**Abstract**

Bozepinib is a potent antitumour compound that shows an IC50 of 0.166 μM against MDA-MB-231 human breast cancer cell line. It is also a very selective drug that presents a therapeutic index (TI) of 11.0 against MDA-MB-231 in relation to the normal MCF-10A. It is important to identify new cancer stem-like cells (CSCs) anticancer drugs to struggle against the resistance and the high risk of relapse in patients. In the present chapter, we show how bozepinib demonstrated selectivity on cancer cells and showed an inhibitory effect over kinases involved in carcinogenesis, proliferation and angiogenesis. Bozepinib inhibits HER-2 signaling pathway and JNK and ERK kinases. In addition, it has an inhibitory effect on AKT and VEGF together with anti-angiogenic and anti-migratory activities. Interestingly, bozepinib suppresses the formation of both mammo- and colonospheres and eliminated ALDH+ CSC subpopulations at a low micromolar range similar to salinomycin. It also induces the downregulation of SOX2, c-MYC and β-CATENIN and upregulation of the GLI-3 Hedgehog signaling repressor. Finally, bozepinib shows in vivo antitumor and anti-metastatic efficacy in xenotransplanted nude mice without presenting subacute toxicity. However, further studies in cancer patients are needed to confirm the therapeutic potential of bozepinib.

**Keywords:** benzoxazepine, bozepinib, cancer stem-like cells, MDA-MB-231, MCF-7, MCF-10A, protein kinases, seven-membered ring

### **1. Introduction**

Expansion of cancer keeps going on as an important health problem in the developed, undeveloped and developing countries. Although major advances have been made in the chemotherapeutic management of some patients, the continued commitment to the laborious task of discovering new anticancer agents remains critically important, in the course of identifying various chemical substances, which may serve as leads for designing novel antitumor agents.

#### **1.1 Cancer stem cells**

Currently, one of the most interesting concepts being explored in cancer research is the theory of cancer stem cells. CSCs can be defined as the

subpopulations of cells within tumors that possess the ability to self-renew and differentiate into the different lines of cancer cells that make up the tumor. This type of cell is proposed as the promoter of resistance against antitumor therapies, being able to maintain benign and malignant tumors, as well as causing relapses [1]. Compared to normal stem cells, CSCs are thought to have no control over their proliferation. These CSCs, present in tumors in small numbers, are characterized by their ability to remain quiescent for long periods of time, capacity for self-renewal, maintenance of growth and heterogeneity of the tumor, affinity for environment resistance to chemotherapy and development of metastases [2].

CSCs are characterized by their ability to form spherical colonies when cultivated in suspension [3]. Al-Hajj et al. managed to demonstrate that the injection of 200 tumoural cells expressing characteristic markers of CSCs was more effective in generating tumors in immunodepressed mice than the injection of 50,000 tumor cells with differentiated cell markers of the same histological lineage [4]. It has been found that multiple molecules related to the characteristic properties of stem cells such as self-renewal and pluripotency and certain enzymatic activities are largely expressed in CSCs, including c-MYC, β-CATENIN [5], SOX-2 [6] and aldehyde dehydrogenase activity (ALDH1) [7]. New strategies for selective and effective cancer therapy can be provided by selectively acting on overregulated pathways or molecules in differentiated cancer cells and/or in CSC populations, but not in normal cells.

#### **1.2 Therapies targeted against cancer stem cells**

Cancer treatment currently targets its proliferation potential, and therefore most treatments target rapidly dividing cells. The presence of CSCs may explain the failure of treatments to eradicate the disease or the recurrence of cancer [1]. CSCs have to remain in a state of quiescence, a state in which the cell does not divide staying in the G0 phase of the cell cycle [8] allows them to survive most anticancer treatments. This characteristic makes relapses possible, even decades after initial treatment, such as in colon or breast cancers [1, 9]. Although current treatments may reduce the tumor size, these effects are transient and generally do not improve patients' survival. For tumors in which CSCs play a role, there are three possibilities. First, the mutation of normal stem cells or CSC progenitor cells can lead to the development of the primary tumor. Second, during chemotherapy, most cells in the primary tumor can be destroyed, but if CSCs are not eradicated, they become refractory CSCs and can lead to the recurrence of the tumor [10]. Third, CSCs can migrate to distant sites of the primary tumor and cause metastasis [11–13]. Theoretically, the identification of CSCs can allow the development of treatment modalities that target these cells rather than rapidly dividing cells [14].

