Article pubs.acs.org/jnp
Eucarobustols A−I, Conjugates of Sesquiterpenoids and Acylphloroglucinols from Eucalyptus robusta Yang Yu,† Li-She Gan,‡ Sheng-Ping Yang,† Li Sheng,† Qun-Fang Liu,† Shao-Nong Chen,§ Jia Li,*,† and Jian-Min Yue*,† †
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China ‡ Institute of Modern Chinese Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China § Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, United States S Supporting Information *
ABSTRACT: Nine new conjugates of sesquiterpenoids and acylphloroglucinols, named eucarobustols A−I (1−9), as well as 11 known analogues were isolated from the leaves of Eucalyptus robusta. The sesquiterpenoid motifs furnishing the new conjugates included four structural types of aristolane (1 and 2), guaiane (3), eudesmane (4), and aromadendrane (5−9) moieties. Compounds 1 and 2 were found to represent the first examples of conjugates of aristolane and acylphloroglucinol units. In turn, compound 3 features a new coupling model of guaiane and acylphloroglucinol via the C-4−C-7′ bond. Compounds 1, 7, and 9 showed inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 1.3, 1.8, and 1.6 μM, respectively.
T
he genus Eucalyptus (Myrtaceae) contains about 700 species globally, of which over 100 species and varieties grow in mainland China.1 Eucalyptus robusta Smith occurs widely in the southern region of the country, and its leaves have been used in Chinese folk medicine for the treatment of dysentery, malaria, and other bacterial diseases.2 Previous studies on the genus of Eucalyptus have afforded an array of structurally diverse conjugates of sesquiterpenoids and acylphloroglucinols with a wide spectrum of biological activities, such as antiviral, antifungal, antibacterial, cytotoxic, HIV-RTase inhibition, granulation inhibition, aldose reductase inhibition, and HGF/c-Met axis inhibition effects.3−25 As part of our continuing efforts to obtain structurally interesting and biologically relevant components from this plant genus,24,25 20 conjugates of sesquiterpenoids and acylphloroglucinols were isolated from the leaves of E. robusta. Among them, nine were identified as new compounds, named eucarobustols A−I (1−9). Compounds 1 and 2 represent the first examples of the conjugates of aristolane and acylphloroglucinol. Compound 3 features a new coupling model of guaiane and acylphloroglucinol via the C-4−C-7′ bond. Compound 1026 was isolated as a natural substance for the first time. Compounds 1−10 showed significant inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) with IC50 values ranging from 1.3 to 5.6 μM. Herein, the isolation, structural elucidation, and biological evaluation of these new natural products are discussed. © XXXX American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Compound 1, a pale, amorphous powder, [α]20D −150 (c 0.1, MeOH), gave a molecular formula of C28H38O5 as determined by its 13C NMR data and the (−)-HRESIMS at m/z 453.2629 [M − H]− (calcd for C28H37O5, 453.2641), requiring 10 indices of hydrogen deficiency (IHD). The IR spectrum indicated the presence of hydroxy (3431 cm−1) and carbonyl (1630 cm−1) Received: February 1, 2016
A
DOI: 10.1021/acs.jnatprod.6b00090 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. 1H NMR Spectroscopic Data of Compounds 1−4a position 1 2 3
4 6 7 8
9
1b
2b
3b
4c
(J in Hz)
(J in Hz)
(J in Hz)
(J in Hz)
3.20 brdd (11.5, 4.8) α 1.90 brd (13.5) β 1.52 m α 2.02 qd (13.5, 2.5) β 1.32 m 1.68 m 0.70 d (9.3) 0.81 brdd (9.3, 7.0) α 2.48 ddd (18.7, 7.0, 2.2) β 2.13 brdd (18.7, 5.0) 5.44 dd (5.0, 2.2)
3.39 brdd (11.0, 5.3) α 2.22 m β 1.64 m α 1.78 m
α 1.62 m β 1.38 m α 1.53 m
β 2.06 m
β 1.53 m 2.39 m 5.19 d (4.0) 2.59 brt (4.0) α 1.59 m
α 2.25 m
α 1.97 m
β 2.01 brdd (18.7, 5.1) 5.62 m
β 1.83 qd (12.0, 3.2) α 1.27 tt (12.0, 3.2) β 2.04 m 2.01 m 1.37 s
β 1.83 m
a 3.17 m b 3.17 m
a 2.29 brdd (11.3, 9.8) b 1.23 m
2.39 m 1.01 d (6.6) 1.01 d (6.6)
1.22 m 0.80 d (6.1) 0.82 d (6.0)
0.98 s
13 14 15 7′
1.19 s 1.44 s 1.01 d (6.8) 10.61 s
1.19 s 1.43 s 1.02 d (6.6) 10.56 s
8′ 9′
10.62 s 3.95 td (11.5, 3.6) a 2.43 td (11.5, 2.0) b 1.51 m
10.53 s 3.79 td (11.0, 3.2) a 2.34 td (11.0, 2.0) b 1.57 ddd (13.2, 11.0, 3.2) 1.82 m 0.97 d (6.0) 1.18 d (6.4)
