Isotopically labelled tropane alkaloids. The synthesis of (RS)-[3′, 3′-2H2]- and (RS)-[1′-13C, 3′, 3′-2H2]- hyoscyamines for metabolism studies in plants

Article abstract of DOI:10.1002/jlcr.543

A synthetic route to isotopically labelled forms of the tropane alkaloid hyoscyamine, including (RS)-[3′, 3′,-²H₂]- (2a) and (RS)-[1′-¹³C, 3′, 3′,-²H₂]- (2b) hyoscyamines, involving the reaction betwe

Full text of DOI:10.1002/jlcr.543

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 191–198.
DOI: 10.1002/jlcr.543
Research Article
Isotopically labelled tropane alkaloids.
0
0
2
The synthesis of (RS)-[3 , 3 - H ]- and
2
0
13
0
0
2
(
for metabolism studies in plants
RS)-[1 - C, 3 , 3 - H ]- hyoscyamines
2
Stephen Patterson and David O’Hagan*
School of Chemistry, Centre for Biomolecular Sciences, University of St.
Andrews, North Haugh, St. Andrews, Fife, KY16 9ST, UK
Summary
A synthetic route to isotopically labelled forms of the tropane alkaloid
0
0
2
hyoscyamine, including (RS)-[3, 3,- H ]- (2a) and (RS)-[1 - C, 3, 3,- H ]- (2b)
0
13
0
0
2
2
2
hyoscyamines, involving the reaction between phenylacetyl tropine and
formaldehyde is described. The isotopically labelled products enable the
metabolism of hyoscyamine to be studied in plants such as Datura
stramonium. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: tropane alkaloids; tropic acid; hyoscyamine; Datura stramonium;
biosynthesis
Introduction
The tropane alkaloids littorine (1) and hyoscyamine (2) are produced by
several plant species including henbane (Hyoscyamus niger), mandrake
(Mandragora officinarum) and thornapple (Datura stramonium). Hyos-
cyamine has a rich folklore as a hallucinogenic alkaloid and has been a
*Correspondence to: D. O’Hagan. School of Chemistry, Center for Biomolecular Sciences,
University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST UK.
E-mail: dol@st-andrews.ac.uk
Copyright # 2002 John Wiley & Sons, Ltd.
Received 22 October 2001
Accepted 24 October 2001

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92
S. PATTERSON AND D. O’HAGAN
topic of medical research primarily due to its potency as a muscarinic
1
antagonist and its myriadic properties in optometery.
We and others have been investigating the biosynthesis of hyoscya-
2
ꢀ5
mine fora numberof yea rs ,
acid estermoiety. It has been shown that litto ri ne ( 1) is the immediate
and in particular the origin of the tropic
6
precursor to hyoscyamine (2). The conversion of littorine (1) to
hyoscyamine (2) involves an intriguing intra-molecular isomerization
ꢀ9
7
shown in Scheme 1. Despite a numberof p ro posals
the rearrangement is unknown.
the mechanism of
Scheme 1. Hyoscyamine (2) is formed by an intramolecular rearrangement of
littorine (1). There is no evidence for the reversible oxidation of hyoscyamine (2)
to aldehyde (3)
Previous results from our laboratory involving isotopic labelling
0
18
experiments with [2 - O, H]-littorine in D. stramonium, demon-
2
10
18
0
strated a 71% retention of O at the C-3 oxygen of hyoscyamine during
the rearrangement of littorine (1) to hyoscyamine (2). Liberation of the
1
8
8
O (29%) could arise either during the rearrangement orhas been
9
suggested as a consequence of the subsequent reversible metabolism of
hyoscyamine (2) by a redox enzyme to aldehyde (3). Clearly if aldehyde
(
18
3) is generated then the O could have exchanged to some extent with
the aqueous medium in the plant cells. In an attempt to delineate these
two processes a synthetic route has been developed which allowed the
0
preparation of hyoscyamine 2 carrying deuterium at the 3 positions.
The level of reversible in vivo oxidation in D. stramonium plants, at the
0
C3 hydroxyl group could then be assessed by monitoring the loss of
0
deuterium at C3 by mass spectrometry. The present paper reports the
0
0
2
preparation of (RS)-[3 , 3 - H ]- and [1 - C, 3 , 3 - H ]-hyoscyamines
0
13
0
0
2
2
2
using a novel two step strategy for hyoscyamine synthesis.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 191–198

