Synthesis of potent lymphocyte function-associated antigen-1 inhibitors labeled with carbon-14 and deuterium, part 1

Article abstract of DOI:10.1002/jlcr.1920

The lymphocyte function-associated antigen-1 (LFA-1) is an essential component in normal immune system function and is a target for drug discovery for its broad therapeutic potential in treating inflammatory diseases. Here, we report the synthesis of thre

Full text of DOI:10.1002/jlcr.1920

Research Article
Received 5 May 2011,
Revised 13 July 2011,
Accepted 19 July 2011
Published online 31 August 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1920
Synthesis of potent lymphocyte function-
associated antigen-1 inhibitors labeled with
carbon-14 and deuterium, part 1
*
Bachir Latli, Denis Byrne, Larry Nummy, Dhileepkumar Krishnamurthy
and Chris H. Senanayake
The lymphocyte function-associated antigen-1 (LFA-1) is an essential component in normal immune system function and is a tar-
get for drug discovery for its broad therapeutic potential in treating inflammatory diseases. Here, we report the synthesis of
three potent antagonists of LFA-1 labeled with carbon-14 and deuterium to support drug metabolism and pharmacokinetics
studies. Carbon-14 labeled (R)-1-acetyl-5-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-5-methyl-imidazolidine-2,4-dione (1) was
prepared in 27% radiochemical yield in two steps and with a specific activity of 2.1 GBq/mmol by using [14C]-phosgene. Carbon-
14 labeled 5-bromopyrimidine was used to prepare (R)-5-(1-piperazinylsulfonyl)-1-(3,5-dichlorophenyl)-3-[4-(5-pyrimidinyl)
benzyl]-3-methyl-1-H-imidazo[1,2a]imidazol-2-one (2) and (R)-1-[7-(3,5-dichlorophenyl)-5-methyl-6-oxo-5-(4-pyrimidin-5-yl-
benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonyl]piperidin-4-carboxylic acid amide (3) via a Suzuki reaction with
the corresponding boronic acid esters in 42% and 67% radiochemical yield and specific activities of 1.85 GBq/mmol and
1.95 GBq/mmol, respectively. Deuterium labeled piperazine was reacted with the sulfonyl chloride derivative (7), followed
by a Suzuki coupling to the pyrimidine boronic ester to give deuterium labeled (2) in 47% yield. Deuterium labeled isonipe-
cotamide was reacted in a similar way with the sulfonyl chloride derivative (14) to furnish deuterium labeled (3) in one step
and in 94% yield.
Keywords: carbon-14; deuterium; LFA-1 antagonists; metabolism; synthesis
binding site of BIRT377 was also identified.17 Although this mole-
Introduction
cule demonstrated good molecular and cellular potency, it was
Adhesion between T cells and antigen-presenting cells is necessary
poorly soluble in aqueous solution and was rapidly metabolized
for the formation of the immunological synapse. This adhesion is
in vitro by human liver microsomes by N-demethylation. Re-
mediated by T cell surface molecules including the lymphocyte
placement of this methyl group with an acetyl, such as that in
function-associated antigen-1 (LFA-1) and the intracellular adhe-
compound (1), led to improved metabolic stability. However,
sion molecule (ICAM) ligands.1–4 To appreciate the physiological
(1) was still prone to phase I metabolic N-deacetylation. The
importance of LFA-1, individuals with leukocyte adhesion defi-
search for compounds with superior pharmacokinetic profiles
ciency lack the ability to clear pathogens from their bodies, suf-
led to the introduction of the five-membered ring at this nitro-
fer from recurrent infections, and die at a young age.5 LFA-1
plays a role in organ transplantation and in the progression of
diseases including rheumatoid arthritis, multiple sclerosis,
Crohn’s diseases, and psoriasis. Because of this broad therapeutic
potential in treating these inflammatory diseases, LFA-1 is a tar-
get for drug discovery. Inhibitors of LFA-1 to its counter-receptors
ICAM-1, ICAM-2, ICAM-3, and other receptors have attracted con-
siderable attention in the pharmaceutical industry.6–13 For exam-
ple, the primary drug resistance is a major problem in the
incurable disease of the bone marrow, multiple myeloma.
Targeting LFA-1 by monoclonal antibodies or by small molecules
was shown to reduce cell adhesion-mediated drug resistance
significantly, which is responsible for this strong primary drug
resistance.14 The inhibition of ICAM-1/LFA-1 interactions was also
shown to play a role in HIV-1 attachment to target cells. Once
ICAM-1 is acquired by HIV-1, it promotes virus infectivity via ligation
of LFA-1, which helps in HIV-1 adsorption into the host cells.15,16
BIRT377 (Figure 1) was the first small molecule reported to
reversibly and specifically inhibit LFA-1 binding to ICAM-1.6 The
gen and the adjacent carbon in compounds (2) and (3).18
Further, replacement of the bromide by a pyrimidine moiety
enhanced the water solubility.19
[14C]-(1) was prepared according to Scheme 1. Thus, (R)-2-
amino-3-(4-bromophenyl)-N-(3,5-dichlorophenyl)-2-methyl-pro-
pionamide hydrochloride (4)20,21 underwent cyclization to
hydantoin by using carbon-14 labeled phosgene under Schotten–
Baumann conditions.22 The isocyanate was postulated as being
formed first and subsequently cyclized to the hydantoin.23 The
product (R)-5-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-5-methyl-
imidazolidine-2,4-dione (5) was easily isolated in methylene
chloride solution. Treatment of this compound in dry THF with
Chemical Development, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury
Road, Ridgefield, CT 06877, USA
*Correspodence to: Bachir Latli, Chemical Development, Boehringer Ingelheim
Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA.
