CHAPMAN ELI (US)
UNIV ARIZONA (US)
WO2020092947A1 | 2020-05-07 |
TRENT A KUNKLE: "ANTIBIOTIC DISCOVERY TARGETING BACTERIAL GROEL/GROES CHAPERONIN SYSTEMS", MASTER'S THESIS, INDIANA UNIVERSITY, US, US, XP055706480, Retrieved from the Internet
WHAT IS CLAIMED IS: 1. A compound of formula I R1 is H, –OH, -OC1-C6 alkyl, –NHC(O)C1-C6 alkyl, -C(O)OC1-C6 alkyl, -C(O)OH, C1-C6 alkyl, -S-heteroaryl, or , wherein each hydrogen atom in C1- C6 alkyl is optionally substituted by –CN, R2 is H or halo, each of R3, R4, R5, and R6 is independently H, –OH, halo, C1-C6 alkyl, -O-C1-C6 alkyl, NO2, or -NH2, R7 is H or C1-C6 alkyl, X is –O-, -S-, -C(R9)(R10)m-, or -C(R9)(R10)mO-, optionally X is –O-, -S-, or C(R9)(CN); R8 is halo, R9 is H or –CN, R10 is H or –CN, m is 1 or 2, and n is 0, 1, or 2; or a pharmaceutically acceptable salt thereof, provided the compound of formula (I) is not . 2. The compound or pharmaceutically acceptable salt of claim 1, having the formula Ia) (Ia), wherein R1, R2, R3, R4, R5, R6, and R7 are defined in accordance with claim 1. 3. The compound or pharmaceutically acceptable salt of claim 1 or 2, wherein R3 is – OH or -O-C1-C6 alkyl. 4. The compound or pharmaceutically acceptable salt of claim 3, wherein R4 is halo. 5. The compound or pharmaceutically acceptable salt of claim 3 or 4, wherein R6 is halo. 6. The compound or pharmaceutically acceptable salt of claim 3 or 4, wherein R6 is chloro. 7. The compound or pharmaceutically acceptable salt of claim 1, wherein R7 is C1-C6 alkyl. 8. The compound or pharmaceutically acceptable salt of claim 7, wherein R7 is methyl. 9. The compound or pharmaceutically acceptable salt of any of the preceding claims, wherein X is –C(H)(CN)-. 10. The compound or pharmaceutically acceptable salt of any of the preceding claims, wherein R8 is chloro. 11. The compound or pharmaceutically acceptable salt of any of the preceding claims, wherein R2 is halo. 12. The compound or pharmaceutically acceptable salt of any of the preceding claims, wherein R2 is chloro. 13. The compound or pharmaceutically acceptable of claim 1, wherein R1 is –S- benzothiazole. 14. The compound of pharmaceutically acceptable salt of claim 1, having the formula Ib) (Ib). 15. The compound of claim 14, wherein R2 is H or halo, each of R3, R4, and R5 is independently H, –OH, halo, -O-C1-C6 alkyl, -NO2, or -NH2, and R7 is H or C1-C6 alkyl. 16. A method of killing or inhibiting the growth of bacteria, said method comprising contacting the bacteria with a compound of the formula I R1 is H, –OH, -OC1-C6 alkyl, –NHC(O)C1-C6 alkyl, -C(O)OC1-C6 alkyl, -C(O)OH, C1-C6 alkyl, -S-heteroaryl, or , wherein each hydrogen atom in C1- C6 alkyl is optionally substituted by –CN, R2 is H or halo, each of R3, R4, R5, and R6 is independently H, –OH, halo, -O-C1-C6 alkyl, -NO2, or - NH2, R7 is H or C1-C6 alkyl, X is –O-, -S-, -C(R9)(R10)m-, or -C(R9)(R10)mO-, R8 is halo, R9 is H or –CN, R10 is H or –CN, m is 1 or 2, and n is 0, 1, or 2; or a pharmaceutically acceptable salt thereof, provided the compound of formula (I) is not . 1 The method of claim 16, having the formula (Ia) (Ia), wherein R1, R2, R3, R4, R5, R6, and R7 are defined in accordance with claim 1. 18. The method of claim 16 or 17, wherein R3 is –OH or -O-C1-C6 alkyl. 19. The method of any of one of claims 16-18, wherein R4 is halo. 20. The method of any of one of claims 16-19, wherein R6 is halo. 21. The method of any of one of claims 16-20, wherein R6 is chloro. 22. The method of any of one of claims 16-21, wherein R7 is C1-C6 alkyl. 23. The method of any of one of claims 16-22, wherein R7 is methyl. 24. The method of any of one of claims 16-23, wherein X is –C(H)(CN)-. 25. The method of any of one of claims 16-24, wherein R8 is chloro. 26. The method of any of one of claims 16-25, wherein R2 is halo. 27. The method of any of one of claims 16-26, wherein R2 is chloro. 28. The method of claim 16, wherein R1 is –S-benzothiazole. 29. The method of claim 16 or 17, wherein the genus of bacteria are selected from a group consisting of Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, and Enterobacter or a combination thereof. 30. The method of claim 29, wherein the bacteria are Enterococcus faecium, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, or a combination thereof. 31. The method of claim 16 wherein the bacteria are contacted with a compound having the structure of formula II: wherein W is O or CHCN; R31 is OH, or OCH3; R32 is halo, optionally Cl or Br; R33 is H, or halo; R34 is H, or halo. In one embodiment W is O or CHCN, R31 is OH, R32 is Cl, and R33 and R34 are independently H, or Cl, or a compound of formula III: : wherein R40 is a compound of the formula wherein R31 is OH or OCH3; R32 and R36 are independently H, Br or Cl, with the proviso that R32 and R36 are not both H. 32. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1-15. 33. A method of killing bacteria in a biofilm comprising contacting the biofilm with a compound of any of the preceding claims. 34. A method of preventing bacteria from forming a biofilm comprising contactinghe bacteria with a compound of any of the preceding claims. 35. The method of claims 33 and 34, wherein the bacteria is from the genus Staphylococcus. |
. In some embodiments, the compound or pharmaceutically acceptable salt of formula I, is formula (Ia) In some embodiments, R 3 is –OH or -O-C 1 -C 6 alkyl. In some embodiments, R 3 is –OH. In some embodiments R 3 is –O-C 1 -C 6 alkyl. In some embodiments, R 4 is halo. In some embodiments, R 4 is a bromine. In some embodiments, R 4 is a chlorine. In some embodiments, R 4 is a fluorine. In some embodiments, R 4 is an iodine. In some embodiments, R 6 is halo. In some embodiments, R 6 is a bromo. In some embodiments, R 6 is a chloro. In some embodiments, R 6 is a fluoro. In some embodiments, R 6 is an iodo. In some embodiments, the compound is the formula (Ib) wherein R 2 , R 3 , R 4 , R 5 , and R 7 are as described herein; or a pharmaceutically acceptable salt thereof. In accordance with one embodiment a compound of formula II: is provided wherein W is O or CHCN; R 31 is OH, or OCH3; R 32 is halo, optionally Cl or Br; R 33 is H, or halo; R 34 is H, or halo. In one embodiment W is O or CHCN, R 31 is OH, R 32 is Cl, and R 33 and R 34 are independently H, or Cl. In one embodiment W is O, R 31 is OH, R 32 is Cl, and R 33 and R 34 are independently H, or Cl. In one embodiment the compound has the structure of wherein R 31 is OH, R 32 is Cl, and R 33 and R 34 are independently H, or Cl. In one embodiment the compounds of I, Ia, Ib and II are used as anti-microbial agents to inhibit replication and/or kill microbial organisms, including bacteria, such as S. aureus or other gram negative bacteria. In one embodiment the compounds of I, Ia, Ib and II are used to inhibiting chaperonin-mediated refolding as measured in the dMDH refolding assay of Example 76. In one embodiment the method of killing or inhibiting the growth of bacteria comprises contacting bacteria with a compound of formula I, Ia, Ib, or II, or a pharmaceutically acceptable salt thereof. In one embodiment a compound of formula III: is provided wherein R 40 is a compound of the formula wherein R 31 is OH or OCH3; R 32 and R 36 are independently H, Br or Cl, with the proviso that R 32 and R 36 are not both H. In one embodiment R 31 is OH, R 36 is H, and R 32 is Br or Cl. In one embodiment R 40 has the structure of wherein R 31 is OH, and R 32 is Br or Cl. In some embodiments, a method of killing or inhibiting the growth of bacteria comprising contacting the bacteria with a compound of formula I, Ia, Ib, II, or III, or a pharmaceutically acceptable salt thereof, is provided. In some embodiments, the bacteria is Gram-positive. In some embodiments, the bacteria is Gram-negative. In some embodiments, the bacteria comprise Gram-positive bacteria, Gram-negative bacteria, or a combination thereof. In some embodiments, the bacteria are capable of forming a biofilm. In some embodiments, the genus of bacteria are selected from a group consisting of Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, and Enterobacter or a combination thereof. In some embodiments, the bacteria are Enterococcus faecium, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, or a combination thereof. In some embodiments, a method of killing or inhibiting the growth of bacteria forming a biofilm is provided. The method comprises contacting the bacteria with a compound of formula I, Ia, Ib, II, III, or a pharmaceutically acceptable salt thereof. In some embodiments, a method of killing or inhibiting the growth of bacteria within a biofilm is provided. The method comprises contacting the biofilm with a compound of formula I, Ia, Ib, II, III or a pharmaceutically acceptable salt thereof. The following represent illustrative embodiments of compounds of the formula I, Ia, or Ib:
