3-Fused Tetracyclic Core

Letter Cite This: Org. Lett. 2018, 20, 6314−6317

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Premnafulvol A: A Diterpenoid with a 6/5/7/3-Fused Tetracyclic Core and Its Biosynthetically Related Analogues from Premna fulva De-Bing Pu,†,§,⊥ Bao-Wen Du,‡,⊥ Wen Chen,† Jun-Bo Gao,§ Kun Hu,§ Nan Shi,‡ Yi-Ming Li,† Xing-Jie Zhang,† Rui-Han Zhang,† Xiao-Nian Li,§ Hong-Bin Zhang,† Fei Wang,*,‡ and Wei-Lie Xiao*,†,§

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Key Laboratory of Medicinal Chemistry for Natural Resource of Ministry of Education and Yunnan Province, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China ‡ Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China § State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China S Supporting Information *

ABSTRACT: Premnafulvol A (1), a unique diterpenoid featuring a 6/5/7/3-fused tetracyclic carbon skeleton, with three biosynthetically related analogues, premnafulvols B−D (2−4), were isolated from the aerial parts of Premna fulva. Structures of 1−4 were established by a combination of extensive spectroscopic analyses, quantum chemical calculations, and X-ray crystallography. Plausible biosynthetic pathways of 1−4 were proposed. Interestingly, 2 and 3 exhibited opposite effects on estrogen biosynthesis in human ovarian granulosa-like KGN cells by modulating the expression of aromatase.

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tetracyclic 6/6/6/3 carbon rings. This paper described the structural elucidation of 1, the plausible biogenetic relationships of 1−4, and the bioactivities of 2 and 3 on estrogen biosynthesis.

strogens play a crucial role in the normal physiology of a variety of tissues. Aromatase is the rate-limiting enzyme that catalyzes the biosynthesis of estrogens by using androgens as substrates.1 Aromatase inhibitors, such as letrozole, and supplementation of estrogen have been developed and used to treat hormone-dependent breast cancer and osteoporosis, respectively, in postmenopausal women.2 The side effects caused by deprivation or supplementation of estrogen to the whole body limit the clinical use of these drugs.3 New aromatase modulators, preferably with tissue-specific effects, are needed to offer greater clinical efficacy and fewer side effects than the currently available drugs. Natural products from traditional medicinal plants are good sources of aromatase modulators with low toxicity.4 However, only a few compounds have been found to modulate the transcription of aromatase,5 which is important for the development of tissue-selective aromatase modulators to treat estrogen-related disease. Premna fulva Craib, as a Zhuang medicine named “zhangu”, is mainly distributed in the Guangxi province and other south and southwest regions of China. Zhangu has been widely used to treat periarthritis, osteoproliferation, pain, and other diseaes.6 However, chemical constituents from the folk medicine have been rarely reported, except for a few common triterpenoids,7 lignans,8 and flavonoids.9 In our current research, four unique isopimarane-type derivatives, premnafulvols A−D (1−4), were successfully isolated from the aerial parts of this medicinal plant. 1 possessed an unprecedented 14,15-cyclo-C-homo-B-norisopimarane carbon core with a tetracyclic 6/5/7/3 ring system, and 2 and 3 had 14,15-cyclo-isopimarane carbon skeletons with rare © 2018 American Chemical Society

1 was initially isolated as an amorphous powder with [α]D22 +9.0 (c 0.12, MeOH). The molecular formula, C20H32O4, was established from its positive HR-ESI-MS data (m/z 359.2199 [M + Na]+) and its 13C NMR spectrum, with five degrees of unsaturation. Its 1H NMR and HSQC spectra clearly showed three tertiary methyl groups (δH 1.03, 1.13, and 1.41). The 13C NMR and DEPT spectra exhibited 20 carbon resonances ascribed to three methyls, eight methylenes (two oxygenated, δC 61.9 and 73.6), four methines, and five quaternary carbons (one ketocarbonyl, δC 207.5; one oxygenated, δC 84.3) (Table S4-1). The aforementioned functional group (one keto-carbonyl) accounted Received: September 6, 2018 Published: September 26, 2018 6314

