Psiguajadials A–K: Unusual Psidium Meroterpenoids as Phosphodiesterase-4 Inhibitors from the Leaves of Psidium guajava

Bioassay-guided fractionation of the ethanolic extract of the leaves of Psidium guajava led to the isolation of 11 new Psidium meroterpenoids, psiguajadials A–K (1–11), along with 17 known ones (12–28). Their structures and absolute configurations were elucidated by spectroscopic methods and comparison of experimental and calculated ECD. Compounds 1 and 2 represent two unprecedented skeletons of 3,5-diformyl-benzyl phloroglucinol-coupled sesquiterpenoid, while 3 is the first example of Psidium meroterpenoids coupling via an oxepane ring. Putative biosynthetic pathways towards 1 and 2 are proposed. Compounds 1–13 and 16–26 exhibited moderate inhibitory activities against phosphodiesterase-4 (PDE4), a drug target for asthma and chronic obstructive pulmonary disease, with IC50 values in the range of 1.34–7.26 μM.

Psiguajadial F (6) displayed a quasi-molecular ion peak at m/z 473.2353 [M -H] − (calcd 473.2333) in HR-ESI-MS, in agreement with the molecular formula C 30 H 34 O 5 . The 1D and 2D NMR analysis revealed that 6 shared the same gross structure with psiguadial B 9 , a known caryolane-based Psidium monoterpenoid. Thus, the structural differences between the two compounds were attributable to the configurational variations on the coupling sites. The relative configuration of the caryolane moiety in 6 (C-1, C-4, C-8, and C-9) was assigned to be the same as those in psiguadial B by NOESY analysis (Fig. 10) and comparison of their 1D NMR data. Consequently, the NOE correlations of H-3α/H-5 and H-9 and H-1′/H-14b indicated that H-5 and H-1′ were αand βoriented in 6, respectively, which were reversed as in psiguadial B. The absolute configuration of 6 was determined by comparison of its ECD spectrum (see Supplementary S7.58) with that of psiguadial B, in which the reversed Cotton effects at 344 and 284 nm indicated that the configuration of C-1′ in 6 was S. Thus, 6 was assigned as depicted.
Psiguajadial G (7) had the molecular formula C 30 H 34 O 5 as determined by HR-ESI-MS. Its 1D NMR data (Tables 3 and 4) were very similar to those of the synthesized caryophyllane-based meroterpenoid, compound  20 27 , which was isolated as a natural product for the first time in the current study and was given a trivial psiguajadial L. The structural difference between 7 and 20 was being due to the configuration of H-1′, which was designated as α-orientation in 7 by NOE correlations of H 3 -14/H-9′ (13′) and H-1′/H-5 and H-7α (Fig. 11). Thus, psiguajadial G (7) was assigned as the C-1′ epimer of 20. The absolute configurations of psiguajadials G (7) and L (20) were established by analysis of their ECD spectra (see Supplementary S7.59 and S7.67), in which the Cotton effects around at 345 nm (positive for 7 and negative for 20) and 284 nm (positive for 7 and negative for 20) indicated that the C-1′ configuration of 7 and 20 were R and S, respectively. These assignments were in agreement with the ECD tendencies of those for guajadial (19) and psidial A (18) 9, 27 , respectively.   Psiguajadials H (8) and I (9) had the identical molecular formula C 30 H 34 O 5 on the basis of their HR-ESI-MS data. Analysis of their 1D and 2D NMR data indicated that both compounds had the same gross structure as the known cadinane-based Pisudium meroterpenoids guajadials C (21) and D (22) 34 , a pair of C-1′ epimers also isolated in the current study. The relative configurations of the cadinane moiety (C-1, C-7, and C-10) in 8 and 9 were established as the same as those in 21 and 22 based on the NOESY analysis and comparison of their 1D NMR data. Thus, the remaining two chiral centers (C-4 and C-1′) formed by the spiro ring junction suggested that 8, 9, 21, and 22 were four stereoisomers. Detailed NOESY analysis of 8 and 9 (Fig. 11) indicated that they had the same configuration at the spirocenter (C-4-C-15 bond α-oriented) but different configuration at C-1′ (H-1′β in 8 and H-1′α in 9). Thus, compounds 8 and 9 were also a pair of C-1′ epimers. The absolute configurations of 8 and 9 were established by analysis of their ECD spectra (see Supplementary S7.60 and S7.61), in which the Cotton effects at 344 nm (negative for 8 and positive for 9) and 284 nm (negative for 8 and positive for 9) indicated that   the C-1′ configuration of 8 and 9 were S and R, respectively. These assignments were in agreement with the ECD tendencies of those for 21 and 22, respectively.
