Synthesis of C-14-labeled novel IKK inhibitor: 2-[14C]-N-(6- chloro-9H-pyrido [3,4-b]indol-8-yl)-3-pyridinecarboxamide

Article abstract of DOI:10.1002/jlcr.926

2-[¹⁴C]-N-(6-Chloro-9H-pyrido [3,4-b]indol-8-yl)-3- pyridinecarboxamide (9A, also referred to as [¹⁴C]-PS-1145) was synthesized from [¹⁴C]-paraformaldehyde in five steps in an overall radiochemical yield of 15%. The key intermediate 1-[¹⁴C]-6-chloro-1, 2,3,4-tetrahydro-β-carboline was obtained by Pictet-Spengler cyclization of chlorotryptamine with [¹⁴C]-paraformaldehyde. Similar reactions were conducted with tryptamine to address the generality of the methodology. Copyright

Full text of DOI:10.1002/jlcr.926

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 323–330.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.926
Research Article
Synthesis of C-14-labeled novel IKK inhibitor: 2-[14C]-
N-(6-chloro-9H-pyrido [3,4-b]indol-8-yl)-3-
pyridinecarboxamide
Yuexian Li* and Shimoga R. Prakash
Department of Drug Metabolism and Pharmacokinetics, Radiochemistry Group,
Millennium Pharmaceuticals, 35 Landsdowne Street, Cambridge, MA 02139, USA
Summary
2-[14C]-N-(6-Chloro-9H-pyrido [3,4-b]indol-8-yl)-3-pyridinecarboxamide (9A; also
referred to as [14C]-PS-1145) was synthesized from [14C]-paraformaldehyde in five
steps in an overall radiochemical yield of 15%. The key intermediate 1-[14C]-6-chloro-
1,2,3,4-tetrahydro-b-carboline was obtained by Pictet–Spengler cyclization of
chlorotryptamine with [14C]-paraformaldehyde. Similar reactions were conducted
with tryptamine to address the generality of the methodology. Copyright # 2005
John Wiley & Sons, Ltd.
Key Words: 2-[14C]-N-(6-chloro-9H-pyrido [3,4-b]indol-8-yl)-3-pyridine-carboxamide;
[14C]-paraformaldehyde
Introduction
The transcription factor NF-kB mediates the expression of a number of pro-
inflammatory cytokines, adhesion molecules, growth factors, and anti-
apoptosis survival proteins. Inhibitors of IkB kinase (IKK) have been sought
as specific regulators of NF-kB. These inhibitors are expected to be of clinical
utility in the treatment of inflammatory or immune-related disorders or
diseases. The remarkable biological activity of the recently discovered IKK
inhibitor, N-(6-chloro-9H-pyrido [3,4-b]indol-8-yl)-3-pyridinecarboxamide ð9Þ1
%
led us to prepare a carbon-14(C-14)-labeled version. This radiolabeled version
was required to assist in metabolite profiling and whole body autoradiography
studies in experimental animals.
*Correspondence to: Y. Li, Department of Drug Metabolism and Pharmacokinetics, Radiochemistry
Group, Millennium Pharmaceuticals, 35 Landsdowne Street, Cambridge, MA 02139, USA. E-mail:
yuexian.li@mpi.com
Copyright # 2005 John Wiley & Sons, Ltd.
Received 11 November 2004
Revised 30 November 2004
Accepted 15 December 2004

324
Y. LI AND S. R. PRAKASH
Results and discussion
As delineated in Scheme 1, the published procedure for synthesis of 8-amino-6-
chloro-b-carboline ð4Þ utilized Pictet–Spengler cyclization of tryptamine with
%
glyoxalic acid followed by chlorination, nitration and subsequent reduction of
the nitro group.1–3 Adaptation of this procedure for synthesis of PS-1145
labeled with C-14 in the beta carboline ring would have required [14C]-
glyoxalic acid. Along with the difficulty of obtaining or preparing
[14C]-glyoxalic acid4,5 one has to contend with problems of regioselectivity
during chlorination.
