First total synthesis of cyclodepsipeptides clavatustide A and B and their enantiomers

Abstract

The enantiopure synthesis of clavatustides A (1) and B (3) were accomplished by a seven step synthetic protocol starting from commercially available (R)-phenyllactic acid. As the optical rotation values of synthetic (R)-clavatustides A and B were not in agreement with the reported values, the corresponding antipodes 2 and 4 were synthesized from (S)-phenyllactic acid to address the issue. Both (R) and (S) clavatustides A and B were evaluated for their anti-proliferative activity against three human cancer cell lines using MTT assay. The cervical cancer cell line (HeLa) was found to be most sensitive to the test compounds in decreasing the cell viability. S-Isomers (2 and 4) were found to exhibit better anti-proliferative activity when compared to their enantiomers (R-isomers) with IC₅₀s 24.5 and 26.8 μM respectively against HeLa cell lines. All the compounds tested had a minimal effect on the cell viability of normal lung cells, indicating that these compounds are not toxic to normal cells.

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1. Introduction

Cyclodepsipeptides show a broad spectrum of biological activities including antiplasmodial, antiviral, insecticidal, cytotoxic and antiproliferative activities. They are currently being evaluated in clinical trials, and are used in refractory cancer therapy in combination with other cytotoxic drugs. Members of this new class of compounds also serve as lead compounds/templates for more pharmacologically potent and toxicologically safe derivatives.^1a

Renewed interest in the structural and pharmacological characterization of naturally occurring cyclic peptides and depsipeptides has recognized that this class of natural products may be useful in the development of new therapeutics.^1b,c Cyclodepsipeptides are cyclic peptides in which one or more of the amide groups are replaced by the corresponding esters (cyclic peptides that has both peptide and ester linkages in proximity). Generally they are secondary metabolites of marine bacteria, fungi, sponge and plants.^1a Cyclic peptides with unique biological activities are ubiquitous in nature.² However; cyclopeptides with an anthranilic acid unit are rare in nature. Anthranilic acid dimers with micromolar affinity for the CCK1 receptors were considered to be an important molecular scaffold in medicinal chemistry.³ Very recently; two novel cyclodepsipeptides clavatustides A (1) and B (3) containing an anthranilic acid dimer and D-phenyllactic acid were isolated by Bin Wu and co-workers from cultured mycelia and broth of Aspergillus clavatus C2WU isolated from Xenograpsus testudinatus.⁴

Both 1 and 3 suppressed the proliferation of hepatocellular carcinoma (HCC) cell lines (HepG2, SMMC-7721 and Bel-7402) in a dose-dependent manner, induced an accumulation of HepG2 cells in G1 phase and reduction of cells in S phase.⁴ In particular, clavatustide B (3) efficiently suppressed cell proliferation in a dose dependent manner on human cancer cell lines, including pancreatic cancer (Panc-1), gastric cancer (MGC-803), colorectal cancer (SW-480), retinoblastoma (WERI-Rb-1) and prostate cancer (PC-3).⁵ The gross structure of 1 and 3 were determined using extensive spectroscopic analysis and absolute configuration of the phenyllactic acid moiety was confirmed as ‘R’. However, it is reported that both these compounds could not be isolated in pure form in spite of iterative purifications.⁴ Purity assessment is perhaps most critical in the case of novel compounds to which a biological activity is ascribed, because trace impurities of high potency can lead to false conclusions. Examples are the historic case of the lead compound sesbanamide “hidden” in sesbanin,^6,7 the more recent findings of inactive leads such as epiquinamide containing the β2-selective nicotinic acetylcholine receptor agonist, epibatidine,⁸ and the lack of in vitro anti-TB potency in high-purity ursolic acid.⁹ Therefore, any step towards the synthesis of pure clavatustides A and B could be rewarding as it will confirm its activity and its structural simplicity would render opportunities to design and synthesize potent new analogs. In this regard we aimed at the total synthesis of cyclodepsipeptides clavatustides A and B (Fig. 1).

Fig. 1 Chemical structures of clavatustides A and B and their isomer.

2. Results and discussion

2.1. Chemistry

In order to synthesize clavatustides A (1) and B (3) in pure form and sufficient quantities, we intended to construct the depsipeptide via macrolactonization approach using key intermediate 5 as shown below (Fig. 2).

Fig. 2 Macrolactonization approach to clavatustides A, B.

Our synthesis towards 5 for clavatustide B began with the acylation of known amine 6 with 2-nitrobenzoyl chloride. Metal mediated reduction of the nitro group in 7 followed by acid amine coupling with N-Boc-Sar-OH afforded intermediate 9 as a mixture of rotamers. (R)-Phenyllactic acid was coupled to the amine obtained after deprotection of the Boc group in 9 to yield compound 10. Debenzylation of compound 10 afforded intermediate 5 in good yields (Scheme 1).

Scheme 1 Macrolactonization strategy towards clavatustide B (3).

However, to our disappointment, Yamaguchi lactonization¹⁰ of compound 5 under different conditions led only to trace amount of product formation along with other impurities in all the cases (Table 1).

Table 1 Attempts made towards Yamaguchi macrolactonization reaction^a

Entry	Reaction conditions	Remarks
a The conversion was found to be sluggish. LC-MS of the crude reaction mixture indicated ∼5–10% of desired product formation along with multiple unknown impurities.
1	Compound 5 (100 mg, 0.21 mmol), 2,4,6-trichlorobenzoyl chloride (0.036 mL, 0.23 mmol), pyridine (0.025 mL, 0.31 mmol), DMAP (38 mg, 0.31 mmol), CH2Cl2, (5 mL) room temperature to reflux, 24 h	The conversion was found to be sluggish, trace of desired product formation
2	Compound 5 (100 mg, 0.21 mmol), 2,4,6-trichlorobenzoyl chloride (0.036 mL, 0.23 mmol), pyridine (0.025 mL, 0.31 mmol), DMAP (77 mg, 0.63 mmol), THF (5 mL), RT, 48 h	The conversion was found to be sluggish. LC-MS of the crude RM indicated ∼5% of desired product formation along with multiple unknown impurities (UV/KMnO4 active)
3	Compound 5 (100 mg, 0.21 mmol), 2,4,6-trichlorobenzoyl chloride (0.036 mL, 0.23 mmol), DMAP (77 mg, 0.63 mmol), THF (10 mL), reflux, 48 h	The conversion was found to be sluggish. LC-MS of the crude RM indicated ∼5–10% of desired product formation along with multiple unknown impurities (UV/KMnO4 active)
4	Compound 5 (100 mg, 0.21 mmol), 2,4,6-trichlorobenzoyl chloride (0.036 mL, 0.23 mmol), NEt3 (0.044 mL, 0.31 mmol), toluene (5 mL), room temperature to reflux, 48 h	The conversion was found to be sluggish, no desired product formation
5	Compound 5 (100 mg, 0.21 mmol), 2,4,6-trichlorobenzoyl chloride (0.036 mL, 0.23 mmol), NEt3 (0.044 mL, 0.31 mmol), DMAP (77 mg, 0.63 mmol), toluene (10 mL), room temperature to reflux, 48 h	The conversion was found to be sluggish, trace of desired product formation

