Expeditive Synthesis of Potent C20-epi-Amino Derivatives of Salinomycin against Cancer Stem-Like Cells.

As a continuation of our studies toward the development of small molecules to selectively target cancer stem cells (CSCs), a library of 18 novel derivatives of salinomycin (Sal), a naturally occurring polyether ionophore, was synthesized with a good overall yield using a one-pot Mitsunobu-Staudinger procedure. Compared to the parent structure, the newly synthesized products contained the mono- or disubstituted C20-epi-amine groups. The biological activity of these compounds was evaluated against human mammary mesenchymal HMLER CD24low/CD44high cells, a well-established model of breast CSCs, and its isogenic epithelial cell line (HMLER CD24high/CD44low) lacking CSC properties. Importantly, the vast majority of Sal derivatives were characterized by low nanomolar activities, comparing favorably with previous data in the literature. Furthermore, some of these derivatives exhibited a higher selectivity for the mesenchymal state compared to the reference Sal and ironomycin, representing a promising new series of compounds with anti-CSC activity.

Introduction

There is an urgent need to develop new cancer therapeutics.[1] Particularly interesting in this context are molecules that could preferentially target a fraction of cancer cells, known as cancer stem cells (CSCs).[2,3] These cells can be refractory to conventional chemotherapy and radiation therapy, leading to disease recurrence and metastasis.[4,5]

Using a high-throughput screening method, Gupta et al. identified the natural product salinomycin (Sal) as a selective inhibitor of breast CSCs among ∼16 000 compounds tested.[6] Since Sal was reported to be active against CSCs of various tissue types,[7,8] intensive studies have been performed to elucidate the mechanism of action (MoA) of Sal. In addition to other effects, Sal has been shown to alter mitochondrial functions, decrease ATP levels, and alter autophagy.[9−11] Moreover, Sal has also been evidenced to induce stress in the endoplasmic reticulum (ER) by altering Ca2+ homeostasis.[12]

Regarding its promising anticancer potential, many research groups have attempted to develop more effective chemical modifications of Sal,[13] particularly through the derivatization of the C1-carboxyl[14−19] or C20-hydroxyl.[20−24] Using a series of chemo- and stereocontrolled reactions, we successfully modified the C20-hydroxyl of Sal to obtain a series of potent C20-amino analogs.[20,21] The highly promising molecule in this group, ironomycin (Figure ), had been found to be approximately 10-fold more active against breast CSCs than the unmodified Sal both in vitro and in vivo.[21]

Figure 1

Molecular structures of the parent natural product salinomycin, a potent C20-amino derivative previously reported by us, and a novel series with C20-epi-amino substituents.

Mechanistically, we have provided robust evidence that Sal, ironomycin, and other analogs exert their activity by accumulating in lysosomes and sequestering iron in this organelle, which can lead to the production of reactive oxygen species (ROS), lipid peroxidation, and cell death reminiscent of ferroptosis.[21] As cells in the mesenchymal state and CSCs are addicted to iron showing a higher load,[25] this cell state has a pronounced vulnerability to cell death induced by ironomycin and Sal.

Given that Sal and ironomycin have been found to target lysosomal iron, we hypothesized that a distinct orientation of the C20-amino substituents might affect its stability and target engagement as well as efficacy and selectivity toward the CSC populations. In this context, the synthetic strategy for the diastereoselective inversion of the absolute configuration at the C20-position of Sal has been reported previously.[26−29] The esters of C20-epi-salinomycin showed a potent activity toward colorectal, gastric, and triple-negative breast cancer cells,[28] while the corresponding C20-epi-carbonates and carbamates were identified as inducers of late apoptosis in colon cancer and necrosis in prostate cancer cells.[29] Jiang and co-workers synthesized a series of C20-epi-N-acyl[26] and C20-epi-triazole[27] analogs of Sal, showing improved properties compared to Sal.

Thus, we were interested in evaluating the effect of combining the presence of an amine at C20 together with the opposite stereochemistry. Here, we describe rapid access to the C20-epi-amino derivatives of Sal (Figure and Scheme ). We evaluated a library of 18 novel Sal derivatives against a well-established model of breast CSCs together with the corresponding cells deprived of stem-like properties (Table ). We identified three compounds that have the ability to preferentially kill the cancer stem-like cells with remarkably low IC50 values that compete favorably with our reference compound ironomycin. All products were also less toxic against the normal breast cell line MCF10A compared to ironomycin, illustrating some degree of improvement (Table ).

Scheme 1

Synthesis of C20-epi-Amino Derivatives of Salinomycin

Reagents and conditions are as follows: (a) DMAP, TMSEtOH, TCFH, CH2Cl2, 0 °C to RT; (b) TPP, DIAD, DPPA, THF, RT, then TPP, H2O, THF, RT; (c) TBAF, THF, RT, then aq. Na2CO3; (d) RCHO, CH2Cl2, RT, then NaBH3CN, MeOH, RT.

Table 1

Antiproliferative Activity (IC50, μM) with Standard Deviation and Selectivity Index (SI) Values of the C20-epi-Amino Derivatives of Salinomycin Measured at 72 h in HMLER CD24low/CD44high, HMLER CD24high/CD44low, and MCF10A Cellsa

	HMLER CD24low/CD44high	HMLER CD24high/CD44low	SI (HMLER)	MCF10A
Sal	1.39 ± 0.10	4.36 ± 0.50	3.1	1.13 ± 0.14
Sal-epiNH2	20.48 ± 3.30	>50	n.d.	>12.5
ironomycin	0.17 ± 0.02	2.28 ± 0.16	13.4	0.12 ± 0.02
1	0.53 ± 0.08	3.25 ± 3.30	6.1	2.61 ± 0.78
2	0.17 ± 0.02	1.71 ± 0.40	10.1	1.56 ± 0.68
3	0.12 ± 0.02	1.83 ± 0.70	15.2	1.46 ± 0.08
4	0.07 ± 0.05	0.77 ± 0.46	11.0	1.30 ± 0.46
5	0.11 ± 0.03	1.19 ± 0.60	10.8	0.46 ± 0.27
6	0.03 ± 0.01	0.74 ± 0.29	24.7	1.50 ± 0.50
7	0.17 ± 0.04	0.69 ± 0.05	4.1	n.d.
8	0.003 ± 0.0005	0.27 ± 0.04	90.0	1.39 ± 0.18
9	0.009 ± 0.0002	0.18 ± 0.02	20.0	0.47 ± 0.19
10	0.85 ± 0.22	6.48 ± 0.50	7.6	n.d.
11	0.33 ± 0.12	2.53 ± 1.00	7.7	3.93 ± 0.29
12	0.30 ± 0.16	3.98 ± 2.20	13.3	1.70 ± 0.44
13	1.92 ± 0.50	10.02 ± 3.60	5.2	n.d.
14	0.12 ± 0.003	0.75 ± 0.12	6.2	4.67 ± 0.73
15	0.10 ± 0.01	1.24 ± 0.02	12.4	4.16 ± 0.57
16	0.07 ± 0.04	0.89 ± 0.28	12.7	1.26 ± 0.18
17	0.06 ± 0.01	0.61 ± 0.01	10.2	1.88 ± 0.11
18	0.08 ± 0.02	0.88 ± 0.54	11.0	1.86 ± 0.31

