Synthesis of 3H, 2H4 and 14C-SCH 417690 (Vicriviroc)

Article abstract of DOI:10.1002/jlcr.3387

Vicriviroc or SCH 417690 is a potent and selective antagonist of the CCR5 receptor. CCR5 receptor antagonists have the potential for the treatment of HIV infections. Four distinct isotopically labelled forms of SCH 417690 were synthesized. Low specific activity [³H]SCH 417690 was prepared for a preliminary absorption, distribution, metabolism and excretion evaluation of the compound and [¹⁴C]SCH 417690 for more definitive absorption, distribution, metabolism and excretion work, including an absorption, metabolism and excretion study in man. In addition, high specific activity [³H]SCH 417690 was prepared for CCR5 receptor binding work and [²H₄]SCH 417690 was prepared as an internal standard for a liquid chromatography-mass spectrometry bioanalytical method. The paper discusses the synthesis of four isotopically labelled forms of SCH 417690.

Full text of DOI:10.1002/jlcr.3387

Research article
Received 07 December 2015,
Revised 27 January 2016,
Accepted 09 February 2016
Published online 15 March 2016 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3387
3
2
14
Synthesis of H, H and C-SCH 417690
4
(Vicriviroc)
D. Hesk,* S. Borges, S. Hendershot, D. Koharski, P. McNamara, S. Ren,
S. Saluja, V. Truong, and K. Voronin
Vicriviroc or SCH 417690 is a potent and selective antagonist of the CCR5 receptor. CCR5 receptor antagonists have the
potential for the treatment of HIV infections. Four distinct isotopically labelled forms of SCH 417690 were synthesized.
3
Low specific activity [ H]SCH 417690 was prepared for a preliminary absorption, distribution, metabolism and excretion
1
4
evaluation of the compound and [ C]SCH 417690 for more definitive absorption, distribution, metabolism and excretion
3
work, including an absorption, metabolism and excretion study in man. In addition, high specific activity [ H]SCH 417690
2
was prepared for CCR5 receptor binding work and [ H
4
]SCH 417690 was prepared as an internal standard for a liquid
chromatography–mass spectrometry bioanalytical method. The paper discusses the synthesis of four isotopically labelled
forms of SCH 417690.
Keywords: tritium exchange; ruthenium catalyst; deuterium; carbon-14
1
3
Introduction
pathway. A second batch of [ H]SCH 417690 was also prepared
at higher specific activity in order to conduct CCR5 receptor
binding studies. Following successful progression of the
compound into development, [ H
SCH 417690 was discovered as the result of optimization of a
lead containing a benzylic methyl group.
1
–8
2
4
]SCH 417690 was prepared
as an internal standard for a liquid chromatography–mass
spectrometry bioanalytical method, supporting the analysis of
samples derived from toxicology and clinical studies. Finally,
1
4
[
C]SCH 417690 was prepared to conduct definitive ADME
14
studies supporting compound registration, including a
C
absorption, metabolism and excretion (AME) study in man. The
paper hence discusses the synthesis of these four isotopically
labelled compounds.
Results and discussion
The size of the benzylic substituent was shown to be the key to
enhanced potency and receptor selectivity in this series.
Optimization around this benzylic methyl functionality led to
the methoxymethyl analogue SCH 417690 1, which had the best
overall profile.
3
Low specific activity [ H]SCH 417690 4 was prepared via Ru
9
(
Ph P) Cl catalysed exchange of piperidine intermediate 2 with
3
3
2
579 mCi, 50 Ci/mL tritiated water as shown in Figure 1.
Using this chemistry, the methylene protons adjacent to the
piperidine nitrogen are selectively exchanged.
9,10
Hence,
SCH 417690 is extremely potent (Ki 2.5 nM) and selective for
the CCR5 receptor with almost no affinity for the muscarinic
receptor or other G-protein–coupled receptors. It has excellent
oral bioavailability in rats and monkeys, reduced affinity for the
73 mCi of crude product 3 was isolated from the reaction, which
was purified by silica gel chromatography and coupled with 4,6-
dimethyl pyrimidine-5-carboxylic acid A under standard 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide/hydroxybenzotriazole
+
hERG K channel and no significant central nervous system or
3
(EDC/HOBt) conditions to generate 28 mCi of crude [ H]SCH
gastrointestinal side effects at an oral dose of 10 mg/kg in rats.
During the course of the progression of the compound from
discovery into development, several isotopically labelled forms
417690 4. Purification by reverse phase HPLC yielded a total
batch of 24 mCi at a specific activity of 1.2 Ci/mmol.
3
of SCH 417690 were prepared. Low specific activity [ H]SCH
4
17690 was prepared to conduct preliminary absorption,
Merck and Co, 126 E. Lincoln Avenue, RY 80R, Rahway, NJ 07065, USA
distribution, metabolism and excretion studies (ADME), which
confirmed the high oral bioavailability observed in cold
pharmacokinetic studies and identified O-demethylation of the
*Correspondence to: David Hesk, Merck and Co, 126 E. Lincoln Avenue, RY 80R,
Rahway NJ 07065, USA.
side chain followed by glucuronidation as the major metabolic E-mail: david.hesk@merck.com
J. Label Compd. Radiopharm 2016, 59 190–196
Copyright © 2016 John Wiley & Sons, Ltd.

