A large scale synthesis of a natural antibiotic, 2,4- diacetylophloroglucinol (DAPG)

Article abstract of DOI:10.1002/ardp.200600139

A natural antibiotic, 2,4-diacetylophloroglucinol (DAPG), has been prepared by the acetylation of phloroglucinol (PhL) with acetic anhydride in the presence of boron trifluoride etherate complex. The crude DAPG is efficiently purified using chromatographi

Full text of DOI:10.1002/ardp.200600139

Arch. Pharm. Chem. Life Sci. 2007, 340, 103–106
J. Zakrzewski et al.
103
Full Paper
A Large Scale Synthesis of a Natural Antibiotic,
2
,4-Diacetylophloroglucinol (DAPG)
Jerzy Zakrzewski, Monika Karpi n´ ska, Zbigniew Mali n´ ski
Institute of Industrial Organic Chemistry, Warsaw, Poland
A natural antibiotic, 2,4-diacetylophloroglucinol (DAPG), has been prepared by the acetylation
of phloroglucinol (PhL) with acetic anhydride in the presence of boron trifluoride etherate com-
plex. The crude DAPG is efficiently purified using chromatographic filtration with boiling
eluent.
Keywords: Antibacterial activity / Antifungal activity / Synthesis /
Received: September 5, 2006; accepted: November 24, 2006
DOI 10.1002/ardp.200600139
Introduction
[13, 15–17, 20], or even 24 h [11, 12, 14, 18]. The reported
yields of DAPG vary from 21% [19] to 90% [12] and 92%
2
,4-Diacetylophloroglucinol (DAPG), a naturally occur- [11] (undoubtedly due to the using MAPG as a starting
ring antibiotic, is known as an antifungal metabolite of material). The crude DAPG was reported as a strongly
many fluorescent pseudomonad bacteria living in the coloured solid material [13, 18, 19, 21, 24] and was puri-
rhizosphere of crop plants [1–4]. Antibacterial activity of fied by means of either crystallisation in water [24],
DAPG against Staphylococcus aureus was well documented methanol/water (pale yellow; after purification) [13, 20],
[
5–10].
The synthesis of DAPG involves the Friedel–Crafts acet- nol/water (nearly colourless) [21], 80% ethanol [23], ben-
ylation of either monoacetylophloroglucinol (MAPG) zene/hexane [23], benzene/petroleum ether 2:1 or by col-
11, 12] or phloroglucinol (PhL) [13–24] in the presence of umn chromatography on silica gel using hexane/ethyl
methanol/water (orange) [22], ethanol/water [18], etha-
[
Lewis acids. Both, PhL and MAPG, should be used as anhy- acetate 5:1 (bright yellow; after purification) [15–17],
drous material [12–14, 18, 20], however, use of the MAPG hexane/ethyl acetate 4:1 [11, 12], hexane/ethyl acetate
monohydrate is also reported [11]. Two acetylating [14], or benzene/ethyl acetate 3:2 [19].
agents, acetic acid and acetic anhydride, were used.
The acetylation of the 50 g (0.4 mol) of PhL is the big-
Acetic acid was used either saturated with a gaseous gest amount reported up to date [20]. The acetylation
boron trifluoride [20–22, 24] or with the commercially reaction itself can be scaled up without any difficulties.
available complexes: acetic acid/boron trifluoride (BF
CH
COOH) complex [13, 18, 20] as well as boron trifluor- crude, strongly coloured DAPG is not so simple. Crystalli-
ide/diethyl ether (BF /Et
O) one [11, 14–17]. Acetic anhy- sation methods together with the typical decolourisation
dride was used in the presence of BF /Et
O complex procedures are not efficient. The crystallisation/decolour-
12, 23] or zinc chloride as catalysts [19]. Recently, the isation of DAPG is a fickle and unrepeatable process.
3
/
However, the scaling up of the purification step of the
3
3
2
3
2
[
application of the mixture of acetic acid and acetic anhy- Herein, we present the synthesis of DAPG starting from
dride has been reported [11]. The temperature of acetyla- 0.7 mol of PhL together with its efficient crystallisation/
tion varied from room temperature [12, 18, 23] to 1508C decolourisation procedure.
[
19]. The time of the reaction was 15 min [19], 2–3h
Results and discussion
Correspondence: Jerzy Zakrzewski, Institute of Industrial Organic
Chemistry, Annopol 6, 03-236 Warsaw, Poland
E-mail: zakrzewski@ipo.waw.pl
In the preliminary experiments, we obtained DAPG by
the acetylation of the anhydrous PhL with either acetic
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104
J. Zakrzewski et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 103–106
acid and CH
3
COOH/BF
3
complex or with acetic anhydride
Anhydrous PhL was acetylated with acetic anhydride
and BF /Et O complex.
