Ixorine , a New Cyclopeptide Alkaloid from the Branches of Ixora brevifolia

The isolation and structure determination of new cyclic peptide alkaloid ixorine, along with five known constituents frangulanine, syringaresinol, cinnamtannin B-1, daucosterol and mannitol from the branches of Ixora brevifolia are described. The cyclic peptide frangulanine is being described for the first time in the Rubiaceae family. The structures were elucidated on their spectral data basis, mainly one(H, C, DEPT) and two-dimensional (COSY, NOESY, HSQC and HMBC) nuclear magnetic resonance (NMR) and by comparison with data from the literature. The mixture of two cyclopeptide alkaloids showed weak activity against Leishmania amazonensis.


Introduction
][11] Ixora brevifolia Benth. is a plant popularly known in Brazil as "ixora-arbórea" and can be found in Brazilian Cerrado.The study of the bark of this species led to the isolation of the steroid β-sitosterol glycoside, seven pentacyclic triterpenes: lupeol, 3β-lupeonil-eicosanate, 3β-lupeonil-estearate, 3β-lupeonil-palmitate, α-amyrin, β-amyrin, 30-hydroxyfriedelan-3-one, and the flavonoid quercetin.The same study reported the antifungal activity of extracts and fractions from this plant. 12ur continuing interest in the research on bioactive constituents of native plants of the Brazilian Cerrado led us to investigate bioactive metabolites of Ixora brevifolia branches.Chemical investigation of its methanolic extract resulted in the isolation and identification of two alkaloids, including a new cyclopeptide alkaloid (1), named ixorine, and the known cyclopeptide alkaloid frangulanine (2), the lignin syringaresinol (3), the proanthocyanidin cinnamtannin B-1 (4) (Figure 1), along with daucosterol and mannitol.The new and known cyclopeptide alkaloids are being described for the first time in the Rubiaceae family.
In addition to the chemical study, several bioassays were performed, as antibacterial, antifungal and antiprotozoal.

General experimental procedures
Chromatography columns were carried out on silica gel 60 (Merck, 70-230 mesh).Analytical thin layer chromatography (TLC) was performed on precoated silica gel plates (TLC Silica gel 60F 254 from Merck).The fractions and compounds were detected in TLC by UV light (254 and 366 nm) and by spraying with p-anisaldehyde-H 2 SO 4 solution, followed by heating at 150 °C or by spraying with Dragendorff solution.
Nuclear magnetic resonance (NMR) experiments were recorded on a VARIAN-MERCURY plus spectrometer operating at 300 and 75 MHz for 1 H and 13 C, respectively.The optical rotations were measured on PerkinElmer 341 and 343 digital polarimeters.The high resolution electrospray ionization mass spectrometry (HRESIMS) were acquired using a ESI-Q-TOF-MS (WATERS) spectrometer.MS analysis was performed on a GC-MS (Thermo-Finnigan, Focus DSQ II) with a quadrupole mass analyzer, using electron impact ionization mode (70 eV).

Plant material
The branches of the plant were collected in May 2010, on Samambaia campus of the Federal University of Goiás (Goiânia, GO, Brazil).A voucher specimen has been deposited in the Herbarium at Universidade Federal de Goiás, Brazil, under the registration number 45523.
A portion of CE (18.2 g) was dissolved in H 2 O/MeOH 7/3 and acidified with a solution of hydrochloric acid (HCl, 2 mol L −1 ) to pH 2-3.The acidic solution was exhaustively extracted with diethyl ether (Et 2 O) to yield the acidic ether fraction (AEF, 1.70 g).The aqueous solution was made basic with ammonium hydroxide (pH 8-9) and extracted with Et 2 O to yield the basic ether fraction (BEF, 43.0 mg) and basic aqueous fraction (BAF, 15.5 g).The BEF (43.0 mg) was purified using silica gel column chromatography, which was eluted with chloroform containing increasing amounts of methanol (up to 50%).It provided 123 fractions (8 mL each), which were pooled together to 21 fractions after TLC analysis (BEF1 to BEF21).BEF6 provided 2 (frangulanine, 2.6 mg) and BEF9, after acetone washing, provided 1 (ixorine, 0.8 mg).Fractions BEF7 (0.6 mg) and BEF8 (1.1 mg) afforded a mixture of these two substances.

