An improved synthesis of isotope labeled 6-hydroxychlorzoxazone

Article abstract of DOI:10.1002/jlcr.1113

[¹³C₆]6-Hydroxychlorzoxazone, a major P450 metabolite of chlorzoxazone, was synthesized from [¹³C₆]benzene in an overall 18% yield. An improved procedure for converting 4-chloro-6- nitrosoresorcinol to 6-hydroxy

Full text of DOI:10.1002/jlcr.1113

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd. Radiopharm 2006; 49: 979–984.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1113
Research Article
An improved synthesis of isotope labeled
6-hydroxychlorzoxazone
Zhigang Jian*, Tapan Ray, Amy Wu and Lawrence Jones
Isotope Chemistry, Early Development, Novartis Pharmaceuticals, East Hanover,
NJ 07936, USA
Summary
[13C6]6-Hydroxychlorzoxazone, a major P450 metabolite of chlorzoxazone, was
synthesized from [13C6]benzene in an overall 18% yield. An improved procedure for
converting 4-chloro-6-nitrosoresorcinol to 6-hydroxychlorzoxazone was developed,
with a yield more than double that previously reported in the literature. Copyright #
2006 John Wiley & Sons, Ltd.
Received 14 June 2006; Revised 29 June 2006; Accepted 30 June 2006
Key Words: 6-hydroxychlorzoxazone; 4-chloro-6-nitrosoresorcinol; chlorzoxazone;
stable isotope
Introduction
P450 enzymes are a group of mixed function monooxidases involved in the
oxidative metabolism of many different compounds, including drugs,
environmental pollutants, steroids, prostaglandins, and fatty acids. Chlorzox-
azone, a compound used therapeutically as a central acting muscle relaxant,
was found to be oxidized to 6-hydroxychlorzoxazone in human liver
microsomes.1 Our own research at Novartis in this field created the need for
stable isotope labeled 6-hydroxychlorzoxazone with a molecular weight at
least 4 mass unit higher than the non-labeled compound.
A quick literature search revealed that synthesis of both of the non-labeled
and the stable isotope labeled 6-hydroxychlorzoxazone have been reported.
The synthesis of C-13 labeled 6-hydroxychlorzoxazone was accomplished
through a 5-step preparation starting from C-13 labeled phenol. Unfortu-
nately, the reported yield was only less than 2%, a result caused by the very
*Correspondence to: Z. Jian, Isotope Chemistry, Early Development, Novartis Pharmaceuticals, B435/
R3149, One Health Plaza, East Hanover, NJ 07936, USA. E-mail: zhigang.jian@novartis.com
Copyright # 2006 John Wiley & Sons, Ltd.

980
Z. JIAN ET AL.
HO
OH
HO
OH
NO
O
HO
Cl
Phosgene/NaOAc
Pd/C/H2, EtOAc
O
N
H
NH2
Cl
Cl
Figure 1. Synthesis of 6-hydroxychlorzoxazone
13C
HO 13C
13C 13C
OH
HO 13C
OH
13C 13C
1. Fuming H2SO4
2. NaOH
13C 13C
DMD/HCl
KOH/Isoamyl Nitrite
13C 13C
13C 13C
13C 13C
13C
13C
13C
Cl
1
3
2
HO 13C
OH
HO 13C
OH
13C
13C 13C
13C 13C
O
HO
Triphosgene/Et3N
13C 13C
Pd/C/H2, THF
O
13C 13C
13C 13C
13C
13C 13C
13C
NH2
13C
Cl
N
H
NO
Cl
Cl
4
6
5
Figure 2. Synthesis of [13C]-labeled 6-hydroxychlorzoxazone
low yield (7%) in the key step, the enzymatic hydroxylation of chorzoxazone.2
Synthesis of non-labeled 6-hydroxychlorzoxazone was achieved by converting
4-chloro-6-nitrosoresorcinol to 6-hydroxychlorzoxazone (Figure 1).1,3 The
reported yields for this synthesis were quite different: while one author claimed
32%,3 the other one only reported 16%,1 both on multi-gram scales. Since we
have developed a way to produce [13C6]resorcinol in our lab, we decided to
carry out the synthesis by a similar route to the non-labeled synthesis.
Results and discussion
The synthesis of [13C6]6-hydroxychlorzoxazone in our lab is outlined in
Figure 2.
[13C6]Benzene (1) was converted to [13C6]resorcinol (2) in 51.7% yield, first
by treating with fuming sulfuric acid, and then by fusing with sodium
hydroxide at high temperature. Chlorination of phenolic compounds is usually
troublesome and mixtures of multi-chlorinated products are often formed
following treatment with chlorine. A recent report claimed selective mono-
chlorination of phenolic compounds when treating a solution of a phenol with
excess of DMD (dimethyldioxirane), in the presence of 1 equivalent of
hydrogen chloride.4 One of the advantages of this method is that the hydrogen
chloride can be generated italics from diluted sulfuric acid and sodium
chloride, which can be accurately weighed so that exactly 1 equivalent of active
chlorine can be generated. Synthesis of [13C6]4-chlororesorcinol (3) following
this procedure was quite successful, we were able to achieve 76% yield of the
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 979–984
DOI: 10.1002/jlcr

