Synthesis of small combinatorial libraries of natural products: Identification and quantification of new long-chain 3-methyl-2-alkanones from the root essential oil of Inula helenium L. (Asteraceae)

Article abstract of DOI:10.1002/pca.2466

Introduction Recently, a potent anti-staphylococcal activity of Inula helenium L. (Asteraceae) root essential oil was reported. Also, bioassay guided fractionation of the oil pointed to eudesmane sesquiterpene lactones and a series of unidentified constituents as the main carriers of the observed activity. Objective To identify nine new constituents (long-chain 3-methyl-2-alkanones) from a fraction of this root essential oil with a low minimum inhibitory concentration value (0.8 μg/mL) by employing a synthetic methodology that leads to the formation of a small combinatorial library of these compounds. Methods The identity of these constituents was inferred from mass spectral fragmentation patterns and GC retention data. A library of 3-methyl-2-alkanones (C₁₁-C₁₉ homologous series) was synthesised in three steps starting from methyl acetoacetate and the corresponding alkyl halides. The synthetic library was also screened for in vitro anti-microbial activity. Results Gas chromatographic analyses of I. helenium essential oil samples with spiked compounds from the synthesised library corroborated the tentative identifications of the long-chain 3-methyl-2-alkanones. The availability of these anti-microbial compounds from this library made it possible to construct GC/FID calibration curves and determine their content in the plant material: 0.08 - 24.2 mg/100 g of dry roots. Conclusion The small combinatorial library approach enabled the first unequivocal identification of long-chain 3-methyl-2-alkanones as plant secondary metabolites, and, also, allowed determination of not only a single compound and biological properties, but those of a group of structurally related compounds. Copyright

Full text of DOI:10.1002/pca.2466

Research Article
Received: 21 February 2013,
Revised: 18 June 2013,
Accepted: 20 June 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/pca.2466
Synthesis of Small Combinatorial Libraries of
Natural Products: Identification
and Quantification of New Long-chain
3-Methyl-2-alkanones from the Root Essential
Oil of Inula helenium L. (Asteraceae)
Niko S. Radulović,a* Marija S. Denića and Zorica Z. Stojanović-Radićb
ABSTRACT:
Introduction – Recently, a potent anti-staphylococcal activity of Inula helenium L. (Asteraceae) root essential oil was reported.
Also, bioassay guided fractionation of the oil pointed to eudesmane sesquiterpene lactones and a series of unidentified
constituents as the main carriers of the observed activity.
Objective – To identify nine new constituents (long-chain 3-methyl-2-alkanones) from a fraction of this root essential oil with
a low minimum inhibitory concentration value (0.8 μg/mL) by employing a synthetic methodology that leads to the formation
of a small combinatorial library of these compounds.
Methods – The identity of these constituents was inferred from mass spectral fragmentation patterns and GC retention data.
A library of 3-methyl-2-alkanones (C11–C19 homologous series) was synthesised in three steps starting from methyl
acetoacetate and the corresponding alkyl halides. The synthetic library was also screened for in vitro anti-microbial activity.
Results – Gas chromatographic analyses of I. helenium essential oil samples with spiked compounds from the synthesised
library corroborated the tentative identifications of the long-chain 3-methyl-2-alkanones. The availability of these
anti-microbial compounds from this library made it possible to construct GC/FID calibration curves and determine their
content in the plant material: 0.08 À 24.2 mg/100 g of dry roots.
Conclusion – The small combinatorial library approach enabled the first unequivocal identification of long-chain
3-methyl-2-alkanones as plant secondary metabolites, and, also, allowed determination of not only a single compound
and biological properties, but those of a group of structurally related compounds. Copyright © 2013 John Wiley & Sons, Ltd.
Supporting information can be found in the online version of this article.
Keywords: Anti-staphylococcal activity; small combinatorial library; 3-methyl-2-alkanones; 3-methyl-2-tetradecanone; root essential
oil; Inula helenium L
characterisation and the opportunity to test the biological activity of
Introduction
thecompoundinanumberofassays. Inthisway, two new minor vol-
A large number of papers dealing with essential oil analysis by
atile compounds, conmaculatin and ternanthranin, were identi-
GC and GC–MS usually disregarded many minor constituents,
fied from two plant species, and they were found to be strong
due to the lack of any positive MS library search hits for the
given mass scan. These compounds could even be the
analgaesics (Radulović et al., 2011, 2012a).
If only a fragment of the structure of an interesting essential
contributors to organoleptic properties and/or biological activity
oil constituent could be inferred from the available data (MS
of the essential oil. A close inspection of the mass spectral data
and retention index (RI)), or if the tentative identification could
of these minor components may give an idea of their identity
to an experienced phytochemist. The knowledge of their retention
data may further strengthen the tentative identification and provide
the rationale for any future work on this matter. There are two
possibilities: (1) to try to isolate in their pure state the constituents
* Correspondence to: N. S. Radulović, Department of Chemistry, Faculty of
Science and Mathematics, University of Niš, Višegradska 33, 18000 Niš,
of interest from a usually complex matrix; or (ii) to perform a
synthesis of the compound with the proposed structure. The latter
approach has several advantages over the more frequent isolation
and identification approach, although it is considered more time-
consuming. The synthetic work gives the desired compound in the
amount that allows unobstructed structural elucidation, spectroscopic
Serbia. E-mail: nikoradulovic@yahoo.com
a
Department of Chemistry, Faculty of Science and Mathematics, University
of Niš, Višegradska 33, 18000, Niš, Serbia
b
Department of Biology and Ecology, Faculty of Science and Mathematics,
University of Niš, Višegradska 33, 18000, Niš, Serbia
Phytochem. Anal. 2013
Copyright © 2013 John Wiley & Sons, Ltd.

