Synthesis,characterization,and biological properties of oxidovanadium(IV) complexes of acetylsalicylhydroxamic acid ( N -acetyloxy-2-hydroxybenzamide) as potential antimicrobials

Abstract

New oxidovanadium(IV) complexes of composition [VO(AcSHA)₂] 1 and [VO(acac)(AcSHA)] 2 are synthesized by reactions of VOSO₄.5H₂O and [VO(acac)₂] with acetylsalicylhydroxamic acid AcSH₂A (C₆H₄(OH)(CONHOCOCH₃)) in a 1:2 molar ratio in absolute ethanol. The compounds are characterized by the Fourier-transform infrared spectroscopy, ultraviolet–visible spectroscopy, electron spin resonance, and mass spectrometry along with elemental analyses, molar conductivity, and magnetic moment measurements. The infrared spectra of the complexes suggest bonding through carbonyl and phenolic oxygen atoms (O,O coordination). The magnetic moment, electron spin resonance, and mass spectra of the complexes indicate that both exist as monomers, and a distorted square pyramidal geometry around vanadium is proposed. The thermal behavior of the complexes is studied by thermogravimetry and differential thermal analysis techniques under an N₂ atmosphere, yielding VO₂ as the decomposition product. The in vitro antimicrobial assays against pathogenic Gram-positive bacteria, Gram-negative bacteria, and fungi (minimum inhibitory concentration method) show the appreciable antimicrobial potential relative to the respective standard drugs, tetracycline hydrochloride, and fluconazole.

Keywords

acetylsalicylhydroxamic acid biological properties oxidovanadium(IV) complexes spectral studies

Introduction

The ever-increasing research interest in oxidovanadium(IV) complexes is due to their prodigious biological and medicinal properties,^1–4 such as insulin-mimetic,^5–8 DNA binding and cleavage activity,^9–11 anticancer,¹² antioxidant,^13–15 antitrypanosomiasis,^16,17 antileishmaniasis,¹⁸ antiamebiasis,¹⁹ antituberculosis,²⁰ and anti-HIV²¹ activity inducing apoptosis, inhibiting cell proliferation and preventing the metastasis of cancer against various cell lines.^22–24 The model studies related to vanadium biochemistry providing information on vanadium metabolism, toxicity, detoxification, and catalytic activity have been reported.²⁵ The bis(maltolato) (BMOV), bis(ethylmaltolato) (BEOV), and bis(allixinato)oxidovanadium(IV) are some of the most studied insulin-enhancing oxidovanadium(IV) complexes.^26–28 A drug containing vitamin A and vanadium(IV) salt having insulin-mimetic antidiabetic properties has been reported.⁸ Oxidovanadium(IV) complexes as YopH tyrosine phosphatase inhibitors, a virulence factor produced by pathogenic species of Yersinia, have also been described.²⁹ The exponential progress in the medicinal chemistry of biologically relevant vanadium complexes for therapeutic applications is evident from numerous reports.^30–33

On the contrary, hydroxamic acids with the functionality R_CC(O)N(R_N)OH (R_C = alkyl/aryl; R_N = H or alkyl/aryl) are an important and key class of weak organic acids that have been well studied as bioligands. The distinctive chelating ability^34,35—due to keto–enol tautomerism exhibited by the hydroxamic and hydroximic forms³⁶—and the vast spectrum of pharmacological, toxicological, and pathological properties of hydroxamic acids have fascinated researchers in the design of new complexes of biological relevance.^37–39

Biologically significant acetylated hydroxamic acids synthesized from salicylhydroxamic acid using various acetylating agents and different methodologies have been reported.^40,41 The reaction of salicylhydroxamic acid with acetyl chloride in the presence of pyridine and ethyl acetate forms acetylsalicylhydroxamic acid (AcSH₂A) where AcSH₂A = C₆H₄(OH)(CONHOCOCH₃), and the acetyl group is located on the hydroxamic acid group. The vanadium hydroxamate complexes derived from acetohydroxamic acid (aceto-HAha), benzohydroxamic acid (HBha), N-phenylbenzohydroxamic acid, 2-hydroxypyridine N-oxide,⁴² nicotinohydroxamato, 4-nitrobenzohydroxamate, and 2-chloro-4-nitrobenzohydroxamate ligands are also well-documented.^43,44

As a part of our continuing work on vanadium hydroxamates,^45–47 we herein report the synthesis of new bis(acetylsalicylhydroxamato) and the mixed-ligand (acetylacetonato)(acetylsalicylhydroxamato)oxidovanadium(IV) complexes using two different vanadium precursors along with their characterization by the Fourier-transform infrared spectroscopy (FTIR), ultraviolet–visible spectroscopy (UV-Vis), electron spin resonance (ESR), and mass spectral techniques. The electrochemical and thermal behavior of the complexes has also been studied. The antimicrobial and antioxidant potential of the complexes has been assayed against some pathogenic bacteria and fungi by minimum inhibitory concentration (MIC) method and DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging method, respectively.

