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Conformation-Based Design and Synthesis of Apratoxin A Mimetics Modified at the α,β-Unsaturated Thiazoline Moiety Yuichi Onda,†,‡ Yuichi Masuda,†,§ Masahito Yoshida,† and Takayuki Doi*,† †

Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan Mitsubishi Tanabe Pharma Corporation, 2-2-50, Kawagishi, Toda-shi, Saitama 335-8505, Japan

‡

S Supporting Information *

ABSTRACT: We have demonstrated design, synthesis, and biological evaluation of apratoxin A mimetics. In the first generation, the moCys moiety was replaced with seven simple amino acids as their 3D structures can be similar to that of apratoxin A. Apratoxins M1−M7 were synthesized using solidphase peptide synthesis and solution-phase macrolactamization. Apratoxin M7, which contains a piperidinecarboxylic acid moiety, exhibited potent cytotoxicity against HCT-116 cells. In the second generation, substitution of each amino acid residue in the tripeptide Tyr(Me)−MeAla−MeIle moiety in apratoxin M7 led to the development of the highly potent apratoxin M16 possessing biphenylalanine (Bph) instead of Tyr(Me), which exhibited an IC50 value of 1.1 nM against HCT-116 cells. Moreover, compared to apratoxin A, apratoxin M16 exhibited a similarly high level of growth inhibitory activity against various cancer cell lines. The results indicate that apratoxin M16 could be a potential candidate as an anticancer agent.

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INTRODUCTION Apratoxin A (1a) was isolated from the marine cyanobacterium Lyngbya majuscula by Luesch et al. in 2001 (Figure 1).1 It is a 25-membered cyclodepsipeptide comprising a proline (Pro); three methylated amino acids [N-methylisoleucine (MeIle), N-methylalanine (MeAla), and O-methyltyrosine (Tyr(Me))]; an α,β-unsaturated modified cysteine residue (moCys); and 3,7-dihydroxy-2,5,8,8-tetramethylnonanoic acid (Dtena). Apratoxin A has been found to exhibit potent cytotoxicity against various cancer cell lines in vitro.1,2 Luesch et al. reported that apratoxin A prevents cotranslational translocation in the early stages of secretory pathways to downregulate both the levels of growth factors and their receptors, including the vascular endothelial growth factor (VEGF) and its receptor (VEGF-R).3 Recently, it was demonstrated that apratoxin A prevents protein translocation by directly targeting Sec61α, the central subunit of the protein translocation channel.4,5 In addition, Shen et al. reported that the apratoxin A oxazoline analogue 2 promotes degradation of Hsp90 client proteins via chaperone-mediated autophagy.6 Notably, 1a has been found to exhibit some antitumor efficacy in vivo but also to be lethally toxic against mice at its therapeutic concentration (0.25 mg/kg a day).1,2c,g,5 However, Luesch’s structure−activity relationship (SAR) studies on 1a led to the development of potent anticancer analogues 3 with lower toxicity, thereby implying the possibility of separating its anticancer activity from its toxicity.2g,7 Total syntheses of apratoxin A (1a) and its analogues have been reported previously.2a,b,g,7,8 Forsyth et al. accomplished the first total synthesis of 1a,8a,b and several groups including the present authors have since reported the synthesis and SAR © 2017 American Chemical Society

studies of apratoxins and their analogues using solutionphase2a,g,7,8 or solid-phase syntheses.2b,8f However, the syntheses of apratoxins and their analogues require multistep processes and result in very low overall yields. In addition, apratoxins are sensitive to acidic conditions8e,9 and have a Michael acceptor in the moCys moiety.7 Consequently, in order to provide new anticancer agents and molecules for mechanism studies, apratoxin A mimetics with better accessibility and stability must be developed. Luesch et al. developed the apratoxin A/E hybrid analogues 3,2g,7 containing a hydroxy group, similar to 1a, and lacking a Michael acceptor, similarly to apratoxin E (1d).2f,h The analogues 3 exhibited greater anticancer potency than 1a in vivo without toxicities potentially caused by irreversible reaction, indicating that the toxicity may be an off-target effect due to the presence of the Michael acceptor in the molecule, which might be prone to nonspecific additions of cellular nucleophiles. We recently reported on the 3D structures of 1a and apratoxin C (1c), which were obtained from NMR experiments in CD3CN, avoiding dehydration of the C-35 OH group in apratoxin A.8i The distance geometry method was used along with rotating-frame Overhauser effect (ROE) and J-coupling data to establish the structural models. The 3D structure of 1a observed in CD3CN was in good accordance with that observed in CDCl3 previously reported.1 The structures of 1a and 1c were found to be very similar, largely because the substitution patterns in their dihydroxylated fatty Received: June 8, 2017 Published: July 6, 2017 6751

DOI: 10.1021/acs.jmedchem.7b00833 J. Med. Chem. 2017, 60, 6751−6765

Journal of Medicinal Chemistry

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Figure 1. Structures of various apratoxin derivatives.

acid moieties (Dtena and Dtrina) constrain their conformations. In addition, we also reported that the oxazoline analogue of apratoxin C (2b) adopts a similar conformation to that of 1c in CD3CN.8l These similarly constrained structures offer a probable explanation for their comparable cytotoxicities. Hence, apratoxin A mimetics should be designed to maintain a conformation similar to that of 1a. The purpose of this study is to develop new apratoxin A mimetics with better accessibility and stability. Our conformation-based design and synthesis of the mimetics allowed significant alteration of the structure without decreasing potency.

However, it still remains unclear which amino acid residue is important for the potent biological activity because a detailed SAR study on the tripeptide Tyr(Me)−MeAla−MeIle moiety has not been conducted. It is known that the moCys moiety can be replaced by other structural motifs because the apratoxin A oxazoline analogue (2a)2a,b is highly cytotoxic, and the apratoxin A/E hybrid analogues 3 possess improved antitumor activity and in vivo tolerability compared to apratoxin A (1a) (Figure 1).2g,7 Thus, the moCys moiety should be replaced with another structural motif because nonspecific Michael additions of cellular nucleophiles to the α,β-unsaturated amide moiety may cause its cytotoxicity.7 Moreover, the α-position of the thiazoline ring is readily epimerized under mildly acidic and basic conditions, and its preparation is one of the most challenging processes in the synthesis of apratoxins. For these reasons, we planned to replace the moCys moiety with more stable and easily accessible amino acid linkers (Figure 2c). The fact that 2a exhibits similar cytotoxicity to that of 1a indicates that the sulfur atom may be substituted with an oxygen atom. An amide bond was chosen instead of the thiazoline ring in order to retain the sp2 character of the thioimidate moiety, being readily prepared and stable under various chemical and physiological conditions. In the 3D structural model of 1a (Figure 2b), a nearly rectangular bend structure is adopted at the thiazoline ring, which could play a critical role in constraining its conformation. To retain this conformational feature, we placed an sp3 carbon adjacent to the nitrogen atom in the amino acid linkers, which allows a bended structure similar to that seen in 1a.

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RESULTS AND DISCUSSION Design of the First-Generation Mimetics: Modification of the moCys Moiety. The structure of apratoxin A (1a) can be divided into three parts in terms of structural features and activity (Figure 2a). It has been reported that the cytotoxicity of apratoxins disappears upon dehydration at C34 and C35 like (E)-dehydroapratoxin A (1e)2f,9 and triethylsilyl (TES) group protection of the hydroxyl group at C35.2b In addition, our structural analyses of 1a and 1c indicated that steric repulsion between the substituents in their fatty acid regions are essential for maintaining their conformations (Figure 2b).8i These data strongly suggest that modification of the Dtena region would result in loss of cytotoxicity. Several alterations of the amino acid residues in the tetrapeptide moiety have also been reported.2b,c,g For example, replacement of either Tyr(Me) with Ala or MeIle with MeAla, significantly reduced the cytotoxicity,2g whereas substitution of Pro with MeAla retained the activity.2c We have also reported that replacement of Tyr(Me) with Tyr(7-azidoheptyl) increased the cytotoxicity.2b 6752



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Synthesis of the First-Generation Mimetics. Apratoxins M1−M7 (4a−4g) were synthesized by solid-phase peptide synthesis followed by macrolactamization in solution, similar to our previously reported synthesis of apratoxin A (1a) (Scheme 1).2b Fmoc−MeIle-supported SynPhase trityl-lanterns 8 (24.5 μmol/lantern)10,2b were treated with 20% piperidine/ DMF. The resulting amine was acylated with Fmoc−MeAla−OH using bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP)11 and N,N-diisopropylethylamine (DIEA) to afford polymer-supported dipeptide 9. After removal of the Fmoc group, condensation with Fmoc−Tyr(Me)−OH was repeatedly performed using PyBroP and DIEA to afford the polymersupported tripeptide 10. After removal of the Fmoc group from 10, condensation with amino acids M1−M7 proceeded smoothly in parallel using 1,3-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt), affording the polymersupported tetrapeptides 11a−11g, respectively. Removal of the Fmoc groups of 11a−11g, followed by coupling with Fmoc− Pro−Dtena (6) prepared by cleavage of the 1,1,1-trichloroethyloxycarbonyl (Troc) group in 7,2b utilizing N-[1-(cyano-2ethoxy-2-oxoethylideneaminooxy)dimethylamino(morpholino)]uronium hexafluorophosphate (COMU)12 and DIEA, afforded the polymer-supported pentapeptides 12a−12g. Finally, cleavage from the polymer support with 30% 1,1,1,3,3,3hexafluoroisopropyl alcohol (HFIP)13 in CH2Cl2 afforded the cyclization precursors 5a−5g in quantitative crude yield with >80% purity (UV 254 nm). Without purification, macrolactamization was successfully performed at 1 mM concentration of the substrate using HATU and DIEA to provide desired 4a−4g in 16−44% total yields from 8. Although we have previously reported that the partial epimerization at the C34 position was observed in the solid-phase total synthesis of 1a,2b no epimerization at the C34 position was observed in the present synthesis of 4a−4g. Thus, the mimetics are more easily accessible than 1a. Cytotoxicity of the First-Generation Mimetics. The cytotoxic activities of apratoxins M1−M7 (4a−4g) against HCT-116 cells were evaluated by a WST assay (Table 1).14 The cytotoxicity of 4b substituted with N-methyl-4-aminobutanoic acid is 8-fold higher than that of 4a substituted with 4-aminobutanoic acid (Table 1, entry 3 vs entry 2), indicating that the NH group in 4a prevents the molecule from adopting a conformation similar to apratoxin A (1a) and interacting with a putative target protein. Conversely, both 4c and 4d, containing 3-aminopropanoic acid and N-methyl-3-aminopropanoic acid, respectively, exhibit very low cytotoxicities (Table 1, entries 4 and 5). Thus, the shorter and less flexible linkers in 4c and 4d are not suitable for retaining cytotoxic activity. 4e substituted with 3-(aminomethyl)benzoic acid shows relatively potent cytotoxicity compared to 4a, 4c, and 4d (entry 6). Moreover, 4f, its N-Me derivative, is slightly more potent than 4e (Table 1, entry 7). Intriguingly, 4g, possessing a 4-piperidinecarboxylic acid linker, exhibits the strongest cytotoxicity (Table 1, entry 8, IC50, 0.12 μM) among the synthetic 4a−4g. Consequently, apratoxin M7 (4g) was chosen as the lead compound for further development of more potent analogues. Conformational Analysis of the First-Generation Mimetics. To investigate the effect of modification on the conformations, the 3D structures of 4a−4g were investigated by NMR studies. We used CD3CN as the NMR solvent to compare the 3D structures of 4a−4g with that of apratoxin A (1a), as we previously reported.8i The 1H and 13C chemical

Figure 2. (a) Apratoxin A (1a) divided into three parts, (b) 3D structure of 1a obtained by distance geometry calculations, (c) modification of the moCys moiety in 1a, and (d) designed structures of apratoxins M1−M7 (4a−4g).

