Letter pubs.acs.org/OrgLett
Cinnamomols A and B, Immunostimulative Diterpenoids with a New Carbon Skeleton from the Leaves of Cinnamomum cassia Lei Zhou,† Yali Tuo,† Yi Hao,‡ Xiaoman Guo,‡ Wei Tang,§ Yongbo Xue,† Junfen Zeng,† Yu Zhou,§ Ming Xiang,† Jianping Zuo,§ Guangmin Yao,*,† and Yonghui Zhang*,† †
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, and ‡School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China § Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China S Supporting Information *
ABSTRACT: Two diterpenoids with an unprecedented diterpene carbon skeleton, cinnamomols A (1) and B (2), were isolated from the leaves of Cinnamomum cassia. 1 and 2 feature a cage-like, rigid, 5/5/5/5/5/6-fused hexacyclic ring system. The structures of 1 and 2 were established by extensive spectroscopic techniques and single-crystal X-ray diffraction, and their plausible biosynthetic pathways were proposed. 1 and 2 exhibited significant in vitro immunostimulative activity, and the mode of action of 1 was investigated.
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harvested at any time. Therefore, the leaves of C. cassia have a definite advantage over the bark as a source. To search for new high-efficiency and low-toxicity immunostimulators from C. cassia, the leaves were phytochemically investigated for the first time, leading to the isolation of two novel diterpenoids with a new carbon skeleton, cinnamomols A (1) and B (2) (Figure 1),
mmune cells play a pivotal role in pathogenesis of cellmediated autoimmune diseases and chronic inflammatory disorders. Immunostimulators that can promote and modulate immune response function are used to treat hypoimmunity related diseases, immunodeficiency disease, chronic infection, and also as adjuvant drugs for tumor treatment in clinics. Representative first-line immunostimulators in clinical use are thymosin α1 (Tα1),1 thymopentin, levamisole, and iRNA. Tα1, a 28 amino acid peptide, was originally isolated from fresh calf thymus tissue in tiny amounts.2 Solid-phase synthesis or genetic engineering could provide Tα1, but the isolation and purification procedures for Tα1 peptide production are very complicated, and the drawbacks are low yields, insufficient purity, and high expense.3 The main administration route for Tα1 so far is injection, which is inconvenient and may cause many side effects such as nausea, fever, dizziness, chest distress, and weakness.4 Moreover, long-term administration of levamisole leads to liver damage and granulocytopenia. The drawbacks for iRNA are injection administration and expense. Traditional Chinese medicines (TCM) that serve as potential plant immunostimulators5 have attracted much attention for their low toxicity, stability, oral administration, availability, and low costs.6 The bark of Cinnamomum cassia has been proven to be a potent immunostimulator to improve body immunity, stimulate antibody production, and prolong the action time of antibodies6 and was reported to possess anti-implement activity.7 However, the immunostimulative components of C. cassia are still unknown to date, although the extract of the bark of C. cassia was reported to stimulate the proliferation of human lymphocytes in vitro.8 Cinnamomum cassia is an evergreen tree, but the bark is nonrenewable and is usually harvested from trees more than 10 years old. The leaves, by contrast, are renewable and can be © 2017 American Chemical Society
Figure 1. Structures of cinnamomols A (1) and B (2).
