**Essential Oil of** *Mentha aquatica var. Kenting Water Mint* **Suppresses Two-Stage Skin Carcinogenesis Accelerated by BRAF Inhibitor Vemurafenib**

### **Chih-Ting Chang 1,2, Wen-Ni Soo 1, Yu-Hsin Chen <sup>3</sup> and Lie-Fen Shyur 1,2,4,\***


Academic Editor: Kyoko Nakagawa-Goto Received: 29 May 2019; Accepted: 22 June 2019; Published: 25 June 2019

**Abstract:** The v-raf murine sarcoma viral homolog B1 (BRAF) inhibitor drug vemurafenib (PLX4032) is used to treat melanoma; however, epidemiological evidence reveals that it could cause cutaneous keratoacanthomas and squamous cell carcinoma in cancer patients with the most prevalent *HRASQ61L* mutation. In a two-stage skin carcinogenesis mouse model, the skin papillomas induced by 7,12-dimethylbenz[a]anthracene (DMBA)/12-*O*-tetradecanoylphorbol-13-acetate (TPA) (DT) resemble the lesions in BRAF inhibitor-treated patients. In this study, we investigated the bioactivity of *Mentha aquatica var. Kenting Water Mint* essential oil (KWM-EO) against PDV cells, mouse keratinocytes bearing *HRASQ61L* mutation, and its effect on inhibiting papilloma formation in a two-stage skin carcinogenesis mouse model with or without PLX4032 co-treatment. Our results revealed that KWM-EO effectively attenuated cell viability, colony formation, and the invasive and migratory abilities of PDV cells. Induction of G2/M cell-cycle arrest and apoptosis in PDV cells was also observed. KWM-EO treatment significantly decreased the formation of cutaneous papilloma further induced by PLX4032 in DT mice (DTP). Immunohistochemistry analyses showed overexpression of keratin14 and COX-2 in DT and DTP skin were profoundly suppressed by KWM-EO treatment. This study demonstrates that KWM-EO has chemopreventive effects against PLX4032-induced cutaneous side-effects in a DMBA/TPA-induced two-stage carcinogenesis model and will be worth further exploration for possible application in melanoma patients.

**Keywords:** BRAF inhibitor; *Mentha aquatica var. Kenting Water Mint*; essential oil; chemoprevention; two-stage skin carcinogenesis

### **1. Introduction**

Cutaneous squamous cell carcinoma (cuSCC) and keratoacanthoma (KA) develop in approximately 20% to 30% of patients who are treated with BRAF (v-raf murine sarcoma viral homolog B1) inhibitors, such as vemurafenib (PLX4032) [1]. Functional studies have demonstrated that these serious side-effects caused during the treatment of PLX4032 are through paradoxical activation of the MAPK signaling pathway of wild-type BRAF cell lines bearing either oncogenic *RAS* mutations or upstream receptor tyrosine kinase activity [2–4]. In a recent study, cuSCC and KAs emerging from patients administrated with BRAF inhibitor were analyzed for oncogenic mutations and activating mutations on *RAS*, especially the *HRAS* isoform was noticed in about 60% of subjects [5]. Among the *RAS* mutants, *HRASQ61L* was the most prevalent, and thus, the genetic *HRASQ61L* mutation of cells (e.g., keratinocytes PDV) was selected to investigate the pre-clinical pathological mechanisms [6]. Meanwhile, the mouse skin model of multiple-stage chemical carcinogenesis is a representable in vivo model for understanding the development of cuSCC [7,8]. Topical exposure of carcinogens, 7,12-dimethyl[a]anthracene (DMBA), as a tumor initiator results in *HRASQ61L* mutation in mouse skin. Subsequently, topical treatment of tumor promoter, 12-*O*-tetradecanoyl-phorbol-13-acetate (TPA) then leads to the formation of lesions, KAs, and the development of SCC. FVB (Friend Virus B NIH Jackson) mice administrated with DMBA/TPA along with BRAF inhibitor, PLX4720, showed a remarkable acceleration in the appearance of lesions, an increase of incidence, and enhanced progression to KAs and SCC which resemble the papillomas induced by BRAF inhibitors in the clinical setting [5].

Tumor development is correlated with proliferation and expansion of not only cancer cells but also stroma, vessels, and infiltrating inflammatory cells and elements [9]. Neoplastic growth is related to a prolonged inflammatory condition induced by extrinsic or intrinsic pathways. The extrinsic pathways are related to a continued inflammatory condition, while the intrinsic pathways are stimulated by genetic transformations, which result in the activation of oncogenes or inactivation of tumor suppressor genes [10]. Cells with an altered phenotype propagate the secretion of inflammatory mediators, thus triggering the formation of a tumor microenvironment (TME) and development of tumors [11]. Recently, immunoinflammatory cells, such as macrophages, have been identified as critical contributors to malignancies in various tumor types, such as melanoma, lung carcinoma, glioma, gastric cancer, and wound-induced skin cancer [12,13].

Numerous studies have demonstrated that essential oils (EOs) of *Mentha* species have antiviral, antimicrobial, antioxidant, anti-inflammatory, and anti-tumor activities [14–18]. The objective of this study was to investigate the bioefficacy of EO from *Mentha aquatica var. citrata Kenting Water Mint* (KWM-EO) against two-stage skin carcinogenesis, with or without PLX4032 irritation, and the underlying molecular mechanisms. The chemical components of KWM-EO were analyzed using GC×GC-TOF MS, and its effect on *HRAS* mutant PDV keratinocyte activity was further investigated. Our in vitro bioassay results demonstrated that KWM-EO treatment suppressed PDV cell viability, colony formation ability, and induced G2/M cell-cycle arrest and cell apoptosis in the presence and absence of PLX4032. KWM-EO also inhibited proinflammatory cell infiltration and papilloma formation in DMBA/TPA-induced two-stage skin carcinogenesis facilitated by PLX4032 in mice.

### **2. Results**

### *2.1. Chemical Compositions of Mentha aquatica var. Kenting Water Mint Essential Oil*

KWM-EO was obtained by hydrodistillation of the aerial parts. The chemical profile of KWM-EO was analyzed by GC×GC-TOF MS. Twenty compounds representing 81.86% of the total content were identified in KWM-EO (Table 1). Monoterpene hydrocarbons accounted for 56.01% of KWM-EO with 22.18% β-ocimene as the most abundant component, and β-pinene and α-pinene accounting for 15.41% and 10.49%, respectively. KWM-EO was identified to contain 15.86% oxygenated monoterpenes, of which eucalyptol (12.87%) was the most abundant.


**Table 1.** Chemical constituents of KWM-EO determined by GC×GC-TOF MS.


**Table 1.** *Cont.*

<sup>a</sup> Chemical abstracts service registry number; <sup>b</sup> Retention time of the first column; <sup>c</sup> Retention time of the second column; <sup>d</sup> KIexp = Kovats indices, retention indices relative to C7–C30 n-alkanes based on the retention time of components separated by the 1st dimension Rtx-5MS column; <sup>e</sup> KILit: Retention indices reported in the literature.

### *2.2. KWM-EO E*ff*ect on PDV Cell Proliferation, Invasion, and Migration*

The PDV cell line is a mouse keratinocyte bearing *HRASQ61L* mutation, which is the most relevant mutation in BRAF inhibitor-induced cutaneous squamous cell carcinoma. The PDV cell viability after treatment with 0 to 100 μg/mL KWM-EO was determined by MTT assay. The cell viability was decreased when KWM-EO concentration increased. When the PDV cells were treated with up to 100 μg/mL of KWM-EO for 24 h, the cell viability was inhibited to 53.31% (Figure 1A). The long-term colony formation ability of PDV cells was determined by treating with KWM-EO alone or in the presence of PLX4032 (PLX). The MEK (mitogen-activated protein kinase kinase) inhibitor, selumetinib (AZD6244), was used as a reference control. As shown in Figure 1B, 0.5 μM PLX4032 treatment promoted the colony formation of PDV cells compared to the vehicle-treated cells. In the presence or absence of PLX4032, KWM-EO treatment showed a dose-dependent effect, and KWM-EO treatment at the high dose of 40 μg/mL revealed a better effect than 0.5 μM AZD6244 treatment. PDV cell invasive ability was investigated by Matrigel coated-transwell assay. The result showed that PLX4032 treatment facilitated cell invasion relative to vehicle treatment, and KWM-EO suppressed the invasive ability on concentration-dependence (Figure 1C). In wound healing assay representing cell migratory ability, 2 μM PLX4032 treatment significantly and time-dependently increased cell migration. The migratory ability of PDV cells was restricted by 50 μg/mL KWM-EO treatment with or without PLX4032 stimulation (Figure 1D).

**Figure 1.** Effect of *Mentha aquatica var. Kenting Water Mint* essential oil (KWM-EO) on PDV cells. (**A**) PDV cells were treated with vehicle or the indicated concentrations of KWM-EO for 24 h. Cell viability (%) was determined by MTT assay. (**B**) PDV cells were incubated with KWM-EO in the presence or absence of 0.5 μM PLX4032 for 6 days, and colony formation was detected by staining cells with crystal violet. (**C**) PDV cells were seeded in Matrigel coated–transwell inserts and incubated with vehicle or KWM-EO in the presence or absence of 2 μM PLX4032 for 24 h. The invasive cells were stained with crystal violet. (**D**) PDV cell migratory ability was examined by wound healing assay. Cells were treated with vehicle or 50 μg/mL KWM-EO in the presence or absence of 2 μM PLX4032, and observed after 0, 6, 12, 24 h. Vehicle controls (C) were obtained from cells treated with 0.5% DMSO. The absorbance at 595 nm was obtained by dissolving crystal violet with 20% acetic acid. The data are representative of three independent experiments and are expressed as mean ± SD. Representative images are shown. *P\** < 0.05, *P*\*\* < 0.01, *P*\*\*\* < 0.001 compared to vehicle control; *P*## < 0.01, *P*### < 0.001 compared to the PLX4032-treated group (ANOVA). AZD: AZD6244 (MEK inhibitor)

### *2.3. KWM-EO Induces Cell-Cycle Arrest and Apoptosis in PDV Cells*

The KWM-EO effect on the PDV cell-cycle machinery was determined using flow cytometry. The analysis demonstrated that PLX4032 treatment alone had no significant effect on the cell-cycle of PDV cells; however, the cell-cycle profile treated with KWM-EO exhibited G2/M arrest. After KWM-EO treatment for 24 h, the percentage of cells in the G2/M phase was raised from 33.0–33.6% to 44.3–45.0%, in the presence or absence of PLX4032 (Figure 2A). According to cell-cycle analysis, an elevated percentage of the sub-G1 population was also observed with KWM-EO and PLX4032+KWM-EO treatment. Thus cell apoptosis was further examined. Cells were stained with annexin V and propidium iodide and analyzed by flow cytometry. The data demonstrated that treatment with 75 μg/mL KWM-EO strongly induced 86.9% and 80.7% apoptotic cells in the presence or absence of PLX4032, respectively (Figure 2B). Western blotting was further used to explore the protein expression profile related to G2/M cell-cycle arrest and cell apoptosis. Cyclin B1-cell division cycle protein 2 (cdc2), also known as M-phase promoting factor (MPF), regulates G2/M transition. Phosphorylation of Thr161 in cdc2 is required for activation of MPF and brings on the onset of mitosis [19]. Treatment with 75 μg/mL KWM-EO for 24 h reduced the protein expression level of cyclin B1 and p-cdc 2 (Thr161), suggesting the inhibition of cell mitosis. Phosphorylation of cdc25C, which is responsible for activation of MPF, was also decreased (Figure 2C). The initiation of apoptotic cell death is needed to activate a group of intracellular cysteine proteases, named caspases. The cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) by caspases is regarded to be a characteristic of cell apoptosis [20]. After treatment with 75 μg/mL KWM-EO for 12 h, both hallmarks of apoptosis, caspase 3 and PARP-1, were cleaved into their activated forms (Figure 2D). Paradoxical MAPK activation is known to be the main reason for cutaneous squamous cell carcinoma induced by BRAF inhibitor in *RAS* mutant cells [21]. KWM-EO (75 μg/mL) treatment for 24 h significantly inhibited ERK and p-ERK expression in PDV cells; while in treatment with 0.5 μM PLX4032, the re-activation of p-MEK and p-ERK was observed, which could be reversed by KWM-EO and MEK inhibitor (Figure 2E).

**Figure 2.** *Cont.*

**Figure 2.** Effect of KWM-EO on cell-cycle and apoptosis in PDV cells. (**A**) PDV cells were exposed to vehicle or 75 μg/mL KWM-EO in the presence or absence of 0.5 μM PLX4032 for 12 or 24 h, then the cell-cycle was analyzed by flow cytometry. (**B**) PDV cells were treated with vehicle or 75 μg/mL KWM-EO in the presence or absence of 0.5 μM PLX4032 for 24 h. Cells were then stained with annexin V and propidium iodide, and the cell apoptosis was detected by flow cytometry. (**C**) PDV cells were treated with 75 μg/mL KWM-EO in the presence or absence of 0.5 μM PLX4032 for 24 h before lysis. The cell lysates were subjected to Western blotting against cell-cycle-related proteins, including p-cdc2 (Thr161), p-cdc25C, and cyclin B1. (**D**) The expression level of apoptosis-related proteins in PDV cells treated with 75 μg/mL KWM-EO in the presence or absence of 0.5 μM PLX4032 for 6 h was examined by Western blotting against PARP-1 and caspase 3. (**E**) Western blotting analysis of MAPK signaling-related proteins (p-ERK, ERK, p-MEK, MEK) in PDV cells treated with 75 μg/mL KWM-EO in the presence or absence of 0.5 μM PLX4032 for 24 h. Actin was used as an internal control in the experiment. Vehicle controls (**C**) were obtained from cells treated with 0.5% DMSO. The data are representative of three independent experiments and are expressed as mean ± SD. N.S. means non significance; *P*\*\*\* < 0.05 compared to vehicle control; *P*## < 0.01, *P*### < 0.001 compared to PLX4032-treated group (ANOVA).

### *2.4. KWM-EO Inhibits Two-Stage Skin Carcinogenesis in FVB Mice*

To investigate the chemopreventive effect of KWM-EO in BRAF inhibitor-induced cutaneous squamous cell carcinoma, a DMBA-initiated and TPA-promoted two-stage skin carcinogenesis model was established, and the study diagram is shown in Supplementary Figure 1. After topical application of 25 μg DMBA and 4 μg TPA (DT) on mouse dorsal skin for 12 weeks, papillomas were successfully induced. If mice were co-treated with 20 mg/kg/BW PLX4032 (DTP), starting at 6 weeks, bigger and more papillomas occurred (Figure 3A). The DT and DTP groups developed papillomas on the skin as early as 5 weeks after TPA treatment. Within 6 to 7 weeks, the tumor incidence in DTP mice was much higher than in DT mice; at 8 weeks, the tumor incidence in the DT and DTP group reached 100%, while KWM-EO treatment delayed and decreased the tumor incidence (Figure 3B). On average, the DT group developed 15.4 papillomas/mouse at 12 weeks, the topical application of 5 mg KWM-EO reduced the average number of papillomas to 7.9/mouse. Under stimulation of PLX4032 in DTP mice, the average number of papillomas was raised to 22.4/mouse which was ameliorated by KWM-EO to 9.1 papillomas/mouse at 12 weeks (*P* < 0.05) (Figure 3C). The dot histogram shows the distribution of papilloma number per mouse and the median in a group (Figure 3D). The papilloma number was significantly decreased by KWM-EO treatment in DTP mice. Mouse body weights were recorded every week during the experimental period, and the results show that the body weights in all treated mice were similar to the sham control mice (Supplementary Figure 2). To examine the toxicology of these applied compounds and essential oil, mouse organ index was calculated, and H&E staining was

executed to observe the organ structure and pathology. The mouse organ index was unchanged for the heart, lung, and kidney within the groups; however, the index of the liver organ was lower in the KWM-EO-treated mice, and the index of the spleen organ was increased in PLX4032-treated mice. The H&E staining result on the organs showed that there were no observable differences between the sham and all the treatment groups (Figure 3F).

**Figure 3.** Effect of KWM-EO on two-stage skin carcinogenesis in mice. (**A**) Representative images of mice from each group at week 12 are shown. Tumor incidence (**B**) and mean number of papillomas (**C**) per group during the experimental period are calculated. (**D**) Papilloma numbers per mouse at week 12 are shown in the dot histogram. (**E**) Organ weights were recorded after mice were sacrificed at week 12. Organ index was calculated by the following formula: *organ index* = (*organ weight* ÷ *body weight*) × 100 empty. *P*\* < 0.05, *P*\*\* < 0.01, *P*\*\*\* < 0.001 compared to sham group (ANOVA). (**F**) Organ tissues were detected by H&E staining. The DT and DTP groups consisted of 5 mice each. Sham, the DT+KWM, and DTP+KWM groups consisted of 8 mice each. The data are presented as mean ± SD. D: DMBA, T: TPA, P: PLX4032. Scale bar: 100 μm.

