A novel five-lipoxygenase activity protein inhibitor labeled with carbon-14 and deuterium

Article abstract of DOI:10.1002/jlcr.3319

2-[4-(3-{(1R)-1-[4-(2-Aminopyrimidin-5-yl)phenyl]-1-cyclopropylethyl}-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl]-N,N-dimethylacetamide (1), is a novel and selective five-lipoxygenase activity protein (FLAP) inhibitor with excellent pharmacokinetics properties

Full text of DOI:10.1002/jlcr.3319

Research article
Received 29 April 2015,
Revised 27 May 2015,
Accepted 27 June 2015
Published online 20 July 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3319
A novel five-lipoxygenase activity protein
inhibitor labeled with carbon-14 and
deuterium
*
Bachir Latli, Matt Hrapchak, Joe J. Gao, Carl A. Busacca, and
Chris H. Senanayake
2-[4-(3-{(1R)-1-[4-(2-Aminopyrimidin-5-yl)phenyl]-1-cyclopropylethyl}-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl]-N,N-dimethylacetamide
(1), is a novel and selective five-lipoxygenase activity protein (FLAP) inhibitor with excellent pharmacokinetics properties.
The availability of a key chiral intermediate allowed the synthesis of [14C]-(1) in six radiochemical steps and in 47% overall
radiochemical yield with a specific activity of 51 mCi/mmol using carbon-14 zinc cyanide. 2-Chloro-N,N-dimethyl-2H6-
acetamide was prepared and condensed with a penultimate intermediate to give [2H6]-(1) in very high yield and in more
than 99% isotopic enrichment.
Keywords: BI665915; FLAP; carbon-14; deuterium; radiosynthesis
labeled (1) could be achieved in just one additional radioactive
Introduction
step (Scheme 1).
Several cyanating conditions were screened.11 The best results
Five-lipoxygenase catalyzes the metabolism of arachidonic acid
into hydroperoxyeicosatetraenoic acid and leukotrienes.1 The
elevated levels of this enzyme have been implicated in several
were obtained with Merck’s procedure of using zinc cyanide and
low levels of palladium and 1,1′-bis(diphenylphosphine)-ferrocene
diseases including cancer.2,3 Thus, inhibition of this enzyme
may be beneficial in treating allergic conditions, cardiovascular
diseases, arthritis, inflammatory bowel disease, psoriasis and
other related ailments.4–7 Compound (1), also known as
BI665915, has high binding potency to FLAP and excellent
pharmacokinetic properties.8,9 Herein, we describe the synthesis
of this compound labeled with carbon-14 and with deuterium to
aid in drug metabolism, pharmacokinetics and bioanalytical
studies.
(DPPF) as ligand.12 However, rapid hydrolysis of the N,N-
dimethylacetamide moiety prevented isolation of (3). Instead
compound (7) was the only product isolated. The hydrolysis of this
dimethylacetamide moiety was observed to be faster than
hydrolysis of the nitrile group. A different protecting group was
then considered (Scheme 2).
The para-methoxybenzyl (PMB) group was selected over the
reported benzyl group because its removal can be achieved
without the use of hydrogen.13 4-Bromo-1H-pyrazole (6) was thus
protected using para-methoxybenzyl chloride and potassium
carbonate in DMF in quantitative yield.14 Cyanation with
carbon-14 zinc cyanide gave [14C]-(9) in 90% yield.12 Hydrolysis
of [14C]-(9) in aqueous sodium hydroxide solution then gave the
acid [14C]-(10) in 85% yield. The acid [14C]-(10) was then activated
with carbonyldiimidazole (CDI) and condensed with (R)-2-(4-
bromophenyl)-2-cyclopropyl-N′-hydroxypropimidamide (11)9 in
DMF at 100 °C to give [14C]-(12) in 89% yield. Removal of the
protecting group was accomplished using anisole and
trifluoroacetic acid in the presence of sulfuric acid in methylene
chloride.15 Suzuki coupling with the boronate (14)9 gave [14C]-
(15) in 82% yield. Finally, alkylation with 2-chloro-N,N-
dimethylacetamide (16) in DMF in the presence of potassium
Results and discussion
The aim in the preparation of radioactive compounds has always
been to minimize the number of radioactive synthetic steps while
installing the radioactive carbon in a position not susceptible to
metabolism. Compound (1) is a relatively complex molecule with
an all-carbon quaternary stereogenic center. Thus, introducing
the radioactive carbon into the oxadiazole moiety using 1H-
pyrazole-4-carboxylic acid and a chiral intermediate available
from in-house synthesis seemed a logical choice (Scheme 1).
