Synthesis of three alpha 7 agonists in labeled form

Article abstract of DOI:10.1002/jlcr.3186

In support of a program to develop an alpha 7 agonist as a treatment for Alzheimer's disease, three drug candidates, 1, 2, and 3, were prepared in labeled forms. Compound 1 was prepared in C-14 labeled form by lithiation of [2,6-¹⁴C₂]2-chloropyridine and subsequent coupling with spirooxirane-2,3'-quinuclidine. When this same coupling was attempted using [3,4,5,6-²H₄]2-chloropyridine, alcohol [²H ₆]-6 was the major product indicating that the primary isotope effect for the lithiation step was significant enough to shift the reaction pathway. Therefore, an alternate site of labeling was used to prepare [²H ₄]-1. [¹³C₅]-2 was prepared in five steps from [¹³C₅]2-furoic acid, but the C-14 labeled compound used [¹⁴C₂]-1 as the starting material instead. [ ¹⁴C₂]-3 was prepared in two steps from [carbonyl- ¹⁴C]nicotinic acid. Copyright

Full text of DOI:10.1002/jlcr.3186

Research Article
Received 18 November 2013,
Revised 14 January 2014,
Accepted 14 January 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3186
Synthesis of three alpha 7 agonists in labeled form
a
b
c
c
*
Charles S. Elmore, Scott Landvatter, Peter N. Dorff, Mark E. Powell,
David Killick,d Timothy Blake,c James Hall,c J. Richard Heys,c John Harding,d
Rebecca Urbanek,c and Glen Ernstc
In support of a program to develop an alpha 7 agonist as a treatment for Alzheimer’s disease, three drug candidates, 1, 2,
and 3, were prepared in labeled forms. Compound 1 was prepared in C-14 labeled form by lithiation of [2,6-14C2]
2-chloropyridine and subsequent coupling with spirooxirane-2,3’-quinuclidine. When this same coupling was attempted
using [3,4,5,6-2H4]2-chloropyridine, alcohol [2H6]-6 was the major product indicating that the primary isotope effect for
the lithiation step was significant enough to shift the reaction pathway. Therefore, an alternate site of labeling was used to
prepare [2H4]-1. [13C5]-2 was prepared in five steps from [13C5]2-furoic acid, but the C-14 labeled compound used [14C2]-1 as
the starting material instead. [14C2]-3 was prepared in two steps from [carbonyl-14C]nicotinic acid.
Keywords: deuterium isotope effect; alpha 7; [carbonyl-14C]nicotinic acid; [13C5]2-furoic acid
Introduction
Results and discussion
Nicotinic receptors are a widely distributed family of ligand-gated In support of a program to develop an alpha 7 agonist as a
ion channels. They are pentameric in structure, made up of various treatment for Alzheimer’s disease, quinucleodine 1 was required
transmembrane subunits, denoted α, β, γ, δ, and ε. Assembly of in C-14 labeled form for initial DMPK studies.12 The synthesis
these subunits in various combinations gives a diversity of followed the medicinal chemistry route.13 The first step consisted
receptors, with a range of electrical and binding properties.1 In of a Johnson–Corey–Chaykovsky reaction14,15 to give racemic
the brain, α4β2 and α7 are the two most prevalent neuronal epoxide 5 in 70% yield. Epoxide 5 was first isolated as an oil that
nicotinic receptor stoichiometries.2 Relative to α4β2, the α7 failed to generate the desired product in the subsequent reaction,
receptor possesses greater sensitivity to alpha-bungarotoxin (a but azeotropic drying of 5 with PhCH3 provided a waxy solid,
nicotinic receptor antagonist),3 lower sensitivities to acetylcholine which performed adequately in the next step. Coupling of epoxide
and nicotine,4 and higher calcium ion permeability. In spite of 5 with [2,4-14C2]2-chloropyridine proceeded smoothly but in a
these differences, both α4β2 and α7 have been shown to be modest crude yield of 46% after silica gel chromatography. Highly
involved in attention, learning, and working memory.5
enantiomerically enriched [14C2]-1 was obtained by crystallization
Nicotine itself is known to have positive effects on cognitive with D-tartaric acid to give an overall yield of 12% (11%
function in humans and nonhumans in various cognition radiochemical yield) (Scheme 1).
models.6,7 These effects are believed to be mediated, at least in
Given this successful preparation, a similar route was
part, by the α7 receptor, as the α7 subtype is highly expressed envisioned for the preparation of a stable isotope-labeled iso-
in regions of the brain that are implicated in cognitive processes, topomer; the intent was to substitute [2H4]2-chloropyridine and
such as the hippocampus, thalamus, and cortex.8 Nicotinic [2H9]trimethylsulfoxonium iodide for [2,4-14C2]2-chloropyridine
receptors are also involved in cognitive deficits seen in and unlabeled trimethylsulfoxonium iodide. As shown in
schizophrenia, as well as associated sensory gating deficits.9
Scheme 2, quinuclidone (4) was treated with [2H9]trimethylsul-
Further highlighting a connection between α7 and cognition, foxonium iodide to afford [2H2]-5 in 50% yield. The lower yield
α7 knockout mice show deficits in processes involving both relative to the reaction of unlabeled material may be a result of
attention and cognition.10 Because of this apparent link,
modulation of α7 receptor function is thought to provide a
means for the treatment of cognitive deficits, both in
neurodegenerative and psychiatric disorders.11
Compound 1, 2, and 3 were identified as orally available, high-
aIsotope Chemistry, DMPK, AstraZeneca Pharmaceuticals LP, 43183 Mölndal, Sweden
bIsosciences, King of Prussia, PA 19406 m, USA
affinity α7 agonists, and so to further probe their biological activity,
they were required in labeled form.
cCNS-Chemistry, AstraZeneca Pharmaceuticals LP, Wilmington, DE 19850, USA
dIsotope Chemistry, DMPK, AstraZeneca Pharmaceuticals LP, Alderley Park, UK
*Correspondence to: Charles S. Elmore, Isotope Chemistry, DMPK, AstraZeneca
Pharmaceuticals LP, 43183 Mölndal, Sweden.