ISOTOPICALLY LABELLED TROPANE ALKALOIDS
193
Results and discussion
0
A synthetic method was developed for the preparation of (RS)-[3 ,
2
0
0
13
- H ] and [1 - C, 3 , 3 - H ]-hyoscyamine as illustrated for (RS)-
2 2
0
0
2
3
0
13
1 - C, 3 , 3 - H ]-hyoscyamine in Scheme 2.
2
0
0
2
[
0
13
Scheme 2. Synthesis of (RS)- [1 - C, 3 , 3 - H ]-hyoscyamine. i HCl(g) 1458C.
0
0
2
2
o
2
3
h. 79% (ii) (a) LDA/THF –78 C, 30 min. (b) [ H ]-formaldehyde(g) and (c)
2
H O, 56%
2
0
13
1 - C]-Phenylacetyl tropine (4a) was prepared following the classical
[
preparation of tropine esters by acid catalysed esterification of tropine
1
5) and [1- C]-phenylacetic acid (6a) using dry HCl gas. The product
3
6
(
ester was dissolved in dilute sulfuric acid, treated with base and
extracted into an organic solvent. The ester was recovered by this
0
13
method in good yield. The resultant [1 - C]-phenylacetyl tropine (4a)
was treated with LDA in dry THF at ꢀ788C. This generates the enolate
anion, which was then quenched with [ H ]-formaldehyde gas. The
gaseous formaldehyde was generated by the depolymerization of
2
11
2
o
paraformaldehyde at 150 C and bubbled into the reaction using a
stream of dry nitrogen. Aqueous workup and extraction into an organic
solvent gave a mixture of hyoscyamine and unreacted phenylacetyl
tropine (44 : 56). The resultant hyoscyamine was purified by preparative
thin layer chromatography (t.l.c.). This synthetic methodology was also
0
0
2
used to prepare a sample of [3 , 3 - H ]-hyoscyamine from unlabelled
2
phenylacetyl tropine. The method is particularly attractive due to the
1
3
2
commercial availability of [1- C]-phenylacetic acid and [ H ]-parafor-
2
maldehyde, and accordingly hyoscyamine can now be prepared in a
variety of labelled combinations.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 191–198

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S. PATTERSON AND D. O’HAGAN
A previous synthetic route to hyoscyamine has involved the
1
2
acid catalysed esterification of tropic acid and tropine. However,
direct esterification is complicated by a competing inter-molecular
esterification of tropic acid, due to its reactive primary alcohol.
Indeed, to circumvent this problem hyoscyamine has recently
been prepared from tropic acid using a four step protection, activa-
1
3
tion, coupling and deprotection sequence. In the context of these
alternative approaches the current method offers a direct synthetic
0
approach, and is amenable to the introduction of isotopes at the 3 -site
of hyoscyamine.
The isotopically labelled hyoscyamines (2a) and (2b) were incubated
at a final concentration of 0.25 mM with transformed root cultures of
1
0,14
D. stramonium over21 days, following established po rtocols.
Aliquots were extracted for analysis at five day intervals between days
1
1 to 21, forGC-MS analysis. In the event the re was no indication of
0
any washout of deuterium from the C-3 position of hyoscyamine (2)
even over these extended time periods and thus there is no evidence that
hyoscyamine (2) is involved in an in vivo reversible oxidation to
aldehyde (3). These and other related results will be reported elsewhere
in a more detailed analysis of the mechanism of the conversion of
littorine (1) to hyoscyamine (2).
Experimental
All isotopically labelled compounds were purchased from The Aldrich
Chemical Co. and used without further purification. THF was dried
over sodium wire and distilled prior to use. NMR spectra were recorded
1
13
on a Brucker Avance 300 MHz ( H at 300.06 MHz, C at 75.45 MHz)
1
13
and Varian Unity Plus 300 MHz ( H at 299.99 MHz, C at 75.43 MHz)
spectrometers. Chemical shifts are quoted relative to CHCl in CDCl
3
3
(
d 7.26 or d 77.0 ppm), and coupling constants are given in Hertz.
H
C
GCMS analyses were conducted using an Agilent 5890 plus gas
chromatograph equipped with a 5973 N mass selective detector and
683 series injector. The carrier gas was helium. Chromatographic
7
separations were performed using a chiral SGE column (fused silica,
cydex-B, 25 mm ꢁ 0.22 mm with a film thickness of 0.25 mm).
Preparative t.l.c. were performed on Whatman K6F silica gel 60 A,
(
(0.25 mm) plates with fluorescent indicator.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 191–198

