A concise SAR-analysis of antimicrobial cationic amphipathic barbiturates for an improved activity-toxicity profile

An amphipathic barbiturate mimic of the marine eusynstyelamides is reported as a promising class of antimicrobial agents. We hereby report a detailed analysis of the structure-activity relationship for cationic amphipathic N,N ′ -dialkylated-5,5-disubstituted barbiturates. The influence of various cationic groups, hydrocarbon linkers and lipophilic side chains on the compounds ’ antimicrobial potency and haemolytic activity was studied. A comprehensive library of 58 compounds was prepared using a concise synthetic strategy. We found cationic amine and guanidyl groups to yield the highest broad-spectrum activity and cationic trimethylated quaternary amine groups to exert narrow-spectrum activity against Gram-positive bacteria. n-Propyl hydrocarbon linkers proved to be the best compromise between potency and haemolytic activity. The combination of two different lipophilic side chains allowed for further fine-tuning of the biological properties. Using these insights, we were able to prepare both, the potent narrow-spectrum barbiturate 8a and the broad-spectrum barbiturates 11lG , 13jA and 13jG , all having low or no haemolytic activity. The guanidine derivative 11lG demonstrated a strong membrane disrupting effect in luciferase-based assays. We believe that these results may be valuable in further development of antimicrobial lead structures.


Introduction
Since the golden age of antibiotics, the developing rate of new agents has decreased notably, while antimicrobial resistance (AMR) has been rising to a global threat [1].The prominence of this problem is well demonstrated by the World Health Organization (WHO) enacting a global action plan on fighting antimicrobial resistance [2].While the action plan is focusing on a framework at many different levels, the need for potent antimicrobials stays.As the antibiotics employed for decades start to lose activity against resistant bacteria, several alternative approaches have been investigated.Among these are combination therapy [3,4], bacteriophage therapy [5], photodynamic therapy [6], antibacterial antibodies [7], phytochemicals [5], nanoparticles [8] and antimicrobial peptides [9,10].
From the above stated list, the short, cationic antimicrobial peptides (AMPs) are an intriguing class of compounds.They constitute the first line of host defense in virtually all eukaryotic species including plants, mammals, insects, etc. [11] They generally feature between 20 and 50% hydrophobic residues and have an overall positive charge (+2 to +9) at neutral pH [12][13][14].Their amphipathic nature is the basis of their most common mode of action, to permeabilize bacterial membranes.AMPs attach to the negatively charged cytoplastic membrane by electrostatic interactions and subsequently disrupt the apolar bilayer with their hydrophobic part [15].It is believed that due to these non-specific interactions, bacterial resistance is less likely to be induced [16].This makes AMPs a promising group of compounds despite their generally lower activity compared to marketed antibiotics [17].
Despite these promising properties, the clinical application of peptide based drugs is often limited by their poor oral uptake and proteolytic instability [18].Therefore, considerable efforts towards the development of synthetic AMP analogues have been made, cumulating in the development of a variety of different groups of analogues [19][20][21][22][23][24][25][26][27].
Focusing on small molecules, we have recently reported substituted barbituric acid derivatives [28], inspired by a family of marine natural products, the eusynstyelamides, [29,30] as peptidomimetics of AMPs.The lead structure 1aG (Fig. 1) from our previous study [28] demonstrated good in vitro and in vivo activity as a proof of principle.
Encouraged by the in vivo activity of 1aG we herein describe an indepth SAR investigation to improve the potency and selectivity of these peptidomimetics.Several series of amphipathic barbiturates with systematically varying substituents were designed and synthesized.Our aim was to assess the qualitative influence of each structural component aside from the barbituric acid on the antimicrobial and haemolytic activity.Once the impact of each component is identified, improved narrow-and broad-spectrum compounds may be prepared.All new compounds were screened for activity against a panel of antibiotic susceptible strains to determine their minimal inhibitory concentration (MIC) values.Cytotoxicity was assessed by determining the EC 50 values for the lysis of human red blood cells (RBC).Promising candidates were investigated for their antibacterial mode of action (MoA), using three luciferase-based assays of the viability and integrity of the cytoplasmic inner and outer membrane of bacterial cells.

