Synthesis of 3-O-Acetyl-11-keto-β-boswellic Acid (AKBA)-Derived Amides and Their Mitochondria-Targeted Antitumor Activities.

In this study, we synthesized a series of amide and mitochondria-targeted derivatives with 3-O-acetyl-11-keto-β-boswellic acid (AKBA) as the parent structure and an ethylenediamine moiety as the link chain. Compound 5e, a mitochondrial-targeting potential derivative, showed significantly stronger antitumor activity than that of AKBA, and it could induce vacuolization of A549 cells and stimulate the production of reactive oxygen species (ROS) in a time- and concentration-dependent manner. The antioxidant N-acetylcysteine (NAC) could inhibit the ROS level but could not suppress vacuolization and cell death induced by 5e. Further studies demonstrated that 5e caused abnormal opening of mitochondrial permeability transition pore (MPTP) and a decrease of mitochondrial membrane potential; additionally, it caused cell cycle arrest in G0/G1 but did not induce apoptosis. 5e represented a compound with improved antiproliferative effects for cancer therapy working through new mechanisms.

Introduction

With increasing incidence and mortality, cancer was the second main cause of deaths in the world, after cardiovascular disease, in 2017.[1] Therefore, the search and preparation of anticancer compounds with high efficiency and low toxicity has been a research hotspot in related fields.

Mitochondria are the key regulators of cell death, and many characteristics of tumor cells, including immortal proliferation potential, insensitivity to antigrowth signals, resistance to apoptosis and inhibition of autophagy, are related to mitochondrial dysfunction.[2−6] The mitochondrial membrane potential of tumor cells is much greater than that of normal cells.[7] Based on such difference, some delocalized lipophilic cations (DLCs) such as triphenylphosphine (TPP), can selectively accumulate in tumor cell mitochondria, so that DLCs are expected to carry small-molecule antitumor active compounds to target the tumor cell.[8−10]

Natural products and their derivatives have increasingly attracted the interests of pharmaceutists for their anticancer potential. Pentacyclic triterpenoids, especially betulin and betulinic acid were conjugated with the TPP to enhance cellular and mitochondrial availability. Ye et al. synthesized a series of TPP conjugates of betulin and betulinic acid with a varying length ester linkage at the OH groups, and the concentration of the most potential compound was increased 3.4-fold compared with betulin in mitochondria.[11] Coincidentally, C-28-TPP conjugated derivatives of betulinic acid with the alkyl/alkoxyalkyl linkers of variable length were up to 10–17 times more active against MCF-7 than for human skin fibroblasts.[12]

As an indispensable active ingredient of boswellic acids, AKBA (Figure ) possesses potent antitumor properties.[13−18] However, the structural modifications on AKBA for antitumor research remained relatively less than other triterpenoids (oleanolic acid or maslinic acid), which might be due to its limited availability.[19]

Figure 1

Structure of pentacyclic triterpenoid AKBA.

To enhance the antitumor activity of AKBA, medicinal chemists have made some structural modifications. Most of these chemical modifications mainly focused on the acetoxy group at C-3 position and the carboxylic acid group at C-24 position, as well as ring A (expansions, cleavages or contractions). When the acetoxy group turned into a hydroxyl group or the propionyloxy group, the antitumor activity of the derivative decreased.[20,21] However, the replacement of acetoxy group by β-amino or an electron-withdrawing group at C-2 together with a 3-oxo-1-en structure could improve cytotoxicity.[22,23] On the other side, when the carboxylic acid group turned into a cyano group or ester group, the antitumor activity was attenuated.[21,24] The transformations of A ring did not significantly improve the antitumor activity, but those derivatives bearing two nitrogen-containing substituents showed better antitumor activity than AKBA.[25,26] Coincidentally, most derivatives of glycyrrhetinic, ursolic, and oleanolic acid with ethylenediamine as the link chain on the carboxyl group possess a potent antiproliferative profile.[27] Herein, a series of novel AKBA derivatives with the ethylenediamine as the link chain on the carboxyl group were designed and synthesized.

Results and Discussion

Chemistry

AKBA was purified from commercial boswellic acids’ extract as the starting material. The preparation of all derivatives was described in Scheme . First, after being coupled with N-Boc-ethylenediamine using HATU as the amide coupling reagent, AKBA generated the intermediate compound 1. Second, the Boc group was removed using TFA to furnish the free amine, which was subsequently reacted with corresponding acyl chloride using TEA as the acid-binding agent to obtain target products 2a–h. Then the ester group was hydrolyzed in methanol by NaOH to achieve target products 3a–h. The compounds with free amine were subsequently reacted with carboxylic acids of different carbon chain lengths using HATU as amide coupling reagent to provide 4a–e. Finally, triphenylphosphine and 4a–e in MeCN was stirred at 80 °C for 48 h to yield 5a–e.

Scheme 1

Synthesis of AKBA Derivatives

Reagents and conditions: (a) DIPEA, HATU, NH2CH2CH2NHBoc, DMF, N2, 0°C to rt. (b) TFA, CH2Cl2, 0°C; TEA, RCl, N2, rt. (c) NaOH, H2O, CH3OH, rt. (d) TFA, CH2Cl2, 0°C; DIPEA, HATU, Br(CH2)nCOOH, CH2Cl2, N2, 0°C to rt. (e) PPh3, MeCN, 80°C.

Biological Activity

Antiproliferative Assays

First, the anticancer activities of the above semisynthetic derivatives of AKBA were evaluated against three human cancer cell lines (PC-3, NCI-H460, and A549) and human bronchial epithelioid cells (HBE) using the MTT assay. Doxorubicin was selected as the positive control drug. The results expressed as IC50 values are shown in Table .

Table 1

Effects of AKBA Analogues on Proliferation of Three Cancer Cell Lines and HBE Cellsa

	IC50 (μM)
compound	A549	NCI-H460	HBE	PC-3
1	>30	>30	>30	>30
2a	7.16 ± 0.27b	7.25 ± 0.14b	18.47 ± 2.73b	6.79 ± 0.04b
2b	10.93 ± 0.43b	14.88 ± 0.06b	4.61 ± 0.26b	14.29 ± 0.29b
2c	12.98 ± 1.01b	7.78 ± 0.43b	13.66 ± 1.30b	13.71 ± 0.22b
2d	>30	>30	>30	>30
2e	16.02 ± 1.57b	14.05 ± 1.56b	5.00 ± 0.13b	9.73 ± 0.15b
2f	18.96 ± 1.25b	14.50 ± 0.06b	12.72 ± 2.18b	17.59 ± 2.69b
2g	16.43 ± 0.38b	10.76 ± 0.11b	5.94 ± 0.26b	13.79 ± 0.06b
2h	12.19 ± 1.44b	12.05 ± 0.74b	4.89 ± 0.15b	12.35 ± 1.06b
3a	13.96 ± 0.61b	28.05 ± 0.44	24.92 ± 0.78	23.57 ± 2.99
3b	12.77 ± 0.53b	18.12 ± 2.12b	25.71 ± 0.64	25.76 ± 2.48
3c	>30	14.30 ± 0.06b	11.02 ± 0.66b	14.33 ± 0.22b
3d	>30	>30	13.79 ± 1.43b	20.26 ± 2.32b
3e	20.39 ± 1.66b	4.31 ± 0.15b	5.74 ± 0.52b	14.03 ± 0.22b
3f	28.80 ± 0.06	14.35 ± 0.06b	14.44 ± 0.48b	28.25 ± 0.21
3g	19.52 ± 1.05b	13.34 ± 0.43b	7.86 ± 1.08b	12.49 ± 0.58b
3h	14.80 ± 0.12b	14.07 ± 0.26b	8.28 ± 0.56b	13.78 ± 0.01b
4a	26.77 ± 1.50	11.89 ± 1.06b	8.06 ± 0.15b	>30
4b	9.36 ± 0.15b	9.36 ± 0.21b	4.88 ± 0.31b	10.53 ± 2.29b
4c	9.73 ± 0.14b	9.31 ± 0.39b	13.08 ± 2.67b	9.33 ± 0.17b
4d	16.85 ± 0.85b	15.80 ± 1.61b	22.52 ± 0.99	14.10 ± 3.89b
4e	>30	>30	>30	>30
5a	>30	25.52 ± 0.46	18.31 ± 3.40b	22.17 ± 2.96
5b	>30	14.13 ± 1.91b	>30	10.23 ± 1.22b
5c	>30	>30	>30	21.51 ± 1.80
5d	18.35 ± 3.45b	>30	11.30 ± 0.49b	17.94 ± 2.14b
5e	1.32 ± 0.03b	2.31 ± 0.37b	2.44 ± 0.04b	2.45 ± 0.31b
DOXc	2.23 ± 0.16	3.14 ± 0.57	2.54 ± 0.16	1.13 ± 0.20
AKBA	26.99 ± 0.82	27.19 ± 1.93	26.23 ± 0.37	27.88 ± 0.54

The values are presented as mean ± standard deviations, and cell viability was assessed after incubation for 48 h.

