Discovery of small-molecule candidates against inflammatory bowel disease

Journal Pre-proof Discovery of small-molecule candidates against inflammatory bowel disease Renren Bai, Xiaokang Jie, Chuansheng Yao, Yuanyuan Xie PII:

S0223-5234(19)30957-2

DOI:

https://doi.org/10.1016/j.ejmech.2019.111805

Reference:

EJMECH 111805

To appear in:

European Journal of Medicinal Chemistry

Received Date: 2 August 2019 Revised Date:

19 October 2019

Accepted Date: 20 October 2019

Please cite this article as: R. Bai, X. Jie, C. Yao, Y. Xie, Discovery of small-molecule candidates against inflammatory bowel disease, European Journal of Medicinal Chemistry (2019), doi: https:// doi.org/10.1016/j.ejmech.2019.111805. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.

Graphic abstract Recent progress in the discovery of small-molecule candidates against inflammatory bowel disease

Renren Bai a, *, Xiaokang Jie a, Chuansheng Yao a, Yuanyuan, Xie a, *

a

College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, P.R. China.

Discovery of small-molecule candidates against inflammatory bowel disease

Renren Bai a, *, Xiaokang Jie a, Chuansheng Yao a, Yuanyuan, Xie a, *

a

College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, P.R. China.

Correspondence: Dr. Renren Bai, College of Pharmaceutical Sciences, Zhejiang University of Technology,

Hangzhou

310014,

China.

Email:

[email protected] ，

[email protected]

Dr. Yuanyuan Xie, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, China. Email: [email protected]

Abstract: Inflammatory bowel disease (IBD) is a chronic and recurrent inflammatory disease in the gastrointestinal tract emerged as a public health challenge worldwide. IBD exhibits a relapsing and remitting course results in negative impacts on both physical and psychological health of IBD patients. Great efforts have been made during the past few years, but relatively limited drugs are currently available for the management of IBD. Clinically, there is a strong demand for new drugs for the treatment of IBD with better efficacy and lower side effects. This review focuses on the drug discovery process of the anti-IBD agents, aiming to introduce the general characteristics of IBD, as well as systematically summarize the recent advances in the discovery of small-molecule candidates and natural products with promising in vivo 1

potential for the treatment of IBD.

Keywords: Inflammatory bowel disease, Crohn’s disease, Ulcerative colitis, Small molecules, Natural products

Contents: 1. Introduction ................................................................................................................ 3 2. General Characteristics of IBD .................................................................................. 4 3. Small molecules and natural products reported with potential anti-IBD activities ... 5 3.1. Small molecules with in vivo anti-IBD activity .................................................. 5 3.1.1. Salicylic acid derivatives .............................................................................. 5 3.1.2. Aminosalicylic acid derivatives.................................................................... 6 3.1.3. TNF-α inhibitors ........................................................................................... 8 3.1.4. IKK2 inhibitors ............................................................................................. 9 3.1.5. LTB4 inhibitors .......................................................................................... 10 3.1.6. CXCR4 antagonists .................................................................................... 11 3.1.7. CCR9 antagonists ....................................................................................... 11 3.1.8. PPAR-γ mediators ...................................................................................... 13 3.1.9. NAE inhibitor ............................................................................................. 13 3.1.10. LANCL2 inhibitors................................................................................... 14 3.1.11. NLRP3 inhibitors ...................................................................................... 14 3.1.12. CB2 agonists ............................................................................................. 15 3.2. Natural products and related derivatives with in vivo anti-IBD activities ........ 16 3.2.1. Natural products with in vivo anti-IBD activities ....................................... 16 3.2.2. Natural product derivatives with in vivo anti-IBD activities ...................... 25 4. Summary and future directions ................................................................................ 27

2

1. Introduction Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammatory disease in the gastrointestinal tract [1]. In the past decade, IBD has emerged as a public health challenge worldwide [2]. In North America and Europe, over 1.5 million and 2 million people suffer from the disease, respectively [3]. The prevalence of IBD is highest in the Western world, affecting up to 0.5% of the general population. Similarly, the incidence of IBD is highest in the Western world, ranging from 10 to 30 per 100,000 [4]. Interestingly, at the turn of the 21st century, newer epidemiological studies have revealed that the incidence of IBD has been increasing in developing countries in South America, Asia, Africa, and Eastern Europe [5]. IBD exhibits a relapsing and remitting course and there is a significant, often dramatic, reduction in quality of life during exacerbations of the disease with remarkable negative impacts on both physical and psychological health of IBD patients [6]. The cause of IBD is very complicated with repeated and intractable conditions. The related complications may even bring serious health threats. In spite of great efforts have been made during the past few years, as well as the increasing number of therapeutic agents tested so far, relatively limited compounds are currently available for the management of IBD [7]. Design of novel biologic therapies to treat IBD has the challenges of addressing potential safety issues, while more traditional small-molecule drugs should be further developed to facilitate patient compliance to treat this chronic, debilitating disease. Although several reviews have been available on the efficacy of therapy of current medical therapies in IBD, the rapidly changing research field needs a more comprehensive and updated review of the recent literature. This review focuses on the drug discovery process of the anti-IBD agents, aiming to introduce the general characteristics of IBD, as well as systematically summarize the recent advances in the discovery of small-molecule drug candidates and natural products lead compounds with promising in vivo potential for the treatment of IBD. 3

2. General Characteristics of IBD IBD is thought to result from inappropriate and ongoing activation of the mucosal immune system driven by the presence of normal luminal flora. This aberrant response is most likely facilitated by defects in both the barrier function of the intestinal epithelium and the mucosal immune system. The cause of disease activity includes many factors, such as environmental influences, genetic features, changes in the intestinal microbiota ecosystem, and immune factors [8]. The essential difference between IBD and inflammatory responses in the normal gut is that IBD is the inability to down-regulate those responses. In healthy people, the intestine becomes inflamed in response to a potential pathogen, then returns to a state of tolerance once the pathogen is eradicated from the gut; while in patients with IBD, inflammation is not down-regulated, the mucosal immune system remains chronically activated, and the intestine remains chronically inflamed [9]. IBD comprises primarily 2 major disorders: Crohn’s disease (CD) and ulcerative colitis (UC). Both forms of idiopathic IBD, empirically defined by clinical, pathological, endoscopic and radiological features, are chronic and uncontrolled inflammation of the gastrointestinal tract [10]. The etiology of CD is complex and it is assumed that the disease develops under the influence of negative environmental determinants and genetic preconditions [11]. Exposure to infections, dietary customs, hygiene, drugs, and vaccinations are all possible pathogenic factors of CD [12]. UC is a long-term condition that results in inflammation and ulcers of the colon and rectum, with primary symptoms of abdominal pain and diarrhea mixed with blood. The exact pathophysiology is unknown, but theories involve immune system dysfunction, genetics, changes in the normal gut bacteria, and environmental factors [13]. As illustrated in Fig. 1, in the case of CD, chronic inflammation can be localized in anywhere in the gastrointestinal tract segment (from gum to bum) and involves the full thickness of the intestinal wall. In terms of UC, it is characteristically restricted to the mucosal surface of the large intestines (colon and rectum), which is more common 4

than CD [14]. The disorder starts in the rectum and generally extends proximally in a continuous manner through the entire colon [11, 12]. Both diseases mainly differ in the localization and size of the segment involved. There are also differences in the clinical image, laboratory test results, and different characteristics of complications. The general characteristics of UC and CD were summarized in Table 1 [14-16].

