Research advances in anti-cancer activities and mechanism of action of neocryptolepine and its derivatives

Abstract

Cancer has been a severe public health and social problem, a leading disease that has diminished the quality of life, and a barrier to improving life expectancy. Neocryptolepine is an indole-quinoline alkaloid isolated from Cryptolepis sanguinolenta which grows in some African countries. This review summarizes the structures of 228 neocryptolepine derivatives, including 84 neocryptolepine derivatives synthesized by our laboratory, and analyzed the cytotoxic effects and mechanism of action at the cellular level. Neocryptolepine derivatives 43, 65, 93, and 96 have good cytotoxicity against gastric cancer AGS cells and the IC₅₀ value reached 43 nM, 148 nM, 2.9 μM, and 4.5 μM, respectively. The IC₅₀ values of compounds 64 and 69 on colorectal cancer HCT116 cells reached 0.33 and 0.35 μM, respectively. In addition, the structure-activity relationship of these compounds is discussed in this review. Topoisomerase II is discussed as a possible inhibition target of neocryptolepine derivatives in several cancer cell lines by binding DNA. The structures of the reported neocryptolepine derivatives and the possible cytotoxic mechanisms are analyzed. This review provides a fundamental reference for anticancer drug development of neocryptolepine and its derivatives as anti-tumor agents.

Main article text

1. INTRODUCTION

The emergence and spread of cancer are considered major threats to global public health by the World Health Organization. Cancer is a non-communicable disease and one of the leading causes of human mortality [1, 2]. The International Agency for Research on Cancer (IARC) reported that the estimated number of cancer cases worldwide in 2018 has risen to 18.1 million new cases, resulting in 9.6 million deaths [3, 4]. In addition, due to the high contribution of Asia to the global population, nearly one-half of the estimated cases and greater than one-half of cancer deaths in men and women occur in Asia. Greater than 60% of cancers occur in low- and middle-income countries and approximately 70% of cancer deaths occur in these countries [5]. The traditional cancer treatment modalitiess include surgery, radiation, and chemotherapy. The negative side effects associated with these treatment options, such as high cost and toxicity, increase the demand for the development of less toxic and less expensive anticancer drugs from natural sources [6]. Because the cancer death rates are rising in developing and industrialized countries, there is an urgent need to explore novel anticancer drugs. Moreover, there is a need to develop new anticancer drugs to treat cancer and generate interest in the pharmaceutical industry [7].

Cancer cells have the ability of uncontrolled proliferation and can migrate to other tissues and organs [8]. Although research and development of anticancer drugs have made significant advances in recent decades, anti-cancer therapy still has challenges. Chemotherapy, radiotherapy, and surgery are important therapeutic methods for cancer patients [9, 10]. Small molecular chemical drugs have many advantages compared to biological drugs and antibodies, including low cost, more forms of administration, and good patient compliance [11]. However, most clinical anticancer drugs are expensive and toxic, and the side effects of chemical drugs cannot be ignored. Although chemotherapy drugs clearly improve overall survival among cancer patients, treatment can be painful. Therefore, it is essential to find drugs with highly effective and low toxicity for cancer treatment.

Natural products have a role in the development of anticancer drugs, which are a significant source of bioactive molecules in anti-cancer drugs [12]. There were 321 anti-cancer drugs approved between 1946 and 2019, among which greater than 30% belong to natural products or their derivatives [13]. There are many anti-cancer drugs that originated from natural products, such as taxol [14], camptothecin [15], vincristine [16], topotecan [17], and etoposide [18] ( Figure 1 ), that have been shown to have good inhibitory effects on cancer. Neocryptolepine, an indoquinoline alkaloid, is derived from Cryptolepis sanguinolenta, which grows in some African countries [19]. Neocryptolepine is widely used in traditional medicine in many Central and Western countries [20–22]. In fact, cryptolepine, isocryptolepine, and neocryptolepine ( Figure 1 ) are the main alkaloids isolated from the roots of C. sanguinolenta. They are mainly used for the treatment of hypertension and fever [23] and have anti-muscarinic [24], anti-bacterial [25], anti-fungal [26], and anti-inflammatory effects [24]. Therefore, neocryptolepine and its derivatives are promising resources for the development of anti-cancer drugs.

Figure 1 |

Chemical structures of FDA-approved anti-cancer drugs (taxol, camptothecin, vincristine, topotecan, and etoposide), cryptolepine, isocryptolepine, and neocryptolepine.

To develop anti-cancer drugs with high efficacy and low toxicity, the cytotoxicity and mechanism of action underlying a series of neocryptolepine derivatives were reviewed in different cancer cells, such as colorectal, gastric, liver, lung, ovarian, breast, and cervical cancer, Ehrlich ascites carcinoma (EAC), and melanoma. Although many studies have reported that neocryptolepine derivatives exert good cytotoxicity, a systematic summary of structure-activity relationships and the mechanism of action for neocryptolepine and its derivatives are lacking. The cytotoxic mechanism underlying neocryptolepine and its derivatives are analyzed in this review ( Figure 2A and 2B ) and the neocryptolepine derivatives structures modified with the neocryptolepine parent nucleus at A, B, C, and D are discussed ( Figure 2B ). This review lays the foundation to develop anticancer drugs with high efficacy and low toxicity from neocryptolepine and its derivatives and provides ideas for developing novel anti-cancer drugs by structure modification based on the structure of neocryptolepine.

Figure 2 |

The cytotoxic effects and potential mechanism of action of neocryptolepine and its derivatives (Figure A was drawn by Figdraw, ID: WSOSO688f1).

2. NATURAL PRODUCTS AS ANTICANCER AGENTS

Cancer is one of the major diseases that threatens human life and health, so the isolation and synthesis of compounds with antitumor activity are areas of intense research. Derivatization of the skeleton structure of some natural products is a common method by which to obtain active compounds. Studies have shown that plants are natural sources of novel drugs with unique medicinal activities [27–31]. Natural products based on plants and their derivatives have been widely used as sources for the development of anticancer agents and have been extensively documented for the treatment of various diseases. Alkaloids, which are nitrogen-containing compounds in plants, fungi, and bacteria, are among the most important natural products [32–35]. Most alkaloids are found in vascular plants and a few alkaloids occur in lower plants [35]. The chemical structures of alkaloids in the same plant are similar. These alkaloids are mainly categorized as follows: pyridines; terpenoids; indoles; steroids; and isoquinolines. Many kinds of alkaloids have been shown to have anti-cancer activity [36]. With further research involving alkaloids, more novel alkaloids with anti-cancer activity have been identified.

2.1 Indole-quinoline alkaloids

Indole-quinoline alkaloids are tetracyclic heterocycles formed by the fusion of indoles and quinoline. Cryptolepine is the natural product of indole-quinoline alkaloids, which were isolated in 1929. The exact chemical structure of cryptolepine was assigned in 1951 as a linearly-fused 5-methyl-10H-indolo [3,2-b]quinoline [37]. Cryptolepine has been investigated as a promising anti-malarial and anti-cancer agent [38]. In fact, indole-quinoline alkaloids have potential as anti-cancer drugs and it has been reported that quinoline and indole-quinoline alkaloids generally have good anti-bacterial and cytotoxic activities [39, 40]. However, different studies have shown that the toxicity of cryptolepine cannot be ignored, which hinders further progress.

