Cyclopaldic acid, seiridin, and sphaeropsidin A as fungal phytotoxins, and larvicidal and biting deterrents against Aedes aegypti (Diptera: Culicidae): Structure-activity relationships

Article abstract of DOI:10.1002/cbdv.201200358

Aedes aegypti L. is the major vector of the arboviruses responsible for dengue fever, one of the most devastating human diseases. From a preliminary screening of fungal phytotoxins, cyclopaldic acid (1), seiridin (2), sphaeropsidin A (4), and papyracillic acid (5) were evaluated for their biting deterrent and larvicidal activities against Ae. aegypti L. Because compounds 1, 2, 4, and 5 exhibited mosquito biting deterrent activities and 1 and 4 demonstrated larvicidal activities, further structure-activity relationship studies were initiated on these toxins. In biting-deterrence bioassays, 1, 2, 4, and 5, 3,8-didansylhydrazone of cyclopaldic acid, 1F, 5-azidopentanoate of cyclopaldic acid A, 1G, the reduced derivative of cyclopaldic acid, 1 H, isoseiridin (3), 2′-O-acetylseiridin (2A), 2′-oxoseiridin (2C), 6-O-acetylsphaeropsidin A (4A), 8,14-methylensphaeropsidin A methyl ester (4B), and sphaeropsidin B (4C) showed activities higher than the solvent control. Sphaeropsidin B (4C) was the most active compound followed by 2A, while the other compounds were less active. Biting-deterrence activity of compound 4C was statistically similar to DEET. In the larvicidal screening bioassays, only compounds 1 and 4 demonstrated larvicidal activities. Based on LD₅₀ values, compound 4 (LD₅₀ 36.8 ppm) was significantly more active than compound 1 (LD₅₀ 58.2 ppm). However, the activity of these compounds was significantly lower than permethrin. Copyright

Full text of DOI:10.1002/cbdv.201200358

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1239
Cyclopaldic Acid, Seiridin, and Sphaeropsidin A as Fungal Phytotoxins, and
Larvicidal and Biting Deterrents against Aedes aegypti (Diptera: Culicidae):
StructureꢀActivity Relationships
by Alessio Cimminoa), Anna Andolfia), Fabiana Avolioa), Abbas Alib), Nurhayat Tabancab),
Ikhlas A. Khanb)c), and Antonio Evidente*a)
a
`
) Dipartimento di Science Chimiche, Universita di Napoli Federico II, Complesso Universitario Monte
SantꢀAngelo, Via Cintia 4, IT-80126 Napoli
(phone: þ39-081-2539178; fax: þ39-081-674330; e-mail: evidente@unina.it)
b) National Center for Natural Products Research, The University of Mississippi, University,
MS 38677, USA
c) Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University,
MS 38677, USA
Aedes aegypti L. is the major vector of the arboviruses responsible for dengue fever, one of the most
devastating human diseases. From a preliminary screening of fungal phytotoxins, cyclopaldic acid (1),
seiridin (2), sphaeropsidin A (4), and papyracillic acid (5) were evaluated for their biting deterrent and
larvicidal activities against Ae. aegypti L. Because compounds 1, 2, 4, and 5 exhibited mosquito biting
deterrent activities and 1 and 4 demonstrated larvicidal activities, further structureꢀactivity relationship
studies were initiated on these toxins. In biting-deterrence bioassays, 1, 2, 4, and 5, 3,8-didansylhydrazone
of cyclopaldic acid, 1F, 5-azidopentanoate of cyclopaldic acid A, 1G, the reduced derivative of cyclopaldic
acid, 1 H, isoseiridin (3), 2’-O-acetylseiridin (2A), 2’-oxoseiridin (2C), 6-O-acetylsphaeropsidin A (4A),
8,14-methylensphaeropsidin A methyl ester (4B), and sphaeropsidin B (4C) showed activities higher
than the solvent control. Sphaeropsidin B (4C) was the most active compound followed by 2A, while the
other compounds were less active. Biting-deterrence activity of compound 4C was statistically similar to
DEET. In the larvicidal screening bioassays, only compounds 1 and 4 demonstrated larvicidal activities.
Based on LD50 values, compound 4 (LD50 36.8 ppm) was significantly more active than compound 1
(LD50 58.2 ppm). However, the activity of these compounds was significantly lower than permethrin.
Introduction. – Aedes aegypti L. (Diptera: Culicidae) is the major vector of dengue
fever virus, which can attain epidemic levels resulting in high rates of human mortality.
Currently, the only effective way of reducing the incidence of dengue fever is to control
the vector mosquito, mainly by application of insecticides to its breeding place [1].
Repellents applied to skin or clothing are recommended as a means of personal
protection against biting arthropods [2]. Toxicants can reduce the densities of mosquito
and the risk of disease transmission. The synthetic repellent DEET (¼ N,N-diethyl-3-
methylbenzamide) has been used widely since the 1950s to protect human beings
against mosquitoes. Synthetic and natural insecticides, including pyrethroids, are
approved by the EPA (Environmental Protection Agency) to be used as repellents, but
only when applied on clothing [3].
However, pyrethroids used to control a wide range of arthropods are considered
dangerous for agriculture and human health because of their toxicity [2]. These
ꢁ 2013 Verlag Helvetica Chimica Acta AG, Zꢂrich

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
problems increased by the resistance developed against pyrethroids or insecticides of
any class and prompted the development of alternative control strategies. Therefore,
great effort has been expended for the development of pest controls using naturally
occurring compounds such as secondary plant and fungal metabolites. These are
anticipated to overcome the problem of resistance to synthetic insecticides and are
generally regarded as more environmentally friendly than their synthetic counterparts
[4].
Many naturally occurring repellents and insecticides have potential for develop-
ment into useful products with lowered risk to mammals and the environment [2][5–
8].