Fig. 1 Apparent and visual pathological features of IBD. Table 1. General comparison of the main characteristics of CD and UC.

3. Small molecules and natural products reported with potential anti-IBD activities 3.1. Small molecules with in vivo anti-IBD activities 3.1.1. Salicylic acid derivatives Recent research reported that NF-κB inhibition is one of the important anti-inflammatory mechanisms of salicylic acid (SA) and aminosalicylic acid derivatives such as salicylate, aspirin, 5-aminosalicylic acid (5-ASA) and sulfasalazine (Fig. 2a) [17, 18]. N-(5-chlorosalicyloyl)phenethylamine (5-CSPA, 1), a SA derivative, effectively ameliorated TNBS-induced rat colitis (Fig. 2b). Rectal administration of 5-CSPA (300 µM) significantly healed the damaged colon in a dose-dependent manner, showing a more effective activity than that of 5-ASA (30 mM). Additionally, 5-CSPA lowered the level of myeloperoxidase (MPO), a biochemical marker of inflammation, to about 60% (100 µM) and 35% (300 µM), which was in accordance with the recovery of the colonic damage. Moreover, 5-CSPA decreased the levels of the inflammatory mediators including NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in the inflamed tissue in a dose-dependent manner [19].

Fig. 2 a. Structure of compound 5-CSPA (1); b. Designing strategy and the chemical structures of compounds 4AP (2) and 4AT (3); c. Structure of compound 4-ASA-Glu (4). 5

3.1.2. Aminosalicylic acid derivatives 3.1.2.1. 4-Aminosalicylic acid derivatives 4-Aminosalicylic acid (4-ASA) is designated as an orphan drug by the FDA for use in mild to moderate UC in patients who are intolerant of sulfasalazine [20]. To improve the safety profile of 4-ASA, amide prodrugs of 4-ASA combined with D-phenylalanine and L-tryptophan were designed and synthesized for targeted drug delivery to the inflamed gut tissue in IBD (Fig. 2c). In a 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis rats model, the amide prodrugs of 4-ASA demonstrated a significant ameliorating effect. Compound 4AP (2) produced a comparable lowering of the clinical activity score (CAS, 0.8±0.09) to sulfasalazine (0.9±0.7) while the effect of compound 4AT (3) on lowering of CAS was moderate (1.3±0.12), but both were distinctly more potent than that of 4-ASA (2.0±0.08). Moreover, the colonic MPO activity of 4AP and 4AT in mU/100 mg tissue was found to be 46.57 and 49.24, respectively. These effects were comparable to that of sulfasalazine (46.63), but much less than 4-ASA (70.5), suggesting a lower neutrophil infiltrate in the inflamed colon [21]. Other than amide-bound and azo-bound prodrugs, glycoside prodrugs of ASA with D-galactose for site-specific delivery to the colon is also a promising strategy for the colon-targeted delivery, based on the observation that glycosides are cleaved by bacterial glycosidase generated in the colonic microflora to release the active ASA. 4-Aminosalicylic acid-β-O-glucoside (4-ASA-Glu, 4) was a colon-specific prodrug realizing targeted release in the colon (Fig. 2d). The amount of 4-ASA liberated from the incubation of 4-ASA-Glu in cecal or colonic contents of colitis rats at 37 oC was 69% or 79% in 12 h, respectively. However, less than 9% 4-ASA was detected from the incubation of 4-ASA-Glu with the homogenates of the stomach or small intestine. In the TNBS-induced rats colitis model, CAS, colon/body weight ratio, macroscopic injury score, histological evaluation and MPO activity all showed that the rats treated with 4-ASA-Glu had an improvement in the pathology than that of 4-ASA [22]. 6

3.1.2.2. 5-Aminosalicylic acid derivatives Mesalamine (5-ASA) is a drug for treating UC, which is often taken as a lead compound for further structure modification. For example, sulfasalazine is a classic 5-ASA derivative, as well as a prodrug of 5-ASA for the treatment of IBD. However, several serious side effects such as blood dyscrasias, hepatotoxicity and hypersensitivity reactions are ascribed to sulfasalazine [23]. Out of the need for the safer option than sulfasalazine, a concept-based mutual prodrug design was adopted and a series of colon-targeted azo conjugates of 5-ASA with essential amino acids including L-tyrosine, D-phenylalanine and L-tryptophan were prepared (Fig. 3). In the Rainsford’s cold stress assay, the conjugates SP (5), TS (6) and ST (7), showed a remarkable reduction in the ulcer index (10.76±0.55 to 13±1.1) compared to the very high ulcer index of 5-ASA (60.03±1.15). These anti-IBD activities were comparable to that of sulfasalazine (9±2). All of the prodrugs displayed significantly lowering of CAS, among which, TS (0.9±0.13) was similar while SP (1.06±0.51) and ST (1.39±0.39) were comparable to sulfasalazine (0.83±0.42) but distinctly more potent than 5-ASA (2.09±0.27) in a TNBS-induced colitis rats model. The prodrug treated groups also demonstrated a remarkable decrease in the colon/body weight ratio (TS> sulfasalazine = ST > SP) compared to the colitis control group. Besides, prodrugs showed comparable MPO activity to sulfasalazine (SP> ST= TS> sulfasalazine) [24].

Fig. 3 Designing strategy and chemical structures of compounds SP (5), TS (6) and ST (7).

3.1.2.3. 7-Aminofurosalicylic acid derivatives Benzofuran derivatives exhibit a promising gastro-protective activity and show dual cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LOX) inhibition. Therefore, benzofuran derivatives are potential candidates as anti-inflammatory agents [20]. To discovery more potent anti-IBD candidates, a series of furo-salicylic acid derivatives, 7

where the 4- and 5-positions of the salicylic acid nucleus were incorporated in a furan ring, were synthesized and evaluated for the treatment of UC. All the synthesized compounds were screened for their anti-ulcerogenic effect on acetic acid-induced UC in rats. Taking orally treated sulphasalazine (5 mg/kg) as the reference drug, the tested compounds 8-11 (Fig. 4) in equimolar doses remarkably reduced the intensity of lesion score, ulcer area, ulcer index and wet weight/length ratio (P<0.01) compared to the control group. Compound 11 was proved to be the most pharmacologically active among the newly synthesized compounds and could be easily broken in the body by hydrolysis. Additionally, azo derivatives 9 and 10 exhibited antibacterial activity against both Gram-positive and Gram-negative bacteria. While compound 8 displayed antibacterial activity against Gram-negative bacteria E. coli and compound 11 showed antibacterial activity against Gram-positive bacteria B. subtilis. Compounds 9 and 10 also showed a promising antifungal activity closely related to the standard, amphotercin B [25].