and a known diterpenoid, perseanol (3).9 Cinnamomols A (1) and B (2) possess an unprecedented, cage-like, 5/5/5/5/5/6fused hexacyclic diterpene skeleton comprising a 9,10dioxatricyclo[5.2.1.04,8]decane and a tricyclo[5.2.1.02,6]decane moieties. Strikingly, new diterpenoids 1 and 2 exhibited remarkable in vitro immunostimulatory activities. This is the first report of the immunostimulative components of C. cassia. Herein, we present the isolation, structure elucidation, plausible biosynthetic pathway, and immunomodulatory activity of 1 and 2 as well as the mode of action of 1. Cinnamomol A (1) was obtained as colorless platelet crystals from H2O (mp 188−189 °C). Its molecular formula was Received: May 2, 2017 Published: May 23, 2017 3029
DOI: 10.1021/acs.orglett.7b01323 Org. Lett. 2017, 19, 3029−3032
Letter
Organic Letters assigned to be C20H30O8 by the 13C NMR data and [M + Na]+ ion at m/z 421.1816 (calcd for C20H30O8Na, 421.1838) in the HRESIMS, indicating six degrees of unsaturation. The 1H NMR spectrum (Table S1) of 1 showed signals of three secondary methyls at δH 0.98 (d, J = 6.9 Hz, H3-19), 1.00 (d, J = 6.9 Hz, H320), and 1.28 (d, J = 7.2 Hz, H3-15), two quaternary methyls at δH 1.29 (s, H3-16) and 1.58 (s, H3-17), and one singlet methine at δH 1.92 (s, H-10). The 13C NMR spectrum of 1 (Table S1) exhibited 20 carbon resonances, assignable by DEPT and HSQC spectra to five methyls, three methylenes, three methines, a ketal carbon, a hemiketal carbon, six oxygenated quaternary carbons, and an aliphatic quaternary carbon. With the six degrees of unsaturation taken into account, the aforementioned data suggested that 1 should be a hexacyclic diterpenoid. The planar structure of 1 was determined by comprehensive interpretations of 1H−1H COSY, HSQC, and HMBC data. As shown in Figure 2, the 1H−1H COSY spectrum of 1 suggested
Figure 3. X-ray crystal structures of 1 and 2.
eter10 of 0.0(1) and the Hooft parameter11 of 0.07(5) for the given coordinate assigned the absolute configuration of 1 to be 1S,2R,5S,6S,7R,8R,9S,10R,11R,12S,13R. Cinnamomol B (2) was isolated as colorless cubic crystals. The [M + Na]+ ion peak at m/z 437.1730 (calcd for C20H30O9Na, 437.1788) in the HRESIMS and 13C NMR data established the molecular formula of 2 to be C20H30O9, which has one more oxygen atom than 1. The 1H NMR spectrum (Table S1) of 2 was similar to that of 1, and the major differences were the absence of H-2 and the presence of singlet methyl of CH3-15 (δH 1.35) in 2, instead of doublet methyl of CH3-15 (δH 1.28) in 1. Comparison of their 13C NMR data (Table S1) revealed that C-2 (δC 86.9) in 2 was shifted about 39.0 ppm downfield from that (δC 47.9) in 1. Thus, 2 was a 2-OH derivative of 1. 2D NMR data (Figure S1) supported this deduction. The structure including the absolute configuration of 2 was finally determined by single-crystal X-ray diffraction using Cu Kα radiation (Figure 3). Cinnamomols A (1) and B (2) possess an unprecedented, cage-like, and rigid 5/5/5/5/5/6 hexacyclic diterpene skeleton, containing a 9,10-dioxatricyclo[5.2.1.04,8]decane moiety and a tricyclo[5.2.1.02,6]decane moiety. The biosynthetic pathways for compounds 1 and 2 could be traced back to a likely biogenetic precursor, perseanol (3).9 As shown in Scheme 1, we proposed the plausible biosynthetic pathways for 1 and 2, involving carbon−carbon bond cleavage and re-formation. First, the ether linkage between C-11 and C-6 of the hemiketal group in 3 is hydrolyzed under the catalysis of acid to afford a ketone intermediate 4. The carbon bond linkage of C-12 and C-13 in 4 is cleaved by a retro-aldol reaction, and then H-12 and 6-OH in 5 are oxidized to form a triketone intermediate 6. Under the catalysis of acid, the intramolecular nucleophilic addition of 12-OH to the carbonyl C-13 in 6 provides a hemiketal intermediate 7. Due to the induction of the carbonyl C-11, a new carbon bond between C-6 and C-10 is constructed by an aldol reaction under the catalysis of an alkaline enzyme and generates an intermediate 8. The enzyme-mediated aldol reaction is the key step to form the carbon bond between C6 and C-10. Under the catalysis of acid, 13-OH attacks the C-11 carbonyl to form a hemiketal moiety, constructing a 1,3-
Figure 2. 1H−1H COSY, key HMBC, and NOESY correlations of 1.
