Synthesis of a high specific activity methyl sulfone tritium isotopologue of fevipiprant (NVP-QAW039)

Article abstract of DOI:10.1002/jlcr.3281

The synthesis of a triple tritiated isotopologue of the CRTh2 antagonist NVP-QAW039 (fevipiprant) with a specific activity >3TBq/mmol is described. Key to the high specific activity is the methylation of a bench-stable dimeric disulfide precursor that is

Full text of DOI:10.1002/jlcr.3281

Research Article
Received 14 January 2015,
Accepted 2 February 2015
Published online 16 April 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3281
Synthesis of a high specific activity methyl
sulfone tritium isotopologue of fevipiprant
(NVP-QAW039)
Van T. Luu,a Jean-Yves Goujon,b† Christian Meisterhans,c
a
a
*
Matthias Frommherz, and Carsten Bauer
The synthesis of a triple tritiated isotopologue of the CRTh2 antagonist NVP-QAW039 (fevipiprant) with a specific activity
>3 TBq/mmol is described. Key to the high specific activity is the methylation of a bench-stable dimeric disulfide precursor
that is in situ reduced to the corresponding thiol monomer and methylated with [3H3]MeONos having per se a high specific
activity. The high specific activity of the tritiated active pharmaceutical ingredient obtained by a build-up approach is
discussed in the light of the specific activity usually to be expected if hydrogen tritium exchange methods were applied.
Keywords: tritium; CRTh2 receptor antagonists; NVP-QAV680; NVP-QAW039; fevipiprant; 3-(2-methyl-7-aza-indolyl) acetic acid;
chemical cross-linking; [3H3]methyl nosylate; palladium catalysis; hydrogen isotope exchange
exchange under basic aqueous thermal conditions with
Introduction
microwave irradiation,15 we desired to develop a safe synthetic
Transmembrane chemoattractant receptor-homologous molecule
approach providing access to
a methyl sulfone tritium
expressed on T-helper type 2 cells (CRTh2) represents an attractive
target to address the need for the efficient oral anti-inflammatory
treatment of allergic asthma. This receptor recognizes
prostaglandin D2 (1, Figure 1) and regulates in a joint system the
pathophysiology of several allergic and chronic inflammatory
conditions.1,2 Ever since the discovery of the nonsteroidal anti-
inflammatory agent indometacin3 (2, Figure 1) to function as a
ligand for CRTh2,4,5 numerous drug discovery initiatives have
been launched aiming to develop orally available CRTh2
antagonists belonging to many different compound classes,
currently in the preclinical or early clinical phase.6 Among these
development compounds, medicinal chemistry literature
recognizes indol-1-yl acetic acids and indol-3-yl acetic acids as
promising drug candidates.7–10 Recently, the 7-azaindole-3-acetic
acid NVP-QAV680 (3, Figure 1) was found to be a highly selective
compound with low nanomolar functional potency for the in vitro
inhibition of CRTh2-driven human eosinophil and Th2 lymphocyte
activation. This compound progressed into human clinical
studies.11 Further structural and electronic optimization of NVP-
QAV680 led to the discovery of the trifluoromethyl derivative
NVP-QAW039 (fevipiprant, 4a, Figure 1), which exhibits improved
potency in human eosinophil and Th2 cell assays.12 Saturation and
competition-binding experiments with CHO–CRTh2 cell
membranes and [3H3]QAW039 revealed that fevipiprant is a slowly
dissociating CRTh2 antagonist that may afford improved clinical
efficacy.13 The synthesis of the tritium isotopologue of fevipiprant
used for these experiments is the subject of this publication. A
detailed discussion of the aforementioned kinetic profiling and
receptor occupancy studies will be published elsewhere.14
isotopologue of our next-generation CRTh2 receptor antagonist
4a without using basic THO as a dangerous tritium source. The
rationale behind this goal was to develop a radioligand of
fevipiprant that can be used under physiological pH in receptor
occupancy experiments without having the risk of THO
formation due to the aforementioned weak proton acidity of
the methyl sulfone moiety. Thus, a precursor suitable for late-
stage introduction of the tritium label as well as well-defined
high specific activity was desired. Fontana and coworkers
described a strategy for the synthesis of a deuterium-labeled
methyl-phenyl-sulfide containing active pharmaceutical ingredient.16
These authors methylated a volatile, thus easily dimerizing thiol using
a low-boiling deuterated methylating agent followed by sulfur
oxidation. As such, we speculated if this strategy can be applied
aNovartis Pharma AG, Novartis Institutes for Biomedical Research, Forum 1
Novartis Campus, Basel CH-4056, Switzerland
bAtlanchim Pharma, 3 rue Aronax, Saint-Herblain Cedex F-44821, France
cRC Tritec AG, Speicherstrasse CH-9053, Teufen, Switzerland
*Correspondence to: Carsten Bauer, Novartis Pharma AG, Novartis Institutes for
Biomedical Research, Forum 1 Novartis Campus, CH-4056 Basel, Switzerland.
E-mail: carsten.bauer@novartis.com
† Present address: Atlanthera, 3 rue Aronax, F-44821 Saint-Herblain Cedex,
France.
Abbreviations: AIBN azaisobutyronitrile; BEMP 2-tert-butylimino-2-diethylamino-
1,3-dimethylperhydro-1,3,2-diazaphosphorine; DDT dithiothreitol; NBS N-bromo
succinimide; NMO N-methylmorpholine N-oxide; MeONos methyl nosylate;
TIPSSH triisopropylsilyl mercaptane.
