PET Imaging of T Cells: Target Identification and Feasibility Assessment

Article abstract of DOI:10.1002/cmdc.201800241

Imaging T cells using positron emission tomography (PET) would be highly useful for diagnosis and monitoring in immunology and oncology patients. There are, however, no obvious targets that can be used to develop imaging agents for this purpose. We evaluated several potential target proteins with selective expression in T cells, and for which lead molecules were available: protein kinase C isozyme θ (PKC θ), lymphocyte-specific protein tyrosine kinase (Lck), zeta-chain-associated protein kinase 70 (ZAP70), and interleukin-2-inducible T-cell kinase (Itk). Ultimately, we focused on Itk and identified a tool molecule with properties suitable for in vivo imaging of T cells: (5aR)-5,5-difluoro-5a-methyl-N-(1-((S)-3-(methylsulfonyl)phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-carboxamide (23). Although it does not have the optimal profile for clinical use, this molecule indicates that it might be possible to develop Itk-selective PET ligands for imaging the distribution of T cells in patients.

Full text of DOI:10.1002/cmdc.201800241

Accepted Article
Title: PET imaging of T cells: Target identification and feasibility
assessment
Authors: Yves P. Auberson, Emmanuelle Briard, Bettina Rudolph,
Klemen Kaupmann, Paul Smith, and Berndt Oberhauser
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201800241
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800241
A Journal of
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10.1002/cmdc.201800241
ChemMedChem
FULL PAPER
PET imaging of T cells: Target identification and feasibility
assessment
Dr. Yves P. Auberson,*[a] Dr. Emmanuelle Briard,[a] Dr. Bettina Rudolph[b], Dr. Klemens Kaupmann,[a] Dr.
Paul Smith,[c] and Dr. Berndt Oberhauser[a]
[a]
Dr. Y. P. Auberson, Dr. E. Briard, Dr. B. Oberhauser, Dr. K. Kaupmann
Global Discovery Chemistry
Novartis Institutes for BioMedical Research
141 Klybeckstrasse, 4057 Basel, Switzerland
E-mail: yves.auberson@novartis.com
Dr. B. Rudolph
Translational Medicine, Pharmacokinetics Sciences
Novartis Institutes for BioMedical Research
141 Klybeckstrasse, 4057 Basel, Switzerland
Dr. P. Smith
[b]
[c]
Autoimmunity, Transplantation & Inflammation
Novartis Institutes for BioMedical Research
Novartis Campus, 4056 Basel, Switzerland
Abstract: Imaging T cells using positron emission tomography
(PET) would be highly useful for diagnosis and monitoring in
immunology and oncology patients. There are however no obvious
targets that can be used to develop imaging agents for this purpose.
We evaluated several potential target proteins with selective
expression in T cells, and for which lead molecules were available:
PKC θ, Lck, ZAP70 and Itk. Ultimately, we focused on Itk
(interleukin-2-inducible T cell kinase) and identified a tool molecule
with properties suitable for in vivo imaging of T cells, (5aR)-5,5-
difluoro-5a-methyl-N-(1-((S)-3-(methylsulfonyl)-phenyl)(tetrahydro-
2H-pyran-4-yl)methyl)-1H-pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydro-
cyclopropa[f]-indazole-3-carboxamide (23). While not having the
optimal profile for clinical use, this molecule indicates that it might be
possible to develop Itk-selective PET ligands for imaging the
distribution of T cells in patients.
Imaging (MRI) or X-ray based Computer Tomography (CT) for
enhanced spatial resolution. In allows diagnosis and monitoring
of disease progression, and is most frequently used for imaging
of tumors, brain and cardiac diseases. PET delivers molecular
and functional information according to the applied tracer and its
biological target. Most PET tracers have been developed for
imaging targets in the brain,[1] or for diagnosing tumors and
monitoring therapy in cancer patients.[2] They provide valuable
information on target expression in health and disease, or about
target engagement by drug candidates, facilitating the selection
of optimized therapeutic doses. In contrast, there are very few
tracers available for receptor occupancy studies in peripheral
tissues.[3]
In oncology, the most frequently used tracer are [18F]-
fluorodeoxyglucose
([18F]FDG)
and
[18F]-fluorothymidine
([18F]FLT), which provide valuable information on tumor
metabolism. While largely accepted as biomarkers of tumor
activity, both [18F]FDG and [18F]FLT have limitations, including
the fact that they do not differentiate well between cancer and
inflammation.[4] Novel T cell-specific imaging agents that could
be used in peripheral organs would effectively support diagnosis
and evaluation of drug candidates in immuno-oncology.[5] They
would also greatly facilitate the therapeutic development of cell-
based therapies such as chimeric antigen receptor (CAR) T
cells[6]
Introduction
Improved tools for imaging T cells and quantifying their organ
distribution would be highly useful for diagnosis and monitoring
disease progression in cancer and immune diseases. They
would open the door to more effective clinical stratification
methods, and allow monitoring of the therapeutic efficacy of
drugs acting on pathways associated with T cell activity. They
would facilitate the selection of clinically effective doses, and
inform on changes in the distribution of T cells in organs that are
not readily accessible for sampling.
In immunology, there are few tracers that can be used to monitor
disease, or to follow immune cell populations in patients. Tracers
allowing the detection of activated microglia by targeting the
translocator protein (TSPO)[7] are an exception. The prototypical
TSPO tracer is [11C]-PK11195.[8] While suffering from a relatively
low signal-to-noise ratio, it is nevertheless used for quantifying
inflammatory responses in various organs.[9] Second-generation
tracers such as [11C]-PBR28,[10] while displaying improved
properties, are sensitive to a single TSPO polymorphism
(rs6971) that strongly degrades their performance in a portion of
human patients.[11] Recently, novel tracers binding to the
purinergic receptor subtype 7 (P2X7R), have shown promise as
tools for imaging inflammation.[12] P2X7R are expressed on
cellular membranes of myeloid-derived cells and have also been
implicated in mediating microglial proliferation at the site of injury.
T cells are a subtype of lymphocytes and play a central role in
cell-mediated immunity. They are divided in several classes
including helper, killer, memory and regulatory
depending on their specific function in the immune system.
Besides their protective role, T cells are implicated in a diverse
set of illnesses, including autoimmune, inflammatory and allergic
diseases. The options for monitoring T cells in vivo, beyond
measuring their concentration in blood samples, are currently
limited. In clinical practice, T cell activity is mostly determined
from blood samples or biopsies.
T cells,
Positron Emission Tomography (PET) is a non-invasive imaging
technique, often used in combination with Magnetic Resonance
1
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There are no low-molecular weight (lmw) tracers for T cells, but
monoclonal antibodies directed against CD4- or CD8-positive T
cells have been shown to allow imaging of lymph nodes and
spleen with high specificity in mice.[13] In the clinic, one study
with radiolabeled anti-CD4 antibodies demonstrated positive
results in rheumatoid arthritis.[14] The use of antibody-based
imaging agents is however limited by the comparatively high
radioactive load associated with the long-lived radioisotopes
required for their labeling (e.g. 89Zr), by the slow kinetics of their
organ distribution, and by their inability to penetrate the blood-
brain barrier.[13,15] A broad and rapid distribution of the imaging
agent in all organs would indeed be important for imaging T cells
in the brain and spinal cord (in multiple sclerosis), in salivary
glands (for Sjögren’s disease), in muscles (for myositis),
pancreas (in diabetes), in the intestine (coeliac disease), in the
lung (sarcoidosis) or in the thyroid (autoimmune thyroiditis).
signal generated by radio-metabolites produced in vivo, and
which can potentially interfere with the signal related to the
parent tracer. Finally, we considered options to introduce a PET
radioisotope on the tracer candidate, at a late stage of its
chemical synthesis, as an essential factor for the feasibility of
the approach.
The conclusion of this selection process showed that T cell
receptor subunits, co-stimulatory receptors and components of
the associated signaling pathways, as well as the corresponding
transcription factors constitute the most T cell specific targets.
There was however no suitable starting point available for the
optimization of lmw ligands for these targets.
In addition, a few function-related proteins were identified, and
these were mostly associated to cytotoxic T cells. Proteins like
protease Granzyme K, which are involved in the T cell response,
might reach high expression levels during inflammation and
therefore be good target candidates for imaging. We however
did not identify selective chemical starting points for the
optimization of PET tracers for these targets either.
Our aim was to develop a low molecular weight PET tracer for
imaging T cells in peripheral organs, and potentially also in the
brain. The first task was to identify candidate cellular targets for
developing selective imaging agents, and the corresponding
chemical starting points for PET tracer optimization.
Our focus therefore shifted towards T cell specific intracellular
signaling pathway components, of which receptor proximal
kinases proved to be the most cell type specific. Intracellular
targets come with a requirement for cell penetration, which is a
challenge for antibodies and other biological agents. In contrast,
lmw tracer candidates generally penetrate cells rapidly, allowing
the evaluation of targets otherwise not exploitable for imaging.
Naturally, these signaling nodes also represent promising
pharmacological targets for immunological indications, which
allowed us to mine the target profiles of a variety of published
and proprietary inhibitors from very diverse chemical series.
Target selection process
The first step for the selection of suitable targets was the
screening of several RNA expression databases for target
proteins fulfilling the following criteria: differential expression in T
cell derived cell lines vs. other immunocytes, selective
expression in T cells isolated from blood[16] as well as a
pronounced expression in T cell rich organs[17] like spleen,
thymus and lymph nodes. For a limited number of targets, mass
spectroscopy-based protein expression data has also been
published,[18] providing an additional source of data for selection.
Imaging probes for targets expressed across all T cell classes
have the potential for being useful in a variety of clinical settings,
addressing both the regulatory role as well as the effector
function of T cells in immunological disorders. We therefore did
not include criteria that would restrict our selection to targets
providing T cell subtype-specificity, i.e. leading to lmw tracers
capable of discerning between different cellular
T
cell
subpopulations. In contrast, we focused on targets described to
have similar mRNA expression levels across the major T cell
subpopulations.
In the second stage of target selection, we focused on the
availability of potent and specific lmw lead molecules, either
from our proprietary compound collection or from the literature.
Information on the target structure-activity-relationship (SAR)
and selectivity profile of the chemical series, when available,
greatly facilitated prioritization.
Scheme 1. Simplified T-cell signalling pathways with the selected radioligand
targets highlighted: Lck and ZAP70 are directly associated with the T-cell
receptor complex, Itk is linked to the signalosome and the downstream signal
transducer PKC θ.