### *2.5. Skin Histology and Epidermal Cell Proliferation in KWM-EO-Treated Mice*

Effect of topically applied KWM-EO on chemically induced skin tumorigenesis was further observed by skin histologic changes. The skin structure was first examined by H&E staining. With DMBA and TPA treatment in the presence or absence of PLX4032, the thickness of the epidermis was elevated compared to the sham group (Figure 4A), while the epidermal hyperplasia was attenuated by repeated treatment with KWM-EO for 12 weeks. The main cell type responsible for hyper-proliferative epidermis was further explored by immunofluorescent staining. Ki67 is a representative marker of cell proliferation, and cytokeratin 14 (K14) is the intermediate filament protein of basal keratinocytes. From the microphotographs, the expression level of ki67 showed remarkable upregulation in the DT and DTP group. After merging both ki67 and K14 staining with DAPI, the hyperplasia of the epidermis arising from basal keratinocytes was seen which was alleviated by KWM-EO topical treatment (Figure 4B). In addition, the paradoxical MAPK activation that could lead to cell proliferation in *RAS* mutant cells treated with BRAF inhibitors was also investigated. The IHC staining result revealed considerable p-ERK protein between the dermis and papilloma, especially in the group with PLX4032 stimulation; and this activation was significantly diminished by KWM-EO treatment (Figure 4C).

**Figure 4.** Effect of KWM-EO on skin and papilloma tissue from mice. (**A**) Skin morphology was examined by H&E staining. (**B**) Abnormal epidermal proliferation was detected by immunofluorescent staining of ki67 (red). Basal keratinocytes were stained with K14 (green), and nuclei were stained with DAPI (blue). (**C**) Histological images of papilloma indicated the paradoxical activation of p-ERK. Representative images are shown. The data are representative of three independent experiments and are expressed as mean ± SD. N.S. means non significance; *P*\*\* < 0.01, *P*\*\*\* < 0.001 compared to DT group; *P*### < 0.001 compared to DTP group (ANOVA). Scale bar: 100 μm.

### *2.6. Anti-Inflammatory E*ff*ect of KWM-EO*

Inflammation is a vital element in the progression of two-stage skin carcinogenesis. COX-2, a pro-inflammatory enzyme commonly observed in inflamed cells or tissues was counter-stained

with pro-inflammatory immune cells, neutrophils (neutrophil elastase+) and macrophages (F4/80+). The overexpression of COX-2 was increased in both DT and DTP mouse dorsal skin. Interestingly, most of the COX-2 proteins were observed colocalized with infiltrated neutrophils, but not macrophages (Figure 5A,B). Upon treatment with KWM-EO, the neutrophil and macrophage infiltration and upregulation of COX-2 were alleviated (Figure 5A,B).

**Figure 5.** Effect of KWM-EO on the inflammatory immune system in skin tissue from the mice two-stage skin carcinogenesis. Immunofluorescent staining of inflammatory mediator COX-2 (red) with neutrophil elastase (green) (**A**) and macrophage marker, F4/80 (green) (**B**). Nuclei were counterstained with DAPI (blue). Representative images are shown. The data are representative of three independent experiments and are expressed as mean ± SD. N.S. means non significance; *P*\* < 0.05, *P*\*\* < 0.01, *P*\*\*\* < 0.001 compared to DT group; *P*## < 0.01, *P*### < 0.001 compared to DTP group (ANOVA). Scale bar: 100 μm.

### **3. Discussion**

EOs have been utilized as fragrances, food flavorings, and folk medicines, among other applications throughout human history. In recent decades, a large number of studies have reported chemical constituent analysis of EOs and investigated their bio-efficacy and the responsible bioactive compounds. EOs from *Mentha* species have been reported to have anti-inflammatory, anti-oxidant, anti-fungal, and anti-bacterial activities [14–18]. The ethanolic extract of *Mentha*×*piperita* L., a cross-species of watermint and spearmint, at 50 and 100 μg/mL, suppressed LPS-induced nitric oxide production in macrophages by 18.85% and 41.88% inhibition, respectively [22]. Anti-cancer cell activity has also been reported for mint EOs. For example, *Mentha*×*piperita* L. extract showed more potent activity against proliferative activity of human MDA-MB-231 breast cancer cells (cell inhibition ratio = 46.53% at 150 μg/mL) than human A375 melanoma cells (cell inhibition ratio = 25.08% at 150 μg/mL) [14]. This study is the first to investigate and observe that KWM-EO can prevent two-stage skin carcinogenesis chemically induced by DMBA/TPA and its acceleration by BRAF inhibitor drug PLX4032. The two-stage skin carcinogenesis mouse model established by DMBA and TPA irritation is considered to be a representative study system through which to explore the pathology and underlying mechanisms of human squamous cell carcinomas [23]. It has also been used to evaluate the BRAFV600E inhibitor drugs, such as vemurafenib-induced cutaneous side-effects, including SCC and KA in patients [3]. We, thus, established this two-stage skin carcinogenesis mouse model, and the bioactivities of KWM-EO were examined. Our results indicated that KWM-EO treatment significantly inhibited papilloma incidence and number in DT and DTP mice. According to the histopathological analysis of skin tissue sections, KWM-EO not only attenuated the abnormal proliferation and hyperplasia of the epidermis but also decreased the inflammatory neutrophil and macrophage infiltration and COX-2 overexpression in neutrophils. Moreover, this study is the first to observe that abnormal epidermis proliferation in DT and DTP mice was mainly contributed by keratinocytes as a co-positively stained marker protein K14 and proliferation marker ki67. Topical administration of KWM-EO can reverse the over proliferation of K14 keratinocytes in DT- or DTP-irritated mouse skins.

A previous review article published by Pandey et al. [24] summarized that EOs of some *Ocimum* species exhibited anti-inflammatory and anti-cancerous properties which contain pinene, β-ocimene, and linalool, the chemical constituents present in KWM-EO. α-Pinene present in KWM-EO by 10.49% was reported to induce cell apoptosis and disrupt mitochondrial potential in B16F10 cells, and it also effectively reduced melanoma lung metastasis [25]. β-Caryophyllene accounted for 2.8% in KWM-EO was a major compound in the EO of *P. missionis*. Pavithra et al. demonstrated that EO from *P. missionis* induced cell death through intrinsic mitochondrial and extrinsic apoptotic pathways in A431 and HaCaT cells [26]. The results from these studies might potentially support part of our observations for the anti-inflammatory and chemopreventive activities of KWM-EO against two-stage skin carcinogenesis.

PDV keratinocytes harboring *HRAS* mutation are commonly found in DT-induced mouse SCC [27]. We adapted this cell model to investigate the in vitro effect and modes of action of KWM-EO. Our data revealed that KWM-EO treatment significantly diminished PDV cell colony formation ability and suppressed reactivation of MEK-ERK signaling stimulated by PLX4032. The PDV cell invasive and migratory abilities were promoted by PLX4032, which were suppressed by KWM-EO treatment. KWM-EO also induced G2/M arrest in PDV cells through deregulating p-cdc2, p-cdc25C, and cyclin B1 proteins. The cell apoptosis induced by KWM-EO was through activation of caspase 3 and PARP-1 after cells were treated for 6 h. These in vitro data support in part the inhibitory activity of KWM-EO in the DT and DTP mouse skin on keratinocyte proliferation and papilloma formation.

In an open-label phase 2 study using a combination of BRAF inhibitor dabrafenib and MEK inhibitor trametinib in patients, the rate of skin lesions was not significantly reduced although a slight decrease in proliferative skin lesions was observed [28]. A previous study revealed that tumor multiplicity and incidence of skin tumors in DT-induced two-stage skin carcinogenesis accelerated by BRAF inhibitor was decreased when a COX-2 inhibitor celecoxib was orally administrated [29]. Our current data show that topical application of KWM-EO attenuated the formation of cutaneous papilloma in mice induced by DMBA/TPA or by DMBA/TPA/PLX4032. The paradoxical MAPK activation induced by PLX4032 in vitro in PDV keratinocytes and in skin of DT and DTP mice was suppressed by KWM-EO. Taken together, the results of this study demonstrate the novel chemopreventive activity of the essential oil of *Mentha aquatica var. citrata Kenting Water Mint* which can be potentially used in preventing BRAF inhibitor drug-induced cutaneous side-effects in cancer patients.

### **4. Materials and Methods**

### *4.1. Mint Cultivation and Distillation of Essential Oils*

A variety of *Mentha aquatica* (Lamiaceae), named *M. aquatica var. Kenting Water Mint* was cultivated in an experimental field at the Taichung District Agricultural Research and Extension Station, Taichung, Taiwan for 2 years. Mature shoots were harvested and subjected to water vapor distillation to collect essential oils. Two kilograms of fresh shoots were distilled with 4 L of water. Mint essential oil was evaporated, passed through a condenser then the oil and hydrosol were collected with a separating funnel. After 1 L of the hydrosol/essential oils were collected, the distillation ended. The hydrosol and essential oil were then separately collected for use in the following experiments. The mint essential oils were stored at −20 ◦C in sealed vials. Essential oils used in in vitro cell-based assays were diluted into different concentrations with DMSO and those used in in vivo animal studies were diluted in acetone.

### *4.2. Chemical Profiling of KWM-EO Composition by GC*×*GC*−*TOF MS*

The samples were analyzed using LECO Pegasus 4D GC×GC−TOF MS (St Joseph, MI, USA). The first dimension capillary column was Restek Rtx-5MS (30 m × 0.25 mm × 0.25 μm) and the second capillary column was Restek Rtx-200 (2 m × 0.25 mm × 0.25 μm). The GC temperature program was set as follows: injection temperature: 280 ◦C; oven temperature: 40 ◦C maintained for 1 min, and increased at a rate of 10 ◦C/min to 310 ◦C and held constant for 8 min. The helium flow rate was set at 1 mL/min. The mass spectrometry temperature was set at 320 ◦C. The ion source temperature was 200 ◦C, and the analysis mass range was 50-800 m/z. KWM-EO was ran in hexane with a dilution of 1 mg/mL. Hexadecane solution, 64.25 μg/mL, was used as an internal standard to monitor the shift of retention time. Compounds were identified by matching the mass spectra fragmentation patterns, and the results were compared with LECO/Fiehn and Wiley Registry 9th Edition mass spectral library and NIST. Linear Kovats index of n-alkanes (C7-C40, C7-C30) were calculated for each compound and compared with the literature to identify the compound ID [30].

### *4.3. Cell Lines and Cell Culture*

PDV cells, which harbor the *HRASQ61L* mutation were obtained from CLS Cell Lines Service (Eppelheim, Germany). Cells were cultured at 37 ◦C in DMEM supplemented with 10% FBS, containing 100 units/mL penicillin and 100 μg/mL streptomycin in a humidified 5% CO2 incubator. Cells were used within 10 passages for this study.

### *4.4. Measurement of Cell Viability*

Cells (5 <sup>×</sup> 103 cells/well in 96-well plates) were treated with vehicle (0.5% DMSO) or 20, 40, 60, 80, and 100 μg/mL KWM-EO for 24 h. Cell viability was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based colorimetric assays according to Scudiero et al. [31]. The viability of the cells treated with vehicle-only was defined as 100% viable. The viability of the cells after treatment with KWM-EO was calculated using the following formula: *cell viability* (%) = *OD*<sup>570</sup> (*treated cells*) ÷ *OD*<sup>570</sup> [*vehicle control*] × 100 empty. The data are presented by three independent experiments with six replicates per experiment.

### *4.5. Colony Formation Assay*

Colony formation was obtained by growing PDV cells (250 cells/well in 24-well plates) treated with 10 and 40 μg/mL KWM-EO in the presence or absence of 0.5 μM PLX4032 for 6 days. The culture medium was refreshed once on day 3. Cells were fixed with chilled methanol and stained with 0.1% crystal violet. Cells retaining crystal violet were dissolved with 20% acetic acid and quantified by measuring absorbance at 595 nm [32]. The data are presented by three independent experiments with three replicates per experiment.

### *4.6. Cell Invasion Assay*

The cell invasion assay was performed by Millicell Cell Culture Inserts (Merck Millipore, United States). For invasion assay, 100 μL Matrigel (300 μg/mL) was applied to an 8-mm polycarbonate membrane filter and incubated in 37 ◦C for 2 h. PDV cells (5 <sup>×</sup> 104) were seeded to Matrigel-coated filters in 200 μL of serum-free medium in triplicate for 16 h. The bottom chamber of the apparatus contained 1 mL medium with 10% FBS as a chemoattractant and 50 and 75 μg/mL KWM-EO, in the presence or absence of 2 μM PLX4032. Cells were allowed to migrate for 24 h at 37 ◦C. After incubation for 24 h, the non-migrated cells on the apical side of the membrane were removed with cotton swabs. The migrated cells on the basal side of the membrane were fixed with cold 100% methanol for 20 min and washed 3 times with PBS. The cells were stained with 0.1% crystal violet and then washed with PBS to remove extra dye solution. Images were captured using a reverse-phase microscope (Zeiss Axiovert 200M). Cells retaining crystal violet were dissolved with 20% acetic acid and quantified by measuring absorbance at 595 nm. The data are presented by three independent experiments with three replicates per experiment.

### *4.7. Wound Healing Assay*

The wound healing assay was performed by using Culture-Insert (ibidi GmbH, Germany). Culture-Inserts were inserted in 24-well plates before cells were seeded. PDV cells were seeded in Culture-Inserts at a density of 5 <sup>×</sup> 105 cells/mL in 70 <sup>μ</sup>L medium. After 16 h, Culture-Inserts were removed which created two cell-free gaps of 500 ± 50 μm. Undetached cells were washed away by PBS, then the remaining attached cells were immersed in 1 mL medium with 50 μg/mL KWM-EO, in the presence or absence of 2 μM PLX4032. Cell migration was observed using a reverse-phase microscope (Zeiss Observer D1) every 6 h. The data are presented by three independent experiments with three replicates per experiment.

### *4.8. Cell-Cycle Analysis*

PDV cells were seeded in 6-well plates at a density of 1 <sup>×</sup> 105 cells/well with respective medium containing 10% FBS for 16 h. To synchronize the cell-cycle, cells were washed with PBS and incubated with fresh medium containing 5% FBS for 8 h, followed by washing with PBS and incubation with fresh medium containing 0.5% FBS for 24 h. The synchronized PDV cells were then treated with 75 μg/mL KWM-EO, 0.5 μM PLX4032, and 0.5 μM PLX4032+75 μg/mL KWM-EO in the medium containing 10% FBS for 12 and 24 h. Both adherent and floating cells were collected, washed with PBS, and fixed with 500 μL ice-cold 70% ethanol overnight at 4 ◦C. Cells were stained with 500 μL propidium iodide (PI) solution, which contained 20 μg/mL PI, 20 μg/mL RNase A, 0.1% Triton X-100 for 30 min at room temperature in the dark and then analyzed by flow cytometry (Flow cytometry BD Accuri C6, United States).

### *4.9. Apoptosis Assay*

Cells were seeded in 6-well plates at a density of 1.5 <sup>×</sup> 105 cells/well for 16 h and then treated with 75 μg/mL KWM-EO, 0.5 μM PLX4032, and 0.5 μM PLX4032+75 μg/mL KWM-EO. After 24 h, both adherent and floating cells were collected and washed with PBS. Apoptotic cells were analyzed by using FITC Annexin V Apoptosis Detection Kit (BD Bioscience, United States) according to the manufacturer's instructions.

### *4.10. Western Blot Analysis*

Cells were treated with KWM-EO at the indicated concentrations in the presence or absence of PLX4032 and lysed in RIPA lysis buffer. Protein concentrations were measured by *DC* protein assay (Bio-Rad, United States). Western blotting was performed as described by Shyur et al. [33]. Primary antibodies ERK 1, cyclin B1, p-cdc2 p34, p-cdc25C, and PARP-1 were purchased from Santa Cruz (Texas, United States). Antibodies phospho-p44/42 MAPK (Erk1/2), MEK1/2, and phospho-MEK1/2 were purchased from Cell Signaling Technology (Massachusetts, United States). Caspase 3 antibody was purchased from GeneTex (Texas, United States).

### *4.11. Two-Stage Skin Carcinogenesis Study*

Female FVB/NJNarl mice (5–6 weeks old) were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and bred in the Laboratory Animal Core Facility (Agricultural Biotechnology Research Center, Academia Sinica, Taiwan). Animals were given a standard laboratory diet and distilled H2O *ad libitum* and kept on a 12-h light/dark cycle at 22 ± 2 ◦C with humidity 55 ± 5%. All experimental protocols were approved by the Institutional Animal Care and Utilization Committee (IACUC: Protocol #18-08-1221), Academia Sinica, Taiwan. Mice were randomized and had their back hair shaved three days before topical application of 25 μg DMBA in 200 μL acetone. The first week after tumor initiation, 4 μg of TPA in 200 μL acetone was topically applied twice a week to the shaved dorsal skin for 12 weeks [5]. Mice were treated with the indicated concentration of KWM-EO (in 200 μL acetone) twice a week by topical application the day after TPA treatment for 12 weeks (Supplementary Figure 1). Tumor size of more than 1 mm diameter was counted every week.