E-mail: chad_elmore@yahoo.com
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C. S. Elmore et al.
chlorine which would afford quinuclidin-3-ol [2H5]-6 in lieu of
the desired spirocycle.
The deuterium isotope effect in the metalation reaction was
tested by treating 2-chloropyridine and [2H4]2-chloropyridine
under identical metalation conditions (Scheme 4). The labeled
reaction was then neutralized with water, while the unlabeled
reaction was neutralized with D2O. Each reaction was monitored
by TLC, and the resulting products were identified by GC/MS.
Unlabeled 2-chloropyridine was recovered quantitatively and
showed incorporation of a single deuterium. However, [2H4]
2-chloropyridine showed [2H4]pyridine as the major product with
the chlorine replaced by a proton. There was also a small amount
of unreacted starting material recovered.
These results confirmed that the original route to the target
compound was not viable for the synthesis of deuterated 1.
Clearly, an alternative route of synthesis was going to have to be
developed, and this route would have to avoid, at the very least,
Scheme 1. Synthesis of [14C2]-1 · D-(À)-tartaric acid salt.
an isotope effect. [2H4]2-chloropyridine was then treated with
lithium tetramethylpiperide followed by dropwise addition of
the labeled epoxide. However, the expected spirocycle [2H5]-1
was not produced. Instead,
a more polar product was
obtained that was identified as [2H6]-6. The apparent route to
this product is shown in Scheme 3; we believe that treatment
of [2H4]2-chloropyridine with lithium tetramethylpiperide gave
halogen-metal exchange instead of deprotonation alpha to the
Scheme 4. Investigation of the route to [2H6]-6.
Scheme 2. Attempted preparation of stable isotope labeled 1.
Scheme 3. Proposed synthetic route to [2H6]-6.
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C. S. Elmore et al.
the use of a pyridine that had a deuterium ortho to the chlorine.
prepared [13C5]2-furoic acid16 and thus started with a simple
The successful route used for the synthesis of stable isotope peptide coupling to give amide [13C5]-7 (Scheme 6). The
labeled 1 is shown in Scheme 5. Quinuclidone (4) was labeled via conversion of methyl 2-furoate to methyl 4-bromofuroate
deuterium exchange with CH3OD and sodium followed by previously reported by Belen’kii and coworkers served as a good
neutralization with D2O. Deuterium incorporation was greater than precedent for the next sequence of reactions.17 Amide [13C5]-7
98% with less than 0.1% unlabeled starting material. This was was dibrominated with AlCl3 in neat Br2. The resulting dibromide
treated as previously described with [2H9]trimethylsulfoxonium
[
[
13C5]-8 was debrominated to give a mixture of monobromide
13C5]-9, dibromide [13C5]-8, and des-bromide [13C5]-7 in a
iodide to afford epoxide [2H4]-5 in 43% yield. Spirocycle ring
formation using unlabeled 2-chloropyridine successfully afforded 50:15:35 ratio. Monobromide [13C5]-9 was isolated by preparative
the final compound in 20% yield.
A second alpha 7 agonist, quinucleodine 2, was also required HPLC and 1H NMR analysis. The Suzuki coupling of [13C5]-9 to give
in stable isotope labeled form with a minimum mass increase of
13C5]-10 proved to be very problematic. The yields of [13C5]-10
HPLC and the site of debromination confirmed by co-elution on
[
4 AMU. The obvious site of labeling—the morpholine ring either varied dramatically from run-to-run, and the reaction gave many
with H-2 or C-13—was not pursued so that we could better by-products; therefore, the reaction was run in several batches
prepare for our projected C-14 synthesis in which we planned and the final compound purified by preparative HPLC to give an
to use [14C]2-furoic acid as the C-14 precursor. We had previously isolated yield of 33% yield. The final reduction proceeded
smoothly to give agonist [13C5]-2 in 80% yield.
While this route did deliver enough of the stable isotope-
labeled compound for initial DMPK uses, it demonstrated that
several of the steps were suboptimal for radiochemical use.
The Suzuki coupling gave an unexpectedly poor and variable
yield and attempts to improve the yield and make the chemistry
more consistent were not successful. The mono-debromination
was also not ideal although re-cycling of the dibromide and
des-bromo compound partially compensated for this. Overall,
we felt that this synthetic route was not ready for use with
C-14, and yet, a rapid delivery of C-14 labeled 2 was required to
support DMPK studies. Therefore, we looked into other options.
We first investigated the Vilsmeier–Haack reaction using C-14
DMF and furan 12 (Scheme 7). Various reaction conditions were
probed and resulted in between 60% and 80% of the radioactivity
in the organic extracts. However, this radioactivity consisted of a
mixture of compounds with the same M+ 1/Z ratio on LCMS as
the target aldehyde. The two major radioactive HPLC peaks were
Scheme 5. Synthesis of [2H4]-1 · 2HBr.