1.66 m 0.97 d (6.6) 1.08 d (6.4)
α 1.52 m β 2.08 m α 1.45 m
6.01 m 2.42 m
1.03 s
11′ 12′ 13′
1.37 m
β 1.37 m 1.74 m 0.66 m 0.66 m
10 12
10′
3.31 m
to C-5 and from H-6 and H-8 to C-10. The HMBC cross-peaks from H-1 (δH 3.20) to C-5, C-9, and C-10 connected C-1 and C-9 with C-10, which allowed the establishment of two six-membered rings in the sesquiterpenoid moiety. The multiple HMBC correlations from H-12 and H-13 to C-6, C-7, and C-11 not only showed the attachment of two C-12 and C-13 methyl groups to the quaternary carbon C-11 but also connected the C-6 and C-7 methines with C-11 to furnish the cyclopropane ring for an aristolane sesquiterpenoid.27 The HMBC correlations from H-9′ to C-1′, C-5′, and C-6′, from H-7′ to C-1′ and C-3′, and from H-8′ to C-3′ and C-5′ then revealed the presence of a 3,5-diformyl-isopentyl phloroglucinol group, which was further connected with the sesquiterpenoid moiety via the C-1−C-9′ bond that was consistent with the correlation of H-1/H-9′ in the 1H−1H COSY spectrum. The relative configuration of 1 was established on the basis of coupling constants (Table 1 and Figure S8, Supporting Information) and ROESY data (Figure 1B and Figures S16 and S17, Supporting Information). The ROESY correlations of H-3α/H-14, H-14/H-15, H-15/H-6, H-6/H-7, and H-7/H-12 showed that H-6, H-7, H-14, and H-15 are cofacial, and these were arranged randomly in an α-orientation. The large coupling constant between H-6 and H-7 (J = 9.3 Hz) revealed that they are cis-configured in the cyclopropane ring because dihedral angles are rigidly fixed by the geometry of the ring system and Jcis is always larger than Jtrans.28,29 The ROESY correlations of H-9′/H-3α and H-9′/H-14 showed that the acylphloroglucinol group occupied a pseudoaxial position on the twisted sixmembered ring of the aristolane moiety and was α-oriented, indicating that the H-1 is β-configured. The large sesquiterpenoid and 3,5-diformyl phloroglucinol moieties hindered the free rotation of the C-1−C-9′ bond as speculated by the observation of the ROESY correlations between H-9′/H-3α, H-9′/H-14, H-10′a/H-1, H-10′a/H-9, and H-10′b/H-9, suggesting that H-9′ took an α-orientation. The structure of 1 was thus elucidated and named eucarobustol A, which is the first example of the conjugate of an aristolane-type sesquiterpenoid and an acylphloroglucinol. Compound 2 was obtained as a pale, amorphous powder and shared the same molecular formula with 1 as determined by the (−)-HRESIMS ion at m/z 453.2637 [M − H]− (calcd for C28H37O5, 453.2641). The 1H and 13C NMR data (Tables 1 and 3) of 2 also showed high similarity to those of 1. Comparison of their NMR data indicated that C-1 and C-2 were shielded by ΔδC −1.7 and −1.7, whereas C-6′ and C-10′ were deshielded by ΔδC +2.2 and +1.8 in 2, respectively, suggesting that it is likely the C-9′ epimer of 1. This was proved by the 2D NMR data (Figures S1 and S23−30, Supporting Information), especially the ROESY spectrum (Figures S1B, S29, and S30, Supporting Information), in which the correlations of H-9′/ H-3α, H-9′/H-14, H-10′a/H-1, and H-10′b/H-2α indicated that H-9′ is β-oriented. Therefore, the structure of compound 2 was assigned and named eucarobustol B. Compound 3, a pale, amorphous powder, gave a molecular formula of C28H40O6 with nine IHDs as established by the (−)-HRESIMS ion at m/z 471.2738 [M − H]− (calcd for C28H39O6, 471.2747). Analysis of the 1H and 13C NMR data (Tables 1 and 3) indicated that 3 is also a conjugate of sesquiterpenoid and acylphloroglucinol units, of which the latter motif is identical to that of eucalyptin A,25 with differences occurring in the sesquiterpenoid part. In the 1H−1H COSY spectrum (Figure 2A and Figure S36, Supporting Information), the mutual coupling cross-peaks showed the presence of a spin
α 1.51 m β 1.76 m
a 4.85 brs b 4.67 brs 1.36 s 1.77 s 0.92 d (6.8) 1.13 s 1.35 s 1.06 d (7.5) a 3.22 d (13.6) 9.96 s b 3.07 d (13.6) 10.52 s 9.95 s 3.47 m
a Chemical shifts (ppm) were referenced to the solvent peaks (δH 7.58 in C5D5N and δH 3.31 in CD3OD). bData were measured in C5D5N at 500 MHz. cData were measured in CD3OD at 500 MHz.
