**Biofilm Inhibition and Eradication Properties of Medicinal Plant Essential Oils against Methicillin-Resistant** *Staphylococcus aureus* **Clinical Isolates**

### **Fethi Ben Abdallah 1,2,\* , Rihab Lagha 1,2 and Ahmed Gaber 1,3**


Received: 16 October 2020; Accepted: 4 November 2020; Published: 6 November 2020

**Abstract:** Methicillin-resistant *Staphylococcus aureus* is a major human pathogen that poses a high risk to patients due to the development of biofilm. Biofilms, are complex biological systems difficult to treat by conventional antibiotic therapy, which contributes to >80% of humans infections. In this report, we examined the antibacterial activity of *Origanum majorana*, *Rosmarinus o*ffi*cinalis*, and *Thymus zygis* medicinal plant essential oils against MRSA clinical isolates using disc diffusion and MIC methods. Moreover, biofilm inhibition and eradication activities of oils were evaluated by crystal violet. Gas chromatography–mass spectrometry analysis revealed variations between oils in terms of component numbers in addition to their percentages. Antibacterial activity testing showed a strong effect of these oils against MRSA isolates, and *T. zygis* had the highest activity succeeded by *O. majorana* and *R. o*ffi*cinalis*. Investigated oils demonstrated high biofilm inhibition and eradication actions, with the percentage of inhibition ranging from 10.20 to 95.91%, and the percentage of eradication ranging from 12.65 to 98.01%. *O. majorana* oil had the highest biofilm inhibition and eradication activities. Accordingly, oils revealed powerful antibacterial and antibiofilm activities against MRSA isolates and could be a good alternative for antibiotics substitution.

**Keywords:** methicillin-resistant *Staphylococcus aureus*; essential oils; *Origanum majorana*; *Rosmarinus o*ffi*cinalis*; *Thymus zygis*; antibacterial; biofilm inhibition and eradication

#### **1. Introduction**

Methicillin-resistant *Staphylococcus aureus* (MRSA) is considered a principal human pathogen and the most common cause of nosocomial infections. MRSA causes several diseases ranging from skin and soft tissue infections to serious invasive infections such as pneumonia, bacteremia, endocarditis and osteomyelitis [1]. The number of MRSA infections, which are more frequently associated with mortality than other bacterial infections, has increased considerably over recent years. *S. aureus* carries 20–40% mortality at 30 days despite appropriate treatment [2].

MRSA poses a high risk to patients due to the development of biofilm [3]. Biofilm is considered as major virulence factor and is an organized structure built by almost all bacteria that is composed of nucleic acids, lipids, proteins, and polysaccharides [4]. Biofilms contribute to >80% of human infections and *S. aureus* is considered as the leading species in biofilm-associated infections [5]. In Biofilm, MRSA like other bacteria, become more persistent in the host organism, environment, and medical

surfaces, and show an increased resistance to antibiotics and host immune factors [6–8], which is an important medical problem. Therefore, the development of novel compounds to treat biofilm is urgently required; plant essentials oils (EOs) that act against bacterial biofilm are of great interest.

EOs are volatile compounds that have been used to combat a variety of infections during hundreds of years as a natural medicine. It has been shown that EOs possess several significant antimicrobial activities such as antibacterial, antiviral, antifungal, and anti-parasitic activities in addition to their antioxidant, antiseptic, and insecticidal properties [9,10].

*Rosmarinus o*ffi*cinalis* L., *Thymus zygis* L., and *Origanum majorana* L. belong to the Lamiaceae family. EOs obtained from aerial parts of the flowering stage of these plants, have been reported for their antibacterial activities against *S. aureus* [11,12] and their antibiofilm activities against uropathogenic *E. coli* [13]. Several reports have shown that tea tree, thyme, and peppermint EOs, are effective against planktonic [14] and biofilm [15,16] MRSA. In addition, Cáceres et al. [17] demonstrated high anti-biofilm activity of thymol-carvacrol-chemotype (II) oil from *Lippia origanoides* against *E. coli* and *S. epidermidis*. However, these oils did not alter the growth rate of planktonic bacteria. The antibacterial effect of EOs, which is manifested by alterations of the bacterial cell wall and cell membrane, depends of their chemical composition [18]. The cell membrane compositions play an important role in the high resistance of Gram-negative bacteria to EOs compared to Gram-positive [19]. The hydrophobic molecules penetrate easily into the cells due to cell wall structure in Gram-positive bacteria and act on the cell wall and within the cytoplasm [20].

This study aimed to investigate the antibacterial, biofilm inhibition, and eradication properties of *O. majorana*, *R. o*ffi*cinalis*, and *T. zygis* medicinal plants' EOs against clinical methicillin-resistant *Staphylococcus aureus*.

#### **2. Results**

#### *2.1. Distribution of the MRSA Isolates*

Thirty clinical MRSA isolates were collected from King Abdulaziz Specialist Hospital, Taif, Saudi Arabia. The isolates were obtained from infection sites: surgical site infection (SSI, *n* = 4), skin and soft tissue (SST, *n* = 12), blood (*n* = 1), nasal (*n* = 8) and burn (*n* = 5). The distribution of isolates based on the type of specimen is presented in Figure 1.

**Figure 1.** Distribution of MRSA isolates.

#### *2.2. Chemical Composition of the Essential Oils*

*O. majorana*, *T. zygis*, and *R. o*ffi*cinalis'* EOs chemical compositions are summarized in Table 1. In total 37 components were detected in these oils: 10 compounds in *R. o*ffi*cinalis* and 31 compounds in each of *O. majorana* and *T. zygis*.

GC-MS results showed variations between these oils regarding the compound numbers and their percentages. The major constituents of *O. majorana* were terpinen-4-ol (25.9%), γ-terpinene (16.9%), linalool (10.9%), sabinene (8%), and α-terpinene (7.7%); those of *R. o*ffi*cinalis* were α-pinene (37.7%), bornyl acetate (9.1%), camphene (7.3%), borneol (5.5%), verbenone (5.4%), camphor (5.2%), and 1,8-cineole (4.7%). However, the main components of *T. zygis* were linalool (39.7%), terpinen-4-ol (11.7%), β-myrcene (8.6%), and γ-terpinene (7.6%).


**Table 1.** Chemical composition of the essential oils.

#### *2.3. Antibacterial Activity of Essential Oils against MRSA*

#### 2.3.1. Disc Diffusion

The antibacterial activity of EOs against MRSA isolates was assessed by the disc diffusion method (Table 2). *T. zygis* EO has shown strong inhibitory activity on 80% of the strains, succeeded by *O. majorana* and *R. officinalis* that demonstrated a strong inhibitory action on 53.33% and 16.66% of the isolates, respectively. Moreover, according to the high percentage of anti-MRSA activity, *T. zygis* and *O. majorana'* EOs have a strong inhibitory action on 80% and 53.33% of the strains, respectively. However, *R. officinalis* had a slight inhibitory action on 46.66% of the strains. Thereby, *T. zygis'* EO appeared as the EO with the highest antibacterial activity, succeeded by *O. majorana* and *R. officinalis*. According to the type of specimen, globally, the same result was found as detected in cases of total isolates. *T. zygis* was regarded as an EO with strong inhibitory activity, succeeded by *O. majorana* and *R. officinalis*.

**Table 2.** Antimicrobial effect of EOs against MRSA isolates using disc diffusion.


*n*: number of isolates.

#### 2.3.2. Determination of MIC and MBC

Antibacterial activity of EOs was assessed by measuring MICs and MBCs for the 30 MRSA isolates and the reference strain. The values of MIC extended from 0.78 mg/mL to 1.56 mg/mL, while the MBC varied from 3.125 mg/mL to 12.5 mg/mL for *O. majorana* EO.

Concerning *T. zygis* EO, the MIC values ranged from 0.39 mg/mL to 0.78 mg/mL, while the MBC value was 3.125 mg/mL. However, The MIC values of *R. o*ffi*cinalis* varied from 0.78 mg/mL to 1.56 mg/mL, but the MBC value was 12.5 mg/mL. Compared to *O. majorana* and *R. o*ffi*cinalis*, *T. zygis* EO showed the greatest antibacterial activity against MRSA isolates. Person correlation (r) showed no significant correlation between the type of specimen and MICs of oils (*p* > 0.05).

#### *2.4. Biofilm Formation*

MRSA strains were tested for their potentialities to form biofilm on a polystyrene surface. Table 3 indicated that 96.66% of the isolates were able to form biofilm and were distributed as follow: 40% were highly positive biofilm producers with OD570 values ranged from 1.175 to 3.635, and 56.66% were low-grade positive with OD570 values extended from 0.113 to 0.87. However, out of the 30 isolates only one strain was isolated from the nasal sample was biofilm negative. Reference *S. aureus* ATCC 25923 was considered as a highly positive biofilm producer. Analysis of variance (ANOVA) indicated that there is no significant effect of the specimen on biofilm formation (*p* > 0.05).


**Table 3.** Biofilm formation ability of MRSA isolates on polystyrene surface.

SST: skin and soft tissue; SSI: surgical site infection.

#### *2.5. Biofilm Inhibition Activity of Essentials Oils*

Biofilm inhibitory activities of *O. majorana*, *T. zygis*, and *R. o*ffi*cinalis'* EOs are summarized in Table 4. MRSA isolates that showed a biofilm formation potential were selected for this investigation. In all, 29 isolates considered as highly positive biofilm and low-grade positive biofilm in addition to the reference strain were used.


**Table 4.**Biofilm inhibition activity of EOs against MRSA isolates.

\* Isolates changed from low-grade positive to biofilm negative after treatment with EOs. \*\* Isolates changed from highly positive to low-grade positive after treatment with EOs. \*\*\* Isolates changed from highly positive to biofilm negative after treatment with EOs.