Psiguajadial J (10) had the molecular formula C 30 H 34 O 5 as determined on the basis of HR-ESI-MS and NMR data. The 1D and 2D NMR analysis revealed that 10 shared the same gross structure with those of guajadials E (23) and F (24) 34 , a pair of C-1′ epimers of cadinane-based Psidium meroterpenoid, indicating that they were stereoisomers with configurational variations occurring at the coupling sites (C-1′, C-3, and C-4). The relative configuration of 10 was established by NOESY experiment and analysis of its coupling constant. The NOE correlations of H-1/H-3, H-9β, and H-11 and H-3/H-9′ (13′), and H-2β indicated that these protons and the benzene ring were cofacial and were arbitrarily assigned as β-orientated. Thus, the NOE correlations of H 3 -15/H-1′, H-2α, Figure 9. Experimental ECD spectrum of 4 and TDDFT calculated ECD spectra for 4a (1R, 4R, 5S, 9S) and enantiomer of 4a.  and H 3 -14 (Fig. 11), assigned these protons or methyl group in α-orientation. The trans-relationship of H-1′ and H-3 were further confirmed by the large coupling constant of H-1′ (J = 11.6 Hz). Thus, 10 was established as a C-3 epimer of 24, with a trans-fused dihydropyran ring. The R-configuration of C-1′ in 10 was established by the Cotton effects at 346 (positive) and 278 nm (positive) in its ECD spectrum (see Supplementary S7.62), which showed the similar tendency to those of 24.
Psiguajadial K (11) had a molecular formula of C 25 H 25 O 5 as determined by its HR-ESI-MS. The 1D (Table 5) and 2D NMR analysis (Fig. 12) revealed that 11 possessed the same gross structure as that of guadial A (26) 10 , indicating that they were stereoisomers with configurational variations occurring at the coupling sites. In the NOESY spectrum, the correlations of H-2/H-7β and H-1′/H-6β and H-7α (Fig. 12) indicated that C-1-C-7 bond was β-oriented at the spirocenter while H-1′ was α-oriented on the pyran ring. Thus, 11 was established as the  14′ 192.7,CH 191.5,CH 192.2,CH 192.3,CH 192.2,CH 192.4,CH 15′ 191.4,CH 192.2,CH 192.5,CH 191.5,CH 191.6,CH 191.5, CH Table 4. 13 C NMR (100 Hz) data of compounds 5-10 in CDCl 3 (δ in ppm). The spectroscopic data of compound 12 was identical to guajavadial C, a new skeleton of bicyclogermacrane-based Psidium meroterpenoid recently isolated from the same plant by Qin et al. 35 . However, after careful analysis of its NMR data, we found the configuration of guajavadial C was incorrect (see Supplementary Fig. S1.2). The cis-relationship of H-5 and H-1′ was assigned by the author only based on the NOE correlation of H-5/H-1′. In fact, on a six-mumbered ring both cis and trans adjacent protons could generate NOE correlations. In this case, the trans-relationship of H 3 -15 and H-5 was firstly assigned by the NOE correlations of H-5/H 3 -13 and H-3β, and H-6/H 3 -15 (see Supplementary Fig. S1.2). Then the NOE correlations of H-9′(13′)/H-3β further assigned the benzene group in β-orientation. Thus, H-1′ was α-oriented and in the trans-position of H-5. In additon, the congfiguration of Δ 1 (10) was revised as E by the observed NOE correlation of H-1/H-9β. The absolute configuration of 12 was established as 4S, 5R, 6R, 7R, 1′S by comparison of its experimental ECD spectrum with those of calculated for its isomers (see Supplementary Fig. S1.3).
Compounds 1-28 were tested for their inhibitory activities against PDE4D2. Rolipram, a well-known PDE4 inhibitor, was used as the positive control (IC 50 0.62 ± 0.03 μM). Most of compounds (1-13 and 16-26) exhibited moderate inhibitory activities with IC 50 values in the range of 1. 34-7.26 μM (Table 6), which may explain the inhibitory activity of the selected fraction and the anti-inflammatory usage of P. guajava in traditional medicine.
Psidium meroterpenoids are a small group of natural products characterized by a 3,5-diformyl-benzyl phloroglucinol moiety coupled with a mono-or sesquiterpenoid unit. In the current study, bioassay-guided fractionation of the ethanolic extract of the leaves of Psidium guajava led to the isolation of 28 Psidium meroterpenoids, including 11 new ones (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Compounds 1 and 2 represent two unprecedented skeletons with the presence of cubebane moiety in this compound class reported for the first time. Compound 3 represents the first example of Psidium meroterpenoid coupling via an oxepane ring, and 4 was the second example featuring the C-14′ coupling site. Compounds 1-13 and 16-26 exhibited moderate inhibitory activities against phosphodiesterase-4 (PDE4), with IC 50 values in the range of 1. 34-7.26 μM, suggesting that this compound class represents a new class of PDE4 inhibitors, which may serve as new structural motifs for designing new PDE4 inhibitors. Investigations of their mechanism of action and selectivity versus other PDEs are ongoing.

Methods
General. Optical rotations were determined on a Perkin-Elmer 341 polarimeter at 20 °C. UV spectra were performed on a Shimadzu UV-2450 spectrophotometer, and ECD spectra were performed on an Applied Photophysics Chirascan spectrometer. Infrared spectra (IR) were measured on a Bruker Tensor 37 infrared spectrophotometer. NMR experiments were carried out on a Bruker AM-400 spectrometer at the temperature thermostatically controlled at 25 °C. Exact mass measurements and molecular formulas were obtained from ESIMS and HR-ESI-MS using a Finnigan LCQ Deca and a Thermo Scientific LTQ Orbitrap XL spectrometers, respectively. Semi-preparative reversed-phase (RP) HPLC was performed with a YMC-pack ODS-A column (10 × 250 mm, S-5 μm) or a Phenomenex Lux cellulose-2 chiral column (  and voucher specimens (FSL201408) have been stored at the School of Pharmaceutical Sciences, Sun Yat-sen University.