Synthesis of empagliflozin, a novel and selective sodium-glucose co-transporter-2 inhibitor, labeled with carbon-14 and carbon-13

Article abstract of DOI:10.1002/jlcr.3240

Empagliflozin, (2S,3R,4R,5S,6R)-2-[4-chloro-3-[[4-[(3S)-oxolan-3-yl]oxyphenyl]methyl]phenyl]-6-(hydroxymethyl)oxane-3,4,5- triol was recently approved by the FDA for the treatment of chronic type 2 diabetes mellitus. Herein, we report the synthesis of car

Full text of DOI:10.1002/jlcr.3240

Research Article
Received 30 June 2014,
Revised 26 August 2014,
Accepted 23 September 2014
Published online 21 October 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3240
Synthesis of empagliflozin, a novel and selective
sodium-glucose co-transporter-2 inhibitor,
labeled with carbon-14 and carbon-13
Matt Hrapchak, Bachir Latli,* Xiao-Jun Wang, Heewon Lee, Scot Campbell,
Jinhua J. Song, and Chris H. Senanayake
Empagliflozin, (2S,3R,4R,5S,6R)-2-[4-chloro-3-[[4-[(3S)-oxolan-3-yl]oxyphenyl]methyl]phenyl]-6-(hydroxymethyl)oxane-3,4,5-
triol was recently approved by the FDA for the treatment of chronic type 2 diabetes mellitus. Herein, we report the synthesis of
carbon-13 and carbon-14 labeled empagliflozin. Carbon-13 labeled empagliflozin was prepared in five steps and in 34% overall
13
6
chemical yield starting from the commercially available α-D-glucose-[ C ]. For the radiosynthesis, the carbon-14 atom was
introduced in three different positions of the molecule. In the first synthesis, Carbon-14 D-(+)-gluconic acid δ-lactone was used
to prepare specifically labeled empagliflozin in carbon-1 of the sugar moiety in four steps and in 19% overall radiochemical yield.
Carbon-14 labeled empagliflozin with the radioactive atom in the benzylic position was obtained in eight steps and in 7% overall
14
radiochemical yield. In the last synthesis carbon-14 uniformly labeled phenol was used to give [ C]empagliflozin in eight steps and
in 18% overall radiochemical yield. In all these radiosyntheses, the specific activities of the final compounds were higher than
53 mCi/mmol, and the radiochemical purities were above 98.5%.
Keywords: empagliflozin; diabetes mellitus; SGLT-2; carbon-14; carbon-13; radiosynthesis
Introduction
phenyl]-6-(hydroxymethyl)oxane-3,4,5-triol, was recently approved
by the FDA for the treatment of diabetes mellitus-2.
Diabetes is among the top ten leading causes of death in the US.
More than 25 million people are inflected with this debilitating
disease and the numbers continue to escalate due to obesity and
Results and discussion
1
an aging population. Globally, 382 million people have diabetes.
This number is expected to rise to 592 million or about 10% of The synthesis of carbon-13 labeled empagliflozin was carried
2
13
the world’s population, by 2035. Type 2 diabetes is the most out starting from the commercially available α-D-glucose-[ C ]
6
common form accounting for more than 90% of all causes in the (1) (Scheme 1). Selective oxidation using Shvo’s catalyst
3
developed world. This disease occurs when the body does not (1-hydroxytetraphenyl-cyclopentadieyl(tetraphenyl-2,4-
properly produce or use insulin. Elevated blood sugar, or hyper- cyclopentadien-1-one)-μ-hydrotetracarbonyldiruthenium) in the
glycemia, is a common effect of uncontrolled diabetes, which over presence of cyclohexanone as a co-oxidant in dimethylformamide
29
time leads to serious damage to many of the body’s organs and (DMF) provided the desired lactone (2) in an 90% yield.
systems, especially the nerves, blood vessels, heart, and kidneys, Protection of the hydroxyl groups using trimethylsilyl chloride in
resulting in blindness, low limb amputation, kidney failure, and the presence of excess N-methylmorpholine (NMM) in THF
4
,5
30
cardiovascular disease. The discovery of the sodium-glucose provided the fully protected lactone (3) in 89% yield.
31
co-transporters (SGLTs), which act to ensure glucose entering
the kidneys finds its way back into the blood stream,
The lithiated anion of the aryl compound (4) was prepared
6–12
has via halogen metal exchange at À78 °C with n-BuLi, and the
attracted huge interest by the pharmaceutical industry to find resulting anion was reacted with the protected sugar (3) at
suitable inhibitors of these co-transporters and hence lower blood À78 °C, followed by treatment with methanesulfonic acid in
13–18
sugar by increasing the amount of sugar excreted in the urine.
Nearly all of the filtered glucose is reabsorbed in the kidneys by to the literature.
SGLT-1 and SGLT-2, with SGLT-2 responsible for about 90% of this gave the labeled [ C
re-absorption. Phlorizin was the first SGLT inhibitor discovered.
methanol at 45 °C to give the desired compound (6) according
3
2,33
.
