Syntheses of a radiolabelled CXCR2 antagonist AZD5069 and its major human metabolite

Article abstract of DOI:10.1002/jlcr.3429

The CXCR2 antagonist AZD5069 has been synthesized in tritium and carbon-14-labelled forms. [³H]AZD5069 was prepared via reductive dehalogenation of an iodinated precursor with tritium gas to provide material with a specific activity of 25.1 Ci/

Full text of DOI:10.1002/jlcr.3429

Research article
Received 11 April 2016,
Revised 18 May 2016,
Accepted 28 June 2016
Published online 27 July 2016 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3429
Syntheses of a radiolabelled CXCR2 antagonist
AZD5069 and its major human metabolite
a
a
b
b
*
Michael J. Hickey, Paul H. Allen, Moya Caffrey, Peter Hansen,
Lee P. Kingstonc and David J. Wilkinsona
The CXCR2 antagonist AZD5069 has been synthesized in tritium and carbon-14-labelled forms. [3H]AZD5069 was prepared
via reductive dehalogenation of an iodinated precursor with tritium gas to provide material with a specific activity of 25.1 Ci/
mmol. [14C]AZD5069 was labelled in the pyrimidine ring from [14C]thiourea in an overall radiochemical yield of 18%. In
addition, a synthetic route to the major metabolite of AZD5069 was developed. The synthesis of this metabolite was
achieved from AZD5069 using a chemoselective Lindgren–Pinnick reaction in order to minimize oxidation of the sulphide
group.
Keywords: carbon-14; tritium; AZD5069; Lindgren–Pinnick
could then be reductively dehalogenated in the presence of
Introduction
tritium gas. The synthesis is outlined in Scheme 1. The route also
The CXC chemokine receptor-2 (CXCR2) is a Gprotein-coupled
provided a versatile method for preparing tritium-labelled
receptor expressed on
a variety of inflammatory cells
compounds where variations to the substituents on the S-benzyl
group were introduced. The use of an iodine as the halogen
precursor was preferred over bromine for reasons of selectivity,
higher conversion and higher isotopic incorporation.5,6
Protection of the acetal synthetic intermediate (4) with p-
methoxybenzyl bromide followed by oxidation of the sulphide
with 3-chloroperbenzoic acid (mCPBA) furnished the sulphone
(5) in 69% yield. Addition of sodium hydrosulphide (NaSH) from
a new, previously unopened bottle, to the sulphone in dimethyl
sulfoxide generated the thiol in situ, which was smoothly
alkylated with 2,3-difluoro-6-iodobenzyl bromide (3) to give the
desired iodinated precursor (6) in good yield after trifluoroacetic
acid deprotection. 2,3-Difluoro-6-iodobenzyl bromide (3) was
conveniently prepared using a directed lithiation approach.
Deprotonation of 1,2-difluoro-4-iodobenzene with freshly
prepared lithium diisopropylamide at À78°C followed by
quenching with solid carbon dioxide and acidic workup afforded
2,3-difluoro-6-iodobenzoic acid (2) in 67% yield as a single
product. The hindered acid was reduced to the corresponding
(monocytes, macrophages, neutrophils) and is known to be
elevated in several inflammatory diseases, including chronic
obstructive pulmonary disease,1 rheumatoid arthritis2 and
psoriasis.3 Inhibition of the CXCR2 chemokine receptor,
therefore, represents an attractive strategy for the treatment of
inflammatory disorders. To support a programme aimed at
developing CXCR2 antagonists,4 AZD5069 (1) (Figure 1) was
identified as a potent, orally active small molecule that
demonstrated selective reversible antagonism of human CXCR2.
To investigate the pharmacokinetic and metabolism profile of
the compound, the preparations of tritium and carbon-14-
labelled AZD5069 were conducted. Metabolism studies of
AZD5069 in human identified the carboxylic acid (14) as a major
circulating metabolite. In order to develop
a
liquid
chromatography mass spectrometry (LCMS) method for
quantitative bioanalysis of the metabolite, the synthesis of an
authentic reference standard and a stable isotope-labelled
internal standard were also prepared.
Results and discussion
aDrug Safety and Metabolism, Innovative Medicines and Early Development
Biotech Unit, AstraZeneca, Cambridge, UK
Tritium labelling
Our initial approach to the preparation of [3H]AZD5069, [3H]-1
was to introduce the tritium label into the pyrimidine ring.
However, deuterium labelling experiments found this position
to be labile rendering it unsuitable for tritium labelling. Stirring
a solution of AZD5069 (0.5 mg) in deuterated ethanol (0.5 mL)
bRespiratory, Inflammation and Autoimmunity, Innovative Medicines and Early
Development Biotech Unit, AstraZeneca, Gothenburg, Sweden
cDrug Safety and Metabolism, Innovative Medicines and Early Development
Biotech Unit, AstraZeneca, Gothenburg, Sweden
for 24 h revealed 16% isotope incorporation into the pyrimidine
ring by H NMR. Addition of triethylamine (5 μL) increased the Medicines and Early Development Biotech Unit, AstraZeneca, Cambridge Science
deuterium incorporation to 80%. Therefore, a more traditional
synthetic approach was adopted using 2,3-difluoro-6-iodobenzyl
*Correspondence to: Michael J. Hickey, Drug Safety and Metabolism, Innovative
1
Park, Cambridgeshire, Cambridge CB4 0WG, UK.
E-mail: michael.j.hickey@astrazeneca.com.
bromide (3) to prepare a suitable iodinated precursor (6) that The preparation of the CXCR2 antagonist AZD5069 is described.
