Sclerin, a New Cytotoxic Cyclononapeptide from Annona scleroderma

A new cytotoxic cyclononapeptide, sclerin, cyclo(–Dab1–Ser2–Tyr3–Gly4–Thr5–Val6–Ala7– Ile8–Pro9–) (1), was isolated from the methanol extract of the seeds of Annona scleroderma, together with the known metabolite, cyclosenegalin A, cyclo(–Pro1–Gly2–Leu3–Ser4–Ala5–Val6–Thr7–) (2). The planar structures for the two compounds were established by comprehensive analysis of NMR and ESI-HRMS data, and the absolute stereochemistry was stablished by Marfey’s method. Compound 1 showed moderate cytotoxic activity against the human prostate carcinoma cell line DU-145 at µM concentration.


Introduction
Terrestrial natural products have played a fundamental role in drug development during the last decades, either directly as drugs or lead structures that were further optimized by medicinal chemistry [1,2]. Within the natural products, cyclic peptides constitute an important class of natural molecules with a great diversity of ring sizes; some of them have been submitted to clinical trials or come near to that phase, because of their attractive pharmacological properties [3,4]. On the other hand, many cyclopeptides represent research tools in molecular biology for investigating several processes involved in cellular regulation [5]. These metabolites have been isolated from higher plants as well as microorganisms and marine sources [3,6]. Phytochemical studies on species of the genus Annona have demonstrated that the plants belonging to this genus produce an amazing variety of cyclopeptide derivatives. Within this genus, Annona scleroderma is distributed in tropical and subtropical latitudes worldwide. In México, A. scleroderma, commonly named "cawesh", "cahuex", or "poshté", grows in warm climates areas, such as Tabasco, Chiapas, Quintana Roo, Nayarit, Michoacán, Yucatán, and Veracruz [7]. This study describes the investigation on seeds of A. scleroderma, leading to the isolation and structural elucidation of a new cyclopeptide, sclerin (1), together with the known metabolite, cyclosenegalin A (2) (Figure 1). Their planar structures were determined based on detailed spectroscopic NMR studies and ESI-HRMS data. The absolute stereochemistry of each amino acid residue in compounds 1 and 2 were determined by Marfey's method [8]. The cytotoxicity bioassays indicated that these compounds possess activity against human prostate cancer cell line DU-145.
Molecules 2019, 24, x FOR PEER REVIEW 3 of 8 the presence of nine amino acid residues in 1. Furthermore, key HMBC correlations between the carbonyl group of residue i with the α protons of residue i + 1 (H-6 and C-1, H-9 and C-5, H2-18 and C-8, H-20 and C-17, H-24 and C-19, H-29 and C-23, H-32 and C-28, H-38 and C-31, and H-2 and C-37) allowed us to determine the planar structure of 1, as shown in Figure 2 and  (1) shows the presence of an unnatural amino acid residue, L-2,4-diaminobutyric acid (Dab), observed in only a few cyclic polypeptides, such as the polymyxins A-E [14].  [15] The absolute stereochemistry of the amino acid residues for compounds 1 and 2 were established by Marfey's method [8]. The acid hydrolysate of sclerin (1) and cyclosenegalin A (2) was derivatized, with Nα- (2,4-dinitro-5- The next cyclopeptide, cyclo(-Pro 1 -Gly 2 -Leu 3 -Ser 4 -Ala 5 -Val 6 -Thr 7 -) (2), was isolated as an optically active powder, [α] 25 D − 4 (c 0.31, MeOH). The molecular formula of 2, C 28 H 47 N 7 O 9 , was established by HRESIMS analysis, where its sodiated molecular ion was observed at m/z 648.3339 (calculated 648.3333 for C 28 H 47 N 7 O 9 Na, [M + Na] + ). Analysis of the 1 H-and 13 C-NMR spectra of 2 indicated the presence of six CH 3 , six CH 2 , and nine CH, as well as seven quaternary carbonyl groups. Detailed analysis of 2D NMR spectroscopy ( 1 H-1 H COSY, HSQC in CD 3 OD, and HMBC in CD 3 OD and CD 3 OH) suggested that the structure of 2 was identical in all to cyclosenegalin A, reported by Wélé et al., and isolated from the methanol extract of the seeds of A. senegalensis (see Supplementary  Materials). [15] The absolute stereochemistry of the amino acid residues for compounds 1 and 2 were established by Marfey's method [8]. The acid hydrolysate of sclerin (1) and cyclosenegalin A (2) was derivatized, with Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (L-FDLA). The retention times of these FDAA amino acid derivatives were established by HPLC monitoring with UV absorption at 340 nm. All FDAA derivatives were identified based on a comparison of their retention times in HPLC with authentic amino acid standards. The absolute stereochemistry of all the amino acid residues of 1 and 2 were identified as L. Thus, the absolute configurations of 1 and 2 can be assigned as 2S, 6S, 9S, 20S, 21R, 20S, 24S, 29S, 32S, 33S, 38S and 2S, 9S, 15S, 18S, 21S, 26S, 27R, respectively. The in vitro cytotoxic activity of sclerin (1) and cyclosenegalin A (2) was assessed by XTT assay, using the prostate cancer cell line DU-145 [16,17]. As shown in Table 2, sclerin (1) and cyclosenegalin A (2) were able to inhibit cell proliferation of the human prostate cancer at µM concentration.

