Synthesis and Anti-Candida Activity of Cobalt(II) Complexes of Benzene-1,2-Dioxyacetic Acid (bdoaH2). X-Ray Crystal Structures of [Co(bdoa)(H2O)3] ⋅3.5H2O and {[CO(phen)3](bdoa)}2⋅24H2O (phen = 1,10-Phenanthroline)

Co(CH3CO2)2⋅4H2O reacts with benzene-1,2-dioxyacetic acid (bdoaH2) to give the Co2+ complexes [Co(bdoa)(H2O)3]⋅H2O (1a) and [Co(bdoa)(H2O)3] ⋅3.5H2O (1b). Subsequent reaction of 1a with 1,10- phenanthroline produces [CO(phen)3] bdoa⋅10H2O (2a) and {[CO(phen)3](bdoa)}2⋅24H2O (2b). Molecular structures of 1b and 2b were determined crystallographically. In 1b the bdoa2-- ligates the metal by two carboxylate oxygens and two ethereal oxygens, whereas in 2b the bdoa2- is uncoordinated. The Mn2+ and Cu2+ complexes [Mn(bdoa)(phen)2]⋅H2O (3) and [Cu(pdoa)(imid)2] (4) were also synthesised, 1a-4 and other metal complexes of bdoa H2 (metal = Mn2+, Co2+ ,Cu2+, Cu+ ) were screened for their ability to inhibit the growth ofhe yeast Candida albicans. Complexes incorporating the 1,10-phenanthroline ligand were the most active.


Introduction
Microbial infections in both humans and animals are diverse in their manifestations, ranging from superficial skin problems, chronic infection of the nails, mouth, throat or vagina to frequently fatal systematic diseases. The persistent use of cytotoxic drugs, corticosteroids, antibiotics and immunosuppressants has resulted in an increase in systemic opportunistic microbial infections. The yeast Candida albicans, a commensal of the human body, is considered to be the most important fungal pathogen. Over 75% of women suffer from vaginal candidosis (thrush) at some stage in their lifetime, and systemic candidosis is often fatal in immunocompromised patients. 1"4 The search for novel antifungal drugs has intensified over the last decade and some literature has highlighted the use of metal complexes as useful antifungal agents. 5 Benzene-1,2-dioxyacetic acid (bdoaH2) contains six potential oxygen donor atoms (four carboxylate oxygens and two ethereal oxygens, To date, K + ,6 Mg2+,7 Ca2+,7 La3+,8 Mn2+,7,9 Ni2+,7 Cu2+,lO, Cu + 12 and Zn 2+ 7 complexes of this aryloxydicarboxylic acid have been structurally characterised. In the present paper we report the full X-ray Benzene-1,2-Dioxyacetic Acid(bdoaH2) crystal structures of two cobalt(ll) complexes with bdoa2". In addition, the anti-Candida activities of bdoaH2 and its Mn2+, Co2+, Cu 2+ and Cu + complexes are presented and this, to our knowledge, is the first report of the biological chemistry of both the diacid and its metal complexes.

Biological studies
Candida albicans (three isolates) were obtained from in St. James's Hospital, Dublin. The isolates were stored on Sabouraud dextrose agar (SDA) plates at 4 C. Solutions of the test complexes were prepared by dissolving the complex (0.02 g) in distilled water (100 cm3) to yield a stock solution at a concentration of 200 gg cm"3. The solution was filter sterilised using a Millipore membrane flter (0.45 tm). Complexes which were insoluble in water were ground to a fine powder and then suspended in sterile distilled water (100 era3).
Susceptibility testing method RPMI-1640 broth medium (Sigma R 7755) was used for the anti-Candida susceptibility testing. The medium (1 dm3) was supplemented with L-glutamine (0.3 g) and morpholinepropanesultbnic acid (MOPS) (34.6 g) and was adjusted to pH 7.0 using sterile NaOH (0.2 M).
The broth microdilution reference method was used. 5 Prior to testing yeast cells were grown on Sabouraud dextrose agar (SDA) at 37 C for 24 h. Cell suspensions were prepared in sterile phosphate Benzene-1,2-Dioxyacetic Acid(bdoaH2) buffered saline (5 cm3) to a density of 0.5 McFarland standard. A 1:100 dilution of the 0.5 McFarland standard yeast suspension was made in RPMI-1640 medium. The cell concentration of the final inoculum was 3.5 x 104 5.0 x 105 cells cm-3. The prepared cell suspension (90 ktl) was dispensed into microtitre plates and to this was added the test stock complex solution (10 lal) to yield working solutions of the test complexes of concentration 20 lag cm"3. Plates were then incubated for 24 h at 37 C with continuous shaking. Each complex was assessed in triplicate and three independent experiments were performed. The resulting nine data points were statistically analysed using ANOVA one-way analysis of variance followed by Tukey's family error rate.

Results and Discussion
The pink complex [Co(bdoa)(H20)3]'H20 (la) was prepared in high yield by reacting cobalt(II) acetate tetrahydrate with bdoaH2, and crystals of composition [Co(bdoa)(H20)3]'3.5H20 (lb) were isolated from the reaction filtrate. The X-ray crystal structure of lb was determined (Fig. 2, Table 2) 16 and the cobalt(lI) ion is seven-coordinate, being bonded to three water molecules, a carboxylate oxygen from each end of the bdoa 2and, much more weakly, to the two ether oxygens. The  [Co(phen)3]bdoa" 10H20 (2a), and crystals of composition {[Co(phen)3](bdoa)}2'24H20 (2b) were isolated from the mother liquor. The X-ray crystal structure of 2b ( Fig. 3 and 4, Table 3) shows that the bdoa 2" and the three water ligands have been displaced from the coordination sphere of the metal in b and replaced by three chelating phenanthrolines. The bdoa 2" simply functions as a counter dianion. The asymmetric unit of 2b contains two independent (but similar) [Co(phen)3] 2+ cations, two independent (and different) bdoa 2" anions and twenty four water molecules. There is nothing remarkable in the geometry of the [Co(phen)3] 2/ cations, although they interact with each other by n-n stacking, forming chains along the a direction (interplanar distance between the N C-C12C ring, coordinated to Col, and the N D-CI2D ring, coordinated to Co2, is-3.5 A). The two bdoa 2" anions differ in that the first is approximately coplanar vhile, in the second, one of the carboxylate groups is almost perpendicular to the phenyl ring. This is likely to be a consequence of the very extensive hydrogen-bonding network which links the anions and all twenty four water molecules into a three-dimensional grid with spaces for the cation chains.  ( The bdoa complexes, the metal-flee ligands and the simple acetate complexes of Co2+, Mn 2+ and Cu 2+ were each tested for their ability to inhibit the growth of three clinical isolates of C. albicans (Table 4).
Although uncomplexed bdoaH2 is essentially inactive against all three isolates coordination to Co2+and Mn 2+ (complexes a and 5) leads to slightly increased activity, with the Co 2+ complex being marginally the better. The equivalent Cu 2+ complex 6   The Co2+, Mn 2+ and Cu 2+ 1,10-phenanthroline adducts 2a, 3 and 7 dramatically inhibit the growth of all isolates, with the Mn 2+ and Cu 2+ species being the more superior. Furthermore, the Mn 2+ 2+ complex 3 is significantly more active than its Cu analogue 7 against isolate 2 Thus, it is evident that complexation of 1,10-phenanthroline to the metal centres in the conversion of the Mn 2+ and Cu 2+ species 5 and 6 to 3 and 7, respectively, greatly enhances their anti-Candida activities, in contrast to this improved 2+ 2+ 2+ activity with the Mn and Cu complexes the Co non-phenanthroline and 1,10-phenanthroline chelated complexes (la and 2a), have very similar activities. "Metal-free" 1,10-phenanthroline showed very high anti-Candida activity and, indeed, this was only surpassed by the activity of the Mn :+ 1,10-phenanthroline complex 3 (on isolates 2 and 3). Benzene-1,2-Dioxyacetic Aaid(bdoaH2) In contrast to the performance of 1,10-phenanthroline the other NN chelating agent used in this study, 2,2'-bipyridine, is inefficient at preventing the growth of Candida cells.Also, the Cu 2+ bipyridine complex 8 is ineffective.
Complexation of the monodentate N donor ligands aniline, imidazole, ammonia and pyridine to Cu 2+ (complexes 9, 4, l0 and I l, respectively) does not improve the poor anti-Candida activity of the respective free ligands. Whereas the tris(triphenylphosphine) Cu + complex 12 is only moderately active against isolate 3 the bis(triphenylphosphine)/phenanthroline Cu + species 13 is relatively active against isolates and 3.
In conclusion, the 1,10-phenathroline molecule, whether it be uncomplexed or chelated to a metal centre, exhibits potent anti-Candida activity. It is also apparent that the nature of the metal centre to which the 1,10-phenathroline is coordinated is also of significance given that the Mn 2/ and Cu 2+ complexes are superior to Co 2+ complex. The fact that the related NN chelating ligand 2,2'-bipyridine and its copper complex both show negligible activity against the yeast serves to highlight the fundamental structural and reactivity differences between 2,2'-bipyridine and l, 10-phenanthroline.