International Journal of Molecular Sciences

Review Ursolic Acid-Based Derivatives as Potential Anti-Cancer Agents: An Update

Vuyolwethu Khwaza , Opeoluwa O. Oyedeji and Blessing A. Aderibigbe * Department of Chemistry, University of Fort Hare, Alice Campus, Alice 5700, Eastern Cape, South Africa; [email protected] (V.K.); [email protected] (O.O.O.) * Correspondence: [email protected]



Received: 25 April 2020; Accepted: 21 May 2020; Published: 18 August 2020


Abstract: Ursolic acid is a pharmacologically active pentacyclic triterpenoid derived from medicinal plants, fruit, and vegetables. The pharmacological activities of ursolic acid have been extensively studied over the past few years and various reports have revealed that ursolic acid has multiple biological activities, which include anti-inflammatory, antioxidant, anti-cancer, etc. In terms of cancer treatment, ursolic acid interacts with a number of molecular targets that play an essential role in many cell signaling pathways. It suppresses transformation, inhibits proliferation, and induces apoptosis of tumor cells. Although ursolic acid has many benefits, its therapeutic applications in clinical medicine are limited by its poor bioavailability and absorption. To overcome such disadvantages, researchers around the globe have designed and developed synthetic ursolic acid derivatives with enhanced therapeutic effects by structurally modifying the parent skeleton of ursolic acid. These structurally modified compounds display enhanced therapeutic effects when compared to ursolic acid. This present review summarizes various synthesized derivatives of ursolic acid with anti-cancer activity which were reported from 2015 to date.

Keywords: ursolic acid; derivatives/analogs; structural modification; anti-cancer activity

1. Introduction At present, cancer remains a significant health problem and is the second leading cause of death worldwide. The Global Cancer Incidence, Mortality and Prevalence (GLOBOCAN) recently reported that in 2018 about 18.1 million people were diagnosed with cancer and more than 9.6 million deaths were reported due to cancer [1]. Made et al. indicated that by 2025, the number of people living with cancer will increase [2]. In general terms, cancer is defined as a tumor resulting from the abnormal proliferation of cells that can spread to various organs of the body. Most of the currently used treatment includes the combination of chemotherapeutic agents, surgery, radiotherapy, or hormone therapy. Despite the above treatment strategies, there are still many limitations associated with cancer treatment such as multi-drug resistance, unselective targeting of the cancer cells, drug toxicity, etc. [3]. The shortage of suitable cancer chemopreventive procedures suitable for improving the therapeutic outcomes during an anticancer treatment has motivated researchers around the world to test the anticancer effect of biomolecules obtained from natural sources. Natural products are one of the main sources of pharmacologically active compounds, and they are potentially useful for the development of drugs. Generally, natural products are more environmentally friendly for frequent use when compared to synthesized drugs [4]. Extensive research has been performed to study the therapeutic effect of drugs derived from plants against a number of cancers, and the outstanding results indicate that most natural active compounds have potent anticancer effects [5]. Natural compounds can inhibit the formation and development of cancer by specifically interacting with multiple cell-signaling pathways [6]. These properties make it possible to affect multiple cancer hallmarks [7]. As a result,

Int. J. Mol. Sci. 2020, 21, 5920; doi:10.3390/ijms21165920 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 5920 2 of 27 about 74% of FDA-approved drugs are either natural products or natural product-derived [8]. Active chemical compounds originally isolated from natural products contain some dominant structures which can be modified to form new effective drugs [9]. A class of natural products called triterpenoids is among the most important group of phytochemicals originally derived from plants, with approximately 20,000 chemical structures confirmed so far [10]. Triterpenoids comprise six isoprene units with a basic molecular formula (C30H48). In terms of biological perspectives, the large group of pentacyclic triterpenoids with basic molecular structures with five membered rings have attracted the attention of many researchers due to their notable broad spectrum of pharmacological activities including anticancer, anti-inflammatory, antioxidant, antiviral, antimicrobial, etc. [11,12]. Ursolic acid (UA) 3-(β-hydroxy-urs-12-en-28-oic acid) is a ubiquitous pentacyclic compound that possesses functional groups such as a carboxylic moiety at C28, β-hydroxy function at C3, and an alkene at C12-C13. UA was first identified in the 1920s from the epicuticular waxes of apples [13,14] and it has been isolated in recent years from many other plant organs. Some plant species containing UA as an active constituent are listed in Table1.

Table 1. Several plant species with (ursolic acid) UA constituent.

Plant Species (Family) Plant Parts Used Bioactivities Bibliography Arctostaphylos uva-ursi Leaves Antitumor, antibacterial [15,16] (L.) Spreng (Ericaceae) Argania spinosa (L.) Fruits, leaves Antibacterial, antifungal [17,18] Skeels (Sapotaceae) Bouvardia ternifolia (Cav.) Schltdl. Aerial parts Anti-Alzheimer [19] (Rubiaceae) Bursera cuneata (Schldl.) Aerial parts (stems and Anti-inflammatory, [20] Engl (Burseraceae) leaves) antihistaminic Catharanthus roseus (L.) G. Leaves Anticancer [21] Don (Apocynaceae) Cornus mas (L.) Fruits Antitumor [22] (Cornaceae) Anti-cancer, Eriobotrya japonica anti-osteoclastic, skin (Thunb.) Lindl Leaves disorder [23–27] (Rosaceae) anti-inflammatory, and anti-arthritic Eucalyptus globulus Antioxidant, Leaves, bark [28,29] (Labill.) (Myrtaceae) neuroprotective Fragrae fragrans (Roxb.) Leaves, fruits, bark Antimycobacterial [30] (Gentianaceae) Ilex aquifolium (L.) Anticancer, antimalarial, Leaves [31] (Aquifoliaceae) antibacterial Lamium album (L.) Antioxidant and Flowers [32] (Lamiaceae) anti-inflammatory Antifungal, Lantana Camara (L.) Leaves antiproliferative, [33–35] (Verbenaceae) anti-diabetes, anxiolytic Lepidozia chordulifera Leaves Antibacterial [36] (Dumort.) (Porellaceae) Ligustrum lucidum (Ait.) Coronary heart disease Fruits [37,38] (Oleaceae) and diabetes Int. J. Mol. Sci. 2020, 21, 5920 3 of 27

Table 1. Cont.

Plant Species (Family) Plant Parts Used Bioactivities Bibliography Malus domestica (sp.) Fruits, leaves Antioxidant [39] (Rosaceae) Malus pumila (Mill.) fruits Antitumor [40] (Rosaceae) Ocimum forskolei Aerial parts (leaves and Antiulcer [41] (Benth.) (Lamiaceae) stems) Induced arthritis, Ocimum sanctum (L.) leaves antiproliferative, [42–44] (Lamiaceae) anti-stress Panax ginseng (C.A. Mey.) Roots and rhizomes Anticancer, antiviral [45] Baill. (Araliaceae) Paulownia tomentosa (Thunb.) Steud. Leaves Anticancer [46] (Scrophulariaceae) Prunella vulgaris (L.) Aerial parts Antiviral, antiestrogenic [47,48] (Lamiaceae) Psidium guajava (L.) Hypoglycaemic, Leaves [49] (Myrtaceae) antimicrobial Rabdosia rubescens (Linn.) Anti-tumour Antitumor [50] (Lamiaceae) Rosmarinus officinalis (L.) Stems and leaves Antidepressant [51] (Lamiaceae) Sambucus australis (Cham. Antibacterial and Aerial parts [52] & Schltdl.) (Adoxaceae) Antioxidant Saurauja roxburghii (Wall.) Leaves Anticancer [53] (Dilleniaceae) Anticancer, Thymus vulgaris (L.) Aerial parts (stems and cardiovascular, [54–56] (Lamiaceae) leaves) antihyperlipidemic, antioxidant, antifungal Tribulus arabicus (Hosni.) Antihyperuricemic, Aerial parts [57,58] (Zygophyllaceae) antioxidant Paulownia tomentos (Thunb.) Steud. Fruits Anticancer [46] (Scrophulariaceae) Punica granatum (Linn.) Flowers Antioxidant, antidiabetic [59,60] (Punicaceae) Uncaria rhynchophylla Stems and hooks Anticancer [61,62] (Gouteng.) (Rubiaceae) Antibacterial, Vitex negundo (L.) Leaves antifeedant against the [63] (Lamiaceae) larvae Ziziphus jujuba (Mill.) Anticancer, anti-obesity, Leaves [64] (Rhamnaceae) and antioxidant

UA possesses various interesting biological activities including anticancer, anti-inflammatory, antimicrobial, antidiabetic properties etc. [65–68]. Some reports have extensively explored the pharmacological properties of UA, as shown in Table2. In terms of the anticancer properties, studies have demonstrated that UA can modulate the cellular transcription factor; growth factor receptors; Int. J. Mol. Sci. 2020, 21, 5920 4 of 27

cytokines inflammatory; and many other molecular targets which regulate the cell proliferation, metastasis, apoptosis, angiogenesis, and autophagy of cancer cell lines through different mechanisms and signaling pathways [69,70] The anticancer effects of UA have been reported for various types of cancers such as endometrial [71,72], pancreatic [73,74], lung [75–77], prostate [78–80], ovarian [81,82], bladder [83], gastric [84,85], and liver cancers [86]. Other UA molecular targets reported for the treatment of cancer include its effect on p53 pathways [87–90]; the canonical pathway (Wnt/β-catenin) [91,92]; Ras signaling [93]; and transcription pathways like nuclear factor kappa light chain enhancer of activated B cells (NF-Kb) [94], Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) [95], and the Signal transducer and activator of transcription 3 (STAT3) family of transcription factors [96–98]. Int.Figure J. Mol. Sci.1 below 2020, 2 depicts1, x FOR PEER the various REVIEW molecular targets regulated by UA. 4 of 28

Figure 1. Multiple molecular targets modulated by UA. Figure 1. Multiple molecular targets modulated by UA. The biopharmaceutical classification system (BCS) classified UA as a class IV drug with limited The biopharmaceutical classification system (BCS) classified UA as a class IV drug with limited pharmacological effect due to its low water solubility and difficulty in permeating some biological pharmacological effect due to its low water solubility and difficulty in permeating some biological membranes. Drugs in this class not only have slow dissolution but also have limited gastrointestinal membranes. Drugs in this class not only have slow dissolution but also have limited gastrointestinal mucosa penetration, which results in their low oral bioavailability [99,100]. Due to the aforementioned mucosa penetration, which results in their low oral bioavailability [99,100]. Due to the reasons, most researchers have developed some new strategies to enhance the biopharmaceutical aforementioned reasons, most researchers have developed some new strategies to enhance the effect of UA via loading in nanoformulations or the structural modification of its structure. Chen et biopharmaceutical effect of UA via loading in nanoformulations or the structural modification of its al. reported various derivatives of UA with potential anticancer activities until 2015 [101]. The main structure. Chen et al. reported various derivatives of UA with potential anticancer activities until contents of the present review provide an update on reported UA derivatives with potential anticancer 2015 [101]. The main contents of the present review provide an update on reported UA derivatives activities reported over the last five years (2015–2020). with potential anticancer activities reported over the last five years (2015–2020). Table 2. Reports of ursolic acid’s significant pharmacological activities. Table 2. Reports of ursolic acid’s significant pharmacological activities. Sr. No. Pharmacological Activities Bibliography Sr. No. Pharmacological Activities Bibliography 1 1Antioxidant Antioxidant [102–104] [102–104] 2 2Antibacterial Antibacterial [52,67,105–110[52,67,105] –110] 3 3Antifungal Antifungal [111–117] [111–117] 4 Anticancer [13,118–123] 4 Anticancer [13,118–123] 5 Antidiabetic [34,124–128] 6 5Anti-inflammatory Antidiabetic [34,124–128] [129–133] 7 6Antiviral Anti-inflammatory [129–133] [134–136] 7 Antiviral [134–136] 2. An overview of Pharmacokinetics of UA and its Derivatives As mentioned earlier, UA is among the most studied triterpenoids and possesses a wide range of pharmacological activities, but its poor water solubility hinders its efficacy and potential as an appropriate therapeutic drug [137,138]. To overcome its poor water solubility, a number of structure– activity relationships (SARs) of the synthesized derivatives have been studied. The structural modification of UA is a suitable approach to enhance its pharmacokinetic profiles. UA and its derivatives have numerous pharmacological activities and most of them have been extensively screened and reviewed in recent published articles and reviews [101,138]. In terms of toxicity, UA and its derivatives are safe phytochemical compounds with low toxicity. In vitro studies have revealed the cytotoxic activity of UA and its derivatives on tumor cell lines and low cytotoxic effects against normal cell lines [139,140]. Additionally, the in vivo toxicity analysis of UA in animals showed no sign of toxicity in Kunming mice (0.2 mL/10 g) [141]. UA displayed a significant anticancer activity in animal models in vivo [142,143]. Several studies have reported the pharmacokinetics of UA and its derivatives in different animal models. Alzate and colleagues used Phoenix WinNonlin software to determine the pharmacokinetic profile of UA administered to Wistar rats at different doses and

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2. An overview of Pharmacokinetics of UA and Its Derivatives As mentioned earlier, UA is among the most studied triterpenoids and possesses a wide range of pharmacological activities, but its poor water solubility hinders its efficacy and potential as an appropriate therapeutic drug [137,138]. To overcome its poor water solubility, a number of structure–activity relationships (SARs) of the synthesized derivatives have been studied. The structural modification of UA is a suitable approach to enhance its pharmacokinetic profiles. UA and its derivatives have numerous pharmacological activities and most of them have been extensively screened and reviewed in recent published articles and reviews [101,138]. In terms of toxicity, UA and its derivatives are safe phytochemical compounds with low toxicity. In vitro studies have revealed the cytotoxic activity of UA and its derivatives on tumor cell lines and low cytotoxic effects against normal cell lines [139,140]. Additionally, the in vivo toxicity analysis of UA in animals showed no sign of toxicity in Kunming mice (0.2 mL/10 g) [141]. UA displayed a significant anticancer activity in animal models in vivo [142,143]. Several studies have reported the pharmacokinetics of UA and its derivatives in different animal models. Alzate and colleagues used Phoenix WinNonlin software to determine the pharmacokinetic profile of UA administered to Wistar rats at different doses and routes (i.e., 1 mg/kg intravenously and 20 and 50 mg/kg orally). The results indicated that the oral bioavailability of UA was significantly different between the two groups that were administered UA orally at doses of 20 mg/kg (2.8%) and 50 mg/kg (1.55%) [144]. Liao et al. developed and validated a rapid, sensitive, and accurate liquid chromatography–mass spectrometry (LC–MS) method for the determination of ursolic acid in rat plasma. In this procedure, rat plasma was acidified with acetic acid and then extracted with a mixture of hexane-dichloromethane-2-propanol (20:10:1, v/v/v). This LC-MS method was successfully used for pharmacokinetic studies after the oral administration of a Lu-Ying extract containing 80.32 mg/kg UA to the rats [145]. Furthermore, UA was established as an internal standard to determine the glycyrrhetic acid and gambogic acid in human plasma to determine their pharmacokinetics using sensitive liquid chromatography-electrospray ionization-mass spectrometry (LC–ESI-MS) [146,147]. However, a study showed that, after the administration of a UA solution, liposome-loaded UA, or chitosan-coated UA liposome (CS-UA-L) to mice models via the intra-gastric route, the content of UA was high in the liver, spleen, and kidney for the group administered the UA solution when compared to those administered with the UA liposome and CS-UA-L groups. However, a significantly greater amount of UA was accumulated at the tumors for the mice treated with CS-UA-L, which was 4.2- and 1.7-fold higher than those administered the UA solution and UA liposome groups, respectively. CS-UA-L significantly and selectively accumulated in the tumor tissues [148]. Interestingly, UA-medoxomil (NX-201), a UA prodrug (a derivative of UA with a structural modification of C-28 position) showed an improved bioavailability about 200 times better than UA in a rodent model. According to an in vivo test performed with a human pancreatic cancer (PANC-1) xenograft Severe Combined Immunodeficient (SCID) mouse model, the tumor growth rate decreased in a dose-dependent approach and a 100 mg/kg dose of NX-201 had an anticancer effect comparable to gemcitabine [149]. Despite the various reports on the pharmacokinetics of UA and its derivatives, there are currently few clinical trials. UA has been loaded into liposomes and there are few reports [150–153].

Clinical Trials of UA A few clinical trials have been conducted to assess the pharmacokinetics of various UA formulations using both healthy volunteers and patients with different types of cancers. UA is currently undergoing phase I trials to investigate its safety and adverse effects in patients. Wang et al. investigated liposomal ursolic acid (LUA) pharmacokinetics, maximum tolerated dose (MTD), and dose-limiting toxicity (DLT) in healthy adult volunteers and patients with advanced solid tumors. A total of 63 subjects (i.e., 35 healthy volunteers, 24 adults, and 4 patients) received a single dose of LUA (11, 22, 37, 56, 74, 98, and 130 mg/m2) administered as a 4 h intravenous infusion. The clinical data indicated that LUA had low toxicity with a MTD of 98 mg/m2 [152]. Other studies demonstrated Int. J. Mol. Sci. 2020, 21, 5920 6 of 27 that incorporating UA into liposomes can increase the ceramide content of the human subject’s skin, with an increase in the hydroxyl-ceramides appearing after three treatments [153]. Zhu et al. also investigated the single and multiple-dose pharmacokinetics and the safety of UA nanoliposomes (UANL) in 24 healthy volunteers and 8 patients with advanced solid tumors. The 24 healthy volunteers were divided into three groups which received a single dose of 37, 74, and 98 mg/m2 of UANL. Eight patients received multiple doses of 74 mg/m2 of UANL daily for 14 days. Ultra-performance liquid chromatograph-tandem mass spectrometry was used to determine the UA plasma concentrations [150]. The UANL was found to be safe and showed apparent linear pharmacokinetics (PK) behavior for a dose level ranging from 37 mg/m2 to 98 mg/m2. The repeated UANL administration indicated no drug accumulation even with 14 days of continuous IV infusion. Patients with solid tumors and healthy volunteers tolerated the IV infusion of UANL very well [150]. These studies clearly suggest that UA hasInt. J. tremendous Mol. Sci. 2020, 2 potential1, x FOR PEER to be REVIEW developed into a potent anticancer drug. 6 of 28

3. Chemistry of UA The structure of a pentacyclic triterpenoid UA comprises C C-30-30 isoprenoid isoprenoid;; it has a low water solubility but is highly soluble in alcoholic NaOH and glacial aceticacetic acid.acid. It is a white crystallin crystallinee solid with a melting point and molecular weight of 284 °C◦C and and 456.70 456.70,032,032 g/mol g/mol,, respectively respectively.. Its maximum UV absorption wavelength is ~450 nm [1 [15454]].. 3.1. UA Derivatives as an Anticancer Agent 3.1. UA Derivatives as an Anticancer Agent Many researchers have studied various strategies to modify the molecular structure of UA and Many researchers have studied various strategies to modify the molecular structure of UA and simultaneously enhance its therapeutic effect. The structure of UA has been broken down into simultaneously enhance its therapeutic effect. The structure of UA has been broken down into functional groups or pharmacophores to classify the major active sites for structural modification functional groups or pharmacophores to classify the major active sites for structural modification (Compound 1, Figure2). These sites are broadly categorized as the carboxylic moiety (C-28), β-hydroxy (Compound 1, Figure 2). These sites are broadly categorized as the carboxylic moiety (C-28), β- function (C-3), and an alkene at C12–C13. The sites have been extensively studied for their potential hydroxy function (C-3), and an alkene at C12–C13. The sites have been extensively studied for their anti-cancer activity. Some derivatives that are not identified in any of the aforementioned three potential anti-cancer activity. Some derivatives that are not identified in any of the aforementioned pharmacophores are separately categorized into miscellaneous groups. The following reports further three pharmacophores are separately categorized into miscellaneous groups. The following reports detailed the literature previously reported under these active sites and their successfully enhanced further detailed the literature previously reported under these active sites and their successfully anticancer activity. enhanced anticancer activity.

30

29 20 19 21 E 12 18 22 11 17 25 26 13 OH D 28 1 9 C 14 2 16 10 O B 8 15 3 A 4 7 5 27 1 HO 6

24 23 Figure 2. Structure of UA indicating the major active sites. Figure 2. Structure of UA indicating the major active sites. 3.1.1. Modification of the Carboxylic Moiety (C-28) 3.1.1. Modification of the Carboxylic Moiety (C-28) Tian et al. synthesized a series of UA derivatives bearing diamine moieties at C-28 and investigated theirTian antiproliferative et al. synthesized potential a against series threeof UA human derivatives cancer cellbearing lines diamine (MCF-7, HeLa, moieties and at A549). C-28 This and studyinvestigated was performed their antiproliferative as a comparative potential analysis against against three the human conventional cancer antitumor cell lines (MCF drug,- gefitinib.7, HeLa, and The derivativesA549). This werestudy prepared was performed by first protectingas a comparative the C3-OH analysis via acetylation, against the followedconventional by esterification antitumor drug with, 2-hydroxyaceticgefitinib. The derivatives acid at the were C-28 prepared position by and first amidation protecting reaction the C3 with-OH amines via acetylation, including followed piperazine, by esterification with 2-hydroxyacetic acid at the C-28 position and amidation reaction with amines including piperazine, N-methylpiperazine and alkane-1, 6-diamines and alkane-1, 4-diamines and alkane-1, and 2-diamines, as shown in Scheme 1 and Scheme 2. The half maximal inhibitory concentration (IC50) values for most of these derivatives were significantly higher when compared to gefitinib. The study revealed that derivatives containing primary amine moieties were more effective when compared to those with secondary or tertiary amine moieties. The antiproliferative activities all of the secondary amines were more active than those of the tertiary amine compounds. Compound 9a (Scheme 2, Table 3) showed a more potent antiproliferative activity than other derivatives [155].

