Pyrrole-2-carboxaldehydes: Origins and Physiological Activities

Pyrrole-2-carboxaldehyde (Py-2-C) derivatives have been isolated from many natural sources, including fungi, plants (roots, leaves, and seeds), and microorganisms. The well-known diabetes molecular marker, pyrraline, which is produced after sequential reactions in vivo, has a Py-2-C skeleton. Py-2-Cs can be chemically produced by the strong acid-catalyzed condensation of glucose and amino acid derivatives in vitro. These observations indicate the importance of the Py-2-C skeleton in vivo and suggest that molecules containing this skeleton have various biological functions. In this review, we have summarized Py-2-C derivatives based on their origins. We also discuss the structural characteristics, natural sources, and physiological activities of isolated compounds containing the Py-2-C group.


Introduction
The factors that contribute to a healthy lifestyle are a topic of interest in modern society. Some diseases are related to lifestyle, including diabetes, heart disease, and cancer. Preventive treatment of such diseases is important. Lifestyle diseases are sometimes strongly connected with a person's diet, and smoking, binge eating, and drinking are strong risk factors for these diseases. Early detection of lifestyle diseases is important and many biomarkers have been developed in recent years for this purpose.
Many biomarkers have been reported for the early detection and determination of the stage of diabetes. In the stages of diabetes, many metabolites are produced by the reaction of glucose and amino acids. This process occurs via the well-known Amadori and Maillard reactions. After a complicated series of reactions, advanced glycation end products (AGEs) are produced. Typical AGEs include carboxymethyllysine, carboxyethyllysine, GA-pyridine, glucospan, arg-pyrimidine, pentosidine, and pyrraline. Pyrraline (1) has a structure that contains a formyl functional group at the 2-position of a pyrrole ring. Interestingly, AGEs are also produced in fermentation procedures. This finding suggests that similar reaction processes might take place under human physiological conditions over time, which is in agreement with the observation that lifestyle diseases mainly occur in the elderly. If such fermentation processes are also occurring over time in other biological systems, AGEs might be observed in other organisms. To investigate this hypothesis, we focused our attention on Py-2-C and examined naturally occurring Py-2-C derivatives. Furthermore, we discuss the biological activities of Py-2-C derivatives, including both in vitro and in vivo studies. We also explore the origins of Py-2-C by summarizing recently reported synthetic and biosynthetic routes for this compound.
The reaction was performed in 1 h under the catalysis of acetic acid, which suggested that this type of reaction might occur under physiological conditions over time (days, months, or years). However, many other reaction products were produced under the reaction conditions (as determined by thin-layer chromatographic analysis, but compounds were not isolated), and separation and purification of the hydroxymethyl derivatives from other byproducts were quite difficult. Under the reaction conditions, 2,4-dinitrophenylhydrazine (DNP) was used to stabilize the reaction products; however, the use of DNP caused further reactions, such as dehydration. This reaction procedure is also applicable to L-lysine. The condensation reaction of L-lysine monohydrochloride with glucose at elevated temperature (105 • C) for 6 h afforded ε-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-L-norleucine (1) at a 0.16% yield based on L-lysine [2]. Py-2-Cs were not isolated under these reaction conditions; however, this type of reaction might also take place in vivo. Under similar reaction conditions, several amino acids have been reacted with glucose (Maillard reaction), and the reducing ability of the products was examined. In the reaction of a γ-amino acid and glucose, γ-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-butyric acid (3) was isolated in low yield [3]. Among the products of the Maillard reaction of 10 amino acids and D-glucose, GABA showed the highest ferricyanide reduction potential (ascorbic acid was used as a standard). However, precise examination of the reducing ability of these Maillard reaction products revealed that the reducing ability reached the highest reducing state at 0.5 h after the start of the Maillard reaction. At this time, the formation of γ-(2-formyl-5hydroxymethyl-pyrrol-1-yl)-butyric acid did not correspond with the reducing ability. The formation of γ-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-butyric acid gradually increased after 1 h and reached a constant level 10 h after the start of the Maillard reaction. This reaction profile suggested the possibility that other Maillard reaction products were produced in the early stages of the reaction, which were responsible for the reducing ability in the early stages. While, the formation of γ-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-butyric acid might decrease the reducing ability. Bearing a formyl group side chain, γ-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-butyric acid has a potential reducing ability for metal ions; however, the reducing ability is not strong enough to reduce metal ions completely. The proposed mechanism for the formation of Py-2-C from glucose and alkylamines (including lysine and γ-butyric acid) has been suggested to occur via the formation of 3-deoxy-Dglucose in the reaction course. In this mechanism, the alkylamine attacks the enol site of 3-deoxy-D-glucose forming an enamino diketone. Then, intramolecular cyclization of the amine moiety with the ketone produces a dihydropyrrole bearing formyl and hydroxy groups on the ring. Dehydration of the dihydropyrrole produces a Py-2-C as the final product. In the synthetic reaction, the initial attack of the alkylamine on the formyl group of glucose (in the open form of glucose) is the trigger for initiating the reaction. The hydration of the imine moiety to a carbonyl compound provided 3-deoxy-D-glucoside as a key intermediate, which was smoothly dehydrated to afford the enone. The Michael addition of the alkylamine to this enone and subsequent ring closure provided the dihydropyrrole. The dehydration of the dihydropyrrole (aromatization) took place to afford Py-2-C (4) as the final product [4][5][6] (Figure 1). The total synthesis of pyrrolemarumine (5) was attained by the regioselective functionalization of 2-formylpyrrole [7]. The biosynthesis of pyrrole consists of amino acids (glycine, proline, serine, threonine, and tryptophan) and dicarboxylic acids (malonic acid, oxaloacetic acid, and succinic acid) as synthons for the construction of the aromatic ring [8]. Another biosynthetic pathway for the formation of a pyrrole ring has been recently reported by Lautru et al. [9]. In this biosynthetic pathway, N-acetylglucosamine was used as the precursor, and after several enzymatic processes, 4-acetamidepyrrole-2-carboxylic acid was isolated. Further reactions were performed to afford congocidine as the final product. Very recently, the biosynthesis of a Py-2-C was attained via enzymatic fixation [10] in which the production of Py-2-C (6) from pyrrole was achieved using Pseudomonas aeruginosa HudA/PA0254 and Segniliparus rotundus CAR ( Figure 1). biosynthesis of a Py-2-C was attained via enzymatic fixation [10] in which the production of Py-2-C (6) from pyrrole was achieved using Pseudomonas aeruginosa HudA/PA0254 and Segniliparus rotundus CAR ( Figure 1).