Initial investigations focused on standard carbonylation of 4-
bromo-1H-pyrazole to 1H-pyrazole-4-carboxylic acid; however, a
large excess of carbon dioxide was required, and the yields were
very poor.10 Protection of 4-bromopyrazole and introduction of
Chemical Development, Boehringer Ingelheim Pharmaceuticals, Inc., 900
the radioactive carboxyl carbon via a nitrile group was then Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-0368, USA
considered. The initial choice was to use N,N-dimethylacetamide
*Correspondence to: Bachir Latli, Chemical Development, Boehringer Ingelheim
moiety as a protecting group because it is part of the molecule.
Pharmaceuticals, Inc. 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-
0368, USA.
pyrazole-4-carboxylic acid (3) could be prepared, access to E-mail: bachir.latli@boehringer-ingelheim.com
If carbon-14 labeled 1-(2-(dimethylamino)-2-oxoethyl)-1H-
J. Label Compd. Radiopharm 2015, 58 390–394
Copyright © 2015 John Wiley & Sons, Ltd.

B. Latli et al.
Scheme 1. Proposed synthesis of [14C]-(1).
Scheme 2. Synthesis of [14C]-(1).
carbonate furnished the target carbon-14 labeled (1) in 84% yield
after chromatographic purification.
Conclusion
For the synthesis of deuterium labeled (1), an efficient route was Efficient and straightforward syntheses were developed for the
developed (Scheme 3). The chiral advanced intermediated (15) preparation of a potent and selective FLAP inhibitor labeled with
was prepared in two steps from (11) by either (i) condensation carbon-14 and deuterium. The critical introduction of carbon-14
with 1H-pyrazole-4-carboxylic acid followed by a Suzuki coupling was achieved using zinc cyanide, and [14C]-(1) was efficiently
to (14), or (ii) by Suzuki coupling between (11) and (14) first, prepared in six radioactive steps in 47% overall radiochemical
followed by condensation with 1H-pyrazole-4-carboxylic acid.9 yield with a specific activity of 51 mCi/mmol, and radiochemical
Both approaches were equally efficient. 2-Chloro-N,N- purity of 98.6%. The preparation of the deuterium labeled 2-
dimethyl-2H6-acetamide, [2H6]-(16) was obtained from the chloro-N,N-dimethylacetamide reagent allowed for the facile
reaction of chloroacetyl chloride and dimethylamine-2H6 in synthesis of deuterium labeled (1) from a very advanced
excellent yield.16 Condensation with (15) as described before intermediate (15) in 84% yield. These key isotopically labeled
provided [2H6]-(1) in 84% yield and with isotopic enrichment materials then enabled critical studies into the metabolism and
of more than 99%.
pharmacokinetics of this potent FLAP inhibitor.
J. Label Compd. Radiopharm 2015, 58 390–394
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

B. Latli et al.
Scheme 3. Synthesis of [2H6]-(1).
7.32(s, 1H), 7.19(d, J = 8.1 Hz, 2H), 6.88(d, J = 8.1 Hz, 2H), 5.19(s, 2H), 3.81
(s, 3H). 13C NMR (CDCl3) δ: 158.89, 139.06, 128.52, 128.24, 126.85,
113.23, 92.45, 55.41, 54.48.
