Improved Total Synthesis and Biological Evaluation of Coibamide A

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Cite This: J. Med. Chem. 2018, 61, 8908−8916

Improved Total Synthesis and Biological Evaluation of Coibamide A Analogues Guiyang Yao,†,§,∥ Wei Wang,†,∥ Lijiao Ao,†,‡,∥ Zhehong Cheng,†,‡ Chunlei Wu,† Zhengyin Pan,† Ke Liu,† Hongchang Li,† Wu Su,*,† and Lijing Fang*,† †

J. Med. Chem. 2018.61:8908-8916. Downloaded from pubs.acs.org by UNIV OF TEXAS AT EL PASO on 10/16/18. For personal use only.

Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China ‡ Shenzhen College of Advanced Technology, University of Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China S Supporting Information *

ABSTRACT: To enable the large-scale synthesis of coibamide A, we developed an improved synthetic strategy for this class of cyclodepsipeptide. The versatility of the synthetic procedure was demonstrated by the preparation of a series of designed coibamide A analogues, which enabled the preliminary structure−activity relationship (SAR) studies for this compound. Although most modifications of coibamide A resulted in decrease or loss of the antiproliferativity, we found that versatile substitution at position 3 was well tolerated. Remarkably, a simplified analogue, [MeAla3-MeAla6]-coibamide (1f), not only showed nearly the same inhibition as coibamide A against the tested cancer cells but also significantly inhibited tumor growth in vivo. The improved synthetic strategy and the relevant trends of SAR disclosed in this study will be valuable for further optimization of the overall profile of coibamide A.

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INTRODUCTION Since the first reports on cyclodepsipeptides in the 1960s, these compounds have gained great attention due to their broad spectrum of biological functions, which spans antitumor, antibiotic, antifungal, immunosuppressant, anti-inflammatory, anti-HIV, and antimalarial activities.1−10 As a highly Nmethylated cyclodepsipeptide, coibamide A was initially isolated by McPhail and co-workers from a marine filamentous cyanobacterium (Figure 1). It exhibited high inhibitory activities (in the nanomolar range) against a number of cancer cell lines.11 Although the cellular and molecular mechanisms of coibamide A remained unknown, it was found that this compound could inhibit the proliferation, migration, and invasion of cancer cells, induce G1 arrest, and promote subsequent cell death.12−14 As a consequence of its potent antiproliferative activity and distinct mechanism of action, coibamide A is regarded as a promising lead agent in cancer drug discovery. Due to the difficulties of isolation from the natural sources, the development of chemical synthesis of coibamide A and its derivatives is highly desired for further pharmaceutical research and development. Structurely, coibamide A is a complex cyclodepsipeptide that has two ester bonds, a proteogenic residue (Ala), and 10 nonproteogenic residues [Me2Val, two © 2018 American Chemical Society

Figure 1. Revised structure of coibamide A (1).

MeSer(Me), two MeLeu, MeThr, MeIle, Tyr(Me), MeAla, and an α-hydroxy acid (HIV)].11 In 2014, the proposed structure of coibamide A was synthesized by He et al. using a [(4 + 1) +3 + 3]-peptide fragment-coupling strategy in solution phase, but the analytical data and biological activity of the synthetic compound were inconsistent with those reported for natural Received: July 20, 2018 Published: September 24, 2018 8908

DOI: 10.1021/acs.jmedchem.8b01141 J. Med. Chem. 2018, 61, 8908−8916

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coibamide A.15 Then, Nabika et al. reported the synthesis of [D-MeAla11]-epimer of coibamide A by macrolactonization between the hydroxyl group of MeThr5 and C-terminal MeAla11 in low yield.16 Recently, our group performed the total synthesis of coibamide A by macrolactamization at Ala8MeIle7 junction, which also revised two stereochemical assignments of the originally proposed structure (Figure 1).17 Snyder et al. later confirmed that our revision, in which both the macrocycle [MeAla11] and the side chain [HIV2] residues were inverted from L to D, was consistent with the authentic natural product and the computational modeling.18 To enable the large-scale synthesis and optimize the overall profile of this molecule, we further elaborate on the synthesis of coibamide A and its derivatives and report herein an improved synthetic strategy and the biological evaluation of a series of analogues of the original structure.

phosphonium reagent PyAOP,27,28 and symmetric anhydride couplings utilizing DCC as the activating agent.29,30 Among them, BTC has shown superiority in the couplings of Nmethylated or aromatic amino acids by converting them into highly reactive and sterically unhindered acid chlorides in situ.31−33 We also demonstrated that increasing the molar ratio of BTC to the amino acids and addition of 1 equiv of HOAt to the BTC activated intermediate could significantly improve the coupling efficiency of unmethylated amino acids.34,35 However, it was envisioned that BTC would not work properly in the coupling of Fmoc-N-Me-Thr-OH because the hydroxyl group is susceptible to side reactions during the strong activation with BTC. Therefore, a series of different protocols were investigated to optimize the coupling of Fmoc-N-Me-Thr-OH and the reagent set of BTC/collidine was used to activate all of the remaining amino acids of coibamide A in this study. The initial amino acid derivatives were synthesized in solution phase prior to incorporation into the peptides on the solid support. As shown in Scheme 1, Fmoc-N-Me-Ser(Me)-

