Potent and selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 labeled with carbon-13 and carbon-14

Article abstract of DOI:10.1002/jlcr.3518

(S)-6-(2-Hydroxy-2-methylpropyl)-3-((S)-1-(4-(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl)ethyl)-6-phenyl-1,3-oxazinan-2-one (1) and (4aR,9aS)-1-(1H-benzo[d]midazole-5-carbonyl)-2,3,4,4a,9,9a-hexahydro-1-H-indeno[2,1-b]pyridine-6-carbonitrile hydrochloride (2) are potent and selective inhibitor of 11β-hydroxysteroid dehydrogenase type 1 enzyme. These 2 drug candidates developed for the treatment of type-2 diabetes were prepared labeled with carbon-13 and carbon-14 to enable drug metabolism, pharmacokinetics, bioanalytical, and other studies. In the carbon-13 synthesis, benzoic-¹³C₆ acid was converted in 7 steps and in 16% overall yield to [¹³C₆]-(1). Aniline-¹³C₆ was converted in 7 steps to 1H-benzimidazole-1-2,3,4,5,6-¹³C₆-5-carboxylic acid and then coupled to a tricyclic chiral indenopiperidine to afford [¹³C₆]-(2) in 19% overall yield. The carbon-14 labeled (1) was prepared efficiently in 2 radioactive steps in 41% overall yield from an advanced intermediate using carbon-14 labeled methyl magnesium iodide and Suzuki-Miyaura cross coupling via in situ boronate formation. As for the synthesis of [¹⁴C]-(2), 1H-benzimidazole-5-carboxylic-¹⁴C acid was first prepared in 4 steps using potassium cyanide-¹⁴C, then coupled to the chiral indenopiperidine using amide bond formation conditions in 26% overall yield.

Full text of DOI:10.1002/jlcr.3518

Potent and Selective Inhibitors of 11-Hydroxysteroid Dehydrogenase Type 1 Labeled
with Carbon-13 and Carbon-14
Bachir Latli*, Matt Hrapchak, Jolaine Savoie, Yongda Zhan, Carl A. Busacca, and Chris H.
Senanayake
Chemical Development, Boehringer Ingelheim Pharmaceuticals, Inc. 900 Ridgebury Road,
P.O. Box 368, Ridgefield, Connecticut 06877-0368, USA
Abstract
(S)-6-(2-Hydroxy-2-methylpropyl)-3-((S)-1-(4-(1-methyl-2-oxo-1,2-dihydropyridin-4-
yl)phenyl)ethyl)-6-phenyl-1,3-oxazinan-2-one (1) and (4aR,9aS)-1-(1H-benzo[d]midazole-5-
carbonyl)-2,3,4,4a,9,9a-hexahydro-1-H-indeno[2,1-b]pyridine-6-carbonitrile hydrochloride
(2) are potent and selective inhibitor of 11-hydroxysteroid dehydrogenase type 1 enzyme.
These two drug candidates developed for the treatment of type-2 diabetes were prepared
labeled with carbon-13 and carbon-14 to enable drug metabolism, pharmacokinetics,
bioanalytical, and other studies. In the carbon-13 synthesis, benzoic-13C6 acid was converted
in seven steps and in 16% overall yield to [13C6]-(1). Aniline-13C6 was converted in seven
steps to 1H-benzimidazole-1-2,3,4,5,6-13C6-5-carboxylic acid and then coupled to a tricyclic
chiral indenopiperidine to afford [13C6]-(2) in 19% overall yield. The carbon-14 labeled (1)
was prepared efficiently in two radioactive steps in 41% overall yield from an advanced
intermediate using carbon-14 labeled methyl magnesium iodide and Suzuki-Miyaura cross
coupling via in situ boronate formation. As for the synthesis of [14C]-(2), 1H-benzimidazole-
5-carboxylic-14C acid was first prepared in four steps using potassium cyanide-14C, then
coupled to the chiral indenopiperidine using amide bond formation conditions in 26% overall
yield.
Key words: 11HSD1, Type-2 diabetes, Radiosynthesis, Carbon-14, Carbon-13
Introduction:
11-Hydroxysteroid dehydrogenase type 1 (11HSD1) is an NADPH-dependent enzyme
expressed in key metabolic tissues.1,2 It catalyzes the conversion of cortisone into active
glucocorticoid cortisol,3 also known as stress hormone, Figure 1.
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O
HO
O
HO
OH
NAD+
NADH
HO
OH
O
HSD11B2
HSD11B1
H
H
H
H
H
H
O
O
NADPH
NADP+
Cortisol
Cortisone
Figure 1: Conversion of cortisone to cortisol
High levels of glucocorticoids were associated with several serious clinical features including
metabolic syndrome like type-2 diabetes.4 The enzyme 11-HSD1 is considered to be critical
in the development of metabolic syndrome and thus a therapeutic target for treating type-2
diabetes.5-11 The clinical candidates (S)-6-(2-hydroxy-2-methylpropyl)-3-((S)-1-(4-(1-methyl-
2-oxo-1,2-dihydropyridin-4-yl)phenyl)ethyl)-6-phenyl-1,3-oxazinan-2-one
(4aR,9aS)-1-(1H-benzo[d]midazole-5-carbonyl)-2,3,4,4a,9,9a-hexahydro-1-H-indeno[2,1-
(1)12
and
b]pyridine-6-carbonitrile hydrochloride (2)13, Figure 2, inhibit the conversion of cortisone to
cortisol with nanomolar potency, an average IC50 of 11 nM in human adipose tissue ex vivo in
case of (1).12 Herein, we describe the synthesis of these two compounds labeled with carbon-
13 and carbon-14.