The selectivity index (SI) was defined as IC50(HMLER CD24high/CD44low)/IC50(HMLER CD24low/CD44high). Each IC50 value was determined in biological triplicate (three independent biological experiments), and each triplicate was determined in at least technical duplicate; n.d., not determined.

Results and Discussion

Synthesis

Although the synthetic access to our key precursor, C20-epi-aminosalinomycin Sal-epiNH, has been previously reported,[26] we were able to improve on this procedure. It was conveniently afforded using a one-pot Mitsunobu and Staudinger methodology (Scheme ), which is important when it comes to scaling up biologically active compounds for therapeutic use. Briefly, in the first step we masked the C1-functionality of Sal by converting the C1-carboxyl to a TMS ethyl ester. Next, using a Mitsunobu–Staudinger reaction sequence, followed by quantitative deprotection of the C1-position with TBAF, we obtained Sal-epiNH on a gram scale.

Having facile access to the starting material, a library of 18 C20-epi-amino derivatives of Sal was synthesized with a good overall yield by means of a chemoselective reductive amination, reacting Sal-epiNH with structurally diverse aldehydes (Scheme ). Specifically, we used aldehydes that differed in polarity and flexibility, selecting both aliphatic and aromatic substrates. We selected aldehydes with shorter or longer aliphatic chains, from 2 to up to 12 carbon atoms, and various benzyl aldehydes substituted at the para-position with a methyl group, a hydroxyl group, or halogens. Importantly, we were able to obtain the corresponding secondary and tertiary amines at the C20-position by varying the amount of aldehyde employed in the reaction mixture. The NMR data of analogs 1–18 obtained as sodium salts are presented in the Supporting Information. The HRMS analysis (Supporting Information) also supported the formation of the products.

Biological Evaluation

The activity of our benchmark compounds Sal and ironomycin, together with those of Sal-epiNH and the new derivatives 1–18, was measured in vitro against transformed human mammary mesenchymal HMLER CD24low/CD44high cells, an established model of human breast CSCs.[30,31] We also evaluated these products for their selectivity toward the corresponding epithelial counterparts (HMLER CD24high/CD44low), which lacked features of CSCs (Table ).

Notably, C20-epi-amino derivatives exhibited higher anti-CSC potentials compared to Sal. Of note, most of these products also showed an improved potency compared to that of the reference ironomycin, among which compounds 4, 6, 8, 9, and 16–18 were the most potent of this series. A deeper analysis of the results revealed that tertiary amine-containing Sal derivatives were essentially less potent than their corresponding secondary amine counterparts, with the exceptions of 8 and 9 bearing diethyl and dipropyl substituents, respectively. These derivatives were the most potent compounds, with remarkably low IC50 values of 3 and 9 nM against mesenchymal HMLER CD24low/CD44high cells, respectively. Remarkably, the antiproliferative activity of 8 and 9 was accompanied by their higher selectivity for the mesenchymal state, with SIs of 90.0 and 20.0, respectively. This is consistent with iron-targeting and the higher iron demand of that cell state. These data indicate the promising therapeutic potential for 8 and 9 in light of preclinical results already obtained for the less potent lead structure ironomycin. With respect to the other tertiary amines, further elongation of the aliphatic chains resulted in reduced antiproliferative activity, with the didodecyl derivative 13 identified as the least potent of the series. This argues in favor of a model whereby an optimal apolar alkane is required for this biological activity. Analogs with alkanes that are too long might be retained in lipid membranes, up to a point where their capacity to accumulate in the lumen of lysosomes and thus engage with iron is reduced. On the other hand, a critically short side chain may not allow the derivative to effectively cross lipid membranes.

Interestingly, this class of compounds showed generally lower toxicities against the normal breast cell line MCF10A (Table ), highlighting the potential for the development of these compounds to target CSCs selectively. All analogs were less active against the MCF10A cell line than reference ironomycin, indicating that an inversion of the absolute configuration at the C20-position may improve not only the antiproliferative activity against cancer cells but also the selectivity.

Although monosubstituted C20-epi-amino analogs 1 and 2 were found to be less effective than their corresponding disubstituted counterparts 8 and 9, compound 6 bearing an n-hexyl aliphatic chain showed potent antiproliferative activity and selectivity for the mesenchymal state of cells. With an IC50 value of 30 nM toward HMLER CD24low/CD44high cells and a good selectivity (SI = 24.7), this compound was the most potent among all secondary amine products. It exhibited a potency comparable to those of 8 and 9, supporting the importance of an optimal overall lipophilicity, which remains comparable for these three derivatives. It is consistent with the capacity of these derivatives to effectively cross lipid membranes to reach their functional target. Finally, C20-epi-amino derivatives 14–18 with a benzyl moiety exhibited comparable activities against HMLER CD24low/CD44high cells, with IC50 values in the range of 60–120 nM. Interestingly, in all cases the introduction of the substituents (methyl, hydroxyl, or halogen) at the para-position resulted in increased selectivity. Thus, the evaluation of the activity of other monosubstituted products (para-position versus ortho- and meta-positions) or multiple-substituted analogs might be worth considering in the future.

Conclusions

In summary, prompted by the improved therapeutic potential of C20-amino analogs of Sal (e.g., ironomycin) and the fact that C20-functionalized epimers retain their efficacy in vitro, we synthesized a series of 18 C20-epi-amino derivatives of Sal that combined both structural modifications using a straightforward and scalable protocol.

