Withanolides from three species of the genus Deprea (Solanaceae). Chemotaxonomical considerations Author Casero, Carina N. Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Instituto Multidisciplinario de Biología Vegetal (IMBIV-CONICET), Casilla de Correo 495, 5000 Córdoba, Argentina Author Oberti, Juan C. Author Orozco, Clara I. Author Cárdenas, Alejandro Author Brito, Iván Author Barboza, Gloria E. Author Nicotra, Viviana E. text Phytochemistry 2015 2015-02-28 110 83 90 http://dx.doi.org/10.1016/j.phytochem.2014.11.015 journal article 10.1016/j.phytochem.2014.11.015 1873-3700 10485784 2.3. Withanolides isolated from Deprea zamorae 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 ). 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 and 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 , 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 and 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 ). Fig. 2. Physangulidine proposed biosynthetic route. The 1 H NMR spectrum of 6 ( 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 ) 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 ) 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. Table 2 1 H NMR spectroscopic data of compounds 4–8 in CDCl. a 3
Position 4 5 6 7 8
2a 5.94 dd (10.0, 2.6) 3.33 dt (20.4, 3.0) 6.12 d (9.7) 4.63 dd (6.5, 1.4) 6.09 dd (9.9, 1.5)
2b – 2.84 ddd (20.4, 4.8, 1.0) – – –
3a 6.80 ddd (10.0, 4.9, 2.6) 5.87 ddd (9.7, 4.8, 3.0) 7.00 dd (9.7, 6.0) 6.63 dd (8.3, 6.5) 7.03 dd (9.9, 4.3)
4 a 3.00 dd (21.4, 4.9) 6.13 dd (9.7, 2.5) 6.25 d (6.0) 6.80 dd (8.3, 1.4) 3.37 dd (4.4, 1.5)
4 b 3.31 br d (21.4) – – – –
6 5.62 dd (5.6, 1.8) 5.61 d (5.4) 4.34 d (2.3) 4.12 dd (2.2, 1.5) 3.45 br s
7 4.22 t (3.8) 4.33 t (4.5) 4.14 t (2.5) 4.24 t (2.3) 4.20 t (2.7)
8 1.37 dd (12.5, 3.8) 1.44 dd (12.6, 4.0) 1.96 m 1.82 m 1.81 dd (11.6, 3.2)
9 2.30 td (12.5, 4.1) 2.44 m 1.91 m 1.85 m 2.08 m
11a 2.47 m 2.03 m 2.06 m 2.43 m 2.06 m
11b 1.30 m 1.15 m 1.29 m 1.43 m 1.28 m
12a 1.93 td (13.0, 3.5) 1.93 m 1.76 m 1.73 m 1.72 m
12b 1.73 dt (13.0, 3.3) 1.71 m 1.56 m 1.66 m 1.60 m
15a 2.37 td (12.8, 5.2) 2.38 td (12.9, 4.8) 2.36 m 2.33 m 2.35 m
15b 1.67 ddd (14.0, 9.5, 4.9) 1.70 m 1.76 m 1.73 m 1.75 m
16a 2.02 m 2.03 m 2.02 m 2.05 m 2.05 m
16b 1.98 m 2.03 m 1.98 m 2.02 m 2.01 m
18 1.20 s 1.20 s 1.23 s 1.22 s 1.21 s
19 1.20 s 1.29 s 1.41 s 1.50 s 1.45 s
20 2.51 m 2.52 m 2.51 m 2.61 m 2.56 m
21a 2.25 dd (14.1, 6.0) 2.24 dd (14.2, 6.1) 2.12 dd (14.3, 6.2) 2.16 dd (13.9, 6.5) 2.16 dd (14.0, 6.5)
21b 1.56 dd (14.1, 11.9) 1.57 dd (14.2, 11.8) 1.55 m 1.56 m 1.57 dd (14.0, 11.7)
