Recent Developments of Gramine: Chemistry and Biological Activity

The natural alkaloid gramine has attracted significant attention in both academic and industrial circles because of its potential and diverse biological activities, including antiviral, antibacterial, antifungal, anti-inflammatory and antitumor activities; application in therapy for Alzheimer’s disease; serotonin-receptor-related activity; insecticidal activity; and application as an algicide. In this review, we focus on the research advances that have been made for gramine-based molecules since their discovery, providing key information on their extraction and separation, chemical synthesis and diverse biological activities. Data regarding their mechanisms of action are also presented. This comprehensive and critical review will serve as a guide for developing more drug candidates based on gramine skeletons.


Introduction
Gramine 1, also known as N,N-dimethyl-1H-indole-3-methylamine (Figure 1), is an indole alkaloid initially isolated from Arundo donax L. and usually plays a defensive role in plants against herbivores [1]. Recently, this alkaloid was also isolated from various raw plants, particularly barley, and it could act as a precursor for the biosynthesis of tryptophan and play a vital role in amino acid metabolism [2]. Gramine has attracted much attention due to its diverse antiviral, antibacterial, anti-inflammatory, antitumor and insecticidal activities [3]. In traditional Chinese medicine, it is also used to control toothache, bad urination, and heart disease [4]. Interestingly, some molecules similar in structure to gramine, such as sumatriptan and rizatriptan, have been approved for clinical trials or for use in clinical practice [5].

Introduction
Gramine 1, also known as N,N-dimethyl-1H-indole-3-methylamine (Figure 1), is an indole alkaloid initially isolated from Arundo donax L. and usually plays a defensive role in plants against herbivores [1]. Recently, this alkaloid was also isolated from various raw plants, particularly barley, and it could act as a precursor for the biosynthesis of tryptophan and play a vital role in amino acid metabolism [2]. Gramine has attracted much attention due to its diverse antiviral, antibacterial, anti-inflammatory, antitumor and insecticidal activities [3]. In traditional Chinese medicine, it is also used to control toothache, bad urination, and heart disease [4]. Interestingly, some molecules similar in structure to gramine, such as sumatriptan and rizatriptan, have been approved for clinical trials or for use in clinical practice [5].  The chemical synthesis of gramine was reviewed by Semenov et al. in 2004 [6]. However, since then, the following features have not yet been reviewed, or they have only been reviewed in a very fragmented fashion: (i) the extraction and separation of gramine; (ii) The chemical synthesis of gramine was reviewed by Semenov et al. in 2004 [6]. However, since then, the following features have not yet been reviewed, or they have only been reviewed in a very fragmented fashion: (i) the extraction and separation of gramine; (ii) the chemical synthesis of gramine and its derivatives; and (iii) the diverse biological activities.
This review aims to provide a detailed account of the chemistry and biological activities of gramine-based molecules that have been explored since their discovery. For the sake of convenience, their extraction and separation are first summarized. Then, the chemical synthesis of gramine is presented according to the key reaction. In the following section, its function and biological activities are presented, including its antiviral, antibacterial, antifungal, anti-inflammatory and antitumor activities; application in therapy for Alzheimer's disease; serotonin-receptor-related activity; insecticidal activity; and application as an algicide. Finally, we offer a conclusion and suggest future research directions on the topic of gramine based on our expertise in this field.

Extraction and Separation
Arundo donax L., a perennial cane, grows in damp soils, and it is widely distributed in mild-temperate subtropical and tropical regions, such as the western Pacific and Mediterranean [7]. In 1935, Orechoff et al. first discovered gramine in A. donax L. and named it donaxin [1]. Interestingly, in 1959, gramine was first discovered in Acer saccharinum, with 1.1 g of prismatic crystals obtained from 3.75 kg of dried maple leaves, which are widely distributed in the United States and southeastern Canada [8]. Then, in 1976, Anderson et al. extracted 85 mg of gramine from 640 g of seeds of Lupinus hartwegii (six weeks old) grown in vermiculite [9]. In 1985, Zú ecules 2023, 28, x FOR PEER REVIEW 2 of 17 the chemical synthesis of gramine and its derivatives; and (iii) the diverse biological activities. This review aims to provide a detailed account of the chemistry and biological activities of gramine-based molecules that have been explored since their discovery. For the sake of convenience, their extraction and separation are first summarized. Then, the chemical synthesis of gramine is presented according to the key reaction. In the following section, its function and biological activities are presented, including its antiviral, antibacterial, antifungal, anti-inflammatory and antitumor activities; application in therapy for Alzheimer's disease; serotonin-receptor-related activity; insecticidal activity; and application as an algicide. Finally, we offer a conclusion and suggest future research directions on the topic of gramine based on our expertise in this field.

