Synthesis of deuterium, tritium, and carbon-14 labeled BIRB 796, a p38 MAP kinase inhibitor

Article abstract of DOI:10.1002/jlcr.873

1-(5-tert-Butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy) naphthalen-1-yl]urea (BIRB 796), currently in clinical trials for the treatment of inflammatory diseases, is a potent inhibitor of p38 MAP kinase. Labeled BIRB 796 with stable and r

Full text of DOI:10.1002/jlcr.873

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 847–856.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.873
Research Article
Synthesis of deuterium, tritium, and carbon-14 labeled
BIRB 796, a p38 MAP kinase inhibitor
Bachir Latli*
Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Research
and Development Center, 900 Ridgebury Road, Ridgefield, CT 06877, USA
Summary
1-(5-tert-Butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-
1-yl]urea (BIRB 796), currently in clinical trials for the treatment of inflammatory
diseases, is a potent inhibitor of p38 MAP kinase. Labeled BIRB 796 with stable and
radioactive isotopes was required for metabolism, distribution, and absorption
studies. We first report the synthesis of carbon-14 labeled BIRB 796 with a specific
activity of 2 GBq/mmol (54.2 mCi/mmol), using [14C]-phosgene under modified
Schotten–Baumann conditions; second the preparation of tritium-labeled BIRB 796
with a specific activity of 659 GBq/mmol (17.81 Ci/mmol) by reductive dehalogenation
2
of iodo-BIRB 796 with tritium gas; and finally, the synthesis of H8-BIRB 796 using
2
morpholine-2,2,3,3,5,5,6,6-2H8 with isotopic enrichment of 98.9 at% H. Copyright
# 2004 John Wiley & Sons, Ltd.
Key Words: p38 MAP kinase; tritium; deuterium; carbon-14; BIRB 796
Introduction
The p38 is a member of the mitogen-activated protein (MAP) kinases that
control many cellular activities.1 It is a stress responsive, and a signal-
transducing kinase.2 Inhibition of p38 MAP kinase leads to the blockage of
the synthesis of several pro-inflammatory cytokines including tumor necrosis
factor-a (TNFa) and interleukin-1b (IL-1b), which mediate the inflammatory
response associated with immunological recognition of infectious agents. The
presence of elevated levels of pro-inflammatory cytokines is associated with
several diseases.3 Therefore inhibitors of this kinase may be used as
therapeutic agents to treat chronic inflammatory diseases and offer a potential
strategy to treat several other diseases including reducing HIV expression
*Correspondence to: B. Latli, Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals,
Research and Development Center, 900 Ridgebury Road, Ridgefield, CT 06877, USA. E-mail:
blatli@rdg.boehringer-ingelheim.com
Copyright # 2004 John Wiley & Sons, Ltd.
Received 20 November 2003
Revised 16 April 2004
Accepted 14 May 2004

848
B. LATLI
Scheme 1. Synthesis of BIRB 796 and [14C]-BIRB 796. (a) sat’d NaHCO3,
Cl-CO-Cl or Cl-14CO-Cl, CH2Cl2, asterisk indicates position of isotope
when combined with standard HIV antiretroviral agents.4 Thus, the
development of p38 inhibitors is a very competitive field and the goal of
many pharmaceutical companies.5
BIRB 796 (Scheme 1), a N,N0-disubstituted urea, is a potent and a selective
inhibitor of human p38 MAP kinase with nanomolar inhibitory activity in cell
culture. It is also one of the slowest dissociating inhibitors against human p38
MAP kinase reported so far.6 BIRB 796 was found to inhibit p38 MAP kinase
by stabilizing a conformation of a kinase that is incompatible with ATP
binding.6 BIRB 796 is currently in clinical trials for the treatment of
inflammatory diseases.7
Results and discussion
N,N0-Disubstituted ureas are easily accessible from primary amines. The
starting primary amines are first activated as isocyanates,8 carbamates, for
example from the reaction of 2,2,2-trichloroethyl chloroformate and amines
(TROC-carbamates);9 as N,N0-carbonyl-imidazole10 or N,N0-carbonyl-1,2,4-
