Syntheses of carnosic acid and carnosol, anti-oxidants in rosemary, from pisiferic acid, the major constituent of Sawara

Article abstract of DOI:10.1248/cpb.58.27

Carnosic acid (2), a major anti-oxidant in rosemary (Rosmarinus officinalis), was synthesized from pisiferic acid (1), the major constituent of Sawara (Chamaecyparis pisifera), via ortho-oxidation of the phenol using metachlorobenzoyl peroxide (mCBPO), chloroacetyl meta-chlorobenzoyl peroxide (CAMCBPO) or 2-iodoxybenzoic acid (IBX). Carnosol (3), another anti-oxidant in rosemary, was synthesized from carnosic acid by oxidation with silver oxide. Potent antibacterial activities against Propionibacterium acnes (ATCC 6919) (minimum inhibitory concentration (MIC) μg/ml) and Staphylococcus aureus ME/GM/TC Resistant (ATCC 33592) (MIC μg/ml) of carnosic acid and carnosol were reported.

Full text of DOI:10.1248/cpb.58.27

January 2010
Chem. Pharm. Bull. 58(1) 27—29 (2010)
27
Syntheses of Carnosic Acid and Carnosol, Anti-oxidants in Rosemary,
from Pisiferic Acid, the Major Constituent of Sawara
Masahiro TADA,* Tomoyuki OHKANDA, and Jun KURABE
Laboratory of Bioorganic Chemistry, Tokyo University of Agriculture and Technology; Fuchu, Tokyo 183–8509, Japan.
Received July 1, 2009; accepted October 8, 2009; published online October 9, 2009
Carnosic acid (2), a major anti-oxidant in rosemary (Rosmarinus officinalis), was synthesized from pisiferic
acid (1), the major constituent of Sawara (Chamaecyparis pisifera), via ortho-oxidation of the phenol using meta-
chlorobenzoyl peroxide (mCBPO), chloroacetyl meta-chlorobenzoyl peroxide (CAMCBPO) or 2-iodoxybenzoic
acid (IBX). Carnosol (3), another anti-oxidant in rosemary, was synthesized from carnosic acid by oxidation with
silver oxide. Potent antibacterial activities against Propionibacterium acnes (ATCC 6919) (minimum inhibitory
concentration (MIC) mg/ml) and Staphylococcus aureus ME/GM/TC Resistant (ATCC 33592) (MIC mg/ml) of
carnosic acid and carnosol were reported.
Key words carnosic acid; carnosol; synthesis; rosemary; anti-Propionibacterium acnes; anti-methicillin-resistant Staphylococ-
cus aureus (MRSA)
Many species of the Salvia family have been used for sized from carnosic acid 2.14,15) This method will thus be ap-
herbs and spices, folk medicines and anti-oxidants for plicable for the synthesis of these anti-oxidant diterpenes of
foods.1) Rosemary and sage, well known herbs in this family, rosemary. The potent antimicrobial activities (MIC, mg/ml)
contain many anti-oxidant diterpenes, e.g. carnosic acid (2), of carnosic acid and carnosol against Propionibacterium
carnosol (3), and rosmanol (4).2,3) The German Commission acnes (ATCC 6919) and Staphylococcus aureus ME/GM/TC
E recognized that the extract from rosemary is effective for resistant (ATCC 33592) were measured to show the potency
indigestion when used internally or to quicken the circulation of the antimicrobial compounds.