O
N
N
O
N
HCl
O
N
N
N
O
OH
(1)
(2)
Figure 2: Structural formula of clinical candidates (1) and (2)
Results and Discussion
The separation of the molecular weights of the isotopically labeled and unlabeled drug
candidate by more than two mass units is highly desirable, so one can easily distinguish
between the drug candidate and its metabolites using mass spectrometry. Benzoic acid-13C6
and aniline-13C6 are suitable building blocks to access several intermediates in the synthesis
of carbon-13 labeled drug candidates. The synthesis of carbon-13 labeled (1) was accomplished
in seven steps in 16% overall yield starting form benzoic acid-13C6 , Scheme 1. The benzoyl
chloride [13C6]-(4) obtained from refluxing benzoic acid [13C6]-(3) in thionyl chloride was
subjected to Friedel-Crafts reaction using aluminum trichloride in dichlorethane and a slow
bubbling of ethylene gas in the reaction mixture at ambient temperature to afford carbon-13
labeled 3-chloropropiophenone [13C6]-(5) in 69% yield.14 Grignard reaction with methylallyl
o
magnesium chloride in the presence of cerium chloride in THF at – 78 C gave the tertiary
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alcohol [13C6]-(6) in 95% yield. The use of cerium chloride has been reported to enhance the
yields of the addition of Grignard reagents to carbonyl compounds by eliminating undesired
side reactions like enolization, reduction, condensation, conjugate addition, and pinacol
coupling.15 Reaction of (S)-4-bromo--methylbenzylisocyanate (7) with the anion generated
from treating this alcohol with lithium hexamethyldisilazane gave a 1:1 mixture of
bromooxazinones [13C6]-(8) and [13C6]-(9).16 The desired bromooxazinone [13C6]-(9) was
easily separated using silica gel chromatography. The enantioselective synthesis of (9) using
asymmetric methyllylation catalyzed by (S)-7,5-difluoro-BINOL has since been reported.17
Oxidation of [13C6]-(9) with meta-choloroperbenzoic acid in methylene chloride to the
epoxide derivative [13C6]-(10) was accomplished in 96% yield. Opening of the oxirane with
super-hydride® in THF afforded alcohol [13C6]-(11) in 96% yield.18 Finally, the
transformation of this alcohol to the final compound [13C6]-(1) was achieved using
Suzuki−Miyaura coupling with the boronic acid derivative (12) in 60% yield after silica gel
purification.17
Scheme 1: Synthesis of [13C6]-(1)
For the synthesis of [13C6]-(2), the benzimidazole (20) was chosen to incorporate the carbon-
13 atoms to avoid the lengthy synthesis and stereochemical issues associated with
indenopiperidine (21), Scheme 2. Thus, aniline-13C6 was iodinated at the para-position in
aqueous sodium bicarbonate with molecular iodine in quantitative yield.19 In order to
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attenuate the electron density of the aromatic ring for mono-nitration, the amine was
protected as the acetamide [13C6]-(15) in 87% yield using acetic anhydride in the presence of
triethylamine and DMAP.20 Reaction of [13C6]-(15) with two equivalents of copper (I)
cyanide in DMF provided the benzonitrile [13C6]-(16) in 93% yield.21 The cooperative nature
of the ortho/para directing acetamide and meta directing nitrile set the stage for a selective
ortho nitration to the acetamide group. As predicted, the nitration of [13C6]-(16) proceeded
smoothly to provide [13C6]-(17) using 1.8 equivalents of nitric acid keeping the internal
temperature of the reaction below 10 ºC.22 Treatment of [13C6]-(17) with 60% H2SO4 at 95 ºC
led to hydrolysis of the nitrile and removal of the protecting group to give 4-amino-5-
nitrobenzoic acid [13C6]-(18) in 77% yield. Reduction of the nitro group to the diamino-acid
[13C6]-(19) was accomplished via transfer hydrogenation with ammonium formate and 10%
Pd/C in refluxing methanol in excellent yield.23 Cyclodehydration of this compound was
performed in the presence of triethyl orthoformate in refluxing toluene to give the desired 5-
benzimidazole carboxylic acid [13C6]-(20) in 92% yield.24 Finally amide bond formation
using propylphosphonic anhydride solution (T3P®, 50% solution in THF) and triethylamine
in acetonitrile between this acid and (4aR,9aS)-2,3,4,4a,9,9a-hexahydro-1H-indeno[2,1-
b]pyridine-6-carbonitrile (21)25 gave [13C6]-(2) in 36% yield after silica gel chromatography.
Scheme 2: Synthesis of [13C6]-(2)
In the carbon-14 synthesis of compound (1), the available intermediate (9)17 was first
o
subjected to ozonolysis at -78 C followed by reductive work up with polystyrene-supported
triphenylphospine (PS-PPh3) to afford the ketone (22) in quantitative yield, Scheme 3.
o
Carbon-14 methyl magnesium iodide was freshly prepared and added to ketone (22) at 0 C
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to give the tertiary alcohol [14C]-(11) in 68% yield. Compound [14C]-(11) was then used in
the Suzuki−Miyaura coupling using in situ formation of the boronate26 of 4-bromo-1-
methypyridine-2-(1H)-one (23) to afford [14C]-(1) with a specific activity of 54.7 mCi/mmol
after silica gel chromatography purification followed by crystallization from acetone and
water. In general, this final transformation was found to give higher amounts of the
desbromo-derivative of (11) on larger scales than the coupling of the boronic acid (12) to the
bromide (11) as described previously in the carbon-13 synthesis.17
Scheme 3: Synthesis of [14C]-(1)
To prepare [14C]-(2), Scheme 4, the cyanation of the commercially available 5-bromo-2-
nitroaniline (24) with K14CN in the presence of CuI and Pd(PPh3)4 proceeded to give the
desired nitrile [14C]-(25) in 56% yield. Hydrolysis of the nitrile was accomplished with 70%
H2SO4 at 95 ºC and gave the acid [14C]-(26) in quantitative yield. Reduction of the nitro
group via transfer hydrogenation as described above proceeded quantitatively to give 3,4-
diaminobenzoic acid [14C]-(19). Treatment of the diamine with triethylorthoformate gave the
desired 5-benzimidazole carboxylic-14C acid [14C]-(20) in 81% yield with a specific activity
of 56.6 mCi/mmol. The commercially available carbon-14 labeled triethylformate would
have provided a shorter synthetic route. However, the high cost of this material and the fact
that at least two equivalents are needed to completed the synthesis of 14C]-(20) was not
considered for the synthesis of [14C]-(2). Amide bond formation with the chiral (21) followed
by hydrogen chloride salt formation gave [14C]-(2) in 49% yield over these two final steps.
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This six-step radiosynthesis of [14C]-(2) was carried out with only one chromatographic
purification, thus decreasing the amount of radioactive waste.
Scheme 4: Synthesis of [14C]-(2)
Conclusion
We have described the synthesis of carbon-13 and carbon-14 labeled of two potent and
selective 11-HSD1 inhibitors. In the carbon-13 synthesis, benzoic-13C6 acid was converted
in seven steps to [13C6]-(1) in 16% overall yield. The preparation included a Friedel-Crafts
reaction using ethylene gas, a cerium-modified Grignard reaction with methylally magnesium
bromide, and a Suzuki-Miyaura cross coupling with a key boronic acid derivative gave
[13C6]-(1) in 60% yield with more than 99% isotopic enrichment and 99% chemical purity.