Although methods are known, allowing for the synthesis of
different deuterium isotopomers of 4a by site-selective H/D
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V. T. Luu et al.
Figure 1. Structures of the potent mediator of allergic inflammation prostaglandin D2 (PGD2, 1), the first nonsteroidal anti-inflammatory agent indomethacin (2), and
chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells antagonists 3 and 4a emerging from the Novartis pipeline.
to prepare tritiated isotopomers of prostaglandin D2 receptor introduced using palladium-catalyzed methods described by
antagonists, which are structurally related to fevipiprant.9
Migita20,21 and Hartwig22 (Scheme 1, step i) and when the
A bioconjugation chemistry-inspired approach17 in which a oxidation of the deprotected thiol 8 (step ii) was completed in
disulfide such as 5 (Figure 2) serves as a stable precursor for acidic water23 (step iii), the disulfide 9 was isolated in 56% yield
late-state tritium isotope introduction has not been described over two steps. Unfortunately, the radical bis-bromination of the
before in the literature. Our strategy was to cleave 5 by Cleland methyl group in 9 with NBS was difficult to control, and most
reduction18 (step 1) and to trap the released thiol with a high likely, the disulfide bridge was cleaved to form an unstable
specific activity [3H3-Me]methyl electrophile19 (step 2). Subsequent sulfenylbromide24, which we could not characterize further. This
oxidation of the phenyl [3H3]-methyl sulfide moiety (step 3) interpretation is supported by the observation that although a
and hydrolysis of the ester (step 4) will generate the tritiated relatively clean reaction was observed during in-process control,
radioligand of the active pharmaceutical ingredient in a safe and the crude post-extraction product was not stable on silica when
efficient way. This retrosynthetic analysis also can be applied the sulfenylbromide was treated with a weak acid. For this
to any other hydrogen isotope labeling of methyl sulfone reason we turned our attention to route B, where radical side-
moieties, which so far followed the aforementioned strategy of chain bromination of 6 followed an established method for the
Fontana et al.
synthesis of 10.25 The use of a safer alternative solvent to CCl4
for this bromination was not investigated. The original radical
initiator (benzoyl peroxide) was replaced by what is considered
to be less explosive (AIBN). Subsequent alkylation of the pyrrolo
nitrogen of the azaindole moiety of heterocycle 11 using
electrophile 10 in the presence of a phosphazene base11 yielded
Results and discussion
Synthesis of compound 5
Two routes A and B (Scheme 1 and Scheme 2, respectively) for 12 in acceptable yields (71%, step ii). The palladium-catalyzed
the synthesis of the disulfide 5 were tested. While the key step carbon–sulfur cross-coupling (step iii) tested in route A was
of route B was realized in analogy with the medicinal chemistry successfully applied to the even more functionalized substrate
synthesis of NVP-QAV68011 (3), route A differs from route B by 13. Under these conditions, silyl-protected 13 was obtained in
the sequence when the thio substituent was introduced into
a good yield (80%). Further deprotection of 13 with
the commercially available starting material 6. Although this alkylammonium fluoride (step iv) gave a mixture of the thiol 14
difference might appear marginal, route A gave the impression and the auto-oxidation product 5. Upon chromatography on
of being superior because the predicted instability of the TIPS silica, both compounds were separated, and the thiol 14 was
protecting group followed by oxidation of the unprotected thiol oxidized to its dimer 5 as developed in route A (step v). The
group should give clean and quick access to the core disulfide of disulfide 5 was characterized as by two-dimensional NMR and
5. Indeed, when the silyl-protected thiol functionality was mass spectrometry (MS). Fourier transform infrared (FT-IR)
Figure 2. Four-step high specific activity retro-synthesis for the tritium isotopologue of the 3-(2-methyl-7-aza-indolyl) acetic acid derivative 4b from its bench-stable
precursor 5. Steps 1–4 are mentioned in the text and are shown in Schemes 1–3.
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V. T. Luu et al.
Scheme 1. Route A for the synthesis of the disulfide 5. (i) TIPSSH, Pd(OAc)2, PPh3, Cs2CO3, toluene, 110 °C, 30 min, n.y.d.; (ii) Bu4NF, THF, 25 °C, 2 h, n.y.d.; (iii) NaBr, NaBrO3,
aqueous HCl, 0 °C, 10 min, 25%; NBS, (PhCO2)2, CCl4, 25 °C, 16 h, decomposition (n.y.d = no yield determined).
Scheme 2. Route B. (i) NBS, AIBN, CCl4, 5 h, reflux, 55%; (ii) 11, BEMP, DMF, 24 h, 25 °C, 71%; (iii) TIPSSH, Pd(OAc)2, PPh3, Cs2CO3, toluene, 30 min, 110 °C, 80%; (iv) Bu4NF,
THF, 2 h, 25 °C, 44%; v) NaBr, NaOBr3, H2O, 10 min, 4 °C, 40%.
spectroscopy unambiguously showed the existence of a sulfide- 17b and 18b exclusively depends on the comparison of their
bridged constitution of 5 by a weak but characteristic stretch HPLC retention times.
absorption at 499 cm-1.
