Promising derivatives of rutaecarpine with diverse pharmacological activities

Rutaecarpine (RUT) is a natural pentacyclic indolopyridoquinazolinone alkaloid first isolated from one of the most famous traditional Chinese herbs, Evodia rutaecarpa, which is used for treating a variety of ailments, including headaches, gastrointestinal disorders, postpartum hemorrhage, amenorrhea, difficult menstruation, and other diseases. Accumulating pharmacological studies showed that RUT possesses a wide range of pharmacological effects through different mechanisms. However, its poor physicochemical properties and moderate biological activities have hampered its clinical application. In this regard, the modification of RUT aimed at seeking its derivatives with better physicochemical properties and more potency has been extensively studied. These derivatives exhibit diverse pharmacological activities, including anti-inflammatory, anti-atherogenic, anti-Alzheimer’s disease, antitumor, and antifungal activities via a variety of mechanisms, such as inhibiting cyclooxygenase-2 (COX-2), acetylcholine (AChE), phosphodiesterase 4B (PDE4B), phosphodiesterase 5 (PDE5), or topoisomerases (Topos). From this perspective, this paper provides a comprehensive description of RUT derivatives by focusing on their diverse biological activities. This review aims to give an insight into the biological activities of RUT derivatives and encourage further exploration of RUT.

In the past few years, there have been several reviews on RUT.In 2008, Lee et al. provided a review on the isolation, synthesis, structure-activity relationship (SAR) studies, pharmacological activities, and metabolism of RUT (Lee et al., 2008).Two years later, Jia et al. summarized its pharmacological effects as a cardiovascular protective agent (Jia and Hu, 2010).In 2015, Son et al. gave an updated review of RUT on its synthesis, biological activities, and SAR of RUT derivatives (Son et al., 2015).In 2019, Tian et al. gave an updated review of RUT as a promising cardiovascular protective agent (Tian et al., 2019).Recently, Li et al. comprehensively summarized the recent progress of RUT as a promising hepatoprotective agent (Li et al., 2020).These aforementioned reviews were either limited to RUT itself (its derivatives were not described) or to one type of pharmacological activity (such as cardiovascular protective activity or hepatoprotective activity), and recent studies were not included.
Previously, a comprehensive description of the derivatives of evodamine (EVO, 2, a congener of RUT, which is also one of the most bioactive alkaloids isolated from Evodia rutaecarpa) was conducted by our group (Li et al. 2022).In this study, we focus on the broad spectrum of intriguing biological activities of RUT derivatives, aiming to provide a reference for further studies of RUT (Figure 2).

Bioactivities of RUT derivatives
2.1 Anti-inflammatory activity

Protecting against alcohol-induced liver injury
Alcoholic liver disease (ALD) represents the main type of chronic liver disease in the world (Seitz et al., 2018).It has been reported that cyclooxygenase-2 (COX-2) plays a vital role in the pathogenesis of ALD (Dunn and Shah, 2016).Enhanced expression of COX-2 can be seen in ALD (Nanji et al., 1997), and downregulating COX-2 can reduce inflammation and oxidative stress (Deol et al., 2018).Hence, an inhibitor of COX-2 may be an appropriate choice for the treatment of ALD.
Previously, Lee and coworkers demonstrated that 10-fluoro-2methoxyrutaecarpine (3) (Figure 3) retained anti-inflammatory activity both in vitro and in vivo (Lee et al., 2019).Based on the results, they further found that 3 exhibited better COX-2 suppression activity and lower cytotoxicity compared to its parent RUT as a COX-2 selective inhibitor.
Another team, Xu and coworkers, introduced a sulfonyl group to RUT to improve the anti-inflammatory effect and obtained a novel analog termed 3-B-RUT (4) (Figure 3) (Xu et al., 2021).Then, they explored its protective effect on alcoholic liver injury (ALI) in vivo and in vitro.The results showed that 4 (20 μg/kg) attenuated ALI and suppressed oxidative stress and liver inflammation, and the effect was comparable to that of RUT (20 mg/kg).Mechanistically, the results of Western blot showed that administration of 4 inhibited the phosphorylation of P65 and IκBα (silencing COX2 also achieves this effect), leading to suppressing the transfer of P65 into the nucleus.As is known, NF-κB commonly refers to the dimer protein formed by the P65/P50 subunits, and it controls DNA transcription, cytokine production, cell survival, and other important cell events; it specifically plays a key role in regulating immune development, immune responses, oxidative stress, and inflammation (Mitchell et al., 2016).Therefore, 4 might suppress oxidative stress and inflammation by regulating the COX-2/NF-κB pathway.The increased anti-inflammatory activity leading to a lower concentration may overcome the undesirable side effects, such as in gastrointestinal or heart diseases.Therefore, 4 was considered to be a promising clinical candidate for the treatment of ALI.