Aniline-13C6 was converted in seven steps to 5-benzimidale carboxylic acid [13C6]-(20) in
52% yield then coupled to a chiral tricyclic indenopiperidine (21) using amide bond
formation conditions to afford [13C6]-(2) with more than 99% chemical purity and isotopic
enrichment. In the carbon-14 synthesis, the availability of the chiral 1,3-oxazinan-2-one
derivative (9) from in house synthesis shortened the synthesis of carbon-14 labeled (1) to just
two radioactive steps. The drug candidate [14C]-(1) was obtained with 97.6% radiochemical
purity and a specific activity of 54.7 mCi/mmol. 5-Benzimidozle carboxylic-14C acid was
prepared in four steps in 45% yield and then coupled under amide bond formation conditions
to the chiral amine (21) followed by salt formation to give [14C]-(2) with 98.6%
radiochemical purity and a specific activity of 56.6 mCi/mmol.
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Experimental Procedures
Experimental:
Liquid chromatography-mass spectrometry (LCMS) was acquired using a medium polar
method: run time 2.0 min, gradient 95% water (0.1% TFA) and 5% MeCN (0.1%TFA) to 5%
water in 1.7 min, hold to 2 min at 5% water, flow 2.5 mL/min; column: Agilent Zorbax C18
SB (4.6 mm× 30 mm, 3.5 μm). The data were acquired on Waters Acquity™ Ultra
Performance LC (Milford, MA, USA). NMR spectra were recorded with a Bruker 400 MHz
and 500 MHz spectrometers using deuterated chloroform or dimethyl sulfoxide as a solvent
and tetramethyl silane as the internal standard (Sigma-Aldrich, USA). HPLC was performed
using an HPLC system comprised of an Agilent 1200, IN/US β-RAM-4, IN-FLOW™
counting solution (LabLogic systems, Inc. Brandon, FL, USA) and using Laura Lite software
for data evaluation for radioactive compounds. HPLC Conditions: Method (a) mobile phase:
gradient A: MeCN (0.1% TFA), B: H2O (0.1% TFA) 20% A to 100% A in 22 min, flow rate:
1 mL/min, column: Eclipse XDB C18 (4.5 mm × 150 mm, 5m); Method (b) mobile phase
gradient 30 to 98 %A, A: MeCN, B: Water 0.2% H3PO4/MeCN in 12 min, flow rate: 1
mL/min, column: Halo C8 (4.6 × 150 mm, 2.7 m); Method (c) mobile phase gradient 5 to
95% MeCN in water (0.1% TFA) in 12 min, column Zorbax XDB C18 (4.6 x 150 mm, 5.0
m); Method (d) mobile phase gradient 5 to 98% MeCN in water (0.1% H3PO4, 0.2%
HClO4) in 10 min, column: Zorbax SB-Phenyl (4.6 x 150 mm, 1.8 m). Chiral HPLC
conditions: Method (a) mobile phase A: MeCM, B: 0.1% HCO2H in water adjusted with
NH4OH to pH 4.0, isocratic 40:60 A/B, detection UV 220 nm, column: Chiralcel OJ-RH (4.6
x 150 mm, 5m); Method (b) mobile Phase: A: Water 0.1% AcOH, buffered to pH = 4.7 with
NH4OH, B: MeCN, Isocratic: A/B 95/5, detection: UV 230 nm, column: Chiralpak AD (4.6 x
250 mm, 5 µm). Liquid scintillation counting was carried out using a Beckman LS6500 and
Ultima GoldTM cocktail (Beckman, Fullerton, CA, USA). [14C]-Methyl Iodide was purchased
from American Radiolabeled Chemicals (St. Louis, MO, USA). Potassium cyanide-14C was
obtained from Quotient Bioresearch Radiochemicals (Cardiff, UK). Benzoic-13C6 acid with
min 99 atom % 13C and aniline-13C6 were purchased from Isotec (Miamisburg, OH, USA).
Synthesis of [13C6]-(1) and [13C6]-(2)
Benzoyl-1,2,3,4,5,6-13C6 chloride, [13C6]-(4): A solution of benzoic-ring-13C6 acid (2.1 g,
16.4 mmol) and thionyl chloride (6.2 mL, 85 mmol) in toluene (10 mL) was heated to reflux
for 8 h. After cooling to room temperature, the resulting yellow solution was concentrated in
vacuo and the residue was treated with toluene (10 mL) and concentrated again. This
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procedure was repeated twice to remove excess thionyl chloride. The residue (2.3 g) was used
as is in the following step.
3-Chloro-1-(phenyl-13C6)propan-1-one, [13C6]-(5): To a suspension of aluminum chloride
(2.2 g, 16.5 mmol) in 1,2-dichloroethane (10 mL) was added a solution of [13C6]-(4) (2.3 g,
16 mmol) in 1,2-dichloroethane (10 mL). The flask containing the benzoyl chloride was
rinsed once with dichloroethane (5 mL) and added to the reaction flask at ambient
temperature to give a brown solution. Ethylene was then bubbled slowly into this solution for
4 h and the resulting solution was stirred for 14 h. LCMS showed no starting material. A
solution of 4 N aqueous HCl (45 mL) was added slowly (exothermic) to give a precipitate,
which dissolved in contact with the organic layer. The aqueous phase was extracted with
ether (80 mL ×3) and washed with water (50 mL ×3), aqueous saturated NaHCO3 (50 mL
×3), brine (50 mL), dried over MgSO4, filtered, and concentrated in vacuo to give 2.1 g of
pale yellow oil which solidified upon standing at room temperature. This crude material was
purified by Combiflash using a 150 g silica gel cartridge and 100% CH2Cl2 to give 1.92 g of
a pale yellow solid in 69% yield. TLC: 20% EtOAc:Hexanes: Rf = 0.44. HPLC method (a):
9.55 min (98%) identical to an Aldrich sample. LCMS: Rt = 1.56 min, MH+ = 175.04 (100%)
.1H NMR (CDCl3) : 8.10-8.25(m, 1H), 7.60-7.85 (m, 2.5H), 7.35-7.42(m, 0.5H), 7.20-
7.34(m, 1H), 3.93(t, J = 7.2 Hz, 2H), 3.46(t, J =7.2 Hz, 2H). 13C NMR(CDCl3) : 196.7(d, J
= 57 Hz), 149.1(m), 136.5(m), 133.4(m), 128.5(m), 41.3(d), 38.6.