*O. majorana* EO demonstrated an antibiofilm activity on 89.66% of the isolates (26 strains) in addition to the reference strain. Among them, 11 isolates (39.93%) were passed from low-grade positive to biofilm negative, three highly positive biofilm isolates (10.34%) become low-grade positive, and one strain isolated from nasal samples was changed from highly positive to biofilm negative after treatment with *O. majorana* EO. The percentage of inhibition ranged from 10.29 to 95.91%.

Concerning *R. o*ffi*cinalis* EO, we detected an antibiofilm effect on 79.31% of the isolates (23 strains). Furthermore, two groups of five isolates (17.24%) were changed. The first group was varied from low-grade positive to biofilm negative, and the second group was passed from highly positive to low-grade positive. In addition, the same isolate that changed from highly positive to biofilm negative under *O. majorana* EO, also became biofilm negative under *R. o*ffi*cinalis* EO. The percentage of inhibition extended from 10.20 to 95.65%.

Antibiofilm activity of *T. zygis* was observed on 62.06% of the isolates (18 strains). The percentage of biofilm inhibition ranged from 11.67 to 91.48%. Moreover, three low-grade positive isolates (10.34%) were changed to biofilm negative, and four highly positive isolates (13.79%) become low-grade positive.

The outcomes of this study indicated that *O. majorana* EO had the greatest antibiofilm activity against MRSA isolates succeeded by *R. o*ffi*cinalis* and *T. zygis*.

Person correlation (r) indicated a non-significant correlation between MIC and antibiofilm of EOs (*p* > 0.05). ANOVA test showed a non-significant effect of the specimen on biofilm inhibition (*p* > 0.05).

#### *2.6. Biofilm Eradication Activity of Essentials Oils*

The results of biofilm eradication activities of EOs are shown in Table 5. The same isolates selected for biofilm inhibitory investigation were used. *O. majorana*, *T. zygis*, and *R. o*ffi*cinalis* EOs showed eradication activities on 41.37% (12 strains) of the MRSA isolates independently of the specimen, including the reference strain. The highest percentage of eradication was recorded with *O. majorana*. The percentage of eradication ranged from 18.31 to 98.01%, from 12.65 to 94.39%, and from 13.45 to 92.69%, respectively, for *O. majorana*, *T. zygis*, and *R. o*ffi*cinalis* EOs.


**Table 5.**Biofilm eradication activity of EOs against MRSA isolates.

\* Isolates changed from low-grade positive to biofilm negative after treatment with EOs. \*\* Isolates changed from highly positive to low-grade positive after treatment with EOs. \*\*\* Isolates changed from highly positive to biofilm negative after treatment with EOs.

Based on biofilm categories and under *O. majorana* EO, five isolates (17.24%) were changed from highly positive to low-grade positive. Furthermore, a low-grade positive (isolate number 6) and a highly positive (isolate number 14) strains became biofilm negative. For *T. zygis*, only isolates numbers 5 and 6 were changed from highly positive and low-grade positive respectively to biofilm negative after treatment, while, *R. o*ffi*cinalis* caused modifications on three isolates. Among them, a low-grade positive (isolate number 28) was changed to biofilm negative, and two highly positive (isolates number 5 and 10) became low-grade positive.

Person correlation (r) indicated a non-significant correlation between MIC and biofilm eradication of EOs (*p* > 0.05). ANOVA test showed a non-significant effect of the specimen on biofilm eradication (*p* > 0.05).

#### **3. Discussion**

Infection caused by MRSA is considered a major public health threat in many countries and MRSA remains the principal cause of hospital and community-acquired infections [21]. This bacterium is accountable for numerous infections related to remarkable morbidity and mortality [22], such as bacteremia, pneumonia, and skin, soft tissue, surgical site, and urinary tract infections [23,24]. This study was conducted on 30 clinical MRSA isolates and results showed variability in the prevalence of the isolates. Indeed, most of the strains (40%) were recovered from SST followed by nasal, burn, SSI, and blood. Akanbi et al. [25] showed that the majority of MRSA strains were isolated from blood, wound, and urine specimens. In addition, Ghebremedhin et al. [26] demonstrated that MRSA was most found in surgical wound infections, succeeded by eye swabs, skin and soft tissue infections.

The ability of MRSA to develop resistance to every antibiotic to which it is exposed makes it a problem to human health [27]. Thereby the development of novel compounds is of great importance. Medicinal plant EOs have been largely used as a natural medicine to combat bacteria, fungi, viruses, and other pathogens [28]. Until now, about 3000 EOs are known, among them 300 are important for industries such as pharmaceutical, food, agronomic, cosmetics, and fragrance. In this work, we investigated the potential antibacterial activities of *O. majorana*, *T. zygis*, and *R. o*ffi*cinalis* medicinal plant EOs against MRSA clinical isolates by disc diffusion, MIC, and MBC techniques. The highest antibacterial activity was observed with *T. zygis*, followed by *O. majorana*, and *R. o*ffi*cinalis* EOs. This result is in agreement with the study of Lagha et al. [13], who showed that *T. zygis* possessed the strongest antimicrobial effect against uropathogenic *E. coli* in contrast to *O. majorana*, and *R. o*ffi*cinalis* EOs. According to biochemical composition, the greater effect of *T. zygis* is owing to linalool (39.7%), which has a strong effect against bacteria and fungi [29]. Regarding *O. majorana* EO, the antibacterial activity can be attributed to the monoterpene alcohol, terpinen-4-ol, as a major compound (25.9%) [30], which was found to be effective against MRSA [31]. According to Cordeiro et al. [32], terpinen-4-ol has a powerful antibacterial effect against *S. aureus*. This compound functions as a bactericidal by obstructing the synthesis of the cell wall. Moreover, other main components such as terpinen-4-ol (11.7%), β-myrcene (8.6%) and γ-terpinene (7.6%) are present in *T. zygis* in addition to linalool (10.9%), γ-terpinene (16.9%) and α-terpinene (7.7%) present in *O. majorana* may enhance the antibacterial effect of these oils. Concerning *R. o*ffi*cinalis* EO, which showed the lowest activity against MRSA isolates, its antibacterial activity may be related to α-pinene (37.7%) as a major constituent. The study of Leite et al. [33] showed antibacterial activity of α-pinene against *S. aureus* and *S. epidermidis*. Other reports [34,35] revealed the antibacterial activity of some EOs against Gram-negative and Gram-positive bacteria when α-Pinene is the major constituent. However, Utegenova et al. [36] demonstrated that α-pinene had low activity against MRSA, indicating that other components were probably responsible. Among them, in this study, 1,8-cineole (4.7%) altered the structure of *E. coli*, *S. enteritidis*, and *S. aureus* [37]. The antibacterial activity of *R. o*ffi*cinalis* could be attributed to the synergistic effect of camphor (5.2%), verbenone (5.4%), and borneol (5.5%) in addition to α-pinene and 1,8-cineole [38]. In general, whole essential oils have an important antimicrobial effect compared to the

major compounds individually or collectively. This suggests that minor constituents are essential and may have a synergistic antibacterial effect [10].

MRSA were tested for their capacities to produce biofilm on polystyrene microplates and results indicated that 40% of the strains were highly positive biofilm and 56.66% were low-grade positive. Out of the 30 isolates, only one strain was biofilm negative, which indicates the high potentiality of the isolates to form biofilm. Biofilm, as a virulence factor that favors the chronicity of *S*. *aureus* infections, is accountable for more 65% of nosocomial infections and 80% of microbial infections [5]. Biofilm is related to various staphylococcal diseases, such as skin and soft tissue infections, nasal colonization, endocarditis and urinary tract infections [39]. Further, biofilm becomes a serious threat in the urology field due to its responsibility for the long persistence of bacteria in the genitourinary tract [40]. The high ability of the investigated isolates to form biofilm confirms the fact that *S. aureus* is the leading species in biofilm-associated infection.

Biofilm has been associated with medical devices and its treatment is becoming increasingly difficult due to the resistance to antibiotics and the immune system in addition to the spread of infection [39]. Thereby, the development of new therapeutic strategies, such as EOs, to inhibit or eradicate biofilm is great of interest. In this work, biofilm inhibitory activity of EOs showed that *O. majorana* had the highest antibiofilm activity (antibiofilm effect on 89.66% of the isolates) against MRSA isolates followed by *R. o*ffi*cinalis* and *T. zygis* that demonstrated activity on 79.31 and 62.06% of the isolates, respectively. EOs also showed a strong potential to inhibit biofilm, with percentage of inhibition ranging from 10.29 to 95.91%, from 10.20 to 95.65%, and from 11.67 to 91.48%, respectively for *O. majorana*, *R. o*ffi*cinalis*, and *T. zygis*. Based on our results, the oil with the highest growth inhibition activity was different from the oil with the highest biofilm inhibition effect, which indicates that the components involved in growth inhibition were different from those associated with biofilm inhibition. According to biochemical specificity, terpinen-4-ol present in *O. majorana* as major compound, has more inhibition of the biofilm formation process by MRSA isolates compared to α-pinene and linalool present, as the main components, in *R. o*ffi*cinalis* and *T. zygis* EOs, respectively.

