Synthesis of covalent [14C]-labeled diarylsulfonylurea (DASU) inhibitors of the processing and release of IL-1

Article abstract of DOI:10.1002/jlcr.602

CP-452,759 and CP-470,947, two covalent [¹⁴C]-labeled diarylsulfonylurea (DASU) inhibitors of the processing and release of IL-1 from human monocytes, have been synthesized. The radiosyntheses utilize [¹⁴C]-labeled phosgene to prepar

Full text of DOI:10.1002/jlcr.602

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 785–794.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.602
Research Article
Synthesis of covalent [14C]-labeled
diarylsulfonylurea (DASU) inhibitors
of the processing and release of IL-1
J. F. Eggler, C. A. Gabel*, K. Zandi, J. D. Weaver, H. McKechney,
M. A. Dombroski, H. Peurano and N. Hawryluk
Pfizer Global Research and Development, Pfizer Inc, Groton, Connecticut 06340,
USA
Summary
CP-452,759 and CP-470,947, two covalent [14C]-labeled diarylsulfonylurea
(DASU) inhibitors of the processing and release of IL-1 from human
monocytes, have been synthesized. The radiosyntheses utilize [14C]-labeled
phosgene to prepare labeled isocyanates which are coupled with an epoxide
sulfonamide to give the [14C]-DASUs. These tools should allow future studies
aimed at purifying and identifying the DASU binding protein involved
in the inhibition of the processing and release of IL-1 from human
monocytes. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: [14C]phosgene; [14C]Àlabeled covalent DASUs; IL-1 inhibitors
Introduction
The cytokine interleukin-1 (IL-1) is a key mediator in the inflammatory
response.1 Levels of this cytokine are elevated in synovial fluid isolated
from inflamed joints of patients with osteoarthritis and rheumatoid
arthritis.2 Furthermore, repeated injection of IL-1 into the joints of
normal rats results in chronic synovitis.3 Aberrant production of this
*Correspondence to: C. A. Gabel, Central Research Division, Eastern Point Road, Pfizer Inc,
Groton, CT 06340, USA. E-mail: christopher a gabel@groton.pfizer.com
Copyright # 2002 John Wiley & Sons, Ltd.
Received 9 January 2002
Revised 22 March 2002
Accepted 16 April 2002

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J. F. EGGLER ET AL.
Cl
OH
H
N
SO2NHCONH
OMe O
SO2NHCONH
GLYBURIDE (IC50 = 15 uM)
CP-424174 (IC50 = 210 nM)
O
O
SO2NHCONH
SO2NHCONH
Cl
F
F
CP-452759 (IC50 = 150 nM)
CP-470947 (IC50 = 100 nM)
Fig. 1. Glyburide and Diarylsulfonylurea (DASU) IL-1 inhibitor IC50S
cytokine is believed to play a major role in inflammatory disease; thus,
modulation of IL-1 is an attractive target for drug research. In an
in vitro assay,4 where maturation and release of IL-1 is induced by
ATP-treatment of human monocytes, we5 and others6 have shown that
glyburide is inhibitory. Extensive SAR studies around glyburide led to
the discovery of potent DASU inhibitors, exemplified by CP-424,174,5
that blocked processing and release of IL-1. However, the mechanism
by which these agents blocked stimulus-coupled processing was not
known. A radiolabeled covalent DASU inhibitor (a radiolabeled
inhibitor containing a reactive functionality) was therefore critical for
pharmacologic studies aimed at identifying DASU binding proteins
involved in the inhibition of IL-1 post-translational processing. This
paper describes the synthesis of two [14C]-labeled DASU epoxide
inhibitors, CP–452,759 and CP-470,947, which should facilitate
identification and characterization of DASU binding proteins involved
in the inhibition of processing and release of IL-1 from human
monocytes (Figure 1).
Results and discussion
Preparation of the [14C]DASUs (Scheme 1)
[14C]CP-452,759 (0.84 mCi at 55 mCi/mmol) and [14C]CP-470,947
(0.58 mCi, 55 mCi/mmol) were prepared by treatment of excess
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 785–794

LABELING OF DASU INHIBITORS
787
O
O
O
SO2NH2
O C
N
Cl
SO2NHCONH
Cl
a
a
+
*
*
F
F
[14C]CP-452759
1
1
2
SO2NH2
O
O
C N
*
+
SO2NHCONH
F
*
F
3
[14C]CP-470947
(a) NaH/THF
* position of label
Scheme 1.
b
b
H2N
Cl
O C
N
Cl
H2N
O
C
*
N
*
4
2
5
3
(b) [14C]phosgene/TEA/toluene
*position of label
Scheme 2.