2.2 Neocryptolepine and its derivatives

Neocryptolepine is a polycyclic quinoline compound and has cytotoxic effects in different cancer cells, including liver cancer, cholangiocarcinoma, lung cancer, EAC, and leukemia. Neocryptolepine causes cell cycle arrest, induces apoptosis, and has a good anti-oxidant effect [7, 41, 42]. Some neocryptolepine derivatives (9 and 10; Figure 3 ) have strong cytotoxicity against the A549 cell line with IC₅₀ of 0.197 and 0.1988 μM, respectively, and IC₅₀ of 0.138 and 0.117 μM against BALB/3T3 cells, respectively [43].

Figure 3 |

The chemical structures of neocryptolepine derivatives, indoquinazoline alkaloids, and indole alkaloids [43–45].

2.3 Other alkaloids

Nine novel indoquinazoline alkaloids (11-19; Figure 3 ) were isolated and characterized from Euodiae fructus and shown to have significant cytotoxicity against HL-60 human promyelocyte leukemia and N-87 human gastric cancer cells [44]. Some synthetic indole derivatives have been confirmed to have promising anti-cancer activities against various human cancer cell lines, such as colorectal cancer HCT116 cells, breast cancer MCF-7 cells, and cervical cancer Hela cells [44]. An indole alkaloid (20; Figure 3 ) isolated from Tabernaemontana catharinensis showed significant cytotoxic activity against laryngeal carcinoma Hep-2 cells (IC₅₀ = 54.47 μg/mL) [45]. Indole-quinoline alkaloids are considered promising frameworks for the development of anti-cancer drugs, which can be further developed into effective anti-cancer drugs [46, 47]. Neocryptolepine and its derivatives also have great potential as cytotoxic agents. This review was mainly focused on the advances in anti-cancer effects of neocryptolepine and its derivatives in different cancer lines by inhibiting proliferation, migration, inducing apoptosis, and arresting cell cycle. A summary of anti-cancer effects and the mechanism of action of neocryptolepine derivatives in various cancer lines is presented.

3. NEOCRYTOLEPINE AND NEOCRYTOLEPINE DERIVATIVES AS ANTI-CANCER AGENTS

3.1 Anti-cancer activity of neocrytolepine and neocrytolepine derivatives against gastric cancer

Alkaloids as the main anti-tumor active substances undergoing broad development and research. The application of alkaloid anti-cancer drugs will reduce the toxicity and side effects of chemotherapeutic drugs and improve the efficacy of anti-tumor activity. To help the preliminary screening of alkaloids of compounds with anti-tumor activity for future studies, we developed a database of alkaloids with anti-tumor activity [DAAA] (http://www.gsbios.com). This database analyzed and extracted the relevant literature and integrated research data and results. This database contains the name, structure, molecular weight, molecular formula, activity, toxicity, patents, and corresponding references of alkaloids. Furthermore, this database contains many neocryptoleepine derivatives and will be used to develop promising compounds with anti-tumor activity.

In our previous work we designed 84 neocryptolepine derivatives ( Figure 4A, B ) and evaluated the cytotoxic activity against gastric cancer AGS cells [19, 48, 49]. Some neocryptolepine derivarives showed good cytoxixc effects against AGS cells. Neocryptolepine derivative 43 had excellent cytoxicity. Specifically, the IC₅₀ of compound 43 was 43 nM for 48 h in AGS cells. In addition, the IC₅₀ of compounds 65, 93, and 96 reached 148 nM, 2.9 μM, and 4.5 μM, respectively. We also assessed the cytotoxicity of neocryptolepine derivatives in liver cancer SMMC7721 cells, colorectal cancer HCT116 cells, and pancreatic cancer PANC-1 cells. The IC₅₀ of neocryptolepine derivatives in these cancer cells are listed in Table 1 . As seen in Table 1 and Figure 5 , in the A-ring substitutions of neocryptolepine, 2-halogenation (compounds 24, 25, and 26) showed better cytotoxicity than other positions (compounds 29, 30, 31, 33, and 35). 4-methyl substitution (compound 32) had better cytotoxic activity than 3- or 2-methyl substitution (compounds 22 and 27). Moreover, 2-methoxy (compound 23) substitution had better cytoxic effects than the 3-methoxy (compound 28). The 9-Cl substitution (compound 39) is better than the 7-, 8-, and 10-Cl substitution (compounds 44, 49 and 52) when the C1 atom in neocryptolepine is substituted. The 10-methyl substitution (compound 46) has better cytotoxicity than the 7-methyl substitution (compound 51) when the substituent group is methyl. When the 2-position substituent group is methoxy (compounds 54-59), 8-F, 8-methyl, and 8-methoxy (compounds 55, 58, 59) have better cytotoxicity than 8-Cl (compound 56) and 8-Br (compound 57) in the A- and D-ring substitutions. When the substituent group is methyl (compounds 60-64), 8-methyl (compound 63) and 8-methoxy (compound 64) have the best cytotoxic effects, and 8-F (compound 60) and 8-Cl (compound 61) are better than 8-Br (compound 62). When the substituent group is the Cl atom (compound 65-69), 8-Br (compound 67) has better cytotoxicity than 8-Cl (compound 66) and 8-methyl (compound 68). These results suggest that neocryptolepine derivatives have good cytotoxicity in AGS cells by regulating the PI3K/AKT/mTOR signaling pathway. Moreover, neocryptolepine derivatives induce cell apoptosis, arrest the cell cycle at G2/M, and inhibit the migration of cancer cells. The identify and verification of the neocryptolepine derivative targets are important and warrant further study.

Figure 4 |

The structure of neocryptolepine derivatives and analogues [26, 48].

Table 1 |

Anti-proliferative effects (IC₅₀, μM) of neocryptolepine and its derivatives (21-105) and CIS on AGS, SMMC7721, HCT116, and PANC-1 cells for 48 h.

