Synthesis of [4-14C]-pelargonidin chloride and [4- 14C]-delphinidin chloride

Article abstract of DOI:10.1002/jlcr.1120

The synthesis of [4-¹⁴C]-pelargonidin chloride and [4- ¹⁴C]-delphinidin chloride via [formyl-¹⁴C]-2-(benzoyloxy)- 4,6-dihydroxybenzaldehyde, ω,4-diacetoxyacetophenone and ω,3,4,5-tetraacetoxyacetophenone is described. The first step comprised labelling of the carbonyl group of 2-(benzoyloxy)-4,6-dihydroxybenzaldehyde, verifying that the coupling with ω,4-diacetoxyacetophenone or ω,3,4,5-tetraacetoxyacetophenone under hydrogen chloride atmosphere resulted in the formation of [4-¹⁴C] labelled anthocyanidins. Copyright

Full text of DOI:10.1002/jlcr.1120

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 1151–1162.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1120
Research Article
Synthesis of [4-14C]-pelargonidin chloride and
[4-14C]-delphinidin chloride
Michael Kraus, Ellen Biskup, Elke Richling and Peter Schreier*
Chair of Food Chemistry, University of Wuerzburg, Am Hubland, D-97074 Wuerzburg,
Germany
Summary
The synthesis of [4-14C]-pelargonidin chloride and [4-14C]-delphinidin chloride via
[formyl-14C]-2-(benzoyloxy)-4,6-dihydroxybenzaldehyde, o,4-diacetoxyacetophenone
and o,3,4,5-tetraacetoxyacetophenone is described. The first step comprised labelling
of the carbonyl group of 2-(benzoyloxy)-4,6-dihydroxybenzaldehyde, verifying that
the coupling with o,4-diacetoxyacetophenone or o,3,4,5-tetraacetoxyacetophenone
under hydrogen chloride atmosphere resulted in the formation of [4-14C] labelled
anthocyanidins. Copyright # 2006 John Wiley & Sons, Ltd.
Received 26 June 2006; Revised 17 July 2006; Accepted 27 July 2006
Key Words: pelargonidin chloride; delphinidin chloride; 14C-labelling; anthocyanidin
Introduction
Anthocyanidins are polyphenolic substances belonging to the flavonoids, a
large and widespread group of secondary metabolites of higher plants.1 The
six major aglycons occurring in nature are cyanidin, delphinidin, malvidin,
pelargonidin, peonidin and petunidin. The structure of the aglycon is the so-
called flavylium cation as shown in Figure 1.
Usually, anthocyanidins occur in nature in their glycosylated forms, i.e. the
anthocyanins. These water soluble substances are responsible for the
characteristic red, blue or purple pigments of many flowers, seeds, leaves,
fruits and vegetables.2 Anthocyanins are also widely distributed in the human
*Correspondence to: Peter Schreier, Chair of Food Chemistry, University of Wuerzburg, Am Hubland,
D-97074 Wuerzburg, Germany. E-mail: schreier@pzlc.uni-wuerzburg.de
Contract/grant sponsor: Deutsche Forschungsgemeinschaft (DFG); contract/grant number: 211/21-1/-3
Copyright # 2006 John Wiley & Sons, Ltd.

1152
M. KRAUS ET AL.
3'
4'
2'
B
5'
8
1
O
2
1'
7
6
6'
A
C
3
4
5
position
3
5
6
7
3'
H
4'
5'
H
color at pH < 2
orange
violet
pelargonidin
delphinidin
cyanidin
1
2
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
H
H
H
H
H
H
OH
OH
OH OH
OH OH
OH OH
OH
H
H
red
peonidin
OH OMe OH
red
malvidin
OH OMe OH OMe violet
OH OMe OH OH violet
petunidin
Figure 1. Structures and colours of major anthocyanin aglycons (anthocyani-
dins) investigated at pH52
diet; consumption has been estimated to range from 180 to 215 mg/day in the
USA3–6 and around 82 mg/day in Finland.7
Anthocyanins have been associated with potentially beneficial effects on
various diseases. In several animal studies, chemopreventive properties of
anthocyanins have been postulated,8–10 raising the question towards the
underlying mechanism of action. Recently, delphinidin and malvidin were
found to be potent inhibitors of the epidermal growth factor receptor (EGFR)11
and of 3’,5’-cyclic adenosine monophosphate (cAMP) phosphodiesterases.12
Thus, as physiologically active compounds anthocyanins and their aglycons
are attractive substances for the production of so-called functional foods.13 In
order to perform risk/benefit evaluations, further studies, in particular,
comprehensive in vivo investigations about their metabolism, bioavailability,
distribution and excretion are required. For such purposes sufficient amounts
of anthocyanidins, preferably also in their 13C and 14C labelled form, are
required. In this paper, we describe the synthesis of [4-14C]-pelargonidin and
[4-14C]-delphinidin chloride based on the preparation of their unlabelled forms.
