Semisynthetic derivatives of ent-kauranes and their antifeedant activity

Article abstract of DOI:10.1016/S0031-9422(01)00252-7

Chemical modification of functional groups on linearol yielded several ent-kaurane derivatives. These compounds were tested for their antifeedant activity against larvae of Spodoptera littoralis. Although linearol did not influence the feeding behaviour o

Full text of DOI:10.1016/S0031-9422(01)00252-7

Phytochemistry 58 (2001) 463–474
www.elsevier.com/locate/phytochem
Semisynthetic derivatives of ent-kauranes and their
antifeedant activity
Maurizio Brunoa,*, Sergio Rossellia, Ivana Pibiria, Franco Piozzia,b,
Maria Luisa Bondıb, Monique S.J. Simmondsc
aDepartment of Organic Chemistry, Palermo University, Viale delle Scienze, Parco d’Orleans II, 90128 Palermo, Italy
bI.C.T.P.N.-C.N.R., via U. La Malfa 153, 90146 Palermo, Italy
cJodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK
Received 28 November 2000; received in revised form 9 May 2001
Abstract
Chemical modification of functional groups on linearol yielded several ent-kaurane derivatives. These compounds were tested for
their antifeedant activity against larvae of Spodoptera littoralis. Although linearol did not influence the feeding behaviour of larvae,
some of its derivatives showed significant antifeedant activity. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Lamiaceae; ent-Kaurane; Diterpenoids; Linearol; Chemical modifications; Spodoptera littoralis; Antifeedant
1. Introduction
skeleton to evaluate their importance in the antifeedant
activity of these compounds.
ent-Kauranes are naturally occurring diterpenoids
isolated from several families, such as Asteraceae and
Lamiaceae. These compounds have attracted interest
because of their structures and their biological activities
as anti-tumorals, anti-HIV and anti-bacterials (Hanson,
1999).
The genus Sideritis (family Lamiaceae) is a rich source
(Gonzales et al., 1990) of ent-kauranes. In a previous
paper we described the isolation and identification (Bondı
et al., 2000) of many ent-kauranes from several Turkish
species of Sideritis. In the same paper, the antifeedant
activity against larvae of Spodoptera frugiperda and S.
littoralis was described. One of the diterpenes, sideroxol
(1), isolated from Sideritis akmanii and S. rubriflora had
potent antifeedant activity against S. frugiperda.
We were able to isolate large amounts of another ent-
kaurane, linearol (2), from species of Sideritis (Bondı et
al., 2000). This provided us with enough starting mate-
rial to plan a structure–activity study in which we would
modify the different functional groups on the ent-kaurane
2. Results and discussion
Linearol (2), possessing an exocyclic double bond
between carbons 16 and 17, was subjected to catalytic
hydrogenation on 10% Pd/C. The reduction that dia-
stereoselectively happened from the sterically less con-
gested side (Piozzi et al., 1971), yielded a mixture of two
compounds. After separation, the less polar compound
was identified as 16-a-H dihydrolinearol (3); the second
one (4), surprisingly was the transacetylated product of
the former, in which the acetyl group migrated from
position 18 to position 3.
Since the antifeedant sideroxol (1) had an epoxy group
we performed an epoxidation on the double bond of line-
arol. Treatment with 3-chloroperbenzoic acid (MCPBA)
in CH2Cl2 allowed us to generate, diastereoselectively, the
epoxy ring (Piozzi et al., 1973; Gonzales et al., 1990). Also
in this case, as described before, we obtained two com-
pounds: epoxylinearol (5) and the 18!3 transacetylated
product (6). Both 5 and 6 were subjected to acetylation
to give the same triacetylderivative (7).
* Corresponding author. Tel.: +39-091-596905; fax: +39-091-
596825.
E-mail address: bruno@dicpm.unipa.it (M. Bruno).
Next, in order to remove the exocyclic double bond
we performed an ozonolysis. Treatment of 2 with ozone
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00252-7

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M. Bruno et al. / Phytochemistry 58 (2001) 463–474
in CH2Cl2 resulted in a mixture of two products. One of
them was the expected ketone (8), the other one showed
in H NMR spectrum a signal at ꢀ 5.01 (br s, 2H) for
(Rodrıguez et al., 1999). Therefore, to evaluate the effect
of different esters on the activity of ent-kauranes, ester-
ification reactions were undertaken on the two free
hydroxyl groups of linearol.
1
acetalic protons and in 13C NMR spectrum two signals at
ppm 116.0 (s) and 93.2 (t) for two carbons bearing two
oxygens. Therefore this compound was assigned the
structure of ozonide (9). Furthermore, 9 was transformed
to ketone (8) by reduction with Zn/AcOH. Isolation of
ozonides is not common, due to their instability; in fact,
we could not store 9 as after a few hours it started to
decompose.
Treatment of linearol with propanoyl chloride, trie-
thylamine (TEA), 4-dimethylaminopyridine (DMAP) in
CH2Cl2 gave 3,7-dipropanoate ester (10) and a mono
1
esterified derivative. The H and 13C NMR spectra of
the latter compound showed signals at ꢀ 3.60 (t, J=3.8
Hz, H-7) and at ꢀ 4.86 (dd, J=11.2 and 5.2 Hz, H-3)
and ppm 76.7 (d, C-7) and 74.2 (d, C-3), clearly indi-
cating that only 3-OH was esterified; therefore, we
assigned to it structure (11). Similarly, we prepared
The composition of ester moieties on neoclerodane
diterpenoids influences their antifeedant activity

M. Bruno et al. / Phytochemistry 58 (2001) 463–474
465
3-mono- and 3,7-diester derivatives (12), (13), (14), (15),
(16) and (17) using different acyl chlorides such as
butanoyl, benzoyl and 4-methoxybenzoyl chlorides.
The same reactions performed with 2-furoyl chloride,
3-nitrobenzoyl chloride, 4-cyanobenzoyl chloride and
4-nitrobenzoyl chloride allowed us to isolate compounds
(18), (19), (20) and (21) in which both hydroxyl groups
were esterified. Monoacyl derivatives were not obtained.
Acylation of linearol with tigloyl chloride was not an
easy transformation. In fact, treatment of 2 with tiglic
acid chloride, TEA, DMAP in CH2Cl2 gave an inse-
1
parable mixture of two compounds. The H and 13C
NMR spectra showed that esterification was achieved
only in position 3 (ꢀH 4.86 dd, H-3; ꢀC 74.3 d/74.4 d C-3)
0
0
and that the ester moiety was the ꢀ3 -4 isomer of tiglate
(ꢀC 137.2 d/137.1 d, C-30; 115.9 t/115.7 t, C-40). There-

