An efficient synthesis of carbon-14-labeled 6-[2-(dimethylamino)ethyl]-14- (1-ethylpropyl)-5,6,7,8-tetrahydroindolo [2,1-α] [2,5]benzodiazocine-11- carboxylic acid using Curtius rearrangement reaction as a key step

Article abstract of DOI:10.1002/jlcr.1842

Here we report an efficient synthesis of ¹⁴C-labeled 6-[2-(dimethylamino)ethyl]-14-(1-ethylpropyl)-5,6,7,8-tetrahydroindolo-[2, 1-α][2,5]benzodiazocine-11-carboxylic acid (1) using the Curtius rearrangement as a key step. The synthesis was init

Full text of DOI:10.1002/jlcr.1842

Research Article
Received 15 July 2010,
Revised 10 September 2010,
Accepted 13 September 2010
Published online 30 November 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1842
An efficient synthesis of carbon-14-labeled
6-[2-(dimethylamino)ethyl]-14-
(1-ethylpropyl)-5,6,7,8-tetrahydroindolo
[2,1-a] [2,5]benzodiazocine-11-carboxylic
acid using Curtius rearrangement reaction
as a key step
Ã
Andy Shiqiang Zhang, Jonathan Z. Ho, and Matthew P. Braun
Here we report an efficient synthesis of 14C-labeled 6-[2-(dimethylamino)ethyl]-14-(1-ethylpropyl)-5,6,7,8-tetrahydroindolo-
[2,1-a][2,5]benzodiazocine-11-carboxylic acid (1) using the Curtius rearrangement as a key step. The synthesis was initiated
by converting the unlabeled aryl carboxylic acid to an aryl amine via Curtius rearrangement reaction. The resulting aryl
amine was then converted to an aryl iodide which was coupled with zinc [14C]cyanide to form an aryl [14C]nitrile.
Subsequent hydrolysis yielded the 14C-labeled carboxylic acid [14C]-1. This overall process represents a mild and potentially
general approach for conversion of unlabeled carboxylic acids to isotopically labeled carboxylic acids.
Keywords: Hepatitis C Virus (HCV); Curtius rearrangement; zinc [14C]cyanide
Instruments
Introduction
Analytical HPLC assay was performed using an Agilent 1100
Hepatitis C is a viral infection of the liver, which was referred to as
series HPLC system with a G1315B diode-ray UV detector, a
the parenterally transmitted ‘non-A, non-B hepatitis’ until the
Packard Flow Scintillation Analyzer 500TR using one of
identification of the causative agent in 1989.1 Hepatitis C Virus
the following methods: (a) Agilent Eclipse XDB-C8 column
(5 m, 4.6 ꢀ 150 mm), 251C, 1.5 mL/min, A = H2O (0.1% TFA),
(HCV), a blood-borne virus, is a major cause of acute hepatitis and
chronic liver disease, including cirrhosis and hepatocellular
B = MeCN, gradient 85A/15B to 20A/80B over 7.5 min and 100B
carcinoma.2 There is yet no adequate treatment for HCV infection.
for 5.5 min; (b) Phenomenex Phenyl-hexyl column (5 m,
Current medicinal chemistry efforts are focusing on the develop-
ment of small molecule inhibitors of the viral RNA polymerase,
4.6 ꢀ 250 mm), 251C, 1.0 mL/min, A: H2O (0.4% ammonium
formate), B: MeCN, isocratic 35B/65A for 20 min followed by
which is thought to be essential for the life cycle of the virus.3 The
100B for 10 min. LC/MS analysis was performed using a Hewlett
HCV polymerase inhibitor program team at Merck is advancing
Packard 1100 series LC with a G1315A diode-ray UV detector, an
new drug candidates for further development and 6-[2-(dimethy-
Agilent Eclipse XDB-C8 column (3.5 m, 3.0 ꢀ 150 mm) and Agilent
single quadrupole MSD detector (G1956B) with the following
lamino)ethyl]-14-(1-ethylpropyl)-5,6,7,8-tetrahydroindolo-[2,1-a][2,5]
benzodiazocine-11-carboxylic acid-11 (1) is one of them.3 To
support the in vitro and in vivo ADME (adsorption, distribution,
metabolism and excretion) studies for 1, the synthesis of a
HPLC method: A = ammonium formate buffer pH 3.5, B = MeCN,
gradient 95A/5B to 5A/95B over 12 min and 5A/95B for 3.5 min.
The amount of radioactivity was determined by liquid scintilla-
tion counting using a BIOSCAN Hidex liquid scintillation counter.
Flash chromatography was performed on silica gel. Preparative
HPLC purification was performed on a Gilson Preparative HPLC
14C-labeled version ([14C]-1) (Figure 1) was carried out and the
chemistry developed for the synthesis is the subject of this paper.
Experimental
Reagents
Merck Research Laboratories, Process Research – Labeled Compound Synthesis
RY80R-104, Rahway, NJ 07065, USA
All reagents were of analytical grade and used without further
purification. Zinc [14C]cyanide (110mCi/mmol) was purchased
from American Radiolabeled Chemicals Inc. and sodium [14C]for-
mate (55 mCi/mmol) was purchased from Amersham Biosciences.
*Correspondence to: Andy Shiqiang Zhang, RY32-205, P.O. Box 2000, Rahway,
NJ 07065, USA.
E-mail: Andy_zhang@merck.com
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Copyright r 2010 John Wiley & Sons, Ltd.