Substitution of the primary hydroxyl group in compound 15
using PBr3 yielded bromide 16 in a good yield. Subsequent
pyrrolo-N alkylation using bromide 16 as discussed for route
B (Scheme 2) resulted in 17a. The radiochemical route to
Synthesis of reference compounds and radiosynthesis
Prior to the radiosynthesis of 4b (Scheme 3), access to unlabeled generate 17b, that is, the chemical cross-linking with MeONos
products 17a and 18a needed to be prepared to serve as reference during the reduction of the homogenous disulfide 5 in a
compounds. These reference compounds served for the solution of around 0.1 M of DTT also required more
characterization of their tritium isotopologues 17b and 18b by investigation. In fact, clean cross-linking formation of 17a was
means of HPLC-retention times comparison during radiosynthesis. observed overnight, when the aforementioned solution of 5
The synthesis of 17a and 18a is shown in Scheme 3 and is was treated with 4 eq of the methylating agent. The same
illustrated using dashed arrows, while the synthesis of 17b and was observed when in a different experiment, two portions
18b starting from the Cleland reduction product 14 is shown in each of 1 eq of the MeONos/Cs2CO3 reaction pair were added
the same scheme but using through-drawn arrows. It was once the spontaneous DTT reduction was completed. Lowering
important for us to synthesize reference compounds 17a and the excess of methylating agent of course was an advantage
18a by two different ways (upper and lower parts of Scheme 3). during work-up. In both cases, 17a synthesized from 5 was
The reason for this was that we desired to synthesize the reference characterized by means of co-injection with 17a synthesized
compounds on a millimolar scale, where all the intermediates from 16. Nevertheless, the sulfur oxidation in 17 was
could be well characterized spectroscopically (sequence problematic, mostly because of the presence of the
15→ 16→ 17a → 18a) in order to use them as reference bis-aza-heterocyle moiety. First, the oxidation of 17a was attempted
compounds in the micromolar-scale tritium synthesis (sequence with 0.8 eq of acidic potassium permanganate, and the reaction
5 → 14→ 17a → 18a) where characterization of the unlabeled, was followed by liquid chromatography (LC)–MS. Theoretically,
relatively unstable intermediate 14 and the tritium intermediates this ratio of the oxidizing agent relative to 17a should give clean
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V. T. Luu et al.
Scheme 3. Tritium and natural abundance isotopologues differ by notation ‘a’ for natural abundance and ‘b’ for the tritium case. Synthesis steps with unlabeled material
are denoted by a dashed arrow, and synthesis steps with labeled material are denoted by a solid line. Upper part, dashed arrow: synthesis of the unlabeled reference
compounds 17a and 18b from 15 via 16. (i) PBr3, Et2O, 0 → 25 °C, 16 h, 81%; (ii) 11 (see Scheme 2 for structure), BEMP, DMF, 16 h, 33%. Lower part, dashed arrow:
Unlabeled optimizations for the radiochemical case were tested in the sequence 5 to 14 and further micromolar downscaling to 17a and 18a. (iii) DTT, NEt3, ACN/THF,
30 min, 25 °C, n.y.d.; (iv) Cs2CO3, MeONos, ACN/THF, 16 h, 25 °C, n.y.d.; (v) OsO4, NMO, acetone/water/tert-butanol, 16 h, 25 °C, n.y.d. Lower part, solid arrow: Radiosynthesis
of 4b from 5 via 14, 17b, and 18b. (vi) DTT, NEt3, ACN/THF, 30 min, 25 °C, n.y.d; (vii) Cs2CO3, [3H3]MeONos, ACN/THF, 3 h, 25 °C, 61% radiochemical yield SA = 2.6 TBq/mmol
(determined by HPLC); (vii) OsO4, NMO, acetone/water/tert-butanol, 16 h, 25 °C, RA-HPLC 78%; (vii) a. 6-M HCl, b. semi-preparative HPLC, 20% radiochemical yield referred
to 17b (n.y.d. = no yield determined; see experimental part for UV purities of post extraction products).
formation of the sulfone 18a. However, when this stoichiometry techniques28 would not have produced the specific activity needed
was used, the oxidation stopped at the level of the sulfoxide. When as shown by Sandham and coworker.15 Indeed, during the
the reaction was forced to completion using a 3.2-fold excess of preparation of this manuscript, two publications emerged
permanganate, about 10–15% of the sulfone-N-oxide by-product describing transition metal-catalyzed HIE with deuterium gas on
was detected in the LC–MS trace. Faced with these results, osmium very relevant model substrates in a safer manner, thus opening a
tetroxide-catalyzed, NMO-mediated oxidation,26 which in other way to circumvent the radioprotection risks that one would have
hands was shown to successfully transform quinolinyl residue to face if THO was used as tritium source. Muri et al.29 for instance
bearing tetrahydrothiophenes to their corresponding sulfones27 applied an iridium(I) complex with an electron-rich N,P – ‘NeoPHOX’
using an excess of oxidant, was tested. Indeed, when these – ligand30 in order to achieve HIE in position ortho–ortho′ of
conditions were applied in a micromolar scale, the reaction was (methylsulfonyl)benzene, 1-(3,5-dimethoxyphenyl)ethanone, and
clean, and 18a was the only product formed.
