Studies towards the development of a PET radiotracer for imaging of the P2Y1 receptors in the brain: synthesis, 18F-labeling and preliminary biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2019.01.006

Purine nucleotides such as ATP and ADP are important extracellular signaling molecules in almost all tissues activating various subtypes of purinoreceptors. In the brain, the P2Y₁ receptor (P2Y₁R) subtype mediates trophic functions l

Full text of DOI:10.1016/j.ejmech.2019.01.006

Accepted Manuscript
Studies towards the development of a PET radiotracer for imaging of the P2Y
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18
receptors in the brain: synthesis, F-labeling and preliminary biological evaluation
Rareş-Petru Moldovan, Barbara Wenzel, Rodrigo Teodoro, Wilma Neumann,
Sladjana Dukic-Stefanovic, Werner Kraus, Peijing Rong, Winnie Deuther-Conrad,
Evamarie Hey-Hawkins, Ute Krügel, Peter Brust
PII:
S0223-5234(19)30006-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.006
EJMECH 11013
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 September 2018
Revised Date: 20 December 2018
Accepted Date: 4 January 2019
Please cite this article as: Rareş.-Petru. Moldovan, B. Wenzel, R. Teodoro, W. Neumann, S. Dukic-
Stefanovic, W. Kraus, P. Rong, W. Deuther-Conrad, E. Hey-Hawkins, U. Krügel, P. Brust, Studies
towards the development of a PET radiotracer for imaging of the P2Y receptors in the brain: synthesis,
1
18
F-labeling and preliminary biological evaluation, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.01.006.
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Graphical abstract

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Studies towards the development of a PET
radiotracer for imaging of the P2Y receptors in the
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brain: synthesis, F-labeling and preliminary
biological evaluation
a,
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Rareş-Petru Moldovan ⃰, Barbara Wenzel , Rodrigo Teodoro , Wilma Neumann , Sladjana
a
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Dukic-Stefanovic , Werner Kraus , Peijing Rong , Winnie Deuther-Conrad , Evamarie
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d§
a§
Hey-Hawkins , Ute Krügel and Peter Brust
a
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Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research,
Permoserstraße 15, 04318 Leipzig, Germany
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BAM Federal Institute for Materials Research and Testing, Richard-Willstätter-Str. 11, 12489
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Berlin, Germany
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Institute of Inorganic Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig,
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04103 Leipzig, Germany
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Rudolf Boehm Institute of Pharmacology and Toxicology, Medical Faculty, Universität
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Leipzig, 04107 Leipzig, Germany
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Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences,
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100700 Beijing, China
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*Corresponding author: R.-P. Moldovan, E-mail address: r.moldovan@hzdr.de, Tel: +49 (0)341
234 179 4634
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Equal contribution
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ABSTRACT Purine nucleotides such as ATP and ADP are important extracellular signaling
molecules in almost all tissues activating various subtypes of purinoreceptors. In the brain, the
P2Y receptor (P2Y R) subtype mediates trophic functions like differentiation and proliferation,
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and modulates fast synaptic transmission, both suggested to be affected in diseases of the central
nervous system. Research on P2Y R is limited because suitable brain-penetrating P2Y R-
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selective tracers are not yet available. Here, we describe the first efforts to develop an F-labeled
PET tracer based on the structure of the highly affine and selective, non-nucleotidic P2Y
allosteric modulator 1-(2-[2-(tert-butyl)phenoxy]pyridin-3-yl)-3-[4-
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R
(trifluoromethoxy)phenyl]urea (7). A small series of fluorinated compounds was developed by
systematic modification of the p-(trifluoromethoxy)phenyl, the urea and the 2-pyridyl subunits of
the lead compound 7. Additionally, the p-(trifluoromethoxy)phenyl subunit was substituted by
carborane, a boron-rich cluster with potential applicability in boron neutron capture therapy
(BNCT). By functional assays, the new fluorinated derivative 1-{2-[2-(tert-
butyl)phenoxy]pyridin-3-yl}-3-[4-(2-fluoroethyl)phenyl]urea (18) was identified with a high
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P2Y R antagonistic potency (IC50 = 10 nM). Compound [ F]18 was radiosynthesized by using
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tetra-n-butyl ammonium [ F]fluoride with high radiochemical purity, radiochemical yield and
molar activities. Investigation of brain homogenates using hydrophilic interaction
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chromatography (HILIC) revealed [ F]fluoride as major radiometabolite. Although [ F]18
showed fast in vivo metabolization, the high potency and unique allosteric binding mode makes
this class of compounds interesting for further optimizations and investigation of the theranostic
potential as PET tracer and BNCT agent.
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Graphical abstract
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Keywords Purine P2Y
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receptors, positron emission tomography, brain PET tracers,
radiometabolites, micellar chromatography, hydrophilic interaction chromatography
Highlights
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A series of fluorinated, non-nucleotidic P2Y R ligands was developed
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Derivative 18 was identified with an 18-fold higher antagonistic potency than the lead
compound 7
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[ F]18 was obtained in high radiochemical purity, yield and molar activities
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INTRODUCTION
Purinoreceptors are a family of plasma membrane proteins found in almost all mammalian
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tissues including the brain. The P1 receptors (R) are activated by extracellular adenosine. The
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P2R are further classified into the P2XR and P2YR families. P2XR are ligand-gated ion
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channels, preferentially activated by extracellular adenosine triphosphate (ATP). P2YR are G
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protein-coupled receptors (GPCR) activated by ATP, adenosine diphosphate (ADP), uridine
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0
triphosphate (UTP) and/or uridine diphosphate (UDP) as main endogenous ligands. Dependent
on the cellular activity of neurons or glial cells in the brain, ATP is released into the extracellular
space where the nucleotide or its breakdown products interact with the corresponding cell
surface purinoreceptors, thereby modulating fast cell-to-cell signaling such as neurotransmitter
11-14
release, or long-term trophic processes like astrogliosis.
So far, eight metabotropic P2YR subtypes have been identified, namely P2Y , P2Y , P2Y ,
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5
, 10, 15-17
P2Y
6
, P2Y11, P2Y12, P2Y13 and P2Y14
.
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In particular, the P2Y R subtype, which has
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higher affinity to ADP than to ATP is involved in various physiological brain functions and
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processes related to mental and neurological disturbances.
Alterations in P2Y R activity or expression are suggested to be part of the pathophysiology of
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neurodegeneration, like Alzheimer’s disease,
of neuroprotection and neuroregeneration in
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epilepsy,
brain trauma and stroke
and of cellular proliferation of brain tumors.
Furthermore, behavioral and cognitive dysfunction in psychiatric diseases is attributed to the
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involvement of P2Y R in synaptic transmission and plasticity.
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The present knowledge on P2Y R in the normal and diseased brain is limited and quantitative data
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on its protein density in brain are rare, mainly because of lack of highly receptor-selective
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compounds that readily pass the blood-brain barrier and allow in vivo imaging of this receptor
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subtype in the brain.
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In chicken and rat brain membranes, a Bmax for the nucleotidic ligand [ S]dATPαS of 37 and
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39 pmol/mg membrane protein, respectively, was reported. Levels of [ S]dATPαS binding in
individual structures (0.047–0.309 pmol/mg wet tissue) in chick brain are suggested to be
comparable to those found for other G protein-linked receptors such as the muscarinic, dopamine
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7, 30
and α2-adrenergic receptors and corresponded to their respective local transcripts.
The
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distribution of [ S]dATPαS specific binding sites was widespread throughout the rat brain,
prominent in several regions including piriform cortex, ectostriatum, cerebellum and some nuclei
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in the telencephalon and mesencephalon.
Areas of high binding capacity in this
autoradiographic brain study match to more recent data of mouse and human brain transcripts,
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which are available from the Allen Brain Atlas. In addition, data from human brain and
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studies on rat
confirm a high distribution of P2Y R at the protein level suggesting that this
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protein target is expressed at high enough local concentrations in health or disease for imaging.