DOI: 10.1021/acs.orglett.8b02845 Org. Lett. 2018, 20, 6314−6317

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Organic Letters

chemical shift calculation. Theoretical ECD curves are mainly applied to identify the correct absolute configuration between possible enantiomers. Comprehensive quantum chemistry calculations (QCCs) determined the remaining stereochemistry of 1, as our initial attempts to obtain single crystals were unsuccessful. To distinguish the overall relative configuration, chemical shifts of isomers 1a and 1b were predicted using the GIAO method12 by DFT calculations in pyridine with the PCM model at the mPW1PW91/6-31G(d,p) level. Experimental and calculated chemical shifts were further statistically analyzed by DP4+ probability. The DP4+ analysis selected 1a (Figures 2 and S12-1)

for one degree of unsaturation, and the remaining four degrees of unsaturation required 1 to possess a tetracyclic ring system. By analysis of NMR spectra, the planar structure of 1 was elucidated as a diterpenoid with a tetracyclic 6/5/7/3 ring system (Figure 1). Key HMBC correlations from H3-20 to C-1/C-5/C-

Figure 1. 1H−1H COSY (bold), selected HMBC (arrow), and key ROESY (double arrow) correlations of 1.

10, from H2-1 to C-3/C-5/C-10, from H2-18 [δHa 3.49 (d, J = 9.6), δHb 3.44 (d, J = 9.6)] to C-3/C-5/C-19 and from H3-19 to C-3/C5/C-18, with the H−H COSY correlations of H2-1/H2-2/H2-3, indicated the presence of a six-membered A ring. H−H COSY correlations of H-5/H2-6/H-7, in combination with the HMBC correlations from H-5 to C-9/C-10, from OH-9 to C-7/C-9/C10, from H2-6 to C-7/C-10, and from H3-20 to C-9 collectively revealed the presence of a five-membered B ring containing C-5, C-6, C-7, C-9, and C-10. H−H COSY correlations of H-14/H15/H2-16 and HMBC correlations from H3-17 to C-13/C-14/C15 and from H2-16 [δHa 4.16 (br d, J = 8.2), δHb 3.78 (t, J = 9.9)] to C-13/C-14/C-15 revealed the presence of a rare three-membered D ring that contained C-13, C-14, and C-15 and indicated that C16 was linked to C-15 and C-17 was connected to C-13. Sevenmembered C ring, composed of C-7, C-8, C-9, C-11, C-12, C-13, andC-14, was established by HMBC correlations of OH-9 with C7/C-9/C-11, of H3-17 with C-12/C-13/C-14, and of H2-6/H-15 with C-8 (δC 207.5), together with H−H COSY correlation of H11 with H-12. Seven-membered C ring was fused with fivemembered B ring through C-7 and C-9 and with three-membered D ring by bridgehead carbons C-13 and C-14 (Figure 1). The planar structure of 1 was established as an unusual 14,15-cyclo-Chomo-B-norisopimarane carbon skeleton. Partial relative configuration of 1 was assigned based on a ROESY experiment (Figure 1). In rings A and B, the observed ROESY correlations of H-5 with H2-18 and OH-9 indicated that these protons were on the same face, whereas ROESY correlations of H3-20 with H-7 and H3-19 suggested that these protons were on the other side. Protons of H-14, H2-16, and H3-17 in ring D were assigned as cis-oriented by the ROESY correlations of H3-17 with H-14 and H2-16. Due to the overlap of some of the aliphatic signals (H-2α, H-6β, H-11α, and H-14β overlapped between 1.69 and 1.59 ppm) and despite the appearance of a strong correlation of H7 with the overlapping region,there was no clear NOE effect(like a key H-7/H-14 interaction) in the seven-membered ring C, which indicated that two independent stereoclusters separated by ring C could be present in this molecule. From a stereochemical perspective, four reasonable absolute configurations [4R5S7R9R10S//13R14S15S (1a), 4S5R7S9S10R// 13S14R15R (−1a), 4R5S7R9R10S//13S14R15R (1b), and 4S5R7S9S10R//13R14S15S (−1b)] were conservatively considered in this structure. Analyses of DP4+10 probability and improved ANN-PRA11 enhanced the probability to identify the correct stereochemical relationship from possible isomers by coupling with GIAO NMR

Figure 2. DFT studies and DP4+ and ANN-PRA analyses for the configuration of 1.

as a more reasonable configuration, with probabilities of 100, 100, and 100% for the 1H and 13C NMR signals and their combination, respectively. Improved ANN-PRA as another statistical analysis method was used to address the predicted and experimental chemical shifts. NMR data were also calculated using the GIAO method at the mPW1PW91/6-31G(d) level in the gas state and the mPW1PW91/6-31G(d,p) level with the PCM model in pyridine. Results show that 1a was the correct configuration based on the analysis of 72 parameters (1D and 2D data for 1H and 13C NMR using TMS, MSTD, and both of them as reference standards; Figures 2 and S13-1). Detected 1b gave the opposite results as an incorrect assignment along with the 72 corresponding parameters (Figures 2 and S13-2). Configurations 1a and (−) 1a were selected for further identification. Calculated ECD curves of 1a and (−)1a were employed to confidently establish the absolute configuration. TDDFT method13 at the B3LYP/6-311+G(d,p) level in methanol with the PCM model was used to simulate the ECD spectra. As a result, the calculated curve of 1a was very similar to the experimental spectrum, which was opposite to that of its enantiomer (Figure 3). MOs of 1a-8 (43.4%) (Figures 4 and S2-1) revealed that the weak Cotton effect (CE) in the experimental curve at 290 nm could be attributed to the positive rotatory strength at 275 nm from MO 92 to 93 involving an n → π* transition; the experimental negative CE at 214 nm could be assigned to the negative rotatory strength

Figure 3. Experimental and calculated ECD spectra and crystal structure of 1. 6315

DOI: 10.1021/acs.orglett.8b02845 Org. Lett. 2018, 20, 6314−6317

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like reaction18 to construct the novel 14,15-cyclo-C-homo-Bnorisopimarane carbon skeleton and finally form 1 (Scheme 1). Scheme 1. Plausible Biosynthetic Pathways of 1−4

Figure 4. Key MOs involved in important transitions of conformer 1a-8.

at 214 nm that was mainly from the π → π* electronic transition from MO 90 to 93. The results of the QCCs collectively confirm that configuration 1a was the most likely structure. To confirm the above speculations, we tested various mixtures and solvent systems to obtain suitable single crystals for XRD experiments. When chloroform was used as a poor solvent to slowly diffuse into a solution of acetone containing 1 at room temperature, suitable crystals of 1 in the form of colorless microcubes were ultimately obtained. XRD of 1 using Cu Kα radiation was successfully performed [Flack parameter = 0.18(8)],14 and these data also confirmed that 1a is the correct structure (Figure 3). The above stereochemical elucidation provides an excellent example of how the overall spatial configuration of organic molecules possessing independent stereoclusters can be established with a remarkable level of confidence by calculated NMR and ECD data. By structure elucidation, 2 and 3 were identified as rare examples of rearranged isopimaranes with a tetracyclic 6/6/6/3 carbon ring system.15,16 Compound 4 was an isopimarane with a tricyclic 6/6/6 carbon ring skeleton. Despite the high similarity between the NMR shifts of 4 and those of the known compound 15,16,18-trihydroxypimara-8(14)-ene17 (Figure S1-1), they had opposite stereochemistries at C-13. Detailed structure elucidations of 2−4 can be found in the Supporting Information, and their structures were ultimately confirmed by single-crystal XRD experiments (Figure 5).

We evaluated the effects of 1−4 on estrogen biosynthesis (Figure S7-1) using previously reported methods.19 2 and 3 exhibited opposite effects on estrogen biosynthesis in KGN cells (human ovarian granulosa tumor cell line). Treatment with 2 decreased the production of 17β-estradiol in a concentrationdependent manner with an IC50 value of 10.68 ± 0.215 μM (Figure 6A). Treatment with 3 significantly promoted 17β-

Figure 6. Effects of 2 and 3 on estrogen biosynthesis in KGN cells. (A,B) Concentration−response curve of 2 or 3 for the modulation of 17βestradiol biosynthesis in KGN cells. (C,D) Time course for the modulation of 17β-estradiol biosynthesis by 2 (10 μM) or 3 (15 μM) in KGN cells. FSK (forskolin), 10 μM; Let (letrozole), 10 nM; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control (n = 3). Figure 5. Crystal structures of 2−4.

estradiol biosynthesis in KGN cells compared to nontreated control cells; its effect was also concentration-dependent, with an EC50 value of 15.49 ± 0.148 μM (Figure 6B). Inhibitory and promotive effects of 2 and 3 on 17β-estradiol biosynthesis were time-dependent (Figure 6C,D). These results indicate that 2 and 3 could modulate the biosynthesis of estrogen in KGN cells in a dose- and time-dependent manner. To examine whether 2 and 3 modulate 17β-estradiol production by regulating aromatase expression, the only enzyme that is able to convert testosterone into 17β-estradiol, we investigated aromatase mRNA and protein expression in KGN cells treated with these two compounds at the indicated

Biosynthetically, (E,E,E)-geranylgeranyl diphosphate (GGPP) is the precursor of polycyclic diterpenoids regardless of the final number of rings. Through the biosynthetically related cyclase, GGPP is cyclized to intermediate a. Intermediate a might further undergo hydroxylation and dehydrogenation to yield 4. Subsequently, 4 might be subjected to dehydration, cyclization, and double-bond migration to yield 2. 2 might undergo oxidation to yield 3 with an α,β-unsaturated ketone and intermediate c with a 10-membered ring. Intermediate c might then undergo an aldol6316

DOI: 10.1021/acs.orglett.8b02845 Org. Lett. 2018, 20, 6314−6317

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concentrations. 2 blocked aromatase mRNA expression in a concentration-dependent manner (Figure 7A). Consistent with

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AUTHOR INFORMATION

Corresponding Authors

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

De-Bing Pu: 0000-0001-5514-304X Hong-Bin Zhang: 0000-0002-2516-2634 Wei-Lie Xiao: 0000-0001-6826-1993 Author Contributions ⊥

D.-B.P. and B.-W.D. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was financially supported by the NSFC (81422046, 21561142003, 21372214, 21762048, and U1702286) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_17R94). We thank Prof. Ariel M. Sarotti of Universidad Nacional de Rosario for providing help with the ANN-PRA analysis.

Figure 7. Effects of 2 and 3 on aromatase expression in KGN. (A,B) Effect of 2 or 3 on aromatase mRNA expression in KGN cells. Results are expressed as fold change relative to levels in untreated cells. GAPDH was used as an internal control. (C,D) Cell lysates were immunoblotted with anti-aromatase or anti-GAPDH antibodies. Quantitative results are depicted. FSK, 10 μM; *p < 0.05, ***p < 0.001 compared with the control (n = 3).

