## 1 Introduction  
DT-diaphorase (NQO1) is a flavoenzyme isolated by [PERSON] and coworkers over 60 years ago [1, 2]. The enzyme is localized mainly in cytosol, but its low level is also detected in the nucleus. In normal human tissues, a high level of this protein is observed in many epithelial cells and in the vascular endothelium and adipocytes. The NQO1 enzyme is highly expressed in most human solid tumors, such as lung, breast, colon, and pancreas. Moreover, an increase in protein expression is detected in preneoplastic lesion and liver cancer [2, 3, 4, 5, 6, 7]. The crystal structure of human NQO1 was described in 2000 [8]. Human NQO1 is a dimer consisting of two identical subunits, between which there are two active sites composed of residues of polypeptide chains. Each dimer is bound to the flavin adenine dinucleotide (FAD) [3, 9]. The molecular mechanism is based on the direct two-electron-mediated reduction of quinone to a hydroquinone scaffold [10, 11]. The role of NQO1 in cancer treatment has been repeatedly analyzed and described in the literature [2, 4, 12, 13]. Dicoumard is a well-known inhibitor of NQO1, which causes an oxidative stress in the cell (Figure 1). It generates numerous side effects, such as disorders of the blood clotting cycle by inhibiting the vitamin K oxidoreductase [14, 15, 16]. In contrast, streptonigrin (ST) is a substrate for NQO1. By-products of the reaction between streptonigrin (ST) and NQO1 are the reactive oxygen species (ROS) that cause the mutation of DNA (Figure 1). The high toxicity of streptonigrin (ST) prevents its use in cancer treatment [17, 18, 19, 20].  
The study of the structure-activity relationship showed that a key fragment necessary to preserve the bioactivity of streptonigrin (ST) is the 5,8-quinolinedione moiety. Further research has shown that the bioactivity depends on the substituents at the C2, C6, and C7 positions of the 5,8-quinolinedione moiety [19]. The introduction of an amine or alkoxyl1 group at the C6 or/ and C7 positions increases the cytotoxicity against leukemia, melanoma, breast, cervix, ovary, and lung cancers [21; 22; 23; 24; 25; 26]. The halogen atom at the C6 and/or C7 positions also influences the antitumor activity. [PERSON] et al. showed that compounds with chloride atom exhibited higher activity than those with bromide atom [27]. Comparing the activity and biochemical properties of different derivatives of streptonigrin showed that a substituent at the C2 position influences their interaction with metal ions [19].  
Many synthetic 5,8-quinolinedione compounds exhibiting biological activity have been described in the literature [21]. Studies on the modification of 5,8-quinolinedione at the C2 position are rarely found in the literature. The introduction of a methyl group at this position, such as 6,7-dichloro-2-methyl-5,8-quinolinedione **2**, significantly affects the biological activity compared with 6,7-dichloro-5,8-quinolinedione **1**. Moreover, derivative **2** was characterized by lower toxicity against a normal cell line than compound **1**[24; 28].  
In this work, we present the synthesis of new derivatives of 6,7-dichloro-5, 8-quinolonedione, which contain formyl, hydroxyl groups, or chlorine atom as a substituent at the C2 position. These derivatives were characterized by the quantum chemical parameters calculated by the DFT method. The in silico bioavailability of compounds was determined using the pkCSM program. They were also tested as substrates for the NQO1 enzyme. The molecular docking study was used to examine the interaction between derivatives and the NQO1 enzyme.