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The unsuccessful results in the final macrolactonization prompted us to look for an alternate method of ring closure and we intended to explore approach-2 (macrolactamization approach) as shown in Fig. 3.

Fig. 3 Macrolactamization approach to clavatustides A, B.

Our synthesis commenced by coupling known (R)-tert-butyl-2-hydroxy-3-phenylpropanoate¹¹ (13) with 2-nitrobenzoic acid using DCC/DMAP conditions to obtain compound 14 in decent yields. The nitro group was smoothly reduced to amine 15 using 10% Pd/C in EtOAc and immediately reacted with 2-nitrobenzoyl chloride to afford compound 16 in good yields. The Abz-Abz-ester 17 having the di-anthranilic acid motif was obtained in high yield by the reduction of nitro group in compound 16.

The key precursors 11 and 12 of clavatustides A and B were obtained in good yields by amide coupling of 17 with N-ethyl glycine and sarcosine respectively as a mixture of rotamers (Scheme 3). Global de-protection of Boc and tert butyl groups of 11 and 12 followed by macrolactamization under amide coupling conditions (T₃P/DIPEA/THF) furnished 1 and 3 in moderate yields. It is noteworthy that cyclization was sluggish using other amide couple reagents such as EDC·HCl/HOBt/NEt₃/THF/RT and HATU/DIPEA/THF/RT.

Scheme 2 Synthesis of Abz-Abz-ester 17.

Scheme 3 Synthesis of (R) – clavatustides A (1) and B (3).

When compiling the final characterization data of 1 and 3, a discrepancy was noted with respect to the reported optical rotations. While the ¹H and ¹³C NMR spectra were in agreement with the reported values, the optical rotations were not only of opposite sign, but incomparable magnitudes (Table 2). Moreover, we found a discrepancy in the melting points between reported and synthesized depsipeptides. To address this discrepancy, we synthesized (S)-isomers of clavatustides A and B from advanced intermediate ent-17 (enantiomer of 17), which was in turn synthesized from (S)-tert-butyl-2-hydroxy-3-phenylpropanoate (13) through the intermediate compounds ent-14, ent-15 and ent-16 as shown in Scheme 4. The key intermediates ent-11 and ent-12 were synthesized from the compound ent-17 by coupling with corresponding amino acids (Scheme 4). Finally ent-11 and ent-12 were lactamized under macrolactamization conditions to afford the target compounds (S)-clavatustides A (2) and B (4) respectively (Scheme 4). The chemistry followed in Scheme 4 was very much similar to that adopted in Schemes 2 and 3.

Table 2 Optical rotations of natural and synthetic clavatustides A and B

Compound	Optical rotations		Melting points
	Reported values	Experimental values (present work)	Reported values	Experimental values (present work)
(R)-Clavatustide A (1)	[α]^24D = +22	[α]^24D = −111.68	187–188 °C	246–248 °C
(S)-Clavatustide A (2)		[α]^24D = +127.12		248–250 °C
(R)-Clavatustide B (3)	[α]^24D = +65	[α]^24D = −129.59	193–194 °C	213–216 °C
(S)-Clavatustide B (4)		[α]^24D = +111.02		210–214 °C

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Scheme 4 Synthesis of (S)-clavatustides A (2) and B (4).

A comparison of optical rotations and melting points of the naturally occurring clavatustides, synthetic clavatustides and their enantiomers are given in Table 2.

2.2. Pharmacology

2.2.1. Anti-proliferative activity. Above synthesized compounds 1, 2, 3 and 4 were evaluated for their anti-proliferative activity against three human cancer cell lines PC3, MDA-MB-231, HeLa and the normal lung stromal cells by using MTT assay. MTT assays were performed with different concentrations of compounds (1–4) in the three human cancer cell lines and the normal lung stromal cells at the 24 h time point to assess the effect on cell viability. There was a dose-dependent decrease in cell viability with all the four compounds in all the cancer cell lines tested. The IC₅₀ values of the test compounds are tabulated in Table 3.

Table 3 Anti-proliferative activity of compounds 1, 2, 3 and 4

Compound	IC50s (in μM)
	PC3	MDA-MB-231	HeLa
(R)-Clavatustide-A [1]	100	74.9	39.6
(S)-Clavatustide-A [2]	98.8	93.4	24.5
(R)-Clavatustide-B [3]	84.7	69.7	43.2
(S)-Clavatustide-B [4]	80.0	68.8	26.8

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Among the three cell lines tested, the cervical cancer cell line (HeLa) was found to be most sensitive to the compounds. The potency of the compounds in inducing cell death was in the order: HeLa > MDA-MB-231 > PC3. Further S-isomers of clavatustides A (2) and B (4) were found to be more active when compared to their enantiomers i.e. R-isomers.

All the compounds tested had a minimal effect on the cell viability of the normal lung cells, indicating that these compounds are not toxic to normal cells (Fig. 4).

Fig. 4 MTT assay against normal lung stromal cells.

3. Conclusions

In conclusion, we have developed a synthetic route capable of yielding pure depsipeptides (1) and (3) in sufficient quantities and good yields for the first time. By synthesizing the antipodes (2) and (4), we have reported correct optical rotations, there by confirming that the optical rotation values reported in the isolation article were incorrect. These compounds were found to be active against HeLa cancer cell line with IC₅₀s ranging between 26.8 and 43.2 μM. All the compounds tested had a minimal effect on the cell viability of the normal lung cells, indicating that these compounds are not toxic to normal cells. The future direction of work will be aimed at the pharmacological evaluation of these compounds in combination therapy along with other cytotoxic drugs and further synthesis of potent analogues.