All derivatives were assessed for their antiproliferative activity and selectivity toward a well-established model of mesenchymal CSCs (HMLER CD24low/CD44high) together with their epithelial counterparts (HMLER CD24high/CD44low) lacking CSC properties. Most of these derivatives were found to be more potent and more selective against the mesenchymal state compared to the references Sal and ironomycin. Specifically, compounds 6, 8, and 9 were identified to be particularly interesting in this context, with IC50 values of 30, 3, and 9 nM, respectively, together with their outstanding selectivities (SIs between 20.0 and 90.0). Concerning a structure–activity relationship (SAR), we found the following: (i) monosubstituted C20-epi-amino derivatives of Sal are essentially more active than their corresponding disubstituted counterparts; (ii) with respect to secondary amine products, n-pentyl and n-hexyl substituents are more potent against the mesenchymal state, (iii) regarding tertiary amine derivatives, elongation of the aliphatic chains results in a decrease of the antiproliferative activity; and (iv) the introduction of the nonpolar or polar substituent at the para-position of the benzyl motif increases the selectivity.

Here, we have reported a convenient synthetic scheme that can readily afford potent derivatives in a short number of steps. Importantly, we describe the most potent and selective derivatives of Sal reported so far. Future work will involve the preclinical evaluation of the most promising compounds in various cancer settings.

Experimental Section

General Information

Detailed descriptions of the general procedures, equipment, and measurement parameters can be found in the Supporting Information. C20-epi-aminosalinomycin (Sal-epiNH) was resynthesized following the previously reported procedure,[26] with slight modifications.

Briefly, in the first step, to a stirred solution of salinomycin (Sal) (3.00 g, 1.0 equiv) in 50 mL of CH2Cl2 in an ice bath were added DMAP (2.38 g, 5.0 equiv), TMSEtOH (2.77 g, 6.0 equiv), and TCFH (1.31 g, 1.2 equiv). The resulting mixture was stirred at RT overnight. The reaction mixture was then concentrated under reduced pressure. Purification on silica gel using the CombiFlash system (0 → 40% EtOAc/n-hexane) gave the C1-EtTMS ester of Sal as a yellow oil (1.70 g, 50% yield).

Next, to a solution of the C1-EtTMS ester of Sal (1.70 g, 1.0 equiv) in 20 mL of anhydrous THF in an ice bath was added TPP (784 mg, 1.5 equiv). After 20 min, DIAD (481 mg, 1.2 equiv) was slowly added to the mixture, followed by DPPA (605 mg, 1.1 equiv). The solution was stirred at RT for 24 h. After this time, TPP (1.57 g, 3.0 equiv) was added to the mixture in one portion, followed by the addition of 0.5 mL of water. The mixture was stirred at RT for next 24 h, and the reaction progress was monitored by TLC. The reaction mixture was then concentrated under reduced pressure. Purification on silica gel using the CombiFlash system (0 → 50% acetone/CHCl3) gave the C1-EtTMS ester of C20-epi-aminosalinomycin as a yellow oil (716 mg, 42% yield).

Finally, the C1-masked C20-epi-aminosalinomycin (716 mg, 1.0 equiv) was dissolved in 15 mL of anhydrous THF at RT. A 1.0 M solution of TBAF in THF (2.53 mL, 3.0 equiv) was then added dropwise. The solution was left to be stirred at RT for next 24 h. The reaction mixture was then concentrated under reduced pressure. Purification on silica gel using the CombiFlash system (0 → 50% acetone/CHCl3) gave the reaction product as a yellow oil. The product was then dissolved in CH2Cl2 and washed with a 0.1 M solution of Na2CO3. The separated organic layers were dried over MgSO4 and concentrated under reduced pressure. The residue was then evaporated several times with n-pentane to quantitatively give the sodium salt of Sal-epiNH as a white amorphous solid (648 mg). The data for the obtained material were in accordance with the literature.

General Procedure for the Preparation of Analogs 1–18

To a stirred solution of Sal-epiNH (50 mg, 0.07 mmol, 1.0 equiv) in 5 mL of CH2Cl2 was added the corresponding aldehyde (1.0 equiv for singly substituted analogs 1–7 and 14–18 or 5.0 equiv for the doubly substituted counterparts 8–13). The solution was stirred at RT for 24 h. After that time, a solution of NaBH3CN (5 mg, 0.08 mmol, 1.2 equiv; in 2 mL of MeOH) was added to the mixture drop by drop. The reaction mixture was stirred further at RT for an additional 30 min. Next, the solvent was evaporated under reduced pressure. Purification of the crude material on silica gel using the CombiFlash system gave the C20-epi-amino analogs 1–18 as white amorphous solids after evaporation with n-pentane. The NMR and HRMS spectra of compounds 1–18 are included in the Supporting Information (Figures S1–S54).

Compound 1

Yield: 35 mg, 70%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.57 in 60% acetone/CHCl3. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.31 (dd, J = 10.7, 5.6 Hz, 1H), 6.12 (d, J = 10.8 Hz, 1H), 4.29 (q, J = 6.8 Hz, 1H), 4.12 (d, J = 10.4 Hz, 1H), 3.77 (dt, J = 13.8, 6.9 Hz, 1H), 3.64 (dd, J = 10.1, 2.1 Hz, 1H), 3.60 (d, J = 10.2 Hz, 1H), 3.37 (dd, J = 11.9, 2.1 Hz, 1H), 2.86 (d, J = 5.6 Hz, 1H), 2.81–2.73 (m, 2H), 2.72–2.68 (m, 1H), 2.67–2.57 (m, 2H), 2.00–0.50 (m, 60H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.4, 184.1, 129.9, 122.7, 110.1, 99.7, 89.2, 76.4, 76.3, 75.5, 75.1, 71.8, 70.2, 67.7, 56.1, 55.5, 51.6, 50.9, 42.8, 40.7, 39.5, 37.5, 36.5, 33.4, 33.1, 33.0, 29.8, 28.6, 28.3, 27.4, 24.2, 21.4, 20.6, 17.9, 17.3, 16.5, 16.4, 15.1, 13.5, 12.8, 12.4, 11.0, 7.0, 6.8 ppm; FT-IR (KBr) 3319, 2963, 2935, 2875, 1714, 1568, 1460, 1407 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C44H75NO10Na+ 800.5289, found 800.5283; [M – Na + 2H]+ Calcd for C44H76NO10+ 778.5464, found 778.5460.