Compounds	SMMC7721	AGS	PANC-1	HCT116	Compounds	SMMC7721	AGS	PANC-1	HCT116
21	27.00 ± 7.10	20.00 ± 0.77	>50	6.30 ± 1.20	64	9.70 ± 2.00	3.60 ± 2.50	18.40 ± 3.00	0.30 ± 0.00
22	24.00 ± 0.00	27.00 ± 7.40	>50	10.10 ± 0.60	65	24.00 ± 0.00	0.15 ± 0.006	>50	27.60 ± 16.30
23	19.00 ± 5.60	14.00 ± 8.30	37.60 ± 0.00	12.50 ± 5.60	66	23.00 ± 0.00	7.10 ± 3.30	37.50 ± 11.80	22.70 ± 0.00
24	10.00 ± 5.50	4.00 ± 0.60	14.00 ± 0.10	3.70 ± 2.40	67	19.00 ± 0.00	2.80 ± 3.60	27.10 ± 0.00	7.10 ± 0.00
25	12.00 ± 6.10	3.30 ± 0.50	8.90 ± 1.60	1.80 ± 0.90	68	26.00 ± 24.00	7.70 ± 5.70	45.80 ± 0.00	7.500 ± 5.30
26	19.00 ± 16.00	5.00 ± 2.50	20.10 ± 5.80	7.60 ± 4.50	69	13.00 ± 0.00	27.00 ± 38.00	>50	0.40 ± 0.00
27	25.00 ± 0.80	14.00 ± 1.60	35.40 ± 13.10	11.40 ± 2.60	70	>50	>50	>50	>50
28	>50	41.00 ± 0.00	>50	25.70 ± 11.20	71	>50	15.00 ± 6.70	24.00 ± 0.00	12.50 ± 3.10
29	22.00 ± 6.80	7.10 ± 0.20	>50	17.70 ± 13.50	72	>50	26.00 ± 0.70	57.70 ± 0.00	49.60 ± 20.60
30	>50	>50	>50	>50	73	>50	13.00 ± 7.0	26.80 ± 4.30	ND
31	37.00 ± 0.00	36.00 ± 0.00	>50	20.50 ± 9.80	74	>50	8.90 ± 0.60	22.60 ± 8.40	20.30 ± 4.40
32	13.00 ± 8.60	3.10 ± 0.30	10.50 ± 5.40	7.20 ± 7.30	75	>50	30.00 ± 5.60	>50	30.70 ± 5.10
33	15.00 ± 8.70	6.00 ± 0.80	25.00 ± 0.10	6.30 ± 3.70	76	>50	28.00 ± 0.00	42.30 ± 0.00	26.00 ± 11.20
34	17.00 ± 0.00	8.60 ± 1.00	>50	22.30 ± 16.80	77	>50	>50	>50	35.50 ± 8.60
35	17.00 ± 0.00	14.00 ± 1.40	35.40 ± 4.20	11.70 ± 0.00	78	>50	9.30 ± 0.60	25.60 ± 0.00	19.80 ± 2.40
36	18.00 ± 11.00	8.10 ± 3.00	18.10 ± 0.00	6.30 ± 4.00	79	>50	14.00 ± 4.70	22.90 ± 0.00	>50
37	28.00 ± 19.00	7.90 ± 0.60	6.90 ± 0.00	10.50 ± 10.50	80	39.00 ± 130	4.50 ± 0.60	38.50 ± 0.00	160.20 ± 108.50
38	28.00 ± 0.00	38.00 ± 14.00	>50	16.50 ± 9.00	81	23.00 ± 1.90	1.50 ± 1.40	>50	20.70 ± 7.30
39	22.00 ± 0.00	18.00 ± 0.00	>50	11.50 ± 0.00	82	15.00 ± 6.20	2.20 ± 0.30	3.30 ± 0.00	7.60 ± 0.30
40	22.00 ± 2.80	17.50 ± 0.00	>50	11.50 ± 0.00	83	27.00 ± 5.80	3.50 ± 0.70	4.90 ± 0.00	13.00 ± 3.10
41	16.00 ± 10.00	3.10 ± 0.80	>50	6.10 ± 3.00	84	29.00 ± 7.60	26.00 ± 18.00	19.70 ± 0.00	8.00 ± 4.10
42	13.00 ± 1.30	11.00 ± 9.40	31.10 ± 21.50	7.60 ± 4.80	85	15.00 ± 1.20	3.30 ± 1.60	13.30 ± 3.40	7.20 ± 0.40
43	52.00 ± 45.00	0.043 ± 0.0039	>50	17.70 ± 0.00	86	38.00 ± 12.00	7.00 ± 0.60	13.80 ± 0.00	15.00 ± 3.40
44	27.00 ± 0.00	>50	>50	37.60 ± 0.00	87	>50	9.90 ± 0.00	>50	35.60 ± 14.30
45	40.00 ± 0.00	>50	>50	45.90 ± 0.00	88	41.00 ± 9.20	16.00 ± 0.00	>50	12.70 ± 0.00
46	12.00 ± 1.20	4.60 ± 0.30	16.60 ± 5.00	7.20 ± 5.60	89	>50	5.10 ± 0.00	>50	ND
47	16.00 ± 2.40	8.40 ± 2.70	35.30 ± 3.00	9.40 ± 4.70	90	>50	7.30 ± 4.40	23.00 ± 16.30	2.90 ± 0.80
48	36.00 ± 0.00	12.00 ± 7.70	>50	42.40 ± 0.00	91	>50	>50	>50	>50
49	38.00 ± 0.00	>50	>50	>50	92	>50	4.20 ± 3.10	4.30 ± 0.00	1.00 ± 0.60
50	30.00 ± 4.80	15.00 ± 0.00	19.90 ± 0.00	40.80 ± 0.00	93	40.00 ± 18.00	2.90 ± 0.10	3.90 ± 0.10	1.90 ± 2.50
51	22.00 ± 12.00	7.80 ± 1.40	42.00 ± 0.00	9.80 ± 2.50	94	>50	>50	>50	>50
52	43.00 ± 10.00	22.00 ± 0.00	>50	>50	95	>50	4.70 ± 0.50	4.40 ± 0.80	2.20 ± 2.40
53	>50	41.00 ± 0.00	>50	>50	96	12.00 ± 0.51	4.50 ± 2.50	3.60 ± 0.90	2.80 ± 0.00
54	2.70 ± 0.00	3.00 ± 0.10	3.80 ± 0.00	1.60 ± 0.20	97	>50	5.70 ± 1.20	8.50 ± 0.00	8.30 ± 0.60
55	13.00 ± 1.10	6.50 ± 1.40	35.60 ± 0.00	6.70 ± 0.30	98	>50	6.40 ± 0.00	15.80 ± 1.50	5.80 ± 2.80
56	15.00 ± 2.40	13.00 ± 3.00	>50	6.60 ± 0.90	99	>50	15.00 ± 0.00	>50	ND
57	19.00 ± 2.70	21.00 ± 12.00	>50	4.10 ± 2.70	100	>50	3.90 ± 0.00	20.10 ± 0.00	27.30 ± 10.70
58	4.60 ± 2.40	2.30 ± 0.80	5.60 ± 0.00	2.10 ± 2.20	101	>50	4.70 ± 0.00	29.90 ± 5.70	16.80 ± 3.80
59	6.50 ± 5.50	2.70 ± 1.50	9.10 ± 1.90	2.90 ± 3.40	102	>50	10.40 ± 2.00	22.00 ± 0.00	3.70 ± 2.00
60	11.00 ± 2.80	4.80 ± 2.20	>50	8.70 ± 0.00	103	>50	>50	>50	>50
61	15.00 ± 7.20	5.00 ± 2.70	26.30 ± 3.30	4.90 ± 3.50	104	>50	>50	>50	ND
62	21.00 ± 13.00	16.00 ± 2.30	>50	9.20 ± 7.40	105	>50	>50	>50	ND
63	8.00 ± 0.50	3.00 ± 1.40	12.90 ± 2.00	2.90 ± 0.90	CIS	>50	15.00 ± 1.40	>50	7.80 ± 1.90

ND, not detected.

Figure 5 |

The structure-activity relationship analysis of neocryptolepine derivatives.

