Development of an Advanced Synthetic Route to Macrosphelides and Its Application to the Discovery of a More Potent Macrosphelide Derivative

The discovery of a more cytotoxic macrosphelide derivative, including its total synthesis and bioassay are described. Application of the Koide protocol to a readily available propagylic alcohol allowed the rapid and practical synthesis of a macrosphelide A skeleton. This strategy enabled the successful improvement of the cytotoxic activity of the macrosphelide derivative.


Introduction
Natural products have been tapped as an endless supply of resources for new drug development, because of their potency and safety [1,2]. Actually, over the past three decades, approximately 50% of new drugs have been developed from natural products [3,4]. However, some natural products with low potency or high toxicity still need derivatization based on their structure-activity relationships (SAR) and chemical modifications [5].

OPEN ACCESS
Since its first isolation in 1995, macrosphelide A (1) has been expected to serve as a new lead compound because of its safety, anticancer activity and unique chemical skeleton [6][7][8][9]. However, its low potency still hampers further developments. To solve this limitation, a variety of approaches including total synthesis [10][11][12][13][14][15][16][17][18][19][20], medicinal chemistry [21][22][23], combinatorial chemistry [24], and fluorinated chemistry [25] have been carried out to establish the SAR and discover more promising derivatives of 1. As a result, it has been possible to improve its various biological activities slightly after extensive efforts, including the confirmation of natural isomer structures and the discovery of potent derivatives such as aza-1, ring size modified-1, fluorinated-1 and thiazoline linked-1. However, it is noteworthy that C3-modified derivatives of 1 without changing the ring skeleton have not been studied extensively [21,26]. Considering the similar potency of macrosphelide A (1) and E (2), it can be hypothesized that the C3 substituent can be altered without loss of activity ( Figure 1) [27,28]. Moreover, modification of this carbon can be carried out to improve lipophilicity and cell permeability or suppress metabolism, such as by esterase mediated hydrolysis. With this possibility for chemical modification in mind, we sought to modify the C3 position of 1, because this SAR data can be combined with other SAR information to develop more potent derivatives. For the purpose of this research, a practical synthetic route to the macrosphelide skeleton had to be established. Herein, we would like to report the discovery of 3-phenyl substituted macrosphelide A (3), which is much more potent than macrosphelide A 1.

Retrosynthesis
The retrosynthesis is outlined in Scheme 1. Considering the convergent synthesis of 3, it was envisioned that esterification of dimeric alcohol 5 with carboxylic acid 4 would produce a unique macrosphelide skeleton efficiently. Scheme 1. Retrosynthesis to 3-phenyl-macrosphelides 3.
Dimeric alcohol 5 was expected to be transformed via a simple deprotection/esterification sequence of bis-protected unsaturated acid 6. Finally, employment of the Koide protocol [29] to the PMB-protected aldehyde 8 was designed to yield the key monomeric acid 6 because this strategy can introduce a three-carbon unit directly using commercially available resources.

Synthesis of Monomer 6 of Macrosphelide A
Synthesis of monomer 6 commenced with the three-carbon homologation of the PMB-protected aldehyde 8 by reaction of 7 [30] (Scheme 2). Because use of normal bases such as LiHMDS or NaHMDS produced the desired addition product 9 in low yield, exploration of conditions for this conversion was carried out. After an intensive screening of bases and solvents, iPrMgCl was found to produce the corresponding propargylic alcohol 9. Although the desired diastereomer 9 was produced as a minor isomer, this facial selectivity problem could be solved via an oxidation/stereoselective reduction sequence. Dess-Martin oxidation of the diastereomeric mixture 9/C3-epi-9 produced alkynone 10 in good yield. With alkynone 10 in hand, stereoselective reduction of the ketone moiety was executed. Based on the reaction pattern of similar systems [17,31], Super-Hydride was chosen to obtain a chelation-controlled reduction product. Gratifyingly, the desired secondary alcohol 9 was obtained exclusively as the desired isomer, which was subjected to the Koide procedure.

Scheme 2. Synthesis of monomer 6.
When the propargylic alcohol 9 in methanol was treated with NaBH4, the desired trans-allylic alcohol 11 could be obtained in excellent yield, as was previously reported for a similar skeleton [32]. Moreover, this reaction could be performed at a more convenient temperature without loss of chemical yield. Considering the mildness of the reducing agent and ease of the reaction handling, this stereoselective reduction sequence is ideal for the efficient synthesis of macrosphelides and their derivatives. Finally, TIPS protection followed by hydrolysis of the allylic alcohol 11 produced 6 in excellent yield. Employing this synthetic route, the key building block 6 could be prepared on multi-gram scale.

Completion of the Synthesis
Completion of synthesis was performed by the iterative ligation of three hydroxy acid fragments (Scheme 3). O-Allylation and Yonemitsu PMB deprotection [33] of the bis-protected carboxylic acid 6 produced hydroxyester 12, which was transformed into diester 5 by Yamaguchi esterification and PMB deprotection. Stepwise esterification of the known carboxylic acid 4 [34] with the alcohol 5, followed by PMB deprotection produced trimeric alcohol 13 in excellent yield. Pd-catalyzed allyl deprotection and Yamaguchi lactonization produced the bis-TIPS protected 3-phenylmacrosphelide A 14.
Finally, treatment of 14 with TBAF at a low temperature produced the target molecule 3. Scheme 3. Total synthesis of 3-phenylmacrosphelide A (3).

