Natthakaln Lomchoeya,
Panomwan Panseetaab,
Pornthip Boonsria,
Nuttapon Apiratikula,
Samran Prabpaic,
Palangpon Kongsaereec and
Sunit Suksamrarn*a
aDepartment of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand. E-mail: sunit@g.swu.ac.th
bDepartment of Chemistry, Chulachomklao Royal Military Academy, Nakornnayok 26001, Thailand
cDepartment of Chemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
First published on 17th May 2018
Six new 14-membered ring cyclopeptide alkaloids, cambodines A–F (1–6), and two known compounds, frangufoline (7) and lotusanine B (8), were isolated from the root bark extract of Ziziphus cambodiana Pierre. Their structures and configurations were established based on 1D and 2D NMR, HRMS, ECD, and X-ray crystallographic data. Compounds 1 and 3 are rare 5(14)-type cyclopeptide alkaloids that possess an imidazolidin-4-one ring in the terminal unit. The cyclopeptides were tested for their in vitro antiplasmodial, antitubercular, and cytotoxic effects against three cancer cell lines. Compound 3 showed significant antiplasmodial activity against the malarial parasite Plasmodium falciparum, with an IC50 value of 6.09 μM.
Several common chemical and spectroscopic characteristics were evident for compounds 1–8. They displayed a blue colour upon staining with anisaldehyde-H2SO4 reagent on TLC.13 Their IR spectra showed absorption bands for amide (3266–3338 and 1632–1685 cm−1) and aryl ether (1221–1237 cm−1) functionalities. The ECD spectra of compounds 1–8 showed intense negative and weak positive Cotton effects in the 236–244 and 279–286 nm regions, respectively, consistent with the (5S,8S,9S)-configurations.32 Their 1H and 13C NMR data agreed well with the published values for 5(14)-scutianine A-type (1–3), 4(14)-integerrine-type (4–6), 4(14)-frangulanine-type (7), and 5(14)-neutral-type cyclopeptide alkaloid (8).
Compound 1 was isolated as a colourless amorphous solid. The HRESITOFMS data showed a protonated ion at m/z 680.3808 [M + H]+, in accordance with a molecular formula C40H49N5O5. The 13C NMR and DEPT data in DMSO-d6 (Table 1) revealed 40 carbon resonances, which were classified as four methyls (δC 10.8, 12.0, 14.5, 15.4), an N-methyl (δC 40.5), four methylenes (δC 24.0, 24.3, 34.2, 66.3), 23 methines (six aliphatic at δC 35.0, 35.5, 53.4, 56.0, 57.4, 69.7, one oxygenated at δC 80.8, two olefinic at δC 125.8 and 126.7 and 14 aromatic at δC 119.5, 121.8, 126.2, 127.6 × 2, 127.9 × 3, 128.4 × 2, 128.8 × 2, 129.5, 130.0), four amide carbonyls (δC 167.4, 168.9, 169.9, 170.7), three aromatic quaternary (δC 131.5, 136.7, and 137.8), and an oxygenated tertiary (δC 154.7). Interpretation of the 1H, 13C NMR, and 2D NMR spectroscopic data and the IR absorption frequencies at 3289, 1682, 1636, and 1241 cm−1 led to the conclusion that 1 was a 5(14)-type cyclopeptide alkaloid in which the cyclic part comprised a phenylalanine, an p-oxystyrylamine with a Z double bond, an isoleucine moiety, in addition to the coupled phenylalanine and N-methylisoleucine moiety as the terminal unit (Fig. 1). The assembly of these fragments was made possible on the basis of correlations in the COSY, HMBC, and NOESY experiments (Fig. 2). The HMBC spectrum showed correlations of the olefinic H-2 (δH 6.23, dd, J = 7.3, 4.5 Hz) to the C-4 carbonyl carbon resonance (δC 168.9) and of the oxygenated methine H-9 (δH 5.84, d, J = 8.1 Hz) and isoleucyl H-5 (δH 3.81, t, J = 8.5 Hz) to