Isolation, Chemical Transformation, and Antifungal Potential of Sesquiterpene Lactones from Inula Racemosa

Article abstract of DOI:10.1007/s10600-020-02989-1

The present study reports the isolation of two eudesmanolide type sesquiterpenoid lactones, viz. alantolactone and isoalantolactone, from the roots of Inula racemosa Hook L. and their chemical transformations using various reagents. All the compounds isol

Full text of DOI:10.1007/s10600-020-02989-1

DOI 10.1007/s10600-020-02989-1
Chemistry of Natural Compounds, Vol. 56, No. 2, March, 2020
ISOLATION, CHEMICAL TRANSFORMATION, AND ANTIFUNGAL
POTENTIAL OF SESQUITERPENE LACTONES
FROM Inula racemosa
Ramandeep Kaur,* K. K. Chahal, and Urvashi
The present study reports the isolation of two eudesmanolide type sesquiterpenoid lactones, viz. alantolactone
and isoalantolactone, from the roots of Inula racemosa Hook L. and their chemical transformations using
various reagents. All the compounds isolated and synthesized were assessed for their in vitro antifungal
potential against Dreschlera oryzae, Fusarium moniliforme, and Alternaria triticina using spore germination
inhibition method. To understand how the structure of sesquiterpene lactones relates to their antifungal
function, structure activity relationships were studied. Isoalantolactone and its derivatives were found to be
more toxic as compared to alantolactone and its derivatives. Among the different synthesized compounds,
the bromoform derivatives were the most, while the methoxy derivatives were the least effective. All the
compounds showed promising results against three test fungi with ED values less than 400 ppm.
50
Keywords: Inula racemosa, alantolactone, isoalantolactone, antifungal studies, sesquiterpene lactones.
Phytopathogenic fungi are the most damaging pests of cereal crops along with insects and are responsible for decrease
in yield as well as quality of harvested crops. Dreschlera oryzae, Fusarium moniliforme, and Alternaria triticina are among
the most damaging fungi causing severe damage to rice and wheat crops [1–3]. Despite the availability of synthetic fungicides,
there is increased interest in their natural alternatives, which are more environment friendly and cause the least effect on
non-target species [4, 5]. In agricultural research, various new compounds have been reported to possess antifungal potential,
which are natural products or natural product derived molecules [6].
Sesquiterpene lactones are an expeditiously developing group of natural products with a wide spectrum of biological
activities [7]. More than 5000 compounds are isolated from the different members ofAsteraceae (Compositae). Inula racemosa
Hook. f. (Pushkaramoola in Hindi), one of the members of the family Asteraceae (Compositae), is a plant of wide medicinal
importance and is distributed all over Africa, Europe, and East Asia. The roots of this plant consist mainly of two major
sesquterpene lactones, alantolactone and isoalantolactone, which possess numerous biological activities, viz. antibacterial,
antifungal, nematicidal, plant growth regulating, etc. [8–10].
To the best of our knowledge, although the antifungal activity of alantolactone and isoalantolactone and their derivatives
have been reported against many groups of fungi [11], no reports are available against the phytopathogenic fungi under study
(Dreschlera oryzae, Fusarium moniliforme, and Alternaria triticina). The present study also describes the structure–activity
relationship (SAR) of parent and synthesized compounds based on their antifungal activity against three test fungi.
Alantolactone (1) and isoalantolactone (2) were isolated from the roots of I. racemosa using an earlier described
method [11]. Different derivatives (1a–1f and 2a–2h) of isolated compounds (1 and 2) were prepared (Scheme 1) and tested
for their antifungal potential.
The antifungal activity of isolated as well as synthesized compounds was tested against three phytopathogenic
fungi using the spore germination inhibition method, and the ED was calculated (Table 1). Comparison of the
50
ED values revealed that isoalantolactone (2) possessed more toxic potential in comparison to alantolactone (1).