466
M. Bruno et al. / Phytochemistry 58 (2001) 463–474
fore, compound (22) was an epimeric mixture at C-20 due
to the formation of a stereogenic center, a consequence of
isomerization. Isomerization of a tigloyl moiety has been
already observed by other authors (Hatakeyama et al.,
1985).
the substitutions on the molecule resulted in a range of
analogue esters that differ in their activity when tested
at 100 ppm. We selected this dose to allow us to better
discriminate among the effects of different substances
although, in our experience, many diterpenes show
activity at 1000 ppm.
A different behaviour was observed when linearol and
tigloyl chloride were refluxed in toluene for 30 min. In
this case an inseparable mixture of di-tiglates (23) and
(24) was obtained, in which the double bond between
carbons 16 and 17 was hydrated to give a tertiary
hydroxyl group in C-16 (ꢀH 1.35, CH3-17; ꢀC 78.6 s/78.7 s,
C-16, 24.3 q, C-17). Compound (23) was an isomer of 24
in which the acetyl group migrated to position 3 and the
tiglic ester was at position 18. Finally, by refluxing line-
arol, TEA and tigloyl chloride in toluene for 24 h two
compounds were isolated. The more polar one was iden-
tified as 3,7-ditigloyl-linearol (25); the other one (26) was
the transesterification isomer of the former with the
tigloyl moiety in position 18 and the acetyl in position 3.
Compounds with a ketone or epoxide group had sig-
nificant antifeedant activity except for 7. Modification
to the substitutions at R (C-3), R1 (C-7) or R2 (C-18)
altered the activity of the compounds but there were no
clear trends in structure–activity relationship. For
example, increasing the length of the substitution at R
(11 vs. 22), when the substitutes at R1 and R2 did not
change, resulted in an increase in antifeedant activity.
However, the activity of compounds varied when simi-
lar substitutes were added to both R and R1, while R2
was an acetyl group. Of the compounds tested, 26 was
the most active; this was the only compound in which
the substitution at R2 was neither an hydroxy nor an
acetyl group. This suggests that the composition and
position of the functional groups at C-3, C-7 and C-18
modulate the activity of the compound. This is sup-
ported by previous research into the antifeedant activity
of neo-clerodanes from Teucrium (Simmonds et al.,
1989; Simmonds and Blaney, 1992), Scutellaria (Cole et
al., 1990) or Salvia (Simmonds et al., 1996). Whilst there
appears to be no clear structure–activity relationship the
3. Biological activity
The effects of compounds (2–8, 10, 11, 13–22, 25–26)
on the feeding behaviour of larvae of S. littoralis are
reported in Table 2. Linearol did not influence the feed-
ing behaviour of S. littoralis. However, modification to
Table 1a
13C NMR spectral data of compoundsa
C
2
3
4
5
6
7
8
9
b
1
38.2, t
26.0, t
71.5, d
41.2, s
37.9, d
26.9, t
76.3, d
47.6, s
49.9, d
38.4, s
17.5, t
33.2, t
43.4, d
38.0, t
44.7, t
154.9, s
103.0, t
65.8, t
11.9, q
17.6, q
172.2, s
20.5, q
38.4, t
26.4, t
72.3, d
41.8, s
38.2, d
27.2, t
77.1, d
48.5, s
51.4, d
38.7, s
18.3, t
26.3, t
39.5, d
39.8, t
44.4, t
34.4, d
15.6, q
66.1, t
11.8, q
18.1, q
171.8, s
21.1, q
38.4, t
23.4, t
74.5, d
41.6, s
37.3, d
26.5, t
77.0, d
48.5, s
51.5, d
38.4, s
18.3, t
26.3, t
39.5, d
39.8, t
44.3, t
34.4, d
15.6, q
64.2, t
12.7, q
18.1, q
171.7, s
21.2, q
38.3, t*
26.4, t
72.1, d
41.8, s
38.1, d
27.4, t
76.6, d
49.0, s
50.1, d
38.8, s
18.9, t
29.3, t
42.4, d
37.2, t*
45.0, t
66.2, s
50.3, t
65.9, t
11.9, q
18.2, q
171.8, s
21.1, q
38.0, t*
23.3, t
74.4, d
41.6, s
37.2, d
26.6, t
76.5, d
49.1, s
49.7, d
38.5, s
18.9, t
29.4, t
42.5, d
37.1, t*
44.9, t
66.3, s
50.3, t
63.8, t
12.9, q
18.2, q
171.8, s
21.2, q
37.5, t*
22.7, t
73.5, d
40.1, s
39.4, d
23.9, t
78.9, d
47.5, s
49.9, d
38.4, s
18.6, t
28.9, t
42.1, d
36.8, t*
44.7, t
65.5, s
50.5, t
64.6, t
12.7, q
18.0, q
170.4, s
20.9, q
170.3, s
20.9, q
169.8, s
20.7, q
37.9, t
26.2, t*
71.7, d
41.5, s
37.7, d
26.4, t*
75.8, d
46.2, s
49.0, d
38.7, s
17.9, t
29.6, t
47.3, d
35.5, t
51.3, t
222.2, s
37.8, t
26.1, t*
71.8, d
41.4, s
37.8, d
26.7, t*
76.1, d
46.6, s
50.0, d
38.5, s
17.3, t
25.6, t
43.3, d
35.9, t
46.5, t
116.0, s
93.2, t
65.8, t
11.7, q
17.8, q
171.8, s
21.0, q
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
OAc
65.7, t
11.8, q
18.2, q
171.8, s
21.0, q
a
CDCl3 solution.
*, These assignments may be reversed.
b

M. Bruno et al. / Phytochemistry 58 (2001) 463–474
467
data from neo-clerodanes suggests that the composition
of the substitution at C-4 (C-18) is important. Many of
the neo-clerodanes with potent antifeedant activity have
an epoxide substitution at C-4 but, to date, no ent-
kauranes with an epoxide at this position have been
tested for antifeedant activity against S. littoralis. How-
ever, sideroxol, one of the most potent ent-kaurane
diterpenoids tested for antifeedant activity in the binary
choice assay used in this study, has an epoxide at C-15–C-
16. Although this compound showed potent antifeedant
activity, this was against larvae of Spodoptera frugiperda
(Feeding Index=54 (4.3), P<0.05) not S. littoralis
(Feeding Index=9 (15.9), (Bondı et al., 2000). In the pre-
sent study no compounds had the C-15–C16 epoxide pre-
sent in sideroxol, but the two compounds with an epoxide
at C-16 (5 and 6) had antifeedant feeding activity
against S. littoralis (Table 2). However, the antifeedant
activity of the ent-kauranes cannot be totally deter-
mined by the presence of an epoxide, as other active
antifeedant compounds (8, 14, 18, 20, 21 and 26) lacked
this group. Whether the presence of a C-15–C-16 epox-
ide or addition of an epoxide at C-4 would enhance the
activity of the ent-kaurenes justifies investigation.
A comparison of the activity of compounds 25 with
26, the most potent antifeedant in the present study,
illustrates how altering the position of substitutions on
the molecule can alter its potency. For example,
exchange of the acetyl group at C-18 (25) with the
tigloyl group at C-3 (26) resulted in a significant
increase in antifeedant activity. This would suggest that
the position of these groups influence the ability of the
molecule to stimulate receptors on the taste neurones of
S. littoralis. The position of these groups alters the
polarity of the molecule, as compound 26 is less polar
Table 1b
13C NMR spectral data of compoundsa
C
10
11
12
13
14
15
16
17
18
b
1
38.1, t*
23.0, t
73.5, d
40.4, s
39.8, d
24.3, t
78.9, d
46.8, s
50.9, d
38.6, s
17.7, t
33.2, t
43.5, d
37.7, t*
44.9, t
154.0, s
103.8, t
65.0, t
12.9, q
18.0, q
170.5, s
20.8, q
38.4, t*
23.0, t
74.2, d
40.5, s
38.0, d
27.0, t
76.7, d
47.9, s
50.0, d
38.6, s
17.8, t
33.5, t
43.5, d
37.7, t*
44.9, t
154.8, s
103.6, t
64.6, t
13.0, q
17.8, q
171.5, s
21.1, q
38.4, t*
23.0, t
74.1, d
40.5, s
38.0, d
27.0, t
76.6, d
47.9, s
50.0, d
38.6, s
17.8, t
33.5, t
43.6, d
37.7, t*
45.0, t
154.8, s
103.6, t
64.7, t
13.0, q
17.8, q
171.4, s
21.1, q
38.1, t*
23.1, t
73.5, d
40.4, s
39.9, d
24.4, t
78.8, d
46.8, s
51.0, d
38.7, s
17.8, t
33.3, t
43.5, d
37.7, t*
45.0, t
154.1, s
103.9, t
65.2, t
12.9, q
18.0, q
170.5, s
20.9, q
38.3, t*
23.0, t
75.1, d
40.7, s
38.1, d
27.0, t
76.5, d
47.9, s
49.9, d
38.6, s
17.8, t
33.4, t
43.5, d
37.7, t*
44.9, t
154.7, s
103.5, t
65.0, t
13.2, q
17.8, q
171.4, s
21.0, q
165.7, s
37.8, t*
22.9, t
38.3, t*
23.1, t
74.7, d
40.7, s
38.1, d
27.0, t
76.5, d
47.9, s
50.0, d
38.6, s
17.8, t
33.4, t
43.5, d
37.7, t*
44.9, t
154.8, s
103.5, t
65.0, t
13.2, q
17.8, q
171.5, s
21.0, q
165.5, s
38.0, t
23.1, t
37.7, t*
23.0, t
74.3, d
40.2, s
39.2, d
23.9, t
79.9, d
46.7, s
50.8, d
38.4, s
17.7, t
33.0, t
43.2, d
37.6, t*
45.0, t
153.6, s
103.8, t
64.5, t
12.9, q
17.7, q
170.4, s
19.5, q
157.9, s
157.6, s
2
3
74.2, d
40.3, s
74.0, d
40.3, s
4
5
39.4, d
23.9, t
39.6, d
24.1, t
6
7
79.7, d
47.0, s
79.4, d
47.1, s
8
9
51.3, d
38.5, s
17.8, t
51.4, d
38.6, s
17.9, t
10
11
12
13
14
15
16
17
18
19
20
OAc
33.1, t
33.2, t
43.3, d
37.7, t*
45.1, t
43.4, d
38.0, t
45.3, t
153.6, s
103.9, t
64.6, t
153.9, s
103.9, t
64.8, t
13.1, q
17.8, q
170.5, s
19.2, q
165.6, s
165.1, s
130.6, s
129.4, d
128.3, d
132.8, d
128.3, d
129.4, d
130.3, s
129.3, d
128.1, d
132.7, d
128.1, d
129.3, d
13.3, q
17.9, q
170.7, s
19.6, q
165.5, s
165.1, s
123.1, s
131.5, d
113.7, d
163.3, s
113.7, d
131.5, d
122.8, s
131.4, d
113.5, d
163.2, s
113.5, d
131.4, d
55.4, q
55.3, q
C=O0
C=O00
10
173.8, s
27.9, t
9.2, q
173.8, s
27.9, t
9.2, q
173.0, s
36.6, t
18.5, t
13.6, q
173.1, s
36.7, t
18.5, t
13.8, q
130.5, s
129.4, d
128.2, d
132.8, d
128.2, d
129.4, d
123.0, s
131.4, d
113.5, d
163.2, s
113.5, d
131.4, d
20
144.7, s
117.5, d
111.7, d
146.1, d
30
40
500
600
100
200
3
173.5, s
27.9, t
9.1, q
172.8, s
36.5, t
18.5, t
13.7, q
144.5, s
117.5, d
111.5, d
145.9, d
400
500
600
0
00
OCH3
OCH3
55.3, q
a
CDCl3 solution.
*, These assignments may be reversed.
b