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N-methylpiperazine and alkane-1, 6-diamines and alkane-1, 4-diamines and alkane-1, and 2-diamines, as shown in Schemes1 and2. The half maximal inhibitory concentration (IC 50) values for most of these derivatives were significantly higher when compared to gefitinib. The study revealed that derivatives containing primary amine moieties were more effective when compared to those with secondary or tertiary amine moieties. The antiproliferative activities all of the secondary amines were more active than those of the tertiary amine compounds. Compound 9a (Scheme2, Table3) showed a more potent antiproliferativeInt. J. Mol. Sci. 20 activity20, 21, x FOR than PEER other REVIEW derivatives [155]. 7 of 28

R1 O 5 HO

d

OH a R1 R 1 b R1 c R O c R1 O O O O HO 1 O 2 3 4 AcO HO 4 HO

e 2 R1 = OH e 3 R1 = Cl 4 R1 = OCH2COOH 5 R = OCH CO N N CH 5 R1 = OCH2CO N N CH3 R1 R1 N N Bn O f 6 R1 = OCH2CO N N Bn O 7 O HO 6 7 R1 = OCH2CO N NH 1 2 HO

SchemeScheme 1. Reagents 1. Reagents and and conditions: conditions: (a ()a Ac) Ac2O,2O, DMAP,DMAP, THF, rt; ( (bb)) (COCI) (COCI)2, 2CH, CH2Cl22Cl. Rt;2. ( Rt;c) HOCH (c) HOCH2 2 COOH, TEA, rt; (d) N-methylpiperazine, EDCI, DMAP, CH2Cl2, 0 °C to rt; (e) Benzylpiperazine, EDCI, COOH,COOH, TEA, TEA, rt; (d rt;) N-methylpiperazine,(d) N-methylpiperazine, EDCI, EDCI, DMAP, DMAP, CHCH2Cl22, ,0 0°C◦C to tort; rt;(e) (Benzylpiperazine,e) Benzylpiperazine, EDCI, EDCI, DMAP, CH2Cl2, 0 °C to rt; (f) 10% Pd/C, H2 anhydrous ethanol, rt. DMAP, CH2Cl2, 0 ◦C to rt; (f) 10% Pd/C, H2 anhydrous ethanol, rt.

O O O O OH O OH O R R1 R1 O b 1 a O b O HO HO HO 9a-c 4 HO 8a-c 9a R = NH(CH ) NH 8a R = NH(CH ) NHBoc 9a R1 = NH(CH2)2NH2 8a R1 = NH(CH2)2NHBoc 9b R = NH(CH ) NH 8b R = NH(CH ) NHBoc 9b R1 = NH(CH2)4NH2 8b R1 = NH(CH2)4NHBoc 9c R = NH(CH ) NH 8c R = NH(CH ) NHBoc 9c R1 = NH(CH2)6NH2 8c R1 = NH(CH2)6NHBoc Scheme 2. Reagents and conditions: (a) N-Boc-Diamine, EDCl, DMAP, CH Cl , 0 C to rt; (b) TFA, Scheme 2. Reagents and conditions: (a) N-Boc-Diamine, EDCl, DMAP, CH2Cl2, 0 2°C to◦ rt; (b) TFA, CH2ClCH2,2 0Cl◦2,C 0 to°C rt. to rt.

A seriesA series of new of new derivatives derivatives of of UA UA bearing bearing oxadiazole,oxadiazole, piperazine, piperazine, and and triazolone triazolone moieties moieties was was designeddesigned by Chiby Chi et al. et a andl. and they they were were evaluated evaluated forfor their anti anticancercancer activit activityy as asHypoxia Hypoxia-inducible-inducible factor-1factorα (HIF-1-1α (HIFα)-1 inhibitors.α) inhibitors The. Thein in vitro vitroresults results revealed that that these these derivatives derivatives had had a significantly a significantly enhancedenhanced antitumor antitumor activity activity by by inhibiting inhibiting the the expression expression of of HIF HIF-1-1α.α Compound. Compound 13b 13b(Table(Table 3) 3) demonstrateddemonstrated the bestthe best potent potent inhibitory inhibitory e ffeffectect against against HIF HIF-1-1αα activityactivity but but was was not not cytotoxic cytotoxic to cancer to cancer cells. These results indicated that the simple esterification of the UA carboxyl moiety can result in a cells.cells. These These results results indicated indicated that that the the simple simple esterificationesterification of of the the UA UA carboxyl carboxyl moiety moiety can canresult result in a in a significantly enhanced inhibitory effect on HIF-1α activity and decreased toxicity. The mechanism of significantly enhanced inhibitory effect on HIF-1α activity and decreased toxicity. The mechanism of action suggested that 13b can also suppress cell proliferation and block cell cycle progression in the action suggested that 13b can also suppress cell proliferation and block cell cycle progression in the G1 G1 phase (Scheme 3) [156]. phase (Scheme3)[156].

Int. J. Mol. Sci. 2020, 21, 5920 8 of 27 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 28

N Int.Int. J. J. Mol. Mol. Sci. Sci. 20 202020, ,2 211, ,x x FOR FOR PEER PEERN REVIEW REVIEW Br 88 of of 28 28 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW N O8 Hof 28 NH N NH N n 2 a b O O O N NN Br HO UA 10 11 N 12 N Br NH NN nn OOHH NH NN N n OH NNHH22 NN NH N NH2 aa N bb a b O O OO Oc O O 13a n = 1, IC50 = 49.4 µM (HRE) HO UA 1100 11 1122 HO UA 10 13b n = 2,1 I1C50 = 36.9 µM (HRE1)2 13c n = 3, IC50 = 46.5 µM (HRE) 13d n = 4, IC = 61.7 µM (HRE) c 50 c 13a n = 1, IC = 49.4 µM (HRE) 13a n = 1, IC5500 = 49.4 µM (HRE) 1133ba nn == 21,, IICC50 == 3469..94 µµMM ((HHRREE)) O 13b n = 2, IC5500 = 36.9 µM (HRE) 1133cb n n = = 3 2, ,I CIC50 = = 4 366.5.9 µ µMM ( H(HRREE)) O 13c n = 3, IC5500 = 46.5 µM (HRE) 1133dc nn == 43,, IICC50 == 6416..75 µµMM ((HHRREE)) N 13d n = 4, IC5500 = 61.7 µM (HRE) n N 50 O OO O O O 13a-d HO N nn N N O n N O Scheme 3. Synthetic route for compounds 10–H13O. Reagents and conditions:1133aa--dd (a) NH NHCOOCH , Scheme 3. Synthetic route for compounds 10–H13O. Reagents and conditions:13a - (da) NH2NHCOOCH2 3, 3

CH(OCCH(OC2H5)23H; Ethanol(EtOH),5)3; Ethanol(EtOH), MeONa, MeONa, reflux, reflux, 4848 h; ( b)) Br(CH Br(CH2)nBr;2)nBr; Potassium Potassium carbonate carbonate (K2CO (K32),CO KI; 3 ), KI;

(c)K (COc) K2SchemeCO,Scheme KI;3, (CHKI; 3. 3.(CH SyntheticSynthetic) CO,3)2CO, reflux, routereflux, route for for 10 10 compounds compoundsh.h. 1010––1313. . Reagents Reagents and and conditions: conditions: ( (aa)) NH NH2NHCOOCH2NHCOOCH3,3 , 2 3 Scheme 3. 3Synthetic2 route for compounds 10–13. Reagents and conditions: (a) NH2NHCOOCH3, CH(OCCH(OC2H2H5)5)3;3 ;Ethanol(EtOH), Ethanol(EtOH), MeONa, MeONa, reflux, reflux, 48 48 h; h; ( (bb)) Br(CH Br(CH2)nBr;2)nBr; Potassium Potassium carbonate carbonate (K (K2CO2CO3),3), KI; KI; CH(OC2H5)3; Ethanol(EtOH), MeONa, reflux, 48 h; (b) Br(CH2)nBr; Potassium carbonate (K2CO3), KI; ((cc)) K K2CO2CO3,3 ,KI; KI; (CH (CH3)3)2CO,2CO, reflux, reflux, 10 10 h. h. Liu etLiu al. et( synthesizedc al.) K 2synthesizedCO3, KI; (CH a3) number2CO,a number reflux, of10 of novelh. novel UAUA derivatives and and evaluated evaluated their their antitumor antitumor activities activities againstagainst two two human human cancer cancer cell cell lines—gastric lines—gastric cancercancer cell cellss (MGC (MGC-803)-803) and and breast breast cancer cancer cellscells (Bcap (Bcap-37)-37) LiuLiu et et al. al. synthesized synthesized a a number number of of novel novel UA UA derivatives derivatives and and evaluated evaluated their their antitumor antitumor activities activities using an MTTLiu et assay al. synthesized in vitro [157]a number. These of novel derivatives UA derivatives were obtaine and evaluatedd by reacting their antitumor UA at C activities-28 with 1,2- using an MTTagainst assaytwo humanin vitro cancer[157 cell ].lines These—gastric derivatives cancer cells (MGC were-803) obtained and breast by cancer reacting cells (Bcap UA- at37) C-28 with against two human cancer cell lines—gastric cancer cells (MGC-803) and breast cancer cells (Bcap-37) dibromoethane,using an MTT 1, 3assay-dibromoethane in vitro [157]., These1,4-dibromoethane derivatives were, and obtaine butyld by bromide reacting inUA a solutionat C-28 with of DMF1,2- and 1,2-dibromoethane,using an MTT 1,3-dibromoethane, assay in vitro [157]. These 1,4-dibromoethane, derivatives were obtaine andd by butyl reacting bromide UA at C in-28 a with solution 1,2- of DMF K2COdibromoethane,3dibromoethane,, followed by 1, reaction1,33--dibromoethanedibromoethane with corresponding, ,1, 1,44--dibromoethanedibromoethane amines., ,and and Most butyl butyl of bromide bromide the derivatives in in a a solution solution exhibited of of DMF DMF moderateand and and K2COdibromoethane,3, followed by1,3-dibromoethane reaction with, 1,4 corresponding-dibromoethane, and amines. butyl bromide Most in of a solution the derivatives of DMF and exhibited to highKK2 2COCOinhibitory33, ,followed followed activities by by reaction reaction when with with compared corresponding corresponding to UA amines. amines.. The Most Mostresults of of the theillustrated derivatives derivatives that exhibited exhibited compound moderate moderate 14 (Ta ble K2CO3, followed by reaction with corresponding amines. Most of the derivatives exhibited moderate moderateto high high inhibitory inhibitory activities activities when whencompared compared to UA. The to results UA. Theillustrated results that illustrated compound that14 (Ta compoundble 14 3) inducedto high cell inhibitory apoptosis activities in MGC when-803 compared cells. to UA. The results illustrated that compound 14 (Table (Table3) induced3) induced cell cell apoptosis apoptosis in in MGC MGC-803-803 cells. cells. 3) induced cell apoptosis in MGC-803 cells. Table 3. IC50 values of UA derivatives modified on the carboxylic moiety (C-28). TableTable 3. 3. IC IC5050 values values of of UA UA derivatives derivatives modified modified on on the the carboxylic carboxylic moiety moiety (C (C--28).28). Table 3. TableIC50 3.values IC50 values of UA of UA derivatives derivatives modifiedmodified on on thethe carboxylic carboxylic moiety moiety (C-28). (C-28).

R R R O OO HO HOHO

CompoCompo BiologicalBiologicalBiological CellCellCell Lines Lines Lines Tested Tested Tested ICReferenceReferenceReference Molecules Molecules Molecules CompoundCompoCompo R R BiologicalBiological CellCell LinesLines TestedTested 50ReferenceReference Molecules Molecules Bibliography BibliographyBibliography undund R ActivityActivity ICIC5050 (µM) (µM)µ ICIC5050 (µM) (µM) µ Bibliography und R ActivityActivity IC50 (µM)( M) IC50 (µM)IC50 ( M) Bibliography und Activity IC50 (µM) MCF-7 (Gefitinib)IC50 (µM) 17.83 ± MCF-7 (Gefitinib)MCF-7 (Gefitinib)17.83 ± MCF-7 7.85(Gefitinib)7.85 17.83 ± OO MCFMCF--77 (8.45 (8.45 ± ± 0.26) 0.26) 7.8517.83 7.85 O Antiproliferati MCF-MCF-77 (8.45 (8.45± 0.26) 0.26)Hela (Gefitinib)7.85 15.40 ± ± O NH Antiproliferati ± Hela (Gefitinib)Hela (Gefitinib)15.40 ± 9a9a O O NH2 2 Antiproliferati MCFHelaHela- 7(8.37 (8.37 (8.45 ± ± 0.11) ±0.11) 0.26) Hela (Gefitinib) 15.40 ± [155][155] 9a 9a O NN NH2 Antiproliferative Hela Hela(8.37 (8.37± 0.11) 0.11) [155] [155] HNH Antiproliferativeve ± Hela (Gefitinib)4.654.6515.40 15.404.65 ± O HNH2 ve A549A549 A549(10.06 (10.06 (10.06± ± 1.39) 1.39) 1.39) 4.65 ± 9a N HelaA549 (10.06(8.37 ±± 0.11)1.39) A549 (Gefitinib)A549 11.02 (Gefitinib) ± [155] H ve ± A549 (Gefitinib)4.65 11.02 ± A549 (10.06 ± 1.39) 3.2711.02 3.27 A549 (Gefitinib)3.27 ± 11.02 ± OO OO O AAnticancernticancerAnticancer 3.27 13b 13b O 2 NN N Anticancer HRE (36.9)HRE (36.9)n.d n.d[156] [156] 13b 2 N N HRE (36.9) n.d [156] 13b 2 N N activityactivity HRE (36.9) n.d [156] O activity O Anticancer 13b 2 N N HREMGCMGC (36.9)--803803 MGCMGC--803(UA)803(UA)MGC-803(UA)n.d [156] OO activityAnticancer MGC-803 MGC-803(UA) O NN Anticancer MGC-803 1414 N AnticancerAnticancer (4.99(4.99 ± ± 0.40) 0.40) 26.5126.51 ± ± 1.126.51 1.1 1.1 [157][157] 14 OO NN (4.99 ± 0.40) 26.51 ± 1.1 [157] 14 O N activityactivity (4.99 0.40) ± [157] activityactivity BcapBcap-MGC-3737 (8.56 (8.56-803 ± ± ±0.44) 0.44) BcapBcapMGC--37(UA)37(UA)Bcap-37(UA)-803(UA) O Bcap-Bcap-3737 (8.56 (8.56± 0.44) 0.44) Bcap-37(UA) N Anticancer ± 31.39 0.85 14 (4.99 ± 0.40) 26.51 ± 1.1 [157] O N activity ± Bcap-37 (8.56 ± 0.44) Bcap-37(UA) 3.1.2. Modification of both β-hydroxy (C-3) and Carboxylic Moiety (C-28) Hua et al. synthesized a series of new UA derivatives by incorporating piperazine and thiourea at the C28 position and evaluated their in vitro cytotoxicity against selected cancer cell Int. J. Mol. Sci. 2020, 21, 5920 9 of 27 lines—namely HCT-116, T24, MGC-803, HepG2, and A549 cells and a normal cell line, HL-7702, using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor (MTT) assay. The in vitro antiproliferative results suggested that most of the derivatives exhibited a potent inhibitory effect. However, one compound displayed an excellent in vitro cytotoxicity on HepG2 cells with an IC50 value of 5.40 µM and significantly induced apoptosis in the HeG2 cell line (compound 15, Table4)[ 139]. Wiemann et al. designed a class of ursolic and oleanolic acid-derived hydroxamates in an attempt to investigate their cytotoxicity, using sulforhodamine B (SRB) assays against several human cancer cell lines (518A2, A2780, A549, FaDu, HT29, MCF-7, and NIH 3T3). The ursolic acid-based derivatives containing at least an OH and NH moiety in the hydroxamate part displayed good cytotoxicity and were significantly less selective to the cancer cells when compared to the oleanolic acid-based compounds. The results showed that compound 16 (Table4) was the most potent compound, with IC50 values ranging from 2.5 to 6.4 µM[158]. Nedopekina et al. synthesized conjugates of triterpenoids UA and betulinic acid with the triphenylphosphonium (TPP+) group; evaluated their cytotoxic activity against two human cancer cell lines; and also studied their ability to induce programmed cancer cell death by employing markers of apoptosis, including the activation of caspase-3, permeabilization of the outer mitochondrial membrane, PARP-1 cleavage, the release of cytochrome c, and the inhibition of the mitochondrial respiratory chain. Two of the conjugates derived from ursolic acid and betulinic acid indicated a good range of IC50 values. Compound 17 (Table4) derived from UA was obtained by the alkylation of the carboxyl group, C-28 of UA, with triethylene glycol dibromide in dimethylformamide (DMF) using K2CO3 at 50 ◦C for 3 h [159]. Jiang et al. synthesized a series of UA derivatives as inhibitors of Nuclear factor kappa B (NF-κB) by introducing long-chain amide moieties at the C-28 position, and the β-hydroxy group C-3 was protected by an acetyl group. Their in vitro anticancer properties and Tumor necrosis factor alpha (TNF-α-induced) NF-κB activation were evaluated against four human cancer cell lines, such as human ovarian cancer cells (SKOV3), lung cancer cells (A549), liver cancer cells (HepG2), and bladder cancer cells (T24). Several compounds exhibited considerable anticancer effects against different cancer cell lines. Compound 18 (Table4) demonstrated the highest potency by inhibiting the growth of SKOV3, A549, HepG2, and T24 cells with IC50 values of 8.95, 5.22, 6.82, and 6.01 µM, respectively. The IC50 values were five-fold to eight-fold lower than the parent UA, and the results showed that compounds with longer diamide side chains showed relatively enhanced activity compared to compounds with shorter diamide side chains. A related mechanism study indicated that compound 18 caused cell cycle arrest at the G1 phase and triggered apoptosis in A549 cells by blocking the NF-κB signaling pathway [94]. Zhang et al. designed and synthesized UA-based tetrazole derivatives and studied their potential antitumor effects as HIF-1α transcriptional inhibitors. Compound 19 was the most potent with (IC50 = 0.8 µM) (Table4). This compound was prepared by mixing the corresponding anhydrides with UA in a solution of trimethylamine and 4-dimethylaminopyridine (DMAP) in ice and stirred overnight at room temperature; the intermediate compound was reacted with SOCl2 in refluxing dichloromethane (DCM). The results suggest that introducing tetrazole at C-28 of UA increased the HIF-1α inhibitory effect; additionally, the introduction of large groups at C-3 is disadvantageous for the synthesis of effective HIF-1α inhibitors [160]. Kahnt et al. studied the ethylenediamine-spaced carboxamides of UA and betulinic acid and further analyzed their cytotoxicity effects against human tumor cells (8505C, A2780, MCF-7, HT29, NIH 3T3, and 518A2). The UA derivatives that demonstrated good in vitro cytotoxicity effects were compounds 20, 21, and 22)[161]. Wang et al. evaluated the antiproliferative activity of UA derivatives containing thiazole, triazole, tetrazolepiperazine, or homopiperazine moiety against two cancer cells, Hela and MKN45. Compound 23 (Table4) displayed a superior antiproliferative activity on both cell lines and induced apoptosis via the intrinsic mitochondrial pathway [162]. Meng et al. designed UA derivatives through multiple synthetic steps and evaluated the synthesized compounds in vitro using an MTT assay on two cancer cell lines (BEL7402 and SGC7901). The results showed a higher inhibitory rate of the synthesized Int. J. Mol. Sci. 2020, 21, 5920 10 of 27 compoundsInt.Int.on J. J. Mol. Mol. both Sci. Sci. 20 2020 cells20, 2, 12,1 x, xFOR whenFOR PEER PEER REVIEW compared REVIEW to the parent compound, UA. The results1010 of of 28 28 of molecular Int.Int. J. J.Mol. Mol. Sci. Sci. 20 202020, 2, 12,1 x, xFOR FOR PEER PEER REVIEW REVIEW 1010 of of 28 28 docking studiesapoptosisapoptosisInt. J. indicatedMol. via Sci.via the 20the20 intrinsic ,intrinsic 2 the1, x FOR role mitochondrial mitochondrialPEER of REVIEW the structural pa pathwaythway [162] [162] design. Meng. Meng and et et al. al. optimizationdesigned designed UA UA derivatives derivatives of UA. through Thethrough10 mostof 28 promising derivative wasmultipleapoptosismultipleapoptosis compound synthetic viasynthetic via the the intrinsic intrinsicsteps steps24, and asandmitochondrial mitochondrial shownevaluated evaluatedin the pathe pa Table thwaysynthesized thwaysynthesized4 [162] [[162]163. Meng.compounds]. Mengcompounds et et al. al. designed designedin in vitro vitro UAusing usingUA derivatives derivatives an an MTT MTT assay throughassay through on on apoptosis via the intrinsic mitochondrial pathway [162]. Meng et al. designed UA derivatives through Wolframtwomultipletwomultiple etcancer cancer al. synthetic synthetic converted cell cell lines lines steps steps (BEL7402 (BEL7402 pentacyclicand and evaluated evaluated and and SGC7901). SGC7901). triterpenoicthe the synthesized synthesized The The results results acids—namelycompounds compounds showed showed in a ina highervitro highervitro oleanolic, using usinginhibitory inhibitory an an MTT MTT betulinicrate rate assay assay of of the onthe on glycyrrhetinic, multiple synthetic steps and evaluated the synthesized compounds in vitro using an MTT assay on synthestwosynthestwo cancer cancerizedized cell compounds cellcompounds lines lines (BEL7402 (BEL7402 on on both both and andcells cells SGC7901). SGC7901).when when compared compared The The results results to to the the showed showedparent parent acompound, acompound,higher higher inhibitory inhibitory UA UA. .The The rate rateresults results of of the ofthe of boswellic, andtwo ursolic cancer acids—intocell lines (BEL7402 their and acetylated SGC7901).piperazinyl The results showed amides a higher by coupling inhibitory themrate of with the rhodamine molecularsynthesmolecularsynthesizedized docking docking compounds compounds studies studies on on indicated bothindicated both cells cells the thewhen when role role compared comparedof of the the structural structural to to the the parent designparent design compound, compound,and and optimization optimization UA UA. .The The of of results UA resultsUA. .The The of of synthesized compounds on both cells when compared to the parent compound, UA. The results of B. To evaluatemostmolecularmostmolecular theirpromising promising docking dockingin vitroderivative derivative studies studiescytotoxicity, wasindicated wasindicated compound compound the the all role role24 24 the , of, as ofas theshown triterpene-homopiperazinyl-rhodamine theshown structural structural in in Table Table design design 4 4 [163] [163] and .and . optimization optimization of of UA UA. .The The derivatives molecular docking studies indicated the role of the structural design and optimization of UA. The mostmostWolfram promisingWolfram promising etderivativeet derivative al. al. converted converted was was compound compound pentacyclic pentacyclic 24 24, as, as triterpenoicshown triterpenoicshown in in Table Table acids acids 4 4[163] —[163]—namelynamely. . oleanolic, oleanolic, betulinic betulinic were subjectedmost to promising RB assays derivative and most was compound of them 24 were, as shown highly in Table toxic 4 [163] against. numerous human cancer cell glycyrrhetinic,glycyrrhetinic,WolframWolfram boswellic et boswellic et al. al. converted,converted ,and and ursolic ursolic pentacyclic pentacyclic acids acids——intointo triterpenoic triterpenoic their their acetylated acetylated acids acids piperazinyl — piperazinyl—namelynamely oleanolic,amides amidesoleanolic, by by coupling betulinic coupling betulinic lines (A2780, A375,Wolfram HT29, et NiH3T3, al. converted MCF7, pentacyclic and SW1736). triterpenoic The ursolicacids—namely acid-homopiperazinyl-rhodamine oleanolic, betulinic themglycyrrhetinic,themglycyrrhetinic, with with rhodamine rhodamine boswellic boswellic B. B. , To ,andTo and evaluate evaluate ursolic ursolic their acids their acids in —in— intovitro vitrointo their cytotoxicity, theircytotoxicity, acetylated acetylated all all piperazinylthe piperazinylthe tr triterpeneiterpene amides amides-homopiperazinyl-homopiperazinyl by by coupling coupling- - glycyrrhetinic, boswellic, and ursolic acids—into their acetylated piperazinyl amides by coupling derivativerhodaminethem (Compoundrhodaminethem with with rhodamine derivativesrhodamine derivatives25, TableB. wereB. wereTo To evaluate subjected4evaluate subjected) was their their one to to RBin RBin ofvitro assaysvitro assays the cytotoxicity, cytotoxicity, and compounds and most most all ofall of the them themthe tr thattr iterpene wereiterpene were showed highly highly-homopiperazinyl-homopiperazinyl toxic toxic a strong aga againstinst- - cytotoxicity them with rhodamine B. To evaluate their in vitro cytotoxicity, all the triterpene-homopiperazinyl- against thenumerousrhodamine tumornumerousrhodamine cell human human derivatives derivatives lines, cancer cancer while were werecell cell subjected linesit subjectedlines was (A2780, (A2780, less to to RB A375, RBcytotoxicA375, assays assays HT29, HT29, and and onNiH3T3, NiH3T3, most most SW1736 of ofMCF7, MCF7, them them cells were and wereand SW1736). [SW1736). highly164 highly]. toxicKahnt toxicThe The aga ursolic agaursolic etinstinst al. synthesized rhodamine derivatives were subjected to RB assays and most of them were highly toxic against acidnumerousacidnumerous-homopiperazinyl-homopiperazinyl human human cancer cancer-rhodamine-rhodamine cell cell lines lines derivative derivative(A2780, (A2780, A375, (ComA375, (Com HT29,pound poundHT29, NiH3T3,25 NiH3T3,25, ,Table Table MCF7,4) MCF7,4) was was oneand oneand of SW1736).of SW1736).the the compounds compounds The The ursolic ursolic that that amine-spacednumerous conjugates human of cancer UA andcell lines 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic (A2780, A375, HT29, NiH3T3, MCF7, and SW1736). The ursolic acid (DOTA). showedacidshowedacid-homopiperazinyl-homopiperazinyl a a strong strong cytotoxicity cytotoxicity-rhodamine-rhodamine against against derivative derivative the the tumor tumor (Com (Com cell cellpound linespound lines, , while25 while 25, Table, Table it it was was4) 4) was lesswas less one cytotoxic onecytotoxic of of the the compoundson oncompounds SW1736 SW1736 cells thatcells that acid-homopiperazinyl-rhodamine derivative (Compound 25, Table 4) was one of the compounds that Two conjugates[164]showed[164]showed. .Kahnt(Compound Kahnt a astrong strong et et al. al. cytotoxicity cytotoxicitysynt synthesized26hesized, 27 against ,againstamine Tableamine the- space-the4space )tumor tumor displayeddd conjugates conjugates cell cell lines lines the , of while,of while UA highestUA itand itand was was 1,4,7,10 1,4,7,10 less cytotoxicityless cytotoxic cytotoxic-tetraazacyclododecane-tetraazacyclododecane on on onSW1736 SW1736 A375 cells cells melanoma- - and showed a strong cytotoxicity against the tumor cell lines, while it was less cytotoxic on SW1736 cells 1,4,7,10[164]1,4,7,10[164]. .Kahnt -Kahnttetraacetic-tetraacetic et et al. al. syntacid syntacidhesized (DOTA).hesized (DOTA). amine amine Two Two- spaceconjugates- spaceconjugatesdd conjugates conjugates (Compoundµ (Compound of of UA UA 26 26and ,andµ ,27 27 ,1,4,7,10 ,1,4,7,10Table Table 4)- tetraazacyclododecane4)- tetraazacyclododecanedisplayed displayed the the highest highest- - 26 A2780 ovarian[164] carcinoma,. Kahnt et al. with synthesized IC50 values amine-space of 1.5d conjugatesM and of 1.7 UA andM, 1,4,7,10 respectively.-tetraazacyclododecane Compound- induced cytotoxicity1,4,7,10cytotoxicity1,4,7,10-tetraacetic-tetraacetic o onn A375 A375 acid acid melanoma melanoma (DOTA). (DOTA). and Two andTwo A2780 conjugatesA2780 conjugates ovarian ovarian (Compound (Compound carcinoma carcinoma 26 ,26 ,with, with,27 27, ,TableIC TableIC5050 values values4) 4) displayed displayed of of 1.5 1.5 µM theµM the andhighest andhighest 1.7 1.7 the death ofµMµM1,4,7,10 A375, ,respectively. respectively.- cellstetraacetic by C Compound ompoundacid apoptosis (DOTA). 26 26 induced induced [Two165 conjugates]. the the Mang death death (Compound et of of al.A375 A375 synthesized cells cells 26 , by27 by ,apoptosis Table apoptosis UA4) displayed [165] [165] derivatives. . Mang Mang the ethighest et al. al. with potential cytotoxicitycytotoxicity o onn A375 A375 melanoma melanoma and and A2780 A2780 ovarian ovarian carcinoma carcinoma, ,with with IC IC5050 values values of of 1.5 1.5 µM µM and and 1.7 1.7 cytotoxicity on A375 melanoma and A2780 ovarian carcinoma, with IC50 values of 1.5 µM and 1.7 anticancer propertiessynthesizedµMsynthesizedµM, ,respectively. respectively. UA UA by derivatives derivatives modifyingC Compoundompound with with 26 26 C-2, potential potentialinduced induced C-3, anti theanti andthecancer cancerdeath death C-28 properties of properties of A375and A375 evaluatedcells by cells by modifying modifying by by apoptosis apoptosis their C C-2,- 2, [165] C in[165]C-3,-3, vitroand. and. Mang Mang C C-cytotoxicity28- 28 et etand and al. al. against µM, respectively. Compound 26 induced the death of A375 cells by apoptosis [165]. Mang et al. evaluatedsynthesizedevaluatedsynthesized their their UA UA in derivativesin derivativesvitro vitro cytotoxicity cytotoxicity with with potential potential against against anti HepG2, antiHepG2,cancercancer BGC BGC properties properties-823,-823, and and by byHeLa HeLa modifying modifying cell cell lines lines C C- 2,using- using2, C C-3,- 3,an andan and MTT MTT C C-28- assay28 assay and and HepG2, BGC-823,synthesized and UA HeLa derivatives cell with lines potential using anti ancancer MTT properties assay Theseby modifying derivatives C-2, C-3, and were C-28 synthesizedand by ThevaluatedThevaluatedeseese derivatives derivatives their their in in vitrowere vitrowere cytotoxicity synthesizedcytotoxicity synthesized against againstby by reacting reacting HepG2, HepG2, UA UA BGC BGC with with-823,-823, Jones Jones and and reagents HeLa reagentsHeLa cell cell in linesin linesacetone acetone using using and anand an MTT then MTTthen assay with assaywith reacting UAevaluated with Jones their reagentsin vitro cytotoxicity in acetone against and HepG2, then BGC with-823, and NH HeLa-OH cell linesHCl; using the an obtained MTT assay compounds NHThNHThese2ese-2OH·HCl- OH·HClderivatives derivatives; ;the the wereobtained wereobtained synthesized synthesized compounds compounds by by reacting were reactingwere then then UA UA reacted reactedwith with Jones withJones with reagents Ac reagentsAc2O22O in in the in thein · acetone presence acetonepresence and andof of thenDMAP DMAPthen with with in in THTHThF.F. eseThe The derivatives intermediate intermediate were compound compound synthesized was was bycondensed condensed reacting withUA with with the the relevant Jonesrelevant reagents amino amino orin or phenolicacetone phenolic and moieties moieties then within in were then reactedNHNH2-2OH·HCl-OH·HCl with; ;the the Acobtained obtained2O in compounds compounds the presence were were then then of DMAPreacted reacted with with in AcTHF. Ac2O2O in in Thethe the presence presence intermediate of of DMAP DMAP compoundin in was NH2-OH·HCl; the obtained compounds were then reacted with Ac2O in the presence of DMAP in condensedEtTH withEtTH3N3F.NF. toThe toThe the afford affordintermediate intermediate relevant the the targeted targeted compound aminocompound compounds. compounds. or was was phenolic condensed condensed Among Among moieties thesewith thesewith the derivatives,the derivatives, relevant relevant in Et aminoN Compoundsamino Compounds to aor orff phenolicord phenolic 2828 the and and moieties moietiestargeted 29 29 were were in in compounds. demonstratedTHF. The intermediate to be more compound effective on was the condensed cell lines when with comparedthe relevant3 to aminothe reference or phenolic drug ,moieties gefitinib in demonstratedEtEt3N3N to to afford afford to the tbehe targetedmore targeted effective compounds. compounds. on the cell Among Among lines thesewhen these derivatives,compared derivatives, to Compounds Compoundsthe reference 28 28drug and and, gefitinib29 29 were were Among theseEt derivatives,3N to afford the Compounds targeted compounds.28 and Among29 were thesedemonstrated derivatives, Compounds to be more28 and e29ffective were on the cell [166]demonstrated[166]demonstrated. . to to be be more more effective effective on on the the cell cell lines lines when when compared compared to to the the reference reference drug drug, ,gefitinib gefitinib demonstrated to be more effective on the cell lines when compared to the reference drug, gefitinib lines when[166] compared[166]. . to the reference drug, gefitinib [166]. [166]. TaTableble 4. 4. Modification Modification of of C C-3-3 and and C C-28-28 positions. positions. TaTableble 4. 4. Modification Modification of of C C-3- 3and and C C-28-28 positions. positions. Table 4.TableModification 4. Modification of of C-3C-3 and and C-28 C-28 positions. positions.