E-mail: bachir.latli@boehringer-ingelheim.com
J. Label Compd. Radiopharm 2011, 54 763–768
Copyright © 2011 John Wiley & Sons, Ltd.

B. Latli et al.
Cl
O
Cl
Cl
O
Cl
during analytical measurement by mass spectrometry. Deuter-
ium labeled (1) was not synthesized because of no further inter-
est in this compound due to its metabolism as mentioned
previously. In the synthesis of [2H8]-(2), the sulfonyl chloride (7)
was reacted with [2H8]-piperazine dihydrochloride monohydrate
to give the bromide derivative (11) in 59% yield. At least two
equivalents of labeled piperazine had to be used to minimize
the double sulfonylation of piperazine. Higher yields were
obtained when using protected piperazine. However, monopro-
tected labeled piperazine was not available commercially. The
bromide derivative (11) was then reacted with 5-(4,4,5,5,-tetra-
methyl-[1,2,3]dioxaborolan-2-yl)-pyrimidine under Suzuki condi-
tions by using sodium carbonate and toluene : ethanol as
solvent to give the desired product in 80% yield (Scheme 2).
Compound [14C]-(3), 1-[7-(3,5-dichlorophenyl)-5-methyl-6-oxo-
5-(4-pyrimidin-5-yl-benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imida-
zole-3-sulfonyl]-piperidine-4-carboxylic acid amide, labeled with
carbon-14, was prepared with a specific activity of 52.8 mCi/
mmol. The synthesis was simple and straightforward, starting
from the in-house available (12) and the commercially available
[14C]-labeled 5-bromopyrimidine, as seen before (Scheme 3). The
boronic ester (13) was prepared in yields ranging from 80% to
90% as described previously by using bis(pinacolato)diboron in
the presence of potassium acetate and catalytic amounts of
[PdCl2(dppf)] in DMF. The same catalyst was used in the second
step, the only radiosynthetic step with potassium carbonate and
dimethoxyethane as solvent. Thus, the resulting boronic ester
was reacted with 5-bromopyrimidine,[2-14C]- to give labeled (3)
in 67% radiochemical yield.
Cl
O
Cl
N
N
O
O
N
*
N
N
O
O
N
H
Br
Br
(1)
BIRT0377BS
Initial hit
Cl
N
Cl
Cl
Cl
O
N
O
N
N
N
N
N
S
N
O
O
S
O
O
*
N
N
N
N
*
N
H
(3)
O
NH
2
(2)
Figure 1. Structures of LFA-1 antagonists. Asterisks indicate position of the radio-
active carbon.
KHMDS at À25 ꢀC, followed by the addition of acetyl chloride,
gave the desired product [14C]-(1) in 27% overall radiochemical
yield and a specific activity of 56.75 mCi/mmol (Scheme 1).
In the synthesis of [14C]-(2) (Scheme 2), the iodo derivative
(6)24 was converted to the sulfinate salt by treatment with cyclo-
pentyl magnesium bromide at À40 ꢀC, and the Grignard’s pro-
duct was quenched with sulfur dioxide.25 Reaction with N-
chlorosuccinimide gave the sulfonyl chloride (7) that was
reacted directly with 1-tert-butoxycarbonyl piperazine and
triethylamine as a base in THF to give (8) in 81% overall yield.
Heating compound (8) with bis(pinacolato)diboron in the presence
of catalytic amounts of [1,1′-bis(diphenylphosphino)-ferrocene]
dichloropalladium [PdCl2(dppf)] and 4.5 equivalent of potassium
acetate in DMF at 80 ꢀC gave the boronate ester (9) in 77% yield
after flash chromatography purification. The availability of car-
bon-14 labeled 5-bromopyrimidine from commercial suppliers26
prompted us to prepare the boronic ester (9) and then couple it
to [14C]-5-bromopyrimidine in one radiosynthesis under Suzuki
conditions to give (10). Removing the protecting group by using
TFA gave the product [14C]-(2) with a specific activity of 50 mCi/
mmol and in 42% radiochemical yield.
Deuterium labeled (3) was prepared from the in-house avail-
able sulfonyl chloride derivative (14) with the pyrimidine already
attached and deuterated isonipecotamide to give deuterated (3)
in one step and in 94% yield.