Experimental procedures
Materials and methods
[ a Bruker 500-MHz
14C]-(1) were recorded on
1-(4-Methoxybenzyl)-1H-pyrazole-4-carbonitrile (9)
NMR spectra of
spectrometer using double encapsulated NMR tubes in deuterated
chloroform. 1H NMR and 13C NMR spectra of non-radioactive compounds
were acquired using Bruker 400 MHz. Liquid scintillation counting was
accomplished using a Beckman LS6500TA and UltimaGoldTM cocktail
(PerkinElmer, Boston, MA). HPLC analysis was performed on an Agilent
1200 instrument. HPLC conditions (a): mobile phase gradient 20 to
100% (MeCN/H2O both with 0.1% TFA) over 20 min, column: Eclipse
XDB C18 (4.5 mm × 150 mm, 5.0 μm), injection volume 10 μL. HPLC
conditions (b): mobile phase A: water 0.2% H3PO4 and B: MeCN/MeOH
(60/40); column: Zorbax SB C18 (4.6 mm × 150 mm, 1.8 μm); gradient
90% A to 50% A in 1 min then to 40% A in 3 min then to 30% A in
5 min to 15% in 7 min to 10% in min to 10% in 8 min to 5%A in 8.5 min
hold at 5% till 12 min. UV detection at 220 nm, flow rate 0.85 mL/min.
Chiral HPLC: column: Chiralpack AD-3 (4.6 mm × 150 mm, 3.0 μm). Mobile
phase A: heptane, B: 1-propanol, isocratic: 20:80 (A:B), flow : 1.5 mL/min;
run time 6 min, detection at 220 nm. Liquid chromatography-mass
spectrometry (LCMS) was acquired using a medium polar method: run
To
a 25-mL round bottomed flask were placed Zn(CN)2 (80 mg,
0.6 mmol), (8) (268 mg, 1 mmol) and DMF (5 mL). The suspension was
degassed three times (vacuum/nitrogen), and then DPPF (75 mg,
0.13 mmol) and Pd2dba3 (50 mg, 0.06 mmol) were added. The suspension
was degassed three times and then heated to 120 °C and stirred for 48 h.
After cooling to room temperature, water (20 mL) and EtOAc (20 mL)
were added to the reaction flask and then filtered through a short pad
of Celite®. The aqueous layer was removed, and the organic layer was
washed with brine (20 mL ×2). The organic phase was dried over MgSO4,
filtered and concentrated in vacuo to give 450 mg of dark viscous oil.
Purification using up to 10% EtOAc in CH2Cl2 and a 40-g disposable silica
gel column gave 210 mg of the pure material as cream colored solid in
98% yield. HPLCa: Rt = 6.9 min, 99.5%. 1H NMR (CDCl3) δ: 7.81(s, 1H),
7.70(s, 1H), 7.21(d, J = 8.1 Hz, 2H), 6.90(d, J = 8.1 Hz, 2H), 5.25(s, 2H), 3.81
(s, 3H). 13C NMR (CDCl3) δ: 160.09, 142.37, 133.87, 129.81, 126.28,
114.57, 113.38, 92.54, 56.33, 55.35.
time 2.0 min, gradient 95% water (0.1% TFA) and 5% MeCN (0.1%TFA) 1-(4-Methoxybenzyl)-1H-pyrazole-4-carbonitrile-14C, [14C]-(9)
to 5% water in 1.7 min, hold to 2 min at 5% water, flow 2.5 mL/min;
The cyanation was run using Zn(14CN)2 (200 mCi, 1.75 mmol, SA = 114.5
column: Agilent Zorbax C18 SB (4.6 mm × 30 mm, 3.5 μm). The data were
mCi/mmol), 4-bromopyrazole (6) (775 mg, 3 mmol), DMF (6 mL), DPPF
acquired on Waters Acquity™ Ultra Performance LC (Milford, MA, USA).
