Synthesis and characterization of tritium labeled 2,3,4,9-tetrahydro-1H-carbazoles as potent DP receptor antagonists

Article abstract of DOI:10.1002/jlcr.803

Tritium labeled 2,3,4,9-tetrahydro-1H-carbazole 1a and 2a were prepared in good yields with a specific activity of 7.0 Ci/mmol (214GBq/mmol) and 4.2 Ci/mmol (155GBq/mmol), respectively. Both compounds have been synthesized in high radiochemical purity by

Full text of DOI:10.1002/jlcr.803

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 107–113.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.803
Research Article
Synthesis and characterization of tritium labeled
2,3,4,9-tetrahydro-1H-carbazoles as potent DP
receptor antagonists
Carl Berthelette*, Michael J. Boyd, Nicolas Lachance, Bruno Roy, Claudio F.
Sturino, John Scheigetz and Robert J. Zamboni
Department of Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research,
!
PO Box 1005, Pointe-Claire/Dorval, Quebec, Canada H9R 4P8
Summary
Tritium labeled 2,3,4,9-tetrahydro-1H-carbazole 1a and 2a were prepared in good
yields with a specific activity of 7.0 Ci/mmol (214 GBq/mmol) and 4.2 Ci/mmol
(155 GBq/mmol), respectively. Both compounds have been synthesized in high
radiochemical purity by catalytic tritium–bromine exchange of the corresponding aryl
bromide precursors. The 6-bromocarbazole precursors 7 and 8 were prepared as a
mixture by a three step process, involving regioselective bromination of 3c with
pyridinium tribromide, oxidation of thioether 4c using m-CPBA and hydrolysis of
acylsultamcarbazole 5c. Finally, HPLC separation of the enantiomers afforded the 6-
bromo precursors 7 and 8 in high diastereomeric ratio (dr 99% and dr 93%
respectively). Copyright # 2004 John Wiley & Sons, Ltd.
Key Words: tritium; labeled compounds; tritium–bromine exchange; tetrahydrocar-
bazole; DP receptor antagonists
Introduction
Radiolabeled compounds provide unique tools to dissect metabolic pathways,
identify molecular targets and determine protein binding parameters. In this
aspect, the position of the radioactive label is selected based on the molecule’s
metabolism profile. In this regard, a tritium labeled compound offers many
advantages over its carbon-14 counterpart, mainly because of its high specific
radioactivity and relative ease of preparation. During the course of our work,
compound 1c and 2c proved to be potent prostaglandin D2 (DP) receptor
antagonists with Ki values of 2.5 nM and 1.7 nM respectively (Presented at the
*Correspondence to: C. Berthelette, Department of Medicinal Chemistry, Merck Frosst Centre for
!
Therapeutic Research, PO Box 1005, Pointe-Claire/Dorval, Quebec, Canada H9R 4P8. E-mail:
carl berthelette@merck.com
Copyright # 2004 John Wiley & Sons, Ltd.
Received 28 July 2003
Revised 12 September 2003
Accepted 27 October 2003

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C. BERTHELETTE ET AL.
39th IUPAC Congress and the 86th Conference of the Canadian Society for
Chemistry, August 10–15, 2003, Ottawa, Canada). As a result, we became
interested in developing synthetic routes to prepare tritium-labeled 2,3,4,9-
tetrahydro-1H-carbazoles, and we report herein a new and efficient radio-
synthesis for such compounds.
Results and discussion
Tritium labeled compounds 1a and 2a were prepared in good yields with high
radiochemical purity from the corresponding 6-bromocarbazole precursors
(Figure 1). Attempts to directly brominate compounds 1c or 2c were
unsuccessful despite the fact that we tried a variety of bromination conditions
(Br2, NBS, pyridinium tribromide, Br2/Pyridine). To overcome this problem,
we studied the bromination of the corresponding thioether 3. Attempts at
bromination of compounds 3a (acid) and 3b (methyl ester) failed to furnish
any of the desired 6-bromocarbazole 4 (Scheme 1). However, the acylsultam-
carbazole 3c, which was originally synthesized to facilitate the HPLC
separation of compound 3 enantiomers, reacted smoothly with pyridinium
tribromide to afford the desired product 4c in quantitative yield (For
mechanistical details on indole bromination and limitations, see ref. 2).