From
a
medicinal chemistry point-of-view, we carefully
considered information on the potential to modulate physico-
chemical properties, non-specific binding, metabolism, and the
pharmacokinetic profile of the chemical leads. The
characteristics of successful PET tracers have been extensively
discussed in the literature,[19, 20] with an emphasis on tracers for
the central nervous system (CNS). Tracers for peripheral
applications face the additional challenge resulting from the
In the majority of the kinase families of interest, only one of the
isoforms has the necessary T cell specificity. The fact that these
isoforms display a high degree of sequence similarity, especially
in the active sites, poses a formidable challenge to the
development of selective PET tracer candidates. For example,
amongst the closely related members of the Tec-kinase family
Txk, Btk, Bmx and Tec show also high mRNA levels in B cells,
2
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10.1002/cmdc.201800241
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FULL PAPER
NH2
N
I
mast cells and the monocytic lineage, leaving only Itk as a truly
T-cell specific target. We thus selected kinases from the T cell
receptor signaling cascade and co-stimulatory pathways,
including Zap70, PKC θ, Lck and Itk (Scheme 1, Table 1) for
further evaluation of available chemical starting points.
NH2
I
CO2Me
N
N
N
O
N
N
c
a
N
b,
N
N
N
O
N
O
6
7
N
O
5
OMs
Boc
O
CO2Me
NH2
O
O
N
Table 1. Expression profile of Itk, Lck, PKC θ and ZAP70 in the immune
compartment.
O
B
O
e
d,
+
NH
NH
O
N
N
O
N
O
N
Itk[a]
+++
Lck[b]
+++
PKC θ [c]
ZAP70[d]
+++
N
H
9
Tissue
8
+++
CD4+ T cells
CD8+ T cells
NK cells
Scheme 3. Synthesis of LCK inhibitor 9. Reagents and conditions: a) 3-iodo-
1H-pyrazolo[3,4-d]pyridine-4-amine, K2CO3, DMF, 90°C, 16 h, quant.; b) 4M
HCl, 100°C, 16 h, quant.; c) Boc anhydride, Et3N, CH2Cl2, RT, 16 h, 38%; d) 8,
Pd(dppf)Cl2, K2CO3, DMSO, 95°C, 1.2 h, quant.; e) HCl 4M in dioxane, RT, 18
h, 24%.
+++
+++
+/-
-
+++
+++
++
+
+
-
+
-
DCs
-
+/-
-
-
B cells
3. ITK inhibitors
Scheme 4 shows the assembly of the tetrahydroindazole Itk
inhibitors (±)14, 15a (eutomer) and 15b (distomer). Intermediate
11 was prepared by reducing phenyl(piperidin-4-yl)methanone
10 to a racemic carbinol, conversion to a benzylic chloride and
alkylation of 4-nitropyrazole, followed by reduction of the nitro
group with Zn/NH4Cl. Subsequent coupling with the carboxylic
acid 12 provided 13. Deprotection of the piperidine, optional
alkylation with fluoroiodoethane and deprotection of the indazole
provided the desired products. The enantiomers of 15 were
separated by chiral chromatography.
Non-immune tissues
-
retina
retina, testis,
muscle
liver
[a] Tyrosine-protein kinase Itk/Tsk, also known as interleukin-2-inducible T cell
kinase; [b] lymphocyte-specific protein tyrosine kinase; [c] protein kinase C,
isozyme θ; [d] zeta-chain-associated protein kinase 70.
Results and Discussion
O
O
Chemistry
N
HO
N
e
f
a -
+
NH2
N
1. PKC θ inhibitors
N
N
10
11
12
N
THP
.HCl
N
H
The synthetic route toward compound 4 is shown in Scheme 2.
Catalytic hydrogenation of 2,2,2-trifluoro-1-(pyrazin-2-yl)ethan-1-
one (1) over platinum oxide led to 1,1,1-trifluoro-2-(piperazin-2-
yl)propan-2-ol (2), which under basic conditions reacted with
substituted butyldimethylsilyl-pyridine 3[21] to afford the target
product.
Cbz
O
O
N
N
14)
i
g,
N
(for
N
H
N
H
N
N
N
15a,b
NH
g-j
(for
)
THP
=
=
13
R
R
H:
N
N
R
(±)14
CH2CH2F: 15a, 15b
Cbz
H
H
N
N
N
N
Scheme 4. Synthesis of Itk inhibitors (±)14, 15a, 15b. Reagents and
conditions: a) Cbz-Cl, K2CO3, THF, 0-25°C, 15 h; b) NaBH4, MeOH, 0-25°C,
15 h; c) SOCl2, DCM, RT, 15 h; d) 4-nitropyrazole, K2CO3,KI, DMF, 100°C, 15
h; e) Zn, NH4Cl, THF/water f) HATU, DIPEA, DMF, 50°C, 4 d; g) Pd/C, H2,
N
N
H
N
F
F
N
N
c
b
a,
+
N
N
O
HO
F
N
H
HO
Si
N
CF3
CF3
F
MeOH, RT,
4 h; h) 1-fluoro-2-iodoethane, K2CO3, DMF, RT, 15 h; i)
F
HCl/dioxane, RT, 15 h; j) chiral prep HPLC (CHIRALPAK® IA, eluent: n-
hexane, 0.1% DEA in EtOH).
F3C
HN
1
2
3
4
Scheme 2. Synthesis of PKC θ inhibitor 4. Reagents and conditions: a)
(CH3)3SiCF3, K2CO3, DMF, RT, 12 h, 60%; b) PtO2, MeOH, H2, 4 bar, RT, 24 h,
quant.; c) Hunig’s base, NMP, 150°C, MW, 2 h, 30%.
The difluorocyclopropyl-constrained tetrahydroindazole 18
(Scheme 5) was prepared analogously, via the THP protected
racemic acid 17[24]. The four stereoisomers were separated by
chiral HPLC; the biochemical potency of the most potent
stereoisomer (exact configuration not determined) is reported.
2. LCK inhibitors
Scheme 3 shows the preparation of LCK inhibitor 9 from the
previously known mesylate 5.[22] Treatment with commercially
available 3-iodo-1H-pyrazolo[3,4-d]pyridine-4-amine in presence
of potassium carbonate led to the formation of compound 6.
Deprotection and reprotection with the more labile Boc group
provides intermediate 7, which reacted with boronate 8[23] to
produce the desired product.
Racemic 19[25] and 20[24] were synthesized according to literature,
and obtained optically pure after chiral separation as described
in the experimental part (Scheme 6). Amide coupling led to
thioether 21, which was then oxidized to the sulfoxide 22 using t-
BuOOH in decane.
3
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FULL PAPER
O
S
HO
+
N
F
N
H
b
a,
O
F
F
Br
N
F
O
N
N
N
H
NH2
N
N
a
N
b
F
F
H
THP
Br
21
N
N
N
N
11
16
HN
17
N
N
Cbz
H
THP
O
O
24
F
N
O
F
O
O
N
e
c,
d,
S
S
O
O
N
H
O
H
3H
N
H
Br
N
N
N
N
N
H
H
H
18
c
F
F
F
N
F
N
3H
N
N
N
Br
F
N
N
H
H
Scheme 5. Synthesis of Itk inhibitor 18. Reagents and conditions: a) BOP,
DIPEA, DMF, RT, 48 h; b) Pd-C, H2, EtOH, RT, 6 h; c) 1-fluoro-2-iodoethan,
K2CO3, DMF, 80°C, 48 h; d) HCl/dioxane, RT, 15 h. e) chiral prep HPLC.
3H]223
O
[
O
25
Scheme 7. Synthesis of [3H2]23. Reagents and conditions: a) Br2, I2, DCM, 0-
25°C, 16 h; b) mCPBA, DCM, 0°C, 1h; c) 3H2, Pd/C, DIEA, DMF, RT.
The stereoisomers resulting from the formation of the sulfoxide
could be separated by chiral HPLC, and the biological data is
given for the more active diastereoisomer. All four stereoisomers
of 23 were obtained after coupling of racemic 19 and 20 and
oxidation of the thioether with mCPBA. They were separated by
chiral chromatography. The isomer with the highest potency in
the biochemical assay was selected for in vivo characterization.
Lead selection and optimization
This section discusses the optimization of the leads selected
after review of all structures available in the literature. After
thorough characterization and identification of weaknesses as
PET tracer candidates, those were primarily optimized to
decrease non-specific binding, while aiming for optimal
selectivity and pharmacokinetic properties.
S
S
H
N
O
O
NH2 HO
+
H
H
a
b
N
N
F
F
F
F
N
N
c
N
N
1. Lead selection for ZAP70
N
N
H
H
19
20
21
The zeta-chain-associated protein kinase 70kDa (ZAP-70) is
directly associated with the T cell receptor and is a central
regulator of T cell activation, it has been an attractive medicinal
chemistry target in immunology for many years.[26] Despite
numerous efforts, no compounds has yet entered advanced
clinical stages, mainly due to selectivity issues. Initial attempts to
optimize the most promising inhibitors rapidly confirmed the
challenge of improving their off-target profile. We did not identify
suitable leads for developing T cell specific ligands, and did not
pursue this target further.
O
O
O
S
O
S
O
H
N
H
N
O
O
H
H
N
N
F
F
F
F
N
N
N
N
N
N
H
H
O
O
22
23
Scheme 6. Synthesis of Itk inhibitors 22 and 23. Reagents and conditions: a)
BOP, DMF, 50°C, 4 h; b) t-BuOOH 6M in decane, RT, 2 h; c) mCPBA, DCM,
0°C, 1 h.
2. Ligands for PKC θ
For the development of a PKC θ ligand as a T cell tracer, a
review of the available chemical matters showed that compound
26[21] (Figure 1) was the most selective inhibitor, and we focused
on its optimization. This inhibitor has high affinity, reasonable
selectivity but a high chromatography hydrophobicity index on
immobilized artificial membranes[19,27] (CHI(IAM)), suggesting
high non-specific binding (Table 2). Initial structure activity
relationship indicated that the fluorine was required to provide
the right torsion angle, and that the chlorine could be substituted
by fluorine without loss of affinity. To reduce non-specific binding,
the (R)-3-methyl-2-butyl-2-ol side-chain was converted into a
1,1,1-trifluoro-2-propyl-2-ol (4), decreasing the pKb value of the
piperazine ring from 8.5 to 6.6 and the CHI(IAM) value from 61
to 39, with only a slight decrease in affinity for PKC θ. Selectivity
versus LATS1, a serine/threonine-protein kinase encoded by the
LATS1 gene in humans, however decreased significantly and
could not be recovered by further derivatization, leading to
discontinuation of this series.
For the preparation of radiolabeled [3H2]-23 (Scheme 7), two
bromine atoms were introduced on the benzene and pyrazole
rings of 21 to provide 24. X-ray analysis of crystalline 24 allowed
assigning its absolute and relative configuration, by comparison
with the published co-crystal structure of a related inhibitor
bound to the kinase domain of Itk.[24] After oxidation with mCPBA,
hydrogenation of the dibromosulfone 25 under standard
conditions could be accomplished without epimerization at the
benzylic position, and allowed unambiguous assignment of the
chiral centers in all related products. The radiosynthesis of [3H2]-
23 was performed under equivalent conditions, using tritium gas.
4
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FULL PAPER
H
N
N
N
NH2
OMe
NH
NH
N
N
F
H
N
N
N
N
N
S
N
N
N
O
NH
H
N
HO
N
Cl
N
O
N
28
HN
O
26
27
H
N
N
O
N
Figure 1. Structures of PKC θ inhibitor 26 and LCK inhibitor 27 selected as
leads for optimization.
