Stereochemical analysis and cytotoxicity of kulokekahilide-2 and its analogues

Tetrahedron 69 (2013) 3045e3053

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Stereochemical analysis and cytotoxicity of kulokekahilide-2 and its analogues Masahiro Umehara a, Takayuki Negishi a, Yukie Maehara a, Yoichi Nakao b, Junji Kimura a, * a

Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5258, Japan Department of Chemistry and Biochemistry, School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 November 2012 Received in revised form 26 January 2013 Accepted 29 January 2013 Available online 8 February 2013

We report the conformational analysis of kulokekahilide-2, a cytotoxic cyclic depsipeptide, and its analogues. We also evaluated their cytotoxicity against human cancer cells. Although both the cis and trans conformations are possible for the amide bond between MePhe and MeGly, only one conformation was observed in DMSO. We also reveal that the configuration at C-43 in kulokekahilide-2 controls intramolecular ester exchange between the 26- and 24-membered cyclic depsipeptides. Kulokekahilide-2 and its analogues were evaluated for their cell-growth inhibition profile and using COMPARE analysis, which suggested a mechanism of action different from that of standard anticancer drugs. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Kulokekahilide-2 Stereochemical analysis Intramolecular ester exchange Cytotoxic evaluation

1. Introduction Linear and cyclic peptides obtained from marine animals, such as the dolastatins1 and kahalalides,2 exhibit cytotoxicity and anticancer cell effect.3 For this reason, the biological activities of many cyclic depsipeptides have been evaluated.4 We recently reported the structure and absolute configuration of kulokekahilide-2 (1), which was isolated from a marine mollusk and shows potent cytotoxic activity. During structure elucidation, different stereoisomers were synthesized for comparison of their NMR spectra.5 Thus, the 26-membered form of kulokekahilide-2 (1) consisting of 43-D-Ala, 37-L-Ile, 34-MeGly, 24-D-MePhe, 21-LAla, 15-D-2-hydroxyisocaproic acid (15-D-Hica) and (5S,6S,7S)-5,7dihydroxy-2,6,8-trimethyl-2,8-decadienoic acid (Dtda), possessed almost the same components as the aurilides6 (from the Japanese sea hare) and lagunamides7 (from marine cyanobacteria). The 24-membered form of kulokekahilide-2 (2), formed from 1 by intramolecular ester exchange in non-polar solvents such as CH2Cl28 is structurally very similar to palau’amide9 from marine cyanobacteria.

* Corresponding author. Tel.: þ81 42 759 6229; fax: þ81 42 759 6493; e-mail address: [email protected] (J. Kimura). 0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2013.01.089

The aurilides, lagunamides and palau’amide have shown cytotoxicity against HeLa and P388 cells, which can be compared with our kulokekahilide-2 analogues. In addition, kulokekahilide-2 and two analogues were also evaluated against a panel of 39 human cancer cell lines. Below, we report the stereochemical analysis of kulokekahilide2 and its analogues and the evaluation of their cytotoxicity. 2. Results and discussion 2.1. Synthesis of kulokekahilede-2 analogues We synthesized several analogues of the following kind other than five stereoisomers (1a: 21-D-Ala, 24-L-MePhe and 43-D-Ala, 1b: 21-D-Ala, 24-L-MePhe and 43-L-Ala; 1c: 21-L-Ala, 24-D-MePhe and 43-L-Ala; 1d: 21-L-Ala, 24-L-MePhe and 43-D-Ala; and 1e: 21-DAla, 24-D-MePhe and 43-D-Ala) to determine the absolute configuration of the natural product 1. These five isomers had been chosen for comparison with related depsipeptides such as the aurilides (Fig. 1).5 Our first target was the halogenated kulokekahilide-2 analogue 1f, substituting 24-D-p-Cl-MePhe for the 24-D-MePhe in natural 1. Halogen substitution can introduce a steric influence on conformation, an electron-withdrawing effect and a hydrophobic effect

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R3

N

N 24

R2 R1

O

21

O

HN

O

R4

O

O

O

H N

O

N H R7

R5

N 43

O

OR8

O

1

5

7

N

R6

N

O

O

O

O

H O H N

N

O

O Compounds

21

43

24

5

R1

R2

R3

R4

R5

R6

R7

R8

1

Me

H

H

Bn

H

Me

H

H

5

Me

H

H

Bn

H

Me

H

MTM

1a

H

Me

Bn

H

H

Me

H

H

1b

H

Me

Bn

H

Me

H

H

H

1c

Me

H

H

Bn

Me

H

H

H

5c

Me

H

H

Bn

Me

H

H

MTM

1d

Me

H

Bn

H

H

Me

H

H

5d

Me

H

Bn

H

H

Me

H

MTM

1e

H

Me

H

Bn

H

Me

H

H

5e

H

H

Bn

H

Me

H

MTM

1f 5f

Me Me

Me H H

H H

p-Cl-Bn p-Cl-Bn

H H

Me Me

H H

H MTM

1g

Me

H

H

Bn

H

Me Me

H

5g

Me

H

H

Bn

H

Me Me

MTM

5g'

Me

H

H

Bn

H

Me Me (S)-MTPA

5g''

Me

H

H

Bn

H

Me Me (R)-MTPA

1h 5h

Me

H H

Bn

H H

H

Bn

H

H

H

Bn

H

MTM

Me

Bn

Fig. 1. Chemical structures of kulokekahilide-2 (1) and its analogues.

that facilitates cell membrane permeation.10 As a result, halogensubstituted analogues are generally more cytotoxic than natural 1. Compound 1 has two consecutive methyl amino acids (24-DMePhe and 34-MeGly), whereas the aurilides,6 lagunamides7 and

1i Fig. 2. Chemical structure of the 18-membered analogue (1i).

palau’amide9 have an additional methyl amino acid (43-L-MeAla) in addition to 24-D-MePhe and 34-MeGly. Therefore, the kulokekahilide-2 analogue (1g: 21-L-Ala, 24-D-MePhe and 43-DMeAla) was synthesized, containing 43-D-MeAla instead of 43-DAla. During isolation, intramolecular ester exchange occurred, converting the 26-membered ring to a 24-membered ring. The resulting structure was similar to palau’amide, which possesses potent cytotoxicity.8 Because the configuration at C-43 was suspected to exert a steric influence on the intramolecular ester exchange in 1, the 43-Phe analogue 1h (21-L-Ala, 24-L-MePhe and 43D-Phe) was synthesized to examine the effect on intramolecular ester exchange. 15-Membered cyclic depsipeptides consisting of four amino acids and Hica without the dihydroxy acid (Dtda), such as sansalvamide A11 and zygosporamide12 from marine fungi, have been reported to exhibit potent cytotoxicity. Therefore, we prepared the 18-membered cyclic depsipeptide 1i, containing five amino acids and Hica but lacking the Dtda group, to evaluate its cytotoxicity (Fig. 2). The synthesis of these analogues (1ae1e) has been reported previously;5 compounds 1fe1i were synthesized using a literature procedure (Scheme 1; see Supplementary data). We also examined the cytotoxicity of the pre-cyclization precursors (3 and 4) and the synthetic intermediates, including the intermediate for the determination of stereochemistry with the protected 5-hydroxy group in Dtda (5 series: 5-methylthiomethyl (5-MTM), protecting group in synthetic steps; 5, 5ce5h, 5g0 ; 5-(S)-2-methoxy-2-

Scheme 1. Synthesis of 1 and its analogues.