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RESULTS AND DISCUSSION Improved Total Synthesis of Coibamide A. First our efforts were focused on determining the optimal resin for the solid-phase peptide synthesis (SPPS) and thus the cyclization point. In our previous total synthesis of coibamide A, aryl hydrazide resin was chosen because the aryl hydrazide linker is stable in both strongly basic and acidic conditions,19 thus allowing the use of an Fmoc-based solid-phase method that is also compatible with trifluoroacetic acid (TFA) deprotection.17 Therefore, the commercially available amino acid, Fmoc-Nmethyl-O-tert-butyl-L-threonine could be applied for successive solid-phase assembly of the main peptidyl chain followed by removal of the tert-butyl (tBu) group with TFA and introduction of MeAla via the ester bond formation for elongation of the branched chain. Another benefit was that 2,5diketopiperazine [DKP, Tyr(Me)10-MeAla11] formation20 during the base-induced removal of Fmoc group from Tyr(Me)10 residue could be avoided by the use of BocTyr(Me)-OH and subsequent removal of the Boc-protecting group under acidic conditions. However, oxidative cleavage of the aryl hydrazide resin was not as effective as expected, possibly because acylation of the hydrazide linker21 occurred partly during the coupling steps with the highly reactive acyl chloride intermediates. Therefore, the 2-chlorotrityl resin (2CTC resin) was selected as the solid support in the current study because it allowed the release of the linear peptide under very mild conditions.22 To avoid the DKP [Tyr(Me)10MeAla11] formation as mentioned above, the cyclization site was chosen at the junction between Tyr(Me)10-MeAla11 (Figure 1). As a consequence, the main peptidyl chain would be assembled by Fmoc-based SPPS starting from the Cterminus of Tyr(Me)10 and ending at the N-terminus of Me2Val1. The use of Fmoc-N-Me-Thr-OH with a free secondary hydroxyl group for MeThr5 residue would allow the formation of the branched peptidyl chain through solidphase esterification with MeAla11 since the hydroxyl group does not have to be protected during the amide bond formation.23 Since N-methylated amino acids are generally difficult to acylate, synthesis of a peptide containing multiple dense Nmethylated amides is very challenging.24 The coupling of Nmethylated amino acids on solid phase often occurs with low yields as well as racemized products. To resolve these problems, various coupling conditions have been developed involving the use of bis(trichloromethyl) carbonate (BTC), benzotriazole-based reagents HATU25 and DIC/HOAt,26

Scheme 1. Synthesis of Fmoc-N-Me-Ser(Me)-OH (2) and Fmoc-N-Me-Val-D-HIV-OH (7)

OH (2) was prepared from Boc-Ser-OH (3) in three steps. Deprotonation of 3 was undertaken with NaHMDS followed by N,O-dimethylation with MeI, providing Boc-N-Me-Ser(Me)-OH (4). Then the Boc group was removed with TFA in CH2Cl2 and the amino group was reprotected with Fmoc-Cl to give Fmoc-N-Me-Ser(Me)-OH (2), a suitable building block for solid phase peptide synthesis. The synthesis of Fmoc-NMe-Val-D-HIV-OH (7) was achieved by following the published procedure17 with minor modifications as depicted in Scheme 1. Allyl ester 5 was prepared by selective Oallylation of (S)-(+)-2-hydroxy-3-methylbutyric acid with allyl bromide and Cs2CO3. To invert the stereochemistry of the hydroxyl group, Mitsunobu reaction of compound 5 and Fmoc-N-Me-Val-OH (6) was carried out in the presence of PPh3/DIAD. After removal of the allyl group with Pd(PPh3)4 and 1,3-dimethylbarbituric acid (DMBA), the second building block 7 was obtained in good yield. We next commenced the assembly of linear peptide 12 on the solid support with the attachment of the carboxyl group of Fmoc-Tyr(Me)-OH on the 2-CTC resin in the presence of N,N-diisopropylethylamine (DIPEA) (Scheme 2). A relatively moderate load level of 0.40 mmol/g was used to facilitate the N-methylated amide bond formation. To avoid truncated products, acetic acid was then added to cap any of the remaining 2-chlorotrityl chloride on the resin after the initial loading. As expected, the Fmoc-based solid phase synthesis of pentapeptide 9 proceeded smoothly employing BTC/collidine condition for the coupling of N-methylated amino acids, Fmoc-N-Me-Leu-OH, Fmoc-N-Me-Ile-OH as well as Fmoc-NMe-Ser(Me)-OH (2), and BTC/collidine/HOAt condition for the coupling of unmethylated amino acid, Fmoc-Ala-OH. The 8909

DOI: 10.1021/acs.jmedchem.8b01141 J. Med. Chem. 2018, 61, 8908−8916

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Scheme 2. Synthesis of Coibamide A (1)a

a

Reagents and conditions: (a) Fmoc-Tyr(Me)-OH, DIPEA, DMF, then acetic acid, DIPEA, DMF; (b) 20% piperdine/DMF; (c) Fmoc-N-Me-LeuOH; BTC, collidine, DIPEA, THF; (d) Fmoc-Ala-OH, BTC, collidine, THF, HOAt, DIPEA, DMF; (e) Fmoc-N-Me-Ile-OH; BTC, collidine, DIPEA, THF; (f) Fmoc-N-Me-Ser(Me)-OH (2); BTC, collidine, DIPEA, THF; (g) Fmoc-N-Me-Thr-OH, PyAOP, OxymaPure, DIPEA, DMF; (h) Fmoc-N-Me-Leu-OH; BTC, collidine, DIPEA, THF; (i) Fmoc-N-Me-Ser(Me)-OH (2), BTC, collidine, DIPEA, THF; (j) Fmoc-N-Me-Val-DHIV-OH (7), BTC, collidine, DIPEA, THF; (k) aq HCHO, NaBH(OAc)3, DCE; (l) Fmoc-N-Me-D-Ala-OH; BTC, collidine, DMAP, DIPEA, THF; (m) TFE/AcOH/DCM (1/1/8); (n) EDCI, HOAt, DIPEA, DCM.