Experimental procedures
Materials and methods
Liquid scintillation counting was accomplished using a Beckman
LS5000TA and ready safe™ cocktail (Beckman, Fullerton, CA,
USA). Radio-TLC was carried out on a BIOSCAN System 200 ima-
ging scanner by using an auto-changer 1000 and WinScan soft-
ware version 2.1a (Bioscan, Inc., Washington, DC, USA). The quan-
tification of the HPLC chromatograms was carried out using an
HPLC system composed of a radiomatic A515 Flo-one/beta
radioactivity flow detector (Packard Instrument Company, Meri-
den, CT, USA), two pumps (HITACHI L-6200A intelligent pump),
a linear UVIS 200, Ultima Flo™ AP cocktail (Packard, Meriden,
CT, USA), and radiomatic 500TR V 3.60 for data evaluation. The
analytical HPLC purity verification for compound (1) was carried
out on a 3MZ-18 column, particle size 3 mm, 4.6 Â 150 mm (Cohe-
sive Technologies, Inc., Franklin, MA, USA). Mobile phase: A
(water), B (methanol), both solvents contain triethylamine (TEA,
10 mM) and use a gradient: 50% to 100%B in 30 min. For com-
pound (2), the HPLC was carried out on an MV-C18 column, par-
ticle size 5 mm, 5 Â 250 mm (Rainin Instrument Company, Inc.,
Woburn, MA, USA). Mobile phase: A (water), B (acetonitrile), both
solvents contain TFA (10 mM) and use a gradient: 40% to 80%B
in 20 min, hold at 80%B to 30 min. For compound (3), HPLC
was carried out on a Zorbax Eclipse XDB-C8 column, particle size
5 mm, 4.6 Â 150 mm (Agilent Technologies, Inc., Santa Clara, CA,
USA). Mobile phase: A (water), (acetonitrile), both solvents
For the deuterium labeled compounds, it was very important
to have the molecular ion peak of these compounds at least five
units higher than the unlabeled compounds to avoid overlap-
ping with ion peaks resulting from the two chlorine atoms
Cl
O
Cl
Cl
Cl
Cl
O
Cl
O
b
N
HN
a
N
O
*
N
O
*
N
H2N
H
O
2 HCl
Br
Br
(5), 100%
14C]-(5), 35%
(1), 85%
[
14C]-(1), 77%
Br
(4)
[
Scheme 1. (a) ClCOCl or Cl14COCl, sat’d NaHCO3, CH2Cl2, 0 ꢀC to rt; (b) KHMDS,
THF, CH3COCl, À25 ꢀC to rt.
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 763–768

B. Latli et al.
Cl
Cl
O
Cl
Cl
O
Cl
Cl
O
Cl
Cl
O
N
N
N
N
N
N
N
N
N
N
N
N
d
c
e
S
N
S
N
O
S
N
O
O
S
O
O
O
O
O
O
B
N
Br
N
N
O
N
*
N
*
N
N
N
N
H
(8), 81%
O
O
O
O
(10), 66%
(2), 93%
O
O
(9), 77%
14C]-(10)
[
14C]-(2), 42% (two steps)
[
b
Cl
Cl
Cl
Cl
Cl
O
Cl
Cl
O
Cl
N
N
O
O
g
N
N
f
N
N
a
N
N
N
O
N
N
N
S
O
O
S
N
O
O
N
Br
S
I
O
N
Cl
Br
Br
N
D8
D8
(6)
(7)
N
H
N
H
[2H8]-(2), 80%
(11), 59%
Scheme 2. (a) CyMgBr, SO2, À40 ꢀC, NCS, À20 ꢀC, THF; (b) Boc-piperazine, TEA, CH2Cl2, rt; (c) bis(pinacolato)diboron, Pd2Cl2(dppf), CH2Cl2, KOAc, DMF, 80 ꢀC; (d) [14C]-5-
bromopyrimidine, K2CO3, PdCl2(dppf), CH2Cl2, DME, 85 ꢀC; (e) TFA, CH2Cl2, rt; (f) [2H8]-piperazine, TEA, THF, À20 ꢀC to rt; (g) 5-pyrimidine boronic acid pinacol ester, PdCl2
(dppf)ÁCH2Cl2, Na2CO3, toluene : EtOH, 85 ꢀC.
contain TFA (10 mM) and use a gradient: 5% to 100%B in 30 min. MEL-TEMPW 3.0 (Laboratory Devices, Inc., Cambridge, MA, USA).