of the blood when used externally. Recently, a major anti-oxi-
dant in rosemary, carnosic acid, has attracted much attention Results and Discussion
because of its neuron protective effects.4) Many other biolog-
Isolation of Pisiferic Acid (1) from Sawara (Chamaecy-
ical activities of carnosic acid have been reported, e.g. nerve paris pisifera) The leaves of Chamaecyparis pisifera were
growth factor forming promoters,5) therapeutic agents for extracted with methanol under reflux for 24 h. The extract
amnesia, dementia, Alzheimer’s disease,6) and lipid absorp- was evaporated and the residue was chromatographed on a
tion inhibitors.7) Despite these various biological activities, silica gel column with hexane–ethyl acetate. The crude frac-
there is no efficient method for synthesizing carnosic acid. tion of pisiferic acid was rechromatographed on a silica gel
Consequently, carnosic acid is produced by extraction from column with hexane–ethyl acetate. Light green crystals of
natural rosemary and is very expensive (ꢀ$630/500 mg).8) pisiferic acid were obtained after crystallization from hexane–
Sawara (Chamaecyparis pisifera) is a popular and useful tree ethyl acetate (0.6% of the leaves). The spectroscopic data of
in Japan and its leaves contain the phenolic diterpene, the isolated pisiferic acid were identical with those of the au-
pisiferic acid (1), as the major constituent.9,10) Pisiferic acid 1 thentic pisiferic acid.9,10)
has the same abietane carbon skeleton as carnosic acid 2
The oxidation of pisiferic acid was examined with three
with a carboxyl group at C-10 and a phenolic hydroxyl group reagents, meta-chlorobenzoyl peroxide (mCBPO), chloroacetyl
at C-12. Carnosic acid 2 has an additional phenolic hydroxyl meta-chlorobenzoyl peroxide (CAMCBPO),11) and 2-iodoxy-
group at C11. Previously we reported an efficient ortho- benzoic acid (IBX).16,17)
oxidation of phenols with meta-chlorobenzoyl peroxide
Synthesis of Carnosic Acid (2) via Oxidation with
(mCBPO)11) and the synthesis of an anti-microbial diterpene, mCBPO Pisiferic acid 1 was treated with 3 molar equiva-
abietaquinone methide, active against drug resistant bacteria lents of mCBPO in methylene chloride for 16 h at ambient
such as methicillin-resistant Staphylococcus aureus (MRSA) temperature. The crude meta-chlorobenzoic acid (mCBA)
and vancomycin-resistant Enterococcus (VRE).12,13) In this monoester (5) of carnosic acid was obtained and the crude
report, we describe the facile synthesis of the major anti- monoester was hydrolyzed with sodium hydroxide and
oxidant in rosemary, carnosic acid 2, from pisiferic acid 1,
which is the major constituent of Sawara (Chamaecyparis
pisifera). The other rosemary anti-oxidants, e.g., carnosol 3,
rosmanol 4, and other antioxidant diterpenes were synthe-
Chart 1. Synthesis of Carnosic Acid from Pisiferic Acid via Oxidation
Fig. 1. Major Diterpenes of Sawara and Rosemary
with mCBPO
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: nrp53885@nifty.com

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Vol. 58, No. 1
sodium borohydride (NaBH4) in methanol. The products was filtered through Celite and the filtrate was washed with
were separated by chromatography to give 2 in 11% yield aqueous saturated NaHCO3. The oxidation product, quinone
from 1. The spectral properties (1H- and 13C-NMR) of the methide C, the tautomeric isomer of ortho-quinone B, could
synthetic carnosic acid were identical to those of the natural be transformed to the lactone via nucleophilic addition of the
carnosic acid reported in the literature.4) The yield of carboxylic acid group to carbon C-7. After purification by
carnosic acid via oxidation with mCBPO was calculated to column chromatography, carnosol 3 was obtained in 67%
1
be more than 50% by H-NMR spectroscopy, however, the yield.
isolated yield was very low (11%), due to the difficulty of
separating carnosic acid from mCBA.
Biological Activities Minimun inhibitory concentration
(MIC mg/ml) of the synthetic carnosic acid 2 and carnosol 3
Synthesis of Carnosic Acid (2) via Oxidation with were measured against Propionibacterium acnes (ATCC
CAMCBPO A solution of CAMCBPO in CH2Cl2 was pre- 6919)18) and Staphylococcus aureus ME/GM/TC Resistant
pared as previously reported and applied to the oxidation of (ATCC 33592)19) to show the potential of these compounds
pisiferic acid 1 at 0 °C for 66 h.11) The products were treated as antibacterial drugs. Both of the synthetic compounds
with NaOH–NaBH4 in methanol as in the oxidation with showed potent antibacterial activities against P. acnes and
mCBPO to give 2 (23.3 mg, 0.07 mmol, 22.4% from 1). The S. aureus (Table 1). The anti-P. acnes activity of synthetic
yield of carnosic acid was not a considerable improvement carnosic acid was more potent than that of the reported activ-
over the previous synthesis with mCBPO.
ity of natural carnosic acid by Weckesser et al.20) As the dif-
Synthesis of Carnosic Acid (2) via Oxidation with IBX ferent species of P. acnes might be used, it would be difficult
Pisiferic acid 1 was oxidized with 1.2 equivalents of IBX in to compare the strength of the two carnosic acid. The isola-
CHCl3–CH3OH (4 : 1) at ambient temperature for 1 h under tion and purification of carnosic acid and carnosol from natu-
argon. As the product was presumed to be the unstable ortho- ral plant is very difficult in general.3) Our synthesis could
quinone (A), it was reduced without isolation by 10 equiva- afford pure carnosic acid reasonably. The MIC of well known
lents of NaBH4 under argon for 4 h. 2-Iodobenzoic acid was antibiotics, Ampicillin and Vancomycin were measured as
easily crystallized in hexane, the mother liquid was evapo- the reference compounds against P. acnes (ATCC 6919) and
rated, and the residue was chromatographed to afford 2 S. aureus respectively.