2-fluoro-5-oxiranyl-benzenesulfonamide (1) with 5-chloro-2-[14C]-
isocyanato-1,3-diisopropylbenzene (2) (1.1 mCi) and 4-[14C]-isocyana-
to-1,2,3,5,6,7-hexahydro-s-indacene (3) (0.9 mCi), respectively, using
sodium hydride in THF.
Preparation of [14C]isocyanates (Scheme 2)
Treatment of commercially available 2,6-diisopropylphenylamine with
NCS/DMF gave 4 in quantitative yield. Treatment of [14C]phosgene
(5 mCi at 55 mCi/mmol) with triethylamine and excess 4-chloro-2,6-
diisopropylphenylamine (4) in warm toluene for 1 hr gave 5-chloro-2-
[14C]-isocyanato-1,3-diisopropylbenzene (2) in quantitative yield (5 mCi).
Longer reaction times lead to lower yields.
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J. F. EGGLER ET AL.
O
O
O
b, c, d, e
O
O
a
f
Br
F
SO2NH2
F
Br
F
6
7
8
O
O
O
O
g
SO2NH2 h, i
SO2NH2
F
SO2NH2
F
Br
Br
F
10
9
1
(a) TsOH, ethylene glycol/toluene, (b) BuLi/THF, (c) SO2, (d) NCS/CH2Cl2,
(e) NH4OH (f) phenyltrimethylammonium tribromide/CH3CN, (g) aqueous HCl/
dioxane, (h) NaBH4/MeOH, (i) NaOH
Scheme 3.
1,2,3,5,6,7-Hexahydro-sindacen-4-ylamine (5)7 was more reactive,
and when treated with [14C]phosgene (35 mCi at 55 mCi/mmol) in
toluene, the reaction was complete in 45 min at room temperature.
Evaporation of the solvent in vacuo resulted in the isolation of
significant amounts of a white solid, which did not dissolve in hexanes.
After silica gel filtration, 9 mCi of [14C] 3 was isolated (>99%
radiochemically pure by radio-TLC).
The synthesis of 2-fluoro-5-oxiranyl-benzenesulfonamide (1) is shown
in Scheme 3. Dean-Stark distillation of a toluene solution of
commercially available 1-(3-bromo-4-fluoro-phenyl)-ethanone (6), ethy-
lene glycol and a trace of p-toluenesulfonic acid gave 2-(3-bromo-4-
fluoro-phenyl)-2-methyl-[1,3]dioxolane (7). Successive treatment of 7
with n-butyllithium in tetrahydrofuran at À788C, sulfur dioxide, N-
chlorosuccinimide in dichloromethane and aqueous ammonium hydro-
xide gave 2-fluoro-5-(2-methyl-[1,3]dioxolan-2-yl)-benzenesulfonamide
(8). Bromination of 8 with phenyltrimethyl-ammonium tribromide in
acetonitrile gave 5-(2-bromomethyl-[1,3]dioxolan-2-yl)-2-fluoro-benze-
nesulfonamide (9) which was treated with aqueous hydrochloric acid in
dioxane to give 5-bromoacetyl-2-fluoro-benzenesulfonamide (10). Re-
duction of 10 with sodium borohydride in methanol, followed by
treatment with dilute sodium hydroxide gave 1.
Experimental
Melting points were taken on a Thomas–Hoover melting point
1
apparatus and are uncorrected. H NMR spectra were obtained on
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 785–794

LABELING OF DASU INHIBITORS
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Bruker (AM 300) or Varian (XL250 or T69) instruments. Routine mass
spectral data were obtained using either a Hewlett-Packard 5989 MS
Engine operated with a particle beam interface and ammonia chemical
ionization (PB/CI) or a VG/Fisons Instruments Platform II spectro-
meter operating with an atmospheric pressure chemical ionization
(APCI) source. [14C] Phosgene was purchased from Amersham. All
reactions were run under a nitrogen atmosphere.
