Synthesis of deleobuvir, a potent hepatitis C virus polymerase inhibitor, and its major metabolites labeled with carbon-13 and carbon-14

Article abstract of DOI:10.1002/jlcr.3294

Deleobuvir, (2E)-3-(2-{1-[2-(5-bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl-1H-indole-6-carboxamido]cyclobutyl}-1-methyl-1H-benzimidazol-6-yl)prop-2-enoic acid (1), is a non-nucleoside, potent, and selective inhibitor of hepatitis C virus NS5B polymerase.

Full text of DOI:10.1002/jlcr.3294

Research Article
Received 28 January 2015,
Revised 10 March 2015,
Accepted 1 April 2015
Published online 11 May 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3294
Synthesis of deleobuvir, a potent hepatitis C
virus polymerase inhibitor, and its major
metabolites labeled with carbon-13 and
carbon-14
*
Bachir Latli, Matt Hrapchak, Maxim Chevliakov, Guisheng Li,
Scot Campbell, Carl A. Busacca, and Chris H. Senanayake
Deleobuvir, (2E)-3-(2-{1-[2-(5-bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl-1H-indole-6-carboxamido]cyclobutyl}-1-methyl-
1H-benzimidazol-6-yl)prop-2-enoic acid (1), is a non-nucleoside, potent, and selective inhibitor of hepatitis C virus NS5B
polymerase. Herein, we describe the detailed synthesis of this compound labeled with carbon-13 and carbon-14. The
synthesis of its three major metabolites, namely, the reduced double bond metabolite (2) and the acyl glucuronide
derivatives of (1) and (2), is also reported. Aniline-13C6 was the starting material to prepare butyl (E)-3-(3-methylamino-4-
nitrophenyl-13C6)acrylate [13C6]-(11) in six steps. This intermediate was then used to obtain [13C6]-(1) and [13C6]-(2) in five
and four more steps, respectively. For the radioactive synthesis, potassium cyanide-14C was used to prepare 1-
cylobutylaminoacid [14C]-(23) via Buchrer–Bergs reaction. The carbonyl chloride of this acid was then used to access both
[
14C]-(1) and [14C]-(2) in four steps. The acyl glucuronide derivatives [13C6]-(3), [13C6]-(4) and [14C]-(3) were synthesized in
three steps from the acids [13C6]-(1), [13C6]-(2) and [14C]-(1) using known procedures.
Keywords: deleobuvir; metabolites; NS5B polymerase; acyl glucoronide; carbon-14; carbon-13
pharmaceutical companies to eradicate this virus.16,17 Deleobuvir
Introduction
is a non-nucleoside, potent, and selective HCV NS5B polymerase
inhibitor developed for the treatment of HCV infection.18–21
Hepatitis C virus (HCV) infection is a serious public health issue in
the world. Each year between three and four million people
become infected.1,2 According to the Centers for Disease Control
(CDC), most at risk are injection drug users, dialysis patients,
people who acquire tattoos or body piercings with non-sterile
instruments, some healthcare workers, people with HIV, and
children born to mothers with hepatitis C. The highest prevalence
of HCV worldwide is in North Africa and South Asia. In Egypt, for
example, one in seven people is infected with the virus.3,4 The
main methods of transmission in this country are blood
transfusions and unsafe medical procedures.5 HCV is highly
heterogeneous, which can be an obstacle for the development
of a universal treatment and a preventive vaccine. Seven major
HCV genotypes and several subtypes have been identified
throughout the world. Subtypes 1a/b account for approximately
70% of all infections in the USA, Europe, China, and Japan.6 These
subtypes are the hardest to treat.7,8 The remainders are generally
genotypes 2, 3, and 4.9 Hepatitis C is a single-stranded RNA virus.
Its genome encoded a single protein of about three thousands
amino acids.10,11 This protein is cleaved into smaller structural
and nonstructural (NS) proteins by proteases like NS3/4A.
Results and discussion
Boehringer Ingelheim has been in the forefront in tackling the
problem of HCV infections by inventing drug candidates that
specifically inhibit certain proteases and polymerases involved in
the replication of this virus. Previously, we reported the synthesis
of several NS3/4A protease inhibitors labeled with radioactive and
stable isotopes.22 In this paper, we describe the synthesis of a potent
inhibitor of NS5B polymerase labeled with carbon-13 and carbon-14
isotopes as well as its major metabolites (Figure 1).
Two major metabolites of deleobuvir were identified in plasma;
a metabolite with a reduced double bond formed in the gastro-
intestinal tract by gut bacteria (2), and an acyl glucuronide (3).
The reduced double bond metabolite (2) was further
metabolized to glucuronide (4).23 The glucuronation is common
for drug candidates containing carboxylic acid, hydroxyl, amino,
Chemical Development, Boehringer Ingelheim Pharmaceuticals, Inc., 900
Polymerases play a major role in viral replication.12 The Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-0368, USA
polymerase NS5B catalyzes the synthesis of a complementary
*Correspondence to: Bachir Latli, Chemical Development, Boehringer Ingelheim
negative-stranded RNA using the positive-stranded RNA genome
Pharmaceuticals, Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-
0368, USA.
as a template.13–15 Direct acting antiviral agents that target
specific steps in the virus replication cycle have been pursued by E-mail: bachir.latli@boehringer-ingelheim.com
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B. Latli et al.
Figure 1. Structures of deleobuvir (1) and its metabolites (2), (3), and (4).
and sulfuryl moieties, and is usually considered a phase two in 96% yield despite the fact that the reaction was slow
metabolism process.24 α,β-Unsaturated acids and ketones and required four equivalents of NCS. Shorter reaction times
undergo enzymatic reduction of the carbon–carbon double were observed when we used freshly crystallized NCS. Removal
bonds, and for certain compounds, for reasons that remain of the acetyl group was accomplished in quantitative yield by
unclear, the reduction of the double bond is often a prerequisite refluxing in 2.5N aqueous HCl. The 2,4-dichloroaniline [13C6]-(8)
for further metabolism.25–27
was isolated as a white solid, and the amino group was then
oxidized to a nitro by heating with 30% hydrogen peroxide
in acetic acid with traces of concentrated sulfuric acid at 80°C
for 14h in 71% yield.32 The oxidation step was necessary for
Synthesis of [13C6]-(1) and [13C6]-(2)
The synthesis of deleobuvir has been reported.28 The the selective SNAr reaction with methylamine. The product
commercially available 2,4-dichloronitrobenzene was used in 2,4-dichloro-nitrobenzene [13C6]-(9) was treated in DMSO with
the synthesis of the benzimidazole right-hand side of (1). This is an aqueous 40% solution of methylamine to give 4-chloro-2-
a suitable region for introducing six 13C atoms. However, methylamino-nitrobenzene [13C6]-(10) in 98% yield. Ligandless
carbon-13 labeled 2,4-dicholoronitrobenzene is not commercially Heck reaction with butyl acrylate gave (E)-(3-methylamino-4-
available. From earlier work, we reported the preparation of 3,5- nitrophenyl)acrylic acid butyl ester [13C6]-(11) in 83% yield,28
dichloroaniline-13C6 from aniline-13C6.29 Similarly, we envisioned (Scheme 1).
if we could oxidize the amino-moiety to a nitro group, we will
Reduction of the nitro group using tin dichloride gave the
have our starting material (Scheme 1). Direct chlorination of diamine derivative [13C6]-(12) in 62% yield. Condensation of this
aniline with N-chlorosuccinimide (NCS) in chloroform gave 2,4- compound with 1-amino-cyclobutanecarbonyl chloride (13) in
dichloroaniline in yields ranging from 40% to 50%.30 Thus, toluene and methanol at 90 °C gave the benzimidazole derivative
aniline-13C6 was first protected as the acetanilide [13C6]-(6) in
[
13C6]-(14) in 67% yield. The acid fragment (15), which is
94% using acetyl chloride and pyridine in methylene chloride.31 available from previous syntheses,28 was treated with thionyl
Treatment of this acetanilide with NCS in trifluoroacetic acid chloride at 0 °C in methylene chloride in the presence of
(TFA) at room temperature gave 2,4-dicholoacetanilide [13C6]-(7) triethylamine. The acyl-chloride (16) generated in situ was then
Scheme 1. Synthesis of [13C6]-(11).
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B. Latli et al.
reacted with the amine fragment [13C6]-(14) and the butyl ester gave the desired acyl glucuronide derivatives.35–39 The products
[13C6]-(17) was isolated in 84% yield. Hydrolysis in aqueous sodium were isolated in most cases by reverse phase chromatography
hydroxide and n-propanol followed by flash chromatography with yields ranging from 44% to 63%. Allyl-D-glucuronate is
purification, then crystallization from methanol-methylene chloride commercially available or can be easily prepared from glucuronic
gave [13C6]-(1) as a white solid in quantitative yield (Scheme 2).
Our first attempts to prepare the metabolite [13C6]-(2) focused
on using [13C6]-(1). We devised a simple procedure that was
tested successfully on the unlabeled butyl ester (17). The
acid and allylbromide according to the literature.40
Synthesis of [14C]-(1), [14C]-(2), and [14C]-(3)
procedure used diimide generated in situ from potassium The compound 1-amiocyclybutycarboxylic acid (23) seems to be
azodicarboxylic acid (PADA) and acetic acid in DMSO at ambient a convenient intermediate that can be accessed in two steps
temperature.33 The method is selective, and only the targeted using carbon-14 labeled potassium cyanide via the Bucherer–
double bond is reduced. However, direct reduction of acid (1) Bergs reaction.41 Conversion of this amino acid to 1-amino-
with PADA for extended periods of time resulted in no reduction cyclobutane carbonyl chloride was accomplished using PCl5
of the double bond. Other direct reductions of the cinnamic and 2-oxazolidone in acetonitrile,28 (Scheme 5).
double bond using nickel dichloride and sodium borohydride
Carbon-14 labeled deleobuvir was then prepared in three steps
or samarium iodide in water failed to give (2).34 With limited as depicted in Scheme 6. The diamine derivative (12) which was
amounts of compound [13C6]-(14), we turned our attention to obtained from the reduction of butyl ester (11) was condensed
[13C6]-(11). The route described in Scheme 2 to prepare [13C6]-(1) with the acyl chloride derivative [14C]-(13) to give the amine
was then followed to prepare the metabolite [13C6]-(2). The only
[
14C]-(14) in 52% yield. Coupling to the acyl chloride (16), as seen
difference is the tandem reduction of the nitro group and the before, gave the butyl ester [14C]-(17). Ester hydrolysis using
double bond with hydrogen in the presence of palladium on aqueous NaOH in n-propanol gave
14C]-(1) in 25%
[
carbon. Compound [13C6]-(2) was synthesized in seven steps radiochemical overall yield and with a specific activity of
and in 21% overall yield from [13C6]-(11) (Scheme 3).