(72%). Oxidation of pisiferic acid with IBX in DMF, fol-
lowed by reduction with NaBH4, produced less carnosic acid oxidant, carnosic acid, from the major constituent of Sawara,
than the oxidation in CHCl3–CH3OH (4 : 1). pisiferic acid, in a two-step one-pot synthesis. Carnosol, an-
In conclusion, we synthesized the major rosemary anti-
Synthesis of Carnosol (3) from Carnosic Acid (2) other anti-oxidant in rosemary, was synthesized from carnosic
Carnosic acid 2 was oxidized with silver oxide in CH2Cl2 for acid. The other anti-oxidant diterpenes of rosemary, ros-
1.5 h at ambient temperature under Ar. The reaction mixture manol and isorosmanol, have previously been synthesized
from carnosic acid.14,15) Synthetic carnosic acid showed po-
tent antibacterial activities against Propionibacterium acnes
and Staphylococcus aureus ME/GM/TC Resistant. Thus,
these syntheses provide efficient methods to synthesize the
anti-oxidant diterpenes of rosemary and these syntheses also
demonstrate the utility of Sawara as a natural resource.9,10)
Experimental
General Procedures NMR spectra were measured on a JEOL alpha-
600 (1H: 600 MHz, 13C: 150.8 MHz) spectrometer in CDCl3 using tetra-
methylsilane as an internal standard (J-values in Hz). IR spectra were meas-
Chart 2. Synthesis of Carnosic Acid from Pisiferic Acid via Oxidation
Table 1. Antimicrobial Activities of Carnosic Acid and Carnosol
with CACBPO
Staphylococcus aureus
ME/GM/TC Resistant
(ATCC 33592) (MIC mg/ml)
Propionibacterium acnes
(ATCC 6919) (MIC mg/ml)
Compounds
Carnosic acid
Carnosol
Vancomycin
Ampicillin
1
10
30
1
10
Not measured
0.1
Not measured
Chart 3. Synthesis of Carnosic Acid from Pisiferic Acid via Oxidation
with IBX
Chart 4. Synthesis of Carnosol from Carnosic Acid

January 2010
29
ured on a JEOL JIR-WINSPEC 50 infrared spectrometer. Mass spectra were yellow powder, mp 185—188 °C (crystallized from hexane–ethyl acetate);
recorded on a JEOL JMS-SX102A spectrometer. Optical rotations were [a]D(MeOH)ꢁꢂ133 (lit. mp 185—190 °C; [a]D ꢂ191).21)
measured on JASCO DIP-360 polarimeter. Melting points were measured on
Synthesis of Carnosol (3) from Carnosic Acid (2) To a solution of 2
a MEL-TEMP (Laboratory Device) and were uncorrected. TLC was carried (60 mg, 0.18 mmol) in CH2Cl2 (5 ml), silver oxide (85 mg, 0.36 mmol) was
out on Silica gel 60 (0.25 mm thickness) with fluorescent indicator added and the mixture was stirred for 1.5 h at ambient temperature under
(Macherey-Nagel). Silica gel (6 nm, BW-127ZH, Fuji Silysia Chemical Ltd.) Ar. The mixture was filtered through Celite with hexane. The filtrate was
was used for column chromatography. washed with NaHCO3, dried and evaporated. The residue was chro-
Pisiferic Acid (1) from Sawara (Chamaecyparis pisifera) Leaves of matographed on a silica gel column with hexane–ethyl acetate (5 : 1), to give
Chamaecyparis pisifera were collected in the Fuchu campus of Tokyo Uni- carnosol 3 (40.2 mg, 0.12 mmol, 67%), yellow powder, mp 212—213 °C
versity of Agriculture and Technology in January. The leaves (1.2 kg) were (crystallized from hexane–ethyl acetate) (lit. synthetic carnosol, 212—
extracted with methanol (2.5 l) under reflux for 24 h. The extract was evapo- 213 °C15); natural carnosol, 219.5 °C22)).
