Synthesis of lead LFA-1 antagonist [14C]spyrocyclic hydantoin

Article abstract of DOI:10.1002/jlcr.1596

Radiolabelled drug lead candidate leukocyte function-associated antigen 1 antagonist [¹⁴C]spyrocyclic hydantoin: 5-(((5S,9R)-9-(4-[ ¹⁴C]-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3, 7-triazaspiro[4.4]nonan-7-yl)methyl)

Full text of DOI:10.1002/jlcr.1596

Research Article
Received 9 December 2008,
Accepted 5 February 2009
Published online 9 April 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1596
Synthesis of lead LFA-1 antagonist
[14C]spyrocyclic hydantoin
Ã
Scott B. Tran, Brad D. Maxwell, Shiang-Yuan Chen, Samuel J. Bonacorsi,
Leslie Leith, Marc Ogan, J. Kent Rinehart, and Balu Balasubramanian
Radiolabelled drug lead candidate leukocyte function-associated antigen 1 antagonist [14C]spyrocyclic hydantoin:
5-(((5S,9R)-9-(4-[14C]-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]nonan-7-yl)methyl)thio-
phene-3-carboxylic acid, 12, was conveniently prepared in three radiochemical steps from (5S,9R)-tert-butyl 9-(4-
bromophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]nonane-7-carboxylate 9. The radiochemical
yield of 12 was 28.5% from the resolved bromide 9. The preparation of the racemic spyrocyclic hydantoin 3 was obtained
via a [312]dipolar cycloaddition reaction between 2 and N-benzyl-N-(methoxymethyl)trimethylsilylmethylamine. The
introduction of [14C] cyanide was completed via a palladium (0) catalyzed reaction by the addition of Zn(14CN)2 to aryl
bromide 9. The radiochemical and chiral purities of 12 determined by high-performance liquid chromatography were
98.7 and 99.7%, respectively. The specific activity of 12 was 87.5 lCi/mg (48.6 mCi/mmol).
Keywords: Zn(14CN)2; leukocyte function-associated antigen 1 antagonist; palladium (0) catalyzed cyanation
reacted in a [312]dipolar cycloaddition with the intermediary
iminium ylide formed from N-benzyl-N-(methoxymethyl)tri-
Introduction
methylsilylmethylamine7 to generate racemic compound 3.
The CD11a/CD18 (also known as leukocyte function-associated
antigen 1 or LFA-1) leukocyte integrin is expressed at high levels
on the cell surface of T lymphocytes and macrophages, where it
mediates homotypic and heterotypic adherence between
leukocytes and other cell types by binding to intracellular
adhesion molecules (ICAM) 1–3 on the conjugate cell.1
Monoclonal antibodies have been used to demonstrate the
critical role of the LFA-1/ICAM interaction in models of
autoimmune and inflammatory diseases,2 and some have
gained regulatory approvals. There has been an intense effort
to take on the more challenging task of identifying small,
biologically active, and orally bioavailable molecule inhibitors of
this LFA-1/ICAM interaction.3–5 The preparation of [14C]spyr-
ocyclic hydantoin 12 (Figure 1) was requested for use in pre/
clinical absorption, distribution, metabolism, and elimination
studies. This report details the preparation and characterization
of radiolabelled drug lead candidate LFA-1 antagonist [14C]spyr-
ocyclic hydantoin 12.
The benzyl protection group was removed to improve the
solubility of 4 in MeOH for supercritical fluid chromatography
(SFC). Reaction of 3 with 1-chloroethyl chloroformate8 followed
by treatment with MeOH and 6 N HCl gave racemic freebase 4.
Racemic 4 analyzed by chiral HPLC (Method B) indicated two
enantiomers to be present with product 4a, retention time
(Rt) = 9.07 min (57.6%), and product 4b, Rt = 15.65 min (42.2%).