the presence of two partial structures of “CH3-15−CH-2−CH23−CH2-4” and isopropyl “CH3-19/CH-18/CH3-20” (black bold lines). HMBC data (blue arrows) of 1 were used to connect the partial structures to establish the right part of 1 as a 1,2,6trihydroxyl-3,7-dimethyltricyclo[5.2.1.02,6]decane motif. The five-membered ring of C-6−C-7−C-10−C-11−C-12, the location of CH3-17 to C-12, and the connection of C-18−C13−C-14−C-8 could be easily assigned by HMBC data of 1. However, there were no useful HMBC correlations to connect C-13 to C-11 and C-12. In consideration of the dramatically downfield chemical shifts of C-12 (δC 90.8), C-13 (δC 107.7), and C-11 (δC 108.1), as well as the hexacyclic ring system required by the six degrees of unsaturation, the ether linkages from C-13 to C-11 and C-12 were proposed. Thus, the left part of 1 was assigned as a 3,4,7-trihydroxyl-8-methyl-1-isopropyl-9,10dioxatricyclo[5.2.1.04,8]. The NOESY correlations of H-14α/H316, H3-16/H-4α, H-4β/H-10, and H-4α/H-2α suggested the cisconfusion of the four rings and the α-orientation of CH3-15 (Figure 2). However, due to many quaternary carbons in 1, the relative configuration was not determined by few NOESY data. Finally, the structure of compound 1 was successfully determined by single-crystal X-ray diffraction analysis using Cu Kα radiation. The crystal structure (Figure 3) showed that 1 possesses a cage-like 5/5/5/5/5/6-fused hexacyclic diterpene skeleton, comprising unusual 9,10-dioxatricyclo[5.2.1.04,8]decane and tricyclo[5.2.1.02,6]decane motifs. The Flack param3030
DOI: 10.1021/acs.orglett.7b01323 Org. Lett. 2017, 19, 3029−3032
Letter
Organic Letters Scheme 1. Proposed Biosynthetic Pathways for 1 and 2
Figure 4. Impact of 1 on ConA-induced splenocyte proliferation in vitro. (A) Murine splenocytes were labeled with CFSE and then cultured with stimuli, ConA with or without cinnamomol A (1) as shown, for 72 h. Dead cells were excluded by TO-PRO-3. CFSE dilutions indicate proliferation. (B) Percentages of cells divided once (purple) or more than once (blue) among living cells for the same cultures shown in (A). Mean ± SD of six replicates are shown; *indicates 0.01 < p < 0.05, and ** indicates p < 0.01.
dioxolane structure, and produces 1. Finally, compound 2 is generated from 1 by an enzyme-mediated oxidation. Compounds 1 and 2 were evaluated for their in vitro immunomodulatory activity in a ConA (concanavalin A)induced splenocytes proliferation assay12 by assessing radiolabeled [3H]-thymidine ([3H]-TdR) incorporation into splenocyte cell DNA. As shown in Table S2, compounds 1 and 2 enhanced the proliferation of ConA-induced murine T cells with enhancement rates ranging from 29 to 64% at five different concentrations from 0.391 to 100 μM. In contrast, the positive control, Tα1, a first-line immnostimulator in the clinic,1 exhibited enhancement rates of 39, 32, 11, 11, and 8% at concentrations of 0.04, 0.2, 1, 5, and 25 μM, respectively. Thus, 1 and 2 had more potency in immunostimulative activities in vitro than that of Tα1. More importantly, 1 and 2 did not show obvious cytotoxity against murine lymphocytes at the test concentrations. To directly interrogate the cell proliferation impacted by cinnamomol A (1), carboxyfluorescein succinimidyl amino ester (CFSE)-labeled splenocytes were incubated with ConA in the absence or presence of different concentrations of 1. CFSE, a fluorescent cell-staining dye, is cell permeable and can covalently bind to intracellular molecules. When cells divide, CFSE labeling is distributed equally between the daughter cells that are half as fluorescent as the parent cells. Cell division can be measured as successive halving of the fluorescence intensity of CFSE.13 We found that the addition of 1 resulted in higher proportions of ConA-responding live cells and more extensive cell division (Figure 4); however, the survival of splenocytes was not impaired by 1 (Figure S2). Thus, cinnamomol A (1) could enhance ConAinduced lymphocyte activation in vitro. To explore specific effects of cinnamomol A (1) on T cells, anti-CD3/anti-CD28 monoclonal antibodies (mAbs) were used to stimulate and expand T cells.14 Thymopentin (Tp-5), a positive control, is an immunomodulatory peptide that has been used to treat patients with immune deficiencies, autoimmune diseases, and cancer.15 Results (Figure 5) showed that both compound 1 and Tp-5 remarkably enhanced the proliferation of CD3/28-induced T cell expansion. 1 was more favorable to promote CD3/28-induced T cell proliferation than Tp-5 at higher concentrations from 0.0977 to 100 μM. T cells play an important role in regulating immune function because various T cell subsets carry out different duties to maintain immune balance. There are two main types of T cells, CD4+ and CD8+ T cells, both of which are critical components of immunity. 1 6 However, one of the T cell subsets,
Figure 5. Impact of 1 on CD3/28-induced splenocyte proliferation in vitro. Control: spleen cells without CD3/CD28. Other groups are stimulated by CD3/CD28. Mean ± SD of three replicates are shown; * indicates 0.01 < p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 compared with Tp-5.