Clause 1. A compound of the formula I
, y , y , , y g n atom in C 1 -C 6 alkyl is optionally substituted by –CN, R 2 is H or halo, each of R 3 , R 4 , R 5 , and R 6 is independently H, –OH, halo, -O-C 1 -C 6 alkyl, - NO 2 , or -NH 2 , R 7 is H or C 1 -C 6 alkyl, X is –O-, -S-, -C(R 9 )(R 10 )m-, or -C(R 9 )(R 10 )mO-, optionally X is –O-, -S-, or -C(R 9 )(CN); R 8 is halo, R 9 is H or –CN, R 10 is H or –CN, m is 1 or 2, and n is 0, 1, or 2; or a pharmaceutically acceptable salt thereof, provided the compound of (closantel) or (rafoxanide). Clause 2. The compound or pharmaceutically acceptable salt of clause 1, having the formula (Ia) (Ia). Clause 3. The compound or pharmaceutically acceptable salt of clause 1 or 2, wherein R 3 is –OH or -O-C 1 -C 6 alkyl. Clause 4. The compound or pharmaceutically acceptable salt of any of the preceding clauses, wherein R 4 is halo. Clause 5. The compound or pharmaceutically acceptable salt of any of the preceding clauses, wherein R 6 is halo. Clause 6. The compound or pharmaceutically acceptable salt of any of the preceding clauses, wherein R 6 is chloro. Clause 7. The compound or pharmaceutically acceptable salt of any of the preceding clauses, wherein R 7 is C 1 -C 6 alkyl. Clause 8. The compound or pharmaceutically acceptable salt of any of the preceding clauses, wherein R 7 is methyl. Clause 9. The compound or pharmaceutically acceptable salt of any of the preceding clauses, wherein X is –C(H)(CN)-. Clause 10. The compound or pharmaceutically acceptable salt of any of the preceding clauses, wherein R 8 is chloro. Clause 11. The compound or pharmaceutically acceptable salt of any of the preceding clauses, wherein R 2 is halo. Clause 12. The compound or pharmaceutically acceptable salt of any of the preceding clauses, wherein R 2 is chloro. Clause 13. The compound or pharmaceutically acceptable of clause 1, wherein R 1 is –S-benzothiazole. Clause 14. The compound of pharmaceutically acceptable salt of clause 1, having the formula (Ib) Clause 15. The compound of clause 14, wherein R 2 is H or halo, each of R 3 , R 4 , and R 5 is independently H, –OH, halo, -O-C 1 -C 6 alkyl, - NO 2 , or -NH 2 , and R 7 is H or C 1 -C 6 alkyl Clause 16. A method of killing or inhibiting the growth of bacteria said method comprising contacting said bacteria with a compound of formula I R 1 is H, –OH, -OC 1 -C 6 alkyl, –NHC(O)C 1 -C 6 alkyl, -C(O)OC 1 -C 6 alkyl, - C(O)OH, C 1 -C 6 alkyl, -S-heteroaryl, or , wherein each hydrogen atom in C 1 -C 6 alkyl is optionally substituted by –CN, R 2 is H or halo, each of R 3 , R 4 , R 5 , and R 6 is independently H, –OH, halo, -O-C 1 -C 6 alkyl, C 1 -C 6 alkyl, -NO 2 , or -NH 2 , R 7 is H or C 1 -C 6 alkyl, X is –O-, -S-, -C(R 9 )(R 10 )m-, or -C(R 9 )(R 10 )mO-, optionally X is –O-, -S-, or -C(R 9 )(CN); R 8 is halo, R 9 is H or –CN, R 10 is H or –CN, m is 1 or 2, and n is 0, 1, or 2; or a pharmaceutically acceptable salt thereof, provided the compound of formula (I) is not . Clause 17. The method of clause 16, having the formula (Ia) (Ia). Clause 18. The method of clause 16 or 17, wherein R 3 is –OH or -O-C 1 -C 6 alkyl. Clause 19. The method of any of the preceding clauses, wherein R 4 is halo. Clause 20. The method of any of the preceding clauses, wherein R 6 is halo. Clause 21. The method of any of the preceding clauses, wherein R 6 is chloro. Clause 22. The method of any of the preceding clauses, wherein R 7 is C 1 - C6 alkyl. Clause 23. The method of any of the preceding clauses, wherein R 7 is methyl. Clause 24. The method of any of the preceding clauses, wherein X is – C(H)(CN)-. Clause 25. The method of any of the preceding clauses, wherein R 8 is chloro. Clause 26. The method of any of the preceding clauses, wherein R 2 is halo. Clause 27. The method of any of the preceding clauses, wherein R 2 is chloro. Clause 28. The method of clause 16, wherein R 1 is –S-benzothiazole. Clause 29. The method of clause 16 or 17, wherein the genus of bacteria are selected from a group consisting of Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, and Enterobacter or a combination thereof. Clause 30. The method of clause 29, wherein the bacteria are Enterococcus faecium, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, or a combination thereof. Clause 31. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of clauses 1-15. Clause 32. A compound selected from the group consisting of , , , , , , , , , ,
, , , , pharmaceutically acceptable salt thereof. Clause 33. A method of killing bacteria in a biofilm comprising contacting the biofilm with a compound of any of the preceding clauses. Clause 34. A method of preventing bacteria from forming a biofilm comprising contacting the bacteria with a compound of any of the preceding clauses. Clause 35. The method of clauses 33 and 34, wherein the bacteria is from the genus Staphylococcus. Those skilled in the art will recognize that the species listed or illustrated herein are not exhaustive, and that additional species within the scope of these defined terms may also be selected. CHEMICAL SYNTHESIS Exemplary chemical entities useful in methods of the description will now be described by reference to illustrative synthetic schemes for their general preparation below and the specific examples that follow. Artisans will recognize that, to obtain the various compounds herein, starting materials may be suitably selected so that the ultimately desired substituents will be carried through the reaction scheme with or without protection as appropriate to yield the desired product. Alternatively, it may be necessary or desirable to employ, in the place of the ultimately desired substituent, a suitable group that may be carried through the reaction scheme and replaced as appropriate with the desired substituent. Furthermore, one of skill in the art will recognize that the transformations shown in the schemes below may be performed in any order that is compatible with the functionality of the particular pendant groups. Abbreviations: The examples described herein use materials, including but not limited to, those described by the following abbreviations known to those skilled in the art:
Example 1 General Synthetic Method. Unless otherwise stated, all chemicals were purchased from commercial suppliers and used without further purification. Reaction progress was monitored by thin-layer chromatography on silica gel 60 F254 coated glass plates (EM Sciences). Flash chromatography was performed using a Biotage Isolera One flash chromatography system and eluting through Biotage KP-Sil Zip or Snap silica gel columns for normal-phase separations (hexanes:EtOAc gradients), or Snap KP- C18-HS columns for reverse-phase separations (H 2 O:MeOH gradients). Reverse- phase high-performance liquid chromatography (RP-HPLC) was performed using a Waters 1525 binary pump, 2489 tunable UV/Vis detector (254 and 280 nm detection), and 2707 autosampler. For preparatory HPLC purification, samples were chromatographically separated using a Waters XSelect CSH C18 OBD prep column (part number 186005422, 130 Å pore size, 5 µm particle size, 19x150 mm), eluting with a H 2 O:CH3CN gradient solvent system. Linear gradients were run from either 100:0, 80:20, or 60:40 A:B to 0:100 A:B (A = 95:5 H 2 O:CH 3 CN, 0.05% TFA; B = 5:95 H 2 O:CH 3 CN, 0.05% TFA. Products from normal-phase separations were concentrated directly, and reverse-phase separations were concentrated, diluted with H 2 O, frozen, and lyophilized. For primary compound purity analyses (HPLC-1), samples were chromatographically separated using a Waters XSelect CSH C18 column (part number 186005282, 130 Å pore size, 5 µm particle size, 3.0x150 mm), eluting with the above H 2 O:CH3CN gradient solvent systems. For secondary purity analyses (HPLC-2) of final test compounds, samples were chromatographically separated using a Waters XBridge C18 column (either part number 186003027, 130 Å pore size, 3.5 µm particle size, 3.0x100 mm, or part number 186003132, 130 Å pore size, 5.0 µm particle size, 3.0x100 mm), eluting with a H 2 O:MeOH gradient solvent system. Linear gradients were run from either 100:0, 80:20, 60:40, or 20:80 A:B to 0:100 A:B (A = 95:5 H 2 O:MeOH, 0.05% TFA; B = 5:95 H 2 O:MeOH, 0.05% TFA). Test compounds were found to be >95% in purity from both RP-HPLC analyses, with the exception of analogs 74 and, which were less pure in the HPLC-2 conditions (91% and 94% pure, respectively). Mass spectrometry data were collected using an Agilent analytical LC-MS at the IU Chemical Genomics Core Facility (CGCF). 