2-(3-Bromo-4-fluoro-phenyl)-2-methyl-[1,3]dioxolane (7)
A solution of 1-(3-bromo-4-fluoro-phenyl)-ethanone (6) (25 g, 0.115 mol)
(Fluorochem), ethylene glycol (20 ml, 0.345 mol) and a trace of p-
toluenesulfonic acid in 200 ml of toluene was heated at reflux for 12 h.
with Dean–Stark separation of water. The reaction was cooled to room
temperature, washed with a dilute sodium bicarbonate solution, dried
over anhydrous Na2SO4 and the solvent was evaporated to give 30 g of 7
(100%) as an oil TLC: CH2Cl2 single spot material Rf 0.40 1H-
NMR (300 MHz, DMSO-D6) d: (1.52, s, 3 H), (3.67, m, 2 H), (3.95, m,
2 H), (7.34, m, 1 H), (7.40, m, 1 H), (7.63, m, 1 H).
*
*
2-Fluoro-5(2-methyl-[1,3]dioxolan-2-yl)-benzenesulfonamide (8)
n-BuLi [1.6 M in hexane, 50 ml (0.08 mol)] was added dropwise to a
solution of 2-(3-bromo-4-fluoro-phenyl)-2-methyl-(1,3)dioxolane (7)
(20.88 g, 0.08 mol) in 200 ml THF at À788C. After stirring for 2 h at
À788C, SO2 was bubbled in for 15 min. The reaction was allowed to
warm to room temperature and stirred overnight. A solution of NCS
(10.8 g, 0.08 mol) in 150 ml of CH2Cl2 was added and the reaction
stirred for 1 h. The volatiles were evaporated in vacuo, and the brown
residue was slurried with CH2Cl2 and filtered. The filtrate was treated
with 200 ml of 30% NH4OH and stirred for 3 h. The CH2Cl2 layer was
separated, dried over anhydrous Na2SO4 and evaporated. The residue
was triturated with CH2Cl2 and filtered to give 8.1 g of 8 (39%); mp
*
*
149–1508C TLC: CH2Cl2/MeOH:9/1 v/v single-spot material Rf 0.5
1H NMR (300 MHz, DMSO-D6) d: (1.60, s, 3 H), (3.70, m, 2 H), (4.05,
m, 2 H), (7.41, t, 1 H), (7.71, broad s, 3 H), (7.84, dd, 1 H).
5-(2-Bromomethyl-[1,3]dioxolan-2-yl)-2-fluoro-benzenesulfonamide (9)
A solution of phenyltrimethylammonium tribromide (8.4 g, 0.0225 mol)
in 20 ml of CH3CN was added dropwise to a solution of 2-fluoro-5
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J. F. EGGLER ET AL.
(2-methyl-[1,3]dioxolan-2-yl)-benzenesulfonamide (8) (5.2 g, 0.022 mol)
in 100 ml of CH3CN. The reaction was stirred at room temperature for
1 h. and the solvent was evaporated in vacuo. The residue was dissolved
in EtOAc, washed with water, dried over anhydrous Na2SO4 and
evaporated. The residue was purified by column chromatography on
silica gel eluting with CH2Cl2/Et2O:9/1 v/v to give 4.6 g of 9 (62%) as an
1H
*
*
oil
TLC: CH2Cl2/Et2O:9/1 v/v single-spot material Rf 0.30
NMR (300 MHz, CDCl3) d: (3.68, s, 2 H), (3.80, m, 2 H), (4.21, m, 2 H),
(5.15, broad s, 2 H), (7.26, t, 1 H), (7.75, m, 1 H), (8.15, dd, 1 H).
5-Bromoacetyl-2-fluoro-benzenesulfonamide (10)
A solution of 5-(2-bromomethyl-[1,3]dioxolan-2-yl)-2-fluoro-benzene-
sulfonamide (9) (1.5 g, 4.4 mmol) in 50 ml of dioxane and 10 ml of 2N
HCl was heated at reflux for 5 h then cooled to room temperature. The
solvent was evaporated in vacuo and the residue dissolved in EtOAc,
washed with H2O and dried over anhydrous Na2SO4. The residue was
purified by column chromatography on silica gel eluting with EtOAc to
give 900 mg of material which was recrystallized from MeOAc to
*
give 700 mg 10 (54%); mp 153–1588C
TLC: CH2Cl2/Et2O:7/3 v/v
1H NMR (300 MHz, DMSO-D6)
*
single-spot material Rf 0.60
d: (5.20, s, 2 H), (7.63, t, 1 H), (7.86, broad s, 2 H), (8.32, m, 2 H).
2-Fluoro-5-oxiranyl-benzenesulfonamide (1)
A solution of 5-bromoacetyl-2-fluoro-benzenesulfonamide (10) (0.5 g,
1.7 mmol) in 20 ml MeOH was cooled to 08C. NaBH4 (26 mg,
0.67 mmol) was added and the reaction was stirred at 08C for 15 min.
