Synthesis of β3 adrenergic receptor agonist LY377604 and its metabolite 4-hydroxycarbazole, labeled with carbon-14 and deuterium

Article abstract of DOI:10.1002/jlcr.935

Synthesis of ¹⁴C-radiolabeled 4-hydroxycarbazole was accomplished starting from aniline-[U-¹⁴C], based on zinc chloride initiated Fischer cyclization of the phenylhydrazone prepared from phenylhydrazine-[U-¹⁴C] and cyclohexane-1,3-dione. The resulting tetrahydrooxocarbazole was subjected to dehydrogenation-aromatization using palladium on carbon. The aromatized 4-hydroxycarbazole-[4b,5,6,7,8,8a- ¹⁴C] was then used for the synthesis of ¹⁴C-labeled β₃ adrenergic receptor agonist LY377604. The introduction of four deuteria in the carbazole fragment of LY377604 accomplished by its initial bromination and subsequent catalytic deuteration of the resulting tetrabromide. A similar approach was used for the conversion of 4-hydroxycarbazole into its tetradeutero-isotopomer. Copyright

Full text of DOI:10.1002/jlcr.935

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 407–419.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.935
Research Article
Synthesis of b3 adrenergic receptor agonist LY377604
and its metabolite 4-hydroxycarbazole, labeled with
carbon-14 and deuterium
Boris A. Czeskis* and William J. Wheeler
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
Summary
Synthesis of 14C-radiolabeled 4-hydroxycarbazole was accomplished starting from
aniline-[U-14C], based on zinc chloride initiated Fischer cyclization of the
phenylhydrazone prepared from phenylhydrazine-[U-14C] and cyclohexane-1,3-dione.
The resulting tetrahydrooxocarbazole was subjected to dehydrogenation–aromatiza-
tion using palladium on carbon. The aromatized 4-hydroxycarbazole-
[4b,5,6,7,8,8a-14C] was then used for the synthesis of 14C-labeled b3 adrenergic
receptor agonist LY377604. The introduction of four deuteria in the carbazole
fragment of LY377604 accomplished by its initial bromination and subsequent
catalytic deuteration of the resulting tetrabromide. A similar approach was used for
the conversion of 4-hydroxycarbazole into its tetradeutero-isotopomer. Copyright #
2005 John Wiley & Sons, Ltd.
Key Words: 4-hydroxycarbazole; b3 adrenergic agonist; LY377604 succinate; carbon-
14-labeled; deuterium-labeled
Introduction
Compound 1, (4-hydroxycarbazole) is a key intermediate in the synthesis of a
number of physiologically active compounds1 including carvedilol2 and
carazolol.3 We were interested in the preparation of 14C-labeled 4-hydro-
xycarbazole to be used in the synthesis of radiolabeled b3 adrenergic agonist
LY377604, a compound with a potential for the treatment of obesity and
diabetes4 (Figure 1). Stable labeled isotopomers of LY377604 and its putative
metabolite 4-hydroxycarbazole ð1Þ were also required as internal standards for
bioanalytical studies.
*Correspondence to: B. A. Czeskis, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis,
IN 46285, USA. E-mail: czeskis@lilly.com
Copyright # 2005 John Wiley & Sons, Ltd.
Received 5 January 2005
Revised 19 January 2005
Accepted 31 January 2005

408
B. A. CZESKIS AND W. J. WHEELER
OH
OH
O
H
N
HN
O
NH
2
N
H
O
N
LY377604
1
Figure 1.
We describe herein the syntheses of radiolabeled and deuterium-labeled
LY377604 and 4-hydroxycarbazole.
Results and discussion
Synthesis of 14C-labeled compounds
There are few methods for the synthesis of 4-hydroxycarbazole ð1Þ presented in
the literature, including the coupling of 2-bromonitrobenzene with 2-
iodoanisole in the presence of copper,5 bacterial oxygenation of heteroaro-
matic precursors,6 and cyclocarbonylation of indole derivatives.7 However, the
most convenient approach for the preparation of 4-hydroxycarbazole seems to
be the classical Fischer indole synthesis.3a,8 Some challenges and findings
during the application of this method for the synthesis of 14C-labeled 4-
hydroxycarbazole, and following preparation of LY377604-[14C] are presented
below.
The synthesis of 14C-labeled 4-hydroxycarbazole (1a) is shown in Scheme 1.