C-7 (δC 169.9), in addition to the NOESY cross-peaks between H-9 and the aromatic resonances H-12 (δH 7.14) and H-22 (δH 7.44, dd, J = 7.7, 2.2 Hz) established the linkage of the p-oxystyrylamine moiety to the phenylalanine unit and the location of this dimeric moiety in the macrocyclic ring. The H-8 methine signal at δH 4.78 (brt, J = 8.8 Hz) showed an HMBC cross-peak to the phenylalanine carbonyl C-26 (δC 167.4), indicating the connection between the phenylalanine units. A prominent fragment ion at m/z 622 and a base peak at m/z 155 in the EIMS spectrum of 1 (the molecular ion of which is presented as 1′ in Fig. 3) indicated the fragmentations of the corresponding N-methylimidazolidin-4-one to produce 1′a and the terminal residue 1′b,33,34 respectively (Fig. 3). The relatively low field chemical shifts of the two diastereotopic protons at δH 3.99 and 3.11 (each d, J = 4.4 Hz, H-41a and H-41b, respectively), δC 66.3 (C-41), an N-methyl (δH 2.13), a sec-butyl group (δH 1.23, m, H-36, δC 35.0; δH 0.99, m, H-37, δC 24.3; δH 0.67, t, J = 6.3 Hz, H-38, δC 12.0; and δH 0.41, d, J = 6.8 Hz, H-39, δC 14.5), and an amide carbonyl carbon at δC 170.7 (C-34) further supported the presence of a 3-substituted 5-(sec-butyl)-1-methylimidazolidin-4-one ring. Furthermore, the correlations of H-8, NH-25 (δH 8.23, d, J = 9.4 Hz), and H-27 (δH 4.58, dd, J = 10.7 and 5.1 Hz) to CO-26 (δC 167.4) in the HMBC spectrum along with the interactions of H-27 to H-30 (δH 7.02), of H-41a to H-28 (δH 2.61 m) and H-30, and of N–CH3 to H-41b in the NOESY experiments permitted the assignment of the linkage between both phenylalanine units to the imidazolidinone group. The HMBC cross-peaks of H-41a to CO-34, C-35 (δC 69.7), and N–CH3 and of H-35 to CO-34 and C-36 (δC 35.0) and the NOESY interaction between H-35 and N–CH3 were also observed. Thus, the structure of cyclopeptide 1, cambodine A, was deduced as a new member of the 5(14)-scutianine A-type cyclopeptides.
No. | δC | δH | ||||||
---|---|---|---|---|---|---|---|---|
1a | 2a | 2b | 3b | 1a | 2a | 2b | 3b | |
a Recorded in DMSO-d6.b Recorded in CDCl3. | ||||||||
1 | 125.8 | 127.5 | 117.5 | 117.5 | 6.60, d (7.3) | 6.63, d (7.3) | 6.44, d (7.1) | 6.45, d (7.4) |
2 | 126.7 | 126.7 | 125.5 | 125.4 | 6.23, dd (7.3, 4.5) | 6.14, dd (7.3, 3.4) | 6.65, dd (9.6, 7.1) | 6.65, dd (9.2, 7.4) |
4 | 168.9 | 169.0 | 167.2 | 167.2 | ||||
5 | 57.4 | 57.3 | 59.3 | 59.3 | 3.81, t (8.5) | 3.81, t (8.9) | 4.08, dd (8.3, 4.1) | 3.98, dd (8.0, 3.9) |
7 | 169.9 | 170.2 | 171.0 | 171.0 | ||||
8 | 56.0 | 55.9 | 56.7 | 57.2 | 4.78, brt (8.8) | 4.83, dt (9.8, 7.9) | 4.78, dd (8.9, 6.7) | 4.70, dd (8.4, 7.1) |
9 | 80.8 | 80.9 | 81.5 | 81.6 | 5.84, d (8.1) | 5.84, d (7.9) | 6.10, d (6.7) | 6.17, d (7.1) |
11 | 154.7 | 154.8 | 155.1 | 155.2 | ||||
12 | 121.8 | 118.1 | 123.1 | 122.9 | 7.14, overlap | 7.16, overlap | 7.32, overlap | 7.35, dd (8.4, 2.1) |
13 | 129.5 | 129.4 | 130.1 | 130.1 | 6.96, d (6.9) | 6.96, overlap | 7.15, overlap | 7.17, overlap |
14 | 131.5 | 131.2 | 132.5 | 132.2 | ||||
15 | 130.0 | 129.9 | 131.9 | 131.8 | 6.93, d (6.9) | 6.95, overlap | 7.11, overlap | 7.12, overlap |
16 | 119.5 | 121.1 | 122.6 | 122.5 | 7.19, overlap | 7.06, overlap | 7.34, overlap | 7.40, dd (8.4, 2.1) |
17 | 35.5 | 37.2 | 35.2 | 35.3 | 1.58, m | 1.45, m | 2.11, m | 2.04, m |
18 | 24.0 | 24.5 | 23.9 | 23.9 | a 1.16, m | a 1.15, m | a 1.25, m | a 1.15, m |
b 0.99, m | b 0.80, m | b 0.80, m | b 0.85, m | |||||