D. Hesk et al.
3
Figure 1. Synthesis of [ H]SCH 417690 (4).
3
High specific activity [ H]SCH 417690 4, needed for receptor of 4 was lower than expected, it is possible that the presence of
binding work, was prepared via the same route as shown in adventitious water present during the preparation of the
Figure 1, with the tritiation step contracted out to Quotient tritiated water contributed to a significant dilution in specific
Bioresearch. Piperidine 2 was reacted with 20 Ci of 90 atom % activity of the tritiated water in the reaction. H NMR analysis
̒9 0 atom
%
̓
3
̒
̓
tritiated water, which generated 1.65 Ci of crude product 3. confirmed the tritium was exclusively incorporated alpha to the
About 300 mCi of this crude was purified by reverse phase HPLC piperidine nitrogen with no evidence for incorporation at other
3
and converted to [ H]SCH 417690 as previously described. After sites in either the piperidine or piperazine rings and is consistent
10
final reverse phase HPLC purification, a total batch of 46 mCi at a with previous results. While the exact mechanism of the
specific activity of 16.4 Ci/mmole was generated, which was reaction is unknown, the requirement for either a primary or
lower than was expected based on the specific activity secondary amine is suggestive of formation of a Ru-N species
generated from the initial synthesis of 4 utilizing 50 mCi/mL with subsequent activation of the adjacent CH bonds.
2
tritiated water. However, the specific activity was still sufficiently
high enough for the intended receptor binding studies, and
The synthesis of [ H
4
]SCH 417690 9 is shown in Figure 2.
2
4
N-Boc-[ H ]-4-piperidone 7 was prepared in a similar manner
while it is unclear why the specific activity of the second batch as previously described with one minor modification whereby
11
2
Figure 2. Synthesis of [ H
4
]SCH 417690 (12).
J. Label Compd. Radiopharm 2016, 59 190–196
Copyright © 2016 John Wiley & Sons, Ltd.
www.jlcr.org

D. Hesk et al.
14
the debenzylation and Boc installation steps to form 6 were and 100 mCi of a newly received 1% aqueous solution of [ C]
combined into a two-step one-pot process in overall 33% yield formaldehyde at 100 °C for 1.5 h, followed by stirring at room
14
from benzylamine trifluoroacetate. Following oxidation with N- temperature overnight. Under these conditions, the [ C]
methylmorpholine-N-oxide (NMO) and tetrapropylammonium formaldehyde generates a mixture of unlabelled, singly labelled
10
14
perruthenate (TPAP), 7 was then reacted with intermediate 8, and doubly C labelled 13 in a statistical ratio of 1:2:1 because
diethylaluminium cyanide and titanium isopropoxide in a two- of the aza-Cope rearrangement of the intermediate butenyl
2
13
step one-pot process to give the [ H
4
]cyano compound 9 in imminium ion. Hence, a 92% yield of crude product 13 was
7
5% yield. The cyano group was then displaced by generated, which was then reacted with Boc anhydride and
methylmagnesium iodide to give compound 10 in 78% yield. Pearlman’s catalyst under hydrogen (50 psi) to give compound
The Boc group was removed by trifluoroacetic acid in methylene 14 in 97% yield. The N-Boc compound 14 was then oxidized with
14
chloride to give compound 11 in 76% yield, which was then NMO and TPAP to give [ C]N-Boc piperidone 15 in 97% yield,
coupled with A under standard HOBt/EDC conditions to which was then treated with intermediate 8, diethylaluminium
2
generate crude [ H
4
]SCH 417690 12. The crude batch was cyanide and titanium isopropoxide to give compound 16 in
purified by silica gel chromatography to yield 700 mg (64%) of 93% yield after purification by silica gel chromatography. The
2
purified [ H ]SCH 417690 in an overall yield of 7.4%. The cyano group was then displaced by an excess of
4
chemical purity as determined by HPLC system 2 was >99%. methylmagnesium iodide to give compound 17 in 90% yield.
+
No unlabelled SCH 417690 was detected by FAB MS analysis.
The Boc group was removed with 50% trifluoroacetic acid in
1
4
14
[
C]SCH 417690 maleate 20 was synthesized from [ C] methylene chloride to give compound 16 in 98% yield. Compound
formaldehyde using a route similar to [ H ]SCH 417690 as shown 16 was finally coupled with A under standard bromotris
2
4
in Figure 3.
(pyrrolyldino)phosphonium hexafluorophosphate conditions to
1
4
14
On initial examination of the route, preparation of [ C] give crude [ C]SCH 417690 19, which was purified by silica gel
methylmagnesium iodide from [ C]methyl iodide appeared to chromatography to generate a 48 mCi batch of [ C]SCH 417690
be an attractive option to introduce the C label and eliminate in 63% yield. A portion of the batch was further purified by reverse
four steps from the route. Unfortunately, all attempts to prepare phase HPLC and used to generate a 25.7 μCi/mg batch of [ C]SCH
and utilize the [ C]Grignard met with limited success with very 417690 maleate 20 that was subsequently used in a C AME study
low yields when attempting to use 1 equivalent of [ C] in man. The radiochemical purity of the final diluted batch as
methylmagnesium iodide, and this approach was abandoned determined by reverse phase HPLC in systems 2 and 3 was >99%.
