Phainanolide A, Highly Modified and Oxygenated Triterpenoid from

Letter pubs.acs.org/OrgLett

Phainanolide A, Highly Modified and Oxygenated Triterpenoid from Phyllanthus hainanensis Yao-Yue Fan,† Li-She Gan,‡ Hong-Chun Liu,† Heng Li,† Cheng-Hui Xu,† Jian-Ping Zuo,† Jian Ding,*,† and Jian-Min Yue*,† †

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China ‡ Institute of Modern Chinese Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China S Supporting Information *

ABSTRACT: Phainanolide A (1), a highly modified triterpenoid incorporating an unprecedented 6/9/6 heterotricyclic system and a highly oxygenated 5,5-spirocyclic ketal lactone, along with three new triterpenoids 2−4 were isolated from Phyllanthus hainanensis. Their structures were completely elucidated by a combination of diverse methods including 2D NMR, quantum chemical NMR and ECD calculations, and NMR data analogy with model compounds. Compounds 1−4 exhibited both remarkable cytotoxic and immunosuppressive activities.

T

he Phyllanthus genus (Euphorbiaceae family), comprising over 600 species distributed mainly in the tropical and subtropical areas of the world,1 has afforded a large number of structurally interesting metabolites.2 Our previous study on the plants of P. hainanensis,3 which are endemic in the hainan island of China, yielded six immunosuppressive agents, phainanoids A−F featuring a highly modified triterpenoid skeleton,4 which has immediately attracted the interests of synthetic chemists.5 In our continuing search for immunosuppressive components, an in-depth investigation on the remaining fractions of the same plant sample led to the further isolation of four highly modified triterpenoids, named phainanolide A (1) and phainanoids G−I (2−4). Compound 1 incorporated an unprecedented 6/9/6 heterotricyclic system in the down-left and a highly oxygenated 5,5-spirocyclic ketal lactone motif in the up-right. Compounds 2−4 are the congeners of our recently discovered immunosuppressive phainanoid type triterpenoids.4 The structures with the absolute configurations of compounds 1−4 were established by a combination of various methods including spectroscopic data analysis, quantum chemical NMR calculation, analogy of the key 13C NMR data with model compounds, and ECD calculation. The in vitro cytotoxic evaluation showed that compounds 1−4 exhibited potent activities against human leukemia HL-60 and human lung adenocarcinoma A-549 cell lines. Compounds 1 and 3 are comparable with the positive control adriamycin against HL-60 cell lines. Immunosuppressive test revealed that compounds 1− 4 also exhibited significant inhibitory effects with IC50 values at nanomolar levels against both the ConA-induced proliferation of T lymphocytes and LPS-induced proliferation of B lymphocytes. Herein, we present a full account of the isolation, structure elucidation, and bioactive evaluation of compounds 1−4. © 2017 American Chemical Society

Phainanolide A (1), a white amorphous solid, has a molecular formula of C42H48O12 as deduced from (−)-HRESIMS ion at m/z 789.3119 [M + HCO2]− (calcd for C43H49O14, 789.3122), suggestive of 19 degrees of unsaturation (DOU). The 1H and 13C NMR data (Tables S1 and S2, Supporting Information (SI)) of 1, with the aid of DEPT experiment and HSQC spectrum, revealed the presence of a 1,2-disubstituted benzene, three double bonds, three carbonyls (one keto at δC 213.3 and two esters at δC173.7 and 173.5), five sp3 methyls (one doublet at δC 22.8 and four singlets at δC 16.4, 17.3, 18.6, and 28.1), seven sp3 methylenes (two oxygenated at δC 78.0 and 72.7), eight sp3 methines (four oxygenated at δC 76.9, 70.9, 69.6, and 64.5), and seven sp3 quaternary carbons (one ketal at δC 108.8 and one oxygenated at δC 86.6). The above-mentioned functionalities accounted for 10 DOU, and the remaining ones thus required the attendance of nine additional rings in compound 1 to fulfill the unsaturation demand, suggesting that compound 1 possessed a decacyclic framework. The existence of a 1,1,2,2-tetrasubstituted cycloReceived: July 18, 2017 Published: August 23, 2017 4580

DOI: 10.1021/acs.orglett.7b02181 Org. Lett. 2017, 19, 4580−4583

Letter

Organic Letters propane ring was first identified by the diagnostic resonances of the shielded one methylene (δH 1.30 and 1.05; δC 14.5) and two quaternary carbons (δC 36.6 and 31.7).6 In addition, three proton resonances at δH 3.89, 3.31, and 2.36 showing no correlations with any carbons in the HSQC spectrum were assigned to the hydroxy groups. The planar structure of 1 was partially assembled by analysis of 2D NMR spectra, especially 1H−1H COSY and HMBC correlations (Figure 1A). Seven structural fragments as drawn

