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Nov 5, 2015 - ... Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa ..... Okinawa Prefecture, Japan, at a depth of 0−1 m in March 2013. Th...
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Isolation and Structure of Kurahyne B and Total Synthesis of the Kurahynes Shinichiro Okamoto, Arihiro Iwasaki, Osamu Ohno, and Kiyotake Suenaga* Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa 223-8522, Japan S Supporting Information *

ABSTRACT: Kurahyne B (2), a new analogue of kurahyne (1), was isolated from the marine cyanobacterium Okeania sp. Its gross structure was elucidated based on spectroscopic analyses, and the absolute configuration was established by total synthesis. Kurahyne B (2) inhibited the growth of both HeLa and HL60 cells, with IC50 values of 8.1 and 9.0 μM, respectively. The growth-inhibitory activity of kurahyne B was the same as kurahyne (1). In parallel, the first total synthesis of kurahyne (1) was also achieved.

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Cosmosil Cholester, MeCN−H2O) to afford 2.2 mg of kurahyne B (2). The molecular formula of 2 was found to be C46H76N6O7 by HRESIMS, which indicated that 2 was a nor-derivative of 1. The NMR spectral data for 2 are summarized in Table 1. The 1 H NMR spectrum of kurahyne B (2) was very similar to that of kurahyne (1). Further analyses of the 1H NMR spectrum revealed the presence of three singlets corresponding to Nmethyl amide substituents (δH 3.16, 3.08, 3.07), one vinyl methyl group (δH 1.85), and six methine groups corresponding to α positions of amino acids (δH 5.29, 5.21, 5.06, 4.77, 4.66, 4.61). Detailed analysis of the 1H NMR, 13C NMR, COSY, HMQC, HMBC, and NOESY spectra revealed the presence of the following seven partial structures: one proline, two Nmethylvalines, one N-methylisoleucine, one isoleucine, 2-(1oxo-propyl)pyrrolidine (Opp), and 2-methyloct-2-en-7-ynoic acid (Moya). The sequence of these partial structures was determined based on NOESY data (Table S1 and Figure 1). Five NOESY correlations, H-7 of Opp/H-2 of Pro, H-5 of Pro/H-2 of N-MeVal-1, N-Me of N-Me-Val-1/H-2 of N-Me-Val-2, N-Me of NMe-Val-2/H-2 of N-Me-Ile, and N-Me of N-Me-Ile/H-2 of Ile, connected these six residues, Opp-Pro-N-Me-Val-1-N-Me-Val2-N-Me-Ile-Ile. On the basis of the molecular formula of 2, we clarified that the Moya moiety was bound to the Ile residue. Finally, a NOESY correlation, H-4 of Moya/H-9 of Moya, and the chemical shift of the vinyl methyl carbon (δC 13.0, C-9 of Moya) supported an E geometry for the C-2/C-3 olefinic bond

econdary metabolites produced by marine organisms have attracted attention due to their remarkable structures and biological activities.1 In particular, some of marine cyanobacterial compounds, such as dolastatin 102 and cryptophycin,3 exhibit antitumor activity and have been studied as drug leads. In an effort to discover novel bioactive substances, we have investigated the constituents of marine cyanobacteria and have reported several compounds such as jahanyne,4 bisebromoamide,5 and biselyngbyaside.6 One of these compounds, kurahyne (1), is an apoptosis-inducing lipopeptide that was isolated from a marine cyanobacterial assemblage in 2014.7 Recently, we isolated a new analogue of kurahyne (1), kurahyne B (2), from the marine cyanobacterium Okeania sp. collected at the coast near Jahana, Okinawa. Its gross structure was elucidated based on spectroscopic analyses, and the absolute configuration was established by total synthesis. Kurahyne B (2) inhibited the growth of human cancer cells to the same extent as kurahyne (1). In parallel, the first total synthesis of kurahyne (1) was also achieved. In this paper, we describe the isolation and structure determination of kurahyne B (2) and the first total syntheses of the kurahynes (1 and 2).

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RESULTS AND DISCUSSION The marine cyanobacterium Okeania sp. (900 g, wet weight) was collected at the coast near Jahana, Okinawa, and extracted with MeOH. The extract was filtered, concentrated, and partitioned between EtOAc and H2O. The organic extract was further partitioned between 90% aqueous MeOH and hexane. The material obtained from the aqueous MeOH portion was subjected to fractionation with reversed-phase column chromatography (ODS silica gel, MeOH−H2O) and repeated reversed-phase HPLC (Cosmosil 5C18-MS-II, MeOH−H2O; © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 29, 2015

A

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Table 1. NMR Data for Kurahyne B (2) in CD3OD position Opp 1 2 3 4 5a 5b 6a 6b 7a 7b Pro 1 2 3a 3b 4a 4b 5a 5b N-Me-Val-1 1 2 3 4 5 N-Me N-Me-Val-2 1 2 3 a

δC,a type 7.7, CH3 33.7, CH2 211.0,c C 66.0, CH 29.1, CH2

δHb (J in Hz) 1.03, t (7.5) 2.56, q (7.5) 4.61, 2.21, 1.84, 2.00, 1.93, 3.83, 3.62,

dd (8.8, 5.2) m m m m m m

4.66, 2.31, 1.96, 2.04, 1.87, 3.80, 3.68,

dd (8.6, 5.3) m m m m m m

170.2, C 61.0, CH 28.5, CH 19.8, CH3 18.7,f CH3 31.35,g CH3

5.06, 2.23, 0.96, 0.78, 3.07,

d (10.8) m d (6.6) d (6.7) s

172.2,e C 59.6, CH 28.7, CH

5.21, d (10.8) 2.31, m

25.7,d CH2 47.2, CH2

172.3,e C 59.6, CH 29.5, CH2 25.8,d CH2 48.0, CH2

Measured at 100 MHz. bMeasured at 400 MHz. cObserved by HMBC.