22 4.70 td (3.6, 1.7) 4.70 td (3.6, 1.8) 4.61 td (3.3, 1.6) 4.73 td (3.6, 1.4) 4.64 td (3.6, 1.6)
23a 2.44 dd (15.5, 1.7) 2.46 dd (15.5, 1.8) 2.25 dd (15.4, 1.6) 2.00 m 2.37 dd (15.4, 1.8)
23b 2.01 m 2.02 m 1.96 m 2.04 m 1.99 m
27 1.15 s 1.15 s 1.12 s 1.16 s 1.16 s
28 1.18 s 1.19 s 1.17 s 1.21 s 1.20 s
a Chemicals shifts ( δ ) downfield from TMS, J couplings (in parentheses) in Hz. 400.13 MHz. Table 3 13 C NMR spectroscopic data of compounds 18 in CDCl. a 3
Position 1 2 3 4 5 6 7 8
18 R and 18 S 18 R and 18 S 18 R
1 203.0 R /203.3 S 209.6 R /209.8 S 204.1 202.2 208.3 203.8 204.7 198.1
2 128.0 39.5 127.7 128.0 39.4 127.3 80.3 130.7
3 144.9 R /144.6 S 125.2/125.5 144.9 144.5 126.1 139.4 124.8 140.7
4 33.3 129.0/129.1 33.2 32.8 128.5 121.3 142.4 54.1
5 141.4 R /140.9 S 144.6 R /144.3 S 135.1 143.4 146.0 152.7 83.0 63.7
6 126.4 R /126.0 S 126.8/127.3 125.1 121.7 122.5 76.3 70.8 75.1
7 64.5 64.7 26.7 64.9 65.3 74.0 73.0 73.0
8 38.9 R /37.9 S 37.8 R /36.8 S 47.5 39.3 38.7 38.4 37.8 37.2
9 34.7 R /35.2 S 33.3 R /33.7 S 43.2 32.7 31.4 39.6 40.1 37.4
10 51.2 R /51.1 S 52.8 52.7 50.6 52.4 52.8 47.7 49.4
11 25.4 24.0/24.3 25.8 24.7 23.5 21.4 24.1 23.1
12 34.6 R /37.0 S 34.4 R /36.8 S 43.2 37.6 37.4 37.9 37.8 37.9
13 58.1 R /58.9 S 58.2 R /59.1 S 79.1 71.1 71.3 71.0 71.0 71.0
14 50.6 R /49.0 S 50.2 R /48.5 S 101.1 88.1 87.7 89.0 89.0 88.9
15 25.7 R /25.2 S 25.3/25.2 27.4 27.7 27.8 27.5 27.3 27.2
16 25.9 R /27.2 S 25.9 R /27.2 S 15.0 33.3 33.3 33.7 33.2 33.5
17 56.8 R /56.8 S 56.9 R /56.7 S 55.0 107.1 107.2 108.0 108.4 108.2
18 101.3 R /103.9 S 101.3 R /103.7 S 106.1 21.8 21.9 22.1 22.4 22.1
19 18.3 R /18.4 S 19.5 R /19.6 S 18.2 18.2 19.3 17.3 15.4 13.1
20 84.9 R /85.2 S 84.8 R /85.2 S 85.0 41.9 42.0 41.9 41.1 41.6
21 21.7 R /24.7 S 21.7 R /24.4 S 25.8 26.6 26.7 26.4 26.7 26.2
22 80.6 R /80.4 S 80.6 R /80.6 S 78.0 74.6 74.7 74.5 74.6 74.4
23 31.2 R /32.0 S 31.2 R /32.0 S 31.1 40.8 40.9 40.8 40.4 40.7
24 148.0 R /148.8 S 148.2 R /148.9 S 155.3 70.7 70.8 70.7 70.9 71.2
25 121.9 R /122.2 S 122.1 R /121.9 S 125.1 47.6 47.7 47.5 47.7 47.6
26 165.6/165.7 165.9 166.5 177.8 177.8 177.3 177.2 177.1
27 12.4/12.5 12.4 56.8 14.2 14.3 14.2 14.3 14.3
28 20.4/20.5 20.4 20.1 27.9 27.9 27.8 28.3 28.1
a Chemical shifts ( δ ) downfield from TMS; 100.03 MHz. Fig. 3. ORTEP diagram of physangulidine D ( 4 ) showing one molecule of the asymmetric unit with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level and H atoms are shown as small spheres of arbitrary radii. 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.