(161 mg, 0.3 mmol) and Pd2dba3 (265 mg, 0.3 mmol). Purification using
The radiochemical purity was measured using a radio-HPLC detector β-
up to 10% EtOAc in CH2Cl2 and a 40-g disposable silica gel column gave
Ram model 4 (LabLogic systems, Inc. Brandon, FL, USA) connected to
640 mg or 180 mCi of the pure material as cream colored solid in 90%
the Agilent instrument using IN-FLOW™ 2:1 liquid scintillation (LabLogic
yield. HPLCa: Rt = 6.9 min, 99%.
systems, Inc. Brandon, FL, USA). Zinc-[14C]cyanide was purchased from
Moravek Biochemicals (Brea, CA, USA). Dimethylamine-2H6 hydrochloride
1-(4-Methoxybenzyl)-1H-pyrazole-4-carboxylic acid (10)
with 99.4 atom % 2H and The rest of the reagents were obtained from
A mixture of the cyano derivative (9) (200 mg, 0.94 mmol) and 1N aq.
NaOH (5 mL) was heated to 110 °C and stirred overnight. LCMS showed
Sigma-Aldrich Company.
the conversion was complete. The solution was filtered through a
Whatman® 0.2-μm PP filter and concentrated in vacuo. The solid residue
Synthesis of [14C]-(1)
was then treated with aq. 1N HCl (5 mL). The first addition of 1-mL aq. HCl
gave a solution then as more aq. HCl was added a white precipitate
4-Bromo-1-(4-methoxybenzyl)-1H-pyrazole (8)
appeared. It was filtered and washed with water (1.5 mL) and dried at
50 °C under reduced pressure to give 196 mg of a white solid in 90%
yield. HPLCa: Rt = 4.75 min, 98%. 1H NMR (DMSO-d6) δ: 12.42(brs, 1H),
8.32(s, 1H), 7.79(s, 1H), 7.26(d, J = 8.1 Hz, 2H), 6.91(d, J = 8.1 Hz, 2H), 5.27
(s, 2H), 3.11(s, 3H). 13C NMR (DMSO-d6) δ: 163.71, 158.93, 140.64, 133.34,
129.42, 128.60, 114.93, 113.94, 55.09, 54.52.
A solution of 4-bromo-1H-pyrazole (6) (1.5 g, 10 mmol) in anhydrous DMF
(20 mL) was added para-methoxybenzyl chloride (2 mL, 14.7 mmol) and
anhydrous K2CO3 (4.2 g, 30 mmol). The resulting suspension was stirred
at room temperature for 14 h. LCMS showed one major product. The
reaction mixture was treated with 50 mL of water and extracted with
EtOAc (100 mL ×2). The combined extracts were dried over MgSO4,
filtered and concentrated in vacuo to give 5.4 g of colorless oil
contaminated with DMF and excess 4-methoxybenzyl chloride.
Purification on 150 g disposable column using CH2Cl2 and EtOAc as
eluent gave 2.65 g of material in 98% yield. HPLCa: Rt = 9.25 min, 95%.
LCMS, Rt = 0.76 min, MH+ = 121.47(100%). 1H NMR (CDCl3) δ: 7.47(s, 1H),
1-(4-Methoxybenzyl)-1H-pyrazole-4-carboxylic-14C acid, [14C]-(10)
The nitrile hydrolysis was run on 180 mCi of [14C]-(9) (196 mg, 0.9 mmol)
and 1N aq. NaOH (5 mL) to give 180 mg of a white solid in 85% yield
(170 mCi). HPLCa: Rt = 4.75 min, 95%.
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B. Latli et al.
phase column separator and concentrated in vacuo to give 1.52 g of a
honey colored oil, which was still contaminated with anisole. Purification
was conducted using a 40-g disposable silica gel column and CH2Cl2,
then 10% to 40% EtOAc:CH2Cl2, followed by 10% MeOH/CH2Cl2 to elute
the entire product. The pure material was collected and concentrated to
give 705 mg of a solid in 98% yield. HPLCa: Rt = 14.3 min, 98% pure using
254 nm, rad purity 98.2%.