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The present study aims to explore strategies for the development of compounds of theranostic
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potential combining properties of an F-labeled PET tracer and of a boron-based neutron capture
therapeutic (BNTC) agent based on the structure of the highly affine and selective, non-
nucleotidic P2Y
(trifluoromethoxy)phenyl)urea (BPTU, 7 Figure 1).
Given the nucleotidic nature of the endogenous P2Y
synthetic ligands with a similar structural skeleton, and even sub-nanomolar affinity and high
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R
allosteric modulator 1-(2-(2-(tert-butyl)phenoxy)pyridin-3-yl)-3-(4-
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R ligand ADP (1, Figure 1), numerous
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5, 37, 38
selectivity for the P2Y R were developed to date.
Although 1 itself is a rather weak and
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non-selective agonist at the P2Y R, chemical modifications of this molecule led to candidates
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applicable to prove P2Y
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R as potential therapeutic target (e.g. compounds 2, 3 and 4, Figure
Since nucleotidic ligands are thought to pass the blood brain-barrier only to a minor
degree because of their negative charge at physiological pH and low capacity of the available
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1A).
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transporters, they cannot be considered for development of PET tracers for brain imaging.
Only a few non-nucleotidic P2Y R ligands with sub-micromolar affinity have been reported so
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far.
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The predominant scaffold, diarylurea, plays a crucial role for binding to the P2Y R
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5
(Figure 1). As an example, compound 5 (K = 10 nM) was reported by Pfizer laboratories.
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Similarly, GlaxoSmithKline laboratories identified compound 6 with lower micromolar affinity
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(K = 0.6 µM), and Bristol-Myers Squibb (BMS) laboratories identified compound 7 (K = 6
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5, 36
nM, Figure 1).
Beside high affinity towards the P2Y R, compound 7 and related derivatives
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were highly selective, with no affinity towards P2Y R, P2Y R, P2Y R, P2Y R and P2Y R
2
6
11
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14
(K > 1000nM).
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Figure 1. Selected P2Y R ligands. (A) ADP (1) and the P2Y R nucleotidic ligands 2-4; (B) non-
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nucleotidic ligands 5-7; (C) and (D) the structures of the P2Y R–3 and P2Y R–7 complexes with
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their orthosteric and allosteric ligand, respectively; the P2Y R is shown in cartoon representation
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and ligands 3 and 7 are shown in sphere representation. For further details see ref. . The picture
is used by courtesy of Springer Nature License Number 4344250476031.
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The functional antagonism and signaling patterns of the novel allosteric modulator 7 in
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combination with orthosteric antagonists was subject of various studies.
Recently, the
binding mode of compound 7 to the P2Y R was elucidated by X-ray crystal structure analysis
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revealing a unique, allosteric binding pocket, located entirely on the outer surface of the GPCR
(Figure 1D). It illustrates that the affinity determination using binding assays with orthosteric
radioligands is biased by the indirect rather than direct competition of allosteric modulators (e.g.
7-9, Figure 2). Furthermore, the reported data highlight the potential of targeting the P2Y R via
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the allosteric binding pocket, e.g., by the non-nucleotidic compound 7 or its analogues.
The low nanomolar P2Y
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R affinity, the high selectivity and the non-nucleotidic nature of these
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diaryl-urea ligands qualify this scaffold as a good starting point for the development of an F-
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labeled tracer for brain PET imaging. Analogously to compounds 8 and 9 (Figure 2), the
introduction of a fluoroethyl or a fluoroethoxy group should be tolerated at the 4-position in
phenyl ring D, enabling a facile S 2 radiofluorination at an aliphatic position.
N
A major challenge in the developmental process of PET tracers for brain imaging, beside the
brain permeability of the target radioactive compound, is the requirement of absence of brain
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penetrant radiometabolites. Wang et al. identified the oxidative hydroxylation of the tert-butyl
group as one of the major metabolic degradation pathways for this type of compounds. To
overcome the risk for such metabolites, the tert-butyl subunit might be replaced by the metabolic
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more stable group like cyclopropy-CF . However, primarily, the fluorinated derivatives should
be synthesized with tert-butyl and isopropyl as substituents. Although not described as a
metabolic labile partial structure for this type of compounds we proposed the replacement of the
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2
urea (subunit C, Figure 2) by a metabolically more stable heterocycle like thiazole. Moreover,
the substitution of the fluorine at the 2-position of the pyridine ring B facilitates the introduction
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of the F-label on an aromatic position which is described to be beneficial regarding metabolic
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stability.
Beside the potential of such a PET tracer as diagnostic tool, it is worthwhile to explore the
therapeutic potential of this scaffold in boron neutron capture therapy (BNCT). Therefore, we
coupled a carborane, an icosahedral boron cluster that can serve as three-dimensional analog of
aromatic hydrocarbons and that has been proposed as neutron capture moiety for BNCT, in
compound 7. Recently, we reported the possibility to replace phenyl rings with a carborane
moiety in established cyclooxygenase (COX) inhibitors to generate highly potent and selective
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inhibitors.
Likewise, our current approach is to replace the p-(trifluoromethoxy)phenyl
subunit of compound 7 (ring D) by an ortho-carborane (partial structure IV, Figure 2). The
successful implementation of a carborane at subunit D without affecting the binding affinity
towards the P2Y
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R will set the stage for introduction of fluorine at the 2-position of the pyridine
(ring B, Figure 2). As a result, a bifunctional P2Y R-ligand is designed with the theranostic
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potential of being used as both F-PET tracer and BNCT agent.
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Figure 2. The structure of compounds 7, 8 and 9 and proposed fluoro derivatization.
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The investigation of the binding affinity of our newly designed ligands is currently hindered by
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the lack of a binding assay for allosteric P2Y R modulators, making the in-vitro characterization
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of these ligands challenging. Here, we used a commercially available functional assay to assess
the antagonistic potency of P2Y R ligands as selection criteria for potential use in PET.
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The successful development of a PET/BNCT tracer will enable the study of the role and
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distribution of the P2Y R in the brain, as well as the involvement of the P2Y R in various cancer
types and brain diseases (e.g. neurodegeneration).
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RESULTS AND DISCUSSION
Chemistry
The synthesis of the lead compound 7 (Figure 2 and Scheme 1) was performed as described by
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BMS with small modifications. The coupling of the 2-tert-butylphenol (10a) with the pyridine
derivative 11 in the presence of Cs CO was followed by reduction of the nitro group (with Pd/C,
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3
H ) to the corresponding amine 12a and subsequent treatment with diphosgene in presence of
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Et N to give the isocyanate key intermediate 13a in 55% yield over three steps. For several
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examples, isopropyl was used instead of a tert-butyl group (Scheme 1, R ) in order to investigate
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the influence of this position on the binding potency or metabolic stability. The herein obtained
isocyanate was coupled without further purification with various amines to give the
corresponding urea derivatives in high yields (>70%, see Experimental Section). The fluoroethyl
compounds 18 and 19 were obtained from the alcohols 14 and 15, respectively, by tosylation and
subsequent fluorination with TBAF. The mono- and tri-fluoroethoxylated derivatives 22 and 23
were obtained starting from the methoxylated intermediate 20 upon deprotection to phenol
derivative 21 and subsequent fluoroalkylation under basic reaction conditions (Scheme 1).
Furthermore, compounds 24 and 25 bearing the fluorine in the 2-position of the phenyl ring were
synthesized.
