Scientific paper Exploration of the Chemical Space of Novel Naphthalene-Sulfonamide and Anthranilic Acid-Based In hi bi tors of Peni cil lin-Bin ding Pro teins Izidor Sosi~,1 Samo Turk,1 Masa Sinreih,1 Nu{a Tro{t,1 Olivier Verlaine,2 Ana Amoroso,2 Astrid Zervosen,3 André Luxen,3 Bernard Joris2 and Stanislav Gobec1 1 Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia 2 Centre d'Ingenierie des Protéines, Institut de Chimie, B6a, Université de Liège, B4000 Sart Tilman, Liège, Belgium 3 Centre de Recherches du Cyclotron, B 30, Université de Liège, B-4000 Sart Tilman, Liège, Belgium * Corresponding author: E-mail: stanislav.gobec@ffa.uni-lj.si Tel: +386-1-476-9500; Fax: +386-1-425-8031 Received: 27-02-2012 Abstract Penicillin-binding proteins are a well established, validated and still a very promising target for the design and development of new antibacterial agents. Based on our previous discovery of several noncovalent small-molecule inhibitor hits for resistant PBPs we decided to additionally explore the chemical space around these compounds. In order to clarify their structure-activity relationships for PBP inhibition two new series of compounds were synthesized, characterized and evaluated biochemically: the derivatives of anthranilic acid and naphthalene-sulfonamide derivatives. The target compounds were tested for their inhibitory activities on three different transpeptidases: PBP2a from methicillin-resi-stant Staphylococcus aureus (MRSA) strains, PBP5fm from Enterococcusfaecium strains, and PBPlb from Streptococcus pneumoniae strains. The most promising results for both of these series of compounds were obtained against the PBP2a enzyme with the IC50 values in the micromolar range. Although these results do not represent a significant breakthrough in the field of noncovalent PBP inhibitors, they do provide useful structure-activity relationship data, and thus a more solid basis for the design of potent and noncovalent inhibitors of resistant PBPs. Keywords: Penicillin-binding proteins, penicillin-resistance, noncovalent inhibitors, optimization, anthranilic acid 1. Introduction The fast development of antibacterial agents in the second half of the 20th century resulted in remarkable advances for humanity. The rate of mortality caused by infectious diseases has decreased dramatically, and it began to appear that we were the winners in the battle against pathogens. However, the appearance of multidrug resistant strains of pathogenic bacteria has become a serious medical issue in modern healthcare. The incidence of so-called 'superbugs' (organisms that are resistant to most of the clinically used antibiotics) is rapidly increasing. In 2004, more than 70% of pathogenic bacteria were estima- ted to be resistant to at least one of the currently available antibiotics.1-3 Approximately 20 new antibiotics were approved from 2000 to 2010, of which just three had novel mechanisms of action: the lipopeptide daptomycin, oxa-zolidinone linezolid, and retapamulin that belongs to the natural class of compounds called pleuromutilins. Therefore, there is a serious and, unfortunately, unmet need for new antibacterial agents to treat these increasing levels of drug-resistant infections.4 Inhibition of bacterial cell-wall synthesis is a well-known mechanism of action of various antibiotics. In the synthesis of peptidoglycan, which is a major component of the cell wall, many enzymes are involved, and all of them are potential targets for antibacterial agents. The last two steps of peptidoglycan biosynthesis are particularly attractive targets for potential antibacterial compounds, as they take place on the external surface of the cytoplasmic membrane and are therefore readily accesible. The transglycosylation and transpeptidation reactions are catalyzed by penicillin-binding proteins (PBPs). Bacteria have multiple PBPs that are generally membrane bound. PBPs can be divided into two classes: high molecular mass (HMM) and low molecular mass (LMM) PBPs. The bifunctional (class A) HMM PBPs catalyze both polymerization of the GlcNAc-MurNAc chains and cross-linking of the adjacent stem peptide, while the monofunctional (class B) enzymes only catalyze transpeptidation (for excellent reviews, see5,6). Inhibition of HMM PBPs leads to cell death. The LMM PBPs are sometimes referred to as the class C PBPs;5 their inhibition is generally not lethal, and it leads to a change in the cell-wall cross-linking pat-tern.5,6 Transpeptidation activities of PBPs are inhibited by P-lactam antibiotics that act as covalent inhibitors (including penicillins, cephalosporins, monobactams, and car-bapenems). Once a PBP is acylated by a P-lactam antibiotic, it can no longer catalyze hydrolysis of the covalent acyl-enzyme intermediate, and it is inactivated; pepti-doglycan transpeptidation can no longer occur, which results in bacterial death. However, bacteria have developed resistance to P-lactams by mechanisms that include: the production of P-lactamases, which can hydrolyze the P-lactams; the use of antibiotic efflux pumps, which can actively secrete these compounds from the periplasm of Gram-negative bacteria; and the production of P-lactam-insensitive PBPs. This last arises from the altered structure of the transpeptidase domain; therefore, covalent complex formation between the enzyme and P-lactams is hindered, which results in less effective antibiotics. The most representative examples are the highly mutated PBP2x from Streptococcus pneumoniae, the acquired low-affinity additionnal PBP in the case of PBP2a from methicil-lin-resistant Staphylococcus aureus (MRSA), and the overproduced low-affinity PBP5fm from Enterococcus faecium.5,7 One of the possibilities to overcome this intrinsic poor acylation efficiency of resistant PBPs is to move away from the classical P-lactam scaffold, and thus to design new noncovalent compounds that bind tightly to the active site without acylation. Noncovalent inhibitors will not require the unfavorable conformational changes in the active site of resistant PBPs that are required for acyla-tion, and they will also not be susceptible to P-lactama-ses.5,8 To date, only a few noncovalent inhibitors of resistant PBPs have been described, and these have shown only medium potency.9,10 The discovery of true potent non-covalent inhibitors of resistant PBPs thus remains a highly demanding and challenging task for medicinal chemistry. Such molecules will either kill the bacteria directly, or will make them more susceptible to existing antibiotics. Recently, we discovered several noncovalent small-molecule inhibitor hits for resistant PBPs (e.g., Figure 1, compounds a, b).11 As the synthetic preparation of new derivatives is relatively straightforward, a wide variety of possible structural modifications is possible to explore the chemical space of these compounds. Therefore, we decided to use these two compounds as a basis for further studies in the field of noncovalent inhibitors of PBPs from penicillin-resistant bacterial strains. a, ICso (PBP2a) = 230 jiM b, ICg0 (PBP5fm) = 930 j.iM Figure 1. Structural formulae of two previously published PBP inhibitors.11 2. Chemistry The previously published series of compounds was very limited, and thus it was not very representative.11 We wanted to additionally explore the chemical space around these compounds, and consequently to clarify their structure-activity relationships for PBP inhibition. Therefore, two new series of compounds were synthesized: the derivatives of anthranilic acid and naphthalene-sulfonamide derivatives. All in all, 19 anthranilic-acid-based compounds and 15 naphthalene-sulfonamide-containing compounds were prepared, characterized and evaluated biochemically. Substituted derivatives of methyl anthranilate I-IV were obtained from unprotected anthranilic acids with the use of SOCl2 in MeOH, while derivatives V-VII were purchased from Sigma Aldrich and used in the subsequent reactions without purification (Table 1). Very successful amide bond formation was achieved by in-situ transformation of the carboxylic group of benzoic acid derivatives VIII-X into acid chlorides using SOCl2, followed by the addition of the corresponding C-protected anthranilic acid derivative I-VII into the reaction mixture (Scheme 1, compounds 1-11). Compounds 10 and 11 were additionally derivatized afterwards, via formation of an alkyl ether on the OH group at the meta position with respect to the carboxamide moiety (Scheme 2). As shown in Scheme 3, esters 1-10 and 12-24 were deprotected with alkaline hydrolysis to yield the target compounds 15-27. Moreover, compounds with an aromatic NO2 group (15-17, 20, 23, 27) were reduced into their corresponding amines by catalytic hydrogenation (28-33). As expected, when catalytic hydrogenation was performed to reduce the nitro group of compounds 16 and Table 1. Structural formulae of the substituted methyl anthranilate derivatives. °2NT^TX°Me ^NH, O WOM, Me 0 -Wo- MeO'^^NHi HOYVOMe 0 iP^OMe BtvV"0Me 0 c'Tr1L°Me 1 II III IV V VI VII Scheme 1. Reagents and conditions: (a) SOCl2, CH2Cl2, py, 45 °C; (b) corresponding methyl anthranilate derivatives I-VII (Table 1), toluene, reflux. Scheme 2. Reagents and conditions: (a) Br(CH2)nCH3, K2CO3, DMF, 50 °C. 20, the concomitant dehalogenation of a halogen at the meta position with respect to the carboxyl group occurred, to yield compounds 29 and 31, respectively.1213 The removal and replacement of aromatic halogen substituents by hydrogen under conditions of hydrogenation over tran- sition metal catalysts presumably involves intermediates formed by oxidative addition to the metal active catalyst, followed by reductive elimination.14 The naphthalene-sulfonamide derivatives were synthesized as shown in Scheme 4. Variously substitu- Scheme 3. Reagents and conditions: (a) 1 M NaOH, dioxane/THF, room temp; (b) H2, Pd/C, MeOH, room temp. Scheme 4. Reagents and conditions: (a) corresponding amine, py, CH2Cl2, room temp; (b) 1 M NaOH, dioxane, room temp. ted sulfonyl chlorides were successfully reacted with sary, the methyl esters were hydrolyzed under alkaline different aromatic amine derivatives, using pyridine as conditions to the carboxylic acids 49-60 in the next a base, to yield the sulfonamides 34-48. When neces- stage. 3. Results and Discussion 3.1. Inhibitory Activities The target compounds were tested for their inhibitory activities on three different transpeptidases: PBP2a from MRSA strain, PBP5fm from E. faecium strain, and PBPlb from S. pneumoniae strain (Tables 2 and 3). The first two enzymes are representatives of the resistant types of transpeptidases. The results were determined as residual activities of the enzymes in the presence of 1 mM of each compound, and also as IC50 values for the most active derivatives. The most promising results for both of these series of compounds were obtained against the PBP2a enzyme. The majority of compounds were much weaker PBPlb inhibitors, and none of the compounds were active against the PBP5fm enzyme. Table 2. PBP inhibitory potencies of anthranilic acid derivatives.3 Compound RAb of PBP2a (%) RAb of PBP5fm (%) RAb of PBP1b (%) 15 77 85 92 16 8 IC50 = 534 ^M 86 82 17 84 96 106 18 94 92 116 19 88 84 108c 20 9 IC50 = 685 ^M 81 99d 21 48 88 99 22 94 84 86 23 64 96 100 24 50 IC50 = 1 mM 86 102 25 5 IC50 = 490 ^M 83 108 26 2 IC50 = 352 ^M 85 112d 27 6 IC50 = 520 ^M 70 91 28 79 86 113 29 83 81 96 30 83 84 80 31 66 85 95 32 67 82 105c 33 51 IC50 = 1 mM 81 97 a Data are means of three independent experiments, each performed in duplicate. The standard deviations were within ±10% of these means. b Except where noted otherwise, the residual activities (RAs) were determined at 1 mM of each compound. c RAs determined at 100 |M of each compound. d RAs determined at 200 ||M of each compound. 