**1. Introduction**

Reactive oxygen species (ROS) are involved in numerous processes, which mediate physiological and pathophysiological signal transductions. Upon unregulated increased ROS production, redox imbalances occur, which cause atherosclerosis, cardiovascular diseases, hypertension, diabetes mellitus, neurodegenerative and immune-inflammatory diseases. In addition, the impact of oxidants and antioxidants in tumor cell proliferation is observed frequently. On the molecular level, ROS causes oxidative stress, which is responsible for damaging cell structures by acting on lipids, membranes, proteins, and DNA. This behavior of ROS in cancer cells, in particular, offers a basis for the prevention of tumor progression and metastasis by ROS scavengers [1,2]. Therefore, antioxidant therapies are sought to selectively inhibit the growth of tumor cells to induce cellular

**Citation:** Sultani, H.N.; Morgan, I.; Hussain, H.; Roos, A.H.; Haeri, H.H.; Kaluderovi´c, G.N.; Hinderberger, D.; ¯ Westermann, B. Access to New Cytotoxic Triterpene and Steroidal Acid-TEMPO Conjugates by Ugi Multicomponent-Reactions. *Int. J. Mol. Sci.* **2021**, *22*, 7125. https://doi.org/10.3390/ijms 22137125

Academic Editor: Paola Perego

Received: 11 May 2021 Accepted: 25 June 2021 Published: 1 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

differentiation and to alter the intracellular redox state [3]. Besides this, antioxidative food supplements enhance the positive outcome of conventional cancer treatments. In the context of cancer therapy, nitroxide-modified natural products have been shown to be useful due to their ability to remove superoxide anions, trap carbon-centered radicals, or to terminate chain reactions [4]. Furthermore, these derivatizations have been applied in bioreductive drugs to initiate additional cytotoxic events, which make them useful as antitumor drugs [5]. [16,17]. Fusidic acid (FA, **2**) is a triterpene acid that belongs to the family of tetracyclic fusidane nor-triterpenes and has been clinically employed as an antibiotic for staphylococcal infections [18]. Additionally, it has been reported that fusidic acid sodium salt, an approved bacteriostatic antibiotic, showed significant cytotoxic effects (in vitro and in vivo) towards various colon cancer cells alone or coupled with 5-fluorouracil [19,20]. Moreover, various studies have been reported that cholic acid (CA, **3**), which is steroidal acid, can be used for the prevention and treatment of colon cancer [21,22].

therapies are sought to selectively inhibit the growth of tumor cells to induce cellular differentiation and to alter the intracellular redox state [3]. Besides this, antioxidative food supplements enhance the positive outcome of conventional cancer treatments. In the context of cancer therapy, nitroxide-modified natural products have been shown to be useful due to their ability to remove superoxide anions, trap carbon-centered radicals, or to terminate chain reactions [4]. Furthermore, these derivatizations have been applied in bioreductive drugs to initiate additional cytotoxic events, which make them useful as

Betulinic acid (BA, **1**, Figure 1) and its congeners are well known for their abilities to act as natural cytotoxic products [6–10]. These triterpenes have been modified quite extensively providing products with enhanced biological activities [11–15]. Modifications of these lupane triterpenoids with nitroxyl radicals have been shown to produce a positive outcome on the cytotoxic activity on several cell lines (e.g., CEM13, U937, MT4)

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 2 of 19

antitumor drugs [5].

Betulinic acid (BA, **1**, Figure 1) and its congeners are well known for their abilities to act as natural cytotoxic products [6–10]. These triterpenes have been modified quite extensively providing products with enhanced biological activities [11–15]. Modifications of these lupane triterpenoids with nitroxyl radicals have been shown to produce a positive outcome on the cytotoxic activity on several cell lines (e.g., CEM13, U937, MT4) [16,17]. Fusidic acid (FA, **2**) is a triterpene acid that belongs to the family of tetracyclic fusidane nor-triterpenes and has been clinically employed as an antibiotic for staphylococcal infections [18]. Additionally, it has been reported that fusidic acid sodium salt, an approved bacteriostatic antibiotic, showed significant cytotoxic effects (in vitro and in vivo) towards various colon cancer cells alone or coupled with 5-fluorouracil [19,20]. Moreover, various studies have been reported that cholic acid (CA, **3**), which is steroidal acid, can be used for the prevention and treatment of colon cancer [21,22]. Based on these promising cytotoxic effects of the natural products **1**–**3**, we prepared a library of BA (**1**), FA (**2**), CA (**3**) conjugates with TEMPO (nitroxide) by utilizing an Ugi multicomponent reaction approach (U-4CR) with the aim of enhancing the cytotoxic potentials of our conjugates. Although only a few reports have been published about the U-4CR modifications on BA (**1**) as anti-inflammatory agents [23,24], none of them investigated the fusion to nitroxide. In a previous communication, we demonstrated that the U-4CR strategy is very well suited to achieve spin-labelled products [25]. In the present study, we use an amino spin-label viz.: 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (**4**, 4-NH2-TEMPO) as a U-4CR counterpart (Scheme 1) allowing for the preparation of the natural acid-TEMPO adducts. The spin-labelled FA derivative **8** acts strongly against two investigated cancer cell lines of prostate cancer (PC3) and colon cancer (HT29) and induces apoptosis by a caspase-dependent mechanism.