RR2 2

OORR2 2 R2 RR1 1 OO O RR1 1 CompComp RBiological1Biological CellCell Lines Lines ReferenceReference Molec Moleculesules BibliogrBibliogr RR1 1 RR2 2 oundound ActiviActivitiesties TestedTested(IC(IC5050µM) µM) (IC(IC5050µM)µM) aphyaphy CompComp BiologicalBiological CellCell Lines Lines ReferenceReference Molec Moleculesules BibliogrBibliogr RR1 1 RR2 2 MGCMGC-803-803 (9.82 (9.82 ± ± MGCMGC-803(UA)-803(UA) 27.0 27.088 ± ± Comp BiologicalBiological CellCell Lines 50Lines ReferenceReference50 Molec Moleculesules Bibliogr Compoundoundound R1 R1 R2R2 ActiviActivitiesties TestedTested(IC(IC50µM)µM) (IC(IC50µM)µM) aphyaphyBibliography ound ActivitiesActivities Tested(ICTested0.29)0.29)(IC 50 µ50M)µM) 0(IC0.29(IC.2950 50µM) µM) aphy S S MGCMGC-803-803 (9.82 (9.82 ± ± MGCMGC-803(UA)-803(UA) 27.0 27.08 8± ± HCTHCTMGC-116-116- 803(18.97 (18.97 (9.82 ± ± ± MGCHCTHCT--803(UA)116(UA)-116(UA) 27.0 8 ± NN NNHH 0.29)0.29) MGC-803(UA)0.290.29 S S 0.53)0.53) 38.738.788 ± ± 0 0.16.16 OO NN MGC-803HCTHCT-116-116 (9.820.29) (18.97 (18.97 0.29)± ± HCTHCT-27.08116(UA)-116(UA)0.29 0.29 15 NN NSHNH Cytotoxicity T24 (19.60 ± 0.43)± T24(UA) 29.29± ± 0.80 [139] 15 OOCHCH3 3 Cytotoxicity HCT-116T24HCT (19.60 (18.97-116 ± (18.970.43)0.53) ± T24(UA)HCTHCT-116(UA) 29.29-116(UA) ± 0.80 [139] HH3C3COOOO NN N NH 0.53)0.53) ± 38.738.78 8± ±0 .160.16 O N T24HepG2HepG2 (19.60 (15.720.53) (15.720.43) ± ± HepG2(UA)HepG2(UA)38.738.788 ±30.21± 30.21±0.160.16 15 1515 OOCOHCCH3H3 CCytotoxicityCytotoxicityytotoxicity T24T24 (19.60 (19.60 ±± ±0.43) 0.43) T24(UA)T24(UA) 29.29 29.29± ± ±0.80 0.80 [139][139] [139] H C O 3 3 15 3H3C O OOCHCH3 3 Cytotoxicity HepG2T240.84) (15.72(19.600.84) ± 0.43)0.84) T24(UA)T24(UA)0.580.58 29.29 29.29 ± 0.800.80 [139] OCH3 HepG2HepG2 (15.72 (15.72± ± ± HepG2(UA)HepG2(UA) 30.21± 30.21±± H3C O OCOHC3H3 A549A549A549HepG2 (20.79 (20.79(20.79 (15.72± ± 0.54) 0.54) ± A549(UA)A549(UA)HepG2(UA)HepG2(UA) 35.79 35.79 30.21± 30.21±± 0.37 0.37 0.58 OCOHCH OCH 0.84)0.84) ± 0.580.58 ± 3 3 3 HL-7702HL-7702 (˃(>˃100)100) A549(UA)HL-7702(˃˃ 35.79100) 0.37 OCH3 HL-77020.84) ( 100) HL-7702(0.58100) ± A549A549 (20.79 (20.79 ± ±0.54) 0.54) A549(UA)A549(UA)HL-7702( 35.79 35.79 ±> ±0.37100) 0.37 A549 (20.79 ± 0.54) 518A2518A2A549(UA) (UA) (UA) 14. 35.7914.77 ± ± 0± 0.1 0.37.1 518A2HL518A2HL-7702-7702 (3.6 (3.6 ( ˃(± 100)˃± 0.1)100) 0.1) HLHL-7702(-7702(˃100)˃100) HL-7702 (˃100) A2780A2780518A2HL (UA) (UA)-7702( (UA) 11.7 11.7˃ 14.7100)± ± 0.6 0.6 0.1 A2780518A2A2780 (2.7 (3.6(2.7 ± ± 0.1) 0.1) 518A2518A2 (UA) (UA) 14. 14.7 7± ±0 .10±.1 518A2518A2 (3.6 (3.6 ±± ±0.1) 0.1) A2780A549A549 (UA)(UA) (UA) 11.7 0.6 A2780 (2.7 0.1) A2780518A2 (UA) (UA) 11.7 14. ±7 0.6 ±± 0 .1 A549A549518A2 (3.9 (3.9 (3.6 ± ± 0.1) 0.1)± 0.1) A2780 (UA)A549 11.7 (UA) ± 0.6 OO HH A2780A549A2780 (2.7 (3.9 (2.7 ± ±0.1)0.1) 0.1) A278015.515.5 (UA) ± ± 1.3 1.3 11.7 ± 0.6 FaDuFaDuA2780 (6.4 (6.4 (2.7 ±± ± 0.4) 0.4)± 0.1) A549A54915.5 (UA) (UA) 1.3 16 1616 NN CCytotoxicityCytotoxicityytotoxicity A549FaDuA549 (3.9 (6.4(3.9 ± ±0.1) 0.4)0.1) FaDuFaDu (UA) (UA)A549 14.2 14.2 (UA)± ± ± 2.0 2.0 [158][158] [158] HHC3COOOO HHOOHH HT29HT29A549 (3.5 (3.5 (3.9 ± ±± 0.3) ±0.3) 0.1) FaDu15.515.5 (UA)± ±1.3 1.3 14.2 2.0 3 FaDuHT29FaDu (6.4 (3.5(6.4 ± ±0.4) 0.3)0.4) HT29HT29 (UA) (UA)15.5 10.6 10.6± 1.3 ± ± 0.3 0.3± 1616 Int. Int. J. J.Mol. Mol. Sci. Sci.O 20 202020, 2, 12,1 x, xFOR FOR PEERN PEERNH REVIEW REVIEW CCytotoxicityytotoxicity MCFMCFFaDu-7-7 (3.3 (3.3 (6.4 ± ± 0.2)± 0.2) 0.4) FaDuFaDuHT29 (UA) (UA) (UA) 14.2 14.2 10.6± ±2.0 2.0 11 0.311 of of 28[158] 28[158] 16Int.Int. J.H J.Mol.3H CMol.3C Sci. OSci.O 20 202020, 2, 12,1 x, xFOR FOR PEER PEEROOH HREVIEW REVIEW Cytotoxicity MCF-7HT29HT29 (3.5 (3.5 (3.3 ± ±0.3) 0.3)0.2) MCFMCFFaDu-7-7 (UA) (UA) (UA) 12.7 12.7 14.2 ± ± 0.1± 0.1± 2.0 11 11 of of 28 28 [158] N NIHNIH 3T3 3T3 (2.5 (2.5± ± ± HT29HT29MCF-7 (UA) (UA) (UA) 10.6 10.6 ± 12.7 ±0.3 0.3 0.1 H3C O OH NIHHT29 3T3 (2.5(3.5 ± 0.6)0.3) ± MCFMCF-7- 7(3.3 (3.3 ± ±0.2) 0.2) NIHNIHNIHHT29 3T3 3T3 3T3 (UA) (UA)18.7 (UA)18.7 (UA)18.7 10.6 ± ±0.3 1.6 MCF0.6)-0.6)7 (3.3 ± ± 0.2) MCFMCF-7- 7(UA) (UA) 12.7 12.7 ± ±0.1 0.1 NIHNIH 3T3 3T3 (2.5 (2.5 ± ± 1.61.6 ± NIHNIHMCF 3T3 3T3-7 (UA)(UA)18.7 (UA)18.7 12.7 ±± ± 0.1 OO BrBr TET21NTET21NNIH0.6)0.6) 3T3 (0.8 (0.8 (2.51 1± ± ± OO BrBr NIHTET21NTET21N 3T3 ((UA)18.7 ˃(˃10)10) ± ClCl P P TET21NTET21N (0.8 (0.81 1± ± 1.61.6 1717 OO OO CCytotoxicityytotoxicity TET21N0.08)0 (0.81.08)0.6) 0.08) TET21NTET21NTET21N ( ˃(10)˃10) (> 10) [159][159] ClCl P P MCF-7(1.6˃˃25) 17 1717 ClCl OO O O CCytotoxicityCytotoxicityytotoxicity 0.08)0.08) ± MCF-7( 25) [159][159] [159] MCFMCF-7(1.59MCF-7(1.59-7(1.59 ± ±0.11) 0.11)0.11) MCFMCFMCF-7(-7(-7(˃25)˃25)> 25) ClCl MCFMCF-7(1.59-7(1.59 ±± ±0.11) 0.11)

