Simple synthesis of deuterated pterosines

Article abstract of DOI:10.1080/00397910801929366

Ptaquiloside, a potent carcinogen present in bracken fern, a plant consumed by farm animals, may be detected in traces by converting it into the bromopterosine. A simple synthesis of bromopterosine d2, to be used as standard in GC/MS or LC/MS analyses, is

Full text of DOI:10.1080/00397910801929366

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Simple Synthesis of Deuterated Pterosines
Gaetano Miele a , Roberta Costi a , Francesco Bonadies b & Rosario Nicoletti
b
a Department of Pharmaceutical Studies, “Sapienza” University of Rome,
Rome, Italy
b Department of Chemistry, “Sapienza” University of Rome, Rome, Italy
Available online: 24 Apr 2008
To cite this article: Gaetano Miele, Roberta Costi, Francesco Bonadies & Rosario Nicoletti (2008): Simple
Synthesis of Deuterated Pterosines, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 38:10, 1553-1559
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Synthetic Communicationsw, 38: 1553–1559, 2008
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910801929366
Simple Synthesis of Deuterated Pterosines
Gaetano Miele,1 Roberta Costi,1 Francesco Bonadies,2
and Rosario Nicoletti2
1Department of Pharmaceutical Studies, “Sapienza” University of Rome,
Rome, Italy
2Department of Chemistry, “Sapienza” University of Rome, Rome, Italy
Abstract: Ptaquiloside, a potent carcinogen present in bracken fern, a plant consumed
by farm animals, may be detected in traces by converting it into the bromopterosine. A
simple synthesis of bromopterosine d2, to be used as standard in GC/MS or LC/MS
analyses, is described.
Keywords: Deuterated compounds, pterosines, ptaquiloside, sesquiterpene glucoside
Ptaquiloside (Pta, I) is a sesquiterpene glucoside, isolated from a variety of
bracken fern (Pteridium aquilinum, var. latiusculum), whose structure was
fully elucidated in 1987.[1] The same substance and several analogs have
been identified in other pteridacae.[2] Bracken fern is a plant widely distribu-
ted all over the world, and it is currently consumed by farm animals. Because
Pta is a potent carcinogen, the consumption of fern causes a number of well-
known syndromes.[3] In large ruminants, chronic enzootic haematuria, the
clinical expression of multiple neoplasia of the urinary bladder, occurs. Pta
has been shown also in laboratory experiments to be carcinogenic,[4] and it
seems to act synergistically with bovine papilloma virus type 2 (BPV-2), a
well-known virus associated with neoplastic pathology of the bovine
urinary bladder.[5] These adverse properties of Pta have been related to its
electrophilic nature and its capability to react with (alkylate)[6] DNA.
The growing public awareness of the risk implied in continuous exposure
to carcinogenic substances present in foods has prompted some researchers to
Received September 6, 2007
Address correspondence to Gaetano Miele, Department of Pharmaceutical Studies,
“Sapienza” University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy. E-mail:
gaetano.miele@uniroma1.it
1553