14
14
14
14
1
4
14
14
12
14
in favour of the longer route.
Hence, [ C]N-benzyl-4-
hydroxypiperidine 13 was prepared via a modification of the Experimental
11
Mannich-type cyclization used to prepare 7, which allowed
Materials
use of one equivalent of the commercially supplied 1% aqueous
1
4
14
solution of [ C]formaldehyde as opposed to the 20% solution
Tritiated water (50 Ci/mL) and [ C]formaldehyde were purchased from
2
2
used when preparing [ H
4
2
]benzyl-4-hydroxypiperidine. Thus, (Quotient Biosciences, Cardiff, UK). [ H ]Formaldehyde was purchased
benzyl-but-3-enyl-amine was treated with camphorsulfonic acid from (Cambridge Isotope Laboratories, Andover MA, USA). Compounds
1
4
Figure 3. Synthesis of [ C]SCH 417690 maleate (20).
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Copyright © 2016 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2016, 59 190–196

D. Hesk et al.
1
,13,14
3
2
4
, 8, 4,6-dimethylpyrimidine-5-carboxylic acid
and unlabelled SCH Synthesis of low specific activity [ H]SCH 417690 (4)
17690 were obtained from (Merck Research Labs, Process Chemistry,
Rahway NJ, USA). Tris-triphenylphosphine ruthenium (II) chloride was (S)-1-((R)-2-methoxy-1-(4-(trifluoromethyl)phenyl)ethyl)-2-methyl-4-
3
purchased from (Alfa, Ward Hill MA, USA). All remaining reagents were (4-methyl-[2- H]piperidin-4-yl)piperazine (3)
purchased from (Sigma Aldrich, Milwaukee WI, USA) or (Acros Organics,
Pittsburgh PA, USA) and were used as received. All H and C steps were
carried out under an atmosphere of argon.
2
14
Compound 2 (30 mg) and tris-triphenylphosphine ruthenium (II) chloride
4 mg) were weighed into a thick walled glass ampoule (6″ h × 0.313″
ID × 0.078″ wall), and dioxane (100 μL) was added. The ampoule was
fitted with a rubber septum, and tritiated water (50 Ci/mL, 579 mCi) was
added via a syringe. The ampoule was frozen in liquid nitrogen,
evacuated and sealed in a flame before being placed in an oil bath at
(
Liquid scintillation counting
1
20 °C for 3 h. At the completion of the reaction, the ampoule contents
Quantitation of radioactivity was performed using (Packard, Downers
Grove, IL, USA) 2200 or 2900 CA liquid scintillation analysers, with
Scintiverse BD cocktail used throughout.
were partitioned between potassium hydroxide solution (1 M, 5 mL)
and methylene chloride (5 mL). The methylene chloride layer was
removed and the aqueous layer extracted with methylene chloride
(
2 × 5 mL). The combined methylene chloride extracts were washed with
water and evaporated to dryness to yield 73 mCi of crude compound 3
with a radiochemical purity of 35% (TLC system 1). The product was
purified on a 1 g Waters Silica Sep-Pak cartridge using a gradient of 6–
Thin layer chromatography
Thin layer chromatography was performed using Whatman LK6DF (Silica
Gel 60) 5 × 20 cm, 0.25 mm plates. The plates were scanned on either a
Bioscan AR2000 or a Bioscan 1000 linear analyser. The following systems
were used:
1
2% 2 M methanolic ammonia in methylene chloride. A total of 30 mCi
of 3 was isolated at a radiochemical purity of 90% (TLC system 1), which
was used directly in the next step.
(
(
4,6-dimethylpyrimidin-5-yl)(4-((S)-4-((R)-2-methoxy-1-
1
2
3
4
5
6
7
Methylene chloride : 2 M methanolic ammonia (90:10)
Methylene chloride : 2 M methanolic ammonia (95:5)
Methylene chloride : methanol (95:5)
Methylene chloride : 7 M methanolic ammonia (99:1)
Methylene chloride : methanol : triethylamine (93:6:1)
Methylene chloride : methanol : triethylamine (94:5:1)
Ethyl acetate : hexanes : triethylamine (30:69:1)
4-(trifluoromethyl)phenyl)ethyl)-3-methylpiperazin-1-yl)-4-
3
3
methyl-[2- H]piperidin-1-yl)methanone [ H]SCH 417690 (4)
Compound 3 was then dissolved in dimethylformamide (DMF) (500 μL)
and
EDC
(12.5 mg,
65 μmol),
HOBt
(7.3 mg,
53.7 μmol)
diisopropylethylamine (DIPEA) (50 μL) and 4,6-dimethylpyrimidine-5-
carboxylic acid (9.3 mg, 60.5 μmol) were added. The reaction was stirred
overnight and analysed by HPLC system 1, which showed complete
reaction. The reaction was partitioned between aqueous potassium
hydroxide solution (1 M, 2 mL) and methylene chloride (2 mL). The layers
were separated, the methylene chloride layer removed and the aqueous
layer further extracted with methylene chloride (3 × 2 mL). The combined
methylene chloride extracts were dried over anhydrous sodium sulphate,
filtered and evaporated to dryness. The batch was purified on a
High performance liquid chromatography
A (Waters, Miford MA, USA) 2695 ‘Alliance’ HPLC system was used. Chemical
purity was determined using a Waters 2487 dual channel ultraviolet
detector and radiochemical purity either using a Packard C150 radioflow
detector with Packard FloScint III cocktail or a IN/US Beta Ram 2 radioflow
detector with IN/US InFlo 3 cocktail. The following systems were used:
9
.4 × 250 mm Extend C18 column with a mobile phase of 0.05 M pH 9
triethylammonium acetate: acetonitrile (50:50), 6 mL/min, 254 nm.
