A new cyclopeptide and other constituents from the leaves of Zanthoxylum rigidum Humb. & Bonpl. ex Willd. (Rutaceae)

Article abstract of DOI:10.1002/hlca.201100476

A new cyclopeptide, cyclozanthoxylane A (1), the lignans cis- and trans-methylpluviatolide, the flavonoid isoquercitrin, along with a mixture of benzoic and cinnamic acid derivatives, were isolated from the MeOH extract of the leaves of Zanthoxylum rigidum (Rutaceae). The structures of the compounds were determined on the basis of 1D- and 2D-NMR, and MS analysis. It is the first time that a natural cyclic peptide has been isolated from the genus Zanthoxylum.

Full text of DOI:10.1002/hlca.201100476

Helvetica Chimica Acta – Vol. 95 (2012)
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A New Cyclopeptide and Other Constituents from the Leaves of
Zanthoxylum rigidum Humb. & Bonpl. ex Willd. (Rutaceae)
by Tereza Auxiliadora Nascimento Ribeiro*a), Letꢀcia Ricette da Silvaa),
Paulo Teixeira de Sousa Ju´niorb), Rosane Nora Castroa), and Ma´rio Geraldo de Carvalhoa)c)
a
ˆ
) Departamento de Quꢀmica, Instituto de Ciencias Exatas, Universidade Federal Rural do Rio de
´
Janeiro, BR 465, Km 07, CEP: 23890-000-Seropedica-RJ, Brasil
(phone/fax: þ 55-21-26822807; e-mail:terezaribeiro20@gmail.com)
b
c
ˆ
) Departamento de Quꢀmica, Instituto de Ciencias Exatas e da Terra, Universidade Federal de Mato
ˆ
´
Grosso, Avenida Fernando Correa n8 2367, 78060-900 Cuiaba-MT, Brasil
´
) Nucleo de Pesquisas de Produtos Naturais, Bloco H, CCS, Universidade Federal do Rio de Janeiro,
˜
Ilha do Fundao, Rio de Janeiro-RJ, Brasil
A new cyclopeptide, cyclozanthoxylane A (1), the lignans cis- and trans-methylpluviatolide, the
flavonoid isoquercitrin, along with a mixture of benzoic and cinnamic acid derivatives, were isolated from
the MeOH extract of the leaves of Zanthoxylum rigidum (Rutaceae). The structures of the compounds
were determined on the basis of 1D- and 2D-NMR, and MS analysis. It is the first time that a natural
cyclic peptide has been isolated from the genus Zanthoxylum.
Introduction. – Zanthoxylum genus (Rutaceae) comprise more than 200 species
widely distributed around the world. This genus has been characterized by the presence
of alkaloids, coumarins, flavonoids, lignans, and mono-, sesqui-, and triterpenes [1].
Many pharmacological activities such as antiplasmodic [2], anti-HIV [3], anti-
inflammatory [4], and antihelmintic [5] properties of Zanthoxylum species have been
reported. Zanthoxylum rigidum is populary known as ꢁmamica de porca (pigꢀs nipple)ꢂ,
or ꢁmamica de cadela (sowꢂs nipple)ꢂ. There is only one phytochemical study reported
on Zanthoxylum rigidum, in which the isolation and identification of lupeol,
campesterol, stigmasterol, sitosterol, sucrose, the flavonoid hesperidine, and the
alkaloids N-methylatanine and 6-acetonyldihydrochelerythrine from the roots were
described [1]. This is the first phytochemical study of the extracts from the leaves of Z.
rigidum, describing the isolation and structure determination of a new cyclic peptide,
named cyclozanthoxylane A (1), two lignans, cis- and trans-methylpluviatolide, the
flavonoid isoquercitrin, along with a mixture of benzoic and cinnamic acid derivatives,
methyl (E)-4-hydroxycinnamate, methyl (Z)-4-hydroxycinnamate, methyl 4-hydroxy-
benzoate, and the methyl 4-hydroxyphenylacetate. Cyclic peptides are widely
distributed in many higher plants, exhibiting many biological activities, including some
of the mentioned above, and they have often been used as models for studies of
structural features of proteins [6][7]. A small number of cyclic peptides from Rutaceae
plants such as evolidine and citrusins from Citrus species [8], and clausenain I and
clausenain B from Clausena anisum-olens [6][9] have been reported. This is the first
time that a natural cyclic peptide has been isolated from the Zanthoxylum genus.
ꢃ 2012 Verlag Helvetica Chimica Acta AG, Zꢄrich