54.5 mCi/mmol. The chemical purity was 98.5% and a
radiochemical purity of 98.7%. The sodium salt of this compound
was easily prepared by a simple treatment with a solution of
aqueous NaOH in THF and then crystallized from acetonitrile/THF.
The intermediate [14C]-(13) was also used to prepare [14C]-(2)
Synthesis of the acyl glucuronide derivatives [13C6]-(3) and
[
13C6]-(4)
To prepare the acyl glucuronide derivatives, the three-step as described in Scheme 7. This metabolite was obtained in 59%
procedure shown in Scheme 4 was used. The acid was first overall yield with a specific activity of 54.5 mCi/mmol and
activated using HATU (1-[bis(dimethylamino)methylene]-1H- radiochemical purity of 98.8%.
1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) in
The synthesis of the glucuronide [14C]-(3) was accomplished
the presence of N-methylmorpholine (NMM), then reacted with as described before using [14C]-(1) and allyl-D-glucuronate. The
allyl-D-glucuronate ((2S,3S,4S,5R,6R/S)-allyl-3,4,5,6-tetrahydroxyte pure material was obtained after preparative HPLC purification
trahydro-2H-pyran-2-carboxylate). Finally, removal of the allyl with a specific activity of 52.3 mCi/mmol and radiochemical
protecting group using palladium tetrakis triphenylphosphine purity of 97.5%.
Scheme 2. Synthesis of [13C6]-(1).
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B. Latli et al.
Scheme 3. Synthesis of [13C6]-(2).
Scheme 4. Synthesis of glucuronides [13C6]-(3) and [13C6]-(4).
Scheme 5. Synthesis of 1-aminocyclobutane-1-carbonyl chloride-14C.
allowed access to [13C6]-(1) and [13C6]-(2) in another five and
four steps, respectively. The other metabolites [13C6]-(3) and
Conclusion
The carbon-13 labeled deleobuvir and its major metabolites
[
13C6]-(4) were then prepared in three steps from [13C6]-(1)
were prepared starting from aniline-13C6. The preparation of and
[
13C6]-(2).
For
carbon-14
synthesis,
1-
the intermediate [13C6]-(11) in six steps and 52% overall yield amiocyclybutycarboxylic acid [14C]-(23) was prepared in two
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B. Latli et al.
Scheme 6. Synthesis of [14C]-(1).
Scheme 7. Synthesis of [14C]-(2).
0.1% TFA in MeCN/methanol, column: Unison UK-C8 (150 mm × 4.6 mm,
3 μm). Liquid chromatography-mass spectrometry (LCMS) were acquired
using either a fast 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
(3.5 μm, 4.6 mm × 30 mm), or a long medium polar method, run time
9.0 min, gradient 95% water (0.1% TFA) and 5% MeCN (0.1% TFA) to 5%
water in 7 min, hold to 9 min at 5% water, flow 1.5 mL/min; column: Agilent
Zorbax Eclipse XDB-C8 (5 μm, 4.6 mm × 150 mm). The data were acquired
on Waters Acquity™ Ultra Performance LC (Milford, MA, USA). The
radiochemical purity was measured using a radio-HPLC detector β-Ram
model 4 or model 3 (LabLogic systems, Inc. Brandon, FL, USA) connected
to the Agilent instrument using IN-FLOW™ 2:1 liquid scintillation (LabLogic
systems, Inc. Brandon, FL, USA). Carbon-14 potassium cyanide was obtained
from Quotient Bioresearch (Cardiff, UK). Aniline-13C6 (99.4 atom % 13C) was
purchased from Isotec-Sigma-Aldrich (Miamisburg, OH, USA). The rest of
the reagents were purchased from Sigma-Aldrich Company.
steps via Bucherer–Bergs reaction. Conversion of this amino acid to
1-amino-cyclobutane carbonyl chloride gave intermediate [14C]-(13)
which was used to prepare [14C]-(1), [14C]-(2) in three radioactive
steps. The metabolite [14C]-(3) was prepared in a three-step
synthesis starting from [14C]-(1) and allyl-D-glucuronate.
Experimental
Materials and methods
The nuclear magnetic resonance (NMR) spectra of radioactive compounds
were recorded on
a Bruker 500 MHz spectrometer using double
1
encapsulated NMR tubes in deuterated dimethyl sulfoxide. H NMRs and
13C NMR spectra of non-radioactive compounds were acquired in
deuterated solvent using Bruker 400 MHz or Bruker Avance 600B
spectrometers at 400.33 MHz and 600.04 MHz for 1H NMR and 100.66 MHz
and 150.88 MHz for 13C NMR, respectively. Liquid scintillation counting
was accomplished using a Beckman LS6500TA and UltimaGoldTM cocktail
(PerkinElmer, Boston, MA, USA). HPLC analysis was performed on an Agilent
1200 instrument. HPLC conditions: (a) mobile phase gradient 20% to 100%
(MeCN/H2O both 0.1% TFA) over 20 min, column: Eclipse XDB C18
(150 mm × 4.5 mm, 5 μm), injection volume 10 μL; (b) mobile phase
Synthesis of [13C6]-(1)
Acetanilide-13C6, [13C6]-(6): To a solution of aniline-13C6 (4.0 g,
gradient 75%A to 25%A in 25 min, A: 0.1% TFA in 95:5 water/methanol; B: 40 mmol) in methylene chloride (50 mL), was added pyridine
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B. Latli et al.
(6 mL). Acetyl chloride (3.13 mL, 44 mmol) was then added The resulting solution was stirred for 3 h at room temperature to
dropwise at 0 °C under nitrogen atmosphere. The reaction was give a precipitate. Water (30 mL) was added and the mixture was
warmed slowly to room temperature and stirred for 14 h. extracted with ethyl acetate (50 mL ×3). The combined extracts
Methylene chloride (50 mL) was added and the reaction was were washed with water (30 mL), brine (30 mL), dried (MgSO4),
washed with aqueous 2 N HCl (100 mL), water (100 mL), brine filtered, and concentrated in vacuo. The residue was purified by
(100 mL), dried over MgSO4, filtered and concentrated in vacuo silica gel chromatography using a gradient 5–50% EtOAc in
to give 5.4 g of an orange solid in 94% yield. Rf = 0.14 in 50% hexanes to give an orange solid, mp = 101–102 °C in 98% yield.
1
1
EtOAc : Hexane. H NMR (CDCl3) δ: 7.49 (m, JC–H = 167.2 Hz, 2H), Rf = 0.54 in 30% EtOAc : Hexanes or Rf = 0.53 in 100% CH2Cl2. H
7.30 (m, JC–H = 335.1 Hz, 1H), 7.29 (m, JC–H = 159.2 Hz, 2H), 2.15 NMR (CDCl3) δ: 8.30 (brs, 1H, NH), 8.10 (m, JC–H = 165.7 Hz, 1H),
(s, 3H). 13C NMR (CDCl3) δ: 168.68, 137.98 (dt, J = 9.7, 63.4 Hz), 6.83 (m, JC–H = 165.8 Hz, 1H), 6.62 (m, JC–H = 170.1 Hz, 1H), 3.02
128.94 (dt, J = 6.8, 56.2 Hz), 124.22 (dt, J = 9.7, 55.6 Hz), 119.97 (t, J = 4.3 Hz, 3H). 13C NMR (CDCl3) δ: 146.71 (t, J = 66.1 Hz),
(dt, J = 6.2, 55.9 Hz), 24.51. LCMS: ES+ m/z: MH+ = 142.11, 100%.
142.92 (m), 130.63 (m), 128.20 (m), 115.77 (t, J = 60.4 Hz), 112.84
(m), 29.81. LCMS, ES+ m/z: MH+ = 192.99, 100%.
