Identification of boholamide A analogue as a potential hypoxia-selective anti-triple-negative breast cancer agent by targeting eEF1A1

Abstract

Triple-negative breast cancer (TNBC) is one of the most aggressive and metastatic forms of breast cancer, for which there are currently no satisfactory therapeutic agents. Here, we reported for the first time that boholamide A, a naturally occurring macrocyclic depsipeptide, exhibited hypoxia-selective anti-TNBC activity against in MDA-MB-231 cells. However, its structure‒activity relationships and target had not yet been elucidated. A series of boholamide A analogues were chemically synthesized and evaluated for anti-TNBC potency. The most promising compound 1j was prepared in 13 linear steps with an overall yield of 7.92%, and exhibited high potency against MDA-MB-231 cells with an IC₅₀ value of 0.15 μmol/L under hypoxic condition. Moreover, 1j significantly inhibited proliferation and migration, and induced apoptosis in MDA-MB-231 cells. Compound 16, a prodrug of 1j, significantly inhibited the tumor volume and tumor weight in xenografts. Furthermore, we identified that 1j covalently targeted eukaryotic translation elongation factor 1alpha 1 (eEF1A1) which might underlie the anticancer activity and hypoxia selectivity of boholamide A analogues. These results suggested that boholamide A analogues represented a promising scaffold for discovering hypoxia-selective anti-TNBC agents, and compound 16 deserved further investigation as a candidate for TNBC treatment.

Graphical abstract

The SAR of boholamide A at multiple sites was explored by total synthesis. The optimized compound 1j potently targeted eEF1A1 and inhibited triple-negative breast cancer growth in vivo.

1. Introduction

Breast cancer is the second leading cause of cancer-related death among women in the United States, following lung cancer, and accounts for 32% of all newly diagnosed female cancers each year¹. Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer with a worse prognosis than other breast cancer types². Its biological characteristics include high proliferative activity, increased immune cell infiltration, basal-like and mesenchymal phenotype, and homologous recombination deficiency, which is partly associated with loss of BRCA1 or BRCA2 function3, 4, 5. TNBC is a heterogeneous disease for which no effective targeted therapies are currently available. Conventional chemotherapy remains the common choice in clinical practice, yet it reduces the risk of recurrence by only approximately 30% in specific patient populations⁶. Due to the lack of effective therapeutic agents, the median overall survival of TNBC patients remains poor, at less than 18 months⁷. Therefore, novel compounds with a unique mechanism are urgently needed to address this clinical challenge.

Boholamide A is a macrocyclic depsipeptide featuring a 4-amino-2,4-pentadienolate (APD) moiety⁸. The unique APD moiety is found in only a few natural products, such as rakicidin (A–I)9, 10, 11, 12, 13, 14, vinylamycin¹⁵, microtermolide A¹⁶, and BE-43547^17,18 (Fig. 1). Due to their unique structures and hypoxia-selective anticancer activity, these compounds have attracted considerable attention from our research group and other investigators19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32. We previously reported the first total synthesis of boholamide A via a 16 step linear route with an overall yield of 5.46%, which prompted a revision of the C6 stereochemistry in the previously proposed structure of natural boholamide A³³.

Figure 1.

Structures of cyclodepsipeptides containing APD moiety.

Recently, an in-house natural products library screening for potential anti-TNBC agents led to the identification of boholamide A (Fig. 1) as an anti-TNBC compound with moderate activity. However, its structure‒activity relationships and molecular target remained unknown. Given its structural novelty compared to previously reported anti-TNBC agents, we employed an efficient combinatorial approach to synthesize a series of boholamide A analogues and evaluated their anti-cancer activity. Notably, the most promising analogue, compound 1j exhibited high selectivity against TNBC cells under hypoxic conditions and low cytotoxicity against normal cells. To reveal the target of compound 1j, we synthesized probe 15 and performed target identification studies. The results indicated that compound 1j directly bound eukaryotic translation elongation factor 1alpha 1 (eEF1A1), and its target protein exhibited complete identity to the natural product BE-43547A₂ which possessed an APD structure. Mechanistic studies revealed that compound 1j disrupted the eEF1A1/FoxO1 interaction, promoted FoxO1 nuclear translocation and consequently suppressed the JAK/STAT3 pathway. Furthermore, compound 16 which was a prodrug of 1j, significantly inhibited the tumor volume and tumor weight in a dose-dependent manner and inhibited breast cancer cell metastasis in vivo.

2. Results and discussion

2.1. The synthesis of boholamide A analogues

Scheme 1 illustrates the retrosynthetic analysis of boholamides (1). Boholamides was synthesized by elimination of alcohol 2. Alcohol 2 could be disconnected into three fragments: alcohol 8, amino acid fragment 7, and amine fragment 5. Alcohol 8 could be prepared by a Mukaiyama aldol reaction of D-camphorsultam derivative 12 with aldehyde 10. Amine fragment 5 was prepared following our previously reported procedure³³, while amino acid fragment 7 was commercially available.

Scheme 1.

Retrosynthetic analysis of boholamides (1).

Our and other groups’23, 24, 25, 26 studies demonstrated that APD moiety served as the pharmacophore for APD-containing cyclolipodepsipeptides. Therefore, we retained the APD moiety and attempted to modify the long lipophilic chain and the unique methoxy group of alcohol 8 in the first-round modification, and designed a total of seven analogues. The synthetic route is shown in Scheme 2.

Scheme 2.

Total synthesis of boholamide A analogues 1a‒1g.

The d-camphorsultam derivative 12a–12g was converted to a silyl enol ether 11a–11g using TBSOTf/Et₃N. Mukaiyama aldol reaction of 11a–11g with aldehydes 10a–10g under BF₃-mediated condition afforded compounds 9a–9g (dr > 30:1, according to ¹H NMR). After the hydrolysis of compounds 9a–9g with LiOH, the alcohol 8a–8g was obtained after protection with benzyl group. The alcohol 8a–8g was then esterified with the amino acid fragment 7 under DIC/DMAP. Compound 6a–6g was hydrogenated under Pd/C and coupled with amine fragment 5 to form the ring precursor 4a–4g. Compound 5 was synthesized following our previously reported procedure³³. After removal of the two protective groups by 20% TFA, compound 3a–3g was obtained by macro-lactamization using HATU/DIPEA. Deprotection of TBDPS with TBAF/HOAc yielded alcohol 2a–2g, which were activated with an ethyl sulfonyl group and eliminated with DBU to afford boholamide A analogues 1a–1g.

As shown in Table 1, boholamide A analogue 1c exhibited the most potent anti-TNBC activity, comparable to that of natural product. Analogue 1c was prepared in 13 linear steps with 6.40% overall yield, representing an improvement over boholamide A, which required 16 linear steps and 5.46% overall yield. Accordingly, in the second-round modification, we started with chiral alcohol 8c, changed the amino acid fragment 7, and synthesized a total of five boholamide A analogues 1h–1l with modification at the C-11 site. The synthetic route was shown in Scheme 3.

Table 1.

Anti-proliferation activity of boholamide analogues 1a‒1g against cancer cells.^a

Compd.	Structure	MDA-MB-231			U118			A549
		IC50 (μmol/L)		HSIb	IC50 (μmol/L)		HSI	IC50 (μmol/L)		HSI
		Normoxia	Hypoxia		Normoxia	Hypoxia		Normoxia	Hypoxia
Boholamide A		2.78 ± 0.32	0.66 ± 0.18	4.21	4.69 ± 1.04	1.44 ± 0.62	3.26	7.53 ± 1.17	2.44 ± 0.77	3.09
1a		4.85 ± 1.65	2.27 ± 1.17	2.14	8.64 ± 3.44	4.09 ± 0.08	2.11	12.37 ± 3.81	3.01 ± 1.31	4.11
1b		8.87 ± 6.20	5.68 ± 1.44	1.56	17.83 ± 8.68	13.14 ± 5.33	1.36	26.22 ± 8.09	11.08 ± 1.31	2.37
1c		2.02 ± 0.41	1.24 ± 0.24	1.63	3.42 ± 1.49	1.55 ± 0.39	2.21	4.09 ± 0.88	1.30 ± 0.55	3.14
1d		6.14 ± 2.77	3.14 ± 1.37	1.96	14.87 ± 8.37	10.29 ± 2.77	1.45	15.72 ± 4.81	8.58 ± 2.55	1.83
1e		＞20	16.34 ± 7.09	>1.22	＞50	＞20	‒	＞50	22.62 ± 13.71	>2.21
1f		5.93 ± 0.17	2.38 ± 0.99	2.49	7.50 ± 0.85	4.49 ± 2.56	1.67	8.46 ± 1.75	2.51 ± 1.98	3.37
1g		4.36 ± 2.23	3.58 ± 1.92	1.21	7.52 ± 3.23	6.36 ± 2.35	1.18	10.16 ± 2.40	3.43 ± 2.38	2.96

^aAll values were the means of three independent experiments.

^bHSI was calculated from: IC₅₀ (normoxia)/IC₅₀ (hypoxia).

Scheme 3.

Total synthesis of boholamide A analogues 1h‒1l.

To explore the biological mechanism of boholamide A analogues, probes 1m and 15 were designed and synthesized (Fig. 5A). Compound 13 was prepared from alcohol 8c by removal of the benzyl group followed by allyl protection. Coupling of 13 with amino acid 7m gave 6m. The allyl group of 6m was removed by Pd(PPh₃)₄, and the resulting intermediate was coupled with amine fragment 5 to afford ring precursor 4m. Compound 4m was converted to compound 1m following the same synthetic route. A click reaction between 1m and commercially available 14, catalyzed by Cu₂O nanoparticles, yielded the boholamide A probe 15³⁴. Compound 16, a prodrug of 1j, was obtained by nucleophilic substitution of methyl piperazine with ethanesulfonyl chloride-activated alcohol 2j (Supporting Information Fig. S1A).

Figure 5.

Boholamide A analogue directly targeted eEF1A1. (A) The synthesis route of 1m and probe 15. (B) The antiproliferative activity of 1m against MDA-MB-231 cells under normoxia and hypoxia. (C) The silver stain picture of the protein enriched by probe 15. (D) The peptide coverage of eEF1A1 after LC‒MS/MS analysis. (E) The unique peptides of eEF1A1 detected by LC‒MS/MS analysis. (F) Western blot analysis to detect the binding of eEF1A1 using probe 15 in cell lysates. (G) Western blot analysis to detect the binding of eEF1A2 using probe 15 in cell lysates. (H) Western blot analysis to detect the competitive binding of probe 15 and 1j to eEF1A1 protein. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005, ∗∗∗∗P < 0.001. Data presented as means ± SD (n = 3).

2.2. Structure‒activity relationship of boholamide A analogues

With the synthesized boholamide A analogues in hand, we first evaluated their effects on human triple-negative breast cancer cell line MDA-MB-231, the human glioblastoma cell line U118 and the human lung cancer cell line A549 under both normoxic and hypoxic conditions. The hypoxia-selective index (HSI) value was calculated by IC₅₀ (normoxia)/IC₅₀ (hypoxia) for evaluating their potency of hypoxia-selective effect. The natural product boholamide A was also included for comparison.

As indicated in Table 1, all the synthesized analogues 1a–1g exhibited higher potency against MDA-MB-231 cells than against U118 and A549 cells, and showed greater anti-cancer activity under hypoxia than under normoxia. The natural product boholamide A demonstrated potent activity against MDA-MB-231 cells (IC₅₀ = 2.78 μmol/L under normoxia, IC₅₀ = 0.66 μmol/L under hypoxia, HSI = 4.21). Boholamide A also displayed hypoxia-selective activity against U118 and A549 cells with HSI value of 3.26 and 3.09, respectively. Removal of the C6 methyl group yielded compound 1a, which was less active than boholamide A against MDA-MB-231, U118 and A549 cells under both normoxia and hypoxia. Further removal of the C4 methyl group produced compound 1b. The anti-TNBC potency of 1b (IC₅₀ = 8.87 μmol/L under normoxia, IC₅₀ = 5.68 μmol/L under hypoxia) was greatly reduced comparing with boholamide A (IC₅₀ = 2.78 μmol/L under normoxia, IC₅₀ = 0.66 μmol/L under hypoxia), especially under hypoxia. The HSI value was also decreased (4.21 vs 1.56). Repositioning the C6 methyl group to the terminal position of the long lipophilic chain generated compound 1c, which was the most active analogue in series 1a–1g against all three cell lines under both conditions However, 1c exhibited slightly less potent activity against MDA-MB-231 cells compared with boholamide A under hypoxia, and the HSI index was also reduced (1.63 vs 4.21). Further relocation of the C4 methyl group to the chain terminus (1d) significantly diminished anti-cancer activity against all cell lines compared with 1c. The sub-series of 1e–1g was modified at the C2 position. Replacement of the methoxyl group in 1b and 1c with a methyl group afforded 1e and 1f, respectively. Compounds 1e and 1f showed weaker anti-cancer effect than 1b and 1c which indicated that the methoxyl group was important for their high anti-cancer activities.

Compound 1c was selected as a new lead compound for further structure-activity relationship studies. Five boholamide A analogues 1h–1l were rapidly synthesized using a combinatorial strategy to explore the effect of the bottom amino acid fragment on the anti-proliferative activity. The anti-proliferative activities of these boholamide analogues against different tumor cells were summarized in Table 2. Compounds 1h–1l exhibited comparable inhibitory effects on MDA-MB-231 cells under hypoxia, indicating that this site could accommodate diverse modifications. Among these, compound 1j displayed the most potent activity against MDA-MB-231 cells, with an IC₅₀ value of 0.15 μmol/L under hypoxia, which was 8.3-fold more potent than 1c. Moreover, the HSI of 1j was substantially higher than that of 1c for MDA-MB-231 (6.73 vs 1.63). Notably, 1j (IC₅₀ = 1.01 μmol/L under normoxia, IC₅₀ = 0.15 μmol/L under hypoxia) was more potent than natural product boholamide A (IC₅₀ = 2.78 μmol/L under normoxia, IC₅₀ = 0.66 μmol/L under hypoxia) against MDA-MB-231 cells.

Table 2.

Anti-proliferation activity of boholamide analogues 1h–1l against cancer cells.^a

Compd.	Structure	MDA-MB-231			U118			A549
		IC50 (μmol/L)		HSIb	IC50 (μmol/L)		HSI	IC50 (μmol/L)		HSI
		Normoxia	Hypoxia		Normoxia	Hypoxia		Normoxia	Hypoxia
1c		2.02 ± 0.41	1.24 ± 0.24	1.63	3.42 ± 1.49	1.55 ± 0.39	2.21	4.09 ± 0.88	1.30 ± 0.55	3.14
1h		1.49 ± 0.38	0.88 ± 0.08	1.69	3.05 ± 1.42	2.88 ± 1.02	1.06	3.39 ± 0.95	1.40 ± 0.13	2.42
1i		7.51 ± 0.61	1.61 ± 0.36	4.66	15.24 ± 10.07	3.98 ± 1.79	3.83	＞50	4.71 ± 2.69	>10.61
1j		1.01 ± 0.23	0.15 ± 0.04	6.73	1.93 ± 0.70	1.00 ± 0.39	1.93	2.09 ± 0.66	0.97 ± 0.23	2.15
1k		1.50 ± 0.35	0.56 ± 0.03	2.68	2.67 ± 0.84	1.67 ± 0.68	1.60	2.63 ± 0.95	1.01 ± 0.15	2.60
1l		10.34 ± 1.38	2.67 ± 0.46	3.87	28.79 ± 8.66	12.08 ± 3.80	2.38	31.79 ± 6.53	10.88 ± 3.33	2.83

^aAll values were the means of three independent experiments.

^bHSI was calculated from: IC₅₀ (normoxia)/IC₅₀ (hypoxia).

2.3. The antiproliferative activity of 1j against MDA-MB-231 cells

Compound 1j showed the most potent inhibitory activity against TNBC cells and excellent hypoxia selectivity. Accordingly, compound 1j was selected for further investigation. To evaluate the selectivity of 1j to cancer cells, an MTT assay was performed to compare its effects on breast cancer cells versus normal cells. The results indicated that 1j significantly inhibited breast cancer cell proliferation in a dose-dependent manner, with greater potency under hypoxia than under normoxia (Fig. 2A and B). Moreover, compound 1j exhibited selectivity for cancer cells with little inhibitory potency on normal 3T3 cells under normoxia (Fig. 2C), which indicated that 1j would show less toxic side effects. Furthermore, to assess the long-term cytotoxicity of 1j, a colony formation assay was conducted. The results showed the number of colonies was clearly decreased after the treatment with 1j in a dose-dependent manner under both normoxia and hypoxia (Fig. 2D–G).

Figure 2.

The antiproliferative activity of 1j against MDA-MB-231 cells. (A, B) The antiproliferative activity against MDA-MB-231 cells of 1j under normoxia and hypoxia. (C) The antiproliferative activity against normal 3T3 cells under normoxia. (D, F) The representative picture and colony formation rate statistical results after treatment of compound 1j with different concentrations under normoxia. (E, G) The representative picture and colony formation rate statistical results after treatment of compound 1j with different concentrations under hypoxia. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005, ∗∗∗∗P < 0.001. Data presented as means ± SD (n = 3).

2.4. Compound 1j induced apoptosis of TNBC cells through mitochondrial pathway

To identify the mechanism of 1j in inhibiting proliferation of TNBC cells, cell apoptosis was analyzed. The results demonstrated that 1j induced apoptosis of TNBC cells in a dose-dependent manner. The percentage of apoptosis after the treatment of 1j were 4.13 ± 1.23, 5.68 ± 1.61, 6.80 ± 0.64, 16.13 ± 0.82, 26.73 ± 2.84 and 86.68 ± 1.07 in MDA-MB-231 cells under hypoxia at a dose of 0, 0.1, 0.2, 0.5, 1 and 2 μmol/L, respectively (Fig. 3A and B). To further explain the mechanism of 1j in inducing apoptosis of TNBC cells, the proteins related to mitochondrial pathway were detected. The results indicated that the apoptosis related protein Bax was significantly increased. Meanwhile, anti-apoptosis related proteins including Bcl-2, Mcl-1 were clearly decreased (Fig. 3C–F).

Figure 3.

Compound 1j induced apoptosis of TNBC cells through mitochondrial pathway. (A, B) The representative of apoptosis picture and statistical results after treatment of compound 1j with different concentrations under hypoxia. Data presented as means ± SD (n = 4). (C, D) The level of apoptosis related proteins after treatment of compound 1j at different concentrations under normoxia. (E, F) The level of apoptosis related proteins after treatment of compound 1j at different concentrations under hypoxia. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005, ∗∗∗∗P < 0.001. Data presented as means ± SD (n = 3).

2.5. Compound 1j inhibited migration of TNBC cells

To detect the effect of 1j on migration of TNBC cells, a Transwell assay was performed. The results indicated that the migration cancer cells were greatly reduced after treatment with 1j at different concentrations (Fig. 4A and B). Moreover, we detected the protein level related to migration of cancer cells including E-cadherin, Vimentin and MMP9. The results showed that Vimentin and MMP9 which promoted cancer metastasis were apparently decreased. E-cadherin which suppressed cancer metastasis was significantly increased under normoxia and hypoxia (Fig. 4C–J).

Figure 4.

Compound 1j inhibited migration of TNBC cells. (A, B) The representative images of migration cells and statistical results after treatment of compound 1j at different concentrations under hypoxia. Data presented as means ± SD (n = 7). (C–F) The level of migration related proteins after treatment of compound 1j at different concentrations under normoxia. (G–J) The level of migration related proteins after treatment of compound 1j at different concentrations under hypoxia. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005, ∗∗∗∗P < 0.001. Data presented as means ± SD (n = 3).

2.6. Boholamide A analogue directly targeted eEF1A1

To investigate the mechanism of boholamide A analogue in MDA-MB-231 cells, the probe 15 was designed and synthesized (Fig. 5A). Firstly, compound 1m with an alkynyl group was synthesized and the anti-TNBC activity was detected. The result suggested the inhibitory effect and hypoxia selectivity of 1m was maintained (Fig. 5B). Probe 15 containing a biotin moiety was then synthesized based on 1m. We performed pull-down assay with probe 15 in cell lysates to precipitate the target protein of boholamide A analogue. After silver staining, a specific band with the molecular mass of about 50 kDa appeared (Fig. 5C). This band was excised, digested with trypsin and detected with LC‒MS/MS. The LC‒MS/MS analysis suggested a total of 16 unique peptides of eEF1A1 were detected, accounting for 43.1% of the total amino acids in the eEF1A1 protein (Fig. 5D and E). After fully considering molecular weight, number of unique peptides, peptide coverage, and excluding non-specific protein, we hypothesized that the direct target protein of 1j might be eukaryotic translation elongation factor (eEF1A1).

