*3.1. Synthesis*

Derivatives **3a–p**, **4**, and **5** (Table 1) were straightforwardly synthesized by nucleophilic substitution of the corresponding amines with enaminone sca ffolds **1** [49] and **2** [50] in methanolic solution, following the reported method [30,33] (Scheme 1). Due to the potential tautomerism of these families of compounds, X-ray crystallography was carried out on two compounds (**3b** and **3k**) to confirm the structures. (1H-NMR and 13C-NMR spectra, Figures S1–S21, and X-ray data of compounds **3b** and **3k** are provided in the Supplementary materials).

**Scheme 1.** Synthesis of **3a–p**, **4**, and **5.**

### *3.2. In Vitro Evaluation of Urease Inhibition*

The capacity of synthesized derivatives of *<sup>N</sup>*,*<sup>N</sup>*-dimethylpyrimidine trione (**3a**–**i** and **4**) and *<sup>N</sup>*,*<sup>N</sup>*-diethyl-thio pyrimidine dione (**3j**–**p** and **5**) to inhibit urease in vitro was examined and compared with that of acetohydroxamic acid **(AHA)** (IC50= 20± 0.4 μM), as a standard compound (Table 1).

Of note, the tested compounds showed potent urease inhibition activity, twelve of them (**3a**, **3d**, **3e**, **3f**, **3h**, **3j**, **3k**, **3l**, **3m, 3n**, **3o,** and **4**) exerting greater activity than the reference acetohydroxamic acid. The most active member between this series is compound **3h** which the structure attributed the pyridine moiety. Separation the pyridine ring from the enaminone functionality in compound **3j** with CH2 group, the activity has been decreased compared to the most active member **3h**. On the other hands, compounds **3k**, and **3o** showed superior activity to their counterparts **3b** and **3g**. Indeed, **3l** and **3o** showed activity similar to that of **3d**, and **3f**. The chloro atom in *para*-position in compound **3m**, the activity has been decreased compared to the *ortho* position of compounds **3f**, and **3n**. Compounds **3c**, **3i** and **3p** with sulfonic, cyclic amide or sulfonamide functionalities shown less activity compared to standard drug. The cyclohexyl group in compound **3e** increases the activity with 1.8 folds than the control. it was observed that in case of compounds **4** and **5** the presence of the *<sup>N</sup>*,*<sup>N</sup>*'-diethyl group and the S atom in the thiobarbituric derivative **5** make them more voluminous compared to its analogs *N,N'-*dimethyl and the "O" atom in the barbituric derivatives **4**. Finally, compounds **3b**, and **3g** were not active.

However, there are some discrepancies regarding whether AHA is or is not a competitive inhibitor to the active site of the urease enzyme [51,52]. Therefore, some limitations could be applied, and we think that comparisons are important.