In accordance with the above discussion, we designed the apratoxin A mimetics, apratoxins M1−M7 (4a−4g) modified at the moCys moiety, as illustrated in Figure 2d. To maintain a 25-membered ring structure similar to apratoxin A (1a), 4-aminobutanoic acid was adopted in apratoxin M1 (4a) as a linker between Tyr(Me) and Dtena moieties. In addition, N-methyl-4-aminobutanoic was included in apratoxin M2 (4b) to prevent possible hydrogen bonding formed by the NH group of 4a. We also designed apratoxins M3 (4c) and M4 (4d) substituted with 3-aminopropanoic acid and N-methyl-3aminopropanoic acid, respectively, which have less flexible linkers than those in 4a and 4b. To mimic an α-methyl-α,βunsaturated acid in the moCys moiety, we designed apratoxins M5 (4e) and M6 (4f) substituted with 3-(aminomethyl)benzoic acid and 3-(N-methylaminomethyl)benzoic acid, respectively. Moreover, 4-piperidinecarboxylic acid was adopted in apratoxin M7 (4g) as a less flexible linker to constrain the conformation. 6753



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Scheme 1. Synthesis of Apratoxins M1−M7 (4a−4g) Utilizing Solid-Phase Peptide Synthesis and Macrolactamization in Solution

and the other contains a trans-amide bond. Luesch et al. reported the existence of two conformers with trans- and cis-amide bonds at the same position in (E)-dehydroapratoxin A (1e).9 As shown in Table 2, the chemical shift data for 4a and 1e are similar. These conformations are designated as the transamide and cis-amide conformers, respectively. Previous NMR analysis has indicated that 1a, 1c, and the oxazoline analogue of apratoxin C (2b) mainly adopt the trans-amide conformation in CDCl3 and CD3CN,8i whereas the major conformer of apratoxin B (1b) (Figure 1) in CDCl3 has a cis-amide bond.9 The apparent difference in the chemical shifts between the two conformers can be explained by the magnetic anisotropic effect derived from the carbonyl carbon of the amide.15 Hence, the presence of trans- and/or cis-amide conformers can be

shifts of the above mimetics were assigned unambiguously by a combination of 2D NMR experiments including correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear multiple-quantum correlation (HMQC), heteronuclear multiple-bond correlation spectroscopy (HMBC), and rotating frame nuclear Overhauser effect spectroscopy (ROESY) (see the Supporting Information). First, we analyzed the 3D structure of apratoxin M1 (4a) as a typical example. Two sets of chemical shifts are observed at a ratio of 1:1 for several 1H and 13C nuclei, indicating that 4a exists as two main conformers in solution. It is noteworthy that a strong ROE between H14 and H18 is only observed in one of the conformers (Figure 3a). This indicates that one conformer of 4a contains a cis-amide bond between Tyr(Me) and MeAla, 6754



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Table 1. Cytotoxic Activity of Apratoxin A (1a) and Apratoxins M1−M7 (4a−g) against HCT-116 Cells and the Proportion of trans- and cis-Amide Conformers entry 1 2 3 4 5 6 7 8

compound apratoxin apratoxin apratoxin apratoxin apratoxin apratoxin apratoxin apratoxin

A (1a) M1 (4a) M2 (4b) M3 (4c) M4 (4d) M5 (4e) M6 (4f) M7 (4g)

IC50 (μM)a

ratio of conformersb trans/cis

0.0028 ± 0.0006 2.6 ± 0.3 0.34 ± 0.05 3.4 ± 0.0 3.1 ± 0.1 0.82 ± 0.16 0.22 ± 0.05 0.12 ± 0.02

>9:1 1:1 1.2:1 1:>9 1:>1.5 2:1 2.8:1 1:2

a IC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SD from the dose−response curves of at least three independent experiments. b trans- and cis-amide bond orientation between Tyr(Me) and MeAla. The ratio was calculated by the intensity of the N-Me signals in 1 H NMR spectrum.

identified by the pattern of the C16 N-Me signals presented by the MeAla moiety. Since the 3JH,H values of each conformer were unambiguously determined by 1H−1H J-resolved 2D NMR spectra, the conformations of the Dtena region in 4a were estimated by a J-based configuration analysis (JBCA)16 method and are described by the Newman projections shown in Figure 3b. The conformations of the Dtena region in both the trans- and cis-amide conformers are similar to that in apratoxin (1a). This indicates that modification of the moCys region does not affect the conformation of the Dtena region, which would be constrained mainly by the steric repulsion of the substituents.8i Molecular modeling of 4a was conducted with the MacroModel (version 9.9)17 program using distance geometry calculations, a conformational search using a 20,000-step Monte Carlo-based torsional sampling with distance and dihedral angle constraints derived from the NMR data. We utilized an OPLS-2005 force field and a generalized Born/solventaccessible surface area (GB/SA) solvent model.18 The lowest energy structures of the trans- and cis-amide conformers are shown in Figure 4. The overall conformations of both conformers are similar to that of 1a (Figure 2b). In detail, the trans-amide conformation of 4a resembles the major conformation of 1a (Figure 4A),8i whereas the cis-amide conformation is similar to that of apratoxin B (1b) (Figure 4B).9 The newly introduced NH group of the 4-aminobutanoic acid moiety in 4a may form an intramolecular hydrogen bond to induce a conformational change. Thus, to investigate the existence of the hydrogen bond in 4a, we performed H−D exchange experiments. After adding 10 μL of D2O to a solution of 4a in CD3CN (0.45 mL), time-dependent 1H signals of each NH and OH proton were tracked. The H−D exchange of the NH in the 4-aminobutanoic acid moiety is much slower than those of the OH in Dtena and the NH in Tyr(Me) (Figure S2), suggesting that the NH of the 4-aminobutanoic acid moiety forms a hydrogen bond. It has been reported that the 1 H chemical shifts for OH and NH groups involved in intermolecular hydrogen bonds with solvent molecules are high-field shifted compared to those forming intramolecular hydrogen bonds at higher temperature.19 To assess whether the observed hydrogen bonding is intermolecular or intramolecular, we measured the 1H NMR spectra at different temperatures. The chemical shift changes of the 1H signal indicate that the

Figure 3. NMR analysis of apratoxin M1 (4a). (a) The trans- and cis-amide conformers. Hydrogen bonding is observed for the H atoms highlighted by a solid ellipse. Hydrogen bonding is not observed for the H atoms highlighted by a dashed ellipse. (b) Newman projections. Conformation of the Dtena region in 4a confirmed by the NMR data measured in CD3CN. 3JH,H values are shown below the Newman projections and “Large” and “Small” refer to the magnitude of the 3JH,H values, resulting in the identification of anti or gauche orientations.

NH of the 4-aminobutanoic acid moiety forms an intramolecular hydrogen bond. Next, we analyzed the tertiary structure of apratoxin M2 (4b) in the same manner as used for apratoxin M1 (4a) (Figure S3). The existence of trans- and cis-amide conformers is observed at a ratio of 1.2:1 (Table 1, entry 3). The lowest energy structure obtained by distance geometry calculations indicates that 4b adopts two conformers, similar to 4a, despite the presence of the N-Me group in the 4-aminobutanoic acid moiety (Figure 4C,D). The potent cytotoxicity of 4b compared to that 6755



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Table 2. Comparison of Selected NMR Data for Apratoxin M1 (4a) and (E)-Dehydroapratoxin A (1e) apratoxin M1 (4a) trans-amide conformera δH (J in Hz)

C/H no. 12 14 15 16 18

a

δC

δH (J in Hz)

2.65 (s, 3H) 31.0 3.36 (m, 1H) 61.2 1.05 (d, J = 6.8, 3H) 14.4 2.89 (s, 3H) 37.2 5.01 (m, 1H) n.d.c apratoxin M1 (4a) cis-amide conformera δH (J in Hz)

C/H no. 12 14 15 16 18

(E)-dehydroapratoxin A (1e) trans-amide conformerb

2.82 4.74 0.66 2.55 5.31

2.85 3.37 1.24 2.90 5.07

(s, 3H) 30.8 (q, J = 6.8, 1H) 60.4 (d, J = 6.8, 1H) 13.8 (s, 3H) 36.7 (ddd, J = 11.2, 9.7, 5.6, 1H) 50.6 (E)-dehydroapratoxin A (1e) cis-amide conformerb

δC

(s, 3H) (q, J = 6.3, 1H) (d, J = 6.6, 3H) (s, 3H) (ddd, J = 10.2, 9.2, 5.7, 1H)

31.1 54.9 15.6 29.1 51.0

δC

δH (J in Hz) 2.78 4.74 0.63 2.59 5.34

δC

(s, 3H) (q, J = 6.8, 1H) (d, J = 6.8, 3H) (s, 3H) (ddd, J = 10.6, 9.2, 4.6, 1H)

30.0 53.8 15.3 28.8 50.2

Measured in CD3CN. bMeasured in CDCl3. See ref 9. cNot detected.

Tyr(Me) and MeAla (Figures 3 and 4). The amide conformation would be regulated by the steric repulsion between the N-Me and α-Me groups in the MeAla residue. Thus, to alter the steric effects, we replaced MeAla at AA2 with Sar in 13d, MeLeu in 13e, or MePhe in 13f. (3) Modification of the Tyr(Me) residue of 1a has been reported to have a significant effect on the activity of the molecule, with substitution of Tyr(Me) with Ala significantly reducing the cytotoxicity2g and substituting Tyr(Me) with Tyr(7-azidoheptyl) increasing the cytotoxicity.2b As the substituent at the para-position in this residue seems to be critical for the cytotoxicity, we substituted Tyr(Me) at AA3 with Phe in 13g, (4-chlorophenyl)alanine (Phe(4-Cl)) in 13h, or biphenylalanine (Bph) in 13i. The second-generation apratoxin A mimetics, apratoxins M8−M16 (13a−13i), were synthesized by a process similar to that used for the synthesis of apratoxin M7 (4g) with substitution of each amino acid in the tripeptide moiety in 4g (Figures S9 and S10). Cytotoxicity of the Second-Generation Mimetics. The cytotoxic activities of the synthetic apratoxins M8−M16 (13a−13i) against HCT-116 cells were evaluated by WST assay (Table 3). Among the mimetics modified at AA1, 13a substituted with MeAla and 13c substituted with MePhe exhibit lower cytotoxicity than 4g (Table 3, entries 1 and 3 vs Table 1, entry 8), whereas 13b substituted with MeVal is as potent as 4g (Table 3 entry 2). This indicates that branched amino acids such as MeIle and MeVal are required at the AA1 position for inducing potent activity. This tendency is similar to that observed for apratoxin A (1a). Replacement of MeIle with MeAla significantly decreased the cytotoxicity, whereas replacement with MeVal retained the activity.2g It is conceivable that the cytotoxic mechanism of 4g could be the same as that of 1a. The mimetics 13d, 13e, and 13f with AA2 substituted with Sar, MeLeu, and MePhe, respectively, exhibit decreased cytotoxicity (Table 3, entries 4−6). Thus, the size of the methyl group on the side chain of MeAla appears to be critical to activity. Among the mimetics modified at AA3, the cytotoxicity of 13g substituted with Phe is slightly lower than that of 4g (Table 3, entry 7), while 13h substituted with Phe(4-Cl) is twice as potent as 4g (Table 3, entry 8). Notably, 13i replaced with Bph is 100-fold more potent than 4g and is superior or equipotent to 1a (Table 3, entry 9 vs Table 1, entries 8 and 1).

of 4a may be due to its higher cell permeability derived from its physicochemical properties. The tertiary structures of apratoxins M3−M7 (4c−4g) were also analyzed (see Figures S4−S8). All the designed mimetics adopt rectangular bend structures as expected. It is noteworthy that all the mimetics adopt trans- and/or cis-amide conformers but in different ratios (Table 1). The most cytotoxic apratoxin M7 (4g) also adopts trans- and cis-amide conformations at a ratio of 1:2 (Table 1, entry 8; Figure 4E,F). However, there is no direct relationship between the cytotoxicities and the conformer ratios observed for the apratoxin A mimetics. The lowest energy structures of the trans- and cis-amide conformers are shown in Figure 4E,F, and their superpositions on apratoxin A were shown in Figure 4G,H. Despite the subtle differences, geometries of substituents (e.g., side chains of amino acids, tertbutyl and methyl groups in Dtena region) in apratoxin M7 (4g) were very similar to those of apratoxin A (1a) (Figure 4G,H). This indicates that the piperidine moiety in 4g could mimic the 3D structure of the moCys in 1a. Design and Synthesis of the Second-Generation Mimetics. In order to develop more potent mimetics, we designed several second-generation mimetics based on 4g. The Dtena moiety has been reported to be essential not only for constraining the conformation of the macrocyclic ring7 but also for potent cytotoxicity.2b,f,9 Although the substitution of Pro with MeAla in apratoxin F (1f) retains the cytotoxicity,2c we decided not to modify Pro because replacing it would allow for greater conformational flexibility, which may result in lower membrane permeability.20 Conversely, substitution of the tripeptide Tyr(Me)−MeAla−MeIle moiety may lead to improved activity because its modification has been shown to drastically influence the biological activity of the molecule.2b,g Moreover, substitutions in the tripeptide moiety can be easily performed by using different Fmoc-amino acids in the solidphase synthesis of apratoxin A mimetics. Thus, we modified the tripeptide moiety in 4g. The modification of each residue was rationalized as follows: (1) In the 3D structural model of 4g (Figure 4E,F), the side chain of the MeIle faces inside the macrocyclic ring. We planned to replace the MeIle at AA1 with MeAla in 13a, MeVal in 13b, or MePhe in 13c, expecting to change the size and/or lipophilicity of the molecule (Figure 5). (2) Our NMR analysis of apratoxins M1−M7 (4a−4g) revealed the existence of conformers with trans- and cis-amide bonds between the 6756



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Figure 5. Design of second-generation apratoxins M8−M16 (13a− 13i) by modification of apratoxin M7 (4g).

Table 3. Cytotoxic Activities of Apratoxins M8−M16 (13a−13i) against HCT-116 Cells and the Proportion of trans- and cis-Amide Conformersa entry

compound

IC50 (μM)b

ratio of conformersc trans/cis

1 2 3 4 5 6 7 8 9

apratoxin M8 (13a) apratoxin M9 (13b) apratoxin M10 (13c) apratoxin M11 (13d) apratoxin M12 (13e) apratoxin M13 (13f) apratoxin M14 (13g) apratoxin M15 (13h) apratoxin M16 (13i)

7.5 ± 2.3 0.13 ± 0.05 0.71 ± 0.12 0.85 ± 0.16 0.36 ± 0.08 1.6 ± 0.3 0.49 ± 0.20 0.069 ± 0.012 0.0011 ± 0.0001

d 1:1 1.3:1 1.2:1 1:2.4 d 1:2.3 1:2 1:2

a

The cytotoxicity of apratoxin A and mimetics M1−M16 in Tables 1 and 3 were evaluated using the HCT-116 cells in the same generation. b IC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SD from the dose−response curves of at least three independent experiments. c The trans- and cis-amide bond orientation between Tyr(Me) and MeAla. The ratios were calculated by the intensity of the N-Me signals in the 1H NMR spectra. dNot determined due to the broad spectrum. Figure 4. 3D structures of apratoxins M1 (4a), M2 (4b), and M7 (4g). (A) The trans-amide conformer of 4a. (B) The cis-amide conformer of 4a. (C) The trans-amide conformer of 4b. (D) The cis-amide conformer of 4b. (E) The trans-amide conformer of 4g. (F) The cis-amide conformer of 4g. (G) Superposition of the trans-amide conformer of 4g (structure in green) and apratoxin A (1a) (structure in yellow). (H) Superposition of the cis-amide conformer of 4g (structure in green) and apratoxin A (1a) (structure in yellow).

