Investigation of Naphthyl–Polyamine Conjugates as Antimicrobials and Antibiotic Enhancers

Abstract

As part of our search for new antimicrobials and antibiotic enhancers, a series of naphthyl- and biphenyl-substituted polyamine conjugates have been synthesized. The structurally-diverse library of compounds incorporated variation in the capping end groups and in the length of the polyamine (PA) core. Longer chain (PA-3-12-3) variants containing both 1-naphthyl and 2-naphthyl capping groups exhibited more pronounced intrinsic antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) (MIC ≤ 0.29 µM) and the fungus Cryptococcus neoformans (MIC ≤ 0.29 µM). Closer mechanistic study of one of these analogues, 20f, identified it as a bactericide. In contrast to previously reported diarylacyl-substituted polyamines, several examples in the current set were able to enhance the antibiotic action of doxycycline and/or erythromycin towards the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli. Two analogues (19a and 20c) were of note, exhibiting greater than 32-fold enhancement in activity. This latter result suggests that α,ω-disubstituted polyamines bearing 1-naphthyl- and 2-naphthyl-capping groups are worthy of further investigation and optimization as non-toxic antibiotic enhancers.

1. Introduction

Host-defense peptides (HDPs), produced by a wide variety of organisms in nature, including microorganisms, plants, invertebrates, and mammals, play essential roles as a first line of protection against viral, fungal, and bacterial infections [1,2,3,4,5,6]. A subset of HDPs are the antimicrobial peptides, small (50 amino acids or less) amphipathic peptides of diverse sequence homology and secondary structures [7]. From a structural perspective, antimicrobial peptides are characterized as containing hydrophobic residues on one side of the molecule and hydrophilic cationic residues on the other. These peptides are thought to act directly on bacterial cell membranes, with the cationic charges aiding electrostatic attraction to the negatively charged cell membrane, followed by hydrophobic residue insertion into the membrane leading to disruption, increased membrane permeability, and, ultimately, cell death [7]. Drug development issues associated with HDPs, including susceptibility to proteolysis, high production costs, and, in some case, low to moderate activity under physiological salt conditions, prevent their direct introduction as clinical agents [8]. In order to overcome these deficiencies, an extensive amount of research has been directed towards the synthesis and biological evaluation of synthetic mimics of antimicrobial peptides, so-called SMAMPs [9,10,11,12,13,14,15]. Numerous different structural classes of SMAMPs have been identified, including shorter peptides (e.g., LTX-109 (1)), sterols (e.g., squalamine (2)), and hydantoins (e.g., 3) (Figure 1) [15,16,17]. Although they cover diverse chemo-types, all three of these examples are thought to act via a general bacterial membrane-targeting mechanism, with insertion leading to membrane disruption [16,18,19]. LTX-109 is one example of a number of different antimicrobial peptides that have entered clinical trials for the treatment of microbial infections [20].

The discovery of squalamine (2), a polyamine-containing aminosterol isolated from the dogfish shark Squalus acanthias, and observation of its broad-spectrum activity towards both Gram-positive and Gram-negative bacteria [21] prompted further investigation of the marine environment in the search for new classes of antimicrobials [22]. The marine sponge natural product ianthelliformisamine C (4) exhibits antimicrobial activity and can also enhance the activity of legacy antibiotics towards drug resistant Gram-negative bacteria [23,24,25]. Ianthelliformisamine C has all the structural attributes of an SMAMP, with the secondary amines of the polyamine fragment being protonated at physiological pH and the terminal cinnamate capping group being lipophilic, defining this as a scaffold worthy of attention [22,26].

We recently reported the synthesis and antimicrobial evaluation of a set of α,ω-diacylaryl substituted polyamine analogues, of which examples 5–7 (Figure 2) are representative [27].

Analogues bearing a single aryl group at each end of a polyamine (PA) chain, e.g., 5, were found to be almost uniformly inactive towards a panel of Gram-positive and Gram-negative bacteria and fungal strains, with only the longer polyamine chain variants (PA-3-10-3 and PA-3-12-3) exhibiting weak-to-potent activity towards methicillin-resistant Staphylococcus aureus (MRSA). Increasing the lipophilicity by inclusion of one or two additional phenyl rings in the capping acid created sets of analogues, e.g., 6 and 7, that demonstrated good to excellent growth inhibition properties towards Staphylococcus aureus (MIC 3.13 µM for both), MRSA (MIC ≤ 0.28 µM and MIC ≤ 0.24 µM, respectively), and Escherichia coli (MIC 2.2 µM and MIC 7.6 µM, respectively). When counter screen cytotoxicity and hemolytic activities were combined with calculated LogP values, it became apparent that optimal selectivity for antimicrobial activity was observed for diaryl-containing capping groups with whole molecule cLogP in the range 7–8.5. Any analogues with cLogP greater than 9–10 appear to breach a ‘second hydrophobicity threshold’ [28], leading to greater disruption of mammalian membranes and inherent toxicity. Mechanism of action studies carried out on the diaryl-analogue 6 identified it as a strong disruptor of bacterial cell membranes and to be bactericidal [27], making it a good starting point for further optimization.

Mindful of not exceeding the second hydrophobicity threshold, we chose to prepare new examples of α,ω-disubstituted polyamines that targeted the cLogP range of approximately 5–9 and incorporated aryl-carboxylic acids 8–12 (Figure 3), which contained variations in shape, size, lipophilicity and position of attachment to the polyamine core.

Herein, we report the synthesis of a set of new α,ω-disubstituted polyamines that explore variation in the size and shape of the chain end-groups within a narrow band of lipophilicity as well as variation in polyamine length. All analogues were evaluated for antimicrobial activities against a set of Gram-positive and Gram-negative bacteria and two fungi strains, and for the ability to enhance the antibiotic action of doxycycline and erythromycin towards the Gram-negative bacteria Pseudomonas aeruginosa and E. coli, respectively.

2. Chemistry

Of the five carboxylic acids required for this study (8–12), two (8 and 10) were commercially available. Carboxylic acids 9, 11, and 12 were prepared by reaction of naphthalen-1-amine (13), naphthalen-2-amine (14), and 4-aminobiphenyl (15) with succinic anhydride, in yields of 45–97% (Scheme 1, Supplementary Materials Figures S1–S3).

Syntheses of the core Boc-protected polyamine scaffolds 16a–f (Figure 4) have been previously described [29,30,31,32]. These six polyamines (PA3-4-3, PA3-6-3, PA3-7-3, PA3-8-3, PA3-10-3, PA3-12-3) contain variations in chain length, lipophilicity, and spatial separation of the dialkylammonium ion positive charges, all of which were considered to potentially have some influence on bioactivity.

Reaction of carboxylic acids 8–12 with Boc-protected polyamines 16a–f utilized coupling reagents EDC·HCl or EDC·HCl/HOBt in anhydrous CH₂Cl₂, and then the products were deprotected using 2,2,2-trifluoroacetic acid (TFA) to create the target compounds as their di-TFA salts (Scheme 2, Figures S4–S32).

The structures of the synthesized α,ω-disubstituted polyamine library are shown in Figure 5.

3. Results and Discussion

The antimicrobial activity of each compound was initially assessed against various bacterial strains, including S. aureus and MRSA, P. aeruginosa, E. coli, Klebsiella pneumoniae, and Acinetobacter baumannii, as well as fungal strains such as Candida albicans and Cryptococcus neoformans (

Table 1. Antimicrobial activities (MIC, µM) and cLogP values of analogues 17–21.

Cmpd	cLogP a	S. a b	MRSA c	P. a d	E. c e	K. p f	A. b g	C. a h	C. n i
17a	5.1	677	>43.3	677	677	>43.3	>43.3	>43.3	>43.3
17b	6.0	200	>41.7	200	200	>41.7	>41.7	>41.7	>41.7
17c	6.5	256	>41.0	>256	256	>41.0	>41.0	>41.0	>41.0
17d	6.9	50	>40.3	>100	100	>40.3	>40.3	>40.3	>40.3
17e	7.8	244	9.72	608	608	>38.9	>38.9	>38.9	38.9
17f	8.7	25	0.29	300	100	>37.6	0.29	>37.6	0.29
18a	4.8	568	>36.3	568	568	>36.3	>36.3	>36.3	>36.3
18b	5.7	>200	>35.2	>200	>200	>35.2	>35.2	>35.2	>35.2
18c	6.2	>200	>34.7	200	200	>34.7	>34.7	>34.7	>34.7
18d	6.6	107	>34.2	>107	>107	>34.2	>34.2	>34.2	>34.2
18e	7.5	104	33.2	518	518	>33.2	>33.2	>33.2	33.2
18f	8.4	3.15	≤0.25	>100	12.5	>32	>32	>32	≤0.25
19a	5.1	34	43.3	677	271	>43.3	>43.3	>43.3	21.7
19b	6.0	65.2	41.7	>130	130	>41.7	>41.7	>41.7	20.9
19c	6.5	16.0	≤0.32	32.0	16.0	>41	>41	>41	≤0.32
19d	6.9	7.9	40.3	126	126	>40.3	>40.3	>40.3	40.3
19e	7.8	30	1.22	608	122	38.9	38.9	38.9	4.86
19f	8.7	100	0.294	300	300	>37.6	0.294	>37.6	0.294
20a	4.8	57	18.16	568	227	>36.3	>36.3	>36.3	>36.3
20b	5.7	>200	>35.2	>200	50	>35.2	>35.2	>35.2	35.2
20c	6.2	25	2.2	>200	25	>34.7	>34.7	>34.7	≤0.27
20d	6.6	13.3	>34.2	>107	107	>34.2	>34.2	>34.2	17.1
20e	7.5	12.95	16.6	518	104	>33.2	>33.2	>33.2	16.6
20f	8.4	3.125	≤0.25	>100	6.3	>32	>32	>32	≤0.25
21a	5.7	300	0.268	300	300	>34.3	>34.3	>34.3	>34.3
21b	6.6	300	2.08	300	300	>33.3	>33.3	>33.3	>33.3
21c	7.1	200	0.256	300	300	>32.8	>32.8	>32.8	>32.8
21d	7.6	300	0.506	300	300	>32.4	>32.4	>32.4	>32.4
21e	8.5	300	0.246	300	300	>31.5	>31.5	31.5	15.73

^a cLogP values calculated in DataWarrior v05.05.00 [33]; ^b S. aureus (ATCC 25,923 or ATCC 29213) with streptomycin (MIC 21.5 µM) and chloramphenicol (MIC 1.5–3 µM) were used as positive controls, and values are presented as the mean (n = 3); ^c MRSA (ATCC 43300) with vancomycin (MIC 0.7 µM) was used as a positive control, and values are presented as the mean (n = 2); ^d P. aeruginosa (PAO1 or ATCC 27853) with streptomycin (MIC 21.5 µM) and colistin (MIC 1 μM) were used as positive controls, and values are presented as the mean (n = 3); ^e E. coli (ATCC 25922) with colistin (MIC 2 µM) was used as a positive control, and values are presented as the mean (n = 2); ^f K. pneumoniae (ATCC 700603) with colistin (MIC 0.2 µM) was used as a positive control, and values are presented as the mean (n = 2); ^g A. baumannii (ATCC 19606) with colistin (MIC 0.2 µM) was used as a positive control, and values are presented as the mean (n = 2); ^h C. albicans (ATCC 90028) with fluconazole (MIC 0.4 µM) was used as a positive control, and values are presented as the mean (n = 2); ⁱ C. neoformans (ATCC 208821) with fluconazole (MIC 26 µM) was used as a positive control, and values are presented as the mean (n = 2).

). As a set, analogues containing a 1-naphthyl substituted end group (17a–f, 18a–f) were predominantly inactive towards all microbes, with the exceptions being the longer polyamine chain variants 17e (MRSA MIC 9.7 µM), 17f (MRSA MIC 0.29 µM, A. baumannii MIC 0.29 µM, C. neoformans MIC 0.29 µM), and 18f (S. aureus MIC 3.15 µM, MRSA MIC ≤ 0.25 µM, C. neoformans MIC ≤ 0.25 µM). In contrast, the 2-substituted naphthyl analogue sets 19a–f and 20a–f contained a greater number of active compounds, with five analogues (19c, 19e, 19f, 20c, and 20f) exhibiting activity towards primarily the Gram-positive bacteria S. aureus and/or MRSA and the fungus C. neoformans. Direct comparison of antimicrobial activities exhibited by corresponding 1-naphthyl vs. 2-naphthyl substituted analogues suggested, however, that there was little to no difference between the series of compounds as far as intrinsic antimicrobial activity was concerned. For example, analogues 17f vs. 19f were equipotent towards the same three microbial strains (MRSA MIC 0.29 µM, A. baumannii MIC 0.29 µM, C. neoformans MIC 0.29 µM), and 18f vs. 20f were essentially identical growth inhibitors towards S. aureus (MIC 3.125 µM), MRSA (MIC ≤ 0.25 µM), E. coli (MIC 6.3–12.5 µM), and C. neoformans (MIC ≤ 0.25 µM). The biphenyl substituted series of analogues 21a–e were noticeably different in their spectrum of antimicrobial activities, with all analogues exhibiting activity towards just MRSA with MIC 0.25–2.1 µM.

