Design, Synthesis and Biological Evaluation of Highly Potent Simplified Archazolids

Abstract The archazolids are potent antiproliferative compounds that have recently emerged as a novel class of promising anticancer agents. Their complex macrolide structures and scarce natural supply make the development of more readily available analogues highly important. Herein, we report the design, synthesis and biological evaluation of four simplified and partially saturated archazolid derivatives. We also reveal important structure‐activity relationship data as well as insights into the pharmacophore of these complex polyketides.


Chemical syntheses and characterization data
2.1 Synthesis of main fragment 27 and 28

Synthesis of ketone 12
To a solution of 1,5-pentanediol (11) (2.00 g, 19.3 mmol, 1.10 eq) in THF (100 mL) at 0 °C was added NaH (60 % in oil, 419 mg, 17.5 mmol, 1.00 eq). The reaction was stirred 30 min at 0 °C and for 1 h at room temperature. TBDPSCl (4.5 mL, 17.5 mmol, 1.00 eq) was added dropwise and the reaction mixture was stirred overnight. The mixture was diluted with Et 2 O (100 mL) and quenched with a saturated solution of NH 4 Cl (50 mL) and water (50 mL). After separation of the organic layer, the aqueous layer was extracted with Et 2 O (3*100 mL). The combined organic layers were dried over MgSO 4 and evaporated in vacuo. The crude mixture was purified by column chromatography (SiO 2 , CH/EtOAc, 4:1) to obtain the desired mono-protected alcohol (2.43 g, 7.10 mmol, 42 %) as a thick colorless oil. DMSO (2.94 mL,43.8 mmol,3.00 eq) was added dropwise to a solution of (COCl) 2 (2.52 mL, 29 mmol, 2.00 eq) in DCM (80 mL) at -78 °C. The mixture was stirred 15 min then the monoprotected alcohol (5.0 g, 14.6 mmol, 1.00 eq) in DCM (4 mL) was added over 15 min. After 1 h, NEt 3 (9.8 mL, 73 mmol, 4.00 eq) was added over 10 min. The reaction was stirred for 1 h at -78 °C then warmed up at 0 °C for 30 min. The reaction was quenched with a slow addition of water (100 mL). After separation of the organic layer, the aqueous layer was extracted with DCM (3*100 mL). The combined organic layers were dried over MgSO 4 and evaporated in vacuo. The crude product was directly used in the next reaction.
To a solution of crude aldehyde (4.70 g, 13.8 mmol, 1.00 eq) in THF (100 mL) at -78 °C was added MeMgBr (3 M in Et 2 O, 9.20 mL, 27.6 mmol, 2.00 eq). The reaction was stirred 30 min, diluted with Et 2 O (80 mL) and quenched with a cold saturated solution of NH 4 Cl (100 mL). After separation of the organic layer, the aqueous layer was extracted with Et 2 O (3*100 mL). The organic layers were combined, dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column chromatography (SiO 2 , CH/EtOAc, 9:1) to give the racemic alcohol (3.83 g, 10.6 mmol, 73 % over 2 steps) as a colorless oil.

Synthesis of aldehyde 15
To a solution of ester 14-E (3.73 g, 9.08 mmol, 1.00 eq) in DCM (150 mL) at -78 °C was added DIBALH (1 M in hexane, 27.3 mL, 3.00 eq) over 15 min. The reaction was stirred for 1 h 30 before being diluted with Et 2 O (100 mL) and quenched with H 2 O (1 mL). After addition of a 3 M aqueous solution of NaOH (1 mL) and an additional H 2 O (2.6 mL), the reaction was stirred for 15 min at room temperature. MgSO 4 was added and the solution was stirred for another 15 min. The solution was filtered and evaporated in vacuo until 150 mL of solvent was left.