4. Experimental

4.1. General

For product purification by flash column chromatography, silica gel (100–200 mesh) and EtOAC in petroleum ether (bp 60–90 °C) were used unless otherwise noted. All solvents were purified and dried by standard techniques, and distilled prior to use. Other commercially available reagents were used as received without further purification unless otherwise indicated. All organic extracts were dried over anhydrous sodium sulfate or magnesium sulfate. ¹H and ¹³C NMR spectra were recorded on a 400 MHz spectrometer (Bruker or Varian) with TMS as an internal reference and CDCl₃/DMSO-d₆ as solvent, unless otherwise indicated. IR spectra were recorded on an SHIMADZU FT-IR spectrometer as KBr pellet. HRMS were acquired on WATERS MICRO MASS Q-TOF. Melting points were measured on Buchi apparatus and were uncorrected. Chiral HPLC was recorded on Waters SFC method station. 100–200 mesh silica gel was used for purification of compounds.

4.2. Chemistry

4.2.1. Benzyl-2-(2-nitrobenzamido)benzoate (7). To an ice cold stirred solution of compound 6 (5.0 g, 22.02 mmol) in CH₂Cl₂ (80 mL) was added pyridine (2.67 mL, 32.99 mmol) followed by drop wise addition of 2-nitrobenzoyl chloride (3.21 mL, 24.22 mmol) over 30 min. After stirring for 3 h at room temperature, the reaction mixture was diluted with CH₂Cl₂ (50 mL) and washed with cold aqueous 1 N HCl (25 mL × 2), cold water (10 mL × 2), dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Column purification (hexane–EtOAc 8/2) afforded compound 7 (6.72 g, 81%) as an off white solid. Mp: 163–166 °C; IR (KBr): ¹H NMR (400 MHz, DMSO-d₆): δ 11.07 (br.s, 1H), 8.17–8.12 (m, 2H), 7.99 (dd, J = 8.0, 1.6 Hz, 1H), 7.86 (m, 1H), 7.79 (m, 1H), 7.74–7.67 (m, 2H), 7.44 (dd, J = 8.0, 2.0 Hz, 2H), 7.40–7.30 (m, 4H), 5.32 (s, 2H): ¹³C NMR (100 MHz, DMSO-d₆): δ 166.6, 163.7, 146.8, 138.4, 135.5, 134.0, 133.9, 131.8, 131.5, 130.6, 128.5, 128.2, 128.1, 124.5, 122.3, 119.8, 66.6: HRMS (ESI): calcd for C₂₁H₁₆N₂NaO₅ [M + Na]⁺ 399.0951; found 399.0958.

4.2.2. Benzyl-2-(2-aminobenzamido) benzoate (8). To a stirred solution of compound 7 (6.0 g, 15.95 mmol) in EtOH/water (1 : 1), were added NH₄Cl (5.12 g, 95.74 mmol), iron powder (3.46 g, 63.82 mmol) at room temperature and heated to 80 °C for 12 h. The catalyst was filtered over celite and washed with EtOAc (100 mL). The combined organic layer was concentrated under reduced pressure and the residue was purified by silica gel column (hexane–EtOAc 9/1) to obtain compound 8 (4.21 g, 76%) as a white solid. Mp: 87–91 °C (hexane : EtOAc = 9 : 1); IR (KBr): 3455, 3325, 3030, 1686, 1663, 1532, 1254 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆): δ 11.40 (s, 1H, D₂O exchangeable), 8.5 (d, J = 8.0 Hz, 1H), 8.03 (dd, J = 7.6, 1.2 Hz, 1H), 7.65 (q, J = 1.6 Hz, 2H), 7.48 (t, J = 1.6 Hz, 2H), 7.42–7.34 (m, 3H), 7.26 (m, 1H), 7.19 (m, 1H), 6.84 (dd, J = 8.4, 0.8 Hz, 1H), 6.67–6.63 (m, 3H), 5.38 (s, 2H): ¹³C NMR (100 MHz, DMSO-d₆): δ 167.4, 150.5, 140.6, 135.5, 134.2, 132.7, 130.6, 128.4, 128.1, 128.0, 127.3, 122.8, 120.7, 117.0, 116.6, 115.1, 113.7, 66.7: HRMS (ESI): calcd for C₂₁H₁₈N₂NaO₃ [M + Na]⁺ 369.1210; found 369.1227.

4.2.3. Benzyl-2-(2-(2-(tert-butoxycarbonyl(methyl)amino)acetamido)benzamido)benzoate (9). To a stirred solution of N-Boc-N-ethyl Gly (2 g, 10.58 mmol) in THF (50 mL) were added DIPEA (5.25 mL, 31.68 mmol), HATU (6 g, 15.87 mmol) at 0 °C and stirred for 30 min. A solution of compound 8 (3.66 g, 10.58 mmol) in THF (20 mL) was added to the reaction mixture at same temperature and stirred for 48 h at room temperature. The reaction mixture was quenched with cold water (40 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layer was washed with water (100 mL), brine (25 mL), dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column (hexane–EtOAc 7/3) to afford compound 6 (3.51 g, 64.1%) as a brown semi solid (mixture of rotamers); mp: 114–117 °C; IR (KBr): 3298, 3156, 2971, 1688, 1656, 1584, 1262 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆): δ 11.20 (br.s, 1H, D₂O exchangeable), 10.87 (br.s, 1H, D₂O exchangeable), 8.38 (dd, J = 8.4, 2.8 Hz, 2H), 8.03 (dd, J = 8.0, 1.6 Hz, 1H), 7.82 (dd, J = 8.0, 1.2 Hz, 1H), 7.67–7.63 (m, 1H), 7.58–7.54 (m, 1H), 7.42 (d, J = 6.8, 1.4 Hz, 2H), 7.37–7.30 (m, 3H), 7.29–7.21 (m, 2H), 5.36 (s, 2H), 3.94 (s, 2H), 2.93 (s, 3H), 1.33 (s, 9H); HRMS (ESI): calcd for C₂₉H₃₁N₃NaO₆ [M + Na]⁺ 540.2105; found 540.2138.

4.2.4. (R)-Benzyl-2-(2-(2-(2-hydroxy-N-methyl-3-phenylpropanamido)acetamido)benzamido)benzoate (10). To a stirred solution of compound 9 (3 g, 5.8 mmol) in CH₂Cl₂ (50 mL) was added trifluoroacetic acid (10 mL) at 0 °C and stirred at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure to obtain (3.2 g, crude) of TFA salt.

In another round bottomed flask was taken D-phenyllactic acid (0.96 g, 5.8 mmol) in THF (30 mL), added DIPEA (2.88 mL, 17.38 mmol), HATU (3.3 g, 8.70 mmol) at 0 °C and stirred for 30 min. A solution of above obtained TFA salt in THF (20 mL) was added to the reaction mixture at 0 °C and stirred for 24 h at room temperature. The reaction mixture was quenched with cold water (45 mL) and extracted with EtOAc (3 × 45 mL). The combined organic layer was washed with water (50 mL), brine (25 mL), dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column (hexane–EtOAc 6/4) to afford compound 10 (2.51 g, 76% overall yield for two steps) as a brown semi solid (mixture of rotamers); mp: 121–125 °C IR (KBr): 3402, 3264, 2925, 1691, 1647, 1583, 1259 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆); δ 11.18 (br.s, 1H), 10.79 (br.s, 1H), 8.36–8.31 (m, 2H), 8.01 (d, J = 7.5 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 7.56 (t, J = 8 Hz, 2H), 7.42 (d, J = 7.5 Hz, 2H), 7.36–7.30 (m, 3H), 7.24 (t, J = 7.5 Hz, 2H), 7.23–7.15 (m, 5H), 5.36 (s, 2H), 4.68 (d, J = 0.9 Hz, 2H), 4.19 (d, J = 16.4 Hz, 1H), 4.04 (br.s, 1H), 3.02 (br.s, 4H), 2.76–2.72 (m, 1H); HRMS (ESI): calcd for C₃₃H₃₁N₃NaO₆ [M + Na]⁺ 588.2105; found 588.2106.

4.2.5. (R)-2-(2-(2-(2-Hydroxy-N-methyl-3-phenylpropanamido) acetamido)benzamido) benzoic acid (5). To a stirred solution of compound 10 (1.8 g, 3.18 mmol) in EtOAc (30 mL) was added Pd/C (10%, 0.5 g) and hydrogenated at 20 psi for 12 h. The reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure to afford compound 5 (1.36 g, 90%) as an off white solid (mixture of rotamers); mp: 130–134 °C IR (KBr): 3439, 3137, 1683, 1645, 1521, 1294 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ 13.73 (br.s, 1H), 11.83 (br.s, 1H, D₂O exchangeable), 10.82, (br.s, 1H, D₂O exchangeable), 8.51 (d, J = 8.39 Hz, 1H), 8.32 (br.s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.56 (t, J = 8.0 Hz, 2H), 7.27–7.15 (m, 7H), 4.56 (br.s, 2H), 4.20 (d, J = 16 Hz, 2H), 3.04 (d, J = 17.0 Hz, 1H), 2. 99 (br.s, 3H), 2.79–2.73 (m, 1H); HRMS (ESI): calcd for C₂₆H₂₅N₃NaO₆ [M + Na]⁺ 498.1636; found 498.1621.

4.2.6. (R)-3-Phenyl-Lac-O^tBu (13). TLC (hexane : EtOAc = 9 : 1); [α]²⁵_D = +9.20 (c 1.0, CHCl₃); SFC: Chiralpak AD-H (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 90 : 10 flow rate = 3 g min⁻¹, t_R = 2.41 min; IR (KBr): 3498, 2979, 1728, 1157 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 7.29–7.22 (m, 5H), 4.33–4.30 (t, J = 5.6 Hz, 1H), 3.08 (dd, J = 14.0, 4.8 Hz, 1H), 2.94 (dd, J = 14.0, 6.4 Hz, 1H), 2.82 (br.s, 1H), 1.40 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): 173.40, 136.67, 129.58, 128.17, 126.64, 82.54, 71.24, 40.51, 27.93.

4.2.7. (S)-3-Phenyl-Lac-O^tBu (13). TLC (hexane : EtOAc = 9 : 1); [α]²⁵_D = −9.09 (c 1.0, CHCl₃); SFC: Chiralpak AD-H (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 90 : 10 flow rate = 3 g min⁻¹, t_R = 2.13 min; IR (KBr): 3500, 2979, 1728, 1158 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 7.30–7.20 (m, 5H), 4.32 (br.s, 1H), 3.08 (dd, J = 14.0 Hz and J = 4.8 Hz, 1H), 2.94 (dd, J = 14.0, 6.4 Hz, 1H), 2.82 (br.s, 1H), 1.43 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): 173.29, 136.62, 129.48, 128.03, 126.49, 82.29, 71.17, 40.39, 27.80.

4.2.8. (R)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-nitrobenzoate (14). A mixture of 2-nitrobenzoic acid (3.6 g, 21.59 mmol), compound 13 (4.0 g, 18.01 mmol), DMAP (0.44 g, 3.6 mmol) and DCC (5.57 g, 27.02 mmol) in CH₂Cl₂ were stirred for 12 h at room temperature. The solids were filtered, washed with CH₂Cl₂ (20 mL) and the combined filtrate was concentrated under reduced pressure. The crude compound was purified by silica gel column (hexane–EtOAc 8/2) to afford compound 14 (4.75 g, 71%) as pale yellow liquid. [α]²⁵_D = −13.40 (c 0.5, CHCl₃); SFC: Lux amylose-2 (4.6 × 250) mm 5 μ, CO₂-0.5% isopropylamine in IPA, 85 : 15 flow rate = 3 g min⁻¹, t_R = 4.41 min; IR (KBr): 3472, 2981, 1738, 1537 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 7.92–7.89 (m, 1H), 7.72–7.67 (m, 1H), 7.66–7.60 (m, 2H), 7.32–7.23 (m, 5H), 5.38 (dd, J = 7.6, 5.6 Hz, 1H), 3.23–3.19 (m, 2H), 1.42 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 167.86, 164.49, 148.14, 135.57, 132.79, 131.87, 130.16, 129.44, 128.40, 127.00, 123.90, 82.73, 74.83, 37.15, 27.85; HRMS (ESI): calcd for C₂₀H₂₂NO₆ [M + H]⁺ 372.1447; found 372.1446.

