**2.2 Synthesis of tetrahydrobenzoxazepine 2,6-dichloropurine** *O,N***-acetals with nitrosulfonyl groups on the nitrogen atom**

The preparation of the *O*,*N*-acetals **1** and **2** was achieved by the microwaveassisted Vorbrüggen one-pot condensation of the cyclic acetals **3a** and **3b** [27] and the commercially available purine base 2,6-dichloropurine, using trimethylsilyl chloride (TMSCl), 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and tin(IV) chloride as the Lewis acid in anhydrous acetonitrile. The reaction mixture was microwaveirradiated at a temperature of 140°C for 5 min (**Figure 6**).

Compounds **11** and **12** were obtained along with the cyclic 2,6-dichloropurine *O,N*-acetals (**1** and **2**, bozepinib) and the acyclic one **13** (when starting from **3b**) in the reaction of purines with **3a** and **3b**, respectively. The mechanism of the reaction of these compounds is important as none of them were previously isolated in the

#### **Figure 6.**

*Reagents and conditions: (a) 3a (1 equiv), 2,6-dichloropurine (1.5 equiv), trimethylsilyl chloride (TMSCl, 1.5 equiv), 1,1,1,3,3,3-hexamethyldisilazane (HMDS, 1.5 equiv) and SnCl4 (1 M solution in CH2Cl2, 1.5 equiv), 140°C, microwave, 5 min; (b) 3b (1 equiv), 2,6-dichloropurine (2.5 equiv), TMSCl (4.0 equiv), HMDS (4.0 equiv) and SnCl4 (1 M solution in CH2Cl2, 4.0 equiv), 140°C, microwave, 5 min.*

**133**

*a*

*b*

*c*

**Table 1.**

*determinations. The treatment time was 48 h.*

*MDA-MB-231 and the non-cancerous cell line MCF-10A.*

*N.D. = not determined.*

*Anti-proliferative activitiesa*

*Taken from Ref. [31].*

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

Compound **2** was resolved into its two enantiomers: [(*R*)-**2**: [α]

column CHIRALPAK® and a mixture of hexane/*t*-BuOMe/*i*PrOH as eluant [28]. From this moment on, the racemic **2** [(*RS*)-**2**] will be named only as bozepinib.

**Table 1** shows the anti-proliferative activity (IC50 values) for **1**, bozepinib and 5-fluorouracil (5-FU) as a reference drug. Compounds were first assayed as antiproliferative agents against the human breast adenocarcinoma cell line MCF-7 (p53 wild type and ras mutated). Compounds **1** and bozepinib were further assayed against the human breast cancer cell line MDA-MB-231 which presents high levels of mutant p53 [28, 30]. The IC50 = 0.166 μM for bozepinib against the human

In order to determine the in vitro therapeutic index of the compounds, they were assayed against the non-cancerous human mammary epithelial cell line MCF-1oA. The TI of a drug is defined as the ratio of the toxic dose to the therapeutic dose (in vitro TI = IC50 non-tumor cell line/IC50 tumor cell line) [28]. Bozepinib is more selective against both human breast adenocarcinoma MCF-7 and MDA-MB-231 cancer cell lines (TIs = 5.14 and 11.0, respectively) in relation to the normal one

As bozepinib is more active and more selective and more active than its isomer **1**, we decided to carry out the separation of bozepinib into their component enantiomers (resolution). (*S*)-**2** shows higher anti-proliferative effect that of (*R*)-**2** in the MCF-7 cell line. However no differences against the MDA-MB-231 cell line were observed (**Table 3**). The enantioselective cytotoxicity indicates that the

**Compound IC50 MCF-7 (μM) IC50 MDA-MB-231 (μM) IC50 MCF-10A(μM) 1** 0.383 ± 0.027 0.280 ± 0.006 1.530 ± 0.198 Bozepinib 0.355 ± 0.011 0.166 ± 0.063 1.825 ± 0.503 5-FUc 4.32 ± 0.020 N.D.b N.D.b

*All experiments were conducted in duplicate and gave similar results. Data are means ± SEM of three independent* 

 *for compounds 1 and bozepinib against the cancerous cell lines MCF-7 and* 

25

corresponding reactions with uracil or 5-FU [27]. We have previously explained the mechanism of the reaction leading to the benzo-fused derivatives **1**, **2**, **11** and **12** [28].

A better understanding of the molecular recognition of the therapeutic targets in many diseases highlights the issue of drug in the design and development of new drugs. The separation of racemates, chiral pool and asymmetric synthesis are the three most used methods for the production of a chiral drug. Since the 1980s there has been a significant increase in the development of chiral pharmaceutical drugs. When patents of racemic drugs expire, pharmaceutical companies can extend patents by developing the synthesis of enantiomers that exhibit the desired biologi-

25

D = +41.0 (c = 0.23, THF)]; using a semipreparative

D = −43.6

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

**2.3 Homochiral drugs**

cal activity [29].

(c = 0.22, THF) and (*S*)-**2**: [α]

cancerous cell line MDA-MB-231 stands out.

**2.4 Biological studies**

(**Table 2**).

corresponding reactions with uracil or 5-FU [27]. We have previously explained the mechanism of the reaction leading to the benzo-fused derivatives **1**, **2**, **11** and **12** [28].
