Synthesis of tritium- and deuterium-labeled budesonide

Article abstract of DOI:10.1002/jlcr.1480

Tritium-labeled budesonide was prepared by the selective reduction of a double bond in the butenylenedioxy side chain using carrier-free tritium and palladium on carbon as a catalyst in absolute ethanol. Although the reduction gave a mixture of the desired product and the expected byproducts resulting from over reduction of the other double bonds in ring A, the desired tritium-labeled budesonide was easily isolated by reverse phase HPLC and with specific activity of 54 Ci/mmol. [D₈]-budesonide was prepared from 16α-hydroxyprednisolone and D₈-butyraldehyde in 1,4-dioxane in the presence of perchloric acid. The isotopic enrichment was found to be more than 99 atom% D. Copyright

Full text of DOI:10.1002/jlcr.1480

Research Article
Received 3 October 2007,
Revised 29 October 2007,
Accepted 1 November 2007
Published online 21 January 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1480
Synthesis of tritium- and deuterium-labeled
budesonide
Ã
Bachir Latli, Dhileep Krishnamurthy, and Chris Senanayake
Tritium-labeled budesonide was prepared by the selective reduction of a double bond in the butenylenedioxy side chain
using carrier-free tritium and palladium on carbon as a catalyst in absolute ethanol. Although the reduction gave a mixture
of the desired product and the expected byproducts resulting from over reduction of the other double bonds in ring A,
the desired tritium-labeled budesonide was easily isolated by reverse phase HPLC and with specific activity of 54 Ci/mmol.
[D8]-budesonide was prepared from 16a-hydroxyprednisolone and D8-butyraldehyde in 1,4-dioxane in the presence of
perchloric acid. The isotopic enrichment was found to be more than 99 atom% D.
Keywords: tritium; deuterium; radiosynthesis; budesonide; 16a-hydroxyprednisolone
Introduction
Results and discussion
Tritium-labeled budesonide with specific activities of 43 and
68 Ci/mmol was synthesized before, although no details were
given.10 An abstract described the preparation of tritium-labeled
budesonide with 36 Ci/mmol by selective reduction of one of
the two double bonds in ring A, and then reintroduction of this
double bond.11 In optimizing the conditions for introducing
tritium on budesonide, attempts were made to reduce
crotonaldehyde first with deuterium gas to butyraldehyde-2H2
and then coupling it to 16a-hydroxyprednisolone. The volatility
of the product (boiling point of 70–801C), the low yield of the
reduction, and the excess butyraldehyde required for the
coupling with 16a-hydroxyprednisolne prevented us from
pursuing this method. A similar approach would have been
to use an unsaturated butyl hemiketal, which would have a
higher boiling point, reduce the unsaturation, and then couple
it to 16a-hydroxyprednisolne using the above conditions.
Other ideas included the preparation of a budesonide analogue
with a terminal unsaturation on the butyl side chain. Reduction
of such a compound with tritium gas would be fast and the
over reduction observed in ring A will be minimized. However,
the presence of acids in the coupling of 3-butenal diethyl
Budesonide, (22R,S)-16a,17a-butylenedioxy-11b,21-dihydroxy-
pregna-1,4-diene-3,20-dione, is a glucocorticosteroid used in
inhalation treatment of asthma, rhinitis, skin, and the treatment
of inflammatory bowel disease.1–4 The mechanism of this anti-
inflammatory activity of budesonide and other glucocorticoster-
oids (GC) is only recently being investigated at the genetic level.