To identify the biologically active and optically desired
enantiomer, optical resolution of the racemate via diastereoiso-
meric salt formation9 was completed to prepare each enantio-
mer. Small-scale fractional crystallization of 4 was attempted
with various chiral acids to form the corresponding diaster-
eoisomeric salt pairs. The results showed that the chiral acid
(1S)-(1) camphor sulfonic acid when used with 0.25–0.5
equivalent to the base produced higher % ee than di-p-
toluoyl-D-tartaric acid, (2S,3S)-2,3-dihydroxysuccinic acid, or (1R)-
(À) camphor sulfonic acid. Freebasing of each diastereoisomeric
salt pair with saturated NaHCO3 gave two separate enantiomers
identified as 4a and 4b (Scheme 2). At this stage, the two stereo
centers at positions 5 and 9 were not known. Each of the
obtained optically enriched materials was sequentially Boc
Results and discussion
The synthesis of [14C]spyrocyclic hydantoin 12 began with a
Knoevenagel reaction6 of 4-bromobenzaldehyde and an active
methylene compound 1 (Scheme 1). The reaction produced two
isomeric products. The trans olefin was the desired and more
thermodynamically stable product. The E/Z ratio by high-
performance liquid chromatography (HPLC) (Method A) was
95:5. The purification to isolate the trans olefin was completed
by slowly adding THF (30 v/v%) at 551C with stirring for 24 h,
which afforded compound 2 in pure form. Compound 2 was
Department of Chemical Synthesis, Radiochemistry Group, Bristol-Myers Squibb
Pharmaceutical Research and Development, PO Box 4000, Princeton, NJ 08543,
USA
*Correspondence to: Scott B. Tran, Department of Chemical Synthesis, Radio-
chemistry Group, Bristol-Myers Squibb Pharmaceutical Research and Develop-
ment, PO Box 4000, Princeton, NJ 08543, USA.
E-mail: scott.tran@bms.com
J. Label Compd. Radiopharm 2009, 52 236–242
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S. B. Tran et al.
protected, cyanated, and Boc deprotected to produce 7 and 8
(Scheme 3). The cyanated compounds 7 and 8 were compared
with the cyanated authentic reference standard (5S,9R;
Rt = 33.1 min) by chiral HPLC (Method B). It was determined
that 7 (Rt = 20.0 min) did not match the authentic desired
material (Rt = 33.1 min) concluding that compound 4a was the
O
Cl
OH
O
N
S
N
O
N
undesired enantiomer. Compound
8 (Rt = 33.1 min), which
Cl
coeluted with the authentic material, indicated that compound
4b was the desired enantiomer.
Large-scale enantiomeric separation of racemic 4 was com-
pleted by SFC to produce the desired enantiomer 4b with 99.4%
ee in 23% yield. Compound 4b was Boc protected with Boc
anhydride to produce 9 in 98% yield. In the presence of a
catalytic amount of Pd(PPh3)4, compound 9 was cyanated with
freshly prepared Zn(14CN)2 to give radioactive 10 in 50% yield.
*
CN
* = [14C]
Figure 1. [14C]12.
Br
O
H
Br
Cl
Cl
O
Cl
Cl
Cl
Cl
O
O
1) Pyrrolidine,
N
N
EtOH, refluxed 24 h,
+
N
N
N
N
Br
O
2) THF (30 %v/v), 55˚C
79%
O
O
1
Z-isomer
2, E-isomer
E/Z = 95 : 5
After purification, E/Z = 99:1
Ph
N
2
O
Cl
Cl
Ph
TFA, THF, rt - 50˚C
N
Me3Si
N
OMe
N
81%
O
Br
Racemic
3
Cl
5S
O
NH
N
9R
N
Cl
O
O
Cl
NH
5
Desired
enantiomer
N
9
1) 1-Chloroethyl chloroformate
1,2-dichloroethane, refluxed, 16 h
N
Br
4a or 4b?
O
Cl
2) MeOH, 6 N HCl
63%
Br
Racemic
4
Cl
5R
O
NH
HPLC (Method A), Rt = 17.37 min
N
HPLC Chiralpak AD (Method B),
Pdt 4a, Rt = 9.07 min, AUC = 57.6%
Pdt 4b, Rt = 15.65 min, AUC = 42.2%
9S
N
Cl
O
Undesired
enantiomer
Br
Scheme 1. Synthesis of the racemic base 4.
J. Label Compd. Radiopharm 2009, 52 236–242
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S. B. Tran et al.
phase: n-hexane:isopropyl alcohol:triethyl amine:TFA (93:7:1:1).
Isocratic, flowrate = 1.5 mL/min.