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With large amounts of compound 12a in our hands, we also prepared the 2-aminothiazole ring
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as previously described. Accordingly, compound 12a was treated with benzoyl isothiocyanate,
hydrolyzed in the presence of LiOH and cyclized with 3-bromo-1,1,1-trifluoropropan-2-one in
the presence of a base at elevated temperature to give compound 26. Bromination at the thiazole
5-position of compound 27 gave the suitable precursor for the subsequent Suzuki coupling. We
also introduced the fluorine atom at the 2-position of a pyridine ring due to the facile
radiofluorination at this position. We chose the 2-fluoropyridin-5-boronic acid and 2-
5
7, 58
fluoropyridin-3-boronic acid as coupling partner for the Suzuki reaction
and thus,
compounds 28 and 29 were obtained in high yields (>70% yield, see Experimental Section)
(Scheme 1). Additionally, the 4-fluorophenyl derivative 30 was synthesized.
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Scheme 1. Synthesis of the fluorinated diaryl-urea derivatives 7, 14-25 and 2-
a
aminothiazoles 28-30
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Reagents and conditions: (a) Cs CO , DMF, 70 °C, 14 h; (b) Pd/C, H ; (c) diphosgene, Et N,
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DCM, 0 °C to rt, 2 h; (d) corresponding substituted aniline, toluene, 80 °C, 1 h; (e) TsCl,
pyridine, DCM, rt, 16 h; (f) TBAF, THF, rt, 16 h; (g) RX, K CO , CH CN, 80 °C, 16 h; (h) i.
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benzoyl isothiocyanate, DCM, 40 °C, 2 h; ii. 2M LiOH/MeOH, 50 °C, 2 h; (i) 3-bromo-1,1,1-
trifluoropropan-2-one, 2,6-lutidine, EtOH, 80 °C, 24 h; (j) NBS, AcOH/THF, 0 °C, 2 h; (k)
Suzuki coupling using [Pd(PPh ) ], Na CO , toluene/EtOH, 95 °C, 16 h; (m) BBr , DCM; TsCl =
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3 4
p-toluenesulfonyl chloride; [Pd(PPh ) ] = tetrakis(triphenylphosphine)palladium(0); TBAF =
tetra-n-butylammonium fluoride; NBS = N-bromosuccinimide.
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14
Apart from this series of fluorinated derivatives shown in Scheme 1, the p-
(trifluoromethoxy)phenyl subunit of compound 7 was replaced with a carborane cluster using
amino-carborane 31 as coupling partner for the isocyanate 13a. Carborane 31 was obtained based
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on a literature protocol from the commercially available ortho-carborane. The formation of the
urea function in compound 32 was much slower (24 h) compared to the other urea derivatives
shown here, probably due to the low nucleophilicity of the amino group of the carborane
derivative 31.
a
Scheme 2. Synthesis of the carborane derivative 32
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a
Reagents and conditions: a) toluene, 80 °C, 24 h.
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In addition, the incorporation of a fluorine atom at the 2-position of the pyridine (ring B,
Figure 2) of 7 was performed. Compound 33 was reacted with 2-tert-butylphenol (10a) in the
presence of a base to give the two regioisomers 34 and 35. After separation of these regioisomers
by flash chromatography and characterization by 2D-NMR spectroscopy, the formation of the
urea group was performed as described above (Scheme 1 and Scheme 2) to afford the 2-
fluoropyridine derivative 36 over three steps. Several further residues (R) were used to form
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t
analogues of 36 including 4- Bu-phenyl (37) and the more hydrophilic 4-hydroxyethanol phenyl
38. Moreover, the non-aromatic adamantyl, cyclohexyl and cyclopentyl rings were implemented
in order to assess the scope and limitation of this subunit regarding the biological activity
(Scheme 2, compounds 39-41). The same synthesis sequence (Scheme 2 (b)-(d)) was carried out
starting from 35 to give 42, a regioisomer of 36. Additionally, the hydrophobic derivative 43 was
synthesized. The molecular structure of 36 was unambiguously confirmed by single-crystal
diffraction analysis to prove the molecular configuration shown in Figure 3 (for crystallographic
data and structure refinement parameters see Supplementary Information).
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Scheme 3. Synthesis of the 2-fluoropyridine compounds 36-43
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Reagents and conditions: (a) Cs CO , DMF, 70 °C, 14 h (34, 43% and 35, 35%); (b) Pd/C, H ;
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(c) diphosgene, Et N, DCM, 0 °C to rt, 2 h; (d) corresponding substituted aniline, toluene, 80 °C,
3
1 h.
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A new series of derivatives was developed by converting the two regioisomeric amines 44 and
50 into the corresponding 2-aminothiazoles 45 (Scheme 4) and 51 (Scheme 5), respectively.
NBS-promoted bromination at the 5-position of the thiazole ring gave the precursors for the
subsequent Suzuki coupling reactions to obtain compounds 46 and 52, which were further
coupled with selected boronic acids to give compounds 47-49 and 53-54 in moderate to high
yields (see Experimental Section).
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Figure 3. ORTEP representation of compound 36. Atomic labeling is shown with 30%
probability displacement ellipsoids (CCDC number 1849365).
a
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Scheme 4. Synthesis of the 2-fluoropyridyl-5-(2-aminothiazoles) 47-49
Br
R
CF3
CF3
CF3
S
S
S
F
NH2
N
N
N
N
(a), (b)
(c)
(d)
F
NH
F
NH
F
NH
O
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N
N
O
O
O
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46
47, R = phenyl
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9, R = 3-pyridyl
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a
Reagents and conditions: (a) i. benzoyl isothiocyanate, DCM, 40 °C, 2 h; ii. 2M LiOH/MeOH,
50 °C, 2 h; (b) 3-bromo-1,1,1-trifluoropropan-2-one, 2,6-lutidine, EtOH, 80 °C, 24 h; (c) NBS,
7
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AcOH/THF, 0 °C, 2 h; (d) Suzuki coupling using [Pd(PPh
3
)
4
], Na
2
CO
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, toluene/EtOH, 95 °C, 16
h.
a
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Scheme 5. Synthesis of the 2-fluoropyridyl-3-(2-aminothiazoles) 53 and 54
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Reagents and conditions: (a) i. benzoyl isothiocyanate, DCM, 40 °C, 2 h; ii. 2M LiOH/MeOH,
3
4
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50 °C, 2 h; (b) 3-bromo-1,1,1-trifluoropropan-2-one, 2,6-lutidine, EtOH, 80 °C, 24 h; (c) NBS,
AcOH/THF, 0 °C, 2 h; (d) Suzuki coupling using [Pd(PPh ) ], Na CO , toluene/EtOH, 95 °C, 16
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h.
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In vitro biological evaluation
Using a commercially available cellular functional assay, the literature-known compound 7
together with the herein described new derivatives were studied for both agonistic and
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antagonistic effects. The agonistic effect was investigated using the known P2Y R agonist 55
1
(MRS2365,
{[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl]-2,3-
dihydroxybicyclo[3.1.0]hex-1-yl]methyl} diphosphoric acid monoester trisodium salt) as
reference compound at a single concentration (20 nM). The antagonistic effect was investigated
3
8
using compound 3 as reference compound at two concentrations, 20 and 100 nM, respectively.
3
6
As expected, these compounds are devoid of any agonistic effect. As shown in Table 1, at the
given concentrations, the lead compound 7 has a low antagonistic effect. At a concentration of
20 nM, only compounds 18 and 38 had a slight antagonistic effect. However, at a concentration
of 100 nM compound 18 strongly inhibited the agonist response, whereas compounds 7, 24, and
40 were only weak antagonists. Within the 2-fluoropyridine series, only compound 40 showed a
slight antagonism at 100 nM. None of the 2-aminothiazole derivatives showed any potency. The
regioisomeric derivatives 42, 43, 51, 53 and 54 were also ineffective. To reinforce the results
from Table 1, lead compound 7, the fluoroethyl derivative 18, the fluoropyridine 38 and the
carborane 32 were subject of a detailed study on antagonist effectivity (Figure 4). Among them,
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compound 18 (IC50 = 10 nM) possesses ~18-fold higher antagonistic potency than the starting
compound 7 (IC50 = 180 nM). The 2-fluoropyridine derivative 38 is characterized by an IC50
value of 490 nM but the carborane 32 failed to have any antagonistic potency at the P2Y R.