Conformational Analysis of the Second-Generation Mimetics. To investigate the effect of the tripeptide modification on the conformations of apratoxins M8−M16 (13a−13i), 3D structural analysis was performed using distance geometry calculations as previously described. We used CD3CN as the NMR solvent to compare each 3D structure of 13a−13i to that of 4g. The NMR analyses of 4a−4g indicate the existence of two conformers with trans- and cis-amide bonds between Tyr(Me) and MeAla. As already discussed, these two conformers can be discriminated by comparison of the NMR signals presented by their N-Me groups. Therefore,

These data suggest that a hydrophobic substituent at the paraposition of the aromatic ring at AA3 is important for potent cytotoxicity. 6757



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might be disrupted by the substituents, resulting in loss of cytotoxicity. We compared the 1H NMR spectra of 13g−13i modified at Tyr(Me). The 1H NMR spectra of 13g−13i are similar to that of 4g, suggesting a similarity in their conformations (Figure 6h−j vs Figure 6a). In the 3D structural models of 1a and 4g, both side chains of the Tyr(Me) residues face outside the macrocyclic structure. We analyzed the 3D structure of 13i by distance geometry calculations in the same way as 4g (Figure S12). The 3D structure of 13i is similar to that of 4g (Figure 7). The substituent at the para-position of the aromatic

Figure 7. Superposition of the lowest energy trans-amide conformers (left) and cis-amide conformers (right) of apratoxin M7 (4g) (structure in yellow) and apratoxin M16 (13i) (structure in green) obtained by distance geometry calculations using NMR data.

ring at AA3 does not affect their conformations. Thus, the differences in the cytotoxicities between 4g and 13g−13i may arise from a difference in their interactions with a putative target protein or their physicochemical properties, such as solubility and cell membrane permeability. Since the side chain of AA3 faces outside of the cyclic frames in both the cis and trans conformers, the AA3 residue might contact with a putative target protein. This might be a reason why replacement of the AA3 dramatically affect the cytotoxicities. In summary, the mimetics showing broad or different 1 H NMR spectra tend to be less active than 4g, suggesting that the observed conformations of 4g (Figure 4) are closely related to the structure that induces cytotoxicity. However, the ratio of trans- and cis-amide conformers does not correlate with the cytotoxicity among the apratoxin mimetics. The side chains of MeIle and MeAla at AA1 and AA2, respectively, play important roles in constraining the conformations of the macrocyclic rings, whereas the side chain of Tyr(Me) at AA3 does not affect the conformation but the cytotoxic activity. Growth Inhibition Assay of Apratoxins M15 and M16 against Various Cancer Cell Lines. The growth inhibition activities of apratoxins A (1), M15 (13h), and M16 (13i) against 10 selected human cancer cell lines (HCT-116, BxPC-3, A549, HuH-7, MKN74, U-87 MG, SK-OV-3, HEC-6, 786-O, and MCF7) were evaluated by CellTiter-Glo2.0 assay after 72 h treatments and their GI50 values are depicted in Table 4. Interestingly, compared to apratoxin A, we found apratoxin M16 exhibited similarly a high level of potency against 8 of 10 cancer cells, such as HCT-116, BxPC-3, A549, HuH-7, MKN74, U-87 MG, SK-OV-3, and HEC-6, except that it was 3-fold less potent against 786-O cells. Both of them did not exhibit potent activity against MCF7 cells in our hands in

Figure 6. Comparison of the 1H NMR signals from the N-Me groups in apratoxins M8−M16 (13a−13i): (a) apratoxin M7 (4g), (b) 13a (MeIle/MeAla), (c) 13b (MeIle/MeVal), (d) 13c (MeIle/MePhe), (e) 13d (MeAla/Sar), (f) 13e (MeAla/MeLeu), (g) 13f (MeAla/MePhe), (h) 13g (Tyr(Me)/Phe), (i) 13h (Tyr(Me)/Phe(4-Cl)), (j) 13i (Tyr(Me)/Bph).

NMR analyses of 13a−13i were also performed by comparison of the signals of the N-Me groups at AA2 (Figure 6). The 1H NMR signals of 13a are very broad, indicating that its 3D structure is rather flexible than that of 4g (part b vs part a of Figure 6). This could be the reason why 13a is much less active than the other mimetics. In contrast, the 1H NMR spectrum of 13b resembles that of 4g (Figure 6c). Because both MeIle in 4g and MeVal in apratoxin 13b are branched amino acids, they could similarly regulate their macrocyclic ring structures. 13c also adopts trans- and cis-amide conformers similar to 4g (Figure 6d). Interestingly, 13d and 13e contain trans- and cis-amide conformers; however, their chemical shifts are different from those of 4g (Figure 6e,f). In contrast, the NMR signals of 13f are too broad to assign (Figure 6g). The above observation indicates that the steric effect around AA2 6758



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M16 indicated similar metabolic stability for mouse and human liver microsomes to apratoxin A. As apratoxin mimetics 3 that lack the Michael acceptor in the moCys moiety were found to possess in vivo antitumor efficacy without toxicity,2g,7 apratoxin M16 could be utilized as a lead compound for the development of anticancer agents or as a chemical tool for chemical biological studies. Our conformation-based design and synthesis of apratoxin A mimetics have realized significant alteration of the structure of the ring component while retaining extremely potent cytotoxicity for cancer cells. It should be noted that our findings highlight the way in which we can convert the complex structures of biologically active natural products to well-designed mimetics.

Table 4. Growth Inhibition Assay of Apratoxins A (1), M15 (13h), and M16 (13i) against 10 Human Cancer Cell Lines GI50 (μM)a cancer cell line tissue

apratoxin A (1)

apratoxin M15 (13h)

apratoxin M16 (13i)

mitomycin Cb

HCT-116 colon BxPC-3 pancreas A549 lung HuH-7 liver MKN74 stomach U-87 MG brain SK-OV-3 ovary HEC-6 uterus 786-O kidney MCF7 breast

0.011 0.0049 0.0064 0.0072 0.0097 0.018 0.031 0.039 0.041 >1

0.21 0.080 0.19 0.18 0.20 0.32 0.41 0.48 >1 >1

0.011 0.0040 0.0063 0.0068 0.0080 0.012 0.018 0.023 0.12 >1

0.20 0.38 0.080 2.0 1.7 1.8 0.10 1.4 0.21 2.5

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EXPERIMENTAL SECTION

All commercially available reagents were used as received. Dry THF and CH2Cl2 (Kanto Chemical Co.) were obtained by passing through activated alumina column with commercially available predried, oxygen-free formulations. All solution-phase reactions were monitored by thin-layer chromatography (TLC) carried out on 0.2 nm E. Merck silica gel plates (60F-254) with UV light, visualized by p-anisaldehyde H2SO4−ethanol solution or phosphomolybdic acid−ethanol solution or ninhydrin−acetic acid−1-butanol solution. 1H NMR spectra (400 and 600 MHz) were recorded on a JEOL JMN-AL400 or a JEOL ECA-600 spectrometer, respectively. Chemical shifts (δ) for 1 H NMR spectra are given from TMS (0.00 ppm) in CDCl3 and from residual nondeuterated solvent peaks in other solvents (CD2Cl2, 5.32 ppm; CD3CN, 1.94 ppm; CD3OD, 3.31 ppm) as internal standards. 13C NMR spectra (100 and 150 MHz) were recorded on a JEOL JMN-AL400 or a JEOL ECA-600 spectrometer. Chemical shifts (δ) for 13C NMR spectra are given from CDCl3 (77.0 ppm), CD2 Cl2 (54.0 ppm), CD 3 CN (118.26, 1.32 ppm), CD3 OD (49.0 ppm) as internal standards. Multiplicities are reported by the following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (double doublet), dt (double triplet), ddd (double double doublet), ddt (double double triplet), dddd (double double double doublet), brs (broad singlet), J (coupling constants in Hertz). Mass spectra and high-resolution mass spectra were measured on JEOL JMS-DX303 (EI), MS-AX500 (FAB) and Thermo Scientific Exactive Plus Orbitrap mass spectrometer (electrospray ionization, ESI). LC analysis was performed on reversed-phase HPLC (Waters LC/MS system ZQ2000) with Waters 2996 ultraviolet detector with X Bridge C18 3.5 μm 4.6 mm × 75 mm column using A, methanol, and B, H2O as solvent (flow rate, 1.1 mL/min; gradient: 0−1 min A 5%; 1−4 min A 5−95%; 4−11 min A 95%; 11−11.1 min A 5%; 11.1−15 min A 5%). IR spectra were recorded on a Shimadzu FTIR-8400. Only the strongest and/or structurally important absorption are reported as the IR data afforded in cm−1. Specific rotations were measured with a JASCO P-1010 polarimeter. The final products were purified by reversed-phase HPLC equipped with Waters 1525 binary pump and Waters 2489 UV−visible detector with a column, YMC-Pack R&D ODS-A 20 mm × 150 mm (flow rate, 10.0 mL/min; elution method, H2O/MeOH = 30:70−5:95 linear gradient 0.0−15.0 min) as their purities are ≥95% detected by UV (214 nm). General Procedure for Immobilization of an Fmoc-AA1-OH onto Trityl Alcohol-Linked Lanterns. Trityl alcohol lanterns (35 μmol/unit) were treated with a solution of 10% AcCl in dry CH2Cl2 at room temperature. After the lanterns were shaken at the same temperature for 4 h, the mixture was filtered. The lanterns were rinsed with dry CH2Cl2 and washed five times each with dry CH2Cl2 to afford trityl chloride lanterns. The resulting lanterns were immediately used for the immobilization of Fmoc-AA1-OH. The trityl chloride lanterns were treated with a solution of Fmoc-AA1-OH (0.10 mmol/unit, 0.10 M) and DIEA (0.035 mL/unit, 0.20 mmol/unit, 0.20 M) in dry CH2Cl2 (1.00 mL/unit) at room temperature and then shaken at the same temperature. After being shaken for 12 h, the reaction mixture was filtered. The resulting lanterns were rinsed with CH2Cl2, and then washed with CH2Cl2

a

GI50 = compound concentration at which a growth inhibition of 50% is achieved. The GI50 values were determined by derivation of the best-fit dose response line of triplicate experiments. bMitomycin C was evaluated as a control.

contrast to the result previously reported.5 These results strongly suggest that apratoxin M16 could induce the potent growth inhibitory activity against cancer cells by the same mechanism as apratoxin A.4,5 On the other hand, apratoxin M15 exhibited at least 10-fold less potent than apratoxin M16. The results indicate that a substituent on the benzene ring in AA3 should significantly affect the potency of its growth inhibitory activity in the piperidine-linked apratoxin mimetics. We also observed no apparent difference in metabolic stability in both mouse and human liver microsomes (37 °C, 35 min) was observed for apratoxin A (81% loss in mouse; 86% loss in human) and apratoxin M16 (84% loss in mouse; 85% loss in human).