3.2 Anti-cancer activity of neocrytolepine and neocrytolepine derivatives against colorectal cancer

Several studies have attempted to synthesize a variety of neocryptolepine derivatives to find leading compounds with superior anti-colorectal cancer efficacy. Boddupally et al. [50] synthesized a series of 11-substituted derivatives of cryptolepine in 2012. Compound 107 ( Figure 6A ) had the strongest anti-cancer activity with IC₅₀ of 0.97 μM against colorectal cancer HCT116 cells and 2.33 μM against Raji lymphoma cells in in vitro cytotoxicity tests. Compound 107 concurrently demonstrated a significant suppression of c-MYC expression [50]. Wang et al. [51] synthesized a series of 11-amino-substituted 5H- and 6H-indolo[2,3-b]quinolines, the anti-proliferative effects of which were assessed in human lung cancer A549 cells and colorectal cancer HCT116 cells. Compound 109 ( Figure 6B ) has good anti-proliferative activities against HCT116 cells (IC₅₀ = 0.195 ± 0.044 μM) and lower toxicity [51]. Wang et al. [51] reported that the 11-chloroneocryptolepines are prone to undergo addition-elimination reactions of the amine nucleophiles of the linear primary amines at 70–130 °C, giving compound 109 good-to-excellent yields. Sidoryk et al. [56] synthesized a series of neocryptolepine derivatives that have an amino acid or a dipeptide at position C-9 and assessed anti-cancer activity in vitro and in vivo. The anti-proliferative effects of 5H-indolo[2,3b]quinoline against Lovo cells was demonstrated by its amino acid and peptide derivatives and cytotoxicity was as high as 0.20 ± 0.40 μM.

Figure 6 |

The synthetic route and structures of compounds 106-123 [50–55].

Wang et al. [52] tested the 84-neocryptolepine and 44-isocryptolepine derivatives and found that the −NH(CH₂)₃NH₂ side chain substituents were the most powerful and potent compounds. Compound 108 ( Figure 6A ) exerts good cytotoxicity with an IC₅₀ of 0.117 μM against HCT116 cells. New anti-proliferative neocryptolepine derivatives 113-118 were effectively synthesized based on neocryptolepine and were explained using spectroscopic techniques in the presence of lithium perchlorate as a Lewis acid catalyst [53]. Compound 118 had the best cytotoxicity against the HCT116 cells and the IC₅₀ was 2.4 μM compared to the reference drug, doxorubicin (IC₅₀ = 10.90 μM). Håheim [54] presented modifications to previous synthetic strategies, which allowed for the realization of novel tetracyclic ring-systems (compounds 119-123; Figure 6F ) along with the N-alkylation of several compounds to furnish novel analogues. Both parent alkaloids of neocryptolepine (compound 8) and isocryptolepine (compound 7) performed good cytotoxicity against HCT116 cells (compound 8: IC₅₀ = 6.22 μM; compound 7: IC₅₀ = 0.67 μM).

A number of studies have been conducted to investigate the anti-colorectal cancer bioactivity of the coumarin compound, 3,3′-(3,4-dichlorobenzylidene)-bis-(4-hydroxycoumarin), which is termed DCH and its derivatives. Matsui et al. [57] showed that cryptolepine induced cell cycle arrest in MG63 cells through the p53-independent activation of p21^WAF1/CIP1 and the activator is mediated through the specific Sp1 site in the promoter region. These findings suggested that cryptolepine arrested the growth of MG63 cells by activating the p21^WAF1/CIP1 promoter through the specific Sp1 site in a p53-independent manner. The unregulated wingless integrated type-1 (WNT)/β-catenin signaling pathway is the cause of >90% of colorectal cancers. Quarshie et al. [58] showed that cryptolepine inhibits WNT3a, a WNT activator-mediated activation of the WNT/β-catenin signaling pathway, which inhibits the proliferation, stemness, and metastasis of colorectal cancer cells. Cryptolepine (compound 6) had excellent anti-cancer effects in colorectal adenocarcinoma epithelial DLD1 cells and colorectal cancer COLO205 cells with IC₅₀ of 2.45 and 1.16 μM after 48 h, respectively. Cryptolepine also reduces WNT3a-induced OCT4 and CD133 expression and suppresses colony formation of the cells. Cryptolepine suppresses cell stemness. Nagy et al. [55] evaluated the cytotoxic activity of 11(4-aminophenylamino) neocryptolepine [APAN] (compound 110; Figure 6C ) in HCT116 cells. Treatment of compound 110 caused cytotoxicity in HepG2 and HCT116 cells with IC₅₀ values of 2.60 and 1.82 μg/mL, respectively. Compound 110 caused damage and severe morphologic alterations in the cells, such as membrane blebbing, cytoplasmic condensation, shrinking of the nucleus with increased condensed chromatin, and removal of microvilli [55].

Abd Elrahman et al. [59] evaluated the cytotoxic activity of 11-(1,4-bisaminopropylpiperazinyl)5-methyl-5H-indolo[2,3-b] quinoline [BAPPN] (compound 112; Figure 6D ). Compound 112 had cytotoxic activity against colorectal cancer cells (IC₅₀ = 23.00 μg/mL) by upregulation of apoptotic proteins (caspase-3 and p53) and downregulation of proliferative proteins (VEGF, PCNA, and Ki67) [59]. In addition, compound 112 induced cell injury and morphologic changes in ultracellular structure, including cellular delayed activity, vanishing of membrane blebbing, microvilli, cytoplasmic condensation, and shrunken nuclei with more condensed chromatin autophagosomes.

In our previous studies, 8-methoxy-2,5-dimethyl-5H-indolo[2,3-b] quinoline (compound 64) was tested for cytotoxicity in colorectal cells. The results showed that compound 64 inhibited the growth of HCT116 and Caco-2 cells, arrested the cell cycle in the G2/M phase, reduced the mitochondrial membrane potential, and induced apoptosis. In addition, western blot analysis suggested that compound 64 may inhibit the proliferatiion of colorectal cancer cells by reglulating the PI3K/AKT/mTOR signaling pathway [60].

3.3 Anti-cancer activity of neocrytolepine and neocrytolepine derivatives against ovarian cancer

Ovarian cancer (OC) is the eighth most common cancer in women worldwide, accounting for 3.7% of all cancers and 4.7% of all cancer deaths [61]. In the early 2000s, the incidence of OC was highest in North America and northern Europe. OC can be subdivided into at least five different histologic subtypes with different identifiable risk factors, origin cells, molecular composition [62], clinical features, and treatment methods. OC is a global problem that is typically diagnosed in the late stages and lacks effective screening strategies [63].

Neocryptolepine and its derivatives have been shown to have biological activity and promising application in the clinical setting [26]. The antitumor and anti-fungal activities suggest that neocryptolepine and its derivatives could be potential anti-cancer drug candidates [26, 49]. It has been shown that neocryptolepine derivatives exhibit significantly anti-proliferative and pro-apoptotic effects in OC cells and high selectivity towards normal cells [22].

