Cytotoxic Meroterpenoids with Rare Skeletons from Psidium guajava Cultivated in Temperate Zone

Three new meroterpenoids, guajavadials A–C (1–3), were isolated from Psidium guajava cultivated in temperate zone. Their structures were established by extensive spectroscopic evidence and electronic circular dichroism (ECD) calculations. Guajavadial A (1) represents a novel skeleton of the 3,5-diformylbenzyl phloroglucinol-coupled monoterpenoid, while guajavadials B (2) and C (3) are new adducts of the 3,5-diformylbenzyl phloroglucinol and a sesquiterpene with different coupling models. The plausible biosynthetic pathways as well as antimicrobial and cytotoxic activities of these meroterpenoids are also discussed. All these isolates exhibited moderate cytotoxicities against five human cancer cell lines, with 3 being most effective with an IC50 value of 3.54 μM toward SMMC-7721 cell lines.

the two structural fragments "a" and "b" via an ether linkage between C-1 and C-3′ . With the aid of extensive analysis of its 2D NMR spectra, the assignments of 1 H and 13 C NMR data of 1 were achieved. The relative configuration of 1 was determined by the analysis of NOESY spectrum (Fig. 3). NOE correlation between Me-7 and H-2 indicated that Me-7 and H-2 were cis-fused with the dihydropyran ring. In addition, diagnostic NOE correlations between Me-7 and H-5b, between H-2 and H-3a, between H-3b and H-1′ , as well as between H-5a and H-6 suggested that these protons were placed on the same side, respectively. Unfortunately, many attempts to prepare a single crystal of this molecule have not been successful so far. Owing to indispensable methods for the stereochemical characterization of natural products 18 , the quantum mechanical calculations was performed to determine the absolute configuration of 1. As illustrated in Fig. 4, the well matched result between the calculated ECD curve and the measured one led to the assignment of its absolute configuration as 1S,2R,4S,6S,1′ S. Based on the above evidence, the structure of 1 was established as shown.
Guajavadial B (2) was obtained as a white amorphous powder.  13 C NMR data with those of 1 suggested that they both possessed the same structural moiety of 3,5-diformylbenzyl phloroglucinol "b". Two spin systems of H-1′ -H-1~H-3 and H-5~H-9 were revealed by the 1 H-1 H COSY spectrum, which in combination with the HMBC correlations from Me-15 to C-3/C-5 and from Me-14 to C-1/C-9/C-10, indicated the existence of a 10-membered ring in 2. Likewise, the HMBC correlations of Me-12/Me-13 with C-6/C-7 suggested the presence of a cyclopropane ring between C-6 and C-7. Thus, the sesquiterpene unit "c" with a 3/10 continuous carbocyclic system was established. Besides, the downfield shifts at δ C 164.4 (C-3′ ) and 88.3 (C-10) suggested an oxygen atom bridging C-10 and C-3′ to form a dihydropyran ring, which was also consistent with the HMBC correlations from H-1′ to C-10/C-2′ /C-3′ and the unsaturation degrees obtained from the HRMS. The NOE correlations between H-7 and Me-14/Me-13, between Me-12 and H-8b, as well as between H-8b and H-1 settled the relative configuration of 2. Furthermore, the coupling constant between H-1′ and H-1 (5.9 Hz) indicated those protons should be placed at the same side, which was further confirmed by NOE correlations of these two protons (Fig. 3). Finally, a good agreement between the calculated ECD and experimental CD spectra suggested the absolute configuration of 2 as 1R,6S,7R,10S,1′ R.
The antimicrobial activities 1-3 toward five microbial strains (P. aeruginosa, S. aureus, E. coli, M. canis, and M. gypseum) were evaluated as described previously 20 . Unfortunately, none of them exhibited obvious antimicrobial activities against three bacteria and two fungi at the concentrations of 100 μg/mL and 250 μg/mL, respectively. In addition, the inhibitory effects of the isolates against five human cancer cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW480) were also tested by MTT method 21 . All these meroterpenoids showed moderate cytotoxic activities ranging from 22.28 to 3.38 μM (Table 3), with 3 being the most effective with an IC 50 value of 3.54 μM toward SMMC-7721 cell lines compared to positive control cisplatin (IC 50 19.82 μM). These result suggest that P. guajava leaves could provide a source of potential therapeutic compounds for both the prevention and treatment of cancer 22 .
In conclusion, a phytochemistry investigation on the leaves of Psidium guajava cultivated in temperate zone led to the discovery of a novel meroterpenoid featuring an unprecedented skeleton of the 3,5-diformylbenzyl phloroglucinol-coupled monoterpenoid, guajavadial A (1), together with two new ones possessed the 3,5-diformylbenzyl phloroglucinol and a sesquiterpene with different coupling models, guajavadials B (2) and C (3). This finding of 1-3 could provide evidence that the plant secondary metabolites can be influenced by the ecological environment. Besides, these isolates might provide challenging natural products for organic synthesis and enrich the diversity of meroterpenoids via hetero-Diels-Alder reactions. All these meroterpenoids did not display antimicrobial activity but exhibited cytotoxicity effects against five human cancer cell lines.