J. Label Compd. Radiopharm 2016, 59 432–438
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M. J. Hickey et al.
Figure 1. Structural formula of AZD5069 and the major human metabolite.
Scheme 1. Synthesis of [3H]AZD5069, [3H]-1.
benzyl alcohol in the presence of borane and boron trifluoride by reverse phase HPLC using a 10 mM ammonia/acetonitrile
etherate,7 and conversion to the benzyl bromide (3) was gradient were hampered due to the slow formation of two
achieved by refluxing with phosphorous tribromide. With the closely related impurities in the HPLC eluant. The two impurities
iodinated precursor (6) in hand, reductive dehalogenation in were identified as isomeric products resulting from a Smiles
the presence of tritium gas and 10% Pd/C showed complete rearrangement8 of [3H] AZD5069 under basic conditions
conversion after 2 h to furnish the crude product with a (Figure 2). The rate of formation of the two impurities was
radiochemical purity of 96%. Initial efforts to purify the material accelerated as the pH increased. To avoid the formation of these
Figure 2. Smiles rearrangement of [3H]AZD5069, [3H]-1 in basic HPLC eluant.
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M. J. Hickey et al.
impurities, the crude material was subsequently purified by hydride in 2-methyl tetrahydrofuran, and the resulting anion
reverse phase HPLC under acidic conditions using an aqueous was reacted with [14C]-8 affording [14C]-10 in 56% radiochemical
trifluoroacetic acid/acetonitrile gradient. Crude [3H]AZD5069 yield after workup and purification by flash chromatography.
[3H]-1 was successfully purified to give the final material with a Palladium-catalysed cross-coupling10 of [14C]-10 with azetidine-
radiochemical purity of >99% and a specific activity of 25.1 Ci/ 1-sulfonamide (11) in the presence of potassium carbonate
mmol. The material was stored as an ethanol solution at À20°C and Xphos, followed by purification by flash chromatography,
to minimize radiochemical decomposition.
provided the penultimate compound in 84% radiochemical
yield. It was important to carry out this cross-coupling reaction
in degassed solvent under a nitrogen atmosphere accompanied
with rapid stirring to ensure that the conversion proceeded in
Carbon-14 labelling
The synthesis of [14C]AZD5069 [14C]-1 is outlined in Scheme 2. high yield. Finally, deprotection with phosphoric acid in aqueous
Introduction of the carbon-14 isotope into the metabolically acetonitrile provided crude [14C]AZD5069
[ a
14C]-1 with
stable pyrimidine ring could be achieved through condensation radiochemical purity of >98%. A portion of the crude material
of either [14C]thiourea or [14C]diethylmalonate, both of which was further purified by reverse phase HPLC in aqueous
were commercially available. Using a slight modification to the trifluoroacetic acid/acetonitrile to give [14C]AZD5069 [14C]-1 as
published procedure by Mathew et al.,9 we were able to a white solid. The radiochemical purity was measured at 99.8%
maximize the radiochemical yield of this condensation by with a specific activity determined by gravimetric analysis of
using[14C]thiourea as the labelled starting material. The reaction 122.1 μCi/mg.
of [14C]thiourea with diethylmalonate in the presence of freshly
Following the success in preparing carbon-14-labelled
prepared sodium ethoxide and ethanol at 80°C for 4 h produced AZD5069, an identical synthetic route was used to synthesize
[2-14C]thiobarbituric acid [14C]-7 as the sodium salt. The salt was the stable labelled internal standard of AZD5069. Condensation
isolated from the reaction mixture by filtration and the filtrate, of commercially available [13C3]diethylmalonate with [13C,15N2]
which contained predominately unreacted [14C]thiourea, was thiourea and subsequent alkylation with 2,3-difluorobenzyl
evaporated and recycled by reacting with fresh diethyl malonate bromide provided a bulk stock of the key [M+6]dihydroxy-
and sodium ethoxide in ethanol. After heating at 80°C for 3 h, a pyrimidine intermediate. Chlorination, followed by insertion of
further batch of [2-14C]thiobarbituric acid sodium salt [14C]-7 the alcohol and azetidine sulphonamide moieties, gave
was isolated to give a total radiochemical yield of 83.4%. The
[
13C4,15N2]-1 with the required six mass units higher than the
combined solids [14C]-7 were alkylated with one equivalent of parent and was successfully used to support MS method
2,3-difluoro benzyl bromide in aqueous acetonitrile. After 24 h, development.
the reaction mixture was filtered to remove mono and bis O-
alkylated impurities, and the filtrate was evaporated to furnish
the pyrimidine diol as a white solid with a radiochemical purity
Metabolite synthesis
of 97%. Without purification, the diol was heated at 105°C in The synthetic route used to prepare the AZD5069 acid
the presence of POCl3 and N,N-dimethylaniline for 10 h to give metabolite (14) is outlined in Scheme 3. Initially, we investigated
the pyrimidine dichloride [14C]-8 (52.3 mCi, 53% radiochemical the synthesis through direct oxidation of the parent compound
yield from [14C]thiourea) after silica gel flash chromatography. (1). Not surprisingly, attempts to prepare the metabolite by
The material had a radiochemical purity of 97.4%. (R)-1-((S)-2,2- oxidation of the primary alcohol to the carboxylic acid proved
Dimethyl-1,3-dioxolan-4-yl)ethanol (9) was treated with sodium challenging due to concomitant oxidation of the sulphide or
Scheme 2. Synthesis of [14C]AZD5069, [14C]-1.