### *4.12. Histopathological and Immunohistochemical Analysis*

Tissues were fixed with 10% formalin, hydrated, and embedded in paraffin. Tissue sections were cut at 4 μm thickness, then deparaffinized following rehydration in a descendant ethanol bath. H&E staining, immunohistochemistry, and immunofluorescent staining followed the previously published protocols [30]. An upright microscope (Carl Zeiss Axio Imager, Z1) was used to observe the expression of targeted proteins. Primary antibodies cytokeratin 14 and CD163 were purchased from Proteintech (Illinois, United States). Ki67 and neutrophil elastase were purchased from Abcam (Cambridge, United Kingdom). Antibody against COX-2 was purchased from Cayman (Michigan, United States). Antibody against F4/80 was purchased from Biolegend (California, United States). Antibody against iNOS was purchased from BD transduction Laboratories (California, United States).

### *4.13. Statistical Analysis*

All the data are expressed as mean ± standard deviation (SD). Statistical analyses were conducted by the Predictive Analysis Suite Workstation (PASW Statistics, United States), and the significant difference between different treatment groups was determined by analysis of variance (ANOVA). *P* values of less than 0.05 were considered statistically significant.

### **5. Conclusions**

This study is the first to prove that KWM-EO has potential for prevention of chemically induced two-stage skin carcinogenesis. Topical application of KWM-EO significantly attenuated the number of papillomas in DMBA-initiated and TPA-promoted mouse skin, with or without co-stimulation with PLX4032. KWM-EO suppressed epidermal hyperplasia and over proliferation of keratinocytes in DMBA/TPA and DMBA/TPA/PLX4032 mice. Notably, KWM-EO treatment diminished MAPK pathway reactivation, pro-inflammatory immune cell infiltration, and COX-2 expression in both DMBA/TPA and DMBA/TPA/PLX4032 mouse skin tissues. Overall, the results in this study provide strong support for the development of KWM-EO into chemopreventive agents for squamous cell carcinoma patients or cancer patients taking BRAF inhibitor therapy.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/24/12/2344/s1, Supplementary Materials Figure 1: The experimental design of two-stage skin carcinogenesis mouse model, Supplementary Materials Figure 2: Mouse body weights recorded every week.

**Author Contributions:** Conceptualization, L.-F.S.; methodology, L.-F.S., C.-T.C., Y.-H.C. and W.-N.S.; validation, C.-T.C. and L.-F.S.; formal analysis, C.-T.C., W.-N.S. and L.-F.S.; investigation, C.-T.C., W.-N.S., Y.-H.C. and L.-F.S.; resources, L.-F.S.; data curation, C.-T.C. and L.-F.S.; writing—original draft preparation, C.-T.C. and L.-F.S.; writing—review and editing, C.-T.C. and L.-F.S.; visualization, C.-T.C. and L.-F.S.; supervision, L.-F.S.; project administration, L.-F.S.; funding acquisition, L.-F.S.

**Funding:** This research was funded by an institutional grant from the Agricultural Biotechnology Research Center, Academia Sinica, Taiwan.

**Acknowledgments:** The authors thank the Metabolomics Core Facility and the Laboratory Animal Facility of Agricultural Biotechnology Research Center, Academia Sinica, Taiwan for their services, and Ms. Miranda Loney, Agricultural Biotechnology Research Center English Editor's Office, Academia Sinica, Taiwan, for English editorial assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


**Sample Availability:** Samples of the compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Synthesis and Cytotoxicity Evaluation of DOTA-Conjugates of Ursolic Acid**

### **Michael Kahnt 1, Sophie Hoenke 1, Lucie Fischer 1, Ahmed Al-Harrasi <sup>2</sup> and René Csuk 1,\***


Academic Editor: Kyoko Nakagawa-Goto Received: 12 May 2019; Accepted: 14 June 2019; Published: 17 June 2019

**Abstract:** In this study, we report the synthesis of several amine-spacered conjugates of ursolic acid (UA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Thus, a total of 11 UA-DOTA conjugates were prepared holding various oligo-methylene diamine spacers as well as different substituents at the acetate units of DOTA including *tert*-butyl, benzyl, and allyl esters. Furthermore, three synthetic approaches were compared for the ethylenediamine-spacered conjugate **29** regarding reaction steps, yields, and precursor availability. The prepared conjugates were investigated regarding cytotoxicity using SRB assays and a set of human tumor cell lines. The highest cytotoxicity was observed for piperazinyl spacered compound **22**. Thereby, EC50 values of 1.5 μM (for A375 melanoma) and 1.7 μM (for A2780 ovarian carcinoma) were determined. Conjugates **22** and **24** were selected for further cytotoxicity investigations including fluorescence microscopy, annexin V assays and cell cycle analysis.

**Keywords:** ursolic acid; DOTA; triterpenoids; cytotoxicity

### **1. Introduction**

Despite all medical advances in tumor therapy, cancer is still one of the most prevalent diseases worldwide, with 9.6 million cancer-related deaths counted in 2018 [1]. The research of novel therapeutic approaches and potent chemotherapeutic agents are important contributions in the battle against cancer. However, diagnosis is a prerequisite for successful treatment since an early detection of cancer cells can often significantly reduce the pathogenicity of a tumor and increase the healing rate. One molecule, which made significant impact on the field of diagnostic imaging in the past decades, is the EDTA-related macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA, Figure 1) [2,3].

DOTA-derivatives and complexes thereof are widely used for molecular imaging, especially for the medical diagnosis of cancer [2,3]. By variation of the coordinated metal ion or substituents, they are applicable for a number of imaging techniques, such as magnetic resonance imaging (MRI) [2–4], positron emission tomography (PET) [2,3,5,6] and single photon emission computed tomography (SPECT) [2,3,7]. Because of this versatility, a crossover application of DOTA-derivatives as multimodal contrast agents for combined imaging modalities such as PET/MRI or PET/CT is possible and has already been described in the literature [2,3].

Although DOTA and derivatives thereof have a wide range of uses in diagnostic imaging, there are virtually no references for applications in the therapy of cancer. Therefore, we decided to prepare possible cytotoxic DOTA-derivatives by linkage with an ursolic acid backbone. Ursolic

acid (UA, Figure 1) is a natural occurring triterpenoic acid with promising pharmacological properties, being widely distributed in various plants and fruits, such as rosemary [8], sage [8], oleander [9], and apples [10,11]. A wide range of biological activities, including antidiabetic, anti-inflammatory, antibacterial, and anticancer effects have been credited to UA and structurally-related derivatives [12–14]. Many structural modifications have been described in the literature starting from ursolic acid with various impacts on cytotoxic properties [12,15–18]. Structure activity investigations concerning modifications at C-3 of UA revealed the presence of an acetyloxy group to be beneficial for obtaining high anti-tumor activity [18]. Furthermore, it has been shown that the modification of C-28 with a piperazine moiety had a positive influence on the cytotoxic properties of ursolic acid [19,20]. Previously, we also have shown oligo-methylene diamine derived carboxamides of ursolic acid to be of high cytotoxicity [21]. Keeping these structure activity relationships in mind, we considered 3-acetyloxy protected and C-28 modified UA derivatives a convenient starting point for the preparation of cytotoxic oligo-methylene diamine spacered DOTA conjugates.

**Figure 1.** Structures of ursolic acid (UA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

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

The synthesis of UA-DOTA conjugates started with the structural modification of cyclen (**2**) as illustrated in Scheme 1. Treatment of **2** with 3 equiv. of sodium bicarbonate and 3 equiv. of the respective bromoacetic ester (**3**–**5**) in dry acetonitrile yielded triple substituted cyclen derivatives **6**–**8**, being ready to be coupled with ursolic acid. We decided to use *tert*-butyl, benzyl and allyl esters as protecting groups. Benzyl, as well as *tert*-butyl bromoacetate, were bought from commercial suppliers. Allyl bromoacetate was prepared from allylic alcohol and bromoacetyl bromide.

**Scheme 1.** Synthesis of DOTA precursors **6**–**8**: (a) NaHCO3, MeCN, 25 ◦C, 48 h, yield: 50% (**6**), 68% (**7**), 63% (**8**).

Ursolic acid has also been modified before coupling with the DOTA precursors (Scheme 2). Derivatization started with the attachment of a spacer moiety using oxalyl chloride and 1-(2-aminoethyl)piperazine in dry dichloromethane affording compound **10**. The terminal amino moiety was further substituted with chloroacetyl chloride in dry dichloromethane to furnish the ursolic acid precursor **11** in excellent yield. Linkage of both precursors was performed in dry acetonitrile in the presence of potassium carbonate and potassium iodide yielding UA-DOTA conjugates **12**–**14**. Allylic esters of the acetate groups were removed by treating compound **14** with [(PPh3)4Pd],

triphenylphosphane and pyrrolidine in acetonitrile at 25 ◦C for 3 days; this procedure gave **15** in almost quantitative yield. Purification of **15** was performed by reversed phase chromatography using MeOH/MeCN/TFA as eluent since the compound was difficult to eluate from normal silica phases. Furthermore, a synthetic approach for **15** starting from either **12** or **13** failed. Hydrogenation of **13** employing palladium catalysis retained the benzyl esters, and the deprotection of tert-butyl esters (as in compound **12**) using TFA/DCM resulted in a partial degradation of the triterpenoic backbone.

**Scheme 2.** Synthesis of ursolic acid chelator conjugates **12**–**15**: (**a**) Ac2O, CH2Cl2, NEt3, 25 ◦C, 2 days, 82%; (**b**) oxalyl chloride, CH2Cl2, DMF, 0–25 ◦C, 1 h, then 1-(2-Aminoethyl)piperazine, CH2Cl2, 25 ◦C, 2 h, yield: 82%; (**c**) chloroacetyl chloride, CH2Cl2, NEt3, 25 ◦C, 30 min, yield: 91%; (**d**) K2CO3, KI, **6** (for **12**) or **7** (for **13**) or **8** (for **14**), MeCN, 25 ◦C, 48 h, yield: 54% (**12**), 82% (**13**), and 80% (**14**); (**e**) [(PPh3)4Pd], PPh3, pyrrolidine, MeCN, 25 ◦C, 3 days, yield: 96%.

The synthetic approach summarized in Scheme 2 can also be applied to various other spacer units. Thus, we decided to alter the amino component. Therefore, ursolic acid was treated with piperazine, ethylene diamine and 2,2 -oxybis(ethylamine), to furnish ursolic carboxamides **16**–**18** (Scheme 3), respectively. Chloroacetyl derivatives **19**–**21** and UA-DOTA conjugates **22**–**27** were prepared analogous to Scheme 2.

**Scheme 3.** Synthesis of ursolic acid DOTA conjugates **22**–**27**: (**a**) oxalyl chloride, CH2Cl2, DMF, 0–25 ◦C, 1 h, then amine, CH2Cl2, 25 ◦C, 2 h, yield: 80% (**16** and **17**), and 78% (**18**); (**b**) chloroacetyl chloride, CH2Cl2, NEt3, 25 ◦C, 0.5–4 h, yield: 94% (**19**), and 91% (**20** and **21**); (**c**) K2CO3, KI, **6** (for **22**, **24** and **26**) or **7** (for **23**, **25** and **27**), MeCN, 25 ◦C, 5 days, yield: 88% (**22**), 72% (**23**), 73% (**24**), 75% (**25**), 74% (**26**), and 62% (**27**).

Additionally, an alternative synthetic approach was established for the preparation of the ethylene diamine-spacered UA-DOTA conjugate **29** (Scheme 4). Therefore carboxamide **28** was prepared either by deacetylation of **17** or directly from UA by amidation with ethylene diamine using EDC and HOBt in dry DMF. In the next step, DOTA-tris(*tert*-butyl ester) (DOTA-3T) was activated by preparing its HOBt ester. Adding **28** to this freshly prepared ester furnished UA-DOTA conjugate **29**. For comparison, compound **29** was also synthesized from **24** by removing the C-3 acetyloxy moiety. Due to the presence of an unprotected hydroxyl moiety at this position, compound **29** offers the possibility for a set of modifications and is therefore considered to be a good starting material for further modifications.

**Scheme 4.** Synthesis of ursolic acid derivative **29**: (**a**) KOH, MeOH, 25 ◦C, 48 h (for **28**) or 24 h (for **29**), yield: 85% (**28**) or 86% (**29**); (**b**) ethylene diamine, HOBt·H2O, EDC·HCl, DMF, 25 ◦C, 24 h, yield: 46% (**c**) DOTA-3T, HOBt·H2O, EDC·HCl, DMF, 25 ◦C, 5 days, yield: 49%.

Both synthetic approaches for the preparation of **29** (Figure 2) hold advantages but also some disadvantages. Although route A (5 steps) is significantly longer than B, but the former route gave the highest overall yield (44%). Approach B is a rather short and quick way to synthesize **29** (2 steps only), but the overall yield (23%) is barely half as high as in route A. Combining routes A and B, as shown in approach C led to compound **29** in 4 steps with an overall yield of 32%. A major difference between the approaches A and B is the availability and preparation of the DOTA precursors. Both, DOTA-tris(tert-butyl ester) and DO3A-tert-butyl ester (**6**) are available from commercial suppliers, with DOTA-3T being almost twice as expensive as **6**. Most advantageous is the one-step synthesis of **6** starting from cylcen (**2**), since **2** is commercially available for a price, being almost tenfold lower than that of **6**.

**Figure 2.** Comparison of synthetic routes **A**, **B**, and **C** for the preparation of UA-DOTA conjugate **29**.

Due to cytotoxicity evaluation, the prepared UA-DOTA conjugates were screened in rhodamine B assays employing a series of human tumor cell lines and non-malignant mouse fibroblasts (NIH 3T3). Results of this investigation are summarized in Table 1.


**Table 1.** Cytotoxicity of UA-DOTA conjugates (**12**–**15**, **22**–**27**, **29**), DOTA precursors (**6**,**7**), ursolic acid (UA), and doxorubicin hydrochloride (DRC): EC50 values from SRB assays after 72 h of treatment are given in μM (n.d. not detected; n.s. not soluble); the values are averaged from three independent experiments each performed in triplicate; confidence interval CI = 95%.

The DO3A-tert-butyl ester (**6**) showed moderate cytotoxicity as indicated by EC50 values between 10 μM and 14 μM, while DO3A-benzyl ester (**7**) showed EC50 values lower than 5 μM. Unfortunately, most of the UA-DOTA conjugates were not soluble in solvents suitable for SRB assays. However, combining **7** with ursolic acid gave cytotoxic conjugate **13**, showing EC50 values below 6 μM. Removal of ester units (as in **15**) resulted in a complete loss of cytotoxicity (EC50 >60 μM for all tumor cells). Compounds **22** and **24**, both holding tert-butyl esters but different spacer units were also highly cytotoxic. Piperazine-spacered conjugate **22** showed the highest cytotoxicity observed in this screening for A375 tumor cells (EC50 = 1.5 ± 0.4 μM), while being quite selective, too (SI (NIH 3T3/A375) = 3.07, Table 2). EC50 values of ethylenediamine-spacered conjugate **24** were below 2.5 μM for all tumor cell lines. The highest cytotoxicity was observed for ovarian carcinoma (A2780, EC50 = 1.7 ± 0.1 μM). Removal of the acetyloxy moiety of **24** (as in **29**) had almost no significant impact on cytotoxicity.


**Table 2.** Selectivity of selected UA-DOTA conjugates (**13**, **22**, **24,** and **29**), DOTA precursors (**6**, **7**), ursolic acid (UA) and doxorubicin hydrochloride (DRC): Selectivity index (SI) is defined as: SI = EC50 (NIH 3T3)/EC50 (tumor cell line).

Because UA-DOTA conjugates **22** and **24** were the most active compounds of this study, these compounds were selected for further cytotoxicity investigations including fluorescence microscopy, annexin V assays, and cell cycle evaluation employing melanoma cells (A375). Microscopic images of A375 cells treated with compound **24** for 24 h showed vital cells (green staining) with some of them having ruptured cell membranes (Figure 3A, white arrows). Further indications of apoptosis have been detected employing flow cytometry and annexin V-FITC/PI staining. After 24 h 63% of the tumor cells treated with **24** were annexin V-FITC-positive, and 51.9% of all cells having died by apoptosis. Additionally, the number of vital cells decreased in comparison to the control from 86.1% to 36.7 % (Figure 3B). An extra investigation of the cell cycle showed a decreased number of cells in G1/G0, G2/M, as well as in S phase. Additionally, a large population of cells has been shifted into the subG1 region (Figure 3C).

**Figure 3.** Extended cytotoxicity investigation after treatment of A375 cells with **24** (4.0 μM) for 24 h: (**A**) Fluorescence microscopic images (scale bar 20 μm), AO and PI were used; (**B**) Annexin V-FITC/PI assay. Examples of density plots determined by flow cytometry (Attune® Cytometric Software v 1.2.5), R1: necrotic, R2: secondary necrotic/late stage apoptotic, R3: vital, R4: apoptotic; (**C**) Representative examples for cell cycle evaluation via ModFit LT 5.0.

Fluorescence microscopic images and density plots of A375 cells treated with **22** for 24 h showed no significant differences in comparison to the control (Supplementary material, Figure S1). Therefore, further investigations were performed with a prolonged incubation time of 48 h. After treating A375 cells with compound **22** for 48 h, subsequent fluorescence microscopic investigations using AO/PI staining showed ruptures of the plasma membrane (Figure 4A, white arrows). Additionally, some necrotic/late stage apoptotic cells were observed, indicated by slightly orange stained nuclei (Figure 4 A, orange arrow). The density plot of A375 cells treated with **22** for 48 h showed a decreased number of vital cells (66.3%) compared to the control (86.0%), while 32.9% of the cells were considered annexin V-FITC-positive. Nearly half of them (17.4% of all cells) have died by apoptosis, and the remaining cells (15.5% of all cells) were secondary necrotic/late stage apoptotic (Figure 4 B). During extra investigations of the cell cycle, some differences compared to the control have been observed. Cells treated with **22** for 48 h showed a quite broad and flat DNA distribution. G1/G0, G2/M, and S phase were drastically reduced, while an increased population of cells with reduced DNA content has been observed in the subG1 region (Figure 4C).

**Figure 4.** Extended cytotoxicity investigation after treatment of A375 cells with **22** (3.0 μM) for 48 h: (**A**) Fluorescence microscopic images (scale bar 20 μm), AO and PI were used; (**B**) Annexin V-FITC/PI assay. Examples of density plots determined by flow cytometry (Attune® Cytometric Software vl 1.2.5), R1: necrotic, R2: secondary necrotic/late stage apoptotic, R3: vital, R4: apoptotic; (**C**) Representative examples for cell cycle evaluation via ModFit LT 5.0.

### **3. Conclusions**

In this study, a series of overall 11 amine-spacered UA-DOTA conjugates have been prepared starting from the natural occurring triterpenoid ursolic acid (UA). We hereby report a synthetic approach to UA-DOTA conjugates, which is applicable for several amine spacers and other triterpenoic backbones, too. Additionally, we compared three synthetic approaches for the preparation of compound **29** in terms of yield, number of steps and precursor availability. This conjugate offers the possibility for further modifications at the C-3 hydroxylic group, which is known to influence cytotoxicity. All of the prepared DOTA conjugates were screened in SRB assays showing some compounds to be of good cytotoxicity. EC50 values were determined to range from 17.3 μM to 1.4 μM. The most active compound of this series was a piperazinyl spacered conjugate **22** showing low EC50 values such as 1.5 ± 0.4 μM for A375 tumor cells and 1.9 ± 0.3 μM for A2780 tumor cells, respectively, while showing good selectivity (SI (NIH 3T3(A375) = 3.07), too. Unfortunately, the selectivity of the other screened conjugates was quite low. Additional cytotoxicity investigations such as fluorescence microscopy, annexin V assays, and cell cycle analyses were performed employing the UA-DOTA conjugates **22** and **24** to gain information about their mode of action. The results of these extended biological testing indicate **24** to induce death of A375 cancer cells by apoptosis. These results hold some starting points for further studies. Conjugate **15** and structural related compounds (holding free carboxylic acids at the DOTA unit) are currently subjects of ongoing investigations regarding their ability to form complexes with metal ions or radioactive isotopes like 68Ga to examine possible future uses as tracer or contrast agents in molecular imaging techniques, such as positron emission tomography (PET) [22].

### **4. Materials and Methods**

### *4.1. General*

NMR spectra were recorded using the Varian spectrometers Gemini 2000 or Unity 500 (Varian GmbH, Darmstadt, Germany) δ given in ppm, *J* in Hz; typical experiments: APT, H-H-COSY, HMBC, HSQC, NOESY), MS spectra were taken on a Finnigan MAT LCQ 7000 (ThermoFisher Scientific, Braunschweig, Germany) electrospray, voltage 4.1 kV, sheath gas nitrogen) instrument. The optical rotations were measured on a Perkin-Elmer polarimeter (Perkin Elmer LAS, Rodgau, Germany) or on a Jasco P-2000 polarimeter (Jasco Germany, Pfungstadt, Germany) at 20 ◦C; TLC was performed on NP or RP18 silica gel (Macherey-Nagel, detection with cerium molybdate or Dragendorff's reagent). Melting points are uncorrected (*Leica* hot stage microscope, or BUCHI melting point M-565), and elemental analyses were performed on a Foss-Heraeus Vario EL (CHNS, Elementar Analysensysteme GmbH, Langenselbold, Germany) unit. IR spectra were recorded on a Perkin Elmer FT-IR spectrometer Spectrum 1000 or on a Perkin-Elmer Spectrum Two (UATR Two Unit; both instruments from Perkin Elmer LAS, Rodgau, Germany). UV-VIS spectra were taken on a Perkin-Elmer Lambda 14 spectrometer or on a Perkin-Elmer Lambda 750 S (UV/VIS/NIR) spectrometer (both instruments from Perkin Elmer LAS, Rodgau, Germany). The solvents were dried according to usual procedures. The purity of the compounds was determined by HPLC and found to be >96%.

### *4.2. Cytotoxicity*

### 4.2.1. Cell Lines and Culture Conditions

The cell lines used are human cancer cell lines: A2780 (ovarian carcinoma), HT29 (colon adenocarcinoma), MCF-7 (breast adenocarcinoma), A375 (melanoma), FaDu (pharynx squamous cell carcinoma) and non-malignant mouse fibroblasts NIH 3T3; all cell lines were obtained from the Department of Oncology (Martin-Luther-University Halle-Wittenberg). Cultures were maintained as monolayers in RPMI 1640 medium with l-glutamine (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) supplemented with 10% heat inactivated fetal bovine serum (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and penicillin/streptomycin (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) at 37 ◦C in a humidified atmosphere with 5% CO2.

### 4.2.2. Cytotoxic Assay (SRB)

The cytotoxicity of the compounds was evaluated using the sulforhodamine-B (Kiton-Red S, ABCR) micro culture colorimetric assay. Cells were seeded into 96-well plates on day 0 at appropriate cell densities to prevent confluence of the cells during the period of experiment. After 24 h, the cells were treated with six different concentrations (1, 3, 7, 12, 20, and 30 μM) minimum. The final concentration of DMSO/DMF never exceeded 0.5%, which was non-toxic to the cells. After a 72-h treatment, the supernatant medium from the 96-well plates was discarded, the cells were fixed with 10% trichloroacetic acid (TCA) and allowed to rest at 4 ◦C. After 24 h fixation, the cells were washed in a strip washer and dyed with SRB solution (100 μL, 0.4%, in 1% acetic acid) for about 20 min. After dying, the plates were washed four times with 1% acetic acid to remove the excess of the dye and allowed to air-dry overnight. Tris base solution (200 μL, 10 mM) was added to each well and absorbance was measured at λ = 570 nm using a 96 well plate reader (Tecan Spectra, Crailsheim, Germany). The EC50 values were averaged from three independent experiments performed each in triplicate calculated from semi logarithmic dose response curves applying a non-linear 4P Hills-slope equation (GraphPad Prism5; variables top and bottom were set to 100 and 0, respectively).

### 4.2.3. AO/PI Dye Exclusion Test

Morphological characteristics of cell death were analyzed employing an AO/PI assay using human cancer cell line A375. Approx. 2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells were seeded in cell culture flasks (25 cm2), and the cells were allowed to grow up for 24 h. After removing of the used medium, the substance loaded fresh medium was reloaded (or a blank new medium as a control). After 24 h and 48 h, the content of the flask was collected and centrifuged (1200 rpm, 4 ◦C), the pellet was gently suspended in phosphate-buffered saline (PBS (w/Ca2<sup>+</sup> and Mg2<sup>+</sup>), 1 mL) and centrifuged again. The PBS was removed, and the pellet gently suspended in PBS (150 μL) again. The analysis of the cells was performed using a fluorescence microscope after having mixed the cell suspension (10 μL) with a solution of AO/PI (5 μg/mL, 10 μL).

### 4.2.4. Annexin V-FITC/PI Assay

Approximately 2 <sup>×</sup> 105 cells (A375) were seeded in cell culture flasks (25 cm2), and the cells were allowed to grow up for 24 h. After removing of the used medium, the substance loaded fresh medium was reloaded (or a blank fresh medium as a control). After 24 h and 48 h, the cells were harvested, centrifuged (1200 rpm, 4 ◦C), and washed twice with PBS (w/Ca2<sup>+</sup> and Mg2<sup>+</sup>, 1 mL). The cells were counted and approximately 1·106 cells were washed with Annexin V binding buffer (BioLegend®, San Diego, USA) and treated with propidium iodide solution (3 μL, 1 mg/mL) and Annexin V-FITC (5 μL, BioLegend®, San Diego, CA, USA) for 15 min in the dark at room temperature. After adding Annexin V binding buffer (400 μL) the suspension was analyzed using Attune® FACS machine. After gating for living cells, the data from detectors BL-1A and BL-3A were collected (20,000 events) in technical triplicates. The assay was performed in duplicates; cell distribution was calculated using Attune® Software (ThermoFisher Scientific, Braunschweig, Germany).

### 4.2.5. Cell Cycle Investigations

Approximately 2 <sup>×</sup> 105 cells (A375) were seeded in cell culture flasks (25 cm2), and the cells were allowed to grow up for 24 h. After removing of the used medium, the substance loaded fresh medium was reloaded (or a blank fresh medium as a control). After 24 h or 48 h, respectively, only the adherent cells were harvested, centrifuged (1200 rpm, 4 ◦C), and washed twice with PBS ((*w*/*w*), 1 mL). The cells were counted and approximately 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells were fixed with ethanol (70%, 4 ◦C, 24 h). After centrifugation (4500 rpm, 4 ◦C) the cells were washed with PBS ((*w*/*w*), 1 mL) and centrifuged. The pellet was resuspended in 1 mL RNAse A containing PI buffer (100μL RNAse (100 mg/mL), 15 μL PI solution (1 mg/mL)) and after incubating for 30 min at room temperature in the dark, cells were analyzed using the Attune® FACS machine; collecting data from the BL-2A channel. Doublet cells were excluded from the measurements by plotting BL-2A against BL-2H. For each cell cycle distribution 20,000 events were collected in technical triplicates, each sample was measured in duplicates. Cell cycle distribution was calculated using ModFitLT™ (Verity Software House, Topsham, ME, USA).

### *4.3. Syntheses*

### 4.3.1. General

Ursolic acid (**1**) was obtained from betulinines (Stˇríbrná Skalice, Czech Republic). 1,4,7,10-Tetraazacyclododecane (cyclen, **2**) was bought from abcr GmbH (Karlsruhe, Germany) in 95% purity. Benzyl bromoacetate (96%) and tert-Butyl bromoacetate (98%) were both purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). DOTA-tris(*tert*-butyl ester) was obtained from TCI Deutschland GmbH (Eschborn, Germany) in 97% purity. Ursolic acid derivatives **10**, **16**–**18** and **28** have been synthesized as previously reported [20,21]. Experimental procedures and full analytical data of these compounds can be found in the supplementary material [23,24].

### 4.3.2. General Procedure A for the Synthesis of DOTA Precursors (**6**–**8**)

To a suspension of cyclen (5.81 mmol) and sodium bicarbonate (17.43 mmol) in dry acetonitrile (100 mL), a solution of the respective bromoacetate (**3**–**5**, 17.43 mmol) in dry acetonitrile (10 mL) was added dropwise under argon atmosphere. The mixture was stirred for 48 h at 25 ◦C. After usual aqueous work-up, the solvent was removed under reduced pressure, and the crude products were subjected to column chromatography (silica gel, chloroform/methanol mixtures) affording DOTA precursors **6**–**8** (50–68%).

### 4.3.3. General Procedure B for the Synthesis of Carboxamides (**10**, **16**–**18**)

Compound **9** (0.5 mmol) was dissolved in dry DCM (10 mL), cooled to 0 ◦C and oxalyl chloride (3.2 mmol) and dry DMF (2 drops) were added. After warming to 25 ◦C, the mixture was stirred for 1 h. The solvent was removed under reduced pressure, re-evaporated with dry THF (4 × 15 mL), and the residue was immediately resolved in dry DCM (10 mL). This mixture was then added dropwise to a solution of the amine (3.0 mmol) in dry DCM (2 mL) and stirred at 25 ◦C for 2 h. After usual aqueous work-up, the solvent was removed under reduced pressure, and the crude products were subjected to column chromatography (silica gel, chloroform/methanol mixtures). Compounds **10** and **16**–**18** were each obtained as colorless solids (78–82%).

### 4.3.4. General Procedure C for the Alkylation with Chloroacetyl Chloride (**11**, **19**–**21**)

Chloroacetyl chloride (2.20 mmol) was added dropwise to a solution of the respective carboxamide (**10**, **16**–**18**; 1.43 mmol) and triethylamine (0.71 mmol) in dry dichloromethane (75 mL). The mixture was stirred at 25 ◦C for 0.5–4 h. After usual aqueous work-up, the solvent was removed under reduced pressure, and the crude products were subjected to column chromatography (silica gel, chloroform/acetone mixtures). Compounds **11** and **19**–**21** were each obtained as colorless solids (91%–94%).

### 4.3.5. General Procedure D for the Synthesis of Ursolic Acid Chelator Conjugates (**12**–**14**, **22**–**27**)

To a solution of the respective chloroacetyl derivative (**11, 19**–**21**; 0.44 mmol) and freshly grounded potassium carbonate (0.83 mmol) in dry acetonitrile (15 mL) was added potassium iodide (0.35 mmol) and the respective DOTA precursor (**6**–**8,** 0.41 mmol in 5 mL dry acetonitrile). The mixture was stirred for 2–5 days at 25 ◦C. After completion of the reaction (as indicated by TLC) the mixture was filtered, and the solvent was removed under reduced pressure. The crude products were subjected to column chromatography (silica gel, chloroform/methanol mixtures) to afford compounds **12**–**14** and **22**–**27** (yield: 54–88%), respectively.

*Allyl bromoacetate* (**5**), To a solution of allyl alcohol (0.62 mol), bromo acetylbromide (0.1 mol) was added dropwise over a period of 30 min at 0 ◦C under argon atmosphere. The mixture was stirred for 1 h at 0 ◦C, warmed to 25 ◦C and stirred for another 3 h. The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane. After usual aqueous work-up, the solvent was removed under reduced pressure, and the crude product was purified by vacuum distillation affording allyl bromoacetate as colorless oil (68%). 1H NMR (400 MHz, CDCl3): δ = 5.88 (*ddt*, *J* = 16.5, 11.0, 5.8 Hz, 1H, CH=CH2), 5.38–5.17 (*m*, 2H, CH=C*H*2), 4.61 (*dt*, *J* = 5.8, 1.4 Hz, 2H, C*H*2CH=CH2), 3.82 (*s*, 2H, BrC*H*2) ppm; 13C NMR (101 MHz, CDCl3): δ = 166.8 (C=O), 131.2 (*C*H=CH2), 119.0 (CH=*C*H2), 66.6 (*C*H2CH=CH2), 25.8 (Br*C*H2) ppm.

*Tri-tert-butyl 2,2 ,2"-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate* (**6**), Compound **6** was prepared from **2** according to general procedure A using *tert*-butyl bromoacetate (**3**). Column chromatography (SiO2, CHCl3/MeOH 9:1) gave **6** (yield: 50%); m.p. 180–182 ◦C (lit.: 181–183 ◦C [25]); Rf = 0.27 (CHCl3/MeOH 95:5); IR (ATR): ν = 2974*w*, 2943*w*, 2912*w*, 2853*w*, 2736*w*, 1718*s*, 1576*w*, 1466*w*, 1453*w*, 1412*w*, 1392*w*, 1368*m*, 1330*w*, 1255*m*, 1218*w*, 1147*s*, 1117*m*, 1099*m*, 1050*w*, 935*m*, 873*m*, 848*m* cm−1; 1H NMR (400 MHz, CDCl3): <sup>δ</sup> = 3.36 (*s*, 4H, 2 <sup>×</sup> CH2 (acetate)), 3.28 (*s*, 2H, CH2 (acetate)), 3.12–3.06 (*m*, 4H, 2 × CH2 (cyclen)), 2.95–2.84 (*m*, 12H, 6 × CH2 (cyclen)), 1.45 (*s*, 18H, 6 × CH3 (*t*-butyl)), 1.45 (*s*, 9H, 3 <sup>×</sup> CH3 (*t*-butyl)) ppm; 13C NMR (101 MHz, CDCl3): <sup>δ</sup> = 170.6 (2 <sup>×</sup> CO, acetate), 169.8 (CO, acetate), 81.9 (Cq, *t*-butyl), 81.8 (2 × Cq, *t*-butyl), 58.4 (2 × CH2, acetate), 51.5 (2 × CH2, cyclen), 51.4 (2 × CH2, cyclen), 49.4 (2 × CH2, cyclen), 49.0 (CH2, acetate), 47.7 (2 × CH2, cyclen), 28.4 (3 × CH3, *t*-butyl), 28.3 (6 <sup>×</sup> CH3, *t*-butyl) ppm; MS (ESI, MeOH): *m*/*z* = 515.3 (100%, [M + H]+), 537.3 (10%, [M <sup>+</sup> Na]+); analysis calcd for C26H50N4O6 (514.71): C 60.67, H 9.79, N 10.89; found: C 60.51, H 9.98, N 10.67.

