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common forms of epithelial malignant tumours of the ovary III–IV stages, including those accompanied by ascites. Additionally, compound 1.57 has been shown to be effective in patients previously treated with other alkylating agents, in particular thiophosfamide. [5- [[4,6-bis(aziridin-1-yl)-1,3,5-triazin-2-yl]-amino]-2,2-dimethyl-1,3-dioxan-5-yl]- methanol 1.57 is less toxic than drugs based on platinum coordination compounds (the standard in the treatment of ovarian cancer) and, unlike them, does not cause complications in the form of adhesions [43].
A significant side effect of [5-[[4,6-bis(aziridin-1-yl)-1,3,5-triazin-2-yl]-amino]- 2,2-dimethyl-1,3-dioxan-5-yl]-methanol 1.57 in the therapeutic regimen is myelosuppression [43], while other side effects are not pronounced, unlike other cytotoxic drugs, which, even when administered intraperitoneally, have haematotoxic, ototoxic, neurotoxic, and nephrotoxic effects [48]. When applied topically (intraperitoneally and intrapleurally), compound 1.57 does not have a sclerosing effect, while the administration of cisplatin leads to complications in the form of adhesions. Feature of [5-[[4,6- bis(aziridin-1-yl)-1,3,5-triazin-2-yl]-amino]-2,2-dimethyl-1,3-dioxan-5-yl]-methanol
1.57, according to the authors, is that this compound does not undergo rapid biotransformation, which is important for intraperitoneal chemotherapy of ovarian cancer [42].
It should be especially noted that to date, despite the clinical testing of [5-[[4,6- bis(aziridin-1-yl)-1,3,5-triazin-2-yl]-amino]-2,2 -dimethyl-1,3-dioxan-5-yl]-methanol
1.57, its molecular cellular mechanisms of action have not been established, as well as its biocompatibility. These studies are key in preclinical trials of potential drugs and should be implemented as a priority.
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CHAPTER 2. NANOFORM OF DRUGS
In the last decade of the twentieth century, there was a ‘boom’ of research related to the study of various nanostructures. This has happened mainly because of significant progress in the development of methods for obtaining nanomaterials. To date, new derivatives of several fullerenes, nanodiamonds, graphene, carbon nanotubes, albumin and liposomes have been synthesised [44–51]. An ideal nanocarrier should not have a harmful effect on normal cells, but should meet the requirements of stability, biocompatibility in vivo and the ability to release the drug depending on external conditions (pH, temperature). In early 2005, the albumin form of the anticancer drug paclitaxel was approved for clinical use. Paclitaxel is loaded into albumin nanoparticles using a high-pressure emulsification process. This soluble form of paclitaxel has been shown to not only eliminate side effects [52], but also provide some additional benefits: improve the efficiency of drug delivery from the bloodstream to the tumour and allow higher drug doses to be administered [52].
In a study on the delivery of an HIV-1 protease inhibitor, an antiretroviral agent that inhibits the HIV-1 replication cycle CGP 70726 [53] pH-sensitive nanoparticles made from a copolymer of methacrylic acid and ethyl acrylate were used [54]. Nanoparticles were synthesised by emulsifying a copolymer solution and a mixture of CGP 70726 with benzyl alcohol. The resulting nanomaterial was orally administered to laboratory animals, and effective drug release was observed, which was confirmed by analysis of plasma samples.
A promising direction in the development of targeted drugs is the use of carbon nanostructures. For example, graphene-based nanomaterials have a great potential for application in various fields: in the development of bioimaging [55], biosensors [56], in antifungal [56] and antiviral therapy [56],. Graphene has a unique structure consisting of sp2-hybridised carbon atoms forming two-dimensional nanolayers [56]. There are several approaches that make it possible to obtain nanomaterials based on graphene [52],[55–58]. For the first time, our scientific group has developed a new scalable method for the synthesis of graphene oxide (GO) enriched with oxygen-containing functional groups [53].