1554
G. Miele et al.
evaluate the possibility that Pta may represent a pollutant of foods derived
from cattle.[7] Methods of detection of this substance at ppb level are changel-
ling: Pta (I), a glucoside, is unstable in both acid and basic conditions. In basic
conditions, Pta undergoes a b-elimination, affording the dienone(II), which is
converted by acids into pterosin B (Ptb, III)[8] (Fig. 1).
These difficulties have been overcome by converting Pta into the stable
Ptb during sample preparation. Detection and quantitative determinations of
this product (an aromatic ketone) are currently achieved using high pressure
liquid chromatography (HPLC)[9] with ultra violet (UV) detection. To our
knowledge, no methods that directly detect the Pta have been published.
The wide interest in this substance, considered a potential dangerous
pollutant of some foods (i.e., milk, cheese, meat), prompted us to study the
whole analytical problem with the aim to use MS in detecting traces of ptaqui-
loside. We found[10] that Ptb can be revealed by gas chromatography/mass
spectrometry (GC/MS), and ptaquiloside may be converted in a bromopter-
osine during sample preparation. Bromopterosine may be detected by GC/
MS as whitness of the presence of ptaquiloside: this procedure appears to
be more reliable than those based on the identification of Ptb, a possible
decay product of Pta in body fluids. Pterosines are naturally occurring
indanones, isolated from plants and certain fungi.[11]
Quantitative analyses by GC/MS require standards, and to this purpose
the best substances are deuterated analogs of analytes. Then we decided to
prepare deuterated Ptb and bromopterosine: a straightward synthesis of
these substances is reported here.
Several synthetic routes have been proposed since 1974 to prepare
pterosine derivatives.[12] The benzene moieties with appropriate substituents
are the starting material to add the (condensed) five-terms ring, and inter
alia, the use of a methacrylic ester in a one-step reaction—PPA as condensing
agent—has been reported.[13] This route seemed to be suitable for a synthesis
Figure 1. Instability of Pta (I) in acid and basic conditions.

Simple Synthesis of Deuterated Pterosines
1555
of deuterate analogs: its drawback was the very low yield reported for the last
step (15%). We succedeed in modifying this reaction by using the anhydride
of methacrilic acid (commercially available) instead of the ester and by
carring out the reaction at room temperature for 5 days. Yields are fairly
good (.80%) in these conditions.
The synthetic route of deuterated substrates is reported in Scheme 1. All
the substances isolated gave satisfactory NMR spectra, and pure bromoptero-
sine-d2 is now currently used in our laboratories for analytical purposes. In the
Scheme 1. (a) Acetyl chloride, ZnCl2, CH2Cl2, 1 h, room temperature, yield 100%;
(b) HCl 37%, CH3OH, 1 night, room temperature, yield 100%; (c) paraformaldheide,
HCl 37%, 2 h, T ¼ 60 8C, yield 87%; (d) acetyl chloride, ZnCl2, CH2Cl2, 4 h, reflux,
yield 100%; (e) lithium alluminium deuteride, THF, 1 h, room temperature, yield
100%; (f) acetyl chloride, CH2Cl2, pyridine, 1 night, room temperature, yield 75%;
(g) PPA, metacrilic anidride, 5 days, room temperature, yield 86%, (h) MsCl,
Et3NH, DMAP, CH2Cl2, room temperature, yield 86.2%; (i) lithium bromide, acetone,
1 night, reflux, yield 90%; (l) PPA, metacrilic anidride, 5 days, room temperature, yield
82%; (m) HCl 37%, CH3OH, 1 night, room temperature, yield 100%.