Pyrrole-2-carboxaldehydes from Fungi
Edible mushrooms are very popular in eastern countries such as China, Korea, and Japan. Edible mushrooms contain many nutrients, including amino acids, proteins, lipids, and carbohydrates. Some mushrooms have been reported to have pharmacological activities. Identification of the key compounds from the extracts of edible mushrooms has attracted much attention from pharmacological companies and scientists.
From the mangrove plant Aegiceras corniculatum, two Py-2-C derivatives (7 and 8) and one indole alkaloid were isolated, and the structures were determined with spectroscopic analyses [11]. Extraction of the freeze-dried powder of Mycoleptodonoides aictchisonii resulted in the isolation of a Py-2-C bearing a carboxylic acid side chain. This compound was isolated by a series of extraction procedures, including hot methanol extraction, filtration, and subsequent extraction with ethyl acetate. The ethyl acetate layer was separated via silica gel column chromatography followed by HPLC separation using an ODS column. The Py-2-C bearing α-iso-butyl-acetic acid at the N-position was isolated along with 10 other compounds. The chemical structure of this Py-2-C (9) was determined by spectroscopic analyses [12]. Pyrrolezanthine (10) was first isolated from the edible mushroom Leccinum extremiorientale in 2011 [13]. The structure of pyrrolezanthine (10) was confirmed by a comparison of the NMR and MS data with the literature data [14]. From the cultivation of Annulohypoxylon lianense, the pyrrole alkaloid ilanepyrrolal (11) was isolated and characterized based on the spectroscopic analyses [15]. From the cultivation of Chaetomium globosum, which is an endophytic fungus in Ginkgo biloba, N-unsubstituted-5-hydroxymethyl-Py-2-C (4) was isolated and characterized [16]. In 2014, from an extract of the edible mushroom makomotake (Zizaria latifolia), three Py-2-Cs have been isolated and identified. Two of the Py-2-Cs (12 and 13) had already been reported (one compound (13) had already been chemically synthesized [5]) [5,17]; however, the Py-2-C (14) was a new compound [18]. The structures of the two known compounds were determined by a comparison with the previously reported spectral data. The molecular formula of the new compound was determined by HRESIMS and the molecular structure was determined using 1 H-and 13 C-NMR spectra, including DEPT, COSY, HMQC, and HMBC techniques. From the endophytic fungus of Xylaria papulis, a Py-2-C (15, popupyrrolal) was isolated and characterized based on the spectroscopic analyses [19]. Cordyceps species have been widely used in traditional medicine, especially for their effects on metabolic pathways. From an extract of Cordyceps militaris, 12 known compounds (including pyrrole alkaloids and nucleotide derivatives) and two new compounds, named cordyrrole A and B, were isolated and characterized. Cordyrrole A (16) is a Py-2-C derivative bearing a six-membered cyclic amide at the N-position of the pyrrole ring [20]. In 2015, from the edible mushroom, Xylaria nigripes, four Py-2-Cs (17)(18)(19)(20) were isolated and identified. Two pyrrole alkaloids had already been reported: the Py-2-Cs pollenpyrroside A (17) and acortatarin A (18). The other two Py-2-Cs (19 and 20) have a tricyclic ring structure comprising a common bicyclic system with 2-formyl-pyrrole and substituted morpholine rings and a

Pyrrole-2-carboxaldehydes from Fungi
Edible mushrooms are very popular in eastern countries such as China, Korea, and Japan. Edible mushrooms contain many nutrients, including amino acids, proteins, lipids, and carbohydrates. Some mushrooms have been reported to have pharmacological activities. Identification of the key compounds from the extracts of edible mushrooms has attracted much attention from pharmacological companies and scientists.
