Chromone Derivatives with α-Glucosidase Inhibitory Activity from the Marine Fungus Penicillium thomii Maire

The fungal strain YPGA3 was isolated from the sediments of the Yap Trench and identified as Penicillium thomii. Eight new chromone derivatives, named penithochromones M−T (1–8), along with two known analogues, 9 and 10, were isolated from the strain. The structures were established by detailed analyses of the spectroscopic data. The absolute configuration of the only chiral center in compound 1 was tentatively determined by comparing the experimental and the calculated specific rotations. Compounds 7 and 8 represent the first examples of chromone derivatives featuring a 5,7-dioxygenated chromone moiety with a 9-carbon side chain. Bioassay study revealed that compounds 6–10 exhibited remarkable inhibition against α-glucosidase with IC50 values ranging from 268 to 1017 μM, which are more active than the positive control acarbose (1.3 mmol).


Introduction
Chromone is a group of oxygen-bearing heterocyclic molecules featured by a benzoannelated γ-pyrone ring. Natural products containing a chromone moiety are widespread in nature and display various biological activities, such as antioxidant, antiviral, and anti-inflammatory activities [1]. In recent years, the chemical study of marine fungi has led to the discovery of numerous new or bioactive compounds, contributing greatly to natural products chemistry [2][3][4][5][6]. Among these metabolites, some chromone derivatives exhibit significant activity, the following are some examples. Pestalotiochromone A, isolated from the fungus Pestalotiopsis neglecta, exhibited potent binding affinities with liver X receptor α with the dissociation equilibrium constant value 6.2 µM [7]. Pestalotiopsones B and F isolated from Diaporthe sp. displayed potential antiviral activities against three influenza A virus subtypes with IC 50 values ranging from 2.56 to 6.76 µM [8]. The rare dihydrothiophene-condensed chromones oxalicumones A, B, D, and E from Penicillium oxalicum showed notable cytotoxicity against several carcinoma cell lines with IC 50 values below 10 µM [9]. The active chromone derivatives may provide a structural basis for the research and development of related new drugs.
In our efforts to search for bioactive molecules from deep-sea fungus [10][11][12][13][14], the fungal strain Penicillium thomii YPGA3 isolated from the sediments at a depth of 4500 m in the Yap Trench was screened out for chemical investigation, and the 1 H NMR spectrum and HPLC-DAD fingerprint of the EtOAc extract of this strain presented information that suggested the presence of chromone derivatives. As a result, eight new chromone derivatives, namely penithochromones M−T (1-8), along with two known analogues, 9 and 10, were obtained by 1 H NMR-guided isolation ( Figure 1). All compounds were evaluated for their inhibitions against α-glucosidase and the antioxidant capacities. Herein, we report the structural elucidation of the new chromone derivatives and the bioactivities.
were obtained by 1 H NMR-guided isolation ( Figure 1). All compounds were evaluated for their inhibitions against α-glucosidase and the antioxidant capacities. Herein, we report the structural elucidation of the new chromone derivatives and the bioactivities.

Results
Penithochromone M (1) was obtained as a light-yellow oil. The HRESIMS data gave a molecular formula of C 17 H 18 O 6 , requiring nine indices of hydrogen deficiency. The 1 H NMR spectrum of 1 showed resonances for two meta-coupled aromatic protons at δ H 6.36 (1H, d, J = 1.9 Hz, H-6) and 6.61 (1H, d, J = 1.9 Hz, H-8), a singlet for an olefinic resonance at δ H 6.25 (1H, s, H-3), a methoxy (δ H 3.84), an oxygenated methine (δ H 4.52), and ten aliphatic protons (Table 1 and Figure S1). The 13 C NMR and HSQC spectra (Figures S2 and S3) displayed 17 carbon resonances including two carbonyl groups including a ketone carbon (δ C 182.0, 177.1), eight aromatic or olefinic carbons, a methoxy (δ C 56.1), an oxygenated methine (δ C 79.9), and five methylenes (δ C 34.0, 32.9, 28.3, 27.3, 22.3). The aforementioned data indicated a chromone derivative, structurally similar to the co-isolated compound penithochromone C (9) [12]. The chromone moiety was assigned to be the same as that of penithochromone C by HMBC correlations from the protons H-6 (δ H 6.36), H-8 (δ H 6.61), and H-3 (δ H 6.25) to the aromatic or olefinic carbons in association with the correlation from the methoxy protons at δ H 3.84 to the oxygenated aromatic carbon C-7 (δ C 165.2) (Figure 2 and Figure S5). The rest resonances are attributed to a seven-carbon unit located at C-2 by analysis of 2D NMR data ( Figure 2 and Figures S3-S6 ). They were connected by the HMBC correlations from H 2 -9 and H 2 -10 to C-11 (δ C 34.0). The