**Figure 1. Figure 1.**  Structures of Betulinic acid ( Structures of Betulinic acid ( **1 1** ), Fusidic acid ( ), Fusidic acid ( **2 2** ), and Cholic acid ( ), and Cholic acid (**3 3** ). ).

Based on these promising cytotoxic effects of the natural products **1**–**3**, we prepared a library of BA (**1**), FA (**2**), CA (**3**) conjugates with TEMPO (nitroxide) by utilizing an Ugi multicomponent reaction approach (U-4CR) with the aim of enhancing the cytotoxic potentials of our conjugates. Although only a few reports have been published about the U-4CR modifications on BA (**1**) as anti-inflammatory agents [23,24], none of them investigated the fusion to nitroxide. In a previous communication, we demonstrated that the U-4CR strategy is very well suited to achieve spin-labelled products [25]. In the present study, we use an amino spin-label viz.: 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (**4**, 4-NH2-TEMPO) as a U-4CR counterpart (Scheme 1) allowing for the preparation of the natural acid-TEMPO adducts. The spin-labelled FA derivative **8** acts strongly against two investigated cancer cell lines of prostate cancer (PC3) and colon cancer (HT29) and induces apoptosis by a caspase-dependent mechanism.

**Scheme 1.** Synthesis of terpenoic acid-TEMPO adducts **6**–**12**. **Scheme 1.** Synthesis of terpenoic acid-TEMPO adducts **6**–**12**.

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

#### *2.1. Chemistry 2.1. Chemistry*

The general synthetic pathway for the preparation of natural acids **1**–**3**-based Ugi products **6**–**12** is outlined in Scheme 1. Natural acid-TEMPO adducts **6**–**11** were synthesized in a single step operation by utilizing the Ugi four-component reaction. These compounds were prepared in moderate to good yields (57–81%) by the reaction of BA (**1**), FA (**2**), or CA (**3**) as the acid component (A), convertible IPB isonitrile **5** [26], or *t*-butyl isocyanide as isonitrile component (B), 4-NH2-TEMPO as amine (C), and paraformaldehyde (D) in the presence of MeOH. Encouraged by our previous results [25] that spin-label TEMPO is not affected under the reaction conditions of the U-4CR, we plan to couple nitroxide comprising amine viz.: 4-NH2-TEMPO (**4**; as amine component) to enhance the cytotoxicity of natural acids **1**–**3**. Moreover, BA (**1**), FA (**2**), and CA (**3**) have a tertiary carboxylic acid, vinyl carboxylic acid, and secondary carboxylic acid groups respectively. We found that the alteration of these acids did not play any significant role in The general synthetic pathway for the preparation of natural acids **1**–**3**-based Ugi products **6**–**12** is outlined in Scheme 1. Natural acid-TEMPO adducts **6**–**11** were synthesized in a single step operation by utilizing the Ugi four-component reaction. These compounds were prepared in moderate to good yields (57–81%) by the reaction of BA (**1**), FA (**2**), or CA (**3**) as the acid component (A), convertible IPB isonitrile **5** [26], or *t*-butyl isocyanide as isonitrile component (B), 4-NH2-TEMPO as amine (C), and paraformaldehyde (D) in the presence of MeOH. Encouraged by our previous results [25] that spin-label TEMPO is not affected under the reaction conditions of the U-4CR, we plan to couple nitroxide comprising amine viz.: 4-NH2-TEMPO (**4**; as amine component) to enhance the cytotoxicity of natural acids **1**–**3**. Moreover, BA (**1**), FA (**2**), and CA (**3**) have a tertiary carboxylic acid, vinyl carboxylic acid, and secondary carboxylic acid groups respectively. We found that the alteration of these acids did not play any significant role in the product yields. To

the product yields. To introduce further chemical diversity via the Ugi synthetic proce-

introduce further chemical diversity via the Ugi synthetic procedure, we additionally prepared fusidic acid-based Ugi product **12** by utilizing Yudin's fluorescent isocyanide [27]. dure, we additionally prepared fusidic acid-based Ugi product **12** by utilizing Yudin's fluorescent isocyanide [27].