The majority of the compounds, 17–21, were evaluated for cytotoxicity towards human kidney epithelial cell line (HEK293) and for hemolytic activity towards human red blood cells (

Table 2. Cytotoxic (IC₅₀, µM) and hemolytic (HC₁₀, µM) properties of analogues 17–21.

Compound	Cytotoxicity a	Hemolysis b	Compound	Cytotoxicity a	Hemolysis b
17a	nt c	nt c	20a	>36.3	>36.3
17b	>41.7	>41.7	20b	>35.2	>35.2
17c	>41.0	>41.0	20c	>34.7	>34.7
17d	>40.3	>40.3	20d	>34.2	>34.2
17e	23.7	>38.9	20e	>33.2	>33.2
17f	>37.6	>37.6	20f	>32.2	>32.2
18a	nt c	nt c	21a	>34.3	31.4
18b	>35.2	>35.2	21b	>33.3	>33.3
18c	>34.7	>34.7	21c	>32.8	>32.8
18d	>34.2	>34.2	21d	>32.4	>32.4
18e	>33.2	>33.2	21e	>31.5	6.32
18f	>32.2	>32.2
19a	26.4	>43.3
19b	41.7	>41.7
19c	20.5	>41.0
19d	40.3	>40.3
19e	4.75	>38.9
19f	>37.6	>37.6

All values presented as the mean (n = 2); ^a Concentration (IC₅₀, µM) of compound at 50% cytotoxicity on HEK293 human embryonic kidney cells with tamoxifen as the positive control (IC₅₀ 24 μM); ^b Concentration (HC₁₀, µM) of compound at 10% hemolytic activity on human red blood cells with melittin as the positive control (HC₁₀ 0.95 µM); ^c Not tested.

). Of the set of compounds tested, only biphenyl 21e was considered to be hemolytic (HC₁₀ 6.3 µM), and the observation of cytotoxicity was limited to the four analogues 17e (IC₅₀ 23.7 µM), 19a (IC₅₀ 26 µM), 19c (IC₅₀ 20.5 µM), and 19e (IC₅₀ 4.75 µM). The latter results, in particular, emphasize that cytotoxicity in the current series is not determined by lipophilicity alone, as other analogues with similar cLogP values (e.g., 17a–d, 18a–e, 20a–e) were deemed non-toxic.

To conduct a detailed analysis of the antibacterial activity and initial assessment of the mechanism of action, analogue 20f was selected due to its strong antibacterial properties, and because it was devoid of any observed cytotoxic or hemolytic effects. To evaluate the kinetics of antibacterial activity, real-time growth inhibition curves were measured for two Gram-positive bacteria (S. aureus (ATCC 25923), MRSA (CF-Marseille) [34]) and the Gram-negative bacterium E. coli (ATCC 25922). The test compound completely inhibited the growth of the Gram-positive bacteria strains at 3.15 μM (3.13 µg/mL) or higher concentrations, with growth observed at the lowest test concentration of 1.57 µM (1.56 µg/mL) (Figure 6A,B). In the case of the Gram-negative bacterium E. coli (Figure 6C), bacterial growth inhibition was observed at test concentrations of 6.29 µM (6.25 µg/mL) or higher. MIC values of 3.15 μM (3.13 μg/mL), 3.15 µM (3.13 µg/mL), and 6.29 µM (6.25 µg/mL) were determined for analogue 20f against S. aureus (ATCC 25923), MRSA (CF-Marseille), and E. coli (ATCC 25922), respectively. These values corresponded to the inhibitory concentrations observed at the 18-hour mark in the real-time growth inhibition curve plots. Additionally, the same values were observed for the minimum bactericidal concentration (MBC) of 20f against all three organisms, indicating its bactericidal activity.

The ability of compounds 17–21 to enhance the antibiotic activity of doxycycline against P. aeruginosa (ATCC 27853) and of erythromycin against E. coli (ATCC 25922) were determined (

Table 3. Antibiotic enhancement activity (MIC μM) of analogues 17–21.

Compound	Dox/P.a a	Eryth/E.c b	Compound	Dox/P.a a	Eryth/E.c b
17a	271 (2.5)	67.7 (10)	20a	28 (20)	nt c
17b	12.5 (16)	100 (2)	20b	25 (>8)	50 (1)
17c	64 (>2.7)	64 (4)	20c	6.25 (>32)	12.5 (2)
17d	126 (1)	31.5 (3)	20d	53.4 (>2)	26.7 (4)
17e	608 (1)	122 (5)	20e	104 (5)	13.0 (8)
17f	300 (1)	50 (2)	20f	12.6 (>8)	3.15 (2)
18a	568 (1)	568 (1)	21a	300 (1)	300 (1)
18b	100 (>2)	200 (1)	21b	300 (1)	300 (1)
18c	12.5 (16)	200 (1)	21c	300 (1)	300 (1)
18d	>107 (1)	107 (1)	21d	300 (1)	300 (1)
18e	207 (2.5)	51.8 (10)	21e	300 (1)	300 (1)
18f	50.3 (>2)	6.29 (2)
19a	16.9 (40)	4.2 (64)
19b	65.2 (>2)	65.2 (2)
19c	4.00 (8)	4.0 (4)
19d	15.7 (8)	15.7 (8)
19e	608 (1)	60.8 (2)
19f	300 (1)	300 (1)

^a Concentration (µM) required to restore doxycycline activity at 4.5 µM against P. aeruginosa (ATCC 27853). Fold change shown in parentheses is the ratio between the intrinsic MIC of the test compound and the combination MIC; ^b Concentration (µM) required to restore erythromycin activity at 10.9 µM against E. coli (ATCC 25922). Fold change shown in parentheses is the ratio between the intrinsic MIC of the test compound and the combination MIC; ^c Not tested.

). In the case of doxycycline, a low-dose fixed concentration of 2 µg/mL (4.5 µM) was used, being 20-fold lower than the intrinsic MIC (40 µg/mL (90 µM)) against P. aeruginosa. Each of the compounds were then evaluated at a range of concentrations, with the upper limit being dependent upon the intrinsic MIC towards P. aeruginosa (Table 1). Two examples of 1-naphthyl-substituted analogues (17b and 18c) were identified as modest enhancers of the action of doxycycline, with MICs of 12.5 µM, representing 16-fold enhancements over their intrinsic MIC values. A further five examples of 2-naphthyl-substituted variants, 19a, 19c, 19d, 20a, 20c, and 20f, exhibited notable levels of enhancement, with MICs of 16.9, 4.0, 15.7, 28, 6.25, and 12.6 µM, respectively. When compared to their intrinsic growth inhibition activities towards P. aeruginosa (Table 1), these represented 40-fold, 8-fold, 8-fold, 20-fold, >32-fold, and >8-fold enhancements, respectively. The ability of the two structurally related 2-naphthyl-substituted PA-3-4-3 (spermine) analogues 19a and 20a to enhance the action of doxycycline towards P. aeruginosa was investigated more closely, revealing a dose-dependent response when the doxycycline concentration was varied from 2 to 8 µg/mL (

Table 4. Dose response enhancement (MIC, µM) of doxycycline towards P. aeruginosa (ATCC 27853) by compounds 19a and 20a.

	Compound
Doxycycline Concentration (μg/mL)	19a a	20a a
2	16.9	28.4
4	8.5	14.2
6	8.5	14.2
8	4.2	3.5

^a Concentration of test compound (µM) required to restore doxycycline activity at the dose specified against P. aeruginosa (ATCC 27853).

).

Evaluation of compounds 17–21 to enhance the antibiotic activity of erythromycin, using a fixed low dose of 8 µg/mL (10.9 µM) against E. coli (ATCC 25922) identified three analogues in particular, 19a (MIC 4.2 µM, 64-fold enhancement), 19d (MIC 15.7 µM, 8-fold), and 20e (MIC 13 µM, 8-fold enhancement), with strong levels of enhancement (MIC ≤ 20 µM). In addition, they were 8–10-fold more active in combination than when tested alone (Table 3).

Taken together, these studies have identified a number of predominantly 2-naphthyl-substituted polyamines as strong enhancers of the antibiotic action of doxycycline and/or erythromycin towards the Gram-negative bacteria P. aeruginosa, and E. coli. It is interesting to compare these results with our previous investigation of α,ω-diacylarylpolyamines, e.g., 6, which revealed them to be active antimicrobials but with weak antibiotic enhancement properties (e.g., doxycycline vs. P. aeruginosa, 4-fold increase to an MIC of 12.5 µM) [27]. The current results lead us to conclude that substituted naphthyl-polyamines may be worthy of further optimization as antibiotic enhancers. It is pertinent to note that Yasuda et al. have previously reported that naphthylacetylspermine (22) (Figure 7), a synthetic analogue of joro spider toxin, renders E. coli sensitive to hydrophobic antibiotics, including novobiocin and erythromycin, albeit weakly, at doses of 64–128 µg/mL [35].

4. Materials and Methods

4.1. Chemistry General Methods

Infrared spectra were run as dry films on an ATR crystal and acquired with a Perkin-Elmer 100 Fourier Transform infrared spectrometer equipped with a Universal ATR Sampling Accessory. Mass spectra were acquired on a Bruker micrOTOF Q II mass spectrometer. Melting points were obtained on an Electrothermal melting point apparatus and are uncorrected. The ¹H, ¹³C NMR, and 2D NMR spectra were recorded at 298 °K on a Bruker AVANCE AVIII 400 MHz spectrometer at 400.13 and 100.62 MHz, using standard pulse sequences. Proto-deutero solvent signals were used as internal references (DMSO-d₆: δ_H 2.50, δ_C 39.52; CD₃OD: δ_H 3.31, δ_C 49.00). For ¹H NMR, the data are quoted as position (δ), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant (J, Hz), and assignment to the atom. Atom positional assignments were made using 2D-NMR data acquired using standard pulse sequences. The ¹³C NMR data are quoted as position (δ) and assignment to the atom. Flash column chromatography was carried out using Davisil silica gel (40–60 μm) or LiChroprep RP-8 (40–63 µm) solid support. Silica gel thin layer chromatography (TLC) was conducted on 0.2 mm thick plates of DC-plastikfolien Kieselgel 60 F₂₅₄ (Merck). Reversed-phase TLC was carried out on 0.2 mm thick plates of DC-Kieselgel 60 RP-18 F₂₅₄S (Merck). All solvents used were of analytical grade or better and/or purified according to standard procedures. Chemical reagents used were purchased from standard chemical suppliers and used as purchased. All samples were determined to be >95% purity. Protected polyamines di-tert-butyl butane-1,4-diylbis((3-aminopropyl)carbamate) (16a), di-tert-butyl hexane-1,6-diylbis((3-aminopropyl)carbamate) (16b), di-tert-butyl heptane-1,7-diylbis((3-aminopropyl)carbamate) (16c), di-tert-butyl octane-1,8-diylbis((3-aminopropyl)carbamate) (16d), di-tert-butyl decane-1,10-diylbis((3-aminopropyl)carbamate) (16e), and di-tert-butyl dodecane-1,12-diylbis((3-aminopropyl)carbamate) (16f) were synthesized using literature procedures [29,30,31,32].

4.1.1. General Procedure A—Diamide Bond Formation

The appropriate Boc-protected polyamine 16a–f (1 equiv.) was added to a solution of carboxylic acid (2.2 equiv.), EDC⋅HCl (2.6 equiv.), HOBt (2.6 equiv.) and DIPEA (4–6 equiv.), that was stirred in anhydrous CH₂Cl₂ (1.5 mL) at 0 °C for 30 min under N₂. The mixture was allowed to come to room temperature and was stirred for a further 20 h under N₂. The reaction mixture was poured into CH₂Cl₂ (20 mL) and washed with saturated NaHCO₃ (2 × 30 mL) followed by H₂O (2 × 30 mL), and it was then dried under reduced pressure and purified by silica gel flash column chromatography (0–20% MeOH/CH₂Cl₂) to create the desired products.

4.1.2. General Procedure B—Diamide Bond Formation

The appropriate Boc-protected polyamine 16a–f (1 equiv.) was added to a solution of carboxylic acid (2.5 equiv.) and EDC·HCl (2.8 equiv.) with DMAP (5 equiv.) stirred in anhydrous CH₂Cl₂ (1.5 mL) at 0 °C for 10 min under N₂. The mixture was allowed to come to room temperature and stirred for a further 12 h under N₂. The reaction mixture was poured into CH₂Cl₂ (20 mL) and washed with saturated NaHCO₃ (2 × 30 mL) followed by H₂O (2 × 30 mL), and it was then dried under reduced pressure and purified by silica gel flash column chromatography (0–20% MeOH/CH₂Cl₂) to create the desired products.

