Synthesis of labeled ambroxol and its major metabolites

Article abstract of DOI:10.1002/jlcr.1694

Ambroxol is a mucolytic agent used in the treatment of respiratory diseases. Herein, we report the synthesis of carbon-14-labeled ambroxol with the radioactive atom(s) either on the benzylic carbon or uniformly in the cyclohexyl ring with specific activities of 59 and 81 mCi/mmol, respectively. We also describe the preparation of deuterium-labeled ambroxol, its deuterium-labeled tetrahydroquinazoline metabolite (DHTQ), carbon-13-labeled 3,5-dibromoanthranilic acid metabolite, as well as an unlabeled O-glucuronide conjugate. Copyright

Full text of DOI:10.1002/jlcr.1694

Research Article
Received 21 July 2010,
Revised 1 September 2009,
Accepted 5 October 2009
Published online 9 November 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1694
Synthesis of labeled ambroxol and its major
metabolitesz
Ã
Bachir Latli,a Matt Hrapchak,a Heinz-Karl Switek,b Daniel M. Retz,c
Dhileepkumar Krishnamurthy,a and Chris H. Senanayakea
Ambroxol is a mucolytic agent used in the treatment of respiratory diseases. Herein, we report the synthesis of carbon-14-
labeled ambroxol with the radioactive atom(s) either on the benzylic carbon or uniformly in the cyclohexyl ring with specific
activities of 59 and 81 mCi/mmol, respectively. We also describe the preparation of deuterium-labeled ambroxol, its
deuterium-labeled tetrahydroquinazoline metabolite (DHTQ), carbon-13-labeled 3,5-dibromoanthranilic acid metabolite, as
well as an unlabeled O-glucuronide conjugate.
Keywords: ambroxol; carbon-14; carbon-13; deuterium; metabolites
3,5-dibromoanthranilamide labeled with carbon-13 was also
prepared. The unlabeled O-glucuronide conjugate of ambroxol
Introduction
Ambroxol is a mucolytic agent, marketed since the late 1970s,
for the treatment of acute and chronic airway disorders
characterized by the production of excess and thick mucus.
The pharmacological and clinical data of ambroxol have recently
been reviewed.1 In Europe, ambroxol is marketed for acute
upper respiration diseases, for the treatment of chronic
was synthesized to aid in the identification and analytical
comparisons with metabolites generated in vivo.
Results and discussion
bronchitis, and for neonatal respiratory distress syndrome.2 It In the synthesis of carbon-14-labeled ambroxol with the
has been proposed as an anti-oxidant and an anti-inflammatory radioactive carbon in the benzylic position, Scheme 2,
drug useful in preventing and treating influenza,3,4 acute anthranilic acid [carboxylic-14C]-, which is easily accessible in
uncomplicated sore throat,5 and other symptoms associated two steps from 1-bromo-2-nitrobenzene by carboxylation with
with rhinovirus infections.6 It is hypothesized that ambroxol has 14CO2 followed by reduction of the nitro group,11 was reduced
a protective effect on pulmonary function after cardiopulmon- to the benzyl alcohol derivative using LAH in THF in 91% yield.
ary bypass.7 Since 2002, ambroxol has been marketed as a Bromination with bromine in acetic acid gave 2-amino-3,5-
lozenge for sore throat due to its local anesthetic effects.
dibromobenzyl alcohol in 38% radiochemical yield after flash
Azmbroxol is a pharmacologically active phase I metabolite of chromatography purification. Oxidation of the benzyl alcohol
the drug bromhexine, marketed since 1965 for the treatment of derivative to the benzaldehyde was accomplished using MnO2
mild respiratory diseases. Oral bromhexine is metabolized in in toluene at 801C in 85% yield. Condensation of this aldehyde
mammals to several compounds, and ambroxol is the most with trans-4-aminocyclohexanol in ethanol followed by reduc-
potent among several metabolites that retain pharmacological tion of the imine with NaBH4 gave crude ambroxol. Purifi-
activity. As shown in Scheme 1, ambroxol is produced from a cation by flash chromatography followed by treatment with a
hydroxylation on the cyclohexyl ring and an N-demethylation.8–12 solution of HCl in dioxane gave the desired material in 51%
Herein, we report the synthesis of two versions of carbon-14-
labeled ambroxol; one with the radioactive carbon in the benzylic
position and the other with the radioactive carbon(s) uniformly
distributed in the cyclohexyl ring to follow the metabolism of this
Research and Development Center 900, Ridgebury Road, P.O. Box 368,
moiety in mammals. Owing to the presence of two bromine
atoms in the molecule, the preparation of labeled ambroxol with
aDepartment of Chemical Development, Boehringer Ingelheim Pharmaceuticals,
Ridgefield, CT 06877-0368, USA
bIsotope Chemistry Group, Drug Metabolism and Pharmacokinetics, Boehringer
Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strasse 65, 88397 Biberach an
der Riss, Germany
stable isotopes requires introducing more than six atomic mass
units (amu) to easily discern it from the unlabeled drug in
analytical studies. Thus, deuterium-labeled ambroxol with M1
11amu was prepared. The metabolite 6,8-dibromo-3-(trans-4-
hydroxycyclohexyl)-1,2,3,4-tetrahydroquinazoline (DHTQ) was
cMoravek Biochemicals, Inc., 577 Mercury Lane, Brea, CA 92621, USA
*Correspondence to: Bachir Latli, Department of Chemical Development,
prepared with M113amu starting from deuterium-labeled
ambroxol and deuterium-labeled formaldehyde. The other
major metabolite, 3,5-dibromoanthranilic acid (DBAA),13 labeled
with carbon-13 was prepared. The potential metabolite
Boehringer Ingelheim Pharmaceuticals, Research and Development Center,
900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-0368, USA.