Fig. 4 Chemical structures of compounds 8-15.

3.1.3. TNF-α inhibitors Tumor necrosis factor-α (TNF-α) is a major pro-inflammatory cytokine involved in IBD. TNF-α perpetuates the inflammatory process by inducing the expression of other pro-inflammatory cytokines and adhesion molecules, which is required for leukocyte attachment and infiltration through the intestinal mucosa, acting as one of the critical steps in the inflammation and tissue injury encountered in IBD [26, 27]. 2-Benzylidene indanone derivative 12 (Fig. 4) showed effective inhibitory activity against TNF-α-induced monocytic-colonic epithelial cell adhesion (IC50 = 0.50 ± 0.05 µM,), 40,000 times more potent than that of 5-ASA (IC50 = 20.4 ± 2.2 mM). Compound 12 also inhibited TNF-α expression at the 10 µM concentration. Oral administration of 12 suppressed TNBS-induced colitis in a dose-dependent manner. 8

Moreover, there was a significant recovery in body weight decrease and colon tissue edematous inflammation. A higher dose (30 mg/kg) of 12 showed a similar recovery effect to that of sulfasalazine (300 mg/kg). The level of MPO was also markedly decreased by 12 in a dose-dependent manner [1]. It is reported that pyridinium salt formation enhances the physicochemical and biological properties. A series of pyridine-linked indanone derivatives were designed and synthesized to discover new small molecules for the treatment of IBD. Compounds 13 and 14 (Fig. 4) displayed obvious inhibition on TNF-α-induced monocyte adhesion to HT-29 human colonic epithelial cells, with 86% and 78% inhibition, respectively, at the concentration of 10 µM. In the TNBS-induced rats colitis model, oral administration of 13 or 14 at 25 mg/kg/day better ameliorated symptoms than sulfasalazine (300 mg/kg). Derivatives 13 and 14 also exhibited remarkable recovery in altered E-cadherin, TNF-α and IL-1β expressions, indicating 13 and 14 as potential agents against IBD [28]. 6-Amino-2,4,5,-trimethylpyridin-3-ol derivative 15 (Fig. 4) is another potent compound targeting TNF-α related pathway. Rats receiving oral administration of 15 (0.1, 1, and 10 mg/kg) clearly showed marked recovery of the TNBS-induced decrease in body weight in a dose-dependent manner. Surprisingly, 0.1 mg/kg dose of compound 15 showed almost comparable body weight recovery than 300 mg/kg dose of sulfasalazine. Colon damage was significantly recovered in rats orally treated with compound 15 in a dose-dependent manner. As low as 1 mg/kg of 15 showed better colon weight recovery than 300 mg/kg of sulfasalazine, while the recovered colon weight at 10 mg/kg was almost the same to the level of control [29].

3.1.4. IKK2 inhibitors The IKK complex is a high molecular weight trimeric complex comprised of IKK1 (IKKα), IKK2 and IKKβ (a NF-κB essential modulator). Evidence has shown that the IKK2 subunit is the major contributor in NF-κB activation arising from pro-inflammatory stimuli. As a consequence, IKK2 inhibitors were likely effective in 9

the treatment of autoimmune and inflammatory disorders such as rheumatoid arthritis and IBD [30]. Compound 16 (Fig. 5) was an effective IKK2 inhibitor with an IC50 of only 9 nM and showed more than 500-fold selective for IKK2 over IKK1. Pharmacokinetic evaluation of 16 in rat and dog revealed that the compound had excellent oral bioavailability with low clearance and an acceptable half-life. In the TNBS-induced colitis model in mice, compound 16 (10 mg/kg) demonstrated clear dose-dependent efficacy, maintaining the body weight and colon length similar to that of the dexamethasone-treated

group

and

healthy

control

mice.

Additionally,

the

histopathological evaluation revealed that a 10 mg/kg dose of compound 16 demonstrated a significant reduction in both inflammation and tissue damage relative to the vehicle group. These results suggest that compound 16 may be effective in treating IBD in humans [31].

Fig. 5 The chemical structures of compounds 16-23.

3.1.5. LTB4 inhibitors Leukotriene B4 (LTB4) is a leukotriene involved in inflammation. LTB4 is a mediator of inflammatory pain and its binding to peroxisome proliferator-activated receptor (PPAR) affects the duration of the inflammatory response to LTB4 [32]. Derivatives of arylalkanoic acids were reported to display promising anti-leukotriene activities.

Among

them,

derivatives

17-19

(Fig.

5),

bearing

a

N-arylethyl-2-arylacetamido group, were significantly active in the inhibition of UC and simultaneously showed distinguished high anti-leukotriene activities [33]. Based on the above results, the arylalkanoic acid side chain was modified and a series of phenethylamido derivatives of arylalkanoic acids were prepared by the same research group. The results of UC inhibition demonstrated that compounds 20-23 (Fig. 5) were more active than that of the reference drug sulfasalazine. Structure-activity relationship (SAR) showed that the change of connecting chain between aromatic ring 10

and carboxyl did not bring important improvement of this activity in comparison with the previous derivatives of arylacetic acids [34].

3.1.6. CXCR4 antagonists Chemokine (C‑X‑C motif) ligand 12 (CXCL12), also known as stromal cell‑derived factor‑1 (SDF-1), is a member of the chemokine family, which consists of low molecular‑weight proteins produced by various types of cells involved in allergic inflammation [35]. CXCL12 binds to Chemokine (C‑X‑C motif) Receptor 4 (CXCR4) and attracts a variety of inflammatory cells (neutrophils, monocytes, and lymphocytes) to local tissues and regulate the release of inflammatory factors that cause inflammatory responses [36]. Therefore, developing CXCR4 antagonists to control and reduce inflammation is a feasible and effective strategy for the treatment of IBD. MSX-122 (Q-122, 24, Fig. 6) is a unique small molecule CXCR4 antagonist, which is a safer partial CXCR4 modulator than other reported CXCR4 antagonists. In a dextran sulfate sodium (DSS)-induced mice colitis model, the degree of epithelial erosion and crypt destruction of DSS/MSX-122-treated mice was significantly suppressed. At day 10, colonic damage and increased inflammatory cell infiltration remained in DSS-treated mice. By contrast, the colons of DSS/MSX-122-treated mice showed signs of recovery. MSX-122 also attenuated the production of TNF-α, IL-1β, and IL-6 and further decreased cytokine concentration [37].

Fig. 6 The chemical structures of compounds 24-29.

3.1.7. CCR9 antagonists Chemokine receptor 9 (CCR9) is a chemokine receptor known to be central for migration of immune cells into the intestine. Its only ligand, Chemokine (C-C motif) ligand 25 (CCL25), is expressed at the mucosal surface of the intestine and is known to be elevated in intestinal inflammation. To date, there are few reports of 11

small-molecule

antagonists

targeting

CCR9

[38].

