Synthesis, Characterization and Molecular Structures of some Bismuth(III) Complexes with Thiosemicarbazones and Dithiocarbazonic Acid Methylester Derivatives with Activity against Helicobacter Pylori

The reactions of bismuth(III) nitrate pentahydrate and bismuth(III) chloride with heterocyclic thiosemicarbazones and derivatives of dithiocarbazonic acid methylester were used to synthesize the respective bismuth(III) complexes, which could be divided into five groups D-H because of their stoichiometrical properties and their molecular structures. The molecular structure and the near coordination sphere of the bismuth(III) central atom of four representative compounds were determined by single-crystal X-ray studies. Bis[1-azepanyl-4-(2-pyridyl)-2,3-diazapenta-1,3-diene-1-thiolato-N′,N3,S]bismuth(III) nitrate (5) belongs to group D. The two tridentate ligands and the nitrate ion surround the bismuth atom. The best description of the coordination sphere appears to be that of a distorted trigonal dodecahedron with one position occupied by the lone pair of the bismuth atom. Bis[1-azepanyl-4-(2-thienyl)-2,3-diazapenta-1,3-diene-1-thiolato-N3,S]bismuth(III) nitrate (9) is assigned to complex type E. Here, two deprotonated ligand molecules are coordinated to the bismuth(III) central atom as bidentate ligands. The structure of this complex can best be described as a distorted trigonal antiprism with a five-coordinated central atom. The two triangular faces are formed by the atoms S(4), N(6), O(11) and S(3), N(4) and the lone pair of the central atom. The two chelate rings are almost perpendicular to each other. Complex molecules of group F form dimeric units with bichloro-bridged bismuth atoms. The structure of di-μ-chlorobis[1-azepanyl-4-(2-pyridyl)-2,3-diazapenta-1,3-diene-1-thiolato-N′,N3,S-chloro]dibismuth(III) (15) can be described as two six-coordinated bismuth atoms, which are bound together via two bridging chlorine atoms. The two bismuth atoms Bi(1) and Bi(1a) and the two bridging chlorine atoms Cl(2) and Cl(2a) form the Bi2Cl2 plane. The two tridentate ligand molecules coordinate via the same atoms as shown in complex 5. In addition, they form two parallel planes, which are perpendicular to the Bi2Cl2 plane. With regard to the center of the Bi(1)-Bi(2) axis they are central point symmetrical, i.e. one pyridine ring lies above and the other beneath the Bi2Cl2 plane. Bismuth(III) chloride and pyridine-2-carboxaldehydethiosemicarbazone 1 b or 2-acetylpyridine-thiosemicarbazone 1 c form complexes of group G. Three chlorine atoms and a bidentate ligand are coordinated to the bismuth(III) central atom. The bidentate ligand bound to the central atom through the N(3) atom and the sulfur atom of the thioketo group. The structure of 18 is completely different from the structures of the bismuth(III) complexes discussed so far and was therefore assigned to group H. The bismuth central atom is coordinated with two ligands, which are bound in different ways. One of them is deprotonated. This ligand is bound to the central atom via the sulfur atom S(3) of the thiolate group and the N(5) atom. An interaction between the sulfur atom of the thiophene ring and the bismuth atom is not possible.The other ligand molecule is not deprotonated. This ligand is bound to the bismuth(III) cation merely via the sulfur atom S(1) of the thioketo group. The best description of the coordination sphere of the bismuth atom is that of a distorted square bipyramidal polyhedron. The square face is formed by the atoms S(3), N(5), Cl(1), the lone pair and the bismuth atom within. The axial positions are occupied by the atoms S(1) and Cl(2). The bond angle between S(1), Bi(1) and Cl(2) differs by about eight degrees from the value determined for a regular square bipyramidal polyhedron of 180 degrees. Some of the newly synthesized bismuth complexes and three ligands have been tested against several strains of Helicobacter pylori bacteria in an agar dilution test. Almost all of the listed bismuth complexes show excellent inhibitory properties with regard to growth of H. pylori already at low concentrations.