Results and discussion
The anthocyanidins, [4-14C]-pelargonidin chloride (1a) and [4-14C]-delphinidin
chloride (2a) were synthesized using 2-(benzoyloxy)-4,6-dihydroxybenzalde-
hyde (4, 4a) (Scheme 1). In the anthocyanidins, the 2,4,6-trihydroxybenzaldehyde
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1151–1162
DOI: 10.1002/jlcr

SYNTHESIS OF [4-14C]-PELARGONIDIN AND [4-14C]-DELPHINIDIN CHLORIDE 1153
O
O
*
*
HO
OH
HO
OH
HO
OBz
[carbonyl-14C]-DMF
BzCl/Et3N
acetone
POCl3/Et2O/CHCl3
43.9-47.5% yield
OH
OH
OH
* = 12C, 3
* = 14C, 3a
* = 12C, 4 (21%)
5
* = 14C, 4a(16-19%)
Scheme 1.
(3) moiety represents the A-ring; the carbonyl carbon atom becomes the 4-
position in the molecule. The B-ring derives from o,4-diacetoxyacetophenone
(6) and o,3,4,5-tetraacetoxyacetophenone (7) in 1 and 2, respectively. In order
to introduce the carbon-14 atom, a Vilsmeyer reaction was carried out
between phloroglucinol (1,3,5-trihydroxybenzene, 5) and [formyl-14C]-di-
methylformamide to yield 3a in 43.9 and 47.5% yield (two separate runs)
after purification by reverse phase high performance liquid chromatography
(RP18-HPLC).14 Acylation of 3 and 3a with benzoyl chloride/EtN3 followed
by HPLC (on silica gel) yielded 2-(benzoyloxy)-4,6-dihydroxybenzaldehyde in
21% (4) and 16–19% (4a, two separate runs), respectively.15 This reaction led
to a mixture of mono- and di-benzoylated products. Because of the small scale,
product purification was performed by RP18-HPLC.
The preparation of unlabelled and labelled pelargonidin chloride (1, 1a) and
delphinidin chloride (2, 2a) were performed by condensation of 4/4a and o,4–
diacetoxyacetophenone (6) or o,3,4,5-tetraacetoxyacetophenone (7), respec-
tively. Anisole was acylated by reaction with a mixture of chloroacetyl
chloride/AlCl3; additional AlCl3 was given to effect conversion of the methyl
ether 8 to the phenol 9 (yield 20%).16 Reaction of 9 with HOAc/Et3N,
followed by acylation with acetic anhydride yielded 6 via 10 in 30% yield
(two steps) (Scheme 2).17 Condensation of 6 with 4 in the presence of
anhydrous HCl/CH3OH/EtOAc, followed by deprotection with KOH/
aqueous methanol yielded pelargonidin chloride (1). The anthocyanidin 1
was crystallized as its HCl salt, converted to the corresponding picrate and
finally recrystallized as its HCl salt (yield 40%).18 Reaction of 6 with 4a in the
same manner, followed by purification using RP18-HPLC yielded [4-14C]-
pelargonidin chloride (1a) with a specific activity of 33.6 mCi/mg; 0.2 mg was
obtained (3.8%).
For the syntheses of 2 and 2a, first of all, acetylation of gallic acid (3,4,5-
trihydroxybenzoic acid) (11) provided 3,4,5-triacetoxybenzoic acid (12) in 79%
yield.19 Conversion of 12 to its acyl chloride 13 (yield, 91%) and subsequent
reaction of 13 with diazomethane yielded the diazoketone 14 (yield, 81%).18,20
Direct conversion of 14 to 7 by reaction with acetic acid was unsuccessful;
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

1154
M. KRAUS ET AL.
HO
RO
H3CO
ClCH2COCl
HOAc/Et3N
AlCl3/heptane
20% yield
CH3CN
40%
OAc
Cl
O
O
8, R = CH3
10
9, R = H
Ac2O
75%
OH
-
AcO
Cl
1. 4 or 4a
CH3OH/EtOAc(2:1)/HCl
+
HO
O
OAc
2. KOH/CH3OH
O
OH
6
*
OH
* = 12C, 1 (40%)
* = 14C, 1a
Scheme 2.