M. Umehara et al. / Tetrahedron 69 (2013) 3045e3053

trifluoromethylphenylacetic acid (MTPA), 5g00 ; 5-(R)-MTPA,13 determination reagent of stereochemistry; see Fig. 1). 2.2. Conformational analysis and study of intramolecular ester exchange The 1H NMR spectrum of 1 in CD2Cl2 showed two conformers corresponding to the cis and trans conformations around the amide bond between MeGly and D-MePhe.5 The conformer ratio was influenced by the solvent. For example, 1H NMR spectra in the less polar solvents CD2Cl2 and CDCl3 showed four peaks at d 2.97, 2.95, 2.91 and 2.71 (in CD2Cl2), corresponding to the cis and trans conformers with a ratio of approximately 1:1. However, in the polar solvents CD3OD and DMSO-d6, only the trans conformer was observed at d 2.80 and 2.64 (in DMSO-d6) (Fig. 3).8

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H-5 (d 5.06), to the methine H-6 (d 1.95). H-6 showed correlations to the C-12 methyl (d 0.71) and the oxymethine proton H-7 (d 3.82). HMBC correlations were observed between the proton at d 3.82 and dc 11.5 (C-12), 75.2 (C-5), 39.5 (C-6), 136.5 (C-8) and 123.3 (C-9), as well as between d 5.06 and dc 139.9 (C-3), 28.6 (C-4), 39.5 (C-6), 11.5 (C-12) and 171.6 (C-42) (Fig. 4). These results revealed that the 26-membered ring of 1 was transformed into a 24-membered ring (2), connected through the 5- and 7-hydroxy groups of the lactone ring. Compounds 1 and 2 gradually interconverted in the less polar solvent, CD2Cl2. In contrast, both 1 and 2 were stable in the polar solvent DMSO-d6: even after 1 was left at room temperature for five months in DMSO, only a slight amount of 2 was detectable. Compounds 1be1g were similarly examined for intramolecular ester exchange in order to investigate the effect of conformation.

Fig. 3. 1H NMR spectra of 1 in several solvents.

For compound 1b, with the configurations opposite to 1 at C-21, 24 and 43, the cis and trans conformations were present in both polar and less polar solvents; the ratio was approximately 2:1 in CDCl3 and approximately 1.5:1 in DMSO-d6. Compound 5, protected with MTM group at C-5 of Dtda, exhibited different conformer ratios in different solvents: 1.6:1 in CDCl3 and 6.8:1 in DMSO-d6. Similar results were reported for lagunamide A, B and C, which showed complex signals in CDCl3 but simple signals, indicating a single conformer, in CD3OD. As mentioned above, the N-methyl group of MeGly and MePhe in 1 appeared as four peaks for the cis and trans isomers in CD2Cl2. However, after a few days, double signals appeared at d 3.05, 2.98, 2.97, 2.95, 2.93, 2.91, 2.74 and 2.71. Detailed examination by HPLC revealed a small peak near the major peak for 1. The minor peak was found to not be an alternate conformer of 1, but a structural isomer (2). In the 1H NMR spectrum of 2, a characteristic peak at d 3.51 (btd, J¼8.7, 5.6 Hz), corresponding to 5-H of Dtda, was downshifted to d 5.06 (btd, J¼6.1, 3.2 Hz), while 7-H (d 5.22, d, J¼9.8 Hz) was upshifted to 3.82 (d, J¼8.4 Hz). COSY analysis revealed connections from the olefinic proton H-3 (d 6.69), via the allylic methylene protons H-4 (d 2.44, 2.58) and the oxymethine

The 1H NMR spectra of 1b (21-D-Ala, 24-L-MePhe and 43-L-Ala) and 1c (21-L-Ala, 24-D-MePhe and 43-L-Ala) were obtained in CD2Cl2: the signals did not change. Conversely, the 1H NMR spectra of 1d (21-L-Ala, 24-L-MePhe and 43-D-Ala), 1e (21-D-Ala, 24-D-MePhe and 43-D-Ala) and 1f (21-L-Ala, 24-D-p-Cl-MePhe and 43-D-Ala) in CD2Cl2 showed a complex spectra indicating intramolecular ester exchange. The 24-membered cyclic depsipeptides (2de2f) could be isolated by ODS-HPLC. However, in the 1H NMR spectra of 1d or 1e immediately after isolation, small signals of 2d or 2e appeared. This result was qualitative, but ester exchange in 1d and 1e may be more rapid than 1 or 1f, in which this phenomenon was not observed. Compounds 1d and 1e had the same relative configuration between C-21 and -24 and the same absolute configuration at C-43. These configurations might be expected to influence the ester equilibrium. To examine this hypothesis, 1g (21-L-Ala, 24-D-MePhe and 43-D-MeAla instead of the 43-D-Ala in 1) and 1h (21-L-Ala, 24-LMePhe and 43-D-Phe instead of the 43-D-Ala in 1d) were examined in a less polar solvent. As expected, the 43-D-MeAla analogue (1g) did not convert into the 24-membered isomer. For 1h, due to the steric hindrance of 43D-Phe, the intramolecular exchange reaction was unexpectedly fast,

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M. Umehara et al. / Tetrahedron 69 (2013) 3045e3053

Fig. 4. Partial structure of 1 and 2 and key COSY and HMBC correlations of 2.

and the 24-membered cyclic depsipeptide (2h) was not isolable. Transesterification of 1h was more rapid than that of 1d or 1e (Fig. 5).

R3

R2

N

N

R1

O HN

O

R4

O

N H R5

O

O NH

R6 O

O

O

O

the 43-position, in contrast to the D-Ala of 1.5 Therefore these compounds do not undergo intramolecular ester exchange. However, this may be due to ring strain, as well as the steric effect of the configuration at C-43. The chemical shift of the 43-NH of compound 1 was determined in various solvents to investigate the intramolecular ester exchange reaction. As shown in Table 1, in polar solvents (CD3OD and DMSOd6), the 43-NH signal was more downfield than in less polar solvents (CD2Cl2 and CDCl3). It seems that 1 experiences little intermolecular interaction because the chemical shifts of 1 did not change at different concentrations. In other words, these spectral differences were due to the solvents. Furthermore, this implies that the polar solvents coordinate to 43-NH and stabilize the conformation, as 1 was locked in one conformation in polar solvents. This phenomenon might be very important for bioactivity,14 and 1 may have a different mechanism of action than other similar cyclic depsipeptides, such as the aurilides, palau’amide and the lagunamides.