Scheme 6. Synthesis of [13C5]-2.
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C. S. Elmore et al.
Scheme 7. Attempted synthesis of [14C]-2.
Scheme 8. Synthesis of [14C2]-2.
Scheme 9. Synthesis of [14C]-3.
broad and in approximately a 2:1 ratio. The same ratio was recovery of radioactivity, but the desired compound represented
observed on different HPLC columns in both MeCN and MeOH less than 10% of the total radioactivity by HPLC. Therefore, this
and with different acid modifiers. The HPLC profile of authentic route was abandoned.
unlabeled aldehyde also gave a broad peak on HPLC that eluted
The need for C-14 labeled 2 was acute, so a secure option was
with the minor peak from the Vilsmeier reaction. Reductive pursued. A small amount of [14C2]-1 was brominated using TFA
amination of the crude reaction mixture afforded quantitative and N-bromosuccinimide to give monobromide [14C2]-13 in
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C. S. Elmore et al.
PFP, 2–20%; method G: Phenomenex Luna C-18(2), 10–20%; method H:
Phenomenex RP-Polar, 13–17%.
good yield (Scheme 8). The bromide was reacted with boronic
acid 14 in a Suzuki reaction to give aldehyde [14C2]-15 in a
quantitative recovery of radioactivity but only 49%
radiochemical purity. The crude reaction mixture was converted
to the final compound in three batches via reductive amination
to give the target compound, [14C2]-2, in good purity after
purification by HPLC.
GC analyses were performed on a Supelco SPB-S 30M × 0.25 mm column
with an injector temperature of 250°C, detector temperature of 280°C, and
an over temperature of 70°C for 2 min then 10°C/min from 2 to 22 min.
Preparative HPLC purifications were performed on a 21.2 × 250 mm column
using MeOH-0.1% TFA as the eluent and at a flow rate of 15 mL/min and were
concluded with a 10 min wash with 100% organic unless otherwise noted
using the following methods: method α: 30 × 100 mm Phenomenex Gemini
NX C18 5 micron, 2–98% over 12min EtOH in 0.5M NH4HCO3 (pH 10);
A third alpha 7 agonist, 3, was desired labeled with C-14 for
evaluation by DMPK. [14C]Nicotinic acid was prepared from 3-
bromopyridine by lithiation with n-BuLi at À70°C and sub- method β: Phenomenex Luna C18(2), 2–40% over 10min then 40–70%
sequent reaction with 14CO2 to provide nicotinic acid [14C]-16
10–20 min; method ɣ: Phenomenex Luna C18, 2–20% over 15 min;
in a 59% yield (Scheme 9).18,19 Coupling of [14C]-16 with amine
20–30% over 15–25 min; method δ: Phenomenex Luna C-18(2), 2–20% over
10 min then 20–35% over 36 min; method ε: Phenomenex Polar-RP, 5–25%
MeCN-0.1% TFA over 40 min.
17 with HATU activation gave an incomplete reaction with many
impurities. No effort was made to improve this step as the yield
Silica gel purifications were performed on a Teledyne ISCO CombiFlash
was deemed sufficient to deliver the requisite amount of
Companion using gradient elution. It is important to note that the
material. Cyclization was achieved by heating amide [14C]-18
quinucleodine ring is very nucleophilic and thus reacts with CH2Cl2 to give
a quarternary amine adduct.
in Eaton’s solution at 130°C overnight.20 The crude yield of the
reaction was good, but during purification by preparative HPLC,
several products containing fractions had to be rejected because
1’-azaspiro[oxirane-2,3’-bicyclo[2.2.2]octane] (5)
of impurities. The target oxazole [14C]-3 was isolated in 33%
A slurry of 7.086 g (177.2mmol) of a 60% w/w dispersion of NaH in mineral
oil in 50 mL of petroleum ether was stirred for 20 min, and then the solvent
was carefully removed. The residue was taken up in 20 mL of DMF, and
38.41 g (174.4mmol) of trimethylsulphoxonium iodide was added to the
slurry. The slurry was stirred for 5h until gas evolution was complete. The
reaction mixture was diluted with 20mL of DMF, and 18.20g (145.6 mmol)
of 3-quinuclidinone in 20 mL of DMF was added. The mixture was stirred for
4.5h and was then diluted with 350 mL of water. The resulting mixture was
extracted four times with CHCl3, and the combined organic extracts were
washed three times with 100 mL of water and then dried (MgSO4). The
solution was filtered and then concentrated under reduced pressure to give
an oil. The oil was diluted with PhCH3 and then concentrated to azeotrope
any remaining water. After drying under high vacuum, 14.11g (70%) of 5
was obtained as a waxy solid. LC/MS (M + H): 140.1 (100%), 142.1 (10%).
1H NMR (500MHz, CDCl3): δ 3.09 (dd, J = 15, 2 Hz, 1H), 2.95 (m,1H), 2.85
(m,4H), 2.72 (m,2H), 1.97 (m,1H), 1.78 (m,2H), 1.56 (m,1H), 1.38 (m,1H). The
purity of the compounds is estimated to be >95% based on the 1H NMR.
yield in high radiochemical purity and in 29% yield at a lower
purity (81% radiochemical purity). The higher purity batch
contained enough material to support the study, so the impure
batch was stored for purification at a later date. The impurities
were not characterized.