CCX282-B

(Vercirnon,

GSK-1605786, 25, Fig. 6), discovered by Chemocentryx and licensed by GlaxoSmithKline, is a small molecule, orally bioavailable, selective, and potent antagonist of human CCR9. CCX282-B was an equipotent antagonist of CCL25-directed chemotaxis of both splice forms of CCR9 (CCR9A and CCR9B) with IC50 values of 2.8 and 2.6 nM, respectively. TNFΔ ARE mice spontaneously develop a syndrome characterized by severe inflammation of the small intestine, bearing many of the hallmarks associated with human CD. Inhibition of CCR9 with CCX282-B results in normalization of CD in the TNFΔARE mice model. Treatment with CCX282-B (50 mg/kg) displayed complete protection from the severe inflammation associated with TNF-α overexpression. 70% of the mice in this treatment group were scored as normal, and the remaining 30% were scored as having moderate inflammation compared to control group [39]. CCX282-B underwent successful Phase II clinical trials for CD, but ultimately demonstrated a lack of efficacy in the Phase III SHIELD trials because of a combination of the patient population selected for study (who were more seriously ill than the Phase II population) and the relatively poor pharmacokinetic (PK) profile. Aimed at producing an orally available CCR9 antagonist with a superior PK profile to CCX282-B, a series of CCR9 antagonists based on a 1,3-dioxoisoindoline skeleton were discovered. These molecules were highly potent at the receptor and have excellent PK properties, resulting in highly significant data being obtained in well-recognized models of IBD. Compounds 26-28 (Fig. 6) exhibited potent anti-IBD effects in the acute DSS-induced colitis model in mice. At the top dose of 100 mg/kg, compounds 26 and 28 showed 58% and 51% reduction in the disease activity index (DAI) relative to control, respectively, while compound 27 provided the most excellent protection against the induced colitis with 86% inhibition [40].

12

3.1.8. PPAR-γ mediators Proliferator-activated receptors (PPARs) are members of the nuclear receptor’s superfamily and functions as a ligand-activated transcriptional regulator of genes involved in several physiological processes. PPAR-γ maintains intestinal mucosal integrity and prevents inflammatory damage during experimental colitis and UC patients [41, 42]. Recently, PPAR-γ has been identified as a target of 5-ASA, suggesting an additional mechanism of anti-inflammatory action in the colon [43]. 3-Hydroxy-4-pyridinecarboxylic acid derivatives (HPs), possessing the ortho hydroxy-carboxylic

acid

functions,

were

reported

to

demonstrate

certain

anti-inflammatory activity. HP 29 (Fig. 6) increased PPAR-γ mRNA transcript levels in human macrophages and directly stimulated PPAR-γ transcriptional activity in CMT-93 cells. Treatment with HP 29 suppressed the DSS-induced colitis by about 56% of colonic MPO activity, similarly to the equal dose of 5-ASA [43]. HP 29 directly stimulated PPAR-γ transcriptional activity in CMT-93 cells transfected with the luciferase reporter construct carrying PPAR-γ-responsive elements (PPAREs). HP 29 was found to be able to prevent LPS-induced TNF-α and IL8 up-regulation on human macrophages at a 10 µM concentration. Moreover, HP 29 reduced tissue levels of IL-1β whereas 5-ASA did not [43].

3.1.9. NAE inhibitors The NEDD8-activating enzyme (NAE) is a ubiquitin-like E1 enzyme that is involved in the regulation of cullin/RING ubiquitin ligases (CRLs). The specific targeting of NAE could modulate the pace of ubiquitination and degradation of inflammation-related proteins such as IκBα and p27. Therefore, targeting NAE represents a potential avenue for the treatment of inflammatory diseases such as IBD [44]. Cyclometalated rhodium(III) complex (30, Fig. 7) is the first metal-based inhibitor of NAE showing in vivo anti-inflammatory activity for the potential treatment of IBD. The results showed that untreated colitic mice showed an average 13

maximal DAI score of 8.3, while colitic mice treated with complex 30 (5 mg/kg) exhibited a considerably lower maximal average DAI score of 5.8 (p < 0.001). The statistical evaluation of the macroscopic score showed that colitic mice treated with complex 30 (p < 0.001) had significantly lower scores compared with the untreated colitic mice. Furthermore, complex 30 and mesalazine decreased inflammation markers TNF-α, MPO, and IL-1β levels in colon tissue samples [45].

Fig. 7 The chemical structures of compounds 30-33.

3.1.10. LANCL2 inhibitors Lanthionine synthetase C-like 2 (LANCL2) is broadly expressed in epithelial cells and the immune system, including thymus, spleen and colon, suggesting a potential role in modulating mucosal immune responses. Therefore, LANCL2 has emerged as a new therapeutic target for treating inflammatory [46]. LANCL2 inhibitor

31

(Fig.

piperazine-1,4-diylbis(6-benzo[d]imidazole-2-yl)pyridine-2-yl)methanone,

7), was

identified as the lead LANCL2-binding compound for treating IBD. The oral treatment with 31 (8 mg/kg/d) in a mouse model of IBD resulted in lowering the DAI, decreasing colonic inflammatory lesions by 4-fold and suppressing inflammatory markers (such as TNF-α, and interferon-γ) in the gut. Furthermore, studies in LANCL2−/− mice demonstrated that loss of LANCL2 abrogated beneficial actions of 31, suggesting high selectivity for this target [47].

3.1.11. NLRP3 inhibitors NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is a cytosolic complex involved in the production of proinflammatory cytokines, such as interleukin (IL)-1β and IL-18. NLRP3 is considered as a pivotal player in regulating the integrity of intestinal homeostasis and it also shapes innate immune responses against commensal bacteria. Overactivation of NLRP3 during intestinal inflammation 14

is associated with a breakdown of the intestinal immune balance, with consequent detrimental effects for the host [48]. Compound 32 (Fig. 7) is a noncytotoxic molecule able to inhibit the activation of the NLRP3 inflammasome. Compound 32 was able to counteract NLRP3 activation through direct irreversible interaction with NLRP3 and partial inhibition of LPS-driven pro-inflammatory gene expression. Rats treated with 32 displayed a significant reduction of macroscopic damage score (4.7±0.9 at 12.5 mg/kg, 3.1±0.7 at 25 mg/kg, and 2.8±0.4 at 50 mg/kg) in a 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis rats model. Treatments with 32 obviously decreased the concentration of the cytokine in colonic tissues (3.5±0.2, 3.3±0.2, and 2.7±0.1 pg/mg tissue at 12.5, 25, 50 mg/kg, respectively). Compound 32, counteracting intestinal inflammation, may represent as a lead compound for the development of novel NLRP3 inflammasome inhibitors [49].

3.1.12. CB2 agonists Cannabinoid (CB) receptor is a G-protein coupled receptor, existing CB1 and CB2 two subtypes. CB1 receptor is localized mostly within the central nervous system (CNS). While CB2 receptor is sparsely expressed in the CNS and predominately expressed on activated immune cells (thymus, B and T cells, macrophages, monocytes) and their activation mediates immune responses and explains their therapeutic potential [50]. Developing CB2 receptors agonists is a novel strategy for the treatment of inflammation. An original series of selective CB2 agonists were reported around the benzo[d]thiazol-2(3H)-one scaffold leading to the discovery of a very potent and selective CB2 agonist 33 (Ki=13.5 nM).