4.1.3. General Procedure C—Boc Deprotection

A solution of tert-butyl-carbamate derivative in CH₂Cl₂ (2 mL) and TFA (0.2 mL) was stirred at room temperature under N₂ for 2 h, and it was followed by solvent removal under reduced pressure. The crude product was purified using C₈ reversed-phase flash column chromatography eluting with 0–100% MeOH/H₂O (0.05% TFA) to create the corresponding polyamine conjugate as the TFA salt.

4.2. Synthesis of Compounds

4.2.1. 4-(Naphthalen-1-ylamino)-4-oxobutanoic Acid (9)

Naphthalen-1-amine (200 mg, 1.40 mmol) and succinic anhydride (140 mg, 1.40 mmol) were stirred in anhydrous CH₂Cl₂ (10 mL) for 9 h under an N₂ atmosphere. The solvent was then removed under reduced pressure, and the crude product was purified by C₈ reversed-phase column chromatography (100% H₂O to 100% MeOH) to create 9 as a pale pink solid (330 mg, 97%). R_f = 0.43 (SiO₂, CH₂Cl₂:10% MeOH); m.p. 165–167 °C; IR (ATR) v_max 3302, 2919, 1711, 1531, 1401, 1176, 915, 773 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ 12.16 (1H, br s, OH), 9.94 (1H, br s, NH-5), 8.12–8.10 (1H, m, H-7), 7.94–7.91 (1H, m, H-10), 7.75 (1H, d, J = 7.8 Hz, H-11), 7.66 (1H, d, J = 7.8 Hz, H-13), 7.55–7.51 (2H, m, H-8, H-9), 7.47 (1H, dd, J = 7.8, 7.8 Hz, H-12), 2.75 (2H, t, J = 6.8 Hz, H₂-2), 2.60 (2H, t, J = 6.8 Hz, H₂-3); ¹³C NMR (DMSO-d₆, 100 MHz) δ 173.9 (C-1), 170.8 (C-4), 133.7 (C-6, C-10a), 128.0 (C-10), 127.8 (C-6a), 125.9 (C-8/C-9), 125.7 (C-12), 125.5 (C-8/C-9), 125.1 (C-11), 122.9 (C-7), 121.6 (C-13), 30.7 (C-2), 29.1 (C-3); (+)-HRESIMS m/z 266.0783 [M+Na]⁺ (calcd for C₁₄H₁₃NNaO₃, 266.0788).

4.2.2. 4-(Naphthalen-2-ylamino)-4-oxobutanoic Acid (11)

Naphthalen-2-amine (200 mg, 1.40 mmol) and succinic anhydride (140 mg, 1.40 mmol) were stirred in anhydrous CH₂Cl₂ (10 mL) for 9 h under an N₂ atmosphere. The solvent was then removed under reduced pressure, and the crude product was purified by C₈ reversed-phase column chromatography (100% H₂O to 100% MeOH) to create 11 as a pink solid (260 mg, 76%). R_f = 0.19 (SiO₂, CH₂Cl₂:10% MeOH); m.p. 170–172 °C; IR (ATR) v_max 3059, 1706, 1653, 1558, 1397, 1257, 824, 742 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ 10.18 (1H, br s, NH-5), 8.30 (1H, s, H-7), 7.85–7.77 (3H, m, H-8, H-11, H-12), 7.57 (1H, dd, J = 8.9, 2.0 Hz, H-13), 7.47–7.43 (1H, m, H-9), 7.40–7.36 (1H, m, H-10), 2.62 (2H, t, J = 6.0 Hz, H₂-2), 2.56 (2H, t, J = 6.0, H₂-3); ¹³C NMR (DMSO-d₆, 100 MHz) δ 173.9 (C-1), 170.4 (C-4), 136.9 (C-6), 133.5 (C-7a), 129.6 (C-11a), 128.3 (C-12), 127.4 (C-11), 127.2 (C-8), 126.3 (C-9), 124.4 (C-10), 119.8 (C-13), 114.8 (C-7), 31.2 (C-2), 29.0 (C-3); (+)-HRESIMS [M+Na]⁺ m/z 266.0784 (calcd for C₁₄H₁₃NNaO₃, 266.0788).

4.2.3. 4-([1,1’-Biphenyl]-4-ylamino)-4-oxobutanoic Acid (12)

4-Aminobiphenyl (500 mg, 2.95 mmol) and succinic anhydride (295 mg, 2.95 mmol) were stirred in anhydrous CH₂Cl₂ (11 mL) for 24 h under an N₂ atmosphere. The solvent was removed under reduced pressure, and the was product purified by recrystallisation from EtOH to create 12 as light orange crystals (356 mg, 45%). R_f = 0.17 (SiO₂, CH₂Cl₂:10% MeOH); m.p. >230 °C; IR (ATR) v_max 3273, 3031, 2934, 2635, 1689, 1651, 1529, 1403, 1186, 832, 750, 685 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ 12.13 (1H, br s, OH), 10.05 (1H, br s, NH-5), 7.70–7.65 (2H, m, H-7), 7.65–7.59 (4H, m, H-8, H-11), 7.46–7.41 (2H, m, H-12), 7.34–7.29 (1H, m, H-13), 2.61–2.57 (2H, m, H₂-2/H₂-3), 2.55–2.52 (2H, m, H₂-2/H₂-3); ¹³C NMR (DMSO-d₆, 100 MHz) δ 173.8 (C-1), 170.1 (C-4), 139.7 (C-10), 138.8 (C-6), 134.6 (C-9), 128.9 (C-12), 127.0 (C-13), 126.9 (C-8/C-11), 126.2 (C-8/C-11), 119.3 (C-7), 31.1 (C-2/C-3), 28.8 (C-2/C-3); (+)-HRESIMS m/z 292.0950 [M+Na]⁺ (calcd for C₁₆H₁₅NNaO₃, 292.0944).

4.2.4. N¹,N⁴-Bis(3-(1-naphthamido)propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (17a)

Following general procedure A, the reaction of 1-naphthoic acid (8) (94 mg, 0.55 mmol), di-tert-butyl butane-1,4-diylbis((3-aminopropyl)carbamate) (16a) (100 mg, 0.25 mmol), EDC·HCl (124 mg, 0.65 mmol), HOBt (87 mg, 0.64 mmol), and DIPEA (0.26 mL, 1.49 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl butane-1,4-diylbis((3-(1-naphthamido)propyl)carbamate) (42 mg, 24%) as a white solid. Following general procedure C, the reaction of a sub-sample of this material (22 mg, 0.031 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 17a as a white solid after purification (12 mg, 52%). R_f = 0.48 (RP-18, MeOH:10% HCl, 7:3); m.p. 238–239 °C; IR (ATR) v_max 3307, 2839, 1646, 1451, 1203, 1116, 1015 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ 8.70–8.65 (4H, m, NH₂-14), 8.21–8.18 (2H, m, H-3), 8.04–7.97 (4H, m, H-6, H-7), 7.62 (2H, dd, J = 7.0, 1.0 Hz, H-9), 7.59–7.53 (6H, m, H-4, H-5, H-8), 3.43–3.39 (4H, m, H₂-11), 3.03–2.97 (8H, m, H₂-13, H₂-15), 1.96–1.89 (4H, m, H₂-12), 1.69–1.64 (4H, m, H₂-16); ¹³C NMR (DMSO-d₆, 100 MHz) δ 168.9 (C-1), 134.5 (C-6a), 133.1 (C-2), 129.9 (C-7), 129.7 (C-2a), 128.2 (C-6), 126.7 (C-4), 126.2 (C-5), 125.3 (C-3/C-8), 125.2 (C-3/C-8), 124.9 (C-9), 46.1 (C-15), 44.8 (C-13), 36.3 (C-11), 26.0 (C-12), 22.7 (C-16); (+)-HRESIMS m/z 511.3068 [M+H]⁺ (calcd for C₃₂H₃₉N₄O₂, 511.3068).

4.2.5. N¹,N⁶-Bis(3-(1-naphthamido)propyl)hexane-1,6-diaminium 2,2,2-trifluoroacetate (17b)

Following general procedure A, the reaction of 1-naphthoic acid (8) (44.0 mg, 0.256 mmol), di-tert-butyl hexane-1,6-diylbis((3-aminopropyl)carbamate) (16b) (50 mg, 0.12 mmol), EDC·HCl (57.9 mg, 0.302 mmol), HOBt (40.8 mg, 0.30 mmol), and DIPEA (0.12 mL, 0.69 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl hexane-1,6-diylbis((3-(1-naphthamido)propyl)carbamate) as a colorless oil (27 mg, 30%). Following general procedure C, the reaction of a sub-sample of this material (18 mg, 0.024 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 17b as a white solid (16 mg, 87%) with no further purification required. R_f = 0.47 (RP-18, MeOH:10% HCl, 7:3); m.p. 225–227 °C; IR (ATR) v_max 3293, 3057, 2944, 2856, 1675, 1542, 1313, 1201, 1132, 786, 721cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.24–8.20 (2H, m, H-3), 8.03 (2H, d, J = 8.2 Hz, H-7), 7.98–7.94 (2H, m, H-6), 7.68 (2H, dd, J = 7.1, 1.4 Hz, H-9), 7.62–7.53 (6H, m, H-4, H-5, H-8), 3.59 (4H, t, J = 6.6 Hz, H₂-11), 3.15 (4H, t, J = 7.4 Hz, H₂-13), 3.07 (4H, t, J = 7.5 Hz, H₂-15), 2.11–2.03 (4H, m, H₂-12), 1.81–1.72 (4H, m, H₂-16), 1.53–1.48 (4H, m, H₂-17); ¹³C NMR (CD₃OD, 100 MHz) δ 173.4 (C-1), 135.2 (C-2/C-6a), 135.0 (C-2/C-6a), 131.9 (C-7), 131.4 (C-2a), 129.6 (C-6), 128.1 (C-4), 127.6 (C-5), 126.6 (C-9), 126.1 (C-3), 126.0 (C-8), 48.9 (C-15), 46.6 (C-13), 37.5 (C-11), 27.8 (C-12), 27.1 (C-17), 27.0 (C-16); (+)-HRESIMS m/z 539.3385 [M+H]⁺ (calcd for C₃₄H₄₃N₄O₂, 539.3381).

4.2.6. N¹,N⁷-Bis(3-(1-naphthamido)propyl)heptane-1,7-diaminium 2,2,2-trifluoroacetate (17c)

Following general procedure A, the reaction of 1-naphthoic acid (8) (43.0 mg, 0.250 mmol), di-tert-butyl heptane-1,7-diylbis((3-aminopropyl)carbamate) (16c) (50 mg, 0.11 mmol), EDC·HCl (56.1 mg, 0.293 mmol), HOBt (39.6 mg, 0.29 mmol), and DIPEA (0.118 mL, 0.677 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl heptane-1,7-diylbis((3-aminopropyl)carbamate) as a colorless oil (45 mg, 54%). Following general procedure C, the reaction of a sub-sample of this material (6 mg, 0.008 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 17c as a colorless gum after purification (5 mg, 81%). R_f = 0.63 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3752, 326, 2927, 2857, 2305, 1678, 1665, 1543, 1199, 1136, 806, 723 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.12–8.09 (2H, m, H-3), 7.90 (2H, d, J = 8.5 Hz, H-7), 7.85–7.82 (2H, m, H-6), 7.55 (2H, dd, J = 7.2, 1.4 Hz, H-9), 7.49–7.40 (6H, m, H-4, H-5, H-8), 3.47 (4H, t, J = 6.6 Hz, H₂-11), 3.03 (4H, t, J = 7.4 Hz, H₂-13), 2.94 (4H, t, J = 7.7 Hz, H₂-15), 1.98–1.90 (4H, m, H₂-12), 1.67–1.59 (4H, m, H₂-16), 1.38–1.33 (6H, m, H₂-17, H₂-18); ¹³C NMR (CD₃OD, 100 MHz) δ 173.5 (C-1), 135.2 (C-2/C-6a), 134.9 (C-2/C-6a), 131.9 (C-7), 131.4 (C-2a), 129.6 (C-6), 128.1 (C-4), 127.5 (C-5), 126.6 (C-9), 126.1 (C-3), 125.9 (C-8), 49.0 (C-15), 46.5 (C-13), 37.5 (C-11), 29.6 (C-18), 27.8 (C-12), 27.3 (C-16/C-17); (+)-HRESIMS m/z 553.3534 [M+H]⁺ (calcd for C₃₅H₄₅N₄O₂, 553.3537).