19 | 10.8 | 11.3 | 11.9 | 11.9 | 0.66, t (7.1) | 0.64, overlap | 0.84, t (7.2) | 0.79, t (7.2) |
20 | 15.4 | 15.2 | 15.8 | 16.4 | 0.65, t (7.3) | 0.64, overlap | 0.75, d (6.8) | 0.67, d (6.9) |
21 | 137.8 | 137.5 | 136.8 | 137.2 | ||||
22,22′ | 128.4 | 128.6 | 128.1 | 128.2 | 7.44, dd (7.7, 2.2) | 7.49, d (6.9) | 7.51, dd (7.5, 1.0) | 7.67, brd (7.2) |
23,23′ | 127.6 | 127.7 | 128.8 | 128.8 | 7.28, overlap | 7.27, t (6.9) | 7.40, overlap | 7.48, brt (7.4) |
24 | 127.9 | 127.8 | 128.9 | 128.9 | 7.26, overlap | 7.22, overlap | 7.45, overlap | 7.31, overlap |
26 | 167.4 | 169.5 | 170.9 | 170.0 | ||||
27 | 53.4 | 53.0 | 54.7 | 63.0 | 4.58, dd (10.7, 5.1) | 4.23, dt (10.8, 7.9) | 4.20, dd (10.4, 4.8) | 3.49, dd (11.9, 4.4) |
28 | 34.2 | 37.5 | 36.6 | 33.2 | 2.61, m | a 2.33, dd (11.2, 7.9) | a 2.88, dd (14.2, 4.8) | a 2.29, brt (12.6) |
b 2.13, brt (11.2) | b 2.45, dd (14.2, 10.4) | b 2.19, dd (12.6, 4.4) | ||||||
29 | 136.7 | 137.7 | 136.3 | 136.6 | ||||
30,30′ | 128.8 | 129.0 | 128.9 | 129.2 | 7.02, overlap | 6.99, overlap | 6.98, dd (8.4, 2.1) | 6.93, dd (7.6, 1.1) |
31,31′ | 127.9 | 127.5 | 128.6 | 128.5 | 7.08, overlap | 7.10, overlap | 7.18, overlap | 7.22, overlap |
32 | 126.2 | 126.0 | 127.0 | 127.0 | 7.07, overlap | 7.08, overlap | 7.22, overlap | 7.24, overlap |
34 | 170.7 | 172.2 | 174.2 | 174.8 | ||||
35 | 69.7 | 69.1 | 69.1 | 71.0 | 2.45, brs | 2.35, brs | 2.50, brs | 2.57, brs |
36 | 35.0 | 36.1 | 37.6 | 36.5 | 1.23, m | 1.52, m | 1.51, m | 1.50, m |
37 | 24.3 | 24.1 | 24.2 | 24.8 | 0.99, m | a 1.15, m | a 0.86, m | 1.28, m |
b 0.82, m | b 0.70, m | |||||||
38 | 12.0 | 10.9 | 11.7 | 12.2 | 0.67, t (6.3) | 0.64, overlap | 0.65, t (6.0) | 0.85, t (7.3) |
39 | 14.5 | 15.4 | 15.5 | 14.9 | 0.41, d (6.8) | 0.46, d (6.7) | 0.62, d (6.9) | 0.77, d (6.8) |
41 | 66.3 | 78.9 | a 3.99, d (4.4) | 2.41, q (5.4) | ||||
b 3.11, d (4.4) | ||||||||
42 | 19.5 | 0.92, d (5.4) | ||||||
3-NH | 7.84, brd (4.5) | 8.05, brd (3.4) | 6.73, d (9.6) | 6.52, d (9.2) | ||||
6-NH | 7.54, brd (8.5) | 7.62, d (8.9) | 6.19, d (8.3) | 5.92, d (8.0) | ||||
25-NH | 8.23, d (9.4) | 7.93, d (8.9) | 6.84, d (8.9) | 8.73, d (8.4) | ||||
33-NH | 7.64, d (7.9) | |||||||
NMe | 40.5 | 34.9 | 36.5 | 39.6 | 2.13, s | 1.79, s | 2.11, s | 1.85, s |
Compound 2 was also obtained as a colourless amorphous solid. Its molecular formula was determined by the HRESITOFMS ion at m/z 668.3807 [M + H]+ and 13C NMR spectroscopic data as C39H49N5O5. Its 1H and 13C NMR data in DMSO-d6 (Table 1) were similar to those of 1, except for the absence of the two geminal protons at C-41 in 1 and the presence of an amide proton NH-33 at δH7.64 (1H, d, J = 7.9 Hz) in 2, which are in agreement with the structural change in the terminal residue (Fig. 1). The NMR data of 2 recorded in CDCl3 (Table 1) were similar to those observed in DMSO-d6, except for the C-1 chemical shift. Information from the HMBC association of H-8 (δH 4.83, dt, J = 9.8, 7.9 Hz) to CO-26 (δC 169.5) and of NH-33 to CO-34 (δC 172.2) in addition to the NOESY interactions of H-27 (δH 4.23, dt, J = 10.8, 7.9 Hz) to NH-25 (δH 7.93, d, J = 8.9 Hz) and H-30 (δH 6.99) and of H-35 (δH 2.35, brs) to NCH3 (δH 1.79, s) (Fig. 2) provided evidence for the exocyclic phenylalanine moiety connecting to the macrocyclic ring at N-25 and the terminal N-methylisoleucine unit. The structure of 2, cambodine C, was therefore also defined as a new 5(14)-scutianine A-type compound.
