Synthesis of Oleanolic Acid-Dithiocarbamate Conjugates and Evaluation of Their Broad-Spectrum Antitumor Activities

Efficient and mild synthetic routes for bioactive natural product derivatives are of current interest for drug discovery. Herein, on the basis of the pharmacophore hybrid strategy, we report a two-step protocol to obtain a series of structurally novel oleanolic acid (OA)-dithiocarbamate conjugates in mild conditions with high yields. Moreover, biological evaluations indicated that representative compound 3e exhibited the most potent and broad-spectrum antiproliferative effects against Panc1, A549, Hep3B, Huh-7, HT-29, and Hela cells with low cytotoxicity on normal cells. In terms of the IC50 values, these OA-dithiocarbamate conjugates were up to 30-fold more potent than the natural product OA. These compounds may be promising hit compounds for the development of novel anti-cancer drugs.


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][2][3][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][8][9][10][11]. Derivatization of OA has yielded a wide variety of novel compounds for anti-cancer investigations (Scheme 1) [11][12][13][14][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.

Scheme 2. Pharmaceutically Important Dithiocarbamates
In recent years, the pharmacophore hybrid strategy has emerged as an essential method for the discovery and modification of lead compounds [28][29][30][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][33][34][35]. In view of the high performance of dithiocarbamate derivatives in structural modification, the synthesis of OA-dithiocarbamate conjugates Scheme 2. Pharmaceutically Important Dithiocarbamates [23][24][25][26][27].
In recent years, the pharmacophore hybrid strategy has emerged as an essential method for the discovery and modification of lead compounds [28][29][30][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][33][34][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][24][25][26][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][8][9][10][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.

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 CS 2 and pyrrolidine in a one-pot manner afforded the target product 3a in an 80% isolated yield. In the "standard" conditions, 2 equiv. of K 3 PO 4 was shown to be essential to yield the desired product 3a (Entries 1-4, Table 1). Lowering the loading of K 3 PO 4 to 1.5 equiv. led to a decreased yield of 3a (Entry 1, Table 1), while replacement of it by K 2 HPO 4 or Li 2 CO 3 resulted in no desired product (Entries 2-3, Table 1). On the other hand, in the presence of 2 equiv. of K 2 CO 3 , product 3a could be isolated with a 62% yield (Entry 4, Table 1). Changing the reaction temperature or using other solvents, such as DMF, CH 3 CN, and EtOH, did not offer better results (Entries 5-8, Table 1). Lower amounts of CS 2 or pyrrolidine resulted in a decreased yield of 3a (Entries 9-10, Table 1). dithiocarbamate conjugates has not yet been documented in the literature [7][8][9][10][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.

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). Variations from the "standard" conditions. a Reaction temperature was raised to 60 °C. b CS2 was used in 3.0 equiv. instead of 4.5 equiv. c 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. Variations from the "standard" conditions. a Reaction temperature was raised to 60 • C. b CS 2 was used in 3.0 equiv. instead of 4.5 equiv. c 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 1 H and 13 C NMR spectroscopy as well as high-resolution mass spectrometry (see the Supplementary Information for details). 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 1 H and 13 C NMR spectroscopy as well as high-resolution mass spectrometry (see the Supplementary Information for details).
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 com- 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 IC 50 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 IC 50 value of compound 3e in LO2 cells was 62.8 µM, which was several times higher than that in the tumor cells.

General Information
All organic solvents were dried and distilled by standard methods prior to use. 1 H and 13 C NMR spectra were recorded on a Bruker AV II-400 spectrometer (BURKERT, Ingelfingen, Germany) at 400 and 100 MHz, respectively. All chemical shifts were reported in δ units with references to the residual solvent resonances of the deuterated solvents for proton and carbon chemical shifts. High Resolution Mass Spectra (HRMS) were obtained on a Thermo Q Exactive™ Focus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (SCIEX, Framingham, Massachusetts, USA). All other chemicals were purchased from either Aldrich (Sigma-Aldrich, Shanghai, China) or Aladdin Chemical Co. (Aladdin Holdings Group Co., Ltd, Shanghai, China) and used as received, unless otherwise specified.
The optical density at 490 nm of each well was measured using a microplate reader (Molecular devices corporation, Sunnyvale, CA. USA) to calculate the percent of cell viability. The inhibition rates were calculated using GraphPad Prism 7.0 software. The seven tested cell lines were obtained from the laboratory of Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University.

Conclusions
In summary, we have synthesized a series of OA-dithiocarbamate derivatives in a twostep protocol at room temperature, offering a readily accessible synthetic route to obtain novel OA derivatives in high yields. Moreover, some of the compounds were shown to be promising hit compounds, with remarkably improved broad-spectrum antiproliferative activities compared to the natural product OA. Mechanistic insights of their activities on certain tumor cell lines are currently underway in our laboratory.