Design of the study
To systematically study the influence of structural components of 1aG [28] (Fig. 1) on the antibacterial and haemolytic activity, we devised several series of compounds based on the general structure shown in Fig. 2. All structures consisted of a central barbituric acid core, which was kept constant.Three structural parts were varied in the design of the compound library: (i) the cationic head groups (blue placeholder in Fig. 2) attached to the nitrogen atoms of the barbiturate core by (ii) a hydrocarbon linker chain (green placeholder) and (iii) the two lipophilic side chains (red placeholders) connected to the barbiturate C-5 carbon.
The influence of the cationic head groups (R; Fig. 2) was investigated by including 1 • amines, methylated 2 • , 3 • and 4 • amines and imine derivatives containing guanidino or pyridinium groups in the library design.The cationic groups were chosen based on the prospect of varying the interactions with bacterial membranes and their ability to cross the latter and accumulate in Gram-negative Escherichia coli (E.coli) [31,32].Compounds with varying cationic groups are found in compound series 1.
The lipophilic side chains (Fig. 2) were hypothesized to influence the compounds' ability to insert into the hydrophobic lipid bilayer of the bacteria.In our library design haloaryls, hetero-aryls, and linear and cyclic hydrocarbons were chosen as lipophilic side chains.The selection was based on results from our previous study [28] and commercial availability.Two different compound series were included in the study; in series 2 the two lipophilic side chains were identical, while in series 3 two different lipophilic side chains were combined resulting in derivatives with mixed side chains.
The hydrocarbon linkers (Fig. 2) were chosen on the premises of investigating the influence of the flexibility and distance of the cationic groups relative to the barbiturate core.Linear hydrocarbon chains of 2-6 carbons length gave flexible linkers, while cyclic hydrocarbon linkers (cyclobutyl and cyclohexyl) gave more restricted analogues.Compounds with varying linkers are included in series 4.
Based on the results from series 4 we prepared a range of compounds included in series 5, having n-propyl linkers.

Synthesis
Our previously reported synthesis of 5,5-dialkylated barbiturates provided amine and guanidine analogues in six or eight synthetic steps, respectively [28].In the present study, the demand for a large library of compounds prompted us to develop a shorter synthetic approach.Both amines and guanidines were successfully obtained in three steps from barbituric acid.
To obtain unsymmetrically 5,5-dialkylated barbituric acids 3k-q a different approach was needed, since mono-alkylation enhances the nucleophilicity of the barbiturate C-5 carbon leading to inevitable dialkylation [37].We investigated several reported methods for selective monoalkylation and in situ reductions [37][38][39], which did not work well in our hands.We therefore decided to use a stepwise approach as shown in Scheme 1-II.Barbituric acid 2 and 3,5-dibromobenzaldehyde were condensed [40] to give compound 4 and subsequent reduction with NaBH 4 in EtOH [41] gave the C-5 mono-substituted derivative 5 in 80% yield.We found 5 being an approximate 2:1 mixture of the keto and enol form.This mixture was alkylated a second time using the conditions employed for 5,5-dialkylation of barbituric acid to deliver intermediates 3k-q.Yields ranged from 5 to 61%, depending on the reactivity of the employed electrophiles.
Starting from intermediates 3, a wide range of N,N ′ -dialkylated barbituric acid derivatives were prepared, employing a range of methods for N-alkylation depending on the availability of reactants Fig. 1.Lead structure 1aG from our previous study [28] with the barbituric acid core highlighted in orange.(Scheme 2).
All compounds synthesized are summarized in Tables 1-5.Compounds denoted with capital A have an amine as a cationic group and those denoted with capital G have cationic guanidino groups, correspondingly.The compounds are grouped into five series (series 1-5) based on their structural variations.
Series 1 (Table 1) encompasses compounds with varying cationic groups, while the C-5 substituents (3,5-dibromobenzyl) and hydrocarbon linker (n-butyl) were kept unchanged.To obtain the methylated amines and pyridinium containing compounds 6a-9a, 5,5-bis(3,5-dibromobenzyl)barbituric acid 3a was N,N ′ -dialkylated with either 1-bromo-4-chlorobutane or 1,4-dibromobutane and Cs 2 CO 3 in acetone (Scheme 2-I).Subsequent S N 2 substitution of the terminal halo substituent with methylated ammonia or pyridine in acetonitrile at elevated temperature led to compounds 6a-9a in 33-88% yield.Having a bromide as leaving group proved to be necessary for substitution with methylamine and dimethylamine.Substitutions were only successful with organic solutions of the amines, while hydrohalo salts of the amines could not be used.An optimized method for preparation of the previously reported 1 • amine 1aA and guanidine 1aG [28] is described in the next paragraph.
Series 4 (Table 4) contained compounds with varying linkers, such as aliphatic chains with 2-6 carbons length as well as cyclic hydrocarbons.The C-5 substituents were set to 3,5-dibromobenzyl and the cationic groups were either amino (A) or guanidino (G) groups.As the relevant linkers were commercially available as N-Boc-amino alcohols, we decided to explore the above Mitsunobu protocol in a stepwise synthetic approach (Scheme 2-III).The 5,5-bis(3,5-dibromobenzyl)barbituric acid 3a was N,N ′ -dialkylated with the appropriate Boc-protected amino alcohol using the Mitsunobu conditions followed by TFA:DCM treatment to obtain compounds 12aA-19aA in 23-73% over 2 steps.Treatment of the amines 12aA-19aA with N,N ′ -Di-Boc-1H-pyrazole-1-carboxamidine and DIPEA or DBU, followed by Boc removal with TFA in DCM delivered the guanidines 12aG-18aG in 14-92% yield.Employing the well-known and cheaper alternative N,N ′ -bis(tert-butoxycarbonyl)-S-methylisothiourea [43,44] led to an inseparable mixture of Boc protected amine and Boc protected guanyl compounds.
or unsymmetrically 13(l,p)A substituted primary amines in 34-76% yield (Scheme 2-IV).The Mitsunobu protocol was evaluated but discarded due to difficulties with purification of some compounds.Treatment of the primary amines with N,N ′ -di-Boc-1H-pyrazole-1carboxamidine and DIPEA, followed by TFA facilitated Boc removal and RP flash chromatography purification yielded the respective guanidines 13(c,e,h,i,j,l,p)G in 20-91% yield.

Table 1
Antimicrobial activity (MIC in μg/mL) against bacterial reference strains and haemolytic activity against human RBC (EC 50 in μg/mL) for compounds in series 1. strains (Tables 1-5).Haemolytic activity against human red blood cells (RBCs), expressed by the EC 50 value, was used as a measurement of cytotoxicity.We have earlier reported compounds 1aA and 1aG [28], which here are used as reference compounds together with the known antibiotic ciprofloxacin as a positive control.The descriptors for amine derivatives (A) and guanidine derivatives (G) are omitted for derivatives with other cationic groups.

Compound series 1: Exploring the cationic head group (R 3 )
First, we set out to investigate the influence of the effective charge of the cationic groups (Table 1, R 3 group and Fig. 2, blue space holders).Upon N-methylation the electron density at the nitrogen increases, as does its basicity, but the polarity decreases.Successive introduction of one (6a), two (7a) or three (8a) methyl groups had no noteworthy influence on the activity against the Gram-positive strains (MIC: 4-8 μg/ mL), but the activity against the Gram-negative strains dropped considerably for compound 8a (MIC: 128-256 μg/mL).It is suggested that, among other factors, the electrostatic interaction between these compounds and bacterial membrane plays an important role in the compound's activity [45].Successive introduction of methyl groups lowers the effective charge of the amine head groups, thus reducing their interaction with the lower charge per area membrane of Gram-negative bacteria compared to Gram-positive strains [46].Additionally, quaternary ammonium compounds (quats or QACs) are known for their impaired ability to cross the outer membrane of Gram-negative Pseudomonas aeruginosa (P.aeruginosa) [47].Recent studies showed generally impaired uptake of compounds containing methylated primary amines in E. coli [31].Despite that presumably lower uptake, secondary (6a) and tertiary amines (7a) were still active against E. coli (MIC: 8 μg/mL).By replacing the quaternary trimethylated ammonium (8a) with a pyridinium group (9a), the activity against the Gram-positive strains improved (MIC: 2-4 μg/mL) and the activity against E. coli was restored (MIC: 8 μg/mL), probably due to increased accumulation [32].Tertiary (7a), EC 50 : 157 μg/mL) and quaternary amines (8a and 9a; both EC 50 : >500 μg/mL) displayed lower haemolytic activity compared to the primary (1aA, EC 50 : 79 μg/mL) and secondary (6a, EC 50 : 73 μg/mL) amines.
The quaternary ammonium compound 8a exhibited narrowspectrum antimicrobial activity against Gram-positive strains and was non-haemolytic.Compared to the above investigated head groups, the recently reported amine (1aA) and guanidine derivatives (1aG) appeared to be the most effective against the Gram-negative strains, thus rendering them suitable for broad-spectrum applications.The consecutively developed compounds were therefore synthesized with either amino or guanidino groups.

Compound series 2: Exploring new lipophilic side chains
In series 2, the influence of heterocyclic, aliphatic and highly brominated lipophilic side chains (Table 2, R 1 /R 2 groups and Fig. 2, red space holders) on the biological activity was examined.Both side chains employed were identical.
The antimicrobial activity for the amine barbiturates 10(b-g)A ranged from MIC: 2-256 μg/mL and for the guanidine barbiturates 10(bg)G from MIC: 1-16 μg/mL against the Gram-positive strains Staphylococcus aureus (S. aureus) and Bacillus subtilis (B.subtilis).Against the Gram-negative strains E. coli and P. aeruginosa both, the amine and guanidine derivatives, showed MIC values of 4 ->256 μg/mL.We included quinoline and 6-bromoquinoline as heterocyclic alternatives.
In the next step we replaced the aromatic side chains by alkyl chains as found in antimicrobial quats [48,49].We decided to incorporate two hexyl chains, which mimic the single long alkyl chain commonly found in quats [50].The amine derivative 10dA (R 1 /R 2 = n-hexyl) showed weak activity against all bacterial strains (MIC: 8-64 μg/mL), whereas the guanidine derivative 10dG showed high antibacterial activity with MIC values of 2-4 μg/mL against all strains except for P. aeruginosa.Haemolysis was still moderate, with EC 50 : 143 μg/mL.Interestingly, the shorter hexyl chains perform just as good as the longer alkyl chains in quats [48], suggesting that the overall hydrophobic bulk is more important than the actual chain length.
Previously, we have found bromo substituents on the phenyl ring having a positive effect on the biological activity, with 3,5-dibromophenyl providing the highest activity [26].We therefore prepared derivatives 10fA and 10fG (R 1 /R 2 = 2,4,5-tribromobenzyl) and 10gA and 10gG (R 1 /R 2 = 2,4,6-tribromobenzyl) being at the far end of the hydrophobicity scale.They all displayed potent antibacterial activity, with MIC values ≤8 μg/mL against all strains.However, haemolytic activity also increased for all these compounds (EC 50 : 27-32 μg/mL).The positioning of the bromines on the phenyl ring had a minor influence on antibacterial activity, with 10gA and 10gG being most potent.
In summary, halogenated heterocycles are promising side chains for narrow-spectrum application.The hydrophobicity of the C-5 substituents had the greatest influence, while the structure being secondary.When exceeding CLogP ≈4.50, the structures mostly became too haemolytic to be of interest for further studies.

Compound series 3: Exploring mixed lipophilic groups
A series of compounds containing two different side chains were prepared to tune lipophilicity and side chain structure with respect to antimicrobial activity and haemolytic activity.We intended to pair the potent 3,5-dibromobenzyl side chain (R 1 ) with side chains (R 2 ) of different varying lipophilicity (Table 3; R 2 group; Fig. 2, red space holders).
The derivative 11lG showed additionally good activity against the Gram-negative E. coli (MIC: 4 μg/mL).The superior performance of 11lG over 11kG may be attributed to the higher average CLogP value of the lipophilic side chain of 11lG.The polar nitrogen atom in the quinolinyl side chain (11lG) might also reduce the compounds' activity.
Next, we tested two hydrocarbon analogues 11m (R 2 = cyclopentyl) and 11n (R 2 = n-hexyl), with comparable average hydrophobicity to 11k and 11l, respectively.Compound 11mA was potent against both Gram-positive strains and non-haemolytic.The amine derivative 11nA was mainly acting against the Gram-positive strains (MIC: 4 μg/mL) but showed 3-fold higher haemolytic activity compared to 11lA.The guanyl derivative 11nG exhibited potent antibacterial activity, with MIC-values of 2-8 μg/mL against all strains tested.Even though its average CLogP was only marginally higher than 11lG, its haemolytic activity was pronouncedly higher (EC 50 : 58 μg/mL).The compounds 11m and 11n indicated that a combination of an aromatic and a hydrocarbon lipophilic side chain leads to higher haemolytic activity, compared to two aromatic side chains.
Upon guanylation, a further improvement in antimicrobial activity was achieved, but haemolytic activity was also increased.Thus, 11oG and 11pG became twice as potent and haemolytic (EC 50 : 36-39 μg/mL), rendering them unfavorable for systemic in vivo treatment.The bromonaphthyl containing 11qG became more potent against S. aureus (MIC: 1 μg/mL), yet haemolytic activity (EC 50 : 58 μg/mL) was still unfavorably high.No clear trend for the antimicrobial activity could be deduced, based on the structure of the lipophilic side chains.
Taken all together, 11kG displayed promising narrow-spectrum activity against Gram-positive strains and absence of haemolytic activity.Compounds 11oG and 11pG are highly potent derivatives but displayed high haemolytic activity.

Compound series 4: Exploring the hydrocarbon linker chain (X)
We incorporated various linear and cyclic hydrocarbon linkers (Tables 4 and X group; Fig. 2, green space holder) between the central scaffold and the cationic residue.3,5-Dibromobenzyl was kept fixed as the lipophilic side chain and the previously reported compounds 1aA and 1aG (both X = n-butyl) served as reference substances for comparison.Shortening or elongating the alkyl chains to 2, 3, 5 or 6 methylene groups (12aA-15aA) led to no significant change in antibacterial activity (MIC: 4-16 μg/mL against all strains).The haemolytic activity increased slightly compared to 1aA (X = n-butyl), except for 13aA (X = n-propyl), which became slightly less haemolytic.So far, guanidine derivatives tended to have a higher haemolytic activity (vide supra) compared to amine derivatives.In contrast, 13aG (X = n-propyl) and

Table 4
Antimicrobial activity (MIC in μg/mL) against bacterial reference strains and haemolytic activity against human RBC (EC 50 in μg/mL) for compounds in series 4. 12aG (X = ethyl) were observed to exhibit 2-fold and 4-fold decreased haemolytic activity, respectively, compared to their amine counterparts.The activity against the Gram-positive strains was slightly improved, whereas the potencies against the Gram-negative P. aeruginosa were retained or a little diminished.The derivatives 14aA and 14aG (X = npentyl) displayed virtually the same MIC and EC 50 values, whereas 15aG (X = n-hexyl) was less potent against P. aeruginosa (MIC: 32 μg/mL) compared to 15aA (X = n-hexyl) (MIC: 16 μg/mL).Both guanylated compounds were less potent than the previously investigated derivative 1aG (X = n-butyl) and their haemolytic levels were comparably high (EC 50 : 29-57 μg/mL).Compounds 14aG (X = n-pentyl) and 15aG (X = n-hexyl) led also to precipitation in the RBC assay upon sample preparation, possibly due to their higher overall hydrophobicity, demonstrating an unfavorable solubility profile.
In summary, compounds with rigid cyclic linkers showed similar or slightly higher potency compared to their linear analogues, but they tended to be more haemolytic.Furthermore, compounds with pentyl and hexyl linkers showed furthermore decreased water solubility.The amine derivatives having ethyl, n-propyl or n-butyl linkers displayed similar antibacterial bioactivity profiles, whereas the equivalent guanidine derivatives displayed descending antimicrobial activity as follows: n-butyl > n-propyl > ethyl.The best balance between high antimicrobial activity and low haemolytic activity was presented by compounds having n-propyl hydrocarbon linker chains.

Compound series 5: Investigating compounds with a n-propyl hydrocarbon linker
In series 5 (Table 5), we studied the effect of the n-propyl linker more closely due to the promising balance between high antimicrobial activity and low haemolytic activity seen in series 4. We selected the lipophilic side chains (R 1 /R 2 ) based on our previous findings.We reasoned that compounds 13c, 13h and 13l would mainly act against Table 5 Antimicrobial activity (MIC in μg/mL) against bacterial reference strains and haemolytic activity against human RBC (EC 50 in μg/mL) for compounds in series 5. a CLogP values were calculated for the respective lipophilic side chains.For non-identical side chains, the value stated is the average of both individual side chains.
b Values given as greater than correspond to the highest concentration (500 μM) tested in the RBC assay.
Using n-propyl linkers clearly had a positive effect and led to development of the potent derivatives 13iA, 13jA, 13jG, 13lG and 13pG with broad-spectrum activity.All five derivatives displayed low haemolytic activity, making them promising candidates for further evaluation.

Trends in haemolytic activity
When examining the haemolytic activity of our compounds we saw a pronounced difference between compounds having n-propyl and n-butyl linkers.The core findings are presented in the following paragraph and a more detailed section on how the structures were compared can be found in chapter 1 of the Supporting Information.
First, we compared guanidyl-and amine-containing compounds with the same linkers and lipophilic side chains.For compounds with n-butyl linkers (4CG -4CA), all guanidyl containing compounds were more haemolytic than their amine counterparts except for when the (4-bromonaphthalen-1-yl)methyl (e) was present.Comparing n-propyl containing derivatives (3CG -3CA), we observed the reversed trend.The guanidyl derivatives were equally to pronouncedly less haemolytic than their amine counterparts.The difference in EC 50 values ranged from 0 μg/mL for side chain combination l to 269 μg/mL for side chain combination j.
Next, we compared the n-butyl with n-propyl linkers in the presence of amine groups (3CA -4CA).For side chain combinations a, e, j and p the compounds were of comparable haemolytic activity regardless of the linker length.Only for side chain combinations i and l the derivatives with n-propyl linkers (3CA) were less haemolytic by 151 and 158 μg/mL, respectively, compared to their n-butyl counterparts (4CA).The difference between n-butyl and n-propyl linkers was most eminent in the presence of guanidyl groups (4CG -3CG).All compounds having npropyl linkers (3CG) were less haemolytic than compounds with n-butyl linkers (4CG).The difference ranged from 25 μg/mL for side chain combination e to an impressive 347 μg/mL for side chain combination j.
This comparison clearly shows that n-propyl linkers not only led to derivatives with good broad-spectrum activity, but also low haemolytic activity.Despite the often noteworthy difference in EC 50 values for the two linkers, no obvious SAR could be delineated.

Summary of SAR analysis
The general trends of our SAR analysis are summarized in Fig. 3.When assessing the potency of the lipophilic side chains and the hydrocarbon linkers, amine and guanidine derivatives were not distinguished, as they generally follow the same trends.
We found that the antimicrobial activity decreased along the line of n-butyl > n-propyl > ethyl and haemolytic activity increased as follows: n-propyl < ethyl < n-butyl.The cyclic hydrocarbons, n-pentyl and nhexyl displayed varying MIC values, but where all too haemolytic to be of any practical use and were therefore excluded from the list.Guanyl compounds with n-butyl linkers were more haemolytic than their amine counterparts.As mentioned before, for ethyl and n-propyl linkers this Fig. 3. Overview over the general trends observed during the SAR investigation.The trends for haemolytic activity were assessed for the average between the respective amines and guanidines.

M.K. Langer et al.
trend was reversed.Based on this, n-propyl seemed to be the best compromise to achieve high antimicrobial activity and moderate haemolytic activity.
In line with our previous findings, the compounds potency and haemolytic activity increased with higher CLogP values of the lipophilic side chains for both, amines and guanidines.Bromines proved to be a good modulator of the hydrophobicity of aryl groups.The structure of the side chains seemed thereby to be secondary.The most potent compounds proved to be too haemolytic for future therapeutic considerations.By combining two lipophilic side chains of different structure and hydrophobicity (11kG and 11lG), antimicrobial potency and haemolytic activity of the compounds could be fine-tuned.
To achieve good broad-spectrum activity, amine or guanidine groups proved to be necessary.Methylated primary amines showed reduced activity against Gram-negative P. aeruginosa alongside reduced haemolytic activity.The least haemolytic cationic groups were the quaternary ammonium compounds in 8a and 9a.Due to its lack of haemolytic activity and high activity against Gram-positive bacterial strains, 8a could proof valuable for narrow-spectrum applications against Gram-positive bacteria.

Selectivity index
A common measurement for the efficiency of antimicrobial agents is the selectivity index (SI) given by the ratio EC 50 /MIC values (for all SI values see Table S1).Our efforts led to promising candidates for narrowas well as broad-spectrum applications.We have grouped them into three groups (Table 6) based on their activity and SI against Grampositive strains (entries 1-4), Gram-positive strains and E. coli (entries 5-7) and all strains tested (entries 8-11), respectively.Compounds were considered active if the MIC values were ≤16 μg/mL.
The first group, 8a, 11kG, 13cG and 13hG (Table 6, entries 1-4), comprised compounds that had a SI ≥ 54 for the Gram-positive strains, while showing no activity against Gram-negative strains and human red blood cells.These properties make them ideal candidates for narrowspectrum application against Gram-positive bacteria.
Compounds in the second group had SI ≥ 40 (Table 6, entries 5-7) against the Gram-positive strains and the Gram-negative E. coli and a medium SI (<20) against Gram-negative P. aeruginosa.Of the three compounds 9, 11lG and 13pG, only pyridinyl derivative 9 (entry 5) did not show measurable haemolytic activity.But despite having moderate EC 50 values (161 and 169 μg/mL), guanyl derivatives 11lG and 13pG had a high SI.
The third group comprises molecules with a SI ≥ 20 (Table 6, entries 8-11) against all four strains.Compounds 13aG and 13jA (entries 8-9) displayed a good overall SI and had also good activity against the Gramnegative P. aeruginosa (MIC: 8 μg/mL).Compounds 13jG and 13iA (entries 10-11) were mildly potent against P. aeruginosa (MIC: 16 μg/ mL), but due to their low haemolytic activity they still display promising SI values.Their absence of cytotoxicity makes them promising candidates, despite their mild activity against Gram-negative P. aeruginosa, keeping in mind that most naturally occurring AMPs display low activity against this Gram-negative strain as well [17].Additionally, group 3 compounds generally matched or outperformed our reference compounds 1aA and 1aG (entries 12-13).

Effect of the counterion on solubility and activity
The counterion of acidic and basic drugs is known to greatly influence their overall physicochemical properties such as solubility, membrane permeability and stability [54,55].From the long list of physiological anions for basic active pharmaceutical ingredients (APIs), hydrochloride salts are predominant [55] and known to improve water solubility [56].
We found that the water solubility of the TFA salts decreased noticeably when the CLogP values of the lipophilic side chains rose beyond 4. To study if we could counteract this trend, we converted selected compounds to HCl salts.Additionally, we wanted to investigate if the counterion affected the biological activity.Table 7 summarizes the re-evaluated MIC and EC 50 values of selected compounds as hydrochloride salts.Water solubility was assessed qualitatively by setting the threshold at 1 mg/mL.Entries 1-3 show that previously not soluble (− ) TFA salts became soluble (+).Compound 13iG (entry 4) and several others (data not shown) remained poorly soluble in water, especially if several bromine substituents were present in the lipophilic side chain.
Hydrochloride salts of the amine derivatives 13iA and 13jA exhibited no change in their MIC values and showed only slightly differing EC 50 values (entries 1-2).No clear trend could be observed whether hydrochloride salts tended to be more or less haemolytic than TFA salts.Surprisingly, the HCl salts of guanyl derivatives 13iG and 13jG displayed improved MIC values against S. aureus (Entries 3-4), while the activity against E. coli remained unchanged.Compound 13jG was the only HCl salt being considerably more haemolytic than its TFA counterpart (Entry 3), for yet undetermined reasons.The deceivingly higher haemolytic activity of derivatives 13iA and 13iG as HCl salts (Entry 2 and 4) can be attributed to the lower molecular weight of the HCl salts.

Mode of action studies
Luciferase-based biosensor assays (viability and membrane integrity)  were performed to explore the mode of action of promising compounds on B. subtilis 168 and E. coli K12 [57].The biosensor-based viability assay measures bacterial viability as light production through recombinantly expressed bacterial luciferase originating from the Photorhabdus luminescens lux operon.The addition of external substrates does not affect the production of light by the bacterial lux operon.The bacterium itself provides the pool of reduced flavin mononucleotide (FMNH2) and long-chain aliphatic aldehydes, which are the substrates responsible for light production.Bacterial luciferase is an excellent real-time sensor for bacterial viability, as NADH, NADPH, and ATP are necessary to constantly top up the substrates' pool.
The biosensor-based membrane integrity assay depends on the luciferase (lucGR gene) originating from the luminous click beetle Pyrophorus plagiophthalamus.In contrast to bacterial luciferase, the light reaction of lucGR is stringently reliant on the substrate D-luciferin, which is added externally.D-luciferin is inadequately crossing intact biological membranes at neutral pH.After the addition of antimicrobial substances, the uptake is explored to determine if the membrane becomes permeable to the substrate D-luciferin.An increase in light production occurs when D-luciferin enters (increased influx) through a compromised membrane.Light production peaks rapidly if membrane integrity is compromised and, thereafter, usually decreases while the ATP from dying cells is consumed.
Based on structural modifications, MIC values, haemolytic activity, and selectivity index, 17 compounds were selected for mode of action studies against B. subtilis 168 (see Supporting Information, Table S2) as they were mainly potent against Gram-positive bacteria.Furthermore, based on their broad-spectrum activity, 14 additional compounds were tested against both, the Gram-positive B. subtilis 168 and the Gramnegative E. coli K12 biosensor strain (see the Supporting Information, Tables S2 and S3).In general, most of the compounds tested affected viability and showed strong membrane disrupting activity against both bacterial strains.However, some of the compounds showed a more pronounced effect on viability and a faster membranolytic effect against B. subtilis compared to E. coli.For most compounds, both viability and membrane integrity were affected when the concentration of the compounds was higher than the MIC value.Additionally, increasing concentrations affected viability and membranolytic activity in increasing rates, indicating a concentration-dependent killing effect.We could not determine any relationship between structure/activity and the mode of action profiles.
We selected the broad-spectrum barbiturate 11lG to exemplify the results of the viability and membrane integrity assay in detail (Fig. 4 and Fig. 5).Barbiturate 11lG clearly affected the viability of B. subtilis (Fig. 4A, left).The membrane integrity assay was performed on the B. subtilis biosensor strain to confirm that the rapid decrease in bacterial viability was caused by membrane damage.Derivative 11lG showed a membrane-related mode of action as light emission decreased rapidly in a dose-dependent manner (Fig. 4B, left), similar to chlorhexidine (CHX) (Fig. 4B, right).The reference control CHX is a bactericidal agent recognized for its cell wall and membrane disruptive properties [58], with MIC values of 1.5 μg/mL against both, B. subtilis 168 and E. coli K12.
The disruptive membrane effect of barbiturate 11lG on B. subtilis was shown at a concentration as low as 6.4 μg/mL, which is approximately 1.6 times higher than its MIC (4 μg/mL) (Fig. 4B, left).The lowest concentration (3.2 μg/mL), which is slightly lower than its MIC value, showed a limited membrane disruption effect and the peak emission did not decline during the assay period.The bacterial concentration for these experiments was approximately 100 times higher than the concentration used in the MIC assay, which could explain why slightly higher concentrations of barbiturate 11lG were needed to affect the viability and membrane integrity.
When it comes to the effects of barbiturate 11lG on the viability and membrane integrity in the Gram-negative E. coli, the picture is somewhat different from that of the Gram-positive B. subtilis.The broadspectrum derivative 11lG affected the viability of the E. coli strain and showed a concentration-dependent killing effect like CHX (Fig. 5A).Although 11lG affected the viability, a much less prominent inner Fig. 4. The effects of 11lG (broad-spectrum) and CHX (positive control) on the kinetics of (A) viability and (B) membrane integrity in B. subtilis 168.Normalized light emission (normalized with a negative, untreated water control) is plotted as relative light units (RLU) over time (seconds).Light emission was measured each second for 180 s after adding the bacterial cell suspension (with 1 mM D-luciferin for the membrane integrity assay) to the analytes in separate wells.The multiples of the MIC values given in parentheses refers only to compound 11lG.The figure shows a representative data set from at least three independent experiments.membrane disruptive effect was observed as only the two highest concentrations (6.4 -12.8xMIC) gave a rise in light emission (and did not decline during the test period) (Fig. 5B, left).The delayed and reduced action of 11lG on the membrane integrity might be due to the outer membrane of E. coli, which probably acts as an additional barrier.
To confirm the assumption about the outer membrane barrier in E. coli, we used the 1-N-phenylnapthylamine (NPN) fluorescent probe to determine whether compound 11lG can affect the outer membrane to become more permeable.The small molecule NPN (219 Da) is weakly fluorescent in an aqueous solution, but when bound to phospholipids, it gives strong fluorescence [59].The hydrophobic NPN cannot efficiently cross the outer membrane of intact E. coli cells, yielding low fluorescence, but if the outer membranes is compromised, NPN can reach the periplasmic space and bind phospholipids of the inner and outer membranes, thus producing increased fluorescence.In this assay, low concentrations (3.2 μg/mL) of barbiturate 11lG led to higher fluorescence levels (Fig. 5C, left), but did not initially give any increase in luminescence in the inner membrane integrity assay (Fig. 5B, left).This phenomenon suggests that most of the cells are intact and viable without having significantly compromised integrity of the inner membrane but have increased permeability of the outer membrane.Upon increasing the concentration of barbiturate 11lG, the fluorescence levels were lower (Fig. 5C, left) indicating either an intact outer membrane or rapid membrane disintegration before the start of the measurement.At the same time the viability of the bacterial cells was clearly reduced (Fig. 5A, left) and the inner membrane integrity was impaired (Fig. 5B, left).
When the 10 μL sample of the NPN assay was spotted on an agar plate after the test period, the viability of the bacterial cells was clearly reduced for concentrations of 25.6-51.2μg/mL (6.4 -12.8xMIC) (see Fig. S4), confirming the bactericidal effect of barbiturate 11lG.Those results strongly suggest that barbiturate 11lG disrupts both the outer and the inner membrane at the same rate when the concentration is high enough.However, it cannot be excluded that higher concentrations of 11lG induce a different mode of action, resulting in the compound crossing the outer membrane without disrupting it.
Our results indicate that the primary mode of action for most of the compounds, including the broad-spectrum barbiturate 11lG, against both the Gram-positive B. subtilis and the Gram-negative E. coli, is the disruption of the membrane integrity in a concentration-depended manner.However, it is known that certain cationic AMPs exhibit a concentration-dependent dual mode of action [60].For example, the N-terminal 1-35 fragment of Bac7 (a proline-arginine-rich AMP) is known to affect the inner membrane at high concentrations and bind to and affect intracellular chaperone protein DnaK and 70S ribosomes at lower concentrations [61][62][63].Therefore, there might also be other targets than the bacterial cytoplasmic membrane, and more work is required to conclude if there is any dual mode of action present or not.

Conclusion
In the present study, we have investigated the qualitative influence of the individual structural components of N,N-dialkylated-5,5-disubstituted amphipathic barbiturates on their bioactivity.We found that npropyl linkers provide the best balance between antibacterial potency and haemolytic activity and n-butyl linkers provide the highest potency.Guanidyl head-groups led to the highest antimicrobial potency, whereas trimethylated amines proved to be attractive for narrow-spectrum application.By choosing the individual components carefully, we were able to prepare several compounds having SI values ≥20 and being active towards two (8a, 11kG, 13cG, 13hG), three (9a, 11lG, 13pG) or all four (13aG, 13iA, 13jA, 13jG) strains of our test panel.The best compounds (13aG, 13jG and 13jA) had an improved selectivity index compared to the initial starting point (1aG).
Studies on the integrity of the membranes and the viability of bacterial cells suggest that our compounds exert their bactericidal activity by disrupting the bacterial cell wall of Gram-positive B. subtilis in a concentration-dependent manner as exemplified by barbiturate 11lG.In Gram-negative E. coli both, the inner and outer membrane, were supposedly rapidly disrupted at higher compound concentration, but a second mechanism of action might be present in addition.
We believe that our detailed analysis can help to devise new amphipathic cationic mimics of antimicrobial peptides.

Experimental section
For a detailed description of all chemical and biological experimental procedures, chemical analysis, and supporting results, see the Supporting Information.Additional raw data is available through the Data-verseNO repository, link: https://doi.org/10.18710/GNTWOG.

Fig. 2 .
Fig. 2. General structure of the tetrasubstituted barbituric acids used in this study.R = cationic group.The individual parts were evaluated in five series, namely screening of the cationic moieties (series 1), lipophilic side chains (series 2 side chain 1 = side chain 2 and series 3 side chain 1 ∕ = side chain 2), hydrocarbon linker chains (series 4) and optimized structures (series 5).

Fig. 5 .
Fig. 5.The effects of 11lG (broad-spectrum) and a CHX (positive control) on the kinetics of (A) viability and (B) inner membrane integrity (C) outer membrane integrity in E. coli K12.Normalized light emission (normalized with a negative, untreated water control) is plotted as relative light units (RLU) over time (seconds) for A and B. For C, normalized fluorescence (normalized with a negative, untreated water control) is plotted as relative fluorescence units (RFU) over time (seconds).Light emission/fluorescence was measured each second for 180 s after adding the bacterial cell suspension (with 1 mM D-luciferin for the inner membrane integrity assay and 20 μM 1-N-phenylnapthylamine for outer membrane integrity assay) to the analytes in separate wells.The multiples of the MIC values given in parentheses refers only to compound 11lG.The figure shows a representative data set from at least three independent experiments.

Table 2
Antimicrobial activity (MIC in μg/mL) against bacterial reference strains and haemolytic activity against human RBC (EC 50 in μg/mL) for compounds in series 2.

Table 6
Selectivity index (SI) of the most promising wide and narrow-spectrum antimicrobials.EC 50 values are given in [μg/mL].
a No SI was calculated if the MIC was >16 μg/mL.b Values given as greater than correspond to the highest concentration (500 μM) tested in the RBC assay.