Small-Molecule Inhibitors of the NusB–NusE Protein–Protein Interaction with Antibiotic Activity

Abstract

The NusB–NusE protein–protein interaction (PPI) is critical to the formation of stable antitermination complexes required for stable RNA transcription in all bacteria. This PPI is an emerging antibacterial drug target. Pharmacophore-based screening of the mini-Maybridge compound library (56 000 molecules) identified N,N′-[1,4-butanediylbis(oxy-4,1-phenylene)]bis(N-ethyl)urea 1 as a lead of interest. Competitive enzyme-linked immunosorbent assay screening validated 1 as a 20 μM potent inhibitor of NusB–NusE. Four focused compound libraries based on 1, comprising 34 compounds in total were designed, synthesized, and evaluated as NusB–NusE PPI inhibitors. Ten analogues displayed NusB–NusE PPI inhibition ≥50% at 25 μM concentration in vitro. In contrast to representative Gram-negative Escherichia coli and Gram-positive Bacillus subtilis species, these analogues showed up to 100% growth inhibition at 200 μM. 2-((Z)-4-(((Z)-4-(4-((E)-(Carbamimidoylimino)methyl)phenoxy)but-2-en-1-yl)oxy)benzylidene)hydrazine-1-carboximidamide 22 showed excellent activity against important pathogens. With minimum inhibitory concentration values of ≤3 μg/mL for Gram-positive Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus and ≤51 μg/mL for Gram-negative Pseudomonas aeruginosa and Acinetobacter baumannii, 22 is a potent lead for a novel antibacterial target. Epifluorescence studies in live bacteria were consistent with 22, inhibiting the NusB–NusE PPI as proposed.

Introduction

Antibiotics are pivotal to modern medicine. They enable clinicians to conduct invasive surgery, treat immune-compromised patients, and carry out blood transfusions on trauma victims with a minimal risk of death due to secondary bacterial infections.^1,2 However, the prevalence of multidrug-resistant bacteria threatens our ability to survive clinically and community-acquired infections. This increasing prevalence of multidrug-resistant bacteria has the very real potential to undermine all of these significant medical advances.^3,4

Antibiotic-resistant bacteria are estimated to result in 48 000 deaths annually in the United States and Europe.^3,5 Of equal concern is that the Food and Drug Administration (FDA) approved only one new antibiotic in 2015, Avycaz (avibactam/ceftazidime), for the treatment of complicated intra-abdominal infections.⁶ This lack of innovation and investment has meant that a number of multidrug-resistant bacterial strains, particularly the “ESKAPE” pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, are extremely challenging to treat and, in some cases, require complex antibiotic cocktails.⁷ Potentially of greater concern is that the current antibiotic development places considerable emphasis on new iterations of existing drugs, and hence these agents are vulnerable to the rapid acquisition of resistance from the dissemination/modification of the preexisting mechanisms.⁸ Clearly, there is a pressing need to develop new antibiotic classes, especially those with a lower inherent resistance susceptibility.^4,8−12 As a result, global strategies, such as “The 10 × 20 Initiative”, seek to combat this crisis, and this initiative has the ambitious target of 10 new antibacterial drugs by 2020.¹³

Key to the development of next-generation antibacterial agents is the identification of new drug targets, and, to this end, there is a growing focus on interrogating the bacterial interactome to identify essential protein–protein interactions (PPIs). These PPI networks can, in principle, be targeted by small-molecule inhibitors.¹⁴⁻¹⁶ To date, the inhibition of PPIs has proved fruitful, with multiple PPI-targeted drugs receiving FDA approval, including Lifitegrast, Venetoclax, and Birinapant.^17,18 These accomplishments have validated PPIs as drug targets, thus opening up opportunities to develop new classes of antibacterial agents.^18,19

A typical PPI is predominantly hydrophobic in nature, with a handful of polar residues located centrally across a protein surface of area 1000–2000 Ų. The polar, and nearby hydrophobic, residues give rise to critical small “hot spots” and impart a significant proportion of the binding energy responsible for the observed PPI.^18,20,21 These hot spots and the presence of a degree of conformational flexibility make targeting PPI an attractive potential therapeutic intervention. One such PPI in the bacterial interactome is the interface between the transcription factors, NusB and NusE.

The NusB–NusE PPI is a critical nucleation point for the formation of the antitermination complex enabling the regulation of bacterial stable (t- and r-) RNA transcription.²² In the Gram-negative model, Escherichia coli, point mutations, for example, nusE100 (R72G)²³ and nusB5 (Y18D),²⁴ result in a reduced protein–protein binding affinity, affecting the formation of the antitermination complex.^24,25 Strains nusE100 and nusB5 are unable to efficiently transcribe the 16S and 23S ribosomal transcripts, which impedes the formation of new ribosomes and leads to reduced growth,^26,27 demonstrating the importance of the NusB–NusE binding interface.

The examination of the Aquifex aeolicus (PDB: 3R2C) and E. coli (3D3B) NusB–NusE heterodimer crystal structures reveals a PPI surface area of ∼1600 Ų (Figure 1A).^25,28 The PPI interface comprises a mixture of hydrophobic and hydrophilic interactions resulting from helix α1 and strand β2 of NusE bridging the two helical bundles of NusB (Figure 1A–C). Because of the complexity of biomacromolecules under physiological conditions, significant differences typically exist between the NMR and X-ray crystallographic structures of the same protein and so we chose to use both the NMR and crystal structures of A. aeolicus and E. coli proteins to reveal the major hydrogen-bonding contributions.^25,28,29 As seen in Figure 1B,C, these occur between NusB E81 (E. coli E81)–NusE H15 (E. coli H15), NusB Y16 (E. coli Y18)–NusE D19 (E. coli D19), and NusB R76 (E. coli E75)–NusE R16 (E. coli R16) interactions (Figure 1B). The NusB E81 (E. coli E81)–NusE H15 (E. coli H15) interaction is absent in the E. coli crystal structure,²⁵ which is consistent with the high relative B factors observed, indicative of the conformational flexibility in those regions in both the A. aeolicus and E. coli protein crystal structures (PDB IDs: 3R2C and 3D3B, respectively). A close examination of the modeled interface highlights a potentially key interaction between the E. coli E81 and H15 residues and reflects the structural information in solution.²⁹ Subsequently, we developed a hybrid NusB–NusE interface using the information from the A. aeolicus NMR study and the crystal structure, as well as the E. coli crystal structure (Figure 1),^25,28,29 which united the structural information from both techniques and two species.

Figure 1.

(A) Cocrystal structure of A. aeolicus NusB–NusE (PDB ID: 3R2C). (B) Enlarged region of the A. aeolicus NusB–NusE PPI interface with the amino acids (as sticks) involved in PPIs, with the black dashed lines indicating key hydrogen-bonding interactions between H15, R16, and D19 of the α1-helix of NusE and Y16, R76, and E81 of NusB. (C) Enlarged region of the E. coli NusB–NusE PPI interface with the amino acids (as sticks) involved in PPIs, with the black dashed lines indicating key hydrogen-bonding interactions between H15, R16, and D19 of the α1-helix of NusE and Y18, E75, and E81 of NusB. (D) Pharmacophore model based on the NusE amino acids responsible for binding with NusB, with 1 fitted into the pharmacophore. Purple sphere: hydrogen bond donor; green sphere: hydrogen bond acceptor; gray sphere: exclusion zone.

Previously, we reported the development of a pharmacophore model, on the basis of the published NMR and X-ray crystallography structures of the NusB–NusE PPI of A. aeolicus and E. coli.³⁰ This model merged key structural information from three different studies and two bacterial species.^25,28,29 Critical to this model was the α1-helix sequence of NusE, which interacts with the binding groove of NusB. Three critical hydrogen bond interactions between the α1-helix of NusE (D19, R16, and H15) and the binding groove of NusB (Y16 (E. coli Y18), R76 (E.coli E75), and E81), as shown in Figure 1B,C, were manually plotted to generate a pharmacophore (Figure 1D). Screening of this pharmacophore against the mini-Maybridge compound library (56 000 molecules) identified 25 hits. A pharmacophore validation was then conducted using a competitive enzyme-linked immunosorbent assay (ELISA)-based screen and a subset of hits, which were synthesized in-house. From the screen 1,1′-((butane-1,4-diylbis(oxy))bis(4,1-phenylene))bis(3-ethylurea), 1 was identified as a 19.8 ± 1.7 μM inhibitor of the NusB–NusE PPI (Figure 2).³⁰

Figure 2.

Chemical structure of 1,1′-((butane-1,4-diylbis(oxy))bis(4,1-phenylene))bis(3-ethylurea) 1 identified through in silico screening of the mini-Maybridge library and ELISA screening of the NusB–NusE interaction as a 20 μM potent inhibitor of the NusB–NusE PPI. Also depicted are the four regions identified for modification and SAR generation.

Herein, we report the computational and biological screening-guided design, synthesis, and characterization of four structural activity relationship libraries, which focus on modifications to four key regions of lead 1, the bis-ether linker region (A), head-group orientation (B), role of asymmetry (C), and head-group functionality (D), to develop inhibitors of the bacterial PPI between NusB–NusE as potential antibacterial agents (Figure 2).

Results and Discussion

In this work, our previously developed pharmacophore was ported to the molecular operating environment (MOE) software and used to perform the docking analysis of 1 with the NusB A. aeolicus (PDB: 3R2C) interface.^31,32 After initial docking of 1 at the NusB interface, the docked system was subjected to a short molecular dynamics cascade (production step of 2 ns at 300 K), which revealed the predicted pose for 1 as “horseshoe-like” that enabled key hydrogen bond interactions with Y16, R76, and E81 consistent with the initial pharmacophore in silico screening of the mini-Maybridge compound library (Figure 3).

Figure 3.

Docking study of lead 1 at the proposed A. aeolicus (PDB: 3R2C) NusB–NusE interface (stick representation, carbon atoms are shown in yellow). The pharmacophore features are shown as spheres, namely, hydrogen bond donor (pink) and hydrogen bond acceptor (green). Predicted hydrogen bond interactions with key residues (green) are shown as dashed lines. Compound 1 shows a horseshoe orientation with one ether oxygen hydrogen bonding with the side chain of Y18 and the adjacent urea moiety hydrogen bonding with E81 (side chain) and R76 (backbone).

On the basis of the above docking study, analogues 10a–c were designed to probe the optimal linker length, whereas 10d would examine the impact of heteroatom incorporation. The remaining analogues in this library, 10e–i, were proposed to explore the optimum turn radius of the “horseshoe” binding conformation (Scheme 1). The synthesis of the focused library commenced with the coupling of 4-nitrophenol 2 under modified Finkelstein conditions with α,ω-dichloro linkers 5a–c to give the corresponding bis-ethers 6a–c. Flow hydrogenation (ThalesNano H-cube) over Raney Ni facilitated a quantitative conversion to the corresponding amines 8a–c. The treatment of these amines with ethyl isocyanate afforded the desired urea analogues 10a–c. In an effort to generate hydrogenation-susceptible linkers (e.g., alkenes 8e and 8f, Scheme 1), the synthesis commenced with the corresponding N-Boc-4-aminophenol 3, followed by coupling with the appropriate α,ω-dichloro linker 5e and 5f to afford 7e and 7f. Boc removal (HCl/dioxane) and coupling with ethyl isocyanate gave the desired urea analogues 10e and 10f. Bis-ureas 10g–i were accessed by an alternative pathway, where 4-aminophenol 4 was treated with ethyl isocyanate, yielding urea 9, followed by coupling with an α,ω-dichloro linker to give the desired compounds. This urea-based library, 10a–f, was evaluated for their ability to inhibit the NusB–NusE PPI using a Bacillus subtilis NusB and a glutathione-S-transferase (GST)-tagged NusE competitive ELISA. These data are presented in Table 1.³⁰

Scheme 1. Reagents and Conditions.

(a) Cs₂CO₃, KI, CH₃CN, 5a–i, reflux, 16 h; (b) ThalesNano H-Cube, 50 mM in 1,4-dioxane, Raney Ni (30 or 70 mm cartridge) 50 °C, 50 bar, 0.5 mL min^–1, recirculated; (c) 4 M HCl in dioxane, sonication, 30 min; (d) ethyl isocyanate, Et₃N, anhydrous tetrahydrofuran (THF), reflux, 16 h.

Table 1. Inhibition of the NusB–NusE Binding by 1 and 10a–i at 25 μM Compound Concentration Using an ELISA.

The examination of the data presented in Table 1 indicated that minor adjustments to the linker length were tolerated with 1, 10a and 10b displaying 52–59% inhibition of the NusB–NusE PPI at 25 μM. However, elongation to heptyl 10c removed the activity, as did the incorporation of an ether linker 10d (Table 1). In keeping with the docking study prediction, the introduction of turn-inducing linkers 10e and 10g afforded an increase of activity to 72 and 65% respectively. Hence, the turn radius appears crucial as the 1,3-disubstituted phenyl derivative 10h and the furan derivative 10i displayed a marked reduction in activity. With analogue 10h, the data suggest that the turn radius was too high for efficient positioning of the urea head groups essential for hydrogen bonding with D75, R76, and E81. Furan 10i also showed a loss in activity, which was most likely a consequence of the introduction of a heteroatom to the linker (cf. 10d). The diminished activity of 10i and 10d, in addition to the visual inspection of the docked compounds, suggested that the hydrophobic cleft shaped by L20, Y79, and V80 of the NusB-binding groove does not tolerate electronegative atoms (Figure 4). This hypothesis was further supported by the improved binding affinity of hydrophobic linkers 10e and 10g.

Figure 4.

Docking studies of 10e at the proposed A. aeolicus NusB–NusE interface, with the stick representation of atoms colored by type. Key amino acids L20, Y79, and V80 that form the hydrophobic cleft, which interacts with the hydrophobic linker region of 10e, are depicted. In addition, the close proximity of R76 (side chain) to the ethylurea head group of 10e is shown.