ISOTOPICALLY LABELLED TROPANE ALKALOIDS
195
Phenylacetyl tropine (4)
A solid phase mixture of phenylacetic acid (408 mg, 3.0 mmol) and
tropine (424 mg, 3.0 mmol) was heated to 1458C under a stream of dry
nitrogen. A stream of dry hydrochloric acid gas was then passed over
the mixture for 3 h with stirring. After cooling the reaction mixture was
dissolved in sulfuric acid (50 mM, 20 ml), and it was then basified with
ammonia solution (35%) and the product was extracted into chloro-
form (4 ꢁ 30 ml). The solvent was removed under reduced pressure to
yield the product as a clear oil (620 mg, 80%).
1
H-NMR (300 MHz) (CDCl ): 7.32–7.18 (5H, m, aromatics), 4.93
3
0
(
1H, m, C3-H), 3.55 (2H, s, C2 -H ), 2.96 (2H, m, C1-H, C5-H), 2.18
2
2
3
(
3H, s, NCH ), 2.03 (2H, dt, J =15.0, JHH=4.3, C2-Ha, C4-Ha),
3
HH
1
.82 (2H, m, C6-Ha, C7-Ha), 1.57 (4H, m C2-Hb, C4-Hb, C6-Hb,
C7-Hb).
1
3
0
C-NMR (75 MHz) (CDCl ): 170.4 (C1 ), 133.9, 129.1, 128.4, 126.8
3
0
(aromatics), 67.8 (C3), 59.5 (C1, C5), 42.1 (C2 ), 40.2 (NCH ), 36.3 (C2,
C4), 25.3 (C6, C7).
3
+
GCMS (EI): 259 (M , 29%), 140 (9.5%), 124 (100%), 94 (19%),
1 (18%), 82 (21%), 67 (9.5%).
9
0
13
1 - C]-Phenylacetyl tropine (4a)
[
The title compound was prepared following the method for phenyla-
13
cetyl tropine using [1- C]-phenylacetic acid (411 mg, 3.0 mmol). Yield
(620 mg, 79%).
1
H-NMR (300 MHz) (CDCl ): 7.32–7.20 (5H, m, aromatics), 4.95
3
2
1H, m, C3-H), 3.56 (2 H, d, J =7.8, C2 -H ), 2.98 (2H, m, C1-H,
0
(
C5-H), 2.19 (3H, s, NCH ), 2.05 (2H, dt, J =15.0, JHH=4.3,
CH
2
2
3
3
HH
C2-Ha, C4-Ha), 1.83 (2H, m, C6-Ha, C7-Ha), 1.59 (4H, m, C2-Hb,C4-
Hb, C6-Hb, C7-Hb).
C-NMR (75 MHz) (CDCl ): 170.4 (C1 ), 133.9, 129.1 128.3, 126.8
1
3
0
3
0
1
(
aromatics), 67.8 (C3), 59.5 (C1/C5), 42.1 (C2 , d, JCC=57.6), 40.2
(
NCH ), 36.3 (C2/C4), 25.3 (C6/C7).
3
+
GCMS (EI): 260 (M , 16%), 140 (8%), 124 (100%), 94 (22%), 91
(24%), 82 (26%), 67 (11%).
(
RS)-Hyoscyamine (2)
n-BuLi (1.6 M in hexanes; 1.9 ml, 3.0 mmol) was added to diisopropyl-
amine (3 mmol, 420 ml) in THF (10 ml) at 08C, and the reaction was
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 191–198

