Design and Synthesis of Malonamide Derivatives as Antibiotics against Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious threat to humans. Most existing antimicrobial drugs, including the β-lactam and quinoxiline classes, are not effective against MRSA. In this study, we synthesized 24 derivatives of malonamide, a new class of antibacterial agents and potentiators of classic antimicrobials. A derivative that increases bacterial killing and biofilm eradication with low cell toxicity was created.


Introduction
Antibiotic resistance is a crucial issue in human health, and the launch of new antimicrobial drugs has become a rare event in the past few years [1][2][3]. The chronic misuse and overuse of β-lactam antibiotics has led to the development of drug-resistant pathogens. For example, S. aureus strains developed resistance to methicillin in the 1950s and soon spread to many hospitals worldwide [4]. Staphylococcus aureus is a virulent pathogen that causes a variety of infections, from minor infections of skin and soft tissue to life-threatening endocarditis, pneumonia and osteomyelitis [5,6]. Due to the limit of new antibiotics on the market, sources of antibacterial agents were explored from known drugs that were originally designed to treat symptoms other than bacterial infection. For example, statins, drugs for hypercholesterolemia, have been reported to reduce the virulence of S. aureus [7,8]. Phenothiazines, a class of antipsychotic agents, were discovered to exhibit antibacterial activity and potentiate antibiotics to eradicate extensively drug-resistant (XDR) Mycobacterium tuberculosis in patients [9,10]. Thus, known drugs represent an alternative source for the discovery and development of a novel series of antibacterial agents.
Recently, we have performed screening assays against bacteria with our compound library which mostly contains kinase inhibitors and their derivatives. SC-78, a small molecule modified from regorafenib and sorafenib [11], multiple-kinase inhibitors, was shown to possess potent antimicrobial activities against Staphylococcus aureus and other Staphylococcus species [12] (Figure 1). From the pharmacological and medicinal chemistry perspective, the structure of SC-78 has crucial drawbacks. It consists of a stick central urea scaffold, on which the aniline groups are substituted with chloride It consists of a stick central urea scaffold, on which the aniline groups are substituted with chloride and trifluoromethane. The symmetry of the whole structure makes SC-78 less soluble, which might influence its bioavailability. The aromatic rings give the molecule a planar structure, which is prone to stack the molecules through π-π interactions. Moreover, urea is a rigid scaffold, which reduces the interaction force between the whole molecule and its target.
These drawbacks stimulated us to explore three modifications: replacement of the urea groups with isosteric amide linkers, replacement of the alkyl group with an alkenyl or benzyl group, and finally systematic modification of aniline derivatives with various functional groups on aromatic rings, thereby generating symmetric and asymmetric malonamide derivatives (Figure 1).

Results
The dimethyl cyclopropane-1,1-dicarboxylate was considered a commercial starting skeleton replacing the urea structure. A hydrolysis step on dimethyl cyclopropane-1,1-dicarboxylate gave a quantitative amount of cyclopropane-1,1-dicarboxyilic acid 2. For the convenience of the generation of the diacyl chloride from 2, thionyl chloride was chosen as a reagent for the source of acyl chloride. At the same time, the ring-opening process of the cyclopropanyl group of 2 was found in the generation of acyl chloride with the present proton source. Finally, acylation of the resulting diacyl chlorides 3 and 4 with substituted anilines was designed to generate the final symmetrical products 5 and 6 (malonamide derivatives) (Scheme 1). The strategy for the synthesis of the asymmetrical malonamide derivatives is shown in Scheme 2. Intermediate diacyl chloride was sequentially reacted with the first aniline substituent. After the half acylation reaction was completed, another aniline substituent was added to the mixture immediately for the completion of the asymmetrical malonate derivative 7. HO

Results
The dimethyl cyclopropane-1,1-dicarboxylate was considered a commercial starting skeleton replacing the urea structure. A hydrolysis step on dimethyl cyclopropane-1,1-dicarboxylate gave a quantitative amount of cyclopropane-1,1-dicarboxyilic acid 2. For the convenience of the generation of the diacyl chloride from 2, thionyl chloride was chosen as a reagent for the source of acyl chloride. At the same time, the ring-opening process of the cyclopropanyl group of 2 was found in the generation of acyl chloride with the present proton source. Finally, acylation of the resulting diacyl chlorides 3 and 4 with substituted anilines was designed to generate the final symmetrical products 5 and 6 (malonamide derivatives) (Scheme 1).