Literature analysis reveals a number of works devoted to the synthesis of GO-based conjugates with various cytostatic drugs (Table 2.1) [58–70]. The literature data presented allow us to highlight the following advantages of creating conjugates based on GO:
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1)the possibility of applying various approaches to the synthesis of conjugates (covalent [65] and non-covalent [66] modifications);
2)the possibility of varying the percentage of loading of cytostatics, as well as the possibility of simultaneous loading of two or more cytostatics, as well as the synthesis of conjugates containing cytostatic drugs and a vector for targeted delivery [68];
3)higher therapeutic efficacy and less toxicity [69,70].
Table 2.1. GO-based conjugates with various cytostatic drugs.
Conjugate |
Characteristics |
Reference |
|
|
|
|
|
Paclitaxel-GO- |
Survival of endometrioid ovarian adenocarcinoma |
|
|
folic acid (pGO- |
|
||
cells (A2780) with the introduction of pGO-FA-PTX is |
[65] |
||
FA-PTX) PTX |
|||
less than 30% |
|
||
loading 18.7% |
|
||
|
|
||
Cisplatin-GO |
The survival rate of ovarian adenocarcinoma (SKOV3) |
|
|
cells was 10 % at a concentration of 25 mg·l−1 |
|
||
|
|
||
|
Survival of liver fibrosis (LX2) and SKOV3 cell lines |
[66] |
|
Carboplatin -GO |
at conjugate concentrations from 60 mg·l−1 to 250 |
|
|
|
mg·l−1 was less than 20 % |
|
|
Continuation of Table 1.1. GO-based conjugates with various cytostatic drugs. |
|||
Chlorambucil- |
The survival rate of MCF-7 cells was 28 % at a CLB- |
|
|
GO-folic acid |
[67] |
||
FAGGO concentration of 500 mg·l−1 |
|||
(CLB-FAGGO) |
|
||
|
|
||
Doxorubicin-GO |
The IC50 values for the PC3 and A2780 cell lines were |
[68] |
|
(GO-DOX) |
0.84 µM and 1.55 µM, respectively |
||
|
|||
|
IC50 values for lung fibroblast cell lines (HEL299) and |
|
|
|
A549 were 6.36 ± 1.20 and 9.72 ± 0.37 μM, |
|
|
Gemcitabine-GO |
respectively. |
[69] |
|
Subcutaneous injection of GEM-rGO into A549 |
|||
GEM-rGO |
|
||
xenograft mice resulted in tumour growth inhibition |
|
||
|
|
||
|
twice as effective as free gemcitabine |
|
|
Tamoxifen-GO |
The IC50 value for MCF-7 cell lines was 108 mg·l−1 |
[70] |
|
|
|
|
|
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The low haemocompatibility of unmodified graphenes with blood components and toxicity complicate the application of these nanostructures in medicine. The paper [71] presents the results of a study of the effect of aqueous GO dispersions on blood parameters. It was shown that the level of total bilirubin in laboratory rats treated with GO dispersion (C = 500 mg kg−1) increased compared to the control, while the level of high-density lipoproteins significantly decreased. However, in rats treated with GO dispersions with C
= 50 or 150 mg kg−1, total bilirubin levels remained normal. In the studied blood samples, no significant changes were found in other analysed parameters (for example, cholesterol, creatinine, etc.). A histological study of the liver of rats treated with a GO dispersion (C = 50 mg kg−1) revealed minor morphological changes, and after an increase in the concentration of the GO dispersion (up to C = 500 mg kg−1), a massive focal accumulation of particles was found in rats in the liver capsule. In rats treated with GO at a dose of 150 mg·kg−1, multifocal nephritis was observed in the kidneys. The authors found that the functionalisation of GO leads to an increase in haemocompatibility and a decrease in the toxicity of its aqueous dispersions.
The authors of [72] showed the possibility of using GO for targeted drug delivery using the example of GO functionalised with 1,3,5-triazine (Scheme 2.1). GO was functionalised with triaminotriazine (TAT) using thionyl chloride to obtain conjugate 1.57. The synthesis strategy shown in Scheme 1.22 includes the stage of formation of acid chlorides and allows controlled functionalisation of graphene using various molecules. GO functionalised with TAT was encapsulated using alginate by uniformly dispersing conjugate 1.57.
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Scheme 2.1
In an effort to reduce toxicity and increase the effectiveness of the antitumour effect of 2,4,6-trisubstituted 1,3,5-triazines in the work [73] the authors successfully performed the functionalisation of C60 fullerene with 2,4,6-trichloro-1,3,5-triazine (Scheme 2.2) through the formation of azafulleroid 1.58 and subsequent substitution of chlorine 1.59. When studying the biological activity of 1.58, 1.59, and the possibility of using these substances as fluorescent probes using confocal laser scanning microscopy, the promise of using these substances for imaging tumour cells was shown [76].
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Scheme 2.2
Ref. [77] describes a method for obtaining a conjugate using a 1,3-dipolar addition reaction between CN derivatives of single-walled carbon nanotubes (CNTs) and tetrazolecontaining Cu(II) complexes (Fig. 2.1).
Fig. 2.1. Conjugate of CNTs with tetrazole-containing Cu(II) complexes.
The authors of this study showed that the zone of inhibition of the growth of E. coli and S. aureus bacteria with the addition of nanomaterial (0.01 g) was about 16 and 20 mm, which indicates its significant antibacterial effectiveness.
Thus, from the data presented in the literature review, it can be concluded that 2,4,6- trisubstituted 1,3,5-triazines exhibit various types of biological activity, including antitumour activity. An interesting method for the formation of active scaffolds based on
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1,3,5-triazines is the introduction of heterocycles (obtaining hybrid molecules), which also makes it possible to vary the substituents and the properties of the final structures. The problems of the emergence of resistance and prolongation of the action of the obtained structures can be solved by conjugation of 2,4,6-trisubstituted 1,3,5-triazines with various nanocarriers. It should be noted that the introduction of the tetrazole ring into the structure of 1,3,5-triazine and the study of 1,3,5-triazine nanoconjugates have practically not been used in the development of antitumour compounds; therefore, the preparation of the above structures can be considered extremely promising.
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CHAPTER 3. RESULTS AND DISCUSSION
3.1. Synthesis of compound 1.57
Considering options for modifying the procedure for the synthesis of [5-[[4,6- bis(aziridin-1-yl)-1,3,5-triazin-2-yl]-amino]-2,2-dimethyl-1,3-dioxane-5-yl]-methanol
1.57 a study was made of the possibility of substitution of the chlorine atom in TXT in the reaction with tris(hydroxymethyl)aminomethane and the possibility of cyclisation of the resulting triol to a dioxane fragment.
It was shown that when the reaction mixture was cooled in an aqueous acetone solution, 2-((4,6-dichloro-1,3,5-triazin-2-yl)amino)-2-(hydroxymethyl)propan-1,3-diol (3.1). The next stage of cyclisation took place in the presence of trifluoromethanesulfonic acid, upon addition of which the yield of the target product was 96 % (Scheme 3.1). The substitution of the 2nd and 4th chlorine atoms for aziridine was carried out in acetone in the presence of a base. Substitution of the third chlorine atom under these conditions was difficult, and the reaction mixture was kept at 40 °C for 24 h. The product was purified by column chromatography (chloroform : methanol = 9:1).
Scheme 3.1
Mass spectrometry and NMR spectroscopy data confirmed the composition and structure of compounds 1.57 and 3.1. The proton signals of the aziridine groups are in the range of 2.33–2.36 ppm, and the signals of 1.42–1.44 ppm confirm the presence of two CH3-groups in the dioxane fragment. The structure of compounds 1.57 and 3.1 was additionally confirmed by X-ray diffraction analysis (Fig. 3.1 and 3.2). The geometry of the triazine fragment of compounds 1.57 and 3.1 is close to that of analogous compounds [75]. In the crystal lattice, a pair of molecules of compound 3.1, being bound by two intermolecular hydrogen bonds between H3 O1 atoms, forming a dimer. Fragments in the
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2,4,6-positions of compounds 1.57 and 3.1 are rotated relative to the 1,3,5-triazine ring at an angle from 60 to 90 degrees.
Fig. 3.1. Structure of 3.1 |
Fig. 3.2 Structure of 1.57 |
3.2.Synthesis of hybrid triazinyltetrazole
3.2.1.Synthesis of starting tetrazole-containing derivatives
The tetrazolyl group, as already noted, is a generally recognised pharmacophore used in the construction of leading compounds with an optimal pharmacokinetic profile. The ability of the endocyclic nitrogen atoms of the tetrazole cycle to form hydrogen bonds involved in the formation of stable enzyme-substrate complexes is a significant factor that determines the metabolic activity of tetrazole-containing substrates. In addition, it should also be noted that tetrazoles exhibit relatively low toxicity compared to their linear counterparts, azides [76]. So, at the next stage of the work, aryltetrazoles were synthesised, which were used as precursors for the synthesis of hybrid triazinyl-tetrazoles.
The most popular methods for the synthesis of 5-substituted tetrazoles are based on the reactions of nitriles with salts and other derivatives of hydrazoic acid. Successfully used in laboratory and industrial synthesis of 5-substituted tetrazoles from nitriles traditional methods using various azides. The process of 1,3-dipolar cycloaddition of dimethylammonium azide to nitrile is accompanied by the synchronous formation of two covalent bonds: C–N and N–N, due to the simultaneous overlap of the molecular orbitals of the nitrogen atoms of the azide dipole and the carbon and nitrogen atoms of the nitrile group, which ensures the formation of a tetrazolyl cycle [26,77]. But it should be noted that as a result of this interaction, not NH-tetrazole 3.2 itself is formed directly, but its
180
dimethylammonium salt, for this reason, to isolate the final product 3.2, acidification is carried out with a solution of hydrochloric acid to pH 1–2 (Scheme 3.2).
Scheme 3.2
The composition and structure of 5-phenyl-NH-tetrazole was proved by mass spectrometry (HRESI+-MS) and 1H, 13C{1H} NMR spectroscopy. A characteristic signal confirming the presence of a tetrazolyl ring is the chemical shift of the carbon atom of the heterocycle in the range δ = 149.2–156.8 ppm, which varies depending on the 5-R substituent. In the case of compound 3.2, the tetrazole carbon atom has a chemical shift δ = 156.2 ppm.
For the synthesis of ethyl esters of 5-phenyl-tetrazol-1-yl and 5-phenyl-tetrazol-2- ylacetic acids, the previously obtained 5-phenyl-NH-tetrazole 3.2 was alkylated with chloroacetic acid ethyl ester in acetonitrile in the presence of a base, triethylamine. The reaction proceeded for 20 h at ~50 °C (Scheme 3.3). As a result, a mixture of 3.3ab isomers, tetrazol-1-yl and tetrazol-2-ylacetic acids, was formed. The data obtained are in good agreement with the literature data [78–80]. Compound 3.3b was recrystallised from isopropyl alcohol and used further for the next steps of the synthesis.
Scheme 3.3
The composition and structure of compound 3.3b was proved by mass spectrometry (HRESI+-MS) and 1H, 13C{1H} NMR spectroscopy. The structure is confirmed by the presence of characteristic signals corresponding to the ester group. For example, the signal of the carbon atom of the carbonyl group (C=O) for ethyl ester of 5-(4-hydroxyphenyl)- 2H-tetrazol-2-ylacetic acid 3.3b has a value of 171.5 ppm. Another important point is the difference in the structure of regioisomers, which is clearly displayed on the NMR spectra.
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