β-Lactams and Ureas as Cross Inhibitors of Prokaryotic Systems

Abstract

β-Lactams in the last thirty years have been viewed as universal acylating agents of serine and cysteine enzymes of both prokaryotic and eukaryotic systems. More recently, their use has been propelled by the COVID-19 pandemic, thus broadening their application as inhibitors of viral enzymes. The urea-based drugs have been extensively studied as inhibitors of the aforementioned enzymes. The focus of this review is the last decade’s drug discovery strategies, as well as new strategies that show utility in the expansion of β-lactams and ureas in the development of new antimicrobial and antiviral drugs.

1. Introduction

The majority of currently approved FDA drugs bind with reversible noncovalent interactions to their biological (molecular) targets. However, in the last decade, there has been increased interest in covalently binding enzyme inhibitors as potential antimicrobials. Covalent drugs incorporate an electrophilic functional group that forms a covalent bond with a nucleophile on their molecular targets. In fact, most compounds that can form covalent bonds with their drug targets were discovered serendipitously through the use of naturally occurring enzyme inhibitors, e.g., amides.

The structural features of amides have been utilized by nature for millennia. One of the most successful examples of naturally occurring amide-based enzyme products targeting proteins is the group of β-lactams. For close to a century, the β-lactams have served as a rich source of marketed drugs and have opened new areas of target-based drug discovery. They are representatives of the mechanism-based covalent modification of a target enzyme as a key step in achieving potent inhibition of that target. They are also an example of how nature utilizes small molecules to modify key catalytic residues at an enzyme active site. Newly developed covalent modulators of non-catalytic amino acid residues have also been successful as anticancer drugs.

More recently, there has been a turn in the direction of drug development towards the utilization of ureas as targeted covalent enzyme inhibitors. Originally, ureas were marginalized by organic/medicinal chemistry since they lacked the natural ubiquity of amides, which were the historical focus of enzyme inhibition research. However, after a slow start as antimicrobials, initiated by Paul Ehrlich, urea-based inhibitors have gained traction in the past twenty years as HIV and other viral inhibitors, as well as anticancer drugs.

Our focus herein will be on the general electrophile–nucleophile interaction utilized by β-lactams and ureas and their bioisosters in the context of developing anti-infectives in the current age of rapid microbial resistance growth.

• General Chemistry and Structure

Most current β -lactams that target microbes modify their targets, mainly proteases, and are viewed as universal acylating agents of prokaryotic and eukaryotic serine and cysteine enzymes through covalent modification [1,2,3]. These targets and mechanisms of action are shared by a broad range of β-lactam structures, with penicillin being the first-identified antimicrobial of this drug class. The value of the β-lactam ring, a small heterocycle which features a reactive electrophilic center, has been demonstrated for a multitude of molecular targets (Figure 1).

There are also other examples, such as clinically relevant Ezetimibe (Figure 2), intended as an inhibitor (acylating agent) of one of the enzymes associated with the mammalian lipid metabolism (Acyl-CoA:cholesterol acyltransferase, ACAT); however, its molecular target has not yet been completely identified. Currently, Ezetimibe’s mechanism of action is accepted to be an inhibition of a transport protein target called Niemann-Pick C1- Like 1 (NPC1L1) [4,5]. Although non-acylating β-lactams, e.g., alkylthio-β-lactams (Figure 2), also contain the β-lactam functionality, their anti-microbial activity it is not based on enzyme-acylating, but on S-alkylation of the bacterial coenzyme A (CoA) [6,7]. The focus of this review will be on the acylating agents, which are the largest β-lactam class developed to target infectious agents (Figure 1).

• Lactams—Cyclic Amides

The stability and acylating function of a simple β-lactam ring is comparable to that of simple acyclic amide. Briefly, the carbonyl group of either the acyclic amide, or its cyclic counterpart, e.g., the β-lactam, engages in hydrogen bonding in the so-called “oxyanion” hole of a given enzyme. This binding allows for stabilization of the negative charge on the oxygen atom of the carbonyl, which secures the general acid–base catalysis, namely an attack of a nucleophile, most often a primary alcohol, such as the one from serine on the amide carbonyl carbon, as the electrophile. The nucleophilic attack on the latter leads to the formation of a tetrahedral intermediate, a species whose collapse results in acylation of the enzyme. This covalent binding of an amide/β-lactam with an enzyme has essential requirements, which have been extensively described in the literature [1,8,9].

Overall, β-lactams exhibit good pharmacokinetics/pharmacodynamics. Thus, they continue to be on the forefront of recently approved antimicrobial drugs, with the newest being the “pan”-antimicrobial cephalosporin Cefiderocol (Figure 3). This cephalosporin is the only one out of the twelve antimicrobials approved in the past five years, with activity against critical class pathogens as defined by the World Health Organization (WHO) [10]. Cefiderocol has a catechol moiety in its side chain which acts as an iron chelator. This allows Cefiderol to be actively transported across the cell membrane via iron transporter channels [11]. This transport process avoids current resistance mechanisms associated with passive diffusion. Furthermore, Cefiderocol is minimally affected by multidrug efflux pumps and is stable against hydrolysis by β-lactamases, including metallo-β-lactamases [11,12]. This ability to bypass several major mechanisms of bacterial resistance makes Cefiderocol suitable for the treatment of Gram-negative bacteria [11,12].

There are a number of other amide-based clinically approved acyclic amides and β-lactam drugs, including lidocaine, paracetamol, β-lactam antibiotics, and β-lactamase inhibitors, e.g., penicillin, clavulanic acid, and monocyclic β-lactams, as well as compounds with various other activities, e.g., ezetimibe, atorvastatin, chloramphenicol, moclobemide, captopril, acetazolamide, ponatinib, methotrexate, and trimethobenzamide. Although the best-known mode of action for β-lactam antibiotics is through inhibition of cell wall synthesis, their activity has also been demonstrated to interfere with bacterial quorum sensing [13]. In addition to their activity against prokaryotes, they have been shown to regulate innate immune responses through their effect on myeloid cells [14].