The initial docking study of 1 indicated that one of the urea moieties adopted an orientation in close proximity to R76 (Figures 3 and 4). This suggested that a modification of the urea moiety may affect the binding affinity of subsequent analogues. As a result, we explored the development of a second library based on 10e. The initial focus turned to the positioning of the pendent urea moieties through the synthesis of the remaining 1,2- and 1,3-substituted ureas. These analogues were synthesized according to Scheme 1, commencing from the corresponding N-Boc-2-phenol and N-Boc-3-phenol to give 10j and 10k, respectively (Table 2). We also examined the effect of installation of a single urea isostere with a retention of one urea moiety, giving asymmetric analogues 13a–i. The synthesis of these asymmetric analogues commenced from mono-urea 9, which was coupled with (Z)-1,4-dichloro-2-butene 5c, giving 11, which, in turn, was treated with a range of substituted phenols to give rise to 13a–i (Scheme 2). The asymmetric 13a–i were screened for their ability to inhibit the NusB–NusE PPI using an ELISA, and the data are presented in Table 2.

Table 2. Inhibition of NusB–NusE Binding Interaction by 10e, 10j, 10k, and 13g–i at 25 μM Compound Concentration Using an ELISA.

Scheme 2. Reagents and Conditions.

(a) Cs₂CO₃, 12a–i, anhydrous dimethylformamide (DMF) 75 °C, 2 h; (b) 4 M HCl in dioxane, room temperature, 2 h.

As demonstrated by the data presented in Table 2, 10j and 10k were significantly less active than 10e, supporting a 1,4-substitution pattern as a requirement for inhibitory activity. Additionally, 13a–i were less active at 25 μM than 10e, indicating that a urea moiety is a curial component of the binding affinity. Within the asymmetrically substituted library, 13i was the most potent compound, inhibiting 50% of binding at 25 μM.

Having identified the crucial role of a urea moiety, the subsequent library investigated a series of urea bioisosteres. As outlined in Scheme 3, compounds 15a–f were synthesized under standard second-order nucleophilic substitution conditions to afford the desired bis-ether derivatives. N-Methylacetamide 16 was accessed by the treatment of 8e with acetyl chloride. Thiourea 17 was synthesized by the reaction of 8e with ethyl isothiocyanate in the presence of triethylamine. Saponification of 15e yielded carboxylic acid 18, which underwent amide coupling with methylamine to give 19. Nitrile 15d provided oxadiazole 20 in two steps, and on treatment with trimethylaluminum and ammonium chloride afforded the imidamide 21. Finally, compound 22 was accessed via a microwave-facilitated imine formation using aldehyde 13f and a catalytic amount of HCl and aminoguanidine. These analogues were screened for their ability to inhibit the NusB–NusE PPI, and the data are presented in Table 3.

Scheme 3. Reagents and Conditions.

(a) K₂CO₃, KI, CH₃CN, reflux, 16 h; (b) N,N′-diisopropylethylamine (DIPEA), acetyl chloride, anhydrous CH₂Cl₂, room temperature, 16 h; (c) ethyl isothiocyanate, triethylamine (TEA), anhydrous THF, reflux, 16 h; (d) 10% aq KOH/THF (2:1), reflux, 1 h; (e) thionyl chloride, four drops of anhydrous DMF, CH₂Cl₂, 40 °C, 4 h; (f) 2 M CH₃NH₂/THF, DIPEA, room temperature, 1 h; (g) hydroxylamine, CH₃CN, reflux, 4 h; (h) acetyl chloride, 3 Å molecular sieves, THF, reflux, 16 h; (i) ammonium chloride, 2 M (CH₃)₃Al/PhCH₃, N₂, 0–80 °C, 16 h; (j) aminoguanidine HCl, cat. 10% HCl, ethanol, μWave, 120 °C, 0.5 h.

Table 3. Evaluation of NusB–NusE Binding Inhibition at 25 μM Using an ELISA 6e, 7e, and 13–21.

^a‘-’ no activity at 25 μM compound concentration.

The moderate activity of 18 aligned with the initial docked conformation (Figure 4), which suggested one of the urea moieties resided within close proximity to R76; however, this result also indicated that for this interaction to occur the ionic moiety must be relatively small (e.g., 18 vs 13b and 13c) (Table 2). Nonetheless, with the exception of 18 and 8e, a biological evaluation of this fourth series of compounds indicated that an amide moiety was required with compounds 15a–f exhibiting ≤43% inhibition. Additionally, the dual nitrogen atoms of the urea moiety appear to be essential for activity with the removal of either the nitrogen α-16 or γ-19 to the aromatic ring (relative to 10a), resulting in a 27 or 15% reduction of NusB–NusE PPI inhibition, respectively. This inference was supported by the acetimidamide 21 being devoid of activity and the reduced activity of 7e. A further bioisosteric replacement of the oxygen 10e with sulfur, 17, abolished activity. However, installation of mono-aminoguanidine 22 or carboxylic acid 18 afforded a similar binding inhibition to lead compound 1.

Having established SAR data based on the four focused libraries developed herein, we evaluated analogues with >50% inhibition in the NusB–NusE binding ELISA as potential inhibitors of bacterial growth. As outlined in Table 4, B. subtilis and E. coli were used as representative Gram-positive and Gram-negative species, respectively.

Table 4. Percentage Inhibition of B. subtilis and E. coli Growth by Bis-Ether Analogues 1, 8e, 10a, 10b, 10e, 10g, 13i, 16, 18, and 22 at 200 μM Compound Concentration.

	percent bacterial growth inhibition at 200 μM			percent bacterial growth inhibition at 200 μM
compound	B. subtilis	E. coli	compound	B. subtilis	E. coli
1		17	10g		19
8e	17	3	13i	37	6
10a	31		16	35	14
10b	8	20	18	15	9
10e	26	23	22	100	100

Pleasingly, all compounds in this analysis exhibited some level of bacterial growth inhibition ranging from mild to excellent at 200 μM across both E. coli and B. subtilis or against a single species. Analogues 1 and 10g exhibited selective inhibition of E. coli at 17 and 19%, respectively. Compound 10a selectively inhibited the growth of B. subtilis at 31%. Notably, the incorporation of a cis-butene linker with 8e, 10e, 13i, 16, 18, and 22 resulted in an antibacterial activity against both Gram-positive and Gram-negative organisms. Of the analogues evaluated herein, 22 showed the greatest activity with 100% inhibition against both B. subtilis and E. coli. Although our ELISA evaluation of these analogues showed promising levels of inhibition of the NusB–NusE PPI, the use of these compounds in bacteria screen reveals a poor correlation between ELISA and phenotypic outcomes, which is most probably a consequence of either a poor uptake or a rapid efflux of these compounds.

As our initial lead 1 has been predicted (but not demonstrated) to be hepatotoxic,³³ we examined a number of analogues in a panel of 11 cancer and 1 normal cell lines. However, we detected no cytotoxicity for our lead 1 or for the related analogues 8e, 10a, 10b, 10e, and 18. Toxicity, at a level comparable to the minimum inhibitory concentration (MIC) values, was noted with analogues 13i and 22 (Table 5), but after a 4-fold increase in exposure times (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay conducted over 72 h; MIC evaluation over 18 h).³⁴ It is important to recognize the difference between chronic and acute use and that this in vitro toxicity determined using human cancer cell lines is not a demonstration of in vivo toxicity. Ultimately, only in vivo evaluation will be the determinant of safety and tolerability.³⁵

Table 5. Growth Inhibition (GI₅₀ μM) Values of Analogues 1, 8e, 10a, 10b, 10e, 13i, 18, and 22 against a Panel of Human and Murine Cancer Cell Lines.

	compound
cell line	1	8e	10a	10b	10e	13i	18	22
HT29	>50	>50	>50	>50	>50	15 ± 0.000	>50	2.1 ± 0.050
U87	>50	>50	>50	>50	>50	29 ± 1.4	>50	2.0 ± 0.10
MCF-7	>50	>50	>50	>50	>50	17 ± 0.82	>50	2.3 ± 0.10
A2780	>50	>50	>50	>50	>50	18 ± 0.91	>50	2.4 ± 0.14
H460	>50	>50	>50	>50	>50	21 ± 2.1	>50	2.2 ± 0.065
A431	>50	>50	>50	>50	>50	17 ± 0.750	>50	2.2 ± 0.11
Du145	>50	>50	>50	>50	>50	13 ± 0.41	>50	1.7 ± 0.12
BE2-C	>50	>50	>50	>50	>50	18 ± 3.2	>50	2.3 ± 0.13
SJ-G2	>50	>50	>50	>50	>50	24 ± 2.6	>50	1.8 ± 0.048
MIA	>50	>50	>50	>50	>50	28 ± 0.82	>50	2.4 ± 0.14
SMA	>50	>50	>50	>50	>50	28 ± 4.4	>50	2.9 ± 0.000
MCF10A	>50	>50	>50	>50	>50	19 ± 0.50	>50	2.8 ± 0.15

With 22 displaying a promising antibacterial activity, it was further examined against four clinically relevant Gram-positive and Gram-negative human isolates (Table 6). The MIC for 22 was determined against community-acquired methicillin-resistant Staphylococcus aureus USA300 (CA-MRSA), Streptococcus pneumoniae D39, Pseudomonas aeruginosa PA14, and Acinetobacter baumannii ATCC19606.

Table 6. MIC of Analogue 22 (μg/mL).

compound	Staphylococcus aureus USA300	Streptococcus pneumonia D39	Pseudomonas aeruginosa PA14	Acinetobacter baumannii ATCC19606
22	≤3	≤3	≤51	≤51

An examination of the data presented in Table 5 shows 22 as highly potent, with an MIC of ≤3 μg/mL (7 μM), against both S. aureus USA300 and S. pneumoniae D39. This result is comparable to that of clinically relevant tetracycline, which has an MIC of 12–96 μg/mL against isolates of S. aureus USA300.³⁶ In addition, 22 showed a promising activity against both P. aeruginosa PA14 and A. baumannii ATCC19606 with an MIC of ≤51 μg/mL (125 μM) against both isolates. Again, this promising result is comparable to the data obtained previously with penicillins, cephalosporins, and carbapenems, which have MICs of 4–16 μg/mL against P. aeruginosa.³⁷

To confirm that compound 22 had a mechanism of action consistent with the inhibition of rRNA transcription through targeting the NusB–NusE interaction, epifluorescence microscopy was performed on B. subtilis strains BS23 and BS61.^38,39 Strain BS23 contains a GFP fusion to the α subunit of the membrane-localized ATP synthase, whereas BS61 contains a GFP fusion to NusB that has a signal restricted to the subnucleoid foci that represent the sites of rRNA synthesis (Figure 5A,D, respectively).^38,39 The treatment of BS23 with 22 (Figure 5C) at 3 μM (1.2 μg/mL) had no effect on ATP synthase localization compared with colistin (Figure 5B), which caused the delocalization of the ATP synthase indicative of a membrane damage. This result confirms that 22 does not target the cell membranes. Furthermore, the lack of a morphological change of the cell outline or filamentation, which is indicative of the cell wall synthesis, cell division, or chromosome segregation defects, suggests that 22 does not affect the cytoplasmic membrane, cell wall integrity, or DNA synthesis. The treatment of BS61 (NusB-GFP) with 22 (Figure 5F) caused a significant delocalization of the NusB-GFP signal, similar to that seen with rifampicin, a known inhibitor of transcription (Figure 5E). The delocalization of the NusB-GFP signal is consistent with the loss of rRNA transcriptional activity similar to that seen on the induction of the stringent response, a bacterial starvation response that results in a massive downshift of the rRNA synthesis,³⁹ supporting the result of our modeling and ELSIA study that 22 is able to target the NusB–NusE interaction in live cells.

Figure 5.

Epifluorescence images of B. subtilis BS23 (GFP fusion to the α subunit of ATP synthase: (A–C)) and BS61 (GFP fusion to NusB: (D–E)). (A) Control, untreated exponentially growing BS23; (B) BS23 treated with colistin (Col); (C) treatment of BS23 with compound 22; (D) control, untreated exponentially growing BS61; (E) BS61 treated with rifampicin (rif); (F) BS61 treated with 22.

The epifluorescence microscopy data are consistent with the ability of 22 to target NusB and inhibit rRNA synthesis in live cells and support our proposed mechanism of action with 22. However, the cytotoxicity of 22, although not inherent within this compound class (cf. 1, 8e, 10a, 10b, and 10e, all of which inhibit the NusB–NusE PPI >50% at 25 μM and show no cytotoxicity; Table 5), suggests that that there is an additional unidentified off-target effect of this analogue.

Conclusions

A screening of our NusB–NusE pharmacophore against the mini-Maybridge compound library (56 000 molecules) and a subsequent ELISA screening identified 1 as an inhibitor of the NusB–NusE PPI. Guided by the molecular modeling approaches, the subsequent development of four focused compound libraries led to the identification of 22 as a potent antibacterial agent active against clinically relevant Gram-positive isolates S. aureus USA300 (methicillin resistant) and S. pneumoniae, with an MIC of ≤3 μg/mL against both strains. In addition, 22 showed a promising activity against problematic Gram-negative isolates P. aeruginosa PA14 and A. baumannii ATCC19606, which have proven to be difficult to treat, with an MIC of ≤51 μg/mL against both isolates. Furthermore, using epifluorescence microscopy, we demonstrated that the mode of action of 22 is consistent with the inhibition of the interaction of NusB with NusE, which would lead to a significant reduction in rRNA synthesis. We believe that 22 is a promising lead compound for the development of next-generation broad-spectrum antibiotic agents, further validating the NusB–NusB protein–protein binding interaction as a potential antibacterial target. However, given the observed cytotoxicity of this analogue, a careful cytotoxicity screening for the retention of this activity should be employed in the further development of this analogue. Notwithstanding this, the lack of cytotoxicity for other analogues within this family that also displayed good levels of NusB–NusE PPI interaction, but only low levels of antibiotic activity, supports the further development of this compound class. Hence, our current focus is aimed at improving the MIC value of 22 and examining the antibacterial effects of subsequent analogues in other clinically problematic bacteria. This represents a new class of antitranscription antibiotic leads with activity against clinically relevant Gram-positive and Gram-negative bacteria strains. As we have demonstrated an antibiotic effect that supports the inhibition of the NusB–NusE PPI, future analogues targeting this interaction should design away from any cytotoxicity liability. Notwithstanding this, an in vivo evaluation of later generations will be the ultimate determination of toxicity.