d,e,f,g

position

δC,a type

4 5 N-Me N-Me-Ile 1 2 3 4a 4b 5 6 N-Me Ile 1 2 3 4a 4b 5 6 NH Moya 1 2 3 4 5 6 7 8 9

19.7, CH3 18.6,f CH3 31.35,g CH3 172.1,e C 58.3, CH 34.5, CH 24.9, CH2 10.9, CH3 15.9, CH3 31.30,g CH3 175.0, 55.3, 37.5, 25.8,

C CH CH CH2

11.0, CH3 16.0, CH3

172.1,e C 132.8, C 136.6, CH 28.1, CH2 28.8, CH2 18.6, CH2 84.5, C 70.0, CH 13.0, CH3

δHb (J in Hz) 0.89, d (6.6) 0.79, d (6.9) 3.08, s

5.29, 2.13, 1.35, 1.29, 0.84, 0.83, 3.16,

d (10.8) m m m m m s

4.77, 1.96, 1.58, 1.20, 0.92, 0.90, 7.78,

dd (9.6, 9.3) m m m m d (6.9) d (9.3)

6.22, 2.30, 1.65, 2.21,

tq (7.6, 1.5) m m m

2.24, m 1.85, d (1.5)

Carbon signals with the same superscript are interchangeable.

which an N-terminal Ile replaces an N-terminal N-Me-Ile. Therefore, we hypothesized that 1 and 2 shared the same absolute configuration. To verify this hypothesis, we carried out the total synthesis of kurahyne B (2) along with the first total synthesis of kurahyne (1). Our retrosynthetic analysis of 1 and 2 is shown in Scheme 1. Kurahynes 1 and 2 are composed of an α,β-unsaturated carboxylic acid 3 and hexapeptide 4a or 4b. Hexapeptides 4a and 4b could be prepared from ethyl ketone 5 by repeated condensation reactions (Scheme 2). Carboxylic acid 3 could be prepared by hydrolysis of the ethyl ester of 3,8 ethyl ketone 5 could be prepared according to a previous report,9 and the other N-Me amino acids could be easily prepared from the corresponding amino acids.10 We connected these moieties in a stepwise manner, starting from the C-terminal residue, ethyl ketone 5.

Figure 1. Gross structure of kurahyne B (2), based on 2D NMR correlations.

in the Moya moiety, thereby completing the gross structure of kurahyne B (2), as shown in Figure 1. As described above, we clarified that the gross structure of kurahyne B (2) was a demethyl analogue of kurahyne (1) in Scheme 1. Retrosynthetic Analysis

B

DOI: 10.1021/acs.jnatprod.5b00662 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Scheme 2. Synthesis of Modified Tetrapeptide 10a

(a) TFA, CH2Cl2, 0 °C, 1 h, rt, 1 h; (b) N-Boc-L-Pro, 11, DIPEA, DMF, rt, 50 min, 84% in 2 steps; (c) NaBH4, MeOH, 0 °C, 1 h, rt, 1 h, 89%; (d) TFA, CH2Cl2, 0 °C, 1 h, rt, 1 h; (e) N-Boc-N-Me-L-Val, 11, DIPEA, DMF, rt, 50 min; (f) DMP, CH2Cl2, 0 °C, 2.5 h, 52% in 3 steps; (g) TFA, CH2Cl2, 0 °C, 1 h, rt, 1 h; (h) N-Boc-N-Me-L-Val, 11, DIPEA, DMF, rt, overnight, quant in 2 steps; (i) TFA, CH2Cl2, 0 °C, 1 h, rt, 1 h; (j) N-Boc-NMe-L-Ile, 11, DIPEA, DMF, rt, overnight, 69% in 2 steps. a

Scheme 3. Syntheses of Kurahyne (1) and Kurahyne B (2)a

(a) TFA, CH2Cl2, 0 °C, 1 h, rt, 1 h; (b) N-Boc-N-Me-L-Ile, 12, DIPEA, DMF, rt, 5 days, 52% in 2 steps; (c) TFA, CH2Cl2, 0 °C, 1.5 h, rt, 1 h; (d) 3, 12, DIPEA, DMF, rt, overnight, 24% in 2 steps; (e) TFA, CH2Cl2, 0 °C, 1.5 h, rt, 1 h; (f) N-Boc-L-Ile, 12, DIPEA, DMF, rt, 5 days, 50% in 2 steps; (g) TFA, CH2Cl2, 0 °C, 1 h, rt, 1 h; (h) 3, 12, DIPEA, DMF, rt, overnight, 27% in 2 steps. a

with carboxylic acid 3 afforded kurahyne (1) (3.3% overall yield in 14 steps from ethyl ketone 5 based on the longest linear sequence) (Scheme 3). As for kurahyne B (2), the condensation of modified tetrapeptide 10 with N-Boc-L-Ile with the use of COMU followed by further condensation with carboxylic acid 3 afforded kurahyne B (2) (3.6% overall yield in 14 steps from ethyl ketone 5 based on the longest linear sequence) (Scheme 3). The condensation reaction of 4a or 4b with carboxylic acid 3 resulted in low yield. These results can be attributed to the steric hindrance of the N-terminus of 4a and 4b. The spectroscopic data of synthetic 1 and 2, including 1H NMR, 13C NMR, specific rotation, and mass spectra, were matched within the margin of error with the data originally reported for natural 17 and 2, respectively. Therefore, the absolute configuration of kurahyne B was established as shown in 2. An MTT assay with HeLa cells and HL60 cells was used to evaluate the growth-inhibitory activities of kurahyne B (2). The cells were treated in 96-well plates with various concentrations of the compound (0.01−10 μg/mL for HeLa cells, 0.001−10 μg/mL for HL60 cells) for 72 h. The data from these assays