1-Chloro-5-methyl-3-(phenyl-13C6)hex-5-en-3-ol, [13C6]-(6): In a two-neck round bottomed
flask was added cerium III chloride (1.5 g, 6.1 mmol) and anhydrous THF (10 mL). The
suspension was stirred for 30 min at room temperature to give a thick slurry. To this mixture,
2-methylallylmagnesium chloride solution (12 mL, 0.5 M in THF) was added slowly to give
an orange mixture. The mixture was cooled to -30 oC and [13C6]-(5) (1.6 g, 4.6 mmol) in THF
o
(10 mL) was added dropwise keeping the internal temperature below -25 C. A colorless
mixture was obtained by the end of the addition. The mixture was stirred at -30 oC for 20
o
min, then warmed to 0 C in an ice bath and stirred for 30 min. The reaction was quenched
with 1N aqueous HCl (150 mL) and extracted with MTBE (200 mL), dried over MgSO4,
filtered and concentrated in vacuo to give 2.1 g of yellow oil. Purification using silica gel
chromatography and CH2Cl2 as eluent gave 1.85 g of product in 95% yield. 1H NMR (CDCl3)
δ: 8.15-8.27(m, 1H), 7.62-7.87(m, 2.5H), 7.37-7.444(m, 0.5H), 7.22-7.36(m, 1H), 4.92 (m,
1H), 4.77(m, 1H), 3.57(ddd, J = 5.5, 11, 11 Hz, 1H), 3.13(ddd, J = 5.5, 11, 11 Hz, 1H),
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2.67(d, J = 13.5 Hz, 1H), 2.54(dd, J = 5, 13.5 Hz, 1H), 2.52(bs, 1H), 2.32(m, 2H), 1.28(s,
3H).
(S)-3-((S)-1-(4-Bromophenyl)ethyl)-6-(2-methylallyl)-6-(phenyl-13C6)-1,3-oxazinan-2-
one, [13C6]-(9): A solution of [13C6]-(6) (1.66 g, 5 mmol) and (S)-1-bromo-4-(1-
isocyanatoethyl)benzene (7) (1.25 g, 4.8 mmol) in anhydrous THF (15 mL) was stirred at
room temperature, then cooled in an ice bath and a solution of LiHMDS in THF (1 M, 5.5
o
ml) was added slowly keeping the internal temperature below 10 C. The reaction solution
was warmed to room temperature and stirred for 10 h. A solution of 2 N aqueous HCl (50
mL) was added and the mixture was extracted with MTBE (100 mL ×2). The combined
extracts were washed with water (100 mL), brine (50 mL), dried over MgSO4, filtered and
concentrated in vacuo to give 2.6 g of foam as a mixture of [13C6]-(8), Rf = 0.45, and [13C6]-
(9), Rf = 0.27 in 30% EtOAc:Hexanes and visualized with 10% solution of phosphomolybdic
acid (PMA) in ethanol. Purification by Combiflash using 150 g disposable silica gel cartridge
and up to 30% EtOAc in Hexanes gave 0.9 g of [13C6]-(9) in 43% yield. LCMS: MH+ (420.3,
1
422,2 1:1). HPLC method (a), Rt = 14.89 min (98%), for [13C6]-(8), Rt = 15.24 min. HNMR
(CDCl3) : 7.65(m, 3H), 7.47(d, J = 8.5 Hz, 2H), 7.10-7.40(m, 2H), 6.64(d, J = 8.5 Hz, 2H),
5.59(q, J = 7.1 Hz, 1H), 4.86(d, J = 8.1 Hz, 2H), 2.91(m, 2H), 2.56(dq, J = 2.0, 13.8 Hz, 2H),
2.1-2.4(m, 2H), 1.64(s, 3H), 1.47(d, J = 7.1 Hz, 3H).
(6S)-3-((S)-1-(4-Bromophenyl)ethyl]-6-((2-methyloxiran-2-yl)methyl)-6-(phenyl-13C6)-
1,3-oxazinan-2-one, [13C6]-(10): To a 100 mL flask containing the alkene [13C6]-(9) (0.5 g,
0.95 mmol) and CH2Cl2 (15 mL) under a nitrogen atmosphere, was added m-CPBA (0.42 g,
1.7 mmol). The resulting colorless solution was stirred at room temperature for 2 h. HPLC,
method (a), indicated the reaction was completed. The reaction was diluted with CH2Cl2 (20
mL) and washed with 30 wt% solution of Na2S2O3 (15 mL), a saturated solution of NaHCO3
(50 mL ×2), dried over MgSO4, filtered and concentrated in vacuo to give 494 mg of a white
foam in 96% yield. HPLC method (a): 12.30 min (98%). LCMS: Rt = 1.71 min, MH+ =
1
436.1, 438.1 (1:1) HNMR(CDCl3) : 7.54(m, 3H), 7.15-7.45(m, 2H), 7.18(d, J = 8.1 Hz,
2H), 7.68(d, J = 8.1 Hz, 2H), 5.65(q, J = 7.1 Hz, 1H), 2.85(m, 2H), 2.61(d, J = 7.1 Hz, 1H),
2.01-2.52(m, 2H), 1.75(d, 1H), 1.55(s, 3H).
(S)-3-((S)-1-(4-Bromophenyl)ethyl)-6-(2-hydroxy-2-methylpropyl)-6-(phenyl-13C6)-1,3-
oxazinan-2-one, [13C6]-(11): To a solution of the epoxide [13C6]-(10) (0.38 g, 0.87 mmol) in
anhydrous THF (10 mL) was added a solution of super-hydride® (LiBHEt3) (1 M in THF, 1
o
mL) dropwise keeping the internal temperature below 25 C. After 2 h of stirring, HPLC
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showed a new product. A solution of 30% (w/w) of hydrogen peroxide in water (1 mL) was
diluted in water (1 mL) and added dropwise while cooling in an ice-bath. After the addition
was completed the ice bath was removed and the reaction was warmed to room temperature.
Ethyl acetate (100 mL) and water (50 mL) were added and the aqueous layer was removed.
The organic layer was washed twice with a 30% solution of Na2S2O3 (40 mL ×2), brine (50
mL) and dried over MgSO4, filtered and concentrated in vacuo to give 0.5 g of white foam.
Purification by Combiflash using a 40 g disposable silica gel cartridge and up to 10%
MeOH/CH2Cl2 gave 0.36 g of product in 95% yield. LCMS: Rt = 1.3 min, MH+ = 439.16 :
441.16 (1:1), 100%. HPLC method (a): Rt= 11.47 min (98%).