smoothly in the presence of DMAP and DIPEA, allowing the coupling to be accomplished in 1 h with a single-coupling cycle. As a result, this BTC method proved to be very useful in the formation of ester bonds on the solid phase, especially for the coupling N-methylated amino acids. After removal of the Fmoc group, cleavage from the resin using a mixture of TFE/AcOH/CH2Cl2, and subsequent semipreparative HPLC purification, linear peptide 13 was obtained in 66% yield. Then macrocyclization between Tyr(Me)10-MeAla11 was performed under selected conditions. The superiority of BOP-Cl over other carboxyl activating reagents has been demonstrated in several cyclization reactions involving the formation of N-methylated amide bonds.36 Surprisingly, poor yield (20%) was obtained when BOP-Cl/DIPEA was used as the activating reagents for the linear precursor 13. Among the other reagents investigated, DPPA/NaHCO3 or PyAOP/OxyPure/DIPEA also proved to be ineffective, resulting in the formation of several side products along with the desired one.25 Optimum cyclization results were achieved with EDCI/HOAt/DIPEA,27,28 affording the cyclized product in modest yield (48%) after purified by semipreparative RP-HPLC. The synthetic 1 was identical to natural coibamide A with respect to all spectroscopic data (Figure S1, Supporting Information) and biological activities. Thus, coibamide A was synthesized in an overall yield of 32%, 2-fold higher compared with our previous work.17 Biological Evaluation of Coibamide A Analogues and Structure−Activity Relationships. Having established an efficient synthetic strategy for this class of compounds, we then exploited it to perform the structure−activity relationship (SAR) studies. A series of coibamide A analogues were designed and synthesized following the optimized protocol depicted in Scheme 1 with minor modifications. For the synthesis of most of the analogues, one or more amino acids were either removed or replaced by another properly protected

coupling of Fmoc-N-Me-Thr-OH with the resin bound amine of MeSer(Me)6 proved to be challenging. The activation reagent BTC is not suitable for this amino acid because the free hydroxyl group is reactive toward BTC. A variety of coupling protocols investigated including the use of HATU/ DIPEA, DIC/HOAt, PyBOP/DIPEA, or BEP/DIPEA failed to drive the reaction to completion, while elimination or epimerization byproducts were detected under these conditions. Finally, the coupling was successfully achieved to form peptide 10 by using the reagent set PyAOP/OxymaPure/ DIPEA, which was reported to be very useful for the coupling of hindered amino acids.27,28 Then the synthesis of peptide 11 was accomplished by using the BTC/collidine condition for the coupling of Fmoc-N-Me-Leu-OH, Fmoc-N-Me-Ser(Me)OH, and Fmoc-N-Me-Val-D-HIV-OH (7). Finally, the Fmoc group of 11 was removed and the reductive methylation was conducted in the presence of formalin and NaBH(OAc)3 in 1,2-dichloroethane (DCE) to afford the desired linear decapeptidyl resin 12. In the following study, BTC-mediated esterification of Fmoc-N-Me-D-Ala-OH with the free secondary hydroxyl group of MeThr5 in resin-bound peptide 12 was examined. The building of an ester bond on the solid phase is always a synthetic challenge, especially in the exceptionally hindered environment such as that of coibamide A. In the previous work, we demonstrated that an acceptable yield could be achieved by using the symmetric anhydride formed by the reaction of the amino acid and DCC in a ratio of 2:1. In this study, we anticipated that the use of BTC could further improve the esterification efficiency by converting the amino acid into acid chloride which is more reactive and sterically less hindered compared with symmetric anhydride. Therefore, Fmoc-N-Me-D-Ala-OH was converted to Fmoc-N-Me-D-Ala-Cl in situ with BTC/collidine in THF and the reaction of FmocN-Me-D-Ala-Cl with the hydroxyl group of MeThr5 proceeded 8910

DOI: 10.1021/acs.jmedchem.8b01141 J. Med. Chem. 2018, 61, 8908−8916

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Figure 2. Structures of coibamide A analogues 1a−r.

amino acid. The substitutions on the N-terminal were introduced by acylation of the methylated amino group instead of the reductive methylation. Another set of coibamide A derivatives were obtained from the cyclized intermediates by amidation or esterification reactions with diverse reactants in solution phase. After semipreparative RP-HPLC purification and lyophilization, all of the analogues 1c−r (Figure 2) were provided in pure form and the analogues with Me2Val1 or MeVal1 residue as the N-terminus were used as their TFA salts. The cytotoxicity of these analogues was evaluated against

three human tumor cell lines [breast MDA-MB-231, NSCLC A549, and pancreas PANC-1] using the MTT assay. The results are listed in Table 1 in comparison to the GI50 (concentration at which the growth is inhibited by 50%) of 1. In the previous work, He et al. reported that the proposed structure of coibamide A, [L-HIV2-L-MeAla11]-diastereomer (1a), exhibited micromolar cytotoxicities (>10 μM) which was 4 orders of magnitude less active than the natural one.15 Nabika et al. demonstrated that the cytotoxicity of the [LHIV2]-epimer of coibamide A (1b) was 3.7- to 8.3-fold less 8911