From the mangrove plant Aegiceras corniculatum, two Py-2-C derivatives (7 and 8) and one indole alkaloid were isolated, and the structures were determined with spectroscopic analyses [11]. Extraction of the freeze-dried powder of Mycoleptodonoides aictchisonii resulted in the isolation of a Py-2-C bearing a carboxylic acid side chain. This compound was isolated by a series of extraction procedures, including hot methanol extraction, filtration, and subsequent extraction with ethyl acetate. The ethyl acetate layer was separated via silica gel column chromatography followed by HPLC separation using an ODS column. The Py-2-C bearing α-iso-butyl-acetic acid at the N-position was isolated along with 10 other compounds. The chemical structure of this Py-2-C (9) was determined by spectroscopic analyses [12]. Pyrrolezanthine (10) was first isolated from the edible mushroom Leccinum extremiorientale in 2011 [13]. The structure of pyrrolezanthine (10) was confirmed by a comparison of the NMR and MS data with the literature data [14]. From the cultivation of Annulohypoxylon lianense, the pyrrole alkaloid ilanepyrrolal (11) was isolated and characterized based on the spectroscopic analyses [15]. From the cultivation of Chaetomium globosum, which is an endophytic fungus in Ginkgo biloba, N-unsubstituted-5-hydroxymethyl-Py-2-C (4) was isolated and characterized [16]. In 2014, from an extract of the edible mushroom makomotake (Zizaria latifolia), three Py-2-Cs have been isolated and identified. Two of the Py-2-Cs (12 and 13) had already been reported (one compound (13) had already been chemically synthesized [5]) [5,17]; however, the Py-2-C (14) was a new compound [18]. The structures of the two known compounds were determined by a comparison with the previously reported spectral data. The molecular formula of the new compound was determined by HRESIMS and the molecular structure was determined using 1 H-and 13 C-NMR spectra, including DEPT, COSY, HMQC, and HMBC techniques. From the endophytic fungus of Xylaria papulis, a Py-2-C (15, popupyrrolal) was isolated and characterized based on the spectroscopic analyses [19]. Cordyceps species have been widely used in traditional medicine, especially for their effects on metabolic pathways. From an extract of Cordyceps militaris, 12 known compounds (including pyrrole alkaloids and nucleotide derivatives) and two new compounds, named cordyrrole A and B, were isolated and characterized. Cordyrrole A (16) is a Py-2-C derivative bearing a six-membered cyclic amide at the N-position of the pyrrole ring [20]. In 2015, from the edible mushroom, Xylaria nigripes, four Py-2-Cs (17)(18)(19)(20) were isolated and identified. Two pyrrole alkaloids had already been reported: the Py-2-Cs pollenpyrroside A (17) and acortatarin A (18). The other two Py-2-Cs (19 and 20) have a tricyclic ring structure comprising a common bicyclic system with 2-formyl-pyrrole and substituted morpholine rings and a ketohexoside ring [21]. From the fruiting bodies of Leccinum extremiorientale, two known Py-2-Cs bearing a butyric acid moiety (3 and 21) and one unknown Py-2-C bearing an acetic acid moiety (22) were isolated and characterized based on 1D-and 2D-NMR spectroscopy and MS [22]. All of these compounds are carboxylic acid derivatives connected by a short-chain (one-or three-carbon) linker to the N-position of the pyrrole ring. From the ethanol extract of the fermented mycelia of Xylaria nigripes, two unknown (23 and 24) and six known Py-2-Cs were isolated along with flavonoid derivatives. The chemical structures of the known Py-2-Cs (25, 26, 27, 3, 21, and 12) were determined based on the spectroscopic analyses [23]. From the endophytic fungus Mollisia sp., among many metabolites, one known Py-2-C (15) was isolated and identified [24]. From the edible mushroom Basidiomycetes-X (Echigoshirayukitake), three Py-2-C derivatives (3, 4, and 28) have been isolated and identified [25]. Two compounds had already been reported and one was a new compound (28) bearing a carbamide functional group on the side chain. From another edible mushroom, Phlebopus portentosus, three unknown (29, 30, and 31) and four known Py-2-Cs (32)(33)(34)(35) were isolated and the molecular formulas of each compound were determined with HRESIMS. The structure determination of the compounds was based on NMR spectroscopic analyses, including COSY and HMBC [26]. Among the known compounds, two compounds (33 and 35) had been previously isolated from the mycelium of Inonotus obliquus [27,28]. One compound (34) was a Ganoderma alkaloid previously isolated from the hypha of deep fermented Ganoderma capense [29]. Compound (33) had already been isolated from Lyceum Chinense in 2013 [30] ( Figure 2).
Py-2-Cs bearing a butyric acid moiety (3 and 21) and one unknown Py-2-C bearing acetic acid moiety (22) were isolated and characterized based on 1D-and 2D-NMR sp troscopy and MS [22]. All of these compounds are carboxylic acid derivatives connec by a short-chain (one-or three-carbon) linker to the N-position of the pyrrole ring. Fr the ethanol extract of the fermented mycelia of Xylaria nigripes, two unknown (23 and and six known Py-2-Cs were isolated along with flavonoid derivatives. The chem structures of the known Py-2-Cs (25, 26, 27, 3, 21, and 12) were determined based on spectroscopic analyses [23]. From the endophytic fungus Mollisia sp., among many m tabolites, one known Py-2-C (15) was isolated and identified [24]. From the edible mu room Basidiomycetes-X (Echigoshirayukitake), three Py-2-C derivatives (3, 4, and 28) h been isolated and identified [25]. Two compounds had already been reported and one w a new compound (28) bearing a carbamide functional group on the side chain. From other edible mushroom, Phlebopus portentosus, three unknown (29, 30, and 31) and f known Py-2-Cs (32)(33)(34)(35) were isolated and the molecular formulas of each compound w determined with HRESIMS. The structure determination of the compounds was based NMR spectroscopic analyses, including COSY and HMBC [26]. Among the known co pounds, two compounds (33 and 35) had been previously isolated from the mycelium Inonotus obliquus [27,28]. One compound (34) was a Ganoderma alkaloid previously i lated from the hypha of deep fermented Ganoderma capense [29]. Compound (33) had ready been isolated from Lyceum Chinense in 2013 [30] (Figure 2).

