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. Author manuscript; available in PMC: 2020 Nov 27.
Published in final edited form as: J Med Chem. 2019 Nov 12;62(22):10245–10257. doi: 10.1021/acs.jmedchem.9b01210

The Structure-Activity Relationship of Peptide Conjugated Chloramphenicol for Inhibiting E. coli

Jiaqing Wang †, Adrianna Shy †, Difei Wu †, Deani L Cooper ‡, Jiashu Xu †, Hongjian He †, Wenjun Zhan †, Shenghuan Sun †, Susan T Lovett ‡, Bing Xu †,*
PMCID: PMC6902879  NIHMSID: NIHMS1060117  PMID: 31670952

Abstract

Intravenous administration of the prodrug, chloramphenicol succinate (CLsu), is ineffective. Recently, we have shown that conjugation of diglycine of CLsu (CLsuGG), not only increases antibiotic efficacy against E. coli but also reduces adverse drug effects against bone marrow stromal cells. Here, we report the synthesis of structural analogs of CLsuGG and their activities against E. coli. These analogs reveal several trends: (i) Except the water-insoluble analogs, the attachment of peptides to CLsu enhances the efficacy of the prodrugs; (ii) negative charges, high steric hindrance in the side chains, or a rigid diester decreases the activities of prodrugs in comparison to CLsuGG; (iii) dipeptides apparently increase the efficacy of the prodrugs most effectively, etc. This work suggests that conjugating peptides to chloramphenicol succinate effectively modulates the properties of prodrugs. The structure-activity relationship of these new conjugates may provide useful insight for expanding the pool of antibiotics.

Graphical Abstract

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INTRODUCTION

Resulting from a large amount of antibiotics used for human and animal treatment, multidrug resistance (MDR) in bacteria remains a serious threat in public health.1–5 Drugs recently developed to thwart emerging antibiotic resistances, such as linezolid and the latest β-lactams, to vancomycin, have already lost effectiveness against some bacterial strains.6–8 An even more serious threat may be the emergence of MDR Gram-negative bacteria that are resistant to essentially all of the available agents.9 Even more discouraging, the development of alternatives to the existing strategies for killing pathogenic bacteria has slowed dramatically over the past decades and fails to keep pace with the outbreak of resistance. Moreover, newer, successfully developed alternatives are strictly reserved to treat only the most serious infections. These factors make the antibiotic supply in the clinical pipeline remain limited.4, 10 Thus, there is an urgent need for developing new antimicrobial approaches against MDR bacterial pathogens.

Different strategies being used to discover and develop novel drugs to fight bacteria, mainly include (1) drug derivatives, which enhance the efficacy and safety of existing antibacterial agents via the modification of the drugs11 or increasing specificity (e.g., efflux pump inhibitors);12 (2) discovery of new antibacterial agents, which involves the development of new tools to discover genomic or target-based antibiotics via previously unexplored mechanisms,11 and classical or whole-cell antibacterial assay to find antibiotics produced by microorganism of different sources;13 (3) bacteriophages or enzybiotics, which utilize bacteriophages or phage-lytic enzymes;14–15 (4) ecology/evolutionary biology approaches, which target the ecology and evolution of antibiotic resistance.16 In comparison to developing a new drug that requires years of extensive testing, modifying existing antibiotics to achieve higher efficacy is safer, faster, and lower-cost. Therefore, developing feasible drug derivatives has become an active research focus.

Recently, the development of antibiotic derivatives has made significant progress. For example, Boger et al. reported that modifying the binding pocket of vancomycin, introducing a quaternary ammonium salt to the peripheral C-terminal, and linking (4-chlorobiphenyl)methyl (CBP) to the vancomycin disaccharide provide dual target binding and three independent action mechanisms, which directly overcome the molecular basis of vancomycin resistance.17–18 The vancomycin analogues provide improvements in antibacterial potency against vancomycin-resistant enterococci and display little propensity for resistance. Kahne et al. reported the synthesis of novobiocin derivatives, which were more potent than novobiocin when being used in combination with polymyxin, the drug of last resort for treating Gram-negative infections.19 The novobiocin analogues not only allow the lower dosage of polymyxin, but also increase its efficacy and safety via inhibiting DNA gyrase, binding LptB, and disrupting the outer membrane. Nolan et al. developed a synthetic siderophore-antibiotic conjugate (Ent-Cipro), which affords targeted antibacterial activity against pathogenic E. coli strains.20 Ent-Cipro provides an excellent selectivity between non-pathogenic and pathogenic E. coli. Ramström and Yan synthesized a series of ciprofloxacin derivatives, which exhibited enhanced antibacterial activities against both sensitive and resistant E. coli.21 In addition, there are reports to conjugate CL to amino acid. For example, in the early years, Khanna et al. reported that the direct conjugation of ploy(amino acids) to the reduced nitro group of CL generated conjugated antigens, which provided conventional ways for the production of antibodies specific for CL.22 Recently, Gu et al. directly attached antimicrobial peptide to CL to form conjugate (i.e., CL-UBI29–41 hybrid), which are tested in mouse models.23

Our recent study shows that conjugating diglycine to a prodrug of antibiotics (i.e., chloramphenicol and ciprofloxacin) drastically accelerates intrabacterial hydrolysis of ester bonds, regenerating the antibiotics against E. coli. Moreover, the ester conjugate of chloramphenicol and diglycine (CLsuGG, 1) also exhibits reduced toxicity to bone marrow cells, a major side effect of CL.24 In addition, mechanistic investigation reveals that 1 shows different hydrolysis rates upon being treated by the mammalian and bacterial esterases. The effects of the structures of conjugated-peptides on intrabacterial hydrolysis, as well as on the antibacterial activity, however, remain unexplored.

To understand the structure-activity relationship of the peptide conjugated chloramphenicol for inhibiting bacteria, we synthesized 34 structural analogs of 1 (Table 1) and examined their activities against E. coli. Our results show that conjugating peptides to CLsu enhances the efficacy of prodrugs to varying degrees, apart from water-insoluble analogs. As shown in Scheme 1, negative charges, high steric hindrance in the side chains of peptides, or a rigid diester (i.e., cyclohexane-1,2-dicarboxylic acid) results in lower activities than that of 1. Moreover, the investigation of conjugated-peptides from single amino acid to pentapeptide indicates that dipeptides are the most effective to increase the efficacy of CLsu. In addition, capping the C-terminal of the peptide with an N-methylacetamide group scarcely lowers the activity of the prodrug in comparison to the uncapped peptide conjugate. D-amino acid residues, in general, are more favorable than L-amino acid residues for increasing the activity of the prodrugs, suggesting that D-peptides affect little on the hydrolysis rate of the ester bond in the prodrugs. Besides establishing conjugation of peptides as a simple and effective way for modulating the properties of the prodrug chloramphenicol (CLsu), the structure-activity relationship of these peptides conjugated prodrugs may provide useful insight for designing peptide conjugates25–29 of other antibiotics.

Table 1.

Structures of chloramphenicol (CL), chloramphenicol succinate (CLsu) and peptide-conjugated prodrugs.

graphic file with name nihms-1060117-t0007.jpggraphic file with name nihms-1060117-t0008.jpg
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Scheme 1.

Scheme 1.

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Illustration of peptide conjugated antibiotics for intrabacterial activation and the trends of the efficiency associated with the structures of the conjugates (LPS: lipopolysaccharide; OM: outer membrane; IM: inner membrane).

RESULTS AND DISCUSSION

Molecular Design.

We designed the peptide conjugated chloramphenicol in a straightforward manner for easy production and convenient optimization. As shown in Table 1, the conjugates have a general formula of antibiotic—succinate—peptide (or amino acid). The peptides fall into 3 main categories: a variety of neutral peptides (1a-1q), from di- to pentapeptides, for studying the influence of numbers of same amino acid residues or the side chains of the amino acid residues on the activity; charged peptides (e.g., arginine, lysine, glutamic acid, or aspartic acid (2a-2j)) for investigating the effect of charges; naphthyl containing peptides (3a-3c) for understanding the roles of strong hydrophobicity. Besides peptide conjugates, there are conjugates bearing a single amino acid (4a), or only capping the C-terminal of CLsu using a methyl (4b), sulfate (4c), or phosphate (4d) group. Moreover, considering the stereochemistry of the peptides as a determining factor for biological activities, we generated D-peptide conjugates in the first two categories (e.g., 1g, 1i, 2b, and 2h).

Synthesis.

As shown in Scheme S1, the commercially available chloramphenicol succinate sodium, after acidification, is suitable for solid phase peptide synthesis to produce all peptide-conjugated analogues of 1. Briefly, being attached to 2-chlorotrityl chloride resin, the Fmoc- protected amino acid, after releasing the Fmoc group, reacts with the next amino acid or CLsu for expanding the chain of the peptide or for introducing chloramphenicol. Then, trifluoroacetic acid cleaves the products from the resin. Dropwise addition of bromotrimethylsilane to the methanol solution of CLsu generates 4b. Solution phase amidation of CLsu with taurine and phosphorylethanolamine results in 4c and 4d, respectively.

Antibacterial activity.