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CONCLUSIONS We have demonstrated the effective development of apratoxin A mimetics by conformation-based design and synthesis. The first-generation mimetics, apratoxins M1−M7 (4a−4g), were designed to replace the moCys moiety with several linear or cyclic amino acid linkers. 4-Aminobutanoic acid, 3-aminopropanoic acid, 3-(aminomethyl)benzoic acid, and their N-Me derivatives as well as piperidinecarboxylic acid were chosen as linkers to maintain 3D structures similar to apratoxin A (1a). These mimetics were efficiently synthesized by a combination of solid-phase peptide synthesis and solution-phase macrolactamization. Among the first-generation mimetics, apratoxin M7 (4g) substituted with piperidinecarboxylic acid exhibited the highest cytotoxicity against HCT-116 cells. The 3D structural analyses of apratoxins M1−M7 using distance geometry calculations indicated the existence of two conformers with trans- and cis-amide bonds between Tyr(Me) and MeAla. Although their ratios do not correlate with the cytotoxicity, we confirmed that the 3D structure of 4g is similar to that of 1a. Next, we designed and synthesized the second-generation mimetics, apratoxins M8−M16 (13a−13i) by substitution of each amino acid in the tripeptide Tyr(Me)−MeAla−MeIle moiety in 4g. Notably, apratoxin M16 (13i), containing Bph instead of Tyr(Me), exhibited similarly high level of growth inhibitory activity against 8 of 10 cancer cell lines as apratoxin A. It should be noted that the moCys moiety was replaced by piperidine carboxylic acid; thereby, this modification facilitates the effective synthesis of potent mimetics. Moreover, apratoxin 6759



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(3 min ×5), MeOH (3 min ×3), and CH2Cl2 (3 min ×3) and were dried in vacuo. The loading amount was determined by gravimetric analysis after cleavage with 30% HFIP/CH2Cl2 (room temperature, 30 min) from the polymer-support: Fmoc-MeIle (71%), Fmoc-MeAla (77%), Fmoc-MeVal (73%), Fmoc-MePhe (74%). General Procedure for the Coupling of Fmoc-AA2-OH to Fmoc-AA1-Linked Lanterns. The Fmoc-AA1-linked lanterns were treated with a solution of 20% piperidine in DMF at room temperature. After being shaken for 30 min, the reaction mixture was filtered and the lanterns were washed with DMF (3 min ×5). The washed lanterns were immediately used for the next reaction. To a suspension of lanterns, Fmoc-AA2-OH (0.15 mmol/unit, 0.15 M) and DIEA (0.052 mL, 0.30 mmol/unit, 0.30 M) in DMF (1.00 mL/unit) was added PyBroP (70 mg/unit, 0.15 mmol/unit, 0.15 M), and the mixture was shaken at room temperature. After being shaken for 24 h, the reaction mixture was filtered and the lanterns were washed with DMF (3 min ×5), CH2Cl2 (3 min ×3), THF/H2O (3:1, 3 min ×3), MeOH (3 min ×3), and CH2Cl2 (3 min ×3) and were dried in vacuo. In the preparation of Fmoc-MeLeu-MeIle-linked lanterns, the coupling reaction was repeated. General Procedure for the Coupling of Fmoc-AA3-OH to Fmoc-AA2-AA1-Linked Lanterns. The polymer-supported dipeptide was treated with a solution of 20% piperidine in DMF at room temperature. After being shaken for 30 min, the reaction mixture was filtered and the lanterns were washed with DMF (3 min ×5). The washed lanterns were immediately used for the next condensation. To a suspension of lanterns, Fmoc-AA3-OH (0.15 mmol/unit, 0.15 M) and DIEA (0.052 mL, 0.30 mmol/unit, 0.30 M) in DMF (1.00 mL/unit) and PyBroP (70 mg/unit, 0.15 mmol/unit, 0.15 M) were added, and the mixture was shaken at room temperature. After being shaken for 24 h, the lanterns were washed with DMF (3 min ×5), and the coupling reaction was repeated. After being shaken for 24 h, the reaction mixture was filtered and the lanterns were washed with DMF (3 min ×5), CH2Cl2 (3 min ×3), THF/H2O (3:1, 3 min ×3), MeOH (3 min ×3), and CH2Cl2 (3 min ×3) and were dried in vacuo. The coupling reaction was repeated. General Procedure for the Synthesis of Polymer-Supported Tetrapeptide. The polymer-supported tripeptides were treated with a solution of 20% piperidine in DMF at room temperature. After being shaken for 30 min, the reaction mixture was filtered and the lanterns were washed with DMF (3 min ×5). The washed lanterns were immediately used for the next condensation. To a mixture of the above lanterns, Fmoc amino acid M1−M7 (0.10 mmol/unit, 0.10 M) and HOBt (20 mg/unit, 0.15 mmol/unit, 0.15 M) in DMF (1.00 mL/unit) was added DIC (0.016 mL/unit, 0.10 mmol/unit, 0.10 M), independently. The mixture was shaken at room temperature. After being shaken for 12 h, the reaction mixture was filtered and the lanterns were washed with DMF (3 min ×5), CH2Cl2 (3 min ×3), THF/H2O (3:1, 3 min ×3), MeOH (3 min ×3), and CH2Cl2 (3 min ×3) and were dried in vacuo to afford polymersupported tetrapeptides. General Procedure for the Synthesis of Polymer-Supported Hexadepsipeptides. The polymer-supported tetrapeptides were treated with a solution of 20% piperidine in DMF at room temperature. After being shaken for 30 min, the reaction mixture was filtered and the lanterns were washed with DMF (3 min ×5). The washed lanterns were immediately used for the next condensation. To a suspension of the above lanterns, acid 6 (40 mg/unit, 0.070 mmol/unit, 0.070 M), DIEA (0.044 mL, 0.25 mmol/unit, 0.25 M) in DMF (1.00 mL/unit), and COMU (43 mg/unit, 0.10 mmol/unit, 0.10 M) were added, and the mixture was shaken at room temperature. After being shaken for 24 h, the reaction mixture was filtered, and the lanterns were washed with DMF (3 min ×5), CH2Cl2 (3 min ×3), THF/H2O (3:1, 3 min ×3), MeOH (3 min ×3), and CH2Cl2 (3 min ×3) and were dried in vacuo to afford polymersupported hexadepsipeptides. The purity of each Fmoc-hexadepsipeptide was determined by LC−MS analysis (254 nm) after cleavage with 30% HFIP/CH2Cl2 (room temperature, 30 min) from the polymersupport (Figure S13).

General Procedure for Cleavage of the Linear Hexadepsipeptide from the Polymer-Support and Synthesis of Apratoxin A Mimetic by Macrolactamization. The polymersupported Fmoc-hexadepsipeptides were treated with a solution of 20% piperidine in DMF at room temperature. After being shaken for 30 min, the reaction mixture was filtered and the lanterns were washed with DMF (3 min ×5). The washed lanterns were immediately used for the next reaction. The polymer-supported hexadepsipeptides were treated with 30% HFIP/CH2Cl2 at room temperature in parallel. After being shaken for 30 h, each reaction mixture was filtered and the lantern was washed with CH2Cl2 (3 min ×3). The combined filtrate was concentrated in vacuo to afford the linear hexadepsipeptide, which was used for next reaction without further purification. To a solution of the linear hexadepsipeptide (1.0 equiv) in dry CH2Cl2 (1.0 mM) were added DIEA (9.0 equiv) and HATU (3.0 equiv) at 0 °C under argon. After being stirred at room temperature for 18 h, the reaction mixture was quenched with H2O at 0 °C. The aqueous layer was extracted with CH2Cl2 and the combined organic layers were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by reversed-phase HPLC to afford the desired apratoxin A mimetic (Figure S14). Apratoxin M1 (4a). Purified by reversed-phase HPLC (9.9 mg, 25%), retention time = 11.5 min. [α]25D −81 (c 0.50, MeOH). 1 H NMR (600 MHz, CD3CN, major rotamer) δ 7.14 (d, J = 8.9 Hz, 2 H), 6.83 (m, 1 H), 6.83 (d, J = 8.9 Hz, 2 H), 6.39 (m, 1 H), 5.24 (d, J = 11.5 Hz, 1 H), 5.01 (m, 1 H), 4.89 (dd, J = 12.5, 2.1 Hz, 1 H), 4.43 (d, J = 8.2 Hz, 1 H), 4.11 (dd, J = 7.8, 7.8 Hz, 1 H), 3.99 (m, 1 H), 3.74 (s, 3 H), 3.62 (m, 1 H), 3.58 (dddd, J = 11.2, 8.2, 6.8, 3.1 Hz, 1 H), 3.36 (m, 1 H), 3.36 (m, 1 H), 2.94 (m, 1 H), 2.89 (s, 3 H), 2.81 (m, 1 H), 2.78 (m, 1 H), 2.65 (s, 3 H), 2.30 (m, 1 H), 2.10 (m, 1 H), 2.09 (ddqdd, J = 12.1, 11.4, 7.1, 3.6, 3.2 Hz, 1 H), 2.04 (m, 1 H), 2.00 (m, 1 H), 1.88 (dq, J = 10.1, 7.1 Hz, 1 H), 1.87 (m, 1 H), 1.80 (m, 1 H), 1.78 (m, 1 H), 1.72 (ddd, J = 14.3, 12.1, 3.2 Hz, 1 H), 1.71 (m, 1 H), 1.54 (m, 1 H), 1.45 (ddd, J = 14.1, 11.2, 3.6 Hz, 1 H), 1.41 (m, 1 H), 1.33 (ddd, J = 14.1, 12.1, 2.1 Hz, 1 H), 1.21 (m, 1 H), 1.13 (ddd, J = 14.4, 11.4, 3.1 Hz, 1 H), 1.05 (d, J = 7.1 Hz, 3 H), 0.99 (d, J = 7.0 Hz, 3 H), 0.93 (d, J = 7.1 Hz, 3 H), 0.92 (d, J = 7.1 Hz, 3 H), 0.87 (m, 3 H), 0.86 (s, 9 H). 13C NMR (150 MHz, CD3CN, major rotamer) δ 176.4, 173.4, 173.2, 171.2, 170.9, 159.6, 131.3, 130.2, 114.7, 78.1, 71.5, 61.2, 60.7, 56.7, 55.7, 50.6, 48.5, 39.5, 39.4, 38.3, 37.2, 35.5, 34.1, 33.6, 31.0, 29.9, 26.3, 26.2, 26.1, 25.7, 20.2, 15.3, 14.7, 14.4, 9.3. 1H NMR (600 MHz, CD3CN, minor rotamer) δ 7.10 (d, J = 8.9 Hz, 1 H), 6.89 (m, 1 H), 6.82 (d, J = 8.9 Hz, 1 H), 6.71 (m, 1 H), 5.11 (ddd, J = 10.5, 9.2, 5.7 Hz, 1 H), 4.92 (d, J = 11.4 Hz, 1 H), 4.85 (dd, J = 12.0, 2.7 Hz, 1 H), 4.74 (q, J = 6.3 Hz, 1 H), 4.27 (dd, J = 8.3, 6.5 Hz, 1 H), 4.04 (d, J = 10.2 Hz, 1 H), 4.02 (m, 1 H), 3.73 (s, 3 H), 3.62 (m, 1 H), 3.52 (dddd, J = 11.6, 10.2, 6.8, 3.1 Hz, 1 H), 3.07 (m, 1 H), 3.07 (m, 1 H), 2.99 (dd, J = 13.6, 5.6 Hz, 1 H), 2.84 (dd, J = 12.7, 9.0 Hz, 1 H), 2.82 (s, 3 H), 2.55 (s, 3 H), 2.29 (m, 1 H), 2.12 (m, 1 H), 2.12 (m, 1 H), 2.07 (dq, J = 6.8, 7.1 Hz, 1 H), 2.06 (m, 1 H), 1.99 (ddqdd, J = 11.1, 10.2, 7.1, 3.4, 3.2 Hz, 1 H), 1.90 (m, 1 H), 1.90 (m, 1 H), 1.89 (m, 1 H), 1.72 (ddd, J = 14.3, 12.1, 3.2 Hz, 1 H), 1.66 (m, 1 H), 1.60 (m, 1 H), 1.41 (ddd, J = 14.3, 10.2, 2.7 Hz, 1 H), 1.32 (ddd, J = 14.1, 11.6, 3.4 Hz, 1 H), 1.30 (m, 1 H), 1.15 (ddd, J = 14.1, 11.1, 3.1 Hz, 1 H), 1.05 (d, J = 7.1 Hz, 3 H), 1.00 (d, J = 7.0 Hz, 3 H), 0.99 (m, 1 H), 0.93 (d, J = 7.1 Hz, 3 H), 0.86 (s, 9 H), 0.85 (m, 3 H), 0.66 (d, J = 6.6 Hz, 3 H). 13C NMR (150 MHz, CD3CN, minor rotamer) δ 177.1, 172.8, 172.0, 171.2, 171.1, 159.5, 131.3, 130.2, 114.6, 78.5, 71.8, 60.2, 58.6, 55.7, 54.9, 51.0, 49.8, 48.5, 40.3, 39.8, 39.7, 37.6, 35.5, 34.7, 33.7, 31.0, 29.9, 29.1, 26.3, 26.2, 25.7, 25.6, 19.9, 15.6, 15.4, 14.2, 9.9; IR (CH2Cl2) 3432, 3319, 2966, 2935, 1742, 1629, 1539, 1513, 1457, 1396, 1372, 1275, 1248, 1223, 1179, 1109, 1075, 1035, 735 cm−1. HRESIMS calcd for C43H69N5O9Na [M + Na]+ 822.4987, found 822.4987. Apratoxin M2 (4b). Purified by reversed-phase HPLC (11.1 mg, 28%), retention time = 10.9 min. [α]19D −117 (c 0.555, MeOH). 1 H NMR (600 MHz, CD3CN, major rotamer) δ 7.14 (d, J = 8.9 Hz, 2 H), 6.83 (d, J = 8.9 Hz, 2 H), 6.81 (m, 1 H), 5.27 (d, J = 11.0 Hz, 6760