Experimental Section

Biology

Plasmid Construction

All of the cloning steps were carried out in E. coli DH5α ( Table S1). The plasmids and bacterial strains used and constructed in this work were confirmed by DNA sequencing and are listed in Table S1. B. subtilisnusB was amplified using primers 5′-AAAGGAGATCTAGACATGAAAGAAGA-3′ and 5′-TTTTCTGGTACCCTATGATTCCC-3′ from purified B. subtilis chromosomal DNA. The nusB mutants were made by PCR splicing using mutant primers (altered bases in lower case) 5′-CTTTGCAGGCACTAgcTCAAATTGATGTC-3′ and 5′-GACATCAATTTGAgcTAGTGCCTGCAAAG-3′ (F15A); 5′-GAATTGGAAGCTCGATgcGATTGCCAATG-3′ and 5′-CATTGGCAATCgcATCGAGCTTCCAATTC-3′ (R70A); and 5′-GATTGCCAATGTTGcCCGTGCGATTTTGC-3′ and 5′-GCAAAATCGCACGGgCAACATTGGCAATC-3′ (D75A).⁴⁰ The amplicons were cut with XbaI and Acc65I and inserted into similarly cut pETMCSIII (Table S1) to produce pNG130, pNG1178, pNG1179, and pNG1180, respectively (Table S1). B. subtilisnusE was amplified using primers 5′-AGGAGGGTCTAGAATGGCAAAAC-3′ and 5′-CTATATTTTAGGTACCAAGTTTAATTT-3′ from B. subtilis chromosomal DNA and ligated into the NdeI and Acc65I sites of pNG651 to give pNG896.

Protein Overproduction and Purification

B. subtilis NusB (wild type and mutant) and NusE-GST were overproduced and purified using a similar approach to that described previously.^15,41 Briefly, E. coli BL21 (DE3) was transformed with one of the protein overproduction plasmids (Table S1) and cultures were grown in an autoinduction medium for 48 h at 25 °C. Following lysis and clarification, the NusB proteins were purified using a 1 mL HisTrap HP column (GE Healthcare) and the GST-tagged NusE was purified using a 1 mL GSTrap column (GE Healthcare). The purified proteins were dialyzed into 20 mM KH₂PO₄, 150 mM NaCl, 30% glycerol, pH 7.8, and stored at −80 °C.

ELISA

Purified full-length B. subtilis NusB was diluted to 250 nM in phosphate-buffered saline (PBS), and 100 μL of the solution was added into NUNC Maxisorp microtitre plate wells. Following overnight incubation with the NusB solution at 4 °C, the wells were washed three times with 300 μL of PBS buffer and blocked with 300 μL of 1% (w/v) bovine serum albumin dissolved in PBS buffer at room temperature. After 2 h blocking, the plates were washed three times with a wash buffer (PBS, 0.05% (v/v) Tween-20). The appropriate inhibitor (Tables 1–4) and 100 μL of affinity-purified GST-tagged NusE at 200 nM were incubated at 37 °C for 15 min, then added to each well, and incubated for 1 h at room temperature. Unbound NusE was removed by washing each well three times in 300 μL of the wash buffer. Rabbit anti-GST primary antibody (100 μL, 1:2000 in PBS) was added to each well and incubated for 1 h at room temperature. After washing, goat-anti-rabbit HRP secondary antibody (1:2000 in PBS) was added to each well, incubated for 1 h at room temperature, and then washed three times in 300 μL of the wash buffer. Visualization of PPI was achieved by adding 100 μL of 3,3′,5,5′-tetramethylbenzidine (liquid substrate system for ELISA, Sigma-Aldrich) to each well. The plate was incubated in a plate reader (FLUOstar Optima) at 37 °C with 200 rpm shaking for 6 min. The optical density of each well was recorded at 600 nm.

Growth Inhibition Assay

The compounds were dissolved to 50 mM in dimethyl sulfoxide (DMSO) and serially diluted in 100 μL of Luria broth (LB) to concentrations of 2.0, 1.0, 0.5, 0.25, 0.125, 0.0625, 0.032, 0.016, 0.008, 0.004, 0.002, and 0.001 mM in a 96-well NUNC MicroWell plate. Strains except S. pneumoniae were grown at 37 °C in 5 mL of LB with shaking until the A₆₀₀ reached 0.6–0.7 AU, and 5 μL of the culture was added to each well. The plate was incubated in the plate reader (FLUOstar Optima) at 37 °C with 200 rpm shaking. S. pneumoniae was grown in brain heart infusion (BHI) broth at 37 °C in the presence of 5% CO₂. A 5 μL of the culture at 0.6–0.7 AU A₆₀₀ was added to each well, and the plate was incubated in the plate reader at 37 °C with shaking only for 10 s preceding an optical density reading. The optical density of the culture was taken every 10 min using LB or BHI as the blank for 16 h at 600 nm. The samples were tested in triplicate, and the growth pattern of each sample was compared to that of the cells exposed to equal amounts of DMSO.

Cytotoxicity Growth Inhibition

All test agents were prepared as stock solutions (20 mM) in DMSO and stored at −20 °C. Cell lines used in the study included MCF-7 (breast carcinoma); HT29 (colorectal carcinoma); U87, SJ-G2 (glioblastoma); SMA (murine glioblastoma); A2780 (ovarian carcinoma); H460 (lung carcinoma); A431 (skin carcinoma); Du145 (prostate carcinoma); BE2-C (neuroblastoma); and MiaPaCa-2 (pancreatic carcinoma) and the non-cancer derived MCF10A breast cell line. All cancer cell lines were incubated in a humidified atmosphere (5% CO₂ at 37 °C) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, Australia) supplemented with fetal bovine serum (10%), sodium pyruvate (10 mM), penicillin (100 IU/mL), streptomycin (100 μg/mL), and l-glutamine (4 mM). The non-cancer MCF10A cell line was cultured in DMEM:F12 (1:1) cell culture media, 5% heat inactivated horse serum, supplemented with penicillin (50 IU/mL), streptomycin (50 μg/mL), 20 mM Hepes, l-glutamine (2 mM) epidermal growth factor (20 ng/mL), hydrocortisone (500 ng/mL), cholera toxin (100 ng/mL), and insulin (10 μg/mL). Growth inhibition was determined by plating cells in duplicate in medium (100 μL) at a density of 2500–4000 cells per well in 96-well plates. On day 0 (24 h after plating), when the cells are in logarithmic growth, medium (100 μL) with or without the test agent was added to each well. After a 72 h drug exposure, growth inhibitory effects were evaluated using the MTT assay and the absorbance was read at 540 nm. An eight-point dose–response curve was produced, from which the GI₅₀ value was calculated, representing the drug concentration at which the cell growth was inhibited by 50% on the basis of the difference between the optical density values on day 0 and those at the end of drug exposure.³⁴

Microscopy

B. subtilis strains BS23 (atpA-gfp)³⁸ and BS61 (nusB-gfp)³⁹ were grown in LB medium at 37 °C with shaking until OD₆₀₀ becomes ∼0.5. At this point, 2 mL of aliquots were transferred to 15 mL tubes, antibiotics/compounds were added (5 μg/mL rifampicin, 10 μg/mL colistin, or 1.2 μg/mL 22), and the cultures were incubated with shaking for a further 30 min. The cells were then imaged by epifluorescence microscopy as detailed.¹⁵

Molecular Modeling

Molecular docking was performed using the docking engine of MOE software (MOE, Montreal, QC, Canada) “MOE-Dock” with “Triangle Matcher” as the ligand placement method. The docked poses were refined using our reported pharmacophore and re-ranked. The highest ranked pose was exported to Accelrys Discovery Studio software. Water and axillary molecules were omitted, and the structure was typed with CHARMM force field and subjected to the implemented standard molecular dynamics cascade (two steps of energy minimization using steepest decent and conjugate gradient methods, heating to 300 K and equilibration for 100 ps). An in vacuo energy minimization procedure was performed. The production phase for the equilibrated system was run for 2 ns at 300 K. The obtained model was analyzed for potential interaction using MOE-LigX module.

Chemistry

General Methods

All reagents were purchased from Sigma-Aldrich, AK Scientific, Matrix Scientific, or Lancaster Synthesis and used without purification. All solvents were redistilled from glass before use.

¹H and ¹³C NMR spectra were recorded on a Bruker Avance AMX 400 spectrometer at 400.13 and 100.62 MHz, respectively, and an Advance AMX 600 spectrometer at 600.21 and 150.92 MHz, respectively. Chemical shifts (δ) are reported in parts per million (ppm) measured relative to the internal standards. Coupling constants (J) are expressed in hertz (Hz). Mass spectra were recorded on a Shimadzu LCMS 2010 EV spectrometer and an Agilent 6100 series single quadrupole LCMS system using a mobile phase of 1:1 acetonitrile/H₂O with 0.1% formic acid. Samples analyzed by Mass Spectrometry User Resource & Research Facility (MSURRF), University of Wollongong, Australia, for high-resolution mass spectrometry (HRMS) analytical HPLC traces were obtained using a Shimadzu system possessing an SIL-20A autosampler, dual LC-20AP pumps, CBM-20A bus module, CTO-20A column heater, and a SPD-20A UV/vis detector. This system was fitted with an Alltima C₁₈ 5 μm 150 mm × 4.6 mm column with solvent A (0.06% trifluoroacetic acid (TFA) in water) and solvent B (0.06% TFA in CH₃CN–H₂O) (90:10). In each case, HPLC traces were acquired at a flow rate of 2.0 mL min^–1 and gradient 10–100 (%B), over 15.0 min, with detections at 220 and 254 nm. All samples returned satisfactory analyses. The compound purity was confirmed by a combination of LC–MS (HPLC), micro-, and/or high-resolution mass spectrometry and NMR analysis. All analogues are ≥95% pure.

Melting points were recorded on a Büchi Melting Point M-565 instrument. IR spectra were recorded on a PerkinElmer Spectrum Two FTIR Spectrometer with the UATR accessories. Thin-layer chromatography (TLC) was performed on Merck 60 F254 precoated aluminum plates with a thickness of 0.2 mm. Column chromatography was performed under “flash” conditions on Merck silica gel 60 (230–400 mesh).

1,3-Bis(4-nitrophenoxy)propane (6a)

General Procedure 1: A suspension of 4-nitrophenol (2) (1.516 g, 10.900 mmol), 1,3-dibromopropane (5a) (0.500 mL, 4.953 mmol), cesium carbonate (3.55 g, 10.900 mmol), and potassium iodide (1.809 g, 10.900 mmol) in acetonitrile (50 mL) was heated at reflux for 16 h. The resulting reaction mixture was then cooled to room temperature, concentrated in vacuo, and diluted with ethyl acetate (50 mL). The solution was washed with 1 M NaOH (2 × 50 mL) and water (50 mL). The organic layer was dried over MgSO₄ and concentrated in vacuo. The crude solid was then recrystallized from 1:1 EtOAc/CH₃OH to afford the title compound (1.08 g, 96%) as a white needle-like crystal (mp 210–211 °C).⁴²

¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (d, J = 9.3 Hz, 4H), 7.18 (d, J = 9.3 Hz, 4H), 4.31 (t, J = 6.2 Hz, 4H), 2.27 (p, J = 6.2 Hz, 2H);

¹³C NMR (101 MHz, DMSO-d₆) δ 164.2, 141.3, 126.3, 115.5, 65.7, 28.5;

LRMS (ESI^–) m/z: 363 (M – H + HCOOH, 100%), 353 (M + Cl, 25%).

1,5-Bis(4-nitrophenoxy)pentane (6b)

Compound 6b was synthesized using general procedure 1 from 4-nitrophenol (2) (1.330 g, 9.570 mmol), 1,5-dibromopentane (5b) (0.600 mL, 4.350 mmol), cesium carbonate (3.120 g, 9.570 mmol), and potassium iodide (1.500 g, 9.570 mmol) in acetonitrile (50 mL) to afford the title compound (1.107 g, 89%) as a white needle-like crystal (mp 102–103 °C).⁴²

¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (d, J = 9.2 Hz, 4H), 7.14 (d, J = 9.3 Hz, 4H), 4.16 (t, J = 6.4 Hz, 4H), 1.88–1.79 (m, 4H), 1.63–1.54 (m, 2H);

¹³C NMR (101 MHz, DMSO-d₆) δ 164.4, 141.1, 126.3, 115.4, 68.9, 28.4, 22.3;

LRMS (ESI^–) m/z: 391 (M – H + HCOOH, 100%), 381 (M + Cl, 27%).

1,7-Bis(4-nitrophenoxy)heptane (6c)

Compound 6c was synthesized using general procedure 1 from 4-nitrophenol (2) (0.594 g, 4.270 mmol), 1,5-dibromoheptane (5c) (0.330 mL, 1.940 mmol), cesium carbonate (1.391 g, 4.270 mmol), and potassium iodide (0.709 g, 4.270 mmol) in acetonitrile (50 mL) to afford the title compound (0.539 g, 74%) as a white solid (mp 117–119 °C).⁴³

¹H NMR (400 MHz, DMSO-d₆) δ 8.19 (d, J = 9.1 Hz, 4H), 7.13 (d, J = 9.1 Hz, 4H), 4.12 (t, J = 6.4 Hz, 4H), 1.84–1.67 (m, 4H), 1.42 (bs, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 164.5, 141.1, 126.4, 115.4, 69.0, 28.8, 28.8, 25.7;

LRMS (ESI^–) m/z: 419 (M – H + HCOOH, 100%), 409 (M + Cl, 60%).

1,3-Bis(4-aminophenoxy)propane (8a)

General Procedure 2: A solution of 1,3-bis(4-nitrophenoxy)propane (6a) (0.100 g, 0.314 mmol) in 1,4-dioxane (60 mL) was recirculated through the ThalesNano H-cube equipped with a 70 mm Raney nickel catalyst (0.5 mL min^–1, 100% H₂, 50 bar, 50 °C). The reaction was monitored by TLC (1:1 EtOAc/hexane). Following the consumption of the starting material (one cycle), the reaction mixture was concentrated in vacuo to afford the title compound (0.078 g, 96%) as a yellow solid (mp 108–109 °C).⁴²

¹H NMR (400 MHz, CDCl₃) δ 6.77–6.70 (m, 4H), 6.67–6.59 (m, 4H), 4.06 (t, J = 6.2 Hz, 4H), 2.17 (p, J = 6.2 Hz, 2H);

¹³C NMR (101 MHz, CDCl₃) δ 152.1, 140.0, 116.4, 115.7, 65.3, 29.6;

LRMS (ESI⁺) m/z: 259 (M + H, 100%).

1,5-Bis(4-aminophenoxy)pentane (8b)

Compound 8b was synthesized using general procedure 2 from 1,5-bis(4-nitrophenoxy)pentane (6b) (0.200 g, 0.577 mmol) in 1,4-dioxane (15 mL) to afford the title compound (0.160 g, 97%) as a yellow solid (mp 74–76 °C).⁴²

¹H NMR (400 MHz, DMSO-d₆) δ 6.63 (d, J = 8.8 Hz, 4H), 6.49 (d, J = 8.8 Hz, 4H), 4.56 (s, 4H), 3.82 (t, J = 6.4 Hz, 4H), 1.76–1.61 (m, 4H), 1.55–1.45 (m, 2H);

¹³C NMR (101 MHz, DMSO) δ 150.5, 142.8, 115.8, 115.4, 68.3, 29.2, 22.8;

LRMS (ESI⁺) m/z: 287 (M + H, 100%).

1,7-Bis(4-aminophenoxy)heptane (8c)

Compound 8c was synthesized using general procedure 2 from 1,7-bis(4-nitrophenoxy)butane (6c) in 1,4-dioxane (20 mL) to afford the title compound (0.215 g, 95%) as a yellow solid (mp 78–79.5 °C).⁴⁴

¹H NMR (400 MHz, CDCl₃) δ 6.82–6.67 (m, 4H), 6.67–6.54 (m, 4H), 3.87 (t, J = 6.5 Hz, 4H), 1.87–1.66 (m, 4H), 1.55–1.34 (m, 6H);

¹³C NMR (101 MHz, CDCl₃) δ 152.4, 139.4, 116.5, 115.8, 68.7, 29.5, 29.3, 26.1;

LRMS (ESI⁺) m/z: 315 (M + H, 100%).

N,N′-[1,3-Propanediylbis(oxy-4,1-phenylene)]bis(N-ethyl)urea (10a)

General Procedure 3: A suspension of 1,3-bis(4-aminophenoxy)propane (8a) (0.090 g, 0.314 mmol) and ethyl isocyanate (0.05 g, 0.691 mmol) in anhydrous THF (50 mL) was heated at reflux for 16 h. The resulting reaction mixture was cooled to room temperature, and the precipitate was collected and washed with diethyl ether (50 mL) to afford the title compound (0.032 g, 25%) as a cream solid (mp 211–212 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (s, 2H), 7.26 (d, J = 9.0 Hz, 4H), 6.82 (d, J = 9.0 Hz, 4H), 4.04 (t, J = 6.3 Hz, 4H), 3.13–3.01 (m, 4H), 2.13–2.02 (m, 4H), 1.03 (t, J = 7.2 Hz, 6H).

¹³C NMR (101 MHz, DMSO-d₆) δ 155.8, 153.5, 134.3, 119.8, 115.0, 64.8, 34.4, 29.2, 16.0;

LRMS (ESI⁺) m/z: 401 (M + H, 100%);

HRMS (ESI⁺): Calcd 401.2189 for C₂₁H₂₉N₄O₀ [M + H]⁺; found, 401.2196.

N,N′-[1,5-Pentanediylbis(oxy-4,1-phenylene)]bis(N-ethyl)urea (10b)

Compound 10b was synthesized using general procedure 3 from 1,5-bis(4-aminophenoxy)pentane (8b) (0.200 g, 0.700 mmol) and ethyl isocyanate (0.11 mL, 1.540 mmol) in THF (50 mL) to afford the title compound (0.100 g, 33%) as an off-white solid (mp 203–205 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.16 (s, 2H), 7.25 (d, J = 9.0 Hz, 4H), 6.79 (d, J = 9.0 Hz, 4H), 6.00–5.89 (m, 2H), 3.90 (t, J = 6.4 Hz, 4H), 3.08 (dq, J = 7.1, 5.6 Hz, 4H), 1.81–1.63 (m, 4H), 1.60–1.45 (m, 2H), 1.03 (t, J = 7.2 Hz, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 155.4, 153.2, 133.6, 119.3, 114.5, 67.5, 33.9, 28.5, 22.3, 15.5;

LRMS (ESI⁺) m/z: 429 (M + H, 100%);

HRMS (ESI⁺): Calcd 429.2502 for C₂₃H₃₃N₄O₄ [M + H]⁺; found, 429.2596.

N,N′-[1,7-Heptanediylbis(oxy-4,1-phenylene)]bis(N-ethyl)urea (10c)

Compound 10c was synthesized using general procedure 3 from 1,7-bis(4-aminophenoxy)heptane (8c) (0.270 g, 0.859 mmol) and ethyl isocyanate (0.15 mL, 1.891 mmol) in THF (50 mL) to afford the title compound (0.077 g, 23%) as an off-white solid (mp 187–189 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (s, 2H), 7.25 (d, J = 8.9 Hz, 4H), 6.78 (d, J = 8.9 Hz, 4H), 5.98–5.95 (m, 2H), 3.88 (t, J = 6.4 Hz, 4H), 3.12–3.05 (m, 4H), 1.75–1.58 (m, 4H), 1.39 (s, 6H), 1.03 (t, J = 7.2 Hz, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 155.9, 153.7, 134.1, 119.8, 115.0, 68.0, 34.4, 29.2, 29.0, 26.0, 16.0;

LRMS (ESI^–) m/z: 501(M + HCOOH(−H), 100%);

HRMS (ESI⁺): Calcd 457.2815 for C₂₅H₃₇N₄O₄ [M + H]⁺; found, 457.2831.

Di-tert-butyl(((oxybis(ethane-2,1-diyl))bis(oxy))bis(4,1-phenylene))dicarbamate (7d)

General Procedure 4: A suspension of tert-butyl(4-hydroxyphenyl)carbamate (3) (0.570 g, 2.660 mmol) and cesium carbonate (0.990 g, 3.030 mmol) in acetonitrile (50 mL) was stirred for 10 min, before the portionwise addition of diethylene glycol di(p-toluenesulfonate) (5d) (0.500 g, 1.210 mmol). The mixture was then stirred at room temperature for 16 h. The resulting mixture was concentrated in vacuo and diluted with ethyl acetate (50 mL). The solution was washed with water (2 × 50 mL) and 1 M sodium hydroxide (50 mL). The organic layer was dried over magnesium sulfate and concentrated in vacuo to afford the title compound (0.545 g, 92%) as an off-white solid (mp 146–153 °C).

¹H NMR (400 MHz, acetone-d₆) δ 8.18 (s, 2H, NH), 7.44 (d, J = 8.8 Hz, 4H), 6.98–6.68 (m, 4H), 4.25–4.08 (m, 4H), 3.98–3.81 (m, 4H), 1.47 (s, 18H);

¹³C NMR (101 MHz, DMSO-d₆) δ 153.6, 152.9, 132.7, 119.7, 114.5, 78.6, 69.1, 67.3, 28.2;

LRMS (ESI^–) m/z: 533 (M + HCOOH(−H), 40%), 413 (100%);

HRMS (ESI⁺): Calcd 489.2601 for C₂₆H₃₇N₂O₇ [M + H]⁺; found, 489.2514.

Di-tert-butyl((but-2-ene-1,4-diylbis(oxy))bis(4,1-phenylene))(Z)-dicarbamate (7e)

Compound 7e was synthesized using general procedure 4 from tert-butyl(4-hydroxyphenyl)carbamate (3) (0.870 g, 4.160 mmol), cesium carbonate (1.360 g, 4.160 mmol), and (Z)-1,4-dichloro-2-butene (5e) (0.125 mL, 1.890 mmol) in acetonitrile (50 mL) to afford the title compound (0.400 g, 45%) an off-white solid (mp 139–145 °C).

¹H NMR (400 MHz, acetone-d₆) δ 8.19 (s, 2H), 7.45 (d, J = 8.8 Hz, 4H), 6.90 (d, J = 9.0 Hz, 2H), 5.88 (t, J = 3.4 Hz, 2H), 4.73 (d, J = 4.1 Hz, 4H), 1.47 (s, 18H);

¹³C NMR (101 MHz, DMSO-d₆) δ 153.8, 153.4, 133.2, 128.9, 120.1, 115.1, 79.1, 66.8, 28.6;

LRMS (ESI^–) m/z: 515 (M + FA – H, 55%), 395 (100);

HRMS (ESI⁺): Calcd 493.2309 for C₂₆H₃₄N₂O₆Na [M + Na]⁺; found, 493.2298.

Di-tert-butyl((but-2-ene-1,4-diylbis(oxy))bis(4,1-phenylene))(E)-dicarbamate (7f)

Compound 7f was synthesized using general procedure 4 from tert-butyl(4-hydroxyphenyl)carbamate (3) (0.750 g, 3.580 mmol), cesium carbonate (1.166 g, 3.580 mmol), and (E)-1,4-dichloro-2-butene (5f) (0.125 mL, 1.890 mmol) in acetonitrile (50 mL) to afford the title compound (0.734 g, 96%) an off-white solid (mp 196–201 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 9.12 (s, 2H), 7.34 (d, J = 8.4 Hz, 4H), 6.85 (d, J = 9.0 Hz, 4H), 6.07–5.95 (m, 2H), 4.71–4.40 (m, 4H), 1.46 (s, 8H);

¹³C NMR (101 MHz, DMSO-d₆) δ 153.8, 153.4, 133.2, 128.9, 120.1, 115.1, 79.1, 66.8, 28.6;

LRMS (ESI^–) m/z: 515 (M + FA – H, 55%), 395 (100);

HRMS (ESI⁺): Calcd 493.2309 for C₂₆H₃₄N₂O₆Na [M + Na]⁺; found, 493.2301.

1,1′-(((Oxybis(ethane-2,1-diyl))bis(oxy))bis(4,1-phenylene))bis(3-ethylurea) (10d)

A suspension of di-tert-butyl(((oxybis(ethane-2,1-diyl))bis(oxy))bis(4,1-phenylene))dicarbamate (7d) (0.400 g, 0.819 mmol) in 4 M HCl in 1,4-dioxane (50 mL) was stirred for 1 h at room temperature. The reaction mixture was then concentrated under a stream of air overnight. The resulting precipitate was diluted with anhydrous THF (50 mL) and heated to reflux for over 10 min. Triethylamine (0.260 mL, 1.970 mmol) and ethyl isocyanate (0.160 mL, 1.970 mmol) were then added to the suspension, and the mixture was heated at reflux for 16 h. The resulting reaction mixture was then concentrated in vacuo and diluted with ethyl acetate (100 mL). The solution was washed with water (2 × 100 mL) and a saturated sodium chloride solution (100 mL). The organic layer was dried over magnesium sulfate and concentrated in vacuo to afford the title compound (0.204 g, 57%) as a white solid (mp 203–207 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (s, 2H), 7.27 (d, J = 8.9 Hz, 4H), 6.82 (d, J = 8.9 Hz, 4H), 5.97 (t, J = 5.4 Hz, 2H), 4.13–3.97 (m, 4H), 3.87–3.66 (m, 4H), 3.14–2.99 (m, 4H), 1.04 (t, J = 7.1 Hz, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 155.9, 153.5, 134.3, 119.8, 115.0, 69.6, 67.8, 34.4, 16.0;

LRMS (ESI⁺) m/z: 431 (M + H, 100%);

HRMS (ESI⁺): Calcd 431.2294 for C₂₂H₃₁N₄O₅ [M + H]⁺; found, 431.2285.

(Z)-4,4′-(But-2-ene-1,4-diylbis(oxy))dianiline hydrochloride (8e)

General Procedure 5: A suspension of di-tert-butyl((but-2-ene-1,4-diylbis(oxy))bis(4,1-phenylene))(Z)-dicarbamate (7e) (0.400 g, 0.819 mmol) in 4 M HCl in 1,4-dioxane (50 mL) was stirred for 1 h at room temperature. The reaction mixture was then concentrated under a stream of air overnight, washed with cold THF (20 mL), and filtered to afford the title compound (0.183 g, 90%) as a brown solid (mp 230–233 °C).

¹H NMR (400 MHz, CD₃OD) δ 7.39–7.29 (m, 4H), 7.18–7.05 (m, 4H), 6.01–5.89 (m, 2H), 4.79 (d, J = 4.0 Hz, 4H);

¹³C NMR (101 MHz, CD₃OD) δ 158.9, 128.1, 123.9, 123.1, 115.7, 64.3;

LRMS (ESI⁺) m/z: 271 (M + H, 100%), 312 (M + CH₃CN + H, 30%);

HRMS (ESI⁺): Calcd 271.1446 for C₁₆H₁₉N₂O₂ [M + H]⁺; found, 271.1455.

(E)-4,4′-(But-2-ene-1,4-diylbis(oxy))dianiline Hydrochloride (8f)

Compound 8f was synthesized using general procedure 5 from di-tert-butyl((but-2-ene-1,4-diylbis(oxy))bis(4,1-phenylene))(E)-dicarbamate (7f) (0.500 g, 1.063 mmol) and 4 M HCl in 1,4-dioxane (50 mL) to afford the title compound (0.231 g, 90%) as a brown solid (mp 105 °C (dec.)).

¹H NMR (400 MHz, CDCl₃) δ 6.80–6.69 (m, 4H), 6.69–6.56 (m, 4H), 6.04–6.02 (m, 2H), 4.48 (dd, J = 2.4, 1.1 Hz, 4H);

¹³C NMR (101 MHz, DMSO-d₆) δ 143.0, 129.0, 116.0, 115.4, 68.3;

LRMS (ESI^–) m/z: 271 (M + H, 33%), 136 (100%);

HRMS (ESI⁺): Calcd 271.1446 for C₁₆H₁₉N₂O₂ [M + H]⁺; found, 271.1443.

(Z)-1,1′-((But-2-ene-1,4-diylbis(oxy))bis(4,1-phenylene))bis(3-ethylurea) (10e)

General Procedure 6: A solution of (Z)-4,4′-(but-2-ene-1,4-diylbis(oxy))dianiline hydrochloride (8e) (0.147 g, 0.428 mmol) and trimethylamine (0.130 mL, 0.942 mmol) in anhydrous THF (30 mL) was stirred for 20 min, followed by the addition of ethyl isocyanate (0.100 mL, 0.942 mmol). The resultant mixture was then heated at reflux for 16 h and cooled to room temperature in vacuo to give an off-white precipitate, which was washed with water (25 mL) and CH₂Cl₂ (25 mL). The precipitate was then dried in vacuo to afford the title compound (0.127 g, 72%) as an off-white solid (mp 210–212 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.19 (s, 2H), 7.27 (d, J = 8.9 Hz, 4H), 6.83 (d, J = 8.9 Hz, 4H), 5.97 (t, J = 5.4 Hz, 2H), 5.82 (t, J = 3.2 Hz, 2H), 4.63 (d, J = 3.5 Hz, 4H), 3.12–3.03 (m, 4H), 1.03 (t, J = 7.2 Hz, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 155.8, 153.1, 134.5, 129.0, 119.8, 115.2, 64.5, 34.4, 16.0;

LRMS (ESI⁺) m/z: 413 (M + H, 100%);

HRMS (ESI⁺): Calcd 413.2189 for C₂₂H₂₉N₄O₄ [M + H]⁺; found, 413.2204.

(E)-1,1′-((But-2-ene-1,4-diylbis(oxy))bis(4,1-phenylene))bis(3-ethylurea) (10f)

Compound 10f was synthesized using general procedure 6 from (E)-4,4′-(but-2-ene-1,4-diylbis(oxy))dianiline hydrochloride (8f) (0.198 g, 0.732 mmol), triethylamine (0.122 mL, 1.537 mmol), and anhydrous THF (50 mL) to afford the title compound (0.156 g, 52%) as an off-white solid (mp 240 °C (dec.)).

¹H NMR (600 MHz, DMSO-d₆) δ 8.20 (s, 2H), 7.27 (d, J = 8.7 Hz, 4H), 6.82 (d, J = 8.7 Hz, 4H), 6.02 (s, 2H), 6.00–5.94 (m, 2H), 4.52 (s, 4H), 3.14–3.01 (m, 4H), 1.04 (t, J = 7.1 Hz, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 155.8, 153.2, 134.4, 128.9, 119.8, 115.2, 67.9, 34.4, 16.0;

LRMS (ESI⁺) m/z: 413 (M + H, 100%);

HRMS (ESI⁺): Calcd 413.2189 for C₂₂H₂₉N₄O₄ [M + H]⁺; found, 413.2198.

1-Ethyl-3-(4-hydroxyphenyl)urea (9)

A mixture of 4-aminophenol (4) (0.800 g, 7.36 mmol) and ethyl isocyanate (0.500 g, 7.034 mmol) in anhydrous THF (50 mL) was then stirred at room temperature for 4 h. The reaction mixture was the concentrated in vacuo, diluted with ether (50 mL), and sonicated for 2 min. The resulting suspension was then filtered and washed with diethyl ether to afford the title compound (1.008 g, 80%) as an off-white solid (mp 165–171 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.94 (s, 1H), 8.04 (s, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H), 5.93 (s, 1H), 3.15–2.95 (m, 2H), 1.03 (t, J = 7.0 Hz, 3H);

¹³C NMR (101 MHz, acetone-d₆) δ 155.5, 152.3, 132.8, 120.3, 115.0, 34.3, 15.1;

LRMS (ESI^–) m/z: 179 (M – H, 100%);

HRMS (ESI⁺): Calcd 181.0977 for C₉H₁₃N₂O₂ [M + H]⁺; found, 181.0983.

1,1′-(((1,2-Phenylenebis(methylene))bis(oxy))bis(4,1-phenylene))bis(3-ethylurea) (10g)

General Procedure 7: A suspension of 1-ethyl-3-(4-hydroxyphenyl)urea (9) (0.225 g, 1.250 mmol), 1,2-bis(bromomethyl)benzene (5g) (0.150 g, 0.570 mmol), cesium carbonate (0.407, 1.250 mmol), and a catalytic amount of potassium iodide in acetone (50 mL) was refluxed for 16 h. The resulting reaction mixture was then concentrated to dryness, and the resulting residue was purified by flash chromatography (10% CH₃OH in CH₂Cl₂) to afford the title compound (0.200 g, 76%) as a white solid (mp 219–220 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (brs, 2H), 7.50 (dd, J = 5.5, 3.5 Hz, 2H), 7.35 (dd, J = 5.6, 3.4 Hz, 2H), 7.27 (d, J = 9.0 Hz, 4H), 6.90 (d, J = 9.0 Hz, 4H), 5.99 (brs, 2H), 5.15 (s, 4H), 3.08 (dt, J = 7.1, 5.7 Hz, 4H), 1.04 (t, J = 7.2 Hz, 6H);

¹³C NMR (400 MHz, DMSO-d₆) δ 155.8, 153.2, 135.9, 134.6, 128.8, 128.3, 119.8, 115.39, 67.7, 34.4, 16.0;

LRMS (ESI⁺) m/z: 463 (M + H, 100%);

HRMS (ESI⁺): Calcd 463.2345 for C₂₆H₃₁N₄O₄ [M + H]⁺; found, 463.2360.

1,1′-(((1,3-Phenylenebis(methylene))bis(oxy))bis(4,1-phenylene))bis(3-ethylurea) (10h)

Compound 10h was synthesized using general procedure 7 from 1-ethyl-3-(4-hydroxyphenyl)urea (9) (0.757 g, 4.200 mmol), 1,3-bis(bromomethyl)benzene (5h) (0.528 g, 2.000 mmol), potassium carbonate (0.580 g, 4.200 mmol), and a catalytic amount of potassium iodide in acetone (50 mL). The resulting residue was purified by flash chromatography (10% EtOAc in hexane) to afford the title compound (0.318 g, 34%) as a white solid (mp 213–214 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.19 (s, 2H), 7.50 (s, 1H), 7.45–7.35 (m, 3H), 7.27 (d, J = 9.0 Hz, 4H), 6.88 (d, J = 9.0 Hz, 4H), 5.97 (t, J = 5.5 Hz, 2H), 5.04 (s, 4H), 3.15–2.97 (m, 4H), 1.03 (t, J = 7.2 Hz, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 155.8, 153.4, 138.1, 134.5, 128.9, 127.5, 127.2, 119.8, 115.4, 69.8, 34.4, 16.0;

LRMS (ESI⁺) m/z: 463 (M + H, 30%) 147 (100);

HRMS (ESI⁺): Calcd 463.2345 for C₂₆H₃₁N₄O₄ [M + H]⁺; found, 463.2358.

1,1′-(((Furan-3,4-diylbis(methylene))bis(oxy))bis(4,1-phenylene))bis(3-ethylurea) (10i)

Compound 10i was synthesized using general procedure 7 from 1-ethyl-3-(4-hydroxyphenyl)urea (9) (1.209 g, 6.708 mmol), 3,4-bis(chloromethyl)furan (5i) (0.500 g, 3.049 mmol), potassium carbonate (0.930 g, 6.708 mmol), and catalytic amount of potassium iodide in acetone (50 mL). The resulting residue was purified by flash chromatography (15% EtOAc in hexane) to afford the title compound (0.318 g, 34%) as a white solid (mp 201–202 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (s, 2H), 7.74 (s, 2H), 7.25 (d, J = 9.0 Hz, 4H), 6.85 (d, J = 9.0 Hz, 4H), 5.98–5.93 (m, 2H), 4.93 (s, 4H), 3.16–3.00 (m, 4H), 1.03 (t, J = 7.2 Hz, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 155.8, 153.2, 142.8, 134.5, 121.4, 119.7, 115.5, 61.2, 34.4, 16.0;

LRMS (ESI⁺) m/z: 453 (M + H, 100%);

HRMS (ESI⁺): Calcd 453.2138 for C₂₄H₂₉N₄O₅ [M + H]⁺; found, 453.2147.

Di-tert-butyl((but-2-ene-1,4-diylbis(oxy))bis(2,1-phenylene))(Z)-dicarbamate (7k)

General Procedure 8: A suspension of (Z)-1,4-dichlorobutene (5e) (0.200 g, 1.622 mmol), tert-butyl(2-hydroxyphenyl)carbamate (4b) (0.641 g, 3.318 mmol), and cesium carbonate (1.081 g, 3.318 mmol) in anhydrous DMF (15 mL) was then heated at 75 °C for 3 h. The resulting reaction mixture was then poured on ice water, and the solution was extracted with ethyl acetate (2 × 100 mL). The organic layer was washed with water (2 × 100 mL), saturated NaHCO₃ (2 × 100 mL), and brine (100 mL). The organic layer was dried over MgSO₄ and concentrated in vacuo. The resulting residue was purified by flash chromatography (2% EtOAc in hexane) to afford the title compound (0.227 g, 34%) as a yellow gum.

¹H NMR (400 MHz, acetone-d₆) δ 8.07 (d, J = 7.6 Hz, 2H), 7.45 (s, 2H), 7.02 (dd, J = 7.7, 1.7 Hz, 2H), 6.98–6.89 (m, 4H), 6.00 (t, J = 3.6 Hz, 2H), 4.85 (d, J = 4.1 Hz, 4H), 1.49 (s, 18H);

¹³C NMR (101 MHz, CDCl₃) δ 171.3, 152.9, 146.3, 128.8, 122.4, 121.8, 111.4, 80.6, 64.7, 28.5;

LRMS (ESI^–) m/z: 515 (M + HCOOH(−H), 55%), 395 (100);

HRMS (ESI⁺): Calcd 493.2309 for C₂₆H₃₄N₂O₆Na [M + Na]⁺; found, 493.2302.

Di-tert-butyl((but-2-ene-1,4-diylbis(oxy))bis(3,1-phenylene))(Z)-dicarbamate (7j)

Compound 7j was synthesized using general procedure 8 from tert-butyl(3-hydroxyphenyl)carbamate (4c) (0.641 g, 3.318 mmol), (Z)-1,4-dichloro-2-butene (5e) (0.200 g, 1.622 mmol), cesium carbonate (1.081 g, 3.318 mmol), and anhydrous DMF (15 mL). The resulting residue was purified by flash chromatography (2% EtOAc in hexane) to afford the title compound (0.339 g, 51%) as a white solid (mp 49–53 °C).

¹H NMR (400 MHz, acetone-d₆) δ 8.35 (s, 2H), 7.31 (s, 2H), 7.16 (t, J = 8.1 Hz, 2H), 7.10 (d, J = 8.2 Hz, 2H), 6.62 (dd, J = 8.1, 1.5 Hz, 2H), 5.90–5.89 (m, 2H), 4.76 (d, J = 4.1 Hz, 4H), 1.47 (s, 18H);

¹³C NMR (101 MHz, acetone-d₆) δ 160.0, 153.6, 141.9, 130.3, 129.3, 111.6, 109.2, 105.8, 80.0, 64.9, 28.5;

LRMS (ESI^–) m/z: 515 (M + HCOOH(−H), 55%), 395 (100);

HRMS (ESI⁺): Calcd 493.2309 for C₂₆H₃₄N₂O₆Na [M + Na]⁺; found, 493.2311.

(Z)-3,3′-(But-2-ene-1,4-diylbis(oxy))dibenzenaminium Chloride (8k)

Compound 8k was synthesized using general procedure 5 from di-tert-butyl((but-2-ene-1,4-diylbis(oxy))bis(2,1-phenylene))(Z)-dicarbamate (7k) (0.150 g 0.319 mmol) and 4 M HCl in dioxane (5 mL) to afford the title compound (0.137 g, 98%) as an off-white solid (mp 241–243 °C).

¹H NMR (400 MHz, CD₃OD) δ 7.45 (t, J = 8.3 Hz, 2H), 7.07 (d, J = 8.9 Hz, 2H), 6.97–6.95 (m, 4H), 5.95 (t, J = 3.4 Hz, 2H), 4.80 (d, J = 3.7 Hz, 4H);

¹³C NMR (101 MHz, CD₃OD) δ 161.1, 132.3, 129.4, 116.1, 115.7, 110.9, 65.7;

LRMS (ESI⁺) m/z: 271 (M + H, 100%).

HRMS (ESI⁺): Calcd 271.1446 for C₁₆H₁₉N₂O₂ [M + H]⁺; found, 271.1440.

(Z)-2,2′-(But-2-ene-1,4-diylbis(oxy))dibenzenaminium Chloride (8j)

Compound 8j was synthesized using general procedure 5 from di-tert-butyl((but-2-ene-1,4-diylbis(oxy))bis(3,1-phenylene))(Z)-dicarbamate (7j) (0.150 g 0.319 mmol) and 4 M HCl in dioxane (5 mL) to afford the title compound (0.0.98 g, 98%) as an off-white solid (mp 157 °C (dec.)).

¹H NMR (600 MHz, CD₃OD) δ 7.46 (t, J = 7.9 Hz, 2H), 7.41 (d, J = 7.8 Hz, 2H), 7.27 (d, J = 8.3 Hz, 2H), 7.10 (t, J = 7.7 Hz, 2H), 6.11–5.95 (m, 2H), 4.96 (d, J = 3.1 Hz, 4H);

¹³C NMR (101 MHz, CD₃OD) δ 148.2, 129.8, 123.6, 122.8, 122.2, 114.3, 64.0;

LRMS (ESI⁺) m/z: 271 (M + H, 100%).

HRMS (ESI⁺): Calcd 271.1446 for C₁₆H₁₉N₂O₂ [M + H]⁺; found, 271.1452.

(Z)-1,1′-((But-2-ene-1,4-diylbis(oxy))bis(3,1-phenylene))bis(3-ethylurea) (10j)

A solution of 8j (0.100 g, 0.291 mmol) and trimethylamine (1.00 mL, 7.275 mmol) in anhydrous THF (20 mL) was heated to reflux, ethyl isocyanate (0.090 mL, 1.164 mmol) was added, and the mixture was refluxed overnight. The resulting reaction mixture was cooled to room temperature, adsorbed onto silica (∼2.00 g), and purified by flash chromatography (50% EtOAc in hexane) to afford the title compound (0.050 g, 42%) as a cream solid (mp 145–152 °C).

¹H NMR (400 MHz, acetone-d₆) δ 7.94 (s, 2H), 6.69 (t, J = 2.0 Hz, 2H), 6.64 (t, J = 8.1 Hz, 2H), 6.42 (d, J = 8.1 Hz, 2H), 6.05 (dd, J = 8.1, 2.2 Hz, 2H), 5.62 (t, J = 5.4 Hz, 2H), 5.43–5.34 (m, 2H), 4.21 (d, J = 3.7 Hz, 4H), 2.72–2.56 (m, 4H), 0.59 (t, J = 7.2 Hz, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 158.5, 155.0, 141.82, 129.3, 128.4, 110.3, 107.0, 104.2, 63.7, 33.9, 15.4;

LRMS (ESI⁺) m/z: 413 (M + H, 100%);

HRMS (ESI⁺): Calcd 413.2189 for C₂₂H₂₉N₄O₄ [M + H]⁺; found, 429.2596.

(Z)-1,1′-((But-2-ene-1,4-diylbis(oxy))bis(2,1-phenylene))bis(3-ethylurea) (10k)

A solution of (Z)-3,3′-(but-2-ene-1,4-diylbis(oxy))dibenzenaminium chloride (8k) (0.100 g, 0.291 mmol) and trimethylamine (1.00 mL, 7.275 mmol) in anhydrous THF (20 mL) was heated to reflux, ethyl isocyanate (0.090 mL, 1.164 mmol) was added, and the reflux was maintained overnight. The resulting reaction mixture was cooled to room temperature, adsorbed onto silica (∼2.00 g), and purified by flash chromatography (50% EtOAc in hexane) to afford the title compound (0.109 g, 91%) as an off-white solid (mp 196–201 °C).

¹H NMR (600 MHz, acetone-d₆) δ 8.25 (dt, J = 7.9, 1.4 Hz, 2H), 7.58 (brs, 2H), 6.94 (dd, J = 7.6, 1.7 Hz, 1H), 6.87–6.80 (m, 4H), 6.33 (brs, 2H), 5.93 (t, J = 3.6 Hz, 2H), 4.77 (d, J = 4.1 Hz, 4H), 3.22 (q, J = 7.2 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H);

¹³C NMR (151 MHz, acetone-d₆) δ 155.9, 147.3, 131.3, 129.4, 121.9, 121.8, 119.4, 112.7, 65.6, 35.0, 15.8;

LRMS (ESI⁺) m/z: 413 (M + H, 100%);

HRMS (ESI⁺): Calcd 413.2189 for C₂₂H₂₉N₄O₄ [M + H]⁺; found, 413.2184.

(Z)-1-(4-((4-Chlorobut-2-en-1-yl)oxy)phenyl)-3-ethylurea (11)

To a warmed (75 °C) solution of (Z)-1,4-dichloro-2-butene (5e) (2.00 g, 16.137 mmol) in anhydrous DMF (10 mL), a suspension of 1-ethyl-3-(4-hydroxyphenyl)urea (9) (1.4454 g, 8.068 mmol) and cesium carbonate (5.252 g, 16.134 mmol) in anhydrous DMF (20 mL) was added dropwise. The reaction mixture was then heated at 75 °C for 2 h, diluted with water (200 mL), and extracted with EtOAc (2 × 200 mL). The organic layer was washed with water (4 × 200 mL), dried over MgSO₄, and concentrated in vacuo. The resulting residue was the recrystallized from (10:1) hexane/ethyl acetate to afford the title compound (0.800 g, 37%) as a brown solid (mp 125 °C (dec.)).

¹H NMR (400 MHz, acetone-d₆) δ 7.63 (s, 1H), 7.44–7.31 (m, 2H), 6.87–6.78 (m, 2H), 5.94–5.73 (m, 2H), 4.69 (d, J = 4.8 Hz, 2H), 4.32 (d, J = 6.9 Hz, 2H), 3.20 (qd, J = 7.2, 5.7 Hz, 2H), 1.09 (t, J = 7.2 Hz, 3H);

¹³C NMR (101 MHz, acetone-d₆) δ 155.4, 153.4, 134.4, 130.1, 128.3, 119.8, 1148, 63.6, 39.2, 34.3, 15.1;

LRMS (ESI⁺) m/z: 269 (M + H, ³⁵Cl, 100%) 271 (M + H, ³⁷Cl, 30%);

HRMS (ESI⁺): Calcd 269.1057 for C₁₃H₁₈ClN₂O₂ [M + H]⁺; found, 269.1058.

(Z)-2-(4-((4-(4-(3-Ethylureido)phenoxy)but-2-en-1-yl)oxy)phenyl)acetimidic Acid (13a)

General Procedure 9: A solution of (Z)-1-(4-((4-chlorobut-2-en-1-yl)oxy)phenyl)-3-ethylurea (11) (0.210 g, 0.714 mmol), 4-hydroxyphenylacetamide (12a) (0.205 g, 1.356 mmol), and cesium carbonate (0.450 g, 1.381 mmol) in anhydrous DMF (10 mL) was heated at 75 °C for 2 h. On cooling, water (100 mL) was added and the solution was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with water (4 × 100 mL), dried over MgSO₄, and concentrated in vacuo. The resulting residue was then adsorbed onto silica (∼1.00 g) and purified by flash chromatography (6.5% CH₃OH in CH₂Cl₂) to afford the title compound (0.110 g, 40%) as a white solid (mp 180–181 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (s, 1H), 7.37 (bs, 1H), 7.26 (d, J = 9.0 Hz, 2H), 7.16 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 8.6, 2H), 6.84 (d, J = 9.0 Hz, 2H), 6.81 (bs, 1H), 5.99–5.91 (m, 1H), 5.86–5.76 (m, 2H), 4.66 (dd, J = 14.1, 3.5 Hz, 4H), 3.14–3.01 (m, 2H), 1.03 (t, J = 7.2 Hz, 3H);

¹³C NMR (101 MHz, DMSO-d₆) 173.0, 157.2, 155.8, 153.1, 134.5, 130.5, 129.2, 129.1, 128.8, 119.8, 115.3, 114.9, 64.5, 64.3, 41.8, 34.4, 16.0;

LRMS (ESI⁺) m/z: 384 (M + H, 100%);

HRMS (ESI⁺): Calcd 384.1923 for C₂₁H₂₆N₃O₄ [M + H]⁺; found, 384.1934.

(Z)-3-(4-((4-(4-(3-Ethylureido)phenoxy)but-2-en-1-yl)oxy)phenyl)propanoic Acid (13b)

Compound 13b was synthesized using general procedure 9 from 3-(4-hydroxyphenyl)propionic acid (12b) (0.225 g, 1.356 mmol), (Z)-1-(4-((4-chlorobut-2-en-1-yl)oxy)phenyl)-3-ethylurea (11) (0.210 g, 0.714 mmol), and cesium carbonate (0.450 g, 1.381 mmol) in anhydrous DMF (10 mL). The resulting reaction mixture was purified by flash chromatography (7.5% CH₃OH in CH₂Cl₂) to afford the title compound (0.137 g, 28%) as an white solid (mp 95–97 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 9.16 (s, 1H), 8.19 (s, 1H), 7.27 (d, J = 9.1 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 6.82 (d, J = 9.0 Hz, 2H), 6.65 (d, J = 8.5, 2H), 5.96 (t, J = 5.2 Hz, 1H), 5.81 (dtt, ³J_CH = 10.9, ³J_CH2 = 6.1, ⁴J_CH2 = 1.4 Hz 1H), 5.65 (dtt, ³J_CH = 11.3, ³J_CH2 = 6.6, ⁴J_CH2 = 1.4 Hz, 1H), 4.62 (dd, J = 27.5, 6.5 Hz, 4H), 3.08 (dq, J = 7.1, 6.5 Hz, 2H), 2.73 (t, J = 7.6 Hz, 2H), 2.56 (t, J = 7.5 Hz, 2H), 1.03 (t, J = 7.2 Hz, 3H);

¹³C NMR (101 MHz, DMSO-d₆) δ 172.6, 156.1, 155.8, 153.1, 134.5, 130.9, 130.1, 129.6, 127.4, 119.8, 115.5, 115.2, 64.3, 60.3, 35.9, 34.4, 29.9, 16.0;

LRMS (ESI⁺) m/z: 399 (M + H, 100%);

HRMS (ESI⁺): Calcd 399.1920 for C₂₂H₂₇N₂O₅ [M + H]⁺; found, 399.1931.

(Z)-3-(4-((4-(4-(3-Ethylureido)phenoxy)but-2-en-1-yl)oxy)phenyl)acetic Acid (13c)

Compound 13c was synthesized using general procedure 9 from 4-hydroxyphenylacetic acid (12c) (0.210 g, 1.356 mmol), (Z)-1-(4-((4-chlorobut-2-en-1-yl)oxy)phenyl)-3-ethylurea (11) (0.210 g, 0.714 mmol), and cesium carbonate (0.450 g, 1.381 mmol) in anhydrous DMF (10 mL). The resulting reaction mixture was purified by flash chromatography (3% CH₃OH in CH₂Cl₂) to afford the title compound (0.100 g, 27%) as a tan solid (mp 92–93 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 9.30 (s, 1H), 8.18 (s, 1H), 7.26 (d, J = 9.0 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 6.69 (d, J = 8.5 Hz, 2H), 5.96 (t, J = 5.5 Hz, 1H), 5.81 (dtt, ³J_CH = 10.9, ³J_CH2 = 6.1, ⁴J_CH2 = 1.4 Hz 1H), 5.65 (dtt, ³J_CH = 11.3, ³J_CH2 = 6.6, ⁴J_CH2= 1.4 Hz, 1H), 4.63 (dd, J = 27.5, 6.5 Hz, 4H), 3.54 (s, 2H), 3.15–2.96 (m, 2H), 1.03 (t, J = 7.2 Hz, 3H);

¹³C NMR (101 MHz, DMSO-d₆) δ 171.4, 156.3, 155.4, 152.6, 134.0, 130.3, 129.8, 126.8, 124.3, 119.3, 115.1, 114.8, 63.8, 60.2, 39.4, 34.0, 15.5;

LRMS (ESI⁺) m/z: 385 (M + H, 100%);

HRMS (ESI⁺): Calcd 385.1763 for C₂₁H₂₅N₂O₅ [M + H]⁺; found, 385.1772.

(R,Z)-2-((tert-Butoxycarbonyl)amino)-3-(4-((4-(4-(3-ethylureido)phenoxy)but-2-en-1-yl)oxy)phenyl)propanoic Acid (13d)

General Procedure 10: To a microwave vial, (tert-butoxycarbonyl)-l-tyrosine (12d) (0.381 g, 1.356 mmol), (Z)-1-(4-((4-chlorobut-2-en-1-yl)oxy)phenyl)-3-ethylurea (11) (0.210 g, 0.714 mmol), and cesium carbonate (0.450 g, 1.381 mmol) were added in anhydrous DMF (2.5 mL). The suspension was subjected to microwave irradiation at 85 °C for 30 min. The resulting reaction mixture was poured into water (50 mL) and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with 1 M NaOH (100 mL), water (4 × 100 mL), and saturated brine (100 mL); dried over MgSO₄ adsorbed onto silica (∼1.00 g); and purified by flash chromatography (50:50 EtOAc/hexane) to afford the title compound (0.300 g, 81%) as a white solid (mp < 50 °C).

¹H NMR (400 MHz, acetone-d₆) δ 8.23 (s, 1H), 7.65 (s, 1H), 7.44–7.27 (m, 2H), 7.06 (d, J = 8.2 Hz, 2H), 6.92–6.80 (m, 2H), 6.75 (d, J = 8.4 Hz, 2H), 6.04 (d, J = 7.7 Hz, 1H), 5.87 (dt, J = 11.6, 5.9 Hz, 1H), 5.79–5.54 (m, 2H), 4.75 (t, J = 5.8 Hz, 2H), 4.68 (d, J = 5.6 Hz, 2H), 4.37–4.32 (m, 1H), 3.24–3.17 (m, 2H), 2.89–3.04 (ddd, J = 21.9, 13.8, 6.7 Hz, 2H), 1.36 (s, 9H), 1.09 (t, J = 7.2 Hz, 3H);

¹³C NMR (101 MHz, acetone-d₆) δ 172.7, 157.2, 156.4, 156.2, 154.44, 135.1, 131.2, 131.1, 128.6, 127.3, 120.9, 120.8, 116.1, 115.7, 79.36, 6.76, 63.8, 56.4, 37.5, 35.3, 29.8, 28.5, 15.6;

LRMS (ESI^–) m/z: 512 (M – H, 100%);

HRMS (ESI⁺): Calcd 514.2553 for C₂₇H₃₆N₃O₇ [M + H]⁺; found, 514.2557.

(S,Z)-2-((tert-Butoxycarbonyl)amino)-3-(4-((4-(4-(3-ethylureido)phenoxy)but-2-en-1-yl)oxy)phenyl)propanoic Acid (13e)

Compound 13e was synthesized using general procedure 10 from (tert-butoxycarbonyl)-l-tyrosine (12e) (0.200 g, 0.712 mmol), (Z)-1-(4-((4-chlorobut-2-en-1-yl)oxy)phenyl)-3-ethylurea (11) (0.109 g, 0.370 mmol), and cesium carbonate (0.232 g, 0.712 mmol) in anhydrous DMF (3 mL) to afford the title compound (0.151 g, 79%) as a white solid (mp < 50 °C).

¹H NMR (400 MHz, acetone-d₆) δ 8.23 (s, 1H), 7.65 (s, 1H), 7.44–7.27 (m, 2H), 7.06 (d, J = 8.2 Hz, 2H), 6.92–6.80 (m, 2H), 6.75 (d, J = 8.4 Hz, 2H), 6.04 (d, J = 7.7 Hz, 1H), 5.87 (dt, J = 11.6, 5.9 Hz, 1H), 5.79–5.54 (m, 2H), 4.75 (t, J = 5.8 Hz, 2H), 4.68 (d, J = 5.6 Hz, 2H), 4.37–4.32 (m, 1H), 3.24–3.17 (m, 2H), 2.89–3.04 (ddd, J = 21.9, 13.8, 6.7 Hz, 2H), 1.36 (s, 9H), 1.09 (t, J = 7.2 Hz, 3H).

¹³C NMR (101 MHz, acetone-d₆) δ 172.7, 157.2, 156.4, 156.2, 154.44, 135.1, 131.2, 131.1, 128.6, 127.3, 120.9, 120.8, 116.1, 115.7, 79.36, 6.76, 63.8, 56.4, 37.5, 35.3, 29.8, 28.5, 15.6;

LRMS (ESI^–) m/z: 512 (M – H, 100%);

HRMS (ESI⁺): Calcd 514.2553 for C₂₇H₃₆N₃O₇ [M + H]⁺; found, 514.2556.

(R,Z)-1-Carboxy-2-(4-((4-(4-(3-ethylureido)phenoxy)but-2-en-1-yl)oxy)phenyl)ethan-1-aminium Chloride (13g)

Compound 13g was synthesized using general procedure 5 from (R,Z)-2-((tert-butoxycarbonyl)amino)-3-(4-((4-(4-(3-ethylureido)phenoxy)but-2-en-1-yl)oxy)phenyl)propanoic acid (13d) (0.150 g, 0.292 mmol) and 4 M HCl in 1,4-dioxane (5 mL) to afford the title compound (0.117 g, 89%) as a brown gum.

¹H NMR (400 MHz, DMSO) δ 9.39 (bs, 1H), 8.37 (bs, 2H), 8.28–8.20 (m, 1H), 7.28 (d, J = 9.0 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.82 (d, J = 9.0 Hz, 2H), 6.71 (d, J = 8.4 Hz, 2H), 6.03 (bs, 1H), 5.95–5.80 (m, 1H), 5.63–5.53 (m, 1H), 4.78 (d, J = 6.7 Hz, 2H), 4.60 (d, J = 5.9 Hz, 2H), 4.27–4.22 (m, 1H), 3.13–2.88 (m, 4H), 1.03 (t, J = 7.2 Hz, 3H);

¹³C NMR (101 MHz, CD₃OD) δ 170.0, 158.8, 158.4, 155.6, 134.3, 132.2, 131.6, 126.7, 125.5, 122.7, 116.9, 116.1, 65.4, 63.0, 55.4, 36.7, 35.7, 15.7;

LRMS (ESI^–) m/z: 412 (M – H, 100%), 448 (M + Cl – H, 85%);

HRMS (ESI⁺): Calcd 414.2029 for C₂₂H₂₈N₃O₅ [M + H]⁺; found, 413.3191.

(S,Z)-1-Carboxy-2-(4-((4-(4-(3-ethylureido)phenoxy)but-2-en-1-yl)oxy)phenyl)ethan-1-aminium Chloride (13h)

Compound 13h was synthesized using general procedure 5 from (S,Z)-2-((tert-butoxycarbonyl)amino)-3-(4-((4-(4-(3-ethylureido)phenoxy)but-2-en-1-yl)oxy)phenyl)propanoic acid (13e) (0.127 g, 0.247 mmol) and 4 M HCl in 1,4-dioxane (5 mL) to afford the title compound (0.104 g, 94%) as a brown gum.

¹H NMR (400 MHz, CD₃OD) δ 7.22 (d, J = 9.0 Hz, 2H), 6.99 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 9.0 Hz, 2H), 6.70 (d, J = 8.5 Hz, 2H), 5.86 (dtt, ³J_CH = 11.2, ³J_CH2 = 6.0, ⁴J_CH2 = 1.3 Hz, 1H), 5.66 (dtt, ³J_CH = 11.2, ³J_CH2 = 6.8, ⁴J_CH2 = 1.6 Hz, 1H), 4.72 (d, J = 6.8 Hz, 2H), 4.62 (d, J = 5.8 Hz, 2H), 3.65 (t, J = 6.5 Hz, 1H), 3.21 (q, J = 7.2 Hz, 2H), 2.94–2.75 (m, 2H), 1.14 (t, J = 7.2 Hz, 3H);

¹³C NMR (101 MHz, CD₃OD) δ 170.0, 158.8, 158.4, 155.6, 134.3, 132.2, 131.6, 126.7, 125.5, 122.7, 116.9, 116.1, 65.4, 63.0, 55.4, 36.7, 35.7, 15.7;

LRMS (ESI⁺) m/z: 414 (M + H, 100%).

HRMS (ESI⁺): Calcd 413.1951 for C₂₂H₂₇N₃O₅ [M]⁺; found, 413.2191.

(Z)-2-(4-((4-(4-(3-Ethylureido)phenoxy)but-2-en-1-yl)oxy)phenyl)ethan-1-aminium (13i)

Compound 13i was synthesized using general procedure 10 from tert-butyl (4-hydroxyphenethyl)carbamate (12i) (0.322 g, 1.356 mmol), (Z)-1-(4-((4-chlorobut-2-en-1-yl)oxy)phenyl)-3-ethylurea (11) (0.210 g, 0.714 mmol), and cesium carbonate (0.450 g, 1.381 mmol) in DMF (3 mL). The resulting reaction mixture was washed with water (3 × 50 mL) and brine (50 mL). The organic layer was dried over MgSO₄ and concentrated in vacuo. The resulting residue was taken up in 4 M HCl in dioxane (5 mL), stirred at room temperature for 2 h, diluted with ether (25 mL), and cooled to 0 °C. The precipitate was then filtered to afford the title compound (0.085 g, 29%) as a white solid (mp 177–179 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (s, 1H), 7.93 (bs, 3H), 7.28 (d, J = 9.0 Hz, 2H), 7.17 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 9.1 Hz, 2H), 6.11 (bs, 1H), 5.89–5.76 (m, 2H), 4.66 (dd, J = 19.5, 4.4 Hz, 4H), 3.07 (q, J = 7.2 Hz, 2H), 3.03–2.91 (m, 2H), 2.84–2.75 (m, 2H), 1.03 (t, J = 7.2 Hz, 3H);

¹³C NMR (101 MHz, DMSO-d₆) δ 157.4, 155.9, 153.0, 134.6, 130.2, 129.8, 129.2, 128.7, 119.6, 115.3, 115.3, 64.5, 64.3, 40.6, 34.4, 32.6, 16.0;

LRMS (ESI⁺) m/z: 370 (M – Cl, 100%);

HRMS (ESI⁺): Calcd 370.2130 for C₂₁H₂₈N₃O₃ [M + H]⁺; found, 370.2133.

(Z)-1,1′-((But-2-ene-1,4-diylbis(oxy))bis(4,1-phenylene))bis(ethan-1-one) (15a)

Compound 15a was synthesized using general procedure 1 from 4-hydroxyacetophenone (14a) (0.130 g, 0.948 mmol), (Z)-1,4-dichloro-2-butene (5e) (0.040 mL, 0.380 mmol), potassium carbonate (0.131 g, 0.948 mmol), and potassium iodide (0.147 g, 0.890 mmol) in acetonitrile (50 mL) to afford the title compound (0.089 g, 73%) as an off-white solid (mp 74 °C (dec.)).

¹H NMR (400 MHz, CDCl₃) δ 7.93 (d, J = 8.9 Hz, 4H), 6.94 (d, J = 8.9 Hz, 4H), 501–5.94 (m, 2H), 4.76 (d, J = 4.1 Hz, 4H), 2.55 (s, 6H);

¹³C NMR (101 MHz, CDCl₃) δ 196.7, 162.2, 130.7, 130.7, 128.3, 114.3, 64.3, 26.4;

LRMS (ESI⁺) m/z: 365 (M + H, 100%), 247 (M + Na, 25%);

HRMS (ESI⁺): Calcd 325.144 for C₂₀H₂₁O₄ [M + H]⁺; found, 325.1449.

(Z)-1,4-Bis(4-chlorophenoxy)but-2-ene (15b)

Compound 15b was synthesized using general procedure 1 from 4-chlorophenol (14b) (0.102 g, 0.800 mmol), (Z)-1,4-dichloro-2-butene (5e) (0.040 mL, 0.380 mmol), potassium carbonate (0.111 g, 0.800 mmol), and potassium iodide (0.147 g, 0.890 mmol) in acetonitrile (50 mL) to afford the title compound (0.115 g, 98%) as a cream solid (mp 37–41 °C).⁴⁴

¹H NMR (400 MHz, DMSO-d₆) δ 7.42–7.23 (m, 4H), 7.08–6.86 (m, 4H), 5.96–5.78 (m, 2H), 4.72 (d, J = 4.2 Hz, 2H);

¹³C NMR (101 MHz, DMSO-d₆) δ 161.6, 134.2, 128.3, 119.10, 115.8, 103.0, 64.3;

LRMS (ESI^–) m/z: 307 (M – H, ³⁵Cl₂, 100%), 309 (M – H, ³⁷Cl₂, 75%), 311 (M – H, ³⁵Cl³⁷Cl, 25%).

(Z)-1,4-Bis(4-methoxyphenoxy)but-2-ene (15c)

Compound 15c was synthesized using general procedure 1 from 4-hydroxyanisole (14c) (1.099 g, 8.870 mmol), (Z)-1,4-dichloro-2-butene (5e) (0.421 mL, 4.030 mmol), potassium carbonate (1.226 g, 8.89 mmol), and potassium iodide (0.147 g, 0.890 mmol) in acetonitrile (50 mL) to afford the title compound (0.723 g, 60%) as an off-white solid (mp 112–122 °C).⁴⁵

¹H NMR (400 MHz, CD₃OD) δ 6.93–6.81 (m, 8H), 5.94–5.83 (m, 2H), 4.65 (d, J = 4.1 Hz, 4H), 3.76 (s, 6H);

¹³C NMR (101 MHz, CD₃OD) δ 154.2, 152.6, 128.4, 115.5, 114.3, 64.6, 54.7;

LRMS (EI, 70 eV) m/z: 300 (M^+•, 10%), 177 (35), 123 (100);

HRMS (ESI⁺): Calcd 301.1440 for C₁₈H₂₁O₄ [M + H]⁺; found, 301.1384.

(Z)-4,4′-(But-2-ene-1,4-diylbis(oxy))dibenzonitrile (15d)

Compound 15d was synthesized using general procedure 1 from 4-hydroxybenzonitrile (15b) (0.095 g, 0.800 mmol), (Z)-1,4-dichloro-2-butene (5e) (0.040 mL, 0.380 mmol), potassium carbonate (0.111 g, 0.800 mmol), and potassium iodide (0.147 g, 0.890 mmol) in acetonitrile (50 mL) to afford the title compound (0.063 g, 57%) as an off-white solid (mp 122–124 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 7.78 (d, J = 8.7 Hz, 4H), 7.14 (d, J = 8.8 Hz, 4H), 5.98–5.85 (m, 2H), 4.84 (d, J = 3.7 Hz, 4H);

¹³C NMR (101 MHz, DMSO-d₆) δ 161.6, 134.2, 128.3, 119.10, 115.8, 103.0, 64.3;

LRMS (ESI^–) m/z: 289 (M – H, 90%), 325 (M + Cl, 100%);

HRMS (ESI⁺): Calcd 291.1133 for C₁₈H₁₅N₂O₂ [M + H]⁺; found, 291.1144.