S
O
N
N
N
N
NH
N
O
H
N
N
N
H
29
30
Table 2. Compound profiling
NH2
O
target
Cpd
IC50
Selectivity
CHI(IAM)
LSE [c]
MDCK[d]
[b]
[nM][a]
Figure 2. Structures of covalent (28) and reversible (29, 30) Itk inhibitors
selected as leads for evaluation.
26
4
0.1
0.45
0.2
1
30x LATS1
61
39
51
37
5.6
5.9
5.7
5.7
3.9/2.0
n.t.
PKC θ
5x
2x
2x
LATS1
Hck
All three compounds were analyzed for potency, inhibition of key
off-target kinases and potential for non-specific binding.
27
9
n.t.
Lck
Lyn
2.5/1.1
The covalent inhibitor 28 has a high affinity for Itk (Table 3), but
we observed that it binds with similar potencies to the Cys-
containing Tec family kinases (Itk, Tec, Bmx, Btk, Txk). Tec
family members are widely expressed in the hematopoietic
compartment, therefore strongly limiting the potential T cell
specificity of 28. We therefore deprioritized this compound for
PET tracer optimization.
[a] IC50 measured by fluorescence using a mobility shift assay with a LC3000
Caliper Life Sciences system[34]; [b] Selectivity with regard to the second
highest affinity target in panels of respectively 111, 24, 164 and 40
pharmacologically-relevant targets; [c] Ligand Specific Efficiency index[19], LSE
= IC50/log(CHI(IAM)); [d] Permeability as Pappx10-6 cm s-1 and efflux ratio in a
Madin Darby Canine Kidney (MDCK) cell line transfected with the human
multidrug resistance (MDR1) gene.
Table 3. Profile of selected Itk inhibitors.
3. Ligands for Lck
As an additional option, we selected the Lck inhibitor A-770041
(27),[29] as starting point for tracer development. Compound 27
displays high affinity for Lck and reasonable selectivity. Despite
an encouraging Ligand Specific Efficiency (LSE) index[19] of 5.7,
its CHI(IAM) value of 51 is relatively high, and its clearance too
Cpd
IC50
[nM]
Selectivity
(fold)[a]
CHI(IAM)
LSE
% HSA
MDR1-
[b]
binding[c]
MDCK[d]
8x IRAK1
3x MAP4K3
15
10
43
46
4.9
4.8
95.4
94.9
1.1/8.8
0.7/6.1
20x IRAK1
38x MAP4K3
18
22
23
28
1.5
slow for imaging (t = 3.2 h in Balb/c mouse). To address these
½
issues, we explored the replacement of the acetylpiperazinyl-
cyclohexyl moiety, and prepared a series of compounds inspired
by the work published by Calderwood et al.[30] Ultimately,
replacement by an isoxazolidine group (9) retained potency and
improved non-specific binding significantly. Unfortunately, the
target selectivity profile of 9, while different from 27, is not
superior to the lead molecule (Table 2). The physicochemical
properties of 9 also remain far from the range desirable for PET
tracers, with a total polar surface area (tPSA) of 129 Å2 and low
permeability. We therefore concluded that this scaffold did not
warrant further investigation and decided to focus on the last
target, Itk.
0.8
0.3
0.8
0.5
31
22x IRAK1
20x IRAK1
30
32
49
29
59
6.2
6.4
5.4
6.4
4.2
85.7
89.2
88.2
93.6
93.2
0.9/11
0.5/6.5
1.5/14
1.0/2.8
1.0/11
1.5x Txk, Bmx
400x IRAK4
4x IRAK1
0.5x Flt3
29
30
12x IRAK1
[a] Selectivity with regard to the second highest affinity target in a panel of 24
related kinases; [b] Ligand Specific Efficiency index, LSE
pIC50/log(CHI(IAM)); [c] binding to Human Serum Albumin binding; [d]
Permeability as Pappx10-6 cm s-1 and efflux ratio in a Madin Darby Canine
Kidney (MDCK) cell line transfected with the human multidrug resistance
(MDR1) gene.
=
4. Ligands for Itk
Among the four targets we selected initially, Itk displays the most
pronounced T cell specific expression pattern. Several highly
potent inhibitors with diverse chemical structures have been
published, and to identify the best starting point for tracer
optimization, we evaluated both the covalent (28)[31] and
reversible inhibitors (29, 30)[28,32] illustrated in Figure 2.
In contrast, reversible inhibitors proved selective with regard to
Tec family members. While aminomethyl-benzimidazole 29 was
remarkable for its biochemical potency, it demonstrated very
little selectivity towards IRAK1/4 and Flt3. We thus focused on
tetrahydroindazole 30, which despite a lower affinity for Itk
offered better kinome selectivity, and a better overall radioligand
5
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10.1002/cmdc.201800241
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property profile. In particular, we were encouraged by its lower
MW and PSA, leading to a ligand efficiency[33] (LE = 0.33)
comparable to the more potent, but heavier 29 (LE = 0.34).
with an IC50 of 1.5 nM. Compound 18 has an almost unchanged
LSE index despite added hydrophobicity.
We then replaced the fluoroethyl-piperidine side-chain by a
tetrahydropyrane, compensating the loss of polarity by placing a
methylsulfoxide on the benzene ring, which adds a strong dipole
The most critical property to be improved in compound 30 is its
very high non-specific binding to cell membranes, illustrated by a
CHI(IAM) value of 59. We hypothesized that this was largely
influenced by its basic amine side-chain. Down-modulating the
pKa of the tertiary amine by introducing a fluoroethyl substituent
(15, Scheme 8) led to a substantial reduction of the CHI(IAM)
value to 43, together with a 3-fold improvement in potency.
Conveniently, the fluoroethyl side-chain represents an easy
point for radiolabeling with 18F, late in the synthesis of the tracer.
in
the
hydrophobic
region,
while
maintaining
the
difluorocyclopropyl group successfully introduced in compound
18. This led to compound 22. Indeed, while these modifications
retained potency and selectivity, they also decreased non-
specific binding dramatically, with a CHI(IAM) value dropping
from 46 to 30. In vivo, this translated into a much more favorable
tissue distribution in spleen, thymus and muscle, with a
blood/organ concentration ratio now close to unity and a
distribution volume reduced to 1.1 L/kg. The observed
elimination half-life of 0.5 h is within an acceptable range for 18F
PET ligands.
N
O
N
N
30
H
N
NH
N
Interestingly, the in vitro metabolite profile of 22 in rat liver
microsomes (data not shown) indicated rapid oxidation to the
corresponding sulfone 23. This led us to re-analyze the samples
of the pharmacokinetic study conducted with 23, confirming that
this transformation was also taking place in vivo, and showing
that 23 actually constitutes a major circulating metabolite one
hour after injection of 22 (Figure 3).
15a,b
F
R
F
F
N
N
F
F
O
O
N
N
N
H
N
H
N
N
NH
N
H
O
N
18
=
=
22
R
R
MeSO:
MeSO2
:
23
F
Scheme 8. Itk radioligand optimization of the tetrahydroindazole series
To better understand the potential of 15 as an imaging tracer,
we investigated its pharmacokinetic parameters and tissue
distribution (Table 4). The study was performed with
a
pharmacological i.v. dose of 1 mg/kg in rats. Unfortunately,
compound 15 showed an unfavorable tissue distribution profile,
suggesting high non-specific binding: Ten minutes after dosing,
15 had already strongly accumulated in the spleen, which is a
critical target organ for the assessment of T cell imaging agents.
This finding prompted us to attempt a more radical change in
molecular properties, and we removed the basic group from the
molecule, a known liability for unspecific binding.
Figure 3. Pharmacokinetic profile of sulfoxide 22 and of its sulfone metabolite
23 after i.v. dosing in rats.
Luckily, compound 23 turned out to be 3-fold more potent than
its parent 22, more than compensating for a slight increase in
non-specific binding. Compound 23 has the best LSE value in
the series (6.4). As the pharmacokinetic profile and tissue
distribution of 22 and 23 proved similar, we selected 23 for
further validation of Itk as a target for T cell imaging.
Table 4. PK parameters and tissue distribution
Cpd
Cl[a]
Vss[b]
t
½
[h] [c]
Tissue/blood ratio after 10
min and 1 h
In vivo profiling
15
22
60
33
4.5
1.1
1.1
0.5
spleen
4.8 10.1
While the preliminary selection of Itk as a target for T cell
imaging was primarily based on published RNA expression data
on relevant cell types and tissue distribution, the optimization of
potency and selectivity of the ligand itself relied on biochemical
kinase inhibition assays. These assays are individually tuned for
robustness and sensitivity, allowing an efficient profiling process.
They however do not allow a precise comparison of affinities
across kinases, and poorly predict the potential effect of binding
to off-target kinases in imaging applications. The influence of off-
target affinities is highly dependent on expression level and
kinase activation states in target tissues. Therefore, while the in
vitro selectivity profile of the Itk-inhibitors was a valuable guide in
the ligand optimization process, a meaningful assessment of T
spleen
thymus
muscle
spleen
thymus
muscle
0.4
0.6
0.8
0.5
0.5
0.8
-
1.1
1.1
0.4
0.9
0.6
23
34
3.5
1.3
[a] Cl: clearance, [mL.min-1.kg-1]; [b] Vss: distribution volume at steady state,
[L.kg-1]; [c] terminal half-life.
It had been previously demonstrated that a further improvement
in potency and selectivity can be achieved with the fusion of a
difluorocyclopropyl group to the tetrahydroindazole moiety.[24]
This was applied to 15, leading to a more potent molecule (18)
6
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cell specificity and sensitivity can only be achieved using in vitro
tissue binding studies with radiolabeled compounds.
of the spleen, which is consistent with labeling of the T cell rich
regions in the white pulp of this organ. The radioactive signal
also decreases in the lymph nodes after T cell depletion. Overall,
Overall, compound 23 had the desired profile for a proof of
concept study using tritium labeling. To achieve high specific
activity, two bromine atoms were introduced at the phenyl and
the pyrazole ring of 21 (Scheme 7, compound 24), allowing the
introduction of two tritium atoms by reductive displacement. The
resulting [3H]2-23 had sufficient specific activity (27.0 Ci/mmol) to
assess the feasibility of imaging T cells in vitro and in vivo.
a
broad and fast tissue distribution of [3H2]-23 related
radioactivity was observed one hour post dose. Measurable
concentrations were detected in 53 out of 55 tissues and were
generally low, reflecting a high systemic clearance rate. Table 5
lists the tissue concentration of total radiolabeled components in
representative tissues, including spleen and lymph nodes.
Our first objective was to demonstrate that [3H]2-23 leads to a
displaceable, selective in vitro radiolabeling of T cell rich tissues
of human and murine origin. Indeed, autoradiography of sections
of human pharyngeal tonsils and mouse inguinal lymph nodes,
incubated with 10 nM [3H]2-23, showed a strong labeling of the
lymphatic tissue from both species. The autoradiographic
resolution achieved with human tonsils allows the assignment of
specific labeling to the lymphatic tissue, while sparing the
tonsillar epithelium and crypts (Figure 4b). Specificity of labeling
could be demonstrated by blocking the signal with an excess of
cold 23, as well as with the structurally unrelated Itk-inhibitor 28.