Subsequent oxidation of the thioether 4c with m-CPBA afforded a 1:1 mixture
of sulfoxide diastereomers 5c in 80% yield. The acylsultamcarbazole
diastereomers 5c were then hydrolyzed with lithium hydroxide in a mixture
of THF and water to obtain the corresponding acid mixture 6 in 70% yield.
Separation of the diastereomers (7, 8) was then carried out using flash
chromatography and normal phase preparative HPLC.
Figure 1.
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TRITIUM LABELED PROSTAGLANDIN D2 RECEPTOR ANTAGONISTS
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Scheme 1.
Having in hand compounds 7 and 8 in high diastereomeric ratio (dr 99%
and dr 93% respectively), we next investigated the catalytic deuterium–
halogen exchange of the 6-bromocarbazole 7 (Scheme 2). Evaluation of
deuterium incorporation by mass spectrometry indicated that 76% of
compound 1b was effectively labeled with deuterium and the remaining 24%
with hydrogen. We postulated that ethanol may play an important role in
donating hydrogen to the palladium in this reaction. Further modifications of
the reaction conditions (solvent, catalyst, temperature, time) failed to improve
the level of deuterium incorporation. Selective exchange of the aromatic
bromine with tritium gas and 10% Pd/C in ethanol proceeded smoothly to
give crude 1a and 2a. After purification, the radiochemical purity was
determined to be 99 and 93% with a specific radioactivity of 7.0 and 4.2 Ci/
mmol, respectively for compounds 1a and 2a. Tritium incorporation evaluated
by mass spectrometry indicated that 24% of compound 1a was labeled with
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C. BERTHELETTE ET AL.
Scheme 2.
tritium. This result was consistent with the observed 7.0 Ci/mmol (24% of
28.8 Ci/mmol in theory) calculated by HPLC using a non-radioactive standard
and a liquid scintillation counter. Despite the fact that precursors 7 and 8
contain a chlorine atom on the benzyl substituent, this halogen reacts much
slower than bromine in this catalytic dehalogenation reaction and we observe
only trace amounts of the tritiated benzyl analogs.
Experimental
General
All reagents and solvents are commercially available and used directly without
any purification. Reactions were performed in a well ventilated fume-hood and
the tritiation was done with a Tri-Sorber instrument (TS-1000) from IN/US
systems. Tritium gas was released from the primary bed containing 100 Ci of
tritium gas stored in the form of U3H3 by heating the uranium bed to 5008C.
Evaporations were carried out on a Buchi evaporator in vacuo at bath
temperatures less than 358C. TLC was performed on Merck kGaA F254
precoated silica plates. Analytical HPLC was performed on a Waters
instrument equipped with a Zorbax C18 column (4.6 Â 150 mm) and peak
detection was done simultaneously by UV (254 nm, Waters 996 Photodiode
Array detector) and a liquid scintillation flow monitor (Packard Radiomatic
150 TR Flow Scintillation Analyzer). Tritium and deuterium incorporation
were determined on a Micromass Quattro LC (electrospray mode) coupled
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TRITIUM LABELED PROSTAGLANDIN D2 RECEPTOR ANTAGONISTS
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with a Waters 2790 HPLC and a RadioHPLC detector (b-ram model 3, IN/
US) using Flo-Scint II (Packard Bioscience #6013529) as liquid scintillation
cocktail. Radioactivity measurements were performed on a Beckman Coulter
LS6000SC liquid scintillation counter using Ultima Gold XR (Packard
Bioscience #6013119) scintillant. A Bruker 500 MHz instrument was used for
proton NMR characterization and chemical shifts are reported in ppm
downfield from TMS.
(1R)-6-bromo-9-(4-chlorobenzyl)-1-{2-[(3aR)-8,8-dimethyl-2,2-dioxidotetrahy-
dro - 3a,6-methano -2,1-benzisothiazol-1(4H)-yl]-2-oxoethyl}-8-(methylthio)-2,3,
4,9-tetrahydro-1H-carbazole (4c)
Compound 3c (500 mg, 0.84 mmol) was dissolved in 7 ml of pyridine at 08C
and pyridine tribromide (670 mg, 2.10 mmol) was added under nitrogen. The
cooling bath was removed and the reaction was stirred for 30 min.