*Tribenzyl 2,2',2"-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate* (**7**). Compound **7** was prepared from **2** according to general procedure A using benzyl bromoacetate (**4**). Column chromatography (SiO2, CHCl3/MeOH 9:1) gave **7** (yield: 68%); Rf = 0.32 (CHCl3/MeOH 95:5); IR (KBr): ν = 2948*w*, 2857*w*, 2738*w*, 1732*s*, 1586*w*, 1498*w*, 1455*m*, 1418*w*, 1381*w*, 1314*w*, 1169*s*, 1096*m*, 1049*m*, 994*m*, 739*s*, 697*s* cm<sup>−</sup>1; UV-Vis (CHCl3): λmax (logε) = 251 nm (2.87), 258 nm (2.86), 263 nm (2.76); 1H NMR (400 MHz, CDCl3): δ = 7.40–7.29 (*m*, 15H, 15 × CH (Bn)), 5.13 (*s*, 4H, 2 × CH2 (Bn)), 5.13 (*s*, 2H, CH2 (Bn)), 3.48 (*s*, 4H, 2 × CH2 (acetate)), 3.41 (*s*, 2H, CH2 (acetate)), 3.12–3.05 (*m*, 4H, 2 × CH2 (cyclen)), 2.93–2.79 (*m*, 12H, <sup>6</sup> <sup>×</sup> CH2 (cyclen)) ppm; 13C NMR (101 MHz, CDCl3): <sup>δ</sup> = 171.1 (2 <sup>×</sup> CO, acetate), 170.3 (CO, acetate), 135.5 (Ci, Bn), 128.8 (CH, Bn), 128.8 (CH, Bn), 128.7 (CH, Bn), 128.7 (CH, Bn), 128.6 (CH, Bn), 66.8 (CH2, Bn), 57.4 (2 × CH2, acetate), 51.9 (2 × CH2, cyclen), 51.7 (2 × CH2, cyclen), 49.6 (2 × CH2, cyclen), 48.8 (CH2, acetate), 47.5 (2 <sup>×</sup> CH2, cyclen) ppm; MS (ESI, MeOH): *<sup>m</sup>*/*<sup>z</sup>* <sup>=</sup> 309.0 (10%, [M <sup>+</sup> 2H]2<sup>+</sup>), 617.4 (100%, [M + H]+), 639.3 (10%, [M + Na]<sup>+</sup>); analysis calcd for C35H44N4O6 (616.76): C 68.16, H 7.19, N 9.08; found: C 67.84, H 7.39, N 8.81.

*Triallyl 2,2',2"-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate* (**8**), Compound **8** was prepared from **2** according to general procedure A using allyl bromoacetate (**5**). Column chromatography (SiO2, CHCl3/MeOH 95:5) gave **8** (yield: 63%); Rf = 0.27 (CHCl3/MeOH 95:5); IR (KBr): ν = 2945*w*, 2858*w*, 2743*w*, 1731*s*, 1673*w*, 1648*w*, 1455*w*, 1420*w*, 1364*w*, 1314*w*, 1179*s*, 1095*m*, 985*s*, 928*s* cm−1; 1H NMR (400 MHz, CDCl3): <sup>δ</sup> = 5.93–5.81 (*m*, 3H, 3 <sup>×</sup> CH (allyl)), 5.32–5.20 (*m*, 6H, 3 <sup>×</sup> CH2 (allyl)), 4.60–4.53 (*m*, 6H, 3 × CH2 (allyl)), 3.49 (*s*, 4H, 2 × CH2 (acetate)), 3.41 (*s*, 2H, CH2 (acetate)), 3.12–3.06 (*m*, 4H, <sup>2</sup> <sup>×</sup> CH2 (cyclen)), 2.96–2.81 (*m*, 12H, 6 <sup>×</sup> CH2 (cyclen)) ppm; 13C NMR (101 MHz, CDCl3): δ = 170.8 (2 × CO, acetate), 170.0 (CO, acetate), 131.7 (2 × CH, allyl), 131.7 (CH, allyl), 119.2 (CH2, allyl), 119.0 (2 × CH2, allyl), 65.5 (2 × CH2, allyl), 65.4 (CH2, allyl), 57.3 (2 × CH2, acetate), 51.7 (2 × CH2, cyclen), 51.6 (2 × CH2, cyclen), 49.4 (2 × CH2, cyclen), 48.5 (CH2, acetate), 47.4 (CH2, cyclen) ppm; MS (ESI, MeOH): *m*/*z* = 234.1 (18%, [M + 2H]2+), 467.3 (100%, [M + H]+), 489.3 (10%, [M + Na]+); analysis calcd for C23H38N4O6 (466.6): C 59.21, H 8.21, N 12.01; found: C 59.03, H 8.44, N 11.78.

*(3*β*) 3-Acetyloxy-urs-12-en-28-oic acid* (**9**), Compound **1** was prepared from ursolic acid according to the procedure given in the literature [26]. Yield: 96%; m.p. 287–290 ◦C (lit.: 289–290 ◦C [27]).

*(3*β*) N-(2-(4-(2-Chloroacetyl)piperazin-1-yl)ethyl)-3-acetyloxy-urs-12-en-28-amide* (**11**). Compound **11** was synthesized from **10** according to general procedure C. Column chromatography (SiO2, CHCl3/acetone 4:1) furnished compound **11** (91%); m.p. 124–129 ◦C; [α]D = +32.3◦ (*c* 0.320, CHCl3); Rf = 0.38 (CHCl3/acetone 4:1); IR (KBr): ν = 3423*s*, 2947*s*, 1734*m*, 1654*s*, 1522*m*, 1458*m*, 1370*m*, 1247*s*, 1150*w*, 1027*m* cm<sup>−</sup>1; 1H NMR (400 MHz, CDCl3): δ = 6.32 (*t*, *J* = 5.0 Hz, 1H, N*H*), 5.28 (*t*, *J* = 3.6 Hz, 1H, 12-H), 4.49 (*dd*, *J* = 10.4, 5.4 Hz, 1H, 3-H), 4.06 (*s*, 2H, 36-H), 3.72–3.47 (*m*, 4H, 34-H, 34 -H), 3.47–3.36 (*m*, 1H, 31-Ha), 3.25–3.15 (*m*, 1H, 31-Hb), 2.58–2.39 (*m*, 6H, 33-H, 32-H, 33 -H), 2.04 (*s*, 3H, Ac), 2.02–1.80 (*m*, 5H, 11-Ha, 11-Hb, 16-Ha, 22-Ha, 18-H), 1.79–1.70 (*m*, 1H, 16-Hb), 1.69–1.21 (*m*, 13H, 15-Ha, 1-Ha, 2-Ha, 2-Hb, 9-H, 6-Ha, 21-Ha, 7-Ha, 22-Hb, 19-H, 6-Hb, 21-Hb, 7-Hb), 1.09 (*s*, 3H, 27-H), 1.08–1.01 (*m*, 2H, 1-Hb, 15-Hb), 0.96–0.94 (*m*, 4H, 20-H, 30-H), 0.93 (*s*, 3H, 25-H), 0.89–0.86 (*m*, 3H, 29-H), 0.86 (*s*, 3H, 23-H), 0.85 (*s*, 3H, 24-H), 0.84–0.79 (*m*, 1H, 5-H), 0.78 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 178.1 (C-28), 171.1 (Ac), 165.2 (C-35), 140.0 (C-13), 125.3 (C-12), 80.9 (C-3), 56.7 (C-32), 55.4 (C-5), 54.2 (C-18), 53.0 (C-33), 52.5 (C-33 ), 48.0 (C-17), 47.6 (C-9), 46.5 (C-34), 42.6 (C-14), 42.4 (C-34 ), 40.9 (C-36), 39.9 (C-19), 39.7 (C-8), 39.3 (C-20) 38.4 (C-1), 37.8 (C-4), 37.5 (C-22), 37.0 (C-10), 35.9 (C-31), 32.8 (C-7), 31.0 (C-21), 28.2 (C-23), 28.0 (C-15), 25.0 (C-16), 23.6 (C-2), 23.6 (C-11), 23.4 (C-27), 21.4 (Ac), 21.3 (C-30), 18.3 (C-6), 17.5 (C-29), 17.1 (C-26), 16.9 (C-24), 15.8 (C-25) ppm; MS (ESI, MeOH): m/z = 686.5 (100%, [M + H]<sup>+</sup>); analysis calcd for C40H64ClN3O4 (686.42): C 69.99, H 9.40, N 6.12; found: C 69.70, H 9.63, N 6.02.

*Tris-t-butyl 2',2"-[10-[2-[4-[2-(3*β*-acetyloxy-urs-12-en-28-oylamino)ethyl]piperazin-1-yl]-2-oxoethyl]-1,4,7,10 tetraazacyclododecane-1,4,7-triyl]triacetate* (**12**), Compound **12** was synthesized from **6** and **11** according to general procedure D. Column chromatography (SiO2, CHCl3/MeOH 9:1) furnished compound

**12** (54%). m.p. 247–250 ◦C (decomp.); [α]D = +18.9◦ (c 0.345, CHCl3); Rf = 0.30 (CHCl3/MeOH 9:1); IR (KBr): ν = 2931*m*, 1727*s*, 1644*s*, 1529*w*, 1455*m*, 1425*w*, 1368*s*, 1306*m*, 1228*s*, 1159*s*, 1105*s*, 1005*m*, 755*m* cm−1; 1H NMR (400 MHz, CDCl3): δ = 6.38 (*s*, 1H, NH), 5.26 (*t*, *J* = 3.4 Hz, 1H, 12-H), 4.46 (*dd*, *J* = 10.5, 5.2 Hz, 1H, 3-H), 3.92–2.04 (*m*, 36H, 34-H, 34 -H, 36-H, 3 × CH2 (acetate), 31-Ha, 31-Hb, 8 × CH2 (cyclen), 32-H, 33-H, 33 -H), 2.01 (*s*, 3H, Ac), 2.00–1.68 (*m*, 4H, 16-Ha, 11-Ha, 11-Hb, 18-H), 1.69–1.14 (*m*, 14H, 16-Hb, 22-Hb, 15-Ha, 1-Ha, 2-Ha, 2-Hb, 9-H, 6-Ha, 21-Ha, 7-Ha, 19-H, 6-Hb, 21-Hb, 7-Hb), 1.42 (*s*, 27H, 9 × CH3 (t-Butyl)), 1.05 (*s*, 3H, 27-H), 1.11–0.95 (*m*, 3H, 1-Hb, 15-Hb, 20-H), 0.91 (*d*, *J* = 6.1 Hz, 3H, 30-H), 0.90 (*s*, 3H, 25-H), 0.85 (*d*, *J* = 6.5 Hz, 3H, 29-H), 0.83 (*s*, 3H, 23-H), 0.82 (*s*, 3H, 24-H), 0.80–0.75 (*m*, 1H, 5-H), 0.74 (*s*, 3H, 26-H) ppm; 13C NMR (100 MHz, CDCl3): δ = 178.0 (C-28), 172.8 (CO, acetate), 171.0 (Ac), 169.8 (C-35), 139.8 (C-13), 125.3 (C-12), 81.9 (Cq, *t*-Butyl), 81.7 (2 × Cq, *t*-Butyl), 80.9 (C-3), 56.8 (C-32), 55.8 (3 × CH2, acetate), 55.3 (C-5), 55.2 (C-36), 53.9 (C-18), 53.4 (8 × CH2, cyclen), 52.2 (C-33, C-33 ), 47.8 (C-17), 47.5 (C-9), 44.3 (C-34, C-34 ), 42.5 (C-14), 39.8 (C-19), 39.7 (C-8), 39.1 (C-20), 38.4 (C-1), 37.8 (C-4), 37.4 (C-22), 36.9 (C-10), 35.7 (C-31), 32.8 (C-7), 31.0 (C-21), 28.2 (C-23), 28.0 (9 × CH3, t-Butyl), 27.9 (C-15), 24.8 (C-16), 23.6 (C-2), 23.5 (C-11), 23.4 (C-27), 21.4 (Ac), 21.3 (C-30), 18.3 (C-6), 17.4 (C-29), 17.1 (C-26), 16.8 (C-24), 15.7 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 593.9 (100%, [M + Na + H]+), 1186.7 (95%, [M + Na]<sup>+</sup>); analysis calcd for C66H113N7O10 (1164.67): C 68.06, H 9.78, N 8.42; found: C 67.75, H 9.97, N 8.51.

*Tribenzyl 2,2',2"-[10-[2-[4-[2-(3*β*-acetyloxy-urs-12-en-28-oylamino)ethyl]piperazin-1-yl]-2-oxoethyl]-1,4,7,10 tetraazacyclododecane-1,4,7-triyl]triacetate* (**13**). Compound **13** was synthesized from **7** and **11** according to general procedure D. Column chromatography (SiO2, CHCl3/MeOH 95:5) furnished compound **13** (82%). m.p. 142–146 ◦C; [α]D = +14.5◦ (c 0.300, CHCl3); Rf = 0.50 (CHCl3/MeOH 9:1); IR (KBr): ν = 3440*s*, 2947*m*, 1734*s*, 1641*s*, 1456*m*, 1371*m*, 1310*w*, 1247*m*, 1197*s*, 1105*m*, 1006*w*, 750*m* cm<sup>−</sup>1; UV-Vis (CHCl3): λmax (logε) = 257 nm (3.99); 1H NMR (400 MHz, CDCl3): δ = 7.36–7.24 (*m*, 15H, CHAr), 6.36–6.28 (*m*, 1H, NH), 5.27 (*t*, *J* = 3.7 Hz, 1H, 12-H), 5.21–5.13 (*m*, 4H, 2 × CH2Bn), 5.12–5.05 (*m*, 2H, CH2Bn), 4.47 (*dd*, *J* = 10.7, 5.1 Hz, 1H, 3-H), 3.72–2.81 (*m*, 12H, 34-H, 34 -H, 36-H, 3 × CH2 (acetate)), 3.40–3.30 (*m*, 1H, 31-Ha), 3.22–3.14 (*m*, 1H, 31-Hb), 2.46–2.33 (*m*, 6H, 32-H, 33-H, 33 -H), 2.81–2.06 (*m*, 16H,8 × CH2 (cyclen)), 2.03 (*s*, 3H, Ac), 2.00–1.68 (*m*, 6H, 16-Ha, 11-Ha, 11-Hb, 18-H, 22-Ha, 16-Hb), 1.68–1.20 (*m*, 13H, 15-Ha, 1-Ha, 2-Ha, 2-Hb, 9-H, 6-Ha, 21-Ha, 7-Ha, 22-Hb, 19-H, 6-Hb, 21-Hb, 7-Hb), 1.06 (*s*, 3H, 27-H), 1.05–0.94 (*m*, 3H, 1-Hb, 15-Hb, 20-H), 0.93 (*brs*, 3H, 30-H), 0.91 (*s*, 3H, 25-H), 0.85 (*d*, *J* = 5.7 Hz, 3H, 29-H), 0.85 (*s*, 3H, 23-H), 0.83 (*s*, 3H, 24-H), 0.82–0.77 (*m*, 1H, 5-H), 0.76 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 177.9 (C-28), 173.5 (CO, acetate), 170.9 (Ac), 170.0 (C-35), 139.7 (C-13), 135.4 (CAr), 135.3 (CAr), 135.2 (CAr), 128.7 (CHAr), 128.6 (CHAr), 128.5 (CHAr), 128.3 (CHAr), 128.3 (CHAr), 128.2 (CHAr), 125.3 (CHAr), 80.8 (C-3), 67.0 (CH2, Bn), 66.8 (CH2, Bn), 56.7 (C-32), 55.4 (C-36), 55.3 (CH2, acetate), 55.2 (C-5), 53.9 (C-18), 53.4 (CH2, cyclen), 52.7 (C-33, C-33 ), 47.7 (C-17), 47.4 (C-9), 42.4 (C-14), 39.7 (C-19), 39.5 (C-8), 39.0 (C-20), 38.3 (C-1), 37.6 (C-4), 37.3 (C-22), 36.8 (C-10), 35.8 (C-31), 32.7 (C-7), 30.9 (C-21), 28.0 (C-23), 27.8 (C-15), 24.8 (C-16), 23.5 (C-2), 23.4 (C-11), 23.2 (C-27), 21.3 (Ac), 21.2 (C-30), 18.1 (C-6), 17.3 (C-29), 17.0 (C-26), 16.7 (C-24), 15.6 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 634 (20%, [M + 2H]2<sup>+</sup>), 645 (100%, [M + H + Na]2+), 1289 (62%, [M + Na]+); analysis calcd for C75H107N7O10 (1266.72): C 71.11, H 8.51, N 7.74; found: C 70.73, H 8.70, N 7.49.