reaction of 14 with HCl/Et2O21 yielded o-chloro-3,4,5-triacetoxyacetophenone
(15) in 87% yield. Subsequent reaction of 15 with KOAc/Ac2O/HOAc yielded
7 in 75% yield.22 Delphinidin chloride (2) and [4-14C]-delphinidin chloride (2a)
were prepared by reaction of 7 with 4 (or 4a) using anhydrous HCl in EtOH/
EtOAc followed by reaction with KOH. Delphinidin chloride (2) was isolated
in 42% yield by crystallization from EtOH/HCl;20 2a was isolated directly from
the reaction mixture (along with its benzoate ester 16). Both were purified by
RP-18 HPLC; treatment of 16 with KOH yielded additional 2a which was
combined with the original 2a isolated directly. The combined yield was 5.5%;
the specific activity was 30.4 Ci/mg (Scheme 3).
Experimental
Reagents
[Formyl-14C]-dimethylformamide with a specific activity of 53 mCi/mmol was
purchased from American Radiolabelled Chemicals ARC, Inc. (St. Louis,
MO, USA) and was used without further purification. 1,3,5-Trihydroxyben-
zene (phloroglucinol), N,N-dimethylformamide (N,N-DMF), benzoyl chlor-
ide, 3,4,5-trihydroxybenzoic acid (gallic acid), phosphorus oxychloride
(POCl3) and 1 N hydrogen chloride (in diethyl ether) were purchased from
Fluka (Deisenhofen, Germany). Triethylamine, potassium acetate, trifluor-
oacetic acid and DiazaldTM were from Sigma Aldrich (Steinheim, Germany).
All chemicals were of analytical grade (purity>98%). The solvents for
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1151–1162
DOI: 10.1002/jlcr

SYNTHESIS OF [4-14C]-PELARGONIDIN AND [4-14C]-DELPHINIDIN CHLORIDE 1155
O
O
O
HO
AcO
AcO
OH
OH
Cl
Ac O
79%
SOCl
91%
HO
AcO
AcO
OH
OAc
OAc
11
12
13
CH N /Et O
81%
OH
O
O
OH
OH
-
N
Cl
AcO
AcO
OAc
1. 4 or 4a
1. HCl/Et O
15 (87%)
+
HO
O
EtOH/EtOAc
2. KOH
AcO
2. HOAc/AcO
KOAc/H SO
75%
AcO
OH
OAc
OAc
*
OR
14
7
* = C, R = H, 2
* = C, R = H, 2a
R = Bz; 16, 16a
Scheme 3.
synthesis were redistilled and stored on molecular sieve (5 A) before use.
Acetonitrile (HPLC grade) was purchased from Fisher Scientific (Loughbor-
ough, UK).
Analyses
The HPLC instrumentation comprised two Knauer 64 pumps, a Knauer
mixing chamber and a Knauer UV/VIS-Detector (Knauer, Berlin, Germany).
A Knauer Eurospher 100 C18 column (250 Â 4.6 mm, 5 mm) was used. The
mobile phase consisted of aqueous 0.1% trifluoroacetic acid (A) and
acetonitrile (B) (v/v). The gradient applied was 1–99% B in 45 min at a flow
rate of 1 ml/min and 25 ml- injection volumes were employed.
The purity of the unlabelled substances was controlled by HPLC diode
array detector (DAD). The HPLC-DAD system was a Hewlett-Packard 1100
HPLC gradient pump and a Hewlett-Packard 1100 photodiode array detector
(Waldbronn, Germany), equipped with a Wisp 710b autosampler (Waters,
Eschborn, Germany). Data acquisition and evaluation were performed with
ChemstationTM software (Hewlett-Packard, Waldbronn, Germany). A
Knauer Europher 100 C18 column (250 Â 4.0 mm, 5 mm) was used. The
mobile phase consisted of aqueous 0.1% formic acid (A) and acetonitrile (B)
(v/v). The gradient applied was 1–99% B in 40 min at a flow rate of 1 ml/min
and 20 ml-injection volumes were employed.
The specific activity of 1a and 2a was determined by liquid scintillation
counting (LSC) using a Rackbeta 1214 LKB Pharmacia scintillation counter
(Wallac, Freiburg, Germany) with 15 ml vessels of polyethylene (Packard
Copyright # 2006 John Wiley & Sons, Ltd.
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M. KRAUS ET AL.