N. S. RADULOVIĆ et al.
Gram-negative Escherichia coli ATCC 8739, Proteus vulgaris ATCC 8427
and Pseudomonas aeruginosa ATCC 9027; as well as a yeast organism
Candida albicans ATCC 10231 and a mold organism Aspergillus niger
ATCC 16404.
be narrowed down to a number of isomers (e.g. by biosynthetic
considerations), the only comprehensive approach in this
situation would be to create a small combinatorial library of all
possible compounds. Such libraries provide the possibility to
investigate structure–activity/property relationships within the
group of synthesized compounds and provide important
(spectral) data for future investigations.
Plant material
Dried roots of I. helenium were purchased from a local herb shop in Niš,
Serbia (manufactured by the Institute Josif Pančić, Belgrade, Serbia). The
plant material was macro- and microscopically examined by one of us
(Z. Stojanović-Radić) to verify the taxonomical identification of the plant
species from which it originated. All tests confirmed the identity and
purity of the material. A voucher specimen was deposited with the
Herbarium of the Faculty of Science and Mathematics, University of Niš,
under the accession number DM0112.
Inula helenium L. (Compositae) is a perennial herb (‘oman’ in
Serbian) used extensively in folk medicine, mostly for the
treatment of respiratory conditions, digestive disorders, urinary
infections and skin disorders, as well as a mosquito repellent
(Tucakov, 1984).
Recently, it has been shown that I. helenium root essential oil
possesses a potent anti-staphylococcal activity that is closely
connected to its ethnopharmacological usage (Stojanović-Radić
et al., 2012). A bioassay-guided fractionation of the oil yielded a
number of chromatographic fractions that had activity that
surpassed that of the oil itself and further enabled the location
of the major active principles – three bacterial cell-membrane
damaging sesquiterpene lactones, alantolactone, isoalantolactone
and diplophyllin (Blagojević and Radulović, 2012; Stojanović-Radić
et al., 2012). However, the chemical composition of another
fraction with a noteworthy minimal inhibitory concentration
(MIC= 0.8μg/mL) remained unknown. This minor fraction (3.5%)
contained an unidentified sesquiterpene aldehyde and a series
ofcompoundswithananalogousfragmentationpatternintheirmass
spectral data. The MS data hinted that the compounds of this series
could be 3-methyl-2-alkanones of varying chain lengths. As the isola-
tion of single compounds from this fraction was impossible (70 mg of
a mixtureofmore than 10 compoundswas available)a combinatorial
library of long-chain 3-methyl-2-alkanones was created.
Essential oil – extraction and fractionation
Air-dried, to constant weight, roots of I. helenium (ca. 100 g) were ground
and subjected to hydrodistillation with ca. 500mL of distilled water for
3.5h using the original Clevenger-type apparatus. The oil obtained was
separated by extraction with diethyl ether and dried over anhydrous
magnesium sulphate. The solvent was evaporated under a gentle stream
of nitrogen at room temperature in order to exclude any loss of the
essential oil and stored at À18 ° C until further analysis. Once the oil yield
was determined, the residue was exposed to vacuum at room
temperature for a short period to eliminate the solvent completely. The
pure oil was then measured on an analytical balance and multiple
gravimetric measurements were taken during 24h to ensure that all of
the solvent had evaporated. The yield of the essential oil was 1.4% (w/w).
Preparative medium-pressure liquid chromatography (MPLC) was
performed with a pump module C-601 and a pump controller C-610
Work-21 pump (Büchi, Switzerland) and was carried out on pre-packed
column cartridges (40 × 75mm) Silica-gel 60 (Büchi), particle size
distribution 40–63 μm. Silica-gel 60 on Al plates, layer thickness 0.2mm
(Kieselgel 60F254, Merck), was used for thin-layer chromatography (TLC).
The spots on TLC were visualised by UV light (254 nm) and by spraying with
50% (v/v) aqueous sulphuric acid or phosphomolybdic acid (12g) in
ethanol (250mL) followed by heating. A sample of the essential oil
(500mg) was subjected to MPLC (gradient diethyl ether: n-hexane, from
pure n-hexane to pure diethyl ether, 100mL). The fractions obtained
(10 mL) were pooled according to TLC and/or GCÀMS analyses.
Thus, in continuation of our previous studies on the volatile
anti-microbial constituents of I. helenium (Blagojević and
Radulović, 2012; Stojanović-Radić et al., 2012), in this work we
report the identification, synthesis and microbiological testing
of nine additional minor constituents that represent secondary
metabolites found for the first time in the Plant Kingdom.
Experimental
Analytical and spectral analyses
Chemicals and reagents
GCÀMS analyses. The GCÀMS analyses of all samples (pure oil, MPLC
fractions, reaction mixtures) were repeated three times using a Hewlett-Packard
6890 N gas chromatograph. The gas chromatograph was equipped with a
fused silica capillary column DB-5 (5% phenylmethylsiloxane, 30 m × 0.25 mm,
film thickness 0.25 μm; Agilent Technologies, USA) and coupled with a 5975B
mass selective detector from the same company. The injector and interface
were operated at 250 and 300 °C, respectively. The oven temperature was
raised from 70 to 290 °C at a heating rate of 5 °C/min and then isothermally held
for 10 min. Helium at 1.0 mL/min was used as a carrier gas. The samples, 1 μL of
the corresponding solutions in diethyl ether (1:100), were injected in a pulsed
split mode (the flow was 1.5 mL/min for the first 0.5 min and then set to
1.0 mL/min throughout the remainder of the analysis; split ratio 40:1). The mass
selective detector was operated at the ionisation energy of 70 eV, in the
35–500 amu range, with a scanning speed of 0.34 s.