In order to expand the diversity of the Ugi products synthesized, the advantage was taken of the isonitrile functionality as illustrated for compounds **7** and **9** (Scheme 2). As demonstrated earlier [26], the secondary amide can be transformed upon acidic treatment to acyl pyrroles, which can easily be cleaved by nucleophiles. Thus, in the presence of camphor sulfonic acid (CSA) both Ugi-products **7** and **9** were transformed to the corresponding acyl pyrroles **13** and **15**, which upon treatment with KOH were converted into the corresponding carboxylates **14** and **16**. However, no selectivity could be obtained for the acetyl moiety in the fusidic acid derivative **15** since the ester moiety (C-16 acetyl group) was cleaved as well, as expected under these conditions. To achieve selectivity in the displacement of the acyl pyrrole, an alternative procedure (DMAP, H2O/*t*-BuOH) was successful and furnished the C-16 acetyl fusidic acid analog **17**. For biological evaluation, we envisioned the preparation of a conjugate with a triphenylphosphine moiety, since this moiety is known to selectively bind to mitochondria membranes. Again, the U-4CR proved to be the synthetic protocol of choice, since in a single step not only the triphenylphosphine moiety but a dye (Yudin's dye/Yudin's isonitrile) can be assembled to form the product in the same synthetic process yielding the fusidic acid analog **18**. In order to expand the diversity of the Ugi products synthesized, the advantage was taken of the isonitrile functionality as illustrated for compounds **7** and **9** (Scheme 2). As demonstrated earlier [26], the secondary amide can be transformed upon acidic treatment to acyl pyrroles, which can easily be cleaved by nucleophiles. Thus, in the presence of camphor sulfonic acid (CSA) both Ugi-products **7** and **9** were transformed to the corresponding acyl pyrroles **13** and **15**, which upon treatment with KOH were converted into the corresponding carboxylates **14** and **16**. However, no selectivity could be obtained for the acetyl moiety in the fusidic acid derivative **15** since the ester moiety (C-16 acetyl group) was cleaved as well, as expected under these conditions. To achieve selectivity in the displacement of the acyl pyrrole, an alternative procedure (DMAP, H*2*O/*t*-BuOH) was successful and furnished the C-16 acetyl fusidic acid analog **17**. For biological evaluation, we envisioned the preparation of a conjugate with a triphenylphosphine moiety, since this moiety is known to selectively bind to mitochondria membranes. Again, the U-4CR proved to be the synthetic protocol of choice, since in a single step not only the triphenylphosphine moiety but a dye (Yudin's dye/Yudin's isonitrile) can be assembled to form the product in the same synthetic process yielding the fusidic acid analog **18**.

*2.2. Characterization of the Nitroxide Conjugated Compounds by Electron Paramagnetic* 

**Scheme 2.** Synthesis of compounds **13**–**18**. **Scheme 2.** Synthesis of compounds **13**–**18**.

*Resonance (EPR) Spectroscopy* 

#### *2.2. Characterization of the Nitroxide Conjugated Compounds by Electron Paramagnetic Resonance (EPR) Spectroscopy* correlation times (), as a simple measure of nitroxide rotational dynamics were monitored and were found to be between 1–2 ns for the synthesized nitroxide adducts, in good

The EPR characteristics, mainly the isotropic hyperfine coupling (Aiso) and isotropic rotational correlation times (), dominate solution-state EPR spectra as a measure of the line spacing and line shape, were obtained by simulations using the Easyspin software package [28]. All synthesized nitroxide adducts show hyperfine couplings (Aiso~45–47 MHz) that are indicative of a water-exposed nitroxide moiety. The isotropic rotational

The radical nature of the synthesized nitroxide conjugates were verified by continu-ous wave (CW) EPR spectroscopy. Figure 2 shows the CW EPR spectra of the nitroxide conjugated compounds **6**–**12**, **14**, **16**, **17**, and **18**. Conventional triplet pattern of TEMPO nitroxide with relative spectral intensities of 1:1:1 can be seen in Figure 2 due to the coupling of the unpaired electron to the *N*-atom which indicates that the nitroxide

The radical nature of the synthesized nitroxide conjugates were verified by continuous wave (CW) EPR spectroscopy. Figure 2 shows the CW EPR spectra of the nitroxide conjugated compounds **6**–**12**, **14**, **16**, **17**, and **18**. Conventional triplet pattern of TEMPO nitroxide with relative spectral intensities of 1:1:1 can be seen in Figure 2 due to the coupling of the unpaired electron to the *N*-atom which indicates that the nitroxide was intact during the EPR measurements. agreement with what can be expected when attached to medium-sized molecules as in this case. During the simulations, the g-values were kept constant at giso~2.005, the commonly found value for piperidine-based nitroxide radicals [29–31]. The numerical values are summarized in Table S1. Altogether, one can state that the EPR parameters clearly show that none of the spin-labelled natural products seems to be aggregated/micellized or non-homogeneously dissolved in aqueous solution.

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 5 of 19

was intact during the EPR measurements.

**Figure 2.** Experimental room temperature CW EPR (X-band, microwave frequency ≈ 9.4 GHz) spectra (black) and the corresponding simulations (red) of the synthesized nitroxide conjugates are **Figure 2.** Experimental room temperature CW EPR (X-band, microwave frequency ≈ 9.4 GHz) spectra (black) and the corresponding simulations (red) of the synthesized nitroxide conjugates are given.

