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, UK

‡ Department of Chemistry, Faculty of Science and Technology, Rambhai Barni Rajabhat

University, Chanthaburi 22000, Thailand

§ Department of Chemistry, Carnegie Mellon University, Pittsburgh PA 15213, USA

⊥ Faculty of Science and Technology, Hatyai University, Songkhla 90110, Thailand

║ School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4041, South Africa

1

ABSTRACT: Nine triterpenoid derivatives were isolated from the heartwood of X. 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 sidechain 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 (RDCs). Dipolar couplings were collected from the sample oriented in compressed

poly (methyl methacrylate) (PMMA) 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-3-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 x 10-5 M, in the NCI59 cell one-dose screen but did not show significant

activity.

2

Xylocarpus is a small genus belonging to the Meliaceae family. There has been debate about

the number of species comprising 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. Xylocarpus 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 antidiarrhoeal,7,8 antiviral,3 antimalarial,9 antisecretory,10

anti-osteoclastogenic,11 anti-inflammatory,12,13 antifungal,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-14-en-3-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. Structures are provided in Figure 1. Compounds 4-9 possess hemiacetal carbons at either

3

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 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) that

permitted differentiation of the two epimers. RDCs provide information of non-local 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

RESULTS AND DISCUSSION

The CH2Cl2 extract of the dried, milled heartwood of X. rumphii was separated using repeated

column chromatography over 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-

4

substituted furan ring at C-17α were observed at H 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 C 78.0 and H 5.91 (s) could be assigned to C-17

and H-17 using the HSQC and HMBC spectra. The H-17 resonance showed correlations with the

C-18 (C 14.4), C-12 (C 26.0), C-13(C 37.9) and C-14 (C 67.6) resonances in the HMBC

spectrum. Ring D comprised a δ-lactone moiety with the C-16 lactone carbonyl resonance (C

169.4) showing correlations in the HMBC spectrum with the diastereotopic H-15 methylene

protons, H 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 (C 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 H 3.76 (3H, s, 7-OMe).12 The typical H-3/H-2/ H-30

coupled system was indicated in the COSY spectrum by resonances at H 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 C-3β and C-30α and were found to be (2S)-methylbutyryloxy and isobutyryloxy

repectively, 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 C 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,30α-diisobutyryloxy 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 three C-23 epimeric xylorumphiins M-O (4-6) and three C-21 epimeric xylorumphiins

P-R (7-9). Compounds 4-9 were acetylated and products were separated using column

chromatography.

5

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 sidechain. The FTIR spectrum showed absorption bands at 3416, 1782 and 1732

cm-1 corresponding to OH stretching, C=O stretching of a 5-membered lactone moiety and C=O

stretching of a saturated ester, respectively. The H-17 resonance (H 5.04, 1H, s) showed

correlations in the HMBC spectrum with the C-20 (C 134.2), C-21 (C 168.0), and C-22 (C

147.8) resonances. The corresponding H-22 resonance (H 7.40, 1H, s) showed coupling in the

COSY spectrum with the H-23 resonance (H 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

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

neighbouring H-2 resonance to H 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 (H 7.04) was deshielded compared to 4a. Again

the neighbouring H-2 proton resonance was deshielded to H 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-

4d 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,

6

which were interchanged in compound 5 as confirmed by HMBC studies. The H-23 resonances

again occurred at H 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. Residual Dipolar Couplings (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% of 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 (∆νQ) 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% of cross-link density. With this

gel, maximum compression was obtained with a ∆νQ of 30 Hz, but after spectra acquisition, most

1DCH signals were barely visible, especially with the C-23 signal difficult to observe. Hence,

other degrees of compression were tested until it was found that 16 Hz of ∆νQ 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 ∆νQ and these values are listed in

Table 4.

7

The structures of the two C-23 epimers were generated using the Shrodinger MacroModel

Suite22 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 0.055 for the (23R) epimer clearly indicates this configuration for compound

5b, while the Q factor for C-the (23S) epimer is 0.110. Both molecules show identical alignment

tensors. The RDC values of the CH bonds in the skeletons arehighly 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. Comparison of the 1DCH values of compound 5a and 5b

are 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 H 6.93 in the 1H NMR

spectrum have the S configuration at C-23 (compounds 4a, 4c, 5a) and compounds with H-23 at

H 7.04 have the R configuration at C-23 (compounds 4b, 4d, 5b). Compounds showing a value

of H 6.93 for H-23 consistently show a value of C 92.3 for C-23, while those showing a value

8

of H 7.04 for H-23 consistently show a value of C 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, 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 B-ring

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 H 6.93, while the C-23 resonance occurred

at C 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 H 4.84 (s) showed

correlations with the C-20 (C 160.2) and C-22 (C 123.9) resonances and the C-23 lactone

carbonyl carbon (C 168.5) showed correlations with the H-21 oxymethine resonance (H 6.97)

and H-22 (H 6.44) resonances. The H-21 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

9

compound 7a was determined using a combination of quantitative NOE experiments and

molecular modeling. A set of 500 ms selective 1D NOE experiments were 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 energy-

minimized 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 Å and 2.65 Å, respectively. In both isomers, H-22 is close to the 18-

methyl group. Based on 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-12 (H 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 (H 1.44),

which was identified as H-12 (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

moiety; this conformation has the C-23 carbonyl projecting to the back, which correlates with the

conformational search.