triazole derivatives11 using carbonyl diimidazole (CDI) or carbonyl di-1,2,4-
triazole (CDT), respectively. Carbon dioxide has been used to prepare ureas,12
and is an attractive way to introduce a carbon-14 on the carbonyl urea.13
Another substitute for [14C]-carbon dioxide is [14C]-phosgene, which is
available commercially as a solution in toluene. The primary disadvantages
of [14C]-phosgene are its instability and excess has to be used to avoid the
generation of symmetrical ureas. On a small scale, excess phosgene is easily
removed by evaporation. This procedure is not acceptable when using
[14C]-phosgene. However, under Schotten–Baumann conditions, excess
phosgene is hydrolyzed to carbonic acid and trapped in a basic aqueous
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LABELING OF BIRB 796, A P38 MAP KINASE INHIBITOR
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phase.14 [14C]-phosgene can also be used to prepare CDI or CDT which are
easily handled as solids and stored for longer periods of time when kept away
from moisture.10,11
In Scheme 1, good yields of the desired urea product were obtained when
phosgene was used to generate the isocyanate derivative of 1-(4-methyl-
phenyl)-3-tert-butyl-2H-aminopyrazole-HCl (1), which is then trapped by the
addition of 4-amino-1-[(2-morpholin-yl)ethoxy]naphthalene (2) to produce
BIRB 796 in 84% chemical yield and the undesired symmetrical pyrazole
urea (3) in 9% chemical yield, both yields are based on pyrazole (1). Only 1.5
equivalent of [14C]-phosgene in toluene was used to obtain the above yields.
Attempts to generate the isocyanate derivative of naphthylamine (2) and
trapping it with the pyrazolamine (1) gave the symmetrical naphthyl urea (4)
as the major product. This discrepancy in reactivity is probably due to the
different nucleophlicity of the pyrazole amine (1) and the naphthyl amine (2).
[14C]-BIRB 796 was easily separated from the symmetrical urea [14C]-(3)
by flash chromatography. Further crystallization from isopropanol gave a
material with about 99% chemical and radiochemical purities and a specific
activity of 54.2 mCi/mmol.
To prepare the tritium-labeled BIRB 796, Scheme 2, the iodo-precursor (6)
was synthesized from 1-(3-iodo-4-methylphenyl)-3-tert-butyl-2H-aminopyrazole
(5), prepared from 3-iodo-4-methylaniline according to the literature,15 and
from naphthyl amine (2) using phosgene as seen before. The conditions for
introducing the tritium atom were first investigated using reductive
dehalogenation with deuterium gas of iodo-BIRB 796 (6) in the presence of
palladium on carbon (10%) and triethylamine in ethanol. The reduction gave
the specifically labeled mono-deuterated BIRB 796. These conditions were
then applied to the synthesis of tritium-labeled BIRB 796. Tritium
incorporation was about 62% or 17.81 Ci/mmol.
In the synthesis of deuterium labeled BIRB 796, Scheme 3, morpholine-
2,2,3,3,4,4,5,5,6,6-2H8 (7) was used to prepare the right-hand side fragment
(13) according to the literature.7 Thus, [2H8]-morpholine was refluxed with
Scheme 2. Synthesis of [2H]-BIRB 796 and [3H]-BIRB 796. (a) sat’d NaHCO3,
2
COCl2, CH2Cl2, (2): (b) H2 or H2, EtOH, Et3N, 10% Pd/C
3
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850
B. LATLI
Scheme 3. Synthesis of [2H8]-BIRB 796. (a) BrCH2CH2OH, K2CO3, CH3CN;
(b) SOCl2, CHCl3; (c) t-BuOK, (t-Boc)2O, THF; (d) K2CO3, CH3CN; (e) 4 N
HCl, 1,4-dioxane; (f) sat’d NaHCO3, EtOAc; (g) sat’d NaHCO3, COCl2,
CH2Cl2, (13)
ethylene bromohydrin and potassium carbonate in acetonitrile,16 and the
product, 4-(2-hydroxyethyl) morpholine (8), was converted to the chloro-
analog (9) by refluxing in thionyl chloride and chloroform. Crystallization
from ethanol gave 1-chloro-2-morpholin-4-yl-ethane-HCl (9) in 71% overall
yield in two steps.17 4-Amino-1-naphthol (10) was first protected at the amino
group using di-tert-butyl dicarbonate, then coupled to compound (9).