To verify the binding of 1j to eEF1A1 protein and to avoid non-specific binding, we performed Western blot assay and selected biotin fragment as a negative control. The results demonstrated that probe 15 specifically bound to eEF1A1 protein rather than biotin fragment (Fig. 5F). Given that eEF1A2 and eEF1A1 share 98% amino acid similarity³⁷, we further assessed the binding specificity of 1j toward eEF1A1 relative to eEF1A2 by conducting a Western blot assay with an eEF1A2-specific antibody. The results indicated that probe 15 exhibited significantly stronger specific binding to eEF1A1 than to eEF1A2 in the same condition (Fig. 5G). Moreover, competitive binding assays confirmed that 1j competed specifically for binding to eEF1A1, indicating that eEF1A1 was a specific target of 1j (Fig. 5H).

2.7. Boholamide A analogue bound to Cys31 of eEF1A1

We then performed a pull-down assay with probe 15 at different concentrations in cell lysates, and the binding affinity of probe 15 with eEF1A1 was verified with Western blot. The binding of probe 15 with eEF1A1 exhibited a dose-dependent manner with the K_i value of 0.667 μmol/L (Fig. 6A). Furthermore, the binding of eEF1A1 with probe 15 indicated a time-dependent saturation curve with the K_obs value of 0.019/min, which was consistent with an irreversible binding mechanism (Fig. 6B). Cellular thermal shift assay (CETSA) suggested that 1j increased the thermal stability of eEF1A1 protein, indicating the binding between 1j and eEF1A1 protein (Fig. 6C). The target of 1j was consistent with the target of BE-43547A₂, an APD-containing cyclolipodepsipeptide¹⁸. Accordingly, we proposed that eEF1A1 might be the stem of hypoxia-selective anti-cancer activity of APD-containing cyclolipodepsipeptides.

Figure 6.

Boholamide A analogue bound to Cys31 of eEF1A1. (A) The specific binding K_i analysis of probe 15 and eEF1A1 using cell lysates and analyzed by Western blot assay. (B) The K_obs determination between probe 15 (10 μmol/L) and eEF1A1 protein using cell lysate and analyzed by Western blot assay. (C) The CESTA analysis using cell lysis after treated with 1j. (D) The covalent docking between 1j and Cys31 of eEF1A1 protein. (E) LC–MS/MS analysis of recombinant eEF1A1 peptide after incubated with 1j. (F) Western blot analysis of the binding affinity after incubating probe 15 with wild-type or mutant eEF1A1 protein respectively.

Based on preliminary results and relevant study references on BE-43547A₂, we hypothesized that 1j covalently reacted with the thiol group of a cysteine residue in eEF1A1¹⁸. To identify the specific amino acid residue directly bound to 1j, we co-incubated the water-soluble derivative of 1j with recombinant eEF1A1 protein and subsequently performed LC‒MS/MS analysis. The results showed that the peptide CGGIDK (m/z = 536.29) was 479.299 Da larger after incubation, with the mass difference exactly matching the molecular weight of 1j. Further analysis of this peptide showed the 479.299 Da mass shift occurred from fragment ion b1 to b5, indicating that the Cys31 residue was the site of covalent modification by 1j (Fig. 6E). The covalent docking assay between 1j and eEF1A1 protein suggested 1j could covalently bind Cys31 of eEF1A1 protein as a Michael acceptor and Ser83 promoted the covalent bond formation by establishing hydrogen bonds with 1j (docking score: −5.138) (Fig. 6D). To further validate the binding of 1j to Cys31 of eEF1A1 protein, we mutated the Cys31 of eEF1A1 to serine and purified the mutant recombinant eEF1A1 protein. Then the Western blot assay was performed after incubating probe 15 with wild-type or mutant eEF1A1 protein respectively. The results showed the binding affinity of probe 15 was significantly reduced after Cys31 of eEF1A1 mutation which indicated that 1j covalently bound to the eEF1A1 protein via Cys31 (Fig. 6F).

eEF1A1 was recognized as a translation elongation factor which catalyzed the GTP-dependent binding of aminoacyl-tRNA (aa-tRNA) to the ribosomeal A-site during the elongation phase of protein synthesis³⁸. In addition to this canonical function, eEF1A1 exhibited noncanonical cellular functions to participate in diverse biological processes beyond translation39, 40. Until now, eEF1A1 has emerged as a key target for multiple therapeutic agents including compounds with non-APD structure. As reported, the natural product Neferine inhibited macrophage glycolytic reprogramming by directly targeting eEF1A1 and disrupting the eEF1A1/ARID3A/PKC-δ complex⁴¹. N-Cinnamoylpyrrole-derived alkaloids targeted eEF1A1 to achieve the anti-neuroinflammatory and antioxidative stress function⁴². Plitidepsin targeted the host protein eEF1A to inhibit SARS-CoV-2 replication and eEF1A could be a druggable target for antiviral research⁴³. Besides that, the number of anti-cancer agents targeting eEF1A were growing, indicating that modulating this crucial translation elongation factor could exhibit therapeutic potential in cancer treatment⁴⁴.

2.8. Boholamide A analogue disrupted the interaction between eEF1A1 and FoxO1 in hypoxia

As reported, eEF1A1 was upregulated under hypoxic conditions and exhibited strong interaction with FoxO1, which is similarly induced by hypoxia. Furthermore, eEF1A1 inhibits the nuclear transport of FoxO1 by directly binding, thereby mediating the JAK/STAT3 signaling pathway⁴⁵. To investigate whether 1j disrupted the interaction between eEF1A1 and FoxO1 protein, we performed co-IP assay. First, we overexpressed Flag-tagged eEF1A1 in MDA-MB-231 cells and performed co-immunoprecipitation using an anti-Flag antibody. Treatment of 1j markedly reduced the amount of FoxO1 co-immunoprecipitated with eEF1A1, indicating that 1j disrupted the eEF1A1–FoxO1 interaction (Fig. 7A). Conversely, when Flag-tagged FoxO1 was overexpressed in MDA-MB-231 cells and subjected to anti-Flag co-immunoprecipitation, treatment of 1j similarly led to a significant decrease in the amount of eEF1A1 pulled down with FoxO1 (Fig. 7B). These co-immunoprecipitation experiments collectively demonstrated that compound 1j inhibited the association between eEF1A1 and FoxO1.

Figure 7.

Boholamide A analogue disrupted the interaction between eEF1A1 and FoxO1 under hypoxia. (A) co-IP analysis of the interaction between eEF1A1 and FoxO1 after overexpressing Flag-tagged eEF1A1. (B) co-IP analysis of the interaction between eEF1A1 and FoxO1 after overexpressing Flag-tagged FoxO1. (C) The protein level of FoxO1 in cytoplasmic. (D) The protein level of FoxO1 in nuclear. (E–H) The protein level of JAK1, JAK2, STAT3 and pSTAT3 after treatment of 1j under hypoxia for 48 h.

To verify whether compound 1j promoted FoxO1 nuclear translocation by disrupting the eEF1A1/FoxO1 interaction and consequently suppressed the JAK/STAT3 pathway, we extracted nuclear proteins and cytoplasmic proteins separately from MDA-MB-231 cells with nuclear and cytoplasmic protein extraction kits to analyze the nuclear translocation of FoxO1. And the results suggested compound 1j increased the level of FoxO1 in the nucleus and decreased the level of FoxO1 in the cytoplasm, indicating that compound 1j significantly increased the nuclear translocation of FoxO1 (Fig. 7C and D).

We further investigated the effects of compound 1j on the downstream JAK/STAT3 signal pathway. The results showed that 1j significantly suppressed levels of JAK1, JAK2, STAT3 and phosphorylation of STAT3 in a dose-dependent manner (Fig. 7E–H). Collectively, these findings suggested 1j inhibited the association between eEF1A1 and FoxO1, promoted FoxO1 nuclear translocation and consequently suppressed the JAK/STAT3 pathway.

2.9. The toxicity evaluation of boholamide A analogue

Since 1j showed significant anti-TNBC activity in vitro, we decided to determine the anti-TNBC effect of 1j in vivo. However, compound 1j exhibited low water solubility, and then compound 1j was transformed to its prodrug 16 following our previously reported strategy (Fig. S1A)35, 36. The results showed that the water solubility was clearly improved and the activity of compound 16 was maintained (Fig. S1B). The preliminary toxicity of compound 16 was evaluated in Balb/c mice. The results suggested no significant alterations were observed in body weight and organ coefficients including liver, spleen, lung, kidney, brain and heart compared with vehicle group after intraperitoneal administration of compound 16 at doses of 20, 40 and 80 mg/kg respectively (Fig. S1C and S1D). Furthermore, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine (CR) which were key indicators of hepatic and kidney function were not clearly increased after intraperitoneal administration of compound 16 at all tested doses, indicating no hepatotoxicity and nephrotoxicity (Fig. S1E). We performed histopathological analyses of major organs (including liver, kidney, heart, spleen, brain and lung) using hematoxylin and eosin (H&E) staining (Supporting Information Fig. S2). The results indicated no significant pathological abnormalities in any examined tissues at the tested doses, further supporting the safety profile of the compound 1j. Collectively, these findings suggested that compound 16 exhibited no obvious toxicity in vivo at the tested doses.

2.10. Compound 16 inhibited tumor growth in xenografts

These results prompted us to evaluate the anti-TNBC efficacy of compound 16 in vivo. Firstly, mouse breast cancer cells 4T1 were cultured and transplanted into the mammary fat pad of Bal-b/c mice to establish breast cancer model. After the model was established, compound 16 was intraperitoneally administrated at different doses. The results indicated that compound 16 significantly inhibited the tumor volume and tumor weights in a dose-dependent manner (Fig. 8A, B, D). Hepatosplenomegaly is commonly associated with breast cancer metastasis. Notably, compound 16 significantly reduced the weight of both spleen and livers, which may suggest compound 16 could inhibit breast cancer metastasis in vivo (Fig. 8E–G). Moreover, although the mice exhibited a reduction in body weight after repeated administration of compound 16, the toxicity remained manageable in vivo (Fig. 8C).

Figure 8.

Compound 16 inhibited TNBC tumor growth in xenografts. (A–D) The tumor picture, tumor volume, body weight and tumor weight of mice after administration of prodrug 16. (E–G) The spleen picture, spleen weight and liver weight of mice after administration of prodrug 16. (H) The level of JAK2, STAT3 and pSTAT3 in tumor tissues. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005, ∗∗∗∗P < 0.001. Data presented as means ± SD (n = 6 in vehicle and 20 mg/kg groups, n = 5 in 40 mg/kg group).

Furthermore, immunohistochemical analysis revealed that treatment with compound 16 significantly decreased the protein levels of JAK2, STAT3 and pSTAT3 in tumor tissues (Fig. 8H). All these results suggested that compound 16 inhibited tumor growth and metastasis in vivo through inhibiting JAK/STAT3 signal pathway.

3. Conclusions

In conclusion, boholamide A was identified as a lead compound for the discovery of potential hypoxia-selective anti-TNBC agents. A library of boholamide A analogues was synthesized through a highly efficient combinatorial strategy. Some preliminary structure‒activity relationships were concluded: (1) the C2 methoxyl group is important for the anti-TNBC activity; (2) the C6 methyl group is not essential, and relocating it to the terminus of the lipophilic chain retains potency; (3) the C11 position accommodates diverse modification. Among these, the most promising compound 1j exhibited an IC₅₀ value of 0.15 μmol/L, which was more potent than the parent natural product boholamide A for TNBC cells. Compound 1j could selectively killed TNBC cells without apparent inhibitory effect on normal cells. Moreover, 1j induced apoptosis and inhibited migration of TNBC cells. Meanwhile, eEF1A1 which was identified as the target of 1j might be the stem of anticancer activity and hypoxia selectivity of boholamide A analogues. Furthermore, the binding site analysis suggested 1j covalently bound Cys31 of eEF1A1 protein. The mechanism research showed 1j significantly inhibited the association between eEF1A1 and FoxO1, promoted FoxO1 nuclear translocation and consequently suppressed the JAK/STAT3 pathway. These results suggested that boholamide A analogues might be a good source for discovery of hypoxia-selective anti-TNBC agents. Compound 16, a prodrug of 1j, exhibited no observable toxicity in vivo at the 20‒80 mg/kg doses. The in vivo efficacy research indicated compound 16 significantly inhibited the tumor volume, tumor weight and metastasis of TNBC with a dose-dependent manner in xenografts. These findings indicated that compound 16 may deserve further investigation as a promising lead compound for ultimate discovery of anti-TNBC drug.

4. Experimental

4.1. Chemistry

General. Unless otherwise mentioned, all reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions. The used solvents were purified and dried according to common procedures. Yields refer to chromatographically and spectroscopically (¹H NMR) homogeneous materials, unless otherwise stated. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. ¹H NMR and ¹³C NMR were recorded on Bruker AV 400 and calibrated using internal references and solvent signals CDCl₃ (δ_H = 7.26 ppm, δ_C = 77.16 ppm), DMSO-d₆ (δ_H = 2.50 ppm, δ_C = 39.52 ppm). ¹H NMR data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants and integration. High-resolution mass spectra (HRMS) were detected by FTICR-MS (Ion spec 7.0T) spectrometer.

(2S,3S,4R)-1-((3aR,6R)-8,8-Dimethyl-2,2-dioxidotetrahydro-3H-3a,6-methanobenzo[c]isothiazol-1(4H)-yl)-3-hydroxy-2-methoxy-4-methylnonan-1-one(9a). To a solution of compound 12a (10 g, 34.80 mmol, 2.0 equiv) in CH₂Cl₂ (170 mL) was added triethylamine (9.86 g, 97.43 mmol, 2.8 equiv) and TBSOTf (23.90 g, 90.47 mmol, 2.6 equiv). The reaction mixture was stirred at room temperature for 12 h. All of the volatile materials were evaporated and extracted with hexane (3 × 40 mL). The combined organic phases were concentrated under reduced pressure to afford the desired compound 11a as a yellow solid, which was dissolved in CH₂Cl₂ (50 mL) at–78 °C under argon atmosphere was added aldehyde 10a (2.23 g, 1.0 equiv).

After stirred for 30 min, BF₃•Et₂O (2.72 g, 19.14 mmol, 1.1 equiv) was added to the reaction mixture. The reaction was stirred at −78 °C for 5 h before saturated aqueous Na₂CO₃ (50 mL) was added. The aqueous phase was extracted with ethyl acetate (2 × 50 mL). The combined organic phase was washed with brine (2 × 10 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 4:1) to obtain 9a (3.83 g, 53% for 2 steps) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 4.42 (d, J = 8.9 Hz, 1H), 3.99 (dd, J = 7.7, 5.0 Hz, 1H), 3.71 (ddd, J = 11.4, 8.8, 2.2 Hz, 1H), 3.60–3.46 (m, 2H), 3.45 (s, 1H), 3.35 (s, 3H), 2.20 (dt, J = 11.7, 4.0 Hz, 1H), 2.12 (dd, J = 13.9, 7.8 Hz, 1H), 1.98 (d, J = 11.6 Hz, 1H), 1.89 (dd, J = 19.6, 6.8 Hz, 3H), 1.40 (dt, J = 18.0, 9.2 Hz, 3H), 1.27 (td, J = 11.7, 10.5, 5.1 Hz, 7H), 1.16 (s, 3H), 0.96 (s, 3H), 0.90–0.81 (m, 6H). ¹³C NMR (100 MHz, CDCl₃₎ δ 172.2, 81.0, 76.2, 65.5, 58.2, 53.4, 48.8, 47.9, 44.6, 38.2, 34.3, 34.0, 33.0, 32.2, 27.1, 26.6, 22.8, 20.7, 20.1, 14.2, 12.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₁H₃₇NO₅SNa⁺ 438.2285, found 438.2288.

(2S,3S)-1-((3aR,6R)-8,8-Dimethyl-2,2-dioxidotetrahydro-3H-3a,6-methanobenzo[c]isothiazol-1(4H)-yl)-3-hydroxy-2-methoxynonan-1-one (9b). The titled compound 9b was obtained following the general procedure described for 9a. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 15:1) to obtain compound 9b (3.92 g, 57% for 2 steps) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 4.35 (d, J = 6.4 Hz, 1H), 3.97 (t, J = 6.3 Hz, 1H), 3.77 (tdd, J = 9.2, 6.2, 2.5 Hz, 1H), 3.49 (d, J = 12.3 Hz, 2H), 3.37 (s, 3H), 2.18–1.99 (m, 3H), 1.97–1.82 (m, 3H), 1.66-1.60 (m, 1H), 1.58–1.47 (m, 1H), 1.45–1.33 (m, 3H), 1.32–1.19 (m, 7H), 1.12 (s, 3H), 0.95 (s, 3H), 0.89–0.77 (m, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 171.1, 83.7, 73.5, 65.4, 58.3, 53.3, 48.7, 47.9, 44.6, 38.2, 33.9, 33.0, 31.8, 29.2, 26.5, 25.4, 22.7, 20.6, 20.0, 14.2. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₀H₃₆NO₅S⁺ 402.2309, found 402.2314.

(2S,3S,4R)-1-((3aR,6R)-8,8-Dimethyl-2,2-dioxidotetrahydro-3H-3a,6-methanobenzo[c]isothiazol-1(4H)-yl)-3-hydroxy-2-methoxy-4-methyldecan-1-one (9c). The titled compound 9c was obtained following the general procedure described for 9a. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 15:1) to obtain compound 9c (4.13 g, 62% for 2 steps) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 4.40 (d, J = 8.9 Hz, 1H), 3.98 (dd, J = 7.7, 5.0 Hz, 1H), 3.70 (ddd, J = 11.3, 8.9, 2.2 Hz, 1H), 3.57–3.39 (m, 2H), 3.34 (s, 3H), 2.23–2.14 (m, 1H), 2.10 (dd, J = 13.9, 7.8 Hz, 1H), 1.98 (d, J = 11.7 Hz, 1H), 1.95–1.78 (m, 4H), 1.39 (dt, J = 18.2, 8.7 Hz, 3H), 1.27–1.20 (m, 9H), 1.14 (s, 3H), 0.94 (s, 3H), 0.90–0.80 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 172.2, 80.9, 76.2, 65.5, 58.1, 53.4, 48.7, 47.9, 44.6, 38.1, 34.2, 34.0, 33.0, 31.9, 29.6, 27.3, 26.5, 22.7, 20.6, 20.0, 14.2, 12.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₂H₃₉NO₅Na⁺ 452.2442, found 452.2438.

(2S,3S)-3-((tert-Butyldimethylsilyl)oxy)-1-((3aR,6R)-8,8-dimethyl-2,2-dioxidotetrahydro-3H-3a,6-methanobenzo[c]iso-thiazol-1(4H)-yl)-2-methoxyundecan-1-one (9d¹). The titled compound 9d was obtained following the general procedure described for 9a. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 15:1) to obtain crude compound 9d as a white solid.

Since compound 9d and raw material 12d had similar polarity, they could not be separated by conventional methods, so the obtained 9d was a mixture, but it did not affect the purification of the next reaction. We protected the hydroxyl group of 9d with TBS to separate the two and collect its data.

To a solution of crude compound 9d (426 mg, 1.13 mmol, 1.0 equiv) in CH₂Cl₂ (10 mL) was added 2,6-lutidine (566 mg, 3.39 mmol, 3.0 equiv) and TBSOTf (597.0 mg, 2.26 mmol, 2.0 equiv). The reaction mixture was stirred at room temperature for 3 h. The aqueous phase was extracted with ethyl acetate (2 × 10 mL). The combined organic phase was washed with brine (2 × 5 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 2:1) to obtain 9d¹ (237 mg) as a white solid. Supporting Information is visible for reaction equations. ¹H NMR (400 MHz, CDCl₃) δ 4.47 (d, J = 3.0 Hz, 1H), 4.03 (dt, J = 9.5, 2.8 Hz, 1H), 3.96 (dd, J = 7.7, 4.4 Hz, 1H), 3.53–3.39 (m, 2H), 3.39 (s, 3H), 2.08 (dd, J = 13.5, 7.7 Hz, 1H), 1.97–1.82 (m, 4H), 1.62 (dtd, J = 13.8, 9.3, 5.1 Hz, 1H), 1.54–1.38 (m, 2H), 1.36 (d, J = 7.4 Hz, 1H), 1.31–1.14 (m, 12H), 1.12 (s, 3H), 0.96 (s, 3H), 0.88 (s, 12H), 0.14 (s, 3H), 0.07 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.6, 85.3, 73.4, 65.2, 59.0, 53.2, 48.6, 47.9, 44.7, 38.3, 32.9, 32.0, 31.3, 29.6, 29.5, 29.3, 26.6, 26.1 (3C), 25.8, 22.8, 20.8, 20.0, 18.3, 14.2, −4.2, −4.9. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₈H₅₄NO₅SSi⁺ 544.3487, found 544.3491.