In slices where [3H]2-23 binding is blocked with unlabeled ltk
inhibitors (Figure 4c,d) the residual radioactivity is uniformly
distributed indicating a low level of unspecific binding.
Figure 4. In vitro autoradiography of human pharyngeal tonsils (serial sections
a-d) and mouse inguinal lymph nodes (sections e-h) showing specific binding
of radiolabeled Itk inhibitor [3H2]-23 to T cell rich tissue in both species; (a, e)
hematoxylin-eosin stain; (b, f) incubation with 10 nM [3H2]-23; (c, d, g, h)
incubation with 10 nM [3H2]-23 in the presence of 10 µM unlabelled 23; (c, g) or
in the presence of 10 µM of a structurally unrelated Itk inhibitor (d, h), 28.
Figure 5. Autoradiographs (3H) and visual scans (vis) of the spleen region in
sagittal whole body sections from mice (1 h after a single intravenous nominal
25 μg/kg [3H2]-23 dose. Slices used for autoradiography are 40 µM thick,
dehydrated, and the darkest areas in the autoradiographs correspond to the
highest concentration of radioactivity. a) naïve mouse (isotype control mAb
pre-treated), upper panel: abdominal region with punctuated pattern of
radioactivity in the white pulp of the spleen consistent with labeling of the T cell
rich areas, lower panel: head and neck region with increased radioactivity in
the thymus (T), submandibular lymph nodes (LN) and the harderian gland
(HG); b) partially T cell depleted mouse (anti CD4 mAb pre-treated): reduced
signal in the T cell areas of the spleen and the submandibular lymph nodes,
no change in the harderian gland, thymus and background signal.
A key parameter for in vivo PET tracer imaging is rapid tissue
distribution combined with rapid elimination of the radioactive
probe, matching the half-live of the radioisotope, without the
formation of interfering radio-metabolites. To prove the suitability
of the tetrahydro-indazole class of Itk-inhibitors as a lead for
further [18F] PET tracer optimization, we evaluated the ability of
[3H2]-23 to label T cells in an in vivo experiment, one hour after
i.v. dosing in male C57/BL6 mice. The T cell specificity of the
signal was confirmed by comparing labeling intensity in T cell
rich organs in naïve and partially T cell depleted (anti-CD4
treated) animals, using quantitative whole body autoradiography
(QWBA, Figure 5). Special focus was put on the signal
distribution in lymphatic organs such as thymus, spleen and
lymph nodes. Visual inspection of the autoradiographic images
Levels above 10 pmol/g in isotype control pre-treated mice were
only observed in the liver (164 pmol/g), and other excretory or
secretory organs like kidney (11.6 pmol/g) and various glands
(e.g. harderian gland 10.3 pmol/g), which is a common finding
for many low molecular weight radioligands. Labeling of
lymphoid tissue (thymus, white pulp of spleen, submandibular
lymph nodes) was in the range 4.47 to 7.03 pmol/g.
does not show
a strong contrast between tissues, and
interpretation is facilitated by the quantification of the
radioactivity signal (Table 5). Nevertheless, a clear punctuated
pattern of radioactivity can be observed in the sagittal sections
7
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ChemMedChem
FULL PAPER
[3H2]-23 and potential metabolites, the observed decrease in
signal in the immune compartments indicates that this tracer
indeed detects the reduction in T cell numbers in partially T cell
depleted mice. There was almost no signal change in the
thymus (+5%), paralleling observations made with CD4 and CD8
targeting agents, which also did not show T cell reduction in the
thymus after anti-CD4 mAb treatment.[13] This is likely due to a
non-saturating dose of GK1.5 antibody, combined with a partial
blood-thymus barrier.
Table 5. Tissue concentration of total radiolabelled components [pmol/g] in
main organs, 1 h after an i.v. dose of 25.0 µg/kg [3H]23 to male control and T
cell depleted mice.
Tissue
Control[a]
T cell depleted[b]
Brain
0.181
1.12
2.70
3.42
7.03
2.50
4.97
2.58
7.63
6.97
0.131
1.37
2.85
3.56
4.14
2.63
5.06
2.37
5.21
7.03
Fat (white)
Heart
Lung
Conclusions
Lymph nodes (submandibular)
Muscle
In this study we have evaluated four potential target proteins for
a pan T cell specific PET tracer. Combining the mRNA
expression data of the potential targets with the selectivity profile
and physico-chemical properties of available ligands led to the
identification of the T cell specific kinase Itk as the most
promising candidate. Itk inhibitors of the tetrahydro-indazole
class could be optimized to sub-nanomolar binding affinities with
favorable parameters for binding specificity and pharmacokinetic
parameters. Specificity of the probe was shown by in vitro
autoradiography of human tonsils and mouse lymph nodes using
tritiated [3H2]-23, confirming selective and displaceable labelling
in lymphatic tissue. Finally, in vivo studies and QWBA in control
and partially T cell-depleted mice demonstrated a reduction of
radioactive signal in the T cell rich regions of the spleen and in
lymph nodes, one hour after dosing of [3H2]-23. This matches
the values of T cell depletion determined by flow cytometry.
Pancreas
Spleen (red pulp)
Spleen (T cell rich region, white pulp)
Thymus
[a] Control animals pre-treated with isotype control antibody. [b] Animals
treated with T cell depleting antibody.
The extent of
T cell depletion in lymphoid tissues was
independently quantified in an identically depleted satellite group
by FACS counting of T cells in spleen and lymph nodes (Figure
6).
While being a useful tool for target validation, 23 does not yet
have the optimal profile required for
a PET tracer. Its
pharmacokinetic half-life is too long for [18F] PET imaging. Using
current methodology, the difluoro-cyclopropyl group already
present in the molecule is not suited for introduction of [18F] with
sufficient specific activity, due to isotopic dilution. Other features
which still need improvement are its low permeation and Pgp-
mediated efflux, which would be prohibitive for imaging T cells in
the CNS compartment.
This work nevertheless demonstrates that imaging T cells in vivo
is feasible and that Itk-selective radiotracers have the potential
to be used for PET imaging in the clinic.
Figure 6. a) Tissue distribution of [3H2]-23 related radioactivity (blood
normalized) at 1 h post-dose in QWBA experiment in a naïve and a partially T
cell depleted mouse (single experiment: no error bars). b) T cell depletion
Experimental Section
measured by FACS analysis of
T
cell content (% CD3+/CD19ˉ of total
lymphocytes) in inguinal lymph node and spleen (satellite groups, n=6,
combined data from 26 h and 30 h after mAb injection, control group treated
with an isotype matched control mAb).
Biology
Autoradiography
Human tonsils were obtained from two patients following tonsillectomy.
The study was conducted in accordance with the ethical principles
originating in the Declaration of Helsinki and approved by the local Ethics
Committee (study PJMR1000244). All subjects provided written informed
consent before participating in the study. Eight week old male C57BL/6
mice were commercially purchased (Harlan, Itingen, Switzerland) and
and inguinal lymph nodes were removed following termination. The study
was conducted in accordance with Swiss federal law for animal
protection and approved by the Veterinary Office of the Canton Basel-
Stadt.
Comparing blood-normalized tissue concentrations of total
radiolabeled components in lymphatic tissue of control mice with
those of partially T cell depleted mice, a signal reduction of 38%
and 29% was observed in submandibular lymph nodes and in
the T cell rich regions (white pulp) of the spleen, respectively
(Figures 5 and 6a). This result matches the T cell depletion
values of 39% and 43% obtained by FACS analysis of lymph
nodes and spleen in identically treated satellite animals (Figure
6b). In contrast, only 4% decrease of radioactive signal was
observed in the T cell poor region of the spleen (red pulp).
Although measured radioactivity does not differentiate between
Frozen sections were cut 20 um thick with a microtome-cryostat and
thaw-mounted on poly-L-lysine coated microscope slides and stored at
8
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−80°C. Before incubation with labelled ligand, the sections were dried
(eBioscience, USA). Cells were washed in fluorescence-activated cell
sorting FACS buffer (PBS containing 2% bovine serum albumin BSA,
5mM ethylenediaminetetraacetic acid), blocked with anti-mouse
CD16/CD32 antibody (Biolegend, USA). Lymphocytes were stained with
CD3 (clone 145-2C11), CD4 (clone RM4-5), CD19 (clone 6D5) and CD8
(clone 53-6.7) fluorochrome-conjugated antibodies 30 min at 4°C.
Washed and resuspended cells were acquired on a Fortessa flow
cytometer (Becton Dickinson, USA). Data were analysed using the
FlowJo software (Flow Jo LLC, USA).
under
a stream of cold air. Target binding autoradiography was
performed according to the following procedure: after 20 min pre-
incubation at RT in Krebs Tris buffer, the sections were incubated for 2 h
at RT in the same buffer (20 mM Tris pH 7.4, 118 mM NaCl, 5.6 mM
glucose. 1.2 mM KH2PO4, 4.78 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4)
supplemented with 10 nM [3H2]-23 (27 Ci/mmol) with and without the
competitor. After incubation, the slides were briefly dipped in ice cold
distilled water followed by two 10 min washes in ice-cold buffer and brief
dipping in ice-cold distilled water to remove salts. The sections were
dried under a stream of cold air. Autoradiograms were generated by
exposing the labelled tissue to a Tritium Phosphor screen medium
(7001489 TR) at RT for 24 h. Data from binding were analysed with the
OptiQuant software of the phosphor-imager cyclone plus.
Chemistry
General methods: 1H NMR spectra were acquired on a Varian 400 MHz.
Chemical shifts (δ) values are given in parts per million (ppm) relative to
the residual solvent peak. Analytical LCMS conditions (%: percent per
volume), column: Phenomenex Synergi® MAX-RP, 4x20 mm, 2.5 μm,
40°C, solvent A: water + 0.1% HCO2H, solvent B: CH3CN, gradient: 5%
B for 0.5 min + from 5% to 95% B over 0.5 min + 95% B for 0.5 min +
from 95% to 5% over 0.5 min + 5% B for 1 min; Flow : 2.0 mL/min
Animal pre-treatment: Eight week old male C57BL/6 mice (Harlan,
Itingen, Switzerland) were randomized to isotype control (rat IgG2b,
BioLegend, USA) or anti-CD4 depleting antibody (clone GK1.5,
BioLegend, USA) treatment groups. Animals received a single 1mg
intravenous injection to induce partial T-cell depletion. Satellite groups
subsequently continued into parallel QWBA or flow cytometry analysis
readouts.