Removal of the Boc-protecting group with hydrogen chloride solution
in dioxane followed by treatment with a saturated solution of sodium
bicarbonate gave the free base (13) in 38% overall yield. Compound (13) was
then reacted with the isocyanate of (1) as seen before to give [2H8]-BIRB 796.
Experimental procedures
Materials and methods
Silica gel TLC was performed for analysis with pre-coated aluminum sheets
and glass plates with fluorescent indicator (EM Separating, Gibbstown, NJ,
and Merck, Germany, respectively). Silica gel 60–200 Mesh (Nominal, I.D.,
grade 62) was obtained from EM Science (Gibbstown, NJ). Melting points
(uncorrected) were recorded on MEL-TEMP1 3.0 (Laboratory Devices Inc.,
USA). All reagents were obtained from Aldrich Chemical Co. (Milwaukee,
WI), except for phosgene, which was purchased from Fluka (Milwaukee, WI)
as a 20% solution in toluene, 4-amino-1-naphthol hydrochloride from Acros,
Copyright # 2004 John Wiley & Sons, Ltd.
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LABELING OF BIRB 796, A P38 MAP KINASE INHIBITOR
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morpholine-2,2,3,3,4,4,5,5,6,6-2H8 from CDN (Pointe-Claire, Quebec, Canada),
and [14C]-phosgene from ViTrax (Placentia, CA). Solvents were of HPLC
grade. Reverse phase separations were accomplished at 358C using HITACHI
L-6200A Intelligent pump and Radiomatic Flo-one\Beta (Packard), and
LINEAR UVIS 200 set at 254 nm and Ultima flo1 AP cocktail (Packard). The
HPLC columns were heated with an Eppendorf CH-30 column heater. The
separation was accomplished on a 3M-Z18, 4.6 ꢀ 150 mm (Cohesive Techno-
logies Inc.) using a gradient 40–85% methanol in water (both solvents contain
10 mM triethylamine) as the mobile phase. The flow rate was 1 ml/min.
Weighing operations were performed on a Mettler MT5 microbalance or on a
PG 503 Delta Range1 (Mettler Toledo) for larger scale. Liquid scintillation
counting was accomplished using a Beckman LS 5000 TA and Ready Safe
cocktail (Beckman, Fullerton, CA). Radio-TLC imaging was carried out on a
BIOSCAN System 200 Imaging Scanner using an auto-changer 1000 and
winScan software V2.1a (Bioscan Inc., Washington, DC). Evaporation of
solvents and volatile components was accomplished at reduced pressure using
.
a Buchi rotary evaporator unless stated otherwise. Mass spectra were acquired
by a Hewlett Packard auto sampler Series 1050, connected to a Micromass
Platform LCZ in the ES mode or by a Finnigan SSQ7000 mass spectrometer in
the Particle Beam-NH3-CI (PB-NH3-CI) mode. NMR spectra were recorded
with a Bruker 400 MHz DPX spectrometer using deuterated chloroform as a
solvent and tetramethylsilane as the internal standard. The proton NMR
spectrum of [14C]-BIRB 796 was recorded using a sample of 10 mg in 0.3 ml of
deuterated chloroform, placed in a Teflon tube liner (Wilmad, cat No. 6005-7).