Pyrrole-2-carbaldehydes from Plant Sources
Py-2-Cs have been shown to be some of the compounds responsible for the arom flavor of raw cane sugar using GC-MS [31]. The detected Py-2-Cs (37-40) all have a cy lactone structure, which is produced by the condensation reaction of the hydroxymet group at the 5-position of the pyrrole ring with the carboxylic acid of the amino acid. Fr roasted chicory root (Cichorium intrybus L.), three Py-2-C lactones (38)(39)(40) were isola and the structures were determined based on the spectroscopic analyses (NMR, IR, U and MS) [32]. A butanol extract of flue-cured Virginia tobacco was fractionized to aff an acidic dichloromethane-soluble fraction. From this fraction, an unknown 2-formylp role acetic acid derivative (41) was isolated and the structure was determined based the spectroscopic analyses [33]. From the root of Pisum saticum, a Py-2-C (42) bearin five-membered lactone at the N-position was isolated and identified based on the spect scopic analyses [34]. This Py-2-C acted as a specific regulator of trigonelline-induced arrest. Py-2-Cs have also been isolated from other sources. For example, from the odor flower Quararibea funebris, the Py-2-C funebral (43) with a lactone chromophore was i lated and characterized by various spectroscopic methods [35]. The total synthesis of nebral was first reported in 1999, which used the Paal-Knorr condensation as a key s to prepare the pyrrole lactone moiety [36]. Another total synthesis of funebral has a been reported that used a chiral nitrone as a key substrate [37]. From the leaves of Magn coco, mangnolamide (44) with a phenylpropanoid chromophore has been isolated a characterized using HREIMS, IR, MS, and 1 H-and 13 C-NMR spectroscopy [38]. The ex structure was determined using NOE and HMBC spectra along with the MS fragmen tion pattern. The total synthesis of magnolamide was achieved in six steps starting fr the condensation of pyrrole-2,5-dicarboxaldehyde with N-(4-bromobutyl) phthalim [36]. From the Formosan plant Zanthoxylum simulans, the pyrrole alkaloid pyrrolezanth (10, 5-hydroxymethyl-1[2-(4-hydroxyphenyl)-ethyl]-1H-Py-2-C) was isolated along w the previously unknown (-)-simulanol, zanthopyranone, and 28 known compounds [ The structure of pyrrolezanthine (10) was determined based on the spectroscopic an yses. Among the unsaturated fatty acids isolated from the seed of Allium fistulosum L (2-formyl-5-hydroxymethylpyrrol-1-yl) butyric acid (3) was identified [39] and the str ture was determined based on a previous report [3]. From the seeds of the sweet chest Castanea sativa, methyl-(5-formyl-1H-pyrrole-2-yl)-4-hydroxybutyrate (45) was isola and the structure was identified based on the spectroscopic analyses [40]. Two new p
From the leaves of Nicotiana tabacum, the pyrrolezanthine (10) was isolated along with a benzofuran derivative (3-acetyl-7-hydroxy-6-methoxy-2-methylbenzofuran-4-carboxylate) [79]. The above data show that Py-2-Cs have been isolated from many fungal and plant sources. In some cases, the same compound has been isolated from different sources and named differently. For example, shensongine A is the same compound as xylapyrroside A (ent-capprisine B), acortatarin A is the same as pollempyrroside B (ent-capparisine A), and shensongine B is the same as xylapyrroside B. All of these compounds have a pyrrolomorpholine spiroketal structure and chiral carbon atoms [80]. In some cases, a stereochemical revision has been performed after the isolation and original characterization.
The sponge Mycale sp. has produced a variety of Py-2-Cs along with other metabolites, which have been summarized in recent review articles [103,104].
The sponge Mycale sp. has produced a variety of Py-2-Cs along with other metabolites, which have been summarized in recent review articles [103,104].  In 2014, seven new compounds, including five new Py-2-Cs (177-181, jiangrines A-E, along with the pyrrolezanthine, 10) were isolated from an Actinobacterium, Jiangella gansuensis. The structures of the compounds were determined mainly based on NMR spectroscopic techniques (COSY, HMBC, and NOE,) and HRESIMS. The precise stereochemical configuration of the glycerol moiety at the 5-position of the pyrrole ring was determined based on the chemical reaction in which the hydroxyl group at the 2-position of glycerol attacks the carbonyl moiety of the aldehyde to form a seven-membered cyclic hemiacetal structure in methanol. From NOE correlation studies, the stereochemical configuration of the glycerol moiety of jiangrine A was determined to be S (for the carbon attached to the ring) and R (for the carbon adjacent to the carbon attached to the ring). From a comparison to the chemical shifts of jiangrine A, the stereochemistries of jiangrines B, C, and D were determined. Under the reaction conditions, the separation of the stereoisomers of jiangrine C and jiangrine D was not achieved. These two compounds were present as a 1:1 mixture and this mixture was used in the biological experiments [105]. A new Py-2-C (182, jiangrine G), along with the known Py-2-Cs jiangrine A (183, revised structure of 117) and pyrrolezanthine (10) were isolated from the fermentation broth of Jiangella alba associated with the traditional Chinese medicinal plant Maytenus austroyunnanensis. The structure of jiangrine G is very similar to jiangrine A, including the stereochemistry of the moiety at the 3-position, except for the hydroxyl moiety on the phenyl ring attached at the 1-position of the pyrrole ring is absent in jiangrine G. The precise determination of the structure of jiangrine G was determined from spectroscopic studies ( 1 H-and 13 C-NMR, HRESIMS, and CD) [106] (Figure 6).
