Carbon-14 labeling of K777·HCl, a therapeutic agent for Chagas disease

Article abstract of DOI:10.1002/jlcr.3107

The antitrypanosomal agent K777·HCl was labeled with carbon-14 to support absorption, distribution, metabolism, and excretion studies of this potential new drug for the treatment of Chagas disease. The radiolabeled compound was prepared in eight steps from [¹⁴C(U)]-(L)-phenylalanine with a specific activity of 54.4 mCi/mmol and an overall radiochemical yield of 4.1%. Copyright

Full text of DOI:10.1002/jlcr.3107

Special Issue Article
Received 29 April 2013,
Revised 5 July 2013,
Accepted 8 July 2013
Published online 19 August 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3107
Carbon-14 labeling of K777•HCl, a therapeutic
agent for Chagas disease†
Sung-Whi Rhee, Wallace W. Bradford, Jeremiah P. Malerich,
*
and Mary J. Tanga
The antitrypanosomal agent K777•HCl was labeled with carbon-14 to support absorption, distribution, metabolism, and
excretion studies of this potential new drug for the treatment of Chagas disease. The radiolabeled compound was prepared
in eight steps from [14C(U)]-(L)-phenylalanine with a specific activity of 54.4 mCi/mmol and an overall radiochemical yield of
4.1%.
Keywords: K777; Chagas disease; Trypanosoma cruzi; vinyl sulfone
Subsequent addition of benzyl alcohol and ethereal HCl
Introduction
provided H-[14C(U)]-Phe-OBn as the HCl salt 3. Reaction of 3
Chagas disease (American trypanosomiasis) is a potentially life-
with phosgene gave the isocyanate 4, which was reacted with
N-methylpiperazine to produce urea 5. Hydrogenolysis of the
threatening disease that affects 10 million people worldwide,
primarily in Latin America.1 The parasite Trypanosoma cruzi,
benzyl ester gave carboxylic acid 6, setting the stage for coupling
with amine 1.8 Treatment of 6 with isobutylchloroformate and
which causes Chagas disease, is transmitted to animals and
people through the fecal matter of the insect vector, triatomine
N-methylmorpholine gave a mixed anhydride that was reacted
or ‘kissing bugs.’ Left untreated, Chagas disease causes serious
with 1 as its hydrotosylate (HOTs) salt to give K777•HOTs. The
heart and digestive problems. The two frontline therapies for
tosylate counterion was exchanged by reaction with ethereal
the treatment of Chagas disease, benznidazole and nifurtimox,
HCl to afford K777 as its hydrochloride salt. A single hot run
are accompanied by significant toxicity.2–4 K777 is a vinyl sulfone
cysteine protease inhibitor, which irreversibly inhibits cruzain, a
key protease required for the viability of T. cruzi. A novel
chemical entity originally synthesized at Khepri Pharmaceuticals
and shown to be active against T. cruzi at the University of
California, San Francisco, K777 has been shown to be safe and
effective in models of acute and chronic Chagas disease.5,6 To
support the preclinical development of the drug candidate,
14C-K777•HCl was prepared for use in absorption, distribution,
metabolism, and excretion studies.
produced 46.1 mg of [14C9]K777•HCl with total activity of
4.11 mCi and specific activity of 54.4 mCi/mmol. The overall
radiochemical yield from [14C(U)]-(L)-phenylalanine was 4.1%.
Experimental
General methods
All reactions were carried out under argon atmosphere by using
commercial reagents of the highest available grade and without further
purification. [14C(U)]-(L)-phenylalanine was purchased from American
Radiolabeled Chemicals, Inc. (St. Louis, MO). Unlabeled intermediates
were analyzed by nuclear magnetic resonance (NMR) (Varian Mercury
VX 300 MHz or Varian 400 MHz) and liquid chromatography-mass
spectrometry (LCMS) (Thermo LCQ Fleet and Finnigan Surveyor System).
Radioassays of labeled products were carried out in 10 mL of Scintisol
cocktail (Isolab Inc.) with internal standards and counted using a
Results and discussion
K777 can be disconnected retrosynthetically (Scheme 1) into
N-methylpiperazine, phosgene (or
a synthetic equivalent),
(L)-phenylalanine, and chiral amine 1, as was described previously
by Somoza.7 The choice of site for radiolabeling was twofold:
(1) the phenylalanine unit is metabolically stable; and (2)
phenylalanine uniformly labeled with carbon-14 is commercially
available. A synthesis from [14C(U)]-(L)-phenylalanine would allow
us to prepare K777 following the established route and with high
specific activity.