Budesonide and other GC play important roles in the expression
of key genes involved in the inflammatory response. GC interact
with an intracellular GC receptor, which shuttles them into the
nucleus and regulates the expression of genes such as those
encoding cytokines, cell adhesion molecules, and metallopro-
teinases known to be critical to both inflammation and the
immune response.5
Budesonide is a 1:1 epimeric mixture,1 resulting from an
asymmetric 16a, 17a-acetal group, see Scheme 1, with epimer
22R having 2–3 times higher topical glucocorticoid potency than
epimer 22S.6 (22R)-epimer has approximately twice the volume
of distribution, clearance, and in vitro liver biotransformation
rate as the 22S-epimer in man.7 In all reported syntheses of
budesonide, a mixture of 1:1 of epimers 22R and 22S is always
isolated.1 This ratio can be modified by using stronger acids in
the coupling of 16a-hydroxyprednisolone to butyraldehyde. For
example, the 22R-epimer was obtained with 99% epimeric
excess when a solution of 70% hydrogen fluoride was used at
01C.8 Budesonide followed the general metabolic pathways
reported for synthetic glucocorticoids. However, 22R- and
22S-epimers gave different metabolites, probably due to
substrate-selective oxidation of the non-symmetric 16a,17a-
acetal substituent.7 16a-Hydroxyprednisolone being the major
budesonide metabolite both in vitro, in rat, in mouse, in human
liver preparations, and in plasma after i.v. administration to
acetal for example with 16a-hydroxyprednisolone gave
a
mixture of products rising from the migration of the terminal
double bond. In this article, carrier-free tritium was used
to reduce a double bond in the alkyl side chain of carbon 22
using 10% palladium on carbon in absolute ethanol. The
precursor: (22R,S)-16a,17a-(2-butenyl)enedioxy-11b,21-dihydrox-
Department of Chemical Development, Boehringer Ingelheim Pharmaceuticals,
Inc. Research and Development Center, 900 Ridgebury Road, Ridgefield, CT
06877, USA
healthy subjects and arising solely from the 22R-epimer.7,9
.
*Correspondence to: Bachir Latli, Department of Chemical Development,
Boehringer Ingelheim Pharmaceuticals, Inc. Research and Development Center,
900 Ridgebury Road, Ridgefield, CT 06877, USA.
Non-enzymatic hydrolysis of budesonide, i.e. using strong
acids over prolonged periods of time, failed to produce 16a-
hydroxyprednisolone.
E-mail: blatli@rdg.boehringer-ingelheim.com
J. Label Compd. Radiopharm 2008, 51 64–67
Copyright r 2008 John Wiley & Sons, Ltd.

B. Latli et al.
OH
OH
O
O
O
O
HO
OH
OH
O
HO
H
1,4-dioxane
HClO4 (70%)
H
H
H
H
O
O
n H2
C3D7CDO (99.6 atom % D)
10% Pd/C
EtOH
1,4-dioxane
HClO4 (70%)
OH
n H
OH
21
O
20
24
C3D7
O
22
18
D
O
25
12
O
23
n H
HO
HO
19
O
O
17
11 13
B
C
16
1
9
2
14
15
8
10
H
H
H
A
H
7
O
5
O
3
6
4
94%
n = 2, D2-Budesonide
n = 3, T2-Budesonide
D8-Budesonide
Scheme 1
ypregna-1,4,23-triene-3,20-dione was prepared from reacting Gibbstown, NJ). NMR spectra were recorded with Bruker
16a-hydroxyprednisolone with crotonaldehyde in 1,4-dioxane 400 MHz spectrometer, using CDCl3 as a solvent and TMS peak
and a solution of 70% perchloric acid1 (Scheme 1). The reduction as a reference. All reagents were obtained from Aldrich Chemical
was first performed using deuterium gas and gave a mixture of Co. (Milwaukee, WI), except 16a-hydroxyprednisolone (technical)
deuterium-labeled budesonide and compounds where the was purchased from SICOR (Milan, Italy) and butyraldehyde-2H8
double bonds in ring A of the steroid were reduced as well. was obtained from CDN (Pointe-Claire, Quebec, Canada).