(R)-3-(1-(4-Bromophenyl)-1-cyclopropylethyl)-5-(1-(4-methoxybenzyl)-
1H-pyrazol-4-yl)-1,2,4-oxadiazole (12)
The acid (10) (233 mg, 1.0 mmol) and CDI (195 mg, 1.2 mmol) were mixed
in anhydrous 1,4-dioxane (2 mL) at ambient temperature. The resulting
solution became cloudy in few minutes. It was heated to 55 °C and stirred
for 1 h before the amino-oxime (12) (275 mg, 0.9 mmol, 94% pure) was
added in one portion and the mixture was heated to 120 °C and stirred
for 14 h. LCMS and HPLCa showed one major product. After cooling to
room temperature, the suspension was concentrated under a stream of
nitrogen, and the residue was dissolved in CH2Cl2 (5 mL) and purified
using Combi-Flash Companion on a 40-g disposable silica gel column
and up to 10% MeOH/CH2Cl2 as eluent. The fractions containing the pure
material were combined and concentrated to give 390 mg of a white
residue. 1H NMR(CDCl3) δ: 8.05(s, 1H), 7.93(s, 1H), 7.42(d, J = 8.5 Hz, 2H),
7.24(d, J = 8.5 Hz, 2H), 7.20(d, J = 8.1 Hz, 2H), 6.89(d, J = 8.1 Hz, 2H), 3.80
(s, 3H), 3.70(s, 2H), 1.65(m, 1H), 1.50(s, 3H), 0.67(m, 1H), 0.28–0.61
(m, 3H). 13C NMR (CDCl3) δ:174.55, 169.37, 158.69, 156.86, 143.03,
138.36, 130.14, 129.37, 127.98, 127.61, 127.31, 125.69, 119.52, 113.27,
106.93, 65.88, 54.95, 54.13, 52.26, 49.42, 44.75, 42.09, 21.23, 20.97, 18.22,
16.94, 1.16, 0.08. LCMS, Rt = 1.38 min, MH +: M+2H+ : 481.1: 483.06
(1:1, 100%).
(R)-5-(4-(1-(5-(1H-pyrazol-4-yl)-1,2,4-oxadiazol-3-yl-3-14C)-1-
cyclopropylethyl) phenyl)pyrimidin-2-amine, [14C]-(15)
The bromide [14C]-(13) (435 mg, 1.2 mmol) was dissolved in isopropanol
(3.7 mL), and a solution of K3PO4 (530 mg, 2.4 mmol) in water (1 mL) was
added. The solution was purged with argon for few minutes. Pd2(dba)3,
(110 mg, 0.12 mmol), tri-tert-butyl phosphonium tetrafluoroborate
(70 mg, 0.24 mmol) and the boronate (14) (335 mg, 1.5 mmol) were
added. The dark mixture was heated in an oil-bath at 90 °C to give a faint
yellow mixture after 20 min at 90 °C, and stirred for 18 h. HPLCa showed a
new single product with the starting material consumed completely. The
new product co-eluted with the product from the unlabeled synthesis.
Water (4 mL) was added to the reaction flask, and the mixture was
extracted with 1:4 EtOH:CHCl3 (10 mL ×3). The combined extracts were
dried over anhydrous Na2SO4, filtered through a column separator and
(R)-3-(1-(4-Bromophenyl)-1-cyclopropylethyl)-5-(1-(4-methoxybenzyl)- concentrated in vacuo to give 0.97 g of a bright yellow solid, which was
1H-pyrazol-4-yl)-1,2,4-oxadiazole-3-14C, [14C]-(12)
used as is in the next step. HPLC, Rt = 7.0 min, 95%.