Fractions containing the purified product were pooled and evaporated
3
1
2
3
Waters Xterra RP18, 100 × 3 mm, 5 μ, 254 nm, 0.05 M pH 8 to dryness to yield a total of 24 mCi of [ H]SCH 417690 4 at radiochemical
purities of 99.7%, 98.8% (HPLC system 1 and TLC system 2) and a specific
triethylammonium acetate (50:50) acetonitrile for 10 min
followed by a step gradient to acetonitrile, 0.5 mL/min.
Agilent Extend C18, 150 × 3 mm, 5 μ, 254 nm, 0.05 M pH 9
activity of 1.2 Ci/mmol.
3
triethylammonium acetate (50:50) acetonitrile for 15 min Synthesis of high specific activity [ H]SCH 417690 (4)
followed by a step gradient to acetonitrile, 0.5 mL/min.
Phenomenex Luna C18(2) 100 × 4.6 mm, 3 μ, 254 nm, 0.05 M
pH 2.5 triethylammonium phosphate (70:30) acetonitrile for
(
(
S)-1-((R)-2-methoxy-1-(4-(trifluoromethyl)phenyl)ethyl)-2-methyl-4-
3
4-methyl-[2- H]piperidin-4-yl)piperazine (3)
10 min followed by a step gradient to acetonitrile, 1 mL/min.
At Quotient Bioresearch, compound
2
(20 mg) and tris-
triphenylphosphine ruthenium (II) chloride (4 mg) were combined in a
tube, and dioxane (100 μL) was added followed by 20 Ci of tritiated water
(
90 atom %). The tube was sealed and heated at 120 °C for 3 h. The tube
Specific activity measurements
was opened, the contents transferred to a flask with ethanol and the
3
14
The chemical concentrations of [ H] and
[
C]SCH 417690 were labile tritium removed by repeated rotary evaporation from ethanol to
determined using an ultraviolet-based HPLC assay (systems 1 and 3)
yield 1.65 Ci of 3 at a radiochemical purity of 23% as determined by
using an authentic standard of SCH 417690 for low specific activity [ H] TLC system 2.
3
1
4
SCH 417690 and a reference standard of SCH 417690 for [ C]SCH
17690. These data were used in conjunction with the radiochemical
About 300 mCi of crude 3 obtained from Quotient Bioresearch was
purified by reverse phase HPLC on a 9.4 × 250 mm YMC basic column
4
concentration to calculate the specific activity values. Mass spectrometry
was used to determine the specific activity value for the high specific
activity batch of [ H]SCH 417690.
with a mobile phase of 0.1% aqueous trifluoroacetic acid : 0.1%
trifluoroacetic acid in acetonitrile (75:25) at a flow of 5 mL/min, 215 nm.
The purified fractions were combined and evaporated to dryness to yield
a total of 54.8 mCi of compound 3. The batch was partitioned between
aqueous potassium hydroxide solution (0.1 M, 2 mL) and methylene
chloride (2 mL). The layers were separated, the methylene chloride layer
removed and the aqueous layer further extracted with methylene
chloride (3 × 2 mL). The combined methylene chloride extracts were
dried over anhydrous sodium sulphate, filtered and evaporated to
3
Mass spectrometry
Mass spectra were acquired on the JEOL MStation magnetic sector mass
+
spectrometer operating in the ESI or FAB ionization mode.
J. Label Compd. Radiopharm 2016, 59 190–196
Copyright © 2016 John Wiley & Sons, Ltd.
www.jlcr.org

D. Hesk et al.
dryness and transferred to a 1 mL V-vial and used directly in the next
step.
(6.83 mL, 53.13 mmol) was then added dropwise and the reaction
continued by stirring overnight at room temperature at which point,
monitoring by TLC system 3 showed complete reaction. The reaction
was quenched using saturated sodium bicarbonate solution and
extracted with methylene chloride. The combined organic layers were
washed with brine, dried over anhydrous sodium sulphate, filtered and
evaporated to dryness. The crude product was purified by silica gel
chromatography using a gradient of 0–10% methanol in methylene
chloride. The product fractions were combined and evaporated to
dryness to yield a total of 1.81 g (75%) of compound 9.