The tritium reduction was similar to the deuterium and gave the Solvents were of HPLC grade. Liquid scintillation counting was
same over-reduced products. The tritium-labeled budesonide carried out on a Beckman LS 5000TA, using Ready Safe cocktail
was isolated by HPLC, and its specific activity of 54 Ci/mmol was (Beckman, Fullerton, CA).
determined by comparison of the UV absorbance with that of a
standard analytical sample and liquid scintillation counting of
the isolated HPLC peak. Deuterium-labeled budesonide with
isotopic enrichment of more than 99% was also prepared as an
internal standard in the quantification of human plasma, urine,
and a tool to enable real characterization of the pharmacoki-
netic parameters of budesonide used in the therapy of asthma
and other diseases. Deuterium-labeled budesonide and deriva-
tives have been prepared before as well.12 [2H8]-Budesonide,
[(22R,S)-(22,23,23,24,24,25,25,25-2H8)-16a,17a-butylenedioxy-11b,21-
dihydroxypregna-1,4-diene-3,20-dione, for example, was pre-
pared from the [2H8]-butyraldehyde.13–15 However, the isotopic
composition was only 80% 2H8, 17% 2H7, and 3% 2H6 as
determined by mass spectrometry. The high content of 2H7 was
explained by an exchange of deuterium atom in the aldehyde
group during the oxidation of butanol-2H10 to butanal-2H8 using
sodium dichromate in concentrated sulfuric acid. The yield of
this oxidation was very poor too, about 15%.14 Butyraldehy-
de-2H8 with isotopic enrichment of more than 99% was
purchased from a commercial supplier and reacted according
to the literature13–15 with 16a-hydroxyprednisolone in 94% yield.
Mass spectroscopy showed that the product has more than 99%
HPLC
Reverse phase separations were accomplished on a Kromasil
C18 column (4.6 Â 150 mm) and a gradient mobile phase
30–50% acetonitrile in water in 30 mins, using Rainin HPLC
equipped with HPXL solvent delivery system and DYNAMAX
absorbance detector. Radio-HPLC was performed using HITACHI
L-6200A intelligent pump and Radiomatic Flo-oneBeta (Packard),
and LINEAR UVIS 200 set at 240 nm and Ultima flo AP cocktail
(Packard). The HPLC column was maintained at 351C with an
Eppendorf CH-30 column heater.
Synthesis
(22R,S)-16a,17a-(2-butenyl)enedioxy-11b,21-dihydroxypregna-1,4,23-
triene-3,20-dione
To a solution of crotonaldehyde (400 mL, 4.8 mmol) and 16a-
hydroxy-prednisolone (190 mg, 0.5 mmol) in dry 1,4-dioxane
(10 mL) were added five drops of 70% HClO4. After stirring for
5 min at room temperature, a solution of 10% K2CO3 was added
(10 mL) and the organic phase was extracted with CH2Cl2. The
combined organic solutions were dried (MgSO4), filtered, and
concentrated in vacuo to give 340 mg of crude product.
Purification by silica gel chromatography using 0–10% MeOH/
CHCl3 as eluent gave 320 mg of an oily residue, which was
crystallized from CH2Cl2:hexane (280 mg) of white crystals,
Rf = 0.7 in 30% MeOH/CHCl3. HPLC: 14.32 min as a 3:2 mixture
of C22-epimers. 1HNMR (CDCl3)(mixture): d 7.25(d, J = 10.1 Hz,
2
2
atom H and only traces of H7.
Experimental procedures
Materials and methods
Silica gel TLC was performed from analysis with pre-coated
aluminum sheets with fluorescent indicator (EM Separating,
J. Label Compd. Radiopharm 2008, 51 64–67
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

B. Latli et al.
Figure 1. Radio- and UV-chromatograms of tritium-labeled budesonide.
1H), 6.32(d, J = 10.1 Hz, 1H), 6.05(s, 1H), 5.95(m, 1H), 5.50(dd, acetonitrile and purified by HPLC. A second purification gave the
J = 4.2, 8.4 Hz), 5.30(dd, J = 7.58, 16.86 Hz), 5.22(d, J = 6.74 Hz), tritium-labeled budesonide of more than 99% radiochemical
4.98(d, J = 4.2 Hz), 4.92(d, J = 6.74 Hz), 4.6(m), 4.3(m), 3.8(m), purity (See Figure 1).