UV detection for all compounds was carried out at 254 nm. Eva- Pre-coated TLC sheets (silica gel 60 F254) and silica gel 60–200
poration of solvents and non-radioactive volatile components Mesh (Nominal, ID, grade 62) for flash chromatography were
was accomplished at reduced pressure by using Büchi rotary eva- obtained from EM Science (Gibbstown, NJ, USA). Phosgene,
porator unless stated otherwise. Mass spectra for non-radioactive 20% solution in toluene was obtained from Fluka (Milwaukee,
compounds were acquired by a Hewlett-Packard auto sampler WI, USA). [2H8]-PiperazineÁ2HClÁH2O, 98 at.%2H was purchased
Series 1100, connected to a Micromass Platform LCZ in the ES from Aldrich. [2H9]-Isonipecotamide, 99 at.%2H was purchased
mode. NMR spectra were recorded with a Bruker 400 MHz DPX from CDN (Pointe Claire, Quebec, Canada). [14C]-Phosgene with
spectrometer by using deuterated chloroform as a solvent and a specific activity of 58 mCi/mmol was obtained from Vitrax (Pla-
tetramethyl silane as the internal standard unless stated other- centia, CA, USA). 5-Bromopyrimidine,[2-14C]- with a specific activ-
wise. Melting points are uncorrected and were obtained using ity of 53 mCi/mmol was purchased from Moravek Biochemicals,
Cl
N
Cl
O
Cl
N
Cl
O
Cl
N
Cl
O
N
N
N
b
a
N
N
N
N
S
N
O
S
N
O
O
O
S
N
O
O
B
O
Br
*
O
N
(12)
O
NH2
(3), 64%
14C]-(3), 67%
(13), 80%
O
NH2
O
NH2
[
Cl
Cl
O
Cl
Cl
N
N
N
O
c
N
N
N
N
S
O
O
N
N
S
O
O
N
D9
Cl
N
(14)
[2H9]-(3), 94%
O
NH2
Scheme 3. (a) Bis(pinacolato)diboron, PdCl2(dppf)ÁCH2Cl2, KOAc, DMF, 80 ꢀC; (b) [14C]-5-bromopyrimidine, K2CO3, PdCl2(dppf)ÁCH2Cl2, DME, 80 ꢀC; (c) [2H9]-isonipecotamide,
THF : DMF (10:1), TEA, rt.
J. Label Compd. Radiopharm 2011, 54 763–768
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

B. Latli et al.
Inc. (Brea, CA, USA). The rest of the reagents were purchased were concentrated under reduced pressure. The residue was
from Aldrich Chemicals Company.
purified by flash chromatography using 1% MeOH/CHCl3 to give
a white solid, which was crystallized from hot hexane to give
222 mg of material. A total activity of 27 mCi was obtained with
a specific activity of 56.75 mCi/mmol. HPLC (tR = 17.4 min) and
Synthesis of (R)-1-actyl-5-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-
5-methyl-imidazolidine-2,4-dione (1) and [14C]-(1) (Scheme 1)
(R)-5-(4-Bromobenzyl)-3-(3,5-dichlorophenyl)-5-methyl-imidazolidine- radio-TLC (plate developed in 5% MeOH/CHCl3) showed the pro-
1
2,4-dione (5): A mixture of (4) (633.4 mg, 1.33 mmol) in methylene duct to be more than 99% pure. H NMR in CDCl3 was identical
chloride (30 mL) and a saturated solution of NaHCO3 (40 mL) was to unlabeled material.
stirred at room temperature until all the amine was dissolved.
Synthesis of (R)-5-(1-piperazinylsulfonyl)-1-(3,5-dichlorophenyl)-
The clear two-phase solution was then cooled in an ice bath to
3-[4-(5-pyrimidinyl)benzyl]-3-methyl-1-H-imidazo[1,2-a]imidazol-
0 ꢀC. Stirring was stopped, and a solution of phosgene in toluene
2-one (2), [2H8]-(2), and [14C]-(2) (Scheme 2)
(1.14 mL, 2 mmol) was added to the organic phase. Stirring was
then resumed, and the reaction was warmed to room tempera- 4-[(R)-5-(4-Bromobenzyl)-7-(3,5-dichlorophenyl)-5-methyl-6-oxo-
ture and stirred for 4 h. The organic phase was then removed, 6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfonyl]-piperazine-1-
and the aqueous was extracted with methylene chloride. The carboxylic acid tert-butyl ester (8): To a solution of (6) (2.83 g,
combined extracts were concentrated in vacuo to give 661 mg 4.9 mmol) in THF (50 mL), stirred at À40 ꢀC under nitrogen atmo-
of a white solid, which was used in the next step without further sphere, was added dropwise a solution of cyclopentyl magne-
purification. Rf = 0.57 in 10% MeOH/CHCl3, tR = 13.25 min. 1H sium bromide (2.94 mL, 5.88 mmol, 2.0 M solution in ether). The
NMR (CDCl3): d 7.50(d, J = 8.26 Hz, 2H), 7.38(t, J = 1.83 Hz, 1H), resulting solution was further stirred at this temperature for