Four milliliter of 1N NaOH was added and the reaction was stirred at
08C for 4 h. The reaction was adjusted to pH 6 by addition of dilute
HCl. The volatiles were evaporated and the residue dissolved in EtOAc,
washed with H2O, dried over anhydrous Na2SO4 and evaporated. The
product was purified by column chromatography on silica gel eluting
*
with CH2Cl2/MeOH:9/1 v/v to give 180 mg of 1 (48%); mp 100–1038C
TLC: CH2Cl2/MeOH:9/1 v/v single-spot material Rf 0.65 1H-NMR
*
(300 MHz, DMSO-D6) d: (2.82, m, 1 H), (3.14, m, 1 H), (4.06, m, 1 H),
(7.40, t, 1 H), (7.56, m, 1 H), (7.66, m, 3 H).
4-Chloro-2-isocyanato-1,3-diisopropyl-benzene (2) (non-labeled)
To a solution of 4-chloro-2,6-diiopropyl-phenylamine (4) (8.9 g,
0.042 mol) and TEA (4.66 g, 0.046 mol) in 125 ml of THF was added a
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LABELING OF DASU INHIBITORS
791
1.93 M solution of phosgene in toluene (23.8 ml, 0.046 mol). The
reaction was stirred at room temperature for 15 min, then at 708C for
15 min. The solvents were evaporated in vacuo. The residue was
dissolved in hexane and purified on a plug of silica gel eluting with 10%
v/v CH2Cl2/hexane to give 9.45 g of non-labeled 2 (95%) as an
1H-
*
*
oil
TLC: Hex/CH2Cl2:2/1 v/v single spot material Rf 0.70
NMR (300 MHz, CDCl3) d: (1.20, d, 12 H), (3.19, septet, 2 H), (7.06, s, 2 H).
4-Isocyanato-1,2,3,5,6,7-hexahydro-s-indacene (3) (non-labeled)
To a solution of 1,2,3,5,6,7-hexahydro-s-indacen-4-ylamine (5)7 (1.77 g,
0.01 mol) and TEA (1.11 g, 0.011 mol) in 25 ml of THF was added a
1.93 M solution of phosgene in toluene (5.7 m, 0.011 mol). The reaction
was stirred at room temperature for 15 min and at 708C for 15 min. The
solvents were evaporated in vacuo. The residue was dissolved in hexane
and purified on a plug of silica gel eluting with 10% v/v CH2Cl2/hexane
*
*
to give 1.79 g of non-labeled 3 (90%); mp 40–418C
TLC: Hex/
1H-NMR (300 MHz,
*
CH2Cl2:2/1 v/v single-spot material Rf 0.70
CDCl3) d: (2.13, t, 4 H), (2.85, m, 8 H), (6.93, s, 1 H).
CP-452,759 (non-labeled)
Sodium hydride [60% dispersion in mineral oil, (23 mg, 0.57 mmol)] was
added to a solution of 2-fluoro-5-oxiranyl-benzenesulfonamide (1)
(113 mg, 0.52 mmol) and 4-chloro-2-isocyanato-1,3-diisopropyl-benzene
(2) (136 mg, 0.57 mmol) in 10 ml of THF. After stirring at room
temperature for 3 h, the solvent was evaporated in vacuo. The residue
was acidified with 2N HCl and extracted with EtOAc. The EtOAc layer
was dried over anhydrous Na2SO4 and evaporated in vacuo. The residue
was purified by column chromatography on silica gel eluting with
CH2Cl2/MeOH:9/1 v/v to give 190 mg of CP-452,759 (81%); mp 126–
1308C TLC: CH2Cl2/MeOH:9/1 v/v single-spot material Rf 0.70 1H-
NMR (300 MHz, DMSO-D6) d: (0.97, m, 12 H), (2.76, m, 1 H), (2.93, m,
1 H), (3.10, m, 1 H), (4.05, m, 1 H), (7.04, s, 2 H), (7.35, m, 1 H), (7.50,
m, 1 H), (7.77, m, 2 H).
*
*
CP-470,947 (non-labeled)
Sodium hydride [60% dispersion in mineral oil, 30 mg (0.75 mmol)] was
added to a solution of 2-fluoro-5-oxiranyl-benzenesulfonamide (1)
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J. F. EGGLER ET AL.
(148 mg, 0.68 mmol) and 4-isocyanato-1,2,3,5,6,7-hexahydro-s-indacene
(3) (150 mg, 0.75 mmol). After stirring at room for 12 h, the solvent was
evaporated in vacuo. The residue was acidified with 2N HCl, then
extracted with EtOAc. The EtOAc layer was dried over anhydrous
Na2SO4 and evaporated in vacuo. The residue was triturated with
*
hexane and filtered to give 210 mg of CP-470,947 (75%); mp 97–1028C
TLC: CH2Cl2/MeOH:9/1 v/v single-spot material Rf 0.65 1H-NMR
*
(300 MHz, DMSO-D6) d : (1.96, m, 4 H), (2.47, m, 4 H), (2.74, m, 5 H),
(3.11, m, 1 H), (4.06, m, 1 H), (6.90, s, 1 H), (7.49, t, 1 H), (7.61, m, 1 H),
(7.78, dd, 1 H), (8.05, s, 1 H).