Synthesis of aldehyde 18
To a solution of protected alcohol (3.64 g, 5.19 mmol, 1.00 eq) in THF (120 mL) at -78 °C was added LiBH 4 (1.68 g, 77.1 mmol, 15.0 eq) in one portion. After stirring 2 h at -78 °C, the mixture was stirred 3 days at room temperature. At 0 °C, water (40 mL) was added followed by careful addition of a saturated solution of NH 4 Cl (5 mL). The mixture was poured to a mixture of water and Et 2 O (1:1, 100 mL). After separation of the organic layer, the aqueous layer was extracted with Et 2 O (3*50 mL). The organic layers were combined, dried MgSO 4 and evaporated in vacuo. Purification by column chromatography (SiO 2 , CH/EtOAc, 4:1) yielded to the diol (3.01 g, 5.09 mmol, 98 %, dr = 4:1) as a thick colorless oil.

Synthesis of aldehyde 21
To a solution of ester 20 (2.38 g, 3.82 mmol, 1.00 eq) in DCM (50 mL) at -78°C was added DIBALH (1 M in hexane, 11.4 mL, 11.4 mmol, 3.00 eq) dropwise. The mixture was stirred for 1 h and warmed up to 0 °C for 45 min. DCM (50 mL) was added followed by H 2 O (0.45 mL), a 3 M aqueous solution of NaOH (0.45 mL) and H 2 O (1.1 mL). After stirring 15 min at room temperature, MgSO 4 was added and the mixture was stirred an additionnal 15 min. After filtration, the solvents were removed in vacuo.

Synthesis of aldehyde 23
To a solution of ester 22 (2.17 g, 3.27 mmol, 1.00 eq) in DCM (70 mL) at -78 °C was added DIBALH (1 M in hexane, 9.81 mL, 9.81 mmol, 3.00 eq) dropwise. The mixture was stirred for 1 h and warmed up to 0 °C. After dilution with DCM (50 mL), H 2 O (0.4 mL) was added followed by a 3 M aqueous solution of NaOH (0.4 mL) and H 2 O (1 mL). After stirring 15 min at room temperature, MgSO 4 was added and the mixture was stirred an additionnal 15 min. After filtration, the solvents were removed in vacuo.

Synthesis of aldehyde 25
To a solution of ester 24 (582 mg, 0.84 mmol, 1.00 eq) in DCM (15 mL) at -78 °C was added DIBALH (1 M in hexane, 2.5 mL, 2.52 mmol, 3.00 eq) dropwise. The mixture was stirred for 1 h and warmed up to 0 °C. The solution was diluted with DCM (20 mL), H 2 O (0.1 mL) followed by a 3 M aqueous solution of NaOH (0.1 mL) and H 2 O (0.1 mL) were added. After stirring 15 min at room temperature, MgSO 4 was added and the mixture was stirred an additionnal 15 min. After filtration, the solvents were removed in vacuo.

Synthesis of aldehyde 34
To a solution of ester 33 (620 mg, 1.01 mmol, 1.00 eq) in DCM (15 mL) at -78 °C was added DIBALH (1 M in hexane, 3.0 mL, 3.04 mmol, 3.00 eq) dropwise. The mixture was stirred for 1 h and warmed up to 0 °C. The reaction was diluted with DCM (15 mL), H 2 O (0.12 mL) was added followed by 3 M NaOH aqueous solution (0.12 mL) and H 2 O (0.3 mL). After stirring 15 min at room temperature, MgSO 4 was added and the mixture was stirred an additionnal 15 min. After filtration, the solvents were removed in vacuo. The crude product was directly use in the next step.

Synthesis of aldehyde 36
To a solution of ester 35 (507 mg, 0.78 mmol, 1.00 eq) in DCM (12 mL) at -78 °C was added DIBALH (1 M in hexane, 2.33 mL, 2.33 mmol, 3.00 eq) dropwise. The mixture was stirred for 1 h and warmed up to 0 °C. The reaction was diluted with DCM (12 mL), H 2 O (0.09 mL) was added followed by 3 M NaOH (0.09 mL) and H 2 O (0.23 mL). After stirring 15 min at room temperature, MgSO 4 was added and the mixture was stirred an additionnal 15 min. After filtration, the solvents were removed in vacuo. The crude product was directly use in the next step.