In this respect, we initiated the screening of some fungal phytotoxins, cyclopaldic
acid (1) [9], seiridin (2) [10][11], sphaeropsidin A (4) [12], obtained from Seiridium
cupressi and Diplodia cupressi, fungi causing different forms of cankers of cypress
(Cupressus sempervirens L.) [13], and papyracillic acid (5), obtained from Ascochyta
agropyrina var. nana, which was proposed as mycoherbicide for the biocontrol of
Elytrigia repens [14] (Fig. 1). We also carried out structureꢀactivity relationship (SAR)
studies on the three fungal phytotoxins 1, 2, and 4, by preparing several known and new
derivatives.
Fig. 1. Structures of cyclopaldic acid, seiridin, isoseiridin, sphaeropsidin A, and papyracillic acid (1–5,
resp.)
Results and Discussion. – Cyclopaldic acid (1), and seiridin (2) and isoseiridin (3),
and sphaeropsidin A (4) were obtained as white crystals, oily compound, and white
needles, respectively, from the culture filtrates of S. cupressi and D. cupressi as
described in [9][11][15].
In an effort to discover new natural compounds with the activity against Ae. aegypti,
several known and new derivatives of 1, 2, and 4 were semisynthesized and evaluated
for their biting-deterrent and larvicidal activities against Ae. aegypti.
Semisynthetic Derivatives of Cyclopaldic Acid (1; Scheme 1). By treatment with
diluted NaOH and according to the well-known Cannizzaro reaction, compound 1 was
converted into isocyclopaldic acid (1A; Scheme 1) [16][17], via the reduction of the

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1241
Scheme 1
a) 2n NaOH, reflux, 15 min. b) Ac2O and anh. AcONa, reflux, 30 min. c) 2,4-(NO2)2ꢀC6H3ꢀNHꢀNH2,
6n HCl, r.t., 1 h. d) Dansyl (¼ 5-(dimethylamino)naphthalene-1-sulfonyl) hydrazine (DNSꢀNHꢀNH2),
808, 3 h. e) Anh. CH2Cl2 and pyridine, N,N’-dicyclohexylcarbodiimide (DCC), 5-azidopentanoic acid, 08,
1 h, r.t., 2 h. f) NaBH4, 08,10 min.
hemiacetal aldehyde at C(3) to a CH2 group and the oxidation of the aldehyde at C(4)
to a COOH group. The acetylation carried out with Ac2O and anhydrous AcONa
allowed the conversion of 1 to the 3-O-acetyl and 3,5,8-O,O’,O’’-triacetyl-8-acetoxy

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
derivatives 1B and 1C (arbitrary atom numbering; Scheme 1) [16][17]. Of the
derivatives 1B and 1C, the first was obtained by the acetylation of the hemiacetale OH
group at C(3), and the latter by the acetylation of both the OH groups at C(3) and
C(5), followed by acetoxylation of C(8) and finally the acetylation of the resultant
hemiacetal OH group at the same C-atom. The reaction of 1 with a acid-saturated
solution of (2,4-dinitrophenyl)hydrazine furnished the corresponding hydrazone 1D
(Scheme 1) [17], by conversion of the CHO group at C(4) to the corresponding Schiffꢀs
base. A similar reaction of 1 with the dansylhydrazine (¼ [5-(dimethylamino)naph-
thalene-1-sulfonyl]hydrazine) allowed the preparation of the mono- and didansylhy-
drazones 1E and 1F (Scheme 1). These were previously reported, when they were used,
together with other derivatives of 1, to test the specificity of the antibodies produced by
rabbit immunization with a conjugate of 1 with bovine serum albumin (BSA) [18].
However, the complete spectroscopic characterization of 1E and 1F is reported here for
the first time. Derivative 1E, as 1D, was formed by the conversion of the CHO at C(4)
to the corresponding Schiffꢀs base but the hydrazine N-atom of 1E was linked to a
dansyl group. The didansyl derivative 1F underwent the same modification as 1E, but
also the opening of the butenolide ring occurred, generating the COO group at C(7a)
and the CHO group at C(3a), which was successively converted to the same Schiffꢀs
base at C(8). By esterification carried out with the 5-azidopentanoic acid and N,N’-
dicyclohexylcarbodiimide (DCC), 1 was converted to the new derivative 1G
(Scheme 1), which, as 1A, underwent the reversible esterification of the hemiacetal
OH group at C(3). Finally by NaBH4 reduction, 1 was converted to the two new
reduced derivatives 1H and 1I (Scheme 1). Derivative 1H displayed the expected
reduction of both the hemiacetalized and lactonized aldehyde at C(3) and the aldehyde
at C(4), the latter was converted to a primary OH group. Derivative 1I was the result of
the same reduction of the aldehyde only at C(4), and the subsequent H2O elimination
from the primary OH group at C(8) and that at C(3), generating the new trisubstituted
dihydrofuran ring fused to the benzofuranone.
Semisynthetic Derivatives of Seiridin (Scheme 2). Seiridin (2), as previously
reported [11], was converted to the 2’-O-acetyl and 3,4-dihydro derivatives 2A and
2B, respectively, by usual acetylation with AC2O and pyridine, and catalytic hydro-
genation (Scheme 2). The former was formed by the reversible esterification of the OH
group at C(2’) (arbitrary atom numbering) of the hydroxyheptyl side chain at C(4) of
the butenolide ring, while the reduction of butenolide ring converted 2 to the
corresponding butanolide 2B. The oxidation of 2 with Jones reagent [19] furnished the
derivative 2C (Scheme 2), which was previously prepared to conjugate seridin to BSA
to form a suitable hapten to elicit specific antibodies by a rabbit immunization [20].
Compound 2C displayed the presence of a C¼O group at position 2’ of the heptyl side
chain at C(4), which represents, differently from 2A, a not reversible modification.
Another modification of the heptyl side chain was the shift of the secondary OH group
from C(2’) to C(3’) in isoseiridin (3), the natural isomer of 2 isolated together with
seiridin from the culture filtrate of S. cupressi.