Diazotization of aniline-[U-14C] hydrochloride (2) and subsequent reduction
of the resulting diazonium salt smoothly gave 14C-labeled phenylhydrazine
hydrochloride (3) when tin (II) chloride was used as a reducing agent.9 Use of
sodium sulfite10 resulted in significantly lower yield of 3. The condensation of
hydrochloride 3 with cyclohexane-1,3-dione in the presence of acetic acid11 or
in aqueous media was not successful suggesting that the generation of a free
base from 3 was necessary. Indeed, when phenylhydrazine obtained by the
alkaline treatment of hydrochloride 3, was reacted with cyclohexane-1,3-
dione, the hydrazone 4 was obtained in satisfactory yield. Initial attempt at the
cyclization of the phenylhydrazone 4 using sulfuric acid11,12 gave just trace
quantities of the desired tetrahydrooxocarbazole 5. A somewhat better result
was obtained using 85% phosphoric acid. A considerably higher yield of 5 was
achieved when zinc chloride in acetic acid3a,8a was used to initiate the
cyclization. The last step in the preparation of 4-hydroxycarbazole-[14C] (1a)
was dehydrogenation–aromatization of the tetrahydroprecursor 5. We
investigated three reagents known from the literature for achieving such a
goal: 2,3-dichloro-1,3-dicyano-1,4-benzoquinone (DDQ),13 Raney nickel,3a,8a
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF b3 ADRENERGIC RECEPTOR
409
1. NaNO ,
aq. HCl
O
2
1. NaOH
*
*
2. SnCl ,
2
aq. HCl
NH HCl
NHNH HCl
2
O
O
2
*
N
3
2
N
H
2.
4
O
OH
ZnCl
10%Pd/C
2
*
*
AcOH
Triglyme
N
H
N
H
5
1a
* = 14C
Scheme 1.
and palladium on carbon.14 In our hands the best, although not very
reproducible, results were achieved using 10% palladium on carbon in
triglyme at 200–2208C. In model experiments non-radiolabeled 4-hydroxy-
carbazole was obtained in up to 90% yields, whereas the dehydrogenation of
14C-labeled compound 5 resulted in poor yields of 1a (10 and 35% after two
attempts, correspondingly).
The synthesis of LY377604-[14C] succinate from 4-hydroxycarbazole-[14C]
(1a) was accomplished based on the previously developed procedure.4 Thus,
reaction of 1a with (S)-glycidyl nosylate (6) in the presence of potassium
carbonate gave (S)-glycidyloxycarbazole (7). The epoxide 7 was coupled with
amine 8 to afford the target radiolabeled compound LY377604-[14C], and
subsequently its succinate (Scheme 2).
The method described above allowed us to obtain 4-hydroxycarbazole-
[4b,5,6,7,8,8a-14C] (5) and then convert it into LY377604-[14C] succinate with
97.5% radiochemical purity in six steps from commercially available aniline-
[U-14C] hydrochloride.
Synthesis of deuterium-labeled compounds
In order for an isotopomer to be a good internal standard in mass
spectrometry it should have a mass of at least four mass units higher than
the parent compound. Our goal was to introduce four deuteria into the
molecule of LY377604 through initial aromatic bromination of this
compound, followed by catalytic reduction of the resulting polybromide with
deuterium. The known examples of halogenation of structurally similar
compounds include carvedilol bromination on microscale using bromine and
potassium carbonate in chloroform to give tribromide isolated by preparative
HPLC,15 and the bromination of carbazole itself with N-bromosuccinimide on
silica gel to provide dibromide as a main product.16
Copyright # 2005 John Wiley & Sons, Ltd.
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410
B. A. CZESKIS AND W. J. WHEELER
O
H N
2
NH
O
2
*
OH
NsO
O
N
O
1.
HN
O
*
6
8
K CO , Me CO
2
3
2
N
H
2. (HOOCCH )
2 2
7
1a
*
(CH COOH)
2
2
OH
O
H
N
HN
O
NH
2
N
O
LY377604-[14C] Succinate
Scheme 2.
In an attempt to reach a high level of bromine incorporation in the molecule
of LY377604, we investigated its bromination with excess of bromine under
acidic conditions. When hydrochloride salt of LY377604 was treated with four
equivalents of bromine at room temperature the reaction provided mainly
1,3,6-tribromosubstituted carbazole derivative 9a. It took almost 10 equiva-
lents of bromine to push the reaction further to form 1,3,6,8-tetrabromo-
derivative 9b (Scheme 3). Surprisingly, the bromination occurred exclusively
onto the carbazole fragment even though two other aromatic rings were
present in the molecule.