The liner was capped with a Teflon plug and inserted into a screw cap NMR
tube (Wilmad 507-TR-8).18
Non-radioactive synthesis
Synthesis of 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-
ethoxy)naphthalen-1-yl]urea (BIRB 796), Scheme 1. To a suspension of
5-tert-butyl-2-p-tolyl-2H-pyrazol-3-ylamine-HCl (1) (443.4 mg, 1.67 mmol) in
CH2Cl2 (30 ml), stirred at room temperature, was added a saturated solution
of NaHCO3 (30 ml). The resulting two-phase clear solution was cooled to 08C
in an ice bath. Stirring was stopped and a solution of 20% phosgene in toluene
(1.43 ml, 2.5 mmol) was added via a syringe to the CH2Cl2 phase. The stirring
was then resumed and continued for 4 h at 08C. 4-(2-Morpholin-4-yl-ethoxy)-
naphthalen-1-ylamine (2) (364 mg, 1.33 mmol) was added in one portion and
the resulting mixture was stirred overnight as the ice melted. The organic phase
was transferred via a syringe to another round bottom flask, and the remaining
aqueous solution was extracted twice with methylene chloride (30 ml ꢀ 2)
(stirring vigorously at room temperature with CH2Cl2). The combined
solutions were concentrated in vacuo and the residue was purified by flash
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852
B. LATLI
chromatography on silica gel using chloroform and methanol (1%) as eluent
to give 100 mg of the symmetrical urea, 1,3-bis-(5-tert-butyl-2-p-tolyl-2H-
pyrazol-3-yl)-urea (3) and 700 mg of the desired product as an off-white solid.
The product was dissolved in 5 ml of isopropanol and about 20 mg of
decolorizing charcoal was added. The mixture was heated to boiling and then
filtered. The resulting colorless solution was crystallized upon standing at
room temperature to give 670 mg of a white powder in 95% yield based
on compound (2), or 76% based on the pyrazole amine (1). Rf=0.52 in 10%
1
MeOH/CHCl3, Rt=14.26 min. H NMR (CDCl3) d: 8.26(m, 1H), 7.81(d, J=
7.53 Hz, 1H), 7.52(m, 2H), 7.31(d, J=8.04 Hz, 1H), 6.91(m, 4H), 6.67(d,
J=8.54 Hz, 1H), 6.61(s, 1H, ex.), 6.47(s, 1H), 6.42(s, 1H), 4.27(t, J=5.52 Hz,
2H), 3.75(t, J=4.52 Hz, 4H), 2.97(t, J=5.52 Hz, 2H), 2.67(t, J=4.52 Hz, 4H),
2.27(s, 3H), 1.31(s, 9H). MS-ES+: MH+ (528, 60%), 265(100%). ES-:
526(11%). PB-NH3-CI: 230.2 (100%), MH+ (528.3, 20%).
Synthesis of mono-deuterated BIRB 796, Scheme 2
1-[5-tert-Butyl-2-(3-iodo-4-methylphenyl)-2H-pyrazol-3-yl]-3-[4-(2-morpholin-
4-yl-ethoxy)-naphthalen-1-yl]-urea (6). To a stirred solution of (5) (36 mg,
0.1 mmol) in methylene chloride (2 ml) was added a saturated solution of
NaHCO3 (2 ml) and the mixture was cooled to 08C in an ice bath. Stirring was
stopped and a 20% solution of phosgene in toluene (100 ml) was added directly
to the organic phase via a syringe. The stirring was resumed and continued for
two hours at this temperature. Compound (2) (22 mg, 0.08 mmol) was then
added and the reaction mixture was warmed to room temperature overnight.
The organic phase was separated and the aqueous layer was extracted twice
with methylene chloride (5 ml). The combined extracts were dried over
MgSO4, filtered and concentrated in vacuo to give 73 mg of a solid residue.
Purification by flash chromatography gave the product in 95% yield, Rf =0.36
in 10% MeOH/CHCl3, Rt=14.23 min (Rt=10.52 for BIRB 796) in HPLC. 1H
NMR (CDCl3) d: 8.23(d, J=7.53 Hz, 1H), 7.74(m, 1H), 7.48(m, 2H), 7.33(d,
J=8.03 Hz, 1H), 6.92 (m, 3H), 6.69(d, J=8.03 Hz, 1H), 6.60(m, 1H), 6.47(s,
1H), 6.37(s, 1H), 4.62(t, J=5.52 Hz, 2H), 3.74(t, J=3.51 Hz, 4H), 2.96(t,
J=5.52 Hz, 2H), 2.66(t, J=3.51 Hz, 4H), 2.34(s, 3H), 1.28(s, 9H). MS-ES+:
MH+ (654.3, 100%), 327.9(85%).