(2S,3S)-1-((3aR,6R)-8,8-Dimethyl-2,2-dioxidotetrahydro-3H-3a,6-methanobenzo[c]isothiazol-1(4H)-yl)-3-hydroxy-2-methylnonan-1-one (9e). The titled compound 9e was obtained following the general procedure described for 9a. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 15:1) to obtain compound 9e (4.22 g, 65% for 2 steps) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 3.80 (dd, J = 7.7, 5.0 Hz, 1H), 3.58–3.51 (m, 1H), 3.49–3.33 (m, 2H), 3.07 (p, J = 6.5 Hz, 1H), 2.49 (s, 1H), 2.01 (qd, J = 13.7, 5.6 Hz, 2H), 1.90–1.74 (m, 3H), 1.43 (ddd, J = 21.3, 8.6, 5.4 Hz, 2H), 1.28 (dt, J = 16.4, 8.4 Hz, 5H), 1.20–1.14 (m, 5H), 1.11 (d, J = 6.9 Hz, 3H), 1.08 (s, 3H), 0.88 (s, 3H), 0.77 (t, J = 6.5 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 175.3, 75.2, 65.2, 53.0, 48.2, 47.6, 45.2, 44.6, 38.4, 35.5, 32.7, 31.6, 29.1, 26.3, 25.3, 22.5, 20.6, 19.8, 14.0 (2C). HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₀H₃₆NO₄S⁺ 386.2360, found 386.2358.

(2S,3S,4R)-3-((tert-Butyldimethylsilyl)oxy)-1-((3aR,6R)-8,8-dimethyl-2,2-dioxidotetrahydro-3H-3a,6-methanobenzo[c]iso-thiazol-1(4H)-yl)-2,4-dimethyldecan-1-one (9f¹). The titled compound 9f¹ was obtained following the general procedure described for 9d¹. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 15:1) to obtain compound 9f¹ as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 4.04 (dd, J = 6.2, 1.8 Hz, 1H), 3.88 (t, J = 6.3 Hz, 1H), 3.51–3.40 (m, 2H), 3.35 (p, J = 6.7 Hz, 1H), 2.04 (dd, J = 6.4, 1.9 Hz, 2H), 1.94–1.80 (m, 3H), 1.54–1.44 (m, 1H), 1.44–1.33 (m, 2H), 1.29–1.11 (m, 16H), 0.96 (s, 3H), 0.92–0.83 (m, 15H), 0.07 (d, J = 9.9 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 174.3, 75.5, 65.7, 53.3, 48.1, 47.8, 46.8, 44.9, 38.8, 36.6, 35.6, 33.1, 32.0, 29.7, 27.8, 26.6, 26.1 (3C), 22.8, 21.1, 20.0, 18.4, 14.7, 14.2, 12.2, −3.9, −4.8. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₈H₅₄NO₄SSi⁺ 528.3538, found 528.3539.

(2S,3S,4R)-3-((tert-Butyldimethylsilyl)oxy)-1-((3aR,6R)-8,8-dimethyl-2,2-dioxidotetrahydro-3H-3a,6-methanobenzo[c]isothi-azol-1(4H)-yl)-2-ethyl-4-methyldecan-1-one (9g¹). The titled compound 9g¹ was obtained following the general procedure described for 9d. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 15:1) to obtain compound 9g¹ as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 4.09 (dd, J = 5.4, 1.6 Hz, 1H), 3.93 (dd, J = 7.7, 4.8 Hz, 1H), 3.53–3.39 (m, 2H), 3.22 (ddd, J = 10.8, 5.3, 2.5 Hz, 1H), 2.11–2.01 (m, 1H), 1.99–1.84 (m, 5H), 1.79 (ddd, J = 13.4, 7.7, 2.5 Hz, 1H), 1.46–1.34 (m, 3H), 1.29–1.11 (m, 13H), 0.96 (s, 3H), 0.93–0.80 (m, 18H), 0.13 (s, 3H), 0.07 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 173.3, 74.3, 65.5, 56.3, 53.4, 48.1, 47.9, 44.8, 38.7, 36.6, 36.1, 33.0, 32.0, 29.7, 27.8, 26.7, 26.1 (3C), 22.8, 21.0, 20.0, 18.6, 18.4, 14.6, 14.2, 13.5, −4.0, −5.3. HRMS(ESI) m/z: [M + H]⁺ calcd for C₂₉H₅₆NO₄SSi⁺ 542.3694, found 542.3699.

Benzyl (2S,3S,4R)-3-hydroxy-2-methoxy-4-methylnonanoate (8a). To a solution of 9a (3.00 g, 7.22 mmol, 1.0 equiv) in THF/MeOH/H₂O (21 mL/7 mL/7 mL) was added LiOH•H₂O (363 mg, 8.66 mmol, 1.2 equiv) at room temperature. After being stirred for 4 h at this temperature, the reaction mixture acidified to pH = 3.0 with aqueous 10% NaHSO₄ and extracted with ethyl acetate (2 × 300 mL). The solvent was evaporated, and the resulting mixture was directly used for the next step without further purification.

The above colorless oil was dissolved in DMF (35 mL) was added K₂CO₃ (2.00g, 14.44 mmol, 2.0 equiv) and BnBr (1.84 g, 10.83 mmol, 1.5 equiv) at room temperature. The mixture was stirred at room temperature for 10 h, and then H₂O (10 mL) was added, and the resultant mixture was extracted with ethyl acetate (3 × 50 mL). The organic phase was washed by brine (4 × 100 mL). Dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified with column chromatography on silica gel (petroleum ether: ethyl acetate = 6:1) to afford compound 8a (1.45 g, 65% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.40–7.30 (m, 5H), 5.23 (d, J = 3.6 Hz, 2H), 3.86 (d, J = 6.4 Hz, 1H), 3.80–3.74 (m, 1H), 3.40 (s, 3H), 2.21 (d, J = 5.7 Hz, 1H), 1.69 (tt, J = 10.2, 5.9 Hz, 1H), 1.39 (ddt, J = 9.4, 6.0, 3.4 Hz, 1H), 1.33–1.15 (m, 7H), 0.92–0.84 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 171.5, 135.5, 128.7 (2C), 128.5, 128.5 (2C), 82.3, 74.8, 66.9, 58.7, 34.35, 33.6, 32.1, 26.8, 22.7, 14.2, 13.6. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₈H₂₈O₄Na⁺ 331.1880, found 331.1878.

Benzyl (2S,3S)-3-hydroxy-2-methoxynonanoate (8b). The titled compound 8b was obtained following the general procedure described for 8a. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 35:1) to obtain compound 8b (1.24 g, 62% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.27 (m, 5H), 5.29–5.12 (m, 2H), 3.85 (dt, J = 13.5, 4.2 Hz, 2H), 3.42 (s, 3H), 2.39 (s, 1H), 1.52–1.39 (m, 3H), 1.29–1.20 (m, 7H), 0.96–0.81 (m, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.5, 135.5, 128.6 (2C), 128.5, 128.4 (2C), 84.1, 72.3, 66.7, 58.8, 32.3, 31.7, 29.2, 25.6, 22.6, 14.1. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₇H₂₆O₄Na⁺ 317.1724, found 317.1720.

Benzyl (2S,3S,4R)-3-hydroxy-2-methoxy-4-methyldecanoate (8c). The titled compound 8c was obtained following the general procedure described for 8a. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 35:1) to obtain compound 8c (1.73 g, 63% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.41–7.28 (m, 5H), 5.52–4.90 (m, 2H), 3.86 (d, J = 6.4 Hz, 1H), 3.81–3.73 (m, 1H), 3.40 (s, 3H), 2.20 (d, J = 5.8 Hz, 1H), 1.69 (dq, J = 12.5, 6.6, 5.4 Hz, 1H), 1.39 (qd, J = 9.1, 8.4, 6.1 Hz, 1H), 1.26–1.17 (m, 9H), 0.93–0.83 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 171.6, 135.5, 128.7 (2C), 128.6, 128.5 (2C), 82.4, 74.9, 66.9, 58.7, 34.4, 33.7, 32.0, 29.6, 27.1, 22.7, 14.2, 13.6. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₉H₃₀O₄Na⁺ 345.2037, found 345.2037.

Benzyl (2S,3S)-3-hydroxy-2-methoxyundecanoate (8d). The titled compound 8d was obtained following the general procedure described for 8a. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 35:1) to obtain compound 8d (0.983 g, 49% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.34 (tdd, J = 10.2, 5.6, 4.1 Hz, 5H), 5.39–5.01 (m, 2H), 3.86 (dt, J = 14.7, 4.4 Hz, 2H), 3.43 (s, 3H), 2.41 (d, J = 6.1 Hz, 1H), 1.43 (ddt, J = 19.4, 10.0, 6.2 Hz, 3H), 1.33–1.15 (m, 11H), 0.87 (t, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.5, 135.5, 128.7 (2C), 128.5, 128.5 (2C), 84.0, 72.3, 66.7, 58.8, 32.3, 31.9, 29.5, 29.5, 29.3, 25.7, 22.7, 14.2. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₉H₃₀O₄Na⁺ 345.2037, found 345.2036.

Benzyl (2S,3S)-3-hydroxy-2-methylnonanoate (8e). The titled compound 8e was obtained following the general procedure described for 8a. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 35:1) to obtain compound 8e (1.27 g, 62% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.43–7.27 (m, 5H), 5.15 (d, J = 1.7 Hz, 2H), 3.71–3.64 (m, 1H), 2.68–2.45 (m, 2H), 1.53–1.24 (m, 10H), 1.22 (d, J = 7.2 Hz, 3H), 0.88 (t, J = 6.6 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 176.0, 136.0, 128.7 (2C), 128.4, 128.3 (2C), 73.5, 66.5, 45.4, 34.9, 31.9, 29.4, 25.6, 22.7, 14.4, 14.2. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₇H₂₆O₃Na⁺ 301.1775, found 301.1776.

Benzyl (2S,3S,4R)-3-hydroxy-2,4-dimethyldecanoate (8f). The titled compound 8f was obtained following the general procedure described for 8a. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 35:1) to obtain compound 8f (1.68 g, 67% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.41–7.28 (m, 5H), 5.15 (s, 2H), 3.62 (dd, J = 7.8, 3.8 Hz, 1H), 2.70 (p, J = 7.3 Hz, 1H), 2.49 (s, 1H), 1.57–1.50 (m, 1H), 1.46–1.35 (m, 1H), 1.27 (h, J = 6.4, 5.1 Hz, 9H), 1.18 (d, J = 7.2 Hz, 3H), 0.91–0.85 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 176.4, 135.9, 128.7 (2C), 128.4, 128.2 (2C), 76.1, 66.5, 43.3, 35.2, 34.0, 32.0, 29.6, 27.3, 22.8, 14.6, 14.2, 13.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₉H₃₀O₃Na⁺ 329.2088, found 329.2088.

Benzyl (2S,3S,4R)-2-ethyl-3-hydroxy-4-methyldecanoate (8g). The titled compound 8g was obtained following the general procedure described for 8a. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 35:1) to obtain compound 8g (1.49 g, 71% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.29–7.14 (m, 5H), 5.07 (s, 2H), 3.52 (q, J = 6.3 Hz, 1H), 2.54–2.39 (m, 2H), 1.58 (dtd, J = 15.3, 13.5, 7.3 Hz, 2H), 1.41 (ddd, J = 12.3, 7.3, 5.2 Hz, 1H), 1.36–1.27 (m, 1H), 1.23–1.15 (m, 9H), 0.80 (td, J = 7.7, 7.2, 4.5 Hz, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 175.9, 135.9, 128.6 (2C), 128.3 (2C), 128.3, 75.2, 66.3, 50.3, 36.1, 33.7, 31.9, 29.6, 27.1, 22.9, 22.7, 14.2, 13.6, 11.7. HRMS(ESI) m/z: [M + Na]⁺ calcd for C₂₀H₃₂O₃Na⁺ 343.2244, found 343.2243.

Benzyl (2S,3S,4R)-3-(((tert-butoxycarbonyl)-l-valyl)oxy)-2-methoxy-4-methylnonanoate (6a). To a solution of compound 7 (2.11 g, 9.73 mmol, 3.0 equiv) and 8a (1.00 g, 3.24 mmol, 1.0 equiv) in CH₂Cl₂ (3.2 mL) was added DMAP (39.6 mg, 0.32 mmol, 0.1 equiv) and DIC (1.35 g, 10.69 mmol, 3.3 equiv) under argon atmosphere at 0 °C. The reaction mixture was stirred for 1.5 h, and diluted with CH₂Cl₂ (50 mL). The suspension was filtered through a pad of silica gel and the filter cake was washed with CH₂Cl₂ (3 × 10 mL) and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 40:1 to 30:1) to obtain compound 6a (1.33 g, 81%) as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.36 (d, J = 4.1 Hz, 5H), 5.17 (d, J = 3.5 Hz, 3H), 4.99 (d, J = 9.4 Hz, 1H), 4.19 (dd, J = 9.6, 4.6 Hz, 1H), 3.96 (d, J = 4.9 Hz, 1H), 3.34 (t, J = 2.4 Hz, 3H), 2.13–2.08 (m, 1H), 1.86 (d, J = 9.7 Hz, 1H), 1.47–1.39 (m, 10H), 1.34–1.06 (m, 7H), 0.99–0.88 (m, 6H), 0.84 (dt, J = 6.2, 2.4 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 171.9, 170.0, 155.8, 135.3, 128.7 (2C), 128.6 (3C), 80.6, 79.7, 76.7, 67.1, 58.6, 58.5, 33.6, 33.3, 32.0, 30.9, 28.4 (3C), 26.6, 22.7, 19.4, 17.1, 14.4, 14.2. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₈H₄₅NO₇Na⁺ 530.3089, found 530.3090.

Benzyl (2S,3S)-3-(((tert-butoxycarbonyl)-l-valyl)oxy)-2-methoxynonanoate (6b). The titled compound 6b was obtained following the general procedure described for 6a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 6b (1.1 g, 87%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.30 (dtd, J = 13.7, 7.3, 6.5, 3.3 Hz, 5H), 5.26–5.08 (m, 3H), 5.01 (d, J = 9.2 Hz, 1H), 4.18 (dd, J = 9.2, 4.6 Hz, 1H), 3.93 (d, J = 3.7 Hz, 1H), 3.35 (s, 3H), 2.14–2.05 (m, 1H), 1.66 (tt, J = 10.3, 6.3 Hz, 1H), 1.40 (s, 10H), 1.24–1.13 (m, 8H), 0.90 (d, J = 6.8 Hz, 3H), 0.81 (t, J = 6.7 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 171.9, 169.3, 155.6, 135.3, 128.6 (2C), 128.5, 128.5 (2C), 81.5, 79.6, 74.5, 66.8, 58.7, 58.5, 31.5, 31.0, 29.3, 28.8, 28.3 (3C), 25.2, 22.5, 19.1, 17.2, 14.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₇H₄₃NO₇Na⁺ 516.2932, found 516.2930.

Benzyl (2S,3S,4R)-3-(((tert-butoxycarbonyl)-l-valyl)oxy)-2-methoxy-4-methyldecanoate (6c). The titled compound 6c was obtained following the general procedure described for 6a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 6c (0.942 g, 72%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.43–7.28 (m, 5H), 5.19 (q, J = 4.2, 3.7 Hz, 3H), 5.03 (d, J = 9.3 Hz, 1H), 4.20 (dt, J = 8.3, 3.4 Hz, 1H), 3.99 (t, J = 4.0 Hz, 1H), 3.34 (s, 3H), 2.12 (q, J = 6.4 Hz, 1H), 1.92–1.84 (m, 1H), 1.44 (s, 9H), 1.37–1.08 (m, 10H), 0.98–0.78 (m, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 171.7, 169.8, 155.6, 135.2, 128.6 (2C), 128.5 (3C), 80.5, 79.5, 76.5, 66.9, 58.5, 58.3, 33.5, 33.2, 31.7, 30.7, 29.3, 28.3 (3C), 26.8, 22.6, 19.2, 17.0, 14.3, 14.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₉H₄₇NO₇Na⁺ 544.3245, found 544.3244.

Benzyl (2S,3S)-3-(((tert-butoxycarbonyl)-l-valyl)oxy)-2-methoxyundecanoate (6d). The titled compound 6d was obtained following the general procedure described for 6a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 6d (0.912 g, 76%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.38–7.28 (m, 5H), 5.28–5.10 (m, 3H), 5.00 (d, J = 9.2 Hz, 1H), 4.21 (dd, J = 9.2, 4.5 Hz, 1H), 3.96 (d, J = 3.7 Hz, 1H), 3.38 (s, 3H), 2.17–2.08 (m, 1H), 1.68 (ddt, J = 13.2, 8.9, 5.8 Hz, 1H), 1.43 (s, 10H), 1.35–1.11 (m, 12H), 0.93 (d, J = 6.8 Hz, 3H), 0.90–0.76 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 172.0, 169.4, 155.7, 135.4, 128.7 (2C), 128.6, 128.6 (2C), 81.6, 79.7, 74.6, 67.0, 58.8, 58.6, 31.9, 31.2, 29.4, 29.4, 29.3, 29.3, 28.4 (3C), 25.4, 22.7, 19.2, 17.2, 14.2. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₉H₄₇NO₇Na⁺ 544.3245, found 544.3244.

Benzyl (2S,3S)-3-(((tert-butoxycarbonyl)-l-valyl)oxy)-2-methylnonanoate (6e). The titled compound 6e was obtained following the general procedure described for 6a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 6e (1.08 g, 82%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.34 (d, J = 3.2 Hz, 5H), 5.18 (d, J = 5.8 Hz, 1H), 5.16–5.04 (m, 2H), 5.00 (d, J = 9.2 Hz, 1H), 4.18 (dt, J = 8.8, 3.8 Hz, 1H), 2.84 (t, J = 7.1 Hz, 1H), 2.06 (dt, J = 17.2, 5.7 Hz, 1H), 1.54 (s, 2H), 1.43 (s, 9H), 1.28–1.21 (m, 8H), 1.15 (dd, J = 7.6, 2.8 Hz, 3H), 0.95–0.89 (m, 3H), 0.88–0.78 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 173.2, 171.9, 155.8, 135.1, 128.6 (2C), 128.4, 128.3 (2C), 79.7, 75.7, 66.5, 58.7, 42.9, 31.7, 31.2, 30.8, 29.1, 28.4 (3C), 24.9, 22.6, 19.3, 17.2, 14.1, 12.7. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₇H₄₃NO₆Na⁺ 500.2983, found 500.2982.

Benzyl (2S,3S,4R)-3-(((tert-butoxycarbonyl)-l-valyl)oxy)-2,4-dimethyldecanoate (6f). The titled compound 6f was obtained following the general procedure described for 6a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 6f (1.35 g, 69%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.48–7.28 (m, 5H), 5.19 (dd, J = 8.4, 3.9 Hz, 1H), 5.06 (s, 2H), 4.99 (d, J = 9.4 Hz, 1H), 4.17 (dd, J = 9.4, 4.4 Hz, 1H), 2.86 (dd, J = 8.2, 7.0 Hz, 1H), 2.05 (d, J = 4.4 Hz, 1H), 1.74 (dq, J = 11.6, 5.6, 4.9 Hz, 1H), 1.42 (s, 9H), 1.32–1.19 (m, 9H), 1.14 (d, J = 7.1 Hz, 3H), 1.09 (dd, J = 8.4, 1.5 Hz, 1H), 0.92 (d, J = 6.8 Hz, 3H), 0.88–0.79 (m, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 173.7, 171.8, 155.8, 135.8, 128.6 (2C), 128.3, 128.2 (2C), 79.6, 78.3, 66.5, 58.7, 42.1, 34.3, 33.5, 31.8, 30.8, 29.4, 28.4 (3C), 27.2, 22.7, 19.4, 17.1, 14.2, 14.1, 13.5. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₉H₄₇NO₆Na⁺ 528.3296, found 528.3295.