Compound 6 was prepared by condensation of the known ethyl ketone 59 with N-Boc-L-Pro. O-(7-Azabenzotriazol-1yl)tetramethyluronium hexafluorophosphate (HATU, 11) was used for the condensation reaction, and 6 was obtained in 84% yield. We anticipated that the direct condensation of 6 with NBoc-N-Me-L-Val would give an undesired cyclized product due to an intramolecular nucleophilic attack of the deprotected amino group of Pro on the ketone group of Opp. Therefore, we reduced the ketone group of 6 by sodium borohydride and obtained the corresponding alcohol 7. The condensation of alcohol 7 with N-Boc-N-Me-L-Val followed by oxidation gave modified dipeptide 8, which was converted into modified tripeptide 9 by condensation with N-Boc-N-Me-L-Val. Modified tripeptide 9 was transformed into modified tetrapeptide 10 by condensation with N-Boc-N-Me-L-Ile (Scheme 2). To synthesize modified pentapeptide 4a, we performed the condensation of modified tetrapeptide 10 with N-Boc-N-Me-LIle by using HATU, but did not obtain the desired modified pentapeptide. To improve the reactivity, we used (1-cyano-2ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholinocarbenium hexafluorophosphate (COMU, 12) as a condensation reagent and obtained 4a. Finally, the condensation of 4a C

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Okinawa Prefecture, Japan, at a depth of 0−1 m in March 2013. The collected cyanobacterium (900 g) was extracted two times with MeOH (3 L). The extract was filtered, and the filtrate was concentrated. The residue was partitioned between EtOAc (3 × 0.3 L) and H2O (0.3 L). The material obtained from the organic layer was partitioned between 90% aqueous MeOH (0.3 L) and hexane (3 × 0.3 L). The aqueous MeOH fraction (1.5 g) was first separated by column chromatography on ODS (15 g) eluted with 40% MeOH, 60% MeOH, 80% MeOH, and MeOH. The fraction (415 mg) eluted with 80% MeOH was subjected to HPLC [Cosmosil 5C18MS-II (ϕ 20 × 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent 85% MeOH] in seven batches to give a fraction that contained kurahyne B (27.5 mg, tR = 26.0 min). This fraction was further separated by HPLC [Cosmosil Cholester (ϕ 20 × 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent 65% MeCN] to give kurahyne B (2) (2.2 mg, tR = 42.1 min, total yield 0.000 24% based on wet weight). Kurahyne B (2): colorless oil; [α]30D −280 (c 0.05, CH3OH); UV (MeOH) λmax (log ε) 207 (3.91) nm; IR (neat) 3314, 2964, 2875, 1652, 1448 cm−1; 1H NMR, 13C NMR, COSY, HMBC, and NOESY data, Tables 1 and S1; HRESIMS m/z 825.5810 [M + H]+ (calcd for C46H77N6O7, 825.5854). Preparation of Carboxylic Acid 3. To a stirred solution of the ethyl ester of 38 (489 mg, 2.69 mmol) in dry THF (2 mL) was added 2 mL of LiOH (176 mg, 4.20 mmol) aqueous solution, and the mixture was stirred at 0 °C for 10 h. After completion of the reaction, it was diluted with H2O (4 mL) and washed with CH2Cl2 (2 × 6 mL). The H2O layer was acidified with 1 M HCl and extracted with EtOAc (3 × 6 mL). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, and concentrated to afford crude carboxylic acid 3 (204 mg). This carboxylic acid 3 was used in the next reaction without purification. 1H NMR (400 MHz, CDCl3) δ 6.87 (tq, J = 7.2, 1.6 Hz, 1H), 2.35 (q, J = 7.2 Hz, 2H), 2.23 (td, J = 6.8, 2.4 Hz, 2H), 1.98 (t, J = 2.4 Hz, 1H), 1.86 (d, J = 1.6 Hz, 3H), 1.69 (quint, J = 7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 173.5, 144.0, 128.1, 83.8, 69.1, 27.8, 27.3, 18.2, 12.2; IR (neat cm−1) 3301, 2934, 2867, 1700, 1685; HRESIMS m/z 153.0905 [M + H]+ (calcd for C9H13O2, 153.0910). Preparation of 6. To a stirred solution of ethyl ketone 59 (2.10 g, 9.24 mmol) in CH2Cl2 (4 mL) was added TFA (2 mL), and the reaction mixture was stirred at 0 °C for 1 h and room temperature (rt) for 1 h. After completion of the reaction, benzene (4 mL) was added to the reaction mixture and concentrated. After repetition of this operation five times, the trifluoroacetate salt of deprotected 5 was afforded and used in the next reaction without purification. To a 200 mL recovery flask were added the trifluoroacetate salt of deprotected 5, N-Boc-L-Pro (2.56 g, 11.0 mmol), and HATU (4.33 g, 11.1 mmol), and the mixture was dissolved in dry DMF (4 mL) under a N2 atmosphere. To the stirred solution was added dry DIPEA (4.83 mL, 33.0 mmol), and the reaction solution was stirred at rt for 50 min. After completion of the reaction, the reaction mixture was diluted with EtOAc (20 mL), washed with 10% aqueous citric acid (20 mL), saturated aqueous NaHCO3 (20 mL), and brine (20 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (EtOAc−hexane (1:1) → EtOAc) to afford compound 6 (2.81 g, 8.66 mmol, 94% in 2 steps) as a pale yellow oil. The ratio of major and minor rotamers is 5:4. [α]23D −112 (c 1.04, CHCl3); IR (neat) 2974, 2877, 1718, 1700, 1696, 1684, 1648, 1436 cm−1; 1H NMR (400 MHz, CDCl3) major δ 4.56 (dd, J = 4.4, 8.8 Hz, 1H), 4.37 (dd, J = 4.0, 8.4 Hz, 1H), 3.69−3.27 (m, 4H), 2.51−2.36 (m, 2H), 2.11−1.63 (m, 8H), 1.32 (s, 9H), 0.96−0.92 (m, 3H); minor δ 4.52 (dd, J = 5.2, 8.8 Hz, 1H), 4.28 (dd, J = 3.2, 8.0 Hz, 1H), 1.28 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 209.5, 171.3, 170.8, 154.4, 153.6, 79.3, 64.0, 57.5, 46.8, 46.7, 33.2, 33.1, 29.0, 28.4, 28.2, 27.8, 27.7, 24.9, 23.9, 23.4, 7.3; HRESIMS m/z 347.1941 [M + Na]+ (calcd for C17H28N2O4Na, 347.1941). Preparation of Alcohol 7. To a stirred solution of compound 6 (67.0 mg, 207 μmol) in dry MeOH (0.5 mL) was added NaBH4 (15.6 mg, 412 μmol) under a N2 atmosphere, and the reaction solution was stirred at 0 °C for 1 h and rt for 1 h. After the completion of the reaction, saturated aqueous NH4Cl (10 mL) was added to the reaction