Semisynthetic Derivatives of Sphaeropsidin A (Scheme 3). Sphaeropsidin A (4) as
described in [21], was converted by usual acetylation and reaction with ethereal CH2N2
to the well-known derivatives 4A and 4B (Scheme 3). Derivative 4A was formed by the
reversible esterification of the hemiketal OH group at C(6), while, in 4B, this OH group

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1243
Scheme 2
a) Ac2O, Pyridine, p-toluenesulfonic acid (TsOH), r.t., 12 h. b) H2/PdBaSO4, MeOH, r.t., 3 h. c) Jones
reagent, Me2CO, 08, 1 min.
was converted to the corresponding C¼O group as consequence of the opening of the
lactone ring and methyl esterification of the COO group at C(10). The same derivative
4B was also accessible via a methylene insertion by CH2N2 on the C(8)¼C(14) bond
generating the fused oxirane ring. By the well-known enantioselective reduction with
NaBH4, as also described in [22], sphaeropsidin A (4) was converted into sphaerop-
sidin B (4C) which is another phytotoxin produced together with the other
sphaeropsidins C–F and sphaeropsidones by both Sphaeropsis sapinea f. sp. cupressi
(¼ D. cupressi) [15] and D. mutila [22]. The synthesized sphaeropsidin B was identified
by comparing its 1H-NMR and ESI mass spectra with those reported in [22].
Sphaeropsidin B so obtained was converted to the new isopropylidene derivative 4D
(Scheme 3), in which the hemiketal OH group at C(6) and that at C(7) led to the
formation of a 2,2-dimethyl-4,5-disubstituted-1,3-dioxolane fused to the pimarane
system through C(6) and C(7). The structure of 4D was confirmed by the significant
downfield shift (Dd 0.97) of HꢀC(7) resonating as broad singlet at d(H) 5.19, noted
1
when its H-NMR spectrum was compared to that of 4C recorded under the same
conditions. The probable alternative isopropylidene derivative that could be formed by
acetalization of the tertiary OH group at C(9) was ruled out for mechanistic reasons,
using a suitable Dreiding model. Similarly, also ketalizations of both OH groups at C(6)
and C(9) was ruled out, as sphaeropsidin A (4) did not give any ketal by reaction
carried out under the same conditions. Finally, the reaction of the hemiketal OH group
at C(6) of 4 with the 5-azidopentanoic acid, already described for the conversion of 1 to
1G, allowed preparation of the new derivative 4E, which was, as 4A, formed by
esterification of the OH group at C(6) with a different acyl group.
The structures of all the new derivatives of cyclopaldic acid and sphaeropsidin A,
1E–1I, and 4D and 4E, respectively, were determined by extensive use of spectroscopic

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Scheme 3
a) Ac2O, Pyridine, TsOH, r.t., 12 h. b) Etheral CH2N2, r.t., 18 h. c) NaBH4, r.t., 2 h. d) Anh. acetone and
anh. CuSO4, reflux, 24 h. e) Anh. CH2Cl2 and pyridine, DCC, 5-azidopentanoic acid, 08, 1 h, r.t, 2 h.
methods as reported in the Exper. Part, and also by comparison of their data with those
of the parent toxins.
Biting-Deterrent Activity Bioassay. In biting-deterrence bioassays, compounds 1–5,
1F–1 H, 2A, 2C, and 4A–4C showed activities above that of the solvent control. Biting-
deterrence index (BDI) values for the active compounds are given in Fig. 2.
Sphaeropsidin B (4C; BDI 0.95) was the most active compound, followed by 2’-O-
acetylseiridin (2A; BDI 0.73), while for the rest of the compounds the BDI values
ranged between 0.3 and 0.69. Biting-deterrence activity of 4C was similar to that of
N,N-diethyl-3-methylbenzamide (DEET) at 20 nmol/cm2 concentration.
StructureꢀActivity Relationships (SARs). The biting-deterrent activity of 1G was
not surprising, as it could be hydrolyzed, according to a lethal metabolism [23], to 1 at
physiological pH. This should also occur with 1B, but they differ in the length of the acyl
residue so that, in 1G, the lipophilicity was increased and membrane crossing was
facilitated. The importance of the presence of the CHO group at C(4), which is in
agreement with the results of a previous SAR study [17], was revealed by the inactivity
of 1C, 1D and 1E, and 1I. This C¼O group in 1G, which was active, was converted to the
corresponding Schiffꢀs base by reaction with dansylhydrazine in 1E, but this could be
reversed to the original functionality by hydrolysis at physiological pH. The same
reaction could occur at C(3) of 1F generating the ortho-dicarbaldehyde moiety which

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1245
Fig. 2. Mean biting-deterrence index (BDI) values (ꢁ SE) of compounds 1–5 and selected derivatives
thereof against Aedes aegypti. All the compounds were tested at 25 nmol/cm2 and evaluated using N,N-
diethyl-3-methylbenzamide (DEET) at 25 nmol/cm2 as positive control and EtOH as solvent control. A
BDI value greater than 0 indicates deterrence relative to EtOH, and a BDI value not significantly
different from 1 shows biting-deterrent effect statistically similar to DEET.
appeared to be a very important structural feature in the above cited SAR study, in
which the phytotoxic and the antimicrobial activities of 1 and of some of its several
derivatives were assayed [17]. The reduction of the same carbaldehyde at group C(4) to
the CH2OH group, as in 1H, seems to preserve the activity. This is in contrast to the
results of the above mentioned SAR study, in which a derivative obtained from the
reduction at C(8) but also at C(1) and C(3) exhibited a significantly decreased activity
[17]. However, the lacking of activity of 1I indicated that the primary OH group at C(8)
should be free and not involved in an ether bond as in the 3,4,5-trisubstituted
dihydrofuran ring.