50
Department of Chemistry, Punjab Agricultural University, Ludhiana, 141004, Punjab, India, e-mail:
ramandeep.saini6@gmail.com. Published in Khimiya Prirodnykh Soedinenii, No. 2, March–April, 2020, pp. 186–190.
Original article submitted January 1, 2019.
0009-3130/20/5602-0207 ©2020 Springer Science+Business Media, LLC
207

TABLE 1. ED Values (ppm) of the Tested Compounds against Alternaria. triticina, Dreschlera oryzae, and Fusarium moniliforme
50
Compound
Compound
A. triticina
D. oryzae
F. moniliforme
A. triticina
D. oryzae
F. moniliforme
270
160
220
385
370
267
180
220
155
250
120
190
360
355
258
165
200
140
280
180
235
395
380
284
196
235
168
184
298
278
206
215
257
270
08
160
270
260
172
185
230
234
18
205
314
287
218
235
287
300
15
1
2b
1a
1b
1c
1d
1e
1f
2
2c
2d
2e
2f
2g
2h
Carbendazim
Propiconazole
12
15
8
2a
O
O
O
O
O
O
O
O
Br
N
OCH
3
Br
N
1b, 2b
1c, 2c
1a, 2a
1d, 2d
c
d
a
b
15
10
1
8
O
12
O
O
4
O
O
11
6
O
O
13
1 or
14
f
e
N
O
N
N
15
N
1
9
O
12
1e, 2e
1f, 2f
6
13
2
14
g
O
O
O
+
HO
HO
OH
2g
2h
a. CHBr , KOH; b. NaBH or Li(LiAl[OC(CH ) ] ; c. Mg/CH OH; d. CH N , (CH ) N;
3
4
3 3 3
3
2
2
3 3
e. CH CHN , (CH ) N; f. CH CH CHN , (CH ) N; g. SeO /TBHP
3
2
3 3
3
2
2
3 3
2
Scheme 1. Reaction scheme of alantolactone (1) and isoalantolactone (2) with different reagents
and their products (1a–1f and 2a–2h)
A similar trend was observed in their derivatives.Among the various synthesized derivatives, dibromocyclopropane derivatives
(1a and 2a) were the most toxic, whereas 13-methoxy-11,13-dihydro derivatives (1c and 2c) were the least toxic. Comparison
of different pyrazoline derivatives (1d–1f and 2d–2f) showed varying toxic effects, and, among these, diazopropane derivatives
(1f and 2f) were found to be most effective against all the fungi under study in comparison with diazomethane (1d and 2d) and
diazoethane derivatives (1e and 2e). The hydroxyl derivatives of isoalantolactone (2g and 2h) showed less toxic effect in
comparison with parent compound (2) against all test fungi. On comparison of the three fungi, the compounds showed the
most toxic effect against D. oryzae and the least effect against F. moniliforme. All the compounds showed less toxic effect as
compared to commercial standard fungicides, carbendazim (3) and propiconazole (4). However, both the isolated compounds
(1 and 2) as well as their derivatives (1a–1f and 2a–2h) showed ED values less than 400 ppm against all the three fungi
50
(Table 1).
Structure–activity relationship (SAR) may be defined as the correlation between the structure of the compound and
its biological activity. SAR relates to the particular functional groups responsible for evoking a response in the test organism
or the site in the test organism. It is well known fact that the change in structure of the compound results in either an increase
208

or a decrease in its biological activity. Alantolactone and isoalantolactone, the two isomeric sesquiterpene lactones isolated
from the roots of I. racemosa, were tested for their antifungal activity against three phytopathogenic fungi. Isoalantolactone
was found to be more toxic as compared to alantolactone against all the tested fungi.