SYNTHESIS OF ISOTOPE LABELED 6-HYDROXYCHLORZOXAZONE
981
desired mono-chlorination product [13C6]4-chlororesorcinol (3). A minor
amount of [13C6]dichlororesorcinol was also detected (8.6%), even when
the reaction was carried out strictly with only 1 equivalent of sodium
chloride.
Our first attempt to prepare 4-chloro-6-nitrosoresorcinol following a
literature procedure, by treating a solution of 4-chlororesorcinol and sodium
ethoxide in ethanol with n-butyl nitrite,3 was unsuccessful. We were not able to
produce any detectable amount of 4-chloro-6-nitrosoresorcinol this way.
Fortunately when the reaction was done with potassium hydroxide and
isoamyl nitrite,5 we were able to prepare [13C6]4-chloro-6-nitrosoresorcinol
(4) in a reasonable yield (60%).
In the reported non-labeled synthesis of 6-hydroxychlorzoxazone,1,3
4-chloro-6-nitrosoresorcinol was first hydrogenated to produce 4-amino-
6-chlororesorcinol, and then the solution of the 4-amino-6-chlororesorcinol
was filtered to remove the palladium catalyst before reacting with phosgene.
Our preliminary experiment with the reduction of 4-chloro-6-nitrosoresorcinol
revealed that the reaction solution was almost colorless when hydrogenation
was completed, but it turned to dark brown quickly when we tried to filter
the reaction mixture, an indication that oxidative by-products had formed
during filtration, even when it was done under a nitrogen atmosphere.
We believe that this is one of the major factors which had led to low and
un-reproducible yield. The other possible factor was the use of phosgene, a
very reactive reagent. We believe that phosgene reacts non-selectively with
both amino and hydroxyl groups in the molecule, although the nucleophilicity
of the amino group in 4-amino-6-chlororesorcinol is stronger than the
two hydroxyl groups. Improvements in yield can be achieved if we can
eliminate the filtration step and if we can use a less active phosgene equivalent.
It turned out to be the case: [13C6]6-hydroxychlorzoxazone (6) was produced in
much higher yield when we eliminated the filtration step and replaced
phosgene with triphosgen, a safer and milder phosgene equivalent.6 Thus,
[13C6]4-chloro-6-nitrosoresorcinol (4) was first catalytically hydrogenated in
THF to the corresponding amine, and then without filtration to remove
the catalyst, triethylamine and a THF solution of stoichiometric amount of
triphosgene was added quickly with exclusion of oxygen. The reaction
proceeded quickly, and after shaking for 30 min at room temperature, [13C6]6-
hydroxychlorzoxazone (6) formed in a much better yield (75%). Initially
we were also concerned about possible over reduction, resulting in the
removal of chlorine from the benzene ring, but it turned out to be unnecessary
because hydrogenation stopped under our reaction conditions (10% Pd/C as
catalyst, THF as solvent, and under 50 psi hydrogen pressure) when 2