2.6(td, J = 5.75, 8.63), 2.48(m), 2.3(m), 1.85(t, J = 8.63 Hz), 1.78(m),
1.5(s, 3H), 1.1–1.4(m), 1.01(s), 0.98(s). CI-MS: MH1 (429, 70%), Synthesis of [2H8]-budesonide
411(100%).
To a mixture of 16a-hydroxyprednisolone (11.7 g, 28 mmol) in
dry 1,4-dioxane (435 mL) was added n-butyraldehyde-2H8 (5.0 g,
(22,23-2H2)-16a,17a-(22R,S)-butylenedioxy-11b,21-dihydroxypregna
-1,4-diene-3,20-dione (2H2-budesonide)
62.5 mmol) and a solution of 70% HClO4 (1.65 mL). The resulting
clear solution was stirred at room temperature for 1 h before it
A mixture of the above compound (2.07 mg, 4.83 mmol), 10% Pd/
C (0.5 mg), a stirring bar, and absolute ethanol (100 mL) in a
2.2 mL glass vessel, was attached to a deuterium manifold. After
degassing the mixture using three cycles of freeze–thaw under
argon atmosphere, 4.95 mmol of deuterium gas was then
introduced. The mixture was warmed to room temperature
and stirred for 2.5 h. The vessel was then removed from the
manifold and filtered through a Gillman PTFE 0.4 mm filter and
washed with 2.0 mL of ethanol. The product was analyzed by
HPLC. Starting material, Rt = 14.32 min, 46%; an unidentified
byproduct, Rt = 15.43 min, 19%; the desired deuterated budeso-
nide, Rt = 17.93 min, 22%; and over reduced products, Rt = 19.29,
11%, and 22.37 min, 2%.
was diluted with CH2Cl2 (600 mL) and washed with a solution of
10% K2CO3 (200 mL), water, and brine. The organic solution was
dried (MgSO4), filtered, and concentrated in vacuo to give 14.4 g
of a viscous oil that was dissolved in CH2Cl2 and precipitated
with hexane. The precipitate was filtered and then purified by
flash chromatography to give 13.5 g. The purity of the product
was only 92% by HPLC analysis. Hence, the product was
crystallized twice from methanol to 98% pure. LC-MS showed
only traces of [2H7]-budesonide. 1H NMR (CDCl3): d 7.28(d,
J = 101 Hz, 1H), 6.31(d, J = 10.1 Hz, 1H), 6.05(br s, 1H), 5.2(d,
J = 6.87 Hz, 0.3 H), 4.92(d, J = 5.5 Hz, 0.55H), 4.65(dd, J = 5.5,
20.6 Hz, 0.3H), 4.51(m), 4.29(dd, J = 4.2, 20.6 Hz, 1H), 4.22(dd,
J = 4.2, 20.6 Hz, 0.3H), 3.05(t, J = 6.87 Hz, 1H), 2.60(td, J = 4.67,
14.1 Hz, 1H), 2.38(dd, J = 4.61, 14.1 Hz, 1H), 2.1–2.25(m),
1.55–1.80(m), 1.48(s, 3H), 1.01(s, 1.15H), 0.95(s, 1.95H).