SRI International, Biosciences Division, 333-Ravenswood Ave, Menlo Park, CA
94025 USA
*Correspondence to: Mary J. Tanga, SRI International, Biosciences Division, 333-
Ravenswood Ave, Menlo Park, CA 94025 USA.
E-mail: mary.tanga@sri.com
Synthetically, the radiosynthesis began with [14C(U)]-(L)-
phenylalanine and required the immediate protection of the
carboxyl group (Scheme 2). To circumvent the protection and
deprotection of the amino group during esterification, an in situ
protection strategy was utilized.8 [14C(U)]-(L)-phenylalanine was
† This article is published in Journal of Labelled Compounds and
Radiopharmaceuticals as a special issue on IIS 2012 Heidelberg Conference,
edited by Jens Atzrodt and Volker Derdau, Isotope Chemistry and Metabolite
Synthesis, DSAR-DD, Sanofi-Aventis Deutschland GmbH, Industriepark Höchst
treated with phosgene to generate oxazolidine dione 2. G876, 65926 Frankfurt am Main, Germany.
J. Label Compd. Radiopharm 2013, 56 461–463
Copyright © 2013 John Wiley & Sons, Ltd.

S.-W. Rhee et al.
Scheme 1. Retrosynthetic analysis.
Scheme 2. Synthesis of [14C9]K777•HCl.
Benzyl (S)-2-isocyanato-3-[14C6]phenyl[14C3]propionate (4)
Beckman LS-6000 liquid scintillation system. Analyses by radio-HPLC were
obtained with a Waters HPLC system (Waters Corporation, MA, USA; 600
Controller, 717 plus autosampler, and 996 photodiode array detector;
column: Agilent Zorbax S-8 C18, 4.6 × 150 mm) with detection by a
Radiomatic 610 TR as a flow scintillation analyzer (PerkinElmer, Inc., MA).
To a suspension of [14C(U)]-(L)-phenylalanine, benzyl ester hydrochloride
(3; 0.70 mmol, 206 mg, 43.8 mCi) in CH2Cl2 (15 mL) was added anhydrous
pyridine (3.09 mmol, 0.25 mL). The mixture was cooled to À40°C and
stirred vigorously while a phosgene solution (11.3 mmol, 20% in toluene,
6.0 mL) was added. The mixture was maintained at À40°C for an
additional 5 h and then washed with ice-cold aqueous HCl (0.5 M,
[14C(U)]-(L)-phenylalanine benzyl ester hydrochloride (3)
[
14C(U)]-(L)-phenylalanine (0.62 mmol, 103 mg, manufacturer specified 10 mL). The aqueous phase was extracted with CH2Cl2 (3 × 20 mL). The
specific activity: 150 mCi/mmol, total activity: 93.6 mCi) was dried under combined organic phases were washed with ice-cold brine (20 mL), and
vacuum for 2 days. Unlabeled (L)-phenylalanine (0.49 mmol, 81.7 mg) the aqueous phase was back-extracted with CH2Cl2 (2 × 20 mL). The
and anhydrous 1,4-dioxane (8 mL) were added to give a solution of
[
combined organic phases were dried over anhydrous MgSO4 and filtered
14C(U)]-(L)-phenylalanine with a calculated specific activity of 85.0 mCi/mmol. through a pad of MgSO4/charcoal/Celite in a sintered glass funnel. The
The solution was cooled in an ice bath, and a phosgene solution
(17 mmol, 20% in toluene, 9 mL) was added. After stirring for 72 h at
room temperature (RT), the mixture became clear and was concentrated
under a stream of argon. The residue was redissolved in anhydrous
1,4–dioxane (9 mL) and cooled to 0°C. Benzyl alcohol (5.8 mmol,
0.6 mL) and ethereal HCl (9.0 mmol, 2 M in Et2O, 4.5 mL) were added.
filtrate was concentrated to give crude 4 (150 mg), which was used
without further purification.