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Helvetica Chimica Acta – Vol. 95 (2012)
Besides other compounds, the structure of this new peptide was elucidated by 1H- and
13C-NMR and MS analysis, and comparison with literature data.
Results and Discussion. – The chromatographic fractionation of the MeOH extract
of the leaves of Z. rigidum afforded methyl (E)-4-hydroxycinnamate [10], methyl (Z)-
4-hydroxycinnamate [10], methyl 4-hydroxybenzoate [11], methyl 4-hydroxyphenyl-
acetate [12], cis- and trans-methylpluviatolide [13], and isoquercitrin [14], besides a
new cyclopeptide, named cyclozanthoxylane A (1). The known compounds were
identified by GC/MS, 1D- and 2D-NMR spectra, and comparison with literature data
[10 – 14].
Compound 1 was isolated as white amorphous powder with a molecular formula of
C23H33N5O5, on the basis of its quasimolecular ion detected by HR-ESI-MS (positive-
ion mode) at m/z 460.2557 ([M þ H]þ; calc. for C23H34N5O5þ, 460.2554), corresponding
to ten degrees of unsaturation. The IR absorption bands were characteristic of amine
(3306 cmꢀ1) and amide C¼O (1652 cmꢀ1) groups. The DEPTQ-NMR spectrum
exhibited signals of five amide CO groups (d(C) 173.2, 172.5, 171.9, 170.5, 169.6),
and four CH groups d(C) (55.6, 54.2, 50.1, 48.4) and one CH2 group d(C) (43.8), which
Table. NMR Data of Cyclozanthoxylane A (1). Recorded in (D6)DMSO; d in ppm, J in Hz.
Position
d(H)
d(C)
HMBC
Gly
Ala1
Leu
CH2(a)
3.89 (dd, J ¼ 14.4, 6.4),
3.24 (dd, J ¼ 14.4, 5.2)
8.67 (t, J ¼ 5.6)
43.8 (t)
CO(Ala1), CO(Gly)
NH
CO
CO(Ala1)
169.6 (s)
HꢀC(a)
Me(b)
NH
4.19 – 4.24 (m)
1.20 (d, J ¼ 7.2)
7.93 – 7.96 (m)
48.4 (d)
18.2 (q)
CO(Ala1), CO(Leu)
CO(Ala1), CH(a)(Ala1)
CO(Ala1), CO(Leu)
CO
173.2 (s)
HꢀC(a)
CH2(b)
HꢀC(g)
Me(d)
Me(d’)
NH
3.98 – 4.03 (m)
1.68 – 1.75 (m), 1.47 – 1.53 (m)
1.56 – 1.61 (m)
54.2 (d)
40.0 (t)
25.0 (d)
23.2 (q)
22.0 (q)
CO(Leu)
CO(Leu)
0.91 (d, J ¼ 6.0)
CH2(b)(Leu)
CH2(b)(Leu)
CO(Ala2)
0.86 (d, J ¼ 6.4)
8.02 (d, J ¼ 7.6)
CO
172.5 (s)
Phe
HꢀC(a)
CH2(b)
4.34 – 4.40 (m)
55.6 (d)
37.2 (t)
CO(Phe), CO(Gly)
CO(Phe), CH(a)(Phe),
C(1’), C(2’), C(6’)
3.12 (dd, J ¼ 14.0, 5.2),
2.85 (dd, J ¼ 14.2, 10.0)
1’
138.2 (s)
129.3 (d)
128.6 (d)
126.8 (d)
2’, 6’
3’, 5’
4’
NH
CO
7.18 – 7.30 (m)
7.18 – 7.30 (m)
7.18 – 7.30 (m)
8.21 (d, J ¼ 8.8)
CO(Gly)
170.5 (s)
Ala2
HꢀC(a)
Me(b)
NH
4.19 – 4.24 (m)
1.31 (d, J ¼ 7.2)
7.93 – 7.96 (m)
50.1 (d)
17.5 (q)
CO(Ala2), CO(Phe)
CO(Ala2), CH(a)(Ala2)
CO(Phe)
CO
171.9 (s)

Helvetica Chimica Acta – Vol. 95 (2012)
937
are compatible to C(a) of a-amino acids. The 1H-NMR spectrum (Table) displayed five
signals at d(H) 8.67 (t, 1 H), 8.21 (d, 1 H), 8.02 (d, 1 H), and a multiplet at 7.93 – 7.96
(2 H) that was identified as two doublets according the TOCSY experiment. The
HMQC spectrum analysis led to the assignment of these signals to the amide NH H-
atoms, compatible with a cyclic system containing five amino acids, including one
glycine, as described above. At higher field, four signals corresponding to Me groups
1
were present. The analysis of H,1H-COSY and selective TOCSY (selecting the HꢀN
signals) spectra allowed identification of the coupling signals of a glycine (Gly), two
alanine (Ala1 and Ala2), one leucine (Leu), and one phenylalanine (Phe) (see the
Table). The following IMPACT-HMBC correlations of the five CO group chemical
shifts were observed: 169.6 (Gly) with NH(Phe) and NCH2(Gly); 170.5 (Phe) with
NH(Ala2) and CH2(Phe); 171.9 (Ala2) with NH(Leu), NCH(Ala2) and Me(Ala2);
172.5 (Leu) with NH(Ala1); 173.2 (Ala1) with NH(Gly), NCH2(Gly), NCH(Ala1), and
Me(Ala1). Additional analysis of HMBC and NOESY spectra (Table and Fig.) allowed
us to propose the sequence of Phe-Gly-Ala1-Leu-Ala2 in the cyclic system. The detailed
MS analysis (Scheme) was used to confirm the structure with this amino acid sequence.
1
The Table contains the complete H- and 13C-NMR assignments of compound 1 that
was elucidated as cyclo(-Phe-Gly-Ala1-Leu-Ala2-) and named cyclozanthoxylane A.
The absolute configurations of the amino acids present in 1 were determined by the
adapted Marfey method [15][16]. HPLC analysis of hydrolyzated derivatives indicated
four types of amino acid present in the mixture, which were identified as glycine, l-
alanine, l-phenylalanine, and l-leucine by comparation with the derivatives of
authentic samples.
Scheme. Proposed Fragments for the Principal Peaks Detected in HR-ESI-MS of 1

