Absolute Configuration of Periplosides C and F ... - ACS Publications

Mar 15, 2017 - Department of Natural Product Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,. People ...
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Absolute Configuration of Periplosides C and F and Isolation of Minor Spiro-orthoester Group-Containing Pregnane-type Steroidal Glycosides from Periploca sepium and Their T‑Lymphocyte Proliferation Inhibitory Activities Luo-Yi Wang,†,§ Jun-Jun Qin,†,‡,§ Zhen-Hua Chen,†,§ Yu Zhou,⊥ Wei Tang,⊥ Jian-Ping Zuo,*,⊥ and Wei-Min Zhao*,† †

Department of Natural Product Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China ‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, People’s Republic of China ⊥ State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Further phytochemical investigation of the root bark of Periploca sepium afforded nine new spiro-orthoester group-containing pregnane-type glycosides termed periplosides O−V and 3-O-formyl-periploside A. The structures of these glycosides along with the absolute configuration of the unique seven-membered formyl acetal-bridged spiro-orthoester function and the 4,6-dideoxy-3-O-methyl-Δ3-2-hexosulosyl moiety were elucidated on the basis of spectroscopic data interpretation and chemical transformation. The absolute configurations of the major compounds periplosides C and F were established by singlecrystal X-ray diffraction analysis. The isolated compounds were evaluated for their inhibitory activities against the proliferation of T-lymphocytes. As a result, periploside C, the most abundant glycoside containing a spiro-orthoester moiety found in the plant, exhibited the most favorite selective index value (SI = 82.5). The length and constitution of the saccharide chain in the periplosides were found to influence the inhibitory activity and the SI value.

C

to inhibit the proliferation of T-lymphocytes in vitro and to exhibit antirheumatoid arthritis and antihepatitis effects in experimental animal models.8,9 Moreover, the structure of periplocoside A, possessing a peroxy group, that was characterized in the 1980s was revised to the structure of periploside C (12), which bears a seven-membered formyl acetal-bridged spiro-orthoester function, assigned on the basis of chemical transformation and X-ray single-crystal diffraction methods.10−13 However, only the relative configurations of the spiro-orthoester group and the 4,6-dideoxy-3-O-methyl-Δ3-2hexosulose function at the C-3 position of the aglycone have been elucidated to date. We herein report the absolute configurations for the major compounds, periplosides C and F, and structural identification of nine new (1−9) and two known (10 and 11) minor spiro-orthoester group-containing

linically important immunosuppressants, e.g., cyclosporin A,1 tacrolimus,2 and rapamycin,3 have afforded undeniable advantages in organ grafts and the treatment of autoimmunological diseases, but they also cause rather serious adverse effects, including renal and liver toxicity, decreased cancer immunosurveillance, and increased susceptibility to infection.4 Therefore, the challenge of developing newgeneration immunosuppressive drugs with high efficacy and less side effects is urgent. Natural products have been demonstrated to be invaluable sources of immunosuppressive agents, and in recent years several compounds with unprecedented chemical structures and potent immunosuppressive activity have been isolated and characterized from Chinese medicinal plants.5−7 The root bark of Periploca sepium Bge. (Asclepiadaceae) has been used to treat rheumatoid arthritis in traditional Chinese medicine. In our previous study, periplocoside A, the major C21 pregnane-type steroidal glycoside found in P. sepium was found © 2017 American Chemical Society and American Society of Pharmacognosy

Received: January 5, 2017 Published: March 15, 2017 1102

DOI: 10.1021/acs.jnatprod.7b00017 J. Nat. Prod. 2017, 80, 1102−1109

Journal of Natural Products

Article

Table 1. 1H NMR (400 MHz) and 13C NMR (100 MHz) Spectroscopic Data for the Aglycone Moieties of Compounds 1−9 in CDCl3 (J in Hz)

C21 pregnane-type steroidal glycosides from the root bark of P. sepium along with their inhibitory activities against the proliferation of T-lymphocytes.

1−6, 8, 9 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

1

H NMR

1.09/1.84, m 1.94/1.65, m 3.66, m 2.42/2.29, m 5.35, 1.99, 1.50, 0.99,

br s m m m

1.55, m 1.53/1.75, m 1.79, m 1.17, m 1.62/1.95, m 0.72, 0.99, 3.75, 1.29,

s s m d (6.3)

7

13

C NMR 37.2 29.2 78.4 38.3 140.1 121.9 31.7 31.7 49.5 36.6 20.4 30.8 45.2 50.9 23.3 38.2 85.3 14.0 19.2 83.0 17.0

1

H NMR

1.13/1.86, m 1.87/1.62, m 4.72, m 2.35/1.90, m 5.38, 1.97, 1.49, 0.99,

br s m m m

1.57, m 1.51/1.75, m 1.80, m 1.16, m 1.64/1.91, m 0.71, 1.01, 3.72, 1.28,

s s m d (6.3)