Compound 3 displayed a sodium adduct molecular ion at m/z 716.3792 [M + Na]+ in the HRESITOFMS, corresponding to the molecular formula C41H51N5O5. Its 1H and 13C NMR data in CDCl3 (Table 1) showed signals similar to those observed for compounds 1 and 2 in the same NMR solvent, except for one fewer methylene carbon and the additional methyl (δC 19.5) and methine resonances (δC 78.9) observed in the DEPT spectra for 3. Similar to those of 1, the observed fragment peaks at m/z 636 and 169 in the EIMS data of 3 suggest the presence of phenylalanine and a 5-(sec-butyl)-1,2-dimethylimidazolidin-4-one as the respective exocyclic units (Fig. 3), which is supported by a molecular mass 14 amu greater than that of 1. The NOE associations of NH-25 (δH 8.73, d, J = 8.4 Hz) to H-9 (δH 6.17, d, J = 7.1 Hz) and H-28a (δH 2.29, brt, J = 12.6 Hz), of H-41 (δH 2.41, q, J = 5.4 Hz) to H-27 (δH 3.49, dd, J = 11.9, 4.4 Hz), H-30 (δH 6.93, dd, J = 7.6, 1.1 Hz), and N–CH3 (δH1.85), and of the latter N–CH3 to H-35 (δH 2.57 brs), together with connectivities of H-28 to C-30 (δC 129.2), of N–CH3 to C-41 (δC 78.9) and C-35 (δC 71.0), and of H-35 to C-36 (δC 36.5) in the HMBC spectrum also supported the linkage between the acyclic part and the macrocyclic ring (Fig. 2). The NOESY associations of NH-6 (δH 5.92, d, J = 8.0 Hz)/H-8 and H-9/H-16 (δH7.40, dd, J = 8.4, 2.1 Hz) confirmed the location of the phenylalanine unit next to the p-oxystyrylamine moiety and the isoleucine fragments in the macrocyclic system. The significantly different C-27 (δC 63.0) and C-41 (δC 78.9) chemical shifts compared to those of 1 (C-27, δC 53.4 and C-41, 66.3) and 2 (C-27, δC 54.7) could be attributed to the presence of the C-41 methyl group. However, the existing data did not permit the establishment of the configuration at C-41. Thus, the structure of 3, cambodine F, was deduced as a new member of the 5(14)-scutianine A-type cyclopeptides possessing a 5-(sec-butyl)-1,2-dimethylimidazolidin-4-one moiety.
The J values of H-1 and H-2 ranging from 7.1–7.4 Hz accounted for the Z geometry of the double bond in compounds 1–3. The intense negative (236 nm) and weak positive (283 nm) Cotton effects present in the CD spectrum of 1, similar to those of 2 and 3, and indicative of the (5S,8S,9S) configuration.32 The H-8/H-9 vicinal coupling constants of 8.1 Hz for 1, 6.7 for 2, and 7.1 for 3 and the relatively shielded shift of the 13C NMR resonance at approximately δC 81 ppm for C-9 permitted assignment of the erythro, i.e., (8S,9S) configuration.13 The 13C NMR chemical shifts of the phenylalanine and the N-methyl isoleucine terminal amino acid units for 1–3, particularly those of stereocenters [1: δC 53.4 (C-27), 69.7 (C-35); 2: δC 53.0 (C-27), 69.1 (C-35); 3: δC 63.0 (C-27), 71.0 (C-35)] were comparable, except for C-27 in 3 as indicated above, with the same terminal dipeptide in paliurine B [δC 51.5 (C-27), 69.9 (C-35)], a 13-membered ring cyclopeptide obtained from Paliurus ramossisimus.35 The stereochemical assignment of the exocyclic dipeptide in paliurine B was determined by the 13C NMR data comparison with those of the comparable synthesized model dipeptide-L-Phe(OMe)-L-Ile(NMe2),35 the L amino acids, or 27S and 35S, in the acyclic part attached to the macrocycles in 1–3 were then suggested, though the DD-dipeptide cannot be ruled out due to no NMR information available. Furthermore, amino acids in Rhamnaceous cyclopeptide alkaloids generally present in L-form whilst the D-amino acid only rarely found. For instances, the D-erythro (8R,9R) of scutianine E,36 and D-threo (8R,9S) of scutianene L,37 were isolated from Scutia buxifolia; whereas epimauritine A and its N-oxide obtained from Z. apetala showed the R-configuration at the terminal unit.38 Formation of the imidazolidinone ring in cyclopeptide has been implicated from the reaction between the amino acid residue and aldehyde.34,39 The imidazolidinone ring in compounds 1 and 3 could therefore arise through the condensation of 2 with the respective corresponding carbonyl compounds without an alteration in the amino acid stereocenter.