carbonyl carbon at δ C 177.1 was adjacent to the methylene CH 2 -14 by HMBC correlation from H 2 -14 to C-15 (δ C 177.1). As the chromone nucleus and the carbonyl carbon C-15 accounted for eight degrees of unsaturation, the remaining one required the presence of an additional ring in the seven-carbon unit. Taking the chemical shifts of H-12 and C-15 and the MS data into consideration, the methine carbon C-12 and the carbonyl carbon C-15 should be connected via an O-atom to form a five-membered lactone ring. HMBC correlations from H 2 -9 to C-2 and C-3 positioned the seven-carbon unit at C-2. Thus, the gross structure of 1 was determined as depicted. The absolute configuration of the only chiral center C-12 in 1 was tentatively determined by comparing the experimental and the calculated specific rotation. Theoretical specific rotations of the model molecules S/R-1 were calculated at the b3lyp/6-31+g(d) level using methanol as solvent. The results (  20 D + 211). Thus, the absolute configuration of C-12 was tentatively assigned as R. Molecules 2021, 26, x FOR PEER REVIEW 3 of 10 seven-carbon unit at C-2. Thus, the gross structure of 1 was determined as depicted. The absolute configuration of the only chiral center C-12 in 1 was tentatively determined by comparing the experimental and the calculated specific rotation. Theoretical specific rotations of the model molecules S/R-1 were calculated at the b3lyp/6-31+g(d) level using methanol as solvent. The results (Table S1) were that the theoretical specific rotation of R- 20 D + 175.60) was nearly the same as the experimental value ([α] 20 D + 211). Thus, the absolute configuration of C-12 was tentatively assigned as R.  The molecular formula of penithochromone N (2) was determined to be C16H18O7 by the HRESIMS data. The 1 H NMR spectrum showed the resonances for a 5,7-dihydroxychromone moiety (δH 6.10, 6.18, 6.33), an oxygenated proton (δH 4.0), and a series of aliphatic protons (Table 1 and Figure S7). The 13  seven-carbon unit at C-2. Thus, the gross structure of 1 was determined as depicted. The absolute configuration of the only chiral center C-12 in 1 was tentatively determined by comparing the experimental and the calculated specific rotation. Theoretical specific rotations of the model molecules S/R-1 were calculated at the b3lyp/6-31+g(d) level using methanol as solvent. The results (Table S1) were that the theoretical specific rotation of R- 20 D + 211). Thus, the absolute configuration of C-12 was tentatively assigned as R.  The molecular formula of penithochromone N (2) was determined to be C16H18O7 by the HRESIMS data. The 1 H NMR spectrum showed the resonances for a 5,7-dihydroxychromone moiety (δH 6.10, 6.18, 6.33), an oxygenated proton (δH 4.0), and a series of aliphatic protons (Table 1 and Figure S7). The 13  seven-carbon unit at C-2. Thus, the gross structure of 1 was determined as depicted. The absolute configuration of the only chiral center C-12 in 1 was tentatively determined by comparing the experimental and the calculated specific rotation. Theoretical specific rotations of the model molecules S/R-1 were calculated at the b3lyp/6-31+g(d) level using methanol as solvent. The results (Table S1)  20 D + 211). Thus, the absolute configuration of C-12 was tentatively assigned as R.  The molecular formula of penithochromone N (2) was determined to be C16H18O7 by the HRESIMS data. The 1 H NMR spectrum showed the resonances for a 5,7-dihydroxychromone moiety (δH 6.10, 6.18, 6.33), an oxygenated proton (δH 4.0), and a series of aliphatic protons (Table 1 and Figure S7). The 13  The molecular formula of penithochromone N (2) was determined to be C 16 H 18 O 7 by the HRESIMS data. The 1 H NMR spectrum showed the resonances for a 5,7-dihydroxychromone moiety (δ H 6.10, 6.18, 6.33), an oxygenated proton (δ H 4.0), and a series of aliphatic protons (Table 1 and Figure S7). The 13 C NMR spectrum exhibited a total of 16 carbon resonances ( Figure S8), including eight aromatic carbons for a benzene ring and a double bond, a carboxylic acid (δ C 177.7), a ketone carbonyl (δ C 183.9), five methylenes (δ C 26.0, 26.3, 35.0, 38.1, 43.2), and an oxygenated methine (δ C 70.0). The above-mentioned information was very similar to that of the co-isolated penithochromone A (10), with obvious distinction due to the presence of an oxygenated methine (δ H 4.0; δ C 70.0) and one less methylene [12]. As the molecular formula of 2 possessed one more O atom than that of penithochromone A, compound 2 was proposed to be hydroxylated derivative of penithochromone A. The hydroxyl group was positioned at C-10 by the COSY correlations from H 2 -9 (δ H 2.81; 2.63) to H-10 (δ H 4.0). Thus, the gross structure of 2 was determined as shown and was secured by 2D NMR analyses (Figure 2 and Figures S9-S12).
Penithochromone O (3) had the molecular formula C 18 H 22 O 7 . The NMR data of 3 were quite similar to those of 2 (Table 1 and Figures S13-S18). The structural differences were found to be the presences of two methoxy groups (δ H 3.65, 3.87; δ C 52.0, 56.5). The one at δ H 3.87 was positioned at C-7 by HMBC correlation from the protons at δ H 3.87 to C-7 (δ C 167.4) (Figure 2), while the other one was positioned at C-15 by the cross peak δ H 3.65/δ C 176.0 in the HMBC spectrum. The structure of 3 was thus determined as depicted.
Penithochromone P (4) was isolated as a light-yellow oil. The HRESIMS data gave a molecular formula of C 18 H 22 O 7 , which was the same as that of 3, suggesting that they were isomers. The NMR spectra showed similar structural features as those of 3 (Tables 1 and 2). Based on analyses of the 1D and 2D NMR data ( Figure 2 and Figures S19-S24), the differences between 4 and 3 were owing to the positions of the hydroxyl in the side chain and the two methoxy groups. With HMBC correlations from the protons at δ H 3.89 to C-5 (δ C 162.1) and the protons at 3.90 to C-7 (δ C 166.3), the two methoxy groups were located at C-5 and C-7, respectively. The hydroxyl group in the side chain was placed at C-13 (δ C 69.3) by the spin system H 2 -9/H 2 -10/H 2 -11 /H 2 -12/H-13/H 2 -14 observed in the COSY spectrum. The NMR data of penithochromone Q (5) were nearly the same as those of 4 (Table 2 and Figures S25-S30). The only difference between 4 and 5 was found by the presence of an additional methoxy group (δ H 3.67, δ C 52.0) in 5, which was placed at C-15 by the HMBC correlation from the methoxy protons at δ H 3.67 to the carbonyl carbon at δ C 174.0. The structure of 5 was further confirmed by detailed analyses of the 2D NMR data (Figure 2).
The NMR data of penithochromone R (6) indicated that 6 was structurally similar to that of compound 4 ( Table 2 and Figures S31-S36). The distinctions were attributed to the absence of the two aromatic methoxy groups, indicating that 6 was the corresponding demethylated derivative. The deduction was corroborated by 2D NMR analyses (Figure 2). Penithochromone S (7), a light-yellow oil, had the molecular formula C 19 H 24 O 7 as provided by HRESIMS data. The NMR data of 7 indicated the structure consisted of a chromone moiety and a side chain (Table 3 and Figures S37-S41). The chromone moiety was determined to be the same as that of 1 by 2D NMR analyses ( Figure 3)  absence of the two aromatic methoxy groups, indicating that 6 was the corresponding demethylated derivative. The deduction was corroborated by 2D NMR analyses ( Figure  2). Penithochromone S (7), a light-yellow oil, had the molecular formula C19H24O7 as provided by HRESIMS data. The NMR data of 7 indicated the structure consisted of a chromone moiety and a side chain (Table 3 and Figures S37−S41). The chromone moiety was determined to be the same as that of 1 by 2D NMR analyses ( Figure 3). As for the side chain, two spin systems made up by H2-9/H-10/H2-11/H2-12 and H2-14/H2-15/H2-16 can be deduced based on the COSY peaks, they were connected by HMBC correlations from H2-11 (δH 1.55) to C-13 (δC 30.3) and H2-12 (δH 1.41) to C-14 (δC 30.2). Additional HMBC correlations from H2-15 (δH 1.62) and H2-16 (δH 2.28) to the carboxylic acid carbon C-17 (δC 177.9) finally established a 9-carbon side chain, which was positioned at C-2 (δC 170.4) by HMBC correlations from H2-9 (δH 2.82, 2.65) to C-2 and C-3 (δC 110.4) and H-10 (δH 4.0) to C-2.   seven-carbon unit at C-2. Thus, the gross structure of 1 was determined as depicted. The absolute configuration of the only chiral center C-12 in 1 was tentatively determined by comparing the experimental and the calculated specific rotation. Theoretical specific rotations of the model molecules S/R-1 were calculated at the b3lyp/6-31+g(d) level using methanol as solvent. The results (Table S1)  20 D + 211). Thus, the absolute configuration of C-12 was tentatively assigned as R.  The molecular formula of penithochromone N (2) was determined to be C16H18O7 by the HRESIMS data. The 1 H NMR spectrum showed the resonances for a 5,7-dihydroxychromone moiety (δH 6.10, 6.18, 6.33), an oxygenated proton (δH 4.0), and a series of aliphatic protons (Table 1 and Figure S7). The 13  seven-carbon unit at C-2. Thus, the gross structure of 1 was determined as depicted. The absolute configuration of the only chiral center C-12 in 1 was tentatively determined by comparing the experimental and the calculated specific rotation. Theoretical specific rotations of the model molecules S/R-1 were calculated at the b3lyp/6-31+g(d) level using methanol as solvent. The results (Table S1)  20 D + 211). Thus, the absolute configuration of C-12 was tentatively assigned as R.  The molecular formula of penithochromone N (2) was determined to be C16H18O7 by the HRESIMS data. The 1 H NMR spectrum showed the resonances for a 5,7-dihydroxychromone moiety (δH 6.10, 6.18, 6.33), an oxygenated proton (δH 4.0), and a series of aliphatic protons (Table 1 and Figure S7). The 13 (Table 3 and Figures S42-S46), 8 was also determined to be a chromone derivative bearing a nine-carbon side chain. The distinctions can be found to be the presence of one trans-double bond [δ H 6.95 (1H, dd, J = 15.7, 7.0 Hz), 5.81 (1H, d, J = 15.7 Hz); δ C 150.6, 123.0] and the absence of two methylenes. The double bond was positioned at C-15 and C-16 by HMBC correlations from H-15 (δ H 6.95) and H-16 (δ H 5.81) to the carboxylic acid carbon C-17 (δ C 170.5). The structure of 8 was supported by detailed analyses of the 2D NMR data (Figure 2).
α-Glucosidase inhibitors can prevent the digestion of carbohydrates and decrease the effect of carbohydrates on blood glucose, and are an effective therapy for patients with type 2 diabetes mellitus. As the known analog 4-(5,7-dimethoxy-4-oxo-4H-chromen-2yl)butanoic acid exhibited moderate inhibitory activity on α-glucosidase [15], and some molecules containing a chromone moiety in the literature showed notable inhibitions against α-glucosidase [16], compounds 1-10 were screened for their α-glucosidase inhibitory effects at an initial concentration of 667 µM. Those with inhibitions more than 25% were further evaluated to determine the IC 50 values ( Table 4). The results showed that compounds 9 and 10 displayed significant inhibitory effects with IC 50 values of 688 and 268 µM respectively, being much more effective than the positive control acarbose (1.33 mM). While compounds 7 and 8 exhibited comparable effects as acarbose with IC 50 values of 917 and 1017 µM, respectively. Based on analysis of the structures and activities of 1-10, the introduction of hydroxyl group in the side chain may decrease the inhibitory effects, as compound 9 was much more active than its hydroxylated derivatives 2 and 6. As compounds containing a phenolic moiety usually display effective antioxidant activity and could neutralize free radicals, thus preventing them from causing damage [17], compounds 1-10 were further evaluated for their antioxidant activity at the initial concentration of 1 mM. The results showed that all tested compounds exhibited very weak inhibitions less than 50% (Table 4).

Conclusions
In the current study, chemical research of the deep-sea fungus Penicillium thomii YPGA3 resulted in the isolation of eight new chromone derivatives named penithochromones M−T (1-8), together with penithochromones C (9) and A (10). The structures were established by analyses of the NMR and HRESI data. Compounds 7 and 8 represent the first examples of chromone derivatives featuring a 5,7-dioxygenated chromone moiety with a 9-carbon side chain. Penithochromones A and C displayed conspicuous inhibition on α-glucosidase with IC 50 values of 268 and 688 µM respectively, being much more effective than the positive control acarbose (1.3 mmol). Our study enriched the family of chromone derivatives and may provide ideas for the development of α-glucosidase inhibitors.