13

C NMR 36.9 27.7 73.9 38.0 139.2 122.9 31.8 31.8 49.5 36.5 20.5 30.9 45.3 51.0 23.4 38.3 85.4 14.1 19.3 83.1 17.0

respectively (Table 2) in the HSQC spectrum. Selective irradiation of the anomeric proton signal at δH 4.38 (d, J = 8.0 Hz) and the methyl doublet at δH 1.36 (d, J = 6.2 Hz) in a 1DTOCSY experiment enabled extraction of the sub-spectrum of one monosaccharide unit from the highly overlapped 1H NMR spectrum of 1, which in concerted analysis of its 1H−1H COSY spectrum revealed the chemical shifts and coupling constants of H-1 (δH 4.38, d, J = 8.0 Hz), H-2 (δH 5.06, dd, J = 9.8, 8.0 Hz), H-3 (δH 3.26, dd, J = 9.8, 3.3 Hz), H-4 (δH 3.86, d, J = 3.3 Hz), H-5 (δH 3.57, m), and H-6 (δH 1.36, d, J = 6.2 Hz) to be discerned of a 2-O-acetyl-β-digitalose residue (Figure S1, Supporting Information). Together with analysis of the 1 H−1H COSY, HSQC, and HMBC data, the remaining monosaccharide units were identified as two β-cymarose and two β-canarose residues using similar 1D-TOCSY experiments via the irradiation of the isolated proton signals. The inter-sugar linkages were then established on the basis of the HMBC correlations between H-1digita (δH 4.38) and C-4cymII (δC 82.8), between H-1cymII (δH 4.73) and C-4canII (δC 87.7), between H1canII (δH 4.49) and C-4cymI (δC 82.4), and between H-1cymI (δH 4.92) and C-4ole (δC 82.4), and the sugar-aglycone linkage was determined according to the correlation signal between H-1canI (δH 4.57) and C-20 (δC 83.0). Therefore, 1 was characterized as shown and assigned the trivial name periploside O. Compound 2 produced a sodium adduct ion at m/z 1377.7198 in the HRESIMS, 42 mass units less than that of 1, which revealed its molecular formula to be C69H110O26, and was suggestive of the loss of one acetyl group from the structure of 1. A detailed comparison of the 1D-NMR data indicated the absence of a 2-O-acetyl group on the terminal βdigitalose unit in 2 as evidenced by the upfield shift of H-2 at δH 3.68 for this monosaccharide unit in 2 when compared with analogous resonance at δH 5.06 in 1 (Table 3). Further treatment of 1 with 1% NaOH in methanol for 10 min at room temperature yielded 2 as confirmed by co-TLC and LC-MS

■

RESULTS AND DISCUSSION Compound 1 was purified as a white amorphous powder with a deduced molecular formula of C71H112O27, according to the sodium adduct ion at m/z 1419.7275 (calcd for C71H112O27Na, 1419.7289) in its HRESIMS and from its 13C NMR data (Tables 1 and 2). The 1H and 13C NMR spectra of 1 exhibited characteristic signals of a 4,6-dideoxy-3-O-methyl-Δ3-2-hexosulosyl moiety and the C21 pregnane-type steroidal aglycone of other periplosides along with diagnostic proton signals at δH 5.14 and 4.74 (d, J = 7.6 Hz) and carbon signals at δC 113.5 and 86.2 of the formyl acetal-bridged spiro-orthoester function (Tables 2 and 3).12,13 The NMR data of 1 were similar to those of periploside C (12) with the only differences arising from the oligosaccharide moiety.10 Five anomeric proton signals were observed at δH 4.38 (d, J = 8.0 Hz), 4.49 (br d, J = 9.0 Hz), 4.57 (br d, J = 9.0 Hz), 4.73 (br d, J = 9.5 Hz), and 4.92 (br d, J = 9.5 Hz) in its 1H NMR spectrum (Table 3), corresponding to the carbon resonances at δC 102.4, 101.3, 100.7, 99.1, and 98.4, 1103

DOI: 10.1021/acs.jnatprod.7b00017 J. Nat. Prod. 2017, 80, 1102−1109

Journal of Natural Products

Article

Table 2. 13C NMR (100 MHz) Spectroscopic Data for the Sugar Moieties of Compounds 1−9 in CDCl3 position 1 2 3 4 5 6 OMe-3 1 2 3 4 5 6 OCH2O-3 1 2 3 4 5 6 OMe-3 1 2 3 4 5 6 OMe-3 1 2 3 4 5 6 OMe-3 1 2 3 4 5 6 OMe-3 1 2 3 4 5 6 -OAc OMe-3

1

2

hexo 97.1 185.9 147.6 118.4 68.7 22.8 54.9 can I 100.7 36.7 78.1 79.0 69.8 17.9 86.2 ole 113.5 36.7 77.5 82.4 69.8 18.0 57.4 cym I 98.4 35.6 76.7 82.4 68.6 18.1 58.1 can II 101.3 38.3 69.4 87.7 70.3 17.7

hexo 97.1 185.9 147.6 118.4 68.7 22.8 54.9 can I 100.7 36.7 78.1 79.0 69.8 17.9 86.2 ole 113.5 36.7 77.5 82.4 69.8 18.0 57.4 cym I 98.4 35.6 76.7 82.4 68.6 18.1 58.1 can II 101.3 38.3 69.4 87.7 70.3 17.7

cym II 99.1 35.5 76.2 82.8 68.7 17.4 58.6 digita 102.4 70.6 81.3 67.7 70.3 16.4 169.5/20.9 57.3

cym II 99.1 35.5 76.2 82.8 68.7 17.4 58.6 digita 104.7 70.6 82.8 67.7 70.3 16.4 57.3

3

4

5

hexo 97.1 185.9 147.6 118.4 68.7 22.8 54.9 can 100.7 36.8 78.1 79.0 69.6 17.9 86.2 ole 113.5 36.7 77.5 82.2 69.8 18.1 57.5 digito 98.3 36.5 66.3 82.1 68.2 18.1

hexo 97.1 185.9 147.6 118.4 68.7 22.8 54.9 can 100.7 36.8 78.1 79.0 69.6 17.9 86.2 ole 113.5 36.7 77.5 82.2 69.8 18.1 57.5 digito 98.3 36.5 66.3 82.1 68.2 18.1

hexo 97.1 185.9 147.7 118.4 68.8 22.9 54.9 can I 100.8 36.8 78.2 79.1 69.7 17.9 86.2 ole 113.6 36.6 77.6 82.2 69.9 18.2 57.5 digito 98.4 37.0 66.7 82.5 68.2 18.2

cym I 98.5 35.6 76.7 83.4 68.0 18.0 57.9 cym II 99.6 35.1 76.4 82.8 68.7 17.9 58.4 digita 102.4 70.7 81.4 67.8 70.2 16.4 169.5/20.9 57.3

cym I 98.5 35.6 76.7 83.4 68.0 18.0 57.9 cym II 99.6 35.1 76.4 82.8 68.7 17.9 58.4 digita 104.7 70.7 82.9 67.8 70.2 16.4

can II 100.3 38.3 69.3 87.7 70.6 17.8

57.3

analysis. Accordingly, the structure of 2 (periploside P) was characterized as shown.

cym I 99.3 35.3 76.7 82.1 69.1 17.8 58.4 cym II 99.7 35.0 75.0 74.8 67.5 18.0 170.3/21.0 58.2

6 hexo 97.2 185.9 147.7 118.4 68.8 22.9 54.9 can 100.8 36.8 78.2 79.1 69.7 17.9 86.3 ole I 113.6 36.7 77.6 82.6 69.9 18.2 57.6 cym I 98.5 35.4 77.0 82.4 68.8 18.2 57.9 cym II 99.7 35.4 77.0 82.4 68.4 18.2 58.0 cym III 99.7 35.4 77.0 82.6 68.3 18.2 58.1 ole II 101.4 35.3 80.5 75.3 71.5 17.9 56.3

7 formyl 160.6

can 100.8 36.9 78.2 79.1 69.7 18.0 86.3 ole 113.6 36.6 77.6 82.6 69.9 18.0 57.7 cym I 98.5 35.8 77.0 82.4 68.8 18.2 57.9 cym II 99.7 35.4 76.9 82.4 68.4 18.2 57.9 cym III 99.7 35.1 76.5 83.5 68.1 18.2 58.5 digita 102.5 70.8 81.5 67.9 70.3 16.5 169.5/21.0 57.4

8

9

hexo 97.2 185.9 147.8 118.4 68.8 22.9 54.9 can 100.8 36.8 78.2 79.1 69.7 18.0 86.3 ole 113.6 36.7 77.6 82.6 69.9 18.2 57.6 cym I 98.4 35.3 77.4 82.4 68.8 18.2 57.9 cym II 99.7 35.2 77.0 82.4 68.4 18.2 58.0 cym III 99.4 33.7 70.7 72.4 76.9 18.3 57.2

hexo 97.2 185.9 147.7 118.4 68.8 22.9 54.9 can 100.8 36.8 78.2 79.2 69.7 18.0 86.4 ole 113.8 36.6 79.2 75.6 70.5 17.8 56.9

Compound 3 gave a molecular formula of C71H112O27 according to the sodium adduct ion at m/z 1419.7284 in its 1104

DOI: 10.1021/acs.jnatprod.7b00017 J. Nat. Prod. 2017, 80, 1102−1109

Journal of Natural Products

Article

Table 3. 1H NMR (400 MHz) Spectroscopic Data for the Sugar Moieties of Compounds 1−9 in CDCl3 (J in Hz) position

1

2

3

4

5

6

hexo 5.04, s 5.78, d (3.0) 4.71, m 1.50, d (6.9) 3.62, s can I 4.57, br d (9.0) 2.20/1.64, m 3.51, m 3.33, t (9.5) 3.38, dq (9.5, 6.2) 1.30, d (6.2) 5.14/4.74 d (7.6) ole 2.44/1.57, m 3.50, m 3.26, t (9.5) 3.57, dq (9.5, 6.2) 1.27, d (6.2) 3.42, s cym I 4.92, br d (9.5) 2.10/1.54, m 3.77, br s 3.23, dd (9.4, 2.8) 3.87, dq (9.4, 6.2) 1.25, d (6.2) 3.42, s can II 4.49, br d (9.0) 2.23/1.60, m 3.53, m 2.95, t (9.5)

hexo 5.04, s 5.78, d (3.0) 4.71, m 1.50, d (6.9) 3.62, s can I 4.57, br d (9.0) 2.20/1.64, m 3.51, m 3.33, t (9.5) 3.38, dq (9.5, 6.2) 1.30, d (6.2) 5.14/4.74 d (7.6) ole 2.44/1.57, m 3.50, m 3.26, t (9.5) 3.57, dq (9.5, 6.2) 1.27, d (6.2) 3.42, s cym I 4.92, br d (9.5) 2.10/1.54, m 3.77, br s 3.23, dd (9.4, 2.8) 3.87, dq (9.4, 6.2) 1.25, d (6.2) 3.42, s can II 4.49, br d (9.0) 2.23/1.60, m 3.53, m 2.95, t (9.5)

hexo 5.04, s 5.78, d (3.0) 4.71, m 1.50, d (6.9) 3.62, s can 4.57, br d (9.0) 2.20/1.64, m 3.50, m 3.33, t (9.5) 3.37, dq (9.5, 6.2) 1.29, d (6.2) 5.14/4.74 d (7.6) ole 2.44/1.56, m 3.49, m 3.27, t (9.5) 3.56, dq (9.5, 6.2) 1.27, d (6.2) 3.42, s digito 4.97, br d (9.2) 2.09/1.66, m 4.21, br s 3.18, dd (9.5, 3.0) 3.78, dq (9.5, 6.2) 1.24, d (6.2)

hexo 5.04, s 5.78, d (3.0) 4.71, m 1.50, d (6.9) 3.62, s can 4.57, br d (9.0) 2.20/1.64, m 3.50, m 3.33, t (9.5) 3.37, dq (9.5, 6.2) 1.29, d (6.2) 5.14/4.74 d (7.6) ole 2.44/1.56, m 3.49, m 3.27, t (9.5) 3.56, dq (9.5, 6.2) 1.27, d (6.2) 3.42, s digito 4.97, br d (9.2) 2.09/1.66, m 4.21, br s 3.18, dd (9.5, 3.0) 3.78, dq (9.5, 6.2) 1.24, d (6.2)

hexo 5.04, s 5.78, d (3.0) 4.71, m 1.50, d (6.9) 3.62, s can I 4.56, br d (9.0) 2.19/1.63, m 3.49, m 3.32, t (9.5) 3.36, dq (9.5, 6.2) 1.28, d (6.2) 5.12/4.72 d (7.6) ole 2.45/1.58, m 3.51, m 3.27, t (9.5) 3.58, dq (9.5, 6.2) 1.28, d (6.2) 3.42, s digito 4.98, br d (9.6) 2.10/1.70, m 4.21, br s 3.19, dd (9.5, 3.0) 3.81, dq (9.5, 6.3) 1.23, d (6.2)

3.27, dq (9.5, 6.2) 1.25, d (6.2) cym II 4.73, br d (9.5) 2.12/1.60, m 3.79, br s 3.21, dd (9.4, 2.6) 3.97, dq (9.4, 6.2) 1.21, d (6.2) 3.44, s digita 4.26, d (7.4) 3.68, m

cym I 4.72, br d (9.5) 2.15/1.60, m 3.80, br s 3.24, dd (9.4, 2.6) 3.96, dq (9.4, 6.2) 1.23, d (6.2) 3.44, s cym II 4.78, br d (9.5) 2.15/1.73, m

3.18, m

3.77, br s

3.15, m

4

cym II 4.73, br d (9.5) 2.12/1.60, m 3.79, br s 3.21, dd (9.4, 2.6) 3.97, dq (9.4, 6.2) 1.21, d (6.2) 3.44, s digita 4.38, d (8.0) 5.06, dd (9.8, 8.0) 3.26, dd (9.8, 3.3) 3.86, d (3.3)

cym I 4.78, br d (9.5) 2.10/1.58, m 3.75, br s 3.21, dd (9.4, 2.6) 3.85, dq (9.4, 6.2) 1.25, d (6.2) 3.42, s cym II 4.72, br d (9.5) 2.10/1.60, m 3.77, br s 3.19, dd (9.4, 2.6) 3.95, dq (9.4, 6.2) 1.20, d (6.2) 3.44, s digita 4.27, d (7.4) 3.68, m

can II 4.55, br d (9.0) 2.22/1.60, m 3.52, m 2.94, t (9.5)

3.27, dq (9.5, 6.2) 1.25, d (6.2)

3.86, d (3.3)

cym I 4.78, br d (9.5) 2.10/1.58, m 3.75, br s 3.21, dd (9.4, 2.6) 3.85, dq (9.4, 6.2) 1.25, d (6.2) 3.42, s cym II 4.72, br d (9.5) 2.10/1.60, m 3.77, br s 3.19, dd (9.4, 2.6) 3.95, dq (9.4, 6.2) 1.20, d (6.2) 3.44, s digita 4.37, d (8.0) 5.06, dd (9.8, 8.0) 3.26, dd (9.8, 3.3) 3.86, d (3.3)

hexo 5.04, s 5.78, d (3.0) 4.71, m 1.51, d (6.9) 3.62, s can I 4.57, br d (9.0) 2.19/1.63, m 3.49, m 3.32, t (9.5) 3.36, dq (9.5, 6.2) 1.28, d (6.2) 5.13/4.73 d (7.6) ole I 2.45/1.58, m 3.51, m 3.27, t (9.5) 3.58, dq (9.5, 6.2) 1.28, d (6.2) 3.42, s cym I 4.92, br d (9.6) 2.10/1.54, m 3.77, br s 3.23, dd (9.4, 2.8) 3.86, dq (9.4, 6.2) 1.22, d (6.2) 3.43, s cym II 4.75, br d (9.5) 2.15/1.60, m 3.80, br s 3.24, m

3.86, d (3.3)

3.18, t (9.5)

5

3.57, m

3.57, m

3.57, m

3.57, m

6 -OAc

1.36, d (6.2) 2.06, s

1.36, d (6.2)

1.35, d (6.2) 2.04, s

1.35, d (6.2)

4.50, dd (9.5, 3.0) 3.95, dq (9.5, 6.3) 1.16, d (6.2) 2.10, s

1 4 5 6 OMe-3 1 2 3 4 5 6 OCH2O-3

2 3 4 5 6 OMe-3 1 2 3 4 5 6 OMe-3 1 2 3 4 5 6 OMe-3 1 2 3 4 5 6 OMe-3 1 2 3

3.18, m

3.30, dq (9.5, 6.2) 1.25, d (6.2)

1105

7

8

9

can I 4.57, br d (9.0) 2.19/1.63, m 3.52, m 3.32, t (9.5) 3.37, dq (9.5, 6.2) 1.28, d (6.2) 5.13/4.74 d (7.6) ole 2.45/1.58, m 3.52, m 3.26, t (9.5) 3.58, dq (9.5, 6.2) 1.30, d (6.2) 3.41, s cym I 4.91, br d (9.6) 2.11/1.62, m 3.77, br s 3.23, dd (9.4, 2.8) 3.86, m

hexo 5.05, s 5.78, d (3.0) 4.71, m 1.51, d (6.9) 3.63, s can I 4.58, br d (9.0) 2.19/1.63, m 3.53, m 3.33, t (9.5) 3.36, dq (9.5, 6.2) 1.27, d (6.2) 5.14/4.74 d (7.6) ole 2.45/1.58, m 3.51, m 3.27, t (9.5) 3.58, dq (9.5, 6.2) 1.29, d (6.2) 3.42, s cym I 4.93, br d (9.6) 2.15/1.61, m 3.62, br s 3.20, dd (9.4, 2.8) 3.59, m

hexo 5.04, s 5.78, d (3.0) 4.71, m 1.51, d (6.9) 3.62, s can I 4.59, br d (9.0) 2.22/1.65, m 3.53, m 3.35, t (9.5) 3.38, dq (9.5, 6.2) 1.32, d (6.2) 5.17/4.78 d (7.6) ole 2.54/1.50, m 3.39, m 3.21, t (9.2) 3.39, dq (9.5, 6.2) 1.34, d (6.2) 3.41, s

1.16, d (6.2) 3.42, s cym II 4.74, br d (9.5) 2.11/1.56, m 3.80, br s 3.24, m

1.23, d (6.2) 3.43, s cym II 4.75, br d (9.5) 2.13/1.63, m 3.81, br s 3.22, m

3.96, m

3.86, m

3.86, m

1.21, d (6.2) 3.43, s cym III 4.75, br d (9.5) 2.15/1.60, m 3.82, br s 3.22, m

1.18, d (6.2) 3.43, s cym III 4.74, br d (9.5) 2.11/1.60, m 3.77, br s 3.19, m

1.21, d (6.2) 3.44, s cym III 4.68, br d (9.5) 2.26/1.61, m 3.56, br s 3.20, m

3.95, m

3.86, m

3.80, m

1.22, d (6.2) 3.42, s ole II 4.49, br d (9.7) 2.30/1.45, m

1.21, d (6.2) 3.43, s digita 4.37, d (8.0) 5.07, dd (9.8, 8.0) 3.26, dd (9.8, 3.3) 3.86, d (3.3)

1.32, d (6.2) 3.42, s

3.30, dq (9.5, 6.1) 1.30, d (6.1)

formyl 8.02, s

3.57, m 1.36, d (6.2) 2.06, s DOI: 10.1021/acs.jnatprod.7b00017 J. Nat. Prod. 2017, 80, 1102−1109

Journal of Natural Products

Article

Table 3. continued position OMe-3

1 3.40, s

2 3.40, s

3 3.40, s

4 3.40, s

5 3.39, s

6 3.38, s

7

8

9

3.40, s

1oleII (δH 4.49) and C-4cymIII (δC 82.6) confirmed that this βoleandrose residue to be the terminal sugar moiety of the oligosaccharide chain in 6. Accordingly, the structure of 6 (periploside T) was characterized as shown. Compound 7 was obtained as a white amorphous powder, and a molecular formula of C66H106O25 was deduced from the sodium adduct ion at m/z 1321.6974 (calcd for C66H106O25Na, 1321.6921) in its HRESIMS and the 13C NMR data. Comparison of the NMR spectra of 7 with those of periploside C (12) revealed the absence of a 4,6-dideoxy-3-O-methyl-Δ3-2hexosulose moiety and the presence of a singlet proton signal at δH 8.02 in its 1H NMR spectrum that corresponded to the carbon signal at δC 160.6 in its HSQC spectrum (Tables 2 and 3). According to the molecular formula and the degree of unsaturation, these signals could be attributed to a formyl group, for which the location was determined to be at C-3 of the aglycone according to the HMBC correlation signal between the formyl proton (δH 8.02) and C-3 (δC 73.9). Thus, 7 was characterized as 3-O-formyl-periploside A. The HRESIMS of 8 (periploside U) gave a [M + Na]+ ion peak at m/z 1231.6638, revealing a molecular formula of C63H100O22, while compound 9 (periploside V) showed a molecular formula of C42H64O13 based on its HRESIMS (m/z 799.4261 [M + Na]+) and 13C NMR data. The 1D-NMR spectra of 8 and 9 both displayed characteristic signals for the 4,6-dideoxy-3-O-methyl-Δ3-2-hexosulosyl moiety, the C21 pregnane-type steroidal aglycone, and the formyl acetal-bridged spiro-orthoester group (Tables 1−3), which indicated them to be structural analogues of periploside C (12). However, their lower molecular weights relative to 12 suggested the presence of truncated oligosaccharide chains in the structures of 8 and 9. The molecular weight of compound 8 was 202 Da less than that of periploside C (12), suggesting its structure to be that of periploside C with the loss of the terminal 2-O-acetyl-βdigitalose unit. This structure was confirmed by the absence of relevant signals in its NMR spectra. Compared to those of periploside M,11 both the 1H and 13C NMR data of 8 exhibited evidence of an additional β-cymarose sugar unit with the anomeric proton at δH 4.68 (br d, J = 9.5 Hz) and the anomeric carbon at δC 99.4. Thus, the structure of 8 was determined as shown. Similarly, the structure of compound 9 was identified as shown, having a further truncated oligosaccharide chain, on the basis of comparisons of its HRESIMS and NMR data to those of periploside N.11−13 Two known compounds, periplosides M (10) and N (11), were also isolated with their structures elucidated by comparing the experimental and reported spectroscopic data.11−13 In a previous investigation, periploside F (13) was prepared by the alkaline hydrolysis of periploside C (12) with 1% NaOH in methanol, and the single-crystal X-ray diffraction methodology used for 13 enabled the establishment of its relative configuration.12,13 In the present investigation, single-crystal Xray diffraction analysis of 13 was performed again with Cu Kα radiation [Flack parameter: 0.10(8)],15 which enabled the final characterization of the absolute configuration of the pregnane skeleton, the seven-membered formyl acetal-bridged spiroorthoester fragment, and also the sugar moieties as Dcanaropyranose, D-oleandrose, D-cymaropyranose, and D-

HRESIMS and from the 13C NMR data (Tables 1 and 2). Comparison of the NMR data of 3 and 1 suggested a structural difference in the oligosaccharide chain. More specifically, the anomeric proton signal of the β-canarose unit at δH 4.49 (br d, J = 9.0 Hz) in the 1H NMR spectrum of 1 was replaced by a new anomeric proton signal at δH 4.97 (br d, J = 9.2 Hz) in 3 and an additional diagnostic proton signal for H-3 (δH 4.21, br s) of a β-digitoxose moiety (Table 3). The occurrence of the βdigitoxose residue was further confirmed via analysis of its 2D NMR spectra. The HMBC correlation signals between H-1digita (δH 4.37) and C-4cymII (δC 82.8), between H-1cymII (δH 4.72) and C-4cymI (δC 83.4), between H-1cymI (δH 4.78) and C-4digito (δC 82.1), and between H-1digito (δH 4.97) and C-4ole (δC 82.2) enabled the establishment of the inter-sugar linkages. Therefore, the structure of 3 (periploside Q) was characterized as shown. Similar to the structural elucidation of compound 2, 4 (periploside R) was determined to lack a 2-O-acetyl group on its terminal β-digitalose unit, on the basis of the comparison of its HRESIMS and NMR data with those of 3. In addition, compound 4 was produced after the treatment of 3 with 1% NaOH in methanol as confirmed by co-TLC and LC-MS analysis. The molecular formula of 5 was assigned to be C70H110O26 on the basis of the HRESIMS (m/z 1389.7200 [M + Na]+) and 13 C NMR data. Analysis of the 1D-NMR spectroscopic data of 5 suggested its structure to be closely related to that of periploside E (previously named periperoxide A),14 with the only difference occurring in the resonance of the terminal sugar unit. The anomeric proton signal at δH 4.35 (d, J = 8.0 Hz) of the terminal 2-O-acetyl-β-digitalose sugar residue in periploside E was replaced by a signal at δH 4.78 (br d, J = 9.5 Hz) in 5 (Table 3).14 Starting from this anomeric proton in the TOCSY spectrum of 5, a continuous coupled set of signals was identified as H-2 (δH 1.73, m), H-3 (δH 3.77, br s), H-4 (δH 4.50, dd, J = 9.5, 3.0 Hz), H-5 (δH 3.95, dd, J = 9.5, 6.3 Hz), and H-6 (δH 1.16, d, J = 6.2 Hz), which established the terminal monosaccharide in 5 as a β-cymarose sugar unit. The downfield chemical shift of H-4 at δH 4.50 suggested that the acetyl group in 5 is attached at OH-4 of the terminal β-cymarose. This was supported by further comparing the NMR data of 5 with those of perisaccharide B,14 which had the same oligosaccharide chain moiety as 5. Therefore, the structure of 5 (periploside S) was assigned as shown. Periploside T (6) was accorded the molecular formula, C70H112O25, according to its HRESIMS and 13C NMR data. The 1D-NMR data of 6 resembled those of periploside C (12) with slight differences in the terminal sugar unit. The anomeric proton signal at δH 4.49 (br d, J = 9.7 Hz) corresponded to the anomeric carbon signal at δC 101.4 in its HSQC spectrum. Further analysis of the 1H−1H COSY and HSQC spectra of 6 allowed the assignment of H-2 (δH 2.30, 1.45, m), H-3 (δH 3.15, m), H-4 (δH 3.18, t, J = 9.5 Hz), H-5 (δH 3.30, dq, J = 9.5, 6.1 Hz), and H-6 (δH 1.30, d, J = 6.1 Hz) of the terminal monosaccharide. These resonances corresponded to C-2 (δC 35.3), C-3 (δC 80.5), C-4 (δC 75.3), C-5 (δC 71.5), and C-6 (δC 17.9), respectively, and revealed the monosaccharide to be a βoleandrose residue. The HMBC correlation signals between H1106

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Figure 1. Alkaline and partial acid hydrolysis of periploside C (12) and ORTEP drawings of the products 13 and 14.

digitalopyranose as shown in Figure 1. Further partial acid hydrolysis of periploside C (12) in ethanol afforded compound 14 as a crystal, and the molybdenum target X-ray diffraction analysis of this crystal revealed its relative configuration. Since compounds 13 and 14 share the same pregnane skeleton and the absolute configuration of this skeleton was determined as described above for 13, the 4,6-dideoxy-3-O-methyl-Δ3-2hexosulose moiety in 14 was deduced to be in the D configuration (Figure 1). Due to the lack of standard sugar samples and a prioritization for bioassays to be conducted on the limited amounts of isolated compounds, the acid hydrolysis of the other new compounds to determine the absolute configuration of each monosaccharide using HPLC or GC analysis of the diastereomeric derivatives was not undertaken. The isolated spiro-orthoester group-containing periplosides (1−11) were assessed for their inhibitory activities against the proliferation of T-lymphocytes in vitro in a preliminary manner, using cyclosporin A as positive control. Periploside C (12), the most abundant spiro-orthoester moiety-bearing component in the root bark of P. sepium, exhibited the best selective index (SI) value. The length and constitution of the saccharide chain in the periplosides influences both the inhibitory activity and

the SI value. However, the substitution of a formyl group in replacement of the 4,6-dideoxy-3-O-methyl-Δ3-2-hexosulose function at the C-3 position of the aglycone may not be critical to this type of activity in this compound class (Table 4). Due to their structural complexity and immunosuppressive effects, the periplosides attracted the attention of synthetic chemists. Recently, Yu and co-workers reported the synthesis of periploside A in a total of 76 steps with the longest linear sequence of 29 steps and a 9.2% overall yield.19 Similar to our bioassay results for 8−11 with truncated saccharide chains, two synthetic periploside analogues with disaccharide chains in Yu’s work also exhibited weak inhibitory activities against Tlymphocyte proliferation. Further in-depth investigations of the structure−activity relationships of the periplosides and their mechanisms of pharmacological action would seem to be necessary.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained with a PerkinElmer 341 polarimeter. The IR spectra were recorded with a PerkinElmer 577 spectrometer. NMR spectra were recorded on a Bruker AM 400 MHz spectrometer with TMS as the internal reference. The HRESIMS data were obtained on a Bruker

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1:1−1:3, v/v) followed by preparative HPLC (CH3CN-H2O, 90:10− 100:0, v/v). Periploside O (1): white amorphous powder; [α]18 D −10 (c 0.41, CHCl3); IR (KBr) νmax 3454, 2935, 1749, 1716, 1637, 1454, 1375, 1313, 1238, 1157, 1095, 1067, 1003 cm−1; 1H and 13C NMR data, see Tables 1−3; HRESIMS m/z 1419.7275 [M + Na]+ (calcd for C71H112O27Na, 1419.7289). Periploside P (2): white amorphous powder; [α]18 D −8 (c 0.20, CHCl3); IR (KBr) νmax 3464, 2937, 1747, 1714, 1639, 1454, 1379, 1157, 1095, 1059 cm−1; 1H and 13C NMR data, see Tables 1−3; HRESIMS m/z 1377.7198 [M + Na]+ (calcd for C69H110O26Na, 1377.7183). Periploside Q (3): white amorphous powder; [α]18 D −3 (c 0.28, CHCl3); IR (KBr) νmax 3448, 2935, 1751, 1718, 1637, 1456, 1375, 1238, 1157, 1095, 1059, 1003 cm−1; 1H and 13C NMR data, see Tables 1−3; HRESIMS m/z 1419.7284 [M + Na]+ (calcd for C71H112O27Na, 1419.7289). Periploside R (4): white amorphous powder; [α]18 D −8 (c 0.15, CHCl3); IR (KBr) νmax 3508, 2935, 1714, 1639, 1456, 1381, 1159, 1094, 1061, 1003 cm−1; 1H and 13C NMR data, see Tables 1−3; HRESIMS m/z 1377.7151 [M + Na]+ (calcd for C69H110O26Na, 1377.7183). Periploside S (5): white amorphous powder; [α]22 D −9 (c 0.25, CHCl3); IR (KBr) νmax 3457, 2935, 1736, 1639, 1456, 1375, 1240, 1160, 1095, 1059, 1003 cm−1; 1H and 13C NMR data, see Tables 1−3; HRESIMS m/z 1389.7200 [M + Na]+ (calcd for C70H110O26Na, 1389.7183). Periploside T (6): white amorphous powder; [α]22 D −25 (c 0.23, CHCl3); IR (KBr) νmax 3462, 2933, 1716, 1639, 1454, 1379, 1317, 1159, 1103, 1059, 1003 cm−1; 1H and 13C NMR data, see Tables 1−3; HRESIMS m/z 1375.7389 [M + Na]+ (calcd for C70H112O25Na, 1375.7390). 3-O-Formyl-periploside A (7): white amorphous powder; [α]22 D + 5 (c 0.34, CHCl3); IR (KBr) νmax 3496, 2935, 1753, 1726, 1454, 1373, 1317, 1236, 1159, 1095, 1057, 1003 cm−1; 1H and 13C NMR data, see Tables 1−3; HRESIMS m/z 1321.6974 [M + Na]+ (calcd for C66H106O25Na, 1321.6921). Periploside U (8): white amorphous powder; [α]22 D −6 (c 0.16, CHCl3); IR (KBr) νmax 3448, 2935, 1720, 1639, 1454, 1373, 1163, 1093, 1059, 1005 cm−1; 1H and 13C NMR data, see Tables 1−3; HRESIMS m/z 1231.6638 [M + Na]+ (calcd for C63H100O22Na, 1231.6604). Periploside V (9): white amorphous powder; [α]22 D −35 (c 0.29, CHCl3); IR (KBr) νmax 3446, 2935, 1716, 1633, 1458, 1383, 1161, 1097, 1066, 972 cm−1; 1H and 13C NMR data, see Tables 1−3; HRESIMS m/z 799.4261 [M + Na]+ (calcd for C42H64O13Na, 799.4245). Alkaline Hydrolysis of Periploside C (12) for the Production of 13. The procedures were identical to those used in ref 12. X-ray Crystallographic Data of 13. Triclinic space group P1, a = 11.5223(5) Å, b = 11.5593(5) Å, c = 14.8540(6) Å, α = 109.544(2)°, β = 94.088(2)°, γ = 113.055(2)°, V = 1668.16(13) Å3, Z = 1, Dx = 1.221 mg/m3, μ(Cu Kα) = 0.76 mm−1, and F(000) = 663. Crystal dimensions: 0.1 × 0.08 × 0.05 mm3. A total of 36 648 reflections were measured with 10 137 independent reflections (Rint = 0.043). Final R1 = 0.036, wR2 = 0.094, Flack parameter = 0.10(8). Crystallographic data have been deposited in the Cambridge Crystallographic Data Center as entry 1450004, and can be obtained, free of charge, on application to the Director, CCDC, 12 Union Rd., Cambridge CB21EZ, U.K. [fax, +44(0)-1233-336033; E-mail, [email protected]]. Partial Acid Hydrolysis of Periploside C (12) for the Production of 14. Periploside C (12, 100 mg) was dissolved with 0.001 N H2SO4 in ethanol and stirred at room temperature until no starting material remained according to TLC detection. The reactant was then partitioned between ethyl acetate and water. The organic layer was evaporated to dryness and the residue was subjected to column chromatography over silica gel and eluted with CH2Cl2− MeOH (20:1, v/v) to produce 14 (35 mg). Compound 14: 1H NMR (CDCl3, 400 MHz) δ 0.72, 0.99 (each 3H, s), 1.24 (3H, t, J = 7.0 Hz), 1.30, 1.36 (each 3H, d, J = 6.2 Hz),

Table 4. In Vitro T-Lymphocyte Proliferation Inhibiting Activities of Compounds 1−14 compound

CC50 (μM)

IC50 (μM)

SIa

periploside O (1) periploside P (2) periploside Q (3) periploside R (4) periploside S (5) periploside T (6) 3-O-formyl-periploside A (7) periploside U (8) periploside V (9) periploside M (10) periploside N (11) periploside C (12) periploside F (13) compound 14 cyclosporin A

>50 3.4 >50 24.6 49.0 71.9 3.9 >100 20.4 3.6 20.0 52.0 >20 64.2 4.5

2.5 1.2 2.0 1.3 5.8 11.7 0.37 8.4 3.7 7.7 8.4 0.63 0.91 2.1 0.08

>20.1 2.8 24.8 18.4 8.4 6.1 10.5 >11.9 5.5 0.5 2.4 82.5 >22.0 31.3 56.3

a

The selectivity index (SI) was defined as the ratio of the concentration of the compound that reduced cell viability to 50% (CC50) to the concentration of the compound required to inhibit the proliferation by 50% relative to the control value (IC50). Daltonics microTOF QII mass spectrometer. Semipreparative HPLC was performed on a Unimicro Technologies 2010 instrument equipped with a Waters X-Bridge C18 column (19 mm × 100 mm, 5 μm). Column chromatography (CC) was performed with silica gel (300−400 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, People’s Republic of China), LiChroprep C18 Lobar (40−63 μm, Merck), CHP-20P MCI (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), Toyopearl HW-40F (Tosoh Corporation, Tokyo, Japan), D801 macroporous resin (Huazhen Scientific Corporation, Shanghai, People’s Republic of China), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). Plant Material. Samples of the root bark of P. sepium, wild grown in Gansu Province, People’s Republic of China, were purchased from Shanghai Yanghetang Chinese Herbal Medicine Co., Ltd. in 2009, and identified by Prof. Jingui Shen of the Shanghai Institute of Materia Medica. A voucher specimen (No. WJ-20091112) has been deposited at Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation. The air-dried root bark of P. sepium (50 kg) was powdered and percolated with 95% EtOH (3 × 25 L) at room temperature to produce a crude extract (6.5 kg), which was then suspended in 30% EtOH and centrifuged. The precipitate (3.1 kg) was washed with petroleum ether (boiling range 60−90 °C), suspended in 70% EtOH. and subsequently applied to column chromatography over D801 macroporous resin and eluted in a gradient of 70%, 80%, 85%, 90%, and 95% EtOH. The 85% EtOH eluent (500 g) was separated by column chromatography over silica gel with a gradient elution of petroleum ether/acetone (6:1−2:1, v/v), yielding fractions 1−9. Dereplication analysis of each fraction by LC-DAD-MS revealed the occurrence of possible new compounds in fractions 6 and 9. Fraction 6 was chromatographed over RP-18 (CH3CN−H2O, 3:1−1:0, v/v) to produce fractions 61−63. Fraction 62 was purified using Toyopearl HW-40F (eluted with 95% EtOH) to produce compound 9 (15 mg). Fraction 63 was further chromatographed over silica gel (CHCl3− MeOH, 1:0−50:1, v/v) yielding fractions 631−636. Fraction 632 was separated using Toyopearl HW-40F (95% EtOH) followed by preparative HPLC (CH3CN−H2O, 85:15−100:0, v/v) to produce 5 (15 mg) and 8 (7 mg). Compound 6 (20 mg) was obtained by preparative TLC (CHCl3−MeOH, 20:1, v/v) from fraction 634. Fraction 635 was subjected to separation over Toyopearl HW-40F CC (95% EtOH) and then silica gel CC (CHCl3−MeOH, 50:1, v/v) to produce compound 7 (38 mg). Fraction 636 was applied to Sephadex LH-20 CC (95% EtOH) to produce compounds 10 (6 mg) and 11 (4 mg). Compounds 1 (35 mg), 2 (18 mg), 3 (37 mg), and 4 (14 mg) were isolated from fraction 9 by silica gel CC (petroleum ether-EtOAc, 1108

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1.51 (3H, d, J = 6.8 Hz), 3.58, 3.77 (each 1H, dq, J = 9.6, 7.0 Hz), 3.63 (3H, s), 3.75 (1H, m), 4.57 (1H, br d, J = 9.8 Hz), 4.71 (1H, m), 4.73, 4.79 (each 1H, d, J = 7.2 Hz), 5.05 (1H, s), 5.35 (1H, br s), 5.78 (1H, d, J = 3.0 Hz); 13C NMR (CDCl3, 100 MHz) δ: 95.6 (CH2), 81.7 (CH, can-3), 64.0 (CH2), 15.0 (CH3), the remaining data were identical to those of periploside B;16 ESIMS m/z 685 [M + Na]+, HRESIMS m/z 685.3934 [M + Na]+ (calcd for C37H58O10Na, 685.3928). X-ray Crystallographic Data of 14. Monoclinic space group P21, a = 12.5069(16) Å, b = 23.641(5) Å, c = 25.417(6) Å, α = 90.00°, β = 98.788(2)°, γ = 90.00°, V = 7426.9(16) Å3, Z = 2, Dx = 1.211 mg/m3, μ(Mo Kα) = 0.71 mm−1, and F(000) = 2930 e−. Crystal dimensions: 0.30 × 0.15 × 0.12 mm3. A total of 27 481 reflections were measured with 21 112 independent reflections (Rint = 0.0606). Final R1 = 0.0761, wR2 = 0.1886. Crystallographic data have been deposited in the Cambridge Crystallographic Data Center as entry 1053879, and can be obtained, free of charge, on application to the Director, CCDC, 12 Union Rd., Cambridge CB21EZ, U.K. [fax, +44(0)-1233-336033; email, [email protected]]. T-Lymphocyte Proliferation Assay. The bioassay methods were identical to those mentioned in the literatures,17,18 and the experimental protocols can also be found in the Supporting Information.

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(7) Fan, Y. Y.; Zhang, H.; Zhou, Y.; Liu, H. B.; Tang, W.; Zhou, B.; Zuo, J. P.; Yue, J. M. J. Am. Chem. Soc. 2015, 137, 138−141. (8) Wan, J.; Zhu, Y. N.; Feng, J. Q.; Chen, H. J.; Zhang, R. J.; Ni, J.; Chen, Z. H.; Hou, L. F.; Liu, Q. F.; Zhang, J.; Yang, L.; Tang, W.; Yang, Y. F.; Nan, F. J.; Zhao, W. M.; Zuo, J. P. Int. Immunopharmacol. 2008, 8, 1248−1256. (9) Zhang, J.; Ni, J.; Chen, Z. H.; Li, X.; Zhang, R. J.; Tang, W.; Zhao, W. M.; Yang, Y. F.; Zuo, J. P. Acta Pharmacol. Sin. 2009, 30, 1144− 1152. (10) Oshima, Y.; Hirota, T.; Hikino, H. Heterocycles 1987, 26, 2093− 2098. (11) Itokawa, H.; Xu, J. P.; Takeya, K.; Watanabe, K.; Shoji, J. Chem. Pharm. Bull. 1988, 36, 982−987. (12) Wang, L. Y.; Chen, Z. H.; Zhou, Y.; Tang, W.; Zuo, J. P.; Zhao, W. M. Phytochemistry 2011, 72, 2230−2236. (13) Wang, L. Y.; Chen, Z. H.; Zhou, Y.; Tang, W.; Zuo, J. P.; Zhao, W. M. Phytochemistry 2013, 95, 445. (14) Feng, J. Q.; Zhang, R. J.; Zhou, Y.; Chen, Z. H.; Tang, W.; Liu, Q. F.; Zuo, J. P.; Zhao, W. M. Phytochemistry 2008, 69, 2716−2723. (15) Flack, H. D. Acta Crystallogr., Sect. A: Found. Crystallogr. 1983, 39, 876−881. (16) Itokawa, H.; Xu, J. P.; Takeya, K. Chem. Pharm. Bull. 1988, 36, 2084−2089. (17) Zhu, Y. N.; Zhao, W. M.; Yang, Y. F.; Zuo, J. P.; et al. J. Pharmacol. Exp. Ther. 2006, 316, 662−669. (18) Zhu, Y. N.; Zhong, X. G.; Feng, J. Q.; Zhao, W. M.; Zuo, J. P.; et al. J. Pharmacol. Exp. Ther. 2006, 318, 1153−1162. (19) Zhang, X.; Zhou, Y.; Zuo, J.; Yu, B. Nat. Commun. 2015, 6, 5879.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00017. NMR spectra of all new compounds (PDF) X-ray crystallographic data for 13 (CIF) X-ray crystallographic data for 14 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel./fax: 86-21-50806052. E-mail: [email protected] (W.Z.). *Tel./fax: 86-21-50806701. E-mail: [email protected] (J.Z.). ORCID

Wei-Min Zhao: 0000-0001-8879-069X Author Contributions §

L.-Y.W., J.-J.Q., and Z.-H.C. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Science and Technology Commission of Shanghai Municipality (No. 15401901200) and the National Natural Science Foundation (No. 81273399).

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REFERENCES

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