CD4 +CD25 +Foxp3 + T cells (Tregs), dampens immune responses by inhibiting the function of effector CD4+ and CD8+ T cells.17 We incubated cinnamomol A (1) with T cells to investigate which T cell populations are involved in immune modulation by 1. As shown in Figure 6, 1 promoted CD4+ T cell differentiation comparably to that of Tp-5 but was more active than CD3/28 alone or in combination with 1. Meanwhile, CD8+ T cells were unaffected by 1. Intriguingly, 1 dramatically decreased the percentage of Tregs in CD3/28-induced T cells and presented no distinction with Tp-5. Therefore, the findings indicated that 1 enhanced immunity in vitro by increasing CD4+ T cell proliferation, while reducing Treg differentiation. In summary, the leaves of C. cassia were phytochemically investigated for the first time, leading to the isolation of two novel diterpenoids, cinnamomols A (1) and B (2), and a known diterpenoid, perseanol (3). Cinnamomols A (1) and B (2) possess an unprecedented, cage-like, rigid, 5/5/5/5/5/6-fused hexacyclic diterpene skeleton, comprising 9,10-dioxatricyclo[5.2.1.04,8]decane and tricyclo[5.2.1.02,6]decane moieties. Cinnamomols A (1) and B (2) exhibited significant in vitro immunostimulative activities compared to those of Tα1 and Tp5 at concentrations over 0.3906 μM. The mode of action studies revealed that 1 enhances immunity in vitro by increasing CD4+ T cell expansion but reducing Tregs. This finding provides a new structural class of immunostimulative agents. 3031
DOI: 10.1021/acs.orglett.7b01323 Org. Lett. 2017, 19, 3029−3032
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Organic Letters
CAS (2008DP173091-2016-01), and the Fundamental Research Funds for the Central Universities (HUST: 2016YXMS148). We thank the Analytical and Testing Center at HUST for spectroscopic data collection.
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Figure 6. Effects of 1 on T cell subsets in spleen by FACS analysis in vitro. (A,B) Cells were stained with CD3, CD4, or CD8. The percentage of T cell population is shown in the column. (C) Representative FACS staining for CD25 and Foxp3 on gated CD4+ T cells, and the percentage of CD4+CD25+Foxp3+ Tregs is displayed. Mean ± SD of three replicates are shown; *indicates p < 0.05.
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REFERENCES
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01323. Experimental procedures; spectroscopic data, 1D and 2D NMR, and HR ESI-MS data of 1 and 2 (PDF) X-ray data for 1 (CCDC 1438196) (CIF) X-ray data for 2 (CCDC 1438197) (CIF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Yongbo Xue: 0000-0001-9133-6439 Guangmin Yao: 0000-0002-8893-8743 Yonghui Zhang: 0000-0002-7222-2142 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (31370372), the Foundation of the Key Laboratory of Plant Resources and Chemistry in Arid Regions, Xinjiang Technical Institute of Physics and Chemistry, 3032
DOI: 10.1021/acs.orglett.7b01323 Org. Lett. 2017, 19, 3029−3032