*2.3. Cytotoxic Activity*  The first set of synthesized spin-labelled adducts **6**–**11** were subjected to fast screening by MTT and CV assays to have an overall view on their potential activity against human cancer cell lines viz.: PC3 (prostate cancer) and HT29 (colon cancer). Two concentrations were employed viz.: 0.1 and 10 μM and compared to the activity of unmodified BA (**1**), FA (**2**), and CA (**3**). As shown in Figure 3 both the betulinic acid derivatives **6** and **7** and the fusidic acid derivatives **8** and **9** showed a significant reduction in cell viability when compared to cholic acid derivatives **10** and **11**. The low anticancer activity of cholic acid and its derivatives can be attributed to the high lipophilic character (log p ~ 2.02) and low water solubility, which makes it difficult to cross membranes effectively to be present in high concentrations in the cytosol of the cancer cells [32]. The EPR characteristics, mainly the isotropic hyperfine coupling (Aiso) and isotropic rotational correlation times (*τc*), dominate solution-state EPR spectra as a measure of the line spacing and line shape, were obtained by simulations using the Easyspin software package [28]. All synthesized nitroxide adducts show hyperfine couplings (Aiso~45–47 MHz) that are indicative of a water-exposed nitroxide moiety. The isotropic rotational correlation times (*τc*), as a simple measure of nitroxide rotational dynamics were monitored and were found to be between 1–2 ns for the synthesized nitroxide adducts, in good agreement with what can be expected when attached to medium-sized molecules as in this case. During the simulations, the g-values were kept constant at giso~2.005, the commonly found value for piperidine-based nitroxide radicals [29–31]. The numerical values are summarized in Table S1. Altogether, one can state that the EPR parameters clearly show that none of the spin-labelled natural products seems to be aggregated/micellized or non-homogeneously dissolved in aqueous solution.

#### *2.3. Cytotoxic Activity*

given.

The first set of synthesized spin-labelled adducts **6**–**11** were subjected to fast screening by MTT and CV assays to have an overall view on their potential activity against human cancer cell lines viz.: PC3 (prostate cancer) and HT29 (colon cancer). Two concentrations were employed viz.: 0.1 and 10 µM and compared to the activity of unmodified BA (**1**), FA (**2**), and CA (**3**). As shown in Figure 3 both the betulinic acid derivatives **6** and **7** and the fusidic acid derivatives **8** and **9** showed a significant reduction in cell viability when compared to cholic acid derivatives **10** and **11**. The low anticancer activity of cholic acid and its derivatives can be attributed to the high lipophilic character (log p ~ 2.02) and low

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 6 of 19

water solubility, which makes it difficult to cross membranes effectively to be present in high concentrations in the cytosol of the cancer cells [32]. vided by the Ugi multicomponent reaction dramatically enhances the anticancer activity of these classes of terpenes.

As expected, FA (**2**) failed to show any activity against the tested cancer cell lines. However, the unanticipated very high activity displayed by its derivatives **8** and **9**, especially when compared to the well-known anticancer activity of BA (**1**) was encouraging [33]. The IC50 values were also determined for the most active compounds against both cell lines used in our appraisal. The IC50 values are illustrated in Table 1, where it is evident that fusidic acid analogue **8** is the most active compound against both cell lines tested. Additionally, another fusidic acid analogue **9** was also active towards HT29 cells with an IC50 of 6.98 ± 0.25 μM (CV). Moreover, the betulinic acid analogue **6** possesses cytotoxic effects towards PC3 (IC50: 7.43 ± 0.72 μM) and HT29 cells (IC50: 8.98 ± 0.43 μM). In addition, it is worthy to note that FA alone possesses no activity when compared to its spin-labelled adducts. Thus, it may clearly be noted that the structure modification pro-

**Figure 3.** Cell viability of HT29 (**A**,**B**) and PC3 (**C**,**D**); cell lines were treated by the investigated compounds for 48 h. Cell viability was determined using CV assay (**A**,**C**) and MTT assay (**B**,**D**). **Figure 3.** Cell viability of HT29 (**A**,**B**) and PC3 (**C**,**D**); cell lines were treated by the investigated compounds for 48 h. Cell viability was determined using CV assay (**A**,**C**) and MTT assay (**B**,**D**).

**Table 1.** IC50 values (μM) for the most active compounds against HT29 and PC3 cell lines determined by MTT and CV assays. **Compound PC3 HT29 CV MTT CV MTT 1** 24.64 ± 1.78 25.43 ± 4.35 24.97 ± 0.57 19.02 ± 2.26 **6** 13.69 ± 0.80 7.43 ± 0.72 13.16 ± 0.97 8.98 ± 0.43 **7** 10.59 ± 0.85 10.54 ± 0.91 13.82 ± 0.29 11.87 ± 0.94 **8** 7.44 ± 0.80 6.00 ± 1.09 8.10 ± 0.43 7.41 ± 0.56 **9** 15.26 ± 1.01 13.85 ± 2.04 6.98 ± 0.25 12.94 ± 1.03 As expected, FA (**2**) failed to show any activity against the tested cancer cell lines. However, the unanticipated very high activity displayed by its derivatives **8** and **9**, especially when compared to the well-known anticancer activity of BA (**1**) was encouraging [33]. The IC<sup>50</sup> values were also determined for the most active compounds against both cell lines used in our appraisal. The IC<sup>50</sup> values are illustrated in Table 1, where it is evident that fusidic acid analogue **8** is the most active compound against both cell lines tested. Additionally, another fusidic acid analogue **9** was also active towards HT29 cells with an IC<sup>50</sup> of 6.98 ± 0.25 µM (CV). Moreover, the betulinic acid analogue **6** possesses cytotoxic effects towards PC3 (IC50: 7.43 ± 0.72 µM) and HT29 cells (IC50: 8.98 ± 0.43 µM). In addition, it is worthy to note that FA alone possesses no activity when compared to its spin-labelled adducts. Thus, it may clearly be noted that the structure modification provided by the Ugi multicomponent reaction dramatically enhances the anticancer activity of these classes of terpenes.

tions for 48 h (AnnV/PI assay).