The next step, catalytic reduction of tetrabromide 9b, was performed using
atmospheric pressure of deuterium in the presence of palladium on carbon and
triethylamine in dimethylformamide. According to mass spectral data (ES+),
the reaction provided a mixture of D4, D3, D2, and D1 isotopomers of
LY377604 in a ratio 7:15:4:1. Although the reason for the isotopic exchange
remains unclear, the obvious approach to overcome this problem was the
elimination of any possible proton donors. Generation of free base (9c) from
the hydrobromide salt 9b and following its re-evaporation with methanol-d4
would exclude internal proton source (HBr, NH, NH2); however deuteration
of the resulting material did not provide better results. The ratio of D4, D3, D2,
D1 isotopomers was 3:9:4:1. Considerable improvement in the isotopic ratio
was achieved when we turned our attention to the catalyst as a possible
external proton source. To remove hydrogen, possibly contained in the
palladium on carbon, the catalyst was pre-activated by triple evacuating and
re-filling with deuterium. As a result, the deuteration reaction gave D4, D3, D2
isotopomers in a ratio 46:9:1, and no D1 isotopomer was detected.
Surprisingly, changing the solvent from dimethylformamide to methanol-d4
led to the formation of D5-compound 10 resulting from the reductive cleavage
O-aromatic bond, an unprecedented reaction to the best of our knowledge.
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF b3 ADRENERGIC RECEPTOR
411
HCl
OH
O
H
N
HN
O
NH
2
O
N
LY377604-[14C] Hydrochloride
Br2,
AcOH
Br
X
HBr
OH
O
H
N
HN
O
NH
2
N
O
Br
Br
9a: X = H
9b: X = Br
D2, Pd/C
Et3N
D
D
OH
O
H
N
HN
D
O
D
NH
2
O
N
LY377604-[d4]
D
D
D2, Pd/C
CD3OD
9b
OH
H
N
HN
D
O
D
D
10
Scheme 3.
Copyright # 2005 John Wiley & Sons, Ltd.
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412
B. A. CZESKIS AND W. J. WHEELER
Br
D
OH
OH
OH
1. D , Pd/C
2
Br
D
Br
2
CD OD/DMF
3
AcOH
Br
D
N
H
N
N
H
2. Et N
3
H
Br
D
1
11
1b
Scheme 4.
The bromination of 4-hydroxycarbazole (1) was performed under condi-
tions similar to those described above for LY377604 (Scheme 4). The resulting
1,3,6,8-tetrabromo-4-hydroxycarbazole (11) appeared to be very unstable
under the basic conditions required for the catalytic reduction. The addition of
triethylamine to a solution of 11 caused formation of a dark mixture, and no
desired product was detected after the deuteration. We found that the reaction
could be accomplished successfully if triethylamine was added after a few
hours of deuteration in the presence of 10% palladium on carbon. The best
isotopic ratio of the resulting product 1b (D4/D3/D2, 6:4:1) was achieved when
the mixture of methanol-d4 and dimethylformamide was used as a solvent. In
dimethylformamide itself this ratio was 2.7:2.4:1.
Experimental
The aniline-[U-14C] hydrochloride was purchased from American Radiola-
beled Chemicals, Inc. The NMR spectra were obtained on a General Electric
QE-300 at 300 (1H) and 75 (13C) MHz, and on Varian mercury-400 at 400 (1H)
and 100 (13C) MHz. Chemical shifts are reported in parts per million (ppm)
downfield from tetramethylsilane. Mass spectra were obtained on Agilent 1100
spectrometer. Microanalytical, IR, and UV data were provided by Physical
Chemistry Department of Lilly Research Laboratories. Flash chromatography
was performed using silica gel 60 (230–400 mesh) or Biotage Flash System.
TLC was performed on precoated plates of silica gel 60 F254. HPLC was
conducted on a Hitachi instrument with UV detection at 220 nm; a Zorbax C8
column (4.6 mm ꢀ 25 cm); isocratic elution with a mobile phase consisting of
aqueous 0.5% monobasic ammonium phosphate and acetonitrile with ratio
65:35 (conditions A), or 55:45 (conditions B), or 45:55 (conditions C), or UV
detection at 230 nm; a Zorbax RX-C8 column (4.6 mm ꢀ 25 cm); gradient
elution (time, ratio 0.013 N trifluoroacetic acid to acetonitrile): 0 min, 60:40;
6 min, 60:40; 7 min, 50:50; 20 min, 50:50; 20.1 min, 60:40; 30 min, 60:40
(conditions D) at a flow rate of 1 ml/min.