groups. Analysis of the 1H and 13C NMR data (Tables 1 and 3) as well as the DEPT and HSQC spectra (Figures S9, S11, and S12, Supporting Information) revealed the presence of a 3,5-diformyl phloroglucinol,24 six methyls, four methylenes, six methines, two sp3 quaternary carbons, and a trisubstituted double bond. The aforementioned data and literature investigation on the compound classes occurring in the Eucalyptus genus suggested that 1 is a conjugate of a sesquiterpenoid and 3,5-diformyl-isopentyl phloroglucinol. The above identified functionalities accounted for seven out of the 10 IHDs thus required, so that the sesquiterpenoid moiety in the conjugate could be proposed as tricyclic. The structure of 1 was then elucidated further using its 2D NMR data (Figure 1). Analysis of the 1H−1H COSY spectrum (Figure 1A and Figure S10, Supporting Information) revealed the presence of two coupling spin systems as drawn with bold bonds. In the HMBC spectrum (Figure 1A and Figures S13−15, Supporting Information), the correlations from H-14 to C-4, C-5, C-6, and C-10 established the connections of C-4, C-6, C-10, and C-14 to the quaternary carbon C-5, which was proved by the correlations from both H-9 (δH 5.44) and H-15 B
DOI: 10.1021/acs.jnatprod.6b00090 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. 1H NMR Spectroscopic Data of Compounds 5−9 position 1 2 3 5 6 7 8 9 10 12 13 14 15 7′ 8′ 9′ 10′ 11′ 12′ 13′ OMe a
5a
6a
7a
8a
9a
(J in Hz)
(J in Hz)
(J in Hz)
(J in Hz)
(J in Hz)
2.20 ddd (11.0, 10.0, 4.4) α 1.59 m β 1.59 m α 1.47 ddd (12.0, 6.0, 3.0) β 1.82 m 1.42 t (10.0) 0.56 m 0.59 m α 1.01 m β 1.86 m α 1.72 brt (12.6) β 1.64 m
1.82 m α 1.64 m β 1.68 m α 1.34 m β 2.02 m 1.84 m 0.51 m 0.53 m α 1.58 m β 1.46 m α 1.51 brt (12.6) β 1.71 m
1.07 s 1.08 s 1.09 s 0.73 s 9.98 s 9.98 s 3.44 dd (12.6, 4.4) a 2.28 td (12.6, 4.4) b 1.09 m 1.24 m 0.79 d (6.6) 0.80 d (6.6) 3.18 s
1.06 s 1.09 s 1.18 s 0.74 s 10.02 s 10.02 s 3.42 dd (12.6, 4.0) a 2.26 td (12.6, 4.0) b 1.29 m 1.22 m 0.80 d (6.4) 0.79 d (6.4)
4.98 m
5.03 m
α 2.01 m β 2.50 dt (15.8, 2.7) 2.13 brd (9.5) 0.42 m 0.44 m α 1.80 m β 0.99 m α 1.63 m β 0.98 m 1.42 m 1.01 s 1.05 s 0.87 d (6.6) 1.17 s 10.04 s 10.05 s 3.20 dd (12.0, 3.5) a 2.36 td (12.0, 2.2) b 1.20 m 1.18 m 0.83 d (6.2) 0.77 d (6.1)
α 2.07 brd (15.9) β 2.37 dt (15.9, 2.6) 2.02 m 0.41 m 0.43 m α 1.79 m β 0.97 m α 1.72 m β 1.02 m 2.00 m 0.94 s 0.73 s 1.03 d (6.7) 1.19 s 10.06 s 10.05 s 3.33 m a 2.41 m b 1.19 m 1.20 m 0.84 d (6.0) 0.77 d (6.0)
α 2.36 m β 2.15 m α 1.33 m β 2.55 ddd (13.2, 9.2, 6.4) 1.14 m 0.92 m α 2.14 m β 1.51 m α 1.69 m β 1.47 m 2.20 m 1.23 s 1.03 s 1.00 d (7.1) 0.81 s 10.07 s 10.06 s 3.71 dd (12.6, 4.5) a 2.24 td (12.6, 5.1) b 1.14 m 1.31 m 0.76 d (6.6) 0.81 d (6.6)
Data were measured in CD3OD at 400 MHz, and chemical shifts (ppm) were referenced to the solvent peak (δH 3.31).
that its structure is closely related to macrocarpal E, possessing a eudesmane sesquiterpenoid and a 3,5-diformyl-isopentyl phloroglucinol moiety.6 The difference between 4 and macrocarpal E was the appendages at C-7, where an isopropenyl group (δH 4.85 and 4.67, each 1H, brs; δC 149.6 and 112.2) was assigned for 4 instead of a 2-propanoyl motif in macrocarpal E. This was supported by the molecular formula of C28H38O5 as assigned for 4 by the (−)-HRESIMS ion at m/z 453.2636 [M − H]− (calcd for C28H37O5, 453.2641), which is 18 mass units less than that of macrocarpal E. The structure of 4 was verified by 2D NMR data analysis (Figure 3 and Figures S50−57, Supporting Information). In particular, the HMBC correlations of H-6/C-11; H-7/C-11, C-12, and C-13; H-8/C-11; H-12/ C-13; and H-13/C-11 and C-12 confirmed the presence of an isopropenyl group at C-7. The relative configuration of 4 was then established by coupling constants (Table 1 and Figure S48, Supporting Information) and ROESY data (Figure 3B and Figures S56 and S57, Supporting Information). The ROESY correlations of H-14/H-8β, H-14/H-9β, and H-15/H-2β showed that they are cofacial and arranged randomly in a β-orientation. The small coupling constant of H-7 (δH 2.59, brt, J = 4.0 Hz) revealed it is equatorial and β-oriented. The H-1 proton was assigned as α-configured by the ROESY correlation network of H-1/H-12b, H-9α/H-12b, and H-13/H-12a. The ROESY correlations of H-1/H-10′a, H-9β/H-10′b, H-9′/H-14, and H-9′/H-9β indicated that H-9′ was β-directed. Thus, the structure of 4 was assigned and named eucarobustol D. Compound 5, obtained as a pale, amorphous powder, gave a molecular formula of C29H42O6 as determined by the (−)-HRESIMS ion at m/z 485.2894 [M − H]− (calcd for C29H41O6, 485.2903) and 13C NMR data. Comprehensive