Cytotoxic Activity
The cytotoxic activities of 1 and 3 are shown in Figure 2. Cell viability was determined with the use of a luminescent cell viability assay kit. After 4 × 10 3 SKOV3 cells were seeded in an opaque-walled 96-well microplate, the cells were treated with 1 and 3 and incubated at 37 °C for 72 h. For equilibration of the microplate, it was incubated for 30 min at room temperature. Thereafter, 100 µL CellTiter-Glo ® reagent was added to 100 µL of medium containing cells and the plate was mixed for 2 min on an orbital shaker to induce cell lysis. Finally, the plate was incubated for 10 min at room temperature and the luminescence was recorded by a GENios reader. From this assay, a remarkable improvement in cytotoxic activity could be observed. When cells were treated with 3 instead of 1, cytotoxicity in the carcinoma cell line was increased (see Figure 2). This result means that C3 modification of the macrosphelide skeleton could contribute to an improvement of cytotoxic activity, which is strongly required for macrosphelide-related anticancer drug development.

General Information
Unless noted otherwise, all starting materials and reagents were obtained from commercial suppliers and were used without further purification. Tetrahydrofuran and Et2O were distilled from sodium benzophenone ketyl. Dichloromethane was freshly distilled from calcium hydride. All solvents used for routine isolation of products and chromatography were reagent grade and glass distilled. Air and moisture sensitive reactions were performed under an argon atmosphere. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck, Darmstadt, Germany) with the indicated solvents. Thin-layer chromatography was performed using 0.25 mm silica gel plates (Merck). 1 H-NMR data were reported in the order of chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet and/or multiple resonance), number of protons, and coupling constant in hertz (Hz). (9). To a solution of methyl propiolate (540 mg, 6.4 mmol) in THF (10 mL), iPrMgCl (2.0 M in THF, 3.2 mL, 6.4 mmol) was added at −20 °C. After stirring at the same temperature for 30 min, a solution of aldehyde 8 (340 mg, 1.8 mmol) in THF (5 mL) was added at −20 °C. The reaction mixture was stirred for 1 h at the same temperature and quenched with aq. NH4Cl. The reaction mixture was extracted with EtOAc and the combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (EtOAc/n-hexane = 1:5 to 1:2) to afford 420 mg (86%) of propargylic alcohol 9 as inseparable diastereomers: (4R,5S,E)-Methyl 4-hydroxy-5-(4-methoxybenzyloxy)hex-2-enoate (11). To a solution of propargylic alcohol 9 (415 mg, 1.5 mmol, mixture of diastereomers) in CH2Cl2 (10 mL), Dess-Martin periodinane (760 mg, 1.8 mmol) was added at room temperature. After stirring for 10 min, the reaction mixture was quenched with saturated aq. NaHCO3, and then extracted with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (EtOAc/n-hexane = 1:5 to 1:2) to afford 380 mg (92%) of slightly unstable ketone 10 as a yellow oil: 1  A solution of the crude mixture from the previous reaction in THF/H2O (3 mL/1 mL) was treated with NaOH (10% in H2O, 0.2 mL) and warmed to 50 °C for 12 h. The reaction mixture was quenched with 1N HCl and then extracted with EtOAc (3 times). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (EtOAc/n-hexane = 1:1 to EtOAc only) to afford 15 mg (65% for 2 steps) of carboxylic acid 6 as a colorless oil.  (12). To a solution of carboxylic acid 6 (480 mg, 1.1 mmol) in DMF (5 mL), Cs2CO3 (740 mg, 2.3 mmol) and allyl bromide (0.15 mL, 1.7 mmol) were added at 0 °C. After stirring for 10 min at the same temperature, the reaction mixture was quenched with saturated aq. NH4Cl. The reaction mixture was diluted with EtOAc and washed with H2O (3 times) and the organic layers was dried over MgSO4, filtered and concentrated in vacuo.

Cell Culture
The human ovarian carcinoma cell line SKOV3 was obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1× penicillin/streptomycin at 37 °C in 5% CO2 atmosphere. Cells were routinely checked for mycoplasma contamination and authenticated using STR DNA technology. These culture check was carried out three times.

Cell Viability
Cell viability was determined by the use of a CellTiter-Glo ® luminescent cell viability kit (Promega Corporation, Madison, WI, USA). After 4 × 10 3 SKOV3 cells were seeded in an opaque-walled 96-well microplate, cells were treated with chemicals and incubated at 37 °C for 72 h. For equilibration of a microplate, it was incubated for 30 min at room temperature (RT). Thereafter, 100 µL of CellTiter-Glo ® reagent was added to 100 µL of medium containing cells and the plate was mixed for 2 min on an orbital shaker for inducing cell lysis. Finally, the plate was incubated for 10 min at RT to stabilize the luminescent signal and recorded by a GENios reader (Tecan, Mä nnedorf, Switzerland). These tests were carried out three times.

Conclusions
The synthesis of a valuable C3-modified macrosphelide derivative was accomplished. The synthetic route includes a direct three-carbon introduction through propiolate addition and a sequential stereoselective reduction process. Employing this efficient synthetic route, biologically enhanced derivatives could be developed and utilized for macrosphelide-related anticancer drug discovery. Full disclosure of more advanced derivatives and the SAR studies of macrosphelides will be reported in due course.

Acknowledgments
This work was also supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, no. PJ0090882014).

Author Contributions
Y.M.H. made 3-phenyl-macrosphelide A and its synthetic intermediates. H.L. and Y.K.S. executed cytotoxic tests. S.M.P. made 3-phenyl-macrosphelide A and wrote this manuscript.

Conflicts of Interest
The authors declare no conflict of interest.