NOE-derived distances using the PANIC26 correction were also obtained and were compared

to the distances from the computer generated structures. qNOE distances were calculated using

10

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 Table 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 sidechain as compound 7. Acetylation of compound 8 yielded compound 8a, the

(21S) monoacetate, with H-21 resonating at H 6.97 as in compound 7a. Compound 9 was

acetylated to yield the diacetate, 9a, which had the same tetracyclic core structure as compound

2, with a (2S)-methylbutyryloxy ester present at C-30, and acetylation of a 3β-hydroxyl group

and the C-21 hemiacetal group had occurred. The same resonance at H 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, 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 (H 6.97) and C-21 (C 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-3-ol, (22S)-

hydroxytirucalla-7,24-diene-3,23-dione, 25-hydroxy-(20S,24S)-epoxydammaran-3-one, 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

11

Compounds 1-6 and xyloccensin E were subjected to the NCI59 panel.18 The compounds did

not show significant activity (Figures 1-6, S3, Supplementary Information).

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a Jasco P-2000

polarimeter and IR spectra were obtained on a Perkin-Elmer (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 regulation at the

NMR probe was maintained at 300K. 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 () referencing the

solvent signal (CDCl3) as internal standard respect to TMS (0 ppm), and coupling constants (J)

are measured in Hz. One-bond proton-carbon residual dipolar couplings (1DCH) were measured

with the F1 proton-coupled J-scaled BIRD HSQC experiment,27 using a J-scaling factor (κ) of 4

and INEPT transfer optimized for 145 Hz 1H-13C coupling constant. A total of 1024 increments

in F1 were used.

Anisotropic conditions were obtained using cross-linked poly(methylmethacrylate) (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, aluminium-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

12

Luang University, Chiang Rai, Thailand, 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 (x 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 fractions by column

chromatography (CC) 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 compound 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 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), 7b (0.3 mg) of 7, 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 (x 3) at room

temperature. The extract was filtered and concentrated under reduced pressure to give a dark red

solid (174.3 g) which was partition with EtOAc to give an EtOAc extract (36.7 g). The EtOAc

13

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-3-ol (10.3 mg), fraction 4 yielded

(22S)-hydroxytirucalla-7,24-diene-3,23-dione (7.9 mg), fraction 6 yielded 25-hydroxy-(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 (2mL) 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 x 10 mL) was added in order to remove the pyridine using a rotary evaporator.

Thereafter, MeOH (5 x 10 mL) was added and evaporated off to remove the remaining toluene.

Xylorumphiin L (3): white amorphous solid; []25D -59 (c 0.2, CHCl3); IR (KBr) max 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; []25D +33 (c 0.001, CHCl3); IR

(KBr) max 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; []25D +46 (c 1.5, CHCl3); IR (KBr) max

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).

14

1,(23S)-di-O-Acetylxylorumphiin M (4c): white solid; []25D +84 (c 2, CHCl3); IR (KBr)

max1774, 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; []25D +38 (c 0.001, CHCl3);

IR (KBr) max1777, 1736 cm-1; 1H and 13C 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; []25D +34 (c 3, CHCl3); IR (KBr)

max 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; []25D +6 (c 0.001, CHCl3); IR (KBr) max

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).

Xylorumphiin O (6): white solid; IR (KBr) max 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; []25D +12 (c 0.001, CHCl3); IR (KBr) max

3408, 1782, 1732 cm-1; 1H and 13C NMR data, see Table 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; []25D +10 (c 0.001, CHCl3); IR (KBr) max

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).

15

1,(21S)-di-O-Acetylxylorumphiin P (7b): white solid; []25D +10 (c 0.001, CHCl3); IR (KBr)

max 1800, 1773, 1735 cm-1; 1H and 13C NMR data, see Tables 3 and 6; HREIMS m/z 781.3047

[M+Na]+ (calcd for C39H50O15Na, 781.3047).