revealed that 2 inhibited the growth of both HeLa cells and HL60 cells, with IC50 values of 8.1 and 9.0 μM, respectively. As a result, kurahyne B inhibited the growth of human cancer cells to the same extent as kurahyne (IC50 values of kurahyne: 3.9 ± 1.1 μM (HeLa) and 1.5 ± 0.1 μM (HL60)).7 In summary, a new analogue of kurahyne (1), kurahyne B (2), was isolated from the marine cyanobacterium Okeania sp. collected at the coast near Jahana, Okinawa. Its gross structure was elucidated based on spectroscopic analyses, and the absolute configuration was established by total synthesis (3.6% overall yield in 14 steps). Kurahyne B (2) inhibited the growth of human cancer cells to the same extent as kurahyne (1). In parallel, the first total synthesis of kurahyne (1) was also achieved (3.3% overall yield in 14 steps).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO DIP-1000 polarimeter. IR spectra were recorded on a JASCO RT/IR-4200 instrument. 1H NMR and 13C NMR spectra were recorded in CDCl3, C6D6 or CD3OD on a JEOL JNM-ECX400 (400 MHz) and JNM-A400 (400 MHz), respectively. 1 H NMR chemical shifts are referenced to CHCl3 observed at δH 7.26, CHD2OD observed at δH 3.31, and C6HD5 observed at δH 7.16. 13C NMR chemical shifts are referenced to CDCl3 observed at δC 77.1, CD3OD observed at δC 49.0, and C6D6 observed at δC 128.06. 1H NMR chemical shifts were assigned using a combination of data from COSY and HMQC experiments. Similarly, 13C NMR chemical shifts were assigned based on HMBC and HMQC experiments. ESI mass spectra were obtained on an LCT Premier EX spectrometer (Waters). The reaction progress was checked on a thin layer chromatography (TLC) plate. TLC was carried out using precoated sheets (E. Merck silica gel 60F254 (Art. 5715)), which, after development, were visualized under UV light at 254 nm and staining in phosphomolybdic acid solution. Silica gel column chromatography was performed using the indicated solvents on Fuji Silisia Chemical Ltd. silica gel (BW820MH) and Wako Pure Chemical Industries, Ltd. silica gel (Wakogel 60N). All nonaqueous reactions were performed under an atmosphere of nitrogen using oven-dried glassware under a N2 atmosphere and standard syringe in septa techniques. Commercially available reagents were used without further purification. All solvents were distilled prior to use: tetrahydrofuran was distilled from Na−benzophenone; DIPEA and MeOH were distilled from CaH2; CH3I was distilled from P2O5. Dess-Martin periodinane (DMP) was prepared from 2-iodobenzoic acid.11 Identification of the Marine Cyanobacterium. A cyanobacterial filament was isolated under a microscope and crushed with freezing and thawing. The 16S rRNA genes were PCR-amplified from isolated DNA using the primer set CYA 106F, a cyanobacterial-specific primer, and 23S 30R, a cyanobacterial-specific primer. The PCR reaction contained DNA derived from a cyanobacterial filament, 12.5 μL of TakaraTaq (TaKaRa Bio), 1.0 μL of each primer (10 pM), and H2O for a total volume of 25 μL. The PCR reaction was performed as follows: initial denaturation for 10 min at 94 °C, amplification by 35 cycles of 1 min at 94 °C, 1 min at 57 °C, and 1 min at 72 °C, and final elongation for 7 min at 72 °C. PCR products were analyzed on agarose gel (1%) in TBE buffer and visualized by ethidium bromide staining. The obtained DNA was sequenced with CYA 106F and 23S 30R primers. These sequences are available in the DDBJ/EMBL/GenBank databases under accession numbers LC89730 and LC089731. From the phylogenetic tree inferred from 739 and 766 bp of 16S rRNA gene sequences revealed that the present cyanobacterium (1303-13, accession nos. LC89730 and LC89731) formed a clade with Okeania sp. Therefore, the cyanobacterium was classified into the genus Okeania. Isolation of Kurahyne B (2). Samples of the marine cyanobacterium Okeania sp. were collected at the coast near Jahana, D