Compound 33 (Fig. 7) has shown a strong

protective effect in the in vivo DSS-induced colitis mice model, with improved body weight, a lower colon weight/size ratio (0.044 ± 0.0024, p=0.004), and a decrease of MPO activity (43%). ADME-Tox profile of compound 33 was acceptable but needed to be improved to consider oral administration. Taking together, these results 15

suggested that benzo[d]thiazol-2(3H)-one scaffold could open new perspectives for the development of new CB2 receptor agonists [51].

3.2. Natural products and related derivatives with in vivo anti-IBD activities 3.2.1. Natural products with in vivo anti-IBD activities 3.2.1.1. Diammonium glycyrrhizinate Ginger, the rhizome of Zingiber officinale, is one of the most commonly used fresh herbs and spices. The ginger extract consists of several phenolic compounds, and the anti-inflammatory effects of phenolic compounds have been demonstrated both in vitro and in vivo [52]. Zingerone (34, Fig. 8) is one of the components of ginger extracts and HPLC quantification showed that the content of zingerone in the ginger extract was 0.1%. The effect of zingerone in mice with TNBS-induced colitis was investigated. Results proved that zingerone improved the TNBS-induced colonic weight/length ratio and histological scores in a dose-dependent manner. TNBS induced colonic ulceration and inflammation, while zingerone suppressed the TNBS-induced colonic injury. In conclusion, zingerone improved TNBS-induced colitis via modulation of NF-κB activity and IL-1β signaling pathway and might be the active component of ginger responsible for the amelioration of colitis [53].

Fig. 8 The chemical structures of natural products 34-43.

3.2.1.2. Propyl-propane thiosulfonate Propyl-propane thiosulfonate (PTSO, 35, Fig. 8) is a component isolated from garlic (Allium sativum) with antioxidant, anti-inflammatory, immunomodulatory, and antimicrobial properties [54]. The anti-inflammatory effects of PTSO were studied in both DNBS- and DSS-induced colitis mice models. The immunomodulatory effects of PTSO (0.1–25 µM) were also shown in Caco-2 and THP-1 cells, reducing the production of pro-inflammatory mediators and downregulating mitogen-activated protein kinases (MAPKs) signaling pathways. This compound displayed beneficial 16

effects in both models of mouse colitis by reducing the expression of different pro-inflammatory mediators and improving the intestinal epithelial barrier integrity. Moreover, PTSO ameliorated the altered gut microbiota composition in DSS-induced colitis mice. In consequence, PTSO can be a potential candidate for the treatment of IBD [55].

3.2.1.3. Diallyl trisulfide Several organosulfur compounds (OSCs) derived from garlic, especially diallyl trisulfide (DATS, 36, Fig. 8), have been considered to contribute to the chemopreventive and cytoprotective activities of garlic [56, 57]. Orally administered DATS for 7 days before and for another 7 days after the treatment of DSS protected against colitis induced by DSS in ICR mice. DATS significantly inhibited the DSS-induced DNA binding of NF-κB, phosphorylation of IκBα and the expression of proinflammatory proteins, such as COX-2 and inducible NO. The DSS-induced DNA binding and phosphorylation at the Tyr 705 residue of signal transducer and activator of transcription 3 (STAT3) were also inhibited by DATS.

In conclusion, DATS

ameliorated the DSS-induced mouse colitis presumably by blocking inflammatory signaling mediated by NF-κB and STAT3 [58].

3.2.1.4. Gallic acid Gallic acid (3,4,5-trihydroxybenzoic acid, 37, Fig. 8) is a type of phenolic acid that is found in various natural products, such as gallnuts, pineapples, sumac, oak bark, green tea, apple peels, tea leaves, grapes, strawberries, bananas, lemons and also in red and white wine [59]. Compound 37 exhibits various beneficial activities including anti-oxidant and anti-inflammatory [60, 61]. In an experimental murine model of UC, 37 displayed remarkable amelioration of the disruption of the colonic architecture, a significant reduction in colonic MPO activity, and a decrease in the expression of inflammatory mediators, such as inducible nitric oxide synthase (iNOS), COX-2, and pro-inflammatory cytokines. In addition, compound 37 reduced the activation and 17

nuclear accumulation of p-STAT3Y705, preventing the degradation of the inhibitory protein IκB and inhibiting of the nuclear translocation of p65-NF-κB in the colonic mucosa, suggesting that compound 37 exerted potentially clinically useful anti-inflammatory effects mediated through the suppression of p65-NF-κB and IL-6/p-STAT3Y705 activation [62].

3.2.1.5. Piperine Piperine (1-peperoylpiperidine, 38, Fig. 8), the primary lipophilic component in black pepper (Piper nigrum) and long pepper (Piper longum), has been reported to display anti-inflammatory activity [63]. Investigation indicated that pre-administration of piperine decreased clinical hallmarks of colitis in DSS-treated mice as measured by body weight loss and assessment of diarrhea, rectal bleeding, colon length, and histology. Inflammatory mediators (CCR2, ICAM-1, IL-1β, IL-6, IL-10, iNOS, MCP-1, and TNF-α) were significantly decreased in mice pretreated with piperine. These results demonstrated that piperine might contribute to the prevention or reduction of colonic inflammation [64, 65].

3.2.1.6. Curcumin Curcumin (39, Fig. 8) is a natural compound found in the plant Curcuma longa, which is used as a food additive known as turmeric. Curcumin possesses both anti-inflammatory and antioxidant properties [66]. Based on its in vitro and in vivo activity, the anti-inflammatory effect of curcumin was investigated in 10 patients suffered from IBD. Overall, all five subjects with proctitis were improved by the end of the study. In the patients with limited ulcerative colitis, serologic indexes of inflammation, sedimentation rate, and C-Reactive Protein (CRP) returned to within normal limits at the conclusion of the study. The CD activity index (CDAI) scores for all completed subjects fell, with a mean reduction of 55 points. However, there were no changes in the indexes of liver or renal function [67].

18

3.2.1.7. 6-Gingerol Ginger (Zingiber officinale) is a globally cultivated crop used in traditional oriental medicine to treat gastrointestinal disorders including stomachaches, abdominal spasm, and nausea and vomiting [68]. 6-Gingerol (40, Fig. 8), a major pungent phenolic component in ginger, has been reported to have various pharmacological properties including anti-inflammatory, anti-cancer, and antioxidant activities [69, 70]. Studies proved that adenosine monophosphate-activated protein kinase (AMPK) was activated by 6-gingerol treatment in vitro. In animal studies, 6-gingerol significantly ameliorated DSS-induced colitis by the restoration of body weight loss, reduction in intestinal bleeding, and prevention of colon length shortening. In addition, 6-gingerol suppressed DSS-elevated production of proinflammatory cytokines (IL-1β, TNF-α, and IL-12) [71]. The anti-IBD activity of 6-gingerol was also confirmed by another reference. It is illustrated that 6-gingerol remarkably suppressed the circulating concentrations of IL-1β and TNF-α, and restored the colonic NO concentration and myeloperoxidase activity to normal in DSS-treated mice. Moreover, 6-gingerol efficiently prevented colonic oxidative damage by increasing the activities of antioxidant enzymes and glutathione level and ameliorated the colonic atrophy [72].