All chemicals and solvents used were of analytical or pharmaceutical
grade. Diethyl ether, n-hexane, methanol, ethanol, carbon tetrachloride
and anhydrous magnesium sulphate, as well as, octanoic, decanoic,
dodecanoic, tetradecanoic and hexadecanoic acids, were purchased
from Sigma-Aldrich (St Louis, MS, USA). 1-Octanol, 1-decanol,
1-dodecanol and 1-tetradecanol were obtained from Carl Roth GmbH +
Co.KG (Karlsruhe, Germany). Sulphuric acid, hydrochloric acid, dimethyl
sulphoxide, potassium hydroxide, sodium, bromine, iodine, red
phosphorus, mercuric oxide, methyl acetoacetate and methyl iodide
were supplied by Merck (Darmstadt, Germany). Triphenyltetrazolium
chloride and Mueller Hinton agar were also supplied by Merck, whereas
Sabouraud dextrose agar was obtained from Difco Laboratories
(Detroit, MI, USA). Microtitre plates were purchased from Carl Roth
GmbH + Co.KG, whereas tetracycline and nystatin, used as positive
controls in the anti-microbial assays, were obtained from Galenika
(Belgrade, Serbia). Chromatographic separations were carried out using
silica gel 60 (particle size distribution 40–63 μm ) purchased from Merck.
The in vitro anti-microbial activity was tested against a panel of
laboratory control strains belonging to the American Type Culture
Collection (MD, USA) including: Gram-positive Staphylococcus aureus
ATCC 6538, Bacillus subtilis ATCC 6633 and Bacillus cereus ATCC 9139;
GC/FID analyses. The GC analyses were carried out using a Agilent
7890A GC system equipped with
a single injector, one flame
ionisation detector (FID) and a fused silica capillary column HP-5MS
(5% phenylmethylsiloxane, 30 m × 0.32 mm, film thickness 0.25 μm;
Agilent Technologies, USA). The oven temperature was programmed from
150 to 300 °C at 15°C/min and then held isothermally at 300 °C for 5 min;
the carrier gas was nitrogen at 3 mL/min; the injector temperature was held
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Copyright © 2013 John Wiley & Sons, Ltd.
Phytochem. Anal. 2013

New Long-Chain 3-Methyl-2-Alkanones From Inula Helenium
at 250 °C. The samples, 1 μL of the corresponding solutions, were injected
in a splitless mode. The parameters of the FID detector were as follows:
heater temperature, 300°C; H2 flow, 30mL/min; air flow, 400 mL/min;
make-up flow 23.5mL/min; signal, 20 Hz.
[C6H9O3]+ (31.5), 116 [C5H8O3]+. (100), 101 (9.2), 87 (59), 74 (14.3), 55
(21.8), 43 [CH3CO]+ (61). The RI and MS data for compounds 3aÀc, 3e
and 3f are given in Supporting information.
Methyl 2-acetyl-3-methylalkanoates (4a-i). Methyl 2-acetyl-
3-methylalkanoates (4a-i) were prepared by alkylation of methyl
2-acetylalkanoates (3a-i) with commercially available methyl iodide follow-
ing an analogous procedure described for methyl 2-acetylalkanoates.
Compound 4a: yield 90%. GC–MS purity: 93.4%, RI (DB-5) = 1507; MS
(EI, 70 eV), m/z (relative intensity, %): 228 [M]+. (0.1), 197 [M À OCH3]+
(1.8), 186 (65), 169 [M À COOCH3]+. (1.8), 157 (3.5), 143 (30.8), 130
[CH3COCH(CH3)COOCH3]+. (60.4), 112 (5.3), 101 (100), 98 (13.6), 88 (7.7),
69 (13.6), 55 (13.3), 43 [CH3CO]+ (43.8).
GC/FID quantification. The quantification of 3-methyl-2-alkanones was
carried out by peak-area integration. All synthesised standards of 3-methyl-
2-alkanones were injected at five different concentrations (0.009, 0.045,
0.18, 0.45 and 0.9 mg/mL) in order to build up five-point GC/FID
calibration curves by plotting compound concentration versus peak area
(C= f (A)). Each sample was analysed for three consecutive runs.
IR measurements. The IR measurements (ATR-attenuated total
reflectance) were carried out using a Thermo Nicolet model 6700 FTIR
instrument (Waltham, USA).
Compound 4b: yield 86%. GC–MS purity: 94.9%, RI (DB-5) = 1608; MS
(EI, 70 eV), m/z (relative intensity, %): 242 [M]+. (0.1), 211 [M À OCH3]+
(1.5), 200 (64.8), 183 [M À COOCH3]+ (1.8), 171 (3.3), 157 (26.7), 143
(22.8), 130 [CH3COCH(CH3)COOCH3]+. (49), 112 (5.2), 101 (100), 98 (13),
88 (7.9), 69 (13.1), 55 (13), 43 [CH3CO]+ (43.1).