**Table 1.** IC<sup>50</sup> values (µM) for the most active compounds against HT29 and PC3 cell lines determined by MTT and CV assays.

Compound **8**, the most promising conjugate, was selected to determine its mode of action against the PC3 cell line based on its cytotoxic effects. To determine the mode of cell death induced by compound **8**, the AnnV/PI assay was performed since the degree of induction of apoptosis by compound **8** can be effectively measured. The assay determines the expression of phosphatidylserine on the cell surface by annexin V (AnnV) stain and the DNA fragmentation by propidium iodide (PI) (Figure 4). Compound **8** was tested at two different concentrations (IC50, 2 × IC50) for 48 h and it was analysed using flow cytometry. It is clearly shown, that this compound increases both early and late apoptosis, only when the PC3 cancer cells were treated with 2 × IC<sup>50</sup> value with a total apoptotic event of 68% compared to the control of 16%. To study the impact of compound **8** on the cell cycle distribution, the DAPI assay was performed as outlined in Figure 5. Based on the results obtained, compound **8** caused a dose-dependent increase in the entrapment of cells in sub G1-phase. This accumulation of the cells in the sub G1-phase of the cell cycle indicates the fragmentation of the DNA that has occurred due to the induction of apoptosis by compound **8**. Compound **8**, the most promising conjugate, was selected to determine its mode of action against the PC3 cell line based on its cytotoxic effects. To determine the mode of cell death induced by compound **8**, the AnnV/PI assay was performed since the degree of induction of apoptosis by compound **8** can be effectively measured. The assay determines the expression of phosphatidylserine on the cell surface by annexin V (AnnV) stain and the DNA fragmentation by propidium iodide (PI) (Figure 4). Compound **8** was tested at two different concentrations (IC50, 2 × IC50) for 48 h and it was analysed using flow cytometry. It is clearly shown, that this compound increases both early and late apoptosis, only when the PC3 cancer cells were treated with 2 × IC50 value with a total apoptotic event of 68% compared to the control of 16%. To study the impact of compound **8** on the cell cycle distribution, the DAPI assay was performed as outlined in Figure 5. Based on the results obtained, compound **8** caused a dose-dependent increase in the entrapment of cells in sub G1-phase. This accumulation of the cells in the sub G1-phase of the cell cycle indicates the fragmentation of the DNA that has occurred due to the induction of apoptosis by compound **8**.

**Figure 4.** The impact of compound **8** on the apoptosis induction in PC3 cells. **8** was tested using IC50, 2 × IC50 concentra-**Figure 4.** The impact of compound **8** on the apoptosis induction in PC3 cells. **8** was tested using IC50, 2 × IC<sup>50</sup> concentrations for 48 h (AnnV/PI assay).

Reyes et al. reported that natural triterpenoic acids induce caspase-dependent apoptosis and in particular, caspase-3 [34]. Furthermore, recent reports showed that treating tumor cells with nitroxides can also induce apoptosis by a caspase activation mechanism [35]. These studies inspired us to investigate the possibility of caspase-3 being involved in the mechanism of action of conjugate **8** to explain the apoptotic mode of cell death, passivate the level of protein expression of the anti-apoptotic protein Bcl-XL and the housekeeping proteins (β-actin and α/β-tubulin). Results illustrated in Figure 6 show that **8** increased the level of caspase-3 significantly after 48 h of incubation, which clearly indicates the induction of apoptosis by activation of the caspase pathway. Expression of the anti-apoptotic protein

Bcl-XL which is a transmembrane molecule in the mitochondria was also measured. After 48 h of incubation, it is clearly evident that the level of Bcl-XL decreases which supports the apoptosis by triggering the caspase-3 activation pathway. **Figure 4.** The impact of compound **8** on the apoptosis induction in PC3 cells. **8** was tested using IC50, 2 × IC50 concentrations for 48 h (AnnV/PI assay). apoptosis and in particular, caspase-3 [34]. Furthermore, recent reports showed that treating tumor cells with nitroxides can also induce apoptosis by a caspase activation mechanism [35]. These studies inspired us to investigate the possibility of caspase-3 being involved in the mechanism of action of conjugate **8** to explain the apoptotic mode of

Reyes et al. reported that natural triterpenoic acids induce caspase-dependent

**Figure 5.** The impact of compound **8** on the distribution of PC3 cells in the cell cycle phases.

Compound **8** was tested using IC50, 2 × IC50 concentrations for 48 h (DAPI assay).

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 7 of 19

to the induction of apoptosis by compound **8**.