Kataria and Chahal [11] also reported the higher fungitoxic potential of isoalantolactone than alantolactone against
three phytopathogenic fungi, Alternaria brassicae, Penicillium italicum, and Rhizoctonia solani. Earlier, the biological activities
of these sesquiterpene lactones were purely attributed to the presence of the α-methylene-γ-lactone moiety [12]. But the
difference in the activity revealed that the other structural features were also important. A comparative analysis of the structure
of these compounds showed that these compounds differ only in the position of the additional double bond. This information
implies that the exocyclic double bond may be responsible for the higher toxicity of the isoalantolactone. These findings
prompted us to explore the role of the conjugated double bond in determining any biological property. Therefore, we attempt
to modify the conjugated double bond in both the sesequiterpene lactones (1 and 2) using different reagents. The reduced
derivatives (1b and 2b) showed only a small increase in toxicity. The addition of the methoxy group and pyrazolines decreases
the activity of the synthesized derivatives (1c–1f and 2c–2f) as compared to the parent compounds (1 and 2), which revealed
that the conjugated double bond plays an important role in the inhibition of spore germination in the test fungi. Further
increase in antifungal activity of pyrazoline derivatives was also supported by previous study [11] which showed that extension
of the carbon chain in the lactone ring increased the inhibition potential of the compound. The increase in activity due to
cycloaddition of bromine (1a and 2a) might be due to the inhibitory effect of halogens. On comparing alantolactone (1) and
isoalantolactone (2) and their derivatives, (1a–1f and 2a–2f), the latter were found to be more toxic as compared to their
alantolactone analogues, which confirmed the importance of exocyclic double bond for the activity.
In general, polar groups like the hydroxyl group (OH) are responsible for the increased activity of the molecules.
Therefore, hydroxyl derivatives of isoalantolactone (2g and 2h) were also prepared.A comparison of ED values showed that
50
hydroxyl derivatives were less active against fungus as compared to parent compound 2, which revealed that addition of the
HO group decreased the antifungal potential instead of increasing it. Nidiry et al. [13] also reported that the presence of an
additional hydroxyl group in alcohol has an adverse effect on the antifungal activity of parent compounds.
EXPERIMENTAL
General. Melting points were determined in open capillaries on a Buchi B-545 melting point apparatus and are
1
13
uncorrected. FT-IR spectra were measured on a Perkin Elmer, Model RX-1 FT-IR spectrophotometer. H NMR and C NMR
spectra were recorded on a Bruker AC (400 MHz) spectrometer or mentioned otherwise as solutions (in CDCl ) using TMS as
3
an internal reference.
Extraction and Isolation of Pure Compounds from Roots of I. racemosa. The dried and powdered roots (250 g) of
Inula racemosa were subjected to Soxhlet extraction using chloroform (1.0 L) as the solvent. The chloroform extract was
distilled to yield a yellow semisolid crude extract (5.0 g). The concentrated extract was subjected to column chromatography
using silver nitrate impregnated silica gel. Alantolactone (1, mp 78°C) and isoalantolactone (2, mp 108°C) were isolated from
hexane (100%) and hexane–dichloromethane (5%) fractions, respectively.
–1
1
Compound 1. IR (KBr, ν, cm ): 1745, 1657, 1457, 1370, 1261, 890, 811. H NMR (400 MHz, CDCl , δ, ppm, J/Hz):
3
1.09 (3H, d, J = 7.6, H-14), 1.19 (3H, s, H-15), 3.57–3.61 (1H, m, H-7), 4.81–4.84 (1H, m, H-8), 5.16 (1H, d, J = 4.1, H-6),
13
6.19 (1H, d, J = 1.9, H-13), 5.63 (1H, d, J = 1.7, H-13). C NMR (100 MHz, CDCl , δ, ppm): 39.46 (C-1), 22.56 (C-2), 37.56
3
(C-3), 32.86 (C-4), 148.94 (C-5), 118.83 (C-6), 41.70 (C-7), 76.45 (C-8), 42.63 (C-9), 32.69 (C-10), 139.84 (C-11), 170.45
(C-12), 121.63 (C-13), 16.74 (C-14), 8.58 (C-15).