T24(6.01T24(6.01 ± ±0.87) 0.87) OO T24(UA)37.88T24(UA)37.88 ± ±1.12 1.12 HH T24(6.01T24(6.01T24(6.01 ± ±0.87) 0.87) T24(UA)37.88 1.12 O O O OAc A549(5.22A549(5.22 ± ±0.65) 0.65) T24(UA)37.88T24(UA)37.88 ± ±1.12 1.12 O HNHN OAc ± ± (C(HC2H)6N) HNH A549(5.22A549(5.22A549(5.22 ± ±0.65) 0.65) A549(UA)A549(UA)A549(UA) 18 OO N N 2 6 OAOcAc HepG2(6.82 ± [94] 18 18 (C(HC2H)62N)6HNH HepG2(6.82± ± A549(UA)A549(UA) [94] [94] 1818 HH3C3C OO OAOcAc HepG2(6.82HepG2(6.82HepG2(6.82 ±1.07) ± HepG2(UA)HepG2(UA)HepG2(UA) [94][94] HHCC OO 1.07)1.07) ± HepG2(UA)HepG2(UA) 3 3 AcAOcO OAOcA c SKOV3(8.951.07)1.07) 1.26) SKOV3(UA)SKOV3(UA)SKOV3(UA) AcAOcO SKOV3(8.95±SKOV3(8.95±± 1.26) 1.26) SKOV3(UA)SKOV3(UA) SKOV3(8.95±SKOV3(8.95± 1.26) 1.26) HH HH OO HNHN HNHN 1919 O O NN NN AAntinti-cancer-cancer HRE(0.8HRE(0.8 ± ±0.2) 0.2) HREHRE (UA (UA) )> >1 00100 [160][160] 1919 OO NN AAntinti-cancer-cancer HRE(0.8HRE(0.8 ± ±0.2) 0.2) HREHRE (UA (UA) >) >1 00100 [160][160] O O NN NN NNNN NN 518A2(2.7518A2(2.7 ± ±0.10) 0.10) 518A2(UA)518A2(UA) 14.7 14.7 ± ±0.1 0.1 518A2(2.7518A2(2.7 ± ±0.10) 0.10) 518A2(UA)518A2(UA) 14.7 14.7 ± ±0.1 0.1 A2780(2.3A2780(2.3 ± ±0.10) 0.10) A2780(UA)A2780(UA) 11.7 11.7 ± ±0.6 0.6 A2780(2.3A2780(2.3 ± ±0.10) 0.10) A2780(UA)A2780(UA) 11.7 11.7 ± ±0.6 0.6 OO HH HT29(1.8HT29(1.8 ± ±0.10) 0.10) HT29(UA)HT29(UA) 10.6 10.6 ± ±0.7 0.7 20 OO HNHN MCFHT29(1.8HT29(1.8-7(2.0 ± ± ±0.10) 0.10)0.10) MCFHT29(UA)HT29(UA)-7(UA) 10.6 12.710.6 ± ± ±0.7 0.10.7 20 NN NNHH MCF-7(2.0 ± 0.10) MCF-7(UA) 12.7 ± 0.1 2020 HH3C3C OO 2 2 MCFMCF-7(2.0-7(2.0 ± ±0.10) 0.10) MCFMCF-7(UA)-7(UA) 12.7 12.7 ± ±0.1 0.1 HHCC OO NNHH2 2 8505C(4.18505C(4.1 ± ±0.40) 0.40) 8505C(UA)8505C(UA) 13.5 13.5 ± ±1.5 1.5 3 3 8505C(4.18505C(4.1 ± ±0.40) 0.40) 8505C(UA)8505C(UA) 13.5 13.5 ± ±1.5 1.5 NIH3T3(2.6NIH3T3(2.6 ± ± NIHNIH 3T3(UA) 3T3(UA) 18.7 18.7 ± ± NIH3T3(2.6NIH3T3(2.6 ± ± NIHNIH 3T3(UA) 3T3(UA) 18.7 18.7 ± ± 0.30)0.30) 1.61.6 0.30)0.30) 1.61.6 518A2(3.2518A2(3.2 ± ±0.10) 0.10) 518A2(UA)518A2(UA) 14.7 14.7 ± ±0.1 0.1 518A2(3.2518A2(3.2 ± ±0.10) 0.10) 518A2(UA)518A2(UA) 14.7 14.7 ± ±0.1 0.1 A2780(2.4A2780(2.4 ±0.10) ±0.10) A2780(UA)A2780(UA) 11.7 11.7 ± ±0.6 0.6 H A2780(2.4A2780(2.4 ±0.10) ±0.10) A2780(UA)A2780(UA) 11.7 11.7 ± ±0.6 0.6 OO H HT29(1.8HT29(1.8 ± ±0.20) 0.20) HT29(UA)HT29(UA) 10.6 10.6 ± ±0.7 0.7 HNHN HT29(1.8HT29(1.8 ± ±0.20) 0.20) HT29(UA)HT29(UA) 10.6 10.6 ± ±0.7 0.7 2121 OO NN NN CCytotoxicityytotoxicity MCFMCF-7(2.7-7(2.7 ± ±0.30) 0.30) MCFMCF-7(UA)-7(UA) 12.7 12.7 ± ±0.1 0.1 [161][161] NN 2121 HH3C3C OO NNHH CCytotoxicityytotoxicity MCFMCF-7(2.7-7(2.7 ± ±0.30) 0.30) MCFMCF-7(UA)-7(UA) 12.7 12.7 ± ±0.1 0.1 [161][161] HHCC OO 8505C(5.48505C(5.4 ± ±0.40) 0.40) 8505C(UA)8505C(UA) 13.5 13.5 ± ±1.5 1.5 3 3 NNHH 8505C(5.48505C(5.4 ± ±0.40) 0.40) 8505C(UA)8505C(UA) 13.5 13.5 ± ±1.5 1.5 NIHNIH 3T3(2.2 3T3(2.2 ± ± NIHNIH 3T3(UA) 3T3(UA) 18.7 18.7 ± ± NIHNIH 3T3(2.2 3T3(2.2 ± ± NIHNIH 3T3(UA) 3T3(UA) 18.7 18.7 ± ± 0.10)0.10) 1.61.6 0.10)0.10) 1.61.6 518A2(2.7518A2(2.7 ± ±0.10) 0.10) 518A2(UA)518A2(UA) 14.7 14.7 ± ±0.1 0.1 518A2(2.7518A2(2.7 ± ±0.10) 0.10) 518A2(UA)518A2(UA) 14.7 14.7 ± ±0.1 0.1 A2780(2.6A2780(2.6 ± ±0.10) 0.10) A2780(UA)A2780(UA) 11.7 11.7 ± ±0.6 0.6 A2780(2.6A2780(2.6 ± ±0.10) 0.10) A2780(UA)A2780(UA) 11.7 11.7 ± ±0.6 0.6 OO H HT29(1.7HT29(1.7 ± ±0.10) 0.10) HT29(UA)HT29(UA) 10.6 10.6 ± ±0.7 0.7 H HT29(1.7HT29(1.7 ± ±0.10) 0.10) HT29(UA)HT29(UA) 10.6 10.6 ± ±0.7 0.7 2222 OO HNHN MCFMCF-7(1.7-7(1.7 ± ±0.10) 0.10) MCFMCF-7(UA)-7(UA) 12.7 12.7 ± ±0.1 0.1 NN NNHH2 2 2222 HH3C3C OO MCFMCF-7(1.7-7(1.7 ± ±0.10) 0.10) MCFMCF-7(UA)-7(UA) 12.7 12.7 ± ±0.1 0.1 HHCC OO NNHH2 2 8505C(3.28505C(3.2 ± ±0.01) 0.01) 8505C(UA)8505C(UA) 13.5 13.5 ± ±1.5 1.5 3 3 8505C(3.28505C(3.2 ± ±0.01) 0.01) 8505C(UA)8505C(UA) 13.5 13.5 ± ±1.5 1.5 NIHNIH 3T3 3T3 (1.3 (1.3 ± ± NIHNIH 3T3(UA) 3T3(UA) 18.7 18.7 ± ± NIHNIH 3T3 3T3 (1.3 (1.3 ± ± NIHNIH 3T3(UA) 3T3(UA) 18.7 18.7 ± ± 0.20)0.20) 1.61.6 0.20)0.20) 1.61.6 HelaHela (Cisplatin) (Cisplatin) 15. 15.1 1± ± OO OO F F HelaHela (Cisplatin) (Cisplatin) 15. 15.1 1± ± NN HelaHela (2.6 (2.6 ± ±1.1) 1.1) 0.90.9 23 OO O O F F Cytotoxicity [162] 23 N N NN Cytotoxicity HelaHela (2.6 (2.6 ± ±1.1) 1.1) 0.90.9 [162] 2323 HH3C3C OO CCytotoxicityytotoxicity MKN45(2.1MKN45(2.1 ± ±0.3) 0.3) MKN45(Cisplatin)MKN45(Cisplatin) 2. 2.8 8 [162][162] HHCC OO N N MKN45(2.1MKN45(2.1 ± ±0.3) 0.3) MKN45(Cisplatin)MKN45(Cisplatin) 2. 2.8 8 3 3 ± ±0 .10.1 ± ±0 .10.1 HHNNN HHNNN BELBEL-7402-7402 (4.49) (4.49) BELBEL-7402-7402 (UA) (UA) >50 >50 2424 CCytotoxicityytotoxicity BELBEL-7402-7402 (4.49) (4.49) BELBEL-7402-7402 (UA) (UA) >50 >50 [163][163] 2424 CCytotoxicityytotoxicity SGCSGC-7901(7.01)-7901(7.01) SGCSGC-7901(UA)-7901(UA) >50 >50 [163][163] SGCSGC-7901(7.01)-7901(7.01) SGCSGC-7901(UA)-7901(UA) >50 >50 CCl l CCl l A375(0.51A375(0.51 ± ±0.05) 0.05) A375(BA)A375(BA) A2780(0.45A375(0.51A375(0.51 ± ± ±0.05) 0.03) 0.05) Et N A2780(0.45 ± 0.03) A375(BA)A375(BA) 2Et2N A2780(0.45A2780(0.45 ± ±0.03) 0.03) A2780(BA)A2780(BA) CElCt2lENt2N OO O O HT29(0.50HT29(0.50 ± ±0.07) 0.07) A2780(BA)A2780(BA) Cl Cl HT29(BA)HT29(BA) OO O O HT29(0.50HT29(0.50 ± ±0.07) 0.07) 2525 O O NENt2Et CCytotoxicityytotoxicity MCF7(0.39MCF7(0.39 ± ±0.04) 0.04) HT29(BA)HT29(BA) [164][164] HHCC OO O O 2 MCF7(BA)MCF7(BA) 2525 3 3 N NO O NENt2Et2 CCytotoxicityytotoxicity MCF7(0.39MCF7(0.39 ± ±0.04) 0.04) [164][164] O O N N H3HC3C OO NiH3T3(0.4NiH3T3(0.40 0± ± MCF7(BA)MCF7(BA) N N N N NiH3T3(0.4NiH3T3(0.40 0± ± NiH3T3(BA)NiH3T3(BA) 0.03)0.03) NiH3T3(BA)NiH3T3(BA) 0.03)0.03) SW1736(BA)SW1736(BA) SW1736SW1736 (n.d) (n.d) SW1736(BA)SW1736(BA) SW1736SW1736 (n.d) (n.d) O O A375(1.5A375(1.5 ± ±0.4) 0.4) XOXO O O A375(1.5A375(1.5 ± ±0.4) 0.4) A375(UA)A375(UA) n.d. n.d. N N XOXO A2780(1.9A2780(1.9 ± ±0.3) 0.3) A375(UA)A375(UA) n.d. n.d. OO N N N N N N A2780(1.9A2780(1.9 ± ±0.3) 0.3) A2780(UA)A2780(UA) 11.7 11.7 ± ±0.6 0.6 N N O O HT29 (5.7 ± 0.5) OO N N N N HT29 (5.7 ± 0.5) A2780(UA)A2780(UA) 11.7 11.7 ± ±0.6 0.6 2626 O O N N N N O O CCytotoxicityytotoxicity HT29HT29 (5.7 (5.7 ± ±0.5) 0.5) HT29HT29 (UA) (UA) 10.6 10.6 ± ±0.7 0.7 [165][165] HHCC OO N N OXOX MCFMCF-7(4.4-7(4.4 ± ±0.7) 0.7) 2626 3 3 O O N N CCytotoxicityytotoxicity HT29HT29 (UA) (UA) 10.6 10.6 ± ±0.7 0.7 [165][165] H3HC3C OO X X= t=B tuBu N N OXOX MCFMCF-7(4.4-7(4.4 ± ±0.7) 0.7) MCFMCF-7(UA)-7(UA) 12.7 12.7 ± ±0.1 0.1 OXOX FaDuFaDu (3.7 (3.7 ± ±0.6) 0.6) MCFMCF-7(UA)-7(UA) 12.7 12.7 ± ±0.1 0.1 X =X t=B utBu FaDuFaDu (3.7 (3.7 ± ±0.6) 0.6) FaDuFaDu (UA) (UA) n.d n.d O O OXOX NIHNIH 3T3(4.6 3T3(4.6 ± ±1.0) 1.0) FaDuFaDu (UA) (UA) n.d n.d O O NIHNIH 3T3(4.6 3T3(4.6 ± ±1.0) 1.0)