Bioscience, Billerica, MA, USA) and Rotiszint Ecoplus1 (Roth, Karlsruhe,
Germany) as scintillation cocktail. Data evaluation was performed by Ultro-
Term1 Software (Wallac, Freiburg, Germany).
1H-NMR spectra were recorded on a Bruker AVANCE 400 NMR
spectrometer (Rheinstetten, Germany). As solvents DMSO-d6, methanol-d4
and CDCl3 (stored on mol sieve, 5 A) were used. Data evaluation was
performed by Mestre-C 4.4.1.0 software.
Mass spectrometrical data were obtained by LC-ESI-MS/MS and GC-MS
analyses. LC-ESI-MS/MS data were recorded with a Finnigan TSQ 7000
apparatus (Finnigan MAT, Bremen, Germany) with an electrospray ionisation
interface (ESI). Aqueous 0.1% acetic acid (A) and acetonitrile (B) (v/v)
was used as mobile phase in an isocratic gradient (50:50). An Applied
Biosystems 140B pump was used. The capillary temperature was 2008C
and the spray capillary voltage was set to 3.2 kV. Nitrogen served as both
sheath (70 psi) and auxiliary gas (10 units). The mass spectrometer was
operated in the full scan mode, m/z 20–400, with a total scan duration of 1.0 s.
MS/MS experiments were performed at a collision energy of 20–35 eV, with
argon (2.0 mTorr) serving as collision gas. The multiplier voltage was set
to 1300 V.
GC-MS was carried out using a Fisons Instrument GC 8060 (Thermo
Electron, Dreieich, Germany) gas chromatograph with split injection (2208C;
1:20) directly coupled to a MD 800 mass spectrometer (Thermo Electron,
Dreieich, Germany). The GC was equipped with a HP 5 capillary column
(30 m  0.25 mm, df = 0.25 mm) and a temperature program running from
608C to 3008C at 58C/min with a gas flow of 2 ml/min helium under constant
pressure was used. Temperature of connecting parts was 2208C, electron
energy for the EI mass spectra was 70 eV. Data evaluation was performed with
XcaliburTM software (Thermo Electron, Dreieich, Germany).
2-(Benzoyloxy)-4,6-dihydroxybenzaldehyde (4)
To a solution of 10 g (0.065 mol) 2,4,6-trihydroxybenzaldehyde 3 in 150 ml dry
acetone kept under nitrogen atmosphere and cooled at 08C, each a solution of
5 g (0.049 mol) triethylamine and 6.8 g (0.048 mol) benzoyl chloride in 30 ml
dry acetone was added dropwise within 30 min. After subsequent stirring for
half an hour at room temperature 50 g of silica gel (0.063–0.2 mm, Merck,
Darmstadt, Germany) was added and the mixture was evaporated to dryness
under reduced pressure. The dry yellow powder was applied to a 5.5 Â 35 cm
glass column filled with silica gel (0.063–0.2 mm, Merck, Darmstadt,
Germany) and preconditioned with benzene. Elution with 1 l benzene
gave fraction 1. The second fraction was eluted using 1 l of a chloroform/
benzene (1+1) mixture; in this fraction the dibenzoylated product was
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1151–1162
DOI: 10.1002/jlcr

SYNTHESIS OF [4-14C]-PELARGONIDIN AND [4-14C]-DELPHINIDIN CHLORIDE 1157
achieved. In fraction 3, eluted with 2 l of a chloroform/ethyl acetate (1+1)
mixture, 3.4 g of 2-(benzoyloxy)-4,6-dihydroxybenzaldehyde (4) was isolated
(3.6 g, 21% yield). Owing to the high purity of the product (HPLC-DAD
control, 99%), no recrystallization process was necessary HY.15 1H-NMR:
DMSO-d6, 400 MHz: d 6.27 (Ar, d, 2.1 Hz , 2H), 6.32 (Ar, d, 2.1 Hz , 2H), 7.59
(Ar, t, 7.6 Hz, 1H), 7.74 (Ar, t, 7.4 Hz, 1H), 8.11 (Ar, d, 7.2 Hz, 1H), 10.00
(CHO, s, 1H), 11.01 (OH, s, 1H), 11.38 (OH, s, 1H). LC-ESIneg-MS/MS (%)
[20 eV ]: 257.0 (100), 213.0 (20), 187.0 (58), 169.0 (16), 145.0 (13), 143.0 (24),
119.0 (19), 111.1 (24).