–1
1
Compound 2. IR (KBr, ν, cm ): 1755, 1641, 1447, 1262, 887, 818. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 0.82
3
(3H, s, H-15), 2.95–3.01 (1H, m, H-7), 4.44 (1H, d, J = 1.4, H-14), 4.77 (1H, d, J = 1.52, H-14), 4.48–4.51 (1H, m, H-8), 5.59,
13
6.13 (each 1H, d, J = 1.52, H-13). C NMR (100 MHz, CDCl , δ, ppm): 36.81 (C-1), 17.68 (C-2), 40.52 (C-3), 148.98 (C-4),
3
46.15 (C-5), 22.69 (C-6), 41.35 (C-7), 76.85 (C-8), 42.18 (C-9), 32.28 (C-10), 142.22 (C-11), 170.68 (C-12), 120.08 (C-13),
106.41 (C-14), 27.48 (C-15).
General Procedure for Reaction of Alantolactone or Isoalantolactone with CHBr . To CHBr (15 mL), aq. KOH
3
3
(10 mL, 50%) was added in the presence of phase transfer catalyst (TEBAC, 100 mg), and the mixture was homogenized for
2 min. Alantolactone or isoalantolactone (1 or 2, 1.0 g) was added to the same reaction mixture and stirred for 5 h. The
completion of reaction was monitored by TLC. The reaction mixture was diluted with water and extracted with ethyl acetate
209

(3 × 50 mL) and dried (Na SO ). Excess solvent was removed under vacuum. The solid pure compounds 1a (yield 0.91 g,
2
4
mp 58°C) or 2a (yield 0.92 g, mp 149°C) were obtained separately in each reaction.
–1
1
Compound 1a. IR (KBr, ν, cm ): 1767, 1458, 1356, 894, 688. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 1.05 (3H,
3
d, J = 7.6, H-14), 1.19 (3H, s, H-15), 1.92 and 2.18 (each 1H, d, J = 8, H-13), 3.16–3.18 (1H, m, H-7), 4.95–4.98 (1H, m, H-8),
13
4.91 (1H, d, J = 3.5, H-6). C NMR (100 MHz, CDCl , δ, ppm): 40.64 (C-1), 22.83 (C-2), 38.10 (C-3), 32.95 (C-4), 151.38
3
(C-5), 115.29 (C-6), 41.26 (C-7), 76.79 (C-8), 42.26 (C-9), 32.83 (C-10), 42.65 (C-11), 171.84 (C-12), 27.27 (C-13), 16.80
(C-14), 28.54 (C-15), 29.66 (C-16).
–1
1
Compound 2a. IR (KBr, ν, cm ): 1762, 1643, 890, 650. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 0.86 (3H, s,
3
H-15), 1.92–2.29 (each 1H, d, J = 7.9, H-13), 2.59–2.65 (1H, m, H-7), 4.80–4.82 (1H, m, H-8), 4.79 (1H, d, J = 1.28, H-14),
13
4.45 (1H, d, J = 1.32, H-14). C NMR (100 MHz, CDCl , δ, ppm): 41.23 (C-1), 22.64 (C-2), 36.70 (C-3), 148.85 (C-4), 46.43
3
(C-5), 23.74 (C-6), 42.77 (C-7), 76.73 (C-8), 42.09 (C-9), 25.67 (C-10), 42.68 (C-11), 172.11 (C-12), 34.64 (C-13), 17.85 (C-14),
106.69 (C-15), 28.97 (C-16).
General Procedure for Reaction of Alantolactone or Isoalantolactone with NaBH . Alantolactone or
4
isoalantolactone (1 or 2, 1.0 g) was taken in round bottomed flask and dissolved in methanol (20.0 mL). To this, sodium
borohydride (2.0 g) was added in batches. The reaction mixture was stirred for 25 min. The extent of completion of the
reaction was monitored by TLC. For workup, a few drops of acetic acid was added, and the reaction mixture was extracted
with water and then with dichloromethane (3 × 50 mL). The organic layer was dried (Na SO ), and excess of solvent was
2
4
distilled off to get pure crystals of compound 1b (yield 0.92 g, mp 172°C) or 2b (yield 0.93 g, mp 288°C) separately in each
reaction.