7.09(d, J = 8.26 Hz, 2H), 7.03(d, J = 1.83 Hz, 2H), 6.61(s, 1H, ex.), 20 min, and then sulfur dioxide was slowly bubbled into the
3.1(dd, J = 13.69, 90.29 Hz, 2H), 1.64(s, 3H). MS-ESÀ: [M-1]À = 427. reaction flask for 5 min at À50 ꢀC. The reaction flask was warmed
(R)-1-Acetyl-5(4-bromobenzyl)-3-(3,5-dichlorophenyl)-5-methyl-imi- gradually to room temperature and was stirred for 1 h. Solvents
dazolidine-2,4-dione (1): To a solution of the aforementioned com- and excess sulfur dioxide were then removed under reduced
pound (621 mg, 1.45 mmol) in anhydrous THF (8 mL), stirred at pressure, and the residue was further dried to give a yellow-
À25 ꢀC under nitrogen, was added a solution of potassium bis orange foam, which was dissolved in dry THF (30 mL) and
(trimethylsilyl)amide (3.25 mL, 1.624 mmol, 0.5 M in toluene) added at À20 ꢀC to a suspension of N-chlorosuccinimide (3 g,
dropwise. The resulting yellow solution was stirred at this tem- 22.05 mmol) in dry THF (30 mL). The resulting mixture was stirred
perature for 10 min before acetyl chloride (114 mL, 1.6 mmol) at this temperature for 20 min to give (7). To this mixture, 1-Boc-
was added dropwise. After stirring at À25 ꢀC for 5 min, the cool- piperazine (2 g, 10.78 mmol) and triethylamine (1.5 mL,
ing bath was removed, and stirring was continued for 90 min at 10.78 mmol) in dry THF (30 mL) were added. The reaction was
room temperature. Most of the solvent was then evaporated, warmed to room temperature and was stirred for 12 h then
and the residue was dissolved in methylene chloride (30 mL). A diluted with ethyl acetate (200 mL). The organic phase was
solution of aqueous HCl (1.0 N, 10 mL) was added, and the mix- washed with aqueous HCl (0.1 N, 200 mL), brine (200 mL), dried
ture was stirred vigorously. The organic phase was removed, over MgSO4, filtered and concentrated in vacuo to give 5.5 g of
and the aqueous was extracted with methylene chloride. The brown foam. Purification by flash chromatography gave 3 g of
1
combined extracts were dried over MgSO4, filtered and concen- pure product. H NMR (CDCl3): d 7.36(m, 2H), 7.32(m, 2H), 7.26
trated in vacuo to give 973 mg of yellow oil. Purification by flash (m, 2H), 6.81(d, J = 8.29 Hz, 2H), 3.81(d, J = 13.91 Hz, 1H), 3.57
chromatography using 1% MeOH/CHCl3 gave 636 mg of pure (brs, 4H), 3.21(d, J = 13.91 Hz, 1H), 3.20(m, 4H), 1.97(s, 3H), 1.45
product as viscous colorless oil. Rf = 0.75 in 10% MeOH/CHCl3. (s, 9H). Rf = 0.31 in 10% EtOAc : CH2Cl2. MS-ES+: [MH]+ = 700.2.
Crystallization from warm hexane gave 531 mg of white crystal-
4-{(R)-7-(3,5-Dichlorophenyl)-5-methyl-6-oxo-5-[4-(4,4,5,5,-tet-
1
line material in 85% yield. tR = 19.6 min. H NMR (CDCl3): d 7.42 ramethyl-[1,3,2]dioxaborolan-2-yl]6,7-dihydro-5H-imidazo[1,2-a]imida-
(d, J = 8.54 Hz, 2H), 7.38(t, J = 1.83 Hz, 1H), 6.95(d, J = 8.54 Hz, zole-3-sulfonyl}-piperazine-1-carboxylic acid tert-butyl ester (9): To a
2H), 6.90(d, J = 1.83 Hz, 2H), 3.50(dd, J = 14.05, 237.40 Hz, 2H), mixture of (8) (0.97 g, 1.4 mmol), potassium acetate (0.61 g,
2.56(s, 3H), 1.90(s, 3H).
6.23 mmol), bis(pinacolato)diboron (0.53 g, 2.1 mmol), PdCl2
[14C]-(5): As described previously, a solution of [14C]-phosgene (dppf)ÁCH2Cl2 (0.17 g, 0.21 mmol), was added dry DMF (10 mL)
(100 mCi, SA = 58 mCi/mmol) in anhydrous toluene (2 mL) was under nitrogen atmosphere, and the resulting dark mixture
added at 0 ꢀC to the propionamide derivative (4) (634 mg, was heated to 80 ꢀC overnight. After cooling to room tempera-
1.33 mmol). After stirring for 10 h at room temperature, the ture, the mixture was filtered through a short pad of CeliteW,
methylene chloride phase was removed, and the aqueous was and the filtrate was washed with water (250 mL), brine (100 mL),
extracted with methylene chloride (2 Â 30 mL). The combined dried over MgSO4, filtered and concentrated in vacuo. The resi-
extracts were concentrated in vacuo, and the residue was puri- due was purified by flash chromatography using 30% EtOAc :
fied by flash chromatography to give 315 mg of a white solid
in 35% radiochemical yield. Rf = 0.16 in 1% MeOH/CHCl3.