DOI: 10.1021/acs.jmedchem.8b01141 J. Med. Chem. 2018, 61, 8908−8916

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To further explore the significance of the aliphatic substitution at position 3/6 and position 10, analogue 1j bearing two free hydroxyl groups, analogue 1i bearing a free hydroxyl group at position 3 and a tBu-protected hydroxyl group at position 6, and analogue 1k bearing a phenolic hydroxyl group at position 10 were synthesized. As a result, one hydroxyl analogue 1i showed slightly decreased cytotoxicity while dihydroxyl analogue 1j had significantly weaker cytotoxicity (GI50 > 2 μM). Free phenolic hydroxyl analogue 1k proved about 40 times less potent than coibamide A. The interesting findings indicate that aliphatic substitutions at position 6 and position 10 are required for the activity and versatile substitution at position 3 is well tolerated. Then, we wanted to explore the possibility of replacing the ester bond in the branched chain of coibamide A with an amide bond since amide bond is more stable than ester bond. The ester linkage of Me2Val1 and D-HIV2 was thus replaced by the amide bond between Me2Val1 and D-Val2, generating analogue 1l, which exhibited 80- to 90-fold reduced potency when compared to coibamide A. Analogue 1m with a Nmethylated amide bond between Me2Val1 and D-MeVal2 had the same level cytotoxicity with analogue 1l. Since the presence of the N-methylated amide bond in analogue 1m would not change the hydrogen bond pattern of the molecule, the decrease in activity is probably attributed to the different metabolic pattern of amide bonds in comparison with ester bond. Finally, to further explore the influence of the size and the N-terminal group of the peptidyl chain outside the macrocycle, a series of analogues 1n−q were designed and synthesized. The short chain analogue 1n with a free amino group as the Nterminal was inactive, while the lipophilic side chain analogue 1o proved 100 times less active than coibamide A, once again demonstrating that the branched peptidyl chain also plays an important role in activity. Substitution of one terminal Nmethyl group of Me2Val1 with proton (1p) or acylation of the terminal N-Me group with Boc-β-Ala-OH (1q) caused a significant reduction in activity (GI50 > 200 nM). However, removal of the Boc protecting group of 1q resulted in the total loss of the activity (1r). As observed for analogues 1n, 1p, and 1r, although the cationic character did not change, the presence of the terminal NH group may alter the hydrogen bond pattern of the molecule, preventing it from adopting the preferred bioactive conformation. In Vivo Activities of [MeAla3-MeAla6]-Coibamide (1f). Since [MeAla3-MeAla6]-coibamide (1f) exhibited nearly the same potency as that of the natural cyclodesipeptide against all three cancer cell lines, it could be used as a simplified analogue of coibamide A for pharmaceutical research. Therefore, we evaluated the in vivo antitumor activity and the toxicity profile of 1f using a human tumor xenograft model. Balb/c nu mice carrying MDA-MB-231 tumor cells at right flank were treated with 0.3 mg/kg 1f by subcutaneous injection every 2 days to determine the antitumor activity in vivo. From day 9, the tumor size of the mice treated with 1f (n = 7) was significantly smaller (P < 0.05) than vehicle control (n = 4) (Figure 3A). At the end of treatment, the tumor mass of treated group was nearly 2-fold smaller than vehicle control (Figure 3C). These results demonstrated the effective tumor suppression of 1f in animals. In the previous study,13 coibamide A (1) at the dose of 0.3 mg/kg inhibited U-87 MG tumor growth in a nude mouse xenograft model of glioblastoma, but it also caused rapid weight loss in some treated mice. In our research, no

Table 1. Comparative Cytotoxicities for Coibamide A Analogues against MDA-MB-231, A549, and PANC-1 Cancer Cell Lines (GI50, nM) compd

MDA-MB-231

A549

PANC-1

1 1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 1m 1n 1o 1p 1q 1r

5.0 ± 2.5 >16000b 545b 7518 ± 245 10809 ± 545 2662 ± 212 5.1 ± 2.2 5.3 ± 3.0 61.6 ± 0.9 20.8 ± 5.7 2056 ± 151 183 ± 12 450 ± 62 415 ± 76 >16000 470 ± 109 236 ± 29 239 ± 90 >16000

5.4 ± 0.9 22800b 19b 20091 NDc 1995 7.3 ± 3.8 12.4 ± 2.1 81.7 ± 5.6 194 ± 55 NDc 222 ± 57 473 ± 65 511 ± 40 NDc 733 ± 125 360 ± 16 443 ± 70 NDc

3.1 NDc NDc 12417 NDc 1906 7.0 32.9 124 46.3 2178 277 601 723 NDc 828 204 415 NDc

a

Values are the mean ± SD from three independent experiments. Published values from refs 15 and 16. cNot determined.

a

b

than that of natural coibamide A.16 To further explore the stereochemical influence on the pharmacological activity, we synthesized [ L -MeAla11]-epimer (1c), [ L -HIV2- D -alloMeThr5]-diastereomer (1d), and [L-HIV2-D-MeSer(Me)3]diastereomer (1e) of coibamide A. All of them showed low cytotoxicity (GI50 > 1.9 μM) against the tested cancer cell lines. Compared with coibamide A (1), the stereotopical changes of the residues belonging to the macrocycle (1c and 1d) resulted in a loss of 3 orders of magnitude in cytotoxicity, indicating that the macrocycle plays a crucial role in activity. However, the activity is not attributable exclusively to the pharmacophore. As illustrated by the activities obtained for 1e, inversion of the configuration of HIV2 and MeSer(Me)3, two residues outside the macrocycle, also caused a significant reduction in activity (GI50 > 2 μM). Collectively, these findings suggest that natural coibamide A has a well-defined conformational structure and is sensitive to the backbone stereotopical modifications. In the following work, we wanted to explore the possibility of replacing the difficultly obtained Fmoc-N-Me-Ser(Me)-OH with commercially available amino acids such as Fmoc-N-MeAla-OH, Fmoc-N-Me-Ser(tBu)-OH or Fmoc-N-Me-Lys(Boc)OH, to examine how this would affect the pharmacological activity. To facilitate the synthesis, analogue 1f/1g/1h was prepared by substitution of MeSer(Me)3 with MeAla/ MeSer(tBu)/MeLys(Boc) and substitution of MeSer(Me)6 with MeAla. As we expected, the analogues 1f and 1g showed almost the same cytotoxicity toward breast MDA-MB-231 cell as observed with coibamide A. And analogue 1h with MeLys(Boc) at position 3 displayed only 12 times less cytotoxicity than coibamide A. Since the alanine scans at positions 3 and 6 revealed that unusual OMe groups were not necessary for activity, MeSer(Me)3 and MeSer(Me)6 were substituted by MeAla in some analogues used for the SAR studies. 8912

