A Domino 10-Step Total Synthesis of FR252921 and Its Analogues, Complex Macrocyclic Immunosuppressants

FR252921, FR252922, and FR256523 are a family of potent macrocyclic polyene immunosuppressive agents with a novel mode of action. However, the lack of an efficient and flexible synthesis has hindered further biological studies, mostly due to the fact that the natural products appear to be kinetic isomers regarding the triene moiety. Herein, we report the development and application of an unprecedented, unique domino Suzuki–Miyaura/4π-electrocyclic ring-opening macrocyclization, resulting in a concise, unified, and stereoselective synthetic route to these complex targets in only 10 steps. This in turn enables ready access to a range of unnatural analogues, among which several compounds showed inhibition of T-lymphocyte proliferation at levels equal or superior to those of the natural products themselves.


General information
Unless otherwise stated, all glassware was oven dried before use and all reactions were carried out under an argon atmosphere using standard Schlenk-techniques. Dry solvents were purchased from Acros Organics or Sigma-Aldrich and used without further purification. All reagents were purchased from commercial sources and were used without further purification unless otherwise stated. Reaction progress was monitored by thin layer chromatography (TLC) performed on aluminum plates coated with Kieselgel F254 with 0.2 mm thickness. Visualization was achieved by ultraviolet light (254 nm) or by staining with potassium permanganate. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck ans co.). Neat infrared spectra were recorded using a Perkin-Elmer Spectrum 100 FT-IR spectrometer. Mass spectra were obtained using a Finnigan MAT 8200 (70 eV), an Agilent 5973 (70 eV), using electrospray ionization (ESI) or electron impact ionization (EI). All 1 H NMR, 13 C NMR NMR were recorded on a BrukerAV-400, AV-500, AV-600 or AV-700 spectrometer in Chloroform-d1 or DMSO-d6.
Purification by column chromatography (silica gel, Heptane/EtOAc = 15:1) afforded 9.62 g (79%) of the product 3 as a colorless oil 1 . The spectra data is in accordance with the literature 2 .
The acid (rac)-7 has been synthesized before by our group 3 . We describe herein an improved synthesis of (rac)-7 through modification of the purification conditions. The bicyclobutene lactone (0.3 M, 34 mL, Et 2 O) was stirred with activated 3 Å molecular sieves (10 g) at -20 °C for 15 min. Then dry HCl (2 M in Et 2 O, 20 mL) was added. The solution was then stirred vigorously at -20 °C for another 24 h. The mixture was then allowed to warm to room temperature, concentrated. Purification by column chromatography (silica gel,
Typical yield obtained using the non-improved purification or crystallization: 44% 3 .
NB:The addition of acetic acid in the eluent is crucial for a facile isolation of acid (rac)-7 by column chromatography.
2) The ester obtained was then dissolved in CH 2 Cl 2 . At 0 °C, diisobutylaluminium hydride (DIBAL-H) (2.5 equiv.) was dropwise added. After 1 h, the mixture was first carefully quenched by sat. Rochelle salt solution. The resulting mixture was allowed to stir vigorously till the organic and aqueous phases separated. Then the mixture was extracted with CH 2 Cl 2 3 times. The combined organic layers were washed by brine, dried over MgSO 4 , filtered and concentrated under reduced pressure. The crude product was used directly in the next step without further purification.

3)
To the solution of the crude alcohol (1.0 equiv.) in CH 2 Cl 2 was added activated MnO 2 (15 equiv. Crude product was purified by column chromatography on silica gel to give the pure ester. 2) The ester obtained was then dissolved in CH 2 Cl 2 . At 0 °C, diisobutylaluminium hydride (DIBAL-H) (2.5 equiv.) was dropwise added. After 1 h, the mixture was first carefully quenched by sat. Rochelle salt solution. The resulting mixture was allowed to stir vigorously till the organic and aqueous phases separated. Then the mixture was extracted with CH 2 Cl 2 3 times. The combined organic layers were washed by brine, dried over MgSO 4 , filtered and concentrated under reduced pressure. The crude product was used directly in the next step without further purification.

Condition of 2 nd generation
We therefore conducted another screening of bases featuring the carbodiimide activation of the acid. However, only a slightly better result was obtained (Table S4). Note: As these studies showed that the combination of pyridine and acyl chloride was key for the selective cis-ester formation (Table S3). We decided to focus on the optimization of this condition. To address the issue of volatility of (rac)-7, we tried to find mild and clean conditions for the in-situ synthesis of (rac)-7, which led us to the condition of 3 rd generation. Note: To achieve a high cis/trans selectivity, the Ghosez's reagent needed to be freshly distilled. room temperature, the mixture was filtered and carefully concentrated in vacuo. The crude acyl chloride was then dissolved in CH 2 Cl 2 and added to a solution of amide alcohol (1.0 equiv.) in CH 2 Cl 2 and pyridine (4.0 equiv.) at 0° C. The reaction was allowed to reach room temperature and stirred overnight. The reaction was quenched with water and extracted 3 times with CH 2 Cl 2 .

No isomerization was observed for the acid chloride formation
The resulting organic layers were combined, dried over Na 2 SO 4 , filtered and concentrated in vacuo. Purification by column chromatography (silica gel, Heptane/EtOAc = 7:3 to 1:1) afforded the cis-isomer as mixture of diastereomers.

Results and discussions
It is possible to infer from the studies of Falk et al. 9 and Cossy et al. 21  We have undertaken computational studies, demonstrating that the (EEZ) isomer of FR252921 is considerably more stable than the natural (EEE) isomer presumably as a result of two intramolecular hydrogen bonding interactions involving the N-15 CONH moiety and the C-11 CO FR252921 thus appears to be a kinetic product compared to its (EEZ)-isomer, meaning that the stereoselective construction of the (EEE)-triene embedded in this compound stands as a considerable challenge.
The methodology we exploited in attempting the synthesis of the FR derivatives allow us to get rid of the aforementioned flaws and gives us a route to exclusively access the desired isomer. Besides, going from the most to the least stable, the macrocycle changes in a way that it starts resembling a circular ribbon being a funnel in the case of EEZ to be almost flat for ZEE. Both strain, dipole moment and solvent can therefore influence the relative stability.