Bacterial resistance to available antibiotics has resulted in limited options for treatment of patients suffering from life-threatening infectious diseases, causing significant burden on healthcare systems and global economic costs [1]. It has been reported that the formation of biofilm is an important reason for the loss of drug sensitivity which resulted in the recurrent infection of living organisms [2]. Biofilm is composed of bacteria enclosed within a matrix of extracellular polymeric substances, which include exopolysaccharides (EPSs), extracellular proteins (EPs) and extracellular DNA (eDNA), they play positive roles in promoting biofilm formation and inducing drug resistance. EPSs suppress host immune responses and confer antibiotic resistance to pathogens; EPs enhance biofilm formation and stability; eDNA coordinates bacterial twitching motility, bacterial aggregation and acquisition of antibiotic resistance genes, inducing the antibiotic resistance [3]. The biofilm network as a protective barrier can protect bacteria from antibiotics and other antimicrobial effect, and then the microbe could survive in a variety of environmental stresses. Studies revealed that the dose of drugs needed to kill bacterial cells encasing biofilms is about 10 to 1000 times that required to remove plankton-laden bacteria [4]. Therefore, the development of novel molecules with biofilm inhibition and excellent antibacterial activity may be an effective strategy to conquer the global drug resistance.

Aminothiazoximone moiety contains electron-rich aminothiazolyl, polar oxime and carbonyl groups, and is an important functional fragment of the third and fourth generation cephalosporins such as cefodizime, cefotaxime, and cefepime [5]. Polar amino group, oxime segment and carbonyl group can exert hydrogen bonds with the residues of intracellular or extracellular proteins, thereby affecting protein synthesis, leading to loss of the enzyme vitality, and ultimately leading to microbial death or reducing biofilm formation and stability. Moreover, these polar fragments can also bind with hydroxyl or carbonyl groups in nucleic acids through hydrogen bonds, thereby interfering with the synthesis of nucleic acids and their normal physiological functions. In addition, the binding of these polar fragments with nucleic acids may also affect the normal expression of eDNA genes, impact their regulation of biofilm establishment factors, reduce the clustering and convulsion effect of bacteria, and thus reduce the construction of biofilm and decrease the scary bacterial resistance [6]. Thiazole as an important functional fragment is widely present in clinical drugs such as antibacterial sulfathiazole and antifungal ravuconazole [7]. The rich electronic property of thiazole facilitates its binding to biologically essential internal targets through noncovalent interactions. Literature also reported that some thiazole-containing molecules exhibited good antibiofilm potential, so aminothiazoximone moiety may be a potential antibacterial and antibiofilm fragment [8].

Six-membered heterocyclic pyrimidinone is an important part of some nucleic acids [9]. The CO and NH of pyrimidinone could enhance the binding affinity and specificity of molecules to target biomolecules through hydrogen bonds and other weak interaction forces, hindering the physiological function of targets, and thereby reducing the viability of microorganisms. Therefore, pyrimidinone is widely used in the design of antibacterial drugs [10].

It was revealed that the hybridization of aminothiazoximone segment with sulfonamides, quinolones, and other bioactive skeletons can produce molecules with potent antibacterial effects and potential antibiofilm ability, but the introduction of aminothiazoximone moiety into pyrimidione has not received much attention [11]. In this work, a series of novel aminothiazoximone-corbelled ethoxycarbonylpyrimidones and their analogs were constructed, hoping to produce some new antibacterial molecules with strong antibiofilm property and low drug resistance. The design of these target molecules was based on the following considerations (Fig. 1):

It is well known that hybridizing the azole ring with other active fragments can provide new backbone molecules with potentially enhanced biological activity [12]. The introduction of unique thiazole can improve the biological activities of some molecules [13], and many efforts have been made to incorporate thiazole into bioactive skeletons such as indole, quinazolone, and purine, as well as berberine to discover potential antimicrobial candidates, some of which compounds exhibited excellent inhibitory effect and pharmacokinetic parameter [14]. However, there is few work to investigate the combination of thiazole and pyrimidinone, therefore, the merger of thiazole into pyrimidinone to produce novel compounds as antibacterial agents is an attractive research direction.

The ethoxycarbonyl group can enhance the lipid solubility of molecule and facilitate the interaction with enzyme through noncovalent bonds, and the incorporation of ethoxycarbonyl group might be beneficial to improve the binding ability to the target.

Piperazine is widely used in the construction of clinical antibacterial drugs such as norfloxacin and ciprofloxacin. Moreover, piperazine as a flexible group can also improve the binding stability between molecule and target by adjusting the molecular conformation [15].

The aromatic groups with various sizes and substituents might affect the spatial arrangement and charge distribution, thereby influencing antibacterial potency [16]. Therefore, five-membered heterocyclyl, phenyl, naphthyl, and large conjugated carbazolyl groups were introduced into the C-4 position of pyrimidinone to investigate their effect on antibacterial activity [17].

The aminothiazoximone-corbelled pyrimidinones with piperazine as a linker were characterized by multiple heteroatoms, which was beneficial for exerting multitargeting antibacterial mechanisms and further mitigating the development of drug resistance.

The aminothiazoximone moiety was replaced by acetylthiazole, and the carbonyl group was further reduced to a hydroxyl group to explore the contribution of the oxime fragment and carbonyl group to antibacterial capacity.

Phenyl group may affect lipid solubility and the halogenated phenyl groups are closely related to the rate of the absorption and transportation of drugs [18]. Therefore, phenyl groups as an isostere of thiazolyl group were introduced to discuss its contribution to antibacterial potency with the aim to enrich the structure-activity relationship.

The structures of the prepared ACEs were identified by NMR and HRMS spectra, and the antimicrobial activity was evaluated to screen the active molecules. Growth suppression, bactericidal kinetics, and drug combination experiments were conducted to further evaluate the antibacterial potential of active molecule. The druglikeness of the highly active compound was also investigated by hemolysis, cell cytotoxicity and drug resistance tests, as well as phototoxicity and ADME calculation. At the same time, the inhibition and clearance rates against the biofilm were evaluated, and the effect of the active molecules on the virulence factors that were beneficial to producing biofilm was also studied. Finally, the possible antibacterial mechanisms were explored from multiple dimensions including membrane activity, metabolic or respiratory arrest, oxidative stress, interaction with DNA.