## 1 Introduction  
Phenothiazazines are completely synthetic heterocyclic systems that have no precursor or equivalent in the world of living nature [1; 2]. Although they were discovered more than 140 years ago, to this day, the molecules are still very popular in the world of medical, pharmaceutical, as well as chemical research [3; 4; 5].  
In the 1950s, phenothiazazines containing dialkylaminoalkyl substituents in their structure triggered revolutionary changes in psychiatry by showing phenomenal antipsychotic activity to treat schizophrenia or manic states. To this day, these drugs are still used in medicine [1]. Scientific research in the area of phenothiazazines is constantly being carried out, revealing many further interesting pharmacological activities, including anticancer [6; 7; 8; 9; 10], antimicrobial [11; 12], antifungal [13], antiviral [14; 15], antioxidant [16], or the reversal of multidrug resistance activities [17]. This topic is so rich that its results are published in numerous review papers each year around the world [18; 19; 20; 21; 22]. In 2006, English researcher [PERSON] named the phenothiazazine \"parent molecule\", which fully reflects the application nature of this chemical compound [22].  
The structure of phenothiazazines is also modified at various levels, which leads to the creation of new, previously undescribed derivatives with interesting biological activities [23]. Such a group of modified phenothiazazines includes dipyridothiazazines having two pyridine rings in their structure instead of benzene rings [21]. Dipyridothiazines have a valuable anticancer effect against numerous cancers, such as melanoma, glioma, lung, colon, breast, and ovarian cancer, consisting of the induction of apoptosis through its mitochondrial activation and the possibility of partial DNA intercalation [21; 24]. Dipyridothiazines also showed significant immunosuppressive and anti-inflammatory activity by inhibiting TNF alpha and interleukin 6 levels, as well as low cytotoxicity towards normal cells [21].  
Dimeric derivatives of dipyridothiazines, in which two dipyridothiazine units were connected by selected organic linkers, such as \(o\)-xylene, \(p\)-xylene, and 2,6-dimethylpyridine (lutidine), have recently shown highly promising anticancer activities in relation to colon (SW480) and breast (MCF 7) cancer lines. Additionally, these compounds were characterized by their relatively low cytotoxicity in relation to normal myoblast (L6) cell lines [25]. Among dimeric dipyridothiazine derivatives, the highest anticancer potential was demonstrated by compounds that contained a linker in their structure, which was 2,6-dimethylpyridine, i.e., lutidine. Additionally, pyridine and its substituted derivatives are known as six-membered heterocycles with broad pharmacological activity profiles [26; 27].  
The idea of this work was to synthesize new dimeric derivatives of dipyridothiazines (1,6-, 1,8-, 2,7-, and 3,6-diazphenothiazazines **1-4**) with an \(m\)-xylene linker (**6-9**) as structural analogs of the early described dimers with the lutidine moiety (**6a-9a**) [25] (Scheme 1, Figure 1) in order to comparatively analyze their biological activity and determine the structure-activity relationship in the area of in vitro anticancer research.  
The structure of the new compounds was clearly confirmed by the NMR, 2D NMR spectroscopy, and mass spectrometry HR MS. In silico analyses of probable molecular targets were performed using the Way2 Drug server. The compounds were tested for anticancer activity in relation to the following cancer cell lines: human primary colon cancer (SW480), human metastatic colon cancer (SW620), human breast adenocarcinoma (MDA-MB-231), human lung carcinoma (A-549), and human glioblastoma (LN-229), and the human immortal keratinocyte cell line from adult human skin (HaCaT). To elucidate the structure-activity relationship, quantum mechanical calculations and a molecular docking study were performed against the selected histone deacetylase target.