Benzyl (2S,3S,4R)-3-(((tert-butoxycarbonyl)-l-valyl)oxy)-2-ethyl-4-methyldecanoate (6g). The titled compound 6g was obtained following the general procedure described for 6a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 6g (0.893 g, 68%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.40–7.26 (m, 5H), 5.21 (dd, J = 8.0, 4.1 Hz, 1H), 5.04 (dd, J = 32.5, 6.9 Hz, 3H), 4.17 (dd, J = 9.4, 4.4 Hz, 1H), 2.72 (ddd, J = 9.8, 7.9, 4.8 Hz, 1H), 2.13–1.99 (m, 1H), 1.72 (td, J = 7.5, 4.2 Hz, 1H), 1.56 (dtdd, J = 23.8, 14.0, 7.5, 2.8 Hz, 2H), 1.43 (s, 9H), 1.38–1.20 (m, 9H), 1.10 (ddd, J = 12.5, 10.3, 5.8 Hz, 1H), 1.00–0.89 (m, 3H), 0.85 (ddd, J = 11.1, 5.4, 3.0 Hz, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 173.1, 171.8, 155.8, 135.8, 128.6 (2C), 128.4 (2C), 128.4, 79.7, 77.5, 66.5, 58.8, 49.6, 34.7, 33.6, 31.9, 30.9, 29.5, 28.4 (3C), 27.2, 22.7, 22.4, 19.5, 17.2, 14.2, 13.8, 11.6. HRMS(ESI) m/z: [M + Na]⁺ calcd for C₃₀H₄₉NO₆Na⁺ 542.3453, found 542.3454.

(2S,3S,4R)-2-Methoxy-4-methyl-1-oxo-1-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)amino)nonan-3-yl (tert-butoxycarbonyl)-L-valinate (4a). To a solution of compound 6a (0.8 g, 1.58 mmol, 1.0 equiv) in MeOH (16 mL) was added Pd/C (10%, 50 mg) under Ar. The suspension was degassed under vacuum and purged with H₂ three times. The mixture was stirred under H₂ balloon at 20 °C for 5 h. The resulting mixture was filtered through a short pad of silica gel, washed with MeOH (2 × 30 mL) and concentrated under reduced pressure. The crude product was used directly for the next step without further purification.

To a solution of above acid and 5 (1.53 g, 3.16 mmol, 2.0 equiv) in CH₂Cl₂ (5 mL) was added EDCI (364 mg, 1.90 mmol, 1.2 equiv) and HOBt (235 mg, 1.74 mmol, 1.1 equiv) under argon atmosphere at 20 °C. The reaction mixture was stirred for 12 h, and then quenched with 1% HCl (100 mL). The aqueous phase was extracted with ethyl acetate (2 × 80 mL). The combined organic phases were dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4a (920 mg, 66% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.66–7.57 (m, 4H), 7.39 (dtt, J = 16.3, 6.3, 3.1 Hz, 6H), 7.20 (d, J = 8.2 Hz, 1H), 6.81 (dd, J = 15.3, 6.0 Hz, 1H), 6.11 (t, J = 4.9 Hz, 1H), 5.95 (dd, J = 15.4, 1.5 Hz, 1H), 5.20 (t, J = 5.2 Hz, 1H), 5.02 (d, J = 9.3 Hz, 1H), 4.60–4.56 (m, 1H), 4.18 (dd, J = 9.3, 4.6 Hz, 1H), 4.00 (dd, J = 5.1, 1.7 Hz, 2H), 3.84 (d, J = 4.8 Hz, 1H), 3.76 (qd, J = 10.2, 4.2 Hz, 2H), 3.46 (s, 3H), 2.13 (h, J = 6.6 Hz, 1H), 2.02 (q, J = 6.6, 6.1 Hz, 1H), 1.47 (s, 10H), 1.42 (s, 10H), 1.29–1.16 (m, 6H), 1.05 (s, 9H), 0.90 (t, J = 6.4 Hz, 6H), 0.86–0.75 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 172.0, 169.1, 168.9, 165.0, 155.8, 141.3, 135.7 (2C), 135.7 (2C), 132.8, 132.6, 130.0 (2C), 128.0 (4C), 124.7, 82.5, 81.9, 79.7, 76.9, 65.0, 59.2, 58.7, 51.5, 42.2, 33.5, 32.1 (2C), 30.8, 28.4 (3C), 28.1 (3C), 26.9 (3C), 26.5, 22.7, 19.4, 19.3, 17.2, 14.8, 14.2. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₈H₇₆N₃O₁₀Si⁺ 882.5295, found 882.5295.

(2S,3S)-2-Methoxy-1-oxo-1-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)amino)nonan-3-yl (tert-butoxycarbonyl)-l-valinate (4b). The titled compound 4b was obtained following the general procedure described for 4a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4b (1.13 g, 81% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (ddt, J = 6.5, 4.7, 1.8 Hz, 4H), 7.44–7.31 (m, 6H), 7.27 (d, J = 9.1 Hz, 1H), 6.80 (dd, J = 15.3, 6.2 Hz, 1H), 6.28 (t, J = 5.1 Hz, 1H), 5.98 (dd, J = 15.4, 1.4 Hz, 1H), 5.35 (dt, J = 9.7, 3.4 Hz, 1H), 5.05 (d, J = 9.2 Hz, 1H), 4.62–4.58 (m, 1H), 4.20 (dd, J = 9.3, 4.8 Hz, 1H), 3.97 (d, J = 5.1 Hz, 2H), 3.85 (d, J = 2.6 Hz, 1H), 3.72 (qt, J = 6.7, 3.0 Hz, 2H), 3.48 (s, 3H), 2.11 (td, J = 14.5, 12.2, 6.2 Hz, 1H), 1.74 (ddt, J = 14.0, 9.5, 5.2 Hz, 1H), 1.43 (d, J = 13.3 Hz, 18H), 1.39–1.34 (m, 1H), 1.26–1.16 (m, 8H), 1.03 (d, J = 6.7 Hz, 9H), 0.92 (d, J = 6.7 Hz, 3H), 0.82 (dd, J = 15.2, 7.0 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 171.9, 169.1, 168.4, 165.0, 155.7, 141.0, 135.6 (2C), 135.6 (2C), 132.7, 132.5, 130.0 (2C), 127.9 (4C), 124.9, 83.1, 82.3, 79.7, 75.4, 65.1, 59.7, 58.7, 51.4, 42.1, 31.7, 31.1 (2C), 29.0, 28.3 (3C), 28.0 (3C), 26.8 (3C), 25.4, 22.5, 19.3, 19.2, 17.4, 14.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₇H₇₃N₃O₁₀SiNa⁺ 890.4958, found 890.4954.

(2S,3S,4R)-2-Methoxy-4-methyl-1-oxo-1-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)amino)decan-3-yl (tert-butoxycarbonyl)-l-valinate (4c). The titled compound 4c was obtained following the general procedure described for 4a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4c (1.62 g, 86% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.61 (td, J = 8.0, 1.7 Hz, 4H), 7.38 (ttd, J = 8.9, 6.4, 6.0, 2.7 Hz, 6H), 7.19 (d, J = 8.2 Hz, 1H), 6.81 (ddd, J = 15.4, 6.1, 1.6 Hz, 1H), 6.14 (t, J = 5.2 Hz, 1H), 5.95 (dd, J = 15.4, 1.8 Hz, 1H), 5.19 (t, J = 5.2 Hz, 1H), 5.02 (d, J = 9.3 Hz, 1H), 4.57 (t, J = 7.2 Hz, 1H), 4.17 (dd, J = 9.4, 4.6 Hz, 1H), 3.99 (d, J = 4.8 Hz, 2H), 3.84 (d, J = 4.8 Hz, 1H), 3.75 (qd, J = 10.2, 4.0 Hz, 2H), 3.45 (s, 3H), 2.13 (q, J = 6.3 Hz, 1H), 2.01 (h, J = 6.5, 5.7 Hz, 1H), 1.44 (dd, J = 18.6, 1.6 Hz, 18H), 1.29–1.16 (m, 10H), 1.05 (s, 9H), 0.90 (t, J = 6.3 Hz, 6H), 0.86–0.77 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 172.0, 169.0, 168.8, 165.0, 155.8, 141.2, 135.7 (2C), 135.6 (2C), 132.7, 132.6, 130.0 (2C), 127.9 (4C), 124.7, 82.4, 81.9, 79.7, 76.8, 65.0, 59.1, 58.7, 51.5, 42.2, 33.5, 33.5, 31.8, 30.7, 29.5, 28.4 (3C), 28.0 (3C), 26.9 (3C), 26.8, 22.7, 19.4, 19.3, 17.2, 14.8, 14.1. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₉H₇₈N₃O₁₀Si⁺ 896.5451, found 896.5454.

(2S,3S)-2-Methoxy-1-oxo-1-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)amino)undecan-3-yl (tert-butoxycarbonyl)-l-valinate (4d). The titled compound 4d was obtained following the general procedure described for 4a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4d (643 mg, 63% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.61 (ddd, J = 8.0, 4.4, 1.6 Hz, 4H), 7.46–7.33 (m, 6H), 7.27–7.22 (m, 1H), 6.81 (dd, J = 15.4, 6.2 Hz, 1H), 6.08 (t, J = 5.0 Hz, 1H), 5.95 (dd, J = 15.4, 1.5 Hz, 1H), 5.42–5.33 (m, 1H), 5.02 (d, J = 9.2 Hz, 1H), 4.62 (dqd, J = 8.8, 4.4, 2.1 Hz, 1H), 4.22 (dd, J = 9.3, 4.7 Hz, 1H), 3.99 (d, J = 5.0 Hz, 2H), 3.86 (d, J = 2.6 Hz, 1H), 3.80–3.67 (m, 2H), 3.50 (s, 3H), 2.12 (h, J = 6.6 Hz, 1H), 1.74 (tt, J = 9.3, 4.5 Hz, 1H), 1.45 (d, J = 15.7 Hz, 19H), 1.28–1.18 (m, 12H), 1.05 (s, 9H), 0.93 (d, J = 6.8 Hz, 3H), 0.91–0.81 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 171.9, 169.1, 168.3, 164.9, 155.7, 141.2, 135.7 (2C), 135.6 (2C), 132.7, 132.6, 130.0 (2C), 128.0 (4C), 124.8, 83.2, 82.4, 79.8, 75.4, 65.2, 59.7, 58.7, 51.3, 42.2, 31.9, 31.2, 29.5, 29.5, 29.3, 28.9, 28.4 (3C), 28.1 (3C), 26.9 (3C), 25.6, 22.7, 19.3, 19.3, 17.4, 14.2. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₉H₇₈N₃O₁₀Si⁺ 896.5451, found 896.5454.

(2S,3S)-2-Methyl-1-oxo-1-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)amino)nonan-3-yl (tert-butoxycarbonyl)-l-valinate (4e). The titled compound 4e was obtained following the general procedure described for 4a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4e (1.395 g, 79% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.61 (dt, J = 8.0, 1.8 Hz, 4H), 7.45–7.32 (m, 6H), 6.82 (dd, J = 15.4, 5.2 Hz, 1H), 6.53 (t, J = 5.3 Hz, 1H), 6.12 (d, J = 8.4 Hz, 1H), 6.01 (dd, J = 15.4, 1.7 Hz, 1H), 5.17–4.95 (m, 2H), 4.70 (q, J = 4.8 Hz, 1H), 4.10 (dd, J = 8.7, 5.2 Hz, 1H), 4.04 (dd, J = 18.3, 5.4 Hz, 1H), 3.91 (dd, J = 18.3, 5.0 Hz, 1H), 3.78–3.66 (m, 2H), 2.50 (p, J = 7.0 Hz, 1H), 2.09 (h, J = 6.6 Hz, 1H), 1.63–1.55 (m, 2H), 1.43 (d, J = 11.1 Hz, 18H), 1.24 (q, J = 7.2 Hz, 8H), 1.10 (d, J = 7.0 Hz, 3H), 1.03 (s, 9H), 0.90 (d, J = 6.8 Hz, 3H), 0.88–0.79 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 172.6, 172.0, 169.0, 165.0, 156.0, 141.6, 135.7 (2C), 135.6 (2C), 133.0, 132.8, 130.0 (2C), 127.9 (4C), 124.3, 82.2, 79.9, 76.5, 65.4, 59.0, 51.6, 44.7, 42.1, 31.7, 30.7, 30.7, 29.2, 28.4 (3C), 28.1 (3C), 26.9 (3C), 24.9, 22.6, 19.3 (2C), 17.4, 14.1, 13.4. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₇H₇₄N₃O₉Si⁺ 852.5189, found 852.5189.

(2S,3S,4R)-2,4-Dimethyl-1-oxo-1-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)amino)decan-3-yl (tert-butoxycarbonyl)-l-valinate (4f). The titled compound 4f was obtained following the general procedure described for 4a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4f (1.12 g, 85% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.64–7.58 (m, 4H), 7.45–7.32 (m, 6H), 6.78 (dd, J = 15.5, 5.3 Hz, 1H), 6.73–6.66 (m, 1H), 6.28 (d, J = 8.2 Hz, 1H), 6.04 (dd, J = 15.4, 1.7 Hz, 1H), 5.17 (dd, J = 9.2, 2.6 Hz, 1H), 4.92 (d, J = 9.4 Hz, 1H), 4.69 (s, 1H), 4.15–3.98 (m, 2H), 3.89 (dd, J = 18.2, 5.0 Hz, 1H), 3.79–3.68 (m, 2H), 2.40 (dt, J = 14.0, 7.1 Hz, 1H), 2.19 (dq, J = 11.5, 7.1 Hz, 1H), 1.76–1.71 (m, 1H), 1.43 (d, J = 11.1 Hz, 18H), 1.28–1.19 (m, 10H), 1.08 (d, J = 6.9 Hz, 3H), 1.03 (s, 9H), 0.91–0.77 (m, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 173.2, 171.3, 168.9, 165.3, 156.4, 141.0, 135.6 (2C), 135.6 (2C), 133.1, 133.0, 129.9 (2C), 127.9 (4C), 124.6, 82.0, 79.9, 78.0, 65.3, 59.1, 51.9, 43.8, 42.0, 34.0, 33.9, 31.8, 29.7, 29.5, 28.4 (3C), 28.1 (3C), 27.2, 26.9 (3C), 22.7, 19.5, 19.3, 17.3, 14.6, 14.1, 13.2. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₉H₇₈N₃O₉Si⁺ 880.5502, found 880.5507.

(3S,4S,5R)-5-Methyl-3-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)carbamoyl)undecan-4-yl (tert-butoxycarbonyl)-l-valinate (4g). The titled compound 4g was obtained following the general procedure described for 4a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4g (529 mg, 62% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (dq, J = 8.0, 1.9 Hz, 4H), 7.38 (dtd, J = 14.1, 6.8, 3.3 Hz, 7H), 6.79 (dd, J = 15.4, 5.2 Hz, 1H), 6.68 (t, J = 5.4 Hz, 1H), 6.25 (d, J = 8.2 Hz, 1H), 6.06 (dd, J = 15.5, 1.7 Hz, 1H), 5.20 (dd, J = 8.7, 2.9 Hz, 1H), 4.93 (d, J = 9.5 Hz, 1H), 4.73 (ddt, J = 10.1, 5.2, 2.5 Hz, 1H), 4.13–3.99 (m, 2H), 3.89 (dd, J = 18.2, 4.9 Hz, 1H), 3.74 (d, J = 4.8 Hz, 2H), 2.32–2.11 (m, 2H), 1.79–1.56 (m, 2H), 1.45 (d, J = 9.5 Hz, 18H), 1.25 (t, J = 11.0 Hz, 10H), 1.04 (s, 9H), 0.86 (ddd, J = 25.5, 12.3, 6.6 Hz, 15H). ¹³C NMR (100 MHz, CDCl₃) δ 172.3, 171.4, 169.0, 165.3, 156.4, 141.2, 135.7 (2C), 135.7 (2C), 133.0, 132.9, 130.0 (2C), 128.0 (4C), 127.9, 124.7, 82.1, 80.0, 77.7, 65.5, 59.2, 52.0, 51.9, 42.1, 34.3, 34.1, 31.9, 29.5, 28.5 (3C), 28.2 (3C), 27.2, 27.0 (3C), 22.7, 22.3, 19.6, 19.4, 17.4, 14.2, 13.5, 12.1. HRMS(ESI) m/z: [M + H]⁺ calcd for C₅₀H₈₀N₃O₉Si⁺ 894.5659, found 894.5662.

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-15-((R)-heptan-2-yl)-3-isopropyl-14-methoxy-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (3a). To a solution of 4a (771 mg, 0.874 mmol, 1.0 equiv) in CH₂Cl₂ (7.0 mL) was added trifluoroacetic acid (1.7 mL) at 0 °C. After stirred for 4 h, the reaction solution was diluted with toluene (7 mL). And the resulting solution was concentrated under reduced pressure. The resulting white solid was dissolved in THF (20 mL), and the solution was added slowly to a suspension of HATU (4.98 g, 13.11 mmol, 15.0 equiv) and DIPEA (3.39 g, 26.22 mmol, 30.0 equiv) in THF (900 mL) over 30 h at room temperature and then continued to stirred another 12 h at room temperature. The solvent was evaporated, diluted with MeOH and EtOAc (v/v = 2:1, 300 mL) and filtered through a short pad of silica gel. The filtrate was concentrated under reduced pressure. The residue was dissolved in EtOAc (300 mL), washed with 1% HCl (80 mL), saturated aqueous solution of NaHCO₃ (60 mL), and brine (60 mL). The organic phase was dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 50:1 to 9:1) to obtain 3a (460 mg, 67% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.41 (d, J = 9.0 Hz, 1H), 8.22 (d, J = 9.8 Hz, 1H), 7.59 (ddd, J = 7.8, 3.5, 1.6 Hz, 4H), 7.48–7.35 (m, 6H), 6.71 (dd, J = 15.1, 3.0 Hz, 1H), 5.92 (dd, J = 15.1, 2.2 Hz, 1H), 5.31–5.20 (m, 1H), 4.70 (dtt, J = 8.9, 6.0, 2.4 Hz, 1H), 4.25 (dd, J = 9.8, 6.9 Hz, 1H), 4.06–3.95 (m, 1H), 3.88 (d, J = 9.7 Hz, 1H), 3.58 (dd, J = 19.4, 8.3 Hz, 3H), 3.33 (d, J = 1.2 Hz, 1H), 3.20 (s, 3H), 2.02–1.90 (m, 1H), 1.84 (q, J = 7.1, 6.6 Hz, 1H), 1.28–1.13 (m, 8H), 0.95 (d, J = 10.1 Hz, 12H), 0.88–0.76 (m, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.1, 168.8, 167.7, 166.5, 141.5, 135.2 (2C), 135.1 (2C), 132.7, 132.6, 130.0, 130.0, 128.0 (4C), 119.5, 78.4, 73.4, 65.5, 57.4, 56.7, 51.5, 44.1, 33.1, 32.9, 31.5, 31.4, 26.6 (3C), 26.6, 22.1, 19.3 (2C), 18.8, 18.3, 14.0. HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₉H₅₈N₃O₇Si⁺ 708.4039, found 708.4039.

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-15-hexyl-3-isopropyl-14-methoxy-1-oxa-4,7,12-triazacyclopen-tadec-9-ene-2,5,8,13-tetraone (3b). The titled compound 3b was obtained following the general procedure described for 3a. Flash column chromatography eluent (dichloromethane/methanol = 50:1 to 9:1) to obtain 3b (870 mg, 82% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.46 (d, J = 8.9 Hz, 1H), 8.26 (d, J = 9.4 Hz, 1H), 7.66–7.60 (m, 4H), 7.57 (t, J = 6.8 Hz, 1H), 7.45 (qd, J = 6.7, 2.5 Hz, 6H), 6.78 (dd, J = 15.1, 2.9 Hz, 1H), 5.92 (dd, J = 15.1, 2.3 Hz, 1H), 5.16 (dt, J = 9.3, 4.4 Hz, 1H), 4.76 (ddt, J = 9.6, 6.8, 3.9 Hz, 1H), 4.33 (dd, J = 9.4, 5.7 Hz, 1H), 4.03 (dd, J = 17.7, 5.9 Hz, 1H), 3.81 (d, J = 9.7 Hz, 1H), 3.69–3.56 (m, 3H), 3.26 (s, 3H), 2.09 (dt, J = 13.1, 6.6 Hz, 1H), 1.64 (ddt, J = 15.2, 10.4, 4.9 Hz, 2H), 1.45–1.31 (m, 2H), 1.25 (d, J = 4.4 Hz, 6H), 1.00 (s, 9H), 0.87 (t, J = 7.1 Hz, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.6, 168.9, 167.7, 166.4, 141.6, 135.2 (2C), 135.1 (2C), 132.6, 132.6, 130.0, 130.0, 127.9 (4C), 119.4, 79.4, 71.7, 65.3, 56.9, 56.8, 51.5, 43.9, 31.2 (2C), 30.1, 28.8, 26.6 (3C), 23.1, 22.0, 19.0, 18.8, 17.7, 13.9. HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₈H₅₆N₃O₇Si⁺ 694.3883, found 694.3882.