Benzyl (S)-[4-methylpiperazin-1-carbonyl]amino-3-[14C6]phenyl[14C3]
propionate (5)
The mixture was allowed to warm to RT. The reaction mixture became A solution of compound 4 (0.47 mmol, 133 mg) in anhydrous THF (20 mL)
cloudy over 72 h, and the crude solid compound 3 was isolated by was cooled to 0°C, and then a solution of N-methylpiperazine (0.65 mmol,
filtration. On cooling to 0°C, additional 3 precipitated. The combined
0.8 mL) in anhydrous THF (0.8 mL) was added. The mixture was stirred at
solids were recrystallized from boiling water to give benzyl ester 3 as a RT for 2 h, and additional N-methylpiperazine (0.15 mmol, 0.2 mL) in THF
white solid (206 mg, 70% yield, total activity 43.8 mCi, and specific (0.2 mL) was added. After an additional 2 h, the reaction mixture was
activity of 61.0 mCi/mmol). HPLC (Zorbax 50% aqueous trifluoroacetic concentrated on a rotary evaporator. The obtained residue was purified
acid (0.1%)/50% MeCN isocratic, 0.5 mL/min flow rate, 210 nm): tr
by column chromatography (silica gel, 2 × 13 cm, EtOAc/MeOH gradient).
5.0 min. Data for cold sample are as follows: 1H NMR (400 MHz, The piperazinyl urea 5 was isolated as viscous oil (131 mg, 48% yield from
methanol-d4) 3.19 (d, J = 6 Hz, 1H), 3.29 (m, 1H), 4.34 (t, J= 7 Hz, 1H), 3). HPLC (Zorbax 5 mM aqueous NH4OH/50% MeCN isocratic, 1.0 mL/min
5.21 (m, 2H), 7.12–7.20 (m, 2H), and 7.24–7.39 (m, 8H). ESI MS (M + H)+ 257.
flow rate, 210 nm): tr 5.1 min. Data for cold sample are as follows: 1H NMR
www.jlcr.org
Copyright © 2013 John Wiley & Sons, Ltd. J. Label Compd. Radiopharm 2013, 56 461–463

S.-W. Rhee et al.
(300 MHz, CDCl3) δ 2.28 (s, 3H), 2.36 (m, 4H), 3.11 (d, J= 6 Hz, 1H), 3.25–3.41 yield, total activity: 4.11 mCi, specific activity: 54.4mCi/mmol, radiochemical
(m, 4H), 4.77–4.94 (m, 2H), 5.09 (d, J = 13 Hz, 1H), 5.18 (d, J = 13 Hz, 1H), purity of >94% determined by radio-HPLC). HPLC (Zorbax 50% aqueous
6.97–7.03 (m, 2H), 7.16–7.24 (m, 3H), and 7.27–7.40 (m, 5H). ESI MS
trifluoroacetic acid (0.1%)/50% MeCN isocratic, 0.5mL/min flow rate,
210 nm): tr 6.3min. Data for cold sample are as follows: 1H NMR (300 MHz,
DMSO-d6) δ 1.75 (1H, m), 1.88 (1H, m), 2.49–2.90 (4H, m), 2.68 (3H, d,
J = 4 Hz), 3.05 (2H, m), 3.04–3.26 (2H, m), 3.30 (2H, d, J= 11 Hz), 3.97–4.15
(2H, m), 4.27 (1H, m), 4.43 (1H, m), 6.45 (1H, d, J = 15 Hz), 6.81 (1H, dd,
J = 15, 4 Hz), 7.13–7.31 (10H, m), 7.61–7.74 (3H, m), 7.82 (2H, d, J = 8 Hz),
8.22 (1H, d, J= 8Hz), 10.85 (1H, br s). 13C NMR (75MHz, DMSO-d6) δ 31.3,
34.7, 37.46, 40.8, 41.9, 48.7, 52.0, 56.4, 125.8, 126.3, 127.1, 128.0, 128.3,
128.4, 129.2, 129.6, 133.6, 138.2, 140.3, 141.2, 147.2, 156.7, and 172.0. ESI
MS (M+ H)+ 575.
(M + H)+ 382.
(S)-2-[4-methylpiperazine-1-carbonyl]amino-3-[14C6]phenyl[14C3]
propionic acid (6)
To a solution of compound 5 (0.34 mmol, 131 mg) in absolute, EtOH
(10 mL) was added Pd/C (40.6 mg, 30% Pd loading on activated carbon).