Results and discussion

The reactions of VOSO₄.5H₂O and VO(acac)₂ with 2 equiv. of AcSH₂A in absolute ethanol in separate experiments gave dark blue complexes of composition [VO(AcSHA)₂] 1 and [VO(acac)(AcSHA)] 2, respectively, in quantitative yields and are consistent with their elemental analysis (Scheme 1).

Scheme 1.

Synthesis of oxidovanadium(IV) complexes.

The molar conductance values of the complexes measured in methanol were 3.92 and 3.90 Scm² mol⁻¹, respectively, suggesting their non-electrolytic nature.⁴⁸ At the room temperature, magnetic moment of 1.70 and 1.72 BM (Bohr magneton) for complexes 1 and 2 is well within the range reported for monomeric oxidovanadium(IV) complexes.⁴⁹

Infrared spectra

A comparison of the infrared (IR) spectra of the complexes with that of the ligand supports their formation. Free AcSH₂A exhibited bands due to ν(OH)_phenolic, ν(N–H), ν(C=O)_acetyl, ν(C=O)_hydroxamic, ν(C–N), and ν(N–O) modes at 3423, 3345, 1773, 1645, 1348, and 954 cm⁻¹, respectively, whereas the complexes [VO(AcSHA)₂] and [VO(acac)(AcSHA)] displayed the respective bands at 3274, 1770, 1603, 1371, and 907 cm⁻¹ and 3392, 1770, 1660–1626, 1348–1342, and 917 cm⁻¹, as presented in Tables 1 and 2, respectively.

Table 1.

IR spectral data of the ligand (AcSH₂A) and the complexes 1 and 2.

Ligand/complex	Bands (cm⁻¹)
(AcSH₂A)	3423, 3345, 3189, 3071, 1773, 1645, 1625, 1596, 1537, 1486, 1348, 1307, 1244, 1141, 1123, 1030, 954, 864, 748, 675, 637, 578, 502, 445, 419
[VO(AcSHA)₂]	3274, 3050, 2941, 2866, 2729, 2583, 1770, 1603, 1463, 1371, 1308, 1231, 1185, 1156, 1097, 1051, 1031, 970, 907, 857, 782, 744, 688, 596, 546, 461, 422
[VO(acac)(AcSHA)]	3392, 3182, 3071, 2793, 2464, 1770, 1660, 1626, 1596, 1490, 1348, 1342, 1213, 1154, 1119, 1032, 1012, 957, 917, 867, 748, 676, 578, 504, 444

Table 2.

Important IR frequencies (cm⁻¹) of the ligand (AcSH₂A) and the complexes 1 and 2.

Ligand/complex	ν(OH)_hydroxamic	ν(N–H)	ν(C=O)_acetyl	ν(C=O)_hydroxamic	ν(C–N)	ν(N–O)
(AcSHA)	3423	3345	1773	1645	1348	954
[VO(AcSHA)₂]	–	3274	1770	1603	1371	907
[VO(acac)(AcSHA)]	–	3392	1770	1660–1626	1348–1342	917

The nonobservance of the band due to the ν(OH) mode in complexes 1 and 2 is indicative of deprotonation of the phenolic hydrogen, involving bonding through the phenolic oxygen. The ν(C=O)_hydroxamic mode in complex 1 shifted to a lower wavenumber by 42 cm⁻¹ and the ν(C–N) mode shifted to a higher wavenumber by 23 cm⁻¹ as compared to the ligand. The absorption band due to ν(C=O)_acetyl occurred at 1770 cm⁻¹, showing an insignificant shift from the ligand and suggesting the nonparticipation of the acetyl group in bonding (see Figure S1(a)–(c) in the supporting information). The shift in the ν(N–O) mode to a lower wavenumber by 47 cm⁻¹ is contrary to earlier reports wherein the ν(N–O) mode shifts to a higher wavenumber upon complexation. The occurrence of the ν(N–H) mode is suggestive of its retention upon complexation. The absorption bands due to the ν(V=O) mode occurred at 970 and 957 cm⁻¹, respectively. However, this observation is in contrast to those for Co(II), Ni(II), Cu(II), and Zn(II) complexes of AcSH₂A wherein tridentate bonding behavior of ligand has been reported.⁴⁰