Antileishmanial activity assay
Promastigote forms of L. amazonensis (MHOM/BR/75/ Josefa strain) were maintained by weekly transfers in Warren's medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 25 °C in a tissue flask.For the experiments, promastigotes (1 × 10 6 parasites mL −1 ) were inoculated in a 24-well plate containing Warren's medium supplemented with 10% inactivated fetal bovine serum with different concentrations of isolated compound, and incubated at 25 °C for 72 h.The cell density for each concentration was determined by counting in a hemocytometer (Improved Double Neubauer).Controls containing 0.5% dimethyl sulfoxide (DMSO from Sigma) and medium alone were also included.The 50% inhibitory concentration (IC 50 ) was determined by logarithm regression analysis of the data obtained. 13orine (1)
The combined use of 1D ( 1 H and 13 C NMR, DEPT) and 2D (COSY and HSQC) spectra allowed attributable unambiguous assignments of all protons and carbons of the amino acids units and the presence of a p-oxygenated Z-styrylamine group (Table 1).
The relative stereochemistry for 1 was proposed from the 1 H NMR coupling constants and NOESY analysis (Figure 2) and is in agreement with that of frangulanine (2). 149][20] In addition, H-3 showed correlation with H-17 and H-18 in the NOESY spectra, while the H-4 exhibited correlation with H-19 and NH-6, and the latter did not showed interaction with H-7, which is consistent with the coupling constant of 6.3 Hz of the H-6, due coupling with H-7.These evidences are also in agreement with L-erythro configuration of β-hidroxyleucine of ixorine. 21The coupling constants of the H-4 (J 4,20 = 9.0; J 4,3 = 8.0 Hz) show that the relative orientation of H-4 and H-20 are also in opposite faces.The correlations of H-20 with H-17, H-22, and H-23, observed in NOESY spectra, indicate that these protons are co-facially oriented.
Although the 14-membered ring is the largest subgroup of cyclopeptide alkaloids, few studies of their biological activities have been reported due to the low amounts present in the plants of origin.The biological properties for this class of substances includes antibacterial, antifungal, antiplasmodial, antinociceptive and immunostimulant activities. 22he alkaloidal mixture 1 and 2 (CF24 fraction) were tested for antibacterial, antifungal and antiprotozoal activities.Antibacterial and antifungal activities were evaluated for the strains Escherichia coli ATCC 25922, However, no significant biological activities were observed.Antiprotozoal activity was evaluated in vitro against promastigotes of Leishmania amazonensis.Treatment with concentrations 5.0, 10.0, 50.0 and 100.0 µg mL −1 of CP24 inhibited the parasite growth by 12.41, 24.13, 48.27 and 63.45%, respectively.The IC 50 value was 54.16 µg mL −1 .Leishmaniasis is regarded as a neglected disease, and nothing was found in literature about assays with these or related cyclic peptides against this protozoal.

Conclusions
The phytochemical study of Ixora brevifolia Benth.has led to the isolation of two cyclopeptide alkaloids, ixorine and frangulanine, along four known constituents.Frangulanine, previously isolated in species of Rhamnaceae family, 14 is reported in Rubiaceae family for the first time.This kind of cyclic peptide alkaloids was isolated only in five species of this family: Canthium anorldianum, Canthium euryoides, Feretia apodanthera, Plectronia odorata and Amaioua guianensis 25,26 and all of these species belong to the subfamily Ixoroideae, as well as Ixora brevifolia.

Figure 1 .
Figure 1.Structures of compounds1-4 isolated from the branches of I. brevifolia.