In an alternative approach, a similar carboline like 1-[14C]-6-methoxy-
1,2,3,4-tetrahydro-b-carboline has been synthesized from commercially
available [14C]-formaldehyde in a yield of 25%.6 We believe that this
low yield is because of instability and volatility of [14C]-formaldehyde.
Commercially available [14C]-paraformaldehyde is more stable and can
easily replace [14C]-formaldehyde in such Pictect–Spengler cyclization
reactions. In order to avoid poor regioselectivity during chlorination, we
reasoned that Pictet–Spengler cyclization of 5-chlorotryptamine hydro-
chloride with [14C]-paraformaldehyde would be a better alternative. Using
this strategy, [14C]-PS-1145 was synthesized from [14C]-paraformaldehyde in
five steps (Scheme 2).
In order to address generality of the methods, these reactions were also
conducted with tryptamine. Cyclization reactions of 5-chlorotryptamine
hydrochloride and tryptamine hydrochloride with [14C]-paraformaldehyde in
3 M acetate buffer provided the desired b-carbolines in yields of 51 and 67%,
NH2
NH
1. OHC-COOH/pH 6.0 buffer
2. HCl/reflux
.HCl
N
H
N
H
1
2
1. NCS/THF
2. NaNO3 /TFA
3. SnCl2 /HCl
N
Cl
N
Pd-C/xylenes
reflux
N
N
H
H
NH2
3
4
Scheme 1.
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF C-14-LABELED NOVEL IKK INHIBITOR
325
1. (*CH2O)n
acetate buffer
2. NaOH
NH
X
NH2
.HCl
X
*
N
H
N
H
2: X = H, * = 12
C
C
C
2A: X = H, * = 14
1: X = H
5: X = Cl
6: X = Cl, * = 12
6A: X = Cl, * = 14
C
N
X
N
Cl
*
*
catalyst
xylenes
NaNO3/TFA
N
N
H
H
.TFA
NO2
3: X = H, * = 12
3A: X = H, * = 14
C
C
8: * = 12
C
C
7: X = Cl, * = 12
C
8A: * = 14
7A: X = Cl, * = 14
C
OH
N
Cl
O
O
C
N
Cl
*
N
H
*
Pt(S)-C/Et3N
HCOOH/EtOH
N
H
NH
N
C
NH2
EDCI/pyridine
9: * = 12
9A: * = 14
C
C
4: * = 12
4A: * = 14
C
C
N
Scheme 2.
respectively. 1,2,3,4-Tetra-b-carbolines were oxidized to b-carbolines by
refluxing with sulfur in xylenes2,7 or Pd-C in xylenes.1 Oxidation with Pd-C
proceeded smoothly. Yields of oxidized products ranged between 60 and
65%. Nitration of 3A with sodium nitrate in TFA provided the nitro
compound (73%), which was reduced with poisoned catalyst to provide
the amine (94%).1,8–10 Finally, [14C]-PS-1145 (2-[14C]-N-(6-chloro-9H-pyrido
[3,4-b]indol-8-yl)-3-pyridinecarboxamide) ð9AÞ was prepared by reacting
1-[14C]-8-amino-6-chloro-b-carboline ð4AÞ; nicotinic acid, and EDCI
(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) in pyridine
(73%).1,11
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Y. LI AND S. R. PRAKASH
Conclusion
Reaction of tryptamines with paraformaldehyde provides a convenient
method of preparing 1,2,3,4-tetrahydro-b-carbolines in reasonable yields.
C-14-labeled novel IKK inhibitor, 2-[14C]-N-(6-chloro-9H-pyrido [3,4-b]indol-
8-yl)-3-pyridine-carboxamide, was synthesized in five steps in an overall
radiochemical yield of 15%.
Experimental
General
All commercial reagents were used as supplied unless otherwise stated. [14C]-Para-
formaldehyde was purchased from Amersham Biosciences. Radioactivity was
quantified by liquid scintillation counting using Beckman LS6500 counter.