Method C: Used for compound 12. YMC-ODS-AQ C18, 4 mm,
4.6 Â 150 mm, detected at 220 nm. Mobile phase A: 0.1% TFA in
water, B: 0.1% TFA in acetonitrile. Gradient: 0–25 min 35%B,
35 min 98%B, 47 min 35%B. Flowrate = 1 mL/min.
H
(1S)-(+) Camphor
sulfonic acid, 0.45 eq.
1)
O
HO S
CH2Cl2 (0.5 M)
Method D: Used for compound 12. Chiralcel OJ-RH,
4.6 Â 150 mm, 5 mm, Chiral Technologies, detected at 220 nm.
Mobile phase: methanol:acetonitrile:0.2% aq. phosphoric acid
(30:30:40). Isocratic, flowrate = 1.0 mL/min.
2) Sat'd NaHCO3
4a
98.8% ee
Racemic
4
Overall 12%
(E)-5-(4-bromobenzylidene)-3-(3,5-dichlorophenyl)-1-methy-
limidazolidine-2,4-dione, 2
H
(1S)-(+) Camphor
sulfonic acid, 0.45 eq.
1)
A reaction flask was charged with 3-(3,5-dichlorophenyl)-
1-methylimidazolidine-2,4-dione 1 (15.1 g, 58.3 mmol), 4-bromo-
benzaldehyde (12.7 g, 68.8 mmol), ethanol (210 mL), and
pyrrolidine (3.9 g, 55.4 mmol). The mixture was heated to reflux
for 24 h. The reaction was monitored by TLC (20% EtOAc/
hexane, product Rf = 0.7, SM Rf = 0.3), HPLC (Method A, product
O
HO S
CH2Cl2 (0.02 M)
Methyl t-butyl ether, 0˚C - rt
2) Sat'd NaHCO3
Overall 14%
Racemic
4
1
4b
96.0% ee
Rt = 14.0 min), and H NMR for the completion of reaction. The
resulting clear solution was cooled slowly to 551C and the
desired E-isomer began to crystallize. At 551C, THF (70 mL) was
added. The thick mixture was stirred at 551C for 24 h. The
product was collected by vacuum filtration as a white solid
Scheme 2. Enantioenrichment by chiral crystallization.
1
The Boc-protecting group on product 10 was removed to give
11 in 92% yield. Then 11 was reacted via a reductive amination
reaction with 5-formyl-3-thiophenecarboxylic acid and sodium
triacetoxyborohydride to give the desired product [14C]spyr-
ocyclic hydantoin 12 in an overall yield of 62% (Scheme 4).
(19.6 g, 79%, E/Z499%). H NMR (500 MHz, DMSO-d6) d 7.88 (d,
2H, J = 8.3 Hz), 7.70 (d, 1H, J = 1.6 Hz), 7.69–7.57 (m, 4H), 6.63 (s,
1H), 3.21 (s, 3H). MS (ESI1 and ESIÀ) m/z not detectable.
Racemic 7-benzyl-9-(4-bromophenyl)-3-(3,5-dichlorophenyl)-
1-methyl-1,3,7-triazaspiro[4.4]nonane-2,4-dione, 3
Experimental
Compound 2 (3.0 g, 7.0 mmol) was dissolved in THF (60 mL). The
General: Radioactivity was measured with a Wallac Model 1409 solution was cooled to 01C and N-methoxymethyl-N-trimethyl-
liquid scintillation counter (Wallac-LKB instruments, Inc.). Count- silylmethyl benzylamine (1.92 g, 8.1 mmol) was added. TFA
ing efficiency was determined by the channels ratio method. (50 mL, 0.63 mmol) was added slowly. The reaction was stirred
Mass spectra were obtained with a Finnigan TSQ or a Finnigan at 01C for 2.5 h and at rt for 40 h. After this period, additional N-
LCQ mass spectrometer. Proton NMR spectra were recorded on methoxymethyl-N-trimethylsilylmethyl
benzylamine
(0.6 g,
a Bruker Advance Ultrashield 300 MHz or a Jeol EC1500 MHz. UV 2.5 mmol) and TFA (50 mL, 0.63 mmol) were added. The reaction
and radiochemical purities were determined by HPLC (Shimadzu was heated to 501C for 2.5 h to drive the reaction to completion.