Synthesis of pelargonidin chloride (1)
o-Chloro-4-hydroxyacetophenone (9). To a solution of 30 g (0.28 mol) anisole
and 36 g (0.32 mol) chloroacetyl chloride in 500 ml n-heptane was added
stepwise 45 g (0.34 mol) AlCl3 and the mixture was subsequently heated to
358C for 90 min. During the following 30 min, again 45 g AlCl3 (0.34 mol) was
added and the solution heated for 2.5 h. The solvent was removed under
reduced pressure and ice was put to the residue. The solution was then
acidified with 200 ml of concentrated hydrochloric acid. After extraction with
250 ml of diethyl ether for three times, the ether fraction was neutralized using
200 ml of a 15% ammonium carbonate solution. After this, the organic phase
was extracted with 250 ml of a 10% sodium carbonate solution. The water
phase was acidified and extracted with diethyl ether for several times. The
ether phase was evaporated and a yellow oil was obtained. The yellow
o-chloro-4-hydroxyacetophenone (9) crystallized from 25% aqueous ethanol
solution (9.5 g, 20% yield).17 1H-NMR: DMSO-d6, 400 MHz: d 5.02 (CH2, s,
2H), 6.84 (Ar, d, 8.8 Hz, 2H), 7.84 (Ar, d, 8.7 Hz, 2H), 10.48 (OH, s, 1H).
LC-ESIneg-MS/MS (%) [20 eV]: 168.7 (100), 132.7 (52), 126.8 (7), 122.9 (27),
104.8 (32), 77.0 (15), 58.6 (4), 34.9 (93).
o-Acetoxy-4-hydroxyacetophenone (10)
A solution of 10 g (0.06 mol) o-chloro-4-hydroxyacetophenone (9) and 7.7 ml
acetic acid in 100 ml anhydrous acetonitrile was cooled on ice. Step by step,
11.2 g (0.11 mol) of triethylamine was added and the mixture was heated under
reflux for 3 h. The solution was diluted with diethyl ether and washed with
hydrochloric acid, water and sodium chloride solution. The organic phase was
dried over sodium sulphate. After evaporating the solvent under reduced
pressure, the solid of 10 was crystallized in water (4.5 g, 40% yield).21
1H-NMR: DMSO-d6, 400 MHz: d 2.15 (CH3, s, 3H), 5.40 (CH2, s, 2H), 7.13 (Ar,
d, 8.8 Hz, 2H), 7.91 (Ar, d, 8.8 Hz, 2H), 10.31 (OH, s, 1H). LC-ESIneg-MS/MS
(%) [20 eV ]: 192.9 (12), 150.9 (100), 132.8 (9), 122.7 (14), 106.8 (12), 104.8
(21), 58.8 (35).
Copyright # 2006 John Wiley & Sons, Ltd.
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1158
M. KRAUS ET AL.
o,4-Diacetoxyacetophenone (6)
o-Acetoxy-4-hydroxyacetophenone (10) (4.9 g, 0.025 mol) solved in 20 ml acetic
anhydride was heated for 2 h under reflux at 1008C and then poured in 100 ml
of boiling water. After boiling for a short time, subsequent cooling down led
to crystallization of o,4-diacetoxyacetophenone (6) (4.0 g, 75% yield).22
1H-NMR: DMSO-d6, 400 MHz: d 2.15 (CH3, s, 3H), 2.31 (CH3, s, 3H), 5.46
(CH2, s, 2H), 7.33 (Ar, d, 8.8 Hz, 2H), 8.03 (Ar, d, 8.8 Hz, 2H). LC-ESIneg-MS/
MS (%) [20 eV]: 237 (7), 194.9 (60), 152.8 (100), 134.7 (13), 106.8 (8).