(
(
[
4,6-dimethylpyrimidin-5-yl)(4-((S)-4-((R)-2-methoxy-1-(4-
trifluoromethyl)phenyl)ethyl)-3-methylpiperazin-1-yl)-4-methyl-
3
3
2- H]piperidin-1-yl)methanone [ H]SCH 417690 (4)
Compound 3 was then dissolved in DMF (100 μL) and EDC (2.49 mg,
3 μmol), HOBt (1.45 mg, 10.7 μmol) DIPEA (10 μL) and 4,6-
1
dimethylpyrimidine-5-carboxylic acid (1.84 mg, 12.1 μmol) were added.
Work up and purification was performed as described for the low specific
3
activity batch to yield 46.5 mCi of [ H]SCH 417690 4 at a specific activity
Tert-butyl-4-((S)-4-((R)-2-methoxy-1-(4-(trifluoromethyl)phenyl)
+
of 16.4 Ci/mmol, which was determined by FAB mass spectrometry. The
2
ethyl)-3-methylpiperazin-1-yl)-4-methyl-[2,6- H ]piperidine-1-
4
radiochemical purity as determined in HPLC systems 2 and 3 was 99.6%
3
carboxylate (10)
and 99.3%. H NMR, 533 MHz, δ2.95 ppm (m), 3.37 ppm (m), 3.34 ppm (m)
and 4.22 ppm (m).
Compound 9 (1.81 g, 3.54 mmol) was added to a flame-dried two-neck
flask and fit with a condenser. Anhydrous tetrahydrofuran (20 mL) was
added, and the resulting solution was stirred at 0 °C for 10 min.
Methylmagnesium iodide (3 M in diethyl ether, 3.54 mL, 10.62 mmol)
was then added dropwise at 0 °C, and the reaction stirred for 30 min at
this temperature, before being heated to 40 °C and stirred for a further
2
Synthesis of [ H
4
]SCH 417690 (12)
2
N-benzyl-[2,6- H ]-4-hydroxypiperidine (5)
4
2
A 20% solution of [ H
4
]formaldehyde in D
2
O (12.9 mL, 93.6 mmol) was
1
.5 h at which point, the reaction was complete by TLC system 3. The
added to benzylamine trifluoroacetate (9 g, 30.5 mmol), the resulting
mixture sonicated for 10 min and then stirred for 1 h at room
temperature. To the resulting clear solution was added
allyltrimethylsilane (7.2 mL, 44.7 mmol), and the reaction was heated at
reaction mixture was added to 25 mL of a 20% (wt/wt) aqueous solution
of sodium citrate at 0 °C. The product was extracted using ethyl acetate
(
3 × 20 mL), the combined ethyl acetate extracts were dried over
anhydrous sodium sulphate filtered and concentrated to a yellow oil.
The crude product was purified by silica gel chromatography using a
gradient of 0–1% methanol in methylene chloride. The product fractions
were combined and concentrated to give 1.38 g (78%) of compound 10.
40 °C overnight. The resulting two-phase mixture was diluted with water
(6 mL), and solid potassium carbonate was added until the pH was
greater than pH 10. The product was extracted with ether (3 × 20 mL),
the layers were combined, dried over anhydrous sodium sulphate,
filtered and evaporated to an oil. The product was purified by silica gel
chromatography using a gradient of 40–50% ethyl acetate in hexane.
The product fractions were combined and evaporated to dryness to yield
a total of 3.49 g (44%) of compound 5.
(
S)-1-((R)-2-Methoxy-1-(4-(trifluoromethyl)phenyl)ethyl)-2-methyl-4-
2
(4-methyl-[2,6- H ]piperidin-4-yl)piperazine (11)
4
Compound 10 (1.38 g, 2.74 mmol) was dissolved in methylene chloride
25 mL) and the solution cooled to 0 °C. A solution of trifluoroacetic acid
(
2
N-Boc-[2,6- H
4
]-4-hydroxypiperidine (6)
(2.8 mL) dissolved in methylene chloride (2.8 mL) was added dropwise
and stirred for 90 min at 0 °C at which point, analysis by TLC system 4
showed complete reaction. Sodium hydroxide solution (25% w/v) was
then added dropwise at 0 °C until pH 12 was reached. The layers were
separated and the product extracted with methylene chloride
Compound 5 (3.49 g, 17.8 mmol) was dissolved in ethyl acetate (40 mL),
and di-tert-Boc-dicarbonate (4.66 g, 21.37 mmol) and Pearlman’s catalyst
(882 mg) were added. The reaction was pressured to 55 psi with
hydrogen and shaken overnight on a Parr shaker. The reaction mixture
was filtered through a pad of celite and evaporated to an oil. The oil
was then partitioned between ether (18 mL) and aqueous potassium
bisulphate solution (0.5 M, 20 mL). The ether layer was removed and
the aqueous fraction further extracted with ether (1 × 20 mL). The
combined ether layers were dried over anhydrous sodium sulphate,
filtered and evaporated to an oil. The product was purified by silica gel
chromatography using a gradient of 1–2% methanol in methylene
chloride. The product fractions were combined and evaporated to an
oil to yield a total of 2.72 g (74%) of compound 6.
(
3 × 30 mL). The combined organic layers were dried over anhydrous
sodium sulphate, filtered and concentrated to yield 836 mg (76%) of
compound 11 as a yellow oil.