P < 0.001 vs AKBA.

DOX was used as positive agent.

As shown in Table , most derivatives showed preferable cytotoxicity compared with AKBA (IC50 = 26.99 ± 0.82 μM(A549)), especially mitochondrial-targeting derivatives 5e (IC50 = 1.32 ± 0.03 μM(A549)), which showed the most potent antiproliferative activity, up to 20-fold stronger than AKBA. Compound 2a (R = cyclopropanecarbonyl, IC50 = 7.16 ± 0.27 μM(A549) vs IC50 = 18.47 ± 2.73 μM(HBE)) possesses better antitumor activity and selectivity than AKBA. For the most part, in compounds 3a–h, the hydrolysis of acetoxy group at C-3 resulted in a varying degree of decrease in anticancer activity compared with compounds 2a–h. The substituent R could influence the activity, and compound 2/3d (R = 2-propylvaleryl) showed a sharp decrease in antiproliferative activity compared with AKBA. When R was carbon chain acyl group, with the carbon chain becoming longer, the antitumor activity of derivatives increased (IC50(A549): 2f > 2g > 2h; 3f > 3g > 3h). Furthermore, for the mitochondrial-targeting derivatives (5a–e), a longer carbon chain showed better activity among mitochondrial-targeting derivatives, with compound 5e possessing the most potent antiproliferative activity, but an excessively short carbon chain (n = 2, 4, 5) resulted in loss of the activity. However, the intermediates of these mitochondrial-targeting derivatives had a different situation, and 4e with the longest carbon chain almost lost its antitumor activity. In addition, when comparing the cell viability of 5e on A549, it was found that A549 line is relatively more sensitive to 5e than PC-3 and NCI-H460 cell lines. Interestingly, a viability test (Figure ) showed that 5e had different performance on A549 line and human bronchial epithelioid (HBE) cell line. 5e was able to suppress the growth of A549 and HBE in a dose-dependent manner at 0, 0.5, 1, 2, 3, and 4 μM, but equal concentrations (0.5, 1, 2 μM) had a greater growth inhibition ratio in A549 than HBE, especially after 72 h of treatment.

Figure 2

Effects of 5e on cell viability of HBE and A549. A, at 24 h; B, at 48 h, and C, at 72 h. **P < 0.01 vs Control, ***P < 0.001 vs Control.

Vacuolization of A549 Induced by 5e

As shown in Figure , fluorescence inverted microscope images showed compound 5e triggers vacuolization of A549 in time- and dose-dependent manners. The electron microscope images (Figure ) showed mitochondrial damage and endoplasmic reticulum swelling in the treated group. The control group had regular nuclei, rich cytoplasm, oval mitochondria, and abundant endoplasmic reticulum. After the treatment with 3 μM 5e for 12 h, small vacuoles and slight swelling of endoplasmic reticulum appeared in the cytoplasm, and the mitochondrial cristae disappeared. At 24 h after treatment, the cytoplasm was sparse, the vacuoles in the cell cytoplasm became larger, the mitochondria became more swollen and vacuolated, and the endoplasmic reticulum was significantly reduced. In other words, the subcellular structure diagram suggested that 5e treatment was able to cause mitochondrial and endoplasmic reticulum damage (Figure ).

Figure 3

Compound 5e triggered vacuolization in time- and dose-dependent manners. A549 cells were treated with 2 μM 5e for different time (12, 24, and 36 h) or indicated concentrations (1, 2, 3, 4 μM) of 5e for 24 h. Scale bar, 20 μm.

Figure 4

Transmission electron microscopy images of microstructural changes of mitochondria in A549 cells incubated with or without 3 μM 5e. Black arrowheads point to the mitochondria and endoplasmic reticulum. Scale bar in the original and enlarged images indicates 1.2 and 0.6 μm, respectively.

Effect of 5e on Intracellular ROS, Mitochondrial Permeability, and Membrane Potential (ΔΨm)

Mitochondria are the primary organelles producing ROS in cells. Their damage can lead to elevated intracellular ROS, which can damage cellular DNA, proteins, and lipids, and induce tumor cell death.[28] Herin, we evaluated the level of ROS. First, the results (Figure A,B) showed that 5e stimulated the production of ROS in a time- and concentration-dependent manner. Then, we found antioxidant NAC could inhibit the level of ROS but could not remarkably suppress vacuolization and cell death (Figure C,D). However, antioxidant NAC could suppress vacuolization and cell death by inhibiting the level of reactive oxygen species in study of chalcomoracin.[29]

Figure 5

ROS generation in 5e treated A549 cells. (A) ROS generation in cells after 12 h exposed to 5e in a dose-dependent manner. (B) ROS generation in A549 cells exposed to 5e in a time-dependent manner. (C) The effect of NAC on accumulation of ROS and cell viability. ROS generation in A549 cell after 12 h exposed to 2 μM 5e with or without 10 mM NAC. MTT assays tested cell viability after 24 h of being exposed to NAC and 5e. (D) The effect of NAC on accumulation of vacuolization. Pictures of A549 cell treated with 2 μM 5e with or without 10 mM NAC for 12 h. Scale bar, 20 μm.**P < 0.01 vs Control, ***P < 0.001 vs Control, ###P < 0.001 vs Control.

A high concentration of ROS will stimulate the abnormal opening of the MPTP. When the MPTP pore is abnormally opened, CoCl2 can enter the mitochondria and quench the hydrolysis of the green fluorescent Calcein (product of Calcein-AM).[30,31] Therefore, we used this method to explore whether 5e causes abnormal opening of MPTP channels. As shown in Figure A, green fluorescence in mitochondria was lost and the shape of the fluorescence changed from filamentous to spherical following treatment with 2/3 μM 5e for 24 h. Subsequently, we performed JC-1 staining on the A549 cells after 5e treatment. The result from flow cytometry quantitative detection indicated that the red fluorescence decreased while the green fluorescence increased (Figure B), suggesting the mitochondrial membrane potential decreased with increasing concentration of 5e (Figure C).

Figure 6

Mitochondrial dysfunction induced by 5e. (A) Mitochondria leakage induced by 5e. Cells were exposed to 5e at indicated doses for 24 h and then stained with calcein-AM in the presence of CoCl2. Images were captured by fluorescence microscopy. Scale bar, 10 μm. (B) Images of changes in Δψm were analyzed using JC-1 following treatment for 24 h. Scale bar, 20 μm. (C) Changes in Δψm were analyzed using JC-1 following treatment for 24 h, detected by flow cytometry. Histograms of JC-1 aggregates are expressed. **P < 0.01 vs control, ***P < 0.001 vs control.

On the basis of the findings above, we show that 5e was able to cause an increase of ROS, an abnormal opening of MPTP channels, and a decrease of mitochondrial permeability and membrane potential, but it was not the root cause of cell death and vacuolization.

5e Arrested the Cell Cycle at the G0/G1 Phase

In this study, we detected the distribution of the cell cycle using flow cytometry to evaluate the effects of 5e (0, 0.5, 1, and 2 μM) on the cell cycle of A549. We found that 5e increased the proportion of cells in G0/G1 phase (Figure ). Interestingly, AKBA also was able to arrest the cell cycle at the G0/G1 phase[32] to inhibit the growth of A549. These results indicated that 5e inhibited the growth of A549 by arresting the cell cycle at the G0/G1 phase.

Figure 7

Effects of 5e on the cell cycle in A549. (A) The cell cycle distribution of A549 using flow cytometry. (B) The percent of A549 in the G0/G1 phase. (C) The percent of A549 in the S phase. (D) The percent of A549 in the G2/M phase. **P < 0.01 vs control, ***P < 0.001 vs control.

Unconspicuous Apoptosis Induced by 5e in the Early Stage

Flow cytometry was used to examine the number of apoptotic cells by annexin V/PI double staining. A549 cells treated for 24 h revealed that 5e (1, 2, and 3 μM) was not able to induce significant apoptosis (Figure A). Western blot indicated that PARP, cleaved PARP, and cleaved caspase 3 were not upregulated (Figure B). Next, we examined the cellular localization of apoptosis-inducing factor (AIF), but AIF was not translocated to the nucleus and distributed widely in the cytoplasm in the presence of 5e (Figure C). In the end, we employed DAPI staining assay to examine the morphological alterations of the apoptotic nuclei, finding that 5e was not able to significantly cause the contraction and rupture of nucleus and increased the ratio of apoptotic nuclei in A549 cells (Figure D). These results suggested that apoptosis was not the main way of cell death induced by 5e at early stages.

Figure 8

Unconspicuous apoptosis induced by 5e in the early stage. (A) The apoptosis of A549 using flow cytometry. Apoptosis after 24 h of being exposed to 5e; A549 was determined with FITC/PI gating strategy in A549. (B) The protein expressions of PARP, Cleaved-PARP, and Cleaved-caspase-3 in A549. The protein expressions in A549 after 24 h of being exposed to 5e was determined using Western blotting. (C) The position of AIF was detected using immunofluorescence assays after treatment with 5e for 24 h. The red fluorescence represents AIF, and the blue fluorescence represents the nucleus. Scale bar, 20 μm. (D) Morphological alterations of the nuclei of A549 in each group. Scale bar, 50 μm.