Dimethyl 4,4′-(But-2-ene-1,4-diylbis(oxy))(Z)-dibenzoate (15e)

Compound 15e was synthesized using general procedure 1 from methyl-4-hydroxybenzoate (14e) (5.51 g, 36.210 mmol), (Z)-1,4-dichloro-2-butene (5e) (1.72 mL, 16.46 mmol), potassium carbonate (5.000 g, 36.21 mmol), and potassium iodide (0.600 g, 3.620 mmol) in acetonitrile (150 mL) to afford the title compound (2.640 g, 45%) as a white solid (mp 78–81 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 7.91 (d, J = 8.9 Hz, 4H), 7.08 (d, J = 8.9 Hz, 4H), 5.95–5.86 (m, 2H), 4.84 (d, J = 4.1 Hz, 4H), 3.82 (s, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 166.3, 162.5, 131.7, 128.8, 122.5, 115.2, 64.6, 52.3;

LRMS (ESI⁺) m/z: 357 (M + H, 100%);

HRMS (ESI⁺): Calcd 357.1338 for C₂₀H₂₁O₆ [M + H]⁺; found, 357.1348.

(Z)-4,4′-(But-2-ene-1,4-diylbis(oxy))dibenzaldehyde (15f)

Compound 15f was synthesized using general procedure 1 from 4-hydroxybenzaldehyde (14f) (0.654 g, 5.324 mmol), (Z)-1,4-dichloro-2-butene (5e) (0.253 mL, 2.420 mmol), potassium carbonate (0.735 g, 5.534 mmol), and potassium iodide (0.040 g, 0.240 mmol) in acetonitrile (50 mL) to afford the title compound (0.538 g, 75%) as a white solid (mp 90–93 °C).

¹H NMR (400 MHz, CDCl₃) δ 9.90 (s, 2H), 7.85 (d, J = 8.7 Hz, 4H), 7.02 (d, J = 8.7 Hz, 4H), 6.05–5.91 (m, 2H), 4.78 (d, J = 3.8 Hz, 4H);

¹³C NMR (101 MHz, CDCl₃) δ 190.9, 163.4, 132.2, 130.5, 128.4, 115.0, 64.6;

LRMS (ESI⁺) m/z: 297 (M + H, 20%), 145 (100);

HRMS (ESI⁺): Calcd 319.0941 for C₁₈H₁₆O₄Na [M + Na]⁺; found, 319.0953.

(Z)-N,N′-((But-2-ene-1,4-diylbis(oxy))bis(4,1-phenylene))diacetamide (16)

A solution of (Z)-4,4′-(but-2-ene-1,4-diylbis(oxy))dianiline hydrochloride (8e) (0.100 g, 0.291 mmol) and DIPEA (0.200 mL, 1.164 mmol) in anhydrous CH₂Cl₂ (25 mL) was stirred for 5 min before adding acetyl chloride (0.166 mL, 2.328 mmol). The reaction mixture was then stirred at room temperature for 16 h. The resulting reaction mixture was then concentrated in vacuo, diluted with ethyl acetate (100 mL), washed with water (2 × 100 mL), and saturated with NaHCO₃ (100 mL). The organic layer was dried over MgSO₄, concentrated in vacuo with the residue, adsorbed onto silica (∼1.00 g), and purified by flash chromatography (2% CH₃OH in CH₂Cl₂) to afford the title compound (0.067 g, 65%) as a white solid (mp 158–164 °C).⁴⁶

¹H NMR (400 MHz, DMSO-d₆) δ 9.77 (s, 2H), 7.46 (d, J = 8.5 Hz, 4H), 6.89 (d, J = 8.5 Hz, 4H), 5.86–5.80 (m, 2H), 4.66 (d, J = 1.5 Hz, 4H), 2.00 (s, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 167.1, 153.2, 132.2, 127.9, 119.8, 114.0, 63.3, 23.2;

MS (ESI⁺) m/z: 355 (M + H, 100%), 377 (M + Na, 25%).

(Z)-1,1′-((But-2-ene-1,4-diylbis(oxy))bis(4,1-phenylene))bis(3-ethylthiourea) (17)

To a solution of (Z)-4,4′-(but-2-ene-1,4-diylbis(oxy))dianiline hydrochloride (8e) (0.159 g, 0.465 mmol) and TEA (0.133 mL, 0.953 mmol) in 15 mL of anhydrous THF, ethyl isothiocyanate (0.113 mL, 1.297 mmol) was added. The resulting mixture was heated at reflux for 6 h under a nitrogen atmosphere, cooled to room temperature, and concentrated in vacuo. The residue was taken up in EtOAc (100 mL); washed with water (100 mL), 1 M HCl (100 mL), and brine (100 mL); dried over MgSO₄; concentrated in vacuo; and then purified by flash chromatography (2% CH₃OH in CH₂Cl₂) to afford the title compound (0.069 g, 33%) as a white solid (mp 152–157 °C).

¹H NMR (400 MHz, acetone-d₆) δ 8.53 (s, 2H), 7.25 (d, J = 8.9 Hz, 4H), 6.96 (d, J = 8.9 Hz, 6H), 5.95–5.89 (m, 2H), 4.78 (d, J = 4.0 Hz, 4H), 3.61 (dq, J = 7.1, 5.6 Hz, 4H), 1.14 (t, J = 7.2 Hz, 6H);

¹³C NMR (101 MHz, acetone-d₆) δ 182.5, 157.5, 132.3, 129.4, 127.6, 116.0, 65.2, 40.2, 14.7;

LRMS (ESI⁺) m/z: 445 (M + H, 100%);

HRMS (ESI⁺): Calcd 445.1732 for C₂₂H₂₉N₄O₂S₂ [M + H]⁺; found, 445.1743.

(Z)-4,4′-(But-2-ene-1,4-diylbis(oxy))dibenzoic Acid (18)

A solution of dimethyl 4,4′-(but-2-ene-1,4-diylbis(oxy))(Z)-dibenzoate (15e) (0.400 g, 1.122 mmol) in 10% KOH: THF (2:1, 100 mL) was refluxed for 1 h. The resulting solution was then acidified (pH 4 and 5) with 0.25 M HCl. The resulting precipitate was collected and washed with water (25 mL) to afford the title compound (0.360 g, 98%) as a white solid (mp 248 °C (dec.)).⁴⁷

¹H NMR (400 MHz, DMSO-d₆) δ 7.89 (d, J = 8.5 Hz, 4H), 7.05 (d, J = 8.5 Hz, 4H), 5.91 (bs, 2H), 4.83 (bs, 4H);

¹³C NMR (101 MHz, DMSO-d₆) δ 167.3, 162.1, 131.8, 128.7, 123.7, 115.0, 64.5;

LRMS (ESI^–) m/z: 327 (M – H, 100%);

HRMS (ESI⁺): Calcd 351.0839 for C₁₈H₁₆O₆Na [M + Na]⁺; found, 351.0853.

(Z)-4,4′-(But-2-ene-1,4-diylbis(oxy))bis(N-methylbenzamide) (19)

To a suspension of (Z)-4,4′-(but-2-ene-1,4-diylbis(oxy))dibenzoic acid (18) (0.228 g, 0.690 mmol) in anhydrous CH₂Cl₂ (10 mL), DMF (four drops) and oxalic chloride (1.5 mL, 2 M in CH₂Cl₂) were added. The solution was then stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuo, taken up in anhydrous THF (50 mL), and 2 M CH₃NH₂ in THF (2.76 mL, 0.171 g, 5.52 mmol) was added. Then, the mixture was stirred at room temperature for 1 h. The resulting reaction mixture was concentrated in vacuo, adsorbed onto silica, and purified by flash chromatography (0.35% NH₄OH, 2.5% CH₃OH in CH₂Cl₂) to afford the title compound (0.106 g, 43%) as an off-white solid (mp 128 −131 °C).

¹H NMR (400 MHz, DMSO-d₆) δ 8.27 (m, 2H), 7.80 (d, J = 8.8 Hz, 4H), 7.02 (d, J = 8.8 Hz, 4H), 5.93–5.86 (m, 2H), 4.80 (d, J = 4.0 Hz, 4H), 2.76 (d, J = 4.5 Hz, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 166.5, 160.7, 129.3, 128.8, 127.5, 114.6, 64.4, 26.6;

LRMS (ESI⁺) m/z: 355 (M + H, 100%);

HRMS (ESI⁺): Calcd 355.1658 for C₂₀H₂₃N₂O₄ [M + H]⁺; found, 445.1743.

(Z)-1,4-Bis(4-(5-methyl-1,2,4-oxadiazol-3-yl)phenoxy)but-2-ene (20)

To a solution of (Z)-4,4′-(but-2-ene-1,4-diylbis(oxy))dibenzonitrile (15d) (0.298 g, 1.026 mmol) in acetonitrile (50 mL) at room temperature, hydroxylamine (1.280 mL, 20.890 mmol) was added, and the mixture heated to reflux for 4 h. On cooling, the mixture was concentrated in vacuo and the residue was adsorbed onto silica (∼1.00 g) and purified by flash chromatography (10% CH₃OH in CH₂Cl₂). The material was then carried through to the next step, without any further characterization.

To a solution of the N-hydroxybenzimidamide intermediate and 3 Å molecular sieves in anhydrous THF (50 mL), acetyl chloride (0.300 mL, 3.97 mmol) was added and heated at reflux for 16 h. The resulting reaction mixture was diluted with a 1:1:1 mixture of CH₃OH/EtOAc/THF (250 mL), and the molecular sieves were washed with THF (2 × 100 mL). The combined organic layers were concentrated in vacuo, and the residue was adsorbed onto silica (∼1.00 g) and purified by flash chromatography (30% EtOAc in hexane) to afford an off-white crystal (30 mg, 7%, two steps).

¹H NMR (400 MHz, DMSO-d₆) δ 7.93 (d, J = 8.9 Hz, 4H), 7.14 (d, J = 8.9 Hz, 4H), 6.02–5.77 (m, 2H), 4.84 (d, J = 4.1 Hz, 4H), 2.64 (s, 6H);

¹³C NMR (101 MHz, DMSO-d₆) δ 177.1, 167.3, 160.5, 128.6, 128.4, 118.8, 115.4, 64.10, 12.0;

LRMS (ESI⁺) m/z: 405 (M + H, 100%), 321 (25);

HRMS (ESI⁺): Calcd 405.1563 for C₂₂H₂₁N₄O₄ [M + H]⁺; found, 405.1559.

(Z)-4,4′-(But-2-ene-1,4-diylbis(oxy))dibenzimidamide (21)

Ammonium chloride (0.450 g, 8.413 mmol) was added to a 2 M solution of trimethylaluminum in toluene (4.05 mL, 8.100 mmol) at 0 °C under a nitrogen atmosphere. The suspension was then warmed to room temperature before dropwise addition of a solution of (Z)-4,4′-(but-2-ene-1,4-diylbis(oxy))dibenzonitrile (15d) (0.401 g, 1.380 mmol) in anhydrous toluene (50 mL). After complete addition, the reaction was then heated to 80 °C for 16 h. The resulting reaction mixture was then cooled to room temperature and poured into a slurry of silica gel (∼2.00 g) and chloroform (20 mL). The silica plug was then washed with 10% CH₃OH in CH₂Cl₂ (200 mL). The filtrate was then concentrated in vacuo, diluted with 100 mL of EtOAc (100 mL), washed with water (100 mL) and brine (100 mL), dried over MgSO₄, and concentrated in vacuo to afford the title compound (0.098 g, 45%) as a white solid (mp 212–213 °C).⁴⁸

¹H NMR (400 MHz, DMSO-d₆) δ 7.79 (d, J = 8.9 Hz, 4H), 7.14 (d, J = 8.9 Hz, 4H), 6.02–5.78 (m, 2H), 4.85 (d, J = 4.2 Hz, 4H);

¹³C NMR (101 MHz, DMSO-d₆) δ 162.0, 134.7, 128.7, 119.6, 116.3, 103.5, 64.7;

MS (ESI⁺) m/z: 325 (M + H, 10%) 269 (100%), 335 (80%).

2-((Z)-4-(((Z)-4-(4-((E)-(Carbamimidoylimino)methyl)phenoxy)but-2-en-1-yl)oxy)benzylidene)hydrazine-1-carboximidamide (22)

General Procedure 11: A solution of (Z)-4,4′-(but-2-ene-1,4-diylbis(oxy))dibenzaldehyde (15f) (0.100 g, 0.337 mmol), aminoguanidine hydrochloride (0.082 g, 0.741 mmol), and a drop of 10% HCl in ethanol (3 mL) was subjected to microwave irradiation at 120 °C for 30 min. The reaction mixture was then concentrated to afford a white precipitate. The precipitate was then suspended in ether (25 mL), sonicated, and filtered to afford the title compound (0.135 g, 98%) as an off-white solid (mp 232 °C (dec.)).

¹H NMR (400 MHz, DMSO-d₆) δ 11.96 (brs, 2H), 8.12 (s, 2H), 7.77 (m, 12H), 7.04 (d, J = 8.4 Hz, 4H), 5.90 (brs, 2H), 4.81 (d, J = 2.2 Hz, 4H);

¹³C NMR (101 MHz, CD₃OD) δ 152.9, 147.6, 139.8, 121.0, 120.0, 118.1, 106.6, 56.0;

LRMS (ESI^–) m/z: 443 (M + Cl, 100%);

HRMS (ESI⁺): Calcd 409.2100 for C₂₀H₂₅N₈O₂ [M + H]⁺; found, 409.2114.

Glossary

Abbreviations

PPI

protein–protein interaction

MIC

minimum inhibitory concentration

FDA

Food and Drug Administration

ESKAPE

Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species

MOE

molecular operating environment

μWave

microwave

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b00273.

• ¹H and ¹³C NMR spectra of the compounds synthesized herein (PDF)

• Plasmids and bacterial strains used and constructed in this work were confirmed by DNA sequencing and are listed in the table (XLSX)

Author Present Address

^# Department of Applied Biology and Chemical Technology, The State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, P. R. China (C.M.).

The authors declare no competing financial interest.