Phenylhydrazine-[U-14C] hydrochloride, 3
To a solution of aniline-[U-14C] hydrochloride ð2Þ (55.3 mCi/mmol, 250 mCi,
4.52 mmol) and aniline hydrochloride (710 mg, 5.48 mmol) in water (3 ml) and
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF b3 ADRENERGIC RECEPTOR
413
concentrated hydrochloric acid (3 ml) at 0–58C (ice bath) was added a solu-
tion of sodium nitrite (800 mg, 11.6 mmol) in water (2.3 ml) dropwise
over the period of 15 min. The reaction mixture was stirred for 1 h, and a
solution of tin(II) chloride (4.0 g, 21.1 mmol) in conc. hydrochloric acid
(5 ml) was added dropwise. After 2 h a bulky precipitate was collected, washed
with water (5 ml), ethanol (3 ml), ethyl ether (10 ml), and dried under vacuum
to give hydrochloride
3 (1.165 g, 80%) as a reddish solid. For
1
the non-radioactive compound (prepared in a model experiment): H-NMR
(DMSO-d6 +D2O, d, ppm): 6.99 (br. d, J=7 Hz, 3H), 7.30 (t, J=7 Hz,
2H).
Cyclohexane-1,3-dione monophenylhydrazone-[phenyl-14C], 4
To a suspension of hydrochloride 3 (1.165 g, 7.98 mmol) in water (12 ml) was
added aqueous sodium hydroxide (5 N, 1.65 ml, 8.25 mmol). The resulting
mixture was added to a solution of cyclohexane-1,3-dione (900 mg, 8.0 mmol)
in water (12 ml) dropwise over the period of 35 min. The reaction mixture was
stirred for 2 h at room temperature. The precipitate was filtered off and dried
under vacuum at 358C to give hydrazone 4 (666 mg, 41%) as a brown solid.
TLC: Rf=0.41 (ethyl acetate/ethanol, 9:1). HPLC (conditions A): Rt=6 min.
The compound co-eluted with an authentic sample of cyclohexane-1,3-dione
monophenylhydrazone by HPLC and TLC under the above conditions. For
1
the non-radioactive compound (prepared in a model experiment): H-NMR
(CDCl3, d, ppm): 2.0 (m, 2H), 2.3–2.7 (m, 6H), 6.80 (d, J=7 Hz, 2H), 6.95 (t,
J=7 Hz, 1H), 7.22 (d, J=7 Hz, 2H).
1,2,3,4-Tetrahydro-9H-carbazol-4-one-[4b,5,6,7,8,8a-14C], 5
To a solution of zinc chloride (3.29 g, 24.1 mmol) in acetic acid (3.3 ml) at 708C
(bath) was added hydrazone 4 (666 mg, 3.26 mmol) in one portion. The
reaction mixture was heated at 1058C (bath) for 4.5 h, then cooled to 708C and
diluted with water (3.7 ml). After further cooling to room temperature, the
reaction mixture was poured into water (16 ml). The precipitate was collected
by filtration, washed with water (5 ml), and dried under vacuum to give ketone
5 (317 mg, 52%) as a dark solid. TLC: Rf=0.79 (ethyl acetate/ethanol, 9:1),
0.21 (hexane/ethyl acetate, 1:1). HPLC (conditions A): Rt=8 min, HPLC
(conditions B): Rt=5 min. The compound co-eluted with an authentic sample
of 1,2,3,4-tetrahydro-9H-carbazol-4-one by HPLC and TLC under the above
conditions. For the non-radioactive compound (prepared in a model
1
experiment): H-NMR (CDCl3, d, ppm): 2.13 (t, J=4 Hz, 2H), 2.50 (m,
2H), 2.97 (t, J=6 Hz, 2H), 7.16 (m, 2H), 7.39 (d, J=7 Hz, 1H), 7.95 (d,
J=7 Hz, 2H).
