Application of Residual Dipolar Couplings and Selective Quantitative

Article pubs.acs.org/jnp

Application of Residual Dipolar Couplings and Selective Quantitative NOE to Establish the Structures of Tetranortriterpenoids from Xylocarpus rumphii Watcharee Waratchareeyakul,†,‡ Erich Hellemann,§ Roberto R. Gil,§ Kan Chantrapromma,⊥ Moses K. Langat,†,∥ and Dulcie A. Mulholland*,†,∥ †

Natural Products Research Group, Department of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford GU2 7XH, U.K. ‡ Department of Chemistry, Faculty of Science and Technology, Rambhai Barni Rajabhat University, Chanthaburi 22000, Thailand § Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States ⊥ Faculty of Science and Technology, Hatyai University, Songkhla 90110, Thailand ∥ School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4041, South Africa S Supporting Information *

ABSTRACT: Nine triterpenoid derivatives were isolated from the heartwood of Xylocarpus rumphii and were identified as xylorumphiins E (1), C (2), L (3), and M−R (4−9). Compounds 4−9 have a hemiacetal group in the triterpenoid side chain, making them impossible to purify. Purification was achieved after acetylation and subsequent separation of the epimeric mixtures of acetates; however differentiaition of the R and S epimers was not possible using standard NMR techniques. In one case, the relative configuration of a remotely located stereocenter with respect to the stereocenters in the main skeleton was unambiguously determined using residual dipolar couplings. Dipolar couplings were collected from the sample oriented in compressed poly(methyl methacrylate) gels swollen in CDCl3. In another case, the relative configuration was determined using 1D selective quantitative NOE experiments. Xylorumphiin K (10), xyloccensin E, taraxer-14-en-3β-ol, (22S)-hydroxytirucalla-7,24-diene-3,23-dione, and 25-hydroxy-(20S,24S)-epoxydammaran-3-one were isolated from the bark of the same plant. Compounds 3−10 are new compounds. Compounds 1−6 and xyloccensin E were tested at one concentration, 1 × 10−5 M, in the NCI59 cell one-dose screen but did not show significant activity. gal,14 antifilarial,15 and insecticidal activity.16 Limonoids have been reported previously from the seeds and seed kernels of X. rumphii.12,17 The aim of this study was to investigate the phytochemistry of the bark and heartwood of X. rumphii. Herein the isolation of seven new tetranortriterpenoid derivatives (3−9), 11 acetylated derivatives (4a−d, 5a, 5b, 6a, 7a, 7b, 8a, and 9a), and two known limonoids (1 and 2) from the heartwood is reported. One new limonoid, xylorumphiin K (10), and the known xyloccensin E, along with three known triterpenoids, taraxer-14en-3β-ol, (22S)-hydroxytirucalla-7,24-diene-3,23-dione, and 25hydroxy-(20S,24S)-epoxydammaran-3-one, were isolated from the bark of the same plant. Structures are provided in Figure 1. Compounds 4−9 possess hemiacetal carbons at either C-23 (4−6) or C-21 (7−9), and due to the equilibration of the hemiacetal epimers in solution, the compounds cannot be purified. In order to enable purification, compounds were

Xylocarpus is a small genus belonging to the Meliaceae family. There has been debate about the number of species constituting this genus, with only three of the 17 names listed in the Plant List having an “Accepted” status.1 The three species are very similar and, consequently, have often been confused. X. granatum J. Koenig, also known as the cannonball, puzzlenut, or cedar mangrove, is a mangrove species found in Africa, Asia, Australasia, and the Pacific Islands. X. moluccensis (Lam.) M. Roem is a second mangrove species whose range stretches from Bangladesh, through Thailand, Indonesia, Malaysia, Papua New Guinea, to northern Australia. In many African floras, X. moluccensis (Lam.) is confused with X. rumphii (Kostel.) Mabb., the third species and the subject of this study, which is restricted to tropical Asia and Australia and does not occur in Africa. X. rumphii does not grow in mangroves, but occurs above the high water level on cliffs, rocks, and sandy upland areas.2 Previous ethnopharmacological investigations of extracts of the Xylocarpus genus have shown antibacterial,3,4 anticancer,3,5 cytotoxic,6 antidiarrheal,7,8 antiviral,3 antimalarial,9 antisecretory,10 antiosteoclastogenic,11 anti-inflammatory,12,13 antifun© 2017 American Chemical Society and American Society of Pharmacognosy

Received: October 4, 2016 Published: January 25, 2017 391

DOI: 10.1021/acs.jnatprod.6b00906 J. Nat. Prod. 2017, 80, 391−402

Journal of Natural Products

Article

Figure 1. Structures of compounds 1−10.

acetylated and the R and S epimers could be purified. However, for the C-23 epimers, use of standard NOESY and ROESY techniques did not permit their differentiation, using proton− proton short-range NOE correlations. Thus, compressed poly(methyl methacrylate) (PMMA) gels swollen in CDCl3 were used in order to orient the sample and measure RDCs (residual dipolar couplings), which permitted differentiation of the two epimers. RDCs provide information on nonlocal character and permit the determination of the relative configuration of stereocenters when NOEs fail to provide a solution. For the C-21 epimers, NOE-derived distances from 1D quantitative NOE experiments involving H-21 and protons from the skeleton, in combination with molecular modeling, permitted the unambiguous determination of the configuration at C-21. Compounds 1−6 and xyloccensin E were submitted for screening against the NCI59 cell panel.18

silica gel, leading to the isolation and identification of nine compounds. Compounds 1 and 2 were identified as the limonoids xylorumphiins E and C, previously isolated from the seeds and seed kernels of this species.12,17 HRMS data of compound 3 showed an [M + Na]+ ion at m/ z 679.3082, indicating a molecular formula of C36H48O11. The FTIR spectrum showed strong absorption bands at 1765 and 1724 cm−1 corresponding to CO stretching of an ester and ketone moiety, respectively. A comparison of the NMR spectra of compound 3 with those of compounds 1 and 2 indicated similarities and typical features of a mexicanolide class of limonoid. Resonances ascribed to protons of the β-substituted furan ring at C-17α were observed at δH 7.62 (1H, br s, H-21), 7.39 (1H, br s, H-23), and 6.50 (1H, br s, H-22), and resonances at δC 78.0 and δH 5.91 (s) could be assigned to C17 and H-17 using the HSQC and HMBC spectra. The H-17 resonance showed correlations with the C-18 (δC 14.4), C-12 (δC 26.0), C-13(δC 37.9), and C-14 (δC 67.6) resonances in the HMBC spectrum. Ring D comprised a δ-lactone moiety with the C-16 lactone carbonyl resonance (δC 169.4) showing

■

RESULTS AND DISCUSSION The CH2Cl2 extract of the dried, milled heartwood of X. rumphii was separated using repeated column chromatography over 392



