1Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia

1Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia

1Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia

1Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia

1Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia

2Chemistry Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia

1Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia

A computer-aided predictions of antioxidant activities were performed with the Prediction Activity Spectra of Substances (PASS) program. Antioxidant activity of compounds 1, 3, 4 and 5 were studied using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and lipid peroxidation assays to verify the predictions obtained by the PASS program. Compounds 3 and 5 showed more inhibition of DPPH stable free radical at 10−4 M than the well-known standard antioxidant, butylated hydroxytoluene (BHT). Compound 5 exhibited promising in vitro inhibition of Fe2+-induced lipid peroxidation of the essential egg yolk as a lipid-rich medium (83.99%, IC50 16.07 ± 3.51 µM/mL) compared to α-tocopherol (α-TOH, 84.6%, IC50 5.6 ± 1.09 µM/mL). The parameters for drug-likeness of these BHT analogues were also evaluated according to the Lipinski’s “rule-of-five” (RO5). All the BHT analogues were found to violate one of the Lipinski’s parameters (LogP > 5), even though they have been found to be soluble in protic solvents. The predictive polar surface area (PSA) and absorption percent (% ABS) data allow us to conclude that they could have a good capacity for penetrating cell membranes. Therefore, one can propose these new multipotent antioxidants (MPAOs) as potential antioxidants for tackling oxidative stress and lipid peroxidation processes.

Reactive oxygen species (ROS) are generally considered responsible for many cell disorders and the development of many undesired processes, including aging [1], inflammatory [2] and many others [3,4,5,6,7]. Phenolic primary antioxidants are the most active dietary antioxidants [8]. The commonly used synthetic antioxidants in foods are butylated hydroxyanisole (BHA) [9], butylated hydroxytoluene (BHT) [10] (Figure 1).

Chemical structures of compounds BHA, BHT, 1 and 2.

BHT (CAS 128-37-0; NCI {"type":"entrez-nucleotide","attrs":{"text":"C03598","term_id":"1466849","term_text":"C03598"}}C03598) was patented in 1947 [11]. It was found that two tert-butyl groups flanking the OH group are required to retain in vivo anti-inflammatory potency [12]. Following the same route, researchers of Parke-Davis have disclosed a new class of potent, selective and orally active COX-2 inhibitors incorporating the 2,6-di-tert-butyl phenol moiety, such as PD 164387 and PD 138387 (Figure 2) [13,14].

Parke-Davis COX-2 inhibitors.

Based on the Parke-Davis COX-2 inhibitors, researchers have investigated their SAR to improve COX-2 selective inhibitors containing a 2,6-di-tert-butylphenol substituent as an antioxidant moiety [15]. Their selectivity might be related to their free-radical scavenging potency [15].

On the other hand, sterically hindered phenols linked with heterocyclic rings have also been extensively studied as dual COX/5-LOX inhibitors [16,17,18]. A combination of radical scavenging properties and anti-inflammatory activity has already been proven for a number of non-steroidal anti-inflammatory drugs (NSAIDs) [19,20]. However, BHT derivatives have become attractive antioxidant or co-antioxidant groups [21]. Therefore, it is no surprise that BHT has been modified to prepare a series of new antioxidants having new medicinal properties in the pharmaceutical industry [12,22,23]. Recently, qualitative SARS and rational-design strategies for antioxidants have been used to combine multiple functions; including various multiple antioxidant properties such as radical-scavenging ability and diversified pharmacological activities to offer hybrid compounds, which can exert multiple pharmacological functions through one molecular framework [24,25,26].

Thiosemicarbazides have been reported to show antibacterial [27], antimicrobial [28,29], anti-toxoplasmagondii [29] and antioxidant [30] activities. To date acyl derivatives of thiosemicarbazide bearing BHT moiety have been rarely synthesized.

Compounds containing the 1,3,4-thiadiazole nucleus have a wide range of pharmacological activities that include antimicrobial [31], antitubercular [32], anticancer [33,34] and antioxidant [35] properties.

1,2,4-Triazoles are an important class of five membered heterocyclic compounds. 1,2,4-Triazole-5-thiones are known for their anti-inflammatory [36], selective COX-2 inhibitor [37] and antimycotic [38] and antioxidant [35] activity.