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 8 of 19

**18** 9.27 ± 0.73 6.19 ± 0.20 16.30 ± 0.87 12.23 ± 0.67

Compound **8**, the most promising conjugate, was selected to determine its mode of action against the PC3 cell line based on its cytotoxic effects. To determine the mode of cell death induced by compound **8**, the AnnV/PI assay was performed since the degree of induction of apoptosis by compound **8** can be effectively measured. The assay determines the expression of phosphatidylserine on the cell surface by annexin V (AnnV) stain and the DNA fragmentation by propidium iodide (PI) (Figure 4). Compound **8** was tested at two different concentrations (IC50, 2 × IC50) for 48 h and it was analysed using flow cytometry. It is clearly shown, that this compound increases both early and late apoptosis, only when the PC3 cancer cells were treated with 2 × IC50 value with a total apoptotic event of 68% compared to the control of 16%. To study the impact of compound **8** on the cell cycle distribution, the DAPI assay was performed as outlined in Figure 5. Based on the results obtained, compound **8** caused a dose-dependent increase in the entrapment of cells in sub G1-phase. This accumulation of the cells in the sub G1-phase of the cell cycle indicates the fragmentation of the DNA that has occurred due

**Figure 6.** Effect of compound **8** on protein expression in PC3 cells (relative to β-actin). **Figure 6.** Effect of compound **8** on protein expression in PC3 cells (relative to β-actin).

The influence of compound **8** (IC50 and 2 × IC50) on the ROS production in PC3 cells was monitored using dihydrorhodamine (DHR) assay for 48 h and the data were analysed with flow cytometry. As shown in Figure 7 compound **8** indeed reduced the level of ROS as anticipated in a dose-dependent manner. β-Actin is a housekeeping protein that is involved in the restriction of the cell motility, structure integrity, and in addition to its resistance to different cellular treatment, which makes it a good choice as a housekeeping protein for western blot analysis [36]. Indeed, after 48 h of incubation, no change in its expression was detected. α/β-Tubulins is another housekeeping protein control used since their expression should remain unchanged. Surprisingly, the behavior of this protein was manifested by a strong elevation of the expression level being obvious after 48 h of incubation as shown in Figure 6. Recent studies report that microtubulin increases during apoptosis and functions as a physical barrier preventing caspase from spreading into the cellular cortex. In addition, it increases phosphatidylserine (PS) externalization which helps the macrophage for efficient clearance [37].

The influence of compound **8** (IC<sup>50</sup> and 2 × IC50) on the ROS production in PC3 cells was monitored using dihydrorhodamine (DHR) assay for 48 h and the data were analysed with flow cytometry.

As shown in Figure 7 compound **8** indeed reduced the level of ROS as anticipated in a dose-dependent manner.

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 9 of 19

**Figure 7.** The impact of compound **8** on ROS produced by PC3. **8** was tested using IC50, 2 × IC50 concentrations for 48 h using DHR assay, fluorescence was detected in FITC channel (ex/em: 488/520 nm). **Figure 7.** The impact of compound **8** on ROS produced by PC3. **8** was tested using IC50, 2 × IC<sup>50</sup> concentrations for 48 h using DHR assay, fluorescence was detected in FITC channel (ex/em: 488/520 nm). **Figure 7.** The impact of compound **8** on ROS produced by PC3. **8** was tested using IC50, 2 × IC50 concentrations for 48 h using DHR assay, fluorescence was detected in FITC channel (ex/em: 488/520 nm). **Figure 7.** The impact of compound **8** on ROS produced by PC3. **8** was tested using IC50, 2 × IC50 concentrations for 48 h using DHR assay, fluorescence was detected in FITC channel (ex/em: 488/520 nm).

#### *2.4. Fluorescent Imaging Study 2.4. Fluorescent Imaging Study 2.4. Fluorescent Imaging Study*

Fluorescent conjugate **12** was initially used to determine if this dye-tagged analogue of fusidic acid conjugate **8** can target the mitochondria, since mitochondria are the major source of ROS generation and therefore is more sensible for ROS manipulation. Unfortunately, after PC3 cancer cells were incubated with **12** (depicted as green color in Figure 8), no mitochondrial targeting was observed. Therefore, we turned our attention to test the mitochondrial targeting of TPP-conjugate **18**. After 24 h of incubation of compound **18** with PC3 cells, a clear mitochondrial targeting was successfully achieved as shown in Figure 9. Additionally, cytotoxic activity of **18** against PC3 and HT29 was found to be with significant effects towards PC3 cancer cell line (IC50: 6.18 ± 0.20 μM, MTT). Fluorescent conjugate **12** was initially used to determine if this dye-tagged analogue of fusidic acid conjugate **8** can target the mitochondria, since mitochondria are the major source of ROS generation and therefore is more sensible for ROS manipulation. Unfortunately, after PC3 cancer cells were incubated with **12** (depicted as green color in Figure 8), no mitochondrial targeting was observed. Therefore, we turned our attention to test the mitochondrial targeting of TPP-conjugate **18**. After 24 h of incubation of compound **18** with PC3 cells, a clear mitochondrial targeting was successfully achieved as shown in Figure 9. Additionally, cytotoxic activity of **18** against PC3 and HT29 was found to be with significant effects towards PC3 cancer cell line (IC50: 6.18 ± 0.20 µM, MTT). Fluorescent conjugate **12** was initially used to determine if this dye-tagged analogue of fusidic acid conjugate **8** can target the mitochondria, since mitochondria are the major source of ROS generation and therefore is more sensible for ROS manipulation. Unfortunately, after PC3 cancer cells were incubated with **12** (depicted as green color in Figure 8), no mitochondrial targeting was observed. Therefore, we turned our attention to test the mitochondrial targeting of TPP-conjugate **18**. After 24 h of incubation of compound **18** with PC3 cells, a clear mitochondrial targeting was successfully achieved as shown in Figure 9. Additionally, cytotoxic activity of **18** against PC3 and HT29 was found to be with significant effects towards PC3 cancer cell line (IC50: 6.18 ± 0.20 μM, MTT). *2.4. Fluorescent Imaging Study*  Fluorescent conjugate **12** was initially used to determine if this dye-tagged analogue of fusidic acid conjugate **8** can target the mitochondria, since mitochondria are the major source of ROS generation and therefore is more sensible for ROS manipulation. Unfortunately, after PC3 cancer cells were incubated with **12** (depicted as green color in Figure 8), no mitochondrial targeting was observed. Therefore, we turned our attention to test the mitochondrial targeting of TPP-conjugate **18**. After 24 h of incubation of compound **18** with PC3 cells, a clear mitochondrial targeting was successfully achieved as shown in Figure 9. Additionally, cytotoxic activity of **18** against PC3 and HT29 was found to be

with significant effects towards PC3 cancer cell line (IC50: 6.18 ± 0.20 μM, MTT).

**Figure 8.** Fluorescent imaging with fluorescent conjugate **12** (ex/em: 488/520 nm) double-stained with MitoTrackerTM Deep Red (ex/em: 596/615 nm) in PC3 cancer cell line. **Figure 8.** Fluorescent imaging with fluorescent conjugate **12** (ex/em: 488/520 nm) double-stained with MitoTrackerTM Deep Red (ex/em: 596/615 nm) in PC3 cancer cell line. **Figure 8.** Fluorescent imaging with fluorescent conjugate **12** (ex/em: 488/520 nm) double-stained with MitoTrackerTM Deep Red (ex/em: 596/615 nm) in PC3 cancer cell line. **Figure 8.** Fluorescent imaging with fluorescent conjugate **12** (ex/em: 488/520 nm) double-stained with MitoTrackerTM Deep Red (ex/em: 596/615 nm) in PC3 cancer cell line.

**Figure 9.** Fluorescent imaging of PC3 cancer cell line stained with TPP-conjugate **18** (ex/em:488/520 nm) and with Mito-TrackerTM Deep Red (ex/em: 596/615 nm). **Figure 9.** Fluorescent imaging of PC3 cancer cell line stained with TPP-conjugate **18** (ex/em:488/520 nm) and with Mito-TrackerTM Deep Red (ex/em: 596/615 nm). **Figure 9.** Fluorescent imaging of PC3 cancer cell line stained with TPP-conjugate **18** (ex/em:488/520 nm) and with Mito-TrackerTM Deep Red (ex/em: 596/615 nm). **Figure 9.** Fluorescent imaging of PC3 cancer cell line stained with TPP-conjugate **18** (ex/em:488/520 nm) and with MitoTrackerTM Deep Red (ex/em: 596/615 nm).

#### **3. Conclusions**

TEMPO radical conjugation to natural products can serve as a potential strategy to obtain new hybrid compounds with novel mechanisms of action. Conjugate **8** revealed a high activity against both PC3 and HT29 cancer cell lines (PC3: IC<sup>50</sup> 6.0 ± 1.1 µM; HT29: IC<sup>50</sup> 7.4 ± 0.6 µM), furthermore, apoptosis was induced through the caspase activation mechanism. In addition, targeting mitochondria (the major source of ROS generation) was successfully achieved with **18**. Moreover, it was clearly demonstrated that utilizing Ugi multicomponent reactions is a powerful synthetic tool that gives access to a wide variety of different analogues via a fairly easy synthetic effort. We envisioned that utilizing the power of MCR, large interesting libraries of natural product TEMPO conjugates for the treatment of cancer can be generated.

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

#### *4.1. Chemistry*

#### 4.1.1. Materials

All commercially available reagents were purchased and used without further purification. Convertible isocyanide 2-isocyano-2-methylpropyl phenyl carbonate "IPB" was synthesized following reported procedures [27]. (3-Aminopropyl)triphenylphosphonium bromide (TPP-NH2) was also synthesized following reported protocols [38]. HPLC grade methanol was used in all Ugi reactions. Analytical thin layer chromatography (TLC) was performed using silica gel 60 F<sup>254</sup> aluminum sheets (Merck, Darmstadt, Germany) and the visualization of the spots has been done under UV light (254 nm) or by developing with a solution of cerium sulfate. Flash column chromatography was performed using silica gel (0.040–0.063 mm). <sup>1</sup>H- and <sup>13</sup>C-NMR spectra were recorded in solutions on a 400 NMR Varian MERCURY-VX 400 at 22 ◦C at 400 MHz and 100 MHz, or an Agilent (Varian, Santa Clara, CA, USA) VNMRS 600 NMR spectrometer at 599.83 MHz and 150.83 MHz respectively. Chemical shifts (δ) are reported in ppm relative to TMS (1H-NMR) and to the solvent signal (13C NMR spectra). The positive-ion high-resolution ESI mass spectra were obtained with an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Schwerte, Germany) equipped with HESI electrospray ion source (positive spray voltage 4 kV, capillary temperature 275 ◦C, source heater temperature 80 ◦C, FTMS resolution 60,000). Nitrogen was used as sheath gas. The instrument was externally calibrated using the Pierce LTQ Velos ESI positive ion calibration solution (product number 88323, ThermoFisher Scientific, Rockford, IL, USA). The data were evaluated using the software Xcalibur 2.7 SP1. Analytical RP-HPLC analysis was performed with an 1100 system (Agilent, Santa Clara, CA, USA) on a reverse-phase C18 column (4.6 × 150 mm, 5 µm) with a PDA detector. A linear gradient from 5% to 100% of solvent B in solvent A over 15–30 min at a flow rate of 0.8 mL min−<sup>1</sup> . Detection was accomplished at 210 nm. Solvent A: 0.1% (*v/v*) formic acid (FA) in water. Solvent B: 0.1% (*v/v*) FA in acetonitrile.

PBS, RPMI 1640, and Trypsin EDTA were from Capricorn Scientific (Ebsdorfergrund, Germany). β-mercaptoethanol was from Bio-Rad (Hercules, CA, USA). Anti-rabbit IgG HRP-linked antibody, α/β-tubulin rabbit Ab, caspase-3 rabbit Ab were purchased from Cell Signalling Technology (Frankfurt/Main, Germany), while Bcl-XL rabbit antibody was obtained from Abcam (Cambridge, UK). DMSO was bought from Duchefa Biochemie (Harleem, The Netherlands). ECL Prime Western Blotting System was supplied by GE Healthcare (Braunschweig, Germany). AnnV/PI, PAGE Ruler, EDTA Solution, Trypan blue, MitoTrackerTM Deep Red and Halt Protease Inhibitor Cocktail were obtained from ThermoFisher Scientific, Schwerte, Germany). Ethanol, Na2HPO4, NaH2PO4, and BSA were bought from Merck, Darmstadt, Germany). Digitonin was from Riedel De Haen (Seelze, Germany). Acetic acid, APS, FCS, glycerol, glycine, methanol, NaOH, penicillin/streptomycin, Roti-quant "5x", TEMED and TRIS were from Roth (Karlsruhe, Germany). Acrylamide/Bisacrylamide was bought from Serva (Heidelberg, Germany). Finally, bromophenol blue, CV, DAPI, MTT, Triton X-100, and Tween-20 were from Sigma Aldrich (St. Louis, MO, USA).

#### 4.1.2. General Procedure A for the Ugi-4CR

To a stirred solution of TEMPO amine **4** (0.1 mmol) in methanol (250 µL, 0.4 M) was added paraformaldehyde (0.1 mmol) and the mixture was stirred for 2 h. After this time the acid (0.1 mmol) and isonitrile (0.1 mmol) were added before stirring was continued for 18 h. The solvent was removed under reduced pressure and the crude material purified by column chromatography to afford the desired products.

Note: due to the paramagnetism of nitroxide moieties, NMR cannot provide information useful for structural elucidation of nitroxide-containing products, therefore, reduction of the paramagnetic center was performed with phenylhydrazine or hydrazobenzene [39,40].

*(4-{[2-(tert-Butylamino)-2-oxoethyl][(1R,3aS,5aR,5bR,9S,11aR)-9-hydroxy-5a,5b,8,8,11a-pentamethyl-1- (prop-1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carbonyl]amino}-2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl* (**6**)

Obtained using the general method **A**, *N*-oxyl amine **4** and paraformaldehyde were used, then betulinic acid and *t*-butyl isonitrile were added. The crude reaction product was purified by silica gel column chromatography (ethyl acetate/hexane 8:2) to yield compound **6** (51 mg, 0.070 mmol, 70%) as red solid. *R<sup>F</sup>* 0.77 (ethyl acetate/hexane 8:2). NMR of the corresponding hydroxylamine after phenylhydrazine reduction. <sup>1</sup>H-NMR (600 MHz, CDCl3) δ 4.72 (s, 1H), 4.59 (s, 1H), 4.35 (d, *J* = 12.6 Hz, 1H), 3.65 (s, 2H), 3.16 (dd, *J* = 11.3, 4.8 Hz, 2H), 2.98 (m, 1H), 2.87–2.81 (m, 1H), 2.24–2.18 (m, 1H), 2.06–1.20 (m, 49H), 0.96 (s, 3H), 0.95 (s, 3H), 0.92 (s, 3H), 0.80 (s, 3H), 0.74 (s, 3H). <sup>13</sup>C-NMR (151 MHz, CDCl3): δ 176.0 (CO), 109.5, 78.9, 55.5, 54.9, 53.0, 50.9, 50.8, 49.3, 47.7, 45.8, 42.8, 42.3, 42.1, 40.8, 38.9, 38.8, 37.3, 36.9, 36.1, 34.3, 32.2, 32.0, 31.6, 29.9, 29.8, 28.7 (4 × CH<sup>3</sup> (TEMPO)), 28.1 (CH<sup>3</sup> (*t*Bu)), 27.5, 25.7, 21.1, 20.1, 19.9, 19.6, 18.3, 16.2, 15.5, 14.8. HRMS (ESI) *m/z*: 723.5890 [M + H]<sup>+</sup> , calcd. for [C45H77N3O4] <sup>+</sup> 723.5914.