HPLC analyses were performed on a Luna C18 (2) column (5 m, 4.6 ꢀ 150 mm)
with a flow rate of 1 ml/min. Solvent systems utilized consisted of A (0.1%
formic acid in 99% water and 1% CH3CN) and B (0.1% formic acid in 5%
water and 95% CH3CN) with a following gradient profile: 5% B for 1 min,
5–100% B over 12 min, and 100% B for 3 min. UV detection was at 254 (or
210) nm and radioactive detection was with a IN/US b-Ram Model 3 flow
detector at a scintillant flow of 3 ml/min. NMR spectra were recorded on a
Varian 600 (or 300) MHz spectrometer. LC–MS analyses were performed on a
ThermoFinnigan LCQ mass spectrometer.
1-[14C]-1,2,3,4-Tetrahydro-b-carboline ð2AÞ
To a stirred suspension of tryptamine hydrochloride (4.8 mmol), [14C]-
paraformaldehyde (10 mCi, 56 mCi/mmol) and paraformaldehyde (3.8 mmol,
0.114 g, specific activity of diluted paraformaldehyde, 2.2 mCi/mmol) in water
(16 ml) was added 3 M acetate buffer (2.4 ml) under nitrogen. The mixture was
heated under nitrogen at 1058C for 4 h. The reaction mixture was
concentrated. The product was isolated by crystallization at 08C. The dried
solid was dissolved in methanol. This solution was basified with 1 N NaOH
and evaporated to dryness. The solid was washed with cold water and dried to
give 0.462 g (67%, 6.7 mCi, 2.2 mCi/mmol) product. HPLC: chemical purity
94% (UV area % at 210 nm); radiochemical purity, 94%. LC–MS/MS: m/z
175 (M þ 1), 173, 144.
1,2,3,4-Tetrahydro-b-carboline ð2Þ
%
Under similar reaction conditions, compound 2 was obtained in 71% yield
%
(0.25 g) from paraformaldehyde (2 mmol). HPLC: 95% pure (UV at 210 nm).
LC–MS/MS: m/z 173 (M þ 1), 144. 1H-NMR ((CD3)2SO): d 7.33 (1H, d), 7.25
(1H, d), 6.97 (1H, t), 6.91 (1H, t), 3.85 (2H, s), 2.97 (2H, t), 2.60 (2H, t).
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SYNTHESIS OF C-14-LABELED NOVEL IKK INHIBITOR
327
1-[14C]-6-Chloro-1,2,3,4-tetrahydro-b-carboline ð6AÞ
To a stirred suspension of 5-chlorotryptamine hydrochloride (3.77 mmol) and
[14C]-paraformaldehyde (175 mCi, 56 mCi/mmol), in water (12.5 ml) was
added 3 M acetate buffer (1.88 ml) under nitrogen. The mixture was heated
under nitrogen at 1058C for 4 h. The reaction mixture was cooled to room
temperature. The product was isolated by crystallization at 08C. The dried
solid was dissolved in methanol. This solution was basified with 1 N NaOH
and evaporated to dryness. The solid was washed with cold water and dried to
give 91 mCi (55 mCi/mmol, 52%) product. HPLC: 99% (UV at 210 nm);
radiochemical purity, 99%. LC–MS/MS: m/z 209 (M þ 1), 207, 178.
6-Chloro-1,2,3,4-tetrahydro-b-carboline ð6Þ
%
The title compound was prepared similarly from paraformaldehyde (2 mmol)
in 65% yield (0.27 g). HPLC: 99% (UV at 210 nm). LC–MS/MS: m/z 207
(M þ 1), 178. 1H-NMR ((CD3)2SO): d 7.32 (1H, s), 7.24 (1H, d), 6.92 (1H, dd),
3.85 (2H, s), 2.95 (2H, t), 2.58 (2H, t).
1-[14C]-b-carboline ð3AÞ
1-[14C]-1,2,3,4-Tetrahydro-b-carboline (2A; 6.7 mCi, 2.2 mCi/mmol) was
suspended in xylenes (15 ml) in a 100 ml round-bottom flask equipped with
a condenser that was open to the atmosphere. 5% Pd/C (0.24 g) was added.