Extraction and Separation
Arundo donax L., a perennial cane, grows in damp soils, and it is widely distributed in mild-temperate subtropical and tropical regions, such as the western Pacific and Mediterranean [7]. In 1935, Orechoff et al. first discovered gramine in A. donax L. and named it donaxin [1]. Interestingly, in 1959, gramine was first discovered in Acer saccharinum, with 1.1 g of prismatic crystals obtained from 3.75 kg of dried maple leaves, which are widely distributed in the United States and southeastern Canada [8]. Then, in 1976, Anderson et al. extracted 85 mg of gramine from 640 g of seeds of Lupinus hartwegii (six weeks old) grown in vermiculite [9]. In 1985, Zúǹiga et al. reported gramine in 34 barley cultivars, ranging in amounts from 0 to 48 mmol/kg [10]. Barley is a major cereal grain that is widely planted in temperate climates. Specifically, the content of gramine in barley leaves decreased with age. Moreover, its concentration in dry barley cultivars could reach up to 8 mg/g [11]. In 2020, an ultrasonic method was used to extract gramine [4]. The extraction rate of gramine from A. donax L. was 1% under the following conditions: ultrasonic power: 600 W, time: 50 min, temperature: 50 °C, liquid-material ratio: 40 mL/g, and pH value: 5.

Chemical Synthesis
Gramine and its derivatives have been synthesized via different methods by the scientific community due to their attractive function and biological activities. With readability in mind, we have summarized these classic and efficient synthetic methods according to their key reaction type.

Acetic-Acid-Catalyzed Reaction
The Mannich reaction (Scheme 1) is widely employed in the synthesis of gramine and its derivatives, with indole, formaldehyde, and dimethylamine used as the raw materials. In 2004, Xu et al. carried out the synthesis of gramine 1 using acetic acid as the catalyst, with the total yield optimized to 95.6% [12]. In 2020, Zhang et al. showed the synthesis of gramine 1 and its analogue 5, which were substituted by methyl, nitro, and methoxy groups, with yields ranging from 70.5% to 95% [13]. In 2014, microwave-assisted technology was introduced to the acetic-acid-catalyzed Mannich reaction, with a high yield of 98.1% [14]. Notably, the reaction time is only 5 min. iga et al. reported gramine in 34 barley cultivars, ranging in amounts from 0 to 48 mmol/kg [10]. Barley is a major cereal grain that is widely planted in temperate climates. Specifically, the content of gramine in barley leaves decreased with age. Moreover, its concentration in dry barley cultivars could reach up to 8 mg/g [11]. In 2020, an ultrasonic method was used to extract gramine [4]. The extraction rate of gramine from A. donax L. was 1% under the following conditions: ultrasonic power: 600 W, time: 50 min, temperature: 50 • C, liquid-material ratio: 40 mL/g, and pH value: 5.

Chemical Synthesis
Gramine and its derivatives have been synthesized via different methods by the scientific community due to their attractive function and biological activities. With readability in mind, we have summarized these classic and efficient synthetic methods according to their key reaction type.

Acetic-Acid-Catalyzed Reaction
The Mannich reaction (Scheme 1) is widely employed in the synthesis of gramine and its derivatives, with indole, formaldehyde, and dimethylamine used as the raw materials. In 2004, Xu et al. carried out the synthesis of gramine 1 using acetic acid as the catalyst, with the total yield optimized to 95.6% [12]. In 2020, Zhang et al. showed the synthesis of gramine 1 and its analogue 5, which were substituted by methyl, nitro, and methoxy groups, with yields ranging from 70.5% to 95% [13]. In 2014, microwave-assisted technology was introduced to the acetic-acid-catalyzed Mannich reaction, with a high yield of 98.1% [14]. Notably, the reaction time is only 5 min.
the chemical synthesis of gramine and its derivatives; and (iii) the diverse biological activities. This review aims to provide a detailed account of the chemistry and biological activities of gramine-based molecules that have been explored since their discovery. For the sake of convenience, their extraction and separation are first summarized. Then, the chemical synthesis of gramine is presented according to the key reaction. In the following section, its function and biological activities are presented, including its antiviral, antibacterial, antifungal, anti-inflammatory and antitumor activities; application in therapy for Alzheimer's disease; serotonin-receptor-related activity; insecticidal activity; and application as an algicide. Finally, we offer a conclusion and suggest future research directions on the topic of gramine based on our expertise in this field.

Extraction and Separation
Arundo donax L., a perennial cane, grows in damp soils, and it is widely distributed in mild-temperate subtropical and tropical regions, such as the western Pacific and Mediterranean [7]. In 1935, Orechoff et al. first discovered gramine in A. donax L. and named it donaxin [1]. Interestingly, in 1959, gramine was first discovered in Acer saccharinum, with 1.1 g of prismatic crystals obtained from 3.75 kg of dried maple leaves, which are widely distributed in the United States and southeastern Canada [8]. Then, in 1976, Anderson et al. extracted 85 mg of gramine from 640 g of seeds of Lupinus hartwegii (six weeks old) grown in vermiculite [9]. In 1985, Zúǹiga et al. reported gramine in 34 barley cultivars, ranging in amounts from 0 to 48 mmol/kg [10]. Barley is a major cereal grain that is widely planted in temperate climates. Specifically, the content of gramine in barley leaves decreased with age. Moreover, its concentration in dry barley cultivars could reach up to 8 mg/g [11]. In 2020, an ultrasonic method was used to extract gramine [4]. The extraction rate of gramine from A. donax L. was 1% under the following conditions: ultrasonic power: 600 W, time: 50 min, temperature: 50 °C, liquid-material ratio: 40 mL/g, and pH value: 5.

