Water Soluble Cyclophosphamide Adducts of Rhodium(II) Keto-Gluconate and Glucuronate. Synthesis, Characterization and In Vitro Cytostatic Assays

The synthesis, characterization and biological assays of two new rhodium carboxylate sugar derivatives and respective cyclosphosphamide adducts are described. The compounds, characterized by 13C and 1H NMR, infrared and UV-visible spectra, presented high water solubility and hydration grades were confirmed given the concordance between thermal and CHN analyses. The adducts were active in vitro against K-562 cells.


INTRODUCTION
Ever since Rosenberg et al. introduced cisplatin in turnout disease therapeutics, researchers became increasingly interested in this field, giving rise to a number of published findings on platinum group metal complexes. In 1972, Bear and co-workers reported that rhodium (II) carboxylates present anti-tumoural activity2. Albeit the promising start, interest in this class of compounds as anti-tumoural agents has somewhat decreased mostly in view of significant toxicity levels. Currently, a number of papers have been issued in an attempt to identify less toxic derivatives 3-8.
One of the best means of obtaining chemotherapeutical metal complexes is to synthesize adducts using ligand molecules that are, by their very nature, biologically active. To this effect, cyclophosphamide (CP) (Fig.l) has been used to obtain adducts with rhodium(II) carboxylates. The compounds were submitted to biological assays and results indicated that the complexes were not active 9. This might be partially due to the fact that these present somewhat low water solubility levels. With views to obtaining compounds that present the appropriate partition coefficient for biological assays, two new rhodium(II) derivatives, Rhz(GU)4 and Rh2(KG)4, rhodium glucuronate and rhodium keto-gluconate ( Fig.l)  Resultant data were compared by means of the statistic chi square test l. When P _< 0.05, differences were deemed to be statistically significant. Preliminar toxicity assay Toxicity was investigated in nine different groups of eight healthy male Balb-C mice, with a single ip dose of up to 200mg/kg. Death and/or toxic effects were sought for, during 60 days. Table presents the results encountered in carbon, nitrogen and hydrogen analyses. Percentages were compatible with the compounds' high water affinity.

RESULTS AND DISCUSSION
The first inflection of TGA curves ( Fig.2) is relative to water lost to Rh2(KG)4xH20 and the hydration grade (x=6-8 H20) depends on freeze drying conditions. Sugar salt, rhodium comgound and respective CP adduct's major infrared assignments are depicted in Table 2. Co, ordination modes are forecasted as of the stretched, asymmetric (--1600 cm-) and symmetric (-1400 cm-l) values, of the coordinated carboxylate groups. Average A(asym-sym) values under 200 cm -I allow for the discarding of the hypothesis of occurrence of complex's monodentate coordination l. Within the 3500-3000 cm-range, the free D-glucuronic acid presents three broad bands centered at 3400, 3280 and 3160 cm -I due to OH stretching. The first two are attributed to intermolecular interactions and the latter might be related to an intramolecular hydrogen bond 2. Usually these bands are shifted to a higher frequency when the sugar ligands coordinate to metals. It is worth noting that such shafts may be influenced by the metal charge and consequently by the covalent character of the metal-ligand bond. For instance, the Rhz(GU)4 highest energy band at 3400 cm -I in 12 sugar free acid, shifts to 3600 cm-in its sodium salt and to 3470 cm in calcium salt remaining at approximately 3400 crn -t for the rhodium (II) dimer. This suggests that the intermolecular hydrogen bonds of the free acids are analogous to those observed in the rhodium derivatives.      All signals in the cyclic derivative (GU) related to both anomeric forms o and 13 (the latter represented by ') were identified and carefully assigned as per literature data 9,14-18. Two possible conformations [15][16][17] (Fig.3) explained the additional shifts (represented by ') encountered in keto-gluconate compounds. This effect was most intense in carbon 6.
In addition, with reference to the keto-gluconate compounds, a carbonyl group that presented an atypical signal at approximately C.ao 97 ppm was identified. In conclusion, the displacement was probably due to a keto-enolic equilibrium. Nevertheless, it is worth emphasizing that other than 31p NMR, few important shifts were observed after axial CP coordination in the H and 13C NMR spectra.  and CP in water solution in view of the higher donor character of D20 in relation to that of CDC13.
Existing literature 9 reports that cyclophosphamide is not a ligand that presents high affinity with rhodium(II) carboxylates. A link via the oxygen P=O was involved when adducts were isolated. In addition, should this have been due to a nitrogen link, the compound would most probably have been pink in colour, although there are exceptions. In support of this hypothesis, the 590 nm band in UV/vis spectra (Table 6) suggests, as observed in other ligands, a coordination through oxygen.
Although data indicate that oxygen coordination is most probable, the alternative nitrogen coordination via hydrogen bonds could not be discarded. Table 7 presents the results of cytotoxic activity.

BIOLOGICAL TESTS
Data was submitted to the statistical chi squared %2 test.
Although all compounds presented higher in vitro activity in relation to the control, no deaths or toxic effects were observed along the 60 days of its vivo tests using doses of up to 200mg/kg. The group treated with adducts showed a significant increase of cytotoxicity in vitro, mostly in the highest doses. The most promising in the series, against the cell line tumour used, was the KG derivative. On the other hand, it is worth noting that Rhz(KG)4(CP)2 presented higher activity than the CP and Rhz(KG)4 from which it is derived. Possibly, once these adducts are administered, a gradual dissociation occurs before acting upon the cancer cells. Should this be the case, both rhodium carboxylates and CP meet each other's requirements as carriers, promoting a synergistic effect in vivo. The relevance of this effect requires further investigation and will be subject to forthcoming studies.