Urceoloids A and B, Two C19 Steroids with a Rearranged Spirocyclic

Mar 5, 2019 - Urceoloids A (1) and B (2), two C19 steroids with a rearranged new carbon ... A plausible biosynthetic pathway for the new carbon skelet...
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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Urceoloids A and B, Two C19 Steroids with a Rearranged Spirocyclic Carbon Skeleton from Urceola quintaretii Yu-Hao Ren,†,‡ Qun-Fang Liu,† Li Chen,†,‡ Shi-Jun He,† Jian-Ping Zuo,† Yao-Yue Fan,*,† and Jian-Min Yue*,†,‡ †

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People’s Republic of China ‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, People’s Republic of China Org. Lett. Downloaded from pubs.acs.org by WASHINGTON UNIV on 03/05/19. For personal use only.

S Supporting Information *

ABSTRACT: Urceoloids A (1) and B (2), two C19 steroids with a rearranged new carbon skeleton by featuring a very unique spiro[4.4]nona-3,6,8-triene system, and a biosynthetically related known steroid (3) were isolated from Urceola quintaretii. Their structures were completely established by a combined method. A plausible biosynthetic pathway for the new carbon skeleton represented by compounds 1 and 2 was proposed. Compounds 2 and 3 showed immunosuppressive activities.

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rearranged tetracyclic C19 steroids, urceoloids A (1) and B (2), as well as their possible biogenetic precursor androstane4,6,8(9),13(14)-tetraene-3,11,16-triketone (3).9 Compounds 1 and 2 possessed an unprecedented carbon skeleton by incorporating a very unique spiro[4.4]nona-3,6,8-triene system. Their structures with absolute configurations were determined on the basis of spectroscopic methods, singlecrystal X-ray diffraction study, and ECD analysis.

teroids are a class of very important natural molecules that exist ubiquitously in the kingdoms of animals, plants, and fungi. This compound category exhibits diverse biological functions and has many potential biomedical applications.1,2 Continuous efforts in exploring structurally diverse and potentially bioactive steroids by scientific community have led to prosperity in this area and even contributed to the establishment of a discipline of steroid chemistry. Up to now, more than 100 drugs developed from steroids have been approved by the FDA for clinical applications, such as hormone-replacement therapy (HPT), treatment for inflammatory diseases, and oncotherapy.3,4 Some steroidal drugs have also been found to exhibit new indications, e.g., allopregnanolone,5 which was initially under investigation for the treatment of super-refractory status epilepticus (SRSE), has recently passed the phase III clinical trials for the treatment of postpartum depression (PPD). Therefore, it is of great significance to further discover natural steroids with novel skeletons and potent bioactivities, which will provide target molecules for organic synthesis, biosynthesis, chemical biology, and pharmacology. The plants of Urceola quintaretii belong to the Urceola genus (Apocynaceae family) that consists of eight species. Some of the plants in this genus have been applied in the remedy of traditional Chinese medicine for the treatment of infantile paralysis, rheumatalgia, injury, and fractures.6 Previous chemical studies on the plants of this genus led to the identification of a limited number of steroids, triterpenoids, and phenolics.7 As part of our ongoing efforts to explore structurally interesting and biologically significant steroids from plant resources,8 the plant sample of U. quintaretii collected from Hainan island of China was subjected to chemical study, which resulted in the isolation of two highly © XXXX American Chemical Society

A plausible biogenetic pathway for the formation of this new carbon skeleton represented by compounds 1 and 2 was proposed. Compound 2 is likely an artifact derived from 1 and/or the corresponding carboxylic acid during the extraction procedures by using ethanol as the solvent. Herein, we present the isolation, structure elucidation, biosynthetic origin, and biological evaluation of the two novel steroids. Urceoloids A (1) and B (2) were assigned molecular formulas of C20H20O4 and C21H22O4 by the protonated molecular ion peaks at m/z 325.1438 [M + H]+ (calcd 325.1440) and m/z 339.1596 [M + H]+ (calcd 339.1596) in Received: February 12, 2019

A

DOI: 10.1021/acs.orglett.9b00539 Org. Lett. XXXX, XXX, XXX−XXX

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resolved all 21 carbon resonances, which were further identified with the aid of HSQC and DEPT spectra as two mutually conjugated keto carbonyls (δC 206.5, 191.5), an ester carbonyl (δC 171.7), four double bonds, three methyls, four sp3 methylenes (one oxygenated, δC 60.6), one sp3 methine, and two sp3 quaternary carbons. The above-mentioned functionalities accounted for seven DOUs, and the remaining four thus required compound 2 to be tetracyclic. The planar structure of compound 2 was successfully constructed by comprehensive interpretation of 2D NMR spectra, especially 1H−1H COSY and HMBC spectroscopic data (Figure 1). Three structural fragments a−c as drawn with

the (+)-HRESI mass spectra, respectively. Both compounds shared 11 degrees of unsaturation (DOUs) and showed a difference of 14 mass units in their molecular formulas, suggesting that they are structural analogues. This deduction was supported by the resemblance of their IR (Figure S5 and S20) and UV (Figure S6 and S21) spectra. Furthermore, comprehensive comparison of the 1H and 13C NMR data (Table 1) of the two compounds indicated that their proton Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compounds 1 and 2 in CDCl3 1 no.