Chemical Synthesis
Gramine and its derivatives have been synthesized via different methods by the scientific community due to their attractive function and biological activities. With readability in mind, we have summarized these classic and efficient synthetic methods according to their key reaction type.

Acetic-Acid-Catalyzed Reaction
The Mannich reaction (Scheme 1) is widely employed in the synthesis of gramine and its derivatives, with indole, formaldehyde, and dimethylamine used as the raw materials. In 2004, Xu et al. carried out the synthesis of gramine 1 using acetic acid as the catalyst, with the total yield optimized to 95.6% [12]. In 2020, Zhang et al. showed the synthesis of gramine 1 and its analogue 5, which were substituted by methyl, nitro, and methoxy groups, with yields ranging from 70.5% to 95% [13]. In 2014, microwave-assisted technology was introduced to the acetic-acid-catalyzed Mannich reaction, with a high yield of 98.1% [14]. Notably, the reaction time is only 5 min. In addition, a great deal of gramine derivatives were also prepared using an aceticacid-catalyzed Mannich reaction. In 1959, compound 8 was synthesized as an interme-diate to prepare γ-carboline analogue with 2-lithium-1-methyl-indole 6 as raw reagents (Scheme 2) [15]. In 1985, some gramine-based quaternary ammonium salts 12a-12c were obtained by Sinhababu et al. [16]. Notably, the reductive cyclization of compound 9 to compound 10 using an Fe/AcOH system provided poor yields. When silica gel was added to toluene, compound 10 could be obtained with yields of 85-96%. In 2011, compound 15 was prepared in a Mannich reaction under ultrasound irradiation, with yields ranging from 69% to 98%, which was milder and more efficient than the traditional stirring method [17].
In 2009, some gramine analogues, such as compounds 17 and 18, containing ester functional groups were prepared and evaluated for their algal inhibition activity (Scheme 3) [18]. Notably, 3,3 -dithiobispropionate was used to provide the ester functional groups in the presence of sodium hydride. In 2018, compound 21 was synthesized using a similar method, and its antibacterial and antifouling activities were studied [19].

Zinc-Chloride-Catalyzed Reaction
Zinc chloride has been widely used in organic reactions, including the Mannich reaction. In 2006, a series of gramine derivatives 24 were obtained at room temperature using ZnCl2 as a catalyst in EtOH (Scheme 4) [20]. In 2015, an immobilized montmorillonite Scheme 3. Synthesis of gramine derivatives 17, 18 and 21 [18,19].

Zinc-Chloride-Catalyzed Reaction
Zinc chloride has been widely used in organic reactions, including the Mannich reaction. In 2006, a series of gramine derivatives 24 were obtained at room temperature using ZnCl 2 as a catalyst in EtOH (Scheme 4) [20]. In 2015, an immobilized montmorillonite (MMT)/ZnCl 2 system was used to synthesize gramine 1 via a microwave-assisted method. The yield reached 93.8% with a reaction time of 5 min, and the yield was still 60% after being recycled three times [21]. In 2016, compound 27 was prepared using the Diels-Alder cycloaddition reaction, with compound 26 as the dienophile. Then, the substituted indole 28 was obtained via acid-catalyzed cyclization and concomitant deprotection, which further derivatized to the gramine analogue 29 via the Mannich reaction [22]. Scheme 3. Synthesis of gramine derivatives 17, 18 and 21 [18,19].

Zinc-Chloride-Catalyzed Reaction
Zinc chloride has been widely used in organic reactions, including the Mannich reaction. In 2006, a series of gramine derivatives 24 were obtained at room temperature using ZnCl2 as a catalyst in EtOH (Scheme 4) [20]. In 2015, an immobilized montmorillonite (MMT)/ZnCl2 system was used to synthesize gramine 1 via a microwave-assisted method. The yield reached 93.8% with a reaction time of 5 min, and the yield was still 60% after being recycled three times [21]. In 2016, compound 27 was prepared using the Diels-Alder cycloaddition reaction, with compound 26 as the dienophile. Then, the substituted indole 28 was obtained via acid-catalyzed cyclization and concomitant deprotection, which further derivatized to the gramine analogue 29 via the Mannich reaction [22]. Scheme 4. Synthesis of gramine derivatives 24 and 29 [20,22]. Scheme 4. Synthesis of gramine derivatives 24 and 29 [20,22].

Ionic-Liquid-Catalyzed Reaction
In 2015, acidic ionic liquid, which is more suitable and reusable than classical acid catalysts, was used as a catalyst to prepare gramine 1 in a Mannich reaction with a yield of 81.6%. Notably, 1-butyl-3-methyl imidazole hydrogen sulfate can be continuously used as a catalyst. After four uses, the yield remained relatively unchanged [23].

Alkylation Reaction
In 1983, some substituted gramines such as compound 49 were synthesized via the alkylation of amines (Scheme 6) [29]. In 2017, some gramine derivatives 56-57 that fused with nitrogen heterocyclic ring were synthesized via a reaction of compounds 52-54 with gramine N-oxide 51 or gramine methyl iodide 50 [30]. However, compounds 50 and 51 could convert to gramine 1 when the temperature was raised to 160 °C in the presence of compound 52.

