**1. Introduction**

Cancer is the second leading cause of human death after cardiovascular disease, and cancer of the digestive system accounts for about 50% of all cancers [1]. According to the global cancer statistics in 2018, gastric cancer is the most common cancer of digestive system tumors, with about 1.03 million new cases of gastric cancer worldwide, ranking fifth in the incidence of malignant tumors and becoming the third leading cause of cancer death [2]. The five-year survival rate of gastric cancer is less than 25%, and we are often powerless for patients with advanced gastric cancer [3,4]. The methods of cancer treatment mainly include radiotherapy, chemotherapy, surgery, and gene therapy, but chemotherapy is a necessary means to treat solid tumors at present. Compared with other cancer treatments, oral chemotherapy drugs have the advantages of low cost and strong patient compliance. However, chemical drugs have large side effects, and drug resistance is difficult to solve [5,6]. Therefore, in order to overcome these obstacles, it is very necessary to develop new and less toxic chemical drugs to treat gastric cancer.

Among natural products, alkaloids are one of the main natural products. Alkaloids were discovered and used as early as 4000 years ago, and alkaloids and their derivatives

**Citation:** Ma, Y.; Tian, Y.; Zhou, Z.; Chen, S.; Du, K.; Zhang, H.; Jiang, X.; Lu, J.; Niu, Y.; Tu, L.; et al. Design, Synthesis and Biological Evaluation of Neocryptolepine Derivatives as Potential Anti-Gastric Cancer Agents. *Int. J. Mol. Sci.* **2022**, *23*, 11924. https://doi.org/10.3390/ ijms231911924

Academic Editors: Marialuigia Fantacuzzi, Barbara De Filippis and Alessandra Ammazzalorso

Received: 18 September 2022 Accepted: 5 October 2022 Published: 7 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

have been used as drug sources to treat various diseases around the world, including the development of anticancer drugs [7]. Traditional alkaloids extracted from plants have played a huge role in the past [8], and more than 5000 alkaloids have been reported since the discovery of the first alkaloid, morphine, in 1805 [9]. A large number of studies have also shown that alkaloids performed excellent cytotoxicity to different cancers, including human melanoma, breast cancer, pancreatic cancer, colorectal cancer, oral cancer, liver cancer, and gastric cancer [10–15]. *Cryptolepis sanguinolenta* is a vine that grows in some African countries, and the roots of this plant have proven to be a rich source of indolinequinoline alkaloids [16]. In recent years, neocryptolepine, a promising natural quinoline indole alkaloid, has attracted much attention. Some neocryptolepine derivatives have strong cytotoxicity to leukemia cells MV4-11, with an IC50 of 42 nM and also to lung cancer cells with an IC50 of 197 nM [17]. Neocryptolepine and its derivatives have a wide range of biological activities, and compounds containing this ring system have antifungal, antibacterial, antiviral, and cytotoxic activity [18–21]. In fact, indolequinoline alkaloids have good anticancer activities, and semi-synthetic analogues of these neocryptolepine can be prepared, which have shown great potential effects of cytotoxic agents [22]. Therefore, indolequinoline alkaloids are considered as a promising framework for drug development and can be further developed as effective anticancer drugs [8,22].

Due to the complex structure of natural products, different chemical components have different anticancer mechanisms [23]. Studies have shown that some alkaloids can induce apoptosis and cell cycle arrest [24]. The activation of cancer signaling pathways is common in the occurrence of cancer [25]. Multiple signaling pathways are involved in the occurrence of gastric cancer [26]. Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) is an important signaling pathway in cells. When the PI3K/AKT signaling pathway is abnormally activated, it may cause the activation of downstream signaling molecules, thus affecting the development of gastric cancer, lung cancer, and other malignant tumors [27–29]. Studies have shown that the development of gastric cancer is related to excessive cell proliferation and inhibition of apoptosis, and activation of the PI3K/AKT cell signaling pathway often prevents programmed cell death [30]. The PI3K/AKT cell signaling pathway plays an important role not only in tumor development but also in tumor therapy, and many new targeted agents are realized by acting on relevant targets of the PI3K/AKT signaling pathway [31,32].