Conclusion
In summary, the synthesis of compounds 1 and 2 labeled with
both C-14 and stable isotope labels and compound 3 with C-14
was successfully completed. During the preparation of
a
deuterated version of 1, an unanticipated product was isolated
which resulted from a change in the rate of deprotonation of 2-
chloropyridine when the protons were substituted with deuterium.
Therefore, an alternate route to deuterated 1 was developed.
(2R,4’S)-3H-1’-azaspiro[furo[2,3-b, 2,6-14C2]pyridine-2,3’-
bicyclo[2.2.2]octane], D-(À)-tartaric acid salt, ([14C2]-1 · D-
(À)-tartaric acid salt)
Experimental
General: [2,6-14C2]2-chloropyridine was obtained from Quotient Bioresearch
(Cardiff CF24 5JQ, UK). Ba14CO3 and [14C]DMF were obtained from American
Radiolabeled Chemicals, Inc. (St. Louis, MO, USA). [13C5]2-furoic acid
was prepared according to the method of Dorff.16 Quinuclidin-3-one,
11, 12, 14, and 17 were provided by AstraZeneca CNS-Chemistry
(Wilmington, DE, USA). All other reagents were obtained from Acros
Organics (Pittsburgh, PA, USA), Sigma-Aldrich (St. Louis, MO, USA),
Fisher (Waltham, MA, USA), and Strem (Newburyport, MA, USA) and
were used without purification.
A solution of 1.6 mL (9.5 mmol) of 2,2,6,6-tetramethylpiperidine in 10 mL
of THF was cooled to À60°C as 7.8 mL of a 2.5 M (19.5 mmol) solution of
n-BuLi in hexanes was added over 10 min. The solution was stirred at
À60°C for 1 h, and then a solution of 250 mCi (4.4 mmol) of [2,6-14C2]2-
chloropyridine and 637 mg (5.61 mmol) of 2-chloropyridine in 4 mL of
THF was added over 20 min. The solution was warmed to À40°C and
was stirred for 40 min. Then, a solution of 1.32 g (9.51 mmol) of 5 in
10 mL of THF was added over 15 min at À40°C, and the reaction was
1H NMR spectra were recorded on Bruker DPX 300, DRX 400, Avance
500, and DRX 600 spectrometers in 2H6-DMSO, or C2H3O2H at 30°C and warmed to À10°C and stirred for 5 h. It was then warmed to room
were referenced to the residual solvent peak. LC/MS analyses were
performed on an HP MSD-1100 using a 4.6 × 100 mm Phenomenex
temperature and stirred overnight and was then heated at 50°C for 6 h.
The reaction mixture was then cooled to 0°C, and 10 mL of water and
Luna-C18(2) column, with a 10–100% gradient of MeOH-0.1% aq formic 10 mL of methyl t-butylether (MTBE) were added. The biphasic mixture
acid over 10 min and ESI.
was filtered through celite, and the celite washed twice with 40 mL of
1:1 MTBE:water. The pH of the aqueous layer was adjusted to 7 with
5 M HCl, and the layers were separated. The aqueous phase was washed
with 10 mL of MTBE, basified to pH 10 with 47% NaOH, and then
extracted three times with 20 mL of CHCl3. The combined chloroform
extracts were concentrated to dryness to give a brown oil, and the oil
was purified by column chromatography on silica gel (85:15 CHCl3:
High-performance liquid chromatography analyses were performed using
an Agilent 1100 series HPLC system using the following conditions unless
otherwise noted: 150 × 4.6 mm column, 20 min gradient elution with MeCN-
0.1% TFA as mobile phase followed by a 5 min wash with 100% organic
mobile phase and a flow rate of 1 mL/min using one of the following
methods: method A: 100 × 4.6 mm Thermo Scientific beta basic C18, isocratic
5% aqueous MeCN with 0.026 M NH4OAc over 30min; method B: YMC Basic, MeOH) to give 0.974 g of a brown solid after drying at reduced pressure.
20–40% MeCN-0.2% diethylamine in water, 1.5 mL/mL; method C: The solid was dissolved in 3 mL of refluxing EtOH, and 678 mg
Phenomenex Luna C-18(2), 5–100% MeOH-0.1% formic acid over 10 min (4.52 mmol) of D-(À)-tartaric acid in 1 mL of water was slowly added.
followed by 3 min at 100%; method D: Phenomenex Luna C18(2), 2–20%;
method E: Phenomenex Luna C18(2), 2–50%; method F: Phenomenex Curosil
After 15 min, the solution was cooled to room temperature. A crystal of
the D-(À)-tartaric acid salt of 1 was added, and the turbid mixture was
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C. S. Elmore et al.
cooled to 0°C. The slurry was filtered, and the solid washed with 2 mL of
cold EtOH. After drying under reduced pressure, 538 mg of [14C]-1 was
obtained as the D-(À)-tartaric acid salt. HPLC showed a 96:4 ratio of R:S
enantiomers (Daicel Chirocel OD 250 × 4.6 mm, 15% EtOH in isohexane).
The solid was recrystallized twice from 2 mL of 1:1 EtOH:water to give
445 mg (12%, 11% radiochemical yield) of [14C2]-1 · D-(À)-tartaric acid
salt which showed less than 0.5% of the S enantiomer to be present.