1-[5-tert-Butyl-2-(3-deutero-4-methylphenyl)-2H-pyrazol-3-yl]-3-[4-(2-morpholin-
4-yl-ethoxy)-naphthalen-1-yl]-urea [2H]-BIRB 796. A mixture of the above
compound (6.75 mg, 10.34 mmol), 10% Pd/C (3.5 mg), and triethylamine
(2.2 ml) in absolute ethanol (0.4 ml) was freeze degassed several times before
deuterium gas was introduced (8.78 mmol). The mixture was warmed to room
temperature and stirred for 3 h. The reaction was then diluted with 10.0 ml
of water to remove all exchangeable deuterium and then extracted with
Copyright # 2004 John Wiley & Sons, Ltd.
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LABELING OF BIRB 796, A P38 MAP KINASE INHIBITOR
853
methylene chloride. The combined methylene chloride extracts were dried over
MgSO4, filtered and concentrated in vacuo. The residue was dissolved in
acetonitrile and analyzed by HPLC. The product, Rt=10.55 min, 64% and
starting material about 36%, Rt=14.23 min, MS-ES: [2H]-BIRB 796 (MH+:
529, 60%), un-reacted iodo-BIRB 796 (MH+:654.2, 20%).
Synthesis of [2H8]-BIRB 796, Scheme 3
4-(2-Hydroxyethyl)-[2,2,3,3,5,5,6,6-2H8]morpholine (8). A mixture of
[2,2,3,3,5,5,6,6-2H8]morpholine (7) (0.5 g, 5.26 mmol), ethyl bromohydrin
(0.71 g, 5.7 mmol), and potassium carbonate (0.9 g, 6.52 mmol) in acetonitrile
(20 ml) was refluxed for 3 h. After cooling to room temperature, water was
added (10 ml) and the aqueous phase was extracted with chloroform. The
combined organic extracts were dried over MgSO4, filtered and concentrated
1
in vacuo to give 2.0 g of product, Rf=0.33 in 10% MeOH/CHCl3. H NMR
(CDCl3) d: 3.65(t, J=5.4 Hz, 2H), 2.58(t, J=5.4 Hz, 2H). MS-ES: MH+
(140.2, 100%), M+ (139.2, 50%), M+ +2 (141.2, 50%).
4-(2-Chloroethyl)-[2,2,3,3,5,5,6,6-2H8]morpholine (9). The above crude pro-
duct (8) and thionyl chloride (3 ml) in chloroform (20 ml) were refluxed for 4 h.
The solution was cooled to room temperature and concentrated in vacuo. The
resulting yellow solid residue (0.92 g) was crystallized from ethanol to give
0.72 g of a white solid as the HCl salt. Mp=174–1758C. MS-ES: MH+ (158.2,
100%), M+(157.2, 50%).
(4-Hydroxy-naphthalen-1-yl)-carbamic acid tert-butyl ester (11). A suspen-
sion of 4-amino-1-naphthol hydrochloride (10) (2.0 g, 10.2 mmol, 90% tech) in
THF (30 ml) was cooled to ꢁ708C (dry-ice-methylene chloride bath) under a
nitrogen atmosphere. Potassium-tert-butoxide (9 ml, 1.0 M solution in THF)
was added dropwise over a 10 min period. The resulting mixture was warmed
to ꢁ108C (dry-ice- ethylene glycol bath) and stirred for 1 h. The reaction was
cooled again to ꢁ708C and di-tert-butyl dicarbonate (2.47 g, 11.31 mmol) was
added in THF (10 ml). The reaction was warmed gradually to room
temperature and stirred overnight. The mixture was concentrated in vacuo,
dissolved in ethyl acetate (100 ml) and washed with water (3 ꢀ 30 ml), brine
(3 ꢀ 30 ml), dried over MgSO4, filtered and concentrated in vacuo to give 3.0 g
of crude material. Rf=0.6 in 10% MeOH/CHCl3. 1H NMR (CDCl3) d: 8.08(d,
1H), 7.85(d, 1H), 7.52(d, 1H), 7.42(t, 1H), 7.20(d, 1H), 7.01(brs, 1H), 6.52(s,
1H), 6.41(brs, 1H), 1.6(s, 9H). ES-, M- (258.1, 100%).