Conclusion

In this study, we synthesized 26 novel AKBA derivatives with ethylenediamine as the link chain on the carboxyl group. Most of these derivatives showed preferable cytotoxicity compared with AKBA, especially mitochondrial-targeting derivatives 5e (up to 20 fold), which showed the most potent antiproliferative activity. 5e could induce vacuolization of A549 cells and cause mitochondrial damage and endoplasmic reticulum swelling. Further studies demonstrated that 5e was able to stimulate the production of ROS in a time- and concentration-dependent manner, the abnormal opening of MPTP channels, and the decrease of mitochondrial membrane potential, but ROS is not the root cause of cell death and vacuolization. 5e inhibited the growth of A549 by arresting the cell cycle at the G0/G1 phase. However, apoptosis was not observed obviously at early stages. This paper designed ethylenediamine-linked substituents and triphenylphosphine salts of AKBA for the first time and attempted to analyze their structure–activity relationship and explore the action mechanism, which may be instructive for the antitumor research and development of structural modification based on AKBA.

Experimental Section

Nuclear magnetic resonance (1H and 13C NMR) spectra were measured on a Bruker Avance DRX-400 or Avance DRX-600 MHz instrument. The chemical shifts were recorded in δ (ppm), and the coupling constants (J) are given in Hz for NMR. HR-electrospray ionization (ESI)-MS was conducted using LTQ Orbitrap XL or Thermo Fisher Q-Exactive instrument. The purity of compounds was established as >95% by HPLC using an Agilent1100 series with a ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm). Frankincense was obtained from the Jinan Jianlian Chinese medicine store in bulk quantities. Compound AKBA was isolated from frankincense, and the purity was at least 98%. The other reagents were obtained commercially and were used without further purification. Column chromatography was carried out on silica gel (300–400 mesh, Qindao Ocean Chemical Company, China). TLC was performed with precoated silica gel GF-254 glass plates (Yantai Jiangyou Chemical Company, China).

Synthesis of N-[2-(tert-Butoxycarbonylamino)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (1)

To a solution of AKBA (2.52 g, 4.92 mmol) and HATU (3.75 g, 9.87 mmol) in DMF (30 mL) was added DIPEA (4.05 mL, 24.6 mmol). The solution was stirred at 0 °C for 10 min, followed by addition of N-boc-ethylenediamine (2.56 mL, 14.7 mmol), and it was stirred in N2 for 12 h. The mixture was extracted by ethyl acetate (3 × 100 mL). Then the supernatant solution was washed successively with saturated NaHCO3 solution (100 mL), HCl solution (1N, 100 mL), and brine (100 mL) and dried over anhydrous Na2SO4. After the product was filtered and concentrated in vacuo, the residue was isolated by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 7:1 to 2:1) to achieve the product 1 as a white solid.

Yield: 69.9%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.35 (s, 1H), 5.55 (s, 1H), 5.32 (t, J = 2.5 Hz, 1H), 4.89 (s, 1H), 3.44–3.24 (m, 4H, −NH–CH2–CH2–NH−), 2.53 (m, 1H), 2.41 (s, 1H), 2.35 (m, 1H), 2.10 (m, 1H), 2.08 (s, 3H, -COCH3), 1.89–1.48 (m, 11H, overlapped), 1.44 (s, 9H, -Boc), 1.40–1.36 (m, 2H), 1.35 (s, 3H), 1.25–1.21 (m, 2H), 1.19 (s, 3H), 1.14 (s, 3H), 1.12 (s, 3H), 1.01 (m, 1H), 0.95 (s, 4H, H-20, H-30), 0.82 (s, 3H), 0.80 (d, J = 6.4 Hz, 3H); 13C NMR (150 MHz, CDCl3): δ 199.3 (C-11), 177.4 (−CONH−), 176.2(−CONH−), 170.3 (−COCH3), 164.8, 130.8, 80.1, 73.9, 60.7, 59.3, 50.6, 46.8, 45.3, 44.0, 41.1, 39.6 (−NH–CH2–CH2–NH−), 39.5 (2C), 37.6, 35.2, 34.2, 33.4, 31.1, 29.1, 28.6 (3C), 27.8, 27.4, 24.7, 24.2, 21.6, 21.4, 20.7, 19.6, 18.6, 17.6, 13.6; HR-ESI-MS m/z: calcd for C39H62N2O6Na+ [M + Na]+: 677.4500; found: 677.4483.

General Procedure for the Synthesis of 2a–h and 3a–h

TFA (1.5 mL) was added dropwise to a stirred solution of 1 (65.5 mg, 0.1 mmol) in CH2Cl2 (2 mL), and the mixture was continued at 0 °C for 0.5 h. After the substrate was completely consumed, the mixture was concentrated in vacuo. To the intermediate dissolved in anhydrous CH2Cl2 (2 mL) was added TEA (0.2 mL) and corresponding acid chloride (0.2 mmol). After stirring in a nitrogen atmosphere at 0 °C for 2 h, the mixture was washed with HCl solution (1N, 15 mL) and extracted by ethyl acetate (2 × 15 mL). The ethyl acetate layers were washed successively with saturated NaHCO3 solution and brine and dried over anhydrous Na2SO4. The mixture was concentrated in vacuo, and the residue was isolated by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 5:1 to 1:1) to afford 2a–h as a white solid.

Compound 2a–h (0.05 mmol) was dissolved in methanol (1 mL) followed by addition of NaOH (4 M, 1 mL) was added. After stirring for 12 h, the pH was adjusted to neutral (aq HCl), the mixture was extracted with ethyl acetate (2 × 10 mL), and the product was isolated by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 5:1 to 1:2) to afford 3a–h as a white solid.

N-[2-(Cyclopropanecarboxamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (2a)

Yield: 69.9%; white solid. 1H NMR (600 MHz, CDCl3): δ 7.00 (s, 1H), 6.59 (s, 1H), 5.54 (s, 1H), 5.29 (s, 1H), 3.51–3.31 (m, 4H, -NH–CH2–CH2–NH−), 2.53–2.51 (m, 1H), 2.41 (s, 1H), 2.37–2.32 (m, 1H), 2.12 (m, 1H), 2.08 (s, 3H, −COCH3), 1.91–1.20 (m, 16H, overlapped), 1.34 (s, 3H), 1.18 (s, 3H), 1.12 (s, 3H), 1.10 (s, 3H, H-25), 1.02 (m, 1H), 0.98 (m, 2H), 0.95 (s, 4H), 0.82 (s, 3H), 0.79 (d, J = 6.4 Hz, 5H). 13C NMR (150 MHz, CDCl3): δ 199.4 (C-11), 177.0 (−CONH−), 175.9 (−CONH−), 170.5 (−COCH3), 165.1 (C-13), 130.7 (C-12), 73.7, 60.6, 59.2, 50.5, 46.8, 45.2, 44.0, 41.1 (C-22, -NHCH2−), 40.2 (−NHCH2−), 39.5 (2C), 37.6, 35.1, 34.2, 33.3, 31.1, 29.1, 27.7, 27.4, 24.6, 24.2, 21.6, 21.3, 20.7, 19.5, 18.6, 17.6, 14.7 13.5, 7.9, 7.8. HR-ESI-MS m/z: calcd for C38H58N2O5Na+ [M + Na]+: 645.4238; found: 645.4218.

N-[2-(Cyclopropanecarboxamido)ethyl]-3-hydroxy-11-oxours-12-en-24-amide (3a)

Yield: 84.3%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.46 (t, J = 5.3 Hz, 1H), 6.37 (t, J = 4.4 Hz, 1H), 5.53 (s, 1H), 4.06 (s, 1H), 3.52–3.27 (m, 4H, -NH–CH2–CH2–NH−), 2.48 (dt, J = 13.2, 3.6 Hz, 1H), 2.42 (s, 1H), 2.38 (m, 1H), 2.12–2.04 (m, 1H), 2.49–1.32 (m, 14H, overlapped), 1.30 (s, 3H), 1.26–1.24 (m, 1H), 1.23 (s, 3H), 1.21–1.19 (m, 1H), 1.16 (s, 3H), 1.09 (s, 3H), 1.02 (m, 1H), 0.96–0.93 (m, 6H), 0.81 (s, 3H), 0.78 (d, J = 6.5 Hz, 2H), 0.75–0.73 (m, 2H). 13C NMR (150 MHz, CDCl3): δ 199.6 (C-11), 177.9 (−CONH−), 175.4 (−CONH−), 165.1 (C-13), 130.7 (C-12), 70.9, 60.8, 59.3, 49.0, 47.6, 45.3, 44.0, 41.2 (−NHCH2−), 41.1, 39.9 (−NHCH2−), 39.5 (2C), 37.7, 34.5, 34.2, 33.4, 31.1, 29.1, 27.7, 27.4, 26.8, 25.1, 21.3, 20.7, 19.6, 18.5, 17.6, 14.8, 13.7, 7.5. HR-ESI-MS m/z: calcd for C36H56N2O4Na+ [M + Na]+: 603.4132; found: 603.4110.