(
(
[
4,6-Dimethylpyrimidin-5-yl)(4-((S)-4-((R)-2-methoxy-1-(4-
trifluoromethyl)phenyl)ethyl)-3-methylpiperazin-1-yl)-4-methyl-
2
2,6- H ]piperidin-1-yl)methanone SCH 417690 (12)
4
Compound 11 (836 mg, 2.07 mmol) was dissolved in anhydrous DMF
(20 mL) and 4,6-dimethylpyrimidine-5-carboxylic acid (315 mg,
2
.07 mmol) DIPEA (0.90 mL, 5.18 mmol), HOBt (419.6 mg, 3.1 mmol) and
2
N-Boc-[2,6- H
4
]-4-piperidone (7)
EDC (595.3 mg, 3.1 mmol) were added and the reaction mixture stirred
at room temperature overnight. Analysis by TLC system 3 showed
complete reaction. The reaction was concentrated to partially remove
the DMF, and methylene chloride (20 mL) and water (15 mL) were added.
The methylene chloride layer was removed, washed with water (5 mL),
dried over anhydrous sodium sulphate, filtered and evaporated to
dryness. The crude product was purified by silica gel chromatography
using a gradient of 0–5% methanol in methylene chloride, The product
fractions were combined and concentrated to give 700 mg (64%) of
Compound 5 (2.72 g, 13.25 mmol) was dissolved in methylene chloride
40 mL) and cooled to 0 °C. 4Ǻ molecular sieves (4.2 g) NMO (2.33 g,
9.87 mmol) and TPAP (233 mg, 0.66 mmol) were added and the reaction
stirred at this temperature for 15 min and then at room temperature for a
further 90 min. The reaction solution was filtered through a pad of celite,
evaporated to dryness and purified by silica gel chromatography using a
gradient of 1–2% methanol in methylene chloride. The product fractions
were combined and evaporated to dryness to yield a total of 2.15 g (80%)
of compound 7.
(
1
2
+
[
H ]SCH 417690 12. Purity (HPLC system 1): 99.4% . FAB MS m/z 538.
4
+
FAB MS m/z 534 observed for reference SCH 417690. No m/z 534 was
observed in the sample of [ H ]SCH 417690.
4
2
Tert-butyl-4-cyano-4-((S)-4-((R)-2-methoxy-1-(4-(trifluoromethyl)
phenyl)ethyl)-3-methylpiperazin-1-yl)[2,6- H
carboxylate (9)
2
4
]piperidine-1-
1
4
Synthesis of [ C]SCH 417690 maleate (20)
Compound 8 (1.46 g, 4.83 mmol) and compound 7 (982 mg, 4.83 mmol)
were dissolved in anhydrous methylene chloride (45 mL). Titanium
isopropoxide (1.44 mL, 4.83 mmol) was added dropwise, and the reaction
14
[
C]-N-Benzyl-4-hydroxypiperidine (13)
Benzyl-but-3-enyl-amine (288 mg, 1.786 mmol) and (1S)-(+)-10-
stirred at room temperature overnight. Diethylaluminium cyanide camphorsulphonic acid (415 mg, 1.786 mmol) were suspended in water
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J. Label Compd. Radiopharm 2016, 59 190–196

D. Hesk et al.
1
4
(
1
1 mL). [ C]formaldehyde in water (1% w/v 100 mCi, 56 mCi/mmol,
.786 mmol) was added to this suspension and the reaction flask quenched by the addition of saturated ammonium chloride solution
equipped with a reflux condenser and heated at 100 °C for 4 h at which (20 mL). The resulting clear solution was extracted with methylene
7 showed complete conversion. The reaction was cooled to 0 °C and
time, analysis in TLC system 1 showed complete reaction. The cooled
reaction was neutralized with saturated sodium bicarbonate solution to
chloride (5 × 20 mL). The combined organic extracts were washed with
brine (50 mL), dried over anhydrous sodium sulphate, filtered and
pH 8 and extracted with methylene chloride (6 × 15 mL). The combined evaporated to dryness. The crude product was purified by silica gel
methylene chloride extracts were washed with brine (5 mL), dried over
anhydrous sodium sulphate, filtered and evaporated to an oil to yield
chromatography with a mobile phase of hexanes : methylene chloride :
triethylamine (20:79:1) to yield 73.3 mCi (90%) of compound 17.
Radiochemical purity by analysis with TLC system 7 was > 97%.
92.8 mCi of compound 13, which was used directly in the next step.