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M. J. Hickey et al.
Scheme 3. Synthesis of AZD5069 acid metabolite, (14).
Detector and a Lab Logic Radioflow Detector Beta-Ram Model 3. The
following conditions were used: Waters Xbridge C18, 5.0 μm, 4.6 × 150
mm, column temperature 40°C, 5% acetonitrile : water (0.1%
trifluoroacetic acid) to 95% acetonitrile : water (0.1% trifluoroacetic acid)
10 min linear gradient, 1 mL/min, UV 254 nm. Quantification of
radioactivity was performed using a Perkin-Elmer TRI-CARB 2500 liquid
scintillation analyser, with Ultima GoldTM cocktail.
cleavage of the diol. The problem was neatly solved through the
synthesis of the aldehyde precursor that was then selectively
oxidized using Lindgren–Pinnick oxidation conditions.11
Protection of the AZD5069 (1) diol as silyl ethers followed by
removal of the primary silyl protecting group12 produced (12)
in a modest 68% yield. Subsequent Dess–Martin periodinane
oxidation cleanly furnished the aldehyde (13) in 95% yield after
purification with no oxidation of the sulphide being observed.
Finally, chemoselective oxidation of the aldehyde with sodium
chlorite followed by in situ deprotection and dimethyl sulfoxide
(DMSO) quench gave the crude acid metabolite (14) with
<10% of sulphoxide and sulphone being observed. Purification
of the crude product by reverse phase preparative
chromatography afforded pure AZD5069 acid metabolite (14).
2,3-Difluoro-6-iodobenzoic acid (2)
A solution of diisopropylamine (5.46 mL, 4.21 g, 41.6 mmol) in dry
tetrahydrofuran (200 mL) was stirred at À78°C under nitrogen. n-
Butyllithium 2.5 M in tetrahydrofuran (16.6 mL, 41.6 mmol) was added
dropwise, and the mixture was stirred for 10 min. 1,2-Difluoro-4-
iodobenzene (10 g, 41.6 mmol) was added over 15 min, and the yellow
solution was stirred at À78°C for 1.5 h. The mixture was poured on to
Using the conditions developed in the preceding texts, the solid carbon dioxide and left to attain room temperature. The mixture
stable labelled internal standard [13C4,15N2] AZD5069 acid
was partitioned between 1 N aqueous sodium hydroxide (60 mL) and
metabolite was successfully prepared from [13C4,15N2]AZD5069
diethyl ether (60 mL). The basic aqueous extract was acidified to pH 2
with concentrated hydrochloric acid and extracted with diethyl ether
for use in quantitative bioanalysis studies.
(3 × 60 mL). The combined organic phases were washed with brine
(50 mL), dried over magnesium sulphate, filtered and evaporated in
Experimental
vacuo to yield the acid (2) as a white solid (7.9 g, 67%).
1H NMR (CDCl3) δ 7.05 (td, J = 8.6, 8.7 Hz, 1H), 7.66 (ddd, J = 8.8, 4.4,
1.9 Hz, 1H), 10.87 (s, 1H)
Materials and methods
[
14C]Thiourea (specific activity 0.75 mCi/mg) was purchased from
2,3-Difluoro-6-iodobenzyl bromide (3)
Quotient Bioresearch (Radiochemicals), Cardiff, UK. All other reagents
and anhydrous solvents were obtained from Sigma-Aldrich and were
used without further purification. Azetidine-1-sulphonamide, (R)-1-((S)-
To a solution of 2,3-difluoro-6-iodobenzoic acid (2) (3 g, 10.56 mmol) in
dry tetrahydrofuran (50 mL) at 0°C under nitrogen was added dropwise
2,2-dimethyl-1,3-dioxolan-4-yl)ethanol and authentic reference standards borane-tetrahydrofuran complex 1.0 M (38 mL, 38 mmol) followed by
were prepared by AstraZeneca Medicinal Chemistry. 1H NMR spectra boron trifluoride etherate (1.33 mL, 1.49 g, 10.56 mmol). The mixture
were recorded on a Varian Unity Inova 400 MHz NMR spectrometer
was heated at 60°C for 2 h and left to stand at room temperature for
unless otherwise stated. Chemical shifts (δ) in ppm are quoted relative 48 h. The mixture was quenched by dropwise addition of methanol
to (CD3)2S=O (δ 2.50) or CDCl3 (δ 7.26). Flash column chromatography
(30 mL). The solution was evaporated to an oil and partitioned between
was performed using prepacked SiliSepTM silica gel cartridges (SiliCycle, 1 N aqueous sodium hydroxide (30 mL) and diethyl ether (2 × 50 mL).
Quebec, Canada). Analytical thin-layer chromatography (TLC) was carried The combined organic extracts were washed with brine (30 mL), dried
out on Merck 5785 Kieselgel 60F254 fluorescent plates. Liquid over magnesium sulphate, filtered and evaporated in vacuo before
chromatography mass spectrometry (LCMS) and specific activity pumping under high vacuum to constant weight. The alcohol was
measurements were obtained by electrospray ionization using a Waters
Acquity UPLC with a Waters Micromass ZQ ESCi probe mass detector.
obtained as a white solid (2.45 g, 86%).
1H NMR (CDCl3) δ 4.83 (d, J = 2.3 Hz, 2H), 7.59 (ddd, J = 8.6, 4.5, 1.9 Hz,
Specific activity was calculated by comparison of the ratio of carbon- 1H), 7.66 (dd, J = 8.8 Hz, 1H)
14/carbon-12 for the tracer against the unlabelled reference.
