Deprea zamorae, Barboza & S. Leiva.

2.3. Withanolides isolated from Deprea zamorae View in CoL

Finally, the EtOH extract of D. zamorae yielded five new withanolides, named physangulidines DH ( 48). This unusual nucleus is characterized by the typical arrangement of withajardins, where C-21 is directly bonded to C-25 resulting in a bicyclic lactone side-chain with a six-membered homocycle. Regarding the steroid nucleus, physangulidines have a ketal functionality at the C-17 position formed through oxidative cleavage of the C13 A C17 bond and subsequent nucleophilic attack of the C-14 and C-7 hydroxy groups ( Fig. 2 View Fig ). Compound 4, C 28 H 36 O 7 Na, showed a peak at m / z 507.2352, corresponding to [M+Na] in the HRESIMS mass spectrum. The 1 H and 13 CNMR spectra of withanolide 4 closely resembled those of physangulidine C, recently isolated from Physalis angulata ( Zhuang et al., 2012) . Characteristic signals assigned to the side-chain and the resonances corresponding to the ketal functionality involving the C17 A C7 and C17 A C14 positions were observed ( Tables 2 View Table 2 and 3 View Table 3 ). Regarding the rings A and B substitution patterns, the 1 H and 13 C NMR spectra of 4 had almost identical signals for all carbons and protons of rings A and B of compound 1, indicating a 1-oxo-2,5-diene-7 a,O-substitution. Confirmation of the structure of 4 and assignment of the configuration at C-7, C-13, C-14, and C-17 were obtained from X-ray diffraction analysis. The diffraction data afforded the structure depicted in Fig. 3 View Fig , where the orientation for the hydroxy group at position 13 was established as a. The R configuration for C-20, C-22, C-24, and C-25 was also evident.

The 1 H and 13 C NMR spectra of withanolides 5–8 ( Tables 2 View Table 2 and 3 View Table 3 ) were closely related to those of 4, showing patterns typical of the physalngulidins. The almost identical 13 C NMR spectroscopic data for rings C, D and the side-chain of compounds 4–8 indicated that structural differences were restricted to substituents in rings A and B. The NMR spectroscopic data of compound 5 suggested a 1-oxo-3,5-diene system in rings A and B. This assumption was confirmed by comparison of the 1 H and 13 C NMR data of 5 and compound 2 ( Tables 1–3 View Table 1 View Table 2 View Table 3 ).

The 1 H NMR spectrum of 6 ( Table 2 View Table 2 ) displayed signals at δ 6.12 ( d, J = 9.7 Hz), 7.00 ( dd, J = 9.7, 6.0 Hz) and 6.25 ( d, J = 6.0 Hz), assigned to three olefinic hydrogens at C-2, C-3 and C-4, respectively. In addition to these resonances, the 1 H NMR spectrum displayed a carbynolic hydrogen signal at δ 4.34 ( d, J = 2.3 Hz) assigned to H-6. The presence of the 1-oxo-2,4-diene-6 b -hydroxy moiety was confirmed by the resonances at δ 203.8, 127.3, 139.4, 121.3, 152.7, 76.3, and 74.0 in the 13 C NMR spectrum, assigned to C1–C7, respectively. The b orientation of hydroxy group at C-6 was established by the strong intensity NOE observed between H-6 with H-4.

Regarding the A/B rings of compound 7, the 1 H and 13 C NMR spectra of 7 ( Table 2 View Table 2 ) exhibited a close resemblance to those of physalin Q isolated from Physalis alkekengi var. francheti ( Makina et al., 1995) possessing the same 1-oxo-3-ene-2 b,5 b -epidioxy-6 b - hydroxy substitution pattern. The 1 H NMR spectrum of 7 showed signals for the two coupled olefinic protons at δ 6.63 ( dd, J = 8.3, 6.5 Hz) and 6.80 ( dd, J = 8.3, 1.4 Hz), assigned to the H-3 and H-4 vicinal protons, respectively. In addition to these resonances, the 1 H NMR spectrum displayed a carbynolic hydrogen signal at δ 4.63 ( dd, J = 6.5, 1.5 Hz) which showed connectivity in the COSY spectrum with the hydrogen resonance at δ 6.80, indicating the hydroxy group at C-2. The signal at δ H 4.12 ( dd, J = 2.2, 1.5 Hz) indicated that the compound 7 also possessed hydroxyl substituent located at C-6, supported by HMBC correlations from H-4 to C-6. The 13 C NMR spectrum of 7 was in agreement with the structure proposed for the resonances at δ 204.7, 80.3, 124.8, 142.4, 83.0, and 70.8 assigned to C1C6, respectively. The b orientation of the C-2/C-5 peroxy bridge was established by the NOE observed between the H-4 and H-22 ( δ 4.73) signals, indicating the cis rings A/B fusion (see Supporting Information), while the b orientation of the hydroxy group at C-6 was established by a cross-correlation peak observed between the H-6 and H-4 resonances in the NOESY experiment.

Finally, the 1 H NMR spectrum of 8 ( Table 2 View Table 2 ) showed characteristic chemical shifts for the 1-oxo-2-ene-4 b,5 b -epoxy system at ring A, with signals for H-2 and H-3 being clearly distinguished at δ 6.09 ( dd, J = 9.9, 1.5 Hz) and δ 7.03 ( dd, J = 9.9, 4.3 Hz), respectively. The correlation observed in the COSY experiment between the pair H-3/H-4 led to the assignment of H-4 at δ 3.37 ( dd, J = 4.4, 1.4 Hz) of the 4 b,5 b -epoxy function. Cis rings A/B fusion was established from a NOESY experiment, where the NOE correlation observed for H-4/H-22 ( δ 4.64) indicated the presence of an epoxy group with a b -orientation (see Supporting Information). The 1 H NMR spectrum of 8 exhibited a signal at δ 3.45 assigned to a carbynolic proton at C-6 indicating a hydroxy group. The cross-correlation peak between the H-6 and H-4 resonances in the NOESY experiment indicated the b orientation of the hydroxy group at C-6. The 13 C NMR spectrum showed the expected chemical shifts for signals corresponding to rings A/B carbons at δ 198.1 (C-1), 130.7 (CH-2), 140.7 (CH-3), 54.1 (CH-4), 63.7 (C-5), 75.1 (CH-6), and 73.0 (CH-7), respectively.

3

The full and unambiguous proton and carbon NMR assignments for compounds 5–8 were confirmed using a combination of DEPT-135, COSY, HSQC, HMBC, and NOESY experiments. Moreover, the high-resolution mass measurements were in agreement with the proposed formulas.