Evaporation of the pyridine to dryness under high vacuum was done prior
to addition of 7 ml of AcOH. The reaction mixture was re-cooled to 08C and
zinc dust (220 mg, 3.4 mmol) was added. The reaction mixture was stirred
overnight. Quenching of the reaction was done by adding 10 ml of HCl 10%,
100 ml of EtOAc and stirred for 30 min. The reaction was extracted twice with
50 ml of EtOAc, washed with brine, dried over Na2SO4 and evaporated. The
1
compound 4c was obtained in 99% yield. H NMR (500 MHz, DMSO-d6) d
7.32 (d, 2H), 7.20 (d, 1H), 6.95 (d, 1H), 6.72 (d, 2H), 6.05 (d, 1H), 5.60 (d, 1H),
3.85 (m, 2H), 3.62 (d, 1H), 3.22 (m, 2H), 2.95-2.80 (m, 2H), 2.50 (m, 1H), 2.28
(s, 3H), 1.90-1.60 (m, 9H), 1.42 (t, 1H), 1.21 (m, 1H), 1.04 (s, 3H), 0.92 (s, 3H).
Mass spectrum for C32H36N2O3S2BrCl: (M-H) 675.4.
(1R)-6-bromo-9-(4-chlorobenzyl)-1-{2-[(3aR)-8,8-dimethyl-2,2-dioxidotetra-
hydro-3a,6-methano-2,1-benzisothiazol-1(4H)-yl]-2-oxoethyl}-8-(methylsulfi-
nyl)-2,3,4,9-tetrahydro-1H-carbazole (5c)
Compound 4c (360 mg, 0.53 mmol) was dissolved in 10 ml of CH2Cl2 under
nitrogen. Addition of mCPBA (114 mg, 0.56 mmol) was done at 08C and the
mixture was allowed to stir for 30 min. The reaction was then quenched by
addition of NaHCO3 sat., extracted twice with 20 ml of CH2Cl2, washed with
brine, dried over Na2SO4 and evaporated. Product 5c was obtained in 80%
yield (1:1 mixture of sulfoxide) after a quick purification through a pad of
silica gel and used directly for the next step.
[(1R)-6-bromo-9-(4-chlorobenzyl)-8-(methylsulfinyl)- 2,3,4,9-tetrahydro-1H-
carbazol-1-yl]acetic acid (6)
Hydrolysis was performed on compound 5c (150 mg, 0.22 mmol) by addition
of lithium hydroxide (260 ml, 2N soln) at 258C in a 5:1 mixture of THF and
water. The reaction was stirred overnight and then quenched by adding HCl
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C. BERTHELETTE ET AL.
2N until pH ꢀ3. The reaction mixture was extracted twice with 50 ml of
EtOAc, washed with brine, dried over Na2SO4 and evaporated. Purification of
the crude compound 6 by flash chromatography using a gradient from 0 to
100% EtOAc in toluene and 2% AcOH afforded the desired 6-bromocarba-
zole precursors 7 and 8 in 70% yield. Further purification by preparative
HPLC using mPorasil column (2 Â 25 cm) at a flow rate of 3.8 ml/min with a
mixture of solvent (20% iPrOH/80% Hexanes/0.2% AcOH) at 275 nm
afforded compound 7 (RT=6.2 min, dr 99%) and compound 8 (RT=8.1 min,
dr 93%). Mass spectrum for C22H21NO3SBrCl: (M-H) 494.3.
[(1R)-6-bromo-9-(4-chlorobenzyl)-8-((R)methylsulfinyl)-2,3,4,9-tetrahydro-
1H-carbazol-1-yl]acetic acid (7)
1H NMR (500 MHz, acetone-d6) d 11.2 (bs, 1H), 7.63 (d, 1H), 7.45 (d, 1H),
7.34 (d, 2H), 6.81 (d, 2H), 5.80 (d, 1H), 5.45 (d, 1H), 3.42 (m, 2H), 2.91 (m,
1H), 2.57 (m, 2H), 2.30 (s, 3H), 1.93-1.81 (m, 4H).
[(1R)-6-bromo-9-(4-chlorobenzyl)-8-((S)methylsulfinyl)-2,3,4,9-tetrahydro-
1H-carbazol-1-yl]acetic acid (8)
1H NMR (500 MHz, acetone-d6) d 11.2 (bs, 1H), 7.61 (d, 1H), 7.45 (d, 1H),
7.27 (d, 2H), 6.74 (d, 2H), 5.63 (d, 1H), 5.37 (d, 1H), 3.35 (m, 3H), 2.64 (d,
2H), 2.60 (s, 3H), 1.93 (m, 2H), 1.81 (m, 2H).