4.2.9. (S)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-nitrobenzoate (ent-14). By using the above procedure ent-14 (5.62 g, 70%) was synthesized from (4.80 g, 21.61 mmol) of ent-13; [α]²⁵_D = +11.84 (c 0.5, CHCl₃); SFC: Lux amylose-2 (4.6 × 250) mm 5 μ, CO₂-0.5% iso propyl amine in IPA, 85 : 15 flow rate = 3 g min⁻¹, t_R = 5.15 min; IR (KBr): 3460, 2979, 1739, 1538 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 7.91–7.89 (m, 1H), 7.72–7.62 (m, 3H), 7.31–7.23 (m, 5H), 5.40–5.36 (m, 1H), 3.23–3.19 (m, 2H), 1.42 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 167.88, 164.51, 148.13, 135.57, 132.81, 131.88, 130.17, 129.45, 128.41, 127.01, 123.92, 82.75, 74.83, 37.16, 27.86; HRMS (ESI): calcd for C₂₀H₂₂NO₆ [M + H]⁺ 372.1447; found 372.1444.

4.2.10. (R)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-aminobenzoate (15). To a stirred solution of compound 14 (4.30 g, 11.58 mmol) in EtOAc was added 10% Pd/C (800 mg). The resulting mixture was hydrogenated at 20 psi pressure for 12 h at room temperature. The catalyst was filtered over celite and washed with EtOAc. The combined filtrate was concentrated under reduced pressure and the crude amine was purified by silica gel column eluting (hexane : EtOAc 9/1) to obtain compound 15 as an off white solid (3.41 g, 86%). TLC (hexane : EtOAc 8/2); [α]²⁵_D = +23.20 (c 0.5, CHCl₃); SFC: Chiralcel OJ-H (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 80 : 20 flow rate = 3 g min⁻¹, t_R = 2.58 min; IR (KBr): 3469, 3356, 2982, 1719, 1692 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 7.89 (dd, J = 8.0, 1.2 Hz, 1H), 7.31–7.27 (m, 4H), 7.24–7.22 (m, 2H), 6.66–6.61 (m, 2H), 5.60 (br.s, 2H, D₂O exchangeable), 5.28 (t, J = 6.4 Hz, 1H), 3.23 (d, J = 6.4 Hz, 2H), 1.40 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 168.88, 167.28, 150.58, 136.28, 134.29, 131.41, 129.48, 128.38, 126.88, 116.54, 116.31, 110.27, 82.15, 73.39, 37.54, 27.89; HRMS (ESI): calcd for C₂₀H₂₄NO₄ [M + H]⁺ 342.1627; found 342.1630; HRMS (ESI): calcd for C₂₀H₂₄NO₄ [M + H]⁺ 342.1705; found 342.1701.

4.2.11. (S)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-aminobenzoate (ent-15). By using the above procedure ent-15 (3.60 g, 87%) was synthesized from (4.50 g, 12.12 mmol) of ent-14; [α]²⁵_D = −18.4 (c 0.5, CHCl₃); SFC: Chiralcel OJ-H (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 80 : 20 flow rate = 3 g min⁻¹, t_R = 3.39 min; IR (KBr): 3468, 3355, 2976, 1721, 1691 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 7.88 (d, J = 8.0, 1.6 Hz, 1H), 7.30–7.21 (m, 6H), 6.65–6.60 (m, 2H), 5.61 (br.s, 2H, D₂O exchangeable), 5.28 (t, J = 6.81 Hz, 1H), 3.24 (d, J = 6.4 Hz, 2H), 1.40 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 168.88, 167.28, 150.58, 136.27, 134.29, 131.40, 129.47, 128.37, 126.88, 116.53, 116.29, 110.24, 82.15, 73.80, 37.53, 27.88; HRMS (ESI): calcd for C₂₀H₂₄NO₄ [M + H]⁺ 342.1705; found 342.1705.

4.2.12. (R)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-(2-nitrobenzamido)benzoate (16). To an ice cold stirred solution of compound 15 (3.20 g, 9.38 mmol), NEt₃ (6.51 mL, 46.88 mmol) in CH₂Cl₂ was added a solution of 2-nitrobenzoyl chloride (1.61 mL, 12.18 mmol) in CH₂Cl₂ slowly over a period of 10 min and stirred for 12 h at room temperature. The reaction mixture was quenched with ice water and extracted with CH₂Cl₂ (3 × 70 mL). The combined organic layer was washed with water, 10% aqueous NaHCO₃ solution, brine, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column (hexane–EtOAc 7/3) to afford compound 16 (3.22 g, 70%) as pale yellow semi solid; [α]²⁵_D = +13.54 (c 0.5, CHCl₃); SFC: Chiralcel OD-H (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 70 : 30 flow rate = 3 g min⁻¹, t_R = 4.06 min; IR (KBr): 3297, 3268, 2981, 1743, 1530, 1275, 1259 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 11.17 (br.s, 1H, D₂O exchangeable), 8.78 (d, J = 8.4 Hz, 1H), 8.08–8.06 (m, 2H), 7.71–7.59 (m, 4H), 7.32–7.17 (m, 6H), 5.29 (dd, J = 7.6, 5.2 Hz, 1H), 3.25–3.22 (m, 2H), 1.37 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 168.10, 167.53, 164.39, 146.92, 140.89, 135.69, 135.07, 133.60, 132.98, 131.05, 130.75, 129.40, 128.50, 128.36, 127.13, 124.67, 123.52, 120.90, 115.40, 82.78, 74.22, 37.34, 27.83; HRMS: calcd for C₂₇H₂₇N₂O₇ [M + H]⁺ 491.1818 found 491.1812.

4.2.13. (S)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-(2-nitrobenzamido)benzoate (ent-16). By using the above procedure ent-16 (3.4 g, 71%) was synthesized from (3.3 g, 9.67 mmol) of ent-15; [α]²⁵_D = −14.28 (c 0.5, CHCl₃); SFC: Chiralcel OD-H (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 70 : 30 flow rate = 3 g min⁻¹, t_R = 3.68 min; IR (KBr): 3447, 3347, 1743, 1691, 1530, 1252 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 11.17 (br.s, 1H, D₂O exchangeable), 8.78 (d, J = 8.4 Hz, 1H), 8.07 (t, J = 8.2 Hz, 2H), 7.70–7.58 (m, 4H), 7.32–7.16 (m, 6H), 5.29 (dd, J = 7.6, 5.6 Hz, 1H), 3.25–3.22 (m, 2H), 1.37 (s, 9H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 168.09, 167.51, 164.37, 146.91, 140.88, 135.68, 135.05, 133.59, 132.96, 131.04, 130.75, 129.39, 128.49, 128.34, 127.11, 124.66, 123.50, 120.87, 115.38, 82.77, 74.21, 37.33, 27.81 ppm; HRMS: calcd for C₂₇H₂₇N₂O₇ [M + H]⁺ 491.1818 found 491.1813.