NMR measurements. The NMR spectra for all 3-methyl-2-alkanones,
excluding 3-methyloctadecan-2-one, were recorded at 200 MHz (1H)
and 50 MHz (13C) using a Varian Gemini 200 spectrometer (Palo Alto,
CA, USA). The NMR spectra of 3-methyloctadecan-2-one were recorded
on a Bruker Avance II + 600 (Fällanden, Switzerland; 1H at 600.13 MHz;
13C at 150.92 MHz). All NMR spectra were measured at 25 °C in deuterated
chloroform. Chemical shifts are reported in parts per million (δ) relative to
residual solvent protons as the internal standard (deuterated chloroform:
7.26 ppm for 1H and 77 ppm for 13C). Scalar couplings are reported in
hertz.
Compound 4c: yield 86%. GC–MS purity: 93.9%, RI (DB-5) = 1709; MS
(EI, 70 eV), m/z (relative intensity, %): 256 [M]+. (0.1), 225 [M À OCH3]+
(1.3), 213 (64.4), 197 [M À COOCH3]+ (1.6), 185 (3.1), 171 (7.6), 157
(25.3), 143 (11.3), 130 [CH3COCH(CH3)COOCH3]+. (48.6), 112 (5), 101
(100), 98 (12.9), 88 (8), 69 (12.8), 55 (12.7), 43 [CH3CO]+ (42.5).
Compound 4d: yield 85%. GC–MS purity: 95.9%, RI (DB-5) = 1810; MS
(EI, 70 eV), m/z (relative intensity, %): 270 [M]+. (0.1), 239 [M À OCH3]+
(1.4), 228 (64.3), 211 [M À COOCH3]+. (1.5), 199 (3), 185 (7.9), 171 (15.0),
157 (15.8), 143 (10.8), 130 [CH3COCH(CH3)COOCH3]+. (48.5), 112 (5), 101
(100), 98 (13.1), 88 (7.9), 69 (12.7), 55 (12.6), 43 43 [CH3CO]+ (42.2).
Compound 4e: yield 82%. GC–MS purity: 95.9%, RI (DB-5) = 1910; MS (EI,
70eV), m/z (relative intensity, %): 284 [M]+. (0.1), 253 [MÀ OCH3]+ (1.3), 242
(64.7), 225 [M À COOCH3]+ (1.4), 213 (2.9), 199 (7.6), 185 (14.9), 171 (2.4),
157 (15.9), 143 (10.7), 130 [CH3COCH(CH3)COOCH3]+. (48.6), 112 (5), 101
(100), 98 (12.9), 88 (7.9), 69 (12.8), 55 (12.7), 43 [CH3CO]+ (42.5).
Compound 4f: yield 84%. GC–MS purity: 95.8%, RI (DB-5) = 2012; MS (EI,
70eV), m/z (relative intensity, %): 298 [M]+. (0.1), 267 [MÀ OCH3]+ (1.2), 256
(67.3), 239 [M À COOCH3]+ (1.2), 213 (6.6), 199 (14.6), 185 (4.2), 171 (2), 157
(16.9), 143 (9.7), 130 [CH3COCH(CH3)COOCH3]+. (52.6), 112 (5.1), 101 (100),
98 (12.6), 88 (8.5), 69 (13.7), 55 (13.7), 43 [CH3CO]+ (45.6).
Synthesis of 3-methyl-2-alkanones
Alkyl halides. Alkyl halides were obtained following two different
standard procedures. A modified Hunsdiecker reaction, where free
carboxylic acid is treated with a mixture of mercuric oxide and bromine
in carbon tetrachloride, was applied for synthesis of odd-numbered alkyl
bromides (Lampman and Aumiller, 1988), whereas even-numbered alkyl
iodides were prepared by reaction of an appropriate alcohol with
phosphorus triiodide generated in situ from red phosphorus and
crystalline iodine (Vogel, 1989). All synthesised alkyl halides were purified
by ‘dry-flash’ column chromatography and obtained in 75–85% yields.
Methyl 2-acetylalkanoates (3a–i). Metallic sodium (5 mmol) was
dissolved in a solution of methyl acetoacetate (2, 5 mmol) in anhydrous
methanol (12 mL). The reaction mixture was then cooled to 0 °C and alkyl
halide (6 mmol) was slowly added dropwise with stirring during 20 min.
After heating the reaction mixture under reflux for 15–25 h, the excess
of methanol was evaporated in vacuo, 100 mL of water was added and
the resulting mixture was extracted several times with diethyl ether.
The organic layers were combined, washed with 10% aqueous solution
of hydrochloric acid, dried over anhydrous magnesium sulphate and
concentrated under reduced pressure to yield crude 3a-i, which were
used without further purification.
Compound 3d: yield 78%. GC–MS purity: 94.2%, RI (DB-5) = 1766; MS
(EI, 70 eV), m/z (relative intensity, %): 256 [M]+. (0.3), 241 [M À CH3]+
(0.2), 225 [M À OCH3]+ (3.5), 214 (25.2), 196 (0.1), 185 (5.2), 171 (12.4),
157 (2.2), 143 (17.8), 129 [C6H9O3]+ (27.2), 116 [C5H8O3]+. (100), 101
(14.2), 87 (76.7), 74 (16.6), 55 (27.2), 43 [CH3CO]+ (70.2).