Regarding the effect of m-substituents, Tetsuto et al. [39] have evaluated the antioxidant activity of different donating substituents on a m-substituted phenol, and found that the m-substituent does not influence the antioxidant activity of a phenol at all. This is probably because a m-substituent shows only a small resonance effect. In contrast to this fact, previous reports found that heterocyclic systems with a halogen substituted at the m-position show greater antioxidant properties than those with other substituents [40,41].

Since we could not find any reports on the use of electron withdrawing groups to enhance the antioxidant activity of BHT, therefore, we used MPAO as an effective strategy to enhance the antioxidant activity of BHT even if a strong electron withdrawing group (m-fluoro substituent) is a basic part of the hybrid molecule.

In the present study four BHT derivatives have been synthesized. The acid-(base-)catalyzed intramolecular dehydrative cyclization reaction of acylthiosemicarbazide 3 to the corresponding 1,3,4-thiadiazole 4 and 1,2,4-triazole 5 are described. Synthesized compounds have been characterized by IR, NMR and mass spectral analysis. X-ray structures of 3 and 4 will be further discussed in this paper. Potential biological effects of new compounds were predicted based on structure-activity relationships with the PASS software. Antioxidant activities predicted by the PASS program were experimentally verified by DPPH and TBARS (thiobarbituric acid reactive substance) assays. Furthermore, a computational study for prediction of absorption, distribution (ADMET) [42] properties of the synthesized compounds was performed by determination of polar surface area (PSA), absorption (ABS) and Lipinski parameters.

Carboxylic acid 1 (Figure 1) was synthesized according to an established solvent-free procedure [43] through the reaction between 2,6-di-tert-butyl-phenol with formaldehyde and thioglycolic acid in the presence of di-n-butylamine. Hydrazide 2 (Figure 1) was synthesized using the indirect hydazination method through the reaction of 3,5-di-t-butyl-4-hydroxybenzyl chloride with the sodium salt of thioglycolic acid hydrazide [44].

The synthesis of thiosemicarbazide 3, 1,3,4-thiadiazole 4 and 1,2,4-triazole 5, described in this study are outlined in Scheme 1. Acid hydrazide 2 was treated with 3-fluorophenylisothiocyanate to give the corresponding acylthiosemicarbazide 3 in good yield. Compound 3 under acidic and basic conditions gave thiadiazole 4 and triazole 5, respectively. The structure of the synthesized compounds was confirmed based on their physical and spectral data. The structures of compounds 3 and 4 were further confirmed by X-ray crystallography.

Synthesis of compounds 3–5.

The IR spectra of all synthesized compounds showed strong absorptions at 3,623–3,653 cm−1, attributed to free ν(Ar-O-H). Acylthiosemicarbazide 3 showed NH stretching bands between 3,285–3,340 cm−1, a C=O stretching band at 1,713 cm−1, and did not show any ν(S-H) band at 2,570 cm−1, while the presence of a C=S stretching band at 1,248 cm−1 indicated that 3 exists in the thione form in the solid-state [45,46]. Compound 4 showed two C=N stretching bands at 1,616 and 1,610 cm−1 attributed to the presence of the thiadiazole ring C=N. Compound 5 did not show any ν(S-H) band at 2,570 cm−1, while the presence of C=S stretching band at 1,252 cm−1 indicated that 5 exists in the thione form in the solid state [42,43].

The 1H-NMR spectra of compound 3, recorded in CDCl3, showed a singlet peak at 8.32 ppm due to NH attached to a phenyl group while the other two singlets at 9.26 ppm and 9.84 were attributed to the hydrazido group NHs. Both appear as a broad band which supports the formation of intramolecular hydrogen bonding [45,47] (Figure 3). The disappearance of the NH-1 and NH-2 groups of 3, combined with a singlet at 11.75 ppm due to the presence of the NH-triazole of 5 suggest that a 1,2,4-triazole-5-thione, and not a 1,2,4-triazole-5-thiol was formed.

Intramolecular hydrogen bonding of thioxo form of 1-acylthiosemicarbazide 3.

In the 13C-NMR spectra, the greatest differences between the acylthiosemicarbazide 3 and its cyclic derivatives 4 and 5 were found at the carbons designated C-8, C-9 and C-10. For C-8 in 3, this carbon was bonded to C-9 (C=O). The electron-withdrawing effects of the oxygen of the C=O greatly deshielded the C-8 and caused it to appear at a high chemical shift of 33.76 ppm. After acid (base)-catalysed intramolecular dehydrative cyclizations of 3, the carbon at the same position (C-8) in 4 and 5 were bonded to the new C-9 due to the formation of thiadiazole and thiadiazole rings, respectively, so it had a new electronic environment. This greatly lowered the chemical shift of the methylene (C-8) to values of 29.96 and 24.68 ppm in 4 and 5, respectively. Interestingly, the heteronuclear 13C-19F coupling constants of compounds 3, 4 and 5 were 972, 984 and 1,004 Hz, respectively.

The crystal structure of molecule 3 is depicted in Figure 4 and the selected bond lengths and angles are given in Table 1. In the crystal the molecule exists in its thione form. The two methylene carbon atoms, C15 and C16, subtend an angle of 100.77 (7) at S1 atom. Several atom pairs of the molecule are connected via N-H…O hydrogen bonding (Table 2) in a bifurcated system to form centro-symmetric dimers. The hydroxyl group is shielded by the two di-tert-butyl residues and therefore is not involved in any hydrogen bonding.

The molecular structures and labeling schemes of 3 (50% probability ellipsoids).

Selected bond lengths [Å] and bond angles [°] for 3 and 4.

Hydrogen-bond geometry for 3 and 4.

Symmetry transformations used to generate equivalent atoms: #1 -x+1,-y+1,-z+1; #2 x+1/2,-y+1/2,-z+1.

Figure 5 presents the molecular structure of compound 4 and Table 1 compiles the selected bond lengths and angles. The thiadiazole ring makes dihedral angles of 14.6 (3)° and 29.6(2)° with the C10-C24 and C1-C6 aromatic rings, respectively. Similar to what was observed in the structure of the semicarbazide 3, the hydroxyl group is not involved in any hydrogen bonding as it is shielded by the two sterically hindered tert-butyl groups. In the crystal, pairs of N3-H…N2 hydrogen bonds (Table 2) link the molecules into centro-symmetric dimers.

The molecular structure and labeling scheme of 4 (50% probability ellipsoids).

The crystal data and structure refinement for compounds 3 and 4 is summarized in Table 3.

Crystal data and refinement parameters for 3 and 4.

The biological activity spectra of the target compounds were obtained using the Prediction of Activity Spectra for Substances (PASS) software [48]. PASS prediction tools are constructed using 20,000 principal compounds from the MDDR database (produced by Accelrys and Prous Science) [49]. The database contains over 180,000 biologically relevant compounds and is constantly updated [49]. PASS web tool has the ability to predict about 4,000 kinds of biological activity on the basis of structural formula with mean accuracy of about 90% [50,51], therefore, it is reasonable to use PASS for finding and optimizing new lead compounds. The PASS training set which has been compiled from various sources including publications, patents, chemical databases, and “gray” literatures consists of over 26,000 biological compounds and includes drugs, lead compounds, drug-candidates, and toxic substances. Algorithm of activity spectrum estimation is based on Bayesian approach. The result of prediction is presented as the list of activities with appropriate Pa and Pi ratio. Pa and Pi are the estimates of probability for the compound to be active and inactive respectively for each type of activity from the biological. It is reasonable that only those types of activities may be revealed by the compound, which Pa > Pi. If Pa > 0.3 the compound is likely to reveal this activity in experiments, but in this case the chance of being the analogue of the known pharmaceutical agents for this compound is also high. A portion of the predicted biological activity spectra (lipid peroxidase inhibitor, antioxidant, free radical scavenger and antiinflammatory) for the synthesized compounds and BHT are given in Table 4.

Part of the predicted biological activity spectra for the compounds 1, 3–5 and BHT.

Pa—probability “to be active”; Pi—probability “to be inactive”.

Drug-likeness can be defined as a delicate balance between various structural features, which determine whether a particular molecule is similar to recognized drugs. It generally means molecules, which contain functional groups and/or have physical properties consistent with most of the known drugs. These properties are; absorption, distribution, metabolism, and excretion in the human body like a drug. Lipinski [52] used these molecular properties in formulating his Rule of Five. The rule states that the most molecules with good membrane permeability have logP < 5, molecular weight