Abstract
Periodontal diseases are inflammatory diseases triggered by pathogenic oral bacterial species, such as Porphyromonas gingivalis. Zafirlukast (ZAF) has displayed antibacterial activity against P. gingivalis. Herein, we report the design, synthesis, and selective antibacterial activity of 14 novel 3rd generation ZAF derivatives. Two 2nd generation ZAF derivatives were tested as they were not previously tested against P. gingivalis ATCC 33277. Most 3rd generation derivatives displayed superior/selective antibacterial activity against P. gingivalis compared to ZAF and its 2nd generation derivatives. Compounds displayed bactericidal activity against P. gingivalis, inhibited biofilm growth, displayed no hemolytic activity, and displayed less cytotoxicity against mammalian cells than ZAF. The superior/selective antibacterial activity of ZAF derivatives against P. gingivalis, increased safety profile, and inhibition of biofilm growth compared to ZAF indicate that compounds, especially 21a, 21b, and 24g, show promise as antibacterial agents for future studies to test their potential for preventing and treating P. gingivalis-induced periodontal diseases.
Keywords: Biofilm, Gum inflammation, Oral dysbiosis, Periodontitis, Toxicity
Graphical Abstract
INTRODUCTION
In 2018, the economic burden due to periodontal diseases caused an estimated loss of $154 B in the United States and €149 B in Europe due to direct (e.g., overall expenses of periodontal care) and indirect (e.g., loss of productivity) costs.1, 2 Periodontal diseases are chronic inflammatory diseases of the gum and bone that support the tooth.3–5 The preventable but more severe and irreversible form of periodontal disease, periodontitis, is associated with dysbiotic dental plaque, known as oral biofilms, that are the major cause of the destruction of periodontal tissue and alveolar bone that support the tooth. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis is a major keystone anaerobic pathogen found below the gum line in plaque.6 P. gingivalis is associated with the initiation and progression of periodontitis, causes tissue damage, increases the pathogenicity of the biofilm community, and is able to invade the weakened epithelial cell layers leading to the development of systemic diseases such as rheumatoid arthritis, Alzheimer’s disease, diabetes, and cancer.7–13 Therefore, P. gingivalis is an ideal target for antibacterial agents for the treatment and prevention of periodontal diseases. Current treatment options for periodontal diseases include scaling and root planning, and surgery to remove the infected tissue with antibiotic adjunctive therapy using doxycycline or a combination of amoxicillin and metronidazole.14 Lack of specificity of the broad-spectrum antibiotics and lack of clear guidelines for choosing an antibiotic that specifically targets pathogenic oral bacterial species such as P. gingivalis can lead to the development of antibiotic resistance by some bacterial species.15 Therefore, it is of interest to develop highly specific antibacterial agents that target pathogenic bacteria without harming commensal bacteria that are critical for oral health and homeostasis.
For the discovery of novel antibacterial agents, an appealing option is to use drug repositioning as a starting point to develop medications in a fast and an affordable way. Drug repositioning is where a medication that is FDA-approved for the treatment of one disease is used to treat a different disease.16 Previously, we discovered that derivatives of the FDA-approved anti-asthma oral medication zafirlukast (ZAF) led to compounds that are highly specific for P. gingivalis only, while displaying less toxicity to oral epithelial cells, which are the first line of defense against invading pathogens.17–20 In the current study, the two most active 2nd generation ZAF derivatives18 were further tested for their anti-P. gingivalis biofilm activity and were used as starting points for further structure optimization on the ZAF indole ring (e.g., alkyl groups and bulky aryl groups) and arylfulfonamide ring (e.g., halogens) with the goal of increasing inhibition of P. gingivalis growth, retaining specificity, and having the ability to inhibit P. gingivalis biofilm growth (Figure 1). Herein, the short 4-step linear synthesis of 14 3rd generation ZAF derivatives is reported. Additionally, we present their antibacterial activity against P. gingivalis ATCC 33277 and other oral bacterial species, their cytotoxic activity against human oral epithelial cells and mammalian cells, as well as their inhibition of biofilm formation and disruption of pre-formed P. gingivalis biofilm growth.
Figure 1.
Chemical structure of zafirlukast (ZAF) and overview of the R1 and R2 substituents present in the two previously published 2nd generation as well as 14 novel 3rd generation ZAF derivatives synthesized herein.
RESULTS AND DISCUSSION
Design and synthesis of 3rd generation ZAF derivatives
In our previous structure-activity relationship (SAR) studies with 1st and 2nd generation ZAF derivatives, the primary focus was the modification of the substituents’ positions on the ZAF benzoyl ring along with bioisosteric replacement of substituents on the indole and arylsulfonamide ring.17, 18 Through these studies we discovered that: (i) modification of the 3-methoxy-4-indoyl organization of ZAF to a 2-methoxy-5-indoyl arrangement increased activity of the ZAF derivatives; (ii) the presence of the cyclopentyl carbamate group at the C5 position of the indole ring of ZAF is not needed for activity, but its complete removal (i.e., having a hydrogen at that position) decreased activity, while its replacement by an electron-withdrawing group (e.g., NO2) at C5 increased activity of the derivatives when compared to the parent ZAF; (iii) removal of the methyl group on the nitrogen of the indole of ZAF decreased activity of the derivatives, and (iv) modification of the substitution pattern on the arylsulfonamide ring of ZAF by removal of the methyl group at C2 decreased activity of the ZAF derivatives, while addition of electron-withdrawing groups (e.g., F at C2 and NO2 at C4) increased antibacterial activity when compared to ZAF. Based on these observations, for the design of the 3rd generation ZAF derivatives, we used two 2nd generation ZAF derivatives (21a and 21g previously 14a and 14e; Scheme 1)18 as starting points. We kept the C5 NO2 group of the indole and positions of the substituents (i.e., substituents at positions C1, C2, and C5) on the benzoyl ring constant. Herein, we synthesized 14 novel 3rd generation ZAF derivatives (21b-f, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g) to (i) examine the effect of various electron-withdrawing groups on the arylsulfonamide ring while keeping the N-methyl group on the indole ring constant, and (ii) investigate the importance of the methyl group on the nitrogen of the indole of ZAF by its replacement with longer alkyl chains or bulky aryl groups.
Scheme 1.
Synthetic scheme for the preparation of 2nd generation ZAF derivatives 21a and 21g and 3rd generation ZAF derivatives 21b-21f, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g. Reaction conditions: (a) Et3SiH, TFA, CH2Cl2, 0 °C to room temperature, 8–63%; (b) MeOH:THF:H2O/5:1:1, KOH, 65 °C, 31–96%; (c) arylsulfonamide, EDC•HCl, DMAP, CH2Cl2, room temperature, 12%-quantitative yields.
We used a linear 4-step synthesis for the preparation of these 14 novel 3rd generation ZAF derivatives (Scheme 1). The commercially available 5-nitroindole was alkylated to form compounds 1-6 (1-methyl-5-nitroindole (1), 1-ethyl-5-nitroindole (2), 1-propyl-5-nitroindole (3), 1-n-butyl-5-nitroindole (4), 1-isobutyl-5-nitroindole (5), and 1-benzyl-5-nitroindole (6)) in 28–72% yields. A condensation reaction “a” between the synthesized 1-alkyl-5-nitroindoles 1-6 with the commercially available (indicated by a $ sign in Scheme 1) methyl-5-formyl-2-methoxybenzoate (7) generated compounds 8-13 in 8–63% yields. The condensation reaction used triethylsilane (Et3SiH), which is a source of hydride, trifluoroacetic acid (TFA), and dichloromethane (CH2Cl2). Hydrolysis of the methyl ester group of 8-13 to a carboxylic acid “b” using methanol (MeOH), tetrahydrofuran (THF), and water in potassium hydroxide (KOH), then afforded compounds 14-19 in 31–96% yields. Finally, amide coupling “c” of the carboxylic acids 14-19 with various commercially available arylsulfonamides (20a-20g) using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC•HCl), 4-dimethylaminopyridine (DMAP), and CH2Cl2 yielded 14 novel 3rd generation ZAF derivatives 21b-f, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g in 12%-quantitative yields. Additionally, two of the lead 2nd generation ZAF derivatives 21a and 21g (previously 14a and 14e),18 were also re-synthesized in quantitative and 82% yields, respectively (Scheme 1), for novel testing against a different strain of P. gingivalis and testing against mono-species biofilms along with the new 3rd generation ZAF derivatives 21b-f, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g synthesized herein.
Anti-P. gingivalis activity and structure-activity relationship (SAR)
With the aim of increasing P. gingivalis ATCC 33277 growth inhibition in planktonic and mono-species biofilms with these novel 3rd generation ZAF derivatives, we set to answer four questions in terms of SAR (Note: The compound number for the ZAF derivatives used to answer the questions are provided into parentheses following the questions). (i) What is the effect of varying the R2 electron-withdrawing group(s) on the arylsulfonamide ring when R1 = Me? (comparing R2 = 2-F (21a), 4-NO2 (21g), 2-Cl (21b), 2-Br (21c), 2,6-diF (21d), 2,4-diF (21e), 3,4-diF (21f)). (ii) Are compounds with R2 = 4-NO2 always more active than those with R2 = 2-F when the R1 substituent is varied? (comparing g (R2 = 4-NO2) vs a (R2 = 2-F) counterparts). (iii) What is the effect of adding a longer linear or larger branched R1 group or a bulkier benzyl group to the nitrogen of the indole ring when R2 = 4-NO2? (comparing R1 = methyl (21g), ethyl (22g), propyl (23g), n-butyl (24g), isobutyl (25g), and benzyl (26g)). (iv) Does changing the substitution pattern on the arylsulfonamide from R2 = 4-NO2 to R2 = 2-F affect the activity trend established in question iii for the various R1 substituents when R2 = 4-NO2? (comparing R1 = methyl (21a), ethyl (22a), propyl (23a), n-butyl (24a), isobutyl (25a) to their counterparts 21g-25g).
To answer the four questions posed and to determine the potential of the 3rd generation ZAF derivatives 21b-f, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g as antibacterial agents compared to the parent ZAF, we first tested them against P. gingivalis ATCC 33277 (Figures 2 and S87 as bar graph representations of the data and Figure S88 as heat map representation of the data). We also tested the two most active 2nd generation ZAF derivatives (21a and 21g) against P. gingivalis ATCC 33277 (Figures 2, S87, and S88), as this strain is more virulent than P. gingivalis 381 that we used in our previous study. To determine the antimicrobial effect of the 16 synthesized ZAF derivatives (including the two 2nd generation compounds), we used a colorimetric water-soluble tetrazolium-1 (WST-1) assay to test the compounds at 1, 10, and 100 μM. Positive controls for this assay were the commercially available tetracycline (Tet, at 2.81 μM, which displayed 33% P. gingivalis growth inhibition) and ZAF (at 25 and 50 μM, which displayed 74% and 88% P. gingivalis growth inhibition, respectively; pale blue, grey, and orange bars, respectively in Figures 2 and S87). We opted to test ZAF at 25 and 50 μM, based on a previous publication using P. gingivalis ATCC 33277, which showed that these concentrations were the minimum concentration that inhibited bacterial growth on titanium discs.19 Additionally, we used Tet as a positive control as it is a common antibiotic used for the treatment of periodontitis and it has been shown to be active against many P. gingivalis species within the range of 2.81 μM.21 It is also important to note that the 2nd and 3rd generation ZAF derivatives were tested in preliminary percent growth inhibition assays with a wide range of concentrations in order to determine their initial activity to be used in future studies and further investigation for lead optimization outside the scope of this preliminary investigation. As these compounds were not previously tested against this specific strain of P. gingivalis, in order to test the ability of the ZAF derivatives to inhibit P. gingivalis growth a wide range, 1–100 μM, of concentrations was used to determine their level of potency against planktonic oral bacterial species. As expected, we did not observe bacterial growth inhibition with dimethyl sulfoxide (DMSO), which we used as a negative control along with bacteria grown in medium only.
Figure 2.
Percent growth inhibition effect of 2nd generation ZAF derivatives 21a and 21g and 3rd generation ZAF derivatives 21b-21f, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g on P. gingivalis ATCC 33277 determined by a colorimetric WST-1 assay. In supplemented brain heart infusion (BHI), planktonic P. gingivalis (108 cells/well) was exponentially grown and incubated under anaerobic conditions and was incubated with different treatments of ZAF derivatives 21a-21g, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g (1, 10, and 100 μM) for 24 h. The cultures were treated with medium only (sterility control) and the experimental cultures were treated with DMSO (vehicle for ZAF and its derivatives, negative control) and 2.81 μM of tetracycline (Tet) or 25 and 50 μM of ZAF. The data represent the average from 42 independent replicates for the controls. For each ZAF derivative, the data represent the average from six independent replicates per condition of the growth inhibitory effect vs bacterial cultures grown in medium only. These data represent the percent growth inhibition 2.5 h after the addition of the WST-1 reagent. Note: The data for 30 min after the addition of the WST-1 reagent are presented in Figure S87. Additionally, the data 2.5 h after the addition of the WST-1 reagent is presented in a visual form as a heat map in Figure S88.
When looking broadly at the data collected 2.5 h after the addition of the WST-1 reagent (Figure 2), we observed that when tested against P. gingivalis ATCC 33277 compared to P. gingivalis 381, the 2nd generation ZAF derivative 21g displayed more activity than 21a against this more virulent strain. When comparing the 16 synthesized ZAF derivatives to the Tet control, we found that, at 100, 10, and 1 μM, nine (21a, 21g, 21b, 21c, 21d, 22g, 23g, 24g, and 25g), four (21g, 22g, 23g, and 24g), and two (23g and 24g) out of the 16 compounds displayed comparable or superior activity when compared to Tet (33% growth inhibition), respectively. When comparing these ZAF derivatives to the parent ZAF, we observed that, at 10 μM, three out of the 16 compounds (22g, 23g, and 24g) displayed comparable/superior activity (77–90% growth inhibition) to ZAF (74% growth inhibition at 25 μM and 88% at 50 μM). At 1 μM, compounds 23g and 24g also displayed comparable/superior activity (60% and 69% growth inhibition, respectively) when compared to ZAF. It is important to note that an increased antibacterial activity of compounds 21a, 21b, 21c, 21d, and 25g was observed 30 min after the addition of WST-1 (Figure S87) at 100 μM with activity between 61–73% growth inhibition. Therefore, these compounds were additionally selected for further biological tests along with the active compounds of 21g, 22g, 23g, and 24g.