Compound 3g: yield 70%. GC–MS purity: 93.2%, RI (DB-5) = 2071; MS (EI,
70 eV), m/z (relative intensity, %): 298 [M]+. (0.4), 283 [M À CH3]+ (0.2), 267
[MÀ OCH3]+ (2.3), 256 (21.9), 241 (0.1), 227 (2), 213 (10.4), 199 (4.1), 185
(2.9), 171 (2.9), 157 (4.1), 143 (13.5), 129 [C6H9O3]+ (26.8), 116 [C5H8O3]+.
(100), 101 (11.4), 87 (64.3), 74 (14.9), 55 (21.7), 43 [CH3CO]+ (59.5).
Compound 3h: yield 71%. GC–MS purity: 93.8%, RI (DB-5) = 2172; MS
(EI, 70 eV), m/z (relative intensity, %): 312 [M]+. (0.5), 297 [MÀ CH3]+ (0.3),
281 [M À OCH3]+ (1.9), 270 (22.1), 241 (2.2), 227 (9.3), 213 (3), 199 (1.5),
185 (3.7), 172 (3), 154 (2), 143 (12.8), 129 [C6H9O3]+ (28.6), 116 [C5H8O3]+.
(100), 101 (9.9), 87 (61.2), 74 (14.7), 55 (21.8), 43 43 [CH3CO]+ (60.2).
Compound 3i: yield 70%. GC–MS purity: 93.5%, RI (DB-5) = 2272; MS
(EI, 70 eV), m/z (relative intensity, %): 326 [M]+. (0.6), 311 [M À CH3]+
(0.3), 295 [M À OCH3]+ (1.5), 284 (21.9), 255 (2.0), 241 (8.9), 227 (1.3),
213 (0.8), 199 (4.1), 185 (4.7), 172 (2.3), 154 (1.6), 143 (12.2), 129
Compound 4 g: yield 80%. GC–MS purity: 95.4%, RI (DB-5) = 2114; MS
(EI, 70 eV), m/z (relative intensity, %): 312 [M]+. (0.2), 281 [M À OCH3]+
(1.1), 270 (73.8), 253 [M À COOCH3]+ (1.1), 227 (5.7), 213 (13.9), 199
(4.3), 185 (1.9), 171 (4.0), 157 (19.4), 143 (8.6), 130 [CH3COCH(CH3)
COOCH3]+. (57.7), 112 (5.5), 101 (100), 98 (12.4), 88 (9), 69 (14.3), 55
(13.4), 43 [CH3CO]+ (43.6).
Compound 4 h: yield 81%. GC–MS purity: 95.2%, RI (DB-5) = 2213; MS
(EI, 70 eV), m/z (relative intensity, %): 326 [M]+. (0.1), 295 [M À OCH3]+
(1), 284 (75.6), 267 [M À COOCH3]+ (1.5), 241 (3.4), 227 (13.7), 213 (3.3),
199 (2.8), 185 (4.0), 171 (2.3), 157 (17.4), 143 (8.3), 130 [CH3COCH(CH3)
COOCH3]+. (60.8), 112 (5.5), 101 (100), 98 (12), 88 (9.3), 69 (14.4), 55
(14.5), 43 [CH3CO]+ (47.6).
Compound 4i: yield 84%. GC–MS purity: 95.6%, RI (DB-5) = 2313; MS
(EI, 70 eV), m/z (relative intensity, %): 340 [M]+. (0.1), 309 [M À OCH3]+
(0.8), 298 (79.7), 281 [M À COOCH3]+ (1.8), 265 (1.2), 255 (5.4), 241
(13.9), 227 (1.5), 213 (3.9), 199 (5.3), 185 (4.1), 171 (1.3), 157 (16.4), 143
(8.7), 130 [CH3COCH(CH3)COOCH3]+. (64.5), 112 (6.4), 101 (100), 98
(11.7), 88 (9.7), 69 (14.3), 55 (15.1), 43 [CH3CO]+ (50.7).
3-Methyl-2-alkanones (1a-i). A slightly modified procedure of Ohnuma
et al. (1989) was applied. A mixture of methyl 2-acetyl-3-methylalkanoates
(4a–i, 3 mmol), 2 mol/L aqueous solution of potassium hydroxide (20 mL)
and ethanol (3.3 mL) was stirred at room temperature for 24 h. The reaction
mixture was then acidified with 10% aqueous solution of hydrochloric acid
and extracted three times with diethyl ether. Combined ether extracts, dried
over anhydrous magnesium sulphate, were concentrated under reduced
pressure. The crude products obtained were fractioned by ‘dry-flash’column
chromatography using a gradient of diethyl ether and n-hexane (from pure
n-hexane to 2% diethyl ether in n-hexane with an increment step of 1%) to
yield pure 3-methyl-2-alkanones.
Phytochem. Anal. 2013
Copyright © 2013 John Wiley & Sons, Ltd.
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N. S. RADULOVIĆ et al.