NOESY correlations of H-5/H-9 and H3-28, H-9/H-30a, and H-30b/H-16α demonstrated that H-9, Me-28, and CH2−30 were in α-orientation. The relative stereochemistry of 5,5spiroketal moiety with an α-orientated 22-OH and a βorientated 24-OH was determined by the NOESY correlations of H-12/H-20, H-16α/H-22, H-17/H-20, H-30b/H-22, H-22/ H-24, and H-26β/H3-27, which was identical to those of phainanoids A−F.4 However, the assembly of components I and II, and the assignment of the relative configuration of C-11′ were much challenged due to the lack of the available HMBC correlation of H-11′/C-1′ and relevant NOESY correlations. The remaining one DOU and the chemical shifts of C-1′, C-8′, C-9′, and C-11′ (δC137.1, 153.3, 173.7, and 64.5, respectively) in the “loose ends” of two components thus required the formation of a nine-membered lactone ring to bridge them into a whole molecule. In order to determine the linkage patterns of two components I and II and the relative configuration of C-11′ in the nine-membered lactone ring, four model compounds without the triterpenoid side chain representing all the four possible isomers were proposed (Figure 2, the details see the

Figure 1. Key 2D NMR correlations for phainanolide A (1).

in bold bonds were delineated by 1H−1H COSY cross-peaks, and six of them except for the fragment (C-10′ to C-12′) of a 3hydroxybutyrate moiety were connected with other functional groups, quaternary carbons, and oxygen atoms by HMBC correlations to accomplish the modified main triterpenoid framework. In detail, the multiple HMBC correlations of H-2/ C-3 and C-1′; H-5 and OH-6/C-7; H3-18/C-7, C-8, C-9, and C-14; H3-19/C-1, C-5, C-9, and C-10; H3-28/C-3, C-4, C-5, and C-29; H2-29/C-3 and C-2′; H2-30/C-12, C-13, C-14, C-15, and C-17; H-4′/C-2′, C-6′, and C-8′; and H-7′/C-3′ allowed the establishment of the phenylpyranotriterpenoid core with a cyclopropyl moiety. The chemical shift of C-23 at δC 108.8 and the mutual HMBC cross-peaks of H-20/C-21; H-22, OH-22, OH-24, and H2-26/C-23; and H3-27/C-24, C-25, and C-26 revealed the presence of a 5,5-spiroketal lactone moiety, which was attached to C-17 by the key 1H−1H COSY correlation of H-17/H-20 and the HMBC correlation of H-17/C-21. The aforementioned deduction delineated the main part (component I) of 1 as a dichapetalin-type triterpenoid with a 5,5spiroketal lactone motif.7 The presence of a 3-hydroxybutyrate motif (component II) in 1 was readily identified by NMR data (Tables S1 and S2, SI), 1H−1H COSY analysis (H2-10′ via H11′ to H3-12′), and HMBC correlations of H2-10′ and H3-12′/ C-9′ (δC 173.7). The relative configuration of the component I in 1 was assigned by the NOESY spectrum (Figure 1B). In the NOESY spectrum, the cross-peaks of H-6β/H3-18 and H3-19, H-16β/ H-17, and H3-19/H-29β revealed that H-6β, H-16β, H-17, Me18, Me-19, and H-29β were cofacial and arbitrarily assigned as β-orientated. The α-orientated assignment of H-5 was based on the large coupling constant (J5,6β = 12.9 Hz). Accordingly, the

Figure 2. Four model compounds designed for the quantum chemical NMR calculation of compound 1.

NMR calculation for compound 1 in SI), and their 13C NMR calculations were carried out by using density functional theory (DFT) at the rmpw1pw91/6-31+g(d,p) level. After calculation, the chemical shifts of the lactone ring associated carbons (C1′−C-12′) were selected for comparing with those of experimental data, respectively. The linear correlation coefficients (R2), root-mean-square deviation (RMSD), and the DP4+ method8 were adopted for evaluation of the results. The calculation results indicated that the model compound 1a showed the highest R2 and lowest RMSD values among the calculated model isomers (Table S3, SI), which favored an ether bond between C-1′ and C-11′, an ester linkage between C-8′ and C-9′, and an α-configuration for H-11′. A significant higher DP4+ probability score (95.89%) (Table S4, SI) again suggested that 1a was the right structure. Additionally, the formation of an ester at the C-8′ of 1 was further corroborated by comparing its C-8′ chemical shift with those of the key carbons in the model compounds (the details see the NMR data analogy of compound 1 with the model compounds in SI). The structure of compound 1 was thus completely assigned. The attendance of interesting H-bonds in the 5,5-spiroketal lactone moiety of phainanoids A−F was demonstrated previously by the chemical shifts and coupling constants of OH-24.4 Compound 1 possessed an additional OH-22α that 4581

DOI: 10.1021/acs.orglett.7b02181 Org. Lett. 2017, 19, 4580−4583

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Organic Letters

20.9). The acetoxy group was readily positioned at C-6 by the markedly downfield shifted H-6 resonance (ΔδH 0.99) as compared with that of phainanoid B, and was confirmed by the key HMBC correlation from H-6 to the carbonyl carbon of the acetyl group (Figure S1A, SI). As the consequence, the C-5 (ΔδC −3.7 ppm) and C-7 (ΔδC −7.3 ppm) were severely upfield shifted due to the γ-gauche effect from the acetyl group. Further examination of ROESY spectrum (Figure S1B, SI) and comparison of the NMR data of 2 with those of phainanoid B supported the common relative configurations at all the stereogenic centers and double bonds for the two cometabolites. Phainanoid H (3) has a sodiated molecular ion peak at m/z 765.3237 [M + Na]+ (calcd for C43H50O11Na, 765.3245) in the (+)-HRESIMS spectrum, consisting with a molecular formula of C43H50O11. Comparison of its 1H and 13C NMR data (Tables S1 and S2, SI) with those for phainanoid B showed high similarities except for the absence of the 25-OH proton signal and the presence of the additional NMR resonances for an ester carbonyl (δC 172.8), an oxygenated methine (δH 3.75; δC 73.8), a methylene (δH 2.41 and 2.54; δC 42.4), a secondary methyl (δH 1.21; δC 19.2), and a methoxy group (δH 3.33; δC 56.6). These extra proton and carbon resonances were assignable to a 3-methoxybutanoyl group, which was verified by the 1H−1H COSY and HMBC spectra (Figure S2A, SI), and was fixed at C-25 to form an ester on the basis of chemical shift changes of C-24 to C-27 as compared with those of phainanoid B. The relative configurations of all the double bonds and stereogenic centers except for that of C-3″ in the C-25 appendage were assigned to be identical with those of phainanoid B by the ROESY data (Figure S2B, SI) and their similar NMR patterns. Phainanoid I (4) shares a molecular formula of C38H42O9 with phainanoid B as deduced from the (−)-HRESIMS ion at m/z 687.2826 [M + HCO2]− (calcd for C39H43O11, 687.2805), indicative of their isomeric nature. Analysis of the 2D NMR spectra, particularly 1H−1H COSY and HMBC correlations (Figure S3A, SI) further revealed that compound 4 also possessed the same gross structure as phainanoid B. The 1D NMR data of 4 (Tables S1 and S2, SI) showed many similarities to those of phainanoid B, and the main differences were associated with the proton and carbon resonances from the 5,5-spiroketal lactone moiety. Compared with phainanoid B, the C-23 of 4 was markedly downfield shifted (ΔδC 4.2 ppm), and in concomitant the small changes were also observed for the neighboring carbons from C-20 to C-27 (Table S2, SI), suggesting that the configuration of C-23 in 4 was inverted. This assignment was supported by ROESY spectrum (Figure S3B, SI), where the ROESY interaction between H-22β and H-24 observed for phainanoid B was absent in 4. The relative configurations of the other stereogenic centers and double bonds were established to be identical with those of phainanoid B by the ROESY correlations and the excellent NMR resemblance. From the biosynthetic standpoints, and in combination with the chemical evidence, the absolute configurations of the highly modified triterpenoid cores of compounds 2−4 were suggested as depicted, and were confirmed by their resembling CD spectra (Figure S4, SI) with those of phainanoids A−F.4 The absolute configuration of C-3″ was tentatively assigned as Sconfigured by biogenetic correlations with the cometabolites of compound 1 and phainanoids E and F that possessed the biogenetically related acyl groups.

severely affected the chemical shift and coupling constant of OH-24 (δ24‑OH 3.31, d, J24,OH = 5.4 Hz in CDCl3) as compared with those of phainanoids A and B (δ24‑OH 2.05 and 2.26, d, J24,OH = 10.9 and 10.1 Hz, respectively, in CDCl3). This was believed to be caused by the formation of a strong intramolecular H-bond between OH-24 and the O atom of the lactone (H-bond angle and length: 100.6° and 2.2 Å, respectively),9 which was likely driven and stabilized by the existence of a more stronger H-bond between OH-22 and OH25 (Figure 3). The dihedral angle of 108.1° between H-24 and

Figure 3. Optimized 3D structure for the 5,5-spiroketal lactone of 1 generated by Hartree−Fock/3-21G showing the dihedral angle of H− C−O−H (blue) and H-bond angles (red) and lengths (Å).

OH-24 generated by Hartree−Fock/3-21G attributed to the smaller coupling constant for the OH-24, which satisfied the Karplus equation.10 The absolute configuration of compound 1 was established by comparing its experimental ECD spectrum with that of the model compound 1a acquired by quantum chemical TDDFT calculation, and the B3LYP/6-31+G(d,p) optimized conformer of model 1a in the 13C NMR calculation was adopted for this computation (the details see the ECD calculation for compound 1 in SI). The tendency of the experimental ECD curves of 1 showed good consistent with that of the calculated ECD spectrum of 1a at the range of 200 to 400 nm (Figure 4), which allowed the assignment of the absolute configuration of compound 1 as depicted. Phainanoid G (2) gives a molecular formula of C40H44O10 as established by the (−)-HRESIMS ion at m/z 729.2922 [M + HCO2]− (calcd for C41H45O12, 729.2911). Analysis of its 1D NMR data (Tables S1 and S2, SI) revealed that compound 2 was an analogue of phainanoid B4 with the evident difference being the presence of an acetyl group (δH 2.18; δC 170.2 and

Figure 4. Experimental CD spectrum of compound 1 (black line) and calculated ECD spectrum of model compound 1a (red line). 4582

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ACKNOWLEDGMENTS Financial supports of the National Natural Science Foundation (Nos. 21532007, U1302222) of the P. R. China and the “Personalized MedicinesMolecular Signature-based Drug Discovery and Development”, Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA12020321) are highly acknowledged. We thank Prof. S.-M. Huang of Hainan University for the identification of the plant materials.

All the isolates were tested for cytotoxic activities against HL60 and A-549 human tumor cell lines in vitro with adriamycin as the positive control. The assay results showed that compounds 1−4 exhibited remarkable activities (Table 1). Table 1. In Vitro Cytotoxic Activity of Compounds 1−4 (IC50± SD in μM) compounds

HL-60

A-549

1 2 3 4 adriamycin

0.079 ± 0.037 10.036 ± 0.307 0.025 ± 0.007 2.340 ± 1.383 0.073 ± 0.015

8.957 ± 0.519 20.900 ± 10.282 11.266 ± 3.049 32.802 ± 3.931 0.324 ± 0.030

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Table 2. Immunosuppressive Activities of Compounds 1−4 LPS-induced B-cell proliferation

compounds

CC50 (nM)

IC50 (nM)

SIa

IC50 (nM)

SIa

1 2 3 4 CsA

356.45 1234.22 19.14 819.33 >1000

364.75 566.83 16.15 218.14 14.21

0.98 2.18 1.19 3.76 >70.37

245.47 456.63 8.24 305.38 352.87

1.45 2.70 2.32 2.68 >2.83

a

The selectivity index (SI) is determined as the ratio of the concentration of the compound that reduced cell viability to 50% (CC50) to the concentration of the compound needed to inhibit the proliferation by 50% relative to the control value (IC50).

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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.7b02181. Tabulated NMR data of 1−4; selected key 2D NMR correlations of 2−4; experimental section; NMR calculation for 1; NMR data analogy of compound 1 with model compounds; ECD calculation for 1; bioassays; and 1D and 2D NMR, MS, and IR spectra of compounds 1−4 (PDF)

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REFERENCES

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Particularly, compounds 1 and 3 exhibited potent activities against HL-60 cell lines with IC50 values of 0.079 and 0.025 μM, respectively, which were comparable to that of the positive control adriamycin. Compounds 1−4 were also evaluated for immunosuppressive activity against the proliferation of T and B lymphocyte in vitro (Table 2) with cyclosporin A (CsA) as the positive control, and all compounds exhibited pronounced activities at nM levels.

ConA-induced T-cell proliferation

Letter

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. 4583

DOI: 10.1021/acs.orglett.7b02181 Org. Lett. 2017, 19, 4580−4583