–1
1
Compound 1b. IR (KBr, ν, cm ): 1728, 1599, 1456, 112, 889. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 1.24 (3H,
3
d, J = 7.1, H-13), 1.26 (3H, s, H-15), 1.14 (3H, d, J = 7.6, H-14), 3.02–3.07 (1H, m, H-7), 4.74–4.77 (1H, m, H-8), 5.18 (1H,
13
d, J = 3.2, H-6). C NMR (100 MHz, CDCl , δ, ppm): 38.76 (C-1), 23.07 (C-2), 38.52 (C-3), 33.05 (C-4), 150.61 (C-5),
3
115.56 (C-6), 42.92 (C-7), 76.98 (C-8), 40.38 (C-9), 32.91 (C-10), 42.25 (C-11), 179.33 (C-12), 10.73 (C-13), 16.88 (C-14),
28.75 (C-15).
–1
1
Compound 2b. IR (KBr, ν, cm ): 2934, 1756, 1640, 1168, 884. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 0.83
3
(3H, s, H-15), 1.22 (3H, d, J = 7.16, H-13), 2.79–2.82 (1H, m, H-11), 2.95–3.01 (1H, m, H-7), 4.44–4.51 (1H, m, H-8), 4.77
13
(2H, br.s, H-14). C NMR (100 MHz, CDCl , δ, ppm): 40.50 (C-1), 22.69 (C-2), 36.80 (C-3), 148.96 (C-4), 46.50 (C-5),
3
34.82 (C-6), 42.24 (C-7), 76.84 (C-8), 41.75 (C-9), 27.47 (C-10), 41.57 (C-11), 179.46 (C-12), 9.29 (C-13), 106.59 (C-14),
17.79 (C-15).
General Procedure for Reaction of Alantolactone or Isoalantolactone with LiAl[OC(CH ) ] . Alantolactone or
3 3 3
isoalantolactone (1 or 2, 2.0 g) was dissolved in dry THF (20.0 mL) and to this was added lithium tri-tert-butoxyaluminohydride
(1.0 g). The reaction mixture was stirred for 15 min and after completion of reaction, THF was removed under vacuum.
For workup, a few drops of glacial acetic acid was added, and the reaction mixture was extracted with water and then with
diethyl ether (3 × 50 mL). Evaporation of solvent afforded a pure compound, 1b (yield 1.91 g, mp 172°C) or 2b (yield 1.93 g,
mp 288°C) for each reaction.
General Procedure for Reaction of Alantolactone or Isoalantolactone with Mg/Methanol. To dry methanol
(50.0 mL), alantolactone or isoalantolactone (1 or 2, 0.5 g) and active Mg turnings (0.5 g) were added with constant stirring
under dry conditions. A mild exothermic reaction was observed after 3 h with the evolution of H gas. The progress of the
2
reaction was monitored by TLC. Unreacted metal was then separated, and the mixture was added to dilute HCl (150 mL) to get
a clear solution; pH was adjusted to around 8.0–9.0 by adding ammonia solution. The reaction mixture was then concentrated,
diluted with water, and extracted with dichloromethane (4 × 20 mL). The organic layer was dried (Na SO ) to afford a
2
4
mixture. In the case of alantolactone, the mixture (0.441 g) was chromatographed over silica gel to get pure compound 1c
(yield 0.32 g, mp 86°C), whereas in the case of isoalantolactone the mixture (0.436 g) obtained on chromatographic isolation
yielded the compound 2c (yield 0.34 g, mp 91°C).
–1
1
Compound 1c. IR (KBr, ν, cm ): 1750, 1660, 1450, 1370, 1262, 1138, 890, 810. H NMR (400 MHz, CDCl ,
3
δ, ppm, J/Hz): 1.04 (3H, d, J = 3.8, H-14), 1.15 (3H, s, H-15), 2.91–2.95 (1H, m, H-7), 3.29 (3H, s, OCH ), 4.79–4.82 (1H, m,
3
13
H-8), 5.10 (1H, d, J = 3.6, H-6). C NMR (100 MHz, CDCl , δ, ppm): 38.75 (C-1), 22.72 (C-2), 37.89 (C-3), 32.90 (C-4),
3
148.90 (C-5), 119.96 (C-6), 42.85 (C-7), 76.74 (C-8), 42.00 (C-9), 32.72 (C-10), 50.43 (C-11), 177.94 (C-12), 72.43 (C-13),
16.84 (C-14), 28.86 (C-15), 59.21 (C-16).