*Triallyl 2,2',2"-[10-[2-[4-[2-(3*β*-acetyloxy-urs-12-en-28-oylamino)ethyl]piperazin-1-yl]-2-oxoethyl]-1,4,7,10 tetraazacyclododecane-1,4,7-triyl]triacetate* (**14**). Compound **14** was synthesized from **8** and **11** according to general procedure D. Column chromatography (SiO2, CHCl3/MeOH 95:5) furnished compound **14** (80%); m.p. 159–163 ◦C (decomp.); [α]D = +14.8◦ (c 0.310, CHCl3); Rf = 0.35 (SiO2, CHCl3/MeOH 9:1); IR (KBr): ν = 3342*s*, 2946*m*, 2852*w*, 1734*s*, 1642*s*, 1522*w*, 1456*m*, 1386*w*, 1310*w*, 1246*m*, 1202*m*, 1106*m*, 1026*w* cm−1; 1H NMR (400 MHz, CDCl3): <sup>δ</sup> = 6.35 (*s*, 1H, NH), 5.97 – 5.83 (*m*, 3H, 3 <sup>×</sup> CH (allyl)), 5.34–5.19 (*m*, 7H, 12-H, 3 × CH2 (allyl)), 4.67–4.55 (*m*, 6H, 3 × CH2 (allyl)), 4.47 (*dd*, *J* = 10.6, 5.3 Hz, 1H, 3-H), 3.72–2.97 (*m*, 14H, 34-H, 34 -H, 36-H, 3 × CH2 (acetate), 31-Ha, 31-Hb), 2.97–2.14 (*m*, 22H, 8 × CH2 (cyclen), 32-H, 33-H, 33 -H), 2.03 (*s*, 3H, Ac), 2.01–1.68 (*m*, 6H, 16-Ha, 11-Ha, 11-Hb, 18-H, 22-Ha, 16-Hb), 1.68–1.19 (*m*, 13H, 15-Ha, 1-Ha, 2-Ha, 2-Hb, 9-H, 6-Ha, 21-Ha, 7-Ha, 22-Hb, 19-H, 6-Hb,

21-Hb, 7-Hb), 1.07 (*s*, 3H, 27-H), 1.06–0.94 (*m*, 3H, 1-Hb, 15-Hb, 20-H), 0.94 (*d*, *J* = 6.5 Hz, 3H, 30-H), 0.92 (*s*, 3H, 25-H), 0.87 (*d*, *J* = 6.5 Hz, 3H, 29-H), 0.85 (*s*, 3H, 23-H), 0.83 (*s*, 3H, 24-H), 0.82–0.78 (*m*, 1H, 5-H), 0.76 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): <sup>δ</sup> = 178.0 (C-28), 173.4 (2 <sup>×</sup> CO, acetate), 173.3 (CO, acetate), 171.1 (Ac), 170.0 (C-35), 139.8 (C-13), 131.9 (2 × CH, allyl), 131.7 (CH, allyl), 125.4 (C-12), 119.0 (CH2, allyl), 118.8 (2 × CH2, allyl), 80.9 (C-3), 66.0 (CH2, allyl), 65.9 (2 × CH2, allyl), 56.8 (C-32), 55.4 (C-36), 55.3 (C-5), 55.2 (3 × CH2, acetate), 53.9 (C-18), 53.6 (8 x CH2, cyclen), 52.7 (C-33, C-33 ), 47.9 (C-17), 47.5 (C-9), 45.0 (C-34, C-34 ), 42.6 (C-24), 39.8 (C-19), 39.7 (C-8), 39.1 (C-20), 38.4 (C-1), 37.8 (C-4), 37.4 (C-22), 37.0 (C-10), 35.9 (C-31), 32.8 (C-7), 31.0 (C-21), 28.2 (C-23), 27.9 (C-15), 24.9 (C-16), 23.6 (C-2), 23.6 (C-11), 23.3 (C-27), 21.4 (Ac), 21.3 (C-30), 18.3 (C-6), 17.4 (C-29), 17.1 (C-26), 16.8 (C-24), 15.7 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 569.8 (100%, [M + Na + H]+), 1138.8 (52%, [M + Na]+); analysis calcd for C63H101N7O10 (1116.54): C 67.77, H 9.12, N 8.78; found: C 67.50, H 9.37, N 8.43.

*2,2',2"-[10-[2-[4-[2-(3*β*-Acetyloxy-urs-12-en-28-oylamino)ethyl]piperazin-1-yl]-2-oxoethyl]-1,4,7,10 tetraazacyclododecane-1,4,7-triyl]triacetic acid* (**15**). Triphenylphosphane (0.038 mmol), [(PPh3)4Pd] (0.013 mmol) and pyrrolidine (0.290 mmol) were added to a solution of compound **14** (0.128 mmol) in acetonitrile (4 mL), and the mixture was stirred for 6 days at 25 ◦C. After filtration, the solvent was removed under reduced pressure, and the crude product was subjected to column chromatography (RP18, MeCN/MeOH/TFA 60:40:0.1) affording compound **15** as colorless solid (96%); m.p. 206–210 ◦C (decomp.); [α]D = +17.1◦ (c 0.315, MeOH); Rf = 0.35 (RP18, ACN/TFA 100:1); IR (ATR): ν = 2925*w*, 1634*s*, 1371*m*, 1245*s*, 1199*s*, 1127*s*, 1026*m*, 829*m*, 800*m*, 719*m* cm−1; 1H NMR (400 MHz, CD3OD): δ = 5.35 (*t*, *J* = 3.6 Hz, 1H, 12-H), 4.47 (*dd*, *J* = 11.0, 5.3 Hz, 1H, 3-H), 3.72–2.93 (*m*, 14H, 34-H, 34'-H, 36-H, 31-Ha, 31-Hb, 3 × CH2 (acetate)), 2.91–2.20 (*m*, 22H, 32-H, 33-H, 33'-H, 8 × CH2 (cyclen)), 2.09–2.06 (*m*, 1H, 18-H), 2.03 (*s*, 3H, Ac), 2.02–1.93 (*m*, 3H, 11-Ha, 11-Hb, 16-Ha), 1.84–1.23 (*m*, 15H, 15-Ha, 22-Ha, 1-Ha, 16-Hb, 2-Ha, 2-Hb, 9-H, 7-Ha, 6-Ha, 21-Ha, 22-Hb, 19-H, 6-Hb, 21-Hb, 7-Hb), 1.15 (*s*, 3H, 27-H), 1.12–0.96 (*m*, 3H, 15-Hb, 1-Hb, 20-H), 0.99 (*s*, 3H, 25-H), 0.97 (*brs*, 3H, 30-H), 0.92 (*d*, *J* = 6.4 Hz, 3H, 29-H), 0.89 (*s*, 3H, 24-H), 0.88 (*s*, 3H, 23-H), 0.87–0.84 (*m*, 1H, 5-H), 0.83 (*s*, 3H,26-H) ppm; 13C NMR (101 MHz, CD3OD): δ = 180.1 (C-28), 172.8 (Ac), 172.5 (CO, acetate), 172.1 (2 × CO, acetate), 171.2 (C-35), 140.2 (C-13), 127.0 (C-12), 82.4 (C-3), 59.8 (CH2, acetate), 59.7 (CH2, acetate), 59.1 (CH2, acetate), 57.7 (C-32), 56.7 (C-5), 55.3 (C-36), 54.4 (C-18), 54.2 (8 × CH2, cyclen), 53.8 (C-33, C-33'), 49.0 (C-17), 48.8 (C-9), 46.2 (C-34. C-34'), 43.4 (C-14), 40.9 (C-8), 40.9 (C-19), 40.3 (C-20), 39.4 (C-1), 38.7 (C-4), 38.7 (C-22), 38.1 (C-10), 37.3 (C-31), 34.0 (C-7), 31.9 (C-21), 29.0 (C-15), 28.6 (C-23), 25.4 (C-16), 24.6 (C-2), 24.5 (C-11), 24.0 (C-27), 21.6 (C-30), 21.1 (Ac), 19.3 (C-6), 18.0 (C-26), 17.8 (C-29), 17.2 (C-24), 16.1 (C-25) ppm; MS (ESI, MeOH, positive ion mode): *m*/*z* = 1018.6 (27%, [M + Na]+), 1034.7 (100%, [M + K]+); MS (ESI, MeOH, negative ion mode): *m*/*z* = 1017.7 (13%, [M <sup>−</sup> 2H + Na]–), 1032.6 (100%, [M – 2H + K]–); analysis calcd for C54H89N7O10 (996.35): C 65.10, H 9.00, N 9.84; found: C 64.82, H 9.21, N 9.61.

*1-(3*β*-Acetyloxy-urs-12-en-28-oyl)-4-(2-chloroacetyl) piperazine* (**19**). Compound **19** has been synthesized from **16** according to general procedure C. Column chromatography (SiO2, CHCl3/acetone/hexanes 95:5:20) furnished compound **19** (94%); m.p. 155–158 ◦C; [α]D = +34.3◦ (*c* 0.370, CHCl3); Rf = 0.66 (CHCl3/acetone 9:1); IR (ATR): ν = 2924*m*, 2871*w*, 1731*m*, 1658*s*, 1455*m*, 1392*m*, 1370*m*, 1243*s*, 1200*m*, 1145*m*, 1025*m*, 985*m*, 752*m* cm−1; 1H NMR (400 MHz, CDCl3): δ = 5.21 (*t*, *J* = 3.6 Hz, 1H, 12-H), 4.52–4.45 (*m*, 1H, 3-H), 4.06 (*s*, 2H, 34-H), 3.73–3.46 (*m*, 8H, 31-H, 31 -H, 32-H, 32 -H), 2.41 (*d*, *J* = 11.6 Hz, 1H, 18-H), 2.24–2.11 (*m*, 1H, 16-Ha), 2.03 (*s*, 3H, Ac), 1.91 (*dd*, *J* = 8.9, 3.6 Hz, 2H, 11-Ha, 11-Hb), 1.80–1.24 (*m*, 15H, 15-Ha, 16-Hb, 22-Ha, 1-Ha, 2-Ha, 2-Hb, 22-Hb, 9-H, 6-Ha, 21-Ha, 7-Ha, 19-H, 6-Hb, 21-Hb, 7-Hb), 1.07 (*s*, 3H, 27-H), 1.12–0.98 (*m*, 3H, 1-Hb, 15-Hb, 20-H), 0.95 (*d*, *J* = 6.2 Hz, 3H, 30-H), 0.93 (*s*, 3H, 25-H), 0.88 (*d*, *J* = 6.4 Hz, 3H, 29-H), 0.85 (*s*, 3H, 23-H), 0.84 (*s*, 3H, 24-H), 0.84–0.77 (*m*, 1H, 5-H), 0.73 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 175.8 (C-28), 171.1 (Ac), 165.5 (C-33), 138.6 (C-13), 125.5 (C-12), 81.0 (C-3), 55.5 (C-5), 55.1 (C-18), 48.8 (C-17), 47.7 (C-9), 46.3 (C-31), 45.5 (C-31 ), 45.1 (C-32), 42.3 (C-32 , C-14), 40.9 (C-34), 39.6 (C-19), 39.6 (C-8), 38.9 (C-20), 38.4 (C-1), 37.8 (C-4), 37.1 (C-10), 34.6 (C-22), 33.1 (C-7), 30.6 (C-21), 28.3 (C-15), 28.2 (C-23), 23.9 (C-27), 23.7 (C-2, C-16), 23.4 (C-11), 21.4 (Ac), 21.4 (C-30), 18.3 (C-6), 17.6 (C-29), 17.0 (C-26), 16.9 (C-24), 15.6 (C-25) ppm; MS

(ESI, MeOH): *m*/*z* = 643.5 (100%, [M + H]+), 665.4 (56%, [M + Na]+), 1307.3 78%, [2M + Na]+); analysis calcd for C38H59ClN2O4 (643.4): C 70.94, H 9.24, N 4.35; found: C 70.72, H 9.51, N 4.09.

*(3*β*) N-(2-(2-Chloroacetyl)aminoethyl)-3-acetyloxy-urs-12-en-28-amide* (**20**). Compound **20** was synthesized from **17** according to general procedure C. Column chromatography (SiO2, CHCl3/acetone/hexanes 95:5:20) furnished compound **20** (91%); m.p. 103–107 ◦C; [α]D = +25.2◦ (*c* 0.300, CHCl3); Rf = 0.40 (CHCl3/acetone 9:1); IR (KBr): ν = 3422*br s*, 2948*s*, 2872*m*, 1734*s*, 1640*s*, 1532*s*, 1456*m*, 1370*m*, 1246*s*, 1148*w*, 1092*w*, 1028*m*, 756*m* cm−1; 1H NMR (400 MHz, CDCl3): δ = 7.43 (*t*, *J* = 5.1 Hz, 1H, NH), 6.28 (*t*, *J* = 5.7 Hz, 1H, NH), 5.32 (*t*, *J* = 3.6 Hz, 1H, 12-H), 4.48 (*dd*, *J* = 9.7, 6.1 Hz, 1H, 3-H), 4.00 (*s*, 2H, 34-H), 3.56–3.46 (*m*, 1H, 31-Ha), 3.41–3.35 (*m*, 2H, 32-H), 3.26–3.18 (*m*, 1H, 31-Hb), 2.04 (*s*, 3H, Ac), 2.03–1.80 (*m*, 5H, 16-Ha, 11-Ha, 11-Hb, 18-H, 22-Ha), 1.80–1.22 (*m*, 14H, 16-Hb, 2-Ha, 2-Hb, 1-Ha, 15-Ha, 9-H, 6-Ha, 21-Ha, 7-Ha, 22-Hb, 19-H, 6-Hb, 21-Hb, 7-Hb), 1.08 (*s*, 3H, 27-H), 1.08–0.95 (*m*, 3H, 1-Hb, 15-Hb, 20-H), 0.94 (*s*, 3H, 30-H), 0.93 (*s*, 3H, 25-H), 0.87 (*d*, *J* = 6.5 Hz, 3H, 29-H), 0.86 (*s*, 3H, 23-H), 0.84 (*s*, 3H, 24-H), 0.84–0.79 (*m*, 1H, 5-H), 0.75 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 180.0 (C-28), 171.1 (Ac), 167.0 (C-33), 139.7 (C-13), 125.9 (C-12), 81.0 (C-3), 55.4 (C-5), 53.8 (C-18), 48.0 (C-17), 47.6 (C-9), 42.6 (C-14), 42.6 (C-34), 41.3 (C-32), 39.9 (C-19), 39.7 (C-8), 39.2 (C-31), 39.2 (C-20), 38.4 (C-1), 37.8 (C-4), 37.4 (C-22), 37.0 (C-10), 32.8 (C-7), 31.0 (C-21), 28.2 (C-23), 27.9 (C-15), 24.9 (C-16), 23.7 (C-2), 23.5 (C-11), 23.4 (C-27), 21.4 (Ac), 21.3 (C-30), 18.3 (C-6), 17.4 (C-29), 17.0 (C-26), 16.8 (C-24), 15.7 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 617.3 (48%, [M + H]+), 639.5 (52%, [M + Na]+), 1255.4 (100%, [2M + Na]+); analysis calcd for C36H57ClN2O4 (617.31): C 70.04, H 9.31, N 4.54; found: C 69.83, H 9.52, N 4.11.

