Synthesis, Spectroscopic, and Biological Studies on New Zirconium(IV) Porphyrins with Axial Ligand

A series of parasubstituted tetraphenylporphyrin zirconium(IV) salicylate complexes (SA/5-SSAZr(IV)RTPP, R = p-H, p-CH3, p-NO2, p-Cl, SA = salicylate, and 5-SSA = 5-sulfosalicylate) have been synthesized, and the spectral properties of free base porphyrins, their corresponding metallated, and axially ligated zirconium(IV) porphyrin compounds were compared with each other. A detailed analysis of ultraviolet-visible (UV-vis), proton nulcear magnetic resonance (1H NMR) spectroscopy, infrared (IR) spectroscopy, and elemental analysis suggested the transformation from free base porphyrins to zirconium(IV) porphyrins. The ability of the metal in this complex for extra coordination of solvent molecules was confirmed by ESI-MS spectra. Besides the fluorescence, cyclic voltammetry, and thermogravimetric studies, the complexes were also screened for antimicrobial and anticancer activities. Among all the complexes, 5-SSAZr(p-NO2TPP) shows high antibacterial activity.


Introduction
Synthesis and functionalization of porphyrins [1] have long been of great interest in the chemistry community because of the vast potentials and demands for porphyrin derivatives in diverse fields, such as materials [2,3], supramolecular chemistry [4,5], biomimetic models [6], catalysis [7,8], photodynamic therapy [9], and ionophores [10]. Porphyrins were reported to exhibit a variety of biological activities. This is due to the fact that natural and synthetic porphyrins have relatively low toxicity in vitro and in vivo and they possess antitumor [11,12] and antioxidant effects [13,14] and have a good potential for metal ions complexation. The ability for numerous chemical modifications and the large number of different mechanisms by which porphyrins affect microbial and viral pathogens place porphyrins into a group of compounds with an outstanding potential for discovery of novel agents, procedures, and materials active against pathogenic microorganisms [15]. Metalloporphyrins are the basis of new antifungal, antiparasitic, and anticancer drugs because modification of the porphyrin periphery confers qualitatively a new spectrum of activities to metalloporphyrins [16,17]. It has been reported that metal complexation alters the various physiological properties, especially the cytotoxic and antitumor activities, of many naturally occurring compounds. Zirconium(IV) porphyrins have gained attention from global researchers due to the peculiar characteristics of this class of compounds. To the best of our knowledge, the chemistry of zirconium(IV) porphyrinates remains underdeveloped, being limited to OEP and TPP, with a small variety of derivatives with different anions to balance the remaining +2 charge and ligands to satisfy the 7-8 coordination sphere. The metal ion in these complexes is oxophilic [18]; thus, it may show preference for carboxylate and other oxygen-bearing anionic ligands. A lot of work is reported on the spectral, electrochemical, and biological properties of zirconium and hafnium phthalocyanines with different outplaned organic ligands [19][20][21][22], but comparatively less work has been done on zirconium(IV) porphyrin complexes with carboxylate as axial ligand which are expected to be biomedically relevant complexes. Salicylic acid and its derivatives are biologically important molecules, and in view of the interesting results obtained from zirconium(IV) phthalocyanines with various out-of-plane ligands, it is considered worthwhile to make Bioinorganic Chemistry and Applications a study of axially substituted zirconium(IV) porphyrin with salicylic acid and its derivatives. We believe that these novel compounds will be fundamental substances for potential applications in the future. With this objective, we aimed at the synthesis, spectroscopic characterization, and biological studies on axially substituted zirconium(IV) porphyrins with salicylic acid and 5-sulfosalicylic acid as axial ligands.

Materials and Instruments.
All the chemicals were of analytical grade and used as received unless otherwise noted. Pyrrole was distilled over potassium hydroxide pellets under vacuum prior to use. All the organic solvents that were used for the synthesis and for chromatographic separations were dried before use. (TBA)PF 6 was recrystallized twice from EtOAc and dried in vacuo prior to use. Elementary analyses (C, H, N, and S) were obtained on a Vario EL III and CHNS-932 Leco Elemental Analyzer. UV-vis spectra were recorded on a T90+ UV/VIS spectrophotometer in the range 350-700 nm. The oscillator strength ( ) of the transitions in absorption spectra were calculated from the expression where is the molar absorption coefficient in dm 3 mol −1 cm −1 and Δ] 1/2 is the full width at half maximum in cm −1 . Infrared spectra were recorded on a Perkin Elmer-spectrum 400 FTIR spectrophotometer using KBr pellets in the range of 4000-400 cm −1 . The 1 H NMR spectra were recorded on a Bruker Avance II 500 (500 MHz) using tetramethylsilane as internal standard and CDCl 3 as solvent. TGA and DTA were recorded on Linseis STA PT-100 thermometer using around 21.87 mg of dry samples at the heating rate of 10 ∘ C/min in an air atmosphere. The cyclic voltammetry measurements were carried out by an Autolab computer-controlled electrochemical measurement system equipped with a potentiostat. A three Cytotoxicity Assay. In vitro cytotoxicity against human cancer cell lines was determined using sulforhodamine B dye assay [24,25]. Both test samples stock solutions were prepared in DMSO.

Synthesis of Macrocycles.
The metal free-base mesotetraphenylporphyrin (TPP) and its parasubstituted derivative (RTPP, R = p-CH 3 , p-NO 2 , p-Cl) were synthesized by the direct condensation of pyrrole with substituted benzaldehydes according to the documented procedure [26]. The purified porphyrin complexes were obtained in yields of less than 25%. (5,10,15,20-tetraarylporphinato)zirconium(IV), Cl 2 Zr(RTPP). Dichloro(5,10,15,20-tetraphenylporphinato)zirconium(IV), Cl 2 Zr(RTPP)s were obtained by the reaction of mesotetraphenylporphyrin and its parasubstituted derivatives, RTPP (R = p-H, p-CH 3 , p-NO 2 , p-Cl) with zirconium(IV)chloride by benzonitrile method [27]. RTPP (0.41 mmol) was boiled with ZrCl 4 (2.64 mmol) in 15 mL of benzonitrile at reflux for 1-3 hours. The reaction course was monitored by absorption spectra of the reaction mixture. The refluxing was stopped when in the electron absorption spectra of the reaction mixture disappeared the absorption bands of RTPP. The complex was isolated in the solid form after distilling benzonitrile off in a vacuum. Then the saturated solution of the complex in chloroform was subjected to chromatography on basic Al 2 O 3 using CHCl 3 as eluent. After chromatography, the solution of the zirconium(IV) complex was treated additionally with gaseous HCl. The purified dichlorozirconium(IV) porphyrin complexes were obtained in yields of around 30%.

Synthesis of Dichloro
(1) Dichloro (5,10,15,20-tetraphenylporphinato) [28]. After concentration, the mixture was dissolved in minimum quantity of CH 2 Cl 2 and extracted four times with distilled water to remove excess salicylic acid/5-sulfosalicylic acid. The lower layer containing compound in CH 2 Cl 2 was collected, and then it was filtered through anhydrous Na 2 SO 4 in order to remove water. The compound was recrystallized from dichloromethane-hexane solution (1 : 1). The same procedure was applied for the synthesis of all axially ligated zirconium porphyrin complexes as described above. The purified axially ligated zirconium porphyrin complexes were obtained in yields of 40-45%. (

Synthesis and Characterization.
The general synthetic routes to peripherally tetrasubstituted tetraphenylporphyrin derivatives (RTPP), their corresponding metallated, and axially ligated zirconium(IV) porphyrins are shown in Schemes 1, 2, and 3, respectively. All of these new zirconium(IV) porphyrins were purified by column chromatography with aluminum oxide as adsorbent and were characterized by spectral data (UV-visible spectroscopy, IR spectroscopy, 1 H NMR spectroscopy, mass spectral data, and elemental analysis). The characterization data of the new compounds are consistent with the assigned formula. All the free base porphyrins, substituted tetraphenylporphyrin zirconium(IV) chlorides, and substituted tetraphenylporphyrin zirconium(IV) salicylates, are water insoluble.  Tables 1 and 2. The UV-vis data shows that many of the absorption bands of the parasubstituted derivatives exhibit small shifts to longer wavelength, that is, bathochromic shift (red shift) as compared to the spectrum of TPP. The most pronounced bathochromic shift occurs in p-NO 2 TPP with paranitro group on mesophenyl ring of the porphyrin but in all the substituted tetraphenylporphyrin, intensities of all the peaks are higher than those of the parent tetraphenylporphyrin. The reason might be that the strong electronwithdrawing-NO 2 group decreased the electronic density of the porphyrin ring. Thus, the - * electron excitation of the porphyrin ring required absorbing the light of smaller energy (longer wavelength). By comparing the UV-vis data in Table 1, it was noticed that in the visible absorption spectra, free base porphyrins show an intense absorption, referred to as the Soret band at around 400 nm and four weaker absorptions known as Q bands between 450 nm and 800 nm. When the metal ion was inserted into the porphyrin ring, the number and intensity of the Q bands were found to decrease and the B (Soret) band showed a slight blue shift (Figure 1). The reason might be that the structural symmetry of zirconium(IV) porphyrin compounds was improved and the energy gap decreased as compared to the free base porphyrins [29]. In axially ligated zirconium(IV) porphyrin complexes, both B-and Q-bands regions of the spectra show slight red shift. The red shift may be due to structural distortion in the porphyrin macrocycle and concomitant electronic coupling of the metalloporphyrin to the salicylate mediated by the zirconium metal ion [18]. The optical absorption spectra of the compounds of the axially ligated zirconium(IV) porphyrins were when recorded in different solvents; only marginal changes of max values, absorption coefficient ( ), and oscillatory strength ( ) were observed. It is found that with the increase in polarity of the solvents, B-and Q-bands in axially ligated Zr(IV) metal derivatives with different porphyrins show a slight red shift with progressive broadening of bands indicating that the magnitude of red shift of B-and Q-bands depends on the nature of the solvent used. This is so since excited state is more polar than the ground state and hence stabilization is greater relative to the ground state in polar solvents. It is observed that for all the axially ligated Zr(IV) derivatives, Band Q-bands exhibit a red shift on increasing the polarity of the solvents in the order: acetone > chloroform > toluene ( Figure 2). The magnitude of change of the " " values in axially ligated Zr(IV) metal derivatives of porphyrins reveal the relative strength of interaction. Infrared spectral data of above porphyrin compounds is listed in Table 3. It is found that the ](N-H) stretching and bending frequencies of free base porphyrins are located at ∼3400-3320 cm −1 and ∼960 cm −1 , respectively [30]. When the zirconium ion was inserted into the porphyrin ring, the N-H vibration frequency of free base porphyrins disappeared and the characteristic ](Zr-N) vibration frequency was found at ∼500-400 cm −1 , which indicated the formation of zirconium(IV) porphyrin compounds [31]. In the spectra of all the axially ligated zirconium(IV) porphyrin complexes, the band due to ](O-H) of the ligand disappeared indicating the coordination of phenolic oxygen to the metal via deprotonation [32]. The band assigned to the ](C-O) stretching show upward shift in the range ∼1264-1249 cm −1 , which is due to the coordination of the phenolic oxygen to Zr(IV) porphyrins [33]. The incorporation of salicylates in Zr(IV) metal derivatives of different porphyrins, that is, SA/5-SSAZrRTPP, is further confirmed by the appearance of Zr-O vibrational frequencies in the range 690-662 cm −1 and 740-719 cm −1 corresponding to the ligation of zirconium to oxygen of phenolic and carboxylic groups of salicylate, respectively. Thus, the zirconium atom in the centre of porphyrin ring coordinates with the salicylate group axially to form six-coordinate complex of Zr(IV) porphyrin.
1 H NMR data of free base porphyrin, their corresponding metallated, and axially ligated zirconium(IV) porphyrin complexes in CDCl 3 at 298 K is listed Table 4. In all the metallated porphyrins, there was absence of signal related to N-H protons and shift in other signals indicating the insertion of zirconium in porphyrin macrocycle [31]. Generally, the presence of Zr(IV) metal in the porphyrin ring shifts the resonances of the porphyrin's protons to downfield accompanied by marginal changes in the pattern. One of the important features of axially ligated Zr(IV) derivatives of porphyrins is that the metal is almost out of the plane of the porphyrin ring which has earlier been reviewed in the literature, responsible for the production of asymmetric environment above and below the plane of the macrocycle which ultimately accounts for the pronounced nonequivalence of the orthoprotons of the phenyl rings. In axially ligated zirconium(IV) porphyrin complexes, the signals of axial salicylic and 5-sulfosalicylic fragment protons are shifted to higher field in comparison to the signals of porphyrin protons and also in comparison to proton signals of free salicylic and 5-sulfosalicylic acids respectively [34]. These positions of protons show that axial ligand is under the influence of -conjugated system of porphyrin macrocycle [22]. This is most probably due to deshielding effect resulting from the -donation of electron density upon bond formation as compared to the shielding effect of the porphyrin.
Mass spectrometric characterization of SA/5-SSAZrRTPP complexes employed ESI as soft ionization technique. The mass spectra of axial ligated zirconium(IV) porphyrins with salicylic acid/5-sulfosalicylic acid are characterized by the presence of the molecular ion peak for monomeric form followed by a degree of fragmentation when employing this technique, which suggested that axial ligand was labile. The substituents such as sulphonates in the axial ligand were labile too. The major losses are 81, 104, and 33 corresponding to the loss of SO 3 H group, salicylic fragment, and H 2 O molecules. The obtained ESI-MS data display that the molecule of axially ligated zirconium(IV) porphyrins contain one or two molecules of solvent coordinated to the central zirconium atom, which are not labile sufficiently and present in all    molecular ions. This fact can be explained by the coordinated unsaturation of zirconium atom [22].
In the present investigation, the variations of emission properties in free base porphyrin p-CH 3 TPP and its corresponding metallated and axially ligated Zr(IV) porphyrins have been studied ( Table 5). The freebase porphyrins exhibit two emission bands at 653 and 715 nm corresponding to Q(0,0) and Q(0,1) transitions, respectively, the intensity of the Q(0,0) being higher than the Q(0,1) transition.  The complexes Cl 2 Zr(p-CH 3 TPP), SAZr(p-CH 3 TPP), and 5-SSAZr(p-CH 3 TPP) are emissive and show intraligand fluorescence comparable to other regular metalloporphyrins with maxima occurring at 653, 660, and 657 nm, respectively. However, the emission bands of axially ligated Zr(IV) porphyrins are blue shifted compared to free base porphyrins. This behavior is attributed to an enhanced spin-orbit coupling induced by the presence of the heavy-atom central metals in zirconium(IV) porphyrins complexes, which leads to a more efficient intersystem crossing from the lowest porphyrin singlet excited state 1 S 1 ( , * ) to the corresponding triplet manifold and thus reduces the probability of fluorescent emission [35]. Thus, the excitation spectrum of fluorescence is in agreement with absorption spectrum (Figure 3). Cyclic voltammetric studies have been carried out for Cl 2 Zr(p-CH 3 TPP) and its salicylate and 5-sulfosalicylate     Table 6). The electrochemical redox processes of the porphyrin and their metallated derivatives arise primarily from the electron system of the macrocycle. Figure 4 shows the cyclic voltammogram of Cl 2 Zr(p-CH 3 TPP) in CH 2 Cl 2 . In the oxidative scan, two redox couples were observed at 0.66 and 1.22 V corresponding to the formation of porphyrin cation radical and porphyrin dication, respectively. There is a wave in the reductive scan at 1.30 V. The wave is characteristic of one electron reduction of the macrocycle. The data clearly demonstrated the formation of the porphyrin radical anion and inability to reduce Zr(IV) metal centre [36].
For the investigated salicylate ligated zirconium(IV) porphyrins, we observed one reversible reduction wave and two reversible oxidation waves. The three waves for the complexes (Table 6) can be attributed to one-electron processes ZrT(p-CH 3 )PPX 0/−1 , ZrT(p-CH 3 )PPX 1+/0 , and ZrT(p-CH 3 )PPX 2+/+1 (X = SA, 5-SA), respectively, corresponding to the one-electron oxidation/reduction processes involving not only the porphyrin ring but also the entire system as a whole, including even the conjugated salicylate/5sulfosalicylate ligand [37].
The central metal atom (zirconium), being electrochemically passive [38], plays the role of a "linkage" connecting theconjugated systems into a whole integral. The zirconium(IV) porphyrin HOMO-LUMO gap can be expressed as Δ , the potential difference between the first oxidation and the first reduction. The differences between the potentials of the first oxidation wave and the first reduction wave ( ox1 − red1 ) are 1.93 V and 1.98 V for SAZr(p-CH 3 TPP) and 5-SSAZr(p-CH 3 TPP), respectively, allow us to assign these waves to oxidation/reduction processes involving the macrocyclic ring. The experimental Δ values of substituted zirconium(IV) porphyrins are in the range of 1.88-1.98 V.
Thermal analysis of SAZrTPP is shown in Figure 5. The TG curve of SAZrTPP ( Figure 5) shows a continuous weight     Our results demonstrated antibacterial activity against most of the zirconium(IV) porphyrin complexes. Among all the complexes prepared, 5-SSAZr(p-NO 2 TPP) was found to be highly potential against all the five bacterial strains with sensitivity ranging from 10 to 15 mm zone of inhibition at 100 g/mL, respectively (Table 7). SAZr(p-CH 3 TPP) complex showed sensitivity only against B. subtilis and M. luteus with zones of inhibition of 6 and 7 mm, respectively, and it was the only other complex after 5-SSAZr(p-NO 2 TPP) complex that showed antibacterial sensitivity against gram positive bacteria (B. subtilis). Other complexes, namely, Cl 2 ZrTPP, SAZrTPP, p-CH 3 TPP, Cl 2 Zr(p-CH 3 TPP), and 5-SSAZr(p-CH 3 TPP) showed sensitivity only against gram negative bacteria E. coli with zones of inhibition ranging from 6 to 14 mm, respectively. The rest of the complexes did not show any sensitivity against any bacterial strain used in the studies (Table 7).

Conclusion
In this paper, we have described the synthesis of pure porphyrins and their subsequent reactions with ZrCl 4 and salicylic acid/5-sulfosalicylic acid so as to get their axially ligated Zr(IV) porphyrins. The structures of above porphyrin compounds were characterized by UV-vis, IR, 1 H NMR,  and elemental analysis. In axially ligated zirconium(IV) porphyrin complexes, bands showed slight red shift corresponding to the structural distortion in the porphyrin macrocycle and concomitant electronic coupling of the metalloporphyrin to the salicylate mediated by the zirconium metal ion. The infrared spectra of these compounds showed that salicylate groups axially ligated to zirconium(IV) porphyrins to form six-coordinate complexes of Zr(IV) porphyrins ( Figure 7). Additionally, the 1 H NMR spectral study of these compounds showed that signals of axial salicylic and 5-sulfosalicylic fragment protons are shifted to higher field in comparison to the signals of porphyrin protons and also in comparison to proton signals of free salicylic and/or 5-sulfosalicylic acids respectively. The ESI mass spectroscopy provided the information regarding the appearance of the molecular ion peak ( / ) and specific fragmentation. Meanwhile, cyclic voltammograms of axially ligated zirconium(IV) porphyrins complexes have shown three waves for the studied complexes which can be attributed to one-electron processes M(4-CH 3 TPP)X 0/−1 , M(4-CH 3 TPP)X 1+/0 , and M(4-CH 3 TPP)X 2+/+1 (X = SA, 5-SA; M = Zr). All these complexes exhibit reversible oxidation and reduction potentials characteristic of the porphyrin. The data of thermogravimetric analysis indicated that the zirconium(IV) porphyrins complexes are stable up to approximately 150 ∘ C. Their possible biological activities were also investigated and most of the samples exhibited moderate antimicrobial activity against test microorganisms. The two complexes screening for anticancer activity showed less than 50% growth inhibition.