3.2.1.8. Embelin Embelin (41, Fig. 8), a major constituent of Embelia ribes Burm., is a naturally occurring alkyl-substituted hydroxy benzoquinone possessing anti-inflammatory, analgesic and antioxidant activities [73]. In an acetic acid-induced rats colitis model, embelin (25 and 50 mg/kg, p.o.) assessed the colonic mucosal injury by clinical, macroscopic, biochemical and histopathological examinations. Embelin treatment significantly decreased CAS, gross lesion score, percent affected area and wet colon weight. The treatment also potently reduced the colonic myeloperoxidase activity, 19

lipid peroxides and serum lactate dehydrogenase [74]. Moreover, in a DSS-induced mice colitis model, embelin significantly attenuated DAI scores and tissue MPO accumulation. Embelin administration also effectively and dose-dependently prevented the shortening of colon length and enlargement of spleen size. Histological examinations indicated that embelin suppressed edema, mucosal damage, and the loss of crypts induced by DSS. Furthermore, embelin inhibited the abnormal secretions and mRNA expressions of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. These results suggest that embelin has a therapeutic value in the treatment of IBD [75].

3.2.1.9. Epigallocatechin-3-gallate Fresh tea leaves are rich in tea polyphenols known as catechins [7,8]. The principal catechins found in tea are epicatechin (EC), epigallocatechin (EGC), epicatechingallate (ECG) and epigallocatechin-3-gallate (EGCG, 42, Fig. 8) [76]. EGCG is the most abundant catechin in green tea. In vitro and animal studies provide strong evidence that EGCG may possess antioxidant and anti-inflammatory properties to affect the pathogenesis of several chronic diseases. Recently, the effect and mechanism of EGCG treatment in rats with acetic acid-induced colitis were reported. In the acetic acid-induced (intrarectal administration) colitis model, EGCG notably improved the DAI (1.1 ± 0.9), colon mucosa damage index (CMDI, 1.5 ± 0.9) and histological scores (4.6 ± 3.1) compared with the placebo (3.9 ± 0.4, p < 0.01; 3.3 ± 0.6, p < 0.05; 9.3 ± 2.8, p < 0.01). Compared with the placebo and SASP groups, the levels of NO (9.1 ± 5.6 mmol/g), malondialdehyde (MDA), TNF-α, IFN-γ (33.3 ± 0.9 PG/mL), and NF-κBp65 in EGCG-treated group were significantly reduced. In summary, EGCG ameliorates mucosal inflammation by inhibiting the production of TNF-α and NF-κB p65 and may be a potential therapeutic agent in IBD [77].

20

3.2.1.10. Celastrol Celastrol (43, Fig. 8), also known as Thunder of God Vine, is a triterpene from the root bark of the Chinese medicinal plant Tripterygium wilfordii. Celastrol possesses multiple biological and pharmacological activities, including immune modulation, anti-inflammatory and anti-tumor activities [78]. In a DSS-induced colitis mice model, celastrol (1 mg/kg, i.p.) ameliorated the severity of colitis, decreased the level of IL-1β, IL-6 and MPO, and upregulated the level of E-cadherin in colitis mice. Moreover, the tunel staining and cleaved caspase-3 immunohistochemistry staining proved that celastrol treatment decreased necrotic cell death. On the mechanism, decreased level of necroptosis factors RIP3 and MLKL, and increased level of active caspase-8 were detected after celastrol treatment. Taken together, celastrol exerted beneficial effects in colitis treatment via suppressing the RIP3/MLKL necroptosis pathway [79].

3.2.1.11. 15,16-Dihydrotanshinone I 15,16-Dihydrotanshinone I (DHT, 44, Fig. 9) is a natural diterpenoid isolated from Chinese herb Danshen (Salvia miltiorrhiza Bunge). Accumulated studies showed that it has a variety of pharmacological activities, such as anti-bacterial and anti-inflammatory effects [80]. DHT (10 and 25 mg/kg) significantly alleviated DSS-induced body weight loss, DAI scores, and improved histological alterations of colon tissues. DHT inhibited the MPO activity, iNOS and COX-2 expression in colon tissues and decreased serum levels of TNF-α, IL-1β, IL-6, and high-mobility group box 1 (HMGB1). In addition, increased expression of iNOS, COX-2, and phosphorylated RIP1, RIP3, MLKL in response to LPS plus Z-VAD (LZ) were also suppressed by DHT [81].

Fig. 9 Chemical structures of natural products 44-49.

3.2.1.12. Ellagic acid 21

Ellagic acid (EA, 45, Fig. 9) is a naturally occurring plant phenol found in certain fruits, nuts and vegetables including berries and pomegranate. Over the last few years, a number of in vitro and in vivo studies have provided evidence of its important pharmacological properties including antioxidant, anti-inflammatory activities [82]. Oral administration of EA (10–20 mg/kg) diminished the severity and extension of the intestinal injuries induced by TNBS. In addition, EA increased mucus production in goblet cells in colon mucosa, decreased neutrophil infiltration and pro-inflammatory proteins COX-2 and iNOS overexpression. Moreover, EA was capable to reduce the activation of p38, JNK and ERK1/2 MAPKs, preventing the inhibitory protein IκB-degradation. In conclusion, EA reduced the damage in a rat model of CD, alleviated the oxidative events and returned pro-inflammatory proteins expression to basal levels probably through MAPKs and NF-κB signaling pathways [83].

3.2.1.13. Berberine Berberine (BBR, 46, Fig. 9) is an isoquinoline alkaloid extracted from numerous plants of Berberis and Coptis, which are used in China for centuries to treat patients with gastroenteritis, abdominal pain, and diarrhea [84, 85]. Berberine, given orally at 40, 20, 10 mg/kg for 10 days, ameliorated all the supposed inflammatory symptoms of the DSS-induced colitis, such as body weight loss, blood hemoglobin reduction, high myeloperoxidase levels, and malondialdehyde content-inflamed mucosa [86]. Additionally, the investigation showed that comparing to 5-ASA alone, 5-ASA plus berberine more potently ameliorated the severity of DSS-induced disease, colon shortening, and colon histological injury [87]. As a well-evaluated anti-IBD natural product, the protective effects of berberine against the colitis were also confirmed in other publications [88, 89].

