Synthesis of [3,5-dichlorobenzenesulfonamide-U-14C)] labeled VLA-4 antagonists

Article abstract of DOI:10.1002/jlcr.804

Radiolabeled tracers were required for the development of a series of VLA-4 antagonists. A method to synthesize [U- ¹⁴C]3,5-dichlorobenzenesulfonyl chloride was developed. From this key intermediate, various tracers were prepared in high yield.

Full text of DOI:10.1002/jlcr.804

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 115–125.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.804
Research Article
Synthesis of [3,5-dichlorobenzenesulfonamide-U-14C)]
labeled VLA-4 antagonists
Nathan X. Yu1,*, Conrad E. Raab1, Dennis C. Dean1, Linus S. Lin2 and
David G. Melillo1
1 Department of Drug Metabolism, Merck Research Laboratories, RY80R-104, PO Box
2000, Rahway, NJ 07065, USA
2 Department of Basic Chemistry, Merck Research Laboratories, RY80R-104, PO Box
2000, Rahway, NJ 07065, USA
Summary
Radiolabeled tracers were required for the development of a series of VLA-4
antagonists. A method to synthesize [U-14C]3,5-dichlorobenzenesulfonyl chloride was
developed. From this key intermediate, various tracers were prepared in high
yield. Copyright # 2004 John Wiley & Sons, Ltd.
Key Words: VLA-4 antagonist; asthma; [U-14C]3,5-dichlorobenzenesulfonyl chloride
Introduction
VLA-4 (a4b1 protein), a key regulator of inflammation and auto-immune
disease response, has been a target for the treatment of a number of
inflammatory conditions including asthma.1 The series of VLA-4 antagonists,
1–5, were investigated as potential drug candidates.2,3 Carbon-14 radiolabeled
tracers of these compounds were required for pharmacokinetic and
metabolism studies (Scheme 1).
We report here a synthetic route to [U-14C]3,5-dichlorobenzenesulfonyl
chloride (6), which can allow the rapid preparation of tracers of 1–5, as well as
other 3,5-dichlorobenzenesulfonamides. [14C]Aniline hydrochloride was cho-
sen as the starting material because it is readily available and reasonably
priced. The amino group, very important in directing aromatic substitution,
was easily removed at the end of the synthesis by a diazotization/
dediazotization sequence.
*Correspondence to: N.X. Yu, Merck Research Laboratories, Department of Drug Metabolism, RY80R-
104, Rahway, NJ 07065, USA. E-mail: xiao yu@merck.com
Copyright # 2004 John Wiley & Sons, Ltd.
Received 26 August 2003
Revised 28 October 2003
Accepted 6 November 2003

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N.X. YU ET AL.
Scheme 1. VLA-4 antagonists
Results and discussion
To prevent possible N-oxidation or bis-sulfonation, aniline was acetylated with
acetic anhydride.4 Acetamide 7 was then sulfonated with chlorosulfonic acid
to give sulfonyl chloride 8,5 which was stirred in an ammonium hydroxide/
acetone solution to provide the corresponding sulfonamide 9.6 Subsequent
ethanolysis under acidic conditions removed the acetyl protecting group.7
Even though unlabeled and small-scale probe reactions proceeded well (50 and
60% yields of 11, respectively), the main batch of chlorination of 10 provided
11 in only 13% radiochemical yield from 9. This reaction provides better yields
of the desired product if the reaction temperature is limited. The lower yield in
the main batch reaction was probably due to a local elevation of the reaction
temperature, resulting in the formation of azo- or azoxybenzenes.8 Deamina-
tion of 11 was performed through initial formation of the diazonium salt 12,
which was heated at 908C in 2-propanol to promote nitrogen loss leading to
sulfonamide 13.9 The reaction of 13 proceeded smoothly with chlorosulfonic
acid and thionyl chloride to give 42 mCi of the target sulfonyl chloride 6 (8.4%
radiochemical yield from aniline).10 Storage of this labeled intermediate in
anhydrous benzene at À278C resulted in less than 5% decomposition over a
3-year period (Scheme 2).