It consists of a stick central urea scaffold, on which the aniline groups are substituted with chloride and trifluoromethane. The symmetry of the whole structure makes SC-78 less soluble, which might influence its bioavailability. The aromatic rings give the molecule a planar structure, which is prone to stack the molecules through π-π interactions. Moreover, urea is a rigid scaffold, which reduces the interaction force between the whole molecule and its target.
These drawbacks stimulated us to explore three modifications: replacement of the urea groups with isosteric amide linkers, replacement of the alkyl group with an alkenyl or benzyl group, and finally systematic modification of aniline derivatives with various functional groups on aromatic rings, thereby generating symmetric and asymmetric malonamide derivatives (Figure 1).

Results
The dimethyl cyclopropane-1,1-dicarboxylate was considered a commercial starting skeleton replacing the urea structure. A hydrolysis step on dimethyl cyclopropane-1,1-dicarboxylate gave a quantitative amount of cyclopropane-1,1-dicarboxyilic acid 2. For the convenience of the generation of the diacyl chloride from 2, thionyl chloride was chosen as a reagent for the source of acyl chloride. At the same time, the ring-opening process of the cyclopropanyl group of 2 was found in the generation of acyl chloride with the present proton source. Finally, acylation of the resulting diacyl chlorides 3 and 4 with substituted anilines was designed to generate the final symmetrical products 5 and 6 (malonamide derivatives) (Scheme 1). The strategy for the synthesis of the asymmetrical malonamide derivatives is shown in Scheme 2. Intermediate diacyl chloride was sequentially reacted with the first aniline substituent. After the half acylation reaction was completed, another aniline substituent was added to the mixture immediately for the completion of the asymmetrical malonate derivative 7. The strategy for the synthesis of the asymmetrical malonamide derivatives is shown in Scheme 2. Intermediate diacyl chloride was sequentially reacted with the first aniline substituent. After the half acylation reaction was completed, another aniline substituent was added to the mixture immediately for the completion of the asymmetrical malonate derivative 7.
Next, to explore the potency of ethane chloride in the malonamide derivative against S. aureus, ethane chloride was replaced by a cyclopropanyl, allyl or benzyl group, and the antibacterial activity and structure-activity relationship was analyzed. The strategy by which ethane chloride was replaced is shown in Scheme 3. Dimethyl malonate 8 was reacted with allyl chloride and benzyl chloride resulting in compound 9. Hydroxylation of the methyl group by sodium methoxide led to compound 10. Subsequently, the di-acid was converted to diacyl chloride 11 by thionyl chloride. Diacyl chloride was used to generate a series of amide-linked derivatives of malonamide 12. Our efforts to design and synthesize novel anti-Staphylococcus molecules from SC-78 resulted in the development of a malonate scaffold as a new structural entity with suppressive activity against bacteria. These malonamide derivatives were assessed on S. aureus NCTC8325 by using the minimum inhibitory concentration (MIC) assay. The MIC values of all the malonamide derivatives are summarized in Tables 1-3. MIC values of each compound were determined by escalating doses, ranging from 0.125 to 64 mg/L.