equivalents of hydrogen were consumed. No products from chlorine removal
were detected.
Copyright # 2006 John Wiley & Sons, Ltd.
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Experimental
Materials and methods
1H NMR Spectra were recorded on a 500 MHz Bruker NMR spectrometer.
Chemical shifts are reported in ppm relative to TMS. The LC-MS analysis was
performed on a LCQ-Advantage mass spectrometer coupled with a Waters
Alliance HPLC instrument. HPLC conditions: column, Phenomenex Polar
RP-C18, 4m, 4.6 Â 150 mm; mobile phase A, 10 mM ammonium acetate with
0.5% acetic acid; mobile phase B, acetonitrile; gradient, 18 min from 80% A–
20% B to 20% A–80% B; flow-rate, 1 ml/min; UV, 297 nm. The chemicals and
solvents were reagent grade obtained from Sigma Aldrich without further
purification. [13C6]Benzene was a Cambridge Isotope Labs product.
[13C6]Resorcinol (2). Fuming sulfuric acid (20%, 32 g) was added drop wise
at room temperature to [13C6]benzene (10 g, 119 mmol) with stirring. The
reaction temperature was raised to 608C after the addition was complete.
More fuming sulfuric acid (66%, 26 g) was added drop wise over 2 h and the
resulting reaction mixture was stirred for 1 h at 908C. The reaction mixture
was then cooled down to room temperature, poured into water (250 ml),
boiled, and neutralized with calcium carbonate (51 g). The hot solution was
filtered and the filtrate was treated with sodium carbonate (13 g). Precipitate
formed while adding sodium carbonate and was removed by filtration. The
filtrate was concentrated to dryness to afford a solid (38.5 g) after drying under
vacuum. The solid was then added portionwise to a melt of sodium hydroxide
(41.5 g) and water (26 ml) at 2008C. The mixture was distilled under reduced
pressure to afford a solid, which was heated at 3208C for 5 h, and then cooled
to room temperature. Water (80 ml) was added to the reaction mixture, the
suspension was acidified with conc. HCl, filtered through a thin layer of celite,
and the filtrate was extracted with diethyl ether (6 Â 30 ml). The combined
diethyl ether extracts were dried over sodium sulfate, filtered, and evaporated
to dryness to afford a dark tar material, which was distilled under high
vacuum, heating with a heat-gun and collecting in an end-trap cooled at liquid
nitrogen temperature to afford (2) as a yellowish solid (7.14 g, 52%). 1H NMR
(CDCl3) d 7.02 (m, d, J=167 Hz, 1 H), 6.34 (m, d, J=160 Hz, 2 H), 6.31 (m, d,
J=159 Hz, 1 H); MS (ESI-): m/z 115 ([M-H]À).
[13C6]4-Chlororesorcinol (3). Preparation of dimethyldioxirane (DMD):
Oxone (240 g, 390 mmol) was added from a solid addition funnel to a mixture
of water (500 ml), acetone (360 ml), and sodium bicarbonate (120 g,
1428 mmol) cooled in an ice bath in 25 min. DMD, along with acetone, was
distilled under slight vacuum (>500 mmHg) to afford a slightly yellow
solution (180 ml).
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 979–984
DOI: 10.1002/jlcr

SYNTHESIS OF ISOTOPE LABELED 6-HYDROXYCHLORZOXAZONE
983
Preparation of [13C6]4-chlororesorcinol (3) : [13C6]resorcinol (2, 600 mg,
5.2 mmol) was added to acetone (6 ml), followed by 10% sulfuric acid (5.2 ml)
and sodium chloride (303 mg, 5.2 mmol). The resulting reaction mixture was
stirred until a homogeneous solution was achieved and then the DMD
solution (180 ml) was added all at once. The reaction mixture was stirred at
room temperature for 10 min, concentrated to remove acetone, and extracted
with diethyl ether (3 Â 15 ml). The combined diethyl ether extracts were dried
over magnesium sulfate, filtered, and evaporated. The brownish residue was
purified by flash chromatography (silica gel column, eluted with hexane/
diethyl ether 3/1) to afford pure [13C6]4-chlororesorcinol (3, 470 mg) as a
colorless solid. An impure solid (205 mg) was also recovered. LC-MS analysis
indicated that the impure material contained [13C6]4-chlororesorcinol (3, 60%)
and [13C6]4,6-dichlororesorcinol (40%). This brings the total yield for [13C6]
1
4-chlororesorcinol (3) to 76%. H NMR (CDCl3) d 7.16 (m, d, J=173 Hz,
1 H), 6.54 (m, d, J=160 Hz, 1 H), 6.39 (m, d, J=160 Hz, 1 H); MS (ESI-): m/z
149 (100, [M-H]À), 151 (33).
[13C6]4-Chloro-6-nitrosoresorcinol (4). To a solution of [13C6]4-chlororesor-
cinol (3, 460 mg, 3.07 mmol)in ethanol (2.4 ml) cooled in an ice bath was added
a solution of potassium hydroxide (240 mg, 6 mmol) in water (0.47 ml),
followed by isoamyl nitrite (422 mg, 3.6 mmol) drop wise with stirring. The
reaction mixture was stirred at room temperature for 1 h before the pH of the
solution was adjusted to 2 with 1 N hydrochloric acid. Precipitate formed
during acidification and was collected by filtration. The filter cake was washed
with 1/1 ethanol/water (3 Â 1 ml) and water (3 Â 1 ml). Crude [13C6]4-chloro-6-
nitrosoresorcinol (4) was obtained (530 mg) after brief drying under vacuum.
The crude (4) was passed through a short silica gel cartridge (Anologix, 4 g
silica gel), eluting with hexane/ethyl acetate (6/4) to afford (4) as a yellow solid
1
(330 mg, 60%). H NMR (CD3OD) d 7.81 (d, J=173, 1 H), 5.88 (d, J=166,
1 H). MS (ESI-): m/z 178 (100, [M-H]À), 180 (34).
[13C6]6-Hydroxychlorzoxazone (6). [13C6]4-Chloro-6-nitrosoresorcinol (4, 180 mg,
1 mmol) was dissolved in anhydrous THF (15 ml) in a hydrogenation bottle.
Catalyst 10% Pd/C (50 mg) was then added. The reaction mixture was
evacuated and purged with nitrogen. This process was repeated three more
times in order to remove all air. The hydrogenation was carried out at 50 psi
under hydrogen until calculated amount of hydrogen has been taken up
(45 ml). Triethylamine (290 ml) was added, followed by a solution of
triphosgene (104 mg, 0.35 mmol) in THF (1.5 ml) under nitrogen atmosphere.
The resulting mixture was allowed to react for 30 min at room temperature,
and then filtered through a thin layer of celite to remove catalyst. The filtrate
was concentrated to afford a light brownish residue which was purified by flash
Copyright # 2006 John Wiley & Sons, Ltd.
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Z. JIAN ET AL.
chromatography (Analogix silica gel cartridge with 4 g silica gel, eluted with
hexane/ethyl acetate 7/3) to afford pure [13C6]6-hydroxychlorzoxazone (6,
105 mg). A second batch (49 mg) of impure (6) was also recovered with 79%
being the desired product when checked by HPLC. The total yield of (6) from
1
(4) is thus 75%. H NMR (CD3OD) d 6.99 (d, J=99, 1 H), 6.67 (d, J=96,
1 H). MS (ESI-): m/z 190 (100, [M-H]À), 192 (33), and no non-labeled (M+0)
species was detected.
Acknowledgements
The authors would like to thank Novartis DMPK management for their
support and other Novartis Isotope Lab colleagues for their technical
assistants.
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Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 979–984
DOI: 10.1002/jlcr