SCL-10A VP/UV-1 detector or Varian Prostar 330 PDA detector The solvent was evaporated to dryness. The dry residue was
and IN/US System b-Ram radiometric flow detector with 0.5-mL diluted with EtOAc (30 mL), washed with water (30 mL), and
flow cell). TLC was performed on 60 F254 silica gel plates (Merck). backextracted with EtOAc (2 Â 30 mL). After separation, the
Flash chromatography was conducted on KP-Sil silica gel organic layer was washed with brine, dried over MgSO4, and
(Biotage). Radiolabelled products were compared with authentic evaporated to dryness. The crude product was triturated in
standards when possible. All reagents and solvents were ACS diethyl ether to give a white solid (3.2 g, 81%). TLC (20% EtOAc/
grade or better.
hexane, product Rf = 0.3) and HPLC (Method A, Rt = 18.6 min). 1H
Specific activity was determined gravimetrically by dissolution of NMR (500 MHz, DMSO-d6) d 7.60 (d, 1H, J = 1.6 Hz), 7.51 (d, 2H,
the weighed sample (ca 0.5 mg) into DMF in a 10-mL volumetric J = 8.8 Hz), 7.40–7.25 (m, 5H), 7.12 (d, 2H, J = 8.3 Hz), 6.70 (d, 2H,
flask. Aliquots of 25 and 50 mL of the solution sample were J = 1.9 Hz), 3.90–3.80 (m, 2H), 3.69 (d, 1H, J = 13.2 Hz), 3.30–3.18
diluted into 10-mL Ecolite cocktail and counted.
(m, 2H), 3.12 (s, 3H), 3.01 (t, 1H, J = 11.6 Hz), 2.82 (d, 1H,
High-performance liquid chromatography: HPLC methods J = 11.6 Hz). MS ESI1 [M1H]1 = 560.03.
described below were used for in process and final product
analyses. Co-injections with authentic samples were used when
Racemic 9-(4-bromophenyl)-3-(3,5-dichlorophenyl)-1-methyl-
1,3,7-triazaspiro[4.4]nonane-2,4-dione, 4
possible. All HPLC purities were measurements of UV and
radiochemical purities.
A solution of racemic 3 (2.0 g, 3.6 mmol) in 1,2-dichloroethane
(30 mL) under a nitrogen atmosphere at 51C was added
dropwise over 15 min 1-chloroethyl chloroformate (1.5 g,
10.5 mmol). The white suspension was stirred at 51C for 1 h
and heated to reflux at 801C for 18 h. The reaction was cooled to
rt and the solution was evaporated to dryness to yield a
light yellow oil. This oil was further concentrated using a
Method A: Used for compounds 2–4 and 10. YMC-ODS-AQ
C18, 3 mm, 4.6 Â 150 mm, detected at 254 nm. Mobile phase A:
0.05% trifluoroacetic acid (TFA) in water, B: 0.05% TFA in
acetonitrile. Gradient: 0 min 25% B, 5 min 55% B, 15 min 95% B,
30 min 95% B, 32 min 25%B. Flowrate = 1 mL/min.
Method B: Used for compounds 4, 4a, 4b, and 7–9. Chiralpak
AD, Daicel, 5 mm, 4.6 Â 250 mm, detected at 254 nm. Mobile
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S. B. Tran et al.
Cl
Zn(CN)2,
10 mol%
Pd(PPh3)4,
DMF, 90˚C,
3h
O
O
Cl
NH
O
O
N
Boc anhydride,
CH2Cl2, TEA
N
O
N
O
N
Cl
N
Cl
98%
47%
Product 4a,
98.8% ee
Br
5
Br
O
Cl
Cl
O
O
O
N
Cl
NH
O
20% TFA/CH2Cl2,
rt
N
NH
N
O
N
Cl
N
N
=
Cl
O
N
66%
Cl
O
6
CN
7
CN
CN
Chiral HPLC (Method B), Rt = 20.0 min
Different from the optically
Undesired
desired authentic sample Rt = 33.1 min.
Conclusion: Compound 7 and therefore 4a = Undesired enantiomer
Cl
Cl
O
O
NH
NH
As above
N
N
O
N
N
Cl
Cl
O
Product 4b,
96.0% ee
Br
CN
8
Chiral HPLC (Method B), Rt = 33.1 min
Matched with the optically
desired authentic sample Rt = 33.1 min.