Xylorumphiin Q (8): white solid; []25D -71 (c 0.003, CHCl3); IR (KBr) max 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; []25D +7 (c 0.001, CHCl3); IR (KBr) max

3389, 1790, 1728, 1645 cm-1; 1H and 13C 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; []25D -4 (c 0.001, CHCl3); IR (KBr) max3419,

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; []25D +25 (c 0.001, CHCl3); IR (KBr) max 3417,

1732 cm-1; 1H and 13C NMR data, see Table 1; HREIMS m/z 671.34268 [M+H]+ (calcd for

C37H51O11, 671.34259).

16

-

Figure 1. Structures of compounds 1-10.

17

Figure 2. Plots of calculated (1DCHcalc) vs. experimental (1DCH

exp), and Q factors, from the SVD

fitting of RDCs data of the 23-epimers of compound 5a.

1DCHcalc1DCH

calc

18

Figure 3. Plots of calculated (1DCHcalc) vs. experimental (1DCH

exp), and Q factors, from the SVD

fitting of RDCs 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.

1DCHcalc

1DCHcalc

19

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.

20

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.

21

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

1 2 3 10position C, type H, mult. (J in Hz) C, type H, mult. (J in Hz) C, type H, mult. (J in Hz) C, type H, mult. (J in Hz)

1 106.8, C 107.7, C 213.6, C 107.7, C2 57.2, CH 2.63, m 53.5, CH 2.91, dd (9.2, 4.3) 52.5, CH 3.08, dd (10.8, 2.4) 53.6, CH 2.88, dd (9.2, 4.2)3 73.9, CH 5.10, d (9.0) 73.9, CH 5.11, d (9.2) 76.6, CH 5.20, d (10.8) 73.7, CH 5.13, d (9.2)4 38.3, C 38.0, C 39.3, C 38.0, C5 40.8, CH 2.60, d (9.9) 40.8, CH 2.64, m 41.8, CH 3.37, dd (9.9, 3.3) 40.8, CH 2.63, dd (10.2, 1.5)6 32.7, CH2 2.35, dd (16.4, 9.9) 32.3, CH2 2.35, m 33.3, CH2 2.47, m 32.3, CH2 2.35, m

2.26, d (16.4) 2.17, m 2.15, m7 174.2, C 174.0, C 174.0, C 174.0, C8 82.4, C 81.8, C 62.8, C 81.8, C9 63.5, CH 1.43, dd (13.4, 2.6) 51.8, CH 2.14, m 47.4, CH 2.26, m 51.8, CH 2.15, m

10 43.9, C 43.1, C 51.5, C 43.1, C11 19.8, CH2 1.90, m 15.2, CH2 2.38, m 16.8, CH2 1.62, m 15.3, CH2 2.35, m

1.67, m 1.82, m 1.80, m12 36.0, CH2 1.83, m 25.1, CH2 2.18, m 26.0, CH2 1.60, m 25.1, CH2 2.17, m

1.32, m 1.41, m 1.40, m13 36.5, C 39.1, C 37.9, C 39.1, C14 46.7, CH 2.20, d (9.3) 159.8, C 67.6, C 159.7, C15 29.2, CH2 3.25, d (19.6) 117.9, CH 6.00, s 35.6, CH2 3.72, d (17.1) 118.0, CH 6.00, s

2.73, dd (19.6, 9.3) 2.90, d (17.1)16 170.4, C 163.8, C 169.4, C 163.7, C17 77.3, CH 5.25, s 81.4, CH 5.02, s 78.0, CH 5.91, s 81.4, CH 5.03, s18 22.4, CH3 1.03* 19.9, CH3 1.21, s 14.4, CH3 1.05, s 19.9, CH3 1.21, s19 21.3, CH3 1.04* 20.7, CH3 1.06, s 17.8, CH3 1.20, s 20.7, CH3 1.06, s20 121.1, C 120.2, C 119.8, C 120.2, C21 141.7, CH 7.52, br s 141.5, CH 7.49, s 142.1, CH 7.62, br s 141.4, CH 7.49, br s22 110.2, CH 6.38, br s 110.2, CH 6.43, s 110.4, CH 6.50, br s 110.2, CH 6.43, br s23 143.2, CH 7.38, br s 143.1, CH 7.41, s 142.6, CH 7.39, br s 143.1, CH 7.41, br s28 24.9, CH3 0.75, s 24.8, CH3 0.78, s 23.6, CH3 0.78, s 24.8, CH3 0.77, s29 22.6, CH3 1.19, s 22.1, CH3 1.24, s 20.9, CH3 0.88, s 22.1, CH3 1.24, s30 76.0, CH 6.16, d (4.2) 76.4, CH 5.55, d (4.3) 70.9, CH 5.34, d (2.4) 76.4, CH 5.57, d (4.2)