Alkylation Reaction
In 1983, some substituted gramines such as compound 49 were synthesized via the alkylation of amines (Scheme 6) [29]. In 2017, some gramine derivatives 56-57 that fused with nitrogen heterocyclic ring were synthesized via a reaction of compounds 52-54 with gramine N-oxide 51 or gramine methyl iodide 50 [30]. However, compounds 50 and 51 could convert to gramine 1 when the temperature was raised to 160 • C in the presence of compound 52.

Three-Component Condensation Reaction
In 2012, some heterocycle-functional gramine analogues 61 were prepared via a convenient three-component reaction using a solvent-and catalyst-free method (Scheme 6) [31]. Inspired by Ke's method, a series of gramine derivatives 65 fused with a pyrimidine ring were synthesized via a one-pot, three-component condensation reaction, providing an efficient, convenient, and green strategy [32].

Other Reactions
In 1990, N, N-dimethylacetamide was used to construct the gramine analogue 69 in the presence of POCl3 with a total yield of 33%. Notably, the intermediate 68 without purification was further reduced by NaBH4 (Scheme 7) [33]. In 2005, some Pt (II) and Pd (II) complexes 72-74 based on a gramine skeleton were synthesized and used as catalysts in a Heck reaction [34]. As shown in Scheme 7, gramine analogues 71 that fused with pyrazole and 3,5-dimethylpyrazole can be easily synthesized using gramine 1 or compound 70. Then, the target compounds 72-74 can be obtained in the presence of Pt(DMSO)2Cl2, Li2PdCl4, and Pd(OAc)2, respectively.

Three-Component Condensation Reaction
In 2012, some heterocycle-functional gramine analogues 61 were prepared via a convenient three-component reaction using a solvent-and catalyst-free method (Scheme 6) [31]. Inspired by Ke's method, a series of gramine derivatives 65 fused with a pyrimidine ring were synthesized via a one-pot, three-component condensation reaction, providing an efficient, convenient, and green strategy [32].

Other Reactions
In 1990, N, N-dimethylacetamide was used to construct the gramine analogue 69 in the presence of POCl 3 with a total yield of 33%. Notably, the intermediate 68 without purification was further reduced by NaBH 4 (Scheme 7) [33]. In 2005, some Pt (II) and Pd (II) complexes 72-74 based on a gramine skeleton were synthesized and used as catalysts in a Heck reaction [34]. As shown in Scheme 7, gramine analogues 71 that fused with pyrazole and 3,5-dimethylpyrazole can be easily synthesized using gramine 1 or compound 70. Then, the target compounds 72-74 can be obtained in the presence of Pt(DMSO) 2 Cl 2 , Li 2 PdCl 4 , and Pd(OAc) 2 , respectively.

Antiviral Activity
Enterovirus 71, widely reported in the Asia-Pacific region, is a classical RNA virus, which could infect the hand, foot and mouth of a human [35]. The prevention of this disease thus far has mainly relied on public health alerts and management [36]. Moreover, no specific medication for enterovirus 71 infections has thus far been used in clinical practice. Usually, some broad-spectrum antiviral drugs, such as type I interferon, ribavirin, and pleconaril, are used for infection treatment of enterovirus 71 [37]. In 2014, some gramine derivatives synthesized by Wei et al. exhibited potential inhibition activity on enterovirus-71-induced cytopathic effects ( Figure 2). The EC50 values for compound 75 against enterovirus 71 in African green monkey kidney cells (Vero) and rhabdomyosarcoma cells (RD) were 7.6 and 9.1 µg/mL, respectively, which were more potent than ribavirin (44.6 and 32.1 µg/mL). Interestingly, it exhibited high selectivity with CC50 values greater than 100 µg/mL. The study of the antiviral mechanism revealed that compound 75 acted in the early stage of the enterovirus 71 lifecycle; this compound could significantly intervene in protein synthesis, RNA replication, and enterovirus-71-induced apoptosis in RD cells [38].

Antiviral Activity
Enterovirus 71, widely reported in the Asia-Pacific region, is a classical RNA virus, which could infect the hand, foot and mouth of a human [35]. The prevention of this disease thus far has mainly relied on public health alerts and management [36]. Moreover, no specific medication for enterovirus 71 infections has thus far been used in clinical practice. Usually, some broad-spectrum antiviral drugs, such as type I interferon, ribavirin, and pleconaril, are used for infection treatment of enterovirus 71 [37]. In 2014, some gramine derivatives synthesized by Wei et al. exhibited potential inhibition activity on enterovirus-71-induced cytopathic effects ( Figure 2). The EC 50 values for compound 75 against enterovirus 71 in African green monkey kidney cells (Vero) and rhabdomyosarcoma cells (RD) were 7.6 and 9.1 µg/mL, respectively, which were more potent than ribavirin (44.6 and 32.1 µg/mL). Interestingly, it exhibited high selectivity with CC 50 values greater than 100 µg/mL. The study of the antiviral mechanism revealed that compound 75 acted in the early stage of the enterovirus 71 lifecycle; this compound could significantly intervene in protein synthesis, RNA replication, and enterovirus-71-induced apoptosis in RD cells [38].