Hexanes as eluant to give 795 mg of white foam in 77% yield.
Rf = 0.23 in 10% EtOAc : CH2Cl2. 1H NMR (CDCl3): d 7.55(d,
[14C]-(1): To a solution of the aforementioned compound J = 7.57 Hz, 2H), 7.36(s, 1H), 7.27(m, 1H), 7.21(m, 2H), 6.90(d,
(315 mg, 0.736 mmol) in anhydrous THF (6 mL) stirred under J = 7.65 Hz, 2H), 3.84(d, J = 13.76 Hz, 1H), 3.57(brs, 4H), 3.24(d,
nitrogen atmosphere at À25 ꢀC was added a solution of KHMDS J = 13.76 Hz, 1H), 3.57(brs, 4H), 3.24(d, J = 13.76 Hz, 1H), 3.23(brs,
(1.65 mmol, 0.824 mmol, 0.5 M in toluene) dropwise. Acetyl chlor- 4H), 1.98(s, 3H), 1.45(s, 9H), 1.26(brs, 12H). MS-ES+: [MH]+ = 746.5.
ide (59 mL, 0.824 mmol) was added. The cooling bath was
4-[(R)-7-(3,5-Dichlorophenyl)-5-methyl-6-oxo-5(4-pyrimidin-5-
removed, and the reaction was warmed to room temperature yl-benzyl)6,7-dihydro-5-H-imidazo[1,2-a]imidazole-3-sulfonyl]-
and stirred for 12 h. An aqueous solution of HCl (10 mL, 1%) piperazine-1-carboxylic acid tert-butyl ester (10): A mixture of
was added and stirred for 15 min. The aqueous phase was the aforementioned boronic acid ester (9) (0.34 g, 0.46 mmol),
extracted with methylene chloride, and the combined extracts 5-bromopyrimidine (74 mg, 0.46 mmol), PdCl2(dppf)ÁCH2Cl2 (40 mg,
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J. Label Compd. Radiopharm 2011, 54 763–768

B. Latli et al.
0.04 mmol), and potassium carbonate (0.32 g, 2.3 mmol) in to room temperature, and most of the solvents were removed
dimethoxyethane (8 mL) was heated at 80 ꢀC under nitrogen under reduced pressure. Ethyl acetate (100 mL) and water
atmosphere for 7 h. The resulting dark mixture was then cooled (50 mL) were added, and the aqueous phase was extracted with
to room temperature, and water (50 mL) was added, and the ethyl acetate (2 Â 100 mL). The combined organic layers were
mixture was extracted with ethyl acetate (2 Â 150 mL). The com- dried over MgSO4, filtered and concentrated in vacuo to give
bined extracts were dried over MgSO4, filtered and concentrated 1.0 g of a foamy residue. Purification by flash chromatography
in vacuo. Purification of the residue by flash chromatography followed by preparative TLC gave 682 mg of pure product in
using 5% to 10% EtOAc : Hexanes followed by crystallization 80% yield. 1H NMR (CDCl3): d 9.17(s, 1H), 8.84(s, 2H), 7.36(m,
from ethyl acetate : hexanes gave 210 mg of white solid in 66% 5H), 7.28(m, 1H), 7.09(d, J = 8.15 Hz, 2H), 3.95(d, J = 13.81 Hz,
1
yield. Rf = 0.27 in 50% EtOAc : Hexanes. H NMR (CDCl3): d 9.17 1H), 3.32(d, J = 13.77 Hz, 1H), 2.02(s, 3H). MS-ES+: MH+ = 606.2:
(s, 1H), 8.84(s, 2H), 7.36(m, 3H), 7.31(m, 3H), 7.08(d, J = 8.10 Hz, 100%, [MH + CAN]+ = 747.2 (20%). MS-ESÀ: [M + OAc]À = 664.0
2H), 3.93(d, J = 13.85 Hz, 1H), 3.60(brs, 4H), 3.32(d, J = 13.85 Hz, (100%). HPLC: tR = 9.8 min, 99.9%.
1H), 3.26(brs, 4H), 2.02(s, 3H), 1.62(s, 3H), 1.45(s, 9H), 1.26(s, 9H).
[14C]-(2): A mixture of the boronic acid ester (9) (239 mg,
MS-ES+: [MH]+ = 698.4.
0.32 mmol), 5-bromopyrimidine,[2-14C]- (16 mCi, 48 mg, 0.3 mmol,
(R)-5-(1-Piperazinylsulfonyl)-1-(3,5-dichlorophenyl)-3-[4-(5-pyr- SA = 53 mCi/mmol), PdCl2(dppf)ÁCH2Cl2 (40 mg, 0.05 mmol),
imidinyl)benzyl]-3-methyl-1-H-imidazo[1,2-a]imidazol-2-one (2): potassium carbonate (221 mg, 1.5 mmol) in DME (10 mL) was
A solution of (10) (64 mg, 0.0916 mmol) in methylene chloride refluxed for 8 h. After cooling to room temperature, water was
(5 mL) and TFA (0.5 mL) was stirred at room temperature for added (40 mL), and the mixture was extracted with ethyl acetate.