system of CHCHCH2CH2CH(CH3)CHCH2CH2 as drawn with bold bonds. In the HMBC spectrum (Figure 2A and Figures S39−41, Supporting Information), the correlations from H-15 to C-3, C-4, and C-5 were used to connect C-3, C-5, and C-15 to the quaternary carbon C-4. The key HMBC correlation from H-6 to C-1 then allowed the construction of a 5,7-fused bicyclic ring system for 3, which was supported by the long-range coupling between the H-1 and H-6 signals in the 1 H−1H COSY spectrum. An 2-propanol group could then be attached to C-7 by the mutual HMBC correlations of H-12/C-7, C-11, and C-13; H-13/C-7, C-11, and C-12; and H-7/C-11. The aforementioned analysis revealed the presence of a guaiane-type sesquiterpenoid moiety in 3. The connection of sesquiterpenoid and acylphloroglucinol moieties via the C-4−C-7′ bond was finally made by the HMBC correlations of H-15/C-7′ and H-7′/C-3, C-4, and C-15. The relative configuration of 3 was established by the NOESY experiment (Figure 2B and Figures S42 and S43, Supporting Information). The H-1 proton was assigned randomly in a β-orientation by the mutual NOESY correlations of H-1/H-2β, H-1/H-3β, H-1/ H-8β, H-1/H-9β, and H-1/H-10. The NOESY correlations of H-2α/H-14, H-9α/H-14, H-8α/H-7, H-9α/H-7, and H-7/ H-14 then showed that H-7 and H-14 are α-oriented. In turn, the NOESY correlations of H-7′b/H-3β, H-7′a/H-6, and H-7′a/H-15 revealed H-15 to be α-oriented. The relative configuration assigned for the sesquiterpenoid unit of 3 was identical to those of macrocarpals D and O.6,10 Therefore, the structure of compound 3 was established as shown and named eucarobustol C. Compound 4 was obtained as a pale, amorphous powder. Analysis of its 1H and 13C NMR data (Tables 1 and 3) showed C
DOI: 10.1021/acs.jnatprod.6b00090 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 3. 13C NMR Spectroscopic Data of Compounds 1−9a position
1b
2b
3b
4c
5c
6c
7c
8d
9c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ OMe
46.7 30.8 28.5 40.1 37.7 34.8 20.4 22.9 124.7 142.1 18.3 30.6 16.0 24.1 16.6 173.2 108.8 177.0 108.3 173.2 105.1 192.4 192.0 34.2 40.5 27.7 25.5 22.5
45.0 29.1 27.6 40.1 37.1 34.7 20.5 23.0 123.2 142.6 18.2 30.6 15.9 24.9 16.6 173.3 108.0 175.3e 108.0 171.9 107.3 192.2 191.8 35.3 42.3 27.6 25.2 22.7
43.9 29.0 39.1 50.8 152.9 122.3 53.3 26.1 35.0 35.8 73.2 28.2 27.8 18.5 26.8 173.5 107.2 168.2 105.5 170.3 106.8 33.9 193.0 206.7 53.2 25.7 23.3 23.3
55.6 22.9 35.5 40.7 151.8 124.1 43.2 23.8 36.4 41.4 149.6 112.2 22.4 23.1 23.5 173.6 108.3 173.6 108.2 172.1 107.2 192.7 192.4 32.9 45.2 28.2 24.8 22.2
51.4 25.2 36.6 49.7 45.1 29.0 27.2 21.2 38.6 82.1 20.9 29.5 18.3 19.0 21.9 172.9 107.8 172.3 107.8 172.3 107.9 192.8 192.6 36.6 35.8 28.5 25.1 22.9 48.5
55.3 25.9 36.5 49.6 43.7 29.6 28.0 20.6 44.1 74.4 22.0 29.6 17.9 31.4 22.5 172.7 107.5 171.8 107.4 171.8 109.3 192.9 192.9 37.7 35.7 28.6 25.1 22.9
154.4 120.4 41.6 51.4 52.9 29.3 29.4 25.1 38.5 38.7 20.8 29.1 16.6 20.5 23.9 172.6 106.9 170.9 106.6 170.7 110.5 193.0 192.8 42.5 37.3 28.5 24.9 22.1
155.7 119.1 44.5 51.9 52.2 28.6 29.4 25.1 38.6 38.6 20.5 29.0 15.9 20.6 22.3 171.5 107.1 171.5 106.7 172.2 109.9 192.9 192.9 40.8 37.6 28.5 24.9 22.2
140.7 36.4 34.0 57.6 141.6 27.7 28.1 24.5 36.0 38.6 22.2 29.6 18.7 22.0 27.6 171.2 106.9 171.2 106.9 171.9 110.6 193.1 192.9 38.1 36.9 29.0 24.7 23.2
a Chemical shifts (ppm) were referenced to the solvent peaks (δC 135.91 in C5D5N and δC 49.15 in CD3OD). bData were measured in C5D5N at 125 MHz. cData were measured in CD3OD at 125 MHz. dData were measured in CD3OD at 100 MHz. eThe resonance data were obtained from the HMBC spectrum.