–1
1
Compound 2c. IR (KBr, ν, cm ): 1770, 1660, 1470, 1380, 1140, 1110, 895, 730. H NMR (400 MHz, CDCl , δ,
3
ppm, J/Hz): 0.80 (3H, s, H-15), 2.93–2.95 (1H, m, H-7), 4.42 (1H, d, J = 1.4, H-14), 4.73 (1H, d, J = 1.52, H-14), 4.43–4.45
210

13
(1H, m, H-8), 3.27 (3H, s, OCH ). C NMR (100 MHz, CDCl , δ, ppm): 37.21 (C-1), 19.65 (C-2), 40.52 (C-3), 149.24 (C-4),
3
3
47.45 (C-5), 23.62 (C-6), 40.25 (C-7), 76.25 (C-8), 41.26 (C-9), 33.75 (C-10), 44.78 (C-11), 170.59 (C-12), 75.30 (C-13),
106.41 (C-14), 27.48 (C-15), 59.30 (C-16).
General Procedure for Reaction of Alantolactone or Isoalantolactone with Diazomethane, Diazoethane, or
Diazopropane. To a solution of lactone (1 or 2, 2.0 g each) in ether containing 2–3 drops of triethylamine was added an excess
ethereal solution of diazomethane, diazoethane, or diazopropane separately. It was kept overnight.After completion of reaction,
the solvent was evaporated to afford crystalline compounds in each case, which were identified as pyrazoline 1d (yield 1.81 g,
mp 98°C), 1e (yield 1.82 g, mp 70°C), or 1f (yield 1.88 g, mp 55°C), or 2d (yield 1.65 g, mp 118°C), 2e (yield 1.72 g,
mp 104°C), or 2f (yield 1.80 g, mp 73°C), respectively, for each reaction.
–1
1
Compound 1d. IR (KBr, ν, cm ): 2924, 1767, 1646, 1554. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 1.13 (3H, d,
3
13
J = 7.6, H-14), 1.28 (3H, s, H-15), 1.54–1.65 (4H, m, H-13, 16), 4.78–4.86 (1H, m, H-8), 5.04 (1H, d, J = 3.4, H-6). C NMR
(100 MHz, CDCl , δ, ppm): 42.19 (C-1), 21.28 (C-2), 38.58 (C-3), 33.01 (C-4), 152.81 (C-5), 113.39 (C-6), 42.07 (C-7),
3
101.55 (C-8), 42.66 (C-9), 28.56 (C-10), 78.38 (C-11), 172.94 (C-12), 32.80 (C-13), 16.80 (C-14), 22.82 (C-15), 78.12 (C-16).
–1
1
Compound 1e. IR (KBr, ν, cm ): 2929, 1763, 1670, 1554, 1170, 888. H NMR (400 MHz, CDCl , δ, ppm, J/Hz):
3
13
1.23 (3H, d, J = 7.8, H-14), 1.26 (3H, s, H-15), 1.57 (3H, d, J = 7.3, H-17), 4.73–4.76 (1H, m, H-8). C NMR (100 MHz,
CDCl , δ, ppm): 38.53 (C-1), 22.74 (C-2), 37.55 (C-3), 33.04 (C-4), 145.48 (C-5), 118.81 (C-6), 37.87 (C-7), 86.59 (C-8),
3
40.79 (C-9), 32.76 (C-10), 76.46 (C-11), 172.75 (C-12), 42.02 (C-13), 18.74 (C-14), 24.68 (C-15), 43.12 (C-16), 16.83 (C-17).