NMR and 13C NMR) of compounds 1aÀf are given in Supporting
Compound 1g: yield 93%. GC–MS purity: 99.1%, RI (DB-5) = 1849; MS
(EI, 70 eV), m/z (relative intensity, %): 254 [M]+. (0.6), 239 [MÀ CH3]+ (0.1),
211 [MÀ CH3CO]+ (0.1), 197 [MÀ C4H9]+ (0.1), 182 [MÀ C4H8O]+. (0.1),
169 (0.1), 155 (0.1), 141 (0.3), 127 (0.6), 109 (0.4), 97 (0.9), 85 (11.4), 72
[C4H8O]+. (100), 71 [CH3COCHCH3]+ (6.5), 57 [C4H9]+ (12.6), 43 [CH3CO]+
(32.3); FTIR-ATR (neat) cmÀ1: 2921, 2852, 1714 (C = O), 1461, 1347, 1119,
715; 1H-NMR (200 MHz, CDCl3) δ 2.50 (h, J = 6.9Hz, 1H, CH), 2.13 (s, 3H,
CH3CO), 1.32–1.22 (m, 24H, 12× CH2), 1.08 (d, J = 6.9Hz, 3H, CHCH3), 0.88
(t, J = 7.0Hz, 3H, CH2CH3); 13C-NMR (50 MHz, CDCl3) δ 212.9 (C = O), 47.2
(CH), 32.9 (CH2), 31.9 (CH2), 29.7 (5× CH2), 29.6 (CH2), 29.5 (CH2), 29.3
(CH2), 27.9 (CH3C = O), 27.2 (CH2), 22.7 (CH2), 16.1 (CH3), 14.1 (CH3).
Compound 1 h: yield 86%. GC–MS purity: 99.4%, RI (DB-5) = 1950; MS
(EI, 70 eV), m/z (relative intensity, %): 268 [M]+. (0.8), 255 [M À CH3]+
(0.1), 225 [M À CH3CO]+ (0.1), 211 [M À C4H9]+ (0.1), 196 [M À C4H8O]+.
(0.1), 169 (0.1), 155 (0.1), 141 (0.1), 127 (0.3), 113 (0.6), 109 (0.5), 97
(1.1), 85 (11.5), 72 [C4H8O]+. (100), 71 [CH3COCHCH3]+ (6.2), 57 [C4H9]+
(11.1), 43 [CH3CO]+ (28.7); FTIR-ATR (neat) cmÀ1: 2920, 2851, 1713
information.
Antimicrobial assay
The anti-microbial activity was evaluated using a microdilution broth
method (Radulović et al., 2012b). Stock solutions of 3-methyl-2-
alkanones were prepared in 0.05% (v/v) aqueous Tween 80 and serial
dilutions tested in the range 37.00–0.14mg/mL. Tetracycline and nysta-
tin served as positive controls, whereas the corresponding solvent
(0.05% (v/v) Tween 80) was used as the negative control.
Results and discussion
In a previous study of I. helenium root oil 45 compounds were identified,
but additional constituents that did not appear in the original total ion
chromatogram (TIC) of the unfractionated oil were detected after a chro-
matographic separation on SiO2 of the oil. One of these fractions (eluting
with 5% diethyl ether in n-hexane), highly active against S. aureus,
contained a sesquiterpene aldehyde and a series of nine compounds
(1a–i) showing regularities in their GC retention behaviour and possessing
analogous mass spectral data. In the mass spectra of compounds 1a–i
(Fig. 1) the base peaks at m/z 72 arising from a probable McLafferty frag-
mentation and the abundant acetyl ion fragment at m/z 43 hinted that
these compounds might constitute a series of 3-methyl-2-alkanones
(Francke, 2010). Additionally, the difference of 14 amu (one CH2 group)
between the molecular ions of any two consecutive compounds in the TIC,
separated by ca. 100 RI units, pointed to the existence of a C11–C19 homolo-
gous series (Fig. 1). Unfortunately, we found no retention data in the literature
on these compounds and relied on the following rule of thumb for the
structure–retention data relationship: the difference between literature RI
values for isomeric compounds 2-tridecanone and 6,10-dimethyl-2-
undecanone was more than 90 units in favour of the first, which is consistent
with the presence of two methyl branches (Setzer et al., 2005; Wang et al.,
2006). Hence, the existence of additional branches in the chain of com-
pounds 1a–i was ruled out because compound 1c from the I. helenium oil
(C = O), 1460, 1352, 1123, 720; 1H-NMR (200 MHz, CDCl3)
δ 2.52
(h, J = 6.9 Hz, 1H, CH), 2.15 (s, 3H, CH3CO), 1.39–1.21 (m, 26H, 13 × CH2),
1.10 (d, J = 6.9 Hz, 3H, CHCH3), 0.90 (t, J = 7.0 Hz, 3H, CH2CH3); 13C-NMR
(50 MHz, CDCl3) δ 213.1 (C = O), 47.2 (CH), 33.0 (CH2), 31.9 (CH2), 29.7
(6 × CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 28.0 (CH3C = O), 27.3 (CH2),
22.7 (CH2), 16.2 (CH3), 14.2 (CH3).