INTRODUCTION
The stomach bacterium Helicobacter pylori (H. pylorl}, which was discovered as late as 1983, is a pathogenic factor in the etiology of chronic gastritis and peptic ulcer. 1 Today, bismuth complexes such as colloidal bismuth citrate (CBS), of which the X-ray crystal structure is known, are used more frequently in the treatment of these diseases. 67 In the course of our research into the development of new compounds with better antibacterial activity we found that thiosemicarbazones would be suited for our intentions. Thiosemicarbazones have been known as potential antiviral and antitumor agents since 1950. The first investigations with thiosemicarbazones were carried out by Domagk, who observed considerable activity against streptococcus. Various thiosemicarbazones have been shown to possess an increased activity against influenza viruses, against a high number of protozoa, plasmodium berghei in mice and also against some types of tumor. 611 Coordination of some thiosemicarbazones with transition metal ions such as copper(ll), nickel(ll) etc. often increases their biological activities. 2 Thiosemicarbazones coordinate as multidentate ligands both to transition metals and nontransition metals. Such complexes could be synthesized with, for example, iron, cobalt, nickel, copper, silicon and lead as central atoms. 31 The first bismuth(Ill) complex with a thiosemicarbazone derivative as ligand was published in 1992. TM We now wish to report syntheses and single-crystal studies of bismuth(Ill) compounds with several thiosemicarbazones and dithiocarbazonic acid methylesters. Some complexes have also been tested for antibacterial activity against strains of H. pylori. Details of the syntheses, crystal structures, and tests of antibacterial activity will be presented herein.
Activity of some bismuth(Ill) complexes against H. pylori. A number of bismuth(Ill) complexes with thiosemicarbazone and dithiocarbazonic acid methylester derivatives as ligands and some ligands themselves have been screened in an agar dilution test for their activity against H. py-Iori. Five different strains of H. pylori are incubated for 24 hours under microaerophilic conditions (5 % CO) in brain heart infusions at 37 C and supplemented with yeast extract (0.25 %), hermin (1%) and horse serum (10 %). The bismuth complexes, having been dissolved in DMSO, are diluted with water in a geometrical dilution series to the corresponding concentrations. These solutions are given onto blood agar plates (4 % sheep blood) in the order of falling concentrations 512 2 pg/ml. 20 pl of the bacterial suspension are spot inoculated on the blood agar plates containing the dilutions of the bismuth complexes. After absorption of the suspensions, the inoculum plates are incubated at 37 C in anaerobic jars with Anaerocult C (Merck No. 16275), about 8-10 % by volume CO2 and 5-7 % by volume O2 for 5-6 days. The lowest concentration of the bismuth complexes leading to complete inhibition of bacterial growth was determined (in /Jg/ml agar). Reading of these minimal inhibitory concentration (MIC) results is rather easy, reliable and reproducible with the agar-dilution method whereas the broth-dilution leads to deviating results due to difficulties interpreting the turbidity. Growth control experiments for nonsupplemented blood agar plates and blood agar plates containing DMSO (2 %) for all strains are included for all strains. could not be observed under the conditions of the agar dilution test, the antibacterial activity of the bismuth complex 6 depends not only upon the ligand's activity itself. In relation to compound 16, the RLC is 172.0 nmol/ml, so this compound is less effective against H.p. bacteria than the ligand. The new bismuth compounds could be used instead of bismuth citrate in the triple-therapy, which consists of a bismuth compound, amoxicillin and metronidazole as an alternative to the combination of the ATP-ase-inhibitor opremazole with either amoxicillin or clarithromycin. 2'21 Both therapeutic regimes lead to an eradication rate of about 85-90 % and have been proven to be the most successful regimes in many different studies.

Results and Discussion
Reaction of bismuth(Ill)chloride (BiCI3) or bismuth(Ill)nitrate Bi(NO3)3 5 H20 with thiosemicarbazones and dithiocarbazonic acid methylester derivatives forms bismuth(Ill)complexes with different molecular structures and stoichiometrical content. Therefore the synthesized bismuth compounds are divided into five groups named D H. It was possible to resolve the molecular structures of four representative complexes (5, 9, 15, 18) by means of crystal structure analysis.
In the bismuth complexes of group D (1-8), two tridentate ligands and one nitrate are coordinated to the bismuth(Ill) central atom, the latter via one of its oxygen atoms. In a keto enol tautomerism, the thioketo group is converted into a thiolate group while the proton of the H-N(2) group is split off. Owing to the additional C=N double bond, a conjugated double binding system is formed in the ligand, which contributes considerably to the stabilization of the complexes. Scheme 2 displays the thioketo-enol tautomerism and the complexation possibilities of the pyridine ring containing ligands.
