Five New Cantharidin Derivatives from the Insect Mylabris cichorii L. and Their Potential against Kidney Fibrosis In Vitro

Five new monoterpenoids including three 1-hydroxymethyl-2-methyl cantharimide-type derivatives (1, 2, and 5) and two 1,2-dimethyl cantharimide-type derivatives (3 and 4), together with three known compounds (6–8) were isolated from the insect Mylabris cichorii Linnaeus. The structures of these new compounds, including their absolute configurations, were characterized by detailed analysis of NMR, chemical derivatization, and quantum chemical ECD calculations. All of the compounds were tested for their biological activity against kidney fibrosis. The results revealed that compounds 2, 4, and 7 could inhibit kidney fibrosis in vitro at 40 μM by inhibiting the expression of fibronectin and collagen I in TGF-β1-induced NRK-52e cells.


Introduction
In recent years, small molecules found in insects have been found to have good biological activities such as antiangiogenic activity [1], renal fibrosis inhibition [2], and COX-2 inhibitory activity [3]. The earliest recording of the blister beetle Mylabris cichorii Linnaeus as a medicinal insect is found in Sheng Nong's Herbal Classic. The dried bodies of M. cichorii have been used as a traditional Chinese medicine for 2000 years, significantly used for the treatment of tumors [4][5][6][7]. Previous studies have revealed cantharidin and its derivatives as a class of small molecules in the genus Mylabris [8][9][10][11]. As a part of our search for characterizing the potent bioactive compounds from insects, research on M. cichorii was carried out. As a result, three new 1-hydroxymethyl-2-methyl cantharimide-type cantharidins (1, 2, and 5), two new 1,2-dimethyl cantharimide-type derivatives (3 and 4), and three known compounds ( Figure 1) were isolated from M. cichorii. Recent studies of monoterpene alkaloids have revealed their powerful anti-fibrotic effects [12][13][14]. Because cantharidin derivatives also resemble such a class of monoterpene alkaloids, we were motivated to conduct a study on kidney fibrosis. Biological evaluation revealed the inhibitory properties of kidney fibrosis thereof. Herein, we report the isolation, structure characterization, and biological evaluation of the isolates.   Figure S2) exhibit 12 carbon signals including an imide group (δC 182.5, 180.9), a carbonyl group (δC 169.7), two oxygenated methines (δC 85.3, 83.2), and two quaternary carbons (δC 61.9, 55.3). The 1 H and 13 C NMR data of 1 are similar to cantharimide J [8], except for the signal of one less methylene group at C-2'. The methylene is oxidized as a carbonyl at the C-2', which could be confirmed by the lower field chemical shift of C-1' and the 13 C NMR data of C-2' at δC 169.9. An HMBC correlation ( Figure 2, Figure S4) of H2-1'/C-2' (δC 169.7) further confirmed our conclusion. The relative configuration of 1 was assigned by ROESY correlations (Figure 3, Figure S6), which show H-3 (δH 4.52), H2-10 (δH 3.73)/H3-11 (δH 1.33), and H2-10 (δH 3.97)/H-6 (δH 4.56), showing identical relative configuration at each chiral center with cantharidin. The absolute configuration of 1 was verified by the quantum chemical electronic circular dichroism (ECD) calculations. It was found that the experimental ECD spectrum of 1 is similar to the calculated one of (1R,2R,3S,6R)-1 ( Figure 4, Figure S8). Thus, the structure of 1 was finally deduced and named cichormide A.    Figure S2) exhibit 12 carbon signals including an imide group (δ C 182.5, 180.9), a carbonyl group (δ C 169.7), two oxygenated methines (δ C 85.3, 83.2), and two quaternary carbons (δ C 61.9, 55.3). The 1 H and 13 C NMR data of 1 are similar to cantharimide J [8], except for the signal of one less methylene group at C-2 . The methylene is oxidized as a carbonyl at the C-2 , which could be confirmed by the lower field chemical shift of C-1 and the 13 C NMR data of C-2 at δ C 169.9. An HMBC correlation ( Figure 2, Figure S4) of H 2 -1 /C-2 (δ C 169.7) further confirmed our conclusion. The relative configuration of 1 was assigned by ROESY correlations (Figure 3, Figure S6), which show H-3 (δ H 4.52), H 2 -10 (δ H 3.73)/H 3 -11 (δ H 1.33), and H 2 -10 (δ H 3.97)/H-6 (δ H 4.56), showing identical relative configuration at each chiral center with cantharidin. The absolute configuration of 1 was verified by the quantum chemical electronic circular dichroism (ECD) calculations. It was found that the experimental ECD spectrum of 1 is similar to the calculated one of (1R,2R,3S,6R)-1 ( Figure 4, Figure S8). Thus, the structure of 1 was finally deduced and named cichormide A.   