Guajadials C-F, four unusual meroterpenoids from Psidium guajava

Abstract Guajadials C-F (1–4), four sesquiterpenoid-based meroterpenoids with unprecedented skeletons were isolated from the leaves of Psidium guajava. Their structures and relative configurations were established by extensive spectroscopic analysis. A possible biosynthetic pathway for 1–4 was also proposed. Graphical abstract Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-012-0102-4 and is accessible for authorized users.


Introduction
Psidium guajava L. (Myrtaceae) is an evergreen shrub grown in tropical and subtropical regions as a food, and is also an indigenous medicinal plant used to treat inflammation, diabetes, hypertension, wounds, pain, fever, vomiting, and diarrhea. 1 In recent years, several sesquiterpene-based meroterpenoids with unusual skeletons have been discovered from the leaves of P. guajava, 2 some of which exhibited significant biological activities including inhibitory effects on proton tyrosine phosphatase 1B (TPT1B) 2b and the growth of human hepatoma cell (HepG2 and HepG2/ADM). 2c,d Plausible biosynthetic pathways 2a,b,d and biomimetic synthesis 2e,3 relative to Psidium meroterpenoids had also stimulated considerable interest in several laboratories. In continuation of our studies on structurally unique and biologically active Psidium meroterpenoids, by which two rare meroterpenoids guajadial 2a and guajadial B 2e had previously been discovered, another four novel meroterpenoids guajadials C-F (1-4) were isolated from the leaves of P. guajava. Herein, we describe the isolation, structure elucidation, plausible biosynthetic pathway, and cytotoxicity of the new compounds.

guajadial E (3) guajadial F (4)
A B C *To whom correspondence should be addressed. E-mail: jkliu@mail.kib.ac.cn C-5, and C-6 and H-15 to C-3, C-4, and C-5, revealed the C-linkages of C-3/C-4/C-5/C-6/C-7, C-1/C-6, and C-15/C-4. The above evidence strongly suggested that compound 1 possessed a sesquiterpenoid moiety (1b) resembled scapanol. 4 Comparison of the 1 H and 13 C NMR data assigned to 1b with those of scapanol indicated that they were similar, except that Me-15 of scapanol was replaced by a methylene and a significant downfield shift (Δ ≈ 10.3 ppm) for C-4 of 1b was observed. The above observation implied that 1b was fused to 1a via C-4 and C-15, which was further confirmed by the HMBC correlations from H-1′ to C-4 and C-15, as well as the degrees of unstauration requiring the existence of an ether bridge between C-4 and C-3′.
The relative configuration of 1 was established by analysis of the ROESY data and proton coupling constants based on computer-generated 3D drawing with minimized energy by MM2 calculation using ChemBio3D software. In the ROESY spectrum, the pseudo-axially oriented proton H-3 at  H 1.45  Table 1). The HMBC correlations from H-7 to C-1/C-5/C-6, Me-14 to C-1/C-9/C-10, and H-15 to C-3/C-4/C-5, and comparison with 1 H and 13 C NMR spectroscopic data of 1, are in agreement with a meroterpenoid possessing the same planar structure as 1. Detailed analysis of its ROESY correlations (Figure 2), among which H-1↔H-11, Me-14↔H-2α/H-8α, and H-15α↔Me-12/H-5 established the relative configuration of the sesquiterpenoid unit identical to that of 1, however, revealed as a major difference from 1, the discrepant orientation of phenyl. The significant ROESY correlations between H-1′ and H-3 fixing the phenyl of 1 as -oriented was no longer observable in the ROESY spectrum of 2, instead, strong correlation between H-1′ and H-5 was observed, indicating the -orientation of phenyl. Consequently, the relative configuration of 2 was determined as shown in Figure 2.
Guajadial E (3) was obtained as amorphous powder, [α] 24 D +  The UV spectrum (MeOH) showed absorptions at  max 277 and 337 (sh) nm. The NMR spectra (Table 2) also displayed signals for partial structure of benzyldiformylphloroglucinol (3a) besides which there were signals for four methyls, three methylenes, five methines, one oxygenated quarternary carbon, and a trisubstituted double bond that could be ascribed to a sesquiterpenoid unit with the aid of HSQC, 1 H-1 H COSY, and HMBC experiments (Figure 1). The HSQC and 1 H-1 H COSY spectra revealed the connections of C-1′/C-3/C-2/C-1/C-10/C-9/C-8/C-7/C-11, C-12/C-11/C-13, and C-10/C-14. In the HMBC spectrum, cross peaks from H-7 to C-1, C-5, and C-6 and Me-15 to C-3, C-4, and C-5 established the C-linkage of C-3/C-4/C-5/C-6/C-7, C-1/C-6, and C-15/C-4. The above evidence allowed the construction of a basic structural unit of a sesquiterpenoid 3b, to which signals assigned were analogous to those of 1b. Different from 1b, the C-3 methylene was replaced by a methine, meanwhile, the C-15 methylene was changed into a methyl group, suggesting a dihydropyran ring was fused to 3b via C-3 and C-4 instead of C-4 and C-15. The linkage between fragments 3a and 3b was further supported by HMBC experiment and molecular formula information. In the HMBC spectrum, correlations of H-1′ with C-2, C-3, and C-4 revealed the connection of C-1′/C-3.
According to the molecular formula information, the oxygen atom leftover was to bridge C-4 and C-3′, to form a dihydropyran ring. The relative configuration for 3 could be deduced by its ROESY spectrum. The ROESY correlations of H-1↔H-11, Me-14↔H-2α/H-8α, Me-15↔H-3, and H-1′↔H-1/Me-12/H-2 established the relative configuration of 3 as shown in The UV spectrum (MeOH) showed absorptions at  max 278 and 342 nm. Careful comparison of its 1 H and 13 C NMR data ( Table 2) with those of 3 suggested that they also share the same planar structure, which was further confirmed by HMBC correlations from H-7 to C-1/C-5/C-6, Me-15 to C-3/C-4/C-5, Me-14 to C-9/C-10/C-1, and H-1′ to C-2/C-3/C-3′/C-7′/C-9′/C-13′. In the ROESY spectrum (Figure 2), correlations of H-1↔H-11, Me-14↔H-3/8α, and Me-15↔H-3 established the relative configuration of the sesquiterpenoid moiety which was identical to that of 3. Meanwhile, the correlation of H-1′↔Me-15 implied that 4 was a C-1′ epimer of 3. The noticeable difference between the chemical shift value of H-2α (3: δ H 1.65; 4: δ H 0.58) also provided evidence that the phenyl had a different orientation because the significant up field shift (Δ ≈ 1.07 ppm) can be explained by the anisotropic effect of phenyl  as shown in the computer-generated 3D drawing. Thus, the relative configuration of 4 was elucidated as shown in Figure 2. Because the key intermediate 3,5-dimethyl-2,4,6-trihydroxybenzophenon had been previously isolated from the same plant, 5 the plausible biogenetic route of 1-4 could be proposed as shown in Scheme 1. The intermediate could be oxidized and then generated carbocation A, then A could attack 5 and 6 to generate tertiary carbocations B-E with resonance stabilization. Further cyclization of B-E could construct compounds 1-4.
Compounds 1-4 were evaluated for their in vitro growth inhibitory effects against five human cancer cell lines (MCF-7, A-549, SMMC-7721, SW480, and HL-60). All of the compounds exhibited positive activity (IC 50 5.59-40.0 μM) toward all five human cancer cell lines except that compound 1 was inactive to MCF-7 (Table 3).

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-012-0102-4 and is accessible for authorized users.