Next, by taking a deeper look into the data, we aimed to answer the four questions posed above. Question i: What is the effect of varying the R2 electron-withdrawing group(s) on the arylsulfonamide ring when R1 = Me? To answer this question, we compared compounds 21a-g at 100 μM concentration and 2.5 h after the addition of the WST-1 reagent. Overall, it was found that compound 21g (R2 = 4-NO2) displayed better P. gingivalis growth inhibition than (>) 21a (R2 = 2-F), which was similar to (≈) 21b (R2 = 2-Cl) (≈) 21d (R2 = 2,6-diF), which was better than (>) 21c (R2 = 2-Br) (>) 21f (R2 = 3,4-diF) and (>) 21e (R2 = 2,4-diF).
Having confirmed the importance of the R2 2-F and 4-NO2 groups, the next set of derivatives prepared kept these two R2 groups constant while varying R1. To verify if ZAF derivatives with R2 = 4-NO2 are always more active than those with R2 = 2-F, we performed a pairwise comparison of 21a-25a vs 21g-25g (question ii). We found that at 10 and 100 μM, R2 = 4-NO2 always yielded more active compounds than R2 = 2-F. At 1 μM, this was also true for compounds 23g and 24g that displayed significant activity, while their 2-fluorinated counterparts 23a and 24a displayed little to no activity at that concentration.
To investigate the importance of the R1 group attached to the nitrogen of the indole when R2 = 4-NO2 (question iii), we compared compounds 22g with R1 = ethyl, 23g with R1 = propyl, 24g with R1 = n-butyl, 25g with R1 = isobutyl, and 26g with R1 = benzyl to compound 21g with R1 = methyl. We observed that replacing the R1 methyl group of 21g by a bulky aromatic benzyl moiety (26g) resulted in an almost complete loss of activity. Thus, the benzylated counterpart 26a with R2 = 2-F was not synthesized to answer question iv. In general, we discovered that substitution of the R1 methyl group on the nitrogen of the indole of 21g with longer/larger alkyl chains (22g-24g) yielded a significant increase in activity when tested at 1, 10, and 100 μM. A decrease in activity was seen with compound 25g (R1 = isobutyl), which indicated the limit of the size of the group that can be attached to the nitrogen of the indole ring. More specifically, compound 22g (R1 = ethyl) was found to display 6, 77, and 81% P. gingivalis growth inhibition at 1, 10, and 100 μM, respectively, which were superior to that of 21g (0, 63, and 56%) at all concentrations tested. As the increase in chain length proved to be beneficial for P. gingivalis growth inhibition, the effect of longer chain lengths was also investigated. At all concentrations tested, compound 23g (R1= propyl) yielded 60–90% growth inhibition, which was similar to the activity observed with 24g (R1 = n-butyl) (with 69–90% growth inhibition). Overall, at 100 μM, it was found that compound 23g (R1 = propyl) displayed better P. gingivalis growth inhibition than (>) 22g (R1 = ethyl) (>) 24g (R1 = n-butyl) (>) 21g (R1 = methyl) (>) 25g (R1 = isobutyl) and (>) 26g (R1 = benzyl).
With the trend established for the substitution patterns of the arylsulfonamides with R2 = 4-NO2 (series g) and various alkyl chains for R1 (question iii), the effect of the arylsulfonamide with R2= 2-F (series a) with these same N-alkyl groups on the indole ring was investigated by generating the counterpart series 22a-25a (question iv). When comparing the N-alkylated compounds 21a-25a with R2 = 2-F within that specific series a, it was found that compound 21a (R1 = methyl) displayed better P. gingivalis growth inhibition than (>) 23a (R1 = propyl) (>) 22a (R1 = ethyl), which was similar to (≈) 24a (R1 = n-butyl) (≈) 25a (R1 = isobutyl). Interestingly, when compared to the trend observed for compounds 21g-25g with R2 = 4-NO2, the trend remained the same with the exception of the methyl groups. The overall SAR for all compounds evaluated summarized based on their activity at 100 μM is presented in Figure 3. Although 100 μM is a high therapeutic concentration, all comparisons were made at the 100 μM concentration (even though some compounds displayed excellent activity at 1 and 10 μM) due to most compounds not being active at the lower concentrations. In the preliminary study, we wanted to make comparisons of each group modified in the structure in order to get a clear picture of which groups were better or worse for inhibiting planktonic P. gingivalis growth. Additionally, there are many ways to test the activity of the ZAF derivatives against oral bacterial species such as minimum inhibitory concentration (MIC) or IC50 assays. In the literature it is common to see both MIC testing and percent growth inhibition assays performed for testing with oral bacterial species.22–26 As this was an initial screening of activity for percent growth inhibition to determine if the compounds were active against P. gingivalis a wide range of concentrations were chosen to determine if the compounds displayed activity, and future studies, outside the scope of this investigation, will be performed to determine the exact dose response of the most active compounds against P. gingivalis. To perform other assays, such as determination of IC50 values, the compounds would have to be tested at a much larger range of 2-fold dilutions, which is impractical while we are currently optimizing the structure of the ZAF derivatives based on the data observed in the additional assays performed in this study. For the most active compounds a much higher range of concentrations would need to be used that are over 200 μM. Whereas for the least active compounds a range of concentrations much less than 1 μM would need to be used in order to determine exact IC50 values for these compounds. For these reasons, we opted to analyze the activity of the compounds in the initial screening by the percent growth inhibition assays.
Figure 3.
Summary of the SAR for the 2nd (21a and 21g) and 3rd generation (21b-21f, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g) ZAF derivatives when tested against P. gingivalis ATCC 33277 at 100 μM.
Antibacterial activity against non-P. gingivalis oral bacterial species
Based on the results of the percent growth inhibition assay presented in Figures 2 and S87, the seven most active 3rd generation ZAF derivatives were established to be 21b, 21c, 21d, 22g, 23g, 24g, and 25g. These compounds, along with two 2nd generation ZAF derivatives (21a and 21g), were then further tested against other Gram-positive and Gram-negative oral bacterial species to determine their antibacterial specificity profile (Figure 4 as a bar graph representation of the data and Figure S89 as a visual heat map representation of the data). The oral bacterial species chosen were a variety of early and late colonizers found in oral biofilm
Figure 4.
Percent growth inhibition effect of ZAF derivatives 21a, 21g, 21b, 21c, 21d, 22g, 23g, 24g, and 25g on A. naeslundii ATCC 49340 (anaerobic), A. actinomycetemcomitans JP2 (anaerobic), F. nucleatum ATCC 25586 (anaerobic), S. sanguinis ATCC 10556 (aerobic), and V. parvula ATCC 10790 (anaerobic) determined by a colorimetric WST-1 assay. Grown supplemented BHI for P. gingivalis, A. naeslundii, A. actinomycetemcomitans, F. nucleatum, and S. sanguinis, or Reinforced Clostridial medium for V. parvula (108 cells/well) was exponentially grown and incubated under aerobic or anaerobic conditions and was treated with ZAF derivatives 21a, 21g, 21b, 21c, 21d, 22g, 23g, 24g, and 25g (1, 10, and 100 μM) for 24 h. The cultures were treated with medium only (sterility control) and the experimental cultures were treated with DMSO (vehicle for ZAF and its derivatives, negative control), 2.81 μM of Tet (for F. nucleatum), the antibiotics penicillin/streptomycin (P/S) 1X (100 U/mL of penicillin and 100 μg/mL of streptomycin for all other oral bacterial species), and 25 and 50 μM of ZAF. The data represent the average from 30 independent replicates for the controls. For each ZAF derivative, the data represent the average from six independent replicates per condition of the growth inhibitory effect vs bacterial cultures grown in medium only. These data represent the percent growth inhibition 30 min (for F. nucleatum as this was the time when the controls reached the standard percent growth inhibition values consistent with the literature) or 2.5 h for the other oral bacterial species after the addition of the WST-1 reagent. Note: A visual representation of the data in the form of a heat map is presented in Figure S89.
s, also known as dental plaque. The early colonizers abundant in periodontal health include Streptococcus sanguinis ATCC 10556 (Gram-positive aerobic), Actinomyces naeslundii ATCC 49340 (Gram-positive anaerobic), and Veillonella parvula ATCC 10790 (Gram-negative anaerobic), while the late colonizers abundant in periodontal disease include Aggregatibacter actinomycetemcomitans JP2 (Gram-negative anaerobic) and Fusobacterium nucleatum ATCC 25586 (Gram-negative anaerobic). In Figures 4 and S89, the percent growth inhibition data for F. nucleatum are presented 30 min after the addition of the WST-1 reagent because this was the time when the standard positive controls of Tet and ZAF reached the percent growth inhibition values consistent with the literature and consistent within the experiments performed in the laboratory. All other oral bacterial species data are presented 2.5 h after the addition of the WST-1 reagent for the same reason. The compounds displayed little to no activity against V. parvula, A. naeslundii, S. sanguinis, and A. actinomycetemcomitans, with the exception of about 40% growth inhibition at 100 μM by 21d against A. naeslundii and S. sanguinis. Compound 21d also displayed 76% growth inhibition against F. nucleatum at 100 μM, but there was no activity seen at 1 or 10 μM.
This bacterium is considered a bridge colonizer, where it can attach to both early colonizing bacterial species abundant in periodontal health and late-stage colonizing pathogenic bacterial species. Therefore, if some of the compounds display activity against F. nucleatum, that will only aid in the ability of these ZAF derivatives to act as a treatment for periodontal diseases. Overall, these data suggest that the 3rd generation ZAF derivatives display specificity against P. gingivalis.
Colony forming unit (CFU) assays
To determine if the nine most active ZAF derivatives (21a, 21g, 21b, 21c, 21d, 22g, 23g, 24g, and 25g) displayed bactericidal activity, colony forming unit (CFU) assays were performed with P. gingivalis ATCC 33277 (Figure 5). P. gingivalis was exposed to all controls and treatments for 24 h in medium followed by bacterial growth in blood agar plates for 5 days under anaerobic conditions and CFUs determination. For positive controls, P. gingivalis was incubated with the antibiotic Tet (2.81 μM) and ZAF (25 and 50 μM). For a negative control bacteria grown with only DMSO were used. P. gingivalis was exposed to ZAF derivatives 21a, 21g, 21b, 21c, 21d, 22g, 23g, 24g, and 25g at 10 and 100 μM. At 100 μM, all nine compounds exhibited bactericidal activity with 100% growth inhibition. At 10 μM, compounds 21g, 21b, and 21c were also bactericidal, compound 21a displayed 96% growth inhibition, and 24g displayed 71% growth inhibition. Compounds 21d, 22g, 23g, and 25g were not bactericidal at 10 μM. At both concentrations tested, the 2nd generation compounds 21a and 21g, along with 3rd generation compounds 21b, 21c, and 24g displayed similar or higher percent growth inhibition than the controls Tet and ZAF (25 and 50 μM). At 100 μM, compound 22g displayed higher percent growth inhibition by CFU assays than the controls and displayed 19% growth inhibition at 10 μM. Based on these results, we decided to continue with further investigation of 21a, 21b, 21c, 21g, 22g, and 24g.
Figure 5.
Bactericidal effect of ZAF derivatives 21a-21d, 21g, 22g, 23g, 24g, and 25g on P. gingivalis ATCC 33277 determined by colony forming unit (CFU) assays. In supplemented BHI, P. gingivalis (108 cells/well) was exponentially grown and incubated under anaerobic conditions and was treated with ZAF derivatives 21a-21d, 21g, 22g, 23g, 24g, and 25g (1 (data not shown), 10, and 100 μM) for 24 h. The bacteria exposed to treatment were diluted (1:400) and spread onto blood agar plates. After 5 days of incubation under anaerobic conditions CFUs were counted. Negative controls were bacteria exposed to only medium or DMSO. Bacteria exposed to antibiotic Tet (2.81 μM) or ZAF (25 and 50 μM) were used as positive controls. For each control and ZAF derivative, the data represent the average from three independent replicates per condition. The data shows the calculated percentage of inhibitory effect by comparison of the treated bacteria groups (CFUs/mL) vs bacteria grown in medium only (CFUs/mL) using the formula 100 × [CFUs/mL of (control) - CFUs/mL (experimental) / CFUs/mL (control)].
Disruption of pre-formed biofilms and prevention of P. gingivalis biofilm formation by ZAF derivatives
Two of the most active 2nd generation ZAF derivatives (21a and 21g) and four of the most active 3rd generation ZAF derivatives (21b, 21c, 22g, and 24g) were first tested to determine their ability to disrupt mono-species biofilms of P. gingivalis ATCC 33277 (Figure 6). P. gingivalis was grown in medium for 48 h with gentle shaking under appropriate anaerobic conditions to form the biofilm. Then the bacteria were exposed to all controls and treatments for 24 h in medium followed by staining with safranin to determine the percentage of biofilm growth. For positive controls, P. gingivalis was incubated with the antibiotic Tet (2.81 μM) and ZAF (50 μM). Bacteria grown with only DMSO were used as a negative control. P. gingivalis was exposed to ZAF derivatives 21a-21c, 21g, 22g, and 24g at 50 and 100 μM. All compounds were compared to the DMSO control to calculate the percentage of biofilm growth where 100% biofilm growth was observed. Unfortunately, at 100 μM, none of the six compounds tested appear to be able to disrupt preformed P. gingivalis biofilms.
Figure 6.
Effect of ZAF derivatives 21a-c, 21g, 22g, and 24g on their ability to disrupt pre-formed biofilms of P. gingivalis ATCC 33277 determined by a safranin staining assay of mono-species P. gingivalis cultures. In supplemented BHI, P. gingivalis (108 cells/well) was exponentially grown and incubated under anaerobic conditions for 48 h with gentle shaking in 96-well plates (100 μL). After biofilm formation, the old medium was removed, cells were washed with phosphate-buffered saline (PBS), and 100 μL of BHI was added to each well. The bacteria were treated with 100 μL of ZAF derivatives 21a-c, 21g, 22g, and 24g (10 and 100 μM) for 24 h. The negative control was bacteria grown in the presence of DMSO. Bacteria exposed to Tet (2.81 μM) or ZAF (50 μM) were used as positive controls. The cells were stained with 0.1% safranin (100 μL) and absorbance was measured at 492 nm. The data show the calculated percentage of biofilm growth by comparison of the treated bacteria groups vs bacteria grown in DMSO only using the formula 100 × [OD (experimental) / OD (DMSO control)].