2-phenylpyridin. Using D2, a 2 × 95% degree of deuteration was
The optimized conditions described earlier were applied to the achieved in the ortho–ortho′ positions of (methylsulfonyl)benzene
radiosynthesis. However, because it was shown that complete and 1-(3,5-dimethoxyphenyl)ethanone, while – probably because
methylation with stoichiometric amounts of [3H3]MeONos would of Ir coordination to the N-heterocycle – only 2 × 50% degree of
be hard to achieve, an inverted stoichiometry was used in the deuteration was obtained in the case of 2-phenylpyridin. Facing
tritiation step where 6 eq of 14 were treated with 1 eq of [3H3] the functional group complexity of NVP-QAW039 (4a, Figure 1)
MeONos. This procedure gave crude 17b with 87% radiochemical having not only an electron-withdrawing trifluoromethyl group in
purity and 61% radiochemical yield (relative to [3H3]MeONos) the meta position of its methyl sulfone moiety but also a pyrrolo-
after final purification. Oxidation to 17b occurred smoothly as pyridine moiety capable of trapping the Ir catalyst by coordination,
developed for the unlabeled case. As shown by Sandham and it only can be speculated that the exposure of esterified
coworker15, the ester hydrolysis of 18b would not have been NVP-QAW039 to this particular complex in a tritium gas atmosphere
possible without a significant loss of tritium label, which is why would yield maximum specific activity of what is maximally possible
4b was generated from 18b under strongly acidic conditions with based on the given structure of fevipiprant, that is, for this catalyst
a radiochemical yield of 20% after final HPLC purification. The 2 TBq/mmol.
specific activity of 4b was determined to be 3.11 TBq/mmol. Thus,
A similar lesson can be learned from the regioselective
it was possible to use a material with 97% tritium labelling per deuteration of simple indole and pyridine substrates using
tritiated position for occupancy studies on cell membranes with ruthenium nanoparticles dispersed in polyvinylpyrrolidone
low receptor density and also to reach sufficient counting (‘RuNp@PVP’).31 In this publication, Chaudret et al. reported HIE
efficiency with a low-energy β-particle radioligand (Emax of on the QAW039-model substrates quinoline, 3-methylpyridine,
3H = 18.6 keV).
and 3-methyl-1H-indole. For quinoline, 2 × 97% deuteration was
observed on C(2) and C(8), and for 3-methylpyridine deuteration
was observed on C(2) and C(6) (80% and 90%, respectively). For
3-methyl-1H-indole, deuteration was observed on C(2) (94%), and
for the methyl-group on C(3) (17%) and on C(7) (42%).
Translating these values to the hypothetical outcome of HIE
Discussion
In the context of the maximal specific activity achieved for 4b, it
must be noted that direct hydrogen-isotope exchange (HIE)
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V. T. Luu et al.
or on Waters Acquity UPLC–MS H Class with PDA SQD2 detector
equipped with a Waters CSH C18 column 1.7 μm (2.1 × 50 mm) using
water/0.1% HCO2H and ACN/0.1% HCO2H as eluent. The LC–MS release
analysis of compound 4b was performed on a Finnigan LCQ-Advantage
MAX equipped with Waters Sunfire C18 (30 × 2.1 mm) using water/ACN/
HCO2H 99:1:0.1 (system A) and water/ACN/HCO2H 5:95:0.1 (system B)
an injection volume of 5–10 mL and a sample concentration of around
3 mg/mL in H2O/EtOH. The specific activity of 4b was calculated from
the distribution of the monotritiated, bitritiated and tritritiated
isotopologues after correction of the molecular ion by the natural
on esterified NVP-QAW039 using Ru nanoparticles as catalyst, a
high degree of tritiation only would be expected on C(6) of the
pyrrolo-pyridine moiety, while only traces of tritium would be found
on the β-C of the acetic acid side chain. Overall, it is unlikely to
obtain a tritium isotopologue of fevipiprant with a specific activity
higher than 1.25 TBq/mmol if this catalyst would have been used.
To summarize for the synthesis of [3H3]fevipiprant, we still
conclude that high specific activity of the targeted isotopologue
only can be achieved by a multistep build-up approach of a
suitable bench-stable precursor and its methylation with a abundance of the (X + 1) element carbon using the commonly accepted
approach.33
high specific activity, commercially available reagent such as
[3H3]MeONos in the later steps of this multistep process.
NMR-spectroscopy
Experimental part
Chemical shifts (δ) are given in ppm referenced to the residual solvent
peak.34 1H-NMR spectra were recorded on Bruker AVANCE 400 and on
Bruker DRX600 with 1.7-mm-cryoprobe TCI-C/N probehead. 3H-NMR
spectra of compound 4b were acquired on Bruker DPX400 with SXI-3H
probehead using 4a as external reference for NMR difference
spectroscopy. 13C-NMR shifts of 5 were extracted from the HSQC data
set as much as this was possible. The processing software in all cases
was XWin-NMR. Proton assignments of the disulfide 5 were based on
HSQC, COSY and ROESY data sets. Coupling constants (J) are given in
Hz and were determined using the multiplet analysis mode of the MNova
software (v8.1.).
General
Solvents, reagents, and registered reference compounds
Commercially available absolute solvents were purchased over molsieve
(4 Å) and stored under argon. Reagents and compound 6 used for the
experimental work were purchased from commercial sources in the
highest available quality. Compound 15 was synthesized as described
elsewhere.32 Compounds 11, 18, and 4a were obtained from the
compound archive of Novartis Pharma AG, Basel, Switzerland. [3H3]
MeONos (radiochemical purity (RCP) > 99% (TLC); specific activity
(SA) > 2.96 TBq/mmol; 185 MBq/mL in toluene) was prepared at RC Tritec
AG, Teufen CH-9053, Switzerland, following a method described by
Pounds, where SA = 3.03 TBq/mmol was reported.19
Optical spectroscopy
FT-IR spectra were acquired on Bruker Vertex 70 FT-IR coupled with a
Hyperion 2000 FT-IT microscope. Raman spectra were acquired on Bruker
MultiRam FT-Rama spectrometer (laser power 300 mW).