DOI: 10.1021/acs.jmedchem.8b01141 J. Med. Chem. 2018, 61, 8908−8916

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the ULTIMAT 3000 instrument (DIONEX). UV absorbance was measured using a photodiode array detector at 220 and 254 nm. The RP-HPLC gradient was started at 10% of B (MeCN), then increased to 100% of B over 30 min (A: 0.1% TFA in water). 1H NMR (13C NMR) spectra were recorded with a Bruker AV400 at 400 (100) MHz. Chemical shifts are referenced to either tetramethylsilane as an internal standard or the signals resulting from the residual solvent. High resolution mass spectra were measured with an ABI Q-star Elite. (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-methoxypropanoic Acid (2). To a stirring solution of Boc-L-Ser-OH (3) (2.05 g, 10 mmol) in anhydrous THF (40 mL) at −78 °C was added sodium bis(trimethylsilyl)amide (11 mL, 11 mmol, 1.0 M in THF). The mixture was stirred at −40 °C for 0.5 h before iodomethane (2.05 mL, 33 mmol) was added. The resulting suspension was stirred at 0 °C for 1 and 17 h at room temperature. The reaction mixture was diluted with water (40 mL) and EtOAc (40 mL), concentrated under reduced pressure, and acidified with 10% citric acid to pH 4. The aqueous layer was extracted with EtOAc (4 × 40 mL), and the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was cooled to 0 °C, and TFA in DCM (50% v/v) (10 mL) was added. After stirring for 2 h, the reaction mixture was concentrated under vacuum and the residue was dissolved in dioxane (20 mL). Then, sodium carbonate aqueous solution (10% w/w, 40 mL) and Fmoc-Cl (2.84g, 11 mmol) in dioxane (40 mL) were added and the mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with water (40 mL), acidified to pH 3 with 1 N hydrochloric acid aqueous solution and extracted with EtOAc (2 × 100 mL). The organic extracts were combined, dried over MgSO4, filtered, and concentrated under vacuum. Flash column chromatography of the crude product (ethyl acetate/hexanes, 40%) afforded compound 2 (3.02 g, 85%) as a pale-yellow oil. 1H NMR and HRMS data were identical to the data previously reported.16 (R)-2-(((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-methylbutanoyl)oxy)-3-methylbutanoic Acid (7). Step 1. DIAD (416 μL, 2.1 mmol) was added to a solution of 5 (291 mg, 2 mmol), Fmoc-N-Me-Val-OH (6) (706 mg, 2 mmol), and PPh3 (525 mg, 2 mmol) in THF (10 mL) at room temperature. After being stirred overnight, the reaction mixture was concentrated under reduced pressure, and the crude product was purified by silica gel chromatography (hexane/EtOAc) to afford a colorless oil. 1H NMR (400 MHz, CDCl3) δ 0.73−1.10 (m, 12H), 2.30−2.20 (m, 2H), 2.92 (s, 1.26H), 2.95 (s, 1.77H), 4.20−4.72 (m, 6H), 4.85 (d, J = 4.2 Hz, 0.45H), 4.89 (d, J = 4.2 Hz, 0.55H), 5.17−5.25 (m, 1H), 5.28 (dd, J = 2.8, 1.4 Hz, 0.45H), 5.32 (dd, J = 2.8, 1.4 Hz, 0.55H), 5.87 (ddt, J = 16.2, 10.5, 5.8 Hz, 1H), 7.30 (t, J = 7.4 Hz, 2H), 7.37 (t, J = 7.3 Hz, 2H), 7.65−7.58 (m, 2H), 7.74 (d, J = 7.5 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 16.87, 18.53, 18.61, 19.53, 27.11, 29.69, 430.13, 6.99, 63.47, 65.32, 67.42, 76.68, 118.47, 119.65,124.58, 124.79, 126.75, 127.36, 131.26, 141.02, 143.60, 156.65, 168.55, 170.26. HRMS (ESITOF) m/z: calcd for C29H36NO6 [M + H]+ 494.2537, found 494.2534. Step 2. DMBA (2.87g, 18.4 mmol) in THF (5 mL) was slowly added to a solution of the product obtained from step 1 (907 mg, 1.84 mmol) and Pd(PPh3)4 (213 mg, 0.184 mmol) in THF (30 mL) at room temperature. After being stirred for 1 h, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc and washed with saturated NH4Cl and brine. The organic phase was dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (hexane/EtOAc) to afford 7 as a colorless oil (700 mg, 84%). 1H NMR (400 MHz, CDCl3) δ 0.69−1.08 (m, 12H), 2.12−2.32 (m, 2H), 2.88 (s, 1.31H), 2.91 (s, 1.69H), 4.17 (d, J = 10.6 Hz, 0.45H), 4.21−4.31 (m, 1H), 4.38−4.58 (m, 2H), 4.64 (d, J = 10.3 Hz, 0.55H), 4.84 (d, J = 4.0 Hz, 0.45H), 4.89 (d, J = 4.0 Hz, 0.55H), 7.30 (t, J = 7.4 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.60 (d, J = 7.7 Hz, 2H), 7.76 (d, J = 7.5 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 16.91, 18.72, 19.63, 20.68, 27.28, 29.76, 30.41, 63.72, 67.80, 76.56, 119.82, 124.74, 124.92, 126.96, 127.55, 141.21, 143.69, 157.13, 170.46, 174.48.

Figure 3. [MeAla3-MeAla6]-coibamide (1f) inhibits subcutaneous xenograft tumor growth in vivo: (A) average tumor volume ± SD of 1f treated group (red, n = 7) and vehicle control (black, n = 4); (B) mean body weight ± SD of 1f treated group and vehicle control; (C) average tumor mass of treatment and control. All of the statistics significance values were calculated by one-way ANOVA: (∗∗) P < 0.01, (∗) P < 0.05.

significant weight loss and side effects were observed in 1f treated group at the same dose (Figure 3B), indicating that 1f might have lower toxicity than 1.

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CONCLUSIONS In summary, an improved synthetic strategy for the synthesis of coibamide A was developed. The key features of this strategy included the following: (1) the 2-CTC resin was selected as the solid support to overcome the low cleavage efficiency due to the acylation of the hydrazide linker; (2) efficient coupling of Fmoc-N-Me-Thr-OH in the presence of the free hydroxyl group was accomplished by using PyAOP/OxyPure/DIPEA reagents; (3) BTC-mediated esterification of Fmoc-N-Me-DAla-OH was applied for effective formation of the ester bond on the solid phase; (4) optimum macrocyclization between Tyr(Me)10-MeAla11 were achieved with EDCI/HOAt/ DIPEA. A new series of coibamide A analogues were synthesized, and the antitumor activities were evaluated by using the MTT assay. Our preliminary SAR studies demonstrated that natural coibamide A has a well-defined conformational structure and is very sensitive to the backbone modifications. Although most modifications resulted in significantly decreased activities, we found that versatile substitution at position 3 was well tolerated. Moreover, a simplified analogue [MeAla3-MeAla6]-coibamide (1f) not only exhibited similar inhibition as coibamide A against the tested cancer cells but also significantly suppressed tumor growth in vivo. Overall, the improved synthetic strategy and the relevant trends of the SAR disclosed in this study will be valuable for further optimization of the overall profile of coibamide A.

