Chemical Synthesis and Antifibrotic Properties of (±)-Cochlearol T, (±)-Ganocochlearin A, and (±)-Cochlearol Y

Abstract

The cochlearols and ganocochlearins are natural products with unique antifibrotic and renoprotective activities in models of kidney disease. They represent compelling lead compounds for pharmacological intervention against kidney disease, often characterized by renal fibrosis. We report a four-step synthesis of (±)-cochlearol T (1) and the first reported syntheses of (±)-ganocochlearin A (2) and (±)-cochlearol Y (3) through a strategy that includes a Robinson annulation and unexpected oxidative aromatization. We also access tricyclic intermediate 12 that represents a formal synthesis of ganocins A–C and ganocochlearins C–D. We investigated the activity of these synthesized compounds in vitro by inducing fibrosis in a human kidney cell line with TGF-β1. The effect on fibrosis was assessed by qPCR and Western blot studies. We detected significantly lower mRNA gene and protein expression of fibrosis markers for all three natural products.

The cochlearols and related ganocochlearins are structurally unique and biologically active natural products isolated from the fruiting bodies of the fungus Ganoderma cochlear (Scheme 1A).¹ To date, over 25 cochlearols and ganocochlearins have been isolated, mostly as racemates. Our interest in these monoterpenoids is motivated by their antifibrotic and renoprotective properties. For example, several members have been reported to exhibit a protective effect in cellular and murine models of renal fibrosis,¹ which is a major contributor to chronic kidney disease that requires eventually dialysis or a kidney transplant.² In addition, in vivo renoprotective effects of cochlearols and ganocochlearins have been shown for murine models of acute kidney injury due to ureteral obstruction, cisplatin nephropathy, and autosomal dominant polycystic kidney disease.³

Scheme 1. Retrosynthetic Analysis for Renoprotective Cochlearol and Ganocochlearin Natural Products.

Therefore, these natural products represent a new opportunity for pharmacological intervention against renal fibrosis and chronic kidney disease. Mechanistically, cochlearols and ganocochlearins have been shown to interfere with TGF-β1-induced renal fibrosis in proximal tubular cells.⁴ Specifically, they inhibit Smad3 phosphorylation which is an essential member of TGF-β1 signaling.⁵ In addition, some cochlearols lower fibronectin overproduction after fibrosis induction with TGF-β1.⁶

Herein we present a unified approach to the synthesis of cochlearols and ganocochlearins, which resulted in the chemical synthesis of (±)-cochlearol T (1) and the first reported synthesis of (±)-ganocochlearin A (2) as well as its diastereomer (±)-cochlearol Y (3). We also evaluated the antifibrotic properties of these newly synthesized compounds in a renal fibrosis model by applying the HK2 human kidney cell line, which represents the proximal tubule.⁷ This nephron segment is a major target for acute kidney injury and progression of kidney disease.⁸ This cell line has been previously published as a model of renal fibrosis when treated with TGF-β1 or cisplatin.⁹

The compelling renoprotective activities of cochlearols and ganocochlearins have motivated several syntheses of these natural products. Notably, cochlearol A was synthesized by Qin,¹⁰ Ishigami,¹¹ and Chandrasekhar,¹² and cochlearol B was synthesized by Sugita,¹³ Schindler,¹⁴ and Hao.¹⁵ Recently, Zhao reported the synthesis of cochlearol T and ganocochlearins C and D.¹⁶

Our retrosynthetic analysis stemmed from the hypothesis that cochlearol T (1) could be accessed by oxidative aromatization of either ganocochlearin A (2) or cochlearol Y (3) (Scheme 1B). We envisaged that the tricyclic core of ganocochlearin A and cochlearol Y could arise from the Robinson annulation of chromanone 4 and methyl vinyl ketone 5.¹⁷ Ultimately, we planned to derive bicyclic chromanone 4 from commercially available acetophenone 6.

Based on Schindler’s recent report of the synthesis of bicycle 4 via a Kabbe condensation,^14,18 we initiated our synthetic studies by coupling 2-hydroxy-5-methoxyacetophenone 6 and sulcatone 7 in the presence of pyrrolidine and 3 Å molecular sieves in ethanol (Scheme 2A). We next explored the Robinson annulation strategy for converting bicycle 4 and methyl vinyl ketone 5 to tricycle 10. Attempts at realizing the Michael addition step of the Robinson annulation were unsuccessful under basic conditions in alcoholic solvents (see Supporting Information for details). In some cases, we observed retro-1,4-addition product 8 in low yield. Acidic conditions also failed to furnish the desired Michael addition product 9. Ultimately, diketone 9 was formed in 44% yield in the presence of NaOH in THF as a 1:1 mixture of inseparable diastereomers.