One study found that a neocryptolepine derivative (compound 134; Figure 7C ) had good cytotoxic activity against OC cells and the IC₅₀ value of compound 134 against SKOV3 cells was 4.60 ± 0.10 μM [64]. Overall, neocryptolepine derivatives have shown potential as promising leading compounds for the treatment of OC [7]. Neocryptolepine derivatives have a significant inhibitory effect on OC cells by cytotoxicity or induction of cell apoptosis. Further research and evaluation are needed for the clinical application of neocryptolepine derivatives, including studies on pharmacokinetics, toxicology, and clinical efficacy. In summary, studies involving the treatment of OC have made progress but there are still many challenges. An anti-cancer study of neocryptolepine and its derivatives may provide novel ideas for the development of anti-OC drugs in the future.

Figure 7 |

The synthesis and structure of neocryptolepine derivatives 124-144 [42, 46, 56, 64].

3.4 Anti-cancer activity of neocrytolepine and neocrytolepine derivatives against liver cancer

Liver cancer is the fourth most common cancer globally. The occurrence of liver cancer is mainly concentrated in cases of cirrhosis, hepatitis B or C virus infection, or non-alcoholic steatohepatitis. The potential liver disease also limits the therapeutic effect [65]. Hepatocellular carcinoma (HCC) is the most common primary liver cancer, accounting for 85–90% of all cases. Since 1980 the global incidence of liver cancer has tripled and the growth rate of cancer for men and women is faster than that of any other cancer [66]. Since 2000 the HCC mortality rate has increased year-after-year, increasing by 3% every year. Until 2019 liver cancer, including HCC, was the fifth and seventh leading cause of cancer deaths in men and women, respectively. In the past decade significant progress has been made in the systemic treatment of advanced HCC. However, newly developed treatment strategies have not achieved widespread success and patients with advanced liver cancer often exhibit resistance to these therapies.

The indole-quinoline alkaloids are representative examples of natural products and the indole-quinoline alkaloids exhibit various biological activities by DNA binding and inhibition of topoisomerase II [22]. The indole-quinoline alkaloids also have cytotoxic, anti-bacterial, antitumor, and molluscicidal effects, as well as anti-protozoan activity, especially against Trypanosoma, Leishmania, and Plasmodium [67–69]. Some studies have suggested that neocryptoolepine derivatives exhibit significant anti-proliferative and pro-apoptotic effects in liver cancer cells. In one study five of seven neocryptoolepine derivatives (compounds 135-139; Figure 7E and 7F ) showed significant efficacy against human liver cancer HepG-2 cells with an IC₅₀ of 27.70 ± 3.80 μM for compound 135. The IC₅₀ of compound 136 is 25.40 ± 3.30 mM [46].

3.5 Anti-cancer activity of neocrytolepine and neocrytolepine derivatives against ascites carcinoma

El-Aarag et al. [70] investigated the toxicity of cryptolepine in EAC cells at the cellular level. The analog (compound 140; Figure 7G ) exerted cytotoxic effects in EAC cells and reduced the ascites volume. Moreover, compound 140 induced oxidative stress in EAC by increasing the level of malonaldehyde and decreasing the level of total antioxidant capacity and catalase activity. Compound 140 also induced apoptosis by elevating the level of caspase-8 expression in EAC. Furthermore, compound 140 decreased the level of AKT and mTOR protein expression and upregulated PTEN expression in EAC [70].

Altwaijry et al. [42] synthesized a series of derivatives and the results of an acute toxicity study in mice showed that the LD₅₀ of compound 144 ( Figure 7G ) was 1000 mg/kg, which indicated that compound 144 could be considered as a low toxicity compound with higher safety margins. The maximum reduction in tumor volume was seen in treatment groups with compound 144 compared to a positive control. In addition, compound 144 might exert anti-tumor activity by reducing the peroxidation of lipids, scavenging the free radicals, and increasing the level of anti-oxidant enzymes.

Nofal et al. [71] studied the anti-tumor effects of APAN in mice (compound 144). Nofal et al. [71] found that compound 144 had ameliorative activity against Ehrlich solid tumors and hepatic toxicity in mice and the greatest improvement occurred with the combined treatment of compound 144 and chemotherapeutic drug, etoposide. TNF-α was highly expressed in the solid tumor and liver tissues of animals with Ehrlich solid tumors (ESTs) and was markedly downregulated in mono- and/or dual-EST-treated groups compared to untreated mice with EST. Therefore, it is of great significance to develop promising leading compounds against anti-ascites cancer from neocryptolepine and its derivatives.

3.6 Anti-cancer activity of neocrytolepine and neocrytolepine derivatives against osteosarcoma

Osteosarcoma is the most common form of primary bone cancer in children and young people [72]. Bone malignancy mainly occurs in adolescent patients. Although current effective treatment options greatly improve patient outcomes, >20% of patients die due to tumor metastasis [73]. One of the most important reasons for osteosarcoma treatment failure is resistance of the tumor to chemotherapy drugs [74]. Neoryptolepine and cryptolepine derived from C. sanguinolenta [42] have shown significant anti-plasmodium and cytotoxic activity and have become an important scaffold for drug discovery. Cryptolepine (compound 5) has been reported to inhibit cancer cell growth in vitro by inhibiting cell cycle progression in human osteosarcoma cells [75]. Compound 5 has been shown to have anti-proliferative activity in vitro against several types of cancer cells (KB, MCF-7, A549, and Lovo) [76]. Further research has identified compound 5 as a potential anti-microbial agent against the most common pathogenic bacteria and fungi that cause cancer and transplant-associated infections in humans [77].

3.7 Anti-cancer activity of neocrytolepine and neocrytolepine derivatives against lung cancer

Lung cancer is one of the cancers with the highest incidence and highest mortality rates [78]. Chemotherapy is still the main treatment modality for lung cancer and compounds extracted and isolated from natural products have an important role in the treatment of lung cancer [79].

Neocryptolepine and its derivatives have been reported to exert good cytotoxic effects in lung cancer in recent decades. Akkachairin and his co-workers found novel compounds containing five-membered ring fused quinoline core structures as anti-cancer and anti-malarial agents. Akkachairin et al. innovated synthetic methods for the synthesis of neocryptolepine derivatives and carbocycle-fused quinolines and the cytotoxicity of all neocryptolepine derivatives (145-167; Figure 8A ) against human lung cancer A549 cells were evaluated using the MTT assay. Among these compounds, compounds 149 (IC₅₀ = 15.58 ± 0.78 μM), 150 (IC₅₀ = 13.63 ± 1.57 μM), 151 (IC₅₀ = 14.89 ± 0.46 μM), 152 (IC₅₀ = 15.31 ± 0.57 μM), 153 (IC₅₀ = 10.80 ± 1.57 μM), and 164 (IC₅₀ = 14.00 ± 1.37 μM) displayed good cytotoxic activity. Compound 153 exhibited the best cytotoxic activity and compound 152 had the greatest selectivity. Topoisomerase II has been shown to be be a target of inhibition by neocryptolepine derivatives in several cancer cells via DNA intercalation [81–83]. Therefore, the mode of inhibiting the proliferation of compound 152 involved inhibition of topoisomerase II in A549 cells as well [7]. Ahmed et al. [53] synthesized a series of neocryptolepine compounds 113-118 ( Figure 6E ) and the cytotoxicity of these compounds in A549 cells was examined using the MTT assay. Compound 113 displayed the best cytotoxic activity against A549 cells with an IC₅₀ of 23.10 μM compared to doxorubicin [53]. Wang et al. [84] designed and synthesized a series of neocryptolepine derivatives and the results of the MTT assay revealed that the compounds had good anti-proliferative activity (IC₅₀ = 0.20–4.54 μM) against A549 cells and lower cytotoxicity against normal fibroblasts (BALB/3T3 cells). Inokuchi and his co-workers obtained a series of neocryptolepine derivatives 168-193 ( Figures 8B and 9A ) by the structural modification of neocryptolepine. The anti-proliferative activities of these compounds were tested in vitro against A549 cells. Most compounds showed good cytotoxicity and 11-(3-amino-2-hydroxy) propylamino derivatives 172 and 173 had the best cytotoxicity with IC₅₀ values of 0.20 and 0.19 μM against the A549 cells, respectively. The computer-assisted database analysis, COMPARE, suggested that compounds 171 and 176 have a mode of action similar to actinomycin D, while compound 175 has a mode of action similar to vincristine sulfate or aclarubicin hydrochloride [52].