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-3-isopropyl-14-methoxy-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (3c). The titled compound 3c was obtained following the general procedure described for 3a. Flash column chromatography eluent (dichloromethane/methanol = 50:1 to 9:1) to obtain 3c (501 mg, 67% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.41 (d, J = 9.0 Hz, 1H), 8.21 (d, J = 9.8 Hz, 1H), 7.69–7.54 (m, 5H), 7.44 (qdd, J = 8.4, 3.3, 1.8 Hz, 6H), 6.74 (dd, J = 15.1, 3.0 Hz, 1H), 5.95 (dd, J = 15.2, 2.2 Hz, 1H), 5.26 (dd, J = 9.8, 1.7 Hz, 1H), 4.73 (ddt, J = 9.5, 6.7, 3.8 Hz, 1H), 4.28 (dd, J = 9.7, 6.9 Hz, 1H), 4.03 (dd, J = 17.5, 5.8 Hz, 1H), 3.90 (d, J = 9.7 Hz, 1H), 3.67–3.53 (m, 3H), 3.23 (s, 3H), 2.03–1.90 (m, J = 7.3 Hz, 1H), 1.87 (q, J = 6.8 Hz, 1H), 1.32–1.20 (m, 9H), 1.17–1.02 (m, 1H), 0.98 (d, J = 11.4 Hz, 12H), 0.91–0.81 (m, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.0, 168.7, 167.6, 166.5, 141.4, 135.1 (2C), 135.0 (2C), 132.6, 132.6, 130.0, 130.0, 127.9 (4C), 119.5, 78.4, 73.4, 65.4, 57.4, 56.6, 51.4, 44.0, 33.1, 32.9, 31.3, 31.2, 28.9, 26.8, 26.6 (3C), 22.0, 19.2, 18.8, 18.2, 13.9, 13.9. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₀H₆₀N₃O₇Si⁺ 722.4196, found 722.4195.

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-3-isopropyl-14-methoxy-15-octyl-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (3d). The titled compound 3d was obtained following the general procedure described for 3a. Flash column chromatography eluent (dichloromethane/methanol = 50:1 to 9:1) to obtain 3d (216 mg, 62% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.55 (d, J = 8.9 Hz, 1H), 8.32 (d, J = 9.4 Hz, 1H), 7.59 (s, 4H), 7.56 (t, J = 6.8 Hz, 1H), 7.44 (dtdd, J = 11.6, 8.6, 6.0, 1.9 Hz, 6H), 6.76 (dd, J = 15.1, 3.0 Hz, 1H), 5.98 (dd, J = 15.2, 2.3 Hz, 1H), 5.14 (dt, J = 9.2, 4.5 Hz, 1H), 4.76 (qd, J = 6.6, 3.7 Hz, 1H), 4.32 (dd, J = 9.4, 5.8 Hz, 1H), 4.10 (dd, J = 17.6, 6.0 Hz, 1H), 3.87 (d, J = 9.7 Hz, 1H), 3.71–3.54 (m, 3H), 3.38 (d, J = 7.4 Hz, 3H), 2.12–1.99 (m, J = 7.2 Hz, 1H), 1.62 (ddt, J = 20.3, 14.6, 6.9 Hz, 2H), 1.24 (s, 12H), 0.99 (s, 9H), 0.90–0.77 (m, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.5, 169.0, 167.8, 166.4, 141.4, 135.1 (2C), 135.0 (2C), 132.6, 132.6, 129.9, 129.9, 127.9 (4C), 119.5, 79.3, 71.8, 65.4, 57.0, 56.8, 51.6, 43.9, 31.2, 31.2, 30.1, 29.0, 28.8, 28.5, 26.6 (3C), 23.1, 22.1, 19.0, 18.8, 17.7, 13.9. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₀H₆₀N₃O₇Si⁺ 722.4196, found 722.4200.

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-15-hexyl-3-isopropyl-14-methyl-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (3e). The titled compound 3e was obtained following the general procedure described for 3a. Flash column chromatography eluent (dichloromethane/methanol = 50:1 to 9:1) to obtain 3e (362 mg, 66% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.23 (d, J = 9.5 Hz, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.62 (td, J = 6.1, 3.0 Hz, 4H), 7.56–7.36 (m, 7H), 6.75 (dd, J = 15.0, 2.8 Hz, 1H), 5.91 (dd, J = 15.0, 2.3 Hz, 1H), 5.13 (dt, J = 10.8, 4.2 Hz, 1H), 4.64 (dtt, J = 8.2, 5.4, 2.7 Hz, 1H), 4.31 (dd, J = 9.5, 5.5 Hz, 1H), 4.04 (dd, J = 17.7, 6.1 Hz, 1H), 3.66 (dd, J = 10.1, 5.8 Hz, 1H), 3.63–3.49 (m, 2H), 2.72 (dq, J = 10.3, 6.9 Hz, 1H), 2.06 (dq, J = 13.3, 6.7 Hz, 1H), 1.54 (ddt, J = 24.6, 15.0, 4.1 Hz, 2H), 1.38 (p, J = 5.1, 4.5 Hz, 2H), 1.30–1.21 (m, 9H), 1.00 (d, J = 10.9 Hz, 12H), 0.86 (dt, J = 7.3, 3.7 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 173.0, 170.0, 169.0, 166.3, 142.4, 135.1 (2C), 135.1 (2C), 132.8, 132.7, 129.9 (2C), 127.9 (4C), 119.1, 74.8, 65.1, 56.8, 51.4, 43.8, 42.0, 31.2, 31.2, 30.1, 28.8, 26.6 (3C), 22.6, 22.0, 19.0, 18.8, 17.6, 15.5, 13.9. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₃₈H₅₅N₃O₆SiNa⁺ 700.3753, found 700.3747.

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-3-isopropyl-14-methyl-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (3f). The titled compound 3f was obtained following the general procedure described for 3a. Flash column chromatography eluent (dichloromethane/methanol = 50:1 to 9:1) to obtain 3f (332 mg, 69% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 9.8 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.62 (ddd, J = 8.1, 4.9, 1.8 Hz, 4H), 7.54 (t, J = 6.8 Hz, 1H), 7.44 (dtt, J = 10.5, 6.6, 2.2 Hz, 6H), 6.72 (dd, J = 15.0, 2.7 Hz, 1H), 5.94 (dd, J = 15.1, 2.3 Hz, 1H), 5.26 (dd, J = 10.4, 1.6 Hz, 1H), 4.67–4.61 (m, 1H), 4.25 (dd, J = 9.8, 6.9 Hz, 1H), 4.03 (dd, J = 17.4, 5.9 Hz, 1H), 3.68–3.49 (m, 3H), 2.79 (dt, J = 14.6, 7.2 Hz, 1H), 1.93 (h, J = 6.9 Hz, 1H), 1.71 (q, J = 7.3, 6.8 Hz, 1H), 1.24 (d, J = 10.0 Hz, 10H), 1.02 (d, J = 7.0 Hz, 3H), 0.98 (d, J = 2.1 Hz, 9H), 0.93 (d, J = 6.8 Hz, 3H), 0.90–0.81 (m, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 173.0, 169.1, 168.6, 166.5, 142.2, 135.1 (2C), 135.1 (2C), 132.8, 132.7, 129.9 (2C), 127.9 (4C), 119.3, 76.4, 65.2, 57.5, 51.2, 44.0, 41.7, 33.5, 33.4, 31.4, 31.2, 28.9, 26.8, 26.6 (3C), 22.1, 19.3, 18.8, 18.3, 16.0, 13.9, 13.3. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₀H₆₀N₃O₆Si⁺ 706.4246, found 706.4251.

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-14-ethyl-3-isopropyl-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacy-clopentadec-9-ene-2,5,8,13-tetraone (3g). The titled compound 3g was obtained following the general procedure described for 3a. Flash column chromatography eluent (dichloromethane/methanol = 50:1 to 9:1) to obtain 3g (289 mg, 58% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (d, J = 9.9 Hz, 1H), 7.90 (d, J = 9.0 Hz, 1H), 7.64–7.52 (m, 4H), 7.47–7.38 (m, 6H), 6.76 (dd, J = 15.1, 2.8 Hz, 1H), 5.98 (dd, J = 15.1, 2.3 Hz, 1H), 5.34–5.27 (m, 1H), 4.69 (ddt, J = 9.6, 6.8, 3.9 Hz, 1H), 4.30–4.21 (m, 1H), 4.09–3.98 (m, 1H), 3.61–3.50 (m, 3H), 2.69 (dt, J = 10.0, 4.7 Hz, 1H), 1.99–1.90 (m, 1H), 1.70 (q, J = 6.9 Hz, 1H), 1.51 (ddd, J = 11.9, 7.2, 4.1 Hz, 1H), 1.37 (td, J = 8.7, 3.9 Hz, 2H), 1.24 (d, J = 9.0 Hz, 10H), 0.99 (s, 9H), 0.93 (d, J = 6.8 Hz, 3H), 0.90–0.81 (m, 9H), 0.76 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.4, 169.1, 168.6, 166.6, 142.2, 135.1 (2C), 135.1 (2C), 132.6, 132.6, 130.0 (2C), 127.9 (4C), 119.4, 75.4, 65.5, 57.4, 51.1, 47.8, 44.1, 33.6, 33.4, 31.5, 31.1, 28.9, 26.8, 26.6 (3C), 22.4, 22.0, 19.4, 18.8, 18.3, 13.9, 13.4, 10.7. HRMS(ESI) m/z: [M + H]⁺ calcd for C₄₁H₆₂N₃O₆Si⁺ 720.4403, found 720.4404.

(3S,14S,15S,E)-15-((R)-Heptan-2-yl)-3-isopropyl-14-methoxy-11-methylene-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1a). To a solution of compound 3a (460 mg, 0.650 mmol, 1.0 equiv) in THF (10 mL) were added HOAc (97.5 mg, 1.63 mmol, 2.5 equiv) and TBAF (1.63 ml, 1 mol/L in THF, 1.63 mmol, 3.0 equiv). The mixture was stirred at room temperature for 24 h, and then concentrated. The residue was purified by column chromatography on silica gel (dichloromethane/methanol = 20:1 to 9:1) to obtain 2a as a white powder.

To a solution of compound 2a (292 mg, 0.622 mmol, 1.0 equiv) in THF (6.2 mL), then triethylamine (157 mg, 1.56 mmol, 3.0 equiv) and ethanesulfonyl chloride (160 mg, 1.24 mmol, 2.0 equiv) were added at 0 °C. After stirred for 2 h, the reaction solution was quenched by addition of H₂O (0.1 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The resulting crude product was dissolved in THF (6.2 mL). To the resulting solution was added DBU (806 mg, 5.3 mmol, 8 equiv) at 20 °C. After stirred for 2.5 h, the solution was dissolved in ethyl acetate (80 mL) and washed successively with 1% HCl (30 mL), saturated aqueous NaHCO₃ (30 mL), brine (30 mL), dried over Na₂SO₄ and concentrated under reduced pressure carefully. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100:1 to 100:6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1a (155 mg, 52% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.17 (s, 1H), 8.24 (d, J = 9.8 Hz, 1H), 7.68 (dd, J = 7.7, 5.3 Hz, 1H), 6.89 (d, J = 15.2 Hz, 1H), 6.11 (d, J = 15.2 Hz, 1H), 5.59 (s, 1H), 5.46 (s, 1H), 5.18 (dd, J = 8.7, 3.0 Hz, 1H), 4.31 (dd, J = 9.8, 7.0 Hz, 1H), 4.13–3.90 (m, 2H), 3.59 (dd, J = 17.2, 7.7 Hz, 1H), 3.30 (s, 3H), 1.98 (tt, J = 15.4, 7.3 Hz, 2H), 1.25 (tdd, J = 16.1, 7.7, 4.1 Hz, 8H), 0.95 (d, J = 6.8 Hz, 3H), 0.89–0.82 (m, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.2, 168.9, 168.0, 167.0, 138.1, 137.4, 119.0, 116.3, 79.0, 74.2, 57.4, 56.8, 44.3, 33.1, 32.7, 31.4, 31.3, 26.3, 22.0, 19.2, 18.2, 14.0, 13.9. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₃H₃₈N₃O₆⁺ 452.2756, found 452.2758.

(3S,14S,15S,E)-15-Hexyl-3-isopropyl-14-methoxy-11-methylene-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1b). The titled compound 1b was obtained following the general procedure described for 1a. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100:1 to 100:6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1b (123 mg, 57% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.13 (s, 1H), 8.33 (d, J = 9.5 Hz, 1H), 7.65 (t, J = 6.5 Hz, 1H), 6.94 (d, J = 15.2 Hz, 1H), 6.03 (d, J = 15.2 Hz, 1H), 5.51 (d, J = 6.3 Hz, 2H), 5.16 (q, J = 5.9 Hz, 1H), 4.40 (dd, J = 9.5, 5.6 Hz, 1H), 4.00 (dd, J = 17.4, 5.4 Hz, 1H), 3.91 (d, J = 7.7 Hz, 1H), 3.58 (dd, J = 17.6, 7.8 Hz, 1H), 3.32 (s, 3H), 2.10 (q, J = 6.5 Hz, 1H), 1.96 (ddd, J = 28.6, 13.9, 7.4 Hz, 1H), 1.79 (q, J = 12.3 Hz, 1H), 1.64 (dt, J = 14.7, 7.0 Hz, 1H), 1.46 (d, J = 9.1 Hz, 1H), 1.39–1.26 (m, 6H), 0.85 (q, J = 6.5 Hz, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.8, 169.1, 167.8, 167.0, 138.3, 137.6, 119.3, 116.9, 80.4, 72.3, 57.2, 56.8, 44.0, 31.1, 31.1, 30.2, 28.6, 23.7, 22.0, 19.0, 17.6, 13.1. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₂H₃₅N₃O₆Na⁺ 460.2419, found 460.2423.

(3S,14S,15S,E)-3-Isopropyl-14-methoxy-11-methylene-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1c). The titled compound 1c was obtained following the general procedure described for 1a. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100:1 to 100:6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1c (109 mg, 47% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.23 (s, 1H), 8.28 (d, J = 9.8 Hz, 1H), 7.68 (dd, J = 7.7, 5.3 Hz, 1H), 6.87 (d, J = 15.2 Hz, 1H), 6.19 (d, J = 15.3 Hz, 1H), 5.61 (s, 1H), 5.45 (s, 1H), 5.16 (dd, J = 8.7, 2.9 Hz, 1H), 4.30 (dd, J = 9.7, 7.1 Hz, 1H), 4.19–4.05 (m, 2H), 3.62–3.54 (m, 1H), 3.34 (s, 3H), 1.98 (dq, J = 13.6, 7.0 Hz, 2H), 1.31–1.21 (m, 9H), 1.16–1.05 (m, 1H), 0.95 (dd, J = 7.2, 2.9 Hz, 3H), 0.88–0.81 (m, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.1, 169.0, 168.1, 167.0, 138.1, 137.4, 119.1, 116.1, 78.9, 74.3, 57.5, 56.8, 44.2, 33.1, 32.7, 31.4, 31.2, 28.9, 26.6, 22.1, 19.2, 18.2, 14.0, 13.9. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₄H₄₀N₃O₆⁺ 466.2912, found 466.2911.

(3S,14S,15S,E)-3-Isopropyl-14-methoxy-11-methylene-15-octyl-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1d). The titled compound 1d was obtained following the general procedure described for 1a. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100:1 to 100:6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1d (87 mg, 67% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.23 (s, 1H), 8.38 (d, J = 9.5 Hz, 1H), 7.66 (dd, J = 7.6, 5.4 Hz, 1H), 6.92 (d, J = 15.2 Hz, 1H), 6.12 (d, J = 15.2 Hz, 1H), 5.54 (s, 1H), 5.49 (s, 1H), 5.14 (dt, J = 7.7, 5.1 Hz, 1H), 4.38 (dd, J = 9.5, 5.7 Hz, 1H), 4.09 (dd, J = 17.3, 5.4 Hz, 1H), 3.99 (d, J = 8.0 Hz, 1H), 3.57 (dd, J = 17.4, 7.7 Hz, 1H), 3.31 (s, 3H), 2.08 (p, J = 6.7 Hz, 1H), 1.65 (p, J = 6.4 Hz, 2H), 1.56 (dt, J = 15.3, 7.9 Hz, 2H), 1.35–1.25 (m, 10H), 0.93 (t, J = 7.3 Hz, 3H), 0.85 (q, J = 6.1 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.7, 169.2, 168.0, 167.0, 138.3, 137.6, 119.3, 116.7, 80.3, 72.4, 57.1, 56.9, 44.0, 31.2 (2C), 30.2, 28.9, 28.9, 28.6, 23.6, 22.1, 19.0, 17.6, 14.0. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₄H₄₀N₃O₆⁺ 466.2912, found 466.2912.

(3S,14S,15S,E)-15-Hexyl-3-isopropyl-14-methyl-11-methylene-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1e). The titled compound 1e was obtained following the general procedure described for 1a. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100:1 to 100:6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1e (93 mg, 61% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.18 (s, 1H), 8.38 (d, J = 9.4 Hz, 1H), 7.64 (t, J = 6.7 Hz, 1H), 6.95 (d, J = 15.0 Hz, 1H), 6.16 (d, J = 15.0 Hz, 1H), 5.45 (s, 1H), 5.33 (s, 1H), 5.04 (dt, J = 10.6, 4.1 Hz, 1H), 4.26 (dd, J = 9.4, 6.1 Hz, 1H), 4.19 (dd, J = 17.2, 5.7 Hz, 1H), 3.52 (dd, J = 17.3, 7.6 Hz, 1H), 2.91 (dt, J = 10.3, 7.0 Hz, 1H), 2.00 (dt, J = 14.8, 7.1 Hz, 2H), 1.59–1.47 (m, 2H), 1.27–1.21 (m, 7H), 1.05 (d, J = 6.9 Hz, 3H), 0.94–0.80 (m, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.6, 169.7, 169.0, 166.8, 138.8, 138.6, 118.8, 117.5, 75.1, 57.2, 43.9, 41.9, 31.3, 31.1, 30.1, 28.8, 22.5, 23.0, 19.0, 17.8, 15.0, 13.9. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₂H₃₆N₃O₅⁺ 422.2650, found 422.2652.

(3S,14S,15S,E)-3-Isopropyl-14-methyl-11-methylene-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1f). The titled compound 1f was obtained following the general procedure described for 1a. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100:1 to 100:6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1f (118 mg, 59% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.94 (s, 1H), 8.24 (d, J = 9.8 Hz, 1H), 7.66 (t, J = 6.6 Hz, 1H), 6.91 (d, J = 15.1 Hz, 1H), 6.13 (d, J = 15.1 Hz, 1H), 5.44 (s, 1H), 5.37 (s, 1H), 5.18 (d, J = 10.2 Hz, 1H), 4.27–4.18 (m, 1H), 4.08 (dd, J = 17.2, 5.6 Hz, 1H), 3.55 (dd, J = 17.2, 7.8 Hz, 1H), 2.89 (dt, J = 13.8, 7.2 Hz, 1H), 1.92 (h, J = 6.5 Hz, 1H), 1.73 (q, J = 6.9 Hz, 1H), 1.62–1.50 (m, 1H), 1.39–1.25 (m, 9H), 1.06 (d, J = 7.0 Hz, 3H), 0.94 (d, J = 6.6 Hz, 3H), 0.86 (dd, J = 10.9, 6.7 Hz, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.5, 169.0, 168.7, 166.9, 138.8, 138.2, 118.6, 117.1, 76.7, 57.7, 44.2, 41.8, 33.5, 31.4, 31.2, 28.9, 26.8, 23.1, 22.0, 19.3, 18.4, 15.4, 13.9, 13.2. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₄H₄₀N₃O₅⁺450.2963, found 450.2966.