One of the therapeutic approaches currently being studied to combat CSCs is related to the signaling pathways involved in the processes of their self-renewal, proliferation and differentiation. This is because the loss of regulation of pathways such as Hedgehog (Hh), Notch and Wnt/β-catenin results in the key processes involved in the characteristics of CSCs. Currently, the therapy directed against these routes represents one of the most promising mechanisms of action against these initiation cells of the tumor [15, 16].

Herein we will discuss the search and biological activity of small synthetic derivatives: racemate **2**, named as bozepinib (isomer of **1**), was selected for anti-CSC studies (**Figure 1**). The information presented in this chapter should be of interest to medicinal chemists and represents an effort to summarize the experimental research and advances in the field of CSCs [17, 18].

**129**

or both.

**Figure 1.**

*Bozepinib: A Promising Selective Derivative Targeting Breast Cancer Stem Cells*

A better understanding of the molecular mechanisms responsible for CSC formation probably lead to the design and synthesis of new anticancer drugs which will be able to eliminate CSC or to halt tumor growth by interfering with important intracellular signaling pathways related with CSC stemness or CSC differentiation

**2. (***RS***)-2,6-Dichloro-9-[1-(***o***- or** *p***-nitrobenzenesulfonyl)-1,2,3,5 tetrahydro-4,1-benzoxazepine-3-yl]-9***H***-purines 1 and 2**

As part of an Anticancer Drug Programme, we were interested in the preparation of the heterocycles **3a-b** (**Figure 2**) that could be useful intermediates for the synthesis of novel bioactive compounds. **Figure 2** shows the synthesis of derivatives **3a-b** [19]. The Mitsunobu reaction is a versatile method for the transformation of aliphatic alcohols into alkylating agents in situ and under mild conditions [20]. A successful Mitsunobu displacement depends on the p*K*a associated with the N▬H bond [21]. Thus, a powerful electron withdrawing group for the amino moiety was

The synthesis of compounds **3a-b** begins with the protection of the hydroxyl group of anthranilic alcohol by the *tert*-butyldimethylsilanyl group to give **4**; the synthesis of sulfonamides **5a** and **5b** was accomplished under the conditions of Fukuyama et al. [22]. Compound **6a** was obtained in a 70% yield under Mitsunobu conditions from **5a** and glycolaldehyde dimethyl acetal. The yield of this reaction depends greatly on the temperature: at rt., it is 19%, increasing up to 70% at 30°C and falling to 40% when the temperature is 40°C (**Figure 2**). When the optimized temperature conditions were applied to **5b**, **6b** was obtained in an 80% yield. After deprotection of the silanyl group of **6a** (and **6b**) with tetra-*n*-butylammonium fluoride (TBAF) in tetrahydrofuran (THF), **7a** was obtained in an 83% yield (and **7b** in 100% yield). Compounds **7a** and **7b** quantitatively afforded the benzo-fused seven-membered *O*,*O*-acetals **3a** and **3b**, respectively, using boron trifluoride

**2.1 Synthesis of tetrahydrobenzoxazepine** *O,O***-acetals with electron** 

**withdrawing groups on the nitrogen atom**

needed such as the *p*- or *o*-nitrobenzenesulfonyl fragment.

diethyl etherate as previously reported [23].

*DOI: http://dx.doi.org/10.5772/intechopen.91423*

*Chemical structures of bezoxazepine derivatives 1 and 2.*

*Bozepinib: A Promising Selective Derivative Targeting Breast Cancer Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.91423*

#### **Figure 1.**

*Translational Research in Cancer*

normal cells.

subpopulations of cells within tumors that possess the ability to self-renew and differentiate into the different lines of cancer cells that make up the tumor. This type of cell is proposed as the promoter of resistance against antitumor therapies, being able to maintain benign and malignant tumors, as well as causing relapses [1]. Compared to normal stem cells, CSCs are thought to have no control over their proliferation. These CSCs, present in tumors in small numbers, are characterized by their ability to remain quiescent for long periods of time, capacity for self-renewal, maintenance of growth and heterogeneity of the tumor, affinity for environment