468
M. Bruno et al. / Phytochemistry 58 (2001) 463–474
than 25. Thus, whether changes in the polarity of the
compound rather than the position or composition of
specific functional groups on the molecule influence
activity is not known.
discs. The discs are dried before being presented to the
insect. Although this type of bioassay is only suitable for
a range of lepidopteran larvae and locusts it does enable
us to provide quantitative comparative data about the
potency of compounds for use in structure–activity rela-
tionship studies. However, the influence that compounds
have on the acceptance or rejection of the discs by insects
could depend on their solubility in the saliva produced
during feeding or in the fluid that bathes their taste
receptors. The composition of these fluids could be
modified by the diet the insects are reared on, which is
why we try and standardise the diet fed to our insects
(Simmonds et al., 1992). As yet we known very little
about the role these fluids have in modulating the ability
of insects to detect compounds in their food and thus
feeding behaviour of leaf chewing insects (Chapman
and de Boer, 1995).
To test the effect of compounds on insect feeding we
usually test compounds dry on an inert substrate (glass–
fibre discs). This avoids interactions between the test
compound and compounds in leaf material but it also
avoids problems associated with having to apply sol-
vents to the insect mouth parts that we know could
themselves modulate feeding by damaging insect taste
neurones, such as high concentrations of ethanol. In our
bioassay we apply a set aliquot (100 ml) of test com-
pounds to the discs and always start with a dose of 100
ppm. The test compound is dissolved in an appropriate
solvent, which will differ among compounds. An aliquot
(100 ml) of the solvent is also applied to the control
Table 1c
13C NMR spectral data of compoundsa
C
19
20
21
22A>22B
<
23>24
<
25
26
b
1
37.9, t*
22.8, t
37.7, t*
22.7, t
38.0, t*
23.0, t
38.3, t*
22.9, t
74.3, d
40.7, s
38.0, d
26.9, t
76.6, d
47.9, s
50.0, d
38.6, s
17.8, t
33.4, t
43.5, d
37.6, t*
44.9, t
154.8, s
103.6, t
64.5, t
13.0, q
17.8, q
171.4, s
21.1, q
38.3, t*
22.8, t
74.4, d
40.6, s
38.0, d
26.9, t
76.6, d
47.9, s
50.0, d
38.6, s
17.8, t
33.4, t
43.5, d
37.6, t*
44.9, t
154.8, s
103.6, t
64.6, t
13.0, q
17.8, q
171.4, s
21.1, q
37.8, t
22.9, t
74.2, d
40.3, s
39.9, d
24.1, t
79.7, d
48.0, s
48.3, d
38.5, s
17.6, t
26.8, t
51.9, d
35.9, t
54.2, t
78.6, s
24.3, q
65.1, t
12.8, q
18.2, q
170.5, s
21.1, q
37.8, t
22.9, t
73.7, d
40.3, s
39.5, d
23.9, t
79.4, d
47.9, s
48.3, d
38.5, s
17.6, t
26.8, t
51.9, d
35.8, t
54.2, t
78.7, s
24.3, q
65.0, t
13.1, q
18.1, q
170.7, s
20.2, q
38.0, t*
23.1, t
73.9, d
40.5, s
39.7, d
24.1, t
79.2, d
47.1, s
51.3, d
38.7, s
17.9, t
33.3, t
43.5, d
37.9, t*
45.3, t
154.1, s
103.8, t
65.2, t
13.2, q
18.0, q
170.7, s
20.3, q
38.1, t*
23.1, t
74.4, d
40.5, s
40.2, d
24.3, t
79.4, d
47.2, s
51.3, d
38.7, s
17.9, t
33.3, t
43.5, d
37.9, t*
45.3, t
154.1, s
103.8, t
65.3, t
12.9, q
18.1, q
170.5, s
21.1, q
2
3
75.3, d
40.5, s
75.0, d
40.3, s
75.5, d
40.6, s
4
5
39.7, d
23.9, t
39.5, d
23.7, t
39.9, d
24.0, t
6
7
81.2, d
46.8, s
80.9, d
46.7, s
81.4, d
47.0, s
8
9
51.3, d
38.5, s
17.8, t
51.1, d
38.4, s
17.6, t
51.5, d
38.6, s
17.9, t
10
11
12
13
14
15
16
17
18
19
20
OAc
33.0, t
32.9, t
33.1, t
43.2, d
37.6, t*
45.1, t
43.0, d
37.5, t*
45.0, t
43.3, d
37.8, t*
45.2, t
153.2, s
104.1, t
64.5, t
153.1, s
104.0, t
64.3, t
153.2, s
104.3, t
64.7, t
13.0, q
17.8, q
170.0, s
19.5, q
163.5, s
163.1, s
132.3, s
124.2, d
148.2, s
127.3, d
129.7, d
134.9, d
132.0, s
124.2, d
148.0, s
127.1, d
129.5, d
134.9, d
12.9, q
17.6, q
169.9, s
19.4, q
163.9, s
163.4, s
134.2, s
129.8, d
132.1, d
116.2, s
132.1, d
129.8, d
133.9, s
129.7, d
131.9, d
116.0, s
131.9, d
129.7, d
117.6, s
117.5, s
13.1, q
17.9, q
170.1, s
19.7, q
163.9, s
163.5, s
136.0, s
130.6, d
123.6, d
150.5, s
123.6, d
130.6, d
135.7, s
130.5, d
123.4, d
150.4, s
123.4, d
130.5, d
0C=O
00C=O
10
173.6, s
44.1, d
137.2, d
115.9, t
16.5, q
173.7, s
43.9, d
137.1, d
115.7, t
16.7, q
166.7, s
128.5, s
136.2, d
12.0, q
14.3, q
167.3, s
128.8, s
137.0, d
12.1, q
14.3, q
167.4, s
128.9, s
136.8, d
12.2, q
14.3, q
167.0, s
128.6, s
136.3, d
12.1, q
14.3, q
20
30
40
500
600
100
167.0, s
129.0, s
136.5, d
12.0, q
14.3, q
167.5, s
129.1, s
136.7, d
12.1, q
14.3, q
167.7, s
129.2, s
136.6, d
12.0, q
14.3, q
167.6, s
129.2, s
136.6, d
12.0, q
14.3, q
2
300
400
500
600
CN0
CN00
a
CDCl3 solution.
*, These assignments may be reversed.
b