The radiochemical purity of the sample was >99% (method A). The
specific activity was determined to be 23 mCi/mmol by gravimetric
analysis. LC/MS(M + H): 217 (100%), 221 (21.2%), 218 (14.6%), 219
(3.1%). 1H NMR (D6-DMSO, 500 MHz): δ 7.92 (d, J = 7.0 Hz, 1H), 7.60 (d,
J = 7.0 Hz, 1H), 6.87 (t, J = 7.0 Hz, 1H), 4.05 (s, 2H), 3.45(d, J = 17 Hz, 1H),
3.34(d, J = 14 Hz, 1H), 3.24 (d, J = 14 Hz, 1H), 3.18 (d, J = 17 Hz, 1H), 3.03
(m, 2H), 2.95 (m, 2H), 2.07 (s, 2H), 1.79 (m, 2H), 1.61 (m, 1H).
>99.5% (on the free base). LC/MS (M + H): 221 (100%), 222 (18.6%), 220
(4.8). (no peak for 217, 218, or 219 was observed. LC/MS detection limit
was 0.1%) 1H NMR (D6-DMSO, 500 MHz): δ 7.96 (dd, J = 7.0, 2 Hz, 1H),
7.65 (dd, J = 7.5, 2 Hz, 1H), 6.92 (dd, J = 7.0, 7.5 Hz, 1H), 3.35 (m, 2H), 2.25
(s, 1H), 2.20 (m, 1H), 1.92 (m, 3H), 1.83 (m, 1H). 13C NMR (D6-DMSO,
150 MHz): δ 165.9, 146.4, 134.2, 118.8, 117.4, 83.0, 58.1 (m), 45.2, 44.8,
36.9 (m), 29.5, 18.5, 18.0.
[
13C4]furan-2-yl(morpholino) )[13C]methanone, [13C5]-7
A solution of 106 mg (0.91 mmol) of [13C5]2-furoic acid16 in 5 mL of
CH2Cl2 was stirred as 2.3 mL (4.5 mmol) of oxalyl chloride and 5 μL of
DMF were added. The solution was stirred for 90 min at room
temperature, and then the solvent was evaporated. The residue was
taken up in 3 mL of CH2Cl2, and 95 μL (1.1 mmol) of morpholine was
added. After 30 min, the solution was diluted with 5 mL of CH2Cl2, and
the resulting solution was washed twice with 10 mL of sat. NaHCO3 and
then with 5 mL of brine. The organic layer was dried (MgSO4) and filtered,
and then the filtrate was concentrated to dryness to give a 107 mg (63%)
of [13C5]-7 as a brown oil. LC/MS: 187 (100%), 186 (8.2%), 188 (6.2%). 1H
NMR (CD3OD, 500 MHz): δ 7.67 (dm, J = 205 Hz, 1H), 7.09 (dm, J = 184 Hz,
1H), 7.03 (dm, J = 173 Hz, 1H), 6.62 (dm, J = 178 Hz, 1H), 3.75 (m, 8H).
[2,2-2H2]Quinuclidin-3-one, [2H2]-4
A solution of sodium methoxide in MeOD prepared from 150mL of CH3OD
and 10.2g of sodium metal21 was stirred as 9.6 g (76.7 mmol) of quinuclidin-
3-one was added and the resulting solution was heated to reflux under an
argon atmosphere as 150 mL of D2O was added. The reaction was heated at
reflux for 2.5h. The MeOH was removed under vacuum, and the water
extracted with chloroform. The chloroform layer was dried over sodium
sulfate, and after filtering, was concentrated to give 9.3 g of a pale, yellow,
and waxy solid. This crude product was partitioned between ether and
D2O/dilute NaOD. The ether layer was removed, dried (MgSO4), and filtered,
and the resulting solution was concentrated under vacuum to give 6.6g
(68%) of [2H2]-4 as a white solid.
(4,5-dibromo[13C4]furan-2-yl)(morpholino))[13C]methanone,
[
13C5]-8
A reaction vessel was charged with 107 mg (0.57 mmol) of [13C5]-7, and
89 μL (1.7 mmol) of bromine was added. The resulting slurry was stirred
to create a homogenous solution which hardened into a semi-solid after
about 1 min. To the top of the hardened mixture was added 230 mg
(1.72 mmol) of AlCl3, and the reaction vessel was sealed. The mixture
was heated for approximately 4 min with a heat gun during which the
semi-solid melted and stirred freely. The reaction was cooled to room
temperature, and 5 mL of water was carefully added. The aqueous
solution was extracted five times with 4 mL of Et2O, and the combined
organic layers were dried (MgSO4) and filtered. Concentration of the
filtrate afforded 192 mg (97%) of [13C5]-8 as a yellow oil. LC/MS: 345
(100%) 347 (50%), 343 (48%) 1H NMR (CD3OD, 500 MHz): δ 7.13 (dm,
J = 184 Hz, 1H), 3.73 (m, 8H).
[2’,2’,3,3-2H4]spirooxirane-2,3’-quinuclidine, [2H4]-5
A slurry of 2.16 g of NaH (60% dispersion in oil) in 45 mL of dry DMF was
stirred as 11.1 g (48.4 mmol) of [2H9]trimethylsulfoxonium iodide22 was
added portionwise. After the addition was complete, the reaction
mixture was stirred an additional 10 min. To this was added 6.6 g
(53 mmol) of [2,2-2H2]quinuclidin-3-one. The reaction was stirred 30 min
and was then poured into 150 mL of D2O. The product was extracted
with ether and concentrated under vacuum, and the residue was dried
by azeotroping with toluene. This gave 2.4 g (43%) of a pale yellow oil.