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Apratoxin M5 (4e). Purified by reversed-phase HPLC [column, YMC-Pack R&D ODS-A 20 mm × 150 mm; flow rate, 10.0 mL/min; elution method, H 2 O/MeOH = 20:80−5:95 linear gradient (0.0−10.0 min), H2O/MeOH = 5:95 isocratic (10.0−15.0 min)] (8.2 mg, 23%); retention time = 8.5 min. [α]25D −54 (c 0.090, MeOH). 1H NMR (600 MHz, CD3CN, major rotamer) δ 7.64 (m, 1 H), 7.61 (m, 1 H), 7.41 (m, 1 H), 7.34 (m, 1 H), 7.28 (br, 1 H), 7.22 (d, J = 8.8 Hz, 2 H), 6.87 (d, J = 8.8 Hz, 2 H), 6.59 (br, 1 H), 5.06 (br, 1 H), 4.98 (d, J = 11.4 Hz, 1 H), 4.92 (dd, J = 12.6, 2.2 Hz, 1 H), 4.85 (dd, J = 14.6, 8.6 Hz, 1 H), 4.27 (br, 1 H), 3.90 (dd, J = 14.6, 4.0 Hz, 1 H), 3.78 (m, 1 H), 3.75 (s, 3 H), 3.39 (br, 1 H), 3.10 (br, 1 H), 2.98 (br, 1 H), 2.94 (br, 3 H), 2.73 (br, 3 H), 2.06 (ddqdd, J = 11.7, 10.8, 6.9, 4.0, 2.2 Hz, 1 H), 1.84 (dq, J = 10.0, 7.4 Hz, 1 H), 1.76 (m, 1 H), 1.74 (ddd, J = 14.4, 12.2, 2.2 Hz, 1 H), 1.52 (ddd, J = 14.0, 11.8, 4.0 Hz, 1 H), 1.31 (ddd, J = 14.0, 11.7, 2.5 Hz, 1 H), 1.07 (ddd, J = 14.4, 10.8, 2.6 Hz, 1 H), 1.07 (d, J = 6.8 Hz, 3 H), 0.99 (d, J = 6.9, 3 H), 0.97 (d, J = 6.9 Hz, 3 H), 0.86 (s, 9 H), 0.74 (br, 3 H), 0.59 (br, 1 H), 0.53 (br, 1 H), 0.15 (m, 3 H). 13C NMR (150 MHz, CD3CN, major rotamer) δ 176.7, 173.2, 172.2, 171.6, 171.0, 168.6, 159.7, 141.4, 136.1, 132.8, 131.6, 129.2, 129.1, 126.1, 114.7, 78.0, 70.2, 61.1, 60.6, 57.6, 54.8, 51.5, 50.9, 48.4, 43.6, 38.6, 38.3, 37.3, 37.2, 35.4, 33.0, 30.7, 30.1, 26.2, 25.9, 25.0, 24.8, 19.6, 15.3, 14.4, 8.62. IR (CH2Cl2) 3380, 2960, 2924, 2852, 1741, 1735, 1729, 1654, 1625, 1513, 1466, 1261, 1178, 1090, 1030, 800 cm−1. HRESIMS calcd for C47H69N5O9Na [M + Na]+ 870.4987, found 870.4952 Apratoxin M6 (4f). Purified by reversed-phase HPLC (12.5 mg, 30%), retention time = 11.5 min. [α]17D −120 (c 0.625, MeOH). 1 H NMR (600 MHz, CD3CN, major rotamer) δ 7.32 (m, 1 H), 7.27 (d, J = 9.1 Hz, 2 H), 6.86 (d, J = 9.1 Hz, 2 H), 5.74 (d, J = 14.3 Hz, 1 H), 5.04 (m, 1 H), 4.97 (d, J = 11.4 Hz, 1 H), 4.89 (dd, J = 12.4, 2.1 Hz, 1 H), 4.13 (d, J = 7.4 Hz, 1 H), 4.12 (m, 1 H), 4.06 (dd, J = 8.0, 8.0 Hz, 1 H), 3.90 (dddd, J = 11.3, 10.3, 7.4, 3.3 Hz, 1 H), 3.75 (s, 3 H), 3.53 (m, 1 H), 3.39 (m, 1 H), 3.38 (d, J = 14.3 Hz, 1 H), 3.08 (m, 1 H), 2.98 (m, 1 H), 2.93 (s, 3 H), 2.77 (s, 3 H), 2.74 (s, 3 H), 2.46 (dq, J = 10.3, 7.0 Hz, 1 H), 2.25 (m, 1 H), 2.13 (ddqdd, J = 12.1, 11.3, 6.8, 3.7, 2.7 Hz, 1 H), 1.99 (m, 1 H), 1.84 (m, 1 H), 1.74 (m, 1 H), 1.72 (m, 1 H), 1.72 (ddd, J = 14.7, 12.1, 2.7 Hz, 1 H), 1.57 (ddd, J = 14.7, 11.8, 3.7 Hz, 1 H), 1.33 (ddd, J = 14.0, 12.1, 2.3 Hz, 1 H), 1.14 (ddd, J = 14.3, 11.3, 3.3 Hz, 1 H), 1.06 (d, J = 6.8 Hz, 3 H), 0.97 (d, J = 6.8 Hz, 3 H), 0.95 (d, J = 7.0 Hz, 3 H), 0.86 (s, 9 H), 0.72 (m, 3 H), 0.62 (m, 1 H), 0.60 (m, 1 H), 0.13 (dd, J = 7.3, 7.3 Hz, 3 H). 13 C NMR (150 MHz, CD3CN, major rotamer) δ 176.2, 173.0, 172.0, 171.3, 170.8, 168.2, 159.4, 140.1, 136.2, 133.7, 131.6, 131.4, 129.3, 128.9, 126.4, 114.6, 77.9, 70.8, 61.0, 60.8, 57.2, 55.8, 51.6, 50.3, 48.3, 45.6, 39.0, 38.8, 37.5, 37.2, 35.2, 34.4, 32.9, 30.5, 30.1, 26.2, 25.4, 25.2, 24.7, 20.2, 15.0, 14.5, 14.3, 8.8. IR (CH2Cl2) 3439, 2962, 2933, 1743, 1647, 1635, 1559, 1513, 1457, 1395, 1294, 1248, 1179, 1085, 1033 cm−1. HRESIMS calcd for C48H71N5O9Na [M + Na]+ 884.5144, found 884.5119 Apratoxin M7 (4g). Purified by reversed-phase HPLC (6.6 mg, 16%), retention time = 11.8 min. [α]25D −82 (c 0.33, MeOH). 1 H NMR (600 MHz, CD3CN, major rotamer) δ 7.10 (d, J = 8.9 Hz, 2 H), 6.97 (m, 1 H), 6.82 (d, J = 8.9 Hz, 2 H), 5.15 (ddd, J = 10.2, 9.0, 5.6 Hz, 1 H), 4.96 (d, J = 11.7 Hz, 1 H), 4.82 (q, J = 6.6 Hz, 1 H), 4.76 (dd, J = 8.9, 4.2 Hz, 1 H), 4.39 (m, 1 H), 4.16 (dd, J = 7.7, 7.7 Hz, 1 H), 3.93 (m, 1 H), 3.91 (m, 1 H), 3.73 (s, 3 H), 3.59 (m, 1 H), 3.56 (m, 1 H), 3.08 (m, 1 H), 2.99 (m, 1 H), 2.82 (m, 1 H), 2.80 (s, 3 H), 2.80 (m, 1 H), 2.67 (m, 1 H), 2.53 (s, 3 H), 2.49 (m, 1 H), 2.26 (m, 1 H), 1.95 (m, 1 H), 1.95 (m, 1 H), 1.87 (m, 1 H), 1.83 (m, 1 H), 1.80 (ddqdd, J = 10.2, 6.8, 6.6, 5.7, 3.1 Hz, 1 H), 1.62 (m, 1 H), 1.62 (m, 1 H), 1.61 (ddd, J = 14.5, 8.8, 5.7 Hz, 1 H), 1.54 (m, 1 H), 1.53 (ddd, J = 14.4, 6.8, 4.2 Hz, 1 H), 1.37 (m, 1 H), 1.35 (ddd, J = 14.2, 9.1, 3.1, 1 H), 1.31 (m, 1 H), 1.23 (ddd, J = 14.2, 10.2, 2.7, 1 H), 1.02 (d, J = 7.0 Hz, 3 H), 1.02 (d, J = 7.0 Hz, 3 H), 0.91 (d, J = 6.6, Hz 3 H), 0.88 (s, 9 H), 0.88 (m, 1 H), 0.84 (dd, J = 7.5, 7.5 Hz, 3 H), 0.67 (d, J = 6.6 Hz, 3 H). 13C NMR (150 MHz, CD3CN, major rotamer) δ 175.8, 174.9, 172.9, 172.2, 171.1, 170.9, 159.7, 131.5, 130.1, 114.8, 79.8, 73.1, 60.9, 58.3, 55.8, 50.6, 48.5, 46.4, 42.8, 41.9, 41.7, 40.3, 38.2, 35.7, 35.0, 32.1, 31.8, 30.0, 29.3, 27.5, 27.1, 26.3, 26.2, 25.9, 20.8, 15.9, 14.3, 14.2, 10.1. 1H NMR (600 MHz, CD3OD, mixture of rotamers)

1 H), 4.99 (m, 1 H), 4.87 (dd, J = 12.5, 2.0 Hz, 1 H), 4.10 (dd, J = 9.3, 7.0 Hz, 1 H), 4.03 (m, 1 H), 3.90 (m, 1 H), 3.74 (s, 3 H), 3.66 (m, 1 H), 3.61 (m, 1 H), 3.36 (m, 1 H), 3.01 (s, 3 H), 2.94 (m, 1 H), 2.92 (s, 3 H), 2.78 (m, 1 H), 2.66 (s, 3 H), 2.58 (dq, J = 9.4, 6.8 Hz, 1 H), 2.43 (m, 1 H), 2.29 (m, 1 H), 2.25 (m, 1 H), 2.09 (m, 1 H), 2.04 (m, 1 H), 2.04 (ddqdd, J = 11.5, 11.0, 6.8, 2.9, 2.5 Hz, 1 H), 1.92 (m, 1 H), 1.87 (m, 1 H), 1.78 (m, 1 H), 1.74 (m, 1 H), 1.66 (ddd, J = 14.1, 12.3, 2.5 Hz, 1 H), 1.56 (m, 1 H), 1.48 (ddd, J = 13.8, 11.0, 2.9 Hz, 1 H), 1.35 (m, 1 H), 1.35 (ddd, J = 14.9, 11.5, 2.2 Hz, 1 H), 1.17 (ddd, J = 14.8, 11.0, 2.9 Hz, 1 H), 1.16 (m, 1 H), 1.03 (d, J = 6.4 Hz, 3 H), 0.93 (d, J = 7.0 Hz, 3 H), 0.93 (d, J = 6.8 Hz, 3 H), 0.88 (m, 3 H), 0.88 (d, J = 7.1 Hz, 3 H), 0.86 (s, 9 H). 13C NMR (150 MHz, CD3CN, major rotamer) δ176.5, 173.0, 172.8, 171.6, 171.0, 159.5, 131.4, 129.9, 114.6, 78.4, 73.3, 60.8, 55.9, 55.8, 49.8, 48.7, 43.8, 40.3, 38.8, 37.6, 37.5, 35.2, 34.5, 33.1, 31.2, 30.1, 30.0, 26.4, 26.0, 25.7, 25.6, 20.8, 14.5, 14.4, 8.9. IR (CH2Cl2) 3463, 3301, 2965, 2935, 2876, 1744, 1631, 1513, 1457, 1396, 1371, 1276, 1248, 1222, 1178, 1097, 1069, 1034, 732 cm−1. HRESIMS calcd for C44H71N5O9Na [M + Na]+ 836.5144, found 836.5119. Apratoxin M3 (4c). Purified by reversed-phase HPLC (17.1 mg, 44%), retention time = 13.1 min. [α]18D −70 (c 0.86, MeOH). 1 H NMR (600 MHz, CD3CN, major rotamer) δ 7.79 (m, 1 H), 7.11 (d, J = 8.8 Hz, 2 H), 6.89 (br, 1 H), 6.82 (d, J = 8.8 Hz, 2 H), 5.27 (ddd, J = 9.5, 9.5, 5.8 Hz, 1 H), 4.89 (d, J = 11.4 Hz, 1 H), 4.84 (dd, J = 12.3, 3.1 Hz, 1 H), 4.82 (q, J = 6.7 Hz, 1 H), 4.31 (d, J = 11.2 Hz, 1 H), 4.22 (dd, J = 8.2, 8.2 Hz, 1 H), 4.04 (m, 1 H), 3.73 (s, 3 H), 3.60 (m, 1 H), 3.54 (m, 1 H), 3.53 (dddd, J = 11.6, 11.2, 9.3, 2.8 Hz, 1 H), 3.19 (m, 1 H), 2.97 (dd, J = 12.7, 9.5 Hz, 1 H), 2.84 (s, 3 H), 2.78 (dd, J = 12.7, 5.8 Hz, 1 H), 2.50 (s, 3 H), 2.30 (m, 1 H), 2.30 (m, 1 H), 2.21 (m, 1 H), 2.13 (dq, J = 9.3, 7.2 Hz, 1 H), 1.96 (ddqdd, J = 11.6, 11.1, 6.7, 3.6, 3.4 Hz, 1 H), 1.95 (m, 1 H), 1.90 (m, 1 H), 1.85 (m, 1 H), 1.83 (m, 1 H), 1.76 (ddd, J = 14.6, 12.4, 3.4 Hz, 1 H), 1.47 (ddd, J = 14.6, 11.6, 2.8 Hz, 1 H), 1.35 (ddd, J = 14.6, 11.1, 3.0 Hz, 1 H), 1.08 (d, J = 6.8 Hz, 3 H), 1.06 (ddd, J = 14.6, 11.6, 3.6 Hz, 1 H), 0.94 (d, J = 7.2 Hz, 3 H), 0.92 (d, J = 6.7 Hz, 3 H), 1.10−0.90 (m, 2 H), 0.87 (s, 9 H), 0.81 (dd, J = 7.4, 7.4, 3 H), 0.59 (d, J = 6.7 Hz, 3 H). 13C NMR (150 MHz, CD3CN, mixture of rotamers) δ 176.1, 172.7, 172.0, 171.8, 171.5, 171.4, 159.7, 131.5, 130.0, 114.9, 114.8, 78.4, 71.6, 60.4, 58.6, 55.9, 55.2, 50.3, 48.9, 47.8, 40.4, 39.3, 38.0, 35.8, 35.6, 35.2, 35.1, 31.1, 30.1, 29.0, 26.4, 26.4, 26.4, 26.1, 25.7, 20.0, 15.6, 14.4, 13.7, 10.1. IR (CH2Cl2) 3428, 3307, 2965, 2876, 1746, 1634, 1513, 1456, 1396, 1371, 1301, 1273, 1248, 1222, 1178, 1100, 1077, 1036, 733 cm −1 . HRESIMS calcd for C42H67N5O9Na [M + Na]+ 808.4831, found 808.4814. Apratoxin M4 (4d). Purified by reversed-phase HPLC (12.2 mg, 31%), retention time = 12.1 min. [α]25D −90 (c 0.61, MeOH). 1 H NMR (600 MHz, CD3CN, major rotamer) δ 7.08 (d, J = 8.9 Hz, 2 H), 6.86 (m, 1 H), 6.80 (d, J = 8.9 Hz, 2 H), 5.25 (dd, J = 9.4, 7.3 Hz, 1 H), 4.85 (d, J = 11.3 Hz, 1 H), 4.79 (dd, J = 11.9, 3.1 Hz, 1 H), 4.75 (q, J = 6.6 Hz, 1 H), 4.13 (dd, J = 7.7, 7.7 Hz, 1 H), 4.01 (m, 1 H), 3.90 (m, 1 H), 3.73 (m, 1 H), 3.72 (s, 3 H), 3.59 (m, 1 H), 2.88 (m, 2 H), 2.86 (s, 3 H), 2.84 (s, 3 H), 2.70 (m, 1 H), 2.63 (m, 1 H), 2.58 (dq, J = 9.1, 7.0 Hz, 1 H), 2.52 (s, 3 H), 2.31 (m, 1 H), 2.31 (m, 1 H), 2.26 (m, 1 H), 1.96 (ddqdd, J = 12.4, 10.6, 6.7, 3.3, 3.1 Hz, 1 H), 1.95 (m, 1 H), 1.94 (m, 1 H), 1.86 (m, 1 H), 1.80 (m, 1 H), 1.73 (ddd, J = 14.2, 12.4, 3.2 Hz, 1 H), 1.47 (ddd, J = 13.7, 10.6, 3.1 Hz, 1 H), 1.36 (ddd, J = 14.2, 11.5, 3.1 Hz, 1 H), 1.30 (m, 1 H), 1.13 (d, J = 6.8 Hz, 3 H), 1.07 (ddd, J = 14.3, 11.3, 3.3 Hz, 1 H), 1.01 (m, 1 H) 0.92 (d, J = 6.7 Hz, 3 H), 0.89 (d, J = 7.0 Hz, 3 H), 0.86 (s, 9 H), 0.85 (dd, J = 7.5, 7.5 Hz, 3 H), 0.39 (d, J = 6.7 Hz, 3 H). 13 C NMR (150 MHz, CD3CN, mixture of rotamers) δ 177.7, 176.9, 172.5, 172.2, 172.0, 171.6, 171.5, 170.8, 170.7, 170.7, 170.2, 159.7, 159.5, 131.5, 131.5, 130.4, 129.9, 114.9, 114.7, 78.7, 78.5, 74.1, 71.4, 60.6, 60.5, 58.7, 58.4, 55.9, 55.8, 54.5, 51.2, 50.8, 48.8, 48.4, 46.3, 45.4, 41.8, 41.6, 40.7, 40.5, 39.1, 38.4, 38.1, 37.5, 36.0, 35.8, 34.8, 34.7, 33.8, 32.7, 32.6, 31.5, 30.9, 30.8, 30.3, 30.1, 29.4, 28.9, 27.3, 26.5, 26.3, 26.2, 26.1, 25.8, 25.8, 20.3, 18.7, 16.5, 16.3, 15.1, 14.9, 14.6, 14.5, 10.5, 10.2. IR (CH2Cl2) 3439, 3285, 2963, 2934, 1745, 1634, 1513, 1456, 1397, 1371, 1273, 1248, 1179, 1101, 1080, 1036, 734 cm−1. HRESIMS calcd for C43H69N5O9Na [M + Na]+ 822.4987, found 822.4964 6761