HO Table 1 NH shifts of 1 in various solventsa

Compounds

21

43

24

R1

R2

R3

R4

R5

R6

2

Me

H

H

Bn

H

Me

2d

Me

H

Bn

H

H

Me

2e

H

Me

H

Bn

H

Me

2f

Me

H

H

p-Cl-Bn

H

Me

2h

Me

H

Bn

H

H

Bn

Fig. 5. Chemical structures of 24-membered analogues.

It is clear that the configuration of the amino acid at C-43 is important in the intramolecular ester exchange reaction of 1 and its analogues. The reaction occurred only with analogues having 43-Dconfiguration and did not occur in the 43-L or D-N-methyl amino acid analogues. Aurilide, aurilide B and C, palau’amide and lagunamide A, B and C consist of five amino acids and two hydroxyacids possessing 27-, 26- or 24-membered rings,6,7,9 with an L-MeAla at

Solvent

21-NH

37-NH

43-NH (ppm)

CD2Cl2 CDCl3 CD3ODb DMSO-d6

7.10 7.07 n.d. 8.63

6.95 7.08 7.67 7.18

6.32 6.33 8.83 8.78

In contrast, 43-NH was detected. a The concentration of each solution was about 0.12 mol/L. b 21-NH was not detected due to rapid exchange.

2.3. Evaluation of cytotoxic properties of kulokekahilide-2 and analogues The cytotoxicities of 1, its synthetic intermediates (3, 4 and 5 series) and related analogues (1ae1i, 2, 2e, 2f and 2h) against HeLa and P388 cells are summarized in Table 2, represented by the IC50 (50% inhibitory concentration) values. Previously, the absolute configuration of 1 at C-21 was found to be essential for cytotoxicity.5 The 26-membered (1, 1e, 1f and 1h) and 24-membered (2, 2e, 2f and 2h) cyclic depsipeptides showed almost equal activity against HeLa and P388, as shown in Table 2. Compounds 1, 1f and 1h, with 21-D-Ala, were more potent than 1e, with 21-L-Ala. The 18membered depsipeptide without Dtda (1i) and the cyclization

M. Umehara et al. / Tetrahedron 69 (2013) 3045e3053 Table 2 Cytotoxicity of kulokekahilide-2, synthetic intermediates and analogues Compounds

1 2 3 4 5 1a 1b 1c 5c 1d 5d 1e 2e 5e 1f 2f 5f 1g 5g 5g0 5g00 1h/2h (1.4:1) 5h 1i

Cell lines

(IC50: mg/mL)

P388

HeLa

0.016 0.0072 10 >10 0.016 >10 10 0.016 0.08 0.4 0.4 4.5 4.5 >10 0.0072 0.016 0.016 0.0072 0.04 0.89 4.47 0.08 0.89 >10

0.0032 0.04 4.5 >10 0.0072 >10 >10 0.0052 0.016 0.039 0.039 2 2 >10 0.0014 0.0014 0.0072 0.0072 0.04 0.89 0.89 0.08 0.89 >10

precursor peptides (3 and 4) showed low potency. These results indicate that ring formation and ring size are important for potent cytotoxicity. Compound (5), protected with a 5-MTM group, and its related analogues (5ce5g) were slightly less cytotoxic than the corresponding cyclic depsipeptides (1 and 1ce1g). However, the MTPA analogues (5g0 and 5g00 ) and the MTM analogue 5h (43-Phe) were less cytotoxic than the deprotected compounds (5, 1h and 2h). These results indicate that the 5-hydroxyl group at Dtda in 1 and its analogues is less important for cytotoxicity, but steric hindrance from the 5-hydroxyl group decreases cytotoxicity. In addition, comparison of 1, 1f and 1g suggests that the amino acid functional groups affect cytotoxicity. In particular, the 24-D-p-Cl-MePhe analogue (1f) exhibits the most potent cytotoxicity against HeLa and P388 cells. Additionally, cytotoxicity may be related to the conformers described above. Thus, 1 and 5 showed a single conformer in polar solvents and were highly cytotoxic. In contrast, 1b showed two conformers and was less cytotoxic. We provided 1 and its analogues (1c: 43-L-Ala and 1g: 43-DMeAla) to the Screening Committee of Anticancer Drugs, who tested these compounds against 39 human cancer cell lines: breast, central nervous system, colon, lung, renal, stomach and prostate cancers, as well as melanoma (Table 3).15 The mean log GI50s or log LC50s of the 39 human cancer cell lines of 1, 1c and 1g were less than 8 and 7, respectively, which indicates potent growth inhibition and cytotoxicity. The delta values (the difference between log GI50 or log LC50 in the most sensitive cell line vs the mean log GI50 or the mean log LC50), the ranges (the difference in log GI50 or log LC50 between the most sensitive and the most resistant cell lines), and the CVs (%) (the coefficient of variation of the log GI50s in all 39 cell lines) indicated that the growth inhibition and cytotoxicity caused by 1, 1c and 1g differed between cell lines (delta>0.5 and range>1.0). In addition, among the three compounds tested, 1g produced growth inhibition with the lowest specificity (the lowest CV in GI50), and 1c induced cell death with the highest specificity (the highest CV in LC50). Compound 1 and 1g showed similar patterns for growth inhibition and cytotoxicity against 39 human cancer cell lines (Fig. 6A), while 1c showed a different pattern. Regression analyses confirmed the distinct cytotoxicity of 1c compared to the other two peptides: lower correlation coefficients

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Table 3 Cytotoxicity of kulokekahilide-2 and its analogues in the 39 cell lines GI50a 1

LC50b 1c

1g

Mean Delta Range

8.64 0.96 2.60

8.42 0.90 2.66

8.73 0.75 1.76

7.93 0.57 1.50

7.36 0.83 2.19

7.97 0.44 1.41

Breast cancer HBC-4 BSY-1 HBC-5 MCF-7 MDA-MB-231

8.72 8.68 8.89 8.64 7.96

8.37 8.62 8.60 8.51 7.80

8.68 8.80 8.77 8.62 8.19

8.16 8.15 8.30 >7.00 >7.00

>6.00 7.64 7.72 >6.00 7.14

8.10 8.21 8.24 >7.00 7.12

Central nervous system cancer U251 8.77 8.70 SF-268 8.74 8.72 SF-295 8.74 7.85 SF-539 8.70 8.68 SNB-75 8.70 8.42 SNB-78 8.66 8.68

8.82 8.82 8.72 8.72 8.64 8.60

8.21 8.23 8.20 8.14 >7.00 >7.00

8.14 8.19 7.21 8.13 7.24 6.92

8.22 8.24 8.18 8.15 7.24 >7.00

Colon cancer HCC2998 KM-12 HT-29 HCT-15 HCT-116 Lung cancer NCI-H23 NCI-H226 NCI-H522 NCI-H460 A549 DMS273 DMS114

