µL 8 M urea and 3.5 µL 4x Laemmli’s sample
buffer containing 2ꢀmercaptoethanol, boiled for 5 min at 95° C and
resolved on 10% SDSꢀPAGE. Inꢀgel visualization of the fluorescent
labeling was performed in the wet gel slabs directly using Cy2
settings.
In situ modification of BTK by ABPs 6 or 7 followed by in vitro
copper(I)-catalyzed click ligation
In situ and post-lysis tetrazine ligation
Ramos cells (±5x106 cells per experiment) were seeded in 6 cm petri
dishes and grown 2 hrs at 37° C, before being exposed to 4 µM ABP
6 or 7 (2 µL 1000x solution in DMSO) in 2 mL fresh medium for 3
hrs at 37° C. As a positive control, cells were exposed to ABP 4 (1
µM) for 3 hrs at 37° C. As a background control, cells were lysed
and subjected to click labeling in the absence of ABP 6 or 7. Next,
the cells were centrifuged for 5 min at 1200 rpm, and washed with
PBS (3x), before flash freezing the cell pellets in liquid nitrogen and
cell lysis in 15 µL lysis buffer. The lysates (50 µg total protein per
experiment) in lysis buffer (10 µL) were diluted with an additional 9
Ramos cells (±5x106 cells per experiment) were seeded in 6 cm petri
dishes and grown 2 hrs at 37° C, before being exposed to 4 µM ABP
8 (2 µL 1000x solution in DMSO) in 2 mL fresh medium for 3 hrs
at 37° C. After centrifugation of the cells for 5 min at 1200 rpm, the
cells were washed with fresh medium for 5 min at 37° C (3x), and
then exposed to 25 µM of tetrazine 24 (4 µL 10x solution in DMSO)
in 4 mL fresh medium for 1 hr at 37° C. As a positive control, cells
were exposed to ABP 4 (1 µM) for 3 hrs at 37° C. As a background
control, cells were lysed and subjected to tetrazine labeling in the
absence of ABP 8. Next, cells were harvested in PBS, centrifuged
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DOI: 10.1039/C5OB00474H
µL buffer containing 6.5 mM CuSO4 (0.58 µL 100 mM in H2O), 6.5
mM THPTA (0.58 µL 100 mM in H2O), 6.5 mM sodium Lꢀ
ascorbate (0.58 µL 100 mM in H2O) and exposed for 1 hr at room
temperature to 25 µM of azide 22 (reaction with 6) or alkyne 23
(reaction with 7) (1 µL 20x solution in DMSO). Next, proteins were
precipitated by c/m precipitation and taken up in 10 µL 8 M urea and 4.
3.5 µL 4x Laemmli’s sample buffer containing 2ꢀmercaptoethanol, 5.
boiled for 5 min at 95° C and resolved on 10% SDSꢀPAGE. Inꢀgel
visualization of the fluorescent labeling was performed in the wet gel
slabs directly using Cy2 settings.
B. E. Schultz, P. A. Sprengeler, L. C. Burrill, R. V.
Mendonca, M. D. Seeney, K. C. K. Scott, P. G. Grothaus,
D. A. Jeffery, J. M. Spoerke, L. A. Honigberg, P. R.
Young, S. A. Dalrymple and J. T. Palmer, Chem. Med.
Chem., 2007, 2, 58.
T. Barf and A. Kaptein, J. Med. Chem., 2012, 55, 6243.
a) Y. Liu, M. P. Patricelli and B. F. Cravatt, PNAS, 1999,
96, 14694. b) B. F. Cravatt, A. T. Wright and J. W.
Kozarich, Annu. Rev. Biochem., 2008, 77, 383. c) M. J.
Niphakis and B. F. Cravatt, Annu. Rev. Biochem., 2014,
83, 341.
Acknowledgements
6.
B. R. Lanning, L. R. Whitby, M. M. Dix, J. Douhan, A. M.
Gilbert, E. C. Hett, T. O. Johnson, C. Joslyn, J. C. Kath, S.
Niessen, L. R. Roberts, M. E. Schnute, C. Wang, J. J.
Hulce, B. Wei, L. O. Whiteley, M. M. Hayward and B. F.
Cravatt, Nat. Chem. Biol., 2014, 10, 760.
L. I. Willems, W. A. van der Linden, N. Li, K.ꢀY. Li, N.
Liu, S. Hoogendoorn, G. A. van der Marel, B. I. Florea, H.
S. Overkleeft, Acc. Chem. Res., 2011, 44, 718.
L. I. Willems, N. Li, B. I. Florea, M. Ruben, G. A. van der
Marel and H. S. Overkleeft, Angew. Chem. Int. Ed., 2012,
51, 4431.
The Netherlands Organization for Scientific Research (NWOꢀCW,
Mozaiek grant to NL) and the European Research Council (ERC,
Advanced Grant to HSO) are acknowledged for the financial
support.
7.
8.
9.
Affiliation
aLeiden Institute of Chemistry, Leiden University, Einsteinweg 55,
b
2300 RA Leiden, the Netherlands. AcertaPharma, Molenweg 79
5349 AC Oss, the Netherlands. cKantonspital St. Gallen,
Rorschacher Strasse 95 St. Gallen, Switserland.
a) A. Turetsky, E. Kim, R. H. Kohler, M. A. Miller and R.
Weissleder, Sci. Rep., 2014, 4, 4782. b) Z. Pan, S. J. Li, H.
Scheerens, L. Honigberg, E. verner, US Pat., US
2008/0214501 A1, 2008.
*h.s.overkleeft@chem.leidenuniv.nl,
m.van.der.stelt@chem.leidenuniv.nl
10.
11.
L. Honigberg, E. Verner, Z. Pan, US Pat., US
2008/0076921 A1, 2008.
The Walter and Eliza Hall institute of medical research,
WO 2012 003544 A1, 2012.
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