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mixture and extracted with EtOAc (3 × 10 mL). The combined organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (EtOAc−hexane, 1:1) to afford alcohol 7 (60.5 mg, 184 μmol, 89%) as a colorless oil. This was an inseparable mixture of the epimers (the ratio is 2:1): [α]23D −41 (c 1.13, CHCl3) (as a mixture of epimers); IR (neat) 3429, 2972, 2934, 2876, 1683, 1653, 1634 cm−1; 1H NMR (400 MHz, CDCl3) major δ 4.52 (dd, J = 7.6, 4.4 Hz 1H), 4.26−4.21 (m, 1H), 3.81 (m, 1H), 3.66−3.34 (m, 4H), 2.13−2.00 (m, 2H), 1.90−1.75 (m, 8H), 1.42 (s, 1.8H), 1.40 (s, 7.2H), 1.06−0.97 (m, 3H); minor δ 4.41 (dd, J = 8.0, 4.4 Hz 1H), 4.26−4.21 (m, 1H), 3.66−3.34 (m, 5H), 2.13−2.00 (m, 2H), 1.90− 1.75 (m, 8H), 1.45 (s, 9H), 1.06−0.97 (m, 3H); 13C NMR (100 MHz, CDCl3) major δ 175.5, 155.3, 154.7, 79.7, 79.6, 62.7, 57.8, 46.8, 46.7, 30.1, 28.7, 28.6, 27.7, 23.7, 10.2, 9.6; minor 175.2, 153.8, 80.1, 62.8, 57.9, 47.2, 45.5, 30.9, 28.6, 28.5, 28.3, 27.6, 10.9, 9.7; HRESIMS m/z 327.2283 [M + H]+ (calcd for C17H31N2O4, 327.2278). Preparation of Modified Dipeptide 8. To a stirred solution of alcohol 7 (62.3 mg, 191 μmol) in CH2Cl2 (0.8 mL) was added TFA (0.5 mL), and the reaction solution was stirred at 0 °C for 1 h and rt for 1 h. After completion of the reaction, benzene (1 mL) was added to the reaction mixture and concentrated. After repetition of this operation five times, the trifluoroacetate salt of deprotected 7 was afforded and used in the next reaction without purification. In a 200 mL recovery flask, the trifluoroacetate salt of deprotected 7, N-Boc-NMe-L-Val (71.0 mg, 285 μmol), and HATU (108 mg, 284 μmol) were added and dissolved in dry DMF (0.2 mL) under a N2 atmosphere. To the stirred solution was added dry DIPEA (0.20 mL, 1.15 mmol), and the reaction solution was stirred at rt for 50 min. After completion of the reaction, it was diluted with EtOAc (3 mL), washed with 10% aqueous citric acid (3 mL), saturated aqueous NaHCO3 (3 mL), and brine (5 mL), dried over anhydrous Na2SO4, and concentrated to afford crude modified dipeptide alcohol (122 mg). To a stirred solution of modified dipeptide alcohol (122 mg) in CH2Cl2 (0.5 mL) was added DMP (117 mg, 266 μmol), and the reaction solution was stirred at 0 °C for 2.5 h. After completion of the reaction, the reaction mixture was diluted with saturated aqueous Na2S2O3 (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layer was washed with saturated aqueous NaHCO3 (10 mL) and brine (10 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (EtOAc−CH2Cl2, 1:1) to afford modified dipeptide 8 (42.0 mg, 96.9 μmol, 52% in 3 steps) as a colorless oil. The ratio of major and minor rotamers is 2:1. [α]23D −138 (c 1.02, CHCl3); IR (neat) 2970, 2875, 1687, 1683, 1652, 1645, 1436, 1309, 1150, 1117 cm−1; 1H NMR (400 MHz, CDCl3) major δ 4.70 (dd, J = 4.4, 8.8 Hz, 1H), 4.63 (dd, J = 4.4, 8.8 Hz, 1H), 4.61 (d, J = 11.2 Hz, 1H), 3.87−3.55 (m, 4H), 2.84 (s, 3H), 2.16−1.99 (m, 6H), 1.98−1.77 (m, 4H), 1.76 (m, 1H), 1.45 (s, 9H), 1.03 (t, J = 7.2 Hz, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H); minor δ 4.38 (d, J = 11.2 Hz, 1H), 2.80 (s, 3H), 1.47 (s, 9H), 1.02 (t, J = 7.2 Hz 3H), 0.93 (d, J = 6.4 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 209.5, 170.3, 170.2, 156.5, 155.6, 80.3, 64.2, 60.8, 58.1, 47.8, 46.9, 33.4, 33.3, 29.8, 29.2, 28.5, 24.9, 24.8, 19.9, 19.5, 18.7, 18.6, 7.4; HRESIMS m/z 460.2786 [M + Na]+ (calcd for C23H39N3O5Na, 460.2782) Preparation of Modified Tripeptide 9. To a stirred solution of compound 8 (541 mg, 1.24 mmol) in CH2Cl2 (3 mL) was added TFA (1.5 mL), and the reaction solution was stirred at 0 °C for 1 h and rt for 1 h. After completion of the reaction, benzene (2 mL) was added to the reaction mixture and concentrated. After repetition of this operation five times, the trifluoroacetate salt of deprotected 8 was afforded and used in the next reaction without purification. To a 100 mL recovery flask were added the trifluoroacetate salt of deprotected 8, N-Boc-N-Me-L-Val (456 mg, 1.97 mmol), and HATU (945 mg, 2.49 mmol), and the mixture was dissolved in dry DMF (2 mL) under a N2 atmosphere. To the stirred solution was added dry DIPEA (1.30 mL, 7.43 mmol), and the reaction solution was stirred at rt for 12 h. After completion of the reaction, the reaction mixture was diluted with EtOAc (10 mL), washed with 10% aqueous citric acid (10 mL), saturated aqueous NaHCO3 (10 mL), and brine (10 mL), dried over

anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (EtOAc−CH2Cl2, 1:1) to afford modified tripeptide 9 (602 mg, quant in 2 steps), as a colorless oil. The ratio of major and minor rotamers is 3:2. [α]23D −112 (c 1.04, CHCl3); IR (neat) 2966, 2938, 2875, 1700, 1695, 1652, 1635 cm−1; 1 H NMR (400 MHz, CDCl3) major δ 5.06 (d, J = 10.8 Hz, 1H), 4.70 (dd, J = 4.4, 8.8 Hz 1H), 4.69 (d, J = 10.8 Hz, 1H), 4.62 (dd, J = 4.0, 8.8 Hz, 1H), 3.89 (m, 1H), 3.81−3.72 (m, 2H) 3.57 (m, 1H), 3.11 (s, 3H), 2.74 (s, 3H), 2.53 (q, J = 7.2 Hz 1H), 2.52 (q, J = 7.2 Hz 1H), 2.38−1.65 (m, 6H), 1.44 (s, 9H), 1.03 (t, J = 7.2 Hz, 3H), 1.02−0.98 (m, 3H), 0.89−0.83 (m, 6H), 0.80−0.78 (m, 3H); minor δ 4.42 (d, J = 10.8 Hz, 1H), 2.73 (s, 3H), 1.46 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 209.4, 171.2, 171.1, 170.9, 169.0, 156.3, 155.3, 80.1, 64.2, 59.9, 58.3, 58.1, 50.7, 47.8, 46.9, 33.3, 30.8, 30.3, 29.5, 28.5, 28.4, 27.9, 27.2, 24.9, 19.8, 19.6, 19.4, 18.2, 7.4; HRESIMS m/z 551.3807 [M + H]+ (calcd for C29H51N4O6, 551.3803). Preparation of Modified Tetrapeptide 10. To a stirred solution of compound 9 (364 mg, 661 μmol) in CH2Cl2 (3 mL) was added TFA (1.5 mL), and the reaction solution was stirred at 0 °C for 1 h and rt for 1 h. After completion of the reaction, benzene (2 mL) was added to the reaction mixture and concentrated. After repetition of this operation five times, the trifluoroacetate salt of deprotected 9 was afforded and used in the next reaction without purification. To a 100 mL recovery flask were added the trifluoroacetate salt of deprotected 9, N-Boc-N-Me-L-Ile (243 mg, 991 μmol), and HATU (502 mg, 1.32 mmol), and the mixture was dissolved in dry DMF (1 mL) under a N2 atmosphere. To the stirred solution was added dry DIPEA (0.70 mL, 4.02 mmol), and the reaction solution was stirred at rt for 12 h. After completion of the reaction, the reaction mixture was diluted with EtOAc (5 mL), washed with 10% aqueous citric acid (5 mL), saturated aqueous NaHCO3 (5 mL), and brine (5 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (EtOAc−CH2Cl2, 1:1) to afford modified tetrapeptide 10 (307 mg, 460 μmol, 69% in 2 steps), as a colorless oil. The ratio of major and minor rotamers is 3:2. [α]23D −141 (c 1.17, CHCl3); IR (neat) 2974, 2933, 2877, 1700, 1696, 1690, 1628 cm−1; 1 H NMR (400 MHz, CDCl3) major δ 5.17 (d, J = 10.8 Hz, 1H), 5.05 (d, J = 11.2 Hz, 1H), 4.75 (d, J = 11.2 Hz, 1H), 4.68 (dd, J = 4.0, 8.8 Hz, 1H), 4.61 (dd, J = 4.4, 8.0 Hz, 1H), 3.85 (m, 1H), 3.81−3.71 (m, 2H), 3.56 (m, 1H), 3.08 (s, 3H), 3.03 (s, 3H), 2.74 (s, 3H), 2.52 (q, J = 7.2 Hz, 1H), 2.50 (q, J = 7.2 Hz, 1H), 2.38−2.21 (m, 2H), 2.18− 1.91 (m, 8H), 1.89−1.72 (m, 3H), 1.42 (s, 9H), 1.02 (t, J = 7.2 Hz, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H), 0.79−0.71 (m, 9H); minor δ 5.18 (d, J = 10.8 Hz, 1H), 4.89 (d, J = 11.2 Hz, 1H), 4.51 (d, J = 11.2 Hz, 1H), 3.10 (s, 3H), 3.02 (s, 3H), 1.45 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 209.4, 171.5, 171.0, 170.2, 169.0, 156.3, 155.3, 80.1, 64.1, 59.4, 58.4, 58.2, 58.1, 47.8, 46.9, 33.3, 33.2, 30.8, 30.5, 29.6, 29.1, 28.6, 28.4, 27.9, 27.7, 27.4, 24.9, 24.3, 19.8, 19.4, 18.3, 17.8, 15.7, 11.0, 7.4; HRESIMS m/z 678.4795 [M + H]+ (calcd for C36H64N5O7, 678.4800). Preparation of Modified Pentapeptide 4a. To a stirred solution of compound 10 (42.0 mg, 63.7 μmol) in CH2Cl2 (0.5 mL) was added TFA (0.2 mL), and the reaction solution was stirred at 0 °C for 1 h and rt for 1 h. After completion of the reaction, benzene (2 mL) was added to the reaction mixture and concentrated. After repetition of this operation five times, the trifluoroacetate salt of deprotected 10 was afforded and used in the next reaction without purification. To a 10 mL pear-shaped flask were added the trifluoroacetate salt of deprotected 10, N-Boc-N-Me-L-Ile (24.3 mg, 99.1 μmol), and COMU (57.3 mg, 133 μmol), and the mixture was dissolved in dry DMF (10 drops) under a N2 atmosphere. To the stirred solution was added dry DIPEA (0.07 mL, 382 μmol), and the reaction solution was stirred at rt for 5 days. After completion of the reaction, the reaction mixture was diluted with EtOAc (5 mL), washed with 10% aqueous citric acid (5 mL), saturated aqueous NaHCO3 (5 mL), and brine (5 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (EtOAc−CH2Cl2, 1:1) to afford modified pentapeptide 4a (24.2 mg, 29.9 μmol, 52% in 2 steps) as a pale yellow oil. The ratio of major and minor rotamers is 3:2. [α]23D −136 (c 1.02, CHCl3); IR E