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 28

apoptosis via the intrinsic mitochondrial pathway [162]. Meng et al. designed UA derivatives through multiple synthetic steps and evaluated the synthesized compounds in vitro using an MTT assay on two cancer cell lines (BEL7402 and SGC7901). The results showed a higher inhibitory rate of the synthesized compounds on both cells when compared to the parent compound, UA. The results of molecular docking studies indicated the role of the structural design and optimization of UA. The most promising derivative was compound 24, as shown in Table 4 [163]. Wolfram et al. converted pentacyclic triterpenoic acids—namely oleanolic, betulinic glycyrrhetinic, boswellic, and ursolic acids—into their acetylated piperazinyl amides by coupling them with rhodamine B. To evaluate their in vitro cytotoxicity, all the triterpene-homopiperazinyl- rhodamine derivatives were subjected to RB assays and most of them were highly toxic against numerous human cancer cell lines (A2780, A375, HT29, NiH3T3, MCF7, and SW1736). The ursolic acid-homopiperazinyl-rhodamine derivative (Compound 25, Table 4) was one of the compounds that showed a strong cytotoxicity against the tumor cell lines, while it was less cytotoxic on SW1736 cells [164]. Kahnt et al. synthesized amine-spaced conjugates of UA and 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid (DOTA). Two conjugates (Compound 26, 27, Table 4) displayed the highest cytotoxicity on A375 melanoma and A2780 ovarian carcinoma, with IC50 values of 1.5 µM and 1.7 µM, respectively. Compound 26 induced the death of A375 cells by apoptosis [165]. Mang et al. synthesized UA derivatives with potential anticancer properties by modifying C-2, C-3, and C-28 and evaluated their in vitro cytotoxicity against HepG2, BGC-823, and HeLa cell lines using an MTT assay These derivatives were synthesized by reacting UA with Jones reagents in acetone and then with NH2-OH·HCl; the obtained compounds were then reacted with Ac2O in the presence of DMAP in Int.Int. J. J.Mol. Mol. Sci. Sci. 20 202020, 2,1 2, 1x, FORx FOR PEER PEER REVIEW REVIEW 1111 of of 28 28 Int.Int.TH J. J.Mol. F.Mol. TheSci. Sci. 20 intermediate202020, 2, 12,1 x, xFOR FOR PEER PEERcompound REVIEW REVIEW was condensed with the relevant amino or phenolic moieties1111 of of 28 28 in Int.Int.Int. J.J. J. Mol.Mol. Mol. Sci.Sci. Sci. 20 202020,, 2,, 12,,1 xx,, x xFORFOR FORFOR PEERPEER PEERPEER REVIEWREVIEW REVIEWREVIEW 1111 of of 28 28 Et3N to afford the targeted compounds. Among these derivatives, Compounds 28 and 29 were Int. J. Mol. Sci.Int.Int.2020 J. J.Mol. Mol., 21 Sci. Sci., 5920 20 202020, 2, 12,1 x, xFOR FOR PEER PEER REVIEW REVIEW 1111 of of 28 28 11 of 27 demonstrated to be more effective on the cell lines when compared to the reference drug, gefitinib Int.Int. J. J.Mol. Mol.O Sci.O Sci. 20 202020, 2, 12,1 x, xFOR FORB rPEERB rPEER REVIEW REVIEW TET21NTET21N (0.8 (0.81 1± ± 1111 of of 28 28 [166]ClCl .O O BrBr TET21NTET21N (0.8 (0.81 1± ± TET21NTET21N (˃ (10)˃10) OO BrBr P P TET21N (0.81 ± 1717 ClCl OOO O O O BrBr CytotoxicityCytotoxicity TET21NTET21N0.08)0.08) (0.8 (0.8 1 1± ± TET21NTET21N (˃ (10)˃10) [159][159] Int.Int. J.C J.Mol.l Mol. Sci. OSci.O 20 202020, 2, 12,1 x, xFOR FOR PEER PEERP P REVIEW REVIEW TET21NTET21N ( ˃(˃10)10) 1111 of of 28 28 1717 ClCllClCl O O P P CytotoxicityCytotoxicity 0.08)0.08) TET21NMCFTET21NMCF-7(-7( ˃(˃ 25)(10)˃25)10) [159][159] 1717 Int. Int. J. J.Mol. Mol. Sci. OSci.O 20 2020 20, 2, 12,1 x,O xFORO FOR PEER PEERP P REVIEW REVIEW CytotoxicityCytotoxicity MCFMCF-7(1.59-07(1.590.08).08) ± ±0.11) 0.11) MCF-7(˃25)˃ 1111 of of 28[159] 28[159] 1717 ClCOl O O O O BrBr CCytotoxicityytotoxicity TET21NTET21N0.08)0.08) (0.8 (0.8 1 1± ± MCF-7(˃ 25) [159][159] CCl l Table 4. MCFCont.MCF-7(1.59-7(1.59 ± ±0.11) 0.11) MCFMCF-7(-7(˃˃25)25) ClClClCl Table 4. Modification of CMCF-3 and-7(1.59 C-28 ±positions. 0.11) TET21NTET21NMCF-7( ( ˃(10)˃25)10) 1717 OO P P CCytotoxicityytotoxicity MCFMCF-7(1.59-7(1.590.08)0.08) ± ±0.11) 0.11) [159][159] OO O O BrBr TET21NTET21N (0.8 (0.81 1± ± ClCl MCFMCF-7(-7(˃25)˃25) ClCl MCFMCF-7(1.59-7(1.59 ± ±0.11) 0.11) TET21NTET21N ( ˃(10)˃10) 1717 OO P P CCytotoxicityytotoxicity T24(6.01T24(6.010.08)0.08) ± ±0.87) 0.87) [159][159] O O O O BrBr O O TET21NT24(6.01TET21NT24(6.01 (0.8± (0.8±0.87) 0.87)1 1± ± T24(UA)37.88T24(UA)37.88˃˃ ± ±1.12 1.12 ClCOl O H H BrBr O O TET21NT24(6.01T24(6.01TET21N (0.8± ± (0.80.87) 0.87)1 1± ± TET21NMCFTET21NMCF-7(-7( (˃ 25)(10)˃25)10) ClCl O O N N P P OAOcAc A549(5.22T24(6.01A549(5.22T24(6.01 ±± ± 0.87)±0.65) 0.87) 0.65) T24(UA)37.88T24(UA)37.88 ± ±1.12 1.12 1717 O O H H OO CCytotoxicityytotoxicity MCFMCF-7(1.59-07(1.59.08)0.08) ± ±0.11) 0.11) T24(UA)37.88T24(UA)37.88TET21NTET21N (˃ (10) ˃± 10)± 1.12 1.12 [159][159] ClCl OO NHHNO(COH(C2)H62N)6HNH P P OAOcAc A549(5.22A549(5.22 ± ±0.65) 0.65) T24(UA)37.88T24(UA)37.88A549(UA)A549(UA) ± ±1.12 1.12 171718 O O O H H(CH ) NH OAc CCytotoxicityytotoxicity A549(5.22A549(5.22HepG2(6.820.08)0.08) ± ± 0.65) 0.65) ± MCFA549(UA)MCF-7(-7(˃25)˃25) [159][159][94] 18 ClCOlO NNO O(C2H62)6NH OAO cA cc A549(5.22T24(6.01A549(5.22HepG2(6.82T24(6.01 ± ± ± 0.87) ±0.65) 0.87) 0.65)± A549(UA) [94] 18 H HC C O O (C(HC2H)6)NHNHO O MCFMCFHepG2(6.82-7(1.59-7(1.59 ± ±0.11) ±0.11) MCFA549(UA)HepG2(UA)MCFA549(UA)-7(-7(˃25)˃25) [94] 18 3 C3 lCl (C(HC2H)622N)66HNH OAOcAc HepG2(6.82 ± T24(UA)37.88T24(UA)37.88HepG2(UA)A549(UA)A549(UA) ± ± 1.12 1.12 [94] 1818 HHC C OO H H MCFMCFHepG2(6.82HepG2(6.82-7(1.59-1.07)7(1.591.07)R ±2 ±0.11) ±0.11) ± HepG2(UA)HepG2(UA) [94][94] 18 H3 C3 OOO N N AcAOcO OA OccA c A549(5.22A549(5.22HepG2(6.82 ± ±0.65) 0.65) ± HepG2(UA) [94] HH3C3C OO (CH ) NH OAOccA c T24(6.01T24(6.011.07)1.07) ± ±0.87) 0.87) SKOV3(UA)HepG2(UA)A549(UA)HepG2(UA)SKOV3(UA)A549(UA) 3 3 (CH2)62N6H O AOcAOcO OA c 1.07)1.07) T24(UA)37.88T24(UA)37.88 ± ±1.12 1.12 1818 H H AAcOcO SKOV3(8.95±SKOV3(8.95±HepG2(6.82HepG2(6.821.07)1.07) 1.26) ±1.26) ± SKOV3(UA)SKOV3(UA) [94][94] OO AcAOcO OAOcA c SKOV3(8.95±A549(5.22SKOV3(8.95±A549(5.22O ± ±0.65) 1.26)0.65) 1.26) SKOV3(UA)SKOV3(UA) H3HC3C OO N N OAOcA c SKOV3(8.95±SKOV3(8.95±T24(6.01T24(6.01 ± ±0.87) 0.87)1.26) 1.26) HepG2(UA)SKOV3(UA)HepG2(UA) (C(HC2H)H62N)6HNHO O SKOV3(8.95±SKOV3(8.95±T24(6.01T24(6.011.07)1.07) ± ±0.87) 0.87)1.26) 1.26) T24(UA)37.88T24(UA)37.88A549(UA)A549(UA) ± ±1.12 1.12 1818 H H O OAHcAHOcO HepG2(6.82HepG2(6.82 ± ± [94][94] OOO N N NHNH OAOcA c A549(5.22A549(5.22 ± ±0.65) 0.65) T24(UA)37.88T24(UA)37.88SKOV3(UA)SKOV3(UA) ± ±1.12 1.12 H3HC3C O OO H H HH NHNH OAOcAc R1 HepG2(UA)HepG2(UA)A549(UA) OOOO N N(C(HC2H)6NH2N)6HNNH HH OAOcAc SKOV3(8.95±A549(5.22SKOV3(8.95±A549(5.22 ± ±0.65) 1.26)0.65) 1.26) A549(UA) 19181819 O (CH(CH) N)NHNH NHHN AntiAnti-cancer -cancer HRE(0.8HepG2(6.82HRE(0.8HepG2(6.821.07)1.07) ± ±0.2) 0.2) ± ± HREHREA549(UA)A549(UA) (UA (UA) >) >1 001 00 [160][94][160][94] OOO O 2 6N2 6N ANcANONcON SKOV3(UA)SKOV3(UA) 18191819 H3HC3C OO NN OAOcA c AntiAnti-cancer -cancer HRE(0.8HepG2(6.82HRE(0.8HepG2(6.82 ± ±0.2) 0.2) ± ± HREHREHepG2(UA)HepG2(UA) (UA (UA) >) >1 00 100 [160][94][160][94] 1919 O O HHNNHHNN AAntiAntinti-cancer-cancer SKOV3(8.95±SKOV3(8.95±HRE(0.8HRE(0.81.07)1.07) ± ±0.2) 1.26)0.2) 1.26) HREHRE (UA (UA) )> >1 00100 [160][160] 19 H3HC3C OOOO NN ANcAONNcON OAOcAc Anti-cancer HRE(0.8 ± 0.2) HREHepG2(UA)HepG2(UA) (UA) > 100 [160] Comp OOO O NNNNNN BiologicalBiological Cell1.07)Cell1.07) Lines Lines ReferenceReferenceSKOV3(UA)SKOV3(UA) Molec Molecules ules Bibliogr Compound1919 R1 R1 HHNRN2RANc2AON cO AAntinti-cancer-cancer SKOV3(8.95±SKOV3(8.95±HRE(0.8HRE(0.8 ± ±0.2) 1.26)0.2) 1.26) HREHRESKOV3(UA)SKOV3(UA) (UA (UA) )> >1 001 00 [160][160]Bibliography ound HNHN ActivitiesActivities Tested(ICTested(ICµ50M)µM) (IC(IC50µM)µM) aphy O OOO NN NN SKOV3(8.95±518A2(2.7SKOV3(8.95±518A2(2.7 ±50 ±0.10) 1.26) 0.10) 1.26) 518A2(UA)518A2(UA) 14.7 5014.7 ± ±0.1 0.1 HHNNNN 518A2(2.7518A2(2.7 ± ±0.10) 0.10) 518A2(UA)518A2(UA) 14.7 14.7 ± ±0.1 0.1 1919 HNHN AAntinti-cancer-cancer A278518A2(2.7518A2(2.7HRE(0.8A278HRE(0.8MGC0(2.30(2.3-803 ± ±0.2)0.10)0.10) (9.820.2)0.10) ± A2780(UA)518A2(UA)518A2(UA)A2780(UA)MGCHREHRE (UA- 803(UA)(UA 11.714.7) 14.711.7>) >1 00±1± 0027.0 ±0.60.1 0.10.6 8 ± [160][160] O OO O NHNH NHNHNN A278A2780(2.30(2.3 ± ±0.10) 0.10) A2780(UA)A2780(UA) 11.7 11.7 ± ±0.6 0.6 O O NNNN A2780(2.3 ± 0.10) A2780(UA) 11.7 ± 0.6 1919 O O HH NN AAntinti-cancer-cancer A2780(2.3518A2(2.7HT29(1.8HRE(0.8A278518A2(2.7HT29(1.8HRE(0.80(2.30.29) ± ± 0.10) ±0.2)0.10) 0.2)0.10) A2780(UA)518A2(UA)HT29(UA)A2780(UA)518A2(UA)HT29(UA)HREHRE (UA (UA 10.60 )11.714.7.29 10.611.7>14.7) >1 ± 001± 0.700 ±0.60.1 0.70.60.1 [160][160] 19 1919 OOO O HNH S NN AAnti-cancerAntinti-cancer-cancer HT29(1.8HRE(0.8HT29(1.8 HRE(0.8HRE(0.8 ± ±0.10)0.2) 0.2)0.10)0.2) HT29(UA)HT29(UA)HREHRE HRE (UA (UA 10.6 (UA)) 10.6>) >1 ±001 >±000.7 0.7100 [160][160] [160] 2020 OOO O NHH NN NN MCFHT29(1.8MCFA2780(2.3HT29(1.8HCT-7(2.0-7(2.0-116 ±± ± 0.10)±0.10)(18.97 0.10) 0.10) ± MCFHT29(UA)MCFA2780(UA)HT29(UA)-7(UA)-HCT7(UA) - 10.6116(UA)12.7 10.6 11.712.7 ±± ± 0.7±±0.1 0.7 0.60.1 OO NHHNN NNHNNHNH A2780(2.3 ± 0.10) A2780(UA) 11.7 ± 0.6 2020 H3HC3C O O NN NN 2 2 MCF518A2(2.7MCF518A2(2.7-7(2.0-7(2.0 ±± ± 0.10)±0.10) 0.10)0.10) MCF518A2(UA)MCF518A2(UA)-7(UA)-7(UA) 14.712.7 14.712.7 ±± ± 0.1±0.1 0.10.1 2020 H C O NN NNNNHH2 2 MCFMCF8505C(4.1-7(2.0-7(2.00.53) ± ± 0.10) 0.40) 0.10) MCFMCF8505C(UA)-7(UA)-7(UA)38.78 12.7 13.5±12.7 0.16 ± ±± 0.1 1.50.1 2020 3H3COOOO N NNH H2 MCF8505C(4.1HT29(1.8MCFHT29(1.8-7(2.0-7(2.0 ± ± ± 0.40)0.10) ±0.10) 0.10) 0.10) MCF8505C(UA)HT29(UA)MCFHT29(UA)-7(UA)-7(UA) 13.510.6 12.7 10.6 12.7 ± ± ± 1.50.7 ±0.1 0.7 0.1 HH3C3C OO HH NNHH2 2 A2780(2.38505C(4.1A2780(2.38505C(4.1 ± ±0.40)0.10) 0.10)0.40) A2780(UA)8505C(UA)A2780(UA)8505C(UA) 13.511.7 11.713.5 ± ±1.50.6 1.50.6 15 H3HC3C OO NN Cytotoxicity 518A2(2.78505C(4.1518A2(2.78505C(4.1T24 (19.60 ±± ± 0.10)0.40) 0.40)±0.10) 0.43) 518A2(UA)8505C(UA)8505C(UA)518A2(UA)T24(UA) 14.713.529.29 13.514.7 ± ± ±0.11.5 1.50.10.80 [139] 2020 OC H3 MCF518A2(2.78505C(4.1MCF518A2(2.78505C(4.1NIH3T3(2.6NIH3T3(2.6-7(2.0-7(2.0 ±± ± 0.10)0.40)±0.10) 0.40)0.10)±0.10) ± MCF518A2(UA)8505C(UA)NIHMCF518A2(UA)8505C(UA)NIH -3T3(UA)7(UA)- 7(UA)3T3(UA) 14.713.512.7 13.514.712.7 18.7 18.7 ±± ± 0.11.5±0.1 ± 1.50.10.1 ± HHCHCO3COOOO HH NNHH2 2 A2780(2.3HT29(1.8518A2(2.7A2780(2.3NIH3T3(2.6HT29(1.8NIH3T3(2.6 ± ±0.10)± 0.10) 0.10)± ± A2780(UA)HT29(UA)NIHA2780(UA)HT29(UA)NIH518A2(UA) 3T3(UA) 3T3(UA) 10.611.7 10.611.7 18.7 14.7±18.7± ± 0.7±0.6 0.7±0.6 ± 0.1 3 3 NN A2780(2.38505C(4.1A2780(2.38505C(4.1NIH3T3(2.6NIH3T3(2.6HepG20.30)0.30) ± ± (15.72 ±0.10) 0.40) 0.40) 0.10)± ± ± A2780(UA)8505C(UA)NIHA2780(UA)8505C(UA)NIHHepG2(UA) 3T3(UA) 3T3(UA)1.61.6 11.713.5 13.511.7 18.7 18.7±30.21± ±0.61.5 ±1.50.6 ± 2020 OCH3 MCFHT29(1.8A2780(2.3MCFHT29(1.8-7(2.0-0.30)7(2.00.30) ± ± ± 0.10) ± 0.10) 0.10) 0.10) MCFHT29(UA)MCFHT29(UA)A2780(UA)-7(UA)-7(UA)1.61.6 10.6 12.7 10.6 12.7 11.7± ± ± 0.7 ±0.1 0.7 0.1 0.6 HHCCOOOO HH NONHCH2H2 0.30)0.30)0.84) ± 1.61.60.58 ± 3 3 OO HH 3 518A2(3.2HT29(1.8HT29(1.8518A2(3.2HT29(1.8NIH3T3(2.6NIH3T3(2.60.30)0.30) ± ±±0.10) 0.10) 0.10) ± ± 518A2(UA)HT29(UA)NIH518A2(UA)HT29(UA)NIHHT29(UA) 3T3(UA) 3T3(UA)1.61.6 10.614.7 10.6 14.7 18.710.6 18.7± ±0.7±0.1 0.7±0.1 ± 0.7 20 2020 NN MCF518A2(3.28505C(4.1MCF8505C(4.1518A2(3.2-7(2.0-7(2.0 ±± ± 0.40)±0.10)0.10) 0.40)0.10)0.10) MCF518A2(UA)8505C(UA)MCF8505C(UA)518A2(UA)-7(UA)-7(UA) 13.514.712.7 13.514.712.7 ±± ± 1.5±0.10.1 1.50.1± 2020 HHC C OO NN NNHH2 2 MCF518A2(3.2A2780(2.4MCF-7(2.0MCF518A2(3.2A2780(2.4A549-7(2.0-0.30)7(2.0 0.30)(20.79 ±±0.10) ± ±0.10)± 0.10) 0.10)0.10)± 0.54) MCFA2780(UA)518A2(UA)MCF518A2(UA)A2780(UA)A549(UA)MCF-7(UA)-7(UA)-7(UA)1.61.6 11.714.7 12.7 35.7914.7 11.712.7 12.7±± ±0.6± 0.1± 0.10.60.1 0.37 0.1 3 3 NNHH A2780(2.4NIH3T3(2.6NIH3T3(2.6 ±0.10) ± ± A2780(UA)NIHNIH 3T3(UA) 3T3(UA) 11.7 18.7 18.7± 0.6 ± ± H3HC3C OO H 2 2 8505C(4.18505C(4.1A2780(2.4 ± ±0.40)±0.10) 0.40) 8505C(UA)A2780(UA)8505C(UA) 13.5 13.511.7 ± ±1.5 1.50.6± [161] O H 8505C(4.1HT29(1.8A2780(2.48505C(4.1518A2(3.28505C(4.1A2780(2.4HT29(1.8HL-7702 ± ±±0.10) 0.20)±0.40)±0.10) ( 0.40)0.20)0.40)˃0.10)100) 8505C(UA)A2780(UA)HT29(UA)A2780(UA)518A2(UA)8505C(UA)HT29(UA)8505C(UA)HL-7702( 10.6 13.511.7 10.611.713.514.7˃ 13.5± ±±100) 0.7 ±1.50.6 0.70.61.50.1 1.5 O NHHN 518A2(3.2HT29(1.80.30)0.30) ±± ± 0.20) 0.10) 518A2(UA)HT29(UA)1.61.6 10.6 14.7 ± ± 0.70.1± OO NHHN NN HT29(1.8NIH3T3(2.6HT29(1.8NIH3T3(2.6 ± ±0.20) 0.20) ± ± NIHHT29(UA)HT29(UA)NIH 3T3(UA) 3T3(UA) 10.6 10.6 18.7 18.7± ±0.7 0.7± ± 2121 OO HNH CytotoxicityCytotoxicity MCFNIH3T3(2.6A2780(2.4HT29(1.8MCFNIH3T3(2.6HT29(1.8A2780(2.4NIH3T3(2.6-7(2.7-7(2.7 ±± ±0.10)± 0.20)±0.10)±0.30) 0.20) 0.30)±0.30) ± MCFA2780(UA)NIHHT29(UA)MCFA2780(UA)HT29(UA)NIH518A2NIH- 7(UA)3T3(UA)- 7(UA)3T3(UA) 3T3(UA) (UA) 10.612.711.7 10.6 11.712.7 18.714. ±18.7± 18.7 ± 0.7±7±0.10.6 0.7 ±0.60.1 ± 0 .1 1.6 [161][161] 2121 H HC C OO O NN NN CytotoxicityCytotoxicity MCF518A2(3.2MCF518A2(3.2518A2-7(2.7-7(2.70.30) (3.6 ±± ± 0.10) ±0.30) 0.10) 0.30)± 0.1) MCF518A2(UA)MCF518A2(UA)-7(UA)-7(UA)1.6 14.712.7 14.7 12.7 ±± ± 0.1±0.1 0.10.1 ± [161][161] 2121 3 3 NN NHNH CytotoxicityCytotoxicity MCFMCF-7(2.7-0.30)7(2.7 ± ± 0.30) 0.30) MCFMCF-7(UA)-7(UA)1.6 12.7 12.7 ± ± 0.1 0.1 [161][161] 2121 H3HC3C OO HH NH CCytotoxicityytotoxicity MCF8505C(5.4HT29(1.8518A2(3.2MCF8505C(5.4HT29(1.8-7(2.70.30)-7(2.70.30) ± ± ±0.40)0.20)± 0.30) 0.20)0.10) 0.40)0.30) MCF8505C(UA)HT29(UA)MCF8505C(UA)HT29(UA)A2780518A2(UA)-7(UA)-7(UA) 1.6(UA)1.6 13.510.6 12.7 10.6 13.5 12.7 11.7 14.7± ± ±1.50.7 ±0.1 0.7 1.5±0.1 0.6 0.1 [161][161] HH3CCOOOO NN NH A2780(2.4A2780(2.4A2780 (2.7 ±0.10) ±0.10) ± 0.1) A2780(UA)A2780(UA) 11.7 11.7 ± ±0.6 0.6 H3HC33C OO NNHH 518A2(3.28505C(5.4518A2(3.28505C(5.4 ±± ±0.40)0.10) 0.40)0.10) 518A2(UA)8505C(UA)518A2(UA)8505C(UA) 13.514.7 13.514.7 ± ±1.50.1 1.50.1± NN NNHH 8505C(5.4A2780(2.4NIH8505C(5.4NIH 3T3(2.2 3T3(2.2 ± ±0.40) 0.10)0.40) ± ± 8505C(UA)NIH8505C(UA)NIHA2780(UA) 3T3(UA) 3T3(UA)A549 13.5 13.5 (UA) 18.711.7 18.7± ±1.5 ±1.5 ± 0.6 2121 HH CCytotoxicityCytotoxicityytotoxicity MCF518A2(3.2HT29(1.8MCFNIH518A2(3.2HT29(1.8A549-7(2.7 -3T3(2.27(2.7 (3.9 ± ±± 0.20)± 0.10)± 0.30) 0.20) 0.10)± 0.30)± 0.1) MCF518A2(UA)HT29(UA)NIHMCF518A2(UA)HT29(UA)- 3T3(UA)7(UA)-7(UA) 10.6 14.7 12.7 10.6 14.712.7 18.7 ± ± ± ± 0.7± 0.1± 0.1 0.7 ±0.10.1 [161][161] HHC3COOOO NN A2780(2.4A2780(2.4NIH 3T3(2.2 ±0.10)± ±0.10) ± A2780(UA)A2780(UA)NIH 3T3(UA) 11.7 11.7 18.7± ±0.6 0.6 ±± 3 O H NNHH HT29(1.8NIHNIH0.10) 3T3(2.2 0.10)3T3(2.2 0.20) ± ± NIHNIHHT29(UA) 3T3(UA) 3T3(UA)15.51.61.6 ± 18.71.3 10.618.7 ± ± 0.7 H NN A2780(2.48505C(5.48505C(5.4A2780(2.4NIH 3T3(2.2 ±±0.10) ±±0.10)0.40) 0.40) ± A2780(UA)8505C(UA)A2780(UA)8505C(UA)NIH 3T3(UA) 11.713.5 13.511.7 18.7± ±0.61.5 1.50.6 ± 21 2121 OO H CCytotoxicityytotoxicity MCFHT29(1.8MCFHT29(1.8FaDu-7(2.7-0.10)7(2.70.10) (6.4± ± ± 0.20) ± 0.30) 0.20) ±0.30) 0.4) MCFHT29(UA)MCFHT29(UA)-7(UA)-7(UA)1.61.6 10.6 12.7 10.6 12.7 ± ± ± 0.7 ±0.1 0.7 0.1± [161][161] 16 H3HC3C OO HNNH N Cytotoxicity 518A2(2.7MCF-7(2.7518A2(2.70.10)0.10) ± ±0.10) 0.10)0.30) 518A2(UA)518A2(UA)FaDuMCF-7(UA) (UA)1.61.6 14.7 14.7 14.2 12.7± ±0.1 ±0.1 2.0 0.1 [158] OO NN NNHH HT29(1.8NIHHT29(1.8NIH 3T3(2.2 0.10)3T3(2.2 ± ±0.20) 0.20) ± ± HT29(UA)NIHHT29(UA)NIH 3T3(UA) 3T3(UA)1.6 10.6 10.6 18.7 ±18.7 ±0.7 0.7± ± 2121 H3C O NN OH CCytotoxicityytotoxicity MCF518A2(2.78505C(5.48505C(5.4MCF8505C(5.4518A2(2.7HT29-7(2.7-7(2.7 (3.5 ±± ± 0.40)±0.10)0.30) 0.40) 0.10)±0.30) 0.3) MCF518A2(UA)8505C(UA)MCF8505C(UA)518A2(UA)8505C(UA)-7(UA)-7(UA) 13.514.712.7 13.514.712.7 13.5 ±± ± 1.5±0.10.1 1.50.1 1.5 [161][161] 2121 HHC C OO NN CCytotoxicityytotoxicity MCFA2780(2.6518A2(2.7MCF518A2(2.7A2780(2.6-7(2.7-0.10)7(2.70.10) ± ±± 0.10)0.30) 0.10)0.30) MCFA2780(UA)518A2(UA)MCF518A2(UA)A2780(UA)HT29-7(UA)-7(UA) (UA)1.61.6 11.714.7 12.7 14.7 11.712.7 10.6 ±± ±0.6±0.1 ± 0.10.60.1 0.3 [161][161] 3 3 NNHH A2780(2.6NIHA2780(2.6NIHMCF 3T3(2.2 3T3(2.2-7 (3.3±± ±0.10) 0.10) ± ±0.2) A2780(UA)NIHA2780(UA)NIH 3T3(UA) 3T3(UA) 11.7 11.7 18.7 18.7± ±0.6 ±0.6 ± ± H3HC3C OO NHNH NIH8505C(5.4A2780(2.68505C(5.4 3T3(2.2 ±± ± 0.40)0.10) 0.40)0.10) 8505C(UA)A2780(UA)8505C(UA)NIH 3T3(UA) 13.511.7 13.5 ±±18.7 ± 1.50.6 1.5 1.6 O O HH 