An enantioselective synthesis of the C3–C21 segment of the macrolide immunosuppressive agent FR252921

Abstract

An enantioselective synthesis of the C3–C21 segment of the novel immunosuppressant FR252921 is described. The C12 and C13 stereogenic centers were constructed by a non-aldol process utilizing optically active 4-phenylbutyrolactone. The C18 stereogenic center was installed using Braun's highly diastereoselective acetate aldol reaction. Other key steps involved Curtius rearrangement and Horner-Wadsworth-Emmons olefination reactions.

Graphical Abstract

Organ transplantation is often the last treatment option for patients with organ failure. Immunosuppressive drugs such as cyclosporin A and FR506 play a critical role for transplant patients by suppressing the immune system's ability to reject transplanted organs.^1,2 For decades, these calcineurin (CN) inhibitors have been the mainstay post-transplantation immunosuppressive regimens. However, long-term use of these drugs is associated with issues ranging from chronic allograft nephropathy and adverse side effects. Thus, development of new immunosuppressive agents with a novel mechanism of action has been a very active area of research.^3,4 In 2003, Fujine and co-workers isolated a novel immunosuppressive agent, FK252921 (1, Figure 1), from the culture both of Pseudomonas Fluorescens, no. 408813.^5,6 This compound exhibits potent inhibition of both lipopolysaccharide-stimulated and anti-CD3 mAb-stimulated splenocyte proliferation in vitro without blocking T-cell activation. Also, FK252921 shows a strong synergistic effect with FK506 in vitro and in vivo.⁷ These results suggest that the target and mechanism of action of FK252921 is different from CN inhibitors.

Figure 1.

Structure of FR252921 (1) and pseudotrienic acid (2).

Not surprisingly, the biology and chemistry of FK252921 attracted immense attention from the synthetic and medicinal communities. FR252921 is a 19-membered macrolactone with two amide linkages. Initial structural work of FR252921 was carried out by Fujine and co-workers and Pohanka and co-workers.^5,6 It appears that FR252921 is related to pseudotrienic acid, which is an acid-catalyzed lactone ring opened product followed by isomerization at C18-22 centers.⁸ A number of synthetic studies and total syntheses of FR252921 have been reported. Falck and co-workers established the absolute configuration through total synthesis.⁹ Subsequently, a number of other total syntheses of this compound were reported.^10-13 In an effort to explore biological properties of derivatives of pseudotrienic acid and FR252921, we sought to develop an asymmetric synthesis of FR252921. Herein, we report our enantioselective synthesis of the C3-C21 fragment of FR252921 where the C12 and C13 stereogenic centers were constructed using a non-aldol protocol developed utilizing readily available optically active 4-phenylbutyrolactone in our laboratory.^{14, 15} The C18 stereogenic center was constructed by a highly diastereoselective acetate aldol reaction as the key step.

Our retrosynthesis of FR252921 is show in Figure 1. In order to incorporate various side chains, we planned to synthesize the protected 19-membered macrocycle 4. We envisioned a late stage intramolecular Horner-Wadsworth-Emmons (HWE) olefination to construct this 19-membered macrocycle of FR252921. The C3-C21 segment 5, containing all stereogenic centers, would be synthesized from carboxylic acid derivative 6, C3-N10 dienamine 7 and β-hydroxyacid 8. This diene derivative would be obtained from 3-Fmoc-aminopropionaldehyde and commercially available triethyl-4-phosphonocrotonate.

As shown in Scheme 1, our synthesis of 12(S), 13(R)-acid segment 6 utilized optically active 4-phenylbutyrolactone 9 as the key starting material. This was prepared in multigram quantities by CBS reduction as described by Corey and coworkers.^16,17 Deprotonation of lactone 9 wih LiHMDS at -78 °C followed by reaction with methyl iodide provided the methylated product 10 in 88% yield. The alkyation is highly diastereoselctive, providing 10 in a 99:1 dr by HPLC analysis. Alkylated lactone 10 was reduced by DIBAL-H and the resulting lactol was treated with carbethoxymethylenetriphenylphosphorane to afford α,β-unsaturated ester 11 in 96% yield. Reaction of 11 with KHMDS in THF at -78 °C furnished the oxa-Michael product 12 in 93% yield and excellent diastereoselectivity by ¹H-NMR (96:4 dr) and HPLC (92:8 dr) analysis. Interestingly, the oxa-Michael reaction of 11 with LiHMDS in THF at -78 °C afforded tetrahydrofuran derivative 12 in 67% yield. Treatment of 12 with a catalytic amount of Zn(OTf)₂ (5 mol%) in the presence of acetic anhydride in toluene at 75 °C afforded the ring opened product 13 in 95% yield.¹⁵ Oxidative cleavage of 13 using a catalytic amount of RuCl₃ and NaIO₄ as a stoichiometric oxidant in a mixture of CH₂Cl₂, acetonitrile and water as described by Sharpless and co-workers¹⁸ afforded carboxylic acid 6 in 82% yield.

Scheme 1. Synthesis of acid segment 6.