Conclusion: Compound 8 and therefore 4b = Desired enantiomer
Scheme 3. Synthesis and chiral HPLC analyses to determine which enantiomer was the desired product.
high-vacuum pump to remove residual benzyl chloride that
Fractional chiral crystallization, 4a
was formed in the first step. An amorphous solid was obtained.
The solid was suspended in MeOH (30 mL) and 6 N HCl (1.3 mL).
The suspension was heated to 501C for 1.5 h and then
evaporated to dryness. The residue was suspended in dichlor-
omethane (90 mL) and water (15 mL) and neutralized to pH = 8.5
with 2 N NaOH (ꢀ7 mL). The layers were separated and the
organic layer was washed with water, brine, dried over sodium
sulfate, and concentrated to a foam. It was purified by silica gel
flash chromatography by eluting with 0–5% MeOH/CH2Cl2
to give a white solid as a racemic mixture (1.1 g, 63%). TLC
(30% EtOAc/hexane, product Rf = 0.3), HPLC (Method A,
Rt = 17.37 min), and chiral HPLC (Method B, Rt = 9.07 min,
The pyrrolidine racemic mixture 4 (0.5 g, 1.06 mmol) was dissolved
in dichloromethane (2.0 mL). To this solution was added (1S)-(1)
camphor sulfonic acid (0.124 g, 0.53 mmol). Scratching the sides of
the flask with a spatula facilitated the crystallization. Over 10 min
the solution became cloudy. It was left at rt for 30min. The
crystallized diastereoisomeric salt was collected by vacuum filtration
to give a white solid (147 mg). 1H NMR (500 MHz, DMSO-d6) d 9.45
(s, 2H), 7.67 (t, 1H, J= 2.2 Hz), 7.62 (d, 2H, J= 8.8 Hz), 7.17 (d, 2H,
J= 8.3 Hz), 6.67 (d, 2H, J= 1.3 Hz), 3.99–3.95 (t, 1H, J= 10.4 Hz),
3.83–3.81 (m, 2H), 3.73 (m, 2H), 3.14 (s, 3H), 2.88 (d, 1H, J=14.3Hz),
2.69–2.62 (m, 1H), 2.39 (d, 1H, J=14.8 Hz), 2.20 (1H, tt), 1.93 (t, 1H,
J= 4.4 Hz), 1.91–1.80 (m, 1H), 1.80 (d, 1H, J= 18.1 Hz), 1.30–1.20 (m,
2H), 1.03 (s, 3H), 0.73 (s, 3H). MS ESI1 [M1H]1 = 470.0. This salt was
freebased with saturated sodium bicarbonate (25 mL) and extracted
with dichloromethane (3 Â 20 mL) to give the optically pure
enantiomer as a white foam (60 mg, yield = 12%). Chiral HPLC
1
57.6%, and Rt = 15.65 min, 42.2%). H NMR (500 MHz, DMSO-d6)
d 7.60 (d, 1H, J = 2.2 Hz), 7.53 (d, 2H, J = 8.8 Hz), 7.10 (d, 2H,
J = 8.3 Hz), 6.70 (d, 2H, J = 1.7 Hz), 3.50 (m, 1H), 3.35–3.30 (m, 2H),
3.30–3.20 (m, 2H), 3.09 (s, 3H), 3.05 (bs, 1H). MS ESI1 [M1H]1
= 470.2.
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S. B. Tran et al.
Cl
O
O
Cl
NH
O
O
N
Boc-anhydride,
CH2Cl2
N
O
N
O
N
Cl
N
Cl
98%
4b
Br
9
Br
9
6 mol%
O
Pd(PPh3)4,
DMF, 90˚C,
36h
Cl
O
O
N
Water
80%
Zn(14CN)2
K14CN
ZnCl2
+
N
N
50%
Cl
O
10
* CN
* = [14C]
O
O
N
HO
1)
HO
S
Cl
H
O
S
Cl
NH
O
N
O
20% TFA/CH2Cl2
92%
N
N
O
N
2) Na(OAc)3BH
62%
Cl
Cl
O
11
*
CN
12
*
CN
Scheme 4. Incorporation of [14C].