Figure S2) exhibit 12 carbon signals including an imide group (δC 182.5, 180.9), a carbonyl group (δC 169.7), two oxygenated methines (δC 85.3, 83.2), and two quaternary carbons (δC 61.9, 55.3). The 1 H and 13 C NMR data of 1 are similar to cantharimide J [8], except for the signal of one less methylene group at C-2'. The methylene is oxidized as a carbonyl at the C-2', which could be confirmed by the lower field chemical shift of C-1' and the 13 C NMR data of C-2' at δC 169.9. An HMBC correlation ( Figure 2, Figure S4) of H2-1'/C-2' (δC 169.7) further confirmed our conclusion. The relative configuration of 1 was assigned by ROESY correlations (Figure 3, Figure S6), which show H-3 (δH 4.52), H2-10 (δH 3.73)/H3-11 (δH 1.33), and H2-10 (δH 3.97)/H-6 (δH 4.56), showing identical relative configuration at each chiral center with cantharidin. The absolute configuration of 1 was verified by the quantum chemical electronic circular dichroism (ECD) calculations. It was found that the experimental ECD spectrum of 1 is similar to the calculated one of (1R,2R,3S,6R)-1 ( Figure 4, Figure S8). Thus, the structure of 1 was finally deduced and named cichormide A.  configuration of 2 at C-1′ was assigned. With this information in hand, the absolute configurations at the rest chiral carbons of 2 were solved by comparison of the theoretical and experimental ECD spectra ( Figure 4, Figure S16). The results showed that the calculated ECD spectrum of (1R,2R,3S,6R,1′R)-2 is in good accordance with that of 2. Thus, the structure of 2 was determined to be [(1R,2R,3S,6R,1′R)-1-hydroxymethyl-2-methyl-3,6-epoxycyclohexane-1,2-dicarboximide]-(1'R)-histidine and named cichormide B.       Figure S10) show 16 carbon signals, which were assigned as one methyl, four methylenes, five methines, and six non-protonated carbons (including 2 carbonyls and 1 carboxyl). These featured NMR signals are similar to those of canthaminomide F [11], suggesting that 2 is a 1-hydroxymethyl-2-methyl cantharimidetype derivative. The key difference is that the substitution of a methyl at C-1 is replaced by a hydroxymethyl, which is supported by the HMBC correlations of H 2 -10/C-2, C-6, and C-7 (Figure 2, Figure S12). Thus, the planar structure of 2 was assigned. The ROESY correlations between H-3 (δ H 4.46), H 2 -10 (δ H 3.69)/H 3 -11 (δ H 1.24), and between H 2 -10 (δ H 3.94)/H-6 (δ H 4.36) suggested that 2 possesses the same relative configuration as compound 1 in the counterpart (Figure 3, Figure S14). The resolution of the configuration at C-1 has been reported in previous literature [11], and the same method was used to determine the configuration of compound 2. The D or L-histidine was, respectively, submitted to a reaction containing compound 5 under 95% EtOH of solvent at 78 • C for 48 h, which led to the generation of 2a (1R,2R,3S,6R,1 R)/2b (1R,2R,3S,6R,1 S) (Supplementary Materials, Scheme S1). A careful comparison of the NMR data and the retention time in the HPLC chromatogram of 2a and 2b with those of compound 2 revealed that 2a is actually the same as 2 (Supplementary Materials, Figures S41-S45). Thus, the absolute configuration of 2 at C-1 was assigned. With this information in hand, the absolute configurations at the rest chiral carbons of 2 were solved by comparison of the theoretical and experimental ECD spectra (Figure 4, Figure S16). The results showed that the calculated ECD spectrum of (1R,2R,3S,6R,1 R)-2 is in good accordance with that of 2. Thus, the structure of 2 was  Figure S18) exhibit 14 signals including an imide group [δ C 183.1 × 2], two oxygenated methines [δ C 85.1 × 2], a carbonyl group (δ C 172.9), and two quaternary carbons (δ C 55.3 × 2). The 13 C NMR spectrum of 3 is similar to that of cantharimide E [8], except for one additional methoxy group at δ C 52.4. The amine group in cantharimide E at C-3 is absent in 3 and replaced by a methoxy group, which could be further confirmed by the HMBC (Figure 2, Figure S20) correlation between H 3 -4 and the carbonyl group δ C 172.9 (C-3 ). In the same manner, the relative configuration of 3 was determined by ROESY correlations (Figure 3, Figure S22) of H 3 -10 or H 3 -11/H-3 (δ H 4.47), H-6 (δ H 4.48), suggesting that 3 possesses the same relative configuration as that of cantharidin. Likewise, the absolute configuration of 3 was determined as (1S,2R,3S,4R)-3 by comparing the calculated ECD spectrum with the experimental one ( Figure 4, Figure S24). Taken together, the structure of 3 was finally identified and named cichormide C.
As mentioned above, cantharidin derivatives have been characterized by the genus Mylabris. It is clear that the side chain attaching to the nitrogen atom could be different amino acid residues. In this study, the side chains belonging to glycine, histidine, alanine, and lysine were observed adding the diversity for the side chain of suan a class of compounds.