2,4-Dichloroacetanilide-13C6, [13C6]-(7): A solution of the
aforementioned acetanilide (5.35 g, 38 mmol) in TFA (120 mL) Butyl (E)-3-(3-methylamino-4-nitrophenyl-13C6)acrylate, [13C6]-
was treated at room temperature with NCS (22 g, 165 mmol). (11): Argon was bubbled in a mixture of [13C6]-(10) (0.94g,
The resulting solution was stirred until completion of the 4.9mmol), tetrabutyl ammonium chloride (2.7 g, 10mmol), Hünig’s
reaction in 5 days. The solution was poured into an aqueous cold base (1.7mL, 10 mmol) and butyl acrylate (1.4mL, 10 mmol) in
solution of NaOH. The pH was adjusted to 9 by the addition of dimethyl acetamide (15 mL) for 10 min. Palladium acetate
aqueous 4 N NaOH. The resulting precipitate was filtered off (110 mg, 0.5mmol) was then added, and the mixture was heated
and washed with water. Purification by flash chromatography to 110 °C for 8 h. The dark mixture was cooled to 50 °C, and 1-
using 20% EtOAc : CH2Cl2 as eluent gave 7.7 g of an off-white methylimidazole (0.78 mL, 10 mmol) was added, and the reaction
solid in 96% yield. Rf = 0.4 in 50% EtOAc : Hexanes. 1H NMR was stirred for 1 h at this temperature. After cooling to room
(CDCl3) δ: 8.25 (m, JC–H = 168.6 Hz, 1H), 7.66 (brs, 1H), 7.34 (m, temperature, the mixture was filtered through a short pad of
JC–H = 164.7 Hz, 1H), 7.20 (m, JC–H = 164.5 Hz, 1H), 2.2 (s, 3H). 13C Celite® and washed with ethyl acetate (50 mL). The filtrate was
NMR (CDCl3) δ: 168.40, 133.68 (m), 127.13–129.84 (m), 121.97– poured into water (20mL). The organic phase was removed and
125.22 (m), 124.75. LCMS: ES+ m/z: MH+ = 209.96, 100%.
washed again with water (15 mL), brine (15 mL), dried (MgSO4),
filtered, and concentrated in vacuo. The residue was purified by
2,4-Dichloroaniline-13C6, [13C6]-(8): A mixture of [13C6]-(7) silica gel chromatography using 1–25% EtOAc : Hexanes to give
(2.1 g, 10 mmol) in 2.5 N aqueous HCl (80 mL) was heated to 166 mg of starting material and 950 mg of product in 83% yield
reflux for 3 h. After cooling to room temperature, the solution based on reacted chloride. Rf = 0.3 in 20 EtOAc: Hexanes as an
was poured into a saturated aqueous solution of NaHCO3 orange solid, mp= 85–86°C. 1H NMR (CDCl3) δ: 8.17 (dq, JH–
(100 mL). The solution was made basic to pH = 8 by adding more H = 7.4, J13C–H = 165.1 Hz, 1H), 8.09 (brs, 1H, NH), 7.61 (m, 1H), 6.90
NaHCO3 solution, and the resulting white solid was filtered off, (dt, J = 7.4, 160.2 Hz, 1H), 6.84 (dq, J = 7.4, 165.4Hz, 1H), 6.52 (dd,
washed with water, and dried in vacuo to give 1.93 g of material J = 5.4, 16.0 Hz, 1H), 4.23 (t, J = 6.7Hz, 2H), 3.06 (t, J= 4.3 Hz, 3H),
1
in quantitative yield. Rf = 0.22 in 20% EtOAc : Hexanes. H NMR 1.70 (dt, J = 6.8, 7.8Hz, 2H), 1.44 (dt, J= 7.4, 7.8Hz, 2H), 0.97 (t,
(CDCl3) δ: 7.24 (m, JC–H = 167.6 Hz,1H), 7.02 (m, JC–H = 164.3 Hz, J = 7.4Hz, 3H). 13C NMR (CDCl3) δ: 164.50, 144.40 (m), 139.89 (m),
1H), 6.69 (m, JC–H = 160.6 Hz, 1H), 4.01 (brs, 2H). 13C NMR (CDCl3) 130.20 (m), 125.50 (m), 123.94 (m), 114.87 (m), 11.81 (m), 111.2
δ: 141.0 (m), 128.32 (m), 127.07 (m), 122.77 (m), 118.91 (m), (m), 62.97, 28.83, 27.90, 17.30, 11.86.
115.73 (m). LCMS: ES+ m/z: MH+ = 168.11, 50%; (MH
+ MeCN)+ = 208.91, 100%.
Butyl (E)-3-(4-amino-3-methylaminophenyl-13C6)acrylate, [13C6]-
(12): A mixture of [13C6]-(11) (0.92 g, 3.6 mmol) and tin chloride
2,4-Dichloro-1-nitrobenzene-13C6, [13C6]-(9): To a solution of dihydrate (3.7 g, 16.4 mmol) in ethyl acetate (20 mL) and ethanol
[
13C6]-(8) (1.4 g, 8.2 mmol) in acetic acid (33 mL), was added a (20 mL) was heated to reflux for 4 h. After cooling to room
solution of 30% hydrogen peroxide (11 mL), followed by a temperature, the solution was poured into an aqueous solution of
concentrated solution of sulfuric acid (0.7 mL). The resulting NaOH (1.0 N, 100 mL) and extracted with ethyl acetate (100 mL ×2).
solution was heated at 80 °C and stirred for 14 h. After cooling The combined extracts were dried over MgSO4, filtered and
to room temperature, the reaction was poured into water concentrated in vacuo to give 0.85 g of dark residue. Purification by
(150 mL) and the resulting precipitate was extracted with ethyl silica gel chromatography using 1–20% EtOAc in methylene chloride
acetate (100 mL ×3). The combined extracts were washed with gave 0.5 g of product in 62% yield; Rf = 0.29 in 20% EtOAc : CH2Cl2. 1H
a saturated solution of NaHCO3 (50 mL), water (50 mL), and NMR (CDCl3) δ: 7.54 (dtd, J= 1.5, 5.5, 15.9 Hz, 1H), 6.79 (ddd, J=7.9,
brine (50 mL), dried over MgSO4, filtered, and concentrated in 13.9, 158.6 Hz, 1H), 6.73 (dt, J= 6.5, 154.7 Hz, 1H), 6.57 (ddd, J=7.6,
vacuo to give 1.15 g of orange oil in 71% yield, which solidified 14.6, 154.7 Hz, 1H), 6.25 (dd, J= 5.1, 15.9 Hz, 1H), 4.11 (t, J=6.7Hz,
upon standing at room temperature; Rf = 0.55 in 30% EtOAc :
2H), 3.50 (brs, 2H), 2.78 (d, J=3.4Hz, 3H), 1.60 (dt, J= 6.9, 13.9 Hz,
1
Hexanes. H NMR (CDCl3) δ: 7.86 (m, JC–H = 167.6 Hz, 1H), 7.57 2H), 1.35 (dt, J= 7.4, 15.1 Hz, 2H), 0.89 (t, J=7.4Hz, 3H). 13C NMR
(m, JC–H = 164.3 Hz, 1H), 7.39 (m, JC–H = 160.6 Hz, 1H). 13C NMR (CDCl3) δ: 166.36 (d, J= 7.01 Hz), 144.24 (d, J= 56.3 Hz), 142.91 (m),
(CDCl3) δ: 145.41 (m), 138.50 (m), 130.90 (m), 128.13 (m), 125.14 (m), 119.17 (m), 113.84 (m), 108.70 (m), 62.57, 51.91, 29.32,
127.11 (m), 125.76 (m).
17.69, 12.23. LCMS, ES+ m/z: MH+ = 255.61 (100%).
4-Chloro-2-methylamino-nitrobenzene-13C6, [13C6]-(10): To a Butyl (E)-3-(2-(1-Aminocyclobutyl)-1-methyl-1H-benzo[d]imi
solution of [13C6]-(9) (1.1 g, 5.7 mmol) in DMSO (10 mL), was dazole-6-yl-3a,4,5,6,7,7a-13C6)acrylate, [13C6]-(14): A mixture
added a solution of methylamine (2.5 mL, 40% solution in water). of [13C6]-(12) (408 mg, 1.6 mmol) and 1-amino-cyclobutane
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B. Latli et al.
carbonyl chloride hydrochloride salt (13) (0.5 g, 2 mmol, 70 wt.%) (50 mL), and the combined extracts were dried (MgSO4), filtered,
in toluene (10 mL) was stirred at room temperature for 30 min and concentrated in vacuo to give 430 mg of an off-white solid.
to give a clear solution. The reaction was then heated to 90 °C Purification by silica gel chromatography and using up to 10%
and stirred for 2 h. A slurry was obtained to which methanol MeOH in methylene chloride gave 359 mg of a pale yellow solid
(4 mL) was added, and the heating was continued for 8 h. in quantitative yield. The solid (200 mg) was further crystallized
After cooling to room temperature, the reaction mixture was from 2 mL of 10% methanol/methylene chloride to give
concentrated under reduced pressure, and the resulting solid 160 mg of a white solid; Rf = 0.1 in 10% MeOH/CH2Cl2, mp 232 °
was treated with ethyl acetate and a saturated solution of C decomposed. 1H NMR (MeOH-d4) δ: 9.07 (s, 2H), 8.04 (d,
aqueous NaHCO3 (100 mL). The organic phase was removed J = 1.2 Hz, 1H), 7.91 (m, 1H), 7.81 (d, J = 8.43 Hz, 1H), 7.73–7.85
and then washed with water (50 mL), brine (30 mL), dried (m, 1.5H), 7.59 (dd, J = 1.5, 8.4 Hz, 1H), 7.51 (m, 1H), 7.36 (m,
(MgSO4), filtered, and concentrated in vacuo to give 0.5 g of 0.5H), 6.53 (dd, J = 5.2, 15.9 Hz, 1H), 3.88 (s, 3H), 3.87 (d,
product. Purification using a 40 g silica gel column and a J = 2.9 Hz, 3H), 3.77 (quin, J = 8.9 Hz, 1H), 3.11 (m, 2H), 2.83 (m,
gradient 10–50% EtOAc:CH2Cl2 gave 0.36 g of product in 2H), 2.02–2.28 (m, 2H), 1.9–2.02 (m, 6H), 1.73 (m, 2H). 13C NMR
67% yield; Rf = 0.31 in 15% MeOH/CH2Cl2. 1H NMR (CDCl3) δ: (DMSO-d6) δ: 167.86, 165.98, 157.98, 145.44, 144.91, 143.34 (m),
7.82 (m, 1H), 7.75 (m, JC–H = 155.5 Hz, 1H), 7.65 (m, 1H), 137.29 (m), 135.05, 128.31, 127.46 (m), 127.03, 123.9 (t,
7.28 (m, 1H), 6.47 (dd, J= 5.2, 15.9 Hz, 1H), 4.22 (t, J= 6.7Hz, J = 55.1 Hz), 121.93 (m), 120.22, 118.29 (m), 117.02, 111.97,
2H), 3.89 (d, J= 2.8 Hz, 3H), 3.01 (m, 2H), 2.27 (m, 2H), 11.31, 110.39, 109.93 (m), 55.24, 54.88, 36.27, 32.77, 31.73,
2.04 (m, 2H), 1.85 (brs, 2H), 1.70 (dt, J= 6.7, 13.9 Hz, 2H), 31.03, 26.02, 14.98. LCMS, fast medium polar, run time 1.75 min,
1.45 (dt, J= 7.5, 15.0 Hz, 2H), 0.97 (t, J= 7.4 Hz, 3H). 13C NMR Rt = 0.96 min, 659.21:661.20 (1:1). HRMS: calculated 659.20716
(CDCl3) δ: 167.43, 143.60 (m), 137.28 (m), 129.22 (m), 125.14 (100%), 660.21051 (30%), 661.20511 (100%), 662.20847 (30%),
(m), 122.08 (m), 120.47 (m), 110.98 (m), 109.24 (m), 64.36, 663.21182 (5%); found 659.20750 (100%), 660.21121 (30%),
55.95, 36.44. 31.27, 30.83, 19.24, 14.15, 13.78. LCMS: ES+ m/z: 661.20547 (100%), 662.20919 (30%), 663.21307 (5%). HPLCa:
MH+ = 334.65 (100%).
Rt = 11.52 min (99.74%).
Synthesis of [13C6]-(2)
Butyl (E)-3-(2-(1-(2-(5-bromo-pyrimidin-2-yl)-3-cyclopentyl-
1-methyl-1H-indole-6-carboxamido)cyclobutyl)-1-methyl-1H
-benzo[d]imidazol-6-yl-3a,4,5,6,7,7a-13C6)acrylate, [13C6]-(17) Butyl 3-(4-amino-3-(methylamino)phenyl-13C6)-propanoate,
: To a suspension of (15) (410 mg, 1 mmol) in methylene chloride
[
13C6]-(18): The cinnamic acid derivative [13C6]-(11) (0.37 g,
(15 mL) was added thionyl chloride (0.1 mL, 1.4 mmol) at 0 °C 1.3 mmol) was divided into two tubes each containing 185 mg
under nitrogen atmosphere. After stirring for 15 min, of Pd/C and 6 mL of methanol and stirred under 200 PSI of
triethylamine (0.43 mL, 3 mmol) was added dropwise to give a hydrogen at 50 °C for 20 h. The mixtures were filtered, combined
dark solution, which was stirred at 0 °C for 2 h and 30 min. The and concentrated in vacuo to give 290 mg of viscous oil.
amine [13C6]-(14) (340 mg, 1 mmol) in methylene chloride Purification using a 12 g disposable column and 20–30%
(8 mL) was then added. After stirring at 0 °C for 1 h, the ice bath EtOAc : CH2Cl2 gave 236 mg in 70% yield. LCMS: ES+ m/z:
1
was removed, and the reaction was warmed to room MH+ = 257.68 (100%). H NMR (CDCl3) δ: 6.62 (dm, J = 152.1 Hz,
temperature and stirred for 16 h. The reaction was poured into 1H), 6.60 (dm, J = 153.9 Hz, 1H), 6.46 (dd, J = 6.2, 152.9 Hz, 1H),
a saturated solution of Na2CO3 (50 mL) and extracted with ethyl 4.06 (t, J = 6.7 Hz, 2H), 3.42 (s, 3H), 2.85 (m, 2H), 2.82 (d,
acetate (50 mL ×3). The combined extracts were dried (MgSO4), J = 7.9 Hz, 3H), 2.59 (m, 2H), 1.58 (m, 2H), 1.36 (m, 2H), 0.91 (t,
filtered, and concentrated in vacuo to give 0.8 g of a solid. J = 7.4 Hz, 3H). 13СNMR (CDCl3) δ: 173.44, 139.18 (t, J = 65.6 Hz),
Purification by silica gel chromatography using 10–50% EtOAc 133.11 (t, J = 56.5 Hz), 132.11 (t, J = 64.6 Hz), 117.71 (t,
in CH2Cl2 gave 614 mg of product in 84% yield as a white solid J = 58.8 Hz), 116.32 (t, J = 59.3 Hz), 110.82 (t, J = 66.4 Hz), 64.49,
with 98% chemical purity. Rf = 0.17 in 40% EtOAc : CH2Cl2. 1H 38.58, 32.22, 31.72, 30.12, 19.17, 14.28.
NMR (CDCl3) δ: 8.93 (brs, 2H), 7.95 (s, 1H), 7.93 (m, 0.5 H), 7.80
(m, 1H), 7.7 (d, J = 8.5 Hz, 1H), 7.65 (m, 1H), 7.53 (m, 0.5H), 7.42 Butyl 3-(2-(1-aminocyclobutyl)-3-methyl-1H-benzo[d]]imidazo
(dd, J = 1.3, 8.5 Hz, 1H), 7.32 (m, 1H), 7.01 (m, 1H), 6.47 (dd, l-6-yl-3a,4,5,6,7,7a-13C6)-propanoate, [13C6]-(19): To a mixture
J = 5.2, 15.9 Hz, 1H), 4.21 (t, J = 6.7 Hz, 2H), 3.97 (d, J = 2.8 Hz, of (23) (182mg, 1.2 mmol) in anhydrous MeCN (5mL) were added
3H), 3.83 (s, 3H), 3.72 (m, 1H), 3.11 (m, 2H), 2.92 (m, 2H), 2.30 2-oxazolidone (46 mg, 0.5mmol) followed by phosphorus
(m, 1H), 1.85–2.15 (m, 8H), 1.62 (m, 4H), 1.62 (brs, NH), 1.44 (m, pentachloride (250 mg, 1.2mmol) and stirred for 2 h. The
2H), 0.97 (t, J = 7.4 Hz, 3H). 13C NMR (CDCl3) δ:167.45, 157.70, aforementioned
diamino-derivative
[
13C6]-(18)
(234 mg,
143.79 (m), 136.80 (m), 129.17 (m), 123.38, 122.21 (m), 121.28, 0.9mmol) was added in MeCN (4mL) and the resulting mixture
119.89 (t), 116.63, 110.60, 109.61 (m), 64.35, 56.33, 36.80, 33.63, was stirred for 5 h at room temperature. Toluene (2mL) was added,
33.34, 31.91, 31.34, 30.83, 26.70, 19.24, 16.25, 13.78. LCMS ES+ and the mixture was heated to 60 °C and stirred overnight to give a
m/z: MH+ = 717.11 (100%).
homogeneous solution. Isopropanol (4mL) was added, and the
solution was stirred at 60 °C for another 4 h. After cooling to room
(E)-3-(2-(1-(2-(5-Bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl- temperature, the solution was concentrated in vacuo to half its
1H-indole-6-carboxamido)-cyclobutyl)-1-methyl-1H-benzo[d] volume then diluted with EtOAc (20 mL) and washed with a
imidazol-6-yl-3a,4,5,6,7,7a-13C6)acrylic acid,
[
13C6]-(1):
A
saturated solution of NaHCO3 (20 mL), brine (15 mL), dried over
mixture of [13C6]-(17) (400 mg, 0.6 mmol) in n-propanol MgSO4, filtered, and concentrated in vacuo to give 380 mg of
(11 mL), water (11 mL), and a 50% solution of NaOH (1 mL) was residue. Purification using a 40 g disposable silica gel column and
heated at 90 °C for 4 h. The reaction was concentrated in vacuo up to 10% MeOH in methylene chloride gave 188 mg of product
and the residue was treated with ethyl acetate (50 mL) and water in 81% yield. LCMS, ESI+, m/z: MH+ = 336.79 (100%). 1H NMR
(50 mL). The aqueous phase was extracted with ethyl acetate (CDCl3) δ: 7.67 (dm, J = 159.5Hz, 1H), 7.15 (dm, J = 157.9Hz, 1H),
www.jlcr.org
Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 250–260

B. Latli et al.
7.10 (dm, J = 155.4 Hz, 1H), 4.07 (t, J = 6.7Hz, 2H), 3.84 (d, J = 2.8Hz, 662.22616 (30%), 663.22076 (100%), 664.22412 (30%),
2H), 3.48 (brs, 3H), 3.09 (m, 2H), 2.97 (m, 2H), 2.69 (m, 2H), 2.25 (m, 665.22747 (5%); found 661.22283 (100%), 662.22655 (30%),
2H), 1.85 (m, 2H), 1.58 (m, 2H), 1.33 (m, 2H), 1.30 (m, 2H), 1.21 (d, 663.22087 (100%), 664.22458 (30%), 665.22807 (5%).
J = 6.27 Hz, 2H), 0.89 (t, J = 7.4Hz, 3H).
Synthesis of acyl glucuronides [13C6]-(3) and [13C6]-(4),
Butyl 3-(2-(1-(2-(5-bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl Scheme 5
-1H-indole-6-carboxamido)cyclobutyl)-1-methyl-1H-benzo[d]]
imidazol-6-yl-3a,4,5,6,7,7a-13C6)-propanoate, [13C6]-(20): To a
(2R,3R,4R,5S,6R)-6-{(E)-3-[2-(1-{[2-(5-Bromo-pyrimidin-2-yl)-
3-cyclopentyl-1-methyl-1H-indole-6-carboxamido]}-cyclobutyl)-
mixture of the acid (15) (358 mg, 0.9 mmol) in CH2Cl2 (7 mL) in
1-methyl-1H-benzo[d]imidazol-6-yl-3a,4,5,6,7,7a-13C6]-acryloy-
an ice bath under nitrogen, was added thionyl chloride (90 μL,
1.2 mmol). After stirring for 15 min, triethylamine (446 μL,
loxy}-3,4,5-trihydroxy-tetrahydro-pyran-2-carboxylic acid,
[
13C6]-(3): To a solution of [13C6]-(1) (145 mg, 0.22 mmol) and
3.2 mmol) was added, and the resulting dark mixture was stirred
for 2 h. The aforementioned amine derivative [13C6]-(19) (0.3 g,
0.9 mmol) was then added in CH2Cl2 (4 mL). The flask was rinsed
with another 4 mL of CH2Cl2 and added to the reaction and stirred
for 2 h at 0 °C, and then, the ice bath was removed and the
reaction was stirred at room temperature for 14 h. LCMS showed
no starting material. Most of the solvent was removed in vacuo
and the residue was dissolved in EtOAc (20 mL) and washed with
a saturated solution of Na2CO3 (15 mL), brine (15 mL), dried over
MgSO4, filtered, and concentrated in vacuo to give 0.65 g of an
off-white foam. Purification on a 40 g disposable silica gel column
and using 100% CH2Cl2 to 50% EtOAc : CH2Cl2 gave 378 mg in 59%
yield. 1H NMR (CDCl3) δ: 8.90 (s, 1H), 8.06 (s, 1H), 7.92 (s, 1H), 7.73 (d,
J = 8.5 Hz, 1H), 7.62 (m, 1H), 7.54 (d, J = 9.6 Hz, 1H), 7.10 (m, 1H), 4.06
(t, J = 6.7 Hz, 2H), 3.92 (d, J = 2.7 Hz, 3H), 3.71 (brs, 4H), 3.45 (s, 3H),
3.05 (m, H), 2.98 (t, J = 7.6 Hz, 2H), 2.65 (m, 2H), 2.24 (brs, 1H), 2.22
(m, 2H), 1.81–1.99 (m, 8H), 1.70 (m, 2H), 1.57 (m, 2H), 1.33 (m, 2H),
0.88 (t, J = 7.4 Hz, 3H). 13С NMR (CDCl3) δ: 173.11, 167.73, 158.75,
157.06, 140.25 (t, J = 62.01 Hz), 136.51 (t. J = 58.72 Hz), 135.34 (t,
J = 56.33), 122.77 (t, J = 58.36 Hz), 119.07 (t, J = 63.9 Hz), 108.85 (t,
J = 64.32 Hz), 64.38, 56.83, 50.50, 36.92, 36.67, 33.80, 32.37, 31.73,
30.65, 29.70, 26.67, 22.85, 19.11, 16.39, 13.70. LCMS long medium
polar method: Rt = 9.38 min, m/z: MH+ = 720.96 (100%).
HATU (100 mg, 0.26 mmol) in anhydrous DMF (2 mL), was
added NMM (30 μL, 0.27 mmol) at room temperature. The
solution was stirred for 14 h. HPLC showed the consumption
of the starting acid (Rt = 12.01 min) and the presence of a new
product (Rt = 14.66 min); LCMS: MH+ = 778.74 (100%). Allyl-D-
glucuronate (103 mg, 0.427 mmol) was then added followed
by NMM (60 μL, 0.545 mmol), and the solution was stirred for
14 h. The solution was diluted with EtOAc (20 mL) and washed
with water (20 mL ×2), brine (20 mL), dried over MgSO4,
filtered, and concentrated in vacuo to give 160 mg of a residue.
The residue was dissolved in THF (3.0 mL). Tetrakis
(triphenylphosphine)-palladium, polymer bound (200–400
mesh, 0.5–0.9 mmol/g, 150 mg) was added followed by
morpholine (100 μL). The mixture was stirred at room
temperature for 3 h. HPLC showed one single product,
Rt = 10.6min. Amberlite IR-120 (H+) (300 mg) was added and the
mixture was stirred for 30 min. Filtration and washing with THF,
then concentration in vacuo gave 140mg of a solid. Purification
by HPLC using YMC-Pack Pro C18 (30 × 30 mm ×150 mm, 5 μm),
mobile phase A: water with 0.1% TFA, B: MeCN, flow rate:
32 mL/min and a gradient: 20% B to 95% B in 19 min, hold at
95% for 1 min, post run 3 min, run time 20 min, and collecting
using ultraviolet 254 nm gave 80 mg of product in 44% yield and
1
with a purity of 95.4%. H NMR (600.3MHz, d6-DMSO) δ: 9.23 (s,
3-(2-(1-(2-(5-Bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl-
1H, NH), 9.18 (s, 2H), 8.25 (s, 1H), 8.01 (m, 0.5H), 7.72–7.85 (m,
1.5H), 7.80 (d, J = 15.8 Hz, 1H), 7.75 (d, J = 8.5Hz, 1H), 7.68 (d,
J = 8.3Hz, 1H), 7.58 (m, 2H), 6.70 (d, J = 15.8Hz, 1H), 5.52 (d,
J = 7.9Hz, 1H), 5.45 (m, 1H), 5.30 (m, 1H), 3.86 (s, 3H), 3.79 (t,
J = 9.5Hz, 1H), 3.78 (s, 3H), 3.71 (t, J = 9.0Hz, 1H), 3.38 (t, J = 9.1Hz,
1H), 3.34 (t, J = 8.8Hz, 1H), 3.29 (m, 1H), 3.01 (m, 2H), 2.71 (q,
J = 9.4Hz, 2H), 2.06 (m, 1H), 1.94 (m, 7H), 1.68 (m, 2H). 13C NMR
(150.92 MHz, DMSO-d6) δ: 171.10, 167.20, 165.8, 158.2, 157.3,
147.5, 143.6 (m), 137.4 (m), 137.3, 135.1, 128.3, 127.6 (m), 127.1,
122.3 (m), 121.4 (m), 120.2, 119.1 (m), 118.4, 118.2, 114.9, 110.6,
110.2, 94.3, 76.6, 75.1, 72.1, 71.0, 55.1, 36.2, 33.2, 33.0, 31.1, 27.1,
13.8.13. LCMS: m/z 1:1 835:837.
1H-indole-6-carboxamido)cyclobutyl)-1-methyl-1H-benzo[d]
imidazol-6-yl-3a,4,5,6,7,7a-13C6)propanoic acid,
[
13C6]-(2):
To a solution of [13C6]-(20) (157 mg, 0.2 mmol) in methanol
(5 mL), was added a solution of 2 N NaOH (1 mL) dropwise. The
resulting mixture was stirred at room temperature for 14 h.
The reaction was treated with acetic acid (0.2 mL) and
concentrated in vacuo to remove most of the methanol. The
residue was dissolved in water (10 mL) and extracted with ethyl
acetate (20 mL ×2). The combined ethyl acetate extracts were
dried over MgSO4, filtered, and concentrated in vacuo. The
residue of 160 mg was purified using a 12 g disposable silica
gel column and up to 10% MeOH/CH2Cl2. The fractions
containing the pure material were combined and concentrated,
then crystallized from hot methanol to give 130 mg of an off- (2R,3R,4R,5S,6R)-6-{3-[2-(1-{[2-(5-Bromo-pyrimidin-2-yl)-3-cycl-
white product in 84% yield. Rf = 0.6 in 20% MeOH/CH2Cl2. HPLC: opentyl-1-methyl-1H-indole-6-carboxamido]}-cyclobutyl)-
1
Rt = 12.14 min (99%). H NMR (DMSO-d6) δ: 9.25 (s, 2H), 9.20 (s, 1-methyl-1H-benzo[d]imidazol-6-yl-3a,4,5,6,7,7a-13C6]-pro-
1H), 8.18 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.65 (m, 1H), 7.55 (m, pyloxy}-3,4,5-trihydroxy-tetrahydro-pyran-2-carboxylic acid,
1H), 7.30 (m, 1H), 7.15 (m, 1H), 3.92 (s, 3H), 5.75 (m, 4H), 3.21
[
13C6]-(4): To a suspension of [13C6]-(2) (366 mg, 0.6 mmol), HATU
(s, 2H), 2.90–3.10 (m, 4H), 2.75 (t, J = 8.1 Hz, 2H), 2.61 (m, 2H), (325 mg, 0.8mmol) in anhydrous acetonitrile (10 mL), was added
2.09 (m, 1H), 1.90 (m, 7H). 13С NMR (DMSO-d6) δ: 173.82, NMM (0.2mL, 1.8 mmol) at room temperature and stirred for
165.88, 157.97, 141.55 (m), 136.95 (m), 134.95 (m), 128.50, 14 h. HPLC showed no starting material. To this white mixture,
126.96, 121.72 (t, J = 55.3 Hz), 118.98, 118.39 (m), 110.32, was added anhydrous CH2Cl2 (10 mL) to give a clear yellow
108.85 (m), 55.15, 48.56, 38.84, 36.25, 32.77, 31.72, 31.06, 30.81, solution. Allyl-D- glucuronate (518 mg, 2.21 mmol) was added
26.02, and 14.99. LCMS, long medium polar method: followed by NMM (0.8mL, 7.2mmol), and the resulting solution
Rt = 8.35 min; m/z: 661.53:663.54 (1:1). LCMS fast medium polar was stirred for 90 min. HPLC showed a new single product. LCMS:
method: Rt = 0.87 min. HRMS: calculated 661.22281 (100%), m/z (879.31:881.35, 1:1, 100%). The reaction was cooled in an ice
J. Label Compd. Radiopharm 2015, 58 250–260
Copyright © 2015 John Wiley & Sons, Ltd.