3.1.1. Anthranilic-Acid-Based Derivatives In our exploration of the chemical space of newly synthesized anthranilic-acid-based derivatives, and to determine the influences of the different substitution patterns on both of the phenyl rings, several functionalities were introduced onto both of these rings (Scheme 3, Table 2). As we previously established that on the phenyl rings the meta position with respect to both the carboxyl moiety and the carboxamide moiety is the most influential for PBP2a inhibition, we focused most of our attention on the exploration of possible substituents on these positions. Unfortunately, most of the new substituents did not improve the PBP2a inhibitory activity of the compounds in comparison with the initial hit a (Figure 1). However, valuable structural data can be obtained on the basis of the results of the enzymatic evaluation of these new compounds. For instance, we have once again confirmed the importance of a large bromine atom at the meta position with respect to the carboxyl group, to maintain moderate PBP2a inhibition (compounds 20, 24-26). The absence of Br at the meta position completely abrogated the inhibitory activity (compare the residual activities between compounds 19 and 20, or 22 and 24, Table 2). Also, the introduction of a methoxy, nitro or amino group at the meta position led to reduced inhibition of PBP2a (e.g. compounds 17, 23 and 32). It was cleary seen that the length of the alkyloxy chain positioned meta with respect to the carboxamide moiety has an important role in PBP2a inhibition. The compound with a butoxy fragment showed better inhibitory activity when compared with its ethoxy analog (compounds 25 and 26; IC50 values of 490 |M and 352 |M, respectively). Moreover, when the chain was removed, the activity was significantly decreased (compound 24; IC50 = 1 mM). It also appears that additional elongation of the chain is not desirable, as the inhibitory activity of the pro-poxy analog a (IC50 = 230 |M) was slightly better than for compound 26 with the butoxy chain. Also, when the nitro group at the meta position with respect to the carboxamide moiety was accompanied by Cl or Br at the appropriate position on the second phenyl ring, this led to compounds with desired inhibitory properties (compounds 16 and 20; IC50 values of 534 |M and 685 |M, respectively). The compounds without the halogen atom (compound 19) or with a hydroxy group (compound 15) positioned meta with respect to the carboxyl group did not show inhibitory activity against PBP2a. The only compound that showed desirable properties and lacked the halogen at the appropriate position was compound 27 (IC50 = 520 |M), which had an additional NO2 moiety positioned para with respect to the carboxamide group. To better understand the binding mode of these compounds, a docking study was performed. The most active compound from this series, compound 26, formed several interactions with the active site. Oxygen from the Table 3: PBP inhibitory potencies of the naphthalene-sulfonamide derivatives.3 Com- R1 pound R2 or R 38 49 50 51 52 54 RAb of RAb of RAb of PBP2a PBP5fm PBPlb (%) (%) (%) OH jö 98 "¿0 11 IC = 425 ^M 100 100 97 90 100 to Br IC_0 = 80 flM 43 H OH J> 53 H O HOJ--Q ICf(| = 74(1 MM >V H H 92 100 55 H 56 H o ixr 98c 97 100 112 7 100 40 92 100 131 30 92 89 111 100 100 98 14 100 5 IC50 = 297 (jM IC50 = 540 |uM 48 N(Me). OH 96 100 99d Com- R1 R2 or R3 RAb of RAb of RAb of pound PBP2a PBP5fm PBP1b (%) (%) (%) 57 N(Me). 92 77 IC50 = 536 ^M 58 N(Me)2 84 89 61 59 N(Me)2 99 99 104 60 N(Me )2 2 U -¿a8' 4 86 1 IC50 = 245 nM IC50 = 416 nM a Data are means of three independent experiments, each performed in duplicate. The standard deviations were within ±10% of these means. b Except where noted otherwise, the residual activities (RAs) were determined at 1 mM of each compound. c RA determined at 100 of compound. d RA determined at 200 ||M of compound. butoxy group formed H-bonds with Thr444 and Asn464, the amide oxygen formed an H-bond with Thr600, and the carboxylic acid group formed either H-bonds with Ser403 and Thr600 or electrostatic interactions with Lys406 (Figure 2). There were additional n-n interactions between the aromatic rings and Tyr446 (not shown, for clarity). However, the benefits of the bromine atom cannot be rationalized from these docking studies. A survey of the literature leads us to hypothesize that the Br is involved in the formation of a halogen bond,15 although this effect could not be confirmed in silico because the docking programs Figure 2. Docked conformation of compound 26 (magenta) into the PBP2a active site. The relevant amino-acid residues are shown as green sticks. do not include a term for halogen bonds in their scoring functions. 3.1. 2. Naphthalene-Sulfonamide-Based Derivatives Better results were obtained with the second series of compounds, the naphthalene-sulfonamide derivatives. Similar to what was seen above, the best results were obtained against the PBP2a enzyme. The most potent of this series was compound 52, with an IC50 of 80 pM against PBP2a. Moreover, as well as inhibition of PBP2a, two of the compounds showed moderate PBPlb inhibition (Table 3). In contrast to the starting compound b, none of the compounds from this series inhibited PBP5fm. When comparing the results presented in Table 3, some structure-activity relationship data can be extracted. For instance, it appears that the carboxyl group on the phenyl ring at the ortho position with respect to the sulfo-namide group was beneficial for the inhibition of PBP2a. Moreover, the best inhibitors additionally had the bromine atom positioned meta with respect to the carboxyl moiety (compounds 52, 56 and 60; IC50 values of 80 pM, 297 pM and 245 pM, respectively). The importance of the bromine atom for improvement of the inhibitory activity is un-disputable if we compare compounds 49 and 52, where the introduction of the Br led to more than 5-fold improved inhibitory activity (IC50 reduced from 425 pM to 80 pM). The influence of the dimethylamino group at position 5 of the naphthalene ring was, on the other hand, less pronounced, as the IC50 was only slightly decreased when the -N(CH3)2 moiety was introduced (compare the IC50 values of compounds 53, 57 and 56, 60). The better inhibitory activities of compounds 49 and 52 in comparison with 53 and 56 indicates that the position of the sulfona-mide moiety on the naphthalene ring also has a role in the inhibition of PBP2a. The better inhibitors were compounds with the sulfonamide moiety at position 2 of the naphthalene ring, as the results showed from two-fold to three-fold increases in the IC50 values for 1-substituted naphthalene derivatives. Compounds 56 and 60 also inhibited the PBPlb enzyme with IC50 values of 540 pM and 416 pM, respectively. Again, it appears that it is the 5-bromo-2-sulfonami-do benzoic acid that contributes most to the binding affinity. Compounds without the bromine atom positioned meta with respect to the carboxylic acid (compounds 53 and 57) did not inhibit PBPlb at all. Contrary to the PBP2a inhibition, for the inhibitory activity against PBPlb, the substitution via position l of the naphthalene ring was favorable, which is clearly seen from a comparison of the inhibitory activities of compounds 52 (residual activity, 40%) and 60 (residual acivity, 1%; IC50 = 416 pM). Similar to PBP2a, the positive role of the dimethylamino group for inhibition of PBP1b was not substantial, as only a slight improvement was observed. Figure 3 shows the plausible binding mode of compound 52 in the active site of PBP2a. The FlexX program predicted 3 hydrogen bonds between the inhibitor and the active site of the enzyme. The sulfonamide oxygen atoms enabled the formation of two H-bonds: one with Thr600 and the other with Asn464. Based on the position of the sulfonamide moiety in the active site, we can postulate that it acts as a natural substrate D-Ala-D-Ala peptide bond mimetic. A third hydrogen bond can be formed between the carboxyl group of the inhibitor and the residue Asn464. This undisputedly confirms the importance of the position of the carboxyl group. All of the above-mentioned amino-acid residues are a part of the conserved motifs that are characteristic of the transpeptidase domain and that define the active site of the enzyme. Additional n-in-teractions were possible between the naphthalene ring and Tyr446 (not shown, for clarity). Similar to what was seen above, no obvious benefits of the bromine atom could be observed from these docking studies. Figure 3. Docked conformation of compound 52 (magenta) into the PBP2a active site. The relevant amino-acid residues are shown as green sticks. We also predicted the binding mode of compounds 56 and 60 (data not shown) into the active site, and compared their positions with the most potent compound (52). The H-bonding patterns remained the same; however, some differences in the naphthalene ring orientation were noted, which possibly contribute to the less favorable inhibition. The influence of the dimethylamino group (compound 60) is not entirely clear, although the docking study suggested that this moiety is responsible for contact with the solvent. 3. 