8505C(5.4A2780(2.6518A2(2.7HT2A2780(2.6518A2(2.78505C(5.4HT29(1.79(1.7 ± ±± ±0.10) ±0.40)0.10)± 0.10)0.40)0.10) 8505C(UA)A2780(UA)518A2(UA)HT29(UA)A2780(UA)518A2(UA)8505C(UA)HT29(UA)MCF-7 (UA) 10.6 13.511.714.7 10.611.713.514.7 12.7 ± ±± 0.7 ±1.50.60.1 0.7 0.61.50.1± 0.1± OO H HT2NIHHT2NIHNIH9(1.7 0.10)9(1.73T3(2.2 0.10)3T3(2.2 3T3 ± ±0.10) 0.10)(2.5 ± ± ± HT29(UA)NIHHT29(UA)NIH 3T3(UA) 3T3(UA)1.61.6 10.6 10.6 18.7 ±18.7 ±0.7 0.7± ± 2222 OO NHNH MCFHT29(1.7HT2518A2(2.7MCFHT2-9(1.77(1.79(1.7-7(1.7 ±± ± 0.10)±0.10) 0.10) 0.10) MCFHT29(UA)MCFHT29(UA)NIH518A2(UA)-7(UA)-7(UA) 3T3 10.612.7 (UA)18.710.6 12.7 14.7±± ± 0.7±0.1 0.7 0.1 ±0.1 OO NHHN NHNH2 A2780(2.6NIHA2780(2.6HT2NIH 9(1.73T3(2.2 3T3(2.2 ± ± ±0.10) 0.10)0.10) ± ± A2780(UA)NIHA2780(UA)HT29(UA)NIH 3T3(UA) 3T3(UA) 11.7 10.611.7 18.7 18.7± ± ±0.6 0.7±0.6 ± 2222 H HC3C O O N 2 MCF518A2(2.7MCF518A2(2.7-7(1.70.10)-7(1.70.10)0.6) ± ± ± ± 0.10) ±0.10) 0.10)0.10) MCF518A2(UA)MCF518A2(UA)-7(UA)-7(UA)1.61.6 14.712.7 14.7 12.7 ±± ± 0.1±0.1 0.10.1± 2222 H3C O NN NNHH2 2 MCFA2780(2.6MCF8505C(3.2-7(1.7-7(1.7 ± ± 0.10) 0.10)0.01)0.10) MCFMCF8505C(UA)A2780(UA)-7(UA)-7(UA) 1.612.7 13.512.7 11.7 ± ±± 0.1 1.50.1 0.6 2222 3H3COO O NNHH2 2 MCF8505C(3.2HT29(1.7MCFHT29(1.7-7(1.70.10)-7(1.70.10) ± ±± 0.01)0.10)± 0.10) 0.10) 0.10) MCF8505C(UA)HT29(UA)MCFHT29(UA)-7(UA)-7(UA)1.61.6 13.510.6 12.7 10.6 12.7 ± ± ± 1.50.7 ±0.1 0.7 0.1 HH3C3C OO HH NNHH2 2 A2780(2.68505C(3.2A2780(2.68505C(3.2 ±± ±0.01)0.10) 0.10)0.01) A2780(UA)8505C(UA)A2780(UA)8505C(UA) 13.511.7 11.713.5 ± ±1.50.6 1.50.6± H3HC3C OO N 518A2(2.78505C(3.2HT29(1.7518A2(2.7 ±± ±0.10)0.01)0.10) 0.10) 518A2(UA)8505C(UA)518A2(UA)HT29(UA) 14.713.5 14.710.6 ± ±0.11.5 0.1 0.7 22 2222 N MCF518A2(2.78505C(3.2NIHMCF8505C(3.2518A2(2.7NIH- 7(1.73T3-7(1.7 3T3 (1.3 ±± ±(1.3 0.10)0.01)±0.10) 0.01)0.10) 0.10)± ± MCF518A2(UA)8505C(UA)NIHMCF8505C(UA)518A2(UA)NIH -3T3(UA)7(UA)- 7(UA)3T3(UA) 14.713.512.7 13.514.712.7 18.7 18.7 ±± ± 0.11.5±0.1 ± 1.50.10.1± ± HHCCOOOO HH NNHH2 2 A2780(2.6HT29(1.7MCF-7(1.7NIHA2780(2.6HT29(1.7NIH 3T3 3T3 ±(1.3 ±0.10)±(1.3 0.10)0.10) ± ± A2780(UA)HT29(UA)NIHA2780(UA)HT29(UA)NIHMCF-7(UA) 3T3(UA) 3T3(UA) 10.611.7 10.611.7 18.7 ±18.7 12.7± ± 0.7±0.6 0.7±0.6 ± 0.1 3 3 NN A2780(2.68505C(3.2NIHA2780(2.68505C(3.2NIH 0.20)3T3 3T30.20) ±(1.3 (1.3±0.10) 0.01) 0.01)0.10) ± ± A2780(UA)8505C(UA)NIHA2780(UA)8505C(UA)NIH 3T3(UA) 3T3(UA)1.61.6 11.713.5 13.511.7 18.7 18.7± ±0.61.5 ±1.50.6 ± 2222 MCF8505C(3.2MCFHT29(1.7-7(1.7-0.20)7(1.70.20) ± ± ± 0.10) 0.10) 0.01)0.10) MCFMCFHT29(UA)8505C(UA)-7(UA)-7(UA)1.61.6 12.7 10.6 12.7 13.5 ± ± ±0.1 0.7 0.1 1.5 HHCCOOOO HH NNHH2 2 HT29(1.70.20)0.20) ± 0.10) HT29(UA)1.61.6 10.6 ± 0.7 3 3 OO HT29(1.7NIHHT29(1.7NIH 0.20)3T3 0.20)3T3 ± (1.3 ± ±0.10)(1.3 0.10) ± ± HeHT29(UA)NIHHeHT29(UA)NIHlala (Cisplatin) 3T3(UA)(Cisplatin) 3T3(UA)1.61.6 10.6 10.6 18.7 15.±18.7 15.±0.71 0.7± 1± ± ± 2222 O O NHNH NIHMCF8505C(3.2MCF8505C(3.2 3T3-7(1.7-7(1.7 (1.3 ±± ± 0.01)±0.10) 0.01)0.10)0.20) HeMCF8505C(UA)HeMCF8505C(UA)laNIHla -(Cisplatin)7(UA) -(Cisplatin)7(UA) 3T3(UA) 13.512.7 13.512.7 15. ±± 18.715. ± 1.5±0.11 1.50.1 1± ± 1.6 O ONN NNHHF 2F HeHelala (Cisplatin) (Cisplatin) 15. 15.11 ± ± 2222 HHC3COOOO O N N 2F MCFHelaMCFHela-7(1.7 -0.20)(2.67(1.7 0.20)(2.6 ± ±1.1) 0.10)± 1.1)0.10) HelaMCFHeMCFla -(Cisplatin)7(UA) (Cisplatin)-7(UA)01.6.91.60 .9 12.7 12.7 15. ± 15. ±0.11 0.1 1± ± 3 O O NNHH2 2F NIHNIH 3T3 3T3 (1.3 (1.3 ± ± NIHNIH 3T3(UA) 3T3(UA) 18.7 18.7 ± ± 2323 H3HC3COOOO OO N N N F F CytotoxicityCytotoxicity 8505C(3.2Hela8505C(3.2Hela (2.6 (2.6 ± ±0.01)1.1) 0.01)1.1) 8505C(UA)8505C(UA)Hela (Cisplatin)0.90 .913.5 13.5 ± ±1.5 15.1 1.5 0.9[162][162] 2323 H HC C O O O O NN N F F CytotoxicityCytotoxicity MKN45(2.1MKN45(2.1HelaHela (2.6 (2.6 ± ± 1.1) ±0.3)1.1) 0.3) MKN45(Cisplatin)MKN45(Cisplatin)00.9.9 2. 2.8 8 [162][162] 23 3 3 N N N N Cytotoxicity 8505C(3.2Hela8505C(3.2HelaHela (2.6 (2.6(2.6 ± ±0.01)1.1) 1.1)0.01) Hela8505C(UA)Hela8505C(UA) (Cisplatin) (Cisplatin)0.90 .913.5 13.5 15.± 15.±1.51 1.5 1± ± ± [162] 23 2323 H3HC C OO NN CCytotoxicityCytotoxicityytotoxicity MKN45(2.1NIHMKN45(2.1NIH 0.20)3T3 0.20)3T3 (1.3 (1.3± ±0.3) ±0.3) ± MKN45(Cisplatin)NIHMKN45(Cisplatin)NIHMKN45(Cisplatin) 3T3(UA) 3T3(UA)1.61.6 18.7 18.7 2. ± 2. 8± 82.8 [162][162] [162] HHC3COOOO O O N N F F MKN45(2.1MKN45(2.1± ± ± 0.3) 0.3) MKN45(Cisplatin)MKN45(Cisplatin) 2. 2.88 HH3C3C OO N MKN45(2.1MKN45(2.1NIHMKN45(2.1HelaHelaNIH 3T3(2.6 3T3(2.6 (1.3± (1.3±1.1) ±0.3)1.1) ±0.3)0.3) ± MKN45(Cisplatin)NIHMKN45(Cisplatin)NIH 3T3(UA) 3T3(UA)± 0±0.90 .10.9 .1 18.7 18.7 2. ± 2. 8± 8 ± 3 3 N 0.20)0.20) ± HelaHela (Cisplatin) (Cisplatin)±1.6 ±01.6 .10 .1 0.1 15. 15.1 1± ± 2323 N N CCytotoxicityytotoxicity ± 0.1 [162][162] HHCCOOOO O O F F MKN45(2.1MKN45(2.10.20)0.20) ± ±0.3) 0.3) MKN45(Cisplatin)MKN45(Cisplatin)±1.6 ±01.6 .10 .1.1 2. 2.8 8 3H3NHNN N N HelaHela (2.6 (2.6 ± ±1.1) 1.1) HelaHela (Cisplatin) (Cisplatin)0.90.9 15. 15.1 1± ± 2323 HHNNN CCytotoxicityytotoxicity ± 0.1 [162][162] HHCHCNONOOO O O N N F F MKN45(2.1MKN45(2.1 ± ±0.3) 0.3) HelaMKN45(Cisplatin)HelaMKN45(Cisplatin) (Cisplatin) (Cisplatin)± 0.1 15. 15. 2.1 2.18± 8± 3H3HNNN N N BELHelaBELHela-7402 -(2.67402 (2.6 ±(4.49) ±(4.49)1.1) 1.1) BELBEL-7402-74020.9 0(UA).9 (UA) >50 >50 2323 OO O O F F CCytotoxicityytotoxicity [162][162] 2424 N N N N CytotoxicityCytotoxicity BELHelaBELHela-7402 -(2.67402 (2.6 ±(4.49) ±(4.49)1.1) 1.1) BELBEL-7402-7402±0 ±0.9 0 (UA).10.9 .1(UA) >50 >50 [163][163] 23242324 H3HCH3CHNNONO CCytotoxicityCCytotoxicityytotoxicityytotoxicity MKN45(2.1SGCBELMKN45(2.1BEL-7402BELSGC--74027901(7.01)--74027901(7.01) (4.49) (4.49)±(4.49) ±0.3) 0.3) MKN45(Cisplatin)MKN45(Cisplatin)SGCBELBELSGCBEL-7402--74027901(UA)--74027901(UA) (UA) (UA) (UA) >50>50 >50 2. 2. 8> 850 [162][163][162][163] 24 2424 HHC C OO N N CytotoxicityCytotoxicity MKN45(2.1SGCMKN45(2.1SGC-7901(7.01)-7901(7.01) ± ±0.3) 0.3) MKN45(Cisplatin)MKN45(Cisplatin)SGCSGC-7901(UA)-7901(UA) >50 >50 2. 2.8 8 [163][163] [163] 2424 3 3 CCytotoxicityytotoxicity SGCSGC-7901(7.01)SGC-7901(7.01)-7901(7.01) SGCSGCSGC-7901(UA)-7901(UA)-7901(UA)± ±0 .10.1 >50 >50 > 50 [163][163] HHNNN BELSGCBELSGC--74027901(7.01)--74027901(7.01) (4.49) (4.49) BELSGCBELSGC--74027901(UA)--74027901(UA) (UA) (UA) >50 >50 2424 ClCl CCytotoxicityytotoxicity ± ±0 .10.1 [163][163] CCl l SGCSGC-7901(7.01)-7901(7.01) SGCSGC-7901(UA)-7901(UA) >50 >50 HCCHNl lNN BELBEL-7402-7402 (4.49) (4.49) BELBEL-7402-7402 (UA) (UA) >50 >50 2424 HCHCNl Nl N CCytotoxicityytotoxicity A375(0.51A375(0.51 ± ±0.05) 0.05) [163][163] A375(0.51SGCA375(0.51SGC-7901(7.01)-7901(7.01) ± ±0.05) 0.05) SGCSGCA375(BA)-7901(UA)-A375(BA)7901(UA) >50 >50 CCl l A2780(0.45A375(0.51BELA2780(0.45A375(0.51BEL-7402-7402 ±(4.49) ± (4.49)0.05)±0.03) 0.05) 0.03) BELBEL-A375(BA)7402-A375(BA)7402 (UA) (UA) >50 >50 Et NEt N A375(0.51A375(0.51 ± 0.05) A375(BA) 2424 2 2 CCytotoxicityytotoxicity A2780(0.45BELA2780(0.45BEL-7402-7402 (4.49)± (4.49)±0.03) 0.03) BELBELA2780(BA)-A375(BA)7402A2780(BA)-A375(BA)7402 (UA) (UA) >50 >50 [163][163] Et2ENt N ± 2424 Cl Cl 2 CCytotoxicityytotoxicity A2780(0.45HT29(0.50A2780(0.45SGCHT29(0.50SGC-7901(7.01)-7901(7.01) ± ± ±0.07) 0.03) 0.07)0.03) SGCSGCA2780(BA)-7901(UA)A2780(BA)-7901(UA) >50 >50 [163][163] O O EtE2Nt2N O O A375(0.51A2780(0.45A2780(0.45A375(0.51 ± ±0.05)± 0.05) 0.03) A2780(BA) CCl l ClECt2lENt2N SGCSGC-7901(7.01)-7901(7.01) SGCSGCA2780(BA)-7901(UA)A2780(BA)-7901(UA) >50 >50 O O HT29(0.50HT29(0.50 ± ±0.07) 0.07) A2780(BA)HT29(BA)A2780(BA)HT29(BA)A375(BA) O ClCl O ± A375(BA) 2525 OO Cl COl O O O NEt CytotoxicityCytotoxicity MCF7(0.39HT29(0.50MCF7(0.39HT29(0.50 ± ±±0.04)0.07) 0.07)0.04) HT29(BA)HT29(BA) [164][164] OO O O NEt2 2 A2780(0.45HT29(0.50HT29(0.50A2780(0.45HT29(0.50 ±± ± 0.07)±0.03) 0.07) 0.03) HT29(BA) O O Et2ENt2N A375(0.51A375(0.51 ± ±0.05) 0.05) HT29(BA)HT29(BA) 25 2525 H HC3Cl l O O O O NENt2Et CytotoxicityCytotoxicity MCF7(0.39MCF7(0.39 ±± ±0.04) 0.04) MCF7(BA)HT29(BA)MCF7(BA)HT29(BA) [164][164] [164] 3 O N N 2 A2780(BA)A2780(BA) 2525 ON N O O NENtE2 t CytotoxicityCytotoxicity MCF7(0.39MCF7(0.39MCF7(0.39 ± ± 0.04) 0.04) A375(BA)A375(BA)MCF7(BA) [164][164] 2525 H3HC3Cl l OO Cl ClO O NENt Et2 CCytotoxicityytotoxicity MCF7(0.39HT29(0.50MCF7(0.39NiH3T3(0.4HT29(0.50NiH3T3(0.4 ± ±0.07)00.04) 0.07) 0.04)0± ± MCF7(BA)MCF7(BA) [164][164] H C OOO O O N N N O O 2 2 MCF7(BA) HH3C3C OO O O N N A2780(0.45A2780(0.45NiH3T3(0.4A375(0.51NiH3T3(0.4 ± ± ±0.03)0 0.05) 0.03)0± ± NiH3T3(BA)MCF7(BA)NiH3T3(BA)MCF7(BA) 3 3 N N NEtN2ENt2N A375(0.51 ± 0.05) HT29(BA)HT29(BA) N N N NiH3T3(0.40NiH3T3(0.4NiH3T3(0.400 0.03)± ± A2780(BA)A2780(BA)NiH3T3(BA) 2525 O O CCytotoxicityytotoxicity MCF7(0.39A375(0.51MCF7(0.39NiH3T3(0.4A375(0.51NiH3T3(0.40.03)0.03) ± ± ±0.05) ±0.04)0 0.05)0.04) 0± ± NiH3T3(BA)A375(BA)NiH3T3(BA)A375(BA) [164][164] Cl Cl NENt2E t2 ± NiH3T3(BA)NiH3T3(BA) HHCCOOOO O O O O A2780(0.45HT29(0.50A2780(0.45HT29(0.500.03)0.03) ± ± 0.07)±0.03) 0.07)0.03) SW1736(BA)NiH3T3(BA)MCF7(BA)A375(BA)NiH3T3(BA)SW1736(BA)MCF7(BA)A375(BA) 3 3 NEtNENt N SW17360.03) (n.d) SW1736(BA) N N 2 2 A2780(0.45A2780(0.45NiH3T3(0.4SW1736NiH3T3(0.4SW17360.03)0.03).03) (n.d) ± (n.d) ±0.03)0 0.03) 0± ± SW1736(BA)A2780(BA)HT29(BA)SW1736(BA)A2780(BA)HT29(BA) 2525 Et ENOt ON CCytotoxicityytotoxicity MCF7(0.39MCF7(0.39 ± ±0.04) 0.04) SW1736(BA)SW1736(BA) [164][164] Cl C2l 2 NENt2Et2 HT29(0.50HT29(0.50SW1736SW1736 (n.d)± (n.d)±0.07) 0.07) SW1736(BA)NiH3T3(BA)A2780(BA)NiH3T3(BA)SW1736(BA)A2780(BA) HHCCOOOO O O O OO SW1736SW1736 (n.d) (n.d) MCF7(BA)MCF7(BA) 3 3 CNl CNl O HT29(0.50A375(1.5HT29(0.50SW1736A375(1.5SW17360.03)0.03) ±(n.d) (n.d)±0.4) 0.07) 0.4)0.07) HT29(BA)HT29(BA) OO N N OOOO NiH3T3(0.4A375(1.5NiH3T3(0.400.4) 0± ± A375(UA) n.d. 2525 O OXOXO NENt Et CCytotoxicityytotoxicity MCF7(0.39MCF7(0.39A375(1.5A375(1.5 ±± ±0.4)±0.04) 0.4)0.04) A375(UA)SW1736(BA)A375(UA)HT29(BA)SW1736(BA)HT29(BA) n.d. n.d. [164][164] Int.Int. J. J.Mol. Mol. Sci. Sci. 20 202020,, 2,O, 12NO,,1 Nxx,, xxFORFOR FORFOR PEERPEER XPEERPEERO O REVIEWREVIEWO REVIEWREVIEW2 2 A375(1.5A375(1.5 ± ±± 0.4) 0.4) A375(UA)NiH3T3(BA)NiH3T3(BA) n.d. 1212 of of 28 28 2525 H3HC3C OO O O XO O NENt Et CCytotoxicityytotoxicity MCF7(0.39A2780(1.9MCF7(0.39A375(1.5A2780(1.9SW1736A2780(1.9A375(1.5SW1736 (n.d)± (n.d)±0.4)±0.3)0.04) 0.4)0.3)0.04)0.3) A2780(UA)A375(UA)MCF7(BA)MCF7(BA) n.d. 11.7 0.6 [164][164] Int.Int. J. J.Mol. Mol. Sci. Sci. 20 202020, 2O,1 N2O, 1Nx,N xFOR FORN N PEER XPEEROXO REVIEW REVIEW2 2 0.03)0.03) A375(UA)A375(UA) n.d. n.d. 1212 of of 28 28 H3HC3CO OOO N N N XOXON N A2780(1.9NiH3T3(0.4A2780(1.9NiH3T3(0.4 ±± ±0.3)0 0.3)0± ± A2780(UA)A2780(UA)A375(UA)A375(UA)MCF7(BA)MCF7(BA) 11.7 11.7 n.d. n.d. ± ± 0.6 0.6± NN N N N N N O O HT29A2780(1.9A2780(1.9HT29 (5.7 (5.7 ±± ± 0.5)0.3) 0.3)0.5) A2780(UA)SW1736(BA)SW1736(BA) 11.7 ± 0.6 OO N N N NONO A2780(1.9NiH3T3(0.4A375(1.5HT29SW1736A2780(1.9A375(1.5NiH3T3(0.4SW1736 (5.7 (n.d) ± ±(n.d) ± 0.4)±00.3) 0.4) 0.5) 0.3)0± ± A2780(UA)A2780(UA)NiH3T3(BA)HT29NiH3T3(BA) (UA) 11.7 11.7 10.6± ± 0.6 0.6 0.7 26 2626 OO NNO O NXNOXONNNN O O CytotoxicityCytotoxicity HT29HT290 (5.7.03)0 (5.7.03) ±± ±0.5) 0.5) HT29A2780(UA)A2780(UA)HT29A375(UA)NiH3T3(BA)A375(UA) NiH3T3(BA)(UA) (UA) 10.611.7 11.710.6 n.d. n.d. ±± ±0.7 0.6 0.60.7± [165][165] OO N N NN N N OO HT29 (5.7 ± 0.5) NIHNIH 3T3(UA) 3T3(UA) 13.1 13.1 ± ± 2626 H3HC3C O O N N O O NNNN N N O OXO X CytotoxicityCytotoxicity MCFA2780(1.9HT29MCF-7(4.4MCFHT29A2780(1.9-7(4.40 -(5.7.03)7(4.40 (5.7.03) ± ±0.5)±0.7)0.3) 0.5)0.3)0.7)0.7) HT29NIHHT29NIHMCF-7(UA)SW1736(BA) 3T3(UA)SW1736(BA) (UA)3T3(UA) (UA) 10.6 10.6 13.1 13.1 12.7± ±0.7 ±0.7 ± 0.1 [165][165] [165] 2626 HHC C OO OO N N ONNO OXOX CytotoxicityCytotoxicity MCFA375(1.5A375(1.5-7(4.4 ± ± ±0.4) 0.7)0.4) HT29NIHMCFHT29NIH 3T3(UA) -(UA) 7(UA) 3T3(UA)(UA) 10.6 10.612.7 13.1 13.1± ±±0.7 0.7±0.1 ± [165][165] 2626 3 3 OO X =X Nt B=Nu OtBOu NNNN CCytotoxicityytotoxicity MCFSW1736SW1736-7(4.4 (n.d) (n.d) ± 0.7) MCFA2780(UA)HT29A2780(UA)HT29SW1736(BA)-7(UA)SW1736(BA) (UA) (UA) 10.612.711.7 10.611.7 ± ±0.7 0.10.6 0.70.6 [165][165] HH3CC OO XNOXNO OXOX MCFFaDuMCFFaDu-7(4.4-7(4.4 (3.7(3.7 ± ±± 0.7) 0.6)0.6)0.7) MCFA375(UA)A375(UA)-7(UA)FaDu1.11.1 12.7 (UA)n.d. n.d. ± 0.1n.d H3HC33C OO X =X t B= utBu NNNNOXOX O O OX MCFFaDuHT29MCFSW1736HT29SW1736- 7(4.4(3.7-(5.77(4.4 (5.7 (n.d)± ±(n.d) ±0.6) ±0.5)0.7) 0.5)0.7) MCF-7(UA) 12.7 ± 0.1 N NX X= =tB tuBu OXO O A2780(1.9FaDuA2780(1.9FaDu (3.7 (3.7 ±± ±0.6)0.3) 0.6)0.3) MCFMCFFaDuFaDu-7(UA)-7(UA) (UA)1.1 (UA)1.1 12.7 12.7 n.d n.d ± ± 0.1 0.1 2626 X =X t=B OutBOu O O ONXN CCytotoxicityytotoxicity NIHFaDuA375(1.5FaDuA375(1.5 3T3(4.6 (3.7 (3.7 ± ± ±0.4)± 0.6) 0.4)0.6)1.0) HT29MCFHT29NIHA375(UA) -(UA)7(UA) (UA) 3T3(UA) 10.6 10.6 12.7n.d. ± 13.1± ±0.7 0.70.1 1.1 [165][165] HHCCOOOO N N OXNOXNOONXONXO OXO X NIHMCFFaDuNIHA375(1.5MCFFaDuA375(1.5 3T3(4.6 -3T3(4.67(4.4 -(3.77(4.4 (3.7 ± ± ± ±0.4) ± 0.6)± 0.7) 0.4)±1.0) 0.6)0.7) 1.0) A2780(UA)A2780(UA)FaDuA375(UA)A375(UA)FaDu (UA) (UA) 11.7 11.7 n.d. n.dn.d. n.d± ± 0.6 0.6 3 3 N ON OX O O A375(2.0 ± 0.1)± FaDu (UA) n.d ± N N OXOXO O O Cytotoxicity NIHA2780(1.9HT29NIHA375(2.0A2780(1.9HT29A375(2.0 3T3(4.6 3T3(4.6 (5.7 (5.7 ±± ± 0.1)±0.5)0.3) 0.1) ±1.0)0.5)0.3) 1.0) FaDuA375(UA)FaDuA375(UA) (UA) (UA) n.d. n.d n.d.n.d 2626 X =X t=B utBu O O O CCytotoxicityytotoxicity NIHNIH 3T3(4.6 3T3(4.6 ± ± 1.0) 1.0) MCFA2780(UA)HT29MCFHT29A375(UA)A375(UA)-7(UA) -(UA)7(UA) (UA) 10.611.712.7 10.612.7n.d. n.d. ± ± ± 0.7 ±0.60.1 0.70.1 [165][165] N N O O XOXONON NIHA2780(1.9NIHFaDuA375(2.0A2780(1.9FaDuA375(2.0 3T3(4.6 3T3(4.6 (3.7 (3.7 ±± 0.1)±0.3)0.6) ±0.1)0.6)1.0)0.3) 1.0) A2780(UA)A2780(UA) 11.7 11.7 ± ±0.6 0.6 HHCCOOOO N N N NONXONX O OXOX MCFA2780(1.7MCFA375(2.0-7(4.4-7(4.4 ± ±0.1)0.7)0.1) 0.7) A375(UA) n.d. 3 3 N XNO O O A2780(1.7HT29A2780(1.7HT29 (5.7 (5.7 ± ±0.5)0.1) 0.1)0.5) A2780(UA)A2780(UA)FaDuFaDu (UA) (UA) 11.7 11.7 n.d n.d± ±0.6 0.6 OO NHNH O O XNO N ± MCFHT29MCFHT29-7(UA) -(UA)7(UA) (UA) 10.612.7 10.612.7 ±± ± 0.7±0.1 0.70.1 2626 OO X =X t=BN OutBNOu N N NNNN O O CCytotoxicityytotoxicity NIHHT29A2780(1.7NIHA2780(1.7HT29A2780(1.7 3T3(4.6 3T3(4.6 (5.7 (5.7 ± ±0.5)0.1) ±0.1)0.5)1.0)0.1) 1.0) HT29HT29A2780(UA) (UA) (UA) 10.6 10.6 11.7± ±0.7 0.7 0.6 [165][165] N N H H N NNOXOX O O XOX HT29FaDuHT29FaDu (2.3(3.7 (2.3(3.7 ± ±0.3)0.6) 0.3)0.6) HT29 (UA) 10.6 ± 0.7 2626 H3HC3CO OOO H H NONO N N N CCytotoxicityytotoxicity MCFMCF-7(4.4-7(4.4 ±± ±0.7) 0.7) HT29HT29 (UA) (UA) 10.6 10.6 ± ±0.7 0.7± [165][165] 2727 HHC C OO N N O OONON N N O OOXOX MCFHT29HT29MCFHT29-7(4.4 -(2.37(4.4 (2.3(2.3 ± 0.3)±0.7)0.3) 0.3)0.7) MCFMCFFaDuHT29FaDu-7(UA)--7(UA) (UA) (UA) 1212.7 1212.7 .7.7n.d .7.710.6n.d ±± ± ±0.10.1 0.10.1 0.7 H3C3 O H HX =X t =B utBu N N OXOX 27 2727 HH33C33C OO O O N N NIHMCFFaDuNIHMCFFaDu 3T3(4.6 -3T3(4.67(1.8 -(3.77(1.8 (3.7 ±± ± ± ±0.6)0.1) ± 1.0)0.6)0.1) 1.0) MCFMCF-7(UA)-7(UA) 12 12.7.7 ± ±0.1 0.1± X =X t B= utBu N OXOX OX MCFMCF-7(UA)-7(UA) 12.7 12.7 ± ±0.1 0.1 H3HC C O O X =X t t=B ututBuu N OX MCFMCF-7(1.8MCF-7(1.8-7(1.8 ± ±0.1) 0.1)0.1) FaDuMCF-7(UA)FaDu (UA) (UA) n.d n.d 12.7 0.1 3 O O OOXOXOXX FaDuFaDu (3.7 (3.7 ± ±0.6) 0.6) FaDuFaDu (UA) (UA) n.d n.d X =X t B=u tBu OXOX NIHFaDuNIHFaDu 3T3(4.6 3T3(4.6 (2.0 (2.0 ± ±0.2) ±0.2)1.0) 1.0) FaDuFaDu (UA) (UA) n.d n.d ± OOOO OXOX NIHFaDuNIHFaDuFaDu 3T3(4.6 3T3(4.6 (2.0 (2.0(2.0 ± ±±0.2) ±0.2)1.0)0.2) 1.0) NIHNIH 3T3(UA) 3T3(UA)FaDu (UA) 13. 13.1 1n.d± ± O O NIHNIH 3T3(1.4 3T3(1.4 ±± ±0.1) 0.1) NIHNIH 3T3(UA) 3T3(UA) 13. 13.1 1± ± NIHNIHNIH 3T3(1.4 3T3(1.43T3(1.4 ± ±0.1) 0.1)0.1) NIH 3T3(UA)1.11.1.1.1 13.1 1.1 ± 1.11.1 ± HH OO NHNH HeLa(9.25)HeLa(9.25) OO HeLa(9.25)HeLa(9.25) 28 2828 NN NN HepG2(21.2)HepG2(21.2) HeLa(Gefitinib) 17.1 2828 OONN HepG2(21.2)HepG2(21.2) [166] OO BGCBGC-823(8.06)BGC-823(8.06)-823(8.06) HepG2(Gefitinib) 20.7 BGCBGC-823(8.06)-823(8.06) HeLa(Gefitinib)HeLa(Gefitinib)BGC-823 Gefitinib 17.1 17.1 19.3 cytotoxicity HeLa(Gefitinib)HeLa(Gefitinib) 17.1 17.1 HHOO cytotoxicitycytotoxicity HepG2(Gefitinib)HepG2(Gefitinib) 20.7 20.7 HHOO cytotoxicitycytotoxicity HepG2(Gefitinib)HepG2(Gefitinib) 20.7 20.7 [166][166] OO HH HeLa(13.8)HeLa(13.8) BGCBGC-823-823 Gefitinib Gefitinib 19.3 19.3 [166][166] OO H HeLa(13.8)HeLa(13.8) BGCBGC-823-823 Gefitinib Gefitinib 19.3 19.3 29 2929 NN NNH HepG2(23.7)HepG2(23.7) 2929 OONN NN HepG2(23.7)HepG2(23.7) OO BGCBGC-823(9.15)BGC-823(9.15)-823(9.15) BGC-823(9.15) BGC-823(9.15)