The appropriately protected amines (15–19) were prepared using procedures
similar to those described in Lin et al.3, allowing for late stage carbon-14
incorporation. Thus, sulfonylation of the amines with 6 gave, in high yield,
esters 20–24, which were saponified to provide the corresponding acids, 1, 2,
25–27. The protecting groups on 25–27 were removed by acidic hydrolysis or
hydrogenation. All five final tracers were purified by preparative HPLC
(Scheme 3).
In summary, we have developed a synthetic route to [U-14C]3,5-
dichlorobenzenesulfonyl chloride (6), from which the sulfonamide tracers
(1–5) were rapidly prepared. The [l4C]sulfonyl chloride (6) is stable in storage
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LABELED VLA-4 ANTAGONISTS
117
Scheme 2. Synthesis of sulfonyl chloride 6
over a long period of time and can be used as an off-the-shelf radiolabeling
reagent.
Experimental
Radioactivity measurements were carried out using a Bioscan Lumi-Scint
liquid scintillation counter with Packard Ultima GoldTM scintillant. Analytical
HPLC measurements were performed on a system consisting of Shimadzu LC-
10ADVP pumps, SPD-10AVP UV detector (215/254 nm), CTO-10ASVP
column oven heated at 308C, SIL-10ADVP auto-injector, SCL-10AVP system
controller and Packard RadiomaticTM 150TR flow monitor controlled by a
Shimadzu Class-VP software. All HPLC analyses were conducted on
4.6 Â 250 mm columns at 1.0 ml/min, eluting with acetonitrile (A) and either
0.1% aqueous trifluoroacetic acid (B) or 0.1% aqueous perchloric acid (C),
and concluded with a 10 min wash of 100% acetonitrile. All retention times
(tR) refer to the radioactive channel. Preparative HPLC was performed on a
system consisting of Gilson 322 pump, UV/VIS-155 detector, and 215 liquid
handler controlled by UniPointTM software. Silica gel chromatography was
performed on a Biotage FlashEluteTM system, using FlashEluteTM cartridges.
Mass spectra were recorded on an HP-1100 LCMSD instrument in API-ES
positive ionization mode. The MS data reported are the masses of the most
1
abundant molecular ions. H NMR spectra were measured at 400 MHz on a
Varian Unity-400 spectrometer.
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Scheme 3. Synthesis of the VLA-4 tracers (1–5)
[U-14C]Aniline hydrochloride was obtained from American Radiolabeled
Chemicals. Amines 15–19 were obtained from Merck Basic Chemistry,
Rahway. Anhydrous solvents were obtained from Aldrich Chemical Co. and
(
dried over 4 A molecular sieves for at least 24 h prior to use. All other reagents
were obtained from Aldrich and used as is. All reactions were carried out
under a nitrogen atmosphere. All radioactive compounds were stored at
À278C.
The identities of all intermediates and the final products were established by
1H NMR and/or by co-elution on HPLC with authentic standards obtained
from Aldrich, TCI America or Merck Basic Chemistry.
[phenyl-U-l4C]N-Acetylaniline (7)
Acetic anhydride (6.5 ml, 68.9 mmol) was added to a solution of [l4C]aniline
(500 mCi, 60 mCi/mmol, 8.33 mmol) in carbon tetrachloride (12 ml), with
stirring. The reaction mixture was refluxed at 858C for 1 h, then cooled to
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LABELED VLA-4 ANTAGONISTS
119
room temperature. The solvent was removed in vacuo. Water (25 ml) was
added. The mixture was refluxed at 1008C for 1 h, then cooled to room
temperature. It was extracted with methylene chloride, dried with anhydrous
sodium sulfate, filtered and counted (500 mCi, 97.1% radiochemical purity).
The solvent was removed in vacuo to give 7 (97% radiochemical yield) as a
white solid, which was analyzed by HPLC (Phenomenex Curosil PFP, 40A/
60B isocratic, tR=5.92 min, 97.1% radiochemical purity). Crude 7 was used in
the following reaction without purification. 1H NMR (CDCl3) d 7.49 (d, 2H, J
=7.9 Hz), 7.31 (t, 2H, J=7.9 Hz), 7.10 (t, 1H, J=7.9 Hz), 2.15 (s, 3H). MS
Calculated for 7 [M+H+]: 136, Found: 136.
[phenyl-U-14C]4-(N-Acetylamino)benzenesulfonamide (9)
Chlorosulfonic acid (10 ml, 150 mmol) was added to 7 (500 mCi, 97.1%
radiochemical purity, 8.09 mmol) at 08C with stirring. The reaction mixture
was heated to 608C for 1 h, then cooled to room temperature and poured onto
crushed ice. A white solid formed, which was isolated by filtration, then
dissolved in acetone (40 ml). At 08C, ammonium hydroxide (10 ml, 28% NH3
in water) was added. The mixture was stirred at room temperature for 1 h, then
counted (400 mCi, 98.6% radiochemical purity) and analyzed by HPLC
(Phenomenex Curosil PFP, 40A/60B isocratic, tR=6.81 min, 98.6% radio-
chemical purity). The solvent was removed in vacuo to give 9 (81%
radiochemical yield) as a white solid, which was used in the following reaction
without purification. 1H NMR (DMSO-d6) d 10.66 (s, 1H), 7.74 (m, 4H), 7.22
(b, 2H), 2.08 (s, 3H). MS Calculated for 9 [M+H+]: 215, Found: 215.
[phenyl-U-14C]4-Aminobenzenesulfonamide (10)
Concentrated hydrochloric acid (8 ml) was added to a solution of 9 (400 mCi,
98.6% radiochemical purity, 6.57 mmol) in ethanol (50 ml) at room
temperature, with stirring. The reaction mixture was refluxed at 908C for
30 min, then counted (400 mCi, 96.9% radiochemical purity) and analyzed by
HPLC (Phenomenex Curosil PFP, 20A/80B isocratic, tR=4.12 min, 96.9%
radiochemical purity). The solvent was removed in vacuo to give 10 (98%
radiochemical yield) as a light yellow solid, which was used in the following
reaction without purification. MS Calculated for 10 [M+Na+]: 195, Found:
195. The identity of 10 was established by co-elution with an authentic sample
of unlabeled material obtained commercially.
[phenyl-U-14C]4-Amino-3,5-dichlorobenzenesulfonamide (11)
The crude 10 (400 mCi, 96.9% radiochemical purity, 6.46 mmol) was dissolved
in water (25 ml) and concentrated hydrochloric acid (25 ml). Aqueous
hydrogen peroxide (30%, 2.5 ml) was added, and the mixture stirred at room
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temperature for 1 h, then extracted with methylene chloride. The
organic layers were dried with anhydrous sodium sulfate, filtered, counted
(385 mCi, 20.6% radiochemical purity), and analyzed by HPLC (Phenomenex
Curosil PFP, 40A/60B isocratic, tR=8.02 min, 20.6% radiochemical
purity). Purification by silica gel chromatography (methylene chloride/ethyl
acetate 4/1) gave 65 mCi of 11 (80% radiochemical purity, 13% radiochemical
yield), which was stored in methanol. The identity of 11 was established
by co-elution with an authentic sample of unlabeled material obtained
commercially.
[phenyl-U-14C]3,5-Dichlorobenzenesulfonamide (13)
A methanol solution of 11 (65 mCi, 80% radiochemical purity, 0.87 mmol)
was dried in vacuo, and the residue dissolved in 2-propanol
(5 ml). Concentrated hydrochloric acid (0.67 ml) was added at room
temperature. After cooling to 08C,
a
solution of sodium nitrite
(190 mg, 2.75 mmol) in water (0.48 ml) was added. The mixture was stirred
at 08C for 30 min, then refluxed at 908C for 2 h. The solution was
counted (61.5 mCi), then concentrated in vacuo. The residue was dissolved
in methanol and purified by preparative HPLC (Zorbax SB-C18,
21.2 Â 250 mm, 215/254 nm, 27A/73B isocratic, flow rate =20 ml/min) to give
42 mCi of 13 (100% radiochemical purity, 81% radiochemical yield) as a
solution in methanol. HPLC analysis: Zorbax SB-C18, 35A/65B isocratic,
tR=12.03 min, 100% radiochemical purity. The identity of 13 was established
by co-elution with an authentic sample of unlabeled material obtained
commercially.
[phenyl-U-14C]3,5-Dichlorobenzenesulfonyl chloride (6)
A methanol solution of 13 (42 mCi, 0.70 mmol) was dried in vacuo.
Chlorosulfonic acid (300 ml) was added to the residue, and the reaction
mixture stirred at 908C for 100 min. Thionyl chloride (50 ml) was then
added, and the mixture stirred at 908C for another 60 min. After cooling to
room temperature, the mixture was poured onto ice and extracted
with methylene chloride. The combined organic layers were quickly washed
with saturated aqueous sodium bicarbonate solution and then brine at 08C.
The solution was dried with anhydrous sodium sulfate and filtered.
The solvent was removed in vacuo, and the residue dissolved in anhydrous
benzene (10 ml) to give 42 mCi of 6 (100% radiochemical purity, 100%
radiochemical yield). HPLC analysis: Phenomenex Curosil PFP, 20A/80B
isocratic, tR=5.52 min, 100% radiochemical purity. The identity of 11 was
established by co-elution with an authentic sample of unlabeled material
obtained commercially.
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LABELED VLA-4 ANTAGONISTS
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[3,5-dichlorophenyl-U-14C]Methyl(2S)-2-({(2S)-1-[(3,5-dichlorophenyl)
sulfonyl]-2-methylazetidin-2-yl}carbonylamino)-3-[4-(2,6-dimethoxyphenyl)
phenyl]propanoate (20)
A benzene solution of 6 (10 mCi, 0.17 mmol) was dried in vacuo. A solution of 15
(75 mg, 0.18 mmol) in methylene chloride (2 ml), tetrahydrofuran (850 ml) and
diisopropylethylamine (425 ml) was added to the residue at 08C, with stirring. The
reaction mixture was allowed to reach room temperature slowly and was then
stirred overnight. The solvent was removed in vacuo, and the residue dissolved in
methanol to give 10 mCi of 20 (95.7% radiochemical purity, 96% radiochemical
yield). HPLC analysis: Phenomenex Curosil PFP, 80A/20B isocratic,
tR=5.41 min, 95.7% radiochemical purity. The identity of 20 was established
by co-elution with an authentic sample obtained from Merck Basic Chemistry.
[3,5-dichlorophenyl-U-14C]Methyl (2S)-2-({(2S)-1-[(3,5-dichlorophenyl)
sulfonyl]-azetidin-2-yl }carbonylamino)-3-[4-(2,6-dimethoxyphenyl)phenyl]
propanoate (21)
A benzene solution of 6 (1.2 mCi, 0.02 mmol) was dried in vacuo. A solution of
16 (8.3 mg, 0.02 mmol) in methylene chloride (240 ml), tetrahydrofuran (100 ml)
and diisopropylethylamine (50 ml) was added to the residue at 08C, with
stirring. A catalytic amount of 4-dimethylaminopyridine was also added. The
reaction mixture was allowed to reach room temperature slowly and was then
stirred overnight. The solvent was removed in vacuo, and the residue dissolved
in methanol to give 1.2 mCi of 21 (100% radiochemical yield). HPLC analysis:
Phenomenex Curosil PFP, 80A/20B isocratic, tR=5.01 min, 99.7% radio-
chemical purity. The identity of 21 was established by co-elution with an
authentic sample obtained from Merck Basic Chemistry.
[3,5-dichlorophenyl-U-14C]Methyl (2S)-2-({(2S)-1-[(3,5-dichlorophenyl)sul-
fonyl]-2-methylazetidin-2-yl }carbonylamino)-3-[4-({4-[(tert-butyl)oxycarbo-
nyl]piperazinyl}-carbonyl)phenyl]propanoate (22)
The reaction of 6 (1.9 mCi, 0.032 mmol) with 17 (17 mg, 0.035 mmol),
following the procedure described for the preparation of 21, yielded 1.9 mCi
of 22 (87.8% radiochemical purity, 88% radiochemical yield). HPLC analysis:
Zorbax Rx-C18, 70A/30C isocratic, tR=7.53 min, 87.8% radiochemical
purity. The identity of 22 was established by co-elution with an authentic
sample obtained from Merck Basic Chemistry.
[3,5-dichlorophenyl-U-14C]Methyl
sulfonyl]-azetidin-2-yl}carbonylamino)-3-[4-({4-[(tert-butyl)oxycarbonyl]
piperazinyl }carbonyl)-phenyl]propanoate (23)
(2S)-2-({(2S)-1-(3,5-dichlorophenyl)
The reaction of 6 (2.0 mCi, 0.033 mmol) with 18 (17 mg, 0.036 mmol),
following the procedure described for the preparation of 21, yielded 2.0 mCi
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of 23 (78.4% radiochemical purity, 78% radiochemical yield). HPLC analysis:
Phenomenex Curosil PFP, 80A/20B isocratic, tR=4.22 min, 78.4% radio-
chemical purity. The identity of 23 was established by co-elution with an
authentic sample obtained from Merck Basic Chemistry.
[3,5-dichlorophenyl-U-14C]Methyl (3R)-3-({(2S)-1-[(3,5-dichlorophenyl)
sulfonyl]-azetidin-2-yl }carbonylamino)-3-[4-({4-[benzyloxycarbonyl]piperazi-
nyl)carbonyl)-phenyl]propanoate (24)
The reaction of 6 (2.7 mCi, 0.045 mmol) with 19 (25 mg, 0.050 mmol),
following the procedure described for the preparation of 21, yielded 2.7 mCi
of 24 (94.0% radiochemical purity, 94% radiochemical yield). HPLC analysis:
Zorbax Rx-C18, 60A/40B isocratic, tR=8.52 min, 94.0% radiochemical
purity. The identity of 24 was established by co-elution with an authentic
sample obtained from Merck Basic Chemistry.
[3,5-dichlorophenyl-U-14C](2S)-2-({(2S)-1-[(3,5-Dichlorophenyl)sulfonyl]-
2-methyl-azetidin-2-yl}carbonylamino)-3-[4-(2,6-dimethoxyphenyl)phenyl]-
propanoic acid (1)
To a solution of methyl ester 20 (8.8 mCi, 95.7% radiochemical purity,
0.14 mmol) in methanol (1.9 ml) and tetrahydrofuran (1.9 ml) was added a
solution of lithium hydroxide monohydrate (45 mg) in water (1.9 ml) at 08C.
After stirring at 08C for 1 h, the solvent was removed in vacuo, and the residue
extracted with ethyl acetate and water. The combined organic layers were
dried with anhydrous sodium sulfate, filtered, counted (8.8 mCi) and analyzed
by HPLC (Phenomenex Curosil PFP, 80A/20B isocratic, tR=4.52 min, 95.3%
radiochemical purity). The tracer was purified by preparative HPLC (Zorbax
Rx-C18, 21.2 Â 250 mm, 215/254 nm, 49A/51B isocratic, flow rate =20 ml/
min) to give 5.3 mCi of 1 (63% radiochemical yield), which was isolated by
solid phase extraction and stored in acetonitrile. HPLC analysis: Zorbax Rx-
C18, 60A/40B isocratic, tR=10.72 min, 100% radiochemical purity. 1H NMR
(CD3OD) d 7.85 (d, 2H), 7.77 (t, 1H), 7.72 (br d, 1H), 7.23 (t, 1H), 7.16 (ABq,
4H), 6.67 (d, 2H), 4.75 (m, 1H), 3.91 (m, 1H), 3.62 (s, 6H), 3.51 (m, 1H), 3.38
(dd, 1H), 3.06 (dd, 1H), 2.18 (m, 1H), 2.03 (m, 1H), 1.62 (s, 3H). MS
Calculated for 1 [M+H+]: 607, Found: 607. The identity of 1 was also
established by co-elution with an authentic sample obtained from Merck Basic
Chemistry.