Antiviral Activity
Enterovirus 71, widely reported in the Asia-Pacific region, is a classical RNA virus, which could infect the hand, foot and mouth of a human [35]. The prevention of this disease thus far has mainly relied on public health alerts and management [36]. Moreover, no specific medication for enterovirus 71 infections has thus far been used in clinical practice. Usually, some broad-spectrum antiviral drugs, such as type I interferon, ribavirin, and pleconaril, are used for infection treatment of enterovirus 71 [37]. In 2014, some gramine derivatives synthesized by Wei et al. exhibited potential inhibition activity on enterovirus-71-induced cytopathic effects ( Figure 2). The EC50 values for compound 75 against enterovirus 71 in African green monkey kidney cells (Vero) and rhabdomyosarcoma cells (RD) were 7.6 and 9.1 µg/mL, respectively, which were more potent than ribavirin (44.6 and 32.1 µg/mL). Interestingly, it exhibited high selectivity with CC50 values greater than 100 µg/mL. The study of the antiviral mechanism revealed that compound 75 acted in the early stage of the enterovirus 71 lifecycle; this compound could significantly intervene in protein synthesis, RNA replication, and enterovirus-71-induced apoptosis in RD cells [38]. Tobacco mosaic virus (TMV), first discovered in tobacco, can infect as many as 400 different crops and lead to enormous economic losses [40]. The commercial pesticide, ningnanmycin, is currently the most competitive antiviral product; however, it only exhibits 50-60% control at a concentration of 500 µg/mL [40]. As shown in Figure 2, compared with the inhibition rate of gramine 1 (23%), bromination analogues (76a and 76b) exhibit higher antiviral activities with inhibition rates of 51% and 39% at 500 µg/mL, respectively. Methylation at the N 1 position could also improve anti-TMV activity, such as in compound 76c. Interestingly, the simultaneous substitution of bromination at the C 6 position and methylation at N 1 position caused a synergistic effect, such as in compound 76d, with an inhibition rate of 61% at 500 µg/mL. In addition, the introduction of aryl substituents at the methylene of the flexible chain could further enhance anti-TMV activity, such as in compounds 76e (62%) and 76f (67%). A study of the anti-TMV mechanism revealed that compound 76 could block the assembly of TMV via cross-linking the TMV capsid protein [39]. Influenza A virus (IAV) is a kind of respiratory pathogen with high infectivity and pathogenicity, belonging to the family of Orthomyxoviridae. In 2021, Zhao et al. studied the inhibition activity of gramine 1 in IAV. Regrettably, no anti-IAV activity was found [41].

Antibacterial Activity
In 2014, Yang et al. reported the antibacterial activity of gramine 1, which could effectively inhibit the growth of Escherichia coli and Staphylococcus aureus with minimum inhibitory concentrations (MICs) of 16.92 and 6.26 µg/mL [42]. Moreover, it could also mildly inhibit the growth of methicillin-resistant S. aureus (MRSA) with an inhibition rate of 82% at 400 µg/mL [43]. In 2018, Feng et al. reported the antibacterial activity of gramine derivatives associated with the acylamino group [19]. Interestingly, compounds 21a and 21b exhibited moderate antibacterial activity against S. aureus with an MIC of 30 µg/mL. The rhizosphere microbiota can reflect the growth and development status of plants, which is valuable for sustainable agriculture. In 2021, Maver et al. found that gramine could regulate the prokaryotic communities of barley rhizosphere microbiota [44]. In the same year, these results were also validated by Schütz et al. [45]. They found that gramine 1 could promote the proliferation of beneficial strains, such as Novosphingobium and Massilia ASVs.

Antifungal Activity
In 2001, Matsuo et al. found that the content of gramine 1 significantly increased in the leaves of barley seedlings after being infected by Blumeria graminis, which indicated its potential antifungal activity [2]. In 2011, Schreiber et al. reported that gramine 1 could effectively decrease the severity of Fusarium graminearum infection in wheat [46]. In 2011, Wollein et al. studied the antifungal activity of gramine derivative 77a against Candida glabris and Aspergillus Niger (Figure 3). Unfortunately, no inhibition activity was found. However, its cyclization product 77b exhibited antifungal activity against C. glabris and A. Niger with the diameters of inhibitory zones being 11 and 10 mm, respectively [47]. In 2019, Lu et al. discovered that compounds 76e and 78 exhibited more than a 90% inhibitory effect in vitro against the plant pathogen Phytophthora piricola at 50 µg/mL via the mycelial growth method [39].

Anti-Inflammatory Activity
Inflammation is loosely defined as a response to invading pathogens or endogenous signals, which plays a vital role in many diseases, especially in some chronic diseases [48]. 5-Lipoxygenase (LOX) enzymes, which are involved in leukotriene biosynthesis with arachidonic acid, could mediate inflammatory reactions [49]. The inhibition of the inflam-