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Helvetica Chimica Acta – Vol. 95 (2012)
Figure. Key NOESY correlations of 1
Experimental Part
General. TLC: silica gel plates 60 F 254 (Whatman). Column chromatography (CC): silica gel (SiO2;
73 – 230 mesh; Silicycle), Sephadex LH-20 (SigmaꢀAldrich). HPLC: Shimadzu Prominence LC-20AT,
equipped with a PDA (photodiode array) detector, RP-18 (4.6 ꢁ 250 mm, 5 mm; Thermo); tR in min. UV
Spectra: (l in nm). IR Spectra: Vertex-70 FT-IT; in KBr discs; ˜n in cmꢀ1. 1D- and 2D-NMR spectra:
Bruker Avance II 400 MHz; d in ppm, with the residual solvent peak as the internal standard, J in Hz.
ESI-MS: QP2010 Plus Shimadzu. HR-ESI-MS (positive-ion mode): Bruker Daltonics, in m/z (rel. %).
´
Plant Material. Leaves of Zanthoxylum rigidum were collected in Pocone, Mato Grosso State, Brazil,
in June 2008. A voucher specimen (No. 38648) is deposited with the herbarium of Universidade Federal
de Mato Grosso, Mato Grosso state, Brazil.
Extraction and Isolation. Dried and powered leaves of Z. rigidum (1.3 kg) were extracted with
hexane and MeOH using a Soxhlet apparatus for 10 h. The extracts were concentrated under vacuum to
furnish the residues from hexane (46.6 g) and MeOH (205.9 g). Part of the MeOH extract (100.0 g) was
dissolved in MeOH/H2O 7:3 and partitioned with hexane, CHCl3, and AcOEt to yield the fractions
ZRFH (35.8 g), ZRFC (1.7 g), ZRFE (3.1 g), and ZRFM (57.1 g). Fr. ZRFC (1.5 g) was subjected to CC
(SiO2; gradient CH2Cl2/AcOEt and AcOEt/MeOH): Fractions A – J. Fr. C (80 mg) was submitted to CC
(SiO2; gradient CH2Cl2/AcOEt): Frs. C1 – C8. Fr. C3 (30 mg) was fractioned by CC (SiO2; gradient
CH2Cl2/MeOH): mixture (6 mg) of methyl 4-hydroxybenzoate (3.91%), methyl (E)-4-hydroxycinnamate
(87.51%), methyl (Z)-4-hydroxycinnamate (6.55%), and methyl 4- hydroxyphenylacetate (2.01%). Fr. C5
(5 mg) was submitted to CC (SiO2; CHCl3/MeOH 19 :1): mixture (3 mg) of cis- (16.38%) and trans-
methylpluviatolide (83.61%). Fr. H (526 mg) was submitted to CC (SiO2; gradient CH2Cl2/MeOH):
cyclozanthoxylane A (1; 4 mg). Fr. ZRFE (1.8 g) was subjected to CC (SiO2; gradient CHCl3/AcOEt and
AcOEt/MeOH): Frs. 1 – 8. Fr. 4 (523 mg) was subjected to CC (SiO2; gradient CHCl3/MeOH): Frs. 4.1 –
4.7. Fr. 4.5 (219 mg) was submitted to CC (SiO2; gradient CHCl3/MeOH): Frs. 4.5A – 4.5F. Separation of
Fr. 4.5C (47 mg) by CC (Sephadex LH-20; MeOH): isoquercitrin (16 mg).
Acid Hydrolis of Cyclozanthoxylane A (1). Compound 1 (1 mg) was dissolved in 6n HCl (1 ml) and
heated at 1108 for 14 h in a reaction vial. After cooling, the soln. was evaporated to dryness under
reduced pressure and redissolved in 100 ml of H2O.
Preparation and Analysis of Marfey Derivatives. To 50 ml of a 50 mm soln. of the respective amino
acid (or to 50 ml of the acid hydrolysate soln.) was added 100 ml of a 1% (w/v) soln. of l-FDAA (Marfeyꢂs
reagent, Na-(5-fluoro-2,4-dinitrophenyl)-l-alaninamide) in acetone. After addition of a 1m NaHCO3
soln. (20 ml), the mixture was incubated for 1 h at 408. The reaction was stopped by the addition of 10 ml
of 2n HCl. The solvents were evaporated to dryness, and the residue was redissolved in MeOH. An
aliquot (20 ml) was analyzed by reversed-phase (RP) HPLC (4.6 ꢁ 250-mm C18 column (5 mm), linear
gradient of MeCN (A) and aq. CF3COOH (B) (pH 3.1) from 28 to 55% A over 35 min; flow rate, 1 ml/
min; detection, at 340 nm). Peaks in the chromatogram were identified by comparing the tR values with
those of the l-FDAA derivatives of the authentic amino acids. The standards derivatives gave the

Helvetica Chimica Acta – Vol. 95 (2012)
939
following tR values [min]: 9.30 (Gly-l-FDAA), 10.94 (l-Ala-l-FDAA), 14.56 (d-Ala-l-FDAA), 15.51 (l-
FDAA), 26.92 (l-Phe-l-FDAA), 27.22 (l-Leu-l-FDAA), 29.52 (d-Phe-l-FDAA), 30.83 (d-Leu-l-
FDAA). The l-FDAA derivatives of the liberated amino acids from 1 showed peaks at 9.45 (Gly-l-
FDAA), 11.10 (l-Ala-l-FDAA), 15.79 (l-FDAA), 27.11 (l-Phe-l-FDAA), 27.37 (l-Leu-l-FDAA).
Cyclozanthoxylane A (Cyclo(l-alanyl-l-leucyl-l-alanyl-glycyl-l-phenylalanyl); 1). White amor-
phous powder. IR (KBr): 3306, 2953, 1652, 1533, 1384, 746. 1H- and 13C-NMR: see the Table. HR-ESI-
MS: 460.2557 ([M þ H]þ, C23H34N5O5þ ; calc. 460.2554).
`
The authors are grateful to FundaÅa˜o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio
´
de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cientꢁfico e Tecnologico (CNPq), Centro
´
´
de Pesquisas do Pantanal (CPP), Instituto Nacional de Areas Umidas (INAU), and CoordenaÅa˜o de
AperfeiÅoamento de Pessoal de Nꢁvel Superior (CAPES) for scholarships and financial support. They are
´
´
also grateful to the NPPNS (Nucleo de Pesquisa em Produtos Naturais e Sinteticos) Depto. de Fꢀsica e
Quꢀmica – FCFRP – USP for the HR-mass spectra.
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Received November 29, 2011