(calcd for C28H39O6, 471.2747) and its 13C NMR data. Comparing the NMR data of 6 with those of macrocarpal A,6 the C-10 signal of 6 was shielded (ΔδC −0.3), whereas its C-14 resonance was deshielded (ΔδC +13.5), suggesting that it is likely the C-10 epimer of the latter, which was supported by the fact that the NMR data of the sesquiterpenoid motif of 6 matched those of its biosynthetic precursor, (−)-epiglobulol.30 This assignment was confirmed by the 2D NMR spectra (Figures S75−81, Supporting Information), and particularly the ROESY data (Figures S3B, S80, and S81, Supporting Information), in which the correlations of H-1/H-9α, H-1/ H-14, H-1/H-15, H-2α/H-14, and H-2α/H-15 showed that H-14 is α-oriented. Therefore, the structure of compound 6 was assigned and named eucarobustol F. Compound 7 was assigned the molecular formula of C28H38O5 from its HRESIMS and 13C NMR data, which is 18 mass units less than that of 6, suggesting that it is a dehydration product of the latter compound. The NMR data (Tables 2 and 3) of 7 showed many similarities to those of 6, especially the presence of proton and carbon signals (δH 4.98, 1H, m; δC 154.4 and 120.4) for an additional trisubstituted double bond and the absence of the oxygenated quaternary carbon of 7 in the sesquiterpenoid part. Analysis of the COSY spectrum (Figure 4A and Figure S88, Supporting Information) allowed the construction of two spin coupling systems of CHCH2 and CH3CHCH2CH2CHCHCH, as drawn with bold bonds. In the HMBC spectrum (Figure 4A and Figures S91−93,
analysis of its NMR data (Tables 2 and 3, and Figures S61−69, Supporting Information) indicated that its structure is closely related with that of an aromadendrane-acylphloroglucinol conjugate, macrocarpal A,5,6 and the difference was the presence of an additional methoxy group (δH 3.18, 3H, s; δC 48.5) instead of the hydroxy group in the terpenoid part of macrocarpal A. This was verified by the key HMBC correlation (Figures S2A, S66, and S67, Supporting Information) from OCH3 to C-10 (δC 82.1). The relative configuration of 5 was established by the coupling constants (Table 2 and Figure S61, Supporting Information) and ROESY experiment (Figures S2B, S68, and S69, Supporting Information). The ROESY correlations of H-1/H-6, H-1/H-15, H-1/10-OCH3, H-2α/H-15, H-3α/H-15, H-6/H-15, H-6/H-12, and H-7/H-12 showed that these protons are on the same side of the molecule and H-1, H-6, H-7, H-15, and OCH3-10 were randomly assigned with an α-orientation. Subsequently, the ROESY correlations of H-14/2β, H-14/3β, H-14/H-5, H-14/8β, and H-8β/H-13 showed that H-5 and H-14 were β-configured. The large coupling constant between H-1 and H-5 (J = 10.0 Hz) supported the five- and sevenmembered rings in the sesquiterpenoid core being trans-fused. The H-9′ proton was assigned as α-directed by the ROESY correlations of H-9′/H-5, H-9′/H-15, H-10′a/H-3β, H-10′b/ H-3β, and H-10′b/H-5. Thus, the structure of compound 5, named eucarobustol E, was established as depicted. Compound 6 gave a molecular formula of C28H40O6 as established by the (−)-HRESIMS ion at m/z 471.2739 [M − H]− D
DOI: 10.1021/acs.jnatprod.6b00090 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 3. 1H−1H COSY (bold lines), selected HMBC (H→C) (A), and key ROESY (H↔H) (B) correlations of 4. Figure 1. 1H−1H COSY (bold lines), selected HMBC (H→C) (A), and key ROESY (H↔H) (B) correlations of 1.
Figure 2. 1H−1H COSY (bold lines), selected HMBC (H→C) (A), and key ROESY (H↔H) (B) correlations of 3.
Supporting Information), the correlations of H-2/C-1, C-4, and C-5; H-5/C-1, C-2, and C-4; H-6/C-4; and H-14/C-1 not only delineated the 5,7-fused bicyclic ring system for the sesquiterpenoid moiety of 7 but also were used to locate the Δ1 double bond. The other linkages of 7 were also confirmed by the HMBC correlations (Figure 4A). The relative configuration of 7 was assigned by the coupling constants and the ROESY data (Figure 4B and Figures S6, S94, and S95, Supporting Information). The ROESY correlations of H-3α/H-15, H-6/ H-12, H-6/H-15, H-7/H-8α, H-7/H-9α, and H-7/H-12
Figure 4. 1H−1H COSY (bold lines), selected HMBC (H→C) (A), and key ROESY (H↔H) (B) correlations of 7.