90 min. It was then poured into aqueous NaOH (1.0 N, 10 mL) The combine organic extracts were dried over MgSO4, filtered
and extracted with methylene chloride. The combined extracts and concentrated in vacuo. The pure product was obtained after
were dried over MgSO4, filtered and concentrated in vacuo. Pur- flash chromatography purification using 30% to 50% EtOAc :
ification by flash chromatography gave 51 mg of the desired pro-
Hexanes, Rf = 0.25 in 50% EtOAc : Hexanes; HPLC and TLC similar
duct in 93% yield. 1H NMR (CDCl3): d 9.16(s, 1H), 8.83(s, 2H), 7.35 to (9). This material was then dissolved in methylene chloride
(m, 3H), 7.31(m, 2H), 7.27(m, 1H), 7.08(d, J = 8.14 Hz, 2H), 3.93(d, (10 mL), and TFA (2 mL) was added. The resulting solution was
J = 13.81 Hz, 1H), 3.46(s, 3H), 3.26(m, 4H), 3.28(d, J = 13.80 Hz, stirred at room temperature for 90 min. Then it was poured into
1H), 3.01(m, 4H), 2.01(s, 3H). MS-ES: MH+: 598.4 (100%). MS-ESÀ: an ice cold solution of NaOH (1.0 N, 40 mL) and extracted with
[M + OAc]À = 656.
methylene chloride. The combined extracts were dried over
(R)-3-(4-Bromobenzyl)-1-(3,5-dichlorophenyl)-3-methyl-5-(pipera- MgSO4, filtered and concentrated in vacuo. The residue was pur-
zine-1-sulfonyl)-1H-imidazo[1,2-a]imidazol-2-one (11): To a solution ified by flash chromatography using 50% EtOAc : Hexanes to give
of (6) (1.41 g, 2.45 mmol) in THF (30 mL), stirred at À40 ꢀC under 80 mg of pure material as a white solid. Total activity was
nitrogen atmosphere, was added dropwise a solution of cyclo- 6.68 mCi and a specific activity of 50 mCi/mmol. 1H NMR was
pentyl magnesium bromide (1.47 mL, 2.94 mmol, 2.0 M solution identical to unlabeled (2).
in ether). The resulting solution was further stirred at this tem-
Synthesis of (R)-1-[7-(3,5-dichlorophenyl)-5-methyl-6-oxo-5-[4-pyri-
perature for 20 min. Then, sulfur dioxide was slowly bubbled into
midin-5-yl-benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfo-
the reaction flask for 5 min at À50 ꢀC. The reaction was then
nyl]-piperidine-4-carboxylic acid amide (3), [2H9]-(3), and [14C]-(3)
warmed gradually to room temperature and was stirred for
(Scheme 3)
another hour. Solvent and excess sulfur dioxide were removed
in vacuo, and the residue was further dried for 1 h under reduced 1-{(R)-7-(3,5-Dichlorophenyl)-5-methyl-6-oxo-5-[4,4,5,5-tetramethyl-
pressure to give a yellow-orange foam, which was used in the [1,3,2]dioxaborolan-2-yl)-benzyl]-6,7-dihydro-5H-imidazo[1,2-a]
next step without further purification. The aforementioned sulfi- imidazole-3-sulfonyl}-piperidine-4-carboxylic acid amide (13):
nate salt was dissolved in dry THF (15 mL) and was added at A mixture of (12) (1.28 g, 2.0 mmol), potassium acetate (0.881 g,
À20 ꢀC to a suspension of N-chlorosuccinimide (0.5 g, 3.67 mmol) 9.0 mmol), PdCl2(dppf)ÁCH2Cl2 (0.245 g, 0.3 mmol), and bis(pina-
in dry THF (15 mL). The resulting mixture was stirred at this tem- colato)diboron (0.9 g, 3.47 mmol) in DMF (20 mL) was heated at
perature for 20 min to give (7), which was then added via cannu- 80 ꢀC under nitrogen atmosphere for 14 h. The dark mixture
lation to
a
flask containing [2H8]-piperazine-hydrochloride was cooled to room temperature and diluted with ethyl acetate
monohydrate (1.0 g, 5.4 mmol), triethylamine (2.2 mL, 15.8 mmol), (300 mL) then poured into water (200 mL). The organic phase
and ethanol (10 mL) at À30 ꢀC. The resulting mixture was warmed was removed, washed twice with water (2 Â 100 mL), brine
to room temperature slowly and then concentrated in vacuo. The (200 mL), dried over MgSO4, filtered and concentrated in vacuo
residue was dissolved in ethyl acetate (200 mL) and was washed to give 1.8 g of a dark solid residue, which was purified by flash
with water (200 mL), brine (100 mL), dried over MgSO4, filtered chromatography using 10% ethanol in ethyl acetate to give
and concentrated in vacuo to give 2.02 g of a yellow solid. Purifi- 1.33 g of a foam contaminated with traces of the starting bis
cation by flash chromatography using chloroform and then 10% (pinacolato)diboron. A second purification using 100% ethyl
methanol/chloroform gave 0.87 g of white foam in 59% yield. 1H acetate as eluant gave 1.1 g of the desired product in 80%
NMR (CDCl3): d 7.38(m, 2H), 7.40(s, 1H), 7.32(m, 1H), 7.25(m, 2H), yield. 1H NMR (CDCl3): d 7.52(d, J = 7.68 Hz, 2H), 7.26(m, 4H),
6.82(d, J = 8.35 Hz, 2H), 3.83(d, J = 13.84 Hz, 1H), 3.22(d, 7.27(m, 1H), 6.89(d, J = 7.80 Hz, 2H), 5.81(d, J = 13.65 Hz, 2H),
J = 13.91 Hz, 1H), 1.98(s, 3H). MS-ES+: [MH]+ = 606.1: 80%, 608.1: 3.27(m, 3H), 3.21(d, J = 13.69 Hz, 1H), 2.85(m, 2H), 2.29(m, 1H),
100%, 610.1: 60%.