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B. A. CZESKIS AND W. J. WHEELER
4-hydroxy-9H-carbazole-[4b,5,6,7,8,8a-14C], 1a
A mixture of ketone 5 (317 mg, 1.7 mmol), 10% palladium on carbon (150 mg)
and 1-dodecene (286 mg, 1.7 mmol) in triglyme (6.7 ml) was evacuated under
vacuum and refilled with argon twice. The reaction mixture was stirred at
2208C (bath) for 21 h. HPLC showed that the ratio of starting material to
product was 10:1. The mixture was filtered through Hyflo super cel. To the
filtrate was added 10% palladium on carbon (125 mg), and the suspension was
evacuated under vacuum and refilled with argon twice. The reaction mixture
was heated at 2208C (bath) for additional 13 h. HPLC showed that the
reaction was still incomplete. The mixture was filtered, fresh catalyst (100 mg)
was added as before, and the heating was continued for 11 h. Most of the
triglyme was evaporated under vacuum. The residue was diluted with ethyl
acetate (50 ml), filtered, concentrated, and subjected to flash chromatography
(eluting with hexane/ethyl acetate, 7:3) to obtain hydroxycarbazole 5 (32 mg,
10%) as light-yellow solid. Subsequent elution with ethyl acetate gave starting
material (129 mg). For hydroxycarbazole 1a, TLC: Rf=0.39 (hexane/ethyl
acetate, 7:3). HPLC (conditions B): Rt=8 min. The compound co-eluted with
an authentic sample of 4-hydroxy-9H-carbazole by HPLC and TLC under the
above conditions. For the non-radioactive compound (prepared in a model
1
experiment): H-NMR (CDCl3, d, ppm): 5.39 (br. s, 1H), 6.63 (d, J=8 Hz,
1H), 7.06 (d, J=8 Hz, 1H), 7.30 (m, 2H), 7.45 (d, J=3.7 Hz, 2H), 8.08 (br. s,
1H), 8.33 (d, J=8 Hz, 1H).
In a separate experiment, starting from ketone 5 (410 mg, 2.19 mmol), 10%
palladium on carbon (205 mg and then 120 mg) and 1-dodecene (369 mg,
2.19 mmol) in triglyme (8.6 ml), hydroxycarbazole 1a (142 mg, 35%) was prepared.
(S)-3-(9H-carbazol-4-yloxy)-1,2-epoxypropane-[carbazole-4b,5,6,7,8,8a-14C], 7
A mixture of hydroxycarbazole 5 (32 mg, 0.173 mmol), (S)-glycidyl 3-
nitrobenzenesulfonate (6) (51 mg, 0.197 mmol) and potassium carbonate
(72 mg, 0.521 mmol) in acetone (1.5 ml) was refluxed for 9 h, and evaporated
under vacuum. The residue was diluted with a warm solution of non-
radioactive (S)-3-(9H-carbazol-4-yloxy)-1,2-epoxypropane (25 mg) in dichlor-
omethane (0.5 ml). Flash chromatography of the resulting mixture (eluting
with hexane/ethyl acetate, 7:3) gave epoxide 7 (59 mg, 82%) as a pale solid.
TLC: Rf=0.39 (hexane/ethyl acetate, 7:3). HPLC (conditions C): Rt=9 min.
The compound co-eluted with an authentic sample of (S)-3-(9H-carbazol-4-
yloxy)-1,2-epoxypropane by HPLC and TLC under the above conditions. For
1
the non-radioactive compound (prepared in a model experiment): H-NMR
(CDCl3, d, ppm): 2.9–3.1 (m, 2H), 3.62 (m, 1H), 4.32 (dd, J=11.0 and 5.4 Hz,
1H), 4.52 (dd, J=11.0 and 3.3 Hz, 1H), 6.71 (d, J=8 Hz, 1H), 7.12 (d,
J=8 Hz, 1H), 7.3–7.5 (m, 4H), 8.12 (br. s, 1H), 8.39 (d, J=8 Hz, 1H).
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF b3 ADRENERGIC RECEPTOR
415
(S)-6-[4-[2-[3-(9H-carbazol-4-yloxy)-2-hydroxypropylamino]-2-methylpropyl]
phenoxy]pyridine-3-carboxamide-[carbazole-4b,5,6,7,8,8a-14C], LY377604-[14C]
A mixture of epoxide 7 (59 mg, 0.245 mmol) and 6-[4-(2-amino-2-methylpro-
pyl)-phenoxy]-pyridine-3-carboxamide (8) (140 mg, 0.49 mmol) in 2-propanol
(3 ml) was heated at 75–808C (bath) for 18 h, and evaporated under vacuum.
Flash chromatography of the residue was performed on a column 25 ꢀ 2 cm,
eluting with one column volume each of hexane/ethyl acetate (1:1) and ethyl
acetate. Elution was continued in a gradient fashion starting with chloroform/
methanol (95:5) and progressing successively to chloroform/methanol (90:10),
chloroform/methanol/ammonium hydroxide (90:10:1), chloroform/methanol/
ammonium hydroxide (85:15:1.5), and finally chloroform/methanol/ammo-
nium hydroxide (80:20:2) yielding LY377604-[14C] (115 mg, 89%) as a white
solid. HPLC (conditions C): Rt=6 min. The compound co-eluted with an
authentic sample of LY377604 by HPLC under the above conditions. For the
non-radioactive compound (prepared in a model experiment): 1H-NMR
(DMSO-d6, d, ppm): 1.05 (s, 6H), 2.70 (s, 2H), 2.9–3.1 (m, 2H), 3.35 (s, 2H),
4.1–4.3 (m, 3H), 6.70 (d, J=8 Hz, 1H), 6.9–7.1 (m, 5H), 7.2–7.4 (m, 4H), 7.45 (d,
J=8 Hz, 1H), 7.51 (s, 1H), 8.05 (br. s, 1H), 8.25 (d, J=8 Hz, 2H), 8.63 (s, 1H).