Compound 2

Yield: 35 mg, 66%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.23 in 66% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.27 (dd, J = 10.7, 5.6 Hz, 1H), 6.08 (d, J = 10.8 Hz, 1H), 4.28 (q, J = 6.7 Hz, 1H), 4.11 (d, J = 10.2 Hz, 1H), 3.78 (dd, J = 11.1, 4.8 Hz, 1H), 3.64 (dd, J = 16.6, 6.3 Hz, 2H), 3.37 (dd, J = 11.9, 2.1 Hz, 1H), 2.97–2.87 (m, 2H), 2.79–2.68 (m, 2H), 2.68–2.61 (m, 1H), 2.11–2.04 (m, 2H), 2.00–0.50 (m, 61H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.6, 184.1, 130.3, 122.0, 110.4, 99.6, 89.22, 76.5, 76.4, 75.4, 75.1, 71.8, 70.2, 67.8, 56.1, 53.3, 51.6, 50.9, 47.6, 40.7, 39.5, 37.7, 36.5, 33.3, 33.2, 33.0, 30.0, 28.6, 28.2, 27.5, 24.8, 24.1, 23.2, 21.3, 20.6, 17.9, 17.4, 16.6, 15.1, 13.5, 12.8, 12.4, 11.0, 7.1, 6.8 ppm; FT-IR (KBr) 3289, 2962, 2933, 2874, 1713, 1660, 1568, 1459, 1407 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C45H77NO10Na+ 814.5440, found 814.5426; [M – Na + 2H]+ Calcd for C45H78NO10+ 792.5620, found 792.5619.

Compound 3

Yield: 36 mg, 68%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.29 in 66% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.31 (dd, J = 10.7, 5.6 Hz, 1H), 6.11 (d, J = 10.8 Hz, 1H), 4.29 (q, J = 6.7 Hz, 1H), 4.12 (d, J = 10.4 Hz, 1H), 3.78 (dd, J = 11.1, 4.8 Hz, 1H), 3.64 (dd, J = 9.9, 1.8 Hz, 1H), 3.60 (d, J = 10.2 Hz, 1H), 3.37 (dd, J = 11.9, 2.1 Hz, 1H), 2.84 (d, J = 5.6 Hz, 1H), 2.80–2.69 (m, 3H), 2.68–2.62 (m, 1H), 2.60–2.52 (m, 1H), 2.00–0.50 (m, 64H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.4, 184.1, 130.0, 122.6, 110.1, 99.7, 89.2, 76.4, 76.3, 75.5, 75.0, 71.8, 70.2, 67.7, 56.1, 55.8, 51.6, 50.9, 48.3, 40.7, 39.4, 37.5, 36.5, 33.5, 33.4, 33.1, 33.0, 29.8, 28.6, 28.3, 27.4, 24.2, 21.4, 20.8, 20.6, 17.8, 17.3, 16.5, 15.1, 14.3, 13.5, 12.8, 12.4, 11.0, 7.0, 6.8 ppm; FT-IR (KBr) 3288, 2960, 2931, 2873, 1713, 1661, 1567, 1459, 1406 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C46H79NO10Na+ 828.5596, found 828.5577; [M – Na + 2H]+ Calcd for C46H80NO10+ 806.5777, found 806.5772.

Compound 4

Yield: 40 mg, 74%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.50 in 66% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.35 (dd, J = 10.7, 5.6 Hz, 1H), 6.15 (d, J = 10.8 Hz, 1H), 4.33 (q, J = 6.8 Hz, 1H), 4.16 (d, J = 9.4 Hz, 1H), 3.81 (dd, J = 11.1, 4.9 Hz, 1H), 3.72–3.60 (m, 2H), 3.41 (dd, J = 12.0, 2.1 Hz, 1H), 2.85 (d, J = 5.6 Hz, 1H), 2.82–2.73 (m, 2H), 2.72–2.65 (m, 1H), 2.62 (dd, J = 11.3, 6.7 Hz, 1H), 2.41 (dd, J = 11.3, 6.6 Hz, 1H), 2.15–2.09 (m, 2H), 2.05–0.50 (m, 62H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.3, 184.1, 130.0, 122.5, 110.2, 99.7, 89.2, 76.4, 76.3, 75.5, 75.0, 71.8, 70.2, 67.7, 56.7, 56.0 (2C), 51.6, 50.8, 40.7, 39.4, 37.6, 36.5, 33.4, 33.2, 33.0, 29.8, 29.7, 28.6, 28.3, 27.4, 24.2, 21.3, 20.8, 20.7, 20.5, 17.8, 17.4, 16.5, 15.1, 13.5, 12.8, 12.4, 11.0, 7.0, 6.8 ppm; FT-IR (KBr) 3289, 2960, 2931, 2874, 1713, 1660, 1568, 1459, 1407 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C46H79NO10Na+ 828.5596, found 828.5578; [M – Na + 2H]+ Calcd for C46H80NO10+ 806.5777, found 806.5771.

Compound 5

Yield: 45 mg, 82%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.30 in 66% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.23 (dd, J = 10.7, 5.6 Hz, 1H), 6.03 (d, J = 10.8 Hz, 1H), 4.21 (q, J = 6.7 Hz, 1H), 4.04 (d, J = 10.4 Hz, 1H), 3.70 (dd, J = 11.1, 4.8 Hz, 1H), 3.62–3.49 (m, 2H), 3.29 (dd, J = 11.9, 2.1 Hz, 1H), 2.76 (d, J = 5.6 Hz, 1H), 2.71–2.60 (m, 3H), 2.60–2.53 (m, 1H), 2.48 (ddd, J = 11.3, 7.5, 6.4 Hz, 1H), 2.02–1.96 (m, 2H), 1.90–0.50 (m, 64H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.4, 184.1, 130.0, 122.6, 110.1, 99.7, 89.2, 76.5, 76.3, 75.5, 75.1, 71.8, 70.2, 67.7, 56.1, 55.8, 51.6, 50.9, 48.6, 40.7, 39.5, 37.5, 36.5, 33.4, 33.2, 33.0, 31.1, 29.9, 29.8, 28.6, 28.3, 27.5, 24.2, 23.1, 21.4, 20.6, 17.9, 17.4, 16.6, 15.1, 14.4, 13.5, 12.8, 12.4, 11.0, 7.0, 6.8 ppm; FT-IR (KBr) 3292, 2961, 2932, 2873, 1713, 1660, 1567, 1459, 1406 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C47H81NO10Na+ 842.5753, found 842.5728; [M – Na + 2H]+ Calcd for C47H82NO10+ 820.5933, found 820.5926.