E-mail: Bachir.latli@boehringer-ingelheim.com
zThis paper is dedicated to Professor John E. Casida on his 80th birthday.
J. Label Compd. Radiopharm 2010, 53 15–23
Copyright r 2009 John Wiley & Sons, Ltd.

B. Latli et al.
O
Br
OH
NH2
Br
DBAA
Br
N
NH2
Br
Bromhexine
OH
Br
N
Br
Br
N
H
N
N
H
3-hydroxy & 4-hydroxy
metabolites
NH2
Br
NH2
Br
Br
OH
Br
N
H
3-hydroxy
metabolites
+
NH2
Br
Ambroxol
OH
Br
N
3-hydroxy
metabolites
N
H
+
Br
DHTQ
Scheme 1. Bromhexine and some of its metabolites as proposed in the literature.
radiochemical yield and with a specific activity of 59.5 mCi/mmol contaminated with about 15% cis-isomer, which was easily
and radiochemical purity of 98.3%. removed by reverse phase HPLC. Labeled ambroxol was isolated
In the second synthesis of carbon-14 ambroxol, Scheme 3, the with a specific activity of 81mCi/mmol, but it was diluted to
radioactive atoms were incorporated in the cyclohexyl portion of 49.4mCi/mmol with commercial unlabeled ambroxol to minimize
the molecule so it can be easily followed during metabolism radiolysis.
studies. Thus, uniformly labeled acetaminophen was reduced to
To prepare deuterium-labeled ambroxol hydrochloride,
N-acetyl-4-aminocyclohexanol under hydrogen gas in the pre- Scheme 4, the commercially available [2H7]-4-aminophenol
sence of 5% Rh/alumina at 50PSI.14–16 The product was crystal- was first converted to [2H4]-acetaminophen using acetic
lized from acetonitrile to enrich the trans-isomer. Removal of the anhydride and catalytic amounts of sodium dodecyl sulfate
acetyl group was accomplished by heating at 1101C for 12h with (SDS) in water.17 Then, reduction under deuterium gas in the
aqueous 10% KOH.14 Condensation with 2-amino-3,5-dibromo- presence of rhodium on alumina at 601C gave N-acetyl-4-amino-
benzaldehyde in ethanol followed by imine reduction in situ with [2H10]-cyclohexanol in quantitative yield. At this stage, instead of
NaBH4, as previously described, gave carbon-14-labeled ambroxol performing a crystallization to remove the cis-isomer, the
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Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 15–23

B. Latli et al.
O
14C
O
14C
Br
14C
14C
Br
LiAlH4
Et2O
OH
2
Br2
MnO2
OH
H
OH
NH
38%
toluene
AcOH:H2O
NH2
NH2
85%
NH2
91%
Br
Br
OH
OH
1. NaBH4
EtOH
14C
EtOH
NH2
Br
14C
Br
N
N
H
NH2
2. HCl
HCl
NH2
Br
Br
51%
OH
Scheme 2. Synthesis of ambroxol, [benzyl, 14C]-.
O
O
NH2
HN
NH2
HN
H2
(MeCO)2O
14C
Rh/Al 2O3
EtOH
Aq. KOH
14C
14C
14C
EtOH
OH
OH
OH
OH
100%
80%
[
14C(U)]-acetaminophen
100%
NH2
14C
14C
N
OH
14C
OH
O
Br
Br
N
H
1. NaBH4
Br
H
OH
HCl
NH2
EtOH
NH2
NH2
EtOH
Br
Br
2. HCl
Br
76%
Scheme 3. Synthesis of ambroxol, [cyclohexyl, 14C(U)]-.
product was taken directly into the next step as a mixture of 801C with a solution of [2H2]-formaldehyde in deuterated water
cis-and trans-isomers. Removal of the acetyl group was achieved to give after purification [2H13]-6,8-dibromo-3-(trans-4-hydroxy-
using either aqueous 2N KOH in 93% yield as seen above, or cyclohexyl)-1,2,3,4-tetrahydroquinazoline ([2H13]-DHTQ) in 49%
2.75N aqueous HCl in 89% yield. Coupling to 2-amino-3,5- yield.18
dibromobenzaldehyde as seen above gave the labeled imine
To prepare labeled DBAA, the major metabolite of ambroxol,
derivative in 92% yield. Reduction of the imine with sodium and the potential metabolite 3,5-dibromoanthranilamide,
borodeuteride in ethanol gave [2H11]-ambroxol as a mixture of Scheme 6, [13C6]-aniline was first brominated to [13C6]-2,4-
almost 1:1 cis: trans isomers. Isolation of the desired trans-isomer dibromoaniline using N-bromosuccinimide in methylene chlor-
was accomplished by HPLC using a C18 reverse phase column ide in 76% yield. This compound was then iodinated with ICl
and acetonitrile and water, which contained 0.1% perchloric acid in acetic acid at 601C to 2,4-dibromo-6-iodo-[13C6]-aniline in
and 0.1% phosphoric acid. The isotopic enrichment of the final 69% yield.19 Cyanation with [13C]-copper cyanide in pyridine at
product was more than 99%.
For the synthesis of the tetrahydroquinazoline metabolite lysis in either 70% aqueous sulfuric acid or in concentrated
1001C gave 2,4-dibromo-[13C7]-benzonitrile in 95% yield. Hydro-
(DHTQ), Scheme 5, [2H11]-ambroxol in methanol was heated to sulfuric acid gave 3,5-dibromo-[13C7]-anthranilic acid or
J. Label Compd. Radiopharm 2010, 53 15–23
Copyright r 2009 John Wiley & Sons, Ltd.
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B. Latli et al.
O
O
2H
2H
NH2
HN
2H
2H
2H
2H
HN
2H
2H
2H
2H
2H
N
O
2H
2H
2H
2H
2H
2H2
2H
2H
2H
2H
2H
2H
2H
2H
2 N KOH
reflux
2H
(MeCO)2O
SDS, H2O
2H
Rh/Al 2O3
MeO2H
2H
2H
2H
2H
OH
93% (1:1 trans:cis)
OH
98%
OH
2H
99%
2H2
2H2
H
H
2H
OH
2H
2H
OH
2H
2H
2H
2H
2H
O
NH2
2H
2H
2H
2H
2H
2H
2H
2H
2H
2H
Br
Br
2H
H
Br
NaB2H4
EtO2H
2H
EtOH
2H
2H
N
2H
N
2H
2H
+
H
2H
NH2
2H
NH2
NH2
Br
OH
Br
Br
92%
2H2
40%
H
2H
OH
2H
2H
2H
2H
2H
Br
2H
2H
RPC
N
2H
H
NH2
Br
Scheme 4. Synthesis of [2H11]-ambroxol.
2H2
2H 2
H
2H
H
2H
OH
2H
2H
2H
OH
2H
2H
2H
2H
2H
2H
2H
C H O
H O-MeOH
Br
2H
Br
2H
2H
2H
N
2H
2H
N
2H
H
N
2H
NH2
H
Br
Br
[2H11]-Ambroxol
49%
Scheme 5. Synthesis of deuterium-labeled tetrahydroquinazoline metabolite (DHTQ).
3,5-dibromo-[13C7]-anthranilamide in 53 and 47% yield, respec- structure in Scheme 7. Ester hydrolysis with 1N aqueous NaOH
tively. Alternatively, 2,4,6-tribromoaniline obtained from in methanol gave mostly ambroxol after cleavage of the ether
bromination of aniline in one step failed to give clean 3,5- linkage to the sugar moiety. The desired product was obtained
dibromo-2-aminobenzonitrile using different cyanation condi- in low yield.
tions. When the amino group was transformed to a nitro group
to help directing the ortho-cyanation, a mixture of products were
obtained instead of the desired 2-nitro-3,5-dibromobenzonitrile.
While the coupling constants between the aromatic protons
of the unlabeled DBAA are about 2 Hz, large coupling constants
are observed for the carbon-13-labeled DBAA between 13C and
Liquid scintillation counting was accomplished using a Beckman
1H. See Figure 1 below.
Lastly, to prepare the unlabeled O-glucuronide conjugate of
ambroxol, Scheme 7, the free base of ambroxol was treated with
acetobromo-D-glucuronic acid methyl ester in the presence of
Ag2CO3 in toluene. Flash chromatography purification provided
the desired product in a 30% yield. The structure was confirmed
by 2D ROESY experiment. An NOE was observed as shown in the
Experimental procedures
Materials and methods
LS6500 and ready safeTM cocktail (Beckman, Fullerton, CA). The
analytical HPLC radiopurity verification was carried out on a
Supelco Discovery C18 (4.6 ꢀ 250 mm). UV detection was at
254 nm. The mobile phase consisted of A (water with 0.1% TFA),
B (acetonitrile with 0.1% TFA), gradient: 0–5 min 0% B, 5–30 min
0–50% B, 30–30.1 min 50–100% B and hold to 40 min. For
deuterium-labeled products, HPLC was carried out on an Eclipse
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B. Latli et al.
NH2
NH2
NH2
NH2
NH2
ICl
AcOH
CuCN
NBS
CH2Cl2
H2SO4
I
Br
NC
Br
Br
HOOC
Br
Pyridine
100 °C
60 °C
H2O
Br
Br
95 °C
Br
Br
76%
69%
95%
53%
H2SO4
95 °C
O
NH2
Br
all carbon atoms13
C
H2N
Br
47%
Scheme 6. Synthesis of 3,5-dibromo-[13C7]-anthranilic acid (DBAA) and the potential metabolite 3,5-dibromo-[13C7]-anthranilamide.
solvent and tetramethyl silane as the internal standard (Cam-
bridge Isotope Laboratories, Andover, MA). Pre-coated TLC
sheets (silica gel 60 F254) were obtained from EM Science
(Gibbstown, NJ). [13C6]-aniline, [2H7]-4-aminophenol, and [2H2]-
formaldehyde ca. 20% wt% in H2O was purchased from Isotec
(St. Louis, MO). All other reagents were purchased from Sigma-
Aldrich.
12
10
8
2
Synthesis
Synthesis of ambroxol, [benzyl, 14C]-
6
DBAA
2-Amino-[14C]-benzyl alcohol: To a solution of anthranilic acid
(265 mCi, 4.5 mmol) in anhydrous ether (30 mL) was added LAH
(300 mg, 7.5 mmol) in ether (7.5 mL) with stirring in 20 min
period. The mixture was refluxed for 1 h then cooled to room
temperature. Excess LAH was destroyed by the addition of ethyl
acetate, 2N aqueous NaOH and water (4 mL each). The organic
phase was separated and the aqueous was extracted three times
with ether. The combined extracts were dried over MgSO4,
filtered and concentrated in vacuo to give 263 mCi. Purification
by silica gel chromatography using methylene chloride:
methanol (15:1) gave 240 mCi of pure material in 91% radio-
chemical yield.
4
2
0
8.1
8.0
7.9
7.8
7.7
7.6
[ppm]
Figure 1. Proton NMR showing aromatic protons of [13C7]-DBAA and unlabeled
DBAA in DMSO-d6. Data were acquired using 500 MHz Bruker spectrometer.
2-Amino-3,5-dibromo-[14C]-benzyl alcohol: The above com-
XDB-C18 (4.6 ꢀ 150 mm). UV detection was at 220 nm. The pound (240 mCi, 4.0 mmol) was dissolved in glacial acetic acid
mobile phase consisted of A (water with 0.1% H3PO4 and 0.1% (6.2 mL) and water (2 mL). A solution of bromine (0.412 mL,
HClO4), B (acetonitrile with 0.1% H3PO4 and 0.1% HClO4), 8.05 mmol) in glacial acetic acid (1.25 mL) was then added
gradient: 0–2 min 15–25% B, 2–3 min 25–30% B, 3–7.5 min dropwise with stirring. A precipitate formed. Water (20 mL) was
30–50% B. For carbon-13-labeled compounds HPLC was carried added and the mixture was stirred for 30 min, filtered, washed
out on a Zorbax Eclipse XDB-C18 (4.6 ꢀ 150 mm). UV detection with water and dried under reduced pressure at 401C. Flash
was at 254 nm. Mobile phase consisted of A (water with 0.1% chromatography purification using EtOAc:CHCl3 as eluent
TFA), B (acetonitrile with 0.1% TFA), gradient: 20–100% B over (5–20%) gave 92 mCi of the desired product in 38% radio-
20 min. For the glucuronide derivative of ambroxol, HPLC was chemical yield.
carried out as described for carbon-13-labeled compounds with
2-Amino-3,5-dibromo-[14C]-benzaldehyde: To the above com-
a gradient 5–100% B over 20 min. Mass spectra were acquired by pound (92 mCi, 1.56 mmol) in anhydrous toluene (26 mL) was
a Hewlett-Packard auto sampler Series 1150, connected to a added MnO2 (0.88 g, 10.1 mmol) and stirred at 801C for 1 h. TLC
Micromass LCZ mass spectrometer in the ES mode. NMR spectra (2:1 CHCl3: EtOAc) indicated no starting material. Filtration of the
were recorded with a Bruker 400 MHz DPXB for the non- mixture gave a clear solution, which was concentrated in vacuo
radioactive compounds and Bruker 300 MHz spectrometers for to give 78 mCi (85% yield) of material used directly in the
carbon-14-labeled compounds using deuterated methanol as a following step.
J. Label Compd. Radiopharm 2010, 53 15–23
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B. Latli et al.
OAc
Br
OAc
OAc
NOE observed
O
OAc
O
O
OAc
OAc
OH
NH2
Br
NH2
Br
Br
O
O
OMe
Br
N
H
N
H
OMe
NOE observed
Ag2CO3
Toluene
30%
OH
O
OH
NH2
1N NaOH
MeOH
Br
O
O
N
H
OH
OH
7%
Br
Scheme 7. Synthesis of unlabeled ambroxol-O-glucuronide Conjugate.
Trans-4-(2-Amino-3,5-dibromo-[14C]-benzylamino)-cyclohexanol:
Trans-4-Aminocyclohexanol,[cyclohexyl-14C(U)]-: The trans-en-
The above aldehyde (78 mCi, 1.32 mmol) and trans-4-aminocy- riched material from above (0.16 g, 1.0 mmol) and potassium
clohexanol (173 mg, 1.5 mmol), which was obtained from the hydroxide (0.5 g, 8.9 mmol) in water (5 mL) was heated to 1101C
HCl salt by neutralizing and extracting with chloroform and for 12 h. After cooling to room temperature, the solution was
isopropanol, in absolute ethanol (2.5 mL) was refluxed at 901C saturated with NaCl and extracted four times with 4:1 chloro-
over a period of 3 h. After cooling to room temperature, NaBH4 form/isopropanol. The combined extracts were dried over
(100 mg, 2.64 mmol) was added and stirred for 12 h. The mixture MgSO4, filtered and concentrated to give 127 mg of material,
was evaporated to dryness and then diluted with aqueous 1N which was used in the next step without further purification.
NaOH and extracted with CH2Cl2. The solvent was then removed
Trans-4-(2-Amino-3,5-dibromobenzylamino)-cyclohexanol,[cyclo-
in vacuo and the residue was purified by silica gel flash hexyl-14C(U)]-: A mixture of the above crude material (0.127 g,
chromatography using 10% methanol in methylene chloride to 1.0 mmol), 2-amino-3,5-dibromobenzaldehyde (0.335 g, 1.2 mmol)
give 44 mCi of ambroxol free base. The free base was dissolved in absolute ethanol (5 mL) was heated to 901C for 5 h. After
in methanol and treated with a solution of 4N HCl in dioxane. cooling to room temperature, NaBH4 (0.09 g, 2.4mmol) was
The precipitate was concentrated and then crystallized from added and the mixture was stirred at room temperature for 12 h.
ethanol/ether to give 40 mCi of material with a specific activity The product was isolated as mentioned above and purified by
1
of 59.5 mCi/mmol. H NMR (500 MHz, CD3OD)d: 7.65 (d, J = 2 Hz, silica gel chromatography using chloroform to elute non-polar
1H), 7.42 (d, J = 2 Hz, 1H), 4.21 (s, 2H), 3.56 (m, 1H), 3.18 (m, 1H), impurities then 5% MeOH/CHCl3 to elute the desired product in
2.22 (m, 2H), 2.07 (m, 2H), 1.51 (m, 2H), 1.36 (m, 2H). HPLC, 76% yield (289 mg) as syrup, or 62 mCi with a specific activity of
Rt = 24.07 min (98.3%), co-elutes with unlabeled commercial 81 mCi/mmol. The product was dissolved in methanol and
sample. MS, m/z: 378.8, 380.8, 382.8 (50%, 100%, 50% treated with 4N HCl in dioxane. After evaporation of the solvent,
respectively); 379.8, 381.8, 383.8 (1:2:1).
the residue was crystallized from ethanol/ether as seen above to
give 35 mCi of material with a specific activity of 81 mCi/mmol.
The specific activity was diluted to 49.4 mCi/mmol by adding
95 mg of ambroxol hydrochloride, purchased from Sigma-
Aldrich, to 153 mg of labeled ambroxol in methanol and the
solution was then concentrated and dried under vacuum. 1H
NMR (500MHz, CD3OD)d: 7.65 (d, J = 2 Hz, 1H), 7.42 (d, J = 2 Hz,
1H), 4.21 (s, 2H), 3.56 (m, 1H), 3.18 (m, 1H), 2.22 (m, 2H), 2.07 (m,
2H), 1.51 (m, 2H), 1.36 (m, 2H). HPLC, 23.5 min (97.4%) co-elutes
with an unlabeled commercial sample. MS: m/z: 378.9, 380.8,
382.2 (1:2:1), 384.8, 386.8, 388.8, 3918, 393.7, 395.8.
Synthesis of ambroxol, [cyclohexyl-14C(U)]-
N-Acetyl-4-aminophenol,[phenyl-14C(U)]-: 4-Aminophenol [14C(U)]-
(428 mCi, 0.61g, 5.59 mmol) in absolute ethanol (10mL) was
added acetic anhydride (0.6g, 5.9mmol) at room temperature.
The solution was stirred for 15 min then concentrated in vacuo to
give 0.859 g of a tan solid. Purification by silica gel flash
chromatography using 10% methanol in methylene chloride gave
428 mCi of a white solid in 100% yield.
N-Acetyl-4-aminocyclohexanol,[cyclohexyl-14C(U)]-: A mixture of
the above compound (0.51 g, 3.33 mmol), 5% Rh on Al2O3 (0.13 g)
in absolute ethanol (6.0 mL) was stirred under 50 psi of hydrogen
Synthesis of [2H11]-ambroxol
for 12 h. The pressure dropped to 37 psi. TLC showed some [2H4]-Acetaminophen: To a mixture of 4-aminophenol-[2H7]
starting material remaining. The hydrogenation bottle was (5.0 g, 43.077 mmol) in water (180 mL) was added SDS (280 mg,
pressurized again to 50 psi and stirred for 3 h. The reaction mixture 0.971 mmol) and the mixture was heated to 651C until all the
was filtered and concentrated in vacuo. The residue was triturated aminophenol was dissolved. To this dark solution acetic
with acetonitrile. Acetonitrile was then removed and the solid was anhydride (6.5 mL, 67.4 mmol) was added and the mixture was
crystallized from acetonitrile (2 mL). Removal of the solvent and stirred at this temperature for 30 min. Then, cooled to room
drying the residue under reduced pressure gave 160 mg of trans- temperature and stirred for 14 h. LC-MS showed no starting
enriched isomer. The mother liquor gave after drying 267 mg of a material. The dark mixture was extracted with ethyl acetate
mixture of cis/trans (3:2). Total of 427 mg or 80% yield.
(3 ꢀ 200 mL), washed with brine, dried (MgSO4), filtered and
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B. Latli et al.
concentrated in vacuo to give 6.78 g of purple solid. 1HNMR 388.05473, found 388.05491. Traces of [2H10]- and [2H9]-
(MeOH-d4): 1.97(s, 3H). 13C NMR (MeOH-d4): 32.35, 115.84(t, ambroxol were observed.
J = 24.42 Hz), 122.97(t, J = 24.54 Hz), 131.45, 155.28, 171.37. LC-
MS one single peak: MH1 156.13.
Synthesis of [2H13]-6,8-Dibromo-(3-trans-4-hydroxycyclohexyl)-
1,2,3,4-tetra-hydroquinazoline ([2H13]-DHTQ)
N-Acetyl-4-amino-[2H10-]-cyclohexanol: A mixture of [2H4]-acet-
aminophen (6.25 g, 40.3 mmol) and Rh on alumina (10% Rh/
Al2O3, 1.25 g) in deuterated methanol (36 mL) was stirred under
a 25 PSI of deuterium gas (99.8 atom % 2H) for 12 h. The mixture
was filtered through a short pad of Celite, rinsed with methanol
and concentrated in vacuo to give 6.98 g of a semi-solid.
Purification by Combi-Flash Companion using 120 g column and
10–100% MeOH/CH2Cl2 gave 4.3 g of the desired product, LC-MS
MH1 = 168.21, and 2.3 g of material with traces of UV active
contaminant. Total yield 98%.
A solution of [2H11]-ambroxol (160 mg, 0.376 mmol) in methanol
(2 mL) was treated with a solution of formaldehyde-2H2, 98
2
2
atom% H, ca. 20 wt.% in H2O (2.0 g, 12.5 mmol). The resultant
was heated to 801C for 2 h. LC-MS showed the presence of new
product MH1: 401–407. After cooling to room temperature, the
solution was concentrated in vacuo to give 221 mg of yellowish
viscous oil. The crude was dissolved in CHCl3 (10 mL) and treated
with a solution of 2N NH4OH (10 mL), extracted with CHCl3, dried
over MgSO4, filtered and concentrated in vacuo to give 180 mg
of material. Purification by flash chromatography using 12 g
disposable silica gel column and 1–5% MeOH/CH2Cl2 gave
75 mg of pure product as a white solid in 49% yield. 1H NMR
(CDCl3) d: 3.90 (bs, 1H), 4.37(bs, 1H), 7.38(d, J = 2.10 Hz, 1H),
6.96(d, J = 2.10 Hz, 1H). 13C NMR (CDCl3) d: 27.44(m), 32.88(m),
49.65(t, J = 20.63 Hz), 51.86(m), 71.88(m), 79.62(m), 108.15,
109.08, 122.97, 128.89, 132.29, 140.00. LCMS: m/z : 401 to 408.
4-Amino-[2H10]-cyclohexanol: A solution of the above material
(1.0g, 6.0mmol) in aqueous KOH (10mL, 2N) was heated to a
gentle reflux for 12 h. The solution was cooled to room
temperature and saturated with NaCl (3.5g) and then extracted
with CHCl3: iPrOH (3:1), dried over MgSO4, filtered and concen-
trated in vacuo to give 0.7 g of a solid as a mixture of cis- and
trans- isomers. 13CNMR (CDCl3) d: 29.05 (m), 32.11 (m), 46.88 (m),
48.15 (m), 64.10 (m), 67.81 (m). LC-MS: ESI, MH1 = 122.86.
Removal of the acetyl group was also performed using aq.
2.75N HCl. So a solution of the above material (1.27 g, 6.0 mmol)
in aq. 2.75N HCl (15 mL) was heated to a gentle reflux for 12 h.
Synthesis of 3,5-dibromo-[13C7]-anthranilic acid ([13C7]-DBAA) and
3,5-3,5-dibromo-[13C7]-anthranilamide
The solution was cooled to room temperature and saturated 2,4-dibromo-[13C6]-aniline: NBS (7.2g, 43mmol) was added to a
with NaCl (3.5 g) and then extracted with CHCl3: iPrOH (3:1), solution of [13C6]-aniline (2.0 g, 21.5mmol) CH2Cl2 (30mL) at 01C.
dried over MgSO4, filtered and concentrated in vacuo to give The mixture was warmed to room temperature over 15min, then
0.85 g (89% yield) of material, which was used as it is in the next stirred overnight. The reaction was quenched by the addition of
step.
2-Amino-3,5-dibromobenzylidene-4-amino-[2H10]-cyclohexanol:
H2O, extracted with CH2Cl2 (10 mLꢀ 3), dried over Na2SO4 and
concentrated giving a light brown solid. The solid was diluted
A mixture of [2H10]-4-aminocyclohexanol (0.7 g, 5.6 mmol) and 2- with EtOH (8 mL), warmed to dissolve the solid, then H2O (7 mL)
amino-3,5-dibromobenzaldehyde (1.4 g, 4.9 mmol) in absolute was added and the mixture was allowed to cool to room
ethanol (10 mL) was heated to 801C and stirred for 3 h. LCMS temperature. The solid was collected by vacuum filtration to give
showed one single product with MH1 = 387.42. The reaction was 2,4-dibromo-[13C6]-aniline (4.1g, 76%) as light brown needles.
1
cooled to room temperature and concentrated in vacuo to give HPLC: 11.1 min. LCMS: m/z 256. H NMR (400 MHz, CDCl3)d: 7.53
1.988 g of a yellow solid, which was used as it is in the next step. (dm, J = 170 Hz, 1H), 7.19 (dm, J = 166 Hz, 1H), 6.64 (m, 1H), 4.59
[2H11]-Trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexanol (bs, 2H). 13C NMR (100MHz, CDCl3)d: 143 (t, J = 64Hz), 134 (t,
([2H11]-Ambroxol): To a solution of the above imine (1.988 g, J = 66Hz), 131 (t, J = 60Hz), 117 (t, J = 60Hz), 110 (t, J = 64 Hz).
5.148 mmol) in ethanol (25 mL), was added sodium borodeuter-
2,4-dibromo-6-iodo-[13C6]-aniline: ICl (0.946 g, 5.84 mmol) in
ide (0.5 g, 11.72 mmol) at room temperature. Excessive gas acetic acid (2 mL) was added to a solution of 2,4-dibromo-[13C6]-
evolution was observed. The reaction was stirred overnight aniline (1.5 g, 5.84 mmol) in acetic acid (10 mL). The mixture was
under nitrogen atmosphere. No starting material was observed heated at 601C for 5 min. Then H2O (5 mL) was added and the
by LC-MS. Concentrated HCl (1.2 mL) was added to decompose mixture was heated to 901C for 1h. The mixture was cooled to
excess NaB2H4, followed by aqueous 2N NH4OH (50 mL) and room temperature and the solid was collected by vacuum
extracted with CHCl3 (100 mL ꢀ 3). The combined extracts were filtration to give 2,4-dibromo-6-iodo-[13C6]-aniline (1.56 g, 69%)
dried over MgSO4, filtered and concentrated in vacuo to give as a light brown solid. HPLC: 15.5min. 1H NMR (400 MHz, CDCl3)d:
1.96 g of a solid. Purification by Combi-Flash on 120 g silicagel 7.69 (dm, J =172 Hz, 1H), 7.53 (dm, J = 172 Hz, 1H), 4.59 (bs, 2H).
column and using up to 20% MeOH/CH2Cl2 gave 0.79 g of 13C NMR (100MHz, CDCl3)d: 143 (t, J= 68Hz), 140 (t, J = 64 Hz), 135
desired product in 40% yield as colorless film. LC-MS: MH1: (t, J = 68 Hz), 109 (t, J= 64Hz), 107 (t, J = 68 Hz), 83 (dd, J = 64, 68).
387–393, unlabeled ambroxol MH1 377–382. The product was
2-Amino-3,5-dibromo-[13C7]-benzonitrile: A mixture of 2-iodo-
purified by Combi-Flash using a 120 g Redi-Sep C-18 column, 4,6-dibromo-[13C6]-aniline (0.75 g, 1.96 mmol) and CuCN (99
which was first preconditioned using about 100 mL of MeCN. atom % 13C, 0.355 g, 3.92 mmol) in pyridine (15 mL) was heated
The compound was dissolved in methanol and applied to a 5 g in a sealed vial at 1001C for 14 h. The mixture was cooled to
C18 cartridge and then eluted using up to 25% MeCN in water room temperature and diluted with EtOAc, washed sequentially
(0.1% H3PO4 and 0.1% HClO4). Tubes containing the desired with H2O, brine, dried over Na2SO4 and concentrated to give a
isomer as judged form HPLC were then combined. HPLC, brown solid. The solid was diluted with 25% EtOAc/Hexane
Rt = 4.48 min, 99.16%. (Cis-isomer Rt = 4.88 min). 1HNMR (CDCl3) (50 mL) and stirred overnight. The filtrate was then concentrated
of the free base: d: 3.76(bs, 1H), 5.34(bs, 2H), 7.08(d, J = 2.20 Hz, to give 2-amino-3,5-dibromo-[13C7]-benzonitrile (0.53 g, 95%) as
1H), 7.45(d, J = 2.20 Hz, 1H). 13CNMR (CDCl3): 29.10(m), 31.77 (m), an off white solid. HPLC: 11.2 min. 1H NMR (400 MHz, CDCl3)d:
49.14(t, J = 20.41 Hz, benzylic CHD), 53.50(m), 68.62(m), 107.25, 7.73 (dm, J = 171 Hz, 1H), 7.48 (dm, J = 169 Hz, 1H), 4.90 (bs, 2H).
109.37, 125.80, 130.24, 131.80, 142.85. MS-HR: MH1 calculated 13C NMR (100 MHz, CDCl3)d: 144 (t, J = 68 Hz), 137 (t, J = 64 Hz),
J. Label Compd. Radiopharm 2010, 53 15–23
Copyright r 2009 John Wiley & Sons, Ltd.
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B. Latli et al.
131 (dd, J = 64, 66 Hz), 113 (d, J = 82 Hz), 107 (t, J = 68 Hz), 106 (t, (110 mg, 0.16 mmol) in MeOH (5 mL) at room temperature. The
J = 64 Hz), 96 (m).
mixture was allowed to stir at room temperature for 14 h, and
2-Amino-3,5-dibromo-[13C7]-anthranilic acid ([13C7]-DBAA): Concen- then concentrated to give dark oil. The crude residue was
trated H2SO4 (5 mL) and H2O (2 mL) was added to 2-amino-3,5- purified by silica gel chromatography (5–15% MeOH/CH2Cl2),
dibromo-[13C7]-benzonitrile (117 mg, 0.41 mmol), the mixture was and then further purified by preparative HPLC (Agilent, 10–95%
heated at 951C overnight. The mixture was cooled to room MeCN/H2O (0.1%TFA)) to give a light yellow oil. LCMS revealed
temperature, poured over ice and the pH was adjusted to 4 with 3N the product was the methyl ester, m/z 568. This oil was then
NaOH. The mixture was extracted with EtOAc and the combined diluted with MeOH (1 mL), THF (1 mL) and 1N NaOH (1 mL) was
organic extracts were washed with brine, dried over Na2SO4 and added at room temperature. The mixture was allowed to stir at
concentrated to give a light brown solid. The solid was recrystallized room temperature for another 14 h to give the desired product
from EtOH/H2O to give 2-amino-3,5-dibromo-[13C7]-benzoic acid and ambroxol. The mixture was concentrated then diluted with
(66 mg, 53%) as a light brown solid. HPLC: 10.4 min. LCMS: m/z 301. water, extracted with Et2O to remove ambroxol. The aqueous
1H NMR (400 MHz, d6-DMSO)d: 13.37 (bs, 1H), 7.84 (dm, J=169Hz, layer, which contained the desired product, was then lyophilized
1H), 7.81 (dm, J= 169 Hz, 1H), 6.88 (bs, 2H). 13C NMR (100 MHz, d6- to give a white solid. The solid was diluted with water, and the
DMSO)d: 168 (d, J= 72 Hz), 147 (t, J=65Hz), 138 (t, J= 65 Hz), 133 (t, pH was adjusted to neutral with 3N HCl solution. The solution
J= 65 Hz), 112 (m), 110 (t, J=65Hz), 105 (t, J=65Hz).
was then passed through a pre-washed SPE (C18) cartridge and
H2SO4 the product was eluted using MeOH. Concentration in vacuo
3,5-Dibromo-[13C7]-anthranilamide:
Concentrated
(10 mL) was added to 2-amino-3,5-dibromo-[13C7]-benzonitrile gave (2S,3S,4S,5R,6R)-6-((1R,4R)-4-(2-amino-3,5-dibromobenzyl-
(405 mg, 1.43 mmol) and the mixture was heated at 951C for amino)-cyclo-hexyloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-car-
14 h. After cooling to room temperature, the mixture was boxylic acid (6mg, 7%) as an off white solid. HPLC: 9.2min. LCMS:
poured into ice and the pH was adjusted to 4 with 3N NaOH. m/z 554. 1H NMR (400 MHz, CD3OD)d: 7.38 (d, J = 2.0Hz, 1H), 7.15
The mixture was extracted with EtOAc and the combined (d, J = 2.0 Hz, 1H), 4.46 (bs, 2H), 4.26 (d, J = 8.0Hz, 1H), 3.79 (m, 2H),
extracts were washed with brine, dried over Na2SO4 and 3.66 (m, 1H), 3.42 (d, J = 9.0 Hz, 1H), 3.28 (m, 2H), 3.05 (dd, J = 8.0,
concentrated to give a light brown solid. The solid was purified 9.0Hz, 1H), 2.56 (m, 1H), 1.99 (m, 4H), 1.27 (m, 2H), 0.77 (m, 2H).
by silica gel flash chromatography (20–100% EtOAc/Hexane)
followed by crystallization from EtOH/H2O to give 2-amino-3,5- Conclusion
dibromo-[13C7]-benzoic acid amide (203 mg, 47%) as a light
1
brown solid. HPLC: 8.2 min. LCMS m/z: 300. H NMR (400 MHz,
We have prepared carbon-14-labeled ambroxol with the radio-
active carbon in either the benzylic position or uniformly
distributed in the cylcohexyl ring with specific activities of 59.5
and 81 mCi/mmol, respectively. Deuterium-labeled ambroxol
with an increase of 11 amu and its metabolite the tetrahydro-
quinazoline (DHTQ) with 13 amu were also prepared. The major
d6-DMSO)d: 8.03 (bs, 1H), 7.78 (dm, J = 82 Hz, 1H), 7.73 (dm,
J = 82 Hz, 1H), 7.45 (bs, 1H), 6.78 (bs, 2H). 13C NMR (100 MHz, d6-
DMSO)d: 169 (d, J = 63 Hz), 146 (t, J = 64 Hz), 137 (t, J = 64 Hz), 131
(t, J = 63 Hz), 117 (q, J = 64 Hz), 110 (t, J = 69 Hz), 105 (t, J = 64 Hz).
metabolite of ambroxol, DBAA as well as the potential
Synthesis of unlabeled O-glucuronide conjugate of ambroxol
metabolite 3,5-dibromoanthranilamide with all carbon C13 were
also prepared. The synthesis of O-glucuronide conjugate of
ambroxol was synthesized as well to be used in the identifica-
tion and in analytical studies.
(2R,3R,4S,5S,6S)-2-((1R,4R)-4-(2-Amino-3,5-dibromobenzylamino)-
cyclohexyloxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl
triacetate: A mixture of ambroxol (250 mg, 0.66 mmol), aceto-
bromo-D-glucuronic acid methyl ester (262 mg, 0.66 mmol), and
Ag2CO3 (182 mg, 0.66 mmol) in toluene (5 mL) was stirred at
room temperature overnight. HPLC showed only recovered
starting material. The mixture was heated at 1001C overnight.
The mixture was cooled to room temperature, filtered (Celite)
and concentrated to give yellow oil. The crude residue was
purified using CombiFlash Companion (40 g disposable silica
gel column and 0–10% MeOH/CH2Cl2) to give a yellow oil,
which was further purified using CombiFlash Companion
(12 g disposable column, 20–60% EtOAc/CH2Cl2) to give
(2R,3R,4S,5S,6S)-2-((1R,4R)-4-(2-amino-3,5-dibromobenzylamino)-
cyclohexyloxy)-6-(methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-
triyl triacetate (140 mg, 30%) as a light yellow solid. HPLC:
13.3 min. LCMS: m/z 694. 1H NMR (400 MHz, CDCl3)d: 7.46 (d,
J = 2.0 Hz, 1H), 7.08 (d, J = 2.0 Hz, 1H), 5.31 (bs, 2H), 5.23 (m, 2H),
4.96 (dd, J = 7.5, 9 Hz, 1H), 4.64 (d, J = 7.5 Hz, 1H), 4.02
(d, J = 9.5 Hz, 1H), 3.76 (m, 2H), 3.75 (s, 3H), 3.62 (m, 1H), 2.46
(m, 1H), 2.03 (s, 3H), 2.01 (s, 6H), 1.95 (m, 4H), 1.42 (m, 2H), 1.12
(m, 2H). 13C NMR (100 MHz, CDCl3)d: 170.7, 169.8, 169.6, 167.8,
144.3, 133.7, 131.8, 127.2, 110.9, 108.8, 99.9, 78.5, 77.7, 73.1, 72.6,
71.9, 70.0, 55.6, 53.3, 51.1, 31.7, 31.0, 30.3, 21.1, 21.0.
Acknowledgement
We are in debt to our colleagues Dr Tom MacGregor and Peter
Grob for critical reading of this manuscript, to Scott Pennino for
obtaining high-resolution mass spectra for compounds labeled
with stable isotopes, and to Dr Nelu Grinberg and Dr Ma Shengli
for HPLC analysis of ambroxol isomers, and Dr Nizar Haddad for
technical assistance.
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