(S)-6-[4-[2-[3-(9H-carbazol-4-yloxy)-2-hydroxypropylamino]-2-methylpropyl]
phenoxy]pyridine-3-carboxamide-[carbazole-4b,5,6,7,8,8a-14C] succinate, LY
377604-[14C] succinate
To a suspension of LY377604-[14C] (115 mg, 0.219 mmol) in ethyl acetate
(0.8 ml) and ethanol (0.2 ml) at 50–608C (bath) was added a solution of
succinic acid (13 mg, 0.11 mmol) in ethanol (0.35 ml). The reaction mixture was
stirred at 808C (bath) for 30 min, and evaporated under vacuum. The residue
was triturated with ethyl ether, and the resulting solid was collected by
filtration and dried under vacuum to give LY377604-[14C] succinate (117 mg,
91%) as a white solid. Radiochemical purity: 97.5% (radio-HPLC). Specific
activity: 22.8 mCi/mg. The compound co-eluted with an authentic sample of
LY377604 succinate by HPLC under the above conditions. For the non-
1
radioactive compound (prepared in a model experiment): H-NMR (DMSO-
d6, d, ppm): 1.09 (s, 6H), 2.35 (s, 2H), 2.77 (s, 2H), 2.9–3.2 (m, 2H), 3.35 (s,
2H), 4.1–4.3 (m, 3H), 6.71 (d, J=8 Hz, 1H), 6.9–7.1 (m, 5H), 7.2–7.4 (m, 4H),
7.45 (d, J=8 Hz, 1H), 7.51 (s, 1H), 8.05 (br. s, 1H), 8.25 (br. d, J=8 Hz, 2H),
8.63 (s, 1H).
(S)-6-[4-[2-[3-(9H-1,3,6,8-tetrabromocarbazol-4-yloxy)-2-hydroxypropyla-
mino]-2-methylpropyl]phenoxy]pyridine-3-carboxamide hydrobromide, 9b
To a solution of LY377604 hydrochloride (561 mg, 1.0 mmol) in acetic acid
(25 ml) was added bromine (500 ml, 9.7 mmol) dropwise. The reaction mixture
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B. A. CZESKIS AND W. J. WHEELER
was stirred at room temperature for 20 h and evaporated under vacuum. The
residue was re-evaporated with toluene (2 ꢀ 3 ml) to leave crude hydrobromide
1
9b (1.4 g). HPLC (conditions D): Rt=14 min. H-NMR (CD3OD, d, ppm):
1.50 (s, 3H), 1.51 (s, 3H), 3.15 (AB q, JAB=13.6 Hz, 2H), 3.49 (dd, J=12.3
and 8.8 Hz, 1H), 3.59 (dd, J=12.3 and 3.5 Hz, 1H), 4.23 (dd, J=9.7 and
4.0 Hz, 1H), 4.37 (dd, J=9.7 and 4.8 Hz, 1H), 4.49 (m, 1H), 7.08 (d, J=7.9 Hz,
1H), 7.10 (d, J=7.5 Hz, 2H), 7.41 (d, J=7.5 Hz, 2H), 7.86 (d, J=1.4 Hz, 1H),
7.88 (s, 1H), 8.34 (dt, J=7.9 and 2.6 Hz, 1H), 8.63 (t, J=2.6 Hz, 1H), 8.71 (d,
J=1.4 Hz, 1H). IR (KBr, n, cmꢁ1): 595, 738, 764, 851, 1016, 1155, 1191, 1294,
1415, 1451, 1502, 1638, 1676, 1709, 3170, 3390. UV (EtOH, lmax, nm): 296
(e 11842), 252 (e 45217).
(S)-6-[4-[2-[3-(9H-1,3,6,8-tetrabromocarbazol-4-yloxy)-2-hydroxypropylamino]
-2-methylpropyl]phenoxy]pyridine-3-carboxamide, 9c
To a solution of hydrobromide 9b (350 mg) in methanol (5 ml) was added 10%
aqueous potassium carbonate (4 ml). The reaction mixture was stirred for
10 min, and extracted with dichloromethane (20 ml). The extract was washed
with brine (2 ml), dried over sodium sulfate, and evaporated under vacuum.