Radiochemical reaction monitoring and purity checks were determined
A mixture of 2,3-difluoro-6-iodobenzyl alcohol (1.5 g, 5.56 mmol) and
phosphorous tribromide (2 mL, 5.7 g, 21.1 mmol) was heated at 80°C for
on a Waters 2695 alliance analytical HPLC fitted with a 996 Diode Array 1 h. The dark mixture was cooled to room temperature, and saturated
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M. J. Hickey et al.
sodium bicarbonate (40 mL) was carefully added dropwise. The mixture HPLC (Xterra C8 RP column, 150 × 19 mm, 5 to 95% acetonitrile/water
was extracted with diethyl ether (2 × 50 mL), and the combined organic
extracts were washed with water (50 mL) and brine (30 mL), dried over
magnesium sulphate, filtered and evaporated in vacuo to yield (3) as a
white solid (1.61 g, 87%).
(0.1% trifluoroacetic acid) gradient over 15 min, 20 mL/min, 254 nm).
Pure factions were combined and freeze dried to afford N-(2-(2,3-
difluoro-6-iodobenzylthio)-6-((2R,3S)-3,4-dihydroxybutan-2-yloxy)pyrimidin-
4-yl)azetidine-1-sulfonamide (6) (0.030 g, 35.5%) as a white solid.
1H NMR ((CD3)2SO) δ 1.27 (d, 3H) 2.14 (quin, 2H) 3.39 (t, 2H) 3.70 (m,
1H) 3.93 (t, 4H) 4.59 (m, 3H) 4.92 (d, 1H) 5.29 (m, 1H) 6.13 (s, 1H) 7.27
(m, 1H) 7.77 (m, 1H) 11.11 (br. s, 1H). LCMS (ES+) m/z: 603 [M+H]+.
1H NMR (CDCl3) δ 4.62 (d, J = 2.5 Hz, 2H), 6.92 (m, 1H), 7.60 (ddd, J = 8.8,
4.7, 2.0 Hz, 1H); 13C NMR (76 MHz, CDCl3) δ 29.9, 93.1, 119.0, 130.61, 134.9,
148.8, 150.4.
N-(2-(2,3-Difluoro-[6-3H]benzylthio)-6-((2R,3S)-3,4-dihydroxybutan-
N-(2-(2,3-Difluorobenzylsulfonyl)-6-((R)-1-((S)-2,2-dimethyl-1,3-
dioxolan-4-yl)ethoxy)pyrimidin-4-yl)-N-(4-methoxybenzyl)
azetidine-1-sulfonamide (5)
2-yloxy)pyrimidin-4-yl)azetidine-1-sulfonamide, [3H]AZD5069, [3H]-1
N-(2-(2,3-Difluoro-6-iodobenzylthio)-6-((2R,3S)-3,4-dihydroxybutan-2-
yloxy)pyrimidin-4-yl)azetidine-1-sulfonamide (6) (5.3 mg, 8.30 μmol)
was dissolved in ethanol (1.0 mL), and triethylamine (20 μL, 133 μmol)
was added followed by 10% palladium on carbon (2.5 mg, 2.35 μmol).
The reaction mixture was cooled to liquid nitrogen temperature,
degassed twice by a freeze-thaw cycle and placed under a partial
atmosphere of tritium gas (73 mbar, 2.5 Ci) for 2 h. The reaction
mixture was filtered, and the labile tritium was removed through
lyophilization with ethanol (2 × 5 mL) to leave crude [3H]AZD5069,
[3H]-1. Ethanol (10 mL) was added, and the solution (240 mCi,
24.0 mCi/mL) was stored at À20°C. The radiochemical purity was
measured at 96%.
Sodium hydride (60% in mineral oil) (0.511 g, 23.23 mmol) was added to a
solution of N-(2-(2,3-difluorobenzylthio)-6-((R)-1-((S)-2,2-dimethyl-1,3-
dioxolan-4-yl)ethoxy)pyrimidin-4-yl)azetidine-1-sulfonamide (4) (6.0 g,
11.61 mmol) in N,N-dimethylformamide (60 mL) at 0°C under an
atmosphere of nitrogen. The reaction mixture was stirred for 15 min;
then, 4-methoxybenzyl chloride (3.16 mL, 23.23 mmol) was added
followed by potassium iodide (2.03 g, 12.2 mmol). After stirring at room
temperature for 18 h, the reaction mixture was diluted with ethyl acetate
(50 mL) and washed with water (50 mL). The aqueous was further
extracted with ethyl acetate (2 × 50 mL), and the combined organic
phases were washed with brine (50 mL) and dried over magnesium
sulphate, filtered and evaporated to give a yellow oil. The crude product
was purified by flash silica chromatography, eluting with a 5 to 20% ethyl
acetate/isohexane gradient. Pure fractions were evaporated to
dryness to afford N-(2-(2,3-difluorobenzylthio)-6-((R)-1-((S)-2,2-dimethyl-
1,3-dioxolan-4-yl)ethoxy)pyrimidin-4-yl)-N-(4-methoxybenzyl)azetidine-
1-sulfonamide (5.61 g, 76%) as a yellow gum that was used directly in the
next step.
3-Chloroperbenzoic acid (mCPBA) (70%) (0.774 g, 3.14 mmol) was
added to N-(2-(2,3-difluorobenzylthio)-6-((R)-1-((S)-2,2-dimethyl-1,3-
dioxolan-4-yl)ethoxy)pyrimidin-4-yl)-N-(4-methoxybenzyl)azetidine-1-
sulfonamide (1 g, 1.57 mmol) in dichloromethane (40 mL). The resulting
mixture was stirred at room temperature for 24 h. The reaction mixture
was diluted with dichloromethane (50 mL) and washed successively with
aqueous sodium thiosulphate solution (3 × 50 mL, 10 g/100 mL),
saturated aqueous sodium bicarbonate (2 × 50 mL) and brine (50 mL).
The organic phase was dried over magnesium sulphate, filtered and
evaporated, and the crude residue was triturated with diethyl ether
and filtered to give N-(2-(2,3-difluorobenzylsulfonyl)-6-((R)-1-((S)-2,2-
dimethyl-1,3-dioxolan-4-yl)ethoxy)pyrimidin-4-yl)-N-(4-methoxybenzyl)
azetidine-1-sulfonamide (5) (0.96 g, 91%) as a white solid. The material
was 90% pure by HPLC and used directly in the next step. LCMS (ES+)
m/z: 669 [M+H]+.
An ethanol solution of N-(2-(2,3-difluoro-[6-3H]benzylthio)-6-((2R,3S)-
3,4-dihydroxybutan-2-yloxy)pyrimidin-4-yl)azetidine-1-sulfonamide [3H]-
1 (24 mCi) was evaporated and dissolved in 0.1% aq trifluoroacetic
acid/acetonitrile 50/50% (0.2 mL) and purified by reverse phase
preparative chromatography (Xterra C8 RP column, 150 × 7.8 mm, 40%
acetonitrile/water (0.1% trifluoroacetic acid) isocratic, then acetonitrile
gradient flush, 3.0 mL/min, 254 nm). The pure fractions were combined
and freeze dried to give [3H]AZD5069 [3H]-1 that was dissolved in
ethanol (10 mL). The activity was measured at 18.4 mCi, and the
radiochemical purity was 99% by HPLC with a specific activity of
25.1 Ci/mmol. LCMS (ES+) m/z: 477, 479 [M+H]+
[2-14C]Thiobarbituric acid sodium salt, [14C]-7
Into a dried 5 mL reactivial fitted with a sure seal septum and stirrer bar
was placed diethyl malonate (282 mg, 1.76 mmol), ethanol (2 mL) and
[
14C]thiourea (98.8 mCi, 131 mg, 1.68 mmol). The mixture was stirred at
80°C in a heat block, and after 10 min, a solution was observed. Freshly
prepared sodium ethoxide (0.8 mL, 1.74 mmol; made from 0.19 g sodium
in 3.8 mL ethanol) was injected in one portion, and the yellow mixture
was heated at 80°C for 4 h. A cream-white solid precipitated. The mixture
was cooled and left at room temperature overnight for 16 h. The solid
was filtered and washed with ice-cold ethanol (0.5 mL) and ether
(0.5 mL), air-dried and collected to give [2-14C]thiobarbituric acid sodium
salt [14C]-7 as a cream-white solid (157 mg). The mother liquor was
evaporated to dryness and heated at 80°C in ethanol (1 mL) with diethyl
malonate (141 mg, 0.88 mmol) and sodium ethoxide (0.4 mL, 0.87 mmol;
made from 0.19 g sodium in 3.8 mL ethanol) for 3 h, cooled and left at
room temperature overnight. The solid was filtered and washed with
ice-cold ethanol (0.5 mL) and ether (0.5 mL), air-dried and collected to
give [2-14C]thiobarbituric acid sodium salt [14C]-7 as a cream solid
(78 mg). The two solids were combined (235 mg, 83.4%) and used
directly in the next reaction.
N-(2-(2,3-Difluoro-6-iodobenzylthio)-6-((R)-1-((S)-2,2-dimethyl-
1,3-dioxolan-4-yl)ethoxy)pyrimidin-4-yl)-N-(4-methoxybenzyl)
azetidine-1-sulfonamide (6)
Sodium hydrosulphide hydrate (0.025 g, 0.45 mmol) was added to a
solution of N-(2-(2,3-difluorobenzylsulfonyl)-6-((R)-1-((S)-2,2-dimethyl-
1,3-dioxolan-4-yl)ethoxy)pyrimidin-4-yl)-N-(4-methoxybenzyl)azetidine-
1-sulfonamide (5) (0.15 g, 0.22 mmol) and stirred in anhydrous DMSO
(5 mL) under nitrogen. After 30 min, 2,3-difluoro-6-iodobenzyl bromide
(3) (0.223 g, 0.67 mmol) was added, and the reaction mixture was stirred
for 1 h, before it was diluted with diethyl ether (50 mL) and washed with
saturated aqueous ammonium chloride (3 × 50 mL) and brine (50 mL).