1556
G. Miele et al.
experimental section, the reactions not yet reported in literature are described
(Scheme 1).
EXPERIMENTAL
Chemistry
Melting points were determined with a Buchi 530 capillary apparatus and are
uncorrected. Infrared (IR) spectra were recorded on a Spectrum-One spectro-
photometer. 1H NMR spectra were recorded on a Bruker AC 400 spec-
trometer, using tetramethylsilane (Me4Si) as an internal standard. All
compounds were routinely checked by thin-layer chromatography (TLC)
and 1H NMR. TLC was performed by using aluminium-baked silica-gel
plates (Fluka F254) and aluminium-baked aluminium oxide plates (Fluka
F254). Concentration of solutions after reactions and extractions involved
the use of a rotatory evaporator operating at a reduced pressure of approxi-
mately 20 Torr. Organic solutions were dried over anhydrous sodium
sulfate. Mass spectra were recorded with gascromatography HP 5890
supplied with mass spectrometer (MS) HP 5971A at 70 eV.
2-(2-Chloromethylphenyl)-ethanol acetate (1)[13]
This compound was prepared as reported in Reference 13.
2-(2-Chloromethyl-phenyl)-ethanol (2)
To a solution of compound 1 (23.5 mmol, 5.0 g) in methanol (35 ml), HCl
37% (1.5 ml) was added. The solution was stirred at room temperature for 1
night. Water was added, and the resulted suspension was extracted with
chloroform (3 Â 50 mL). The organic extracts were collected, washed with
brine (3 Â 100 mL), and dried. Evaporation of the solvent gave 4.58 g of
compound 2 (yield 100%). IR: n 3325 cm21 (OH); 1H NMR (CDCl3):
d3.08 (t, 2H, CH2), 3.97 (t, 2H, CH2), 4.7 (s, 2H, CH2), 7.28–7.43 (m, 4H,
1
benzene H). H NMR (CDCl3) d3.00 (t, 2H, CH2), 4.09 (t, 2H, CH2), 4.64
(s, 2H, CH2), 4.85 (s, 2H, CH2), 7.04 (m, 1H, benzene H), 7.23–7.28
(m, 2H, benzene H).
5-chloromethyl Isochromane (3)[13]
This compound was prepared as reported in Reference 13.
2-(2,6-Bis-chloromethylphenyl) Ethanol Acetate (4)[13]
This compound was prepared as reported in Reference 13.

Simple Synthesis of Deuterated Pterosines
2(2,6-Diethyl-phenyl)-ethanol-d2 (5)
1557
A solution of compound 4 (11.5 mmol, 3.0 g) in anhydrous tetrahydrofuran
(8.0 ml) was slowly added to a suspension of lithium aluminum deuteride
(34.5 mmol, 1.45 g) in anhydrous tetrahydrofuran (11.0 ml). The reaction
was stirred at room temperature for 1 h and diluted with water. The suspension
was filtered, and the residue was washed with THF. The organic solvent was
evaporated, and then the acqueous layer was extracted with chloroform. The
organic extracts were collected, washed with brine, and dried. The evaporation
of the solvent gave 1.75 g of compound 5 (yield 100%). IR: n 3328 cm21
(OH); 1H NMR (CDCl3): d 2.41 (m, 4H, CH2), 3.03 (t, 2H, CH2), 3.82
(t, 2H, CH2), 7.09 (m, 3H, benzene H).
Methanesulfonic Acid 2-(2,6-Diethyl-phenyl)-ethyl Ester-d2 (6)
A solution of methanesulfonyl chloride (5.99 mmol, 0.46 ml) in dichloro-
methane (31.5 ml) was slowly added to a solution of compound 5 (0.9 g,
5.99 mmol), triethylamine (12.0 mmol, 1.6 ml), and a catalytic amount of
DMAP in dichloromethane (31.5 ml). The reaction was stirred at room
temperature for 1 night. Water was added, and the suspension was extracted
with chloroform. The organic extracts were collected, washed with brine,
and dried. The evaporation of the solvent gave the crude product (1.10 g),
which was chromatographed on a silica-gel column (ethyl acetate/
n-hexane, 1:2, as eluent) to obtain 0.72 g of compound 6 (yield 86.2%).
1
IR: See inter alia n 1351 cm21 (SO2); H NMR (CDCl3): d 2.41 (m, 4H,
CH2), 2.93 (s, 3H, CH3), 3.21 (t, 2H, CH2), 4.35 (t, 2H, CH2), 7.08–7.14
(m, 3H, benzene H).
2-(2-Bromo-ethyl)-1,3-diethyl-benzene-d2 (7)
Lithium bromide (2.19 mmol, 0.19 g) was added to a solution of compound
6 (2.19 mmol, 0.5 g) in acetone (8.0 ml). The reaction was stirred at
room temperature for 27 h. Lihium bromide was added (2.19 mmol, 019 g)
and then refluxed for 3 h. After evaporation of the solvent, the crude
product was diluted with water and extracted with chloroform. The organic
layers were washed with brine and dried. The evaporation of the solvent
gave a crude yellow oil, which was chromatographed on a silica-gel
column (ethyl ether/n-hexane, 1:10, as eluent) to obtain 0.72 g of
1
compound 7 (yield 90%), mp 65 8C (n-hexane). H NMR (CDCl3): d 2.40
(m, 4H, CH2), 3.28 (m, 2H, CH2), 3.46–3.48 (m, 2H, CH2), 7.08–7.15
(m, 3H, benzene H).

