Mixed Ligand Complexes Derived from Isoniazid and Amino Acids; Preparation, Characterization and Anti-Tuberculosis Assay

Introduction

Isoniazid is an organic compound that available in different pharmaceutical forms, which used to prevent and treat both latent and active tuberculosis [1]. Tuberculosis is a public health disease that caused several millions deaths annually worldwide [2]. The number of infected people in this disease have increased, in particularly immunocompromised patients. These include those who co-infected with human immunodeficiency virus (HIV), and human who are receiving anti-tumour therapy or diabetes [3-5]. Therefore, this critical situation has stimulated scientist to develop and explore new strategies in drug discovery to generate new agents with best performance for human health. The aim of this work is explore the influence of incorporating isoniazid in complexation, as a ligand, on the activity of the prepared complexes against tuberculosis bacteria. A series of metal-based complexes were prepared and characterised by mixing isoniazid (the active drug) with some amino acids as bio-active materials in presence of the central metal ion that used as a linkage between drug and amino acids. The bio-assay study shows a positive activity against normal tuberculosis bacteria. However, the complexes exhibit no activity against the resistance tuberculosis bacteria.

Experimental

Chemicals were obtained commercially (Aldrich) and used as received using salt sources; CrCl₃.6H₂O, Fe(NO₃)₃.6H₂O, CoCl₂.6H₂O, NiCl₂.6H₂O and Cu(NO₃)₂.6H₂O. Solvents used in the synthesis were distilled from appropriate drying agent immediately prior to use.

Physical measurements

FTIR spectra were recorded as KBr discs using a Shimadzu 8400 S FTIR spectrophotometer from 4000-400 cm^-1. Electronic spectra were measured from 200-1100 nm for 10^-3 M solutions in DMSO at 25 °C using a Shimadzu 1700 spectrophotometer. HPLC analysis was performed on a Shimadzu LC-2010 AHT in Ibn Sina Company, Ministry of Industry, Baghdad, IRAQ.  Melting points were obtained on an Electro-thermal Stuart melting point SMP40apparatus and are uncorrected.  Elemental analyses (C, H, N) for metal complexes were carried out on a Perkin-Elmer 2400 Series-II analyzer at Materials Research Centre, Ministry for Science and Technology, Baghdad, IRAQ. Metals were determined using a Shimadzu (A.A) 680 G atomic absorption spectrophotometer. Chloride content was determined using potentiometer titration method on a 686-Titrp processor-665Dosimat-Metrohm Swiss. Conductivity measurements were made with DMSO solutions using a Philips PW at 25^°C for complex concentration of 10^-3 M. Magnetic susceptibility were measured with a Brucker magnet B.M.6 apparatus at 20°C. TGA analyses were performed by Linseis instrument. Biological assay of the complexes was conducted in Baghdad Health City, Tuberculosis Treatment Centre where the drug resistance bacteria and ordinary one are obtained from. Primary isolation and culturing of mycobacterium from sputum specimens were followed according to standard procedures [6]. Drug susceptibility testing against isoniazid was performed by the proportional method on Löwenstein-Jensen media at concentrations of 0.2, 0.4 and 2.0 μg/ml, respectively. Drug resistance was defined as greater than 1% growth in the presence of 0.1 μg of isoniazid per millilitre [7, 8].

NaHPO₄ standard buffer solution (0.02M) and acetonitrile were used in the HPLC analysis. For each ligand, a series of three dilutions in water ranged between 0.0001-0.01M were used to perform a calibration curve against the peak area with the following operational conditions;(i) column C₁₈ with dimensions 250*4.6mm and pore 5µm, (ii) the mobile phase was [89% NaH₂PO₄ (0.02M) buffer standard solution pH=5 + 11% acetonitrile] for isoniazid determination and 5% acetonitrile in water solution for amino acid determination, (iii) the flow rate was 1.5 ml/min for isoniazid, while 1 ml/min for amino acids and, (iv) the UV detector wave length was 259 nm in isoniazid and 210 nm for amino acids. The injection volume was 20 µL and the operational temperature was 40°C. Alanine, glycine and isoniazid showed peaks with retention time 6.23, 6.74 and 5.40 minute, respectively.