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δ 7.30−7.04 (m, 2 H), 6.93−6.71 (m, 2 H), 5.70−0.64 (m, 65 H); 13C NMR (150 MHz, CD3OD, mixture of rotamers) δ 177.6, 176.5, 176.3, 173.7, 173.5, 172.7, 172.6, 172.0, 171.5, 171.4, 170.5, 169.4, 160.3, 160.2, 132.4, 131.6, 131.5, 131.4, 129.9, 129.9, 129.6, 127.0, 115.1, 115.0, 81.0, 79.1, 73.4, 73.2, 61.3, 61.1, 60.9, 59.3, 58.2, 56.5, 55.7, 55.7, 52.3, 51.5, 49.8, 47.0, 46.9, 44.9, 43.2, 43.0, 42.7, 42.5, 42.5, 41.9, 40.4, 39.7, 38.4, 38.4, 37.8, 36.1, 35.9, 35.4, 35.3, 34.5, 33.8, 33.1, 32.8, 32.2, 31.9, 30.9, 30.8, 30.7, 30.5, 30.4, 30.2, 29.6, 28.1, 27.6, 27.3, 27.0, 26.9, 26.7, 26.6, 26.5, 26.4, 26.3, 25.7, 25.4, 24.2, 23.7, 21.8, 20.6, 15.9, 15.6, 15.4, 15.0, 14.8, 14.6, 14.4, 13.3, 11.6, 10.7, 10.2, 9.8. IR (CH2Cl2) 3481, 2963, 2934, 1743, 1636, 1559, 1540, 1513, 1457, 1395, 1247, 1179, 1080, 1034, 730 cm−1. HRESIMS calcd for C45H71N5O9Na [M + Na]+ 848.5144, found 848.5144. (2S,3S,5S,7S)-7-(((S)-1-(((9H-Fluoren-9-yl)methoxy)carbonyl)pyrrolidine-2-carbonyl)oxy)-3-hydroxy-2,5,8,8-tetramethylnonanoic acid (6). To a solution of 72b (100 mg, 0.134 mmol, 1.0 equiv) in THF (1.3 mL) and aqueous 1.0 M NH4OAc (1.61 mL, 1.61 mmol, 12 equiv) was added Zn dust (88 mg, 1.34 mmol, 10 equiv) at 0 °C. After being stirred for 2 h at 0 °C to room temperature, the mixture was quenched with H2O at 0 °C. The aqueous layer was extracted with AcOEt, and the combined organic layers were dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (0% to 5% MeOH in CHCl3) to afford 6 (62.7 mg, 0.111 mmol, 82%) as a white amorphous solid. [α]28D −46.9 (c 2.56, CHCl3). 1H NMR (400 MHz, CDCl3, mixture of rotamers) δ 7.78−7.71 (m, 2 H), 7.65−7.55 (m, 2 H), 7.42−7.35 (m, 2 H), 7.34−7.28 (m, 2 H), 4.92 (dd, J = 11.95, 1.71 Hz, 1 H), 4.53−4.13 (m, 5 H), 3.85−3.41 (m, 3 H), 2.48−2.39 (m, 1 H), 2.34−2.20 (m, 1 H), 2.07−1.60 (m, 6 H), 1.36−1.22 (m, 1 H), 1.16 (d, J = 7.3 Hz, 3 H), 1.12−1.03 (m, 1 H), 0.97 (d, J = 6.3 Hz, 3 H), 0.89 (s, 9 H). 13C NMR (100 MHz, CDCl3, mixture of rotamers) δ 172.5, 155.4, 144.0, 143.8, 141.40, 141.37, 127.8, 127.7, 127.2, 127.1, 125.2, 125.1, 119.98, 119.96, 78.3, 77.2, 70.7, 67.8, 59.5, 47.1, 46.5, 46.2, 39.4, 37.3, 34.6, 29.8, 25.9, 24.7, 24.4, 20.3. IR (CHCl3) 3470, 2695, 1738, 1706, 1478, 1452, 1427, 1359, 1341, 1219, 1195, 1179, 1125, 1089, 988, 758, 741 cm−1. HRESIMS calcd for C33H43NO7Na [M + Na]+ 588.2932, found 588.2905. Apratoxin M8 (13a). Purified by reversed-phase HPLC (6.9 mg, 16%), retention time = 10.0 min. [α]19D −86 (c 0.35, MeOH). 1 H NMR (600 MHz, CD3CN, mixture of rotamers) δ 7.22−7.06 (m, 2 H), 6.90−6.74 (m, 2 H), 5.58−3.44 (m, 13 H), 3.36−1.87 (m, 16 H), 1.85−0.66 (m, 30 H); 13C NMR (150 MHz, CD3CN, mixture of rotamers) δ 165.0, 159.7, 159.6, 132.3, 131.6, 130.1, 129.7, 129.0, 114.8, 114.7, 79.9, 73.3, 60.3, 55.9, 51.6, 50.8, 49.9, 48.2, 47.9, 46.6, 42.4, 40.2, 38.3, 35.7, 35.5, 31.8, 31.4, 30.9, 30.4, 30.0, 29.6, 27.2, 26.4, 26.3, 26.2, 26.0, 23.7, 20.5, 17.2, 16.2, 15.7, 14.8, 14.5. 1H NMR 600 MHz, CD3OD, mixture of rotamers) δ 7.21−7.09 (m, 2 H), 6.90− 6.79 (m, 2 H), 5.70−0.64 (m, 59 H). 13C NMR (150 MHz, CD3OD, mixture of rotamers) δ 177.5, 176.8, 176.4, 176.3, 173.7, 173.6, 173.5, 172.9, 172.7, 172.2, 171.4, 160.4, 160.3, 131.7, 131.6, 130.0, 129.4, 115.0, 81.2, 79.7, 73.1, 73.0, 60.7, 56.2, 55.7, 55.7, 52.6, 51.4, 51.3, 49.9, 48.4, 47.0, 46.8, 44.2, 42.8, 42.7, 41.9, 40.1, 39.5, 39.2, 39.0, 37.9, 35.8, 35.8, 34.2, 32.5, 31.5, 30.8, 30.1, 30.0, 29.2, 27.7, 27.3, 26.8, 26.6, 26.5, 26.4, 26.2, 25.5, 21.6, 20.6, 16.2, 15.9, 15.5, 14.7, 14.5. IR (CH2Cl2) 3429, 3293, 2957, 1734, 1636, 1513, 1448, 1397, 1370, 1301, 1248, 1181, 1103, 1035, 734 cm−1. HRESIMS calcd for C42H65N5O9Na [M + Na]+ 806.4674, found 806.4660. Apratoxin M9 (13b). Purified by reversed-phase HPLC (7.3 mg, 18%), retention time = 11.0 min. [α]26D −103 (c 0.365, MeOH). 1 H NMR (600 MHz, CD3CN, mixture of rotamers) δ 7.23−7.03 (m, 2 H), 6.92−6.75 (m, 2 H), 5.50−3.42 (m, 13 H), 3.37−0.58 (m, 50 H). 13C NMR (150 MHz, CD3CN, mixture of rotamers) δ 174.8, 172.8, 172.2, 172.1, 171.6, 171.0, 164.9, 159.7, 159.6, 132.2, 131.6, 131.5, 131.5, 129.8, 129.7, 126.7, 114.8, 114.8, 114.7, 114.7, 114.6, 80.2, 79.7, 78.3, 73.1, 72.8, 66.6, 62.8, 60.8, 60.7, 60.4, 59.6, 55.9, 55.5, 53.1, 51.4, 51.1, 50.6, 48.7, 47.2, 46.4, 45.6, 44.4, 42.0, 41.9, 40.3, 39.3, 38.1, 35.7, 35.6, 35.5, 33.2, 32.5, 31.7, 31.4, 31.0, 30.7, 30.4, 30.2, 30.1, 29.3, 28.2, 26.8, 26.5, 26.3, 26.3, 26.2, 26.1, 25.2, 25.1, 23.3, 20.9, 20.4, 20.0, 20.0, 19.8, 19.3, 18.8, 15.8, 15.7, 15.3, 14.6, 14.4, 14.3, 14.1. 1 H NMR (600 MHz, CD3OD, mixture of rotamers) δ 7.23−7.07