δH, mult (J, Hz)

1 2α 2β 3 4 5 6 7 8 9 10 11 12α 12β 13 14 15 16 17α 17β 18 19

2.50, 2.50, 1.34, 1.33,

1′

3.50, s

2′

5.78, 2.77, 3.31, 3.73,

m m m t (9.1)

6.67, d (5.3) 6.71, d (5.3)

2.71, d (15.6) 2.63, d (15.6)

6.31, s s s s ddd (2.6, 1.6, 1.6)

2 δC 129.8 35.3 49.3 172.2 71.9 152.9 128.1 149.1 147.0 137.2 191.7 51.8 46.0 170.6 127.0 206.4 51.4 28.6 12.6 51.8

δH, mult (J, Hz) 5.78, 2.75, 3.32, 3.71,

m m m t (9.2)

6.69, d (5.3) 6.70, d (5.3)

2.71, d (15.7) 2.61, d (15.7)

6.30, s 2.49, s 2.49, s 1.34, s 1.33, ddd (2.7, 1.6, 1.6) a 3.92, dq (10.8, 7.1) b 3.96, dq (10.8, 7.1) 1.09, t (7.1)

δC 129.9 35.2 49.3 171.7 72.0 153.3 128.0 149.2 147.0 137.1 191.5 51.9

Figure 1. Key 2D NMR correlations for urceoloid B (2). 46.0 170.8 126.9 206.5 51.4

bold bonds were established by the 1H−1H COSY spectrum. The connectivity of the structural fragments a−c, the quaternary carbons, and the incorporated oxygen atoms was then achieved by the HMBC correlations as well as the chemical shifts of the related carbons and protons. In the HMBC, the correlations of H2-2 and H-3/C-5; H-3/C-6 and C-9; H-7/C-8 and C-9; and H3-19/C-1, C-5, and C-10 enabled the establishment of the five-membered A and B rings that furnished a novel 5,5-spirocyclic triene moiety. The ring D bearing an angular methyl group at C-13 was assembled by the HMBC networks of H3-18/C-12, C-13, C-14, and C-17; H217/C-16 (δC 206.5) and C-15; and H-15/C-14. The sixmembered C ring was then established to fuse the B and D rings by HMBC correlations from H3-18 to C-12; from H-15 to C-8; and from H2-12 to C-11 (δC 191.5) and C-9. An ethoxycarbonyl moiety was attached to C-3 as corroborated by the key HMBC correlations from H2-1′ and H-3 to the ester carbonyl carbon C-4 (δC 171.7). Two keto group at C-11 and C-16 in the multiple conjugated system were recognized by chemical shifts at δC 191.5 and 206.5, respectively. The planar structure of 2 was delineated as drawn. The relative configuration of 2 was partially assigned on the basis of NOESY spectrum recorded in C5D5N (Figure 2). The strong correlations of H3-18/H-12β and H3-18/H-17β suggested that these protons were cofacial and were arbitrarily assigned as β-oriented, which are consistent with those in the common steroids.1b The H-12α and H-17α were then distinguished consequently and verified by the distinct NOESY correlation between them. The NOESY correlations of H-6/H-3 and H-6/H3-19 indicated that these protons were in the same side, which, however, favored two stereoisomers 2a and 2b (Figure 2), and left the arrangement for the spiro Aand B-ring system and the assignment for the relative configurations of C-3 and C-5 impeded. To our delight, the

28.6 12.6 60.6

14.4

and carbon resonances were nearly compatible except for the minor differences resulting from the presence of a methoxycarbonyl group (δH 3.50, 3H, s; δC 172.2, 51.8) in 1 and an ethoxycarbonyl group (δH 3.92 and 3.96, both 1H, dq, J = 10.8, 7.1 Hz; δH 1.09, 3H, t, J = 7.1 Hz; δC 171.7, 60.6 and 14.4) in 2, respectively, which was in agreement with their molecular formulas assigned above. The aforementioned analysis further indicated that two compounds possessed an identical basic framework and substitution patterns and were only different at the terminal ester moieties. In order to effectively elucidate the structures of two compounds, we first began to work on compound 2 due largely to the fact that it was obtained as quality crystals. A full set of 2D NMR spectra for compound 2 were thus acquired for the structural determination. Urceoloid B (2) was obtained as yellow crystals (mp 169− 171 °C). The IR spectrum suggested the presence of carbonyl (1719, 1700, and 1652 cm−1) and olefinic (1618 cm−1) groups. The 1H NMR spectrum (Table 1) displayed the characteristic signals of two methyls (δH 1.33 and 1.34, both 3H), four olefinic protons (δH 6.69, 6.70, 6.30, 5.78, both 1H), and an ethoxycarbonyl group. The 13C NMR spectrum (Table 1) B

