Synthesis and characterization of some new thallium(III) macrocyclic complexes and their biological studies

Elemental analysis

All the resulting solids are soluble in CHCl₃, CH₂Cl₂, CH₃OH and DMSO. The analytical data of the complexes of thallium(III) metal complexes are given in Table 1.

Absorption spectroscopy

The B and the Q bands in the spectrum of typical porphyrin both arise from π-π* transitions and can be explained by considering the four frontier orbitals (Milgrom, 1997) (HOMO and LUMO orbitals) (the Gouterman four-orbital model). From the UV-Vis absorption values given in the Experimental section, it can be clearly observed that variation of the peripheral substituents on the porphyrin ring causes minor changes in the intensity and wavelengths of these absorptions, and their spectral data in different solvents (Table 2) shows that many of the absorption bands of para-substituted derivatives exhibit bathochromic (red shift) as compared to the unsubstituted porphyrin. The electronic absorption spectra of aquo-meso-tetraaryl porphyrinato thallium(III) hydroxide (OH)(H₂O)T1(III)tpp and (OH)(H₂O)T1(III)t(4-Y)PP, Y=-CH₃, -OCH₃ are of normal two-banded type. It is apparent from the spectra that there is no major distortion of the macrocycle ring upon metalation and that the large metal atom is situated above rather than in the plane of the porphyrin ring. Both B bands (350–450 nm) and Q bands (500–700 nm) were found to be red-shifted in all thallium porphyrins (Figure 1). Also, the molar absorbance of both the main Soret and the Q bands of the metalloporphyrins are higher than the corresponding values for the free-base porphyrin (Tables 2 and 3). According to earlier observations (Horvath et al., 2004, 2006; Valicsek et al., 2004, Valicsek et al., 2011) this type of spectral properties is unambiguously characteristic for OOP (out of plane) or Sat (sitting atop) complexes, confirming the expectations based on the size (95 pm ionic radius) of Tl(III). Not only metalation but also axial coordination is accompanied by red shifts of the characteristic absorption bands. As the corresponding values for B(0,0) itself indicate, the larger OOP distance in case of axially ligated porphyrin results in the higher dome distortion of the porphyrin ligand. For the Q(0,0) band the effect of the axial ligand is much stronger, indicating that the energy of the S₁ state is more influenced by this structural change. The f values (oscillator strength) of some of the metalated porphyrin and their axially ligated derivatives (Table 3) show that change in polarity of the solvent does not significantly alter the position of transitions but result in an enhanced full width at half maximum (fwhm) (1/2) value indicating weak interactions with the solvent.

Figure 1:

UV visible spectra of free base porphyrin and thallium(III) porphyrin.

(a) UV-visible overlapped B-bands of H₂tpp and (OH)(H₂O)Tltpp. (b) UV-visible overlapped Q-bands of H₂tpp and (OH)(H₂O)Tltpp.

The optical absorption spectra of axially ligated Tl(III) porphyrins when recorded in different solvents show only marginal change in λ_max values. The spectrum of the complex SATl(III)t(4-CH₃)PP is shown in Figure 2.

Figure 2:

UV-visible overlapped B and Q bands of SATlt(4-CH₃)PP.

Infrared spectroscopy

The infrared (IR) spectral data of various synthesized complexes of thallium porphyrins are listed in Table 4. It is found that the ν(N-H) stretching frequency of free base porphyrins are located at ~3400–3320 cm^-1 (Sun et al., 2011) and disappeared when thallium metal ion was inserted into the porphyrin ring. There is an additional Tl-N stretch vibration at 400–500 cm^-1 in metalated complexes indicating the formation of Tl(III) porphyrins. The band in the complexes at 1633 cm^-1 [ν_asymmetric(OCO)] are assigned to the chelating bidentate salicylate ligand. The bidentate nature of salicylates in the IR spectra of the complexes was confirmed on the basis of Stoilovo et al. results that unidentate CO₂ exhibits three bands (COO deformation) at 920–720 cm^-1 and a strong band at 540 cm^-1 (Suen et al., 1992). All these four bands were absent in axially ligated thallium porphyrin complexes. Further, the band observed near 1724 cm^-1 is assigned to ν(C=O) stretching frequency. The frequency corresponding to free O-H group is observed at 3418 cm^-1 confirming the coordination of axial ligand through carboxylate oxygen atoms. The decrease in vibrational frequency of OH group is due to the weakening of O-H bond as -CO₂ in its vicinity from salicylate ring ligates to the metal atom.

¹H NMR spectroscopy

The thallium porphyrin complexes with salicylates(SA/5-SSA) as axial ligands all exhibit sharp ¹H NMR spectra. The characteristic data for the NMR of the free base porphyrins, corresponding metalated complexes of thallium(III) and their axially ligated derivatives are summarized in Table 5.

¹H NMR spectra are fruitful in clarifying the insertion of metal ion in the porphyrin core as the signal related to N-H protons was found to be absent, and also there was a shift in the signals of other protons in the NMR spectra of metalated porphyrins. In the axially ligated derivatives of thallium porphyrins, the singlet at 5.0 ppm of medium intensity was observed assigned to the -OH group which confirms that axial ligation was taking place through the oxygen atom of carboxylate group. All the δ values correspond to the proton signals of axial ligands in the axially ligated complexes that exhibited upfield shift when compared with the signals of porphyrin protons and the proton signal of free axial ligands. This upfield shift is attributed to the ring current effect (Lu et al., 1999) of the porphyrin macrocycle.

²⁰⁵Tl NMR spectroscopy

The range for ²⁰⁵Tl NMR chemical shift values is very large, i.e. about 7000 ppm. As far as oxidation states are concerned, for Tl(I) complexes chemical shift values are in the range -200 to +200 ppm, and for Tl(III) these lie in the range +2000 to +3000 ppm. ²⁰⁵Tl NMR spectra of SATl(III)t(4-CH₃)PP when recorded in CDCl₃ exhibits a sharp resonance at δ 2588 ppm which confirms the presence of Tl(III) ion in the complex. On comparing this value with the chemical shift value of the complexes already reported in literature (Ma et al., 2003), it was found that this value has shifted towards lower frequency which can be attributed to the presence of four electron-releasing -CH₃ groups on the meso-phenyl ring. This parameter causes shielding of the thallium nucleus and hence shifts towards lower frequency (Figure 3).

Figure 3:

²⁰⁵Tl NMR spectrum of the complex SATlt(4-CH₃)PP.

Thermal analysis

The thermogravimetric (TG) curve of one of the synthesized complex 5-SSATl(III)t(4-H)pp (Figure 4) shows a continuous weight loss starting from 150°C to 750°C. The curve shows an initial weight loss of a sulphosalicylate group and one phenyl ring at 158.2°C (obs. wt. loss=27.7%, calc. wt. loss=27.2%). This is followed by a loss of three phenyl rings and thallium metal ion at 408.5°C (obs. wt. loss=40.2%, calc. wt. loss=40.2%). There are some exothermal peaks observed in the range of 300–600°C showing major weight loss in this region. It is a continuous decomposition process; the small exothermic peaks correspond to the loss of chains of the porphyrin ring (Zhuang et al., 2009), and the large exothermic peaks at nearly 580°C corresponds to the collapse of the porphyrins skeleton. The residual mass was 0 at 751.2°C.

Figure 4:

Thermogravimetric analysis (TGA)-differential thermal analysis (DTA) curve of 5-SSATltpp.

Fluorescence spectroscopy

The variation of emission properties in free base porphyrin H₂t(4-Y)PP (Y: -CH₃, -OCH₃) and their axially ligated Tl(III) porphyrins have been investigated by means of fluorescence spectroscopy; data are given in Table 6. Porphyrins show two emission bands, a strong Q(0,0) band at higher energy accompanied by a weak Q(0,1) band at a lower energy. The free-base porphyrins H₂t(4-CH₃)PP, with excitation at 450nm, exhibits two emission bands at 620 and 672 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. Meso-subsitution of the porphyrin ring leads to red shift of all the emission bands relative to that of unsubstituted tetraphenylporphyrins. However, the emission bands of axially ligated Tl(III) porphyrins are blue shifted (Horvath et al., 2006) compared to free base porphyrins. This behavior is attributed to an enhanced spin-orbit coupling induced by the presence of the heavy central metal atom in thallium porphyrin complexes, which leads to a more efficient S₁→T₁ intersystem crossing and thus reduces the probability of fluorescent emission (Knöra and Strasser, 2002). Thus, the excitation spectrum of fluorescence is in agreement with the absorption spectrum (Figure 5).

Figure 5:

In vitro cytotoxicity of 5-SSATlt(4-OCH₃)PP complex against human cancer cell lines.

Mass spectrometry

The mass spectra of some of the investigated axially ligated thallium porphyrin complexes are characterized by the presence of the molecular ion peak followed by different specific fragmentation pattern. The molecular ion peak of the investigated complexes was found to be 44 units less than the corresponding molecular weight of the complex, which is attributed to the loss of stable CO₂ molecule from the complex. The major losses are 304, 615, 616 and 81. Besides the molecular ion, an identical peak m/z=304 fragment, corresponding to the tetrapyrrole moiety, appears in each spectrum (Cosma et al., 2006). Two groups of peaks were observed (main signals accompanied by isotopic patterns confirming the chemical composition of the ions) at m/z 615 and 616 that correspond to the fragments of H₂tpp+1 and H₂tpp+2 (H₂tpp=meso-tetraphenyl porphyrin), and the peak at 81 corresponds to the SO₃H group.

In addition, the intensities of the registered peaks are significantly higher. The base peak of thallium porphyrin complexes was observed 100% intensity giving evidence about the stability of complexes of thallium porphyrins.

Antibacterial studies

Antibacterial activity of the synthesized Tl(III) porphyrin complexes was tested by agar well diffusion method. The free base prophyrins, their corresponding metalated and axially ligated thallium(III) porphyrin complexes viz., H₂t(4-OCH₃)PP, H₂t(4-CH₃)PP, (OH)(H₂O)Tlt(4-CH₃)PP, (OH)(H₂O)Tlt(4-OCH₃)PP, SATlt(4-CH₃)PP, 5-SSATlt(4-CH₃)PP and 5-SSATlt(4-OCH₃)PP were tested at three concentrations (10^-3, 10^-4 and 10^-5m) against five bacterial strains viz., B. subtilis, Micrococcus luteus, S. aureus, Pseudomonas florescens and E. coli. The thallium(III) complex 5-SSATlt(4-OCH₃)PP was found sensitive only to B. subtilis (Gram positive) and P. florescens (Gram negative) bacteria at the concentrations of 10^-3 and 10^-4m. Other complex SATlt(4-CH₃)PP) showed sensitivity against B. subtilis and M. luteus. The remaining complexes, viz., H₂t(4-CH₃)PP, (OH)(H₂O)Tlt(4-CH₃)PP and 5-SSATlt(4-CH₃)PP showed activity only against Gram negative bacteria E. coli with zone of inhibition ranging from 6 to 14 mm. The rest of the complexes did not show any activity against bacterial strains. The results were observed in dose-dependent manner as demonstrated in Table 7.

Anticancer activity

In the present work, the cytotoxicities of 5-SSATl(III)t (4-OCH₃)PP, SATl(III)t(4-OCH₃)PP and SATl(III)t(4-CH₃)PP were evaluated against four human cancer cell lines, viz., breast (MCF-7), leukemia (THP-1), prostate (PC-3) and lung (A549) at different concentrations. One of the complexes, namely, 5-Sulphosalicylato (meso-tetra-p-methoxyporphyrinato)thallium(III) (MB-5) shows significant activity (Figure 5) against lung and leukemia, classifying these complexes as chemotherapeutically significant. We also tested other synthesized compounds against the above mentioned panel of cancer cell lines; however, none of the compounds exhibited cytotoxicity even at 100 μm concentration (results not shown).

Materials and instrumentation

All the chemicals were of analytical grade and were used as received unless otherwise noted. All the reagents were purchased from Himedia (Mumbai, India). 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. Elemental analysis (C, H, N and S) was performed on a Vario EL III and CHNS-932 LECO Elemental Analyzer (IIIM, Jammu). UV-Vis spectra were recorded on a Systonic 119 UV Visible spectrophotometer (University of Jammu, Jammu) in the range 350–700 nm. The oscillator strength (f) of the transitions in absorption spectra were calculated from the expression

f=4.33×10-9εΔ ν½

where ε is the molar absorption coefficient in dm³ mol^-1 cm^-1 and Δν_½ is the fwhm in cm^-1. IR spectra were recorded on a Perkin Elmer-spectrum 400 FTIR spectrophotometer using KBr pellets in the range of 4000–400 cm^-1. The ¹H NMR spectra were recorded on a Bruker Avance II 500 (500 MHz) using tetramethylsilane as internal standard and CDCl₃ as solvent. ²⁰⁵Tl NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer in CDCl₃. TGA and DTA were recorded on Linseis STA PT-100 thermometer using dry samples at the heating rate of 10°C min^-1 in an air atmosphere. Fluorescence measurements were performed on Synergy MX BIOTEK Multimode Reader. The porphyrin solution prepared in CH₂Cl₂ was 10^-6m.

Biological studies

Synthesis of macrocycles

H₂tPP and H₂t(4-Y)TPP (Y=-CH₃ and -OCH₃) were synthesized. The synthesis of the porphyrins has been adapted from the synthesis reported by Adler et al. (1967). The appropriate benzaldehyde is refluxed for 30 min with 0.08 mL of pyrrole in 12 mL of propionic acid in a 50-mL round bottom flask fitted with a water condenser. The pyrrole must be from a freshly opened bottle or have recently been purified by vacuum distillation. After refluxing, the reaction mixture is cooled to room temperature, and 10 mL of cold methanol is added. The deep-purple solid is filtered by vacuum filtration with a Buchner funnel. The flask and crude solid is washed with three portions of cold methanol followed by boiling distilled water. The crystals are air-dried on the Buchner funnel for 15 min, dried in a vacuum desiccator and purified by column chromatography (Scheme 1). The syntheses of the substituted porphyrins, i.e. H₂t(4-CH₃)TPP and H₂t(4-OCH₃)TPP, are performed in a similar manner. Average yields are about 40 mg (23%). All the benzaldehydes are commercially available. The resulting porphyrins can be characterized by visible absorption or ¹H NMR spectroscopy.

Scheme 1:

Synthesis of meso-tetraphenylporphyrins and their corresponding metalated and axially ligated derivatives.

Synthesis of aquo-meso-(5,10,15, 20-tetraarylporphyrinato)thallium(III)hydroxide, [(OH)(H₂O)Tltpp and (OH)(H₂O)Tlt(4-Y)PP] (Y: -CH₃, -OCH₃)

A mixture of meso-tetraphenylporphyrins 0.0068 mmol in CH₂Cl₂ (10 mL) and Tl(OAC)₃ 0.12 mmol in MeOH (2.5 mL) was refluxed for 1 h. After concentration, the residue was dissolved in CHCl₃, dried with anhydrous Na₂SO₄ and filtered. The filtrate was concentrated and recrystallized from CH₂Cl₂-MeOH (1:5 v/v) yielding purple solid of complex, (0.037 mmol, 55%), which was again dissolved in CH₂Cl₂-ether [1:1 (v/v)] and layered with MeOH to get purple crystals of thallium(III) porphyrins (Scheme 1).

Synthesis of axially ligated Tl(III) Porphyrins: Tlt(4-Y)PP, SA and Tlt(4-Y)PP, 5-SSA

(OH)(H₂O)Tl(III)t(4-Y)PP 0.15 mmol in 30 mL CHCl₃ was treated with respective salicylates 0.56 mmol in 25 mL CH₃OH and stirred under reflux for 12 h. After concentration, the mixture was dissolved in minimum quantity of CH₂Cl₂ and extracted four times with distilled water to remove excess unreacted salicylates (SA, 5-SSA). The resulting solution was then filtered through anhydrous Na₂SO₄ in order to remove water molecule. The CH₂Cl₂ layer was then concentrated to dryness, producing purple prism. The same procedure was applied for the synthesis of all axially ligated complexes of thallium porphyrins. The overall route for the synthesis of axially ligated thallium (III) porphyrin complexes is given in Scheme 1.