Article

Table 1. NMR Data for Limonoids 1−3 and 10 (500 MHz, CDCl3) 1 position

δC, type

1 2 3 4 5 6

106.8, 57.2, 73.9, 38.3, 40.8, 32.7,

C CH CH C CH CH2

7 8 9 10 11

174.2, 82.4, 63.5, 43.9, 19.8,

C C CH C CH2

12

36.0, CH2

13 14 15

36.5, C 46.7, CH 29.2, CH2

16 17 18 19 20 21 22 23 28 29 30 1-OH 7-OMe 3-acyl 1′ 2′ 3′ 4′ 5′ 30-acyl 1″ 2″ 3″ 4″

170.4, 77.3, 22.4, 21.3, 121.1, 141.7, 110.2, 143.2, 24.9, 22.6, 76.0,

C CH CH3 CH3 C CH CH CH CH3 CH3 CH

52.0, CH3 177.7, 34.2, 18.5, 20.2,

C CH CH3 CH3

2

δH, mult. (J in Hz) 2.63, m 5.10, d (9.0) 2.60, d (9.9) 2.35, dd (16.4, 9.9) 2.26, d (16.4)

1.43, dd (13.4, 2.6) 1.90, 1.67, 1.83, 1.32,

m m m m

2.20, d (9.3) 3.25, d (19.6) 2.73, dd (19.6, 9.3) 5.25, s 1.03a 1.04a 7.52, 6.38, 7.38, 0.75, 1.19, 6.16,

br s br s br s s s d (4.2)

3.69, s

2.65, m 1.10, d (6.9) 1.21, d (7.1)

δC, type 107.7, 53.5, 73.9, 38.0, 40.8, 32.3,

C CH CH C CH CH2

174.0, 81.8, 51.8, 43.1, 15.2,

C C CH C CH2

25.1, CH2 39.1, C 159.8, C 117.9, CH 163.8, 81.4, 19.9, 20.7, 120.2, 141.5, 110.2, 143.1, 24.8, 22.1, 76.4,


52.1, CH3 175.9, 41.3, 16.4, 26.4,

C CH CH3 CH2

11.7, CH3 175.2, 33.5, 17.7, 19.3,

C CH CH3 CH3

2.53, septet (7.5) 1.04a 0.91, d (7.5)

176.6, 34.3, 19.2, 19.0,

C CH CH3 CH3

3

δH, mult. (J in Hz) 2.91, dd (9.2, 4.3) 5.11, d (9.2) 2.64, m 2.35, m 2.17, m

2.14, m 2.38, 1.82, 2.18, 1.41,

m m m m

s s s s s d (4.3) s s

2.27, 1.13, 1.64, 1.40, 0.89,

m d (7.1) m m t (7.4)

174.0, 62.8, 47.4, 51.5, 16.8,

C C CH C CH2

1.62, m

26.0, CH2

1.60, m


52.4, CH3 176.0, 41.6, 17.6, 26.7,

C CH CH3 CH2

11.8, CH3 174.9, 33.8, 18.8, 16.3,

2.45, m 1.10, d (7.0) 1.10, d (7.0)

5″ a

C CH CH C CH CH2

169.4, 78.0, 14.4, 17.8, 119.8, 142.1, 110.4, 142.6, 23.6, 20.9, 70.9,

5.02, s 1.21, s 1.06, s

10

δH, mult. (J in Hz)

213.6, 52.5, 76.6, 39.3, 41.8, 33.3,

37.9, C 67.6, C 35.6, CH2

6.00, s

7.49, 6.43, 7.41, 0.78, 1.24, 5.55, 3.90, 3.69,

δC, type

C CH CH3 CH3

3.08, dd (10.8, 2.4) 5.20, d (10.8) 3.37, dd (9.9, 3.3) 2.47, m

2.26, m

3.72, d (17.1) 2.90, d (17.1) 5.91, s 1.05, s 1.20, s 7.62, 6.50, 7.39, 0.78, 0.88, 5.34,

br s br s br s s s d (2.4)

3.76, s

2.39, 1.16, 1.81, 1.49, 1.02,

m d (7.2) sextet (7.2) sextet (7.2) t (7.2)

2.50, m 1.13, d (6.3) 1.15, d (6.6)

δC, type 107.7, 53.6, 73.7, 38.0, 40.8, 32.3,

C CH CH C CH CH2

174.0, 81.8, 51.8, 43.1, 15.3,

C C CH C CH2

25.1, CH2 39.1, C 159.7, C 118.0, CH 163.7, 81.4, 19.9, 20.7, 120.2, 141.4, 110.2, 143.1, 24.8, 22.1, 76.4,


52.2, CH3 175.8, 41.2, 16.4, 26.3,

C CH CH3 CH2

11.7, CH3 176.2, 40.9, 15.9, 26.6,

C CH CH3 CH2

11.9, CH3

δH, mult. (J in Hz) 2.88, dd (9.2, 4.2) 5.13, d (9.2) 2.63, dd (10.2, 1.5) 2.35, m 2.15, m

2.15, m 2.35, 1.80, 2.17, 1.40,

m m m m

6.00, s

5.03, s 1.21, s 1.06, s 7.49, 6.43, 7.41, 0.77, 1.24, 5.57, 3.51, 3.69,

br s br s br s s s d (4.2) s s

2.27, 1.13, 1.65, 1.38, 0.89,

m d (7.0) m m t (7.4)

2.23, 1.05, 1.65, 1.33, 0.90,

m d (6.8) m m t (7.4)

Assignments for positions with identical superscripts are interchangeable.

correlations in the HMBC spectrum with the diastereotopic H15 methylene protons, δH 3.72 (1H, d, J = 17.1 Hz, H-15α) and 2.90 (1H, d, J = 17.1 Hz, H-15β), which, in turn, showed correlations with the C-13, C-14, and C-8 (δC 62.8) resonances. The chemical shifts for C-8 and C-14 indicated the presence of an 8,14-epoxide. Ring A was rearranged as shown by the characteristic carbomethoxy resonance at δH 3.76 (3H, s, 7OMe).12 The typical H-3/H-2/H-30 coupled system was indicated in the COSY spectrum by resonances at δH 5.20 (1H, d, J = 10.8 Hz, H-3), 3.08 (1H, dd, J = 10.8, 2.4 Hz, H-2), and 5.34 (1H, d, J = 2.4 Hz, H-30). Ester groups were present at C3β and C-30α and were found to be (2S)-methylbutyryloxy and

isobutyryloxy, respectively, as in compounds 1 and 2. The H-2 and H-30 resonances showed a correlation in the HMBC spectrum with a keto carbonyl resonance at δC 213.6, which was assigned as C-1. All other resonances could be assigned from 2D NMR spectra and are given in Table 1. Compound 3, xylorumphiin L, was similar to xyloccensin G, the 3β,30αdiisobutyryloxy derivative isolated previously from X. moluccensis.19 Six tetranortriterpenoids were isolated as hemiacetals, which could not be purified due to the equilibration of the hemiacetal epimers in solution, hence giving complex spectra. These included C-23 epimeric xylorumphiins M−O (4−6) and C-21 393