Pelargonidin chloride (1)
2-(Benzoyloxy)-4,6-dihydroxybenzaldehyde (4) (2.03 g, 7.86 mmol) and o,4-
diacetoxyacetophenone (6) (2.03 g, 8.6 mmol) were dissolved in 30 ml
methanol/ethyl acetate (2+1) mixture. For 1 h, dry hydrogen chloride gas
was bubbled into the solution and the mixture was stirred at room temperature
for 24 h. After removing of the solvent by centrifugation, the purple residue of
5-(benzoyloxy)pelargonidin chloride was suspended in 30 ml of a basic
methanol/water solution (0.25 g potassium hydroxide in 5 ml methanol/water
1:1) under ice cooling and stirred for 1 h at 0–4 8C. The blue solution was
acidified with 16.5 ml of concentrated hydrochloric acid. The colour changed
to purple again and 1 crystallized in brownish crystals that were recovered
after centrifugation. For recrystallization via the picrate it was solved in 1%
HCI solution containing 50% ethanol, added to a solution of picric acid in
50% aqueous ethanol and heated to 608C for 2 min. The picrate crystallized
over night in a freezer. The crystals were dried and washed with dry diethyl
ether. To regenerate 1, methanol, saturated with hydrogen chloride gas, was
added to the picrate. Dry diethyl ether was added in excess (ꢀ6 fold of
methanol) and 1 crystallized in crude crystals. The crystallization process was
repeated leading to 967 mg (40% yield) pelargonidin chloride 1 with 95%
1
purity (HPLC-DAD control). H-NMR: DMSO-d6,400 MHz: d 6.86 (Ar, d,
1.8 Hz, 1H), 6.97 (Ar, d, 1.4 Hz, 1H), 7.11 (Ar, d, 9.1 Hz, 2H), 8.54 (Ar, d,
9.1 Hz, 2H), 8.84 (CH, s, 1H), 11.31 (OH, s, 1H), 12.14 (OH, s, 1H), 12.20
(OH, s, 1H), 12.35 (OH, s, 1H). LC-ESIneg-MS/MS (%) [20 eV]: 270.9 (93),
196.8 (19), 168.9 (17), 158.9 (8), 152.9 (8), 144.8 (16), 140.7 (19), 120.8 (100),
92.9 (10).
Synthesis of delphinidin chloride (2)
3,4,5-Triacetylbenzoic acid (12). A solution of 50 g (0.3 mol) 3,4,5-trihydrox-
ybenzoic acid (gallic acid) (11) in 200 ml acetic anhydride was heated under
reflux at 1108C for 3 h. The solution was then poured into 1 l hot water.
During cooling 3,4,5-triacetylbenzoic acid (12) crystallized. After filtering and
washing with water, the white crystals were dried over silica gel. The yield was
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

SYNTHESIS OF [4-14C]-PELARGONIDIN AND [4-14C]-DELPHINIDIN CHLORIDE 1159
62 g (79%).18 1H-NMR: DMSO-d6, 400 MHz: d 2.30 (CH3, s, 6H), 2.33 (CH3,
s, 3H), 7.75 (Ar, s, 2H), 13.38 (COOH, s, 1H). LC-ESIneg-MS/MS (%) [20 eV]:
296.1 (3), 251.8 (50), 209.7 (100), 168.0 (36), 166.7 (79), 149.4 (5), 125.0 (5),
59.0 (8).
3,4,5-Triacetylbenzoic acid chloride (13). 3,4,5-Triacetylbenzoic acid (12)
(25 g, 0.084 mol) was refluxed with 30 ml of thionyl chloride at 858C for 1 h.
The excess of thionyl chloride was removed by evaporating under reduced
pressure. After drying, the pale yellow residue was washed with diethyl ether.
The raw product 12 was recrystallized from xylene. The yield was 23.8 g
(91%).18 1H-NMR: DMSO-d6, 400 MHz: d 2.31 (CH3, s, 6H), 2.32 (CH3, s,
3H), 7.89 (Ar, s, 2H). GC-MS (70 eV): 268 (34), 237 (18), 227 (19), 226 (71),
209 (22), 185 (28), 184 (99), 153 (51), 152 (19), 43 (100).
o-Diazo-3,4,5-triacetoxyacetophenone (14). Diazomethane was generated
from an etheric solution of DiazaldTM (13.6 g) in the presence of 13 g
potassium hydroxide dissolved in 13 ml water and 45 ml ethanol. 3,4,
5-Triacetylbenzoic acid chloride (13) (5 g, 0.016 mol), dissolved in 12 ml
chloroform and cooled to À108C, was poured step by step within 30 min into
the diazomethane solution (À108C), yielding a yellow powder of o-diazo-3,4,5-
Triacetoxyacetophenone (14) which was collected and washed with diethyl
ether. The product (4.2 g, 81% yield) was stored in the freezer at À248C.20
1H-NMR: CDCl3, 400 MHz: d 2.30 (CH3, s, 6H), 2.31 (CH3, s, 3H), 5.83 (CH,
s, 1 H), 7.52 (ar, s, 2H). Owing to instability no mass data are available.