Scheme 2. Total Synthesis of (±)-Cochlearol T, (±)-Ganocochlearin A, and (±)-Cochlearol Y, and Formal Synthesis of (±)-Ganocins A–C and (±)-GanocochlearinsC–D.

With the Michael addition realized, we explored the second Aldol condensation step of the Robinson annulation. Treatment with Lewis acids or secondary amines did not result in the formation of the desired tricycle 10.¹⁸ To our delight, subjection of diketone 9 to KOH in EtOH at an elevated temperature led to the formation of Robinson annulation product 10 in 38% yield and 1:1 dr. Under these reaction conditions, we also formed tricyclic intermediate 12 in 9% yield, which constitutes a formal total synthesis of ganocins A–C and ganocochlearins C–D.^16a

We then explored the demethylation of tricycle 10 with the expectation that we would form (±)-ganocochlearin A (2) and (±)-cochlearol Y (3). Serendipitously, upon exposure of protected phenol 10 to demethylation conditions that include p-methoxythiophenol and K₂CO₃ at 190 °C in NMP as the solvent, we formed the natural product (±)-cochlearol T (1) in 93% yield. The spectroscopic data obtained for our synthetic sample of 1 were identical with the data reported in the original isolation paper.⁵ We hypothesize that under these reaction conditions, tricycle 10 undergoes demethylation, followed by enolization and oxidation of the resulting dienolate 11 to form the bis-phenolic natural product 1. Presumably, phenol 12 is formed from diketone 9 via a similar oxidative aromatization. To our knowledge, our result constitutes the shortest reported synthesis of (±)-cochlearol T (4 steps from commercially available starting materials).

Although we were pleased to complete an expedient synthesis of cochlearol T (1), we recognized the need for a revised protecting group strategy to access the unaromatized natural products ganocochlearin A (2) and (±)-cochlearol Y (3). We therefore commenced with the pyrrolidine mediated Kabbe condensation of 2,5-dihydroxyacetophenone 13 and sulcatone 7 to furnish chromanone 14 (Scheme 2B). Protection of the phenol resulted in TBS-protected hydroxy chromanone 15. We formed diketone 16 in 37% yield and 1:1 dr upon exposure of chromanone 15 to methyl vinyl ketone 5 and NaOH in THF. Subsequent aldol condensation with KOH in refluxing EtOH resulted in three products in a combined 24% yield: (±)-ganocochlearin A (2), its C1-epimer (±)-cochlearol Y (3), and desilylated phenol 17 in a ratio of 1:1:3. Our characterization data for ganocochlearin A (2)² and cochlearol Y (3)⁵ matched in all respects to the data reported in the original isolation papers.

Subsequently, we evaluated the newly synthesized (±)-cochlearol T, (±)-cochlearol Y, and (±)-ganocochlearin A for their antifibrotic activities in a cell culture model of renal fibrosis. To test for the antifibrotic effects of the newly synthesized molecules, we induced fibrosis in the HK2 cells with TGF-β1 and treated them with either (±)-cochlearol T, (±)-cochlearol Y, or (±)-ganocholearin A to reduce fibrosis. Performing qPCR and Western blot studies for the three natural products, we identified a lower mRNA gene and protein expression for the fibrosis markers collagen 4A1 (Col4A1), vimentin, and fibronectin (FN) (Scheme 3). Specifically, treatment with 10 μM (±)-cochlearol T reduced Col4A1 (TGF-β1 2.35 ± 0.38 vs TGF-β1+CochT 0.66 ± 0.15, p < 0.0002), vimentin (TGF-β1 1.88 ± 0.48 vs TGF-β1+CochT 0.65 ± 0.4, p < 0.01), and FN (TGF-β1 6.88 ± 0.1.84 vs TGF-β1+CochT 1 ± 0.5, p < 0.001) mRNA expression by almost more than two-thirds (Scheme 3A). Western-blot studies for cochlearol T showed that FN, vimentin, and α-SMA protein expression were induced with TGF-β1. With the exception of α-SMA protein, bands were absent in untreated HK2 cells (Scheme 3B). As little as 1 μM of (±)-cochlearol T prevented the upregulation of fibrosis protein markers when treated with TGF-β1 underlining a strong antifibrotic role for the synthetic (±)-cochlearol T. Similarly, addition of (±)-cochlearol Y to HK2 cells treated with TGF-β1 displayed a lower mRNA expression for Col4A1 (TGF-β1 2.81 ± 0.22 vs TGF-β1+CochY 1.1 ± 0.2, p < 0.0001), vimentin (TGF-β1 2.2 ± 0.28 vs TGF-β1+CochY 1.22 ± 0.1, p < 0.001), and FN (TGF-β1 2.16 ± 0.31 vs TGF-β1+CochT 0.6 ± 0.37, p < 0.001), lowering mRNA expression of fibrosis markers by almost more than 50% (Scheme 3C). Studying protein expression of fibrosis markers after (±)-cochlearol Y treatment revealed that vimentin expression continued at a much lower expression levels with 1, 5, and 10 μM dosages, whereas α-SMA protein bands disappeared with 5 and 10 μM (±)-cochlearol Y dosages (Scheme 3D). Finally, addition of (±)-ganocochlearin A to HK2 cells treated with TGF-β1 displayed a lower mRNA expression for Col4A1 (TGF-β1 2.54 ± 0.24 vs TGF-β1+GanoA 0.6 ± 0.23, p < 0.0001), vimentin (TGF-β1 3.73 ± 0.47 vs TGF-β1+GanoA 0.93 ± 0.12, p < 0.0001), and FN (TGF-β1 11.31 ± 2.49 vs TGF-β1+GanoA 0.39 ± 0.2, p < 0.0001), lowering mRNA expression of fibrosis markers by more than two-thirds (Scheme 3E). Studying protein expression of fibrosis markers after (±)-ganocochlearin A treatment revealed that FN expression disappeared with 5 and 10 μM dosages, whereas α-SMA protein bands disappeared with as little as 1 μM (Scheme 3F).

