*2.3. Neuroprotective Activity*

In 2011, Akamatsu et al. first confirmed the intrinsic neuroprotective effect of compound **1** [39]. In the photochemical-induced thrombotic occlusion model of cynomolgus monkeys, compound **1** of 10 mg/kg improved the neurologic deficit by 29% and cerebral hemorrhage by 51%, after treatment for 24 h, in comparison to the saline control group [29]. Then, Shibata et al. further explored the mechanism of reducing brain damage [66]. Compound **1** inhibited the expression of 4-NHE and neutrophil cytosolic factor 2 (Ncf2) after treatment for 1–3 h. Additionally, 4-NHE is an oxidized product of lipid peroxidation, Ncf2 can stimulate the NADPH oxidase complex to produce SOD, and their levels would increase in the infarction area [67]. Therefore, compound **1** reduced lipid peroxidation and the generation of SOD, in cerebral infarction, to possess neuroprotective activity [66]. Moreover, Ito et al. evaluated the activation of MMP-9 with compound **1** treatment in a mouse model. Compared with the control group, compound **1**, of 10 mg/kg, inhibited the expression of MMP-9, which could digest the endothelial basal lamina and open the blood–brain barrier, causing neuro-inflammation [32]. Compound **1** showed less basal membrane damage and functional breakdown of the blood–brain barrier. Therefore, compound **1** reduced neuronal damage by inhibiting MMP-9 expression. In 2018, Huang et al. investigated the anti-inflammatory and antiapoptosis mechanisms of compound **1** for neuroprotective effects [68]. Compound **1**, of 10 mg/kg, decreased the expression of NF-κB, TNF-<sup>α</sup>, and NLRP3-positive cells, which involved the alleviation of neuroinflammation. Meanwhile, compound **1** reduced the expression of cleaved-caspase-3, suggesting the inhibition in cell death progress. Therefore, compound **1** treatment demonstrated less necrosis of neurons and high neuroprotective activity in the peri-ischemic area. The results showed that the neuroprotective activity of compound **1** was attributed to the anti-oxidative, anti-inflammatory, and anti-apoptosis mechanisms in cerebral infarction.

In 2014, Matsumoto et al. found that congener **18** (10 mg/kg) (Figure 9) alleviated neuritis symptoms in a rat model presenting neuroprotective activity [63]. Shi et al. investigated the therapeutic effect of congener **18** in the neurovascular unit (NVU) and neurovascular trophic coupling damage [69]. Congener **18** ameliorated the NVU dissociation between pericyte, basal lamina, and astrocytic foot. It also improved the endothelial neuroprotective support for the outsider neurons. Moreover, congener **18** decreased the expression of TNF-<sup>α</sup>, 4-HNE, 8-OHdG, and cleaved caspase-3. Therefore, neuroprotective activity of congener **18** was due to its anti-inflammatory, anti-oxidative, and anti-apoptotic

mechanisms. Congener **18**, especially, had therapeutic potential for diabetic neuropathy symptoms [70]. Congener **18** (30 mg/kg) improved the mechanical allodynia, thermal hyperalgesia symptoms, and neurological degeneration of DN in a streptozotocin-induced diabetes mouse model.

**Figure 9.** The structure of congener **18**.

#### *2.4. Effects on IgA Nephropathy and Acute Kidney Injury*

IgA nephropathy (IgAN) has become a primary chronic glomerulonephritis worldwide, featuring mesangial cell proliferation and the deposition of IgA [71]. It causes a gradual decline in renal function, and 30% of patients will develop to the end stage of renal disease [72–74]. Kemmochi et al., (2012) investigated the therapeutic effects of compound **1** against IgAN [75]. In a mouse IgAN model, compound **1** (10 mg/kg) slightly reduced the deposition of IgA, but it had no effect on the serum concentration of IgA. The results suggested that compound **1** might inhibit the progression of IgAN through reducing the deposition of IgA in the glomerular mesangium. However, it was not effective in decreasing IgA production and treatment for terminal IgAN, indicating the limited therapeutic ability in IgAN.