• Bioisosters of Amides—Ureas

A short-fall of amide-based structures is that they have limited stability in vivo. Therefore, use of these compounds requires replacing the initial amide-based lead compound with a peptide mimic, i.e., a bioisoster. There are different heterocyclic molecules that are broadly used in drug design as amide bioisosters. Their properties are elegantly described in recent perspectives and reviews [15,16,17]. The focus herein is on the urea-based scaffold commonly used in drug development for the treatment of bacterial and viral infections.

The earliest example of a urea compound developed as an anti-infective having anti-parasitic (anti-trypanosome) activity was developed by the Nobel laureate Paul Ehrlich (Figure 4). Recently, some compounds that were developed for mammalian (human) diseases (e.g., cancer) have been re-purposed as anti-microbials, including anti-virals, via exploitation of the mammalian metabolic pathways that are hijacked by the viruses.

The ability of urea to form stable hydrogen bonds with a drug target is the core quality that allows for specificity in drug action. Modification of the physicochemical properties of the molecule by increasing its stability also leads to the development of new drugs [15,16]. It is the use of various types of substituents on the urea nitrogen atoms that significantly alters the conformational preferences of the urea derivatives as they appear to have a wide variety of drug targets [16]. Ureas typically are more tempered electrophiles (as compared to β-lactams) covalently inhibiting individual serine hydrolases with excellent potency and selectivity [18,19,20,21]. Ureas’ utility as antimicrobials are being further expanded through the testing of currently FDA-approved urea-based drugs which have potential for drug repurposing. These drugs are anticancer agents (e.g., the kinase inhibitors Sorafenib and Regorafenib, Figure 5), stearoyl-CoA desaturase 1, as well as inhibitors of the insulin-like growth factor I receptor.

2. Microbe-Specific Targeting by β-Lactams and Ureas

2.1. Gram-Positive Staphylococcus aureus and Enterococcus faecalis

Despite the increasing resistance to antibiotics, β-lactams, particularly the anti-staphylococcal penicillins (ASPs), e.g., nafcillin, oxacillin, and flucloxacillin and the first-generation cephalosporins, e.g., cefazolin, remain the primary treatments for methicillin-sensitive Staphylococcus aureus (MSSA) infections, including bacteremia and infective endocarditis, although with restrictions [22,23]. An overview of the structure–function relationship for β-lactams and ureas with activity against Staphylococcus aureus and Enterococcus faecalis is shown in Table 1. Similarly, Enterococcus faecalis clinical isolates are resistant against most of the clinically relevant β-lactams with only the third- and fourth-generation cephalosporins and the carbapenems remaining that are efficacious. Monocyclic β-lactams substituted on the lactam nitrogen with the sulphonyl benzene pharmacophore were prepared recently in search of novel antimicrobial and antiviral compounds [24]. Compound 1 (Table 1) demonstrated one of the best antimicrobial activities of all compounds tested against both Gram-positive and Gram-negative organisms, with an MIC of 1 μg/mL for S. aureus. The N-phenylsulfonyl ureas (e.g., 1, Table 1) have moderate antiviral activity, but are not promising enough for further optimization of the scaffold as antivirals [24].

Beyond involvement with penicillin binding proteins (PBP) as the mode of action, kinase inhibitors, such as Sorafenib and Regorafenib (Figure 5, Table 6) FDA-approved anticancer agents), have demonstrated antibacterial and antiviral activity. The Sorafenib analog PK150 (Table 1) has demonstrated antimicrobial activity against MSSA strains and a 10-fold enhanced anti-MRSA activity. In addition, PK150 is as effective in the elimination of pre-existing biofilms as urea 2 (Table 1) with better activity than either Sorafenib or Regorafenib (3 µM for both Sorafenib and Regorafenib) [25]. Additionally, PK150 is an inhibitor of demethylmenaquinone methyltranferase (MenG) biosynthesis, which is involved in S. aureus menaquinone metabolism. Furthermore, it is not affected by environmentally acquired resistance as it causes over-activation of SpsB, an S. aureus signal peptidase I enzyme [25]. PK150 also shows oral bioavailability and antibacterial activity against several pathogenic strains at submicromolar concentrations [25].

Triclocarban belongs to the same chemotype as Sorafenib and Regorafenib, but was FDA approved specifically as an antimicrobial. Triclocarban’s mechanism of action, which is similar to Sorafenib, has MenG as its molecular target [26]. However, due to its numerous side effects, and its lack of timely degradation in the environment, it was banned by the FDA in 2016 for use in hygiene consumer products [27,28,29]. The development of triclocarban analogs with reduced toxicity led to the development of a series of diarylureas, some of which can dissipate pre-existing S. aureus biofilms. The most promising diarylurea in this library is the thiofluoro-substituted urea 2 (Table 1) that has increased selectivity against MRSA (MIC, 0.05 μg/mL) [30].

Other substitutions include PQ 401 (Table 1), an inhibitor of insulin-like growth factor I receptor (IGF-1R) signaling, which is important in both breast cancer and osteosarcoma [31]. PQ 401, which has demonstrated bactericidal activity against S. aureus strains, including MRSA, functions by disruption of the lipid bilayer [32]. Meanwhile, incorporating an aminoguanidine group into the diarylurea scaffold, such as in compounds 3 and 4 (Table 1), results in compounds that interfere with cell wall biosynthesis and produce re-sensitization of vancomycin-resistant S. aureus (VRSA) to vancomycin [33]. In high concentrations, these guanidinolated ureas also have S. aureus anti-biofilm activity [34]. Alternatively, substituting one of the aryl groups in the diarylureas with a pyrazoyl group led to the preparation of pyrazoyl-ureas 5 (Table 1) [35]. These compounds, when evaluated for antimicrobial activity against S. aureus, E. coli, and B. subtilis, demonstrated a moderate bacteriostatic/bacteriocidal activity of 250 μg/mL for all organisms tested. Furthermore, pyrazoyl-substituted ureas of type 6 (Table 1) demonstrated good antimicrobial activity against both Gram-positive and Gram-negative bacteria with S. aureus MICs in the 0.8 mg/mL range for both the trifluoromethyl- and chloro-substituted aromatic ring [36]. The pyrazoyl-substituted diarylureas have additional activities, such as antifungal and anti-malarial activity [37]. Diarylureas 7 and 8 also demonstrate activity against Enterococcus faecalis (MIC = 31.3 µg/mL, Table 1) and Klebsiella pneumoniae (MIC = 31.3 µg/mL, Table 2). Interestingly, both 7 and 8 are the only ones from the ureas described that contain a hydroxy group and a Michael acceptor. Whether this chemical difference affects their activity against these organisms remains to be answered [38]. In all cases, a disubstituted urea is necessary for activity and active compounds require, in addition to the pyrazole scaffold, an N-aryl substituent preferably 3,4 or 3,5-disubstituted with halogen atoms and/or CF₃ groups. Unfortunately, these ureas demonstrate low bioavailability in vivo, a result that could be attributed to low solubility [36].

Table 1. Representative examples of Gram-positive bacteria with recognized/reported β-lactams and ureas as antimicrobials: Staphylococcus aureus and Enterococcus faecalis. The structures of the lactams and ureas associated with a given microorganism with their corresponding references (bolded, in parentheses) are shown.

Microorg.	Structure—β-Lactam/Reference	Structure—Urea/Reference
S. aureus	Cephazolin (first-generation cephalosporin) [22] Ceftaroline (first-generation cephalosporin) [23] Cefiderocol (approved for medical use in the United States in November 2019, and in the European Union in April 2020) [10,11,12] 1 [24] MIC = 1 mg/mL	Triclocarban (TCC) [26,27,28,29] 2 [25,30] S. aureus (ATCC, MIC50 = 0.5 µg/mL) as well as MRSA (MIC50 = 0.05 µg/mL) PK150 [25] MIC 0.3 µM for MSSA, MIC 0.03 µM for MRSA. PQ 401 [31,32]
MRSA/ Biofilm		3 [33,34] 4 [33,34] MIC values for 3 and 4 between 2.0 and 4.0 µg/mL as compared with vancomycin (from 0.5 to 1 µg/mL). Molecular target: cell wall synthesis
S. aureus		5 [35] MIC = 250 mg/mL Compound 5 was evaluated as an antibacterial and antifungal agent against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 8236F800), and Candida albicans (ATCC 885-653), showing a moderate antimicrobial activity. 6 [36] IC50 ranging from 0.054 to 4.24 μM
E. faecalis	Cefepime—fourth-generation cephalosporin [39] Meropenem [40]	7 [38] 8 [38]

Table 1. Representative examples of Gram-positive bacteria with recognized/reported β-lactams and ureas as antimicrobials: Staphylococcus aureus and Enterococcus faecalis. The structures of the lactams and ureas associated with a given microorganism with their corresponding references (bolded, in parentheses) are shown.

Microorg.	Structure—β-Lactam/Reference	Structure—Urea/Reference
S. aureus	Cephazolin (first-generation cephalosporin) [22] Ceftaroline (first-generation cephalosporin) [23] Cefiderocol (approved for medical use in the United States in November 2019, and in the European Union in April 2020) [10,11,12] 1 [24] MIC = 1 mg/mL	Triclocarban (TCC) [26,27,28,29] 2 [25,30] S. aureus (ATCC, MIC50 = 0.5 µg/mL) as well as MRSA (MIC50 = 0.05 µg/mL) PK150 [25] MIC 0.3 µM for MSSA, MIC 0.03 µM for MRSA. PQ 401 [31,32]
MRSA/ Biofilm		3 [33,34] 4 [33,34] MIC values for 3 and 4 between 2.0 and 4.0 µg/mL as compared with vancomycin (from 0.5 to 1 µg/mL). Molecular target: cell wall synthesis
S. aureus		5 [35] MIC = 250 mg/mL Compound 5 was evaluated as an antibacterial and antifungal agent against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 8236F800), and Candida albicans (ATCC 885-653), showing a moderate antimicrobial activity. 6 [36] IC50 ranging from 0.054 to 4.24 μM
E. faecalis	Cefepime—fourth-generation cephalosporin [39] Meropenem [40]	7 [38] 8 [38]

2.2. Gram-Negative Multi-Drug Resistant Bacteria: Proteus mirabilis, Klebsiella pneumoniae, and Acinetobacter baumannii

Resistance to β-lactam drugs is even more pronounced with respect to Gram-negative pathogenic bacteria, such as Proteus mirabilis, Klebsiella pneumoniae, and Acinetobacter baumannii. While some strains of P. mirabilis are sensitive to ampicillin, in general, broad-spectrum penicillins (e.g., ticarcillin and piperacillin), first-, second-, and third-generation cephalosporins (e.g., imipenem and aztreonam), carbapenems, and piperacillin/tazobactam are required for use in the treatment of extended-spectrum β-lactamase-producing P. mirabilis bacteremia [39]. With respect to the treatment of K. pneumoniae infections, the use of β-lactams is limited to third- and fourth-generation cephalosporins and carbapenems. The only clinically used monobactam, aztreonam, has good activity against many Gram-negative bacteria; however, its activity against some of the most problematic multidrug-resistant (MDR) strains of P. aeruginosa and A. baumannii is limited [40]. Interestingly, coupling of the aztreonam side chain free acid with a bis-catechol siderophore mimetic (lactam 9, Table 2) significantly improves the activity against the MDR strains of Gram-negative bacteria that are of most significant concern [41].