N-[2-(Cyclohexanecarboxamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (2b)

Yield: 79.0%; white solid. 1H NMR (600 MHz, CDCl3): δ 7.32 (s, 1H), 6.76 (s, 1H), 5.52 (s, 1H), 5.28 (s, 1H), 3.47–3.29 (m, 4H, -NH–CH2–CH2–NH−), 2.51–2.49 (m, 1H), 2.39 (s, 1H), 2.22 (m, 1H, −COCH−), 2.33 (m, 1H), 2.07 (m, 1H), 2.06 (s, 3H, −COCH3), 1.89–1.24 (m, 25H, overlapped), 1.32 (s, 3H), 1.23 (s, 3H), 1.16 (s, 3H), 1.08 (s, 3H), 1.01–0.98 (m, 1H), 0.93 (s, 4H), 0.81 (s, 3H), 0.78 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.4 (C-11), 178.8 (−CONH−), 177.2 (−CONH−), 170.4 (−COCH3), 165.1 (C-13), 130.6 (C-12), 73.7, 60.6, 59.2, 50.5, 46.7, 45.2, 45.0 (−COCH−), 44.0, 41.1, 40.8 (−NHCH2−), 40.4 (−NHCH2−), 39.5, 39.4, 37.5, 35.0, 34.1, 33.2, 31.1, 29.7, 29.6, 29.0, 27.7, 27.3, 25.7 (2C), 24.5, 24.2, 21.6, 21.3, 21.2, 20.6, 19.4, 18.5, 17.6, 13.4. HR-ESI-MS m/z: calcd for C41H64N2O5Na+ [M + Na]+: 687.4707; found: 687.4701.

N-[2-(Cyclohexanecarboxamido)ethyl]-3-hydroxy-11-oxours-12-en-24-amide (3b)

Yield: 90.6%; white solid. 1H NMR (400 MHz, CDCl3): δ 6.43 (s, 1H), 6.31 (s, 1H), 5.53 (s, 1H), 4.06 (s, 1H), 3.48–3.29 (m, 4H, -NH–CH2–CH2–NH−), 2.49–2.46 (m, 1H), 2.42 (s, 1H), 2.38–2.35 (m, 1H, −COCH−), 2.13–2.04 (m, 2H), 1.87–1.17 (m, 25H, overlapped), 1.29 (s, 3H),1.16 (s 3H), 1.23 (s, 3H), 1.15 (s, 3H), 1.01 (m, 1H), 0.92 (s, 4H), 0.79 (s, 3H), 0.77 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 199.6 (C-11), 178.0 (−CONH-, −CONH−), 165.1 (C-13), 130.7 (C-12), 70.9, 60.7, 59.3, 49.0, 47.5, 45.6, 45.3, 44.0, 41.3 (−NHCH2−), 41.1, 39.8 (−NHCH2−), 39.5 (2C), 37.7, 34.4, 34.2, 33.4, 31.1, 29.9, 29.8, 29.1, 27.7, 27.3, 26.8, 25.9 (3C), 25.2, 21.3, 20.7, 19.6, 18.5, 17.6, 13.6. HR-ESI-MS m/z: calcd for C39H63N2O4+ [M + H]+: 623.4782; found: 623.4767.

N-[2-(Benzamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (2c)

Yield: 78.7%; white solid. 1H NMR (400 MHz, CDCl3): δ 7.80 (d, J = 7.3 Hz, 2H), 7.66 (s, 1H), 7.42 (m, 3H), 6.64 (s, 1H), 5.49 (s, 1H), 5.30 (s, 1H), 3.64–3.34 (m, 4H, -NH–CH2–CH2–NH−), 2.49–2.45 (m, 1H), 2.36 (s, 1H), 2.32 (m, 1H), 2.06 (m, 1H), 2.04 (s, 3H, −COCH3), 1.84–1.14 (m, 15H, overlapped), 1.30 (s, 3H), 1.08 (s, 3H), 1.04 (s, 3H), 1.02 (s, 3H), 0.97 (m, 1H), 0.91 (s, 4H), 0.78 (s, 3H), 0.76 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 199.3 (C-11), 177.2 (−CONH−), 170.4 (−COCH3), 168.8 (−CONH−), 165.1 (C-13), 133.8, 131.8, 130.6 (C-12), 128.7 (2C), 127.2 (2C), 73.7, 60.5, 59.1, 50.4, 46.7, 45.1, 43.9, 41.0, 40.8 (−NHCH2−), 40.4 (−NHCH2−), 39.4 (2C), 37.5, 35.0, 34.1, 33.1, 31.0, 29.0, 27.6, 27.3, 24.5, 24.1, 21.5, 21.3, 20.6, 19.4, 18.3, 17.5, 13.4. HR-ESI-MS m/z: calcd for C41H58N2O5Na+ [M + Na]+: 681.4238; found: 681.4223.

N-[2-(Benzamido)ethyl]-3-hydroxy-11-oxours-12-en-24-amide (3c)

Yield: 87.4%; white solid. 1H NMR (600 MHz, CDCl3): δ 7.79 (d, J = 8.5 Hz, 2H), 7.48 (t, J = 8.0 Hz, 1H), 7.42–7.37 (m, 3H), 6.43 (t, J = 4.9 Hz, 1H), 5.50 (s, 1H), 4.08 (s, 1H), 3.65–3.40 (m, 4H, -NH–CH2–CH2–NH−), 2.45–2.43 (m, 1H), 2.39 (s, 1H), 2.35 (m, 1H), 2.06 (m, 1H), 1.84–1.15 (m, 15H, overlapped), 1.28 (s, 3H), 1.21 (s, 3H), 1.05 (s, 3H), 1.04 (s, 3H), 1.01–0.97 (m, 1H), 0.92 (s, 4H), 0.79 (s, 3H), 0.76 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.6 (C-11), 178.5 (−CONH−), 168.7 (−CONH−), 165.2 (C-13), 133.9, 131.8, 130.6 (C-12), 128.8 (2C), 127.2 (2C), 70.8, 60.6, 59.2, 48.9, 47.5, 45.2, 44.0, 41.1, 40.8 (−NHCH2−), 40.6 (−NHCH2−), 39.4 (2C), 37.6, 34.4, 34.1, 33.3, 31.1, 29.0, 27.6, 27.3, 26.8, 25.2, 21.3, 20.7, 19.6, 18.4, 17.6, 13.6. HR-ESI-MS m/z: calcd for C39H57N2O4+ [M + H]+: 617.4313; found: 617.4302.

N-[2-(2-Propylpentanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (2d)

Yield: 79.5%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.64 (s, 1H), 6.50 (s, 1H), 5.52 (s, 1H), 5.28 (s, 1H), 3.52–3.23 (m, 4H, -NH–CH2–CH2–NH−), 2.51–2.49 (m, 1H), 2.39 (s, 1H), 2.36 (m, 1H), 2.07 (m, 2H), 2.06 (s, 3H, −COCH3), 1.84–1.18 (m, 23H, overlapped), 1.33 (s, 3H), 1.16 (s, 3H), 1.09 (s, 3H), 1.08 (s, 3H), 1.02–0.97 (m, 1H), 0.92 (s, 3H), 0.88–0.83 (m, 7H), 0.80 (s, 3H), 0.76 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.4 (C-11), 178.1 (−CONH−), 176.6 (−CONH−), 170.3 (−COCH3), 165.0 (C-13), 130.6 (C-12), 73.8, 60.6, 59.2, 50.4, 47.6, 46.6, 45.2, 43.9, 41.8 (−NHCH2−), 41.0, 39.6, 39.5, 39.4 (−NHCH2−), 37.5, 35.3 (2C), 35.0, 34.1, 33.2, 31.0, 29.0, 27.6, 27.3, 24.4, 24.1, 21.6, 21.3, 21.0 (2C), 20.6, 19.3, 18.5, 17.6, 14.3 (2C), 13.3. HR-ESI-MS m/z: calcd for C42H68N2O5Na+ [M + Na]+: 703.5020; found: 703.5003.