The two diagnostic Cotton effect bands at about 240 (intense negative) and 280 nm (weak positive) displayed in the ECD spectrum which could be attributed to the transition of CC double bond conjugation with benzene ring or adjacent amide group, and characterized for the existence of L-amino acids in the 14-membered ring cyclopeptide.32,40,41 Comparison of the experimental ECD spectrum of 1 to the qualitative calculated ECD data of (5S,8S,9S,17S,27S,35S,36S)-1 and the (5R,8R,9R,17R,27R,35R,36R)-1 (Fig. 4A), indicated 1 to be in agreement with that of all S configuration on the amino acid residues. Calculation of the ECD spectra of individual epimer of 1 were also performed (Fig. 4B) and revealed that the major Cotton effect contribution with opposite sign observed at the band near 238 nm for 9S/9R and at around 245 nm for 8S/8R epimer pair. Similar Cotton effects were found for compounds 2 (negative 236 nm and positive 283 nm bands) and 3 (negative 236 nm and positive 282 nm bands) and comparable to the theoretical ECD ones (Fig. 4C) allowed the same absolute configuration assignment as for 1. From these evidences, including their negative specific rotations, the stereochemical structures of compounds 1–3 were then deduced.
Compound 4, a minor compound from the same fraction of compound 1, was isolated as a colourless amorphous solid and given a molecular formula C37H36N4O5, as deduced from its positive ion HRESITOFMS at m/z 617.2751 [M + H]+. The 13C NMR and DEPT spectra (CDCl3) disclosed 37 carbon resonances, consisting of an N-methyl (δC 39.8), two methylenes (δC 37.5, 67.1), 23 methines (two olefinic at δC 121.2, 125.2, two oxymethine at δC 73.1, 81.6, 19 aromatic at δC 121.9, 123.2, 126.3, 126.9 × 2, 128.2 × 4, 128.6 × 2, 128.7 × 3, 129.1 × 3, and 130.2), four aromatic quaternary (δC 131.9, 136.0, 138.1, 139.0), an oxygenated tertiary (δC 155.3), three amide carbonyls (δC 167.7, 167.8 and 172.1) and no signal observed in the aliphatic region (Table 2, Fig. 1). A 1D and 2D NMR extensive data analysis in addition to a comparison with previously described values evidenced the spin system of a p-oxystyrylamine, a β-hydroxyphenylalanine, a phenylalanine, and a 1-methylimidazolidin-4-one, which is derived from the phenylalanine of the 4(14)-integerrine-type cyclopeptide alkaloid. Interestingly, a broad three-bond singlet at δH1.59 was assigned to the styrylamine double bond H-1, H-2, and NH-3 by the HMBC of H-1 to C-2 (δC 125.5), C-14 (δC 131.9) and C-15 (δC 131.0) and of H-2 to C-14 and the NOESY of H-3 to H-5 (δH 4.51, brt, J = 8.1 Hz) connectivities (Fig. 2). The presence of the β-hydroxyphenylalanine was characterized by the OH absorption frequency at 3447 cm−1 in the IR data, along with the correlations from the oxymethine doublet resonance H-17 (δH 4.94, J = 7.6 Hz) to a carbonyl carbon C-4 at δC 167.8 and an aromatic carbon at δC 126.9 (C-18) in the HMBC spectrum, in addition to the NOESY connectivities of H-17 to NH-6 (δH 6.37, brd, J = 8.5 Hz). As for other cyclopeptides, the HMBC correlations of H-1 to C-14 and C-15, of H-5 to CO-7 (δC 167.7), and of H-9 (δH 5.90, d, J = 7.3 Hz) to C-22 (δC 128.2) and of the NOESY interactions of H-5 to NH-3 and of H-9 to H-16 (δH 7.29) suggested that the phenylalanine and β-hydroxyphenylalanine were placed next to each other and attached to the oxystyrylamine in the cyclic ring. A 5-benzyl-1-methylimidazolidin-4-one group was determined by the analysis of the 1D and 2D NMR spectra of 4: the presence of the two diastereotopic protons at δH 3.66 and 2.70 (each d, J = 4.3 Hz, H-34a and H-34b), δC 67.1; an N-methyl (δH 1.64, s), a ring multiplet methine proton resonance at δH 2.69 (H-27), δC 66.5; a benzyl group (δH 2.67, m, H-28, δC 37.5; δH 7.24–7.47, ArH, δC 126.3–138.1) and an amide carbonyl at δC 172.1 (C-26). A series of connectivities was observed: a diastereotopic proton at δH 3.66 showed NOESY correlations to H-9 and H-27 and of H-27 to N–CH3, and the HMBC connections of H-8 (δH 4.87, d, J = 7.3 Hz) to carbonyl carbon C-26, of H-27 to C-29 and of H-28 to C-30 showed that the imidazolidin-4-one ring was connected to the macrocyclic ring at C-8 of the phenylalanine. The structure of 4, cambodine B, was thus elucidated as a new member of the 4(14)-integerrine-type cyclopeptides.