In the previous studies, in addition to numerous activity tests and mechanistic studies on the parent structure of neocryptolepine, a great deal of work has been done on the derivatives with the **C11** position substitution of neocryptolepine. Some studies have found that neocryptolepine derivatives have good antibacterial, anti-proliferative, and antifungal activities [33–35]. For example, in 2009, Ibrahim El Sayed et al. introduced a long aminoalkyl chain substitution at the **C11** position of neocryptolepine. A series of derivatives were prepared and further tested for their inhibitory activity against Plasmodium. Among them, the IC50 of the most active compound was 0.043 μM [36]. In 2012, Li Wang et al. reported the effects of derivatives obtained by modifying the **C11** position with a variety of amino alkyl chains on the human leukemia MV4-11 cell line in anti-proliferation experiments. The experimental results showed that most of the derived molecules had good anti-proliferation activity, but they also had high cytotoxicity [35,37]. Therefore, after performing the cytotoxicity study of 8-chloroneocryptolepine, the substitution at **C11** might be ideal to improve the inhibitory activity of 8-chloroneocryptolepine on cancer cells.

As a result, we aimed to perform the modification of the **C11** position of 8-chloroneocryptolepine, and a series of neocryptolepine derivatives were synthesized. The cytotoxic effects of neocryptolepine derivatives on liver cancer SMMC7721 and gastric cancer AGS cells were evaluated in vitro. The results showed that compounds **C5** and **C8** exhibited strong cytotoxicity against gastric cancer cells and may be promising lead compounds in the treatment of gastric cancer.

### **2. Results and Discussion**

#### *2.1. Chemistry*

As shown in Scheme 1, in previous work, the structure of neocryptolepine was optimized by structural modification, and 8-chloroneocryptolepine performed good anti-fungal effects. The active functional group piecing strategy has been widely used in the field of derivative synthesis and structure optimization of antitumor compounds [24]. Moreover, the introduction of amino long-chain alkanes at the **C11** position of neocryptolepine could improve the cytotoxic effect of the compound [36,37]. Therefore, the cytotoxicity evaluation of a series of derivatives, by introducing an active functional group to **C11** of 8-chloroneocryptolepine, is a promising strategy for the development of a lead compound with anti-tumor drugs.

**Scheme 1.** Design strategy of target compounds in this study.

The synthesis of neocryptolepine derivatives and intermediates was shown in Scheme 2. Intermediate I was easily obtained with a yield of more than 80%. Indoles, trichloroacetyl chloride, and tetrahydrofuran were acylated to obtain intermediate I. Subsequently, intermediate II was obtained with the reaction of intermediate I and *N*-methylaniline, and the yield was more than 60%. Intermediate III was obtained by the reaction of intermediate II with diphenyl ether, and intermediate IV was obtained by the interaction with phosphorus oxychloride, with a yield of more than 60%. Intermediate IV reacted with the corresponding alcohol hydroxyl compound in N, N-dimethylformamide (DMF) to obtain the corresponding compounds **A1–A10**, and with the corresponding phenylhydrazine compound to obtain compounds **B1–B9**, with the corresponding amide compound to obtain compounds **C1–C10**, **D1–D6**. The structures of these compounds can be found in Table 1. It is important to note that the commercially available raw materials were obtained through the synthesis of intermediates and final products with a good yield. The structures of the target compounds were confirmed by 1H and 13C NMR and MS.

**Scheme 2.** Synthetic route of a series of neocryptolepine derivatives. Reagents and conditions: (a) trichloroacetyl chloride, pyridine, KOH, 85% (b) N-chlorosucccinimide, 1,4-dimethylpiperazine, CH2Cl2, 0 ◦C, 2 h; trichloroacetic acid, RT, 2 h, 64%; (c) diphenyl ether, reflux, 1.3 h; (d) POCl3, toluene, reflux, 6–12 h, 70%; (e) amino derivatives or hydroxyl, DMF; (f) KOH; (g) ethanol, heat.

**Table 1.** Chemical structures of neocryptolepine derivatives.



**Table 1.** *Cont.*

#### *2.2. Cytotoxic Activity In Vitro and Structure-Activity Relationship (SAR)*

The cytotoxicity of intermediate IV and neocryptolepine derivatives on gastric cancer AGS cells and hepatoma SMMC7721 cells was determined by MTT assay. Based on our results of cytotoxicity, the structure–activity relationship was studied for neocryptolepine derivatives (as shown in Figure 1). Firstly, the **C11** position of 8-chloroneocryptolepine was substituted with ether groups. However, in Table 2, the results suggest that the IC50 of compounds **A1–A10** was greater than 50 μM for SMMC7721, but some of the compounds had a strong cytotoxicity on AGS cells. According to the cytotoxicity results, the *para*-site substitution of F atom (**A4**) is better than the *meta*-site (**A3**) and *ortho*-site (**A2**) substitution, and the methoxy *ortho*-site (**A5**) substitution is more cytotoxic than the *meta* (**A6**) and *para*-site (**A7**) substitution, as well as the dimethoxy (**A8**) substitution, on benzene ring.