Alleviating acute kidney injury
Acute kidney injury (AKI) is a clinical syndrome caused by multiple factors, including ischemic-reperfusion injury, sepsis, and drug toxicity (such as cisplatin).Similarly, inflammation and oxidative stress have been shown to play critical roles in the pathogenesis of AKI (Liu et al., 2020).
Liu et al. found that phosphodiesterase 4B (PDE4B), a key enzyme modulator in immune and inflammation-related diseases, was significantly upregulated in the serum of AKI patients and participated in the progression of AKI (Liu et al., 2020).Therefore, they synthesized and obtained a series of 3-aromatic sulphonamidesubstituted RUT derivatives, and compound 5 (Figure 4) exhibited the best renoprotective and anti-inflammatory activity (Liu et al., 2020).Using target prediction (performed by Discovery Studio 2017 software), molecular docking (pdb code: 5O0J), and cellular thermal shift assay (CETSA), they identified PDE4B as the target of 5. Therefore, 5 might serve as a therapeutic candidate for AKI via the PDE4B pathway.

Anti-atherogenic activity
Atherosclerosis is a chronic disease featuring lipid deposition and an inflammatory response (Libby et al., 2011).It is well known that nitric oxide (NO) and tumor necrosis factor (TNF)-α play vital roles in inflammation (Kaltenmeier et al., 2022).Furthermore, the    NLRP3 inflammasome, a protein complex associated with the inflammatory progression of atherosclerosis, has attracted much interest in the pathogenesis of atherosclerosis (Abderrazak et al., 2015;Guo et al., 2015;Grebe et al., 2018).Therefore, compounds with reduced effects on NO, TNF-α, or NLRP3 that alleviate vascular inflammation would be beneficial for anti-atherogenic progression.
Previously, Lee et al. (2017) reported that compound 3 could also inhibit the production of NO and TNF-α in lipopolysaccharide (LPS)-

FIGURE 6
General SAR observations for anti-inflammatory activity.

FIGURE 7
Structure and activities of the representative compounds 7 and 8.

FIGURE 8
Structure and activities of the representative compounds 9 and 10. stimulated RAW264.7 macrophages.In addition, no cytotoxicity was observed toward RAW264.7 macrophages at a concentration of 20 µM.Although Luo et al. first demonstrated that 5-deoxy-rutaecarpine (R3, 6) (Figure 5), a novel derivative of RUT, could alleviate atherosclerotic burden in Apoe −/− mice fed a high-fat diet, pharmacokinetics studies showed that 6 had better oral bioavailability (F% = 27.89)than RUT (F % = 0.07) in male rats via gastric gavage at a dosage of 20 mg/kg.Subsequently, they explored its anti-atherosclerotic mechanisms (Luo et al., 2020).The results showed that 6 inhibited NLRP3 inflammasome activation through NF-κB, and the MAPK signaling pathway blocked atherosclerotic progression via debilitating NLRP3 inflammasomerelated inflammation.
Based on the aforementioned information, we found that i) modification of the D-ring of RUT, especially the introduction of a sulfonyl group to the D-ring of RUT, seemed beneficial to enhance its anti-inflammatory activity; and ii) removal of the carbonyl group from the C-ring of RUT may improve its oral bioavailability (Figure 6).

Anti-Alzheimer's disease activity
Alzheimer's disease (AD) is an incurable and long-term neurodegenerative brain disorder, and its pathogenic mechanism is still not fully understood.Currently, many hypotheses, including low levels of acetylcholine (AChE) and butyrylcholinesterase (BChE), aggregation of tau protein, and deposition of amyloid βprotein (Aβ) plaques, etc., have been proposed to explain the pathophysiology of AD (Wilson et al., 2012).Currently, the development of anti-AD drugs mainly focuses on reducing the levels of AChE and/or BChE, Aβ, and tau proteins.

As AChE inhibitors
Although 6 was demonstrated to exhibit anti-atherogenic activity, it is worth noting that it was first synthesized by Decker in early 2005, and its cholinesterase inhibitory activity was evaluated (Decker, 2005).The results showed that 6 exhibited moderate inhibitory activity toward both AChE and BChE with IC 50 values of 3.4 μM and 0.5 μM, respectively.
In 2010, the research group of Huang synthesized a series of novel RUT derivatives and evaluated their AChE inhibitory activity (Wang et al., 2010).Most of the derivatives were found to be selective for AChE over BChE.In particular, compound 7 showed the highest inhibitory activity toward AChE and also demonstrated the highest selectivity index (SI) over BChE.However, it showed a cytotoxic effect on SH-SY5Y cells.Considering that the dehydrorutaecarpine moiety had better potency than derivatives with the RUT moiety, they reported another series of dehydrorutaecarpine derivatives (He et al., 2013).All these synthetic compounds, particularly 8, showed higher inhibitory activity against AChE, Aβ aggregation, and oxidative stress compared to RUT.Notably, 8 showed a lower cytotoxic effect on SH-SY5Y cells (Figure 7).

As PDE5 inhibitors
Phosphodiesterases (PDEs), being responsible for hydrolyzing two second messengers, cyclic GMP (cGMP) and cyclic AMP (cAMP), can affect neuronal cell survival (Tsunada et al., 2021).Thus, PDEs play important roles in neurodegenerative diseases, including AD (Zuccarello et al., 2020).Among the different PDEs, PDE5 specifically hydrolyzes cGMP.Recently, an upregulated PDE5 expression has been found in the brains of mild AD patients, and studies have confirmed that PDE5 inhibitors exhibit therapeutic effects on AD patients by improving learning and memory, and reversing cognitive impairment (Boccia et al., 2011;Hosseini-Sharifabad et al., 2012;Al-Amin et al., 2014).Therefore, several researchers are developing anti-AD drugs by reducing PDE5 levels.
Considering RUT containing a quinoline structure, which has been demonstrated to be a privileged structure for the PDE5 inhibitor (Bi et al., 2004), Huang et al. decided to use RUT as the lead compound to develop PDE5 inhibitors for the treatment of AD (Huang et al., 2020).Thus, a series of RUT derivatives were synthesized, and their PDE5 inhibitory activities were tested.As a result, 11 showed the most potent PDE5 inhibitory activity with an IC 50 value of 0.086 μM.Moreover, it also exhibited good effects against scopolamine-induced cognitive impairment in vivo.These results might provide significant instruction for further developing PDE5 inhibitors derived from RUT (Figure 9).

Anti-tumor activity 2.3.1 As topoisomerase inhibitors
It is well known that topoisomerases are important targets in tumor therapy, and their inhibitors, including camptothecin, irinotecan, topotecan, etc., are widely used in clinic applications.In 2006, Xu et al. revealed that RUT itself did not show any significant activity against topoisomerases I and II (Topo I and Topo II) despite its significant cytotoxicity on several cancer cell lines (Xu et al., 2006).However, Kim et al. found that two derivatives of RUT (12 and 13) not only exhibited cytotoxicity against selected human cancer cell lines (GI 50 values of 5 μM, 3 μM, and 3 μM for 15b against U251, SKOV3, and DU145 cell lines, respectively; GI 50 values of 2.5 μM, 2 μM, and 2 μM for 15c against U251, SKOV3, and DU145 cell lines, respectively) (Baruah et al., 2004) but also showed inhibitory activities against Topo I and Topo II (Kim et al., 2012) (Figure 10).

Targets undefined
Huang et al. synthesized two sets of RUT derivatives with amine groups to improve their solubility (Huang et al., 2017).The results showed that the solubility of the compounds with the amine moieties connected to the indole-N atom ( 14) was improved significantly.Moreover, cell viability on HeLa cells and reduction of metabolic activity were in the same range or better for these derivatives compared to the chemically unaltered parent compounds (Figure 11).

Antifungal activity
To explore the potential applications of RUT and its derivatives against phytopathogenic fungi (Botrytis cinerea, Fusarium graminearum, Fusarium oxysporum, Magnaporthe oryzae, Rhizoctonia solani, and Sclerotinia sclerotiorum), various kinds of RUT derivatives were designed and synthesized by Yang et al. (2022), and their antifungal abilities against the aforementioned six phytopathogenic fungi were evaluated.The results showed that imidazole derivatives of RUT (A1, 15) exhibited broad-spectrum inhibitory activities against B. cinerea, F. graminearum, F. oxysporum, M. oryzae, R. solani, and S. sclerotiorum, with EC 50 values of 5.97 mg/mL, 12.72 mg/mL, 16.58 mg/mL, 1.97 mg/mL, and 2.87 mg/mL, respectively.However, RUT showed no activity against these six phytopathogenic fungi (the inhibition rate was 0% at 100 mg/mL).Moreover, the curative effects of 15 against S. sclerotiorum were 94.7%, 81.5%, 80.8%, and 65.0% at different concentrations of 400 mg/mL, 200 mg/mL, 100 mg/mL, and 50 mg/mL, respectively, in in vivo experiments, which was far more effective than the positive drug azoxystrobin.Notably, no phytotoxicity of 15 on oilseed rape leaves was observed even at a high concentration of 400 mg/mL.Therefore, 15 was considered a novel leading compound for the development of antifungal agents (Figure 12).

Conclusion
In summary, the diverse biological activities of RUT derivatives have been presented in this review.Undoubtedly, RUT and its derivatives exhibit diverse pharmacological activities, indicating that this class of alkaloids shows great potential in medicinal chemistry (Figure 13).Although there are currently no RUTbased drugs available in the market, research and development of RUT and its derivatives should be continued.
Nevertheless, the future directions of RUT and its derivatives could be summarized as follows: i) more studies should be carried out to clarify the similarities and differences between RUT and EVO.Both of them contain a pentacyclic scaffold, and their derivatives possess diverse pharmacological activities, including antitumor, anti-AD, and antifungal activities.However, based on the studies, it seems that RUT derivatives show better activity against inflammation and cardiovascular disease, while EVO derivatives show better activity against cancer.Zhang and coworkers explored the activity of the main bioactive indoloquinazoline alkaloids isolated from Euodia rutaecarpa on the aryl hydrocarbon receptor (AHR) (Zhang et al., 2018).The results showed that RUT was more efficient than EVO.These findings revealed that the methyl substitute at the N-14 atom was a key factor affecting AHR activation.However, similar studies addressing the underlying reasons for their different biological activities are rare.ii) The underlying reasons for one compound exhibiting different biological activities should be reinforced, as observed in the case of compounds 3 and 4. In other words, detailed mechanism research related to the activity should be reinforced.iii) The structural modification of RUT to obtain a variety of sufficient derivatives still needs to be carried out: on one hand, to improve its physicochemical properties or selectivity, and on the other hand, to broaden the scope of compounds with diverse pharmacological activities.Moreover, SARs between different biological activities need to be summarized.iv) How to increase the bioavailability for clinical practice should be given more attention.Most of the compounds introduced in this article have not reached clinical trials; so, their physiological functions have not been clinically confirmed, which could mislead or cause bad effects.

FIGURE 3
FIGURE 3Structure of the representative compounds 3 and 4 with anti-inflammatory activity.

FIGURE 4
FIGURE 4Structure of the representative compound 5 with anti-inflammatory activity.

FIGURE 2
FIGURE 2Congeners of RUT and EVO isolated from Evodia rutaecarpa.

FIGURE 5
FIGURE 5Structure of the representative compound 6 with anti-inflammatory activity.

FIGURE 9
FIGURE 9Structure and activities of the representative compound 11.

FIGURE 11
FIGURE 11Structure and activities of the representative compound 14.