A. S. Zhang et al.
(0.1% TFA) B: MeCN, gradient 85A/15B to 50A/50B over 30 min)
to give amine 4 (110 mg, 0.264 mmol, 499% UVA 220 nm) in
61% yield.
2-(14-cyclohexyl-11-iodo-7,8-dihydroindolo[2,1-a][2,5]benzodiazo-
cin-6(5H)-yl)-N,N-dimethylethanamine (5)
A solution of conc. H2SO4 (85 mL) in water (0.51 mL) was added
to a mixture of amine 4 (85 mg, 0.20 mmol) in diglyme (0.57 mL)
at 01C followed by a solution of NaNO2 (21.1 mg, 0.306 mmol) in
water (0.12 mL). The reaction was stirred at 01C for 20 min. A
solution of NaI (640 mg, 4.08 mmol) in water (0.5 mL) was added
to the reaction mixture and stirred at 01C for 10 min. The
reaction was then warmed up to ambient temperature and aged
40 min to completion based on HPLC assay using method (a)
(tR = 6.44 min). The reaction was diluted with CH2Cl2 (10 mL),
saturated Na2S2O3 (5 mL) and 5% sodium bicarbonate (5 mL).
After extraction, the organic was washed with saturated Na2S2O3
(10 mL), brine (10 mL), dried over Na2SO4 and concentrated
under vacuum. The resulting crude iodide 5 was purified using
flash silica gel chromatography (CH2Cl2:MeOH:Et3N = 90:10:0.1)
to give iodide 5 (94 mg, 0.09 mmol, 50.8% UVA 220 nm) in 45%
yield.
Figure 1. 14C Labeled carboxylic acid 1.
system using the following methods: (a) Phenomenex Curosil
PFP (5 m, 21.2 ꢀ 250 mm), 20 mL/min, A: H2O (0.1% HClO4),
B: MeCN, isocratic 61A/39B for 20 min; (b) ACE C8 (5 m,
21.2 ꢀ 250 mm), 20 mL/min, A: H2O (0.1% HClO4), B: MeCN,
gradient 64A/36B to 61A/39B over 20 min.
1-[(f14-cyclohexyl-6-[2-(dimethylamino)ethyl]-5,6,7,8-tetrahydroin-
dolo-[2,1-a][2,5]benzodiazocin-11-ylgcarbonyl)oxy]pyridine-2(1H)-
thione (2)
14-cyclohexyl-6-[2-(dimethylamino)ethyl]-5,6,7,8-tetrahydroindolo-
[2,1-a][2,5]benzodiazocin-11-carbonitrile (6)
A mixture of iodide 5 (190 mg, 0.36 mmol), Zn(14CN)2 (specific
activity (SA) = 110 mCi/mmol, 46 mg, 0.38 mmol) and Pd(PPh3)4
(38 mg, 0.033 mmol) in DMF (4.0 mL) was degassed with a
stream of N2 for 5 min before heating at 851C for 7 h. The
progress of the reaction was monitored by HPLC using method
(a) (tR = 5.70 min). Upon completion, the workup was effected by
diluting the reaction mixture with EtOAc (15 mL) and extracting
with 5% aqueous sodium bicarbonate (4 ꢀ 5 mL) followed by
brine (10 mL). The organic solution was dried with Na2SO4 and
filtered. The organic solvent was removed under vacuum and
the resulting residue was re-dissolved in methanol (5.0 mL).
Liquid scintillation counting showed 13 mCi. Crude 6 was
analyzed by HPLC using method (a) (tR = 5.72 min) to show
92.7% radiochemical purity of radioactivity and corresponds to
66% yield for the reaction (13 mCi, SA = 55 mCi/mmol,
0.24 mmol).
Triethylamine (100 mL, 0.717 mmol) was added to a mixture of
acid 1 (105 mg, 0.236 mmol), mercatopyridine N-oxide (100 mg,
0.786 mmol) in DMF (1.0 mL) at ambient temperature followed
by TBTU (128 mg, 0.400 mmol). The reaction mixture was stirred
for 40 min and the progress was monitored by HPLC using
method (a) (tR = 5.70 min). The reaction was then partitioned
between 5% sodium bicarbonate solution (10 mL) and ethyl
acetate (10 mL). The organic layer was washed with 5% sodium
bicarbonate (3 ꢀ 10 mL) and brine (10 mL), dried over Na2SO4
and removed solvent under vacuum to give
0.179 mmol, 92.9% UVA 220 nm) in 76% yield.
2 (99 mg,
14-cyclohexyl-6-[2-(dimethylamino)ethyl]-5,6,7,8-tetrahydroindolo-
[2,1-a][2,5]benzodiazocin-11-amine (4)
A slurry of acid 1 (190 mg, 0.43 mmol) in benzene (10 mL) and
DMF (10 mL) was sonicated for 5 min. Oxalyl chloride (2.77 mL,
2M in CH2Cl2, 5.54 mmol) was added dropwise to the suspension
and stirring was continued at ambient temperature for 40 min.
The solvent was removed under vacuum and the residue was
re-dissolved in DMF (3.0 mL). The DMF solution of acid chloride
was added to a solution of NaN3 (305 mg, 4.69 mmol) in water
(3.0 mL) slowly in an ice-water bath. The reaction was monitored
by HPLC using method (a) (tR = 3.75 min).
The reaction was extracted with benzene (4 ꢀ 5 mL) and the
combined organic layers were dried with Na2SO4 and filtered.
The benzene solution was heated at 601C for 20 min. Conc. HCl
(3.0 mL) was added to the reaction and stirred at 801C for 3 h.
The two layers of the reaction were separated. The organic layer
was washed with water (4 mL). The combined aqueous layers
were added NaOH to pH = 9, and extracted with CH2Cl2
(3 ꢀ 5 mL). The combined organic layers were extracted with
brine (10 mL), separated and dried with Na2SO4. The organic
solvent was removed under vacuum and the reslulting
crude amine 4 was purified with preparative reversed-phase
HPLC (ACE C18 5 u 21.2 ꢀ 250 mm column, 20 mL/min, A: H2O
6-[2-(dimethylamino)ethyl]-14-(1-ethylpropyl)-5,6,7,8-tetrahy-
droindolo-[2,1-a][2,5]benzodiazocine-11-carboxylic acid-11-
[14C] ([14C]-1)
A solution of nitrile 6 (5 mCi, SA = 55 mCi/mmol, 0.091 mmol) in
conc. HCl (2.0 mL) was heated at reflux temperature of 1201C for
7 h. Upon completion based on HPLC assay using method (a)
(tR = 4.88 min), the reaction was neutralized with 4M NaOH till
pH = 7 and purified twice using preparative HPLC method (a)
and (b) to give 1.8 mCi [14C]-1 in 36% yield. The radiochemical
purity was determined to be 98.9% based on HPLC analysis
using method (b) (tR = 12.29 min). [14C]-1 was co-injected with
the authentic sample by HPLC analysis using method (b) and
shows as one peak. The identity of [14C]-1 was also confirmed by
LC/MS (ESI1) [M1H]1 448.2. The SA of [14C]-1 was calculated to
be 53 mCi/mmol by using its MS profile from that of unlabeled
compound 1 (ESI1) [M1H]1 446.2. This is in line with the
claimed specific activity of 110 mCi/mmol for the commercial
zinc [14C]cyanide.
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 163–167