2. Antimicrobial Activity Evaluation For the compounds that showed enzyme inhibitory activity, their in-vitro antibacterial activities were determined using a panel of different Gram-positive and Gram-negative bacterial strains. These data are given in Table 4. Table 4. MIC determinations from the antimicrobial testing. Bacterial strain 16 20 25 26 27 Compound 49 52 53 56 57 60 Escherichia coli ATCC 8739 >128 >128 >128 >128 >256 >128 >128 >128 >128 >128 >128 Proteus mirabilis ATCC 29936 >128 >128 >128 >128 >256 >128 >128 >128 >128 >128 >128 Klebsiella pneumoniae ATCC 13883 >128 >128 >128 >128 >256 >128 >128 >128 >128 >128 >128 Citrobacter freundii ATCC 8090 >128 >128 >128 >128 >256 >128 >128 >128 >128 >128 >128 Pseudomonas aeruginosa ATCC 27853 >128 >128 >128 >128 >256 >128 >128 >128 >128 >128 >128 Micrococcus luteus ATCC 9341 >128 >128 >128 32 128 >128 >128 >128 >128 >128 >128 Bacillus subtilis ATCC 6633 128 >128 64 8 128 >128 128 >128 128 >128 64 Listeria innocua ATCC 33090 128 >128 64 4 128 >128 64 >128 128 >128 64 Listeria monocytogenes ATCC 14780 128 >128 64 4 128 >128 32 >128 128 >128 64 Staphylococcus epidermidis ATCC 12228 128 >128 8 4 256 >128 128 >128 64 >128 64 Staphylococcus aureus ATCC 25923 128 >128 128 64 128 >128 128 >128 64 >128 128 Staphylococcus aureus PL1 (inducible MRSA) 128 >128 8 4 128 >128 128 >128 128 >128 128 Staphylococcus aureus ATCC 43300 (MRSA) 128 >128 8 2 128 >128 128 >128 128 128 64 Enterococcus faecalis ATCC 7937 128 >128 128 32 128 128 128 >128 128 128 64 Enterococcus faecalis ATCC 29212 128 >128 128 32 128 128 128 >128 128 128 64 Enterococcus faecium ATCC 19434 128 >128 128 64 >256 128 128 >128 128 128 128 Enterococcus hirae ATCC 9790 128 >128 128 64 128 128 128 >128 128 128 128 Most of the compounds from both of the series were unfortunately found to be weak inhibitors of bacterial growth, with minimum inhibitory concentrations (MICs) around 128 |g/mL. This might be ascribed to their poor on-target activities. Nevertheless, some compounds showed good antibacterial activities; e.g., compounds 25 and 26. This latter showed antibacterial activity against Listeria innocua, Listeria monocytogenes, Staphylococcus epi-dermidis and Bacillus subtilis strains, with MICs of 4 |g/mL or 8 |g/mL. Interestingly, compound 26 also prevented the growth of one strain (PL1) of the inducible met-hicillin-resistant S. aureus and the MRSA strain ATCC 43300, with MICs of 4 |g/mL and 2 |g/mL, respectively. Even more intriguing, compounds 25 and 26 prevented the growth of the two S. aureus strains with PBP2a, from 14fold to 32-fold more efficiently than the S. aureus ATCC 25923 strain, which is sensitive to pencillin and devoid of PBP2a (Table 4). However, as the MIC values for 25 and 26 are lower than the corresponding IC50 values for each of these compounds, we suggest that these compounds show their antibacterial activities in vitro through actions on other bacterial targets, besides interacting with PBPs. 4. Conclusions Although these synthesized compounds showed only moderate inhibitory activities (IC50 generally >100 |M), and although the data do not represent a significant breakthrough in the field of noncovalent PBP inhibitors, these data do provide useful additional structural information, and thus a more solid basis for the design of potent and noncovalent inhibitors of resistant PBPs. All of the new knowledge gathered in this field should even- tually lead to the development of effective antibacterial agents that target a validated and very promising bacterial target. Our future plans include the synthesis of new structurally related derivatives to further improve the inhibitory potencies against PBPs, as well as the obtaining of co-crystal structures of the inhibitor-enzyme complexes that will provide an excellent basis for further rational structure-based developments of noncovalent PBP inhibitors. 5. Supporting Information Detailed descriptions of the materials and methods and all experimental procedures used to prepare the intermediates and final compounds reported in the manuscript are available in the supporting information. Also, the enzymatic inhibition assays, antimicrobial activity evaluation and the data regarding the computational work are fully disclosed in the supplementary data. 6. Abbreviations PBP, penicillin-binding protein; HMM, high molecular mass; LMM, low molecular mass; MRSA, methicil-lin-resistant Staphylococcus aureus »We thank the European Union (European Community Sixth Framework Programme) via the EUR-INTA-FAR project for the financial support for this research. We thank Andréa Dessen (IBS, Grenoble, France) for PBPlb.« 6. Re fe ren ces 1. H. W. Boucher, G. H. Talbot, J. S. Bradley, J. E. Edwards Jr., D. Gilbert, L. B Rice, M. Scheld, B. Spellberg, J. Bartlett, Clin. Infect. Dis. 2009, 48, 1-12. 2. A. L. Demain, S. Sanchez, J. Antibio. 2009, 62, 5-16. 3. L. L. Silver, Clin. Microbiol. Rev. 2011, 24, 71-109. 4. M. S. Butler, M. A. Cooper, J. Antibio. 2011, 64, 413-425. 5. P. Macheboeuf, C. Contreras-Martel, V. Job, O. Dideberg, A. Dessen, FEMS Microbiol. Rev. 2006, 30, 673-691. 6.E. Sauvage, F. Kerff, M. Terrak, J. A. Ayala, P. Charlier, FEMS Microbiol. Rev. 2008, 32, 234-258. 7. C. Contreras-Martel, C. Dahout-Gonzales, A. Dos Santos Martins, M. Kotnik, A. Dessen, J. Mol. Biol. 2009, 387, 899-909. 8. D. Lim, N. C. Strynadka, Nat. Struct. Biol. 2002, 9, 870-876. 9. L. Miguet, A. Zervosen, T. Gerards, F. A. Pasha, A. Luxen, M. Distéche-Nguyen, A. Thomas, J. Med. Chem. 2009, 52, 5926-5936. 10. A. Zervosen, W-P. Lu, Z. Chen, R. E. White, T. P. Demuth, J-M. Frere, Antimicrob. Agents Chemother. 2004, 48, 961-969. 11. S. Turk, O. Verlaine, T. Gerards, M. Zivec, J. Humljan, I. Sosie, A. Amoroso, A. Zervosen, A. Luxen, B. Joris, S. Gobec, PLoS ONE 2011, 6(5), e19418. 12. R. Baltzly, A. P. Phillips, J. Am. Chem. Soc. 1946, 68(2), 261-265. 13. A. R. Pinder, Synthesis 1980, 6, 425-452. 14. F. A. Carey, R. J. Sundberg, Advanced organic chemistry, Part B: Reactions and synthesis, fourth ed., Springer, New York, 2001. 15. A. R. Voth, P. Khuu, K. Oishi, P. S. Ho, Nature Chem. 2009, 1, 74-79. Povzetek Penicilin vezoči proteini (PBP) so uveljavljena, validirana ter še vedno obetavna tarča za načrtovanje in razvoj novih protimikrobnih učinkovin. Na osnovi naših, pred kratkim odkritih nekovalentnih inhibitorjev (zadetkov) PBP iz rezi-stentnih sevov smo se odločili dodatno raziskati kemijski prostor teh spojin. Z namenom dodobra razjasniti odnos med strukturo in delovanjem smo sintetizirali ter biokemijsko ovrednotili dve seriji spojin: derivate antranilne kisline ter naf-talen-sulfonamidne derivate. Spojinam smo določili inhibitorno aktivnost na treh različnih transpeptidazah, in sicer na PBP2a iz na meticilin odpornega Staphylococcus aureus (MRSA), PBP5fm iz Enterococcus faecium (sev D63r) ter na PBPlb iz seva Streptococcus pneumoniae. Najbolj obetavne rezultate pri obeh serijah spojin smo dobili na encimu PBP2a z IC50 vrednostmi v mikromolarnem območju. Čeprav ti rezultati ne predstavljajo signifikantnega preboja na področju nekovalentnih inhibitorjev PBP, pa zagotovo nudijo uporabne podatke o odnosu med strukturo in delovanjem ter tako tudi boljšo osnovo za nadaljnje načrtovanje močnih, nekovalentnih inhibitorjev PBP iz rezistentnih bakterij. Supporting information Exploration of the Chemical Space of Novel Naphthalene-Sulfonamide and Anthranilic Acid-Based In hi bi tors of Peni cil lin-Bin ding Pro teins 1 Faculty of Pharmacy, University of Ljubljana, A{ker~eva 7, 1000 Ljubljana, Slovenia 2 Centre for Protein Engineering, University of Liège, Institut de Chimie B6a, B-4000 Sart Tilman, Liège, Belgium 3 Laboratory of Organic Chemsitry, University of Liège, Institut de Chimie B6a, B-4000 Sart Tilman, Liège, Belgium Izidor Sosi~,1 Samo Turk,1 Ma{a Sinreih,1 Nu{a Tro{t,1 Olivier Verlaine,2 Ana Amoroso,2 Astrid Zervosen,3 André Luxen,3 Bernard Joris2 and Sta ni slav Go bec1 Corresponding author: e-mail: stanislav.gobec@ffa.uni-lj.si Tel: +386-1-476-9500; Fax: +386-1-425-8031; 1. Materials and Methods 1.1. Chemistry The reagents and solvents were obtained from commercial sources (Fluka, Sigma-Aldrich, Acros Organics, Alfa Aesar, Fluorochem). The solvents were distilled before use, while the other chemicals were used as received. Analytical TLC was performed on Merck silica gel (60F254) pre-coated plates (0.25 mm), with the compounds visualised under UV light and/or stained with the relevant reagent. Column chromatography was performed on Merck silica gel 60 (mesh 70-230), using the indicated solvents. Yields refer to the purified products, and they were not optimised. All of the melting points were determined on a Reichert hot-stage apparatus, and are uncorrected. 1H NMR spectra were recorded on a Bruker Avance 300 DPX spectrometer at 302 K, and are reported in ppm using tetramethylsilane or solvent as internal standard (DMSO-d6 at 2.50 ppm, CDCl3 at 7.26 ppm). The coupling constants (J) are in Hz, and the splitting patterns are designated as: s, singlet; bs, broad singlet; d, doublet; dd, double doublet; td, triple doublet; t, triplet; dt, double triplet; and m, multiplet. 13C NMR spectra were recorded on a Bruker Avance 400 DPX spectrometer at 302 K, and are reported in ppm using solvent as internal standard (DMSO-d6 at 39.5 ppm). Mass spectra data and high-resolution mass measurements were performed on a VG-Analytical Autospec Q mass spectrometer. The purities of all assayed compounds were determined with elemental analyes or HPLC. The analyses are indicated by the sym- bols of the elements, and they were within ±0.4% of the theoretical values. The purities of the compounds determined by HPLC were >95%. Elemental analyses were performed on a 240 C Perkin-Elmer C, H, N analyser. HPLC was performed on an Agilent Eclipse C18 column (4.6 x 50 mm, 5 pm), with a flow rate of 1.0 mL/min, detection at 254 nm, and an eluent system of: A = 0.1% TFA in H2O; B = MeOH. The following gradient was applied: 0-3 min, 40% B; 3-18 min, 40% B ^ 80% B; 18-23 min, 80% B; 23-30 min, 80% B ^ 40% B. The run time was 30 min, at a temperature of 25 °C. 1. 2. Enzymatic Inhibition Assays for Low-Affinity PBP2a and PBP5fm PBP2a from S. aureus ATCC 43300 and PBP5fm from E. faecium D63r were over-expressed and purified as described previously.1,2 Each of the purified PBPs (2.5 pM) were first incubated with 1 mM of each of the potential inhibitors, in 100 mM phosphate buffer, 0.01% Triton X-100,3 pH 7, for 4 h at 30 °C. Then, 25 pM fluorescein-labeled ampicillin4 was added to detect the residual penicillin-binding activity (the residual activity). The samples were further incubated for 30 min at 37 °C in a total volume of 20 pL. Denaturation buffer was added (0.1 M Tris/HCl, pH 6.8, containing 25% glycerol, 2% SDS, 20% P-mercaptoethanol and 0.02% bromophenol blue) and the samples were heated to 100 °C for 1 min. The samples were then loaded onto a 10% SDS-acrilamide gel (10 x 7 cm), and electrophoresis was performed for 45 min at 180 V (12 mA). Detection and quantification of the residual activities were carried out with Molecular Image FX equipment and Quantity One software (BioRad, Hercules, CA, USA). Three independent experiments, each performed in duplicate, were carried out for each inhibitor. 1. 3. Enzymatic Inhibition Assays for PBPlb PBPlb (0.2 pM) was incubated with 1 mM of each of the potential inhibitors, in sodium phosphate buffer (pH 7.0) in the presence of 100 mM D-alanine, 0.01% Tri-ton-X-1003 and 0.01 mg/mL BSA, for 60 min at 25 °C. The residual activities were determined from the initial rates of hydrolysis of the thioester 2-(2-benzamidopropa-noylthio) acetic acid (5 mM) used as the reporter substrate. After preincubation, the initial rate of thioester hydrolysis and of spontaneous hydrolysis (e[Ae]412 nm = 13,600 M-1 cm-1) were measured in the presence of 1 mM DTNB using a 96-well microtiter plate and a Power Wave microtiter plate reader (Bio-Tek Instruments; total volume, 150 pL). Three independent experiments, each performed in duplicate, were carried out. The activity of PBP1b in the absence of the compounds (residual activity, 100%) was measured by performing six replicates on each plate. If inhibition was detected, the residual activity was measured over a range of concentrations, from which the IC50 values were determined, using nonlinear regression analysis and Sigma Plot (Systat software), with the fitting of the data to the equation y = y0 + (a x b)/(b + x). 1. 4. Antibacterial Activity Determination of the antibacterial activities was carried out on microtiter plates, in Mueller-Hinton broth and with a 200 pL final volume, following the recommended procedures of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standard Institute (CLSI).5,6 The compounds were dissolved in Mueller-Hinton broth just before use. Inocula were prepared for each strain by resuspen-ding isolated colonies from 18-h-cultured plates. Equivalents of 0.5 MacFarland turbidity standards (approximately 1 x 108 CFU/mL) were prepared in saline solution (0.085% NaCl) and then diluted 200-fold in Mueller-Hin-ton broth. The MICs were determined as the lowest dilution of product that showed no visual turbidity. 