[3,5-dichlorophenyl-U-14C](2S)-2-({(2S)-1-[(3,5-Dichlorophenyl)sulfonyl]
azetidin-2-yl }carbonylamino)-3-[4-(2,6-dimethoxyphenyl)phenyl]propanoic
acid (2)
The saponification of 21 (1.2 mCi, 99.7 radiochemical purity, 0.02
mol), following the procedure described for the preparation of 1,
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yielded 1.2 mCi of 2 (96.9% radiochemical purity, 97% radiochemical
yield). HPLC analysis: Phenomenex Curosil PFP, 80A/20B iso-
cratic, tR=4.32 min, 96.9% radiochemical purity. MS Calculated for
2 [M+H+]: 593, Found: 593. The identity of 2 was established by
co-elution with an authentic sample obtained from Merck Basic
Chemistry.
[3,5-dichlorophenyl-U-14C](2S)-2-({(2S)-1-[(3,5-Dichlorophenyl)sulfonyl]-
2-methyl-azetidin-2-yl }carbonylamino)-3-[4-({4-[(tert-butyl)oxycarbonyl]
piperazinyl }carbonyl)-phenyl]propanoic acid (25)
The saponification of 22 (1.9 mCi, 87.8% radiochemical purity, 0.028 mmol),
following the procedure described for the preparation of 1, yielded 1.9 mCi of
25 (86.7% radiochemical purity, 99% radiochemical yield), which was used in
the next reaction without purification. HPLC analysis: Zorbax Rx-C18, 50A/
50C isocratic, tR=11.03 min, 86.7% radiochemical purity. The identity of 25
was established by co-elution with an authentic sample obtained from Merck
Basic Chemistry.
[3,5-dichlorophenyl-U-14C](2S)-2-({(2S)-1-[(3,5-Dichlorophenyl)sulfonyl]
azetidin-2-yl }carbonylamino)-3-[4-({4-[(tert-butyl)oxycarbonyl]piperazinyl}-
carbonyl)phenyl]propanoic acid (26)
The saponification of 23 (2.0 mCi, 78.4% radiochemical purity, 0.026
mmol), following the procedure described for the preparation of 1,
yielded 2.0 mCi of 26 (78.0% radiochemical purity, 99% radiochemical
yield), which was used in the next reaction without purification.
HPLC analysis: Phenomenex Curosil PFP, 80A/20B isocratic, tR=5.25 min,
78.0% radiochemical purity. The identity of 26 was established by
co-elution with an authentic sample obtained from Merck Basic
Chemistry.
[3,5-dichlorophenyl-U-14C](3R)-3-({(2S)-1-[(3,5-Dichlorophenyl)sulfonyl]
azetidin-2-yl }carbonylamino)-3-[4-({4-[benzyloxycarbonyl]piperazinyl}carbo-
nyl)phenyl]propanoic acid (27)
The saponification of 24 (2.7 mCi, 94.0% radiochemical purity, 0.042
mmol), following the procedure described for the preparation of 1,
yielded 2.1 mCi of 27 (97.2% radiochemical purity, 80% radiochemical
yield), which was used in the next reaction without purification.
HPLC analysis: Zorbax Rx-C18, 50A/50B isocratic, tR=10.62 min,
97.2% radiochemical purity. The identity of 27 was established by
co-elution with an authentic sample obtained from Merck Basic
Chemistry.
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[3,5-dichlorophenyl-U-14C](2S)-2-({(2S)-1-[(3,5-Dichlorophenyl)sulfonyl]-
2-methyl-azetidin-2-yl }carbonylamino)-3-[4-(piperazinylcarbonyl)phenyl]
propanoic acid (3)
A solution of 25 (1.9 mCi, 86.7% radiochemical purity, 0.027 mmol) in ethyl
acetate (5 ml) was saturated with hydrogen chloride gas by bubbling. The
reaction mixture was stirred at room temperature for 15 min, and then the
solvent removed in vacuo. The residue was dissolved in acetonitrile, counted
(0.9 mCi), and analyzed by HPLC (Zorbax Rx-C18, 50A/50C isocratic,
tR=3.21, 97.5% radiochemical purity). The tracer was purified by preparative
HPLC (Zorbax Rx-C18, 21.2 Â 250 mm, 215/254 nm, 25A/75C isocratic, flow
rate =20 ml/min) to give 0.59 mCi of 3 (35% radiochemical yield), which was
isolated as an acetonitrile solution after solid phase extraction. HPLC analysis:
Zorbax Rx-C18, 29A/71C isocratic, tR=14.82 min, 99.8% radiochemical
1
purity. H NMR (CD3OD) d 7.85 (br d, 1H), 7.82–7.78 (m, 3H), 7.40 (ABq,
4H), 4.75 (m, 1H), 3.91 (m, 1H), 3,83 (br, 4H), 3.70 (m, 1H), 3.35 (dd, 1H),
3.25 (br, 4H), 3.19 (dd, 1H), 2.42 (m, 1H), 2.09 (m, 1H), 1.55 (s, 3H). MS
Calculated for 3 [M+H+]: 583, Found: 583. The identity of 3 was also
established by co-elution with an authentic sample obtained from Merck Basic
Chemistry.