394

a

2.43, m 1.09, d (7.1) 1.07, d (7.0)

2.46, m 1.10, d (6.9) 1.07a

m d (7.0) m m t (7.4)

2.29, 1.12, 1.63, 1.38, 0.87,

2.29, 1.12, 1.63, 1.38, 0.87,

m d (7.1) m m t (7.5)

2.16, s

2.17, s

4b dd (9.2, 4.2) d (9.2) dd (10.3, 1.5) m m m m m m m s s s s s s s s d (4.2) s s

2.90, 5.11, 2.59, 2.35, 2.13, 2.11, 2.39, 1.82, 2.48, 1.33, 5.98, 5.06, 1.24, 1.06, 7.42, 7.04, 0.77, 1.23, 5.51, 3.59, 3.69,

2.90, dd (9.2, 4.2) 5.12, d (9.2) 2.59, dd (10.3, 2.0) 2.35, m 2.15, m 2.12, m 2.39, m 1.85, m 2.50, m 1.36, m 5.98, s 5.04, s 1.27, s 1.06, s 7.40, s 6.93, s 0.78, s 1.23, s 5.52, d (4.2) 3.58b 3.69, s

4a dd (9.2, 4.4) d (9.2) m m m m m m m m s s s s br s br s s s d (4.4)

2.46, m 1.14a 1.06a

2.28, m 1.09a 1.60, m 1.36, m 0.85a

3.69, s 2.14, s 2.17, s

4.07, 5.08, 2.60, 2.33, 2.15, 2.10, 2.38, 1.86, 2.51, 1.37, 6.07, 5.03, 1.27, 1.06, 7.39, 6.93, 0.76, 1.12, 5.55,

4c

4d

2.47, m 1.15b 1.06b

2.27 m 1.13a 1.63, m 1.41, m 0.87b

3.69, s 2.15 s 2.16 s

4.06, dd (9.0, 4.5) 5.09b 2.60, m 2.33, m 2.14, m 2.10, m 2.38, m 1.84, m 2.51, m 1.34, m 6.07, s 5.06a 1.25, s 1.06, s 7.42, br s 7.04, br s 0.77, s 1.12, s 5.56, d (4.5)

Assignments for positions with identical superscripts are interchangeable. bSuperimposed with impurity.

5″

5′ 30-acyl 2″ 3″ 4″

15 17 18 19 22 23 28 29 30 1-OH 7-OMe 1-OAc 23-OAc 3-acyl 2′ 3′ 4′

12

9 11

2 3 5 6

position

Table 2. 1H NMR Data for 4a−d, 5a,b, and 6a (500 MHz, J in Hz, CDCl3)

2.30, 1.12, 1.62, 1.37, 0.86,

m d (7.1) m m t (7.4)

2.46, m 1.09, d (7.0) 1.06, d (6.9)

2.17, s

2.91, dd (9.2, 4.2) 5.12, d (9.2) 2.58, dd (10.1, 1.6) 2.34, m 2.15, m 2.12, m 2.39, m 1.84, m 2.46, m 1.35, m 5.98b 5.03b 1.27, s 1.06, s 7.39, br s 6.93, br s 0.77, s 1.24, s 5.51, d (4.2) 4.30, s 3.69, s

5a dd (9.2, 4.2) d (9.2) dd (10.0, 1.7) m m m m m m m s s s s s s s s d (4.2) s s

2.29, 1.12, 1.62, 1.38, 0.87,

m d (7.1) m m t (7.4)

2.47, m 1.08, d (6.9) 1.08, d (6.9)

2.15, s

2.90, 5.12, 2.58, 2.34, 2.14, 2.10, 2.38, 1.83, 2.47, 1.32, 5.97, 5.06, 1.24, 1.06, 7.42, 7.03, 0.77, 1.23, 5.50, 4.28, 3.69,

5b

2.22 m 1.06a 1.63 m 1.40 m 0.87a

2.29 m 1.13, d (7.0) 1.63 m 1.40 m 0.87 m

2.17 s

2.88, m 5.14, d (9.3) 2.58, dd (10.2, 1.5) 2.34, m 2.14, m 2.12, t (10.5) 2.39, m 1.84, m 2.46, m 1.35, m 5.98, s 5.05, s 1.27, s 1.06, s 7.39, br s 6.93, br s 0.77, s 1.24, s 5.53, d (4.0) 4.19b 3.69, s

6a

Journal of Natural Products Article



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especially with the C-23 signal difficult to observe. Hence, other degrees of compression were tested until it was found that 16 Hz of ΔνQ produced the appropriate anisotropy to measure RDCs. These two molecules showed strong alignment in the gels, and this is why it was necessary to use weaker alignment conditions, which means using less compression and a less cross-linked PMMA gel. Measurement of 1DCH values could be performed with the compression degree that gave 16 Hz of ΔνQ, and these values are listed in Table 4. The structures of the two C-23 epimers were generated using the Shrodinger MacroModel Suite,22 and their geometries were further refined by DFT (B3LYP/6-31G) using Gaussian 09.23 Each structure showed one energetically preferred rotamer for the side chain, making the analysis of RDCs very straightforward. Singular value decomposition (SVD) fitting of the RDC data of 5a and 5b to both structures led to assignment of the configuration of C-23 in 5a as S and in 5b as R. SVD fittings were performed using the MSpin24 software package from Mestrelabs Research. The quality of the fittings was scored using the Cornilescu quality factor Q. The lower the Q factor, the better the fitting. Figure 2 shows the calculated vs experimental 1DCH from the fitting of the RDC data of compound 5a to the epimeric structures at C-23. The (23S) configuration shows a Q factor of 0.081 vs Q = 0.177 for the (23R) epimer, clearly indicating a (23S) configuration for compound 5a. Figure 3 shows the fitting results for compound 5b, in which a Q factor of 0.055 for the (23R) epimer clearly indicates this configuration for compound 5b, while the Q factor for the (23S) epimer is 0.110. Both molecules show identical alignment tensors. The RDC values of the CH bonds in the skeletons are highly similar. Only the RDC for the bond H-23−C-23 is different. This is because the rigid skeleton of the molecules dominates the orientation of the sample, and only the orientation at C-23 changes, as seen in the 3D structure provided in the Supporting Information. A comparison of the 1 DCH values of compound 5a and 5b is presented in Figure 4. Only the signals that could be extracted from both compounds are shown. Using these results it is clear that the compounds that show H-23 at δH 6.93 in the 1H NMR spectrum have the S configuration at C-23 (compounds 4a, 4c, 5a), and compounds with H-23 at δH 7.04 have the R configuration at C-23 (compounds 4b, 4d, 5b). Compounds showing a value of δH 6.93 for H-23 consistently show a value of δC 92.3 for C-23, while those showing a value of δH 7.04 for H-23 consistently show a value of δC 93.0 for C-23 (see Tables 2 and 3). These differences in chemical shift may sound small to discriminate unambiguously the epimeric structures at C-23. However, the structural geometry of rings C and D and the C-17 lactone moiety is highly similar in all of these compounds, and the inversion of configuration at C-23 leads to a subtle but unique chemical shift for H-23 and C-23 in each epimer. The A- and Bring substituents are too far to introduce ambiguities in these chemical shift values. Of course, care must be taken when collecting the NMR spectra in terms of solvent purity and sample temperature regulation. Only one acetate derivative, compound 6a, was isolated on acetylation of compound 6. The molecular formula of C39H52O14 in conjunction with NMR data indicated that compound 6 only differed from compounds 4 and 5 in that (2S)-methylbutyryloxy ester units were present at both C-3 and C-30 in 6a. The H-23 resonance occurred at δH 6.93, while the

epimeric xylorumphiins P−R (7−9). Compounds 4−9 were acetylated, and products were separated using column chromatography. The acetylation mixture of compound 4 yielded four compounds, 4a−d. NMR spectra showed that rings A−D were the same as those for the limonoid 2, xylorumphiin C. Compounds 4a and 4b were monoacetates; however, compounds 4c and 4d were diacetates. The HRMS data of compound 4a gave an [M + Na]+ ion at m/z 753.3088, indicating a molecular formula of C38H50O14. Subtracting the formula of rings A−D for limonoid 2 left a fragment of C6H5O4 for the acetylated side chain. The FTIR spectrum showed absorption bands at 3416, 1782, and 1732 cm−1 corresponding to OH stretching, CO stretching of a five-membered lactone moiety, and CO stretching of a saturated ester, respectively. The H-17 resonance (δH 5.04, 1H, s) showed correlations in the HMBC spectrum with the C-20 (δC 134.2), C-21 (δC 168.0), and C-22 (δC 147.8) resonances. The corresponding H22 resonance (δH 7.40, 1H, s) showed coupling in the COSY spectrum with the H-23 resonance (δH 6.93, 1H, s), indicating that a Δ20,22 double bond was present, the lactone carbonyl carbon occurred at C-21, and the acetylated hemiacetal carbon occurred at C-23. Compound 4c had the identical chemical shift for H-23 as 4a, but the hemiacetal hydroxyl group at C-1 was also acetylated, as shown by a downfield proton shift of the neighboring H-2 resonance to δH 4.07 (1H, dd, J = 9.2, 4.4 Hz). Compound 4b was found to be the C-23 epimer of 4a, and, likewise, compound 4d was the 1-O-acetyl derivative of 4b. The H-23 resonance (δH 7.04) was deshielded compared to 4a. Again the neighboring H-2 proton resonance was deshielded to δH 4.06 (1H, dd, J = 9.0, 4.5 Hz) in 4d. Thus, we had two pairs of C-23 stereoisomers, compounds 4a/4c and 4b/4d, but it was not possible, at this stage, to differentiate the (23S) and (23R) epimers. NMR data for compounds 4a−d are shown in Tables 2 and 3. Acetylation of compound 5 yielded an epimeric mixture of monoacetates, compounds 5a and 5b. These compounds differed from 4a and 4b in the ester moieties present at C-3 and C-30, which were interchanged in compound 5 as confirmed by HMBC studies. The H-23 resonances again occurred at δH 6.93 and 7.03 for compounds 5a and 5b, respectively. The configuration at C-23 for compounds 5a and 5b could not be determined by standard NOE methods due to the lack of short-range NOE interactions between H-23 and the protons of the main skeleton. RDCs were used to solve the problem due the fact that they can correlate the relative orientation of stereocenters regardless of the distance between them.20 A PMMA gel with 0.3 M% cross-link density was used first with compound 5b. After spectra acquisition, RDCs for 5b could not be seen due to the high degree of alignment of the molecule. This meant that a gel with 0.2% cross-linker had to be synthesized. It is known that the degree of alignment depends on the amount of cross-linker.21 With the new PMMA gel, RDCs were successfully acquired for compound 5b at a degree of gel compression where a quadrupolar splitting (ΔνQ) of 17 Hz of the 2H NMR signal of CDCl3 was observed. From the anisotropic HSQC spectra, the total splitting (1TCH) values were extracted, and then 1DCH values were acquired from the difference of 1TCH and 1JCH. 1DCH values for compound 5b are listed in Table 4. Compound 5a was diffused in a PMMA gel with 0.2% cross-link density. With this gel, maximum compression was obtained with a ΔνQ of 30 Hz, but after spectra acquisition, most 1DCH signals were barely visible, 395


396

C CH CH3 CH2 CH3

176.2, C 34.2, CH 19.1, CH3

176.1, 41.1, 16.6, 26.3, 11.7,

168.8, C 20.9, CH3

23-OAc

3-acyl 1′ 2′ 3′ 4′ 5′ 30-acyl 1″ 2″ 3″

52.2, CH3

7-OMe 21-OAc

C CH CH C CH CH2 C C CH C CH2 CH2 C C CH C CH CH3 CH3 C C CH CH CH3 CH3 CH

4a

107.7, 53.4, 73.6, 38.0, 40.8, 32.5, 173.7, 81.6, 51.5, 43.1, 15.3, 25.1, 39.2, 159.6, 117.4, 162.5, 78.9, 19.9, 20.7, 134.2, 168.0, 147.8, 92.3, 24.8, 22.1, 76.5,

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 1-OAc

position C CH CH C CH CH2 C C CH C CH2 CH2 C C CH C CH CH3 CH3 C C CH CH CH3 CH3 CH

C CH CH3 CH2 CH3

176.4, C 34.2, CH 19.1, CH3

176.0, 41.1, 16.6, 26.3, 11.7,

169.1, C 20.8, CH3

52.2, CH3

107.7, 53.4, 73.7, 38.0, 40.8, 32.5, 173.7, 81.5, 51.5, 43.1, 15.3, 25.1, 39.3, 159.6, 117.5, 162.6, 79.2, 19.9, 20.7, 134.2, 168.4, 148.3, 93.0, 24.8, 22.1, 76.5,

4b C CH CH C CH CH2 C C CH C CH2 CH2 C C CH C CH CH3 CH3 C C CH CH CH3 CH3 CH C CH3 CH3

C CH CH3 CH2 CH3

176.5, C 34.1, CH 19.1, CH3

175.7, 41.1, 16.7, 26.3, 11.6,

168.8, C 20.8, CH3

108.6, 47.9, 72.7, 37.8, 40.6, 32.4, 173.5, 82.7, 50.5, 45.2, 15.1, 25.1, 39.3, 158.6, 118.0, 162.2, 78.8, 19.9, 21.2, 134.2, 168.0, 147.8, 92.3, 24.4, 21.8, 76.0, 167.5, 22.3, 52.2,

4c C CH CH C CH CH2 C C CH C CH2 CH2 C C CH C CH CH3 CH3 C C CH CH CH3 CH3 CH C CH3 CH3

C CH CH3 CH2 CH3

176.2, C 34.2, CH 19.1, CH3

175.8, 41.0, 16.7, 26.2, 11.6,

169.2, C 20.8, CH3

108.6, 47.9, 72.7, 37.8, 40.5, 32.4, 173.5, 82.7, 50.4, 45.2, 15.1, 25.1, 39.3, 158.6, 118.0, 162.3, 79.1, 19.9, 21.2, 134.2, 168.5, 148.4, 93.0, 24.4, 21.8, 76.0, 167.6, 22.3, 52.2,

4d C CH CH C CH CH2 C C CH C CH2 CH2 C C CH C CH CH3 CH3 C C CH CH CH3 CH3 CH

C CH CH3 CH3

176.2, C 41.1, CH 16.6, CH3

176.3, 34.2, 19.1, 19.0,

168.8, C 20.8, CH3

52.2, CH3

107.7, 53.3, 73.9, 38.0, 40.9, 32.5, 173.7, 81.5, 51.5, 43.1, 15.3, 25.1, 39.2, 159.8, 117.4, 162.7, 78.9, 19.9, 20.7, 134.1, 168.0, 147.8, 92.3, 24.8, 22.2, 76.5,

5a

Table 3. 13C NMR Data for 4a−d, 5a,b, 6a, 7a,b, 8a, and 9a (125 MHz, CDCl3)

C CH CH3 CH3

176.3, C 41.2, CH 16.6, CH3

176.3, 34.2, 19.1, 19.0,

169.2, C 20.8, CH3

52.2, CH3


5b 107.7, 53.3, 73.9, 38.0, 40.8, 32.5, 173.7, 81.4, 51.4, 43.1, 15.2, 25.1, 39.3, 159.8, 117.4, 162.7, 79.2, 19.9, 20.7, 134.1, 168.4, 148.4, 93.0, 24.8, 22.2, 76.5,

C CH CH3 CH2 CH3 175.9, C 40.9, CH 15.8, CH3

176.3, 41.1, 16.6, 26.2, 11.6,

168.8, C 20.8, CH3

52.2, CH3


6a 107.8, 53.4, 73.8, 38.0, 40.8, 32.5, 173.7, 81.5, 51.5, 43.1, 15.3, 25.1, 39.2, 159.8, 117.4, 162.7, 78.9, 19.9, 20.7, 134.1, 168.0, 147.8, 92.3, 24.8, 22.2, 76.5,

C CH CH3 CH3

176.9, C 34.2, CH 19.0, CH3

176.2, 34.2, 19.3, 19.1,

52.3, CH3 168.7, C 20.6, CH3

C CH CH C CH CH2 C C CH C CH2 CH2 C C CH C CH CH3 CH3 C CH CH C CH3 CH3 CH

7a 107.6, 53.3, 73.8, 37.9, 40.7, 32.3, 174.0, 81.4, 51.1, 43.2, 15.4, 24.9, 39.4, 159.3, 117.6, 161.9, 80.0, 20.7, 20.8, 160.2, 93.1, 123.9, 168.5, 24.9, 22.1, 76.5,

C CH CH3 CH3

176.8, C 34.1, CH 19.0, CH3

175.6, 34.1, 19.3, 19.1,

C CH CH C CH CH2 C C CH C CH2 CH2 C C CH C CH CH3 CH3 C CH CH C CH3 CH3 CH C CH3 CH3 C CH3

7b 108.4, 47.8, 72.9, 37.7, 40.4, 32.2, 173.5, 82.4, 50.1, 45.3, 15.2, 24.9, 39.5, 158.1, 118.1, 161.6, 79.9, 20.7, 21.4, 160.2, 93.1, 124.0, 168.5, 24.5, 21.8, 76.1, 167.3, 22.3, 52.3, 168.7, 20.6,

C CH CH3 CH2 CH3 176.8, C 34.2, CH 19.1, CH3

175.9, 41.2, 16.3, 26.5, 11.8,

52.3, CH3 168.7, C 20.6, CH3


8a 107.6, 53.3, 73.7, 38.0, 40.7, 32.3, 174.0, 81.4, 51.1, 43.2, 15.4, 24.8, 39.4, 159.4, 117.6, 161.9, 80.0, 20.7, 20.8, 160.2, 93.1, 123.9, 168.5, 24.9, 22.1, 76.6,

176.6, C 40.8, CH 16.4, CH3

170.2, C 21.1, CH3

52.3, CH3 168.7, C 20.6, CH3


9a 107.6, 53.3, 74.0, 37.6, 40.7, 32.2, 174.1, 81.3, 51.1, 43.2, 15.3, 24.9, 39.4, 159.3, 117.7, 162.0, 80.0, 20.6, 20.8, 160.2, 93.1, 124.0, 168.5, 24.7, 22.0, 76.4,

Journal of Natural Products Article


26.7, CH2 11.8, CH3

8a

19.1, CH3

7b 7a 6a

26.5, CH2 11.8, CH3 26.3, CH2 11.7, CH3

5b 5a

26.3, CH2 11.6, CH3 18.9, CH3

4d

1

DCH from 5a (Hz) −13.02 −22.84 −33.81 37.19 −48.53 12.59 −2.74 −24.09 39.15 7.46 −60.61

1

DCH from 5b (Hz) −11.16 −21.78 −27.06 39.53 −46.24 NA NA −27.01 0.00 7.66 −62.63

C-23 resonance occurred at δC 92.3, clearly indicating the (23S) configuration for the compound. Compounds 7−9 occurred as C-21 epimers and were acetylated, as above, to obtain pure compounds for analysis. Compound 7 was acetylated to give acetates 7a and 7b. Compound 7a, a monoacetate, had the same tetracyclic core structure as compound 2, but with isobutyryloxy ester moieties present at both C-3 and C-30. The H-17 resonance at δH 4.84 (s) showed correlations with the C-20 (δC 160.2) and C-22 (δC 123.9) resonances, and the C-23 lactone carbonyl carbon (δC 168.5) showed correlations with the H-21 oxymethine resonance (δH 6.97) and H-22 (δH 6.44) resonances. The H21 resonance showed a correlation with the acetoxy carbonyl resonance. Thus, compound 7a is a Δ20(22),23,21-lactone. Compound 7b was the 1-O-acetyl derivative of compound 7a. The “other” C-21 epimers were not isolated, probably due to small amounts present. NMR data are shown in Tables 3 and 5. The configuration at C-21 of compound 7a was determined using a combination of quantitative NOE experiments and molecular modeling. A set of 500 ms selective 1D NOE experiments was used to obtain the appropriate NOE interactions.25 The two epimeric structures at C-21 (Figure 5) were generated in the same way as for compounds 5a and 5b (vide supra). As for compounds 5a and 5b, each epimeric configuration at C-21 yielded only one energetically preferred conformation, which should show unique NOE interactions for that epimer. These two structures were further energyminimized by DFT (B3LYP/6-31G). As a result of this computational analysis, for the (21R) isomer, the distance between H-21 and the methylene protons H-12a,b is ∼3.9 Å, while for the (21S) isomer it is 2.33 and 2.65 Å, respectively. In both isomers, H-22 is close to the 18-methyl group. On the basis of this computational analysis, the quantitative results of the NOE interaction between H-22 and H-12a,b would be enough to determine the configuration at C-21. To obtain these NOE interactions experimentally, 1.8 mg of 7a was dissolved in CDCl3 and H-17, H-21, and H-22 were selectively excited for the 1D NOE experiments. Selective excitation of H-17 showed a strong NOE interaction with H-12β (δH 2.26). The spectra from the selective excitation of H-22 gave an NOE interaction with CH3-18, while the experiment with selective excitation of H-21 gives an NOE interaction with one of the methylene protons at C-12 (δH 1.44), which was identified as H-12α (the spectra are shown in Supporting Information S2, Figures 1−3). This is a key interaction to confirm the (21S) configuration of compound 7a (Figure 5). The interaction between H-22 and H-18 is indicative of the preferred conformation of the lactone

19.1, CH3

4c

2 3 5 6 15 17 18 22 23 29 30

19.0, CH3

4b

carbon

19.0, CH3

4a

Table 4. Experimental RDC Values (1DCH) for 5a and 5b

4″ 5″

position

Table 3. continued

Article

19.1, CH3

19.1, CH3

9a


397



Article

Figure 2. Plots of calculated (1DCHcalc) vs experimental (1DCHexp) and Q factors from the SVD fitting of RDC data of the 23-epimers of compound 5a.

Figure 3. Plots of calculated (1DCHcalc) vs experimental (1DCHexp) and Q factors from the SVD fitting of RDC data of the C-23 epimers of compound 5b. The experimental values for C-5 and C-22 are highly similar (−27.06 and −27.01 Hz, respectively), and due to a good fitting for the (23R) epimer, these two values collapse into a single dot in the left correlation plot.

diacetate 9a, which had the same tetracyclic core structure as compound 2, with a (2S)-methylbutyryloxy ester present at C30, and acetylation of a 3β-hydroxyl group and the C-21 hemiacetal group had occurred. The same resonance at δH 6.97 for H-21 as found for compound 7a indicated a (21S) configuration. For compounds 7a, 7b, 8a, and 9b, the same structural situation as the one described above for the epimers at C-23 applies. The structural geometry of rings C and D and the C-17 lactone unit is highly similar in all of these compounds. The same proton and carbon chemical shifts observed for H-21 (δH 6.97) and C-21 (δC 93.1) clearly indicate the same (21S) configuration at C-21 as determined by quantitative NOE analysis. The EtOAc extract of the bark yielded sitosterol, stigmasterol, taraxer-14-en-3β-ol, (22S)-hydroxytirucalla-7,24diene-3,23-dione, 25-hydroxy-(20S,24S)-epoxydammaran-3one, xyloccensin E, and compound 10, xylorumphiin K, a new limonoid. This compound differed from compound 2 only in that the 30α-ester moiety was a (2S)-methylbutyryloxy unit. The configuration at C-2 of the ester has been confirmed previously by X-ray analysis.17

moiety; this conformation has the C-23 carbonyl projecting to the back, which correlates with the conformational search. NOE-derived distances using the PANIC26−28 correction were also obtained and were compared to the distances from the computer-generated structures. qNOE distances were calculated using two references (ref: H-2 to H-3 and H-17 to H-12β). Calculated vs experimental distances are shown in Table 6. Cornilescu quality factors (Q), χ2, and N/χ2 were calculated using the distances H-2 to H-3 and H-17 to H-12β as a distance reference for both diastereomers. The error was assumed to be 0.5 Å for all measurements. The results for references H-2 to H-3 and H-17 to H-12β are presented in Tables 7 and 8, respectively. Regardless of the distance reference used, a lowest Q factor for the (21S) epimer, confirming the configuration at C-21 for compound 7a, was observed. In addition, a low χ2 and a high N/χ2 were obtained. Compound 8 has the same tetracyclic core structure and ester groups as compound 4, but the same C-17 side chain as compound 7. Acetylation of compound 8 yielded compound 8a, the (21S) monoacetate, with H-21 resonating at δH 6.97 as in compound 7a. Compound 9 was acetylated to yield the 398



Article

fractions by column chromatography and eluted with gradient elution using n-hexane, EtOAc, and MeOH (collecting 75 mL fractions, which were combined based on similarities on TLC), to afford five fractions (C1−C5). Fraction C2, a pale yellow, viscous oil (124.2 mg), was subjected to repeated column chromatography starting with n-hexane and increasing polarity with EtOAc to give compounds 1−3. Separation of fraction C3, a pale yellow, viscous oil (511.1 mg), gave a mixture of compounds 4 and 5 (102.7 mg combined) and compound 6 (35.1 mg). Acetylation of a mixture containing 4 and 5 (102.7 mg) yielded, after separation, 4a (4.5 mg), 4b (7.6 mg), 4c (7.3 mg), 4d (4.9 mg), 5a (7.5 mg), and 5b (24.3 mg). Acetylation of 6 (35.1 mg) yielded, after separation, 6a (11.6 mg). The other epimer was not isolated. Similarly, fraction C4, a pale yellow, viscous oil (368.6 mg), gave epimeric mixtures of 7 (8.8 mg), 8 (7.0 mg), and 9 (4.0 mg). Acetylation of 7 (8.8 mg), 8 (7.0 mg), and 9 (4.0 mg), respectively, followed by separation, led to the isolation of two acetylated derivatives, 7a (3.6 mg) and 7b (0.3 mg), of 7 and one acetylated derivative of compounds 8 (8a, 3.2 mg) and 9 (9a, 1.9 mg). Air-dried bark (4.0 kg) of X. rumphii was extracted with MeOH for 7 days (3×) at room temperature. The extract was filtered and concentrated under reduced pressure to give a dark red solid (174.3 g), which was partitioned with EtOAc to give an EtOAc extract (36.7 g). The EtOAc extract was purified by column chromatography and eluted with gradient elution of n-hexane, EtOAc, and MeOH (collecting 75 mL fractions, which were combined based on similarities on TLC), to afford 11 fractions (B1−B11). Fraction B1 yielded a mixture of sitosterol and stigmasterol (30.5 mg), fraction 3 yielded taraxer-14-en-3β-ol (10.3 mg), fraction 4 yielded (22S)hydroxytirucalla-7,24-diene-3,23-dione (7.9 mg), fraction 6 yielded 25hydroxy-(20S,24S)-epoxydammaran-3-one (26.3 mg), fraction 8 yielded xylorumphiin K (10) (9.8 mg), and fraction 10 yielded xyloccensin E (53.4 mg). General Acetylation Procedure. Compounds or fractions to be acetylated were dissolved in pyridine (2 mL) in a round-bottomed flask, Ac2O (2 mL) was added, and the reaction was left to stand overnight. MeOH (10 mL) was added to the reaction mixture to remove unreacted Ac2O. Toluene (4 × 10 mL) was added in order to remove the pyridine using a rotary evaporator. Thereafter, MeOH (5 × 10 mL) was added and evaporated off to remove the remaining toluene. Xylorumphiin L (3): white, amorphous solid; [α]25D −59 (c 0.2, CHCl3); IR (KBr) νmax 1765, 1724 cm−1; 1H and 13C NMR data, see Table 1; HREIMS m/z 679.3082 [M + Na]+ (calcd for C36H48O11Na, 679.3089). (23S)-O-Acetylxylorumphiin M (4a): white, amorphous solid; [α]25D +33 (c 0.001, CHCl3); IR (KBr) νmax 3416, 1782, 1732 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HREIMS m/z 753.3088 [M + Na]+ (calcd for C38H50NaO14, 753.3093). (23R)-O-Acetylxylorumphiin M (4b): white solid; [α]25D +46 (c 1.5, CHCl3); IR (KBr) νmax 3431, 1776, 1730 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HREIMS m/z 753.3088 [M + Na]+ (calcd for C38H50NaO14, 753.3093). 1,(23S)-Di-O-Acetylxylorumphiin M (4c): white solid; [α]25D +84 (c 2, CHCl3); IR (KBr) νmax 1774, 1735 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HREIMS m/z 795.3201 [M + Na]+ (calcd for C40H52NaO15, 795.3198). 1,(23R)-Di-O-acetylxylorumphiin M (4d): white, amorphous solid; [α]25D +38 (c 0.001, CHCl3); IR (KBr) νmax 1777, 1736 cm−1; 1H and 13 C NMR data, see Tables 2 and 3; HREIMS m/z 795.3176 [M + Na]+ (calcd for C40H52NaO15, 795.3198). (23S)-O-Acetylxylorumphiin N (5a): white, amorphous solid; [α]25D +34 (c 3, CHCl3); IR (KBr) νmax 3430, 1781, 1732, 1641 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HREIMS m/z 753.3080 [M + Na]+ (calcd for C38H50NaO14, 753.3093). (23R)-O-Acetylxylorumphiin N (5b): white solid; [α]25D +6 (c 0.001, CHCl3); IR (KBr) νmax 3439, 1779,1733 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HREIMS m/z 753.3095 [M + Na]+ (calcd for C38H50NaO14, 753.3093).

Figure 4. Comparison of 1DCH values of 5a and 5b. Note the outlier in red color corresponding to the epimeric center, a key RDC value to discriminate the configuration at C-23. The values for C-17 and C-18 are not shown since these two values were not measured for 5b.

Compounds 1−6 and xyloccensin E were subjected to the NCI59 panel.18 The compounds did not show significant activity (Figures 1−6, S3, Supporting Information).

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a Jasco P-2000 polarimeter, and IR spectra were obtained on a PerkinElmer (2000 FTIR) spectrometer using KBr disks. 1H, 13C, and 2D NMR spectra were recorded on a Bruker AVANCE III NMR spectrometer, operating at 500.13 MHz for 1H, 125.76 MHz for 13C, and 76.77 MHz for 2H, using standard experiments from the Bruker Pulse programs library. Temperature at the NMR probe was maintained at 300 K. CDCl3 was kept free of HCl by storing it in the dark with silver foil, molecular sieves, and K2CO3. Chemical shifts are reported in ppm (δ) referencing the solvent signal (CDCl3) as internal standard with respect to tetramethylsilane (0 ppm), and coupling constants (J) are measured in Hz. One-bond proton−carbon residual dipolar couplings (1DCH) were measured with the F1 protoncoupled J-scaled BIRD HSQC experiment,29 using a J-scaling factor (κ) of 4 and INEPT transfer optimized for a 145 Hz 1H−13C coupling constant. A total of 1024 increments in F1 were used. Anisotropic conditions were obtained using cross-linked PMMA gels swollen in CDCl3 using the reversible compression/relaxation method as described previously.21 Column chromatographic separations were carried out using silica gel (Merck Art. 9385). TLC was carried out on 0.2 mm silica gel, aluminum-backed plates (Merck Art.5554). The plates were developed using anisaldehyde spray reagent and heating. Plant Material. X. rumphii was collected in Chanthaburi Province, Thailand. The plant specimen was prepared by Associate Professor Surat Laphookhieo, School of Science, Mae Fah Luang University, Chiang Rai, Thailand, and identified by Professor James Maxwell, Chiang Mai University Herbarium, Chiang Mai, Thailand, and the voucher specimen was deposited at Chiang Mai University Herbarium, Chiang Mai, Thailand (voucher number laphookhieo 9). Extraction and Isolation. Air-dried heartwood (5.0 kg) of X. rumphii was extracted with CH2Cl2 for 7 days (3×) at room temperature. The mixture was filtered and concentrated under reduced pressure to give the crude extract (28.9 g), which was separated into 399



Article

Table 5. 1H NMR Data for 7a, 7b, 8a, and 9a (500 MHz, J in Hz, CDCl3) position 2 3 5 6a 6b 9 11a 11b 12β 12α 15 17 18 19 21 22 28 29 30 1-OH 1-OAc 7-OMe 21-OAc 3-acyl 2′ 3′ 4′ 5′ 30-acyl 2″ 3″ 4″

7a 2.92, 5.05, 2.64, 2.38, 2.18, 2.14, 2.46, 1.92, 2.26, 1.44, 5.98, 4.84, 1.30, 1.08, 6.97, 6.44, 0.80, 1.24, 5.38,

dd (9.2, 4.2) d (9.2) m m m m m m m m s s s s s s s s d (4.2)

7b 4.11, 5.04, 2.63, 2.37, 2.19, 2.08, 2.46, 1.94, 2.28, 1.45, 6.07, 4.83, 1.30, 1.08, 6.97, 6.45, 0.79, 1.12, 5.42,