(Method B, Rt = 9.07 min, ee = 98.8%). [a]D = 20.7–76.081, (55.8 mg, 0.26 mmol). The reaction was stirred at rt for 3 h. It was
conc. = 0.700 w/v% in methanol. 1H NMR (500 MHz, CD3OD) d diluted with dichloromethane (5 mL), washed with water
7.41–7.38 (dd, 2H, J= 2.3, 9.1 Hz), 7.28 (t, 1H, J= 1.9 Hz), 7.04 (d, 2H, (20 mL), and extracted with dichloromethane (2 Â 20 mL). The
J= 8.5 Hz), 6.60 (d, 2H, J= 1.9 Hz), 3.61–3.56 (m, 1H), 3.51 (t, 1H, layers were separated and the organic layer was washed with
J= 9.1 Hz), 3.28–3.19 (m, 3H), 3.07 (s, 3H). MS ES1 [M1H]1 =470.2. brine, dried over MgSO4, and concentrated to dryness to give a
white solid (119 mg, 98%). TLC (5% MeOH/dichloromethane,
Fractional chiral crystallization, 4b
Rf = 0.8). 1H NMR (500 MHz, DMSO-d6) d 7.65 (t, 1H, J = 1.7 Hz),
7.57 (d, 2H, J = 7.7 Hz), 7.15 (d, 2H, J = 6.0 Hz), 6.80 (d, 2H,
J = 9.3 Hz), 4.07–4.05 (m, 1H), 3.93–3.84 (m, 3H), 3.70 (d, 1H,
J = 12.0 Hz), 3.10 (s, 3H), 1.48 (s, 9H). MS (ESI1 and ESIÀ) m/z not
detectable.
The pyrrolidine racemic mixture 4 (0.4 g, 0.85 mmol) was
dissolved in dichloromethane (45 mL). To this solution was
added (1S)-(1) camphor sulfonic acid (80.0 mg, 0.34 mmol). To
the stirred solution was added t-butyl methyl ether (5 mL)
dropwise. It was left unstirred at rt for 2 h for the precipitation to
occur. The crystallized diastereoisomeric salt was collected by
vacuum filtration to give a white solid (88 mg). This salt was
freebased with saturated sodium bicarbonate (20 mL) and
extracted with dichloromethane (3 Â 15 mL). The organic layer
was concentrated under reduced pressure to give the optically
pure enantiomer as a white thin oil (54.2 mg, yield = 13.6%).
Chiral HPLC (Method B, Rt = 15.65 min, ee = 95.8%). 1H NMR:
Same as compound 4a. MS ES1 [M1H]1 = 470.2.
Preparation of 6
In a pressure tube under argon was added compound 5
(119 mg, 0.21 mmol) and DMF (2.0 mL). The solution was purged
with argon for 10 min. To this was quickly added Zn(CN)2
(12.3 mg, 0.11 mmol) and tetrakis(triphenylphosphine)palladium
(0) (24.3 mg, 0.021 mmol). The reaction mixture was heated to
901C for 3 h. It was cooled to rt, washed with 10% LiCl (5 mL),
and extracted with EtOAc (3 Â 10 mL). After the layers were
separated, the organic layer was washed with water, brine, dried
over MgSO4, and concentrated to dryness. The crude product
was purified by silica gel chromatography by eluting with a
gradient 0–50% EtOAc/hexane to give a white solid (50.6 mg,
Preparation of 5
To a solution of compound 4a (99.2 mg, 0.21 mmol) in
dichloromethane (2 mL) was added di-tert-butyl dicarbonate
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S. B. Tran et al.
47%). TLC (40% EtOAc/hexane, Rf = 0.3). 1H NMR (500 MHz, pre-purged DMF (4 mL). The mixture was carefully purged with
DMSO-d6) d 7.92 (d, 2H, J = 7.1 Hz), 7.71 (s, 1H), 7.47 (d, 2H, N2 for 15 min. It was heated to 901C for 36 h and monitored by
J = 7.1 Hz), 6.88 (d, 2H, J = 12.1 Hz), 4.34–4.20 (m, 1H), 4.10–3.85 TLC (40% EtOAc/hexane, product Rf = 0.3) and HPLC (Method A,
(m, 3H), 3.81 (d, 1H, J = 12.1 Hz), 3.19 (s, 3H), 1.51 (s, 9H). MS (ESI1 Rt = 15.2 min). The DMF in the suspension was carefully reduced
and ESIÀ) m/z not detectable.