A. S. Zhang et al.
In an effort to develop a more efficient synthesis for [14C]-1,
we pursued a decarboxylation/14C-carboxylation route utilizing
the readily available unlabeled 1 as starting material. Aromatic
carboxylic acid groups are usually stable to metabolism, which
was confirmed in the subsequent drug metabolism studies
using [14C]-1. A route using decarboxylative bromination of 1
was initiated to convert carboxylic acid 1 to aryl bromide 3 via
ester 2 (Scheme 2).4 While formation of ester 2 was high
yielding, the radical rearrangement reaction of 2–3
was unsuccessful under numerous conditions (thermal, light,
variation of solvent). Based on previous literature reports,4
radical decarboxylative halogenation on functionalized aromatic
Results and discussion
In order to maintain metabolic stability of the label, it was
required that the 14C label be installed within the core of
the molecule instead of the side chain because of potential
N-demethylation process. Prior synthetic routes developed by
Process and Medicinal Chemistry were evaluated for 14C label
incorporation. This analysis yielded a 9-linear-step sequence
from [14C]bromoacetic acid as the most efficient route which
would satisfy label position constraints (Scheme 1). Unfortu-
nately, this was projected to be a time-consuming route that
was anticipated to result in low overall radiochemical yield.
Scheme 1.
Scheme 2.
J. Label Compd. Radiopharm 2011, 54 163–167
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