Unlike IgAN, acute kidney injury (AKI) could cause rapid reduction in renal function [76]. The pathological condition of the kidney retained toxins and wastes, causing toxicosis and disorder in fluid, electrolyte, and acid–base balance [77]. It is estimated that 22% of hospitalized adults suffered from AKI [78]. Compound **1** showed less damage of ischemia-reperfusion in thrombolysis therapy. Meanwhile, ischemia-reperfusion played a major role in AKI renal damage. Therefore, in 2021, Shibata et al. studied the efficacy of compound **1** in renal damage [79]. Compound **1** improved the parameters of renal function (blood–urea nitrogen, creatinine levels in serum, creatinine clearance, and fractional excretion of sodium) and renal tubule damage. The therapeutic effect of compound **1** was derived from anti-inflammatory and anti-oxidative activities. The inhibition to sEH elevated the EET level, which inhibited tubular dysfunction and inflammatory factors, such as NF-κB, TNF-<sup>α</sup>, IL-6, and IL-1β. ROS production was also reduced after compound **1** treatment. Thus, the suppression of peroxidation led to less renal cell injury.

#### *2.5. Effects on Cancer: Non-Small Cell Lung Cancer*

The essence of cancer is the abnormal proliferation and differentiation of cells, which are dependent on angiogenesis [80]. Therefore, anti-cancer agents could identify and inhibit angiogenesis, thus representing an approach for cancer therapy [81]. Many patents on angiogenesis inhibitors have been published, such as angiostatin, endostatin, and thrombospondin. Therein, angiostatin is a hidden fragment of plasminogen with grea<sup>t</sup> antiangiogenic properties [82]. Congener **7** reduced vascular formation, along with proliferation and migration, to inhibit tumor growth and possessed plasminogen activation activity, causing a conformational change of plasminogen to dissolve thrombus [83]. Ohyama et al., (2004) reported that congener **7** also promoted the autoproteolytic of plasmin, inducing extensive fragmentation of the catalytic domain [83]. After urokinased-catalyzed plasminogen was activated by congener **7**, the catalytic domain of plasmin (activated plasminogen) rapidly degraded into 68–77 kDa fragments. These fragments blocked proliferation, migration, and

vascular formation of endothelial cells, at concentrations of 0.3–10 μg/mL, meaning they provide potential applications of congener **7** for cancer treatment.

As the most common lung cancer, non-small cell lung cancer (NSCLC) accounts for approximately 80–85% of lung cancer diseases [84]. The clinical drugs for treating NSCLC are the epidermal growth factor receptor (EGFR) and EGFR-targeted tyrosine kinase inhibitors (TKIs). However, more than 80% patients gradually showed drug resistance after about 1 year of treatment with EGFR-TKI. Thus, it is necessary to discover new anti-tumor agents for treating NSCLC [85]. In 2022, Yan et al. observed the effects of compound **1** on erlotinib-resistant NSCLC and explored the underlying mechanism [86]. NSCLC cells were sensitive to compound **1** with IC50 = 7.45 ± 0.57 μM in vitro, especially for erlotinib-resistant NSCLC H1975 cells (IC50 = 9.22 ± 0.84 μM); meanwhile, compound **1** was relatively safe for normal cells. The accumulation of cleaved-PARP, cleaved-caspase-3, Bax, and the reduction in Bcl-2 revealed that compound **1** induced the cell apoptosis of NSCLC cells [86]. Then, they discovered the underlying mechanism of treating erlotinib-resistant NSCLC. Compound **1** induced mitochondria-mediated apoptosis, leading to increased ROS and reduced GSH. Thus, compound **1** caused apoptosis of erlotinib-resistant NSCLC cells [86,87]. In addition, compound **1** also inhibited the PI3K/Akt signaling pathway and the EGFR/PI3K/Akt/mTOR pathway. The abnormal activation of the PI3K/Akt pathway could cause TKI resistance, as well as invasiveness and migration of NSCLC [85]. Moreover, compound **1** showed high binding affinity to EGFRT790M/L858R in molecular modeling, meaning compound **1** selectively exhibited anticancer activity on erlotinib-resistant NSCLC cells. Finally, compound **1** (10 mg/kg) had consistent anti-cancer effects in nude mice, meaning that it showed potential for erlotinib-resistant NSCLC therapy. In 2022, Feng et al. further observed that compound **1** downregulated the levels of CD4K and Cyclin D1 to arrest the cell cycle of PC9 cells at the G0/G1 phase [88]. Compound **1**, especially, inhibited the viability and proliferation of PC9 cells through the inhibition of the NF-κB signaling pathway. The results indicated that compound **1** had excellent anti-cancer activity on *EGFR*-mutant NSCLC cells, but it had weak or no effect on wild-type EGFR cells. It can be concluded that compound **1** might depend on the EGFR status to induce apoptosis of NSCLC cells.