3
2
in the presence of BF
3
/Et O complex at 958C for 4 h. The
2
The typical work-up procedure – decomposition of the complex of DAPG was decomposed with potassium acet-
DAPG complex with various hydrolyzing agents follow- ate to give the crude DAPG, usually a dark orange to
ing by the extraction – yields the crude DAPG. Various strongly red-coloured solid. The hot, saturated solution
methods of its purification (crystallization, column chro- of crude DAPG was purified using the filtration through
matography, boiling with or filtration through sorbents) a coarse-sized silica gel bed placed in a heated column.
were investigated.
Below, we summarise our findings:
The use of PhL as a starting material seems to be more
This procedure allows to manufacture decolourised
DAPG in 80–85% yield and 95.5–96% purity.
1
The structure of DAPG was confirmed by MS, HRMS, H-
1
3
reasonable. Although MAPG is commercially available as NMR, C-NMR, IR, and UV spectra. They are consistent
a fine reagent (Aldrich, Germany), PhL is a more popular, with the literature data [12, 13], however slight differ-
1
industrially manufactured intermediate.
There is no difference between the acetylation with trum are noticed.
either acetic acid and CH COOH/BF complex or with As a typical Friedel-Crafts reaction, acetylation of PhL
acetic anhydride and BF /Et O complex. The quantity and with acetic anhydride leads directly to a complex of a
quality (appearance, colour) of the crude DAPG in both product – DAPG – and a Lewis acid catalyst – BF . Com-
preparations were similar. The acetylation with acetic plexes of BF with carbonyl compounds are well known
anhydride in the presence of BF /Et O complex seems to [25–27]. The structures of these complexes involve coor-
be more convenient due to the availability of BF /Et O, its dination of BF to the lone pair of a carbonyl oxygen
lower price and its simple purification by means of atom. In order to isolate the complex, in a separate
vacuum distillation. experiment the step of the decomposition of the complex
ences in signals attributed to OH groups in H NMR spec-
3
3
3
2
3
3
3
2
3
2
3
Crystallisation procedures and decolourisation of by the hydrolysis was replaced by the removing of all
DAPG were not efficient, because the DAPG is dissolved volatiles from the reaction mixture. The dark brown
efficiently only in polar solvents (acetone, methanol) and glassy residue slowly solidified and a yellow solid was
coloured impurities are not adsorbed enough on used obtained. The mass of the residue confirms that two
solid phases.
molecules of BF are coordinated to two carbonyl oxygen
3
Our preliminary experiments confirmed the advan- atoms (DAPG62 BF3).
tage of the column chromatography as a tool for the
decolourisation and purification of DAPG. Typical col-
The authors thank the Polish State Committee for Scientific
umn chromatography processes used in the preliminary Research (grant 3 T09B 023 27) for financial support.
experiments with the fine, flash silica gel and mixtures
of hexane and ethyl acetate as eluents were very efficient
–
white, pure DAPG was obtained. However, the column
Experimental
chromatography is not practical when the enlargement
of the scale is projected. The attempts to scale up the
chromatographic process inevitably leads to the enor-
mous costs and tedious procedure. Moreover, DAPG dis-
solves poorly in a non-polar eluent such as hexane:ethyl
acetate mixtures. The preliminary dissolving of DAPG in
acetone (as proposed in [12]) causes worse separation of
the product and impurities.
General
Commercially available phloroglucinol dihydrate (Aldrich, Ger-
many) (115 g, 0.71 mol) was placed in a round-bottomed flask
equipped with a Dean–Stark adapter and a reflux condenser.
Benzene (500 mL) was introduced into the flask. Azeotropic dis-
tillation was continued until no more water had been collected
in the Dean–Stark adapter (26 mL). Benzene was evaporated
under reduced pressure using a rotary evaporator. Anhydrous
PhL was stored in a vacuum desiccator over silica gel with moist-
ure indicator (blue gel, Merck 1925; Merck, Germany) or molecu-
Finally, the following procedure was chosen (Scheme
):
1
lar sieves 4A. Commercially available BF
3 2
/Et O complex (Aldrich)
was purified by means of distillation under reduced pressure
(
56–588C/28 mmHg).
Hexane used for the purification of DAPG should be of 95%
purity (minimum).
GLC: Varian 3300 (Varian, Palo Alto, CA., USA), DB1 (30 m), t =
k
9
08C (39) t (208C/min) t 1908C (88C/min) t 2708C (59), t
i
= 2708C,
t
d
= 2808C (FID).