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(neat) 2962, 2874, 1718, 1700, 1635, 1628, 1575, 1436 cm−1; 1H NMR (400 MHz, CDCl3) major δ 5.26 (d, J = 10.8 Hz, 1H), 5.20 (d, J = 10.8 Hz, 1H), 5.07 (d, J = 7.2 Hz, 1H), 4.79 (d, J = 10.8 Hz, 1H), 4.70 (dd, J = 4.0, 8.8 Hz, 1H), 4.62 (dd, J = 4.8, 8.0 Hz, 1H), 3.88 (m, 1H), 3.82−3.71 (m, 2H), 3.58 (m, 1H), 3.11 (s, 3H), 3.08 (s, 3H), 3.07 (s, 3H), 2.75 (s, 3H), 2,54 (q, J = 7.2 Hz, 1H), 2.52 (q, J = 7.2 Hz, 1H), 2.37−2.25 (m, 3H), 2.20−2.06 (m, 5H), 2.05−1.96 (m, 4H), 1.90−1.76 (m, 4H), 1.46 (s, 9H), 1.05 (t, J = 7.2 Hz, 3H), 0.99 (m, 6H), 0.92−0.78 (m, 12H), 0.75−0.73 (m, 6H); minor δ 5.28 (d, J = 10.8 Hz, 1H), 4.54 (d, J = 10.8 Hz, 1H), 3.12 (s, 3H), 3.09 (s, 3H), 3.05 (s, 3H), 2.76 (s, 3H), 1.48 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 209.3, 173.7, 171.0, 170.9, 170.2, 169.1, 156.3, 79.8, 64.2, 58.0, 57.9, 56.8, 55.1, 47.8, 46.9, 33.3, 30.9, 30.6, 28.6, 28.4, 27.9, 27.7, 27.5, 24.9, 24.3, 24.2, 19.6, 19.4, 18.4, 17.9, 15.9, 11.3, 10.9, 7.4; HRESIMS m/z 827.5625 [M + Na]+ (calcd for C43H76N6O8Na, 827.5617). Preparation of Modified Pentapeptide 4b. To a stirred solution of compound 10 (53.4 mg, 70.9 μmol) in CH2Cl2 (0.5 mL) was added TFA (0.2 mL), and the reaction solution was stirred at 0 °C for 1.5 h and rt for 1 h. After completion of the reaction, benzene (1 mL) was added to the reaction mixture and concentrated. After repetition of this operation five times, the trifluoroacetate salt of deprotected 10 was afforded and used in the next reaction without purification. In a 10 mL pear-shaped flask, the trifluoroacetate salt of deprotected 10, N-Boc-L-Ile (24.0 mg, 104 μmol), and COMU (46.4 mg, 107 μmol) were added and dissolved in dry DMF (7 drops) under a N2 atmosphere. To the stirred solution was added DIPEA (0.05 mL, 282 μmol), and the reaction solution was stirred at rt for 5 days. After completion of the reaction, the reaction mixture was diluted with EtOAc (5 mL), washed with 10% aqueous citric acid (5 mL), saturated aqueous NaHCO3 (5 mL), and brine (5 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel (EtOAc−CH2Cl2, 1:1) to afford modified pentapeptide 4b (27.3 mg, 34.1 μmol, 50% in 2 steps), as an oil. [α]23D −197 (c 1.46, CHCl3); IR (neat) 2964, 2933, 2877, 1706, 1637, 1675, 1456, 1448 cm−1; 1H NMR (400 MHz, CDCl3) δ 5.26 (d, J = 10.4 Hz, 1H), 5.20 (d, J = 10.8 Hz, 1H), 5.12 (m, 1H), 5.07 (d, J = 10.8 Hz, 1H), 4.70 (dd, J = 4.0, 8.8 Hz, 1H), 4.63 (dd, J = 4.8, 8.4 Hz, 1H), 4.44 (m, 1H), 3.95 (m, 1H), 3.85−3.71 (m, 2H), 3.58 (m, 1H), 3.11 (s, 3H), 3.01 (s, 3H), 3.06 (s, 3H), 2.54 (q, J = 7.6 Hz, 1H), 2.52 (q, J = 7.6 Hz, 1H), 2.37−1.49 (m, 12H), 1.42 (s, 9H), 1.04 (t, J = 7.2 Hz, 3H), 1.00 (d, J = 6.4 Hz, 3H), 0.95−0.78(m, 19H), 0.75−0.73 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 209.3, 173.9, 171.1, 171.0, 170.2, 169.2, 156.1, 79.8, 64.2, 59.4, 58.1, 58.0, 56.9, 55.1, 47.9, 47.0, 37.4, 33.4, 33.4, 30.9, 30.7, 30.6, 28.6, 28.4, 27.9, 27.7, 27.5, 24.9, 24.8, 24.2, 24.1, 19.6, 19.4, 18.4, 18.0, 15.9, 15.6, 11.3, 11.0, 7.5; HRESIMS m/z 813.5469 [M + Na]+ (calcd for C42H74N6O8Na, 813.5460). Preparation of Kurahyne (1). To a stirred solution of compound 4a (14.3 mg, 17.4 μmol) in CH2Cl2 (0.5 mL) was added TFA (0.3 mL), and the reaction solution was stirred at 0 °C for 1.5 h and rt for 1 h. After completion of the reaction, benzene (1 mL) was added to the reaction mixture and concentrated. After repetition of this operation five times, the trifluoroacetate salt of deprotected 4a was afforded and used in the next reaction without purification. To a 10 mL pear-shaped flask were added the trifluoroacetate salt of deprotected 4a, carboxylic acid 3 (5.32 mg, 34.8 μmol), and COMU (15.1 mg, 35.2 μmol), and the mixture was dissolved in dry DMF (7 drops) under a N2 atmosphere. To the stirred solution was added dry DIPEA (0.05 mL, 282 μmol), and the reaction solution was stirred at rt for 12 h. After completion of the reaction, the reaction mixture was diluted with EtOAc (1 mL), washed with 10% aqueous citric acid (1 mL), saturated NaHCO3 (1 mL), and brine (1 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography on ODS (100% MeOH) followed by HPLC [Cosmosil 5C18 MS-II (ϕ 20 × 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent; 80% MeOH−H2O] to afford kurahyne (1) (3.9 mg, 4.1 μmol, 27% in 2 steps) as a colorless oil. [α]23D −270 (c 0.07, MeOH) (lit. [α]29D −258 (c 0.20, MeOH));7 IR (neat) 3314, 3254, 2964, 2827, 2874, 1734, 1684, 1635, 1447 cm−1; NMR data, see Table S2; HRESIMS m/z 839.6015 [M + H]+ (calcd for C47H79N6O7 839.6005).