1-OH 3.90, s 3.51, s7-OMe 52.0, CH3 3.69, s 52.1, CH3 3.69, s 52.4, CH3 3.76, s 52.2, CH3 3.69, s3-acyl

1' 177.7, C 175.9, C 176.0, C 175.8, C2' 34.2, CH 2.65, m 41.3, CH 2.27, m 41.6, CH 2.39, m 41.2, CH 2.27, m3' 18.5, CH3 1.10, d (6.9) 16.4, CH3 1.13, d (7.1) 17.6, CH3 1.16, d (7.2) 16.4, CH3 1.13, d (7.0)4' 20.2, CH3 1.21, d (7.1) 26.4, CH2 1.64, m 26.7, CH2 1.81, sextet (7.2) 26.3, CH2 1.65, m

1.40, m 1.49, sextet (7.2) 1.38, m5' 11.7, CH3 0.89, t (7.4) 11.8, CH3 1.02, t (7.2) 11.7, CH3 0.89, t (7.4)

30-acyl1" 175.2, C 176.6, C 174.9, C 176.2, C2" 33.5, CH 2.53, septet (7.5) 34.3, CH 2.45, m 33.8, CH 2.50, m 40.9, CH 2.23, m3" 17.7, CH3 1.04* 19.2, CH3 1.10, d (7.0) 18.8, CH3 1.13, d (6.3) 15.9, CH3 1.05, d (6.8)4" 19.3, CH3 0.91, d (7.5) 19.0, CH3 1.10, d (7.0) 16.3, CH3 1.15, d (6.6) 26.6, CH2 1.65, m

1.33, m5" 11.9, CH3 0.90, t (7.4)

*Assignments for positions with identical superscripts are interchangeable.

22

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

position 4a 4b 4c 4d 5a 5b 6a2 2.90, dd (9.2, 4.2) 2.90, dd (9.2, 4.2) 4.07, dd (9.2, 4.4) 4.06, dd (9.0, 4.5) 2.91, dd (9.2, 4.2) 2.90, dd (9.2, 4.2) 2.88, m3 5.12, d (9.2) 5.11, d (9.2) 5.08, d (9.2) 5.09** 5.12, d (9.2) 5.12, d (9.2) 5.14, d (9.3)5 2.59, dd (10.3, 2.0) 2.59, dd (10.3, 1.5) 2.60, m 2.60, m 2.58, dd (10.1, 1.6) 2.58, dd (10.0, 1.7) 2.58, dd (10.2, 1.5)6 2.35, m 2.35, m 2.33, m 2.33, m 2.34, m 2.34, m 2.34, m

2.15, m 2.13, m 2.15, m 2.14, m 2.15, m 2.14, m 2.14, m9 2.12, m 2.11, m 2.10, m 2.10, m 2.12, m 2.10, m 2.12, t (10.5)11 2.39, m 2.39, m 2.38, m 2.38, m 2.39, m 2.38, m 2.39, m

1.85, m 1.82, m 1.86, m 1.84, m 1.84, m 1.83, m 1.84, m12 2.50, m 2.48, m 2.51, m 2.51, m 2.46, m 2.47, m 2.46, m

1.36, m 1.33, m 1.37, m 1.34, m 1.35, m 1.32, m 1.35, m15 5.98, s 5.98, s 6.07, s 6.07, s 5.98** 5.97, s 5.98, s17 5.04, s 5.06, s 5.03, s 5.06* 5.03** 5.06, s 5.05, s18 1.27, s 1.24, s 1.27, s 1.25, s 1.27, s 1.24, s 1.27, s19 1.06, s 1.06, s 1.06, s 1.06, s 1.06, s 1.06, s 1.06, s22 7.40, s 7.42, s 7.39, br s 7.42, br s 7.39, br s 7.42, s 7.39, br s 23 6.93, s 7.04, s 6.93, br s 7.04, br s 6.93, br s 7.03, s 6.93, br s 28 0.78, s 0.77, s 0.76, s 0.77, s 0.77, s 0.77, s 0.77, s29 1.23, s 1.23, s 1.12, s 1.12, s 1.24, s 1.23, s 1.24, s30 5.52, d (4.2) 5.51, d (4.2) 5.55, d (4.4) 5.56, d (4.5) 5.51, d (4.2) 5.50, d (4.2) 5.53, d (4.0)

1-OH 3.58** 3.59, s 4.30, s 4.28, s 4.19**7-OMe 3.69, s 3.69, s 3.69, s 3.69, s 3.69, s 3.69, s 3.69, s1-OAc 2.14, s 2.15 s

23-OAc 2.17, s 2.16, s 2.17, s 2.16 s 2.17, s 2.15, s 2.17 s3-acyl

2' 2.29, m 2.29, m 2.28, m 2.27 m 2.46, m 2.47, m 2.29 m3' 1.12, d (7.1) 1.12, d (7.0) 1.09* 1.13* 1.09, d (7.0) 1.08, d (6.9) 1.13, d (7.0)4' 1.63, m 1.63, m 1.60, m 1.63, m 1.06, d (6.9) 1.08, d (6.9) 1.63 m

1.38, m 1.38, m 1.36, m 1.41, m 1.40 m5' 0.87, t (7.5) 0.87, t (7.4) 0.85* 0.87** 0.87 m

30-acyl2" 2.43, m 2.46, m 2.46, m 2.47, m 2.30, m 2.29, m 2.22 m3" 1.09, d (7.1) 1.10, d (6.9) 1.14* 1.15** 1.12, d (7.1) 1.12, d (7.1) 1.06*4" 1.07, d (7.0) 1.07* 1.06* 1.06** 1.62, m 1.62, m 1.63 m

1.37, m 1.38, m 1.40 m5" 0.86, t (7.4) 0.87, t (7.4) 0.87*

*Assignments for positions with identical superscripts are interchangeable.**Superimposed with impurity

23

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

position 4a 4b 4c 4d 5a 5b 6a 7a 7b 8a 9a1 107.7, C 107.7, C 108.6, C 108.6, C 107.7, C 107.7, C 107.8, C 107.6, C 108.4, C 107.6, C 107.6, C2 53.4, CH 53.4, CH 47.9, CH 47.9, CH 53.3, CH 53.3, CH 53.4, CH 53.3, CH 47.8, CH 53.3, CH 53.3, CH3 73.6, CH 73.7, CH 72.7, CH 72.7, CH 73.9, CH 73.9, CH 73.8, CH 73.8, CH 72.9, CH 73.7, CH 74.0, CH4 38.0, C 38.0, C 37.8, C 37.8, C 38.0, C 38.0, C 38.0, C 37.9, C 37.7, C 38.0, C 37.6, C5 40.8, CH 40.8, CH 40.6, CH 40.5, CH 40.9, CH 40.8, CH 40.8, CH 40.7, CH 40.4, CH 40.7, CH 40.7, CH6 32.5, CH2 32.5, CH2 32.4, CH2 32.4, CH2 32.5, CH2 32.5, CH2 32.5, CH2 32.3, CH2 32.2, CH2 32.3, CH2 32.2, CH2

7 173.7, C 173.7, C 173.5, C 173.5, C 173.7, C 173.7, C 173.7, C 174.0, C 173.5, C 174.0, C 174.1, C8 81.6, C 81.5, C 82.7, C 82.7, C 81.5, C 81.4, C 81.5, C 81.4, C 82.4, C 81.4, C 81.3, C9 51.5, CH 51.5, CH 50.5, CH 50.4, CH 51.5, CH 51.4, CH 51.5, CH 51.1, CH 50.1, CH 51.1, CH 51.1, CH10 43.1, C 43.1, C 45.2, C 45.2, C 43.1, C 43.1, C 43.1, C 43.2, C 45.3, C 43.2, C 43.2, C11 15.3, CH2 15.3, CH2 15.1, CH2 15.1, CH2 15.3, CH2 15.2, CH2 15.3, CH2 15.4, CH2 15.2, CH2 15.4, CH2 15.3, CH2

12 25.1, CH2 25.1, CH2 25.1, CH2 25.1, CH2 25.1, CH2 25.1, CH2 25.1, CH2 24.9, CH2 24.9, CH2 24.8, CH2 24.9, CH2

13 39.2, C 39.3, C 39.3, C 39.3, C 39.2, C 39.3, C 39.2, C 39.4, C 39.5, C 39.4, C 39.4, C14 159.6, C 159.6, C 158.6, C 158.6, C 159.8, C 159.8, C 159.8, C 159.3, C 158.1, C 159.4, C 159.3, C15 117.4, CH 117.5, CH 118.0, CH 118.0, CH 117.4, CH 117.4, CH 117.4, CH 117.6, CH 118.1, CH 117.6, CH 117.7, CH16 162.5, C 162.6, C 162.2, C 162.3, C 162.7, C 162.7, C 162.7, C 161.9, C 161.6, C 161.9, C 162.0, C17 78.9, CH 79.2, CH 78.8, CH 79.1, CH 78.9, CH 79.2, CH 78.9, CH 80.0, CH 79.9, CH 80.0, CH 80.0, CH18 19.9, CH3 19.9, CH3 19.9, CH3 19.9, CH3 19.9, CH3 19.9, CH3 19.9, CH3 20.7, CH3 20.7, CH3 20.7, CH3 20.6, CH3