The activities of iso-, 2’-oxo- and 2’-O-acetylseiridin (3, 2C, and 2A, resp.) indicated
that the presence and the position of the OH group of the hydroxyheptyl side chain at
C(4), in contrast to the results of a previous SAR study in which the phytotoxic and the
antimicrobial activities of 1 and 3, and of several of their derivatives were tested [24],
are not important features for the activity. On the contrary, in agreement with the
mentioned SAR study, the lacking of activity of 2B showed that the presence of an
unaltered butenolide ring, which was converted to a butanolide ring in 2B, is very
important for the activity. The increased activity of 2’-O-acetylseiridin (2A) with
respect to 2, was probably due to its increased lipohilicity and facilitated membrane
crossing. Then according to the lethal metabolism [23] it probably was hydrolyzed at
physiological pH.

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The activity found for 8,14-methylenesphaeropsidin A methyl ester (4B), which is
in total disagreement with the results of the previous SAR studies [15][21], indicated
that the lactone ring, the hemiketal OH group at C(6), and the C(8)¼C(14) bond are
not relevant for the activity. The activity of the 6-O-acetylsphaeropsidin (4A) is in
agreement with the lethal metabolism [23] as, after crossing the membrane, it could be
hydrolyzed to 4 at physiological pH. This did not occur with another reversible ester,
4E, which contains the 5-azidopentanoyl residue. The increased activity of sphaerop-
sidin B (4C) suggested that the presence of a secondary OH group at C(7) is more
significant compared to the C¼O group in 4. This finding is in total disagreement with
the previously SAR study in which a C(7)¼O group was reported to be important to
impart phytotoxic, antifungal, and antibacterial activity [15][21]. However, the OH at
group C(7) must be free, because, if not (as in 4D), the activity was lost.
Larvicidal Activity Bioassay. In larvicidal bioassays, only sphaeropsidin A (4) and
cyclopaldic acid (1) showed larvicidal activity, while all the other compounds were
inactive up to the dose of 100 ppm. The LD50 and LD90 values for 1 and 4 are compiled
in the Table. Based on 95% CIs, compound 4 (LD50 36.8 (33.05–41.0)) was significantly
more potent than 1 (LD50 58.2 (50.9–66.8)) at 24-h posttreatment, whereas the LD50
value of permethrin, used as a positive control, was 0.0031 (0.0016–0.0047) ppm.
Although mortality increased, there were no significant differences in the results of 24-
and 48-h posttreatment.
Table. Toxicity of Cyclopaldic Acid (1) and Sphaeropsidin A (4) against First-Instar Larvae of Ae.
aegypti 24-h Posttreatment
Compound
LD50a) (95% CIb)) [ppm]
LD90 (95% CI) [ppm]
c2
88.25
132.0
DF
Cyclopaldic acid (1)
Sphaeropsidin A (4)
Permethrinc)
58.2 (50.9–66.8)
36.8 (33.05–41.0)
0.0031
120.1 (110.2–154.5)
75.4 (65.4–90.7)
73
73
a) LD50 and LD90 values are (given in ppm) dosages which kill 50 and 90% of the population,
respectively. b) Confidence interval. c) Positive standard, purity 46.1 and 53.2% for cis and trans,
respectively.
Conclusions. – In this work, biting-deterrent activities of the fungal phytotoxins
cyclopaldic acid (1), papyracillic acid (5), sphaeropsidin A (4), and seiridin (2), as well
as larvicidal activities of compounds 1 and 4 were evaluated. A series of derivatives of 1,
2, and 4 were semisynthesized and assayed for further structureꢀactivity relationship
studies. The bioassay results indicate that structural features responsible for the activity
of compounds 1, 2, and 4 were the presence of a CHO or CH2OH group at C(4), an
unaltered butenolide ring, and the presence of a C¼O or a secondary OH group at
C(7). Sphaeropsidin B (4C) was the most active biting deterrent, followed by 2’-O-
acetylseiridin (2A) which also showed good larvicidal activity. Compounds 2A and 4C,
which can also be prepared in only one step in high yield from 2 and 4, respectively,
could be considered as potential natural biting deterrents against Ae. aegypti.
The authors thank Dr. Pierluigi Mazzei for recording the NMR spectra at the laboratory
CERMANU centre, University of Naples Federico II. The authors are grateful to Dr. John F. Parcher,

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1247
National Center for Natural Products Research, The University of Mississippi, University, MS 38677,
USA, for English corrections and suggestions. This was financially supported by the Italian Ministry of
University and Research (MIUR), in part by USDA/ARS grant No. 56-6402-1-612, and Deployed War-
Fighter Protection Research Program Grant funded by the U.S. Department of Defense through the
Armed Forces Pest Management Board.
Experimental Part
General. Anal. and prep. TLC: silica gel (Merck, Kieselgel 60, F254, 0.25 and 0.5 mm, resp.) plates;
visualization by exposure to UV light or by spraying with 10% H2SO4 in MeOH and then 5%
phosphomolybdic acid in EtOH, followed by heating at 1108 for 10 min. Column chromatography (CC:
silica gel (Merck, Kiesegel 60, 0.0063–0.200 mm). UV Spectra: in MeOH on a PerkinꢀElmer Lambda 25
UV/VIS spectrophotometer; l in nm (log e). IR Spectra: as glassy film on a PerkinꢀElmer Spectrum One
FT-IR spectrometer; ˜n in cmꢀ1 1H-NMR Spectra: at 600 or 400 MHz, resp., in CDCl3, on Bruker
.
spectrometers; the same solvent used as internal standard; d in ppm rel. to solvent peak; J in Hz. ESI-MS:
Agilent Technologies (Milan, Italy) 6120 quadrupole LC/MS instruments; in m/z.
Fungal strain. D. cupressi was purchased from Centraalbureau voor Schimmelcultures of Baarn (The
Netherlands), strain 261.85 CBS, and deposited with the collection of Dipartimento di Protezione delle
`
Piante, Universita di Sassari, Italy. S. cupressi, (strain No. 1022), isolated from an infected cypress tree at
Kos (Greece), was used for toxins production. The isolate was grown in liquid culture under conditions as
described in [25]. Ascochyta agropyrina var. nana was deposited with the culture collection of All-
Russian Research Institute of Plant Protection, Pushkin, Saint Petersburg, Russia.