Preparation of Kurahyne B (2). To a stirred solution of compound 4b (17.2 mg, 21.5 μmol) in CH2Cl2 (0.5 mL) was added TFA (0.3 mL), and the reaction solution was stirred at 0 °C for 1 h and rt for 1 h. After completion of the reaction, benzene (1 mL) was added to the reaction mixture and concentrated. After repetition of this operation five times, the trifluoroacetate salt of deprotected 4b was afforded and used in the next reaction without purification. To a 10 mL pear-shaped flask were added the trifluoroacetate salt of deprotected 4b, carboxylic acid 3 (6.53 mg, 42.7 μmol), and COMU (18.4 mg, 43.0 μmol), and the mixture was dissolved in dry DMF (7 drops) under a N2 atmosphere. To the stirred solution was added dry DIPEA (0.03 mL, 122 μmol), and the reaction solution was stirred at rt for 12 h. After completion of the reaction, the reaction mixture was diluted with EtOAc (1 mL), washed with 10% aqueous citric acid (1 mL), saturated aqueous NaHCO3 (1 mL), and brine (1 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography on ODS (100% MeOH) followed by HPLC [Cosmosil Cholester (ϕ 20 × 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent; 65% MeCN−H2O] to afford kurahyne B (2) (4.6 mg, 5.6 μmol, 26% in 2 steps) as a colorless oil. [α]23D −250 (c 0.24, MeOH); IR (neat) 3312, 3261, 2962, 2937, 2875, 1725, 1635 cm−1; NMR data, see Table S3; HRESIMS m/z 847.5679 [M + Na]+ (calcd for C46H76N6O7Na, 847.5668). Cell Growth Analysis. All cells were obtained from RIKEN Cell Bank. HeLa cells were cultured at 37 °C with 5% CO2 in DMEM (Nissui) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin, 300 μg/mL L-glutamine, and 2.25 mg/mL NaHCO3. HL60 cells were cultured at 37 °C with 5% CO2 in RPMI (Nissui) supplemented with 10% heat-inactivated FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin, 300 μg/mL Lglutamine, and 2.25 mg/mL NaHCO3. HeLa cells were seeded at 2 × 104 cells/well in 96-well plates (Iwaki) and cultured overnight. HL60 cells were seeded at 1 × 105 cells/well in 96-well plates. Various concentrations of compounds were then added, and cells were incubated for 72 h. Cell proliferation was measured by the MTT assay. Adriamycin was used as positive control (IC50 value 0.5 μM (HeLa cells)).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00662. 1 H and 13C NMR data for 1−10. COSY, HMBC, HMQC, and NOESY data for 2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail (K. Suenaga): [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24310160 and 20436992) for the Promotion of Science, the Uehara Memorial Foundation, and the Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research.

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REFERENCES

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