4.2.14. (R)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-(2-aminobenzamido) benzoate (17). To a stirred solution of compound 16 (3.1 g, 6.32 mmol) in EtOAc was added 10% Pd/C (600 mg) and the resulting mixture was hydrogenated at 20 psi pressure for 12 h at room temperature. The catalyst was filtered over celite and washed with EtOAc. The combined filtrate was concentrated under reduced pressure and the crude amine was purified by silica gel column (hexane–EtOAc 7/3) to obtain compound 17 (2.59 g, 89%) as an off white solid; [α]²⁵_D = +17.60 (c = 0.5, CHCl₃); SFC: Chiralcel OD-H (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 60 : 40 flow rate = 4 g min⁻¹, t_R = 5.17 min; IR (KBr): 3445, 3347, 2978, 1736, 1671 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 11.53 (br.s, 1H, D₂O exchangeable), 8.79 (d, J = 8.8 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.60–7.56 (m, 2H), 7.33–7.22 (m, 6H), 7.10 (t, J = 7.6 Hz, 1H), 6.72–6.68 (m, 2H), 5.74 (br.s, 2H, D₂O exchangeable), 5.36–5.33 (m, 1H), 3.30–3.22 (m, 2H), 1.41 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 168.27, 168.07, 167.81, 149.80, 141.96, 135.87, 134.79, 132.81, 131.06, 129.41, 128.48, 127.69, 127.07, 122.41, 120.41, 117.41, 116.75, 115.63, 115.00, 82.62, 74.23, 37.39, 27.87; HRMS (ESI): calcd for C₂₇H₂₉N₂O₅ [M + H]⁺ 461.2076; found 461.2079.

4.2.15. (S)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-(2-aminobenzamido)benzoate (ent-17). By using the above procedure ent-17 (2.71 g, 90%) was synthesized from (3.2 g, 6.52 mmol) of ent-16; [α]²⁵_D = −13.00 (c 0.5, CHCl₃); SFC: Chiralcel OD-H (4.6 × 250) mm 5 μ, CO₂-0.5% NEt₂ (DEA) in MeOH, 60 : 40 flow rate = 4 g min⁻¹, t_R = 4.5 min; IR (KBr): 3447, 3346, 2976, 1721, 1691 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 11.53 (br.s, 1H, D₂O exchangeable), 8.80–8.78 (m, 1H), 8.09–8.07 (m, 1H), 7.63–7.55 (m, 2H), 7.33–7.24 (m, 4H), 7.23–7.20 (m, 2H), 7.12–7.08 (m, 1H), 6.71–6.67 (m, 2H), 5.74 (br.s, 2H, D₂O exchangeable), 5.36 (dd, J = 7.6, 5.2 Hz, 1H), 3.30–3.23 (m, 2H), 1.41 (s, 9H); ¹³C NMR (100 MHz, CDCl₃): δ 168.25, 168.05, 167.79, 149.79, 141.95, 135.85, 134.77, 132.79, 131.04, 129.40, 128.46, 127.67, 127.05, 122.39, 120.40, 117.42, 116.72, 115.60, 114.98, 82.60, 74.21, 37.37, 27.85; HRMS (ESI): calcd for C₂₇H₂₉N₂O₅ [M + H]⁺ 461.2076; found 461.2072.

4.2.16. (R)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-(2-(2-(tert-butoxycarbonyl(ethyl)amino)acetamido)benzamido)benzoate (11). To a stirred solution of N-Boc-N-ethyl Gly (0.529 g, 2.60 mmol) in THF were added DIPEA (1.43 mL, 8.66 mmol), HATU (1.23 g, 3.26 mmol) at 0 °C and stirred for 30 min. A solution of compound 17 (1.0 g, 2.17 mmol) in THF (15 mL) was added to the reaction mixture at 0 °C and stirred for 48 h at room temperature. The reaction mixture was quenched with cold water (25 mL) and extracted with EtOAc (3 × 25 mL). The combined organic layer were washed with water, brine (25 mL), dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column (hexane–EtOAc 7/3) to afford compound 11 (1.05 g, 75%) as a brown semi solid (mixture of rotamers); [α]²⁵_D = +6.94 (c 0.5, CHCl₃); SFC: Chiralpak IC (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 60 : 40 flow rate = 3 g min⁻¹, t_R = 5.32 min; IR (KBr): 3447, 2978, 1748, 1741, 1694 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆, VT NMR, 90 °C): δ 11.0 (br.s, 1H, D₂O exchangeable), 10.8 (bs, 1H, D₂O exchangeable), 8.44–8.39 (m, 2H), 8.0 (d, J = 6.8 Hz, 1H), 7.77 (d, J = 6.8 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.30–7.19 (m, 7H), 5.34 (t, J = 6.4 Hz, 1H), 3.97 (s, 2H), 3.30 (q, J = 7.0 Hz, 2H), 3.22 (d, J = 6.4 Hz, 2H), 1.32 (s, 18H), 1.06 (t, J = 7.2 Hz, 3H); HRMS (ESI): calcd for C₃₆H₄₄N₃O₈ [M + H]⁺ 646.3128; found 646.3122.

4.2.17. (S)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-(2-(2-(tert-butoxycarbonyl(ethyl)amino)acetamido)benzamido)benzoate (ent-11). By using the above procedure ent-11 (1.1 g, 78%) was synthesized as mixture of rotamers from (1.0 g, 2.17 mmol) of ent-17; [α]²⁵_D = −7.05 (c 0.5, CHCl₃); SFC: Chiralpak IC (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 60 : 40 flow rate = 3 g min⁻¹, t_R = 7.39 min; IR (KBr): 3466, 3307, 2977, 1745, 1694 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆, VT NMR, 90 °C): δ 11.0 (br.s, 1H, D₂O exchangeable), 10.8 (br.s, 1H, D₂O exchangeable), 8.44–8.39 (m, 2H), 7.99 (d, J = 7.6 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H), 7.30–7.19 (m, 7H), 5.34 (t, J = 6.4 Hz, 1H), 3.91 (s, 2H), 3.30 (q, J = 7.2 Hz, 2H), 3.22 (d, J = 6.8 Hz, 2H), 1.32 (s, 18H), 1.06 (t, J = 7.0 Hz, 3H); HRMS (ESI): calcd for C₃₆H₄₄N₃O₈ [M + H]⁺ 646.3128; found 646.3127.