(m, 2 H), 6.90−6.78 (m, 2 H), 5.39−0.59 (m, 63 H). 13C NMR (150 MHz, CD3OD, mixture of rotamers) δ 177.6, 176.6, 176.3, 176.2, 173.7, 173.6, 172.7, 172.6, 172.0, 171.5, 170.4, 160.3, 131.6, 131.5, 129.9, 129.6, 115.1, 115.0, 80.9, 79.0, 73.4, 73.2, 61.4, 61.2, 61.1, 60.7, 56.4, 55.7, 51.5, 49.6, 46.9, 44.9, 43.1, 43.0, 42.6, 42.4, 41.9, 40.4, 39.7, 38.4, 38.3, 37.8, 36.0, 35.9, 33.8, 32.8, 32.1, 31.8, 30.9, 30.8, 30.5, 30.4, 30.2, 29.8, 29.6, 28.0, 27.5, 27.1, 26.6, 26.5, 26.5, 26.3, 25.4, 21.7, 21.6, 20.6, 20.5, 19.6, 19.1, 15.8, 15.4, 14.5, 13.4. IR (CH2Cl2) 3470, 3293, 2957, 2931, 1742, 1627, 1513, 1451, 1370, 1248, 1179, 1098, 1034, 947, 730 cm−1. HRESIMS calcd for C44H69N5O9Na [M + Na]+ 834.4987, found 834.4975. Apratoxin M10 (13c). Purified by reversed-phase HPLC (10.8 mg, 24%), retention time = 11.7 min. [α]27D −68 (c 0.54, MeOH). 1 H NMR (600 MHz, CD3CN, mixture of rotamers) δ 7.50−6.57 (m, 10 H), 5.80−3.36 (m, 13 H), 3.33−0.02 (m, 45 H). 13C NMR (150 MHz, CD3CN, mixture of rotamers) δ 176.1, 175.6, 175.0, 174.7, 172.8, 172.4, 171.8, 171.1, 170.9, 170.7, 169.4, 167.7, 165.6, 159.7, 159.6, 138.5, 131.6, 131.5, 131.4, 131.3, 130.4, 130.1, 129.8, 129.6, 129.6, 129.4, 129.2, 127.9, 127.4, 114.8, 114.7, 114.7, 80.3, 78.2, 72.9, 72.9, 61.1, 60.5, 60.3, 58.0, 56.5, 55.9, 55.9, 55.8, 55.5, 50.4, 48.1, 48.1, 46.4, 46.3, 44.1, 42.9, 42.3, 42.2, 41.9, 41.7, 41.6, 41.5, 39.9, 39.5, 39.0, 38.7, 38.4, 38.2, 37.3, 35.6, 35.5, 34.9, 34.0, 32.3, 32.3, 31.7, 30.2, 29.9, 29.7, 28.9, 27.9, 27.4, 27.2, 27.0, 26.5, 26.4, 26.3, 26.3, 25.9, 25.8, 25.3, 21.7, 20.6, 15.9, 15.7, 14.6, 14.3. IR (CH2Cl2) 3477, 3286, 2956, 2934, 1739, 1652, 1645, 1634, 1544, 1513, 1455, 1393, 1300, 1248, 1180, 1100, 1034, 945, 733, 700 cm−1. HRESIMS calcd for C48H69N5O9Na [M + Na]+ 882.4987, found 882.4971. Apratoxin M11 (13d). Purified by reversed-phase HPLC (8.1 mg, 20%), retention time = 11.8 min. [α]25D −73 (c 0.41, MeOH). 1 H NMR (600 MHz, CD3CN, mixture of rotamers) δ 7.19−7.05 (m, 2 H), 6.89−6.60 (m, 2 H), 5.32−3.23 (m, 14 H), 3.21−0.67 (m, 49 H). 13C NMR (150 MHz, CD3CN, mixture of rotamers) δ 176.2, 174.3, 172.0, 171.4, 171.3, 169.9, 168.4, 159.6, 159.2, 131.6, 131.3, 114.7, 114.3, 78.7, 74.3, 61.0, 60.8, 55.8, 52.2, 51.2, 48.4, 45.8, 44.9, 42.9, 41.8, 40.5, 40.1, 38.2, 37.5, 37.4, 36.4, 36.0, 35.5, 33.5, 30.6, 30.0, 29.2, 28.3, 27.9, 27.5, 26.8, 26.5, 26.5, 26.2, 25.9, 25.5, 25.2, 24.9, 17.8, 16.3, 16.1, 14.4, 11.4. IR (CH2Cl2) 3451, 3301, 2959, 2930, 1741, 1646, 1513, 1442, 1369, 1300, 1247, 1181, 1037, 954, 822 cm−1. HRESIMS calcd for C44H69N5O9 [M + Na]+ 834.4987, found 834.4975. Apratoxin M12 (13e). Purified by reversed-phase HPLC (8.3 mg, 19%), retention time = 13.5 min. [α]25D −101 (c 0.415, MeOH). 1 H NMR (600 MHz, CD3CN, mixture of rotamers) δ 7.22−6.62 (m, 5 H), 5.53−3.37 (m, 13 H), 3.34−0.05 (m, 58 H). 13C NMR (150 MHz, CD3CN, mixture of rotamer) δ 175.9, 174.9, 172.9, 172.2, 170.8, 169.9, 159.7, 131.6, 130.1, 114.8, 79.7, 79.6, 73.1, 60.8, 58.2, 58.1, 57.7, 55.9, 55.8, 50.7, 48.6, 46.4, 42.9, 42.1, 40.3, 39.9, 38.3, 35.7, 35.6, 35.1, 35.0, 32.4, 30.2, 30.1, 30.0, 29.9, 27.6, 27.2, 26.5, 26.3, 26.2, 26.1, 26.0, 25.5, 25.1, 23.8, 21.9, 20.7, 15.7, 15.5, 14.7, 14.6, 11.4, 10.3, 9.7. IR (CH2Cl2) 3466, 3289, 2958, 2932, 2874, 1745, 1656, 1640, 1632, 1513, 1462, 1444, 1392, 1367, 1274, 1248, 1179, 1095, 1036, 947, 831, 731 cm−1. HRESIMS calcd for C48H77N5O9Na [M + Na]+ 890.5614, found 890.5597. Apratoxin M13 (13f). Purified by reversed-phase HPLC (12.1 mg, 27%), retention time = 14.8 min. [α]20D −90 (c 0.61, MeOH). 1 H NMR (600 MHz, CD3CN, mixture of rotamers) δ 7.51−6.53 (m, 10 H), 5.72−3.35 (m, 13 H), 3.32−0.16 (m, 51 H). 13C NMR (150 MHz, CD3CN, mixture of rotamers) δ 173.4, 172.2, 170.8, 170.4, 170.2, 159.4, 139.2, 131.8, 131.6, 131.0, 130.9, 130.7, 130.5, 130.0, 129.9, 129.3, 127.8, 115.0, 114.7, 114.3, 79.9, 72.8, 61.6, 61.1, 58.5, 56.0, 55.9, 50.0, 49.6, 48.4, 46.5, 42.5, 42.0, 38.7, 36.7, 35.8, 35.7, 34.9, 34.7, 31.9, 31.4, 31.0, 30.4, 30.3, 30.2, 28.1, 27.2, 26.6, 26.4, 26.3, 26.2, 15.8, 15.2, 14.7, 14.2, 13.8, 10.4. 1H NMR (600 MHz, CD3OD, mixture of rotamers) δ 7.43−6.58 (m, 9 H), 5.80−0.03 (m, 64 H). 13 C NMR (150 MHz, CD3OD, mixture of rotamers) δ 177.5, 176.5, 176.2, 175.9, 175.8, 174.2, 174.0, 173.5, 172.9, 172.4, 171.8, 171.1, 170.9, 170.8, 160.3, 160.2, 159.9, 138.7, 131.7, 131.5, 131.4, 131.0, 130.8, 130.5, 129.9, 129.9, 129.4, 127.9, 127.3, 115.3, 115.0, 114.9, 114.5, 82.7, 80.9, 80.6, 79.0, 73.4, 73.2, 63.4, 61.8, 61.5, 61.2, 61.1, 59.4, 59.2, 57.8, 55.7, 55.7, 51.1, 50.3, 49.8, 47.0, 46.8, 44.8, 43.1, 43.1, 6762



Article

at 5 × 103 cells/100 μL/well fresh culture medium in a 96-well clear bottom plate and incubated at 37 °C under 5% CO2 for 24 h before the assays. Each apratoxin A mimetic was dissolved in DMSO at concentrations ranging from 0.01 to 100 μM. A volume of 1 μL of the resultant solution was added to the above-mentioned 100 μL cell culture, resulting in various concentrations of the compound (0.1−1000 nM) or solvent control (DMSO 1%). After a 48 h incubation at 37 °C under 5% CO2, 10 μL of WST-8 reagent solution (Cell Count Reagent SF, Nacalai Tesque, Inc.)14 was added to the cell culture. The cell culture was then incubated at 37 °C under 5% CO2 for 2 h. Colorimetric determination of WST-8 was conducted at 450 nm with an optional reference wavelength at 595 nm using a microplate reader (model 680, Bio-Rad, Hercules, CA). The absorbance obtained upon the addition of the vehicle was considered as 100%. Data are expressed from the dose−response curves at three independent experiments. IC50 values were calculated by probit analysis using the PriProbit 1.63 software.21 Evaluation of Sensitivity to Different Cancer Cell Lines. A panel of 10 cancer cell lines were obtained and cultured as summarized in Table S1 (Supporting Information). The growth inhibitory activities of apratoxin A, M15, and M16 against the cancer cell lines were assayed by measuring the amount of ATP in the cells using CellTiter-Glo (Promega, Madison, WI) as reported previously.22 In brief, the cells were incubated in 384-well plates at a density of 1 × 103 to ∼3 × 103 cells/ well with a medium volume of 40 μL for 24 h at 37 °C under 5% CO2. The cells were then treated with 0.1 μL of compound solutions at final concentration ranges of 1 μM to 0.03 nM (10-point dose) using an ADS-348-8 multistage-dispense station (Biotec, Tokyo, Japan). The vehicle solvent (DMSO) was used as a control at a maximum concentration of 0.25%. After a 72 h incubation at 37 °C under 5% CO2, 10 μL of CellTiter-Glo reagent solution was added to the medium and the plate was mixed with a plate mixer and incubated for 10 min at 30 °C. The luminescence was measured using an EnSpire plate reader (PerkinElmer). Absorbance for the control well (C) and test well (T) was measured along with that for the test well at time 0 (T0). Cell growth inhibition (% growth) by each concentration of the drug was calculated as 100[(T − T0)/(C − T0)] and the GI50 values were analyzed using Morphit software (The Edge Software Consultancy, Guildford, U.K.). Molecular Modeling Based on NMR Data. NMR measurements for tertiary structural analysis were conducted using a NMR spectrometer (600 MHz for 1H) at 298 K using samples. 3JH,H values were determined by 1D 1H spectra and 1H−1H J-resolved 2D NMR spectra. According to a J-based configuration analysis (JBCA) method,16 3JH,H coupling constants for clearly antioriented vicinal protons (3JH,H ≥ 10 Hz) were interpreted as dihedral angle constraints. Molecular modeling was performed on the MacroModel (version 9.9) program17 by the distance geometry method. We used an OPLS2005 force field and a generalized Born/solvent-accessible surface area (GB/SA) solvent model.18 The calculations were conducted in a chloroform environment. To find 3D structures that were in agreement with the experimental data and also had low energies in a given force field, we selected a protocol that comprised two steps. First, a conformational search was performed using Monte Carlo-based torsional sampling with 1H−1H distance constraints (force constant, 10 kJ mol−1 Å−2) at 20 000 iterations with 500 times of energy minimization. Then, energy minimization was applied to each found structure without constraints. Measurement of Metabolic Stability. Pooled liver microsomes from mouse and human sources were purchased from Sekisui Medical Co., Ltd. (Japan). An incubation mixture with a final volume of 0.1 mL consisted of microsomal protein in a 3.25 mmol/L β-NADPH solution dissolved in a 125 mmol/L phosphate buffer and 100 nmol/L test compound in CH3CN. The concentration of liver microsomal protein was 0.1 mg/mL. The mixture was incubated at 37 °C for 35 min. The reaction was terminated by the addition of methanol (0.4 mL). All incubations were made in duplicate. The quenched solution was set for 30 min at −20 °C and was centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was analyzed by LC−MS/MS (Shimadzu LC-20A/