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S. PATTERSON AND D. O’HAGAN
stirred for 30 min. The reaction was then cooled to –788C and
phenylacetyl tropine (518 mg, 2 mmol) in THF (5 ml) was added slowly
over 5 min and then the reaction was stirred for a further 30 min. The
reaction mixture was warmed to ꢀ208C and formaldehyde gas,
generated by heating paraformaldehyde (300 mg, 10.0 mmol) to
1
508C, was bubbled through the mixture with a stream of dry nitrogen,
until all of the paraformaldehyde had sublimed. After warming to
ambient temperature the reaction was quenched by the addition of
water (10 ml). The aqueous layer was separated and extracted into
diethylether(4 ꢁ 30 ml). The organic extracts were combined and
evaporated under reduced pressure to yield a pale orange gum
(480 mg) which was purified over silica gel (CHCl : MeOH : NEt ,
3 3
75 : 25 : 1) to yield hyoscyamine (121 mg, 21%) and recovered phenyla-
cetyl tropine (164 mg). For GCMS analysis the TMS derivative was
1
5
prepared using MSTFA.
1
H-NMR (300 MHz) (CDCl ): 7.39–7.22 (5H, m, aromatics), 4.99
3
3
0
0
(
1H, t, J =5.2, C3-H), 4.16 (1H, m, C3 -Ha), 3.79 (2H, m, C3 -Hb,
C2 -H), 3.02 (1H, m, C1/C5-H), 2.90 (1H, m, C1/C5-H), 2.18 (3H, s,
NCH ), 2.05 (2H, m, C2-Ha, C4-Ha), 1.72 (3H, m, C6-Ha, C7-Ha,
HH
0
3
C6/C7-Hb), 1.59 (1H, m, C2/C4-Hb), 1.54 (1H, m, C2/C4-Hb), 1.18
(
1H, m, C6/C7-Hb).
1
3
0
C-NMR (75 MHz) (CDCl ): 172.3 (C1 ), 135.6, 128.8, 128.1, 127.7
3
0
(
(
(
aromatics), 68.1 (C3), 64.1 (C3 ), 59.6 (C1/C5), 59.5 (C1/C5), 54.4
0
C2 ), 40.2 (NCH ), 36.3 (C2/C4), 36.1 (C2/C4), 25.4 (C6/C7), 24.8
3
C6/C7).
NB where two carbon numbers are given for diastereomeric carbon
e.g. C6/C7 the assignment is uncertain.
+
GCMS (EI): 361 (M , 17%), 140 (7%), 124 (100%), 104 (7%), 94
(11%), 82 (13%), 73 (10%), 67 (7%).
0
0
2
(
RS)-[3 , 3 - H ]-Hyoscyamine (2a)
2
2
The title compound was prepared as (RS)-hyoscyamine using [ H ]-
2
formaldehydegas (538 mg, 16.8 mmol). The reactionyielded a paleorange
oil (520 mg) which was estimated to be 38% hyoscyamine by GC analysis.
Aliquots were purified by preparative t.l.c. (CHCl : MeOH : Net ,
3
3
7
of product was recovered from 30 mg of the oil).
5 : 25 : 0.5), and the silica extracted into chloroform/methanol (10 mg
1
H-NMR (300 MHz) (CDCl ): 7.38–7.24 (5 H, m, aromatics), 5.03
3
3
1 H, t, J =5.4, C3-H), 3.78 (1H, s, C2 -H), 3.04 (1H, m, C1/C5-H),
0
(
HH
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 191–198