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B. Latli et al.
bath under nitrogen and tetrakis triphenyl phosphine Pd(0) Butyl (E)-3-(4-amino-3-(methylamino)phenyl)acrylate (12): To
(64 mg, 0.055 mmol) was added followed by morpholine a solution of (11) (2.8 g, 10 mmol) in ethyl acetate (60 mL) and
(0.145 mL, 1.7mmol). The resulting solution was stirred for 2 h at ethanol (60 mL), was added tin chloride dihydrate (11.3 g,
0 °C. HPLC and LCMS showed one single product corresponding 50 mmol), and the resulting mixture was heated to reflux for
to the acid m/z 1:1, 839:841. A solution of 2 N HCl (5mL) was added 14 h. After cooling to room temperature, the reaction was
followed by water (12 mL) at 0 °C. The resulting mixture was stirred poured into a saturated aqueous solution of Na2CO3 (250 mL)
at room temperature for 2 h then concentrated in vacuo to remove and extracted with EtOAc (250 mL ×2). The combined extracts
most of the organic solvents. The mixture was then decanted to were washed with brine (300 mL ×2), dried over MgSO4, filtered,
remove the water (HPLC of aqueous showed no product). The solid and concentrated in vacuo to give 2.76 g of a viscous honey-
residue showed the desired product. Purification using a 43-g C18 colored oil. Purification on 150 g disposable silica gel column
column, which was first preconditioned with 50% water/MeCN and using up to 20% EtOAc in CH2Cl2 gave 1.86 g of a bright yellow
0–50% MeCN. Water (0.1% formic acid). The crude product was material in 75% yield. This solidified upon standing at room
first loaded on C18 cartridge using methanol, and then most of temperature. LCMS (fast medium polar method): Rt = 0.86 min,
the methanol was dried overnight. The fractions containing the MH+ = 249.24 (100%). TLC, Rf = 0.31 in 20% EtOAc:CH2Cl2. 1H
pure material were combined, and most of the organic solvent NMR (CDCl3) δ: 7.62 (d, J = 16.0 Hz, 1H), 6.91 (dd, J = 1.0, 8.1 Hz,
was removed in vacuo, and the resulting white mixture (product 1H), 6.83 (d, J = 1.0 Hz, 1H), 6.68 (d, J = 8.1 Hz, 1H), 6.29 (d,
+ water) was freeze dried to give 293 mg of product in 63% yield. J = 16.0 Hz, 1H), 4.19 (t, J = 6.2 Hz, 2H), 3.56 (s, 3H), 2.89 (m, 3H),
1HNMR (400.33 MHz, DMSO-d6) δ: 9.11 (s, 2H), 9.07 (s, 1H, NH), 1.70 (m, 2H), 1.45 (m, 2H), 0.96 (t, J = 6.2 Hz, 3H). 13С NMR (CDCl3)
8.05 (s, 1H), 7.68 (d, J= 8.5 Hz, 1H), 7.75 (d, J= 8.5 Hz, 1H), 7.53 (m, δ: 167.87, 145.77, 138.37, 137.46, 126.78, 120.78, 115.43, 114.02,
1H), 7.32 (m, 1H), 7.08 (m, 1H), 5.41 (d, J= 8.1 Hz, 1H), 5.35 (s, OH), 109.69, 64.13, 30.88, 19.25, 13.79.
3.86 (s, 3H), 3.75 (t, J= 9.5 Hz, 1H), 3.71 (s, 3H), 3.35 (d, J=9.3Hz,
1H), 3.29 (t, J= 8.9 Hz, 1H), 3.20 (t, J= 8.6 Hz, 1H), 3.18 (s, OH), 2.01 Butyl
(E)-3-(2-(1-aminocyclobutyl)-1-methyl-1H-benzo[d]
14C]-(14):
solution of
(m, 4H), 2.73 (m, 4H), 2.53 (m, 2H), 1.90 (m, 6H), 1.64 (m, 2H). 13С imidazol-6-yl-2-14C)acrylate,
[
A
NMR (100.66 MHz, DMSO-d6) δ: 171.2, 169.7, 166.0, 157.9, 156.0, phosphorous pentachloride (300 mg, 1.4 mmol) and 2-
139.9 (m), 136.9 (m), 134.1 (m), 128.5, 127.0, 122.0, 121.9 (m), 120.2, oxazolidone (250 mg, 2.9 mmol) in anhydrous acetonitrile
118.4 (m), 110.3, 108.9 (m), 109.1, 94.1, 76.1, 75.6, 72.1, 71.3, 55.2, (10 mL) was stirred at room temperature for 14 h. The acid
36.3, 35.9, 32.8, 31.7, 31.6, 30.8, 26.0, 14.9. HPLC: Rt =10.35min,
[
14C]-(23) (154 mg, 1 mmol) was added in one portion, and the
96.55%. LCMS (fast medium polar method) Rt = 0.76 min, 839.26 resulting mixture was stirred for 6 h. The diamine (12) (240 mg,
(MH+): 837.47 (MÀ). HRMS: calculated 837.25355 (100%), 838.25691 0.97 mmol) was then added and stirring was continued for
(30%), 839.25151 (100%), 840.25486 (30%), 841.25822 (5%); found 12 h. Toluene (1 mL) was added, and the mixture was stirred at
837.25429 (100%), 838.25846 (30%), 839.25185 (100%), 840.25589 65 °C for 4 h. Isopropanol (3 mL) was added, and stirring was
(30%), 84125913 (5%).
continued for another 3 h. TLC (10% MeOH/CH2Cl2) showed no
starting material. The mixture was cooled to room temperature
and concentrated to remove most of the solvents. Then, a
saturated solution of NaHCO3 (50 mL) was added, and the
aqueous layer was extracted with ethyl acetate (50 mL ×2). The
Synthesis of [14C]-(1)
5,7-Diazaspiro[3,4]octane-6,8-dione-8-14C,
[ a
14C]-(22): In
24 mL screw–cap vial, were placed ammonium carbonate combined extracts were dried over Na2SO4, filtered, and
(730 mg, 7.6 mmol), potassium cynanide-14C (224 mCi, concentrated in vacuo. The residue was purified on a 40-g
SA = 59 mCi/mmol, 3.8 mmol). Methanol (3.5 mL), water (3.5 mL), RediSep™ column and up to 10% MeOH in methylene chloride
and cyclobutanone (290 mg, 3.83 mmol) were then added, and to give 180 mg of a solid, which was used as is in the next step.
the resulting solution was heated at 65 °C for 24 h. Another
aliquot of cyclobutanone (300 μL) was added, and heating was Butyl (E)-3-(2-(1-(2-(5-bromopyrimidin-2-yl)-3-cyclopentyl-1-
continued for another 3 h. After cooling the reaction in an ice methyl-1H-indole-6-carboxamido)cyclobutyl)-1-methyl-1H-
bath, aqueous HCl (4.75 mL, 6 N) was added slowly to give a benzo[d]imidazol-6-yl-2-14C)acrylate,
[
14C]-(17): To
a
white precipitate, which was filtered and dried under reduced suspension of (15) (262 mg, 0.7 mmol) in anhydrous methylene
pressure overnight. The product was obtained as an off-white chloride (8 mL), was added thionyl chloride (66 μL, 0.9 mmol) at
solid in 55% radiochemical yield (300 mg, 124 mCi).
0 °C under nitrogen atmosphere. After stirring for 15 min,
triethylamine (0.32 mL, 2.3 mmol) was added dropwise to give
1-Amino-cyclobutane-1-carboxylic-14C acid, [14C]-(23): The a dark solution in few minutes. The stirring was continued for
hydantoin derivative [14C]-(22) (300 mg, 2 mmol) in an aqueous 2.5 h. The amine [14C]-(14) (180 mg, 0.55 mmol) was added in
solution of NaOH (5.0 N, 6 mL) was heated in a screw–cap vial methylene chloride (6 mL). The flask containing this amine
at 110 °C for 24 h. After cooling in an ice bath, the mixture was was rinsed one more time with methylene chloride (6 mL)
transferred to
a
100 mL round bottomed flask, and
a
and added to the reaction flask. After stirring at 0 °C for 1 h,
concentrated solution of HCl (5 mL, 12 N) was added dropwise the ice bath was removed, and the reaction was warmed
in a 30 min period. The resulting mixture was concentrated in slowly to room temperature and stirred for 14 h. TLC (30%
vacuo to dryness. n-Propanol (60 mL) was added, and the EtOAc : CH2Cl2) showed all the starting material was consumed.
mixture was stirred for 12 h to dissolve the amino acid. The Most of the solvent was removed under reduced pressure, and
mixture was filtered through a sintered funnel and washed with the residue was dissolved in ethyl acetate. A saturated solution
n-propanol. The solvent was removed under reduced pressure, of Na2CO3 (30 mL) was added and extracted with methylene
and the solid residue was further dried at 40 °C for 12 h. The chloride (25 mL ×3). The combined extracts were dried
product was obtained as white solid (0.51 g, crude) and was used (Na2SO4), filtered, and concentrated in vacuo to give 0.49 g of
as it is in the next step without further characterization.
a foam. Purification using a 40 g disposable silica gel column
www.jlcr.org
Copyright © 2015 John Wiley & Sons, Ltd. J. Label Compd. Radiopharm 2015, 58 250–260

B. Latli et al.
and 10–45% ethyl acetate in methylene chloride as eluent 6.49 (dd, J = 2.1, 8.1 Hz, 1H), 4.09 (t, J = 6.5 Hz, 2H), 3.28 (s, 3H),
gave 280 mg of an off-white solid or 22 mCi in 57% yield and 2.87 (t, J = 8.1 Hz, 2H), 2.85 (s, 3H), 2.60 (t, J = 8.1 Hz, 2H), 1.60
a specific activity of 55.3 mCi/mmol. Crystallization from (m, 2H), 1.36 (m, 2H), 0.92 (t, J = 6.5 Hz, 3H). 13C NMR (CDCl3) δ:
acetonitrile gave 264 mg of
radiochemical purity of 97%.
a
fluffy white solid with 173.40, 139.22, 133.28, 132.05, 117.67, 116.49, 110.68, 64.27,
36.55, 30.99, 30.93, 30.71, 19.15, 13.73.