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EXPERIMENTAL SECTION

General Information. Analytical RP-HPLC was performed on an Agilent 1260 infinity system equipped with a DAD-UV detector using Agilent Poroshell 120, EC-C18 column (4.6 mm × 100 mm, 2.7um). The RP-HPLC gradient was started at 10% of B (MeCN), then increased to 100% of B over 20 min (A: 0.1% TFA in water) with a flow rate of 0.5 mL/min. The purity of the compounds (>95%) was determined by HPLC. Semipreparative RP-HPLC was performed on 8913

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HRMS (ESI-TOF) m/z: calcd for C26H32NO6 [M + H]+ 454.2224, found 454.2211. Solid-Phase Peptide Synthesis. The synthesis was carried out on a 0.16 mmol scale. Each cycle of amino acid addition involved deprotection, amino acids activation, coupling, and chloranil test. All couplings are performed with single coupling cycles. General Methods. Method A: Removal of Fmoc Group. A solution of 20% piperidine in DMF (3 mL) was added to the resin, and the resulting suspension was shaken for 10 min. Then the solution was removed from the resin. Again, a solution of 20% piperidine in DMF (3 mL) was added to the resin and the resulting suspension was shaken for another 10 min. The solution was drained, and the resin was washed with DMF (6 × 3 mL) and dry THF (3 mL). Method B: Coupling of N-Methylated Amino Acid. N-Methylated amino acid (4.0 equiv) and BTC (1.64 equiv) were dissolved in dry THF (2 mL). Collidine (12 equiv) was added dropwise to the THF solution. After activating for 1 min, the suspension was added to the deprotected resin followed by addition of DIPEA (20.0 equiv). The mixture was shaken for an appropriate time until a negative chloranil test was observed. After this time, the solution was drained and the resin was rinsed with DMF (4 × 3 mL). Method C: Coupling of Unmethylated Amino Acid. Unmethylated amino acid (4.0 equiv) and BTC (1.64 equiv) were dissolved in dry THF (1 mL). Upon dropwise addition of collidine (12 equiv), a mixture of HOAt (4.0 equiv), DIPEA (20 equiv) in DMF (1 mL) was added. The resulting solution was stirred for 3 min before it was transferred to the deprotected resin. The mixture was shaken until a negative chloranil test was observed. After this time, the resin was rinsed with DMF (4 × 3 mL). Method D: Chloranil Test. During the coupling reaction, a few resin beads were taken out and rinsed with DMF (2 × 1 mL). To the resin were added 2 drops of a 2% solution of acetaldehyde and 2 drops of a 2% solution of chloranil in DMF. The resulting suspension was allowed to stand for 5 min at room temperature. Blue- to greenstained beads indicated the presence of secondary amines. Method E: Coupling of Fmoc-N-Me-Thr-OH. To a solution of Fmoc-N-Me-Thr-OH (6.0 equiv), PyAOP (6.0 equiv), and OxymaPure (6.0 equiv) was added DIPEA (12 equiv). After activating for 3 min, the solution was poured to the deprotected peptidyl resin. The mixture was shaken for an appropriate time until a negative chloranil test was observed. After this time, the solution was drained and the resin was rinsed with DMF (4 × 3 mL). Method F: Esterification. N-Methylated amino acid (4.0 equiv) and BTC (1.64 equiv) were dissolved in dry THF (2 mL). Collidine (12 equiv) was added dropwise to the THF solution. After activating for 2 min, the suspension was added to the deprotected resin followed by addition of DIPEA (20.0 equiv) and DMAP (1 equiv). The reaction mixture was shaken for 1 h to complete the coupling. After this time, the solution was drained and the resin was rinsed with DMF (4 × 3 mL). Method G: Reductive Amination for N-Terminal Methylation. Formalin (20.0 equiv) in DCE (2 mL) and NaBH(OAc)3 (15.0 equiv) in DCE (2 mL) were added to the resin (1 equiv). After the reaction mixture was shaken for 3 h at room temperature, the resulting peptidyl resin was washed sequentially with DCE (3 × 3 mL), MeOH (3 × 3 mL), and dry THF (2 × 3 mL). Synthesis of Peptidyl Resin 8. 2-CTC resin (400 mg, 0.98 mmol/g) was preswelled for 20 min in DCM in a manual solid phase peptide synthesis vessel (10 mL). After the solvent was drained, Fmoc-Tyr(Me)-OH (97 mg, 0.16 mmol) and DIPEA (300 μL, 1.8 mmol) in dry DMF (3 mL) were added to the resin. The mixture was agitated for 2 h and before the solvent was drained. The resin was rinsed with DMF (4 × 3 mL) and dry DMF (3 mL). Then a mixture of acetic acid (200 μL, 1.32 mmol) and DIPEA (500 μL, 3 mmol) in dry DMF (2 mL) was added to cap the remaining 2-chlorotrityl chloride on the resin. The mixture was agitated for 0.5 h. Then the solvent was drained and the resin was washed with DMF (4 × 3 mL). The resin loading was determined to be 0.40 mmol/g in the case of complete reaction of Fmoc-Tyr(Me)-OH with the resin.