n.d = not determined. BA = Betulinic acid. n.dn.d = =not not determined. determined. BA BA = =Be Betulinictulinic acid acid.. .. n.dn.d = =not not determined. determined. BA BA = =Be Betulinictulinic acid acid. .

3.1.3.3.1.3. Modification Modification o of fβ β-Hyd-Hydroxyroxy (C (C-3-3 Po Position)sition) 3.1.3.3.1.3. Modification Modification o fo βf -βHyd-Hydroxyroxy (C (C-3- 3Po Position)sition) FontanaFontana et et al. al. synthesized synthesized derivatives derivatives of of UA UA and and oleanolic oleanolic acid acid with with a amodified modified oxidation oxidation state state FontanaFontana et et al. al. synthesized synthesized derivatives derivatives of of UA UA and and oleanolic oleanolic acid acid with with a amodified modified oxidation oxidation state state andand lipophilicity lipophilicity at at C C-3- 3and and C C-28-28 positions positions which which were were screened screened in in vitro vitro on on hepatocarcinoma hepatocarcinoma cell cell andand lipophilicity lipophilicity at at C C-3- 3and and C C-28-28 positions positions which which were were screened screened in in vitro vitro on on hepatocarcinoma hepatocarcinoma cell cell lineslines——namelynamely HepG2, HepG2, HA22T/VGH HA22T/VGH,, and,, and Hep Hep3B.3B. UA UA derivatives derivatives containing containing three three carbons carbons as as the the side side lineslines——namelynamely HepG2, HepG2, HA22T/VGH HA22T/VGH, and, and Hep Hep3B.3B. UA UA derivatives derivatives containing containing three three carbons carbons as as the the side side chainchain at at the the C C-3-3 position position of of UA UA were were synthesized synthesized in in stereoisomeric stereoisomeric forms forms using using the the Barbier Barbier––GrignardGrignard chainchain at at the the C C-3- 3position position of of UA UA were were synthesized synthesized in in stereoisomeric stereoisomeric forms forms using using the the Barbier Barbier–Grignard–Grignard methodmethod and and (Compounds (Compounds 30 30 and and 31 31,, ,Table,Table TableTable 5)5) 5)5) werewere werewere foundfound foundfound toto toto bebe bebe effeeffe effeeffectivective against against all all three three cancer cancer cell cell methodmethod and and (Compounds (Compounds 30 30 and and 31 31, Table, Table 5) 5) were were found found to to be be effe effectivective against against all all three three cancer cancer cell cell lineslines;; ;;these these compounds compounds inhibited inhibited cell cell growth growth and and induce inducedd the the inhibition inhibition of of NF NF-κB-κB activation activation in in the the lineslines; these; these compounds compounds inhibited inhibited cell cell growth growth and and induce inducedd the the inhibition inhibition of of NF NF-κB-κB activation activation in in the the cellcell lines lines [167] [167].. .. cellcell lines lines [167] [167]. . XuXu et et al al.. ..isolated isolated triterpenoids triterpenoids from from the the acorn acorn-starch/licorice-starch/licorice and and reacted reacted them them with with 3,4,5 3,4,5- - XuXu et et al al. .isolated isolated triterpenoids triterpenoids from from the the acorn acorn-starch/licorice-starch/licorice and and reacted reacted them them with with 3,4,5 3,4,5- - methoxybenzoicmethoxybenzoic acid acid under under dicyclohexyl dicyclohexyl carbodiimide carbodiimide ( DCC (DCC)/DMAP)/DMAP conditions conditions and and evaluated evaluated their their methoxybenzoicmethoxybenzoic acid acid under under dicyclohexyl dicyclohexyl carbodiimide carbodiimide (DCC (DCC)/DMAP)/DMAP conditions conditions and and evaluated evaluated their their cytotoxicitycytotoxicity in in four four cells cells,, ,,including including A A-549,-549, MCF MCF-7,-7, H1975, H1975, and and BGC BGC-823.-823. Most Most of of the the synthesized synthesized cytotoxicitycytotoxicity in in four four cells cells, ,including including A A-549,-549, MCF MCF-7,-7, H1975, H1975, and and BGC BGC-823.-823. Most Most of of the the synthesized synthesized compoundscompounds indicated indicated a asignificant significant cytotoxic cytotoxic activity activity in in all all the the four four c ellcell lines lines and and a alower lower toxicity toxicity in in compoundscompounds indicated indicated a asignificant significant cytotoxic cytotoxic activity activity in in all all the the four four c ellcell lines lines and and a alower lower toxicity toxicity in in thethe normal normal cell cells,,s Human,, Human Hair Hair Dermal Dermal Papilla Papilla Cells Cells (HHDPC) (HHDPC) when when compar compareded to to the the positive positive control control,, ,, thethe normal normal cell cells,s Human, Human Hair Hair Dermal Dermal Papilla Papilla Cells Cells (HHDPC) (HHDPC) when when compar compareded to to the the positive positive control control, , mitomycinmitomycin C. C. The The UA UA derivative derivative containing containing 3,4,5 3,4,5-methoxy-methoxy-phenacyl-phenacyl at at the the C C-3-3 position position (c (compoundompound mitomycinmitomycin C. C. The The UA UA derivative derivative containing containing 3,4,5 3,4,5-methoxy-methoxy-phenacyl-phenacyl at at the the C C-3- 3position position (c (compoundompound 3232,, ,Figure,Figure FigureFigure 3,3, 3,3, TableTable TableTable 5)5) 5)5) waswas waswas the the derivative derivative with with a asignificant significant antiproliferative antiproliferative effect effect,, with,, with IC IC5050 values values in in 3232, Figure, Figure 3, 3, Table Table 5) 5) was was the the derivative derivative with with a asignificant significant antiproliferative antiproliferative effect effect, with, with IC IC50 50values values in in thethe range range of of 6.07 6.07––22.2722.27 µM µM [140] [140].. .. thethe range range of of 6.07 6.07–22.27–22.27 µM µM [140] [140]. .

Int. J. Mol. Sci. 2020, 21, 5920 12 of 27

3.1.3. Modification of β-Hydroxy (C-3 Position) Fontana et al. synthesized derivatives of UA and oleanolic acid with a modified oxidation state and lipophilicity at C-3 and C-28 positions which were screened in vitro on hepatocarcinoma cell lines—namely HepG2, HA22T/VGH, and Hep3B. UA derivatives containing three carbons as the side chain at the C-3 position of UA were synthesized in stereoisomeric forms using the Barbier–Grignard method and (Compounds 30 and 31, Table5) were found to be e ffective against all three cancer cell lines; these compounds inhibited cell growth and induced the inhibition of NF-κB activation in the cell lines [167]. Xu et al. isolated triterpenoids from the acorn-starch/licorice and reacted them with 3,4,5-methoxybenzoic acid under dicyclohexyl carbodiimide (DCC)/DMAP conditions and evaluated their cytotoxicity in four cells, including A-549, MCF-7, H1975, and BGC-823. Most of the synthesized compounds indicated a significant cytotoxic activity in all the four cell lines and a lower toxicity in the normal cells, Human Hair Dermal Papilla Cells (HHDPC) when compared to the positive control, mitomycin C. The UA derivative containing 3,4,5-methoxy-phenacyl at the C-3 position (compound 32, Figure3, Table5) was the derivative with a significant antiproliferative e ffect, with IC50 values in the rangeInt. J. Mol. of Sci. 6.07–22.27 2020, 21, xµ FORM[ 140PEER]. REVIEW 13 of 28

OH OH O O HO

HO 30 31

OH O O H3CO

H3CO 32 OCH3 Figure 3. Fontana and Xu ‘s UA derivatives. Figure 3. Fontana and Xu ‘s UA derivatives.

Table 5.5. ModificationModification ofof β-hydroxy-hydroxy (C-3).(C-3).

Biological Cell Lines Tested Reference Molecules CompoundCompound Cell Lines Tested (IC50µM) Reference Molecules (IC50µM) BibliographyBibliography ActivityActivity (IC50µM) (IC50µM) HA22THA22T/VGH(31./VGH(31.0 1.5)0 ± 1.5) HA22THA22T/VGH(UA)/VGH(UA) > 100 ˃ 100 ± 30 Cytotoxicity HepG2(28.0 2.0) HepG2(UA) > 100 [167] 30 Cytotoxicity HepG2(28.± 0 ± 2.0) HepG2(UA) ˃ 100 [167] Hep3B(32.5 2.5) Hep3B(UA) > 100 Hep3B(32.± 5 ± 2.5) Hep3B(UA) ˃ 100 HA22T/VGH(31.0 1.5) HA22T/VGH(UA) >100 HA22T/VGH(31.0± ±1.5) HA22T/VGH(UA) ˃100 31 Cytotoxicity HepG2(28.0 2.0) HepG2(UA) > 100 [167] 31 Cytotoxicity HepG2(28.± 0 ± 2.0) HepG2(UA) ˃ 100 [167] Hep3B(32.5 2.5) Hep3B(UA) > 100 Hep3B(32.± 5 ± 2.5) Hep3B(UA) ˃ 100 n.d = not determined. A549 (6.07 ± 0.91) A549 (mitomycin C) 28.14 ± 3.41 H1975(10.64 ± 1.94) H1975(mitomycin C) 34.51 ± 3.06 MCF-7(mitomycin C) 44.08 ± 4.01 32 Cytotoxicity MCF-7(22.27± 3.51) [140] BGC-823 (mitomycin C) 37.94 ± BGC-823 (17.10 ± 1.04) 2.88 t-HSC/Cl-6 (29.12 ± 3.71) t-HSC/Cl-6 (mitomycin C) n.d

n.d = not determined.

3.1.4. Modification of miscellaneous groups Wu et al. synthesized UA derivatives bearing an aminoguanidine moiety and investigated them as HIF-1α inhibitors and anticancer agents in human cancer cell lines. The majority of these compounds showed a potent inhibition of the HIF-1α transcriptional effect; among these derivatives, compound 35b (Scheme 4) was found to be the most potent inhibitor of HIF-1α expression in hypoxic conditions (IC50 4.0 µM), with no significant cytotoxicity noted against any cell lines tested [168].

Int. J. Mol. Sci. 2020, 21, 5920 13 of 27

Table 5. Cont.

Biological Compound Cell Lines Tested (IC µM) Reference Molecules (IC µM) Bibliography Activity 50 50 A549 (6.07 0.91) A549 (mitomycin C) 28.14 3.41 ± ± H1975(10.64 1.94) H1975(mitomycin C) 34.51 3.06 ± ± 32 Cytotoxicity MCF-7(22.27 3.51) MCF-7(mitomycin C) 44.08 4.01 [140] ± ± BGC-823 (17.10 1.04) BGC-823 (mitomycin C) 37.94 2.88 ± ± t-HSC/Cl-6 (29.12 3.71) t-HSC/Cl-6 (mitomycin C) n.d ± n.d = not determined.

3.1.4. Modification of Miscellaneous Groups Wu et al. synthesized UA derivatives bearing an aminoguanidine moiety and investigated them as HIF-1α inhibitors and anticancer agents in human cancer cell lines. The majority of these compounds showed a potent inhibition of the HIF-1α transcriptional effect; among these derivatives, compound 35b (Scheme4) was found to be the most potent inhibitor of HIF-1 α expression in hypoxic conditions Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 14 of 28 (IC50 4.0 µM), with no significant cytotoxicity noted against any cell lines tested [168].

OH OH R OH O a b O O UA HO 33 34 O O

c

35a R1 = , IC50 = 37.8 µM (HRE)

35b R1 = Cl , IC50 = 4.0 µM (HRE) R1 OH

35c R1 = , IC50 = 75.4 µM (HRE) O Br H H N N 2 N NH 35a-c

Scheme 4. Synthesis of compounds 33–35. Reagents and conditions: (a) Jones reagent acetone, 0 ◦C, 5 h,Scheme 90%; (b )4. Aldehydes, Synthesis of 5% compounds NaOH, absolute 33–35 EtOH,. Reagents r.t 2 h,and 30–75%; conditions: (c) 37% (a) HCl, Jones absolute reagent EtOH, acetone, reflux, 0 °C 8, 5 h,h, 34–65%. 90%; (b) Aldehydes, 5% NaOH, absolute EtOH, r.t 2 h, 30–75%; (c) 37% HCl, absolute EtOH, reflux, 8 h, 34–65%. Gu et al. designed and synthesized a series of new ursolic acid-based derivatives through the conjugationGu et ofal. UAdesigned with quinoloneand synthesized and oxadiazole a series of motifs new andursolic investigated acid-based their derivati anticancerves through activity. the Theseconjugation compounds of UA were with investigated quinolone and in vitro oxadiazole on human mot cancerifs and cell investigated lines-namely their MDA-MB-231, anticancer activity Hela, . andThese SMMC-7721, compounds and were the resultsinvestigated indicated in vitro that on compounds human cancer36–38 cellexhibited lines-namely a strong MDA growth-MB inhibitory-231, Hela, effandect against SMMC- the7721, three and cancer the cellresults lines. indicated Among these that c derivatives,ompounds compound36–38 exhibited38b exhibited a strong the growth most potentinhibitory antitumor effect activity,against the with three IC cancervalues cell of0.61 lines. Among0.07 (MDA-MB-231), these derivatives, 0.36 compound0.05 (HeLa), 38b and exhibited 12.49 50 ± ± the0.08 mostµM potent (SMMC-7721) antitumor when activity compared, with toIC the50 values positive of 0.61 control, ± 0.07 etoposide (MDA-MB [169-231),] (Scheme 0.36 ±5 0.05). (HeLa), ±and 12.49 ± 0.08 µM (SMMC-7721) when compared to the positive control, etoposide [169] (Scheme 5).

O O OH OH OH R O O a O b HO 36 N O 37 38a-d

38a R = H, IC50 = 0.75 ± 0.05 µM (MDA-MB-231) 38c R = Cl, IC50 = 1.36 ± 0.03 µM (MDA-MB-231) = 0.37 ± 0.04 µM (HeLa) = 1.22 ± 0.08 µM (HeLa) = 13.40 ± 0.08 µM (SMMC-77212) = 14.62 ± 0.05 µM (SMMC-77212)

38b R = OCH3, 38c R = F, IC50 = 0.90 ± 0.10 µM (MDA-MB-231) IC50 = 0.61 ± 0.07 µM (MDA-MB-231) = 1.87 ± 0.03 µM (HeLa) = 0.36 ± 0.05 µM (HeLa) = 13.34 ± 0.13 µM (SMMC-77212) = 12.49 ± 0.08 µM (SMMC-77212) Scheme 5. Synthesis of compounds 36–38. Reagents and conditions: (a) Jones reagent acetone, 0 °C, 5 h; (b) EtOH, substituted o-amino benzaldehyde, KOH, reflux under N2 atmosphere for 24 h.

Mendes et al. synthesized ring-A-cleaved UA derivatives and evaluated their antiproliferative activity against three lung cancer cell lines—namely H460, H322, and H460+/+—using 2D or 3D culture models. Three of these newly designed UA derivatives bearing a cleaved ring-A with a secondary amide at C-3 (compounds 43a–c, Scheme 6) demonstrated significantly enhanced antiproliferative effects in the 2D systems. These compounds possessed a potent anticancer activity and the

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 14 of 28

OH OH R OH O a b O O UA HO 33 34 O O

c

35a R1 = , IC50 = 37.8 µM (HRE)

35b R1 = Cl , IC50 = 4.0 µM (HRE) R1 OH

35c R1 = , IC50 = 75.4 µM (HRE) O Br H H N N 2 N NH 35a-c

Scheme 4. Synthesis of compounds 33–35. Reagents and conditions: (a) Jones reagent acetone, 0 °C, 5 h, 90%; (b) Aldehydes, 5% NaOH, absolute EtOH, r.t 2 h, 30–75%; (c) 37% HCl, absolute EtOH, reflux, 8 h, 34–65%.

Gu et al. designed and synthesized a series of new ursolic acid-based derivatives through the conjugation of UA with quinolone and oxadiazole motifs and investigated their anticancer activity. These compounds were investigated in vitro on human cancer cell lines-namely MDA-MB-231, Hela, and SMMC-7721, and the results indicated that compounds 36–38 exhibited a strong growth inhibitory effect against the three cancer cell lines. Among these derivatives, compound 38b exhibited the most potent antitumor activity, with IC50 values of 0.61 ± 0.07 (MDA-MB-231), 0.36 ± 0.05 (HeLa), Int. J.and Mol. 12.49 Sci. 2020 ± 0.08, 21 ,µM 5920 (SMMC-7721) when compared to the positive control, etoposide [169] (Scheme 14 of 27 5).

