Syntheses of Coordination Compounds of (±)-2-Amino-3-(4-Hydroxyphenyl)Propionic Acid, Mixed Ligand Complexes and Their Biological Activities

Coordination compounds of (±)-2-amino-3-(4-hydroxyphenyl)propionic acid and their mixed ligand complexes with 1,10-phenantroline were synthesized, characterized using electronic and infrared spectral analyses and magnetic susceptibility. The compounds were evaluated for antimicrobial and antioxidant activities. Four different assays were applied for evaluating antioxidant capacity of the compounds. The results obtained indicated a diametric square planar geometry for both cobalt (±)-2-amino-3-(4-hydroxyphenyl)propionic acid complex and its mixed ligand complex. It was suggested that for the binary cobalt(II) complex, the phenolic substituent coordinated with neighbouring central metal ions. However, for the ternary cobalt(II) complex it was suggested it was deprotonated. Octahedral geometry was proposed for both copper complexes. Square planar geometry was indicated for the nickel (±)-2-amino-3-(4-hydroxyphenyl)propionic acid complex and a mixture of square planar and octahedral geometry for the nickel mixed ligand complex. The cobalt mixed ligand complex elicited the highest activity for all the antioxidant assays. In most cases the binary complexes exhibited better antimicrobial activities relative to their ternary counterparts.


Introduction
A key part of research in medicinal chemistry involves the finding of compounds which may serve as lead for therapeutic use. These include coordination compounds, because of their unique properties and broad range of applications; including anti-microbial, -cancer and -oxidant [1] [2] [3] [4]. In combination with a variety of ligands transition metals, form mixed ligand complexes. Some of these have been shown to exhibit anti-biotic and -cancer activities [5] [6]. In this regard, extensive applications have been found for 1,10-phenanthroline, a bidentate ligand that forms very stable chelates with many first-row transition metals [7].
Various metabolic processes in living systems and environmental stresses generate various reactive species. These include free radicals and mainly reactive oxygen species (ROS) [8] [9] [10] [11]. Increased level of ROS can damage structure of biomolecules and modify their functions and lead to cellular dysfunction and possibly cell death. The cumulative effect of increased ROS can therefore increase oxidative stress which may be manifested in the form of a variety of health problems such as cancer, age related disease and cardiovascular diseases [8] [9] [10] [11]. As a result of pathologies associated with the attack of free radicals, there is a general need for antioxidant compounds. These compounds are capable of delaying or inhibiting the oxidation processes which occur under the influence of atmospheric oxygen or reactive oxygen species [12] [13] [14]. As such in some cases they are considered to be involved in the defense mechanism against pathogens resulting from such activities. In cases in which endogenous factors are unable to ensure the control and complete protection of an organism against the reactive oxygen species, the need for exogenous antioxidant compounds arises.
This may be obtained as nutritional supplements, pharmaceutical products and some fruits. A class of compound with potential antioxidant activity is the phenols and polyphenols [12] [13] [14].
Previous studies have indicated that some cancer cells are related to some microorganisms [15] [16]. Additionally, reports have shown that high levels of reactive oxygen species have been detected in most cancers, where they promote tumor development and progression [16] [17]. One of the principal public health problems of this century is the emergence of the resistant microorganisms, increasing the numbers of infections caused by microorganisms no longer susceptible to the common medicines used to treat them [17] [18]. Studies of potential lead compounds with antimicrobial activity with antimicrobial and antioxidant activities having minimal side effects are considered needful. Therefore it was considered to synthesize coordination compound of (±)-2-amino-3-(4-hydroxyphenyl)propionic acid (Figure 1), its mixed ligand complexes with 1,10-phenantroline ( Figure 2) and to investigate the antimicrobial and antioxidant activities of these compounds.
The compounds were characterized using infrared, electronic spectrophotoscopic method and magnetic susceptibility.    The equations of the reactions are given in Equations (1)-(6).