General method for preparation of complexes

The complexes were prepared adopting a similar method and as follows;

To a solution of (0.01 mole) of the metal salt dissolved in 10-20 ml of deionized water, was added gradually with stirring a mixture of (0.01 mole) of each ligand dissolved in 10 ml of deionized water. The reaction mixture was left stirring for 4 h, and then the solution was allowed to evaporate on a water bath. The concentrated solution was left to stand and a coloured solid that formed was filtered, washed with cold water (3 ml), and then recrystallized from EtOH/H₂O. The coloured solid was dried under vacuum. Melting points, colours and yields of the products are listed in Table (1).

Table 1: physical properties of the ligands and prepared complexes.

Comp.	Yield %	Colour	m.p. °C	Free Cl–/mole	Micro analysis Found (calcd.) %					HPLC retention time (min.)	m S.cm2 .mol-1
					M	C	H	N	Cl
Cr	68	Purple	185	1	10.89(10.47)	27.66(27.38)	4.43(4.27)	14.66(14.42)	22.27(21.79)	–	39.1
Fe	59	Light- brown	275	–	9.35(9.12)	22.12(21.79)	4.22(3.98)	18.76(18.37)	–	–	111.8
Co	65	Pink	155	1	12.64(12.28)	28.34(28.10)	4.76(4.43)	15.02(14.76)	15.21(14.87)	–	60.9
Cu	53	Green- blue	330	–	11.70(11.24)	24.34(24.07)	4.64(4.38)	18.06(17.81)	–	–	54.7
Ni	77	Light- blue	280	2	12.10(11.82)	27.24(26.97)	5.20(5.02)	14.44(14.25)	14.62(14.18)	–	48.2
Iso	–	–		–					–	5.9	–
Ala	–	–		–					–	6.2	–
Gly	–	–		–					–	6.8	–

 

Results and Discussion

The mixed ligand complexes were obtained in good yields in a one-pot approach using an aqueous medium.  The complexes were prepared from the reaction of Cr⁺³, Co⁺², Cu⁺², Fe⁺³, and Ni⁺² with the amino acid ligands (alanine and glycine) and isoniazid. The mixed ligand complexes were prepared from mixing of 10 mmole of metal salt and 10 mmole of each ligand in an aqueous medium. A range of techniques were used to confirm the entity of the prepared compounds. These include FT-IR spectra (Table 2), UV–Vis (Table 3), HPLC, elemental analyses, chloride content, conductance, melting point and magnetic moment measurements. The conductance measurements in DMSO indicated that all complexes are electrolyte [9], see Table (1), complexes are electrolyte. These measurements agree well with the following formulas:

The physico-chemical data indicated the formation of complexes with the general formulae [M(Gly)(Alan)(Ison)(H₂O)_nCl_w]Y (M = Cr⁺³, w = 2, n = 1, Y = Cl; Fe⁺³_,w = 0, n = 3, Y = 3(NO₃^–);Co⁺², w = 1, n = 2, Y = Cl; Cu⁺², w = 0, n = 3, Y = 2(NO₃^–) and Ni⁺², w = 0, n = 3, Y = 2Cl). Complexes were isolated as solids, air-stable that showed solubility in high polar solvents such; H₂O, DMF, DMSO, and insoluble in the low polar ones. The coordination spheres of the complexes were assumed from their spectra (FTIR, UV-Vis), magnetic susceptibility, HPLC, metal and chloride content and TGA, see Figure 1.

Figure 1: The proposed structures of; A) Cr(III)-complex, B) Co(II)-complex, C) Ni(II)-complex, D) Fe(III)-complex and E) Cu(II)-complex. Click here to View figure

 

HPLC and chloride content analysis

HPLC technique was used to clarify the amount of the unreacted organic ligands after complex formation. The HPLC data of glycine (gly), alanine (alan), and isoniazid (ison) have shown retention times of 6.75, 6.23and 5.88 min., respectively. A calibration curve based on a series of standard organic ligands in aqueous solutions was established. The HPLC data indicated that; there is no free ligands remain after the reaction of 1 equivalent of metal ion in an aqueous solution with a mixture of 1 equivalent of each ligand, confirming the involvement of the three ligands in complexation, equation (1):

The analytical data of checking on the ionic chloride content using AgNO₃ solution indicated the precipitation of a 1 equivalent of chloride ion in the Co(II) and Cr(III) complexes, and 2 equivalents in the Ni(II). Therefore, the previous stoichiometry equation for these complexes can be redraw as follows, equation (2-4):

FT-IR spectra

The FTIR data of the complexes and their ligands are listed in Table (2). The FTIR spectra of the free alanine and glycine amino acid display peaks at 1593, 1412 and 1595 and 1412 cm^-1assigned to ν_as(COO^–) and ν_s(COO^–), respectively. These bands were shifted to higher frequencies in the spectra of Cr(III), Co(II), Fe(III) and Ni(II) (Figures (1, 2, 4 and 5)) and appeared in the range 1595-1618 and 1412-1418 cm^-1 for ν_as(COO^–) and ν_s(COO^–), respectively. However, in the Cu(II)-complex, Figure (3), these bands were shifted to lower frequencies and appeared at 1580 and 1416 cm^-1. The shift to higher wave number, compared with that in the free amino acid ligands, can be attributed to a weak linkage between the carboxylate groups in the amino acids and metal ion. While, shifting to lower wave number, compared with that in the free amino acid ligands, is related to delocalisation of metal electron density (t_2g) to the π-system of the ligands [10]. The differences between the asymmetric and symmetric vibration of -COO^– group for both amino acids in all complexes is laying between 164-206 cm^-1. This is in accordance with results indicated the carboxylate group coordinated to a metal ion in a monodentate fashion [11]. The spectra of complexes indicated peaks around ca. 3100 cm^-1, similar to that recorded in the spectra of amino acid ligands, which assigned to ν(NH₃⁺) group. This result indicated that amino acid ligands behaved as a zwitterion species upon complexation. Therefore, they behave as neutral ligands.

The FTIR spectrum of isoniazid shows peak at 3304 cm^-1 related to ν(N-H) group. This peak appeared as a broaden in the spectra of the complexes, indicating the involvement of the nitrogen atom in complexation. Further, the FTIR spectrum indicated peak at 1668 cm^-1 assigned to ν(C=O) group. This band was shifted to lower frequency in all complexes, except that for Cu(II)-complex, which shifted to higher frequency. This shifting indicated the involvement of the isoniazid moiety in complexation. More, the isoniazid moiety coordinate to the metal centre as a neutral species. The spectra of the complexes revealed new peaks in the range 400-430 and 459-511 cm^-1 that assigned to ν(M-N) and ν(M-O), respectively, which indicated that both the nitrogen of isoniazid and carboxylate of amino acids were coordinated to the metal centre [12]. Further, the spectra of ligands and complexes indicated a strong hydrogen bonding that appeared as medium broad band above 3400 cm^-1. Further, this band includes the (OH) and (NH) stretching of the water and the amine fragments. In the Fe(III) and Cu(II) complexes, the spectra indicated peaks at 1517-1560 and1334-1342 cm^-1 related to the ionic NO₃^–group, see Table (2).

Table 2: IR frequencies (cm^–1) of the compounds.

Comp.	νasy(COO-)	νsym(COO-)	νasy(NH3+)Ala, Gly	νsy (NH3+)Ala, Gly	Ala ν (NH3+)	Gly- ν (NH3+)	ν(N-H)	ν(C=O)	ν(M-N)	ν(M-O)	Δ(νas-νsy)
Ala	1593	1412	–	2604
Gly	1595	1412	3159 and 3078	2823	2604		3304
Ison								1668
Cr	1618	1412	3103	2617, 2729	2823	1649		1668	415	496	206
Co	1595	1418	-3123	2619, 2870	2617	2729		1668	415	496	177
Cu	1580	1416	3200 and 3000	2610, 2830	2619	2870		1668	415	496	164
Fe	1608	1418	3196 and 3062	2610, 2839	2610	2830		1668	415	496	190
Ni	1595	1412	3127 and 3055	-, 2866	2610	2839		1668	415	496	183

 

Electronic spectra and magnetic moment measurement

The electronic spectra of the complexes exhibited bands related to the π → π* and n → π*transitions. The Cr(III)-complex showed additional peaks at 552 and 683 nm related to⁴A₂g^(F)→⁴T₁g^(F) and ⁴A₂g^(F)→⁴Tg^(F) transitions, respectively indicating an octahedral arrangement about metal centre [13]. The magnetic moment value 3.73 BM confirmed octahedral geometry around metal ion [12]. The Co(II)-complex, showed additional peaks at 462 and 502 nm, type ⁴T₁g^(P)→⁴T₁g^(P) and ⁴T₁g^(F)→⁴A₁g^(F), respectively. This result with the magnetic moment value 4.45 BM confirmed an octahedral geometry around Co atom [14]. The Cu(II)-complex, Figure (8), showed additional peaks at 710 nm correlated to ²Eg→²T₂g transition that confirmed a distorted octahedral arrangement about the Cu atom. The magnetic moment value 1.72 BM confirmed octahedral geometry around metal ion [15]. The Fe(III) and Ni(II)-complexes, Figures (9 and 10), showed additional peaks at 520, 380 and 630 nm, type ⁶A₁g→²Eg^(G), ³A₂g→³T₁g^(P) and A₂g→³T₁g, respectively. The magnetic moment values 4.33 and 2.88 BM confirmed distorted octahedral geometries around Fe(III) and Ni(II) centres, respectively [16] (see Table 3).

Table 3: Electronic spectral data in DMSO solutions. Click here to View table

 

Thermal decomposition of Fe(III) and Ni(II)complexes

The TGA thermal analysis curve for [Fe(Ala)(Gly)(Ison)(H₂O)₃](NO₃)₃ complex is shown in Figure 2. The complex decomposes in three steps, from 90 to 594.9ºC. The complex undergoes a rapid decomposition and weight loss initially at 160.3 ºC (obs. = 12.0833%, calc.=11.9518%) related to loss (3H₂O+NH₃) molecules. This mass loss could be attributed to the elimination of and ammonia species. This is in agreement with results reported previously in literature [16-18]. The second decomposition step refers to the loss of (C₆H₅N+CO+N₂H₂+3CO₂+O₂+CH₃CN) segment with weight loss (obs. =57.1328, calc. =56.8975%), at 323ºC). The final weight of the compound observed with 28.8015 (calc.=30.43775%) related to (Fe+ H₃O+3NO).

Figure 2: TGA thermogram of Fe(III)-complex in nitrogen with heating rate of 20 °C min–1.  Click here to View figure

 

The TGA thermal analysis curve for [Ni(Ala)(Gly)(Ison)(H₂O)₃]Cl₂complex is depicted in Figure 3. The complex decomposed in three steps, from 95 to 593.7ºC. The complex undergoes a rapid decomposition and weight loss at (obs. = 11.8463%, calc.=11.2028%). The mass loss occurred may relate to losing of 3H₂O molecules, which related to the loss of three aqua water molecules. This is in agreement with results reported previously in literature [9,10]. The second and third decomposition steps refer to the loss of (2CO₂+2N₂+CO) and (C₆H₅N+Cl₂) molecules, with weight loss (obs. = 35.3517, calc. = 35.2697 %), at 454.5 ºC) and (obs. = 30.5574, calc. = 31.1203 %), at 593.7 ºC, respectively.

Figure 3: TGA thermogram of Ni(II)-complex in nitrogen with heating rate of 20 °C min–1. Click here to View figure

 

The differences in the calculated and observed weight may be related to oxidation upon thermal decomposition. This is in agreement with results reported previously in literature [19, 20].

Biological assay

The biological assay data of the complexes against sensitive and resistance tuberculosis bacteria are listed in Table (4). The obtained results indicated the complexes have a similar activity, compared with the original isoniazid drug which is active against sensitive bacteria and did not affect the resistance ones, see Figure 4. This may indicate that upon testing complexes, in vivo or in vitro, they intend to liberate the isoniazid drug in to the biological system. This will lead to the formation of more stable complexes in which the amino acids act as a bidentate chelate ligand. Further, this result may indicate the need to examine the formation of complexes at different pH and elucidate the geometry and stability of complexes, forming upon testing stable complexes containing isoniazid ligand.

Figure 4: Biological assay of the prepared complexes;6- growth controller, 1- Fe complex, 2- Cr complex, 3-Ni complex, 4- Co complex and 5-Cu complex. Click here to View figure

 

Table 4: Biological assay of the prepared complexes.

Compound	Bacteria growth
	Resistance	Sensitivity
Cr	–	+
Co	–	+
Cu	–	+
Fe	–	+
Ni	–	+
Iso	–	+

Conclusion

In this paper, the synthesis of mixed ligands complexes derived from two amino acids and isoniazid ligands are reported. The complexes were prepared by mixing with stirring an equivalent amount of each amino acids and isoniazid with the appropriate metal chloride or nitrate salts. Physico-chemical analysis indicated the formation of six coordinate species in which the geometry around metal centre is a distorted octahedral. Bio-assay study revealed these complexes showed a similar effect against tuberculosis bacteria, compared with the isoniazid compound.

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