Scheme 3. Biological Effects of (±)-Cochlearol T, (±)-Ganocochlearin A, and (±)-Cochlearol Y on Fibrosis Markers.

Interestingly, (±)-cochlearol T showed low toxicity in a cell viability assay (Scheme 4A). Treatment with TGF-β1 lowered cell viability to approximately 80%, which was not further decreased by 1 or 5 μM (±)-cochlearol T. Only the 10 μM dose of (±)-cochlearol T showed an additive negative effect on cell viability from approximately 80% to 70% (Scheme 4A). Given the convincing mRNA and protein expression data of (±)-cochlearol T (Scheme 3A,B), we investigated the dose–response of a more impactful fibrosis injury using TGF-β1 10 vs 20 ng/mL and applying a higher (±)-cochlearol T dose with 10 vs 20 μM (Scheme 4B). Applying the regular fibrosis injury with 10 ng/mL TGF-β1, the 20 μM (±)-cochlearol T dose reduced the Col4A1 mRNA expression by more than two-thirds compared to the 10 μM dose (TGF-β1 2.44 ± 0.21 vs TGF-β1+CochT 10 μM 0.74 ± 0.09 vs CochT 20 μM 0.11 ± 0.03, p < 0.0001). This effect was also seen with the higher TGF-β1 dose of 20 ng/mL, which resulted in more pronounced fibrosis injury as indicated by the 3–4-fold higher Col4A1 mRNA expression (TGF-β1 8.96 ± 0.96 vs TGF-β1+CochT 10 μM 4.08 ± 0.56 vs CochT 20 μM 1.21 ± 0.27, p < 0.0001). Similar effects were seen for the higher (±)-cochlearol T dose monitoring vimentin mRNA at 10 ng/mL TGF-β1 (TGF-β1 1.41 ± 0.1 vs TGF-β1+CochT 10 μM 0.54 ± 0.09 vs CochT 20 μM 0.24 ± 0.07, p < 0.0001) and at 20 ng/mL TGF-β1 (TGF-β1 3.19 ± 0.23 vs TGF-β1+CochT 10 μM 1.44 ± 0.17 vs CochT 20 μM 0.5 ± 0.1, p < 0.0001). The 20 μM (±)-cochlearol T dose reduced Col4A1 (p < 0.0001) and vimentin mRNA (p < 0.0001) significantly more compared to the 10 μM dose. Given the reduction of cell viability with the 10 μM dose, future studies are necessary to confirm the clinical use of higher than 10 μM dosages of (±)-cochlearol T.

Scheme 4. Dose-Dependent Effects on Renal Fibrosis and Cell Viability of (±)-Cochlearol T.

In conclusion, we completed the total synthesis of three related natural products, (±)-cochlearol T (1), (±)-ganocochlearin A (2), and (±)-cochlearol Y (3). Key steps in our synthesis include strategic Robinson annulations of differentially protected chromanones and an unexpected oxidative aromatization upon demethylative conditions. Importantly, the synthesized natural compounds display in vitro antifibrotic activities and affect the mRNA and protein expression of fibrosis markers. We anticipate the expediency of our total syntheses will enable the generation of analogues for in-depth studies of renoprotective activities in animal models of chronic kidney disease.

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.4c00497.

• Experimental details, characterization data, and spectral data (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.