Determination of Total Phenol Content
The method of determining the total phenolic content was described by Singleton and Rossi, 1965, Gulcin et al., (2004) [19] [20] using the folin ciocalteu's phenol reagent which is an oxidizing reagent. To a mixture of 0.1 ml of sample and 0.9 ml of water was added 0.2 ml of folin-ciocalteu's phenol reagent and the resulting mixture voltexed. After 5 minutes of standing, 1.0 ml of 7% (w/w) Na 2 CO 3 solution then added and the solution was then distilled to 2.5 ml before incubation for 90 min at room temperature. The absorbance against a negative control containing 1 ml of water in place of the sample was then taken at 750 nm. The standard used was the Gallic acid at 0.1 mg/ml in order to determine Gallic acid Equivalent (GAE) of sample, after preparing a calibration curve. Distilled water was used as blank.

DPPH Assay
The radical scavenging ability of the compounds was determined using the stable radical DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate) as described by Brand-Williams et al., (1995) [21]. The reaction of DPPH with an antioxidant compound which can donate hydrogen, leads to its reduction (Blois, 1958) [22]. where: A blank = the absorbance of the control reaction (containing all reagents except the test compound); A sample = the absorbance of the test compound.
Sample concentration providing 50% inhibition (IC 50 ) was calculated from the graph plotting inhibition percentage against concentration of the compound. in a water bath at 95˚C for 90 mins. The mixture was then allowed to stand and cool to room temperature and the absorbance measured at 695 nm against a blank which consisted of the reacting mixture containing distilled water in place of the compounds. The antioxidant activities of the extracts were expressed as an ascorbic acid equivalent.
All measurements were taken at room temperature with samples protected from direct sunlight. The reducing power was expressed as equivalent concentration (EC) which is defined as the concentration of antioxidant that gave a ferric reducing ability equivalent to that of the ascorbic acid standard.

Antimicrobial Methodology
The organisms used were five Gram-positive and three Gram-negative bacteria and two fungi. These were S. aureus, S. epidermidis, B. subtilis 12, B. subtilis 82, Clostridium sp., K. pneumonia, P. aeruginosa, E. coli, C. albicans and C. pseudotropicalis, respectively. The agents were dissolved in water at room temperature or hot water as appropriate to give a concentration of 40 mg/ml. The resulting solutions were used to soak sterile Whatman No 2 discs (diameter of 6 mm) and allowed to dry in an oven at 50˚C. The discs were then utilized to determine antibacterial and antifungal activities as previously described by Aiyelabola et al.
2012 [24]. Discs that were impregnated with imipenem and chlorhexidine were used as positive controls for bacteria and fungi respectively. Zones of inhibition were used as indices of antimicrobial actions.

Infrared Spectra
The relevant vibrational frequencies in the infrared spectra of the ligands and complexes and their assignments are summarized in Table 1. Information about the metal ions coordination was obtained by comparing the IR frequencies of the ligands with those of the metal complexes.

(±)-2-Amino-3-(4-Hydroxyphenyl)Propionic Acid Complexes
In the spectrum of (±)-2-amino-3-  were observed at lower frequencies than expected. It is however suggested that this may be as a result of intramolecular hydrogen bonding between the hydrogen atom of the positively charged amine substituent and the oxygen atom of phenol [25] [26]. The deprotonation of the cationic amine and consequently the loss of this hydrogen bond is suggested by the resultant increment in these absorption frequencies in the spectra of the complexes ( Table 1). The occurrence of the ν(N-H) of the amino substituent at higher energies at about 40 cm −1 in case of the cobalt(II) and copper(II) complexes as compared to the free ligand suggest the bonding of amino nitrogen atom to the metal ion. Thus indicating deprotonation followed by coordination through the nitrogen atom [26]. This is in con-