Special equipment and glassware
(4-(Bromomethyl)-3-(trifluoromethyl)phenyl)(methyl)sulfane (16).
At 0 °C, PBr3 (385 μmol, 106 mg) was added to a solution of the
benzylalcohol 15 (95 mg, 38.5 mmol) in Et2O (500 μL), and the reaction
mixture was stirred overnight at room temperature. Then, 0.2-M NaOH
(1 mL) was added, the biphasic system was passed over a liquid/liquid
extraction cartridge (ChemElut, 3 mL) and the cartridge was washed with
Et2O. The combined organic phases were evaporated under reduced
pressure, and the residue was purified on silica (eluent hexane/EtOAc
10:1). After evaporation of the product fractions, 90 mg (81%) of pure
16 (Rf = 0.70 (hexane/EtOAc 10:1)) was obtained. 1H-NMR (400 MHz,
DMSO-d6) δ 7.66 (d, J = 8.2 Hz, 1H), 7.57 (dd, J = 8.3, 2.0 Hz, 1H), 7.51
(d, J = 2.0 Hz, 1H), 4.76 (s, 2H), 2.55 (s, 3H). ESI-MS (cation mode) m/z 205
[C9H8F3S+].
All air and moisture sensitive reactions were carried out in dried
glassware under inert gas atmosphere. Microwave assistant synthesis
was performed using CEM Microwave Discover apparatus ramping with
200 W at 110 °C.
Flash chromatography, TLC, and solid-phase extraction
Analytical TLC was run on Merck TLC Silica 60 plates with fluorescence
indicator (merckmilipore.com) and was visualized at 254 nm or with iodine
vapors. Column chromatography was performed using silica gel 60 (Merck,
70–230 mesh) or on high-purity grade silica (Fluka, 230–400 mesh). Solid-
phase extraction cartridges were purchased either at Agilent (ChemElut)
or at Phenomenex (StrataX).
HPLC and RA-HPLC
Methyl 2-(2-methyl-1-(4-(methylthio)-2-(trifluoromethyl)benzyl)-1H-
pyrrolo[2,3-b]pyridin-3-yl)acetate (17a) by N-alkylation of
azaindole 11 with benzylbromide 16. Under Ar, BEMP (52 μL,
176 μmol) was added to a solution of 16 (51 mg, 0.176 mmol) and 11
(40 mg, 176 μmol) in dimethylformamide (DMF) (3.0 mL). The reaction
was stirred for 16 h, then adjusted with HCl (5%) to pH 7.5, diluted with
water and extracted with EtOAc (2 × 5 mL). The combined organic layers
were dried over Na2SO4, filtered and evaporated to dryness. The crude
material was purified on silica (10 g, eluent hexane/EtOAc 4:1). After
evaporation of the product fractions, 24 mg of colorless oil was obtained.
1H-NMR (400 MHz, DMSO-d6) δ 8.14 (dd, J = 4.7, 1.5 Hz, 1H), 7.92 (dd,
J = 7.8, 1.5 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.35 (dd, J = 8.3, 2.0 Hz, 1H),
7.11 (dd, J = 7.8, 4.7 Hz, 1H), 6.10 (d, J = 8.3 Hz, 1H), 5.62 (s, 2H), 3.62
(s, 3H), 3.30 (d, J = 9.5 Hz, 2H), 2.47 (s, 3H), 2.24 (s, 3H). ESI-MS (cation mode)
m/z 409.0 [MH+].
HPLC for in-process control of compounds 15, 16, 17ab, 18ab, and 4b
were run on Agilent 1200 Series HPLC system with ChemStation Plus
spectra software (v9) using a Sunfire C18-RP column (4.6 × 250 mm). The
HPLC release analysis of [3H3]MeONos and of compound 18b was
performed on a modular Gilson HPLC system equipped with a Macherey
& Nagel Nucleodur C18-Gravity column (5 μm, 4.6 × 150 mm). For all
compounds, solvent systems A (water plus 0.05–0.1% TFA) and solvent
systems B (Acetonitrile (ACN) plus 0.1–0.05% TFA) were used. For HPLC
release analysis of compound 4b, a 0.30 mg/mL sample (H2O/EtOH 1 : 1,
v/v) was prepared and analyzed on Agilent 1290 Infinity using a Waters
XBridge RP18 column (3.5 μm, 2.1 × 100 mm) (flow 1.0 mL/min). For RA-
HPLC, a Berthold LB509 radio detector with a 100-μL Z-cell was used in
all cases. Scintillation cocktails were either Perkin Elmer’s Flow-Scint A
at 3.0 mL/min or Zinser’s Quickzint Flow 302 at 2.8mL/min. In most cases,
ultraviolet (UV) detection was done at 215–220 nm, except [3H3]MeONos,
which was analyzed at 254 nm.