(S)-6-(2-Hydroxy-2-methyl-propyl)-3-((S)-1-[4-(1-methyl-2-oxo-1,2-dihydropyridin-4-
yl)phenyl]ethyl)-6-(phenyl-13C6)-1,3-oxazinan-2-one, [13C6]-(1): To a solution of [13C6]-
(11) (360 mg, 0.82 mmol) in isopropanol (5 mL), were added (1,1'-bis(diphenylphosphino-
ferrocene)dichloropalladium II) complex with dichloromethane (1:1) (PdCl2(dppf).CH2Cl2,
10 mg, 12 mol), potassium acetate (246 mg, 2.5 mmol), and (12) (140 mg, 0.95 mmol). The
mixture was degassed three times introducing nitrogen gas each time and then heated to 80
o
oC under nitrogen and stirred for 14 h. After cooling to 50 C, a solution of N-acetyl-L-
cysteine (160 mg) in water (10 mL) was added and stirred for 30 min to chelate the
palladium. The mixture was then cooled to room temperature, water (30 mL) was added and
extracted with EtOAc (100 mL ×2). The combined organic extracts were washed with brine
(100 mL), dried over MgSO4, filtered and concentrated in vacuo to give 0.85 mg of a viscous
oil. The crude material was purified using 40 g disposable silica gel cartridge and up to 20%
EtOAc in CH2Cl2 to remove non-polar impurities, then up to 20% MeOH in CH2Cl2 to elute
the desired product (230 mg, 60% yield). LCMS: Rt = 1.34 min MH+ = 467.29 (100%).
HRMS: [C2213C6H33N2O4]+,(M+H)+, calculated: 467.26361, found 467.26360. MS/MS:
449.25329 (M-H2O)+, 423.27378 (M-CO2)+, 409.22155(M-C3H6O)+. HPLC method (a): Rt =
7.3 min, 99.5%; chiral HPLC (method a) 99%. 1H NMR (CDCl3) : 7.32(dm, J = 78 Hz, 4H),
7.30(m, 3H), 7.01(d, J = 8.51 Hz, 2H), 6.71(d, J = 1.63 Hz, 1H), 6.35(dd, J = 2.04, 7.04 Hz,
1H), 5.69(dd, J = 7.04, 14.0 Hz, 1H), 3.57(s, 3H), 2.92(m, 1H), 2.39-2.47(m, 2H), 2.15-
13
2.32(m, 6H), 1.54(d, J = 7.0 Hz, 3H), 1.17(s, 3H), 1.14(s, 3H). C NMR (CDCl3) : 163.2,
153.0, 151.2, 142.8(dt), 140.7, 138.1, 136.3, 128.8(m), 127.6(m), 126.5(m), 125.0(m), 116.7,
105.3, 84.1(m), 71.1, 53.8, 53.2, 37.4, 36.2, 33.2, 31.9, 30.4, 15.4.
4-Iodoaniline-13C6, [13C6]-(14): A 100 ml round bottomed flask was charged with aniline-
13C6 (2.0 g, 20.2 mmol), sodium bicarbonate (2.6 g, 30.3 mmol) and 10 mL of water. The
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mixture was stirred in an ice-water bath. Iodine (5.2 g, 20.2 mmol) was added portionwise in
a 30 min period. The resulting deep brown color mixture was stirred vigorously for 6 h, then
filtered using a Buchner funnel and the brown filter cake was washed with ice cold water (20
mL ×3) until the filtrate was colorless. The solid was dried under high vacuum for six h to
give 4.5 g of [13C6]-(14) in 99% yield; HPLC (method c) 8.7 min, 98%, starting aniline (2.61
1
min). H NMR (DMSO-d6, 400 MHz) δ: 7.43-7.50 and 7.03-7.09(dm, J = 163.8 Hz, 2H),
6.28-6.24 and 6.64-6.58(dm, J = 158.8 Hz, 2H), 5.27(brs, 2H); 13C NMR (DMSO-d6, 100
MHz) δ: 148.3(td, J = 60.6, 9.3 Hz), 137.0(m), 116.5(m), 75.7(tdt, J = 61.4, 9.0, 1.7 Hz);
LCMS (ESI+) m/z 226.15 at 1.26 min.
N-(4-Iodophenyl-13C6)acetamide, [13C6]-(15): [13C6]-(14) (4 g, 17.7 mmol) was dissolved
CH2Cl2 (55 mL) to give a dark brown solution. The reaction was chilled in an ice-water bath
and triethylamine (3 mL, 21.2 mmol) was added. Acetyl chloride (1.3 mL, 18.6 mmol) was
then added dropwise; slight fuming was observed. The reaction mixture was stirred for 30
min and then water (100 mL) was added. The lower brown organic phase was cut. The
aqueous phase was extracted with CH2Cl2 (50 mL). The combined organic phases were dried
over MgSO4 and filtered through a phase separator cartridge. The brown filtrate was
concentrated in vacuo to a brown solid. The solid was dried under high vacuum for 3 h to
1
afford [13C6]-(15) (4.16 g, 87%) as a brown solid. HPLC (method c): 13.23 min, 98%. H
NMR (DMSO-d6, 400 MHz) δ: 9.96 (1H, s, NHAc), 7.54(dm, J = 167.0 Hz, 2H), 7.34(dm, J
= 163.3 Hz, 2H), 1.96(s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ: 168.4(s), 139.0(td, J =
62.6, 10.1 Hz), 137.2(ddd, J = 60.4, 55.9, 7.2 Hz), 121.1(m), 86.1(tdt, J = 60.6, 10.1, 2.4 Hz),
24.0(m); LCMS (ESI+) m/z 267.00 at 1.39 min.
N-(4-Cyanophenyl-13C6)acetamide, [13C6]-(16): [13C6]-(15) (4 g, 15 mmol) was dissolved in
DMF (40 mL) to give a brown solution. Copper cyanide (2.7 g 29.8 mmol) was added giving
a suspension. The dark brown suspension was stirred at 140 ºC for 3 h. The vessel was
removed from the oil bath and cooled to room temperature. To the dark brown mixture was
added 20 mL of aqueous ammonium hydroxide. The mixture was stirred vigorously for one
hour then, 50 mL of ethyl acetate was added. After one hour, the mixture was filtered through
a 3 cm high bed of Celite® in a 60 mL sintered glass funnel. The cake was rinsed with
EtOAc (50 mL ×2). The phases were separated and the aqueous phase was extracted with
EtOAc (50 mL ×2). The combined clear brown organic phases were washed with 100 mL of
saturated aqueous sodium chloride (the aqueous phase turned dark blue after mixing). The
organic phase was dried over MgSO4 and filtered through a phase separator cartridge yielding
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a clear brown filtrate. The filtrate was concentrated in vacuo to a brownish grey solid of
[13C6]-(16) (2.32 g, 93%). HPLC (method c) 8.9 min, 98%. 1H NMR (DMSO-d6, 400 MHz)
13
δ: 10.31(1H, s, NHAc), 7.68(dm, J = 166.4 Hz, 4H), 2.02(s, 3H); C NMR (DMSO-d6, 100
MHz) δ: 168.1(s), 143.4(td, J = 61.2, 10.5 Hz), 133.15(td, J = 58.9, 6.8 Hz), 118.9(td, J =
59.7, 6.5 Hz), 104.62(td, J = 59.6, 10.5 Hz), 24.23(m). LCMS (ESI+) m/z 166.19 at 0.61 min.