No. | δC | δH | ||||
---|---|---|---|---|---|---|
4 | 5 | 6 | 4 | 5 | 6 | |
1 | 121.2 | 117.8 | 116.9 | 6.59, brs | 6.46, d (7.6) | 6.47, d (7.6) |
2 | 125.2 | 125.4 | 125.6 | 6.59, brs | 6.66, dd (9.5, 7.6) | 6.37, brt (7.6) |
4 | 167.8 | 166.8 | 166.5 | |||
5 | 57.5 | 59.3 | 55.4 | 4.51, brt (8.1) | 4.01, dd (8.5, 4.4) | 4.44, ddd (11.2, 8.1, 3.4) |
7 | 167.7 | 168.2 | 168.1 | |||
8 | 57.8 | 57.9 | 57.6 | 4.87, d (7.3) | 5.09, d (7.6) | 4.95, d (7.2) |
9 | 81.6 | 80.7 | 81.7 | 5.90, d (7.3) | 5.99, d (7.6) | 5.91, d (7.2) |
11 | 155.3 | 155.0 | 155.4 | |||
12 | 123.2 | 123.3 | 123.2 | 7.26, overlap | 7.32, overlap | 7.34, overlap |
13 | 130.2 | 130.2 | 130.4 | 7.05, dd (6.8, 1.3) | 7.13, t (7.1) | 7.14, overlap |
14 | 131.9 | 132.3 | 132.2 | |||
15 | 131.0 | 131.6 | 131.7 | 7.10, dd (6.8, 1.3) | 7.10, t (7.1) | 7.14, overlap |
16 | 121.9 | 122.8 | 122.5 | 7.29, overlap | 7.34, overlap | 7.34, overlap |
17 | 73.1 | 34.4 | 36.7 | 4.94, d (7.6) | 2.16, m | a 3.40, dd (14.8, 3.4) |
b 2.61, dd (14.8, 11.2) | ||||||
18a | 139.0 | 136.9 | ||||
18,18′ | 126.9 | 23.9 | 128.8 | 7.24, overlap | 1.17, m, 0.88, m | 7.09, d (8.7) |
19,19′ | 128.7 | 11.7 | 128.5 | 7.03, overlap | 0.81, t (6.3) | 7.19, brt (8.7) |
20 | 128.7 | 16.5 | 127.1 | 7.24, overlap | 0.70, d (6.8) | 7.07, overlap |
21 | 136.0 | 136.2 | 136.0 | |||
22,22′ | 128.2 | 128.2 | 128.2 | 7.46, overlap | 7.52, dd (7.7, 1.9) | 7.46, overlap |
23,23′ | 128.6 | 128.8 | 128.8 | 7.19, overlap | 7.39, t (6.9) | 7.39, overlap |
24 | 129.1 | 129.2 | 129.0 | 7.39, overlap | 7.41, overlap | 7.11, overlap |
26 | 172.1 | 172.8 | 172.3 | |||
27 | 66.5 | 70.4 | 66.1 | 2.69, m | 2.48, d (2.6) | 2.39, m |
28 | 37.5 | 35.8 | 37.6 | 2.67, m | 1.29, m | a 2.66, dd (13.8, 8.9) |
b 1.72, dd (13.8, 11.2) | ||||||
29 | 138.1 | 24.7 | 138.2 | 1.08, m | ||
30 | 129.1 | 11.9 | 129.1 | 7.39, overlap | 0.77, t (7.4) | 7.00, brd (6.1) |
31 | 128.2 | 14.7 | 128.2 | 7.24, overlap | 0.55, d (6.8) | 7.26, overlap |
32 | 126.3 | 126.2 | 7.47, overlap | 7.22, overlap | ||
33 | 67.6 | a 3.83, br d (3.9) | ||||
b 3.22, br d (3.9) | ||||||
34 | 67.1 | 67.0 | a 3.66, d (4.3) | a 3.56, d (4.7) | ||
b 2.70, d (4.3) | b 2.43, d (4.7) | |||||
3-NH | 6.59, brs | 6.50, d (9.5) | 6.51, d (9.9) | |||
6-NH | 6.37, brd (8.5) | 5.81, d (8.5) | 6.40, d (8.1) | |||
NMe | 39.8 | 41.1 | 39.5 | 1.64, s | 2.11, s | 1.48, s |
Compound 5 was also obtained as a colourless amorphous solid from the detannified EtOAc-soluble fraction of Z. cambodiana. Its molecular formula was deduced as C31H40N4O4 from its positive ion HRESITOFMS at m/z 555.2929 [M + H]+. A detailed analysis of the 1D, DEPT and 2D spectroscopic data suggested that 5 also possessed a 4(14)-type cyclopeptide containing the same macrocyclic ring and terminal unit as for 1 (Fig. 1). The main differences in their 13C NMR data (Table 2) are the absence of the intermediate phenylalanine resonances in 5 compared to that of 1. A series of correlations of H-8 (δH 5.09, d, J = 7.6 Hz) to C-26 (δC 172.8), of H-33a (δH 3.83, brd, J = 3.9 Hz) to C-27 (δC 70.4), and of H-27 (δH 2.48, d, J = 2.6 Hz) to CO-26, C-29 (δC 24.7) and C-31 (δC 14.7) displayed in the