Electronic spectra

The UV-Vis spectra of the ligand (AcSH₂A) recorded in absolute ethanol displayed an absorption band at 319 nm attributed to an intra-ligand π → π* transition. Complexes 1 and 2 exhibited bands at 270, 308, 478, and 735 nm and 305, 316, 425, and 720 nm in the UV-Vis region, respectively (Table 3). The high-energy intra-ligand bands at 270 and 308 nm and 305 and 316 nm occurred in complexes 1 and 2, respectively, are contrary to previous reports.^49,50 The bands appeared in the visible region may be assigned to ²B₂ → ²A₁ (3dxy → 3dz²) and ²B₂ → ²E (3dxy → 3dxz, 3dyz) d–d transitions, respectively, and a plausible square pyramidal geometry around vanadium is proposed.^51,52

Table 3.

UV-Vis spectral data of the ligand (AcSH₂A) and the complexes 1 and 2.

Ligand/complex	Λ (nm)
(AcSH₂A)	319
[VO(AcSHA)₂]	270, 308, 478, 735
[VO(acac)(AcSHA)]	305, 316, 425, 720

Mass spectra

The electrospray ionization (ESI) mass spectrum of complex 1 exhibited a molecular ion peak at m/z (%), that is, 479 (4.3), corresponding to [VO(AcSHA)₂ + Na]⁺. The most abundant peak at 218 (100) corresponded to [AcSHA + Na]⁺, which is presented in Table 4.

Table 4.

Mass spectral data of the complex [VO(AcSHA)₂] 1.

Complex	m/z	Intensity (%)
[VO(AcSHA)₂ + Na]⁺	479	4.3
[VO(AcSHA)₂ − 6H]⁺	451	21.7
[2AcSHA]	390	8.6
[2(AcSHA) − 2CH₃ − 4H]⁺	354	60.8
[VO(AcSHA)]⁺	262	34.7
[AcSHA + K]⁺	234	20
[AcSHA + Na]⁺	218	100

The other structurally important fragment ions at m/z (%) 451 (21.7), 390 (8.6), 354 (60.8), 262 (34.7), and 234 (20) corresponded to [VO(AcSHA)₂ − 6H]⁺, [2AcSHA], [2AcSHA − 2CH₃ − 4H]⁺, [VO(AcSHA)]⁺, and [AcSHA + K]⁺, respectively, as clearly shown in Scheme 2 (see Figure S2 in the supporting information).

Scheme 2.

Mass spectral fragmentation pattern of complex 1.

The mass spectrum of complex 2 displayed the molecular ion and the base peak at m/z (%) 360 (18) and 302 (100) corresponding to [VO(acac)(AcSHA)] and [VO(acac)(AcSHA) − OCOCH₃ − H]⁺, respectively (Table 5).

Table 5.

Mass spectral data of complex [VO(acac)(AcSHA)] 2.

Complex	m/z	Intensity (%)
[VO(acac)(AcSHA)]	360	18
[VO(acac)(AcSHA) − OCOCH₃ − H]⁺	302	100
[AcSHA + K⁺ − 4H]⁺	230	25.4
[VO(acac) + K⁺ − 3H]⁺	202	21.8

The other fragment ions at m/z (%) 230 (25.4) and 202 (21.8) corresponded to [AcSHA + K⁺ − 4H]⁺ and [VO(acac) + K⁺ − 3H]⁺, respectively (Scheme 3) (see Figure S3 in the supporting information).

Scheme 3.

Mass spectral fragmentation pattern of complex 2.

ESR spectra

At the room temperature, X-band ESR spectra of complexes 1 and 2 displayed the well-resolved typical eight lines due to the interaction of an unpaired electron of the vanadium(IV) center with its own nucleus, that is, I = 7/2 indicative of the mononuclear oxidovanadium(IV). The nuclear magnetic quantum numbers analogous to these lines are −7/2, −5/2, −3/2, −1/2, +1/2, +3/2, +5/2, and +7/2 from low to high field. The complex 1 showed g‖ = 1.94 and A‖ (×10⁻⁴ cm⁻¹) = 163 and g┴ = 1.98 and A┴ (×10⁻⁴ cm⁻¹) = 65. The complex 2 exhibited g‖ = 1.96 and A‖ = 161 and g┴ = 1.98 and A┴ = 67. The g_iso values calculated from the spectra for complexes 1 and 2 of same magnitude 1.96 are close to the spin-only value (free electron value of 2.0023; Table 6).

Table 6.

ESR spectral data of oxidovanadium(IV) complexes 1 and 2.