Anti-Inflammatory Activity
Inflammation is loosely defined as a response to invading pathogens or endogenous signals, which plays a vital role in many diseases, especially in some chronic diseases [48]. 5-Lipoxygenase (LOX) enzymes, which are involved in leukotriene biosynthesis with arachidonic acid, could mediate inflammatory reactions [49]. The inhibition of the inflammatory factor nitric oxide (NO) has also been considered to be an anti-inflammatory strategy [50]. In 2017, Magalhães et al. reported that gramine 1 can inhibit the activity of LOX, with an IC 25 value of 119 µg/mL. Moreover, it can effectively scavenge 34% of nitric oxide radicals at 1 mg/mL [51]. It has been suggested that the nuclear translocation of nuclear factor kappa B (NF-KB) and signal transducer and activator of transcription 3 (STAT 3) could transcribe inflammatory genes [52]. The activation of the tyrosine kinase phosphorylation of the epidermal growth factor receptor (EGFR) may initiate the Janus Kinases (JAKs) and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathways [53]. In 2018, Ramu et al. reported that gramine 1 exhibited an antiinflammatory activity via the intervention of EGFR/PI3K/Akt/mTOR/IKK/NF-κB and JAK/STAT3 signaling pathways [54]. In 2021, Lu et al. discovered that gramine 1 could inhibit the release of pro-inflammatory mediators, including interleukin-1β (IL-1β), IL-6, tumor necrosis factor (TNF)-α, and NO secreted from lipopolysaccharide (LPS)-induced microglia. Moreover, it was found to reduce the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In vivo behavioral and histological experiments indicated that gramine 1 could alleviate microglia activation and promote motor functional recovery via the NF-κB pathway [55].

Antitumor Activity
Oral squamous cell carcinoma (OSCC), as one of the most lethal tumors, is one of the top six human malignancies [56]. 7,12 Dimethylbenz[a]anthracene (DMBA) induced OSCC in a hamster buccal pouch (HBP), which is widely used as an animal model [57]. In 2014, Kumar et al. reported that gramine 1 exhibited a potential chemopreventive effect on DMBAinduced HBP, which might be attributed to its anti-lipid peroxidative, antioxidant potential, and retrieval effects as well as detoxifying potential [58]. In 2017, they found that gramine 1 decreased angiogenesis and induced apoptosis in HBP by regulating transforming growth factor (TGF)-β signals [59]. In 2018, they further proved that gramine 1 could activate the operation of key cancer suppressor proteins p21, p53 as well as Gsk-3β, which explained the anti-proliferation effects [54]. In 2012, Ke et al. reported the anti-proliferation activity of compound 61 against human gastric cancer, human lung cancer, and human hepatocellular liver cancer cell lines (Figure 4). Among these, compound 61b, with an IC 50 value of 5.7 µg/mL, could inhibit the growth of BGC-823 human gastric cells [31].  In 2021, Zhang et al. also reported the anti-gastric cancer activity of a series of gramine-based hybrid molecules, including 79. Compound 79c exhibited good activity against MGC803 human gastric cancer cells (IC50 = 3.74 µM). A study of its mechanism revealed that compound 79c could cause cell cycle arrest, induce mitochondria-mediated apoptosis, and inhibit metastasis [60]. In 2023, gramine-loaded, polyvinyl-alcohol-coated iron oxide nanoparticles were prepared by Alnaim et al. and exhibited a good anti-proliferation activity against HCT-116 human colon cancer cells, with an IC50 value of 25 µg/mL. The study of its mechanism indicated that these nanoparticles could destroy the equilibrium of redox pathways and cause cell apoptosis [61]. In 2021, Zhang et al. also reported the anti-gastric cancer activity of a series of graminebased hybrid molecules, including 79. Compound 79c exhibited good activity against MGC803 human gastric cancer cells (IC 50 = 3.74 µM). A study of its mechanism revealed that compound 79c could cause cell cycle arrest, induce mitochondria-mediated apoptosis, and inhibit metastasis [60]. In 2023, gramine-loaded, polyvinyl-alcohol-coated iron oxide nanoparticles were prepared by Alnaim et al. and exhibited a good anti-proliferation activity against HCT-116 human colon cancer cells, with an IC 50 value of 25 µg/mL. The study of its mechanism indicated that these nanoparticles could destroy the equilibrium of redox pathways and cause cell apoptosis [61].

Alzheimer's Disease Therapy
Alzheimer's disease (AD), as a neurodegenerative disease, is an enormous burden on society [62]. The physiological characteristics of AD, including amyloid β peptide (Aβ) and neurofibrillary tangles (NFTs) caused by τ protein aggregation, are important therapeutic targets [63]. The increasing amount of phosphorylated τ protein causes it to self-aggregate into NFT, depending on the activity of kinase and phosphatase. Specifically, Ser/Thr phosphatase plays an important role in the dephosphorylation of the τ protein [64]. In addition, the reduction in cytosolic Ca 2+ in neurons can block the development of AD, such as in the successful commercialization of memantine [65]. In 2016, Lajarín-Cuesta et al. reported that compound 80 could reduce the entry of Ca 2+ via voltage-gated calcium channels (VGCC) and maintain the action of Ser/Thr phosphatase 2A (PP2A), which decreased τ hyperphosphorylation ( Figure 5) [66]. In 2018, their group reported the activity of N-benzylated gramines 81 that dissipate the overload of neuronal Ca 2+ [67]. The introduction of a nitro group in benzyl could greatly improve the blocking effect of VGCC with an IC 50 of 1.8 µM for compound 81e. Moreover, Lajarín-Cuesta et al. synthesized a gramine derivative 82, which could restore 78% of the PP2A activity and blockade 40% of VGCC [68]. Previous research indicated that neuroinflammation was a therapeutic target in neurodegenerative diseases, such as AD [48]. The activation of NF-κB in neurons will promote their survival. In 2021, Yang et al. reported that gramine 1 prevented the apoptosis of PC12 cells, inhibited neuroinflammation via the NF-κB signaling pathway, and ultimately promoted the treatment of associated central nervous diseases such as AD [55]. In 2023, Jadhav et al. found that gramine 1 could remarkably restore memory in an amnesia model induced by scopolamine, indicating its potential in therapy for AD [69].