4,6-Dichloro-2-(2,3-difluorobenzylthio)[2-14C]pyrimidine, [14C]-8
The organic phase was dried over magnesium sulphate, filtered and [2-14C]Thiobarbituric acid sodium salt [14C]-7 (235 mg, 1.40 mmol) was
evaporated to give a colourless oil. The crude product was purified by
flash silica chromatography, eluting with isohexane 67%/ethyl acetate
33%. The pure fractions were combined and evaporated to dryness to
afford N-(2-(2,3-difluoro-6-iodobenzylthio)-6-((R)-1-((S)-2,2-dimethyl-1,3-
dioxolan-4-yl)ethoxy)pyrimidin-4-yl)-N-(4-methoxybenzyl)azetidine-1-
sulfonamide (0.107 g, 63%) as a white solid. LCMS (ES+) m/z: 763 [M+H]+.
dissolved in acetonitrile (3.0 mL) and water (3.0 mL) and stirred under
nitrogen at room temperature. 2,3-Difluorobenzyl bromide (289 mg,
1.40 mmol) in acetonitrile (1.5 mL) was added dropwise, and the mixture
was stirred at room temperature for a total of 24 h. A white solid
precipitated. The mixture was cooled in an ice bath, then filtered. The
solid contained mostly peralkylated material. The filtrate was evaporated
The solid was dissolved in trifluoroacetic acid (2.0 mL) and in vacuo and repeated with 3 × 4 mL of acetonitrile. The sample was dried
dichloromethane (2.0 mL) and stirred at room temperature for 2 h before at 40°C under high vacuum to a white solid 395 mg that was taken on
evaporating to dryness. The crude product was purified by preparative
directly to the next reaction. The material co-chromatographed with an
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M. J. Hickey et al.
authentic sample by HPLC and had a radiochemical purity of 93%. A
column, 150 × 19 mm, 40% acetonitrile in water (0.1% trifluoroacetic
acid) isocratic, then acetonitrile gradient flush, 20 mL/min, 10 × 0.66 mL
mixture of (2,3-difluorobenzylthio)[2-14C]pyrimidine-4,6-diol (395 mg,
1.05 mmol), phosphorous oxychloride (1.7 mL, 18.2 mmol) and N,N- injections). Pure factions were combined and freeze dried to give [14C]
dimethylaniline (0.31 mL, 2.46 mmol) were heated at 105°C for 10 h,
and the dark mixture was left to cool overnight. A small aliquot was
AZD5069 [14C]-1 as a white solid (89.2 mg, 122.1 μCi/mg, 10.9 mCi) with
a radiochemical purity of 99.8% by HPLC and a specific activity of
quenched in water/acetonitrile (1:1). HPLC analysis showed no starting 59.3 mCi/mmol by mass spectrometry.
material remaining. The reaction mixture was carefully quenched by
1H NMR ((CD3)2SO) δ 1.21 (d, J = 6.36 Hz, 3H), 2.14 (quin, J = 7.68 Hz,
adding to warm water (15 mL) and extracted with ethyl acetate 2H), 3.39 (br. s, 2H), 3.63–3.70 (m, 1H), 3.92 (t, J = 7.70 Hz, 4H), 4.42–4.56
(3 × 15 mL). The combined extracts were washed with brine, dried
(magnesium sulphate) and concentrated to a brown oil, dissolved in
dichloromethane (3 mL) and purified by flash chromatography on silica
eluting with 1% ethyl acetate in isohexane. Fractions containing product
were combined and evaporated yielding a pale yellow oil [14C]-8
(m, 2H), 4.62 (t, 1H), 4.92 (d, J = 5.17 Hz, 1H), 5.18–5.25 (m, 1H), 6.12 (s,
1H), 7.13–7.19 (m, 1H), 7.29–7.44 (m, 2H), 11.11 (br. s, 1H). LCMS (ES+)
m/z: 479 [M+H]+.
N-(6-((2R,3S)-3-(tert-Butyldimethylsilyloxy)-4-hydroxybutan-2-
(276 mg, 52.3 mCi, 53% radiochemical yield from [14C]thiourea). The yloxy)-2-(2,3-difluorobenzylthio)pyrimidin-4-yl)azetidine-1-
material co-chromatographed with an authentic sample by HPLC and
had a radiochemical purity of 97.4%.
sulfonamide (12)
AZD5069 (1) (4.23 g, 8.88 mmol), tert-butylchlorodimethylsilane (5.35 g,
35.51 mmol), and imidazole (3.02 g, 44.38 mmol) were dissolved in N,N-
dimethylformamide (30 mL) to give a colourless solution. The mixture
was stirred overnight. TLC (isohexane 75%/ethyl acetate 25%) showed
complete disappearance of the starting material and formation of a
single product. The reaction mixture was partitioned between ethyl
acetate (200 mL) and 0.5 M aqueous hydrochloric acid (200 mL). The
organic phase was collected, and the aqueous phase was extracted with
another portion of ethyl acetate (150 mL). The combined organic phases
were washed with water (2 × 100 mL), brine (50 mL) and dried over
sodium sulphate. Filtration and evaporation of the solvent in vacuo gave
8 g of crude product as a yellow syrup. The crude product was taken up
in dichloromethane (20 mL), applied onto a silica column and eluted with
isohexane 88%/ethyl acetate 12% to isohexane 84%/ethyl acetate 16%.
Fractions containing product were combined and concentrated under
reduced pressure to yield the bis-silyl ether, 5.70 g, 91% as a clear oil.
1H NMR (500 MHz, CDCl3) δ 0.03 (2s, 6H), 0.07 (2s, 6H), 0.89 (2s, 18H),
1.25 (d, 3H), 2.25 (p, 2H), 3.55 (ddd, 2H), 3.94 (td, 1H), 4.01 (t, 4H), 4.39
(dd, 2H), 5.42 (qd, 1H), 6.26 (s, 1H), 6.95 (bs, 1H), 6.99–7.1 (m, 2H), 7.21
(t, 1H).
4-Chloro-2-(2,3-difluorobenzylthio)-6-((R)-1-((S)-2,2-dimethyl-1,3-
dioxolan-4-yl)ethoxy) [2-14C]pyrimidine, [14C]-10
A mixture of (R)-1-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethanol (9) (98 mg,
0.67 mmol) and 4,6-dichloro-2-(2,3-difluorobenzylthio)[2-14C]pyrimidine
[
14C]-8 (~39.4 mCi, 206 mg, 0.67 mmol) were stirred at 0°C in 2-methyl
tetrahydrofuran (6 mL) under nitrogen. Sodium hydride in oil (34.6 mg,
0.87 mmol) was added, and the mixture was stirred for 18 h overnight
at room temperature. TLC (isohexane 96%/ethyl acetate 4%) showed
no starting material and major product as monochloride. The mixture
was treated with brine (30 mL) and extracted with ethyl acetate
(2 × 30 mL), evaporated to an oil and purified by flash chromatography
on silica eluting with isohexane 96%/ethyl acetate 4%. Pure fractions
were combined and evaporated in vacuo to [14C]-10 as a clear oil
(156 mg, 21.9 mCi, 56% radiochemical yield). The material co-
chromatographed with an authentic sample by HPLC and had a
radiochemical purity of 98.2%. LCMS (ES+) m/z: 419 [M+H]+.
N-(2-(2,3-Difluorobenzylthio)-6-((2R,3S)-3,4-dihydroxybutan-2-
yloxy)[14C]pyrimidin-4-yl)azetidine-1-sulfonamide, [14C]AZD5069,
[14C]-1
The oil (5.70 g, 8.08 mmol) was dissolved with pyridine (4.6 mL,
56.87 mmol) and tetrahydrofuran (55 mL) in a 50-mL plastic bottle to give
a colourless solution. To this solution was added HF-pyridine (4.6 mL,
30.63 mmol). The bottle was closed, and the contents were stirred for
2.5 h, monitoring the reaction by TLC (isohexane 67%/ethyl acetate
33%). The reaction was stopped at 95% conversion by partitioning the
reaction mixture between ethyl acetate and 1 M aqueous hydrochloric
acid (150 mL/150 mL). The organic phase was collected, and the aqueous
phase was extracted with another portion of ethyl acetate (150 mL). The
combined organic extracts were washed with 0.5 M aqueous
hydrochloric acid (100 mL) and a solution of saturated sodium carbonate
(50 mL) and brine before drying over sodium sulphate. Filtration and
evaporation in vacuo yielded a pale yellow oil. The oil was dissolved in
25 mL of dichloromethane, applied onto a silica column and eluted with
isohexane 84%/ethyl acetate 16% to isohexane 75%/ethyl acetate 25%.
Pure fractions were combined and evaporated to give (12) as a white
solid foam (3.60 g, 75%).
Into
difluorobenzylthio)-6-((R)-1-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)[2-14C]
pyrimidine
14C]-10 (21.9 mCi, 156 mg, 0.37 mmol), azetidine-1-
a
dry flask under nitrogen was added 4-chloro-2-(2,3-
[
sulfonamide (11) (102 mg, 0.75 mmol), potassium carbonate (77 mg,
0.56 mmol), tris(dibenzylideneacetone)dipalladium(0) (34 mg, 0.04 mmol),
2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (35 mg, 0.07 mmol)
and butyl acetate (5 mL). The red mixture was degassed twice and stirred
vigorously under nitrogen for 10 min at room temperature, then for 1 h at
100°C during which time the reaction mixture became a pale brown
colour. The reaction mixture was cooled, butyl acetate (10 mL) added
followed by brine. The mixture was filtered through 0.5 μ filter, and the
layers were separated. The organic layer was washed with 1 M aqueous
hydrochloric acid, saturated sodium bicarbonate solution, brine and dried
over magnesium sulphate, concentrated under vacuum to a dark red oil
and purified by flash chromatography on silica eluting with isohexane
90%/ethyl acetate 10%. Pure fractions were collected, combined and
concentrated to furnish a pale yellow oil (162 mg, 18.4 mCi, 84%
radiochemical yield). The material had a radiochemical purity of 98.6%.
LCMS (ES+) m/z: 519 [M+H]+.
1H NMR (500 MHz, CDCl3) δ 0.11 (s, 3H), 0.12 (s, 3H), 0.93 (s, 9H), 1.29 (d,
3H), 1.89 (t, 1H), 2.26 (p, 2H), 3.61 (t, 2H), 3.86 (q, 1H), 3.98–4.05 (m, 4H),
4.33–4.43 (m, 2H), 5.25–5.33 (m, 1H), 6.27 (s, 1H), 6.96 (bs, 1H), 7–7.1 (m,
2H), 7.21–7.26 (m, 1H).
The oil (18.4 mCi, 162 mg, 0.31 mmol) was dissolved in acetonitrile
(2 mL), water (0.4 mL) and phosphoric acid (36.0 mg, 0.31 mmol) and
was heated under nitrogen at 50°C for 24 h. The pale yellow solution
was evaporated under reduced pressure, and the residue was dissolved
N-(6-((2R,3R)-3-(tert-Butyldimethylsilyloxy)-4-oxobutan-2-yloxy)-
2-(2,3-difluorobenzylthio)pyrimidin-4-yl)azetidine-1-sulfonamide
(13)
in ethanol (10 mL). The crude solution of [14C]AZD5069 [14C]-1, which A solution of N-(6-((2R,3S)-3-(tert-butyldimethylsilyloxy)-4-hydroxybutan-
had a total radioactivity of 18.4 mCi by liquid scintillation counting and
a radiochemical purity of 98%, was stored in the fridge. LCMS (ES+) m/
z: 479 [M+H]+.