HRMS and MS data were determined using an AMD 604 appa-
ratus (AMD Intectra GmbH, Germany). H-NMR and C-NMR
Scheme 1. Synthetic pathway to DAPG.
1
13
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Arch. Pharm. Chem. Life Sci. 2007, 340, 103–106
Synthesis of 2,4-Diacetylophloroglucinol
105
spectra were recorded on a Varian UNITY-plus 200 spectrometer.
IR and UV spectra were determined using FT/IR Jasco 420 (Jasco;
Tokyo, Japan ) and Pye-Unicam SP-8 100 apparatus (Pye Unicam
Ltd. Cambridge, England).
3:1): 98.8% (GLC), m. p.: 168–1708C (161–1638C [15–17], 173–
1748C [13]).
HRMS for
10 10 5
C H O , 210 (M): Found: 210.05211, Calc.:
210.05282; MS (EI, 70 eV, m/z, int%): 210 (50, M), 195 (100), 177
1
(
75), 69 (43), 67 (38), 43 (92); H NMR (d, ppm, acetone-d
MHz): 2,64 (s, 6H, 2 COCH ), 5,90 (s, 1H, Ar-H), 16.37 (brs, 1H, O …
H… O); C-NMR (d, ppm, acetone-d , 50 MHz): 32.84 (2 CH ),
95,63 (Ar-H), 104.68 (2 C-COCH ), 170,0 (Car-OH), 172.76 (2 Car-
6
, 200
3
2
,4-Diacetylophloroglucinol (DAPG)
13
6
3
A reactor of 2 L-capacity, equipped with a mechanical stirrer, a
reflux condenser protected against humidity with a drying tube
containing anhydrous calcium chloride, a thermometer, and a
3
–
1
OH), 204,63 (2 C=O); IR k [cm ], KBr): 3432, 1622 (C=O); UV kmax
[nm], e: 270 (33675), 331 (6675), 378 (2800).
dropping funnel was fed with a freshly distilled BF
plex (771.7 g, 5.43 mol, 689 mL). Acetic anhydride (136.2 g,
.34 mol, 126 mL) was added dropwise at ambient temperature.
No thermal effect was observed. Anhydrous PhL (85.6 g,
.68 mol) was added in portions. The temperature was raised up
3 2
/Et O com-
2
To BF
,4-Diacetylophloroglucinol62BF
3
1
3 2
/Et O complex (56.8 g, 0.4 mol, 50.7 mL), acetic anhydride
(
9.2 g, 0.09 mol, 7.6 mL) was added dropwise at ambient tem-
0
perature. Then anhydrous PhL (6.3 g, 0.05 mol) was added. The
brown-orange reaction mixture was stirred and heated at 958C
for 4 h. The volatiles were distilled off under reduced pressure:
to 388C. The brown-orange reaction mixture was stirred and
heated at 958C for 4 h. The reaction mixture was allowed to cool
to the ambient temperature, and the aqueous potassium acetate
solution (402 g/4.1 mol of anhydrous potassium acetate in 1 L of
distilled water) was added dropwise. The resulting yellow emul-
sion was extracted with diethyl ether (56400 mL). The aqueous
layer was thoroughly separated and washed with two portions
of diethyl ether (26150 mL). The saturated sodium bicarbonate
solution was carefully added dropwise (intense foaming) to the
combined organic layers. The etheral layer was washed thrice
with distilled water (36500 mL) and dried over anhydrous mag-
nesium sulphate. The drying agent was filtered off and the ether
filtrate was evaporated. The crude orange DAPG (146.5 g) was air
dried. The purity: 89.2% (GLC). The crude DAPG was purified by
means of filtration through a bed of silica gel. Silica gel (Kiesel-
gel 60, 0.2–0.5 mm, 35–70 mesh, Merck 7733, 4:1 w/w in rela-
tion to the portion of crude DAPG) was suspended in hexane/
ethyl acetate 9:1. The suspension was placed in a wide glass col-
umn surrounded with a heating tape, heated enough to main-
tain the temperature of both the silica gel bed and the solution
close below the boiling point of the hexane:ethyl acetate 9:1.
The portion of the crude DAPG (50 g) was dissolved in boiling
hexane:ethyl acetate 9:1 (2.5 L) and the solution was introduced
at the top of the silica gel bed.
4
2.5 g of distillate, t = 60–838C/15 mmHg, t cub f 1058C. The
dark-brown glassy residue (in a refrigerator slowly solidified to
yellow solid) was obtained: 16.6 g, 96% of calculated DAPG 2 BF
mass.
3
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the hot eluent (total: about 5 L hexane:ethyl acetate 9:1/50 g of
crude DAPG) was passed through the column, the near-colour-
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1
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