In 2014, seven new compounds, including five new Py-2-Cs (177-181, jiangrines A-E, along with the pyrrolezanthine, 10) were isolated from an Actinobacterium, Jiangella gansuensis. The structures of the compounds were determined mainly based on NMR spectroscopic techniques (COSY, HMBC, and NOE,) and HRESIMS. The precise stereochemical configuration of the glycerol moiety at the 5-position of the pyrrole ring was determined based on the chemical reaction in which the hydroxyl group at the 2-position of glycerol attacks the carbonyl moiety of the aldehyde to form a seven-membered cyclic hemiacetal structure in methanol. From NOE correlation studies, the stereochemical configuration of the glycerol moiety of jiangrine A was determined to be S (for the carbon attached to the ring) and R (for the carbon adjacent to the carbon attached to the ring). From a comparison to the chemical shifts of jiangrine A, the stereochemistries of jiangrines B, C, and D were determined. Under the reaction conditions, the separation of the stereoisomers of jiangrine C and jiangrine D was not achieved. These two compounds were present as a 1:1 mixture and this mixture was used in the biological experiments [105]. A new Py-2-C (182, jiangrine G), along with the known Py-2-Cs jiangrine A (183, revised structure of 117) and pyrrolezanthine (10) were isolated from the fermentation broth of Jiangella alba associated with the traditional Chinese medicinal plant Maytenus austroyunnanensis. The structure of jiangrine G is very similar to jiangrine A, including the stereochemistry of the moiety at the 3-position, except for the hydroxyl moiety on the phenyl ring attached at the 1-position of the pyrrole ring is absent in jiangrine G. The precise determination of the structure of jiangrine G was determined from spectroscopic studies ( 1 H-and 13 C-NMR, HRESIMS, and CD) [106] (Figure 6).
A Py-2-C derivative (100 µ g/mL) derived from makomotake (Zizania latifolia) showed moderate NQO1 induction activity in a Hepa 1c1c7 cell line and no cytotoxic effect was observed under the reaction conditions. The three Py-2-Cs (12)(13)(14) have been subjected to some bioassays, including the suppression of osteoclast formation (which has been
Administration of a Cordyceps millitaris extract (100 mg/kg, 300 mg/kg body weight) reduced the body weight gain and food efficiency ratio in C58BL/6J mice fed a high-fat diet. Among the compounds isolated from Cordyceps millitaris, cordyrrole A (16) showed a significant reduction in adipocyte differentiation and pancreatic lipase activity in 3T3-L1 cells [20] (Table 1).
Four Py-2-Cs (17)(18)(19)(20) isolated from Xylaria nigripes and structurally related synthetic derivatives have shown moderate to strong antioxidant effects. Rat A7r5 vascular smooth muscle cells were subjected to t-butyl hydroperoxide-induced oxidative stress after pretreatment for 4 h with Py-2- Cs (17, 18, and 20) at varying concentrations. The administration of Py-2-Cs attenuated cell death in a concentration-dependent manner. Among these compounds, 20 showed the strongest activity and a synthetic diastereomer of 17 also showed similar strong activity [21] (Table 1).
From the edible mushroom Phlebopus portentosus, seven Py-2-Cs (29-35) were isolated and characterized. SH-SY5Y cells were pretreated with each of these compounds or Nacetylcysteine (NAC) at 10 µM for 2 h prior to treatment with H 2 O 2 (10 µM). The Py-2-C with an ethylene bridge between the 3-position of the indole ring and the N-position of the pyrrole carboxaldehyde (35) showed the strongest neuroprotective activity, which was almost the same as NAC. The improvement in the cell viability for NAC and 36 was 24.5% and 26.5%, respectively. The other compounds showed moderate to mild activity (5.8%-15.7%). None of these compounds showed significant acetylcholine esterase inhibitory activity under the reaction conditions [26] (Table 1).

Physiological Activities of Pyrrole-2-carbaldehydes Originating from Plants
The anti-platelet aggregation activity of pyrrolezanthine (10) was examined along with other metabolites. Some metabolites showed anti-platelet activity but 10 did not show any activity [14]. The antioxidative activity of magnolamide (44) and a structurally related analog of magnolamide was investigated in the copper-induced oxidation of freshly prepared human LDL lipids by measuring the absorbance at 234 nm of the conjugated dienes in the presence or absence of Py-2-Cs. The IC 50 values of magnolamide (44) and the structurally related analog were 9.7 ± 2.8 and 16.9 ± 2.3, respectively. The IC 50 values of resveratrol and probucol under the same conditions were 13.1 ± 2.6 and 8.7 ± 1.4, respectively. These results showed that mangnolamide (44) had almost the same inhibitory activity against Cu + -induced LDL oxidation as probucol and resveratrol [108].
Forty-six compounds were characterized from a methanol extract of Lobelia chinensis, including the Py-2-C lobechine (61). Although 61 showed no inhibitory activity against HSV-1 replication or inhibition of superoxide generation by human neutrophils in response to N-formyl-methionyl-leucyl phenylalanine/cytochalasin B, 61 showed moderate elastase release inhibitory activity. The IC 50 value of 66 for elastase release inhibition was 25.01 ± 6.95 µM (the IC 50 value of the positive control LY294002 was 2.64 ± 0.29 µM) [52].