1
Table 1. Results from cellular functional assays investigating antagonistic and agonistic
effects of selected compounds
a
b
Agonism
Antagonism
1
2
3
4
R
R
R
R
(
100
(
20 nM)
(20 nM)
nM)
t
7
Bu
H
H
H
H
-12
-14
-10
26
22
73
t
1
8
9
Bu
i
1
Pr
H
H
NA
-14
15
t
2
2
2
2
2
3
4
5
Bu
H
H
H
H
H
H
H
H
-15
-14
NA
NA
-5
NA
4
t
Bu
-12
6
t
Bu
16
4
i
Pr
-18
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t
2
2
3
8
9
0
Bu
H
H
H
H
H
H
-11
NA
NA
-20
7
-5
t
Bu
-14
-15
t
Bu
-1
t
3
3
3
2
6
7
Bu
H
F
F
H
H
H
NA
-9
1.5
-17
3
NA
-19
-11
t
Bu
t
Bu
NA
t
3
3
4
4
8
9
0
1
Bu
F
F
F
F
H
H
H
H
-7
18
-7
7
NA
-7
t
Bu
-8
t
Bu
NA
NA
26
2
t
Bu
6
t
4
4
2
3
Bu
F
F
H
H
-1
-16
-13
-24
-21
t
Bu
t
4
6
Bu
F
H
NA
-21
-4
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4
4
4
5
7
8
9
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Bu
F
F
F
F
H
H
H
H
NA
NA
NA
NA
-1
-14
10
6
t
Bu
10
0
t
Bu
t
Bu
-27
-16
t
5
5
3
4
Bu
F
F
H
H
NA
NA
-3
-17
-16
t
Bu
11
a
b
1
2
% of control agonist response using 55 as reference compound (n = 2); % inhibition of
control agonist response using 3 as reference compound (n = 2); NA = not available.
3
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Figure 4. P2Y
1
R antagonistic properties of compounds 7, 18, 32 and 38. (N.E. = no effect)
2
3
4
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6
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8
9
0
1
2
Radiochemistry
As derivative 18 demonstrated the strongest antagonistic response, it was selected for F-
1
8
1
8
labeling. The new radioligand [ F]18 was prepared by nucleophilic substitution of the tosylate
1
8
group of precursor 16 (Scheme 1) using anhydrous [ F]TBAF in tert-BuOH (Scheme 6). Under
conventional heating at 90 °C and 10 min reaction time, radiochemical yields (RCY) of 58.0 ±
6.4% (n = 5) were achieved for the labeling process. According to radio-TLC analysis, two
radioactive by-products were observed in the reaction mixture accounting for about 15% of total
1
8
radioactivity. Interestingly, when the classical K[ F]F-K2.2.2-carbonate complex system and
1
1
1
acetonitrile (CH CN) at 90 °C were used, a considerably lower RCY was achieved (< 9%). The
3
precursor 16 remained stable under all conditions tested as proven by HPLC.
1
8
Scheme 6. F-Radiolabeling of compound 16
1
3
4
1
8
1
The isolation of [ F]18 was performed by semi-preparative RP-HPLC (Figure 5A). The
collected product was purified using solid-phase extraction on an RP cartridge and formulated in
sterile isotonic saline containing 10% of EtOH for better solubility. Analytical radio- and UV-
HPLC of the final product, spiked with the nonlabeled reference compound, confirmed the
15
16
17
18
19
20
21
22
1
8
identity of [ F]18 (Figure 5B). Finally, the formulated radiotracer was obtained with a
radiochemical purity of 99%, in a radiochemical yield (EOB) of 28.2 ± 0.8% (n = 4, decay
corrected), and molar activities (EOS) in the range of 153–283 GBq/µmol using starting
≥
1
8
activities of 2-3 GBq. The logD value of [ F]18 was experimentally determined by the
shake-flask method to be 3.64 ± 0.33 (see Experimental Section).
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Figure 5. A: Semi-preparative radio- and UV-HPLC chromatograms of [ F]18 (conditions:
3
4
5
6
Reprosil-Pur C18-AQ, 250 x 10 mm, 62% CH CN/20 mM aq NH OAc , 4.0 mL/min). B:
3
4
.
1
8
Analytical radio- and UV-HPLC chromatograms of the final product of [ F]18 spiked with the
nonradioactive reference 18 (conditions: Reprosil-Pur C18-AQ, 250 x 4.6 mm, gradient with
3 4
eluent mixture of CH CN/20 mM aq NH OAc, 1.0 mL/min).
1
8
7
8
9
0
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2
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4
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6
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8
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2
3
Metabolism studies of [ F]18 in vivo
1
8
Metabolism of [ F]18 in vivo was investigated in plasma and brain samples obtained from
CD-1 mice (mice lacking the cluster of differentiation 1 molecules) in two independent
experiments at 30 min after injection of the radioligand. At this time point, the activity in plasma
and brain accounted for 1.7 and 1.7% ID/g respectively. Sample analysis was performed by
micellar chromatography (MLC) and RP-HPLC. MLC allows a direct injection of samples into
the HPLC system without eliminating the tissue matrix, whereas samples for RP-HPLC analysis
need to be treated with a mixture of methanol/water to precipitate the proteins, resulting in
recoveries of about 70% and 60% of activity for plasma and brain samples, respectively.
Data obtained with both methods indicated a fast metabolic degradation of the radiotracer.
Radio-MLC and radio-RP-HPLC chromatograms of plasma and brain samples showed only
1
1
1
1
1
1
1
1
1
1
2
2
2
2
1
8
negligible amounts of [ F]18 and the formation of a main radiometabolite representing more
than 85% of total activity (Figure 6). Due to the high amount of activity found in the brain at this
time point (1.7% ID/g), it can be excluded that the activity found there is related to
contaminations with residual blood. The very fast elution in both chromatography systems
indicates a high polarity of the radiometabolite. Moreover, the observed low recovery during the
1
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extraction procedure indicates free [ F]fluoride as the main radiometabolite that partially binds
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to the precipitated proteins. To verify this assumption, brain homogenate samples were
investigated for the first time with hydrophilic interaction chromatography (HILIC). Due to the
use of a polar stationary phase and a mobile phase with a high content of organic solvent, the
6
0
retention of analytes is prolonged with their increasing polarity. Therefore, the elution order in
HILIC is more or less the opposite of the elution order in RP-HPLC as illustrated by
1
8
chromatogram (c) in Figure 6C. Accordingly, [ F]18 elutes at a very short retention time of
1
8
about 3 min followed by the much more polar [ F]fluoride at about 14 min. The assumption of
defluorination is supported by comparing chromatogram (b) in Figure 6C obtained from a brain
1
8
sample spiked with [ F]fluoride and chromatogram (a) representing a corresponding non-spiked
1
1
1
1
1
1
1
1
1
1
2
2
2
brain sample. A potential mechanism for radiodefluorination of a fluoroethyl chain bound to an
6
1-64
aromatic ring was already proposed by Lee et al.
involving an α-carbon hydroxylation
followed by oxidation and slow -elimination with the formation of the halide. As fluoride does
α
1
8
not pass the blood-brain barrier in a considerable extent, the [ F]fluoride detected in the brain
may be due to local metabolism or to degradation during sample preparation. The latter can be
excluded, because the stability of the radiotracer has been proven under the conditions used for
6
5
MLC. Drug metabolism in the brain is a phenomenon that is currently under investigation. In
particular, the central expression and function of several cytochrome P450 containing enzymes
(CYP) enzymes support this hypothesis. Despite low CYP levels in the brain with approximately
6
5
0.5-2% of those in the liver, it is conceivable that a CYP-mediated metabolism influences
radiotracer stability because of its negligible injected mass. This is in agreement with Coenen et
6
6
18
al. who determined a certain level of [ F]fluoride in mouse brain samples. Thereupon Welch et
6
7
al. postulated a metabolic process within the brain as a possible explanation.