Mixed Ligand Complexes
The ν(O-H) band observed at 3205 cm −1 in the spectrum of (±)-2-amino-3-(4hydroxyphenyl)propionic acid was shifted to higher frequencies in these complexes, similar to their binary counterpart. Although compound 2 did not exhibit this frequency, it is suggested that this may be an indication of the deprotonation of the phenolic substituent by the base [25] [26]. The N-H stretching frequency observed as a strong band at 3126 cm −1 in the spectrum of (±)-2-amino-3-(4-hydroxyphenyl)propionic acid was shifted to lower regions in the cobalt complex suggesting the addition of the Lewis base to the metal ion [25] [26]. This indicates the elongation of the N-H bond. In the case of the copper(II) complex this band shifted to higher frequency suggestive of reduction of bonds, this also serves as evidence of coordination of the nitrogen atom to the metal ion. Similar to that observed for compound 5 the spectrum for compound 6 did not exhibit this frequency; this has been attributed to the anomalous behavior of nickel which was also corroborated by its observed magnetic moment [27].
In this instance, for the mixed ligand complexes the ν sym (COO − ) absorption was shifted to higher position by (136) cm −1 while the band caused by ν asym (COO − ) lowered by (49 -30) cm −1 as compared with the free ligand. This therefore indicates the coordination of the carboxylate group to the central metal ion [26]. The monodentate nature of the COO − functionality is suggested by the hypsochromic shifts in the ν asym (COO − ) frequency and bathochromic shift for the ν sym (COO − ) frequency for compounds 2, 4 and 6 indicated the monodenticity of the carboxylate ion on coordination [29] [30] [31] [32].
Additionally further confirmation of the coordination of amino nitrogen and carboxylate oxygen is given by the observance of metal-nitrogen (M-N) and metal-oxygen at 600 and 500 cm −1 respectively. Evidence for the coordination of 1,10-phenanthroline and thus the formation of mixed ligand complexes, was observed in the shift in the ν(C=N) ring vibrations of the uncoordinated 1,10-phenanthroline (

Electronic Spectra
The electronic spectra of the ligands showed two absorption bands at 265, 286 nm for (±)-2-amino-3-(4-hydroxyphenyl)propionic acid 181.19 attributed to π → π* and n → π* transitions and that of 1,10-phenanthroline at 310, 324 nm attributed to π → π* and n → π* transitions of the major chromophores, −NH 2 , −CN and COO − [25]. However on coordination, shifts in the bands were observed. Measurements of the effective magnetic moments of coordination compounds can be used to estimate the number of unpaired electrons. The stereochemistry and bond type of the coordinated metal ion can then be determined with the aid of valence bond or crystal field theory [27].
The electronic spectrum of compound 1 exhibited two bands at 512 nm and 735 nm corresponding to 2 A 1g → 2 B 2g and 2 A 1g → 2 E 1g respectively On the basis of simplest model three spin-allowed bands are expected in a four coordinate cobalt(II) complex, that is, 4 A 2 (F) → 4 T 2 (F), 4  Usually such complexes show two bands between 830 and 625 nm, which can be assigned to 4

A 2 (F) → 4 T 2 (F) and 4 A 2 (F) → 4 T 1 (F), respectively; 4 A 2 (F) → 4 T 1 (P) is
usually observed as a well-defined shoulder at about 550 nm [31]. The UV-visible spectrum of complex (1) exhibited bands at 512 nm and 735 nm corresponding to 2 A 1 g → 2 B 2 g and 2 A 1 g → 2 E 1 g respectively. Absorption at 259 nm is ascribed to transitions π → π* and n → π of the ligand [32] [33]. The spectrum for the cobalt(II) complex of the mixed ligand, compound 2, showed two bands at 460 nm and 620 nm corresponding to the transitions 3 A 2 g → 3 T 2 g and 4 A 2 g → 3 T 1 g respectively for octahedral geometry [27]. Absorption at 259 and 318 nm is associated with π → π* transition of the ligand [43] [44] Its magnetic moment of 1.066 BM is lower than that expected for Co(II) ion may be ascribed to antiferromagnetism which is due to the interaction between electron spins on neighbouring metal ions or polymerization. This is similar to that obtained for compound 1 [34] [35] [36].
The electronic spectrum of compound 3 showed a well resolved band at 499 nm and a weak band at 517 nm typical for a tetragonally distorted octahedral configuration and may be assigned to 2 B 1g → 2 A 1g and 2 B 1g → 2 E g transitions [27]. The observed magnetic moment 2.17 BM is therefore indicative of an unpaired electron as expected for a d 9 system. And is consistent with an octahedral geometry and is in agreement with previous reports [27] [37].
The spectrum for compound 4 exhibited absorption band at 620 nm attributed to 2 E g → 2 T 2 g at the d-d transition for octahedral geometry. Absorption at 259 and 330 nm are associated with π → π* and n → π * transitions of the ligands [27] [33]. Its magnetic moment of 2.40 BM suggests Jahn Teller distorted octahedral geometry. This is in agreement with previous reports [38] [39].
The spectrum of compound 5 elicited bands at 476 and 512 nm attributed to 3 A 2g → 3 T1 g and 3 A 2g → 3 T 2g 3 A 2g (F) → 5 T 1g (F) and 3 A 2g (F) → 5 T 1g (P) suggestive of an octahedral geometry respectively suggestive of an octahedral geometry. Absorption at 259 nm is associated with π → π* and n → π * transitions of the ligand [27] [33]. There is no clear cut distinction between the magnetic moments of tetrahedral and octahedral nickel(II) complexes. This is because they both have the same number of unpaired electrons in the d-orbital. On the other hand the square planar complex is however diamagnetic. A magnetic moment of 2.22 BM was obtained for compound 5 indicative of a mixture of octahedral geometry [27]. The nickel(II) complex of the mixed ligands showed a band at 636 nm which corresponds to the transition 3 A 2g → 3T 1g for octahedral geometry [27]. Absorption at 259 and 332 nm is associated with π → π* transition of the ligand. The complex elicited a magnetic moment of 1.66 BM. This may be interpreted as an indication of a low-spin-high-spin equilibrium mixture with 15% -20% paramagnetic nickel(II) species and agrees with what was reported by Woods and Patel (1994) to be octahedral in solution [27] [40]. We further propose an octahedral geometry for compounds 3, 4 and 6. Additionally a square planar geometry is suggested for compound 5.

Antioxidant Activity
Four assays for evaluating antioxidant capacity of the compounds was applied namely DPPH, TRAP, TAC and FRAP. The results obtained are presented in Tables 2-5.

Total Phenolic Content
It is well known that phenolic compounds are potential antioxidants; free radical scavengers, as such there should be a close correlation between the content of phenolic compounds and antioxidant activity. In this present study, the total phenolic content of the compounds was investigated with compound 2 having the highest total phenolic content followed by compound 1 and compound 5 with the least activity. Order of activity is as follows 2 > 1 > 6 > 3 > 4 > 5. Thus indicating that compound 1 may have the most outstanding antioxidant activity. This is not quite surprising and supports the hypothesis that the phenolic OH in the case of compound 2 is deprotonated and not coordinated to any central atom [12] [13] [14]. Therefore it is more readily available relative to compound 1. As a consequence the result obtained serves to corroborate the deprotonation of the hydroxyl moiety in compound 2.

DPPH Radical Scavenging Activity
DPPH has been widely used for free radical scavenging assessment due to its ease and convenience. In this study all the synthesized compounds were found to be effective DPPH radical scavengers. The activity of the complexes was more superior to the standard, ascorbic acid (IC 50 = 1.603), with order of activity; 2 > 1 > 6 > 3 > 4 > 5. This result positively correlates with that obtained with total phenolic content.

FRAP
The ferric ion reducing antioxidant power assay is used to determine the electron donating ability, of the matrix. The FRAP assay uses antioxidants as reductants in a redox-linked colorimetric method with absorbance measured with a spectrophotometer [23]. The principle of this method is based on the reduction of a colourless ferric-tripyridyltriazine complex to its blue ferrous coloured form owing to the action of electron donation in the presence of antioxidants. In this case compound 2 exhibited the best activity. Order of activity was 2 > 1 > 4 > 6 > 3 > 5.

Total Antioxidant Capacity Using Phosphomolybdenum Method
This method is based on the reduction of molybdenum(VI) to molybdenum(V) by the compounds and the subsequent formation of a green phosphate/Molybdenum(V) complex at an acidic pH [43]. The observed activity was of the order 2 > 1 > 6 > 3 > 4 > 5.     [44]. For all the assays compound 2 exhibited the highest activity. This is may be unexpected as two molecules of the primary ligand is proposed for this compound in relation to compound 1 which is proposed to have three phenolic −OH. We propose that these phenolic −OH in compound 1 are coordinated to neighbouring central metal ion. This is supported by the magnetic moment and infrared spectra. The result obtained for the four assays indicated that compound 5 exhibited the least activity. The reason for this is not readily evident. However, this suggests that the geometry assumed by a coordination compound is a function of the central metal ion and as a consequence, its functionality [45] [46] [47]. The result obtained for the antioxidant properties of the compounds may therefore as a result be ascribed to the varying molecular structures assumed by each compound. The results obtained further validates the anti ̶ oxidant activity of phenolic substituents [12] [13] [14]. Further studies on the antioxidant are however suggested for this compound.
For the entire four assays compound 5 exhibited the least activity, this observed activity validates the proposed geometry for the complex. The lower activity of compounds 3 and 4, copper complexes with octahedral geometry in comparison with compound 6, the mixed ligand complex, alludes to the individualistic nature of the central metal ion in defining the geometry of a given complex and as a consequence its activity [45] [46]. This may be exemplified by the activity of cisplatin, with platinum as the central metal ion, the use of other metal ions as the central metal resulted in their null or lower activity in comparison with cisplatin [47] [48].

Antibacterial Activity Testing
A comparison of the antimicrobial activity of the complexes against the reference antibiotic demonstrated that the standards drug used imipenem and chlorhexidine elicited significantly better antimicrobial than the synthesized complexes and ligands, ( Table 6). The ligands (±)-2-amino-3-(4-hydroxyphenyl)propionic acid did not exhibit any activity while o-phenantroline exhibited significant antimicrobial activity (Table 6). On coordination however the complexes had enhanced antimicrobial activity in some cases. This increase in antibacterial activity can be explained on the basis of Overtone's concept and Tweedy's chelation theory [49] [50]. The lipid membrane that surrounds the cell favors the passage of lipid soluble materials. As such this may be a factor for antimicrobial activity. On coordination, the polarity of the metal ion is reduced to a greater extent due to the overlap of the ligand orbitals and partial sharing of the positive charge of the metal ion with the donor groups. Furthermore, it increases the delocalization of π electrons over the whole chelate ring and hence enhances the liposolubility of the complexes. This increased liposolubility enhances the penetration of the complexes into the lipid membrane and the complex interferes in the normal activities of the bacteria [49] [50].  The cobalt(II) (±)-2-amino-3-(4-hydroxyphenyl)propionic acid complex elicited the best activity among all the complexes. On the other hand compound 6 exhibited the least activity of all the compounds. In some cases, chelation enhanced the activity of the coordination compounds relative to the ligand [49] [50]. The cobalt(II) (±)-2-amino-3-(4-hydroxyphenyl)propionic acid complex exhibited better activity than the mixed ligand complex with the exception of the Clostridium. Mostly the (±)-2-amino-3-(4-hydroxyphenyl)propionic acid were more active than their mixed ligand counterparts. In some cases, however the mixed ligand complexes exhibited a fairly better active compared with the 2-amino-3-(4-hydroxyphenyl)propionic acid complexes. The compounds were however not as effective as the standards imipenem and chlorhexidine against bacteria and fungi.

Conclusion
Coordination compounds of (±)-2-amino-3-(4-hydroxyphenyl)propionic acid and their mixed ligand complex with 1,10-phenantroline were synthesized, characterized. The primary ligand coordinated via the nitrogen atom of the amino moiety and the oxygen atom of the carboxylate ion. On the other hand, the secondary ligand coordinated via the nitrogen atoms of the molecule. The result obtained indicated a diametric square planar geometry for both cobalt(II) (±)-2-amino-3-(4-hydroxyphenyl)propionic acid complex and its mixed ligand complex. Octahedral geometry was proposed for both copper complexes. Square planar for the nickel(II) (±)-2-amino-3-(4-hydroxyphenyl)propionic acid complex and a mixture of square planar and octahedral geometry for the nickel mixed ligand complex. Although phenolic substituents have been related with anti-microbial and -oxidant activities, in this case the results obtained for both activities do not correlate with each other. Thus, this further corroborates the fact that the mode of activity of both biological activities may vary.