Five Py-2-Cs (3, 4, 21, 65, and morrole A (66)) isolated from Morus alba fruits were subjected to biological assays using RAW 264.7 cells. By measuring the production of nitric oxide (NO), TNF-α, and IL12, two butanoic acid derivatives bearing hydroxymethyl (3) or methoxymethyl (21) substituents at the 5-position of the pyrrole ring were shown to cause significant macrophage activation and also stimulated phagocytic activity in RAW 264.7 cells. Three other Py-2-Cs (4, 65, and 66) did not show significant activity [60]. Morrole A (66), which has the bulky substituent CH(CH 3 )-CH(CH 3 )-OH on the hydroxymethyl moiety did not show significant activity in any of the assays. The less bulky CH 3 derivative showed significant activity, which suggested the importance of the influence of steric hindrance on the activity (Table 2).
From Capparis spinosa, five alkaloids, including two new and two known Py-2-Cs (18, 37, 47, and 58), were isolated. The two new Py-2-Cs were named capparisine A (18) and capparisine B (58). The inhibitory effects of these compounds on the apoptosis (HL-7702 human hepatocyte cell line) induced by Act D (200 ng/mL) and TNF-α (2 ng/mL) were examined by evaluating the nuclei size and the average number of mitochondrial masses. None of these compounds showed any apoptosis inhibitory activity on the human hepatocyte cell line HL-7702 [49] ( Table 2).
Acortatarins A (18) and B (74) showed inhibition of ROS generation induced in highglucose-stimulated mesangial cells. In a fluorescent assay using 2 ,7 -dichlorofluorescein, 18 and 74 showed the highest inhibition activity (statistically meaningful) at 10 and 50 µM, respectively. After mesangial cells were pretreated with 18 for 1 h, the cells were incubated with a high concentration of glucose for 1-24 h. The fluorescent intensity showed a decrease (ca. 50% compared with the high glucose-treated cells) in the pretreated cells at various times (1, 3, 6, 12, and 24 h) [66]. The morpholine motif may plausibly be in part responsible for these results ( Table 2).
The hemerocallisamine derivatives (75 and 76) isolated from the daylily [69,70] were tested for their inhibitory activity against 42-mer amyloid β-protein aggregation and the effect on nerve growth factor using PC12 cell lines. Hemellocallisamine I (75) did not show any significant effects in either test [73] (Table 2).
Shensongines A (19) and C (77), isolated from an anti-arrhythmic TCM, showed cardiovascular activity by shortening the action potential duration in rat myocardial cell lines at 10 -6 mol/L [75]. Shensongine B (20) and pollenopyrroside B (18) did not show any cardiovascular activity. Shensongine A and shensongine C might affect ion channels, i.e., inhibit L-type calcium channel opening or facilitate potassium channel opening.
Pyrrolezanthine (10) isolated from Nicotiana tabacum showed moderate cytotoxicity against lung cancer A-549 and human colon cancer SW480 cell lines with IC 50 values of 38.3 and 33.7 µM, respectively [79].
The inhibitory activities of 95 (isolated from Selaginella delicatula) and delicatulines were evaluated against HBV surface antigen and HBV DNA in HepAD38 cells; however, all of these compounds showed very weak or no inhibitory activity against HBV [85].
The compound 5-ethoxymethyl-pyrrolemarumine (96), isolated from Hosta plantaginea, showed significant anti-inflammatory activity against LPS-induced stress. The IC 50 value was 8.6 ± 0.7 µM, which was very close to the value for the positive control parthenolide (4.96 ± 0.5 µM). Analysis of the mechanism of action revealed an interaction of 108 with iNOS protein [86] (  [87]. A similar result was observed in a hot-plate experiment; a posterior injection of the compounds (2 mg/BW) significantly increased the pain threshold of the mice on the hot-plate, especially for compound 92 after 60 min (<0.01 vs. control), and the threshold for 92 was very close to that of morphine (<0.01 vs. control) [87] (Table 2).
From Moringa oleifera seeds, seven new Py-2-Cs (99-105) and four known Py-2-Cs (5, 10, 96, and 106) were isolated and characterized [88]. An oxygen deprivation experiment was carried out in the presence of 5, 99, and 103 at 0.1 µM using PC12 cell lines. Significant attenuation of cell death was observed for all compounds compared with the control (absence of the compound). Western blot analyses of Nrf2 and NFκB revealed that 99 and 103 upregulated Nrf2, and 5, 99, and 103 downregulated NFκB (Table 2).