1558
6-(2-Bromo-ethyl)-5,7-diethyl-2-methyl-indan-1-one-d2 (8)
G. Miele et al.
[13]
MS data m/z (abundance) 282–284 (25), 203 (100), 189 (35), 161 (7).
Acetic Acid 2-(2,6-Diethyl-phenyl)-ethyl Ester-d2 (9)
Acetyl chloride (6.70 mmol, 0.47 ml) was added to a solution of compound 5
(0.85 g, 5.58 mmol) and pirydine (6.70 mmol, 0.54 ml) in anhydrous dichlor-
omethane (44 ml). The reaction was stirred at room temperature for 1 night,
diluted with water, and extracted with chloroform. The organic extracts
were washed with brine and dried. The evaporation of the solvent gave
1.12 g of crude product, which was chromatographed on a silica-gel column
(ethyl acetate/n-hexane, 1:4, as eluent) to obtain 0.81 g of compound 9
(yield 75%). IR: n 1736 cm21 (CO); 1H NMR (CDCl3): d 2.13 (s, 3H,
COCH3), 2.41 (m, 4H, CH2D), 3.04–3.08 (m, 2H, CH2), 4.20–4.24 (m, 2H,
CH2), 7.07–7.10 (m, 3H, benzene H).
Acetic Acid 2-(4,6-Diethyl-2-methyl-3-oxo-indan-5-yl)-ethyl
Ester-d2 (10)
Methacrilic anhydride (3.83 mmol, 0.59 g, 0.61 ml) was added to a suspension of
compound 9 (2.57 mmol, 0.5 g) in PPA (15.7 g). The reaction was stirred for 5
days, diluted with water and ice, and extracted with chloroform. The organic
layers were collected, washed with brine, and dried. The evaporation of the
solvent gave the crude product (580 mg), which was chromatographed on a
silica-gel column (ethyl ether/n-hexane, 1:7, as eluent) to obtain compound
10 (yield 86%). 1H NMR (CDCl3): d 1.33 (d, 3H, CH3), 2.09 (s, 3H, COCH3),
2.48 (m, 2H, CH2), 3.62–2.73 (m, 4H, CH2D), 3.08–3.12 (t, 2H, CH2), 3.27–
3.33 (q, 1H, CHCO), 4.18–4.22 (t, 2H, CH2), 7.15 (s, 1H, benzene H).
5,7-Diethyl-6-(2-hydroxy-ethyl)-2-methyl-indan-1-one-d2 (11)
HCl 37% (0.1 ml) was added to a solution of compound 10 (0.28 mmol,
0.06 g) in methanol (2 ml). The solution was stirred at room temperature for
1 night. Water was added, and the resulting suspension was extracted with
chloroform (3 Â 50 mL). The organic extracts were collected, washed with
brine (3 Â 100 mL), and dried. The evaporation of the solvent gave 0.06 g
1
of compound 11 (yield 100%); IR: n 3325 cm21 (OH); H NMR (CDCl3):
d 1.33 (d, 3H, CH3), 2.48 (m, 2H, CH2), 2.61–2.72 (m, 4H, CH2D), 3.07
(t, 2H, CH2), 3.30 (q, 1H, CH), 3.81 (t, 2H, CH2), 7.15 (s, 1H, benzene H);
mass data m/z (abundance) 220 (50); 203 (71); 189 (100); 161 (8); 130 (17).

Simple Synthesis of Deuterated Pterosines
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1559
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