*(3*β*) N-(2-(2-(2-Chloroacetyl)aminoethoxy)ethyl)-3-acetyloxy-urs-12-en-28-amide* (**21**). Compound **21** has been synthesized from **18** according to general procedure C. Column chromatography (SiO2, CHCl3/acetone/hexanes 95:5:20) furnished compound **21** (91%); m.p. 94–97 ◦C; [α]D = +33.7◦ (*c* 0.335, CHCl3); Rf = 0.35 (CHCl3/acetone 9:1); IR (KBr): ν = 3426*br s*, 2928*m*, 2872*m*, 1734*m*, 1638*s*, 1528*m*, 1458*w*, 1386*w*, 1248*s*, 1124*w*, 1028*m* cm−1; 1H NMR (400 MHz, CDCl3): δ = 7.00–6.90 (*m*, 1H, NH), 6.22 (*t*, *J* = 5.0 Hz, 1H, NH), 5.30 (*t*, *J* = 3.6 Hz, 1H, 12-H), 4.49 (*dd*, *J* = 10.0, 5.7 Hz, 1H, 3-H), 4.06 (*s*, 2H, 36-H), 3.57–3.47 (*m*, 7H, 32-H, 31-Ha, 33-H, 34-H), 3.31–3.21 (*m*, 1H, 31-Hb), 2.04 (*s*, 3H, Ac), 2.02–1.76 (*m*, 5H, 16-Ha, 11-Ha, 11-Hb, 18-H, 22-Ha), 1.77–1.21 (*m*, 14H, 16-Hb, 15-Ha, 1-Ha, 2-Ha, 2-Hb, 9-H, 6-Ha, 21-Ha, 7-Ha, 22-Hb, 19-H, 6-Hb, 21-Hb, 7-Hb), 1.09 (*s*, 3H, 27-H), 1.08–0.95 (*m*, 3H, 1-Hb, 15-Hb, 20-H), 0.94 (*d*, *J* = 6.2 Hz, 3H, 30-H), 0.93 (*s*, 3H, 25-H), 0.88 (*d*, *J* = 6.2 Hz, 3H, 29-H), 0.86 (*s*, 3H, 23-H), 0.85 (*s*, 3H, 24-H), 0.84–0.80 (*m*, 1H, 5-H), 0.78 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 178.4 (C-28), 171.1 (Ac), 166.1 (C-35), 139.9 (C-13), 125.6 (C-12), 80.9 (C-3), 70.0 (C-33), 69.4 (C-32), 55.4 (C-5), 54.0 (C-18), 48.0 (C-17), 47.6 (C-9), 42.8 (C-36), 42.6 (C-14), 39.9 (C-29), 39.8 (C-34), 39.7 (C-8), 39.2 (C-31), 39.2 (C-20), 38.5 (C-1), 37.8 (C-4), 37.4 (C-22), 37.0 (C-10), 32.8 (C-7), 31.0 (C-21), 28.2 (C-23), 28.0 (C-15), 25.0 (C-16), 23.7 (C-2), 23.6 (C-11), 23.4 (C-27), 21.4 (Ac), 21.4 (C-30), 18.3 (C-6), 17.4 (C-29), 17.1 (C-26), 16.9 (C-24), 15.7 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 661.4 (60%, [M + H]+), 685.5 (86%, [M + Na]+), 1343.3 (100%, [2M + Na]<sup>+</sup>); analysis calcd for C38H61ClN2O5 (661.37): C 69.01, H 9.30, N 4.24, Cl 5.36; found: C 68.80, H 9.61, N 4.01.

*Tri-tert-butyl 2,2',2"-[10-[2-[4-(3*β*-acetyloxy-urs-12-en-28-oyl)piperazin-1-yl]-2-oxoethyl]-1,4,7,10 tetraazacyclododecane-1,4,7-triyl]triacetate* (**22**). Compound **22** was synthesized from **6** and **19** according to general procedure D. Column chromatography (SiO2, CHCl3/MeOH 95:5) furnished compound **22** (88%); m.p. 237–240 ◦C (decomp.); [α]D = +15.0◦ (c 0.3, CHCl3); Rf = 0.42 (CHCl3/MeOH 9:1); IR (ATR): ν = 2927*w*, 2928*w*, 2871*w*, 2829*w*, 1726*s*, 1646*m*, 1453*m*, 1424*w*, 1368*s*, 1305*m*, 1227*s*, 1160*s*, 1105*s*, 1004*m*, 975*m*, 754*m* cm<sup>−</sup>1; 1H NMR (400 MHz, CDCl3): δ = 5.19 (*t*, *J* = 3.5 Hz, 1H, 12-H), 4.47 (*dd*, *J* = 9.8, 6.0 Hz, 1H, 3-H), 3.93 – 2.05 (*m*, 32H, 31-H, 31 -H, 32-H, 32 -H, 8 × CH2 (cyclen), 3 × CH2 (acetate), 34-H), 2.40 (*d*, *J* = 11.0 Hz, 1H, 18-H), 2.19–2.09 (*m*, 1H, 16-Ha), 2.02 (*s*, 3H, Ac), 1.93–1.85 (*m*, 2H, 11-Ha, 11-Hb), 1.82–1.67 (*m*, 3H, 15-Ha, 16-Hb, 22-Ha), 1.65–1.21 (*m*, 12H, 2-Ha, 2-Hb, 22-Hb, 1-Ha, 9-H, 21-Ha, 6-Ha, 7-Ha, 19-H, 21-Hb, 6-Hb, 7-Hb), 1.43 (*s*, 9H, CH3 (*t*Butyl)), 1.42 (*s*, 9H; CH3 (*t*Butyl)), 1.42 (*s*, 9H, CH3 (*t*Butyl)), 1.10–0.96 (*m*, 3H, 1-Hb, 15-Hb, 20-H), 1.05 (*s*, 3H, 27-H), 0.92 (*d*, *J* = 6.3 Hz, 3H, 30-H), 0.91 (*s*, 3H, 25-H), 0.86 (*d*, *J* = 6.3 Hz, 3H, 29-H), 0.84 (*s*, 3H, 23-H), 0.82

(*s*, 3H, 24-H), 0.82–0.76 (*m*, 1H, 5-H), 0.71 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 175.9 (C-28), 172.8 (CO, acetate), 172.7 (CO, acetate), 171.0 (Ac), 170.7 (C-33), 138.7 (C-13), 125.1 (C-12), 81.9 (Cq, *t*Butyl), 81.7 (Cq, *t*Butyl), 81.7 (Cq, *t*Butyl), 81.0 (C-3), 55.8 (CH2, acetate), 55.8 (CH2, acetate), 55.7 (CH2, cyclen), 55.4 (C-5), 55.1 (C-18), 48.8 (C-17), 47.6 (C-9), 44.6 (C-31, C-31 , C-32, C-32 ), 42.3 (C-14), 41.7 (C-34), 39.5 (C-8), 39.4 (C-19), 38.8 (C-20), 38.3 (C-1), 37.8 (C-4), 37.0 (C-10), 34.4 (C-22), 33.1 (C-7), 30.5 (C-21), 28.3 (C-15), 28.2 (C-23), 28.1 (CH3, *t*Butyl), 28.0 (CH3, *t*Butyl), 23.8 (C-27), 23.6 (C-2, C-16), 23.4 (C-11), 21.4 (Ac), 21.3 (C-30), 18.2 (C-6), 17.5 (C-29), 17.0 (C-26), 16.8 (C-24), 15.6 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 1143.7 (100%, [M + Na]<sup>+</sup>); analysis calcd for C64H108N6O10 (1121.60): C 68.54, H 9.71, N 7.49; found: C 68.31, H 10.03, N 7.27.

*Tribenzyl 2,2',2"-[10-[2-[4-(3*β*-acetyloxy-urs-12-en-28-oyl)piperazin-1-yl]-2-oxoethyl]-1,4,7,10 tetraazacyclododecane-1,4,7-triyl]triacetate* (**23**). Compound **23** was synthesized from **19** and **7** according to general procedure D. Column chromatography (SiO2, CHCl3/MeOH 95:5) furnished compound **23** (72%); m.p. 157–161 ◦C; [α]D = +4.2◦ (c 0.300, CHCl3); Rf = 0.37 (CHCl3/MeOH 9:1); IR (ATR): ν = 2945*w*, 2836*w*, 1730*s*, 1638*m*, 1454*m*, 1424*w*, 1392*m*, 1370*m*, 1302*m*, 1243*s*, 1194*s*, 1105*s*, 1005*m*, 966*m*, 743*m*, 697*s* cm−1; UV-Vis (CHCl3): λmax (logε) = 246 nm (3.96), 294 nm (3.47), 364 nm (3.23); 1H NMR (400 MHz, CDCl3): <sup>δ</sup> = 7.39–7.26 (*m*, 15H, 15 <sup>×</sup> CH (Bn)), 5.33–4.97 (*m*, 7H, 12-H, 3 × CH2 (Bn)), 4.45 (*dd*, *J* = 10.1, 5.9 Hz, 1H, 3-H), 4.02–2.05 (*m*, 34H, 31-H, 31'-H, 32-H, 32'-H, 8 × CH2 (cyclen), 3 × CH2 (acetate), 34-H, 16-Ha, 18-H), 2.02 (*s*, 3H, Ac), 1.95–1.65 (*m*, 5H, 11-Ha, 11-Hb, 15-Ha, 16-Hb, 22-Ha), 1.65–1.09 (*m*, 12H, 2-Ha, 2-Hb, 22-Hb, 1-Ha, 9-H, 21-Ha, 6-Ha, 7-Ha, 19-H, 21-Hb, 6-Hb, 7-Hb), 1.03 (*s*, 3H, 27-H), 1.08–0.95 (*m*, 3H, 1-Hb, 15-Hb, 20-H), 0.93 (*d*, *J* = 6.0 Hz, 3H, 30-H), 0.85 (*d*, *J* = 6.3 Hz, 3H, 29-H), 0.85 (*s*, 3H, 25-H), 0.82 (*s*, 3H, 23-H), 0.79 (*s*, 3H, 24-H), 0.79–0.71 (*m*, 1H, 5-H), 0.66 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 175.9 (C-28), 173.6 (CO, acetate), 171.0 (Ac), 170.8 (C-33), 138.6 (C-13), 135.5 (Ci, Bn), 135.3 (Ci, Bn), 128.7 (CH, Bn), 128.7 (CH, Bn), 128.6 (CH, Bn), 128.6 (CH, Bn), 128.4 (CH, Bn), 128.4 (CH, Bn), 125.1 (C-12), 81.0 (C-3), 67.1 (CH2, Bn), 66.9 (CH2, Bn), 55.7 (CH2, acetate), 55.4 (CH2, cyclen), 55.2 (C-5), 55.1 (C-18), 48.7 (C-17), 47.6 (C-9), 44.8 (C-31, C-31', C-32, C-32'), 42.2 (C-14), 42.0 (C-34), 39.5 (C-19), 39.5 (C-8), 38.8 (C-20), 38.3 (C-1), 37.7 (C-4), 36.9 (C-10), 34.5 (C-22), 33.1 (C-7), 30.6 (C-21), 28.2 (C-15), 28.1 (C-23), 23.6 (C-2, C-16), 23.5 (C-27), 23.4 (C-11), 21.4 (Ac), 21.3 (C-30), 18.2 (C-6), 17.5 (C-29), 17.0 (C-26), 16.8 (C-24), 15.5 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 1245.8 (100%, [M + Na]<sup>+</sup>); analysis calcd for C73H102N6O10 (1223.65): C 71.65, H 8.40, N 6.87; found: C 71.42, H 8.69, N 6.56.

*Tri-tert-butyl 2,2',2"-[10-[2-[2-(3*β*-acetyloxy-urs-12-en-28-oylamino)ethyl]amino-2-oxoethyl]-1,4,7,10 tetraazacyclododecane-1,4,7-triyl]triacetate* (**24**). Compound **24** was synthesized from **20** and **6** according to general procedure D. Column chromatography (SiO2, CHCl3/MeOH 95:5) furnished compound **24** (73%); m.p. 254–257 ◦C (decomp.); [α]D = +33.8◦ (c 0.300, CHCl3); Rf = 0.40 (CHCl3/MeOH 9:1); IR (KBr): ν = 2971*m*, 2929*m*, 2829*w*, 1727*s*, 1668*m*, 1520*m*, 1454*m*, 1426*w*, 1368*s*, 1307*m*, 1228*s*, 1159*s*, 1106*s*, 1027*m*, 1006*m*, 975*m*, 755*m* cm<sup>−</sup>1; 1H NMR (400 MHz, CDCl3): δ = 8.03 (*s*, 1H, NH), 7.08 (*s*, 1H, NH), 5.46 (*t*, *J* = 3.4 Hz, 1H, 12-H), 4.47 (*dd*, *J* = 9.8, 6.1 Hz, 1H, 3-H), 3.80–2.05 (*m*, 28H, 31-H, 32-H, 34-H, 3 × CH2 (acetate), 8 × CH2 (cyclen)), 2.44–2.38 (*m*, 1H, 18-H), 2.02 (*s*, 3H, Ac), 2.00–1.67 (*m*, 6H, 15-Ha, 11-Ha, 11-Hb, 16-Ha, 16-Hb, 22-Ha), 1.66–1.19 (*m*, 12H, 1-Ha, 2-Ha, 2-Hb, 22-Hb, 9-H, 6-Ha, 7-Ha, 21-Ha, 19-H, 6-Hb, 21-Hb, 7-Hb), 1.44 (*s*, 9H, CH3 (*t*Butyl)), 1.43 (*s*, 9H, CH3 (*t*Butyl)), 1.43 (*s*, 9H, CH3 (*t*Butyl)), 1.15–0.98 (*m*, 3H, 20-H, 1-Hb, 15-Hb), 1.04 (*s*, 3H, 27-H), 0.91 (*s*, 3H, 25-H), 0.90 (*d*, *J* = 6.1 Hz, 3H, 30-H), 0.88 (*d*, *J* = 6.1 Hz, 3H, 29-H), 0.84 (*s*, 3H, 23-H), 0.83 (*s*, 3H, 24-H), 0.82–0.77 (*m*, 1H, 5-H), 0.74 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 178.7 (C-28), 172.4 (CO, acetate), 171.7 (C-33), 171.1 (Ac), 139.0 (C-13), 125.5 (C-12), 82.1 (Cq, *t*Butyl), 82.1 (Cq, *t*Butyl), 82.1 (Cq, *t*Butyl), 81.1 (C-3), 56.4 (3 × CH2, acetate), 55.8 (C-34), 55.8 (CH2, cyclen), 55.4 (C-5), 52.3 (C-18), 47.7 (C-9), 47.6 (C-17), 42.2 (C-14), 40.0 (C-32), 39.7 (C-19), 39.7 (C-8), 38.5 (C-20), 38.4 (C-31, C-1), 37.8 (C-4), 37.4 (C-22), 37.0 (C-10), 32.9 (C-7), 31.2 (C-21), 28.2 (C-23), 28.2 (CH3, *t*Butyl), 28.1 (CH3, *t*Butyl), 28.1 (CH3, *t*Butyl), 27.9 (C-15), 24.4 (C-16), 23.7 (C-2), 23.5 (C-27), 23.4 (C-11), 21.5 (C-30), 21.4 (Ac), 18.4 (C-6), 17.2 (C-29), 17.0

(C-26), 16.9 (C-24), 15.7 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 1117.7 (100%, [M + Na]+); analysis calcd for C62H106N6O10 (1095.56): C 67.97, H 9.75, N 7.67; found: C 67.68, H 10.02, N 7.41.

*Tribenzyl 2,2',2"-[10-[2-[2-(3*β*-acetyloxy-urs-12-en-28-oylamino)ethyl]amino-2-oxoethyl]-1,4,7,10 tetraazacyclododecane-1,4,7-triyl]triacetate* (**25**). Compound **25** was synthesized from **7** and **20** according to general procedure D. Column chromatography (SiO2, CHCl3/MeOH 95:5) furnished compound **25** (75%); m.p. 142–146 ◦C; [α]D = +20.5◦ (c 0.390, CHCl3); Rf = 0.36 (CHCl3/MeOH 9:1); IR (ATR): ν = 2945*w*, 2832*w*, 1732*s*, 1663*m*, 1519*w*, 1454*m*, 1370*m*, 1305*m*, 1245*s*, 1194*s*, 1176*s*, 1105*s*, 1007*m*, 965*m*, 747*m*, 697*s* cm−1; UV-Vis (CHCl3): λmax (logε) = 241 nm (3.89), 295 nm (3.29), 364 nm (3.08); 1H NMR (400 MHz, CDCl3): δ = 8.20 (*t*, *J* = 4.5 Hz, 1H, NH), 7.39–7.27 (*m*, 15H, CH (Bn)), 7.01 (*t*, *J* = 4.9 Hz, 1H, NH), 5.48 (*s*, 1H, 12-H), 5.26–5.05 (*m*, 6H, CH2 (Bn)), 4.47 (*dd*, *J* = 10.0, 5.7 Hz, 1H, 3-H), 3.77–2.07 (*m*, 28H, 31-Ha, 31-Hb, 32-H, 34-H, 3 × CH2 (acetate),8 × CH2 (cyclen)), 2.41 (*d*, *J* = 10.7 Hz, 1H, 18-H), 2.02 (*s*, 3H, Ac), 2.00–1.64 (*m*, 6H, 16-Ha, 16-Hb, 11-Ha, 11-Hb, 22-Ha, 15-Ha), 1.64–1.19 (*m*, 12H, 1-Ha, 2-Ha, 2-Hb, 22-Hb, 9-H, 6-Ha, 7-Ha, 21-Ha, 6-Hb, 19-H, 21-Hb, 7-Hb), 1.17–0.97 (*m*, 3H, 20-H, 1-Hb, 15-Hb), 1.04 (*s*, 3H, 27-H), 0.89 (*d*, *J* = 6.3 Hz, 6H, 29-H, 30-H), 0.87 (*s*, 3H, 25-H), 0.83 (*s*, 3H, 23-H), 0.81 (*s*, 3H, 24-H), 0.80–0.76 (*m*, 1H, 5-H), 0.72 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 178.8 (C-28), 173.2 (CO, acetate), 172.0 (C-33), 171.0 (Ac), 139.0 (C-13), 135.4 (Ci, Bn), 135.4 (Ci, Bn), 135.3 (Ci, Bn), 128.8 (CH, Bn), 128.8 (CH, Bn), 128.7 (CH, Bn), 128.6 (CH, Bn), 125.6 (12-H), 81.0 (3-H), 67.3 (CH2, Bn), 67.3 (CH2, Bn), 67.2 (CH2, Bn), 56.8 (CH2, acetate), 55.4 (C-34), 55.3 (C-5), 55.3 (CH2, cyclen), 52.3 (C-18), 47.7 (C-17), 47.6 (C-9), 42.2 (C-14), 40.1 (C-32), 39.8 (C-19), 39.7 (C-8), 38.5 (C-20), 38.5 (C-31, C-1), 37.8 (C-4), 37.4 (C-22), 37.0 (C-10), 32.9 (C-7), 31.2 (C-21), 28.2 (C-23), 28.0 (C-15), 24.5 (C-16), 23.7 (C-2), 23.5 (C-27), 23.4 (C-11), 21.4 (Ac), 21.4 (C-30), 18.4 (C-6), 17.2 (C-29), 17.1 (C-26), 16.8 (C-24), 15.6 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 1219.8 (100%, [M + Na]+); analysis calcd for C71H100N6O10 (1197.61): C 71.21, H 8.42, N 7.02; found: 70.93, H 8.56, N 6.82.