o-Chloro-3,4,5-triacetoxyacetophenone (15). o-Diazo-3,4,5-triacetoxyaceto-
phenone (14) (3.0 g, 9.77 mmol), dissolved in 16 ml dioxane was cooled to
108C. Then 12 ml of 1 N hydrogen chloride solved in diethyl ether was added
to the solution and the mixture was stirred for 30 min at 108C. After a strong
development of nitrogen, the diethyl ether was removed in a stream of
nitrogen; dioxane was evaporated under reduced pressure. The pale yellow
residue was solved in dichloromethane and washed with water. The organic
phase was dried with magnesium sulphate and evaporated under vacuum. The
residue was dissolved in ethyl acetate/petrol ether (1/1, v/v). The solution was
diluted with petrol ether and 15 was obtained as a fine crystalline powder
(2.6 g, 87% yield).21 1H-NMR: DMSO-d6, 400 MHz: d 2.32 (CH3, s, 6H),
2.34 (CH3, s, 3H), 5.17 (CH2, s, 2H), 7.87 (Ar, s, 2H). LC-ESIpos-MS/MS (%)
[20 eV]: 329.9 (7), 287.7 (12), 245.8 (100), 203.8 (30).
o,3,4,5-Tetracetoxyacetophenone (7). A mixture of 1 g (5.4 mmol) o-chloro-
3,4,5-triacetoxyacetophenone (15), 3.9 ml (42 mmol) acetic anhydride, 2 ml
concentrated sulphuric acid, 3.9 ml glacial acetic acid and 1.53 g potassium
acetate was kept at 1008C under reflux for 3 h and then poured into a mixture
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of water/ice. The precipitate was filtered and crystallized from ethyl acetate/
cyclohexane (1:1). The yield of 7 was 0.8 g (75% yield).22 1H-NMR: DMSO-
d6, 400 MHz: d 2.15 (CH3, s, 3H), 2.32 (CH3, s, 6H), 2.35 (CH3, s, 3H), 5.43
(CH2, s, 2H), 7.85 (Ar, s, 2H). LC-ESIpos-MS/MS (%) [20 eV]: 353.1 (4), 310.8
(7), 268.9 (100), 226.9 (27).
Delphinidin chloride (2). In a solution of 0.25 g (0.71 mmol) o-3,4,5-
tetraacetoxyacetophenone (7) and 0.2 g (0.78 mmol) 2-(benzoyloxy)-4,6-dihy-
droxybenzaldehyde (4) in 5 ml absolute ethanol and 5 ml ethyl acetate, kept
under argon, dry hydrogen chloride gas was applied for 1 h. After 24 h
brownish crystals of 5-(benzoyloxy)-delphinidin chloride were obtained by
centrifugation (5.000 g). The cleavage of the benzoyl group took place by
adding 2.5 ml of an oxygen-free aqueous solution of 10% sodium hydroxide at
08C under argon atmosphere. After keeping at 0–48C for 15 min the blue
solution was stored for 4 h at room temperature. After adding 5 ml of ethanol
and 5 ml of concentrated hydrochloric acid, the mixture was kept for 4 days at
48C. Then, 10 ml of concentrated hydrochloric acid was added and the mixture
was stored for two further days at 48C leading to the crystallization of 2.20
After centrifugation, the residue was solved in a small amount of methanol,
saturated with hydrogen chloride gas. By addition of absolute diethyl ether, in
1
total, 220 mg (42% yield) of 2 was obtained. H-NMR: methanol-d6, 400
MHz: d 5.90 (Ar, d, 2.2Hz, 1H), 5.94 (Ar, d, 2.2 Hz, 1H), 6.14 (CH, s, 1H),
6.62 (Ar, s, 2H). LC-ESIpos-MS/MS (%) [20 eV]: 303.0 (66), 257.0 (31), 229.0
(100), 201.0 (24), 173.0 (36), 163.2 (12), 153.0 (42), 149.8 (15), 148.9 (11), 145.0
(22), 127.1 (17), 125.0 (20), 121.0 (22), 107.1 (10), 79.1 (11), 69.1 (10).