After obtaining the pure conjugates, we tested these analogues for their antibacterial activity against a wild type E. coli strain (K-12). As shown in Table 2, the first category, relatively hydrophilic neutral peptides conjugated to CLsu (1a-1q), exhibits higher inhibitory activity than other categories of the conjugates, with MICs ranging from 20 to 200 μM, which is comparable to that of CL but much lower than that of CLsu (standard compounds). Our previous work demonstrates that diglycine conjugating CLsu apparently increases the efficacy of the prodrugs most effectively.30 The replacement of the C-terminal glycine on 1 by leucine, alanine, or serine generates 1a, 1b, and 1c, which all exhibit a MIC of 20 μM. Switching the location of glycine and serine in 1c produces 1d, which exhibits a MIC (40 μM) twice of that of 1c. These results suggest that directly linking the carboxyl group of CLsu by a glycine residue is important for the high activity of the prodrugs. Capping the carboxylic acid of 1 by N-methylacetamide forms 1e, which exhibits the MIC of 20 μM. The activity of 1e is comparable to that of 1, indicating that capping the C-terminal of 1 by N-methylacetamide has little effect on the hydrolysis of prodrugs.

Table 2.

The minimum inhibitory concentration (MIC) of peptides-conjugated CLsu against E. coli (K-12).

# Compound MIC (μM) # Compound MIC (μM)
- CL 20 1q CLsu-GGGGG 40
- CLsu >200 2a CLsu-rr 200
1a CLsu-Gl 20 2b CLsu-kk 200
1b CLsu-Ga 20 2c CLsu-KK 200
1c CLsu-Gs 20 2d CLsu-ffrr 200
1d CLsu-sG 40 2e CLsu-ffkk 200
1e CLsu-GGNHMe 20 2f CLsu-GK 20
1f CLsu-GGGG 20 2g CLsu-ee >200
1g CLsu-aa 80 2h CLsu-dd >200
1h CLsu-AA 100 2i CLsu-DD 200
1i CLsu-aaa 80 2j CLsu-GD 20
1j CLsu-AAA 100 3a CLsu-ff 100
1k CLsu-GGf 20 3b CLsu-ff(2-NaI) >200
1l CLsu-GGF 20 3c CLsu-ff(2-NaI)GG >200
1m CLsu-GFG 20 4a CLsu-a >200
1n CLsu-FGG 80 4b CLsu-OMe >200
1o CLsu-GGff 20 4c CLsu-Tau 40
1p CLsu-GGFF 20 4d CLsu-ep 100
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Di-D-alanine and di-L-alanine replace the diglycine in 1 to generate 1g and 1h, which exhibit MIC values of 80 μM and 100 μM, respectively. Being considerably higher than that of 1 but close to each other, the MIC values of 1g and 1h suggest that the side chains, on the amino acid residues of the peptides, likely affect the hydrolysis of prodrugs by the esterases. 1i and 1j, containing three alanine residues and being enantiomers, exhibit of the MIC values of 80 and 100 μM, respectively. These results indicate that D-alanine slightly enhances the activity of the prodrugs and agree with our previous observation that dipeptide (i.e., diglycine) is optimal for enhancing the antibacterial efficacy of the conjugates.

Inserting one phenylalanine in 1 at a different position of produces 1k, 1l, 1m, and 1n, which exhibit MIC values of 20, 20, 20, and 80 μM, respectively. While the activity of 1l is comparable to that of 1, the activity of 1n is ten times lower than that of 1. This result indicates that phenylalanine should be away from the ester linkage to favor the hydrolysis of the prodrug. The activities of 1k and 1m are comparable to that of 1, indicating that the position of phenylalanine affects little on the activities of the prodrugs when glycine is the first residue connected to succinate. The attachment of two D- or L-diphenylalanine in the carboxylic acid end of 1 generates 1o and 1p, both of which exhibit a MIC value of 20 μM, which is similar with that of 1k and 1l, indicating that the number of phenylalanine may affect little on the hydrolysis. While the conjugate (1f), containing four glycine residues, shows a comparable activity (MIC = 20 μM) to that of 1, the conjugate (1q), containing five glycine residues and exhibiting the MIC of 40 μM, is considerably less active than 1. This result, again agrees with our earlier conclusions, that diglycine is optimal for enhancing the antibacterial efficacy of CL.

Considering the electrostatic interactions with negatively charged bacterial membrane,31 we designed the second category of conjugates (2a-2j) by attaching charged peptides to CLsu. The incorporation of two D-arginine and two D-lysine residues generates 2a and 2b, with the MIC values of 200 and 200 μM, respectively, which are similar with 1g (80 μM) and 1h (100 μM) in the first category. With two L-lysine residues in the conjugates, the MIC of 2c is 200 μM. This result indicates that the D- and L-lysine residues would have similar influences on the activities of the prodrugs. Moreover, the insertion of two D-phenylalanine residues in 2a and 2b generates 2d and 2e, which still shows comparable activity with that of 2a and 2b, with both the MIC values of 200 μM. Using one glycine to replace one L-lysine in 2c leads to 2f, which shows much higher inhibitory activity (20 μM) than 2c (200 μM).

Besides introducing the above positively charged peptides, we also attached various negatively charged peptides in the conjugates and compared their antibacterial efficiency with the positively charged ones. Containing glutamic acid or aspartic acid residues, 2g, 2h, and 2i all exhibit high MIC values (2g: >200 μM; 2h: >200 μM; 2i: 200 μM), indicating multiple negative charges decrease the activities of the prodrugs. This notion is supported by the lower MIC value (20 μM) of 2j than that of 2i. These results are consistent with the idea that the negatively charged bacterial membrane may electrostatically repel the prodrugs bearing multiple negative charges.

Introducing either hydrophilic neutral peptides or charged peptides to CLsu results in water soluble conjugates. To understand the roles of solubility, we created conjugates attached with hydrophobic peptides or steric hindrance in side chains to CLsu (3a-3c). The direct attachment of diphenylalanine in the carboxylic acid end of CLsu generates 3a, which shows low inhibitory activity against E. coli with a MIC value of 100 μM. Furthermore, attaching a hydrophobic amino acid (i.e., naphthylalanine) produces 3b, which is more hydrophobic than 3a, hardly inhibits E. coli growth, and the concentration is higher than 200 μM. In addition, the insertion of diphenylalanine and naphthylalanine in 1 forms 3c, which also results in a MIC value higher than 200 μM. These observations imply that the incorporation of hydrophobic peptides significantly impairs the efficiency of prodrugs, if the solubility of the conjugates is low. This observation is reasonable since low solubility increases difficulty to enter bacterial membranes and decreases the rate of hydrolysis of the ester bond by the esterases.

Besides peptide conjugates, there are conjugates bearing a single alanine (4a), or only capping the C-terminal of CLsu with a methyl (4b), sulfate (4c), or phosphate group (4d). 4a exhibits a high MIC value (>200 μM), implying that the direct attachment of one amino acid residue to CLsu has little influence on the hydrolysis of the prodrugs. Blocking the carboxyl group with methyl group generates water-insoluble 4b with two ester bonds. The high MIC value of 4b (>200 μM) suggests that multiple ester bonds and/or low solubility likely reduces the rate of hydrolysis by esterases. 4c and 4d exhibit the MIC of 40 and 100 μM, respectively, which are lower than the prodrug (i.e., CLsu (>200 μM)) but still higher than 1. The result is not only consistent with the fact that conjugating taurine32–33 plays a role in boosting cellular uptake, but also supports the conjugation of peptides to prodrugs as an effective way for enhancing efficacy of the prodrugs. Although the antibacterial activity of these peptide conjugated CL, like CL itself, is not as potent as that of some other broad-spectrum antibiotics (e.g., ciprofloxacin and trimethoprim with MIC of 0.01 μg/mL, 1 μg/mL and 0.6 μg/mL against E. coli K-12, respectively),34–36 they not only show improved potency in the comparison of CLsu, the clinically failed prodrug of CL,37 but also exhibit better water-solubility than that of CL. This discovery may provide potentially clinical application for intravenous injection of the new prodrugs of CL.

Hydrolysis rate.

To further understand the effects of the structures of conjugated-peptides on hydrolysis, as well as on the antibacterial activity, first, we compared the antibacterial activity of 1c, 1d, 2b and 2c before and after the treatment of commercially available mammalian esterase (porcine liver esterase, PLE) (1U/mL) and found the addition of exogenous PLE hardly changes the inhibitory efficacy of 1c, 1d, 2b and 2c (Figure 1A). Then we measured the hydrolysis rate of the ester bond of 1c, 1d, 2b and 2c by E. coli or HepG2 (a hepatocyte cell which overexpress mammalian esterases38) lysates, after normalizing their activities. As shown in Figure 1B, catalyzed by the lysate of either E. coli or HepG2, all peptide conjugated prodrugs are able to undergo complete hydrolysis for regenerating the active drug, which further supports our conclusion that the attachment of peptides to CLsu enhances the efficacy of the prodrugs. In detail, under the condition of E. coli lysates, the different conjugated peptides (with slightly different apparent first-order rate constant of 1c: 3.763 h−1, 1d: 1.267 h−1, 2b: 0.317 h−1 and 2c: 0.310 h−1) leads to different inhibitory activity of prodrugs with the tendency that the faster hydrolysis results in higher efficacy (1c: 20 μM, 1d: 40 μM, 2b: 200 μM and 2c: 200 μM).

Figure 1.

Figure 1.

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(A) The antibacterial activity of 1c, 1d, 2b and 2c before and after the treatment of PLE; [PLE] = 1 U/mL. (B) The hydrolysis curve of 1c, 1d, 2b and 2c with the addition of the lysate of HepG2 cells or the lysate of E. coli (K-12); [1c] = [1d] = [2b] = [2c] = 200 μM, [HepG2 lysate] = [E. coli lysate] = 0.1 U/mL. (C) The static light scattering signals of the solution of 1c, 1d, 2b and 2c before and after the addition of PLE; [1c] = [1d] = [2b] = [2c] = 200 μM, [PLE] = 1 U/mL, t = 24h. (D) The transmission electron microscopy images of 1c, 1d, 2b and 2c before and after the addition of PLE; [1c] = [1d] = [2b] = [2c] = 500 μM, [PLE] = 1 U/mL, t = 24h, scale bar = 500 nm.