Synthesis of tritium-labeled budsonide
As seen before in the reduction with deuterium, a mixture
of (22R,S)-16a,17a-(2-butenyl)enedioxy-11b,21-dihydroxypregna-
1,4,23-triene-3,20-dione (2.0 mg, 4.67 mmol), 10% Pd/C (0.55 mg),
and a stirring bar in absolute ethanol (100 mL) in a 2.2 mL glass
vessel was introduced tritium gas (4.4 mmol). The specific activity
of tritium used was decay corrected and found to be 54 Ci/
mmol. The total activity of tritium in the vessel was 237 mCi. The
mixture was stirred for 3 h at room temperature and then
transferred to a 20 mL vial and diluted with 1.0 mL of water. The
mixture was filtrated through a Sep Pak (a reverse phase C18
cartridge that was washed with 5.0 mL of ethanol and 15 mL of
water prior to use) and washed with 10 mL of water to elute any
exchangeable tritium. The reaction products were then eluted
with acetonitrile and collected in five vials. The first vial was
0.5 mL and the rest each 1.0 mL per vial. The vials were then
counted for radioactivity. Most of the radioactivity was in vial 2
Conclusion
Budesonide, a glucocorticosteroid used in the treatment of
asthma, labeled with deuterium and tritium was needed to
perform metabolism and pharmacokinetics research. The
deuterium-labeled budesonide was prepared from 16a-hydro-
xyprednisoline and [2H8]-butyraldehyde. Tritium was incorpo-
rated by the reduction of a double bond in credesonide side
chain. Although the reduction gave a mixture of over reduced
products, tritium-labeled budesonide was easily isolated by
reverse phase HPLC (Figure 1). The specific activity of this
material was 54 Ci/mmol.
Acknowledgement
(97 mCi) and vial 3 (11 mCi). The aqueous phase contained We thank Dr Pan Peng for the LC-MS analysis of the deuterated
60 mCi of labile tritium. A fraction of vial 2 was diluted with budesonide.
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 64–67

B. Latli et al.
References
[8] T. Uszycka-Horawa, S. Jadwiga, W. Kroszczynski, Patent WO 92/
11280, 1992.
˚
[9] S. Edsbacker, P. Andersson, C. Lindberg, J. Paulson, A. Ryrfeldt,
¨
´
[1] A. Thalen, R. Brattsand, Drug Res. 1979, 29, 1687–1690.
´
A. Thalen, Drug Metab. Dispos. 1987, 15, 403–411.
[2] M. J. Ashton, C. Lawrence, J.-A. Karlsson, K. A. J. Stuttle,
C. G. Newton, B. Y. J. Vacher, S. Webber, M. J. Withnall, J. Med.
Chem. 1996, 39, 4888–4896.
[3] B. Kongkathip, N. Kongkathip, P. Khunnavutimanotum, U. Sakee,
Chem. Lett. 1998, 27, 1207–1208.
[4] K. N. Hofer, Ann. Pharmacotherapy 2003, 37, 1457–1464.
[5] W. J. Lukiw, R. P. Pelaez, J. Martinez, N. G. Bazan, Proc. Natl. Acad.
Sci. USA 1998, 95, 3914–3919.
˚
[10] G. Josson, A. Astrom, P. Andersson, Drug Metab. Dispos. 1995, 23,
¨
137–142.
¨
[11] M. Skrinjar, P. Strom, J. Label. Compd. Radiopharm. 1998, 41, 1030
(Abstract).
˚
´
[12] S. Edsba¨cker, P. Andersson, C. Lindberg, A. Ryrfeldt, A. Thalen, Drug
Metab. Dispos. 1987, 15, 412–417.
[13] Y. N. Li, B. Tattam, K. F. Brown, J. P. Seale, J. Chromatogr. B 1996,
683, 259–268.
´
[14] A. Thalen, Acta Pharm. Suec. 1982, 19, 327–354.
[15] K. Kronkvist, M. Gustavsson, A. K. Wendel, H. Jaegfeldt,
˚
[6] I. Elovaara, M. Lalla, E. Spare, T. Lehtimaki, P. Dastidar, Neurology
¨ ¨
1998, 51, 1703–1708.
¨
´
¨
[7] R. Brattsand, A. Thalen, K. Roempke, L. Kallstrom, E. Gruvstad,
¨
J. Chromatogr. A 1998, 823, 401–409.
J. Steroid Biochem. 1982, 16, 779–786.
J. Label Compd. Radiopharm 2008, 51 64–67
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org