Acid [14C]-(10) (100 mCi, ≈1.92 mmol) and CDI (350 mg, 2.16 mmol) were
mixed in anhydrous 1,4-dioxane (4 mL) at room temperature to give a
1,2,4-oxadiazol-3-14C-5-yl)-1H-pyrazol-1-yl)-N,N-dimethylacetamide,
solution. It was heated to 55 °C and stirred for 1 h before (R)-2-(4-
bromophenyl)-2-cyclopropyl-N-hydroxypropionamide (11) (577 mg,
1.92 mmol) was added in one portion and the mixture was heated to
(R)-2-(4-(3-(1(4-(2-Aminopyrimidin-5-yl)phenyl)-1-cyclopropylethyl)-
[14C]-(1)
To a solution of the pyrazole derivative [14C]-(15) (454 mg, 1.2 mmol) and
120 °C and stirred overnight. TLC showed the oxime was all consumed
and the presence of a non-polar product which eluted very fast on TLC
using 5% MeOH/CH2Cl2. Most of the dioxane was removed by a stream
of argon, and the residue was dissolved in CH2Cl2 (10 mL) and washed
with water (10 mL). The organic phase was removed, and the aqueous
was extracted with CH2Cl2 (10 mL ×3). The combined extracts were dried
over anhydrous Na2SO4, filtered and concentrated in vacuo to give 1.27 g
of a honey colored oil, which was used as is in the next step. HPLCa,
Rt = 19.7 min, 98.5% at 254 nm.
2-chloro-N,N-dimethylacetamide (16) (0.27 mL, 2.4 mmol) in anhydrous
DMF (15 mL) was added K2CO3 (669 mg, 4.8 mmol), and the mixture
was stirred overnight at room temperature. HPLCb showed only one
major peak that co-elutes with unlabeled sample. Water (40 mL) was
added, and the mixture was extracted with CH2Cl2 (40 mL ×3). The
combined extracts were dried over Na2SO4, filtered through a column
separator and concentrated under reduced pressure at 40 °C (to remove
DMF) to give 1.36 g of a honey colored residue,. The crude product was
purified using silica gel flash chromatography on a 40-g disposable
column and up to 10% MeOH/CH2Cl2. The fractions containing purified
material were combined and concentrated in vacuo to give 340 mg of
foam in 84% yield and 97% radiochemical purity. A second purification
using a 40-g silica gel disposable column and up to 10% MeOH/CH2Cl2
gave 186 mg of an off-white solid (20.5 mCi) with a specific activity of
51 mCi/mmol. HPLCb: Rt = 6.0 min, 98.6% radio-purity, UV at 220 nm,
(R)-3-(1-(4-Bromophenyl)-1-cyclopropylethyl)-5-(1H-pyrazol-4-yl)-
1,2,4-oxadiazole (13)
A solution of the protected pyrazole (12) (274 mg, 0.57 mmol), anisole
(0.62 mL, 5.7 mmol) and trifluoroacetic acid (0.22 mL, 2.87 mmol) in
CH2Cl2 (3 mL) was stirred at room temperature. Concentrated sulfuric
acid (0.1 mL, 18.4 M) was added at room temperature, and the resulting
was stirred for 14 h. The reaction solution was added dropwise to a
saturated solution of NaHCO3 (5 mL) at 0 °C and then extracted with
CH2Cl2 (10 mL ×2). The combine extracts were concentrated in vacuo at
40 °C to distil off the anisole and to give 340 mg of oil. HPLCa,
Rt = 11.5 min (100% at 254 nm), 1H NMR (CDCl3) δ: 8.16(s, 2H), 7.44
(d, J = 8.5 Hz, 2H), 7.24(d, J = 8.5 Hz, 2H), 1.67(m, 1H), 1.53(s, 3H), 0.70
1
purity: 98.7%. Chiral purity: ee = 98.5%. H NMR (CDCl3, 500 MHz): δ 8.61
(s, 2H), 8.20(s, 1), 8.10(s, 1H), 7.51(ABq, J = 8.5 Hz, 4H), 5.20(s, 2H), 5.01
(s, 2H), 3.10(s, 3H), 2.85(s, 3H), 2.71(m, 1H), 1.52(s, 3H), 0.72(m, 1H), 0.54
(m, 1H), 0.42(m, 1H), 0.31(m, 1H).
Synthesis of [2H6]-(1)
(m, 1H), 0.32–0.61(m, 3H). LCMS, Rt = 1.02 min: 360.37: 362.38 (1:1), 100%. 2-Chloro-N,N-dimethylacetamide-2H6, [2H6]-(16)
To a three-necked round-bottomed flask were introduced dimethyl-2H6-
amine hydrochloride (1.75 g, 20 mmol) and methylene chloride (20 mL).