to about 1 mL by a stream of N2 at 601C. The mixture was
washed with 10% LiCl (20 mL) and extracted into EtOAc
(3 Â 30 mL). The layers were separated and the organic layer
was washed with brine, dried over sodium sulfate, and
concentrated to dryness. The crude product was purified by
silica gel flash chromatography eluting with 10–50% EtOAc/
hexane and then with 5% MeOH/dichloromethane to afford a
pure white solid (463 mg), radiochemical purity = 99.4%, specific
activity = 84.7 mCi/mg, total activity = 39.2 mCi, radiochemical
Preparation of 7
Compound 6 (51 mg, 0.098 mmol) was dissolved in dichloro-
methane (1.5 mL). To this solution was slowly added TFA
(300 mL) and it was stirred for 30 min. The reaction solution was
evaporated to dryness. The residue was dissolved in dichlor-
omethane (3 Â 6 mL) and saturated NaHCO3 was added. The
layers were separated and the organic layer was washed with
brine, dried over MgSO4, and concentrated to give the product
1
yield = 49%. H NMR was the same as that reported for 6.
as
a thin film (27 mg, 66%). Chiral HPLC (Method B,
4-[14C]((5S,9R)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-
1,3,7-triazaspiro[4.4]nonan-9-yl)benzonitrile, 11
Rt = 20.07 min). 1H NMR (400 MHz, DMSO-d6) d 7.84 (d, 2H,
J = 8.1 Hz), 7.62 (s, 1H), 7.37 (d, 2H, J = 8.2 Hz), 6.71 (t, 2H,
J = 0.7 Hz), 3.73–3.69 (m, 1H), 3.41–3.26 (m, 4H), 3.13 (s, 3H). MS
ESI1 [M1H]1 = 415.1.
To a solution of compound 10 (457 mg, 0.89 mmol) in CH2Cl2
(3 mL) was slowly added 30% TFA in CH2Cl2 (5 mL). The reaction
was stirred at rt for 1.5 h while monitoring by TLC (5%MeOH/
CH2Cl2, product Rf = 0.2). The reaction solution was evaporated
to dryness. The resulting oil was dissolved in dichloromethane
(3 Â 20 mL) and saturated sodium bicarbonate (20 mL) was
added to pH = 8–9. The layers were separated and the organic
layer was washed with brine, dried over sodium sulfate, and
concentrated. The reaction produced a white sticky foam
(338 mg, 92%), which was used in the next reaction without
Resolution of racemic 4 by SFC
The pyrrolidine racemic mixture 4 was resolved by preparative
SFC on a Chiralpaks AD-H, 3 Â 25 cm, 5 mm at 351C eluted with
30% methanol/CO2 as the mobile phase. The conditions were:
pressure 100 bar, flowrate 80 g/min, injection amount
100–300 mg, run time 15 min, and throughput 1.0 g/h. The
compound
4 (5.2 g, UV purity = 99.7%) was dissolved in
1
further purification. H NMR (300 MHz, DMSO-d6) d 7.84 (d, 2H,
methanol at a concentration of 100 mg/mL and used for
multiple injections. The two enantiomers were separated,
collected, and concentrated to dryness. Chiral HPLC analysis
(Method B) indicated that the first SFC eluted peak indeed
contained the desired enantiomer 4b (Rt = 15.65 min) (1.30 g,
yield = 23%, 99.44% ee).
J = 8.1 Hz), 7.62 (s, 1H), 7.37 (d, 2H, J = 8.2 Hz), 6.71 (t, 2H,
J = 0.7 Hz), 3.73–3.69 (m, 1H), 3.41–3.26 (m, 4H), 3.13 (s, 3H). MS
ESI1 [M1H]1 = 417.1.