1,1,1-Trifluoro-2-(pyrazin-2-yl)propan-2-ol (1): A suspension of acetyl
pyrazine (1.0 g, 8.2 mmol), trimethyl(trifluoromethyl)silane (1.7 g, 12.3
mmol) and K2CO3 (1.7 g, 12.3 mmol) in DMF (10 mL) was stirred at RT
for 12 h. The reaction mixture was extracted with EtOAc and the
combined organic layers were washed with water, dried using sodium
sulfate, filtered and concentrated. The residue was purified on silica gel
by flash chromatography (hexane/EtOAc, gradient: 100:0 to 85:15) to
afford the desired product (0.9 g, 60%) as yellow oil: 1H NMR (400 MHz,
CDCl3): δ 1.81 (s, 3 H), 5.48 (bs, 1 H), 8.59-8.61 (m, 1 H), 8.69 (d, J =
2.4 Hz, 1 H), 8.89 (s, 1 H).
Animal dosing: The vehicle used for intravenous bolus administration of
[3H]23 was an ethanol/0.9% saline solution (5/95, w/w). Mice were dosed
with a single intravenous tracer dose of nominal 25 µg/kg [3H]23 (50
MBq/kg) 24 h after antibody pre-treatment.
QWBA (Quantitative Whole Body Autoradiography): In brief, mice (n = 1
per time point) were sacrificed one hour post [3H]-23 dosing, after deep
anaesthesia (3-5% isoflurane, 95-97% O2). The carcasses were deep-
frozen by transfer into a heptane/dry ice mixture, where they were kept at
−70°C for approximately 15-30 min. The carcasses were then stored in a
deep-freezer below −20°C for at least 24 h; thereafter, they were rapidly
shaved and stored again below −20°C; all subsequent procedures were
performed at temperatures below −20°C to minimize diffusion of
radiolabeled material in the tissues. Blood samples were collected from
the sublingual vein of each mouse before freezing, for quality control
purposes. In these samples, radioactivity was determined by liquid
scintillation counting. The frozen carcasses were embedded in a mold on
a microtome stage by adding an ice-cold aq. solution of 2% low viscosity
sodium carboxymethylcellulose; the embedding block was frozen for
approx. 45 min in a heptane/dry ice mixture at −70°C followed by a
temperature stabilization overnight in a −20°C freezer. The animals were
sectioned in the sagittal plane using a CM3600 cryomicrotome (Leica
Microsystems GmbH, D-Nussloch) according to the method of Ullberg,[35]
using disposable blades (Feather Inc.). Several lengthwise 40 μm
sections were taken at varying depths; based on sectioning of the
requisite organs, tissues and body fluids. A block of [3H] radiolabeled
standards, containing 9 standards prepared in blood and assayed by
liquid scintillation counting, was sectioned in the same manner and on
the same day as the mice were sectioned. The sections were dehydrated
in the cryomicrotome at −23°C for at least 48 h and placed under
controlled light conditions in direct and close contact with Fuji BAS
MS2025 imaging plates (Fuji Photo FilmCo., Ltd., J-Tokyo, provided by
GE Healthcare) for 7 d at RT in a lead shielding box. After exposure, the
plates used for [3H]autoradiography were transferred into a Fuji BAS
5000 TR phosphorimager (Fuji Photo Film Co., Ltd., J-Tokyo) and
scanned with 50 μm scanning steps to produce an autoradiogram.
Concentrations of total radiolabeled components in tissues/matrices were
determined by comparative densitometry and digital analysis of the
autoradiograms using an MCID/Analysis image analyzer (Imaging
Research, St. Catharines, Ontario, Canada)
1,1,1-Trifluoro-2-(piperazin-2-yl)propan-2-ol (2): Platinum(IV) oxide
(250 mg) was added to a suspension of 1,1,1-trifluoro-2-(pyrazin-2-
yl)propan-2-ol (500 mg, 2.6 mmol) in MeOH (15 mL). The resulting
mixture was stirred under hydrogen (4 bar) for 24 h at RT, cooled to RT,
and filtered through celite. The celite cake was washed with MeOH and
the filtrate concentrated. The crude product (450 mg) was used as such
in the next step. LC–MS: tR=0.11 min, m/z: 199.2 [M+H].
2-(4-(3,5-Difluoro-6-(1h-pyrazolo[3,4-b]pyridin-3-yl)pyridin-2-
yl)piperazin-2-yl)-1,1,1-trifluoropropan-2-ol (4): A suspension of 2 (120
mg, 0.6 mmol), 3-(4-(tert-butyldimethylsilyl)-3,5,6-trifluoropyridin-2-yl)-1H-
pyrazolo[3,4-b]pyridine[22] 3 (200 mg, 0.5 mmol) and Hünig’s base in
NMP (8 mL) was stirred for 2 h at 150°C. The reaction mixture was
cooled to RT, diluted in EtOAc and washed with water. The organic layer
was extracted, washed with brine, dried over MgSO4, filtered and
concentrated. The crude product was purified by preparative HPLC
(column: Kinetec® EVO C18, 21.2x150 mm,
5 µm, solvent A:
MeOH+0.1%TFA, solvent B: CH3CN, flow: 15mL/min, gradient: from 15%
B to 25% B over 2 min + from 25% B to 55% B over 8 min) to afford 50
mg (21%, yellow solid) of the desired product as a racemic mixture. The
residue was purified by chiral preparative HPLC (column: CHIRALPAK®
IA, 10x250 mm, 5 µm, solvent A: hexane, solvent B: EtOH, flow: 8
mL/min, isocratic: A:B 60:40) to afford enantiomerically pure 4 (13 mg,
yellow solid): : LC–MS: tR=0.48 min, m/z: 429.3 [M+H]. 1H NMR (300
MHz, CD3OD): δ 1.98 (s, 3 H), 3.39-3.53 (m, 2 H), 3.63-3.67 (m, 1 H).
3.77-3.80 (m, 1 H), 3.92-3.96 (m, 1 H), 4.07-4.12 (m, 1 H), 4.33-4.38 (m,
1 H), 7.75 (dd, J = 4.8 Hz, 4.8 Hz, 1 H), 7.95 (dd, J = 8.1 Hz, 8.1 Hz, 1 H),
9.02 (m, 1 H), 9.26 (dd, J = 8.1 Hz, 1.5 Hz, 1 H).
Methyl 2-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)isoxa-
zolidine-2-carbonyl)benzoate (6): Methyl 2-(4-((methylsulfonyl)oxy)-
isoxazolidine-2-carbonyl)benzoate[23] (760 mg, 0.7 mmol), 3-iodo-1H-
pyrazolo[3,4-d]pyridine-4-amine (500 mg, 1.9 mmol) and K2CO3 (668 mg,
1.5 mmol) were dissolved in DMF (10 mL) at 0°C. The resulting
suspension was heated to 90°C for 16 h. Water was added and the
reaction mixture was extracted with EtOAc. The organic layers were
washed with brine, dried with anhyd. Na2SO4, filtered and concentrated
Flow cytometry Mice were sacrificed 25 hours post anti-CD4 antibody
treatment and single cell suspensions generated from lymph nodes and
spleen tissues via 70μm sieves (Becton Dickinson, USA). Lymphocytes
were isolated from whole blood using red blood cell RBC lysis buffer
9
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under reduced pressure to give crude 6, which was used in the next step
without further purification. LC–MS: tR=1.37 min, m/z: 494.95 [M+H].
argon at 0 °C. The reaction mixture was stirred at RT overnight,
quenched on ice and extracted with EtOAc. The combined organic layers
were washed with brine, dried over anhyd. Na2SO4 and concentrated
under reduced pressure to give 33 (7 g, quantitative). LC–MS: tR=1.55
min, m/z: 326.0 [M+H].
3-Iodo-1-(isoxazolidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine:
Compound 6 (500 mg, crude) was dissolved in 10 mL of 4M HCl in dioxane at
0°C. The resulting suspension was stirred at 100°C for 16 h. The reaction
mixture was treated with sat. aq. NaHCO3 until pH=8 and EtOAc was added.
The layers were separated and the organic extract was washed with brine,
dried over anhyd. Na2SO4, and concentrated to afford the crude title product
which was further used without purification. LC–MS: tR=0.587 min, m/z: 332.85
[M+H].
Benzyl
4-(chloro(phenyl)methyl)piperidine-1-carboxylate
(34):
Thionyl chloride (3.12 mL, 43.05 mmol) was added dropwise to a stirred
solution of 33 (7 g, 21.527 mmol) in CH2Cl2 (100 mL) under argon. The
reaction mixture was stirred overnight at RT, excess solvent removed
under reduced pressure. The residue was dissolved in CH2Cl2 and
washed with water and brine. The organic layer was dried over anhyd.
Na2SO4 and concentrated under reduced pressure to give 34 (7 g,
94.6%). LC–MS: tR=1.69 min, m/z: 344.2 [M+H] which was directly used
in the next step.
tert-Butyl 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)isoxa-
zolidine-2-carboxylate (7): 3-Iodo-1-(isoxazolidin-4-yl)-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (300 mg, 0.9 mmol) was dissolved in CH2Cl2 (30 mL) and
Boc anhydride (236 mg, 1.1 mmol) and NEt3 (228 mg, 2.2 mmol) were added
at 0°C under a nitrogen atmosphere. The suspension was stirred at RT for 16
h. The reaction mixture was diluted with EtOAc and water, and the layers were
separated. The organic layer was washed with brine, dried over anhyd.
Na2SO4 and concentrated to afford the crude product which was purified by
flash chromatography on silica gel (eluent: hexane to hexane/EtOAc 1:1) to
give 7 as a white solid (150 mg, 38%). LC–MS: tR=1.42 min, m/z: 432.90
[M+H]. 1H NMR (300 MHz, DMSO-d6): δ 1.44 (s, 9 H), 3.91-3.94 (m, 2 H), 4.1-
4.3 (m, 2 H), 5.22-5.32 (m, 1 H), 8.2 (s, 1 H).
Benzyl
4-((4-nitro-1H-pyrazol-1-yl)(phenyl)methyl)piperidine-1-
carboxylate (35): 4-Nitro-1H-pyrazole (1 g, 8.72 mmol) was added to a
stirred mixture of 34 (3 g, 8.72 mmol), K2CO3 (2.4 g, 17.44 mmol) and KI
(2.85 g, 17.44 mmol) in DMF under argon. The resulting reaction mixture
was heated at 100 °C overnight, cooled to RT, quenched with ice and
extracted with EtOAc. The combined organic layer was washed with
brine, dried over anhyd. Na2SO4 and concentrated in vacuo. The residue
was purified by column chromatography (60-120 mesh silica, solvent:
EtOAc/hexane 1:1) to give yellow solid 35 (1.25 g, 34%). LC–MS: tR=1.65
min, m/z: 421.05 [M+H]. 1H NMR (300 MHz, CDCl3): δ 1.1-1.43 (m, 4 H),
2.6-2.9 (m, 2 H), 4.1-4.16 (m, 2 H), 4.77 (d, J = 10.8 Hz, 1 H), 5.12 (s, 2
H), 5.31 (s, 1 H), 7.27-7.45 (m, 10 H), 8.11 (s, 1 H), 8.17 (s, 1 H).
tert-Butyl 4-(4-amino-3-(3-methoxy-4-(1-methyl-1H-indole-2-carbox-
amido)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)isoxazolidine-2-
carboxylate: Compound 7 (100 mg, 0.1 mmol), the boronate ester 8[23]
(141 mg, 0.2 mmol) and K2CO3 (80 mg, 0.3 mmol) were dissolved in
DMSO (10 mL) and argon was bubbled through the suspension for 10
min. Pd(dppf)Cl2 (19 mg, 0.01 mmol) was added and the degasification
with argon was repeated. The reaction mixture was heated at 95°C for
1.2 h. After completion, the suspension was cooled to RT and water was
added. The precipitate was isolated, washed with water and the filtrate
extracted with EtOAc. The organic layers were washed with brine, dried
over anhyd. Na2SO4 and concentrated to give the crude title product. LC–
MS: tR=1.58 min, m/z: 585.20 [M+H].
Benzyl
4-((4-amino-1H-pyrazol-1-yl)(phenyl)methyl)piperidine-1-
carboxylate (11): Zn powder (1.63 g, 29.73 mmol) was added to a
stirred mixture of 35 (2.5 g, 5.95 mmol) and NH4Cl (1.93 g; 29.73 mmol)
in THF : water (2:1, 30 mL) at 0°C. The resulting reaction mixture was
stirred overnight at RT. and then filtered through celite. The filtrate
obtained was diluted with water and extracted using EtOAc (3 x 30 ml).
The combined organic layers were washed with brine, dried over anhyd.
Na2SO4 and concentrated under reduced pressure to give crude 11 (2.3
g, quantitative) as red gum. LC–MS: tR=1.366 min, m/z: 391.55 [M+H].
N-(4-(4-Amino-1-(isoxazolidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-2-methoxyphenyl)-1-methyl-1H-indole-2-carboxamide (9): 80 mg
6,6-Dimethyl-1-(tetrahydro-2H-pyran-2-yl)-4,5,6,7-tetrahydro-1H-
indazole-3-carboxylic acid (12): To a stirred solution of 6,6-dimethyl-
4,5,6,7-tetrahydro-1H-indazole-3-carboxylic acid[28] (2 g, 9 mmol) in
toluene (20 mL) was added 3,4-dihydro-2H-pyran (0.83 g, 9.9 mmol) and
TFA (0.1 mL). The resulting reaction mixture was refluxed for 4 h and
then diluted with AcOEt. The organic layer was washed with Na2CO3
solution, dried over anhyd. Na2SO4 and concentrated in vacuo. The
crude compound purified by chromatography (60-120 mesh silica, 10%
AcOEt in hexane as eluent) to obtain the protected intermediate (2.05 g,
74.3%) which was directly subjected to hydrolysis. The compound was
dissolved in THF/MeOH/water (1:1:1, 45 mL) and LiOH.H2O was added
(2.61 g, 65.3 mmol). The resulting reaction mixture was stirred at room
temperature for 4 h. The excess solvents were removed in vacuo, the
remaining aqueous phase was acidified using aq. citric acid solution. The
compound was extracted with AcOEt, the organic layer was dried over
anhyd. Na2SO4 and concentrated under reduced pressure to get pale
solid 12 (1.6 g, 88%). LC–MS: tR= 1.389 min, m/z: 279.3 [M+H]; 1H
NMR (300 MHz, DMSO-d6): δ 0.91 (s, 3 H), 0.93 (s, 3 H), 1.48-1.63 (m, 4
H), 1.65-1.70 (m, 1 H), 1.93-1.98 (m, 2 H), 2.11-2.32 (m, 1 H), 2.40 (s, 2
H), 2.56-2.72 (m, 2 H), 3.55-3.65 (m, 1 H), 3.81-3.90 (m, 1 H), 5.39 (dd, J
= 2.7 Hz, 2.4 Hz, 1 H), 12.3 (bs, 1 H).
crude
tert-butyl
4-(4-amino-3-(3-methoxy-4-(1-methyl-1H-indole-2-
carboxamido)-phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)isoxazolidine-2-
carboxylate were dissolved in 3 mL 4M HCl in dioxane at 0°C. The
resulting suspension was stirred at RT for 16 h, then concentrated under
reduced pressure. The crude product was purified by preparative HPLC
(column: Zorbax C18, 21.2x150 mm, 5 µm, solvent A: water, solvent B:
CH3CN), flow: 20 mL/min, gradient: from 10% B to 20% B in 2 min then
up to 80% B in 8 min) to afford 9 as a white solid (16 mg, 24%): LC-MS:
1
tR=1.44 min, m/z: 485.10 [M+H]. H NMR (400 MHz, DMSO-d6): δ 3.34-
3.58 (m, 2 H), 3.95 (s, 3 H), 4.03 (s, 3 H), 4.08-4.12 (m, 2 H), 5.73-5.81
(m, 1 H), 7.12-7.16 (m, 1 H), 7.29-7.36 (m, 4 H), 7.58 (d, J = 8.4 Hz, 1 H),
7.69 (d, J = 8 Hz, 1 H), 8.11 (d, J = 8 Hz, 1 H), 8.27 (s, 1 H), 9.43 (s, 1 H).
Benzyl 4-benzoylpiperidine-1-carboxylate (32): Benzyl chloroformate
(3.8 mL, 26.5 mmol) was added dropwise to a stirred solution of
phenyl(piperidin-4-yl)methanone hydrochloride 10 (5 g, 22.15 mmol) and
K2CO3 (9.17 g, 66.45 mmol) in THF at 0 °C. The mixture was stirred
under argon overnight at RT, then quenched with ice cold water and
extracted with EtOAc. The combined organic layers were washed with
brine, dried over anhyd. Na2SO4 and concentrated under reduced
pressure to give 32 (7 g, 97.76%); 1H NMR (300 MHz, CDCl3): δ 1.65-
1.86 (m, 4 H), 2.92-3.08 (m, 2 H), 3.39-3.5 (m, 1 H), 4.2-4.3 (m, 2 H), 5.2
(s, 2 H), 7.3-7.4 (m, 5 H), 7.45-7.51 (m, 2 H), 7.55-7.61 (m, 1 H), 7.92-
7.96 (m, 2 H).
Benzyl
4-((4-(6,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-4,5,6,7-
tetrahydro-1H-indazole-3-carboxamido)-1H-pyrazol-1-yl)(phenyl)-
methyl)piperidine-1-carboxylate ((±)-13): DIPEA (2.05 mL, 11.78
mmol) was added to a stirred solution of 12 (1.64 g, 5.89 mmol) and
HATU (3.92 g, 11.78 mmol) in DMF (25 mL) at RT. The resulting reaction
mixture was stirred for 10 min then 11 (2.3 g, 5.89 mmol) was added in
portions. The reaction mixture was heated to 50 °C for 4 days, cooled to
Benzyl
4-(hydroxy(phenyl)methyl)piperidine-1-carboxylate
(33):
Sodium borohydride (2.46 g, 64.92 mmol) was added in portions to a
stirred solution of compound 32 (7 g, 21.6 mmol) in MeOH (70 mL) under
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800241
ChemMedChem
FULL PAPER
0 °C and quenched with ice. The mixture was extracted with EtOAc and
the combined organic layer was washed with water and brine, dried over
anhyd. Na2SO4, filtered and concentrated in vacuo. The residue was
purified using column chromatography (60-120 mesh silica, solvent:
EtOAc/hexane 1:1) to obtain (±)-13 (3.5 g, 65%). LC–MS: tR=1.758 min,
m/z: 651.25 [M+H].
(0.3 g, 45%) which was directly hydrolysed by dissolving the crude
product in THF/methanol/water (1:1:1, 9 mL) and adding LiOH.H2O
(0.184 g, 4.4 mmol). The resulting reaction mixture was stirred at RT for
14 h, excess solvents were removed under reduced pressure, and the aq.
layer acidified with aq. citric acid. The compound was extracted with
EtOAc, the organic layer dried over anhyd. Na2SO4, filtered and
concentrated under reduced pressure to obtain crude (±)-16 (0.25 g,
90.8%) as gummy solid. LC–MS: tR= 1.467 min, m/z: 313.05 [M+H]; 1H
NMR (300 MHz, DMSO-d6): δ 1.38 (s, 3 H), 1.56-1.70 (m, 2 H), 1.75-2.0
(m, 4 H), 2.08 (s, 2 H), 2.1-2.22 (m, 2 H), 2.78-2.82 (m, 1 H), 3.59-3.63
(m, 1 H), 3.80-3.90 (m, 1 H), 5.41 (dd, J = 2.6 Hz, 2.4 Hz, 1 H).
6,6-Dimethyl-N-(1-(phenyl(piperidin-4-yl)methyl)-1H-pyrazol-4-yl)-1-
(tetrahydro-2H-pyran-2-yl)-4,5,6,7-tetrahydro-1H-indazole-3-
carboxamide ((±)-37): Pd-C (10%) (350 mg) was added to solution of 13
(3.5 g, 5.38 mmol) in MeOH (35 mL) and the mixture was stirred under
H2 atmosphere (balloon pressure) for 4 h. The mixture was filtered
through celite and concentrated under reduced pressure to give crude
(±)-37 (2.5 g, 90%) as brown solid. LC–MS: tR = 1.39 min, m/z: 517.70
[M+H]. The compound was used in the next step without further
purification.
Benzyl
4-((4-(5,5-difluoro-5a-methyl-1-(tetrahydro-2H-pyran-2-yl)-
1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-carboxamido)-1H-
pyrazol-1-yl)(phenyl)methyl)piperidine-1-carboxylate
(diastereomeric mixture 39): BOP (595 mg, 1.345 mmol) was added to
a solution of (±)-16 (280 mg, 0.9 mmol) in DMF (3 mL) followed by
DIPEA (0.312 mL, 1.8 mmol). The reaction mixture was stirred for 10 min
and (±)-11 (525 mg, 1.35 mmol) was added in portions. The reaction
mixture was stirred at RT for 48 h, cooled to 0 °C, quenched using ice
cold water and extracted with EtOAc. The combined organic layers were
washed with brine, dried over anhyd. Na2SO4, filtered and concentrated
under reduced pressure. The crude compound was purified by column
chromatography (60-120 mesh silica, solvent: EtOAc/hexane 1:1) to give
diastereomeric mixture 39 (280 mg, 46%). LC–MS: tR=1.70 min, m/z:
685.3 [M+H].
6,6-Dimethyl-N-(1-(phenyl(piperidin-4-yl)methyl)-1H-pyrazol-4-yl)-
4,5,6,7-tetrahydro-1H-indazole-3-carboxamide ((±)-14): Trifluoroacetic
acid (1 mL) was added dropwise to a stirred solution of compound (±)-37
(150 mg, 0.29 mmol) in CH2Cl2 (5 mL) at 0 °C and the mixture was then
stirred at RT for 1 h. The excess solvent was removed under reduced
pressure and the residue purified by prep HPLC (column: Phenomenex
Gemini® NX-C18, 19x150 mm, 5 µm, eluent A: 0.1% HCOOH in water,
eluent B: CH3CN, flow: 20 mL/min, gradient: 30% B for 2 min + from 30%
B to 80% B over 8 min) to give (±)-14 (12 mg, brown solid). LC–MS:
tR=0.328 min, m/z: 433.4 [M+H]. 1H NMR (400 MHz, CD3OD): δ 1.0 (s, 6
H), 1.15-1.42 (m, 6 H), 1.58-1.60 (m, 2 H), 2.50-2.65 (m, 6 H), 2.98-3.02
(m, 1 H), 7.28-7.30 (m, 1 H), 7.31-7.36 (m, 2 H), 7.48-7.50 (m, 2 H), 7.65
(m, 1 H), 8.12 (m, 1 H).
5,5-Difluoro-5a-methyl-N-(1-(phenyl(piperidin-4-yl)methyl)-1H-
pyrazol-4-yl)-1-(tetrahydro-2H-pyran-2-yl)-1,4,4a,5,5a,6-
hexahydrocyclopropa[f]indazole-3-carboxamide
(diastereomeric
mixture 17): Pd-C (10%) (50 mg) was added to a stirred solution of 39
(270 mg, 0.394 mmol) in EtOH (5 mL) and the mixture was hydrogenated
under balloon pressure of hydrogen for 6 h. The reaction mixture was
filtered through celite and concentrated under reduced pressure to give
the crude diastereomeric mixture 17 (190 mg, 88%) as brown solid. LC–
MS: tR=1.37 min, m/z: 551.25 [M+H].
N-(1-((1-(2-Fluoroethyl)piperidin-4-yl)(phenyl)methyl)-1H-pyrazol-4-
yl)-6,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-4,5,6,7-tetrahydro-1H-
indazole-3-carboxamide ((±)-38): Solid K2CO3 (156 mg, 1.16 mmol)
was added to a solution of (±)-37 (200 mg, 0.387 mmol) in DMF (5 mL)
followed by 1-fluoro-2-iodoethane (101 mg, 0.58 mmol). The resulting
reaction mixture was stirred overnight at RT, then diluted with water and
extracted with EtOAc (3 x 10 mL). The combined organic layers were
washed with brine, dried over anhyd. Na2SO4 and concentrated under
reduced pressure to give compound (±)-38 (100 mg, 45.9 %) which was
directly used for the next step. LC–MS: tR=1.27 min, m/z: 563.1 [M+H].
5,5-Difluoro-N-(1-((1-(2-fluoroethyl)piperidin-4-yl)(phenyl)methyl)-1H-
pyrazol-4-yl)-5a-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,4,4a,5,5a,6-
hexahydrocyclopropa[f]indazole-3-carboxamide
(diastereomeric
mixture 40): 1-Fluoro-2-iodoethane (0.053 mL, 0.654 mmol) was added
to a mixture of 17 (180 mg, 0.327 mmol) and K2CO3 ( 90 mg, 0.654
mmol) in DMF (3 mL). The resulting reaction mixture was heated at 80 °C
for 1 h, cooled to RT, diluted with water and extracted with EtOAc. The
combined organic layers were washed with brine, dried over anhyd.
Na2SO4 and concentrated under reduced pressure to give crude 40 (195
mg, quantitative) LC–MS: tR=1.36 min, m/z: 597.3 [M+H] which was
directly used in the next step.
N-(1-((1-(2-Fluoroethyl)piperidin-4-yl)(phenyl)methyl)-1H-pyrazol-4-
yl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide
(enantiomers 15a,b): HCl in dioxane (3 mL) was added to a solution of
(±)-38 (85 mg, 0.151 mmol), in dioxane (2 mL). The reaction mixture was
stirred overnight at RT and excess solvent removed under reduced
pressure to obtain (±)-15 (50 mg, 69.15%). 40 mg of the racemic mixture
were resolved using chiral prep. HPLC (column: Chiral Pak IA®,10x250
mm, 5µm, solvent A: n-hexane, solvent B: EtOH+0.1% DEA, flow: 6
mL/min, isocratic A:B 30:70) to obtain enantiomerically pure 15a (first
peak, 10.2 mg) and 15b (second peak, 11 mg). Analytical data of (±)-15:
5,5-difluoro-N-(1-((1-(2-fluoroethyl)piperidin-4-yl)(phenyl)methyl)-1H-
pyrazol-4-yl)-5a-methyl-1,4,4a,5,5a,6-
hexahydrocyclopropa[f]indazole-3-carboxamide
(stereoisomers
1
LC–MS: tR=1.35 min, m/z: 479.65 [M+H]. H NMR (400 MHz, CDCl3): δ
18a-d): A solution of 40 (180 mg, 0.30 mmol) in dioxane/HCl (5 mL) was
stirred over night at RT. The excess solvents was removed in vacuo. The
crude product was further purified using prep HPLC (column:
Phenomenex Kinetex® C18, 21.5x250mm, 5.0µm, solvent A:
water+0.02% TFA, solvent B: CH3CN:MEOH 1:1, flow: 20 mL/min,
gradient: from 50% B to 60% B over 2 min + from 60% B to 80% B over 8
min) to obtain 18 as a racemic mixture of diastereoisomers (80 mg,
51.61%). The four stereoisomers were separated using chiral prep HPLC
(Chiral Pak® IA, 10x250 mm, 5 μm, solvent A: n-hexane, solvent B: 0.1%
DEA in EtOH:i-PrOH 3:1, isocratic A:B 65:35) to get 18a (first peak, 7.5
mg, 4.9%), 18b (second peak, 7 mg, 4.6%), 18c (third peak, 3 mg, 2%),
18d (fourth peak, 5 mg,3.3%). LC–MS: tR=1.33 min, m/z: 513.25 [M+H],
no separation of diastereoisomers. 1H NMR (mixture of stereoisomers,
400 MHz, CDCl3): δ 1.2-1.4 (m, 4 H), 1.4 (s, 3 H), 1.99-2.09 (m, 3 H), 2.4-
2.51 (m, 1 H), 2.61-2.63 (m, 1 H), 2.64-2.77 (m, 2 H), 2.92-3.0 (m, 2 H),
3.05-3.1 (m, 1 H), 3.1-3.22 (m, 1 H), 3.23-3.28 (m, 1 H), 4.47-4.49 (m, 1
1.0 (s, 6 H), 1.35-1.50 (m, 3 H), 1.52-1.65 (m, 3 H), 2.0-2.1 (m, 2 H),
2.40-2.42 (m, 1 H), 2.6-2.8 (m, 6 H), 2.95-3.0 (m, 2 H), 4.45-4.46 (m, 1 H),
4.60-4.61 (m, 1 H), 4.79-4.81 (d, J = 10.8 Hz, 1 H), 7.28-7.36 (m, 2 H),
7.43-7.45 (m, 2 H), 7.52 (s, 1 H), 8.22 (s, 1 H), 8.56 (bs, 1 H).
5,5-Difluoro-5a-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,4,4a,5,5a,6-
hexahydrocyclopropa[f]indazole-3-carboxylic acid ((±)-16): To
a
stirred solution of ethyl 5,5-difluoro-5a-methyl-1,4,4a,5,5a,6-
hexahydrocyclo-propa[f]indazole-3-carboxylate[24] (0.5 g, 1.95 mmol) in
toluene (10 mL) was added 3,4-dihydro-2H-pyran (0.328 g, 3.9 mmol)
and TFA (0.018 g). The resulting reaction mixture was refluxed for 14 h
and then diluted with EtOAc. The organic layer was washed with sat. aq.
Na2CO3, dried over anhyd. Na2SO4 and concentrated under reduced
pressure. The crude compound was purified by chromatography (60-120
mesh silica, 15% AcOEt in hexane) to obtain the protected intermediate
11
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10.1002/cmdc.201800241
ChemMedChem
FULL PAPER
H), 4.59-4.61 (m, 1 H), 4.76 (d, J = 11.2 Hz, 1 H), 7.27-7.34 (m, 3 H),
(21):
Enantiomerically
pure
(4aS,5aR)-5,5-difluoro-5a-methyl-
7.41-7.44 (m, 2 H), 7.50 (s, 1 H), 8.14 (s, 1 H), 8.51 (s, 1 H).
1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-carboxylic acid was
prepared by separating the corresponding ethyl ester[24] by chiral HPLC
(column: ChiralPak® IA, 10x250 mm,
5
μm, solvent:
isocratic
(3-(Methylthio)phenyl)(tetrahydro-2H-pyran-4-yl)methanol ((±)-41): n-
BuLi (1.6 M in hexane, 24 mL) was added to a solution of (3-
bromophenyl)(methyl)sulfane (7 g, 34.8 mmol) in anhyd. THF (70 mL) at
−78°C followed by dropwise addition of tetrahydro-2H-pyran-4-
carbaldehyde (3.97 g, 34.8 mmol). The reaction mixture was slowly
brought to RT and allowed to stir for another 2 h. The reaction mixture
was quenched using sat. aq. NH4Cl at 0 °C, and extracted with EtOAc.
The combined organic layers were washed with brine, dried over anhyd.
MgSO4, filtered and concentrated in vacuo. The residue was purified
using column chromatography (60-120 mesh silica, solvent:
EtOAc/hexane 3:7) to give (±)-41 (6 g, 73.1%) as pale yellow liquid. 1H
NMR (300 MHz, CDCl3): δ 1.1-1.3 (m, 4 H), 1.89-1.93 (m, 2 H), 2.5 (s, 3
H), 3.24-3.42 (m, 2 H), 3.74-4.05 (m, 2 H), 4.31-4.34 (m, 1 H), 7.04-7.08
(m, 1 H), 7.18-7.29 (m, 3 H).
hexane/isopropanol/methanol 80:10:10, flow: 20 ml/min, first eluting
peak), and subsequent hydrolysis. BOP (0.581 g, 1.315 mmol) was
added to a solution of the chiral acid (0.2 g, 0.876 mmol) in 3 mL anhyd.
DMF followed by DIPEA (0.31 mL, 1.75 mmol). After 10 min a solution of
19a (0.265 g, 0.876 mmol) in anhyd. DMF was added slowly. The
reaction mixture was stirred at 50°C for 4 h. After cooling to RT, the
mixture was quenched with ice water and extracted with EtOAc (3 x 20
mL). The combined organic layers were washed with water and brine,
dried over anhyd. Na2SO4 and concentrated under reduced pressure to
give crude 21 (250 mg, 28%) which was directly used for the next step.
LC–MS: tR=1.548 min, m/z: 514.20 [M+H].
(5aR)-5,5-Difluoro-5a-methyl-N-(1-((1S)-(3-
(methylsulfinyl)phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-
pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-
carboxamide (22a,b): A solution of 21 (150 mg, 0.292 mmol) in 6 M t-
butyl hydroperoxide in decane (3 mL) was stirred at RT for 1 h. The
excess of solvents was removed under reduced pressure to give a crude
product which was purified by reverse phase prep. HPLC (column:
Phenomenex LUNA® C18, 21.2x250 mm, 5 μm, solvent A: water+0.1%
TFA, Solvent B: CH3CN, flow: 20mL/min, gradient: from35% B to 45% B
over 2 min + from 45% B to 65% B over 8 min) to afford 60 mg of the
crude product. The diastereoisomers were separated by chiral prep
HPLC (column: ChiralPak® IA, 10x250 mm, 5 µm, solvent: hexane/i-
PrOH isocratic 65:35, flow: 9 mL/min) to afford 22a (peak 1, 28 mg, 18%)
and 22b (peak 2, 29 mg, 19%) as pale solids. LC–MS: tR =1.42 min, m/z:
4-(Chloro(3-(methylthio)phenyl)methyl)tetrahydro-2H-pyran ((±)-42):
Thionyl chloride (2.2 mL, 30.2 mmol) was dropwise added to a solution of
(±)-41 (6 g, 25.2 mmol) in anhyd. CH2Cl2 (60 mL) and the resulting
reaction mixture was stirred at RT for 2 h. Excess of solvents was
removed under reduced pressure and the crude product was dissolved in
CH2Cl2 and washed with water and brine. The organic layer dried over
anhyd. Na2SO4 and concentrated under reduced pressure to get crude
(±)-42 as yellow oil (6.2 g, 96%) which was directly used for the next step.
1H NMR (300 MHz, CDCl3): δ 1.2-1.5 (m, 4 H), 2.02-2.12 (m, 1 H), 2.5 (s,
3 H), 3.23-3.43 (m, 2 H), 3.86-3.92 (m, 1 H), 4.04-4.13 (m, 1 H), 4.52 (d,
J = 9 Hz, m 1 H), 7.08-7.12 (m, 1 H), 7.17-7.31 (m, 3 H).
1
530.15 [M+H]. H NMR (400 MHz, CD3OD): δ 1.2-1.36 (m, 4 H), 1.37 (s,
1-((3-(Methylthio)phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-4-nitro-
1H-pyrazole ((±)-43): Solid K2CO3 (6.68 g, 48.4 mmol) and KI (4 g, 24.2
mmol) were added to a solution of 4-nitropyrazole (2.73 g, 24.2 mmol) in
60 mL anh. DMF followed by (±)-42 (6.2 g, 24.2 mmol). The resulting
reaction mixture was heated at 100 °C for 14 h. The reaction mixture was
cooled to RT, quenched with ice water and extracted using EtOAc (3 x 50
mL). The combined organic layers were washed with water and brine,
dried over anhyd. Na2SO4 and concentrated under reduced pressure to
give crude product which was purified by silica gel chromatography
(EtOAc/hexane 1:4) to yield (±)-43 (4 g, 50%) as yellow oil. 1H NMR (300
MHz, CDCl3): δ 1.2-1.22 (m, 2 H), 1.62-1.68 (m, 2 H), 1.9-2.0 (m, 1 H),
3.07-3.15 (m, 2 H), 3.26-3.34 (m, 2 H), 4.25 (d, J = 10.8 Hz, 1 H), 6.4-
6.53 (m, 3 H), 6.66 (s, 1 H), 7.37 (s, 1 H), 7.94 (s, 1 H).
3 H), 1.62-1.67 (m, 1 H), 2.78-2.80 (m, 4 H), 3.03-3.2 (m, 4 H), 3.3-3.45
(m, 2 H), 3.86-3.93 (m, 2 H), 5.08 (d, J = 10.8 Hz, 1 H), 7.55-7.58 (m, 1
H), 7.59-7.65 (m, 1 H), 7.68 (s, 1 H), 7.72-7.74 (m, 1 H), 7.89 (d, J = 4.4
Hz, 1 H), 8.14 (s, 1 H).
(5aR)-5,5-Difluoro-5a-methyl-N-(1-((S)-3-
(methylsulfonyl)phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-
pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-
carboxamide (23): mCPBA (67 mg, 0.39 mmol) was added to a solution
of 21 (100 mg, 0.194 mmol) in anhyd. CH2Cl2 (3 mL) at 0 °C. The
reaction mixture stirred at 0 0C for 1 h, then diluted with CH2Cl2 and
quenched with sat. aq. NaHCO3. The organic layer was separated,
washed with brine, then dried over anhyd. Na2SO4 and concentrated
under reduced pressure. The crude product was purified by reverse
phase prep HPLC (column: Zorbax XDB® C18, 21.2x150 mm, 5 μm,
solvent A: water+0.1% TFA, solvent B: CH3CN, flow: 18mL/min, gradient:
from 30% B to 40% B over 2 min + from 40% B to 70% B over 8 min) to
afford 23 (14.5 mg, 14%) as a white solid. LC–MS: tR =1.46 min, m/z:
1-((3-(Methylthio)phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-
pyrazol-4-amine (enantiomers 19a,b): A saturated aqueous solution of
NH4Cl (10 mL) was added to a solution of (±)-44 (4 g; 12 mmol) in THF
(40 mL) followed by Zn dust (3.9 g, 60 mmol). The reaction mixture was
stirred at RT for 3 h until TLC analysis indicated completion of the
reaction. The reaction mixture was filtered through a celite bed and
washed with EtOAc. The organic layer was separated, dried over anhyd.
Na2SO4 and concentrated under reduced pressure to give crude product
(±)-19 which was further purified using reverse phase prep HPLC
(column: Phenomenex Kinetex® C18, 21.2x150mm, 5 µm, solvent A:
water+0.05% TFA, solvent B: CH3CN/MeOH 1:1, flow: 18mL/min,
gradient: from 20% B to 30% B over 2 min + from 30% B to 70% B over 8
min) to afford 1.5 g brown solid (1.50 g) which was further resolved using
chiral prep HPLC (Lux® Cellulose-4, 10x250 mm, 5 µm, solvent A:
hexane, solvent B: 0.1% DEA in EtOH/MEOH/i-PrOH, 50:25:25, isocratic
A:B 65:35) to afford the pale solids (R)-19a (peak 1, (R)-enantiomer, 550
mg, 15%) and (S)-19b (peak 2, (S)-enantiomer, 550 mg, 15%),
respectively. LC–MS: tR =0.6 min, m/z: 303.9 [M+H]. Assignment of the
absolute configuration was based on the X-ray crystal structure of 25,
which was synthesized from (S)-19b.
1
546.20 [M+H]. H NMR (400 MHz, CDCl3): δ 1.2-1.36 (m, 4 H), 1.4 (s, 3
H), 1.59-1.62 (m, 1 H), 2.7-2.78 (m, 2 H), 3.04 (s, 3 H), 3.05-3.2 (m, 2 H),
3.28-3.41 (m, 3 H), 3.89-3.96 (m, 2 H), 4.81 (d, J = 11.2 Hz, 1 H), 7.54-
7.59 (m, 2 H), 7.77-7.82 (m, 1 H), 7.85-7.88 (m, 1 H), 8.04 (s, 1 H), 8.18
(s, 1 H), 8.59 (bs, 1 H).
(5aR)-N-(5-Bromo-1-((S)-(2-bromo-5-(methylthio)phenyl)(tetrahydro-
2H-pyran-4-yl)methyl)-1H-pyrazol-4-yl)-5,5-difluoro-5a-methyl-
1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-carboxamide (24): A
catalytic amount of I2 was added to a solution of 21 (300 mg, 0.584
mmol) in anhyd. CH2Cl2 (10 mL) at 0 °C followed by dropwise addition of
Br2 (0.187 g, 1.16 mmol). The reaction mixture was stirred overnight at
RT. Additional Br2 (0.187, 1.16 mmol) was added and the reaction
mixture was kept at RT for an additional 12 h. The reaction was
quenched with aq. Na2S2O3 and extracted with dichloromethane (3 x 10
mL). The combined organic layers were washed with brine, dried over
anhyd. Na2SO4 and concentrated under reduced pressure. The crude
product was purified by reverse phase prep HPLC (column: Phenomenex
Gemini® NX-C18, 21.2x150 mm, 5 µ, solvent A: water+0.1% formic acid,
solvent B: CH3CN, flow: 20mL/min, gradient: from 30% B to 40% B over
(5aR)-5,5-Difluoro-5a-methyl-N-(1-((S)-(3-
(methylthio)phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol-4-
yl)-1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-carboxamide
12
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800241
ChemMedChem
FULL PAPER
2 min + from 40% B to 80% B over 8 min) to afford 24 (50 mg, 13 %).
LC–MS: tR =1.681 min, m/z: 672.0 [M+H] which was directly used in the
next step.
S. Ostrowitzki, V. Raymont, J. R. Brasic, N. Parkar, D. Umbricht, R. F.
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a) P. Sharma, A. Mukherjee. Ann Transl Med. 2016, 4(3), 53; b) C. L.
Charron, A. L. Farnsworth, P. D. Roselt, R. J. Hicks, C. A. Hutton,
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1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-carboxamide (25):
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0.074 mmol) in anhyd. CH2Cl2 (3 mL) at 0 0C. The resulting reaction
mixture was stirred at 0 0C for 1 h, diluted with CH2Cl2 and quenched with
sat. aq. NaHCO3. The organic layer was separated, washed with brine,
dried over anhyd. Na2SO4 and concentrated under reduced pressure.
The crude product was purified by reverse phase prep. HPLC (column:
Waters XBridge® C18, 21.2x150 mm, 5 μm, solvent A: water, solvent B:
CH3CN, flow: 15mL/min, gradient: from 30% B to 40% B over 2 min +
from 40% B to 90% B over 8 min), to afford 25 (22 mg, 43%) as
colourless gum. LC–MS: tR =1.53 min, m/z: 703.9 [M+H]. 1H NMR (300
MHz, CDCl3): δ 1.14-1.32 (m, 5 H), 1.4 (s, 3 H), 1.56-1.58 (m, 1 H), 2.72-
2.78 (m, 2 H), 3.02 (s, 3 H), 3.06-3.38 (m, 5 H), 3.9-3.96 (m, 2 H), 5.79 (d,
1 H), 7.69-7.79 (m, 2 H), 8.26-8.28 (m, 2 H), 8.58 (d, J = 2.1 Hz, 1 H).
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EtOAc. The organic layer was separated, washed with water, dried over
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LC–MS: tR = 3.70 min, m/z: 545.9 [M+H]. The product was analyzed on
chiral column and eluted at the same tR as the authentic sample. No
epimerization could be detected.
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The authors have the pleasure to acknowledge the support of Dr.
Bernard Pirard for molecular modeling; Dr. Ina Dix for X-ray
analysis; Emanuele Mauro, Prasad P.W. Appukuttan and Nilesh
M Shirode for synthesis, as well as the skilled support of Carsten
Bauer for radiolabeling; Luca Gianolla for the QWBA study,
Catherine Huck for flow cytometry, Dr. Grazyna Wieczorek for
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Entry for the Table of Contents
Illuminating T cells in vivo. Four kinases were selected as potential targets for imaging T cell populations by Positron Emission
Tomography (PET). Ultimately, the optimization of Itk inhibitors allowed the identification of 23 which, once radiolabeled, proved
capable of quantifying T cell concentrations in vivo. This indicates that PET imaging agent that bind to Itk might ultimately allow
measuring the distribution of T cells in patients.
15
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