Compound 1i: yield 85%. GC–MS purity: 99.5%, RI (DB-5) = 2051; MS
(EI, 70 eV), m/z (relative intensity, %): 282 [M]+. (0.7), 267 [M À CH3]+
(0.1), 239 [M À CH3CO]+ (0.1), 225 [M À C4H9]+ (0.1), 210 [M À C4H8O]+
(0.1), 183 (0.1), 169 (0.1), 155 (0.1), 141 (0.3), 127 (0.6), 109 (0.5), 97
(1.1), 85 (11.5), 72 [C4H8O]+. (100), 71 [CH3COCHCH3]+ (6.2), 57 [C4H9]+
(11.1), 43 [CH3CO]+ (28.7); FTIR-ATR (neat) cmÀ1: 2919, 2852, 1713
(C = O), 1461, 1352, 1124, 720; 1H-NMR (600 MHz, CDCl3)
δ 2.52
(h, J = 6.9 Hz, 1H, CH), 2.15 (s, 3H, CH3CO), 1.39–1.21 (m, 28H, 14 × CH2),
1.10 (d, J = 6.9 Hz, 3H, CHCH3), 0.90 (t, J = 7.0 Hz, 3H, CH2CH3); 13C-NMR
(151 MHz, CDCl3) δ 213.1 (C = O), 47.2 (CH), 33.0 (CH2), 31.9 (CH2), 29.7
(7 × CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 28.0 (CH3C = O), 27.3 (CH2),
22.7 (CH2), 16.2 (CH3), 14.2 (CH3). Spectroscopic data (RI, MS, IR, 1H
Figure 1. TIC chromatogram of the fraction of I. helenium root essential oil (5% diethyl ether in n-hexane) showing peaks corresponding to an
unidentified sesquiterpene aldehyde (RI 1578) and 3-methyl-2-alkanones (1a–i) and the mass spectrum of 3-methyltetradecan-2-one (1e).
wileyonlinelibrary.com/journal/pca
Copyright © 2013 John Wiley & Sons, Ltd.
Phytochem. Anal. 2013

New Long-Chain 3-Methyl-2-Alkanones From Inula Helenium
Figure 2. Synthetic route to 3-methyl-2-alkanones (1a–i)
fraction that corresponds to this molecular mass has an RI value of ca. 50 units
less that the straight-chain isomer.
methyl 2-acetylalkanoates (3a–i) that were further methylated, in an
analogous manner. Subsequent tandem mild basic hydrolysis and decar-
boxylation of the methylation products 4a–i yielded 3-methyl-2-
alkanones (1a–i) (as depicted in Fig. 2). These three step transformations
were achieved in 50–65% overall yields. The structural assignments of
the target molecules were undertaken by spectral means (1H- and 13C-
NMR, IR, MS). After the co-injection of the nine synthesised compounds
with the root essential oil of I. helenium, the originally proposed hypoth-
esis was unambiguously corroborated by these methods. All nine
3-methyl-2-alkanones were found herein for the first time in the Plant
Kingdom. Additionally, four of them (C13, C17 À C19) are reported here
to occur in a living organism for the first time.
The GC/FID calibration curves for the synthesised 3-methyl-2-alkanones
were built up in order to quantify these compounds in the plant material.
For example, the calibration curve (amount = 1.3285 × 10À4 × area + 5.0526 ×
10À3) for 3-methyl-2-octadecanone, in the concentration range 0.009-
0.9 mg/mL, had an excellent correlation coefficient R2 = 0.99989. All other
3-methyl-2-alkanones from this series had comparable curves and similar
response factors. The content of 3-methyl-2-alkanones determined per
100 g of dry I. helenium roots was within the range 0.08–24.2 mg (Table 1).
The contribution of the identified 3-methyl-2-alkanones to the overall
anti-microbial action of the original fraction of I. helenium essential oil
was evaluated in microdilution assays against six pathogenic bacterial
and two fungal strains. Surprisingly, 3-methyl-2-alkanones did not show
A detailed literature survey showed that long-chain 3-methyl-2-
alkanones had rarely been reported in samples of natural origin, with
no reference to their existence in the Plant Kingdom. 3-Methyl-2-
decanone was found in territorial marking fluids of the male Bengal
tiger, Panthera tigris (Burger et al., 2008), whereas this compound and
its homologue (3-methyl-2-undecanone) were detected in the normal
urine of male wolves, Canis lupus (Raymer et al., 1986). Three longer chain
homologues, 3-methyl-2-tridecanone, 3-methyl-2-tetradecanone and
3-methyl-2-pentadecanone, were found in Dufour glands’ secretions
of a species of desert-dwelling ants of the Cataglyphis bicolor group (Gökçen
et al., 2002). Motivated by the interesting properties of these ketones and
their limited natural occurrence, the tentative structure assignment was
confirmed by comparing the chromatographic and spectral properties of
these, up to now unknown, I. helenium constituents to that of synthetic
3-methyl-2-alkanones. It was virtually impossible to isolate these
compounds from the complex oil fraction matrix and the synthesis gave
the opportunity to ascertain to what extent these compounds had con-
tributed to the previously observed strong anti-staphylococcal activity
of the fraction in question.
Commercially available methyl acetoacetate (2) was the starting mate-
rial in this synthetic effort. The route began with a base-catalysed alkyl-
ation of β-ketoester 2 with an appropriate halogen alkane to obtain
Table 1. GC/FID calibration range, calibration curve equation, correlation coefficient (R2) and quantitative results for each
3-methyl-2-alkanone
Designation
Compound name
Calibration
range (mg/mL)
Calibration curve equation
Correlation
Content
coefficient (R2) (mg/100 g of dry roots)
C = 1.3807 × 10À4A + 5.0699 × 10À3
C = 1.3690 × 10À4A + 5.0678 × 10À3
C = 1.3579 × 10À4A + 5.0626 × 10À3
C = 1.3542 × 10À4A + 5.0606 × 10À3
C = 1.3436 × 10À4A + 5.0595 × 10À3
C = 1.3379 × 10À4A + 5.0566 × 10À3
C = 1.3336 × 10À4A + 5.0544 × 10À3
C = 1.3379 × 10À4A + 5.0531 × 10À3
C = 1.3285 × 10À4A + 5.0526 × 10À3
0.9997
0.9997
0.9998
0.9999
0.9999
0.9997
0.9998
0.9999
0.9999
0.32
0.21
5.1
2.1
24.2
2.5
4.5
0.08
0.54
0.009–0.9
1a
1b
1c
1d
1e
1f
1 g
1 h
1i
3-methyldecan-2-one
3-methylundecan-2-one
3-methyldodecan-2-one
3-methyltridecan-2-one
3-methyltetradecan-2-one
3-methylpentadecan-2-one
3-methylhexadecan-2-one
3-methylheptadecan-2-one
3-methyloctadecan-2-one
Table 2. Anti-microbial activity of 3-methyl-2-alkanones
Fungal strain
Compounda MIC (mg/mL)
Nystatin MIC
(μg/mL)
1a
1b
1c
1d
1e
1f
1 g
1 h
1i
Candida albicans
3.70
3.70
3.70
3.70
3.70
3.70
3.70
3.70
3.70
0.78
MIC, minimum inhibitory concentrations.