1

1c

1g

7.89 8.72 8.72 >7.00 8.72

7.72 8.48 8.51 6.66 8.70

8.30 8.70 8.74 7.72 8.80

7.11 8.10 8.21 >7.00 8.22

7.08 7.28 7.34 >6.00 8.08

7.08 8.13 8.24 7.09 8.30

8.77 8.66 8.74 8.77 8.72 9.08 9.60

8.70 7.82 8.70 8.52 8.72 8.77 9.32

8.74 8.70 8.85 8.80 8.80 9.42 9.43

8.07 8.10 8.22 8.19 8.21 8.33 8.49

>6.00 7.14 8.10 7.43 8.17 8.15 8.05

>7.00 8.15 8.24 8.20 8.24 8.41 8.22

Melanoma LOX-IMVI Ov OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8 SKeOV-3

8.74

8.74

8.74

8.21

8.17

8.19

9.37 8.70 8.52 8.68 8.68

8.70 8.70 8.17 8.32 8.72

9.48 8.74 8.74 8.74 8.70

8.28 8.19 7.51 8.07 8.12

8.09 8.08 7.36 7.27 8.15

8.39 8.24 8.25 8.23 8.19

Renal cancer RXF-631L ACHN

7.74 8.64

7.72 7.44

8.70 8.70

7.21 7.74

7.17 6.02

8.07 8.13

Stomach cancer St-4 MKN1 MKN7 MKN28 MKN45 MKN74

8.66 8.74 8.68 8.62 8.70 8.68

7.92 8.68 8.68 8.57 8.48 8.77

8.70 8.80 8.72 8.68 8.66 8.70

8.09 8.20 8.18 8.15 8.05 8.21

7.18 8.18 7.96 >6.00 >6.00 8.15

8.17 8.24 8.21 8.15 7.89 8.21

Prostate cancer DU-145 PC-3

8.74 8.68

8.74 8.57

8.74 8.70

8.23 7.11

8.15 >6.00

8.24 >7.00

a b

50% growth inhibitory concentration. 50% lethal concentration.

in the LC50 were observed for 1 versus 1c (0.530) and 1c versus 1g (0.617), in contrast to the high value for 1 versus 1g (0.837). These results suggest that the configuration at the C-43 could affect the mechanism of action of kulokekahilide-2 qualitatively without a remarkable reduction in its cytotoxicity (Fig. 6B). Furthermore, COMPARE analyses by the Screening Committee of Anticancer Drugs produced a list of standard anticancer drugs with a similar growth inhibition pattern to the drug tested through regression

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M. Umehara et al. / Tetrahedron 69 (2013) 3045e3053

Fig. 6. Differential activity patterns for 1, 1c and 1g against 39 human cancer cell lines. Cytotoxicity of kulokekahilide-2 (1), the 43-L-Ala analogue (1c), and the 43-D-MeAla analogue (1g) against the 39 human cancer cell lines. (A) Growth inhibition for each peptide in each cell line (left panel) was represented as a difference between the logarithm of the 50% growth inhibitory concentration (GI50) in base 10 and the mean log10(GI50)s in the 39 lines. Cytotoxicity (right panel) was calculated similarly, using 50% lethal concentration (LC50) in each cell line. The mean GI50 and LC50 for each analogue is shown under the respective abbreviation. Br: breast cancer cells, CNS: central nervous system cancer cells, Co: colon cancer cells, Lu: lung cancer cells, Me: melanoma cells, Ov: ovary cancer cells, Re: renal cancer cells, St: stomach cancer cells, and xPg: prostate cancer cells. (B) Regression analyses of GI50 (left panel) and LC50 (right panel) for 1, 1c, and 1g. The Pearson product-moment correlation coefficient (r) between the two peptides is shown in each panel. Note that the regression analyses indicated significant (P < 0.001) correlations in all pairs of tested peptides for both GI50 and LC50.

M. Umehara et al. / Tetrahedron 69 (2013) 3045e3053

analyses of the GI50. COMPARE analyses of 1, 1c and 1g revealed the similarity of actinomycin-D (correlation coefficient 0.723) and doxorubicin (0.601) to 1, Taxol (0.729) and actinomycin-D (0.621) to 1c, and bleomycin (0.639) and actinomycin-D (0.621) to 1g. However, these correlation coefficients were limited to the range 0.50e0.75, despite the potent cytotoxicities of 1, 1c and 1g. This indicates that, although these cyclic depsipeptides may have a similar mechanism of action to these anticancer drugs, they may instead have a novel biological mechanism, different from conventional anticancer drugs. 3. Conclusion We synthesized several new kulokekahilide-2 analogues and investigated their conformational preferences and cytotoxicity compared to kulokekahilide-2. The key position for intramolecular ester exchange of kulokekahilide-2 (1) was C-43, which locked 1 into a single conformation in polar solvents. This phenomenon influenced the cytotoxicity. In addition, the structureeactivity relationships (SARs) indicated that the cyclic structure of 1 is critical for its cytotoxicity. Furthermore, protection of the 5-OH of Dtda in 1 had little influence on the cytotoxicity. Moreover, preliminary biological evaluation revealed that 1 showed potent cytotoxicity against 39 human cancer cell lines, exhibiting differential growth inhibition; thus, 1 may have a different mechanism of action than standard anticancer drugs, the aurilides, palau’amide and the lagunamides. 4. Experimental section 4.1. General procedures NMR spectra were measured on a JEOL JNM-ECP500 spectrometer, and recorded at 500 MHz for 1H and 126 MHz for 13C. Chemical shifts are reported in d parts per million calibrated using residual undeuterated solvent as an internal reference and J values are given in hertz (Hz). Mass spectra were measured on a JEOL MS700 mass spectrometer. NBA (m-nitrobenzyl alcohol) was used as a matrix for the FABMS measurement. Optical rotations were measured on a Perkin Elmer-340 digital spectropolarimeter. IR spectra are reported in wavenumbers (cm1) and were measured on a HORIBA FTIR-720 infrared spectrometer. 4.2. Spectral data of new compounds 4.2.1. Compound 5g. Characteristic signals of two conformations are shown, but some protons could not be determined due to overlap. [a]20 D þ9.9 (c 0.34, CHCl3); IR (KBr disk) 3309, 2962, 1735, 1647, 1535, 1458, 1284, 1049, 748 cm1; (major rotamer); 1H NMR (500 MHz, CDCl3): d¼0.75 (d, 3H, J¼7.1 Hz), 0.76 (d, 3H, J¼7.8 Hz), 0.86 (d, 3H, J¼6.4 Hz), 0.91 (d, 3H, J¼6.9 Hz), 0.92 (d, 3H, J¼7.3 Hz), 1.37 (d, 3H, J¼6.9 Hz), 1.57 (br d, 3H, J¼6.6 Hz), 1.53 (br s, 3H), 1.74 (m, 1H), 1.79e1.87 (overlapping, 2H), 1.82 (m, 1H), 1.87 (br s, 3H), 2.05 (s, 3H), 2.16 (m, 1H), 2.89 (s, 3H), 2.92 (s, 3H), 3.05e3.13 (overlapping, 2H), 3.11 (s, 3H), 3.44 (d, 1H, J¼17.4 Hz), 4.09 (d, 1H, J¼17.4 Hz), 4.54 (s, 2H), 4.69e4.72 (overlapping, 2H), 4.76 (dd, 1H, J¼9.2, 8.5 Hz), 5.03 (d, 1H, J¼10.1 Hz), 5.52e5.56 (overlapping, 1H), 5.55 (dd, 1H, J¼10.5, 5.7 Hz), 6.45 (d, 1H, J¼8.5 Hz), 7.13e7.19 (overlapping, 5H), 7.46 (d, 1H, J¼9.2 Hz); 13C NMR (126 MHz, CDCl3): dc¼10.9 (q), 11.1 (q), 11.1 (q), 12.4 (q), 13.0 (q), 13.5 (q), 13.7 (q), 15.1 (q), 17.0 (q), 21.4 (q), 23.1 (q), 29.9 (q), 34.8 (t), 35.3 (q), 40.7 (t), 44.2 (d), 51.3 (t), 53.2 (d), 53.7 (d), 72.9 (d), 73.8 (t), 81.3 (d), 125.7 (d), 126.5 (d), 127.5 (s), 128.0 (d, 2C), 129.3 (d, 2C), 132.1 (s), 136.6 (s), 143.4 (d), 168.2 (s), 169.6 (s), 171.5 (s), 172.7 (s); (minor rotamer); 1H NMR (500 MHz, CDCl3): d¼1.81 (br s, 3H), 2.11 (s, 3H), 5.02 (d, 1H, J¼10.1 Hz), 5.47 (dd, 1H, J¼10.8, 5.3 Hz), 7.13e7.19