N-[2-(2-Propylpentanamido)ethyl]-3-hydroxy-11-oxours-12-en-24-amide (3d)

Yield: 87.9%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.54 (s, 1H), 6.38 (s, 1H), 5.52 (s, 1H), 4.06 (s, 1H), 3.51–328 (m, 4H, -NH–CH2–CH2–NH−), 2.49–2.46 (m, 1H), 2.42 (s, 1H), 2.38–2.34 (m, 1H), 2.11–2.03 (m, 2H), 2.04 (s, 1H), 1.90–1.24 (m, 23H, overlapped), 1.30 (s, 3H), 1.23 (s, 3H), 1.16 (s, 3H), 1.08 (s, 3H), 1.01–0.97 (m, 1H), 0.94 (s, 3H), 0.87 (t, J = 7.5 Hz, 7H, H-20, −CH2–CH3), 0.80 (s, 3H), 0.78 (d, J = 6.3 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 199.6 (C-11), 178.1 (−CONH−), 171.9 (−CONH−), 165.0 (C-13), 130.7 (C-12), 70.9, 60.7, 59.3, 49.0, 47.7, 47.5, 45.3, 44.0, 41.5 (−NHCH2−), 41.1, 39.8 (−NHCH2−), 39.5 (2C), 37.7, 35.4, 35.3, 34.4, 34.2, 33.4, 31.1, 29.1, 27.7, 27.4, 26.8, 25.1, 21.3, 21.1, 21.0, 20.7, 19.6, 18.6, 17.6, 14.3, 14.2, 13.6. HR-ESI-MS m/z: calcd for C40H67N2O4+ [M + H]+: 661.4915; found: 661.4907.

N-[2-(4-Methylbenzamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (2e)

Yield: 85.6%; white solid. 1H NMR (600 MHz, CDCl3): δ: 7.70 (d, J = 7.9 Hz, 2H), 7.21 (d, J = 7.6 Hz, 2H), 6.48 (s, 1H), 5.51 (s, 1H), 5.32 (s, 1H), 3.65–3.39 (m, 4H, -NH–CH2–CH2–NH−), 2.50–2.48 (m, 1H), 2.37 (s, 4H, H-9, phenmethyl), 2.33 (m, 1H), 2.07 (m, 1H), 2.06 (s, 3H, −COCH3), 1.87–1.17 (m, 15H, overlapped), 1.32 (s, 3H), 1.10 (s, 3H), 1.05 (s, 3H), 1.02 (s, 3H), 1.00 (m, 1H), 0.93 (s, 4H), 0.80 (s, 3H), 0.78 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.3 (C-11), 177.2 (−CONH−), 170.4 (−COCH3), 168.7 (−CONH−), 165.0 (C-13), 142.3, 131.0, 130.7 (C-12), 129.5 (2C), 127.2 (2C), 73.8, 60.5, 59.2, 50.5, 46.8, 45.1, 43.9, 41.1 (C-22, -NHCH2−), 40.4 (−NHCH2−), 39.5 (2C), 37.6, 35.0, 34.2, 33.2, 31.1, 29.0, 27.7, 27.3, 24.6, 24.1, 21.6 (2C), 21.3 (C-40), 20.7, 19.5, 18.3, 17.6, 13.5. HR-ESI-MS m/z: calcd for C42H60N2O5Na+ [M + Na]+: 695.4394; found: 695.4380.

N-[2-(4-Methylbenzamido)ethyl]-3-hydroxy-11-oxours-12-en-24-amide (3e)

Yield: 88.9%; white solid. 1H NMR (600 MHz, CDCl3): δ 7.76–7.65 (m, 2H), 7.45 (s, 1H), 7.21 (d, J = 7.5 Hz, 2H), 6.59 (s, 1H), 5.50 (s, 1H), 4.10 (s, 1H), 3.62–3.38 (m, 4H, -NH–CH2–CH2–NH−), 2.45–2.43 (m, 1H), 2.39 (s, 1H), 2.37 (s, 3H, phenmethyl), 2.06–2.04 (m, 2H), 1.82–1.15 (m, 15H, overlapped), 1.27 (s, 3H), 1.22 (s, 3H), 1.03(s, 3H), 1.01 (s, 3H), 1.00–0.98 (m, 1H), 0.92 (s, 4H, H-20, H-30), 0.78 (s, 3H, H-28), 0.76 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.7 (C-11), 171.4 (−CONH−), 165.3 (−CONH−), 165.2 (C-13), 142.5, 130.6, 129.5 (3C), 127.3 (2C), 70.7, 60.6, 59.2, 49.0, 47.6, 45.2, 44.0, 41.1, 40.7 (−NHCH2–CH2–NH−), 39.4 (2C), 37.6, 34.4, 34.1, 33.2, 31.1, 29.0, 27.7, 27.3, 26.8, 25.2, 21.7, 21.3, 20.7, 19.6, 18.3, 17.6, 14.4. HR-ESI-MS m/z: calcd for C40H59N2O4+ [M + H]+: 653.4289; found: 653.4280.

N-[2-(Hexanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (2f)

Yield: 77.5%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.61 (s, 1H), 5.53 (s, 1H), 5.30 (s, 1H), 3.47–3.35 (m, 4H, -NH–CH2–CH2–NH−), 2.53–2.50 (m, 1H), 2.40 (s, 1H), 2.33 (m, 1H), 2.25 (m, 2H, −COCH2−), 2.08 (m, 1H), 2.07 (s, 3H, −COCH3), 2.03–1.20 (m, 21H, overlapped), 1.34 (s, 3H), 1.18 (s, 3H), 1.13 (s, 3H), 1.09 (s, 3H), 1.01 (m, 1H), 0.94 (s, 4H), 0.87 (t, J = 6.7 Hz, 3H, −CH2–CH3), 0.81 (s, 3H), 0.79 (d, J = 6.4 Hz, 3H). 13C NMR (151 MHz, CDCl3): δ 199.4 (C-11), 177.2 (−CONH−), 175.8 (−CONH−), 170.4 (−COCH3), 165.0 (C-13), 130.7 (C-12), 73.7, 60.6, 59.2, 50.6, 46.8, 45.2, 44.0, 41.1, 40.7 (−NHCH2−), 40.4 (−NHCH2−), 39.5 (2C), 37.6, 36.3 (−COCH2−), 35.1, 34.2, 33.3, 31.6, 31.1, 29.0, 27.7, 27.4, 25.7, 24.6, 24.2, 22.5, 21.6, 21.3, 20.7, 19.5, 18.6, 17.6, 14.1 (−CH2–CH3), 13.5. HR-ESI-MS m/z: calcd for C40H64N2O5Na+ [M + Na]+: 675.4707; found: 675.4703.

N-[2-(Hexanamido)ethyl]-3-hydroxy-11-oxours-12-en-24-amide (3f)

Yield: 77.7%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.53 (s, 1H), 6.45 (s, 1H), 5.52 (s, 1H), 4.06 (s, 1H), 3.46–3.31 (m, 4H, -NH–CH2–CH2–NH−), 2.47–2.45 (m, 1H), 2.42 (s, 1H, H-9), 2.36 (m, 1H), 2.17 (m, 2H, −COCH2−), 2.06 (m, 1H), 1.88–1.17 (m, 21H, overlapped), 1.29 (s, 3H), 1.23 (s, 3H), 1.16 (s, 3H), 1.07 (s, 3H), 0.99 (m, 1H), 0.93 (s, 4H), 0.87 (t, J = 7.0 Hz, 3H, −CH2–CH3), 0.80 (s, 3H), 0.77 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.7 (C-11), 178.1 (−CONH−), 175.2 (−CONH−), 165.3 (C-13), 130.6 (C-12), 70.8, 60.6, 59.2, 48.9, 47.5, 45.2, 44.0, 41.1, 40.9 (−NHCH2−), 39.8 (−NHCH2−), 39.4 (2C), 37.6, 36.8 (−COCH2−), 34.4, 34.1, 33.3, 31.6, 31.1, 29.0, 27.7, 27.3, 26.8, 25.6, 25.2, 22.6, 21.3, 20.7, 19.5, 18.5, 17.6, 14.1 (−CH2–CH3), 13.6. HR-ESI-MS m/z: calcd for C38H63N2O4+ [M + H]+: 611.4782; found: 611.4780.

N-[2-(Heptanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (2g)

Yield: 85.5%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.97 (s, 1H), 6.62 (s, 1H),5.51 (s, 1H), 5.27 (s, 1H), 3.42–3.28 (m, 4H, -NH–CH2–CH2–NH−), 2.50–2.48 (m, 1H), 2.38 (s, 1H), 2.32 (m, 1H), 2.18 (t, J = 7.6 Hz, 2H, −COCH2−), 2.06 (m, 1H), 2.05 (s, 3H, −COCH3), 1.87–1.18 (m, 23H, overlapped), 1.31 (s, 3H), 1.15 (s, 3H), 1.09 (s, 3H), 1.06 (s, 3H), 1.01–0.98 (m, 1H), 0.91 (s, 4H), 0.84 (t, J = 6.7 Hz, 3H, −CH2–CH3), 0.79 (s, 3H), 0.77 (d, J = 6.4 Hz, 3H). 13C NMR (151 MHz, CDCl3): δ 199.3 (C-11), 176.8 (−CONH−), 175.5 (−CONH−), 170.4 (−COCH3), 165.1 (C-13), 130.6 (C-12), 73.7, 60.5, 59.2, 50.4, 46.7, 45.2, 43.9, 41.0 (C-22, -NHCH2−), 39.8 (−NHCH2−), 39.4 (2C), 37.5, 36.6 (−COCH2−), 35.0, 34.1, 33.2, 31.7, 31.0, 29.1, 29.0, 27.6, 27.3, 25.9, 24.4, 24.1, 22.6, 21.5, 21.3, 20.6, 19.3, 18.5, 17.5, 14.2 (−CH2–CH3), 13.4. HR-ESI-MS m/z: calcd for C41H66N2O5Na+ [M + Na]+: 689.4864; found: 689.4854.

