A New Imidazole Alkaloid and Other Constituents from Pilocarpus
grandiflorus and their Antifungal Activity

Rejane C. de Souzaa, João B. Fernandesa, Paulo C. Vieiraa, M. Fátima das G. F. da Silvaa,
Marizete F. P. Godoyb, Fernando C. Pagnoccab, Odair C. Buenob, M. José A. Heblingb,
and José R. Piranic

a Departamento de Quı́mica, Universidade Federal de São Carlos, Caixa Postal 676, São Carlos, SP,
Brazil

b Centro de Estudos de Insetos Sociais, Universidade Estadual Paulista, Rio Claro, SP, Brazil
c Departamento de Botanica, Instituto de Biociencias, Universidade de São Paulo, São Paulo – SP,

Brazil

Reprint requests to Prof. Dr. João Batista Fernandes. Fax: +55-16-3351-8350.
E-mail: djbf@power.ufscar.br

Z. Naturforsch. 60b, 787 – 791 (2005); received September 17, 2004

The stems of Pilocarpus grandiflorus have afforded the new imidazole alkaloid 4,6-dehydro-
1,2,4,5-tetrahydro-2,5-dioxopilocarpine in addition to the 17 known compounds germanicol, β -
amiryn, ocotillone, stigmast-4-en-3-one, 3β -hydroxy-stigmast-5-en-7-one, 6β -hydroxy-stigmast-4-
en-3-one, β -sitosterol, scopoletin, 3-(1’,1’-dimethylallyl)-scopoletin, elisin, dictamine, 4-methoxy-2-
quinolone, platydesmine, syringaresinol, syringaldehyde, syringic acid and vanillic acid. Their struc-
tures were elucidated on the basis of chemical and spectroscopic evidence. The phenolic compounds
vanillic acid and syringaldehyde and the furoquinoline alkaloid platydesmine exhibited antifungal ac-
tivity against Leucoagaricus gongylophorus, the symbiotic fungus of leaf-cutting ants (Atta sexdens
rubropilosa).

Key words: Pilocarpus grandiflorus, Jaborandi, Imidazole Alkaloid,
Leucoagaricus gongylophorus, Atta sexdens rubropilosa

Introduction

Pilocarpus commonly known as “Jaborandi” is a
shrub belonging to the Rutaceae and comprises 16
neotropical species spread over tropical and subtrop-
ical America. In Brazil, the occurrence of thirteen
species [1] has been reported. This genus has long been
known as the most important source of the imidazole
alkaloid pilocarpine. Imidazole alkaloids are histamine
derivatives found only in the genus Casimiroa and Pi-
locarpus [2]. The alkaloid content in some Pilocar-
pus (0.5 – 1.0%) consists mainly of the imidazole alka-
loid pilocarpine, together with small amounts of pilo-
sine and related structures. Pilocarpine salts are valu-
able in ophthalmic practice and used in eyedrops as
miotics and for the treatment of glaucoma [3].

Leaf-cutting ants of the genera Atta and Acromyr-
mex are among the most polyphagous agricultural pests
of the South American countries. These ants cut veg-
etables for substrate of their symbiotic food fungus and
produce consistent economic damage to the harvesting
of the cultivated plants. The agricultural pest control

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practices generally involve toxic bait applications dam-
aging the environment [4]. An alternative control for
these insects can be achieved by inhibiting the growth
of their symbiotic fungus through more selective and
less toxic natural products than the baits existent in the
market.

In this paper, we report the isolation of 18 com-
pounds, the structural elucidation of a new imidazole
alkaloid and the evaluation of their activities as in-
hibitors of the growth of the symbiotic fungus Leuco-
agaricus gongylophorus of leaf-cutting ants, Atta sex-
dens rubropilosa.

Results and Discussion

In continuation of our search for compounds to be
used in the control of leaf-cutting ants [5 – 15], we
have investigated the dichloromethane extract of the
stems of P. grandiflorus leading to the isolation of a
new imidazole alkaloid (1) in addition to the known
compounds germanicol (2) [16], β -amiryn (3) [17],
ocotillone (4) [18], β -sitosterol (5) [19], stigmast-



788 R. C. de Souza et al. · Pilocarpus grandiflorus and their Antifungal Activity

4-en-3-one (6) [20], 3β -hydroxy-stigmast-5-en-7-one
(7) [21], 6β -hydroxy-stigmast-4-en-3-one (8) [22], sy-

ringaresinol (9) [23], syringaldehyde (10) [24], sy-
ringic acid (11) [25], vanillic acid (12) [26], scopoletin



R. C. de Souza et al. · Pilocarpus grandiflorus and their Antifungal Activity 789

Table 1. 1H (400 MHz) and 13C (100 MHz) NMR data of 1
(CDCl3, δ ).