HMBC spectrum, together with the interactions of H-33a to H-9 (δH 5.99, d, J = 7.6 Hz), H-22′ (δH 7.52, dd, J = 7.7, 1.9 Hz) and N–CH3 (δH 2.11) and of H-27 to N–CH3, H-29 (δH 1.08, m) and H-31 (δH 0.55, d, J = 6.8 Hz) observed in the NOESY spectrum indicated that the 5-(sec-butyl)-1-methylimidazolidin-4-one ring was the end fragment of the system (Fig. 2). The structure of 5, cambodine D, was thus established as an additional member of the 4(14)-integerrine-type cyclopeptides.
Compound 6 was isolated as colourless needles and displayed a sodium adduct molecular ion [M + Na]+ at m/z 623.2606 in the HRESITOFMS, corresponding to a molecular formula of C37H36N4O4. Its 1H and 13C NMR data (Table 2, Fig. 1) were almost identical to those of compound 4, with the difference being the presence of the two germinal proton resonances at δH 3.40 (dd, J = 14.8 and 3.4 Hz, H-17a) and δH 2.61 (dd, J = 14.8 and 11.2 Hz, H-17b) (δC 36.7) of the ring-bound amino acid in 6, instead of the oxymethine signal in 4. In the HMBC spectrum, the connectivities of the resonance at δH 2.61 to C-5 (δC 55.4) and a carbonyl carbon at δC 166.5 (C-4), of NH-6 at δH 6.40 (d, J = 8.1 Hz) to C-5, and of an aromatic signal at δH 7.09 (d, J = 8.7 Hz, H-18) to C-17 confirmed that the phenylalanine was bound to p-oxystyrylamine and β-hydroxyphenylalanine in the cyclic structure. Its EIMS spectrum showed the base peak at m/z 509 and a fragment ion peak at m/z 227, indicating the existence of a 5-benzyl-1-methylimidazolidin-4-one unit as the end residue. This was supported by the presence of two geminal protons at δH 3.56 (d, J = 4.7 Hz, H-34a) and δH 2.43 (d, J = 4.7 Hz, H-34b), a singlet N–CH3 at δH 1.48 (δC 39.5), and a multiplet methine resonance of H-27 at δH 2.39 (δC 66.1), in addition to a carbonyl carbon signal at δC 172.3 (C-26) and a benzyl group in its NMR data. Compound 6 exhibited similar 2D NMR (HMBC and NOESY) correlations, both at the cyclic and the terminal ring, to those of compound 4. A remarkable upfield-shifted N–CH3 resonance at δH 1.48 in 6 could be due to an anisotropic effect arising from the benzyl group when compared with compound 5 (δH 2.11) that has a sec-butyl moiety. The structure of 6, cambodine E, was thus elucidated as an analogue of cyclopeptide 4.
The X-ray crystal structure of 6 supported a skeleton comprised of a Z-styrylamine, two phenylalanines and a 5-benzyl-1-methylimidazolidin-4-one subunit and also confirmed the trans arrangement between H-8 and H-9 displayed in the macrocyclic motif (Fig. 5). The ECD spectrum of 6, as well as the spectra of the other cyclopeptides indicated above, provided the 5S, 8S and 9S configuration assignments at the amino acid residues of the cyclic part. The imidazolidinone configuration at 27S was then subsequently decisively assigned. The similarities in the NMR data on the terminal ring of 6 compared with those of 4 and 5, associated with the calculated ECD spectra of 4–6 (Fig. 6) were in accordance with those observed ECD value have led to the conclusion that cyclopeptides 4–6 contribute the same stereochemistry both at the macrocyclic ring and at the imidazolidinone unit.