M. Bruno et al. / Phytochemistry 58 (2001) 463–474
469
littoralis. Further research is required to understand the
molecular mechanism associated with the observed
behavioural responses to these and other diterpenoids
and whether different species of insects will show similar
behavioural responses to these compounds.
4. Experimental
4.1. General experimental procedures
IR spectra (film) were registred on a Shimadzu FT–IR
1
8300 spectrophotometer. H NMR spectra were recor-
ded in CDCl3 solution using a Bruker AC 250 E appara-
tus at 250 MHz and chemical shifts were reported with
respect to residual CHCl3 (ꢀ 7.27). 13C NMR spectra were
recorded in CDCl3 on the same apparatus at 62.7 MHz
and chemical shifts were reported with respect to solvent
signals (ꢀCDCl 77.0). 13C NMR assignments were deter-
3
mined by DEPT spectra. MS were recorded on a Finnigan
TSQ70 instrument (70 eV, direct inlet). Melting points
were determined on a Kofler block and are uncorrected.
Merck Si gel no. 7734 (70–230 mesh) deactivated with
15% H2O wt./v was used for column chromatography.
An ozone generator Fischer 500 M has been used to per-
form ozonolysis. Linearol was isolated from the following
species: Sideritis akmanii, S. niveotomentosa, S. brevidens,
S. rubriflora and S. gulendamii (Bondı et al., 2000).
CH2Cl2 was dried by distillation over calcium
hydride. Toluene was dried by distillation over molecular
sieves (3 A).
Table 2
Effect of compounds (100 ppm) on the feeding behaviour of final sta-
dium larvae of Spodoptera littoralis (n=10–15)
Feeding Indexa
Compound
Mean
(SEM)
2
6.8
ꢀ9.4
19.5
39.0
47.7
22.6
42.4
ꢀ37.5
ꢀ1.6
0.0
(12.91)
(10.22)
(21.34)
(19.06)*
(25.06)*
(12.76)
(18.88)*
(18.64)#
(18.93)
(29.81)
(11.76)*
(12.54)
(14.07)
(12.27)
(19.57)*
(13.32)
(11.55)*
(17.82)*
(25.91)
(8.21)
3
4
5
6
7
8
4.2. Reduction of linearol (2) to compounds 3 and 4
10
11
13
14
15
16
17
18
19
20
21
22
25
26
To a solution of 2 (50 mg) in MeOH (20 ml) was
added Pd/C 10% (30 mg). This solution was subjected
to a H2 pressure (3 atm) in a Parr apparatus for 24 h.
After filtration, the solvent was evaporated to give a
mixture which was resolved by CC (silica gel, petrol–
EtOAc 3:2 as eluent) yielding compound 3 (34 mg) and
4 (14 mg) in order of increasing polarity.
27.7
23.4
22.0
ꢀ6.0
48.3
21.5
44.6
34.0
36.6
ꢀ15.5
74.1
Compound 3: amorphous solid. IR ꢁmax (film) cmꢀ1
:
3423, 2970, 2930, 2840, 1712, 1438, 1373, 1255, 1172,
1039, 1018, 979. EI–MS m/z (rel. int.): 364 [M]+ (1), 346
[M–H2O]+ (5), 328 [M–2(H2O)]+ (14), 304 [M–AcOH]+
(20), 286 [M–AcOH–H2O]+ (56), 268 [M–AcOH–
(12.93)**
a
Feeding Index ((CꢀT)/(C+T))%; significant antifeedant activity
*P<0.05, **P<0.01; significant phagostimulant activity #P<0.05,
Wilcoxon matched-pairs test.
1
2(H2O)]+ (100), 255 (42), 235 (83), 217 (36). H NMR
(CDCl3): ꢀ 3.52 (dd, 1H, J=9.0 and 5.4 Hz, H-3b), 3.59
(t, 1H, J=3.0 Hz, H-7a), 1.01 (d, 3H, J=6.4 Hz, Me-
17), 4.07 (d, 1H, J=11.6 Hz, HA-18), 3.96 (d, 1H,
J=11.6 Hz, HB-18), 0.75 (s, 3H, Me-19), 1.05 (s, 3H,
Me-20), 2.09 (s, 3H, OAc). 13C NMR: Table 1a.
Despite these limitations in our knowledge the present
study has shown that ent-kauranes can have potent
antifeedant activity in a binary choice bioassay. For
example, compound 26 at 100 ppm has elicited a higher
Feeding Index than ajugarin I (Feeding Index=43 (7.3),
Simmonds et al., 1989) but not a high as Jodrellin B
(Feeding Index=100 (0.0), Cole et al., 1990) against S.
Compound 4: amorphous solid. IR ꢁmax (film) cmꢀ1
:
3430, 2970, 2930, 2840, 1715, 1438, 1373, 1258, 1170,
1036, 1020, 985. EI–MS m/z (rel. int.): [M]+ absent, 346
[M–H2O]+ (2), 328 [M–2(H2O)]+ (2), 304 [M–AcOH]+

470
M. Bruno et al. / Phytochemistry 58 (2001) 463–474
(4), 286 [M–AcOH–H2O]+ (10), 268 [M–AcOH–
3.84 (d, 1H, J=11.9 Hz, HA-18), 3.44 (d, 1H, J=11.9
Hz, HB-18), 0.75 (s, 3H, Me-19), 1.04 (s, 3H, Me-20),
1.95 (s, 3H, OAc), 1.94 (s, 3H, OAc), 1.92 (s, 3H, OAc).
13C NMR: Table 1a.
1
2(H2O)]+ (10), 255 (100), 241 (30), 213 (21). H NMR
(CDCl3): ꢀ 4.91 (dd, 1H, J=11.2 and 5.4 Hz, H-3b),
3.60 (t, 1H, J=3.0 Hz, H-7a), 1.01 (d, 3H, J=6.4 Hz,
Me-17), 3.33 (d, 1H, J=12.4 Hz, HA-18), 2.99 (d, 1H,
J=12.4 Hz, HB-18), 0.67 (s, 3H, Me-19), 1.08 (s, 3H,
Me-20), 2.07 (s, 3H, OAc). 13C NMR: Table 1a.
4.5. Ozonolysis of 2 to compounds 8 and 9
A current of O2/O3 (30 l/h) was bubbled into a solution
of 2 (300 mg) in dry CH2Cl2 at ꢀ78 ꢂC for 30 min. After
evaporation of the solvent under reduced pressure, the
residue was purified by CC (silica gel, gradient elution
from petrol ether–AcOEt 2:3 to pure AcOEt) giving, in
order of increasing polarity, 87 mg of compound 8 and
186 mg of compound 9.
4.3. Epoxidation of linearol (2) to compounds (5) and (6)
To a solution of 2 (250 mg) in CH2Cl2 (15 ml) was
added MCPBA 70% (170 mg). After stirring overnight,
the solution was extracted with a saturated aqueous
solution of NaHCO3 (3ꢁ10 ml) and washed with H2O
(3ꢁ10 ml). The organic layer was dried with dry Na2SO4
and evaporated under reduced pressure giving a residue
which was purified by CC (silica gel, diethyl ether as elu-
ent) yielding compounds 5 (140 mg) and 6 (92 mg).
ꢂ
Compound 8: white crystals, mp 155–158 C. IR ꢁmax
(film) cmꢀ1: 3433, 2935, 2870, 1732, 1720, 1371, 1251,
1047, 837, 734. EI–MS m/z (rel. int.): 364 [M]+ (1), 346
[M–H2O]+ (4), 328 [M–2(H2O)]+ (5), 304 [M–AcOH]+
(6), 286 [M–AcOH–H2O]+ (18), 268 [M–AcOH–
2(H2O)]+ (10), 235 (15), 217 (11), 119 (13), 107 (20), 95
(26), 43 (100). 1H NMR (CDCl3): ꢀ 3.51 (dd, 1H, J=9.0
and 5.2 Hz, H-3b), 3.66 (t, 1H, J=3.8 Hz, H-7a), 2.37
(m, 1H, H-13), 2.10 (br s, 2H, H-15), 3.99 (d, 1H,
J=11.6 Hz, HA-18), 3.92 (d, 1H, J=11.6 Hz, HB-18),
0.72 (s, 3H, Me-19), 1.06 (s, 3H, Me-20), 2.02 (s, 3H,
OAc). 13C NMR: Table 1a.
Compound 5: amorphous solid. IR ꢁmax (film) cmꢀ1
:
3480, 3267, 3040, 2931, 2866, 1714, 1384, 1371, 1255,
1049, 1018. EI–MS m/z (rel. int.): 378 [M]+ (1), 360 [M–
H2O]+ (5), 318 [M–AcOH]+ (36), 300 [M–H2O–AcOH]+
(55), 282 [M–AcOH–2(H2O)]+ (97), 270 (100), 249 (96),
1
231 (41), 121 (85). H NMR (CDCl3): ꢀ 3.54 (dd, 1H,
J=9.0 and 5.2 Hz, H-3b), 3.73 (t, 1H, J=3.8 Hz, H-7a),
2.91 (d, 1H, J=4.5 Hz, HA-17), 2.82 (d, 1H, J=4.5 Hz,
HB-17), 4.06 (d, 1H, J=11.8 Hz, HA-18), 3.99 (d, 1H,
J=11.8 Hz, HB-18), 0.77 (s, 3H, Me-19), 1.08 (s, 3H,
Me-20), 2.08 (s, 3H, OAc). 13C NMR: Table 1a.
Compound 9: white solid, mp 88–92. IR ꢁmax (film)
cmꢀ1: 3445, 2937, 2870, 1735, 1372, 1248, 1040, 1005,
956, 734. EI–MS m/z (rel. int.): [M]+ absent, 364 [M–
CH2O2]+ (1), 346 [M–CH2O2–H2O]+ (1), 304 [M–
CH2O2–AcOH]+ (3), 286 [M–CH2O2–AcOH–H2O]+
(5), 268 [M–CH2O2–AcOH–2(H2O)]+ (3), 245 (7), 227
Compound 6: amorphous solid. IR ꢁmax (film) cmꢀ1
:
3388, 3040, 2933, 2868, 1724, 1369, 1250, 1053, 1024,
1004, 953, 736. EI–MS m/z (rel. int.): 378 [M]+ (1), 360
[M–H2O]+ (2), 318 [M–AcOH]+ (4), 300 [M–AcOH–
H2O]+ (12), 282 [M–AcOH–2(H2O)]+ (22), 270 (100),
255 (40), 213 (11), 182 (12), 159 (12), 145 (19), 119 (22).
1H NMR (CDCl3): ꢀ 4.90 (dd, 1H, J=11.2 and 5.2 Hz,
H-3b), 3.72 (t, 1H, J=3.8 Hz, H-7a), 2.91 (d, 1H,
J=4.6 Hz, HA-17), 2.79 (d, 1H, J=4.6 Hz, HB-17), 3.29
(d, 1H, J=12.6 Hz, HA-18), 2.96 (d, 1H, J=12.6 Hz,
HB-18), 0.67 (s, 3H, Me-19), 1.07 (s, 3H, Me-20), 2.05 (s,
3H, OAc). 13C NMR: Table 1a.
1
(10), 145 (12), 119 (10), 107 (15), 91 (20), 43 (100). H
NMR (CDCl3): ꢀ 3.48 (dd, 1H, J=9.0 and 5.2 Hz, H-
3b), 3.55 (t, 1H, J=3.8 Hz, H-7a), 5.01 (br s, 2H, H-17),
3.91 (d, 1H, J=11.6 Hz, HA-18), 3.89 (d, 1H, J=11.6
Hz, HB-18), 0.67 (s, 3H, Me-19), 0.98 (s, 3H, Me-20),
2.00 (s, 3H, OAc). 13C NMR: Table 1a.
4.6. Transformation of compound 9 into compound 8
Twenty milligrams of ozonide 9 were dissolved in 10
ml of glacial acetic acid and added to zinc powder. After
30 min the conversion of 9 to 8 was complete. This was
confirmed by TLC comparison with an authentic sample
of 8.
4.4. Acetylation of compounds 5 and 6
Fifty milligrams of compounds 5 and 6 were treated
separately with 5 ml of a mixture of pyridine–acetic
anhydride (3:2) yielding, after evaporation under reduced
pressure, the same compound 7 (98% yield).
4.7. General esterification procedure
Compound 7: amorphous solid. IR ꢁmax (film) cmꢀ1
:
3040, 2939, 2868, 1734, 1732, 1371, 1246, 1030, 962,
902, 734. EI–MS m/z (rel. int.): 462 [M]+ (1), 402 [M–
AcOH]+ (2), 342 [M–2(AcOH)]+ (5), 329 (2), 300 (2),
282 [M–3(AcOH)]+ (23), 269 (10), 119 (8), 95 (12), 43
(100). H NMR (CDCl3): ꢀ 4.65 (dd, 1H, J=11.2 and
5.2 Hz, H-3b), 4.79 (t, 1H, J=3.8 Hz, H-7a), 2.80 (d,
1H, J=4.5 Hz, HA-17), 2.72 (d, 1H, J=4.5 Hz, HB-17),
Linearol (150 mg) was solubilized in 10 ml of dry
CH2Cl2 and added with 1 eq. of DMAP (56 mg), 25 eq.
of TEA (1.5 ml) and the appropriate acyl chloride (4 eq.)
at room temperature under argon atmosphere. After
stirring overnight, the reaction was subjected to usual
work-up adding H2O and extracting with AcOEt;
finally, the organic layer was dried over Na2SO4 and
1

M. Bruno et al. / Phytochemistry 58 (2001) 463–474
471
evaporated under reduced pressure. Generally, the resi-
Compound 13: amorphous solid. IR ꢁmax (film) cmꢀ1
:
due was purified by CC (silica gel, mixtures of petrol–
AcOEt as eluent). This procedure gave the following
ester derivatives:
2931, 2875, 2856, 1732, 1658, 1460, 1371, 1232, 1193,
1174, 1041, 873, 754. EI–MS m/z (rel. int.): [M]+
absent, 414 [M–CH3CH2CH2COOH]+ (6), 354 [M–
CH3CH2CH2COOH–AcOH)]+ (2), 326 [M–2(CH3CH2
CH2COOH)]+ (82), 266 [M–2(CH3CH2CH2COOH)–
Compounds 10 and 11. Treatment of 2 with propa-
noyl chloride gave a mixture of two compounds which
were separated by CC (silica gel, petrol ether–AcOEt 4:1
as eluent) giving 125 mg of 10 and 47 mg of 11.
1
AcOH]+ (100), 253 (29), 185 (20). H NMR (CDCl3): ꢀ
4.78 (dd, 1H, J=11.2 and 5.2 Hz, H-3b), 4.77 (t, 1H,
J=3.8 Hz, H-7a), 2.71 (m, 1H, H-13), 2.16 (dt, 1H,
J=17 and 2.2 Hz, HA-15), 2.12 (dt, 1H, J=17 and 2.2
Hz, HB-15), 4.82 (br s, 1H, HA-17), 4.81 (br s, 1H, HB-
17), 3.90 (d, 1H, J=11.7 Hz, HA-18), 3.54 (d, 1H,
J=11.7 Hz, HB-18), 0.83 (s, 3H, Me-19), 1.11 (s, 3H,
Me-20), 2.03 (s, 3H, OAc), 2.28 (t, 4H, J=7.3 Hz, H-20
and H-200), 0.97 (t, 3H, J=7.5 Hz, H-40), 0.93 (t, 3H,
J=7.5 Hz, H-400). 13C NMR: Table 1b.
Compounds 14 and 15. Treatment of 2 with benzoyl
chloride gave a mixture of two compounds which were
separated by CC (silica gel, petrol ether–AcOEt 4:1,
petrol–AcOEt 3:2 as eluent) giving 37 mg of 14 and 180
mg of 15.
Compound 10: amorphous solid. IR ꢁmax (film) cmꢀ1
:
2937, 2858, 1732, 1660, 1462, 1371, 1230, 1195, 1178, 1150,
1070, 1041, 1024, 875. EI–MS m/z (rel. int.): 474 [M]+ (1),
400 [M–CH3CH2COOH]+ (9), 340 [M–CH3CH2COOH–
AcOH)]+ (2), 326 [M–2(CH3CH2COOH)]+ (93), 283 (2),
266 [M–2(CH3CH2COOH)–AcOH]+ (82), 253 (28), 185
1
(22), 119 (31), 105 (28), 43 (100). H NMR (CDCl3): ꢀ
4.77 (dd, 1H, J=11.2 and 5.2 Hz, H-3b), 4.77 (t, 1H,
J=3.8 Hz, H-7a), 2.70 (m, 1H, H-13), 2.17 (dt, 1H,
J=17 and 2.2 Hz, HA-15), 2.13 (dt, 1H, J=17 and 2.2
Hz, HB-15), 4.82 (br s, 1H, HA-17), 4.81 (br s, 1H, HB-
17), 3.92 (d, 1H, J=11.8 Hz, HA-18), 3.53 (d, 1H,
J=11.8 Hz, HB-18), 0.83 (s, 3H, Me-19), 1.09 (s, 3H,
Me-20), 2.02 (s, 3H, OAc), 2.31 (q, 2H, J=7.5 Hz, H-
20), 1.16 (t, 3H, J=7.5 Hz, H-30), 020 .30 (q, 2H, J=7.5 Hz,
H-200), 1.11 (t, 3H, J=7.5 Hz, H-3 ). 13C NMR: Table 1b.
Compound 14: white crystals, mp 176–180 ꢂC. IR
ꢁmax (film) cmꢀ1: 3527, 2933, 2858, 1743, 1716, 1655,
1600, 1450, 1273, 1250, 1176, 1115, 943, 736, 711. EI–
MS m/z (rel. int.): 466 [M]+ (1), 448 [M–H2O]+ (2), 344
[M–C6H5COOH]+ (2), 326 [M–C6H5COOH–H2O]+
(35), 284 [M–C6H5COOH–AcOH]+ (10), 266 [M–
C6H5COOH–AcOH–H2O]+ (37), 176 (22), 149 (47),
Compound 11: amorphous solid. IR ꢁmax (film) cmꢀ1
:
3435, 2937, 2858, 1730, 1660, 1462, 1423, 1371, 1230,
1180, 1041, 1024. EI–MS m/z (rel. int.): 418 [M]+ (1), 400
[M–H2O]+ (2), 344 [M–CH3CH2COOH)]+ (2), 326 [M–
CH3CH2COOH–H2O]+ (37), 284 [M–CH3CH2COOH–
AcOH]+ (10), 266 [M–CH3CH2COOH–AcOH–H2O]+
1
105 (93), 84 (100). H NMR (CDCl3): ꢀ 5.09 (dd, 1H,
J=11.2 and 5.2 Hz, H-3b), 3.59 (t, 1H, J=3.8 Hz, H-
7a), 2.67 (m, 1H, H-13), 2.26 (br s, 2H, H-15), 4.81 (br s,
1H, HA-17), 4.79 (br s, 1H, HB-17), 4.17 (d, 1H, J=11.7
Hz, HA-18), 3.56 (d, 1H, J=11.7 Hz, HB-18), 0.98 (s,
3H, Me-19), 1.11 (s, 3H, Me-20), 2.03 (s, 3H, OAc), 8.00
(dd, 2H, J=7.0 and 1.6 Hz, H-20 and H-60), 7.41 (td, 2H,
J=7.0 and 1.6 Hz, H-30 and H-50), 7.51 (tt, 1H, J=7.0
and 1.6 Hz, H-40). 13C NMR: Table 1b.
1
(42), 251 (23), 149 (32), 121 (35), 93 (35), 43 (100). H
NMR (CDCl3): ꢀ 4.86 (dd, 1H, J=11.2 and 5.2 Hz, H-
3b), 3.60 (t, 1H, J=3.8 Hz, H-7a), 2.69 (m, 1H, H-13),
2.26 (br s, 2H, H-15), 4.83 (br s, 1H, HA-17), 4.80 (br s,
1H, HB-17), 4.08 (d, 1H, J=11.6 Hz, HA-18), 3.54 (d,
1H, J=11.6 Hz, HB-18), 0.84 (s, 3H, Me-19), 1.09 (s,
3H, Me-20), 2.07 (s, 3H, OAc), 2.32 (q, 2H, J=7.5 Hz,
H-20), 1.14 (t, 3H, J=7.5 Hz, H-30). 13C NMR: Table 1b.
Compounds 12 and 13. Treatment of 2 with butanoyl
chloride gave a mixture of two compounds which were
separated by CC (silica gel, petrol ether–AcOEt 4:1 as
eluent) giving 50 mg of 12 and 120 mg of 13.
Compound 15: white crystals, mp 175–178 ꢂC. IR ꢁmax
(film) cmꢀ1: 3060, 2933, 2860, 1743, 1716, 1658, 1600,
1450, 1276, 1249, 1176, 1114, 879, 738, 711. EI–MS m/z
(rel. int.): [M]+ absent, 448 [M–C6H5COOH]+ (3), 388
[M–C6H5COOH–AcOH]+ (2), 326 [M–2(C6H5COOH)]+
(58), 266 [M–2(C5H6COOH)–AcOH]+ (70), 251 (32),
Compound 12: amorphous solid. IR ꢁmax (film) cmꢀ1
:
1
3440, 2937, 2858, 1725, 1660, 1458, 1425, 1367, 1235,
1185, 1020. EI–MS m/z (rel. int.): 432 [M]+ (1), 414 [M–
H2O]+ (1), 344 [M–CH3CH2CH2COOH)]+ (2), 326
[M–CH3CH2CH2COOH–H2O]+ (30), 284 [M–CH3CH2
CH2COOH–AcOH]+ (7), 266 [M–CH3CH2CH2COOH–
AcOH-H2O]+ (50), 149 (42), 121 (30), 93 (20), 71 (33), 43
(100). 1H NMR (CDCl3): ꢀ 4.87 (dd, 1H, J=11.2 and 5.2
Hz, H-3b), 3.60 (t, 1H, J=3.8 Hz, H-7a), 2.69 (m, 1H, H-
13), 2.27 (br s, 2H, H-15), 4.83 (br s, 1H, HA-17), 4.80 (br s,
1H, HB-17), 4.08 (d, 1H, J=11.7 Hz, HA-18), 3.53 (d, 1H,
J=11.7 Hz, HB-18), 0.83 (s, 3H, Me-19), 1.09 (s, 3H, Me-
20), 2.07 (s, 3H, OAc), 2.27 (t, 2H, J=7.5 Hz, H-20), 0.95
(t, 3H, J=7.5 Hz, H-40). 13C NMR: Table 1b.
185 (32), 119 (18), 105 (100). H NMR (CDCl3): ꢀ 5.02
(dd, 1H, J=11.2 and 5.2 Hz, H-3b), 5.02 (t, 1H, J=3.8
Hz, H-7a), 2.75 (m, 1H, H-13), 2.35 (dt, 1H, J=17 and
2.2 Hz, HA-15), 2.20 (dt, 1H, J=17 and 2.2 Hz, HB-15),
4.83 (br s, 1H, HA-17), 4.76 (br s, 1H, HB-17), 3.80 (d,
1H, J=11.7 Hz, HA-18), 3.60 (d, 1H, J=11.7 Hz, HB-
18), 0.99 (s, 3H, Me-19), 1.21 (s, 3H, Me-20), 1.12 (s,
3H, OAc), 8.05 (dd, 2H, J=7.0 and 1.6 Hz, H-20 and H-
60), 7.46 (td, 2H, J=7.0 and 1.6 Hz, H-30 and H-50), 7.54
(tt, 1H, J=7.0 and 1.6 Hz, H-40), 7.96 (dd, 2H, J=7.0
and 1.6 Hz, H-200 and H-600), 7.40 (td, 2H, J=7.0 and 1.6
Hz, H-300 and H-500), 7.51 (tt, 1H, J=7.0 and 1.6 Hz, H-
400). 13C NMR: Table 1b.

472
M. Bruno et al. / Phytochemistry 58 (2001) 463–474
Compounds 16 and 17. Treatment of 2 with 4-meth-
Compound 19. Treatment of 2 with 3-nitrobenzoyl
chloride gave 220 mg of 19. Amorphous solid. IR ꢁmax
(film) cmꢀ1: 3085, 3064, 2937, 2864, 1747, 1732, 1658,
1616, 1537, 1479, 1440, 1260, 1234, 1136, 1041, 837,
775, 719. EI–MS m/z (rel. int.): [M]+ absent, 493 [M–
NO2C6H4COOH]+ (3), 433 [M–NO2C6H4COOH–
AcOH]+ (3), 326 [M–2(NO2C6H4COOH)]+ (53), 266
[M–2(NO2C6H4COOH)–AcOH]+ (100), 251 (45), 150
oxybenzoyl chloride gave a mixture of two compounds
which were separated by CC (silica gel, petrol ether–
AcOEt 4:1, petrol ether–AcOEt 3:2 as eluent) giving 60
mg of 16 and 175 mg of 17.
Compound 16: white crystals, mp 187–190 ꢂC. IR
ꢁmax (film) cmꢀ1: 3508, 2931, 2856, 1740, 1700, 1654,
1606, 1579, 1512, 1317, 1270, 1256, 1167, 1101, 1029,
869, 771, 736. EI–MS m/z (rel. int.): 496 [M]+ (2), 478
[M–H2O]+ (6), 418 [M–H2O–AcOH]+ (16), 344 [M–
CH3OC6H5COOH]+ (3), 326 [M–CH3OC6H5COOH–
H2O]+ (32), 284 [M–CH3OC6H5COOH–AcOH]+ (18),
266 [M–CH3OC6H5COOH–AcOH–H2O]+ (80), 253
(60), 135 (100). 1H NMR (CDCl3): ꢀ 5.06 (dd, 1H, J=11.2
and 5.2 Hz, H-3b), 3.58 (t, 1H, J=3.8 Hz, H-7a), 2.67 (m,
1H, H-13), 2.26 (br s, 2H, H-15), 4.81 (br s, 1H, HA-17),
4.78 (br s, 1H, HB-17), 4.16 (d, 1H, J=11.7 Hz, HA-18),
3.56 (d, 1H, J=11.7 Hz, HB-18), 0.97 (s, 3H, Me-19), 1.14
(s, 3H, Me-20), 2.03 (s, 3H, OAc), 7.95 (d, 2H, J=6.8 Hz,
H-20 and H-60), 6.90 (d, 2H, J=6.8 Hz, H-30 and H-50),
3.83 (s, 3H, OME0). 13C NMR: Table 1b.
1
(67), 119 (24), 83 (86). H NMR (CDCl3): ꢀ 5.00 (dd,
1H, J=11.2 and 5.2 Hz, H-3b), 5.03 (t, 1H, J=3.8 Hz,
H-7a), 2.72 (m, 1H, H-13), 2.33 (dt, 1H, J=17 and 2.2
Hz, HA-15), 2.17 (dt, 1H, J=17 and 2.2 Hz, HB-15),
4.80 (br s, 1H, HA-17), 4.73 (br s, 1H, HB-17), 3.86 (d,
1H, J=11.7 Hz, HA-18), 3.54 (d, 1H, J=11.7 Hz, HB-
18), 1.00 (s, 3H, Me-19), 1.20 (s, 3H, Me-20), 1.17 (s,
3H, OAc), 8.81 (br s, 1H, H-20), 8.41 (br d, 1H, J=7.5,
H-40), 7.70 (t, 1H, J=7.5, H-50), 8.37 (br d, 1H, J=7.5,
H-60), 8.70 (br s, 1H, H-200), 8.34 (br d, 1H, J=7.5, H-
400), 7.60 (t, 1H, J=7.5, H-500), 8.24 (br d, 1H, J=7.5, H-
600). 13C NMR: Table 1c.
Compound 20. Treatment of 2 with 4-cyanobenzoyl
chloride gave 232 mg of 20. Amorphous solid. IR ꢁmax
(film) cmꢀ1: 3064, 2937, 2862, 2231, 1744, 1716, 1658,
1610, 1569, 1309, 1276, 1234, 1176, 1041, 1015, 862, 767,
736. EI–MS m/z (rel. int.): [M]+ absent, 326 [M–
2(NCC6H4COOH)]+ (24), 266 [M–2(NCC6H4COOH)–
ꢂ
Compound 17: white crystals, mp 83–86 C. IR ꢁmax
(film) cmꢀ1: 3064, 2935, 2860, 1743, 1708, 1658, 1606,
1581, 1512, 1461, 1317, 1270, 1257, 1166, 1101, 1029,
877, 771, 736. EI–MS m/z (rel. int.): [M]+ absent, 478
[M–CH3OC6H5COOH]+ (3), 326 [M–2(CH3OC6H5-
COOH)]+ (76), 266 [M–2(CH3OC5H6COOH)–AcOH]+
(100), 251 (28), 135 (96). 1H NMR (CDCl3): &delta;
4.97 (dd, 1H, J=11.2 and 5.2 Hz, H-3b), 4.97 (t, 1H,
J=3.8 Hz, H-7a), 2.73 (m, 1H, H-13), 2.32 (dt, 1H,
J=17 and 2.2 Hz, HA-15), 2.18 (dt, 1H, J=17 and 2.2
Hz, HB-15), 4.82 (br s, 1H, HA-17), 4.74 (br s, 1H, HB-
17), 3.76 (d, 1H, J=11.7 Hz, HA-18), 3.60 (d, 1H,
J=11.7 Hz, HB-18), 0.97 (s, 3H, Me-19), 1.20 (s, 3H,
Me-20), 1.20 (s, 3H, OAc), 8.00 (d, 2H, J=6.8 Hz, H-20
and H-60), 6.94 (d, 2H, J=6.8 Hz, H-30 and H-50), 7.91
(d, 2H, J=6.8 Hz, H-200 and H-600), 6.87 (d, 2H, J=6.8
Hz, H-300 and H-500), 3.84 (s, 3H, OME0), 3.82 (s, 3H,
OME00). 13C NMR: Table 1b.
1
AcOH]+ (44), 251 (20), 185 (16), 130 (100). H NMR
(CDCl3): ꢀ 4.96 (dd, 1H, J=11.2 and 5.2 Hz, H-3b), 4.98
(t, 1H, J=3.8 Hz, H-7a), 2.70 (m, 1H, H-13), 2.28 (dt, 1H,
J=17 and 2.2 Hz, HA-15), 2.13 (dt, 1H, J=17 and 2.2 Hz,
HB-15), 4.78 (br s, 1H, HA-17), 4.71 (br s, 1H, HB-17),
3.79 (d, 1H, J=11.7 Hz, HA-18), 3.52 (d, 1H, J=11.7
Hz, HB-18), 0.94 (s, 3H, Me-19), 1.15 (s, 3H, Me-20),
1.14 (s, 3H, OAc), 8.12 (br d, 2H, J=6.8 Hz, H-20 and
H-60), 7.75 (br d, 2H, J=6.8 Hz, H-30 and H-50), 8.00 (br
d, 2H, J=6.8 Hz, H-200 and H-600), 7.66 (br d, 2H, J=6.8
Hz, H-300 and H-500). 13C NMR: Table 1c.
Compound 21. Treatment of 2 with 4-nitrobenzoyl
chloride gave 260 mg of 21. Amorphous solid. IR ꢁmax
(film) cmꢀ1: 3057, 2935, 2862, 1743, 1722, 1656, 1606,
1529, 1344, 1276, 1234, 1118, 1103, 1041, 1014, 873,
842, 785, 736, 719. EI–MS m/z (rel. int.): [M]+ absent,
493 [M–NO2C6H4COOH]+ (1), 433 [M–NO2C6H4-
COOH–AcOH]+ (2), 326 [M–2(NO2C6H4COOH)]+
(40), 266 [M–2(NO2C6H4COOH)–AcOH]+ (100), 251
Compound 18. Treatment of 2 with 2-furoyl chloride
gave 187 mg of 18. Amorphous solid. IR ꢁmax (film) cmꢀ1
:
3124, 3064, 2937, 2862, 1744, 1714, 1660, 1579, 1569,
1471, 1394, 1299, 1232, 1180, 1120, 1041, 1012, 975, 885,
763, 736, 702. EI–MS m/z (rel. int.): [M]+ absent, 438 [M–
C4H3OCOOH]+ (2), 326 [M–2(C4H3OCOOH)]+ (50),
266 [M–2(C4H3OCOOH)–AcOH]+ (52), 251 (16), 185
(14), 95 (100). 1H NMR (CDCl3): ꢀ 4.95 (dd, 1H,
J=11.2 and 5.2 Hz, H-3b), 4.91 (t, 1H, J=3.8 Hz, H-
7a), 2.67 (m, 1H, H-13), 2.25 (dt, 1H, J=17 and 2.2 Hz,
HA-15), 2.10 (dt, 1H, J=17 and 2.2 Hz, HB-15), 4.77 (br
s, 1H, HA-17), 4.70 (br s, 1H, HB-17), 3.77 (d, 1H,
J=11.7 Hz, HA-18), 3.54 (d, 1H, J=11.7 Hz, HB-18),
0.88 (s, 3H, Me-19), 1.11 (s, 3H, Me-20), 1.37 (s, 3H,
OAc), 7.13 (m, 1H, H-30), 6.47 (m, 1H, H-40), 7.54 (m,
1H, H-50), 70.004 (m, 1H, H-300), 6.42 (m, 1H, H-400), 7.49
(m, 1H, H-5 ). 13C NMR: Table 1b.
1
(57), 185 (35), 150 (82). H NMR (CDCl3): ꢀ 5.02 (dd,
1H, J=11.2 and 5.2 Hz, H-3b), 5.04 (t, 1H, J=3.8 Hz,
H-7a), 2.77 (m, 1H, H-13), 2.33 (dt, 1H, J=17 and 2.2
Hz, HA-15), 2.20 (dt, 1H, J=17 and 2.2 Hz, HB-15),
4.85 (br s, 1H, HA-17), 4.77 (br s, 1H, HB-17), 3.86 (d,
1H, J=11.7 Hz, HA-18), 3.59 (d, 1H, J=11.7 Hz, HB-
18), 1.00 (s, 3H, Me-19), 1.22 (s, 3H, Me-20), 1.21 (s,
3H, OAc), 8.23 (br d, 2H, J=6.8 Hz, H-20 and H-60),
8.33 (br d, 2H, J=6.8 Hz, H-30 and H-50), 8.11 (br d,
2H, J=6.8 Hz, H0-0200 and H-600), 8.24 (br d, 2H, J=6.8
Hz, H-300 and H-5 ). 13C NMR: Table 1c.

M. Bruno et al. / Phytochemistry 58 (2001) 463–474
473
1
Compound 22. Treatment of 2 with tigloyl chloride
gave 92 mg of 22 an unsemixture of two compounds in a
3:2 ratio. Amorphous solid. IR ꢁmax (film) cmꢀ1: 3531,
2981, 2935, 2877, 1732, 1656, 1639, 1458, 1371, 1250,
1193, 1049, 995, 977, 873, 756. EI–MS m/z (rel. int.):
444 [M]+ (1), 426 [M–H2O]+ (3), 344 [M–C4H7
COOH]+ (8), 326 [M–C4H7COOH-H2O]+ (92), 284
[M–C4H7COOH–AcOH]+ (23), 266 [M–C4H7COOH–
H2O–AcOH]+ (100), 253 (32), 223 (14), 83 (40). 1H
NMR (CDCl3): ꢀ 4.86 (dd, 1H, J=11.2 and 5.2 Hz, H-
3b), 3.57 (t, 1H, J=3.8 Hz, H-7a), 2.66 (m, 1H, H-13),
2.25 (br s, 2H, H-15), 4.80 (br s, 1H, HA-17), 4.77 (br s,
1H, HB-17), 4.07 (d, 1H, J=11.6 Hz, HA-18), 3.45 (d,
0.6H, J=11.6 Hz, HB-18, 22A epimer), 3.49 (d, 0.4H,
J=11.6 Hz, HB-18, 22B epimer), 0.81 (s, 3H, Me-19),
1.07 (s, 3H, Me-20), 2.04 (s, 3H, OAc), 3.11 (br quint,
1H, J=7.0 Hz, H-20), 5.87 (m, 1H, H-30), 5.08 (br d, 1H,
J=10.0 Hz, HA-40), 5.12 (br d, 1H, J=17.2 Hz, HB-40),
1.24 (d, 1.8H, J=7.0 Hz, Me-5, 22A epimer), 1.25 (d,
1.2H, J=7.0 Hz, Me-50, 22B epimer). 13C NMR:
Table 1c.
Compounds 23 and 24. Treatment of 2 (150 mg) with
287 mg of tigloyl chloride in refluxing toluene for 30
min gave 140 mg of an unresolvable mixture of 23 and
24 in a 55:45 ratio. Amorphous solid. IR ꢁmax (film)
cmꢀ1: 3435, 2935, 2868, 1733, 1708, 1652, 1380, 1270,
1139, 1072, 1028, 732. EI–MS m/z (rel. int.): 544 [M]+
(1), 526 [M–H2O]+ (3), 426 [M–H2O–C4H7COOH]+
(4), 366 [M–H2O–C4H7COOH–AcOH]+ (15), 344 [M–
2(C4H7COOH)]+ (7), 326 [M–H2O–2(C4H7COOH)]+
(33), 284 [M–2(C4H7COOH)–AcOH]+ (97), 266 [M–
2(C4H7COOH)–H2O–AcOH]+ (100), 225 (20). 1H
NMR (CDCl3): ꢀ 4.88 (dd, 0.55H, J=11.2 and 5.2 Hz,
H-3b of 23), 4.82 (dd, 0.45H, J=11.2 and 5.2 Hz, H-3b
of 24), 4.78 (t, 1H, J=3.8 Hz, H-7a), 1.35 (s, 3H, Me-
17), 3.83 (d, 0.55H, J=12.0 Hz, HA-18 of 23), 3.78 (d,
0.45H, J=12.0 Hz, HA-18 of 24), 3.58 (d, 0.55H,
J=12.0 Hz, HB-18 of 23), 3.54 (d, 0.45H, J=12.0 Hz,
HB-18 of 24), 0.84 (s, 1.35H, Me-19 of 24), 0.86 (s,
1.65H, Me-19 of 23), 1.13 (s, 3H, Me-20), 1.99 (s, 1.65H,
OAc of 23), 1.82 (s, 1.35H, OAc of 24), 6.69 (qq, 1.55H,
J=7.0 and 1.0 Hz, H-30 and H-300 of 23 and H-300 of 24),
6.82 (qq, 0.45H, J=7.0 and 1.0 Hz, H-30 of 24), 1.77 (br
d, 6H, J=7.0 Hz, Me-40 and Me-400), 1.71 (br s, 6H, Me-
50 and Me-500). 13C NMR: Table 1c.
2(C4H7COOH)–AcOH]+ (100), 251 (26), 226 (15). H
NMR (CDCl3): ꢀ 4.80 (dd, 1H, J=11.2 and 5.2 Hz, H-
3b), 4.77 (t, 1H, J=3.8 Hz, H-7a), 2.71 (m, 1H, H-13),
2.22 (dt, 1H, J=17 and 2.2 Hz, HA-15), 2.10 (dt, 1H,
J=17 and 2.2 Hz, HB-15), 4.81 (br s, 1H, HA-17), 4.76
(br s, 1H, HB-17), 3.78 (d, 1H, J=11.8 Hz, HA-18), 3.56
(d, 1H, J=11.8 Hz, HB-18), 0.87 (s, 3H, Me-19), 1.13 (s,
3H, Me-20), 1.84 (s, 3H, OAc), 6.86 (qq, 1H, J=7.0 and
1.0 Hz, H-30), 6.79 (qq, 1H, J=7.0 and 1.0 Hz, H-300),
1.76 (br d, 6H, J=7.1 Hz, Me-40 and Me-400), 1.78 (br s,
6H, Me-50 and Me-500). 13C NMR: Table 1c.
Compound 26. Amorphous solid. IR ꢁmax (film)
cmꢀ1: 2925, 2856, 1745, 1709, 1653, 1630, 1269, 1238,
1140, 1074, 1026, 756, 732. EI–MS m/z (rel. int.): 526
[M]+ (1), 426 [M–C4H7COOH]+ (10), 366 [M–
C4H7COOH–AcOH]+ (20), 326 [M–2(C4H7COOH)]+
(80), 266 [M–2(C4H7COOH)–AcOH]+ (100), 251 (30),
226 (20). 1H NMR (CDCl3): ꢀ 4.89 (dd, 1H, J=11.2 and
5.2 Hz, H-3b), 4.77 (t, 1H, J=3.8 Hz, H-7a), 2.71 (m,
1H, H-13), 2.21 (dt, 1H, J=17 and 2.2 Hz, HA-15), 2.12
(dt, 1H, J=17 and 2.2 Hz, HB-15), 4.81 (br s, 1H, HA-
17), 4.76 (br s, 1H, HB-17), 3.87 (d, 1H, J=12.0 Hz, HA-
18), 3.59 (d, 1H, J=12.0 Hz, HB-18), 0.87 (s, 3H, Me-
19), 1.14 (s, 3H, Me-20), 2.01 (s, 3H, OAc), 6.73 (qq,
2H, J=7.0 and 1.0 Hz, H-30 and H-300), 1.75 (br d, 6H,
J=7.1 Hz, Me-40 and Me-400), 1.74 (br s, 6H, Me-50 and
Me-500). 13C NMR: Table 1c.
4.8. Antifeedant bioassay
Compounds were exposed to final stadium larvae of
Spodoptera littoralis in a binary choice test using glass-
fibre discs (Simmonds et al., 1990). All compounds were
tested at 100 ppm as this is the dose used to compare
activity among compounds (Rodrıguez et al., 1999;
Bondı et al., 2000). The majority of compounds were
tested against insects from 2–3 generations, therefore,
each compound was tested against either 10 or 15
insects. All discs were treated with 100 ml sucrose (0.05
M), and then the treatment discs were treated with 100
ml of one of the test compounds. The control discs were
treated with an aliquot (100 ml) of the solvent used to
dissolve the test compound. Larvae were taken from a
colony fed on a wheat-germ based diet (Simmonds et
al., 1992). Larvae 24–36 h into the final stadium were
removed from food 2–3 h prior to the bioassay. These
larvae were placed individually in Petri dishes (9.1 cm
diam.) with a pair of discs: one treatment (T) and one
control (C) disc. The discs were dried and weighed
before and after being exposed to the insects. The
amount eaten of each disc was used to calculate a
Feeding Index [(CꢀT)/(C+T)]%. A positive Index
indicates an antifeedant and a negative Index a phagos-
timulant. The data did not differ significantly among
generations and were pooled for analysis using the Wil-
coxon matched-pairs test.
Compounds 25 and 26. Treatment of 2 (150 mg) with
287 mg of tigloyl chloride and 1.5 ml of TEA in reflux-
ing toluene for 24 h gave a mixture of two compounds
which were separated by CC (silica gel, gradient elution
from petrol ether to petrol ether–AcOEt 3:2) giving 50
mg of 25 and 70 mg of 26.
Compound 25. Amorphous solid. IR ꢁmax (film)
cmꢀ1: 2931, 2858, 1745, 1707, 1653, 1630, 1265, 1236,
1137, 1074, 1021, 732. EI–MS m/z (rel. int.): 526 [M]+
(1), 426 [M–C4H7COOH]+ (8), 366 [M–C4H7COOH–
AcOH]+ (10), 326 [M–2(C4H7COOH)]+ (90), 266 [M–