2
3
4
1
8
2
Figure 6. Representative radio chromatograms of a mouse plasma and brain sample of [ F]18 at
30 min p.i.. A: Micellar chromatography (conditions: Reprosil-Pur C18-AQ, 250 x 4.6 mm,
2
5
6
2
2
gradient: 3-30-3% PrOH/100 mM SDSaq, 10 mM Na HPO4aq; flow: 0.75 mL/min). B: Reversed
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phase chromatography (conditions: Reprosil-Pur C18-AQ, 250 x 4.6 mm, gradient: 10-90-10%
MeCN/20 mM NH OAc ; flow 1.0 mL/min). C: Hydrophilic interaction chromatography; a:
4
aq
radio chromatogram of a mouse brain sample, b: radio chromatogram of a mouse brain sample
1
8
18
18
spiked with [ F]fluoride, c: chromatogram of a mixture of [ F]18 and [ F]fluoride (conditions:
Nucleodur HILIC, 250 x 4.6 mm, 74% MeCN/20 mM NH OAc , flow 1.0 mL/min).
4
aq
1
8
6
7
8
9
0
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2
3
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Finally, the observed strong radiodefluorination of [ F]18 was rather unexpected, considering
that a 2-fluoroethyl aryl group is thought to be slightly more stable than the corresponding 2-
fluoroethoxy aryl analogue (compound 22, Scheme 1) which is known to be metabolized quite
1
8
fast via CYP-mediated O-dealkylation. Recently, we reported on the development of an F-
6
8
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
labeled radiotracer for imaging the oxytocin receptor, which also contains a fluoroethyl
function and did not demonstrate such a fast metabolic degradation. Nevertheless, the metabolic
1
8
stability of a particular F-bearing functional group considerably depends on the entire
molecular structure of the compound. As reviewed by Kirchmair and co-workers, the prediction
of potential target sites within a compound for metabolic degradation is challenging and needs
6
9
further research.
These results might support but do not prove the brain-penetrant nature of this class of
1
8
compounds, however the fast metabolization of [ F]18 hinders any further investigation of the
1
8
P2Y R in vivo with PET. Several methods to increase the metabolic stability of F-labeled
1
5
3
tracers are described in the literature. One option might be a deuterated fluoroethyl chain to
1
8
decelerate the defluorination process. Alternatively, the F-label could be introduced at an
aromatic ring, e.g. at the 2-pyridine position.
CONCLUSIONS
In summary, a novel, small series of fluorinated compounds was synthesized to develop an
1
8
F-PET radiotracer for P2Y R imaging in the brain by modifying the structure of the highly
1
affine and selective P2Y R ligand 7. Additionally, the boron-rich derivative 32 was designed by
1
replacing the p-(trifluoromethoxy)phenyl subunit with an ortho-carborane as potential tool for
BNCT for cancer treatment. Preliminary in vitro investigations revealed a strong antagonistic
1
8
effect for the fluorinated compound 18. Therefore, 18 was selected for F-labeling. A manual
1
8
radiosynthesis procedure was developed to generate [ F]18 with suitable radiochemical
parameters. First in vivo experiments performed with CD-1 mice revealed a fast metabolism with
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nearly no intact [ F]18 at 30 minutes post injection in plasma and brain samples. The main
1
8
radiometabolite was identified by HILIC to be [ F]fluoride. Future efforts will focus on the
1
8
development of an analogue of [ F]18 with a deuterated fluoroethyl chain or investigation of
alternative labeling positions (e.g. the 2-pyridine position) to obtain metabolically more stable
compounds suitable for the in vivo mapping of the P2Y R.
1
6
7
EXPERIMENTAL SECTION
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Chemistry
General methods
1
1
1
1
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1
2
2
2
2
2
2
2
2
2
2
3
3
Unless otherwise noted, moisture-sensitive reactions were conducted under dry nitrogen or
argon. All chemicals and reagents were purchased from commercially available sources and used
without further purification. Thin layer chromatography (TLC): Silica gel 60 F254 plates (Merck
KGaA, Darmstadt, Germany). Flash chromatography (fc): Silica gel 60, 40-64
µm (Merck).
Room temperature (rt) was 21 °C. MS: MAT GCQ (Thermo Finnigan MAT GmbH, Bremen,
1
13
19
Germany). H, C and F NMR spectra were recorded on VARIAN "MERCURY plus" (300
1
13
19
MHz for H NMR, 75 MHz for C NMR, 282 MHz for F NMR) and VARIAN "MERCURY
1
13
19
plus" and BRUKER DRX-400 (400 MHz for H NMR, 100 MHz for C NMR, 377 MHz for F
NMR); in ppm related to tetramethylsilane; coupling constants (J) are given with 0.1 Hz
δ
resolution. Multiplicities of NMR signals are indicated as follows: s (singlet), d (doublet), t
(triplet), m (multiplet), dd (doublet of doublets), dt (doublet of triplets). ESI/Ion trap mass
spectra (LRMS) were recorded with a Bruker Esquire 3000 plus instrument (Billerica, MA,
USA). High resolution mass spectra were recorded on a FT-ICR APEX II spectrometer (Bruker
Daltonics; Bruker Corporation, Billerica, MA, USA) using electrospray ionization (ESI) in
positive ion mode. The purity of all the tested compounds was
[unless otherwise noted: Jasco, MD-2010Plus, LG-2080-04S, DG-2080-54, AS-2055Plus, LC-
NetII/ADC, = 254 nm, column ReproSil-Pur Basic C18-HD (250 x 4.6 mm, 5 m, Dr. Maisch
GmbH), gradient MeCN/20mM NH OAc from 10/90 to 90/10, (v/v) over 35 min, flowrate 1
≥95% as determined by HPLC
λ
µ
4
aq
mL/Min].
Procedures and compound characterization
General procedure A: 2-Halo-3-nitropyridine (1 eq, 7.3 mmol) in DMF (6 mL) was treated
with 2-isopropylphenol (1 eq, 7.3 mmol) and Cs CO (1.5 eq, 10.9 mmol) and heated at 70 °C
2
3
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for 14 h. Afterwards, the reaction mixture was cooled to room temperature, and DMF was
evaporated. The residue was dissolved in ethyl acetate (EA, 20 mL) and washed with 5% LiCl aq
sol. (3 × 7 mL) and brine (3 × 7 mL). Drying (MgSO ) and removal of solvent afforded a brown
4
solid which was recrystallized from ethanol to afford the corresponding ether as yellow needles.
General procedure B: A solution of nitro compound (1 eq, 7.4 mmol) in MeOH/THF (1:4, 16
2
mL) was reduced under H atmosphere (1 atm) in the presence of Pd/C (10 mol%) overnight at
room temperature. The reaction mixture was filtered over Celite and concentrated to afford a
white solid which was recrystallized from EA to afford the corresponding amine as a white
powder.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
General procedure C: A solution of dichloromethane (DCM, 10 mL) containing Et N (3 eq,
3
13 mmol) was added dropwise to a solution of the amine (1 eq, 4.4 mmol) and diphosgene (0.8
eq, 3.5 mmol) in DCM (20 mL) at 0 °C. After the addition was completed, the resulting mixture
was stirred at 0 °C for 45 min and additional 2 hours at room temperature. The organic phase
was then washed with 0.5 M HCl (3 × 20 mL), 1 N NaOH (2 × 10 mL), and brine. Drying
(MgSO ) and removal of solvent afforded the corresponding isocyanate as a yellow solid. This
4
material was used for the subsequent reaction without further purification
General procedure D: The substituted aniline (1 eq, 0.2 mmol) was added to a solution of
isocyanate (1 eq, 0.2 mmol) in toluene (1 mL) under argon. The resulting mixture was stirred at
80 °C for 1.5 h. The solvent was removed by rotary evaporation, and the crude product was
purified by flash chromatography to give the diaryl urea as colorless solid.