[4-(2-[2,2,3,3,5,5,6,6-2H8]Morpholin-4-yl-ethoxy)-naphthalen-1-yl]carbamic
acid tert-butyl ester (12). A mixture of 4-t-Boc-amino-1-naphthol (0.5 g,
2.5 mmol), the above chloroethylmorpholine (9) (667.5 mg, 2.57 mmol) and
potassium carbonate (1.3 g, 9.2 mmol) in acetonitrile (20 ml) was refluxed
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854
B. LATLI
overnight. The resulting brown mixture was cooled to room temperature and
water was added. The organic layer was extracted with ethyl acetate (150 ml),
washed with water, brine, and then dried over MgSO4. Filtration and concen-
tration in vacuo gave 0.87 g of a brown solid, which was purified by flash
chromatography to give 0.76 g of a brown residue, Rf=0.33 in 10% MeOH/
1
CHCl3. H NMR (CDCl3) d: 8.26(d, 1H), 7.85(d, 1H), 7.72–7.45(m, 3H), 6.75
(d, 1H), 6.64(br s, 1H), 4.28(t, J=5.52 Hz, 2H), 2.91(t, J=5.52 Hz, 2H), 1.51
(s, 9H). MS-ES: MH+ (381.4, 90%), M+ (380.3, 20%), M+ +2 (382.4, 50%).
4-(2-[2,2,3,3,5,5,6,6-2H8]Morpholin-4-yl-ethoxy)-naphthalen-1-ylamine (13). A
solution of the above material (0.74 g, 1.94 mmol) in dioxane (20 ml) was
stirred with a dioxane solution of HCl (4.0 M, 2 ml) for 14 h. The resulting
suspension was concentrated in vacuo to give a cream colored solid (0.78 g),
which was dissolved in a saturated solution of NaHCO3 and extracted with
ethyl acetate (30 ml). The ethyl acetate solution was dried over MgSO4, filtered
and concentrated in vacuo to give 0.64 g of a brown residue. Flash
chromatography gave 330 mg of a brown residue, Rf=0.34 in 10% MeOH/
CHCl3. 1H NMR (CDCl3) d: 8.25(m, 1H), 7.82(m, 1H), 7.42(m, 2H), 6.68(br s,
2H), 4.20(t, J=5.52 Hz, 2H), 2.91(t, J=5.52 Hz, 2H). MS-ES: MH+ (281.3,
100%), M+ (280.3, 25%), M+ +2 (282.3, 70%), M+ +3 (283.3, 10%).
1-(5-tert-Butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-[2,2,3,3,4,4,5,5,6,6-2H8]-mor-
pholin-4-yl-ethoxy)-naphthalen-1-yl]-urea, [2H8]-BIRB 796. Using the same
procedure as before, compound (1) (390 mg, 1.47 mmol), was reacted with a
20% phosgene solution in toluene (1.2 ml, 2.1 mmol), and the isocyanate
derivative was reacted with (13) (329 mg, 1.175 mmol) in a bi-phasic solution
of methylene chloride and a saturated solution of NaHCO3. The crude
product (0.83 g) was purified by flash chromatography using 50%
EtOAc:CH2Cl2 to remove the symmetrical urea (3) and then 15% methanol:
methylene chloride to elute the desired product (0.73 g). This product was
dissolved in hot isopropanol (10 ml) with decolorizing charcoal, cooled,
filtered, and concentrated in vacuo. Crystallization from isopropanol gave
0.4 g of a white solid. Mp : 152–1538C, Rt=10.67 on HPLC, Rf=0.47 in 10%
1
MeOH/CHCl3. H NMR (CDCl3) d: 8.25(d, J= 8.54 Hz, 1H), 7.85(d, J=
8.54 Hz, 1H), 7.52(m, 2H), 7.32(d, J= 8.53 Hz, 1H), 6.95(m, 4H), 6.66
(d, J= 8.53 Hz, 1H), 6.59(s, 1H), 6.41(d, J= 3.51 Hz, 2H), 4.31(t, J= 5.52 Hz,
2H), 2.98(t, J=5.52 Hz, 2H), 2.27(s, 3H), 1.31(s, 9H). MS-ES: MH+ (536.5,
50%), M+ (535.4, 8%), M+ +2 (537.5, 20%), M+ +3 (538.5, 5%).