42.8, 42.5, 42.4, 41.8, 39.7, 38.9, 38.5, 38.2, 37.0, 36.3, 36.1, 35.9, 35.5, 35.1, 35.0, 33.7, 32.5, 32.1, 31.8, 31.1, 30.7, 30.7, 30.5, 30.4, 30.3, 30.2, 30.0, 29.6, 29.2, 28.2, 27.5, 27.3, 27.2, 26.6, 26.5, 26.4, 26.3, 26.3, 26.2, 26.2, 25.9, 25.8, 25.4, 23.7, 21.8, 21.7, 20.6, 20.3, 15.4, 15.2, 14.8, 14.1, 13.5, 11.4, 10.3, 10.3, 9.7, 9.7. IR (CH2Cl2) 3466, 3298, 2960, 2932, 2876, 1744, 1631, 1513, 1453, 1395, 1369, 1248, 1180, 1095, 1033, 949, 701 cm−1. HRESIMS calcd for C51H75N5O9Na [M + Na]+ 924.5457, found 924.5454. Apratoxin M14 (13g). Purified by reversed-phase HPLC (8.5 mg, 22%), retention time = 12.1 min; [α]26D −102 (c 0.425, MeOH). 1 H NMR (600 MHz, CD3CN, major rotamer) δ 7.38−6.66 (m, 6 H), 5.44−3.48 (m, 10 H), 3.39−0.46 (m, 52 H). 13C NMR (150 MHz, CD3CN, mixture of rotamer) δ 175.8, 174.8, 172.9, 172.7, 172.2, 171.4, 171.0, 170.8, 138.2, 138.0, 130.5, 130.5, 129.5, 129.5, 129.4, 129.4, 127.8, 127.7, 79.7, 78.3, 73.1, 72.8, 60.9, 60.7, 58.2, 55.7, 50.5, 48.7, 48.5, 46.5, 46.4, 44.3, 43.0, 42.3, 42.0, 42.0, 41.8, 41.5, 41.2, 39.3, 38.3, 38.2, 35.7, 35.6, 35.0, 33.2, 32.4, 31.8, 31.5, 30.2, 30.1, 30.0, 29.3, 27.8, 27.2, 26.8, 26.5, 26.3, 26.2, 26.2, 26.1, 25.4, 25.1, 20.9, 20.4, 15.8, 15.7, 14.7, 14.5, 14.4, 14.2, 10.2, 9.8. IR (CH2Cl2) 3471, 3284, 2964, 2932, 2875, 1743, 1634, 1546, 1447, 1395, 1370, 1319, 1273, 1181, 1101, 1079, 1013, 947, 734, 702 cm−1. HRESIMS calcd for C44H69N5O8Na [M + Na]+ 818.5038, found 818.5026. Apratoxin M15 (13h). Purified by reversed-phase HPLC (10.6 mg, 26%), retention time = 12.8 min. [α]25D −104 (c 0.530, MeOH). 1 H NMR (600 MHz, CD3CN, mixture of rotamers) δ 7.44−6.57 (m, 5 H), 5.47−3.46 (m, 10 H), 3.42−0.54 (m, 52 H). 13C NMR (150 MHz, CD3CN, mixture of rotamer) δ 176.4, 175.8, 174.8, 172.9, 172.4, 172.2, 171.2, 171.0, 170.8, 137.1, 136.9, 133.1, 133.1, 132.2, 129.4, 129.3, 79.8, 78.4, 73.1, 72.8, 60.9, 60.7, 58.3, 55.6, 50.2, 48.7, 48.5, 46.5, 46.4, 44.3, 43.0, 42.3, 42.0, 41.8, 41.5, 40.3, 39.3, 38.3, 38.2, 35.7, 35.6, 35.0, 33.2, 32.4, 31.8, 31.5, 30.3, 30.1, 30.0, 29.3, 27.8, 27.2, 26.8, 26.5, 26.4, 26.3, 26.2, 26.2, 26.1, 25.4, 25.1, 20.9, 20.4, 16.1, 15.8, 14.7, 14.5, 14.4, 14.2, 10.2, 9.8. IR (CH2Cl2) 3480, 3285, 2965, 2936, 2875, 1743, 1634, 1540, 1492, 1448, 1395, 1369, 1340, 1272, 1181, 1096, 1016, 947, 735 cm−1. HRESIMS calcd for C44H68N5O8ClNa [M + Na]+ 852.4649, found 852.4636. Apratoxin M16 (13i). Purified by reversed-phase HPLC (10.5 mg, 24%), retention time = 13.8 min. [α]27D −86 (c 0.53, MeOH). 1 H NMR (600 MHz, CD3CN, major rotamer) δ 7.65−7.52 (m, 4 H), 7.47−7.42 (m, 2 H), 7.38−7.27 (m, 3 H), 6.99 (d, J = 9.6 Hz, 1 H), 5.24 (ddd, J = 9.6, 8.9, 6.1 Hz, 1 H), 4.96 (d, J = 11.3 Hz, 1 H), 4.84 (q, J = 6.7 Hz, 1 H), 4.77 (dd, J = 9.0, 4.2 Hz, 1 H), 4.38 (m, 1 H), 4.16 (dd, J = 7.9, 7.9 Hz, 1 H), 3.92 (m, 1 H), 3.90 (m, 1 H), 3.67 (m, 1 H), 3.60 (m, 1 H), 3.56 (m, 1 H), 3.10 (dd, J = 12.9, 8.9 Hz, 1 H), 3.08 (m, 1 H), 2.93 (dd, J = 12.9, 6.1 Hz, 1 H), 2.81 (s, 3 H), 2.81 (m, 1 H), 2.67 (m, 1 H), 2.55 (s, 3 H), 2.52 (m, 1 H), 2.26 (m, 1 H), 1.96 (m, 1 H), 1.93 (m, 1 H), 1.87 (m, 1 H), 1.83 (m, 1 H), 1.80 (ddqdd, J = 10.1, 7.1, 6.2, 5.7, 3.6 Hz, 1 H), 1.65 (m, 1 H), 1.64 (m, 1 H), 1.61 (ddd, J = 14.5, 8.9, 5.7 Hz, 1 H), 1.54 (m, 1 H), 1.53 (ddd, J = 14.5, 7.1, 4.0 Hz, 1 H), 1.37 (m, 1 H), 1.34 (ddd, J = 14.3, 9.1, 3.6 Hz, 1 H), 1.31 (m, 1 H), 1.23 (ddd, J = 13.6, 10.1, 2.6 Hz, 1 H), 1.02 (d, J = 6.8 Hz, 3 H), 1.02 (d, J = 6.8 Hz, 3 H), 0.92 (d, J = 6.2 Hz, 3 H), 0.91 (m, 1 H), 0.88 (s, 9 H), 0.83 (dd, J = 7.4, 7.4 Hz, 3 H), 0.67 (d, J = 6.7 Hz, 3 H). 13C NMR (150 MHz, CD3CN, major rotamer) δ 175.8, 174.9, 172.7, 172.2, 171.1, 170.9, 141.6, 141.6, 140.5, 140.4, 137.5, 131.1, 130.0, 128.4, 128.1, 128.0, 127.9, 127.8, 79.8, 73.0, 60.7, 58.3, 55.7, 50.4, 48.3, 46.3, 42.9, 41.9, 41.8, 40.7, 38.2, 35.7, 34.9, 32.3, 31.6, 30.0, 29.2, 27.6, 27.1, 26.3, 26.2, 25.9, 20.8, 15.7, 14.4, 14.3, 10.1. IR (CH2Cl2) 3473, 3287, 2964, 2932, 2875, 1743, 1634, 1543, 1486, 1449, 1395, 1370, 1341, 1318, 1273, 1246, 1181, 1100, 1080, 1009, 948, 847, 761, 733, 699 cm−1. HRESIMS calcd for C50H73N5O8Na [M + Na]+ 894.5351, found 894.5341. Cytotoxicity Assay. Human colon adenocarcinoma HCT-116 cells were kindly provided by Prof. Yoshiteru Oshima at the Graduate School of Pharmaceutical Sciences in Tohoku University. They were cultured in an RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) containing 10% fetal bovine serum (Equitech-Bio, Inc., Texas), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Nacalai Tesque, Inc., Kyoto, Japan) at 37 °C under 5% CO2. For the cytotoxicity assay, near-confluent cultures of the cells were plated 6763



Article

API 4000, AB Sciex Pte. Ltd.). For metabolic stability determination, the chromatogram was analyzed for parent compound disappearance from the reaction mixture.

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osine; Dtena, 3,7-dihydroxy-2,5,8,8-tetramethylnonanoic acid; VEGF, vascular endothelial growth factor; VEGF-R, vascular endothelial growth factor receptor; SAR, structure−activity relationship; ROE, rotating-frame nuclear Overhauser effect; TES, triethylsilyl, PyBroP: bromo-tris-pyrrolidino-phosphonium hexafluorophosphate; DIEA, N,N-diisopropylethylamine; DIC, 1,3-diisopropylcarbodiimide; HOBt, 1-hydroxybenzotriazole; Troc, 1,1,1-trichloroethyloxycarbonyl; COMU, N-[1(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino(morpholino)]uronium hexafluorophosphate; HFIP, 1,1,1,3,3,3hexafluoroisopropyl alcohol; JBCA, J-based configuration analysis; GB/SA, Born/solvent-accessible surface area; WST, water-soluble tetrazolium salts; TLC, thin-layer chromatography; HATU, 2-(1-oxy-7-azabenzotriazol-3-yl)-1,1,3,3-tetramethylguanidium hexafluorophosphate; HPLC, high performance liquid chromatography

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00833. Magnetic anisotropic effect of amides, conformational analysis of apratoxin M1−M7, synthetic scheme for apratoxin M8−M16, building blocks for the synthesis of apratoxin M8−M16, comparison of 1H NMR spectra of apratoxin M8−M16, conformational analysis of apratoxin M16, LC−MS analysis of linear Fmoc-hexadepsipeptides, summary of yields, IC50 values against HCT-116 cells and ratios of trans/cis amide bond orientation in apratoxin M1−M16, spectral assignment, and 1H and 13C NMR spectra (PDF) Molecular formula strings of compounds 1a, 4a−4g, and 13a−13i (CSV) IC50 and ratio of trans/cis conformers b of apratoxin (CSV) trans- and cis-amide NMR data analysis of 4a and 1e (CSV) IC50 and ratio of trans/cis conformers c of apratoxin (CSV) Cancer cell tissues and GI50 values and apratoxins and mitomycin (CSV)

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REFERENCES

(1) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Corbett, T. H. Total structure determination of apratoxin A, a potent novel cytotoxin from the marine cyanobacterium Lyngbya majuscula. J. Am. Chem. Soc. 2001, 123, 5418−5423. (2) (a) Ma, D.; Zou, B.; Cai, G.; Hu, X.; Liu, J. O. Total synthesis of the cyclodepsipeptide apratoxin A and its analogues and assessment of their biological activities. Chem. - Eur. J. 2006, 12, 7615−7626. (b) Doi, T.; Numajiri, Y.; Takahashi, T.; Takagi, M.; Shin-ya, K. Solidphase total synthesis of (−)-apratoxin A and its analogues and their biological evaluation. Chem. - Asian J. 2011, 6, 180−188. (c) Tidgewell, K.; Engene, N.; Byrum, T.; Media, J.; Doi, T.; Valeriote, F. A.; Gerwick, W. H. Evolved diversification of a modular natural product pathway: apratoxins F and G, two cytotoxic cyclic depsipeptides from a Palmyra collection of Lyngbya bouillonii. ChemBioChem 2010, 11, 1458−1466. (d) Gutierrez, M.; Suyama, T. L.; Engene, N.; Wingerd, J. S.; Matainaho, T.; Gerwick, W. H. Apratoxin D, a potent cytotoxic cyclodepsipeptide from Papua New Guinea collections of the marine cyanobacteria Lyngbya majuscula and Lyngbya sordida. J. Nat. Prod. 2008, 71, 1099−1103. (e) Thornburg, C. C.; Cowley, E. S.; Sikorska, J.; Shaala, L. A.; Ishmael, J. E.; Youssef, D. T. A.; McPhail, K. L. Apratoxin H and apratoxin A sulfoxide from the Red Sea cyanobacterium Moorea producens. J. Nat. Prod. 2013, 76, 1781− 1788. (f) Matthew, S.; Schupp, P. J.; Luesch, H. Apratoxin E, a cytotoxic peptolide from a Guamanian collection of the marine cyanobacterium Lyngbya bouillonii. J. Nat. Prod. 2008, 71, 1113−1116. (g) Chen, Q.−Y.; Liu, Y.; Luesch, H. Systematic chemical mutagenesis identifies a potent novel apratoxin A/E hybrid with improved in vivo antitumor activity. ACS Med. Chem. Lett. 2011, 2, 861−865. (h) Wu, P.; Cai, W.; Chen, Q.-Y.; Xu, S.; Yin, R.; Li, Y.; Zhang, W.; Luesch, H. Total synthesis and biological evaluation of apratoxin E and its C30 epimer: configurational reassignment of the natural product. Org. Lett. 2016, 18, 5400−5403. (3) (a) Luesch, H.; Chanda, S. K.; Raya, R. M.; DeJesus, P. D.; Orth, A. P.; Walker, J. R.; Izpisúa Belmonte, J. C.; Schultz, P. G. A functional genomics approach to the mode of action of apratoxin A. Nat. Chem. Biol. 2006, 2, 158. (b) Liu, Y.; Law, B. K.; Luesch, H. Apratoxin A reversibly inhibits the secretory pathway by preventing cotranslational translocation. Mol. Pharmacol. 2009, 76, 91−104. (4) Paatero, A. O.; Kellosalo, J.; Dunyak, B. M.; Almaliti, J.; Gestwicki, J. E.; Gerwick, W. H.; Taunton, J.; Paavilainen, V. O. Apratoxin kills cells by direct blockade of the Sec61 protein translocation channel. Cell Chem. Biol. 2016, 23, 561−566. (5) Huang, K.-C.; Chen, Z.; Jiang, Y.; Akare, S.; Kolber-Simonds, D.; Condon, K.; Agoulnik, S.; Tendyke, K.; Shen, Y.; Wu, K.-M.; Mathieu, S.; Choi, H.-w.; Zhu, X.; Shimizu, H.; Kotake, Y.; Gerwick, W. H.; Uenaka, T.; Woodall-Jappe, M.; Nomoto, K. Apratoxin A shows novel pancreas-targeting activity through the binding of Sec 61. Mol. Cancer Ther. 2016, 15, 1208−1216.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Takayuki Doi: 0000-0002-8306-6819 Present Address §

Y.M.: Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, 514-8507, Japan. Notes

The authors declare the following competing financial interest(s): A patent application has been submitted based on the results described in this manuscript. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Prof. Yoshiteru Oshima and Dr. Teigo Asai at the Graduate School of Pharmaceutical Sciences in Tohoku University for allowing us to use their facility for the cell-based assay. The authors also thank Prof. Motoki Takagi at Fukushima Medical University for evaluation of sensitivity to different cancer cell lines. Financial contribution from JSPS KAKENHI Grant JP15H05837 in Middle Molecular Strategy, Grant JP26282208, and the Platform Project for Supporting in Drug Discovery and Life Science Research from Japan Agency for Medical Research and Development (AMED) are gratefully acknowledged.

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ABBREVIATIONS USED moCys, α,β-unsaturated modified cysteine residue; 3D, threedimensional; Bph, biphenylalanine; Pro, proline; MeIle, N-methylisoleucine; MeAla, N-methylalanine; Tyr(Me), O-methyltyr6764



Article

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