O O OH OH OH R O O a O b HO 36 N O 37 38a-d

38a R = H, IC50 = 0.75 ± 0.05 µM (MDA-MB-231) 38c R = Cl, IC50 = 1.36 ± 0.03 µM (MDA-MB-231) = 0.37 ± 0.04 µM (HeLa) = 1.22 ± 0.08 µM (HeLa) = 13.40 ± 0.08 µM (SMMC-77212) = 14.62 ± 0.05 µM (SMMC-77212)

38b R = OCH3, 38c R = F, IC50 = 0.90 ± 0.10 µM (MDA-MB-231) IC50 = 0.61 ± 0.07 µM (MDA-MB-231) = 1.87 ± 0.03 µM (HeLa) = 0.36 ± 0.05 µM (HeLa) = 13.34 ± 0.13 µM (SMMC-77212) = 12.49 ± 0.08 µM (SMMC-77212) SchemeScheme 5. Synthesis5. Synthesis of of compounds compounds 3636–38.. Reagents Reagents and and conditions: conditions: (a) (Jonesa) Jones reagent reagent acetone, acetone, 0 °C, 0 ◦C, 5 h; (b5) h; EtOH, (b) EtOH, substituted substituted o-amino o-amino benzaldehyde, benzaldehyde, KOH, KOH, reflux reflux under under N2 atmosphere N2 atmosphere for 24 for h. 24 h. Mendes et al. synthesized ring-A-cleaved UA derivatives and evaluated their antiproliferative activity againstMendes threeet al. synthesized lung cancer ring cell- lines—namelyA-cleaved UA derivatives H460, H322, and and evaluated H460+/+ their—using antiproliferative 2D or 3D culture models.activity Three against of three these lung newly cancer designed cell lines UA—namely derivatives H460, H322, bearing and aH460 cleaved+/+—using ring-A 2D or with 3D culture a secondary amidemodels. at C-3 Three (compounds of these newly43a –desc, Schemeigned UA6) derivatives demonstrated bearing significantly a cleaved ring enhanced-A with a antiproliferative secondary Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 28 effectsamide in theat C 2D-3 systems.(compounds These 43a compounds–c, Scheme 6) possessed demonstrated a potent significantly anticancer enhanced activity antiproliferative and the preliminary effects in the 2D systems. These compounds possessed a potent anticancer activity and the mechanismpreliminary of action mechanism showed of that action compound showed that43c induced compound apoptosis 43c induced through apoptosis the activation through of the caspase-7 andactivation caspase-8 of andcaspase the-7 decrease and caspase in Bcl-2-8 and [170 the ].decrease in Bcl-2 [170].

R O 1 O R O OH 1 O O a b HO HO UA 39 O 40

c 43a R2 = CH3 43b R2 = (CH3)2 R1 = F 43c R2 = (CH3)3 R O 1 O O R1 O R O 1 O R2HNOC e HOOC d

O 43a-c 41 42 O

Scheme 6. a b Scheme 6.Reagents Reagents and conditions: conditions: (a) (Selectfluor®) Selectfluor, dioxane,®, dioxane, nitromethane, nitromethane, 80 °C, 80 24◦ h;C, (b 24) Jones h; ( ) Jones reagent, acetone, ice; (c) m-CPBA 77%, CHCl , r.t., 120 h; (d) p-toluenesulfonic acid monohydrate, reagent, acetone, ice; (c) m-CPBA 77%, CHCl3, 3r.t., 120 h; (d) p-toluenesulfonic acid monohydrate, CHCH2Cl22Cl,2 r.t.,, r.t., 24 24 h; h; (e) (e) RR22NH22, ,dry dry THF, THF, Et Et3N,3N, T3P T3P (50 (50wt% wt% in THF), in THF), ice. ice.

BorkoaBorkoa et et al. al. synthesized synthesized the novel novel triterpenic triterpenic derivatives derivatives of dihydrobetulonic, of dihydrobetulonic, betulonic, betulonic, and and ursonicursonic acid. acid. All All of of these these compoundscompounds were were tested tested for for their their in vitroin vitro cytotoxiccytotoxic activity activity against against human human −/− / cancercancer cell cell lines—namely lines—namely CCRF-CEM,CCRF-CEM, CEM CEM-DNR,-DNR, HCT116, HCT116, HCT116 HCT116 p53 p53, K562,− −, K562,K562-TAX, K562-TAX, A549, A549, U2OS,U2OS, and and two two noncancerous noncancerous fibroblastsfibroblasts such such as as BJ BJ and and MRC MRC-5.-5. Compounds Compounds 45 and45 46and were46 thewere most the most effectiveeffective on on the the CCRF-CEM CCRF-CEM cellcell lines and and less less toxic toxic in innon non-cancerous-cancerous fibroblasts. fibroblasts. These These derivatives derivatives triggered apoptosis via the intrinsic pathways. The ursonic acid derivative 45 was synthesized by the triggered apoptosis via the intrinsic pathways. The ursonic acid derivative 45 was synthesized by the bromination of UA using copper (II) bromide in a solution of ethyl acetate (EtOAc) and methanol bromination of UA using copper (II) bromide in a solution of ethyl acetate (EtOAc) and methanol (MeOH) at room temperature. Compound 46 was obtained by the nucleophilic substitution of (MeOH)bromoketone at room 43 by temperature. potassium thiocyanate Compound (KSCN)46 wasin DMSO obtained at 90 °C by, as the sho nucleophilicwn in Scheme substitution7 [171]. of bromoketone 43 by potassium thiocyanate (KSCN) in DMSO at 90 ◦C, as shown in Scheme7[171].

OH OH OH O Br NCS a O b O O O O 44 45 46

Scheme 7. Reagents and conditions: (a) CuBr2, EtOAc, MeOH, r.t., 3 h; (b) KSCN, DMSO, 90 °C, 24 h.

Fan and colleagues synthesized UA hybrid compound 49 (Scheme 8) by reacting UA with Jones reagent at 0 °C in dimethyl ketone to obtain a C-3 oxidized derivative. The oxidized compound was then reacted with benzaldehyde using ethanolic potassium hydroxide (KOH) via the Claisen Schmidt condensation reaction at room temperature to achieve a benzylidine hybrid compound. The benzylidine compound was then reacted with an indole and substituted with a benzaldehyde in EtOAc at room temperature for 2 h to achieve compound 49. The anticancer potential of the

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 28

preliminary mechanism of action showed that compound 43c induced apoptosis through the activation of caspase-7 and caspase-8 and the decrease in Bcl-2 [170].

R O 1 O R O OH 1 O O a b HO HO UA 39 O 40

c 43a R2 = CH3 43b R2 = (CH3)2 R1 = F 43c R2 = (CH3)3 R O 1 O O R1 O R O 1 O R2HNOC e HOOC d

O 43a-c 41 42 O

Scheme 6. Reagents and conditions: (a) Selectfluor®, dioxane, nitromethane, 80 °C, 24 h; (b) Jones

reagent, acetone, ice; (c) m-CPBA 77%, CHCl3, r.t., 120 h; (d) p-toluenesulfonic acid monohydrate, CH2Cl2, r.t., 24 h; (e) R2NH2, dry THF, Et3N, T3P (50 wt% in THF), ice.

Borkoa et al. synthesized the novel triterpenic derivatives of dihydrobetulonic, betulonic, and ursonic acid. All of these compounds were tested for their in vitro cytotoxic activity against human cancer cell lines—namely CCRF-CEM, CEM-DNR, HCT116, HCT116 p53−/−, K562, K562-TAX, A549, U2OS, and two noncancerous fibroblasts such as BJ and MRC-5. Compounds 45 and 46 were the most effective on the CCRF-CEM cell lines and less toxic in non-cancerous fibroblasts. These derivatives triggered apoptosis via the intrinsic pathways. The ursonic acid derivative 45 was synthesized by the bromination of UA using copper (II) bromide in a solution of ethyl acetate (EtOAc) and methanol Int. J.(MeOH) Mol. Sci. 2020 at room, 21, 5920 temperature. Compound 46 was obtained by the nucleophilic substitution of15 of 27 bromoketone 43 by potassium thiocyanate (KSCN) in DMSO at 90 °C, as shown in Scheme 7 [171].

OH OH OH O Br NCS a O b O O O O 44 45 46

Scheme 7. Reagents and conditions: (a) CuBr2, EtOAc, MeOH, r.t., 3 h; (b) KSCN, DMSO, 90 ◦C, 24 h. Scheme 7. Reagents and conditions: (a) CuBr2, EtOAc, MeOH, r.t., 3 h; (b) KSCN, DMSO, 90 °C, 24 Fanh. and colleagues synthesized UA hybrid compound 49 (Scheme8) by reacting UA with Jones reagent at 0 C in dimethyl ketone to obtain a C-3 oxidized derivative. The oxidized compound Fan and◦ colleagues synthesized UA hybrid compound 49 (Scheme 8) by reacting UA with Jones wasreagent then reacted at 0 °C in with dimethyl benzaldehyde ketone to obtain using a ethanolic C-3 oxidized potassium derivative. hydroxide The oxidized (KOH) compound via the was Claisen Schmidtthen reacted condensation with benzaldehyde reaction at using room ethanolic temperature potassium to achieve hydroxide a benzylidine (KOH) via the hybrid Claisen compound. Schmidt The benzylidinecondensationInt. J. Mol. Sci. compound 20 20 reaction, 21, x FOR was atPEER then room REVIEW reacted temperature with an to indole achieve and a substitutedbenzylidine with hybrid a benzaldehyde compound.16 Theof in 28 EtOAc at roombenzylidine temperature compound for 2 h was to achieve then reacted compound with an49 indole. The anticancer and substituted potential with of a the benzaldehyde compound in against gliomaEtOAccompound cells at wasroom against studied. temperature glioma The cells compound for was 2 studied. h to achieve demonstrated The compound compound a demonstrated good 49. The inhibition anti acancer good of cellinhibitio potential proliferationn ofof cell the and inducedprolifer apoptosisation and when induced compared apoptosis to when the parentcompared compound, to the parent UA compound, [172]. UA [172].

CHO R R R R R O CrO3,HOSO4 O R O R Ace,98% UA O KOH,MeOH,rt, 92% O 47 48

Br

H N OHC R = OH R L-proline Br R R

O

O N H 49 Scheme 8. Synthesis of UA hybrid compounds. Scheme 8. Synthesis of UA hybrid compounds. An aromatic heterocyclic compound containing carbazole has attracted attention due its potential An aromatic heterocyclic compound containing carbazole has attracted attention due its anticancerpotential activity anticancer [173 activity]. Gu et [173] al. synthesized. Gu et al. synthesized a series of a carbazole series of carbazole derivatives derivatives of UA. Among of UA. these derivatives,Among these compound derivatives,50 (Figure compound4) showed 50 (Figure a significant 4) showed cytotoxic a significant activity cytotoxic against activity the hepatocarcinoma against the cell line,hepatocarcinoma HepG2, with cell an line IC50, HepG2value of, with 1.26 an 0.17IC50 µvalueM[174 of ].1.26 Zhang ± 0.17 et µM al. [174] employed. Zhang the et biotransformational. employed ± of UAthe by biotransformationMucor spinosus ASof UA 3.3450, by Mucor and threespinosus novel AS 3.3450 compounds, and three were novel isolated. compounds Compound were isolated.51 (Figure 4), identifiedCompound as 3β ,51 7β (Figure-dihydroxy-ursolic 4), identified acid-28-etha-none as 3β, 7β-dihydroxy indicated-ursolic a acid stronger-28-etha cytotoxic-none indicated activity againsta the tumorstronger cell cytotoxic lines (Hela,activity K562,against and the tumor KB) when cell lines compared (Hela, K562 to the, and parent KB) when compound compared UA to the [175 ]. A numberparent of compound NO-donating UA [175] ursolic. A acid-basednumber of NO benzylidene-donating ursolic derivatives acid-based with benzylidene various substitutions derivatives were synthesizedwith various by Zhangsubstitutions et al. were They synthesized were further by Zhang analyzed et al. They for their werein further vitro analycytotoxicityzed for their against in the vitro cytotoxicity against the HepG-2, MCF-7, HT-29, and A549 cancer cell lines. Most of these HepG-2, MCF-7, HT-29, and A549 cancer cell lines. Most of these derivatives revealed a weaker derivatives revealed a weaker inhibitory effect when compared to UA; compound 52 (Figure 4) inhibitory effect when compared to UA; compound 52 (Figure4) showed the most potent activity showed the most potent activity against HT-29 (IC50 = 4.28 µM). The further investigation of its againstanticancer HT-29 (ICmode50 = of4.28 actionµM). revealed The further that it investigation induced the of apoptosis its anticancer of HT- mode29 cell of lines action in revealeda dose- that it induceddependent the apoptosismanner and of indicated HT-29 cell cell lines cycle in arrest a dose-dependent at the G1 phase manner, which andled to indicated cell apoptosis cell cycle and arrest also induced apoptosis via the mitochondria-mediated pathways [176].

Int. J. Mol. Sci. 2020, 21, 5920 16 of 27 at the G1 phase, which led to cell apoptosis and also induced apoptosis via the mitochondria-mediated pathwaysInt. J. Mol. Sci. [176 2020]., 21, x FOR PEER REVIEW 17 of 28

R = NH R N R O O R = CH3 R 50 by Gu et al. HO OH 51 by S. Zhang et al

p-Cl R = NH O R R Cl R O O

O 52 by Jin et al. N 53 by T. Zhang et al.

R = ONO2 Figure 4. GuGu’s,’s, S. S. Zhang’s, Zhang’s, Jin’s Jin’s and and T. T. Zhang’s work on UA derivatives.

Jin et al. synthesized UA-based quinoline derivatives bearing thiadiazole, hydrazide, or oxadiazole Jin et al. synthesized UA-based quinoline derivatives bearing thiadiazole, hydrazide, or oxadiazolemoieties. The moietiesin vitro. Theantiproliferative in vitro antiproliferative activity on activity tumor on cells tumor such cells as SMMC-7721, such as SMMC MDA-MB-231,-7721, MDA- MBand-231 HeLa, and revealed HeLa revealed that quinoline-based that quinoline derivatives-based derivatives bearing carboxylbearing carboxyl moieties moieties or hydrazide or hydrazide moieties moietiesexhibited exhibited a significant a significant anticancer anti activitycancer activity against against all the all cancer the cancer cell lines cell line whens when compared compared to the to positivethe positive control, control, etoposide. etoposide Furthermore,. Further amore, pharmacological a pharmacologicalin vitro analysisin vitro revealedanalysis that revealed compound that c53ompound(Figure4 )53 exhibited (Figure 4) an exhibited antiproliferative an antiproliferative activity against activity HeLa against cell linesHeLa bycell cell lines cycle by arrestcell cycle at arrestthe G0 at/G1 the phase, G0/G1 decreasingphase, decreasing the mitochondrial the mitochondrial membrane membrane potential, potential, inducing inducing intracellular intracellular ROS ROSgeneration, generation, intervening intervening with the Ras with/Raf /mitogen-activated the Ras/Raf/mitogen protein-activated kinase kinase protein (MEK) kinase/extracellular kinase (signal-regulatedMEK)/extracellular kinase signal (ERK)-regulated signaling kinase pathways (ERK) assignaling MEK kinase pathways inhibitors, as MEK and kinase finally inhibitors inducing, and the finallyapoptosis induc of HeLaing the cell apoptosis lines. The of molecular HeLa cell docking lines. analysis The molecular also revealed docking compound analysis53 alsocapability revealed to ceompoundffectively bind53 capability with the to active effectively site of MEKbind with [177]. the active site of MEK [177]. The 1C values of compound 35b, 38b, 43a–b, 45, 46 and 50–53 on different cancer cell lines are The 1C50 values of compound 35b, 38b, 43a–b, 45, 46 and 50–53 on different cancer cell lines are shown in Table 6 6.. Table 6. Modification of miscellaneous groups. Table 6. Modification of miscellaneous groups. Compound Activity Cell Lines Tested (IC50µM) Reference Molecules (IC50µM) Bibliography Cell Lines Tested Compound35b ActivityCytotoxicity HRE (4.0)Reference HRE(UA) Molecu>100les (IC50µM) Bibliography [168] (IC50µM) MDA-MB-231 (0.61 0.07) MDA-MB-231(UA) > 40 ± 35b Cytotoxicity HeLaHRE (0.36 (4.0)0.05) HeLaHRE(UA) (UA) >40 >100 [168] 38b Cytotoxicity ± [169] SMMC-7721(12.49 0.08) SMMC-7721(UA) >40 MDA-MB-231 (0.6± 1 ± QSG-7701( > 40) MDAQSG-7701(UA)-MB-231(UA n.d ) > 40 0.07) HeLa (UA) >40 38b Cytotoxicity HeLa (0.36 ± 0.05) [169] SMMC-7721(UA) >40 SMMC-7721(12.49± 0.08) QSG-7701(UA) n.d QSG-7701( > 40) H460(4.5 ± 0.4) 43a H322(6.8 ± 1.5) H460(6.7 ± 0.5) H460(5.3 ± 0.3) H460(UA) 14.8 ± 0.6 43b Cytotoxicity H322(7.3 ± 1.0) H322(UA) 15.3 ± 2.8 [170] H460 LKB1+/+(7.8 ± 1.1) H460 LKB1+/+(UA) 21.1 ± 1.6 H460(2.6 ± 0.9) 43c H322(3.3 ± 0.9) H460 LKB1+/+(4.4 ± 0.6)

Int. J. Mol. Sci. 2020, 21, 5920 17 of 27

Table 6. Cont.

Compound Activity Cell Lines Tested (IC50µM) Reference Molecules (IC50µM) Bibliography H460(4.5 0.4) ± 43a H322(6.8 1.5) H460(UA) 14.8 0.6 ± ± H460(6.7 0.5) H322(UA) 15.3 2.8 [170] ± ± H460 LKB1+/+(UA) 21.1 1.6 H460(5.3 0.3) ± ± 43b Cytotoxicity H322(7.3 1.0) ± H460 LKB1+/+(7.8 1.1) ± H460(2.6 0.9) ± 43c H322(3.3 0.9) ± H460 LKB1+/+(4.4 0.6) ± CCRF-CEM (3.6) CEM-DNR (21.8) HCT116 (28.4) / HCT116 p53− −(29.8) K562 (38.8) 45 CCRF-CEM14 (44) 10.4 K562-TAX (25.1) CEM-DNR (44) 34.0 [171] A549 (27.6) HCT116(44) 34.0 U2OS (20.7) / HCT116 p53− −(44) 49.7 BJ (49.5) K562 (44) >50 MRC-5(29.3) K562-TAX (44) 35.5 Cytotoxicity CCRF-CEM (4.7) A549 (44) >50 CEM-DNR (28.2) U2OS (44) >50 HCT116 (32.1) BJ (44) >50 / HCT116 p53− −(32.3) MRC-5(44) >50 K562 (34.7) 46 K562-TAX (29.0) A549 (42.5) U2OS (33.1) BJ (> 50) MRC-5(39.8) SMMC-7721 (1.08 0.22) SMMC-7721(Doxorubicin) 0.62 0.16 50 Cytotoxicity ± ± [174] HepG2 (1.26 0.17) HepG2 (Doxorubicin) 0.77 0.12) ± ± Hela (1.06) Hela (UA) 14.2 51 Cytotoxicity K562 (28.7) K562 (UA) 52.7 [175] KB(35.6) KB 3(UA) 42.9 HepG-2(65.8 6.3) HepG-2 (UA) 44.35 4.9 ± ± MCF-7(> 100) MCF-7 (UA) > 100 52 Cytotoxicity [176] HT-29(4.28 3.5) HT-29 (UA) > 100 ± A549(78.39 5.6) A549 (UA) > 100 ± MDA-MB-231 (1.84 0.13) MDA-MB-231 (Etoposide) 5.26 1.21 ± ± MEK HeLa (1.18 0.03) HeLa (Etoposide) 2.98 0.42 53 ± ± [177] inhibitors SMMC-7721 (17.48 0.10) SMMC-7721 (Etoposide) 3.48 0.35 ± ± QSG-7701(40.59 2.89) QSG-7701(Etoposide) 28.75 3.28 ± ± n.d = not determined.

4. Insights and Future Directions This review reports various strategies that have been used to design UA-based derivatives with enhanced anticancer activity when compared to UA or conventional drugs used as controls in most of the studies reported over the past five years. Many studies on pentacyclic triterpenoids have shown that the C-2 position, β-hydroxyl (C-3), and carboxylic moieties (C-28) of UA were the major sites for the modification of its structure. Most researchers modified the molecular structure of UA around the three sites, resulting in the improvement in the chemical or physical activities of the UA molecule. The derivatives were grouped according to their active sites of modifications. Their corresponding IC50 values are listed in the tables against reference compounds, usually UA or a conventional drug. The derivatives were tested against various cancer cell lines in vitro using different cell lines of colon, prostate, gastric, leukaemia, lung, breast, pancreas, skin, glioblastoma, and renal cells. Most of the derivatives demonstrated an improved antiproliferative activity when compared to their respective reference molecules, revealing that UA structural modification significantly enhanced their antiproliferative activity. Int. J. Mol. Sci. 2020, 21, 5920 18 of 27

Despite the great progress which has been made, there are still a lot of research gaps, such as the synthesis of ursolic acid-based hybrid compounds via hybridization with known chemotherapeutic scaffolds, bioavailability studies, and toxicological studies. Furthermore, more evaluation in vivo is lacking and there is a pressing need to evaluate the synthesized compounds in vivo.

Funding: This research was funded by the South African Medical Research Council (Self-Initiated Research), National Research Foundation South Africa, Sasol Inzalo Foundation, and Govan Mbeki Research and Development Centre (GMRDC), University of Fort Hare. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

FDA Food and Drug Administration UA Ursolic acid P53 Tumor protein p53 Wnt Wnt/β-catenin pathways Ras Retrovirus-associated DNA sequences TRAIL TNF-related apoptosis-inducing ligand STAT3 Signal transducers and activators of transcription PK Pharmacokinetic UV Ultraviolet HIF-1α Hypoxia-inducible factor 1-alpha DMF Dimethylformamide MTT Dye compound 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay SRB sulforhodamine B assay PARP-1 Poly [ADP-ribose] polymerase 1 NF-kB Nuclear factor kappa-B DMAP 4-Dimethylaminopyridine DCM Dichloromethane THF Tetrahydrofuran DCC N,N0-Dicyclohexylcarbodiimide DMSO Dimethyl sulfoxide FROS Reactive oxygen species MEK Mitogen-activated extracellular signal-regulated kinase

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