3051

(overlapping, 5H); 13C NMR (126 MHz, CDCl3): dc¼12.5 (q), 13.9 (q), 127.5 (s), 140.9 (d); HR-FABMS: [MþNa]þ m/z 922.4936 (m/z 922.4976 calcd for C47H73N5O10SNa). 4.2.2. Compound 1g. Characteristic signals of two conformations are shown, but some protons could not be determined due to overlap. [a]20 D þ7.5 (c 0.50, MeOH); IR (KBr disk) 3317, 2962, 1732, 1647, 1535, 1458, 1284, 1095, 744 cm1; (major rotamer); 1H NMR (500 MHz, CD2Cl2): d¼0.75 (d, 3H, J¼7.0 Hz), 0.86 (dd, 3H, J¼7.4, 7.4 Hz), 0.89 (d, 3H, J¼7.4 Hz), 0.91 (d, 3H, J¼6.6 Hz), 0.94 (d, 3H, J¼6.8 Hz), 1.04 (m, 1H), 1.08 (d, 3H, J¼6.6 Hz), 1.34 (d, 3H, J¼7.4 Hz), 1.50 (m, 1H), 1.62 (d, 3H, J¼6.1 Hz), 1.62 (s, 3H), 1.67 (m, 1H), 1.70 (m, 1H), 1.79 (s, 3H), 1.80 (m, 1H), 1.94 (m, 1H), 2.10 (m, 1H), 2.10 (m, 1H), 2.10 (m, 1H), 2.63 (s, 3H), 3.01 (s, 3H), 3.03 (m, 3H), 3.04 (s, 3H), 3.15 (m, 1H), 3.61 (m, 1H), 3.86 (br d, 1H, J¼15.1 Hz), 4.03 (br d, 1H, J¼15.1 Hz), 4.57 (br t, 1H, J¼8.0 Hz), 4.84 (d, 1H, J¼11.0 Hz), 4.84 (dq, 1H, J¼9.0, 6.8 Hz, H-21), 4.85 (dd, 1H, J¼6.5, 2.5 Hz), 5.23 (q, 1H, J¼7.4 Hz), 5.46 (dd, 1H, J¼10.0, 5.7 Hz), 5.60 (bqd, 1H, J¼7.3, 1.8 Hz), 6.86e6.91 (overlapping, 1H), 6.89 (d, 1H, J¼7.8 Hz), 7.15e7.30 (overlapping, 5H), 7.47 (d, 1H, J¼9.0 Hz); 13C NMR (126 MHz, CD2Cl2): dc¼9.5 (q), 11.2 (q), 11.3 (q), 12.5 (q), 13.2 (q), 14.7 (q), 15.7 (q), 17.4 (q), 22.0 (q), 23.3 (q), 25.0 (t), 25.0 (t), 30.1 (t), 31.5 (q), 31.9 (q), 35.5 (t), 36.2 (d), 36.5 (q), 41.0 (t), 41.4 (d), 45.3 (d), 52.4 (t), 52.4 (d), 54.2 (d), 54.5 (d), 69.1 (d), 73.4 (d), 83.6 (d), 126.4 (d), 127.3 (d), 127.7 (s), 128.7 (d, 2C), 129.8 (d, 2C), 132.9 (s), 136.8 (d), 142.3 (d), 167.2 (s), 169.2 (s), 170.7 (s), 171.2 (s), 173.0 (s), 173.1 (s), 173.4 (s); (minor rotamer); 1H NMR (500 MHz, CD2Cl2): d¼0.69 (d, 3H, J¼7.0 Hz), 0.80 (d, 3H, J¼7.0 Hz), 0.91 (d, 3H, J¼7.3 Hz), 0.94 (d, 3H, J¼6.5 Hz), 0.94 (d, 3H, J¼6.5 Hz), 0.95 (dd, 3H, J¼7.4 Hz), 1.36 (m, 1H), 1.36 (m, 1H), 1.39 (d, 3H, J¼7.4 Hz), 1.53 (s, 3H), 1.57 (m, 1H), 1.61 (d, 3H, J¼6.1 Hz), 1.76 (m, 1H), 1.87 (m, 1H), 1.87 (s, 3H), 1.88 (m, 1H), 1.93 (m, 1H), 2.20 (m, 1H), 2.36 (ddd, 1H, J¼16.8, 8.8, 4.4 Hz), 2.90 (s, 3H), 2.96 (s, 3H), 3.08 (m, 1H), 3.14 (s, 3H), 3.15 (dd, 1H, J¼14.5, 8.6 Hz), 3.33 (d, 1H, J¼17.7 Hz), 3.66 (m, 1H), 4.06 (d, 1H, J¼17.7 Hz), 4.61 (br t, 1H, J¼7.3 Hz), 4.84 (q, 1H, J¼7.4 Hz), 4.94 (dd, 1H, J¼9.1, 8.3 Hz), 4.96 (d, 1H, J¼10.3 Hz), 5.11 (dd, 1H, J¼8.9, 3.3 Hz), 5.29 (br t, 1H, J¼7.7 Hz), 5.56 (bqd, 1H, J¼6.7, 1.1 Hz), 6.42 (d, 1H, J¼8.1 Hz), 6.98 (bdd, 1H, J¼7.7, 5.7 Hz), 7.15e7.30 (overlapping, 5H), 7.47 (d, 1H, J¼9.0 Hz); 13C NMR (126 MHz, CD2Cl2): dc¼11.0 (q), 11.4 (q), 11.7 (q), 12.7 (q), 13.2 (q), 14.4 (q), 15.4 (q), 16.7 (q), 21.9 (q), 23.3 (q), 24.7 (t), 25.0 (d), 30.3 (q), 32.2 (t), 32.7 (q), 35.3 (t), 36.4 (q), 38.1 (d), 40.0 (d), 40.7 (t), 45.0 (d), 51.5 (t), 53.5 (d), 54.3 (q), 56.5 (d), 72.7 (d), 72.8 (d), 83.6 (d), 126.1 (d), 126.8 (d), 128.4 (d, 2C), 129.2 (s), 129.9 (d, 2C), 132.8 (s), 137.5 (s), 142.9 (d), 168.7 (s), 169.7 (s), 170.1 (s), 170.5 (s), 171.0 (s), 172.8 (s), 173.9 (s); HR-FABMS: [MþNa]þ m/z 862.4949 (m/z 862.4942 calcd for C45H69N5O10Na). Ratio of each isomer is 1.1:1. 4.2.3. Compound 5h. Characteristic signals of two conformations are shown, but some protons could not be determined due to overlap. [a]20 D þ8.2 (c 0.49, CHCl3); IR (KBr disk) 3359, 2962, 1712 (sh), 1670, 1520, 1454, 1053, 744 cm1; 1H NMR (500 MHz, CDCl3): d¼0.66 (d, 3H, J¼7.3 Hz), 0.75 (d, 3H, J¼6.8 Hz), 0.90 (d, 3H, J¼6.8 Hz), 0.91 (d, 3H, J¼6.4 Hz), 1.12 (d, 3H, J¼6.9 Hz), 1.45e1.47 (overlapping, 2H), 1.89 (br s, 3H), 2.11 (s, 3H), 2.14 (m, 1H), 2.14 (m, 1H), 2.55 (m, 1H), 2.79 (s, 3H), 2.90e2.94 (overlapping, 2H), 2.92 (d, 1H, J¼17.3 Hz), 2.98 (m, 1H), 3.07e3.14 (overlapping, 1H), 3.18e3.23 (overlapping, 1H), 3.51 (s, 3H), 3.98 (d, 1H, J¼17.3 Hz), 4.32 (dd, 1H, J¼9.4, 5.3 Hz), 4.33e4.36 (overlapping, 2H), 4.86 (d, 1H, J¼11.5 Hz), 4.94 (bdt, 1H, J¼9.4, 6.8 Hz), 5.20 (dq, 1H, J¼8.6, 6.9 Hz), 5.20e5.24 (overlapping, 1H), 5.28 (dd, 1H, J¼10.9, 2.6 Hz), 5.39 (dd, 1H, J¼8.8, 7.2 Hz), 6.63 (ddd, 1H, J¼11.4, 4.5, 1.4 Hz), 7.00 (d, 1H, J¼9.4 Hz), 7.14e7.30 (overlapping, 10H), 7.76 (d, 1H, J¼9.4 Hz, C-43eNH), 8.00 (d, 1H, J¼8.6 Hz); 13C NMR (126 MHz, CDCl3): dc¼9.7 (q), 9.7 (q), 11.5 (q), 12.9 (q), 13.0 (q), 14.1 (q), 15.6 (q), 16.6 (q), 21.2 (q), 23.2 (q), 25.0 (t), 30.6 (t), 32.2 (q), 33.9 (q), 35.9 (t), 37.5 (t), 37.6 (d), 41.5 (t), 43.9

