Phenoxyacetohydrazide Schiff Bases: β-Glucuronidase Inhibitors

Phenoxyacetohydrazide Schiff base analogs 1–28 have been synthesized and their in vitro β-glucouoronidase inhibition potential studied. Compounds 1 (IC50 = 9.20 ± 0.32 µM), 5 (IC50 = 9.47 ± 0.16 µM), 7 (IC50 = 14.7 ± 0.19 µM), 8 (IC50 = 15.4 ± 1.56 µM), 11 (IC50 = 19.6 ± 0.62 µM), 12 (IC50 = 30.7 ± 1.49 µM), 15 (IC50 = 12.0 ± 0.16 µM), 21 (IC50 = 13.7 ± 0.40 µM) and 22 (IC50 = 22.0 ± 0.14 µM) showed promising β-glucuronidase inhibition activity, better than the standard (D-saccharic acid-1,4-lactone, IC50 = 48.4 ± 1.25 µM).

The present work aimed to investigate the potential activity of a series of aryl hydrazide-hydrazones as in vitro β-glucouoronidase inhibitors. In our designed analogues substituted phenoxy-acetohydrazides were treated with different aromatic aldehydes to scrutinize their potential activity. The earlier reported literature [25] showed that β-glucouoronidase is a lysosomal enzyme, present in many organs like the spleen, kidney, lung, bile, serum and urine, etc., where its specific task is to catalyze the cleavage of glucuronosyl-O-bonds [26][27][28]. It degrades glucuronic acid-containing glycosaminoglycans, like heparan sulfate, chondroitin sulfate and dermatan sulfate [29]. An elevated level of β-glucouoronidase was observed in various types of malignancies, such as breast, lung and gastrointestinal tract carcinomas, and melanomas. Its high expression also observed in bronchial tumors [30]. On the other hand, mucopolysaccharidosis type VII (MPS VII; Sly Syndrome) is caused by the deficiency of human β-glucuronidase [31]. The circulating level of β-glucuronidase is also useful as a lysosomal enzyme in children affected by leprosy. In borderline tuberculoid patients and lepromatous patients higher activity of this enzyme was also observed.

Chemistry
Lead identification is a well defined tool in drug design and discovery. Our research group has been involved for a decade in lead discovery programs in search of novel therapeutic agents. We have earlier reported Schiff bases of different classes of organic compounds in the search for lead molecules with different biological activities [32][33][34]. Earlier, our group reported the leishmanicidal and β-glucurinodase inhibition potential of hydrazides derived from the corresponding esters [35][36][37][38]. In view of the formerly reported work we synthesized hydrazide Schiff bases and screened their potential biological activities [39][40][41][42]. Acylhydrazide Schiff base derivatives 1-28 were synthesized from an acylhyrazide by condensing it with different aromatic aldehydes and acetophenones under reflux conditions in ethanol for 2-3 h (Scheme 1). The crude products (Table 1) were further recrystallized from methanol and needle-like crystals were obtained in most of the cases. The starting acylhydrazide was synthesized from ester of ethyl 2-(4-chloro-2-methylphenoxy) acetate by refluxing with hydrazine hydrate.