N-(2-Nitro-4-cyanophenyl-13C6)acetamide [13C6]-(17): To a two-neck 100 mL round
bottomed flask equipped with a thermometer, was added 8 mL of sulfuric acid. The vessel
was chilled in an ice-water bath with the sulfuric acid reaching 5 oC. To the sulfuric acid was
added [13C6]-(16) (2.5 g, 15 mmol) portion wise. In a separate flask sulfuric acid (2.43 ml,
44.7 mmol) was added and chilled in an ice-water bath and nitric acid (2.14 ml, 33.5 mmol)
was added. The contents were mixed. To the substrate-sulfuric acid mixture was added 3 mL
of the sulfuric acid-nitric acid mixture dropwise via a pipet. The temperature spiked to 20 deg
o
then cooled to 5 C over a 20 minute period. HPLC analysis after 45 minutes showed
consumption of the starting material at 8.8 min and product peak eluting at 10.6 min. LCMS
showed the desired [M]+ m/z 211 at 0.82 min. The dark brown mixture was poured into 200
grams of ice. Upon stirring, a brown solid precipitated, which was extracted from the aqueous
phase with EtOAc (50 mL ×3). The combined organic extracts were dried over MgSO4 and
filtered through a phase separator cartridge. The dark brown filtrate was concentrated in
vacuo to afford 3 g of product in 95% yield as a dark grey-brown solid. This material (87%
1
purity by HPLC, method c) was carried forward for the next step without purification. H
NMR (DMSO-d6, 400 MHz) δ: 10.50(s, 1H, NHAc), 8.50(dm, J = 173.7 Hz, 1H), 8.12(dq, J
= 170.9, 8.6 Hz, 1H) 7.84(dq, J = 170.2, 8.6 Hz, 1H) 2.05(s, 3H). 13C NMR (DMSO-d6, 100
MHz) δ: 168.1(s), 143.4(td, J = 61.2, 10.5 Hz), 138.5(td, J = 59.2, 6.5 Hz), 133.15 (td, J =
58.9, 6.8 Hz), 129.4(dt, J = 59.4, 6.7 Hz), 123.3(dt, J = 59.4, 7.1 Hz), 118.9 (td, J = 59.7, 6.5
Hz), 104.62 (td, J = 59.6, 10.5 Hz), 24.23 (m); LCMS (ESI+) m/z 210.10 at 0.82 min.
3-Amino-4-nitrobenzoic-13C6 acid, [13C6]-(18): To a 100 mL single neck round bottomed
flask was charged [13C6]-(17) (3 g, 14.2 mmol) followed by 10 mL of water. The vessel was
chilled in an ice-water bath and sulfuric acid (15 ml, 275.8 mmol) was added in one portion.
The suspension became homogenous and was warm to the touch. The vessel was equipped
with a reflux condenser and submerged in a preheated 140 oC oil bath. The reaction mixture
was stirred for three h then the vessel was cooled to room temperature and the contents
poured into 200 g of ice. The mixture was agitated with a glass stir rod forming a dark black
tar. The organics were extracted with EtOAc ((50 mL ×3). The combined organic phases
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were dried over MgSO4 and filtered through a phase separator cartridge. Prolonged drying
on the rotovap gave [13C6]-(18) as solid (2.45 g, 77%). HPLC (method c) 8.5 min, 98%;
LCMS (ESI+) m/z 187.09 at 0.54 min.
3,4-Diaminobenzoic-13C6 acid, [13C6]-(19): To a 100 mL single neck round bottomed flask
were placed [13C6]-(18) (2.45 g, 13 mmol) and 10% Palladium on carbon (277 mg) followed
by methanol (20 mL). To this black suspension was added ammonium formate (4.15 g, 65.1
mmol) in one portion. After stirring for 4 h at 70 ºC, the catalyst was filtered off on a 1 cm
tall bed of Celite®. The cake was rinsed with 50 mL of methanol. The filtrate was
concentrated in vacuo to give [13C6]-(19) (2g, 97%) as a dark brown solid. HPLC (method c)
2.5 min 98%. 1H NMR (DMSO-d6, 400 MHz) δ: 7.06(dt, J = 159.3, 6.5 Hz, 1H), 6.99(dt, J =
13
156.7, 6.8 Hz, 1H), 6.42(dq, J = 155.5, 7.3 Hz, 1H), 5.11(br s, 2H), 3.33(br s, 1H); C NMR
(DMSO-d6, 100 MHz) δ: 166.4, 140.1(dddd, J = 63.1, 63.0, 6.3, 3.4 Hz), 133.5(t, J = 64.1
Hz), 120.3(td, J = 58.3, 5.4 Hz), 118.3(td, J = 59.5, 6.9 Hz), 115.2(t, J = 63.0 Hz),
112.6(dddd, J = 63.9, 60.9, 5.4, 4.1 Hz); LCMS (ESI+) m/z 158.97 at 0.24 min.
1H-Benzo-13C6-[d]imidazole-5-carboxylic acid, [13C6]-(20):
In a 100 mL single neck
round bottomed flask was charged [13C6]-(19) (1.2 g, 7.53 mmol) followed by toluene (10
mL) and DMF (10 mL) to give a brown red solution. To the reaction mixture was added
triethyl orthoformate (2.55 mL, 15 mmol). The vessel equipped with a reflux condenser and
nitrogen inlet then submerged in a preheated 120 ºC oil bath. LCMS after 2 h showed
consumption of the diamine and benzimidazole at 0.23 min with m/z 168.98. The vessel was
cooled to room temperature and the solvent removed in vacuo leaving a dark solid that
weighed 1.16 g (92%). Due to the insolubility of this material in common solvents (except
DMSO), no purification was performed. The material was carried onto the amide formation
step as is. HPLC 2.5 min; 1H NMR (DMSO-d6, 400 MHz) δ: 12.4(s, 1H), 8.33(d, J = 8.3 Hz,
1H), 8.16(dm, J = 163.0 Hz, 1H), 7.79(dm, J = 163.0 Hz, 1H), 7.61(dm, J = 163.0 Hz, 1H);
LCMS (ESI+) m/z 168.98 at 0.23 min.
