**1. Introduction**

Many microorganisms use nitrogen from urease for growth (urea amidohydrolase, EC: 3.5.1.5). Urease is an enzyme that catalyzes the hydrolysis of urea into ammonia and carbon dioxide. There is a link between health complications and ammonia production by urease [1]. In animals and humans, low pH of the stomach allows microbial strains to survive, multiply, and grow, resulting in pyelonephritis, hepatic coma, gastric carcinoma, gastric lymphoma, kidney stones, and peptic ulcer complications [2,3]. The treatment of bacterial infection with therapeutics has often proven ineffective due to drug resistance. Thus, there is a clear need for alternatives or novel treatments. Given the

involvement of ureases in various diseases, pharmaceutical research has channeled considerable efforts into discovering potent and safe urease inhibitors [4–7].

Molecules with a binding site that can chelate metals are an interesting challenge and could be a promising line of action to prevent the adverse effects of ureolytic bacterial infections in humans [8]. In this regard, many types of potent urease inhibitors have been designed [9], such as dihydropyrimidines [10], urea derivatives [11], semicarbazones [12], Schiff bases [13], hydroxamic acid derivatives [14], piperazines [15], biscoumarines [16], benzimidazoles [17], and sulfonamides [18].

Enaminone molecular scaffolds have been found in many synthetic drugs and natural products [19,20]. Specially, pyrimidine-based enamines have demonstrated an array of biological activities owing to the presence of the alkenylamine moiety (R2NCH = CH−) in their structures, and are capable of strong bonding with metal ion chelates in biological systems [21,22].

The barbituric and thiobarbituric acids [(thio)pyrimdine trione analog derivatives] have been reported urease inhibitors [12]. These can be considered privileged structures because they have antifungal [23], antimicrobial [24,25], anti-adiponectin [26], anti-sclerosis [27], anti-convulsing [28,29], antiglycation [30], and α-glucosidase inhibitory [30] properties. Several compounds **A, B,** and **C** have been reported, and showed anti-urease activity comparable to acetohydroxamic acid (Figure 1) [31,32].

**Figure 1.** Some examples of bacterial urease inhibitors.

In this context, and as a continuation of our extended medicinal chemistry program based on the barbituric and thiobarbituric acid moieties [30,33–44], here we examined the in vitro anti-urease activity of a collection of 1,3-dimethylbarbiturate-enamine compounds and their analogs, thiobarbiturate derivatives. In addition, molecular docking studies were performed to evaluate the molecular interactions of the newly synthesized compounds with selected drug targets (PDB ID 4GY7).

### **2. Materials and Methods**

The synthesis and the full characterization of compounds **3a**, **3d**, **3h**, **3j–p**, **4,** and **5** (Table 1) have been previously described by our group [30,33]. The rest of the compounds studied were prepared following the previously described method. The yields and full characterization are provided in the Supplementary materials.

### *2.1. In Vitro Urease Inhibition Assay Protocol*

The urease inhibition assay was performed spectrophotometrically following the manufacturer's instructions [45–47]. The source of urease is Jack bean Urease and the full protocol provided in the supplementary materials. In the present study, urease was preincubated with the inhibitors for a period of 15 min, which proved to be sufficient in our studies.


**Table 1.** Urease inhibition capacity of compounds **3a–p**, **4**, and **5**.


**Table 1.** *Cont*.