A. S. Zhang et al.
carboxylic acids can be problematic, therefore, we abandoned 14C-labeled reagents and a couple of one- or two-pot multiple-
this approach.
step reactions. The method is thus efficient with regard to
We next turned our attention to accessing the aryl amine 4 isotope use, and in the present case, reduced the number of
from carboxylic acid 1, which could serve as a precursor to aryl synthetic steps relative to the de novo route. It is particularly
halide 3, using a re-arrangement strategy. After a variety of attractive in cases where the carboxylic acid containing
approaches5 were explored, we found that Curtius rearrange- synthetic target is readily available but synthetic precursors
ment6,7 was most efficient and provided the desired product in are not. This methodology also has the potential of general
61% yield (Scheme 3).
application to other 14C-labeled compounds with aromatic
Diazonium salts have been shown to be efficient cross carboxylic acid functionality. The direct carbonylation of the
coupling partners with a variety of nucleophiles under Pd(0)- diazonium salt would provide significant additional improve-
catalyzed coupling conditions. Accordingly, we initially tried to ment of the route and this is the subject of further investigation
make [14C]-1 directly through the diazonium salt 4 using Pd- which will be reported in due course. Owing to the complexity
catalyzed carbonylation reaction conditions (Scheme 4) but this of the molecule, using a Grignard intermediate to make [14C]-1
route failed to yield any product.8,9 Owing to study timeline was not explored. Using [14C]KCN or [14C]CuCN to convert the
restraints, we pursued the more lengthy route of converting diazonium salt directly to 6 was not explored, either.
amine 4 to aryl iodide 5 through the diazonium salt and this
quickly provided 5 in 45% yield.10 A subsequent cyanation Acknowledgements
reaction gave nitrile 6 in 66% yield.11 The nitrile was hydrolyzed12
using concentrated HCl to give the final product [14C]-1 in 36%
yield after preparative HPLC purification (Scheme 5).
The route developed for converting unlabeled carboxylic
acid 1 to its 14C-labeled version [14C]-1 involved two steps using
The authors thank Frank Narjes and his group from IRBM for
providing unlabeled aryl carboxylic acid 1 and Allen Jones and
Rosemary Marques in the LCS analytical group for final analysis
of aryl carboxylic acid [14C]-1.
Scheme 3.
Scheme 4.
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 163–167

A. S. Zhang et al.
Scheme 5.
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