The antifungal and antiviral activity of two Py-2-Cs, 5-octadecyl Py-2-C and (6 Z)-5-(6 -hexeicosenyl) Py-2-C (129), (6 Z)-5-(6 -heneicosenyl)pyrrole-2-carboxaaldehyde (137), and other metabolites were examined for antifungal and antiviral activity. Compounds 136 and 137 did not show any significant activity. Compound 136 showed hypoglycemic activity in normal rats at an oral dose of 30 mg/kg body weight. At the same concentration, a reduction in the glucose level was also observed, which was almost equivalent to that of gilbenclamide administered orally at 30 µg/kg. Similar results were obtained in alloxantreated diabetic rats [93] (Table 3).  112, 115, 117, 123, 126, 127, and 131) isolated from the sponge Mycale Cecilia collected in the Gulf of California (Mexico) were tested for cytotoxic effects against various tumor cell lines (prostate carcinoma, DU-145 and LN-caP; ovarian adenocarcinoma IGROV; breast adenocarcinoma SK-BR3; melanoma SK-MEL-28; lung adenocarcinoma A549; chronic myelogenous leukemia K-562; pancreatic carcinoma PANC-1; colon adenocarcinoma HT-29 and LOVO; colon adenocarcinoma resistant to doxorubicin LOVO-DOX; and cervix epithelial adenocarcinoma HeLa). These compounds showed moderate cytotoxic effects (the GI 50 value was the highest for 0.2 µg/mL of 148 against LN-caP). In A-549 cells, only one compound (142) showed a moderate cytotoxic effect, and in LN-caP and SK-MEL cells, moderate cytotoxic effects were observed for 12 compounds. The compound 123, which bears a nitrile functional group at the terminal carbon of the side chain, showed moderate inhibitory activity against Leishmania mexicana; however, compounds bearing a nitrile group on the side chain (152, 153, 154, and 123) did not show strong activity against most of the cancer cell lines, but showed mild and selective inhibitory activity against PANC-1, LOVO, and HeLa cells. Other metabolites containing a nitrile group have shown strong biological activity [104]. The nitrile functional group is quite interesting in the pharmaceutical standpoint of view. It is well known that other metabolites bearing nitrile group in the molecule sometimes showed strong biological activities [110]. In an investigation of the cytotoxic effects of Py-2-Cs containing 21 carbon atoms, including tri-unsaturated (141), di-unsaturated (144), mono-unsaturated (148), and non-saturated (109) compounds, the mono-unsaturated Py-2-C (148) showed the strongest activity when compared with the other compounds (141, 144, and 109). The structure-activity relationship, including the effects in different tumor cells, of compounds 141, 144, and 109 has not yet been clearly established [95] (Table 3).
A lipophilic extract of the marine sponge Mycale sp. showed inhibition of the activation of HIF-1 in a T47D (human breast cancer)-based reporter assay. Bioassay-guided isolation of the extract afforded 18 new lipophilic Py-2-Cs (155-172) and eight known Py-2- Cs (115, 116, 123, 126, 127, 141, 152, 136, and 153). The effect of these compounds on HIF-1 activation was investigated quantitatively. Two compounds (157 and 158) showed strong activation of HIF-1 with IC 50 values of 7.8 (95% CI, 6.8-8.8 µM) and 8.8 µM (95% CI, 7.6-9.9 µM, respectively). Four compounds (152, 156, 159, and 164) showed moderate activation with IC 50 values of 10-20 µM. Seven compounds (126, 141, 166, 168, 169, 170,  and 172) showed weak activation with IC 50 values of 20-30 µM. The other 13 Py-2- Cs  (115, 116, 123, 127, 153, 155, 160-163, 165, 167, and 171) did not show any HIF-1 activation at 30 µM (<50% highest concentration in these experiments). The mechanism of action of compounds 157 and 168 was examined to determine the effect on hypoxia-induced vascular endothelial growth factor (VEGF) secretion using the T47D cell line. The Py-2-C (150) bearing a nitrile group at the terminus of the lipophilic side chain showed VEGF suppression at a concentration of 30 µM. The mechanism of action was investigated using T47D cells. Two compounds inhibited the T47D respiration in a concentration-dependent fashion and did not affect the mitochondrial electron transport complexes II, III, and IV. These compounds disrupted mitochondrial respiration at complex I [96]. Focusing on the diene compounds (155, 156, 157, and 158) bearing a nitrile group at the terminal carbon, two compounds (157, 158) showed strong HIF-1 activity, whereas 156 showed moderate activity. In contrast, 155 showed no activity. The number of carbons is the same in the alkyl chains of 155 and 156; however, the number of carbons in the alkyl chain between the pyrrole ring and the C=C double bond in the side chain is 2 in 155 and 14 in 156. For 157 and 158, the numbers of carbons in the alkyl chain between the pyrrole ring and the C=C double bond in the side chain are 10 and 11, respectively. These differences suggested the importance of the distance between the two functional groups for the physiological activity. Similar results were also observed for compounds containing one double bond in the chain (159,  160, 161, 162, and 163) and bearing a nitrile group at the terminal carbon. Moderate activity was observed for 159; however, the other compounds (160-163) did not show any activity. There are 19 carbons in the alkyl chain at the 2-position of 159, and the other compounds (160-163) have a longer chain length than 159. The numbers of carbons in the alkyl chain between the pyrrole ring and the C=C double bond of 159, 160, 161, 162, and 163 are 11, 13, 15, 6, and 6, respectively. This observation also suggested the importance of the length of the alkyl chain between the pyrrole ring and the C=C double bond in the side chain. Regarding the triene derivatives 171, 172, and 141, two compounds (171 and 141) showed weak HIF-1 activity, while compound 172 with a 9-carbon alkyl chain between the pyrrole ring and the double bond in the side chain did not show HIF-1 activity. The importance of the length of the alkyl chain between the pyrrole ring and the nitrile functional group in the side chain can also be observed in the saturated alkyl nitrile compounds (123, 152, 153,  164, and 165). Compounds 152 and 164 showed moderate HIF-1 activity, whereas 123, 153, and 165 did not show any HIF-1 activity. The numbers of carbons in the alkyl chains of 164, 152, 165, 123, and 153 are 15,17,18,19, and 20, respectively. The clear asynechia might be present in 152 and 165, which resulted from the different chain lengths of these compounds (Table 3).
A 2:1 mixture of Py-2-Cs (172 and 173) isolated from Mycale phyllophia was tested for cytotoxic activity against the L5178Y cell line (mouse lymphoma cell) in vitro. The IC 50 value (growth inhibition) of this mixture was 1.8 µg/mL [97].
