**1. Introduction**

Natural products and their derivatives have a long history in cancer therapy and are important for drug development. Efficient and mild synthetic routes for bioactive natural product derivatives are of current interest for drug discovery [1–4]. Recently, pentacyclic triterpenes have been identified as the main biologically active components in many traditional Chinese medicines [5,6]. Among them, oleanolic acid (OA) is the most abundant and cheap; thus, OA and its derivatives have been widely investigated for their diverse biological activities, including their anti-cancer, anti-inflammatory, anti-HIV, antibacterial, anti-diabetic, and anti-hepatotoxic effects, among others [7–11]. Derivatization of OA has yielded a wide variety of novel compounds for anti-cancer investigations (Scheme 1) [11–15]; however, poor pharmacokinetic properties, low cell selectivities, limited bioavailabilities, and synthetic complexity have hindered further clinical application [7]. Therefore, methods for readily accessible modification of OA to enhance its polarity and anti-proliferative activity are urgently required.

Dithiocarbamates are an important class of sulfur-containing organic compounds with a wide range of applications in both academia and industry [16–27]. They serve as fungicides and pesticides in agriculture [17–19], vulcanization agents in the rubber industry [20], radical chain transfer agents in polymerization [21], effective ligands in coordination chemistry [22], and, last but not least, as biologically important structural motifs in medicinal chemistry (Scheme 2) [23–27].

**Citation:** Tang, L.; Zhang, Y.; Xu, J.; Yang, Q.; Du, F.; Wu, X.; Li, M.; Shen, J.; Deng, S.; Zhao, Y.; et al. Synthesis of Oleanolic Acid-Dithiocarbamate Conjugates and Evaluation of Their Broad-Spectrum Antitumor Activities. *Molecules* **2023**, *28*, 1414. https://doi.org/10.3390/molecules 28031414

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

Received: 4 January 2023 Revised: 27 January 2023 Accepted: 30 January 2023 Published: 2 February 2023

**Copyright:** © 2023 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/).

**Scheme 1.** Oleanolic Acid and Its Derivatization for Antitumor Medicinal Chemistry [11–15].

**Scheme 2.** Pharmaceutically Important Dithiocarbamates [23–27].

In recent years, the pharmacophore hybrid strategy has emerged as an essential method for the discovery and modification of lead compounds [28–31]. Covalently combining two known pharmacophores yields a novel hybrid molecule, which can possess integrated advantages for optimizing certain biological activities and overcoming the deficiencies of a single drug [32–35]. In view of the high performance of dithiocarbamate derivatives in structural modification, the synthesis of OA-dithiocarbamate conjugates may enhance the polarities and antitumor properties of the reaction products in a readily accessible manner [7,23–27]. The structural modifications of OA have mainly focused on the C-3 hydroxyl and C-28 carboxyl groups (Scheme 1) [7]. The C-28 carboxyl group can easily be esterified by alcohols or amidated by amines; however, the preparation of OA-dithiocarbamate conjugates has not yet been documented in the literature [7–11]. In order to simplify the synthetic route and control the polarity of target molecules, ethylidene was chosen as a linker between OA and dithiocarbamates.

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

To establish the optimal reaction conditions, we prepared key intermediate **2,** as previously described [36,37]. Under the "standard" conditions, the reaction of **2** with CS2 and pyrrolidine in a one-pot manner afforded the target product **3a** in an 80% isolated yield. In the "standard" conditions, 2 equiv. of K3PO4 was shown to be essential to yield the desired product **3a** (Entries 1–4, Table 1). Lowering the loading of K3PO4 to 1.5 equiv. led to a decreased yield of **3a** (Entry 1, Table 1), while replacement of it by K2HPO4 or Li2CO3 resulted in no desired product (Entries 2–3, Table 1). On the other hand, in the presence of 2 equiv. of K2CO3, product **3a** could be isolated with a 62% yield (Entry 4, Table 1). Changing the reaction temperature or using other solvents, such as DMF, CH3CN, and EtOH, did not offer better results (Entries 5–8, Table 1). Lower amounts of CS2 or pyrrolidine resulted in a decreased yield of **3a** (Entries 9–10, Table 1).

**Table 1.** Optimization of Reaction Conditions.


Variations from the "standard" conditions. *<sup>a</sup>* Reaction temperature was raised to 60 ◦C. *<sup>b</sup>* CS2 was used in 3.0 equiv. instead of 4.5 equiv. *<sup>c</sup>* Pyrrolidine was used in 1.5 equiv. instead of 2.0 equiv.

With the optimal reaction conditions in hand, the substrate scope was subsequently investigated, and the results are compiled in Figure 1. The replacement of the H-atom of the pyrrolidine ring with other substituents, such as methyl, dimethyl, hydroxy, and hydroxymethyl, worked well, affording the corresponding products **3b**–**3e** in 69–85% yields. Among them, hydroxyl containing products were obtained at slightly lower yields. This reaction was also tolerant of fused-ring substrates, such as hexahydroisoindoline and isoindoline, resulting in **3f** and **3g** with 77% and 90% yields, respectively.

To further enhance the structural diversity of products, various types of piperidinederived substrates were also examined, and all of them were compatible with the established reaction conditions. First, methyl-, hydroxy-, hydroxymethyl-, hydroxyethyl-, and phenyl-substituted piperidines reacted smoothly to give **3h**–**3m** in 70–88% yields. Then, methyl-, hydroxyethyl-, phenyl-, and aryl-substituted piperazines were also viable substrates, affording **3n**–**3s** in 71–89% yields. Moreover, thiomorpholine was also compatible, leading to the formation of **3t** in 72% yield. Gratifyingly, the mild reaction conditions, high yields of products, and good functional group tolerances clearly demonstrated the advantages of our pharmacophore hybrid strategy for the structural modification of OA. The

isolated compounds **3a**–**3t** were fully characterized by 1H and 13C NMR spectroscopy as well as high-resolution mass spectrometry (see the Supplementary Information for details).

**Figure 1.** Synthesis of OA-Dithiocarbamate Derivatives.

Having obtained a series of structurally diverse OA-dithiocarbamates, we next performed a systematic biological evaluation to examine whether introducing an extra dithiocarbamate group could improve antitumor activities. These compounds were evaluated by MTT assay against human pancreatic cancer (Panc1), human lung cancer (A549), human hepatoma cell (Hep3B), human hepatoma cell (Huh-7), human colon cancer (HT-29), and human cervical cancer (Hela) cells, with the widely used anticancer drugs fluorouracil, docetaxel, and cisplatin as positive controls (Table 2). Most of the compounds exhibited remarkable antiproliferative activities, and the IC50 values of ten selected compounds were less than 50 μM on certain tumor cell lines. Among them, compounds **3e**, **3i**, **3j,** and **3l** were shown to be excellent, with broad-spectrum antitumor activities as well as being up to 30-fold more potent than the natural product OA and the positive controls; this might be ascribed to the introduction of hydroxyl groups. Particularly, compound **3p** was also found to be a promising hit compound that was 20-fold more potent than the natural product OA against HT-29 cells. Moreover, the cytotoxicities of compounds **3a**-**3t** were also evaluated in human normal hepatocytes (LO2) to determine whether these compounds preferred killing tumor cells over normal cells. Excitingly, the IC50 value of compound **3e** in LO2 cells was 62.8 μM, which was several times higher than that in the tumor cells.

**Table 2.** In Vitro Cytotoxicity Data of OA and Its Derivatives.



**Table 2.** *Cont.*

*<sup>a</sup>* Concentration inhibiting 50% of cell growth for 48 h exposure period of tested samples. *<sup>b</sup>* ND, not determined.