This finding corroborates the recent data of Cordeiro et al. [32] showing that the strongest antibiofilm activity of terpinen-4-ol was against *S. aureus*. Other studies have also demonstrated that this compound possesses an excellent potential against biofilm formed by some pathogenic bacteria like *Pseudomonas aeruginosa* [41], *Streptococcus mutans*, *Lactobacillus acidophilus* [42], *Porphyromonas gingivalis*, and Fusobacterium nucleatum [43]. Biofilm inhibition properties of *O. majorana*, *R. o*ffi*cinalis*, and *T. zygis* EOs against MRSA suggest that the addition of these oils before biofilm formation eliminates planktonic cells and may reduce the polystyrene surface adherence, which becomes less susceptible to cell adhesion. Additionally, the modification of MRSA surface proteins caused by their interactions with oils inhibits the adhesion of this bacterium to the polystyrene surface, which is the initial attachment phase [44].

Preformed biofilms are difficult to eradicate by conventional antibiotic therapy. However, in the present study, *O. majorana*, *T. zygis*, and *R. o*ffi*cinalis* EOs showed high biofilm eradication activities on 41.37% of the MRSA isolates. *O. majorana* EO had the strongest effect, with the percentage of eradication going up to 98.01%, and seven isolates were changed their biofilm phenotype. It seems that the monoterpenoid terpinen-4-ol has an excellent potential to eradicate mature biofilm than α-pinene and linalool. The activity of these oils on mature biofilms was lower than their capacity to inhibit the formation of biofilms. This can be explained by the fact that the major constituents in these oils have an effect on the biofilm formation process more than on mature biofilm. This is in agreement with the report of Cordeiro et al. [32], showing that terpinen-4-ol is more efficient in inhibiting the formation of *S. aureus* biofilms than in breaking or eliminating mature biofilms. Many EOs, such as tea tree [45], eucalyptus [46], and cinnamon oil [47] have shown their effective ability to remove biofilm. Moreover, *R. o*ffi*cinalis* EO has reduced the quantity of *S. aureus* biofilm to 60.76% [48]. In general, EOs diffuse through polysaccharide matrix of the preformed biofilm and destabilize it because of higher intrinsic antimicrobial activities [44].

#### **4. Materials and Methods**

#### *4.1. Bacterial Strains*

Thirty clinical MRSA isolates were collected from King Abdulaziz Specialist Hospital, Taif, Saudi Arabia. The isolates were identified as *S. aureus*, as described previously [49]. The methicillin resistance phenotype was performed by the Vitek 2 system (bioMérieux, Durham, North CA, USA) in accordance with the British Society for Antimicrobial Chemotherapy (BSAC). Each isolate was considered as methicillin-resistant when the minimum inhibitory concentration (MIC) breakpoint of oxacillin was >2 mg/L and cefoxitin >4 mg/L. [50]. *S. aureus* ATCC 25922 was used as control.

#### *4.2. Medicinal Plants Essential Oils*

Three commercial EOs extracted from medicinal plants were investigated. These EOs were bought from Laboratoires OMEGA Pharma (Groupe Perrigo)—Phytosun Arôms (Châtillon, France) and kept at 4 ◦C in dark glass bottles till used. These oils were extracted from twigs of *R. o*ffi*cinalis* L. (M14302), and from the aerial parts of flowering stage of *T. zygis* L. subsp. *zygis* (M13184) and *O. majorana* L. (74K100C6). These EOs were carefully chosen for their antibacterial and/or antibiofilm actions, as stated previously [11–13] and their usage in traditional medicine.

#### *4.3. Gas Chromatography—Mass Spectrometry Analysis*

The GC-MS analysis was performed as described previously [51].

#### *4.4. Antibacterial Activity of Essential Oils*

#### 4.4.1. Disc Diffusion

The agar disc diffusion method was used to evaluate the antibacterial activities of the EOs [52]. Briefly, an overnight cultures of MRSA cells grown at 37 ◦C were diluted to a density of 0.5 McFarland standards turbidity (DENSIMAT, Bio-merieux, Marcy l'Etoile, France) and were streaked onto Mueller–Hinton agar (MHA) plates using a sterile swab. A sterile filter disc (diameter 6 mm) was placed and then was impregnated by *R. o*ffi*cinalis*, *T. zygis*, and *O. majorana* EOs (10 µL /disc). The plates were maintained at 4 ◦C for 2 h and then incubated at 37 ◦C for 24 h. After incubation, the antibacterial activity was evaluated by determining the zone of growth inhibition throughout the discs.

Inhibitory action was categorized according to the zone of inhibition (ZI) as described previously [13–53]. The experiment was performed in triplicate, and the mean diameter of the inhibition zone was documented.

#### 4.4.2. Minimum Inhibitory Concentration and Minimum Bactericidal Concentration

The minimal inhibition concentration (MIC) and the minimal bactericidal concentration (MBC) were assessed in triplicates on 96-well microtiter plates (Nunc, Roskilde, Denmark) as described previously [54]. A bacterial suspension at a density of 0.5 McFarland standards turbidity was prepared from an overnight culture. Then, a serial two-fold dilution for each EOs (50 mg/mL stock solution) was made in 5 mL of nutrient broth with concentration ranged from 0.012–50 mg/mL.

Each well of the 96-well plates contains 95 µL of nutrient broth and 5 µL of the bacterial inoculum. A 100 µL aliquot from the stock solutions of each EO was added into the first well. Then, 100 µL from the serial dilutions were transferred into the consecutive well. The negative control well contains 195 µL of nutrient broth without EO and 5 µL of the bacterial inoculum. The final volume in each well was 200 µL, and the plates were incubated at 37 ◦C for 18–24 h.

The MIC was defined as the lowest concentration of the EO at which the MRSA cells growth is inhibited. The MBC was determined by subculturing 20 µL from clear wells of the MIC test on MHA. MBC was defined as the lowest concentration of EOs, required to kill ≥99.9% of the initial bacterial inoculum [55].

#### *4.5. Biofilm Formation*

Biofilm formation by MRSA isolates was determined using crystal violet assay on U-bottomed 96-well microtiter plates, as detailed previously [56]. Each MRSA strain was tested three times. Wells with sterile TSB only were worked as controls. The optical density of each well was measured at 570 nm (OD570) using an automated Multiskan reader (GIO. DE VITA E C, Rome, Italy). Biofilm formation was interpreted as highly positive (OD570 ≥ 1), low-grade positive (0.1 ≤ OD570 < 1), or negative (OD570 < 0.1).

#### *4.6. Biofilm Inhibition*

EOs were tested for their potential to prevent biofilm formation by MRSA isolates. For the experiment, 100 µL of the EOs emulsified in TSB supplement with 2% glucose were put in the U-bottomed 96-well microtiter plate, including 100 µL of bacterial suspensions (10<sup>8</sup> CFU/mL) in each well. The final concentrations of the EOs were equal to MIC, and the final volume was 200 µL per well. The analyzes were performed three times. After incubation of microplates at 37 ◦C for 24 h, the formed biofilm was measured by crystal violet as described previously [56]. For the Controls wells, the inoculums volume and EOs were replaced by TSB and sterile water, respectively. Inhibition of biofilm was determined from the formula described by Jadhav et al. [57].

$$\% \text{ Inhibition} = 100 - \left(\frac{\text{OD570 sample}}{\text{OD570 control}} \times 100\right)$$

#### *4.7. Biofilm Eradication*

In order to eradicate the preformed biofilm at the maturation stage (48 h biofilms), the plates were incubated for 48 h, the medium was changed after 24 h, and EOs were added at the same concentrations and at the last 24 h. Biofilms formed by bacteria that did not undergo any treatment were used as controls. Experimentally, the plates were incubated for 24 h at 37 ◦C to allow for biofilm attachment and growth. The following day, the non-adhered cells were removed from each well, and the adhered biofilm was rinsed two times with PBS. Then, 200 µL of TSB (2% glucose) with final concentrations of the EOs equivalent to MIC was added, and the plates were incubated for 24 h [44]. The biofilm was assessed by crystal violet, and eradication of biofilm was calculated as described in Section 4.6. The experiment was carried out in triplicate.

#### *4.8. Statistical Analysis*

Statistical analysis was performed using analysis of variance (ANOVA). Pearson's simple linear correlation coefficient (r) and their significance (*p*) were assessed using IBM SPSS (v20).

#### **5. Conclusions**

The outcomes of this study support the medical application of *O. majorana*, *T. zygis*, and *R. o*ffi*cinalis* EOs for the prevention and/or treatment of MRSA infections and diseases as an alternative to or combined with antibiotics. These EOs, provided from Laboratoires OMEGA Pharma–Phytosun Arôms (Châtillon, France), are used orally and in high concentrations (doses), corroborating their non-toxic effect. Generally, the therapeutic application of EOs is limited by their solubility, skin-sensitization synonymous allergic contact dermatitis, and their physicochemical stability due to the volatile components and the conversion of components by cyclization, isomerization, oxidation, or dehydrogenation reactions. Further adequate in vitro testing or in vivo preclinical experiments are warranted to establish safety, efficacy, potential adverse effects, and interaction with other drugs of *O. majorana*, *T. zygis*, and *R. o*ffi*cinalis* EOs before including them in clinical practice.

**Author Contributions:** F.B.A. conceived and designed the experiments; F.B.A. and R.L. performed the experiments; A.G. funding acquisition; F.B.A. writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors appreciated Taif University Researchers Supporting Project number (TURSP-2020/39), Taif University, Taif, Saudi Arabia.

**Acknowledgments:** The authors are thankful to Taif University for supplying essential facilities and acknowledge the support of Taif University Researchers Supporting Project number (TURSP-2020/39), Taif University, Taif, Saudi Arabia.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### **Bromo-Cyclobutenaminones as New Covalent UDP-***N***-Acetylglucosamine Enolpyruvyl Transferase (MurA) Inhibitors**

**David J. Hamilton 1,2, Péter Ábrányi-Balogh <sup>2</sup> , Aaron Keeley <sup>2</sup> , László Petri <sup>2</sup> , Martina Hrast <sup>3</sup> , Tímea Imre <sup>4</sup> , Maikel Wijtmans <sup>1</sup> , Stanislav Gobec <sup>3</sup> , Iwan J. P. de Esch <sup>1</sup> and György Miklós Keser ˝u 2,\***


Received: 8 October 2020; Accepted: 30 October 2020; Published: 3 November 2020

**Abstract:** Drug discovery programs against the antibacterial target UDP-*N*-acetylglucosamine enolpyruvyl transferase (MurA) have already resulted in covalent inhibitors having small threeand five-membered heterocyclic rings. In the current study, the reactivity of four-membered rings was carefully modulated to obtain a novel family of covalent MurA inhibitors. Screening a small library of cyclobutenone derivatives led to the identification of bromo-cyclobutenaminones as new electrophilic warheads. The electrophilic reactivity and cysteine specificity have been determined in a glutathione (GSH) and an oligopeptide assay, respectively. Investigating the structure-activity relationship for MurA suggests a crucial role for the bromine atom in the ligand. In addition, MS/MS experiments have proven the covalent labelling of MurA at Cys115 and the observed loss of the bromine atom suggests a net nucleophilic substitution as the covalent reaction. This new set of compounds might be considered as a viable chemical starting point for the discovery of new MurA inhibitors.

**Keywords:** covalent inhibitor; MurA; cyclobutenaminone; antibacterial; irreversible

#### **1. Introduction**

MurA (UDP-*N*-acetylglucosamine enolpyruvyl transferase) is a key enzyme in the peptidoglycan biosynthesis that catalyzes the transfer of phosphoenolpyruvate (PEP) to UDP-*N*-acetylglucosamine (UNAG) [1]. Targeting the catalytic site of MurA leads to the inactivation of the enzyme that increases the osmotic vulnerability of bacteria [2]. MurA is a preferred antibacterial target, as there is no human orthologue for the enzyme. Known MurA inhibitors contain a three- (**1**,**3**), five-(**2**,**4**–**9**) or occasionally six-membered (**10**) heterocycle equipped with a halogen atom leaving group (**6**,**10**), or an epoxide ring (**1**,**3**) that are prone to nucleophilic substitution. Other inhibitors contain a double bond (**2**,**4**,**5**,**7**–**9**) that is available for Michael addition (Figure 1) [3–6].

Our attention was drawn to the cyclobutenone scaffold, as it also harbors a ring with electrophilic character. Cyclobutenones have received relatively little attention in the literature [7–12], and cyclobutyl compounds, in general, are underrepresented in most (fragment) screening libraries [13]. The ring strain of the cyclobutenone unit suggests a substantial reactivity as an electrophile [10,12,14]. Given the foreseen use as covalent fragments, the reactivity and stability in biological assays need to be balanced. Therefore, the electrophilic reactivity was carefully modulated by incorporating an amine functionality in the electrophilic core, giving cyclobutenaminones. As an additional advantage, appending the amine group to the core provides further chemical handles for growing any hit fragments. Last, cyclobutenaminones contain a double bond enabling the incorporation of, e.g., halogen atoms, that can target nucleophilic amino acid side chains, especially that of cysteines.

**Figure 1.** Known MurA inhibitors with small heterocyclic scaffolds.

In continuation of our interests in finding new MurA inhibitors and in the use of the cyclobutyl motif in drug discovery [6,15–17], here we describe cyclobutenaminone derivatives with carefully-modulated electrophilic character as new warheads for covalently targeting the Cys115 residue in the active site of MurA.

#### **2. Results and Discussion**

‐ ‐ The synthesis of a small set of cyclobutenaminones was accomplished using a strategy based on that from Brand et al. (Scheme 1) [18]. The sequence began with ethoxyacetylene (**11**), which underwent a [2+2] cycloaddition [18–21] with the in situ generated ketene formed via the base-mediated HCl elimination from isobutyryl chloride. The two methyl groups were incorporated so as to restrict the nucleophilic character of enaminones (**14a**–**e**) to but one position in, e.g., the electrophilic bromination. The sequence furnished ethoxyenone (**12**) in moderate yield, which was transformed by acidic hydrolysis to 2,2-dimethylcyclobutane-1,3-dione (**13**) in high yield [18–21]. Next, dione **13** was condensed with various amines in the presence of AcOH as catalyst at 65 ◦C to generate the desired enaminones (**14a**–**e**) in moderate yields [18,22]. Bromination of all enaminones was achieved via electrophilic substitution using Br<sup>2</sup> and base at 0 ◦C to produce bromoenaminones **15a**–**e** [18,22]. Selected compounds were subjected to *N*-acylation via deprotonation of the secondary enaminone in tetrahydrofurane (THF) at –78 ◦C by sodium bis(trimethylsilyl)amide (NaHDMS), followed by subsequent trapping by the relevant acid chloride. Conceivably, the acylation of **14a** to **16a** could also take place at the nucleophilic vinylic position. However, the correct regiochemistry was proven by 2D NMR experiments. The acylations resulted in the corresponding *N*-acyl-cyclobutenaminones (**16a** and **17a**–**b**) in good yields.

‐

‐ ‐

‐ **Scheme 1.** General synthesis route to various (bromo)enaminones and subsequent *N*-acylation of secondary enaminones. Table 1 shows the different substituents introduced (R1, R<sup>2</sup> ), while R<sup>3</sup> = Me or Ph.

**Table 1.** Structures and biological activity of synthesized compounds.


‐ ‐ ‐ <sup>a</sup> RA% refers to the remaining activity in the MurA (*E. coli*) biochemical assay with a fragment concentration of 500 µM with 30 min preincubation at 37 ◦C; <sup>b</sup> PMB: 4-methoxy-benzyl.

‐ ‐

‐

‐

α β α β

−

μ ‐ ‐

A library of thirteen fragments was prepared, containing nonbrominated (six) and brominated (seven) cyclobutenaminones, all of which are novel to the best of our knowledge (Table 1). This library was then screened against MurA from *E. coli* in order to identify possible starting points for the future development of covalent MurA inhibitors. The screening showed that the cyclobutenaminones with a vinylic proton (**14a**–**d**) do not give any substantial inhibition. Indeed, the amine substituent selected for balancing reactivity and stability (vide supra) will likely deactivate the double bond by its electron-donating character. We postulated that *N*-trifluoroethylation or *N*-acylation of the nitrogen atom might reactivate the system towards nucleophiles by withdrawing electron density from the conjugated system, but the results on **14e** and **16a** did not support this postulate. As an intermediate in the synthetic route, ethoxycyclobutenone (**12**) was also tested as the ethoxy unit could serve as an improved leaving group, but to no avail. Next, bromination of the vinylic position was explored for activation, bearing in mind that this modification has been successfully applied already in Diels-Alder reactions for improving reactivity [23]. Gratifyingly, several brominated cyclobutenaminones (**15d**, **17a**–**b**) inhibit MurA from *E. coli* at the 500 µM screening concentration (RA < 15%). Compounds containing amines alkylated with small substituents (**15a**–**c**) do not show any affinity to the protein, but the incorporation of the 4-methoxybenzyl group (**15d**) increases the affinity to IC<sup>50</sup> = 363 µM. Turning attention to electron withdrawing groups once again, it was found that *N*-trifluoroethylation has no effect (**15e**), but the *N*-acetyl- and *N*-benzoyl-methylamino derivatives (**17a** and **17b**) substantially inhibit MurA activity. The time-dependent IC<sup>50</sup> values of these compounds after 30 min are 138 µM and 128 µM, respectively—a substantial effect for such small fragments, with the latter possessing only thirteen heavy atoms. The time dependency of the IC<sup>50</sup> values (see Supplementary Table S1) and the enhanced electrophilicity caused by the electron-withdrawing substituents suggest a covalent mechanism of action, which was confirmed by proving the labeling on Cys115 by MS/MS measurements for both compounds (Scheme 2E,F, Supplementary Figure S1). The MIC (Minimal Inhibitory Concentrations) values for the antimicrobial action of all compounds were determined against *S. aureus* (ATCC 29213) and *E. coli* (ATCC 25922) bacterial strains. These values were > 625 µM, implying that although MurA inhibition is clearly related to antibiotic action, more finetuning on these structures is needed on the path to a potential new class of antibiotics.

In order to characterize this new electrophilic chemotype, the cysteine reactivity of compound **17a** was evaluated in a GSH (glutathione)-based cysteine surrogate assay (Scheme 2A,B) [15]. The reaction of **17a** with GSH gives an adduct in the HPLC-MS-based assay (M + H<sup>+</sup> = 535 Da], suggesting loss of the Br atom, and the conjugation reaction could be characterized with a rate constant of kGSH = 0.0128 (M min)−<sup>1</sup> . Next, the selectivity of **17a** was explored using a nonapeptide assay (Scheme 2C,D) [15]. The KGDYHFPIC nonapeptide contains a cysteine but also other nucleophilic residues i.e., lysine, tyrosine, aspartate, proline, and histidine. As such, the nonapeptide can help to assess the selectivity between different biologically-relevant nucleophiles. In the case of **17a**, only the thiol group of the oligopeptide reacts with the warhead, indicating a high degree of cysteine specificity. To evaluate if the warhead is not too reactive for standard assay conditions, the aqueous stability of the compound (**17a**) was also investigated in PBS buffer (pH 7.4) [15]. The stability proves to be appropriate for biological investigations, as the t1/<sup>2</sup> value for the aqueous degradation was determined to be 36.5 h at room temperature. The stability and bioavailability of these structures is also supported by the fact that interestingly, the rather unique bromocyclobutenaminone core has been incorporated both in the α4β1/α4β7 integrin antagonist prodrug, Zaurategrast [24,25], which progressed to phase II clinical trials [26], as well as in related compounds [22].

‐ ‐ ‐ ‐ ‐ **Scheme 2.** Labelling of (**A**) glutathione (GSH), (**C**) KGDHFPIC nonapeptide and (**E**) MurA with fragment **17a** together with (**B**) the measured consumption of the fragment (blue columns) and the increasing amount of the GSH-adduct (orange columns) in the GSH-assay, (**D**) the MS/MS spectrum of the Cys-labelled nonapeptide indicating the Cys-labelling together with the theoretical and observed ion peaks and (**F**) the MS/MS spectrum of the digested MurA fragment (amino acids 104–120) labelled on Cys115 together with the theoretical and observed ion peaks. For **E** the 1UAE X-ray structure has been used [27].

#### **3. Materials and Methods**

#### *3.1. Synthesis and Characterisation of Compounds*

‐

‐ ‐ ‐ All starting materials were obtained from commercial suppliers (primarily being Sigma-Aldrich (Swijndrecht, The Netherlands), Fluorochem (Hadfield, Derbyshire, UK) and CombiBlocks (San Diego, CA, USA)) and used without purification. Anhydrous Et2O, dichloromethane (DCM), acetonitrile (MeCN) and tetrahydrofurane (THF) were obtained by passing through an activated alumina column prior to use. All other solvents used were used as received unless otherwise stated. All reactions were carried out under a nitrogen atmosphere unless mentioned otherwise. TLC analyses were performed using Merck F<sup>254</sup> (Merck KGaA, Darmstadt, Germany or VWR International B.V., Amsterdam, The Netherlands) aluminum-backed silica gel plates and visualized with 254 nm UV light or a potassium permanganate stain. Flash column chromatography was executed using Silicycle Siliaflash F<sup>60</sup> silica gel (SiliCycle Inc., Quebec City, QC, Canada or Screening Devices, Amersfoort, The Netherlands) or by means of a Teledyne Isco CombiFlash (Teledyne Isco Inc., Lincoln, NE, USA or Beun de Ronde, Abcoude, The Netherlands) or a Biotage Isolera equipment using Biotage SNAP columns (Biotage AB, Uppsala, Sweden). All HRMS spectra were recorded on a Bruker micrOTOF mass spectrometer (Bruker Corp., Billerica, MA, USA) using ESI in positive-ion mode. All NMR spectra were recorded on either a Bruker Avance 300, Bruker Avance 500, or Bruker Avance 600 spectrometer (Bruker Corp., Billerica, MA, USA or Fällanden, Switzerland). The peak multiplicities are defined as follows: s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quartet; p, pentet; dd, doublet of doublets; dt, doublet of triplets; td, a triplet of doublets; m, multiplet; app, apparent. The spectra were referenced to the internal solvent peak as follows: CDCl<sup>3</sup> ( <sup>1</sup>H = 7.26 ppm, <sup>13</sup>C = 77.16 ppm), DMSO-d6 (1H = 2.50 ppm, <sup>13</sup>C = 39.52 ppm). IUPAC names were adapted from ChemDraw Professional 16.0 (PerkinElmer). Purities were measured

with the aid of analytical LC−MS using a Shimadzu LC-20AD liquid chromatography pump system (Shimadzu Corp., Kyoto, Japan or 's Hertogenbosch, The Netherlands) with a Shimadzu SPDM20A diode array detector (Shimadzu Corp., Kyoto, Japan) with the MS detection performed with a Shimadzu LC-MS-2010EV mass spectrometer (Shimadzu Corp., Kyoto, Japan) operating in both positive and negative ionization mode. The column used was an XBridge (C18) 5 µm column (50 mm × 4.6 mm) (Waters Corp., Milford, MA, USA or Phenomenex, Utrecht, The Netherlands). The following solutions are used for the eluents. Solvent A: H2O (+0.1% HCOOH) and solvent B: MeCN (+0.1% HCOOH). The eluent program used is as follows: flow rate: 1.0 mL/min, start 95% A in a linear gradient to 10% A over 4.5 min, hold 1.5 min at 10% A, in 0.5 min in a linear gradient to 95% A, hold 1.5 min at 95% A, total run time: 8.0 min. Compound purities were calculated as the percentage peak area of the analysed compound by UV detection at 254 nm.

#### *3.2. GSH Reactivity and Aqueous Stability Assay*

The assay was adapted from our former publication [15].

For the glutathione assay, 500 µM solution of the fragment (PBS buffer pH 7.4, 10% MeCN, 250 µL) with 200 µM solution of indoprofen (Merck KGaA, Darmstadt, Germany) as internal standard was added to 10 mM glutathione (Merck KGaA, Darmstadt, Germany) solution (dissolved in PBS buffer, 250 µL) in a 1:1 ratio. The final concentration was 250 µM fragment, 100 µM indoprofen, 5 mM glutathione and 5% MeCN (500 µL). The final mixture was analyzed by HPLC-MS (Shimadzu LCMS-2020) after 0, 1, 2, 4, 20, 25, 48, 72 h time intervals. Degradation kinetics were also investigated respectively using the previously described method, applying pure PBS buffer instead of the glutathione solution. In this experiment, the final concentration of the mixture was 250 µM fragment, 100 µM indoprofen and 5% MeCN. The AUC (area under the curve) values were determined via integration of HPLC spectra then corrected using the internal standard. The fragment AUC values were applied for ordinary least squares (OLS) linear regression and for computing the important parameters (kinetic rate constant, half-life time) a programmed excel (Visual Basic for Applications) was utilized. The data are expressed as means of duplicate determinations, and the standard deviations were within 10% of the given values.

The calculation of the kinetic rate constant for the degradation and corrected GSH-reactivity is as follows:

The reaction half-life for pseudo-first-order reactions is t1/<sup>2</sup> = ln2/k, where k is the reaction rate. In the case of competing reactions (reaction with GSH and degradation), the effective rate for the consumption of the starting compound is keff = kdeg + kGSH. When measuring half-lives experimentally, the t1/2(eff) = ln2/(keff) = ln2/(kdeg + kGSH). In our case, the corrected kdeg and keff (regarding blank and GSH-containing samples, respectively) can be calculated by linear regression of the data points of the kinetic measurements. The corrected kGSH is calculated by keff − kdeg, and finally, the half-life time is determined using the equation t1/2(GSH) = ln2/kGSH.

#### *3.3. Oligopeptide Selectivity Assay*

The assay was adapted from our former publication [15].

For the nonapeptide assay, a 2 mM solution of the fragment (PBS buffer pH 7.4 with 20% MeCN) was added to 200 µM nonapeptide solution (PBS buffer pH 7.4) in a 1:1 ratio. The final assay mixture contained 1 mM fragment, 100 µM peptide and 10% MeCN. Based on the GSH reactivity, the applied incubation time was 24 h.

#### *3.4. LC-MS*/*MS Measurement and Data Analysis of the Nonapeptide Reactivity Assay*

A Sciex 6500 QTRAP triple quadrupole—linear ion trap mass spectrometer, equipped with a Turbo V Source in electrospray mode (AB Sciex Pte. Ltd., Framingham, MA, USA) and an Agilent 1100 Binary Pump HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with an autosampler was used for LC-MS/MS analysis. Data acquisition and processing were performed using Analyst software

version 1.6.2 (AB Sciex Pte. Ltd., Framingham, MA, USA). Chromatographic separation was achieved by Purospher STAR RP-18 endcapped (50 mm × 2.1 mm, 3µm) LiChocart® 55-2 HPLC Cartridge (Merck KGaA, Darmstadt, Germany). The sample was eluted with gradient elution using solvent A (0.1% HCOOH in water) and solvent B (0.1% HCOOH in MeCN). Flow rate was set to 0.5 mL/min. The initial condition was 5% B for 2 min, followed by a linear gradient to 95% B for 6 min, followed by holding at 95% B 6–8 min; and from 8 to 8.5 min back to the initial condition with 5% eluent B and held for 14.5 min. The column temperature was kept at room temperature and the injection volume was 10 µL. Nitrogen was used as the nebulizer gas (GS1), heater gas (GS2), and curtain gas with the optimum values set at 35, 45 and 45 (arbitrary units). The source temperature was 450 ◦C and the ion spray voltage set at 5000 V. The declustering potential value was set to 150 V. Information Dependent Acquisition (IDA) LC-MS/MS experiment was used to determine if the fragment binding was specific to thiol residues or not. An enhanced MS scan was applied as the survey scan and enhanced product ion (EPI) was the dependent scan. The collision energy in EPI experiments was set to 30 eV with a collision energy spread (CES) of 10 V. The identification of the binding position of the fragments to the nonapeptide was performed using GPMAW 4.2. software.

#### *3.5. Tryptic Digestion of MurA*

The tryptic digestion method was adapted from our former publication [15].

Briefly, 50 µL of MurA (42 µM) and 10 µL 0.2% (*w*/*v*) RapiGest SF (Waters Corp., Milford, MA, USA) solution buffered with 50 mM ammonium bicarbonate (NH4HCO3) were mixed (pH = 7.8). 4.5 µL of 45 mM DTT (~200 nmol) in 100 mM NH4HCO<sup>3</sup> was added and the mixture kept at 37.5 ◦C for 30 min. After cooling the sample to room temperature, 7.5 µL of 100 mM iodoacetamide (750 nmol) in 100 mM NH4HCO<sup>3</sup> was added and the mixture placed in the dark at room temperature for 30 min. The reduced and alkylated protein was then digested by 10 µL (1 mg mL−<sup>1</sup> ) trypsin (the enzyme-to-protein ratio was 1:10) (Sigma, St Louis, MO, USA). The sample was incubated at 37 ◦C overnight. To degrade the surfactant, 7 µL of HCOOH (500 mM) solution was added to the digested HDAC8 sample to obtain the final 40 mM (pH ≈ 2) solution which was incubated at 37 ◦C for 45 min. For LC-MS analysis, the acid-treated sample was centrifuged for 5 min at 13,000 rpm.

#### *3.6. LC-MS*/*MS Measurements on Digested MurA*

A QTRAP 6500 triple quadruple—linear ion trap mass spectrometer, equipped with a Turbo V source in electrospray mode (AB Sciex Pte. Ltd., Framingham, MA, USA) and an Agilent 1100 Binary Pump HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with an autosampler was used for LC-MS/MS analysis. Data acquisition and processing were performed using Analyst software version 1.6.2 (AB Sciex Pte. Ltd., Framingham, MA, USA). Chromatographic separation was achieved by using the Discovery® BIO Wide Pore C-18-5 (250 mm × 2.1 mm, 5 µm). The sample was eluted with a gradient of solvent A (0.1% HCOOH in water) and solvent B (0.1% HCOOH in MeCN). The flow rate was set to 0.2 mL min−<sup>1</sup> . The initial conditions for separation were 5% B for 7 min, followed by a linear gradient to 90% B for 53 min, followed by 90% B for 3 min; over 2 min back to the initial conditions with 5% eluent B retained for 10 min. The injection volume was 10 µL (300 pmol on the column).

An Information-Dependent Acquisiton (IDA) LC-MS/MS experiment was used to identify the modified tryptic MurA peptide fragments. Enhanced MS scan (EMS) was applied as the survey scan and an enhanced product ion (EPI) was the dependent scan. The collision energy in EPI experiments was set to rolling collision energy mode, where the actual value was set on the basis of the mass and charge state of the selected ion. Further IDA criteria: ions greater than: 400.00 *m*/*z*, which exceeds 106 counts, exclude former target ions for 30 s after 2 occurrence(s). In EMS and in EPI mode, the scan rate was 1000 Da/s as well. Nitrogen was used as the nebulizer gas (GS1), heater gas (GS2), and curtain gas with the optimum values set at 50, 40 and 40 (arbitrary units). The source temperature was 350 ◦C and the ion spray voltage was set at 5000 V. The declustering potential value was set to 150 V. GPMAW 4.2. software was used to analyse a large number of MS-MS spectra and identify the modified tryptic MurA peptides.

#### *3.7. MurA Assay*

MurAEC protein was recombinant, expressed in *E. coli.* [28] The inhibition of MurA was monitored with the colorimetric malachite green method in which orthophosphate generated during the reaction is measured. MurA enzyme (*E. coli*) was pre-incubated with the substrate UNAG and compound for 30 min at 37 ◦C. The reaction was started by the addition of the second substrate PEP, resulting in a mixture with a final volume of 50 µL. The mixtures contained: 50 mM Hepes, pH 7.8, 0.005% Triton X-114, 200 µM UNAG, 100 µM PEP, purified MurA (diluted in 50 mM Hepes, pH 7.8) and 500 µM of each tested compound dissolved in DMSO. All compounds were soluble in the assay mixtures containing 5% DMSO (*v*/*v*). After incubation for 15 min at 37 ◦C, the enzyme reaction was terminated by adding Biomol® reagent (100µL) and the absorbance was measured at 650 nm after 5 min. All of the experiments were run in duplicate. Remaining activities (RAs) were calculated with respect to similar assays without the tested compounds and with 5% DMSO. The IC<sup>50</sup> values, the concentration of the compound at which the remaining activity was 50%, were determined by measuring the remaining activities at seven different compound concentrations. The data are expressed as means of duplicate determinations, and the standard deviations were within 10% of the given values. A time-dependent inhibition assay was also performed. The IC<sup>50</sup> values were determined at 0, 15 and 30 min of pre-incubation.

#### *3.8. Antimicrobial Testing (MIC Determination)*

Antimicrobial testing was carried out by the broth microdilution method in 96-well plate format following the CLSI guidelines and European Committee for Antimicrobial Susceptibility Testing recommendations. Bacterial suspension of specific bacterial strain, equivalent to 0.5 McFarland turbidity standard, was diluted with cation-adjusted Mueller Hinton broth to obtain a final inoculum of 10<sup>5</sup> CFU/mL. Compounds dissolved in DMSO and inoculum were mixed together and incubated for 20–24 h at 37 ◦C. After incubation the minimal inhibitory concentration (MIC) values were determined by visual inspection as the lowest dilution of compounds showing no turbidity. The MICs were determined against *S. aureus* (ATCC 29213) and *E. coli* (ATCC 25922) bacterial strains. Tetracycline was used as a positive control on every assay plate, showing a MIC of 0.5 µg/mL and 1 µg/mL for *S. aureus* and *E. coli*, respectively.

#### *3.9. Chemical Syntheses*

#### 3-Ethoxy-4,4-dimethylcyclobut-2-en-1-one (**12**)

To a stirred solution of isobutyryl chloride (10.7 mL, 102 mmol) and ethoxyacetylene **11** (50.0 mL, 205 mmol, 40% wt in hexanes) in Et2O (128 mL), Et3N (21.4 mL, 154 mmol) was added slowly over 5 min. The mixture was stirred at rt for 30 min before being heated at 40 ◦C for 24 h. The mixture was then allowed to cool. The precipitate was filtered and the filtrate was concentrated in vacuo. The crude product was purified over silica gel with a gradient of 10–40% EtOAc/cHex to afford the title compound **2** (8.90 g, 62% yield) as a yellow oil.

<sup>1</sup>H NMR (500 MHz, Chloroform-*d*) δ 4.78 (s, 1H), 4.21 (q, *J* = 7.1 Hz, 2H), 1.45 (t, *J* = 7.1 Hz, 3H), 1.24 (s, 6H). <sup>13</sup>C NMR (126 MHz, Chloroform-*d*) δ 194.1, 190.4, 102.4, 69.4, 60.1, 19.7, 14.3. LC-MS: RT = 3.33 min, 99+% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 141. HRMS calculated for C8H13O<sup>2</sup> <sup>+</sup> [M + H]<sup>+</sup> = 141.0910, found 141.0923.

#### 2,2-Dimethylcyclobutane-1,3-dione (**13**)

To a flask containing enol ether **12** (8.40 g, 59.9 mmol) was added HCl (2.0 M in H2O, 45.0 mL, 90.0 mmol) in one portion and the mixture was stirred vigorously at rt for 24 h. The product was

extracted with DCM (3×). The organic layers were combined, dried over MgSO4, and concentrated in vacuo to afford the title compound **3** (6.20 g, 92% yield) as a flaky brown solid.

<sup>1</sup>H NMR (600 MHz, Chloroform-*d*) δ 3.92 (s, 2H), 1.28 (s, 6H). <sup>13</sup>C NMR (151 MHz, Chloroform-*d*) δ 207.0, 73.0, 60.4, 17.6. LC-MS: RT = 1.78 min, 98% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 113. HRMS calculated for C6H9O<sup>2</sup> <sup>+</sup> [M + H]<sup>+</sup> = 113.0597, found 113.0603.

#### *3.10. General Procedure A: Enaminone Formation*

To a solution of dione **13** (1.0 eq) in THF (0.50 M) was added amine (1.1 eq), AcOH (1.1 eq) and a spatula of Na2SO4. The reaction mixture was stirred at 65 ◦C for the indicated time. The reaction mixture was allowed to cool to rt. The solids were filtered and the filtrate concentrated in vacuo. The residue was taken up in EtOAc and washed with satd. aq. Na2CO<sup>3</sup> and brine. The organic layer was dried over Na2SO4, filtered and concentrated *in vacuo*. The crude product was purified over silica gel using the indicated gradient of MeOH/DCM to afford the product enaminone.

#### 3-(Methylamino)-4,4-dimethylcyclobut-2-en-1-one (**14a**)

This compound was prepared according to General Procedure **A** using dione **13** (388 mg, 3.46 mmol), MeNH<sup>2</sup> (2.0 M in THF, 1.90 mL, 3.81 mmol) and a reaction time of 40 h. Purification over silica gel using a gradient of 0–10% MeOH/DCM afforded the title compound **14a** (350 mg, 81%) as a pale brown solid.

Rotamers are observed in ratio ca. 1.0:0.1 in Chloroform-*d*. Only peaks corresponding to the major rotamer are reported. <sup>1</sup>H NMR (500 MHz, Chloroform-*d*) δ 5.63 (br s, 1H), 4.59 (s, 1H), 2.99 (d, *J* = 5.0 Hz, 3H), 1.24 (s, 6H). <sup>13</sup>C NMR (126 MHz, Chloroform-*d*) δ 192.2, 178.8, 95.7, 58.5, 31.8, 20.3. LC-MS: RT = 3.20 min, 99+% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 126. HRMS calculated for C7H12NO<sup>+</sup> [M + H]<sup>+</sup> = 126.0913, found 126.0912.

#### 3-(Dimethylamino)-4,4-dimethylcyclobut-2-en-1-one (**14b**)

This compound was prepared according to General Procedure **A** using dione **13** (200 mg, 1.78 mmol), Me2NH (2.0 M in THF, 0.20 mL, 1.96 mmol) and a reaction time of 40 h. Purification over silica gel using a gradient of 0–10% MeOH/DCM afforded the title compound **14b** (191 mg, 77% yield) as a brown crystalline solid.

Rotamers are observed in ratio 1.0:1.0 in Chloroform-*d*. All peaks for both rotamers are reported. <sup>1</sup>H NMR (500 MHz, Chloroform-*d*) δ 4.53 (s, 1H), 3.07 (s, 3H), 2.99 (s, 3H), 1.32 (s, 6H). <sup>13</sup>C NMR (126 MHz, Chloroform-*d*) δ 190.9, 178.4, 96.0, 58.2, 40.1, 39.4, 21.2. LC-MS: RT = 2.34 min, 99 + % (254 nm), *m*/*z* [M + H]<sup>+</sup> = 140. HRMS calculated for C8H14NO<sup>+</sup> [M + H]<sup>+</sup> = 140.1064, found 140.1066.

3-(Diethylamino)-4,4-dimethylcyclobut-2-en-1-one (**14c**)

This compound was prepared according to General Procedure **A** using dione **13** (200 mg, 1.78 mmol), Et2NH (0.20 mL, 1.96 mmol) and a reaction time of 40 h, followed by an additional portion of Et2NH (0.10 mL, 0.89 mmol) and stirring for a further 5 h. Purification over silica gel using a gradient of 0–10% MeOH/DCM afforded the title compound **14c** (175 mg, 59% yield) as a brown oil.

Rotamers are observed in ratio 1.0:1.0 in Chloroform-*d*. All peaks for both rotamers are reported. <sup>1</sup>H NMR (500 MHz, Chloroform-*d*) δ 4.54 (s, 1H), 3.34 (q, *J* = 7.2 Hz, 2H), 3.27 (q, *J* = 7.2 Hz, 2H), 1.33 (s, 6H), 1.25 (t, *J* = 7.2 Hz, 3H), 1.22 (t, *J* = 7.2 Hz, 3H). <sup>13</sup>C NMR (126 MHz, Chloroform-*d*) δ 190.9, 177.5, 95.6, 58.4, 44.6, 44.1, 21.4, 14.2, 12.3. LC-MS: RT = 3.06 min, 99+% (254 nm), *m*/*z* [M+H]<sup>+</sup> = 168. HRMS calculated for C10H18NO<sup>+</sup> [M + H]<sup>+</sup> = 168.1377, found 168.1382.

3-((4-Methoxybenzyl)(methyl)amino)-4,4-dimethylcyclobut-2-en-1-one (**14d**)

This compound was prepared according to General Procedure **A** using dione 13 (200 mg, 1.78 mmol), (4-methoxybenzyl)-*N*-methylamine (0.29 mL, 1.96 mmol) and a reaction time of 40 h. Purification over silica gel using a gradient of 0–10% MeOH/DCM afforded the title compound **14d** (330 mg, 75% yield) as a viscous brown oil.

Rotamers are observed in ratio 1.0:1.0 in Chloroform-*d*. All peaks for both rotamers are reported. <sup>1</sup>H NMR (600 MHz, Chloroform-*d*) δ 7.18–7.12 (m, 2H), 6.93–6.88 (m, 2H), 4.70 (s, 1H), 4.59 (s, 1H), 4.42 (s, 2H), 4.29 (s, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 2.95 (s, 3H), 2.80 (s, 3H), 1.38 (s, 6H), 1.34 (s, 6H). <sup>13</sup>C NMR (151 MHz, Chloroform-*d*) δ 190.9, 190.8, 178.4, 178.3, 159.6, 129.1, 128.7, 126.9, 126.7, 114.5, 114.4, 96.2, 95.9, 58.4, 58.3, 56.2, 55.4, 55.4, 55.3, 36.6, 36.3, 21.4, 21.1. LC-MS: RT = 3.62 min, 99+% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 246. HRMS calculated for C15H20NO<sup>2</sup> <sup>+</sup> [M + H]<sup>+</sup> = 246.1489, found 246.1489.

#### 4,4-Dimethyl-3-(methyl(2,2,2-trifluoroethyl)amino)cyclobut-2-en-1-one (**14e**)

This compound was prepared according to General Procedure **A** using dione **13** (140 mg, 1.25 mmol), (2,2,2-trifluoroethyl)-methylamine (0.14 mL, 1.37 mmol) and a reaction time of 16 h. Purification over silica gel using a gradient of 0–10% MeOH/DCM afforded the title compound **14e** (197 mg, 76% yield) as a brown oil.

Rotamers are observed in ratio 1.0:0.8 in Chloroform-*d*. All peaks for both rotamers are reported. <sup>1</sup>H NMR (500 MHz, Chloroform-*d*) δ 4.67 (s, 1H), δ 4.70 (s, 1H), 3.80 (q, *J* = 8.6 Hz, 2H), 3.75 (q, *J* = 9.0 Hz, 2H), 3.20 (s, 3H), 3.09–3.10 (m, 3H), 1.35 (s, 6H), 1.32 (s, 6H).13C NMR (151 MHz, Chloroform-*d*) δ 190.63, 179.51, 179.47, 124.47 (q, *J* = 281.9 Hz), 123.70 (q, *J* = 281.9 Hz), 99.72, 99.03, 54.29 (q, *J* = 34.1 Hz), 53.51 (q, *J* = 34.1 Hz), 39.06, 21.19, 21.08. LC-MS: RT = 3.30 min, 99+% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 208. HRMS calculated for C9H13F3NO<sup>+</sup> [M + H]<sup>+</sup> = 208.0944, found 208.0947.

#### *3.11. General Procedure B: Bromination*

A solution of enaminone (1.0 eq) and Et3N (2.0 eq) in THF (0.10 M) at 0 ◦C was treated dropwise with a solution of Br<sup>2</sup> (1.1 eq) in THF (2.0 mL). The reaction mixture was stirred at 0 ◦C for the indicated time. The mixture was diluted with EtOAc and washed with satd. aq. NaHCO<sup>3</sup> and brine. The organic layer was dried over MgSO4, filtered and concentrated *in vacuo*. The crude product was purified over silica gel using the indicated gradient of MeOH/EtOAc followed by reversed-phase chromatography on C18 silica gel using the indicated gradient of MeCN/H2O to afford the product bromoenaminone.

#### 2-Bromo-4,4-dimethyl-3-(methylamino)cyclobut-2-en-1-one (**15a**)

This compound was prepared according to General Procedure B using enaminone **14a** (80 mg, 0.64 mmol) and a reaction time of 1 h. Purification over silica gel using a gradient of 0–10% MeOH/EtOAc and over reversed-phase C18 silica gel using a gradient of 0–100% MeCN/H2O (+0.1% HCOOH) afforded the title compound **15a** (81 mg, 62% yield) as a pale yellow solid.

Rotamers are observed in ratio 1.0:0.6 in Chloroform-*d*. All peaks for both rotamers are reported. <sup>1</sup>H NMR (600 MHz, Chloroform-*d*) δ 5.97 (br s, 1H), 5.55 (br s, 1H), 3.30 (d, *J* = 5.2 Hz, 3H), 3.12 (d,*J* = 5.2 Hz, 3H), 1.38 (s, 6H), 1.24 (s, 6H). <sup>13</sup>C NMR (151MHz, Chloroform-*d*) δ 187.8, 185.9, 177.6, 175.5, 72.8, 70.5, 59.1, 58.7, 31.6, 31.4, 20.8, 19.9. LC-MS: RT = 2.80 min, 99+% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 204 (light isotope). HRMS calculated for C7H11NOBr<sup>+</sup> [M + H]<sup>+</sup> = 205.9998 (heavy isotope), found 205.9997.

#### 2-Bromo-4,4-dimethyl-3-(methylamino)cyclobut-2-en-1-one (**15b**)

This compound was prepared according to General Procedure B using enaminone **14b** (80 mg, 0.58 mmol) and a reaction time of 1 h. Purification over silica gel using a gradient of 0–10% MeOH/EtOAc and over reversed-phase C18 silica gel using a gradient of 0–100% MeCN/H2O (+0.1% HCOOH) afforded the title compound **15b** (59 mg, 47% yield) as a pale yellow solid.

Rotamers are observed in ratio 1.0:1.0 in Chloroform-*d*. All peaks for both rotamers are reported. <sup>1</sup>H NMR (600 MHz, Chloroform-*d*) δ 3.35 (s, 3H), 3.07 (s, 3H), 1.32 (s, 6H). <sup>13</sup>C NMR (151 MHz, Chloroform-*d*) δ 186.5, 174.5, 70.3, 58.7, 40.7, 39.6, 20.9. LC-MS: RT = 3.06 min, 99+% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 218 (light isotope). HRMS calculated for C8H13NOBr<sup>+</sup> [M + H]<sup>+</sup> = 218.0175 (light isotope), found 218.0182.

#### 2-Bromo-3-(diethylamino)-4,4-dimethylcyclobut-2-en-1-one (**15c**)

This compound was prepared according to General Procedure B using enaminone **14c** (80 mg, 0.48 mmol) and a reaction time of 1 h. Purification over silica gel using a gradient of 0–10% MeOH/EtOAc and over reversed-phase C18 silica gel using a gradient of 0–100% MeCN/H2O (+0.1% HCOOH) afforded the title compound **15c** (49 mg, 42% yield) as a colourless oil.

Rotamers are observed in ratio 1.0:1.0 in Chloroform-*d*. All peaks for both rotamers are reported. <sup>1</sup>H NMR (600 MHz, Chloroform-*d*) δ 3.64 (q, *J* = 7.2 Hz, 1H), 3.35 (q, *J* = 7.2 Hz, 1H), 1.30 (t, *J* = 7.2 Hz, 3H), 1.28 (t, *J* = 7.2 Hz, 3H). <sup>13</sup>C NMR (151 MHz, Chloroform-*d*) δ 186.6, 173.9, 69.7, 58.8, 45.7, 43.0, 21.1, 14.4, 14.2. LC-MS: RT = 3.78 min, 97% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 248 (heavy isotope). HRMS calculated for C10H17NOBr<sup>+</sup> [M + H]<sup>+</sup> = 246.0488 (light isotope), found 246.0488.

2-Bromo-3-((4-methoxybenzyl)(methyl)amino)-4,4-dimethylcyclobut-2-en-1-one (**15d**)

This compound was prepared according to General Procedure B using enaminone **14d** (100 mg, 0.41 mmol) and a reaction time of 2 h. Purification over silica gel using a gradient of 0–10% MeOH/EtOAc and over reversed-phase C18 silica gel using a gradient of 0–100% MeCN/H2O (+0.1% HCOOH) afforded the title compound **15d** (51 mg, 39% yield) as a pale yellow oil.

Rotamers are observed in ratio 1:0.6 in Chloroform-*d*. All peaks for both rotamers are reported. <sup>1</sup>H NMR (600 MHz, Chloroform-*d*) δ 7.25–7.22 (m, 2H), 7.17–7.14 (m, 2H), 6.95–6.91 (m, 4H), 4.75 (s, 2H), 4.41 (s, 2H), 3.82 (s, 3H), 3.82 (s, 3H), 3.16 (s, 3H), 2.95 (s, 3H), 1.39 (s, 6H), 1.35 (s, 6H). <sup>13</sup>C NMR (151 MHz, Chloroform-*d*) δ 186.71, 186.68, 174.72, 174.28, 159.90, 159.82, 129.52, 128.88, 127.15, 125.90, 114.70, 114.60, 70.51, 70.45, 58.96, 58.93, 56.64, 55.50, 55.47, 54.65, 37.43, 36.27, 21.23, 20.94. LC-MS: RT = 4.24 min, 99+% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 324 (light isotope). HRMS calculated for C15H19NO2Br<sup>+</sup> [M + H]<sup>+</sup> = 326.0573 (heavy isotope), found 326.0573.

2-Bromo-4,4-dimethyl-3-(methyl(2,2,2-trifluoroethyl)amino)cyclobut-2-en-1-one (**15e**)

This compound was prepared according to General Procedure B using enaminone **14e** (80 mg, 0.39 mmol) and a reaction time of 1 h. Purification over silica gel using a gradient of 0–10% MeOH/EtOAc and over reversed-phase C18 silica gel using a gradient of 0–100% MeCN/H2O (+0.1% HCOOH) afforded the title compound **15e** (32% yield) as a pale yellow solid.

Rotamers are observed in ratio 1:0.4 in Chloroform-*d*. All peaks for both rotamers are reported. <sup>1</sup>H NMR (600 MHz, Chloroform-*d*) δ 4.27 (q, *J* = 8.3 Hz, 2H), 3.79 (q, *J* = 8.3 Hz, 2H), 3.46 (s, 3H), 3.22 (s, 3H), 1.37 (s, 6H), 1.34 (s, 6H). <sup>13</sup>C NMR (151 MHz, Chloroform-*d*) δ 186.51, 175.87, 175.66, 123.95 (q, *J* = 282.1 Hz), 123.51 (q, *J* = 282.1 Hz), 74.86, 74.20, 59.70, 59.61, 54.57 (q, *J* = 34.0 Hz), 52.41 (q, *J* = 34.0 Hz), 40.05, 39.13, 20.93, 20.84. LC-MS: RT = 3.86 min, 99+% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 286 (light isotope). HRMS calculated for C9H12NOF2Br<sup>+</sup> [M + H]<sup>+</sup> = 286.0049 (light isotope), found 286.0044.

#### *3.12. General Procedure C: Enaminone N-Acylation*

To a solution of enaminone (1.0 eq) in THF (0.025 M) at −78 ◦C was added NaN(SiMe3)<sup>2</sup> (1.0 M in THF, 1.5 eq) dropwise. The mixture was stirred at this temperature for 90 min before dropwise addition of the acid chloride (1.2 eq). The reaction mixture was stirred for a further 2 h at −78 ◦C. Brine was added slowly at this temperature whilst stirring vigorously and the mixture was allowed to warm to rt. The volatiles were removed in vacuo and the residue was partitioned between EtOAc and water. The organic layer was washed with brine, dried over Na2SO4, and concentrated *in vacuo*. The crude product was purified over silica gel to afford the product *N*-acyl-enaminone.

#### *N*-(4,4-dimethyl-3-oxocyclobut-1-en-1-yl)-*N*-methylbenzamide (**16a**)

This compound was prepared according to General Procedure C using enaminone **14a** (80 mg, 0.64 mmol) and BzCl (0.09 mL, 0.77 mmol). Purification over silica gel using a gradient of 20–80% EtOAc/cHex afforded the title compound **16a** (72% yield) as a white solid.

No significant rotamers are observed in NMR spectra. <sup>1</sup>H NMR (500 MHz, Chloroform-*d*) δ 7.56–7.52 (m, 3H), 7.49–7.45 (m, 2H), 4.83 (s, 1H), 3.35 (s, 3H), 1.41 (s, 6H). <sup>13</sup>C NMR (151 MHz, Chloroform-*d*) δ 194.2, 175.9, 171.1, 134.1, 132.0, 129.0, 127.9, 111.8, 62.7, 36.7, 21.4. LC-MS:RT = 3.85 min, 99% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 230. HRMS calculated for C14H16NO<sup>2</sup> <sup>+</sup> [M + H]<sup>+</sup> = 230.1176 found 230.1171.

The regiochemistry of the acylation (i.e., acylation of N atom and not of vinyl position) was proven by 2D NMR (HSQC + HMBC)—the singlet signal counting for 1H has an associated <sup>13</sup>C signal in HSQC and thus the vinyl position remains unsubstituted in the product.

*N*-(2-bromo-4,4-dimethyl-3-oxocyclobut-1-en-1-yl)-*N*-methylbenzamide (**17a**)

This compound was prepared according to General Procedure C using bromoenaminone **15a** (80 mg, 0.39 mmol) and BzCl (0.06 mL, 0.47 mmol). Purification over silica gel using a gradient of 20–70% EtOAc/cHex afforded the title compound **17a** (86 mg, 71% yield) as a white solid.

No significant rotamers are observed in NMR spectra. <sup>1</sup>H NMR (600 MHz, Chloroform-*d*) δ 7.62–7.54 (m, 3H), 7.53–7.46 (m, 2H), 3.61 (s, 3H), 1.43 (s, 6H). <sup>13</sup>C NMR (151 MHz, Chloroform-*d*) δ 191.4, 174.6, 170.1, 133.5, 132.5, 129.1, 128.5, 87.8, 63.5, 39.0, 21.2. LC-MS: RT = 4.55 min, 99+% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 308 (light isotope). HRMS calculated for C14H15BrNO<sup>2</sup> <sup>+</sup> [M + H]<sup>+</sup> = 310.0260 (heavy isotope), found 310.0254.

*N*-(2-bromo-4,4-dimethyl-3-oxocyclobut-1-en-1-yl)-*N*-methylacetamide (**17b**)

This compound was prepared according to General Procedure C using bromoenaminone **15a** (6 mg, 0.03 mmol) and AcCl (0.002 mL, 0.03 mmol). Extraction with DCM (5 mL) and water (2 × 1 mL) afforded the title compound **17b** (5.9 mg, 80% yield) as a white solid.

No significant rotamers are observed in NMR spectra. <sup>1</sup>H NMR (500 MHz, DMSO-*d6*) δ 3.19 (s, 3H), 1.90 (s, 3H), 1.11 (s, 6H). <sup>13</sup>C NMR (125 MHz, DMSO-*d6*) δ 186.3, 184.9, 175.3, 68.7, 40.9, 30.5, 20.8, 20.1. LC-MS: RT 1.53 min, 97% (254 nm), *m*/*z* [M + H]<sup>+</sup> = 246 (heavy isotope). HRMS calculated for deacetylated C7H11BrNO<sup>+</sup> [M-acetyl + H]<sup>+</sup> = 204.0018 (light isotope), found 204.0018.

#### **4. Conclusions**

A new electrophilic warhead chemotype, the bromocyclobutenaminone scaffold, was designed as a thiol-labelling agent. It was shown that the incorporation of the bromine atom in the cyclobutenaminone core, sometimes in conjunction with an electron withdrawing group on the nitrogen atom, turns the inactive fragments into novel and useful covalent probes. The investigation of a set of compounds against MurA protein from *E. coli* led to the identification of fragments with moderate inhibitory activity. These compounds represent promising starting points for hit optimization studies and fragment growing might lead to new and potent MurA inhibitors.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-8247/13/11/362/s1, Table S1. Results of time-dependent IC<sup>50</sup> measurements of **17b**; Figure S1. The MS/MS spectrum of **17b** modified MurA enzyme peptide [amino acids 104–120] together with the annotation of the peaks; NMR, LC-MS and HRMS data of key compound **17a**.

**Author Contributions:** Conceptualization, S.G., I.J.P.d.E. and G.M.K.; methodology, P.Á.-B., M.W.; investigation, D.J.H., A.K., L.P., M.H. and T.I.; writing—original draft preparation, P.Á.-B., D.J.H. and M.W., writing—review and editing, P.Á.-B., D.J.H., M.W., S.G., I.J.P.d.E. and G.M.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was funded by H2020 MSCA FragNet (project 675899), SNN 125496, OTKA PD124598 and 2018-2.1.11-TÉT-SI-2018-00005, and the Slovenian Research Agency core funding P1-0208.

**Acknowledgments:** We thank Hélène Barreteau for *E. coli* MurA plasmid and Hans Custers for HRMS measurements. The authors are grateful for Krisztina Németh and Pál Szabó for contributing to the analytical experiments.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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