(R)-3-(1-(4-Bromophenyl)-1-cyclopropylethyl)-5-(1H-pyrazol-4-yl)-
1,2,4-oxadiazole-3-14C, [14C]-(13)
The mixture was cooled to À30 °C and an aqueous solution of NaOH
To a solution of [14C]-(12) (963 mg, 2 mmol), anisole (2.2 mL, 20 mmol) (20 wt%, 20 mL) was added. Chloroacetyl chloride (1.8 mL, 22 mmol)
and trifluoroacetic acid (0.77 mL, 10 mmol) in CH2Cl2 (10 mL) was added was dissolved in CH2Cl2 (10 mL) and added dropwise keeping the
dropwise a concentrated solution of H2SO4 (0.35 mL, 6.5 mmol) at room internal temperature below À25 °C. The reaction mixture was stirred for
temperature under argon atmosphere. The resulting was stirred for
14 h. HPLCa showed 2:1 conversion of product to starting material. An
30 min below À15 °C and then transferred to a separatory funnel. The
organic phase was removed, and the yellowish aqueous layer was
additional 150 μL of concentrated H2SO4 was added, and the mixture washed with CH2Cl2 (20 mL ×2). The combined organic layers were
was stirred overnight. HPLC showed that most of the starting material washed with brine (30 mL), dried over MgSO4, filtered and concentrated
was converted to desired product. The reaction mixture was added
dropwise to a saturated solution of NaHCO3 (40 mL) at °C. After gas
evolution ceased, the mixture was extracted with CH2Cl2 (20 mL ×3),
under reduced pressure to give 2.75 g of colorless oil in 97% yield. Rf in
10% MeOH/DCM = 0.6, co-eluted with unlabeled sample. 1H NMR (CDCl3)
δ: 4.15(s, 2H). 13C NMR (CDCl3) δ: 166.5, 41.18, 36.7(m), 34.9(m). 102144-
and the combined extracts were dried over Na2SO4, filtered through a 056. LCMS: Rt = 0.98 min, MH+ 130.16 (100%).
J. Label Compd. Radiopharm 2015, 58 390–394
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B. Latli et al.
[3] P. Aparoy, K. K. Reddy, P. Reddanna, Curr. Med. Chem. 2012; 19,
3763–3778.
[4] D. Pettersen, Ö. Davidsson, C. Whatling, Bioorg. Med. Chem. Lett.
2015, 25, 2606–2612.
(R)-2-(4-(3-(1-(4-(2-Aminopyrimidin-5-yl)phenyl)-1-cyclopropylethyl)-
1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl]-N,N-dimethyl-2H6-acetamide,
[2H6]-(1)
To a solution of (15) (0.6 g, 1.6 mmol) and [2H6]-(16) (0.34 mL, 2.9 mmol)
in anhydrous DMF (10 mL) was added K2CO3 (0.45 g, 3.2 mmol), and the
mixture was stirred for 14 h at room temperature. HPLC showed only
one major peak that co-elutes with a standard sample. Water (20 mL)
was added, and the mixture was extracted with CH2Cl2 (20 mL ×3). The
combined extracts were dried over Na2SO4, filtered and concentrated
under reduced pressure at 40 °C. The residue was purified using a
disposable 120-g silica gel column and up to 10% MeOH in CH2Cl2. The
fractions containing the pure product were combined and concentrated
under reduced pressure to give 650 mg of foam. This foam was heated
with hexanes (15 mL) and after cooling to room temperature the mixture
was filtered and washed with hexanes (20 mL) to give after further drying
625 mg of a white solid in 84% yield. HPLCb: Rt = 6.2 min, 99.2%. Chiral
HPLC ee% = 98.5%. LCMS, Rt = 0.77 min, MH+ = 464.94, 100%. HRMS:
[C24H20D6N8O2+H+] calculated 465.26281, found 465.26252. 1H NMR
(CDCl3, 400 MHz): δ 8.52(s, 2H), 8.19(s, 1), 8.06(s, 1H), 7.45(q, J = 8.5 Hz,
4H), 5.12(s, 2H), 5.02(s, 2H), 1.72(m, 1H), 1.52(s, 3H), 0.71(m, 1H), 0.49
(m, 1H), 0.42(m, 1H), 0.37(m, 1H). 13C NMR (CDCl3, 103MHz) δ: 174.76,
169.16, 164.18, 160.94, 155.21, 143.47, 138.38, 132.33, 131.63, 126.60,
124.63, 123.45, 107.54, 52.17, 42.07, 35.8(m), 35.4(m), 21.35, 18.33, 1.14,
0.01.