DOI: 10.1021/acs.orglett.9b00539 Org. Lett. XXXX, XXX, XXX−XXX

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Figure 4. ECD spectra of compounds 1 and 2.

inspired us to propose a plausible biosynthetic pathway for compounds 1 and 2 with 3 as the precursor (Scheme 1). The Scheme 1. Hypothetical Biosynthetic Pathways of 1 and 2 Figure 2. Two possible stereoisomers of urceoloid B (2) with key NOESY correlations

quality crystals of 2 were obtained from the recrystallization in an optimized binary solvent system (MeOH/H2O, 9:1), which facilitated single-crystal X-ray diffraction study with the anomalous scattering of Cu Kα radiation. The X-ray crystallography results not only confirmed the planar structure for compound 2 and excluded the stereoisomer 2b from consideration but also unambiguously determined its absolute configuration as 3R,5R,13S with an excellent Flack parameter of 0.00(5) (Figure 3).

oxidative cleavage of Δ4 double bond of 3 would produce intermediate i, which would lead to the generation of a key intermediate ii after a cascade of enzyme-catalytic reduction processes subsequently.10 The intermediate ii would be transformed into the intermediate iii by an enzymatic intramolecular aldol reaction, which would readily be transformed into a key carbocation intermediate iv via an acidcatalytic dehydration process. The highly reactive intermediate iv would undergo a 1,2-alkyl shift reaction followed by E1 elimination reaction to furnish the unique spiro[4.4]nona3,6,8-triene motif in v. The carboxylic acid intermediate v would subsequently be modified by methyltransferase11 to afford compound 1. Compound 2 was proposed as an artifact derived from compound 1 and/or the carboxylic acid v during the extraction procedures by using ethanol as the solvent. We failed to make a definite conclusion for the presence of compounds 1 and 2 in the crude extract by LC−ESIMS analysis due likely to their very low concentrations and/or the low abundances of their molecular ion peaks (Figure S27). Thus, we cannot fully exclude the possibility that 1 was produced by methylation of the corresponding carboxylic acid

Figure 3. ORTEP drawing of urceoloid B (2).

Urceoloid A (1) was obtained as pale yellow powder. As the structurally closely related congener of 2, its structure was thus assigned by analogy of the spectroscopic data with those of 2. The only difference for both compounds occurred at C-3, where compound 1 had a methoxycarbonyl group and compound 2 possessed an ethoxycarbonyl group. The ECD curves of 1 were highly compatible to those of 2 (Figure 4), which allowed the assignment of the absolute configuration of 1 (3R,5R,13S) as the same of 2. This assignment is consistent with the biosynthetic considerations. Compounds 1 and 2 represented an unprecedented rearranged carbon skeleton of C19 steroids featuring a unique spiro[4.4]nona-3,6,8-triene system, and compound 3 shared the same C- and D-ring system as those of 1 and 2, which C

DOI: 10.1021/acs.orglett.9b00539 Org. Lett. XXXX, XXX, XXX−XXX

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v during the purification processes where MeOH was applied. However, the whole purification was conducted under neutral and mild conditions where the methylation is unlikely to have occurred. Compounds 2 and 3 were evaluated for immunosuppressive activities against the proliferation of T and B lymphocyte cells in vitro with cyclosporin A (CsA) as the positive control. Two compounds showed weak to moderate activities against the tested two cells with IC50 values ranging from 6.49 to 35.39 μM (Table S2). In summary, the discovery of compounds 1 and 2 with a rearranged spirocyclic carbon skeleton from the plants of U. quintaretii is of great importance in the research field of steroids. Although a few of steroids with rearranged nucleus were found from marine and fungal origins,2g−j the steroids with rearranged skeletons were rarely founded from the plant kingdom. Moreover, the exploration for facile and mild synthetic methods to construct spirocyclic framework has long been attractive to the community of organic chemistry.12 This finding and the proposed biosynthetic routes for compounds 1 and 2 will inspire and facilitate the synthesis of natural products with such a unique spirocyclic system, especially for steroids.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00539. Immunosuppressive activity data for 1−3; tabulated NMR data of 2 in C5D5N; experimental section; X-ray crystallographic data for 2; and 1D and 2D NMR, MS, IR, and UV spectra of compounds 1 and 2; 1D NMR, MS spectra, and the specific rotation data of compound 3; LC−ESI(+)MS chromatogram of ethanolic extract (PDF) Accession Codes

CCDC 1893166 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] . *E-mail: [email protected]. ORCID

Jian-Min Yue: 0000-0002-4053-4870 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation (Nos. 21772213 and 21532007) and the Drug Innovation Major Project (2018ZX09711001-001-005) of P.R. China are highly acknowledged. We thank Prof. S.-M. Huang (Hainan University) for the identification of the plant materials. D

DOI: 10.1021/acs.orglett.9b00539 Org. Lett. XXXX, XXX, XXX−XXX