Isolation, Structural Characterization and Antidiabetic Activity of New Diketopiperazine Alkaloids from Mangrove Endophytic Fungus Aspergillus sp. 16-5c

Six new DIKETOPIPERAZINE alkaloids aspergiamides A–F (1–6), together with ten known alkaloids (7–16), were isolated from the mangrove endophytic fungus Aspergillus sp. 16-5c. The structures of the new compounds were elucidated based on 1D/2D NMR spectroscopic and HR-ESIMS data analyses. The absolute configurations of aspergiamides A-F were established based on the experimental and calculated ECD data. All the compounds were evaluated for the antidiabetic activity against α-glucosidase and PTP1B enzyme. The bioassay results disclosed compounds 1 and 9 exhibited significant α-glucosidase inhibitory with IC50 values of 18.2 and 7.6 μM, respectively; compounds 3, 10, 11, and 15 exhibited moderate α-glucosidase inhibition with IC50 values ranging from 40.7 to 83.9 μM; while no compounds showed obvious PTP1B enzyme inhibition activity.


Introduction
Type 2 diabetes mellitus (T2DM) is a metabolic disorder disease and is characterized by an imbalance of blood glucose levels due to defects in insulin secretion. Increased blood glucose levels in T2DM may induce hypertension, atherosclerosis, and other metabolic disorder diseases [1]. It is estimated that 425 million people have diabetes, and about 90% account for type 2 diabetes. According to the World Health Organization (WHO), diabetes will reach the seventh cause of death by 2030 [2]. Although many antidiabetic drugs with different mechanisms can be available from the market, the inevitable side effects such as weight gain, liver damage, and allergic reactions hinder their application [3]. PTP1B is an intracellular non-transmembrane enzyme, which plays a major negative regulator role in insulin function by dephosphorylation of tyrosine-phosphorylated proteins [4]. Studies have shown that overexpression of protein tyrosine phosphatase 1B (PTP1B) can lead to the dephosphorylation of insulin receptors and insulin receptor substrates, resulting in insulin resistance and ultimately inactivating the entire insulin signaling pathway. Therefore, PTP1B inhibitors have attracted particular attention as potential therapeutic agents against diabetes [5]. In addition, α-glucosidase, a traditional antidiabetic target responsible for the hydrolysis of polysaccharides to monosaccharides, is still considered an important direction to discover new chemical entities for the treatment of non-insulin-dependent T2DM [6]. Therefore, the development of PTP1B and α-glucosidase inhibitors could be a promising strategy in discovering novel antidiabetic drug candidates.
Diketopiperazine (DKP) alkaloids are important secondary metabolites of microbes. Among these alkaloids, the diketopiperazine moiety is biosynthesized from different amino acids and formed as the smallest cyclic dipeptide skeleton. Diketopiperazine alkaloids have many important biological activities, such as antiviral, anti-tumor, antibacterial, Diketopiperazine (DKP) alkaloids are important secondary metabolites of microbes. Among these alkaloids, the diketopiperazine moiety is biosynthesized from different amino acids and formed as the smallest cyclic dipeptide skeleton. Diketopiperazine alkaloids have many important biological activities, such as antiviral, anti-tumor, antibacterial, and so on [7,8]. Indole diketopiperazine alkaloids belong to the subclass of DKPs are the condensation products of a complete tryptophan with a second amino acid-like Ltryptophan, L-proline, L-phenylalanine, or L-leucine [9]. Indole diketopiperazine alkaloids have attracted widespread attention not only because of their unique structure but also because of the wide range of biological activities, such as antiviral [10], anticancer [11], antioxidant [12], and insecticidal activities [13]. Marine fungi are important sources of diketopiperazine alkaloids. According to statistics, between 2000 and 2019, as many as 155 indole diketopiperazine alkaloids were identified from marine fungi alone [14].
As part of our aims for exploring biological active natural products [15], the secondary metabolites from fungus Aspergillus sp. 16-5c were investigated. Chemical investigation yielded six new diketopiperazine alkaloids, including five indole diketopiperazine alkaloids, aspergiamides A-E (1-5) and one 4-quinazolinone like diketopiperazine alkaloids aspergiamide F (6), together with ten known alkaloids (7-16) from the culture extracts of the fungus 16-5c ( Figure 1). These compounds were evaluated the antidiabetic potential by screening the enzyme inhibition of α-glucosidase and PTP1B. Herein, we describe the isolation and structure elucidation of the new compounds, as well as their bioactivities.

Structural Elucidation
Compound 1 was obtained as a pale yellow powder.
Compound 2 was isolated as a white powder and assigned a molecular formula of C 21 H 23 O 4 N 3 (12 degrees of unsaturation) based on the HRESIMS and 1D NMR ( Figures S8-S14). The 13 C NMR (Table 1) displayed 21 carbon signals, consisting of two methyls, three methylenes (one olefinic carbon), seven methines, and nine quaternary carbons (including one carboxyl carbonyl carbon, two amide carbonyl carbons). The 1 H NMR spectrum (Table 1)  , two methyls at δ H 1.54 (s, 3H) and 1.55 (s, 3H), two aliphatic methylenes at δ H 2.21 (m, 2H) and 2.48 (m, 2H), one aliphatic methine at δ H 4.25 (t, J = 5.6 Hz, 1H) were also recorded. Cumulative analyses of 1D and 2D NMR spectroscopic data revealed that 2 possessed a similar planar structure as that of 1. A major difference was the replacement of a hydroxymethylene group in 1 (δ C 62.5) by a carboxyl group in 2 (δ C 176.3), and the chemical shift of H-19 downfield from 1.67 in 1 to 2.84 in 2 due to the deshielding effect of the carboxyl group, indicating that compound 2 is the oxidation product of 1. Then, the planar structure of 2 was further verified by analyzing 2D NMR, as shown in Figure 2. The 1 H-1 H COSY correlation of H-18 (δ H 2.21)/H-19 (δ H 2.48) suggested the existence of -CH 2 -CH 2 -fragment. The chemical shift of C-20 (δ C 176.3), combined with HMBC correlation from H-18 to C-20 and from H-19 to C-20, indicated a carboxyl group was located at C-20 to form a -CH 2 -CH 2 -COOH moiety. Moreover, key HMBC correlations from H-19 to C-12 (δ C 56.1) suggested the moiety was connected with C-12, as shown in Figure 1. ∆8 double bond was identified as Z configuration by the lack of NOE effect between H-8 and NH-14 [17]. Furthermore, the absolute configuration of C-12 was determined to be R based on the same negative sign of its specific rota- MeOH)). Therefore, the structure of compound 2 was identified and named aspergiamide B. 154.8°, c 0.1, MeOH), as that of compound 1 ([α] − 171.4°, c 0.1, MeOH)). Therefore, the structure of compound 2 was identified and named aspergiamide B.   Compound 3 was isolated as a pale yellow powder. It gave a molecular formula of C 21 H 23 O 3 N 3 , as established by the HRESIM and NMR data, showing 12 degrees of unsaturation. The 13 C NMR and HSQC spectra displayed 21 carbon signals, consisting of two methyls, three methylenes (one olefinic carbon), seven methines, and nine quaternary carbons (including one carboxyl carbonyl carbon, two amide carbonyl carbons). Analysis of the 1 H NMR spectrum of 3 revealed a set of adjacent aromatic protons at δ H 7.07 (m), 7.12 (m), 7.31 (d, J = 7.9), 7.41, (d, J = 8.0), indicating the occurrence of two ortho-substituted benzene ring. Four olefinic protons at δ H 5.11 (m), 6.11 (dd, J = 17.2, 10.8), 7.23 (s), two methylenes at δH 2.11 (m, 1H), 2.34 (dd, J = 13.0, 5.9), 3.53 (d, J = 13.2), 3.92 (dd, J = 13.2, 4.9), two aliphatic methines at δ H 4.51 (m) and 4.72 (dd, J = 11.7, 5.9). All the above data were indicative of a prenyl containing 2, 3-disubstituted indole diketopiperazine skeleton. The 1 H-1 H COSY correlation of H-18 (δ H 2.11, 2.34)/H-19 (δ H 4.51)/H-20 (δ H 3.53, 3.92) suggested an aliphatic fragment of -CH 2 -CH-CH 2 -, combined with key HMBC correlations from H-19 to C-12 (δ C 58.7), from H-18 to C-13 (δ C 168.2), from H-20 to C-10 (δ C 160.9) suggested the aliphatic fragment was connected with diketopiperazine ring at C-12 and N-11 to form a five-member ring. The chemical shift of C-19 (δ C/H 68.6, 4.51; CH), together with the HRESIMS result, indicated a hydroxyl was connected with C-19. The geometry of the ∆8 double bond was Z configuration according to the chemical shift of H-8 (δ H 7.23) together with the lack of NOE effect between H-8 and NH-14 [17]. The NOESY correlation observed between H-12 and H-19 indicated that H-12 and H-19 were on the same face of the molecule (Figure 4), implying the existence of two possible relative configurations. Therefore, compound 3 had only one pair of enantiomers (12S, 19S-3 or 12R, 19R-3). The absolute configuration of 3 was determined by ECD calculation (Figure 3). The calculated data of 12S, 19S-3 showed good agreement with the experimental ECD of 3. Thus, the structure of 3 was assigned and named aspergiamide C.
Investigation on the biological activities of prenylated DKP alkaloids disclosed antioxidant activity in the literature [27,28]. It is reported that excessive accumulation of free radical and synchronous reduced the antioxidant defense mechanisms and can induce complications of diabetes mellitus [15]. In order to find potent antidiabetic natural products, all the compounds (1-16) were screened for the enzyme inhibitory activities against α-glucosidase and PTP1B (Table 3). Compared with the positive control acarbose (408 µM), all of the tested compounds, except compounds 5, 12, and 13, showed good to moderate inhibitory activity to α-glucosidase. Especially, compounds 1 and 9 exhibited significant α-glucosidase inhibitory with IC 50 values of 18.2 and 7.6 µM, respectively. Compounds 3, 10, 11, and 15 showed moderate α-glucosidase inhibition with IC 50 values ranging from 40.7 to 83.9 µM. However, these compounds have no obvious PTP1B inhibition at a concentration of 100 µg/mL (Table 3). These outcomes expanded the chemical and biological diversity of DKPs alkaloids and may provide new molecules for antidiabetic drug discovery.