General procedure E: To a solution of the corresponding alcohol (1 eq, 0.25 mmol) in 4 mL
DCM, 4-toluenesulfonyl chloride (60 mg, 1.2 eq, 0.30 mmol) and pyridine (1 mL) were added
and the reaction mixture was stirred overnight at room temperature. The reaction was quenched
by addition of 20 mL 1 M HCl solution. The aqueous solution was extracted with DCM (2 x 20
mL) and the combined organic phases were washed with NaHCO (20 mL), brine (10 mL) and
3
4
dried over MgSO . Evaporation of the solvent under reduced pressure afforded the
corresponding tosylate as colorless solid.
General procedure F: A solution of tosylate (1 eq, 0.09 mmol) in THF (2 mL) was treated
with a 1M TBAF solution in THF (180 µL, 2 eq, 0.18 mmol) and stirred overnight at room
temperature. Saturated aqueous NaHCO (5 mL) was added and the mixture was washed with
3
4
EA (3 x 5 mL). The combined organic phases were dried (MgSO ) and concentrated by rotary
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evaporation. The remaining residue was purified by column chromatography (silica) to give the
fluorinated derivative as colorless solid.
General procedure G: A mixture of amine (e.g. 12a, 1 eq, 2.06 mmol) and benzoyl
isothiocyanate (1.1 eq, 2.27 mmol) was refluxed in 10 mL DCM for 2 h. Elimination of the
solvent under reduced pressure afforded a thick yellowish oil which was redissolved in a 2M
2
LiOH/MeOH (3:1, 15 mL) and kept at 50 °C for 2 h. H O (20 mL) and EA (20 mL) were added
with stirring, and the phases were separated in a separatory funnel. The organic phase was
washed with aqueous saturated NaHCO3 and NaCl solutions, dried over MgSO4 and
concentrated under reduced pressure to give the corresponding thiourea (e.g. 1-(2-[2-(tert-
butyl)phenoxy]pyridin-3-yl)thiourea (56)) as tan solid, which was used without purification in
the next step.
1
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To a solution of the thiourea (1 eq, 1.65 mmol) obtained above and 3-bromo-1,1,1-
trifluoropropan-2-one (1.3 eq, 21.5 mmol) in EtOH (15 mL), 2,6-lutidine (2 eq, 3.3 mmol) was
added and the mixture was refluxed under argon for 24 h. The solvent was evaporated, 20 mL
2M HCl was added and the mixture was washed with EA (2 x 15 mL). The combined organic
3
phases were washed 1 x 15 mL NaHCO and 1 x 10 mL NaCl aqueous saturated solutions, dried
over MgSO and concentrated under reduced pressure to give a yellow solid which was further
4
washed with 3 x 10 mL n-hexane. Upon solvent evaporation under reduced pressure, 2-
aminothiazole (e.g. 26, 82% over 3 steps) was obtained as a light grey solid.
General procedure H: To a solution of 2-aminothiazole (e.g. 26, 1 eq, 0.52 mmol) in 10 mL
AcOH/THF (1:5) at 0 °C, NBS (1.1 eq, 0.58 mmol) was added and the reaction was stirred for 2
h at 0 °C. EA (15 mL) was added and the organic solution was washed with 15 mL H O,
2
concentrated aqueous NaHCO and NaCl solutions dried over MgSO and concentrated under
3
4
reduced pressure. The resulting solid was purified by column chromatography (silica, EA/IH,
1:18) to give 5-bromo-2-amino-thiazole (e.g. 27, 198 mg, 82%) as colorless solid.
General procedure I: Under argon, 5-bromo-2-amino-thiazole (e.g. 27, 1 eq, 0.11 mmol),
boronic acid (1.5 eq, 0.17 mmol), [Pd(PPh ) ] (0.1 eq, 0.01 mmol) and Na CO (3 eq, 0.33
3
4
2
3
mmol) were added to 5 mL toluene/EtOH (2:1) at rt and the mixture was refluxed (95 °C)
overnight. The solvent was eliminated by rotary evaporation, and the resulting residue was
purified by column chromatography on silica. Target compounds were obtained as colorless
solids.
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2-[2-(tert-Butyl)phenoxy]pyridin-3-amine (12a): colorless solid, 55% yield over 2 steps; TLC
(silica gel, EA/IH, 2:8): R = 0.55.
f
1
H NMR (400 MHz, CDCl3):
δ = 7.59 (dd, J = 4.9, 1.7 Hz, 1H), 7.44 (dd, J = 7.8, 1.8 Hz, 1H),
7.25 – 7.16 (td, J = 7.6, 1.5 Hz, 1H), 7.11 (td, J = 7.6, 1.5 Hz, 1H), 7.04 (dd, J = 7.6, 1.6 Hz, 1H),
6.93 (dd, J = 7.9, 1.5 Hz, 1H), 6.83 (dd, J = 7.6, 4.9 Hz, 1H), 3.95 (s, 2H), 1.42 (s, 9H).
1
3
C NMR (100 MHz, CDCl3):
122.6, 122.0, 119.2, 30.5.
δ = 152.9, 152.0, 141.1, 136.2, 132.2, 127.4, 127.0, 124.3,
1-{2-[2-(tert-Butyl)phenoxy]pyridin-3-yl}-3-[4-(trifluoromethoxy)phenyl]urea (7): General
procedure D (colorless solid, 95% yield)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
R
HPLC: t = 26.1 min, >99 %.
M. p. = 152 °C.
TLC (silica gel, EA/IH, 3:7): R = 0.50.
f
1
3
H NMR (400 MHz, CDCl ): δ = 8.53 (dd, J = 8.0, 1.7 Hz, 1H), 7.79 (dd, J = 4.9, 1.7 Hz, 1H),
7.49 (s, 1H), 7.46 – 7.37 (m, 1H), 7.32 (s, 1H), 7.30 – 7.22 (m, 2H), 7.22 – 7.05 (m, 4H), 6.94
(dd, J = 7.9, 4.9 Hz, 1H), 6.87 – 6.76 (m, 1H), 1.29 (s, 9H).
1
3
C NMR (100 MHz, CDCl
3
): δ = 153.0, 152.9, 152.3, 145.8, 141.5, 140.6, 136.2, 127.8,
127.7, 127.3, 125.2, 124.2, 123.1, 122.7, 122.2, 122.1, 119.1, 34.75, 30.63.
1
9
F NMR (282 MHz, CDCl3):
δ = –58.56.
+
HRFT-MS (ESI+): m/z = 446.1686 (calcd. 446.1686 for C H F N O [M+H] ).
2
3
23
3
3
3
1-{2-[2-(tert-Butyl)phenoxy]pyridin-3-yl}-3-[4-(2-hydroxyethyl)phenyl]urea (14): General
procedure D (colorless solid, 72% yield)
M. p. = 192 °C.
TLC (silica gel, EA/IH, 1:1): R = 0.38.
f
1
H NMR (400 MHz, CDCl3):
δ = 8.54 (dd, J = 8.0, 1.7 Hz, 1H), 7.66 (dd, J = 4.9, 1.7 Hz, 1H),
7.39 (dd, J = 7.6, 1.9 Hz, 1H), 7.34 – 7.22 (m, 2H), 7.15 – 7.07 (m, 4H), 6.92 (dd, J = 8.0, 4.9
Hz, 1H), 6.78 (dd, J = 7.7, 1.7 Hz, 1H), 3.71 (t, J = 6.7 Hz, 2H), 2.73 (t, J = 6.7 Hz, 2H), 1.30 (s,
9H).
1
3
C NMR (100 MHz, CDCl3):
δ = 153.7, 153.6, 152.7, 141.6, 139.4, 136.8, 136.7, 133.9,
129.6, 127.5, 127.3, 127.2, 125.1, 124.9, 123.0, 120.3, 119.0, 63.3, 38.4, 34.6, 30.5.
+
HRFT-MS (ESI+): m/z = 406.2125 (calcd. 406.2125 for C H N O [M+H] ).
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4-{3-{2-[2-(tert-Butyl)phenoxy]pyridin-3-yl}ureido}phenethyl 4-methylbenzenesulfonate
(16) General Procedure E (colorless solid, quantitative)
M. p. = 190 °C.
TLC (silica gel, EA/IH, 1:1): R
f
= 0.80.
1
H NMR (400 MHz, CDCl3):
δ
= 8.58 (dd, J = 8.0, 1.7 Hz, 1H), 7.78 (dd, J = 4.9, 1.7 Hz, 1H),
7.67 (d, J = 8.3 Hz, 2H), 7.41 (dd, J = 7.7, 1.9 Hz, 1H), 7.39 (s, 1H) 7.28 (s, 1H), 7.21 (d, J = 8.3
Hz, 2H) 7.20 – 7.10 (m, 2H), 7.03 (d, J = 8.4 Hz, 2H), 6.96 (dd, J = 8.0, 4.9 Hz, 1H), 6.91 (s,
1H), 6.82 (dd, J = 7.8, 1.6 Hz, 1H), 4.15 (t, J = 7.0 Hz, 2H), 2.88 (t, J = 7.0 Hz, 2H), 2.43 (s,
3H), 1.28 (s, 9H).
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1
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3
C NMR (100 MHz, CDCl ): δ = 153.0, 152.7, 152.4, 144.9, 141.5, 140.3, 136.4, 133.0,
132.9, 130.0, 129.9, 127.9, 127.6, 127.4, 127.2, 125.1, 124.5, 123.2, 122.2, 119.1, 77.5, 77.1,
76.8, 70.5, 34.8, 34.7, 30.6, 21.7.
+
FT-MS (ESI+): m/z = 560.3 (calcd. 560.2 for C31
H
34
N
3
O
5
S [M+H] ).
1-{2-[2-(tert-Butyl)phenoxy]pyridin-3-yl}-3-[4-(2-fluoroethyl)phenyl]urea (18) General
Procedure F (colorless solid, 62% yield).
HPLC: t = 40.2 min, purity >99 % (Reprosil-Pur C18-AQ, 250 x 4.6 mm, gradient: 10-90-
R
10% MeCN/20 mM NH OAc ; flow 1.0 mL/min, 60 min).
4
aq
M. p. = 135 °C.
TLC (silica gel, EA/IH, 2:8): R = 0.35.
f
1
H NMR (400 MHz, CDCl3):
δ = 8.61 (dd, J = 7.9, 1.7 Hz, 1H), 7.79 (dd, J = 4.9, 1.7 Hz, 1H),
7.42 (dd, J = 7.7, 1.9 Hz, 1H), 7.36 – 7.26 (m, 3H), 7.24 – 7.09 (m, 4H), 6.99 (dd, J = 8.0, 4.9
Hz, 1H), 6.84 (dd, J = 7.8, 1.6 Hz, 1H), 6.59 (s, 1H), 4.58 (dt, J = 47.0, 6.5 Hz, 2H), 2.96 (dt, J =
23.7, 6.4 Hz, 2H), 1.28 (s, 9H).
1
3
C NMR (100 MHz, CDCl
3
): δ = 153.1, 152.6, 152.5, 140.4, 135.9, 134.6, 130.4, 127.7,
127.5, 127.3, 125.1, 124.6, 123.2, 119.2, 85.1, 82.9, 36.6, 36.4, 30.7
1
9
F NMR (282 MHz, CDCl3):
δ = –216.10.
+
HRFT-MS (ESI+): m/z = 408.2082 (calcd. 408.2082 for C24
H
3 2
27FN O [M+H] ).
1-{2-[2-(tert-Butyl)phenoxy]pyridin-3-yl}-3-(4-methoxyphenyl)urea (20) General procedure
D (colorless solid, 95%).
f
TLC (silica gel, EA/IH, 2:8): R = 0.32.
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H NMR (400 MHz, CDCl3): δ = 8.63 (dd, J = 8.0, 1.7 Hz, 1H), 7.77 (dd, J = 4.9, 1.7 Hz, 1H),
7.40 (dd, J = 7.7, 1.9 Hz, 1H), 7.25 – 7.07 (m, 4H), 6.97 (dd, J = 8.0, 4.9 Hz, 1H), 6.90 – 6.76
(m, 3H), 6.46 (s, 1H), 3.76 (s, 3H), 1.22 (s, 9H).
1-{2-[2-(tert-Butyl)phenoxy]pyridin-3-yl}-3-(4-hydroxyphenyl)urea (21) A solution of
AlCl (1.0 g, 6 eq, 7.7 mmol) in EtSH (30 mL) was added slowly to a solution of 20 (0.5 g, 1 eq,
3
1.3 mmol) in DCM (20 mL) at 0 °C under argon. The reaction was stirred for 2 hours at room
temperature, quenched by addition of saturated aqueous NaHCO
3
solution (30 mL) and extracted
with DCM (3 x 20 mL). The combined DCM solutions were washed once with saturated aqueous
NaCl solution, dried over MgSO
1.1 mmol, 85%).
4
and the solvent was evaporated to give 21 as gray solid (0.4 g,
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3
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M. p. = 213 °C.
TLC (silica gel, EA/IH, 2:8): R
f
= 0.10.
1
H NMR (400 MHz, CDCl3):
δ
= 8.55 (dd, J = 8.0, 1.7 Hz, 1H), 7.66 (dd, J = 4.9, 1.7 Hz, 1H),
7.47 – 7.37 (m, 2H), 7.26 – 7.07 (m, 4H), 6.97 (dd, J = 8.0, 4.9 Hz, 1H), 6.80 (dd, J = 7.8, 1.5
Hz, 1H), 6.78 – 6.70 (m, 2H), 1.32 (s, 9H).
1
3
3
C NMR (100 MHz, CDCl ): δ = 154.5, 153.0, 152.9, 141.7, 139.2, 127.6, 127.5, 127.2,
125.5, 125.0, 123.1, 119.1, 115.9, 34.7, 30.5.
+
24 3 3
HRFT-MS (ESI+): m/z = 378.1812 (calcd. 378.1812 for C22H N O [M+H] ).
1-{2-[2-(tert-Butyl)phenoxy]pyridin-3-yl}-3-[4-(2,2,2-trifluoroethoxy)phenyl]urea
(23):
CF CH OTs (134 mg, 4 eq, 0.52 mmol) was reacted with 17 (50 mg, 1 eq, 0.13 mmol) in the
3
2
2 3
presence of K CO (55 mg, 3 eq, 0.40 mmol) in MeCN (5 mL) at 82 °C under argon for 16
hours. Saturated aqueous NaHCO solution (10 mL) was added and the mixture was washed with
3
EA (3 x 10 mL). The combined EA fractions were washed with H O (1 x 10 mL), saturated
2
aqueous NaHCO (1 x 10) mL and dried over MgSO . The solvent was removed by rotary
3
4
evaporation and the remaining residue was purified by flash chromatography (silica, EA/IH 1:9)
to give 23 (29 mg, 0.06 mmol, 48%) as colorless solid.
HPLC: t = 24.4 min, >99 %.
R
M. p. = 165 °C.
TLC (silica gel, EA/IH, 2:8) R = 0.55.
f
1
H NMR (400 MHz, CDCl3):
δ = 8.60 (d, J = 7.5 Hz, 1H), 7.79 (s, 1H), 7.41 (d, J = 7.4 Hz,
1H), 7.37 – 7.07 (m, 5H), 7.06 – 6.54 (m, 4H), 4.29 (q, J = 8.1 Hz, 2H), 1.26 (s, 9H).