(3S,14S,15S,E)-14-Ethyl-3-isopropyl-11-methylene-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1g). The titled compound 1g was obtained following the general procedure described for 1a. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100:1 to 100:6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1g (93 mg, 56% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92 (s, 1H), 8.18 (d, J = 9.9 Hz, 1H), 7.66 (dd, J = 7.7, 5.4 Hz, 1H), 6.87 (d, J = 15.1 Hz, 1H), 6.16 (d, J = 15.1 Hz, 1H), 5.42 (s, 2H), 5.23 (dd, J = 10.1, 1.7 Hz, 1H), 4.25 (dd, J = 9.9, 7.3 Hz, 1H), 4.07 (dd, J = 17.2, 5.4 Hz, 1H), 3.57 (dd, J = 17.2, 7.8 Hz, 1H), 2.77 (td, J = 10.2, 4.2 Hz, 1H), 1.93 (h, J = 8.0, 6.8 Hz, 1H), 1.72 (h, J = 6.9 Hz, 1H), 1.53 (ddt, J = 12.1, 7.2, 4.1 Hz, 2H), 1.46–1.20 (m, 8H), 1.05 (dtd, J = 18.1, 11.7, 10.8, 5.2 Hz, 2H), 0.98–0.92 (m, 3H), 0.85 (dd, J = 10.3, 6.7 Hz, 12H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.4, 169.0, 168.7, 167.0, 138.6, 137.7, 118.7, 116.3, 76.1, 57.6, 48.8, 44.3, 33.6, 33.5, 31.5, 31.2, 28.9, 26.7, 22.4, 22.1, 19.3, 18.4, 13.9, 13.3, 11.1. HRMS(ESI) m/z: [M + H]⁺ calcd for C₂₅H₄₂N₃O₅⁺ 464.3119, found 464.3120.

Benzyl (2S,3S,4R)-3-(((tert-butoxycarbonyl)-l-phenylalanyl)oxy)-2-methoxy-4-methyldecanoate (6h). To a solution of compound 7h (1.08 g, 4.65 mmol, 2.5 equiv) and 8c (600 mg, 1.86 mmol, 1.0 equiv) in CH₂Cl₂ (3.12 mL) was added DMAP (22.7 mg, 0.186 mmol, 0.1 equiv) and DIC (658 mg, 52.1 mmol, 2.8 equiv) under argon atmosphere at 0 °C. The reaction mixture was stirred for 1.5 h, and diluted with CH₂Cl₂ (50 mL). The suspension was filtered through a pad of silica gel and the filter cake was washed with CH₂Cl₂ (3 × 10 mL) and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 40:1 to 30:1) to obtain compound 6h (879 mg, 83%) as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.43–7.09 (m, 10H), 5.17 (d, J = 11.1 Hz, 3H), 4.84 (d, J = 8.6 Hz, 1H), 4.49 (q, J = 7.1 Hz, 1H), 3.94 (d, J = 5.8 Hz, 1H), 3.37 (s, 3H), 3.08 (dd, J = 14.1, 5.8 Hz, 1H), 2.84 (dd, J = 14.1, 7.1 Hz, 1H), 1.93–1.86 (m, 1H), 1.38 (s, 9H), 1.30–1.04 (m, 10H), 0.91–0.82 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 171.4, 170.0, 155.1, 136.4, 135.3, 129.5 (2C), 128.6 (4C), 128.6, 128.4 (2C), 126.8, 80.5, 79.7, 76.8, 67.0, 58.4, 54.4, 37.7, 33.5, 33.2, 31.8, 29.4, 28.3 (3C), 26.9, 22.7, 14.2, 14.1. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₃₃H₄₇NO₇Na⁺ 592.3245, found 592.3240.

Benzyl (2S,3S,4R)-3-(((tert-butoxycarbonyl)-l-alanyl)oxy)-2-methoxy-4-methyldecanoate (6i). The titled compound 6i was obtained following the general procedure described for 6h. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 6i (799 mg, 87%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.40–7.24 (m, 5H), 5.18–5.11 (m, 3H), 4.99 (d, J = 8.0 Hz, 1H), 4.22 (p, J = 7.4 Hz, 1H), 3.90 (d, J = 6.8 Hz, 1H), 3.34 (s, 3H), 1.95–1.86 (m, 1H), 1.42 (s, 9H), 1.32–1.06 (m, 13H), 0.92–0.78 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 172.6, 170.2, 155.1, 135.3, 128.7 (4C), 128.6, 80.4, 79.7, 76.2, 67.1, 58.5, 49.3, 33.4, 33.2, 31.8, 29.4, 28.4 (3C), 26.9, 22.7, 18.4, 14.1, 13.9. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₇H₄₃NO₇Na⁺ 516.2932, found 516.2931.

Benzyl (2S,3S,4R)-3-(((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)oxy)-2-methoxy-4-methyldecanoate (6j). The titled compound 6j was obtained following the general procedure described for 6h. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 6j (787 mg, 79%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.43–7.27 (m, 5H), 5.24–5.16 (m, 3H), 5.15–5.09 (m, 1H), 4.16–4.05 (m, 1H), 4.00 (d, J = 5.8 Hz, 1H), 3.35 (s, 3H), 1.95–1.79 (m, 1H), 1.44 (s, 9H), 1.39–1.16 (m, 10H), 0.97 (s, 9H), 0.92 (d, J = 6.8 Hz, 3H), 0.87 (t, J = 6.9 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 171.5, 169.9, 155.5, 135.3, 128.6 (2C), 128.5 (2C), 128.5, 80.6, 79.6, 76.7, 66.9, 62.0, 58.5, 34.3, 33.6, 33.4, 31.8, 29.3, 28.3 (3C), 26.9, 26.4 (3C), 22.6, 14.4, 14.1. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₃₀H₄₉NO₇Na⁺ 558.3402, found 558.3399.

Benzyl (2S,3S,4R)-3-(((tert-butoxycarbonyl)-l-leucyl)oxy)-2-methoxy-4-methyldecanoate (6k). The titled compound 6k was obtained following the general procedure described for 6h. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 6k (847 mg, 85%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.42–7.28 (m, 5H), 5.15 (s, 3H), 4.81 (d, J = 9.0 Hz, 1H), 4.25 (td, J = 9.3, 5.1 Hz, 1H), 3.91 (d, J = 6.6 Hz, 1H), 3.34 (s, 3H), 1.95–1.87 (m, 1H), 1.74–1.58 (m, 1H), 1.52 (ddd, J = 13.8, 8.6, 5.2 Hz, 1H), 1.42 (s, 9H), 1.37–1.03 (m, 11H), 0.87 (dd, J = 21.2, 6.9 Hz, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 172.7, 170.2, 155.4, 135.4, 128.7 (2C), 128.6 (2C), 128.5, 80.5, 79.7, 76.2, 67.0, 58.6, 52.3, 41.6, 33.5, 33.3, 31.8, 29.4, 28.4 (3C), 27.0, 24.8, 23.0, 22.7, 21.8, 14.2, 14.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₃₀H₄₉NO₇Na⁺ 558.3402, found 558.3398.

Benzyl (2S,3S,4R)-3-(((tert-butoxycarbonyl)glycyl)oxy)-2-methoxy-4-methyldecanoate (6l). The titled compound 6l was obtained following the general procedure described for 6h. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 6l (722 mg, 81%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.40–7.25 (m, 5H), 5.21–5.09 (m, 3H), 4.81 (t, J = 5.6 Hz, 1H), 3.87 (d, J = 7.2 Hz, 1H), 3.77 (dd, J = 18.1, 6.1 Hz, 1H), 3.58 (dd, J = 18.3, 5.3 Hz, 1H), 3.37 (s, 3H), 1.97–1.88 (m, 1H), 1.43 (s, 9H), 1.30–1.18 (m, 10H), 0.90–0.79 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 170.4, 169.7, 155.5, 135.3, 128.9 (2C), 128.7 (2C), 128.6, 80.1, 79.8, 76.2, 67.1, 58.6, 42.2, 34.7, 33.3, 31.8, 29.4, 28.4 (3C), 26.9, 22.6, 14.1, 13.7. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₆H₄₁NO₇Na⁺ 502.2776, found 502.2774.

(2S,3S,4R)-2-Methoxy-4-methyl-1-oxo-1-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)amino)decan-3-yl (tert-butoxycarbonyl)-l-phenylalaninate (4h). To a solution of compound 6h (569.7 mg, 1.0 mmol, 1.0 equiv) in MeOH (10 mL) was added Pd/C (10%, 30 mg) under N₂. The suspension was degassed under vacuum and purged with H₂ three times. The mixture was stirred under H₂ balloon at 20 °C for 2 h. The resulting mixture was filtered through a short pad of silica gel, washed with MeOH (2 × 30 mL) and concentrated under reduced pressure. The crude product was used directly for the next step without further purification.

To a solution of above acid and 5 (728 mg, 1.5 mmol, 1.5 equiv) in CH₂Cl₂ (5 mL) was added EDCI (230 mg, 1.2 mmol, 1.2 equiv) and HOBt (148.6 mg, 1.1 mmol, 1.1 equiv) under argon atmosphere at 20 °C. The reaction mixture was stirred for 12 h, and then quenched with 1% HCl (100 mL). The aqueous phase was extracted with ethyl acetate (2 × 80 mL). The combined organic phases were dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4h (840 mg, 89% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.61 (d, J = 7.1 Hz, 4H), 7.39 (ddd, J = 17.3, 7.3, 2.9 Hz, 6H), 7.30–7.09 (m, 6H), 6.82 (ddd, J = 15.4, 5.8, 2.2 Hz, 1H), 6.26 (d, J = 5.6 Hz, 1H), 5.97 (d, J = 15.4 Hz, 1H), 5.19 (td, J = 5.3, 2.1 Hz, 1H), 5.06 (d, J = 8.6 Hz, 1H), 4.52 (q, J = 7.5, 6.9 Hz, 2H), 4.01 (qdd, J = 18.3, 6.0, 3.0 Hz, 2H), 3.86–3.71 (m, 3H), 3.43–3.38 (m, 3H), 3.15 (dd, J = 14.3, 6.1 Hz, 1H), 2.92 (dd, J = 13.8, 7.2 Hz, 1H), 2.08–1.94 (m, 1H), 1.48 (d, J = 2.2 Hz, 9H), 1.41–1.35 (m, 9H), 1.24 (d, J = 13.4 Hz, 10H), 1.05 (d, J = 2.3 Hz, 9H), 0.92–0.82 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 171.9, 169.2, 169.1, 165.1, 155.3, 141.0, 136.5, 135.7 (2C), 135.6 (2C), 132.8, 132.7, 130.0, 130.0, 129.5 (2C), 128.5 (2C), 127.9 (4C), 126.9, 124.6, 82.4, 81.6, 79.8, 77.0, 64.9, 59.1, 54.8, 52.0, 42.2, 37.8, 33.5 (2C), 31.9, 29.5, 28.3 (3C), 28.2 (3C), 26.9 (4C), 22.7, 19.3, 14.3, 14.2. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₅₃H₇₇N₃O₁₀SiNa⁺ 966.5271, found 966.5268.

(2S,3S,4R)-2-Methoxy-4-methyl-1-oxo-1-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)amino)decan-3-yl (tert-butoxycarbonyl)-l-alaninate (4i). The titled compound 4i was obtained following the general procedure described for 4h. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4i (965 mg, 82% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (ddt, J = 8.4, 7.0, 1.6 Hz, 4H), 7.39 (dddd, J = 12.5, 8.5, 5.9, 2.4 Hz, 6H), 7.14 (d, J = 8.1 Hz, 1H), 6.81 (dd, J = 15.4, 5.9 Hz, 1H), 6.20 (t, J = 5.1 Hz, 1H), 5.95 (dd, J = 15.4, 1.5 Hz, 1H), 5.13 (t, J = 5.4 Hz, 2H), 4.57–4.48 (m, 1H), 4.24 (p, J = 8.5, 7.9 Hz, 1H), 3.99 (qd, J = 18.5, 5.1 Hz, 2H), 3.84–3.71 (m, 3H), 3.44 (s, 3H), 2.02 (dd, J = 12.5, 6.2 Hz, 1H), 1.44 (d, J = 18.3 Hz, 18H), 1.31–1.19 (m, 13H), 1.05 (s, 9H), 0.91 (d, J = 6.8 Hz, 3H), 0.84 (t, J = 6.7 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 173.0, 169.2, 169.2, 165.1, 155.2, 141.1, 135.7 (2C), 135.6 (2C), 132.8, 132.7, 130.0 (2C), 128.0 (4C), 124.6, 82.5, 81.5, 79.7, 76.5, 65.0, 59.1, 51.7, 49.4, 42.2, 33.4, 33.4, 31.9, 29.5, 28.4 (3C), 28.1 (3C), 26.9 (4C), 22.7, 19.3, 18.4, 14.3, 14.2. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₇H₇₃N₃O₁₀SiNa⁺ 890.4958, found 590.4952.

(2S,3S,4R)-2-Methoxy-4-methyl-1-oxo-1-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)amino)decan-3-yl (S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoate (4j). The titled compound 4j was obtained following the general procedure described for 4h. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4j (1.02 g, 81% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (tt, J = 7.7, 1.6 Hz, 4H), 7.46–7.33 (m, 6H), 7.21 (d, J = 8.2 Hz, 1H), 6.82 (dd, J = 15.4, 6.1 Hz, 1H), 6.11 (t, J = 5.0 Hz, 1H), 5.97 (dd, J = 15.3, 1.5 Hz, 1H), 5.22 (t, J = 4.8 Hz, 1H), 5.10 (d, J = 9.9 Hz, 1H), 4.57 (s, 1H), 4.08 (d, J = 9.9 Hz, 1H), 3.99 (d, J = 5.1 Hz, 2H), 3.88 (d, J = 4.4 Hz, 1H), 3.78 (q, J = 5.4, 4.4 Hz, 2H), 3.47 (s, 3H), 1.99 (s, 1H), 1.47 (s, 9H), 1.41 (s, 9H), 1.27–1.18 (m, 10H), 1.06 (s, 9H), 0.93 (d, J = 18.1 Hz, 12H), 0.84 (t, J = 6.7 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 171.8, 169.1, 168.9, 165.1, 155.7, 141.4, 135.8 (2C), 135.7 (2C), 132.9, 132.7, 130.1, 130.0, 128.0 (2C), 128.0 (2C), 124.7, 82.5, 82.4, 79.8, 77.2, 65.1, 62.2, 59.3, 51.6, 42.3, 34.4, 33.8, 33.7, 31.9, 29.6, 28.4 (3C), 28.2 (3C), 26.9((3C), 26.8, 26.6 (3C), 22.7, 19.4, 14.9, 14.2. HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₀H₈₀N₃O₁₀Si⁺ 910.5608, found 910.5605.

(2S,3S,4R)-2-Methoxy-4-methyl-1-oxo-1-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)amino)decan-3-yl (tert-butoxycarbonyl)-l-leucinate (4k). The titled compound 4k was obtained following the general procedure described for 4h. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4k (802 mg, 86% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.68–7.58 (m, 4H), 7.39 (tt, J = 8.8, 4.6 Hz, 6H), 7.12 (d, J = 8.1 Hz, 1H), 6.81 (dd, J = 15.4, 5.8 Hz, 1H), 6.18 (t, J = 5.1 Hz, 1H), 5.96 (dd, J = 15.5, 1.3 Hz, 1H), 5.16 (t, J = 5.2 Hz, 1H), 4.98 (d, J = 8.9 Hz, 1H), 4.62–4.47 (m, 1H), 4.25 (td, J = 9.3, 4.7 Hz, 1H), 4.00 (qd, J = 18.5, 5.0 Hz, 2H), 3.79 (dtd, J = 15.0, 10.4, 4.5 Hz, 3H), 3.45 (s, 3H), 1.99–1.94 (m, 1H), 1.74–1.55 (m, 3H), 1.47 (s, 9H), 1.42 (s, 9H), 1.32–1.18 (m, 10H), 1.06 (s, 9H), 0.96–0.80 (m, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 172.9, 169.3, 169.2, 165.1, 155.6, 141.1, 135.7 (2C), 135.7 (2C), 132.9, 132.8, 130.0 (2C), 128.0 (4C), 124.7, 82.5, 82.0, 79.8, 76.6, 65.0, 59.4, 52.5, 51.9, 42.22, 41.2, 33.6, 33.6, 31.9, 30.0, 28.5 (3C), 28.2 (3C), 27.0 (4C), 24.9, 23.0, 22.7, 21.9, 19.4, 14.4, 14.2. HRMS (ESI) m/z: [M + H]⁺ calcd for C₅₀H₈₀N₃O₁₀Si⁺ 910.5608, found 910.5612.

(2S,3S,4R)-1-(((R,E)-5-((2-(tert-Butoxy)-2-oxoethyl)amino)-1-((tert-butyldiphenylsilyl)oxy)-5-oxopent-3-en-2-yl)amino)-2-methoxy-4-methyl-1-oxodecan-3-yl (tert-butoxycarbonyl)glycinate (4l). The titled compound 4l was obtained following the general procedure described for 4h. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4l (985 mg, 79% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (t, J = 7.0 Hz, 4H), 7.50–7.32 (m, 6H), 7.17 (d, J = 8.2 Hz, 1H), 6.81 (dd, J = 15.3, 6.0 Hz, 1H), 6.23 (t, J = 5.2 Hz, 1H), 5.96 (d, J = 15.3 Hz, 1H), 5.11 (q, J = 7.0, 6.3 Hz, 2H), 4.54 (d, J = 8.0 Hz, 1H), 3.98 (dd, J = 5.1, 3.3 Hz, 2H), 3.89–3.69 (m, 5H), 3.43 (s, 3H), 2.00–1.96 (m, 1H), 1.46 (s, 9H), 1.41 (s, 9H), 1.23 (d, J = 8.3 Hz, 9H), 1.05 (s, 10H), 0.90 (d, J = 6.7 Hz, 3H), 0.84 (t, J = 6.6 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.0, 169.3, 169.1, 165.1, 155.7, 141.0, 135.6 (2C), 135.5 (2C), 132.8, 132.7, 129.9 (2C), 127.9 (4C), 124.7, 82.2, 81.3, 79.7, 76.6, 65.0, 59.1, 51.5, 42.5, 42.1, 33.4, 33.2, 31.8, 29.4, 28.3 (3C), 28.0 (3C), 26.8 (3C), 26.8, 22.6, 19.3, 14.2, 14.1. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₆H₇₁N₃O₁₀SiNa⁺ 876.4801, found 876.4797.

(3S,11R,14S,15S,E)-3-Benzyl-11-(((tert-butyldiphenylsilyl)oxy)methyl)-14-methoxy-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (3h). To a solution of 4h (815 mg, 0.863 mmol, 1.0 equiv) in CH₂Cl₂ (7 mL) was added trifluoroacetic acid (1.7 mL) at 0 °C. After stirred for 4 h, the reaction solution was diluted with toluene (7 mL). And the resulting solution was concentrated under reduced pressure. The resulting white solid was dissolved in THF (20 mL), and the solution was added slowly to a suspension of HATU (4.92 g, 12.95 mmol, 15.0 equiv) and DIPEA (4.1 mL, 25.9 mmol, 30.0 equiv) in THF (900 mL) over 18 h at room temperature and then continued to stirred another 12h at room temperature. The solvent was evaporated, diluted with MeOH and EtOAc (v/v = 2:1, 300 mL) and filtered through a short pad of silica gel. The filtrate was concentrated under reduced pressure. The residue was dissolved in EtOAc (300 mL), washed with 1% HCl (80 mL), saturated aqueous solution of NaHCO₃ (60 mL), and brine (60 mL). The organic phase was dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 50:1 to 9:1) to obtain 3h (458 mg, 69% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.45 (dd, J = 9.2, 6.1 Hz, 2H), 7.64–7.59 (m, 4H), 7.44 (ddt, J = 13.5, 10.2, 4.6 Hz, 6H), 7.32–7.16 (m, 5H), 6.73 (dd, J = 15.1, 3.0 Hz, 1H), 5.91 (dd, J = 15.2, 2.3 Hz, 1H), 5.27 (dd, J = 9.7, 1.8 Hz, 1H), 4.78–4.72 (m, 1H), 4.65 (td, J = 9.8, 4.0 Hz, 1H), 3.97–3.88 (m, 2H), 3.64 (d, J = 6.6 Hz, 2H), 3.36 (s, 2H), 3.25 (s, 3H), 3.02 (dd, J = 13.9, 4.1 Hz, 1H), 2.85 (dd, J = 13.8, 10.5 Hz, 1H), 1.89 (q, J = 7.1 Hz, 1H), 1.30–1.21 (m, 10H), 0.98 (d, J = 11.7 Hz, 12H), 0.85 (t, J = 6.7 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.5, 168.3, 167.7, 166.4, 141.3, 136.8, 135.1 (2C), 135.0 (2C), 132.6, 132.6, 129.9, 129.9, 128.9 (2C), 128.2 (2C), 127.9 (4C), 126.6, 119.5, 78.4, 73.6, 65.5, 56.7, 53.5, 51.4, 43.9, 38.7, 33.1, 33.0, 31.2, 28.8, 26.7, 26.6 (3C), 22.1, 18.8, 13.9, 13.7. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₄H₆₀N₃O₇Si⁺ 770.4196, found 770.4194.