The mixture was heated in xylenes at 1438C with good stirring until LC–MS/
MS indicated complete conversion (2 days). 5% Pd/C (0.12 g) was added twice
a day to hasten the reaction. The reaction mixture was cooled to room
temperature and methanol (150 ml) was added. The mixture was filtered
through a pad of celite. The solvents were evaporated to give the desired
(4.36 mCi, 2.2 mCi/mmol, 65%) product.y HPLC: 95% (UV at 210 nm);
radiochemical purity, 96%. LC–MS/MS: m/z 171 (M þ 1), 169.
b-Carboline ð3Þ
%
Oxidation of 1,2,3,4-tetrahydro-b-carboline (2; 0.5 mmol) using similar
conditions provided 0.058 g (68%) of product. HPLC: 96% (UV at 210 nm).
%
1
LC–MS/MS: m/z 169 (M þ 1). H-NMR ((CD3)2SO): d 11.62 (NH, s), 8.90
(1H, s), 8.33 (1H, d), 8.24 (1H, d), 8.10 (1H, d), 7.58 (1H, t), 7.52 (1H, d), 7.24
(1H, t).
1-[14C]-6-Chloro-b-carboline ð7AÞ
1-[14C]-6-Chloro-1,2,3,4-tetrohydro-b-carboline (6A; 29.8 mCi, 55 mCi/mmol)
was suspended in xylenes (4.3 ml) in a 25 ml round-bottom flask equipped with
y The low recovery was probably due to adsorption of the product on the catalysts.
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Y. LI AND S. R. PRAKASH
a condenser that was open to the atmosphere. 5% Pd/C (0.054 g) and PtO2
(0.01 g) were added. The mixture was heated in xylenes at 1438C with good
stirring until LC–MS/MS indicated complete conversion (3 days). Pd/C
(0.027 g) was added three times a day to hasten the reaction. The same scale
reaction was repeated twice (total activities, 61.2 mCi, 55 mCi/mmol). The
reaction mixtures were cooled to room temperature and combined. Methanol
(180 ml) was added. The mixture was filtered through a pad of celite.
The solvents were evaporated to give 55 mCi (55 mCi/mmol, 60%) product
(see footnote y). HPLC: 93% (UV at 210 nm); radiochemical purity, 96%.
LC–MS/MS: m/z 205 (M þ 1), 203.
6-Chloro-b-carboline ð7Þ
%
Following the similar conditions, oxidation of 6-chloro-1,2,3,4-tetrohydro-b-
carboline (6; 1 mmol) provided 0.127 g (62%) product. HPLC: 97% (UV at
%
210 nm). LC–MS/MS: m/z 203 (M þ 1). 1H-NMR ((CD3)2SO): d 8.96 (1H, s),
8.38 (1H, d), 8.35 (1H, d), 8.15 (1H, dd), 7.66 (1H, d), 7.54 (1H, dd).
1-[14C]-6-Chloro-8-nitro-b-carboline ð8AÞ
1-[14C]-6-Chloro-b-carboline (7A; 55 mCi, 55 mCi/mmol) and NaNO3
(2 mmol) were taken in a 25 ml round-bottom flask. Trifluoroacetic acid
(3 ml) was added at 08C under nitrogen. The mixture was stirred at room
temperature for 18 h. The solvent was evaporated. The residue was washed
with cold water and ether. The solid was dried to give the product (40 mCi,
55 mCi/mmol, 73%). HPLC: 98% (UV at 254 nm) and radiochemical purity,
99%. LC–MS/MS: m/z 250 (M þ 1), 248.
6-Chloro-8-nitro-b-carboline ð8Þ
%
6-Chloro-b-carboline (7; 0.5 mmol) was nitrated similarly to provide 0.15 g
%
(83%) product. HPLC: 98% (UV at 210 nm). LC–MS/MS: m/z 248 (M þ 1).
1H-NMR ((CD3)2SO): d 13.18 (NH, s), 9.23 (1H, s), 9.16 (1H, d), 8.77 (1H, d),
8.72 (1H, d), 8.63 (1H, d).