Reduction of (6) with Et
3
SiH and BF
]-(7) after re-crystallization from ethanol
3 2
OEt
13
6
1
4
19
The molecule itself was known since 1835 and is a naturally
occurring compound isolated from the bark of pear, apple, cherry,
Chemical Development, Boehringer Ingelheim Pharmaceuticals, Inc., 900
2
0,21
and other fruit trees.
This compound was superseded by better Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-0368, USA
22–24
and more selective synthetic analogs like canagliflozin,
,
2
5,26
27,28
*Correspondence to: Bachir Latli, Chemical Development, Boehringer Ingelheim
Pharmaceuticals, Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT, 06877-
dapagliflozin,
and empagliflozin
(Figure 1).
Empagliflozin, also known as jardiance and BI 10773, (2S,3R,
0368, USA.
4
R,5S,6R)-2-[4-chloro-3-[[4-[(3S)-oxolan-3-yl]oxyphenyl]methyl] E-mail: bachir.latli@boehringer-ingelheim.com
J. Label Compd. Radiopharm 2014, 57 687–694
Copyright © 2014 John Wiley & Sons, Ltd.

M. Hrapchak et al.
Figure 1. Sodium glucose co-transporter 2 (SGLT-2) inhibitors.
1
3
Scheme 1. Synthesis of [
6
C ]empagliflozin.
3
4
in 42% yield. The crystallization removed any α-anomer formed be sluggish and yields were inconsistent. Hence in the second
31
14
during this final reduction of the anomeric methoxy group.
synthesis of [ C]empagliflozin, we considered the preparation
The synthetic route described above was applied to the first of 5-bromo-2-chlorobenzoic acid (15) with the label on the
1
4
synthesis of [ C]empagliflozin, (Scheme 2). Carbon-14 D-(+)- carboxyl carbon. We envisioned that starting from 5-bromo-2-
gluconic acid δ-lactone (8) labeled specifically at carbon-1 of chloro-iodobenzene (13) we could obtain the desired benzoic
the sugar was custom made. Reaction of the protected acid (15) in two steps via the nitrile (14). Carbon-14 cuprous
31
14
tetrahydro-2H-pyran-2-one (9) with the anion of the aryl (10),
cyanide (Cu CN) was reacted with (13) in DMF at 100 °C,
prepared via halogen metal exchange at À20 °C, followed by (Scheme 3). Column chromatography afforded the desired product
treatment with 4 N HCl (solution in 1,4-dioxane) in MeOH at (15) and 4-chloro-5-iodobenzonitrile up to 89:11 ratios. Hydrolysis
4
5 °C, and finally reduction with Et SiH and AlCl gave pure using 50% KOH gave the desired 5-bromo-2-chlorobenzoic acid
3 3
14
[
C]-(12) after crystallization from ethanol with radiochemical (15) which can be crystallized from methanol and water. Friedel
purity of 99.2%, a specific activity of 57.5 mCi/mmol, and in Crafts reaction was performed using freshly prepared acid chloride
9% overall radiochemical yield. and the phenyl-3-(S)-hydroxytetrahydrofuran (19), prepared from
However, the synthesis of the lactone (8) is somewhat phenol (17) and (R)-3-hydroxytetrahydrofuran (18) to give the
problematic as the oxidation of carbon-14 labeled glucose can desired benzophenone (20) in a 41% yield. Reduction of the
1
35
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J. Label Compd. Radiopharm 2014, 57 687–694

M. Hrapchak et al.
1
4
Scheme 2. First synthesis of [ C]empagliflozin (12), asterisk indicates position of carbon-14.
1
4
Scheme 3. Second synthesis of [ C]empagloflizin (23).
carbonyl was accomplished in quantitative yield using Et SiH in the in 51% yield. The final product (23) was obtained in 7.1% overall
3
.
3
presence of BF OEt , and carbon-14 labeled empagliflozin was radiochemical yield (4.57mCi) with
a specific activity of
2
obtained as described before after re-crystallization from ethanol 54.27 mCi/mmol and a radiochemical purity of 99%.
J. Label Compd. Radiopharm 2014, 57 687–694
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

M. Hrapchak et al.
1
4
The third synthesis of [ C]empagliflozin was completed in five HPLC Conditions: (a) Mobile Phase gradient 20 to 100% (MeCN/H
2
O both
0 mM TFA) over 30 min, column: Zorbax SB C8 4.5 × 150 mm; (b) Mobile
Phase gradient 20 to 100% (MeCN/H O both 10 mM TFA) over 20 min; (c)
0% A to 60% A in 7 min, then to 5% A in 8 min. A = water (0.1% TFA),
14
1
synthetic steps, starting from [U- C]phenol (24) as the
radioactive starting material (Scheme 4). Mitsunobu’s reaction
with (R)-3-hydroxy-tetrahydrofuran (18) proceeded smoothly to
give the desired ether (25) in a 76% yield with the desired S-
configuration as described above. Friedel Crafts acylation with
freshly prepared 5-bromo-2-chlorobenzoyl chloride (26) gave
the desired benzophenone (27) in quantitative yield. Reduction
2
7
B = MeCN. Flow rate: 1.2 mL/min, UV detection at 224 nm, column: Zorbax
Eclipse XDB C8 (3.5 μm, 4.6 mm × 150 mm). The chemical purity of prepared
compounds was greater than 98%. The radiochemical purity was measured
using a radio-HPLC detector β-Ram model 4 or model 3 (LabLogic systems,
™
Inc. Brandon, FL) connected to the Agilent instrument using IN-FLOW 2:1
to compound (28) as reported above was accomplished in a liquid scintillation (LabLogic systems, Inc. Brandon, FL). The radiochemical
3% yield. Then, reaction of the protected sugar (22) with the purity of the final compounds was greater than 98%. For enantiomeric purity,
lithiated anion of (28), followed by treatment with the mobile phase consisted of isocratic water with 0.1% AcOH, pH 7.0 with
9
NH
4
OH and MeCN (99/1). Flow rate at 2.0 mL/min for 20 min and UV
methanesulfonic acid in methanol and reaction with triethyl
silane as described above furnished the final product (29) after
crystallization from ethanol in the amount of 36.0 mCi with a
specific activity of 53.4 mCi/mmol. The radio-purity was found
to be 99.4%, with an 18% overall radio-chemical yield.
Chiral HPLC showed that carbon-14 labeled empagliflozin did
not contain any of the (R)-diastereomer of the hydroxyl-
tetrahydrofuran moiety (30) (Figure 2). The same finding was
observed from the synthesis described in Scheme 3. See Figure 3.
detection at 230 nm and using ChromTech Chiral AGP column (5μm,
14
4.0 × 150 mm, ID 4.6 mm). [U- C]Phenol was purchased PerkinElmer (Boston,
14
MA). Cuprous[ C]cyanide was purchased from Quotient Bioresearch (Cardiff,
UK). D-(+)-Gluconic acid δ-lactone [2- C] was custom made by Moravek
Biochemicals (Brea, CA). (R)-3-Hydroxytetrahydrofuran was obtained from
CODEXIS® (Redwood City, CA). The rest of the reagents were purchased from
Sigma-Aldrich Company.
14
13
Synthesis of [ C ]-empagliflozine
6
1
3
[
6
C ]-(3R,4S,5S,6R)-3,4,5-Trihydroxy-6-hydroxymethyl-tetrahydro-
Experimental
2H-pyran-2-one (2)
1
3
Cyclohexanone (20 mL) was added to
6
[ C ]-α-D-glucose (540 mg,
Materials and methods
2
.90 mmol) and Shvo’s catalyst (40 mg) at room temperature. The
NMR spectra of radioactive compounds were recorded on a Bruker
mixture was stirred at 45 °C for 16 h. The mixture was transferred to a
centrifuge tube and centrifuged at 3000 rpm for 5 min. The solvent was
decanted off, and the solid was dried under high vacuum giving
500-MHz spectrometer using double encapsulated NMR tubes in deuterated
dimethyl sulfoxide. Liquid scintillation counting was accomplished using a
TM
+
Beckman LS6500TA and UltimaGold cocktail (PerkinElmer, Boston, MA).
482 mg of (2) in 90% yield as an off white solid. LCMS m/z: 185 (MH ).
1
Pre-coated TLC sheets (silica gel 60 F254) were obtained from EM Science
H NMR (500 MHz, D COD) δ: 5.81 (1H, m), 5.41 (2H, m), 4.88 (1H, m),
3
13
(Gibbstown, NJ). HPLC analysis was performed on an Agilent 1200 instrument.
3
4.10 (1H, bd, J = 97 Hz), 3.83 (1H, m). C NMR (100 MHz, D COD) δ:
1
4
Scheme 4. Third synthesis of [ C]empagliflozin (29).
Figure 2. Empagliflozin and its (R)-3-phenoxy-tetrahydrofuran diastereomer.
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M. Hrapchak et al.
1
4
71.9 (d, J = 54 Hz), 81.3 (t, J = 41 Hz), 73.9 (t, J = 41 Hz), 71.5 (ddd, J = 3, (15 mL). The organic layer was washed sequentially with aq. Na
4
1, 54 Hz), 67.9 (dt, J = 3, 41 Hz), 60.2 (d, J = 41 Hz). (1 g/20 mL water) and brine (15 mL), dried over Na SO ,
2
HPO
and
4
2
concentrated. The residue was diluted with heptane (20 mL) again and
concentrated. The residue was purified by flash chromatography (0–10%
EtOAc/Hexane) to give 130 mCi or 1.06 g of (9) as a colorless oil in 87% yield.
1
3
[
6
C ]-(3R,4S,5R,6R)-3,4,5-Tris-trimethylsilyloxy-6-trimethylsilyloxymethyl-
tetrahydro-2H-pyran-2-one (3)
TMSCl (1.7 g, 15.6 mmol) was added slowly to a solution of the above
lactone (480 mg, 2.60 mmol) and NMM (2.1 g, 20.8 mmol) in THF (10 mL)
at 0 °C. The mixture was warmed to room temperature and stirred for
(
2S,3R,4R,5S,6R)-2-{4-Chloro-3-[4-(tetrahydro-furan-3-yloxy)-benzyl]
1
4
phenyl}-6-hydroxymethyl-tetrahydro-2H-pyran-[2- C]-3,4,5-triol
12). A solution of (10) (0.75 g, 1.8 mmol) was dissolved in THF (10 mL) in a
round bottom flask under N
and cooled to À20 °C. Then, i-PrMgCl/LiCl
1.3 M in THF, 1.45 mL, 1.89 mmol) was slowly added, keeping internal
(
2
0 h. The reaction mixture was diluted with toluene and H
layer was washed sequentially with aq. Na HPO (1 g/20 mL), brine, dried
over Na SO , and concentrated. The residue was diluted with heptane
2
O. The organic
2
2
4
(
2
4
temperature below À10 °C. The mixture was stirred at À7 °C for 40 min.
and concentrated. The residue was purified by flash chromatography on
The reaction was cooled to À20 °C and a solution of the protected lactone
Combi-Flash® Companion (40 g, 0–10% EtOAc/Hexane) to give 1.1 g of (3)
1
(9) (75 mCi, 1.5 mmol) in THF (3 mL) was added, keeping temperature
as a colorless oil in 89% yield. H NMR (500 MHz, DMSO-d
6
) δ: 4.30
below À10 °C. The reaction was stirred at À10 °C for 1.5 h then, warmed
to 0 °C over 30 min and stirred at 0 °C for 20 min. The reaction was cooled
to À10 °C, and MeOH (15 mL) was added slowly, keeping internal
(
0
1H, bd, J = 67 Hz), 3.94 (3H, m), 3.61 (2H, m), 0.15 (9H, s), 0.14 (9H, s),
.13 (9H, s), 0.09 (9H, s).
1
3
[
6
C ]-(2S,3R,4S,5S,6R)-2-{4-Chloro-3-[4-(tetrahydro-furan-(3S)-yloxy)- temperature below 5 °C. A solution of 4 N HCl in dioxane (1 mL, 4 mmol)
benzyl]phenyl}-6-hydroxymethyl-2-methoxytetrahydro-2H-pyran-3,4, was added and the solution was concentrated. Methanol (10 mL) was
added followed with a solution of 4 N HCl in dioxane (1.0 mL, 1.0 mmol)
until pH ≈ 2.5. The mixture was stirred at 45 °C for 2 h and slowly cooled
to 22 °C and stirred for 6 h at 22 °C. The mixture was quenched by addition
5
-triol (6)
A solution of 3(S)-[3-(5-bromo-2-chloro-benzyl)-phenoxy]-tetrahydrofuran
(4) (685 mg, 1.9 mmol) in THF (2 mL) was added to a solution of n-BuLi
(1.0 mL, 2.3 mmol) in THF (5 mL) at À78 °C. After stirring for 1 h, the lactone
(3) (1.1 g, 2.3 mmol) in THF (3 mL) was added dropwise (5 min). The mixture
of 5% Na
extracts were washed with brine and dried over Na
to a residue. This residue (40 mCi, 0.77 mmol), MeCN (2 mL), and CH
2 mL) were added. In a separate flask, AlCl (205 mg, 1.54 mmol) was added
followed by CH Cl (3 mL) and cooled to 0 °C. MeCN (3 mL) was added
slowly, keeping internal temperature below 20 °C. The slurry turned into
solution after the addition was completed. Et SiH (242 mg, 2.08 mmol)
2
CO
3
(20 mL) and extracted with EtOAc (20 mL × 3). The combined
SO and concentrated
Cl
2
4
2
2
was stirred for 1.5 h at À78 °C. The reaction was quenched by addition of
AcOH/H O (300 μL/10 mL), diluted with EtOAc (20 mL). The organic layer
was washed with brine, dried over Na SO , and concentrated giving (5).
The residue was diluted with MeOH (10 mL), then MeSO H (125 μL) was
added and the mixture was stirred at 45 °C for 2.5 h. The reaction mixture
was cooled to room temperature and quenched by addition of NaHCO
10 mL). The mixture was extracted with EtOAc (10 mL × 3). The combined
extracts were washed with brine and dried over Na SO and concentrated
giving 720 mg of (6) in 79% yield as an off white foam. LCMS m/z: 509
(
3
2
2
2
2
4
3
3
was then added. A solution of the starting material was slowly added
keeping internal temperature below 15 °C. The solution was warmed to
3
(
2
0 °C and stirred for 30 min. The mixture was cooled to 0 °C, and water
10 mL) was slowly added with rapid stirring, keeping internal temperature
below 15 °C. The mixture was stirred for 15 min and extracted with EtOAc
20 mL × 3). The combined EtOAc extracts were dried over Na SO and
concentrated giving crude [ C]-(12). The crude material was purified by
silica gel chromatography (0–15%MeOH/CH Cl ) to give 19 mCi of product
as a white foam. Further crystallization from ethanol gave 16.5 mCi of
2
4
(
+
(M + Na ), which was used as it is in the next step.
(
2
4
14
1
3
[
6
C ]-(2S,3R,4R,5S,6R)-2-{4-Chloro-3-[4-(tetrahydro-furan-3(S)-yloxy)-
2
2
benzyl]phenyl}-6-hydroxymethyl-tetrahydro-2H-pyran-3,4,5-triol (7)
.
3
BF
OEt
2
(162 mg, 1.13 mmol) was added dropwise to a solution of (6) material as a white solid after drying under high vacuum with a specific
1
(250 mg, 0.51 mmol) and Et
3
SiH (177 mg, 1.53 mmol) in CH
2
Cl
2
/MeCN activity of 57.5 mCi/mmol and a radiochemical purity of 99.2%. HNMR
was identical to unlabeled standard.
(0.5 mL/2 mL) at À25 °C. The mixture was warmed slowly to 10 °C in 2 h
period. The mixture was cooled to À10 °C, and quenched by addition
of a saturated aqueous solution of NaHCO (10 mL), extracted with EtOAc
10 mL × 3). The combined EtOAc extracts were concentrated, then [ C]Empagliflozin labeled in the benzylic position
diluted with EtOH and concentrated giving off white foam. The foam
was diluted with EtOH (2.5 mL) warmed, covered, and stirred overnight. 5-Bromo-2-chlorobenzo-[ C]nitrile (14). A suspension of Cu CN
The solid was collected by vacuum filtration giving 98 mg of (7) in 42% (200 mCi, 340 mg, 3.7 mmol) in DMF (5 mL) was added to 5-bromo-2-
3
14
(
14
14
(
b)
yield as a white solid. HPLC (retention time): 5.9 min. LCMS m/z: 457 chloroiodobenzene (13) (1.2 g, 3.7 mmol) in DMF (5 mL) at room
+
1
(MH ). H NMR (500 MHz, DMSO-d
6
) δ: 7.37 (1H, d, J = 8.5 Hz), 7.33 (1H,
temperature; the mixture was heated to 100 °C overnight. The reaction
mixture was cooled to room temperature, diluted with EtOAc (20 mL),
filtered through Celite®, washed sequentially with water (5 mL × 4), brine,
filtered through a membrane filter, and concentrated giving a light
bs), 7.23 (1H, bd, J = 8.0 Hz), 7.11 (2H, d, J = 8.0 Hz), 6.82 (2H, d,
J = 8.0 Hz), 4.93 (3H, m), 4.80 (1H, bs), 4.42 (1H,bs), 4.00 (1H, d,
J = 15 Hz), 3.96 (1H, d, J = 15 Hz), 3.83 (3H, m), 3.74 (2H, m), 3.56 (1H, m),
13
3
.10 (1H,m), 3.00 (1H,m), 2.18 (1H, m), 1.93 (1H,m). C NMR (100 MHz, brown solid. The solid was purified by silica gel chromatography (5%
14
DMSO-d
1
6
) δ: 155.5, 139.7 (d, J = 52 Hz), 137.8 (d, J = 3 Hz), 131.6, 130.9,
EtOAc/Hexane) to give a 90:10 mixture of 5-bromo-2-chlorobenzo-[ C]
14
29.7, 128.7 (d, J = 3 Hz), 127.5, 115.2, 81.2 (d, J = 42 Hz), 80.4 (d, nitrile (14) and the undesired 2-chloro-5-iodobenzo-[ C]nitrile (280 mg,
J = 40 Hz), 78.4 (t, J = 40 Hz), 77.0, 74.7 (t, J = 40 Hz), 72.4, 70.3 (t,
J = 40 Hz), 66.5, 61.5 (d, J = 44 Hz), 37.7, 32.5.
68 mCi) in 34% yield as a white solid.
14
5
-Bromo-2-chlorobenzoic acid-[carboxyl- C] (15). The above mixture
(280 mg, 1.29 mmol) was diluted with 50% KOH (2.0 mL) and n-propanol
2.0 mL). The mixture was heated at 100 °C overnight. Then 4 N HCl (6 mL)
1
4
Synthesis of [ C]empagliflozin
(
1
4
[
C]Empagliflozin labeled in the sugar moiety
was added, and the mixture was allowed to cool to room temperature.
The solid was collected by vacuum filtration to give a light brown solid.
(
3R,4S,5R,6R)-3,4,5-Tris-trimethylsilyloxy-6-trimethylsilyloxymethyl- The solid was diluted with MeOH (2.5 mL) warmed to 70 °C, then filtered,
1
4
tetrahydro-2H-pyran-[2- C]-2-one (9). Trimethylsilyl chloride (1.98 g, the liquid was heated to 70 °C, then water (8 mL) was added, and the
1
14
8.0 mmol) was added slowly to a solution of [ C]-D-(+)-gluconic acid δ- mixture was stirred at 70 °C for 0.5 h. The solution was allowed to cool to
lactone (8), (150 mCi, ≈ 3.0 mmol) and NMM (2.44 g, 24.0 mmol) in THF
15 mL) at 0 °C. The mixture was warmed to room temperature and
stirred for 20 h. The mixture was diluted with toluene (20 mL) and H
room temperature, and the solid was collected by vacuum filtration to give
305 mg of (15) in quantitative yield as an off white solid. HPLC: (retention
time) 7.6 min, co-eluted with an unlabeled commercial material.
(
2
O
J. Label Compd. Radiopharm 2014, 57 687–694
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

M. Hrapchak et al.
(
(
S)-3-Phenoxy-tetrahydrofuran (19). Mitsunobu coupling of phenol dissolved. The solution was allowed to cool to room temperature with
17) (753 mg, 8.0 mmol) and (R)-3-hydroxytetrahydrofuran (18) (775 mg,
stirring. Then unlabeled empagliflozin seeds (ca. 1 mg) were added,
8
.8 mmol) according to the general procedure gave (S)-3-phenoxy-
and the mixture was stirred overnight. The solid was collected by
vacuum filtration to give [ C]empagliflozin (35 mg, 23%) as white solid.
The mother liquor was concentrated and purified by silica gel
+
14
tetrahydrofuran (540 mg, 41%) as a colorless oil. LCMS m/z: 165 (MH ).
1
H NMR (500 MHz, CDCl
3
) δ: 7.28 (2H, m), 6.96 (1H, m), 6.87 (2H, m), 4.93
(
2
7
1H, ddd, J = 2, 4.5, 8 Hz), 4.00 (2H, m), 3.90 (1H, ddd, J = 4.5, 8, 8 Hz), chromatography (12 g Cartridge, 0-10% MeOH/CH Cl ) to give 84 mg,
2 2
13
.18 (2H, m). C NMR (100 MHz, CDCl
3.2, 67.2, 33.0.
3
) δ: 157.4, 129.6, 121.0, 115.4,
or 56% as a colorless glass. A total of 119 mg or 14 mCi were obtained
in 50% overall yield. The specific activity was 54.27 mCi/mmol;
radiochemical purity of 98.87%, and chemical purity of 99.16%. HPLC
(c)
(
[
S)-(5-Bromo-2-chlorophenyl)-(4-tetrahydrofuran-3-yloxyphenyl)- (retention time): 4.9 min.
1
4
C]methanone (20). Oxalyl chloride (160 μL, 1.89 mmol) was added
dropwise to a mixture of (15) (300 mg 1.27 mmol) and DMF (50 μL) in THF
10 mL) at room temperature. The mixture was stirred for 1.5 h then,
concentrated. The residue was diluted with heptane (15 mL) and
concentrated to give a light yellow oil. The oil was diluted with CH Cl
5 mL) and cooled to 0 °C, then AlCl (173.3 mg, 1.30 mmol) was added
followed by (19) (213.5 mg, 1.30 mmol) in CH Cl (3 mL). A second portion
of AlCl (173.3 mg, 1.30 mmol) was added, and the mixture was stirred
overnight. HPLC revealed incomplete conversion; therefore, an additional
portion of AlCl (173.3 mg, 1.30 mmol) was added, and the mixture was
stirred for an additional 4 h. The reaction was quenched by the addition
of water (15 mL) and extracted with CH Cl (20 mL × 3). The combined
extracts were dried over Na SO , filtered through a membrane filter and
14
[
(
U- C]Empagliflozin labeled in the phenyl group
(
14
S)-3-[U- C]Phenoxy-tetrahydrofuran (25). DIAD (890 mg, 4.4 mmol)
was added dropwise to a solution of [U- C]phenol (24) (200 mCi, ≈
.0 mmol), Ph
14
2
2
(
3
4
(
3
P (1.2 g, 4.4 mmol), and (R)-3-hydroxytetrahydrofuran (18)
2
2
387 mg, 4.4 mmol) in THF (15 mL). The solution was stirred overnight
b
3
at room temperature (HPLC showed product:phenol ratio of 70:30). A
second portion of Ph P (600 mg, 2.2 mmol), (R)-3-hydroxytetrahydrofuran
193 mg, 2.2 mmol) in THF (2 mL), and DIAD (445 mg, 2.2 mmol) was
added. The reaction mixture was stirred for 2 days. The reaction mixture
was concentrated and purified by Combi-Flash® Companion (40 g, 5–50%
EtOAc/Hexane) to give 500 mg (151 mCi) of (25) in 76% yield as a
3
3
(
2
2
2
4
(b)
concentrated to give a brown oil. The crude oil was purified by silica gel
chromatography (40 g, 10–30% EtOAc/hexanes) to give (20) (201 mg,
colorless oil. HPLC (retention time): 8.9
(c)
28 mCi) in 41% yield as colorless oil. HPLC (retention time): 10.9 min.
(
[
S)-(5-Bromo-2-chlorophenyl)-[3(S)-(tetrahydro-furan-3-yloxy)-
14
U- C]phenyl]-methanone (27). Oxalyl chloride (460 mg, 3.6 mmol) was
1
4
(
(
S)-3-[4-[(5-Bromo-2-chlorophenyl)-[ C]methyl]phenoxy]tetrahydrofuran
added dropwise to a solution of 5-bromo-2-chlorobenzoic acid (730 mg,
3.1 mmol) and DMF (100 μL) in THF (15 mL) at room temperature. After
stirring for 2 h, the reaction mixture was concentrated. The residue was
diluted with heptane (25 mL) and concentrated and dried under high
vacuum. The residue was diluted with CH Cl (10 mL) cooled to 0 °C, then
.
21). BF
material (201 mg, 0.52 mmol) and Et
8.0 mL) MeCN (8.0 mL) at room temperature. The mixture was stirred
overnight. The reaction was quenched by addition of 1 N NaOH
5 mL) and stirred for 20 min. The mixture was diluted with water
and extracted with CH Cl (20 mL × 3). The combined extracts were
dried over Na SO filtered through membrane filter, and
concentrated to give 240 mg (28 mCi) of (21) as a colorless oil. HPLC
3
OEt
2
(93.0 μL, 0.79 mmol) was added to a mixture of the above
3
SiH (435.2 μL, 2.62 mmol) in MeCN
(
2
2
(
AlCl (400 mg, 3.0 mmol) was added. The mixture was warmed to room
3
2
2
temperature and (25) (500 mg, 3.05 mmol) in CH Cl (10 mL) was added
followed by a second portion of AlCl3 (400 mg, 3.0 mmol). The reaction
mixture was stirred overnight. The reaction was quenched by addition of
2
2
2
4
,
a
(c)
(retention time): 12.5 min.
H2O (10 mL), filtered through Celite®, and extracted with CH Cl2
2
(
20 mL × 3). The combined extracts were dried over Na
2 4
SO and
(
[
(
2S,3R,4R,5S,6R)-2-{4-Chloro-3-[4-(tetrahydro-furan-(3S)-yloxy)- concentrated giving 1.18 g (150 mCi) of (27) as a colorless oil in quantitative
1
4
(b)
C]benzyl]phenyl}-6-hydroxymethyl-tetrahydro-2H-pyran-3,4,5-triol
23). n-BuLi 2.5 M in hexanes (0.3 mL, 0.65 mmol) was added to a solution
yield. HPLC (retention time): 14.3 min.
14
of (21) (240.0 mg, 0.65 mmol) in THF (3 mL) at À78 °C. After stirring for 3(S)-[3-(5-Bromo-2-chlorobenzyl)-[U- C]phenoxy]-tetrahydrofuran
.
1
h, (22) (303.5 mg, 0.65 mmol) in THF (1 mL) was added dropwise. The (28). BF OEt (567 mg, 4.0 mmol) was added dropwise to a solution of
3 2
mixture was stirred at À78 °C for 1.5 h and then quenched by addition
of AcOH/water (0.1 mL/3 mL). The mixture was warmed to room
temperature then diluted with EtOAc (10 mL) and brine (5 mL). The
3 2 2
(27) (1.18 g, 3.6 mmol) and Et SiH (1.26 g, 10.9 mmol) in CH Cl :MeCN
(5 mL:20 mL) at room temperature. The mixture was stirred overnight.
The mixture was filtered and quenched by addition of 1 M NaOH
2 4
2 2 2
organic material was dried over Na SO and concentrated. The residue (5 mL). The mixture was diluted with CH Cl (10 mL) and H O (10 mL)
was diluted with MeOH (4 mL), and then MeSO
3
H (0.1 mL, 1.16 mmol) and stirred for 20 min. The organic material was removed by syringe,
was added, and the mixture was heated at 45 °C for 2 h. The mixture
was cooled to room temperature then quenched by addition of a
2 2
and the aqueous layer was extracted with CH Cl (10 mL × 2). The
combined extracts were dried over Na SO and concentrated giving
2
4
saturated solution of NaHCO
EtOAc (10 mL × 3); the combined extracts were dried over Na
concentrated to give a light yellow oil. The oil was diluted with CH
3
(10 mL). The mixture was extracted with
SO and time): 16.6 min.
Cl
1.02 g (139.5 mCi) of (28) in 93% yield as a colorless oil. HPLC (retention
(b)
2
4
2
2
(
1 mL), then heptane (4 mL) was added to precipitate the product out (2S,3R,4R,5S,6R)-2-{4-Chloro-3-[4-(tetrahydrofuran-3(S)-yloxy)-benzyl]-
14
of solution. The precipitate was washed with heptane then diluted with
CH
concentrated to give (3R,4S,5S,6R)-2-{4-chloro-3-[4-(tetrahydro-furan-
[
U- C]phenyl}-6-hydroxymethyl-tetrahydro-2H-pyran-3,4,5-triol (29).
2 2
Cl (20 mL) and transferred to a tarred flask. The solution was
n-BuLi (1.3 mL, 3.34 mmol) was added to a solution of (28) (1.02 g,
2
3
.78 mmol) in THF (10 mL) at À78 °C. After stirring for 1 h, (22) (1.56 g,
1
4
(3S)-yloxy)-[ C]benzyl]-phenyl}-6-hydroxymethyl-2-methoxy-tetrahydro-
.34 mmol) in THF (5 mL) was added dropwise (5 min). The mixture was
2
6
H-pyran-3,4,5-triol (161 mg, 51%) as a white foam. HPLC (retention time)
.5 min. A solution of BF
stirred for 1.5 h at À78 °C. The reaction was quenched by addition of
AcOH:H O (0.6 mL:20 mL), diluted with EtOAc (30 mL). The organic layer
was washed with brine, dried over Na SO , and concentrated. The
residue was diluted with MeOH (10 mL), then MeSO H (200 μL) was
(c)
.
3
OEt
2
(142 mg, 1.0 mmol) was added dropwise
SiH
(1.0 mL)/MeCN (4.0 mL) at À25 °C. The
2
to a solution of the above material (161 mg, 0.33 mmol) and Et
116.3 mg, 1.0 mmol) in CH Cl
3
2
4
(
2
2
3
mixture was allowed to warm to À10 °C over 1 h, then held at À10 °C
added, and the mixture was stirred at 45 °C for 2.5 h. The reaction mixture
for 0.5 h. The reaction was quenched by addition of saturated solution
was cooled to room temperature and quenched by addition of a
of NaHCO
extracts were dried over Na
was diluted with EtOH (2.0 mL) warmed until all the material was
3
(10 mL), extracted with EtOAc (20 mL × 3), and the combined saturated solution of NaHCO3 (10 mL). The mixture was extracted with
SO and concentrated. The crude residue EtOAc (10 mL × 3). The combined extracts were washed with brine and
dried over Na SO and concentrated giving (3R,4S,5S,6R)-2-{4-chloro-3-
2
4
2
4
www.jlcr.org
Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 687–694

M. Hrapchak et al.
1
4
Chiral purity determination of [ C]
empagliflozin
14
To determine if [ C]empagliflozin (Figure 3) contained any of the
diastereomer shown in Figure 2, especially the product produced
from Scheme 3 and Scheme 4. We are not concerned about the
small amounts of α-anomer formed at the sugar moiety which were
usually removed during crystallization. Unlabeled empagliflozin
(2 mg) was dissolved in methanol (10 mL). The diastereomer (30)
(
2 mg) was dissolved in methanol (10 mL). A mixture of 0.5 mL of
14
each of the solutions was mixed. [ C]-Empagliflozin, 1.0 mM
solution in EtOH, 200 μL was diluted with 300 μL MeOH.
HPLC conditions: System: Agilent 1100 (Quaternary Pump);
Column: ChromTech Chiral AGP 4.0 mm × 150 mm ID: 4.6 mm
particle size 5 μm; column temperature: 15 °C; mobile phase: A,
HPLC water with 0.1% AcOH, pH 7.0 with NH
elution mode: isocratic 99/1 A/B; flow rate: 2.0 mL/min. Run time:
0 min; detection: UV at 230 nm; injection: 5 μL. HPLC showed no
4
OH; B, HPLC MeCN;
2
detected amounts of diastereomer (30).
Conclusion
Carbon-13 labeled empagliflozin was prepared in five steps and
in 34% overall chemical yield starting from the commercially
13
6
available α-D-glucose-[ C ]. For the radiosynthesis, the carbon-14
atom was introduced in three different positions of empagliflozin.
In the first synthesis, carbon-14 D-(+)-gluconic acid δ-lactone was
used according to the literature to prepare carbon-14 specifically
labeled empagliflozin in carbon-1 of the sugar moiety. This four
step-synthesis gave the desired material in 19% overall
radiochemical yield and with a specific activity of 57.5 mCi/mmol
and a radiochemical purity of 99.2%. Carbon-14 labeled empagli-
flozin with the radioactive atom in the benzylic position was
obtained in eight steps and in 7% overall radiochemical yield with
a specific activity of 54.3 mCi/mmol and a radiochemical purity of
9
8.9%. In the last synthesis carbon-14 uniformly labeled phenol
14
was used to give [ C]empagliflozin in eight steps and in 18% overall
radiochemical yield with a specific activity of 53.4 mCi/mmol and a
radiochemical purity of 98.6%. Labeled empagliflozin was indis-
pensable in DMPK and other studies.
Figure 3. (a) Chiral HPLC of empagliflozin, (b) mixture of empagliflozin and (30),
1
4
and (c) [ C]empagliflozin.
Acknowledgements
14
[
4-(tetrahydro-furan-(3S)-yloxy)-benzyl]-[U- C]phenyl}-6-hydroxymethyl-
We are in debt to our colleagues Keith Fandrick and Carl Busacca
2
-methoxy-tetrahydro-2H-pyran-3,4,5-triol (1.07 g) as an off white foam. for reading this manuscript, and to Moravek Biochemicals for help
.
14
A solution of BF
3
OEt
2
(930 mg, 6.6 mmol) was added dropwise to a in the synthesis of [ C]empagliflozin labeled in the sugar moiety.
SiH (765 mg,
/MeCN (2.5 mL/10 mL) at À25 °C. The mixture was Conflict of Interest
solution of the above material (1.07 g, 2.2 mmol) and Et
.6 mmol) in CH Cl
warmed slowly to À10 °C over a period of 2 h. The reaction was
quenched by addition of saturated NaHCO
(10 mL) and extracted with The authors did not report any conflict of interest.
EtOAc (20 mL × 3). The combined EtOAc extracts were dried over
Na SO and concentrated, then diluted with EtOH and concentrated
2 mL × 2) giving an off white foam. The crude material was purified
by Combi-Flash Companion (40 g, 0–15% MeOH/CH Cl ) to give the
3
6
2
2
3
2
4
References
(
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2
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vacuum filtration giving [ C]empagliflozin (304 mg, 35.78 mCi) as a
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14
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[
[
[
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[
[
www.jlcr.org
Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 687–694