4.2.18. (R)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-(2-(2-(tert-butoxycarbonyl(methyl)amino)acetamido) benzamido)benzoate (12). To a stirred solution of N-Boc-Sar-OH (0.493 g, 2.60 mmol) in THF (50 mL) were added DIPEA (1.43 mL, 8.68 mmol), HATU (1.23 g, 3.26 mmol) at 0 °C stirred for 30 min. A solution of compound 17 (1.0 g, 2.17 mmol) in THF (15 mL) was added to the reaction mixture at 0 °C and stirred for 48 h at room temperature. The reaction mixture was quenched with cold water (25 mL) and extracted with EtOAc (3 × 25 mL). The combined organic layer was washed with water, brine (15 mL), dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column (hexane–EtOAc 7/3) to afford compound 12 (1.05 g 76%) as a thick liquid (mixture of rotamers), [α]²⁵_D = +6.00 (c 0.5, CHCl₃); SFC: Chiralpak IC (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 55 : 45 flow rate = 3 g min⁻¹, t_R = 4.9 min; IR (KBr): 3271, 2978, 1748, 1694, 1253 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆, VT NMR, 90 °C): δ 11.03 (br.s, 1H, D₂O exchangeable), 10.83 (br.s, 1H, D₂O exchangeable), 8.43 (d, J = 8.0 Hz, 1H), 8.36 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H), 7.30–7.19 (m, 7H), 5.34 (t, J = 6.4 Hz, 1H), 3.88 (s, 2H), 3.21 (d, J = 6.4 Hz, 2H), 2.88 (s, 3H), 1.25 (s, 18H); HRMS (ESI): calcd for C₃₅H₄₂N₃O₈ [M + H]⁺ 632.2972; found 632.2975.

4.2.19. (S)-1-tert-Butoxy-1-oxo-3-phenylpropan-2-yl-2-(2-(2-(tert-butoxycarbonyl(methyl)amino)acetamido) benzamido)benzoate (ent-12). By using the above procedure ent-12 (1.07 g, 78%) was synthesized as mixture of rotamers from ent-17 (1.0 g, 2.17 mmol). [α]²⁵_D = −6.36 (c 0.5, CHCl₃); SFC: Chiralpak IC (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 55 : 45 flow rate = 3 g min⁻¹, t_R = 5.91 min. IR (KBr): 3305, 2978, 1744, 1695, 1264 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆, VT NMR, 90 °C): δ 11.03 (br.s, 1H, D₂O exchangeable), 10.8 (br.s, 1H, D₂O exchangeable), 8.43 (d, J = 8.4 Hz, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.01–7.98 (m, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.70–7.65 (m, 1H), 7.57–7.53 (m, 1H), 7.30–7.19 (m, 7H), 5.35 (t, J = 6.4 Hz, 1H), 3.93 (s, 2H), 3.22 (d, J = 6.4 Hz, 2H), 2.89 (s, 3H), 1.32 (s, 18H); HRMS (ESI): calcd for C₃₅H₄₂N₃O₈ [M + H]⁺ 632.2972; found 632.2975.

4.2.20. (R)-Clavatustide A (1). To a stirred solution of compound 11 (0.9 g, 1.39 mmol) in CH₂Cl₂ (50 mL) was added trifluoroacetic acid (5 mL) at 0 °C and stirred at room temperature for 12 h. The reaction mixture was concentrated in vacuo and the TFA salt was dissolved in dry THF (10 mL) and cooled to 0 °C. DIPEA (0.92 mL, 5.57 mmol) followed by T₃P (50% in EtOAc) (0.66 g, 2.09 mmol) were added to the reaction mixture at 0 °C and stirred at room temperature for 12 h. The reaction was quenched with cold water (25 mL) and extracted with EtOAc (3 × 45 mL). The combined organic layer was washed with water; brine (40 mL) dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column (hexane–EtOAc 1/1) to afford (R)-clavatustide A (1) (230 mg, 35%) as an off white solid. Mp: 246–248 °C; [α]²⁴_D = −111.68 (c 0.5, MeOH); SFC: Chiralpak AS-H (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 60 : 40 flow rate = 4 g min⁻¹, t_R = 3.3 min; IR (KBr): 3275, 1727, 1690, 1660 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 10.21 (br.s, 1H, D₂O exchangeable), 8.58 (d, J = 8.4 Hz, 1H), 7.85 (br.s, 1H, D₂O exchangeable), 7.64 (d, J = 7.2 Hz, 1H), 7.57–7.46 (m, 3H), 7.38–7.30 (m, 6H), 7.22 (d, J = 7.2 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H), 5.47 (dd, J = 8.8, 6.2 Hz, 1H), 5.25 (d, J = 14.8 Hz, 1H), 3.97–3.92 (m, 1H), 3.49–3.44 (m, 1H), 3.31–3.21 (m, 3H), 1.02 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 169.50, 167.90, 167.43, 167.09, 138.09, 135.83, 134.50, 132.42, 132.14, 129.63, 129.36, 128.69, 127.54, 127.26, 126.38, 126.30, 125.87, 123.03, 122.84, 121.71, 71.56, 51.60, 43.24, 37.62, 13.64; HRMS (ESI): calcd for C₂₇H₂₆N₃O₅ [M + H]⁺ 472.1872; found 472.1873.