1.98(s, 3H), 1.84(m, 2H), 1.27(brs, 12H).
1{(R)-[7-(3,5-Dichlorophenyl)-5-methyl-6-oxo-5-(4-pyrimidin-5-
[2H8]-(2): A mixture of the aforementioned compound (0.7 g,
1.132 mmol), 5-pyrimidine boronic acid pinacol ester (0.546 g, yl-benzyl)-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-sulfony}-
2.65 mmol), PdCl2(dppf)ÁCH2Cl2 (28 mg, 3 mol%), aqueous piperidine-4-carboxylic acid amide (3). A mixture of the
Na2CO3 (2.0 M solution, 2 mL) in toluene (8 mL) and ethanol aforementioned boronic ester (344.3 mg, 0.5 mmol), PdCl2
(4 mL) was heated at 85 ꢀC for 4 h. The mixture was then cooled (dppf)ÁCH2Cl2 (45 mg, 0.05 mmol), 5-bromopyrimidine (72 mg,
J. Label Compd. Radiopharm 2011, 54 763–768
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

B. Latli et al.
0.44 mmol), and potassium carbonate (346 mg, 2.5 mmol) in DME and specific activities of 50 mCi/mmol and 52.86 mCi/mmol,
(10 mL) was refluxed for 14 h under nitrogen atmosphere. After respectively. Deuterium labeled (2) and (3) were prepared using
cooling to room temperature, water was added, and the organic the commercially available deuterium labeled piperazine and
phase was extracted with ethyl acetate (2 Â 50 mL), dried over deuterium labeled isonipecotamide.
MgSO4, filtered and concentrated in vacuo to give 420 mg of a
dark residue, Rf = 0.40 in 20% EtOH/EtOAc or 0.27 in 5% MeOH/
CHCl3. Purification by flash chromatography using 50% EtOAc :
Acknowledgements
Hexanes then 100% EtOAc gave 205 mg of pure product as
1
cream-colored solid in 64% yield. H NMR (CDCl3): d 9.16(s, 1H),
We wish to thank Mr. Scott Leonard for his help with NMR of
8.83(s, 2H), 7.36(m, 3H), 7.31(m, 2H), 7.27(m, 1H), 7.09(d,
radioactive compounds and Dr. Fenghe Qiu for obtaining mass
spectra of deuterium labeled compounds.
J = 8.07 Hz, 2H), 5.53(d, J = 15.65 Hz, 2H), 3.93(m, 2H), 3.78(m,
1H), 3.28(d, J = 13.78 Hz, 1H), 2.86(m, 2H), 2.31(m, 1H), 2.06(m,
2H), 2.01(s, 3H), 1.90(m, 2H). MS-ES: [MH]+ (640.1, 100%), (642.0,
80%), MS-ESÀ: [M + OAc]À = 698.1.
References
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tamide-2,2,3,3,4,5,5,6,6-2H9 (205.5 mg, 1.5 mmol, 99 at.%2H) was
added in one portion, and the resulting mixture was warmed
to room temperature and stirred overnight under nitrogen. The
mixture was concentrated under reduced pressure, and the resi-
due was diluted with ethyl acetate (150 mL) and washed with an
aqueous solution of HCl (0.1 N, 50 mL) and brine (50 mL). The
organic phase was dried over MgSO4, filtered and concentrated
in vacuo to give a white solid. Purification by flash chromatogra-
phy using ethyl acetate as eluant and then 10% methanol : ethyl
acetate gave 640 mg of a white solid. Crystallization from warm
ethyl acetate and hexanes gave 610 mg of a white solid in 94%
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1
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1
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Conclusion
Three LFA-1 antagonists labeled with carbon-14 and deuterium
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 763–768