The reaction mixture was degassed twice and exposed to hydrogen gas
(1 atm) for 16 h. The remaining hydrogen gas was removed in vacuo, and
the mixture was filtered through a pad of Celite, rinsed with additional
ethanol (2 × 10 mL), and concentrated under vacuum. Crude 6 (100 mg,
total activity: 20.5 mCi, specific activity: 59.8 mCi/mmol) was isolated as
a pale brown solid, which was used without further purification. HPLC
(Zorbax 5 mM aqueous NH4OH/50% MeCN isocratic, 1.0 mL/min flow
rate, 210 nm): tr 3.1 min. Data for cold sample are as follows: 1H NMR
(DMSO-d6) δ 2.15 (3H, s), 2.18 (4H, m), 2.84–2.94 (1H, dd, J = 15, 11 Hz),
2.95–3.04 (1H, dd, J = 15, 5 Hz), 3.14–3.30 (4H, m), 4.17 (1H, m), 6.65 (1H,
d, J = 8 Hz), and 7.17–7.27 (5H, m). ESI MS (M + H)+ 292.
SUPPORTING INFORMATION
A radio-HPLC trace of [14C9]K777•HCl is provided.
Acknowledgements
4-Methyl-N-((S)-1-oxo-3-[14C6]phenyl-1-(((S,E)-5-phenyl-1-(phenylsulfonyl)
pent-1-en-3-yl)amino) [14C3]propan-2-yl)piperazine-1-carboxamide
hydrotosylate ([14C9]K777•HOTs)
Financial support from the National Institute of Allergy and
Infectious Diseases (NIAID), Division of Microbiology and Infectious
Disease (DMID) (Contract HHSN2722011000221, J. Mirsalis, H. J.
Parish, PIs) is gratefully acknowledged.
A solution of compound 6 (0.34 mmol, 100 mg) in THF (12 mL) and
dimethylformamide (3 mL) was cooled to 0°C. N-Methylmorpholine
(0.41 mmol, 0.5 mL) was then added, followed by isobutyl chloroformate
(0.43 mmol, 0.9 mL) in THF (0.9 mL). The reaction mixture was maintained
at or below À20°C for 2 h. Vinyl sulfone 19–11 (0.374 mmol, 177 mg) was
added as a solid in one portion, followed by dropwise adding of a
solution of N-methylmorpholine (0.41 mmol, 0.5 mL) in THF (0.5 mL).
The temperature was rigorously kept between À15°C and À30°C to
minimize epimerization of the phenylalanine residue. After stirring for
2 h, saturated aqueous NaHCO3 (20 mL) was added. The mixture was
extracted with ethyl acetate (3 × 20 mL), washed with brine (2 × 10 mL),
dried over MgSO4, and filtered. Concentration gave crude K777 free base
(220 mg) as brown viscous oil. The oil was dissolved in acetonitrile (4 mL),
and p-toluenesulfonic acid monohydrate (0.348 mmol, 66.2 mg) was
added. The solution was allowed to stand for 16 h at RT. The resulting
pale brown precipitate was washed with ether (20 mL) and air-dried.
The precipitate was recrystallized twice from boiling acetonitrile to give
Conflict of Interest
The authors did not report any conflict of interest.
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[
14C9]K777•HOTs (68 mg, 27% from 5) as a white solid.
4-Methyl-N-((S)-1-oxo-3-[14C6]phenyl-1-(((S,E)-5-phenyl-1-(phenylsulfonyl)
pent-1-en-3-yl)amino) [14C3]propan-2-yl)piperazine-1-carboxamide
hydrochloride ([14C9]K777•HCl)
[
14C9]K777•HOTs (68 mg) was dissolved in anhydrous CH2Cl2 (10 mL). The
solution was cooled in an ice bath, and ethereal HCl (2 M, 10 mL) was
added. The mixture was stirred and allowed to slowly warm to RT
overnight. The mixture was concentrated, and the residue was washed
with diethyl ether (25mL) and hexanes (25 mL). The solid was isolated by
centrifugation, and after removal of the supernatant, it was dried under
high vacuum. [14C9]K777•HCl was obtained as a white solid (46.1mg, 82%
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J. Label Compd. Radiopharm 2013, 56 461–463
Copyright © 2013 John Wiley & Sons, Ltd.
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