Synthesis, Characterization and Solution Chemistry of trans-Indazoliumtetrachlorobis(Indazole)Ruthenate(III), a New Anticancer Ruthenium Complex. IR, UV, NMR, HPLC Investigations and Antitumor Activity. Crystal Structures of trans-1-Methyl-Indazoliumtetrachlorobis-(1-Methylindazole)Ruthenate(III) and its Hydrolysis Product trans-Monoaquatrichlorobis-(1-Methylindazole)-Ruthenate(III)

Besides intensive studies into the synthesis of the complex trans-Hlnd[RuCl4(ind)2] (Ind = indazole) 1, which differs remarkably from the usual method for the complexes of the HL[RuCl4L2] - type, competitive products and hydrolysis of this species are described. Stability and pseudo-first-order rate constant under physiological conditions of complex 1 in comparison with the analogous imidazole complex trans-Hlm[RuCl4im2] (Im = imidaZole) ICR were examined by means of HPLC, UV and conductivity measurements (Kobs.(1) = 1.55 × 10-4 s-1; Kobs.(ICR) = 9.10 × 10-4 s-1). An attempt was made to elucidate the bonding conditions in 1 by studying the reactions of Ru(lll) and the two N-methyl isomers of indazole. It can be expected that bonding in the unsubstituted ligand should occur via the N2 nitrogen. The molecular structures of the complex trans-H(1-Melnd)[RuCl4(1-Melnd)2] × 1H2O (1-Melnd = 1-methylindazole) 6 and its hydrolysis product in aqueous solution [RuCl4(H2O)(1-Melnd)2] 7 were determined crystallographically. After anisotropic refinement of F values by least squares, R is 0.053 for 6 and 0.059 for 7. Both complexes crystallize with four molecules in a unit cell of monoclinic symmetry. The space group is P2.1/n for 6 with cell dimensions a = 10.511Å, b = 13.87Å, c = 19.93Å, and β = 98.17° and C2/c for 7 with a = 19.90Å, b = 10.94Å, c = 8.490Å and β = 96.74 ° The fact that the aqua species 7 could be isolated after dissolving 6 in a water/acetone solution confirmed the theory of many Ru(lll) complexes being initially transformed, under physiological conditions, into aqua complexes in a first and often rate-determining hydrolysis step. Compounds 1 and ICR are potent antitumor agents which exhibit activity against a variety of tumor cells and experimental tumor models in animals, including autochthonous colorectal tumors. Clinical studies with 1 are in preparation.


Introduction
Today the development of ruthenium complexes as an alternative to platiniferous tumor inhibitors is of special interest-. Ruthenium complexes with the general formula HL [RuCIL], with L nitrogen hetemcycle, show antitumor activity in different systems in vitro and in vivo and outstanding activity in an autochthonous colorectal tumor model with a tumor reduction of about 70 % to 90% ,-. The water-soluble anionic complexes are characterized by transstanding heterocycles. The first complex of the type mentioned above we synthesized was trans-HIm[RuCl,(im)] (ICR), with two imidazoles as organic ligands . Now we present the analogous indazole complex trans-Hlnd [RuCl,(ind)] (1), which shows higher antitumor activity than ICR in many test systems. The structures of ICR and 1 are shown in Figure 1. trans-Hlnd[RuCl(ind)] (1) is not only active against transplantable tumors but also in a model system with acetoxymethylmethylnitrosamine (AMMN) induced tumors in the colon of rats. These autochthonous tumors induced by intrarectal application of this carcinogen are highly comparable to human colon tumors in their histological appearance as well as in their behaviour against chemotherapeutics. The development of such a new type of drug is of special interest because tumors of the colon account for a high percentage of cancer mortality today. Cl  Extensive studies have been made into the fact that ruthenium complexes with good antitumor effects undergo numerous conversions in vivo and are transformed into the species which is active at the affected area by metabolic processes .T herefore Ru(lll) complexes are better handled as "prodrugs", which are converted in situ into the corresponding, more labile, species. These complexes are able to dissociate one or more non-nitrogen ligands, which in our case are the reactive CI leaving groups, to undergo bonding with the target molecule. It is supposed that in the first step HO-molecules coordinate at the free positions until bonding to a biomolecule. In this paper, the results of these investigations for complex 1 and its derivatives are reported in comparison with the hydrolysis of ICR as well as extensive studies into its synthesis. We discuss the structure of the analogous complex trans-H(1-Melnd)[RuCl(1-Melnd)] x 1HO 6 and its hydrolysis product [RuCla(HO)(1-Melnd)] 7 with 1-methylindazole as organic ligand L.

Materials and Methods
Materials. "RuCI x 3 HO" was obtained from Degussa (Frankfurt, FRG) and was used after purification according to methods which are listed in the following section; indazole was provided by Degussa (Frankfurt / Main), and 1-and 2-methylindazole were produced by reduction of N-substituted o-nitrobenzylidenamine with lithium aluminium hydride according to the method of Bakke and Skjervolds.
General instructions. The purification methods for "RuCI x 3 HO" (production of pure Ru(lll) solutions with different properties) were as follows: Method I: Diluted ruthenium solution. 8 g (28.34 mmol) of RuCI x 3 H=O were heated for one hour under reflux in 50 ml of 1 N HCI and ethanol (50 ml). After cooling the dark red solution down to 40C, the alcohol and the produced acetaldehyde were removed under reduced pressure. The residues were filtered and filled up with 1 N HCI to give a volume of 100 ml. The solution thus obtained contains 28.3 mmol of ruthenium, which is present in the oxidation state +111 exclusively.
The procedure is as described under method I, but instead of 1 N HCI concentrated HCI (12 N) was used.
Method II1" Ruthenium solution in 1 N ethanolic HCI. The procedure is as described under method I, but instead of 1 N HCI solution 1 N ethanolic HCI solution was used and preparation is carried out under nitrogen atmosphere. The alcoholic hydro-chloric acid was produced by letting HCI gas stream through absolute ethanol. The concentration was determined by titration against 1 N NaOH.
Complex Syntheses. The preparation of the complexes with indazole, 1-, and 2-methylindazole as nitrogen ligands can be carried out by the following methods and is highly dependent on exact reaction conditions. The quantity of the heterobases varies according to the desired products and the used ligands. With indazole, five different ruthenium complexes could be isolated and characterized, two of them are new types not yet mentioned in the literature. Crystallization of the complexes was difficult because of hydrolysis in most of the suitable solvents. All new compounds gave satisfactory elemental analyses.
trans-Hlnd[RuCl,(ind)=] (1). General method: Indazole (6.0 g; 51 mmol) was dissolved in 80 ml of 12 N HCI at 70 C. 20 ml of the Ru(lll) solution (method II) were added to the hot solution and the mixture was stirred and heated for about 15 min at 80-90 C. An ochre-colored solid separated from the solution almost immediately. Stirring was continued until ambient temperature was reached and then the crude material was separated and stirred with water for about two hours to remove the HCI residues. Then the solid was filtered and washed with ethanol and diethylether and dried in vacuo. To optimize the yield, the solution was stirred again for several hours at about 80-90C. The purification of the separating solid was carried out as mentioned above. The yield of the microcrystalline product 1 was: 2.96 g (87 %) (decomp.: 248 C; A= 44.0 [-cmmol-]). Anal.
At room temperature, the sham of 1 is between 10% and 25 %, but at low temperatures in the range between 0C and 5 C, the share of this competitive product could be kept below 8 %.
The addition of concentrated HCI and a higher reaction temperature is necessary in the synthesis of complex 9. Anal. Calcd for C=HNCI0ORu = x 2HCI: C, 32.62; H, 3.25;N,9.51;CI,36.11;Ru,17.16. Found: C,32.80;H,3.40;N,9.13 Physical measurements. Electronic spectra were recorded on a Perkin Elmer Lambda 9 UVNIS/NIR spectrophotometer in connection with a Perkin Elmer 7300 Professional Computer. Infrared spectra were recorded with Csl-pellets on a Perkin Elmer 983 G infrared spectrophotometer or a Bruker IFS 113 for spectra in the far IR region. The H NMR spectra were taken on a Bruker Ac 200 MHz spectrophotometer and a Bruker AMX 500 MHz spectrophotometer at a temperature of 293 K. Dimethyl-d-sulfoxide was used as solvent. Conductivity measurements were carried out using an LF191 (WTW) digital conductometer with an LS1/'1"-1.5 platinum electrode in dimethylformamide (DMF). The cell constant was calibrated to 12.88 mS with 0.1 M KCI solution in bidistilled water. Measurements were carried out with 0.001 molar solutions at a temperature of lengths and angles are given in Tables 2 and 4 for 6 and in Tables 3 and 5

Results and Discussion
Investigations into the synthesis of the complex Hlnd/[RuCl4(ind)=] (1). Considering the antitumor activity of the complex ICR against tumors of the colon and rectum, which has already been tested with good success, the analogous indazole complex 1 is of major interest for clinical use because of its even higher activity. After the failure to produce complex I in the usual way that was used in the case of the complexes of the HL[RuCl]-type we have synthesized so far and which have already been described in earlier publicationsTM, the reaction procedures for indazole and Ru(lll) solutions were intensively studied. First we copied the synthesis for the imidazole complex ICR and dissolved indazole in 8 N HCI while slightly heating and, after cooling down, the diluted Ru(lll) solution (method I) was added to the solution with stirring. Then the mixture was examined over 48 hours by means of HPLC measurements. After ten minutes, the first sample was taken and immediately put onto the column. The procedure was repeated every ten minutes over a period of 6 hours. The indazolium cation (Hind/), which is predominant, could be detected at a retention time of about 3 minutes. Besides, three other product peaks could also be detected. Their increasing percentages throughout the reaction were determined by peak areas (Fig. 2). The composition of the reaction mixture measured by HPLC after 24 h showed that three ruthenium complexes are formed in different shares: A (10 %), B (57 %) and C (33 %).
In a second experiment, 2 mg of the solid which was isolated from the reaction mixture after 24 hours by filtration, was dissolved in H_O bidest, and separated under the usual conditions on an analytical column. The HPLC chromatogram of the isolated solid is shown in Fig. 3. The percentage composition is in good agreement with that of the liquid phase. The different fractions were collected, the solvent removed under reduced pressure, dried in vacuo, and then observed by means of UV-, IRand H NMR spectroscopy. By making a comparison with the IR spectra of pure KHPO, the IR absorptions caused by residues of the added buffer could be assigned. The H NMR measurements were carried out in DO or in [d6]DMSO to distinguish between the presence and absence of an aqua complex. The signal at a retention time of about three minutes can undoubtedly be assigned to the Hind* cation by means of comparative UV measurements with free indazole. Also, the H NMR of this fraction shows the characteristic chemical shifts of the indazolium cation Hind (5 8.04[s, 1H, 3-H]; 7.74 [d,1H,7.52[d,1H,7.33[t,1H,7.09[t, 1H, 6-HI. As expected, no highfield-shifted signals caused by anionic ruthenium-bonded species can be observed. The following structures could be resolved for the complexes A, B and C: (2). If the bis-aqua complex C: (Hlnd*)[RuOCI6(HO)-(ind)] 2 was present in the solid with an average sham of at least 30 %, which is indicated by HPLC experiments, the calculated value for carbon would be 1-1.5 % higher and the nitrogen value would be about 0.5 % lower.
Finally we succeeded in transforming the product mixture into the pure complex I by treating it with concentrated HCI at a raised reaction temperature. For that, 60 ml of 12 N HCI in portions of 10 ml were given to the reaction mixture over a period of 6 hours. A sample for HPLC was taken each time before adding the acid. The addition of the acid lead to a dramatic change in the composition of the solution. The results are summarized in Fig. 4.  dropping 20 ml of the acid into the reaction mixture leaves merely 8 % of this product. In contrast, the concentration of the desired complex 1 increased and after the addition of 60 ml of HCI it prevailed in the solution, which at that time had a brown colour. At the beginning of the addition the aqua complex C reacted with a slight increase in its concentration, but when further raising the CI concentration, its formation becomes increasingly hindered. This could be seen in further HPLC investigations. The beige-coloured solid which had formed throughout the addition of the 12 N HCI was separated by filtration, washed with HO and ethanol/diethylether (1:1) and dried in vacuo.
The HPLC of this product is shown in Fig. 5a. It is evident that complex 1 provides the main product after the treatment with concentrated HCI. Disturbing residues of the oxo-bridged complex 2 and the aqua complex C, which forms out of 2, could be avoided by further treatment of the solid product with 6 N HCI at 90 C. A HPLC chromatogram of this product after usual purification shows nearly complete transformation (Fig. 5b). It could be proved that complex I can be made available from the oxo complex 2 by raising the temperature and adding concentrated HCI. This knowledge lead us to the general method for the synthesis of 1, which turns out in a remarkably high purity and which is described in the section dealing with complex synthesis.
Bonding in complex 1. Bonding conditions in complex 1 were elucidated by studying the interactions between ruthenium(Ill) and the two N-methyl isomers of indazole. In a neutral or acid solution, the indazole molecule exists in a mesomer equilibrium: the five-membered ring of the indazole can achieve bonding to the metal with its N-1 or with the N-2 nitrogen. To clarify the situation, both N-methyl derivatives were synthesized in high purity according to the method of Bakke and SkjervoldTM. While in the case of the 1-methyl derivative the complex trans-H(1-Me-Ind)[RuCl,(1-Melnd=)] (6) could easily be prepared according to our usual method for complexes of the HL[RuClL=]-type, the 2-methylindazole did not show any tendency towards binding to the metal, even if the parameters of the reaction (molar proportions, temperature, pH, time of reaction) were altered. Only the oxo-species (2-MelndH)[RuOCl0] x 2 HCI (9) could be obtained when a concentrated Ru(lll) solution was used. So it can be expected that the unsubstituted indazole in complex 1 is linked to the ruthenium through the N2-nitrogen.
HPLC investigations into the solution chemistry of 1 in comparison with ICR. HPLC is an important method to prove the purity of a new drug before it can undergo clinical studies. We investigated the hydrolysis reactions of the two active compounds 1 and ICR, and the newly formed hydrolysis products were examined. Stability of the complexes in physiological saline was observed by means of HPLC over a period of two days and by conductivity measurements. While the decomposition of ICR is about 4 % per hour, that of [RuCI4(ind)] 1 is less than 0.9 % per hour. The pseudo-first-order rate constant of hydrolysis of ICR at 22 C was determined to be 9.10 x 10 s and for complex 1, it is 1.55 x 10 s Half-lifes were calculated to be 12.7 hours in the case of ICR and 74.3 hours in the case of 1. These results are in agreement with experiments carried out under slightly changed conditions1. The hydrolysis of ICR causes the formation of three new products while in the case of the indazole complex there is only one. The hydrolysis products were analysed by comparative HPLC measurements, UV spectroscopy and conductivity measurements.   [.RuCl(im)]. This fact seems to be important in view of the remarkably high stability of 1 in the hydrolysis experiments. The HPLC investigations into this complex have shown that only one product is formed over a period of 10 hours. The evaluation of UVand conductivity measurements suggests that this product is probably an aqua complex.
UV spectroscopy and conductivity measurements. In dimethylformamide, ICR equals a 1:1 electrolyte with "slow" cations and anions ( = 52 cmmol 1 at 25 C). Dissolved in water (pH 6.0), it also equals a 1:1 electrolyte, with the molar conductivity A increasing over the time of measurement from 68 Pzcmmolto 143 zcmmol This increase can be attributed to a relatively slow exchange of a CI ion for HO and the simultaneous formation of an aqua complex. Since CI itself has a higher conductivity than the complex anion, the molar conductivity increases: HIm/[RuCl,(im)]-+ HO ---) [RuCl(HO)(im)] + Him* + CI-However, prolonged observation periods over 2 days showed that conductivity still increases very slowly up to values in the range of 2:1 electrolytes. This can be attributed to the formation of another aqua complex, with two chloride ions replaced by water. The formation of a trisimidazole complex, which is responsible for the lowered pH of 4.2 of ICR in aqueous solution, could be a second possibility and is in good agreement with the results of the HPLC investigations:
The formation of compound [RuCla(im)a] is further elucidated by UV measurements over a hydrolysis period of 14 hours at ambient temperature. We observed UV absorption directly after dissolving ICR in water and during 14 hours of hydrolysis respectively. When comparing the results with the absorption spectrum of [RuCl(im)a] in water, the transformation can be seen. The absorption at 347.4 nm, which is characteristic of ICR, is continuously shifted to 336 nm, which is identical with the characteristic absorption band of the pure trisimidazole complex.
As expected, the molar conductivity of the complex Hlnd/[RuCl(ind)] (1) in DMF equals a 1:1 electrolyte (44 4cmmol4). This value remained almost constant over 24 hours. When the complex is dissolved in water, it shows, from the very beginning, high molar conductivity (307 4cmmol4), which increases very slightly to 360 lcmmol4 after 24 hours.
Molecular structures of the complexes 6 and 7. The initial formation of aqua complexes in the hydrolysis reaction of complex type HL[RuCI4] could be confirmed by the isolation of complex 7, which is produced fro m complex 6 dissolved in an aqueous medium. We could crystallize both compounds and resolve the X-ray crystal structures, which are shown in Figs. 7 and 8. Because of the fact that the aqua species 7 is formed out of complex 6 in a hydrolysis reaction in which a HO molecule is substituted for a CI ligand, a comparative examination of these two compounds seems reasonable. In both cases, the ruthenium is placed in the center of a slightly distorted octahedron, in which the two trans-standing 1-methylindazoles are arranged in the axial positions (6: N(1)-Ru(1)-N(2)" 179.8; 7: N(2)-Ru(1)-N(2a)" 174.3), and the four CI atoms in complex 6 are in the equatorial positions, which in the case of complex 7 are occupied by three CI atoms and the HO molecule. Bonding to the metal is via the N2-nitmgen of the organic ligand. Compound 6 crystallizes with four molecules in the unit cell of space group P2.1/n, while in the aqua species 7 the four molecules are in a unit cell of space group C2/c (see Tables 1 and 2).     (10) -5(7) 200(9) "the estimated standard deviations are shown in parentheses.

Length" Bonds Angles
The N-Ru-N angle in 6 amounts to 179.8. In 7, the bond angle is about 174 , a slight reduction in comparison to 6. The C atoms C(1) and C(la) are shifted towards the center, and an optimal sterical positioning respective of the methyl groups on the opposite side is the result. There is a slight reduction of the CI(la)-Ru(1)-CI(1) and N(2a)-Ru(1)-N(2) axes towards the HO. Considering the orientation of the CI-Ru-CI angles, a similar development can be observed: In compound 6 the CI(1)-Ru(1)-CI(2) and CI(3)-Ru(1)-CI(4) angles are 87.6 and the angles CI(2)-Ru(1)-CI(3) and CI(1)-Ru(1)-CI(4) are both 92.4. In contrast to this, the angles in 7, which surround the HO, are reduced to 86.3 (Cl(la)-Ru(1)-O(1)), (CI(1)-Ru(1)-O(1)). The angles on the opposite side are both still 93.7 ((CI(2)-Ru(1)-CI(1)) and (Cl(2)-Ru(1)-Cl(la)). This shows that in accordance with our investigations into the solution chemistry of compound 6 the formation of an aqua complex in a first hydrolysis step does not reduce the stability of the organic ligands and the substitution of another CI leaving group is likely to occur to give the bis-aqua complex.
Biological tests. After the successful synthesis of the indazole complex I with good yields and the necessary high purity, the positive results in biological test systems could be confirmed. Fig. 9 shows the results of the therapy of autochthonous colorectal tumors with complex 1 along with cisplatin, 5-fluorouracil and ICR, which has been the most active ruthenium species until now. The antitumor activity could even be surpassed with the indazole complex 1, which effected a tumor reduction up to 90%. The 1-methyl complex 6 achieved similar T/C-values as 1 in the P388 leukemia. In this test system, complex 3 recently reached a good T/C-value of 160 % using a single dose of 75 mg/kg (0.116 mmol/kg) on three subsequent days after tumor transplantation.  Figure 9. Test results of ICR and trans-Hlnd[RuCl(ind)] 1 in autochthonous colorectal tumors of the rat, compared to cisplatin and 5-fluorouracil. Doses were applied twice a week over ten weeks.
The reduction of tumor volume represented by the shaded columns is statistically significant compared to the control group.
Concluding Remarks. The galenic behaviour of water-soluble ruthenium complexes is easier to manage, because adjuvants are not necessary in parenteral clinical applications. Furthermore the pseudo-first-order rate constants under physiological conditions show that these ruthenium compounds are sufficiently stable for intravenous infusion. This is a considerable advantage for later administration in the clinic. Another point is that freshly prepared solutions of ICR do not inhibit DNA polymerisation, whereas aged solutions, which had enough time to form aqua complexes, are able to inhibit DNA polymerisation to a high extent. This makes it likely that many of the ruthenium compounds are only "prodrugs" and hydrolysis inside the tumor cell is necessary for activation.
As part of our program to synthesize water-soluble tumor-inhibiting ruthenium complexes, we have developed a synthesis for 1 with sufficient yield and high purity. The complex shows higher antitumor activity in all test systems than ICR, which has been the most active compound so far. The aquation of the anticancer complexes ICR , and 1 were studied extensively by means of UV-, IRand NMR-spectmscopy as well as HPLCand conductivity measurements. The data obtained shows that these compounds undergo aquation in aqueous solution, forming different hydrolysis products. For ICR we observed the formation of a neutral complex [RuCl(im)] as well as monoaquaand diaqua adducts. Due to the formation of the trisimidazole complex, the [RuCI4(im)] anion decreases continuously throughout hydrolysis. In contrast, complex 1 shows no tendency towards forming a trisindazole complex. The halflife of 1 was calculated to be 74.3 hours.
In the case of aquation of active complex 6, we could resolve the resulting monoaqua species 7 crystallographically as well as the initial complex 6. For the RuClL-type prodrugs, this is the first time that a hydolysis product could be isolated to enrich the discussion of the relationship between the mode of action and structure. In case preclinical antitumor activity in colorectal tumors can be confirmed in the clinic, this would mean a considerable progress for the chemotherapy of cancer.