**Figure 1.** Structure-activity relationship analysis of neocryptolepine derivatives. Different colours indicated different substituted functional groups, and different arrows indicated different cytotoxic activities after functional group substitution.


**Table 2.** Antiproliferative activities (IC50, μM) of compounds **A1–A10** on SMMC7721 and AGS cells for 48 h.

In 2016, Masashi Okada et al. performed an anti-proliferative activity assay on breast cancer MDA-MB-453 cells, colorectal cancer WiDr Cells, and ovarian cancer SKOv3 cells by introducing amino long-chain alkanes at the **C11** position. The experimental results showed that the introduction of amino long-chain alkanes at the **C11** position was beneficial to the improvement of anti-proliferative activity [38]. Based on the above structural modification and cytotoxicity, the **C11** position of 8-chloroneocryptolepine was substituted by hydrazine group to obtain compounds **B1–B9**. According to the cytotoxicity results in Table 3, the substitution of hydrazine group (**B1–B9**) was more cytotoxic than that of ether group (**A1–A10**). The results show that, in hydrazine group substitution, the substitution of F or methyl group increased its activity. Moreover, mono-substituted (**B2–B4**) F atom on benzene rings was more cytotoxic than disubstituted (**B5**) F atoms, and *meta*-substituted (**B3**) F atom was more cytotoxic than *para*-substituted (**B4**) F atom. The substitution of *ortho* (**B6**) methyl groups on benzene rings was better than that of *meta* (**B7**) and *para* (**B8**) groups.

**Table 3.** Antiproliferative activities (IC50, μM) of compounds **B1–B9** on SMMC7721 and AGS cells for 48 h.


Compounds **C1–C10** were synthesized by substituting **C11** sites with amide groups through electron iso-arrangement. In Table 4, by comparison and verification of experimental results, compounds **C5** and **C8** were found to be more active against AGS cells, and their possible mechanisms were studied at the cellular level. Moreover, as shown in Table 5, the cytotoxic effects of compounds **C5** and **C8** were significantly better than the positive drug cisplatin and the parent nucleus 8-chloroneocryptolepine. According to the cytotoxicity results in Table 4, the *para*-F (**C5**) atomic substitution of benzene ring was more cytotoxic than the *ortho* (**C3**) and *meta*-position (**C4**), and the *meta*-position (**C7**) and *para*-methoxy (**C8**) substitution were better than the *ortho*-substitution (**C6**). The increase in alkyl side chain (**C9** and **C10**) showed better cytotoxicity against AGS cells than compounds **A1–A10**. The structure–activity relationship of compounds **C1–C10** can also be found in Figure 1.


**Table 4.** Antiproliferative activities (IC50, μM) of compounds **C1–C10** on SMMC7721 and AGS cells for 48 h.

**Table 5.** Antiproliferative activities (IC50, μM) of compounds **D1-D6** and E1 on SMMC7721 and AGS cells for 48 h.


Further evaluation of the cytotoxicity of 8-chloroneocryptolepine on gastric cancer AGS and liver cancer SMMC7721 cells revealed that its cytotoxicity was not ideal (as shown in Table 5). Finally, the **C11** position of 8-chloroneocryptolepine was substituted with a sulfonamide group to obtain compounds **D1–D6**. However, in Table 5, the cytotoxicity results suggested that the cytotoxicity for SMMC7721 cells was very weak. Therefore, the substitution of the sulfonamide group at the **C11** position was not an ideal choice. However, in the cytotoxicity results of AGS cells, the *meta*-positional (**D2**) substitution of F atom was less potent (or less cytotoxic) than *ortho*- (**D1**) and *para*-substitutions (**D3**). The *ortho*-methyl substitution (**D4**) in benzene ring was more cytotoxic than the *para*-methyl (**D5**) substitution.