*Tri-tert-butyl 2,2',2"-[10-[2-[2-[2-(3*β*-acetyloxy-urs-12-en-28-oylamino)ethoxy]ethyl]amino-2-oxoethyl]-1,4,7,10 tetraazacyclododecane-1,4,7-triyl]triacetate* (**26**). Compound **26** was synthesized from **6** and **21** according to general procedure D. Column chromatography (SiO2, CHCl3/MeOH 95:5) furnished compound **26** (74%); m.p. 263–266 ◦C (decomp.); [α]D = +21.9◦ (c 0.315, CHCl3); Rf = 0.38 (CHCl3/MeOH 9:1); IR (ATR): ν = 2972*m*, 2930*m*, 1726*s*, 1166*m*, 1523*w*, 1453*m*, 1368*s*, 1307*m*, 1228*s*, 1160*s*, 1106*s*, 1026*m*, 1006*m*, 975*m*, 755*m* cm−1; 1H NMR (400 MHz, CDCl3): δ = 7.82 (*t*, *J* = 5.6 Hz, 1H, NH), 6.46 (*t*, *J* = 5.1 Hz, 1H, NH), 5.33 (*t*, *J* = 3.7 Hz, 1H, 12-H), 4.47 (*dd*, *J* = 10.2, 5.9 Hz, 1H, 3-H), 3.56–3.30 (*m*, 9H, 32-H, 31-Ha, 33-H, 36-H, 34-H), 3.28–3.16 (*m*, 1H, 31-Hb), 3.15–2.05 (*m*, 22H, 3 × CH2 (acetate), 8 × CH2 (cyclen)), 2.02 (*s*, 3H, Ac), 2.02–1.21 (*m*, 19H, 18-H, 16-Ha, 11-Ha, 11-Hb, 22-Hb, 16-Hb, 15-Ha, 1-Ha, 2-Ha, 2-Hb, 9-H, 6-Ha, 21-Ha, 7-Ha, 22-Hb, 19-H, 6-Hb, 21-Hb, 7-Hb), 1.44 (*s*, 9H, CH3 (*t*Butyl)), 1.43 (*s*, 18H, CH3 (*t*Butyl)), 1.06 (*s*, 3H, 27-H), 1.05–0.94 (*m*, 3H, 1-Hb, 15-Hb, 20-H), 0.92 (*s*, 3H, 25-H), 0.92 (*d*, *J* = 6.2 Hz, 3H, 30-H), 0.86 (*d*, *J* = 6.5 Hz, 3H, 29-H), 0.84 (*s*, 3H, 23-H), 0.83 (*s*, 3H, 24-H), 0.82–0.77 (*m*, 1H, 5-H), 0.76 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 178.3 (C-28), 173.0 (CO, acetate), 172.5 (CO, acetate), 172.1 (C-35), 171.1 (Ac), 139.3 (C-13), 125.7 (C-12), 82.1 (Cq, *t*Butyl), 82.0 (Cq, *t*Butyl), 81.9 (Cq, *t*Butyl), 81.0 (C-3), 69.5 (C-33), 69.0 (C-32), 56.5 (C-36), 55.9 (CH2, acetate), 55.8 (CH2, acetate), 55.7 (CH2, cyclen), 55.4 (C-5), 53.4 (C-18), 47.8 (C-17), 47.6 (C-9), 42.4 (C-14), 39.8 (C-19), 39.7 (C-8), 39.2 (C-31), 39.0 (C-34), 39.0 (C-20), 38.4 (C-1), 37.8 (C-4), 37.3 (C-22), 37.0 (C-10), 32.9 (C-7), 31.1 (C-21), 28.2 (CH3, *t*Butyl), 28.0 (CH3, *t*Butyl), 28.0 (C-23), 28.0 (C-15), 24.9 (C-16), 23.7 (C-2), 23.5 (C-11), 23.4 (C-27), 21.4 (Ac), 21.4 (C-30), 18.3 (C-6), 17.3 (C-29), 17.0 (C-26), 16.8 (C-24), 15.7 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 1161.7 (100%, [M + Na]<sup>+</sup>); analysis calcd for C64H110N6O11 (1139.6): C 67.45, H 9.73, N 7.37; found: C 67.31, H 9.87, N 7.09.

*Tribenzyl 2,2',2"-[10-[2-[2-[2-(3*β*-acetyloxy-urs-12-en-28-oylamino)ethoxy]ethyl]amino-2-oxoethyl]-1,4,7,10 tetraazacyclododecane-1,4,7-triyl]triacetate* (**27**). Compound **27** has been synthesized from **7** and **21** according to general procedure D. Column chromatography (SiO2, CHCl3/MeOH 95:5) furnished compound **27** (62%); m.p. 137–141 ◦C; [α]D = +15.4◦ (c 0.350, CHCl3); Rf = 0.35 (CHCl3/MeOH 9:1); IR (ATR): ν = 2945*w*, 2830*w*, 1731*s*, 1662*m*, 1523*w*, 1454*m*, 1390*m*, 1370*m*, 1305*m*, 1245*s*, 1193*s*, 1105*s*,

1026*m*, 1008*m*, 967*m*, 747*m*, 697*m* cm−1; UV-Vis (CHCl3): λmax (logε) = 244 nm (3.73), 294 nm (3.36), 363 nm (3.12); 1H NMR (400 MHz, CDCl3): δ = 8.02 (*t*, *J* = 5.4 Hz, 1H, NH), 7.37–7.27 (*m*, 15H, CH (Bn)), 6.48 (*t*, *J* = 5.1 Hz, 1H, NH), 5.32 (*t*, *J* = 3.5 Hz, 1H, 12-H), 5.24–5.14 (*m*, 4H, CH2 (Bn)), 5.14–5.05 (*m*, 2H, CH2 (Bn)), 4.46 (*dd*, *J* = 10.2, 5.7 Hz, 1H, 3-H), 3.59–3.33 (*m*, 9H, 32-H, 33-H, 31-Ha, 34-H, 36-H), 3.33–2.01 (*m*, 23H, 31-Hb, 8 × CH2 (cyclen), 3 × CH2 (acetate)), 2.02 (*s*, 3H, Ac), 2.00–1.17 (*m*, 19H, 18-H, 16-Ha, 11-Ha, 11-Hb, 22-Ha, 16-Hb, 15-Ha, 1-Ha, 2-Ha, 2-Hb, 9-H, 6-Ha, 22-Hb, 7-Ha, 21-Ha, 19-H, 6-Hb, 7-Hb, 21-Hb), 1.05 (*s*, 3H, 27-H), 1.04 – 0.94 (*m*, 3H, 1-Hb, 15-Hb, 20-H), 0.90 (*s*, 3H, 25-H), 0.90 (*d*, *J* = 5.8 Hz, 3H, 30-H), 0.85 (*d*, *J* = 6.6 Hz, 3H, 29-H), 0.84 (*s*, 3H, 23-H), 0.82 (*s*, 3H, 24-H), 0.81–0.76 (*m*, 1H, 5-H), 0.75 (*s*, 3H, 26-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 178.3 (C-28), 173.3 (CO, acetate), 173.1 (CO, acetate), 172.3 (C-35), 171.1 (Ac), 139.3 (C-13), 135.4 (Ci, Bn), 135.3 (Ci, Bn), 128.7 (CH, Bn), 128.7 (CH, Bn), 128.7 (CH, Bn), 128.6 (CH, Bn), 128.5 (CH, Bn), 125.7 (C-12), 81.0 (C-3), 69.6 (C-33), 68.9 (C-32), 67.2 (CH2, Bn), 67.2 (CH2, Bn), 56.9 (C-36), 55.4 (3 × CH2, acetate), 55.4 (C-5), 55.3 (8 × CH2, cyclen), 53.3 (C-18), 47.8 (C-17), 47.6 (C-9), 42.4 (C-14), 39.8 (C-19), 39.7 (C-8), 39.2 (C-31, C-34), 38.9 (C-20), 38.4 (C-1), 37.8 (C-4), 37.3 (C-22), 36.9 (C-10), 32.9 (C-7), 31.1 (C-21), 28.2 (C-23), 28.0 (C-15), 24.8 (C-16), 23.6 (C-2), 23.5 (C-11), 23.4 (C-27), 21.4 (Ac), 21.4 (C-30), 18.3 (C-6), 17.3 (C-29), 17.1 (C-26), 16.8 (C-24), 15.7 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 1263.9 (100%, [M + Na]+); analysis calcd for C73H104N6O11 (1241.67): C 70.62, H 8.44, N 6.77; found: C 70.41, H 8.69, N 6.41.

*(3*β*) N-(2-Aminoethyl)-3-hydroxy-urs-12-en-28-amide* (**28**). Method A: The synthesis was performed according to the procedure given in the Supplementary material in 82%. *Method B*: Ursolic acid (100 mg, 0.20 mmol), HOBt·H2O (37 mg, 0.24 mmol) and EDC·HCl (46 mg, 0.24 mmol) were dissolved in dry DMF (5 mL), and the mixture was stirred for 30 min at 25 ◦C. Ethylene diamine (55 μL, 0.82 mmol) was added to the mixture, and stirring was continued for 24 h at 25 ◦C. Usual aqueous work-up followed by column chromatography (silica gel, CHCl3/MeOH/NH4OH 90:10:0.1) gave **28** (46%). Analytical data of this compound can be found in the supplementary material.

*Tri-tert-butyl 2,2',2"-[10-[2-[2-(3*β*-hydroxy-urs-12-en-28-oylamino)ethyl]amino-2-oxoethyl]-1,4,7,10 tetraazacyclododecane-1,4,7-triyl]triacetate* (**29**). Method A: To a solution of DOTA-tris(tert-butyl ester) (58 mg, 0.10 mmol) in dry DMF (8 mL) were added HOBt·H2O (29 mg, 0.19 mmol) and EDC·HCl (29 mg, 0.15 mmol). After stirring for 30 min at 25 ◦C, a solution of **28** (71 mg, 0.14 mmol) in dry DMF (2 mL) was added and stirring was continued for 5 days. After usual aqueous work-up, the solvent was removed under reduced pressure and the crude product was subjected to column chromatography (silica gel, CHCl3/MeOH 9:1) yielding compound **29** as colorless solid. Yield: 49%. Method B: Compound **24** (50 mg, 0.10 mmol) was dissolved in methanol (7 mL) and a solution of potassium hydroxide (12 mg, 0.21 mmol) in methanol (1 mL) was added. The mixture was stirred at 25 ◦C for 24 h. After completion of the reaction (as indicated by TLC) and usual work-up, the solvent was removed under reduced pressure, and the residue was subjected to column chromatography (silica gel, CHCl3/MeOH 9:1) affording **29** (yield: 86%); m.p. 136–139 ◦C; [α]D = −44.4◦ (c 0.330, MeOH); Rf = 0.29 (CHCl3/MeOH 9:1); IR (KBr): ν = 3300*br w*, 2973*w*, 2928*m*, 2869*w*, 1728*s*, 1668*m*, 1525*m*, 1455*m*, 1425*w*, 1368*s*, 1307*m*, 1227*s*, 1159*s*, 1121*m*, 1106*s*, 1047*w*, 1006*w* cm−1; 1H NMR (400 MHz, CDCl3): δ = 8.84 (*s*, 1H, NH), 7.55 (*s*, 1H, NH), 5.36 (*t*, *J* = 3.5 Hz, 1H, 12-H), 3.59–2.05 (*m*, 28H, 31-H, 32-H, 33-H, 3 × CH2 (acetate), 8 × CH2 (cyclen)), 3.20 (*dd*, *J* = 11.1, 4.8 Hz, 1H, 3-H), 2.34 (*d*, *J* = 11.0 Hz, 2H, 18-H), 2.03–1.96 (*m*, 1H, 16-Ha), 1.95–1.83 (*m*, 3H, 11-Ha, 11-Hb, 16-Hb), 1.83–1.72 (*m*, 1H, 22-Ha), 1.64–1.21 (*m*, 13H, 1-Ha, 15-Ha, 2-Ha, 2-Hb, 22-Hb, 6-Ha, 9-H, 7-Ha, 21-Ha, 19-H, 6-Hb, 7-Hb, 21-Hb), 1.45 (*s*, 9H, CH3 (*t*Butyl)), 1.43 (*s*, 18H, CH3 (*t*Butyl)), 1.05 (*s*, 3H, 27-H), 1.04–0.97 (*m*, 3H, 1-Hb, 15-Hb, 20-H), 0.97 (*s*, 3H, 23-H), 0.89 (*d*, *J* = 6.3 Hz, 3H, 30-H), 0.89 (*s*, 3H, 25-H), 0.85 (*d*, *J* = 6.4 Hz, 3H, 29-H), 0.76 (*s*, 3H, 24-H), 0.75 (*s*, 3H, 26-H), 0.72–0.68 (*m*, 1H, 5-H) ppm; 13C NMR (101 MHz, CDCl3): δ = 178.4 (C-28), 172.4 (CO, acetate), 171.8 (C-33), 138.9 (C-13), 125.3 (C-12), 82.2 (Cq, *t*Butyl), 82.1 (Cq, *t*Butyl), 82.1 (Cq, *t*Butyl), 79.2 (C-3), 56.0 (CH2, acetate), 55.9 (C-34), 55.8 (CH2, cyclen), 55.3 (C-5), 52.5 (C-18), 47.8 (C-9), 47.5 (C-17), 42.2 (C-14), 40.0 (C-32), 39.7 (C-8), 39.6 (C-19), 38.9 (C-4), 38.7 (C-20), 38.7 (C-31, C-1), 37.4 (C-22), 37.1 (C-10), 33.2 (C-7), 31.3 (C-21), 28.3 (C-23), 28.2 (CH3, *t*Butyl), 28.2 (CH3, *t*Butyl), 28.1 (CH3,

*t*Butyl), 28.0 (C-15), 27.4 (C-2), 24.2 (C-16), 23.6 (C-27), 23.5 (C-11), 21.5 (C-30), 18.6 (C-6), 17.2 (C-29), 17.1 (C-26), 15.8 (C-24), 15.7 (C-25) ppm; MS (ESI, MeOH): *m*/*z* = 1075.7 (100%, [M + Na]+); analysis calcd for C60H104N6O9 (1053.53): C 68.40, H 9.95, N 7.98; found: C 60.09, H 10.11, N 7.83.

**Supplementary Materials:** Supplementary data related to this article including experimental procedures for compounds **10**, **17**, **18**, and **28**, Figure S1: Extended cytotoxicity investigation after treatment of A375 cells with **22** (3.0 μM) for 24 h, representative NMR spectra and calculation of ADMET parameters for compounds **22** and **24** can be found online.

**Author Contributions:** M.K.; and R.C. conceived and designed the experiments; M.K. performed the experiments; S.H. and L.F. performed the biological assays and experiments; M.K.; A.A-H.; and R.C. analyzed the data and wrote the paper.

**Funding:** We acknowledge the financial support within the funding program Open Access Publishing by the German Research Foundation (DFG).

**Acknowledgments:** We would like to thank R. Kluge for measuring the ESI-MS spectra and D. Ströhl and his team for the NMR spectra. Thanks are also due to V. Simon for measuring the IR an UV-VIS spectra and optical rotations. The cell lines were kindly provided by Th. Müller (Dept. of Haematology/Oncology, Martin-Luther Universität Halle-Wittenberg).

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


**Sample Availability:** Samples of all compounds are available from the authors.

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*Article*