a
In the tested concentrations, none of the compounds (1a–i) were active against Staphylococcus aureus, Bacillus subtilis, Bacillus
cereus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa and Aspergillus niger.
Phytochem. Anal. 2013
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/pca

N. S. RADULOVIĆ et al.
any anti-staphylococcal activity in the concentrations tested. Furthermore,
3-methyl-2-alkanones manifested selectivity toward just one fungal strain –
Candida albicans (MIC= 3.7 mg/mL, Table 2). This implies that the very high
activity against S. aureus of this oil fraction comes from the still unidentified
sesquiterpene aldehyde with a mass spectrum resembling that of
bicyclogermacrenal (Stojanović-Radić et al., 2012). The observed anti-candi-
dal activity of 3-methyl-2-alkanones (C. albicans is a yeast species that can
be found in the soil; Marples, 1966; Hube, 2004) suggests that these
compounds could be plant-root defence metabolites against attack of such
pathogens and a number of previous studies on related compounds con-
firm this (McDowell et al., 1988; Fridman et al., 2005; Ntalli et al., 2011).
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identify a key enzyme in the biosynthesis of methylketones. Plant Cell
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contents of ants of the Cataglyphis bicolor group. J Chem Ecol 28: 71–87.
Hube B. 2004. From commensal to pathogen: stage- and tissue-
specific gene expression of Candida albicans. Curr Opin Microbiol
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Marples MJ. 1966. Some observations on the ecology of Candida
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McDowell PG, Lwande W, Deans SG, Waterman P. 1988. Volatile resin
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Ntalli NG, Manconi F, Leonti M, Maxia A, Caboni P. 2011. Aliphatic
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Conclusion
In conclusion, the identification and synthesis of nine additional con-
stituents from a fraction of I. helenium root essential oil with a low MIC
value (0.8 μg/mL) against S. aureus were reported here. The existence of
a C11–C19 homologous series of 3-methyl-2-alkanones was corroborated
by synthesis in three steps with the overall yields of 50–65% starting from
methyl acetoacetate. The construction of GC/FID calibration curves
(R2 = 0.9997–0.9999) enabled the determination of the content of
3-methyl-2-alkanones in the plant material: 0.08–24.2 mg/100 g of dry
roots. The in vitro anti-microbial activity assay revealed that 3-methyl-2-
alkanones were not active against S. aureus at the tested concentrations,
but manifested a selectivity toward one fungal strain – C. albicans
(MIC = 3.7 mg/mL). Finally, these long-chain 3-methyl-2-alkanones have a
rather restricted occurrence in samples of natural origin and this is the
very first report of their occurrence in the Plant Kingdom.
Radulović N, Đorđević N, Denić M, Pinheiro MM, Fernandes PD, Boylan F.
2012a.
A novel toxic alkaloid from poison hemlock (Conium
SUPPORTING INFORMATION
maculatum L., Apiaceae): identification, synthesis and antinociceptive
activity. Food Chem Toxicol 50: 274–279.
Supporting information can be found in the online version of
this article.
Radulović N, Denić M, Stojanović-Radić Z, Skropeta D. 2012b. Fatty and
volatile oils of the gypsywort Lycopus europaeus L. and the
Gaussian-like distribution of its wax alkanes. J Am Oil Chem Soc
89: 2165–2185.
Acknowledgements
Raymer J, Wiesler D, Novotny M, Asa C, Seal US, Mech LD. 1986. Chemical
scent constituents in urine of wolf (Canis lupus) and their dependence
on reproductive hormones. J Chem Ecol 12: 297–314.
Setzer WN, Noletto JA, Lawton RO, Haber WA. 2005. Leaf essential oil
composition of five Zanthoxylum species from Monteverde, Costa
Rica. Mol Divers 9: 3–13.
Stojanović-Radić ZZ, Čomić LR, Radulović NS, Blagojević PD, Denić M,
Miltojević AB, Rajković J, Mihajilov-Krstev TM. 2012. Antistaphylococcal
activity of Inula helenium L. root essential oil: eudesmane sesqui-
terpene lactones induce cell membrane damage. Eur J Clin Microbiol
31: 1015–1025.
The authors acknowledge the Ministry of Education, Science and
Technological Development of Serbia for the financial support
(Project No. 172061). The authors are grateful to Professor
Svetlana Simova, from the Institute of Organic Chemistry with
Centre of Phytochemistry, Bulgarian Academy of Sciences, for
the NMR measurements that were performed as a part of the
young researchers’ project.
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Phytochem. Anal. 2013