(5-bromo[13C4]furan-2-yl)(morpholino) )[13C]methanone,
[
[2’,2’,3,3-2H4]spiro[furo[2,3-b]pyridine-2,3’-quinuclidine HBr
13C5]-9
salt, [2H4]-1 · 2HBr
A suspension of 100 mg (0.29 mmol) of [13C5]-8, 57 mg (0.87 mmol) of Zn,
50 μL of AcOH, and 0.19 mL of water was stirred and heated at 85°C for
10 min. The reaction was cooled to room temperature, and 5 mL of
NaHCO3 was added. The slurry was then extracted four times with 3 mL
of CH2Cl2, and the organic layer was dried (MgSO4). The drying agent
was removed by filtration, and the filtrate was concentrated to dryness
to afford 50 mg of a pale yellow oil which contained 50% monobromide
A solution of 1.30 mL (7.68 mmol) of tetramethylpiperidine in 9 mL of dry
THF cooled to À60°C under argon was stirred as 3.08 mL of a 2.5 M
solution (7.71 mmol) of n-butyl lithium in hexanes was added, and the
reaction was stirred for 1 hour at À60°C. To this was added 0.668 mL
(7.1 mmol) of 2-chloropyridine dropwise over 15 min. The reaction
temperature was allowed to rise to À45°C over 45 min and then 1.0 g
(6.98 mmol) of [2’,2’,3,3--2H4]spirooxirane-2,3’-quinuclidine in 4 mL of
THF was added dropwise over 15 min. The reaction was warmed to
À20°C during which it darkened. The temperature was maintained
between À20°C and À15°C for 4 h. The reaction was neutralized by the
addition of 10 mL of D2O and 10 mL of MTBE. The mixture was vigorously
stirred and was then filtered through celite. The celite was rinsed with 1:1
D2O/MTBE, and the pH of the filtrate was adjusted to 7.0 by addition of
DCl in D2O. The layers were separated, and the aqueous layer was
washed with 10 mL of MTBE. The aqueous layer was adjusted to pH 10
by the addition of NaOD in D2O. This was extracted with chloroform,
and the combined organic extracts were dried (Na2SO4). The solution
was filtered and concentrated under vacuum to give 900 mg. The crude
[
13C5]-9, 15% dibromide [13C5]-8, and 35% desbromide [13C5]-7 (method
C). The compound was purified by preparative HPLC (method α) to give
17 mg (22%) of [13C5]-9 as a white solid. LC/MS: 265 (100%), 267 (98%)
266 (24%), 264 (13%). 1H NMR (CD3OD, 500 MHz):
δ 7.77 (dm,
J = 212 Hz, 1H), 7.09 (dm, J = 184 Hz, 1H), 3.74 (m, 8H). 13C NMR (CD3OD,
125 MHz): δ 159.5 (d), 147.5 (dd), 143.8 (d), 118.4 (dd), 101.6 (dd).
(4-((2R,4’S)-3H-1’-azaspiro[[13C4]furo[2,3-b]pyridine-2,3’-
bicyclo[2.2.2]octane]-5-yl)furan-2-yl)(morpholino)[13C]-
methanone, [13C5]-10
product was purified by flash chromatography (85:15 chloroform/MeOH A stirred solution of 55 mg (0.21 mmol) of 11, 28 mg (0.11 mmol) of
on silica) to afford 363 mg of product as a reddish oil that crystallized
upon standing. This was converted to its bis-HBr salt by treatment of
an ethanol solution of the free base with an excess of 48% HBr in ethanol.
This was stirred 2 h. Diethyl ether was added and the precipitate washed
[
13C5]-9, 45 mg (0.42 mmol) of Na2CO3, 1 mL of dimethoxyethane,
0.4 mL of ethanol, and 0.3 mL of water was sparged with N2 for 20 min
after which 3.71 mg (5.3 μmol) of bis(triphenylphosphine)palladium(II)
chloride was added. The solution was warmed to 55°C, and after 3 h, an
with ether. This gave 523 mg (20%) of [2H4]-1 · 2HBr as an off white solid. additional 25 mg (0.1 mmol) of 11 was added. The solution was heated
The purity by HPLC was 98.9% (method B), and the purity by GC was
overnight. The reaction mixture was concentrated to dryness, and the
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C. S. Elmore et al.
residue was taken up in 30 mL of CHCl3:MeOH (1:1) and then filtered. The
filtrate was concentrated to dryness, and the residue purified by
preparative HPLC (method β). Product containing fractions were pooled,
concentrated to half volume, and passed through an Oasis HLB SepPak.
The SepPak was washed with water, sat NaHCO3, and water, and the
compound was eluted with MeOH. The resulting solution was
concentrated to dryness to give 14 mg (33%) of [13C5]-10. A similar
isolation procedure afforded 14 mg of starting bromide [13C5]-9.
(R)-5-(4-(morpholinomethyl)furan-2-yl)-3H-1’-azaspiro[furo
[2,3-b, 2,6-14C2]pyridine-2,3’-bicyclo[2.2.2]octane], [14C2]-2
A solution of 1.3 mCi (49% radiochemical purity, 0.023 mmol) of [14C2]-15
in 6 mL of MeOH was stirred as 0.33 mL of morpholine was added. After
10 min, 0.25 mL (4.37 mmol) of acetic acid was added, and after a further
10 min, 613 mg (2.89 mmol) of sodium triacetoxyborohydride was added.