4-Chloro-2-[14C]-isocyanato-1,3-diisopropylbenzene (2)
4-Chloro-2,6-diisopropylphenylamine (4) hydrochloride (0.045 g,
0.18 mmol, 2 eq) was dispersed in ether, and triethylamine (57 ml,
0.41 mmol, 4.5 eq) was added. The solution was filtered through a
cotton plug, and most of the ether evaporated in vacuo. The residual
clear colorless oil was taken up in dry toluene (3 ml) and fitted to a
vacuum manifold. The solution was degassed, cooled in a liquid
nitrogen bath and evacuated. An ampoule containing [14C]phosgene gas
(5 mCi at 55 mCi/mmol) was fitted to the manifold, and the system
evacuated. The manifold was then isolated from the pump, the ampoule
broken, and the [14C]phosgene allowed to vacuum transfer into the
cooled reaction vessel. After 20 min, the cold bath was removed, and the
reaction mixture warmed to room temperature. It was then backflushed
with nitrogen, and heated to 808C for 1 h, after which time, no starting
material remained (radio-TLC). The mixture was cooled to room
temperature and concentrated in vacuo. The yellow oil was dissolved in
hexanes and filtered through a small plug of silica gel (hexanes) to give
5 mCi 5-chloro-2-[14C]-isocyanato-1,3-diisopropylbenzene (2), >99%
radiochemically pure by radio-TLC (hexanes).
4-[14C]-isocyanato-1,2,3,5,6,7-hexahydro-s-indacene (3)
[14C]Phosgene (35 mCi at 57 mCi/mmol) as a solution in toluene, 35 ml
was cooled to 08C, and triethylamine (0.50 ml, 3.5 mmol, 6 eq) and
1,2,3,5,6,7-hexahydro-s-indacen-4-ylamine (5)7 (0.17 g, 0.1 mmol, 1.6 eq)
were added as a solution in toluene (2 ml). After 15 min, the reaction
was removed from the ice bath and let warm to room temperature over
30 min after which time radio-TLC analysis indicated desired product
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LABELING OF DASU INHIBITORS
793
was present in 93%. The reaction mixture was concentrated in vacuo,
dissolved in hexanes and filtered through a plug of silica gel (hexanes).
Concentration of the filtrate gave 9 mCi 4-[14C]-isocyanato-1,2,3,
5,6,7-hexahydro-s-indacene (3), which was >99% pure by radio-TLC
(hexanes).
[14C]-CP-452,759
The 5-Chloro-2-[14C]-isocyanato-1,3-diisopropylbenzene (2) (1.08 mCi
at 55 mCi/mmol) and 2-fluoro-5-oxiranylbenzenesulfonamide (1)
(95 mg, 0.022 mmol, 1.1 eq) were dissolved in freshly distilled THF
and sodium hydride (2 mg) was added. After 5 min, radio-TLC
indicated complete reaction. The reaction mixture was concentrated in
vacuo, dissolved in water (5 ml) and extracted with ether. The aqueous
phase was then acidified with 2N HCl, and extracted with ethyl acetate.
The combined ethyl acetate layers were dried, filtered, concentrated and
the residue chromatographed (silica gel, EtOAc) to give 0.84 mCi
[14C]CP-452,759 (>99% pure radio-TLC, EtOAc).
[14C]-CP-470,947
4-[14C]-Isocyanato-1,2,3,5,6,7-hexahydro-s-indacene (3) (0.9 mCi at
57 mCi/mmol) was dissolved in THF (5 ml), and 2-fluoro-5-oxiranyl-
benzenesulfonamide (1) (4.2 mg, 0.19 mmol, 1.2 eq) was added, followed
by sodium hydride (2 mg). After 15 min, no starting material remained
by radio-TLC. The reaction mixture was concentrated in vacuo, and the
residue was dispersed in 0.2 M HCl (5 ml) and extracted with ethyl
acetate (3 Â 5 ml). The combined organic layers were dried, filtered,
concentrated and the residue chromatographed (EtOAc) to give
0.58 mCi [14C]CP-470,947 (>99% pure by radio-TLC, EtOAc).
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J Label Compd Radiopharm 2002; 45: 785–794