1
4
[
C]N-Boc-4-hydroxypiperidine (14)
(S)-1-((R)-2-Methoxy-1-(4-(trifluoromethyl)phenyl)ethyl)-2-methyl-4-
14
(4-methyl-[2- C]piperidin-4-yl)piperazine (18)
Compound 13 (92.8 mCi, 1.66 mmol) was dissolved in ethyl acetate
40 mL), and di-tert-butyl dicarbonate (507 mg, 2.32 mmol) and 20%
palladium hydroxide on carbon (100 mg) were added. The flask was
(
Compound 17 (73.3 mCi, 1.309 mmol) was dissolved in anhydrous
methylene chloride (6 mL) and cooled to 0 °C. To this solution was added
a solution of trifluoroacetic acid in methylene chloride (12 mL of a 50%
attached to a Parr apparatus, pressured to 50 psi with hydrogen and
shaken overnight at which point, analysis by TLC system 5 showed v/v solution) and the reaction stirred for 2 h at 0 °C at which point,
complete reaction. The suspension was filtered through a pad of celite analysis in TLC system 7 showed complete reaction. The reaction was
and evaporated to an oil. The oil was dissolved in diethyl ether (40 mL),
washed with aqueous sodium bisulphite solution (1 M, 20 mL), dried over
anhydrous sodium sulphate, filtered and evaporated to an oil to yield
quenched by the addition of 25% sodium hydroxide solution until a pH
of 12 was achieved. The product was extracted with methylene chloride
(4 × 50 mL), the combined organic extracts were washed with saturated
crude compound 14. The crude compound was purified by silica gel sodium bicarbonate solution (50 mL), dried over anhydrous sodium
chromatography using a mobile phase of hexane : methylene chloride :
sulphate, filtered and evaporated to an oil. A total of 71.8 mCi (98%) of
triethylamine (20:75:5). Fractions containing the purified product were compound 18 was isolated at a radiochemical purity of 98.7% as
pooled and evaporated to an oil to yield 89.8 mCi (97%) of compound 14.
determined by TLC system 7.
1
4
[
C]N-Boc-4-piperidinone (15)
(
(
4,6-Dimethylpyrimidin-5-yl)(4-((S)-4-((R)-2-methoxy-1-
4-(trifluoromethyl)phenyl)ethyl)-3-methylpiperazin-1-yl)-4-
Compound 14 (89.8 mCi, 1.60 mmol) was dissolved in anhydrous
14
methylene chloride (15 mL) and molecular sieves (4Ǻ, 100 mg) added. methyl-[2- C]piperidin-1-yl)methanone SCH 417690 (19)
The flask was cooled to À5 °C and NMO (288 mg, 2.46 mmol) followed
Compound 18 (71.8 mCi, 1.280 mmol) bromotris(pyrrolyldino)
phosphonium hexafluorophosphate (836 mg, 1.795 mmol) and 4,6-
dimethylpyrimidine-5-carboxylic acid (292 mg, 1.903 mol) were dissolved
by TPAP (28 mg, 0.08 mmol) were added. The reaction was stirred at
À5 °C for 5 min and then stirred at room temperature for 90 min at which
point, analysis by TLC system 6 showed complete reaction. The reaction
in anhydrous methylene chloride (20 mL). DIPEA (0.34 mL, 1.932 mmol)
mixture was filtered through a pad of celite and evaporated to dryness to
was added and the reaction stirred overnight at room temperature at
which point, analysis in TLC system 6 showed about 90% product. The
reaction was diluted with methylene chloride (50 mL) and washed with
yield 86.7 mCi (97%) of compound 15 in the form of a white solid, which
was used directly in the next step.
1
M sodium hydroxide solution (50 mL). The aqueous phase was back
Tert-butyl-4-cyano-4-((S)-4-((R)-2-methoxy-1-(4-(trifluoromethyl)
phenyl)ethyl)-3-methylpiperazin-1-yl)[2- C]piperidine-1-
carboxylate (16)
1
4
extracted with methylene chloride (2 × 20 mL), the combined organic
phase washed with brine (30 mL), dried over anhydrous sodium sulphate,
filtered and evaporated to dryness. A total of 69.3 mCi of crude [ C]SCH
14
Compounds 6 (492 mg, 1.627 mmol) and 15 (86.7 mCi, 1.548 mmol) were 417690 17 was isolated. The batch was purified by silica gel
dissolved in anhydrous methylene chloride (8 mL). Titanium chromatography using a mobile phase of ethyl acetate : hexane (50: 50)
isopropoxide (0.6 mL, 2.016 mmol) was added dropwise over 10 min
and the resulting pale yellow solution stirred overnight at room
followed by methylene chloride : methanol : triethylamine (96:3:1).
Fractions containing the purified product were pooled to yield 48 mCi
1
4
temperature. Diethylaluminium cyanide solution in toluene (1 M, (67%) of [ C]SCH 417690 19. Radiochemical purity analysis by HPLC
1
.9 mL) was added, the reaction volume was concentrated to
systems 2 and 3 was >95%. A 30 mCi portion was removed from this
batch and further purified by reverse phase HPLC using an Agilent
approximately to half the original volume and the reaction stirred
overnight at room temperature at which point, analysis by TLC system Extend C18 column, 250 × 9.4 mm 5 μ, with a mobile phase of 0.05 M
showed complete reaction.
pH 9 triethylammonium acetate: acetonitrile (45:55) at a flow rate of
The reaction was diluted with ethyl acetate (30 mL), and water (2 mL) 5 mL/min, 254 nm. The collected fractions were pooled, partially
3
was added dropwise over 20 min, and the resulting suspension was evaporated to remove the acetonitrile and extracted with methylene
stirred for 2 h at room temperature. The reaction mixture was filtered chloride (4 × 20 mL). The combined extracts were washed with brine
through a pad of celite, the pad was rinsed with ethyl acetate (10 mL), dried over anhydrous sodium sulphate, filtered and evaporated
(
(
3 × 20 mL). The combined organic phase was washed with brine
5 mL), dried over anhydrous sodium sulphate, filtered and evaporated
to dryness to yield 24.6 mCi at a specific activity of 57.9 mCi/mmol of
1
4
[
C]SCH 417690 19.
to dryness. The crude compound was purified by silica gel
chromatography with a mobile phase of hexanes : methylene chloride :
triethylamine (20:79:1) to yield 81 mCi (93%) of compound 16.