1-[14C]-8-Amino-6-chloro-b-carboline ð4AÞ
1-[14C]-6-Chloro-8-nitro-b-carboline (8A; 6.6 mCi, 55 mCi/mmol), 6-chloro-8-
nitro-b-carboline (0.18 mmol, 0.065 g, specific activity of diluted carboline,
22 mCi/mmol), and 5% Pt(S)/C (0.0326 g) were taken in a 25 ml round-bottom
flask. Ethanol (2.28 ml), triethylamine (1.5 mmol, 0.21 ml), and formic acid
(1.2 mmol, 0.052 ml) were added at 08C under nitrogen. The mixture was
warmed to room temperature and heated at 788C for 5 h. The reaction mixture
was cooled, diluted with methanol (23 ml), and filtered through a pad of celite.
The filtrate was basified with an aqueous solution of 1 M Na2CO3 to about pH
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF C-14-LABELED NOVEL IKK INHIBITOR
329
10. The solvents were evaporated. The residue was washed with water and
dried to give 6.2 mCi (22 mCi/mmol, 94%) product. HPLC: 97% (UV at
254 nm), radiochemical purity, 98%. LC–MS/MS: m/z 220 (M þ 1), 218.
8-Amino-6-chloro-b-carboline ð4Þ
%
Reduction of 6-chloro-8-nitro-b-carboline (8; 0.2 mmol) provided the desired
(0.434 g, 100%) product. HPLC: 94% (UV at 254 nm). LC–MS/MS: m/z 218
%
1
(M þ 1). H-NMR ((CD3)2SO): d 11.30 (NH, s), 8.94 (1H, s), 8.30 (1H, d),
8.03 (1H, d), 7.50 (1H, s), 6.76 (1H, s), 5.70 (NH2, s).
2-[14C]-N-(6-Chloro-9H-pyrido [3,4-b]indol-8-yl)-3-pyridinecarboxamide (PS-
1145) ð9AÞ
[14C]-8-Amino-6-chloro-b-carboline (4A; 6.2 mCi, 22 mCi/mmol), nicotinic
acid (0.39 mmol), and EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride, 0.849 mmol) were taken in a 25 ml round-bottom flask.
Pyridine (3 ml) was added under nitrogen. The mixture was stirred at room
temperature for 5 h. The solvent was evaporated. To the residue was added
methanol (0.2 ml) and saturated NaHCO3 (3 ml). The mixture was stirred at
room temperature for 0.5 h, then cooled in an ice bath. The solid product was
isolated by centrifugation and dried. The residue was extracted with
dichloromethane and methanol (1:1). The solution was concentrated and
chromatographed on a silica gel column using a mixture of dichloromethane
and methanol as eluants to provide the (4.6 mCi, 22 mCi/mmol, 74%) product.
HPLC: 99.5% pure (UV at 254 nm) and radiochemical purity, 99.7%.
1
LC–MS/MS: m/z 325 (M þ 1), 323. H-NMR ((CD3)2SO, 600 MHz): d 11.58
(NH, s), 10.68 (NH, s), 9.25 (1H, s), 8.97 (1H, s), 8.85 (1H, d), 8.45 (1H, d),
8.38 (1H, d), 8.30 (1H, s), 8.18 (1H, d), 7.80 (1H, s), 7.64 (1H, dd).
N-(6-Chloro-9H-pyrido [3,4-b]indol-8-yl)-3-pyridinecarboxamide (PS-1145)
ð9Þ
%
Condensation of 8-amino-6-chloro-b-carboline (4, 0.3 mmol) similarly with
nicotinic acid provided 0.0678 g (70%) product. HPLC: 99% (UV at 254 nm).
%
1
LC–MS/MS: m/z 323 (M þ 1). H-NMR ((CD3)2SO): d 11.58 (NH, s), 10.68
(NH, s), 9.25 (1H, s), 8.97 (1H, s), 8.85 (1H, d), 8.45 (1H, d), 8.38 (1H, d), 8.30
(1H, s), 8.18 (1H, d), 7.80 (1H, s), 7.64 (1H, dd).
Acknowledgements
Thanks are due to Process Research Department (Xianglin Shi) and
Inflammation Medicinal Chemistry Departments of Millennium Pharmaceu-
ticals for provision of unlabeled intermediates. Thanks are also due to Ian
Copyright # 2005 John Wiley & Sons, Ltd.
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Y. LI AND S. R. PRAKASH
Parsons of Analytical Chemistry Department for providing 600 MHz NMR
spectra of labeled compounds.
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