2. Computational The computational analysis was performed on a workstation with 4 dual-core AMD Opteron 2.0 GHz processors, with 16 GB RAM, 4 320-GB hard drives, in RAID10 array and with nVidia GeForce 7900 graphic cards. The workstation has Fedora 7 64-bit installed. Docking was performed with FlexX 3.0 (BioSolveIT GmbH).7 For docking into the PBP2a crystal structure, 1VQQ was used. The active site was defined as the area within 10 A of Lys406. The maximum allowed overlap volume vas set at 20 A3. For base placement, Triangle Matching was used, and the program generated a maximum of 200 solutions per iteration, and 200 per fragmentation. 3. Experimental Procedures 3.1. General Procedure for the Preparation of Methyl Esters I-IV To an ice-cooled solution of an appropriate anthrani-lic acid (1.0 mmol) in MeOH (15 mL), SOCl2 (1.78 g, 15 mmol) was slowly added, with continuous stirring. The mixture was stirred at room temperature for 2 days. After the reaction was complete (monitored by TLC), the solvent was evaporated, and saturated aqueous NaHCO3 (20 mL) was added to the residue. The aqueous phase was extracted with EtOAc (3 x 20 mL), and the combined organic phases were washed with brine (2 x 20 mL), dried over Na2SO4, filtered, and removed under reduced pressure. The resulting residue was purified by column chroma-tography to provide the corresponding methyl anthranila-tes I-IV. Methyl 2-amino-5-nitrobenzoate (I).8 Column chromatography (EtOAc/hexane = 1/3); Yellow crystals (needles); Yield: 56%; Rf 0.48 (EtOAc/hexane = 1/1); Mp: 166.0-167.0 °C (lit [23] 167.0-169.0 °C); 1H NMR (300 MHz, CDCl3): S 3.95 (s, 3H, CH3), 6.52 (br s, 2H, NH2), 6.69 (d, J =9.2 Hz, 1H, Ar-H), 8.15 (dd, J = 9.2, 2.7 Hz, 1H, Ar-H), 8.86 (d, J = 2.7 Hz, 1H, Ar-H); 13C NMR (100 MHz, CDCl3): S 52.16, 109.20, 116.21, 128.90, 129.21, 137.24, 154.71, 167.23; HRMS (ESI) m/z calcd for C8H9N2O4 [M+H]+ 197.0562, found 197.0562. 8 9 2 4 Methyl 2-amino-5-chloro-3-methylbenzoate (II).9 Column chromatography (CH2Cl2/MeOH = 20/1); Brown crystals (needles); Yield: 61%; Rf 0.83 (CH2Cl2/ MeOH = 20/1); Mp: 35.5-37.5 °C (lit [24] 33.0-35.0 °C); 1H NMR (300 MHz, DMSO-d6): S3.80 (s, 3H, CH3), 6.59 (br s, 2H, NH2), 7.25 (d, J = 2.3 Hz, 1H, Ar-H), 7.57 (d, J = 2.3 Hz, 1H, Ar-H); 13C NMR (100 MHz, CDCl3): S 17.22, 51.67, 110.78, 119.88, 124.89, 128.08, 134.42, 147.54, 167.97; HRMS (ESI) m/z calcd for C9H11NO2Cl [M+H]+ 200.0478, found 200.0472. Methyl 2-amino-4,5-dimethoxybenzoate (III).10 Column chromatography (EtOAc/hexane = 1/1); White crystals (prisms); Yield: 79%; R 0.43 (EtOAc/hexane = 1/1); Mp: 124.0-124.5 °C (lit [25] 127.0-129.0 °C); 1H NMR (300 MHz, DMSO-d6): S 3.54 (s, 3H, CH3), 3.74 (s, 6H, 2 x OCH3), 6.36 (br s, 2H, NH2), 6.43 (s, 1H, Ar-H), 7.13 (s, 1H, Ar-H); 13C NMR (100 MHz, CDCl3): S 51.23, 55.65, 56.24, 99.20, 101.87, 112.37, 140.44, 147.03, 154.66, 168.08; HRMS (ESI) m/z calcd for C9H10NO3 [M+H-CH3OH]+ 180.0661, found 180.0647. Methyl 2-amino-5-hydroxybenzoate (IV).11 Column chromatography (EtOAc/hexane = 1/1); Pale brown crystals (prisms); Yield: 57%; Rf 0.35 (EtOAc/ hexane = 1/1); Mp: 159.0-161.0 °C (lit [2(5] 160.0-162.0 °C); 1H NMR (300 MHz, CDCl3): 8 3.76 (s, 3H, CH3), 6.05 (br s, 2H, NH2), 6.64 (d, J = 8.8 Hz, 1H, Ar-H), 6.80 (dd, J = 8.8, 2.9 Hz, 1H, Ar-H), 7.10 (d, J = 2.9 Hz, 1H, Ar-H); 13C NMR (100 MHz, CDCl3): 8 51.64, 111.05, 116.01, 118.20, 123.08, 144.97, 145.96, 168.07; HRMS (ESI) m/z calcd for C8H10NO3 [M+H]+ 168.0661, found 168.0653. 8 10 3 3. 2. General Procedure for the Preparation of Compounds 1-11. To a solution of an appropriate carboxylic acid VIII-X (1.0 mmol) in CH2Cl2 (10 mL), pyridine (99 mg, 1.25 mmol) and SOCl2 (773 mg, 6.5 mmol) were slowly added. After stirring at 45 °C for 2 h, the solvent was removed under reduced pressure. The reaction mixture was dissolved in toluene, and then the corresponding methyl anthranilate derivative I-VII (1.25 mmol) was added and the reaction mixture was stirred at 100 °C for 3 h. After the reaction was complete (monitored by TLC), the solvent was evaporated, then an aqueous solution of Na2CO3 (10%, 10 mL) was added, and the aqueous layer was extracted with CH2Cl2 (3 x 10 mL). The combined organic phases were washed with brine (2 x 20 mL) and dried over Na2SO4. The solvent was evaporated, and the pure products 1-11 were obtained by crystallization from the corresponding solvent. Methyl 5-hydroxy-2-(3-nitrobenzamido)benzoate (1). Crystallization from EtOH; Yellow crystals (needles); Yield: 55%; Rf 0.35 (EtOAc/hexane = 1/1); Mp: 210.5-212.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.83 (s, 3H, CH3), 7.08 (dd, J = 8.9, 2.9 Hz, 1H, Ar-H), 7.35 (d, J = 2.9 Hz, 1H, Ar-H), 7.88 (t, J = 8.0 Hz, 1H, Ar-H), 7.99 (d, J = 8.9 Hz, 1H, Ar-H), 8.33-8.36 (m, 1H, Ar-H), 8.44-8.47 (m, 1H, Ar-H), 8.72 (t, J = 1.6 Hz, 1H, Ar-H), 9.80 (br s, 1H, OH), 11.08 (br s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6): 8 52.42, 116.15, 120.57, 121.54, 121.94, 124.74, 126.27, 130.27, 130.55, 133.25, 135.98, 147.91, 153.92, 162.49, 167.17; HRMS (ESI) m/z calcd for C15H11N2O6 [M-H]- 315.0617, found 315.0618; Anal. calcd. for C15H12N2O6: C, 56.96; H, 3.82; N, 8.86. Found: C, 56.92; H, 3.651; N, 8.80. Methyl 5-chloro-2-(3-nitrobenzamido)benzoate (2). Crystallization from EtOH; White crystals (needles); Yield: 74%; Rf 0.55 (EtOAc/hexane = 1/1); Mp: 187.0-188.5 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.90 (s, 3H, CH3), 7.39 (dd, J = 8.4, 2.1 Hz, 1H, Ar-H), 7.93 (t, J = 5.0 Hz, 1H, Ar-H), 8.02 (d, J = 8.4 Hz, 1H, Ar-H), 8.36-8.38 (m, 1H, Ar-H), 8.49-8.52 (m, 2H, Ar-H), 8.74 (t, J = 1.8 Hz, 1H, Ar-H), 11.64 (br s, 1H, NHCO); 13C NMR (100 MHz, CDCl3): 8 52.88, 113.50, 120.40, 122.74, 123.57, 126.62, 130.10, 132.08, 132.85, 136.15, 141.35,142.06, 148.59, 163.13, 168.60; HRMS (ESI) m/z calcd for C15H12N2O5Cl [M+H]+ 335.0435, found 335.0428; Anal. calcd. for C15H11N2O5Cl: C, 53.83; H, 3.31; N, 8.37. Found: C, 53.60; H, 2.94; N, 8.18. Methyl 4,5-dimethoxy-2-(3-nitrobenzamido)benzoate (3). Crystallization from EtOAc; Fluorescent yellow crystals (needles); Yield: 69%; Rf 0.26 (EtOAc/hexane = 3/5); Mp: 189.5-192.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.82 (s, 3H, CH3), 3.88 (s, 6H, 2 x OCH3), 7.48 (s, 1H, Ar-H), 7.92 (t, J = 8.2 Hz, 1H, Ar-H), 8.22 (s, 1H, Ar-H), 8.35-8.38 (m, 1H, Ar-H), 8.46-8.49 (m, 1H, Ar-H), 8.73 (t, J = 2.1 Hz, 1H, Ar-H), 11.74 (br s, 1H, NHCO); 13C NMR (100 MHz, CDCl3): 8 52.44, 56.04, 56.19, 103.31, 106.98, 112.01, 122.71, 126.28, 129.96, 132.60, 136.58, 137.28, 144.47, 148.57, 154.08, 162.89, 168.81; HRMS (ESI) m/z calcd for C17H17N2O7 [M+H]+ 361.1036, found 361.1021; Anal. calcd. for C17H16N2O7: C, 56.67; H, 4.48; N, 7.77. Found: C, 56.86; H, 4.33; N, 7.78. Methyl 5-chloro-3-methyl-2-(3-nitrobenzamido)ben-zoate (4). Crystallization from EtOH; White crystals (needles); Yield: 50%; Rf 0.79 (EtOAc/hexane = 1/1); Mp: 153.0-155.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.30 (s, 3H, CH3), 3.72 (s, 3H, CH3), 7.69 (s, 2H, 2 x Ar-H), 7.86 (t, J = 8.1 Hz, 1H, Ar-H), 8.37-8.40 (m, 1H, Ar-H), 8.44-8.48 (ddd, J = 8.1, 2.1, 1.0 Hz, 1H, Ar-H), 8.77 (t, J = 2.1 Hz, 1H, Ar-H), 10.41 (br s, 1H, NHCO); 13C NMR (100 MHz, CDCl3): 8 19.43, 52.82, 122.75, 123.89, 126.58, 128.24, 130.03, 131.31, 133.33, 135.66, 135.75, 135.84, 137.76, 148.43, 163.08, 167.18; HRMS (ESI) m/z calcd for C16H14N2O5Cl [M+H]+ 349.0591, found 349.0599; Anal. calcd. for C16H13N2O5Cl: C, 55.10; H, 3.76; N, 8.03. Found: C, 55.32; H, 3.58; N, 8.03. Methyl 2-(4-fluoro-3-nitrobenzamido)benzoate (5). Crystallization from EtOH; Yellow crystals (needles); Yield: 81%; Rf 0.56 (EtOAc/hexane = 1/1); Mp: 136.0-138.0 °C; 1H NMR (300 MHz, CDCl3): 8 4.00 (s, 3H, CH3), 7.16-7.21 (m, 1H, Ar-H), 7.43-7.49 (m, 1H, Ar-H), 7.64 (td, J = 8.6, 1.6 Hz, 1H, Ar-H), 8.12 (dd, J = 8.0, 1.6 Hz, 1H, Ar-H), 8.29-8.34 (m, 1H, Ar-H), 8.79 (dd, J = 7.2, 2.4 Hz, 1H, Ar-H), 8.86 (d, J = 8.6 Hz, 1H, Ar-H), 12.27 (br s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6): 852.50, 119.04, 119.24 and 119.46 (1C, 2JCF = 21.6 Hz), 121.92, 124.18, 125.45 and 125.47 (1C, 4JCF = 1.6 Hz), 130.54, 131.09 and 131.13 (1C, 3JCF = 4.0 Hz), 133.91, 134.63 and 134.73 (1C, 2JCF = 10.3 Hz), 136.79 and 136.86 (1C, 3JCF = 7.7 Hz), , 138.87, 155.23 and 157.89 (1C, 1JCF = 265 Hz), 161.85, 167.58; HRMS (ESI) m/z calcd for , C15H12N2O5F [M+H]+ 319.0730, found 319.0724; Anal. calcd. for C15H11N2O5F: C, 56.61; H, 3.48; N, 8.80. Found: C, 56.80; H, 3.24; N, 8.71. Methyl 5-bromo-2-(4-fluoro-3-nitrobenzamido)ben-zoate (6). Crystallization from EtOH; White crystals (needles); Yield: 76%; Rf 0.74 (EtOAc/hexane = 1/1); Mp: 187.5-188.5 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.87 (s, 3H, CH3), 7.81-7.91 (m, 2H, Ar-H), 8.06 (d, J = 2.4 Hz, 1H, Ar-H), 8.18 (d, J = 8.9 Hz, 1H, Ar-H), 8.32-8.37 (m, 1H, Ar-H), 8.69 (dd, J = 7.2, 2.4 Hz, 1H, Ar-H), 11.32 (br s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6): 8 52.77, 115.97, 119.28 and 119.49 (1C, 2JCF = 21.3 Hz), 121.87, 124.44, 125.54 and 125.56 (1C, 4JCF = 1.9 Hz), 130.82 and 130.85 (1C, 3JCF = 4.0 Hz), 1C2.64, 134.81 and 134.92 (1C, 2JCF = 10.4 Hz), 136.31, 136.81 and 136.89 (1C, 3Jcf = 8.0 Hz), 137.76, 155.31 and 157.97 (1C, 1JCF = 265) Hz), 162.03, 166.16; HRMS (ESI) m/z calcd Cor C15H9N2O5FBr [M-H]- 394.9679, found 394.9688; Anal. calcd. for C15H10N2O5FBr: C, 45.36; H, 2.54; N, 7.05. Found: C, 45.37; H, 2.35; N, 7.00. Methyl 5-chloro-2-(3-hydroxybenzamido)benzoate (7). Crystallization from EtOH; Yellowish crystals (prisms); Yield: 47%; Rf 0.52 (EtOAc/hexane = 1/1); Mp: 196.0-200.5 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.91 (s, 3H, CH3), 7.03 (dt, J = 6.6, 1.5 Hz, 1H, Ar-H), 7.35-7.40 (m, 3H, Ar-H), 7.75 (dd, J = 9.0, 2.7 Hz, 1H, Ar-H), 7.96 (d, J = 2.7 Hz, 1H, Ar-H), 8.57 (d, J = 9.0 Hz, 1H, Ar-H), 9.89 (br s, 1H, OH), 11.44 (br s, 1H, NHCO); 13C NMR (100 MHz, CDCl3): 8 52.85, 114.74, 116.48, 119.09, 119.48, 121.90, 127.88, 130.25, 130.58, 134.69, 135,94,140.19, 156.31, 165.47, 168.02; HRMS (ESI) m/z calcd for C15H13NO4Cl [M+H]+ 306.0533, found 306.0533; Anal. calcd. for C15H12NO4Cl: C, 58.93; H, 3.96; N, 4.58. Found: C, 58.68; H, 3.71; N, 4.56. Methyl 2-(3-hydroxybenzamido)benzoate (8). Crystallization from EtOH; White crystals (needles); Yield: 52%; Rf 0.46 (EtOAc/hexane = 1/1); Mp: 199.0-206.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.90 (s, 3H, CH3), 7.02-7.04 (m, 1H, Ar-H), 7.24 (td, J = 7.0, 1.0 Hz, 1H, Ar-H), 7.36-7.40 (m, 3H, Ar-H), 7.65-7.71 (m, 1H, Ar-H), 8.02 (dd, J = 8.4, 1.5 Hz, 1H, Ar-H), 8.60 (d, J = 8.4 Hz, 1H, Ar-H), 9.88 (br s, 1H, OH), 11.58 (br s, 1H, NHCO); 13C NMR (100 MHz, CDCl3): 8 52.54, 114.64, 115.21, 119.18, 119.29, 120.48, 122.75, 130.20, 130.98, 134.88, 136.43, 141.69, 156.11, 165.37, 169.08; HRMS (ESI) m/z calcd for C15H14NO4 [M+H]+ 272.0923, found 272.0914; Anal. calcd. for C15H13NO4: C, 66.41; H, 4.83; N, 5.16. Found: 66.19; H, 4.577; 1ST, 5.08. Methyl 2-(3-hydroxybenzamido)-5-nitrobenzoate (9). Crystallization from EtOH; Yellow crystals (prisms); Yield: 77%; Rf 0.46 (EtOAc/hexane = 1/1); Mp: 209.0-212.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.98 (s, 3H, CH3), 7.06-7.09 (m, 1H, Ar-H), 7.38-7.44 (m, 3H, Ar-H), 8.54 (dd, J = 9.3, 2.7 Hz, 1H, Ar-H), 8.75 (d, J = 2.7 Hz, 1H, Ar-H), 8.86 (d, J = 9.3 Hz, 1H, Ar-H), 9.98 (br s, 1H, OH), 11.88 (br s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6): 8 53.26, 114.06, 116.64, 117.53, 119.84, 120.65, 126.14, 129.22, 130.21, 134.84, 141.49, 145.52, 157.87, 165.10, 166.63; HRMS (ESI) m/z calcd for C15H11N2O6 [M-H]- 315.0617, found 315.0619; HPLC purity: 95.35%, retention time: 19.14 min. Methyl 5-bromo-2-(3-hydroxybenzamido)benzoate (10). Crystallization from EtOH; Beige crystals (prisms); Yield: 77%; Rf 0.52 (EtOAc/hexane = 1/1); Mp: 175.0-177.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.91 (s, 3H, CH3), 7.02-7.05 (m, 1H, Ar-H), 7.35-7.40 (m, 3H, Ar-H), 7.87 (dd, J = 9.0, 2.4 Hz, 1H, Ar-H), 8.09 (d, J = 2.4 Hz, 1H, Ar-H), 8.52 (d, J = 9.0 Hz, 1H, Ar-H), 9.90 (br s, 1H, OH), 11.45 (br s, 1H, NHCO); 13C NMR (100 MHz, CDCl3): 8 52.85, 114.72, 115.20, 116.78, 119.11, 119.49, 122.15, 130.25, 133.53, 135.94, 137.57, 140.65, 156.31, 165.47, 167.92; HRMS (ESI) m/z calcd for C15H11NO4Br [M-H]- 347.9871, found 347.9881; HPLC purity: 98.81%, retention time: 21.87 min. Methyl 2-(3-hydroxy-4-nitrobenzamido)benzoate (11). Crystallization from EtOH; Yellow crystals (prisms); Yield: 72%; Rf 0.59 (EtOAc/hexane = 1/1); Mp: 158.0-161.0 °C; 1H NMR (300 MHz, CDCl3): 8 3.99 (s, 3H, CH3), 7.18 (td, J = 7.2, 1.0 Hz, 1H, Ar-H/), 7.60-7.67 (m, 2H, Ar-H), 7.83 (d, J = 1.8 Hz, 1H, Ar-H), 8.11 (dd, J = 8.2, 1.5 Hz, 1H, Ar-H), 8.27 (d, J = 9.0 Hz, 1H, Ar-H), 8.87 (dd, J = 8.2, 1.0 Hz, 1H, Ar-H), 10.60 (br s, 1H, OH), 12.20 (br s, 1H, NHCO); 13C NMR (100 MHz, CDCl3): 8 52.68, 115.32, 118.49, 119.47, 120.40, 123.40, 125.737, 131.02, 134.95, 135.05, 141.04, 143.02, 155.02, 162.93, 169.08; HRMS (ESI) m/z calcd for C15H13N2O6 [M+H]+ 317.0774, found 317.0775; Anal. calcd. for C15H12N2O6: C, 56.96; H, 3.82; N, 8.86. Found: C, 57.31; H, 3.51; N, 8.89. 3. 3. General Procedure for the Preparation of Compounds 12-14. To a solution of compounds 10 or 11 (1.0 mmol) in DMF (5 mL), K2CO3 (601 mg, 5.0 mmol) was added, followed by the addition of an appropriate alkyl bromide (1.4 mmol) under argon. The reaction mixture was stirred at 50 °C for 24 h. After the reaction was complete, EtOAc was added and the organic layer was washed with water (3 x 10 mL) and brine (3 x 10 mL), dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude products were purified by column chromatography to provide pure compounds 12-14. Methyl 5-bromo-2-(3-ethoxybenzamido)benzoate (12). Column chromatography (EtOAc/hexane = 1/3); White crystals (needles); Yield: 49%; Rf 0.42 (EtOAc/he- xane = 1/3); Mp: 122.0-123.5 °C; 1H NMR (300 MHz, DMSO-d6): S 1.37 (t, J = 6.9 Hz, 3H, OCH2CH3), 3.89 (s, 3H, CH3), 4.12 (q, J = 6.9 Hz, 2H, OCH2CH3), 7.19-7.23 (m, 1H, Ar-H), 7.45-7.48 (m, 1H, Ar-H), 7.50-7.53 (m, 2H, Ar-H), 7.87 (dd, J = 8.7, 2.4 Hz, 1H, Ar-H), 8.08 (d, J = 2.4 Hz, 1H, Ar-H), 8.47 (d, J = 8.7 Hz, 1H, Ar-H), 11.42 (br s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6): S 14.50, 52.87, 63.31, 112.97, 114.83, 118.33, 118.92, 119.53, 123.01, 130.15, 132.69, 135.43, 136.68, 139.13, 158.73, 164.56, 166.64; HRMS (ESI) m/z calcd for C17H17NO4Br [M+H]+ 378.0341, found 378.0331. Anal. calcd. for C17H16NO4Br: C, 53.99; H, 4.26; N, 3.70. Found: C, 53.91; H, 3.87; N, 3.68. Methyl 5-bromo-2-(3-butoxybenzamido)benzoate (13). Column chromatography (EtOAc/hexane = 1/3); Yellowish crystals (needles); Yield: 65%; Rf 0.50 (EtOAc/hexane = 1/3); Mp: 94.5-99.0 °C; 1H NMR (300 MHz, DMSO-d6): S0.95 (t, J = 7.2 Hz, 3H, OCH2CH2CH2CH3), 1.45-1.50 (m, 2H, OCH2CH2CH2CH3), 1.72-1.776 (m, 2H, OCH2CH2CH2CH3), 3.89 (s, 3H, CH3), 4.06 (t, J = 6.4 Hz, 2H, OCH2CH2CH2CH3), 7.21-7.23 (m, 1H, Ar-H), 7.45-7.48 (m, 1H, Ar-H), 7.49-7.52 (m, 2H, Ar-H), 7.87 (dd, J = 9.0, 2.4 Hz, 1H, Ar-H), 8.08 (d, J = 2.4 Hz, 1H, Ar-H), 8.46 (d, J = 9.0 Hz, 1H, Ar-H), 11.42 (br s, 1H, NHCO); 13C NMR (100 MHz, CDCl3): S 13.83, 19.20, 31.18, 52.77, 67.91, 113.00, 114.95, 116.67, 118.84, 119.23, 122.09, 129.80, 133.46, 135.87, 137.49, 140.85, 159.57, 165.60, 167.85; HRMS (ESI) m/z calcd for C19H21NO4Br [M+H]+ 406.0654, found 406.0643. Anal. calcd. for C19H20NO4Br: C, 56.17; H, 4.96; N, 3.45. Found: C, 5&57; H, 4.69; N, 3.43. Methyl 2-(3-butoxy-4-nitrobenzamido)benzoate (14). Column chromatography (CH2Cl2); Yellowish crystals (needles); Yield: 66%; Rf 0.74 (CH2Cl2); Mp: 82.0-84.0 °C; 1H NMR (300 MHz, DMSO-d6): S0.95 (t, J = 7.5 Hz, 3H, OCH2CH2CH2CH3), 1.39-1.52 (m, 2H, OCH2CH2CH2CH3), 1.71-1.80 (m, 2H, OCH2CH2CH2 CH3), 3.828 (s, 3H, CH3), 4.28 (t, J = 6.3 Hz, 2H, OCH2CH2CH2CH3), 7.31 (td, J = 7.8, 1.0 Hz, 1H, Ar-H), 7.62 (dd, J = 8.4, 3.5 Hz, 1H, Ar-H), 7.71 (td, J = 8.1, 1.5 Hz, 1H, Ar-H), 7.83 (d, J = 1.5 Hz, 1H), 8.01 (dd, J = 7.8, 1.5 Hz, 1H, Ar-H), 8.07 (d, J = 8.4 Hz, 1H, Ar-H), 8.38 (dd, J = 8.1, 1.0 Hz, 1H, Ar-H), 11.46 (br s, 1H, NHCO); 13C NMR (100 MHz, CDCl3): S 13.70, 19.03, 30.82, 52.62, 69.61, 114.09, 115.23, 118.01, 120.30, 123.26, 125.78, 131.06, 134.99, 139.75, 141.24, 141.64, 152.52, 163.51, 169.17; HRMS (ESI) m/z calcd for C19H20N2O6Na [M+Na]+ 395.1219, found 395.1202. Anal. calcd. for C19H20N2O6: C, 61.28; H, 5.41; N, 7.52. Found: C, 61.47; H, 5^7; N, 7.36. 3. 4. General Procedure for the Synthesis of Compounds 15-27. Alkaline Hydrolysis. To a stirred solution of the protected methyl anthra-nilates 1-10 and 12-14 (0.5 mmol) in dioxane/THF mixture (1:1, 2 mL), 1 M NaOH (1 mL) was added, and the reaction mixture stirred until the starting material had completely reacted (monitored by TLC). The solvent was then evaporated under reduced pressure, the residue diluted with H2O (10 mL), and washed with EtOAc (2 x 10 mL). The aqueous phase was acidified to pH 2 using 1 M HCl, and extracted with EtOAc (3 x 10 mL). The combined organic phases were washed with brine (3 x 10 mL), then dried over Na2SO4, filtered, and evaporated to dryness, to provide compounds 15-27. If necessary, these were additionally purified by column chromatography or crystallization. 5-Hydroxy-2-(3-nitrobenzamido)benzoic acid (15). Yellow crystals (prisms); Yield: 86%; Rf 0.19 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 248.0-251.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 7.08 (dd, J = 8.9, 3.0 Hz, 1H, Ar-H), 7.43 (d, J = 3.0 Hz, 1H, Ar-H), 7.88 (t, J = 8.0 Hz, 1H, Ar-H), 8.32-8.37 (m, 2H, Ar-H), 8.44-8.48 (m, 1H, Ar-H), 8.73 (t, J = 1.6 Hz, 1H, Ar-H), 9.71 (br s, 1H, OH), 11.86 (br s, 1H, NHCO), resonance for COOH missing; 13C NMR (100 MHz, DMSO-d6): 8 116.68, 119.37, 120.87, 121.56, 122.60, 126.25, 130.60, 131.99, 133.19, 136.07, 147.91, 153.21, 161.87, 169.53; HRMS (ESI) m/z calcd for C14H9N2O6 [M-H]- 301.0461, found 301.0460; Anal. calcd. for C14H10N2O6: C, 55.63; H, 3.33; N, 9.27. Found: C, 55.42; H, 3.07; N, 9.15. 5-Chloro-2-(3-nitrobenzamido)benzoic acid (16). Crystallization from CH3CN; White crystals (prisms); Yield: 61%; Rf 0.67 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 257.0-259.0 °C; 1H NMR (300 MHz, DM-SO-d6): 87.34 (dd, J = 8.6, 2.2 Hz, 1H, Ar-H), 7.92 (t, J = 8.0 Hz, 1H, Ar-H), 8.07 (d, J = 8.6 Hz, 1H, Ar-H), 8.35-8.38 (m, 1H, Ar-H), 8.49 (ddd, J = 8.0, 1.8, 1.0 Hz, 1H, Ar-H), 8.73-8.75 (m, 2H, Ar-H), 12.46 (br s, 1H, NHCO), 14.10 (br s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6): 8 115.58, 119.32, 121.56, 123.24, 126.76, 130.76, 132.76, 133.27, 135.18, 138.58, 141.43, 147.87, 162.48, 169.37; HRMS (ESI) m/z calcd for C14H8N2O5Cl [M-H]- 319.0122, found 319.0123; Anal. calcd. for C14H9N2O5Cl: C, 52.43; H, 2.83; N, 8.74. Found: C, 52.26; H, 2.61; N, 8.73. 4,5-Dimethoxy-2-(3-nitrobenzamido)benzoic acid (17). Crystallization from CH3CN/MeOH mixture (5/1); Yellow crystals (prisms); Yield: 51%; Rf 0.27 (CH2Cl2/ MeOH/AcOH = 5/1/0.1); Mp: 277.5-280.0 °C; 1H NMR (300 MHz, DMSO-d6): 83.80 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 7.51 (s, 1H, Ar-H), 7.91 (t, J = 8.0 Hz, 1H, Ar-H), 8.36-8.38 (m, 1H, Ar-H), 8.42 (s, 1H, Ar-H), 8.46-8.50 (m, 1H, Ar-H), 8.73 (t, J = 1.9 Hz, 1H, Ar-H), 12.51 (br s, 1H, NHCO), resonance for COOH missing; 13C NMR (100 MHz, DMSO-dt): 8 55.54, 55.58, 103.39, 112.81, 119.84, 121.52, 126.50, 130.77, 133.17, 135.88, 136.07, 144.06, 147.99, 153.03, 162.08, 169.83; HRMS (ESI) m/z calcd for C16H13N2O7 [M-H]- 345.0723, found 345.0718; Anal. calcd. for C16H14N2O7: C, 55.49; H, 4.07; N, 8.09. Found: C, 55.60; H, 3.98; N, 7.76. 5-Chloro-3-methyl-2-(3-nitrobenzamido)benzoic acid (18). Column chromatography (CH2Cl2/MeOH/AcOH = 9/1/0.1); White crystals (prisms); "Yield: 53%; Rf 0.34 (CH2Cl2/MeOH/AcOH = 9/1/0.1); Mp: 203.0-205.5 °C; 1H NMR (300 MHz, DMSO-dt): 8 2.27 (s, 3H, CH3), 7.63-7.69 (m, 2H, Ar-H), 7.86 (t, J = 8.0 Hz, 1H, Ar-H), 8.36-8.40 (m, 1H, Ar-H), 8.45 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 8.79 (t, J = 2.1 Hz, 1H, Ar-H), 10.44 (br s, 1H, NHCO), resonance for COOH missing; 13C NMR (100 MHz, DMSO-dt): 8 17.70, 122.24, 122.94, 126.18, 127.24, 130.26, 131.06, 133.00, 133.91, 135.68, 141.05, 147.74, 156.63, 166.33, 169.22; HRMS (ESI) m/z calcd for C15H10N2O5Cl [M-H]- 333.0278, found 333.0281; Anal. calcd. for C15H11N2O5Cl: C, 53.83; H, 3.31; N, 8.37. Found: C, 53.74; H, 3^0; N, 8.26. 2-(4-Fluoro-3-nitrobenzamido)benzoic acid (19). Column chromatography (CH2Cl2/MeOH/AcOH = 9/1/0.1); Yellow crystals (prisms); "Yield: 63%; Rf 0.46 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 224.0-225.0 °C; 1H NMR (300 MHz, DMSO-dt): 8 7.21 (td, J = 7.8, 1.2 Hz, 1H, Ar-H), 7.31 (d, J = 8.7 Hz, 1H, Ar-H), 7.61-7.68 (m, 1H, Ar-H), 8.06 (dd, J = 7.8, 1.5 Hz, 1H, Ar-H), 8.10 (dd, J = 8.7, 2.1 Hz, 1H, Ar-H), 8.48 (d, J = 2.1 Hz, 1H, Ar-H), 8.62 (dd, J = 8.1, 1.2 Hz, 1H, Ar-H), 12.36 (br s, 1H, NHCO), resonance for COOH missing; 13C NMR (100 MHz, DMSO-dt): 8117.09, 119.52 and 119.74 (1C, 2Jcf = 21.8 Hz), 119.88, 122.96, 125.54 and 125.57 (1C, 4Jc'f = 1.9 Hz), 124.97, 131.12 and 131.16 (1C, 3JCF = 4.2 Hc), 133.35, 133.98 and 134.08 (1C, 2JCF = 10.5 Hz), 136.57 and 136.64 (1C, 3JCF = 7.8 Hz), 140.78, 153.73 and 156.49 (1C, 1JCF = 263 ' Hz), 162.41, 170.12; HRMS (ESI) m/z calcd for C14H9N2O5F [M-H]- 303.0461, found 303.0466; HPLC purity: 99.13%, retention time: 18.04 min. 5-Bromo-2-(4-fluoro-3-nitrobenzamido)benzoic acid (20). Crystallization from EtOAc; Yellow crystals (prisms); Yield: 42%; Rf 0.13 (CH2Cl2/MeOH/AcOH = 9/1/0.1); Mp: 286.0-288.0 °C; 1H NMR (300 MHz, DMSO-dt): 87.31 (d, J = 8.8 Hz, 1H, Ar-H), 7.84 (dd, J = 8.8, 2.5 Hz, 1H, Ar-H), 8.05-8.12 (m, 2H, Ar-H), 8.47 (d, J = 2.5 Hz, 1H, Ar-H), 8.55 (d, J = 9.0 Hz, 1H, Ar-H), 12.09 (br s, 1H, NHCO), resonance for COOH missing; 13C NMR (100 MHz, DMSO-dt): 8 114.40, 118.74 and 118.95 (1C, 2Jcf = 21.4 Hz), 119.60, 122.00, 124.59 and 124.61 (1C, 4Jcf = 2.0 Hz), 124.66, 133.11 and 133.14 (1C, 3JCF = 4.3 Hz), 133.38, 136.45 and 136.56 (1C, 2JCF = 10.8 Hz), 136.59, 139.87 and 139.95 (1C, 3JCF = 8.5 Hz), 153.83 and 156.49 (1C, 1JCF = 267 Hz), 162.37, 168.74; HRMS (ESI) m/z calcd for C14H7N2O5BrF [M-H]- 380.9619, found 380.9668; HPLC purity: 100.00%, retention time: 22.72 min. 5-Chloro-2-(3-hydroxybenzamido)benzoic acid (21). White crystals (needles); Yield: 84%; Rf 0.38 (CH2Cl2/MeOH/AcOH = 9/1/0.1); Mp: 277.5-28a0 °C; 1H NMR (300 MHz, DMSO-dt): 8 7.00-7.04 (m, 1H, Ar-H), 7.35-7.39 (m, 3H, Ar-H), 7.72 (dd, J = 9.0, 2.7 Hz, 1H, Ar-H), 7.99 (d, J = 2.7 Hz, 1H, Ar-H), 8.72 (d, J = 9.0 Hz, 1H, Ar-H), 9.88 (br s, 1H, OH), 12.05 (br s, 1H, NHCO), resonance for COOH missing; 13C NMR (100 MHz, DMSO-dt): 8 113.95, 117.28, 118.32, 119.25, 121.63, 126.35, 129.99, 130.31, 133.78, 135.55, 139.84, 157.77, 164.66, 168.66; HRMS (ESI) m/z calcd for C14H9NO4Cl [M-H]- 290.0220, found 290.0223; HPLC purity: 100.00%, retention time: 18.89 min. 2-(3-Hydroxybenzamido)benzoic acid (22). White crystals (needles); Yield: 72%; Rf 0.47 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 219.0-223.0 °C; 1H NMR (300 MHz, DMSO-dt): 8 7.00-7.04 (m, 1H, Ar-H), 7.20 (td, J = 7.2, 1.0 Hz, 1H, Ar-H), 7.35-7.39 (m, 3H, Ar-H), 7.62-7.69 (m, 1H, Ar-H), 8.06 (dd, J = 8.0, 1.6 Hz, 1H, Ar-H), 8.71 (dd, J = 8.4, 1.0 Hz, 1H, Ar-H), 9.86 (br s, 1H, OH), 12.11 (br s, 1H, NHCO), 13.82 (br s, 1H, COOH); 13C NMR (100 MHz, DMSO-dt): 8 113.93, 116.28, 117.24, 119.10, 119.72, 122.78, 129.96, 131.21, 134.25, 135.87, 141.10, 157.76, 164.63, 169.93; HRMS (ESI) m/z calcd for C14H10NO4 [M-H]- 256.0610, found 256.0615; HPLC purity: 100.00%, retention time: 14.34 min. 2-(3-Hydroxybenzamido)-5-nitrobenzoic acid (23). Column chromatography (CH2Cl2/MeOH/AcOH = 9/1/0.1); Yellow crystals (prisms); Yield: 54%; Rf 0.27 (CH2Cl2/MeOH/AcOH = 9/1/0.1); Mp: 267.0-268.0 °C; 1H NMR (300 MHz, DMSO-dt): 87.04-7.10 (m, 1H, Ar-H), 7.33-7.42 (m, 3H, Ar-H), 8.48 (dd, J = 9.3, 2.7 Hz, 1H, Ar-H), 8.80 (d, J = 2.7 Hz, 1H, Ar-H), 8.93 (d, J = 9.3 Hz, 1H, Ar-H), 9.95 (br s, 1H, OH), 12.80 (br s, 1H, NHCO), resonance for COOH missing; 13C NMR (100 MHz, DMSO-dt): 8 114.10, 116.53, 117.47, 119.75, 119.90, 126.57, 129.05, 130.12, 134.98, 141.20, 146.29, 157.86, 165.06, 168.45; HRMS (ESI) m/z calcd for C14H9N2O6 [M-H]- 301.0461, found 301.0469; HPLC purity: 99.18%, retention time: 17.00 min. 5-Bromo-2-(3-hydroxybenzamido)benzoic acid (24). Beige crystals (needles); Yield: 70%; Rf 0.85 (CH3CN/MeOH/H2O = 3/1/0.1); Mp: 277.0-281.0 °C; 1H NMR (300 MHz, DMSO-dt): 87.00-7.05 (m, 1H, Ar-H), 7.33-7.41 (m, 3H, Ar-H), 7.84 (dd, J = 9.0, 2.7 Hz, 1H, Ar-H), 8.11 (d, J = 2.7 Hz, 1H, Ar-H), 8.64 (d, J = 9.0 Hz, 1H, Ar-H), 9.93 (br s, 1H, OH), 12.03 (br s, 1H, NHCO), resonance for COOH missing; 13C NMR (100 MHz, DMSO-d6): 8 113.94, 114.14, 117.27, 118.56, 119.26, 121.88, 129.99, 133.20, 135.54, 136.66, 140.22, 157.77, 164.66, 168.58; HRMS (ESI) m/z calcd for C14H9NO4Br [M-H]- 333.9715, found 333.9711; HPLC purity: 100.00%, retention time: 19.54 min. 5-Bromo-2-(3-ethoxybenzamido)benzoic acid (25). White crystals (prisms); Yield: 82%; Rf 0.60 (CH2Cl2/MeOH/AcOH = 9/1/0.1); Mp: 226.5-229.0 °C; 1H NMR (300 MHz, DMSO-d6): 81.37 (t, J = 6.9 Hz, 3H, OCH2CH3), 4.12 (q, J = 6.9 Hz, 2H, OCH2CH3), 7.177.23 (m, 1H, Ar-H), 7.44-7.51 (m, 3H, Ar-H), 7.84 (dd, J = 9.0, 2.4 Hz, 1H, Ar-H), 8.12 (d, J = 2.4 Hz, 1H, Ar-H), 8.64 (d, J = 9.0 Hz, 1H, Ar-H), 12.06 (br s, 1H, NHCO), resonance for COOH missing; 13C NMR (100 MHz, DMSO-d6): 8 14.47, 63.25, 112.60, 114.26, 118.47, 118.81, 118.98, 121.89, 130.12, 133.20, 135.57, 136.64, 140.10, 158.72, 164.40, 168.59; HRMS (ESI) m/z calcd for C16H15NO4Br [M+H]+ 364.0184, found 364.0185; HPLC purity: 99.05%, retention time: 21.20 min. 5-Bromo-2-(3-butoxybenzamido)benzoic acid (26). White crystals (prisms); Yield: 94%; Rf 0.65 (CH2Cl2/MeOH/AcOH = 9/1/0.1); Mp: 206-209.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 0.95 (t, J = 7.5 Hz, 3H, OCH2CH2CH2CH3), 1.42-1.50 (m, 2H, OCH2CH2CH2 CH3), 1.69-1.78 (m, 2H, OCH2CH2CH2CH3), 4.05 (t, J = 6.4 Hz, 2H, OCH2CH2CH2CH3X 7.18-77.24 (m, 1H, Ar-H), 7.45-7.52 (m, 3H, Ar-H), 73.83 (dd, J = 9.0, 2.4 Hz, 1H, Ar-H), 8.12 (d, J = 2.4 Hz, 1H, Ar-H), 8.64 (d, J = 9.0 Hz, 1H, Ar-H), 12.12 (br s, 1H, NHCO), resonance for COOH missing; 13C NMR (100 MHz, DMSO-d6): 8 13.60, 18.65, 30.57, 67.32, 112.75, 114.23, 118.40, 118.88, 118.93, 121.85, 130.08, 133.21, 132.56, 136.56, 140.12, 158.90, 164.38, 168.61; HRMS (ESI) m/z calcd for C18H17NO4Br [M-H]- 390.0341, found 390.0342; Anal. calcd. for C18H18NO4Br: C, 55.12; H, 4.63; N, 3.57. Found: C, 55.27; H, 41.43; IN, 3.50. 2-(3-Butoxy-4-nitrobenzamido)benzoic acid (27). White crystals (prisms); Yield: 74%; Rf 0.92 (CH3CN/MeOH/H2O = 3/1/0.1); Mp: 174.0-176.5 °C; 1H NMR (300 MHz, DMSO-d6): 8 0.95 (t, J = 7.5 Hz, 3H, OCH2CH2CH2CH3), 1.37-1.52 (m, 2H, OCH2CH2CH2 CH3), 1.70-1.80 (m, 2H, OCH2CH2CH2CH3), 428 (2, J = 6.3 Hz, 2H, OCH2CH2CH2CH3), 7.26 (td, J= 7.8, 1.0 Hz, 1H, Ar-H), 7.62 (dd, J = 8.4, 1.2 Hz, 1H, Ar-H), 7.69 (td, J = 8.1, 1.2 Hz, 1H, Ar-H), 7.82 (d, J = 1.5 Hz, 1H), 8.04-8.09 (m, 2H, Ar-H), 8.62 (dd, J = 8.1, 1.0 Hz, 1H, Ar-H), 12.14 (br s, 1H, NHCO), resonance for COOH missing; 13C NMR (100 MHz, DMSO-d6): 813.53, 18.52, 30.28, 69.14, 113.65, 118.81, 120.19, 123.58, 125.41, 131.30, 134.10, 139.63, 140.36, 141.41, 149.73, 151.23, 163.07, 169.79; HRMS (ESI) m/z calcd for C18H17N2O6 [M-H]- 357.1087, found 357.1084; HPLC purity: 100.00%, retention time: 19.50 min. 3. 5. General Procedure for the Synthesis of Compounds 28-33. Reduction of Nitro Group. To a solution of the appropriate compound 15-17, 20, 23, or 27 (1.0 mmol) in MeOH (10 mL), 10% Pd/C was added, and the mixture was hydrogenated for 4 h at room temperature. The suspension was then filtered through a pad of celite, and washed with MeOH (10 mL). The solvent was removed under reduced pressure, yielding the pure products 28-33. 2-(3-Aminobenzamido)-5-hydroxybenzoic acid (28). Off-white solid; Yield: 72%; Rf 0.34 (CH3CN/ MeOH/H2O = 3/1/0.1); Mp: 175.0-178.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 6.75 (dd, J = 7.5, 1.6 Hz, 1H, Ar-H), 6.97-7.05 (m, 2H, Ar-H), 7.13-7.18 (m, 2H, Ar-H), 7.44 (d, J = 2.9 Hz, 1H, Ar-H), 8.50 (d, J = 9.0 Hz, 1H, Ar-H), 9.46 (s, 1H, OH), 12.19 (s, 1H, NHCO), resonances for NH2 and COOH missing; 13C NMR (100 MHz, DMSO-d6): 8 112.44, 113.55, 116.80, 116.86, 119.18, 120.31, 121.15, 129.10, 133.28, 135.78, 149.10, 152.20, 164.67, 169.82; HRMS (ESI) m/z calcd for C14H11N2O4 [M-H]- 271.0719, found 271.0720; HPLC purity: 95.23%, retention time: 4.01 min. 2-(3-Aminobenzamido)benzoic acid (29). Beige crystals (needles); Yield: 54%; Rf 0.67 (CH3CN/MeOH/H2O = 3/1/0.1); Mp: 224.5-227.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 6.79 (dd, J = 7.6, 1.5 Hz, 1H, Ar-H), 7.03-7.07 (m, 1H, Ar-H), 7.16-7.22 (m, 3H, Ar-H), 7.64 (td, J = 8.6, 1.6 Hz, 1H, Ar-H), 8.05 (dd, J = 7.9, 1.5 Hz, 1H, Ar-H), 8.70-8.74 (m, 1H, Ar-H), 12.07 (s, 1H, NHCO), resonances for NH2 and COOH missing; 13C NMR (100 MHz, DMSO-d6): 8 112.47, 113.54, 116.25, 117.27, 119.62, 122.55, 129.25, 131.21, 134.15, 135.34, 141.28, 149.23, 165.40, 169.93; HRMS (ESI) m/z calcd for C14H11N2O3 [M-H]- 255.0770, found 255.0774; HPLC purity: 95.69%, retention time: 9.11 min. 2-(3-Aminobenzamido)-4,5-dimethoxybenzoic acid (30). The product was additionally purified by crystallization from CH2Cl2; Brown crystals (prisms); Yield: 41%; Rf 0.40 (CH3CN/MeOH/H2O = 3/1/0.1); Mp: 225.0-227.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.76 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 6.73-6.78 (m, 1H, Ar-H), 7.10-7.19 (m, 3H, Ar-H), 7.54 (s, 1H, Ar-H), 8.50 (s, 1H, Ar-H), 13.14 (br s, 1H, NHCO), resonances for NH2 and COOH missing; 13C NMR (100 MHz, DMSO-d6): 8 55.31, 55.41, 102.69, 112.58, 113.78, 113.81, 113.97, 116.65, 128.88, 136.03, 136.16, 142.90, 148.91, 153.83, 164.70, 169.67; HRMS (ESI) m/z calcd for C16H15N2O5 [M-H]- 315.0981, found 315.0984; Anal. calcd. for C16H16N2O5: C, 60.75; H, 5.10; N, 8.86. Found: C, 60.76; H, 5.42; N, 8.60. 2-(3-Amino-4-fluorobenzamido)benzoic acid (31). Brown solid; Yield: 77%; Rf 0.36 (CH2Cl2/Me-OH/AcOH = 9/1/0.1); Mp: 270.0-272.5 °C; 1H NMR (300 MHz, DMSO-d6): S 6.97-7.03 (m, 1H, Ar-H), 7.18 (td, J = 7.8, 1.2 Hz, 1H, Ar-H), 7.47-7.55 (m, 1H, Ar-H), 7.61-7.68 (m, 2H, Ar-H), 8.04 (dd, J = 7.8, 1.5 Hz, 1H, Ar-H), 8.68 (d, J = 8.5 Hz, 1H, Ar-H), 12.01 (br s, 1H, NHCO), resonances for NH2 and COOH missing; 13C NMR (100 MHz, DMSO-d6): S 114.91 and 114.99 (1C, 3JCF = 7.5 Hz), 115.85 and 116.04 (1C, 2JCF = 19.5 Hz), 116.20 and 116.27 (1C, 3Jcf = 6.4 Hz), 119.677, 122.50, 125.62, 130.62, 130.94 and 130.97 (1C, 4JCF = 2.8 Hz), 134.27, 136.91 and 137.04 (1C, 2Jcf = 13.3 Hz), 141.36, 151.37 and 153.79 (1C, 1JCF = 242 Hz), 164.16, 169.95; HRMS (ESI) m/z calcd for C14HnN2O3F [M-H]- 273.0823, found 273.0968; Anal. calcd. for C14H12N2O3F: C, 61.31; H, 4.04; N, 6.93. Found: C, 61.03; H, 4.-43; N, 6.97. 5-Amino-2-(3-hydroxybenzamido)benzoic acid (32). Brown crystals (prisms); Yield: 78%; Rf 0.69 (MeOH); Mp: 241.0-245.0 °C; 1H NMR (300 MHz, DMSO-d6): S 6.78 (dd, J = 8.9, 3.0 Hz, 1H, Ar-H), 6.92-7.01 (m, 1H, Ar-H), 7.24-7.38 (m, 4H, Ar-H), 8.34 (d, J = 8.9 Hz, 1H, Ar-H), 9.78 (br s, 1H, OH), 12.23 (br s, 1H, NHCO), resonances for NH2 and COOH missing; 13C NMR (100 MHz, DMSO-d6): S 113.85, 115.71, 117.10, 118.23, 119.72, 119.86, 129.48, 129.60, 130.65, 136.80, 143.77, 157.58, 163.34, 167.26; HRMS (ESI) m/z calcd for C14H12N2O4Na [M+Na]+ 295.0695, found 295.0692; HPLC purity: 96.02%, retention time: 15.64 min. 2-(4-Amino-3-butoxybenzamido)benzoic acid (33). Brown crystals (prisms); Yield: 97%; Rf 0.60 (CH2Cl2/MeOH/AcOH = 9/1/0.1); Mp: 189.0-191.0 °C; 1H NMR (300 MHz, DMSO-d6): S0.96 (t, J = 7.5 Hz, 3H, OCH2CH2CH2CH3), 1.39-1.51 (m, 2H, OCH2CH2CH2 CH3), 1.70-1.80 (m, 2H, OCH2CH2CH2CH3), 4.03 (t, J = 6.3 Hz, 2H, OCH2CH2CH2CH3), 6.68 (d, J = 8.7 Hz, 1H, Ar-H), 6.90 (td, J = 7^, 1.2 Hz, 1H, Ar-H), 7.25 (td, J = 7.2, 1.5 Hz, 1H, Ar-H), 7.42-7.49 (m, 2H, Ar-H), 8.00 (dd, J = 7.5, 1.5 Hz, 1H, Ar-H), 8.63 (dd, J = 8.1, 1.2 Hz, 1H, Ar-H), 12.25 (br s, 1H, NHCO), resonances for NH2 and COOH missing; 13C NMR (100 MHz, DMSO-d6): S 13.74, 18.76, 30.83, 67.36, 110.46, 112.23, 117.98, 120.59, 120.85, 122.65, 124.61, 129.81, 131.18, 141.24, 141.45, 144.74, 164.38, 170.34; HRMS (ESI) m/z calcd for C18H20N2O4Na [M+Na]+ 351.1321, found 351.1320; HPLC purity: 94.31%, retention time: 20.28 min. 3. 6. General Procedure for the Preparation of Compounds 34-48. The corresponding sulfonyl chloride (1.0 mmol) was dissolved in CH2Cl2 (5 mL) and slowly added to a solution of the appropriate amine (1.2 mmol) in CH2Cl2 (5 mL), followed by addition of Py (237 mg, 3.0 mmol) after 10 min. The reaction mixture was stirred for 24 h at room temperature. After the reaction was complete (monitored by TLC), 2 M HCl (10 mL) was added and the product extracted with CH2Cl2 (3 x 20 mL). The combined organic phases were washed with brine (2 x 30 mL), dried over Na2SO4, filtered, and evaporated under reduced pressure. The resulting crude products were purified by crystallization or column chromatography, to yield compounds 34-48. Methyl 2-(naphthalene-2-sulfonamido)benzoate (34). Column chromatography (EtOAc/hexane = 1/5); White crystals (needles); Yield: 49%; Rf 0.29 (EtOAc/hexane = 1/3); Mp: 126.5-128.0 °C; 1H NMR (300 MHz, DMSO-d6): ¿3.78 (s, 3H, CH3), 7.11-7.17 (m, 1H, Ar-H), 7.49-7.55 (m, 2H, Ar-H), 7.62-7.83 (m, 4H, Ar-H), 8.01 (dd, J = 7.4, 1.4 Hz, 1H, Ar-H), 8.08 (d, J = 8.9 Hz, 1H, Ar-H), 8.13-8.18 (m, 1H, Ar-H), 8.53 (d, J = 1.7 Hz, 1H, Ar-H), 10.52 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): 8 52.55, 118.03, 119.99, 121.84, 123.94, 127.77, 1627.78, 128.46, 129.24, 129.29, 129.59, 130.92, 131.45, 134.34, 134.36, 135.55, 138.38, 167.45; HRMS (ESI) m/z calcd for C18H16NO4S [M+H]+ 342.0800, found 342.0785; HPLC purity: 100.00%, retention time: 19.95 min. Methyl 3-(naphthalene-2-sulfonamido)benzoate (35). Column chromatography (EtOAc/hexane = 1/2); White crystals (prisms); Yield: 42%; Rf 0.15 (EtOAc/ hexane = 1/3); Mp: 192.5-193.5 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.78 (s, 3H, CH3), 7.36 (ddd, J = 8.0, 7.5, 0.4 Hz, 1H, Ar-H), 7.39-7.43 (m, 1H, Ar-H), 7.57 (dt, J = 7.5, 1.5 Hz, 1H, Ar-H), .7.62-7.72 (m, 2H, Ar-H), 7.73 (m, 1H, Ar-H), 7.76 (dd, J = 8.7, 1.8 Hz, 1H, Ar-H), 7.99 (d, J = 8.0 Hz, 1H, Ar-H), 8.09 (d, J = 8.7 Hz, 1H, Ar-H), 8.11-8.15 (m, 1H, Ar-H), 8.45 (d, J = 1.8 Hz, 1H, Ar-H), 10.68 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): 8 52.21, 120.18, 121.76, 124.25, 124.59, 127.72, 127.78, 128.00, 129.04, 129.19, 129.56, 129.70, 130.45, 131.43, 134.22, 136.05, 138.06, 165.53; HRMS (ESI) m/z calcd for C18H16NO4S [M+H]+ 342.0800, found 342.0797; HPLC purity: 100.00%, retention time: 16.94 min. Dimethyl 5-(naphthalene-2-sulfonamido)isophthalate (36). Column chromatography (EtOAc/hexane = 1/2); White amorphous solid; Yield: 72%; Rf 0.46 (EtOAc/he-xane = 1/1); Mp: 211.5-213.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 3.83 (s, 6H, 2 x CH3), 7.62-7.74 (m, 2H, Ar-H), 7.7 7 (dd, J = 8.7, 1.9 Hz, 1H, Ar-H), 7.96-8.03 (m, 3H, Ar-H), 8.08 (t, J = 1.5 Hz, 1H, Ar-H), 8.09-8.17 (m, 2H, Ar-H), 8.47 (d, J = 1.5 Hz, 1H, Ar-H), 10.94 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): S 52.11, 52.57, 116.55, 118.01, 121.61, 124.67, 127.80, 127.82, 128.08, 129.18, 129.24, 129.77, 130.57, 131.11 (2C), 131.44, 134.30, 138.77, 164.72, 165.92; HRMS (ESI) m/z calcd for C20H18NO6S [M+H]+ 400.0855, found 400.0858; HPLC purity: 96.08%, retention time: 18.39 min. Methyl 5-bromo-2-(naphthalene-2-sulfonamido)ben-zoate (37). Column chromatography (Et2O/petroleum ether = 1/3); White crystals (prisms); Yield: 71%; Rf 0.19 (Et2O/petroleum ether = 1/3); Mp: 128.0-130.5 °C; 1H NMR (300 MHz, DMSO-d6): S 3.76 (s, 3H, CH3), 7.43 (dd, J = 8.8, 0.5 Hz, 1H, Ar-H), 7.65-7.75 (m, 3H, Ar-H), 7.76 (dd, J = 8.8, 2.2 Hz, Ar-H), 7.88 (dd, J = 2.2, 0.5 Hz, 1H, Ar-H), 8.00-8.04 (m, 1H, Ar-H), 8.10 (dd, J = 8.7, 0.5 Hz, 1H, Ar-H), 8.15-8.18 (m, 1H, Ar-H), 8.52-8.54 (m, 1H, Ar-H), 10.45 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): S 52.79, 115.93, 120.98, 121.84, 122.71, 127.81, 128.42, 129.30, 129.33, 129.66, 131.47, 133.04, 134.42, 135.48, 136.69, 137.26, 166.02; HRMS (ESI) m/z calcd for C18H15NO4SBr [M+H]+ 419.9905, found 419.9913; HPLC purity: 99.75%, retention time: 23.92 min. N-(3-Hydroxyphenyl)naphthalene-2-sulfonamide (38). Column chromatography (EtOAc/hexane = 1/1); Brown solid; Yield: 96%; Rf 0.13 (EtOAc/hexane = 1/2); Mp:121.0-125.0 °C; 1H NMR (300 MHz, DMSO-d6): S 6.36 (d, J = 7,9 Hz, 1H, Ar-H), 6.52-6.62 (m, 2H, Ar-H), 6.95 (t, J = 8.1 Hz, 1H, Ar-H), 7.60-7.73 (m, 2H, Ar-H), 7.77 (d, J = 8.8 Hz, 1H, Ar-H), 8.00 (d, J = 7.5 Hz, 1H, Ar-H), 8.05-8.17 (m, 2H, Ar-H), 8.40-8.44 (m, 1H, Ar-H), 9.39 (br s, 1H, OH), 10.25 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): S 107.99, 112.14, 112.50, 122.92, 128.86, 129.06, 129.09, 130.03,130.36, 130.58, 131.28, 132.39, 135.31, 136.79, 139.31, 158.28; HRMS (ESI) m/z calcd for C16H14NO3S [M+H]+ 300.0694, found 300.0696; HPLC purity: 97.50%, retention time: 12.48 min. Methyl 2-(naphthalene-1-sulfonamido)benzoate (39). Column chromatography (EtOAc/hexane = 1/2); White crystals (prisms); Yield: 61%; Rf 0.30 (EtOAc/hexane = 1/2); Mp: 191.5-192.5 °C; 1H NMR (300 MHz, DMSO-d6): S 3.74 (s, 3H, CH3), 7.08 (ddd, J = 7.9, 7.3, 1.2 Hz, 1H, Ar-H), 7.39-7.42 (m, 1H, Ar-H), 7.46-7.52 (m, 1H, Ar-H), 7.62-7.69 (m, 2H, Ar-H), 7.73-7.78 (m, 2H, Ar-H), 8.07-8.10 (m, 1H, Ar-H), 8.24-8.27 (m, 1H, Ar-H), 8.28 (dd, J = 7.4, 1.2 Hz, 1H, Ar-H), 8.50-8.55 (m, 1H, Ar-H), 10.87 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): S 52.61, 117.12, 119.03, 123.49, 123.59. 124.44, 126.94, 127.11, 128.44, 129.29, 130.39, 130.87, 133.12, 133.70, 134.40, 135.06, 138.46, 167.58; HRMS (ESI) m/z calcd for C18H16NO4S [M+H]+ 342.0800, found 342.080; HPLC purity: 99.13%, retention time: 19.35 min. Methyl 3-(naphthalene-1-sulfonamido)benzoate (40). Crystallization from EtOAc/hexane mixture (1/2); White crystals (needles); Yield: 64%; Rf 0.49 (EtOAc/ hexane = 1/1); Mp: 173.0-174.5 °C; 1H NMR (300 MHz, DMSO-d.): S3.78 (s, 3H, CH3), 7.27-7.34 (m, 2H, Ar-H), 7.46-7.54 (m, 1H, Ar-H), 7.59-7.70 (m, 3H, Ar-H), 7.75 (ddd, J = 8.6, 6.9, 1.4 Hz, 1H, Ar-H), 8.07 (dd, J = 7.9, 1.0 Hz, 1H, Ar-H), 8.19-8.25 (m, 2H, Ar-H), 8.71 (d, J = 8.6 Hz, 1H, Ar-H), 10.94 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d.): S 52.20, 118.96, 122.98, 123.98, 124.02, 124.41, 127.02, 127.24, 128.25, 129.10, 129.65, 129.96, 130.37, 133.69, 133.84, 134.62, 137.97, 165.50; HRMS (ESI) m/z calcd for C18H16NO4S [M+H]+ 342.0800, found 342.0807; HPLC purity: 100.00%, retention time: 16.15 min. Dimethyl 5-(naphthalene-1-sulfonamido)isophthalate (41). Crystallization from EtOAc/hexane mixture (1/2); Off-white crystals (needles); Yield: 91%; Rf 0.37 (EtOAc/ hexane = 1/1); Mp: 208.5-209.5 °C; 1H NMR (300 MHz, DMSO-d.): S 3.82 (s, 6H, 2 x CH3), 7.62-7.71 (m, 2H, Ar-H), 7.7 7 (ddd, J = 7.7, 6.8, 1.3 Hz, 1H, Ar-H), 7.88 (d, J = 1.5 Hz, 2H, Ar-H), 8.01 (t, J = 1.5 Hz, 1H, Ar-H), 8.08 (dd, J = 8.1, 1.3 Hz, 1H, Ar-H), 8.22-8.27 (m, 2H, Ar-H), 8.70 (d, J = 8.3 Hz, 1H, Ar-H), 11.25 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d.): S 52.11, 52.55, 116.54, 118.01, 122.52, 124.02, 124.43, 127.13, 127.15, 128.43, 129.19, 130.00, 130.57, 131.03, 133.48, 133.73, 134.91, 138.64, 164.69, 165.92; HRMS (ESI) m/z calcd for C20H18NO6S [M+H]+ 400.0855, found 400.0859; HPLC purity: 96.41%, retention time: 17.72 min. Methyl 5-bromo-2-(naphthalene-1-sulfonamido)ben-zoate (42). Column chromatography (EtOAc/hexane = 1/5); Brown crystals (prisms); Yield: 44%; Rf 0.21 (EtOAc/ hexane = 1/3); Mp: 151.0-153.0 °C; 1H NMR (300 MHz, DMSO-d.): S 3,71 (s, 3H, CH3), 7.34 (d, J = 8.8 Hz, 1H, Ar-H), 7.62-7.79 (m, 4H, Ar-H), 7.83 (d, J = 2.4 Hz, 1H, Ar-H), 8.08-8.13 (m, 1H, Ar-H), 8.22-8.31 (m, 2H, Ar-H), 8.52 (d, J = 8.5 Hz, 1H, Ar-H), 10.72 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d.): S 52.84, 115.58, 120.08, 121.90, 123.54, 124.47, 126.92, 127.16, 128.48, 129.30, 130.23, 132.97, 133.07, 133.72, 135.15, 136.75, 137.34, 166.15; HRMS (ESI) m/z calcd for C18H15NO4SBr [M+H]+ 419.9905, found 419.9892; HPLC purity: 89.86%, retention time: 23.37 min. N-(3-Hydroxyphenyl)naphthalene-1-sulfonamide (43). Column chromatography (EtOAc/hexane = 2/1); Yellow crystals (needles); Yield: 71%; Rf 0.53 (EtOAc/ hexane = 2/1); Mp: 137.0-139.5 °C; 1H NMR (300 MHz, DMSO-d.): S 5.83 (ddd, J = 8.1, 2.1, 0.9 Hz, 1H, Ar-H), 6.20 (t, J= 2.1 Hz, 1H, Ar-H), 6.38 (ddd, J = 8.1, 2.1, 0.9 Hz, 1H, Ar-H), 6.82 (t, J = 8.1 Hz, 1H, Ar-H), 7.66 (dd, J = 8.1, 7.4 Hz, 1H, Ar-H), 7.74-7.81 (m, 1H, Ar-H), 7.89 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H, Ar-H), 8.12 (dd, J = 7.4, 1.1 Hz, 1H, Ar-H), 8.20 (d, J = 8.1, 1.1 Hz, 1H, Ar-H), 8.39 (d, J = 8.1 Hz, 1H, Ar-H), 8.60-8.65 (m, 1H, Ar-H), resonances for OH and NH missing; 13C NMR (100 MHz, DMSO-d6): 8 106.19, 107.45, 112.54, 124.11, 124.54, 127.45, 127.57, 129.09, 129.34, 129.75, 130.08, 131.00, 133.67, 136.05, 150.05, 150.36; HRMS (ESI) m/z calcd for C16H14NO3S [M+H]+ 300.0694, found 300.0684; HPLC purity: 100.00%, retention time: 12.79 min. Methyl 2-(5-(dimethylamino)naphthalene-1-sulfona-mido)benzoate (44). Column chromatography (EtOAc/hexane = 1/3); Yellow crystals (needles); Yield: 56%; Rf 0.24 (EtOAc/ hexane = 1/3); Mp: 146.0-148.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 2.80 (s, 6H, N(CH3)2), 3.77 (s, 3H, CH3), 7.05-7.12 (m, 1H, Ar-H), 7.22-7.27 (m, 1H, Ar-H), 7.-41 (dd, J = 8.3, 1.0 Hz, 1H, Ar-H), 7.50 (ddd, J = 8.3, 7.2, 1.5 Hz, 1H, Ar-H), 7.59-7.67 (m, 2H, Ar-H), 7.78 (dd, J = 8.1, 1.5 Hz, 1H, Ar-H), 8.17 (dd, J = 8.6, 1.2 Hz, 1H, Ar-H), 8.28 (dd, J = 7.3, 1.2 Hz, 1H, Ar-H), 8.48 (dd, J = 8.6, 1.5 Hz, 1H, Ar-H), 10.85 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): 8 44.93 (2C), 52.65, 115.36, 116.64, 117.69, 118.59, 123.36, 123.49, 128.52, 128.53, 128.87, 130.27, 130.79, 130.90, 133.52, 134.48, 138.69, 151.61, 167.66; HRMS (ESI) m/z calcd for C20H21N2O4S [M+H]+ 385.1222, found 385.1217; HPLC purity: 99.62%, retention time: 20.21 min. Methyl 3-(5-(dimethylamino)naphthalene-1-sulfona-mido)benzoate (45). Column chromatography (EtOAc/hexane = 1/3); Fluorescent yellow crystals (prisms); Yield: 72%; Rf 0.15 (EtOAc/hexane = 1/3); Mp: 140.0-141.5 °C; 1H NMR (300 MHz, DMSO-d6): 8 2.80 (s, 6H, N(CH3)2), 3.78 (s, 3H, CH3), 7.25 (d, J = 7.3 Hz, 1H, Ar-H), 7.29-7.33 (m, 2H, Ar-H), 7.48-7.53 (m, 1H, Ar-H), 7.57-7.66 (m, 3H, Ar-H), 8.22 (dd, J = 7.3, 1.1 Hz, 1H, Ar-H), 8.35 (d, J = 8.7 Hz, 1H, Ar-H), 8.45 (d, J = 8.5 Hz, 1H, Ar-H), 10.91 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): 845.82 (2C), 53.39, 116.42, 116.95, 119.02, 119.836, 124.31, 124.40, 125.35, 129.71, 129.74, 130.81, 131.14, 131.30, 131.61, 134.66, 138.66, 152.46, 167.16; HRMS (ESI) m/z calcd for C20H21N2O4S [M+H]+ 385.1222, found 385.1225; HPLC purity: 100.00%, retention time: 15.23 min. Dimethyl 5-(5-(dimethylamino)naphthalene-1-sulfona-mido)isophthalate (46). Column chromatography (EtOAc/hexane = 1/3); Amorphous white solid; Yield: 43%; Rf 0.44 (EtOAc/he-xane = 1/1); Mp: 207.5-209.5 °C; 1H NMR (300 MHz, DMSO-d6): 8 2.80 (s, 6H, N(CH3)2), 3.83 (s, 6H, CH3), 7.27 (d, J = 7.5 Hz, 1H, Ar-H), 7.59-7.69 (m, 2H, Ar-H), 7.89 (d, J = 1.5 Hz, 2H, Ar-H), 8.02 (t, J = 1.5 Hz, 1H, Ar- H), 8.23 (dd, J = 7.5, 1.0 Hz, 1H, Ar-H), 8.34 (d, J = 8.5 Hz, 1H, Ar-H), 8.46 (d, J = 8.5 Hz, 1H, Ar-H), 11.21 (br s, IH, NH); 13C NMR (100 MHz, DMSO-d6): 844.99 (2C), 52.14, 52.56, 115.50, 116.92, 118.33, 118.36, 122.47, 123.55, 128.50, 128.74, 128.86, 129.84, 130.54, 130.61, 131.04, 133.95, 138.75, 151.25, 164.73, 165.89; HRMS (ESI) m/z calcd for C^H^NPgS [M+H]+ 443.1277, found 443.1263; HPLC purity: 95.04%, retention time: II.71 min. Methyl 5-bromo-2-(5-(dimethylamino)naphthalene-1-sulfonamido)benzoate (47). Column chromatography (Et2O/petroleum ether = 1/2); Fluorescent yellow crystals (needles); Yield: 41%; Rf 0.25 (Et2O/petroleum ether = 1/2); Mp: 148.0-150.0 °C; 1H NMR (300 MHz, DMSO-d6): 82.81 (s, 6H, N(CH3)2), 3.74 (s, 3H, CH3), 7.26 (d, J = 7.4 Hz, 1H, Ar-H), 7.35 (d, J = 8.9 Hz, 1H, Ar-H), 7.59-7.66 (m, 2H, Ar-H), 7.69 (dd, J = 8.9, 2.1 Hz, 1H, Ar-H), 7.85 (d, J = 2.1 Hz, 1H, Ar-H), 8.15 (d, J = 8.6 Hz, 1H, Ar-H), 8.24 (dd, J = 7.4, 1.0 Hz, 1H, Ar-H), 8.49 (d, J = 8.6 Hz, 1H, Ar-H), 10.72 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): 8 44.95 (2C), 52.90, 115.30, 115.40, 117.71, 119.51, 121.41, 123.52, 128.51, 128.60, 128.90, 130.15, 130.91, 132.99, 133.44, 136.84, 137.61, 151.63, 166.27; HRMS (ESI) m/z calcd for C^H^N^SBr [M+H]+ 463.0327, found 463.0319; HPLC purity: 95.41%, retention time: 20.17 min. 5-(Dimethylamino)-N-(3-hydroxyphenyl)naphthalene-1-sulfonamide (48). Column chromatography (EtOAc/hexane = 1/1); Fluorescent yellow crystals (prisms); Yield: 97%; Rf 0.30 (EtOAc/hexane = 1/1); Mp: 82.0-86.0 °C; 1H NMR (300 MHz, DMSO-d6): 8 2.80 (s, 6H, N(CH3)2), 6.31 (dd, J = 8.1, 2.1 Hz, 1H, Ar-H), 6.44-6.53 (m, 2H, Ar-H), 6.91 (t, J = 8.1 Hz, 1H, Ar-H), 7.25 (d, J = 7.5 Hz, 1H, Ar-H), 7.57-7.64 (m, 2H, Ar-H), 8.19 (d, J = 7.5 Hz, 1H, Ar-H), 8.37 (d, J = 8.6 Hz, 1H, Ar-H), 8.44 (d, J = 8.6 Hz, 1H, Ar-H), 9.35 (br s, 1H, OH), 10.50 (br s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): 8 44.98 (2C), 105.57, 109.13, 110.37, 115.20, 118.61, 123.45, 128.07, 128.90, 128.94, 129.64, 129.70, 129.96, 134.83, 138.65, 151.37, 157.72; HRMS (ESI) m/z calcd for C18H19N2O3S [M+H]+ 343.1116, found 343.1121; HPLC purity: 98.61%, retention time: 8.22 min. 3. 7. General procedure for the Preparation of Compounds 49-60. Alkaline hydrolysis. To a stirred solution of the corresponding protected sulfonamide derivative (1.0 mmol) in dioxane (2 mL), 1 M NaOH (1.5 mL) was added, and the reaction mixture stirred at room temperature until the starting material had completely reacted (monitored by TLC). The solvent was then evaporated under reduced pressure, and the residue was diluted with H2O (10 mL) and washed with EtOAc (2 x 10 mL). The aqueous phase was acidified to pH 2 using 1 M HCl, and extracted with EtOAc (3 x 10 mL). The combined organic extracts were washed with brine (3 x 10 mL), then dried over Na2SO4, filtered, and evaporated to dryness, to provide compounds 49-60. If necessary, these were additionally purified by column chromatography. 2-(Naphthalene-2-sulfonamido)benzoic acid (49). Off-white solid; Yield: 67%; Rf 0.40 (CH2Cl2/ MeOH/AcOH = 5/1/0.1); Mp: 223.0-225.0 °C; 1H NMR (300 MHz, DMSO-d6): 5 7.04-7.12 (m, 1H, Ar-H), 7.49-7.60 (m, 2H, Ar-H), 7.63-7.74 (m, 2H, Ar-H), 7.76 (dd, J = 8.7, 2.1 Hz, 1H, Ar-H), 7.86 (dd, J = 8.0, 1.3 Hz, 1H, Ar-H), 7.97-8.03 (m, 1H, Ar-H), 8.08 (d, J = 8.7 Hz, 1H, Ar-H), 8.13-8.19 (m, 1H, Ar-H), 8.59 (d, J = 1.2 Hz, 1H, Ar-H), 11.26 (br s, 1H, NH), resonance for COOH missing; 13C NMR (100 MHz, CD3COCD3-d6): 5117.62, 120.32, 123.92, 124.89, 129.63, 129.79, 130.75, 131.03, 131.18, 131.51, 133.54, 133.91, 136.55, 136.85, 138.16, 142.67, 171.43; HRMS (ESI) m/z calcd for C17H12NO4S [M-H]- 326.0487, found 326.0479; HPLC purity: 97.65%, retention time: 18.02 min. 3-(Naphthalene-2-sulfonamido)benzoic acid (50). Brown amorphous solid; Yield: 57%; Rf 0.43 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 226.0-228.0 °C; 1H NMR (300 MHz, DMSO-d6): 57.33 (ddd, J = 8.1, 7.5, 0.5 Hz, 1H, Ar-H), 7.38 (ddd, J = 8.1, 2.2, 1.4 Hz, 1H, Ar-H), 7.55 (dt, J = 7.5, 1.4 Hz, 1H, Ar-H), 7.62-7.72 (m, 3H, Ar-H), 7.77 (dd, J = 8.7, 1.8 Hz, 1H, Ar-H), 7.98-8.01 (m, 1H, Ar-H), 8.09 (d, J = 8.7 Hz, 1H, Ar-H), 8.11-8.14 (m, 1H, Ar-H); 8.44 (d, J = 1.8 Hz, 1H, Ar-H), 10.64 (br s, 1H, NH), 13.00 (br s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6): 5 120.51, 121.80, 123.93, 124.80, 127.71, 127.79, 127.96, 129.02, 129.17, 129.47, 129.54, 131.44, 131.65, 134.21, 136.14, 137.92, 166.60; HRMS (ESI) m/z calcd for C17H12NO4S [M-H]- 326.0487, found 326.0499; HPLC purity: 100.00%, retention time: 14.12 min. 5-(Naphthalene-2-sulfonamido)isophthalic acid (51). Off-white solid; Yield: 81%; Rf 0.18 (CH2Cl2/Me-OH/AcOH = 5/1/0.1); Mp: 273.0-277.0 °C; 1H NMR (300 MHz, DMSO-d6): 5 7.61-7.73 (m, 2H, Ar-H), 7.74-7.80 (m, 1H, Ar-H), 7.92-7.96 (m, 2H, Ar-H), 8.00 (d, J = 8.1 Hz, 1H, Ar-H), 8.05-8.16 (m, 3H, Ar-H), 8.45 (m, 1H, Ar-H), 10.85 (br s, 1H, NH), 13.27 (br s, 2H, COOH); 13C NMR (100 MHz, DMSO-d6): 5 117.32, 118.07, 121.66, 123.98, 125.23, 127.82 (2C), 127.99, 129.14, 129.21, 129.71, 131.45, 132.17, 134.27, 135.92, 138.45, 165.90, 167.14; HRMS (ESI) m/z calcd for C18H12NO6S [M-H]- 370.0385, found 370.0374; HPLC purity: 99.30%, retention time: 12.45 min. 5-Bromo-2-(naphthalene-2-sulfonamido)benzoic acid (52). Brown solid; Yield: 61%; Rf 0.36 (CH2Cl2/ MeOH/AcOH = 5/1/0.1); Mp: 200.0-202.5 °C; 1H NMR (300 MHz, DMSO-d6): 5 7.51 (dd, J = 8.8, 0.5 Hz, 1H, Ar-H), 7.65-7.74 (m, 3H, Ar-H), 7.76 (dd, J = 8.9, 2.0 Hz, 1H, Ar-H), 7.93 (dd, J = 2.3, 0.5 Hz, 1H, Ar-H), 8.00-8.04 (m, 1H, Ar-H), 8.10 (d, J = 8.9 Hz, 1H, Ar-H), 8.15-8.19 (m, 1H, Ar-H), 8.60 (d, J = 2.0 Hz, 1H, Ar-H), 11.24 (br s, 1H, NH), resonance for COOH missing; 13C NMR (100 MHz, DMSO-d6): 5 114.92, 118.99, 120.56, 121.62, 127.82, 127.84, 128.59, 129.35 (2C), 129.81, 131.46, 133.47, 134.44, 135.27, 136.87, 138.82, 168.28; HRMS (ESI) m/z calcd for C17H11NO4SBr [M-H]- 403.9592, found 403.9609; HPLC17pu1r1ity: 499.74%, retention time: 22.10 min. 2-(Naphthalene-1-sulfonamido)benzoic acid (53). Yellow solid; Yield: 52%; Rf 0.40 (CH2Cl2/ MeOH/AcOH = 5/1/0.1); Mp: 232.0-235.0 °C; 1H NMR (300 MHz, DMSO-d6): 5 6.98-7.05 (m, 1H, Ar-H), 7.43-7.49 (m, 2H, Ar-H), 7.64-7.69 (m, 2H, Ar-H), 7.73 (ddd, J = 8.6, 6.9, 1.5 Hz, 1H, Ar-H), 7.79-7.83 (m, 1H, Ar-H), 8.07-8.10 (m, 1H, Ar-H), 8.24-8.28 (m, 1H, Ar-H), 8.35 (dd, J1 = 7.4, 1.2 Hz, 1H, Ar-H), 8.53 (dd, J = 8.6, 0.8 Hz, 1H, Ar-H), 11.68 (br s, 1H, NH), resonance for COOH missing; 13C NMR (100 MHz, DMSO-d6): 5 115.59, 117.01, 122.78, 123.30, 124.45, 126.89, 1277.13, 128.49, 129.35, 130.60, 131.44, 132.93, 133.73, 134.51, 135.12, 139.73, 169.89; HRMS (ESI) m/z calcd for C17H12NO4S [M-H]- 326.0487, found 326.0497; HPLC purity: 100.00%, retention time: 17.07 min. 3-(Naphthalene-1-sulfonamido)benzoic acid (54). White amorphous solid; Yield: 97%; Rf 0.41 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 195.0-196.5 °C; 1H NMR (300 MHz, DMSO-d6): 5 7.25-7.32 (m, 2H, Ar-H), 7.47-7.52 (m, 1H, Ar-H), 7.59-7.71 (m, 3H, Ar-H), 7.75 (ddd, J = 8.5, 6.9, 1.4 Hz, 1H, Ar-H), 8.05-8.10 (m, 1H, Ar-H), 8.20-8.25 (m, 2H, Ar-H), 8.72 (d, J = 8.5 Hz, 1H, Ar-H), 10.90 (br s, 1H, NH), 12.91 (br s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6): 5 119.30, 122.70, 124.07, 124.25, 124.42, 127.03, 127.29, 128.24, 129.11, 129.43, 129.93, 131.61, 133.71, 133.94, 134.59, 137.82, 166.60; HRMS (ESI) m/z calcd for C17H12NO4S [M-H]- 326.0487, found 326.0502; HPLC purity: 100.00%, retention time: 13.28 min. 5-(Naphthalene-1-sulfonamido)isophthalic acid (55). Off-white amorphous solid; Yield: 72%; Rf 0.16 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 298.0-300.0 °C; 1H NMR (300 MHz, DMSO-d6): 5 7.60-7.88 (m, 5H, Ar-H), 7.99-8.03 (m, 1H, Ar-H), 8.05-8.12 (m, 1H, Ar-H), 8.20-8.27 (m, 2H, Ar-H), 8.67-8.74 (m, 1H, Ar-H), 11.16 (br s, 1H, NH), 13.25 (br s, 2H, COOH); 13C NMR (100 MHz, DMSO-d6): 5 122.66 (2C), 123.93, 124.43, 124.62, 127.12, 127.19, 128.38, 129.18, 129.89, 132.09 (2C), 133.64, 133.73, 134.81, 138.33, 165.87 (2C); HRMS (ESI) m/z calcd for C18H14NO6S [M+H]+ 372.0542, found 372.0541; HPLC purity: 99.64%, retention time: 11.89 min. 5-Bromo-2-(naphthalene-1-sulfonamido)benzoic acid (56). Brown amorphous solid; Yield: 86%; Rf 0.35 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 187.0-189.5 °C; 1H NMR (300 MHz, DMSO-d7): S 7.40 (dd, J =8.9, 0.5 Hz, 1H, Ar-H), 7.63-7.70 (m, 3H, Ar-H), 7.74 (ddd, J = 8.6, 6.9, 1.6 Hz, 1H, Ar-H), 7.88 (dd, J = 2.5, 0.5 Hz, 1H, Ar-H), 8.08-8.12 (m, 1H, Ar-H), 8.26-8.29 (m, 1H, Ar-H), 8.32 (dd, J = 7.4, 1.2 Hz, 1H, Ar-H), 8.51 (dd, J = 8.6, 1.0 Hz, 1H, Ar-H), 11.72 (br s, 1H, NH), resonance for COOH missing; 13C NMR (100 MHz, DMSO-d7): S 114.42, 118.08, 119.46, 123.30, 124.51, 126.86, 1277.23, 128.61, 129.43, 130.56, 132.79, 133.47, 133.78, 135.28, 136.94, 139.00, 168.54; HRMS (ESI) m/z calcd for C17H13NO4SBr [M+H]+ 405.9749, found 405.9744; HPLC purity: 98.01%, retention time: 21.33 min. 2-(5-(Dimethylamino)naphthalene-1-sulfonamido)ben-zoic acid (57). Green amorphous solid; Yield: 87%; Rf 0.51 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 190.5-192.0 °C; 1H NMR (300 MHz, DMSO-d7): S2.78 (s, 6H, N(CH3)2), 6.77-6.87 (m, 1H, Ar-H), 7.16-7.36 (m, 3H, Ar-H), 7.49-7.64 (m, 2H, Ar-H), 7.76-7.80 (m, 1H, Ar-H), 8.24-8.35 (m, 2H, Ar-H), 8.37-8.43 (m, 1H, Ar-H), resonances for NH and COOH missing; 13C NMR (100 MHz, DMSO-d7): S44.96 (2C), 115.09, 116.52, 117.80, 118.73, 120.50, 123.45, 127.82, 128.93, 128.95, 129.21, 129.50, 131.06, 133.06, 135.90, 142.84, 151.31, 169.59; HRMS (ESI) m/z calcd for C19H17N2O4S [M-H]- 369.0909, found 369.0904; HPLC purity: 99.26%, retention time: 17.01 min. 3-(5-(Dimethylamino)naphthalene-1-sulfonamido)ben-zoic acid (58). Yellow crystals (needles); Yield: 87%; Rf 0.40 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 226.5-228.5 °C; 1H NMR (300 MHz, DMSO-d7): S2.80 (s, 6H, N(CH3)2), 7.23-7.33 (m, 3H, Ar-H), 7.45-7.52 (m, 1H, Ar-H), 7.56-7.66 (m, 3H, Ar-H), 8.21 (dd, J= 7.4, 1.1 Hz, 1H, Ar-H), 8.34-8.38 (m, 1H, Ar-H), 8.42-8.47 (m, 1H, Ar-H), 10.86 (br s, 1H, NH), 12.93 (br s, 1H, COOH); 13C NMR (100 MHz, DMSO-d7): S 44.97 (2C), 115.29, 118.40, 119.13, 122.50, 123.46, 124.09, 128.27, 128.84, 128.90, 129.41, 129.74, 130.26, 131.60, 134.38, 137.91, 151.44, 166.61; HRMS (ESI) m/z calcd for C19H17N2O4S [M-H]- 369.0909, found 369.0900; HPLC purity: 99.26%, retention time: 17.01 min. 5-(5-(Dimethylamino)naphthalene-1-sulfonamido)iso-phthalic acid (59). Yellow crystals (needles); Yield: 89%; Rf 0.18 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 283.5-286.0 °C; 1H NMR (300 MHz, DMSO-d7): ¿2.80 (s, 6H, N(CH3)2), 7.26 (dd, J = 7.6, 0.5 Hz, 1H, Ar-H), 7.58-7.67 (m, 2H, Ar-H), 7.85 (d, J = 1.4 Hz, 2H, Ar-H), 8.01 (t, J = 1.4 Hz, 1H, Ar-H), 8.21 (dd, J = 7.3, 1.1 Hz, 1H, Ar-H), 8.32-8.37 (m, 1H, Ar-H), 8.43-8.48 (m, 1H, Ar-H), 11.11 (br s, 1H, NH), 13.25 (br s, 1H, COOH); 13C NMR (100 MHz, DMSO-d7): 8 44.96 (2C), 115.37, 118.25, 122.57 (2C), 123.46, 124.52, 128.43, 128.77, 128.92, 129.70, 130.48, 132.13 (2C), 134.07, 138.41, 151.50, 165.91 (2C); HRMS (ESI) m/z calcd for C20H17N2O6S [M-H]- 413.0807, found 413.0800; HPLC purity: 99.75%, retention time: 9.57 min. 5-Bromo-2-(5-(dimethylamino)naphthalene-1-sulfona-mido)benzoic acid (60). Yellow crystals (needles); Yield: 97%; Rf 0.43 (CH2Cl2/MeOH/AcOH = 5/1/0.1); Mp: 186.5-189.0 °C; 1H NMR (300 MHz, DMSO-d7): 82.78 (s, 6H, N(CH3)2), 7.15-7.21 (m, 2H, Ar-H), 7.26 (dd, J = 8.8, 2.6 Hz, 1H, Ar-H), 7.47-7.59 (m, 2H, Ar-H), 7.82 (d, J = 2.6 Hz, 1H, Ar-H), 8.18 (dd, J = 7.3, 1.1 Hz, 1H, Ar-H), 8.33-8.42 (m, 2H, Ar-H), resonances for NH and COOH missing; 13C NMR (100 MHz, DMSO-d7): 8 45.06 (2C), 110.52, 115.00, 118.42, 119.55, 122.(75, 123.47, 127.47, 128.37, 128.78, 129.05, 129.23, 132.89, 134.02, 137.37, 144.20, 151.18, 167.89; HRMS (ESI) m/z calcd for C19H18N2O4SBr [M+H]+ 449.0171, found 449.0160; HPLC purity: 99.86%, retention time: 22.20 min. 4. References 1.E. Sauvage, F. Kerff, E. Fonzé, R. Herman, B. Schoot, J. P. Marquette, Y. Taburet, D. Prevost, J. Dumas, P. Stefanic, J. Coyette, P. Charlier, Cell. Mol. Life Sci. 2002, 5, 1223-1232. 2. S. Lemaire, Y. Glupczynski, V. Duval, B. Joris, P. M. Tul-kens, F. Van Bambeke, Antimicrob. Agents Chemother. 2009, 53, 2289-2297. 3. B. Y. Feng, B. K. Shoichet, Nat. Protoc. 2006, 1, 550-553. 4. B. Lakaye, C. Damblon, M. Jamin, M. Galleni, S. Lepage, B. Joris, J. Marchandbrynaert, C. Frydrych, J-M. Frere, Bioc-hem. J. 1994, 300, 141-145. 5. European Committee for Antimicrobial Susceptibility Testing (EUCAST). Determination of Minimum Inhibitory Concentrations (MICs) of Antibacterial Agents by Broth Dilution. Clin. Microbiol. Infect. 2003, 9, 1-7. 6. Clinical Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard, 8th ed., M07-A8, 2009, 29(2), pp 1-65. 7. M. Rarey, B. Kramer, T. Lengauer, G. Klebe, J. Mol. Biol. 1996, 271, 470-489. 8.M. Makosza, M. Bialecki, J. Org. Chem. 1998, 73, 4878-4888. 9.W. Dong, J. Xu, L. Xiong, X. Liu, Z. Li, Chin. J. Chem. 2009, 27, 579-586. 10.G. Kumaraswamy, N. Jena, M. N. V. Sastry, B.A. Kumar, 11. O. Hara, K. Sugimoto, Y. Hamada, Tetrahedron 2004, 60, Org. Prep. Proced. Int. 2004, 3t, 341-345. 9381-9390.