Methods
General Experimental Procedures. Optical rotations were measured on a JACSO P-1020 polarimeter.
UV spectra were taken on a Shimadzu UV-2401PC spectrometer. IR spectra were determined on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. ECD spectra were recorded with an Applied Photophysics

GC-MS Analysis of 4 and 5.
The air-dried and powdered leaves of Psidium guajava (100 g) were hydrodistilled for 2 h using a Clevenger apparatus to obtain the essential oil (0.5 mL). An Agilent 7890A gas chromatograph was used with helium as a carrier gas at a flow rate of 2.2 mL/min, 20:1 split injection (injector temperature 250 °C, injection volume 1.0 μL), using an Agilent HP-5 MS column (50 m × 0.32 mm, 0.52 μ m film thickness) and a temperature program from 40 °C to 250 °C at a rate of 2 °C/min, followed by 5 °C/min until 250 °C (10 min hold). The coupled mass spectrometer was an Agilent model 5975C system, transfer line temperature 280 °C, source temperature 230 °C, ionization potential 70 eV, and a scan range of 30-400 atomic mass units (amu). Compounds were identified by comparison of experimental spectra with the reference spectra in NIST14 data base. About ECD Calculation of 1-3. The theoretical calculations of 1-3 were performed using Gaussian 09 23 . Briefly, the 3D structures of 1-3 were first established according to the NOESY spectra. Their 3D structures were then performed in the SYBYL 8.1 program by using MMFF94s molecular force field. All the obtained conformers were optimized using DFT at the B3LYP/6-31+ G(d) level in gas phase. Further optimization at the B3LYP/6-311G(2d,p) level led the dihedral angles to be got. The optimized conformations were used for the ECD calculations using time dependent Density Functional Theory (TDDFT). The ECD spectra were produced by SpecDis 1.6 software 24 . The calculated ECD spectra of 1-3 were subsequently compared with the experimental ones.
The ECD spectra were simulated by overlapping Gaussian functions for each transition according to: where σ represents the width of the band at 1/e height, and Δ E i and R i are the excitation energies and rotational strengths for transition i, respectively. σ = 0.30 eV and R vel were used in this work.
Preparation of microbial inocula. The inocula of bacteria were prepared from 24 h old agar cultures.
The absorbance was read at 600 and 530 nm, respectively, and adjusted with sterile physiological solution to an absorbance of 0.10 (0.5 McFarland standards). These solutions corresponded to 10 8 CFU/mL for bacteria. From the prepared bacterial solutions, other dilutions were prepared to give a final concentration of 10 6 CFU/mL. Conidia suspensions of dermatophyte species were prepared from 10 days old cultures. The number of conidia   was determined using a spectrophotometer and adjusted with 0.9% sterile saline solution to an absorbance of 0.600 at 450 nm, corresponding to a final concentration of 4 × 10 3 spores/mL.

Determination of minimum inhibitory concentration (MIC). The MICs of the isolated compounds
were determined in 96-well micro-titre plates by the broth microdilution method. The 96-well plates were prepared by dispensing into each well 100 μL of Mueller Hinton broth for bacteria and Sabouraud dextrose broth for fungi. The samples were initially prepared in 10% DMSO in broth medium at 400 μg/mL for the tested compounds or 50 μg/mL for the reference antibiotics. A volume of 100 μL of each test sample was added into the first wells of the micro-titre plate (whose wells were previously loaded with 100 μL of broth medium). Serial two-fold dilutions of the test samples were made and 100 μL of the inocula were added into the respective wells. This gave final concentration ranges of 100 to 0.781 μg/mL for the compounds and 12.5 to 0.097 μg/mL for reference substances. The plates were sealed with parafilm, then agitated with a plate shaker to mix their contents and incubated at 35 °C for 24 h for bacteria and at 28 °C for 5 days for dermatophytes. For bacteria, MICs were determined upon addition of 50 μL (0.2 mg/mL) p-iodonitrotetrazolium chloride (INT, Sigma-Aldrich). Viable bacteria reduced the yellow dye to a pink color. For dermatophytes, MICs were determined by visualizing the turbidity of the wells. The MIC was defined as the lowest concentration where no color or turbidity change was observed, indicating no visible growth of microorganism. All the tests were performed in triplicates. Gentamycin for bacteria and griseofulvin for dermatophytes were used as positives controls, respectively.
All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT), supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO 2 . Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO). Briefly, 100 μL of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 10 5 cells/mL in 100 μL of medium. Each cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin as positive control. After the incubation, MTT (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS-50% DMF after removal of 100 μL of medium. The absorbance of the lysate was measured at 595 nm in a 96-well Microtiter plate reader (Bio-Rad 680). Guajavadial