(E)-3-(2-(1-(2-(5-Bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl- Butyl 3-(2-(1-aminocyclobutyl)-1-methyl-1H-benzo[d]imidazol-
1H-indole-6-carboxamido)cyclobutyl)-1-methyl-1H-benzo[d] 6-yl-2-14C)propanoate, [14C]-(19): To (13) (172mg, 1mmol) in
imidazol-6-yl-2-14C)acrylic acid, [14C]-(1): To a mixture of the anhydrous acetonitrile (10 mL), was added the diamine (18)
butyl ester
[
14C]-(17) (264 mg, 0.35 mmol) in n-propanol (226 mg, 1 mmol) in one portion, and the mixture was stirred for
(6 mL), was added a solution of NaOH (6 mL, 0.2 N), and the 14 h. Toluene (1 mL) was added, and the mixture was heated at
mixture was heated at 90 °C for 6 h. After cooling to room 65 °C and stirred for 6 h. Isopropanol (3 mL) was added, and the
temperature, the reaction mixture was treated with acetic acid mixture was heated for another 3 h at 65 °C. The mixture was
(0.25 mL) to give a fluffy white solid. The mixture was treated concentrated to remove most of the solvents, and the residue
with ethyl acetate (30 mL) and water (20 mL). The aqueous phase was dissolved in a saturated aqueous solution of NaHCO3 (40 mL)
was extracted with ethyl acetate (30 mL ×2), and the combined and EtOAc (50 mL). The aqueous layer was extracted one more time
extracts were dried (Na2SO4), filtered, and concentrated in vacuo with EtOAc (50 mL), and the combined organic extracts were dried
to give 300 mg of an off-white solid. Purification using a 40 g over Na2SO4, filtered through a column separator, and concentrated
disposable silica gel column and up to 10% MeOH/CH2Cl2 gave in vacuo to give 0.44 g of a residue. Purification using a 40 g
160 mg of a white solid. The solid was crystallized form hot disposable silica gel column, and up to 4% methanol in methylene
methanol to give 117 mg of a white solid or 9.3 mCi with a chloride gave 260 mg of a pale yellow solid in 84% yield and in 98%
specific activity of 54.5 mCi/mmol with radio purity of 99% and chemical purity.
chemical purity of 98%. The mother liquor was concentrated
and recrystallized from hot methanol to give 85 mg of an Butyl
off-white solid or 6.8 mCi with 97% radiochemical purity.
3-(2-(1-(2-(5-bromopyrimidin-2-yl)-3-cyclopentyl-1-
methyl-1H-indole-6-carboxamido)cyclobutyl)-1-methyl-1H-
benzo[d]imidazol-6-yl-2-14C)propanoate, [14C]-(20): To a
Sodium (E)-3-(2-(1-(2-(5-bromopyrimidin-2-yl)-3-cyclopentyl- suspension of (15) (314 mg, 0.8 mmol) in anhydrous methylene
1-methyl-1H-indol-6-carboxamido)cyclobutyl]-1-methyl-1H- chloride (8 mL), was added thionyl chloride (76 μL, 1 mmol) at
benzo[d]imidazol-6-yl-2-14C)acrylate, [14C]-(1-NA): An aqueous 0 °C under nitrogen. After stirring for few minutes,
solution of NaOH (2N, 75μL, 0.15 mmol) was added to a triethylamine (385 μL, 2.8 mmol) was added slowly to give a
suspension of [14C]-(1) (95 mg, 0.15 mmol, 8 mCi) in THF (1.0mL). clear solution. The solution was stirred for 2 h at 0 °C. The
The mixture was stirred for 15min to give a solution, which was aforementioned amine (260 mg, 0.8 mmol) was dissolved in
warmed in an oil bath to 65 °C. Acetonitrile (0.5 mL) was added to CH2Cl2 (8 mL) and added at 0 °C to the reaction flask, and the
give a cloudy solution. Few crystals of (1), ca; 1 mg in 24:1 MeCN :
solution was stirred for 1 h at this temperature, then warmed
water (0.5 mL) were added. The mixture was stirred at this to room temperature and stirred for 14 h. TLC (10%
temperature for 1 h then acetonitrile (3 mL) was added, and the MeOH/CH2Cl2) showed that the starting amine was completely
mixture was stirred for 30 min then allowed to cool to room consumed. A saturated solution of Na2CO3 (35 mL) was added
temperature. The solid was collected by vacuum filtration, washed to the reaction flask, and the mixture was stirred vigorously.
with 24:1 MeCN : water (3 mL), and then dried under reduced The methylene chloride layer was removed, and the aqueous
pressure for 14 h at room temperature then at 40 °C for 2.5 h to give was extracted (30 mL ×2) with methylene chloride. The
87 mg of a white solid in 88% yield. A total of 7 mCi was obtained combined organic extracts were dried over Na2SO4, filtered
with a specific activity of 54.5 mCi/mmol and with 98.7% through a phase separator column, and concentrated in vacuo
1
radiochemical purity. HPLCb, Rt = 17.3min. H NMR (DMSO-d6) δ: to give 0.7 g of a honey-colored residue. Purification by silica
9.39 (s, 1H), 9.17 (s, 2H), 8.20 (s, 1H), 7.74 (d, J= 8.5 Hz, 1H), 7.62 gel chromatography using a 40 g disposable silica gel column
(dd, J= 1.8, 8.3 Hz, 1H), 7.57 (d, J= 8.3 Hz, 1H), 7.56 (s, 1H), 7.34 (dd, and using 10–50% EtOAc : CH2Cl2 as eluent gave 220 mg of a
J= 1.8, 8.3 Hz, 1H), 7.28 (d, J= 15.8 Hz, 1H), 6.44 (d, J= 15.8 Hz, 1H), yellow solid in 48% yield and in 98% chemical purity.
3.86 (s, 3H), 3.76 (s, 3H), 3.71 (m, 1H), 2.99 (m, 2H), 2.75 (m, 2H),
2.04 (m, 1H), 1.96 (m, 2H), 1.94 (m, 1H), 1.85 (m, 4H), 1.64 (m, 2H).
3-(2-(1-(2-(5-Bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl-
1H-indole-6-carboxamido)cyclobutyl)-1-methyl-1H-benzo[d]
imidazol-6-yl-2-14C)propanoic acid, [14C]-(2): A mixture of
Synthesis of [14C]-(2)
[
14C]-(20) (220 mg, 0.23 mmol) was treated with 0.2 N aqueous
Butyl 3-(4-amino-3-(methylamino)phenyl)propanoate (18): A NaOH (4 mL) and n-propanol (4 mL). The cloudy mixture was
mixture of the cinnamic acid derivative (11) (3 g, 10.8 mmol) and heated at 90 °C and stirred for 6 h. HPLC showed the reaction
Pd/C (10%, 3 g) in methanol (75 mL) was stirred under 200 PSI of to be complete, and only one major peak corresponding to
hydrogen at 50 °C for 20 h in Parr apparatus. After cooling to
[
14C]-(2) was observed. The reaction solution was cooled to
room temperature, the mixture was filtered through a short room temperature and treated with acetic acid (0.17 mL). The
pad of Celite® and concentrated in vacuo to give 2.8 g of a dark solution was then concentrated in vacuo to remove most of
residue. Purification using a 150 g disposable silica gel column the solvents and to give a white precipitate. The mixture was
and up to 30% EtOAc : CH2Cl2 gave 2.25 g of material in 84% treated with water (20 mL) and extracted with methylene
yield. LCMS (fast medium polar method): Rt at 0.64 min chloride. The emulsion was broken by adding methanol (2 mL).
(MH+ = 250.52, 100%). TLC, Rf = 0.7 in 20% EtOAc : CH2Cl2. 1H Then, the organic phase was removed and concentrated in
NMR (CDCl3) δ: 6.63 (d, J = 8.1 Hz, 1H), 6.52 (d, J = 2.1 Hz, 1H), vacuo to give 0.22 g of beige solid. The residue was further dried
J. Label Compd. Radiopharm 2015, 58 250–260
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

B. Latli et al.
[14] R. T. Mosley, T. E. Edwards, E. Murakami, A. M. Lam, R. L. Grice, J. Du,
M. J. Sofia, P. A. Furman, M. J. Otto, J. Virol. 2012, 86, 6503–6511.
[15] C. A. Lesburg, M. B. Cable, E. Ferrari, Z. Hong, A. F. Mannarino,
P. C. Weber, Nat. Struct. Biol. 1999, 6, 937–943.
[16] Q. M. Wang, M. X. Du, M. A. Hockman, R. B. Johnson, X.-L. Sun, Drugs
Future 2000, 25, 933–944.
[17] J. J. Kohler, J. H. Nettles, F. Amblard, S. J. Hurwitz, L. Bassit, R. A. Stanton,
M. Ehteshami, R. F. Schinazi, Infect. Drug Res. 2014, 4, 41–56.
[18] S. Zeuzem, V. Soriano, T. Assselah, J.-P. Bronowicki, A. W. Lohse,
B. Müllhaupt, M. Schuchmann, M. Bourlière, M. Buti, S. K. Roberts,
E. J. Gane, J. O. Stern, R. Vinisko, G. Kukolj, J.-P. Gallivan, W.-O. Böcher,
F. J. Mensa, N. Engl. J. Med. 2013, 369, 630–639.
[19] S. Zeuzem, T. Assselah, P. Angus, J.-P. Zarski, D. Larrey, B. Müllhaupt,
E. J. Gane, M. Schuchmann, A. W. Lohse, S. Pol, J.-P. Bronowicki,
S. K. Roberts, K. Arasteh, F. Zoulim, M. Heim, J. O. Stern, G. Kukolj,
G. Nehmiz, C. Haefner, W. O. Boecher, Gastroenterology 2011, 141,
2047–2055.
under vacuum at 32 °C to give 193 mg of beige solid. HPLC
showed the product to be 82% pure. Purification of this material
using 40 g disposable silica gel column and 2 to 20% methanol in
methylene chloride gave 113 mg of a white solid. The specific
activity was calculated to be 54.5mCi/mmol, and a total of
9.53 mCi was obtained. HPLCb, Rt = 17.2min with radiochemical
1
purity of 98.8%. H NMR (500 MHz, DMSO-d6) δ: 9.41 (s, 1H), 9.20
(s, 2H), 8.21 (s, 1H), 7.74 (d, J = 8.5Hz, 1H), 7.62 (d, J = 8.4Hz, 1H),
7.48 (d, J = 8.2Hz, 1H), 7.22 (d, J = 0.8Hz, 1H), 7.02 (d, J = 8.3 Hz,
1H), 3.86( s, 3H), 3.75–3.65 (m, 1H), 3.71 (s, 3H), 3.33 (brs, 2H), 3.03
(m, 2H), 2.76 (t, J = 7.7Hz, 2H), 2.76 (t, J = 9.1 Hz, 2H), 2.25
(t, J = 8.2 Hz, 2H), 2.01 (m, 2H), 1.90 (m, 6H), 1.70(m, 2H).