[(1R)-6-3H-9-(4-chlorobenzyl)-8-((R)methylsulfinyl)-2,3,4,9-tetrahydro-1H-
carbazol-1-yl]acetic acid (1a)
Pd/C (10%, 6.0 mg) was added to a solution of compound 7 (1.8 mg, 2.6 mmol)
in EtOH (0.5 ml). The reaction mixture was degassed by four freeze/
thaw cycles and then stirred overnight with tritium gas (3 Ci). Excess tritium
was re-adsorbed on the secondary uranium bed followed by filtration of the
reaction through a short pad of celite. Co-evaporation with EtOH (3 Â 3 ml) in
order to remove labile tritium was done prior to purification by HPLC.
Preparative HPLC using a Novapak C18 column (7.8 Â 300 mm) with a
mixture of solvent (82% MeOH/18% H2O/0.1% AcOH) at a flow rate of
4.5 ml/min afforded the desired compound 1a (RT=9 min) in 99% radio-
chemical purity (analytical HPLC measurements) with a specific activity of
7.0 Ci/mmol. Unlabelled (1c), used as a reference compound, had
1
the following spectroscopic data: H NMR (500 MHz, DMSO-d6) d 7.71
(d, 1H), 7.65 (d, 1H), 7.35 (d, 2H), 7.27 (t, 1H*), 6.72 (d, 2H), 5.62 (d, 1H),
5.38 (d, 1H), 2.80 (d, 1H), 2.65-2.50 (m, 1H), 2.38-2.28 (m, 2H), 2.35 (s, 3H),
1.92-1.75 (m, 4H). *denotes the aromatic proton at the 6-position of
the 2,3,4,9-tetrahydrocarbazole. Optical rotation: +121.38 (c=0.39 in
methanol).
Copyright # 2004 John Wiley & Sons, Ltd.
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TRITIUM LABELED PROSTAGLANDIN D2 RECEPTOR ANTAGONISTS
113
[(1R)-6-3H*-9-(4-chlorobenzyl)-8-((S)methylsulfinyl)-2,3,4,9-tetrahydro-1H-
carbazol-1-yl]acetic acid (2a)
Compound 2a was prepared in the same way as compound 1a. Preparative
HPLC using a Novapak C18 column with a mixture of solvent (90% MeOH/
10% H2O/0.1% AcOH) at a flow rate of 4.5 ml/min afforded the desired
compound 2a (RT=13 min) in 93% radiochemical purity (analytical HPLC
measurements) with a specific activity of 4.2 Ci/mmol. Unlabelled (2c), used as
a reference compound, had the following spectroscopic data: 1H NMR
(500 MHz, DMSO-d6) d 7.66 (d, 1H), 7.63 (d, 1H), 7.34 (d, 2H), 7.28 (t, 1H*),
6.69 (d, 2H), 5.42 (d, 1H), 5.24 (d, 1H), 3.20 (d, 1H), 2.8 (d, 1H), 2.68-2.54 (m,
2H), 2.58 (s, 3H), 2.47-2.39 (m, 1H), 1.90-1.75 (m, 4H). *denotes the aromatic
proton at the 6-position of the 2,3,4,9-tetrahydrocarbazole. Optical rotation:
À231.98 (c=0.31 in methanol).
Conclusion
In summary, we have developed an efficient synthetic pathway for the
synthesis of tritium labeled 2,3,4,9-tetrahydro-1H-carbazoles 1a and 2a by use
of a catalytic tritium–halogen exchange reaction on the corresponding 6-
bromocarbazole precursors. A three step sequence was performed to afford the
6-bromocarbazole precursors in good overall yields. Both tritiated compounds
1a and 2a were obtained in high radiochemical and diastereomeric purity as
well as in high radiospecific activity. Covalent binding assays and metabolism
studies were performed on both compounds and these results will be reported
in due course.
Acknowledgements
We would like to thank Dwight Macdonald (Tri-Sorber consultant), Stephen
Day (RadioHPLC and Scintillation counter) and Carmai Seto (MS tritium
incorporation calculation) for invaluable assistance.
References
1. Labelle M, Sturino C, Roy B. Prostaglandin D2 receptor antagonists. International
Patent WO 01/79169 A2, 2001.
2. Lachance N, Chan WY. J Heterocyclic Chem 2003; 40: 289–295 and references
cited therein.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 107–113