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C NMR (100 MHz, CDCl3): δ = 155.4, 152.6, 152.4, 141.4, 140.3, 131.5, 127.6, 127.4,
127.3, 125.5, 125.0, 124.6, 124.6 (m, 1C), 123.1, 119.2, 116.2, 66.3 (d, J = 44.8 Hz, 1C), 34.7,
30.6.
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F NMR (282 MHz, CDCl3):
HRFT-MS (ESI+): m/z = 482.1662 (calcd. 482.1662 for C24
1-{2-[2-(tert-Butyl)phenoxy]pyridin-3-yl}-3-[4-(2-fluoroethoxy)phenyl]urea
obtained by the same procedure as compound 23 with Br(CH ) F as alkylating reagent. (colorless
δ = –74.39.
+
25 3 3 3
H F N O [M+H] ).
(22)
was
2
2
solid, 82% yield).
HPLC: t = 23.3 min, >99 %.
R
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2
2
3
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M. p. = 179 °C.
TLC (silica gel, EA/IH, 2:8): R = 0.13.
f
1
H NMR (400 MHz, CDCl3):
δ = 8.60 (dd, J = 8.0, 1.7 Hz, 1H), 7.76 (dd, J = 4.9, 1.7 Hz, 1H),
7.41 (d, J = 1.6 Hz, 1H), 7.40 (dd, J = 7.7, 1.8 Hz, 1H), 7.21 (d, J = 8.9 Hz, 2H), 7.14 (tdd, J =
8.9, 6.2, 2.4 Hz, 2H), 6.95 (dd, J = 7.9, 4.9 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 6.85 – 6.79 (m,
1H), 4.84 – 4.57 (m, 2H), 4.24 – 4.03 (m, 2H), 1.25 (s, 9H).
1
3
3
C NMR (100 MHz, CDCl ): δ = 156.5, 153.9, 152.6, 152.5, 141.4, 140.1, 130.4, 127.7,
127.3, 127.1, 125.6, 124.9, 124.7, 123.1, 119.1, 115.7, 83.1, 80.8, 81.9 (d, J = 171.0 Hz, 1C),
67.5 (d, J = 20.5 Hz, 1C). 34.6, 30.6.
1
9
F NMR (282 MHz, CDCl3):
HRFT-MS (ESI+): m/z = 424.2031 (calcd. 424.2031 for C24
1-[4-(2-Hydroxyethyl)phenyl]-3-[2-(2-isopropylphenoxy)pyridin-3-yl]urea (15): To
δ = –224.30.
+
H
3 3
27FN O [M+H] ).
a
solution of isocyanate (13b) (50 mg, 1 eq, 0.2 mmol) in toluene (1 mL) under argon, 2-(4-
aminophenyl)ethanol (27.4 mg, 1 eq, 0.2 mmol) was added. The resulting mixture was stirred at
80 °C for 1.5 h. The solvent was removed by rotary evaporation, and the crude product was
purified by column chromatography (silica, EA/IH, 3:17) to give 15 (53 mg, 0.13 mmol, 67%) as
colorless solid.
TLC (silica gel, EA/IH, 1:1): R
f
= 0.38.
1
H NMR (400 MHz, CDCl
3
):
δ
= 8.54 (dd, J = 7.9, 1.6 Hz, 1H), 7.75 (s, 1H), 7.68 (dd, J = 4.9,
1.7 Hz, 1H), 7.57 (s, 1H), 7.38 – 7.29 (m, 1H), 7.24 – 7.11 (m, 4H), 7.06 (s, 2H), 6.97 – 6.82 (m,
2H), 3.75 (t, J = 6.3 Hz, 2H), 3.21 – 2.87 (m, 1H), 2.73 (t, J = 6.3 Hz, 1H), 1.11 (d, J = 6.9 Hz,
6H).
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C NMR (100 MHz, CDCl3):
δ = 153.5, 152.8, 150.6, 140.8, 139.6, 136.2, 134.7, 129.8,
126.9, 126.9, 125.9, 124.0, 122.2, 121.5, 118.9, 63.5, 38.5, 27.2, 23.1.
+
HRFT-MS (ESI+): m/z = 392.1969 (calcd. 392.1969 for C H N O [M+H] ).
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4-{3-[2-(2-Isopropylphenoxy)pyridin-3-yl]ureido}phenethyl
4-methylbenzenesulfonate
(17) was obtained according to general procedure E (95% yield, colorless solid).
M. p. = 128 °C.
TLC (silica gel, EA/IH, 2:8): R
f
= 0.12.
1
H NMR (400 MHz, CDCl3):
δ
= 8.56 (dd, J = 7.9, 1.7 Hz, 1H), 7.68 (m, 4H), 7.53 (s, 1H),
7.33 (dd, J = 7.2, 2.2 Hz, 1H), 7.30 – 7.23 (m, 4H), 7.23 – 7.13 (m, 2H), 7.01 (d, J = 8.4 Hz, 2H),
6.92 (dt, J = 6.9, 3.6 Hz, 2H), 4.21 – 4.07 (t, J = 6.9 Hz, 2H), 3.11 – 2.95 (m, 1H), 2.86 (t, J = 6.9
Hz, 2H), 2.42 (s, 3H), 1.10 (d, J = 6.9 Hz, 6H).
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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1
3
C NMR (100 MHz, CDCl3):
δ = 152.9, 150.6, 144.9, 141.0, 139.6, 137.1, 132.9, 131.8,
129.9, 129.7, 127.9, 127.1, 127.0, 126.7, 125.8, 124.0, 122.3, 120.8, 118.9, 70.7, 34.8, 27.2, 23.1,
21.7.
+
HRFT-MS (ESI+): m/z = 546.2056 (calcd. 387.9231 for C H N O S [M+H] ).
3
0
32
3
5
1-[4-(2-Fluoroethyl)phenyl]-3-[2-(2-isopropylphenoxy)pyridin-3-yl]urea (19) was obtained
according to general procedure F as colorless solid, 82% yield.
R
HPLC: t = 23.8 min, >99 %.
M. p. = 124 °C.
TLC (silica gel, EA/IH, 2:8): R = 0.12.
f
1
3
H NMR (400 MHz, CDCl ): δ = 8.58 (dd, J = 7.9, 1.7 Hz, 1H), 7.73 (dd, J = 4.9, 1.7 Hz, 1H),
7.41 (s, 1H), 7.39 – 7.29 (m, 3H), 7.21 (m, 3H), 7.02 – 6.90 (m, 2H), 6.83 (s, 1H), 4.58 (dt, J =
47.0, 6.5 Hz, 2H), 3.08 – 2.97 (m, 1H), 3.02 –2.91 (dt, J = 15.4, 4.9 Hz, 2H), 1.14 (d, J = 6.9 Hz,
2H).
1
3
3
C NMR (100 MHz, CDCl ): δ = 152.9, 152.7, 150.5, 140.8, 139.9, 136.3, 130.1, 127.1,
127.0, 126.9, 125.9, 123.9, 122.3, 122.1, 119.0, 84.9, 83.2, 36.4 (d, J = 20.3 Hz), 27.3, 23.1,
20.7.
1
9
F NMR (282 MHz, CDCl
3
): δ = –216.00 (sep).
+
HRFT-MS (ESI+): m/z = 394.1925 (calcd. 394.1925 for C H FN O [M+H] ).
2
3
25
3
2
1-{2-[2-(tert-Butyl)phenoxy]pyridin-3-yl}-3-[2-fluoro-4-(trifluoromethoxy)phenyl]urea
(24) was obtained according to general procedure D as colorless solid, quantitative yield.
28