N-[2-(Heptanamido)ethyl]-3-hydroxy-11-oxours-12-en-24-amide (3g)

Yield: 87.4%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.35 (s, 1H), 6.16 (s, 1H), 5.54 (s, 1H), 4.08 (s, 1H), 3.47–3.33 (m, 4H, -NH–CH2–CH2–NH−), 2.51–2.48 (m, 1H), 2.43 (s, 1H), 2.40 (m, 1H), 2.17 (m, 2H, −COCH2−), 2.08 (m, 1H), 1.90–1.17 (m, 23H, overlapped), 1.31 (s, 3H), 1.24 (s, 3H), 1.17 (s, 3H), 1.09 (s, 3H), 0.99 (m, 1H), 0.94 (s, 4H), 0.87 (t, J = 7.0 Hz, 3H, −CH2–CH3), 0.81 (s, 3H), 0.79 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.6 (C-11), 177.9 (−CONH−), 175.0 (−CONH−), 165.1 (C-13), 130.7 (C-12), 70.9, 60.7, 59.2, 49.0, 47.5, 45.3, 44.0, 41.1 (C-22, -NHCH2−), 39.9 (−NHCH2−), 39.5 (2C), 37.7, 36.9 (−COCH2−), 34.4, 34.2, 33.4, 31.8, 31.1, 29.2, 29.1, 27.7, 27.3, 26.8, 25.9, 25.2, 22.7, 21.3, 20.7, 19.6, 18.6, 17.6, 14.3 (−CH2–CH3), 13.6. HR-ESI-MS m/z: calcd for C39H64N2O4Na+ [M + Na]+: 647.4758; found: 647.4752.

N-[2-(Octanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (2h)

Yield: 81.5%; white solid. 1H NMR (600 MHz, CDCl3): δ 8.16 (s, 1H), 6.81 (s, 1H), 5.54 (s, 1H), 5.29 (s, 1H), 3.52–3.38 (m, 4H, -NH–CH2–CH2–NH−), 2.53–2.51 (m, 1H), 2.41 (s, 1H), 2.38 (m,2H,-COCH2−), 2.33 (m, 1H), 2.10 (m, 1H), 2.08 (s, 3H, −COCH3), 1.91–1.18 (m, 25H, overlapped), 1.34 (s, 3H), 1.18 (s, 3H), 1.15 (s, 3H), 1.08 (s, 3H), 1.01 (m, 1H), 0.94 (s, 4H), 0.88 (t, J = 6.4 Hz, 3H, −CH2–CH3), 0.82 (s, 3H, H-28), 0.80 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.4 (C-11), 177.7 (−CONH−), 176.7 (−CONH−), 170.5 (−COCH3), 165.4 (C-13), 130.7 (C-12), 73.7, 60.6, 59.3, 50.5, 46.8, 45.3, 44.0, 41.3 (−NHCH2−), 41.1, 40.1 (−NHCH2−), 39.6, 39.5, 37.6, 35.6 (−COCH2−), 35.1, 34.2, 33.2, 31.5, 31.1, 29.1, 27.7, 27.4, 25.8, 25.7, 24.6, 24.2, 22.5 (2C), 21.6, 21.3, 20.7, 19.5, 18.6, 17.6, 14.1 (−CH2–CH3), 13.5. HR-ESI-MS m/z: calcd for C42H68N2O5Na+ [M + Na]+: 703.5020; found: 703.5007.

N-[2-(Octanamido)ethyl]-3-hydroxy-11-oxours-12-en-24-amide (3h)

Yield: 79.9%; white solid. 1H NMR (400 MHz, CDCl3): δ 6.44 (m, 2H), 5.51 (s, 1H), 4.05 (s, 1H), 3.45–3.27 (m, 4H, -NH–CH2–CH2–NH−), 2.48–2.44 (m, 1H), 2.41 (s, 1H), 2.36 (m, 1H), 2.15 (m, 2H, −COCH2−), 2.06 (m, 1H), 1.89–1.15 (m, 25H, overlapped), 1.29 (s, 3H), 1.22 (s, 3H), 1.15 (s, 3H), 1.07 (s, 3H), 0.98 (m, 1H), 0.92 (s, 4H), 0.85 (t, J = 6.7 Hz, 3H, −CH2–CH3), 0.79 (s, 3H), 0.77 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 199.6 (C-11), 178.0 (−CONH−), 175.0 (−CONH−), 165.1 (C-13), 130.7 (C-12), 70.8, 60.7, 59.2, 48.9, 47.5, 45.2, 44.0, 41.1 ((C-22, -NHCH2−)), 39.8 (−NHCH2−), 39.5 (2C), 37.7, 36.9 (−COCH2−), 34.4, 34.1, 33.3, 31.9, 31.1, 29.5, 29.2, 29.0, 27.7, 27.3, 26.8, 26.0, 25.1, 22.8, 21.3, 20.7, 19.6, 18.5, 17.6, 14.3 (−CH2–CH3), 13.6. HR-ESI-MS m/z: calcd for C40H66N2O4Na+ [M + Na]+: 661.4915; found: 661.4905.

General Procedure for the Synthesis of 4a–e and 5a–e

TFA (2 mL) was added dropwise to a stirred solution of 1 (102.0 mg, 0.16 mmol) in CH2Cl2 (2 mL), and the mixture was continued at 0 °C for 0.5 h. After the substrate was completely consumed, the mixture was concentrated in vacuo as the next reaction substrate. To a solution of corresponding acid (0.3 mmol) and HATU (238.1 mg, 0.62 mmol) in CH2Cl2 (2 mL) was added DIPEA (0.3 mL, 1.6 mmol). The solution was stirred at 0 °C for 10 min, followed by addition of the previous substrate, and it was stirred in N2 for 12 h. The mixture was extracted by ethyl acetate (3 × 10 mL). Then organic solution was washed successively with saturated NaHCO3 solution, HCl solution (1N), and brine and the dried over anhydrous Na2SO4. After the product was filtered and concentrated in vacuo, the mixture was isolated by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 4:1 to 1:1) to achieve the product 4a–e as a white solid.

A solution of 4a–e (0.05 mmol) and triphenylphosphine (40.0 mg, 0.15 mmol) in MeCN (2 mL) was stirred at 80 °C for 48 h. The solvent was subsequently removed under diminished pressure, and the mixture was isolated by flash column chromatography on silica gel (DCM:MeOH= 60:1 to 10:1) to achieve the product 5a–e as a white solid.

N-[2-(3-Bromopropanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (4a)

Yield: 68.6%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.91 (s, 1H, -NH−), 6.44 (s, 1H, -NH−), 5.55 (s, 1H), 5.30 (s, 1H), 3.64 (t, J = 6.4 Hz, 2H, −CH2Br), 3.49–3.37 (m, 4H, -NH–CH2–CH2–NH−), 2.80–1.21 (m, 21H, overlapped), 2.08 (s, 3H, −COCH3), 1.34 (s, 3H), 1.19 (s, 3H), 1.14 (s, 3H), 1.10 (s, 3H), 1.03 (m, 1H), 0.94 (s, 4H), 0.82 (s, 3H), 0.80 (d, J = 6.3 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ199.1 (C-11), 178.2(−CONH−), 170.4 (−CONH-, −COCH3), 165.0 (C-13), 130.7 (C-12),73.2, 60.5, 59.3, 50.6, 47.0, 45.2, 44.0, 41.8, 41.1, 39.6, 39.5, 39.3, 37.6, 35.0, 34.2 (2C), 33.3 (2C), 31.1, 29.1, 27.7, 27.5, 24.8, 24.1, 21.5, 21.3, 20.7, 19.7, 18.5, 17.6, 13.6. HR-ESI-MS m/z: calcd for C37H57BrN2O5Na+ [M + Na]+: 711.3343; found: 711.3333.

N-[2-(3-Triphenylphosphoniopropanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide Bromide(5a)

Yield: 55.1%; white solid. 1H NMR (600 MHz, CDCl3): δ 8.85 (s, 1H), 7.82–7.70 (m, 15H), 7.08 (s, 1H), 5.50 (s, 1H), 5.31 (s, 1H), 3.68–3.23 (m, 6H, -NH–CH2–CH2–NH-, −CH2Br), 2.88–1.15 (m, 21H, overlapped), 2.03 (s, 3H, −COCH3), 1.31 (s, 3H), 1.20 (s, 3H), 1.12 (s, 3H), 1.06 (s, 3H), 0.98 (m, 1H), 0.93 (s, 4H), 0.78 (d, J = 6.5 Hz, 6H). 13C NMR (150 MHz, CDCl3): δ 199.7 (C-11), 175.8 (−CONH−), 170.4 (−COCH3), 169.8 (−CONH−), 164.9 (C-13), 135.6, 133.8 (d, J = 12.7 Hz), 130.9 (d, J = 13.0 Hz), 130.7 (C-12), 118.0 (d, J = 86.7 Hz), 74.2, 65.8, 60.8, 59.2, 50.7, 46.9, 45.3, 43.9, 41.1, 40.0, 39.5, 39.1, 37.6, 35.2, 34.2, 33.2, 31.1, 29.1, 28.9, 27.7, 27.3, 24.5, 24.4, 21.6, 21.3, 20.7, 20.3, 19.8, 18.6, 17.6, 13.3. HR-ESI-MS m/z: calcd for C55H72N2O5P+ [M-Br]+: 871.5173; found: 871.5165.

N-[2-(5-Bromopentanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (4b)

Yield: 71.6%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.45 (s, 1H), 6.37 (s, 1H), 5.54 (s, 1H), 5.30 (s, 1H), 3.48–3.28 (m, 6H, -NH–CH2–CH2–NH-, −CH2Br), 2.54–1.21 (m, 25H, overlapped), 2.08 (s, 3H, −COCH3), 1.34 (s, 3H), 1.18 (s, 3H), 1.12 (s, 3H), 1.09 (s, 3H), 1.03 (m, 1H), 0.94 (s, 4H), 0.82 (s, 3H), 0.79 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.3 (C-11), 177.0 (−CONH−), 174.0 (−CONH−), 170.4 (−COCH3), 165.0 (C-13), 130.7 (C-12), 73.6, 60.6, 59.2, 50.5, 46.8, 45.2, 44.0, 41.1, 40.9, 40.0, 39.5 (2C), 37.6, 35.6, 35.1, 34.2, 33.3 (2C), 32.3, 31.1, 29.1, 27.7, 27.4, 24.6, 24.4, 24.1, 21.6, 21.3, 20.7, 19.5, 18.6, 17.6, 13.5. HR-ESI-MS m/z: calcd for C39H61N2O5+ [M-Br]+: 637.4570; found: 637.4570.

N-[2-(5-Triphenylphosphoniopentanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide Bromide(5b)

Yield: 50.1%; white solid. 1H NMR (600 MHz, CDCl3): δ 9.27 (s, 1H), 7.79–7.66 (m, 15H), 7.31 (s, 1H), 5.49 (s, 1H), 5.25 (s, 1H), 3.68–3.20 (m, 6H, -NH–CH2–CH2–NH-, −CH2Br), 2.60–1.14 (m, 25H, overlapped), 2.02 (s, 3H, −COCH3), 1.30 (s, 3H), 1.10 (s, 3H), 1.06 (s, 3H), 1.02 (s, 3H), 0.96 (m, 1H), 0.92 (s, 4H), 0.77 (d, J = 8.5 Hz, 6H). 13C NMR (150 MHz, CDCl3): δ 199.7 (C-11), 175.6 (−CONH−), 174.3 (−CONH−), 170.5 (−COCH3), 165.0 (C-13), 135.4 (d, J = 2.9 Hz), 133.7 (d, J = 10.0 Hz), 130.7 (d, J = 12.3 Hz), 130.6 (C-12), 118.4 (d, J = 86.1 Hz), 74.2, 60.7, 59.1, 50.6, 46.6, 45.2, 43.9, 41.1, 40.9, 39.4 (2C), 39.2, 37.5, 35.1, 34.1, 33.7, 33.2, 31.1, 29.1, 27.7, 27.3, 24.4, 24.1, 22.3, 22.0, 21.6, 21.3, 20.8, 20.6, 19.3, 18.4, 17.6, 13.2. HR-ESI-MS m/z: calcd for C57H76N2O5P+ [M-Br]+: 899.5486; found: 899.5480.

N-[2-(6-Bromohexanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (4c)

Yield: 67.7%; white solid. 1H NMR (600 MHz, CDCl3): δ 7.68 (s, 1H), 6.68 (s, 1H), 5.53 (s, 1H), 5.28 (s, 1H), 3.48–3.34 (m, 6H, -NH–CH2–CH2–NH-, −CH2Br), 2.52–1.21 (m, 27H, overlapped), 2.07 (s, 3H, −COCH3), 1.33 (s, 3H), 1.17 (s, 3H), 1.13 (s, 3H), 1.08 (s, 3H), 1.01 (m, 1H), 0.93 (s, 4H), 0.81 (s, 3H), 0.79 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.4 (C-11), 177.3 (−CONH−), 175.4 (−CONH−), 170.5 (−COCH3), 165.3 (C-13), 130.6 (C-12), 73.6, 60.5, 59.2, 50.5, 46.8, 45.2, 44.0, 41.1, 40.5 (2C), 39.5 (2C), 37.5, 35.9, 35.0, 34.2, 33.7, 33.2, 32.5, 31.1, 29.1, 27.9, 27.7, 27.4, 25.1, 24.6, 24.1, 21.6, 21.3, 20.7, 19.5, 18.6, 17.6, 13.4. HR-ESI-MS m/z: calcd for C40H63N2O5+ [M-Br]+: 651.4732; found: 651.4723.

N-[2-(6-Triphenylphosphoniohexanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide Bromide (5c)

Yield: 61.7%; white solid. 1H NMR (600 MHz, CDCl3): δ 8.46 (s, 1H), 7.81–7.68 (m, 15H), 7.13 (s, 1H), 5.47 (s, 1H), 5.26 (s, 1H), 3.51–3.24 (m, 6H, -NH–CH2–CH2–NH-, −CH2Br), 2.45–1.15 (m, 27H, overlapped), 2.00 (s, 3H, −COCH3), 1.30 (s, 3H), 1.13 (s, 6H), 1.04 (s, 3H), 0.97 (m, 1H), 0.91 (s, 4H), 0.78 (s, 3H), 0.76 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.7 (C-11), 175.8 (−CONH−), 174.9 (−CONH−), 170.5 (−COCH3), 165.1 (C-13), 135.4 (d, J = 3.0 Hz), 133.7 (d, J = 9.9 Hz), 130.7 (d, J = 12.7 Hz), 130.6 (C-12), 118.5 (d, J = 86.2 Hz), 74.2, 60.7, 59.2, 50.6, 46.7, 45.2, 43.9, 41.1, 40.9, 39.4, 39.1, 37.5, 36.0, 35.1, 34.1, 33.2, 31.0, 29.8, 29.7, 29.0, 27.7, 27.3, 24.8, 24.3, 22.8, 22.4, 21.8, 21.6, 21.3, 20.6, 19.4, 18.5, 17.6, 13.3. HR-ESI-MS m/z: calcd for C58H78N2O5P+ [M-Br]+: 913.5643; found: 913.5635.

N-[2-(8-Bromooctanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (4d)

Yield: 78.3%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.84 (s, 1H), 6.52 (s, 1H), 5.53 (s, 1H), 5.29 (s, 1H), 3.45–3.31 (m, 6H, -NH–CH2–CH2–NH-, −CH2Br), 2.52–1.20 (m, 31H, overlapped), 2.07 (s, 3H, −COCH3), 1.33 (s, 3H), 1.17 (s, 3H), 1.13 (s, 3H), 1.08 (s, 3H), 1.00 (m, 1H), 0.93 (s, 4H), 0.81 (s, 3H), 0.79 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.3 (C-11), 177.0 (−CONH−), 175.2 (−CONH−), 170.4 (−COCH3), 165.1 (C-13), 130.7 (C-12), 73.7, 60.6, 59.2, 50.4, 46.7, 45.2, 44.0, 41.1, 40.9, 40.1, 39.5, 39.4, 37.5, 36.5, 35.0, 34.2, 34.1, 33.2, 32.9, 31.1, 29.2, 29.0, 28.6, 28.1, 27.7, 27.4, 25.8, 24.6, 24.1, 21.6, 21.3, 20.7, 19.4, 18.6, 17.6, 13.4. HR-ESI-MS m/z: calcd for C42H67BrN2O5Na+ [M + Na]+: 781.4126; found: 781.4121.

N-[2-(8-Triphenylphosphoniooctanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide Bromide(5d)

Yield: 76.8%; white solid. 1H NMR (600 MHz, CDCl3): δ 7.82–7.69 (m, 15H), 7.28 (s, 1H), 5.49 (s, 1H), 5.27 (s, 1H), 3.53–3.29 (m, 6H, -NH–CH2–CH2-NH-, −CH2Br), 2.47–1.18 (m, 31H, overlapped), 2.03 (s, 3H, −COCH3), 1.31 (s, 3H), 1.15 (s, 6H, H-23), 1.14 (s, 3H), 1.04 (s, 3H), 0.98 (m, 1H), 0.92 (s, 4H), 0.79 (s, 3H), 0.77(d, J = 5.9 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.7 (C-11), 176.0 (−CONH−), 170.5 (−CONH-, −COCH3), 165.1 (C-13), 135.4, 133.8 (d, J = 10.0 Hz), 130.8 (d, J = 13.0 Hz), 130.7 (C-12), 118.7 (d, J = 85.9 Hz), 74.3, 60.8, 59.2, 50.6, 46.7, 45.2, 43.9, 41.1, 40.4, 39.9, 39.5 (2C), 37.5, 35.1, 34.2, 33.1, 31.1, 29.9, 29.8, 29.6, 29.1, 27.9, 27.7, 27.3, 27.1, 25.4, 24.3, 22.8, 22.4, 21.6, 21.3, 20.7, 19.4, 18.5, 17.6, 13.3. HR-ESI-MS m/z: calcd for C60H82N2O5P+ [M-Br]+:941.5956; found: 941.5945.

N-[2-(11-Bromoundecanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amide (4e)

Yield: 70.6%; white solid. 1H NMR (600 MHz, CDCl3): δ 6.41 (s, 1H), 6.33 (s, 1H), 5.53 (s, 1H), 5.30 (s, 1H), 3.48–3.27 (m, 6H, -NH–CH2–CH2–NH-, −CH2Br), 2.53–1.20 (m, 37H, overlapped), 2.07 (s, 3H, −COCH3), 1.34 (s, 3H), 1.17 (s, 3H), 1.11 (s, 3H), 1.09 (s, 3H), 1.00 (m, 1H), 0.94 (s, 4H), 0.81 (s, 3H), 0.79 (d, J = 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3): δ 199.3 (C-11), 176.8 (−CONH−), 175.0 (−CONH−), 170.4 (−COCH3), 165.0 (C-13), 130.7 (C-12), 73.8, 60.6, 59.2, 50.5, 46.7, 45.2, 44.0, 41.2, 41.1, 39.8, 39.5 (2C), 37.6, 36.9, 35.1, 34.3, 34.2, 33.3, 33.0, 31.1, 29.6, 29.5 (3C), 29.1, 28.9, 28.3, 27.7, 27.4, 25.9, 24.6, 24.1, 21.6, 21.3, 20.7, 19.5, 18.6, 17.6, 13.4. HR-ESI-MS m/z: calcd for C45H73BrN2O5Na+ [M + Na]+: 823.4595; found: 823.4586.

N-[2-(11-Triphenylphosphonioundecanamido)ethyl]-3-acetoxy-11-oxours-12-en-24-amidebromide(5e)

Yield: 64.1%; white solid. 1H NMR (400 MHz, CDCl3): δ 8.51 (s, 1H), 7.83–7.70 (m, 15H), 7.24 (s, 1H), 5.50 (s, 1H), 5.25 (s, 1H), 3.61–3.24 (m, 6H, -NH–CH2–CH2–NH-, −CH2Br), 2.46–1.21 (m, 37H, overlapped), 2.04 (s, 3H, −COCH3), 1.31 (s, 3H), 1.14 (s, 6H), 1.04 (s, 3H), 0.98 (m, 1H), 0.93 (s, 4H), 0.79 (s, 3H), 0.77 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 199.6 (C-11), 176.1 (−CONH−), 175.8 (−CONH−), 170.3 (−COCH3), 165.2 (C-13), 135.3 (d, J = 2.5 Hz), 133.4 (d, J = 9.9 Hz), 130.6 (d, J = 12.5 Hz), 130.3 (C-12), 118.4 (d, J = 86.1 Hz), 74.1, 60.5, 59.1, 50.3, 46.5, 45.0, 43.8, 40.9, 40.4, 39.2 (3C), 37.3, 36.1, 34.9, 33.9, 32.9, 30.8, 30.3, 30.2, 28.8 (2C), 28.7 (2C), 28.4, 27.5, 27.1, 25.7, 24.1, 24.0, 22.7, 22.4(d, J = 3.9 Hz), 21.3, 21.0 (d, J = 7.7 Hz), 20.4, 19.3, 18.3, 17.3, 13.1. HR-ESI-MS m/z: calcd for C63H88N2O5P+ [M-Br]+: 983.6425; found: 983.6403.

Biological Assays

Cell Viability Assay

Cells plated in 96-well plates were incubated 24 h. After drug treatment for 48 h, 10 μL of MTT solution (5 mg/mL) was added into each well and placed back in the incubator for 4 h. Then, the MTT-containing medium was aspirated, and 100 μL of DMSO was added per well. The well plate was shaken for 1 min to detect the absorbance produced at 570 nm using a microplate reader (Bio Tek, CA, U.S.A.). All experiments were performed three times independently.

Fluorescence Inverted Microscope

A549 cells plated in 24-well plates were treated with 2 μM 5e for 12, 24, 36 h or 5e of different concentrations (1, 2, 3, 4 μM) for 24 h. The cell samples were photographed under a fluorescence inverted microscope (Olympus, JPN).

Transmission Electron Microscopy

A549 cells plated in 100 mm dishes were incubated 48 h. Then A549 were treated with 3 μM 5e for 12 or 24 h. The cells were collected to fix in 2.5% glutaraldehyde at 4 °C for 24 h. After a series of graded alcohols dehydration steps and investment in resin, the cell samples were photographed under a transmission electron microscopy (JEM-1200EX II, JPN)

Detection of Intracellular ROS

A549 cells plated in 6-well plates were treated for specified time (the pretreatment of NAC for 2 h). After removal of the solution, 1 mL of FBS-free medium containing 10 μM DCFH-DA was used in staining for 0.5 h in the incubator, followed by two washes with PBS. The cells were collected and dissolved with PBS to be analyzed by flow cytometry (Becton Dickinson, U.S.A.).

Monitoring MPTP Opening

A549 cells plated in 20 mm glass bottom dishes were treated with 5e for 24 h. Then, A549 were cultured with 1.5 μM calcein-AM and 5 mM CoCl2 in F12K medium in the incubator for 20 min.[31] A549 cells were washed thrice using PBS and detected under a laser confocal microscopy (Zeiss, Germany) with a green fluorescence.

Measurement of Mitochondria Membrane Potential

A549 cells plated in 6-well plates were incubated with 5e for 24 h at 37 °C. A549 were cultured with the JC-1 at concentration of 2.5 μg/mL for 30 min. After removal of the solution, the cells washed twice with PBS. Fluorescence inverted microscope (Olympus, JPN) was used to take pictures of cell samples with green and red fluorescenceand. Finally, the cells were collected and dissolved with PBS to be analyzed by flow cytometry (Becton Dickinson, U.S.A.).

Flow Cytometric Determination of the Cell Cycle Stage

A549 cells plated in 6-well plates were incubated with 5e for 24 h at 37 °C. Then the cells were collected to fix in 70% alcohol at 4 °C overnight. Finally, A549 were cultured with RNase A at 37 °C for 0.5 h, and PI at 4 °C for 0.5 h. The fractions of the cells were analyzed by flow cytometry (Becton Dickinson, U.S.A.).

Analysis of Cell Apoptosis

A549 plated in 6-well plates were incubated with 5e for 24 h at 37 °C. Then A549 were cultured with binding buffer containing 3 μL of annexin V and 3 μL of PI for 0.5 h. Measurements were performed using flow cytometry (Becton Dickinson, U.S.A.).[31]

Western Blot

A549 cells treated with 5e or DMSO for 24 h were washed with PBS, cells were lysed on ice in RIPA lysis solution for 20 min and the supernatant was generated by centrifugation. The protein samples were loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membranes by electrophoresis. The membranes were closed with 5% nonfat milk in Tris-buffered saline containing Tween 20 (TBST) for 4 h and then washed thrice with TBST. Next, the membranes containing the target proteins were incubated overnight at 4 °C with the appropriate primary antibody.[31] After they were washed thrice with TBST, the corresponding secondary antibody was added, and the strips were incubated at 25 °C for 1 h. ECL luminescent solution was applied to the strips, and the immunoblotted bands were detected using a gel imager (Millipore, Germany).

Immunofluorescent Staining

A549 cells plated in 20 mm glass bottom dishes were incubated with 5e for 24 h at 37 °C. A549 were collected to fix with methanol/acetone (1:1) for 10 min and washed thrice with PBS at 4 °C. Then, the cells were stained with 2 μg/mL DAPI (Sigma-Aldrich, shanghai, China) staining solution for 10 min at 37 °C. The cell samples were photographed under a fluorescence inverted microscope.

A549 plated in 20 mm glass bottom dishes were incubated with 5e for 24 h at 37 °C. The cells were collected to fix with methanol/acetone (1:1) for 10 min and washed thrice with PBS at 4 °C. Then, 5% bovine serum albumin was closed at room temperature for 1 h. After they were washed with PBS, the primary antibody against AIF was added and incubated at 4 °C overnight, followed Alexa Fluor 594-conjugated secondary antibody was added and closed for 1 h.[31] Finally, the cell nucleus was stained with DAPI for 10 min. The cell samples were photographed under a laser confocal microscopy.