With regard to the activity of diarylureas, there are several representatives of this class of compounds with activity against Gram-negative bacteria. The chloro, fluoro-, and trifluorpmethyl-substituted diarylureas 10 and 11 (Table 2) demonstrated activity against P. mirabilis that is comparable to that of ciprofloxacin [42]. In compound 12 (Table 2), one of the aryl groups of the prototypic diarylurea was substituted by an adamantane group, a feature that increases the lipophilicity of the compound [43]. Compound 12 (Table 2) demonstrated very good selectivity against A. baumannii (94% inhibition at 32 μg/mL) as compared with the rest of the bacteria tested, such as S. aureus, K. pneumonia, E. coli, P. aeruginosa, and C. albicans. Using several other analogs containing the adamantane group, it was determined that these compounds bind to the active site of enoyl-(acyl-carrier-protein) reductase (ENR). Of the library of adamantine-based analogs screened, compound 12 (Table 2) remains the best compound against A. baumannii, with analogs of diarylureas 7 and 8 (Table 1 and Table 2) exhibiting good activity against E. faecalis (MIC = 31.3 µg/mL, Table 1) and K. pneumoniae (MIC = 31.3 µg/mL, Table 2).

Table 2. Representative examples of Gram-negative bacteria with recognized/reported β-lactams and ureas as antimicrobials: Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumannii. The structures of the lactams and ureas associated with a given microorganism with their corresponding references (bolded, in parentheses) are shown.

Microorg.	Structure—β-Lactam/Reference	Structure—Urea/Reference
P. mirabilis	Cefepime [39] fourth-generation cephalosporin Meropenem [39]	10 [42] 11 [42]
K. pneumoniae	Cefepime [40] fourth-generation cephalosporin Meropenem [40] Aztreonam [41]	7 [38] 8 [38]
A. baumannii	Aztreonam [41] 9 [41] Aztreonam with siderophore mimetic	12 [43]

Table 2. Representative examples of Gram-negative bacteria with recognized/reported β-lactams and ureas as antimicrobials: Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumannii. The structures of the lactams and ureas associated with a given microorganism with their corresponding references (bolded, in parentheses) are shown.

Microorg.	Structure—β-Lactam/Reference	Structure—Urea/Reference
P. mirabilis	Cefepime [39] fourth-generation cephalosporin Meropenem [39]	10 [42] 11 [42]
K. pneumoniae	Cefepime [40] fourth-generation cephalosporin Meropenem [40] Aztreonam [41]	7 [38] 8 [38]
A. baumannii	Aztreonam [41] 9 [41] Aztreonam with siderophore mimetic	12 [43]

2.3. Mycobacterium tuberculosis (Mtb)

Until over a decade ago, Mycobacterium tuberculosis (Mtb) was considered refractory to β-lactam antibiotics. However, more recent studies show that carbapenems have anti-Mtb activity and are now used in the treatment of disease caused by MDR and extremely drug-resistant (XDR) Mtb strains [44,45]. Following this recognition of efficacy, further drug screening (8900 β-lactams) by a consortium of pharmaceutical companies, the NIH, and academic institutions identified about 1600 compounds that exhibited activity against Mtb, including many minus the necessity for the presence of a β-lactamase inhibitor [46]. Representative structures of cephalosporins from this work (lactam 13, which is active against replicating (R), and 14, which is active against both R and non-replicating (NR) Mtb) are shown in Table 3 [46]. Identification of β-lactams that are cidal for both R and NR Mtb was an additional reason to propel this massive screen undertaking [46,47]. Among the cephalosporins developed, six have a pyrithione-leaving group and are dual-acting compounds with activity against both NR and R Mtb. The release of the pyrithione upon cleavage of the β-lactam ring by β-lactamases, and/or engagement of the β-lactam ring with its target, allows for these two parts of the molecule to contribute to antitubercular activity.

A new family of anti-Mtb agents consists of C4-phenylthio β-lactams with the 15 scaffold (Table 3) [48]. This family shares similarities to the monocyclic structures 20 (Table 4) with the majority of structures having a benzylthio substituent at C4 and a urea moiety at N1 [49]. Lactams such as 15 having a phenylthio group at C4, and a urea moiety at N1, have shown activity against R and NR Mtb, as well as in macrophages [48]. Lactams with the 20 scaffold (Table 4) have antiviral activity as inhibitors of human cytomegalovirus (HCMV) protease with acceptable anti-HCMV activity [49]. Improvements upon the physicochemical properties of the monocyclic β-lactams 15 (Table 3) in our laboratories are ongoing.

Table 3. Representative examples with recognized/reported β-lactams and ureas as antimicrobials: Mycobacterium tuberculosis (Mtb). The structures of the lactams and ureas associated with Mtb and the corresponding references (bolded, in parentheses) are shown.

Microorg.	Structure—β-Lactam/Reference	Structure—Urea/Reference
Mtb	Meropenem [44,45] 13 [46,47] 14 [46,47] (TBBL-0000316) 15 [48] R = o-F, MIC 1.5 µg/mL (M. tuberculosis pathogenic strain H37Rv)	16 [50] MIC 6.25 µg/mL (M. tuberculosis pathogenic strain H37Rv) 17 [50] MIC 3.125 µg/mL (M. tuberculosis pathogenic strain H37Rv) 18 [31] MIC 6.0 µg/mL (M. tuberculosis pathogenic strain H37Rv) 19 [31] MIC 5.2 µg/mL (M. tuberculosis pathogenic strain H37Rv)

Table 3. Representative examples with recognized/reported β-lactams and ureas as antimicrobials: Mycobacterium tuberculosis (Mtb). The structures of the lactams and ureas associated with Mtb and the corresponding references (bolded, in parentheses) are shown.

Microorg.	Structure—β-Lactam/Reference	Structure—Urea/Reference
Mtb	Meropenem [44,45] 13 [46,47] 14 [46,47] (TBBL-0000316) 15 [48] R = o-F, MIC 1.5 µg/mL (M. tuberculosis pathogenic strain H37Rv)	16 [50] MIC 6.25 µg/mL (M. tuberculosis pathogenic strain H37Rv) 17 [50] MIC 3.125 µg/mL (M. tuberculosis pathogenic strain H37Rv) 18 [31] MIC 6.0 µg/mL (M. tuberculosis pathogenic strain H37Rv) 19 [31] MIC 5.2 µg/mL (M. tuberculosis pathogenic strain H37Rv)

Nitro-substituted diarylurea 16 and methoxy-substituted diarylurea 17 (Table 3) inhibited Mtb H37Rv with an MIC of 6.25 μg/mL and 3.12 μg/mL, respectively [50]. These MIC values are higher than those of isoniazid (0.05–0.2 μg/mL), the most often used drug as a control for evaluation of anti-Mtb activity, but are encouraging for further compound development. Nitro-substituted diarylurea 18 and methyl-substituted diaryl urea 19 (Table 3) have similar MIC values of 6.0 and 5.2 µg/mL, respectively, to compounds 16 and 17 (Table 3) against Mtb H37Rv. Interestingly, these diaryl-substituted ureas proved to be selective inhibitors of mycolic acid biosynthesis [51].

In summary, the predominant development of antimicrobials continues to be focused on the bacterial molecular targets exploring dual action, e.g., anti-Mtb inhibitors of both the PBPs and β-lactamases. Cefiderocol is the only antibiotic, out of the 12 approved since 2017, able to target all the pathogens considered critical by the WHO. The rest of the antibiotics are under development in Phase 1 to 3 clinical trials. As of this year (2023), from a total of 27 compounds, only four have new mechanisms of action, only six are considered innovative enough to meet the WHO criteria for the treatment of bacteria-resistant organisms, and only two of these six target the most difficult organisms to treat, such as A. baumannii and P. aeruginosa. Most of the drugs under development are the next generation of existing classes.

3. β-Lactams and Ureas as Antiviral Agents

3.1. Herpesviridae (DNA viruses): Human Cytomegalovirus (HCMV)

Early examples of monocyclic β-lactams as antivirals are monocyclic structures of type 20 (Table 4) having a carbamoyl moiety at N1. This scaffold contains at C-4 a side chain, thiomethyl (21 and 22, Table 4), or a one-carbon chain (23, Table 4), with either a phenyl or a heterocyclic ring [49,52,53]. These lactams were all developed as inhibitors of human cytomegalovirus (HCMV) serine protease. In the 21/22 series (heterocyclic thiomethyl side chain at C-4), the N-methyl thiotetrazole-containing compounds 22 (Table 4) demonstrated the best activity in the enzymatic assay. Members of the 23 series (C4 carbon-chain-substituted lactams) use both tri- and tetrasubstituted carbamoyl functionalities, which result in effective inhibitors of HCMV protease. However, for all compounds tested only modest antiviral activity was found in plaque reduction assays [53]. Further development led to a library of monocyclic β-lactams of type 24 (Table 4) that function as covalent and noncovalent HCMV protease inhibitors, e.g., 1-acylazetidines of scaffold type 25 (Table 4) [54,55]. Two of the 1-acylazetidines (of the 25 series) have activity as low micromolar inhibitors of HCMV replication (EC50, 0.6–7.0 mM) [55].

Shortly after the synthesis of the first mono-β-lactams (20, Table 4) to be used as inhibitors of the HCMV viral serine protease, an investigation of disubstituted ureas as inhibitors of that HMCV enzyme followed [49,56]. This novel primaquine-containing series consists of ureas having different alkyl, or a combination of alkyl and aryl, substituents, e.g., compounds 26 and 27 (Table 4). Those having an aryl group as one of the substituents on the urea moiety demonstrate the best inhibitory activity against HMCV with compounds 26 and 27 being the most active of the series (EC₅₀ = 1.2–13.4 μM). However, their activity is lower than that of ganciclovir (a purine-based FDA-approved antiviral against HCMV, which preferentially inhibits viral DNA polymerases more than cellular DNA polymerases) [56]. Another representative of the disubstituted ureas with one aryl and a piperidine as the second urea substituent is compound A939572 (Table 4). It was developed originally as an inhibitor of stearoyl-CoA desaturase 1 (SCD1, IC50 < 4 nM) [57]. SCD1 is an enzyme involved in lipid metabolism. Its high activity in humans has been correlated with elevated plasma triglyceride levels and it has been observed to be expressed in abnormally high levels in cell renal cell carcinoma (ccRCC) [58,59].

Table 4. Representative examples of Herpesviridae (DNA viruses) with recognized/reported β-lactams and ureas as antimicrobials: human cytomegalovirus (HCMV). The structures of the lactams and ureas associated with a given microorganism with their corresponding references (bolded, in parentheses) are shown.

Virus	Structure—β-Lactam/Reference	Structure—Urea/Reference
HCMV	20 [49] 21 [52] 22 [52] The most active compound of the series. 23 [53]     24 [54,55]         25 [55] EC50 0.6−7.4 μM	26 [56] (R1 = Ar, R2 = H); 27 (R1 = Ar, R2 = H) (EC50 = 1.2–13.4 μM) A939572 [57,58,59,60,61,62] IC50 < 4 nM (mSCD1)

Table 4. Representative examples of Herpesviridae (DNA viruses) with recognized/reported β-lactams and ureas as antimicrobials: human cytomegalovirus (HCMV). The structures of the lactams and ureas associated with a given microorganism with their corresponding references (bolded, in parentheses) are shown.

Virus	Structure—β-Lactam/Reference	Structure—Urea/Reference
HCMV	20 [49] 21 [52] 22 [52] The most active compound of the series. 23 [53]     24 [54,55]         25 [55] EC50 0.6−7.4 μM	26 [56] (R1 = Ar, R2 = H); 27 (R1 = Ar, R2 = H) (EC50 = 1.2–13.4 μM) A939572 [57,58,59,60,61,62] IC50 < 4 nM (mSCD1)

3.2. Flaviviridae, Picornaviridae, Retroviridae, and Coronaviridae (Single-Stranded Positive Sense RNA Viruses): DENV, ZKV, WNV, HCV, HRV, HIV, and SARS-CoV-2

Peptide-β-lactams 28 (Table 5) are reported as inhibitors of NS2BNS3 protease, a serine protease important in Dengue (DENV) and West Nile (WNV) virus replication [63]. Tripeptide-β-lactams are considerably more active as compared with the di-peptide-β-lactams, wherein the (3S)-β-lactam moiety displayed the highest activity. The IC₅₀ and EC₅₀ values of the S-tripeptide-β-lactams 28 (Table 5) are in the lower micromolar range in both biochemical and cellular assays (IC50, 1.9–2.8 μM). These compounds demonstrated two distinct binding modes to the NS2BNS3 protease: a covalent but reversible acylation of the active site serine by the β-lactams and another that leads to cleavage of the peptide at the C3 amide. Determining these two distinct modes of action on the two different amide bonds is the first experimental evidence that the benzyloxyphenylglycine in flaviviral protease inhibitors is positioned at the prime site of the enzyme [63]. Spirolactams of type BSS-730A (Table 5) have been recently evaluated for anti-HIV activity [64]. Compound BSS-730A inhibited both HIV-1 and HIV-2 replication with an IC₅₀ of 13 ± 9.59 nM, the best antiviral activity of a β-lactam. This potency may be attributed to its ability to affect all stages of the HIV replicative cycle. BSS-730A also affects multidrug-resistant HIV isolates, with a median 2.4-fold higher IC₅₀ relative to control isolates [64].

Other β-lactams containing purines, which are well established as potent antiviral agents, have been synthesized [65,66,67]. The idea behind molecular hybridization is to identify novel combinations of biologically active compounds able to circumvent drug resistance. This is envisioned to occur by linking bioactive units to moieties that are recognized and actively transported into mammalian cells. Hybrid systems also allow for the design of new organic structures through selective modification of one or both of the individual parts. The hybrid monocyclic β-lactam/purine hybrid structures 29 and 30 (Table 6) included the lactam ring that was subsequently opened (compounds 31 and 32, Table 6) [67]. This is similar to the approach reported for HCMV inhibition (Table 4) [54,55]. These two types of purine-containing compounds (20 in total) were evaluated against nine viruses. Eight of the β-lactam/purine hybrids and two of the compounds with an opened lactam ring showed very good antiviral activity in the micromolar range against five viruses (RSV, ChikV, CMV, HBV, and CoxV), with the best activity against RSV (the EC50 for the β-lactams was 2.24–8.05 μM and the EC50 for the opened lactam structures was 1.92–23.55 μM) [67].

C-4-diethoxyphosphoryl β-lactams of type 33 (Table 6) have been synthesized [68] and evaluated for their antiviral (against both DNA and RNA viruses) as well as their antimicrobial and cytostatic activity against several cancer cell lines. The isomers of the most promising structures, 33c and 33t (Table 6), differ in activity, i.e., the cis-isomer 33c has activity against influenza virus (H1N1) at an EC50 of 8.3–12 μM, depending on the type of assay used, while the trans-isomer 33t showed activity against a representative of the coronaviruses (229E) at a higher concentration (EC50, 45 μM). The trans-33t lactam also demonstrated activity against the drug-resistant S. aureus (HEMSA 5) strain as an adjuvant of oxacillin with a significant ability to enhance the efficacy of this antibiotic toward that strain, presumably by binding to the S. aureus β-lactamase, thus sparing the oxacillin from hydrolysis [68].

Table 5. Representative examples of Flaviviridae, Retroviridae, Picornavididae, and Coronaviridae (single-stranded positive-sense RNA viruses) with recognized/reported β-lactams and ureas as antimicrobials. The structures of the lactams and ureas associated with a given microorganism with their corresponding references (bolded, in parentheses) are shown.

Virus	Structure—β-Lactam/Reference	Structure—Urea/Reference
Hepatitis C virus Flaviviridae	28 [63]	38 [57,58,59,60,61,62] A939572 [59,60,61] Boceprevir (SCH 503034) [69,70,71,72] (Ki = 14 nM, EC90 = 0.35 mM)
Dengue virus Flaviviridae		A939572 [59,60,61] 39 [73] IC50 = 91 mM 40 [73] IC50 = 110 mM 41 [74] IC50 = 26 mM 42 [74] IC50 = 24 mM
West Nile virus Flaviviridae	28 [63]	39 [73] IC50 = 51 mM 40 [73] IC50 = 71 mM
Zika virus Flaviviridae		41 [74] IC50 = 28 mM 42 IC50 = 19 mM [74] 43 IC50 = 35 mM [75] 44 [75] ZIKV MTase IC50 = 23–48 mM ZIKV EC50 = 1.67–25 mM ASN 25 [76,77] Suramin [78]
Rhinovirus Picornaviridae		45 [79,80,81,82] 46 [79,80,81,82] 47 [83]
HIV Retroviridae	BSS-730A [64]	Ritonavir [84] 38 [85,86,87,88]
Coronaviruses SARS-CoV-2 Coronaviridae	34 and 35 [89,90] 36 and 37 [89,90]	38 [85,86,87,88]

Table 5. Representative examples of Flaviviridae, Retroviridae, Picornavididae, and Coronaviridae (single-stranded positive-sense RNA viruses) with recognized/reported β-lactams and ureas as antimicrobials. The structures of the lactams and ureas associated with a given microorganism with their corresponding references (bolded, in parentheses) are shown.

Virus	Structure—β-Lactam/Reference	Structure—Urea/Reference
Hepatitis C virus Flaviviridae	28 [63]	38 [57,58,59,60,61,62] A939572 [59,60,61] Boceprevir (SCH 503034) [69,70,71,72] (Ki = 14 nM, EC90 = 0.35 mM)
Dengue virus Flaviviridae		A939572 [59,60,61] 39 [73] IC50 = 91 mM 40 [73] IC50 = 110 mM 41 [74] IC50 = 26 mM 42 [74] IC50 = 24 mM
West Nile virus Flaviviridae	28 [63]	39 [73] IC50 = 51 mM 40 [73] IC50 = 71 mM
Zika virus Flaviviridae		41 [74] IC50 = 28 mM 42 IC50 = 19 mM [74] 43 IC50 = 35 mM [75] 44 [75] ZIKV MTase IC50 = 23–48 mM ZIKV EC50 = 1.67–25 mM ASN 25 [76,77] Suramin [78]
Rhinovirus Picornaviridae		45 [79,80,81,82] 46 [79,80,81,82] 47 [83]
HIV Retroviridae	BSS-730A [64]	Ritonavir [84] 38 [85,86,87,88]
Coronaviruses SARS-CoV-2 Coronaviridae	34 and 35 [89,90] 36 and 37 [89,90]	38 [85,86,87,88]

As is well documented in the literature, the SARS-CoV-2 main protease (M^pro), a cysteine protease, is a molecular target for developing SARS-CoV-2 treatments. Recently, the sulfoxide- and sulfone-penicillins 34 and 35 (Table 5) were synthesized and their molecular target -(M^pro) enzyme confirmed by mass-spectrometry (MS) [89]. It is interesting to note the very subtle structural difference between the compound 36 (Table 5), which demonstrates M^pro inhibition, and the corresponding penicillin V sulfone, 37 (Table 5), which is inactive against this protease. The ability of the C6-phenoxyacetyl ether oxygen to function as a hydrogen bond acceptor/Lewis acid/conformation restrictor may be important for efficient M^pro inhibition, as supported by preliminary molecular docking studies [90]. Compound 36 inhibited M^pro (IC₅₀, ~6.5 μM), with further optimization leading to derivatives of compound 36 having an IC50 of ~0.6mM, superior to its corresponding sulfoxide (IC50, ~22.9 mM). Neither Penicillin V nor its benzyl ester inhibited M^pro efficiently [90].

There are also urea-based drugs that are specifically FDA-approved as antiviral agents, e.g., ritonavir (Table 5) and boceprevir (Table 5). Ritonavir, a disubstituted urea (Table 5) initially approved as an HIV protease inhibitor, has also been shown to inhibit cytochrome P450-3A4 [84]. Clinically, it is usually used in combination with other HIV or Hepatitis C (Table 5) drugs, e.g., Lopinavir, to treat the corresponding infections. The urea-based compounds 38 (Table 5), similarly substituted at the urea moiety, are inhibitors of cyclophilins, which are peptidyl–prolyl cis/trans isomerases (PPIase) that catalyze the interconversion of the peptide bond [85]. It has been shown that human cyclophilins, mainly CypA, play an important role in the life cycle of viruses, including HIV, HCV, DENV, Japanese encephalitis virus, yellow fever virus, coronaviruses, HBV, cytomegalovirus, influenza A virus, and enteroviruses [85,86]. Thus, human cyclophilin inhibitors are an attractive target in antiviral therapy though blockage of an enzyme essential for viral replication. The currently known active site inhibitors of cyclophilins are cyclosporine A (CsA) and non-immunosuppressive macrocyclic analogs of CsA and sanglifehrin A (SfA). These two inhibitors have shown effectiveness against HIV, HCV, and HBV replication [86]. Alispovir, a CsA analog (synthesized from cyclosporin), has demonstrated potent anti-HCV in vivo activity but its clinical development did not progress beyond Phase III clinical trials due to severe adverse events unrelated to cyclophilin inhibition [87,88]. A fragment-based drug discovery (FBDD) approach was used to identify compounds 38 (Table 5) as cyclophilin inhibitors [85] which show antiviral activity for HIV (EC_50s ranging from 3.6 to 15 μM, Table 5), HCV (EC_50s ranging from 0.4 to 8.4 μM, Table 5), and coronaviruses (EC_50s ranging from 7.2 to 71.5 μM, Table 5). These inhibitors appear to be non-cytotoxic at their effective concentrations [85].

An HCV protease inhibitor, Boceprevir (Table 5), played a valuable role in the treatment of patients with an HCV genotype 1 infection. However, production was discontinued by the manufacturer in 2015 due to the superiority of newer agents (e.g., ledipasvir/sofosbuvir) [69]. The relevance of Boceprevir was considered historical until, during the search for effective treatments against SARS-CoV-2, it was demonstrated that Boceprevir inhibits SARS-CoV-2 (Table 5) by inhibiting its main protease [70,71,72].

Viruses are known to hijack critical metabolic pathways in host cells to convert their activity for their replication. Flaviviruses, such as DENV and HCV, hijack lipid metabolic pathways for their energy and substrate requirements. It was shown that SCD1 is critical to virus replication and infectious particle formation [60]. Compound A939572 (Table 5) has been demonstrated to have antiviral activity against HCV and DENV infection in a cell culture [60]. Inhibition of SCD1 continues to be targeted for new drug development [61,62]. What is notable, with respect to the use of the urea-based inhibitor A939572, is that it targets the activity of the host enzyme, rather than the viral enzymes, which is a potential way to slow down the development of viral drug resistance, a too common occurrence with the conventional virus-targeted antivirals.

Incorporating a sulfonamide as well as a carboxylic acid group in one of the aryl substituents in the scaffold of the disubstituted ureas such as PK150 (Table 1) and compounds 10 and 11 (Table 1) using the FBDD resulted in compounds 39 and 40 (Table 5) having activity against DENV and WNV NS5 methyltransferase [73]. The optimization of the fragment linking hit led to another series of diarylureas with compounds 41 and 42 (Table 5) with improved IC50s (DENV IC₅₀ = 26 μM and 24 μM, respectively) as compared with compounds 39 (DENV IC₅₀ = 91 µM; WNV IC₅₀ = 51 µM) and 40 (DENV IC₅₀ = 110 µM; IC₅₀ = 71 µM). Interestingly, compounds 41 and 42 also demonstrate activity against ZIKV (IC₅₀ = 28 μM and 19 μM, respectively (Table 5)) [74].

Compound 43 (Table 5) is an example of the discovery of a hit compound using a structure-based virtual screening approach using the ZIKV NS5-MTase (43 IC50 35 μM) [75]. Optimization of compound 43 led to compounds 44 (Table 5) with improved activity against ZIKV (44 ZIKV Mtase IC50 23–48 μM; ZIKV EC50 1.67–25 μM) [75]. Diarylureas of type ASN-25 (Table 5) exhibit nanomolar to picomolar activity against ZIKV in vitro (IC₅₀ = 160.3 nM, 189.2 pM, 317.7 pM, and 26.9 nM, respectively) [76]. In a continuation of their work, the authors prepared compound ASN-25, which exhibited an excellent IC₅₀ = 85.1 pM [77]. It is interesting to note that it was shown earlier by a high-throughput screening method that a host kinase is a key regulatory factor in Zika infection [91].

3.3. Pneumoviridae and Orthomixoviridae (Single-Stranded Negative-Sense RNA Viruses): RSV and IV

Other representatives of diarylureas containing pyrazoyl groups are compounds of type 45 and 46 (Table 5) as well as compound 47 (Table 5 and Table 6). Compound 46 is structurally related to BIRB 796 (Doramapimod), a pan p38 MAP kinase inhibitor with a K_d of 0.1 nM in THP-1 cells [79]. Initially developed as p38 MAP kinase inhibitors, three derivatives of the 46 scaffold are the most active of the series with an IC₅₀ < 5 μM against the p38α enzyme [80]. They were also described as antivirals against several viruses, including influenza [81,82]. Compound 47 (Table 5 and Table 6) has been described as representative of pyrazoyl ureas for the treatment of inflammatory diseases of the respiratory system (asthma, COPD), and also for the treatment, or prevention, of inflammation mediated by viral infectious diseases (such as influenza virus, rhinovirus, or RSV) [83].

RNAi-based small-molecule repositioning revealed clinically approved urea-based kinase inhibitors (UBKIs) to be broadly active antivirals. Regorafenib and Sorafenib (Figure 1, Table 6) exhibited a superior therapeutic window of high IV antiviral activity and low cytotoxicity. Both UBKIs appeared to block a cell signaling pathway involved in IV replication after internalization, yet prior to vRNP uncoating. Interestingly, both compounds were also active against unrelated viruses including cowpox virus (CPXV), hantavirus (HTV), herpes simplex virus 1 (HSV-1), and vesicular stomatitis virus (VSV) [78]. Pyrazoyl-ureas, such as compound 47 (Table 5 and Table 6), have demonstrated anti-IV activity as well [83].

Table 6. Representative examples of Pneumoviridae (RSV) and Orthomixoviridae (IVA and IVB) (single-stranded negative-sense RNA viruses) recognized/reported β-lactams and ureas as antimicrobials. The structures of the lactams and ureas associated with a given microorganism with their corresponding references (bolded, in parentheses) are shown.

Virus	Structure—β-Lactam/Reference	Structure—Urea/Reference
RSV Pneumoviridae	29 [67] 30 [67] 31 [67] 32 [67]	47 [83]
Influenza virus Orthomixoviridae	33c [68] 33t [68]	Regorafenib [78] Sorafenib [78] 47 [83]

Table 6. Representative examples of Pneumoviridae (RSV) and Orthomixoviridae (IVA and IVB) (single-stranded negative-sense RNA viruses) recognized/reported β-lactams and ureas as antimicrobials. The structures of the lactams and ureas associated with a given microorganism with their corresponding references (bolded, in parentheses) are shown.

Virus	Structure—β-Lactam/Reference	Structure—Urea/Reference
RSV Pneumoviridae	29 [67] 30 [67] 31 [67] 32 [67]	47 [83]
Influenza virus Orthomixoviridae	33c [68] 33t [68]	Regorafenib [78] Sorafenib [78] 47 [83]

4. Conclusions

Recent advances in drug design with potential as pan-antimicrobials are based on the two “classical” electrophilic chemotypes, i.e., β-lactams and urea-based derivatives and their bioisosteres. The illustrative β-lactam and urea covalent protein-reactive drugs discussed represent important advances since they target two commonly present enzyme active-site nucleophiles (serine and cysteine); thus, they are not restricted to a specific taxonomic kingdom affecting fungi, bacteria, and viruses, i.e., pan-activity. The included utility of this enzyme-targeted approach is illustrated by the ability of β-lactams to inhibit representatives of the ESKAPE pathogens. Currently, the focus is on the development of new moieties added to the β-lactam scaffold which exhibit siderophore activity. These new lactams engage with the microbes’ active transport systems, rather than cell entry via diffusion. In addition, their siderophore’s metal chelating properties target an alternative system essential for microbe survival, enhancing the ability of these compounds, especially when used in combination with chemotherapy, to affect drug-resistant organisms since the siderophore moieties allow for evasion of the bacterial efflux pumps as well as β-lactamases. To date, this type of β-lactam structure has found utility in Mtb treatment, including activity against the latent Mtb. This exploitation of the lactam scaffold and ureas takes advantage of the carbonyl group in the lactams and ureas as an electrophile that inhibits serine and cysteine hydrolases via catalytic modification of these active-site residues, resulting in a covalent inhibition of a variety of target proteins. Unfortunately, the selectivity of the β-lactam towards the serine enzymes appears to be rather poor. In contrast, ureas, as more tempered electrophiles, covalently inhibit individual serine hydrolases with excellent potency and selectivity. The advantage of the ureas is that they represent a simple chemical core permitting modifications directed toward the electrophilicity of the carbonyl group, as well as the number of hydrogens on the urea moiety. Thus, they allow for rapid preparation of suitable chemical probes for activity-based protein profiling (ABPP). The ability to inhibit and/or detect changes in the activity and localization of specific enzymes in tissues or whole organisms that contain many related enzymes with similar substrate specificities remains a primary goal of contemporary ABPP research. For example, triazole ureas appear to be some of the scaffolds capable of generating probes with moderate selectivity for serine enzymes. It might be that the urea scaffold, explored a century ago by Paul Erlich, will allow us to achieve the features necessary for better enzyme selectivity. Currently, more than 40 covalent drugs are on the market with many of them in clinical trials. Several ureas have been utilized as antimicrobials at the turn of this century, but their mechanism of action has not been unveiled until recently. Several very successful repurposing studies of ureas from anti-cancer to antimicrobial (antiviral) have occurred, with follow-up FDA approval for use as antivirals. The lessons that might be learned from these protein-reactive drug classes could assist in the further design of the next generation of antimicrobials, especially in the current age of elevated microbial resistance towards the clinically relevant antimicrobials.