(4aR,9aS)-1-(1H-benzo-13C6-[d]midazole-5-carbonyl)-2,3,4,4a,9,9a-hexahydro-1-H-
indeno[2,1-b]pyridine-6-carbonitrile hydrochloride, [13C6]-(2): The amine (21) (0.69 g,
1.8 mmol) was diluted with MeCN (10 mL), then the acid [13C6]-(20) (0.97 g, 1.8 mmol) and
Et3N (0.72 g, 7.1 mmol) were added. T3P® (1.1 mL, 50% solution in THF, 1.95 mmol) was
added to the reaction mixture and the solution was stirred for 2 h. The reaction mixture was
concentrated to a minimum volume and then quenched by addition 1N NaOH (5 mL). The
mixture was diluted with EtOAc (20 mL). The organic phase was removed and washed with
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water (10 mL), brine (10 mL), dried over Na2SO4 and concentrated to give a dark brown
sticky solid. The residue was purified using by Combiflash Companion (40g silica gel
cartridge, 5-10% MeOH/CH2Cl2). Only the pure fractions were combined and concentrated.
The residue was diluted with MeCN/Water (10 mL, 1:1) and transferred to a tarred vial and
lyophilized to give 220 mg as an off white solid in 36% yield. HPLC (method c) 7.6 min
1
(99.3%); chiral HPLC (method b) 99% ee. H NMR (DMSO-d6, 400 MHz) δ: 12.7 (br, 1H),
8.41(t, J = 7.6 Hz, 1H), 8.30(S, 1H), 7.70(br, 1H), 7.58-7.66(m, 3H), 7.45(br, 1H), 7.25(br,
1H), 3.27(br, 1H), 3.11(br, 1H), 3.01(br, 2H), 1.74(br, 1H), 1.52(q, J =12.5 Hz, 1H), 1.30(dq,
J = 3.0, 13.0 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz) δ: 170.2, 147.5, 145.8, 143.5 (m),
138.4, 138.1 (m), 13.8 (m), 129.9, 127.1, 126.2, 120.6 (m), 119.1 (m), 114.2 (m), 109.2, 59.7,
41.2, 32.6, 32.1, 27.8, 23.1, 20.8. LCMS (ESI+) m/z 349.2 (MH+, 100%), LCMS (ESI-) m/z
347.5 (M-,100%), 0.54 min. HRMS [C1513C6H19N4O]+, MH+ calculated 349.1755, found
349.1741. HPLC (method c): 99.3%. Chiral purity (method b): 99%.
Synthesis of [14C]-(1) and [14C]-(2)
(S)-3-((S)-1-(4-Bromophenyl)ethyl)-6-(2-oxopropyl)-6-phenyl-1,3-oxazinan-2-one, (22):
Ozone was bubbled through a solution of (9) (250 mg, 0.6 mmol) in 10 % MeOH/CH2Cl2 (10
mL) at -78 ºC until a blue color persisted. Then O2 was bubbled through for 10 min followed
by N2 for 5 min. PS-PPh3 (1 g, 1.8 mmol) was added, the mixture was allowed to warm to -
20 °C. The mixture was filtered and concentrated, then diluted with 50 % EtOAc/Hexane (30
mL) filtered and concentrated to give 250 mg of (22) in 99% yield as a light yellow solid.
1
HPLC method (b): 6.66 min (98%). LCMS: m/z 416, H NMR (CDCl3, 400 MHz) δ: 7.35
(m, 5H), 7.17 (d, J = 8.5 Hz, 2H), 6.66 (d, J = 8.5 Hz, 2H), 5.57 (q, J = 7.1 Hz, 1H), 3.01 (d,
J = 14.5 Hz, 1H), 2.92 (d, J = 14.5 Hz, 1H), 2.90 (m, 1H), 2.59 (m, 1H), 2.36 (m, 2H), 2.08
(s, 3H), 1.48 (d, J = 7.1 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ: 205.5, 152.9, 140.9, 138.1,
131.3, 129.0, 128.6, 128.2, 124.8, 121.2, 81.3, 55.0, 52.8, 36.1, 32.5, 29.6, 15.2.
(6S)-3-((S)-1-(4-Bromophenyl)ethyl)-6-(2-hydroxy-2-methylpropyl-2-14C)-6-phenyl-1,3-
oxazinan-2-one, [14C]-(11): [14C]-MeI (500 mCi, at 59 mCi/mmol, 8.5 mmol), was cooled
in a dry ice bath and placed under argon. The seal of the ampule was broken, then Et2O (8
mL) was added. The solution was transferred via cannulation into a flask containing a
mixture of magnesium turnings (206 mg, 8.5 mmol) in Et2O (5 mL) at room temperature. A
cold finger was attached to the flask and the mixture was stirred for 1 h and used as is in
Et2O. In a separate reaction flask, THF (10 mL) was added to CeCl3 (1.5 g, 6.3 mmol), and
the mixture was stirred at 0 °C for 1 h. Compound (22) (2.6 g, 6.25 mmol) in THF (15 mL)
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was added via syringe, and the mixture was stirred for 1h. Then, 14CH3MgI in Et2O (13 mL)
was added and the mixture was stirred for 0.5 h. The reaction was quenched by addition of
1N aqueous HCl (25 mL) and diluted with EtOAc (50 mL). The organic material was
washed with a saturated solution of NaHCO3 (20 mL), brine (20 mL), filtered through a
membrane filter and concentrated to give a dark brown foam. The crude product was diluted
with 20% Et2O/Hexanes (30 mL) and stirred for 14 h. The solid was collected by filtration
and washed with 20% Et2O/Hexanes to give [14C]-(11) (1.85 g, 252 mCi) as a solid in 68%
yield. HPLC method (b): 6.56 min (98%).
(S)-6-(2-Hydroxy-2-methylpropyl-2-14C)-3-((S)-1-(4-(1-methyl-2-oxo-1,2-
dihydropyridin-4-yl)phenyl)ethyl)-6-phenyl-1,3-oxazinan-2-one, [14C]-(1):
[14C]-(11)
(81.4 mCi, 600 mg, 1.4 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane)
(356 mg, 1.4 mmol), PdCl2(dppf).CH2Cl2 (57 mg, 0.07 mmol), and KOAc (406 mg, 4.14
mmol) was diluted with IPA (10 mL) and heated to 80 °C for 3.5 h. The mixture was cooled
to room temperature, then (23) (263 mg, 1.4 mmol) in IPA (5 mL) was added followed by
K2CO3 (760 mg, 5.5 mmol), and H2O (300 µL). The mixture was heated and stirred
overnight at 80 °C, then cooled to room temperature, and partitioned between EtOAc (30 mL)
and H2O (30 mL). The organic layer was removed and washed with brine (20 mL), filtered
through a phase separation column and concentrated giving light brown oil. The crude
residue was purified using a CombiFlash (40 g silica gel cartridge, 0-10 % MeOH/CH2Cl2).
The desired fractions were combined and concentrated to afford 383 mg of a solid (49 mCi)
in 60% yield with radiochemical purity of 95%. The solid was further crystallized from
acetone/water twice to afford 12 mCi of [14C]-(1) as a white solid and with a specific activity
of 54.7 mCi/mmol. HPLC method (b): 3.51 min (97.6%). 1H NMR in CDCl3 was identical to
unlabeled material.17
3-Amino-4-nitrobenzonitrile-14C [14C]-(25): 5-Bromo-2-nitroaniline (24) (387 mg, 1.74
mmol), K14CN (100 mCi at 59 mCi/mmol, 1.74 mmol), CuI (65 mg, 0.34 mmol), and
Pd(Ph3P)4 (196 mg, 0.17 mmol) in DMF (5 mL) was evacuated and purged with nitrogen
three times, then heated to 100 ºC for 12 h. The mixture was concentrated then diluted with
EtOAc/H2O (20 mL, 1:1), filtered through Celite®, washed with H2O (5 mL), brine (5 mL),
filtered through a membrane filter and concentrated to give a dark oil. The crude residue was
purified using Combiflash Companion (40g silica gel cartridge, 25-50% EtOAc/Hexane) the
pure fractions were combined and concentrated to give [14C]-(25) (57.5 mCi, 159 mg, 56%)
as a yellow solid. HPLC (method d): 5.29 min (98%).
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3-Amino-4-nitrobenzoic-14C acid, [14C]-(26): H2SO4 (1.5 mL) was added to a mixture of
[14C]-(25) in H2O (1.0 mL) at room temperature. The mixture was stirred at 95 ºC for 3h. An
additional 0.5 mL of water was added and the mixture was stirred at 95 ºC for 14 h. Ice was
added to the reaction mixture, and exctracted with EtOAc (5 mL ×4), the combined extracts
were filtered through a membrane filter and concentrated to give [14C]-(26) (57.5 mCi, 180
mg, 100%) as an orange solid. HPLC (method d): 4.51 min (98%).
3,4-Diaminobenzoic-14C acid, [14C]-(19): 10% Pd/C (20 mg) in MeOH (1 mL) was added
to a solution of [14C]-(26) (159 mg, 0.97 mmol) in MeOH (4 mL). Then, ammonium formate
was added. The mixture was heated to reflux for 4h, then cooled to room temperature,
filtered through Celite® and concentrated to give [14C]-(19) (51 mCi, 150 mg, 100%) as a
brown solid. HPLC (method d): 2.09 min (98%).
1H-Benzo[d]imidazole-5-carboxylic-14C acid, [14C]-(20): Triethyl orthoformate (296 mg, 2
mmol) was added to a mixture of [14C]-(19) (150 mg, 0.97 mmol) in 1:1 DMF:toluene (4
o
mL). The mixture was heated at 120 C for 2h. The mixture was concentrated to give a
brown solid. The solid was diluted with H2O (2.5 mL) and stirred for 14 h. The solid was
collected by vacuum filtration, and washed with H2O (5 mL) to give [14C]-(20) (41.3 mCi,
130 mg, 81%) as a brown solid. HPLC (method d): 3.03 min (98%).
(4aR,9aS)-1-(1H-Benzo[d]midazole-5-carbonyl-14C)-2,3,4,4a,9,9a-hexahydro-1-H-
indeno[2,1-b]pyridine-6-carbonitrile hydrochloride, [14C]-(2): To the amine (21) (159 mg,
0.8 mmol) was added acid [14C]-(20) (130 mg, 0.8 mmol), EDC (184 mg, 0.96 mmol), HOBt
(147 mg, 0.96 mmol), and DIPEA (362 mg, 2.8 mmol) in DMF (2.5 mL). The mixture was
stirred for 14 h at room temperature. The mixture was diluted with water (2.5 mL) and
CH2Cl2 (5 mL) and treated with 1N NaOH (5 mL) and the mixture was stirred for 20 min.
Then 1N HCl was added to adjust the pH 9-10. The organic layer was removed and then
washed with water (10 mL ×3), filtered through a membrane filter and concentrated to give
an oil. This residue was diluted with EtOAc (3 mL) warmed to dissolve the oil and stirred for
14 h. The solid was collected by vacuum filtration to give a brown solid (156 mg) in 56%
yield. The above material (136 mg, 0.4 mmol) was suspended in absolute ethanol (1 mL) and
o
heated to 75 C. A solution of HCl in ethanol (6.55 M, 63 L, 0.41 mmol) was added and
stirring was continued for 10 min. The mixture was removed from the heat after 5 min. Few
seeds of (2) in ethanol (0.2 mL) were added and the mixture was stirred overnight. The solid
was collected by vacuum filtration to give 110 mg of a light beige solid in 49% yield. Total
activity 16.43 mCi at 56.6 mCi/mmol. The mother liquor was concentrated and counted to
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give 4.2 mCi as a brow solid. 1H NMR (DMSO-d6, 500 MHz) δ: 13.84 (br, 2H), 9.42(s, 1H),
7.83(s, 2H), 7.65(s, 1H), 7.54(d, J = 7.81 Hz, 2H), 7.42(br, 1H), 4.75(br, 1H), 3.02(m, 2H),
2.02(m, 1H), 1.62(br, 1H), 1.51(m, 1H), 1.32(m, 1H), 1.25(m, 1H). Chiral purity (method b)
98.6%, and radiochemical purity 98.6%.
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Potent and Selective Inhibitors of 11-Hydroxysteroid Dehydrogenase Type 1 Labeled
with Carbon-13 and Carbon-14
Bachir Latli*, Matt Hrapchak, Jolaine Savoie, Yongda Zhan, Carl A. Busacca, and Chris H.
Senanayake
(S)-6-(2-Hydroxy-2-methylpropyl)-3-((S)-1-(4-(1-methyl-2-oxo-1,2-dihydropyridin-
4-yl)phenyl)ethyl)-6-phenyl-1,3-oxazinan-2-one
(1)
and
(4aR,9aS)-1-(1H-
benzo[d]midazole-5-carbonyl)-2,3,4,4a,9,9a-hexahydro-1-H-indeno[2,1-b]pyridine-6-
carbonitrile hydrochloride (2) are potent and selective inhibitor of 11-hydroxysteroid
dehydrogenase type 1 enzyme. These two drug candidates developed for the treatment
of type-2 diabetes were prepared labeled with carbon-13 and carbon-14.
O
N
N
O
N
HCl
O
N
N
N
O
OH
(1)
(2)
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