[3,5-dichlorophenyl-U-14C](2S)-2-({(2S)-1-[(3,5-Dichlorophenyl)sulfonyl]
azetidin-2-yl }carbonylamino)-3-[4-(piperazinylcarbonyl)phenyl]propanoic
acid (4)
A solution of 26 (2.0 mCi, 78.0% radiochemical purity, 0.026 mmol) in ethyl
acetate (10 ml) was saturated with hydrogen chloride gas by bubbling. The
reaction mixture was stirred at room temperature for 15 min, and then the
solvent removed in vacuo. The residue was dissolved in acetonitrile, counted
(2.0 mCi), and analyzed by HPLC (Phenomenex Curosil PFP, 20A/80B to
50A/50B gradient over 15 min, tR=12.6, 35.1% radiochemical purity). The
tracer was purified by preparative HPLC (Zorbax Rx-C18, 21.2 Â 250 mm,
215/254 nm, 23A/77B isocratic, flow rate=20 ml/min) to give 0.34 mCi of 4
(22% radiochemical yield), which was isolated as an acetonitrile solution after
solid phase extraction. HPLC analysis: Zorbax Rx-C18, 30A/70B isocratic,
tR=7.22 min, 100% radiochemical purity. MS Calculated for 4 [M+H+]: 569,
Found: 569. The identity of 4 was established by co-elution with an authentic
sample obtained from Merck Basic Chemistry.
[3,5-dichlorophenyl-U-14C](3R)-3-({(2S)-1-[(3,5-Dichlorophenyl)sulfonyl]
azetidin-2-yl }carbonylamino)-3-[4-(piperazinylcarbonyl)phenyl]propanoic
acid (5)
Acid 27 (2.1 mCi, 97.2% radiochemical purity, 0.034 mmol) was dissolved in 2-
propanol (8 ml) and 1 M hydrochloric acid (2 ml). Platinum(IV) oxide (8 mg)
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LABELED VLA-4 ANTAGONISTS
125
was added, and the reaction mixture stirred at room temperature under
hydrogen gas (1 atm) for 1 h. Then the mixture was filtered through celite,
rinsed with 2-propanol, counted (2.0 mCi), and analyzed by HPLC analysis
(Zorbax Rx-C8, 50A/50B isocratic, tR=7.12, 43.2% radiochemical purity).
The tracer was purified by preparative HPLC (Waters XTerra RP-18,
19 Â 300 mm, 215/254 nm, 25A/75C isocratic, flow rate=20 ml/min) to give
0.6 mCi of 5 (29% radiochemical yield), which was isolated as an acetonitrile
solution after solid phase extraction. HPLC analysis: Waters XTerra RP-18,
1
30A/70C isocratic, tR=13.42 min, 99.0% radiochemical purity. H NMR
(CD3OD) d 8.09 (s, 1H), 7.84 (s, 2H), 7.50 (ABq, 4H), 5.39 (m, 1H), 4.50 (m,
1H), 4.0–2.6 (m, 12H), 2.26 (m, 2H). MS Calculated for 3 [M+H+]: 569,
Found: 569. The identity of 5 was also established by co-elution with an
authentic sample obtained from Merck Basic Chemistry.
Acknowledgements
We would like to acknowledge Noel Byrne, Yolanda E. Jakubowski, Herb J.
Jenkins, Anson Chang and Allen N. Jones for analysis of the tracers.
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