Radioactive synthesis
Synthesis of 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-
ethoxy)naphthalen-1-yl][14C]urea (BIRB 796), Scheme 1. To a suspension of
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LABELING OF BIRB 796, A P38 MAP KINASE INHIBITOR
855
(1) (531 mg, 2.0 mmol) in CH2Cl2 (30 ml) was added a saturated solution of
NaHCO3 (30 ml) and the mixture was stirred at room temperature. The
resulting two-phase clear solution was cooled to 08C in an ice bath and stirring
was stopped. Once the phases were separated, [14C]-phosgene (150 mCi in
2.5 ml of anhydrous toluene) was added in one portion to the methylene
chloride phase via a syringe. Stirring was then resumed and continued for 4 h
at 08C. Compound (2) (440 mg, 1.61 mmol) was added in one portion and the
resulting mixture was stirred overnight while warming to room temperature.
The organic phase was removed using a syringe fitted with a long needle. The
remaining aqueous phase was extracted twice with methylene chloride
(30 ml ꢀ 2). The combined solutions were concentrated in vacuo and the
residue was purified by flash silica gel chromatography using 30% ethyl
acetate in methylene chloride as eluent to first separate the undesired side
product [14C]-(3), (87 mg, 6.0 mCi), Rf=0.85 in 10% MeOH/CHCl3. The
desired [14C]-BIRB 796 was obtained by eluting with methanol in chloroform
(10%), Rf=0.52 in 10% MeOH/CHCl3, Rt=10.79 min using radio-HPLC
system. The purification gave 885 mg of pure product and 93 mg of 85% pure
[14C]-BIRB 796. Crystallization of pure [14C]-BIRB 796 from 3 ml of
isopropanol gave 394 mg (first crop) of a white powder. The total activity of
the crystallized product was 40.5 mCi and the specific activity was determined
to be 54.2 mCi/mmol.
Preparation of tritium-labeled 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-
(2-morpholin-4-yl-ethoxy)naphthalen-1-yl]urea, Scheme 2. Using the same
procedure as in the synthesis of mono-deuterated BIRB 796, tritium
(8.95 mmol, 482 mCi) was introduced and the reaction mixture was stirred at
room temperature for 3 h. The reaction vessel was then frozen in liquid
nitrogen and evacuated (no measurable gas was noticed). The reaction was
diluted with water and extracted with methylene chloride as before. After the
work-up, methanol was added to the residue and evaporated under reduced
pressure to remove any exchangeable tritium. A total activity of 90 mCi was
obtained. HPLC analysis showed 54% of product and 45% of starting iodo-
BIRB796. No tritium exchange was observed on the iodo-BIRB796.
Purification of about 10 mCi fraction of this material by HPLC gave
5.6 mCi of pure product with a specific activity of 17.81 Ci/mmol.
Conclusion
Simple and straightforward syntheses of carbon-14-, tritium-, and deuterium-
labeled BIRB 796 with isotopic enrichment of 87% (2.0 GBq/mmol), 62%
(659 GBq), 98.9% respectively, were accomplished. Labeled BIRB 796 was
needed for ADME studies.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 847–856

856
B. LATLI
Acknowledgements
My thanks to Clark Perry, and to my colleagues Jeff Song (Chemical
Development Department) for providing compounds (1) and (2), Tom
Gilmore for compound (5), John Regan, Neil Moss, Glenn Van Moffaert,
Matt Hrapchack, Scott Leonard (Medicinal Chemistry Department), and
Edward Harris (Mass Spec Laboratory).
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Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 847–856