Production, Extraction, and Purification of Cyclopaldic Acid, Seiridin, Isoseiridin, Sphaeropsidin A,
and Papyracillic Acid (1–5, resp.). Cyclopaldic acid (1), seiridin and isoseridin (2 and 3, resp.) were
obtained as white crystals and as oily compound, resp., from the purification of the org. extracts of S.
cupressi [9][11]. Sphaeropsidin A (4) was obtained as white needles from the purification of the org.
extract of the culture filtrates of D. cupressi as described in [15]. Papyracillic acid (5) was obtained as
white crystals from A. agropyrina var. nana solid culture as described in [14].
Preparation of Cyclopaldic Acid Derivatives 1A–1D. Isocyclopaldic, 3-O-acetyl-, 3,5,8-O,O’,O’’-
triacetyl-8-acetoxy-, and 8-(2’,4’-dinitro)phenylhydrazone cyclopaldic acid, 1A–1D, resp., were prepared
from 1 as described in [9][16][17].
8-Dansylhydrazone and 3,8-Didansylhydrazone of Cyclopaldic Acid (¼ N’-[(3,5-Dihydroxy-7-
methoxy-6-methyl-1-oxo-1,3-dihydro-2-benzofuran-4-yl)methylidene]-5-(dimethylamino)naphthalene-1-
sulfonohydrazide and 3-[(2-{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl}hydrazinylidene)methyl]-2-
[(2-{[5-(dimethylamino)naphthalen-1-yl]sulfonyl}hydrazinylidene)methyl]-4-hydroxy-6-methoxy-5-
methylbenzoic Acid; 1E and 1F, resp.). To compound 1 (20 mg) dissolved in a CH2Cl2/MeOH 1:1 (20 ml)
was added 5-(dimethylamino)naphthalene-1-sulfonohydrazide (40 mg). The reaction was carried out at
808 for 3 h. When all starting material was reacted, the solvent was evaporated under reduced pressure.
The residue (58 mg) was purified by prep. TLC (CHCl3/iPrOH 9 :1) to give 1E (Rf 0.44; 5 mg) and 1F (Rf
0.50; 10 mg) as homogeneous yellow amorphous solid.
Data of 1E. UV: 382 (3.10), 287 (3.36), 255 (3.48). IR: 3175, 1744, 1615, 1572, 1341. 1H-NMR
(CDCl3): 10.8 (br. s, OHꢀC(5)); 8.62–7.56 (m, 6 H (naphtyl)); 8.38 (br. s, HOꢀC(3)); 8.25 (s,
CH(8)¼O); 7.15 (br. s, NH); 6.4 (s, HꢀC(3)); 3.98 (s, MeO); 2.90 (br. s, Me2N); 2.03 (s, MeꢀC(6)). ESI-
MS: 992 ([2 MþNa]þ ), 508 ([MþNa]þ ), 486 ([MþH]þ ).
Data of 1F. UV: 328 (3.51), 265 (3.83). IR: 3380, 1728, 1620, 1569, 1451. 1H-NMR (CDCl3): 10.8 (br.
s, OHꢀC(5)); 8.66–7.08 (m, 12 H (naphtyl)); 8.04, 8.03 (2s, HꢀC(3), HꢀC(8)); 7.18, 6.48 (2 br. s, 2 NH);
3.94 (s, MeO); 2.88, 2.84 (2s, Me2N); 1.93 (s, MeꢀC(6)). ESI-MS: 771 ([MþK]þ ), 755 ([MþNa]þ ), 733
([MþH]þ ).
7-Formyl-1,3-dihydro-6-hydroxy-4-methoxy-5-methyl-3-oxo-2-benzofuran-1-yl 6-Azidopentanoate
(1G). To compound 1 (32 mg) dissolved in anh. CH2Cl2 (4.5 ml) and pyridine (100 ml) were added
N,N’-dicyclohexylcarbodiimide (DCC; 70 mg) and 5-azidopentanoic acid (70 ml). The reaction was left at
08 for 1 h and then at r.t. for 2 h. The reaction was stopped by evaporation under N2. The residue

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
(100 mg) was purified by prep. TLC (CHCl3/iPrOH 97:3) to yield 1G (Rf 0.76; 35 mg). Homogeneous
amorphous solid. UV: 371 (2.15), 328 (2.29), 261 (2.56). IR: 3300, 2087, 1772, 1640, 1618, 1583, 1489.
1H-NMR (CDCl3): 12.18 (s, HOꢀC(5)); 9.88 (s, HꢀC(8)); 7.64 (s, HꢀC(3)); 4.24 (s, MeO); 3.32 (t, J ¼ 6.5,
CH2(5’)); 2.48 (t, J¼6.3, CH2(2’)); 2.18 (s, MeꢀC(6)); 1.78–1.75, 1.68–1.65 (2m, CH2(3’), CH2(4’)). ESI-
MS: 749 ([2 MþNa]þ ), 364 ([MþH]þ ).
Preparation of Reduced Derivatives of Cyclopaldic Acid: 5-Hydroxy-4-(hydroxymethyl)-7-methoxy-
6-methyl-2-benzofuran-1(3H)-one (1H) and 5-Hydroxy-3-methoxy-4-methyl-6,7a-dihydro-2H-fu-
ro[2,3,4-cd][2]benzofuran-2-one (1I). Compound 1 (40 mg) dissolved in MeOH (3 ml) was reduced
with NaBH4 (80 mg) under stirring at 08. The reaction was stopped after 6 min with ice, and the soln. was
neutralized with HCl (1n) and extracted with AcOEt (3ꢂ10 ml). The org. extracts, combined and dried
(Na2SO4), were purified by TLC (CHCl3/iPrOH 80 :20) to give 1H (Rf 0.71; 11.5 mg) and 1I (Rf 0.6;
10.5 mg) as homogeneous amorphous solids.
Data of 1H. UV: 265 (2.88). IR: 3362, 2912, 2857, 1736. 1H-NMR (CDCl3): 5.06 (s, CH2(3)); 4.67 (s,
CH2(8)); 3.93 (s, MeO); 2.10 (s, MeꢀC(6)). ESI-MS: 225 ([MþH]þ ), 223 ([MꢀH]ꢀ ).
Data of 1I. UV: 2.59 (3.52). IR: 3351, 2923, 2851, 1722, 1599. 1H-NMR (CDCl3): 6.07 (br. s, HꢀC(3));
4.85 (dd, J¼13.4, 2.6, HaꢀC(8); 4.76 (dd, J¼13.4, 2.6, HbꢀC(8)); 3.95 (s, MeO); 2.11 (s, MeꢀC(6)). ESI-
MS: 223 ([MþH]þ ), 221 ([MꢀH]ꢀ ).
Preparation of Seiridin Derivatives 2A–2C. 2’-O-Acetyl-, 3,4-dihydro-, and 2’-oxoseiridin (2A–2C,
resp.) were prepared, according to the procedures previously reported [11][20].
Preparation of Sphaeropsidin A Derivatives 4A and 4B. The 6-O-acetylsphaeropsidin A (4A) and
8,14-methylensphaeropsidin A methyl ester (4B) were prepared as described in [21].
Preparation of Sphaeropsidin B (¼(7b,13a)-6,7,9-Trihydroxy-6,20-epoxypimara-8(14),15-dien-20-
one; 4C). Fifteen mg of 4 was dissolved in MeOH (2 ml) and reduced with NaBH4 (17.3 mg) under
stirring at r.t. The reaction was stopped after 2 h by adding ice and HCl (1n), adjusting the pH to
neutrality. The soln. was extracted with AcOEt (3ꢂ10 ml). The org. extracts, combined and dried
(Na2SO4), were purified by prep. TLC (hexane/acetone 7:3) to yield 4C. Homogeneous oily compound
(Rf 0.3; 10.3 mg) which was identified by 1H-NMR and ESI-MS data identical to those reported in [22].
Preparation of (6b,13a)-9-Hydroxy-7,20-dioxo-6,20-epoxypimara-8(14),15-dien-6-yl 6-Azidopenta-
noate (4E). To 4 (31 mg) dissolved in anh. CH2Cl2 (3 ml) and pyridine (75 ml) were added under stirring
DCC (96 mg) and 5-azidopentanoic acid (90 ml). The reaction was left at 08 for 1 h and then for 2 h at r.t.
The reaction was stopped by evaporation under N2. The crude residue (175 mg) was purified by CC
(CHCl3/iPrOH 97:3) to yield 4E (Rf 0.75; 19.3 mg). Homogeneous amorphous solid. UV: 294 (2.05), 247
(3.09). IR: 3483, 2939, 2873, 2093, 1771, 1725, 1632, 1456. 1H-NMR (CDCl3): 6.54 (d, J¼1.2, HꢀC(14));
5.80 (dd, J¼17.5, 10.5, HꢀC(15)); 5.06 (d, J¼17.5, HaꢀC(16)); 5.06 (d, J¼10.5, HbꢀC(16)); 3.33 (t, J¼6.5,
CH2(5’)); 2.87 (s, HꢀC(5)); 2.63–2.50 (m, CH2(4’), CH2(3’)); 2.22 (br. d, J¼13.2, HaꢀC(1)); 1.90 (dt, J¼
14.1, 3.4, HaꢀC(3); 1.84–1.80 (m, CH2(2’), HaꢀC(2), HaꢀC(12)); 1.75–1.64 (m, HbꢀC(1), HbꢀC(2),
HbꢀC(3), HaꢀC(11), HbꢀC(12)); 1.25 (m, HbꢀC(11); 1.19 (s, Me(18)); 1.10 (s, Me(19)); 1.09 (s, Me(17)).
ESI-MS: 965 ([2 MþNa]þ ), 494 ([MþNa]þ ), 470 ([MꢀH]ꢀ ).
(7b,13a)-6,7,9-Trihydroxy-6,7-O,O-isopropylidene-6,20-epoxypimara-8(14),15-dien-20-one (4D). To
compound 4 (20 mg) dissolved in anh. acetone (20 ml) was added under stirring anh. CuSO4 (400 mg).
The soln. was heated under reflux. After 24 h, the reaction was stopped by filtration, and the clear soln.
was evaporated under reduced pressure. The residue (20 mg) was purified by TLC (CHCl3/iPrOH 95 :5)
to yield 4D (Rf 0.54; 3 mg). Homogeneous amorphous solid. UV: 246 (3.42). IR: 3300, 1744, 1709, 1621,
1
1462, 1454, 1374, 1220. H-NMR (CDCl3): 6.86 (s, HꢀC(7)); 5.82 (dd, J¼17.4, 11.3, HꢀC(15)); 5.19 (s,
HꢀC(14)); 5.09 (d, J¼17.4, HaꢀC(16)); 5.07 (d, J¼11.3, HbꢀC(16)); 2.72 (s, HꢀC(5)); 2.23 (br. d, J¼9.7,
HaꢀC(1)); 1.89 (br. d, J¼15.6, HaꢀC(3)); 1.83 (m, HbꢀC(3), HaꢀC(12)); 1.60 (m, HbꢀC(1), CH2(2),
HbꢀC(12)); 1.60, 1.25 (2s, Me2C); 1.37 (br. d, J¼12.2, HaꢀC(11)); 1.29–1.25 (m, HbꢀC(11)); 1.20 (s,
Me(18)); 1.19 (s, Me(19)); 1.09 (s, Me(17)). ESI-MS: 411 ([MþNa]þ ), 369 ([MþNa-C3H6]þ ).
Insects. Ae. aegypti used in K&D (¼ Klun&Debboun) bioassays were from a laboratory colony
maintained using standard procedures [26] since 1952 at the Mosquito and Fly Research Unit at the
Center for Medical, Agricultural and Veterinary Entomology, United States Department of Agriculture,
Agriculture Research Service, Gainesville, Florida. We received the eggs and stored these in our
laboratory (Biological Field Station, The University of Mississippi, Abbeville, MS 38601) until needed.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1249
Mosquitoes were reared to the adult stage by feeding the larvae on a larval diet of 2% slurry of 3 :2 Beef
Liver powder (Now Foods, Bloomingdale, Illinois) and Brewerꢀs yeast (Lewis Laboratories Ltd.,
Westport, CT). The eggs were hatched by placing a piece of a paper towel with eggs in a cup filled with
100 ml of deionized H2O containing a small quantity of larval diet and maintained under vacuum (ca.
1 h). Larvae were removed from vacuum and held overnight in the cup. These larvae were then
transferred into 500-ml cups (ca. 50–100 larvae per cup) filled with H2O. Larval diet was added every day
until pupation, and the mosquitoes were kept in an environment controlled room. Both the larvae and
adults were maintained 27ꢁ28 and 70ꢁ5% rel. humidity in a photoperiod regimen of 12 :12 (L :D) h.
The adults were fed on cotton pads moistened with 10% sucrose soln. placed on the top of screens of 4-l
cages. Seven- to 14-day-old mated females used in these bioassays were deprived of sucrose for 24 h prior
to the test; but they had free access to water-soaked cotton.
Mosquito-Biting Bioassays. Bioassays were conducted using a six-celled in vitro K&D module
bioassay system developed by Klun et al. [27] for quant. evaluation of biting-deterrent properties of
candidate compounds. Here we use feeding deterrent in the sense of Dethier et al. (1960) [28], i.e., a
chemical that inhibits feeding when present in a place where the insects feed in its absence. This is
contrasted by repellent, which is a chemical that causes insects to make oriented movement away from its
source. The K&D system consists of a six-well reservoir with each of the 4ꢂ3-cm wells containing 6 ml of
feeding soln. As described by Ali et al. [29], we used CPDA 1þATP soln. instead of human blood.
CPDA-1 and ATP preparations were freshly made on the day of the test and contained a red dye that
allowed for identification of mosquitoes that had fed on the soln. (see below). DEET (97% purity N,N-
diethyl-meta-toluamide) was obtained from SigmaꢀAldrich and used as a positive control. Molecular
biology-grade EtOH was obtained from Fisher Scientific Chemical Co. (Fairlawn, NJ 07410). All
compounds and DEET treatments were prepared in EtOH. All the compounds and the positive control
DEET were tested at a concentration of 25 nmol/cm2 except in doseꢀresponse study. The stock solns.
were kept in a refrigerator at 3–48. Treatments were prepared fresh at the time of bioassay.
During the bioassay, temp. of the soln. in the reservoirs, covered with a collagen membrane, was
maintained at 37.58 by circulating water through the reservoir with a temp.-controlled circulatory bath.
This CPDA-1þATP soln. membrane unit simulated a human host for mosquito feeding. The test
compounds and controls were randomly applied to six 4ꢂ3-cm marked portions of Nylon organdy strip,
which was positioned over the six, membrane-covered wells. A Teflon separator was placed between the
treated cloth and the six-celled module. A six-celled K&D module containing five females per cell was
positioned over the six wells, trap doors were opened, and mosquitoes were allowed access at the module
for a 3-min period, after which they were collected back into the module. Mosquitoes were squashed and
the presence of red dye (or not) in the gut was used as an indicator of feeding. A replicate consisted of six
treatments: four test compounds, DEET (a standard bite-deterrent compound), and 95% EtOH as
solvent control. Five replicates were conducted per day using new batches of mosquitoes for each. Total
15 replications were conducted for each treatment. Bioassays were conducted between 13 :00 and
16 :00 h.
Larval Bioassays. Bioassays were conducted by using the bioassay system described by Pridgeon
et al. [30] to determine the larvicidal activity of isolated fungal toxins activity against 1-d old Ae. aegypti
larvae. In brief, the eggs were hatched under vacuum (1 h) by placing a piece of a paper towel with eggs in
a cup filled with 100 ml of deionized water containing a small quantity of larval diet. Larvae were
removed from vacuum and held overnight in the cup in a temp.-controlled chamber maintained at 27ꢁ28
and 70ꢁ5% rel. humidity at a photoperiod regimen of 12 :12 (light:dark) h. Five 1-d-old first instar Ae.
aegypti were added to each well of 24-well plates placed on illuminated light box by using a disposable
22.5-cm Pasteur pipette with a droplet of water. Fifty ml of larval diet (2% slurry of 3 :2 Beef Liver powder
(Now Foods, Bloomingdale, Illinois) and Brewerꢀs yeast (Lewis Laboratories Ltd., Westport, CT)) were
added to each well by using a Finnpipette stepper (Thermo Fisher, FI-Vantaa). All chemicals to be tested
were diluted in DMSO. Eleven ml of the test chemical was added to the labeled wells, and, in control
treatments, 11 ml of DMSO alone were added. Each well had a total volume of 1.1 ml. After the
treatment, the plates were swirled in clockwise and counterclockwise motions, and front and back, and
side to side five times to ensure even mixing of the chemicals. Larval mortality was recorded 24-, 48- and
72-h posttreatment. Larvae that showed no movement in the well after manual disturbance of water by a

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
pipette tip were recorded as dead. A series of dosages was used in each treatment to obtain a range of
mortality. Treatments were repeated 15 times in each extract. Permethrin used as positive control was
obtained from SigmaꢀAldrich.
Data Analyses. Proportion not biting (PNB) was calculated using the procedure described by Ali and
co-workers [29]. Since the K&D module bioassay system can handle only four treatments along with
negative and positive controls, to make direct comparisons among more than four test compounds and to
compensate for variation in overall response among replicates, repellency was quantified as Biting-
Deterrence Index (BDI). The BDI values were calculated using the following formula:
ꢀ
ꢁ
Â
Ã
PNBi;j;kꢀPNBc;j;k
PNBd;j;kꢀPNBc;j;k
BDIi;j;k
¼
where PNBi,j,k denotes the proportion of females not biting test compound i for replication j and day k
(i¼1–4, j¼1–5, k¼1–3), PNBc,j,k denotes the proportion of females not biting the solvent control for
replication j and day k (j¼1–5, k¼1–3), and PNBd,j,k denotes the proportion of females not biting in
response to DEET (positive control) for replication j and day k (j¼1–5, k¼1–3). This formula makes
an adjustment for inter-day variation in response and incorporates information from the solvent control
as well as the positive control. A BDI value of 0 indicates an effect similar to EtOH, while a value
significantly greater than 0 indicates biting-deterrent effect relative to EtOH. BDI Values not
significantly different from 1 are statistically similar to DEET. BDI Values were analyzed using SAS Proc
ANOVA (SAS Institute, 2007), and means were separated using the RyanꢀEinotꢀGabrielꢀWelsch
Multiple Range Test. The LD50 values for larvicidal data were calculated by using SAS, Proc Probit.
Control mortality was corrected by using Abbottꢀs formula.
REFERENCES
[1] D. F. Farias, M. G. Cavalheiro, S. M. Viana, G. P. G. De Lima, C. B. Da Rocha-Bezerra, N. M. P. S.
Ricardo, A. F. U. Carvalho, J. Am. Mosquito Control Assoc. 2009, 25, 386.
[2] D. Hoel, J. W. Pridgeon, U. R. Bernier, K. Chauhan, K. Meepagala, C. L. Cantrell, Wing Beats 2010,
21(1), 29.
[3] C. E. Schreck, G. A. Mount, D. A. Carlson, J. Med. Entomol. 1982, 19, 143.
[4] A. Evidente, in ꢃComprehensive Bioactive Natural Productsꢀ, Ed. V. K. Gupta, Studium Press LLC,
Houston, 2010, Vol. 1, pp. 333.
[5] S. W. Carter, Pestic. Sci. 1989, 27, 361.
[6] F. E. Dayan, C. L. Cantrell, S. O. Duke, Bioorg. Med. Chem. 2009, 14, 4022.
[7] D. R. Bissinger, C. S. Apperson, D. E. Sonenshine, D. W. Watson, R. M. Roe, Exp. Appl. Acarol.
2009, 48, 239.
[8] B. W. Bissinger, R. M. Roe, Pestic. Biochem. Physiol. 2010, 96, 63.
[9] A. Graniti, L. Sparapano, A. Evidente, Plant Pathol. 1992, 41, 563.
[10] L. Sparapano, A. Evidente, A. Ballio, A. Graniti, G. Randazzo, Experientia 1986, 42, 627.
[11] A. Evidente, G. Randazzo, A. Ballio, J. Nat. Prod. 1986, 49, 593.
[12] A. Evidente, L. Sparapano, A. Motta, F. Giordano, O. Fierro, S. Frisullo, Phytochemistry 1996, 42,
1541.
[13] A. Evidente, A. Andolfi, A. Cimmino, A. M. Abouzeid, in ꢃSustainable Agriculture: Technology,
Planning and Managementꢀ, Eds. A. Salazar, I. Rios, Nova Science Publishers, New York, 2010,
pp. 177.
[14] A. Evidente, A. Berestetskiy, A. Cimmino, A. Tuzi, S. Superchi, D. Melck, A. Andolfi, J. Agric. Food
Chem. 2009, 57, 11168.
[15] A. Evidente, V. Venturi, M. Masi, G. Degrassi, A. Cimmino, L. Maddau, A. Andolfi, J. Nat. Prod.
2011, 74, 2520.
[16] J. H. Birkinshaw, H. Raistrick, D. J. Ross, C. E. Stickings, Biochem. J. 1952, 50, 610.
[17] L. Sparapano, A. Evidente, Nat. Toxins 1995, 3, 156.
[18] G. Del Sorbo, A. Evidente, F. Scala, Nat. Toxins 1994, 2, 136.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1251
[19] A. Bowers, T. G. Halsall, E. R. H. Jones, A. A. Lemin, J. Chem. Soc. 1953, 2548.
[20] A. Evidente, A. Andolfi, L. DꢀApice, D. Iannelli, F. Scala, Nat. Toxins 1997, 5, 14.
[21] L. Sparapano, G. Bruno, O. Fierro, A. Evidente, Phytochemistry 2004, 65, 189.
[22] A. Evidente, L. Sparapano, O. Fierro, G. Bruno, F. Giordano, A. Motta, Phytochemistry 1997, 45,
705.
[23] K. A. Hassal, ꢃBiochemistry and Uses of Pesticidesꢀ, Verlag Chemie, Weinheim, 1990, pp. 58, 72,
304, 429, and 497.
[24] L. Sparapano, A. Evidente, Nat. Toxins 1995, 3, 166.
[25] A. Ballio, A. Evidente, A. Graniti, G. Randazzo, L. Sparapano, Phytochemistry 1988, 27, 3117.
[26] J. W. Pridgeon, K. Meepagala, J. J. Becnel, G. G. Clarck, R. M. Pereira, K. J. Linthicum, J. Med.
Entomol. 2007, 44, 263.
[27] J. A. Klun, M. Kramer, M. Debboun, J. Am. Mosquito Control Assoc. 2005, 21, 64.
[28] V. G. Dethier, B. L. Browne, C. N. Smith, J. Econ. Entomol. 1960, 53, 134.
[29] A. Ali, C. L. Cantrell, U. R. Bernier, S. O. Duke, J. C. Schneider, I. Khan, J. Med. Entomol. 2012, 49,
1370.
[30] J. W. Pridgeon, J. J. Becnel, G. G. Clark, K. J. Linthicum, J. Med. Entomol. 2009, 46, 335.
Received October 22, 2012