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M. Umehara et al. / Tetrahedron 69 (2013) 3045e3053

(d), 53.3 (t), 53.3 (d), 54.5 (d), 58.9 (d), 73.3 (d), 73.6 (t), 75.3 (d), 82.3 (d), 126.7 (d), 127.5 (s), 131.7 (s), 135.5 (s), 135.9 (s), 139.3 (d), 166.5 (s), 167.4 (s), 170.5 (s), 170.9 (s), 171.8 (C-42), 172.1 (C-23), 174.8 (C-20); (minor rotamer); 1H NMR (500 MHz, CDCl3): d¼0.68 (d, 3H, J¼7.4 Hz), 0.78 (d, 3H, J¼6.8 Hz), 0.78 (t, 3H, J¼6.8 Hz), 0.89 (d, 3H, J¼6.8 Hz), 0.92 (d, 3H, J¼6.9 Hz), 1.20 (d, 3H, J¼7.1 Hz), 1.45e1.47 (overlapping, 6H), 1.91 (br s, 3H), 2.11 (s, 3H), 2.18 (m, 1H), 2.18 (m, 1H), 2.39 (s, 3H), 2.59 (m, 1H), 2.80 (d, 1H, J¼15.4 Hz), 2.97 (dd, 1H, J¼13.7, 6.5 Hz), 3.07e3.14 (overlapping, 2H), 3.09 (m, 1H), 3.18e3.23 (overlapping, 1H), 3.41 (s, 3H), 4.28 (dd, 1H, J¼8.9, 7.5 Hz), 4.33e4.36 (overlapping, 2H), 4.71 (d, 1H, J¼15.4 Hz), 4.82e4.87 (overlapping, 1H), 4.83 (d, 1H, J¼11.3 Hz), 4.89 (ddd, 1H, J¼8.6, 7.1, 6.5 Hz), 5.09 (dq, 1H, J¼7.9, 7.1 Hz), 5.18 (m,1H), 5.20e5.24 (overlapping, 1H), 6.67 (ddd, 1H, J¼11.6, 4.4, 1.2 Hz), 6.73 (d, 1H, J¼9.0 Hz), 7.14e7.30 (overlapping, 10H), 7.34 (d, 1H, J¼8.6 Hz), 8.11 (d, 1H, J¼7.9 Hz); 13C NMR (126 MHz, CDCl3): dc¼9.6 (q), 9.9 (q), 11.1 (q), 12.9 (q), 13.0 (q), 14.1 (q), 15.0 (q), 15.9 (q), 21.1 (q), 23.2 (q), 25.1 (t), 30.6 (t), 32.0 (q), 35.8 (t), 35.9 (q), 37.8 (d), 38.7 (t), 41.5 (t), 44.8 (d), 52.5 (t), 53.3 (d), 56.4 (d), 58.5 (d), 73.5 (t), 73.8 (d), 75.5 (d), 82.4 (d), 126.7 (d), 127.4 (s), 131.5 (s), 135.2 (d), 136.2 (s), 139.6 (d), 166.6 (s), 168.6 (s), 170.5 (s), 171.3 (s), 172.2 (s), 173.9 (s), 175.3 (s); HR-FABMS: [MþNa]þ m/z 984.5123 (m/z 984.5132 calcd for C52H75N5O10SNa). Ratio of each isomer is 1.1:1. 4.2.4. Mixture of compounds 1h and 2h. Characteristic signals of two conformations are shown, but some protons could not be determined due to overlap. [a]20 D 55.5 (c 0.84, MeOH); IR (KBr disk) 3352, 2965, 1716 (sh), 1643, 1518, 1454, 1277, 1119, 744 cm1; 1 H NMR (500 MHz, CD2Cl2): d¼0.71 (d, 3H, J¼7.1 Hz), 0.71 (d, 3H, J¼7.1 Hz), 0.88 (d, 3H, J¼6.4 Hz), 1.20 (d, 3H, J¼6.9 Hz), 1.22 (d, 3H, J¼7.0 Hz), 1.36 (br s, 3H), 1.40 (d, 3H, J¼6.8 Hz), 1.41 (br s, 3H), 1.42 (d, 3H, J¼7.4 Hz), 1.56 (br d, 3H, J¼7.1 Hz), 1.57 (br d, 3H, J¼7.0 Hz), 1.61 (br s, 3H), 1.61 (br s, 3H), 1.62 (br d, 3H, J¼6.8 Hz), 1.88 (s, 3H), 1.88 (s, 3H), 1.88 (s, 3H), 1.90 (s, 3H), 1.93e1.98 (overlapping, 4H), 2.19 (s, 3H), 2.60 (s, 3H), 2.71 (s, 3H), 2.75 (d, 1H, J¼15.4 Hz), 2.76 (s, 3H), 2.81 (d, 1H, J¼14.5 Hz), 2.82 (br d, 1H, J¼17.2 Hz), 2.91e3.32 (overlapping, 16H), 2.92 (d, 1H, J¼15.7 Hz), 3.28 (s, 3H), 3.32 (s, 3H), 3.34 (m, 1H), 3.38 (s, 3H), 3.46 (m, 1H), 3.52 (s, 3H), 3.80 (d, 1H, J¼17.2 Hz), 3.84 (d, 1H, J¼9.2 Hz), 3.85 (d, 1H, J¼8.9 Hz), 4.00 (dd, 1H, J¼8.6, 7.8 Hz), 4.07 (d, 1H, J¼14.5 Hz), 4.08e4.12 (overlapping, 2H), 4.20 (dd, 1H, J¼9.1, 6.5 Hz), 4.67 (d, 1H, J¼15.4 Hz), 4.71 (d, 1H, J¼15.7 Hz), 4.92 (d, 1H, J¼11.2 Hz), 4.95 (d, 1H, J¼11.5 Hz), 4.98 (dq, 1H, J¼7.9, 7.4 Hz), 5.07 (dq, 1H, J¼7.4, 6.8 Hz), 5.18 (dd, 1H, J¼8.7, 7.7 Hz), 5.23e5.27 (overlapping, 1H), 5.25 (dd, 1H, J¼9.2, 2.7 Hz), 5.26 (dd, 1H, J¼9.2, 2.1 Hz), 5.23e5.27 (overlapping, 2H), 5.31e5.36 (overlapping, 2H), 5.37 (dd, 1H, J¼9.1, 4.3 Hz), 5.52 (dd, 1H, J¼9.7, 6.3 Hz), 5.58 (dd, 1H, J¼9.9, 6.1 Hz), 6.49 (d, 2H, J¼7.4 Hz), 6.60 (d, 1H, J¼8.8 Hz), 6.70 (d, 1H, J¼8.8 Hz), 6.73e6.79 (overlapping, 2H), 6.82 (bdd, 1H, J¼11.6, 4.1 Hz), 6.87 (d, 1H, J¼8.6 Hz), 6.92 (bdd, 1H, J¼10.9, 3.8 Hz), 7.18e7.34 (overlapping, 40H); 13C NMR (126 MHz, CD2Cl2): dc¼10.0 (q), 10.3 (q), 10.6 (q), 10.7 (q), 12.8 (q), 12.8 (q), 13.2 (q), 13.2 (q), 16.4 (q), 16.9 (q), 17.4 (q), 17.5 (q), 21.7 (q), 21.7 (q), 21.8 (q), 21.8 (q), 23.1 (q), 23.2 (q), 23.4 (q), 23.4 (q), 32.1 (q, 2C), 32.4 (q, 2C), 33.5 (q), 34.2 (q), 35.7 (t), 35.9 (t), 35.9 (t), 36.0 (q), 36.5 (t), 36.6 (t), 36.6 (q), 36.8 (t), 37.7 (t), 38.0 (t), 40.4 (d), 40.6 (d), 40.7 (d, 2C), 41.2 (t), 41.5 (t), 41.7 (t), 41.8 (t), 44.6 (d), 45.6 (d, 2C), 45.7 (d), 53.0 (t), 53.0 (t), 53.4 (t), 53.6 (t), 53.8 (d), 54.3 (d), 56.0 (d), 56.7 (d), 58.4 (d), 58.5 (d), 58.8 (d), 59.1 (d), 68.8 (d), 68.9 (d), 72.1 (d), 72.3 (d), 73.1 (d), 73.2 (d), 75.7 (d), 76.1 (d), 80.6 (d), 80.6 (d), 82.6 (d), 82.9 (d), 123.3 (d), 123.7 (d), 132.1 (s), 132.2 (s), 136.2 (s), 136.2 (s), 136.2 (s), 136.3 (s), 136.4 (s), 136.5 (s), 136.9 (s, 2C), 137.0 (s), 137.0 (s), 139.7 (d), 139.9 (d), 140.4 (d), 141.1 (d), 166.2 (s), 166.2 (s), 166.5 (s), 166.7 (s), 167.5 (s), 168.1 (s), 168.6 (s), 169.2 (s), 170.4 (s), 170.5 (s), 170.6 (s), 170.6 (s), 170.7 (s), 170.9 (s), 171.0 (s), 171.2 (s), 171.2 (s), 171.7 (s), 172.0 (s), 172.5 (s), 173.4 (s), 173.5 (s), 173.7 (s), 174.1 (s), 174.8 (s),