Biotage chromatography of the residue (column 40S, eluting with dichlor-
omethane/methanol/ammonium hydroxide, 92:8:0.8) gave 9c (111 mg) as a
colorless solid. TLC: Rf=0.42 (dichloromethane/methanol/ammonium hydro-
1
xide, 90:10:1). HPLC (conditions D): Rt=14 min. H-NMR (CD3OD, d,
ppm): 1.23 (s, 3H), 1.25 (s, 3H), 2.88 (AB q, JAB=13.2 Hz, 2H), 3.11 (m, 2H),
4.22 (m, 2H), 4.29 (m, 1H), 6.99 (d, J=8.8 Hz, 1H), 7.05 (d, J=8.3 Hz, 2H),
7.35 (d, J=8.3 Hz, 2H), 7.83 (d, J=1.4 Hz, 1H), 7.85 (s, 1H), 8.27 (dd, J=8.8
and 2.4 Hz, 1H), 8.64 (d, J=2.4 Hz, 1H), 8.68 (d, J=1.4 Hz, 1H). IR (KBr, n,
cmꢁ1): 541, 741, 851, 1071, 1165, 1202, 1257, 1373, 1415, 1451, 1480, 1594,
1668, 3439. UV (EtOH, lmax, nm): 295 (e 14759), 252 (e 57052), 224 (e 53653).
Analysis calculated for C31H28Br4N4O4: C, 44.31; H, 3.36; N, 6.67. Found: C,
44.13; H, 3.40; N, 6.31.
(S)-6-[4-[2-[3-(9H-1,3,6,8-tetradeuteriocarbazol-4-yloxy)-2-hydroxypropyla-
mino]-2-methylpropyl]phenoxy]pyridine-3-carboxamide, LY377604-[d4]
A flask containing 10% palladium on carbon (20 mg) was placed under
vacuum and refilled with deuterium 3 times. To the resulting catalyst
suspended in dimethylformamide (0.5 ml), a solution of tetrabromide 9b
(150 mg) and triethylamine (300 ml, 2.15 mmol) in dimethylformamide (3.5 ml)
was added. The reaction mixture was placed under vacuum and refilled with
deuterium 3 times, and then vigorously stirred under balloon pressure of
deuterium for 48 h. The catalyst was filtered off, rinsed with ethyl acetate
(10 ml). The filtrate was washed with water (2 ml), and brine (2 ml), dried over
sodium sulfate, and evaporated under vacuum. Biotage chromatography of
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF b3 ADRENERGIC RECEPTOR
417
the residue (column 12 M, eluting with dichloromethane/methanol/ammonium
hydroxide, 92:8:0.8) gave LY377604-[d4] (45 mg, 80% over two steps) as a
white solid. TLC: Rf=0.33 (dichloromethane/methanol/ammonium hydro-
xide, 90:10:1). HPLC (conditions D): Rt=6 min. 1H-NMR (CD3OD, d, ppm):
1.17 (s, 3H), 1.18 (s, 3H), 2.88 (AB q, JAB=13.2 Hz, 2H), 3.06 (m, 1H), 3.18
(m, 1H), 4.28 (m, 2H), 4.33 (m, 1H), 6.95 (d, J=8.5 Hz, 3H), 7.29 (d,
J=8.5 Hz, 2H), 7.30 (s, 1H), 7.33 (s, 1H), 8.26 (dd, J=8.5 and 2.5 Hz, 1H),
8.33 (s, 1H), 8.65 (d, J=2.5 Hz, 1H). IR (KBr, n, cmꢁ1): 594, 886, 1097, 1203,
1258, 1281, 1373, 1418, 1482, 1595, 1668, 3409. UV (EtOH, lmax, nm): 319 (e
6647), 285 (e 19735), 242 (e 59507). MS (ES+, m/z, %): 529 (100, M+1), 528
(19), 527 (2). HRMS (AP+): calculated for C31H28D4N4O4: 529.2749. Found:
529.2723. The compound co-eluted with an authentic sample of non-labeled
LY377604 by TLC and HPLC under the above conditions.