General Experiment Procedures
Optical rotation was determined on a PerkinElmer 241 polarimeter (Waltham, MA, USA), using a sodium lamp operating at 589 nm. The IR spectrum was measured on a Bruker IFS55 spectrometer (Billerica, MA, USA), using a chloroform solution to place a film of the compounds on the NaCl disk. NMR spectra were performed on Bruker AVANCE 600 MHz instruments at 298 K, and coupling constants are given in Hz. NMR experiments, COSY, HSQC, and HMBC, were acquired using standard pulse sequences. 3 J H,H values were measured from 1D 1 H-NMR. NMR data were processed using Topspin and MestReNova software (v 11.01, Santiago de Compostela, Spain). Mass spectra were recorded on a VG AutoSpec FISON spectrometer (Danvers, MA, USA). HPLC (High performance liquid chromatography) separations were carried out with an LKB 2248 system (LKB-Producter AB, Bromma, Sweden) that was equipped with a photodiode array detector. All of the solvents used were HPLC-grade. HPLC chromatography was monitored by TLC, performed on AL Si gel Merck 60 F254 (Kenilworth, NJ, USA). TLC plates were visualized by UV light (365 nm) and phosphomolybdic acid solution 10 wt% in ethanol.

Plant Material
The seeds of A. scleroderma were collected from municipality of Ignacio de la Llave, Veracruz, (México) during May 2015, and identified by taxonomists in the Institute for Biological Research at Veracruz University. After collection, the vegetable material was dried at room temperature for one week and then triturated using a steel blender.

Cell Culture
DU-145 (human prostate cancer) was maintained in culture medium containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 • C in air with 95% humidity and 5% CO 2 . Cells were periodically tested for Mycoplasma infection using the MycoAlert © Mycoplasma detection kit (Lonza, Basel, Switzerland), as well as the Venor © GeM Advance Mycoplasma PCR detection Kit (Minerva Biolabs, Berlin, Germany), and found to be negative.

Cytotoxic Assay
The effect of the two compounds in the proliferation of human prostate cancer cell line was determined as previously described by using the XTT (sodium 3 -[1-(phenylaminocarbonyl)-3,4tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) cell proliferation kit (Roche Molecular Biochemicals, Mannheim, Germany) as previously described [11,12]. Cells (5.0 × 10 3 in 100 µL) were incubated in RPMI-1640 culture medium containing 10% heat-inactivated FBS, in the absence and in the presence of the indicated compounds at a concentration range of 10 −3 to 10 −9 M, in 96-well flat-bottomed microtiter plates, and following 72 h of incubation at 37 • C in a humidified atmosphere of air/CO 2 (19/1), the XTT assay was performed. Measurements were done in triplicate, and each experiment was repeated three times. The IC 50 (50% inhibitory concentration) value, defined as the drug concentration required to cause 50% inhibition in the cellular proliferation with respect to the untreated controls, was determined for each compound.

Conclusions
In the present study, the structure of one new cyclopeptide, sclerin (1), together with the known metabolite, cyclosenegalin A (2), were unambiguously determined by the combined use of spectroscopic and Marfey's method. Sclerin (1) contains nine amino acid residues, being, thus, one of the atypical examples of cyclic nonapeptide isolated of genus Annona. In addition, it is important to highlight that 1 possesses an unnatural amino acid residue, L-2,4-diaminobutyric acid (Dab) observed in few natural metabolites. The cytotoxic activity of these compounds, sclerin (1) and cyclosenegalin A (2), was tested against DU-145 human prostate cancer cell line, resulting in 1 IC 50 27.3 ± 4.19 µM and 2 IC 50 54.9 ± 2.35 µM.