Design of acetaminophen regioisomers
Our design and development strategy adopted by us in this study is the regioisomerism, changed the phenol moiety of para- for ortho-position. In Fig. 3, the differences on BDEOH and BDENH between acetaminophen and chloramidaphen are clearly showed. Both compounds have these electronics properties completely different between para (NAPSQI and NAPQI) and ortho of N-acetyl-o-benzosemiquinone imine and N-acetyl-o-benzoquinone imine (NAOSQI and NAOQI).
The BDEOH values for these compounds were 85.64 and 81.66 kcal mol-1 and a difference of 3.97 kcal mol-1 was observed between them [18]. Therefore, the regioisomer derivative has a lowest quenching energy than on a first hydrogen transfer. These results indicate that the regioisomerism increased the quenching capacity, which could be related to better biological activity [20]. In addition, the BDENH values for these two molecules were 77.30 and 90.72 kcal mol-1. This second hydrogen abstraction shows a higher difference of 13.41 kcal mol-1 for ortho-regioisomer.
From Table 1, electron-donating groups (EDGs) at the 5-position (–CH3, –N(CH3)2, –OCH3, and –SCH3) show the lowest BDEOH values of 80.01 (3), 71.80 (4), 76.91 (5), and 76.89 kcal/mol (6), while electron-withdrawing groups (EWGs) at the same position (–F, –Cl, and –NO2) decreased the BDEOH values for 80.13 (7), 80.64 (8), and 84.58 kcal/mol (9). These results show that all regioisomers would be more reactive than acetaminophen, however, –N(CH3)2, –OCH3, and –SCH3 would be more effective due to high difference when compared to acetaminophen of 13.83, 8.72, and 8.74 kcal/mol, respectively. In addition, EDGs at the 5-position (–CH3, –N(CH3)2, –OCH3, and –SCH3) show higher BDENH values of 89.93 (3), 92.67 (4), 93.48 (5), and 93.29 kcal/mol (6). In the same way, EWGs (–F, –Cl, and –NO2) increased BDENH values for 91.52 (7), 91.69 (8), and 92.81 kcal/mol (9). These results show that all regioisomers would be less toxic than acetaminophen [21].
These results are in agreement to nucleophilicity (HOMO), electrophilicity (LUMO), and chemical stability (GAP) [22]. In addition, due to the high chemical stability of halogen on drug metabolism process [23], we choice the chlorinated derivative for synthesis and toxic evaluation. This compound shows a difference of -5.00 and -14.38 kcal/mol for BDEOH and BDENH values, respectively. Therefore, our methodology shows that new acetaminophen derivative can be a good strategy in drug design for the safer compounds because of the highest values for quinone production as compared to acetaminophen [18]. In fact, ACP toxicity is related to the level of toxic intermediate metabolites such as N-acetyl-p-benzoquinone-imine (NAPQI) as main chemical mechanism [9,14,16]. So, the ability to predict the toxicity profile of CAP is critical to rational pharmaceutical drug design and development. In addition, a similar electronic mechanism is involved for other phenol derivatives [24].
Synthesis of chloramidaphen
After that, CAP was synthesized from 5-chloro-2-aminophenol using acetic acid, sodium carbonate, and water, as acylating group, alkaline catalytic, and solvent medium, respectively. After recrystallization on methanol, a mp 182.6-184oC [183-184oC] [25] was observed. It is soluble in methanol, ethanol, dimethylformamide, acetone, and ethyl acetate and practically insoluble in water, petroleum ether, and pentane. 1H-NMR (300 MHz; CDCl3) d (ppm) 8.43 (1H, s, H-O), 7.43 (1H, s, H-N), 7.09 (2H, dd, 6.0 and 3.0 Hz, H-4 and H-6), 6.95 (1H, d, 6.0 Hz, H-3), and 2.29 (3H, s, CH3). 13C NMR (75 MHz; CDCl3) d (ppm) 170.52 (C=O), 147.29 (C-2), 126.91 (C-6), 126.46 (C-1), 125.03 (C-5), 121.74 (C-4), 120.82 (C-3), and 23.81 (CH3). This compound was used on toxicological evaluations.