5-(((5S,9R)-9-(4-[14C]-cyanophenyl)-3-(3,5-dichlorophenyl)-1-
methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]nonan-7-yl)methyl)-
thiophene-3-carboxylic acid, 12
(5S,9R)-tert-butyl 9-(4-bromophenyl)-3-(3,5-dichlorophenyl)-
1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]nonane-7-carboxy-
late, 9
To a 50-mL, pear-shaped flask containing a stir bar under
nitrogen was charged 11 (338 mg, 0.81 mmol) and 1,2-dichlor-
oethane (10 mL). To the homogeneous solution was added 5-
formyl-3-thiophenecarboxylic acid (153 mg, 1.0 mmol) and the
reaction was stirred for 15 min. To the reaction flask at rt was
added sodium sulfate (338 mg, 2.4 mmol). The mixture was
stirred for 20 h. To this reaction mixture was added sodium
triacetoxyborohydride (310 mg, 1.46 mmol) at rt over a 5-min
period. The reaction was allowed to stir at rt for 3 h during which
it became slightly homogeneous. After 3 h the reaction was
quenched with water (30 mL) and stirred for 15 min. The
reaction was extracted with 1,2-dichloroethane (3 Â 20 mL).
After the layers were separated, the organic layer was washed
with brine, dried over sodium sulfate, and concentrated to
dryness. The dry crude product was triturated with MeOH (5 mL)
and stirred for 30 min. The insoluble residue was collected by
filtration to give a white solid (280 mg, 62%). TLC (5% MeOH/
dichloromethane, product Rf = 0.3) and HPLC (Method C,
Rt = 9.1 min, radiochemical purity = 97.3%). This material was
further purified by silica gel flash chromatography eluting with
0–8% MeOH/dichloromethane to give product 12 [radiochemi-
cal purity = 98.7%, chiral HPLC (Method D), Rt = 13.4 min, chiral
purity = 99.7%, specific activity = 87.5 mCi/mg or 48.6 mCi/mmol].
1H NMR (300 MHz, DMSO-d6) d 8.08 (s, 1H), 7.82 (d, 2H,
J = 8.2 Hz), 7.60 (s, 1H), 7.37 (d, 2H, J = 8.3 Hz), 7.32 (s, 1H), 6.70
Prepared using the same procedure as for the preparation of 5.
Preparation of Zn(14CN)2
To
a 10-mL centrifuge tube was added ZnCl2 (282 mg,
2.07 mmol) dissolved in water (2 mL). K14CN (Amersham, batch
CFQ30087, 100 mCi, SA = 54.9 mCi/mmol, 118.6 mg, 1.82 mmol)
was dissolved in water (2 mL) and the solution was added slowly
to the solution of ZnCl2. Zn(14CN)2 precipitated out almost
immediately. The reaction was stirred for 1 h. The mixture was
centrifuged for 10 min at 2500 rpm. The water layer was
withdrawn by pipet. The insoluble residue was washed with
water (1 mL) and then with acetone (2 mL) and dried under high
vacuum for 16 h to give the product as a white solid (126.5 mg,
80.4 mCi).
(5S,9R)-tert-butyl 9-(4-[14C]-cyanophenyl)-3-(3,5-dichlorophenyl)-
1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]nonane-7-carboxy-
late, 10
To a 10-mL centrifuge tube containing dry Zn(14CN)2 (126 mg,
1.07 mmol, 80.4 mCi) under N2 was quickly added compound 9
(1.22 g, 2.15 mmol) and tetrakis(triphenylphosphine)palladium
(0) (148.7 mg, 6 mol%) followed by the addition of the N2
J. Label Compd. Radiopharm 2009, 52 236–242
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

S. B. Tran et al.
(s, 2H), 4.07–4.0 (m, 2H), 3.89 (d, 1H, J = 13.2 Hz), 3.32 (s, 2H), 3.11
(m, 4H), 2.85 (d, 1H, J = 11.6 Hz). MS [MÀH] = 555.05.
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P. M. Davis, P. Balimane, K. Patel, Z. Yang, P. Marathe, J. Postelneck,
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Acknowledgement
We wish to thank Dominique Potin and Michele Launay of Cerep
(France), Murali Dhar and Zili Xiao of Bristol-Myers Squibb
Medicinal Chemistry, and Douglas Mcleod and Albert DelMonte
of Bristol-Myers Squibb Process Research and Development for
their helpful discussion and providing us valuable intermediates
and analytical reference standards. In addition, thanks are due to
Dauh-Rurng Wu of Chemical Synthesis Group for providing us
the chiral separation work by SFC.
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J. Label Compd. Radiopharm 2009, 52 236–242