19 20.7, CH3 20.7, CH3 21.2, CH3 21.2, CH3 20.7, CH3 20.7, CH3 20.7, CH3 20.8, CH3 21.4, CH3 20.8, CH3 20.8, CH3

20 134.2, C 134.2, C 134.2, C 134.2, C 134.1, C 134.1, C 134.1, C 160.2, C 160.2, C 160.2, C 160.2, C21 168.0, C 168.4, C 168.0, C 168.5, C 168.0, C 168.4, C 168.0, C 93.1, CH 93.1, CH 93.1, CH 93.1, CH22 147.8, CH 148.3, CH 147.8, CH 148.4, CH 147.8, CH 148.4, CH 147.8, CH 123.9, CH 124.0, CH 123.9, CH 124.0, CH23 92.3, CH 93.0, CH 92.3, CH 93.0, CH 92.3, CH 93.0, CH 92.3, CH 168.5, C 168.5, C 168.5, C 168.5, C28 24.8, CH3 24.8, CH3 24.4, CH3 24.4, CH3 24.8, CH3 24.8, CH3 24.8, CH3 24.9, CH3 24.5, CH3 24.9, CH3 24.7, CH3

29 22.1, CH3 22.1, CH3 21.8, CH3 21.8, CH3 22.2, CH3 22.2, CH3 22.2, CH3 22.1, CH3 21.8, CH3 22.1, CH3 22.0, CH3

30 76.5, CH 76.5, CH 76.0, CH 76.0, CH 76.5, CH 76.5, CH 76.5, CH 76.5, CH 76.1, CH 76.6, CH 76.4, CH1-OAc 167.5, C 167.6, C 167.3, C

22.3, CH3 22.3, CH3 22.3, CH3

7-OMe 52.2, CH3 52.2, CH3 52.2, CH3 52.2, CH3 52.2, CH3 52.2, CH3 52.2, CH3 52.3, CH3 52.3, CH3 52.3, CH3 52.3, CH3

21-OAc 168.7, C 168.7, C 168.7, C 168.7, C20.6, CH3 20.6, CH3 20.6, CH3 20.6, CH3

23-OAc 168.8, C 169.1, C 168.8, C 169.2, C 168.8, C 169.2, C 168.8, C20.9, CH3 20.8, CH3 20.8, CH3 20.8, CH3 20.8, CH3 20.8, CH3 20.8, CH3

3-acyl1' 176.1, C 176.0, C 175.7, C 175.8, C 176.3, C 176.3, C 176.3, C 176.2, C 175.6, C 175.9, C 170.2, C2' 41.1, CH 41.1, CH 41.1, CH 41.0, CH 34.2, CH 34.2, CH 41.1, CH 34.2, CH 34.1, CH 41.2, CH 21.1, CH3

3' 16.6, CH3 16.6, CH3 16.7, CH3 16.7, CH3 19.1, CH3 19.1, CH3 16.6, CH3 19.3, CH3 19.3, CH3 16.3, CH3

4' 26.3, CH2 26.3, CH2 26.3, CH2 26.2, CH2 19.0, CH3 19.0, CH3 26.2, CH2 19.1, CH3 19.1, CH3 26.5, CH2

5' 11.7, CH3 11.7, CH3 11.6, CH3 11.6, CH3 11.6, CH3 11.8, CH3

30-acyl1" 176.2, C 176.4, C 176.5, C 176.2, C 176.2, C 176.3, C 175.9, C 176.9, C 176.8, C 176.8, C 176.6, C2" 34.2, CH 34.2, CH 34.1, CH 34.2, CH 41.1, CH 41.2, CH 40.9, CH 34.2, CH 34.1, CH 34.2, CH 40.8, CH3" 19.1, CH3 19.1, CH3 19.1, CH3 19.1, CH3 16.6, CH3 16.6, CH3 15.8, CH3 19.0, CH3 19.0, CH3 19.1, CH3 16.4, CH3

4" 19.0, CH3 19.0, CH3 19.1, CH3 18.9, CH3 26.3, CH2 26.3, CH2 26.5, CH2 19.1, CH3 19.1, CH3 19.1, CH3 26.7, CH2

5" 11.6, CH3 11.7, CH3 11.8, CH3 11.8, CH3

24

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

Carbon

1DCH from 5a (Hz) 1DCH from 5b (Hz)