Synthesis of C3-N10 dienamine segment 7 is shown in Scheme 2. Fmoc-protected aminopropanol 14 was oxidized with IBX in ethyl acetate at 80 °C for 14 h to provide the corresponding aldehyde in 95% yield. Horner-Wadsworth-Emmons olefination was carried out by deprotonation of commercially available triethyl-4-phosphonocrotonate with LiHMDS in THF at 0 °C followed by addition of aldehyde at 0 °C for 1 h which resulted in α,β-unsaturated ester 15 as the major product in 56% yield.¹⁹ DIBAL-H reduction of ester 15 in CH₂Cl₂ at 0 °C for 1 h, provided alcohol 16 in 63% yield. Alcohol 16 was protected as the TBS-ether using TBSCl and imidazole in the presence of a catalytic amount of DMAP at 23 °C for 12 h to provide TBS-ether in 93% yield. Removal of the Fmoc group by exposure to diethylamine in CH₃CN at 23 °C for 2 h afforded desired dienamine 7 in 96% yield.

Scheme 2.

Synthesis of dieneamine segment 7.

For the synthesis of β-hydroxy acid fragment 8, we initially explored the feasibility of a NaBH₄/tartaric acid reduction of the corresponding β-keto ester. As shown in Scheme 3, β-keto ester 18 was prepared by aldol reaction of the corresponding enolate of t-BuOAc with known²⁰ aldehyde 17, followed by oxidation of the resulting racemic alcohol with IBX in ethyl acetate, providing 18 in 83% yield over 2-steps. The NaBH₄/tartaric acid complex was prepared as described by Yatagai and Ohnuki.^20,21 Reduction of keto ester with freshly prepared NaBH₄/tartaric acid complex (1:1) in THF at -20 °C for 65 h provided β-hydroxy ester 19 in 48% yield. However, the reduction product showed 57% ee. We then prepared NaBH₄/tartaric acid complex (1:1) in THF and let the complex age for 144 h. Reduction of 18 with this complex for 114 h, provided product 19 in 68% ee but the yield was only 20%. Reduction of 18 with a freshly prepared NaBH₄/tartaric acid complex (1:2 mixture) at -20 °C for 39 h did not improve enantioselectivity and β-hydroxy ester 19 was obtained in only 23% yield. This result was inadequate for our synthesis and we explored an alternate acetate aldol route.

Scheme 3.

Synthesis of acid 8 by chiral reduction.

As shown in Scheme 4, we investigated Braun's asymmetric acetate aldol reaction to install the C18-(R)-alcohol configuration. Chiral acetate 20 was prepared as described by Braun and co-workers.²² Acetate derivative 20 was deprotonated with LiHMDS in THF at -78 °C to 0 °C for 5 min. This lithium enolate was cooled to -78 °C and then treated with MgBr₂ at -78 °C for 30 min and the resulting magnesium enolate was reacted with aldehyde 17 at -100 °C for 1.5 h.²³ This provided aldol product 21 in 94% yield and a diastereomeric ratio of 96:4 (by HPLC analysis). Removal of the chiral auxiliary by LiOH hydrolysis at 23 °C for 12 h afforded acid 7 in 71% isolated yield.

Scheme 4.

Braun's aldol route to acid 8.

The assembly of the C3-C21 macrocyclic core is shown in Scheme 5. Coupling of carboxylic acid 6 with amine 7 in the presence of EDCI, HOBt, and N-methylmorpholine (NMM) in a mixture (1:1) of CH₂Cl₂ and CH₃CN at 0 °C to 23 °C for 12 h afforded the coupling product 22 in 77% yield. Selective hydrolysis of the ethyl ester in the presence of an acetate group was achieved by exposure of 22 to Me₃SnOH in dichloroethane (DCE) at 80 °C for 12 h.²⁴ This provided acid 23 selectively in 60% yield. A small amount of deacetylation product was observed. Acid 23 was converted to the corresponding amine by Curtius rearrangement.^25,26 Thus, acid 23 was treated with diphenylphoryl azide and diisopropylethylamine (DIPEA) in CH₂Cl₂ at 40 °C for 30 min. The reaction mixture was then reacted with fluorenyl alcohol at 40 °C for 12 h to provide amine 24. Amine 24 was coupled with acid 8 in the presence of HATU, HOBt and NMM in a mixture (1:1) of CH₂Cl₂ and CH₃CN at 23 °C for 12 h to provide our desired C3-C21 segment 5 in 55% yield.

Scheme 5.

Synthesis of the C3-C20 segment of FR252921.

In conclusion, we have synthesized the C3-C21 segment of FR252129 in a highly stereoselective manner. The C12 and C13 stereogenic centers were installed using optically active 4-phenylbutyrolactone as the key starting material. The key reactions involved stereoselective methylation, oxa-Michael reaction of an α,β-unsaturated ester followed by acyloxonium ion-mediated ring opening. The dienamine segment was synthesized from Fmoc-protected aminopropionaldehyde and Horner-Wadsworth-Emmons olefination with ethyl phosphonocrotonate. For the C18-stereogenic center, we explored chiral borane reduction with NaBH₄ and tartaric acid. However, enantioselectivity and product yield were moderate. We then utilized Braun's acetate aldol reaction to construct the C18-sterogenic center efficiently. Further work toward the total synthesis of FR252129 is in progress.

Figure 2.

Retrosynthesis of FR252921.

Highlights.

• Synthesis of the C3–C21 segment of the immunosuppressant FR252921 is achieved.

• The C12 and C13 chiral centers were set by a diastereoselective non-aldol process.

• Non-aldol route includes, alkylation, oxo-Michael reaction and ring opening.

• The C18 stereogenic center was installed using Braun's acetate aldol reaction.

• Chiral reduction of β-keto-ester with NaBH₄ and tartaric acid was investigated.