No. 1H 13C
2 – 152.4
4 – 132.2
5 – 162.4
6 5.23 d (11) 112.3
7 4.76 dddd (11.5; 7; 5.4; 2) 35.7
8b 4.08 dd (9; 2) 71.7
8a 4.42 dd (9; 5.4)
10 – 177.7
11 2.67 ddd (8; 7; 5) 45.4
12b 1.38 m 19.5
12a 1.84 m
13 1.01 t (7.4) 12.2
N-CH3 3.07 s 25.9

(13) [27], 3-(1’,1’-dimethylallyl)-scopoletin (14) [28],
elisin (15) [29], dictamine (16) [30], 1-methyl-4-
methoxy-2-quinolone (17) [31] and platydesmine
(18) [32].

The known compounds were identified by compar-
ison of their physical and spectral data with those al-
ready reported in the literature. The new imidazole
alkaloid had its structure determined on the basis of
chemical and spectral evidence. The new alkaloid 1
had spectroscopic characteristics of the known imi-
dazole alkaloid pilocarpine. The HREIMS spectrum
displayed a molecular ion at m/z 238.0935 in accor-
dance with the molecular formula C11H14O4N2. The
1H NMR spectrum (Table 1) showed signals at δ =
1.01 (3H, t, J = 7.4 Hz), δ = 1.84 (1H, m) and δ =
1.38 (1H, m) indicating an ethyl group; two double-
doublets at δ = 4.42 (1H, dd, J = 9.0, 5.4 Hz) and
δ = 4.08 (1H, dd, J = 9.0, 2.0 Hz) for protons of oxy-
genated carbons at δ = 71.7 and other lactone carbon
at δ = 177.8 and the signals at δ = 2.67 (1H, ddd,
J = 8.0, 7.0, 5.0 Hz), δ = 4.76 (1H, dddd, J = 11.5,
7.0, 5.4, 2.0 Hz) indicating a pattern similar to pi-
locarpine. The 13C NMR spectrum for 1 (Table 1)
showed eleven carbons in agreement with the imi-
dazole alkaloid structure. The signals at δ = 177.7,
162.4, 152.4, 132.2 and 112.3 suggesting a change in
the structure of pilocarpine with two additional car-
bonyl groups and a double bond. The absence of a
signal in the aromatic region and the signal at δ =
5.23 (1H, d, 11.0 Hz) in addition to the two carbonyl
observed in the 13C NMR spectrum suggests a mod-
ification of the imidazole ring. This is supported by
HMBC spectrum (Table 2), which showed a correla-
tion of the N-methyl group to the carbonyl C-2 and
C-9 at δ = 152.4 (3J) and 162.4 (3J), and from H-6 to

Table 2. Correlations in the HMBC and G-NOESY two-
dimensional observed for compound 1.

H — HMBC — G-NOESY
2JC−H

3JC−H H

6 4 5; 8; 11 8b; N-CH3
7 6; 11 4; 10 8a; 8b; 11
8a 6 7; 8b; 11
8b 7 6; 10; 11 6; 7; 8a
11 7; 12 6; 13 7; 8a; 12a
12a 11; 13 7; 10
12b 11; 13 7; 10
13 12 11
N-CH3 2; 4 6

Table 3. Evaluation of the growth inhibitory activity of
dichloromethane extract and compounds of P. grandiflorus.

Extract/compounds Amount Growth inhibition
(µg ml−1) (%)

Dichloromethane extract 1000 100
Germanicol (2) 100 0
β -Amyrin (3) 100 0
β -Sitosterol (5) 100 0
Stigmast-4-en-3-one (6) 100 0
3β -Hydroxystigmast-5-en-7-one (7) 60 20
Syringaresinol (9) 50 0
Syringaldehyde (10) 50 80
Vanillic acid (12) 50 80
Dictamine (16) 40 40
1-Methyl-4-methoxy-2-quinolone (17) 60 0
Platydesmine (18) 50 80

the C-4 and C-5 at δ = 132.2 (3J) and 162.4 (3J). In
the HSQC spectrum, the carbon at δ = 35.7 was cor-
related with the signal at δ = 4.76. This value can be
explained by anisotropic effects of the carbonyl group
and the double bond. The G-NOESY experiments (Ta-
ble 3) showed correlations of H-6 with the N-3-methyl
group and H-8, requiring a cis configuration for the
double bond.

The dichloromethane extract of the stems as well as
fractions of P. grandiflorus showed strong inhibitory
activity on the growth of L. gongylophorus. Among
the 18 isolated compounds, the phenolic compounds
syringaldehyde (10) and vanillic acid (12) and the furo-
quinoline alkaloid platydesmine (18) exhibited higher
inhibition of the fungal growth at 50 µg ml−1.