Biochemical changes upon chloramidaphen exposure
Analyzing the results of AST, ALT, creatinine and urea levels of rats treated with 300 mg/kg of CAP (Table 2), our findings shows that all AST levels measure were increased on days 8 and 15 both treated and control groups and were significant different of day 0 measure (p < 0.05) and samples of treated group were different of control group on day 15 (p < 0.05). Besides that, there was no significant difference in the ALT, urea and creatinine levels of both treated and control rats throughout the experiment.
The biochemistry parameters for 2000 mg/kg dose were showed on Table 3. The results show that AST levels measure were increased on days 8 and 15 both treated and control groups and were significant different of day 0 measure (p < 0.05). On the other hand, ALT level was decreased to control group on day 15 (p < 0.05 vs day 0). In addition, no more significant difference in the ALT, urea and creatinine levels were found in both treated and control groups.
The knowledge of normal range or expected values for hematologic and serum chemistry can be a useful tool for interpreting toxicology and safety studies [26]. It is important to notice that several factors can modify these values, like age, diet, handling and environment [27].
Analysis for biochemistry parameters, the toxicological effects were evident to aspartate transaminase (AST, formerly referred to as serum glutamic-oxaloacetic transaminase, SGOT). Here, we showed that high AST levels on both treated groups (300 mg/kg and 2000 mg/kg) and controls groups. These results were different of the values already found in others studies [26-28]. We hypothesized that the corn oil and other vegetable oil may be altering liver physiology by alteration of triglycerides synthesis and hepatic lipid peroxidation in some conditions [29-33]. Thus, these findings showed that the CAP presented neither hepatotoxic effect nor renal toxicity, since that there is no significant change in urea and creatinine.
To be one more way to study the hepatotoxic effect of the drug, we also calculated the alterations in the relation AST/ALT levels to additionally suggest liver toxicity, but these levels are not significant. Our results for 300 and 2000 mg/kg show low levels of AST and ALT. In both doses, there were few differences among control and treated with chloramidaphen or 300 and 2000 mg/kg. None animal died for 15 days. However, an increase of ALT levels on Swiss-Webster mice treated with 400 and 750 mg/kg acetaminophen was showed of 1552 ± 501 to 4030 ± 829 mU/L. The survivors at 24 hours after acetaminophen treatment was 8 (n = 10) for 400 mg/kg and 4 (n = 10) for 750 mg/kg [34]. In fact, chloramidaphen seems to have a much safer therapeutic index when compared to its relative drug acetaminophen, which have oral LD50 of 338 mg/kg (oral, mouse) [35].
In addition, both acetaminophen regioisomers, i.e. 2-hydroxyacetanilide (2) and 3-hydroxyacetanilide (10) were appointed as nonhepatotoxic, however reactive metabolites as arylate hepatic proteins for hydroxilated derivatives of 3-hydroxy-acetanilide (11) was detected (36-41). Their oxidation by rat liver P450 leads to the formations of two main products of para-aromatic hydroxylation that subsequently can be further oxidized to their respective ortho (12) and para-benzoquinone (13) derivatives and form glutathione adduct (42-44), as showed in Fig. 4. Likewise, 2-hydroxyacetanilide (2) that is metabolized to 2',5'-dihydroxyacetanilide (11) was low hepatotoxic in mice (37,38,45), maybe due to the changes on chemical reactivity of phenol and acetamide moieties. Thus, the chlorinated derivative (8) in position 5 can reduce the formation of hydroxylated derivatives and the formation of quinones, reducing toxicity.
In accordance to our biological study, some evidence was observed that ortho-regioisomers may be an alternative approach for design and development of low toxic derivative of acetaminophen. The substantial protection afforded by chloramidaphen can be related to decrease of electrophilic mechanism involved for metabolism and toxicity of quinone-imine species due to its high BDENH values. These mechanisms need to be further investigated.