Radiochemical purity by analysis with TLC system 7 was >99%.
(
(
4,6-Dimethylpyrimidin-5-yl)(4-((S)-4-((R)-2-methoxy-1-(4-
trifluoromethyl)phenyl)ethyl)-3-methylpiperazin-1-yl)-4-methyl-
14
[2- C]piperidin-1-yl)methanone maleate SCH 417690 maleate (20)
1
4
In a 10 mL centrifuge tube, [ C]SCH 417690 19, (22.1 mCi, 204.6 mg,
0.382 mmol) and unlabelled SCH 417690 (518.1 mg, 0.971 mmol) were
dissolved in methylene chloride (5 mL) and thoroughly mixed before
the methylene chloride was removed by evaporation to generate a white
Tert-butyl-4-((S)-4-((R)-2-methoxy-1-(4-(trifluoromethyl)phenyl)
ethyl)-3-methylpiperazin-1-yl)-4-methyl-[2- C]piperidine-1-
carboxylate (17)
1
4
Compound 16 (81 mCi, 1.446 mmol) was dissolved in anhydrous foam. The foam was dissolved in isopropyl acetate (1.25 mL) and the
tetrahydrofuran (10 mL) and the resulting solution cooled to À5 °C. solution heated to 75 °C. Maleic acid (157.2 mg, 1.354 mmol) was
Methylmagnesium iodide (3 M, 1.45 mL, 4.34 mmol) was added dropwise suspended in isopropyl acetate (2.5 mL) and the suspension heated to
over 20 min and the resulting suspension stirred at this temperature for 75 °C at which point, complete solution was obtained. The maleic acid
1
4
30 min before being heated to 40 °C for 90 min. Analysis in TLC system
solution was then added to the solution of [ C]SCH 417690 together
J. Label Compd. Radiopharm 2016, 59 190–196
Copyright © 2016 John Wiley & Sons, Ltd.
www.jlcr.org

D. Hesk et al.
with a 0.5 mL rinse of isopropyl acetate and the combined solution
stirred for 1 h at 75 °C. The tube was allowed to cool to room temperature
and then stored overnight at 3 °C. The solid was collected by
centrifugation (3000 rpm for 15 min), washed with isopropyl acetate
[5] E. J. Corey, K. Bakshi, S. Shibata, J. Am. Chem. Soc. 1987, 109, 5551–
5553.
[
6] J. Tagat, S. McCombie, D. Nazareno, Y. Xiao, R. Steensma, J. Strizki, B.
Baroudy, K. Cox, J. Lachowicz, G. Varty, S. Lin, S. Xu, L. Wojcik, M.
Murray, N. Valtuno, J. Med. Chem. 2001, 44, 3343–3346.
1
4
(0.5 mL) and dried under vacuum to yield 17.65 mCi, 706 mg of [ C]
[
[
7] H. Ahlbrecht, W. Raab, C. Vonderheid, Synthesis 1979, 2, 127–129.
8] J. Strizki, C. Tremblay, S. Xu, J. Wojcik, N. Wagner, W. Gonsiorek, W.
Hipkin, C. Chou, C. Pugliese-Sivo, Y. Xiao, Antimicrob. Agents
Chemother. 2005, 49, 4911–4919.
SCH 417690 maleate 20. Radiochemical purity as determined in HPLC
systems 2 and 3 was 99.5% and 99.3%, and the specific activity was
16.74 mCi/mmol, 25.76 μCi/mg.
[
9] W. J. S. Lockley, D. Hesk, J. Lab. Comp. Radiopharm. 2010, 53, 704–
7
15.
Acknowledgements
[
10] a) E. Alexakis, M. J. Hickey, J. R. Jones, L. P. Kingston, W. J. S. Lockley,
A. N. Mather, T. Smith, D. J. Wilkinson, Tetrahedron Lett. 2005, 46,
4291–4293;
The authors would like to thank Dr T. M. Chan, Dr Mary Senior
and Dr Pradip Das for NMR and mass spectrometry analysis.
b) D. Hesk, K. Voronin, P. McNamara, P. Royster, D. Koharski, S.
Hendershot, S. Saluja, V. Truong, T. M. Chan, J. Lab. Comp.
Radiopharm. 2007, 50, 131–137.
[
11] S. Ren, P. McNamara, P. Royster, J. Lee, S. Saluja, D. Koharski, S.
Hendershot, V. Truong, J. Lab. Comp. Radiopharm. 2007, 50, 643–
648.
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Copyright © 2016 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2016, 59 190–196