Antifungal activities have been described for pheno-
lic compounds against Cladosporium herbarum [33],
Aspergillus flavus and A. parasiticus [34], Geotrichum
candidum, Coriolus versicolor, Phanerochaete chryso-
sporium and Mycelia sterilia [35].

Grayer and Harbone [36] suggested that furoquino-
line alkaloids might also play a role in the defense of
plants against potentially pathogenic fungi. The result



790 R. C. de Souza et al. · Pilocarpus grandiflorus and their Antifungal Activity

of our search confirmed the antifungal activity for this
compound against L. gongylophorus.

Experimental Section
General experimental procedures

1H NMR (400 MHz) and 13C NMR (100 MHz): Bruker
ARX-400 spectrometer, in CDCl3 containing TMS as int.
standard. EIMS (70 eV): VG Platform II instrument and
Shimadzu QP5000. HRMS: Autospec-Micromass EBE. CC
were performed on silica gel 60 H (0.04 – 0.005 mm) and
sı́lica gel (0.063 – 0.2 mm), respectively. Analytical TLC
were performed on precoated Merck F254 sı́lica gel plates
and visualized on UV (254 – 360 nm) and by spraying with
vaniline-H2SO4.

Plant material

The stems of P. grandiflorus was collected in Poços
D’antas, municı́pio de Murici, Alagoas state, Brazil in 1993.
Identification of the plant was done by Professor Dr. José R.
Pirani, and a voucher is deposited in the Herbarium of São
Paulo University, Biosciences Institute.

Extraction and isolation

Stems of P. grandiflorus were dried and powdered (5.2 kg)
and extracted with hexane, dichloromethane and methanol
respectively. The dichloromethane extract (11.7 g) was sub-
mitted to vacuum column chromatography over silica gel us-
ing hexane, dichloromethane, ethyl acetate and methanol as
eluents.

The dichloromethane fr. (3.8 g) was subjected to Column
Chromatography over silica gel using dichloromethane, ethyl
acetate and methanol at different rations of increasing po-
larity, furnishing 23 fr. Fr. 4-6 were purified by column Lo-
bar Si60 using Hex-EtOAc-MeOH as eluents to yield 2 – 3
(16.3 mg), 5 (107.9 mg), 6 (16.0 mg) and 7 (7.0 mg). Fr. 22
was subjected to CC using gradient elution with Hex-AcOEt
to yield the compounds 10 (4.5 mg), 13 (5.0 mg) and 14
(3.0 mg).

The ethyl acetate fr. (11 g) was subjected to chromatog-
raphy on a silica gel column, with dichloromethane, ethyl
acetate and methanol as eluents, furnishing 17 fr. Fr. 2 was
subjected to CC using gradient elution with Hex-AcOEt to
yield the compounds 4 (1 mg), 8 (0.7 mg), 15 (1.3 mg), 16
(20.5 mg) and 17 (5.6 mg). The Fr. 3 was purified by CC us-
ing gradient elution with Hex-AcOEt to give the compounds
16 (5.0 mg) and 18 (8.3 mg). Fr. 13 was subjected to CC
using gradient elution with CH2Cl2-AcOEt-MeOH furnish-
ing 5 fr. These fractions were further chromatographed by
prep. TLC (silica gel, Hex-EtOAc, 8:2) to give 16 (6.0 mg),
9 (42.5 mg), 12 (7.7 mg), 10 (5.5 mg), 11 (6.0 mg) and 1
(6.0 mg).

Fungicidal assay

The fungus L. gongylophorus Singer was isolated from
Atta sexdens rubropilosa Forel nest, kept in culture media
and the fungicidal activity performed according to estab-
lished protocols [37].

4,6-Dehydro-1,2,4,5-tetrahydro-2,5-dioxopilocarpin (1)

Yellow oil. [α]25
D +19.6 (c 0.005, CHCl3). – UV (MeOH):

λmax(lgε) = 284 nm (3.98), 242 (3.46). – IR (film): ν̃ =
3444, 2967, 2932, 1767, 1731, 1604, 1442, 1382, 1175,
1121, 1065, 1019, 704, 641 cm−1. – 1H NMR and 13C NMR:
Table 1. – HRMS (EI, 70 eV): found 238.0938 [M+],
C11H14N2O4 requires 238.0954. – MS (EI, 70 eV): m/z
(%) = 238 (47) [M+], 209 (20), 192 (56), 180 (93), 165 (53),
151 (67), 139 (35), 126 (60), 94 (100), 81 (62), 68 (83).

Acknowledgements

The authors are grateful to Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP), Conselho
Nacional de Desenvolvimento Cientı́fico e Tecnológico-
PRONEX (CNPq), and Financiadora de Estudos e Projetos
(FINEP) for financial support.

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