2-yloxy)-2-(2,3-difluorobenzylthio)pyrimidin-4-yl)azetidine-1-sulfonamide
(12) (3.6 g, 6.09 mmol) and pyridine (0.49 mL, 6.09 mmol) in
dichloromethane (60 mL) was stirred to give a colourless solution. To
this solution was added solid Dess–Martin periodinane (3.10 g,
A portion of the ethanol solution (6.60 mL, 12.1 mCi) was concentrated
to dryness, dissolved in 0.1% aq trifluoroacetic acid 50%/acetonitrile 50% 7.31 mmol). The yellow solution was stirred at room temperature for
(8 mL) and subjected to preparative chromatography (Xterra C8 RP 1 h to give a milky solution. Diethyl ether (200 mL) was added followed
J. Label Compd. Radiopharm 2016, 59 432–438
Copyright © 2016 John Wiley & Sons, Ltd.
www.jlcr.org

M. J. Hickey et al.
by saturated sodium thiosulphate solution (100 mL) and saturated
sodium hydrogen carbonate solution (100 mL). The mixture was stirred
Conclusion
vigorously for 30 min, and the phases were allowed to separate. The The synthesis of radiolabelled AZD5069 has been described.
[3H]AZD5069 was prepared through reductive dehalogenation
organic phase was collected, and the aqueous phase was further
extracted with ether (100 mL). The combined organic phases were
of an iodinated precursor. The reduction with tritium gas was
washed with 0.5 M aqueous hydrochloric acid (2 × 100 mL), saturated
rapid, clean and high yielding with ~87% incorporation of the
sodium carbonate solution (50 mL) and brine (50 mL). The organic
tritium isotope. The synthetic route was also applicable for
solution was dried over sodium sulphate and filtered. Removal of the
preparing high specific activity tritium labels where changes
solvent under reduced pressure gave a red oil 3.70 g that became
to the S-benzyl group substituents were made. The synthesis
more and more coloured over time. The oil was dissolved in
of [14C]AZD5069 was performed in six steps from [14C]thiourea
dichloromethane (20 mL) and applied onto a silica column. The column
with an overall radiochemical yield of 18%. The synthetic route
used was also applied to the synthesis of an M+6
was eluted with isohexane 85%/ethyl acetate 15% to isohexane 80%/
ethyl acetate 20%. Pure fractions were combined and evaporated to
give (13) as a clear gum (2.90 g, 95%) which was used directly in the stable labelled isotopomer using [13C3]diethylmalonate with
[13C,15N2]thiourea to achieve the desired mass increase. Finally,
next reaction.
1H NMR (500 MHz, (CD3)2SO) δ 0.01 (s, 3H), 0.03 (s, 3H), 0.87 (s, 9H), 1.20
(d, 3H), 2.07–2.17 (m, 2H), 3.90 (t, 4H), 4.44 (d, 1H), 4.48 (d, 2H), 5.54 (qd,
1H), 6.11 (s, 1H), 7.13–7.19 (m, 1H), 7.3–7.37 (m, 1H), 7.40 (t, 1H), 9.57 (s,
1H), 11.16 (s, 1H).
chemoselective oxidation of an aldehyde precursor under
Lindgren–Pinnick oxidizing conditions furnished the AZD5069
acid metabolite.
References
(2R,3R)-3-(6-(Azetidine-1-sulfonamido)-2-(2,3-difluorobenzylthio)
pyrimidin-4-yloxy)-2-hydroxybutanoic acid, AZD5069 acid (14)
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A solution of N-(6-((2R,3R)-3-(tert-butyldimethylsilyloxy)-4-oxobutan-2-
yloxy)-2-(2,3-difluorobenzylthio)pyrimidin-4-yl)azetidine-1-sulfonamide
(13) (1.60 g, 2.72 mmol) in tert-butanol (9 mL) was stirred to give a
colourless solution. The reaction mixture was diluted with water (3 mL),
and sodium dihydrogen phosphate monohydrate (750 mg, 5.44 mmol)
and 2-methyl-2-butene (2 M in tetrahydrofuran) (2.72 mL, 5.44 mmol)
were added. The solution was cooled in an ice bath and stirred for
5 min before sodium chlorite (614 mg, 5.44 mmol) was added. An
immediate colour change from colourless to yellow/green was observed.
After 1 h, DMSO (4 mL) was added, and the mixture was filtered. The
crude product was purified by reverse phase preparative
chromatography using the following conditions: Kromasil C8 RP column,
10 μm, 5 to 50% acetonitrile in water (0.2% acetic acid) over 20 min,
254 nm. The pure fractions containing product were combined and
freeze-dried to yield AZD5069 acid (14) as off-white solid (0.9 g, 67%)
with a chemical purity of 99.5%.
1H NMR (500 MHz, (CD3)2SO) δ 1.21 (d, 3H), 2.13 (p, 2H), 3.91 (t, 4H),
4.25 (d, 1H), 4.48 (q, 2H), 5.38–5.45 (m, 1H), 5.60 (bs, 1H), 6.10 (s, 1H),
7.16 (dd, 1H), 7.34 (dd, 1H), 7.41 (t, 1H), 11.14 (bs, 1H), 12.74 (bs, 1H).
LCMS (ES+) m/z: 491 [M+H]+.
[11] B. O. Lindgren, T. Nilsson, S. Husebye, Ø. Mikalsen, K. Leander, C. G.
Swahn, Acta Chem. Scand. 1973, 27, 888–890.
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J. Label Compd. Radiopharm 2016, 59 432–438