Complex	g‖	g┴	∆g = g┴ − g‖	$g_{i s o} = \frac{2 g ⊥ + g ‖}{3}$	A‖ (×10⁻⁴ cm⁻¹)	A┴ (×10⁻⁴ cm⁻¹)
[VO(AcSHA)₂]	1.94	1.98	0.04	1.96	163	65
[VO(acac)(ACSHA)]	1.96	1.97	0.01	1.96	161	67

The observed order A‖ > A┴ and g┴ > g‖ suggests that the unpaired electron lies in the d_xy orbital corresponding to the square pyramidal geometry around vanadium in complexes 1 and 2 in agreement with the reports on oxidovanadium(IV) chelates.^53,54

Cyclic voltammetry

The free ligand (AcSH₂A) displayed one reductive wave and one oxidative wave at −0.61 and −0.51 V, respectively. The complex 1 exhibited two reduction waves at −0.48 and +0.69 V and one oxidation wave at −0.551 V. The complex [VO(acac)(AcSHA)] 2 exhibited reductive and oxidative waves at +0.62 and −0.26 V, respectively (Table 7 and Figures 1 –3).

Table 7.

Cyclic voltammetric data of ligand (AcSH₂A) and oxidovanadium(IV) complexes 1 and 2.

Ligand/complex	E_pa (V)	E_pc (V)	ΔE (V)	I_pa (A)	I_pc (A)	I_pa/I_pc
(AcSH₂A)	−0.51	+0.61	1.110	0.85	3.83	0.22
[VO(AcSHA)₂]	−0.551	+0.690	−1.250	1.25	4.01	0.31
[VO(acac)(AcSHA)]	−0.260	+0.62	−0.880	3.30	5.09	0.65

Figure 1.

Cyclic voltammogram of the free ligand (AcSH₂A).

Figure 2.

Cyclic voltammogram of the complex 1 [VO(AcSHA)₂].

Figure 3.

Cyclic voltammogram of the complex 2 [VO(acac)(ACSHA)].

The peak-to-peak separation value (ΔE_p = 79 mV) is indicative of a single-step pseudo-reversible one-electron transfer process and also adjudged from the peak current ratio I_pa/I_pc. The electrode process can be represented as

[{VO}^{IV} L] \to [{VO}^{V} L]

The E_1/2 value of 519 mV may be assigned to metal-centered oxidation of V^IV to V^V lying within the range reported for related complexes.⁵⁵ Based upon physicochemical and various spectral techniques, a distorted square pyramidal geometry in both the mononuclear complexes has been proposed around the vanadium (Figures 4 and 5).

Figure 4.

Chemcraft structure of the complex 1 [VO(AcSHA)₂].

Figure 5.

Chemcraft structure of the complex 2 [VO(acac)(ACSHA)].

Thermogravimetric studies

The complexes 1 and 2 have shown the thermal stability up to 60°C and 85°C, respectively, after which these complexes went decomposition in two steps. The mass loss of 45% in the first step in complex 1 in the temperature range of 60°C–427°C analogous to the dissociation of one acetylsalicylhydroxamate ligand yielding [VO(AcSHA)] as the likely unstable intermediate. The mass loss of 36.9% in the second step, between 427°C and 646°C, accounted for the decomposition of another AcSH₂A ligand to yield VO₂ as the final residue. The formation of VO₂ as thermolyzed product of vanadium(IV) hydroxamate complexes has been authenticated by recording the IR spectra of the residue. The sharp absorption bands appeared at ~992 cm⁻¹ due to ν(V=O) mode and the other bands appeared at 675, 658, 620, 520, and 322 cm⁻¹ are in line with earlier reports on the IR spectra of VO₂.⁵⁶ The thermal decomposition pattern of complex 1 is presented in Scheme 4.

Scheme 4.

Thermal decomposition pattern of complex 1.

A broad exothermic peak at 556.0°C in differential thermal analysis (DTA) curve corroborated thermal decomposition in thermogravimetry. The mass loss of 57.73% in complex 2 in the first step in the temperature range of 85°C–400°C accounted for the removal of one acetylsalicylhydroxamate ligand yielding [VO(acac)] as the probable unstable intermediate. The mass loss of 27.57% in the second step, between 400°C and 617°C corresponded to the decomposition of the acetylacetonate ligand to yield VO₂ as the final residue. The thermal decomposition pattern of complex 2 is presented in Scheme 5.

Scheme 5.

Thermal decomposition pattern of complex 2.

The two-step decomposition in the thermogravimetric analysis (TGA) is accompanied by an exothermic inflection at 133°C and a broad peak at 577°C in the DTA curve (Table 8 and Figures 6 and 7).

Table 8.

Thermal data of oxidovanadium(IV) complexes 1 and 2.

Complex	Initial decomposition temperature	Stages of decomposition	Observed mass loss (%)	Decomposition temperature range (°C)	Peak nature in DTA (exothermic, °C)	Decomposition product
[VO(AcSHA)₂]	60°C	I	45	60–427	–	[VO(AcSHA)]
[VO(AcSHA)₂]	60°C	II	36.9	427–646	556	VO₂
[VO(acac)(AcSHA)]	85°C	I	57.73	85–400	133	[VO(acac)]
[VO(acac)(AcSHA)]	85°C	II	27.57	400–617	577	VO₂

DTA: differential thermal analysis.

Figure 6.

TGA and DTA curves of complex 1 [VO(AcSHA)₂].

Figure 7.

TGA and DTA curves of complex 2 [VO(acac)(ACSHA)].

Biological studies

Literature contains numerous reports on the transport of the pharmacologically active vanadium(IV) and (V) complexes as well as the role of the ligand in various redox and uptake processes, wherein the active species formed depend upon the thermodynamic stability of vanadium complexes and the potential of ligand to cross the cell membrane in bodily fluids at physiological pH.^57–69 The oxidovanadium(IV) hydroxamates^{43,45–47,70,71} have drawn considerable attention owing to their interesting biological activity over the last few years. In pursuit of new vanadium-based antimicrobials, the antimicrobial activity of the complexes 1 and 2 has been assayed.

Antibacterial activity

The uncoordinated ligand (AcSH₂A) showed inhibitory effects against the test bacteria at MIC 125 µg/mL. The complex 1 affected the growth inhibition of Bacillus cereus and Escherichia coli at MIC 31.25 µg/mL, while that of Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella typhi occurred at 15.63 µg/mL, which may be attributed to the difference in the diffusion of the complex through the cell wall of these bacteria. The complex 2 has been found to be quite effective against E. coli and S. typhi at MIC 31.25 µg/mL compared to B. cereus, P. aeruginosa, and S. aureus at 62.5 µg/mL. The reference drug tetracycline hydrochloride inhibited test bacteria at MIC 3.90–15.63 µg/mL (Table 9 and Figure 8).

Table 9.

Antibacterial activity data determined by the MIC method.

Ligand/complex	MIC ± SD (μg/mL)
	Gram-positive			Gram-negative
	B. cereus	S. aureus	E. coli	P. aeruginosa	S. typhi
(AcSH₂A)	125 ± 0.3	125 ± 0.5	125 ± 0.2	125 ± 0.04	125 ± 0.4
[VO(AcSHA)₂]	3.90 ± 0.2	15.63 ± 0.4	31.25 ± 0.05	15.63 ± 0.3	15.63 ± 0.05
[VO(acac)(AcSHA)]	62.5 ± 0.45	31.25 ± 0.02	62.5 ± 0.5	62.5 ± 0.2	62.5 ± 0.5
Tetracycline hydrochloride	7.84 ± 0.02	15.63 ± 0.01	15.63 ± 0.03	7.84 ± 0.4	15.63 ± 0.2

MIC: minimum inhibitory concentration; SD: standard deviation.

Figure 8.

Bar diagram showing the in vitro antibacterial activity studies of the free ligand and the complexes 1 and 2.

The complex 1 is quite effective against all the test bacteria and highly effective against B. cereus better than the standard drug. The growth inhibition by complex 1 is prominent against S. aureus (Gram-positive) and S. typhi (Gram-negative) relative to the free ligand and the complex 2. The enhanced inhibitory activity of the complexes can be ascribed to the reduction of the polarity in the metal ion and increased delocalization of π electrons over the whole chelate ring, thereby increasing the lipophilicity and allowing efficient diffusion of the complex into the bacterial cell wall.^72,73

Antifungal activity

The antifungal activity of the free ligand and the complexes 1 and 2 has been studied against Rhizoctonia solani and Fusarium sambucinum. The ligand (AcSH₂A) inhibited these fungi with MIC values of 62.5 and 15.62 μg/mL, respectively. The complexes 1 and 2 have shown enhanced inhibitory effects against the test fungi at MIC values of 7.81, 15.62, and 31.25 μg/mL, respectively (Table 10 and Figure 9). The standard drug fluconazole inhibited these fungi at MIC 3.90 μg/mL.

Table 10.

Antifungal activity determined by the MIC method.

Ligand/complex	MIC ± SD (μg/mL)
Ligand/complex	R. solani	F. sambucinum
(AcSH₂A)	62.5 ± 0.3	15.62 ± 0.04
[VO(AcSHA)₂]	7.81 ± 0.01	15.62 ± 0.2
[VO(acac)(AcSHA)]	31.25 ± 0.5	31.25 ± 0.3
Fluconazole	3.90 ± 0.2	3.90 ± 0.01

MIC: minimum inhibitory concentration; SD: standard deviation.

Figure 9.

Bar diagram showing the in vitro antifungal activity studies of the free ligand and the complexes 1 and 2.

Antioxidant activity

The antioxidant activity of the free ligand and the complexes 1 and 2 studied by the DPPH method showed a low scavenging potential (∼4.9%–26.3%) for the ligand, which was significantly enhanced (∼29.4%–45.3% and ∼10.4%–43.28%) in complexes through the oxidation of V(IV)O²⁺ to V(V)O⁺. The complex 1 exhibited high activity compared to the free ligand and the complex 2 (Table 11).

Table 11.

Antioxidant activity data of the free ligand and the complexes 1 and 2 using DPPH.

Ligand/complex	Scavenging potential (%)
(AcSH₂A)	~4.9–26.3
[VO(AcSHA)₂]	~29.4–45.3
[VO(acac)(AcSHA)]	~10.4–43.28

This is in agreement with the fact that large conjugated systems upon chelation with metal ions promote the trapping of free radicals, which results in discoloration from the purple DPPH radical solution to a yellow solution showing scavenging of DPPH radicals by hydrogen donation.

Conclusion

The oxidovanadium(IV) complexes of composition [VO(AcSHA)₂] and [VO(acac)(AcSHA)] derived from acetylsalicylhydroxamic acid [C₆H₄(OH)CONHOCOCH₃] have been synthesized and thoroughly characterized by various physicochemical and spectroscopic techniques (FTIR, UV-Vis, ESR) and mass spectrometry. IR data showed the disappearance of the ν(OH)_phenolic, lowering of the ν(C=O)_hydroxamic, and a shift of the ν(N–O) and ν(C–N) modes to a higher frequency, without any significant change in ν(C=O)_acetyl. This indicates that the acetylsalicylhydroxamic acid acts as a bidentate chelating ligand involving bonding through carbonyl and phenolic oxygen atoms (O,O). From spectral studies, a distorted square pyramidal geometry may tentatively be proposed around vanadium. The complexes are electrochemically active and exhibit single-step pseudo-reversible one-electron transfer processes. The thermal behavior of complexes 1 and 2 studied by the thermogravimetric technique and showed a two-step decomposition to yield VO₂ as the residue. The in vitro antibacterial assay of the free ligand and the complexes 1 and 2 has shown that the growth inhibition by complex 1 is prominent against S. aureus (Gram-positive) and S. typhi (Gram-negative) relative to the free ligand and the complex 2. The in vitro antifungal activity of the ligand and complexes evaluated against R. solani and F. sambucinum showed that the complex 1 exhibits good inhibitory effects against both the fungi in contrast to complex 2. The results reveal these new complexes as promising antimicrobial agents to be explored further for medicinal applications. It has been further elucidated that the complex 1 exhibited high antioxidant activity compared to the free ligand and the complex 2.

Experimental details

Materials and methods

All chemicals of reagent grade were used. Vanadyl sulfate (VOSO₄.5H₂O; Merck) was used as procured. [VO(acac)₂] and acetylsalicylhydroxamic acid [C₆H₄(OH)CONHOCOCH₃] were synthesized by reported methods,^74,75 and characterized by microanalyses and IR spectroscopy. The vanadium content in the complexes was determined as V₂O₅ by decomposing the complex with a mixture of concentrated H₂SO₄ and concentrated HNO₃. The carbon, hydrogen, and nitrogen analyses were obtained using the CHN elemental analyzer (FLASH 2000; Thermo Scientific). The molar conductance (10⁻³ M solution in methanol) of the complexes was obtained using a conductivity bridge (Type CM-82T; Elico) at 25°C ± 0.1°C. At the room temperature, magnetic susceptibility was measured by Gouy’s method,⁷⁶ using Hg[Co(NCS)₄] as a calibrant. The IR spectra were recorded as KBr pellets using a FTIR spectrophotometer (PerkinElmer). Electronic spectra were recorded using a 100 Bio UV-Vis spectrophotometer (Varian Cary) in absolute ethanol. ESI mass spectra were recorded using a Micromass Quattro-TOF (Waters), with a mass range of 4000 amu in quadrupole and 20,000 amu in Tandem time-of-flight (TOF). At the room temperature, powder X-band ESR spectra were recorded using an E112 ESR spectrometer (Varian). The cyclic voltammetric measurements were performed at 25°C on an Autolab Potentiostat 128N electrochemical analyzer using a single compartmental cell of volume 10–15 mL, comprising a three-electrode system of a Pt-disk as the working electrode, Pt-wire as an auxiliary electrode, and Ag/AgCl electrode as the reference electrode. Potassium chloride (KCl; 0.4 M) was used as the supporting electrolyte in the methanol/H₂O (5:95) electrolyte system. Thermograms (TGA and DTA curves) of the complexes were recorded using a Diamond TG/DTA thermogravimetric analyzer (PerkinElmer) at a heating rate of 10°C/min from ambient temperature to 800°C under an N₂ atmosphere.

Synthesis

Synthesis of (AcSH₂A)

To pyridine (15 mL) was added salicylhydroxamic acid (5 g, 32.7 mmol), and the reaction mixture was stirred at room temperature to form an orange-colored solution. After cooling to 0°C, acetyl chloride (2.4 mL, 34 mmol) was added. The mixture was stirred at 0°C for 30 min, followed by stirring at room temperature.^75,77 To this solution, ethyl acetate (30 mL), 5% HCl (90 mL), and a saturated solution of sodium chloride (120 mL) were added. The aqueous phase was separated from the organic phase through separating funnel and dried over magnesium sulfate. The solvent was removed using a rotary evaporator to afford a cream-colored solid that was recrystallized from ethyl acetate.

Yield = 2.2 g (32%). Decomposition Temp. = 135°C. The formation of acetylsalicylhydroxamic acid [N-(acetoxy)-2-hydroxybenzamide] was established by the elemental analysis, IR, and ¹H nuclear magnetic resonance (NMR) spectroscopy. Anal. calcd for [C₆H₄(OH)CONHOCOCH₃]: C, 55.20; H, 4.90; N, 7.28; found: C, 54.85; H, 4.65; N, 7.00%. IR (KBr disk): 1805, 1795, 1650, 1620, 1200 cm⁻¹. ¹H NMR (MHz CDCl₃): δ 2.45 (s, 3H, CH₃) and 7–8 (m, 4H, ArH).

Synthesis of [VO(AcSHA)₂] from VOSO₄.5H₂O

To a solution of VOSO₄.5H₂O (1 g, 3 mmol) in absolute ethanol (10 mL) was added 2 equiv. of AcSH₂A (1.5 g, 7 mmol) dissolved in absolute ethanol (10 mL). The reaction mixture was stirred for 1 h and then refluxed for 8–10 h. The excess solvent was removed by distillation. To the concentrate was added petroleum ether (20 mL), and the product was dried under vacuum. A dark blue solid was obtained.

Yield = 1.2 g (67%), Decomposition Temp. = 145°C, Mol. Mass [C₁₈H₁₆N₂O₉V] = 457, Anal. calcd for [C₁₈H₁₆N₂O₉V]: C, 50.05; H, 4.35; N, 3.29; V; 14.32; found: C, 49.56; H, 4.00; N, 3.00; V, 13.98%. Λ_M (MeOH): 3.92 Scm² mol⁻¹; µ_eff (293 K): 1.70 BM.

Synthesis of [VO(acac)(AcSHA)] from [VO(acac)₂]

To a solution of VO(acac)₂ (0.5 g, 1 mmol) in absolute ethanol (10 mL) was added 2 equiv. of AcSH₂A (1 g, 5 mmol) dissolved in absolute ethanol (10 mL). The reaction mixture was stirred for 1 h and then refluxed for 8–10 h. The excess solvent was removed by distillation. To the concentrate was added petroleum ether (20 mL), and the product was dried under vacuum whereupon a dark blue complex was formed.

Yield = 0.50 g (72%), Decomposition Temp. = 157°C, Mol. Mass [C₁₄H₁₅NO₇V] = 360, Anal. calcd for [C₁₄H₁₅NO₇V]: C, 50.40; H, 4.27; N, 3.25; V; 14.50; found: C, 50.06; H, 3.95; N, 3.05; V, 13.90%. Λ_M (MeOH): 3.90 Scm² mol⁻¹; µ_eff (293 K): 1.72 BM.

Biological assay

Antibacterial activity

The free ligand AcSH₂A and the oxidovanadium(IV) complexes [VO(AcSHA)₂] and [VO(acac)(AcSHA)] were screened in vitro for the antibacterial and antifungal activity against the selected Gram-positive bacteria (S. aureus and B. cereus), Gram-negative bacteria (E. coli, P. aeruginosa, and S. typhi), and fungi (R. solani and F. sambucinum) at different concentrations in dimethyl sulfoxide (DMSO) employing the MIC method.^78,79 The MIC is the lowest concentration of the antimicrobial agent that prevents the development of visible growth of microbes after overnight incubation. The samples were tested in triplicate.

Method

A sterile flat bottom 96-well microtiter plate was labeled as 1–7, in which wells 1–5 are allotted for the test sample, 6 for the positive control (broth + standard antibiotic (2 μL) + microorganism), and 7 for the negative control (broth + microorganism). Then, 30 µL of media was added to each well, including the positive and negative controls. A stock solution of the sodium salt of Resazurin dye as an indicator (0.02% (wt/mol); Sigma Aldrich)⁸⁰ was prepared in distilled water, filtered, sterilized, and stored at 4°C for 1 week. This dye (5 µL) was added to each well. The test samples were added in twofold dilution to wells (1–5). The plates were then wrapped with aluminum foil and incubated at 37°C for 24 h in the case of bacteria and at 28°C for 72 h for fungi. A change in color in the wells of the positive control indicated the proper growth of the isolate, and no change in color of wells of the negative control indicated the absence of contaminants. The results were compared with the standard antibacterial drug tetracycline hydrochloride and the antifungal drug fluconazole.

Statistical analysis

Statistical analysis was performed using the standard t-test, and p < 0.05 was considered significant. The data are represented as standard deviation.

Antioxidant activity

The free radical scavenging activity of the complexes is measured in terms of hydrogen donation or radical scavenging ability using the stable DPPH radical as described by Blois.⁸¹ DPPH is usually used as a substrate to evaluate antioxidant activity. DPPH (1,1-diphenyl-2-picrylhydrazyl) having an unpaired electron displays a strong absorption band at 517 nm, which decreases stoichiometrically upon pairing of the electron.^82,83 A change in the absorbance produced in the reaction is widely used to test the ability of molecules to act as free radical scavengers. The newly synthesized oxidovanadium complexes in +4 oxidation state can donate an electron to reduce the DPPH radical to DPPH (α,α-diphenyl-β-picrylhydrazine), and the VO(IV) ion is oxidized to VO(V). The stock solutions of the complexes (0.001 g/mL) were prepared in DMSO, from which different concentrations (20–100 µL) being prepared. A solution of DPPH (0.1 mmol) in methanol was prepared, and 1 mL of this solution was added to each of the test solutions. The mixture was shaken vigorously and incubated for 30 min. The absorbance was measured at 517 nm. All the tests were carried out in triplicate. Ascorbic acid was used as a standard or positive control. The ability to scavenge the DPPH radical was calculated using the following equation

I (%) = (\frac{A_{control} - A_{sample}}{A_{control}}) \times 100

where A_control is the absorbance of the control, and A_sample is the absorbance of the sample.

Supplemental Material

Supplementary_material_CH-19-0331 – Supplemental material for Synthesis, characterization, and biological properties of oxidovanadium(IV) complexes of acetylsalicylhydroxamic acid (N-acetyloxy-2-hydroxybenzamide) as potential antimicrobials

Supplemental material, Supplementary_material_CH-19-0331 for Synthesis, characterization, and biological properties of oxidovanadium(IV) complexes of acetylsalicylhydroxamic acid (N-acetyloxy-2-hydroxybenzamide) as potential antimicrobials by Bhanu Priya, Abhishek Kumar and Neeraj Sharma in Journal of Chemical Research

Footnotes

Acknowledgements

The authors thank the IIT Mandi for recording Fourier-transform infrared spectroscopy (FTIR), mass spectra, and elemental analyses. The authors gratefully acknowledge the support of the Department of Biotechnology, Himachal Pradesh University, Shimla, for providing laboratory facilities to carry out biological studies.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Neeraj Sharma

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

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Synthesis,characterization,and biological properties of oxidovanadium(IV) complexes of acetylsalicylhydroxamic acid ( N -acetyloxy-2-hydroxybenzamide) as potential antimicrobials

Abstract

Keywords

Introduction

Results and discussion

Infrared spectra

Electronic spectra

Mass spectra

ESR spectra

Cyclic voltammetry

Thermogravimetric studies

Biological studies

Antibacterial activity

Antifungal activity

Antioxidant activity

Conclusion

Experimental details

Materials and methods

Synthesis

Synthesis of (AcSH2A)

Synthesis of [VO(AcSHA)2] from VOSO4.5H2O

Synthesis of [VO(acac)(AcSHA)] from [VO(acac)2]

Biological assay

Antibacterial activity

Method

Statistical analysis

Antioxidant activity

Supplemental Material

Supplementary_material_CH-19-0331 – Supplemental material for Synthesis, characterization, and biological properties of oxidovanadium(IV) complexes of acetylsalicylhydroxamic acid (N-acetyloxy-2-hydroxybenzamide) as potential antimicrobials

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

ORCID iD

Supplemental material

References

Supplementary Material

Synthesis of (AcSH₂A)

Synthesis of [VO(AcSHA)₂] from VOSO₄.5H₂O

Synthesis of [VO(acac)(AcSHA)] from [VO(acac)₂]