4.2.7. N¹,N⁸-Bis(3-(1-naphthamido)propyl)octane-1,8-diaminium 2,2,2-trifluoroacetate (17d)

Following general procedure A, the reaction of 1-naphthoic acid (8) (41.3 mg, 0.240 mmol), di-tert-butyl octane-1,8-diylbis((3-aminopropyl)carbamate) (16d) (50 mg, 0.11 mmol), EDC·HCl (54.3 mg, 0.283 mmol), HOBt (38.3 mg, 0.28 mmol), and DIPEA (0.114 mL, 0.653 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl octane-1,8-diylbis((3-aminopropyl)carbamate) as a colorless oil (37 mg, 44%). Following general procedure C, the reaction of a sub-sample of this material (13 mg, 0.017 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 17d as a colorless gum after purification (4 mg, 30%). R_f = 0.63 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3743, 3274, 3057, 2935, 2861, 1675, 1542, 1468, 1200, 1132, 784, 721 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.26–8.21 (2H, m, H-3), 8.03 (2H, d, J = 8.6 Hz, H-7), 7.98–7.94 (2H, m, H-6), 7.68 (2H, dd, J = 6.9, 1.2 Hz, H-9), 7.62–7.52 (6H, m, H-4, H-5, H-8), 3.60 (4H, t, J = 6.7 Hz, H₂-11), 3.17 (4H, t, J = 7.5 Hz, H₂-13), 3.06 (4H, t, J = 7.8 Hz, H₂-15), 2.12–2.03 (4H, m, H₂-12), 1.78–1.70 (4H, m, H₂-16), 1.50–1.39 (8H, m, H₂-17, H₂-18); ¹³C NMR (CD₃OD, 100 MHz) δ 173.5 (C-1), 135.2 (C-6a), 135.0 (C-2), 132.0 (C-7), 131.4 (C-2a), 129.6 (C-6), 128.1 (C-4), 127.6 (C-5), 126.6 (C-9), 126.1 (C-3), 126.0 (C-8), 49.0 (C-15), 46.6 (C-13), 37.5 (C-11), 30.0 (C-18), 27.8 (C-12), 27.4 (C-16/C-17), 27.3 (C-16/C-17); (+)-HRESIMS m/z 567.3680 [M+H]⁺ (calcd for C₃₆H₄₇N₄O₂, 567.3694).

4.2.8. N¹,N¹⁰-Bis(3-(1-naphthamido)propyl)decane-1,10-diaminium 2,2,2-trifluoroacetate (17e)

Following general procedure A, the reaction of 1-napthoic acid (8) (78 mg, 0.45 mmol), di-tert-butyl decane-1,10-diylbis((3-aminopropyl)carbamate) (16e) (100 mg, 0.21 mmol), EDC·HCl (103 mg, 0.54 mmol), HOBt (72 mg, 0.53 mmol), and DIPEA (0.22 mL, 1.26 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl decane-1,10-diylbis((3-(1-naphthamido)propyl)carbamate) (120 mg, 72%) as a colorless oil. Following general procedure C, the reaction of a sub-sample of this material (99 mg, 0.13 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 17e (93 mg, 87%) as a yellow oil with no further purification required. R_f = 0.27 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3306, 2944, 2832, 1685, 1448, 1113, 1022 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.23–8.20 (2H, m, H-3), 8.00 (2H, d, J = 8.3 Hz, H-7), 7.99–7.91 (2H, m, H-6), 7.65 (2H, dd, J = 7.0, 0.7 Hz, H-9), 7.58–7.50 (6H, m, H-4, H-5, H-8), 3.58 (4H, t, J = 6.6 Hz, H₂-11), 3.14 (4H, t, J = 7.4 Hz, H₂-13), 3.03 (4H, t, J = 7.8 Hz, H₂-15), 2.09–2.02 (4H, m, H₂-12), 1.75–1.67 (4H, m, H₂-16), 1.42–1.33 (12H, m, H₂-17, H₂-18, H₂-19); ¹³C NMR (CD₃OD, 100 MHz) δ 173.5 (C-1), 135.2 (C-2/C-6a), 135.0 (C-2/C-6a), 131.9 (C-7), 131.4 (C-2a), 129.6 (C-6), 128.1 (C-4), 127.6 (C-5), 126.6 (C-9), 126.1 (C-3), 126.0 (C-8), 49.1 (C-15), 46.6 (C-13), 37.6 (C-11), 30.4 (C-18/C-19), 30.2 (C-18/C-19), 27.8 (C-12), 27.5 (C-17), 27.4 (C-16); (+)-HRESIMS m/z 595.3998 [M+H]⁺ (calcd for C₃₈H₅₁N₄O₂, 595.4007).

4.2.9. N¹,N¹²-Bis(3-(1-naphthamido)propyl)dodecane-1,12-diaminium 2,2,2-trifluoroacetate (17f)

Following general procedure A, the reaction of 1-naphthoic acid (8) (52 mg, 0.30 mmol), di-tert-butyl dodecane-1,12-diylbis((3-aminopropyl)carbamate) (16f) (71 mg, 0.14 mmol), EDC·HCl (69 mg, 0.36 mmol), HOBt (47 mg, 0.35 mmol), and DIPEA (0.14 mL, 0.80 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl dodecane-1,12-diylbis((3-(1-naphthamido)propyl)carbamate) (43 mg, 37%) as a white wax. Following general procedure C, the reaction of a sub-sample of this material (19 mg, 0.023 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 17f (18 mg, 92%) as a white wax with no further purification required. R_f = 0.15 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3326, 2944, 2834, 1654, 1450, 1246, 1116, 1022 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.23–8.20 (2H, m, H-3), 8.00 (2H, d, J = 8.2 Hz, H-7), 7.95–7.92 (2H, m, H-6), 7.66 (2H, dd, J = 7.0, 1.0 Hz, H-9), 7.59–7.50 (6H, m, H-4, H-5, H-8), 3.58 (4H, t, J = 6.6 Hz, H₂-11), 3.15 (4H, t, J = 7.4 Hz, H₂-13), 3.04 (4H, t, J = 7.8 Hz, H₂-15), 2.09–2.02 (4H, m, H₂-12), 1.75–1.68 (4H, m, H₂-16), 1.43–1.30 (16H, m, H₂-17, H₂-18, H₂-19, H₂-20); ¹³C NMR (CD₃OD, 100 MHz) δ 173.4 (C-1), 135.2 (C-2/C-6a), 135.0 (C-2/C-6a), 131.9 (C-7), 131.4 (C-2a), 129.6 (C-6), 128.1 (C-4), 127.5 (C-5), 126.6 (C-9), 126.1 (C-3), 125.9 (C-8), 49.1 (C-15), 46.6 (C-13), 37.6 (C-11), 30.6 (C-18/C-19/C-20), 30.5 (C-18/C-19/C-20), 30.2 (C-18/C-19/C-20), 27.8 (C-12), 27.5 (C-17), 27.4 (C-16); (+)-HRESIMS m/z 623.4321 [M+H]⁺ (calcd for C₄₀H₅₅N₄O₂, 623.4320).

4.2.10. N¹,N⁴-Bis(3-(4-(naphthalen-1-ylamino)-4-oxobutanamido)propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (18a)

Following general procedure B, the reaction of carboxylic acid 9 (76 mg, 0.31 mmol), di-tert-butyl butane-1,4-diylbis((3-aminopropyl)carbamate) (16a) (50 mg, 0.12 mmol), EDC·HCl (67 mg, 0.35 mmol), and DMAP (76 mg, 0.62 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl butane-1,4-diylbis((3-(4-(naphthalen-1-ylamino)-4-oxobutanamido)propyl)carbamate) as a colorless oil (54 mg, 53%). Following general procedure C, the reaction of a sub-sample of this material (26 mg, 0.030 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 18a as a colorless oil after purification (22 mg, 83%). R_f = 0.47 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3278, 1676, 1202, 1160, 799, 766 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.04 (2H, dd, J = 9.0, 2.0 Hz, H-10), 7.89 (2H, dd, J = 7.0, 2.0 Hz, H-7), 7.76 (2H, d, J = 8.5, Hz, H-13), 7.59–7.57 (2H, m, H-11), 7.56–7.50 (4H, m, H-8, H-9), 7.46 (2H, t, J = 7.8 Hz, H-12), 3.32–3.28 (4H, m, H₂-15), 2.92–2.89 (4H, m, H₂-2), 2.83 (4H, t, J = 7.0 Hz, H₂-17), 2.65–2.62 (4H, m, H₂-3), 2.57–2.54 (4H, m, H₂-19), 1.82–1.75 (4H, m, H₂-16), 1.36–1.32 (4H, m, H₂-20); ¹³C NMR (CD₃OD, 100 MHz) δ 176.3 (C-1), 174.3 (C-4), 135.7 (C-6), 134.3 (C-10a), 130.2 (C-6a), 129.4 (C-7), 127.6 (C-13), 127.4 (C-8/C-9), 127.3 (C-8/C-9), 126.6 (C-12), 124.0 (C-11), 123.6 (C-10), 47.8 (C-19), 46.0 (C-17), 36.6 (C-15), 32.0 (C-2), 31.6 (C-3), 27.7 (C-16), 23.9 (C-20); (+)-HRESIMS m/z 653.3807 [M+H]⁺ (calcd for C₃₈H₄₉N₆O₄, 653.3810).

4.2.11. N¹,N⁶-Bis(3-(4-(naphthalen-1-ylamino)-4-oxobutanamido)propyl)hexane-1,6-diaminium 2,2,2-trifluoroacetate (18b)

Following general procedure B, the reaction of carboxylic acid 9 (71 mg, 0.29 mmol), di-tert-butyl hexane-1,6-diylbis((3-aminopropyl)carbamate) (16b) (50 mg, 0.12 mmol), EDC·HCl (62 mg, 0.32 mmol), and DMAP (71 mg, 0.58 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl hexane-1,6-diylbis((3-(4-(naphthalen-1-ylamino)-4-oxobutanamido)propyl)carbamate) as a colorless oil (97 mg, 92%). Following general procedure C, the reaction of a sub-sample of this material (44 mg, 0.050 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 18b as an orange oil after purification (39 mg, 86%). R_f = 0.47 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3279, 3050, 1671, 1542, 1201, 1132, 800, 777 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.01 (2H, dd, J = 9.0, 2.5 Hz, H-10), 7.85 (2H, dd, J = 7.5, 3.5 Hz, H-7), 7.71 (2H, d, J = 8.5, Hz, H-13), 7.57 (2H, dd, J = 7.5, 1.0 Hz, H-11), 7.51–7.47 (4H, m, H-8, H-9), 7.42 (2H, t, J = 7.8 Hz, H-12), 3.31–3.26 (4H, m, H₂-15), 2.88 (8H, t, J = 6.5 Hz, H₂-2, H₂-17), 2.61–2.53 (8H, m, H₂-3, H₂-19), 1.82–1.75 (4H, m, H₂-16), 1.33–1.26 (4H, m, H₂-20), 0.91–0.87 (4H, m, H₂-21); ¹³C NMR (CD₃OD, 100 MHz) δ 176.3 (C-1), 174.2 (C-4), 135.7 (C-6), 134.4 (C-10a), 130.0 (C-6a), 129.4 (C-7), 127.5 (C-13), 127.33 (C-8/C-9), 127.27 (C-8/C-9), 126.5 (C-12), 123.7 (C-11), 123.6 (C-10), 48.7 (C-19), 46.0 (C-17), 36.5 (C-15), 32.0 (C-2), 31.5 (C-3), 27.8 (C-16), 26.8 (C-20/C-21), 26.7 (C-20/C-21); (+)-HRESIMS [M+H]⁺ m/z 681.4104 (calcd for C₄₀H₅₃N₆O₄, 681.4123).

4.2.12. N¹,N⁷-Bis(3-(4-(naphthalen-1-ylamino)-4-oxobutanamido)propyl)heptane-1,7-diaminium 2,2,2-trifluoroacetate (18c)

Following general procedure B, the reaction of carboxylic acid 9 (68 mg, 0.29 mmol), di-tert-butyl heptane-1,7-diylbis((3-aminopropyl)carbamate) (16c) (50 mg, 0.11 mmol), EDC·HCl (60 mg, 0.31 mmol), and DMAP (69 mg, 0.57 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl heptane-1,7-diylbis((3-(4-(naphthalen-1-ylamino)-4-oxobutanamido)propyl)carbamate) as a colorless oil (94 mg, 95%). Following general procedure C, the reaction of a sub-sample of this material (82 mg, 0.092 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 18c as a colorless oil after purification (59 mg, 69%). R_f = 0.42 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3279, 3053, 1671, 1537, 1201, 1131, 800, 777 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.05 (2H, dd, J = 9.5, 1.5 Hz, H-10), 7.87 (2H, dd, J = 7.5, 2.5 Hz, H-7), 7.74 (2H, d, 8.0 Hz, H-13), 7.61 (2H, dd, J = 7.5, 1.0 Hz, H-11), 7.55–7.48 (4H, m, H-8, H-9), 7.45 (2H, t, J = 8.0 Hz, H-12), 3.33–3.30 (4H, m, H₂-15), 2.91 (8H, t, J = 6.5 Hz, H₂-2, H₂-17), 2.65–2.61 (8H, m, H₂-3, H₂-19), 1.85–1.79 (4H, m, H₂-16), 1.42–1.35 (4H, m, H₂-20), 1.01–0.98 (6H, m, H₂-21, H₂-22); ¹³C NMR (CD₃OD, 100 MHz) δ 176.2 (C-1), 174.1 (C-4), 135.7 (C-6), 134.3 (C-10a), 130.0 (C-6a), 129.4 (C-7), 127.5 (C-13), 127.3 (C-8/C-9), 127.2 (C-8/C-9), 126.5 (C-12), 123.7 (C-11), 123.6 (C-10), 48.8 (C-19), 46.0 (C-17), 36.6 (C-15), 32.0 (C-2), 31.6 (C-3), 29.4 (C-22), 27.7 (C-16), 27.0 (C-20/C-21), 26.9 (C-20/C-21); (+)-HRESIMS [M+H]⁺ m/z 695.4265 (calcd for C₄₁H₅₅N₆O₄, 695.4279).