CSCs are characterized by their ability to form spherical colonies when cultivated in suspension [3]. Al-Hajj et al. managed to demonstrate that the injection of 200 tumoural cells expressing characteristic markers of CSCs was more effective in generating tumors in immunodepressed mice than the injection of 50,000 tumor cells with differentiated cell markers of the same histological lineage [4]. It has been found that multiple molecules related to the characteristic properties of stem cells such as self-renewal and pluripotency and certain enzymatic activities are largely expressed in CSCs, including c-MYC, β-CATENIN [5], SOX-2 [6] and aldehyde dehydrogenase activity (ALDH1) [7]. New strategies for selective and effective cancer therapy can be provided by selectively acting on overregulated pathways or molecules in differentiated cancer cells and/or in CSC populations, but not in

Cancer treatment currently targets its proliferation potential, and therefore most treatments target rapidly dividing cells. The presence of CSCs may explain the failure of treatments to eradicate the disease or the recurrence of cancer [1]. CSCs have to remain in a state of quiescence, a state in which the cell does not divide staying in the G0 phase of the cell cycle [8] allows them to survive most anticancer treatments. This characteristic makes relapses possible, even decades after initial treatment, such as in colon or breast cancers [1, 9]. Although current treatments may reduce the tumor size, these effects are transient and generally do not improve patients' survival. For tumors in which CSCs play a role, there are three possibilities. First, the mutation of normal stem cells or CSC progenitor cells can lead to the development of the primary tumor. Second, during chemotherapy, most cells in the primary tumor can be destroyed, but if CSCs are not eradicated, they become refractory CSCs and can lead to the recurrence of the tumor [10]. Third, CSCs can migrate to distant sites of the primary tumor and cause metastasis [11–13]. Theoretically, the identification of CSCs can allow the development of treatment

modalities that target these cells rather than rapidly dividing cells [14].

One of the therapeutic approaches currently being studied to combat CSCs is related to the signaling pathways involved in the processes of their self-renewal, proliferation and differentiation. This is because the loss of regulation of pathways such as Hedgehog (Hh), Notch and Wnt/β-catenin results in the key processes involved in the characteristics of CSCs. Currently, the therapy directed against these routes represents one of the most promising mechanisms of action against

Herein we will discuss the search and biological activity of small synthetic derivatives: racemate **2**, named as bozepinib (isomer of **1**), was selected for anti-CSC studies (**Figure 1**). The information presented in this chapter should be of interest to medicinal chemists and represents an effort to summarize the experi-

resistance to chemotherapy and development of metastases [2].

**1.2 Therapies targeted against cancer stem cells**

these initiation cells of the tumor [15, 16].

mental research and advances in the field of CSCs [17, 18].

**128**

*Chemical structures of bezoxazepine derivatives 1 and 2.*

A better understanding of the molecular mechanisms responsible for CSC formation probably lead to the design and synthesis of new anticancer drugs which will be able to eliminate CSC or to halt tumor growth by interfering with important intracellular signaling pathways related with CSC stemness or CSC differentiation or both.

### **2. (***RS***)-2,6-Dichloro-9-[1-(***o***- or** *p***-nitrobenzenesulfonyl)-1,2,3,5 tetrahydro-4,1-benzoxazepine-3-yl]-9***H***-purines 1 and 2**

#### **2.1 Synthesis of tetrahydrobenzoxazepine** *O,O***-acetals with electron withdrawing groups on the nitrogen atom**

As part of an Anticancer Drug Programme, we were interested in the preparation of the heterocycles **3a-b** (**Figure 2**) that could be useful intermediates for the synthesis of novel bioactive compounds. **Figure 2** shows the synthesis of derivatives **3a-b** [19]. The Mitsunobu reaction is a versatile method for the transformation of aliphatic alcohols into alkylating agents in situ and under mild conditions [20]. A successful Mitsunobu displacement depends on the p*K*a associated with the N▬H bond [21]. Thus, a powerful electron withdrawing group for the amino moiety was needed such as the *p*- or *o*-nitrobenzenesulfonyl fragment.

The synthesis of compounds **3a-b** begins with the protection of the hydroxyl group of anthranilic alcohol by the *tert*-butyldimethylsilanyl group to give **4**; the synthesis of sulfonamides **5a** and **5b** was accomplished under the conditions of Fukuyama et al. [22]. Compound **6a** was obtained in a 70% yield under Mitsunobu conditions from **5a** and glycolaldehyde dimethyl acetal. The yield of this reaction depends greatly on the temperature: at rt., it is 19%, increasing up to 70% at 30°C and falling to 40% when the temperature is 40°C (**Figure 2**). When the optimized temperature conditions were applied to **5b**, **6b** was obtained in an 80% yield. After deprotection of the silanyl group of **6a** (and **6b**) with tetra-*n*-butylammonium fluoride (TBAF) in tetrahydrofuran (THF), **7a** was obtained in an 83% yield (and **7b** in 100% yield). Compounds **7a** and **7b** quantitatively afforded the benzo-fused seven-membered *O*,*O*-acetals **3a** and **3b**, respectively, using boron trifluoride diethyl etherate as previously reported [23].

#### **Figure 2.**

*Reagents and conditions: (a) o-O2N-C6H4-SO2Cl (1.1 equiv), TEA (1.5 equiv), CH2Cl2, reflux, 24 h, for 5a; p-O2N-C6H4-SO2Cl (0.5 equiv), CH2Cl2, rt., 3 h, for 5b; (b) HOCH2CH(OMe)2 (1 equiv), diisopropyl azodicarboxylate (DIAD, 1.1 equiv), PPh3 (1.2 equiv), anhydrous THF, 21 h; (c) TBAF (1 equiv), THF, rt., 1 h; (d) BF3·OEt2 (2 equiv), anhydrous Et2O, rt., 7 days for 3a; when these conditions were used to obtain 3b, the yield was 67%; p-H3C-C6H4-SO3H (0.03 equiv), anhydrous toluene, 110°C, 2 h under argon for 3a and 3b [19].*

Until compounds **3a** and **3b** were obtained under the optimal conditions described above, other conditions were studied giving rise to unwanted products. Product **5b** was obtained using a twofold excess of the *o*-aminobenzyl silanyl ether **4**, and these conditions were most important for the preparation of this compound (**5b, Figure 2**). Actually, the derivative **8** was isolated when the reaction was carried out using triethylamine (TEA) as a hydrochloride acid scavenger and 1.1 equiv. of the sulfonyl chloride was added (**Figure 3**) [19].

A reasonable explanation implied the previous ionization of the sulfonamide hydrogen atom of **5b** to give a <sup>−</sup>NSO2 anion which reacts more rapidly than the starting amine to give **8**. Disulfonimides have been stated [24] to be by-products in reactions of sulfonyl halides with primary amines and ammonia. The *o*-nitro group might sterically hinder the <sup>−</sup>NSO2 anion, and therefore the analogous side product was not isolated.

We also investigated other conditions to synthesize **6b** (**Figure 4**) [19]. The isopropyl alkylated derivative **9** (38%) was obtained together with the expected acetal **6b** (31%) when an excess of glycolaldehyde dimethyl acetal (4.3 equiv) was added. Such a compound could be interpreted by the transesterification reaction of glycolaldehyde dimethyl acetal and diisopropyl azodicarboxylate (DIAD), with the concomitant leaving of isopropanol. A similar process was previously reported for diethyl azodicarboxylate (DEAD) but not when DIAD was used [25].

The *O*,*O*-acetals **3a** and **3b** were formed after treatment of the *p*-toluenesulfonic acid-mediated cyclization from the acyclic acetals **7a** and **7b**, using anhydrous

**131**

**Figure 5.**

**Figure 3.**

**Figure 4.**

*5b → 6b) [19].*

*Bozepinib: A Promising Selective Derivative Targeting Breast Cancer Stem Cells*

toluene as solvent. When neutral and mild conditions using triphenylphosphine/ carbon tetrachloride were employed, the substitution of the hydroxyl group by the chlorine took place, and compounds **10a** and **10b** were obtained instead of the cyclic derivatives **7a** and **7b** (**Figure 5**) [26]. Compounds **7a** and **7b** present bulky groups that limit their conformational motions, making them rigid structures. The oxygen atoms of the acetalic groups cannot act as nucleophiles against the benzylic position (with the triphenylphosphine ether) due to steric hindering. Hydrogen atoms of the methylene groups of both compounds (**7a** and **7b**) are diastereotopic

*Reagents and conditions: (a) p-O2N-C6H4-SO2Cl (1.1 equiv), TEA (1.5 equiv) and anhydrous CH2Cl2, rt., 5 h* 

*Reagents and conditions: (a) HOCH2CH(OMe)2 (4.3 equiv), DIAD (1.2 equiv), PPh3 (1.2 equiv) and anhydrous THF, rt., 18; h; when 1.2 equiv. of HOCH2CH(OMe)2 were used, see* **Figure 2** *(conversion* 

*Reagents and conditions: (a) Ph3P (1 equiv), CCl4, 110°C, 30 min [19].*

*when 0.5 equiv. of p-O2N-C6H4-SO2Cl was used, see* **Figure 2** *(conversion 4 → 5b) [19].*

*DOI: http://dx.doi.org/10.5772/intechopen.91423*

protons (*J*gem = 12.6–14.1 Hz) [19].

*Bozepinib: A Promising Selective Derivative Targeting Breast Cancer Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.91423*

toluene as solvent. When neutral and mild conditions using triphenylphosphine/ carbon tetrachloride were employed, the substitution of the hydroxyl group by the chlorine took place, and compounds **10a** and **10b** were obtained instead of the cyclic derivatives **7a** and **7b** (**Figure 5**) [26]. Compounds **7a** and **7b** present bulky groups that limit their conformational motions, making them rigid structures. The oxygen atoms of the acetalic groups cannot act as nucleophiles against the benzylic position (with the triphenylphosphine ether) due to steric hindering. Hydrogen atoms of the methylene groups of both compounds (**7a** and **7b**) are diastereotopic protons (*J*gem = 12.6–14.1 Hz) [19].

**Figure 3.**

*Translational Research in Cancer*

Until compounds **3a** and **3b** were obtained under the optimal conditions described above, other conditions were studied giving rise to unwanted products. Product **5b** was obtained using a twofold excess of the *o*-aminobenzyl silanyl ether **4**, and these conditions were most important for the preparation of this compound (**5b, Figure 2**). Actually, the derivative **8** was isolated when the reaction was carried out using triethylamine (TEA) as a hydrochloride acid scavenger and 1.1 equiv. of

*Reagents and conditions: (a) o-O2N-C6H4-SO2Cl (1.1 equiv), TEA (1.5 equiv), CH2Cl2, reflux, 24 h, for 5a; p-O2N-C6H4-SO2Cl (0.5 equiv), CH2Cl2, rt., 3 h, for 5b; (b) HOCH2CH(OMe)2 (1 equiv), diisopropyl azodicarboxylate (DIAD, 1.1 equiv), PPh3 (1.2 equiv), anhydrous THF, 21 h; (c) TBAF (1 equiv), THF, rt., 1 h; (d) BF3·OEt2 (2 equiv), anhydrous Et2O, rt., 7 days for 3a; when these conditions were used to obtain 3b, the yield was 67%; p-H3C-C6H4-SO3H (0.03 equiv), anhydrous toluene, 110°C, 2 h under argon for 3a and 3b [19].*

A reasonable explanation implied the previous ionization of the sulfonamide hydrogen atom of **5b** to give a <sup>−</sup>NSO2 anion which reacts more rapidly than the starting amine to give **8**. Disulfonimides have been stated [24] to be by-products in reactions of sulfonyl halides with primary amines and ammonia. The *o*-nitro group might sterically hinder the <sup>−</sup>NSO2 anion, and therefore the analogous side product

We also investigated other conditions to synthesize **6b** (**Figure 4**) [19]. The isopropyl alkylated derivative **9** (38%) was obtained together with the expected acetal **6b** (31%) when an excess of glycolaldehyde dimethyl acetal (4.3 equiv) was added. Such a compound could be interpreted by the transesterification reaction of glycolaldehyde dimethyl acetal and diisopropyl azodicarboxylate (DIAD), with the concomitant leaving of isopropanol. A similar process was previously reported for

The *O*,*O*-acetals **3a** and **3b** were formed after treatment of the *p*-toluenesulfonic

acid-mediated cyclization from the acyclic acetals **7a** and **7b**, using anhydrous

diethyl azodicarboxylate (DEAD) but not when DIAD was used [25].

the sulfonyl chloride was added (**Figure 3**) [19].

**130**

was not isolated.

**Figure 2.**

*Reagents and conditions: (a) p-O2N-C6H4-SO2Cl (1.1 equiv), TEA (1.5 equiv) and anhydrous CH2Cl2, rt., 5 h when 0.5 equiv. of p-O2N-C6H4-SO2Cl was used, see* **Figure 2** *(conversion 4 → 5b) [19].*

#### **Figure 4.**

*Reagents and conditions: (a) HOCH2CH(OMe)2 (4.3 equiv), DIAD (1.2 equiv), PPh3 (1.2 equiv) and anhydrous THF, rt., 18; h; when 1.2 equiv. of HOCH2CH(OMe)2 were used, see* **Figure 2** *(conversion 5b → 6b) [19].*

#### **Figure 5.**

*Reagents and conditions: (a) Ph3P (1 equiv), CCl4, 110°C, 30 min [19].*