Synthesis of aldehyde 38
To a solution of ester 37 (120 mg, 0.18 mmol, 1.00 eq) in DCM (10 mL) at -78 °C was added DIBALH (1 M in hexane, 0.53 mL, 0.53 mmol, 3.00 eq) dropwise. The mixture was stirred for 1 h and warmed up to 0 °C. The reaction was diluted with DCM (10 mL), H 2 O (0.09 mL) was added followed by aqueous solution of 3 M NaOH aqueous solution (0.09 mL) and H 2 O (0.23 mL). After stirring 15 min at room temperature, MgSO 4 was added and the mixture was stirred an additionnal 15 min.
After filtration, the solvents were removed in vacuo. The crude product was directly use in the next step.

Synthesis of methyl ether 46a
To a solution of ketone 44a (84 mg, 90 µmol, 1.00 eq) in MeOH (3 mL) and THF (1 mL) at 0 °C was added NaBH 4 (123.6 mg, 360 µmol, 4.00 eq) and the solution was warmed up to room temperature. After 3 h, the reaction was diluted with EtOAc (5 mL) and quenched carefully with a saturated solution of NH 4 Cl (4 mL) at 0 °C. After separation of the organic layer, the aqueous layer was extracted with EtOAc (3* 10 mL). The organic layers were combined, dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column chromatography (SiO 2 , CH/EtOAc, 50:1) to give the alcohol as a thick oil (67 mg, 73 µmol, 86 %, dr = 8:1).
To a solution of carboxylic acid (23 mg, 27 µmol, 1.00 eq) in THF (0.8 mL) at 0 °C was added the HF-pyr stock solution (0.34 mL). The reaction was stirred for 6 h at 0 °C. The reaction was quenched with a saturated solution of NaHCO 3 (10 mL) and diluted with DCM (3 mL). After separation of the organic layer, the aqueous layer was extracted with DCM (3*5 mL). The combined organic layers were washed with brine (5 mL), dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column chromatography (SiO 2 , CH/EtOAc, 3:2) to give the precursor for Shiina esterification (6.3 mg, 8.6 µmol, 32 %).