8a

dd (9.2, 4.2) d (9.2) m m m m m m m m s s s s s s s s d (4.2)

3.71, s 2.24, s

2.15, s 3.71, s 2.24, s

2.46, m 1.15, d (7.1) 1.09, d (6.9)

2.42, m 1.07 1.05, d (7.1)

2.90, 5.10, 2.61, 2.37, 2.17, 2.09, 2.44, 1.93, 2.25, 1.44, 5.98, 4.86, 1.30, 1.07, 6.97, 6.44, 0.79, 1.24, 5.39, 3.56,

dd (9.2, 4.1) d (9.2) m m m m m m m m s s s s s s s s d (4.1) s

9a 2.90, dd (9.2, 4.4) 5.08, d (9.2) 2.60, m 2.37, m 2.16, m 2.09, m 2.45, m 1.92, m 2.23, m 1.44, m 5.99, s 4.83 s 1.30, s 1.07, s 6.97, s 6.45, s 0.79, s 1.23, s 5.40, d (4.4) 3.72, s

3.71, s 2.24, s

3.71, s 2.25, s

2.46, m 1.13, d (7.1) 1.10, d (6.9)

2.28, 1.11, 1.61, 1.38, 0.88,

1.99, s

2.46, m 1.07 1.05, d (7.1)

2.42, m 1.08 1.05, d (7.2)

5″

m d (7.0) m m t (7.4)

2.26, 1.02, 1.58, 1.37, 0.89,

m d (7.0) m m t (7.4)

Figure 5. Computer-generated C-12 epimeric structures of compound 7a. Calculated distances are shown in red, while NOE-derived distances are shown in black. For clarity only a fragment of the structure is shown. Xylorumphiin O (6): white solid; IR (KBr) νmax 3405, 1733 cm−1; HREIMS m/z 725.3143 [M + Na]+ (calcd for C37H50O13Na, 725.3144); epimeric mixture was acetylated to yield 6a. (23S)-O-Acetylxylorumphiin O (6a): white solid; [α]25D +12 (c 0.001, CHCl3); IR (KBr) νmax 3408, 1782, 1732 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HREIMS m/z 767.3250 [M + Na]+ (calcd for C39H52O14Na, 767.3249).

Xylorumphiin P (7): white solid; epimeric mixture, which was acetylated to yield 7a and 7b. (21S)-O-Acetylxylorumphiin P (7a): white solid; [α]25D +10 (c 0.001, CHCl3); IR (KBr) νmax 3430, 1638 cm−1; 1H and 13C NMR data, see Tables 3 and 6; HREIMS m/z 739.2935 [M + Na]+ (calcd for C37H48O14Na, 739.2936). 1,(21S)-Di-O-Acetylxylorumphiin P (7b): white solid; [α]25D +10 (c 0.001, CHCl3); IR (KBr) νmax 1800, 1773, 1735 cm−1; 1H and 13C 400



■

Table 6. Interproton Distances (Å) for Compound 7

atoms

experimental distances; ref: H-2 to H-3

experimental distances; ref: H-17 to H-12β

DFTcalculated distances (C21R isomer)

DFTcalculated distances (C21S isomer)

2.27

2.17

2.27

2.27

2.56

2.44

2.50

2.44

3.20

3.05

2.55

3.01

3.73

3.55

3.92

3.83

3.86

3.68

3.77

3.77

2.42

2.30

4.12

2.17

3.25

3.10

3.98

2.55

4.19

3.99

3.99

4.04

H2− H3 H17− H12β H17− H21 H17− H22 H17− H15 H21− H12α H21− H12β H21− H22

*Tel: +44 (0) 1483 68 6751. E-mail: [email protected]. uk. ORCID

Dulcie A. Mulholland: 0000-0001-7778-5617 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS W.W. thanks the National Science and Technology Development Agency, Thailand, for a Ph.D. scholarship to study at the University of Surrey, the National Research Council of Thailand for financial support, Associate Professor S. Laphookhieo, Mae Fah Luang University, Thailand, for plant specimen preparation, Professor J. Maxwell, Chiang Mai University Herbarium, Thailand, for plant identification, the National Cancer Institute for NCI59 cancer cell line screening, and Mr. C. Sparrow, Oxford University, for MS analysis. NMR instrumentation at Carnegie Mellon University was partially supported by the NSF (CHE-0130903 and CHE-1039870). R.R.G. gratefully acknowledges support from the NSF (CHE1111684).

Q

χ2

N/χ2

0.215 0.087

15.694 2.583

0.510 3.098

■

Table 8. Quality factors for Both C-21 Diasteromers (Compound 7) Using Ref. H-17 to H-12β C-21R C-21S

Q

χ2

N/χ2

0.241 0.074

17.857 1.676

0.448 4.773

REFERENCES

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NMR data, see Tables 3 and 6; HREIMS m/z 781.3047 [M + Na]+ (calcd for C39H50O15Na, 781.3047). Xylorumphiin Q (8): white solid; [α]25D −71 (c 0.003, CHCl3); IR (KBr) νmax 3410, 1763, 1731 cm−1; 1H and 13C NMR data, see Tables 3 and 6; HREIMS m/z 711.2985 [M + Na]+ (calcd for C36H48O13Na, 711.2987). (21)S-O-Acetylxylorumphiin Q (8a): white solid; [α]25D +7 (c 0.001, CHCl3); IR (KBr) νmax 3389, 1790, 1728, 1645 cm−1; 1H and 13 C NMR data, see Tables 3 and 6; HREIMS m/z 753.3086 [M + Na]+ (calcd for C38H50O14Na, 753.3093). Xylorumphiin R (9): white solid, epimeric mixture, which was acetylated to yield 9a. (21S)-O-Acetylxylorumphiin S (9a): white solid; [α]25D −4 (c 0.001, CHCl3); IR (KBr) νmax 3419, 1781, 1733 cm−1; 1H and 13C NMR data, see Tables 3 and 6; HREIMS m/z 725.2784 [M + Na]+ (calcd for C36H46O14Na, 725.2785). Xylorumphiin K (10): amorphous, white solid; [α]25D +25 (c 0.001, CHCl3); IR (KBr) νmax 3417, 1732 cm−1; 1H and 13C NMR data, see Table 1; HREIMS m/z 671.34268 [M + H]+ (calcd for C37H51O11, 671.34259).

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Table 7. Quality Factors for Both C-21 Diasteromers (Compound 7) Using Ref H-2 to H-3 C-21R C-21S

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00906. Detailed isolation procedure and MS, IR, and NMR spectra for all compounds (PDF) Computational data (PDF) NCI 59 screening data (PDF) 401



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NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on January 25, 2017, with two missing references. The corrected version was reposted on January 27, 2017.

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Application of Residual Dipolar Couplings and Selective Quantitative

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