2 -13.02 -11.16

3 -22.84 -21.78

5 -33.81 -27.06

6 37.19 39.53

15 -48.53 -46.24

17 12.59 NA

18 -2.74 NA

22 -24.09 -27.01

23 39.15 0.00

29 7.46 7.66

30 -60.61 -62.63

25

Table 5. 1H NMR Data for 7a, 7b, 8a and 9a (500 MHz, J in Hz, CDCl3)

position 7a 7b 8a 9a

2 2.92, dd (9.2, 4.2) 4.11, dd (9.2, 4.2) 2.90, dd (9.2, 4.1) 2.90, dd (9.2, 4.4)3 5.05, d (9.2) 5.04, d (9.2) 5.10, d (9.2) 5.08, d (9.2)5 2.64, m 2.63, m 2.61, m 2.60, m6a 2.38, m 2.37, m 2.37, m 2.37, m6b 2.18, m 2.19, m 2.17, m 2.16, m9 2.14, m 2.08, m 2.09, m 2.09, m

11a 2.46, m 2.46, m 2.44, m 2.45, m11b 1.92, m 1.94, m 1.93, m 1.92, m12β 2.26, m 2.28, m 2.25, m 2.23, m12α 1.44, m 1.45, m 1.44, m 1.44, m15 5.98, s 6.07, s 5.98, s 5.99, s17 4.84, s 4.83, s 4.86, s 4.83 s18 1.30, s 1.30, s 1.30, s 1.30, s19 1.08, s 1.08, s 1.07, s 1.07, s21 6.97, s 6.97, s 6.97, s 6.97, s22 6.44, s 6.45, s 6.44, s 6.45, s28 0.80, s 0.79, s 0.79, s 0.79, s29 1.24, s 1.12, s 1.24, s 1.23, s30 5.38, d (4.2) 5.42, d (4.2) 5.39, d (4.1) 5.40, d (4.4)

1-OH 3.56, s 3.72, s1-OAc 2.15, s7-OMe 3.71, s 3.71, s 3.71, s 3.71, s21-OAc 2.24, s 2.24, s 2.24, s 2.25, s3-acyl

2' 2.46, m 2.46, m 2.28, m 1.99, s3' 1.15, d (7.1) 1.13, d (7.1) 1.11, d (7.0)4' 1.09, d (6.9) 1.10, d (6.9) 1.61, m

1.38, m5' 0.88, t (7.4)

30-acyl2" 2.42, m 2.46, m 2.42, m 2.26, m3" 1.07 1.07 1.08 1.02, d (7.0)4" 1.05, d (7.1) 1.05, d (7.1) 1.05, d (7.2) 1.58, m

1.37, m5" 0.89, t (7.4)

26

Table 6. Interproton Distances for Compound 7. All Distances are in Angstroms.

AtomsExperimental

DistancesRef.: H-2 to H-3

Experimental Distances

Ref.: H-17 to H-12β

DFT Calculated Distances

(C-21R isomer)

DFT Calculated Distances

(C-21S isomer)H2-H3 2.27 2.17 2.27 2.27

H17-H12β 2.56 2.44 2.50 2.44

H17-H21 3.20 3.05 2.55 3.01

H17-H22 3.73 3.55 3.92 3.83

H17-H15 3.86 3.68 3.77 3.77

H21-H12α 2.42 2.30 4.12 2.17

H21-H12β 3.25 3.10 3.98 2.55

H21-H22 4.19 3.99 3.99 4.04

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

Q χ2 N/ χ2

C-21R

0.215 15.694 0.510

C-21S 0.087 2.583 3.098

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

Q χ2 N/ χ2

C-21R

0.241 17.857 0.448

C-21S 0.074 1.676 4.773

ASSOCIATED CONTENT

Supporting Information. Three files are provided: S1 gives the detailed isolation

procedure and MS, IR, and NMR spectra for all compounds; S2 gives computational data; S3

27

gives NCI 59 screening data. This material is available free of charge via the Internet at

http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

* Tel: +44 (0) 1483 68 6751. E-mail: d.mulholland@surrey.ac.uk

Author Contributions

The manuscript was written through contributions of all authors and all authors have given

approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

Dr. W. Waratchareeyakul 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 James Maxwell, Chiang Mai

University Herbarium, Thailand for plant identification, the National Cancer Institute for NCI59

cancer cell line screening, 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). RRG gratefully acknowledges support from the NSF (CHE-

1111684).

28

REFERENCES

(1) http://www.theplantlist.org/tpl1.1/search?q=xylocarpus (accessed 14/07/2016)

(2) http://www.mangrovewatch.org.au/ (accessed 14/07/2016)

(3) Pudhom, K.; Sommit, D.; Nuclear, P.; Ngamrojanavanich, N.; Petsom, A. J. Nat. Prod.

2009, 72, 2188-2191.

(4) Pudhom, K.; Sommit, D.; Nuclear, P.; Ngamrojanavanich, N.; Petsom, A. J. Nat. Prod. 2010,

73, 263-266.