Figure 8 |

The synthetic route of compounds 145-167 and compounds 168-185 [7, 80].

Figure 9 |

The chemical structures and synthetic route of neocryptolepine derivatives 186-212 [43, 52, 80, 86].

3.8 Anti-cancer activity of neocrytolepine and neocrytolepine derivatives against breast cancer

Neocryptolepine has become a focus of research because of its good cytotoxicity and is considered a promising backbone for drug development. Some studies have reported that neocryptolepine has good cytotoxic effects on a variety of cancer cells, including lung cancer, cholangiocarcinoma, liver cancer, and leukemia [56]. Neocryptolepine arrests the cell cycle at the G2/M phase, induces apoptosis, and affects mitochondrial function [48]. In recent years it has been shown that some neocryptolepine derivatives have better anti-proliferative activity against cancer cells, such as lung, breast, colorectal, and oral epidermoid cancer, after the structural modification of neocryptolepine [46, 53, 55, 56].

Cryptolepine has been demonstrated to have good cytotoxic activity in breast cancer by affecting cyclins D1, D2, and D3 and cyclin E, which regulate the cell cycle [22, 49, 85]. Plant-derived 5-Me-indolo[2,3-b]quinolines with substituents at C11 and C2 were assayed for anti-proliferative activity against several cancer cells. Specifically, the 11-(3-aminopropylamino)-substituted compounds 194 and 195 ( Figure 9B ) had the best cytotoxic activity against breast cancer MDA-MB-453 cells (IC₅₀ = 0.30–0.50 μM) [52, 80, 87]. A synergistic effect was observed with an electron-donating group, such as methoxyl at C2 in compound 195 (IC₅₀ = 0.20 μM) [60, 71, 88]. Further modification of the terminal free amino group of the lariat attachment at C11 into the corresponding acylamides and 2,3-dihydrobenzo[e] [1, 3] thiazin-4-ones was not effective with respect to anti-proliferative activity [26]. However, the novel compounds may have other unique mode of actions, which would also be an interesting possibility to examine in corollary studies [89].

3.9 Anti-cancer activity of neocrytolepine and neocrytolepine derivatives against leukemia

Leukaemia is a malignant clonal disease originating from hematopoietic stem cells with a complex pathogenesis involving multiple biological, physical, chemical, genetic, and other hematologic factors, such as myelodysplasia, myelofibrosis, multiple myeloma [90]. The most common and studied leukemias are acute myeloid leukemia (AML), acute lymphoblastic leukemia/lymphoma (ALL), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), and chronic myeloid leukemia (CML) [91]. The alkaloids, which differ in the orientation of the indole and quinoline rings, have good cytotoxic activity against cancer cells and possess anti-bacterial and anti-parasitic properties. As early as 1991, Harker et al. [92] investigated the cytotoxic effects of cryptolepine and neocryptolepine on HL-60/MX2 cells and mitoxantrone-resistant HL-60/MX2 cells. Dassonneville et al. [41] assessed the cytotoxicity and effects of these compounds on the cell cycle in mouse and human leukemia cells. Cryptolepine and neocryptolepine were shown to significantly arrest mouse leukemia P388 cells at the G2/M phase. In human leukemia HL-60 cells, cryptolepine treatment resulted in the appearance of a sub-G1 peak below the diploid DNA content and representative of the apoptotic cell population. However, the use of an HL-60/MX2 cell line resistant to the anti-cancer drug, mitoxantrone, indicated that topoisomerase II may not be the primary cellular target of these alkaloids because both alkaloids were only two-fold less toxic to drug-resistant HL-60rMX2 cells than parental cells. The study also examined the ability of these drugs to induce apoptosis in human leukemia HL-60 cells. Western blot analysis showed that cryptolepine induced cleavage of poly-ADP-ribose polymerase and cryptolepine and neocryptolepine induced cytochrome C release from mitochondria. Cleavage of poly-ADP-ribose polymerase was associated with a strong activation of the caspase pathway, which correlated with the significant sub-G1 peak seen in cryptolepine-treated cell cycle experiments. Despite activation of the caspase cascade reaction, the investigators did not detect inter-nucleosome cleavage of DNA in these alkaloid-treated HL-60 cells.

Zhou et al. [86] synthesized quinoline derivatives 198-207 ( Figure 9C ) by modification and compound 200 showed the most potent inhibition (50% inhibition at 0.44 mM in the cell-free telomeric report amplification protocol [TRAP]). Compound 200 is highly selective for telomerase and K562 cells have significant cell growth arrest and the cell senescence phenotype after 35 days of treatment with compound 200. Compound 200 reproducibly inhibited telomerase activity of cancer cells, leading to telomere shortening and subsequent cellular senescence. As early as 1988 Kaczmarek et al. [93] synthesized tetracyclic or pentacyclic benzo-iso-alpha-carboline-based compounds and evaluated the anti-tumor properties. The results clearly showed that the size and shape of the compounds had a significant influence on the biological activity. Among the compounds, compound 208 ( Figure 9C ), a neocryptolepine-like compound with a linear tetracyclic portion bearing two methyl groups at both N-5 and C-11 positions, was the best cytotoxic compound. Further studies showed that compound 208 significantly inhibited tumor growth in mice using the P388 and B16 cell-derived xenograft tumour model in vivo.