β-Glucuronidase
Synthetic acyl hydrazides Schiff bases 1-28 were screened for their in vitro potential as β-glucoronidase inhibitors. The in vitro β-glucornidase inhibitory potential was evaluated by using the literature protocol [43]. Compounds 1-28 showed diversified β-glucoronidase inhibitory activities, with IC 50 values ranging between 9.20-30.7 µM. Compounds 1, 5, 7, 8, 11, 12, 15, 21, and 22 showed excellent β-glucoronidase inhibitory activities, with IC 50 values of 9.20 ± 0.32, 9.47 ± 0.16, 14.7 ± 0.19, 15.4 ± 1.56, 19. ± 0.62, 30.7 ± 1.49, 12.0 ± 0.16, 13.7 ± 0.40, and 22.0 ± 0.14 µM, respectively, and the remaining compounds exhibited no activity ( Table 2). It was observed that both the substituents' nature and their position at the benzilidine part have great importance in the β-glucoronidase inhibition activity of a compound, and apparently the acylium part does not take part in the activity (Figure 1). The best activity was shown by compound 1 (IC 50 = 9.20 ± 0.32 µM, fivefold better than the standard D-saccharic acid-1,4-lactone, IC 50 value 48.4 ± 1.25 µM) which has a methoxy group at the ortho position. Surprisingly, a marked decline in activity (to the point of being inactive) was observed in analog 2 which has a methoxy residue at the para position instead of the ortho position as in compound 1. This huge difference in the activities of compounds 1 and 2 clearly indicates that a specific group at a specific position of the benzylidine phenyl ring part plays a vital role in making a potent β-glucornidase inhibitor in this type of compounds. The excellent activity of compound 5 (IC 50 = 9.47 ± 0.16 µM) having an ortho nitro group on the phenyl ring as compared to the inactivity of its closely related derivative 6 having a meta nitro group on phenyl ring proves our hypothesis that a suitable group at a suitable position of the phenyl ring of benzilidine part of molecules is a prerequisite for β-glucornidase inhibitory potential in these N-acylhydrazone Schiff bases. Comparison of activity of chloro-containing compounds 7 (IC 50 = 14.7 ± 0.19 µM), 8 (IC 50 = 15.4 ± 1.56 µM), and 9 (inactive) demonstrated that the nature and location of a substitution is important for β-glucornidase inhibitory potential. Dichloro-substituted compounds 9 and 10 were found to be completely inactive which further proves our hypothesis. Compound 11 (IC 50 = 19.6 ± 0.62 µM) having an ortho fluoro group showed excellent activity, but a little less than analogous chloro compounds 7 and 8. We also evaluated the effect of heterocyclic ring-containing derivatives, and it was observed that the five membered heterocyclic thiophene ring-containing derivative 12 (IC 50 30.7 ± 1.49 µM) produced remarkable activity, while on the other hand five membered heterocyclic rings like furan and its methyl derivatives 13 and 14 were found to be completely inactive. Almost all mono-, di-and trihydroxy substituted compounds 16, 17, 18, 19, and 20 found to be completely inactive, but unexpectedly compound 15 (IC 50 = 12.0 ± 0.16 µM) which bears 2,3-dihydroxy substitution, was found to be very efficient and displayed remarkable activity, better than the standard, but, compound 16, also a 3,4-dihydroxy derivative did not show any activity. N-acylhydrazones Schiff base 21 (IC 50 = 13.7 ± 0.40 µM) synthesized from 1-napthaldehyde was found to be more active than 22 (IC 50 = 22.0 ± 0.14 µM) which was synthesized from 2-napthaldehyde, both without any substitution. Remaining compounds 23-28 were found to be completely inactive. This pattern of activity reveals that the substituent and its position on the phenyl ring of benzylidine part is a driving force for β-glucornidase inhibition activity.
In conclusion, a number of potential lead molecules has been identified as β-glucuronidase inhibitors. Compounds 1, 5, 7, 8, 11, 12, 15, 21, and 22 demonstrated excellent activity and it is anticipated that by slight synthetic modification in these molecules, some new most active β-glucuronidase inhibitors can be developed.

Biological Assays
β-Glucuronidase (E.C. 3.2.1.31 from bovine liver, G-0251) and p-nitrophenyl-β-D-glucuronide (N-1627) were purchased From Sigma Chemical Co. (St. Louis, MO, USA). Anhydrous Na 2 CO 3 and all other reagents of standard grade were obtained from E. Merck. The anhydrous EtOH and CHCl 3 used in the experiments were dried employing the standard methods. All other solvents and reagents like the benzoyl chloride were of standard grade.

Assay for β-D-Glucuronidase
β-D-Glucuronidase inhibition was determined by measuring the absorbance of the p-nitrophenol which is produce from the substrate at 405 nm. The total reaction volume was 250 µL. The reaction mixture contains 5 µL of test compound solution, 185 µL of 0.1 M acetate buffer, and 10 µL of enzyme, and it was incubated at 37 °C for 30 min. The plates were read on a multiplate reader at 405 nm after the addition of 50 µL of 0.4 mM p-nitrophenyl-β-D-glucuronide. All assays were performed in triplicate.

Conclusions
A number of potential lead molecules 1, 5, 7, 8, 11, 12, 15, 21, and 22 have been identified as β-glucuronidase inhibitors and it is anticipated that by slight synthetic modification in these molecules, some new most active β-glucuronidase inhibitors can be developed.