Compound 17a by alkylation of the in situ generated thiol 14 with
an excess of MeONos. A mixture of disulfide 5 (10 mg, 12.7 μmol), DTT
(19.6 mg, 127.1 μmol) and NEt3 (0.88 μL, 6.3 μmol) in ACN/Tetrahydrofurane
(THF) (5 : 2, v/v, 1.40 mL) was stirred under Ar for 30 min until a yellow
LC–MS
LC–MS analysis of purified unlabeled compounds was acquired on a
Waters ZQ2000 single-stage quadrupole mass spectrometer equipped solution was obtained. Subsequently, Cs2CO3 (16.6 mg, 50.8 μmol) and
with an Ascentis Express C18 column (2.7 μm, 2.1 × 30 mm) using MeONos (11.4 mg, 50.8 μmol) were added to the reaction and stirring
water/0.05% HCO2H/3.75 mM (NH4)OAc and ACN/0.04% HCO2H as eluent was maintained for a total time of 16 h. Subsequently, the reaction
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V. T. Luu et al.
mixture was diluted with water (2 mL) and extracted with EtOAc
Subsequently, water (10 mL) was added and the reaction mixture was
(3 × 2 mL). The combined organic phases were dried over Na2SO4, filtered, extracted with EtOAc (2 × 10 mL). The combined organic layers were dried
and evaporated to dryness in order to yield 11 mg of product (64% UV over Na2SO4, filtered and evaporated under reduced pressure, and the
purity), which was characterized by HPLC using the reference product residue was purified by chromatography on silica gel (cyclohexane/EtOAc
17a prepared as described in the previous section.
90:10) in order to yield 430 mg (71%) of compound 12. 1H-NMR (400 MHz,
CDCl3): δ 8.27 (dd, J = 4.8, 1.5 Hz, 1H), 7.92 (dd, J = 7.8, 1.5 Hz, 1H), 7.85 (d,
J = 2.1 Hz, 1H), 7.44 (dd, J = 8.4, 2.0 Hz, 1H), 7.13 (dd, J = 7.8, 4.7 Hz, 1H),
6.29 (d, J = 8.4 Hz, 1H), 5.68 (s, 2H), 3.76 (2, 3H), 3.71 (s, 3H), 2.25 (s, 3H).
ESI-MS (cation mode) m/z 441.03 [MH+].
Methyl 2-(2-methyl-1-(4-(methylsulfonyl)-2-(trifluoromethyl)benzyl)-
1H-pyrrolo[2,3-b]pyridin-3-yl) acetate (18a). Compound 17a
(0.41 mg, 1 μmol) synthesized by alkylation of azaindole 11 with
benzylbromide 16 was dissolved in a solution of NMO (1.3 mg, 9 μmol)
in acetone/water (2 : 1 v/v, 300 μL). Then, an aliquot (30 μL, 0.6 μmol) of
a solution of 5% OsO4 tert-butanol was added and the reaction mixture
(c (17a) = 3 mM) was incubated at room temperature overnight.
Afterwards, the reaction mixture was diluted with EtOAc (3 mL) and
washed with brine (2 × 1 mL). The combined organic phases were dried
over Na2SO4, filtered and evaporated to yield 18a (90% UV purity), which
was characterized by co-injection with an authentic reference sample.
Methyl 2-(2-methyl-1-(2-(trifluoromethyl)-4-((triisopropylsilyl)thio)benzyl)-
1H-pyrrolo[2,3-b]pyridin-3-yl)acetate (13). A sealable tube was charged
with Pd(OAc)2 (10 mg, 0.02 mmol), PPh3 (30 mg, 0.11 mmol), Cs2CO3 (195 mg,
0.59 mmol), compound 12 (200 mg, 0.45 mmol) and dry toluene (4 mL). After
degassing the suspension, TIPSSH (115 mg, 0.59 mmol) was added and the
sealed reaction mixture was exposed to microwave irradiation (t = 30 min;
T = 110 °C, Pmax = 200 W). After cooling at room temperature, aqueous NH4Cl
(1%, 5 mL) was added the suspension was extracted with EtOAc (2 × 10 mL);
the combined organic layers were dried over Na2SO4, filtered and evaporated
under reduced pressure. The residue was purified by chromatography on silica
gel (eluent cyclohexane/EtOAc 95:5) to yield 210 mg (80%) of oily compound
13 (81% UV). ESI-MS (cation mode) m/z 551.23 [MH+].
Triisopropyl((4-methyl-3-(trifluoromethyl)phenyl)thio)silane (7) and
4-methyl-3-(trifluoromethyl)-benzenethiol (8). A microwave tube was
charged with Pd(OAc)2 (24 mg, 10 μmol), PPh3 (140 mg, 530 μmol),
Cs2CO3 (902 mg, 2.77 mmol, 1.3 eq.) and 6 (500 mg, 2.13 mmol). Then,
the reaction mixture was suspended in dry toluene (10 mL) and the
mixture was degassed. TIPSSH (527 mg, 2.77 mmol) was added, the tube
was sealed via a septum and the reaction mixture was heated for 30 min
under microwave irradiation (T = 110 °C, Pmax = 200 W). After cooling at
room temperature, aqueous NH4Cl (1%, 5 mL) was added and the
reaction mixture was extracted with Et2O (2 × 15 mL). The combined
organic layers were dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by chromatography on silica
gel (cyclohexane/Et2O 100/0 to 90/10) to yield 380 mg of impure 7 as a
colorless oil (Rf = 0.6 (cyclohexane 100%)). Then, about 300 mg of this
oil was dissolved in THF (10 mL), Bu4NF (480 mg, 1.63 mmol) was added
and the reaction mixture was stirred at room temperature for 2 h.
Subsequently, aqueous NH4Cl (1%, 5 mL) was added and the reaction
mixture was extracted with Et2O (2 × 15 mL). The combined organic
phases were dried over Na2SO4 and evaporated under reduced pressure
to obtain a pale-yellow oil. According to LC–MS analysis, this product
consisted out of 7 and 8 in a 7:3 ratio. Rf = 0.2 (cyclohexane 100%).
ESI-MS (anion mode) m/z 191.0 [C8H6F3S]ꢀ, 381 [C16H11F6S2].
Methyl 2-(1-(4-mercapto-2-(trifluoromethyl)benzyl)-2-methyl-1H-
pyrrolo[2,3-b]pyridin-3-yl)acetate (14). Bu4NF (370 mg, 1.26 mmol)
and compound 13 (370 mg, 0.67 mmol) were dissolved in THF (12 mL)
and the solution was stirred at room temperature for 2 h. Subsequently,
aqueous NH4Cl (1%, 12 mL) was added and the reaction mixture was
extracted with EtOAc (2 × 15 mL). The combined organic layers were
dried over Na2SO4, evaporated under reduced pressure and the residue
was purified by chromatography on silica gel (gradient cyclohexane/
EtOAc 95:5 to 60:40). After evaporation of the product fractions
(Rf = 0.4 (cyclohexane/EtOAc = 75:25)), compound 14 (91% UV) was
obtained as a pale-yellow solid in moderate yields (100 mg, 44%)
together with a small amount of disulfide 5 (40 mg, 15%). 1H-NMR
(400 MHz, CDCl3) δ 8.24 (dd, J = 4.8, 1.5 Hz, 1H), 7.89 (dd, J = 7.9, 1.6 Hz,
1H), 7.59 (d, J = 1.9 Hz, 1H), 7.17 (dd, J = 8.2, 2.0 Hz, 1H), 7.10 (ddd, J = 7.8,
4.7, 3.1 Hz, 1H), 6.25 (dd, J = 8.3, 5.7 Hz, 1H), 5.66 (s, 2H), 3.73 (s, 2H), 3.69
(s, 3H), 3.50 (s, 1H), 2.22 (s, 3H).
Dimethyl 2,2′-(1,1′-((disulfanediylbis(2-(trifluoromethyl)-4,1-phenylene))bis-
(methylene))bis(2-methyl-1H-pyrrolo[2,3-b]pyridine-3,1-diyl))diacetate
(5). Compound 14 (120 mg, 0.30 mmol) was suspended in ice-cold water
(3 mL). Sodium bromide (3 mg, 0.03 mmol) and sodium bromate (15 mg,
0.09 mmol) were added, followed by the dropwise addition of 1-N HCl
(0.12 mL). The reaction mixture was stirred for 10 min and then extracted
with EtOAc (2 × 20 mL). The combined organic were washed with
saturated Na2S2O3 (1 × 10 mL) and brine (1 × 10 mL), dried over Na2SO4,
filtered and evaporated. The residue was purified by chromatography on
silica (gradient cyclohexane/EtOAc 95:5 to 70:30). After evaporation of
the product fractions (Rf = 0.45 (cyclohexane/EtOAc = 75:25)), compound
5 (48 mg, 40%) was obtained, which crystallized upon standing at room
temperature. 1H-NMR (600 MHz, DMSO-d6) δ 8.12 (dd, J = 4.7, 1.6 Hz, 2H),
7.91 (dd, J = 7.9, 1.6 Hz, 2H), 7.86 (d, J = 2.1 Hz, 2H), 7.62 (dd, J = 8.3,
2.1 Hz, 2H), 7.10 (dd, J = 7.8, 4.7 Hz, 2H), 6.21 (d, J = 8.4 Hz, 2H), 5.63
(s, 4H), 3.82 (s, 4H), 3.60 (s, 6H), 2.21 (s, 6H). 13C-NMR {HSQC} (DMSO-d6)
δ 142.9, 133.8, 129.0, 128.1, 126.5, 117.2, 53.1, 29.8, 11.2. IR (neat, 1/cm):
3048, 3011 (ν(Car–H)), 2958, 2927 (ν(C–H), 1737 (ν(C¼O)), 1309
(ν(Car–CF3)),1160, 1117 (ν(CF3). Raman (neat, 1/cm): 3075 (ν(Car–H),
2949 (ν(C–H)), 1734 (ν(C¼O), 1605, 1560 (ν(Car–Car), 499 (ν(S–S)). ESI-MS
(cation mode) m/z 787.4 [MH+].
1,2-Bis(4-methyl-3-(trifluoromethyl)-phenyl)disulfane (9). At 0 °C, the
total amount of the crude product obtained in the previous step was
suspended in water (2.5 mL). NaBr (5 mg, 0.05 mmol.) and NaBrO3
(25 mg, 0.16 mmol) were added, followed by the dropwise addition of
1 N HCl (0.2 mL). Then, the reaction mixture was stirred for 10–12 min
and extracted with EtOAc (2 × 20 mL). The combined organic layers were
washed with saturated Na2S2O3 (1 × 10 mL) and brine (1 × 10 mL), dried
over Na2SO4 and evaporated under reduced pressure. The residue was
purified by chromatography on silica gel (cyclohexane/Et2O 100/0 to
95/5) to yield compound
9 (90 mg) as a colorless oil. Rf = 0.44
(cyclohexane 100%). 1H-NMR (400 MHz, CDCl3): δ 7.52 (s, 2H), 7.33
(d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.0 Hz, 2H), 2.26 (s, 6H).
4-Bromo-1-(bromomethyl)-2-(trifluoromethyl)benzene (10). Under
Ar, 4-bromo-1-methyl-2-(trifluoromethyl)benzene (6, 1.5 g, 6.3 mmol)
was dissolved in CCl4 (20 mL). NBS (1.35 g, 7.6 mmol) and AIBN (0.3 mmol,
50 mg) were added and the suspension was refluxed for 5 h in the dark.