#### **3. Preparation of Compound 1 and Staplabin Congeners**

Hu et al. isolated SMTP congeners from *S. microspora* in 2001 [25]. They found that the use of amino acids and amino alcohols significantly increased the production of congeners, and the obtained products were related to the type of added amino acid. The production of compound **1** and congeners **3**, **5**, and **7** increased by 7 to 45-fold, with the addition of Orn, Phe, Leu, Trp, and Lys at 100 mg/mL, which acted as precursors in culture. Therefore, the addition of precursors was an important procedure in the preparation of compound **1** and staplabin congeners.

In 2012, Nishimura et al. isolated a new compound designated pre-SMTP from fungus *S. microspora*, which directly afforded SMTP congeners by reacting nonenzymatically (phthalaldehydes reaction) [89,90]. Pre-SMTP accumulated, with limited amounts of amine, in medium with *S. microspora*, and it consumed rapidly after increased amine feeding. Meanwhile, SMTP-0, as well as congeners **3**, **7**, **19,** and **20** were afforded, by reaction of pre-SMTP, with ammonium chloride, *<sup>L</sup>*-phenylalanine, *L*-tryptophan, L-glutamine, and L-glutamic acid, respectively. Thus, it is available to synthesize a variety of congeners, with different *N*-linked side-chain structures, through nonenzymatic reaction between pre-SMTP and an amine (Figure 10).

In 2013, Su et al. investigated the fermentation conditions of compound **1** isolated from *S. longispora* FG216 [91]. The results showed that the optimized fermentation conditions were as follows: 0.5% ornithine hydrochloride addition, 28 ◦C culture temperature, and 7 d (Figure 11a). The yield of compound **1** increased up to 1.98 g/L. In 2015, Wang et al. designed a metabolic regulation strategy to improve the production of compound **1** [92]. The results indicated that the carbon skeleton of compound **1** was synthesized through

the shikimate and mevalonate pathways. Therefore, the addition of precursor shikimic acid and precursor sodium acetate increased the yield of compound **1** by 10.4%–14.6% (Figure 11b,c). Glucose and ornithine were the essential skeleton and structural core of compound **1**, respectively, which involved the synthesis of compound **1**. Along with 20 g/L glucose and 4.32 g/L *L*-ornithine provision, compound **1** increased up to 82.2 and 95.9 g/L (Figure 11d). During the fermentation, a transformation was observed from ornithine, FGFC3, and FGFC2 into compound **1**. Further research is needed for promoting transformation from ornithine to compound **1**, which will be able to increase production substantially (Figure 11e).

**Figure 10.** Synthesis of SMTP-0, as well as congeners **3**, **7**, **19,** and **20,** based on pre-SMTP (phthalaldehydes reaction). Pre-SMTP (100 μg/mL in acetone) was incubated with (**a**) 5 mg/mL ammonium acetate in acetic acid (1.5%, *v/v*); (**b**) 5 mg/mL *<sup>L</sup>*-phenylalanine in acetic acid (1.5%, *v/v*); (**c**) 5 mg/mL *L*-tryptophan in acetic acid (1.5%, *v/v*); (**d**) 5 mg/mL *<sup>L</sup>*-glutamine in acetone-water-acetic acid (50:50:1); (**e**) 5 mg/mL *<sup>L</sup>*-glutamic acid in acetone-water-acetic acid (50:50:1).

**Figure 11.** The optimized method for a compound **1** culture. On fermentaion basal medium, inducers (or precursor), and conditions: (**a**) 0.5% ornithine hydochloride, 28 ◦C, 7 d, 1.98 g/L; (**b**) (i) 0.1 g/L shikimic acid, yield increased by 10.4%; (ii) 3 mmol/L sodium acetate, yield increased; (**c**) 0.09 mmol/L cerulenin, yield increased by 14.6%; (**d**) (i) 20g/L glucose, 82.2 g/L; (ii) 4.32 g/L, L-ornithine, 95.9 g/L; (iii) and (**e**) need further research.

Yin et al., (2017) studied the biosynthesis pathway in *S. longispora* FG216 [93]. The results were that three reported core genes and the nitrate reductase (NR) gene copy were the isoindolinone biosynthetic gene cluster in *S. longispora* FG216. NR is the rate-limiting enzyme of nitrate reduction. Therefore, nitrate reductase possibly played a role in the balance of ammonium ion concentration. Moreover, four new derivatives, **21**–**24**, were obtained by various amino supplements in *S. longispora* FG216 (Figure 12).

**Figure 12.** The structures of derivatives **21**–**24**.