–1
1
Compound 1f. IR (KBr, ν, cm ): 2965, 1745, 1656, 1555, 1169, 870. H NMR (400 MHz, CDCl , δ, ppm, J/Hz):
3
0.96 (3H, t, J = 7.2, H-18), 1.22 (3H, d, J = 7.7, H-14), 1.27 (3H, s, H-15), 1.33–1.37 (2H, m, H-17), 1.52–1.84 (2H, m, H-13),
13
4.74–4.76 (1H, m, H-8), 5.37 (1H, d, J = 3.7, H-6). C NMR (100 MHz, CDCl , δ, ppm): 38.5 (C-1), 20.10 (C-2), 36.42 (C-3),
3
35.70 (C-4), 144.20 (C-5), 122.80 (C-6), 36.97 (C-7), 81.60 (C-8), 41.21 (C-9), 30.6 (C-10), 76.90 (C-11), 172.33 (C-12),
46.00 (C-13), 19.92 (C-14), 25.4 (C-15), 59.77 (C-16) 28.13 (C-17), 9.00 (C-18).
–1
1
Compound 2d. IR (KBr, ν, cm ): 3084, 1761, 1643, 1549, 1212, 891. H NMR (400 MHz, CDCl , δ, ppm, J/Hz):
3
0.86 (3H, s, H-15), 1.23–1.33 (2H, m, H-16), 2.31–2.37 (2H, m, H-13), 4.45 (1H, d, J = 1.20, H-14), 4.79 (1H, d, J = 1.35,
13
H-14), 5.52–5.54 (1H, m, H-8). C NMR (100 MHz, CDCl , δ, ppm): 42.02 (C-1), 20.42 (C-2), 41.30 (C-3), 149.02 (C-4),
3
46.29 (C-5), 21.86 (C-6), 34.58 (C-7), 78.03 (C-8), 43.33 (C-9), 22.64 (C-10), 106.55 (C-11), 173.11 (C-12), 36.72 (C-13),
17.80 (C-14), 103.15 (C-15), 78.75 (C-16).
–1
1
Compound 2e. IR (KBr, ν, cm ): 3078, 1763, 1676, 1549, 1216, 891. H NMR (400 MHz, CDCl , δ, ppm, J/Hz):
3
13
1.10 (3H, s, H-15), 1.50 (3H, d, J = 7.2, H-17), 3.94–3.96 (1H, m, H-16), 4.50–4.52 (1H, m, H-8), 4.75 (2H, br.s, H-14). C NMR
(100 MHz, CDCl , δ, ppm): 41.54 (C-1), 24.83 (C-2), 40.51 (C-3), 149.40 (C-4), 46.40 (C-5), 26.22 (C-6), 36.83 (C-7), 76.64
3
(C-8), 42.01 (C-9), 34.34 (C-10), 106.62 (C-11), 173.46 (C-12), 43.57 (C-13), 120.11 (C-14), 22.69 (C-15), 46.24 (C-16),
19.03 (C-17).
–1
1
Compound 2f. IR (KBr, ν, cm ): 3082, 2964, 1769, 1707, 1527, 1216, 893. H NMR (400 MHz, CDCl , δ, ppm,
3
J/Hz): 1.65–1.89 (3H, m, H-13, 16), 1.10 (3H, t, J = 5.6, H-18), 1.24 (3H, s, H-15), 4.47, 4.79 (each 1H, br.s, H-14), 4.84–4.88
13
(1H, m, H-8), 5.16 (2H, d, J = 3.8, H-6), 6.66 (dq, J = 1.96, 7.7, H-17). C NMR (100 MHz, CDCl , δ, ppm): 38.58 (C-1),
3
22.69 (C-2), 37.79 (C-3), 149.07 (C-4), 56.4 (C-5), 24.16 (C-6), 33.76 (C-7), 77.37 (C-8), 42.73 (C-9), 29.69 (C-10), 106.58
(C-11), 171.21 (C-12), 45.69 (C-13), 109.43 (C-14), 21.65 (C-15), 58.95 (C-16), 23.10 (C-17), 10.39 (C-18).