4.2.13. N¹,N⁸-Bis(3-(4-(naphthalen-1-ylamino)-4-oxobutanamido)propyl)octane-1,8-diaminium 2,2,2-trifluoroacetate (18d)

Following general procedure B, the reaction of carboxylic acid 9 (67 mg, 0.28 mmol), di-tert-butyl octane-1,8-diylbis((3-aminopropyl)carbamate) (16d) (50 mg, 0.11 mmol), EDC·HCl (59 mg, 0.31 mmol), and DMAP (67 mg, 0.55 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl octane-1,8-diylbis((3-(4-(naphthalen-1-ylamino)-4-oxobutanamido)propyl)carbamate) as a colorless oil (69 mg, 69%). Following general procedure C, the reaction of this material (69 mg, 0.076 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 18d as a colorless oil after purification (37 mg, 54%). R_f = 0.40 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3052, 1655, 1542, 1201, 1132, 799, 777 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.05 (2H, dd, J = 8.0, 1.5 Hz, H-10), 7.88 (2H, dd, J = 7.5, 2.0 Hz, H-7), 7.75 (2H, d, J = 8.0 Hz, H-13), 7.62 (2H, dd, J = 7.5, 1.0 Hz, H-11), 7.56–7.49 (4H, m, H-8, H-9), 7.46 (2H, t, J = 8.0 Hz, H-12), 3.34–3.31 (4H, m, H₂-15), 2.95–2.91 (8H, m, H₂-2, H₂-17), 2.67–2.62 (8H, m, H₂-3, H₂-19), 1.86–1.80 (4H, m, H₂-16), 1.45–1.39 (4H, m, H₂-20), 1.07–1.01 (8H, m, H₂-21, H₂-22); ¹³C NMR (CD₃OD, 100 MHz) δ 176.2 (C-1), 174.1 (C-4), 135.7 (C-6), 134.3 (C-10a), 130.0 (C-6a), 129.4 (C-7), 127.4 (C-13), 127.3 (C-8/C-9), 127.2 (C-8/C-9), 126.5 (C-12), 123.6 (C-11), 123.5 (C-10), 48.9 (C-19), 46.0 (C-17), 36.6 (C-15), 32.0 (C-2), 31.6 (C-3), 29.8 (C-22), 27.8 (C-16), 27.2 (C-20/C-21), 27.1 (C-20/C-21); (+)-HRESIMS [M+2H]²⁺ m/z 355.2250 (calcd for C₄₂H₅₈N₆O₄, 355.2254).

4.2.14. N¹,N¹⁰-Bis(3-(4-(naphthalen-1-ylamino)-4-oxobutanamido)propyl)decane-1,10-diaminium 2,2,2-trifluoroacetate (18e)

Following general procedure B, the reaction of carboxylic acid 9 (63 mg, 0.26 mmol), di-tert-butyl decane-1,10-diylbis((3-aminopropyl)carbamate) (16e) (50 mg, 0.10 mmol), EDC·HCl (55 mg, 0.29 mmol), and DMAP (63 mg, 0.52 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl decane-1,10-diylbis((3-(4-(naphthalen-1-ylamino)-4-oxobutanamido)propyl)carbamate) as a colorless oil (67 mg, 72%). Following general procedure C, the reaction of a sub-sample of this material (27 mg, 0.029 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 18e as a colorless oil after purification (26 mg, 93%). R_f = 0.23 (RP-18, MeOH: 10% HCl, 7:3); IR (ATR) v_max 2937, 1673, 1538, 1202, 1133, 800, 776, 721 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.05 (2H, dd, J = 8.0, 1.5 Hz, H-10), 7.88 (2H, dd, J = 7.4, 2.4 Hz, H-7), 7.76 (2H, d, J = 8.5, Hz, H-13), 7.62 (2H, d, J = 7.5 Hz, H-11), 7.56–7.50 (4H, m, H-8, H-9), 7.46 (2H, t, J = 8.0 Hz, H-12), 3.34–3.30 (4H, m, H₂-15), 2.95–2.91 (8H, m, H₂-2, H₂-17), 2.68–2.61 (8H, m, H₂-3, H₂-19), 1.86–1.79 (4H, m, H₂-16), 1.47–1.40 (4H, m, H₂-20), 1.14–1.08 (12H, m, H₂-21, H₂-22, H₂-23); ¹³C NMR (CD₃OD, 100 MHz) δ 176.3 (C-1), 174.2 (C-4), 135.8 (C-6), 134.4 (C-10a), 130.1 (C-6a), 129.5 (C-7), 127.5 (C-13), 127.34 (C-8/C-9), 127.26 (C-8/C-9), 126.5 (C-12), 123.7 (C-11), 123.6 (C-10), 49.0 (C-19), 46.1(C-17), 36.6 (C-15), 32.0 (C-2), 31.6 (C-3), 30.3 (C-23), 30.1 (C-22), 27.8 (C-16), 27.4 (C-20/C-21), 27.2 (C-20/C-21); (+)-HRESIMS m/z 737.4699 [M+H]⁺ (calcd for C₄₄H₆₁N₆O₄, 737.4749).

4.2.15. N¹,N¹²-Bis(3-(4-(Naphthalen-1-ylamino)-4-oxobutanamido)propyl)dodecane-1,12-diaminium 2,2,2-trifluoroacetate (18f)

Following general procedure B, the reaction of carboxylic acid 9 (58 mg, 0.24 mmol), di-tert-butyl dodecane-1,12-diylbis((3-aminopropyl)carbamate) (16f) (57 mg, 0.11 mmol), EDC·HCl (52 mg, 0.27 mmol), and DMAP (60 mg, 0.49 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl dodecane-1,12-diylbis((3-(4-(naphthalen-1-ylamino)-4-oxobutanamido)propyl)carbamate) as a colorless oil (62 mg, 58%). Following general procedure C, the reaction of a sub-sample of this material (26 mg, 0.027 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 18f as a pale-yellow oil after purification (20 mg, 75%). R_f = 0.20 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3083, 2927, 1667, 1537, 1201, 1132, 800, 775 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.05 (2H, dd, J = 8.0, 1.5 Hz, H-10), 7.88 (2H, dd, J = 7.5, 2.0 Hz, H-7), 7.75 (2H, d, J = 8.0 Hz, H-13), 7.61 (2H, dd, J = 7.5, 1.0 Hz, H-11), 7.56–7.50 (4H, m, H-8, H-9), 7.46 (2H, t, J = 7.8 Hz, H-12), 3.34–3.30 (4H, m, H₂-15), 2.95–2.91 (8H, m, H₂-2, H₂-17), 2.68–2.62 (8H, m, H₂-3, H₂-19), 1.86–1.80 (4H, m, H₂-16), 1.48–1.41 (4H, m, H₂-20), 1.21–1.08 (16H, m, H₂-21, H₂-22, H₂-23, H₂-24); ¹³C NMR (CD₃OD, 100 MHz) δ 176.2 (C-1), 174.2 (C-4), 135.7 (C-6), 134.3 (C-10a), 130.0 (C-6a) 129.4 (C-7), 127.5 (C-13), 127.3 (C-8/C-9), 127.2 (C-8/C-9), 126.5 (C-12), 123.7 (C-11), 123.5 (C-10), 49.0 (C-19), 46.0 (C-17), 36.6 (C-15), 32.0 (C-2), 31.6 (C-3), 30.5 (C-22/C-23/C-24), 30.4 (C-22/C-23/C-24), 30.1 (C-22/C-23/C-24), 27.7 (C-16), 27.3 (C-20/C-21), 27.2 (C-20/C-21); (+)-HRESIMS [M+2H]²⁺ m/z 383.2575 (calcd for C₄₆H₆₆N₆O₄, 383.2567).

4.2.16. N¹,N⁴-Bis(3-(2-naphthamido)propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (19a)

Following general procedure A, the reaction of 2-napthoic acid (10) (94 mg, 0.55 mmol), di-tert-butyl butane-1,4-diylbis((3-aminopropyl)carbamate) (16a) (100 mg, 0.25 mmol), EDC·HCl (124 mg, 0.65 mmol), HOBt (87 mg, 0.64 mmol), and DIPEA (0.26 mL, 1.49 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl butane-1,4-diylbis((3-(2-naphthamido)propyl)carbamate) (91 mg, 51%) as a white solid. Following general procedure C, the reaction of a sub-sample of this material (72 mg, 0.10 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 19a as a beige solid after purification (71 mg, 96%). R_f = 0.33 (RP-18, MeOH:10% HCl, 7:3); m.p. 132–135 °C; IR (ATR) v_max 3332, 2948, 2835, 1674, 1639, 1544, 1435, 1313, 1201, 1133, 1018, 721 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.38 (2H, br s, H-3), 7.96–7.87 (8H, m, H-4, H-7, H-8, H-9), 7.61–7.53 (4H, m, H-5, H-6), 3.57 (4H, t, J = 6.4 Hz, H₂-11), 3.14–3.10 (8H, m, H₂-13, H₂-15), 2.08–2.01 (4H, m, H₂-12), 1.90–1.87 (4H, m, H₂-16); ¹³C NMR (CD₃OD, 100 MHz) δ 171.2 (C-1), 136.4 (C-7a), 134.0 (C-3a), 132.2 (C-2), 130.0 (C-4), 129.5 (C-8), 129.1 (C-3/C-6), 129.0 (C-3/C-6), 128.8 (C-7), 128.0 (C-5), 124.8 (C-9), 48.2 (C-15), 46.5 (C-13), 37.5 (C-11), 27.9 (C-12), 24.4 (C-16); (+)-HRESIMS m/z 511.3071 [M+H]⁺ (calcd for C₃₂H₃₉N₄O₂, 511.3068).

4.2.17. N¹,N⁶-Bis(3-(2-naphthamido)propyl)hexane-1,6-diaminium 2,2,2-trifluoroacetate (19b)

Following general procedure A, the reaction of 2-naphthoic acid (10) (44.0 mg, 0.256 mmol), di-tert-butyl hexane-1,6-diylbis((3-aminopropyl)carbamate) (16b) (50.0 mg, 0.12 mmol), EDC·HCl (57.9 mg, 0.302 mmol), HOBt (40.8 mg, 0.30 mmol), and DIPEA (0.12 mL, 0.69 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl hexane-1,6-diylbis((3-(2-naphthamido)propyl)carbamate) as a colorless oil (48.0 mg, 54%). Following general procedure C, the reaction of a sub-sample of this material (6 mg, 0.0081 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 19b as a colorless gum (6 mg, 97%) with no further purification required. R_f = 0.43 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3747, 3325, 2922, 2853, 2354, 1635, 1541, 1465, 1199 cm-¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.39 (2H, br s, H-3), 7.98–7.88 (8H, m, H-4, H-7, H-8, H-9), 7.62–7.55 (4H, m, H-5, H-6), 3.57 (4H, t, J = 6.5 Hz, H₂-11), 3.09 (4H, t, J = 7.2 Hz, H₂-13), 3.05 (4H, t, J = 8.0 Hz, H₂-15), 2.06–1.99 (4H, m, H₂-12), 1.80–1.74 (4H, m, H₂-16), 1.54–1.50 (4H, m, H₂-17); ¹³C NMR (CD₃OD, 100 MHz) δ 171.3 (C-1), 136.5 (C-7a), 134.1 (C-3a), 131.5 (C-2), 130.0 (C-4), 129.5 (C-8), 129.1 (C-3/C-6), 129.0 (C-3/C-6), 128.9 (C-7), 128.0 (C-5), 124.7 (C-9), 49.0 (C-15), 46.4 (C-13), 37.5 (C-11), 28.0 (C-12), 27.2 (C-16/C-17), 27.1 (C-16/C-17); (+)-HRESIMS m/z 539.3387[M+H]⁺ (calcd for C₃₄H₄₃N₄O₂, 539.3381).

4.2.18. N¹,N⁷-Bis(3-(2-naphthamido)propyl)heptane-1,7-diaminium 2,2,2-trifluoroacetate (19c)

Following general procedure A, the reaction of 2-naphthoic acid (10) (42.6 mg, 0.247 mmol), di-tert-butyl heptane-1,7-diylbis((3-aminopropyl)carbamate) (16c) (50 mg, 0.11 mmol), EDC·HCl (56.1 mg, 0.293 mmol), HOBt (39.6 mg, 0.29 mmol), and DIPEA (0.118 mL, 0.677 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl heptane-1,7-diylbis((3-(2-naphthamido)propyl)carbamate) as a colorless oil (48 mg, 58%). Following general procedure C, the reaction of a sub-sample of this material (12 mg, 0.016 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 19c as a colorless gum after purification (6 mg, 48%). R_f = 0.33 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3311, 2927, 2857, 1674, 1639, 1544, 1505, 1434, 1312, 1200, 1179, 1131, 876, 832, 799, 780, 762, 721 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.43 (2H, s, H-3), 8.03–7.90 (8H, m, H-4, H-7, H-8, H-9), 7.66–7.56 (4H, m, H-5, H-6), 3.59 (4H, t, J = 6.5 Hz, H₂-11), 3.11 (4H, t, J = 7.0 Hz, H₂-13), 3.05 (4H, t, J = 7.5 Hz, H₂-15), 2.09–2.02 (4H, m, H₂-12), 1.81–1.73 (4H, m, H₂-16), 1.51–1.47 (6H, m, H₂-17, H₂-18); ¹³C NMR (CD₃OD, 100 MHz) δ 171.2 (C-1), 136.4 (C-7a), 134.1 (C-3a), 132.2 (C-2), 130.0 (C-4), 129.5 (C-8), 129.1 (C-3/C-6), 129.0 (C-3/C-6), 128.8 (C-7), 128.0 (C-5), 124.8 (C-9), 49.0 (C-15), 46.4 (C-13), 37.5 (C-11), 29.7 (C-18), 27.9 (C-12), 27.3 (C-16/C-17), 27.2 (C-16/C-17); (+)-HRESIMS m/z 553.3520 [M+H]⁺ (calcd for C₃₅H₄₅N₄O₂, 553.3537).

4.2.19. N¹,N⁸-Bis(3-(2-naphthamido)propyl)octane-1,8-diaminium 2,2,2-trifluoroacetate (19d)

Following general procedure A, the reaction of 2-naphthoic acid (10) (41.3 mg, 0.240 mmol), di-tert-butyl octane-1,8-diylbis((3-aminopropyl)carbamate) (16d) (50 mg, 0.11 mmol), EDC·HCl (54.3 mg, 0.283 mmol), HOBt (38.3 mg, 0.28 mmol), and DIPEA (0.114 mL, 0.653 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl octane-1,8-diylbis((3-(2-naphthamido)propyl)carbamate) as a colorless oil (44 mg, 52%). Following general procedure C, the reaction of a sub-sample of this material (18 mg, 0.024 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 19d as a colorless gum after purification (16 mg, 84%). R_f = 0.33 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3716, 2927, 2343, 1739, 1463, 1263, 801 cm-¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.40 (2H, s, H-3), 7.99–7.88 (8H, m, H-4, H-7, H-8, H-9), 7.62–7.56 (4H, m, H-5, H-6), 3.57 (4H, t, J = 6.5 Hz, H₂-11), 3.09 (4H, t, J = 7.4 Hz, H₂-13), 3.02 (4H, t, J = 7.8 Hz, H₂-15), 2.10–1.99 (4H, m, H₂-12), 1.80–1.69 (4H, m, H₂-16), 1.50–1.43 (8H, m, H₂-17, H₂-18); ¹³C NMR (CD₃OD, 100 MHz) δ 171.2 (C-1), 136.4 (C-7a), 134.1 (C-3a), 132.2 (C-2), 130.0 (C-4), 129.5 (C-8), 129.1 (C-3/C-6), 129.0 (C-3/C-6), 128.9 (C-7), 128.0 (C-5), 124.7 (C-9), 49.0 (C-15), 46.4 (C-13), 37.5 (C-11), 30.0 (C-18), 27.9 (C-12), 27.5 (C-16/C-17), 27.4 (C-16/C-17); (+)-HRESIMS m/z 567.3691 [M+H]⁺ (calcd for C₃₆H₄₇N₄O₂, 567.3694).

4.2.20. N¹,N¹⁰-Bis(3-(2-naphthamido)propyl)decane-1,10-diaminium 2,2,2-trifluoroacetate (19e)

Following general procedure A, the reaction of 2-napthoic acid (10) (78 mg, 0.45 mmol), di-tert-butyl decane-1,10-diylbis((3-aminopropyl)carbamate) (16e) (100 mg, 0.21 mmol), EDC·HCl (103 mg, 0.54 mmol), HOBt (72 mg, 0.53 mmol), and DIPEA (0.22 mL, 1.26 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl decane-1,10-diylbis((3-(2-naphthamido)propyl)carbamate) (99 mg, 59%) as a colorless oil. Following general procedure C, the reaction of a sub-sample of this material (78 mg, 0.098 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 19e (76 mg, 94%) as a yellow oil with no further purification required. R_f = 0.14 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3307,2944, 2832, 1685, 1448, 1115, 1022 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.40 (2H, br s, H-3), 7.98–7.89 (8H, m, H-4, H-7, H-8, H-9), 7.62–7.54 (4H, m, H-5, H-6), 3.57 (4H, t, J = 6.5 Hz, H₂-11), 3.08 (4H, t, J = 7.2 Hz, H₂-13), 3.01 (4H, t, J = 7.7 Hz, H₂-15), 2.07–2.00 (4H, m, H₂-12), 1.75–1.67 (4H, m, H₂-16), 1.43–1.34 (12H, m, H₂-17, H₂-18, H₂-19); ¹³C NMR (CD₃OD, 100 MHz) δ 171.1 (C-1), 136.4 (C-7a), 134.1 (C-3a), 132.2 (C-2), 130.1 (C-4), 129.5 (C-8), 129.1 (C-3/C-6), 129.0 (C-3/C-6), 128.8 (C-7), 128.0 (C-5), 124.8 (C-9), 49.0 (C-15), 46.4 (C-13), 37.6 (C-11), 30.4 (C-18/C-19), 30.2 (C-18/C-19), 27.8 (C-12), 27.5 (C-17), 27.4 (C-16); (+)-HRESIMS m/z 595.3993 [M+H]⁺ (calcd for C₃₈H₅₁N₄O₂, 595.4007).

4.2.21. N¹,N¹²-Bis(3-(2-naphthamido)propyl)dodecane-1,12-diaminium 2,2,2-trifluoroacetate (19f)

Following general procedure A, the reaction of 2-naphthoic acid (10) (55 mg, 0.32 mmol), di-tert-butyl dodecane-1,12-diylbis((3-aminopropyl)carbamate) (16f) (75 mg, 0.15 mmol), EDC·HCl (73 mg, 0.38 mmol), HOBt (51 mg, 0.38 mmol), and DIPEA (0.15 mL, 0.86 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl dodecane-1,12-diylbis((3-(2-naphthamido)propyl)carbamate) (35 mg, 28%) as a colorless oil. Following general procedure C, the reaction of a sub-sample of this material (20 mg, 0.024 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 19f (16 mg, 78%) as an orange oil with no further purification required. R_f = 0.12 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3308, 2945, 2833, 1655, 1450, 1246, 1115, 1022 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.43 (2H, s, H-3), 8.01–7.91 (8H, m, H-4, H-7, H-8, H-9), 7.65–7.57 (4H, m, H-5, H-6), 3.59 (4H, t, J = 6.5 Hz, H₂-11), 3.11 (4H, t, J = 7.3 Hz, H₂-13), 3.04 (4H, t, J = 7.8 Hz, H₂-15), 2.08–2.02 (4H, m, H₂-12), 1.77–1.70 (4H, m, H₂-16) 1.46–1.32 (16H, m, H₂-17, H₂-18, H₂-19, H₂-20); ¹³C NMR (CD₃OD, 100 MHz) δ 171.2 (C-1), 136.4 (C-7a), 134.1 (C-3a), 132.2 (C-2), 130.0 (C-4), 129.5 (C-8), 129.1 (C-3/C-6), 129.0 (C-3/C-6), 128.8 (C-7), 128.0 (C-5), 124.7 (C-9), 49.1 (C-15), 46.4 (C-13), 37.5 (C-11), 30.7 (C-18/C-19/C-20), 30.5 (C-18/C-19/C-20), 30.3 (C-18/C-19/C-20), 27.9 (C-12), 27.5 (C-17), 27.4 (C-16); (+)-HRESIMS m/z 623.4306 [M+H]⁺ (calcd for C₄₀H₅₅N₄O₂, 623.4320).

4.2.22. N¹,N⁴-Bis(3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (20a)

Following general procedure B, the reaction of carboxylic acid 11 (62 mg, 0.25 mmol), di-tert-butyl butane-1,4-diylbis((3-aminopropyl)carbamate) (16a) (48 mg, 0.12 mmol), EDC·HCl (55 mg, 0.29 mmol), and DMAP (62 mg, 0.51 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl butane-1,4-diylbis((3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)carbamate) as a pale-pink oil (84 mg, 82%). Following general procedure C, the reaction of a sub-sample of this material (16 mg, 0.019 mmol) in CH₂Cl₂ (2.0 mL) with TFA (0.2 mL) created the di-TFA salt 20a as a colorless oil after purification (15 mg, 90%). R_f = 0.28 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3278, 1676, 1202, 1160, 799, 766 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.18 (2H, d, J = 2.0 Hz, H-7), 7.80–7.73 (6H, m, H-8, H-11, H-12), 7.54 (2H, dd, J = 9.0, 2.0 Hz, H-13), 7.46–7.42 (2H, m, H-9), 7.40–7.36 (2H, m, H-10), 3.32–3.28 (4H, m, H₂-15), 2.87 (4H, t, J = 7.0 Hz, H₂-17), 2.80–2.77 (4H, m, H₂-2), 2.70–2.66 (4H, m, H₂-19), 2.61–2.58 (4H, m, H₂-3), 1.84–1.77 (4H, m, H₂-16), 1.46–1.42 (4H, m, H₂-20); ¹³C NMR (CD₃OD, 100 MHz) δ 176.5 (C-1), 173.2 (C-4), 137.5 (C-6), 135.2 (C-7a), 131.9 (C-11a), 129.7 (C-12), 128.7 (C-11), 128.4 (C-8), 127.6 (C-9), 126.0, (C-10), 121.2 (C-13), 117.4 (C-7), 47.9 (C-19), 45.8 (C-17), 36.4 (C-15), 32.4 (C-2), 31.3 (C-3), 27.8 (C-16), 24.0 (C-20); (+)-HRESIMS m/z 653.3823 [M+H]⁺ (calcd for C₃₈H₄₉N₆O₄, 653.3810).

4.2.23. N¹,N⁶-Bis(3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)hexane-1,6-diaminium 2,2,2-trifluoroacetate (20b)

Following general procedure B, the reaction of carboxylic acid 11 (71 mg, 0.29 mmol), di-tert-butyl hexane-1,6-diylbis((3-aminopropyl)carbamate) (16b) (50 mg, 0.12 mmol), EDC·HCl (62 mg, 0.32 mmol), and DMAP (71 mg, 0.58 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butylhexane-1,6-diylbis((3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)carbamate) as a pale brown oil (53 mg, 50%). Following general procedure C, the reaction of a sub-sample of this material (26 mg, 0.030 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 20b as a yellow oil after purification (24 mg, 88%). R_f = 0.32 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3279, 1671, 1542, 1201, 1132, 800, 777 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.20 (2H, d, J = 2.0 Hz, H-7), 7.78–7.71 (6H, m, H-8, H-11, H-12), 7.54 (2H, dd, J = 8.5, 2.0 Hz, H-13), 7.43 (2H, td, J = 7.5, 1.5 Hz, H-9), 7.37 (2H, td, J = 7.5, 1.5 Hz, H-10), 3.35–3.30 (4H, m, H₂-15), 2.96 (4H, t, J = 6.8 Hz, H₂-17), 2.80 (4H, t, J = 6.5 Hz, H₂-2), 2.63–2.59 (8H, m, H₂-3, H₂-19), 1.88–1.82 (4H, m, H₂-16), 1.31–1.27 (4H, m, H₂-20), 0.83–0.81 (4H, m, H₂-21); ¹³C NMR (CD₃OD, 100 MHz) δ 176.5 (C-1), 173.2 (C-4), 137.6 (C-6), 135.3 (C-7a), 131.9 (C-11a), 129.6 (C-12), 128.7 (C-11), 128.5 (C-8), 127.6 (C-9), 126.0, (C-10), 121.1 (C-13), 117.3 (C-7), 48.7 (C-19), 45.8 (C-17), 36.4 (C-15), 32.4 (C-2), 31.2 (C-3), 27.9 (C-16), 26.8 (C-20/C-21), 26.6 (C-20/C-21); (+)-HRESIMS [M+H]⁺ m/z 681.4106 (calcd for C₄₀H₅₃N₆O₄, 681.4123).

4.2.24. N¹,N⁷-Bis(3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)heptane-1,7-diaminium 2,2,2-trifluoroacetate (20c)

Following general procedure B, the reaction of carboxylic acid 11 (68 mg, 0.29 mmol), di-tert-butyl heptane-1,7-diylbis((3-aminopropyl)carbamate) (16c) (50 mg, 0.11 mmol), EDC·HCl (60 mg, 0.31 mmol), and DMAP (69 mg, 0.57 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl heptane-1,7-diylbis((3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)carbamate) as a colorless oil (64 mg, 65%). Following general procedure C, the reaction of a sub-sample of this material (44 mg, 0.049 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 20c as a colorless oil after purification (42 mg, 93%). R_f = 0.29 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3289, 3027 1666, 1537, 1201, 1131, 800, 723 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.20 (2H, d, J = 2.0 Hz, H-7), 7.77–7.71 (6H, m, H-8, H-11, H-12), 7.54 (2H, dd, J = 9.0, 2.0 Hz, H-13), 7.42 (2H, td, J = 7.5, 1.3 Hz, H-9), 7.36 (2H, td, J = 8.3, 1.5 Hz, H-10), 3.35–3.30 (4H, m, H₂-15), 3.00 (4H, t, J = 7.0 Hz, H₂-17) 2.81–2.78 (4H, m, H₂-2), 2.67 (4H, t, J = 8.0 Hz, H₂-19), 2.62–2.59 (4H, m, H₂-3), 1.89–1.83 (4H, m, H₂-16), 1.37–1.29 (4H, m, H₂-20), 0.81–0.76 (6H, m, H₂-21, H₂-22); ¹³C NMR (CD₃OD, 100 MHz) δ 176.6 (C-1), 173.2 (C-4), 137.6 (C-6), 135.3 (C-7a), 131.9 (C-11a), 129.6 (C-12), 128.6 (C-8/C-11), 128.5 (C-8/C-11), 127.6 (C-9), 126.0, (C-10), 121.0 (C-13), 117.2 (C-7), 48.9 (C-19), 45.8 (C-17), 36.4 (C-15), 32.4 (C-2), 31.2 (C-3), 29.4 (C-22), 27.9 (C-16), 27.01 (C-20/C-21), 26.96 (C-20/C-21); (+)-HRESIMS [M+H]⁺ m/z 695.4283 (calcd for C₄₁H₅₅N₆O₄, 695.4279).

4.2.25. N¹,N⁸-Bis(3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)octane-1,8-diaminium 2,2,2-trifluoroacetate (20d)

Following general procedure B, the reaction of carboxylic acid 11 (67 mg, 0.27 mmol), di-tert-butyl octane-1,8-diylbis((3-aminopropyl)carbamate) (16d) (50 mg, 0.11 mmol), EDC·HCl (59 mg, 0.31 mmol) and DMAP (67 mg, 0.55 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl octane-1,8-diylbis((3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)carbamate) as a colorless oil (40 mg, 40%). Following general procedure C, the reaction of this material (40 mg, 0.044 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 20d as a pale purple oil after purification (37 mg, 90%). R_f = 0.28 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 3288, 3061 1669, 1553, 1200, 1132, 800, 721 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.20 (2H, d, J = 2.0 Hz, H-7), 7.77–7.71 (6H, m, H-8, H-11, H-12), 7.55 (2H, dd, J = 9.0, 2.0 Hz, H-13), 7.44–7.40 (2H, m, H-9), 7.39–7.34 (2H, m, H-10), 3.36–3.30 (4H, m, H₂-15), 3.01 (4H, t, J = 7.0 Hz, H₂-17), 2.82–2.78 (4H, m, H₂-2), 2.71 (4H, t, J = 8.0 Hz, H₂-19), 2.62–2.59 (4H, m, H₂-3), 1.90–1.84 (4H, m, H₂-16), 1.42–1.36 (4H, m, H₂-20), 0.85–0.80 (8H, m, H₂-21, H₂-22); ¹³C NMR (CD₃OD, 100 MHz) δ 176.5 (C-1), 173.2 (C-4), 137.6 (C-6), 135.3 (C-7a), 131.9 (C-11a), 129.6 (C-12), 128.6 (C-8/C-11), 128.4 (C-8/C-11), 127.5 (C-9), 125.9, (C-10), 121.0 (C-13), 117.2 (C-7), 48.9 (C-19), 45.8 (C-17), 36.3 (C-15), 32.3 (C-2), 31.2 (C-3), 29.7 (C-22), 27.9 (C-16), 27.14 (C-20/C-21), 27.07 (C-20/C-21); (+)-HRESIMS [M+2H]²⁺ m/z 355.2252 (calcd for C₄₂H₅₈N₆O₄, 355.2254).

4.2.26. N¹,N¹⁰-Bis(3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)decane-1,10-diaminium 2,2,2-trifluoroacetate (20e)

Following general procedure B, the reaction of carboxylic acid 11 (63 mg, 0.26 mmol), di-tert-butyl decane-1,10-diylbis((3-aminopropyl)carbamate) (16e) (50 mg, 0.10 mmol), EDC·HCl (55 mg, 0.29 mmol), and DMAP (63 mg, 0.52 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl decane-1,10-diylbis((3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)carbamate) as a colorless oil (78 mg, 83%). Following general procedure C, the reaction of a sub-sample of this material (41 mg, 0.044 mmol) in CH₂Cl₂ (2.0 mL) with TFA (0.2 mL) created the di-TFA salt 20e as a colorless oil after purification (38 mg, 89%). R_f = 0.17 (RP-18, MeOH: 10% HCl, 7:3); IR (ATR) v_max 3293, 1679, 1538, 1202, 1132, 721 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.21 (2H, d, J = 2.0 Hz, H-7), 7.78–7.72 (6H, m, H-8, H-11, H-12), 7.55 (2H, dd, J = 8.5, 2.0 Hz, H-13), 7.44–7.39 (2H, m, H-9), 7.37–7.33 (2H, m, H-10), 3.36–3.30 (4H, m, H₂-15), 3.02 (4H, t, J = 7.0 Hz, H₂-17), 2.81–2.74 (8H, m, H₂-2, H₂-19), 2.62–2.59 (4H, m, H₂-3), 1.90–1.83 (4H, m, H₂-16), 1.49–1.41 (4H, m, H₂-20), 0.96–0.88 (12H, m, H₂-21, H₂-22, H₂-23); ¹³C NMR (CD₃OD, 100 MHz) δ 176.5 (C-1), 173.2 (C-4), 137.7 (C-6), 135.3 (C-7a), 132.0 (C-11a), 129.7 (C-12), 128.7 (C-11), 128.5 (C-8), 127.6 (C-9), 126.0 (C-10), 121.1 (C-13), 117.3 (C-7), 49.1 (C-19), 45.9 (C-17), 36.4 (C-15), 32.4 (C-2), 31.3 (C-3), 30.2 (C-23), 30.0 (C-22), 27.9 (C-16), 27.32 (C-20/C-21), 27.28 (C-20/C-21); (+)-HRESIMS m/z 737.4736 [M+H]⁺ (calcd for C₄₄H₆₁N₆O₄, 737.4749).

4.2.27. N¹,N¹²-Bis(3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)dodecane-1,12-diaminium 2,2,2-trifluoroacetate (20f)

Following general procedure B, the reaction of carboxylic acid 11 (58 mg, 0.24 mmol), di-tert-butyl dodecane-1,12-diylbis((3-aminopropyl)carbamate) (16f) (57 mg, 0.11 mmol), EDC·HCl (52 mg, 0.27 mmol), and DMAP (60 mg, 0.49 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl dodecane-1,12-diylbis((3-(4-(naphthalen-2-ylamino)-4-oxobutanamido)propyl)carbamate) as a colorless oil (81 mg, 76%). Following general procedure C, the reaction of a sub-sample of this material (28 mg, 0.029 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 20f as a colorless oil after purification (26 mg, 90%). R_f = 0.13 (RP-18, MeOH:10% HCl, 7:3); IR (ATR) v_max 2916, 1769, 1671, 1503, 1202, 1037, 802 cm⁻¹; ¹H NMR (CD₃OD, 400 MHz) δ 8.21 (2H, d, J = 1.5 Hz, H-7), 7.79–7.72 (6H, m, H-8, H-11, H-12), 7.56 (2H, dd, J = 9.0, 2.0 Hz, H-13), 7.44–7.40 (2H, m, H-9), 7.38–7.34 (2H, m, H-10), 3.36–3.30 (4H, m, H₂-15), 3.02 (4H, t, J = 7.0 Hz, H₂-17), 2.81–2.75 (4H, m, H₂-2, H₂-19), 2.62–2.59 (4H, m, H₂-3), 1.90–1.84 (4H, m, H₂-16), 1.51–1.44 (4H, m, H₂-20), 1.04–0.99 (16H, m, H₂-21, H₂-22, H₂-23, H₂-24); ¹³C NMR (CD₃OD, 100 MHz) δ 176.5 (C-1), 173.2 (C-4), 137.6 (C-6), 135.3 (C-7a), 131.9 (C-11a), 129.6 (C-12), 128.6 (C-8/C-11), 128.4 (C-8/C-11), 127.5 (C-9), 125.9 (C-10), 121.0 (C-13), 117.2 (C-7), 49.1 (C-19), 45.8 (C-17), 36.4 (C-15), 32.4 (C-2), 31.2 (C-3), 30.4 (C-22/C-23/C-24), 30.3 (C-22/C-23/C-24), 30.1 (C-22/C-23/C-24), 27.9 (C-16), 27.3 (C-20/C-21), 27.2 (C-20/C-21); (+)-HRESIMS [M+2H]²⁺ m/z 383.2578 (calcd for C₄₆H₆₆N₆O₄, 383.2567).

4.2.28. N¹,N⁴-Bis(3-(4-([1,1’-biphenyl]-4-ylamino)-4-oxobutanamido)propyl)butane-1,4-diaminium 2,2,2-trifluoroacetate (21a)

Following general procedure A, the reaction of carboxylic acid 12 (74 mg, 0.27 mmol) with di-tert-butyl butane-1,4-diylbis((3-aminopropyl)carbamate) (16a) (50 mg, 0.12 mmol), EDC·HCl (62 mg, 0.32 mmol), HOBt (44 mg, 0.32 mmol), and DIPEA (0.13 mL, 0.75 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl butane-1,4-diylbis((3-(4-([1,1’-biphenyl]-4-ylamino)-4-oxobutanamido)propyl)carbamate) as a pink solid (65 mg, 60%). Following general procedure C, the reaction of a sub-sample of this material (20 mg, 0.022 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 21a as a yellow solid after purification (15 mg, 73%). R_f = 0.37 (RP-18, MeOH:10% HCl, 5:1); m.p. 203–205 °C; IR (ATR) v_max 3290, 3099, 3033, 2831, 1669, 1599, 1531, 1200, 1178, 1130, 835, 764, 720 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ 10.08 (2H, br s, NH-5), 8.61–8.47 (4H, m, NH₂-18), 8.11 (2H, t, J = 6.0 Hz, NH-14), 7.71–7.68 (4H, m, H-7), 7.65–7.58 (8H, m, H-8, H-11), 7.46–7.40 (4H, m, H-12), 7.35–7.29 (2H, m, H-13), 3.13 (4H, dt, J = 6.4, 6.2 Hz, H₂-15), 2.93–2.82 (8H, m, H₂-17, H₂-19), 2.61 (4H, t, J = 7.0 Hz, H₂-2/H₂-3), 2.43 (4H, t, J = 7.0 Hz, H₂-2/H₂-3), 1.72 (4H, tt, J = 7.4, 6.4 Hz, H₂-16), 1.63–1.54 (4H, m, H₂-20); ¹³C NMR (DMSO-d₆, 100 MHz) δ 172.0 (C-1), 170.5 (C-4), 139.7 (C-10), 138.8 (C-6), 134.6 (C-9), 128.9 (C-12), 127.0 (C-13), 126.8 (C-8), 126.2 (C-11), 119.3 (C-7), 46.1 (C-19), 44.5 (C-17), 35.5 (C-15), 31.5 (C-2/C-3), 30.1 (C-2/C-3), 26.1 (C-16), 22.6 (C-20); (+)-HRESIMS m/z 705.4118 [M+H]⁺ (calcd for C₄₂H₅₃N₆O₄, 705.4123).

4.2.29. N¹,N⁶-Bis(3-(4-([1,1’-biphenyl]-4-ylamino)-4-oxobutanamido)propyl)hexane-1,6-diaminium 2,2,2-trifluoroacetate (21b)

Following general procedure A, the reaction of carboxylic acid 12 (69 mg, 0.26 mmol) with di-tert-butyl hexane-1,6-diylbis((3-aminopropyl)carbamate) (16b) (50 mg, 0.12 mmol), EDC·HCl (58 mg, 0.30 mmol), HOBt (41 mg, 0.30 mmol), and DIPEA (0.12 mL, 0.69 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl hexane-1,6-diylbis((3-(4-([1,1’-biphenyl]-4-ylamino)-4-oxobutanamido)propyl)carbamate) as a pink solid (54 mg, 48%). Following general procedure C, the reaction of a sub-sample of this material (20 mg, 0.021 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 21b as a yellow solid after purification (15 mg, 74%). R_f = 0.43 (RP-18, MeOH:10% HCl, 5:1); m.p. 200–202 °C; IR (ATR) v_max 3296, 3031, 2947, 2832, 2528, 1681, 1657, 1637, 1529, 1198, 1173, 1123, 1000, 759, 718 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ 10.08 (2H, br s, NH-5), 8.54–8.29 (4H, m, NH₂-18), 8.12 (2H, t, J = 5.8 Hz, NH-14), 7.71–7.65 (4H, m, H-7), 7.65–7.58 (8H, m, H-8, H-11), 7.46–7.40 (4H, m, H-12), 7.35–7.29 (2H, m, H-13), 3.14 (4H, dt, J = 6.4, 6.2 Hz, H₂-15), 2.86 (4H, t, J = 7.2 Hz, H₂-17), 2.77 (4H, t, J = 7.8 Hz, H₂-19), 2.61 (4H, t, J = 6.8 Hz, H₂-2/H₂-3), 2.44 (4H, t, J = 6.8 Hz, H₂-2/H₂-3), 1.71 (4H, tt, J = 7.2, 6.7 Hz, H₂-16), 1.53–1.43 (4H, m, H₂-20), 1.23–1.15 (4H, m, H₂-21); ¹³C NMR (DMSO-d₆, 100 MHz) δ 172.1 (C-1), 170.6 (C-4), 139.7 (C-10), 138.8 (C-6), 134.5 (C-9), 128.9 (C-12), 127.0 (C-13), 126.8 (C-8), 126.1 (C-11), 119.2 (C-7), 46.7 (C-19), 44.5 (C-17), 35.4 (C-15), 31.4 (C-2/C-3), 30.1 (C-2/C-3), 26.2 (C-16), 25.4 (C-21), 25.3 (C-20); (+)-HRESIMS m/z 733.4415 [M+H]⁺ (calcd for C₄₄H₅₇N₆O₄, 733.4436).

4.2.30. N¹,N⁷-Bis(3-(4-([1,1’-biphenyl]-4-ylamino)-4-oxobutanamido)propyl)heptane-1,7-diaminium 2,2,2-trifluoroacetate (21c)

Following general procedure A, the reaction of carboxylic acid 12 (66 mg, 0.25 mmol) with di-tert-butyl heptane-1,7-diylbis((3-aminopropyl)carbamate) (16c) (50 mg, 0.11 mmol), EDC·HCl (56 mg, 0.29 mmol), HOBt (39 mg, 0.29 mmol), and DIPEA (0.12 mL, 0.69 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl heptane-1,7-diylbis((3-(4-([1,1’-biphenyl]-4-ylamino)-4-oxobutanamido)propyl)carbamate) as a pink solid (52 mg, 50%). Following general procedure C, the reaction of a sub-sample of this material (20 mg, 0.021 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 21c as a yellow solid after purification (14 mg, 68%). R_f = 0.51 (RP-18, MeOH:10% HCl, 5:1); m.p. 178–180 °C; IR (ATR) v_max 3288, 3034, 2939, 2859, 2527, 1658, 1637, 1533, 1198, 1173, 1134, 763, 719 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ 10.08 (2H, br s, NH-5), 8.48–8.33 (4H, m, NH₂-18), 8.12 (2H, t, J = 5.8 Hz, NH-14), 7.71–7.65 (4H, m, H-7), 7.65–7.57 (8H, m, H-8, H-11), 7.46–7.40 (4H, m, H-12), 7.35–7.29 (2H, m, H-13), 3.14 (4H, dt, J = 6.4, 6.2 Hz, H₂-15), 2.86 (4H, t, J = 7.0 Hz, H₂-17), 2.75 (4H, t, J = 7.0 Hz, H₂-19), 2.61 (4H, t, J = 6.6 Hz, H₂-2/H₂-3), 2.44 (4H, t, J = 6.6 Hz, H₂-2/H₂-3), 1.71 (4H, tt, J = 7.0, 6.4 Hz, H₂-16), 1.53–1.42 (4H, m, H₂-20), 1.20–1.10 (6H, m, H₂-21, H₂-22); ¹³C NMR (DMSO-d₆, 100 MHz) δ 172.3 (C-1), 170.6 (C-4), 139.7 (C-10), 138.8 (C-6), 134.5 (C-9), 128.9 (C-12), 127.0 (C-13), 126.8 (C-8), 126.1 (C-11), 119.2 (C-7), 46.7 (C-19), 44.4 (C-17), 35.4 (C-15), 31.4 (C-2/C-3), 30.1 (C-2/C-3), 28.0 (C-21/C-22), 26.2 (C-16), 25.7 (C-21/C-22), 25.4 (C-20); (+)-HRESIMS m/z 747.4566 [M+H]⁺ (calcd for C₄₅H₅₉N₆O₄, 747.4592).

4.2.31. N¹,N⁸-Bis(3-(4-([1,1’-biphenyl]-4-ylamino)-4-oxobutanamido)propyl)octane-1,8-diaminium 2,2,2-trifluoroacetate (21d)

Following general procedure A, the reaction of carboxylic acid 12 (65 mg, 0.24 mmol) with di-tert-butyl octane-1,8-diylbis((3-aminopropyl)carbamate) (16d) (50 mg, 0.11 mmol), EDC·HCl (54 mg, 0.28 mmol), HOBt (38 mg, 0.28 mmol), and DIPEA (0.11 mL, 0.63 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl octane-1,8-diylbis((3-(4-([1,1’-biphenyl]-4-ylamino)-4-oxobutanamido)propyl)carbamate) as a pink solid (56 mg, 53%). Following general procedure C, the reaction of a sub-sample of this material (20 mg, 0.021 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 21d as a yellow solid after purification (14 mg, 67%). R_f = 0.51 (RP-18, MeOH:10% HCl, 5:1); m.p. 182–184 °C; IR (ATR) v_max 3411, 3292, 3049, 2937, 2860, 2255, 1674, 1534, 1200, 1173, 1129, 1024, 1003, 764, 719 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ 10.08 (2H, br s, NH-5), 8.48–8.34 (4H, m, NH₂-18), 8.13 (2H, t, J = 6.0 Hz, NH-14), 7.71–7.65 (4H, m, H-7), 7.65–7.57 (8H, m, H-8, H-11), 7.46–7.40 (4H, m, H-12), 7.35–7.29 (2H, m, H-13), 3.15 (4H, dt, J = 6.4, 6.0 Hz, H₂-15), 2.87 (4H, t, J = 7.0 Hz, H₂-17), 2.76 (4H, t, J = 7.5 Hz, H₂-19), 2.61 (4H, t, J = 6.8 Hz, H₂-2/H₂-3), 2.43 (4H, t, J = 6.8 Hz, H₂-2/H₂-3), 1.71 (4H, tt, J = 7.0, 6.4 Hz, H₂-16), 1.53–1.41 (4H, m, H₂-20), 1.19–1.08 (8H, m, H₂-21, H₂-22); ¹³C NMR (DMSO-d₆, 100 MHz) δ 172.2 (C-1), 170.6 (C-4), 139.7 (C-10), 138.8 (C-6), 134.5 (C-9), 128.9 (C-12), 127.0 (C-13), 126.8 (C-8), 126.1 (C-11), 119.2 (C-7), 46.8 (C-19), 44.4 (C-17), 35.4 (C-15), 31.4 (C-2/C-3), 30.0 (C-2/C-3), 28.3 (C-21/C-22), 26.2 (C-16), 25.8 (C-21/C-22), 25.4 (C-20); (+)-HRESIMS m/z 761.4739 [M+H]⁺ (calcd for C₄₆H₆₁N₆O₄, 761.4749).

4.2.32. N¹,N¹⁰-Bis(3-(4-([1,1’-biphenyl]-4-ylamino)-4-oxobutanamido)propyl)decane-1,10-diaminium 2,2,2-trifluoroacetate (21e)

Following general procedure A, the reaction of carboxylic acid 12 (61 mg, 0.23 mmol) with di-tert-butyl decane-1,10-diylbis((3-aminopropyl)carbamate) (16e) (50 mg, 0.10 mmol), EDC·HCl (51 mg, 0.27 mmol), HOBt (36 mg, 0.27 mmol), and DIPEA (0.11 mL, 0.63 mmol) in CH₂Cl₂ (1.5 mL) created di-tert-butyl decane-1,10-diylbis((3-(4-([1,1’-biphenyl]-4-ylamino)-4-oxobutanamido)propyl)carbamate) as a pale yellow solid (56 mg, 57%). Following general procedure C, the reaction of a sub-sample of this material (20 mg, 0.020 mmol) in CH₂Cl₂ (2 mL) with TFA (0.2 mL) created the di-TFA salt 21e as a yellow solid after purification (15 mg, 74%). R_f = 0.34 (RP-18, MeOH:10% HCl, 5:1); m.p. 184–186 °C; IR (ATR) v_max 3287, 3099, 3033, 2930, 2854, 1668, 1599, 1533, 1200, 1175, 1130, 764, 720 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ 10.09 (2H, br s, NH-5), 8.49–8.37 (4H, m, NH₂-18), 8.14 (2H, t, J = 6.0 Hz, NH-14), 7.71–7.66 (4H, m, H-7), 7.64–7.57 (8H, m, H-8, H-11), 7.46–7.39 (4H, m, H-12), 7.34–7.28 (2H, m, H-13), 3.15 (4H, dt, J = 6.4, 6.2 Hz, H₂-15), 2.93–2.83 (4H, m, H₂-17), 2.82–2.72 (4H, m, H₂-19), 2.62 (4H, t, J = 6.8 Hz, H₂-2/H₂-3), 2.44 (4H, t, J = 6.8 Hz, H₂-2/H₂-3), 1.72 (4H, tt, J = 7.0, 6.4 Hz, H₂-16), 1.53–1.42 (4H, m, H₂-20), 1.20–1.07 (12H, m, H₂-21, H₂-22, H₂-23); ¹³C NMR (DMSO-d₆, 100 MHz) δ 172.3 (C-1), 170.6 (C-4), 139.7 (C-10), 138.8 (C-6), 134.5 (C-9), 128.9 (C-12), 127.0 (C-13), 126.8 (C-8), 126.1 (C-11), 119.2 (C-7), 46.9 (C-19), 44.4 (C-17), 35.4 (C-15), 31.4 (C-2/C-3), 30.1 (C-2/C-3), 28.8 (C-21/C-22/C-23), 28.5 (C-21/C-22/C-23), 26.2 (C-16), 25.9 (C-21/C-22/C-23), 25.5 (C-20); (+)-HRESIMS m/z 789.5055 [M+H]⁺ (calcd for C₄₈H₆₅N₆O₄, 789.5062).

4.3. Antimicrobial Assays

The susceptibility of bacterial strains S. aureus (ATCC 25,923 or 29213), E. coli (ATCC 25922), and P. aeruginosa (ATCC 27853) to antibiotics and compounds was determined according to previously reported protocols [27]. Additional antimicrobial evaluation against MRSA (ATCC 43300), Klebsiella pneumoniae (ATCC 700603), Acinetobacter baumannii (ATCC 19606), Candida albicans (ATCC 90028), and Cryptococcus neoformans (ATCC 208821) was undertaken at the Community for Open Antimicrobial Drug Discovery at The University of Queensland (Australia) according to their standard protocols as reported previously [27,36].

4.4. Determination of the MICs of Antibiotics in the Presence of Synergizing Compounds

Restoring enhancer concentrations were determined using previously reported protocols [27].

4.5. Cytotoxicity Assays

Cytotoxicity assays were conducted using the protocols previously reported [27,36].

4.6. Hemolytic Assays

Hemolysis assays were conducted using the protocols previously reported [27,36].

4.7. Real-Time Growth Curves

Solutions of compound 20f at concentrations of 2, 4, and 16 µg/mL were each tested in triplicate against S. aureus (ATCC 25923), MRSA (CF-Marseille), and E. coli (ATCC 25922) following previously reported protocols [27].

4.8. Minimum Bactericidal Concentration Test

MBC’s were determined following previously reported protocols [27].

5. Conclusions

A series of α,ω-disubstituted polyamines bearing naphthyl and biphenyl capping groups were prepared and evaluated for antimicrobial properties, as well as for the ability to enhance the action of doxycycline and erythromycin towards Gram-negative bacteria. Several analogues were identified as exhibiting pronounced antibacterial activity towards MRSA, with some examples also possessing antifungal activity towards C. neoformans. One particular analogue, 20f, was found to act as a bactericide. In contrast to previously reported structurally related disubstituted polyamines, the naphthyl-containing analogues were able to restore the action of legacy antibiotics with two examples, enhancing activity over 32-fold. These current results suggest that naphthyl capped polyamines may represent a particularly useful scaffold to further explore in the search for non-toxic antibiotic enhancers.