Synthesis of analogue 6
To a solution of DMSO (25 µL, 354 µmol, 10.0 eq), sulfur trioxide pyridine complex (17 mg, 106 µmol, 3.00 eq) and DIEA (25 µL, 141 µmol, 4.00 eq) in DCM (3 mL) at 0 °C was added alcohol 47a (27 mg, 35 µmol, 1.00 eq). The solution was stirred at 0 °C for 1.5 h. After this time the reaction was quenched with aqueous saturated solution of NaHCO 3 (3 mL) and diluted with DCM (3 mL). After separation of the organic layer, the aqueous layer was extracted 3 times with DCM (5 mL).The organic layers were combined, dried over MgSO 4 and evaporated in vacuo until 200 mbar. The crude product was then directly used in the next reaction. The crude aldehyde was diluted in tert-butanol (2.5 mL) and 2-methylbut-2-ene (0.15 mL) and cooled at 0 °C. A solution of NaClO 2 (10.2 mg, 113 µmol, 3.20 eq), KH 2 PO 4 (19 mg, 141 µmol, 4.00 eq) in H 2 O (2.5 mL) was added to the reaction mixture and the reaction was stirred for 1 h at room temperature. Saturated aqueous solution of NaCl (4 mL) was added and DCM (4 mL). After separation of the organic layer, the aqueous layer was extracted with DCM (3*5 mL). The organic layers were combined, dried over MgSO 4 and evaporated in vacuo.
The crude carboxylic acid was diluted in MeOH (2.5 mL) and K 2 CO 3 was added (14.7 mg, 106 µmol, 3.00 eq). The reaction was stirred for 3 h at room temperature. The reaction was quenched with NaHCO 3 (2 mL) and diluted with DCM (3 mL). After separation of the organic layer, the aqueous layer was extracted with DCM (3*5 mL). The combined organic layers were washed with brine (5 mL), dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column chromatography (SiO 2 , CH/EtOAc, 3:2) to give the precursor for Shiina esterification (5 mg, 7 µmol, 20 % over 3 steps).
MNBA (10.5 mg, 30.6 µmol, 5.00 eq), DMAP (5.2 mg, 43 µmol, 7.00 eq) and 3Å MS were dried for 1 h under high vacuum before DCM was added (3 mL). The seco acid was diluted in DCM (4 mL) and added to the solution over 20 h. Two hours after completion of the addition, the reaction was quenched at 0 °C with pH7 Buffer (3 mL). After separation of the organic layer, the aqueous layer was extracted with DCM (3*5 mL). The combined organic layers were washed with brine (5 mL), dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column chromatography (SiO 2 , CH/EtOAc, 50:1) to give the desired macrolactone (4.3 mg, 6 µmol, 86 %).
To a solution of carboxylic acid (33 mg, 40 µmol, 1.00 eq) in THF (1 mL) at 0 °C was added HFpyr stock solution (0.5 mL). The reaction was then stirred for 6 h at 0 °C. The reaction was quenched with a saturated solution of NaHCO 3 (10 mL) and diluted with DCM (3 mL). After separation of the organic layer, the aqueous layer was extracted with DCM (3*5 mL). The combined organic layers were washed with brine (5 mL), dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column chromatography (SiO 2 , CH/EtOAc, 3:2) to give the precursor for Shiina esterification (12.1 mg, 17 µmol, 42 %).
MNBA (29 mg, 84 µmol, 5.00 eq), DMAP (14 mg, 117 µmol, 7.00 eq) and 3Å MS were dried for 1 h under high vacuum before DCM was added (6 mL). The seco acid was diluted in DCM (8 mL) and added to the solution over 20 h. Two hours after completion of the addition, the reaction was quenched at 0 °C with pH7 Buffer (3 mL). After separation of the organic layer, the aqueous layer was extracted with DCM (3*5 mL). The combined organic layers were washed with brine (5 mL), dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column chromatography (SiO 2 , CH/EtOAc, 50:1) to give the desired macrolactone (9.8 mg, 14 µmol, 83 %).

Synthesis of analogue 8
To a solution of DMSO (20 µL, 208 µmol, 10.0 eq), sulfur trioxide pyridine complex (13 mg, 84 µmol, 3.00 eq) and DIEA (20 µL, 112 µmol, 4.00 eq) in DCM (3 mL) at 0 °C was added alcohol 47b (21 mg, 28 µmol, 1.00 eq). The solution was stirred at 0 °C for 1.5 h. After this time the reaction was quenched with a saturated solution of NaHCO 3 (3 mL) and diluted with DCM (3 mL). After separation of the organic layer, the aqueous layer was extracted with DCM (3*5 mL).The organic layers were combined, dried over MgSO 4 and evaporated in vacuo until 200 mbar. The crude product was then directly used in the next reaction.
The crude aldehyde was diluted in tert-butanol (2 mL) and 2-methylbut-2-ene (0.1 mL) and cooled at 0 °C. NaClO 2 (8 mg, 89 µmol, 3.20 eq), KH 2 PO 4 (15 mg, 111 µmol, 4.00 eq) in H 2 O (2 mL) were added to the solution and the reaction was stirred for 1 h at room temperature. Saturated aqueous solution of NaCl (2 mL) was added and diluted with DCM (2 mL). After the separation of the organic layer, the aqueous layer was extracted with DCM (3*5 mL), the organic layers were combined, dried over MgSO 4 and evaporated in vacuo.
The crude carboxylic acid was diluted in MeOH (2 mL) and K 2 CO 3 was added (11.2 mg, 84 µmol, 3.00 eq). The reaction was stirred for 3 h at room temperature. The reaction was quenched with a saturated solution of NaHCO 3 (2 mL) and diluted with DCM (3 mL). After separation of the organic layer, the aqueous layer was extracted with DCM (3*5 mL). The combined organic layers were washed with brine (5 mL), dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column chromatography (SiO 2 , CH/EtOAc, 3:2) to give the precursor for Shiina esterification (9.5 mg, 13 µmol, 46 % over three steps).
MNBA (22.6 mg, 66 µmol, 5.00 eq), DMAP (11 mg, 92 µmol, 7.00 eq) and 3Å MS were dried for 1 h under high vacuum before DCM was added (5 mL). The seco acid was diluted in DCM (7 mL) and added to the solution over 20 h. Two hours after completion of the addition, the reaction was quenched at 0 °C with buffer pH7 (3 mL). After separation of the organic layer, the aqueous layer was extracted with DCM (3*5 mL). The combined organic layers were washed with brine (5 mL), dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column chromatography (SiO 2 , CH/EtOAc, 50:1) to give the desired macrolactone (7 mg, 10 µmol, 77 %).
Ketone 27 (230 mg, 0.27 mmol, 1.00 eq) was diluted in THF (4 mL) and dried over 3Å MS for 30 min. The solution was then cooled down at -78 °C, LiTMP (2.3 mL, 0.54 mmol, 2.00 eq) was added dropwise and the mixture was stirred for 30 min at -78 °C and warmed up to -50 °C for 20 min. The enolate solution was cooled down at -78 °C and aldehyde 63 (88 mg, 0.40 mmol, 1.50 eq, dried over 3Å MS in 0.4 mL THF) was added dropwise. After 2 h, the reaction mixture was diluted with DCM (10 mL) and quenched with a saturated solution of NaHCO 3 (10 mL) at 0 °C. After the separation of the organic layer, the aqueous layer was extracted with DCM (3*15 mL). The organic layers were combined, dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column chromatography (SiO 2 , CH/EtOAc, 30:1 to 10:1) give the aldol product (262 mg, 0.25 mmol, 92 %).