We next investigated the ability of these six selected ZAF derivatives to inhibit mono-species biofilm growth of P. gingivalis ATCC 33277 (Figure 7). Although these compounds cannot disrupt pre-formed biofilms, they can however be tested for their ability to prevent oral biofilm from forming, which is known as dental plaque that is home to many oral bacterial species.27–31 As these compounds show specificity with bactericidal activity against planktonic P. gingivalis, this would mean that it would kill P. gingivalis before it can even form a biofilm and it would help slow down biofilm formation. This could have many downstream effects for the pathogenicity of the biofilm and could help inhibit the growth of other pathogenic oral bacterial species for which the growth is enhanced by P. gingivalis.6, 7, 30, 32, 33 P. gingivalis was grown in medium and exposed to different treatments for 48 h with gentle shaking under anaerobic conditions, followed by staining with safranin to determine the percentage of biofilm growth. P. gingivalis was incubated with Tet (2.81 μM) and ZAF (50 μM) as positive controls. Bacteria grown with DMSO only were utilized as a negative control. P. gingivalis was exposed to ZAF derivatives 21a-21c, 21g, 22g, and 24g at 10, 25, 50, and 100 μM. All compounds were compared to the DMSO control to calculate the percentage of biofilm growth where 100% biofilm growth was observed. All compounds tested displayed the ability to inhibit growth of P. gingivalis biofilm with at least two of the concentrations tested. Overall, compound 21g displayed the least activity at inhibiting biofilm growth with at least 86% of biofilm growth at all concentrations tested. At 100 μM, compound 24g displayed the same activity of 57% biofilm growth as the controls Tet and ZAF with 57% and 58%, respectively. At 50 μM, compounds 21a, 21b, and 24g displayed similar activity to the controls with 61%, 58%, and 63% biofilm growth. Compounds 21a (52%) and 21b (55%) displayed increased activity compared to the controls at 25 μM. Additionally, the most active compound 21b displayed better activity than the parent drug ZAF with 53% biofilm growth at concentrations as low as 10 μM. With the ability to inhibit P. gingivalis biofilm growth, it is next important to determine if these active compounds have any toxic effect to oral epithelial cells and mammalian cells as activity is not the only determining factor when developing an antibacterial agent.
Figure 7.
Effect of ZAF derivatives 21a-c, 21g, 22g, and 24g on their ability to inhibit growth of P. gingivalis ATCC 33277 biofilm determined by a safranin staining assay of mono-species cultures. In supplemented BHI (100 μL), P. gingivalis (108 cells/well) was exponentially grown and incubated under anaerobic conditions and exposed to different treatments (100 μL) for 48 h with gentle shaking in 96-well plates. The bacteria were treated with ZAF derivatives 21a-c, 21g, 22g, and 24g (10, 25, 50, and 100 μM). Negative controls were bacteria grown with DMSO only. Bacteria exposed to Tet (2.81 μM) or ZAF (50 μM) were used as positive controls. The cells were stained with 0.1% safranin (100 μL) and absorbance was measured at 492 nm. The data show the calculated percentage of biofilm growth by comparison of the treated bacteria groups vs bacteria grown in DMSO only using the formula 100 × [OD (experimental) / OD (DMSO control)].
Cytotoxic effect of ZAF derivatives on oral epithelial cells
Oral epithelial cells are the body’s first line of defense against invading periodontopathogenic bacterial species. For a compound to be a promising lead candidate it needs to have selective activity against the targeted pathogen to not disrupt the host cells defense mechanisms along with not disturbing the commensal bacterial species found in periodontal health. Therefore, the three most active 3rd generation ZAF derivatives 21b, 21c, and 24g were evaluated at 10 and 100 μM for their cytotoxic effect against immortalized oral keratinocyte (OKF6) cells by using flow cytometry and visualization by microscopy (Figure 8). When tested at 10 μM, compounds 21b and 21c displayed bactericidal activity in CFU assays against P. gingivalis and compound 24g displayed 71% growth inhibition at this concentration along with bactericidal activity at 100 μM. Therefore, the compounds were tested against OKF6 cells at concentrations from 1- to 10-fold higher than their P. gingivalis percent growth inhibition activity. Microscopy was used to visualize the OKF6 cell morphology and density to examine the effect of the various treatments on the cells. In the cytotoxicity assay, the no treatment control and DMSO control wells had a higher density of OKF6 cells, which were spatially close together and had an elongated and flat epithelial-like morphology.34 Wells with cells treated with staurosporine (8 μM, STS, a pro-apoptotic chemical) and ZAF (25 μM) had a lower density of cells. Wells treated with positive controls were in direct contrast to the negative controls as the OKF6 cells were smaller in size, more round in shape, and farther apart from one another. Wells containing compounds 21c and 24g had the highest density of cells, followed by compound 21b. At 10 μM, compounds 21c and 24g had a higher cell density than the positive controls STS and ZAF, which indicated low toxicity to OKF6 cells. Cell morphology of the wells containing compound 24g at 10 μM resembled the elongated and flat OKF6 cells of the negative controls indicating little to no cytotoxicity, with only a slight decrease in cell density at 100 μM. Although the wells containing compounds 21b and 21c at 100 μM indicate a slight increase in cytotoxicity with round OKF6 cells spread farther apart from one another, this concentration is 10-fold higher than their bactericidal activity and display less cytotoxicity compared to the commercially available STS that was tested at a much lower concentration of 8 μM. The concentrations that we tested ZAF derivatives 21b, 21c, and 24g at are much higher than what is needed to inhibit 100% P. gingivalis growth. Thus these compounds should be safe at the dose that would be needed for treatment.
Figure 8.
Effect of ZAF derivatives 21b, 21c, and 24g on cell morphology of oral epithelial cells (OKF6) visualized under a microscope (Note: the images represent the entire well). OKF6 cells treated with medium only or 1% DMSO were used as negative controls, while cells treated with 8 μM of staurosporine (STS) or 25 μM of ZAF were used as positive controls. Cells were treated with 10 and 100 μM of ZAF derivatives 21b, 21c, and 24g for 24 h.
In these cytotoxicity studies on ZAF derivatives 21b, 21c, and 24g, 9–63% cell viability at the concentration of 10 μM, and 5–23% cell viability at 100 μM were observed (yellow bars in Figure 9, Table S1). All compounds tested displayed less cytotoxicity than the control STS. At 10 μM, compounds 21c and 24g displayed less cytotoxicity than the parent drug ZAF (41% cell viability). Overall, at 10 μM, it was found that compound 24g (R1 = n-butyl and R2 = 4-NO2) displayed less cytotoxicity than (<) 21c (R1 = methyl and R2 = 2-Br), which was less toxic than (<) 21b (R1 = methyl and R2 = 2-Cl). At 100 μM, a similar trend was observed. Overall, with compounds 21c and 24g, the desired effect of these 3rd generation ZAF derivatives achieved the goal of increasing P. gingivalis growth inhibition with decreased cytotoxicity at their bactericidal concentration of 10 μM, compared to the most active 2nd generation ZAF derivatives and ZAF parent drug.
Figure 9.
Effect of ZAF derivatives 21b, 21c, and 24g on apoptosis and necrosis of oral epithelial cells (OKF6) evaluated in a cell viability assay. OKF6 cells treated with medium only or 1% DMSO were used as negative controls, while cells were treated with 8 μM of staurosporine (STS) or 25 μM of ZAF were used as positive controls. Cells were treated with 10 and 100 μM of ZAF derivatives 21b, 21c, and 24g for 24 h and the cytotoxic effect was established by flow cytometry analysis (FACS) using an FITC-Annexin V apoptosis detection kit (BD, Pharmingen). Two independent experiments analyzing at least 10,000 events per condition in duplicate were used to generate the FACS data.
Cytotoxic effect of ZAF derivatives on mammalian cell lines
Although there was a decrease in the cytotoxic effect of 21c compared to the most active 2nd generation ZAF derivatives and the parent compound ZAF with OKF6 cells, there was a slight increase in cytotoxicity with compound 21b. Therefore, another crucial parameter to consider when developing antibacterial drugs is to ensure they are not toxic to mammalian cells. The most active halogenated compounds 21a (R2 = 2-F), 21b (R2 = 2-Cl), and 21c (R2 = 2-Br) were tested against three different mammalian cell lines: human embryonic kidney (HEK-293), human bronchus normal (BEAS-2B), and liver hepatocellular carcinoma (HepG2) cells along with ZAF as a control (Figures S90 and S91). In instances where >100% cell survival was observed, the data displayed in Figure S90 were normalized to 100% cell survival (Note: the non-normalized data are presented in Figure S91). When tested against P. gingivalis ATCC 33277 in CFU assays compounds 21a-c displayed 96% growth inhibition (for 21a) or displayed a bactericidal effect with 100% growth inhibition (for 21b and 21c) at 10 μM. Each compound was tested against the mammalian cell lines in a concentration range of 0–62.6 μg/mL (i.e., concentrations at least up to 10-fold higher than those displaying complete growth inhibition). Against HEK-293 and HepG2, compounds 21a, 21b, and 21c exhibited no toxicity up to 62.6 μg/mL (11- to 13-fold of their bactericidal concentrations). Against BEAS-2B, no toxicity was observed with compounds 21a, 21b, and 21c up to 62.6 μg/mL (11- to 13-fold of their bactericidal concentrations), 31.3 μg/mL (6-fold of their bactericidal concentrations), and 31.3 μg/mL (6-fold of their bactericidal concentrations), respectively. ZAF displayed no toxicity up to 31.3 μg/mL, 62.6 μg/mL, and 62.6 μg/mL for HEK-293, BEAS-2B, and HepG2, respectively. With little to no toxicity (>76% cell survival) at all concentrations tested against mammalian cells, these data are very encouraging.
Hemolytic effect of N-alkyl ZAF derivatives
Due to the promising antibacterial activity of the N-alkylated 3rd generation ZAF derivatives, we wanted to confirm that they would display no or reasonable (<50%) hemolytic activity against murine red blood cells (mRBCs). We tested compounds 23a, 23g, 24a, and 24g at concentrations ranging from 3.13 to 100 μM (Figure 10, Table S2). The negative control was phosphate-buffered saline (PBS) only and the positive control was Triton™ X-100 (1% v/v, 2 μL). Additionally, amphotericin B (data not shown) was tested and displayed no hemolytic activity (0%) at concentrations up to 27 μg/mL. Compounds 23a (R1 = propyl) and 24a (R1 = n-butyl) displayed ≤25% hemolysis of mRBCs at concentrations up to 100 μM. Compounds 23g (R1 = propyl) and 24g (R1 = n-butyl) displayed ≤15% hemolysis at concentrations up to 100 μM. We found that the parent compound ZAF displayed high hemolytic activity with 72% lysis of mRBCs at 100 μM. Therefore, with little to no hemolytic activity, the most active 3rd generation ZAF derivatives are highly promising for further development as anti-P. gingivalis.
Figure 10.
Bar graph depicting the dose-dependent hemolytic activity of compounds 23a (dark green bars), 23g (dark yellow bars), 24a (pink bars), 24g (dark purple bars), and ZAF (grey bars) against murine red blood cells (mRBCs). mRBCs were treated and incubated for 1 h at 37 °C with the compounds at concentrations ranging from 3.13 to 100 μM. Triton™ X-100 (1% v/v) was used as a positive control (100% hemolysis, not shown).
CONCLUSION
In summary, 14 novel 3rd generation ZAF derivatives (21b-f, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g) were synthesized. Seven of these compounds (21b-d, 22-25g), in addition to the two most active 2nd generation ZAF derivatives (21a and 21g) displayed increased and selective antibacterial activity against P. gingivalis ATCC 33277. Modifications to the ZAF structure included testing the effects of various electron-withdrawing groups on the arylsulfonamide on the C2 and C4 positions of the benzene ring (e.g., halogens, difluoro, and nitro groups). Substitution of the N-methyl of the indole ring with various alkyl or bulky aryl groups were also performed. Increased antibacterial activity against P. gingivalis in percent growth inhibition assays, was observed with compounds containing either halogens (e.g., F and Cl) at the C2 position or electron-withdrawing groups (e.g., NO2) at the C4 position on the arylsulfonamide ring. Additionally, longer alkyl chains on the nitrogen of the indole ring of compounds with R2 = 4-NO2 increased antibacterial activity, while branched chains and bulky substituents negatively affected activity. Superior activity was observed with R2 = 4-NO2 and various linear N-alkyl chains compared to the decreased activity with derivatives containing R2 = 2-F and various N-alkyl chains. While branched chains and bulky substituents on the nitrogen of the indole ring negatively affected activity. The most active compounds 21a, 21b, and 21c displayed bactericidal activity at concentrations as low as 10 μM, along with increased antibacterial activity of compounds 21a and 24g compared to the parent ZAF and control Tet when tested against P. gingivalis. Interestingly, compounds 21a, 21b, and 24g displayed increased antibacterial activity compared to the controls with inhibition of P. gingivalis biofilm growth at 25, 10, and 50 μM, respectively. When tested against OKF6 cells, compounds 21c and 24g achieved the overall goal of increasing activity against P. gingivalis while decreasing cytotoxicity compared to the most active 2nd generation ZAF derivatives and the ZAF and Tet controls. Additionally, little to no cytotoxicity against HEK-293, BEAS-2B, and HepG2 was observed with 21a, 21b, and 21c. In addition, little to no hemolytic activity was observed with N-alkylated compounds 23a, 23g, 24a, and 24g. In the future, outside the scope of this study, as there are over 700 bacterial species found in the oral cavity, with only a small number of those bacterial species being pathogenic that lead to inflammation and bone destruction, it would be interesting to evaluate these compounds in multi-species biofilm studies. Important multi-species biofilms to test will be the dual species of F. nucleatum and P. gingivalis as well as the tri-species biofilms of S. sanguinis, P. gingivalis, and A. actinomycetemcomitans. These bacterial species have important synergistic (F. nucleatum enhances the growth of P. gingivalis by providing a carbon dioxide rich microenvironment) and antagonistic interactions (S. sanguinis produced hydrogen peroxide to inhibit the growth of P. gingivalis and A. actinomycetemcomitans) that are vital to the progression or the regression of periodontal diseases. The development of the multi-species biofilms tests is currently underway in our laboratory and will be presented in future studies. Additionally, it will be interesting to see how these promising molecules will fare in in vivo studies using animal models of P. gingivalis-induced periodontal disease.
EXPERIMENTAL SECTION
Materials and instrumentation.
The intermediates of the zafirlukast (ZAF) derivatives with 1-methyl-5-nitroindole (compounds 1, 8, and 14) were resynthesized as previously reported17, 18 and the yields that we obtained from these reactions in the current study are reported in Scheme 1. Compounds 21a and 21g (previously 14a and 14e, respectively, in our previously published manuscript with our 2nd generation ZAF derivatives)18 were also re-synthesized for new biological biofilms studies and for testing against a different strain of P. gingivalis. For this study, all chemicals that were used for the synthesis of the intermediates (including 7 and 20a-20g), ZAF derivatives, and for biological evaluation were purchased from Oakwood Chemical (San Diego, CA), Combi-Blocks (San Diego, CA), TCI America (Portland, OR), Sigma Aldrich (St. Louis, MO), Synthonix (Wake Forest, NC), Matrix Scientific (Columbia, SC), Ricca Chemical Company (Arlington, TX), Acros Organics (Geel, Belgium), Beantown Chemical (Hudson, NH), Chem-Impex (Wood Dale, IL), Alfa Aesar (Ward Hill, MA), and used without further purification. All chemical reactions were monitored by thin layer chromatography (TLC) using glass plates coated with Merk silica gel 60 F254. UV light was used to visualize the chromatographic bands on the TLC plates. Silica gel column chromatography with SiliaFlash® F60 (40–63 μM, SiliCycle, Québec, Canada) was used to purify compounds. Varian 500 (VNMRS500) or 400 (MR400) MHz spectrometers were used to record 1H NMR spectra at 500 or 400 MHz, respectively. A Varian 400 MHz spectrometer was used to record all 13C NMR spectra at 100 MHz. All NMR spectra chemical shifts (d) are given in parts per million (ppm). All coupling constants (J) are given in Hertz (Hz), and the abbreviations used for signal shape are singlet (s), doublet (d), triplet (t), multiplet (m), doublet of doublets (dd), doublet of triplets (dt), and triplet of doublets (td). High-resolution mass spectrometry (HRMS) was performed on an AB SCIEX TripleTOF™5600 mass spectrometer to record mass spectra data for all final compounds (21b-f, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g). The HRMS [M+H]+ signals for all compounds synthesized were consistent with the calculated molecular weights. To further confirm purity of all final compounds 21b-f, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g, an Agilent Technologies 1260 Infinity HPLC system (Santa Clara, CA, USA) was used. Purity of the compounds was confirmed using the following HPLC method - method A: Flow rate = 0.5 mL/min; l = 254 nm; column = Vydac Denali C18 column, 250 × 4.6 mm, 120 Å, 5 μm; Eluents: A = H2O + 0.1% trifluoroacetic acid, B = MeCN; gradient profile: starting from 5% B, increasing from 5% B to 100% C over 10 min, hold at 100% B for 10 min, decreasing from 100% B to 5% B in 3 min. Prior to each injection, the HPLC column was equilibrated for 10 min with 5% B. All compounds were ≥95% pure.
Synthesis of compound 2 (SGT1629).
A solution of 5-nitroindole (3.0 g, 18.5 mmol) in anhydrous DMF (15 mL) was cooled to 0 °C and treated with NaH (60% in mineral oil, 1.48 g, 37.0 mmol). The reaction mixture was allowed to stir at room temperature for 1 h. The mixture was cooled to 0 °C, iodoethane (2.98 mL, 37.0 mmol) was then slowly added, and the resulting mixture was stirred at room temperature for 1 h. The reaction was quenched by pouring onto ice and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with NaHCO3, H2O, and brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/3:1, Rf 0.46) to yield compound 2 (1.70 g, 48%) as a yellow solid: 1H NMR (500 MHz, CDCl3, Figure S1) d 8.58 (dd, J1 = 2.2 Hz, J2 = 0.5 Hz, 1H, aromatic), 8.10 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.34 (dt, J1 = 9.1 Hz, J2 = 0.7 Hz, 1H, aromatic), 7.25 (d, J = 3.3 Hz, 1H, aromatic), 6.66 (dd, J1 = 3.3 Hz, J2 = 0.9 Hz, 1H, aromatic), 4.21 (q, J = 7.4 Hz, 2H, NCH2CH3), 1.49 (t, J = 7.4 Hz, 3H, NCH2CH3); 13C NMR (100 MHz, CDCl3, Figure S2) d 138.7, 131.9, 130.4, 128.0, 118.5, 117.3, 109.3, 104.2, 41.7, 15.6.
Synthesis of compound 3 (SGT1630).
A solution of 5-nitroindole (3.0 g, 18.5 mmol) in anhydrous DMF (15 mL) was cooled to 0 °C and treated with NaH (60% in mineral oil, 1.48 g, 37.0 mmol). The reaction mixture was allowed to stir at room temperature for 1 h. The mixture was cooled to 0 °C, 1-iodopropane (3.59 mL, 37.0 mmol) was then slowly added, and the resulting mixture was stirred at room temperature for 1 h. The reaction was quenched by pouring onto ice and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with NaHCO3, H2O, and brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.81) to yield compound 3 (2.71 g, 72%) as a brown liquid: 1H NMR (500 MHz, CDCl3, Figure S3) d 8.57 (d, J = 2.3Hz, 1H, aromatic), 8.09 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.33 (dd, J1 = 9.1 Hz, J2 = 0.7 Hz, 1H, aromatic), 7.23 (d, J = 3.2 Hz, 1H, aromatic), 6.66 (dt, J1 = 3.3 Hz, J2 = 0.8 Hz, 1H, aromatic), 4.12 (t, J = 7.1 Hz, 2H, NCH2CH2CH3), 1.88 (sextet, J = 7.4 Hz, 2H, NCH2CH2CH3), 0.93 (t, J = 7.4 Hz, 3H, NCH2CH2CH3); 13C NMR (100 MHz, CDCl3, Figure S4) d 141.6, 139.0, 131.2, 127.8, 118.4, 117.2, 109.4, 104.0, 48.7, 23.7, 11.6.
Synthesis of compound 4 (SGT1631).
A solution of 5-nitroindole (3.0 g, 18.5 mmol) in anhydrous DMF (15 mL) was cooled to 0 °C and treated with NaH (60% in mineral oil, 1.48 g, 37.0 mmol). The reaction mixture was allowed to stir at room temperature for 1 h. The mixture was cooled to 0 °C, 1-iodobutane (4.20 mL, 37.0 mmol) was then slowly added, and the resulting mixture was stirred at room temperature for 1 h. The reaction was quenched by pouring onto ice and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with NaHCO3, H2O, and brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/3:1, Rf 0.53) to yield compound 4 (1.54 g, 38%) as a brown liquid: 1H NMR (500 MHz, CDCl3, Figure S5) d 8.57 (d, J = 2.2 Hz, 1H, aromatic), 8.09 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.33 (d, J = 9.1 Hz, 1H, aromatic), 7.23 (d, J = 3.3 Hz, 1H, aromatic), 6.65 (dd, J1 = 3.3 Hz, J2 = 0.8 Hz, 1H, aromatic), 4.15 (t, J = 7.2 Hz, 2H, NCH2CH2CH2CH3), 1.82 (p, J = 7.7 Hz, 2H, NCH2CH2CH2CH3), 1.33 (sextet, J = 7.7 Hz, 2H, NCH2CH2CH2CH3), 0.93 (t, J = 7.4 Hz, 3H, NCH2CH2CH2CH3); 13C NMR (100 MHz, CDCl3, Figure S6) d 141.7, 139.0, 131.2, 127.9, 118.5, 117.3, 109.4, 104.0, 46.8, 32.5, 20.3, 13.8.
Synthesis of compound 5 (SGT1633).
A solution of 5-nitroindole (3.0 g, 18.5 mmol) in anhydrous DMF (15 mL) was cooled to 0 °C and treated with NaH (60% in mineral oil, 1.48 g, 37.0 mmol). The reaction mixture was allowed to stir at room temperature for 1 h. The mixture was cooled to 0 °C, 1-iodo-2-methylpropane (4.25 mL, 37.0 mmol) was then slowly added, and the resulting mixture was stirred at room temperature for 1 h. The reaction was quenched by pouring onto ice and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with NaHCO3, H2O, and brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.89) to yield compound 5 (1.12 g, 28%) as a yellow solid: 1H NMR (500 MHz, CDCl3, Figure S7) d 8.57 (d, J = 2.3 Hz, 1H, aromatic), 8.09 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.32 (dt, J1 = 9.1 Hz, J2 = 0.7 Hz, 1H, aromatic), 7.20 (d, J = 3.3 Hz, 1H, aromatic), 6.66 (dd, J1 = 3.2 Hz, J2 = 0.8 Hz, 1H, aromatic), 3.94 (d, J = 7.4 Hz, 2H, NCH2), 2.18 (septet, J = 7.3 Hz, 1H, CH(CH3)2), 0.92 (d, J = 6.7 Hz, 6H, CH(CH3)2); 13C NMR (100 MHz, CDCl3, Figure S8) d 141.6, 139.3, 131.7, 127.8, 118.4, 117.3, 109.6, 103.9, 54.7, 29.9, 20.4 (2CH3).
Synthesis of compound 6 (SGT1632).
A solution of 5-nitroindole (3.0 g, 18.5 mmol) in anhydrous DMF (15 mL) was cooled to 0 °C and treated with NaH (60% in mineral oil, 1.11 g, 27.8 mmol). The reaction mixture was allowed to stir at room temperature for 1 h. The mixture was cooled to 0 °C, benzyl bromide (3.30 mL, 27.8 mmol) was then slowly added, and the resulting mixture was stirred at room temperature for 1 h. The reaction was quenched by pouring onto ice and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with NaHCO3, H2O, and brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/3:1, Rf 0.58) to yield compound 6 (3.10 g, 66%) as a tan solid: 1H NMR (500 MHz, CDCl3, Figure S9) d 8.59 (dd, J1 = 2.3 Hz, J2 = 0.6 Hz, 1H, aromatic ), 8.06 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.33–7.27 (m, 4H, aromatic), 7.26 (d, J = 3.3 Hz, 1H, aromatic), 7.09–7.18 (m, 2H, aromatic), 6.72 (dd, J1 = 3.3 Hz, J2 = 0.9 Hz, 1H, aromatic), 5.35 (s, 2H, NCH2); 13C NMR (100 MHz, CDCl3, Figure S10) d 142.0, 139.2, 136.4, 131.7, 129.2 (2CH), 128.3, 128.1, 127.0 (2CH), 118.4, 117.6, 109.8, 104.6, 50.8.
Synthesis of compound 9 (SGT1639).
A solution of compound 2 (0.20 g, 1.05 mmol) and methyl-5-formyl-2-methoxybenzoate (0.20 g, 1.05 mmol) in anhydrous CH2Cl2 (5 mL) was cooled to 0 °C in an ice-H2O bath. Then Et3SiH (0.5 mL, 2.94 mmol) was added, followed by TFA (0.2 mL, 2.10 mmol). The mixture was stirred at 0 °C for 10 min and was allowed to warm to room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with NaHCO3, H2O, and brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.60) to yield compound 9 (0.24 g, 63%) as a yellow solid: 1H NMR (500 MHz, CDCl3, Figure S11) d 8.46 (dd, J1 = 2.2 Hz, J2 = 0.6 Hz, 1H, aromatic), 8.09 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.69 (d, J = 2.5 Hz, 1H, aromatic), 7.35 (dd, J1 = 8.6 Hz, J2 = 2.4 Hz, 1H, aromatic), 7.30 (dd, J1 = 9.2 Hz, J2 = 0.7 Hz, 1H, aromatic), 6.91 (d, J = 8.7 Hz, 1H, aromatic), 6.90 (s, 1H, aromatic), 4.14 (q, J = 7.4 Hz, 2H, NCH2CH3), 4.06 (s, 2H, CH2Ar), 3.87 (s, 3H, ArOCH3), 3.85 (s, 3H, ArCO2CH3), 1.44 (t, J = 7.4 Hz, 3H, NCH2CH3); 13C NMR (100 MHz, CDCl3, Figure S12) d 167.0, 157.9, 141.4, 139.2, 133.8, 132.1, 131.9, 128.6, 127.3, 120.3, 117.7, 117.6, 116.8, 112.5, 109.3, 56.4, 52.3, 41.6, 30.4, 15.6.
Synthesis of compound 10 (SGT1640).
A solution of compound 3 (0.20 g, 0.98 mmol) and methyl-5-formyl-2-methoxybenzoate (0.19 g, 0.98 mmol) in anhydrous CH2Cl2 (5 mL) was cooled to 0 °C in an ice-H2O bath. Then Et3SiH (0.4 mL, 2.74 mmol) was added, followed by TFA (0.1 mL, 1.96 mmol). The mixture was stirred at 0 °C for 10 min and was allowed to warm to room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with NaHCO3, H2O, and brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.73) to yield compound 10 (0.24 g, 63%) as a dark yellow solid: 1H NMR (500 MHz, CDCl3, Figure S13) d 8.46 (dd, J1 = 2.3 Hz, J2 = 0.5 Hz, 1H, aromatic), 8.08 (dd, J1 = 9.1 Hz, J2 = 2.2 Hz, 1H, aromatic), 7.68 (d, J = 2.5 Hz, 1H, aromatic), 7.34 (dd, J1 = 8.6 Hz, J2 = 2.5 Hz, 1H, aromatic), 7.29 (d, J = 9.1 Hz, 1H, aromatic), 6.91 (d, J = 8.6 Hz, 1H, aromatic), 6.88 (app. t, J = 1.1 Hz, 1H, aromatic), 4.06 (s, 2H, CH2Ar), 4.04 (t, J = 7.2 Hz, 2H, NCH2CH2CH3), 3.87 (s, 3H, ArOCH3), 3.85 (s, 3H, ArCO2CH3), 1.83 (sextet, J = 7.4 Hz, 3H, NCH2CH2CH3), 0.90 (t, J = 7.4 Hz, 3H, NCH2CH2CH3); 13C NMR (100 MHz, CDCl3, Figure S14) d 166.9, 157.9, 141.3, 139.6, 133.7, 132.1, 131.9, 129.4, 127.2, 120.2, 117.54, 117.46, 116.8, 112.5, 109.5, 56.3, 52.2, 48.5, 30.3, 23.8, 11.6.
Synthesis of compound 11 (SGT1641).
A solution of compound 4 (0.70 g, 3.21 mmol) and methyl-5-formyl-2-methoxybenzoate (0.62 g, 3.21 mmol) in anhydrous CH2Cl2 (5 mL) was cooled to 0 °C in an ice-H2O bath. Then Et3SiH (1.4 mL, 8.98 mmol) was added, followed by TFA (0.5 mL, 6.41 mmol). The mixture was stirred at 0 °C for 10 min and was allowed to warm to room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with NaHCO3, H2O, and brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.60) to yield compound 11 (0.10 g, 8%) as a yellow liquid (which contained some methyl-5-formyl-2-methoxybenzoate that was removed in the next synthetic step): NCH2CH2CH2CH3); 13C NMR (100 MHz, (CD3)2SO, Figure S16) d 166.2, 156.5, 140.2, 134.8, 133.3, 132.5, 130.4, 126.4, 119.8, 116.54, 116.47, 116.0, 113.2, 112.7, 110.4, 55.8, 51.8, 31.9, 29.0, 19.4, 13.5.
Synthesis of compound 12 (SGT1660).
A solution of compound 5 (0.20 g, 0.92 mmol) and methyl-5-formyl-2-methoxybenzoate (0.18 g, 0.92 mmol) in anhydrous CH2Cl2 (5 mL) was cooled to 0 °C in an ice-H2O bath. Then Et3SiH (0.4 mL, 2.57 mmol) was added, followed by TFA (0.1 mL, 1.83 mmol). The mixture was stirred at 0 °C for 10 min and was allowed to warm to room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with NaHCO3, H2O, and brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.72) to yield compound 12 (0.11 g, 36%) as a yellow solid: 1H NMR (500 MHz, CDCl3, Figure S17) d 8.45 (d, J = 2.2 Hz, 1H, aromatic), 8.08 (dd, J1 = 9.1 Hz, J2 = 2.2 Hz, 1H, aromatic), 7.68 (d, J = 2.5 Hz, 1H, aromatic), 7.34 (dd, J1 = 8.6 Hz, J2 = 2.5 Hz, 1H, aromatic), 7.28 (d, J = 9.1 Hz, 1H, aromatic), 6.90 (d, J = 8.6 Hz, 1H, aromatic), 6.87 (app. t, J = 1.1 Hz, 1H, aromatic), 4.06 (s, 2H, CH2Ar), 3.87 (d, J = 7.4 Hz, 2H, NCH2), 3.87 (s, 3H, ArOCH3),3.84 (s, 3H, ArCO2CH3), 2.14 (septet, J = 7.0 Hz, 1H, CH(CH3)2), 0.90 (t, J = 6.7 Hz, 6H, CH(CH3)2); 13C NMR (100 MHz, CDCl3, Figure S18) d 166.9, 157.9, 141.3, 139.9, 133.7, 132.1, 131.9, 129.9, 127.1, 120.2, 117.6, 117.4, 116.8, 112.5, 109.7, 56.3, 54.5, 52.2, 30.3, 29.9, 20.4.
Synthesis of compound 13 (SGT1642).
A solution of compound 6 (0.70 g, 2.77 mmol) and methyl-5-formyl-2-methoxybenzoate (1.08 g, 2.77 mmol) in anhydrous CH2Cl2 (5 mL) was cooled to 0 °C in an ice-H2O bath. Then Et3SiH (1.2 mL, 7.77 mmol) was added, followed by TFA (0.4 mL, 5.55 mmol). The mixture was stirred at 0 °C for 10 min and was allowed to warm to room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with NaHCO3, H2O, and brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.59) to yield compound 13 (0.23 g, 20%) as a yellow solid: 1H NMR (500 MHz, CDCl3, Figure S19) d 8.47 (dd, J1 = 2.3 Hz, J2 = 0.5 Hz, 1H, aromatic), 8.05 (dd, J1 = 9.1 Hz, J2 = 2.2 Hz, 1H, aromatic), 7.68 (d, J = 2.4 Hz, 1H, aromatic), 7.35 (dd, J1 = 8.6 Hz, J2 = 2.5 Hz, 1H, aromatic), 7.32–7.27 (m, 3H, aromatic), 7.08–7.05 (m, 2H, aromatic), 6.95 (app. t, J = 1.1 Hz, 1H, aromatic), 6.90 (d, J = 8.6 Hz, 1H, aromatic), 5.28 (s, 2H, NCH2Ar), 4.07 (s, 2H, CH2Ar), 3.87 (s, 3H, ArOCH3), 3.85 (s, 3H, ArCO2CH3); 13C NMR (100 MHz, CDCl3, Figure S20) d 166.9, 157.9, 141.6, 139.8, 136.5, 133.7, 131.93, 131.87, 129.8, 129.2, 128.3, 127.5, 126.9, 120.3, 118.0, 117.9, 116.8, 112.5, 109.9, 56.3, 52.2, 50.7, 30.4.
Synthesis of compound 15 (SGT1644).
A solution of compound 9 (0.23 g, 0.62 mmol) in MeOH:THF:H2O/10:2:2 (14 mL total) was stirred and treated with KOH pellets (0.25 g, 4.37 mmol). The resulting mixture was refluxed at 65 °C for 2 h. The organic solvents were removed in vacuo after the reaction was complete. The resulting mixture was acidified to pH 1 with 1 N aqueous HCl and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated to yield compound 15 (0.17 g, 75%) as a yellow solid: 1H NMR (500 MHz, CDCl3, Figure S21) d 8.38 (d, J = 2.2 Hz, 1H, aromatic), 8.09 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 8.06 (d, J = 2.5 Hz, 1H, aromatic), 7.49 (dd, J1 = 8.5 Hz, J2 = 2.5 Hz, 1H, aromatic), 7.30 (d, J = 9.2 Hz, 1H, aromatic), 7.00 (d, J = 8.2 Hz, 1H, aromatic), 6.99 (s, 1H, aromatic), 4.15 (q, J = 7.4 Hz, 2H, NCH2CH3), 4.10 (s, 2H, CH2Ar), 4.05 (s, 3H, ArOCH3), 1.46 (t, J = 7.3 Hz, 3H, NCH2CH3); 13C NMR (100 MHz, CDCl3, Figure S22) d 165.5, 156.8, 135.3, 134.7, 133.9, 128.7, 127.1, 117.8, 117.6, 116.9, 116.7, 112.1, 109.4, 57.0, 41.6, 30.5, 15.6.
Synthesis of compound 16 (SGT1645).
A solution of compound 10 (0.20 g, 0.52 mmol) in MeOH:THF:H2O/10:2:2 (14 mL total) was stirred and treated with KOH pellets (0.21 g, 3.66 mmol). The resulting mixture was refluxed at 65 °C for 2 h. The organic solvents were removed in vacuo after the reaction was complete. The resulting mixture was acidified to pH 1 with 1 N aqueous HCl and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated to yield compound 16 (0.17 g, 88%) as a yellow solid: 1H NMR (500 MHz, CDCl3, Figure S23) d 8.38 (d, J = 2.2 Hz, 1H, aromatic), 8.08 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 8.06 (d, J = 2.5 Hz, 1H, aromatic), 7.48 (dd, J1 = 8.5 Hz, J2 = 2.5 Hz, 1H, aromatic), 7.30 (d, J = 9.2 Hz, 1H, aromatic), 6.99 (d, J = 8.6 Hz, 1H, aromatic), 6.97 (app. t, J = 1.0 Hz, 1H, aromatic), 4.10 (s, 2H, CH2Ar), 4.06 (t, J = 7.2 Hz, 2H, NCH2CH2CH3), 4.05 (s, 3H, ArOCH3), 1.85 (sextet, J = 7.4 Hz, 2H, NCH2CH2CH3), 0.92 (t, J = 7.4 Hz, 3H, NCH2CH2CH3); 13C NMR (100 MHz, CDCl3, Figure S24) d 165.5, 156.8, 141.3, 139.7, 135.2, 134.6, 133.8, 129.5, 127.0, 117.8, 117.6, 116.68, 116.66, 112.1, 109.6, 57.0, 48.6, 30.4, 23.8, 11.7.
Synthesis of compound 17 (SGT1646).
A solution of compound 11 (90 mg, 0.23 mmol) in MeOH:THF:H2O/5:1:1 (7 mL total) was stirred and treated with KOH pellets (89 mg, 1.59 mmol). The resulting mixture was refluxed at 65 °C for 2 h. The organic solvents were removed in vacuo after the reaction was complete. The resulting mixture was acidified to pH 1 with 1 N aqueous HCl and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated to yield compound 17 (71 mg, 82%) as a yellow solid: 1H NMR (500 MHz, CD3OD, Figure S25) d 8.30 (dd, J1 = 2.3 Hz, J2 = 0.6 Hz, 1H, aromatic), 7.97 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.66 (d, J = 2.4 Hz, 1H, aromatic), 7.42 (d, J = 9.1 Hz, 1H, aromatic), 7.39 (dd, J1 = 8.6 Hz, J2 = 2.5 Hz, 1H, aromatic), 7.17 (app. t, J = 1.0 Hz, 1H, aromatic), 6.99 (d, J = 8.6 Hz, 1H, aromatic), 4.13 (t, J = 7.1 Hz, 2H, NCH2CH2CH2CH3), 4.04 (s, 2H, CH2Ar), 3.91 (s, 1H, OH), 3.81 (s, 3H, ArOCH3), 1.76–1.70 (m, 2H, NCH2CH2CH2CH3), 1.27–1.20 (m, 2H, NCH2CH2CH2CH3), 0.86 (t, J = 7.5 Hz, 3H, NCH2CH2CH2CH3); 13C NMR (100 MHz, (CD3)2SO, Figure S26) d 156.4, 140.2, 139.1, 132.8, 132.6, 132.5, 130.4, 126.4, 121.0, 116.6, 116.5, 116.0, 113.0, 112.5, 110.4, 55.8, 45.5, 31.9, 29.1, 19.4, 13.5.
Synthesis of compound 18 (SGT1648).
A solution of compound 12 (0.10 g, 0.25 mmol) in MeOH:THF:H2O/10:2:2 (14 mL total) was stirred and treated with KOH pellets (99 mg, 1.77 mmol). The resulting mixture was refluxed at 65 °C for 2 h. The organic solvents were removed in vacuo after the reaction was complete. The resulting mixture was acidified to pH 1 with 1 N aqueous HCl and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated to yield compound 18 (81 mg, 84%) as a yellow solid (which contained some methyl-5-formyl-2-methoxybenzoate that was removed in the next synthetic step): 1H NMR (500 MHz, (CD3)2SO, Figure S27) d 8.41 (d, J = 2.3 Hz, 1H, aromatic), 8.00 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.68 (d, J = 9.2 Hz, 1H, aromatic), 7.54 (d, J = 2.5 Hz, 1H, aromatic), 7.47 (s, 1H, aromatic), 7.42 (dd, J1 = 8.6 Hz, J2 = 2.4 Hz, 1H, aromatic), 7.05 (d, J = 8.6 Hz, 1H, aromatic), 4.10 (s, 2H, CH2Ar), 4.04 (d, J = 7.4 Hz, 2H, NCH2), 3.77 (s, 3H, ArOCH3), 2.10 (septet, J = 7.2 Hz, 1H, CH(CH3)2), 0.84 (d, J = 6.7 Hz, 6H, CH(CH3)2); 13C NMR (100 MHz, (CD3)2SO, Figure S28) d 166.4, 162.6, 140.2, 134.3, 132.8, 132.54, 132.45, 130.9, 128.7, 121.8, 121.0, 116.5, 116.0, 113.0, 112.5, 110.7, 56.4, 55.8, 29.3, 19.7 (2CH3).
Synthesis of compound 19 (SGT1647).
A solution of compound 13 (0.20 g, 0.46 mmol) in MeOH:THF:H2O/10:2:2 (14 mL total) was stirred and treated with KOH pellets (0.18 g, 3.25 mmol). The resulting mixture was refluxed at 65 °C for 2 h. The organic solvents were removed in vacuo after the reaction was complete. The resulting mixture was acidified to pH 1 with 1 N aqueous HCl and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated to yield compound 19 (60 mg, 31%) as a yellow solid: 1H NMR (500 MHz, CD3OD, Figure S29) d 8.33 (d, J = 2.1 Hz, 1H, aromatic), 7.94 (dd, J1 = 9.2 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.64 (d, J = 2.5 Hz, 1H, aromatic), 7.39 (dd, J1 = 8.5 Hz, J2 = 2.6 Hz, 1H, aromatic), 7.37 (d, J = 9.1 Hz, 1H, aromatic), 7.24–7.20 (m, 3H, aromatic), 7.20–7.15 (m, 1H, aromatic), 7.09–7.07 (m, 2H, aromatic), 6.99 (d, J = 8.6 Hz, 1H, aromatic), 5.35 (s, 2H, NCH2Ar), 4.06 (s, 2H, CH2Ar), 3.90 (s, 1H, OH), 3.80 (s, 3H, ArOCH3); 13C NMR (100 MHz, CDCl3, Figure S30) d 165.5, 156.8, 141.6, 139.9, 136.4, 135.2, 134.5, 133.8, 130.0, 129.3, 128.3, 127.3, 126.9, 118.0, 117.8, 117.2, 116.7, 112.1, 110.0, 57.0, 50.7, 30.4.
Synthesis of compound 21b (SGT1649).
A solution of compound 14 (synthesized as previously published)17 (30 mg, 0.09 mmol), 2-chlorobenzenesulfonamide (20b; 20 mg, 0.10 mmol), EDC•HCl (27 mg, 0.14 mmol), and DMAP (19 mg, 0.16 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.17) to yield compound 21b (18 mg, 40%) as an orange solid: 1H NMR (500 MHz, CDCl3, Figure S31) d 10.66 (s, 1H, NH), 8.29 (dd, J1 = 7.9 Hz, J2 = 1.7 Hz, 1H, aromatic), 8.20 (d, J = 2.2 Hz, 1H, aromatic), 8.09 (dd, J1 = 9.1 Hz, J2 = 2.2 Hz, 1H, aromatic), 7.89 (d, J = 2.5 Hz, 1H, aromatic), 7.56 (dd, J1 = 8.6 Hz, J2 = 2.5 Hz, 1H, aromatic), 7.53–7.42 (m, 3H, aromatic), 7.29 (d, J = 9.1 Hz, 1H, aromatic), 7.03 (d, J = 8.6 Hz, 1H, aromatic), 6.55 (s, 1H, aromatic), 4.08 (s, 3H, NCH3), 3.71 (s, 5H, CH2Ar, ArOCH3); 13C NMR (100 MHz, (CD3)2SO, Figure S32) d 155.5, 140.3, 139.8, 135.9, 135.2, 133.1, 132.3, 131.8 (2CH), 131.7, 130.8, 128.9, 127.61, 127.59, 125.8 (2CH), 119.8, 116.7, 116.3, 112.3, 110.5, 56.0, 36.6, 32.9; m/z calcd for C24H20ClN3O6S 513.9490; found 515.4124 [M+H]+ and sodium adduct 537.3946 (Figure S33); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 13.03 min (98% pure, Figure S34).
Synthesis of compound 21c (SGT1650).
A solution of compound 14 (synthesized as previously published)17 (30 mg, 0.09 mmol), 2-bromobenzenesulfonamide (20c; 25 mg, 0.10 mmol), EDC•HCl (27 mg, 0.14 mmol), and DMAP (19 mg, 0.16 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.25) to yield compound 21c (26 mg, 53%) as an orange solid: 1H NMR (500 MHz, CDCl3, Figure S35) d 10.72 (s, 1H, NH), 8.33 (dd, J1 = 7.9 Hz, J2 = 1.7 Hz, 1H, aromatic), 8.20 (d, J = 2.2 Hz, 1H, aromatic), 8.10 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.89 (d, J = 2.5 Hz, 1H, aromatic), 7.69 (dd, J1 = 7.9 Hz, J2 = 1.3 Hz, 1H, aromatic), 7.56 (dd, J1 = 8.6 Hz, J2 = 2.6 Hz, 1H, aromatic), 7.49 (td, J1 = 7.7 Hz, J2 = 1.3 Hz, 1H, aromatic), 7.42 (qd, J1 = 8.5 Hz, J2 = 1.8 Hz, 1H, aromatic), 7.29 (d, J = 9.1 Hz, 1H, aromatic), 7.03 (d, J = 8.6 Hz, 1H, aromatic), 6.55 (s, 1H, aromatic), 4.08 (s, 3H, NCH3), 3.71 (s, 5H, CH2Ar, ArOCH3); 13C NMR (100 MHz, (CD3)2SO, Figure S36) d 155.6, 140.3, 139.8, 136.0, 135.2, 132.6, 131.9 (2CH), 129.1, 128.1, 125.8 (2CH), 119.8, 119.2 (2C), 116.7, 116.3, 112.4, 110.5 (2CH), 109.6, 56.1, 36.6, 32.9; m/z calcd for C24H20BrN3O6S 558.4030; found 559.1314 [M+H]+ (Figure S37); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 13.03 min (98% pure, Figure S38).
Synthesis of compound 21d (SGT1662).
A solution of compound 14 (synthesized as previously published)17 (30 mg, 0.09 mmol), 2,6-difluorobenzenesulfonamide (20d; 20 mg, 0.10 mmol), EDC•HCl (27 mg, 0.14 mmol), and DMAP (19 mg, 0.16 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.29) to yield compound 21d (11 mg, 25%) as an yellow solid: 1H NMR (500 MHz, CDCl3, Figure S39) d 10.64 (s, 1H, NH), 8.33 (d, J = 2.8 Hz, 1H, aromatic), 8.08 (dd, J1 = 9.3 Hz, J2 = 2.4 Hz, 1H, aromatic), 7.90 (d, J = 2.4 Hz, 1H, aromatic), 7.56–7.51 (m, 1H, aromatic), 7.48 (dd, J1 = 8.6 Hz, J2 = 2.6 Hz, 1H, aromatic), 7.26 (d, J = 9.2 Hz, 1H, aromatic), 7.01 (t, J = 8.7 Hz, 2H, aromatic), 6.84 (d, J = 8.6 Hz, 1H, aromatic), 6.88 (s, 1H, aromatic), 4.05 (s, 3H, NCH3), 4.03 (s, 2H, CH2Ar), 3.76 (s, 3H, ArOCH3); 13C NMR (100 MHz, (CD3)2SO, Figure S40) d 164.1, 157.9, 148.8 (2C), 140.2, 139.6, 131.4, 129.2, 129.13 (2CH), 129.10, 129.06, 126.3, 126.0, 117.5, 116.6, 116.5, 115.9 (2CH), 112.2, 110.4, 55.9, 43.5, 32.8; m/z calcd for C24H19F2N3O6S 515.4878; found 516.1034 [M+H]+ and sodium adduct 538.0856 (Figure S41); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 13.03 min (99% pure, Figure S42).
Synthesis of compound 21e (SGT1663).
A solution of compound 14 (synthesized as previously published)17 (30 mg, 0.09 mmol), 2,4-difluorobenzenesulfonamide (20e; 20 mg, 0.10 mmol), EDC•HCl (27 mg, 0.14 mmol), and DMAP (19 mg, 0.16 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.41) to yield compound 21e (16 mg, 35%) as an yellow solid: 1H NMR (500 MHz, CDCl3, Figure S43) d 10.57 (s, 1H, NH), 8.33 (d, J = 2.2 Hz, 1H, aromatic), 8.18–8.14 (m, 1H, aromatic), 8.08 (dd, J1 = 9.1 Hz, J2 = 2.4 Hz, 1H, aromatic), 7.87 (d, J = 2.5 Hz, 1H, aromatic), 7.47 (dd, J1 = 8.5 Hz, J2 = 2.1 Hz, 1H, aromatic), 7.26 (d, J = 9.1 Hz, 1H, aromatic), 7.05–7.01 (m, 1H, aromatic), 6.97 (d, J = 8.6 Hz, 1H, aromatic), 6.92–6.88 (m, 1H, aromatic), 6.87 (s, 1H, aromatic), 4.05 (s, 3H, NCH3), 4.03 (s, 2H, CH2Ar), 3.76 (s, 3H, ArOCH3); 13C NMR (100 MHz, (CD3)2SO, Figure S44) d 155.3, 140.2, 139.6 (2C), 133.6, 133.5, 132.9, 131.4, 129.1, 126.3, 116.5, 116.4, 115.9, 112.3, 112.2, 110.4 (2CH), 106.2, 105.94, 105.91, 105.7, 55.9, 32.8, 28.9; m/z calcd for C24H19F2N3O6S 515.4878; found 516.1027 [M+H]+ and sodium adduct 538.0848 (Figure S45); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 17.41 min (99% pure, Figure S46).
Synthesis of compound 21f (SGT1651).
A solution of compound 14 (synthesized as previously published)17 (30 mg, 0.09 mmol), 3,4-difluorobenzenesulfonamide (20f; 20 mg, 0.10 mmol), EDC•HCl (27 mg, 0.14 mmol), and DMAP (19 mg, 0.16 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.18) to yield compound 21f (12 mg, 26%) as an amber solid: 1H NMR (500 MHz, CDCl3, Figure S47) d 10.43 (s, 1H, NH), 8.19 (d, J = 2.0 Hz, 1H, aromatic), 8.10 (dd, J1 = 9.1 Hz, J2 = 2.2 Hz, 1H, aromatic), 7.99–7.94 (m, 2H, aromatic), 7.92–7.89 (m, 1H, aromatic), 7.54 (dd, J1 = 8.6 Hz, J2 = 2.5 Hz, 1H, aromatic), 7.31 (d, J = 9.1 Hz, 2H, aromatic), 7.00 (d, J = 8.7 Hz, 1H, aromatic), 6.58 (s, 1H, aromatic), 4.06 (s, 3H, NCH3), 3.73 (s, 5H, CH2Ar, ArOCH3); 13C NMR (100 MHz, (CD3)2SO, Figure S48) d 172.0, 164.1, 154.9 (2C), 140.2, 139.8, 134.6, 131.8, 131.7, 131.5, 128.3, 128.2, 125.9, 120.4 (2CH), 117.5, 116.8, 116.62, 116.58, 116.4, 110.5, 55.5, 37.1, 32.9; m/z calcd for C24H19F2N3O6S 515.4878; found 516.1763 [M+H]+ and sodium adduct 536.1524 (Figure S49); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 13.03 min (97% pure, Figure S50).
Synthesis of compound 22a (SGT1652).
A solution of compound 15 (30 mg, 0.08 mmol), 2-fluorobenzenesulfonamide (20a; 18 mg, 0.10 mmol), EDC•HCl (26 mg, 0.14 mmol), and DMAP (19 mg, 0.15 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.46) to yield compound 22a (50 mg, quant.) as a yellow solid: 1H NMR (500 MHz, (CD3)2SO, Figure S51) d 12.25 (s, 1H, NH), 8.43 (dd, J1 = 2.4 Hz, J2 = 0.5 Hz, 1H, aromatic), 8.00 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.95 (t, J = 7.7 Hz, 1H, aromatic), 7.81–7.75 (m, 1H, aromatic), 7.65 (dd, J1 = 9.2 Hz, J2 = 0.5 Hz, 1H, aromatic), 7.49 (s, 1H, aromatic), 7.47–7.43 (m, 1H, aromatic), 7.30 (d, J = 2.3 Hz, 1H, aromatic), 7.06 (d, J = 8.6 Hz, 1H, aromatic), 4.24 (q, J = 7.3 Hz, 2H, NCH2CH3), 4.07 (s, 2H, CH2Ar), 3.78 (s, 3H, ArOCH3), 1.35 (t, J = 7.3 Hz, 3H, NCH2CH3); 13C NMR (100 MHz, (CD3)2SO, Figure S52) d 164.0, 157.0, 155.3, 140.2, 138.7, 132.9, 131.2, 129.8, 129.1, 126.4, 124.7, 117.5, 117.2, 117.0, 116.6, 116.4, 116.0, 112.2, 110.3, 109.6, 55.9, 40.7, 29.0, 15.4; m/z calcd for C25H22FN3O6S 511.5244; found 512.1273 [M+H]+ and sodium adduct 534.1097 (Figure S53); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 13.03 min (97% pure, Figure S54).
Synthesis of compound 22g (SGT1653).
A solution of compound 15 (30 mg, 0.08 mmol), 4-nitrobenzenesulfonamide (20g; 20 mg, 0.10 mmol), EDC•HCl (26 mg, 0.14 mmol), and DMAP (19 mg, 0.15 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.27) to yield compound 22g (37 mg, 82%) as an orange solid: 1H NMR (500 MHz, (CD3)2SO, Figure S55) d 12.19 (s, 1H, NH), 8.45 (d, J = 8.9 Hz, 2H, aromatic), 8.40 (d, J = 2.3 Hz, 1H, aromatic), 8.20 (d, J = 9.0 Hz, 2H, aromatic), 7.99 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.64 (d, J = 9.1 Hz, 1H, aromatic), 7.48 (s, 1H, aromatic), 7.46 (dd, J1 = 8.6 Hz, J2 = 2.4 Hz, 1H, aromatic), 7.33 (d, J = 2.3 Hz, 1H, aromatic), 7.06 (d, J = 8.6 Hz, 1H, aromatic), 4.23 (q, J = 7.3 Hz, 2H, NCH2CH3), 4.06 (s, 2H, CH2Ar), 3.80 (s, 3H, ArOCH3), 1.33 (t, J = 7.3 Hz, 3H, NCH2CH3); 13C NMR (100 MHz, (CD3)2SO, Figure S56) d 165.1, 155.5, 150.3, 144.6, 140.2, 138.6, 133.5, 133.0, 129.8, 129.4, 129.3, 126.4, 124.4, 121.6, 116.5, 116.4, 115.9, 112.3, 110.3, 56.0, 40.7, 28.9, 15.3; m/z calcd for C25H22N4O8S 538.5310; found 539.1226 [M+H]+ and sodium adduct 561.1050 (Figure S57); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 17.77 min (99% pure, Figure S58).
Synthesis of compound 23a (SGT1654).
A solution of compound 16 (30 mg, 0.08 mmol), 2-fluorobenzenesulfonamide (20a; 17 mg, 0.10 mmol), EDC•HCl (25 mg, 0.13 mmol), and DMAP (18 mg, 0.15 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.46) to yield compound 23a (39 mg, 91%) as a yellow solid: 1H NMR (500 MHz, (CD3)2SO, Figure S59) d 12.25 (s, 1H, NH), 8.42 (d, J = 2.3 Hz, 1H, aromatic), 7.99 (dd, J1 = 9.2 Hz, J2 = 2.4 Hz, 1H, aromatic), 7.95 (t, J = 7.3 Hz, 1H, aromatic), 7.81–7.74 (m, 1H, aromatic), 7.66 (d, J = 9.2 Hz, 1H, aromatic), 7.47 (s, 1H, aromatic), 7.44 (m, 3H, aromatic), 7.29 (d, J = 2.3 Hz, 1H, aromatic), 7.05 (d, J = 8.6 Hz, 1H, aromatic), 4.17 (t, J = 7.0 Hz, 2H, NCH2CH2CH3), 4.07 (s, 2H, CH2Ar), 3.78 (s, 3H, ArOCH3), 1.79 (sextet, J = 7.3 Hz, 2H, NCH2CH2CH3), 0.80 (t, J = 7.4 Hz, 3H, NCH2CH2CH3); 13C NMR (100 MHz, (CD3)2SO, Figure S60) d 159.5, 157.0, 155.3, 140.2, 139.1, 136.5, 133.1, 132.9, 131.2, 130.4, 129.1, 126.3, 124.8, 124.7, 117.2, 117.0, 116.44, 116.37, 115.9, 112.2, 110.4, 59.7, 55.9, 47.3, 28.9, 23.1, 11.0; m/z calcd for C26H24FN3O6S 525.5514; found 526.1448 [M+H]+ and sodium adduct 548.1272 (Figure S61); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 17.86 min (98% pure, Figure S62).
Synthesis of compound 23g (SGT1655).
A solution of compound 16 (30 mg, 0.08 mmol), 4-nitrobenzenesulfonamide (20g; 19 mg, 0.10 mmol), EDC•HCl (25 mg, 0.13 mmol), and DMAP (18 mg, 0.15 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.26) to yield compound 23g (22 mg, 49%) as an orange solid: 1H NMR (500 MHz, CDCl3, Figure S63) d 10.49 (s, 1H, NH), 8.35 (d, J = 9.1 Hz, 2H, aromatic), 8.32 (d, J = 8.8 Hz, 2H, aromatic), 8.30 (d, J = 2.3 Hz, 1H, aromatic), 8.06 (dd, J1 = 9.1 Hz, J2 = 2.2 Hz, 1H, aromatic), 7.90 (d, J = 2.4 Hz, 1H, aromatic), 7.45 (dd, J1 = 8.6 Hz, J2 = 2.5 Hz, 1H, aromatic), 7.28 (d, J = 9.1 Hz, 1H, aromatic), 6.95 (d, J = 8.6 Hz, 1H, aromatic), 6.93 (s, 1H, aromatic), 4.03 (t, J = 7.2 Hz, 2H, NCH2CH2CH3), 4.04 (s, 2H, CH2Ar), 4.02 (s, 3H, ArOCH3), 1.83 (sextet, J = 7.3 Hz, 2H, NCH2CH2CH3), 0.90 (t, J = 7.4 Hz, 3H, NCH2CH2CH3); 13C NMR (100 MHz, CDCl3, Figure S64) d 162.6, 156.7, 150.9, 144.6, 141.3, 139.6, 135.8, 134.4, 132.7, 130.3, 129.5, 127.0, 124.2, 118.3, 117.6, 116.6, 116.5, 112.3, 109.6, 56.9, 48.6, 30.3, 23.8, 11.7; m/z calcd for C26H24N4O8S 552.5580; found 553.1369 [M+H]+ and sodium adduct 575.1195 (Figure S65); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 17.82 min (95% pure, Figure S66).
Synthesis of compound 24a (SGT1657).
A solution of compound 17 (30 mg, 0.08 mmol), 2-fluorobenzenesulfonamide (20a; 16 mg, 0.09 mmol), EDC•HCl (24 mg, 0.13 mmol), and DMAP (17 mg, 0.14 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.63) to yield compound 24a (24 mg, 56%) as a yellow solid: 1H NMR (500 MHz, CDCl3, Figure S67) d 10.57 (s, 1H, NH), 8.33 (d, J = 2.3 Hz, 1H, aromatic), 8.14 (td, J1 = 7.6 Hz, J2 = 1.8 Hz, 1H, aromatic), 8.06 (dd, J1 = 9.1 Hz, J2 = 2.2 Hz, 1H, aromatic), 7.88 (d, J = 2.5 Hz, 1H, aromatic), 7.61–7.57 (m, 1H, aromatic), 7.44 (dd, J1 = 8.6 Hz, J2 = 2.6 Hz, 1H, aromatic), 7.33–7.30 (m, 1H, aromatic), 7.27 (d, J = 9.1 Hz, 1H, aromatic), 7.17–7.13 (m, 1H, aromatic), 6.96 (d, J = 8.6 Hz, 1H, aromatic), 6.89 (s, 1H, aromatic), 4.05 (t, J = 7.3 Hz, 2H, NCH2CH2CH2CH3), 4.05 (s, 3H, ArOCH3), 4.02 (s, 2H, CH2Ar), 1.76 (p, J = 7.6 Hz, 2H, NCH2CH2CH2CH3), 1.30 (sextet, J = 7.7 Hz, 2H, NCH2CH2CH2CH3), 0.91 (t, J = 7.4 Hz, 3H, NCH2CH2CH2CH3); 13C NMR (100 MHz, CDCl3, Figure S68) d 162.7, 156.7, 141.3, 139.6, 136.4, 136.3, 135.5, 134.2, 132.6, 132.3, 129.4, 127.0, 124.7, 124.6, 118.8, 117.6, 117.3, 117.1, 116.6, 112.3, 109.5, 56.9, 46.7, 32.5, 30.3, 20.3, 13.8; m/z calcd for C27H26FN3O6S 539.5784; found 540.1576 [M+H]+ and sodium adduct 562.1398 (Figure S69); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 18.26 min (95% pure, Figure S70).
Synthesis of compound 24g (SGT1656).
A solution of compound 17 (30 mg, 0.08 mmol), 4-nitrobenzenesulfonamide (20g; 19 mg, 0.09 mmol), EDC•HCl (24 mg, 0.13 mmol), and DMAP (17 mg, 0.14 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.45) to yield compound 24g (28 mg, 62%) as a yellow solid: 1H NMR (500 MHz, CDCl3, Figure S71) d 10.49 (s, 1H, NH), 8.35 (d, J = 9.0 Hz, 2H, aromatic), 8.32 (d, J = 9.1 Hz, 2H, aromatic), 8.29 (d, J = 2.3 Hz, 1H, aromatic), 8.05 (dd, J1 = 9.1 Hz, J2 = 2.2 Hz, 1H, aromatic), 7.90 (d, J = 2.5 Hz, 1H, aromatic), 7.45 (dd, J1 = 8.6 Hz, J2 = 2.5 Hz, 1H, aromatic), 7.28 (d, J = 9.1 Hz, 1H, aromatic), 6.95 (d, J = 8.6 Hz, 1H, aromatic), 6.92 (s, 1H, aromatic), 4.06 (t, J = 7.2 Hz, 1H, aromatic), 4.04 (s, 2H, CH2Ar), 4.03 (s, 3H, ArOCH3), 1.77 (p, J = 7.5 Hz, 2H, NCH2CH2CH2CH3), 1.30 (sextet, J = 7.6 Hz, 2H, NCH2CH2CH2CH3), 0.92 (t, J = 7.4 Hz, 3H, NCH2CH2CH2CH3); 13C NMR (100 MHz, CDCl3, Figure S72) d 162.6, 156.7, 150.9, 144.6, 141.3, 139.6, 135.8, 134.4, 132.6, 130.3, 129.4, 126.9, 124.2, 118.3, 117.6, 116.6, 116.5, 112.3, 109.6, 56.9, 46.7, 32.5, 30.3, 20.3, 13.8; m/z calcd for C27H26N4O8S 566.5850; found 567.1532 [M+H]+ and sodium adduct 589.1355 (Figure S73); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 13.02 min (99% pure, Figure S74).
Synthesis of compound 25a (SGT1659).
A solution of compound 18 (30 mg, 0.08 mmol), 2-fluorobenzenesulfonamide (20a; 16 mg, 0.09 mmol), EDC•HCl (24 mg, 0.13 mmol), and DMAP (17 mg, 0.14 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.29) to yield compound 25a (22 mg, 52%) as a yellow solid: 1H NMR (500 MHz, (CD3)2SO, Figure S75) d 12.24 (s, 1H, NH), 8.41 (d, J = 7.7 Hz, 1H, aromatic), 7.98 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.95 (t, J = 7.7 Hz, 1H, aromatic), 7.82–7.75 (m, 1H, aromatic), 7.67 (d, J = 9.1 Hz, 1H, aromatic), 7.45 (s, 1H, aromatic), 7.44 (m, 3H, aromatic), 7.28 (d, J = 2.3 Hz, 1H, aromatic), 7.06 (d, J = 8.6 Hz, 1H, aromatic), 4.07 (s, 2H, CH2Ar), 4.03 (d, J = 7.3 Hz, 2H, NCH2), 3.78 (s, 3H, ArOCH3), 2.09 (septet, J = 7.2 Hz, 1H, CH(CH3)2), 0.82 (d, J = 6.7 Hz, 6H, CH(CH3)2); 13C NMR (100 MHz, (CD3)2SO, Figure S76) d 164.2, 159.6, 155.3, 140.2, 139.4, 136.6, 133.0, 131.2, 130.9, 129.1, 126.3, 124.8, 117.4, 117.3, 117.1, 116.5, 116.3, 115.9, 115.3, 112.2, 110.7, 55.9, 52.9, 29.2, 28.9, 19.7; m/z calcd for C27H26FN3O6S 539.5784; found 540.1599 [M+H]+ and sodium adduct 562.1422 (Figure S77); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 13.03 min (98% pure, Figure S78).
Synthesis of compound 25g (SGT1661).
A solution of compound 18 (30 mg, 0.08 mmol), 4nitrobenzenesulfonamide (20g; 19 mg, 0.09 mmol), EDC•HCl (24 mg, 0.13 mmol), and DMAP (17 mg, 0.14 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.41) to yield compound 25g (5 mg, 12%) as a yellow solid: 1H NMR (500 MHz, CD3OD, Figure S79) d 8.40 (d, J = 8.9 Hz, 2H, aromatic), 8.31 (d, J = 2.2 Hz, 1H, aromatic), 8.27 (d, J = 9.0 Hz, 2H, aromatic), 8.02 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.49 (d, J = 9.2 Hz, 1H, aromatic), 7.48 (d, J = 2.2 Hz, 1H, aromatic), 7.45 (dd, J1 = 8.7 Hz, J2 = 1.8 Hz, 1H, aromatic), 7.23 (s, 1H, aromatic), 7.06 (d, J = 8.5 Hz, 1H, aromatic), 4.09 (s, 2H, CH2Ar), 3.99 (d, J = 7.4 Hz, 2H, NCH2), 3.91 (s, 3H, ArOCH3), 2.15 (septet, J = 7.3 Hz, 1H, CH(CH3)2), 0.89 (d, J = 6.7 Hz, 6H, CH(CH3)2); 13C NMR (100 MHz, (CD3)2SO, Figure S80) d 165.1, 155.5, 150.2, 144.6, 140.1, 139.4, 133.4, 133.0, 130.9, 129.32, 129.26, 126.2, 124.4, 121.6, 116.4, 116.2, 115.9, 112.2, 110.6, 56.0, 52.9, 29.2, 28.8, 19.7; m/z calcd for C27H26N4O8S 566.5850; found 567.1539 [M+H]+ and sodium adduct 589.1361 (Figure S81); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 18.11 min (99% pure, Figure S82).
Synthesis of compound 26g (SGT1658).
A solution of compound 19 (30 mg, 0.07 mmol), 4-nitrobenzenesulfonamide (20g; 17 mg, 0.09 mmol), EDC•HCl (22 mg, 0.12 mmol), and DMAP (16 mg, 0.13 mmol) in anhydrous CH2Cl2 (3 mL) was stirred at room temperature overnight. The reaction was quenched with H2O and extracted with CH2Cl2 (3 × ). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The crude product obtained was purified by column chromatography (SiO2 gel, Hexanes:EtOAc/1:1, Rf 0.42) to yield compound 26g (29 mg, 66%) as a yellow solid: 1H NMR (500 MHz, CDCl3, Figure S83) d 10.49 (s, 1H, NH), 8.35 (d, J = 9.1 Hz, 2H, aromatic), 8.32 (d, J = 9.1 Hz, 2H, aromatic), 8.31 (d, J = 2.3 Hz, 1H, aromatic), 8.02 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H, aromatic), 7.90 (d, J = 2.4 Hz, 1H, aromatic), 7.46 (dd, J1 = 8.6 Hz, J2 = 2.5 Hz, 1H, aromatic), 7.32–7.27 (m, 3H, aromatic), 7.23 (d, J = 9.5 Hz, 1H, aromatic), 7.05 (dd, J1 = 7.9 Hz, J2 = 2.2 Hz, 2H, aromatic), 6.97 (s, 1H, aromatic), 6.96 (d, J = 8.7 Hz, 1H, aromatic), 5.27 (s, 2H, NCH2Ar), 4.05 (s, 2H, CH2Ar), 4.04 (s, 3H, ArOCH3); 13C NMR (100 MHz, CDCl3, Figure S84) d 162.6, 156.7, 150.8, 144.6, 141.6, 139.8, 136.3, 135.8, 134.2, 132.6, 130.3, 129.9, 129.2, 128.3, 127.2, 126.9, 124.2, 118.3, 117.9, 117.0, 116.6, 112.3, 110.0, 56.9, 50.7, 30.3; m/z calcd for C30H24N4O8S 600.6020; found 601.1373 [M+H]+ and sodium adduct 623.1203 (Figure S85); Purity of the compound was further confirmed by RP-HPLC method A: Rt = 17.81 min (97% pure, Figure S86).
Organisms and growth conditions.
All oral bacterial strains were grown from a frozen stock on blood agar plates (BBL, Becton Dickinson, Sparks, MD) and incubated in appropriate aerobic or anaerobic conditions at 37 °C for 24 h or 3 days, respectively, as previously reported.17, 18 In this study the following bacterial strains were used: Porphyromonas gingivalis ATCC 33277 (anaerobic), Actinomyces naeslundii ATCC 49340 (anaerobic), Aggregatibacter actinomycetemcomitans JP2 (anaerobic), Fusobacterium nucleatum ATCC 25586 (anaerobic), Streptococcus sanguinis ATCC 10556 (aerobic), and Veillonella parvula ATCC 10790 (anaerobic). After 3 days of growth, a liquid culture was started for each strain in 3 mL of Brain Heart Infusion (BHI) broth supplemented with 5 μg/mL of hemin and 1 μg/mL of menadione for P. gingivalis, A. naeslundii, A. actinomycetemcomitans, F. nucleatum, and S. sanguinis, or Reinforced Clostridial medium with 60% sodium lactate pH 7 for V. parvula. After 24 h, the liquid cultures were inoculated with the corresponding fresh growth medium and allowed to reach logarithmic growth for 3–4 h, the time at which antimicrobial activity of different treatments was evaluated. All 96-well plates for experiments using oral bacterial species used in this study were VWR Tissue Culture Plates 96-wells-F, sterile flat-bottom plates.
The human embryonic kidney cell line HEK-293 (ATCC CRL-1573), the human bronchus normal cell line BEAS-2B (ATCC CRL-9609), and the liver hepatocellular carcinoma HepG2 (ATCC HB-8065) were kind gifts from the laboratories of Dr. Matthew S. Gentry (University of Kentucky, Lexington, KY, USA), Dr. David K. Orren (University of Kentucky, Lexington, KY, USA), and Dr. Vincent Venditto (University of Kentucky, Lexington, KY, USA), respectively. Mammalian cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (from ATCC) with 10% fetal bovine serum (FBS) (from ATCC) and 1% Pen/Strep (from ATCC). Cell lines were incubated at 37 °C and 5% CO2 and passaged by trypsinization with 0.05% trypsin:0.53 mM EDTA (from ATCC). Cell confluency was determined by using a Nikon Eclipse TS100 microscope (Minato, Tokyo, Japan).
Effect of zafirlukast (ZAF) derivatives on the viability of different bacterial strains.
The antimicrobial effect of ZAF derivatives 21a-g, 22a, 22g, 23a, 23g, 24a, 24g, 25a, 25g, and 26g was investigated using a colorimetric water-soluble tetrazolium-1 (WST-1) assay as previously reported.17, 18, 35 The WST-1 cell proliferation reagent acts as a surrogate marker for cell proliferation and viability. All bacterial strains were logarithmically grown in appropriate broth (90 μL) using 96-well plates (108/well) and were incubated with different treatments for 24 h. Then 10 μL of WST-1 reagent (Roche, Mannheim, Germany) was added to each well. The treated plates were then incubated for 2.5 h under the appropriate culture conditions and the absorbance was measured at 405 nm, with a reference wavelength at 600 nm, by using a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA). The data for percent growth inhibition assays with P. gingivalis 2.5 h after the addition of the WST-1 reagent are presented in Figures 2 and S88. Whereas the data for 30 minutes after the addition of the WST-1 reagent are presented in Figure S87. The optical densities (ODs) at 405 nm of bacterial metabolic activity were used to calculate the percentage of inhibitory effect (i.e., % growth inhibition) under different experimental conditions by utilizing the following formula: 100 × [OD (control) - OD (experimental) / OD (control)]. For F. nucleatum and P. gingivalis, negative controls were bacteria exposed to only medium (sterility control) or DMSO, and positive controls were bacteria exposed to 2.81 μM of tetracycline (Tet, equivalent to 1.25 μg/mL, a standard concentration usually used for this control) and ZAF at 25 and 50 μM diluted in DMSO (concentrations that previously showed antimicrobial effect against P. gingivalis). The 2nd and 3rd generation ZAF derivatives were tested at 1, 10, and 100 μM. For testing against the other oral bacterial species of A. naeslundii, A. actinomycetemcomitans, S. sanguinis, and V. parvula, control cultures were treated with medium only (sterility control), exposed to DMSO (vehicle for ZAF and its derivatives, negative control), or treated with penicillin/streptomycin 1X (100 U/mL of penicillin and 100 μg/mL of streptomycin) or ZAF at 25 and 50 μM diluted in DMSO as positive controls. These data are presented in Figures 4 and S89.
Colony forming unit (CFU) assays.
The bactericidal effect of the most active ZAF derivatives (21a-d, 21g, 22g, 23g, 24g, and 25g), which exhibited the best antimicrobial potential at lower concentrations as determined by the WST-1 assay, were further investigated by measuring colony forming units per milliliter (CFUs/mL) after different treatments. P. gingivalis (108/well) was grown under anaerobic conditions in the appropriate medium in 96-well plates. The bacteria were exposed to only medium or DMSO as negative controls, and exposed to 2.81 μM of Tet and ZAF at 25 and 50 μM diluted in DMSO as positive controls. The bacteria were also treated with ZAF derivatives (1, 10, and 100 μM) dissolved in DMSO. After 24 h, the treated bacteria were diluted (1:400) and 100 μL of the diluted samples 21a-d, 21g, 22g, 23g, 24g, and 25g were plated and spread onto blood agar plates. The number of CFUs were counted after 5 days of incubation on the blood agar plate in an anaerobic chamber. These data are presented in Figure 5.
Disruption of pre-formed biofilms of P. gingivalis.
The most active ZAF derivatives (21a-c, 21g, 22g, and 24g), which exhibited the best antimicrobial potential at lower concentrations as determined by the WST-1 assay, were further investigated by determining their ability to disrupt pre-formed biofilms of P. gingivalis ATCC 33277. Liquid cultures of P. gingivalis were grown as described above. Then P. gingivalis (1 × 108 bacteria/well)27, 31, 36 was grown for 48 h with gentle shaking under anaerobic conditions in supplemented BHI medium in flat-bottom VWR® multiwell cell culture 96-well plates (100 μL). The 96-well plates were gently shaken on a Scilogex MX-M Microplate Mixer (Scilogex LLC, Rocky Hill, CT). After biofilm formation, the old medium was removed and the biofilm was washed three times with phosphate-buffered saline (PBS), and then 100 μL of BHI was added to each well. Then, the bacteria were exposed to 100 μL of different treatments for 24 h. The bacteria were exposed to only medium or DMSO as negative controls, and exposed to 2.81 μM of Tet and 50 μM of ZAF diluted in DMSO as positive controls. The bacteria were also treated with ZAF derivatives (50 and 100 μM) dissolved in DMSO. The old medium was removed, the biofilm was washed three times with PBS, and 100 μL of 0.1% safranin was added to each well. After 15 min, the excess safranin was removed by washing four times with PBS. To quantify the amount of biofilm, 100 μL of 95% EtOH was added to each well and after 5 min (time to solubilize the safranin), the EtOH was transferred to a new 96-well plate and the absorbance was measured at 492 nm, with a reference wavelength at 600 nm, by using a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA). The percentage of biofilm growth was determined by utilizing the following formula: 100 × [OD600 (experimental) / OD600 (DMSO control)]. These data are presented in Figure 6.
Inhibition of P. gingivalis biofilm growth.
The most active ZAF derivatives (21a-c, 21g, 22g, and 24g) were further investigated by determining their ability to inhibit biofilm growth of P. gingivalis ATCC 33277. Liquid cultures of P. gingivalis were grown as described above. P. gingivalis was logarithmically grown in supplemented BHI (100 μL) using flat-bottom VWR® multi-well cell culture 96-well plates (1 × 108 bacteria/well)27, 31, 36 and was exposed to different treatments (100 μL) for 48 h with gentle shaking under anaerobic conditions. The bacteria were exposed to only medium or DMSO as negative controls, and exposed to 2.81 μM of Tet and 50 μM of ZAF diluted in DMSO as positive controls. The bacteria were also treated with ZAF derivatives (10, 25, 50, and 100 μM) dissolved in DMSO. The old medium was removed and the biofilm was washed three times with PBS, and 100 μL of 0.1% safranin was added to each. After 15 min, the excess safranin was removed by washing four times with PBS. To quantify the amount of biofilm, 100 μL of 95% EtOH was added to each well and after 5 min (time to solubilize the safranin), the EtOH was transferred to a new 96-well plate and the absorbance was measured at 492 nm, with a reference wavelength at 600 nm, by using a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA). The percentage of biofilm growth was determined by utilizing the following formula: 100 × [OD600 (experimental) / OD600 (DMSO control)]. These data are presented in Figure 7.
Cytotoxic effect of ZAF derivatives on oral epithelial cells.
The immortalized oral keratinocyte cell line OKF6/hTERT (OKF6) were used in viability assays34 to evaluate the cytotoxic effect of ZAF derivatives 21b, 21c, and 24g. The OKF6 cells were cultured in keratinocyte-serum free medium (SFM) supplemented with bovine pituitary extract (25 μg/mL) and recombinant epidermal growth factor (0.2 ng/mL) (Ker-SFM). The cells were grown at 37 °C in a humidified incubator with 5% CO2. The cells were treated with negative controls of medium only (sterility control) or DMSO and positive controls were 8 μM of staurosporine (STS) or 25 μM of ZAF. Then a 1:1 ratio of cell suspension and trypan blue were mixed and 10 μL were counted in an automated cell counter (Countess II FL, Life Technologies, Singapore) to determine the percentage of alive and dead cells. The counted cells were used to establish the volume needed to be added to each well of 24-well plates to provide 105 cells/well. To perform the viability assays, cells were seeded in 24-well plates overnight in Ker-SFM medium and the cells were further exposed to ZAF derivatives (10 and 100 μM) dissolved in DMSO or appropriate negative and positive controls for 24 h. The morphology and density of the treated cells were documented by using a light inverted microscope Nikon Eclipse Ti-Series (Nikon Instruments Inc., Melville, NY). One well from each control treatment was visualized along with one well from each 10 μM concentration of the ZAF derivatives, 21b, 21c, and 24g. These data are presented in Figure 8. Flow cytometry analysis (FACS) was used to establish the cytotoxic effects of ZAF derivatives. OKF6 cells were harvested by trypsinization 24 h after exposure to treatments, washed with phosphate-buffered saline (PBS), pelleted at 1100 rpm for 5 min, and were further labeled with 5 μL of FITC-Annexin V and 5 μL of propidium iodide (BD Pharmingen, San Jose, CA) for 15 min at room temperature. The FACS analyses were done for at least 10,000 acquired events in a flow cytometer FACSCalibur (Becton Dickinson, San Jose, CA). These data are presented in Figure 9 as well as in Table S1.
Cytotoxic effect of ZAF derivatives on mammalian cell lines.
Mammalian cytotoxicity assays were performed as previously described with minor modifications.37 HEK-293, BEAS-2B, and HepG2 cells were cultured as described in the section “organisms and growth conditions” above and were counted by a hemocytometer when cells were about 80% confluent in flasks. The mammalian cells were plated in 96-well microtiter plates at concentrations of 10,000 cells per well for HEK-293 and 3,000 cells per well for BEAS-2B and HepG2. The plates were then incubated at 37 °C and 5% CO2 for 16 h to allow time for the cells to adhere to the wells. The medium was removed and fresh medium with ZAF or compounds 21a-c at 0–62.6 μg/mL were added. The compounds were previously prepared in stock solutions at 1000 × of the intended tested concentrations. The cells were treated with negative controls of medium only (sterility control) or 0.1% DMSO, and the positive control contained 20% Triton™ X-100. The percentage of cell survival was evaluated after 24 h of incubation via addition of resazurin (10 μL of 10 mM solution) for 6 h. A SpectraMax M5 plate reader was used to detect live cells by a color change from purple to pink depicting the conversion of the compound to resorufin, which could be quantified at λ560 absorption and λ590 emission. The percent cell survival rates were calculated by using the following equation: % cell survival = [(fluorescence of sample) - (fluorescence of negative control with medium only)] × 100 / [(fluorescence of DMSO negative control) - (fluorescence of negative control with medium only)]. These experiments were performed in quadruplicate. The normalized data are presented in Figure S90 (Note: For instances where >100% cell survival was observed, the data was displayed as 100% cell survival). The corresponding non-normalized data are presented in Figure S91.
Hemolytic effect of N-alkyl ZAF derivatives.
The hemolytic activity of ZAF and its derivatives 21a-c was determined by using methods previously described with minor modifications.38 In 4 mL of PBS, murine whole blood was suspended and centrifuged at 1,000 rpm for 10 min at room temperature to obtain the murine red blood cells (mRBCs). Then the mRBCs were washed 4 × in PBS and resuspended in the same buffer to a final concentration of 1 × 107 erythrocytes/mL. A two-fold serial dilution of ZAF and compounds 21a, 21b, and 21c was prepared using 100 μL of PBS buffer in Eppendorf tubes followed by the addition of 100 μL of mRBC suspension that produced the final concentrations of compounds as 3.13–100 μM and 5 × 106 erythrocytes/mL, respectively. The tubes were incubated at 37 °C for 1 h. The negative control was tubes with 200 μL of PBS and the positive control was Triton™ X-100 (1% v/v, 2 μL). The percentage of hemolysis was calculated using the equation: % hemolysis = [(absorbance of sample) - (absorbance of negative control)] × 100 / (absorbance of positive control). These data are presented in Figure 10 as well as in Table S2.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by a National Institutes of Health (NIH) F31 fellowship DEO29661 (to K.C.H.). We thank the UK PharmNMR Center (in the College of Pharmacy) for NMR support. We thank Dr. Octavio A. Gonzalez (University of Kentucky) for discussions as well as help with reagents and use of instruments. We thank Dr. Nishad Thamban Chandrika for his help with HPLC and HRMS. We thank Dr. J. G. Rheinwald (Harvard Medical School) for sharing the oral keratinocyte cell line OKF6/hTERT (OKF6).
ABBREVIATIONS
- BEAS-2B
human bronchus normal
- BHI
brain heart infusion
- CFU
colony forming unit
- CH2Cl2
dichloromethane
- DMAP
4-dimethylaminopyridine
- DMSO
dimethyl sulfoxide
- EDC•HCl
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
- Et3SiH
triethylsilane
- FACS
fluorescence-activated cell sorting
- FITC
fluorescein isothiocyanate
- FDA
food and drug administration
- HEK-293
human embryonic kidney
- HepG2
liver hepatocellular carcinoma
- HPLC
high-performance liquid chromatography
- KOH
potassium hydroxide
- MeOH
methanol
- mRBC
murine red blood cell
- NMR
nuclear magnetic resonance
- OD
optical density
- OKF6
immortalized oral keratinocyte cells
- PBS
phosphate-buffered saline
- P/S
penicillin/streptomycin
- SAR
structure-activity relationship
- STS
staurosporine
- Tet
tetracycline
- TFA
trifluoroacetic acid
- THF
tetrahydrofuran
- WST-1
water-soluble tetrazolium-1
- ZAF
zafirlukast
Footnotes
SUPPORTING INFORMATION AVAILABILITY
The supporting information includes Tables for cytotoxicity of ZAF derivatives on OKF6 cells (Table S1) and for % hemolysis caused by ZAF derivatives (Table S2). It also includes all 1H and 13C nuclear magnetic resonance (NMR) as well as HRMS spectra along with high-performance liquid chromatography (HPLC) traces for the final compounds tested. Figures for 30 min % growth inhibition of P. gingivalis with ZAF derivatives, heat maps for % growth inhibition of P. gingivalis and other oral bacterial species with ZAF derivatives, as well as normalized and non-normalized cytotoxicity data for ZAF derivatives on mammalian cell lines are also presented. The Molecular Formula Strings are provided for all compounds synthesized in the CSV file. This material is available free of charge via the Internet.
CONFLICTS OF INTEREST
The authors report no conflicts of interest related to this work.
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