allowed for the arbitrary assignments of H-6, H-7, and H-15 in an α-orientation. Consequently, the ROESY correlations of H-8β/H-10 and H-9α/H-14 showed that H-10 is β-oriented. The large coupling constant between H-5 and H-6 (J = 9.5 Hz) revealed that H-5 is β-directed, which was supported by the ROESY correlations of H-5/H-8β and H-5/H-13. The H-9′ E
DOI: 10.1021/acs.jnatprod.6b00090 J. Nat. Prod. XXXX, XXX, XXX−XXX
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proton was α-configured, as evidenced by the ROESY correlation network of H-9′/H-5, H-9′/H-15, H-10′a/H-3β, H-10′b/ H-3β, and H-10′b/H-5. Thus, the structure of compound 7 was established and named eucarobustol G. Compound 8 was found to share the same molecular formula and very similar NMR data with 7, indicating them to be stereoisomers. Compared to those of 7, the 1H and 13C NMR data (Tables 2 and 3) showed variations in the vicinity of C-9′, especially that the C-5, C-15, C-6′, and C-9′ of 8 were upfield shifted (ΔδC −0.7, −1.6, −0.6, and −1.7, respectively), whereas C-3 and C-4 of 8 were downfield shifted (ΔδC +2.9 and +0.5, respectively), suggesting that compound 8 is the C-9′ epimer of 7. This deduction was proved by analysis of the 2D NMR data (Figures S101−108, Supporting Information), in particular the ROESY spectrum, in which the correlations (Figure 5 and
especially the HMBC spectrum (Figures S5A and S117−119, Supporting Information), in which the correlations of H-2/C-1, H-3/C-1, H-6/C-1, H-14/C-1, H-7/C-5, H-15/C-5, and H-9′/ C-5 were used to establish a Δ1(5) double bond. The relative configuration of 9 was confirmed by the ROESY data (Figures S5B and S120, Supporting Information). Thus, the ROESY correlations of H-3α/H-15, H-6/H-15, H-7/H-8α, H-7/H-12, H-14/H-2α, and H-14/H-9α showed that H-6, H-7, H-14, and H-15 are cofacial and these were arranged randomly in an α-orientation, whereas the ROESY correlations of H-8β/H-10, H-8β/H-13, and H-9β/H-10 revealed that H-10 is β-oriented. The H-9′ proton was assigned in an α-configuration by the ROESY cross-peaks of H-9′/H-6, H-9′/H-15, and H-3β/ H-10′a. Thus, the structure of compound 9 was assigned and named eucarobustol I. Compound 10 was isolated as a natural product for the first time. Its structure was identified by comparing the 1H and 13 C NMR and ESIMS data with the reported data for this compound when obtained as a semisynthetic product.26 The absolute configurations of 7 and 8 were assigned by comparing their experimental ECD curves with those of the quantum chemical TDDFT calculated results (Figure 6).25 The tendencies of experimental ECD curves of compounds 7 and 8 roughly matched the calculated ECD data, which allowed the assignments of the absolute configurations of 7 and 8 as depicted. From biosynthetic considerations, the absolute configurations of compounds 5, 6, 9, and 10 were inferred as drawn due to the fact that they share the same biosynthetic components of a 3,5-diformyl-isopentyl phloroglucinol moiety and the sesquiterpenoids of an aromadendrane type. Ten known compounds, macrocarpal A,5 macrocarpal B,6 macrocarpal C,6 macrocarpal D,6,10 macrocarpal E,6 macrocarpal O,10 macrocarpal L,10 macrocarpal M,10 eucalyptone,10,21 and macrocarpal N,10 were also isolated from this plant. Their structures were identified by comparison of their spectroscopic data with values reported in the literature. Protein tyrosine phosphatase 1B has emerged as an important therapeutic target in the treatment of type 2 diabetes and obesity.31,32 In our continuing search for PTP1B inhibitors,33,34 compounds 1−10 showed significant inhibitory activities (Table 4), and oleanolic acid was used as the positive control. Compounds 1−10 showed significant inhibitory activities against PTP1B with IC50 values ranging from 1.3 to 5.6 μM. Although only a limited number of conjugates were tested, it is speculated from the cases of two pairs of epimers (1/2 and 7/8) that the alteration of the relative configuration
Figure 5. Key ROESY (H↔H) correlations of 8.