A set of derivatives 13-26 were obtained in which chloride, nitro, hydroxyl, trifluoromethyl, ethynyl and trifluoromethoxyl substituents were attached to a phenyl ring on both sides of the malonate scaffold. The analysis of antibacterial activity of 17, 20, 21 and 22 with electron-donating substituents, such as hydroxyl and ethynyl on the phenyl ring, against S. aureus NCTC8325 demonstrated diminished suppressive activity (MIC > 8 mg/L). On the other hand, compounds 14, 15, 25 and 26 with electron-withdrawing substituents, such as trifluoromethoxyl and trifluoromethyl groups, increased the potency against S. aureus NCTC8325. The presence of the dichloride substituent in the phenyl ring led to an increase in activity relative to the monochloride substituent. These results Our efforts to design and synthesize novel anti-Staphylococcus molecules from SC-78 resulted in the development of a malonate scaffold as a new structural entity with suppressive activity against bacteria. These malonamide derivatives were assessed on S. aureus NCTC8325 by using the minimum inhibitory concentration (MIC) assay. The MIC values of all the malonamide derivatives are summarized in Tables 1-3 the potency against S. aureus NCTC8325. The presence of the dichloride substituent in the phenyl ring led to an increase in activity relative to the monochloride substituent. These results suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity.
We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives. suggest that electron-donating groups are less important than electron-withdrawing groups for antibacterial activity. We next tested the hypothesis that the asymmetric substituents on both sides of the malonate scaffold might induce potent antibacterial activity with the symmetric substituents. Therefore, derivatives 28, 29 and 30 were further synthesized and their antibacterial activity was evaluated. The results are shown in Table 2. The asymmetric malonamide derivatives exhibited almost equal antibacterial activity to the symmetric malonamide derivatives.  Next, we tested whether ethane chloride is important in antibacterial activity. Cyclopropanyl, allyl and benzyl substituents were introduced into the center of the malonate scaffold to replace ethane chloride, resulting in a series of compounds as shown in Table 3. The loss of activity upon the replacement of ethyl chloride by the cyclopropanyl group may result from the bending of two carbonyl moieties, thereby losing interaction with the target. Derivatives with rigid benzyl and allyl groups show reduced activity, which is potentially connected to steric effects. We conclude that ethane chloride connected to malonate is crucial for the activity against S. aureus NCTC8325.
To explore whether the synthesized compounds overcome antibiotic-resistant Staphylococcus aureus, we investigated the activity of compounds 26, 27, 28, 29, 33, 36, 37, 38 and 39 in S. aureus NCTC8325. As shown in Table 4, the inhibitory potency of the compounds against MRSA ATCC33592 was almost the same as against S. aureus NCTC8325, except for compound 33, which was neither effective against S. aureus NCTC8325 nor MRSA ATCC33592.
Compound 26 was selected for study of its pharmacological properties and evaluation of its drug-like potential. In-vitro study of antibacterial activity against various bacteria showed that 26 had excellent cell inhibitory effect on various antibiotic-resistant strains. Moreover, compound 26 inhibited cell growth on a K562 human erythroleukemic cell line with an IC50 of 20 mg/L. The selective ratio value of antibacterial versus cell line was over 40, indicating a good selectivity between humans and bacteria (Table 5).
Many clinical reports indicated that biofilm formation is an important factor contributing to S. aureus infections [13]. Bacteria in the biofilm usually exhibited less susceptibility to antibiotics, rendering treatment difficult [14]. To examine whether compound 26 was active against bacteria in biofilm, the minimal bacteria eradication concentrations (MBECs) of 26 and a wide range of antibiotics against MRSA ATCC33591 in biofilm were assessed. As the results show in Table 6, only compound 26 and rifampicin were active against MRSA 33591 in biofilm, but the MBEC values are much higher than their MIC values. Thus, we further tested whether 26 increased the susceptibility of MRSA toward these antibiotics. Among all the pairs of combinations, only rifampicin potentiated compound 26 in the MRSA ATCC33591 strain, while the others showed no synergistic effect (  Next, we tested whether ethane chloride is important in antibacterial activity. Cyclopropanyl, allyl and benzyl substituents were introduced into the center of the malonate scaffold to replace ethane chloride, resulting in a series of compounds as shown in Table 3. The loss of activity upon the replacement of ethyl chloride by the cyclopropanyl group may result from the bending of two carbonyl moieties, thereby losing interaction with the target. Derivatives with rigid benzyl and allyl groups show reduced activity, which is potentially connected to steric effects. We conclude that ethane chloride connected to malonate is crucial for the activity against S. aureus NCTC8325.
To explore whether the synthesized compounds overcome antibiotic-resistant Staphylococcus aureus, we investigated the activity of compounds 26, 27, 28, 29, 33, 36, 37, 38 and 39 in S. aureus NCTC8325. As shown in Table 4, the inhibitory potency of the compounds against MRSA ATCC33592 was almost the same as against S. aureus NCTC8325, except for compound 33, which was neither effective against S. aureus NCTC8325 nor MRSA ATCC33592.
Compound 26 was selected for study of its pharmacological properties and evaluation of its drug-like potential. In-vitro study of antibacterial activity against various bacteria showed that 26 had excellent cell inhibitory effect on various antibiotic-resistant strains. Moreover, compound 26 inhibited cell growth on a K562 human erythroleukemic cell line with an IC50 of 20 mg/L. The selective ratio value of antibacterial versus cell line was over 40, indicating a good selectivity between humans and bacteria (Table 5).
Many clinical reports indicated that biofilm formation is an important factor contributing to S. aureus infections [13]. Bacteria in the biofilm usually exhibited less susceptibility to antibiotics, rendering treatment difficult [14]. To examine whether compound 26 was active against bacteria in biofilm, the minimal bacteria eradication concentrations (MBECs) of 26 and a wide range of antibiotics against MRSA ATCC33591 in biofilm were assessed. As the results show in Table 6, only compound 26 and rifampicin were active against MRSA 33591 in biofilm, but the MBEC values are much higher than their MIC values. Thus, we further tested whether 26 increased the susceptibility of MRSA toward these antibiotics. Among all the pairs of combinations, only rifampicin potentiated compound 26 in the MRSA ATCC33591 strain, while the others showed no synergistic effect (Table 6).  Next, we tested whether ethane chloride is important in antibacterial activity. Cyclopropanyl, allyl and benzyl substituents were introduced into the center of the malonate scaffold to replace ethane chloride, resulting in a series of compounds as shown in Table 3. The loss of activity upon the replacement of ethyl chloride by the cyclopropanyl group may result from the bending of two carbonyl moieties, thereby losing interaction with the target. Derivatives with rigid benzyl and allyl groups show reduced activity, which is potentially connected to steric effects. We conclude that ethane chloride connected to malonate is crucial for the activity against S. aureus NCTC8325.
To explore whether the synthesized compounds overcome antibiotic-resistant Staphylococcus aureus, we investigated the activity of compounds 26, 27, 28, 29, 33, 36, 37, 38 and 39 in S. aureus NCTC8325. As shown in Table 4, the inhibitory potency of the compounds against MRSA ATCC33592 was almost the same as against S. aureus NCTC8325, except for compound 33, which was neither effective against S. aureus NCTC8325 nor MRSA ATCC33592.
Compound 26 was selected for study of its pharmacological properties and evaluation of its drug-like potential. In-vitro study of antibacterial activity against various bacteria showed that 26 had excellent cell inhibitory effect on various antibiotic-resistant strains. Moreover, compound 26 inhibited cell growth on a K562 human erythroleukemic cell line with an IC50 of 20 mg/L. The selective ratio value of antibacterial versus cell line was over 40, indicating a good selectivity between humans and bacteria (Table 5).
Many clinical reports indicated that biofilm formation is an important factor contributing to S. aureus infections [13]. Bacteria in the biofilm usually exhibited less susceptibility to antibiotics, rendering treatment difficult [14]. To examine whether compound 26 was active against bacteria in biofilm, the minimal bacteria eradication concentrations (MBECs) of 26 and a wide range of antibiotics against MRSA ATCC33591 in biofilm were assessed. As the results show in Table 6, only compound 26 and rifampicin were active against MRSA 33591 in biofilm, but the MBEC values are much higher than their MIC values. Thus, we further tested whether 26 increased the susceptibility of MRSA toward these antibiotics. Among all the pairs of combinations, only rifampicin potentiated compound 26 in the MRSA ATCC33591 strain, while the others showed no synergistic effect (Table 6).  Next, we tested whether ethane chloride is important in antibacterial activity. Cyclopropanyl, allyl and benzyl substituents were introduced into the center of the malonate scaffold to replace ethane chloride, resulting in a series of compounds as shown in Table 3. The loss of activity upon the replacement of ethyl chloride by the cyclopropanyl group may result from the bending of two carbonyl moieties, thereby losing interaction with the target. Derivatives with rigid benzyl and allyl groups show reduced activity, which is potentially connected to steric effects. We conclude that ethane chloride connected to malonate is crucial for the activity against S. aureus NCTC8325.
To explore whether the synthesized compounds overcome antibiotic-resistant Staphylococcus aureus, we investigated the activity of compounds 26, 27, 28, 29, 33, 36, 37, 38 and 39 in S. aureus NCTC8325. As shown in Table 4, the inhibitory potency of the compounds against MRSA ATCC33592 was almost the same as against S. aureus NCTC8325, except for compound 33, which was neither effective against S. aureus NCTC8325 nor MRSA ATCC33592.
Compound 26 was selected for study of its pharmacological properties and evaluation of its drug-like potential. In-vitro study of antibacterial activity against various bacteria showed that 26 had excellent cell inhibitory effect on various antibiotic-resistant strains. Moreover, compound 26 inhibited cell growth on a K562 human erythroleukemic cell line with an IC50 of 20 mg/L. The selective ratio value of antibacterial versus cell line was over 40, indicating a good selectivity between humans and bacteria (Table 5).
Many clinical reports indicated that biofilm formation is an important factor contributing to S. aureus infections [13]. Bacteria in the biofilm usually exhibited less susceptibility to antibiotics, rendering treatment difficult [14]. To examine whether compound 26 was active against bacteria in biofilm, the minimal bacteria eradication concentrations (MBECs) of 26 and a wide range of antibiotics against MRSA ATCC33591 in biofilm were assessed. As the results show in Table 6, only compound 26 and rifampicin were active against MRSA 33591 in biofilm, but the MBEC values are much higher than their MIC values. Thus, we further tested whether 26 increased the susceptibility of MRSA toward these antibiotics. Among all the pairs of combinations, only rifampicin potentiated compound 26 in the MRSA ATCC33591 strain, while the others showed no synergistic effect (Table 6).  Next, we tested whether ethane chloride is important in antibacterial activity. Cyclopropanyl, allyl and benzyl substituents were introduced into the center of the malonate scaffold to replace ethane chloride, resulting in a series of compounds as shown in Table 3. The loss of activity upon the replacement of ethyl chloride by the cyclopropanyl group may result from the bending of two carbonyl moieties, thereby losing interaction with the target. Derivatives with rigid benzyl and allyl groups show reduced activity, which is potentially connected to steric effects. We conclude that ethane chloride connected to malonate is crucial for the activity against S. aureus NCTC8325.
To explore whether the synthesized compounds overcome antibiotic-resistant Staphylococcus aureus, we investigated the activity of compounds 26, 27, 28, 29, 33, 36, 37, 38 and 39 in S. aureus NCTC8325. As shown in Table 4, the inhibitory potency of the compounds against MRSA ATCC33592 was almost the same as against S. aureus NCTC8325, except for compound 33, which was neither effective against S. aureus NCTC8325 nor MRSA ATCC33592.
Compound 26 was selected for study of its pharmacological properties and evaluation of its drug-like potential. In-vitro study of antibacterial activity against various bacteria showed that 26 had excellent cell inhibitory effect on various antibiotic-resistant strains. Moreover, compound 26 inhibited cell growth on a K562 human erythroleukemic cell line with an IC50 of 20 mg/L. The selective ratio value of antibacterial versus cell line was over 40, indicating a good selectivity between humans and bacteria (Table 5).
Many clinical reports indicated that biofilm formation is an important factor contributing to S. aureus infections [13]. Bacteria in the biofilm usually exhibited less susceptibility to antibiotics, rendering treatment difficult [14]. To examine whether compound 26 was active against bacteria in biofilm, the minimal bacteria eradication concentrations (MBECs) of 26 and a wide range of antibiotics against MRSA ATCC33591 in biofilm were assessed. As the results show in Table 6, only compound 26 and rifampicin were active against MRSA 33591 in biofilm, but the MBEC values are much higher than their MIC values. Thus, we further tested whether 26 increased the susceptibility of MRSA toward these antibiotics. Among all the pairs of combinations, only rifampicin potentiated compound 26 in the MRSA ATCC33591 strain, while the others showed no synergistic effect (Table 6).  Next, we tested whether ethane chloride is important in antibacterial activity. Cyclopropanyl, allyl and benzyl substituents were introduced into the center of the malonate scaffold to replace ethane chloride, resulting in a series of compounds as shown in Table 3. The loss of activity upon the replacement of ethyl chloride by the cyclopropanyl group may result from the bending of two carbonyl moieties, thereby losing interaction with the target. Derivatives with rigid benzyl and allyl groups show reduced activity, which is potentially connected to steric effects. We conclude that ethane chloride connected to malonate is crucial for the activity against S. aureus NCTC8325.
To explore whether the synthesized compounds overcome antibiotic-resistant Staphylococcus aureus, we investigated the activity of compounds 26, 27, 28, 29, 33, 36, 37, 38 and 39 in S. aureus NCTC8325. As shown in Table 4, the inhibitory potency of the compounds against MRSA ATCC33592 was almost the same as against S. aureus NCTC8325, except for compound 33, which was neither effective against S. aureus NCTC8325 nor MRSA ATCC33592.
Compound 26 was selected for study of its pharmacological properties and evaluation of its drug-like potential. In-vitro study of antibacterial activity against various bacteria showed that 26 had excellent cell inhibitory effect on various antibiotic-resistant strains. Moreover, compound 26 inhibited cell growth on a K562 human erythroleukemic cell line with an IC50 of 20 mg/L. The selective ratio value of antibacterial versus cell line was over 40, indicating a good selectivity between humans and bacteria (Table 5).
Many clinical reports indicated that biofilm formation is an important factor contributing to S. aureus infections [13]. Bacteria in the biofilm usually exhibited less susceptibility to antibiotics, rendering treatment difficult [14]. To examine whether compound 26 was active against bacteria in biofilm, the minimal bacteria eradication concentrations (MBECs) of 26 and a wide range of antibiotics against MRSA ATCC33591 in biofilm were assessed. As the results show in Table 6, only compound 26 and rifampicin were active against MRSA 33591 in biofilm, but the MBEC values are much higher than their MIC values. Thus, we further tested whether 26 increased the susceptibility of MRSA toward these antibiotics. Among all the pairs of combinations, only rifampicin potentiated compound 26 in the MRSA ATCC33591 strain, while the others showed no synergistic effect (Table 6).  Next, we tested whether ethane chloride is important in antibacterial activity. Cyclopropanyl, allyl and benzyl substituents were introduced into the center of the malonate scaffold to replace ethane chloride, resulting in a series of compounds as shown in Table 3. The loss of activity upon the replacement of ethyl chloride by the cyclopropanyl group may result from the bending of two carbonyl moieties, thereby losing interaction with the target. Derivatives with rigid benzyl and allyl groups show reduced activity, which is potentially connected to steric effects. We conclude that ethane chloride connected to malonate is crucial for the activity against S. aureus NCTC8325.
To explore whether the synthesized compounds overcome antibiotic-resistant Staphylococcus aureus, we investigated the activity of compounds 26, 27, 28, 29, 33, 36, 37, 38 and 39 in S. aureus NCTC8325. As shown in Table 4, the inhibitory potency of the compounds against MRSA ATCC33592 was almost the same as against S. aureus NCTC8325, except for compound 33, which was neither effective against S. aureus NCTC8325 nor MRSA ATCC33592.
Compound 26 was selected for study of its pharmacological properties and evaluation of its drug-like potential. In-vitro study of antibacterial activity against various bacteria showed that 26 had excellent cell inhibitory effect on various antibiotic-resistant strains. Moreover, compound 26 inhibited cell growth on a K562 human erythroleukemic cell line with an IC50 of 20 mg/L. The selective ratio value of antibacterial versus cell line was over 40, indicating a good selectivity between humans and bacteria (Table 5).
Many clinical reports indicated that biofilm formation is an important factor contributing to S. aureus infections [13]. Bacteria in the biofilm usually exhibited less susceptibility to antibiotics, rendering treatment difficult [14]. To examine whether compound 26 was active against bacteria in biofilm, the minimal bacteria eradication concentrations (MBECs) of 26 and a wide range of antibiotics against MRSA ATCC33591 in biofilm were assessed. As the results show in Table 6, only compound 26 and rifampicin were active against MRSA 33591 in biofilm, but the MBEC values are much higher than their MIC values. Thus, we further tested whether 26 increased the susceptibility of MRSA toward these antibiotics. Among all the pairs of combinations, only rifampicin potentiated compound 26 in the MRSA ATCC33591 strain, while the others showed no synergistic effect (Table 6).

0.5
Next, we tested whether ethane chloride is important in antibacterial activity. Cyclopropanyl, allyl and benzyl substituents were introduced into the center of the malonate scaffold to replace ethane chloride, resulting in a series of compounds as shown in Table 3. The loss of activity upon the replacement of ethyl chloride by the cyclopropanyl group may result from the bending of two carbonyl moieties, thereby losing interaction with the target. Derivatives with rigid benzyl and allyl groups show reduced activity, which is potentially connected to steric effects. We conclude that ethane chloride connected to malonate is crucial for the activity against S. aureus NCTC8325.
To explore whether the synthesized compounds overcome antibiotic-resistant Staphylococcus aureus, we investigated the activity of compounds 26, 27, 28, 29, 33, 36, 37, 38 and 39 in S. aureus NCTC8325. As shown in Table 4, the inhibitory potency of the compounds against MRSA ATCC33592 was almost the same as against S. aureus NCTC8325, except for compound 33, which was neither effective against S. aureus NCTC8325 nor MRSA ATCC33592.
Compound 26 was selected for study of its pharmacological properties and evaluation of its drug-like potential. In-vitro study of antibacterial activity against various bacteria showed that 26 had excellent cell inhibitory effect on various antibiotic-resistant strains. Moreover, compound 26 inhibited cell growth on a K562 human erythroleukemic cell line with an IC 50 of 20 mg/L. The selective ratio value of antibacterial versus cell line was over 40, indicating a good selectivity between humans and bacteria ( Table 5).
Many clinical reports indicated that biofilm formation is an important factor contributing to S. aureus infections [13]. Bacteria in the biofilm usually exhibited less susceptibility to antibiotics, rendering treatment difficult [14]. To examine whether compound 26 was active against bacteria in biofilm, the minimal bacteria eradication concentrations (MBECs) of 26 and a wide range of antibiotics against MRSA ATCC33591 in biofilm were assessed. As the results show in Table 6, only compound 26 and rifampicin were active against MRSA 33591 in biofilm, but the MBEC values are much higher than their MIC values. Thus, we further tested whether 26 increased the susceptibility of MRSA toward these antibiotics. Among all the pairs of combinations, only rifampicin potentiated compound 26 in the MRSA ATCC33591 strain, while the others showed no synergistic effect (Table 6).

Discussion
There have been concerns about the threat of MRSA to global health in recent years because of the shortage of drugs that are effective against S. aureus, leading to lower survival in patients. S. aureus has also developed various mechanisms to evade the toxicity of antibiotic agents, such as β-lactamase and efflux pumps. The rate of development of antibiotic resistance is faster than the development of antibacterial agents with novel structures. After the latest antibacterial drugs, including linezolid, daptomycin and retapamulin, were approved for clinical application, no new antibiotic with a novel chemical entity has been introduced to the market. The gap in new antibiotic discovery highlights the need to develop new chemical backbones for new classes of antibiotic agents. In this study, we designed a series of agents from the lead compound, SC-78, with a urea backbone. Through pharmacochemical modification of the backbone, the malonate moiety exhibited a high potential and efficient chemical core for the development of a series of compounds. Our new compounds have several advantages: (1) At present, no resistance to compound 26 was observed to develop in vitro, suggesting a different inhibition mechanism from existing antibiotics and that it avoids the traditional antibiotic evasion strategies of MRSA; (2) in addition to providing a new core for an antibiotic agent, compound 26 alone exhibited high activity in vitro against several (at least three) MRSA strains. Compound 26 also showed excellent activity in killing MRSA inside biofilm. Interestingly, combinations of current antibiotics and 26 showed no synergy in eradicating MRSA in biofilm, with the exception of rifampicin; (3) we synthesized a set of malonamide derivatives via a two-step procedure. These efficient synthetic procedures applied commercially available chemicals and reagents to obtain a large set of malonamide derivatives that can be used in an animal study. From the structure-activity relationship analysis, a phenyl ring connected with an electron-withdrawing group, such as trifluoro and nitro, is important for biological activity. Replacement with a hydroxyl group resulted in loss of antimicrobiota activity. On the other hand, ethane chloride in the middle of the malonate moiety exhibited greater activity than allyl, benzyl and cyclopropanyl groups.
In conclusion, we developed a short synthetic route for the preparation of a series of malonamide derivatives. Several agents showed promising antibacterial growth activity and repressed biofilm formation. Further exploration of the detailed mechanisms by which 26 overcome MRSA is ongoing. From the drug development point of view, the new scaffold described herein can guide the development of more potent agents and might provide therapeutic options for fighting infectious diseases.

General Procedure for the Synthesis of 13-26
Cyclopropane-1,1-dicarboxylic acid (1 equiv.) was slowly added to a mixture of thionyl chloride (14 equiv.) and one drop of water without any solution, and the reaction mixture was stirred at 80 • C for 16 h. The intermediate was cooled to ambient temperature and concentrated under reduced pressure. A mixture of aniline derivative (2.5 equiv.) and pyridine (1 equiv.) in anhydrous THF was added dropwise to an ice-cold solution of the intermediate in anhydrous THF (15-20 mL). The reaction mixture was stirred at ambient temperature for 2 h. The product was extracted with ethyl acetate three times and the combined organic extracts were washed with brine, dried over MgSO 4 , and concentrated. The crude mixture was purified by flash column chromatography (EtOAc/hexane = 1/4 to 1/1) to give 13 to 26 (yield: 4-30%).

General Procedure for Synthesis of Compounds 27-29
Cyclopropane-1,1-dicarboxylic acid (1 equiv.) was slowly added to a mixture of thionyl chloride (14 equiv.) and one drop of water without any solution, and the reaction mixture was stirred at 80 • C for 16 h. The intermediate was cooled to ambient temperature and concentrated under reduced pressure. A mixture of aniline derivative (1.2 equiv.) and pyridine (1 equiv.) in anhydrous THF was added dropwise to an ice-cold solution of the intermediate in anhydrous THF (15-20 mL). After 30 min, another aniline derivative (1.8 equiv.) and pyridine (1 equiv.) was added to the mixture. The reaction mixture was stirred at ambient temperature for 2 h. The product was extracted with ethyl acetate three times and the combined organic extracts were washed with brine, dried over MgSO 4 , and concentrated. The crude mixture was purified by flash column chromatography (EtOAc/hexane = 1/4 to 1/1) to give 27-29 (yield: 20-45%).