175.1 (s), 175.3 (s), 175.5 (s); HR-FABMS: [MþNa]þ m/z 924.5128 (m/z 924.5099 calcd for C50H71N5O10Na). 4.2.5. Compound 1i. Some protons could not be determined due to overlap. [a]20 D þ7.0 (c 0.50, MeOH); IR (KBr disk) 3298, 2924, 1643, 1535, 1458, 1126, 744 cm1; 1H NMR (500 MHz, CDCl3): d¼0.87 (dd, 3H, J¼9.1, 9.1 Hz), 0.88 (d, 3H, J¼6.6 Hz), 0.94 (d, 3H, J¼6.6 Hz), 0.91 (d, 3H, J¼6.8 Hz), 1.14 (m, 1H), 1.40 (br d, 3H, J¼6.5 Hz), 1.46 (d, 3H, J¼7.4 Hz), 1.51 (m, 1H), 1.60 (m, 1H), 1.74e1.85 (overlapping, 2H), 2.10 (m, 1H), 2.60 (s, 3H), 3.06 (d, 1H, J¼17.4 Hz), 3.07 (dd, 1H, J¼12.9, 6.0 Hz), 3.19 (dd, 1H, J¼12.9, 10.7 Hz), 3.31 (s, 3H), 4.24 (bdq, 1H, J¼7.4, 3.9 Hz), 4.33 (dd, 1H, J¼9.2, 9.1 Hz), 4.95 (d, 1H, J¼17.4 Hz), 5.15e5.19 (overlapping, 2H), 5.28 (dd, 1H, J¼9.7, 3.5 Hz), 6.56 (br d, 1H, J¼3.3 Hz), 7.20e7.33 (overlapping, 7H); 13C NMR (126 MHz, CDCl3): dc¼10.5 (q), 15.3 (q), 16.8 (q), 17.0 (q), 21.3 (q), 23.5 (q), 24.7 (d), 25.0 (t), 32.1 (q), 35.3 (t), 35.8 (q), 35.8 (d), 40.6 (t), 43.8 (d), 50.1 (d), 51.3 (t), 55.8 (d), 56.7 (d), 73.3 (d), 127.4 (d), 128.8 (d, 2C), 129.0 (d, 2C), 135.9 (s), 168.7 (s), 169.1 (s), 170.6 (s), 171.9 (s), 173.1 (s), 174.1 (s); HR-FABMS: [MþH]þ m/z 602.3542 (m/z 602.3554 calcd for C31H48N5O7). Acknowledgements We thank the Screening Committee of Anticancer Drugs, which is supported by a Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology, Japan, for biological evaluation of compounds 1, 1c and 1g. This study was funded by the Research Institute of Aoyama Gakuin. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2013.01.089. References and notes 1. (a) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tuinman, A. A.; Boettner, F. E.; Kizu, H.; Schmidt, J. M.; Baczynskyj, L.; Tomer, K. B.; Bontems, R. J. J. Am. Chem. Soc. 1987, 109, 6883e6885; (b) Bai, R.; Pettit, G. R.; Hamel, E. Biochem. Pharmacol. 1990, 40, 1859e1864; (c) Margolin, K.; Longmate, J.; Synold, T. W.; Gandara, D. R.; Weber, J.; Gonzalez, R.; Johansen, M. J.; Newman, R.; Baratta, T.; Doroshow, J. H. Invest. New Drugs 2001, 19, 335e340. 2. (a) Hamann, M. T.; Otto, C. S.; Scheuer, P. J.; Dunbar, D. C. J. Org. Chem. 1996, 61, 6594e6600; (b) Rao, K. V.; Na, M.; Cook, J. C.; Peng, J.; Matsumoto, R.; Hamann, M. T. J. Nat. Prod. 2008, 71, 772e778; (c) Shilabin, A. G.; Hamann, M. T. Bioorg. Med. Chem. 2011, 19, 6628e6632. 3. (a) Pettit, G. R.; Kamano, Y.; Dufresne, C.; Cerny, R. L.; Herald, C. L.; Schmidt, J. M. J. Org. Chem. 1989, 54, 6005e6006; (b) Bai, R.; Friedman, S. J.; Pettit, G. R.; Hamei, E. Biochem. Pharmacol. 1992, 43, 2637e2645; (c) Akaji, K.; Hayashi, Y.; Kiso, Y.; Kuriyama, N. J. Org. Chem. 1999, 64, 405e411. 4. (a) Tanaka, A. K.; Valero, V. B.; Takahashi, H. K.; Straus, A. H. J. Antimicrob. Chemother. 2007, 59, 487e492; (b) Narayanaswamy, V. K.; Albericio, F.; Coovadia, Y. M.; Kruger, H. G.; Maguire, G. E. M.; Pillay, M.; Govender, T. J. Pept. Sci. 2011, 17, 683e689; (c) Nollmann, F.; Dowling, A.; Kaiser, M.; Deckmann, K.; € sch, S.; Ffrench-Constant, R.; Bode, H. B. Beilstein J. Org. Chem. 2012, 8, Gro € ttel, S.; Sasse, F.; 528e533; (d) Plaza, A.; Garcia, R.; Bifulco, G.; Martinez, J. P.; Hu € ller, R. Org. Lett. 2012, 14, 2854e2857. Meyerhans, A.; Stadler, M.; Mu 5. (a) Nakao, Y.; Yoshida, W. Y.; Takada, Y.; Kimura, J.; Yang, L.; Susan, L. M.; Scheuer, P. J. J. Nat. Prod. 2004, 67, 1332e1340; (b) Takada, Y.; Umehara, M.; Katsumata, R.; Nakao, Y.; Kimura, J. Tetrahedron 2012, 68, 659e669. 6. (a) Suenaga, K.; Mutou, T.; Shibata, T.; Itoh, T.; Kigoshi, H.; Yamada, K. Tetrahedron Lett. 1996, 37, 6771e6774; (b) Mutou, T.; Suenaga, K.; Fujita, T.; Itoh, T.; Takada, N.; Hayamizu, K.; Kigoshi, H.; Yamada, K. Synlett 1997, 199e201; (c) Takahashi, T.; Nagamiya, H.; Doi, T.; Griffith, P. G.; Bray, A. M. J. Comb. Chem. 2003, 5, 414e428; (d) Suenaga, K.; Mutou, T.; Shibata, T.; Itoh, T.; Fujita, T.; Takada, N.; Hayamizu, K.; Takagi, M.; Irifune, T.; Kigoshi, H.; Yamada, K. Tetrahedron 2004, 60, 8509e8527; (e) Suenaga, K.; Kajiwara, S.; Kuribayashi, S.; Handa, T.; Kigoshi, H. Bioorg. Med. Chem. Lett. 2008, 18, 3902e3905. 7. (a) Tripathi, A.; Puddick, J.; Prinsep, M. R.; Rottmann, M.; Tan, L. T. J. Nat. Prod. 2010, 73, 1810e1814; (b) Tripathi, A.; Puddick, J.; Prinsep, M. R.; Rottmann, M.; Chan, K. P.; Chen, D. Y.; Tan, L. T. Phytochemistry 2011, 72, 2369e2375; (c) Tripathi, A.; Fang, W.; Leong, D. T.; Tan, L. T. Marine Drugs 2012, 10, 1126e1137; (d) Dai, L.; Chen, B.; Lei, H.; Wang, Z.; Liu, Y.; Xu, Z.; Ye, T. Chem. Commun. 2012, 8697e8699.

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