(5) Uddin, S. J.; Nahar, L.; Shilpi, J. A.; Shoeb, M.; Borkowski, T.; Gibbons, S.; Middleton, M.;

Byres, M.; Sarker, S. D. Phytother. Res. 2007, 21, 757-761.

(6) Yin, S.; Wang, X. N.; Fan, C. Q.; Lin, L. P.; Ding, J.; Yue, J. M. J. Nat. Prod. 2007, 70, 682-

685.

(7) Rouf, R.; Uddin, S. J.; Shilpi, J. A.; Alamgir, M. J. Ethnopharmacol. 2007, 109, 539-542.

(8) Uddin, S. J.; Shilpi, J. A.; Alam, S. M. S.; Alamgir, A.; Rahman, M. T.; Sarker, S. D. J.

Ethnopharmacol. 2005, 101, 139-143.

(9) MacKinnon, S.; Durst, T.; Arnason, J. T.; Angerhofer, C.; Pezzuto, J.; SanchezVindas, P. E.;

Poveda, L. J.; Gbeassor, M. J. Nat. Prod. 1997, 60, 336-341.

(10) Lakshmi, V.; Singh, N.; Shrivastva, S.; Mishra, S. K.; Dharmani, P.; Mishra, V.; Palit, G.

Phytomedicine 2010, 17, 569-574.

(11) Wisutsitthiwong, C.; Buranaruk, C.; Pudhom, K.; Palaga, T. Biochem. Bioph. Res. Co. 2011,

415, 361-366.

29

(12) Sarigaputi, C.; Sommit, D.; Teerawatananond, T.; Pudhom, K. J. Nat. Prod. 2014, 77, 2037-

2043.

(13) Ravangpai, W.; Sommit, D.; Teerawatananond, T.; Sinpranee, N.; Palaga, T.; Pengpreecha, S.;

Muangsin, N.; Pudhom, K. Bioorg. Med. Chem. Lett. 2011, 21, 4485-4489.

(14) Du, S. J.; Wang, M. G.; Zhu, W.; Qin, Z. H. Nat. Prod. Res. 2009, 23, 1316-1321.

(15) Misra, S.; Verma, M.; Mishra, S. K.; Srivastava, S.; Lakshmi, V.; Misra-Bhattacharya, S.

Parasitol. Res. 2011, 109, 1351-1360.

(16) Wu, J.; Xiao, Q.; Zhang, S.; Li, X.; Xiao, Z. H.; Ding, H. X.; Li, Q. X. Tetrahedron 2005, 61,

8382-8389.

(17) Sarigaputi, C.; Nuanyai, T.; Teerawatananond, T.; Pengpreecha, S.; Muangsin, N.; Pudhom,

K. J. Nat. Prod. 2010, 73, 1456-1459.

(18) National Cancer Institute DTP Developmental Therapeutics Program. 2016,

https://dtp.cancer.gov/discovery_development/nci-60/methodology.html (accessed 18/2/2016)

(19) Taylor, D. A. H. Phytochemistry 1983, 22 (5), 1297-1299.

(20) Gil, R. R.; Griesinger, C.; Navarro-Vázquez, A.; Sun, H., Structural Elucidation of Small

Organic Molecules Assistedby NMR in Aligned Media, in Structure Elucidation on Organic

Chemistry: The Search for the Right Tools; Cid, M. M.; Bravo, J.; Wiley-VCH Verlag GmBH &

Co. KGaA; Weinheim, 2015; pp 279-324.

(21) Gil, R. R.; Gayathri, C.; Tsarevsky, N. V.; Matyjaszewski, K. J. Org. Chem. 2008, 73 (3),

840-8.

30

(22) MacroModel, version 9.9. Schrödinger, LLC; New York, NY, USA: 2011

(23) Frisch, M. J. T.; G, W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.;

Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.;

Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.;

Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,T.; Honda, Y.; Kitao, O.; Nakai, H.;

Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.;

Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;

Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene,

M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R.

E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;

Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.;

Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian09,

Revision B.01; Gaussian, Inc.: Wallingford, CT, 2009.

(24) Navarro-Vazquez, A. Magn. Reson. Chem.: MRC 2012, 50 Suppl 1, S73-9.

(25) Stott, K.; Stonehouse, J.; Keeler, J.; Hwang, T.-L.; Shaka, A. J. J. Am. Chem. Soc. 1995,

117 (14), 4199-4200.

(26) Hu, H.; Krishnamurthy, K.; J. Magn. Reson. 2006, 183, 173-177.

(27) Thiele, C. M.; Bermel, W.; J. Magn. Reson. 2012, 216, 134-43.

31