Figures S6, S107, and S108, Supporting Information) of H-9′/ H-3β, H-9′/H-5, H-10′a/H-15, and H-10′b/H-5 indicated that H-9′ is β-oriented. Therefore, the structure of compound 8 was assigned as shown and named eucarobustol H. Compound 9 also shared the identical molecular formula and very similar NMR data (Tables 2 and 3) with those of 7, suggesting that they are stereoisomers. Comprehensive analysis of the NMR data of 9 showed that the only structural difference was the presence of a persubstituted double bond (two quaternary sp2 carbons at δC 141.6 and 140.7) instead of the trisubstituted double bond of 7. This was verified by the 2D NMR data (Figures S114−120, Supporting Information),
Figure 6. Experimental (black line) and B3LYP/6-311++G(2d,2p)//B3LYP/6-31+G(d)-calculated (red line) ECD spectra (200−400 nm) of 7 and (220−400 nm) of 8. F
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using CH3CN−H2O (90:10) with 0.3% HOAc as the mobile phase, to afford 1 (3.2 mg), 2 (2.0 mg), and 3 (20.6 mg). Subfraction C8-5-11 (126.0 mg) was subjected to RP-18 semipreparative HPLC (CH3CN− H2O−HOAc, 90:10:0.3) to afford 5 (3.0 mg), 9 (18.2 mg), and 10 (2.2 mg). Fractions C8-3 (4.6 g), C8-4 (5.1 g), and C8-6 (5.6 g) were chromatographed over a column of RP-18 silica gel (H2O−MeOH, 3:7−0:1) to give three subfractions, C8-3-8, C8-4-9, and C8-6-8, respectively. Subfraction C8-3-8 (67.0 mg) was subjected to RP-18 semipreparative HPLC (CH3CN−H2O−HOAc, 76:24:0.3) to afford 7 (3.8 mg) and 8 (5.6 mg). Subfraction C8-4-9 (58.0 mg) was subjected to RP-18 semipreparative HPLC (CH3CN−H2O−HOAc, 76:24:0.3) to afford 6 (4.0 mg). Subfraction C8-6-8 (36.0 mg) was subjected to RP-18 semipreparative HPLC (CH3CN−H2O−HOAc, 90:10:0.3) to afford 4 (2.1 mg). Eucarobustol A (1): pale, amorphous powder; [α]20D −150 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (4.37), 277 (4.38), 287 (4.36), 391 (3.82) nm; IR (KBr) νmax 3431, 2958, 2922, 2870, 1630, 1460, 1377, 1306, 1167, 997, 974, 843, 565 cm−1; 1H and 13C NMR see Tables 1 and 3; (−)-ESIMS m/z 453 [M − H]−; (−)-HRESIMS m/z 453.2629 [M − H]− (calcd for C28H37O5, 453.2641). Eucarobustol B (2): pale, amorphous powder; [α]20D −86 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 205 (4.18), 215 (4.19), 276 (4.32), 389 (3.69) nm; IR (KBr) νmax 3431, 2954, 2924, 2868, 1631, 1456, 1377, 1311, 1169, 1043, 553 cm−1; 1H and 13C NMR see Tables 1 and 3; (−)-ESIMS m/z 453 [M − H]−; (−)-HRESIMS m/z 453.2637 [M − H]− (calcd for C28H37O5, 453.2641). Eucarobustol C (3): pale, amorphous powder; [α]20D +18 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 211 (4.34), 277 (4.53), 291 (4.39), 385 (3.42) nm; IR (KBr) νmax 3433, 2958, 2927, 2871, 1624, 1460, 1377, 1313, 1167, 603 cm−1; 1H and 13C NMR see Tables 1 and 3; (+)-ESIMS m/z 455 [M − H2O + H]+; (−)-ESIMS m/z 471 [M − H]−; (−)-HRESIMS m/z 471.2738 [M − H]− (calcd for C28H39O6, 471.2747). Eucarobustol D (4): pale, amorphous powder; [α]20D +46 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 202 (4.29), 218 (4.28), 276 (4.26), 290 (4.23), 390 (3.79) nm; IR (KBr) νmax 3433, 2958, 2927, 2871, 2135, 1628, 1446, 1375, 1292, 1163, 1101, 1012, 908, 557 cm−1; 1 H and 13C NMR see Tables 1 and 3; (−)-ESIMS m/z 453 [M − H]−; (−)-HRESIMS m/z 453.2636 [M − H]− (calcd for C28H37O5, 453.2641). Eucarobustol E (5): pale, amorphous powder; [α]20D −78 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (4.32), 275 (4.42), 292 (4.32), 390 (3.78) nm; IR (KBr) νmax 3431, 2964, 2908, 1795, 1631, 1450, 1261, 1095, 1020, 872, 800, 702, 561 cm−1; 1H and 13C NMR see Tables 2 and 3; (−)-ESIMS m/z 485 [M − H]−; (−)-HRESIMS m/z 485.2894 [M − H]− (calcd for C29H41O6, 485.2903). Eucarobustol F (6): pale, amorphous powder; [α]20D −67 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (4.25), 276 (4.38), 293 (4.28), 389 (3.72) nm; IR (KBr) νmax 3431, 2954, 2927, 2870, 1631, 1452, 1377, 1311, 1171, 1128, 895, 590 cm−1; 1H and 13C NMR see Tables 2 and 3; (+)-ESIMS m/z 455 [M − H2O + H]+; (−)-ESIMS m/z 471 [M − H]−; (−)-HRESIMS m/z 471.2739 [M − H]− (calcd for C28H39O6, 471.2747). Eucarobustol G (7): pale, amorphous powder; [α]20D +33 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (4.26), 276 (4.44), 292 (4.32), 389 (3.61) nm; CD (MeOH) 222 (Δε −2.70), 275 (Δε −2.46), 310 (Δε +0.67), 353 (Δε +1.02); IR (KBr) νmax 3419, 2954, 2918, 2868, 1633, 1448, 1379, 1311, 1188, 839, 800, 613 cm−1; 1 H and 13C NMR see Tables 2 and 3; (−)-ESIMS m/z 453 [M − H]−; (−)-HRESIMS m/z 453.2635 [M − H]− (calcd for C28H37O5, 453.2641). Eucarobustol H (8): pale, amorphous powder; [α]20D +39 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (4.36), 276 (4.53), 290 (4.41), 388 (3.63) nm; CD (MeOH) 208 (Δε +6.23), 275 (Δε +2.29), 310 (Δε −0.11), 360 (Δε −0.46); IR (KBr) νmax 3433, 2954, 2927, 2868, 1631, 1452, 1377, 1309, 1176, 1126, 841, 802, 613 cm−1; 1H and 13C NMR see Tables 2 and 3; (−)-ESIMS m/z 453 [M − H]−; (−)-HRESIMS m/z 453.2629 [M − H]− (calcd for C28H37O5, 453.2641).