General Procedure for Allylic Oxidation of Isoalantolactone Using SeO /TBHP. Selenium dioxide (10.0 mg) was
2
added to tert-butyl hydroperoxide (3.0 mL, 70%), and the mixture was stirred for 30 min to which a solution of isoalantolactone
(2, 2.0 g) in dichloromethane (25.0 mL) was added. The reaction mixture was stirred for 4 h followed by dilution with cold
water and extraction with dichloromethane (2 × 25 mL). The combined organic extracts were washed with water and dried
(Na SO ). Evaporation of the solvent afforded a mixture (1.9 g) of two compounds, which were chromatographed over silica
2
4
gel to give pure compounds 2g (yield 0.6 g, mp 144°C) and 2h (yield 0.2 g, mp 161°C).
–1
1
Compound 2g. IR (KBr, ν, cm ): 3454, 2935, 1747, 1662, 1263, 1162, 1084, 952, 906, 820. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 0.82 (3H, s, H-15), 2.98–3.04 (1H, m, H-7), 4.58 (1H, t, J = 7.52, H-3), 4.50–4.53 (1H, m, H-8), 5.61
3
13
and 6.13 (each 1H, br.s, H-14), 4.33 and 4.99 (each 1H, br.s, H-13). C NMR (100 MHz, CDCl , δ, ppm): 37.00 (C-1), 35.68
3
(C-2), 73.26 (C-3), 150.13 (C-4), 40.98 (C-5), 29.04 (C-6), 40.21 (C-7), 76.91 (C-8), 40.43 (C-9), 27.05 (C-10), 142.09 (C-11),
170.74 (C-12), 120.31 (C-13), 109.83 (C-14), 16.94 (C-15).
–1
1
Compound 2h. IR (KBr, ν, cm ): 3436, 2930, 1729, 1659, 1420, 1264, 1162, 822. H NMR (400 MHz, CDCl , δ,
3
ppm, J/Hz): 0.94 (3H, s, H-15), 3.32–3.38 (1H, m, H-7), 4.53–4.58 (1H, m, H-8), 4.43 (1H, br.s, H-3), 4.89 and 5.10 (each 1H,
13
br.s, H-14), 5.65 (1H, d, J = 2.28, H-13), 6.13 (1H, d, J = 2.76, H-13). C NMR (100 MHz, CDCl , δ, ppm): 29.06 (C-1),
3
211

30.28 (C-2), 74.90 (C-3), 147.90 (C-4), 75.34 (C-5), 32.96 (C-6), 36.86 (C-7), 77.11 (C-8), 36.97 (C-9), 35.45 (C-10), 141.99
(C-11), 171.00 (C-12), 120.64 (C-13), 112.69 (C-14), 21.65 (C-15).
In vitro Screening of Compounds for Fungitoxicity.Antifungal activity was tested by the spore germination inhibition
technique. Spore suspension was prepared from a ten-day-old culture of the test fungi (Alternaria triticina, Fusarium moniliforme,
and Drechslera oryzae) with sterilized water. The experiment was replicated three times for each concentration as well as
control. A negative control containing only water was also maintained. These slides were placed in Petri plates lined with
moist filter paper and were incubated at 24 1°C. The number of spores germinated was counted, and the percent spore
germination inhibition was calculated by the formula
% Spore germination inhibition = (Spore germination in control – Spore germination in treatment/
Spore germination in control) × 100.
The fungicides carbendazim (Bavistin 50WP) and propiconazole (Tilt 25 EC) were used as standards, and activity
was expressed in terms of ED values.
50
In general, we can establish that alantolactone and isoalantolactone may act as good starting molecules to prepare
natural product based fungicides. Hence it can be concluded that the sesquiterpene lactones present in I. racemosa root extract
possess significant antifungal activity, and with a small modification in the structures we can enhance the activity of these
natural derived biomolecules to get a better natural pesticide molecule.
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