Wang et al. [52] synthesized a series of neocryptolepine (210 and 211) and isocryptolepine derivatives (212; Figure 9C ). The anti-proliferative activity of these compounds were then tested against MV4-11, HCT116, A549, and BALB/3T3 cells in vitro. The cytotoxicity was much better in MV4-11 cells than HCT116 and A549 cells [52]. The anti-proliferative effects of ester groups in vitro and structure-activity relationship studies of 5-methyl-5H-indolo[2,3-b]quinoline (neocryptolepine) derivatives was described by Lu et al. [94]. C-2 and/or C-9 ester-substituted neocryptolepine derivatives were synthesized, starting from indole-3-carboxylates with ester groups and N-methylaniline. Various aminoalkylamino substituents were further attached to the C-11 position of these ester-substituted neocryptolepine and anti-proliferative assays in vitro were carried out by varying the substituents at the C-11 position of neocryptolepine and the position of the ester group in the A and/or D rings. The anti-proliferative activity of the compounds was improved by introducing an ester substituent at the C-9 position ( Figure 5 ). Among the compounds, methyl 11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate (compound 213; Figure 10A ) was the most potent compound with an IC₅₀ value of 0.04 μM against human leukemia MV4-11 cells. Shaban et al. [43] prepared a series of 11-substituted neocryptolepine derivatives with branched ω-aminoalkylamino chains with different linkage lengths between the two nitrogen atoms and evaluated the anti-proliferative activity using MV4-11, A549, HCT116, and BALB/3T3 cells. All the synthesized compounds showed potent anti-proliferative activity against MV4-11 cells compared to 11-chloro substituted precursors in vitro. Due to the diversification of ω-amino alkyl amino chains, the 11-(3-amino-2-hydroxy)propylamino substituted compounds (214 and 215; Figure 10B ) were shown to have the best anti-proliferative activity with mean IC₅₀ values of 0.04 μM and 0.06 μM against MV4-11 cells, respectively. Peng et al. [87] described the design and synthesis of a series of novel 11-aminochromeno[2,3-b]indole derivatives and evaluated the anti-proliferative activity of these derivatives using MV4-11, A549, HCT116, and BALB/3T3 cell lines. Compound 216 ( Figure 10C ) showed good anti-proliferative activity against MV4-11 leukemia cells with an IC₅₀ of 0.12 μM [87].

Figure 10 |

Synthetic route of compounds 213-219 [43, 64, 87, 94].

3.10 Anti-cancer activity of neocrytolepine and neocrytolepine derivatives against other cancer

As natural products with good anticancer activity, neocryptolepine and its derivatives are manifested in the treatment of common generalized cancers such as gastric cancer, colorectal cancer and liver cancer [49, 55]. Several studies have shown that neocryptolepine still exhibited good cytotoxicity in other cancers, such as melanoma, cholangiocarcinoma, cervical cancer, and oral epithelial cancer [22]. In addition, researchers obtained different neocryptolepine derivatives by structural modification of neocryptoleine. Overall, neocryptolepine and its derivatives have great prospects for the development of antitumor drugs and provide promising ideas for the combination therapy of cancer.

3.10.1 Melanoma

Melanoma is a highly malignant skin tumor caused by melanocytes or primitive nevus cells of the skin. The incidence of melanoma is increasing year-after-year and is difficult to treat [95]. Early-stage melanoma can be successfully treated with surgical intervention but advanced metastatic melanoma has a poor prognosis and requires targeted therapy, chemotherapy, and immunotherapy [96]. The efficacy of current treatments is limited by drug resistance or immune tolerance in melanoma patients [97, 98]. In recent years, the application of neocryptolepine and its derivatives in the treatment of melanoma has received increasing attention. Majhi et al. [64] obtained a series of derivatives by structural modification of neocryptolepine and evaluated the cytotoxic activity in various cancer cells. Compounds 6, 217, 218, and 219 ( Figures 1 and 10D ) had better cytotoxicity in melanoma B16F10 cells [64]. Wang et al. [52] also obtained a variety of new derivatives by the introduction of different groups in the parent nucleus of cryptolepine or neocryptolepine. Compound 175 showed strong cytotoxic activity against melanoma LOX-IMVI cells with an IC₅₀ of 0.75 μM [52].

3.10.2 Cervical cancer

Cervical cancer is one of the most common malignant tumors among women worldwide and has high morbidity and mortality rates [99]. Although traditional treatments, such as surgery, radiotherapy, and chemotherapy, have achieved some efficacy in the treatment of cervical cancer, many challenges still exist, such as side effects and drug resistance [100, 101]. Therefore, searching for novel leading compounds with high efficiency and low toxicity is a strategy to develop drugs for the treatment of cervical cancer [102]. Neocryptolepine and its derivatives have gradually shown potential for the treatment of cervical cancer because of unique pharmacologic effects and good anti-cancer activity [103]. Cybulski et al. [103] obtained a series of indoquinoline derivatives and had good inhibitory effects in pancreatic cancer. The IC₅₀ of compounds 220 and 221 ( Figure 11A ) were 808.75 ± 91.29 and 203.15 ± 35.28 nM against cervical cancer Hela cells and the molecular docking results also verified the target affinity [103]. The potential of neocryptolepine derivatives to inhibit the progression of cervical carcinogenesis has also been demonstrated in a study published by Majhi et al. [64]. Compounds 6 and 139 showed significant cytotoxicity in Hela cells [64]. In the future, studies involving neocryptolepine and its derivatives in the treatment of cervical cancer will continue to deepen and be optimized.

Figure 11 |

Synthesis routes of neocryptolepine derivatives 220-228 [103–106].

3.10.3 Cholangiocarcinoma

Cholangiocarcinoma (CCA) is a highly malignant solid tumor of the digestive system and is the second most common primary hepatobiliary malignancy after HCC. The incidence and mortality of CCA have gradually increased worldwide in the last decade and surgical resection remains the most desirable radical therapy for CCA patients [107, 108]. However, the majority of patients are diagnosed in mid- to late-stage because of the atypical clinical presentation and insidious onset of this cancer. Patients with end-stage CCA are usually unable to undergo surgical resection or local intervention. The National Comprehensive Cancer Network (NCCN) guidelines recommend gemcitabine combined with cisplatin (GC) as first-line chemotherapy for end-stage CCA [109]. Therefore, it is necessary to search for effective drugs for the treatment of CCA and the continuous exploration of neocryptolepine derivatives has provided such a possibility. Neocryptolepine derivatives 151, 152, 153, 161, and 164 exhibit selective anti-cancer activity against CCA HuCCA-1 cells in vitro as demonstrated by Akkachairin et al. [7], who showed good cytotoxicity with and IC₅₀ of 13.13 ± 1.82 μM, 13.39 ± 1.68 μM, 13.63 ± 0.00 μM, 13.53 ± 0.42 μM, and 9.65 ± 1.57 μM respectively. Moreover, topoisomerase II has been shown to be a target of these compounds by DNA binding in several cancer cells based on several studies involving C. alba and C. neoformans.

3.10.4 Oral epidermal cancer

In addition to good antitumor potential against solid tumors of organs, neocryptolepine and its derivatives have good inhibitory ability against some specific types of epidermal cancers. The incidence of oral epidermal cancer ranks fifth among all cancers globally and the prevalence has increased with the aging population [110]. Surgical intervention is usually the main choice for the treatment of oral squamous cell carcinomas (OSCC). However, after successful surgery, the 5-year survival rate for OSCC patients is still very low [111, 112]. In addition, long-term survivors may experience severe complications related to occlusion, swallowing, and word articulation, along with facial distortion and psychological disorders [113–115]. Therefore, non-surgical treatment options, such as immunotherapy and gene therapy, are being actively investigated [111]. Currently, basic and clinical research are focused on the development of anti-cancer drugs with selective antitumor effects, especially for advanced or recurrent tumors. Several studies have demonstrated that neocryptolepine and its derivatives have the potential for development of oral epidermoid carcinoma. Sidoryk et al. [104] obtained a series of neocryptolepine derivatives and the results of cytotoxic assays demonstrated that a variety of compounds possess cytotoxic activity against oral epidermoid carcinoma KB cells. Compound 222 ( Figure 11B ) had the strongest inhibition against KB cells compared to other neocryptolepine derivatives and the IC₅₀ against KB cells was 0.38 ± 0.11 μM [104]. The neocryptolepine derivatives synthesized by Li et al. [77] also had good antiproliferative effects in oral epidermal cancer KB cells with IC₅₀ values of 0.08, 0.31, 0.15, 0.15, 0.64, and 0.36 μM for compounds 223-228 ( Figure 11C-E ) against KB cells, respectively [77]. These results also provided a new strategy for the optimization of neocryptolepine and provided promising leading compounds for the treatment of oral epidermal cancer.