[5] R. N. Young, Eur. J. Med. Chem. 1999; 34, 671–685.
[6] M. Peters-Golden, W. R. Henderson, N. Engl. J. Med. 2007; 357,
1841–1854.
[7] (a) C. D. Funk, Nat. Rev. Drug Discov. 2005; 4, 664–672; (b) E. Osher, G.
Weisinger, R. Limor, K. Tordjman, N. Stern, Mol. Cell. Endcrinol. 2006;
252, 201-206.
[8] H. Takahashi, D. Riether, A. Bartolozzi, T. Bosanac, V. Berger, R. Binetti,
J. Broadwater, Z. Chen, R. Crux, S. De Lombaert, R. Dave, J. A. Dines, T.
Fadra-Khan, A. Flegg, M. Garrigou, M.-H. Hao, J. D. Huber, J.M.
Hutzler, S. Kerr, A. Kotey, W. Liu, H. Y. Lo, P. L. Loke, P. E. Mahaney,
T. M. Morwick, S. Napier, A. Olague, E. Pack, A. Padyana, D. S.
Thomson, H. Tye, L. Wu, R. M. Zindell, A. Abeywardane, and T.
Simpson, J. Med. Chem. 2015; 58, 1669–1690.
[9] K. R. Fandrick, J. A. Mulder, N. D. Patel, J. Gao, M. Konrad, E. Archer, F.
G. Buono, A. Duran, R. Schmid, J. Daeubler, J.-N. Desrosiers, X. Zeng,
S. Rodriguez, S. Ma, B. Qu, Z. Li, D. R. Fandrick, N. Grinberg, H. Lee, T.
Bosanac, H. Takahashi, Z. Chen, A. Bartolozzi, P. Nemoto, C. A.
Busacca, J. J. Song, N. K. Yee, P. E. Mahaney, and C. H. Senanayake,
J. Org. Chem. 2015; 80, 1651–1660.
[10] (a) M. F. Hawthorne, Metallacarborane Chelates, US patent
5066479A1, 1991; (b) H. Schoen, Justus Liebigs Ann. Chem., 1959;
625, 55–60.
[11] M. Sundermeier, A. Zapf, M. Beller, Eur. J. Inorg. Chem. 2003; Issue 19,
3513–3526.
[12] P. E Maligres, M. S. Waters, F. Fleitz, D. Askin, Tetrahedron Lett. 1999;
40, 8193–8195.
References
[13] R. Jones, J. Am. Chem. Soc. 1949; 71, 3994–4000.
[14] C. Subramanyam, Synth. Commun. 1995; 25, 761–774.
[15] I. T. Forbes, C. N. Johnson, M. Thompson, J. Chem. Soc. Perkin Trans 1,
1992; 2, 275–281.
[1] (a) J. F. Evans, A. D. Fergusson, R. T. Mosley, J. H. Hutchinson, Trends
Pharm. Sci., 2007; 29, 72–78; (b) C. D. Funk, Science, 2001; 294,
1871–1875.
[2] (a) B. Samuelsson, Science 1983; 220, 568–575; (b) J. M. Drazen, E.
Israel, P. M. O’bryne, N. Engl. J. Med. 1999; 340, 197–206.
[16] M. Kem, N. V. Nguyen, D. J. Cross, J. Org. Chem. 1981; 46, 5188–5192.
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