Scheme 2. Thioketo-enol tautomerism and complexation possibilities of thiosemicarbazones containing a pyridine heterocycle Iol. 2, No. 5, 1995 Synthesis Characterization andMolecular Structures The ligands are bound, as tridentate ligands, to the central atom through the nitrogen atom of the pyridine ring, the nitrogen atom of the C N double bond and the sulfur atom of the thiolate group. The non-bound thiosemicarbazone and dithiocarbazonic acid methylester derivatives can have different stereoisomeric forms (e.g. cis-trans-isomerism with respect to the C =N double bond). Therefore, the 1H NMR spectra of the free ligands show several signals for the same proton. In the 1H NMR spectra of the bismuth(Ill) complexes of type D there is, in each case, exactly one signal for the aromatic and magnetically equivalent protons; i.e., the bound ligand has exactly the stereoisomeric form that has been mentioned above. For the four protons of the pyridine ring H-7 and H-10, two doublets are observed and for H-8 and H-9 one doublet in the range between 7.5 and 9.0 ppm. The pyridine nitrogen binds through the free electron pair. The decreased electron density in the ring system effects a deshielding of the protons and thus a shift to low field by 0.1 0.4 ppm. This downfield shift can be observed in the complexes of group D and F.
The structure of compound 5, a typical representative of the complex group D, can be described as follows. Two deprotonated molecules of 1 g are configurated to the bismuth(Ill) cation as tridentate ligands. Except for the hexamethyleneimine ring, the ligand lies on the same plane. The bismuth atom is seven coordinated, as shown in Figure 1. The best description appears to be that of a distorted trigonal dodecahedron with one position occupied by the lone pair of the bismuth atom. Due to this, the Bi(1)-S(2) bond distance near to this electron pair is somewhat longer than the Bi(1)-S(1) bond distance. All of the calculated Bi-N distances differ from each other, the distances between the central bismuth atom and the Natom of the pyridine rings (N(1), N(5)) are longer than the distances between Bi(1) and the C=N bounded N(2) and N(6). The bonding distance between Bi (1)  Metal Based Drugs W. Opferkueh and B.K. Keppler In the bismuth complexes of group E each time two thiosemicarbazone or dithiocarbazonic acid methylester derivatives are coordinated to the bismuth(Ill) central atom as bidentate ligands via the N(3) atom and the S atom of the thiolate group. There is no coordinative bond between the sulfur atom in the thiophene ring or the oxygen atom in the furan ring and the bismuth central atom. The 1H NMR spectra of the bismuth complexes from group E show one doublet for the ring protons H-7 and H-9 and a doublet of doublets for the proton H-8 in the range between 6.5-8.1 ppm. In contrast to the free ligands, the shift of the signals is negligible. Molar conductivities A M, measured in DMF, lie in the range of 47.6-60.6 [Scm2mol1] and prove that this complex behaves as a 1:1 electrolyte. 28 Compound 9 is assigned to complex type E, its structure is given in Figure 2. Here, two molecules of 1 k, deprotonated in positions N(4) and N(6), are coordinated to the bismuth(Ill) central atom as bidentate ligands. The complex bonds are formed by the sulfur atoms of the thiolate groups S(3) and S(4) and by the nitrogen atoms N(4) and N(6). With the exception of the hexamethyleneimino ring, the whole ligand lies within a single plane. The structure of the complex can best be described as a distorted trigonal antiprism, while the central atom is fivecoordinated. The two triangular faces are formed by the atoms S(4), N(6), O(1 1) and S(3), N(4) and the lone pair of the central atom. The two chelate rings are almost perpendicular to each other. The Bi(1)-O(11) distance of 289 pm is considered to be an intermolecular interaction, as mentioned before in compound 5. The methanol molecules required for crystal formation can be found in the gaps of the crystal lattice. In the FD mass spectrum, a peak is obtained at m/z 769 for the fragment [(Lig)2Bi+]. This is determined by deducting the mass for the methanol molecule and a nitrate ion from the molar peak. The complexes of group F are dimeric bismuth(Ill) complexes. The two bismuth central atoms are bridged via two chlorine atoms. The tridentate chelate ligands are perpendicular to the Bi2CI2-plane and can have a parallel or antiparallel arrangement. The proton of the N(2)-H group shows a singlet 1H NMR signal, which can be observed in the range between 9.5 and 1.5 ppm in the case of the free ligands, it disappears completely in the case of the bismuth complexes of groups D and F. In the 1H NMR spectra, most of the bismuth(Ill) complexes of group F show a second, weak signal for the protons H-IO and H-13 and in some cases also for the protons H-9 and H-14. This additional signal is caused by the different possibilities in which the tridentate ligand can bind to the bismuth atom, as shown in Scheme 2. Complex 15 is an example of a dimeric bismuth(Ill) complex of group F. Its structure, as shown in Figure 3, can be described as two six-coordinated bismuth atoms, which are bound together via two bridging chlorine atoms. ORTEP plot of the dimeric unit of Di-p-chlorobis[1-azepanyl-4-12-pyridyl)-2,3diazapenta-1,3-diene--thiolato-N', N3,S-chloro]dibismuth(lll) ( 1 5) The two bismuth atoms Bi(1) and Bi(la) and the two bridging chlorine atoms (31(2) and Cl (2a) form the Bi2CI plane. The non-bridging chlorine atoms C1(1), Cl(la) almost lie in the Bi2CI2 plane. The greater bond length Bi(1)-CI(2) of 279.1 pm in comparison to 258.5 pm in the case of Bi(1)-CI(1) is an indication of the bridging function of the C1(2) atom. The Bi(1)-Cl(2a) distance was determined as 316.2 pm. This is a relatively great bond length for a Bi-CI bond but can be considered as a covalent bond. 1 The two tridentate ligand molecules coordinate via the same atoms as shown in complex 5. In addition, they form two parallel planes, which are perpendicular to the Bi2CI plane. With regard to the center of the Bi(1 )-Bi(2) axis they are central point symmetrical, i.e. one pyridine ring lies above and the other beneath the BiCI2 plane. group has the deprotonated form. For the N(2)-H proton, singulets are observed at 11.78 ppm (19) and at 11.35 ppm (20). If the 13C NMR spectrum of 1 b is compared with the lzC NMR spectra of the bismuth complexes 1 and 19, synthesized with these ligands, it can be demonstrated that the ligand is bound to the bismuth(Ill) central atom in different ways. While the carbon signals in complex 19 show only a minor shift against the free ligand, they show markedly changed chemical shifts in complex 1. The complex bond to the N(3) atom, which is formed during the complexation of 1 b to the bismuth(Ill) cation, effects a polarisation of the C=N azomethine bond. Due to the reduced electron density, the C(4) signal is shifted by 5 ppm to the lower field. The shift into the high field of the C(1) signal is caused by the ketoenol tautomerism at the C(1) atom. The signals of the ring carbon atoms show a marked tendency towards the lower field. Yet our attempts to resolve the molecular structure of this group of bismuth complexes by crystal structure analyses have been unsuccessful.
On analysis of the reaction product of ligand 1 k and bismuth(Ill) chloride, we were surprised to obtain a complex of a fifth group H, the structure of which differs from the other four groups in several ways. The structure of 18 is completely different from the structures of the bismuth(Ill) complexes discussed so far. The bismuth central atom is coordinated with two ligands of 1 k, which are bound in different ways. One of the two ligand molecules is deprotonated. This ligand is bound to the central atom via the sulfur atom S(3) of the thiolate group and the N(5) atom. The thiophene ring is cis-configurated with regard to the N(5)=C(15) bond, with the result that an interaction of S(4) with Bi(1) is not possible. The second molecule of 1 k is not deprotonated. This ligand is bound to the bismuth(Ill) cation merely via the sulfur atom S(1) of the thioketo group. The value for the distance between Bi(1) and S(1) of 299.2 pm corresponds to an intermolecular interaction and not to a covalent bond. The best description of the coordination sphere of the bismuth atom is that of a distorted square bipyramidal polyhedron. The square face is formed by the atoms S(3), N(5), C1(1), the lone pair and the bismuth atom within. The axial positions are occupied by the atoms S(1) and C1(2). The bond angle between S(1), Bi(1) and C1(2) differs by about eight degrees from the value determined for a regular square bipyramidal polyhedron of 180 degrees. In the H NMR spectrum, a singulet can be observed at 9.42 ppm, which is caused by the proton of the N(2)-H group of the protonated ligand. For the protons of the pyridine rings and the H-5 protons, three different signals are mostly found. The different signals can be produced by the possible Z,E isomerism within the protonated ligand on the one hand and by the difference in the binding of the ligands to the R. Diemero U. Dittes, B. Nuber, V.