After cooling, the solvent was evaporated under reduced pressure and
the residue was purified by chromatography on silica gel (eluent
cyclohexane 100%). Evaporation of the product fractions yielded 1.1 g
(55%) of pure 10. 1H-NMR (400 MHz, CDCl3): δ 7.81 (d, J = 2.0 Hz, 1H),
7.70 (dd, J = 8.3, 2.0 Hz, 1H), 7.49 (d, J = 8.3 Hz, 1H), 4.60 (s, 2H).
Methyl 2-(2-methyl-1-(2-(trifluoromethyl)-4-(([3H3]methyl)thio)benzyl)-
Methyl 2-(1-(4-bromo-2-(trifluoromethyl)benzyl)-2-methyl-1H-
1H-pyrrolo[2,3-b]pyridin-3-yl) acetate (17b). In vial A, disulfide
5
pyrrolo[2,3-b]pyridin-3-yl)acetate (12). Under Ar, azaindole 11 (5.3 mg, 6.7 μmol) and DTT (10.1 mg, 66 μmol) were solubilized in ACN/THF
(300 mg, 1.37 mmol) and compound 10 (525 mg, 1.65 mmol) were (7:3, 1.1 mL). Then, NEt3 (0.5 μL, 4 μmol) in THF (300 μL) was added. The
dissolved in dry DMF (6 mL). BEMP (450 mg, 1.65 mmol) was added and solution was quickly stirred and kept about 30 min at room temperature. In
the reaction mixture was stirred at room temperature for 24 h. parallel, an aliquot of a stock solution of [3H3]MeONos (about 2.4 μmol,
J. Label Compd. Radiopharm 2015, 58 188–195
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

V. T. Luu et al.
6.95 GBq, SA = 3.0 TBq/mmol) was evaporated to dryness in vial B. The
residue in vial B was mixed with Cs2CO3 (2.66 mg, 8.2 μmol). An aliquot of
the thiolate solution from vial A (0.75 mL) was added and the mixture was
incubated for 3 h at room temperature. Afterwards, the reaction solvent
was removed, the crude product was dissolved in THF (400 μL) and TFA
(10 μL) was added. This product solution (6.95 GBq, 87% RA-HPLC) was
injected in portions (11 × 35 μL) on a reversed-phase HPLC column and the
product was eluted with ACN/water. Pure product fractions were combined.
The pH was set to 9.5 using NaHCO3 and the product was adsorbed on a
StrataX cartridge (100 mg). After washing the cartridge with water
(2 × 2 mL), the free base of the product was eluted with EtOH (30 mL) and
characterized as such by comparison of the HPLC retention times with those
of unlabeled 17a. The product solution contained 4.22 GBq of 4b (61%
radiochemical yield, 96% RA-HPLC, SA = 2.6 TBq/mmol).
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Methyl 2-(2-methyl-1-(4-([3H3]methylsulfonyl)-2-(trifluoromethyl)benzyl)-
1H-pyrrolo[2,3-b]pyridin-3-yl) acetate (18b). Compound 17b (0.31 mg,
0.75 μmol, 1.0 GBq) was dissolved in a solution of NMO (0.45 mg, 3.4 μmol)
in acetone/water (2:1 v/v, 90 μL). Then, an aliquot (8 μL, 0.15 μmol) of a solution
of 5% OsO4 in tert-butanol was added and the reaction mixture (c (17b)
= 7.5 mM) was incubated at room temperature overnight. The reaction mixture
was evaporated, and the product (78% purity RA-HPLC) was characterized by
comparison of the HPLC retention times with those of unlabeled 18a. Crude
18b was used in the next step without further purification.
2-(2-Methyl-1-(4-([3H3]methylsulfonyl)-2-(trifluoromethyl)benzyl)-1H-
pyrrolo[2,3-b]pyridin-3-yl)acetic acid (4b). Crude active material 18b
obtained in the previous step was dissolved in 6-M HCl (300 μL) and kept
at room temperature for 2 h. Then, the solution was lyophilized and the
residue was purified by semi-preparative HPLC in order to yield 199 MBq
of pure 4b (20% radiochemical yield referred to 18b, 98.2% purity RA-
HPLC, SA = 3.1121 TBq/mmol) stored in EtOH (14 mL). The purified
product was characterized by comparison of the HPLC retention times
with those of unlabeled 4a. ESI-MS (cation mode) m/z 427.2 [MH+, [3H0]
C
19H14T3F3N2O4S]] (0.83%), 429.3 [MH+, [3H1]C19H14T3F3N2O4S]] (0.90%),
431.2 [MH+, [3H2]C19H14T3F3N2O4S]] (3.79%), 433.2 [MH+, [3H2]
19H14T3F3N2O4S]] (100%). 3H-NMR (400 MHz, DMSO-d6): δ 3.27.
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Acknowledgements
The authors would like to thank the NIBR Analytical Science
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Department for the acquisition of high-quality 2D-NMR data and [19] S. Pounds, Preparation of carrier-free [methyl-3H]methyl nosylate
and its use as a radiochemically-stable methylating reagent. In
Synthesis and Applications of Isotopically Labelled Compounds (Eds.:
D. C. Dean, C. N. Filer, K. E. McCarthy), Wiley, Chichester, 2004,
63–66.
optical spectroscopy data of the disulfide 5 and the acquisition
of H-NMR data of 4b. C. B. would like to thank David Sandham
(NIBR, Cambridge, MA, USA) and Thomas Moenius (NIBR, Basel,
CH) for having provided feedback on parts of this manuscript.
3
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Conflict of Interest
Synthesis of [3H3]MeONos will not be disclosed.
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