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-14-methoxy-3-methyl-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (3i). The titled compound 3i was obtained following the general procedure described for 3h. Flash column chromatography eluent (dichloromethane/methanol = 50:1 to 9:1) to obtain 3i (501 mg, 72% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.46 (dd, J = 8.8, 3.3 Hz, 2H), 7.62 (dd, J = 7.3, 4.0 Hz, 4H), 7.52 (t, J = 6.6 Hz, 1H), 7.44 (qq, J = 8.6, 4.5, 3.0 Hz, 6H), 6.75 (dd, J = 15.1, 3.0 Hz, 1H), 5.90 (dd, J = 15.1, 2.2 Hz, 1H), 5.23 (d, J = 9.6 Hz, 1H), 4.78–4.70 (m, 1H), 4.43 (p, J = 7.4 Hz, 1H), 3.91 (q, J = 7.6, 6.5 Hz, 2H), 3.71–3.52 (m, 3H), 3.24 (s, 3H), 1.88 (q, J = 7.0 Hz, 1H), 1.38–1.17 (m, 13H), 0.98 (d, J = 11.9 Hz, 12H), 0.86 (t, J = 6.7 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.8, 168.0, 167.7, 166.5, 141.2, 135.1 (2C), 135.0 (2C), 132.7, 132.6, 130.0, 130.0, 127.9 (4C), 119.5, 78.4, 73.2, 65.5, 56.7, 51.4, 48.0, 44.0, 33.3, 32.9, 31.1, 28.8, 26.7, 26.6 (3C), 22.0, 19.2, 18.8, 13.9, 13.6. HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₈H₅₆N₃O₇Si⁺ 694.3883, found 694.3881.

(3S,11R,14S,15S,E)-3-(tert-Butyl)-11-(((tert-butyldiphenylsilyl)oxy)methyl)-14-methoxy-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (3j). The titled compound 3j was obtained following the general procedure described for 3h. Flash column chromatography eluent (dichloromethane/methanol = 50:1 to 9:1) to obtain 3j (609 mg, 77% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (d, J = 8.8 Hz, 1H), 8.07 (d, J = 10.2 Hz, 1H), 7.63–7.56 (m, 5H), 7.50–7.41 (m, 6H), 6.72 (dd, J = 15.1, 3.0 Hz, 1H), 5.95 (d, J = 15.2 Hz, 1H), 5.38 (d, J = 9.7 Hz, 1H), 4.71 (d, J = 7.8 Hz, 1H), 4.33 (d, J = 10.1 Hz, 1H), 4.12 (dd, J = 17.5, 5.9 Hz, 1H), 3.91 (d, J = 9.7 Hz, 1H), 3.65 (d, J = 6.6 Hz, 2H), 3.54 (dd, J = 17.5, 7.6 Hz, 1H), 3.24 (s, 3H), 1.87 (q, J = 6.9 Hz, 1H), 1.39–1.20 (m, 8H), 1.12 (d, J = 11.3 Hz, 2H), 1.03–0.90 (m, 21H), 0.85 (t, J = 6.6 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.9, 167.7, 167.5, 166.3, 141.5, 135.1 (2C), 135.0 (2C), 132.6, 132.6, 130.0 (2C), 128.0 (4C), 119.5, 78.0, 73.2, 65.3, 60.2, 56.4, 51.6, 44.0, 34.9, 33.0, 32.9, 31.1, 28.9, 26.75, 26.6 (6C), 22.0, 18.8, 14.0, 13.9. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₁H₆₂N₃O₇Si⁺ 736.4352, found 736.4354.

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-3-isobutyl-14-methoxy-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (3k). The titled compound 3k was obtained following the general procedure described for 3h. Flash column chromatography eluent (dichloromethane/methanol = 50:1 to 9:1) to obtain 3k (442 mg, 74% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.44 (d, J = 9.1 Hz, 1H), 8.35 (d, J = 9.1 Hz, 1H), 7.61 (ddd, J = 7.9, 3.8, 1.7 Hz, 4H), 7.52 (dd, J = 7.5, 5.9 Hz, 1H), 7.49–7.38 (m, 6H), 6.74 (dd, J = 15.1, 3.0 Hz, 1H), 5.89 (dd, J = 15.2, 2.3 Hz, 1H), 5.24 (dd, J = 9.7, 1.8 Hz, 1H), 4.73 (dp, J = 9.3, 3.8 Hz, 1H), 4.44 (td, J = 9.6, 5.0 Hz, 1H), 3.95–3.85 (m, 2H), 3.66–3.53 (m, 3H), 3.23 (s, 3H), 1.86 (q, J = 7.5, 6.9 Hz, 1H), 1.63–1.50 (m, 2H), 1.43 (ddd, J = 13.5, 8.9, 5.1 Hz, 1H), 1.35–1.15 (m, J = 6.4, 5.8 Hz, 10H), 0.99 (s, 9H), 0.92 (dd, J = 20.1, 6.6 Hz, 6H), 0.85 (q, J = 6.6 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.3, 168.3, 167.7, 166.4, 141.4, 135.1 (2C), 135.0 (2C), 132.6, 132.6, 129.9, 129.9, 127.9 (4C), 119.4, 78.3, 73.0, 65.5, 56.7, 51.4, 50.6, 44.0, 42.0, 33.1, 32.9, 31.1, 28.8, 26.6, 26.6 (3C), 24.1, 22.6, 22.0, 21.1, 18.8, 13.9, 13.6. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₁H₆₂N₃O₇Si⁺ 736.4352, found 736.4347.

(11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-14-methoxy-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (3l). The titled compound 3l was obtained following the general procedure described for 3h. Flash column chromatography eluent (dichloromethane/methanol = 50:1 to 9:1) to obtain 3l (533 mg, 83% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.48 (d, J = 8.9 Hz, 1H), 8.40 (t, J = 6.2 Hz, 1H), 7.62 (ddd, J = 8.0, 4.5, 1.6 Hz, 4H), 7.54 (t, J = 6.6 Hz, 1H), 7.43 (dddd, J = 15.8, 8.4, 4.2, 1.9 Hz, 6H), 6.78 (dd, J = 15.1, 3.3 Hz, 1H), 5.99 (dd, J = 15.2, 2.2 Hz, 1H), 5.14 (dd, J = 9.4, 2.1 Hz, 1H), 4.73 (dh, J = 6.2, 3.5 Hz, 1H), 3.99–3.82 (m, 4H), 3.73–3.56 (m, 3H), 3.23 (s, 3H), 1.87 (q, J = 6.5, 5.3 Hz, 1H), 1.31–1.21 (m, 10H), 0.99 (s, 9H), 0.92 (d, J = 6.9 Hz, 3H), 0.89–0.81 (m, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.7, 168.2, 167.7, 166.6, 141.5, 135.1 (2C), 135.1 (2C), 132.7, 132.6, 129.9, 129.9, 127.9 (4C), 120.0, 78.8, 74.2, 65.3, 56.7, 51.6, 44.4, 41.0, 33.6, 33.1, 31.3, 28.9, 26.8, 26.6 (3C), 22.1, 18.8, 13.9, 13.8. HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₇H₅₄N₃O₇Si⁺ 680.3726, found 680.3723.

(3S,14S,15S,E)-3-Benzyl-14-methoxy-11-methylene-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1h). To a solution of compound 3h (312 mg, 1.0 mmol, 1.0 equiv) in THF (10 mL) were added HOAc (72 mmg, 1.2 mmol, 3.0 equiv) and TBAF (1.2 mL, 1 mol/L in THF, 1.2 mmol, 3.0 equiv). The mixture was stirred at room temperature for 24 h, and then concentrated. The residue was purified by column chromatography on silica gel (dichloromethane/methanol = 100:3 to 9:1) to obtain 2h as a white powder.

To a solution of compound 2h (140 mg, 0.263 mmol, 1.0 equiv) in THF (2.48 mL), then triethylamine (80 mg, 0.79 mmol, 3.0 equiv) and ethanesulfonyl chloride (65 mg, 0.658 mmol, 2.5 equiv) were added at 0 °C. After stirred for 2 h, the reaction solution was quenched by addition of water (0.1 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The resulting crude product was dissolved in THF (5.0 mL). To the resulting solution was added DBU (321 mg, 2.11 mmol, 8 equiv) at 20 °C. After stirred for 2.5 h, the solution was dissolved in ethyl acetate (80 mL) and washed successively with 1% HCl (3 mL), saturated aqueous NaHCO₃ (3 mL), brine (3 mL), dried over Na₂SO₄ and concentrated under reduced pressure carefully. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100: 1 to 100: 6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1h (82 mg, 57% for 3 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.29 (s, 1H), 8.57 (d, J = 9.5 Hz, 1H), 7.56 (dd, J = 7.6, 5.3 Hz, 1H), 7.30–7.15 (m, 5H), 6.85 (d, J = 15.3 Hz, 1H), 6.22 (dd, J = 15.3, 3.2 Hz, 1H), 5.61 (s, 1H), 5.44 (s, 1H), 5.15 (dd, J = 8.5, 3.2 Hz, 1H), 4.68 (td, J = 9.8, 4.3 Hz, 1H), 4.19 (d, J = 8.9 Hz, 1H), 4.05 (dd, J = 17.8, 5.0 Hz, 1H), 3.35 (d, J = 3.1 Hz, 1H), 3.30 (s, 3H), 3.03 (dd, J = 13.7, 4.4 Hz, 1H), 2.85 (dd, J = 13.7, 10.1 Hz, 1H), 1.93 (s, 1H), 1.42–1.21 (m, 10H), 0.94 (d, J = 7.1 Hz, 3H), 0.85 (t, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.6, 168.5, 168.3, 167.0, 138.0, 137.4, 136.8, 129.0 (2C), 128.1 (2C), 126.6, 119.3, 116.0, 78.9, 74.7, 56.9, 53.5, 44.1, 38.8, 33.1, 32.7, 31.2, 28.8, 26.5, 22.0, 13.9, 13.8. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₈H₄₀N₃O₆Si⁺ 514.2912, found 514.2907.

(3S,14S,15S,E)-14-Methoxy-3-methyl-11-methylene-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1i). The titled compound 1i was obtained following the general procedure described for 1h. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100: 1 to 100: 6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1i (115 mg, 51% for 3 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.14 (s, 1H), 8.45 (d, J = 8.8 Hz, 1H), 7.65 (dd, J = 7.7, 5.2 Hz, 1H), 6.90 (d, J = 15.2 Hz, 1H), 6.06 (d, J = 15.2 Hz, 1H), 5.57 (s, 1H), 5.48 (s, 1H), 5.14 (dd, J = 8.4, 3.3 Hz, 1H), 4.56–4.44 (m, 1H), 4.06–3.94 (m, 1H), 3.89 (dd, J = 17.4, 5.2 Hz, 1H), 3.57 (dd, J = 17.4, 7.7 Hz, 1H), 3.30 (s, 3H), 1.95–1.90 (m, 1H), 1.33–1.19 (m, 13H), 0.94 (d, J = 6.8 Hz, 3H), 0.86 (t, J = 6.9 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.0, 168.2, 168.1, 167.1, 138.1, 137.3, 119.1, 116.6, 79.1, 74.3, 57.0, 47.8, 44.3, 33.3, 32.7, 31.2, 28.9, 26.5, 22.1, 19.4, 14.0, 13.8. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₂H₃₆N₃O₆⁺ 438.2599, found 438.2595.

(3S,14S,15S,E)-3-(tert-Butyl)-14-methoxy-11-methylene-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1j). The titled compound 1j was obtained following the general procedure described for 1h. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100: 1 to 100: 6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1j (57 mg, 49% for 3 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.23 (s, 1H), 8.11 (d, J = 10.3 Hz, 1H), 7.73–7.63 (m, 1H), 6.92 (d, J = 15.2 Hz, 1H), 6.11 (d, J = 15.2 Hz, 1H), 5.52 (d, J = 13.8 Hz, 2H), 5.29 (dd, J = 9.2, 2.2 Hz, 1H), 4.37 (d, J = 10.3 Hz, 1H), 4.13 (dd, J = 17.0, 5.7 Hz, 1H), 4.02 (d, J = 8.3 Hz, 1H), 3.55 (dd, J = 17.2, 7.7 Hz, 1H), 3.30 (s, 3H), 2.00–1.82 (m, 1H), 1.58 (dddd, J = 34.8, 12.4, 8.8, 6.6 Hz, 2H), 1.35–1.22 (m, 8H), 0.98–0.90 (m, 12H), 0.88–0.83 (m, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.1, 167.9, 167.8, 166.7, 138.2, 137.7, 118.8, 117.1, 78.5, 73.9, 60.2, 56.5, 44.2, 35.0, 33.1, 32.7, 31.1, 28.9, 26.7, 26.6 (3C), 22.0, 14.1, 13.9. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₅H₄₂N₃O₆⁺ 480.3069, found 480.3065.

(3S,14S,15S,E)-3-Isobutyl-14-methoxy-11-methylene-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1k). The titled compound 1k was obtained following the general procedure described for 1h. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100: 1 to 100: 6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1k (105 mg, 51% for 3 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.21 (s, 1H), 8.37 (d, J = 9.4 Hz, 1H), 7.63 (dd, J = 7.7, 5.3 Hz, 1H), 6.88 (d, J = 15.2 Hz, 1H), 6.06 (d, J = 15.2 Hz, 1H), 5.59 (s, 1H), 5.47 (s, 1H), 5.15 (dd, J = 8.8, 2.9 Hz, 1H), 4.50 (td, J = 9.6, 4.9 Hz, 1H), 4.00 (d, J = 8.8 Hz, 1H), 3.91 (dd, J = 17.4, 5.3 Hz, 1H), 3.59 (dd, J = 17.4, 7.8 Hz, 1H), 3.30 (s, 3H), 1.92 (d, J = 7.2 Hz, 1H), 1.61–1.50 (m, 2H), 1.45 (dq, J = 9.8, 5.6 Hz, 1H), 1.25 (d, J = 17.1 Hz, 10H), 0.94 (d, J = 6.8 Hz, 3H), 0.92–0.78 (m, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.5, 168.5, 168.0, 167.0, 138.0, 137.2, 119.0, 116.3, 78.9, 73.9, 56.8, 50.5, 44.3, 42.2, 33.2, 32.7, 31.1, 28.8, 26.5, 24.1, 22.6, 22.1, 21.1, 13.9, 13.7. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₅H₄₂N₃O₆⁺ 480.3069, found 480.3063.

(14S,15S,E)-14-Methoxy-11-methylene-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1l). The titled compound 1l was obtained following the general procedure described for 1h. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100: 1 to 100: 6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1l (100 mg, 43% for 3 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (s, 1H), 8.48 (dd, J = 7.1, 5.1 Hz, 1H), 7.66 (q, J = 6.1 Hz, 1H), 6.93 (d, J = 15.2 Hz, 1H), 6.11 (d, J = 15.3 Hz, 1H), 5.52 (d, J = 7.7 Hz, 2H), 5.13 (dd, J = 6.8, 5.3 Hz, 1H), 4.02 (dd, J = 10.5, 6.9 Hz, 2H), 3.87 (ddd, J = 36.4, 17.4, 5.4 Hz, 2H), 3.61 (dd, J = 17.5, 7.1 Hz, 1H), 3.31 (s, 3H), 1.90–1.80 (m, 1H), 1.26–1.21 (m, 10H), 0.86 (dt, J = 7.2, 3.4 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.0, 168.7, 167.8, 167.5, 138.2, 137.5, 119.8, 116.9, 79.7, 75.3, 57.1, 44.6, 41.0, 33.4, 32.7, 31.2, 28.9, 26.3, 22.1, 14.2, 13.9. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₁H₃₄N₃O₆⁺ 424.2443, found 424.2437.

Allyl (2S,3S,4R)-3-hydroxy-2-methoxy-4-methyldecanoate (13). To a solution of compound 8c (1.0 g, 3.10 mmol, 1.0 equiv) in MeOH (30 mL) was added Pd/C (10%, 100 mg) under N₂. The suspension was degassed under vacuum and purged with H₂ three times. The mixture was stirred under H₂ balloon at 20 °C for 5 h. The resulting mixture was filtered through a short pad of silica gel, washed with MeOH (2 × 30 mL) and concentrated under reduced pressure. The crude product was used directly for the next step without further purification.

The above colorless oil was dissolved in DMF (30 mL) was added K₂CO₃ (857.0 mg, 6.2 mmol, 2.0 equiv) and allyl bromide (402.0 μL, 4.65 mmol, 1.5 equiv) at room temperature. The mixture was stirred at room temperature for 10 h, and then H₂O (10 mL) was added, and the resultant mixture was extracted with ethyl acetate (3 × 50 mL). The organic phase was washed by brine (4 × 10 mL). Dried over MgSO₄, filtered and concentrated under reduced pressure. The residue was purified with column chromatography on silica gel (petroleum ether: ethyl acetate = 10:1 to 6:1) to afford compound 13 (810.0 mg, 96% for 2 steps) as a colorless oil. ¹H NMR (400 MHz,CDCl₃) δ 5.93 (ddt, J = 16.6, 10.4, 5.9 Hz, 1H), 5.35 (dq, J = 17.2, 1.6 Hz, 1H), 5.25 (dd, J = 10.6, 1.5 Hz, 1H), 4.68 (d, J = 5.6 Hz, 2H), 3.81 (d, J = 6.5 Hz, 1H), 3.75 (t, J = 4.9 Hz, 1H), 3.40 (s, 3H), 2.25 (d, J = 5.1 Hz, 1H), 1.76–1.67 (m, 1H), 1.42–1.35 (m, 1H), 1.27–1.22 (m, 9H), 0.89 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.9 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 171.3, 131.7, 118.9, 82.3, 74.6, 65.6, 58.5, 34.2, 33.6, 31.8, 29.5, 27.0, 22.6, 14.0, 13.4. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₁₅H₂₈O₄Na⁺ 295.1880, found 295.1878.

Allyl (2S,3S,4R)-3-(((S)-2-((tert-butoxycarbonyl)amino)pent-4-ynoyl)oxy)-2-methoxy-4-methyldecanoate (6m). The titled compound 6m was obtained following the general procedure described for 6a. Flash column chromatography eluent (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 6m (1.21 g, 88%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.85 (ddt, J = 16.5, 9.7, 5.9 Hz, 1H), 5.32–5.23 (m, 2H), 5.18 (d, J = 10.3 Hz, 1H), 5.09 (t, J = 5.3 Hz, 1H), 4.57 (d, J = 5.9 Hz, 2H), 4.33 (dt, J = 8.9, 4.8 Hz, 1H), 3.85 (d, J = 5.9 Hz, 1H), 3.31 (s, 3H), 2.64 (dd, J = 5.1, 2.7 Hz, 2H), 1.98 (d, J = 2.6 Hz, 1H), 1.84 (dd, J = 9.2, 4.8 Hz, 1H), 1.36 (s, 9H), 1.29–1.11 (m, 9H), 1.09–1.02 (m, 1H), 0.84 (d, J = 6.6 Hz, 3H), 0.81–0.74 (m, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 169.8 (2C), 155.0, 131.6, 119.1, 80.3, 79.9, 78.7, 76.7, 71.7, 65.9, 58.5, 51.8, 33.3, 33.1, 31.7, 29.2, 28.2 (3C), 26.8, 22.5, 22.4, 14.2, 14.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₅H₄₁NO₇Na⁺ 490.2776, found 490.2775.

(2S,3S,4R)-2-Methoxy-4-methyl-1-oxo-1-(((R,E)-2,2,14,14-tetramethyl-9,12-dioxo-3,3-diphenyl-4,13-dioxa-10-aza-3-silapentadec-7-en-6-yl)amino)decan-3-yl (S)-2-((tert-butoxycarbonyl)amino)pent-4-ynoate (4m). To a solution of compound 6m (1.21 g, 2.59 mmol, 1.0 equiv) in THF (25 mL) was added N-methylaniline (555.1 mg, 5.18 mmol, 2.0 equiv) and Pd(PPh₃)₄ (448 mg, 0.388 mmol, 0.15 equiv) under N₂. After stirred for 3.0 h, the solution was dissolved in ethyl acetate (80 mL) and washed successively with 1% HCl (10 mL), brine (10 mL), dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was used directly for the next step without further purification.

