Potent and selective CC chemokine receptor 1 antagonists labeled with carbon-13, carbon-14, and tritium

Article abstract of DOI:10.1002/jlcr.3635

1-(4-Fluorophenyl)-1H-pyrazolo[3,4-c]pyridine-4-carboxylic acid (2-methanesulfonyl-pyridin-4-ylmethyl)-amide (1) and its analogs (2) and (3) are potent CCR1 antagonists intended for the treatment of rheumatoid arthritis. The detailed syntheses of these 3

Full text of DOI:10.1002/jlcr.3635

Received: 14 April 2018
DOI: 10.1002/jlcr.3635
Revised: 3 May 2018
Accepted: 4 May 2018
R E S E A R C H A R T I C L E
Potent and selective CC chemokine receptor 1 antagonists
labeled with carbon‐13, carbon‐14, and tritium
Bachir Latli
| Matt Hrapchak | Maxim Cheveliakov | Jonathan T. Reeves |
Maurice Marsini | Carl A. Busacca | Chris H. Senanayake
Chemical Development, Boehringer
1‐(4‐Fluorophenyl)‐1H‐pyrazolo[3,4‐c]pyridine‐4‐carboxylic
acid
(2‐
Ingelheim Pharmaceuticals, Inc.,
Ridgefield, CT, USA
methanesulfonyl‐pyridin‐4‐ylmethyl)‐amide (1) and its analogs (2) and (3) are
potent CCR1 antagonists intended for the treatment of rheumatoid arthritis.
The detailed syntheses of these 3 compounds labeled with carbon‐13 as well
as the preparation of (1) and (2) labeled with carbon‐14, and (1) labeled with
tritium, are described.
Correspondence
Bachir Latli, Chemical Development,
Boehringer Ingelheim Pharmaceuticals,
Inc. 900 Ridgebury Road, PO Box 368,
Ridgefield, CT 06877‐0368, USA.
Email: bachir.latli@boehringer‐ingelheim.
com
KEYWORDS
carbon‐13, carbon‐14, radiosynthesis, tritium, CCR1 antagonists
1 | INTRODUCTION
Thus, for carbon‐13 synthesis, fluorobenzene‐13C6
([13C6]‐(4)) was converted to 4‐nitrofluorobenzene‐13C6
CCR1 is a G‐protein coupled receptor that is involved in
the migration and activation of leukocytes to sites of
inflammation.1,2 Therefore, it is a therapeutic target for
the treatment of inflammatory diseases.3-12 By restricting
immune cell trafficking to inflammation sites, scientists
hoped to reduce disease severity and pain by using small
molecule antagonists of CCR1.13 In this manuscript, we
describe the synthesis of potent and selective antagonists
of CCR1 labeled with carbon‐13, carbon‐14, and tritium.
([13C6]‐(5)) by using nitric acid in TFAA in 82% yield
after separation of about 10% of the undesired 2‐
nitrofluorobenzene.16 Reduction of the nitro group was
accomplished in quantitative yield by using tin
chloride dissolved in ethanol and ethyl acetate at 80°C
to furnish [13C6]‐(6) in quantitative yield. Conversion to
the hydrazine [13C6]‐(7) was completed in 82% yield by
using sodium nitrate, tin chloride, and 6 M aqueous
HCl.17 Finally, condensation of [13C6]‐(7) with 3,‐5‐
dibromopyridine‐4‐carbaldehyde (8) gave the bromide
[13C6]‐(9) in 82% yield. This key bromide intermediate
was then transformed to the acid [13C6]‐(10) by using iso-
propyl magnesium chloride and lithium chloride solution
in tetrahydrofuran (THF; turbo‐Grignard) and carbon
dioxide in 81% yield (Scheme 1).
With this intermediate in hand, the 3 drug candidates
labeled with carbon‐13 were then obtained via amide
bond formation with the amines 11, 12, and 13 respec-
tively using known reaction conditions like 1,1′‐
carbonyldiimidazole (CDI) activation of the acid or using
propylphosphonic anhydride (T3P) and N‐methyl
morpholine (NMM) in N‐methyl‐2‐pyrrolidone (NMP)
as shown in Scheme 2.
2 | RESULTS AND DISCUSSION
These 3 CCR1 antagonists are structurally similar and
have a common aryl acid moiety, (1‐(4‐fluorophenyl)‐
1H‐pyrazolo[3,4‐c]pyridine‐4‐carboxylic acid (10) coupled
via an amide bond to (2‐methylsulfonyl‐4‐pyridyl)‐
methaneamine (11), 1‐(2‐(methylsulfonyl)pyridin‐4‐yl)
cyclopropan‐1‐amine (12),14 and (S)‐1‐(2‐(methylsulfonyl)
pyridin‐4‐yl)propan‐1‐amine (13),15 respectively (Figure 1).
Our efforts were concentrated on the synthesis of this
acid as a common intermediate to access these com-
pounds labeled with carbon‐13 and carbon‐14.
J Label Compd Radiopharm. 2018;1–9.
wileyonlinelibrary.com/journal/jlcr
Copyright © 2018 John Wiley & Sons, Ltd.
1

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LATLI ET AL.
FIGURE 1 CCR1 antagonists
SCHEME 1 Synthesis of aryl carboxylic
acid [13C6]‐10
SCHEME 2 Synthesis of carbon‐13
labeled CCR1 antagonists

LATLI ET AL.
3
58.3 mCi/mmol and radiochemical purities of 99.9% and
99.5%, respectively (Scheme 4).
To prepare tritium labeled (1), 6‐chloronicotinonitrile
(15) was converted to 6‐(methylsulfonyl)nicotinonitrile
(16) in 78% yield, and then reduced under carrier‐free tri-
tium gas and palladium on carbon in methanol in the
presence of 2 N aqueous HCl to the amine [3H2]‐(11).
Coupling to the acid, (10) gave tritium labeled (1) with
a specific activity of 42.1 Ci/mmol and radiochemical
purity of more than 98% (Scheme 5).
SCHEME 3 Synthesis of aryl acid intermediate [14C]‐(10)
3 | CONCLUSION
For carbon‐14 synthesis, the bromide (9)18 was either
subjected to cyanation reaction by using zinc cyanide‐14C,
followed by nitrile hydrolysis to [14C]‐(10) in 87% overall
yield, or subjected to turbo‐Grignard conditions described
before and carbon‐14 carbon dioxide albeit in poor yield
(Scheme 3).
Amide bond formation with amines (11) and (12) as
described before gave the desired carbon‐14 labeled ana-
logs (1) and (2) with specific activities of 59.5 and
1‐(4‐Fluorophenyl)‐1H‐pyrazolo[3,4‐c]pyridine‐4‐carboxylic
acid (2‐methanesulfonyl‐pyridin‐4‐ylmethyl)‐amide (1)
and its analogs (2) and (3) are potent CCR1 antagonists
intended for the treatment of rheumatoid arthritis. These
compounds labeled with carbon‐13 were prepared
starting from fluorobenzene‐13C6 ([13C6]‐(4)), which was
converted in 3 steps to 4‐fluorobenzene hydrazine
([13C6]‐(7)) in 80% overall yield and then condensed
with 3,5‐dibromo‐4‐pyridinecarboxaldehyde (8) to
SCHEME 4 Synthesis of [14C]‐(1) and
[14C]‐(2)
SCHEME 5 Synthesis of [3H2]‐(1)

4
LATLI ET AL.
afford
4‐bromo‐1‐(4‐fluorophenyl‐13C6)‐1‐H‐pyrazolo
software for data evaluation for radiolabeled compounds.
High‐performance liquid chromatography conditions,
unless indicated otherwise: mobile phase: gradient A:
MeCN (0.1% TFA), B: H2O (0.1% TFA) 20% A to 100% A
in 22 minutes, flow rate: 1 mL/min, column: Eclipse
XDB C18 (4.5 mm × 150 mm, 2.7 μm). Liquid scintilla-
tion counting was carried out by using a Beckman
LS6500 and Ultima Gold™ cocktail (Beckman, Fullerton,
CA, USA).
[3,4‐c]pyridine ([13C6]‐(9)) in 80% yield. Treatment with
turbo‐Grignard (isopropyl magnesium chloride and lith-
ium chloride solution) followed by carbonylation with
carbondioxide gave the acid ([13C6]‐(10)) in 81% yield.
Amide bond formation with amines (11), (12), and (13)
gave [13C6]‐(1), [13C6]‐(2), and [13C6]‐(3) in good yields
and in 99% isotopic enrichment. In carbon‐14 synthesis,
bromide (9) was converted to acid [14C]‐(10) by using
either turbo‐Grignard and [14C]‐CO2 in 34% yield or via
a 2‐step synthesis utilizing Zn(14CN)2 followed by hydro-
lysis in 87% overall yield. Amide bond formation afforded
[14C]‐(1) and [14C]‐(2) in 48% to 76% yield respectively
with specific activities higher than 58 mCi/mmol and
radiochemical purities higher than 99%. Finally, the
amine [3H2]‐(11) was obtained from the reduction of
4‐cyano‐2‐methanesulfonylpyridine (16) under tritium
gas in the presence of Pd/C and 2 N HCl in methanol
and then coupled to the acid (10) to give [3H2]‐(1)
with a specific activity of 42 Ci/mmol and 98% radio-
chemical purity.
5 | EXPERIMENTAL PROCEDURES
5.1 | Synthesis of carbon‐13 labeled
analogs
5.1.1 | 1‐Fluoro‐4‐nitrobenzene‐13C6,
[13C6]‐(5)
A solution of trifluoroacetic anhydride (TFAA, 13.2 mL,
94 mmol) and 40% nitric acid (5.9 g, 37.5 mmol) was pre-
pared by adding TFAA to HNO3 at low temperature (ice
bath). This solution was then added at room temperature
to flurorobenzene‐13C6 ([13C6]‐(4)) (2 g, 19.4 mmol) and
stirred at room temperature for 2 hours. The reaction
was poured into water (200 mL) and extracted with
CH2Cl2 (200 mL × 2), dried over MgSO4, filtered, and
concentrated in vacuo to give 3.1 g of viscous oil. TLC:
in 10% EtOAc:hexanes: Rf of desired product: 0.4, Rf of
the 2‐nitrofluorobenzene byproduct = 0.22. The crude
material was purified by Combi‐Flash by using 150‐g dis-
posable silica gel column and up to 10% EtOAc:hexanes.
The tubes containing the desired product were combined
and concentrated to give 2.35 g of material, which solidi-
fied at room temperature, in 82% yield. The byproduct 1‐
fluoro‐2‐nitrobenzene‐13C6 eluted later and gave 0.4‐g of a
semisolid product in 11% yield. 1H‐NMR (CDCl3,
400 MHz) δ: 8.31 (dm, J = 166.95 Hz, 2H), 7.22 (dm,
J = 164.52 Hz, 2H). 13С‐NMR (CDCl3, 100 MHz) δ:
165.5 (tdd, J = 9.71, 71.1, 257 Hz), 144.35 (dt, J = 7.95,
68.83 Hz), 126.27 (dt, J = 9.8, 71.64 Hz), 116.42 (m).
19F‐NMR (CDCl3, 376 MHz) δ: −101.5 (d).
4 | MATERIALS AND METHODS
Fluorobenzene‐13C6 ([13C6]‐(4)) with isotopic enrichment
of 99% and chemical purity of 99.7% was purchased from
Cambridge Isotope Laboratories, Inc (Tewksbury, MA,
USA). Zinc cyanide‐14C with a specific activity of
115.5 mCi/mmol was obtained from Moravek Biochemi-
cals (Brea, CA, USA). Carbonylation using carbon‐14 car-
bon dioxide and coupling to the amine (11) were
performed at Moravek Biochemicals (Brea, CA, USA).
Tritium reduction of a nitrile derivative (16) was per-
formed by American Radiolabeled Chemicals (Saint
Louis, MO, USA). 3,‐5‐Dibromopyridine‐4‐carbaldehyde
(8) and 6‐chloronicotinonitrile (15) were purchased from
Sigma‐Aldrich (WI, USA). Ultra performance liquid chro-
matography‐mass spectrometry (UPLCMS) was acquired
by using a medium polar method: run time 2.0 minutes,
gradient 95% water (0.1% TFA), and 5% MeCN
(0.1%TFA) to 5% water in 1.7 minutes, hold to 2 minutes
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). Nuclear magnetic resonance
(NMR) spectra were recorded with a Bruker 400 and
500‐MHz spectrometers by using deuterated NMR sol-
vents (Sigma‐Aldrich, USA). High‐performance liquid
chromatography (HPLC) was performed by using an
HPLC system composed of an Agilent 1200, IN/US
β‐RAM‐4, IN‐FLOW™ counting solution (LabLogic sys-
tems, Inc. Brandon, FL, USA) and using Laura Lite
5.1.2 | 4‐Fluoroaniline‐13C6, [13C6]‐(6)
A mixture of [13C6]‐(5) (2.6 g, 17.7 mmol) and tin chloride
dihydrate (16 g, 71 mmol) in ethyl acetate (40 mL) and
ethanol (40 mL) was heated to reflux at 85°C and stirred
for 4 hours. The reaction was then cooled to room tem-
perature and poured into 200 mL of 1 N aqueous NaOH
solution and extracted with ethyl acetate (200 mL × 3).
The combined extracts were dried over MgSO4, filtered,
and concentrated in vacuo to give 2.1 g (100% yield) of a
colorless oil, turned amber after standing in the air.

LATLI ET AL.
5
1
TLC: 10% EtOAc:hexanes, base lane. This material was
used as it is in the next step. 1H‐NMR (CDCl3,
400 MHz) δ: 6.95 (dm, J = 150.7 Hz, 2H), 6.71 (dm,
J = 154.1 Hz, 2H), 3.61 (brs, 2H). 13C‐NMR (CDCl3,
100 MHz) δ: 158.3 (m), 155.2 (m),142.5 (m), 116.4 (m).
19F‐NMR (CDCl3, 376 MHz) δ: −126.2 (d).
TLC in 20% EtOAc:CH2Cl2: Rf = 0.62. H‐NMR (CDCl3,
400 MHz) δ: 9.02 (s, 1H), 8.52 (s, 1H), 8.28 (s, 1H), 7.72
(dm, J = 163.2 Hz, 2H), 7.34 (dm, J = 167.3 Hz, 2H).
13C‐NMR (CDCl3, 100 MHz) δ: 163.1 (ddt, J = 10.3,
71.6, 143.1 Hz), 138.5, 137.5, 136.2, 135.4, 135.1 (dt,
J = 9.1, 65.1 Hz), 126.2, 125.2 (dt, J = 8.16, 65.1 Hz),
121.3, 116.6 (m).
5.1.3 | (4‐Fluorophenyl‐13C6)hydrazine,
[13C6]‐(7)
5.1.5 | 1‐(4‐Fluorophenyl‐13C6)‐1H‐
pyrazolo[3,4‐c]pyridine‐4‐carboxylic acid,
[13C6]‐(10)
A mixture of [13C6]‐(6) (2.1 g, 18 mmol) and 20 mL of 6 N
aq. HCl was stirred in an ice bath, while a solution of
sodium nitrite (1.3 g, 18 mmol) in water (10 mL) was
added dropwise in a 25‐minute period. The resulting
yellow solution was stirred for 30 minutes at 0°C before
a solution of tin chloride hydrate (10.2 g, 44.3 mmol) in
20 mL of 6 N aqueous HCl solution was added dropwise
in a 30‐minute period. The resulting mixture was stirred
for 14 hours. The solid was filtered and washed with con-
centrated HCl (10 mL) and dried under reduced pressure
to give 2.95 of a solid material. The free base was isolated
by treating the solid in water with 40% KOH until pH
is about 8, extracted with EtOAc, and concentrated to
give 2.1 g of material in 82% yield. TLC in 10% MeOH/
CH2Cl2: Rf = 0.8 for product and Rf = 0.7 for starting ani-
[13C6]‐(9) was placed in a dry 25‐mL round bottom flask
equipped with magnetic stirrer (0.51 g, 1.68 mmol). Tetra-
hydrofuran (3.5 mL) was added to the flask under N2, and
the resulting slurry was cooled to −20°C (internal probe).
A solution of iPrMgCl and LiCl (2.0 M in THF, 1.26 mL)
was added with such a rate to keep the internal
T < −10°C. In about 1.5 hour, the slurry became brown
solution. High‐performance liquid chromatography sam-
ple was taken after 2.5 hours. Several drops were
quenched with MeOH (1 mL). High‐performance liquid
chromatography indicated about 6% of starting material
left. The reaction was stirred at −10°C for an additional
1 hour. Carbon dioxide was bubbled through the reaction
solution until there was no rise in the temperature. Ini-
tially, the temperature rose from −10°C to 15°C then
start dropping. Carbon dioxide was bubbled for 5 addi-
tional minutes after the temperature started dropping.
The flask was taken out of the cooling bath, and the
resulting violet solution was stirred for 14 hours at 20 to
25°C. One drop of the reaction mixture was added to
1 mL of MeOH to prepare an HPLC sample. High‐perfor-
mance liquid chromatography indicated 93% product
and 6% of des‐bromo compound. Isopropyl acetate
(IPAC, 3.2 mL) was added in 1 portion at 25°C forming
fine off‐white slurry. A solution of 2.6 M aqueous
HCl (0.1 mL) was added to the reaction mixture and
heated to 55°C (internal probe). More aqueous HCl
(2.4 mL) was added dropwise. The color of the reaction
turned to yellow, then a slurry started to form and
became thick. The reaction was removed from the
heating bath and stirred at 25°C for 1 hour. The solid
was filtered and washed with IPAC (10 mL) and water
(10 mL), providing a yellow solid, which was further
dried in a vacuum oven overnight at 45°C to give
350 mg of product in 80% yield. Ultra performance liquid
chromatography‐mass spectrometry: tR = 0.69 minutes,
MH+ = 264.1 (100%). High‐performance liquid chroma-
tography: tR = 4.58 minutes (99.8%). TLC: R f = 0.12 in
10% MeOH/DCM. 1H‐NMR (DMSO‐d6, 400 MHz) δ:
13.69 (s, 1H, exchangeable), 9.46 (s, 1H), 8.90 (s, 1H),
8.72 (s, 1H), 7.91 (dm, J = 175.1 Hz, 2H), 7.51 (dm,
1
line. H‐NMR (DMSO‐d6, 400 MHz) δ: 9.35 (s, 1H), 7.18
(dm, J = 136.1 Hz, 2H), 6.92 (dm, J = 156.1 Hz, 2H),
3.33 (s, 2H). 13C‐NMR (DMSO‐d6, 100 MHz) δ: 158.35
(m), 155.4 (m), 142.98 (m), 115.6 (m).
5.1.4 | 4‐Bromo‐1‐(4‐fluorophenyl‐13C6)‐
1H‐pyrazolo[3,4‐c]pyridine, [13C6]‐(9)
[13C6]‐(7) (2.1 g, 16 mmol) was added to a solution of 3,5‐
dibromopyridine‐4‐carbaldehyde (8) (4.24 g, 16 mmol)
and NMP (20 mL) as a slurry in NMP (8 mL) with stirring
at room temperature. After stirring for 2 hours, HPLC
showed no starting aldehyde and a new product at
12.9 minutes. Solid Cs2CO3 (14.8 g, 43 mmol) was added
portion wise, followed by copper iodide (164 mg,
0.9 mmol), and the mixture was heated to 90°C and
stirred for 4 hours. The reaction mixture turned dark.
Water (55 mL) was added at 90°C, and the mixture was
cooled slowly to room temperature (3‐h period). The mix-
ture was filtered, washed with water (20 mL), and dried
under suction for 12 hours to give 5.1 g of a yellow fine
powder. This material was heated to 80°C with 25 mL
of toluene, then heptane (50 mL) was added slowly, keep-
ing the temperature at around 80°C. After the addition
was completed, the mixture was cooled to room tempera-
ture slowly and then stirred for 14 hours. Filteration of
the solid and washing with heptane (40 mL), then drying
at 61°C for 3 hours gave 4.2 g of a yellow‐brown solid.

6
LATLI ET AL.
J = 170.4 Hz, 2H). 13С‐NMR (DMSO‐d6, 100 MHz) δ:
166.2, 162.2 (ddt, J = 10.2, 71.1, 244.8 Hz), 154.5, 151.2,
137.2, 138.9, 135.1 (dt, J = 9.12, 65.8 Hz), 134.7, 126.3,
125.1 (dt, J = 6.86, 63.5 Hz), 116.7 (m). HRMS, calculated
264.08746, found 264.08740.
forming
a
light‐brown thick reaction mixture.
Propylphosphonic anhydride (604 mg, 0.95 mmol, 50%
in EtOAc) was added dropwise with cooling (water
bath). The reaction was heated at 70°C for 2.5 hours.
High‐performance liquid chromatography showed com-
plete conversion. Water (1.6 mL) was added to the reac-
tion mixture at 70°C and allowed to stirr at room
temperature for 1 hour. The formation of an off‐white
residue was observed. The reaction vessel was cooled
for 1 hour by using an ice bath to complete the crystal-
lization. The residue was filtered by using a paper filter,
washed with water (10 mL), then IPAC (5 mL). The
solid was air‐dried on the filter for 3 hours and then
in a vacuum oven for 14 hours at 60°C to give 220 mg
of material in 76% yield. Ultra performance liquid chro-
matography‐mass spectrometry: 0.73 minute (100%),
MH+ = 458.11. High‐performance liquid chromatogra-
phy: tR = 2.48 minutes (99.8%). 1H‐NMR (DMSO‐d6,
400 MHz) δ: 9.75 (s, 1H, NH), 9.73 (s, 1H), 9.42 (s,
1H), 8.94 (s, 1H), 8.68 (d, 1H), 8.66 (d, 1H), 7.92 (dm,
2H), 7.80 (d, 1H), 7.53 (d, 1H), 6.98 (dm, 2H), 3.28 (s,
3H), 1.61 (m, 4H). 13C‐NMR (DMSO‐d6, 100 MHz) δ:
166.2, 161.8, 160.2 (dt), 158.2, 156.3, 150.3, 138.6,
137.7, 135.3 (m), 135.1, 126.7, 124.9 (t), 124.1, 122.9,
121.4, 116.7 (m), 115.1, 39.8, 34.5, 20.4. HRMS:
[C1613C6H19N5O3SF]+, calculated 458.13884, found
458.13852.
5.1.6 | 1‐(4‐Fluorophenyl‐13C6)‐N‐(1‐(2‐
(methylsulfonyl)pyridin‐4‐yl)methyl)‐1H‐
pyrazolo[3,4‐c]pyridine‐4‐carboxamide,
[13C6]‐(1)
1,1′‐Carbonyldiimidazole (75 mg, 0.45 mmol) was added
to a mixture of [13C6]‐(10) (94 mg, 0.36 mmol) in DMF
(5 mL) to give a solution. After stirring at room tempera-
ture for 10 minutes, the reaction was heated to 60°C. A
sample was taken with the tip of a pipet after 45 minutes
and treated with 1 mL of MeOH and 3 drops of
triethylamine and checked by HPLC; no starting acid
was observed and UPLCMS
1 single peak, MH
+
= 278.57 corresponding to the methyl ester. The amine
TFA‐salt (11) (118 mg, 0.4 mmol) was added in 1 portion
at 60°C and stirred for 1 hour. High‐performance liquid
chromatography showed a new product and UPLC‐MS:
MH+ = 432.65 corresponding to the product. Water
(10 mL) was added dropwise at 60°C, and the tempera-
ture was then ramped to room temperature slowly. The
precipitate was filtered, washed with water (10 mL) and
heptane (10 mL), and left to dry under vacuum overnight
to give 125 mg as an off‐white solid in 81% yield. Ultra
performance liquid chromatography‐mass spectrometry:
tR = 1.33, MH+ = 432.65 (100%). High‐performance liquid
chromatography: tR = 6.32 minutes (99%). 1H‐NMR
(СDCl3, 400 MHz) δ: 9.65 (s, 1H), 9.12 (s, 1H), 8.75 (d,
2H), 8.63 (d, 1H), 8.10 (s, 1H), 7.70 (dm, 2H), 7.71 (t,
1H), 7.28 (dm, 2H), 4.84 (d, 2H), 3.32 (s, 3H). 13С‐NMR
(СDCl3, 100 MHz) δ: 162.35, 161.5 (dt), 160.1, 158.03,
150.45, 148.2, 137.47 (d), 135.03, 130.60, 130.07, 129.43
(t), 125.51, 125.12 (d), 121.4, 118.1, 116.27 (m), 42.63,
40.15. HRMS: (MH+), calculated 432.12281, found
432.12303.
5.1.8 | (S)‐1‐(4‐Fluorophenyl‐13C6)‐N‐1‐(2‐
(methylsulfonyl)pyridin‐4‐yl)propyl)‐1H‐
pyrazolo[3,4‐c]pyridine‐4‐carboxamide,
[13C6]‐(3)
N‐Methyl morpholine (5 mL) was added to a mixture of
[13C6]‐(10) (346 mg, 1.3 mmol) and the amine
camphorsulfonic acid‐salt (13) (0.59 g, 1.32 mmol) in
NMP (5 mL), and the solution was stirred for 15 minutes.
Propylphosphonic anhydride (50% solution in EtOAc,
1.1 mL, 1.85 mmol) was added dropwise at room temper-
ature. The solution was then heated to 60°C and stirred
for 90 minutes. High‐performance liquid chromatography
indicated about 30% of remaining starting material.
One more equivalent of the amine and 1.4 equivalent of
T3P were added successively and stirred for 14 hours.
Water was added slowly, and the mixture was cooled to
room temperature overnight. The aqueous was extracted
with EtOAc (150 mL × 2), and the combined extracts
were washed with water (100 mL × 3), dried over MgSO4,
filtered, and concentrated in vacuo to give 887.5 mg of
a yellow residue. The crude product was crystallized
from 2‐butanone (3 mL) and heptane (3 mL) by dissolv-
ing first the solid in 2‐butanone at 75°C, then adding
heptane slowly and cooling to room temperature. The
5.1.7 | 1‐(4‐Fluorophenyl‐13C6)‐N‐(1‐(2‐
(methylsulfonyl)pyridin‐4‐yl)cyclopropyl)‐
1H‐pyrazolo[3,4‐c]pyridine‐4‐carboxamide,
[13C6]‐(2)
The acid [13C6]‐(10) (170 mg, 0.63 mmol) and amine
HCl‐salt (12) (241 mg, 0.95 mmol) were placed in a
15‐mL round‐bottom flask, and the flask was flushed
with N2. N‐Methyl‐2‐pyrrolidone (0.6 mL) was added
via syringe under N2 forming a suspension. N‐Methyl
morpholine (522 mg, 5.07 mmol) was added to the sus-
pension at room temperature and stirred for 30 minutes,

LATLI ET AL.
7
product was filtered and dried in vacuo to give 513 mg of
product more than 99% pure in 85% yield. Ultra perfor-
mance liquid chromatography‐mass spectrometry:
tR = 0.82 minute (100%), MH+ = 458.38. High‐
performance liquid chromatography: Eclipseе, XDB‐C18
(4.6 × 150 mm, 1.8 μm). A = 0.2% H3PO4 and 60‐mM
NH4PF6 in water, B MeCN, flow rate 1.5 mL/min, run
time 12 minutes, column temperature 65°C, DAD
210 nm, Rt = 4.81 minutes, 99.5%. Chiral HPLC: column
Chiralcel AD‐H (0.46 × 250 mm), mobile phase: A
heptane with 0.1% acetic acid; B = EtOH isocratic A/B:
50/50. Flow 1 mL/min, run time 16 minutes, column
temperature 20°C 5‐μL injection, DAD 220 nm. The sam-
ple was prepared in methanol. 1H‐NMR (DMSO‐d6,
400 MHz) δ: 9.41 (s, 1H), 9.37 (d, 1H), 8.95 (s, 1H), 8.75
(d, 1H), 8.64 (s, 1H), 8.17 (s, 1H), 7.91 (dm, 2H), 7.82
(dd, 1H), 7.51 (dm, 2H), 5.18 (m, 1H), 3.30 (s, 3H), 1.93
(m, 2H), 1.01 (t, 3H). 13C‐NMR (DMSO‐d6, 100 MHz) δ:
164.7, 161.1 (dt), 158.1, 155.8, 153.4, 150.2, 138.6, 137.5,
135.3 (dt), 135.1, 126.7, 125.9, 124.2 (dt), 121.6, 118.1,
116.6 (m), 39.7, 35.6, 28.3, 11.1. HRMS: calculated
460.15449, found 460.15429.
solution at 55 to 60°C. Stirring was continued for
1.5 hour. Water (10 mL) was added at a rate to maintain
the batch at 55 to 60°C and held for 10 minutes at about
60°C. The mixture was then cooled down to room tem-
perature over 2 hours and held for at least 1 hour at this
temperature. The solid was filtered and washed with
water (2 mL × 3) and heptane (2 mL) and then dried
under vacuum to give 280 mg (38.6 mCi) after flash
chromatography purification. The product was obtained
as a light yellow solid, with a radiopurity of 99.9% by
using the following HPLC conditions: Supelco Discovery
C18 (4.6 × 250 mm), flow rate; 1 mL/min, mobile phase:
A: water, 0.1% TFA; B: MeCN, 0.1% TFA, 10 to
30 minutes, 20‐100%B held to 40 minutes. MS: MH
+
=
426.24 (100%), 427.27 (20%). Specific activ-
ity = 59.5 mCi/mmol. 1H‐NMR (DMSO‐d6, 500 MHz)
δ: 9.65 (t, 1H, NH), 9.45 (s, 1H), 8.93 (s, 1H), 8.74 (s,
2H), 8.08 (s, 1H), 7.94 (m, 2H), 7.77 (d, 1H), 7.47 (m,
2H), 4.75 (d, 2H), 3.30 (s, 3H).
5.2.3 | 1‐(4‐Fluorophenyl)‐1H‐pyrazolo[3,4‐
c]pyridine‐4‐carbonitrile‐14C, [14C]‐14
A mixture of (9) (0.5 g, 1.73 mmol) and zinc cyanide‐14C
(100 mCi, SA = 115.5 mCi/mmol) in anhydrous DMF
(8.0 mL) in a screw cap 24 mL vial was degassed 3 times,
and Argon was introduced in every cycle. Dppf (261 mg,
0.5 mmol) and Pd2(dba)3 (916 mg, 0.21 mmol) were
introduced to the reaction vial and degassed 3 more
times. The mixture was heated to 120°C and stirred for
14 hours. A solution of 2 N NH4OH (50 mL) was added,
and the mixture was extracted with EtOAc. The com-
bined extracts were filtered through a short pad of silica
gel and rinsed with EtOAc. The solution was concen-
trated to give a dark residue (0.45 g), which was used
as is in the next step.
5.2 | Carbon‐14 synthesis
5.2.1 | 1‐(4‐Fluoro‐phenyl)‐1H‐
pyrazolo[3,4‐c]pyridine‐4‐carboxylic acid‐
14С, [14С]‐(10)
4‐Bromo‐1‐(4‐fluoro‐phenyl)‐1H‐pyrazolo[3,4‐c]pyridine
(9) (1.1 g, 3.62 mmol) in THF (10 mL) was cooled to 0°C.
A solution of i‐PrMgCl.LiCl (1.3 M THF, 2.89 mL,
3.75 mmol) was added slowly. The resulting solution
was stirred at 0°C for 1 hour. Carbonation was performed
at −20°C for 2 hours. Then, a solution of aqueous 3 N
HCl (12 mL) was added. The aqueous was extracted with
CH2Cl2, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by silica gel chromatog-
raphy by using 3:1 CH2Cl2:MeOH to give 81 mCi of prod-
uct in 38% yield. The product coeluted on TLC and HPLC
with the unlabeled (10).
5.2.4 | 1‐(4‐Fluorophenyl)‐1H‐pyrazolo[3,4‐
c]pyridine‐4‐carboxylic‐14C acid, [14C]‐(10)
Sulfuric acid (20 mL) and water (30 mL) were added to
the above crude nitrile (0.42 g, 1.74 mmol). The mixture
was heated to 124°C and stirred for 4 hours. The resulting
mixture was filtered off and washed with water. The
aqueous solution was cooled in an ice bath and treated
with aqueous 4 N NaOH (100 mL) until a precipitate
appeared. The mixture was extracted with EtOAc
(150 mL × 3), and the combined extracts were dried over
MgSO4, filtered, and concentrated in vacuo to give
400 mg (90 mCi) of a yellowish powder in 89% yield.
High‐performance liquid chromatography coeluted with
unlabeled (10).
5.2.2 | 1‐(4‐Fluoro‐phenyl)‐1H‐
pyrazolo[3,4‐c]pyridine‐4‐carboxylic‐14C‐
acid‐(2‐methanesulfonyl‐pyridin‐4‐
ylmethyl)‐amide, [14С]‐(1)
The above acid (81 mCi, 1.4 mmol) and CDI (291 mg,
1.79 mmol) were stirred in DMF (6 mL) for 20 minutes
at room temperature. The temperature of the mixture
was slowly raised to about 60°C. After 2 hours at this
temperature, the amine‐TFA salt (11) (441 mg,
1.46 mmol) in DMF (1.5 mL) is added to the above

8
LATLI ET AL.
1
give 5.1 g of a solid. H‐NMR (CDCl3, 400 MHz) δ: 9.21
(d, 1H), 8.62 (d, 1H), 7.95 (d,1H), 3.32 (s, 3H). 13С‐NMR
(CDCl3, 100 MHz) δ: 161.3, 152.4, 131.8, 123.7, 122.1,
119.6, 43.7 ppm.
5.2.5 | 1‐(4‐Fluorophenyl)‐N‐{1‐[2‐
methylsulfonyl)pyridin‐4‐yl]cyclopropyl}‐
1H‐pyrazolo[3,4‐c]pyridine‐4‐carboxamide‐
14C, [14C]‐(2)
N‐Methyl morpholine was added to a mixture of the
above acid (0.4 g, 1.54 mmol) and the HCl‐salt of amine
(12) (0.5 g, 2.0 mmol) in NMP (5 mL), and the solution
was stirred for 15 minutes. Propylphosphonic anhydride
(3.0 mL, 50% solution in EtOAc) was added dropwise
at room temperature. The solution was then heated to
65°C and stirred overnight. High‐performance liquid
chromatography showed that about 4% of the acid was
left. Another batch of T3P (0.5 mL) was added, and the
dark mixture was stirred for another 3 hours. Water
(40 mL) was added, and the mixture was cooled to room
temperature. The mixture was extracted with CH2Cl2
(50 mL × 3), and the combined extracts were dried
over Na2SO4, filtered, and concentrated in vacuo to give
5.6 g of an NMP solution of the product. The organic
phase was heated to 65°C, and water 7 mL was added
slowly. The mixture was stirred at 65°C for 1 hour and
then at room temperature for 14 hours. The mixture
was filtered, washed with water (20 mL), then with hep-
tane (10 mL). The solid was dried to give 470 mg of an
off‐white solid in 76% yield, 90% pure. Further purifica-
tion by silica gel chromatography by using 40‐g dispos-
able silica gel column and up to 5% MeOH/CH2Cl2
afforded 230 mg of a white solid, 29.5 mCi. Ultraviolet
and rad indicated a chemical and radiochemical purity
of more than 99% and with a specific activity of
58.3 mCi/mmol.1H‐NMR (DMSO6, 500 MHz) δ: 9.74 (s,
1H), 9.42 (s, 1H), 8.94 (s, 1H), 8.69 (s, 1H), 8.66 (d, 1H),
7.92 (dd, 1H), 7.81 (s, 1H), 7.51 (m, 3H), 3.28 (s, 3H),
1.60 (brs, 4H). High‐performance liquid chromatography:
column Zorbax Eclipse XDB C18 (4.6 × 50 mm, 1.8 μm).
Mobile phase: A: water (0.1% H3PO4, 0.2% HClO4); B:
MeCN: MeOH (19:10), tR = 6.37 minutes, 99.4%, rad
HPLC 99.5%.
5.3.2 | (2‐Methylsulfonyl‐4‐pyridyl)‐
ditritio‐methanamine hydrochloride,
[3H2]‐(11)
Methanol (0.5‐1.0 mL) and aqueous HCl (2.0 M, 25 μL)
were added to a mixture of (16) (9 mg, 39.7 μmol) and
Pd/C (10%, 10 mg). The mixture was stirred under
1 atm of tritium gas for 20 hours. High‐performance liq-
uid chromatography showed complete conversion to
desired product. A batch of 1.75 Ci of crude product
was obtained.
5.3.3 | 1‐(4‐fluorophenyl)‐N‐[(2‐
methylsulfonyl‐4‐pyridyl)‐ditritio‐methyl]
pyrazolo[3,4‐c]pyridine‐4‐carboxamide,
[3H2]‐(1)
N‐Methyl morpholine (50 μL, 0.44 mmol) was added to a
mixture of the acid (10) (4.24 mg, 16.5 μmol) and
[3H2]‐(11) (3.74 mg, 16.5 μmol) in NMP (2 mL), and the
solution was stirred for 15 minutes. Propylphosphonic
anhydride (50% solution in EtOAc, 0.3 mL) was added
dropwise at room temperature. The solution was then
heated to 65°C and stirred overnight. After cooling to
room temperature, the solution was treated with
water (10 mL) and extracted with EtOAc (5 mL × 3).
The combined extracts were dried over Na2SO4, filtered,
and concentrated in vacuo. The residue with some
NMP was diluted in 6 mL of methanol and saved at
−20°C. A portion of this material (1 mL, about
300 mCi) was purified by semipreparative HPLC to give
175 mCi of product with a specific activity of 42.1 Ci/
mmol. Semipreparative conditions: system Agilent 1100
using Zorbax XDB C18 (4.6 × 150 mm, 5 μm) column,
50‐μL injection size, 2‐mL/min flow rate, mobile phase
A: water (0.1% TFA) and B: MeCN; gradient 0 to
6 minutes 80% A, 6 to 15 minutes 80% A to 5% A. High‐
performance liquid chromatography, tR = 6.76 minutes
(98.5%). Column: Halo С18 (4.6 × 150 mm, 2.7 μm) gradi-
ent 10% to 95% MeCN: water (0.1% TFA) in 15 minutes, c‐
eluted with unlabeled material. 3H‐NMR (DMSO‐d6,
5.3 | Tritium synthesis
5.3.1 | 4‐Cyano‐2‐methylsulfonylpyridine
(16)
A slurry of 2‐chloro‐4‐cyanopyridine (15) (5 g, 36.1 mmol)
and sodium methane sulfinate (4.4 g, 43 mmol) in NMP
(10 mL) was heated to 125°C and stirred for 14 hours.
After cooling to 35°C, water (30 mL) was added slowly.
The mixture was cooled to room temperature and stirred
for 4 hours. The mixture was filtered, washed with water
(50 mL), and dried under suction. The off‐white solid was
further dried in a vacuum oven at 65°C for 48 hours to
1
1
427 MHz) δ: H‐decoupled, 4.72(s); H‐coupled, 4.72 (d,
J = 8.5 Hz).
ORCID
Bachir Latli http://orcid.org/0000-0002-0024-9792

LATLI ET AL.
9
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