3.2.1.14. Baicalin Baicalin (7-glucuronic acid, 5,6-dihydroxyflavone, 47, Fig. 9) is a flavonoid compound originally isolated from the root of the Chinese herb Huangqin (Scutellaria 22

baicalensis Georgi). Baicalin is clinically proven to be safe and is used as an anti-inflammatory traditional Chinese medicine [90]. Pretreatment with baicalin (100 mg/kg) in mice with DSS-induced colitis ameliorated the severity of colitis and significantly decreased the DAI (baicalin group, 3.33± 0.52 vs. DSS group, 5.67±1.03). Baicalin (100 mg/kg) also repressed IRF5 protein expression and promoted IRF4 protein expression in the lamina propria mononuclear cells and induced macrophage polarization to the M2 phenotype. In summary, baicalin alleviated DSS-induced colitis by modulating macrophage polarization to the M2 phenotype [91].

3.2.1.15. Rutin and quercetin Flavonoids are plant secondary metabolites ubiquitously distributed and reported to display anti-oxidative and anti-inflammatory activities in cellular and rodent models. They are also known to be inhibitors of several enzymes activated in certain inflammatory conditions. Rutin (48, Fig. 9), 3-O-rhamnosylglucosyl-quercetin, widely occurs in various foods, including buckwheat, parsley, tomatoes, and apricots, and is one of the most common naturally occurring flavonoids with various biological activities. Rutin and its aglycone, quercetin (3,30,40,5,7-pentahydroxyflavone, 49, Fig. 9), have been proved to exert numerous pharmacological activities including suppression of cellular immune and inflammatory responses [92]. In a DSS-induced colitis model, 0.1% of rutin-fed mice showed marked suppression of shortening by 73% (P < 0.05), whereas the 0.1% of quercetin exhibited only 18% inhibition. As for bodyweight, the model group followed 5% of DSS administration for 1 week, decreased by 9.6 g (P < 0.001) as compared with the control. However, dietary feeding of 0.1% of quercetin and rutin attenuated body weight loss by 21% (P < 0.05) and 54% (P < 0.01), respectively. Moreover, dietary rutin and quercetin even at a low dose, especially rutin, was found to attenuate the production of the critical proinflammatory mediator genes IL-1β, IL-6, GM-CSF, and 23

iNOS. Therefore, rutin may be useful for the prevention and treatment of IBD and colorectal carcinogenesis via attenuation of pro-inflammatory cytokine production [93].

3.2.1.16. Diammonium glycyrrhizinate Diammonium glycyrrhizinate (DG, 50, Fig. 10) is a substance extracted and purified from Glycyrrhiza uralensis Fisch, which is a traditional Chinese medicinal herb used for the treatment of hepatitis due to its anti-inflammatory effect, resistance to biologic oxidation, membranous protection and weak steroidal action. In an acetic acid-induced colitis evaluation, after DG treatment (40 mg/kg), the positive percentage and density of NF-κB, TNF-α and ICAM-1 were reduced significantly (P < 0.01). Both DAI and MPO activity were greatly recovered, indicating that DG might be a promising drug candidate for the treatment of IBD [94].

Fig. 10 Chemical structures of natural products 50 and 51.

3.2.1.17. Methyl protodioscin Dioscoreaceae, a kind of yam plants, is recommended as a treatment for rheumatic conditions, biliary colic, irritable bowel syndrome, diverticulitis and intestinal inflammation. Methyl protodioscin (MPD, 51, Fig. 10) is a member of the furostan saponin family, which broadly exists in Dioscoreaceae plant [95]. In vitro studies showed that MPD significantly increased crypt formation and restored intestinal barrier dysfunction induced by pro-inflammatory cytokines. MPD increased the percentage of survival from high-dose DSS-treated (4%) mice and accelerated mucosal healing and epithelial proliferation in low-dose DSS-treated (2.5%) mice characterized by marked reduction in NF-κB activation, pro-inflammatory cytokines expression and bacterial translocation. Moreover, MPD protected colonic mucosa from C. rodentium-induced colonic inflammation and bacterial colonization [96]. 24

3.2.2. Natural product derivatives with in vivo anti-IBD activities 3.2.2.1. Corticosteroid prodrugs Glucocorticoids are frequently used in the treatment of inflammatory bowel disease. A limitation to their use is that they undergo absorption from the gastrointestinal tract (GIT) before reaching the colon causing severe systemic side effects. To reduce the side effects of corticosteroid, the 21-OH group of corticosteroid is connected to 5-ASA via a primary alkyl ester to afforded a series of corticosteroid prodrugs (Fig. 11). The design sought to exploit the selective reduction of an azo linker in the colon, releasing a latent prodrug that subsequently undergoes lactamization liberating the steroid. Prodrug 52 was an anti-inflammatory agent as effectively as prednisolone in a murine DSS colitis model but did not cause thymic atrophy, a marker for systemic steroid effects. Oral treatment of 52 attenuated weight loss and DAI scores relative to the vehicle-treated group. However, with respect to DSS-induced shortening of the colon, compound 52 was significantly more effective than prednisolone, demonstrating that compound 52 has significant potential in the treatment of IBD with reduced steroid side effects. This design could be applied to target other hydroxyl-bearing therapeutics to the colon [97].

Fig. 11 Designing strategy and the chemical structure of compound 52.

3.2.2.2. Resveratrol prodrugs The naturally occurring polyphenols exert antiplatelet, antioxidant, antitumor, and anti-inflammatory activities. Among polyphenols, resveratrol (1), naturally occurring in grapes and wine, has been reported to ameliorate the inflammation status in mice and rats IBD models [98]. However, like other phenolics, resveratrol is rapidly absorbed and conjugated by phase II enzymes to yield mostly sulfate and glucuronate derivatives, which reduces resveratrol delivery to the colon and decreases its topical 25

effectiveness. A series of resveratrol prodrugs were designed and prepared based on the hypothesized that increasing the delivery of resveratrol to the colon could improve its anti-inflammatory effects in the colon mucosa (Fig. 12). These prodrugs prevented the rapid metabolism of resveratrol and delivered higher quantities of resveratrol to the colon and they reduced mucosal barrier imbalance and prevented diarrhea. Mice in the control group had an average colon length of 8.9 cm, which was reduced to 6.3 cm in the DSS group. The colon length shortening was significantly prevented in the groups treated with compounds 53 or 54, which had colon lengths of 7.7 and 8.2 cm, respectively. MPO activity was induced 30-fold in the mice colon mucosa upon DSS administration, but this increase was only significantly reduced upon pretreatments with compounds 53 (3.7-fold) and 54 (7.2-fold) [99].

Fig. 12 Designing strategy and the chemical structure of compounds 53 and 54.

3.2.2.3. Andrographolide hybrid Andrographolide sulfonate, approved as an anti-inflammatory drug in China for years, has shown significant activity in TNBS-induced colitis in mice [100]. Likewise, α-lipoic acid (LA) has been identified as a potential remedy of IBD [101]. To obtain compounds with more potent anti-IBD activity, the andrographlide-lipoic acid hybrid (55) was synthesized (Fig. 13). Compound 55 treatment led to obvious reductions in DAI, macroscopic score and CMDI associated with TNBS administration. Compound 55 inhibited the inflammatory response via lowering the level of inflammatory cytokines and MPO activity. 55 also attenuated the expression of p-p65, p-IκBα and COX-2 in the colitis mice. The alleviation of colon injury by 55 treatment was also evidenced by the increased expression of PPAR-γ. These results indicated that compound 55 showed the potential effect in the treatment of IBD [102].