Synthesis of Peptidyl Resin 9. The Fmoc group of peptidyl resin 8 (400 mg, 0.40 mmol/g, 0.16 mmol, 1 equiv) was removed according to method A. Fmoc-N-Me-Leu-OH (227 mg, 0.64 mmol, 4 equiv) was coupled to the deprotected resin according to method B. The Fmoc group of the resulting resin was removed according to method A. Fmoc-Ala-OH (197 mg, 0.64 mmol) was coupled to the deprotected resin according to method C. Removal of Fmoc group and amino acid [Fmoc-N-Me-Ile-OH (227 mg, 0.64 mmol, 4 equiv), and Fmoc-N-Me-Ser(Me)-OH (228 mg, 0.64 mmol, 4 equiv) coupling procedures were repeated until peptidyl resin 9 was obtained. Synthesis of Peptidyl Resin 10. The Fmoc group of peptidyl resin 9 was removed according to method A. Fmoc-N-Me-Thr-OH (227 mg, 0.64 mmol) was coupled to the deprotected resin according to method E. Synthesis of Peptidyl Resin 11. The Fmoc group of peptidyl resin 10 was removed according to method A. Amino acid [Fmoc-NMe-Leu-OH (227 mg, 0.64 mmol, 4 equiv), Fmoc-N-Me-Ser(Me)OH (228 mg, 0.64 mmol, 4 equiv), and Fmoc-N-Me-Val-D-HIV-OH (290 mg, 0.64 mmol, 4 equiv)] coupling procedures were repeated until peptidyl resin 11 was obtained. Synthesis of Peptidyl Resin 12. The Fmoc group of peptidyl resin 11 was removed according to method A. Reductive methylation was then conducted according to method G. Synthesis of Peptide 13. According to method F, to a solution of Fmoc-N-Me-D-Ala-OH (312 mg, 0.96 mmol) and BTC (114 mg, 0.38 mmol) in THF (2 mL) was added collidine (441 μL, 3.3 mmol) at 0 °C. The resulting mixture was stirred at room temperature for 2 min. The suspension was then transferred to peptidyl resin 12 followed by the addition of DIPEA (834 μL, 4.8 mmol) and DMAP (24.5 mg, 0.2 mmol). The reaction mixture was agitated for 1 h to complete the coupling. Then the resin was washed with DCM (2 × 3 mL) and DMF (4 × 3 mL). The Fmoc group was removed according the method A. A solution of TFE and HOAc in DCM (TFE/HOAc/ DCM = 1/1/8) was added to the deprotected resin. The resulting mixture was shaken at room temperature for 24 h. Then the resin was removed by filtration through a disposable propylene filter and washed with CH2Cl2 (20 mL). The organic solution was concentrated under reduced pressure and the residue was purified by semipreparative RP-HPLC. After lyophilization, peptide 13 was obtained as a white powder (138 mg, 66% with respect to the first loading of the resin). HRMS (ESI) m/z, calcd for C65H113N10O17 [M + H]+ exact mass, 1305.8280; found, 1305.8274. Synthesis of Coibamide A (1). To a solution of EDCI (19.2 mg, 0.10 mmol), HOAt (13.6 mg, 0.10 mmol), and DIPEA (70 μL, 0.40 mmol) in CH2Cl2 (30 mL) was added the linear peptide 13 (13 mg, 0.01 mmol) in CH2Cl2 (1 mL) at 0 °C. After being stirred at 0 °C for 1 h, the reaction mixture was allowed to warm up to room temperature and the stirring was continued for 36 h. The reaction mixture was concentrated in vacuo and the residue was purified by semipreparative RP-HPLC. After being concentrated, the residue was diluted with CH2Cl2 (20 mL) and washed successively with saturated NaHCO3 (5 mL), water (5 mL), and brine (5 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was dried under vacuum to give the final product 1 (6.2 mg, 48%). The resulting synthetic compound and the previous reported compound17 revealed a perfect overlap of the NMR spectra (Figure S1). Synthesis of Analogues 1c−r. Compounds 1c−r were prepared using similar procedures for the preparation of compound 1 as described above. All analogues were purified by semipreparative RPHPLC. Additional synthetic procedures and chemical data for these compounds are available in Supporting Information. Cell Growth Inhibition Assay. Breast cancer MDA-MB-231 cells, lung cancer A549 cells, and pancreas cancer PANC-1 cells were plated in flat-bottomed 96-well microplates. Twelve hours after seeding, fresh medium containing different concentration gradient of compounds 1c−r or vehicle control (DMSO) was added. Cells were further incubated for 72 h and then treated with medium containing MTT (final concentration 0.5 mg/mL) and incubated for another 4 h. Then the medium was removed carefully, and DMSO (100 μL) was 8914

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added to each well. After the samples were shaken gently for 2 min and incubated at 37 °C for 30 min, the absorbance in each well at 490 nm was measured by microtiter plate reader (Thermol Multiskan GO). Experiments were independently repeated three times, and growth inhibition rate was calculated as follows: growth inhibition rate (%) = [1 − OD(compounds)/OD(controls)] × 100. The growth inhibition (GI50) for each compound was defined as the concentration of drug leading to a 50% reduction in cancer cells compared with controls. These cell lines were purchased from Core Facility of Stem Cell Research, Shanghai Institute of Biological Science CAS. Nude Mice Xenograft Experiments. Balb/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. 1 ×107 MDA-MB-231 tumor cells were subcutaneously injected in the right flank. When tumor size reached ∼100 mm3 (L × W × 1/2W), 0.3 mg/kg [MeAla3-MeAla6]-coibamide (1f) was subcutaneously injected around the tumor every 2 days. Equal volume of 20% DMSO/80% sterile saline solution was used as vehicle control.13 Tumor size and animal weight were measured before each injection. After 11 injections, mice were sacrificed and tumors were weighed and peeled for further experiment. All experiments were performed under the guideline of Institutional Ethical Committee of Animal Experimentation of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.