The reaction was stirred for 1 h and was then concentrated to near
dryness. The residue was taken up in 5 mL of MeOH and was purified
by preparative HPLC (method δ) in five batches. The product containing
fractions were combined and concentrated to approximately half
volume. The solution was basified with NaOH to pH > 11 and was
extracted three times with 40 mL of CHCl3. The combined organic layers
were concentrated to dryness to give 0.43 mCi (67%) of [14C2]-2 as a
white solid. HPLC analysis showed the compound to have a 99.5%
radiochemical purity (methods d and f). The specific activity of the
compound was determined to be 26.6 mCi/mmol by LC/MS. LC/MS: 382
(100%), 383 (30.7), 386 (26.1), 384 (7.3%), 387 (6.0%). 1H NMR (500 MHz,
D3COD): δ 8.17 (s, 1H), 7.93 (s, 1H), 7.86 (s, 1H), 6.83 (s, 1H), 3.90 (br s.,
2H), 3.78 (m, 4H), 3.62 (m, 3H), 3.45 (m, 2H), 3.39 (m, 3H), 2.80 (br s.,
4H), 2.45 (t, J = 6.4 Hz, 1H), 2.37 (br s., 1H), 2.09 (m, 2H), 1.98 (dddd,
J = 10.8, 10.6, 10.6, 1.7 Hz, 1H).
(R)-5-(5-(morpholinomethyl)[13C4]furan-3-[13C]-yl)-3H-1’-
azaspiro[furo[2,3-b]pyridine-2,3’-bicyclo[2.2.2]octane],
[
13C5]-2
A solution of 23 mg (0.057 mmol) of [13C5]-10 in 0.4 mL of THF was slowly
added to a preformed solution of 0.24 mL of 1 M (0.24 mmol) LiAlH4 in
THF and 6.4 μL (0.12 mmol) of concentrated H2SO4 at room temperature.
The reaction mixture was stirred for 20 min, and then 0.2 mL of water was
added to the solution. The resulting slurry was filtered through a 0.45 μm
syringe filter, and the filter was rinsed with 1 mL of water and 1 mL of
THF. The sample was then purified by preparative HPLC (method ɣ). Pure
fractions were pooled and concentrated to approximately half volume.
The residue was then loaded onto a 50 mg Oasis HLB SepPak and the
column was sequentially washed with 20 mL of water, 20 mL of saturated [carbonyl-14C]nicotinic acid, [14C]-16
NaHCO3 solution, and 40 mL of H2O. The compound was eluted with
A stirred solution of 342 mg (2.16 mmol) of 3-bromopyridine in 25 mL of
10 mL of MeOH, and the MeOH was evaporated to afford 19 mg of a
yellow oil. The oil was diluted with 2 mL of ether, and the resulting slurry
was sonicated for 2 min. The resulting white solid was collected by
filtration to give 19 mg (80%) of [13C5]-2 after drying under vacuum
overnight. LC/MS: 387 (100%), 388 (21%), 386 (6.7%), 385 (0.1%). 1H
NMR (500 MHz, CDCl3, 13C decoupled): δ 8.11 (s, 1H), 7.58 (s, 1H), 7.50
(s, 1H), 6.46 (s, 1H), 3.75 (t, J = 4.6 Hz, 4H), 3.55 (m, 2H), 3.40 (t, J = 9 Hz,
2H), 2.8 (m, 6H), 2.52 (m, 4H), 2.26 (m, 1H), 2.01 (m, 1H), 1.68 (t,
J = 7.4 Hz, 2H), 1.47 (m, 1H).
diethyl ether was cooled to À70°C as 1.2 mL (1.92 mmol) of n-
butyllithium was added over 10 min. The solution was stirred for 1 h at
À70°C and was then cooled in a N2(l) bath and evacuated to 0.1 mm
Hg. Approximately 56 mCi (1 mmol) of 14CO2 (generated from 202 mg
(1.01 mmol, 56 mCi/mmol) of Ba14CO3 and 20 mL of H2SO4) was
transferred via gas transfer to the reaction flask,23 and the solution was
then warmed to À70°C and was stirred for 1 h. The solution was then
warmed to room temperature, and 15 mL of water was added. The layers
were separated, and the aqueous layer was concentrated to dryness to
give 33 mCi (58%) of [14C]-16. The compound was used directly in the
next reaction.