(S)-4-[2-hydroxy-3-[3-(4-deuteriophenyl)-2-methylprop-2-ylamino]propyloxy]
-9H-1,3,6,8-tetradeuteriocarbazole, 10
A flask containing 10% palladium on carbon (20 mg) was placed under
vacuum and refilled with deuterium 3 times. To the resulting catalyst
suspended in methanol-D4 (0.5 ml), a solution of tetrabromide 9b (150 mg)
in methanol-d4 (2.5 ml), and triethylamine (300 ml, 2.15 mmol) were succes-
sively added. The reaction mixture was placed under vacuum and refilled with
deuterium 3 times, and then vigorously stirred under balloon pressure of
deuterium for 16 h. The catalyst was filtered off, and rinsed with ethyl acetate
(10 ml). The filtrate was evaporated under vacuum. The residue was diluted
with ethyl acetate, washed with water (2 ml), and brine (2 ml), dried over
sodium sulfate, and evaporated under vacuum. Biotage chromatography of
the residue (column 12 M, eluting with dichloromethane/methanol/ammonium
hydroxide, 93:7:0.7) gave 10 (33 mg, 78% over two steps). TLC: Rf=0.50
(dichloromethane/methanol/ammonium hydroxide, 90:10:1). HPLC (condi-
1
tions D): Rt=11 min. H-NMR (CD3OD, d, ppm): 1.13 (s, 3H), 1.14 (s, 3H),
2.80 (AB q, JAB=12.3 Hz, 2H), 3.03 (m, 1H), 3.13 (m, 1H), 4.26 (m, 2H), 4.29
(m, 1H), 7.23 (s, 4H), 7.32 (s, 1H), 7.33 (s, 1H), 8.32 (s, 1H). MS (ES+, m/z,
%): 393 (100, M+1).
9H-4-hydroxy-1,3,6,8-tetrabromocarbazole, 11
To a solution of 4-hydroxycarbazole (1) (183 mg, 1.0 mmol) in acetic acid (15 ml)
was added bromine (420 ml, 8.15 mmol) dropwise. The reaction mixture was
stirred at room temperature for 20 h and evaporated under vacuum to give crude
bromide 11 (485 mg, 97%) as a grayish solid. 1H-NMR (CD3OD, d, ppm): 7.70
(s, 1H), 7.76 (d, J=1.8 Hz, 1H), 8.39 (d, J=1.8 Hz, 1H). IR (KBr, n, cmꢁ1): 536,
735, 848, 1053, 1198, 1241, 1267, 1282, 1327, 1419, 1457, 1480, 1556, 1603, 1630,
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418
B. A. CZESKIS AND W. J. WHEELER
3443, 3492. UV (EtOH, lmax, nm): 291, 252, 232. Analysis calculated for
C12H5Br4NO: C, 28.89; H, 1.00; N, 2.81. Found: C, 29.45; H, 1.09; N, 2.69.
9H-4-hydroxy-1,3,6,8-tetradeuteriocarbazole, 1b
To a solution of bromide 11 (140 mg, 0.28 mmol) in methanol-d4 (1.5 ml) and
dimethylformamide (1.0 ml) was added a suspension of 10% palladium on
carbon (30 mg) in dimethylformamide (0.5 ml). The reaction mixture was
placed under vacuum and refilled with deuterium 3 times, and then vigorously
stirred under balloon pressure of deuterium for 2.5 h. Triethylamine (300 ml,
2.15 mmol) was added dropwise. The resulting mixture was placed under
vacuum and refilled with deuterium 3 times, and then vigorously stirred under
balloon pressure of deuterium for 4 h. The catalyst was filtered off, rinsed with
ethyl acetate (10 ml). The filtrate was washed with water (1 ml), and brine
(1 ml), dried over sodium sulfate, and evaporated under vacuum. Biotage
chromatography of the residue (column 12 M, eluting with hexane/ethyl
acetate, 75:25) gave 1b (43 mg, 82%) as a white solid. TLC: Rf=0.34 (hexane/
ethyl acetate, 70:30). NMR (CD3OD, d, ppm): 7.17 (s, 1H), 7.31 (s, 1H), 8.24
(s, 1H). IR (KBr, n, cmꢁ1): 462, 597, 756, 806, 902, 974, 1038, 1172, 1203, 1258,
1303, 1337, 1420, 1477, 1576, 1601, 1631, 3223, 3399. UV (EtOH, lmax, nm):
333, 284, 244, 224. MS (ES+, m/z, %): 188 (100, M+1), 187 (66), 186 (16).
Analysis calculated for C12H9NO: C, 76.97; H, 4.85; N, 7.48. Found: C, 76.33;
H, 5.09; N, 7.39.
Acknowledgements
The authors are grateful to Mary Peters and Jack Fisher of Chemical Process
Research and Development for providing non-radioactive samples of 7, 8, and
LY377604.
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