4. CONCLUSION AND PERSPECTIVE

Overall, neocryptolepine and its derivatives exhibit broad-spectrum anti-tumor activity in different cancer cells, including gastric, colorectal, liver, lung, and esophageal cancer. Compounds 43, 65, 93, and 96 exert excellent cytotoxicity in gastric cancer cells and these compounds inhibit cell migration and proliferation, arrest the cell cycle, and induce cell apoptosis. Compound 43 showed better cytotoxicity in AGS cells with an IC₅₀ of 43 nM. Moreover, compound 65 showed good anti-cancer activity in human gastric cancer organoid and low toxicity in mice.

As shown in Table 2 and Figure 12 , most neocryptolepine derivatives exert good cytotoxicity by binding DNA or inhibiting topoisomerase II. Many compounds induce apoptosis of cancer cells and increase the levels of apoptotic protein expression, such as p53, caspase 8, and caspase 3. The PI3K/AKT and Wnt/β-catenin cell signaling pathways have an important role in growth inhibition of neocryptolepine derivatives. In a structure-activity relationship analysis, the double substitution of the B and D rings was more common in the substitution of the neocryptolepine as the parent nucleus. As seen in Figure 5 , the hydrazine and amide linkers have an important role in exerting good cytotoxicity in the B ring substitution. The 8-position F, methyl, or methoxy substitution is important to maintain the cytotoxicity in A and D ring substitutions. Among the B and D ring-substituted compounds, the IC₅₀ of compounds 93 and 96 against gastric cancer AGS cells were 2.9 and 4.5 μM, respectively.

Table 2 |

Preclinical studies of neocryptolepine and its derivatives in different types of cancers.

Type of cancer	Compounds	Experiments		Effects and mechanisms	References
		In vivo	In vitro
Gastric cancer	Neocryptolepine derivative 43	NA	AGS	E-cadherin↑, CDK1↓, Cleased-caspase3↑, PI3KCA↓, p-AKT↓, AKT↓	[19]
Gastric cancer	Neocryptolepine derivative 65, 93, 96	NA	AGS, HGC27, MKN45, SGC7901	CDK1↓, Cyclin B1↓, caspase 3↑, PI3KCA↓, p-AKT↓, AKT↓, mTOR↓, p-mTOR↓	[48, 49]
Colorectal cancer	Compound 6	NA	HCT116	β-catenin↓, c-MYC↓, WNT3a↓, WISP1↓	[58]
Colorectal cancer	Compound 64	NA	HCT116, Caco-2	CDK1↓, Cyclin B1↓, PI3KCA↓, p-AKT↓, AKT↓, mTOR↓, p-mTOR↓	[60]
Colorectal cancer	Compound 107	NA	HCT116	c-MYC↓	[50]
Colorectal cancer	Compound 108	NA	HCT116	NA	[52]
Colorectal cancer	Compound 109	NA	HCT116	DNA intercalation	[51]
Colorectal cancer	Compound 110	NA	HCT116	caspase3↑, p53↑, PCNA↓, Ki-67↓, VEGF↓	[55]
Ovarian cancer	Compound 134	NA	OVCAR3	DNA binding	[64]
Liver cancer	Compound 135 and 136	NA	HepG-2	DNA binding	[46]
Ehrlich ascites carcinoma	Compound 140	Xenograft mice for Ehrlich ascites carcinoma cells	Ehrlich ascites carcinoma cells	Malonaldehyde↑, caspase-8↑, mTOR↓, PTEN↑, AKT↓	[70]
Ehrlich ascites carcinoma	Compound 144	The cells were relocated in the peritoneum of Swiss albino female mice	EAC cells	Induced apoptosis, cell cycle arrest, TNF-α↑	[42, 71]
Lung cancer	Compound 152	NA	A549	Inhibition of topoisomerase II	[7]
Lung cancer	Compound 113	NA	A549	NA	[53]
Lung cancer	Neocryptolepine derivatives 168-193	NA	A549	Interacting with DNA	[52]
Breast cancer	Compounds 194 and 195	NA	MDA-MB-453	Interacting with DNA	[52, 80, 87]
Leukemia	Compound 200	NA	K562	Telomerase inhibitors	[86]
Leukemia	Compound 208	Xenograft mice for P388, L1210 cells	P388, L1210	NA	[93]
Leukemia	Compounds 214 and 215	NA	MV4-11	Inhibition of topoisomerase II	[43]
Leukemia	Compound 216	NA	MV4-11	NA	[87]
Melanoma	Compounds 6, 217, 218, and 219	NA	B16F10	DNA binding	[64]
Melanoma	Compound 175	NA	LOX-IMVI	Interacting with DNA	[52]
Cervical cancer	Compounds 220 and 221	NA	Hela	binding to the topoisomerase II–DNA complex	[103]
Cervical cancer	Compounds 6 and 139	NA	Hela	DNA binding	[64]
Cholangiocarcinoma	Compounds 151, 152, 153, 161, and 164	NA	HuCCA-1	NA	[7]

Figure 12 |

The potential mechanism underlying the cytotoxic effects of neocryptolepine and its derivatives (this Figure was drawn by Figdraw, ID: SWRYU0002d).

It is also very promising to develop more effective and less toxic anti-tumor drugs by structural modification and optimization of the parent nucleus with active neocryptolepine derivatives. Although progress has been made in the field of anti-tumor research involving neocryptolepine derivatives and some neocryptolepine derivatives with good activity have been found, there are still many challenges. As seen in Table 2 , the cytotoxicity and mechanism of action of most neocryptolepine derivatives have only been evaluated at the cellular level. It is crucial to further determine the anti-tumor activity of neocryptolepine derivatives at the animal and organoid levels, and even in clinical trials. There is no toxicity data on normal cells and animals or pharmacokinetic data in vivo in the anti-tumor studies involving neocryptolepine derivatives. Therefore, there is a long way to go before clinical trials and clinical use of neocryptolepine derivatives. Finally, it is extremely important to further clarify the underlying molecular mechanism and target of the anti-tumor effect of neocryptolepine derivatives, which is also a focus in the future.

In conclusion, this review summarized the structure and cytotoxicity of neocryptolepine and its derivatives in the last few years. We found many neocryptolepine derivatives exert good cytoxicity and the cytotoxic effects in vitro and the toxicity and anti-cancer activity in vivo were evaluated. Some neocryptolepine derivatives in this review can be used as leading compounds for the development of anti-cancer drug in future studies.

CONFLICTS OF INTEREST

The authors declare no competing financial interests.

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