Two new (174 and 175) and five known 5-alkyl-pyrrole carboxaldehydes (123, 138,  153, 154, and 164) bearing a nitrile group at the end of the carbon chain were assayed for inhibitory activity against PTP1B. Almost all the compounds showed inhibitory activity. The compounds micalenitrile-15 (174) and micalenitrile-16 (175) showed significant PTP1B131 inhibitory activity with IC 50 values of 8.6 ± 1.9 and 10.0 ± 0.2 µM, respectively (IC 50 = 3.6 ± 0.2 µM for the positive control ursolic acid). The known compound 138, which has a very similar structure to micalenitrile-16 (175) except for the length of the side chain (which is two carbons longer), showed stronger inhibitory activity (IC 50 = 3.1 ± 0.1 µM) compared with micalenitrile-16. This IC 50 value was almost the same as that of the positive control (ursolic acid; IC 50 = 3.6 ± 0.2 µM). A long alkyl side chain and the presence of a nitrile functional group might play important roles in the PTP1B inhibitory activity [99].
Two Py-2-Cs (39 and 48) isolated from a rice-supplemented liquid medium, and other metabolites isolated from a solid or liquid medium, were tested for the ability to affect the interaction between S. albospinus RLe7 and the fungus Coniochaeta sp. Fle4, which is also an endophyte of the same plant (Lychnophors ericoides). Neither of these compounds showed any antifungal activity nor did they show an inducing effect on fungal pigmentation [101] ( Table 3).
Cinerol I, isolated from the sponge Dysidea cinera, belongs to the nitrogeneous meroterpenoid family and contains a Py-2-C moiety [98]. The inhibitory activity of cinerols, including cinerol I (176), toward six kinases (PTP1B, ACL, SHP1, SHP2, JAK-2, and ACCI) has been studied, and cinerol I (176) did not show clear inhibitory activity against any of the kinases tested. The cytotoxic effect of cinerols at a 32 µM concentration has also been investigated using human melanoma A375 and human embryonic kidney HEK293 non-tumor cell lines. However, 176 did not show cytotoxic effects against these cell lines.
The cell viabilities of three compounds (jiangrine A (183 revised structure of 177), jiangrine G (182), and 10) were investigated in RAW 264.7 cells, and a slight inhibitory effect on the cell viability was observed for 10 at a 100 µM concentration. The addition of these compounds to LPS-treated raw cells decreased NO production along the i-NOS expression (dose range: 3.3-33 µM). The anti-inflammatory effects of these compounds were examined by measuring the cytokines TNF-α, IL-1β, and IL-6. The production of IL-1β and IL-6 was suppressed by the addition of 183 and 182; however, the production of TNF-α was not observed in the LPS-treated raw 264.7 cells. The phosphorylation of p38 and p65 was inhibited by the addition of 183 and 182. In addition, these compounds did not affect the ERK1/2 and JNK pathways, which indicated that 183 and 182 suppressed the production of IL-1β and IL-6 by blocking the p38 pathway. Compound 10, at low (3.3 µM) to high (33 µM) concentrations, suppressed the production of TNF-α. The production of IL-1β and IL-6 was decreased only at low to medium (11 µM) concentrations of 10. In contrast, the production of IL-1β and IL-6 increased at a high concentration (33 µM) of 10, which suggested that 10 had a low concentration threshold [106].

Conclusions
Py-2-Cs have been isolated from many fungi, plants, and microorganisms. However, there is a clear structural difference between the pyrrole-2-carbaldehydes from microorganisms and plants (fungi). In most cases, the pyrrole-2-carbaldehydes isolated from microorganisms are N-unsubstituted pyrrole-2-carbaldehydes, whereas most pyrrole-2carbaldehydes isolated from plants are N-substituted. Common substrates, such as 10, have been isolated from many species. The pyrrole-2-carbaldehydes (17-20, 58, 74, and 77) bearing a morpholine chromophore will attract much attention for their potential use in pharmaceutical applications. In addition, a recent report [111] concerning the use of pyrrole-2-carbaldehydes (3, 4, and 8) as biomarkers has indicated that pyrrole-2-carbaldehydes may have other future possible uses. The structure-activity relationships between the carbon chain length and the physiological activity are interesting to elucidate. For example, (as described above) Py-2-Cs bearing a nitrile group at the end of the carbon chain (152,  164, 123, 165, 152, and 164) showed moderate activity (10-20 µM) in the inhibition of the activation of HIF-1 in a T47D (human breast cancer)-based reporter assay, while 123 and 165 showed very weak activity (30 µM < 50%). The numbers of carbons in the alkyl chains of 152, 164, 123, and 165 are 17, 15, 19, and 18, respectively. A discontinuity is found between the carbon numbers 17 and 18. However, the reason for this discontinuity is unclear at present. Molecular calculation studies should be performed to explore this discontinuity. The isolation and identification of Py-2-Cs that show high physiological activities based on the original physiological assays are challenging, and the assay systems in most cases are not comprehensive. Thus, a particular assay system might miss active compounds, which may be found using a different assay system. In addition, most research has been performed by natural product and physiological scientists. To pursue knowledge of the structure-activity relationships, the use of computational chemistry is highly desirable. As described above, Py-2-Cs have the potential for use in various physiological fields, and further studies will provide new avenues of the use for Py-2-Cs in many academic and industrial areas, including pharmaceutical science.