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N,N′-diisopropylcarbodiimide; DIAD, diisopropyl azodicarboxylate; DIPEA, N,N′-diisopropylethylamine; DMAP, 4dimethylaminopyridine; DMBA, 1,3-dimethylbarbituric acid; DMF, N,N′-dimethylformamide; DPPA, diphenylphosphoryl azide; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOAt, 1-hydroxy-7-aza-benzotriazole; OxymaPure, ethyl cyanoglyoxylate-2-oxime; PPh3, triphenylphosphine; PyAOP, (3-hydroxy-3H-1,2,3-triazolo[4,5-b]pyridinato-O)tri-1pyrrolidinylphosphonium hexafluorophosphate; PyBOP, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; TFE, 2,2,2-trifluorethanol; TFA, trifluoroacetic acid; THF, tetrahydrofuran

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(1) Kuranaga, T.; Enomoto, A.; Tan, H.; Fujita, K.; Wakimoto, T. Total synthesis of theonellapeptolide Id. Org. Lett. 2017, 19, 1366− 1369. (2) Murai, M.; Kaji, T.; Kuranaga, T.; Hamamoto, H.; Sekimizu, K.; Inoue, M. Total synthesis and biological evaluation of the antibiotic lysocin E and its enantiomeric, epimeric, and N-demethylated analogues. Angew. Chem., Int. Ed. 2015, 54, 1556−1560. (3) Martin, M. J.; Rodriguez-Acebes, R.; Garcia-Ramos, Y.; Martinez, V.; Murcia, C.; Digon, I.; Marco, I.; Pelay-Gimeno, M.; Fernandez, R.; Reyes, F.; Francesch, A. M.; Munt, S.; Tulla-Puche, J.; Albericio, F.; Cuevas, C. Stellatolides, a new cyclodepsipeptide family from the sponge Ecionemia acervus: isolation, solid-phase total synthesis, and full structural assignment of stellatolide A. J. Am. Chem. Soc. 2014, 136, 6754−6762. (4) Kikuchi, M.; Konno, H. Total synthesis of callipeltin B and M, peptidyl marine natural products. Org. Lett. 2014, 16, 4324−4327. (5) Pelay-Gimeno, M.; Garcia-Ramos, Y.; Jesus Martin, M.; Spengler, J.; Molina-Guijarro, J. M.; Munt, S.; Francesch, A. M.; Cuevas, C.; Tulla-Puche, J.; Albericio, F. The first total synthesis of the cyclodepsipeptide pipecolidepsin A. Nat. Commun. 2013, 4, 2352−2362. (6) Sleebs, M. M.; Scanlon, D.; Karas, J.; Maharani, R.; Hughes, A. B. Total synthesis of the antifungal depsipeptide petriellin A. J. Org. Chem. 2011, 76, 6686−6693. (7) Pohle, S.; Appelt, C.; Roux, M.; Fiedler, H. P.; Sussmuth, R. D. Biosynthetic gene cluster of the non-ribosomally synthesized cyclodepsipeptide skyllamycin: deciphering unprecedented ways of unusual hydroxylation reactions. J. Am. Chem. Soc. 2011, 133, 6194− 6205. (8) Tripathi, A.; Puddick, J.; Prinsep, M. R.; Rottmann, M.; Tan, L. T. Lagunamides A and B: cytotoxic and antimalarial cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 2010, 73, 1810−1814. (9) Zampella, A.; Sepe, V.; Luciano, P.; Bellotta, F.; Monti, M. C.; D’Auria, M. V.; Jepsen, T.; Petek, S.; Adeline, M. T.; Laprevote, O.; Aubertin, A. M.; Debitus, C.; Poupat, C.; Ahond, A. Homophymine A, an anti-HIV cyclodepsipeptide from the sponge Homophymia sp. J. Org. Chem. 2008, 73, 5319−5327. (10) Boger, D. L.; Keim, H.; Oberhauser, B.; Schreiner, E. P.; Foster, C. A. Total synthesis of HUN-7293. J. Am. Chem. Soc. 1999, 121, 6197−6205. (11) Medina, R. A.; Goeger, D. E.; Hills, P.; Mooberry, S. L.; Huang, N.; Romero, L. I.; Ortega-Barria, E.; Gerwick, W. H.; McPhail, K. L. Coibamide A, a potent antiproliferative cyclic depsipeptide from the Panamanian marine cyanobacterium Leptolyngbya sp. J. Am. Chem. Soc. 2008, 130, 6324−6325. (12) Wan, X.; Serrill, J. D.; Humphreys, I. R.; Tan, M.; McPhail, K. L.; Ganley, I. G.; Ishmael, J. E. ATG5 promotes death signaling in response to the cyclic depsipeptides coibamide A and apratoxin A. Mar. Drugs 2018, 16, 77. (13) Serrill, J. D.; Wan, X.; Hau, A. M.; Jang, H. S.; Coleman, D. J.; Indra, A. K.; Alani, A. W.; McPhail, K. L.; Ishmael, J. E. Coibamide A, a natural lariat depsipeptide, inhibits VEGFA/VEGFR2 expression

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b01141. NMR and HRMS spectra of the new compounds and synthetic procedures of analogues 1c−r (PDF) Molecular formula strings and some data (CSV)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*W.S.: e-mail, [email protected]; phone, (+86)755-86585203. *L.F.: e-mail, [email protected]; phone, (+86)755-86392257. ORCID

Wu Su: 0000-0001-9958-3434 Present Address

§ G.Y.: Department of Chemistry, Technical University of Berlin, Strasse des 17, Juni 124, 10623 Berlin, Germany.

Author Contributions ∥

G.Y., W.W., and L.A. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors appreciate Peking University Shenzhen Graduate School for the assistance of Mass facility. This work was supported by the National Natural Science Foundation of China (Grants 21672254, 21778068, 21502219, and 21432003) and Shenzhen Sciences & Technology Innovation Council (Grants JCYJ20170413165916608 and JCYJ2017081815358196).

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ABBREVIATIONS USED BEP, 2-bromo-1-ethylpyridinium tetrafluoroborate; BOP-Cl, phosphoric acid bis(2-oxooxazolidide) chloride; BTC, bis(trichloromethyl)carbonate; CH3CN, acetonitrile; DCC, N,N′dicyclohexylcarbodiimide; DCM, dichloromethane; DIC, 8915

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