The intensities of the static light scattering signals increase drastically at different magnitudes after the addition of PLE to the solutions of 1c, 1d, 2b and 2c for 24 h (Figure 1C), agreeing with the transmission electron microscopy (TEM) images, which show increased amount of nanoparticles after the addition of PLE (Figure 1D). Besides confirming that the precursors become active antibiotics (i.e., CL) after the hydrolysis catalyzed by esterases, these results indicate that soluble precursors are able to undergo intrabacterial hydrolysis to form CL. Because of the poor solubility of CL, such a conversion enhances the retention of CL inside E. coli.

Furthermore, we examined the serum stability of CLsu, 1c, 1d, 2b, 2c and several other most effective conjugates (e.g., 1k, 1o and 2f). We incubated CLsu and these conjugates in human serum (from human male AB plasma) at the concentration at 200 μM, 37 °C for 2 h and 24 h, respectively (Table S2). The LC-MS results show that all the conjugates hydrolyzed to form CL after 24 h while the conversion of CLsu only achieved less than 15% after 24 h. Furthermore, 1c, 1k and 2f were able to hydrolyze completely within 2 h. The discovery is consistent with that the faster hydrolysis results in higher efficacy and further confirms that the attachment of peptides to CLsu would enhance the efficacy of prodrugs. In addition, the results also show that the different conjugated-peptides may lead to slightly different stability in serum environment. Therefore, these conjugates may achieve adequate serum concentration in a short time if being administrated intravenously, which may address the shortcoming of CLsu and its clinical failure. However, the balance between serum stability outside of bacteria and targeting hydrolysis inside bacteria remains optimization, which may be achieved by further investigation the peptide-antibiotic conjugates.

Uptake mechanism.

Our previous work demonstrated that diglycine conjugated CLsu enters the bacteria via multiple paths, including ydgR (i.e., inner membrane oligopeptide transporters involving in the uptake of di- and tripeptides),39 fepA (i.e., siderophore transporter),40 and passive diffusion. We chose several representative peptides conjugating CLsu (e.g., 1c, 1d, 2b and 2c) to study the effects of conjugated peptides on the uptake of the prodrugs. In addition to testing the activities of these conjugates against E. coli mutants that have ydgR or fepA deleted, we also measured the activities of these conjugates against E. coli mutants that have ompF (i.e., outer membrane porin transporters mediating the non-specific diffusion of small solutes)41 or acrA (i.e., the periplasmic lipoprotein component of multidrug efflux pumps)42–43 deleted. As shown in Figure 2, we compared the activities of 1c, 1d, 2b and 2c against ydgR, fepA, ompF, acrA mutants and wild-type E. coli. When the mutants are treated by 1c, 1d, 2b and 2c, the deletion of ydgR, fepA, or ompF reduces the viability of bacteria at different magnitudes and hardly rescues the bacteria, suggesting ydgR, fepA, and ompF are unlikely to be the major contributors for the precursors entering the bacteria, and mutation of one transporter may not lead to drug resistance of these precursors. However, the deletion of acrA significantly reduces the viabilities of the mutant treated by the conjugates (1c: 30%, 1d: 40%, 2b: 20%, 2c: 50%), indicating that acrA likely plays a key role in pumping out the precursors, thus agreeing with the suggestion that acrA contributes to bacterial resistance.44

Figure 2.

Figure 2.

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The antibacterial activity of 1c, 1d, 2b and 2c against ydgR, fepA, ompF transporter deletion mutants or acrA efflux pump deletion mutant of E. coli.

Intrabacterial hydrolysis.

To examine the role of different bacterial esterases on the hydrolysis of different peptides conjugated to CLsu, we measured the activities of 1c, 1d, 2b and 2c against eight E. coli mutants that have one of the bacterial esterase genes (i.e., bioH, yjfP, frsA, ybfF, yfbB, ypfH, yeiG or yqiA)45–47 deleted. As shown in Figure 3, the deletion of the cytoplasmic esterase of E. coli reduces the antibacterial activities of 1c, 1d, 2b and 2c moderately, at about 20%, 10%, 20% and 20%, respectively. These studies confirm that various esterases in bacterial cytoplasm convert precursors to the active antibiotic agent (i.e., CL) after the precursors enter E. coli.

Figure 3.

Figure 3.

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The antibacterial activity of 1c, 1d, 2b and 2c against esterase (bioH, yjfP, frsA, ybfF, yfbB, ypfH, yeiG or yqiA) deletion mutants of E. coli.

Cytotoxicity study.

From previous in vitro cytotoxicity studies against HS-5 (a bone marrow stromal cell line), HepG2 (a hepatocyte cell line), and HEK293 (a kidney cell line) cells, we found that diglycine conjugating CLsu likely would reduce the major adverse effect of CL (i.e., bone marrow suppression), whereas it hardly alters the cytotoxicity of CL against HepG2 and HEK293. To assess the major side effects of these conjugates, we compared the cytotoxicities of the most effective conjugates with MIC values of 20 μM (i.e., 1a, 1b, 1c, 1e, 1f, 1k, 1l, 1m, 1o, 1p, 2f, and 2j), the control compounds (i.e., ciprofloxacin, trimethoprim, and telithromycin) against HS-5 and HEK293 cells. As shown in Figure 4A and 4B, like prodrug CLsu, most of the conjugates show lower cytotoxicity than CL against HS-5 cells, further confirming that peptides-conjugated CLsu likely would reduce the major adverse effect of CL. Meanwhile, ciprofloxacin (Cipro), trimethoprim (TMP), and telithromycin (TEL) also show relatively low cytotoxicity against HS-5 cells at the concentration of around MIC values. In addition, all conjugates show about the same cytotoxicity as CL against HEK293 cells, further confirming that peptides-conjugated CLsu scarcely alter the cytotoxicity of CL against HEK293. Furthermore, we increased the concentration of these conjugates to 10-fold and 100-fold of MIC values (i.e., 200 μM and 2 mM) to examine their cytotoxicities against HS-5 cells. As shown in Figure 4C and 4D, all of them still show relatively lower cytotoxicity than CL against HS-5 cells, implying the possibility of clinical administration.

Figure 4.

Figure 4.

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Cell viability of (A) HS-5 cells and (B) HEK293 cells incubated with 1a, 1b, 1c, 1e, 1f, 1k, 1l, 1m, 1o, 1p, 2f and 2j for 24 h. [CL] = [CLsu] = [1a] = [1b] = [1c] = [1e] = [1f] = [1k] = [1l] = [1m] = [1o] = [1p] = [2f] = [2j] = 20 μM; [Cipro] = [TMP] = [TEL] = 0.5 μM. (C) Cell viability of HS-5 cells incubated with CL, CLsu, 1a, 1b, 1c, 1e, 1f, 1k, 1l, 1m, 1o, 1p, 2f and 2j for 24 h. [CL] = [CLsu] = [1a] = [1b] = [1c] = [1e] = [1f] = [1k] = [1l] = [1m] = [1o] = [1p] = [2f] = [2j] = 200 μM; [Cipro] = [TMP] = [TEL] = 5 μM. (D) Cell viability of HS-5 cells incubated with CL, CLsu, 1a, 1b, 1c, 1e, 1f, 1k, 1l, 1m, 1o, 1p, 2f and 2j for 24 h. [CL] = [CLsu] = [1a] = [1b] = [1c] = [1e] = [1f] = [1k] = [1l] = [1m] = [1o] = [1p] = [2f] = [2j] = 2000 μM; [Cipro] = [TMP] = [TEL] = 50 μM.

CONCLUSION

In summary, we have developed a series of peptide-conjugated prodrugs of chloramphenicol via an ester bond, which modulate the properties of the prodrugs. The antibacterial activity study of these conjugates against E. coli demonstrated the structure-activity relationship of these peptide conjugated chloramphenicols, which may provide a powerful way for developing potential antibiotic agents that treat bacterial infections. In addition, further hydrolysis investigation of the conjugates suggests that rapid intrabacterial hydrolysis is a useful way to increase the retention of active drugs inside bacteria and enhance the efficacy of prodrugs, which deserves further exploration. Additionally, our investigation showed that acrA efflux pumps lead to the bacterial resistance of these peptide-conjugated prodrugs, and various cytoplasmic esterases contributed to form active antibiotic agents after precursors entered E. coli, which indicates the future direction of molecular design based on the functions of bacterial esterases48–49 or bacterial efflux pumps50. This work may also provide a potential strategy to design functional peptides51–53 or peptidomimetic54–58 modified prodrugs. For example, conjugating tissue-specific targeting peptides to antibiotic agents and regenerating the active antibiotics in the target tissues via rapid hydrolysis might be an effective approach to achieve targeted treatment of bacterial infections with lower adverse effects towards others tissues.

EXPERIMENTAL SECTION

General Information.

2-Chlorotrityl chloride resin (1.0–1.2 mmol/g), HBTU and Fmoc protected amino acids were purchased from GL Biochem (Shanghai, China). Other chemical reagents and solvents were purchased from Fisher Scientific. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and Gibco Penicillin-Streptomycin were purchased from Life Technologies. All precursors were purified with Agilent 1100 Series Liquid Chromatograph system, equipped with an XTerra C18 RP column and Variable Wavelength Detector. The LC-MS spectra were obtained with a Waters Acquity Ultra Performance LC with Waters MICROMASS detector.

Synthesis of Desired Compounds.

We synthesized CLsu by acidifying the commercially available chloramphenicol succinate sodium. Chloramphenicol succinate sodium (150 mg) was dissolved in distilled water (3 mL), and HCl (1 M) was added dropwise until the pH of the mixture was adjusted to 2.0. The precipitate was washed several times with distilled water and dried for further use.

We used solid phase peptide synthesis (SPPS)59 for the synthesis of all pepetide-conjugated prodrugs. 2-chlorotrityl chloride resin (500 mg, 0.5 mmol) was swelled in 10 mL of DCM for 20 min. The attachment of the first Fmoc protected amino acid (0.5 mmol) to the resin was achieved by adding N,N-diisopropylethylamine (DIPEA) (413 μL, 2.5 mmol) to the beads in the reaction vessel, which was allowed to shake at room temperature for 1 h. After that, the reaction solution was drained, followed by washing with DMF (10 mL × 3) and DCM (10 mL × 3). The unreacted 2-chlorotrityl chloride moieties were capped with a solution of methanol/DIPEA/DCM (v/v/v: 3/1/16) for 30 min. The beads were washed with DMF (10 mL × 3). The Fmoc group was removed by treating beads with 20% piperidine/DMF (v/v) solution for 20 min at room temperature. The solution was drained and washed with DMF (10 mL × 3). The beads were reacted with the next Fmoc protected amino acid or CLsu (0.5 mmol) by adding HBTU (190 mg, 0.5 mmol) and N,N-diisopropylethylamine (DIPEA) (413 μL, 2.5 mmol) to the beads in the reaction vessel, which was allowed to shake at room temperature for 1 h, and the solution was drained. Then the product was cleaved from resin with 10 mL of trifluoroacetic acid for 2 h. The solution was collected, and the remaining beads were washed with 5 mL of trifluoroacetic acid three times. All the solution was combined and trifluoroacetic acid was removed with the N2 flow. The residue was then precipitated with diethyl ether. The crude product was purified by reverse phase HPLC using HPLC grade acetonitrile and water with supplement of 0.1% trifluoroacetic acid as the eluents. The purity of the compounds was determined to be >95% by LC-MS with a Waters Acquity Ultra Performance LC (Table S1). The NMR spectra were obtained on a Varian Unity Inova 400 instrument (Figure S1–S34 and S69–S102).

Compound 1a.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 8.0 Hz, 1H), 8.16 (m, 3H), 8.04 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 6.44 (m, 1H), 5.03 (s, 1H), 4.22 (m, 3H), 4.08 (m, 2H), 3.73 (m, 2H), 2.44 (m, 4H), 1.56 (m, 3H), 0.84 (m, 6H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 174.34 (CO carboxyl), 172.63 (CO carboxyl), 171.47 (CONH amide), 169.26 (CONH amide), 164.02 (CONH chloramphenicol), 150.65 (CCH phenyl), 146.99 (CNO2 phenyl), 127.87 (CH phenyl), 123.30 (CH phenyl), 69.85 (CH chloramphenicol), 66.67 (CH chloramphenicol), 63.38 (CH2 chloramphenicol), 53.90 (CH), 50.25 (CH2), 42.08 (CH2), 30.05 (CH2), 29.33 (CH2), 24.61 (CH2), 23.19 (CH), 21.73 (CH3); ESI-MS m/z calcd. for C23H30Cl2N4O10 [M]+: m/z = 592.13, found [M-H]− 591.27.

Compound 1b.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 12.0 Hz, 1H), 8.16 (m, 3H), 8.10 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 6.44 (s, 1H), 5.03 (s, 1H), 4.20 (m, 3H), 4.09 (m, 2H), 3.72 (m, 2H), 2.45 (m, 4H), 1.25 (d, J = 8.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 174.38 (CO carboxyl), 172.66 (CO carboxyl), 171.51 (CONH amide), 169.04 (CONH amide), 164.05 (CONH chloramphenicol), 150.67 (CCH phenyl), 147.02 (CNO2 phenyl), 127.89 (CH phenyl), 123.33 (CH phenyl), 69.88 (CH chloramphenicol), 66.70 (CH chloramphenicol), 63.42 (CH2 chloramphenicol), 53.91 (CH, chloramphenicol), 47.84 (CH), 42.11 (CH2), 30.07 (CH2), 29.35 (CH2), 17.63 (CH3); ESI-MS m/z calcd. for C20H24Cl2N4O10 [M]+: m/z = 550.09, found [M-H]− 549.28.

Compound 1c.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 12.63 (s, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.15 (m, 3H), 7.99 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 12.0 Hz, 2H), 6.44 (s, 1H), 6.20 (s, 1H), 5.02 (s, 1H), 4.24 (m, 3H), 4.09 (m, 1H), 3.77 (m, 2H), 3.69 (m, 1H), 3.60 (m, 1H), 2.43 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.58 (CO carboxyl), 172.23 (CO carboxyl), 171.46 (CONH amide), 169.34 (CONH amide), 164.04 (CONH chloramphenicol), 150.66 (CCH phenyl), 147.01 (CNO2 phenyl), 127.88 (CH phenyl), 123.32 (CH phenyl), 69.88 (CH chloramphenicol), 66.69 (CH chloramphenicol), 63.44 (CH2 chloramphenicol), 61.72 (CH2), 54.95 (CH), 53.92 (CH, chloramphenicol), 42.17 (CH2), 30.07 (CH2), 29.36 (CH2); ESI-MS m/z calcd. for C20H24Cl2N4O11 [M]+: m/z = 566.08, found [M-H]− 565.27.

Compound 1d.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.53 (m, 1H), 8.15 (m, 3H), 8.01 (m, 1H), 7.64 (m, 2H), 6.43 (m, 1H), 5.03 (d, J = 8.0 Hz, 1H), 4.22 (m, 6H), 3.73 (m, 2H), 3.57 (m, 2H), 2.48 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.70 (CO carboxyl), 171.49 (CO carboxyl), 171.38 (CONH amide), 170.83 (CONH amide), 164.04 (CONH chloramphenicol), 150.67 (CCH phenyl), 147.01 (CNO2 phenyl), 127.89 (CH phenyl), 123.32 (CH phenyl), 69.85 (CH chloramphenicol), 66.69 (CH chloramphenicol), 63.38 (CH2 chloramphenicol), 62.16 (CH2), 55.49 (CH), 53.90 (CH, chloramphenicol), 41.12 (CH2), 30.15 (CH2), 29.35 (CH2); ESI-MS m/z calcd. for C20H24Cl2N4O11 [M]+: m/z = 566.08, found [M-H]− 565.27.

Compound 1e.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 8.0 Hz, 1H), 8.22 (m, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.11 (m, 1H), 7.63 (d, J = 8.0 Hz, 2H), 6.44 (s, 1H), 5.02 (s, 1H), 4.21 (m, 2H), 4.09 (m, 1H), 3.72 (m, 3H), 3.63 (d, J = 4.0 Hz, 1H), 2.57 (d, J = 4.0 Hz, 2H), 2.45 (d, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.63 (CO carboxyl), 171.90 (CO carboxyl), 171.53 (CONH amide), 169.69 (CONH amide), 164.05 (CONH chloramphenicol), 150.66 (CCH phenyl), 147.02 (CNO2 phenyl), 127.88 (CH phenyl), 123.33 (CH phenyl), 69.89 (CH chloramphenicol), 66.70 (CH chloramphenicol), 63.48 (CH2 chloramphenicol), 53.93 (CH, chloramphenicol), 49.02 (CH2), 42.42 (CH2), 30.08 (CH2), 29.32 (CH2), 25.86 (CH3); ESI-MS m/z calcd. for C20H25Cl2N5O9 [M]+: m/z = 549.10, found [M-H]− 548.20.

Compound 1f.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 8.0 Hz, 1H), 8.16 (m, 6H), 7.63 (d, J = 8.0 Hz, 2H), 6.44 (s, 1H), 5.02 (s, 1H), 4.20 (m, 2H), 4.09 (m, 2H), 3.74 (dd, J = 4.0, 8.0 Hz, 8H), 2.46 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.71 (CO carboxyl), 171.82 (CO carboxyl), 171.58 (CONH amide), 169.90 (CONH amide), 169.62 (CONH amide), 164.14 (CONH chloramphenicol), 150.75 (CCH phenyl), 147.10 (CNO2 phenyl), 127.97 (CH phenyl), 123.42 (CH phenyl), 69.98 (CH chloramphenicol), 66.78 (CH chloramphenicol), 63.55 (CH2 chloramphenicol), 54.02 (CH chloramphenicol), 42.65 (CH2), 42.51 (CH2), 42.24 (CH2), 41.07 (CH2), 30.16 (CH2), 29.40 (CH2); ESI-MS m/z calcd. for C23H28Cl2N6O12 [M]+: m/z = 650.11, found [M-H]− 649.20.

Compound 1g.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 12.0 Hz, 2H), 8.13 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 6.44 (s, 1H), 5.03 (s, 1H), 4.31 (m, 1H), 4.17 (m, 3H), 4.09 (m, 1H), 2.44 (m, 4H), 1.23 (d, J = 4.0 Hz, 3H), 1.19 (d, J = 8.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 174.50 (CO carboxyl), 172.76 (CO carboxyl), 172.66 (CONH amide), 170.94 (CONH amide), 164.13 (CONH chloramphenicol), 150.79 (CCH phenyl), 147.11 (CNO2 phenyl), 128.00 (CH phenyl), 123.43 (CH phenyl), 69.91 (CH chloramphenicol), 66.79 (CH chloramphenicol), 63.38 (CH2 chloramphenicol), 53.97 (CH chloramphenicol), 48.29 (CH), 47.88 (CH), 30.12 (CH2), 29.41 (CH2), 18.72 (CH3), 17.54 (CH3); ESI-MS m/z calcd. for C21H26Cl2N4O10 [M]+: m/z = 564.10, found [M-H]− 563.35.

Compound 1h.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 12.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.12 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 6.44 (s, 1H), 5.03 (s, 1H), 4.31 (m, 1H), 4.18 (m, 3H), 4.09 (m, 1H), 2.44 (m, 4H), 1.26 (d, J = 8.0 Hz, 3H), 1.18 (d, J = 8.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 174.41 (CO carboxyl), 172.61 (CO carboxyl), 172.54 (CONH amide), 170.82 (CONH amide), 164.07 (CONH chloramphenicol), 150.65 (CCH phenyl), 147.02 (CNO2 phenyl), 127.89 (CH phenyl), 123.32 (CH phenyl), 69.93 (CH chloramphenicol), 66.70 (CH chloramphenicol), 63.47 (CH2 chloramphenicol), 53.95 (CH chloramphenicol), 48.15 (CH), 47.81 (CH), 30.05 (CH2), 29.36 (CH2), 18.65 (CH3), 17.46 (CH3); ESI-MS m/z calcd. for C21H26Cl2N4O10 [M]+: m/z = 564.10, found [M-H]− 563.06.

Compound 1i.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.12 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 4.0 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 6.43 (s, 1H), 5.02 (s, 1H), 4.21 (m, 5H), 4.09 (m, 1H), 2.45 (m, 4H), 1.22 (m, 9H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 174.33 (CO carboxyl), 172.69 (CO carboxyl), 172.28 (CONH amide), 171.14 (CONH amide), 164.02 (CONH chloramphenicol), 150.67 (CCH phenyl), 147.00 (CNO2 phenyl), 127.89 (CH phenyl), 123.33 (CH phenyl), 69.84 (CH chloramphenicol), 66.68 (CH chloramphenicol), 63.37 (CH2 chloramphenicol), 53.87 (CH chloramphenicol), 49.01 (CH3 methanol), 48.64 (CH), 48.08 (CH), 47.83 (CH), 30.01 (CH2), 29.29 (CH2), 18.44 (CH3), 17.50 (CH3); ESI-MS m/z calcd. for C24H31Cl2N5O11 [M]+: m/z = 635.14, found [M-H]− 634.30.

Compound 1j.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 12.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.11 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 4.0 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 6.43 (s, 1H), 5.02 (s, 1H), 4.22 (m, 5H), 4.08 (m, 1H), 2.44 (m, 4H), 1.22 (m, 9H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 174.45 (CO carboxyl), 172.76 (CO carboxyl), 172.40 (CONH amide), 171.26 (CONH amide), 164.18 (CONH chloramphenicol), 150.74 (CCH phenyl), 147.13 (CNO2 phenyl), 127.99 (CH phenyl), 123.43 (CH phenyl), 70.07 (CH chloramphenicol), 66.80 (CH chloramphenicol), 63.68 (CH2 chloramphenicol), 54.07 (CH chloramphenicol), 49.12 (CH3 methanol), 48.73 (CH), 48.24 (CH), 47.94 (CH), 30.15 (CH2), 29.44 (CH2), 18.56(CH3), 17.62(CH3); ESI-MS m/z calcd. for C24H31Cl2N5O11 [M]+: m/z = 635.14, found [M-H]− 634.30.

Compound 1k.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 8.0 Hz, 1H), 8.16 (m, 4H), 8.03 (dd, J = 4.0, 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.27 (m, 2H), 7.20 (m, 3H), 6.44 (s, 1H), 5.02 (s, 1H), 4.41 (m, 1H), 4.20 (m, 2H), 4.08 (m, 1H), 3.68 (m, 4H), 3.04 (m, 1H), 2.88 (m, 1H), 2.46 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.14 (CO carboxyl), 172.61 (CO carboxyl), 171.71 (CONH amide), 169.56 (CONH amide), 169.05 (CONH amide), 164.05 (CONH chloramphenicol), 150.65 (CCH phenyl), 147.01 (CNO2 phenyl), 137.85 (CCH2 phenyl), 129.52 (CH phenyl), 128.62 (CH phenyl), 127.87 (CH phenyl), 126.86 (CH phenyl), 123.32 (CH phenyl), 69.89 (CH chloramphenicol), 66.68 (CH chloramphenicol), 63.45 (CH2 chloramphenicol), 53.90 (CH chloramphenicol), 42.52 (CH2), 41.97 (CH2), 37.19 (CH2), 30.06 (CH2), 29.29 (CH2); ESI-MS m/z calcd. for C28H31Cl2N5O11 [M]+: m/z = 683.14, found [M-H]− 682.30.

Compound 1l.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 8.0 Hz, 1H), 8.16 (m, 4H), 8.03 (dd, J = 4.0, 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.27 (m, 2H), 7.20 (m, 3H), 6.43 (s, 1H), 5.02 (s, 1H), 4.41 (m, 1H), 4.20 (m, 2H), 4.08 (m, 1H), 3.67 (m, 4H), 3.04 (dd, J = 4.0, 8.0 Hz, 1H), 2.87 (dd, J = 8.0, 4.0 Hz, 1H), 2.46 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.14 (CO carboxyl), 172.61 (CO carboxyl), 171.71 (CONH amide), 169.56 (CONH amide), 169.05 (CONH amide), 164.05 (CONH chloramphenicol), 150.65 (CCH phenyl), 147.01 (CNO2 phenyl), 137.85 (CCH2 phenyl), 129.52 (CH phenyl), 128.61 (CH phenyl), 127.87 (CH phenyl), 126.86 (CH phenyl), 123.32 (CH phenyl), 69.89 (CH chloramphenicol), 66.69 (CH chloramphenicol), 63.45 (CH2 chloramphenicol), 53.90 (CH chloramphenicol), 42.52 (CH2), 41.97 (CH2), 37.19 (CH2), 30.06 (CH2), 29.29 (CH2); ESI-MS m/z calcd. for C28H31Cl2N5O11 [M]+: m/z = 683.14, found [M-H]− 682.39.

Compound 1m.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.50 (d, J = 12.0 Hz, 1H), 8.36 (s, 1H), 8.16 (m, 2H), 8.06 (m, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.20 (m, 5H), 6.44 (s, 1H), 5.01 (s, 1H), 4.53 (m, 1H), 4.21 (m, 2H), 4.08 (m, 1H), 3.73 (m, 3H), 3.56 (m, 1H), 3.03 (d, J = 12.0 Hz, 1H), 2.76 (t, J = 12.0, 1H), 2.43 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.62 (CO carboxyl), 171.80 (CO carboxyl), 171.53 (CONH amide), 171.44 (CONH amide), 169.08 (CONH amide), 164.05 (CONH chloramphenicol), 150.63 (CCH phenyl), 147.01 (CNO2 phenyl), 138.20 (CCH2 phenyl), 129.56 (CH phenyl), 128.45 (CH phenyl), 127.87 (CH phenyl), 126.68 (CH phenyl), 123.32 (CH phenyl), 69.92 (CH chloramphenicol), 66.69 (CH chloramphenicol), 63.49 (CH2 chloramphenicol), 54.13 (CH), 53.94 (CH chloramphenicol), 42.32 (CH2), 41.06 (CH2), 38.03 (CH2), 30.01 (CH2), 29.30 (CH2); ESI-MS m/z calcd. for C28H31Cl2N5O11 [M]+: m/z = 683.14, found [M-H]− 682.17.

Compound 1n.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.50 (d, J = 8.0 Hz, 1H), 8,29 (t, J = 4.0 Hz, 1H), 8.17 (m, 3H), 8.05 (dd, J = 8.0, 4.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.21 (m, 5H), 6.43 (s, 1H), 5.01 (s, 1H), 4.50 (m, 1H), 4.20 (m, 2H), 4.07 (m, 1H), 3.74 (m, 4H), 3.04 (dd, J = 4.0, 8.0 Hz, 1H), 2.76 (m, 1H), 2.35 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.53 (CO carboxyl), 171.94 (CO carboxyl), 171.48 (CONH amide), 171.29 (CONH amide), 169.46 (CONH amide), 164.07 (CONH chloramphenicol), 150.64 (CCH phenyl), 147.01 (CNO2 phenyl), 138.36 (CCH2 phenyl), 129.54 (CH phenyl), 128.43 (CH phenyl), 127.88 (CH phenyl), 126.63 (CH phenyl), 123.32 (CH phenyl), 69.93 (CH chloramphenicol), 66.69 (CH chloramphenicol), 63.51 (CH2 chloramphenicol), 54.54 (CH), 53.93 (CH chloramphenicol), 42.22 (CH2), 41.00 (CH2), 37.86 (CH2), 30.13 (CH2), 29.35 (CH2); ESI-MS m/z calcd. for C28H31Cl2N5O11 [M]+: m/z = 683.14, found [M-H]− 682.30.

Compound 1o.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 8.0 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.15 (m, 3H), 7.99 (m, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.21 (m, 11H), 6.43 (d, J = 4.0 Hz, 1H), 5.02 (s, 1H), 4.53 (m, 1H), 4.42 (m, 1H), 4.20 (m, 2H), 4.09 (m, 1H), 3.69 (m, 3H), 2.99 (m, 5H), 2.71 (m, 2H), 2.44 (m, 2H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.08 (CO carboxyl), 172.60 (CO carboxyl), 171.69 (CONH amide), 171.43 (CONH amide), 169.60 (CONH amide), 168.74 (CONH amide), 164.05 (CONH chloramphenicol), 150.64 (CCH phenyl), 147.01 (CNO2 phenyl), 138.10 (CCH2 phenyl), 137.81 (CH phenyl), 129.60 (CH phenyl), 129.52 (CH phenyl), 128.63 (CH phenyl), 128.41 (CH phenyl), 127.87 (CH phenyl), 126.87 (CH phenyl), 126.65 (CH phenyl), 123.32 (CH phenyl), 69.89 (CH chloramphenicol), 66.68 (CH chloramphenicol), 63.45 (CH2 chloramphenicol), 53.96 (CH chloramphenicol), 42.50 (CH2), 42.10 (CH2), 37.94 (CH2), 37.06 (CH2), 30.05 (CH2), 29.28 (CH2); ESI-MS m/z calcd. for C37H40Cl2N6O12 [M]+: m/z = 830.21, found [M-H]− 829.29.

Compound 1p.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (m, 1H), 8.31 (m, 1H), 8.14 (m, 3H), 7.98 (m, 2H), 7.62 (m, 2H), 7.23 (m, 10H), 6.42 (d, J = 16.0 Hz, 1H), 5.00 (d, J = 16.0 Hz, 1H), 4.46 (m, 4H), 4.14 (m, 2H), 3.68 (m, 3H), 3.55 (m, 1H), 2.97 (m, 3H), 2.68 (m, 1H), 2.47 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.08 (CO carboxyl), 172.60 (CO carboxyl), 171.68 (CONH amide), 171.42 (CONH amide), 169.59 (CONH amide), 168.73 (CONH amide), 164.04 (CONH chloramphenicol), 150.64 (CCH phenyl), 147.01 (CNO2 phenyl), 138.10 (CCH2 phenyl), 137.82 (CH phenyl), 129.60 (CH phenyl), 129.52 (CH phenyl), 128.63 (CH phenyl), 128.41 (CH phenyl), 127.87 (CH phenyl), 126.87 (CH phenyl), 126.65 (CH phenyl), 123.32 (CH phenyl), 69.88 (CH chloramphenicol), 66.68 (CH chloramphenicol), 63.44 (CH2 chloramphenicol), 53.95 (CH chloramphenicol), 42.49 (CH2), 42.09 (CH2), 37.94 (CH2), 37.05 (CH2), 30.05 (CH2), 29.28 (CH2); ESI-MS m/z calcd. for C37H40Cl2N6O12 [M]+: m/z = 830.21, found [M-H]− 829.29.

Compound 1q.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 12.54 (s, 1H), 8.51 (d, J = 12.0 Hz, 1H), 8.16 (m, 7H), 7.64 (d, J = 8.0 Hz, 2H), 6.43 (d, J = 4 Hz, 1H), 6.20 (s, 1H), 5.03 (s, 1H), 4.21 (m, 2H), 4.11 (m, 1H), 3.74 (s, 10H), 2.46 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.63 (CO carboxyl), 171.73 (CO carboxyl), 171.49 (CONH amide), 169.84 (CONH amide), 169.63 (CONH amide), 169.53 (CONH amide), 169.47 (CONH amide), 164.05 (CONH chloramphenicol), 150.66 (CCH phenyl), 147.01 (CNO2 phenyl), 127.88 (CH phenyl), 123.32 (CH phenyl), 69.89 (CH chloramphenicol), 66.69 (CH chloramphenicol), 63.47 (CH2 chloramphenicol), 53.93 (CH chloramphenicol), 42.56 (CH2), 42.43 (CH2), 42.13 (CH2), 40.97 (CH2), 30.07 (CH2), 29.30 (CH2); ESI-MS m/z calcd. for C25H31Cl2N7O13 [M]+: m/z = 707.14, found [M-H]− 706.22.

Compound 2a.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.55 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 1H), 7.59 (m, 4H), 6.45 (s, 1H), 5.03 (s, 1H), 4.32 (m, 2H), 4.20 (m, 4H), 4.09 (m, 2H), 3.08 (m, 4H), 2.44 (m, 4H), 1.64 (m, 8H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.62 (CO carboxyl), 172.01 (CO carboxyl), 171.18 (CONH amide), 164.13 (CONH amide), 164.10 (CO trifluoroacetic acid) 158.95 (NH2C(NH)2), 157.10 (CONH amide), 150.61 (CCH phenyl), 147.02 (CNO2 phenyl), 127.87 (CH phenyl), 123.33 (CH phenyl), 109.99 (CF3 trifluoroacetic acid), 69.93 (CH chloramphenicol), 66.68 (CH chloramphenicol), 63.53 (CH2 chloramphenicol), 53.93 (CH chloramphenicol), 52.28 (CH), 51.92 (CH), 49.01 (CH3 methanol), 40.93 (CH2), 40.70 (CH2), 30.02 (CH2), 29.84 (CH2), 29.30 (CH2), 28.46 (CH2), 25.56 (CH2), 25.34 (CH2); ESI-MS m/z calcd. for C27H42Cl2N10O10 [M]+: m/z = 736.25, found [M-3H]3- 733.43.

Compound 2b.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.55 (d, J = 8.0 Hz, 1H), 8.17 (t, J = 8.0 Hz, 3H), 8.05 (d, J = 8.0 Hz, 1H), 7.70 (s, 6H), 7.64 (d, J = 8.0 Hz, 2H), 6.45 (s, 1H), 6.24 (d, J = 8.0 Hz, 1H), 5.03 (s, 1H), 4.19 (m, 5H), 2.74 (m, 4H), 2.44 (m, 4H), 1.53 (m, 6H), 1.34 (m, 6H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.84 (CO carboxyl), 172.65 (CO carboxyl), 172.22 (CONH amide), 171.16 (CONH amide), 164.10 (CONH amide), 150.64 (CCH phenyl), 147.02 (CNO2 phenyl), 127.88 (CH phenyl), 123.34 (CH phenyl), 69.89 (CH chloramphenicol), 66.69 (CH chloramphenicol), 63.45 (CH2 chloramphenicol), 53.91 (CH chloramphenicol), 52.48 (CH), 52.00 (CH), 39.17 (CH2), 39.02 (CH2), 32.01 (CH2), 30.74 (CH2), 30.06 (CH2), 29.35 (CH2), 27.14 (CH2), 26.92 (CH2), 22.79 (CH2), 22.63 (CH2); ESI-MS m/z calcd. for C27H42Cl2N6O10 [M]+: m/z = 680.23, found [M-3H]3- 677.27.

Compound 2c.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 12.61 (s, 1H), 8.56 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 3H), 8.06 (d, J = 8.0 Hz, 1H), 7.73 (s, 6H), 7.63 (d, J = 12.0 Hz, 2H), 6.45 (s, 1H), 6.24 (d, J = 8.0 Hz, 1H), 5.02 (s, 1H), 4.20 (m, 5H), 2.74 (s, 4H), 2.43 (m, 4H), 1.51 (m, 6H), 1.35 (m, 6H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.84 (CO carboxyl), 172.60 (CO carboxyl), 172.21 (CONH amide), 171.15 (CONH amide), 164.13 (CONH amide), 150.62 (CCH phenyl), 147.02 (CNO2 phenyl), 127.88 (CH phenyl), 123.34 (CH phenyl), 69.98 (CH chloramphenicol), 66.70 (CH chloramphenicol), 63.61 (CH2 chloramphenicol), 53.97 (CH chloramphenicol), 52.46 (CH), 52.02 (CH), 39.12 (CH2), 39.01 (CH2), 31.99 (CH2), 30.71 (CH2), 30.07 (CH2), 29.40 (CH2), 27.11 (CH2), 26.92 (CH2), 22.79 (CH2), 22.60 (CH2); ESI-MS m/z calcd. for C27H42Cl2N6O10 [M]+: m/z = 680.23, found [M-3H]3- 677.42.

Compound 2d.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 12.71 (s, 1H), 8.52 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.14 (m, 3H), 8.05 (m, 2H), 7.62 (m, 3H), 7.57 (t, J = 4.0 Hz, 1H), 7.19 (m, 14H), 6.42 (m, 1H), 5.01 (s, 1H), 4.50 (m, 2H), 4.32 (m, 1H), 4.18 (m, 3H), 4.08 (m, 1H), 2.94 (m, 8H), 2.67 (m, 2H), 2.32 (m, 4H), 1.75 (m, 2H), 1.57 (m, 6H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.58 (CO carboxyl), 172.51 (CO carboxyl), 171.68 (CONH amide), 171.53 (CONH amide), 171.22 (CONH amide), 171.11 (CONH amide), 164.11 (CONH amide), 157.10 (NH2C(NH)2), 157.07 (NH2C(NH)2), 150.58 (CCH phenyl), 147.01 (CNO2 phenyl), 138.16 (CCH2 phenyl), 137.88 (CCH2 phenyl), 129.59 (CH phenyl), 129.48 (CH phenyl), 128.45 (CH phenyl), 128.39 (CH phenyl), 127.86 (CH phenyl), 123.32 (CH phenyl), 69.99 (CH chloramphenicol), 66.68 (CH chloramphenicol), 54.17 (CH2 chloramphenicol), 53.95 (CH chloramphenicol), 52.34 (CH), 52.00 (CH), 30.13 (CH2), 29.32 (CH2), 25.56 (CH2), 25.25 (CH2); ESI-MS m/z calcd. for C45H60Cl2N12O12 [M]+: m/z = 1030.38, found [M-3H]3- 1027.59.

Compound 2e.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.52 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 8.0 Hz, 2H), 8.06 (m, 3H), 7.70 (s, 5H), 7.62 (d, J = 8.0 Hz, 2H), 7.19 (m, 10H), 6.44 (s, 1H), 5.01 (s, 1H), 4.49 (m, 2H), 4.19 (m, 5H), 3.03 (m, 1H), 2.93 (m, 1H), 2.73 (m, 6H), 2.32 (m, 4H), 1.67 (m, 4H), 1.52 (m, 4H), 1.35 (m, 2H), 1.24 (s, 2H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.77 (CO carboxyl), 172.76 (CO carboxyl), 172.50 (CONH amide), 171.52 (CONH amide), 171.15 (CONH amide), 171.06 (CONH amide), 164.09 (CONH amide), 150.60 (CCH phenyl), 147.02 (CNO2 phenyl), 138.20 (CCH2 phenyl), 137.99 (CCH2 phenyl), 129.60 (CH phenyl), 129.49 (CH phenyl), 128.45 (CH phenyl), 127.87 (CH phenyl), 126.71 (CH phenyl), 126.60 (CH phenyl), 123.33 (CH phenyl), 69.98 (CH chloramphenicol), 66.69 (CH chloramphenicol), 63.65 (CH2 chloramphenicol), 54.18 (CH), 53.95 (CH chloramphenicol), 52.31 (CH), 52.15 (CH), 52.04 (CH), 37.66 (CH2), 31.95 (CH2), 30.79 (CH2), 30.14 (CH2), 29.34 (CH2), 27.11 (CH2), 26.94 (CH2), 26.90 (CH2), 22.77 (CH2), 22.67 (CH2), 22.48 (CH2); ESI-MS m/z calcd. for C45H60Cl2N8O12 [M]+: m/z = 974.37, found [M-3H]3- 971.37.

Compound 2f.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 12.67 (s, 1H), 8.52 (m, 1H), 8.13 (m, 4H), 7.63 (m, 5H), 6.44 (d, J = 8.0 Hz, 1H), 6.22 (t, J = 4.0 Hz, 1H), 5.03 (s, 1H), 4.15 (m, 4H), 3.73 (m, 2H), 2.75 (dd, J = 8.0, 4.0 Hz, 2H), 2.44 (m, 4H), 1.71 (m, 2H), 1.54 (m, 2H), 1.33 (m, 2H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.82 (CO carboxyl), 172.62 (CO carboxyl), 171.56 (CONH amide), 169.34 (CONH amide), 164.08 (CONH amide), 150.64 (CCH phenyl), 147.02 (CNO2 phenyl), 127.88 (CH phenyl), 123.33 (CH phenyl), 69.92 (CH chloramphenicol), 66.70 (CH chloramphenicol), 63.50 (CH2 chloramphenicol), 53.95 (CH chloramphenicol), 51.88 (CH), 42.16 (CH2), 39.06 (CH2), 30.92 (CH2), 30.07 (CH2), 29.35 (CH2), 26.90 (CH2), 22.69 (CH2); ESI-MS m/z calcd. for C23H32Cl2N5O10 [M]+: m/z = 608.15, found [M-2H]2- 606.33.

Compound 2g.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 3H), 8.06 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 6.44 (s, 1H), 6.19 (d, J = 4.0 Hz, 1H), 5.02 (s, 1H), 4.20 (m, 5H), 2.42 (m, 4H), 2.26 (t, J = 8.0 Hz, 4H), 1.92 (m, 2H), 1.75 (m, 2H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 174.43 (CO carboxyl), 174.11 (CO carboxyl), 173.50 (CO carboxyl), 172.64 (CO carboxyl), 171.81 (CONH amide), 171.16 (CONH amide), 164.05 (CONH amide), 150.63 (CCH phenyl), 147.01 (CNO2 phenyl), 127.87 (CH phenyl), 123.32 (CH phenyl), 69.89 (CH chloramphenicol), 66.69 (CH chloramphenicol), 63.45 (CH2 chloramphenicol), 53.91 (CH chloramphenicol), 51.99 (CH), 51.60 (CH), 30.46 (CH2), 30.41 (CH2), 30.04 (CH2), 29.32 (CH2), 28.02 (CH2), 26.50 (CH2); ESI-MS m/z calcd. for C25H30Cl2N4O14 [M]+: m/z = 680.11, found [M-H]− 679.23.

Compound 2h.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 12.44 (s, 3H), 8.52 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.05 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 6.44 (s, 1H), 5.04 (s, 1H), 4.60 (m, 1H), 4.50 (m, 1H), 4.22 (m, 2H), 4.10 (t, J = 8.0 Hz, 1H), 2.66 (m, 4H), 2.43 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.62 (CO carboxyl), 172.54 (CO carboxyl), 172.09 (CO carboxyl), 172.05 (CO carboxyl), 171.25 (CONH amide), 170.96 (CONH amide), 164.06 (CONH amide), 150.65 (CCH phenyl), 147.01 (CNO2 phenyl), 127.89 (CH phenyl), 123.32 (CH phenyl), 69.89 (CH chloramphenicol), 66.68 (CH chloramphenicol), 63.53 (CH2 chloramphenicol), 53.91 (CH chloramphenicol), 49.62 (CH), 48.97 (CH), 36.57 (CH2), 36.23 (CH2), 30.14 (CH2), 29.31 (CH2); ESI-MS m/z calcd. for C23H26Cl2N4O14 [M]+: m/z = 652.08, found [M-H]− 651.15.

Compound 2i.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 12.40 (s, 2H), 8.52 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0Hz, 1H), 8.16 (d, J = 8.0 Hz, 2H), 8.01 (d, J = 4.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 6.44 (s, 1H), 6.21 (s, 1H), 5.03 (s, 1H), 4.58 (dd, J = 8.0, 4.0 Hz, 1H), 4.49 (m, 1H), 4.22 (m, 2H), 4.10 (m, 1H), 2.66 (m, 2H), 2.55 (m, 2H), 2.42 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.59 (CO carboxyl), 172.11 (CO carboxyl), 172.05 (CO carboxyl), 171.25 (CONH amide), 170.90 (CONH amide), 164.08 (CONH amide), 150.62 (CCH phenyl), 147.02 (CNO2 phenyl), 127.88 (CH phenyl), 123.32 (CH phenyl), 69.97 (CH chloramphenicol), 66.69 (CH chloramphenicol), 63.62 (CH2 chloramphenicol), 53.95 (CH chloramphenicol), 49.62 (CH), 49.00 (CH), 36.56 (CH2), 30.15 (CH2), 29.35 (CH2); ESI-MS m/z calcd. for C23H26Cl2N4O14 [M]+: m/z = 652.08, found [M-H]− 651.25.

Compound 2j.

1H NMR (400 MHz, DMSO-d6 25 °C, ppm): δ 8.51 (d, J = 4.0 Hz, 1H), 8.17 (m, 4H), 7.63 (m, 2H), 6.43 (d, J = 4.0 Hz, 1H), 5.03 (s, 1H), 4.54 (m, 1H), 4.22 (m, 2H), 4.09 (m, 1H), 3.16 (d, J = 4.0 Hz, 3H), 2.65 (m, 2H), 2.44 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.63 (CO carboxyl), 172.60 (CO carboxyl), 172.05 (CO carboxyl), 171.47 (CONH amide), 169.17 (CONH amide), 164.05 (CONH amide), 150.64 (CCH phenyl), 147.02 (CNO2 phenyl), 127.88 (CH phenyl), 123.32 (CH phenyl), 69.91 (CH chloramphenicol), 66.69 (CH chloramphenicol), 63.49 (CH2 chloramphenicol), 53.93 (CH chloramphenicol), 49.01 (CH), 42.09 (CH2), 36.42 (CH2), 30.06 (CH2), 29.32 (CH2); ESI-MS m/z calcd. for C21H24Cl2N4O12 [M]+: m/z = 594.08, found [M-H]− 593.32.

Compound 3a.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.50 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.17 (m, 2H), 8.07 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.22 (m, 10H), 6.44 (s, 1H), 5.01 (s, 1H), 4.53 (m, 1H), 4.43 (m, 1H), 4.20 (m, 2H), 4.09 (m, 1H), 2.98 (m, 3H), 2.69 (t, J = 12.0 Hz, 1H), 2.33 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.12 (CO carboxyl), 172.51 (CO carboxyl), 171.66 (CONH amide), 170.88 (CONH amide), 164.05 (CONH amide), 150.63 (CCH phenyl), 147.01 (CNO2 phenyl), 138.24 (CCH2 phenyl), 137.80 (CCH2 phenyl), 129.57 (CH phenyl), 129.53 (CH phenyl), 128.60 (CH phenyl), 128.37 (CH phenyl), 127.89 (CH phenyl), 126.87 (CH phenyl), 126.59 (CH phenyl), 123.33 (CH phenyl), 69.94 (CH chloramphenicol), 66.70 (CH chloramphenicol), 63.49 (CH2 chloramphenicol), 53.91 (CH chloramphenicol), 37.92 (CH2), 37.06 (CH2), 30.14 (CH2), 29.38 (CH2); ESI-MS m/z calcd. for C33H34Cl2N4O10 [M]+: m/z = 716.17, found [M-H]− 715.39.

Compound 3b.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.49 (d, J = 8.0 Hz, 1H), 8.34 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 8.0 Hz, 2H), 8.01 (dd, J = 8.0, 12.0 Hz, 2H), 7.83 (m, 3H), 7.74 (s, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.44 (m, 3H), 7.15 (m, 9H), 6.43 (s, 1H), 6.18 (d, J = 4.0 Hz, 1H), 4.99 (s, 1H), 4.56 (m, 2H), 4.43 (m, 1H), 4.21 (m, 2H), 4.07 (m, 1H), 3.24 (m, 1H), 3.11 (m, 1H), 2.99 (m, 1H), 2.88 (m, 1H), 2.76 (m, 1H), 2.62 (m, 1H), 2.30 (m, 4H), 1.23 (s, 1H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 173.03 (CO carboxyl), 172.47 (CO carboxyl), 171.39 (CONH amide), 171.30 (CONH amide), 170.84 (CONH amide), 164.05 (CONH amide), 150.61 (CCH phenyl), 147.00 (CNO2 phenyl), 138.24 (CCH2 naphthyl), 137.97 (CCH2 phenyl), 135.43 (CCH2 phenyl), 133.39 (C naphthyl), 132.30 (C naphthyl), 129.60 (CH phenyl), 129.50 (CH phenyl), 128.39 (CH phenyl), 128.28 (CH phenyl), 128.11 (CH phenyl), 128.04 (CH phenyl), 127.90 (CH phenyl), 127.87 (CH phenyl), 127.84 (CH phenyl), 126.64 (CH naphthyl), 126.48 (CH naphthyl), 126.37 (CH naphthyl), 125.90 (CH naphthyl), 123.31 (CH phenyl), 69.96 (CH chloramphenicol), 66.70 (CH chloramphenicol), 63.53 (CH2 chloramphenicol), 54.08 (CH), 54.01 (CH), 53.91 (CH chloramphenicol), 37.93 (CH2), 37.76 (CH2), 37.31 (CH2), 30.11 (CH2), 29.35 (CH2); ESI-MS m/z calcd. for C46H45Cl2N5O11 [M]+: m/z = 913.25, found [M-H]− 912.45.

Compound 3c.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.50 (d, J = 8.0 Hz, 1H), 8.24 (m, 2H), 8.15 (m, 2H), 8.08 (m, 2H), 8.01 (d, J = 8.0 Hz, 1H), 7.82 (m, 3H), 7.75 (s, 1H), 7.62 (m, 2H), 7.44 (m, 2H), 7.15 (m, 10H), 6.43 (s, 1H), 5.00 (s, 1H), 4.68 (m, 1H), 4.48 (m, 2H), 4.19 (m, 2H), 4.07 (m, 1H), 3.75 (d, J = 4.0 Hz, 4H), 3.23 (m, 1H), 3.00 (m, 2H), 2.86 (m, 1H), 2.76 (m, 1H), 2.61 (m, 1H), 2.30 (m, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.49 (CO carboxyl), 171.49 (CO carboxyl), 171.44 (CONH amide), 171.37 (CONH amide), 170.89 (CONH amide), 169.39 (CONH amide), 164.07 (CONH amide), 150.60 (CCH phenyl), 147.01 (CNO2 phenyl), 138.23 (CCH2 naphthyl), 137.98 (CCH2 phenyl), 135.72 (CCH2 phenyl), 133.39 (C naphthyl), 132.25 (C naphthyl), 129.56 (CH phenyl), 129.51 (CH phenyl), 128.41 (CH phenyl), 128.28 (CH phenyl), 127.88 (CH phenyl), 127.82 (CH phenyl), 126.63 (CH naphthyl), 126.50 (CH naphthyl), 126.29 (CH naphthyl), 125.79 (CH naphthyl), 123.32 (CH phenyl), 69.98 (CH chloramphenicol), 66.70 (CH chloramphenicol), 63.55 (CH2 chloramphenicol), 54.36 (CH), 54.29 (CH), 53.99 (CH), 53.95 (CH chloramphenicol), 42.24 (CH2), 41.00 (CH2), 38.04 (CH2), 37.81 (CH2), 37.74 (CH2), 30.11 (CH2), 29.35 (CH2); ESI-MS m/z calcd. for C50H51Cl2N7O13 [M]+: m/z = 1027.29, found [M-H]− 1026.49.

Compound 4a.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 8.0 Hz, 1H), 8.18 (m, 3H), 7.63 (d, J = 12.0 Hz, 2H), 6.43 (m, 1H), 5.03 (s, 1H), 4.14 (m, 5H), 2.44 (m, 4H), 1.24 (m, 3H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 174.69 (CO carboxyl), 172.62 (CO carboxyl), 171.06 (CONH amide), 164.13 (CONH amide), 150.78 (CCH phenyl), 147.10 (CNO2 phenyl), 127.98 (CH phenyl), 123.40 (CH phenyl), 69.93 (CH chloramphenicol), 66.78 (CH chloramphenicol), 63.48 (CH2 chloramphenicol), 54.00 (CH chloramphenicol), 48.00 (CH), 30.00 (CH2), 29.37 (CH2), 17.72 (CH3); ESI-MS m/z calcd. for C18H21Cl2N3O9 [M]+: m/z = 493.07, found [M-H]− 492.29.

Compound 4b.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.52 (d, J = 4.0 Hz, 1H), 8.17 (m, 2H), 7.62 (m, 2H), 6.43 (d, J = 4.0 Hz, 1H), 6.22 (d, J = 4.0 Hz, 1H), 5.01 (s, 1H), 4.24 (m, 2H), 4.11 (m, 1H), 3.59 (d, J = 4.0 Hz, 3H), 2.55 (s, 4H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.87 (CO carboxyl), 172.19 (CO carboxyl), 164.17 (CONH amide), 150.65 (CCH phenyl), 147.12 (CNO2 phenyl), 127.94 (CH phenyl), 123.43 (CH phenyl), 70.07 (CH chloramphenicol), 66.78 (CH chloramphenicol), 63.90 (CH2 chloramphenicol), 54.03 (CH chloramphenicol), 52.01 (CH3), 29.09 (CH2), 28.84 (CH2); ESI-MS m/z calcd. for C16H18Cl2N2O8 [M]+: m/z = 436.04, found [M-H]− 435.15.

Compound 4c.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (d, J = 12.0 Hz, 1H), 8.16 (m, 2H), 7.77 (s, 1H), 7.64 (m, 2H), 6.44 (d, J = 4.0 Hz, 1H), 5.03 (s, 1H), 4.14 (m, 5H), 3.29 (m, 2H), 2.55 (m, 2H), 2.33 (m, 2H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.54 (CO carboxyl), 170.68 (CONH amide), 164.03 (CONH amide), 150.70 (CCH phenyl), 147.02 (CNO2 phenyl), 127.92 (CH phenyl), 123.33 (CH phenyl), 69.85 (CH chloramphenicol), 66.71 (CH chloramphenicol), 63.33 (CH2 chloramphenicol), 53.97 (CH chloramphenicol), 50.93 (CH2), 36.04 (CH2), 30.36 (CH2), 29.45 (CH2); ESI-MS m/z calcd. for C17H21Cl2N3O10S [M]+: m/z = 529.03, found [M-H]− 528.25.

Compound 4d.

1H NMR (400 MHz, DMSO-d6, 25 °C, ppm): δ 8.51 (m, 1H), 8.16 (m, 2H), 8.03 (m, 1H), 7.63 (m, 2H), 6.43 (m, 1H), 5.02 (d, J = 8.0 Hz, 1H), 4.21 (s, 2H), 4.10 (m, 1H), 3.79 (m, 2H), 3.25 (m, 2H), 2.47 (m, 2H), 2.37 (m, 2H); 13C NMR (100 MHz, DMSO-d6, 25 °C, ppm): δ 172.59 (CO carboxyl), 171.40 (CONH amide), 164.05 (CONH amide), 150.66 (CCH phenyl), 147.02 (CNO2 phenyl), 127.87 (CH phenyl), 123.32 (CH phenyl), 69.89 (CH chloramphenicol), 66.69 (CH chloramphenicol), 64.18 (CH2), 64.13 (CH2 chloramphenicol), 63.45 (CH chloramphenicol), 53.93 (CH2), 30.10 (CH2), 29.32 (CH2); ESI-MS m/z calcd. for C17H22Cl2N3O11P [M]+: m/z = 545.04, found [M-H]− 544.24.

Supplementary Material

Supplemental information

ACKNOWLEDGMENT

This work is partially supported by NIH (R21AI130560) and NSF (DMR-1420382).

ABBREVIATIONS USED

E. coli

Escherichia coli

MDR

multidrug resistance

LPS

lipopolysaccharide

OM

outer membrane

IM

inner membrane

MIC

minimum inhibitory concentration

CL

chloramphenicol

CLsu

chloramphenicol succinate

PLE

porcine liver esterase

HBTU

O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate

DIPEA

N,N-diisopropylethylamine

Footnotes

Supporting Information.

This material is available free of charge via the Internet at http://pubs.acs.org.

1H NMR spectra, 13C NMR spectra, LC-MS traces and mass spectrometry of all compounds, and experimental details

Molecular formula strings and some data

The authors declare no competing financial interest.

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