(2R,4’S)-5-bromo-3H-1’-azaspiro[furo[2,3-b, 2,6-14C2]
pyridine-2,3’-bicyclo[2.2.2]octane], [14C2]-13
[carboxyl-14C]-N-(2-oxo-2-((2R,4’S)-3H-1’-azaspiro[furo[2,3-
b]pyridine-2,3’-bicyclo[2.2.2]octane]-5-yl)ethyl)
nicotinamide, [14C]-18
A solution of 73 mg (0.41 mmol) of N-bromosuccinimide in 1 mL of TFA
was slowly added to 51 mg (0.13 mmol, 3.1 mCi, 23 mCi/mmol) of
14C2]-1, and the resulting solution was stirred and heated at 80°C for
A solution of 26.4 mCi (0.46 mmol) of [14C]-16 in 3 mL of DMF was stirred
as 1.27 g (3.34 mmol) of HATU and 0.80 mL (5.7 mmol) of triethylamine
were added. After 10 min, 324 mg (0.85 mmol) of 17 was added, and
the reaction mixture was stirred for 3 h. The reaction mixture was then
diluted with 50 mL of 1 M NaOH and 50 mL of CHCl3, and the layers were
separated. The organic layer was washed three times with 50 mL of 1 M
NaOH and was then dried (MgSO4). The drying agent was removed by
filtration, and the resulting yellow solution was concentrated to dryness
to give 16 mCi of [14C]-18 (40% yield corrected for purity) as a yellow
oil. The oil was heated at 130°C for 1 h under vacuum with stirring to
drive off residual solvents. HPLC analysis of the oil showed it to have a
65% radiochemical purity (method d). The aqueous layer consisted of
8 mCi of [14C]nicotinic acid which was isolated by solid phase extraction
for future use.
[
2 h. The solvent was removed, and the residue partitioned between
5 mL of CHCl3 and 3 mL of sat NHCO3(aq). The organic layer was removed,
and the aqueous layer was extracted three times with 4 mL of CHCl3. The
combined organic layers were concentrated to dryness to give 2.4 mCi
(73%) of [14C2]-13. HPLC analysis showed the radiochemical purity to
be 94.3% (method d).
4-((2R,4’S)-3H-1’-azaspiro[furo[2,3-b, 2,6-14C2]pyridine-2,3’-
bicyclo[2.2.2]octane]-5-yl)furan-2-carbaldehyde, [14C2]-15
A solution of 51mg (0.17 mmol, 4.0mCi, 23 mCi/mmol) of [14C2]-13 in 8 mL
of dimethoxyethane was stirred as a solution of 100mg (0.24 mmol) of
NaCO3 in 4 mL of water and 2.8mL of EtOH was added. N2 was bubbled
through the solution for 5min, and then 126 mg (0.90 mmol) of 5-
formylfuran-3-ylboronic acid and 19mg (0.027 mmol) of dichlorobis
(triphenylphosphine)palladium(II) were added. N2 was bubbled through
(2R,4’S)-5-(2-[14C]-2-(pyridin-3-yl)oxazol-5-yl)-3H-1’-azaspiro
[furo[2,3-b]pyridine-2,3’-bicyclo[2.2.2]octane], [14C]-3
the solution for 10 additional minutes, and then the reaction mixture was A solution of 10.2 mCi (0.18 mmol, 65% radiochemical purity) of [14C]-18
stirred at room temperature for 20 min, at 60°C for 3h, and at 100°C for in 3 mL of Eaton’s solution (7.7 wt% P2O5 in MeSO3H)20 was stirred at
2h. The reaction mixture was concentrated to dryness, and 10 mL of 50% 130°C overnight. After cooling to room temperature, the solution was
MeOH in CHCl3 was added. The solution was then concentrated to dryness. diluted with 50 mL of water and basified with 50% NaOH. The solution
The residue was taken up in 10 mL of PhCH3 to give 4.0mCi (49% yield was extracted three times with 50 mL of CHCl3 to give 10 mCi. The solution
based on radiochemical purity) and then the solution concentrated to was concentrated to dryness and then purified batchwise by preparative
dryness. This was repeated three times. HPLC shows the purity of the HPLC (method ε). The pure product containing fractions were combined
product to be 49.9% (method e).
and concentrated to approximately 50% volume. They were diluted with
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Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

C. S. Elmore et al.
10mL of sat NaHCO3(aq) and extracted three times with 40mL of CHCl3. The
combined organic extracts were dried (MgSO4) and filtered, and then the
filtrate was concentrated to dryness to give 2.2mCi (33%) of [14C]-3 as a
white solid. HPLC analysis (methods G and H) showed the radiochemical
purity to be 99.3%. The specific activity of the compound was determined
to be 56 mCi/mmol by LC/MS. A second batch of material was isolated in
the same manner from less pure column fractions to give 2.4mCi at a
radiochemical purity of 81.7%. LC/MS (M+ 1): 363 (100%), 364 (23%), 361
(11%), 362 (3.1%), 365 (3.1%). 1H NMR (500 MHz, MeOD): δ 9.25 (d,
J = 1.5 Hz, 1H), 8.66 (dd, J = 4.9, 1.5Hz, 1H), 8.48 (dt, J = 7.9, 1.8Hz, 1H), 8.43
(d, J= 1.8 Hz, 1H), 8.04 (d, J = 1.8Hz, 1H), 7.63 (s, 1H), 7.60 (dd, J = 7.3,
4.9 Hz, 1H), 3.60 (m, 1H), 3.24 (m, 2H), 3.09 (d, J = 15.0 Hz, 1H), 2.97 (m, 2H),
2.87 (m, 2H), 2.20 (m, 1H), 2.07 (br s., 1H), 1.80 (m, 2H), 1.62 (m, 1H). 13C
NMR (126 MHz, CD3OD): δ 167.0, 150.4, 150.2, 146.5, 142.3, 133.9, 130.7,
124.2, 123.7, 122.8, 121.5, 118.1, 88.1, 61.9, 46.0, 45.4, 37.9, 31.2, 21.6, 20.7.
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Acknowledgement
The authors would like to thank Dr John McCauley for purification
support.
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Conflict of Interest
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The authors did not report any conflict of interest.
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