To a solution of above acid and 5 (1.88 g, 3.89 mmol, 1.5 equiv) in CH₂Cl₂ (25 mL) was added EDCI (596 mg, 3.11 mmol, 1.2 equiv) and HOBt (385 mg, 2.85 mmol, 1.1 equiv) under argon atmosphere at 20 °C. The reaction mixture was stirred for 12 h, and then quenched with 1% HCl (100 mL). The aqueous phase was extracted with ethyl acetate (2 × 80 mL). The combined organic phases were dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 9:1 to 5:1) to obtain 4m (1.49 g, 64% for 2 steps) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (ddd, J = 8.3, 5.0, 1.7 Hz, 4H), 7.45–7.33 (m, 6H), 7.21 (d, J = 8.3 Hz, 1H), 6.80 (dd, J = 15.4, 6.1 Hz, 1H), 6.18 (t, J = 5.1 Hz, 1H), 5.95 (dd, J = 15.4, 1.5 Hz, 1H), 5.37 (d, J = 8.4 Hz, 1H), 5.20 (t, J = 5.2 Hz, 1H), 4.63–4.58 (m, 1H), 4.39 (dt, J = 9.0, 5.0 Hz, 1H), 3.98 (dd, J = 5.1, 2.4 Hz, 2H), 3.84 (d, J = 5.2 Hz, 1H), 3.76 (d, J = 4.4 Hz, 2H), 3.45 (s, 3H), 2.76–2.61 (m, 2H), 2.00–1.94 (m, 2H), 1.46 (s, 9H), 1.43 (s, 9H), 1.30–1.16 (m, 10H), 1.05 (s, 9H), 0.92 (d, J = 6.8 Hz, 3H), 0.83 (t, J = 6.6 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.1, 169.1, 169.0, 165.0, 155.2, 141.1, 135.6 (4C), 132.8, 132.7, 130.0 (2C), 127.9 (4C), 124.7, 82.4, 81.8, 80.1, 79.2, 77.4, 71.8, 65.1, 59.2, 52.1, 51.5, 42.2, 33.5, 33.3, 31.8, 29.5, 28.4 (3C), 28.1 (3C), 26.9 (3C), 26.8, 22.7, 22.4, 19.3, 14.6, 14.1. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₉H₇₃N₃O₁₀SiNa⁺ 914.4958, found 914.4954.

(3S,11R,14S,15S,E)-11-(((tert-Butyldiphenylsilyl)oxy)methyl)-14-methoxy-15-((R)-octan-2-yl)-3-(prop-2-yn-1-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (3m). The titled compound 3m was obtained following the general procedure described for 3a. Flash column chromatography eluent (dichloromethane/methanol = 50:1 to 9:1) to obtain 3m (744 mg, 62% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.57 (d, J = 9.1 Hz, 1H), 8.46 (d, J = 9.0 Hz, 1H), 7.61 (ddt, J = 6.0, 4.1, 1.7 Hz, 5H), 7.44 (qdd, J = 8.4, 3.3, 1.8 Hz, 6H), 6.74 (dd, J = 15.1, 3.0 Hz, 1H), 5.94 (dd, J = 15.1, 2.3 Hz, 1H), 5.20 (dd, J = 9.7, 1.8 Hz, 1H), 4.73 (ddt, J = 9.4, 6.5, 3.7 Hz, 1H), 4.55 (dt, J = 9.2, 6.6 Hz, 1H), 4.01 (dd, J = 17.6, 5.8 Hz, 1H), 3.92 (d, J = 9.7 Hz, 1H), 3.63 (d, J = 6.2 Hz, 2H), 3.56 (dd, J = 17.6, 7.8 Hz, 1H), 3.23 (s, 3H), 2.99 (t, J = 2.6 Hz, 1H), 2.65–2.52 (m, 2H), 1.87 (q, J = 6.8 Hz, 1H), 1.26 (d, J = 22.4 Hz, 10H), 0.99 (s, 9H), 0.96 (d, J = 7.0 Hz, 3H), 0.86 (t, J = 6.6 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.6, 168.4, 167.8, 166.5, 141.5, 135.1 (4C), 132.7, 132.6, 130.0 (2C), 127.9 (4C), 119.5, 79.5, 78.4, 74.1, 74.0, 65.5, 56.7, 51.4, 51.2, 44.1, 33.1, 32.9, 31.3, 28.9, 26.8, 26.6 (3C), 23.1, 22.1, 18.8, 14.0, 13.7. HRMS (ESI) m/z: [M + H]⁺ calcd for C₄₀H₅₆N₃O₇Si⁺ 715.3883, found 715.3879.

(3S,14S,15S,E)-14-Methoxy-11-methylene-15-((R)-octan-2-yl)-3-(prop-2-yn-1-yl)-1-oxa-4,7,12-triazacyclopentadec-9-ene-2,5,8,13-tetraone (1m). The titled compound 1m was obtained following the general procedure described for 1a. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol = 100: 1 to 100: 6), t-BuOH (3 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 1m (132 mg, 60% for 2 steps) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.13 (s, 1H), 8.56 (d, J = 9.1 Hz, 1H), 7.70 (dd, J = 7.6, 5.1 Hz, 1H), 6.89 (d, J = 15.2 Hz, 1H), 6.14 (d, J = 15.2 Hz, 1H), 5.60 (s, 1H), 5.47 (s, 1H), 5.13 (dd, J = 8.2, 3.4 Hz, 1H), 4.61 (dt, J = 8.7, 6.5 Hz, 1H), 4.09–3.98 (m, 2H), 3.56 (dd, J = 17.2, 7.8 Hz, 1H), 3.30 (s, 3H), 2.95 (d, J = 2.4 Hz, 1H), 2.59 (m, J = 6.6, 2.9 Hz, 2H), 1.92 (m, 1H), 1.40–1.09 (m, 10H), 0.94 (d, J = 7.0 Hz, 3H), 0.86 (t, J = 6.6 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.8, 168.5, 168.1, 167.1, 138.3, 137.3, 119.0, 116.3, 79.6, 79.1, 75.2, 73.8, 57.0, 51.1, 44.3, 33.1, 32.6, 31.2, 28.8, 26.6, 23.0, 22.1, 14.0, 13.9. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₄H₃₅N₃O₆Na⁺ 484.2419, found 484.2421.

N-(2-(2-(2-(2-(4-(((3S,14S,15S,E)-14-Methoxy-11-methylene-15-((R)-octan-2-yl)-2,5,8,13-tetraoxo-1-oxa-4,7,12-triazacyclo-pentadec-9-en-3-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazole-4-yl)pentanamide (15). To a solution of compound 1m (17.8 mg, 0.039 mmol, 1.0 equiv) in MeCN: MeOH = 2: 1 (3.0 mL in total) were added commercially available 14 (20.6 mg, 0.046 mmol, 1.2 equiv) and Cu₂O-NP (4.0 mg; for the synthesis, see Adv Synth Catal 2010;352:1600–4). The reaction mixture was stirred at RT for 24 h. The mixture was filtered through a pad of silica gel and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel chromatography (dichloromethane/methanol = 50:1 to 9:1) to obtain 15 (29 mg, 84%) as a white powder. ¹H NMR (400 MHz, DMSO-d₆) δ 9.25 (s, 1H), 8.64 (d, J = 9.2 Hz, 1H), 7.84 (s, 2H), 7.63 (t, J = 6.4 Hz, 1H), 6.88 (d, J = 15.2 Hz, 1H), 6.43 (s, 1H), 6.36 (s, 1H), 6.16 (d, J = 15.2 Hz, 1H), 5.60 (s, 1H), 5.46 (s, 1H), 5.14 (dd, J = 8.4, 3.3 Hz, 1H), 4.70 (td, J = 9.4, 4.7 Hz, 1H), 4.46 (t, J = 5.2 Hz, 2H), 4.30 (dd, J = 7.8, 5.0 Hz, 1H), 4.16–4.10 (m, 1H), 4.10–4.07 (m, 1H), 3.99 (dd, J = 17.3, 5.2 Hz, 1H), 3.76 (t, J = 5.2 Hz, 2H), 3.49 (d, J = 4.6 Hz, 9H), 3.38 (t, J = 6.0 Hz, 2H), 3.30 (s, 3H), 3.17 (q, J = 5.8 Hz, 2H), 3.08 (dt, J = 14.5, 4.4 Hz, 2H), 2.98 (dd, J = 14.7, 9.6 Hz, 1H), 2.81 (dd, J = 12.4, 5.0 Hz, 1H), 2.57 (d, J = 12.5 Hz, 1H), 2.06 (t, J = 7.4 Hz, 2H), 1.92 (s, 1H), 1.65–1.40 (m, 4H), 1.36–1.18 (m, 12H), 0.92 (d, J = 6.7 Hz, 3H), 0.85 (t, J = 6.9 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.1, 169.5, 168.6, 168.2, 167.1, 162.7, 142.1, 138.2, 137.4, 123.3, 119.1, 116.2, 79.0, 74.7, 69.7, 69.6 (2C), 69.5, 69.2, 68.8, 61.0, 59.2, 56.9, 55.4, 52.2, 49.3, 44.2, 40.2, 39.9, 38.4, 35.1, 33.1, 32.7, 31.2, 28.8, 28.2, 28.0, 26.5, 25.3, 22.1, 14.0, 13.8. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₂H₆₇N₆O₁₁SNa⁺ 928.4573, found 928.4571.

(3S,14S,15S,Z)-3-(tert-Butyl)-14-methoxy-11-((4-methylpiperazin-1-yl)methyl)-15-((R)-octan-2-yl)-1-oxa-4,7,12-triazacyclopentadec-10-ene-2,5,8,13-tetraone (16). To a solution of compound 2j (825.0 mg, 1.66 mmol, 1.0 equiv) in THF (6.2 mL), then triethylamine (336 mg, 3.32 mmol, 2.0 equiv) and ethanesulfonyl chloride (320 mg, 2.49 mmol, 1.5 equiv) were added at 0 °C. After stirred for 2 h, the reaction solution was quenched by addition of water (0.1 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure. The resulting crude product was dissolved in THF (6.2 mL). To the resulting solution was added N-methyl piperazine (1.84 mL, 16.6 mmol, 10 equiv) at 20 °C. After stirred for 4 h, the solution was dissolved in ethyl acetate (80 mL) and washed successively with saturated aqueous NaHCO₃ (30 mL) and brine (30 mL), dried over Na₂SO₄ and concentrated under reduced pressure carefully. The crude product was purified by column chromatography on silica gel dichloromethane/methanol = 100: 1 to 8: 1), t-BuOH (10 mL) was added to the eluent and concentrated to remove CH₂Cl₂ and MeOH. The residual solution was lyophilized to give 16 (452 mg, 47% for 2 steps) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.29 (s, 1H), 7.36 (d, J = 9.8 Hz, 1H), 6.46 (s, 1H), 5.37 (t, J = 8.0 Hz, 1H), 5.11 (t, J = 6.2 Hz, 1H), 4.52 (dd, J = 13.6, 9.2 Hz, 2H), 3.86 (d, J = 5.7 Hz, 1H), 3.52 (dd, J = 17.3, 7.9 Hz, 1H), 3.46 (s, 3H), 3.38 (dd, J = 15.4, 4.3 Hz, 1H), 3.13–3.00 (m, 2H), 2.94 (dd, J = 17.2, 8.2 Hz, 1H), 2.47 (s, 8H), 2.30 (s, 3H), 2.18 (p, J = 6.6 Hz, 1H), 1.57–1.47 (m, 1H), 1.36–1.20 (m, 9H), 1.02 (s, 9H), 0.94 (d, J = 6.8 Hz, 3H), 0.89–0.82 (m, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 171.8, 169.5, 169.2, 167.5, 135.5, 113.4, 80.4, 76.1, 62.9, 61.5, 58.8, 55.3 (2C), 52.7 (2C), 46.0, 44.6, 36.0, 35.5, 33.5, 33.3, 31.9, 29.6, 26.9 (3C), 26.7, 22.7, 15.6, 14.2. HRMS (ESI) m/z: [M + H]⁺ calcd for C₃₀H₅₄N₅O₆⁺ 580.4069, found 580.4071.

4.2. Cell culture

MDA-MB-231, Huh-7, U118, A549, 4T1 and 3T3 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and penicillin (100 μg/mL), streptomycin (100 μg/mL) at 37 °C including 5% CO₂.

4.3. MTT assay

Cancer cells including MDA-MB-231, Huh-7, U118 and A549 were seeded into 96 well plate individually. After cell attachment, boholamide A derivatives were added with different concentrations and incubated for 72 h. Then 20 μL MTT solution (5 mg/mL) was added and incubated with additional 4 h. After that the supernatant was discarded, the precipitate was dissolved with DMSO and the absorbance at 570 nm was measured. The IC₅₀ was calculated with GraphPad Prism 5 and repeated for 3 times.

4.4. Colony formation assay

MDA-MB-231 cells were seeded into 24 well plate with 200 cells per well. After cell attachment, compounds were added with different concentrations and treated for 7–10 days. The cells were fixed with 4% paraformaldehyde for 20 min, then stained with crystal violet for 30 min. After that the number of clones were counted.

4.5. Cell apoptosis assay

MDA-MB-231 cells in logarithmic growth phase were digested, counted and seeded into 24 well plate. Compounds were added with different concentrations and treated for 48 h. Then the cells were collected, washed with PBS buffer and resuspended with binding buffer. After that, cells were stained with 5 μL Annexin Ⅴ-FITC and 5 μL propidium iodide (PI) for 15 min at room temperature in the dark. Then the cells were analyzed by flow cytometry.

4.6. Trans-well assay

MDA-MB-231 cells treated with compounds for 48 h were collected and counted. Then 3 × 10⁴ cells were resuspended with serum-free medium and seeded into the upper chambers. The lower layer was supplied with culture containing 20% fetal bovine serum. After 48 h, cells in upper chamber were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet for 30 min. Then cells on the inside bottom of the upper chamber were erased with a cotton swab, and the migration cells on the outside bottom were calculated under a microscope.

4.7. Transfection

The CDS regions of eEF1A1 and FoxO1 genes were cloned into the pCMV plasmid respectively. Then the cells were prepared and transfected with corresponding plasmid using Lipofectamine 3000 Transfection Reagent.

4.8. Western blot assay

MDA-MB-231 cells were collected and lysed with RIPA buffer. Proteins in supernatant were collected, boiled with loading buffer, separated by SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies (1:1000) and subsequently secondary antibodies (1:5000). Then the membranes were washed with PBST buffer and detected with electrochemiluminescence (ECL) reagent.

4.9. Pull-down assay

MDA-MB-231 cell lysates were collected and incubated with probe 15 for 2 h at 4 °C. The protein was precipitated with methanol for 40 min at −20 °C. After centrifugation, then the precipitated proteins were collected, dissolved with PBS containing 0.1% SDS buffer and incubated with streptavidin beads for overnight at 4 °C. After that the streptavidin beads were washed for five times with PBS buffer, and the bead-bound proteins were eluted, separated by SDS-PAGE, and visualized by silver staining or Western blot assay.

4.10. Kinetic determination of eEF1A1 binding

MDA-MB-231 cell lysate was incubated with probe 15 at different concentrations for 2 h. Then the pull-down assay was performed and the precipitated proteins were analyzed by Western blot. The gray value of eEF1A1 protein band was analyzed by Image J. The K_i value was obtained through nonlinear curve fitting. Then MDA-MB-231 cell lysate was incubated with probe 15 at 10 μmol/L for different times. In the following, the pull-down assay was performed and the precipitated proteins were analyzed by Western blot. The gray value of eEF1A1 protein band was analyzed by Image J. And K_obs in binding eEF1A1 was calculated by Eq. (1):

[eEF1A1]t = [ eEF1A1]0 × E‒ K_obs × t (1)

where [eEF1A1]0 means the total concentration of input-eEF1A1, and [eEF1A1]t means the concentration of eEF1A1 at time t.

4.11. Molecular docking assay

The crystal structure of eEF1A1 (PDB ID: 6ZMO) was retrieved from the RCSB Protein Data Bank. The three-dimensional structure of compound 1j was generated using ChemDraw software and subsequently energy-minimized to adopt its lowest-energy conformation. The optimal binding pose of the ligand was selected based on the highest docking score.

4.12. Protein purification

pET28a-eEF1A1 and pET28a-eEF1A1 mutation plasmids were transformed into E. coli strain BL21 respectively. Then E. coli strain BL21 were cultured with LB medium supplement kanamycin at 37 °C. After the OD value at 600 nm reached 0.5–0.8, IPTG (0.5 mmol/L) was added and cultured at 16 °C for another 16–20 h. Then the bacterial cells were collected after centrifuged at 5000 rpm for 30 min, lysed with lysis buffer and cracked with ultrasonic crusher. Then the protein supernatant was obtained, Ni-beads column was used to bind His tagged eEF1A1 protein. After washed with wash buffer, eEF1A1 protein was eluted with elution buffer. In the following the eEF1A1 protein was desalted and quantified with BCA assay.

4.13. CESTA assay

MDA-MB-231 cultured in hypoxia was collected and lysed with RIPA buffer. After centrifuged at 12,000×g for 15 min, the cell lysate was obtained and incubated with 1j for 1 h at room temperature. Then, the lysates were heated individually for 3 min at different temperatures (35 to 70 °C). After cooling for 5 min on ice, the lysates were centrifuged at 12,000×g for 10 min and the protein supernatant was analyzed by Western blot assay.

4.14. Binding site assay

Recombinant human eEF1A1 protein (50 μg) was incubated with compound 1j at room temperature for 16 h. Then the mixture was separated with SDS-PAGE and the specific eEF1A1 band was excised and digested with trypsin. After that, the binding site was analyzed using Triple-quad Ion-trap and Orbitrap fusion (Thermo Fisher Scientific, USA).

4.15. Co-immunoprecipitation assay

MDA-MB-231 cells transfected with flag-labeled pCMV-eEF1A1 or pCMV-FoxO1 plasmid were collected and lysed with RIPA buffer. After centrifuged at 12,000×g for 15 min at 4 °C, the cell lysate was obtained and incubated with IgG or Flag antibody at 4 °C overnight. Then 20 μL protein A + G Agarose was added and incubated at 4 °C overnight. After washing with 1 × PBS buffer for 5 times, the Agarose was collected and resuspended with 1 × loading buffer. After boiled for 10 min at 100 °C, the samples were developed to Western blot assay.

4.16. Nuclear and cytoplasmic protein extraction

MDA-MB-231 cells were seeded into 6-well plate and incubated with compound 1j for 48 h. Then the cells were collected and the protein were extracted by nuclear and cytoplasmic protein extraction kit according to the manufacturer’s protocol.

4.17. Toxicity assay

Balb/c mice were randomly divided into two groups with 4 mouse each group. Then compound 16 was administrated intraperitoneally at dose of 20, 40 and 80 mg/kg respectively. Then the body weight of mice was recorded every day. After 72 h, the organ weights of mice were measured and the ALT, AST, CR level in plasma were examined using ELISA kit.

4.18. Animal model assay

The protocol and experiment were approved by Ethics of Animal Experiments Committee of Nankai University of China. Bal-b/c mice were purchased from Charles River and feed in a standard SPF mouse room. 4T1 cells (2 × 10⁵ cells) were injected into breast fat pad on the right side. After the tumor formation, the mice were randomly divided into 3 groups and intraperitoneally administered with compound 16 every two days. Body weight and tumor volume were monitored throughout the experiment.

4.19. Immunohistochemical assay

The tumor tissues were fixed with 4% paraformaldehyde. After gradient dehydration, the tissues were embedded in paraffin and subsequently sectioned into 4 μm-thick slices. Then the slides were deparaffinized, hydrated and incubated with primary antibodies including JAK2, STAT3 and pSTAT3 at 4 °C overnight. After washed with PBST for 3 times, the slides were incubated with corresponding secondary antibody for 1 h at room temperature. In the following, the slides were developed by DAB, counterstained with hematoxylin and sealed with neutral balsam.

4.20. H&E staining

The tissues were fixed with 4% paraformaldehyde. After gradient dehydration, the tissues were embedded in paraffin and subsequently sectioned into 4 μm-thick slices. Then the slides were deparaffinized, hydrated and dyed with hematoxylin. After that the slides were immersed in 1% hydrochloric acid alcohol, dyed with eosin, dehydrated and sealed with neutral balsam. Then the slides were observed under microscope.

4.21. Statistical analysis

All the data were analyzed using GraphPad Prism. The differences between groups were analyzed using Student t test. Data are represented as the mean ± SD. The test results were considered statistically significant at ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005, ∗∗∗∗P < 0.001.

Author contributions

Quan Zhang and Yahui Ding designed the research; Guangju Liu, Fengyuan Zhang, Fangzhi Han, Sisi Chen, Junjie Ou, Renfeng Qiao, Yixuan Jiang, Shaojie Miao, Wei Lin, and Yahui Ding performed experiment; Guangju Liu, Fengyuan Zhang, Fangzhi Han, Sisi Chen, Junjie Ou, Yixuan Jiang, Shaojie Miao, Wei Lin, Yahui Ding and Quan Zhang analyzed data; Guangju Liu, Yahui Ding and Quan Zhang wrote the manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Appendix A. Supporting information

The following is the Supporting Information to this article: