Synthesis of Bis(Isoxazol-4-Ylmethylsulfanyl)Alkanes and Some Metal Complexes as a Hepatoprotective Agents

Purpose: This research is devoted to designing the synthesis of sulfanyl-substituted 3,5-dimethylisoxazoles, which contain structural analogues of the SAM drug in the molecule. SAM (S-adenosyl-L-methionine), formed in the biosynthetic process, is used as an effective hepatoprotective drug. Complexation and hepatoprotective properties of the combinatorial series of bis(isoxazolylsulfanyl)ethane have been studied. Methods: Bis(isoxazol-4-ylmethylsulfanyl)alkanes were synthesized using the one-pot method. The structures of compounds were established by one-dimensional (1H,13C) and two-dimensional (COSY, HCQS, HMBC) NMR spectroscopy, mass-spectrometry and X-ray diffraction. The biological activity of the combinatorial series of sulfanyl derivatives of diketones, azoles and their metal complexes has been studied by in vivo method. Simulation of the animal associated processes was carried out in accordance with the principles of bioethics. Screening studies of hepatoprotective activity were carried out in a model of acute CC14 intoxication after a single injection intraperitoneally as a 50% solution in olive oil. The pharmacologically known hepatoprotective drug SAM served as a control. Results: Two-step synthesis of novel α,ω-bis(isoxazol-4-ylmethylsulfanyl)alkanes was carried out via the multicomponent reaction between 2,4-pentandione, CH2O and α,ω-dithiols, then the resulting α,ω-bis(1,3-diketone-2-ylmethylsulfanyl)alkanes were transformed by hydroxyl amine to obtain bis-isoxasole derivatives. Promising precursor 1,2-bis(isoxazol-4-ylmethylsulfanyl)ethane was converted to metal complexes by interaction with PdCl2 or CuCl. The obtained compounds were found to be practically non-toxic compounds (1001 – 3000 mg/kg) according to the classification of K.K. Sidorov, but copper complex refers to low-toxic compounds substances (165 mg/kg). Compounds of sulfanyl ethane series demonstrate hepatoprotective activity. Conclusion: Palladium(II) complex being almost non-toxic possesses hepatoprotective activity comparable to the drug like SAM.


Introduction
Currently the use of organometallic complexes in medicine is considered as an innovative approach, due to their unusual activity in biological systems. 1-3 From the standpoint of metal-ligand homeostasis, organic complexes with essential metals forming part of the active site of many enzymes, 4-7 are very promising for the treatment of pathological states. 8 Recently 9 it has been found that baicolin-copper complex is effective hepatoprotective agent unlike baicalin itself. Breakthrough event was the discovery in the late 20thcentury therapeutic properties of cisplatin, 10 and other platinum complexes against cancer, 11,12 which, unfortunately, have significant toxic side effects. 13,14 Later it was shown that the less toxic palladium(II) complexes were also effective for the treatment of cancer. Nowadays there is the interest of researchers to look for low-toxic organometallic complexes with an effective anti-tumor [15][16][17][18] or hepatoprotective activity. 19 We have previously reported the one-pot effective synthesis of α,ω-bis(1,3-diketone-2ylmethylsulfanyl)alkanes, 20 which are promising precursors for methylsulfanyl substituted α,ω-bispyrazoles with pronounced inhibitory effects on alphaamylase activity. [21][22][23] Taking into account, that isoxazoles exhibit pharmacological properties, [24][25][26][27] and sulfanyl substituted isoxazoles are polydentate ligands, 28,29 our aim was to carry out the synthesis of novel complexes of Pd(II) and Cu(I) with 1,2-bis[(3,5-dimethylisoxazol-4yl)methylsulfanyl] ligands and examine the toxicological and hepatoprotective properties of metallo-complexes and precursors thereof.

General procedures and materials
The reaction products were characterized by 1 H and 13 C NMR spectra that were recorded on spectrometers Bruker Avance 400 NMR (400.13 MHz and 100.62 MHz) and Bruker Ascend III HD 500 (500.17 MHz and 125.78 MHz), internal standard TMS, solvent DMSO-d6. The homo-and heteronuclear 2D experiments were performed by the standard pulse sequences of Bruker. IR spectra were recorded on a Bruker Vertex-70V FTIR and Specord M80 spectrometers. Electrospray ionization (ESI) mass spectra were obtained on a HPLC massspectrometer LCMS-2010EV (Shimadzu) in positive and negative ions mode at the corona discharge needle ionizing electrode and ionizing capillary potential of -3.5 kV. Sample solution (direct syringe sample inlet) under ESI conditions was in methanol (acetonitrile), mobile phase was acetonitrile/water, 95/5. Mass-spectra was recorded on a device MALDI TOF Autoflex III firm Bruker (compounds 2a-e) with sinapinic acid as a matrix (see Supplementary data). Elemental analysis was performed on a Carlo Erba 1106 elemental analyzer. Melting points were determined on a Kofler hot-stage microscope and utilized uncorrected. Individuality and purity of synthesized compounds were controlled by means of TLC on Silufol UV-254 plates; I2 was used as developer.

Crystal Structure Determination and Refinement
The X-ray diffraction experiments of 2c and 2d singlecrystals were carried out by a Bruker SMART 1000 CCD area detector using graphite monochromated MoeKa radiation at 100 K. All calculations were performed on an IBM PC/AT using the SHELXTL software Atomic coordinates, bond lengths, bond angles, and thermal parameters have been deposited at the Cambridge Crystallographic Data Centre (CCDC). X-ray diffraction data of 2c and 2d single-crystal was collected on a XCalibur Eos diffractometer with graphite monochromated Mo-Kα radiation (λ=0.71073 Å). Collection and processing of data performed with using the program CrysAlis Pro Oxford Diffraction Ltd., Version 1.171.36.20. The structure was solved by direct methods as implemented in the program SHELXS-97. 30, 31 The refinement was carried out using SHELXL-97. The structure was refined by a fullmatrix least-square technique using anisotropic thermal parameters for nonhydrogen atoms and a riding model for hydrogen atoms.

Biological assay
Simulation of animal-related processes was carried out in accordance with rules of laboratory practice (GLP) and the ethical norms of the Geneva Convention (1971). Conditions of experiment and keeping animals were carried out according to modern requirements. 32 The tissue was fixed in a 10% solution of neutral formalin for light-optical examination of the liver. For further histological treatment it was used samples of 5-7 mm thickness which were cut from a large proportion of the liver by cross-sectional dissection and subjected to standard treatment on the histological complex MICROM (Carl Zeiss, Germany). Samples were dehydrated in alcohols with increasing concentration, followed by pouring into paraffin blocks. The studies were carried out on mice with a line BALB/CJ weighing 20 -23 g (mice are provided by the Bashkir State Medical University Vivarium, Ufa, Russia). The animals were kept in 10 cells in a cage in standard vivarium conditions at an air temperature of 18 -22 °C and a relative humidity of 50 to 65%. During the process there were free access to water and feed (~ 5 g/day).

Chemistry
The key substrates for the three step synthesis of target metallo-complexes of Pd(II) and Cu(I) was α,ω-

bis[(pentane-2,4-dione-3-yl)methylsulfanyl]alkanes 1a-e
produced by a n-BuONa mediated multicomponent reaction (MCR) between 2,4-pentanedion, CH2O and α,ω-dithiols. 20 The yield of products 1a-e was decreased with increasing the aliphatic chain of α,ω-dithiols from 97 to 54%. Substrates 1a-e was successfully converted into α,ω-bis[sulfanylmethyl(3,5-methylisoxazol-4yl)]alkanes 2a-e with high yields through the interaction between 1a-e and hydroxylamine hydrochloride in refluxing ethanol during 2 h (Figure 1). It should be noted, that in the indicated above two-step sequence, the total yield of 2a is higher than in the case of one-pot four-component reaction between 2,4pentandione, CH2O, 1,2-ethanedithiol and NH2OH·HCl. 21 According to X-ray data (Figure 2), compound 2c is crystallized in the monoclinic and 2d -in orthorhombic crystalline system. It was found that bis(3,5dimethylizoxazol) rings are in the cis-conformation with respect to S-(C)n-S fragment for compound 2c and transconfiguration for compound 2d. The crystallographic data for compounds 2с and 2d are collected in Table 1. Among the synthesized 1,2-bis[sulfanylmethyl(3,5dimethylisoxazol-4-yl)]alkanes 2a-e, 2a is the most promising for practical application taking into account the production efficiency and availability of the starting reagents. So that, 1,2-bis[sulfanylmethyl(3,5-dimethylisoxazol-4yl)]ethane 2a was then transformed to Pd(II) complex 3 by reaction with PdCl2 in CH3CN. According NMR analysis, Pd(II) complex 3 in solution is the mixture of diastereomers (see Supplementary data). Using CuCl under the same reaction conditions Cu(I) complex 4 was also produced in 97% yield (Figure 3). According elemental analysis for complex 3 the ligand-metal ratio was 1:1 and for 4 as 1:2. In IR spectra of complexes 3 and 4 there are signals of M-S: bonds is Pd-S in region 334 cm -1 and Cu-S in region 320 cm -1 Biology  Combinatorial series of compounds 1a, 2a, 3, and 4 having the same alkylsulfanyl chain and different cyclic fragments of substitutes were assessed as hepatoprotective agents. They were used as solutions in TWIN oil. As known, the SAM drug (S-adenosyl-L-methionine 5, Figure 4) being produced via biosynthetic process is used for treatment of large group of diseases associated with the hepatotoxic action of the chemicals or the alcohol causing morphological changes in liver tissue, metabolism disorder or the toxic liver damages 33-35 SAM drug is considered as non-toxic sulfanyl-containing hepatoprotectors. 36 It provides a stability of hepatocytes. 37 Moreover, the transsulfuration (synthesis and turnover of glutathione and taurine, as well as conjugation and detoxication of bile acids and other xenobiotics), aminoproliliration and transmethylation processes are activated. Obviously SAM is a powerful antioxidant due to its sulfur atoms and heterocyclic fragments in the structure. 38,39 As seen, compounds 2a-e, 3, 4 also contain these units.

Parameters of acute toxicity
To get reliable results acute toxicity was determined with Litchfield and Wilcoxon method modified by Prozorovskiy. 40 As a result of determining the comparative evaluation of acute toxicity in albino mice after intraperitoneal injection and oral administration, it was established that compounds 1a, 2a and 3 are assigned to the group of virtually non-toxic compounds (1001 -3000 mg/kg) according to the Sidorov classification (Table 2). 40 After oral administration, LD50 value does not differ essentially from those of intraperitoneal administration. Thus, structure-activity relationship shows that sulphanyl bis-diketone 1a is less toxical compound (Sidorov classification, non-toxic group). It is not trivial fact that Pd(II) cis-chelate S,S-complex 3 also refers to group being not toxic. As seen from the Table 1, Cu(I) cischelate S,S-complex 4 is more toxical compound (Sidorov classification, low-toxic group). For this reason, compound 4 is not promising to treat liver diseases.

The model of acute hepatitis
Screening studies of compounds 1a and 3 were carried out on a model of acute toxicity in vivo. Simulation of animal-related processes was carried out with the principles of bioethics. The animals received single dose of CCl4 0.2 mL/kg intraperitoneally as a 50 percent solution of olive oil. The compound was administered intraperitoneally in a dose of 25 mg/kg 1 hour before the injection of CCl4. As control was used SAM 5 (ademetionine), a pharmacologically known hepatoprotective drug in a dose of 25 mg/kg. The control group received 0.2 mL of saline solution (Table 3). Thus, compound 3 in dose of 25 mg/kg has a strong antitoxic effect on the model of acute intoxication of CCl4. In other words the compound 3 in the dose of 25 mg/kg causes significant lowering of lethality from 50 percent to 0 percent, compared with untreated animals in control group. Biochemical analysis of blood were taken on the 10-th day of observation of acute hepatitis, caused with CCl4, to control the development of cytolytic syndrome and evaluate degree of liver injury. The complex 3 at dose 25 mg/kg resulted in a significant lowering (p<0.05) alanine aminotransferase by 70% comparing with untreated group of animals. At the same time the reference preparation ademetionine at the same dose resulted a significant lowering only by 53% (p<0.05) (Table 4). Compounds 1a and 2a are less active according to this indicator. A significant lowering of aspartate aminotransferase between control and experimental groups was not recorded. By the 10 day of observation in all groups with an acute hepatitis the level of conjugated bilirubin raised. Compared to the intact group of animals figures receiving compound 3by 20.2%, 2aat 55.9%, 1a -58.8%, SAM -25.9% in the group with the drug 3 -less often.  Note: * -significant differences between indicators of intact animals, ** -significant differences from that of group CCl4

Histological examination
Staining with hematoxylin, eosin, by standard methods on histological complex MICROM. Histological activity index (HAI) was defined. Evaluation System indicators protein dystrophy, inflammatory infiltration, hyaline drop dystrophy -a 4-point scale.
Administering CCl4 without treatment led to gross structural changes in the form of large-drop dystrophy of hepatocytes, lymphohistiocytic infiltration of the liver structure. Compounds pretreatment and heptral at dose 25 mg/kg led to less expression of morphological changes of liver structures: reduction of inflammatory infiltration, necrosis of hepatocytes, hepatocyte degeneration reduction degree. Semi-quantitative method for assessing the degree of activity of pathological processes in the liver showed: 1) Significant reduction in HAI compared with results of control group was observed during therapy with Compound 3 and heptral. Compounds 1a and 2a had no significant digits. 2) The degree of fatty liver among white mice, treated with Compound 3 was minimal, hepatocytes with fatty inclusions are located only on the periphery of the hepatic lobule. Other animal groups 1a and 2a had moderate degree -1/3 -1/4 the length of the hepatic beams, hearths cirrhosis, liver tissue was sealed. A new sensibly nontoxic (IV class) compound cis-S,Sdichloride-1,6-(3,5-dimethylisoxazol-4-yl)-2,5dithiahexane palladium(II) complex 3 with hepatoprotective activity in laboratory animals (white mice) at a dose of 25 mg/kg intraperitoneally on acute hepatitis model induced by carbon tetrachloride was discovered. Compound 3 exeled the reference preparation ademetionine (SAM) for indications: a) animal survival (100%, SAM -80%); b) biochemical (ALT, AST, bilirubin direct) parameters; c) histological (liver parenchyma lesions are minimal) parameters.
On the basis of biochemical tests (ALT, AST, bilirubin) and histological compounds displayed hepatoprotective activity which decreased in the number of 3 ˃ 1a ˃ 2a.

Ethical Issues
The study was carried out under ethical principles. Permission from the Local Ethics Committee of Bashkir state medical university is presented in supporting information.