1 H-NMR spectra were recorded on a Bruker 300 MHz spectrometer in the CGCF. Chemical shifts are reported in parts per million and calibrated to the d6-DMSO solvent peaks at 2.50 ppm. For the new analogs synthesized and evaluated in this study (45-117), the general protocols for the amide coupling, nitro-to-amine reduction, methoxy-to-hydroxy deprotection, and methyl ester-to-carboxyl deprotection reactions are presented below, with compound characterizations for each analog following. Scheme 1. Reagents and conditions: a) X = Cl: pyridine, CH 2 Cl2; b) X = OH: SOCl2, 60°C, 1 h, then concentrate and add CH 2 Cl2, R 1 -arylamine, and pyridine; c) Tin a) & b) General procedure for the amide coupling reactions. To stirring mixtures of the respective anilines (1 eq.) in anhydrous CH 2 Cl2 were added the respective R 2 -COCl (1.2 eq.) reagents and pyridine (1.2 eq.). Note that for any analogs where the R 2 -CO 2 H starting materials were only commercially available, the acids were first converted to the acid chlorides by stirring in thionyl chloride at 60°C for 1 h, then concentrating. The reactions were allowed to stir at room temperature for 18 h, then were either diluted with hexanes and the precipitates filtered, rinsed with water, collected, and purified by chromatography, or purified directly from the reactions. Flash chromatographic purification (either normal- phase with hexanes:EtOAc gradients, or reverse-phase with water:MeOH gradients) afforded the products as solids. If necessary, products were further purified by preparatory RP-HPLC (water:CH 3 CN gradients), concentrated, and lyophilized. Refer to the characterization data presented below for 1 H-NMR, MS, and HPLC purity information for individual compounds. c) General procedure for the nitro reduction reactions to give amine-bearing analogs 70-72. Tin powder (3 eq.) was added slowly to a stirring mixtures of 70- 72 (1 eq.) in a 10% mixture of HCl in AcOH. The reactions were stirred for 2 h, then purified directly by reverse-phase chromatography (water:MeOH gradients). Product fractions were collected and lyophilized, then re-purified by preparatory RP-HPLC (water:CH 3 CN gradients), concentrated, and lyophilized. Refer to the characterization data presented below for 1 H-NMR, MS, and HPLC purity information for individual compounds. d) General procedure for the methoxy deprotection step to give hydroxylated analogs. To stirring mixtures of the respective methoxy analogs (1 eq.) in anhydrous CH 2 Cl2 was added BBr3 (stock of 1 M in CH 2 Cl2, 3 eq.). The reactions were allowed to stir at R.T. (under Ar) for 18 h and then quenched with MeOH. Flash chromatographic purification (either normal-phase with hexanes:EtOAc gradients, or reverse-phase with water:MeOH gradients) afforded the products as solids. If necessary, products were further purified by preparatory RP-HPLC (water:CH3CN gradients), concentrated, and lyophilized. Refer to the characterization data presented below for 1 H-NMR, MS, and HPLC purity information for individual compounds. Example 2 Analog 45: 3,5-dibromo-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-methoxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.14 (s, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.84 (d, J = 2.3 Hz, 1H), 7.83 (s, 1H), 7.47-7.53 (m, 3H), 7.37-7.43 (m, 2H), 6.03 (s, 1H), 3.85 (s, 3H), 2.32 (s, 3H); MS (ESI) C23H15Br2Cl2N2O 2 [M-H]- m/z expected = 580.9, observed = 580.7; HPLC-1 = >99%; HPLC-2 = >99%. Example 3 Analog 46: 3,5-dibromo-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-hydroxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.71 (s, 1H), 8.23 (d, J = 2.2 Hz, 1H), 8.04 (d, J = 2.1 Hz, 1H), 7.89 (s, 1H), 7.56 (s, 1H), 7.48-7.54 (m, 2H), 7.37-7.44 (m, 2H), 6.05 (s, 1H), 2.28 (s, 3H); MS (ESI) C 22 H 13 Br 2 Cl 2 N 2 O 2 [M-H]- m/z expected = 566.9, observed = 566.7; HPLC-1 = 99%; HPLC-2 = 99%. Example 4 Analog 47: 3-bromo-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-methoxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.07 (s, 1H), 7.89 (s, 1H), 7.82 (dd, J = 8.0, 1.5 Hz, 1H), 7.68 (dd, J = 7.7, 1.4 Hz, 1H), 7.47-7.54 (m, 3H), 7.37-7.43 (m, 2H), 7.23 (t, J = 7.8 Hz, 1H), 6.03 (s, 1H), 3.86 (s, 3H), 2.34 (s, 3H); MS (ESI) C 23 H 16 BrCl 2 N 2 O 2 [M-H]- m/z expected = 503.0, observed = 502.9; HPLC-1 = 98%; HPLC-2 = 98%. Example 5 Analog 48: 3-bromo-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 12.75 (br s, 1H), 10.58 (s, 1H), 7.94-8.01 (m, 1H), 7.75 (dd, J = 7.8, 1.2 Hz, 1H), 7.64 (s, 1H), 7.50 (s, 1H), 7.41-7.48 (m, 2H), 7.31-7.39 (m, 2H), 6.89 (t, J = 7.9 Hz, 1H), 5.99 (s, 1H), 2.22 (s, 3H); MS (ESI) C22H14BrCl2N2O 2 [M-H]- m/z expected = 489.0, observed = 488.8; HPLC-1 = >99%; HPLC-2 = >99%. Example 6 Analog 49: 5-bromo-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-methoxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 9.99 (s, 1H), 8.14 (s, 1H), 7.96 (d, J = 2.4 Hz, 1H), 7.74 (dd, J = 8.8, 2.4 Hz, 1H), 7.47- 7.53 (m, 3H), 7.35-7.43 (m, 2H), 7.24 (d, J = 8.8 Hz, 1H), 6.02 (s, 1H), 3.98 (s, 3H), 2.34 (s, 3H); MS (ESI) C23H16BrCl2N2O 2 [M-H]- m/z expected = 503.0, observed = 502.8; HPLC-1 = 97%; HPLC-2 = 97%. Example 7 Analog 50: 5-bromo-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 12.22 (s, 1H), 10.46 (s, 1H), 8.27 (s, 1H), 8.07 (d, J = 2.6 Hz, 1H), 7.61 (dd, J = 8.8, 2.6 Hz, 1H), 7.46-7.54 (m, 3H), 7.36-7.42 (m, 2H), 7.01 (d, J = 8.8 Hz, 1H), 6.02 (s, 1H), 2.33 (s, 3H); MS (ESI) C22H14BrCl2N2O 2 [M-H]- m/z expected = 489.0, observed = 488.89; HPLC-1 = >99%; HPLC-2 = >99%. Example 8 Analog 51: 3,5-dichloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2 - methylphenyl)-2-methoxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.13 (s, 1H), 7.86 (d, J = 2.6 Hz, 1H), 7.84 (s, 1H), 7.71 (d, J = 2.6 Hz, 1H), 7.47-7.53 (m, 3H), 7.37-7.43 (m, 2H), 6.03 (s, 1H), 3.87 (s, 3H), 2.32 (s, 3H); MS (ESI) C23H15Cl4N2O 2 [M-H]- m/z expected = 493.0, observed = 492.9; HPLC-1 = 96%; HPLC-2 = 98%. Example 9 Analog 52: 3,5-dichloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2 - methylphenyl)-2-hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.68 (s, 1H), 7.98 (d, J = 2.5 Hz, 1H), 7.77 (d, J = 2.5 Hz, 1H), 7.72 (s, 1H), 7.49 (s, 1H), 7.41-7.48 (m, 2H), 7.30-7.38 (m, 2H), 5.98 (s, 1H), 2.23 (s, 3H); MS (ESI) C22H13Cl4N2O 2 [M-H]- m/z expected = 479.0, observed = 478.9; HPLC-1 = >99%; HPLC-2 = 99%. Example 10 Analog 53: 3-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-methoxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.07 (s, 1H), 7.90 (s, 1H), 7.67 (td, J = 7.9, 1.5 Hz, 2H), 7.47-7.54 (m, 3H), 7.37-7.43 (m, 2H), 7.30 (t, J = 7.8 Hz, 1H), 6.03 (s, 1H), 3.88 (s, 3H), 2.34 (s, 3H); MS (ESI) C 23 H 16 Cl 3 N 2 O 2 [M-H]- m/z expected = 457.0, observed = 456.9; HPLC-1 = 98%; HPLC-2 = >99%. Example 11 Analog 54: 3-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 12.83 (br s, 1H), 10.65 (s, 1H), 7.99 (dd, J = 8.1, 1.4 Hz, 1H), 7.77 (s, 1H), 7.67 (dd, J = 7.8, 1.4 Hz, 1H), 7.56 (s, 1H), 7.48-7.54 (m, 2H), 7.37-7.44 (m, 2H), 7.01 (t, J = 8.0 Hz, 1H), 6.05 (s, 1H), 2.29 (s, 3H); MS (ESI) C22H14Cl3N2O 2 [M-H]- m/z expected = 443.0, observed = 442.9; HPLC-1 = >99%; HPLC-2 = 99%. Example 12 Analog 55: 4-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-methoxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 9.92 (s, 1H), 8.19 (s, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.46-7.53 (m, 3H), 7.34-7.42 (m, 3H), 7.19 (dd, J = 8.4, 1.9 Hz, 1H), 6.01 (s, 1H), 4.02 (s, 3H), 2.34 (s, 3H); MS (ESI) C23H16Cl3N2O 2 [M-H]- m/z expected = 457.0, observed = 456.9; HPLC-1 = 98%; HPLC-2 = >99%. Example 13 Analog 56: 4-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 12.42 (br s, 1H), 10.42 (s, 1H), 8.29 (s, 1H), 7.99 (d, J = 9.1 Hz, 1H), 7.45-7.56 (m, 3H), 7.34-7.43 (m, 2H), 7.03-7.11 (m, 2H), 6.02 (s, 1H), 2.33 (s, 3H); MS (ESI) C22H14Cl3N2O 2 [M-H]- m/z expected = 443.0, observed = 442.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 14 Analog 57: 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-methoxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 9.94 (s, 1H), 8.08 (s, 1H), 7.78 (d, J = 2.7 Hz, 1H), 7.57 (dd, J = 8.9, 2.7 Hz, 1H), 7.41- 7.47 (m, 3H), 7.30-7.36 (m, 2H), 7.24 (d, J = 8.9 Hz, 1H), 5.96 (s, 1H), 3.93 (s, 3H), 2.28 (s, 3H); MS (ESI) C23H16Cl3N2O 2 [M-H]- m/z expected = 457.0, observed = 456.9; HPLC-1 = 98%; HPLC-2 = >99%. Example 15 Analog 58: 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.55 (s, 1H), 8.28 (s, 1H), 7.95 (d, J = 2.8 Hz, 1H), 7.46-7.54 (m, 4H), 7.36-7.42 (m, 2H), 7.06 (d, J = 8.8 Hz, 1H), 6.02 (s, 1H), 2.33 (s, 3H); MS (ESI) C22H14Cl3N2O 2 [M- H]- m/z expected = 443.0, observed = 442.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 16 Analog 59: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-methoxy-5-methylbenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.06 (s, 1H), 8.28 (s, 1H), 7.76 (d, J = 2.0 Hz, 1H), 7.46-7.54 (m, 3H), 7.36-7.42 (m, 3H), 7.16 (d, J = 8.5 Hz, 1H), 6.01 (s, 1H), 3.99 (s, 3H), 2.36 (s, 3H), 2.31 (s, 3H); MS (ESI) C24H19Cl2N2O 2 [M-H]- m/z expected = 437.1, observed = 437.0; HPLC-1 = 98%; HPLC-2 = >99%. Example 17 Analog 60: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-hydroxy-5-methylbenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 11.71 (s, 1H), 10.52 (s, 1H), 8.33 (s, 1H), 7.81 (d, J = 1.9 Hz, 1H), 7.47-7.54 (m, 3H), 7.36-7.42 (m, 2H), 7.26 (dd, J = 8.5, 2.0 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.01 (s, 1H), 2.33 (s, 3H), 2.27 (s, 3H); MS (ESI) C23H17Cl2N2O 2 [M-H]- m/z expected = 423.1, observed = 423.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 18 Analog 61: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-methoxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.04 (s, 1H), 8.27 (s, 1H), 7.94 (dd, J = 7.7, 1.7 Hz, 1H), 7.55-7.63 (m, 1H), 7.47-7.53 (m, 3H), 7.36-7.43 (m, 2H), 7.27 (d, J = 8.3 Hz, 1H), 7.10-7.17 (m, 1H), 6.01 (s, 1H), 4.01 (s, 3H), 2.36 (s, 3H); MS (ESI) C 23 H 17 Cl 2 N 2 O 2 [M-H]- m/z expected = 423.1, observed = 423.0; HPLC-1 = 996%; HPLC-2 = 99%. Example 19 Analog 62: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-3-methoxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 9.98 (s, 1H), 7.61 (s, 1H), 7.44-7.57 (m, 6H), 7.37-7.44 (m, 2H), 7.18 (ddd, J = 8.1, 2.6, 0.9 Hz, 1H), 6.04 (s, 1H), 3.83 (s, 3H), 2.28 (s, 3H); MS (ESI) C23H17Cl2N2O 2 [M- H]- m/z expected = 423.1, observed = 423.0; HPLC-1 = 98%; HPLC-2 = 98%. Cl N Example 20 Analog 63: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-4-methoxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 9.83 (s, 1H), 7.92-7.98 (m, 2H), 7.61 (s, 1H), 7.48-7.54 (m, 3H), 7.37-7.43 (m, 2H), 7.04- 7.10 (m, 2H), 6.03 (s, 1H), 3.84 (s, 3H), 2.28 (s, 3H); MS (ESI) C23H17Cl2N2O 2 [M-H]- m/z expected = 423.1, observed = 423.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 21 Analog 64: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 11.97 (br s, 1H), 10.56 (s, 1H), 8.32 (s, 1H), 8.00 (dd, J = 7.9, 1.7 Hz, 1H), 7.36-7.54 (m, 6H), 6.96-7.06 (m, 2H), 6.02 (s, 1H), 2.34 (s, 3H); MS (ESI) C 22 H 15 Cl 2 N 2 O 2 [M- H]- m/z expected = 409.1, observed = 409.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 22 Analog 65: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-3-hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 9.89 (s, 1H), 9.79 (s, 1H), 7.61 (s, 1H), 7.48-7.54 (m, 3H), 7.36-7.54 (m, 3H), 7.29-7.36 (m, 2H), 6.99 (ddd, J = 7.8, 2.4, 1.2 Hz, 1H), 6.03 (s, 1H), 2.28 (s, 3H); MS (ESI) C22H15Cl2N2O 2 [M-H]- m/z expected = 409.1, observed = 408.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 23 Analog 66: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-4-hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.15 (s, 1H), 9.71 (s, 1H), 7.81-7.88 (m, 2H), 7.61 (s, 1H), 7.47-7.54 (m, 3H), 7.37-7.43 (m, 2H), 6.83-6.91 (m, 2H), 6.02 (s, 1H), 2.27 (s, 3H); MS (ESI) C 22 H 15 Cl 2 N 2 O 2 [M-H]- m/z expected = 409.1, observed = 409.0; HPLC-1 = 96%; HPLC-2 = 95%. Example 24 Analog 67: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-nitrobenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.39 (s, 1H), 8.15-8.21 (m, 1H), 7.72-7.94 (m, 4H), 7.47-7.55 (m, 3H), 7.37-7.44 (m, 2H), 6.04 (s, 1H), 2.30 (s, 3H); MS (ESI) C 22 H 14 Cl 2 N 3 O 3 [M-H]- m/z expected = 438.0, observed = 438.0.6; HPLC-1 = 95%; HPLC-2 = 99%. Example 25 Analog 68: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-3-nitrobenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.39 (s, 1H), 8.78 (t, J = 1.9 Hz, 1H), 8.43-8.50 (m, 1H), 8.36-8.53 (m, 1H), 7.86 (t, J = 8.0 Hz, 1H), 7.63 (s, 1H), 7.48-7.57 (m, 3H), 7.38-7.45 (m, 2H), 6.05 (s, 1H), 2.30 (s, 3H); MS (ESI) C22H14Cl2N3O3 [M-H]- m/z expected = 438.0, observed = 438.0; HPLC-1 = 97%; HPLC-2 = 99%. Example 26 Analog 69: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-4-nitrobenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.34 (s, 1H), 8.35-8.42 (m, 2H), 8.15-8.23 (m, 2H), 7.94 (s, 1H), 7.48-7.56 (m, 3H), 7.38-7.44 (m, 2H), 6.05 (s, 1H), 2.30 (s, 3H); MS (ESI) C 22 H 14 Cl 2 N 3 O 3 [M-H]- m/z expected = 438.0, observed = 438.0; HPLC-1 = 98%; HPLC-2 = 98%. Example 27 Analog 70: 2-amino-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)benzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 9.73 (s, 1H), 7.67 (dd, J = 8.0, 1.2 Hz, 1H), 7.60 (s, 1H), 7.47-7.55 (m, 3H), 7.36-7.44 (m, 2H), 7.17- 7.26 (m, 1H), 6.73-6.80 (m, 1H), 6.55-6.64 (m, 1H), 6.37 (br s, 2H), 6.03 (s, 1H), 2.27 (s, 3H); MS (ESI) C22H16Cl2N3O [M-H]- m/z expected = 408.1, observed = 408.0; HPLC-1 = >99%; HPLC-2 = >99% Example 28 Analog 71: 3-amino-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)benzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 9.85 (s, 1H), 7.54 (s, 1H), 7.41-7.48 (m, 3H), 7.29-7.37 (m, 4H), 7.22-7.29 (m, 1H), 6.93-7.00 (s, 1H), 6.25 (br s, 2H), 5.97 (s, 1H), 2.21 (s, 3H); MS (ESI) C22H16Cl2N3O [M-H]- m/z expected = 408.1, observed = 408.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 29 Analog 72: 4-amino-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)benzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 9.19 (s, 1H), 7.41- 7.48 (m, 2H), 7.37 (s, 1H), 7.20-7.28 (m, 3H), 7.10-7.18 (m, 2H), 6.31-6.39 (m, 2H), 5.78 (s, 1H), 5.54 (br s, 2H), 2.01 (s, 3H); MS (ESI) C 22 H 16 Cl 2 N 3 O [M-H]- m/z expected = 408.1, observed = 408.0; HPLC-1 = 95%; HPLC-2 = 96%. Example 30 Analog 73: 2-bromo-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)benzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.18 (s, 1H), 7.69- 7.76 (m, 2H), 7.61 (dd, J = 7.4, 1.5 Hz, 1H), 7.47-7.55 (m, 4H), 7.37-7.47 (m, 3H), 6.04 (s, 1H), 2.33 (s, 3H); MS (ESI) C22H14BrCl2N2O [M-H]- m/z expected = 473.0, observed = 472.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 31 Analog 74: 3-bromo-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)benzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.13 (s, 1H), 8.13 (t, J = 1.7 Hz, 1H), 7.93-7.97 (m, 1H), 7.82 (ddd, J = 8.0, 1.9, 0.9 Hz, 1H), 7.60 (s, 1H), 7.48-7.55 (m, 4H), 7.38-7.44 (m, 2H), 6.04 (s, 1H), 2.28 (s, 3H); MS (ESI) C 22 H 14 BrCl 2 N 2 O [M-H]- m/z expected = 473.0, observed = 472.8; HPLC-1 = >99%; HPLC-2 = 91%. Example 32 Analog 75: 4-bromo-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)benzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.08 (s, 1H), 7.88- 7.94 (m, 2H), 7.73-7.79 (m, 2H), 7.61 (s, 1H), 7.48-7.54 (m, 3H), 7.37-7.43 (m, 2H), 6.04 (s, 1H), 2.28 (s, 3H); MS (ESI) C22H14BrCl2N2O [M-H]- m/z expected = 473.0, observed = 472.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 33 Analog 76: 2-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)benzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.19 (s, 1H), 7.72 (s, 1H), 7.64 (dd, J = 7.2, 1.8 Hz, 1H), 7.55-7.61 (m, 1H), 7.43-7.55 (m, 5H), 7.36- 7.43 (m, 2H), 6.04 (s, 1H), 2.32 (s, 3H); MS (ESI) C22H14Cl3N2O [M-H]- m/z expected = 427.0, observed = 427.0; HPLC-1 = 98%; HPLC-2 = >99%. Example 34 Analog 77: 3-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)benzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.13 (s, 1H), 8.00 (t, J = 1.7 Hz, 1H), 7.91 (dt, J = 7.8, 1.3 Hz, 1H), 7.66-7.72 (m, 1H), 7.55-7.62 (m, 2H), 7.48-7.54 (m, 3H), 7.37-7.44 (m, 2H), 6.04 (s, 1H), 2.28 (s, 3H); MS (ESI C22H14Cl3N2O [M-H]- m/z expected = 427.0, observed = 426.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 35 Analog 78: 4-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)benzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.08 (s, 1H), 7.95- 8.01 (m, 2H), 7.59-7.66 (m, 3H), 7.48-7.54 (m, 3H), 7.37-7.43 (m, 4H), 6.04 (s, 1H), 2.28 (s, 3H); MS (ESI) C 22 H 14 Cl 3 N 2 O [M-H]- m/z expected = 427.0, observed = 426.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 36 Analog 79: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-2-fluorobenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 9.99 (s, 1H), 7.70-7.82 (m, 2H), 7.56-7.66 (m, 1H), 7.47-7.54 (m, 3H), 7.30-7.44 (m, 4H), 6.03 (s, 1H), 2.31 (s, 3H); MS (ESI) C 22 H 14 Cl 2 FN 2 O [M-H]- m/z expected = 411.0, observed = 411.0; HPLC-1 = 99%; HPLC-2 = 95%. Example 37 Analog 80: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-3-fluorobenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.09 (s, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.76 (dt, J = 9.8, 2.0 Hz, 1H), 7.56-7.65 (m, 2H), 7.43-7.54 (m, 4H), 7.37-7.43 (m, 2H), 6.04 (s, 1H), 2.29 (s, 3H); MS (ESI) C 22 H 14 Cl 2 FN 2 O [M-H]- m/z expected = 411.0, observed = 410.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 38 Analog 81: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)-4-fluorobenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.02 (s, 1H), 8.00-8.08 (m, 2H), 7.61 (s, 1H), 7.48-7.54 (m, 3H), 7.34-7.44 (m, 4H), 6.04 (s, 1H), 2.28 (s, 3H); MS (ESI) C22H14Cl2FN2O [M-H]- m/z expected = 411.0, observed = 411.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 39 Analog 82: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)benzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.00 (s, 1H), 7.94- 7.99 (m, 2H), 7.48-7.65 (m, 7H), 7.38-7.43 (m, 2H), 6.04 (s, 1H), 2.29 (s, 3H); MS (ESI) C 22 H 15 Cl 2 N 2 O [M-H]- m/z expected = 393.1, observed = 393.0; HPLC-1 = 96%; HPLC-2 = >99%. Example 40 Analog 83: N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2- methylphenyl)acetamide. 1 H-NMR (300 MHz, d6-DMSO) δ 9.41 (s, 1H), 7.74 (s, 1H), 7.46-7.53 (m, 2H), 7.42 (s, 1H), 7.34-7.39 (m, 2H), 5.98 (s, 1H), 2.24 (s, 3H), 2.09 (s, 3H); MS (ESI) C17H13Cl2N2O [M-H]- m/z expected = 331.0, observed = 331.0; HPLC-1 = 99%; HPLC-2 = 98%. Example 41 Analog 5-chloro-N-(4-(cyano(phenyl)methyl)phenyl)-2- methoxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.28 (s, 1H), 7.71-7.77 (m, 2H), 7.51-7.59 (m, 2H), 7.32-7.44 (m, 7H), 7.20 (d, J = 8.8 Hz, 1H), 5.78 (s, 1H), 3.86 (s, 3H); MS (ESI) C 22 H 16 ClN 2 O 2 [M-H]- m/z expected = 375.1, observed = 375.0; HPLC-1 = 97%; HPLC-2 = 96%. Example 42 Analog 5-chloro-N-(4-(cyano(phenyl)methyl)phenyl)-2- hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 11.79 (br s, 1H), 10.50 (s, 1H), 7.92 (d, J = 2.7 Hz, 1H), 7.70-7.77 (m, 2H), 7.46 (dd, J = 8.8, 2.7 Hz, 1H), 7.38-7.44 (m, 6H), 7.30-7.38 (m, 1H), 7.00 (d, J = 8.8 Hz, 1H), 5.80 (s, 1H); MS (ESI) C21H14ClN2O 2 [M-H]- m/z expected = 361.1, observed = 361.0; HPLC-1 = 98%; HPLC-2 = 97%. Example 43 Analog 86: 5-chloro-N-(3-chloro-4-(4-chlorophenoxy)phenyl)-2- methoxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.42 (s, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.67 (dd, J = 8.9, 2.5 Hz, 1H), 7.60 (d, J = 2.6 Hz, 1H), 7.56 (dd, J = 8.8, 2.7 Hz, 1H), 7.37-7.45 (m, 2H), 7.22 (dd, J = 8.8, 1.2 Hz, 2H), 6.91-6.98 (m, 2H), 3.88 (s, 3H); MS (ESI) C 20 H 13 Cl 3 NO 3 [M-H]- m/z expected = 420.0, observed = 419.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 44 Analog 87: 5-chloro-N-(3-chloro-4-(4-chlorophenoxy)phenyl)-2- hydroxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 11.65 (br s, 1H), 10.55 (s, 1H), 8.07 (d, J = 2.5 Hz, 1H), 7.89 (d, J = 2.6 Hz, 1H), 7.67 (dd, J = 8.8, 2.5 Hz, 1H), 7.47 (dd, J = 8.8, 2.7 Hz, 1H), 7.38-7.45 (m, 2H), 7.23 (d, J = 8.8 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.92-6.99 (m, 2H); MS (ESI) C 19 H 11 Cl 3 NO 3 [M-H]- m/z expected = 406.0, observed = 405.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 45 Analog 88: 5-chloro-N-(4-(4-chlorophenoxy)phenyl)-2-methoxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.24 (s, 1H), 7.71-7.80 (m, 2H), 7.59 (d, J = 2.7 Hz, 1H), 7.54 (dd, J = 8.8, 2.8 Hz, 1H), 7.38-7.46 (m, 2H), 7.20 (d, J = 8.9 Hz, 1H), 7.02-7.09 (m, 2H), 6.96-7.02 (m, 2H), 3.87 (s, 3H); MS (ESI) C 20 H 14 Cl 2 NO 3 [M-H]- m/z expected = 386.0, observed = 386.0; HPLC-1 = 99%; HPLC-2 = >99%. Example 46 Analog 89: 5-chloro-N-(4-(4-chlorophenoxy)phenyl)-2-hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 11.86 (br s, 1H), 10.47 (s, 1H), 7.95 (d, J = 2.7 Hz, 1H), 7.70-7.77 (m, 2H), 7.39-7.50 (m, 3H), 7.05-7.11 (m, 2H), 6.98-7.05 (m, 3H); MS (ESI) C 19 H 12 Cl 2 NO 3 [M-H]- m/z expected = 372.0, observed = 371.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 47 Analog 90: 5-chloro-N-(3-chloro-4-phenoxyphenyl)-2-methoxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.40 (s, 1H), 8.07 (d, J = 2.5 Hz, 1H), 7.65 (dd, J = 8.8, 2.5 Hz, 1H), 7.61 (d, J = 2.7 Hz, 1H), 7.56 (dd, J = 8.8, 2.5 Hz, 1H), 7.32- 7.41 (m, 2H), 7.22 (d, J = 8.8 Hz, 1H), 7.17 (d, J = 8.8 Hz, 1H), 7.07-7.14 (m, 1H), 6.88-6.95 (m, 2H), 3.88 (s, 3H); MS (ESI) C 20 H 14 Cl 2 NO 3 [M-H]- m/z expected = 386.0, observed = 386.0; HPLC-1 = 97%; HPLC-2 = >99%. Example 48 Analog 91: 5-chloro-N-(3-chloro-4-phenoxyphenyl)-2-hydroxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 11.66 (br s, 1H), 10.54 (s, 1H), 8.07 (d, J = 2.5 Hz, 1H), 7.90 (d, J = 2.7 Hz, 1H), 7.65 (dd, J = 8.9, 2.6 Hz, 1H), 7.47 (dd, J = 8.8, 2.7 Hz, 1H), 7.34-7.43 (m, 2H), 7.17 (d, J = 8.8 Hz, 1H), 7.08-7.15 (m, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.90-6.97 (m, 2H); MS (ESI) C19H12Cl2NO3 [M-H]- m/z expected = 372.0, observed = 371.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 49 Analog 92: 5-chloro-2-methoxy-N-(4-phenoxyphenyl)benzamide. 1 H- NMR (300 MHz, d 6 -DMSO) δ 10.22 (s, 1H), 7.70-7.77 (m, 2H), 7.60 (d, J = 2.7 Hz, 1H), 7.54 (dd, J = 8.8, 2.7 Hz, 1H), 7.33-7.41 (m, 2H), 7.21 d, J = 8.8 Hz, 1H), 7.07-7.14 (m, 1H), 7.01-7.06 (m, 2H), 6.94-7.00 (m, 2H), 3.88 (s, 3H); MS (ESI) C20H15ClNO3 [M-H]- m/z expected = 352.1, observed = 352.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 50 Analog 93: 5-chloro-2-hydroxy-N-(4-phenoxyphenyl)benzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 11.87 (br s, 1H), 10.48 (s, 1H), 7.96 (d, J = 2.7 Hz, 1H), 7.67-7.76 (m, 2H), 7.47 (dd, J = 8.8, 2.7 Hz, 1H), 7.35-7.43 (m, 2H), 7.09-7.16 (m, 1H), 6.97-7.08 (m, 5H); MS (ESI) C19H13ClNO3 [M-H]- m/z expected = 338.1, observed = 338.0; HPLC-1 = 99%; HPLC-2 = >99%. Example 51 Analog 94: N-(4-(benzo[d]thiazol-2-ylthio)-3-chlorophenyl)-5-chloro-2- methoxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.71 (s, 1H), 8.22 (d, J = 2.1 Hz, 1H), 7.92-7.99 (m, 2H), 7.79-7.88 (m, 2H), 7.64 (d, J = 2.6 Hz, 1H), 7.59 (dd, J = 8.8, 2.6 Hz, 1H), 7.47 (td, J = 7.7, 1.3 Hz, 1H), 7.31-7.39 (m, 1H), 7.24 (d, J = 8.8 Hz, 1H), 3.89 (s, 3H); MS (ESI) C 21 H 13 Cl 2 N 2 O 2 S 2 [M-H]- m/z expected = 459.0, observed = 458.8; HPLC-1 = >99%; HPLC-2 = >99%. Example 52 Analog 95: N-(4-(benzo[d]thiazol-2-ylthio)-3-chlorophenyl)-5-chloro-2- hydroxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 11.45 (s, 1H), 10.73 (s, 1H), 8.25 (d, J = 2.1 Hz, 1H), 7.91-8.00 (m, 2H), 7.81-7.88 (m, 3H), 7.42-7.51 (m, 2H), 7.31-7.39 (m, 1H), 7.05 (d, J = 8.8 Hz, 1H); MS (ESI) C20H11Cl2N2O 2 S2 [M- H]- m/z expected = 445.0, observed = 444.9; HPLC-1 = 97%; HPLC-2 = 97%. Example 53 Analog 96: N-(4-(benzo[d]thiazol-2-ylthio)phenyl)-5-chloro-2- methoxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.58 (s, 1H), 7.89-7.97 (m, 3H), 7.76-7.86 (m, 3H), 7.62 (d, J = 2.6 Hz, 1H), 7.57 (dd, J = 8.9, 2.6 Hz, 1H), 7.45 (td, J = 7.7, 1.3 Hz, 1H), 7.29-7.37 (m, 1H), 7.23 (d, J = 8.9 Hz, 1H), 3.89 (s, 3H); MS (ESI) C 21 H 14 ClN 2 O 2 S 2 [M-H]- m/z expected = 425.0, observed = 424.9; HPLC-1 = 98%; HPLC-2 = >99%. Example 54 Analog 97: N-(4-(benzo[d]thiazol-2-ylthio)phenyl)-5-chloro-2- hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 11.56 (br s, 1H), 10.68 (s, 1H), 7.90-7.98 (m, 3H), 7.89 (d, J = 2.6 Hz, 1H), 7.78-7.86 (m, 3H), 7.41-7.52 (m, 2H), 7.29-7.37 (m, 1H), 7.04 (d, J = 8.8 Hz, 1H); MS (ESI) C 20 H 12 ClN 2 O 2 S 2 [M- H]- m/z expected = 411.0, observed = 410.9; HPLC-1 = >99%; HPLC-2 = >99%. Example 55 Analog 98: N-(4-benzylphenyl)-5-chloro-2-methoxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 10.13 (s, 1H), 7.59-7.65 (m, 2H), 7.58 (d, J = 2.6 Hz, 1H), 7.53 (dd, J = 8.8, 2.6 Hz, 1H), 7.25-7.32 (m, 2H), 7.16-7.24 (m, 6H), 3.90 (s, 2H), 3.87 (s, 3H); MS (ESI) C21H17ClNO 2 [M-H]- m/z expected = 350.1, observed = 350.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 56 Analog 99: N-(4-benzylphenyl)-5-chloro-2-hydroxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 11.92 (br s, 1H), 10.40 (s, 1H), 7.96 (d, J = 2.7 Hz, 1H), 7.58-7.64 (m, 2H), 7.46 (dd, J = 8.8, 2.7 Hz, 1H), 7.14-7.33 (m, 7H), 7.00 (d, J = 8.8 Hz, 1H), 3.92 (s, 2H); MS (ESI) C 20 H 15 ClNO 2 [M-H]- m/z expected = 336.1, observed = 336.0; HPLC-1 = 96%; HPLC-2 = 97%. Example 57 Analog 100: 5-chloro-2-methoxy-N-(4-phenethylphenyl)benzamide. 1 H- NMR (300 MHz, d6-DMSO) δ 10.12 (s, 1H), 7.58-7.63 (m, 3H), 7.54 (dd, J = 8.8, 2.7 Hz, 1H), 7.13-7.31 (m, 8H), 3.88 (s, 3H), 2.86 (s, 4H); MS (ESI) C 22 H 19 ClNO 2 [M-H]- m/z expected = 364.1, observed = 364.1; HPLC-1 = 98%; HPLC-2 = 99%. Example 58 Analog 101: 5-chloro-2-methoxy-N-(4-phenethylphenyl)benzamide. 1 H- NMR (300 MHz, d 6 -DMSO) δ 11.93 (s, 1H), 10.36 (s, 1H), 7.97 (d, J = 2.7 Hz, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.46 (dd, J = 8.8, 2.7 Hz, 1H), 7.13-7.31 (m, 7H), 7.01 (d, J = 8.8 Hz, 1H), 2.87 (s, 4H); MS (ESI) C 21 H 17 ClNO 2 [M-H]- m/z expected = 350.1, observed = 350.0; HPLC-1 = 97%; HPLC-2 = 94%. Example 59 Analog 102: N-(4-(benzyloxy)phenyl)-5-chloro-2-methoxybenzamide. 1 H- NMR (300 MHz, d6-DMSO) δ 10.06 (s, 1H), 7.58-7.66 (m, 3H), 7.53 (dd, J = 8.8, 2.8 Hz, 1H), 7.29-7.48 (m, 5H), 7.19 (d, J = 8.8 Hz, 1H), 6.96-7.03 (m, 2H), 5.09 (s, 2H), 3.88 (s, 3H); MS (ESI) C21H17ClNO3 [M-H]- m/z expected = 366.1, observed = 366.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 60 Analog 103: N-(4-(benzyloxy)phenyl)-5-chloro-2-hydroxybenzamide. 1 H- NMR (300 MHz, d 6 -DMSO) δ 12.02 (br, s, 1H), 10.34 (s, 1H), 7.98 (d, J = 2.6 Hz, 1H), 7.55-7.64 (m, 2H), 7.43-7.50 (m, 3H), 7.29-7.43 (m, 3H), 6.97-7.07 (m, 3H), 5.10 (s, 2H); MS (ESI) C 20 H 15 ClNO 3 [M-H]- m/z expected = 352.1, observed = 352.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 61 Analog 104: N-(4-acetamidophenyl)-5-chloro-2-methoxybenzamide. 1 H- NMR (300 MHz, d 6 -DMSO) δ 10.11 (s, 1H), 9.91 (s, 1H), 7.58-7.66 (m, 3H), 7.50- 7.57 (m, 3H), 7.20 (d, J = 8.9 Hz, 1H), 3.88 (s, 3H), 2.02 (s, 3H); MS (ESI) C 16 H 14 ClN 2 O 3 [M-H]- m/z expected = 317.1, observed = 317.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 62 Analog 105: N-(4-acetamidophenyl)-5-chloro-2-hydroxybenzamide. 1 H- NMR (300 MHz, d6-DMSO) δ 11.96 (s, 1H), 10.37 (s, 1H), 9.95 (s, 1H), 7.98 (d, J = 2.7 Hz, 1H), 7.54-7.64 (m, 4H), 7.46 (dd, J = 8.8, 2.7 Hz, 1H), 7.00 (d, J = 8.8 Hz, 1H), 2.03 (s, 3H); MS (ESI) C15H12ClN2O3 [M-H]- m/z expected = 303.1, observed = 303.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 63 Analog 106: methyl 4-(5-chloro-2-methoxybenzamido)benzoate. 1 H-NMR (300 MHz, d6-DMSO) δ 10.54 (s, 1H), 7.92-7.98 (m, 2H), 7.82-7.88 (m, 2H), 7.60 (d, J = 2.7 Hz, 1H), 7.56 (dd, J = 8.8, 2.7 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 3.87 (s, 3H), 3.83 (s, 3H); MS (ESI) C16H13ClNO4 [M-H]- m/z expected = 318.1, observed = 318.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 64 Analog 107: methyl 4-(5-chloro-2-hydroxybenzamido)benzoate. 1 H-NMR (300 MHz, d 6 -DMSO) δ 11.63 (br, s, 1H), 10.66 (s, 1H), 7.93-8.01 (m, 2H), 7.84- 7.90 (m, 3H), 7.47 (dd, J = 8.8, 2.7 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H), 3.84 (s, 3H); MS (ESI) C 15 H 11 ClNO 4 [M-H]- m/z expected = 304.0, observed = 304.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 65 Analog 108: 4-(5-chloro-2-methoxybenzamido)benzoic acid. 1 H-NMR (300 MHz, d6-DMSO) δ 12.77 (br, s, 1H), 10.50 (s, 1H), 7.89-7.96 (m, 2H), 7.79-7.87 (m, 2H), 7.60 (d, J = 2.6 Hz, 1H), 7.56 (dd, J = 8.8m 2.6 Hz, 1H), 7.21 (d, J = 8.8 Hz, 1H), 3.88 (s, 3H); MS (ESI) C15H11ClNO4 [M-H]- m/z expected = 304.0, observed = 304.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 66 Analog 109: 4-(5-chloro-2-hydroxybenzamido)benzoic acid. 1 H-NMR (300 MHz, d 6 -DMSO) δ 12.78 (br s, 1H), 11.65 (br s, 1H), 10.63 (s, 1H), 7.92-7.98 (m, 2H), 7.89 (d, J = 2.7 Hz, 1H), 7.81-7.87 (m, 2H), 7.47 (dd, J = 8.8, 2.7 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H); MS (ESI) C 14 H 9 ClNO 4 [M-H]- m/z expected = 290.0, observed = 290.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 67 Analog 110: 5-chloro-N-(4-(cyanomethyl)phenyl)-2-methoxybenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.26 (s, 1H), 7.70-7.76 (m, 2H), 7.60 (d, J = 2.7 Hz, 1H), 7.52-7.57 (m, 1H), 7.29-7.35 (m, 2H), 7.20 (d, J = 8.9 Hz, 1H), 4.00 (s, 2H), 3.88 (s, 3H); MS (ESI) C 16 H 12 ClN 2 O 2 [M-H]- m/z expected = 299.1, observed = 299.0; HPLC-1 = 98%; HPLC-2 = 98%. Example 68 Analog 111: 5-chloro-N-(4-(cyanomethyl)phenyl)-2-hydroxybenzamide. 1 H-NMR (300 MHz, d6-DMSO) δ 11.82 (br s, 1H), 10.47 (s, 1H), 7.95 (d, J = 2.7 Hz, 1H), 7.69-7.76 (m, 2H), 7.47 (dd, J = 8.8, 2.7 Hz, 1H), 7.30-7.39 (m, 2H), 7.01 (d, J = 8.8 Hz, 1H), 4.02 (s, 2H); MS (ESI) C15H10ClN2O 2 [M-H]- m/z expected = 285.0, observed = 285.0; HPLC-1 = 98%; HPLC-2 = 98%. Example 69 Analog 112: 5-chloro-2-methoxy-N-(4-methoxyphenyl)benzamide. 1 H- NMR (300 MHz, d 6 -DMSO) δ 10.05 (s, 1H), 7.58-7.66 (m, 3H), 7.53 (dd, J = 8.8, 2.8 Hz, 1H), 7.20 (d, J = 8.9 Hz, 1H), 6.88-6.95 (m, 2H), 3.88 (s, 3H), 3.74 (s, 3H); MS (ESI) C 15 H 13 ClNO 3 [M-H]- m/z expected = 290.1, observed = 290.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 70 Analog 113: 5-chloro-2-hydroxy-N-(4-methoxyphenyl)benzamide. 1 H- NMR (300 MHz, d 6 -DMSO) δ 12.04 (br s, 1H), 10.34 (s, 1H), 7.99 (d, J = 2.7 Hz, 1H), 7.56-7.63 (m, 2H), 7.46 (dd, J = 8.8, 2.7 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.92-6.97 (m, 2H), 3.75 (s, 3H); MS (ESI) C 14 H 11 ClNO 3 [M-H]- m/z expected = 276.0, observed = 276.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 71 Analog 114: 5-chloro-N-(4-hydroxyphenyl)-2-methoxybenzamide. 1 H- NMR (300 MHz, d6-DMSO) δ 9.94 (s, 1H), 9.27 (s, 1H), 7.59 (d, J = 2.8 Hz, 1H), 7.45-7.55 (m, 3H), 7.19 (d, J = 8.8 Hz, 1H), 6.69-6.76 (m, 2H), 3.88 (s, 3H); MS (ESI) C14H11ClNO3 [M-H]- m/z expected = 276.0, observed = 276.0; HPLC-1 = 99%; HPLC-2 = 99%. Example 72 Analog 115: 5-chloro-2-hydroxy-N-(4-hydroxyphenyl)benzamide. 1 H- NMR (300 MHz, d6-DMSO) δ 12.12 (S, 1H), 10.25 (s, 1H), 9.37 (s, 1H), 8.00 (d, J = 2.6 Hz, 1H), 7.41-7.51 (m, 3H), 6.99 (d, J = 8.8 Hz, 1H), 6.72-6.81 (m, 2H); MS (ESI) C13H9ClNO3 [M-H]- m/z expected = 262.0, observed = 262,0; HPLC-1 = >99%; HPLC-2 = >99%. Example 73 Analog 116: 5-chloro-2-methoxy-N-phenylbenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 10.19 (s, 1H), 7.68-7.74 (m, 2H), 7.60 (d, J = 2.7 Hz, 1H), 7.51-7.57 (m, 1H), 7.30-7.38 (m, 2H), 7.21 (d, J = 8.8 Hz, 1H), 7.06-7.13 (m, 1H), 3.88 (s, 3H); MS (ESI) C 14 H 13 ClNO 2 [M+H] + m/z expected = 262.1, observed = 262.0; HPLC-1 = 99%; HPLC-2 = >99%. Example 74 Analog 117: 5-chloro-2-hydroxy-N-phenylbenzamide. 1 H-NMR (300 MHz, d 6 -DMSO) δ 11.87 (br s, 1H), 10.42 (s, 1H), 7.96 (d, J = 2.6 Hz, 1H), 7.66-7.74 (m, 2H), 7.47 (dd, J = 8.8, 2.7 Hz, 1H), 7.33-7.42 (m, 2H), 7.11-7.18 (m, 1H), 7.01 (d, J = 8.8 Hz, 1H); MS (ESI) C 13 H 9 ClNO 2 [M-H]- m/z expected = 246.0, observed = 246.0; HPLC-1 = >99%; HPLC-2 = >99%. Example 75 Protein expression and purification. E. coli GroEL and GroES purification. E. coli GroEL was expressed from a trc-promoted and Amp(+) resistance marker plasmid in DH5α. E. coli cells. GroES was expressed from a T7-promoted and Amp(+) resistance plasmid in E. coli BL21 (DE3) cells. Transformed colonies were plated onto Ampicillin-treated LB agar and incubated for 24 h at 37°C. Cells were then grown at 37°C in Ampicillin-treated LB medium until an OD 600 of 0.5 was reached, then were induced with 0.8 mM IPTG and continued to grow for 2-3 h at 37°C. The cultures were centrifuged at 8,000 rpm and the cell pellets were collected and re-suspended in Buffer A (50 mM Tris-HCl, pH 7.4, and 20 mM NaCl) supplemented with EDTA-free complete protease inhibitor cocktail (Roche). The combined suspension was lysed by sonication, the lysate was centrifuged at 14,000 rpm, and the clarified lysate was passed through a 0.45 μm filter (Millipore). Anion exchange purification. The filtered lysate was loaded onto a GE HiScale Anion exchange column (Q Sepharose fast flow anion exchange resin) that was equilibrated with 2 column volumes of Buffer A. The loaded column was washed with 4 column volumes of Buffer A containing 30% of Buffer B (50 mM Tris-HCl, pH 7.4, and 1 M NaCl), then bound protein was eluted with a 30-60% gradient elution of Buffer B. Protein-containing fractions, as identified by SDS- PAGE, were collected, spin concentrated using a 10 kDa Amicon Ultra-15 centrifugal filter (EMD Millipore), and dialyzed overnight with 10 kDa SnakeSkin™ dialysis tubing (Thermo Scientific) at 4°C in 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl solution. Size exclusion chromatography. The dialyzed protein was loaded onto a Superdex 200 column (HiLoad 26/600, GE) that was equilibrated with 2 column volumes of 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl solution. The loaded column was eluted with 3 column volumes of 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl solution. Protein-containing fractions, as identified by SDS-PAGE, were collected, spin concentrated using a 10 kDa Amicon Ultra-15 centrifugal filter (EMD Millipore), and dialyzed overnight with 10 kDa SnakeSkin™ dialysis tubing (Thermo Scientific) at 4°C in 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl solution. The final protein concentration was determined by Coomassie Protein Assay Kit (Thermo Scientific). Batches of GroEL and GroES proteins for testing were stored at 4°C for up to one month then discarded. Human HSP60 purification. Human HSP60 (mtHSP60) was expressed from a T7-promoted plasmid in Rosetta™ 2 (DE3) in E. coli cells. For human HSP60 purification, a pET21-HSP60 plasmid with an N-terminal octa-Histidine tag was transformed into Rosetta™ 2 (DE3) E. coli cells for over-expression. Cells were grown at 37°C in LB / ampicillin / chloramphenicol medium until an OD 600 of 0.5 was reached, then cultures were induced with 0.5 mM IPTG and continued to grow for 2-3 h at 25°C. Cells were centrifuged at 14,000 rpm, and the cell pellet was suspended in 50 mL of lysis buffer composed of 100 mM Tris-HCl, pH 7.7, 10 mM MgSO4, 1 mM β-ME, 5% glycerol, 0.1% triton X-100, 1500 Units DNAase, 50 ^g/ml lysozyme, and one tablet of EDTA-free complete protease inhibitor cocktail (Roche). Cells were homogenized and passed through a microfluidizer, washing with buffer containing 10 mM Tris-HCl, pH 7.7, 5% glycerol, and 0.1% triton X-100. 1 st Nickel column purification and His-tag cleavage: The cell lysate was centrifuged at 14,000 rpm, then the clarified lysate was supplemented with 10 mM imidazole, passed through a 0.2 μm filter, and loaded onto a nickel-agarose resin column that was equilibrated with 2 column volume of 20 mM Tris-HCl pH, 7.7, 5% glycerol, 200 mM NaCl, and 10 mM imidazole. The sample loaded column was washed with 6 column volumes of 50 mM imidazole, then bound HSP60 was eluted with 500 mM imidazole. Fractions that were enriched with the His-tagged mtHSP60 were collected, concentrated, dialyzed at room temperature for 2 h in 4 L of 20 mM Tris-HCl, pH 7.7, 200 mM NaCl and 5% glycerol. Proteolytic cleavage of the His-tag was next performed by addition of His-tagged TEV protease at a 1:10 (w:w) ratio, while dialyzing over night at 4°C against 4 L of 20 mM Tris-HCl, pH 7.7, 200 mM NaCl, and 5% glycerol buffer. 2 nd Nickel column purification: The protein sample was loaded onto a second nickel-agarose resin column that was equilibrated with 20 mM Tris-HCl, pH 7.7, 5% glycerol, 10 mM NaCl, and 10 mM imidazole. With this column, undigested His-tagged mtHSP60 can be separated from digested His-tag removed mtHSP60. The unbound fractions enriched with His-tag cleaved mtHSP60 were collected, and anion exchange chromatography was performed on the same day. Anion exchange purification of His-tag removed mtHSP60: The protein sample was next loaded onto an anion-exchange column that was equilibrated with 20 mM Tris-HCl, pH 7.7, and 5% glycerol. Bound proteins were eluted from the column with a linear gradient of 100-400 mM NaCl. Fractions enriched with mtHSP60 were collected, concentrated, and dialyzed in storage buffer (20 mM Tris-HCl, pH 7.7, 300 mM NaCl, 5% glycerol, and 10 mM MgCl2) using 10 kDa SnakeSkin™ dialysis tubing (Thermo Scientific). The concentration of protein was determined by Coomassie Protein Assay Kit (Thermo Scientific). Batches of HSP60 protein for testing were stored at 4°C for up to two weeks, then discarded. Human HSP10 purification: Human HSP10 (mtHSP10) was expressed from a T7-promoted (pET3a-HSP10) plasmid in Rosetta™ 2 (DE3) pLysS cells. Cells were grown at 37°C in LB / kanamycin / chloramphenicol medium until an OD 600 of 0.5 was reached, then were induced with 0.5 mM IPTG and continued to grow for 2-3 h at 37°C. The culture was centrifuged at 14,000 rpm, and the cell pellet was re-suspended in Buffer A (50 mM sodium acetate, pH 4.5, and 20 mM NaCl), supplemented with EDTA-free complete protease inhibitor cocktail (Roche®) and lysed by sonication. Clarified cell lysate was loaded on a cation exchange column (SP Sepharose fast flow resin, GE) and eluted with a linear NaCl gradient using Buffer B (50 mM sodium acetate, pH 4.5, and 1 M NaCl). Fractions containing HSP10 were concentrated, dialyzed with storage buffer (50 mM Tris- HCl, pH 7.4, and 300 mM NaCl) using 10 kDa SnakeSkin™ dialysis tubing (Thermo Scientific) and re-purified on a Superdex 200 column (HiLoad 26/600, GE) in storage buffer. The concentration of protein was determined by Coomassie Protein Assay Kit (Thermo Scientific). Protein was stored at 4°C in 50 mM Tris- HCl, pH 7.4, and 150 mM NaCl. Batches of HSP10 protein for testing were stored at 4°C for up to three weeks, then discarded. Example 76 Evaluation of compounds ability to inhibit GroEL/ES and HSP60/10- mediated dMDH refolding assays. Reagent preparation: For these assays, four primary reagent stocks were prepared: 1) GroEL/ES-dMDH or HSP60/10-dMDH binary complex stock; 2) ATP initiation stock; 3) EDTA quench stock; 4) MDH enzymatic assay stock. Denatured MDH (dMDH) was prepared by 2-fold dilution of MDH (5 mg/ml, soluble pig heart MDH from Roche, product #10127248001) with denaturant buffer (7 M guanidine-HCl, 200 mM Tris, pH 7.4, and 50 mM DTT). MDH was completely denatured by incubating at room temperature for 45 min. The binary complex solutions were prepared by slowly adding the dMDH stock to a stirring stock with GroEL (or HSP60) in folding buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 10 mM MgCl2, and 1 mM DTT), followed by addition of GroES (or HSP10). The binary complex stocks were prepared immediately prior to dispensing into the assay plates and had final protein concentrations of 83.3 nM GroEL (Mr 800 kDa) or HSP60 (Mr 400 kDa), 100 nM GroES or HSP10 (Mr 70 kDa), and 20 nM dMDH in folding buffer. For the ATP initiation stock, ATP solid was diluted into folding buffer to a final concentration of 2.5 mM. Quench solution contained 600 mM EDTA (pH 8.0). The MDH enzymatic assay stock consisted of 20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, and 1 mM DTT). Assay Protocol: First, 30 µL aliquots of the GroEL/ES-dMDH or HSP60/10-dMDH binary complex stocks were dispensed into clear, 384-well polystyrene plates. Next, 0.5 µL of the compound stocks (10 mM to 4.6 µM, 3- fold dilutions series in DMSO) were added by pin-transfer (V&P Scientific). The chaperonin-mediated refolding cycles were initiated by addition of 20 µL of ATP stock (reagent concentrations during refolding cycle: 50 nM GroEL or HSP60, 60 nM GroES or HSP10, 12 nM dMDH, 1 mM ATP, and compounds of 100 µM to 46 nM, 3-fold dilution series). The refolding reactions were incubated at 37°C. The incubation time was determined from refolding time-course control experiments until they reached ~90% completion of refolding cycle – generally ~20-40 min for GroEL/ES, and ~40-60 min for HSP60/10). Next, the assay was quenched by addition of 10 µL of the EDTA to final concentration of 100 mM. Enzymatic activity of the refolded MDH was initiated by addition of 20 µL MDH enzymatic assay stock (20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer, 50 mM Tris pH 7.4, 50 mM KCl, 1 mM DTT), and followed by measuring the NADH absorbance in each well at 340 nm using a Molecular Devices SpectraMax Plus384 microplate reader (NADH absorbs at 340 nm, while NAD + does not). A340 nm measurements were recorded at 0.5 minutes (start point) and at successive time points until the amount of NADH consumed reached ~90% (end point, generally between 20-35 minutes). The differences between the start and end point A 340 values were used to calculate the % inhibition of the GroEL/ES or HSP60/10 machinery by the compounds. IC 50 values for the test compounds were obtained by plotting the % inhibition results in GraphPad Prism 6 and analyzing by non- linear regression using the log (inhibitor) vs. response (variable slope) equation. Results presented represent the averages of IC 50 values obtained from at least quadriplicate (duplicate of duplicate) replicates. For results, see Tables 1, 2, and 3. Example 77 Counter-screening compounds for inhibition of native MDH enzymatic activity. Reagent Preparations & Assay Protocol: This assay was performed as described above for the GroEL/ES-dMDH refolding assay; however, the assay protocol differed in the sequence of compound addition to the assay plates. The refolding reactions were allowed to proceed for 45 min at 37°C in the absence of test compounds (complete refolding of MDH occurs), then quenched with the EDTA stock. Compounds were then pin-transferred into the plates after the EDTA quenching step; thus, compounds effects are only possible by inhibiting the fully- refolded MDH reporter substrate. Next enzymatic activity of the refolded MDH was initiated by addition of 20 µL MDH enzymatic assay stock (20 mM sodium mesoxalate and 2.4 mM NADH in reaction buffer, 50 mM Tris pH 7.4, 50 mM KCl, 1 mM DTT), and followed by measuring the NADH absorbance in each well at 340 nm using a Molecular Devices SpectraMax Plus384 microplate reader (NADH absorbs at 340 nm, while NAD + does not). A340 nm measurements were recorded at 0.5 minutes (start point) and at successive time points until the amount of NADH consumed reached ~90% (end point, generally between 20-35 minutes). Compounds were tested in 8-point, 3-fold dilution series (62.5 µM to 29 nM during the reporter reaction) in clear, flat-bottom 384-well microtiter plates. DMSO was used as a negative control, and previously discovered native MDH inhibitors were used as positive controls. IC50 values for the test compounds were obtained by plotting the % inhibition results in GraphPad Prism 6 and analyzing by non-linear regression using the log (inhibitor) vs. response (variable slope) equation. Results presented represent the averages of IC 50 values obtained from at least quadriplicate (duplicate of duplicate) replicates. Example 78 Evaluation of compounds for inhibition of bacterial cell proliferation. Growth Media: S. aureus bacteria were grown in Brain Heart Infusion (BHI) broth/agar (Becton, Dickinson and Company). All liquid cultures were grown in BHI media supplemented with 25 mg/L Ca 2+ and 12.5 mg/L Mg 2+ to mimic free physiological concentrations of these cations. A 10 mg/mL Ca 2+ stock solution was prepared by dissolving 3.68 g of CaCl2 ^2H 2 O in 100 mL of deionized water, and a 10 mg/mL Mg 2+ stock solution was prepared by dissolving 8.36 g of MgCl2 ^6H 2 O in 100 mL deionized water. Both stock solutions were filter- sterilized using 0.2 µm pore size cellulose-acetate filters. 2.5 mL of the sterile 10 mg/mL Ca 2+ stock and 1.25 mL of the sterile 10 mg/mL Mg 2+ stock solutions were added per 1 L of autoclaved BHI medium to obtain 25 mg/L Ca 2+ and 12.5 mg/L Mg 2+ ions, respectively. General Assay Protocol: Stock bacterial cultures were streaked onto BHI agar plates and grown overnight at 37°C. Fresh aliquots of broth were inoculated with single bacterial colonies and the cultures were grown overnight at 37°C with shaking (240 rpm) in cation adjusted BHI media. The following morning, the overnight cultures were sub-cultured (1:5 dilution) into fresh aliquots of cation adjusted BHI media and grown at 37°C for 1-2 hours with shaking. After 2 h, cultures were diluted into fresh media to achieve final OD600 readings of 0.017. Aliquots of these diluted cultures (30 µL) were added to clear, flat-bottom, 384- well polystyrene plates that were stamped with 0.5 µL of test compounds in 20 µL media. Compounds were evaluated in dose-response format where the inhibitor concentration range during the proliferation assay was 100 µM to 46 nM (3-fold dilution series). Plates were sealed with "Breathe Easy" oxygen permeable membranes (Diversified Biotech) and left to incubate at 37°C without shaking (stagnant assay). OD 600 nm readings were taken generally at 6-8 h, when bacteria had reached log-phase growth. A second set of baseline control plates were prepared analogously, but without any bacteria added, to correct for possible compound absorbance and/or precipitation. Plates were then read at 600 nm using a Molecular Devices SpectraMax Plus384 microplate reader. EC 50 values for the test compounds were obtained by plotting the OD600 results in GraphPad Prism and analyzing by non-linear regression using the log(inhibitor) vs. response (variable slope) equation. For results, see Tables 1, 2, and 3. Example 79 Evaluating compound effects on kidney cell viability. Evaluation of compound cytotoxicities to HEK 293 kidney cells was performed using an Alamar Blue-based viability assay. HEK 293 cells were maintained in MEM medium (Corning Cellgro, 10-009 CV) supplemented with 10% FBS (Sigma, F2242). All assays were carried out in 384-well plates (BRAND cell culture grade plates, 781980). Cells at 80% confluence were harvested and diluted in growth medium, then 45 µL of the HEK 293 cells (1,500 cells/well) were dispensed per well, and plates were sealed with "Breathe Easy" oxygen permeable membranes (Diversified Biotech) and incubated at 37°C, 5% CO 2 , for 24 h. The following day, 1 µL aliquots of the compound stocks (10 mM to 4.6 µM, 3-fold dilutions in DMSO) were pre-diluted by pin-transfer into 25 µL of growth medium. Then, 15 µL aliquots of the diluted compounds were added to the cell assay plates to give inhibitor concentration ranges of 100 µM to 46 nM during the assay (final DMSO concentration of 1% was maintained during the assay). Plates were sealed with "Breathe Easy" oxygen permeable membranes and incubated for an additional 48 h at 37°C and 5% CO 2 . The Alamar Blue reporter reagents were then added to a final concentration of 10%, the plates incubated at 37°C and 5% CO 2 , and sample fluorescence (535 nm excitation, 590 nm emission) was read using a Molecular Devices FlexStation II 384-well plate reader (readings taken between 4-24 h of incubation so as to achieve signals in the 30-60% range for conversion of resazurin to resorufin). Cell viability was calculated as per vendor instructions (Thermo Fisher - Alamar Blue cell viability assay manual). Cytotoxicity CC 50 values for the test compounds were obtained by plotting the % resazurin reduction results in GraphPad Prism and analyzing by non-linear regression using the log(inhibitor) vs. response (variable slope) equation. See Tables 1, 2, and 3. Example 80 Control compounds, calculation of IC50 / EC50 / CC50 values, and statistical considerations. For all assays, DMSO was used as negative control. For the GroEL/ES and HSP60/10-mediated dMDH refolding assays, and native MDH enzymatic activity counter-screens, a panel of our previously discovered and reported chaperonin inhibitors were used as positive controls: e.g. suramin and compound 2h-p from Abdeen et. al 2016; compounds 20R, 20L, and 28R from Abdeen et. al 2018; and closantel and rafoxanide from Kunkle et. al 2018. For the human cell viability assays, control compounds include the aforementioned compounds as well as other protein homeostasis inhibitors, such as bortezomib (proteasome inhibitor); VER- 155008 (HSP70 inhibitor); and ganetespib and 17-DMAG (HSP90 inhibitors). See Table 1. All IC 50 / EC 50 / CC 50 results reported are averages of values determined from individual dose-response curves in assay replicates as follows: 1) Individual I/E/CC 50 values from assay replicates were first log-transformed and the average log(I/E/CC 50 ) values and standard deviations (SD) calculated; 2) Replicate log(I/E/CC50) values were evaluated for outliers using the ROUT method in GraphPad Prism (Q of 10%); and 3) Average I/E/CC 50 values were then back- calculated from the average log(I/E/CC50) values. For compounds where log(I/E/CC 50 ) values were greater than the maximum compound concentrations tested (i.e. >1.8, >2.0, and >2.4 – or >63, >100, and >250 µM, respectively), results were represented as 0.1 log units higher than the maximum concentrations tested (i.e., 1.9, 2.1, and 2.5 – or 79, 126, and 316 µM, respectively) so as not to overly bias comparisons because of the unavailability of definitive values for these inactive compounds. Table 1: Control Samples. Compilation of IC 50 / EC 50 / CC 50 results for compound 1 and purchased antibiotics tested in the GroEL/ES and HSP60/10- mediated dMDH refolding assays and the native MDH counter-screen; the S. aureus bacterial proliferation assay; and the HEK 293 kidney cell viability / cytotoxicity assay.
Table 1
Table 2: Compounds related to formula I assay results. Compilation of IC 50 / EC 50 /50 results for compounds 45-83 tested in the GroEL/ES and HSP60/10-mediated dMDHolding assays and the native MDH counter-screen; the S. aureus bacterial proliferationay; and the HEK 293 kidney cell viability / cytotoxicity assay.
Table 3: Compounds related to formula II assay results. Compilation of IC 50 / EC 50 / results for compounds 57-117 tested in the GroEL/ES and HSP60/10-mediated dMDHing assays and the native MDH counter-screen; the S. aureus bacterial proliferation and the HEK 293 kidney cell viability / cytotoxicity assay.
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81
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