[Formyl-14C]-2,4,6-trihydroxybenzaldehyde (3a). 2,4,6-Trihydroxybenzene (5)
(30 mg, 0.24 mmol) was dissolved in an 1.5 ml diethyl ether/chloroform (2:1)
mixture, dimethylformamide (18.1 ml; 0.21 mmol) unlabelled and [formyl-14C]-
dimethylformamide, 2500mCi) were added and mixed under ice cooling. After
solvation, 22 ml phosphoroxychloride (POCl3) was added. Then a nitrogen
atmosphere was created and the mixture was stirred on ice for 1 h. After stirring
for half an hour at room temperature, 11 ml of POCl3 was added. Under
nitrogen atmosphere and light protection, the mixture was heated at 608C for
4 h. After removal of the solvent, ice was added under sonification. The
solution was adjusted to pH 6 using sodium hydroxide and then lyophilized. To
the residue 4 ml methanol was added and the mixture, after 2 min of
sonification, centrifuged (5000 g, 10 min). The supernatant was purified by
RP18-HPLC yielding 17.4 mg (43.9%) and 15.9 mg (47.5%) (two separate
runs) of the 14C-labelled 2,4,6-trihydroxybenzaldehyde 3a.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1151–1162
DOI: 10.1002/jlcr

SYNTHESIS OF [4-14C]-PELARGONIDIN AND [4-14C]-DELPHINIDIN CHLORIDE 1161
[Formyl-14C]-2-(benzoyloxy)-4,6-dihydroxybenzaldehyde (4a). The obtained
3a was dissolved in 700 ml anhydrous acetone and 9 ml anhydrous triethylamine
and 7.5 ml anhydrous benzoyl chloride were added to the solution which was
stored on ice. After stirring under nitrogen atmosphere for 1 h at 0–48C and
removing of the solvent, the residue was solved in 2 ml methanol. Purification
by RP18-HPLC led to 4.2 mg/5 mg (yields 16%/18.9%, two separate runs) of
[formyl-14C]-2-(benzoyloxy)-4,6-dihydroxybenzaldehyde 4a.
[4-14C]-Pelargonidin chloride (1a). Five mg of [formyl-14C]-2-(benzoyloxy)-
4,6-dihydroxybenzaldehyde (4a) (0.018 mmol, first preparation) and 5 mg
(0.020 mmol) o,4-diacetoxyacetophenone (6) (molar ratio of 1:1.1) were solved
in 500 ml methanol/ethyl acetate. For 1 h, dry HCI gas was transfered into the
solution with a fused silica capillary. Afterwards, the mixture was stirred for
24 h at room temperature. After removing the solvent, the purple residue was
suspended in 80 ml of a basic methanol/water solution (0.25 g potassium
hydroxide in 5 ml methanol/water 1:1) under ice cooling and stirred for 1 h at
0–48C. The blue solution was acidified with 51 ml concentrated hydrochloric
acid. The colour changed to purple again. After freezing and lyophilizing, the
residue was dissolved in methanol and injected for purification to RP18-
HPLC. The pelargonidin chloride 1a was detected at 520 nm. The yield of
the 14C-radiolabelled pelargonidin chloride was 0.2 mg (3.8%). The specific
activity was 33.6 mCi/mg.
[4-14C]-Delphinidin chloride (2a). [Formyl-14C]-2-(benzoyloxy)-4,6-dihydro-
xybenzaldehyde (4a) (4.7 mg, 0.018 mmol) and 7.1 mg (0.020 mmol) o,3,4,5-
tetracetoxyacetophenone (7) were dissolved in each of 100 ml ethanol and ethyl
acetate. Under ice cooling the mixture was saturated with hydrogen chloride
gas. The mixture was kept at room temperature for 24 h yielding [4-14C]-
delphinidin chloride (2a) and [4-14C]-5-benzoyldelphinidin chloride (HPLC-
DAD control). After removing of the solvent, the residue was dissolved in
methanol, saturated with hydrogen chloride. The solution was subjected to
preparative HPLC to yield 2a. The remaining [4-14C]-5-benzoyldelphinidin
chloride was deprotected by resolving in 14 ml oxygen-free 10% sodium
hydroxide solution and 100 ml ethanol at 08C. The solution was stored under
argon atmosphere for half an hour at room temperature. Then 20 ml
concentrated hydrochloric acid was added. Under inert atmosphere the
solution was stored for 4 days at 48C. Purification was performed by HPLC to
achieve the [4-14C]-delphinidin chloride 2a. At the end, 0.33 mg (5.5%) with a
specific activity of 30.4 mCi/mg was obtained. 1H-NMR: methanol-d4,
400 MHz: d 5.90 (Ar, d, 2.2 Hz, 1H), 5.94 (Ar, d, 2.2 Hz, 1H), 6.14 (CH, s,
1H), 6.62 (Ar, s, 2H).
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Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (DFG)
within the FLAVONET project (Schr 211/21-1/-3). The authors thank Dr
Rdest, Institute of Microbiology, for kindly providing laboratory space to
perform the radio syntheses. Dr R. Vic¸ik and Dr. A. Valotis are thanked for
measuring NMR data and specific activities of the synthesized anthocyanidins,
respectively.
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Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1151–1162
DOI: 10.1002/jlcr