Biological Evaluation
The renal protection of all the isolates was carried out in TGF-β1-induced rat renal proximal tubular cells. To exclude the possibility that the biological effects of the compounds are caused by cytotoxicity, a CCK-8 assay was first carried out ( Figure 5). The results showed that the other compounds had slight toxicity toward rat renal proximal tubular cells (NRK-52e) except for 5, 6, and 8. Hence, the renal protection property of compounds 1-4 and 7 were evaluated ( Figure 6). As presented in Figure 7, compounds 2, 4, and 7 were found to reduce the expression of fibronectin and collagen I in a dose-dependent manner in TGF-β1-induced NRK-52e cells. Since fibronectin and collagen I are components of the extracellular matrix and overexpression of the extracellular matrix is considered to be the hallmark of renal fibrosis, our current finding disclosed that cantharidin derivatives might be potent agents in renal protection. To our knowledge, this is the first time that cantharidin derivatives have been found to possess biological activity in renal fibrosis.

General Procedures
Optical rotations were recorded on an Anton Paar MCP-100 digital polarimeter. UV and CD spectra were measured on a Chirascan instrument (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were collected by a Bruker Avance III 600 MHz or a 500

Insect Material
M. cichorii were collected from Henan province, China, in July 2020, and identified by Prof. Dang-Rong Yang from the Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China. A voucher specimen (CHYX-0643) was deposited at the School of Pharmaceutical Sciences, Shenzhen University Health Science Center, China.

Extraction and Isolation
The air-dried powdered M. cichorii (9 kg) were extracted with 50% aqueous EtOH (4 × 45 L, 24 h each) at room temperature. The combined extracts were concentrated to obtain a crude extract (1.5 kg), which was then divided into six parts (Fr.A-Fr.F) by using a macroporous adsorbents Rohmhass AMBERLITE TM XAD 16N column eluted with gradient aqueous EtOH (0:100-100:0). Fr.B (20.0 g) was isolated by an MCI gel CHP 20P column eluted with gradient aqueous MeOH (3-100%) to afford five fractions ( NRK-52e cells were treated with TGF-β1 (10 ng/mL) for 48 h in the absence or presence of 40 µM compounds. Cell lysates were prepared with RIPA buffer (Beyotime, Shanghai, China) containing 1 × protease inhibitor cocktail (Roche, Mannheim, Germany), 1 × phosphatase inhibitor cocktails, 0.1 mM PMSF, and quantified protein samples using the BCA assay (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein extracts were separated by 8% SDS-PAGE and transferred to PVDF membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 5% BSA, then with the indicated antibodies overnight at 4 • C, and were then followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature. The bands were visualized and measured via the ECL kit (Pierce, Hercules, CA, USA) and analysis system

Conclusions
In conclusion, our study resulted in the characterization of cantharidin derivatives from the title material, adding new facets for cantharidin structure diversity. Furthermore, biological comparison found that cantharidin derivatives with an oxygen atom instead of a nitrogen atom at position 8 and an alkyl group at C-1 and C-2 are toxic. To date, a limited number of cantharidin derivatives (<50) have been investigated from the Mylabris species and their potential for kidney fibrosis has not been described. Finally, the anti-fibrotic activity of cantharimide-type derivatives might provide new insight into the biological profiling of chemicals from M. cichorii, an excellent alternative resource for new pharmaceuticals.