Serotonin Receptor-Related Activity
Serotonin (5-HT, 83), with seven known subtypes of receptors, is an important neurotransmitter and regulatory molecule that can regulate various physiological functions, such as appetite, sleep, mentation, emotions, etc. (Figure 6) [70,71]. Notably, different agonists and antagonists can be used in different receptor subtypes to treat different diseases [72]. Many studies have shown that gramine 1 is a 5-HT receptor antagonist due to its structural similarities. After being induced by dorsal swim interneurons (DSIs), 5-HT can mediate neurotransmission and neuromodulation. Gramine 1 can cause antagonistic effects by blocking the fast DSI-dorsal flexion neuron (DFN) excitatory postsynaptic poten-

Serotonin Receptor-Related Activity
Serotonin (5-HT, 83), with seven known subtypes of receptors, is an important neurotransmitter and regulatory molecule that can regulate various physiological functions, such as appetite, sleep, mentation, emotions, etc. (Figure 6) [70,71]. Notably, different agonists and antagonists can be used in different receptor subtypes to treat different diseases [72]. Many studies have shown that gramine 1 is a 5-HT receptor antagonist due to its structural similarities. After being induced by dorsal swim interneurons (DSIs), 5-HT can mediate neurotransmission and neuromodulation. Gramine 1 can cause antagonistic effects by blocking the fast DSI-dorsal flexion neuron (DFN) excitatory postsynaptic potential (EPSP) and reducing the depolarization induced by 5-HT [73]. In addition, gramine 1 could decrease the 5-HT-induced metamorphosis of the gastropod mollusc, Ilyanassa obsolete, reducing it from 48% to 20% [74]. Litosch et al. reported that gramine 1 also antagonized the stimulation of cycloenzyme via 5-HT [75]. Renaud et al. found that gramine 1 could significantly delay the cleavage of sea urchins by stimulating calcium efflux from fertilized eggs and reducing the level of cAMP [76]. 5-HT can regulate the contraction time of the pharynx and increase the eating speed of C. elegans when food resources are sufficient. 5-HT can raise the rate of pharyngeal contraction by reducing the time needed for pharyngeal action. However, gramine 1 exhibited the opposite effect, which increases the duration of action potential [77]. In 2004, Froldi et al. found that gramine 1 as the antagonistic to 5-HT 2A receptor was an effective vasodilator [78].

Serotonin Receptor-Related Activity
Serotonin (5-HT, 83), with seven known subtypes of receptors, is an important neurotransmitter and regulatory molecule that can regulate various physiological functions, such as appetite, sleep, mentation, emotions, etc. (Figure 6) [70,71]. Notably, different agonists and antagonists can be used in different receptor subtypes to treat different diseases [72]. Many studies have shown that gramine 1 is a 5-HT receptor antagonist due to its structural similarities. After being induced by dorsal swim interneurons (DSIs), 5-HT can mediate neurotransmission and neuromodulation. Gramine 1 can cause antagonistic effects by blocking the fast DSI-dorsal flexion neuron (DFN) excitatory postsynaptic potential (EPSP) and reducing the depolarization induced by 5-HT [73]. In addition, gramine 1 could decrease the 5-HT-induced metamorphosis of the gastropod mollusc, Ilyanassa obsolete, reducing it from 48% to 20% [74]. Litosch et al. reported that gramine 1 also antagonized the stimulation of cycloenzyme via 5-HT [75]. Renaud et al. found that gramine 1 could significantly delay the cleavage of sea urchins by stimulating calcium efflux from fertilized eggs and reducing the level of cAMP [76]. 5-HT can regulate the contraction time of the pharynx and increase the eating speed of C. elegans when food resources are sufficient. 5-HT can raise the rate of pharyngeal contraction by reducing the time needed for pharyngeal action. However, gramine 1 exhibited the opposite effect, which increases the duration of action potential [77]. In 2004, Froldi et al. found that gramine 1 as the antagonistic to 5-HT2A receptor was an effective vasodilator [78]. It is well known that 5-HT is the direct precursor of melatonin (MT), which can enhance the activation of the 5-HT1A receptor in the hypothalamus. There are three subtypes of MT receptor, MT1, MT2, and MT3, which are closely related to some mental disorders It is well known that 5-HT is the direct precursor of melatonin (MT), which can enhance the activation of the 5-HT 1A receptor in the hypothalamus. There are three subtypes of MT receptor, MT 1 , MT 2 , and MT 3 , which are closely related to some mental disorders [79]. The EC 50 values of gramine 1 on MT 1 and 5-HT 1A were 1.36 and 0.47 mM, respectively [72]. Simultaneously, some derivatives 84a-84c exhibited more potential activity as displayed in Figure 6, with the strongest EC 50 value of 0.23 mM against 5-HT 1A . Orchard et al. found that gramine 1 was also an antagonist of the octopusamine-2 receptor, which was used to block the increase in cyclic adenosine monophosphate mediated by octopamine [80].
Molecules 2023, 28, x FOR PEER REVIEW 13 of 17 [79]. The EC50 values of gramine 1 on MT1 and 5-HT1A were 1.36 and 0.47 mM, respectively [72]. Simultaneously, some derivatives 84a-84c exhibited more potential activity as displayed in Figure 6, with the strongest EC50 value of 0.23 mM against 5-HT1A. Orchard et al. found that gramine 1 was also an antagonist of the octopusamine-2 receptor, which was used to block the increase in cyclic adenosine monophosphate mediated by octopamine [80].

Algicides
The red tide and marine pollution caused by the large-scale flooding of algae seriously threatened the sustainable stability of the marine ecosystem [83]. Prymnesium par-

Conclusions and Perspectives
This review presents a wealth of information on gramine alkaloids, including their extraction, chemical synthesis, and diverse biological activities that have been discovered since their first isolation in 1935 ( Figure 8A). Firstly, the source, distribution and extraction technology of gramine are briefly summarized. Then, their chemical synthesis methods are described according to their key reaction type. Clearly, gramine skeletons can be easily obtained. Modifications with various bioactive moieties provide many potential molecules. Indeed, some drugs similar in structure to gramine have been successfully marketed, such as sumatriptan and rizatriptan. Additionally, a literature search revealed that their biological activity mechanisms have also been carefully discussed. Of course, there remains much unreported information regarding pharmacological activity in gramine skeletons. For example, Xu et al. recently reported their therapeutic potential in pathological cardiac hypertrophy via an interaction with Runt-related transcription factor 1 [85]. Kozanecka-Okupnik et al. synthesized some triazole-bearing gramine analogues, which exhibited a protective activity against hemolysis induced by oxidative stress [86]. For medicinal chemists, gramine-based drugs will be used in the long term due to their simple structures and desirable activities. medicinal chemists, gramine-based drugs will be used in the long term due to their simple structures and desirable activities. New perspectives for gramine-based medicinal chemistry research can be divided into the following topics ( Figure 8B): (a) introduction of key pharmacophores via the design of hybrid molecules; (b) structural optimization for defects such as poor pharmacokinetics and bioavailability; (c) elucidation of structure-activity relationships; (d) drug combination and drug resistance research; (e) in-depth exploration of diverse molecular mechanisms and targets, such as the application of multi-omics analysis; (f) efficient delivery forms of drug molecules based on gramine skeletons; and the (g) discovery and expansion of diverse pharmacological activity.  New perspectives for gramine-based medicinal chemistry research can be divided into the following topics ( Figure 8B): (a) introduction of key pharmacophores via the design of hybrid molecules; (b) structural optimization for defects such as poor pharmacokinetics and bioavailability; (c) elucidation of structure-activity relationships; (d) drug combination and drug resistance research; (e) in-depth exploration of diverse molecular mechanisms and targets, such as the application of multi-omics analysis; (f) efficient delivery forms of drug molecules based on gramine skeletons; and the (g) discovery and expansion of diverse pharmacological activity. gramine and its derivatives; and (iii) the diverse biological activprovide a detailed account of the chemistry and biological activolecules that have been explored since their discovery. For the extraction and separation are first summarized. Then, the chemis presented according to the key reaction. In the following secogical activities are presented, including its antiviral, antibactematory and antitumor activities; application in therapy for Alzin-receptor-related activity; insecticidal activity; and application offer a conclusion and suggest future research directions on the our expertise in this field.
ion erennial cane, grows in damp soils, and it is widely distributed pical and tropical regions, such as the western Pacific and Medrechoff et al. first discovered gramine in A. donax L. and named gly, in 1959, gramine was first discovered in Acer saccharinum, rystals obtained from 3.75 kg of dried maple leaves, which are United States and southeastern Canada [8]. Then, in 1976, Anmg of gramine from 640 g of seeds of Lupinus hartwegii (six weeks [9]. In 1985, Zúǹiga et al. reported gramine in 34 barley cultivars, 0 to 48 mmol/kg [10]. Barley is a major cereal grain that is widely ates. Specifically, the content of gramine in barley leaves deer, its concentration in dry barley cultivars could reach up to 8 rasonic method was used to extract gramine [4]. The extraction nax L. was 1% under the following conditions: ultrasonic power: erature: 50 °C, liquid-material ratio: 40 mL/g, and pH value: 5.
ivatives have been synthesized via different methods by the scitheir attractive function and biological activities. With readabilmarized these classic and efficient synthetic methods according .
ed Reaction n (Scheme 1) is widely employed in the synthesis of gramine and le, formaldehyde, and dimethylamine used as the raw materials. out the synthesis of gramine 1 using acetic acid as the catalyst, ized to 95.6% [12]. In 2020, Zhang et al. showed the synthesis of gue 5, which were substituted by methyl, nitro, and methoxy ng from 70.5% to 95% [13]. In 2014, microwave-assisted technole acetic-acid-catalyzed Mannich reaction, with a high yield of eaction time is only 5 min.
thods based on the Mannich reaction.