The two cyclopeptides 7 and 8 were identified as the 4(14)-type cyclopeptide alkaloids, frangufoline19 or daechuine S,32 or sanjoinine A42 and lotusanine B,20 respectively, by detailed examinations of their 1D and 2D NMR and MS spectroscopic data along with comparison to their reported values (Fig. 1, ESI†). The 13C NMR resonances of the chiral carbons observed for 7 were in good agreement with the literature data for frangufoline whose stereochemistry was proven to be all S configurations by the analysis of each amino acid residue in its acid hydrolysate and confirmed by total synthesis.19,43 The ECD spectrum of 7 exhibited strong negative (239 nm) and positive (287 nm) Cotton effects that also supported the same (5S,8S,9S)-configurations at the 14-membered ring and its negative specific rotation ([α]39D −218.5), which led to the conclusion that 7 has the same structure and configuration as the reported frangufoline. In a manner similar to that observed for the other cyclopeptide alkaloids, the 13C NMR chemical shifts at the chiral carbons of 8 are almost identical to those of amaiouine, a structurally related cyclopeptide isolated from Amaioua guianensi that was different from 8 by having a phenylalanine in place of a leucine amino acid in the macrocyclic ring. The absolute configurations of amaiouine were established to be all S according to its X-ray crystallographic data.44 Therefore, our observed ECD spectrum (strong negative and weak positive bands at 237 and 283 nm, respectively) was also in accordance with the all S configurations at the amino acid residues for 8. It should be noted that compound 8 showed negative optical activity, although the previously reported lotusanine B was a racemate. Frangufoline has been isolated from many plants belonging to the Rhamnaceae, Celastraceae and Sterculiaceae families,32 whereas lotusanine B was obtained from Ziziphus lotus.20
The in vitro antimalarial effect against Plasmodium falciparum of compounds 1–8 was evaluated.45,46 Only cambodine F (3) showed interesting antiplasmodial activity, with an IC50 of 6.09 μM, which could be accounted for by the importance of the rare 5-(sec-butyl)-1,2-dimethylimidazolidin-4-one unit. All compounds were considered inactive against Mycobacterium tuberculosis,47 except for cambodines B (4) and C (2), and lotusanine B (8) which showed weak activity, with MICs of 81.0, 149.7 and 93.8 μM, respectively. All compounds were also tested in vitro for cytotoxic properties against three human cancer cell lines: epidermoid carcinoma of the mouth (KB), breast cancer (BC-1), and small cell lung cancer (NCI-H187) cells.48 Of the tested compounds, cambodine A (1) was moderately active against the BC cells at IC50 11.1 μM, and cambodines C–E (2, 5, 6) exhibited weak activity towards the NCI cells, with IC50 values of 20.7, 36.7 and 35.0 μM, respectively. None of the compounds were toxic to the non-cancerous Vero cells, except for 6, which showed weak action with an IC50 of 48.2 μM. To date, only two studies on the in vitro cytotoxicity of natural rhamnaceous cyclopeptide alkaloids have been reported,38,49 although the weak activity of some representative synthetic 13- and 15-membered ring cyclopeptides was described recently.50
A portion of the MeOH extract (30.7 g) was fractionated by quick column chromatography (silica gel, 170 g), eluting with EtOAc–CH2Cl2 (50:50), EtOAc, MeOH and H2O–MeOH (50:50) to yield four major fractions (Fr. J–M). Fr. J (0.8 g) was applied to a silica gel column (20 g) and eluted with a MeOH–CH2Cl2 (1:99–30:70) gradient system to yield 10 subfractions (J1–J10). Subfraction J2 was purified by CC over silica gel (eluted with MeOH–CH2Cl2, 3:97–15:85 gradient system) followed by a Sephadex LH-20 column (eluted with MeOH) to yield 7 (11 mg) as colourless needles. Fr. K (11 g) was chromatographed on a silica gel column (16 g) to give six subfractions (K1–K6). Subfraction K2 (163 mg) was separated on a silica gel column (7 g) eluting with EtOAc–CH2Cl2 (5:95–15:85) to give more of frangufoline (7) (16.3 mg) and lotusanine B (8) (7.5 mg).
Another portion of the MeOH extract (225 g) was suspended in 1% NaCl (100 mL) and then partitioned with EtOAc (6 × 150 mL), combined and concentrated to give a detannified EtOAc soluble fraction (12.7 g) as a pale brown paste. This detannified EtOAc soluble fraction was chromatographed on a silica gel column (150 g) to give six major fractions (Fr. N–S). Fr. O (1.2 g) was further separated on a gel column (29 g), eluted with EtOAc–CH2Cl2 (5:95–13:87), to provide 17 subfractions. Compound 5 (29.4 mg) was separated after the subfraction O12 (85 g) was rechromatographed on silica gel (8 g), eluting with EtOAc–CH2Cl2 (10:90). Subfraction O14 (203 mg) was separated on a Sephadex LH-20 column, eluting with MeOH–CH2Cl2, 50:50 to give compounds 3 (7.0 mg) and 6 (35.2 mg).
Footnote |
† Electronic supplementary information (ESI) available. CCDC 1469415. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c7ra13050c |
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