Synthesis of [14C]-(3)
[20] D. Larrey, A. W. Lohse, V. de Ledinghen, C. Trepo, T. Gerlach,
J.-P. Zarski, A. Tran, P. Mathurin, R. Thimme, K. Arastéh, C. Trautwein,
A. Cerny, N. Dikopoulos, M. Schuchmann, M. H. Heim, G. Gerken,
J. O. Stern, K. Wu, N. Abdallah, B. Girlich, J. Scherer, F. Berger,
M. Marquis, G. Kukolj, W. Böcher, J. Steffgen, J. Hepatol. 2012, 57, 39–46.
[21] D. Larrey, A. W. Lohse, C. Trepo, J. P. Bronowicki, K. Arasteh, M.
Bourliere, J. L. Calleja, J. O. Stern, G. Nehmiz, N. Abdallah, K. L. Berger,
M. Marquis, J. Steffgen, G. Kukolj, Antimicrob. Agents Chemother.
2013, 57, 4727–4735.
[22] B. Latli, M. Hrapchak, V. Gorys, M. Llinàs-Brunet, S. S. Campbell,
J. Song, C. H. Senanayake, J. Labelled Compd. Radiopharm. 2014,
57, 350–357.
[23] L.-Z. Chen, J. P. Sabo, E. Philip, L. Rowland, Y. Mao, B. Latli, D. Ramsden,
D. A. Mandarino, R. S. Sane, Antimicrob. Agents Chemother. 2015, 59,
25–37.
(2S,3S,4S,5R,6S)-6-(((E)-3-(2-(1-(2-(5-Bromopyrimidin-2-yl)-3-
cyclopentyl-1-methyl-1H-indole-6-carboxamido)cyclobutyl)-
1-methyl-1H-benzo[d]imidazole-6-yl-2-14C)acryloyl)oxy)-3,4,5-
trihydroxytetrahydro-2H-puran-2-carboxylic acid,
[
14C]-(3):
NMM (19.5 mg, 0.2 mmol) was added to a suspension of [14C]-
(1) (42 mg, 0.06 mmol, 3.1mCi) and HATU (36.7 mg, 0.1 mmol) in
MeCN (2mL) at room temperature. The mixture was stirred for
14 h. HPLC showed complete consumption of the starting acid.
The mixture was diluted with CH2Cl2 (3mL) and D-allyl
glucuronate (43 mg, 0.2mmol) and NMM (19 mg, 0.2 mmol) were
added, and the mixture was stirred overnight. A second portion of
D-allylglucuronate (43 mg, 0.2 mmol) and NMM (19 mg, 0.2 mmol)
were added and the mixture was stirred for 48 h. HPLC revealed
consumption of the active ester. Pd(PPh3)4 (6.7 mg, 0.01 mmol)
and morpholine (10 mg, 0.12 mmol) were added. The reaction
mixture was stirred for 2h, then HCl (2.0 M, 2 mL) was added, and
the mixture was stirred overnight. The mixture was filtered and
concentrated to remove most of the volatiles. The crude product
was purified by HPLC, and the desired fractions were combined
and lyophilized to give 482 μCi (7.7 mg) with a specific activity
of 52.3 mCi/mmol and radiochemical purity of 97.5%, which
co-eluted with unlabeled sample on HPLCb, Rt = 10.37 min.
[24] S. L. Regan, J. L. Maggs, T. G. Hammond, C. Lambert, D. P. Williams,
B. K. Park, Biopharm. Drug Dispos. 2010, 31, 367–395.
[25] E. Caspi, M. Galli Kienle, K. R. Varma, L. J. Mulheirn, J. Am. Chem. Soc.
1970, 92, 2161–2163.
[26] O. Berséus, I. Björkhem, Eur. J. Bio. 1967, 2, 503–507.
[27] J. K. Hilunen, E. J. Davis, Biochem. J. 1981, 194, 427–432.
[28] (a)Y. Zhang, B. Z. Lu, G. Li, S. Rodriguez, J. Tan, H.-X. Wei, J. Liu,
F. Roschangar, F. Ding, W. Zhao, B. Qu, D. Reeves, N. Grinberg,
H. Lee, G. Heckmann, O. Niemeier, M. Brenner, Y. Tsantrizos,
P. L. Beaulieu, A. Hossain, N. Yee, V. Farina, C. H. Senanayake,
Org. Lett. 2014, 16, 4558–4561; (b)A. Khodabocus, Z.-H. Lu,
C. H. Senanayake, H. Wei, Y. Zhang, Process for preparing
2,3-disubstituted indoles. 2010, US patent 7642352B2; (c)Y. S. Tsantrizos,
C. Chabot, P. Beaulieu, C. Brochu, T. A. Stammers, B. Thavonekham,
J. Rancourt, 3-(2-{1-[(3-Cyclopentyl-1-methyl-2-pyridin-2-yl-1H-indole-6-
carbonyl)amino]cyclobutyl}-1-methyl-1H-benzoimidazol-5-yl)acrylic
acid; hepatitis C virus infection; synergistic with another inhibitor of
HCV polymerase or immunomodulatory agent such as interferons.
2009, US patent 7582770B2; (d)P. L. Beaulieu, G. Fazal, S. Goulet,
G. Kukolj, M. Poirier, Y. S. Tsantrizos, E. Jolicoeur, J. Gillard, M.-A. Poupart,
J. Rancourt, Viral polymerase inhibitors. 2009, US patent 0087409A1.
[29] B. Latli, M. Hrapchak, D. Krishnamurthy, C. H. Senanayake, J. Labelled
Compd. Radiopharm. 2008, 51, 283–285.
[30] S. J. Harwood, J. Labelled Compd. Radiopharm. 2004, 4, 869–874.
[31] G. Madegard, P. Mestre, P. Raimond, J.-P. Noel, J. Labelled Compd.
Radiopharm. 1995, 36, 1123–1132.
[32] M. Sienkowska, V. Benin, P. Kaszynski, Tetrahedron 2000, 56, 165–173.
[33] M. Souljoughian, P. G. Williams, H. Morimoto, D. R. Goodlett, R. B. van
Breemenal, J. Chem. Soc. Chem. Commun. 1993, 22, 414–416.
[34] (a)J. M. Concellón, H. Rodríguez-Solla, Chem. Eur. J. 2002, 8, 4493–4497;
(b)J. M. Khurana, P. Sharma, Bull. Chem. Soc. Jpn. 2004, 77, 549–552.
[35] J. A. Perrie, J. R. Harding, D. W. Holt, A. Johnston, P. Meath, A. V. Stachulski,
Org. Lett. 2005, 7, 2591–2594.
Conflict of Interest
The authors did not report any conflict of interest.
References
[1] World Health Organization, Hepatitis C, fact sheet N°164, July 2012.
[2] M. G. Ghany, D. B. Strader, D. L. Thomas, L. B. Seeff, Hepatol. 2009, 49,
1335–1374.
[3] (a)M. Yahia, Nature 2011, 474, S12–S13; (b) A. Fouad, D. Sabry,
R. Ahmed, M. Kamal, S. Abd Allah, S. Marzouk, M. Amin, R. Abd El Aziz,
A. El Badri, H. Khattab, D. Helmy, Int. J. Gen. Med. 2013, 6, 127–134.
[4] M. K. Sanaa, Am. J. Gastroenterol. 2008, 103, 1283–1297.
[5] A. Maheshwari, P. J. Thuluvath, Clin. Liver Dis. 2010, 14, 169–176.
[6] A. I. Kim, S. Saab, Am. J. Med. 2005, 118, 808–815.
[7] L. Gravitz, Nature 2011, 474, S2–S4.
[8] T. Nakano, G. M. Lau, Liver Int. 2011, 32, 339–345.
[9] C. Wartelle-Bladou, G. Le Folgoc, M. Bourliere, L. J. Lecomte, Viral.
Hepat. 2012, 19, 525–536.
[10] T. L. Tellinghuisen, M. J. Evans, T. Von Hahn, S. You, C. M. Rice, J. Virol.
2007, 81, 8853–8867.
[36] H. Juteau, Y. Gareau, M. Labelle, Tetrahedron Lett. 1997, 38, 1481–1484.
[37] A. El Aloui, F. Schmidt, C. Monneret, J.-C. Florent, J. Org. Chem. 2006,
71, 9628–9636.
[38] C. Gautier, J. Legoult, S. Rondeau, A. Pichette, Tetrahedron Lett. 2009,
50, 988–991.
[11] A. A. Kolykhalov, K. Mihalik, S. M. Feinstone, C. M. Rice, J. Virol. 2000,
74, 2046–2051.
[39] J. R. Kenny, J. L. Maggs, X. Meng, D. Sinnott, S. E. Clarke, B. K. Park,
A. S. Stachulski, J. Med. Chem. 2004, 47, 2816–2825.
[12] L. Gerber, T. M. Welzel, S. Zeuzem, Liver Int. 2013, 33, Issue
Supplement s1, 85–92.
[40] E. R. Bowkett, J. R. Harding, J. L. Maggs, B. K. Park, J. A. Perrie,
A. V. Stachulski, Tetrahedron 2007, 63, 7596–7605.
[41] H. T. Bucherer, W. Steiner, J. Prakt. Chem. 1934, 140, 291–316.
[13] S. Bressanelli, L. Tomei, A. Roussel, I. Incitti, R. L. Vitale, M. Mathieu,
R. De Francesco, F. A. Rey, Proc. Natl. Acad. Sci. U. S. A. 1999, 96,
13034–13039.
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 250–260