4.2.21. (S)-Clavatustide A (2). (S)-Clavatustide A (2) was synthesized from ent-11 (0.9 g, 1.39 mmol) by using the above procedure (221 mg, 34%); mp: 248–250 °C; [α]²⁴_D = +127.12 (c 0.5, MeOH); SFC: Chiralpak AS-H (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 60 : 40 flow rate = 4 g min⁻¹, t_R = 2.31 min; IR (KBr): 3277, 1727, 1690, 1651 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 10.22 (br.s, 1H, D₂O exchangeable), 8.55 (d, J = 8.4 Hz, 1H), 7.92 (br.s, 1H, D₂O exchangeable), 7.62 (d, J = 8.0 Hz, 1H), 7.57–7.46 (m, 3H), 7.39–7.31 (m, 6H), 7.21 (d, J = 7.6 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 5.47 (dd, J = 8.2, 6.0 Hz, 1H), 5.24 (d, J = 14.8 Hz, 1H), 3.95–3.93 (m, 1H), 3.46 (dd, J = 14.0, 8.0 Hz, 1H), 3.31–3.20 (m, 3H) 1.01 (t, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 169.49, 167.86, 167.47, 167.11, 138.04, 135.78, 134.53, 132.40, 132.09, 129.61, 129.26, 128.67, 127.47, 127.25, 126.46, 126.19, 125.92, 123.02, 122.81, 121.60, 71.53, 51.57, 43.22, 37.61, 13.61; HRMS (ESI): calcd for C₂₇H₂₆N₃O₅ [M + H]⁺ 472.1872; found 472.1876.

4.2.22. (R)-Clavatustide B (3). To a stirred solution of compound 12 (0.90 g, 1.42 mmol) in CH₂Cl₂ (50 mL) was added trifluoroacetic acid (5 mL) at 0 °C and stirred at room temperature for 12 h. The reaction mixture was concentrated in vacuo, TFA salt dissolved in dry THF (10 mL) and cooled to 0 °C. DIPEA (0.94 mL, 5.65 mmol) followed by T₃P (50% in EtOAc) (0.68 g, 2.10 mmol) were added to the reaction mixture at 0 °C and stirred at room temperature for 12 h. The reaction was quenched with cold water (25 mL) and extracted with EtOAc (3 × 45 mL). The combined organic layer was washed with water (25 mL), brine (25 mL), dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column (methanol–CH₂Cl₂ = 1/9) afforded (R)-clavatustide B (3) (210 mg, 32%) as off white solid. Mp: 213–216 °C; [α]²⁴_D = −129.59 (c 0.51, MeOH); SFC: Chiralpak AS-H (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 60 : 40 flow rate = 4 g min⁻¹, t_R = 4.21 min; IR (KBr): 3306, 3211, 2925, 1721, 1690 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 10.14 (br.s, 1H, D₂O exchangeable), 8.55 (d, J = 8.4 Hz, 1H), 7.86 (br.s, 1H, D₂O exchangeable), 7.69 (d, J = 6.8 Hz, 1H), 7.58–7.45 (m, 3H), 7.37–7.31 (m, 6H), 7.23 (d, J = 7.6 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 5.53 (t, J = 7.6 Hz, 1H), 5.34 (d, J = 14.4 Hz, 1H), 3.43 (dd, J = 13.2, 8.0 Hz, 1H), 3.29 (dd, J = 13.6, 7.2 Hz, 1H), 3.12 (d, J = 14.4 Hz, 1H), 3.06 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 169.65, 167.45, 167.32, 167.20, 137.97, 135.46, 134.68, 132.55, 132.09, 129.52, 128.65, 127.37, 127.28, 126.37, 126.32, 126.03, 123.06, 122.86, 121.73, 71.39, 54.11, 37.15, 35.74; HRMS (ESI): calcd for C₂₆H₂₄N₃O₅ [M + H]⁺ 458.1716; found 458.1713.

4.2.23. (S)-Clavatustide B (4). (S)-Clavatustide-B (4) was synthesized from ent-12 (0.92 g, 1.45 mmol) by using the above procedure (215 mg, 33%); mp: 210–214 °C; [α]²⁴_D = +111.02 (c 0.517, MeOH); SFC Chiralpak AS-H (4.6 × 250) mm 5 μ, CO₂-0.5% DEA in MeOH, 60 : 40 flow rate = 4 g min⁻¹, t_R = 2.41 min; IR (KBr): 3307, 3212, 2925, 1722, 1691, 1661 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 10.14 (br.s, 1H, D₂O exchangeable), 8.55 (d, J = 8.0 Hz, 1H), 7.84 (br.s, 1H, D₂O exchangeable), 7.69 (d, J = 6.8 Hz, 1H), 7.58–7.45 (m, 3H), 7.37–7.31 (m, 6H), 7.23 (d, J = 7.6 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 5.53 (t, J = 7.6 Hz, 1H), 5.34 (d, J = 14.4 Hz, 1H), 3.44–3.41 (m, 1H), 3.32–3.29 (m, 1H), 3.12 (d, J = 14.8 Hz, 1H), 3.06 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 169.65, 167.46, 167.30, 167.21, 137.95, 135.44, 134.69, 132.54, 132.07, 129.51, 128.64, 127.32, 127.28, 126.36, 126.32, 126.03, 123.05, 122.85, 121.69, 71.37, 54.10, 37.14, 35.73, HRMS (ESI): calcd for C₂₆H₂₄N₃O₅ [M + H]⁺ 458.1716; found 458.1716.

4.3. Pharmacology

4.3.1. Anti-proliferative activity – cell viability assay/MTT assay. Human cancer cell lines namely: PC3 (prostate carcinoma), HeLa (cervical carcinoma) and MDA-MB-231 (breast carcinoma) were procured from ATCC, USA. Normal lung stromal cells were isolated from adult rat lung. Cells were cultured in DMEM with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO₂. Cell viability was determined by quantification of 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases. In brief, 2 × 10⁴ cells per well were plated in a 96-well plate and treated with varying concentrations of the compounds (1–4) for 24 h. Following this, MTT was added to a final concentration of 100 μg per well and further incubated for 3 h at 37 °C. The formazan dye crystals formed were solubilized in DMSO and the plate was incubated at room temperature for 1 h. The absorbance was measured at 595 nm in an ELISA microplate reader (Biotek, NY, USA). All samples were assayed in triplicate in three independent experiments. Absorbance values plotted are the mean from three independent experiments and the results are expressed as percentage of the control, which was considered to be 100%.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

The authors express their profound thanks to the management of GVK Biosciences Private Limited for financial support. The encouragement rendered by Dr Sudhir Kumar Singh throughout this work is deeply acknowledged. Our sincere gratitude to Dr K. Muralidharan, Mr Santosh Guduru for analytical support and special thanks to Mr Bhupathi Rajasekhara Rao for his help.

References and notes

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Footnote

† Electronic supplementary information (ESI) available: ¹H, ¹³C NMR and chiral HPLC spectra for all compounds 1–9. DEPT, COSY, HMBC and HSQC for both antipodes of 1 and 2. See DOI: 10.1039/c6ra08861a

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