rated and the residue was extracted with ethyl acetate and water. Organic
Methods of Anti-microbial Activities in vitro Assays Minimun in-
layer was evaporated and the residue was chromatographed on a short col- hibitory concentration (MIC mg/ml) of the synthetic carnosic acid 2 and
umn of silica gel with hexane–ethyl acetate (3 : 1). The crude fraction of carnosol 3 were performed under conditions described in the literatures for
pisiferic acid was chromatographed on a silica gel column with hexane– each assay against Propionibacterium acnes (ATCC 6919)18) and Staphylo-
ethyl acetate (3 : 1) again. Crystallization from ethyl acetate–hexane gave coccus aureus ME/GM/TC Resistant (ATCC 33592).19) Two compounds 2
light green crystals (7.3 g, 0.6% of the leaves) of 1. The isolated pisiferic and 3 were tested at half-log concentrations ranging from 0.03 to 100 mg/ml
acid; green solid; mp 174—180 °C was found to be identical with authentic
in the P. acnes and S. aureus in vitro growth inhibition assays.
Propionibacterium acnes (ATCC 6919): Culture Medium: Reinforced
Clostridial Medium, Vehicle: 1% DMSO, Incubation Time/Temp: 2 d at
1
pisiferic acid by the H-NMR (600 MHz, CDCl3) and 13C-NMR (150 MHz,
CDCl3).10)
Synthesis of Carnosic Acid (2) by Oxidation with mCBPO To the so- 37 °C, Incubation Volume: 3 ml, Time of Assessment: 2 d, Quantitation
lution of 1 (200 mg: 0.63 mmol) in methylene chloride (10 ml), 3 equivalent Method: Turbidity measurement and plating count of subculture.
molar of mCBPO (590 mg: 1.9 mmol) was added. The mixture was allowed
Staphylococcus aureus ME/GM/TC Resistant (ATCC 33592): Culture
at ambient temperature for 16 h. The reaction mixture was evaporated and Medium: Mueller-Hinton Broth, Vehicle: 1% DMSO, Incubation Time/
the residue was dissolved in ethyl acetate. To the solution, hexane was added Temp: 20 h at 37 °C, Incubation Volume: 1 ml, Time of Assessment: 1 d,
to give white crystals of mCBA. After filtration of the precipitate, the solu- Quantitation Method: Turbidity. Measurement.
tion was dissolved in MeOH and treated with NaBH4 at ambient temperature
for 1 h. The solution was evaporated and the residue was chromatographed
Acknowledgement We are grateful to Professor M. Kuroyanagi of Hi-
on a silica gel column with ethyl acetate–hexane to give a mixture of 11- roshima Prefectural University for providing the authentic pisiferic acid.10)
carnosyl m-chlorobenzoate 5, light yellow powder; 1H-NMR (600 MHz,
CDCl3) d: 8.20 (1H, s), 8.10 (1H, dd, Jꢁ7.5, 1.8 Hz), 7.60 (1H, dd, Jꢁ8.4,
1.8 Hz), 7.45 (1H, t, Jꢁ8.4 Hz), 6.69 (1H, s), 3.35—3.32 (1H, m), 2.97—
2.86 (3H, m), 2.40—2.33 (1H, m), 1.91—1.87 (2H, m), 1.61—1.59 (2H, m),
1.52—1.48 (1H, m), 1.34—1.25 (2H, m), 1.20 (3H, d, Jꢁ6.6 Hz), 1.17 (3H,
d, Jꢁ6.6 Hz), 1.01 (3H, s), 0.89 (3H, s); 13C-NMR (150 MHz, CDCl3) d:
180.17, 163.70, 140.06, 136.69, 135.71, 134.81, 133.62, 130.97, 130.32,
129.94, 128.43, 124.77, 119.2, 53.82, 48.36, 41.59, 34.32, 34.29, 32.59,
31.99, 27.64, 22.94, 22.75, 21.16, 20.05, 18.54.
The monoesters mixture was dissolved in methanol (9 ml)–1% NaOH
(1 ml) and then 3 equivalent molar of NaBH4 (72 mg, 1.9 mmol) was added.
The mixture was heated under reflux for 2 h. The reaction mixture was acidi-
fied with 1 M-HCl and extracted with brine–hexane. The organic layer was
washed with brine, dried over MgSO4 and evaporated. The residue was chro-
matographed on a silica gel column with hexane–ethyl acetate (10 : 1) to
give 2 (23 mg: 0.07 mmol, 11% from pisiferic acid).
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