Table 4. Inhibitory Activities of Compounds 1−10 against PTP1B compound 1 2 3 4 5 6 7 8 9 10 oleanolic acid
IC50 ± SD in μM 1.3 4.3 4.3 2.9 4.1 5.6 1.8 3.0 1.6 4.5 2.3
± ± ± ± ± ± ± ± ± ± ±
0.13 0.46 0.36 0.31 0.76 0.38 0.13 0.46 0.15 0.48 0.15
of H-9′ will affect the resultant activity, which could provide useful information for future structural modification. Compounds 3 and 5−10, eucalyptone, and macrocarpals A−E, L−N, and O were tested for the cytotoxic activities against the HL-60 (human leukemia) and A-549 (human lung adenocarcinoma) cell lines, with adriamycin being used as the positive control, but none of them were active (IC50 < 10 μM).
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were obtained on a PerkinElmer 341 polarimeter at room temperature. UV spectra were measured on a Shimadzu UV-2550 spectrophotometer. CD spectra were obtained on a JASCO 810 spectrometer. IR spectra were recorded on a PerkinElmer 577 spectrometer with KBr disks. NMR spectra were measured on Bruker AM-400 or AM-500 NMR spectrometers. ESIMS and HRESIMS were carried out on a Bruker Daltonics Esquire 3000 Plus instrument and a WatersMicromass Q-TOF Ultima Global mass spectrometer, respectively. Silica gel (300−400 mesh, Qingdao Marine Chemical Plant, Qingdao, People’s Republic of China), C18 reversed-phase silica gel (150−200 mesh, Merck), MCI gel (CHP20P, 75−150 μM, Mitsubishi Chemical Industries Ltd.), and Sephadex LH-20 gel (Amersham Biosciences) were used for column chromatography. Precoated silica gel GF254 plates (Qingdao Marine Chemical Plant) were used for TLC. Semipreparative HPLC was performed on a Waters 1525 pump equipped with a Waters 2489 detector and a YMC-Pack ODS-A column (250 × 10 mm, S-5 μm). All solvents used for column chromatography were of analytical grade (Shanghai Chemical Reagents Company, Ltd.), and solvents used for HPLC were of HPLC grade (J&K Scientific Ltd.). Plant Material. The leaves of E. robusta were collected at Guangxi Province, People’s Republic of China, in August 2009 and authenticated by Prof. Shao-Qing Tang of Guangxi Normal University, People’s Republic of China. A voucher specimen has been deposited in Shanghai Institute of Materia Medica, Chinese Academy of Sciences (accession number: ER-2009-1Y). Extraction and Isolation. The air-dried powder of leaves of E. robusta (5.0 kg) was extracted with 95% ethanol three times at room temperature to give a crude extract (550 g). The crude extract was suspended in water (2 L) and then extracted with EtOAc (6 L) three times. The EtOAc extract (215 g) was subjected to passage over a column of macroporous adsorbent resin (SP-700, 5 kg, dried weight) eluted with aqueous EtOH (0−100%), to yield four fractions (A−D) after removing the solvents. Fraction C (100 g) was separated over a MCI gel column (H2O−MeOH, 1:0−0:1) to give eight fractions, C1−C8. Fraction C8 (48 g) was subjected to a silica gel column eluted with petroleum ether−acetone (20:1 to 1:1) to give nine fractions C8-1−C8-9. Subfraction C8-5 (3.2 g) was then chromatographed over a column of C18 reversed-phase (RP-18) silica gel (H2O−MeOH, 3:7−0:1) to give 12 subfractions C8-5-1−C8-5-12. Subfraction C8-5-10 (152.0 mg) was separately subjected to RP-18 semipreparative HPLC, G
DOI: 10.1021/acs.jnatprod.6b00090 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Eucarobustol I (9): pale, amorphous powder; [α]20D −57 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (4.45), 275 (4.54), 291 (4.43), 390 (3.67) nm; IR (KBr) νmax 3423, 2918, 2135, 1655, 1452, 1377, 1315, 1167, 899, 611 cm−1; 1H and 13C NMR see Tables 2 and 3; (−)-ESIMS m/z 453 [M − H]−; (−)-HRESIMS m/z 453.2635 [M − H]− (calcd for C28H37O5, 453.2641). PTP1B Inhibitory Activity Assay. PTP1B inhibitory activity was measured as reported previously.33,34 Cytotoxicity Assay. Cytotoxic activities were evaluated against the HL-60 and A-549 cell lines as previously reported.35
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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.jnatprod.6b00090. Calculated ECD data of 7 and 8; original NMR spectra of eucarobustols A−I (1−9) (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +86-21-50806718. Fax: +86-21-50807088. E-mail:
[email protected] (J.-M. Yue). *Tel: +86-21-50801552. Fax: +86-21-50800721. E-mail:
[email protected] (J. Li). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation (21272245; 21532007) and the Foundation (2012CB721105) from the Ministry of Science and Technology of the People’s Republic of China. We thank Prof. S.-Q. Tang of Guangxi Normal University, People’s Republic of China, for the identification of the plant material.
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REFERENCES
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DOI: 10.1021/acs.jnatprod.6b00090 J. Nat. Prod. XXXX, XXX, XXX−XXX