ISOTOPICALLY LABELLED TROPANE ALKALOIDS
197
2
.92 (1H, m, C1/C5-H), 2.20 (3H, s, NCH ), 2.07 (2H, m, C2-Ha, C4-
3
Ha), 1.75 (3H, m, C6-Ha, C7-Ha, C6/C7-Hb), 1.60 (1H, m, C2/C4-Hb),
1
.55 (1H, m, C2/C4-Hb), 1.15 (1H, m, C6/C7-Hb).
C-NMR (75 MHz) (CDCl ): 172.0 (C1 ), 135.7, 128.7, 128.0, 127.6
1
3
0
3
0
(aromatics), 67.4 (C3), 59.8 (C1/C5), 59.7 (C1/C5), 54.5 (C2 ), 39.8
(NCH ), 35.6 (C2/C4), 35.8 (C2/C4), 25.1 (C6/C7) 24.6 (C6/C7). NB
The signal forC3 was below the detectable threshold due to J
3
0
1
CD
coupling.
+
GCMS (EI): 363 (M , 9%), 140 (7%), 124 (100%), 106 (8%), 94
13%), 82 (15%), 73 (13%), 67 (8%).
Mass abundances: 361 (M, 0.14%), 362 (M+1, 0.58%), 363 (M+2,
(
7
7.26%), 364 (M+3, 22.02%).
0
13
RS)-[1 - C, 3, 3 - H ]-Hyoscyamine (2b)
2
0
0
2
(
1
The title compound was prepared as (RS)-hyoscyamine using [1- C]-
3
2
phenylacetyl tropine (620 mg, 2.38 mmol) and [ H ]-formaldehyde gas
2
(382 mg, 11.9 mmol). The reaction yielded a pale orange gum (506 mg)
which was estimated to be 56% hyoscyamine by GC analysis. An
aliquot was purified by preparative t.l.c. (CHCl : MeOH : NEt ,
3
3
7
of product was recovered from 30 mg of the oil).
5: 2 5 : 0.5), and the silica extracted with chloroform/methanol (11 mg
1
H-NMR (300 MHz) (CDCl ): 7.36-7.23 (5 H, m, aromatics), 5.01
3
2
1 H, m, C3-H), 3.76 (1 H, d, J =7.4, C2 -H), 3.02 (1H, m, C1/C5-H),
0
(
CH
2
.90 (1H, m, C1/C5-H), 2.18 (3H, s, NCH ), 2.05 (2H, m, C2-Ha, C4-
3
Ha), 1.72 (3H, m, C6-Ha, C7-Ha, C6/C7-Hb), 1.59 (1H, m, C2/C4-Hb),
.54 (1 H, m, C2/C4-Hb), 1.18 (1 H, m, C6/C7-Hb).
1
1
3
0
C-NMR (75 MHz) (CDCl ): 172.2 (C1 ), 135.3, 128.8, 128.1, 127.7
3
1
aromatics), 68.1 (C3), 59.6, 59.5 (C1/C5), 54.2 (d, J =56, C2 ), 40.3
0
(
(
CC
0
NCH ), 36.4, 36.1 (C2/C4), 25.4, 24.9 (C6/C7). NB The signal forC3
3
1
was below the detectable threshold due to J coupling.
CD
+
GCMS (EI): 364 (M , 10%), 140 (6%), 124 (100%), 106 (8%), 94
(
12%), 82 (14%), 67 (8%).
Mass abundances: 361 (M, 0.06%), 362 (M+1, 0.14%), 363 (M+2,
.16%), 364 (M+3, 74.78%), 365 (M+4, 23.86%).
1
Acknowledgements
We thank the EPSRC fora studentship (S.P.).
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 191–198

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S. PATTERSON AND D. O’HAGAN
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