474
M. Bruno et al. / Phytochemistry 58 (2001) 463–474
Piozzi, F., Savona, G., Marino, M.L., 1973. Cyclic carbonate formed
Acknowledgements
during performic acid oxidation of atractyligenin. Tetrahedron 29,
621–624.
This paper was supported by MURST research funds.
We thank Mrs. M. Yule and Dr. P. Green for support
in rearing the insects. The insects bioassays were carried
out at RBG Kew under a MAFF, Plant Health Great
Britain licence.
Piozzi, F., Venturella, P., Bellino, A., Paternostro, M.P., 1971.
Revised structures for candicandiol and epicandicandiol. Chem.
Ind. 1971, 962.
Rodrıguez, B., Rodrıguez, B., de la Torre, M.C., Simmonds, M.S.J.,
Blaney, W.M., 1999. From a phagostimulant natural product to
semisynthetic antifeedants against Spodoptera littoralis larvae: che-
mical transformations of the neoclerodane diterpenoid scutegalin B.
J. Nat. Prod. 62, 594–600.
References
Simmonds, M.S.J., Blaney, W.M., 1992. Labiate-insect interactions:
effects of labiate-derived compounds on insect behaviour. In: Har-
vey, R.M., Reynolds, T. (Eds.), Advances in Labiate Science. Royal
Botanic Garden, Kew, England, pp. 375–392.
Bondı, M.L., Bruno, M., Piozzi, F., Baser, K.H.C., Simmonds,
M.S.J., 2000. Diversity and antifeedant activity of diterpenes from
Turkish species of Sideritis. Biochem. Syst. Ecol. 28, 299–303.
Chapman, R.F., de Boer, G., 1995. Regulatory Mechanisms in Insect
Feeding. Chapman & Hall, New York.
Simmonds, M.S.J., Blaney, W.M., Ley, S.V., Savona, G., Bruno, M.,
Rodrıguez, B., 1989. The antifeedant activity of clerodanes from
Teucrium. Phytochemistry 28, 1069–1071.
Cole, M.D., Anderson, J.C., Blaney, W.M., Fellows, L.E., Ley, S.V.,
Sheppard, R.N., Simmonds, M.S.J., 1990. Neo-clerodane insect
antifeedant from Scutellaria galericulata. Phytochemistry 29, 1793–
1976.
Simmonds, M.S.J., Blaney, W.M., Fellows, L.E., 1990. Behavioral and
electrophysiological study of antifeedant mechanisms associated
with polydroxy alkaloids. J. Chem. Ecol. 16, 3167–3196.
Simmonds, M.S.J., Blaney, W.M., Schoonhoven, L.M., 1992. Effects
of larval diet and larval age on the responsiveness of taste neurones
to sucrose. J. Insect. Physiol. 38, 249–257.
Gonzales, A.G., Luis, J.G., Ravelo, A.G., 1990. Plantas Ibericoamer-
icanas Fuentes de Moleculas Bioactivas, Vol. 2, Labiatae. Romero,
Tenerife, Spain.
Simmonds, M.S.J., Blaney, W.M., Esquivel, B., Rodriguez-Hahn, L.,
1996. Effect of clerodane-type diterpenoids isolated from Salvia spp.
on the feeding behaviour of Spodoptera littoralis. Pestic. Sci. 47, 17–
23.
Hanson, J.R., 1999. Diterpenoids. Nat. Prod. Rep. 16, 209–219.
Hatakeyama, S., Matsui, Y., Suzuki, M., Sakurai, K., Takano, S.,
1985. Enantioselective synthesis of (+)-citreoviral using asymmetric
hydroxylation of tiglate esters. Tetrahedron Lett. 26, 6485–6488.