Compound 6

Yield: 37 mg, 70%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.31 in 66% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.31 (dd, J = 10.7, 5.6 Hz, 1H), 6.11 (d, J = 10.7 Hz, 1H), 4.29 (q, J = 6.7 Hz, 1H), 4.12 (d, J = 10.3 Hz, 1H), 3.78 (dd, J = 11.0, 4.9 Hz, 1H), 3.64 (dd, J = 10.0, 1.7 Hz, 1H), 3.60 (d, J = 10.1 Hz, 1H), 3.37 (dd, J = 11.9, 1.7 Hz, 1H), 2.84 (d, J = 5.6 Hz, 1H), 2.79–2.69 (m, 3H), 2.68–2.62 (m, 1H), 2.56 (dt, J = 11.3, 6.8 Hz, 1H), 2.08–2.02 (m, 2H), 2.00–0.50 (m, 66H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 217.8, 183.5, 129.4, 122.0, 109.5, 99.1, 88.6, 75.9, 75.7, 74.9, 74.5, 71.2, 69.6, 67.1, 55.5, 55.2, 51.0, 50.3, 48.0, 40.1, 38.9, 36.9, 35.9, 32.8, 32.6, 32.4, 31.7, 30.7, 29.2, 28.0, 27.7, 26.9, 26.8, 23.6, 22.6, 20.8, 20.0, 17.3, 16.8, 16.0, 14.5, 13.8, 12.9, 12.3, 11.8, 10.4, 6.5, 6.2 ppm; FT-IR (KBr) 3297, 2958, 2930, 2872, 2858, 1714, 1668, 1565, 1459, 1406 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C48H83NO10Na+ 856.5909, found 856.5889; [M – Na + 2H]+ Calcd for C48H84NO10+ 834.6090, found 834.6084.

Compound 7

Yield: 60 mg, 85%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.40 in 66% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.31 (dd, J = 10.7, 5.6 Hz, 1H), 6.11 (d, J = 10.8 Hz, 1H), 4.28 (dd, J = 13.5, 6.7 Hz, 1H), 4.12 (d, J = 10.4 Hz, 1H), 3.77 (dd, J = 11.1, 4.7 Hz, 1H), 3.64 (dd, J = 10.1, 2.0 Hz, 1H), 3.59 (d, J = 10.2 Hz, 1H), 3.37 (dd, J = 11.9, 2.1 Hz, 1H), 2.83 (d, J = 5.6 Hz, 1H), 2.79–2.73 (m, 1H), 2.74–2.68 (m, 2H), 2.68–2.61 (m, 1H), 2.55 (ddd, J = 11.3, 7.4, 6.3 Hz, 1H), 2.10–2.03 (m, 2H), 2.00–0.50 (m, 78H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.1, 183.9, 129.7, 122.4, 109.9, 99.5, 89.0, 76.2, 76.0, 75.3, 74.8, 71.6, 70.0, 67.4, 55.8, 55.5, 51.3, 50.6, 48.3, 40.5, 39.2, 37.3, 36.3, 33.2, 32.9, 32.8, 32.3, 31.2, 30.0 (3C), 29.9, 29.7, 29.6, 28.4, 28.1, 27.5, 27.2, 24.0, 23.1, 21.1, 20.3, 17.6, 17.2, 16.3, 14.9, 14.3, 13.3, 12.6, 12.2, 10.8, 6.8, 6.6 ppm, one signal overlapped; FT-IR (KBr) 3288, 2960, 2927, 2854, 1713, 1568, 1459, 1406 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C54H95NO10Na+ 940.6848, found 940.6829; [M – Na + 2H]+ Calcd for C54H96NO10+ 918.7029, found 918.7024.

Compound 8

Yield: 41 mg, 76%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.42 in 33% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.28 (dd, J = 11.0, 1.6 Hz, 1H), 5.98 (dd, J = 11.0, 4.2 Hz, 1H), 4.22 (q, J = 6.7 Hz, 1H), 4.11 (d, J = 10.2 Hz, 1H), 3.79 (dd, J = 11.1, 4.8 Hz, 1H), 3.63 (d, J = 10.1 Hz, 2H), 3.38 (dd, J = 12.0, 2.1 Hz, 2H), 2.75 (td, J = 11.2, 3.3 Hz, 1H), 2.69–2.64 (m, 2H), 2.56–2.43 (m, 3H), 2.31 (dq, J = 13.4, 6.7 Hz, 2H), 2.20–0.50 (m, 61H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.4, 183.7, 125.6, 123.9, 110.9, 98.0, 87.3, 76.1, 75.8, 75.1, 74.9, 71.2, 69.5, 67.1, 56.4, 55.7, 51.0, 50.1, 44.4, 40.4, 38.6, 36.7, 35.9, 33.0, 32.6, 32.4, 29.8, 28.0, 27.6, 26.9 (2C), 23.6, 21.1, 19.9, 17.3, 16.8, 15.7, 14.6, 13.7 (2C), 12.8, 12.2, 12.0, 10.4, 6.5, 6.2 ppm; FT-IR (KBr) 3300, 2962, 2933, 2874, 1714, 1566, 1458, 1405 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C46H79NO10Na+ 828.5596, found 828.5582; [M – Na + 2H]+ Calcd for C46H80NO10+ 806.5777, found 806.5774.

Compound 9

Yield: 45 mg, 79%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.57 in 33% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.28 (dd, J = 11.0, 1.7 Hz, 1H), 6.00 (dd, J = 11.0, 4.2 Hz, 1H), 4.22 (q, J = 6.8 Hz, 1H), 4.11 (d, J = 10.3 Hz, 1H), 3.79 (dd, J = 11.1, 4.8 Hz, 1H), 3.68–3.59 (m, 2H), 3.37 (dd, J = 12.0, 2.1 Hz, 1H), 3.30 (dd, J = 4.2, 1.8 Hz, 1H), 2.77–2.70 (m, 1H), 2.69–2.63 (m, 2H), 2.52–2.41 (m, 1H), 2.40–2.25 (m, 4H), 2.00–0.50 (m, 65H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 219.0, 184.2, 126.3, 124.4, 111.6, 98.7, 87.9, 76.7, 76.4, 75.6, 75.4, 71.8, 70.0, 67.7, 57.9, 56.3, 53.7 (2C), 51.6, 50.8, 41.1, 39.2, 37.3, 36.5, 33.6, 33.3, 33.0, 30.4, 28.6, 28.2, 27.5, 24.1, 22.1 (2C), 21.7, 20.5, 17.9, 17.5, 16.3, 15.2, 13.4, 12.8, 12.6, 12.1 (2C), 11.0, 7.1, 6.7 ppm; FT-IR (KBr) 3297, 2960, 2933, 2873, 1714, 1566, 1459, 1406 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C48H83NO10Na+ 856.5909, found 856.5892; [M – Na + 2H]+ Calcd for C48H84NO10+ 834.6090, found 834.6089.

Compound 10

Yield: 30 mg, 55%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.59 in 33% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.29 (dd, J = 11.0, 1.7 Hz, 1H), 6.01 (dd, J = 11.0, 4.2 Hz, 1H), 4.20 (q, J = 6.7 Hz, 1H), 4.11 (d, J = 10.2 Hz, 1H), 3.79 (dd, J = 11.1, 4.8 Hz, 1H), 3.66–3.60 (m, 2H), 3.37 (dd, J = 12.0, 2.1 Hz, 1H), 3.30 (dd, J = 4.2, 1.7 Hz, 1H), 2.77–2.64 (m, 3H), 2.51–2.35 (m, 3H), 2.34–2.25 (m, 2H),2.00–0.50 (m, 69H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.9, 184.2, 126.1, 124.4, 111.6, 98.7, 87.9, 76.9, 76.4, 75.6, 75.3, 71.8, 69.9, 67.6, 57.7, 56.2, 51.6, 51.4 (2C), 50.8, 41.1, 39.2, 37.4, 36.5, 33.6, 33.3, 32.9, 31.5 (2C), 30.2, 28.6, 28.2, 27.5, 24.2, 21.7, 21.2 (2C), 20.5, 17.9, 17.5, 16.3, 15.2, 14.5 (2C), 13.4, 12.9, 12.6, 11.0, 7.1, 6.7 ppm; FT-IR (KBr) 3300, 2960, 2933, 2873, 2860, 1712, 1566, 1457, 1406 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C50H87NO10Na+ 884.6222, found 884.6212; [M – Na + 2H]+ Calcd for C50H88NO10+ 862.6403, found 862.6410.

Compound 11

Yield: 35 mg, 60%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.62 in 33% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.29 (dd, J = 11.0, 1.6 Hz, 1H), 6.01 (dd, J = 11.0, 4.2 Hz, 1H), 4.19 (q, J = 6.7 Hz, 1H), 4.11 (d, J = 10.3 Hz, 1H), 3.79 (dd, J = 11.0, 4.7 Hz, 1H), 3.69–3.60 (m, 2H), 3.37 (dd, J = 12.0, 1.9 Hz, 1H), 3.29 (dd, J = 4.1, 1.6 Hz, 1H), 2.70 (tdd, J = 10.3, 9.3, 3.1 Hz, 3H), 2.45 (ddd, J = 22.3, 13.0, 7.1 Hz, 2H), 2.39–2.25 (m, 3H), 2.00–0.50 (m, 73H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.9, 184.2, 126.1, 124.4, 111.6, 98.7, 87.9, 77.0, 76.4, 75.6, 75.3, 71.8, 69.9, 67.7, 57.8, 56.3, 51.7 (2C), 51.6, 50.8, 41.1, 39.2, 37.4, 36.5, 33.6, 33.3, 32.9, 30.3 (2C), 30.1, 29.0 (2C), 28.6, 28.2, 27.5, 24.2, 23.2 (2C), 21.7, 20.5, 17.9, 17.5, 16.3, 15.2, 14.5 (2C), 13.4, 12.9, 12.6, 11.0, 7.1, 6.7 ppm; FT-IR (KBr) 3297, 2959, 2931, 2872, 2860, 1714, 1567, 1459, 1406 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C52H91NO10Na+ 912.6535, found 912.6524; [M – Na + 2H]+ Calcd for C52H92NO10+ 890.6716, found 890.6724.

Compound 12

Yield: 50 mg, 82%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.64 in 33% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.29 (dd, J = 11.0, 1.6 Hz, 1H), 6.01 (dd, J = 11.0, 4.2 Hz, 1H), 4.20 (q, J = 6.7 Hz, 1H), 4.12 (d, J = 10.3 Hz, 1H), 3.78 (dt, J = 13.2, 6.5 Hz, 1H), 3.68–3.59 (m, 2H), 3.38 (dd, J = 12.0, 1.9 Hz, 1H), 3.29 (dd, J = 4.1, 1.6 Hz, 1H), 2.72 (ddd, J = 11.4, 8.5, 3.4 Hz, 2H), 2.68–2.63 (m, 1H), 2.52–2.43 (m, 1H), 2.43–2.36 (m, 2H), 2.36–2.27 (m, 2H), 2.00–0.50 (m, 77H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.9, 184.2, 126.1, 124.4, 111.6, 98.7, 87.9, 77.0, 76.4, 75.6, 75.3, 71.8, 69.9, 67.7, 57.8, 56.3, 51.7 (2C), 51.6, 50.8, 41.1, 39.2, 37.4, 36.5, 33.6, 33.3, 33.0, 32.5 (2C), 30.0, 29.3 (2C), 28.6, 28.2, 27.8 (2C), 27.5, 24.2, 23.4 (2C), 21.7, 20.5, 17.9, 17.6, 16.3, 15.2, 14.5 (2C), 13.5, 12.9, 12.6, 11.0, 7.1, 6.8 ppm; FT-IR (KBr) 3286, 2960, 2931, 2873, 2859, 1713, 1568, 1459, 1407 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C54H95NO10Na+ 940.6848, found 940.6839; [M – Na + 2H]+ Calcd for C54H96NO10+ 918.7029, found 918.7038.

Compound 13

Yield: 21 mg, 45%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.79 in 33% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 6.28 (dd, J = 11.0, 1.6 Hz, 1H), 6.00 (dd, J = 11.0, 4.2 Hz, 1H), 4.20 (q, J = 6.6 Hz, 1H), 4.11 (d, J = 10.3 Hz, 1H), 3.79 (dd, J = 11.0, 4.6 Hz, 1H), 3.71–3.59 (m, 2H), 3.38 (dd, J = 12.0, 1.9 Hz, 1H), 3.29 (dd, J = 4.1, 1.5 Hz, 1H), 2.75 (dd, J = 11.1, 3.2 Hz, 1H), 2.71–2.64 (m, 2H), 2.47 (dd, J = 12.5, 3.6 Hz, 1H), 2.43–2.38 (m, 1H), 2.36 (d, J = 8.1 Hz, 1H), 2.34–2.25 (m, 2H), 2.00–0.50 (m, 101H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.9, 184.2, 126.1, 124.4, 111.6, 98.7, 87.9, 76.9, 76.4, 75.6, 75.3, 71.7, 69.9, 67.6, 57.7, 56.3, 51.7 (2C), 51.6, 50.8, 41.1 (2C), 39.2, 37.4, 36.5, 33.6, 33.3, 32.9, 32.5 (2C), 30.6, 30.4 (3C), 30.3, 30.2 (4C), 30.0 (3C), 29.9, 29.3, 28.6, 28.2, 28.1 (2C), 27.5, 24.2, 23.3 (2C), 21.7, 20.5, 17.9, 17.6, 16.3, 15.2, 14.5 (2C), 13.4, 12.9, 12.6, 11.0, 7.1, 6.8 ppm; FT-IR (KBr) 3295, 2958, 2926, 2872, 2854, 1714, 1566, 1459, 1405 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C66H119NO10Na+ 1108.8726, found 1108.8715; [M – Na + 2H]+ Calcd for C66H120NO10+ 1086.8907, found 1086.8920.

Compound 14

Yield: 48 mg, 75%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.65 in 50% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 7.19 (d, J = 4.4 Hz, 4H), 7.12 (dt, J = 5.9, 4.2 Hz, 1H), 6.28 (dd, J = 10.7, 5.6 Hz, 1H), 6.09 (d, J = 10.7 Hz, 1H), 4.18 (q, J = 6.7 Hz, 1H), 4.04 (d, J = 10.4 Hz, 1H), 3.83 (d, J = 12.7 Hz, 1H), 3.74–3.63 (m, 2H), 3.61–3.50 (m, 2H), 3.26 (dd, J = 11.8, 1.8 Hz, 1H), 2.90 (d, J = 5.5 Hz, 1H), 2.73–2.65 (m, 1H), 2.64–2.54 (m, 2H), 2.00–0.50 (m, 57H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.4, 184.1, 141.5, 129.3, 128.8 (4C), 127.3, 123.1, 110.0, 99.7, 89.3, 76.5, 76.3, 75.5, 75.0, 71.8, 70.2, 67.7, 56.1, 55.1, 52.5, 51.6, 50.9, 40.7, 39.4, 37.6, 36.5, 33.4, 33.1, 33.0, 29.7, 28.6, 28.2, 27.4, 24.2, 21.3, 20.5, 17.8, 17.4, 16.5, 15.1, 13.5, 12.9, 12.4, 11.0, 7.0, 6.8 ppm; FT-IR (KBr) 3317, 2961, 2933, 2874, 1713, 1567, 1495, 1458, 1406 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C49H77NO10Na+ 862.5440, found 862.5432; [M – Na + 2H]+ Calcd for C49H78NO10+ 840.5620, found 840.5628.

Compound 15

Yield: 43 mg, 76%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.60 in 50% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CD2Cl2) δ 7.12 (td, J = 7.9, 1.6 Hz, 1H), 6.98 (d, J = 6.3 Hz, 1H), 6.77–6.72 (m, 2H), 6.43 (dd, J = 10.7, 5.5 Hz, 1H), 6.29 (d, J = 10.8 Hz, 1H), 4.29 (q, J = 6.7 Hz, 1H), 4.13 (d, J = 10.5 Hz, 1H), 4.05 (d, J = 13.5 Hz, 1H), 3.97 (d, J = 13.4 Hz, 1H), 3.77 (dd, J = 11.1, 4.8 Hz, 1H), 3.63 (dd, J = 16.2, 6.0 Hz, 2H), 3.38 (dd, J = 11.9, 1.9 Hz, 1H), 3.10 (d, J = 5.4 Hz, 1H), 2.78 (dd, J = 11.0, 2.9 Hz, 1H), 2.73 (dd, J = 10.8, 2.4 Hz 1H), 2.66 (dd, J = 10.2, 7.4 Hz, 1H), 2.00–0.50 (m, 58H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 218.4, 184.3, 158.4, 129.4, 129.1, 127.7, 125.1, 123.5, 119.7, 116.7, 108.8, 99.9, 89.8, 76.5, 76.2, 75.8, 75.0, 71.8, 70.2, 67.8, 56.0, 55.1, 51.6, 51.2, 50.8, 40.5, 39.3, 37.8, 36.5, 33.4, 33.1, 32.9, 29.7, 28.6, 28.3, 27.4, 24.3, 21.4, 20.5, 17.8, 17.4, 16.5, 15.1, 13.5, 12.9, 12.4, 10.9, 7.0, 6.7 ppm; FT-IR (KBr) 3433, 3320, 2953, 2932, 2872, 1713, 1563, 1456, 1428, 1405 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C49H77NO10Na+ 878.5389, found 878.5381; [M – Na + 2H]+ Calcd for C49H79NO10+ 856.5569, found 856.5575.

Compound 16

Yield: 51 mg, 89%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.63 in 50% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CDCl3) δ 7.15 (d, J = 7.9 Hz, 2H), 7.08 (d, J = 8.2 Hz, 2H), 6.49 (dd, J = 10.7, 5.6 Hz, 1H), 6.19 (d, J = 10.8 Hz, 1H), 4.39 (dd, J = 13.8, 6.9 Hz, 1H), 4.24 (d, J = 10.3 Hz, 1H), 3.90 (dd, J = 11.0, 4.9 Hz, 1H), 3.85 (d, J = 12.5 Hz, 1H), 3.72 (dd, J = 15.5, 7.2 Hz, 2H), 3.56 (d, J = 10.0 Hz, 1H), 3.34–3.29 (m, 1H), 3.03 (d, J = 5.7 Hz, 1H), 2.87 (td, J = 11.0, 3.3 Hz, 1H), 2.68 (dd, J = 11.0, 2.6 Hz, 1H), 2.64–2.59 (m, 1H), 2.31 (s, 3H), 2.20–0.50 (m, 57H) ppm; 13C NMR (101 MHz, CDCl3) δ 216.8, 184.1, 137.7, 136.3, 129.0 (2C), 128.2 (2C), 122.6, 109.3, 99.1, 88.7, 75.7, 75.6, 74.8, 74.5, 71.5, 69.8, 67.1, 55.2, 54.6, 51.6, 51.2, 50.4, 40.1, 38.9, 37.2, 36.0, 32.9, 32.5, 32.4, 29.7, 29.0, 27.9 (2C), 26.9, 23.9, 21.1, 20.8, 19.9, 17.5, 17.0, 16.0, 14.6, 13.2, 12.5, 11.8, 10.6, 6.7, 6.5 ppm; FT-IR (KBr) 3295, 2961, 2931, 2874, 1713, 1567, 1458, 1406 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C50H79NO11Na+ 876.5596, found 876.5593; [M – Na + 2H]+ Calcd for C50H80NO11+ 854.5777, found 854.5777.

Compound 17

Yield: 70 mg, 88%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.67 in 50% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CDCl3) δ 7.22 (dd, J = 8.5, 5.6 Hz, 2H), 6.95 (dd, J = 12.0, 5.3 Hz, 2H), 6.48 (dd, J = 10.7, 5.6 Hz, 1H), 6.20 (d, J = 10.8 Hz, 1H), 4.37 (q, J = 6.6 Hz, 1H), 4.23 (d, J = 10.3 Hz, 1H), 3.89 (dd, J = 11.1, 4.9 Hz, 1H), 3.85 (d, J = 11.9 Hz, 1H), 3.74–3.68 (m, 2H), 3.56 (d, J = 10.1 Hz, 1H), 3.30 (dd, J = 11.9, 1.9 Hz, 1H), 3.00 (d, J = 5.6 Hz, 1H), 2.87 (td, J = 10.9, 3.1 Hz, 1H), 2.67 (dt, J = 7.1, 3.6 Hz, 1H), 2.62 (dd, J = 10.2, 7.4 Hz, 1H), 2.20–0.50 (m, 57H) ppm; 13C NMR (101 MHz, CDCl3) δ 216.8, 184.1, 161.9 (d, J = 191.9 Hz, 1C), 136.4, 129.9 (d, J = 6.2 Hz, 1C), 128.8, 122.8, 115.1 (d, J = 20.2 Hz, 1C), 109.3, 99.1, 88.8, 75.7, 75.7, 74.9, 74.5, 71.6, 69.8, 67.2, 55.3, 54.3, 51.3, 51.0, 50.4, 40.1, 38.9, 37.3, 36.0, 34.1, 32.9, 32.5, 32.4, 29.0, 27.9, 26.9, 24.0, 22.3, 20.9, 20.0, 17.5, 17.0, 16.0, 14.6, 14.1, 13.2, 12.5, 11.8, 10.6, 6.7, 6.5 ppm; FT-IR (KBr) 3321, 2962, 2932, 2874, 1713, 1567, 1510, 1459, 1406 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C49H76FNO10Na+ 880.5345, found 880.5337; [M – Na + 2H]+ Calcd for C49H77FNO10+ 858.5526, found 858.5532.

Compound 18

Yield: 34 mg, 58%. Isolated as a white amorphous solid, >95% pure by NMR and a single spot by TLC; R = 0.58 in 50% EtOAc/n-hexane. Strain green with PMA; 1H NMR (400 MHz, CDCl3) δ 7.18 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 6.42 (dd, J = 10.7, 5.6 Hz, 1H), 6.14 (d, J = 10.8 Hz, 1H), 4.30 (q, J = 6.7 Hz, 1H), 4.17 (d, J = 10.2 Hz, 1H), 3.83 (dd, J = 10.6, 4.2 Hz, 1H), 3.78 (d, J = 12.9 Hz, 1H), 3.68–3.61 (m, 2H), 3.49 (d, J = 10.1 Hz, 1H), 3.23 (d, J = 11.6 Hz, 1H), 2.93 (d, J = 5.6 Hz, 1H), 2.80 (td, J = 10.9, 3.1 Hz, 1H), 2.61 (dd, J = 11.0, 2.4 Hz, 1H), 2.55 (dd, J = 10.2, 7.4 Hz, 1H), 2.20–0.50 (m, 57H) ppm; 13C NMR (101 MHz, CDCl3) δ 216.7, 184.2, 139.1, 132.5, 129.7 (2C), 128.7, 128.4 (2C), 122.8, 109.2, 99.0, 88.7, 75.7, 75.6, 74.9, 74.5, 71.5, 69.8, 67.1, 55.2, 54.3, 51.2, 51.0, 50.4, 40.1, 38.8, 37.3, 35.9, 32.9, 32.5, 32.4, 29.7, 28.9, 27.9 (2C), 23.9, 20.8, 19.9, 17.4, 17.0, 16.0, 14.6, 13.2, 12.5, 11.8, 10.6, 6.7, 6.5 ppm; FT-IR (KBr) 3322, 2962, 2932, 2874, 1713, 1663, 1567, 1492, 1459, 1407 cm–1. HRMS (ESI+) m/z [M + H]+ Calcd for C49H76ClNO10Na+ 896.5050, found 896.5043; [M – Na + 2H]+ Calcd for C49H77ClNO10+ 874.5231, found 874.5238.

Cell Culture

HMLER cells naturally repressing E-cadherin, obtained from human mammary epithelial cells infected with a retrovirus carrying hTERT, SV40, and the oncogenic allele H-rasV12, were cultured in DMEM/F12 (Gibco, 31331–028) supplemented with 10% FBS, 10 μg/mL insulin (Sigma-Aldrich, I0516), 0.5 μg/mL hydrocortisone (Sigma-Aldrich, H0888), and 0.5 μg/mL puromycin (Life Technologies, A11138-02); cells were a generous gift from Alain Puisieux (INSERM). All cells were incubated at 37 °C with 5% CO2. HMLER CD44low/high cells stained with CD24-APC and CD44-PE antibodies were sorted by FACS using an Aria IIu (BD Biosciences) to obtain isolated CD24low/CD44high and CD24high/CD44low cell populations. HMLER CD24low/CD44high cells were supplemented with 10 ng/mL human epidermal growth factor (EGF, Miltenyi Biotec, 130-093-750, 100 ng/mL), while HMLER CD24high/CD44low cells were grown without EGF. MCF10A cells (ATCC, CRL-10317) were cultured in DMEM/F12 supplemented with 10% horse serum (Invitrogen, 16050-122), 10 μg/mL insulin, 10 ng/mL EGF, 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin (Sigma-Aldrich, C8052), and 1 × PenStrep (Invitrogen, 15070-063).

Cell Viability Assay (IC50)

The cell viability assay was carried out by plating 1000 cells per well in 96-well plates. The cells were treated for 72 h in a range between 12 nM and 50 μM or 0.3 nM and 4 μM using serial dilutions following the manufacturer’s protocol. Very briefly, the CellTiter-Blue reagent (G8081, Promega) was added to the wells after 72 h of treatment, and cells were incubated for 3 h before fluorescence intensities (λex = 560/20 nm; λem = 590/10 nm) were recorded using a PerkinElmer Wallac 1420 Victor2 microplate reader.

The IC50 cell viability curves were plotted using the Prism 8 software for the synthesized compounds against HMLER CD24low/CD44high and the isogenic cell line HMLER CD24high/CD44low; MCF10A cells are included in the Supporting Information (Figures S55–S56).