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(1) Simpson, E. R. J. Mol. Endocrinol. 2000, 25, 149. (2) Johnston, S. R. D.; Dowsett, M. Nat. Rev. Cancer 2003, 3, 821. (3) (a) Davison, S.; Davis, S. R. Clin. Endocrinol. 2003, 58, 249. (b) Smith, I. E.; Dowsett, M. N. Engl. J. Med. 2003, 348, 2431. (4) Balunas, M. J.; Kinghorn, A. D. Planta Med. 2010, 76, 1087. (5) Khan, S. I.; Zhao, J.; Khan, I. A.; Walker, L. A.; Dasmahapatra, A. K. Reprod. Biol. Endocrinol. 2011, 9, 91. (6) Pei, J.; Chen, S. L. Zhongguo Zhiwu Zhi; Science Press: Beijing, 1982; Vol. 65, p 97. (7) Song, W.; Si, S. L.; Xu, X. J.; Pu, Q. L.; Pannell, L. K.; Highet, R. J. Planta Med. 1991, 57, 93. (8) Niu, K. Y.; Wang, L. Y.; Liu, S. Z.; Zhao, W. M. J. Asian Nat. Prod. Res. 2013, 15, 1. (9) (a) Chen, G. Y.; Dai, C. Y.; Wang, T. S.; Jiang, C. W.; Han, C. R.; Song, X. P. ARKIVOC 2010, 179. (b) Dai, C. Y.; Chen, G. Y.; Zhu, G. Y.; Fang, H. X.; Jiang, C. W. Zhongcaoyao 2007, 38, 34. (10) Grimblat, N.; Zanardi, M. M.; Sarotti, A. M. J. Org. Chem. 2015, 80, 12526. (11) Zanardi, M. M.; Sarotti, A. M. J. Org. Chem. 2015, 80, 9371. (12) Lodewyk, M. W.; Siebert, M. R.; Tantillo, D. J. Chem. Rev. 2012, 112, 1839. (13) Srebro-Hooper, M.; Autschbach, J. Annu. Rev. Phys. Chem. 2017, 68, 399. (14) Flack, H. D.; Bernardinelli, G. Chirality 2008, 20, 681. (15) Lin, A. S.; Lin, C. R.; Du, Y. C.; Lubken, T.; Chiang, M. Y.; Chen, I. H.; Wu, C. C.; Hwang, T. L.; Chen, S. L.; Yen, M. H.; Chang, F. R.; Wu, Y. C. Planta Med. 2009, 75, 256. (16) Jiang, Z. Y.; Yang, C. T.; Hou, S. Q.; Tian, K.; Wang, W.; Hu, Q. F.; Huang, X. Z. Planta Med. 2016, 82, 742. (17) Wenkert, E.; Ceccherelli, P.; Raju, M. S.; Polonsky, J.; Tingoli, M. J. Org. Chem. 1979, 44, 146. (18) Liang, C. Q.; Shi, Y. M.; Luo, R. H.; Li, X. Y.; Gao, Z. H.; Li, X. N.; Yang, L. M.; Shang, S. Z.; Li, Y.; Zheng, Y. T.; Zhang, H. B.; Xiao, W. L.; Sun, H. D. Org. Lett. 2012, 14, 6362. (19) Guo, J. J.; Yuan, Y.; Lu, D. F.; Du, B. W.; Xiong, L.; Shi, J. G.; Yang, L. J.; Liu, W. L.; Yuan, X. H.; Zhang, G.L.; Wang, F. Toxicol. Appl. Pharmacol. 2014, 279, 23.

its inhibitory effect on aromatase transcription, 2 decreased aromatase protein expression by 60% at a concentration of 25 μM (Figure 7C). Conversely, 3 significantly stimulated the mRNA and protein expression of aromatase in a dose-dependent manner, consistent with its stimulatory effect on estrogen biosynthesis (Figure 7B,D). These results indicate that 2 and 3 regulate 17βestradiol production by modulating aromatase expression in KGN cells. In summary, four biosynthetically related isopimarane-type derivatives 1−4 were isolated from P. fulva. 1 was an unprecedented 14,15-cyclo-C-homo-B-norisopimarane carbon skeleton with a 6/5/7/3-fused tetracyclic system and provided a good example in which the stereochemical relationship between independent stereoclusters can be established by employing affordable QCCs. 2 and 3, with a tetracyclic 6/5/7/3 ring system, showed opposite effects on estrogen biosynthesis by modulating the expression of aromatase, providing a new scaffold for promising drug leads to treat estrogen-related diseases.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b02845. Structure elucidation of 2−4; NMR data of 1−4; experimental section; NMR, MS, UV, and IR spectra for 1−4; computational data of 1 and 3 (PDF) Accession Codes

CCDC 1830670 (1), 1830247 (2), 1830248 (3), and 1830671 (4) contain 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 data_request@ccdc. cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: + 44 1223 336033. 6317

DOI: 10.1021/acs.orglett.8b02845 Org. Lett. 2018, 20, 6314−6317