Fig. 13 Designing strategy and the chemical structure of compound 55. 26

4. Summary and future directions As a chronic inflammatory disease, the number of people suffering from IBD is huge and still increasing. Although IBD is not a direct lethal disease, long-term and recurrent episodes cause great pain to the patients, and may also have the risk of cancer and other diseases. Clinically, there is a strong demand for novel drugs with better efficacy and lower side effects for the effective treatment of IBD. This review summarized the recent anti-IBD candidate compounds in the drug discovery phase. Their related targets include NF-κB, TNF-α, LTB4, IKK2, CCR9, CXCR4, PPAR-γ, NAE, LANCL2, NLRP3, CB2, etc. Researchers and patients may raise a question: since there are plenty of potential targets for the treatment of IBD and many drug candidates have been developed based on the targets above, what underlies and accounts for the lack of new effective treatments? In fact, the answer may be implied in the confusion itself. The numerous targets related to IBD obviously illustrate the complexity of the deep pathogenesis and mechanism of IBD. Although the disease can be effectively relieved, it is difficult to cure or control the conditions of IBD by acting only single target or one part of its pathways. Therefore, multi-drug combination, or design and discovery of multi-target drugs blocking the multiple signaling pathways triggering IBD, will be a more promising strategy for the management of IBD symptoms and achieving desired therapeutic effects. In terms of natural products possessing in vivo anti-IBD activities, they exhibit promising efficacy in animal models and are hopeful sources of novel IBD therapeutic agents [64, 73]. Although natural products are probable to face the issues of low content, the difficulty of extraction and separation, low efficacy and unclear mechanism, they can be taken as effective lead compounds to provide more choices and starting points for the development of anti-IBD drugs.

Acknowledgments This project was supported by the National Natural Science Foundation of China, 27

NSFC (Grant No. 81803340 and 21576239).

Abbreviations AMPK, Adenosine Monophosphate-activated Protein Kinase; ASA, Aminosalicylic Acid; 4-ASA, 4-Aminosalicylic Acid; 5-ASA, 5-Aminosalicylic Acid; 4-ASA-Glu, 4-Aminosalicylic Acid-β-O-Glucoside; BBR, Berberine; CAS, Clinical Activity Score; CB, Cannabinoid; CCR9, Chemokine receptor 9; CCL25, Chemokine (C-C motif) Ligand 25; CD, Crohn’s Disease; CDAI, CD Activity Index; CMDI, Colon Mucosa Damage Index; CNS, Central Nervous System; COX-2, Cyclooxygenase 2; CRP, C-Reactive

Protein;

CRL,

Cullin/RING

Ubiquitin

Ligase;

5-CSPA,

(5-Chlorosalicyloyl)phenethylamine; CXCL12, Chemokine (C‑X‑C motif) Ligand 12; CXCR4, Chemokine (C‑X‑C motif) Receptor 4; DAI, Disease Activity Index; DATS,

Diallyl

Trisulfide;

DG,

Diammonium

Glycyrrhizinate;

DHT,

15,16-Dihydrotanshinone I; DNBS, 2,4-Dinitrobenzenesulfonic Acid; DSS, Dextran Sulfate Sodium; EA, Ellagic Acid; EC, Epicatechin; ECG, Epicatechingallate; EGC, Epigallocatechin; EGCG, Epigallocatechin-3-gallate;

GIT, Gastrointestinal Tract;

HMGB1, High-Mobility Group Box 1; HP, 3-Hydroxy-4-pyridinecarboxylic Acid; IBD, Inflammatory Bowel Disease; iNOS, inducible Nitric Oxide Synthase; LA, α-Lipoic Acid; LANCL2, Lanthionine Synthetase C-Like 2; 5-LOX, 5-Lipoxygenase; LTB4, Leukotriene B4; LZ, LPS plus Z-VAD; MAPKs, Mitogen-Activated Protein Kinases;

MDA,

Malondialdehyde;

MPO,

Myeloperoxidase;

MPD,

Methyl

Protodioscin; NAE, NEDD8-Activating Enzyme; NALP3, NACHT, LRR and PYD Domains-containing Protein 3; NF-κB, Nuclear Factor kappa-light-chain-enhancer of activated B cells; OSCs, Organosulfur Compounds; PK, Pharmacokinetic; PPAR, Peroxisome Proliferator-Activated Receptor; PTSO, Propyl-Propane Thiosulfonate; SA, Salicylic Acid; SAR, Structure-Activity Relationship; STAT3, Signal Transducer and Activator of Transcription 3; TNBS, 2,4,6-Trinitrobenzene Sulphonic Acid; TNF-α, Tumor Necrosis Factor-α; UC, Ulcerative Colitis;

28

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40

Table 1. General comparison of the main characteristics of CD and UC.

Ulcerative Colitis (UC)

Clinical features

Incidence sites of the disease Distribution of disease Intestinal complications

Characteristics of internal symptoms

Fever; Diarrhea; Rectal bleeding; Weight loss; Signs of malnutrition

Limited to the colon Continuous distribution

Crohn’s Disease (CD) Fever; Diarrhea; Abdominal pain; Rectal bleeding; Weight loss; Signs of malnutrition; Perianal disease; Abdominal mass; Growth failure in children & adolescents Throughout the GI tract (Colon and ileum are common sites) Discontinuous distribution (skip lesions)

Cancer

Stricture; Fistulas; Cancer

Superficial ulceration; Friability; Affecting the mucosa and submucosa.

Deep ulceration with submucosal extension; Friability; Cobblestone appearance; Affects all layers including the muscularis propria (transmural).

Fig. 1 Apparent and visual pathological features of IBD.

Fig. 2 a. The structure of compound 5-CSPA (1); b. Designing strategy and the chemical structures of compounds 4AP (2) and 4AT (3); c. The structure of compound 4-ASA-Glu (4).

Fig. 3 Designing strategy and chemical structures of compounds SP (5), TS (6) and ST (7).

Fig. 4 Chemical structures of compounds 8-15.

Fig. 5 The chemical structures of compounds 16-23.

Fig. 6 The chemical structures of compounds 24-29.

Fig. 7 The chemical structures of compounds 30-33.

Fig. 8 The chemical structures of natural products 34-43.

Fig. 9 The chemical structures of natural products 44-49.

Fig. 10 Chemical structures of natural products 50 and 51.

Fig. 11 Designing strategy and the chemical structure of compound 52.

Fig. 12 Designing strategy and the chemical structure of compounds 53 and 54.

Fig. 13 Designing strategy and the chemical structure of compound 55.

Fig. 14 Parts of the plenty of potential targets involved in IBD.

Highlights: There is a strong demand for novel and safe drugs for the treatment of IBD; General characteristics and pathological features of IBD were reviewed; Potential anti-IBD targets were summarized; Anti-IBD small-molecules, natural products and prodrugs were reviewed.

Declaration of interests ☐ √The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: