Withanolide Structural Revisions by 13C NMR Spectroscopic Analysis

Feb 19, 2016 - Atta-ur-Rahman; Dur-e-Shahwar; Naz , A.; Choudhary , M. I. Phytochemistry 2003, 63, 387– 390 DOI: 10.1016/S0031-9422(02)00727-6...
0 downloads 0 Views 1MB Size
Article pubs.acs.org/jnp

Withanolide Structural Revisions by Inclusive of the γ‑Gauche Effect

13

C NMR Spectroscopic Analysis

Huaping Zhang and Barbara N. Timmermann* Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, United States S Supporting Information *

ABSTRACT: A classic withanolide is defined as a highly oxygenated C28 ergostanetype steroid that is characterized by a C22-hydroxy-C26-oic acid δ-lactone in the ninecarbon side chain. Analysis of the reported 13C NMR data of classic withanolides with hydroxy groups (C-14, C-17, and C-20) revealed that (1) a hydroxy (C-14 or C-17) substituent significantly alters the chemical shifts (C-7, C-9, C-12, and C-21) via the γ-gauche effect; (2) the chemical shift values (C-9, C-12, and C-21) reflect the orientation (α or β) of the hydroxy moiety (C-14 or C-17); (3) a double-bond positional change in ring A (Δ2 to Δ3), or hydroxylation (C-27), results in a minuscule effect on the chemical shifts of carbons in rings C and D (from C-12 to C18); and (4) the 13C NMR γ-gauche effect method is more convenient and reliable than the traditional approach (1H NMR shift comparisons in C5D5N versus CDCl3) to probe the orientation of the hydroxy substituent (C-14 and C-17). Utilization of these rules demonstrated that the reported 13C NMR data of withanolides 1a−29a were inconsistent with their published structures, which were subsequently revised as 1−16 and 12 and 18−29, respectively. When combined, this strongly supports the application of these methods to determine the relative configuration of steroidal substituents.

W

orientation of the hydroxy group at C-14 significantly affects the 13C chemical shift of the γ-positioned C-12 via the γ-gauche effect. This observation was successfully applied to determine the 14-hydroxy orientation in withanolides.18 Numerous 17-hydroxy withanolides have been reported: a 17α-hydroxy group was observed in withanone (31),16,19 14βhydroxywithanone (32),20,21 and cinerolide (33);22 whereas a 17β-hydroxy group was observed in 17-epiwithanone (34)23,24 and withanolide E (35)16,25 (Figure 3). Among them, X-ray diffraction crystallography has confirmed the structures of 31,19 32,20,21 33,22 and 35,25 which provides a solid basis for trustworthy NMR data analysis. In addition, the routine usage of 2D NMR techniques over the past 30 years has generated a literature 13C NMR database of fully assigned classic withanolides (including 31−35 and those from our own laboratory) containing C-17 oxygenation. Close inspection of the data set has revealed that 13C NMR chemical shifts could be utilized to determine the precise orientation of a 17-hydroxy moiety in an unmodified withanolide, a feat not yet explored in the literature. Herein, we illustrate that C-14, C-17, and C-20 hydroxy groups induce the γ-gauche effects on the chemical shift of carbons (C-7, C-9, C-12, and C-21) in classic withanolides. Application of these deviations has led to the conclusion that the reported structures of several oxygenated (C-14, C-17, or C-20) withanolides 1a−29a (Figures 1 and 2) are in need of revision. The majority of these withanolides were reported

ithanolides are a family of highly oxygenated C28 ergostane-type steroids that are primarily present in Solanaceae, which include the Jaborosa, Physalis, Tubocapsicum, and Withania genera.1 Approximately 900 withanolides have been reported from natural sources, where diverse oxygenation in the steroid skeleton (from C-1 to C-7 and from C-11 to C19) as well as in the side chain (from C-20 to C-28) has been observed. These include approximately 550 classic (also called unmodified) withanolides, which contain a four-ringed steroidal nucleus adhered to a nine-carbon side chain incorporating a C22-hydroxy-C26-oic acid δ-lactone functionality (1−116, Figures 1−3), where oxygenation at C-14, C-17, and C-20 is commonly encountered.2 Withanolides have gained significant interest in the scientific community due to their biological activities inclusive of antitumor, anti-inflammatory, immunomodulatory, and insect-antifeent activities.1,2 Recently, we reported the isolation and characterization of approximately 70 withanolides from Datura wrightii Regel,3 Jaborosa caulescens var. bipinnatif ida (Dunal) Reiche,4 Physalis coztomatl Dunal,5 P. hispida (Waterf.) Cronquist,6 P. longifolia Nutt.,7,8 Vassobia brevif lora (Sendtn.) Hunz.,9 and Withania somnifera (L.) Dunal.10,11 Consequently, these isolates were examined against the reported 13C NMR data, and discrepancies were observed in the literature, which resulted in the structural revisions of coagulansin A (30a)5,12 to withacoagulin D (30),13 and withasomnilide (31a)14,15 to withanone (31),16 respectively (Figure 3). Chemical shift-based 13C NMR analysis is an extremely powerful tool to probe the orientation of a substituent on the rigid ring system of the steroid skeleton.17 For instance, © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 23, 2015

A

DOI: 10.1021/acs.jnatprod.5b00648 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Structures of withanolides (1a−16a) (claimed to contain a C14−O−C20 bridge) and their respective revisions (1−16).

Figure 2. Structures of reported withanolides (17a−29a) and their respective revisions (12, 18−29).

B

DOI: 10.1021/acs.jnatprod.5b00648 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 3. continued

C

DOI: 10.1021/acs.jnatprod.5b00648 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 3. continued

D

DOI: 10.1021/acs.jnatprod.5b00648 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 3. Reported withanolides 30, 30a, 31, 31a, and 32−116 utilized for 13C NMR comparisons.

tional isomers (with a Δ3 functionality): 17-epiwithanolide K (2),50,41 withanolides I and K (40, 42),16 and withacoagulin F (15),13 respectively (Figures 1, 3 and Table S2, Supporting Information). Section II: γ-Gauche Effect Rules of Steroid Elucidation. Oxygenation at C-14, C-17, or C-20 significantly affects the 13C NMR chemical shift of carbons in rings B−D due to the γ-gauche effect, which is summarized in the following subsections. (IIa) The 14-Hydroxy Group Affects the Chemical Shifts of C-7, C-9, and C-12 in Rings B−D. Both the OH14α and OH-14β groups shield the γ-positional C-7 (∼4−5 ppm decrease) and C-9 (∼3−7 ppm decrease) by the γ-gauche effect. However, differences caused by OH-14α or OH-14β include the following: (1) OH-14α shields C-12 (∼4−7 ppm decrease), whereas OH-14β does not; (2) the shielding effect on C-9 by OH-14α (∼7 ppm decrease) is greater than OH-14β (∼3−5 ppm decrease), and this is due to the axial−axial positions of H-9 and OH-14α. These empirical rules are consistent with the published data, as revealed by the 13C NMR comparison of different types of steroids 43−64 (Figure 3 and Table S3, Supporting Information): androstanes [testosterone (43),51 14α-hydroxytestosterone (44),52,53 androst-4-ene-3,17dione (45),51 and 14α-hydroxyandrost-4-ene-3,17-dione (46)51]; pregnanes [3β-hydroxy-5β-pregnan-20-one (47),54 5β-pregnane-3,20-dione (50),54 and progesterone (53);54 their corresponding 14α-hydroxy derivatives: 3β,14α-dihydroxy-5β-pregnan-20-one (48),54 14α-hydroxy-5β-pregnane3,20-dione (51),54 and 14α-hydroxyprogesterone (54);54 or 14β-hydroxy derivatives: 3β,14β-dihydroxy-5β-pregnan-20-one (49),55 14β-hydroxy-5β-pregnane-3,20-dione (52),55 and 14βhydroxyprogesterone (55)56]; cholestane 56;57 cardenolide 57;58 and withanolides [withacoagulin E (14),13 withanolide G (39),16 withaferin A (58),59 physapubenolide (59),18 5,6deoxywithanolide D (60),34 ixocarpanolide (61),60 14αhydroxyixocarpanolide (62),60 physalolactone B (63),61 and 1-deacetyl-14α-hydroxyphysalolactone B (64)62]. Therefore, the 14-hydroxy group affects the chemical shifts (C-7, C-9, and C-12), and the chemical shifts (C-9 and C-12) can be utilized to accurately determine the orientation of the 14-hydroxy group in steroids. (IIb) The 17-Hydroxy Group Affects the Chemical Shifts of C-12 and C-21 (>3 ppm). Prior to the widespread

(1993−2012) from the genera Ajuga, Jaborosa, Physalis, Tubocapsicum, and Withania: specifically, the species A. parvif lora Benth. [28-hydroxycoagulin D (16a)26]; J. bergii Hieron. [jaborosalactol 23 (28a)27] and J. leucotricha (Speg.) Hunz. [jaborosalactone 8 (27a)28]; P. peruviana L. [27deoxycoagulin (2a),29 2,3-dihydro-3β-O-β-D-glucopyranosylcoagulin C (6a),30 and 2,3-dihydro-5α,6β-dihydroxywithanolide H (17a)31]; T. anomalum (Franch. & Sav.) Makino [tubocapsanolide D (29a)32]; W. coagulans (Stocks) Dunal [coagulin (1a),33 27-deoxycoagulin (2a),34 coagulins B−G (13a,35 3a,35 14a,35 15a,35 9a,36 4a36), I−K (5a,37 10a,37 11a37), M−O (12a,38 8a,38 18a38), and R (7a),39 coagulansin B (19a),12 withacoagulides A and B (21a and 20a),40 withacoagulin A (22a),13 and 14β,15β-epoxy-17β,20-dihydroxy-1oxowitha-3,5,24-trienolide (26a)41]; and W. somnifera (L.) Dunal [withanolide P (23a),16,42 withaoxylactone (24a),43 and 14α,15α-epoxywithaferin A (25a)44]. As a result, withanolides 1a−29a were revised as 1−16 and 12 and 18−29, respectively (Figures 1 and 2).



RESULTS AND DISCUSSION Section I: General Rules for Classic Withanolide-Based 13 C NMR Analysis. (Ia) It has been well documented that hydroxylation at the C-27 peripheral position of classic withanolides (from a CH3 to a CH2OH functionality) induces minuscule effects on the carbon chemical shifts in the rings A− D (from C-1 to C-19) (chemical shift value difference < 0.5 ppm),16 which is consistent with the fact that a similar chemical environment produces similar NMR chemical shift values.45 This can be illustrated through 13C NMR comparison of five structurally similar withanolide pairs: namely, withanone (31)16 and withanolides B, G, I, and J (37,46 39,16 40,16 4116,47) against their corresponding 27-hydroxy derivatives (36,48 38,49 13,16 9,16 3012,13) (Figure 3 and Table S1, Supporting Information). (Ib) Similarly, the positional change of a double bond from Δ2 to Δ3 in ring A has little effect on the carbon chemical shifts in rings C−D (from C-12 to C-18) and the side chain (from C-20 to C-28) (chemical shift value difference < 0.5 ppm). This was exemplified by 13C NMR comparison of four pairs of withanolide isomers, withanolides F, G, and J (3,47 39, 16 41 16,47 ) and withacoagulin E (14), 13 with a Δ 2 functionality, against their corresponding double-bond posiE

DOI: 10.1021/acs.jnatprod.5b00648 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

application of 13C NMR analysis, 1H NMR chemical shift comparison (in C5D5N versus CDCl3) of methyl groups (H318 and H3-21) was used universally to determine the orientation of the 17-hydroxy group in steroids.63 It was observed that both the OH-17α and OH-17β shield the γpositioned C-12 (∼7−9 ppm decrease), as demonstrated by the 13 C NMR comparison of steroids 65−87: androstanes [6564 and its 17β-hydroxy derivatives (4351,64 and 6664)], pregnane (67);65 cholestanes [68,65 and the related 17β-hydroxy (69)66 and 17α-hydroxy (70)67 derivatives]; and withanolides (31, 34, 37, 58, and 71−87) (Figure 3, Tables S1, S3, and S4, Supporting Information). This analysis compared the data of each classic withanolide against their corresponding 17-hydroxy derivatives: (1) withanolide B (37)46 and the corresponding 17α-hydroxy (31)16 and 17β-hydroxy (34)23,24 derivatives; (2) withanolide A (71)16 and its 17α-hydroxy derivative (72);16 (3) daturalactone-4 (73)68 and its 17β-hydroxy derivative (74);16 (4) withaferin A (58)59 against its 17α-hydroxy derivative (75);69 (5) 5β,6β-epoxy-1-oxowitha-3,24-dienolide (76)59 against jaborosalactone L (77);70 (6) 16α-acetoxy5β,6β-epoxy-1-oxowitha-2,24-dienolide (78)71 and its 17αhydroxy derivative (79);71 (7) 5,6-deoxywithaferin A (80)72 against its 17α-hydroxy derivative (81);73 (8) 27-hydroxy-1oxowitha-2,5,24-trienolide (82)74 against cilistol A (83);75 (9) 5α,6β-dihydroxywithaferin A (84)43,76 against cilistadiol (85);77 and (10) 6α-chloro-5β-hydroxywithaferin A (86)10 against 6αchloro-5β,17α-dihydroxywithaferin A (87).10 In addition, there are insignificant chemical shift differences (3 ppm) on the γpositioned C-21 methyl group in the side chain of classic withanolides, where the OH-17α shields C-21 (∼4 ppm decrease), whereas OH-17β does not. This can be represented through the C-21 chemical shift (ppm) comparison of structurally related withanolides, such as (1) withanolide B (37, 13.3)46 and the corresponding 17α-hydroxy (31, 9.6),16 17β-hydroxy (34, 13.5),23,24 14α,17α-dihydroxy (88, 9.7),21 and 14β,17α-dihydroxy (32, 10.0)21 derivatives; (2) daturalactone-4 (73, 13.6)68 and its 17β-hydroxy derivative (74, 13.6);16 (3) withaferin A (58, 13.3)59 and its 17α-hydroxy (75, 9.4),69 4,27-dideoxy-17α-hydroxy (77, 9.4),32 and 4,27dideoxy-3-ene (76, 13.3)59 derivatives; (4) 16α-acetoxy5β,6β-epoxy-1-oxowitha-2,24-dienolide (78, 13.7)71 and its 17α-hydroxy derivative (79, 9.5);71 (5) 5,6-deoxywithaferin A (80, 13.3)72 and its 17α-hydroxy derivative (81, 9.1);73 (6) 27hydroxy-1-oxowitha-2,5,24-trienolide (82, 13.3)74 against cilistol A (83, 10.1);75 (7) 4-deoxy-5α,6β-dihydroxywithaferin A (84, 13.2)43,76 against cilistadiol (85, 9.9);77 and (8) 6α-chloro5β-hydroxywithaferin A (86,10 13.3) against 6α-chloro-5β,17αdihydroxywithaferin A (87,10 9.4) (Tables S1−S5, Supporting Information). Therefore, a 17-hydroxy group affects the chemical shifts (C-12 and/or C-21), and the chemical shift value of the C-21 methyl group can be utilized to accurately distinguish between 17-hydroxy isomers in classic withanolides. (IIc) The 14,17-Dihydroxy Group Shields C-12 (>7 ppm). The presence of a 14,17-dihydroxy group produces an additive γ-gauche effect on C-12. It was observed that 14α,17α-,

14α,17β-, and 14β,17α-dihydroxy withanolides produce distinctly different C-12 chemical shifts (∼27, 30, 34 ppm, respectively) from each other. (Section III covers the revision of questionable structures purported to be 14β,17β-dihydroxy withanolides.) This is supported by the reported C-12 shift (ppm) of withanolides containing (1) a 14α,17α-dihydroxy [withacoagulin D (30, 27.6),13 cinerolide (33, 27.6),22 withanolides J and K (41,16,47 27.1; 42,16 26.5), 14αhydroxywithanone (88,21 28.1), and 17-epiwithanolide E (89,16 26.5)]; (2) a 14α,17β-dihydroxy [17-epiwithanolide K (2, 29.8),41 withanolides E and F (3,47 30.1; 35,16 30.1), 28hydroxywithanolide E (90,81 30.0), 2,3-dihydro-3β,28-dihydroxywithanolide F (91,82 30.0), 28-hydroxywithanolide F (92,79 30.2), and 4β-hydroxywithanolide F (93,83 30.2)]; and (3) a 14β,17α-dihydroxy [14β-hydroxywithanone (32,21 34.3 ppm), 14-epiwithanolide J (94,40 34.8 ppm), and 14epiwithanolide K (95,78 33.2 ppm)] group, in the literature (Figure 3 and Table S5, Supporting Information). Therefore, the C-12 chemical shift value reflects the precise orientation of the 14-hydroxy and 17-hydroxy groups in classic 14,17dihydroxy steroids. (IId) The 20-Hydroxy Group Shields C-16 (>4 ppm). In classic withanolides, a present 20-hydroxy group shields the γpositioned C-16 (∼4−6 ppm decrease) due to the γ-gauche effect. This was elucidated through the C-16 chemical shift (ppm) comparison of withanolides and structurally related 20hydroxy derivatives: (1) withanone (31,16 37.1) against its 20hydroxy derivative (72,16 32.7); (2) withanolide B (39,46 27.3) against its 20-hydroxy derivative (71,16 21.8); (3) withaferin A (58,59 27.2) against withanolide D (96,84 21.9); (4) 17αhydroxywithaferin A (75,69 36.3) against philadelphicalactone A (97,80 32.0); (5) 27-hydroxy-1-oxowitha-2,5,24-trienolide (82,74 27.3) against its 20-hydroxy derivative (98,34 21.9); (6) physagulin D (99,85 27.3) against its 20-hydroxy derivative (100,86 22.5); and (7) 6α-chloro-5β-hydroxywithaferin A (86,10 27.2) against 27-deoxy-6α-chloro-5β,20-dihydroxywithaferin A (101,87 21.9) (Figure 3 and Table S6, Supporting Information). Therefore, the 20-hydroxy group shields C-16 (>4 ppm), and this can be utilized to assign the C-16 chemical shift value in the presence of a 20-hydroxy group in steroids. Section III Analysis of Inconsistencies (Structure, 13C NMR Data) of Reported Classic Withanolides. Compounds 1a−29a (Figures 1 and 2) are a group of structurally related classic withanolideswith similar oxygenation (C-14, C-17, or C-20) patternsthat were previously isolated and characterized in the literature, such as coagulin (1a),33 27deoxycoagulin (2a),34,29 and coagulins B−G, I−K, M−O, and R (13a,35 3a,35 14a,35 15a,35 9a,36 4a,365a,37 10a,37 11a,37 12a,38 8a,38 18a,38 and 7a39). Among them, withanolides 1a− 5a and 7a−15a (as well as 24a−26a) were reported from the same research group.33−39,41,43,44 In addition, withanolides 1a− 16a were claimed to contain an uncommon C(14)−O−C(20) bridge linkage, a structural feature that is believed to be specific to the Withania coagulans species.78,88 Inconsistencies were observed between the reported NMR data and their respective structures 1a−29a, after the aforementioned 13C NMR chemical shift rules were applied to validate these reported structures. On the basis of the reported structures, one would expect to observe similar 13C NMR data for rings C and D (from C-12 to C-18) when compared with the values of (1) nor-27-hydroxy withanolides (2a, 3a, 14a, and 15a) against their corresponding 27-hydroxy derivatives (1a, 4a, 13a, and 9a) and (2) F

DOI: 10.1021/acs.jnatprod.5b00648 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 13C NMR Comparison of Selected Withanolides (1a, 42, 2a, 2, 7a, 7, 8a, 20a, 20, 22a, and 106)a C

1a33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 solv.

211.5 39.7 121.3 129.2 140.5 127.2 25.0 33.0 35.1 52.4 21.0 26.4 51.3 85.7 32.7 34.0 88.9 20.0 18.5 80.9 20.2 81.0 32.6 156.3 124.5 166.3 56.1 20.1 CDCl3

4216 39.8 121.5 129.4 140.7 127.4 25.2 34.2 34.9 52.6 20.7 26.5 51.4 85.9 33.0 34.2 89.2 20.4 18.9 79.6 20.2 81.0 32.4 151.6 120.9 12.2 20.7 CDCl3

2a34

2a29

250

7a39

750

8a38

20a13

2040

22a13

10690

211.1 39.6 121.0 129.3 139.6 127.3 26.4 33.6 36.2 52.3 22.1 30.8 54.3 84.0 33.9 36.3 88.0 20.3 17.3 78.5 17.7 81.4 33.7 150.5 121.0 165.7 12.3 20.0 CDCl3

210.2 39.4 121.9 128.7 140.2 127.9 25.4 33.4 35.1 52.4 21.4 29.9 53.9 81.9 34.2 35.7 87.4 20.2 19.0 78.1 20.3 80.8 31.7 150.7 120.1 165.9 12.0 19.7 DMSO-d6

210.5 39.7 121.2 129.5 140.5 127.9 25.9 33.9 36.1 52.3 21.8 30.1 53.8 83.1 34.2 37.9 87.9 19.9 20.5 79.1 20.6 79.8 32.4 150.4 121.5 165.9 12.4 20.6 CDCl3

210.3 47.1 67.9 40.1 134.7 125.2 25.1 35.5 35.2 52.1 21.5 29.8 54.1 82.0 34.0 36.1 87.0 19.7 17.8 78.2 19.7 81.0 31.7 151.8 120.5 167.5 11.1 19.8 CDCl3 + CD3OD

209.7 47.6 68.6 40.0 135.4 125.9 25.9 36.2 35.9 53.1 22.2 30.4 54.1 82.5 34.6 37.1 87.9 20.6 18.4 78.7 19.1 81.5 32.5 152.3 121.4 166.0 12.4 20.7 CDCl3

211.2 46.3 75.3 38.3 134.1 126.6 25.9 32.7 36.1 53.3 22.5 32.5 54.4 82.8 76.1 47.9 89.1 20.7 18.3 79.2 19.8 81.7 35.0 150.8 121.3 166.8 12.4 20.0 C5D5N

210.5 40.1 121.8 129.7 141.2 128.7 26.2 32.8 34.6 53.0 22.4 32.8 54.6 82.8 76.2 48.1 89.2 20.9 20.2 79.4 20.1 81.7 35.2 150.8 121.4 166.8 12.5 20.1 C5D5N

210.6 40.2 122.3 129.8 141.4 128.9 26.2 32.9 34.8 53.2 22.6 32.9 54.8 82.9 76.4 48.2 89.3 21.1 20.4 79.5 20.1 81.9 35.3 150.9 122.0 166.9 12.6 20.2 C5D5N

210.2 39.5 121.4 129.1 140.1 126.5 28.9 30.5 39.5 52.1 22.8 30.4 53.6 151.1 117.0 39.8 88.0 21.1 19.9 75.8 19.4 80.2 31.8 150.3 121.0 165.5 12.2 20.5 CDCl3

203.9 127.8 145.3 33.2 135.1 124.1 28.7 32.0 41.5 50.4 23.9 30.5 53.3 151.2 117.1 39.7 88.1 21.0 18.7 75.8 19.5 80.3 31.8 150.4 121.0 165.5 12.3 20.6 CDCl3

a

The assignments of some carbons of 1a, 2a, 7a, and 8a were switched. More details about the 13C NMR data of withanolides 1a−29a reported in the literature and their revised structures 1−16, 12, and 18−29 or structurally related withanolides are listed in Tables S8−S11 in the Supporting Information.

withanolides with a Δ3 functionality (1a, 2a, 9a, and 15a) against their corresponding Δ2 isomers (4a, 3a, 13a, and 14a). However, among 1a−4a, 9a, and 13a−15a, there were more than 5−10 ppm discrepancies between the reported chemical shifts of C-15 (25.0 in 1a33 against 35.0 in 3a35), C-17 (85.7 in 1a33 against 78.5 in 2a34), and C-20 (74.7 in 9a36 against 84.5 in 13a35) (Table S7, Supporting Information). In addition, the C-7 chemical shifts were reported to be around 26 ppm in a number of 1-oxo-5-ene-14-hydroxy withanolides with (1) a 2ene functionality [withacoagulins D and E (30,13 25.4; 14,13 26.3); withanolides F, G, H, and J (3,47 25.1; 39,16 25.3; 13,16 25.3; and 41,47 24.9, respectively)] or (2) a 3-ene functionality [withacoagulins C and F (20,13 26.2; 15,13 27.2), withanolides I and K (40,16 25.6; 42,16 25.2)] (Figures 1−3, Tables S1, S5, and S8−S10, Supporting Information). Theoretically, the C-7 chemical shifts of 1a−4a, 9a, and 13a−15a should also be in the 26 ppm region, yet they were reported in the 31−37 ppm range (Table S7, Supporting Information). These inconsistencies initiated concern about the validity of the structures of 1a− 29a, which were subsequently revised in the following section. (IIIa) Structure Revisions of Classic Withanolides Containing a C(14)−O−C(20) Bridge Linkage (1a−16a). In 1993, the structure of the first natural withanolide purported to contain a C(14)−O−C(20) bridge linkage, coagulin (1a),33 was deduced from EIMS and the chemical shift values of three oxygenated quaternary carbons (reported as C-14, C-17, and C20 at δ 88.9, 85.7, and 80.9, respectively). Although this

assignment satisfied the degree of unsaturation required by HREIMS, there was no further evidence to support the presence of this uncommon bridge moiety. In addition, this proposed linkage required that the 17-hydroxy group be assigned in the β-orientation.33 However, the C-12 chemical shift (26.4 ppm) of 1a33 indicates that the 17-hydroxy group is α-oriented. In fact, the 13C NMR data from C-1 to C-22 (including those carbons in rings A−D) of 1a are superimposable on those of another W. coagulans withanolide,40 namely, withanolide K (42),16 which contains a 14α,17α,20trihydroxy functionality (Table 1 and Table S8, Supporting Information). This implies that 1a and 42 contain an identical steroid nucleus. As such, the structure of 1a was revised as 27hydroxywithanolide K (1) (Figure 1). Perhaps the reporting group concluded structure 1a (as opposed to 1) due to two factors: (1) a [M − H2O]+ peak, not a molecular ion peak [M] + , was observed by EIMS, which is common in polyoxygenated withanolides,89−91 and (2) although 2D NMR spectroscopic methods (including 1H−1H COSY, HMQC, and HMBC in the paper33) were applied, the 13C NMR data of 1a (C-2, C-6, C-7, C-14, C-15, C-16, and C-17) seem inaccurately assigned. For example, due to the γ-gauche and β-effects of the 14-hydroxy group, the C-7 and C-15 chemical shifts of 1a should be 25.0 and 32.7 ppm as opposed to the reported values (34.0 and 25.0 ppm33) (Tables S7 and S8, Supporting Information). G

DOI: 10.1021/acs.jnatprod.5b00648 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(IIIb) Structural Revisions of Withanolides Containing 14-OH and/or 17-OH Groups (17a−29a). The structure of withanolide 17a31 with a 14α-hydroxy functionality was reported as a stereoisomer of 12 with a 14β-hydroxy group. However, the 13C NMR data of 17a are superimposable on those of 12a (Table S9, Supporting Information). Therefore, the structure of 17a was also revised as 12 (Figure 2). The structure of coagulin O (18a)38 was reported as 11, although this characterization was not supported by its 13C NMR data. Specifically, the C-9 and C-12 chemical shifts (38.4 and 41.2 ppm) of 18a imply the presence of a 14β-hydroxy, rather than the reported 14α-hydroxy group. Therefore, the structure of 18a was revised as 18 (14-epicoagulin O) (Figure 2 and Table S9, Supporting Information). Similarly, the structure of coagulansin B (19a),12 which was reported to contain a 14αhydroxy functionality but with similar C-9 and C-12 chemical shifts (38.2 and 39.0 ppm), was revised as 19 (14epicoagulansin B) (Figure 2 and Table S10, Supporting Information). Withacoagulide B (20a)40 and withacoagulin C (20),13 both recently isolated from W. coagulans, were reported as a pair of 15-epi stereoisomers. However, the 13C NMR data of 20a (15βhydroxy) and 20 (15α-hydroxy) are superimposable (Table 1 and Table S10, Supporting Information). The reported NOE correlation between the two adjacent protons (H-15 and H16α in the five-membered-ring D) in 20a represents insufficient evidence that should not have been utilized to propose the reported H-15α (or OH-15β)13 orientation assignment.96,97 Combined, the structure of 20a was revised as 20 (Figure 2). In the same paper, the structure of withacoagulide A (21a)40 was reported as a Δ2 isomer of 20a. The 13C NMR data (rings C and D, as well as the side chain) of 20a and 21a are superimposable (Table S10, Supporting Information); therefore the structure of 21a (reported to possess leishmanicidal activity40) was revised as 21 (14α,15α,17β,20-tetrahydroxy-1oxowitha-2,5,24-trienolide, Figure 2), a known withanolide isolated from W. coagulans and characterized by multiple independent research groups from all over the world.13,47,93 The structure of withacoagulin A (22a)13 was reported to contain a 14-ene-17β,20-dihydroxy functionality, where the 17β-hydroxy group was deduced from the pyridine-induced 1H NMR high-frequency shift for both C-18 and C-21 methyl groups.13 However, the 13C NMR data of rings C and D and the side chain of 22a are superimposable on those of 14-ene17α,20-dihydroxy withanolides [withanolide L (106)90 and withacoagulin H (107)78] and quite different from those of 14ene-17β,20-dihydroxy withanolides [5α,6β,17β,20-tetrahydroxy-1-oxowitha-2,14,24-trienolide (108),16,42,92 3β,17β,20trihydroxy-1-oxowitha-5,14,24-trienolide (109), 26 and 4β,5β,6α,17β,20-pentahydroxy-1-oxowitha-2,14,24-trienolide (110)98] (Figure 3, Table 1, and Table S10, Supporting Information). For example, the C-12 chemical shifts (ppm) were reported as 30.4, 30.5, and 30.8 in 22a,13 106,13 and 107,78 but 36.4, 36.3, and 35.7 in 108,16 109,26 and 110,98 respectively. Therefore, the structure of 22a (reported to possess immunosuppressive activity13) was revised as 22 [a Δ3 isomer of withanolide L (106)] (Figure 2). In summary, examination of the NMR data of withanolides reported from W. coagulans revealed that (1) many of its isolates (2a−4a, 7a, 10a, 13a−15a, 18a, 20a, and 21a), which were claimed to be new structures at their time of publication, were in fact known compounds previously reported from the same species (2−4, 7, 10, 13−15, 18, 20, and 21); (2) the

The structure of 27-deoxycoagulin (2a) was reportedly deduced through EIMS and the 13C NMR data comparison between 2a and 1a.29,34 However, there are more than 5 ppm differences (C-15, C-17, and C-20) between 2a and 1a (Table S7, Supporting Information). Similar to the case of 1a, the C-7, C-12, and C-15 chemical shifts (reported as 33.9, 26.4, and 30.8 ppm,29,34 Table S7, Supporting Information) of 2a were switched and are actually 26.4, 30.8, and 33.9 ppm, respectively. In fact, the 13C NMR data of 2a are superimposable on those of another withanolide isolated from W. coagulans, namely, 17epiwithanolide K (2),41 which contains a 14α,17β,20-trihydroxy functionality (Table 1 and Table S8, Supporting Information). As such, the structure of 2a should be revised as 2 (Figure 1). Following the same rationale, the structures of coagulins C (3a,35 a Δ2 isomer of 2a), G (4a,36 a Δ2 isomer of 1a), I (5a,37,92 a 5α,6β-dihydroxy derivative of 3a), N (8a,38 a 15αhydroxy derivative of 6a), and R (7a,39 the aglycone of 6a) and withanolide glycoside 6a (2,3-dihydro-3β-O-β-D-glucopyranosyl derivative of 3a)30 were revised as withanolide F (3),13,48,78,93 27-hydroxywithanolide F (4),78 withanolide S (5, 5α,6βdihydroxywithanolide F),16,42 15α-hydroxycoagulin L (8), 2,3dihydro-3β-hydroxywithanolide F (7, the aglycone of 6),50 and coagulin L (6),37 respectively (Figure 1 and Table 1). These six structural revisions were achieved by comparing the NMR data of (1) 3a against 3, 7, and 28-hydroxywithanolide F (92);79 (2) 4a against 3, 30, and 92; (3) 5a against 3 and phyperunolides B and D (102 and 103);94 (4) 6a against 6; (5) 7a against 7; and (6) 8a against withacoagulin C (20)13 and coagulin H (104)37 (Figures 1−3, Table 1, Tables S8 and S9, Supporting Information). It should be pointed out that the structure of coagulansin A (30a),12 isolated from W. coagulans, was reported as 4, although this characterization was not supported by its 13C NMR data. Furthermore, the structure of 30a was recently revised as 30 (Figure 3) after 13C NMR data analysis.5 The structure of coagulin F (9a) was purported as a 17deoxy derivative of 1a, which was reportedly deduced by NMR comparison between 9a and 1a.36 Similar to the case of 1a, the C-7 and C-12 chemical shifts of 9a (reported as 31.5 and 25.2)36 were switched and are actually 25.2 and 31.5 ppm, respectively (Table S9, Supporting Information). Furthermore, due to the γ-gauche effect caused by the C-20 hydroxy group, the C-16 chemical shift of 9a is 21.9 ppm rather than the reported value (30.2 ppm36). In fact, the 13C NMR data of 9a are superimposable on those of 27-hydroxywithanolide I (9)16 (Table S9, Supporting Information). Therefore, the structure of 9a was revised as 9 (Figure 1). Similarly, the structures of coagulins B (13a,35 a 17-deoxy derivative of 4a), D (14a,35 a 17-deoxy derivative of 3a), E (15a,35 a 17-deoxy derivative of 2a), J (10a,37 a 2,3-dihydro-3β-hydroxy derivative of 9a), K (11a,37 a 27-deoxy-3β-O-β-D-glucopyranosyl derivative of 10a), and M (12a,38 a 17-deoxy-3,4-dihydro-5α,6β-dihydroxy derivative of 1a) and 28-hydroxycoagulin D (16a)26 were revised as withanolide H (13),16,78 withacoagulins E (14)13 and F (15),13 2,3-dihydro-3β,27-dihydroxywithanolide I (10),95 2,3dihydro-3β-O-β-D-glucopyranosylwithanolide I (11), 3,4-dihydro-5α,6β,27-trihydroxywithacoagulin E (12), and 28-hydroxywithacoagulin E (16), respectively. These seven structural revisions were achieved by comparing the NMR data of (1) 10a against 10; (2) 11a against 3β-O-β-D-glucopyranosyl-2,3dihydro-27-hydroxywithanolide I (105);62 (3) 12a against 14 and 15; (4) 13a against 13; (5) 14a against 14; (6) 15a against 15; and (7) 16a against 14 (Figures 1 and 3 and Table S9, Supporting Information). H

DOI: 10.1021/acs.jnatprod.5b00648 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

functionality, was revised as 27, with a 14α,17α-dihydroxy group (Figure 2). Following the same logic and reasoning, the structure of jaborosalactol 23 (28a, C-9: 35.5; C-12: 28.2 ppm)27 was revised as 28, based on 13C NMR comparison with structurally related withanolides, such as jaborosalactone E (112, C-9: 43.3; C-12: 40.2 ppm),101 withanolide C (113, with a 14α-hydroxy group, C-9: 34.9),102 and phyperunolide C (114, with a 14α-hydroxy group, C-9: 35.7)94 (Figure 2 and Table S11, Supporting Information). The structure of tubocapsanolide D (29a)32 was reported to contain a 17α-hydroxy functionality. However, the reported C21 chemical shift value (13.0 ppm) suggests that a 17β-hydroxy group is present in 29a. This is supported by comparing the C21 chemical shift of 29a against those of related withanolides, such as withaferin A (58, 13.3 ppm),59 17α-hydroxywithaferin A (82, 9.4 ppm),69,103 5β,6α-dihydroxywithaferin A (115, 13.3 ppm),43,76 and tubocapsanolide F (116, 9.9 ppm)32 (Figure 3 and Table S11, Supporting Information). As such, the structure of 29a was revised as 29 (Figure 2).

coagulin-related withanolides (2a−16a) were primarily characterized by comparing their NMR data against those of the inaccurately characterized structure of coagulin (1a). To date, a total of 16 withanolides (1a−16a) were claimed in the literature to contain the uncommon C(14)−O−C(20) bridge moiety. Our analysis concluded that no such C(14)−O−C(20) bridge is present in any of these reported withanolides, and as a result the structures of 1a−16a were revised as 1−16, respectively (Figure 1). In 1975, the structure of withanolide P (23a)99 was reported to contain a 14α,17β-dihydroxy functionality, which was proposed on the basis of the pyridine-induced 1H NMR shifts of two methyl groups (C-18 and C-21)42 and the fact that a 14β-hydroxy withanolide had never been documented [the first 14β-hydroxy withanolide, 14β-hydroxywithanone (32),21 was published in 1981]. Although both compounds contain identical rings A−D, the 13C NMR data of 23a16 (C-9: 39.0 ppm) are quite different from those of withanolide F (3, C-9: 35.2 ppm, Table S8, Supporting Information). However, the 13 C NMR data of 23a are similar to those of the withanolide F steroisomer 95, with a 14β,17α-dihydroxy functionality (C9:39.0 ppm, Table S10, Supporting Information). Furthermore, the C-21 chemical shift of 23a (10.3 ppm, Table S10, Supporting Information) is similar to that of the 14β,17αdihydroxy withanolide 32 (10.0 ppm, Table S5, Supporting Information) but quite different from that of the 17β-hydroxy withanolide 34 (13.5 ppm, Table S4, Supporting Information).23,24 These chemical shifts suggest the presence of a 14β,17α-dihydroxy functionality in 23a. Therefore, the structure of 23a was revised as 23 (Figure 2). The structure of withaoxylactone (24a)43 was reported to have a 14α,15α-epoxy functionality, which was proposed based on its H-15 chemical shift value in conjunction with the fact that there was no report of 14β,15β-epoxy withanolides at the time of the publication.43 However, the C-9 and C-12 chemical shifts (39.8 and 38.7 ppm, Table S10, Supporting Information) suggest a β-orientation of the 14,15-epoxide in 24a, which is further supported by 13C NMR comparisons against a nor14,15-epoxide withanolide [viscosalactone B (111),100 C-9: 42.9; C-12: 39.2 ppm, Table S10, Supporting Information]. Therefore, the structure of 24a was revised as 24 (Figure 2). Following the same rationale, the structures of 14α,15αepoxywithaferin A (25a)44 and 14β,15β-epoxywithanoide I (26a)41 were revised as 25 and 26, respectively (Figure 2 and Table S10, Supporting Information). The structure of jaborosalactone 8 (27a)28 was reported to contain an uncommon 14β,17β-dihydroxy functionality that was determined on the basis of C-14 and C-17 high-frequency chemical shifts (88.0 and 88.1 ppm), which were believed to be characteristic of a 14β,17β-dihydroxy functionality.28 However, cinerolide (33), a 14α,17α-dihydroxy withanolide confirmed through X-ray crystallographic analysis,22 was reported to have high-frequency C-14 and C-17 chemical shifts (87.7 and 87.1 ppm, Table S5, Supporting Information). This example illustrates that observed high-frequency C-14 and C-17 chemical shifts do not necessitate the presence of a 14β,17βdihydroxy functionality. Furthermore, the C-12 chemical shift (27.4 ppm) reported in 27a suggests the presence of a 14α,17α-dihydroxy group, which is further supported by its C-9 and C-21 chemical shifts (35.0 and 9.3 ppm, Table S11, Supporting Information) in conjunction with 13C NMR data comparison of related withanolides (84, 85, 102, and 103). Therefore, the structure of 27a, with a 14β,17β-dihydroxy



CONCLUSIONS Analysis of the 13C NMR data of classic withanolides (either reported in the literature or obtained in our own laboratory) containing hydroxy groups at C-14, C-17, or C-20 revealed that (1) a hydroxy substituent at either C-14 or C-17 significantly alters the chemical shifts of C-7, C-9, C-12, and C-21 via the γgauche effect; (2) the chemical shifts of C-9, C-12, and C-21 reflect the orientation (α or β) of the hydroxy group at C-14 or C-17; (3) a double-bond positional change in ring A (Δ2 to Δ3) or hydroxylation at C-27 results in a minuscule effect on the chemical shifts of carbons in rings C and D (from C-12 to C18); and (4) compared to the traditional method of 1H NMR shift comparison (in C5D5N versus CDCl3), which generated inconclusive structures of 22a and 23a, applying the γ-gauche effect in the 13C NMR chemical shift analysis provided a far more convenient and reliable approach to probe the orientation of the hydroxy groups at C-14 and C-17. Utilization of these empirical rules demonstrated that the reported 13C NMR data of withanolides 1a−29a were inconsistent with their published structures, which were subsequently revised as 1−16 and 12 and 18−29, respectively. Our analysis provides an amendment to the structures of more than 3% of the total withanolides reported to date and strongly supports the future application of these methods to determine the correct relative configuration of steroidal substituents.



EXPERIMENTAL SECTION



ASSOCIATED CONTENT

General Experimental Procedures. NMR spectra were recorded with a Bruker AV-400 or AV-500 instrument with a cryoprobe used for 1 H NMR, APT, COSY, HSQC, HMBC, and NOESY/ROESY experiments. Source of Withanolides. A series of withanolides with/without 14-hydroxy, 17-hydroxy, and 20-hydroxy functional groups obtained from our own research work3−11,14 were used for 13C NMR study/ comparison and to make the present structural revisions.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00648. I

DOI: 10.1021/acs.jnatprod.5b00648 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

13



(22) Maldonado, E.; Alvarado, V. E.; Torres, F. R.; Martinez, M.; Perez-Castorena, A. L. Planta Med. 2005, 71, 548−553. (23) Lal, P.; Misra, L.; Sangwan, R. S.; Tuli, R. Z. Naturforsch., B: J. Chem. Sci. 2006, 61, 1143−1147. (24) Habtemariam, S.; Gray, A. I. Planta Med. 1998, 64, 275−276. (25) Lavie, D.; Shakked, Z.; Glotter, E.; Kirson, I.; Rabinovich, D. J. Chem. Soc., Chem. Commun. 1972, 877−878. (26) Nawaz, H. R.; Malik, A.; Muhammad, P.; Ahmed, S.; Riaz, M. Z. Naturforsch., B: J. Chem. Sci. 2000, 55, 100−103. (27) Nicotra, V. E.; Gil, R. R.; Vaccarini, C.; Oberti, J. C.; Burton, G. J. Nat. Prod. 2003, 66, 1471−1475. (28) Misico, R. I.; Veleiro, A. S.; Burton, G.; Oberti, J. C. Phytochemistry 1997, 45, 1045−1048. (29) Ahmad, S.; Malik, A.; Muhammad, P.; Gul, W.; Yasmin, R.; Afza, N. Fitoterapia 1998, 69, 433−436. (30) Ahmad, S.; Malik, A.; Afza, N.; Yasmin, R. J. Nat. Prod. 1999, 62, 493−494. (31) Ahmad, S.; Malik, A.; Yasmin, R.; Ullah, N.; Gul, W.; Khan, P. M.; Nawaz, H. R.; Afza, N. Phytochemistry 1999, 50, 647−651. (32) Hsieh, P. W.; Huang, Z. Y.; Chen, J. H.; Chang, F. R.; Wu, C. C.; Yang, Y. L.; Chiang, M. Y.; Yen, M. H.; Chen, S. L.; Yen, H. F.; Luebken, T.; Hung, W. C.; Wu, Y. C. J. Nat. Prod. 2007, 70, 747−753. (33) Atta-ur-Rahman; Abbas, S.; Dur-e-Shahwar; Jamal, S. A.; Choudhary, M. I. J. Nat. Prod. 1993, 56, 1000−1006. (34) Atta-ur-Rahman; Dur-e-Shahwar; Naz, A.; Choudhary, M. I. Phytochemistry 2003, 63, 387−390. (35) Atta-ur-Rahman; Shabbir, M.; Dur-e-Shahwar; Choudhary, M. I.; Voelter, W.; Hohnholz, D. Heterocycles 1998, 47, 1005−1012. (36) Atta-ur-Rahman; Choudhary, M. I.; Qureshi, S.; Gul, W.; Yousaf, M. J. Nat. Prod. 1998, 61, 812−814. (37) Atta-ur-Rahman; Yousaf, M.; Gui, W.; Qureshi, S.; Choudhary, M. I.; Voelter, W.; Hoff, A.; Jens, F.; Naz, A. Heterocycles 1998, 48, 1801−1811. (38) Atta-ur-Rahman; Choudhary, M. I.; Yousaf, M.; Gul, W.; Qureshi, S. Chem. Pharm. Bull. 1998, 46, 1853−1856. (39) Atta-ur-Rahman; Shabbir, M.; Yousaf, M.; Qureshi, S.; Dur-eShahwar; Naz, A.; Choudhary, M. I. Phytochemistry 1999, 52, 1361− 1364. (40) Kuroyanagi, M.; Murata, M.; Nakane, T.; Shirota, O.; Sekita, S.; Fuchino, H.; Shinwari, Z. K. Chem. Pharm. Bull. 2012, 60, 892−897. (41) Choudhary, M. I.; Dureshahwar; Parveen, Z.; Jabbar, A.; Ali, I.; Atta-ur-Rahman. Phytochemistry 1995, 40, 1243−1246. (42) Glotter, E.; Abraham, A.; Guenzberg, G.; Kirson, I. J. Chem. Soc., Perkin Trans. 1 1977, 341−346. (43) Choudhary, M. I.; Abbas, S.; Jamal, S. A.; Atta-ur-Rahman. Heterocycles 1996, 42, 555−563. (44) Choudhary, M. I.; Hussain, S.; Yousuf, S.; Dar, A.; Mudassar; Atta-ur-Rahman. Phytochemistry 2010, 71, 2205−2209. (45) Bremser, W. Anal. Chim. Acta 1978, 2, 355−365. (46) Evans, W. C.; Grout, R. J.; Mensah, M. L. K. Phytochemistry 1984, 23, 1717−1720. (47) Abdeljebbar, L. H.; Humam, M.; Christen, P.; Jeannerat, D.; Vitorge, B.; Amzazi, S.; Benjouad, A.; Hostettmann, K.; Bekkouche, K. Helv. Chim. Acta 2007, 90, 346−352. (48) Misra, L.; Lal, P.; Sangwan, R. S.; Sangwan, N. S.; Uniyal, G. C.; Tuli, R. Phytochemistry 2005, 66, 2702−2707. (49) Gupta, M.; Bagchi, A.; Ray, A. B. J. Nat. Prod. 1991, 54, 599− 602. (50) Vande Velde, V.; Lavie, D.; Budhiraja, R. D.; Sudhir, S.; Garg, K. N. Phytochemistry 1983, 22, 2253−2257. (51) Hunter, A. C.; Watts, K. R.; Dedi, C.; Dodd, H. T. J. Steroid Biochem. Mol. Biol. 2009, 116, 171−177. (52) Peart, P. C.; Reynolds, W. F.; Reese, P. B. J. Mol. Catal. B: Enzym. 2013, 95, 70−81. (53) Yildirim, K.; Saran, H.; Dolu, O. F.; Kuru, A. J. Chem. Res. 2013, 37, 566−569. (54) Hu, S.; Genain, G.; Azerad, R. Steroids 1995, 60, 337−352. (55) Habermehl, G. G.; Hammann, P. E.; Wray, V. Magn. Reson. Chem. 1985, 23, 959−963.

C NMR data (after assignment adjustment) of reported steroids 1a−31a, 2, 3, 5−7, 9, 10, 13−15, 18, 20, 21, and 30−116 in the literature (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +01-785-864-4844. Fax: +01-785-864-5326. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors are grateful to Mr. R. J. Gallagher and Dr. C.-M. Cao for their assistance in the preparation of the manuscript. This study was supported by the University of Kansas Research Investment Council grant #2506007-700 (awarded to B.N.T.). We are grateful to Dr. L.-H. Hu from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for providing the 13C NMR data of withanolide L (106).



REFERENCES

(1) Zhang, H.; Samadi, A. K.; Cohen, M. S.; Timmermann, B. N. Pure Appl. Chem. 2012, 84, 1353−1367. (2) Zhang, H.; Cao, C. M.; Gallagher, R. J.; Timmermann, B. N. Nat. Prod. Res. 2014, 28, 1941−1951. (3) Zhang, H.; Bazzill, J.; Gallagher, R. J.; Subramanian, C.; Grogan, P. T.; Day, V. W.; Kindscher, K.; Cohen, M. S.; Timmermann, B. N. J. Nat. Prod. 2013, 76, 445−449. (4) Zhang, H.; Cao, C. M.; Gallagher, R. J.; Day, V. W.; Montenegro, G.; Timmermann, B. N. Phytochemistry 2014, 98, 232−235. (5) Zhang, H.; Cao, C. M.; Gallagher, R. J.; Day, V. W.; Kindscher, K.; Timmermann, B. N. Phytochemistry 2015, 109, 147−153. (6) Cao, C. M.; Zhang, H.; Gallagher, R. J.; Day, V. W.; Kindscher, K.; Grogan, P.; Cohen, M. S.; Timmermann, B. N. J. Nat. Prod. 2014, 77, 631−639. (7) Zhang, H.; Samadi, A. K.; Gallagher, R. J.; Araya, J. J.; Tong, X. Q.; Day, V. W.; Cohen, M. S.; Kindscher, K.; Gollapudi, R.; Timmermann, B. N. J. Nat. Prod. 2011, 74, 2532−2544. (8) Zhang, H.; Motiwala, H.; Samadi, A.; Day, V.; Aube, J.; Cohen, M.; Kindscher, K.; Gollapudi, R.; Timmermann, B. Chem. Pharm. Bull. 2012, 60, 1234−1239. (9) Samadi, A. K.; Tong, X.; Mukerji, R.; Zhang, H.; Timmermann, B. N.; Cohen, M. S. J. Nat. Prod. 2010, 73, 1476−1481. (10) Tong, X. Q.; Zhang, H.; Timmermann, B. N. Phytochem. Lett. 2011, 4, 411−414. (11) Zhang, H.; Hagan, K.; Patel, O.; Tong, X. Q.; Day, V. W.; Timmermann, B. N. J. Chem. Crystallogr. 2014, 44, 169−176. (12) Jahan, E.; Perveen, S.; Fatima, I.; Malik, A. Helv. Chim. Acta 2010, 93, 530−535. (13) Huang, C. F.; Ma, L.; Sun, L. J.; Ali, M.; Arfan, M.; Liu, J. W.; Hu, L. H. Chem. Biodiversity 2009, 6, 1415−1426. (14) Zhang, H.; Timmermann, B. N. Curr. Top. Phytochem. 2014, 12, 41−68. (15) Ali, M.; Shuaib, M.; Ansari, S. H. Phytochemistry 1997, 44, 1163−1168. (16) Gottlieb, H. E.; Kirson, I. Org. Magn. Reson. 1981, 16, 20−25. (17) Hammann, P. E.; Kluge, H.; Habermehl, G. G. Magn. Reson. Chem. 1991, 29, 133−136. (18) Glotter, E.; Sahai, M.; Kirson, I.; Gottlieb, H. E. J. Chem. Soc., Perkin Trans. 1 1985, 2241−2245. (19) Bandhoria, P.; Gupta, V. K.; Amina, M.; Satti, N. K.; Dutt, P.; Suri, K. A. J. Chem. Crystallogr. 2006, 36, 153−159. (20) Nittala, S. S.; Frolow, F.; Lavie, D. J. Chem. Soc., Chem. Commun. 1981, 178−179. (21) Nittala, S. S.; Lavie, D. Phytochemistry 1981, 20, 2741−2748. J

DOI: 10.1021/acs.jnatprod.5b00648 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(92) Kirson, I.; Gunzberg, G.; Gottlieb, H. E.; Glotter, E. J. Chem. Soc., Perkin Trans. 1 1980, 531−534. (93) Maurya, R.; Akanksha; Jayendra; Singh, A. B.; Srivastava, A. K. Bioorg. Med. Chem. Lett. 2008, 18, 6534−6537. (94) Lan, Y. H.; Chang, F. R.; Pan, M. J.; Wu, C. C.; Wu, S. J.; Chen, S. L.; Wang, S. S.; Wu, M. J.; Wu, Y. C. Food Chem. 2009, 116, 462− 469. (95) Ramaiah, P. A.; Lavie, D.; Budhiraja, R. D.; Sudhir, S.; Garg, K. N. Phytochemistry 1984, 23, 143−149. (96) Snyder, S. A.; Brill, Z. G. Org. Lett. 2011, 13, 5524−5527. (97) Luo, H. F.; Zhang, L. P.; Hu, C. Q. Tetrahedron 2001, 57, 4849−4854. (98) Sahai, M.; Neogi, P.; Ray, A. B.; Oshima, Y.; Hikino, H. Heterocycles 1982, 19, 37−40. (99) Abraham, A.; Kirson, I.; Lavie, D.; Glotter, E. Phytochemistry 1975, 14, 189−194. (100) Pelletier, S. W.; Gebeyehu, G.; Nowacki, J.; Mody, N. V. Heterocycles 1981, 15, 317−320. (101) Veleiro, A. S.; Burton, G.; Gros, E. G. Phytochemistry 1985, 24, 1799−802. (102) Bessalle, R.; Lavie, D. Phytochemistry 1992, 31, 3648−3651. (103) El Bouzidi, L.; Mahiou-Leddet, V.; Bun, S. S.; Larhsini, M.; Abbad, A.; Markouk, M.; Fathi, M.; Boudon, M.; Ollivier, E.; Bekkouche, K. Pharm. Biol. 2013, 51, 1040−1046.

(56) Templeton, J. F.; Kumar, V. P. S.; Cote, D.; Bose, D.; Elliott, D.; Kim, R. S.; LaBella, F. S. J. Med. Chem. 1987, 30, 1502−1505. (57) Zhang, W.; Liu, W. K.; Che, C. T. Chem. Pharm. Bull. 2003, 51, 1009−1011. (58) Warashina, T.; Noro, T. Phytochemistry 1994, 37, 801−806. (59) Pelletier, S. W.; Mody, N. V.; Nowacki, J.; Bhattacharyya, J. J. Nat. Prod. 1979, 42, 512−521. (60) Vasina, O. E.; Maslennikova, V. A.; Abdullaev, N. D.; Abubakirov, N. K. Chem. Nat. Compd. 1986, 22, 560−565. (61) Kirson, I.; Glotter, E.; Ray, A. B.; Ali, A.; Gottlieb, H. E.; Sahai, M. J. Chem. Res. (S) 1983, 120−121. (62) Ahmad, S.; Yasmin, R.; Malik, A. Chem. Pharm. Bull. 1999, 47, 477−480. (63) Demarco, P. V.; Farkas, E.; Doddrell, D.; Mylari, B. L.; Wenkert, E. J. Am. Chem. Soc. 1968, 90, 5480−5486. (64) Hanson, J. R.; Nasir, H.; Parvez, A. Phytochemistry 1996, 42, 411−415. (65) Iida, T.; Omura, K.; Sakiyama, R.; Kodomari, M. Chem. Phys. Lipids 2014, 178, 45−51. (66) Mappus, E.; Renaud, M.; Rolland de Ravel, M.; Grenot, C.; Cuilleron, C. Y. Steroids 1992, 57, 122−134. (67) Seo, Y.; Rho, J. R.; Cho, K. W.; Shin, J. J. Nat. Prod. 1996, 59, 1196−1199. (68) Ma, C. Y.; Williams, I. D.; Che, C. T. J. Nat. Prod. 1999, 62, 1445−1447. (69) Choudhary, M. I.; Yousuf, S.; Nawaz, S. A.; Ahmed, S.; Atta-urRahman. Chem. Pharm. Bull. 2004, 52, 1358−1361. (70) Lavie, D.; Gottlieb, H. E.; Pestchanker, M. J.; Giordano, O. S. Phytochemistry 1986, 25, 1765−1766. (71) Habtemariam, S.; Gray, A. I.; Waterman, P. G. Phytochemistry 1993, 34, 807−11. (72) Nittala, S. S.; Lavie, D. Phytochemistry 1981, 20, 2735−2739. (73) Llanos, G. G.; Araujo, L. M.; Jimenez, I. A.; Moujir, L. M.; Vazquez, J. T.; Bazzocchi, I. L. Steroids 2010, 75, 974−981. (74) Fang, S. T.; Liu, X.; Kong, N. N.; Liu, S. J.; Xia, C. H. Nat. Prod. Res. 2013, 27, 1965−1970. (75) Zhu, X. H.; Takagi, M.; Ikeda, T.; Midzuki, K.; Nohara, T. Phytochemistry 2001, 56, 741−745. (76) Kuroyanagi, M.; Shibata, K.; Umehara, K. Chem. Pharm. Bull. 1999, 47, 1646−1649. (77) Niero, R.; Da Silva, I. T.; Tonial, G. C.; Camacho, B. D. S.; Gacs-Baitz, E.; Delle Monache, G.; Delle Monache, F. Nat. Prod. Res. 2006, 20, 1164−1168. (78) Ihsan-ul-Haq; Youn, U. J.; Chai, X. Y.; Park, E. J.; Kondratyuk, T. P.; Simmons, C. J.; Borris, R. P.; Mirza, B.; Pezzuto, J. M.; Chang, L. C. J. Nat. Prod. 2013, 76, 22−28. (79) Fang, S. T.; Liu, J. K.; Li, B. Steroids 2012, 77, 36−44. (80) Su, B. N.; Misico, R.; Park, E. J.; Santarsiero, B. D.; Mesecar, A. D.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn, A. D. Tetrahedron 2002, 58, 3453−3466. (81) Dinan, L. N.; Sarker, S. D.; Sik, V. Phytochemistry 1997, 44, 509−512. (82) Nawaz, H. R.; Riaz, M.; Malik, A.; Khan, P. M.; Ullah, N. J. Chem. Soc. Pak. 2000, 22, 138−141. (83) Shingu, K.; Miyagawa, M.; Yahara, S.; Nohara, T. Chem. Pharm. Bull. 1993, 41, 1873−1875. (84) Cordero, C. P.; Morantes, S. J.; Paez, A.; Rincon, J.; Aristizabal, F. A. Fitoterapia 2009, 80, 364−368. (85) Shingu, K.; Yahara, S.; Nohara, T.; Okabe, H. Chem. Pharm. Bull. 1992, 40, 2088−2091. (86) Zhao, J.; Nakamura, N.; Hattori, M.; Kuboyama, T.; Tohda, C.; Komatsu, K. Chem. Pharm. Bull. 2002, 50, 760−765. (87) Nittala, S. S.; Vande Velde, V.; Frolow, F.; Lavie, D. Phytochemistry 1981, 20, 2547−2552. (88) Chen, L. X.; He, H.; Qiu, F. Nat. Prod. Rep. 2011, 28, 705−740. (89) Blunt, J. W.; Stothers, J. B. Org. Magn. Reson. 1977, 9, 439−464. (90) Glotter, E.; Lavie, D.; Kirson, I.; Abraham, A. Tetrahedron 1973, 29, 1353−1364. (91) Kirson, I.; Gottlieb, H. E. J. Chem. Res. (S) 1980, 338−339. K

DOI: 10.1021/acs.jnatprod.5b00648 J. Nat. Prod. XXXX, XXX, XXX−XXX