Synthesis of methyl ester 67
To a solution of alcohol 66 (60 mg, 98 µmol, 1.00 eq) in DCM (4 mL) at 0 °C was added DMP (23 mg, 54 µmol, 1.30 eq). The solution was warmed up to room temperature and stirred for 2 h. After this time the reaction was cooled to 0 °C again, diluted with Et 2 O (6 mL) and filtered through celite. The solvents were evaporated and this sequence was repeated two times. The crude aldehyde was then diluted in tert-butanol (2 mL) and 2-methylbut-2-ene (2 mL) and cooled at 0 °C. NaClO 2 (28.5 mg, 310 µmol, 3.20 eq), KH 2 PO 4 (53.6 mg, 390 µmol, 4.00 eq) and H 2 O (20 µL) were added, the reaction was stirred for 1 h at room temperature. Saturated solution of NaCl (4 mL) was added followed by Et 2 O (5 mL). After separation of the organic layer, the aqueous layer was extracted with Et 2 O (3*5 mL). The organic layers were combined, dried over MgSO 4 and evaporated in vacuo.
The crude aldehyde was diluted in tert-butanol (2 mL) and 2-methylbut-2-ene (0.1 mL) and cooled at 0 °C. A solution of NaClO 2 (15 mg, 168 µmol, 3.20 eq), KH 2 PO 4 (28 mg, 209 µmol, 4.00 eq) in H 2 O (2 mL) was added to the solution. The reaction was stirred for 1 h at room temperature. Saturated solution of NaCl (5 mL) was added followed by Et 2 O (5 mL). After separation of the organic layer, the aqueous layer was extracted with Et 2 O (3*5 mL). The organic layers were combined, dried over MgSO 4 and evaporated in vacuo.

Synthesis of methyl ester 71
To a solution of alcohol 57 (60 mg, 98 µmol, 1.00 eq) in DCM (4 mL) at 0 °C was added DMP (23 mg, 54 µmol, 1.30 eq). The solution was warmed up to room temperature and stirred for 2 h. After this time the reaction was cooled to 0 °C again, diluted with Et 2 O (4 mL) and filtered through celite. The solvents were evaporated and this sequence was repeated two times.

Biological assays
3.1 MTT assay Table 1: Antiproliferative activity of test compounds in 1321N1 astrocytoma cells determined by the MTT assay.