(12) United States Patent (10) Patent No.: US 9,079,870 B2 Chakrapani Et Al

US00907987OB2

(12) United States Patent (10) Patent No.: US 9,079,870 B2 Chakrapani et al. (45) Date of Patent: Jul. 14, 2015

(54) THIOL MEDIATED/ACTIVATED PRODRUGS (56) References Cited OF SULFUR DIOXIDE (SO) HAVING ANTI-BACTERALACTIVITY U.S. PATENT DOCUMENTS 6,380.429 B1* 4/2002 Smith ...... 564.87 (71) Applicant: INDIAN INSTITUTE OF SCIENCE 2009/O163697 A1* 6/2009 Crich et al...... 530/345 EDUCATION AND RESEARCH-PUNE, Pune (IN) FOREIGN PATENT DOCUMENTS WO WO96,06838 * 3/1996 (72) Inventors: Harinath Chakrapani, Pune (IN); Satish Ramesh Malwal, Shrirampur OTHER PUBLICATIONS (IN) (CAS143) Chemical Abstract Registry No. 889794-14-3, indexed in the Registry File on STN CAS Online Jun. 28, 2006.* (73) Assignee: Indian Institute of Science Education Malwaletal. Synthesis and antimycobacterial activity of prodrugs of and Research-Pune, Pune (IN) Sulfur dioxide (SO2). Bioorganic & Medicinal Chemistry Letter, 2012, 22, 3603-3606.* Crich et al. One-Pot Synthesis of Dissymmetric Diamides Based on (*) Notice: Subject to any disclaimer, the term of this the Chemistry of Cyclic Monothioanhydrides. Scope and Limitations patent is extended or adjusted under 35 and Application to the Synthesis of Glycodipeptides. Journal of U.S.C. 154(b) by 0 days. Organich Chemistry, 2009, 74,3886-3893 (including supplementary information).* (21) Appl. No.: 13/690,842 Crich et al. Amino Acid and Peptide Synthesis and Functionalization by the Reaction of Thioacids with 2,4-Dinitrobenzenesulfonamides. Organic Letters, 2007, 9,4423-4426 (including supplementary infor (22) Filed: Nov.30, 2012 mation).* Ito et al. A medium-term ratliver bioassay for rapid in vivo detection of carcinogenic potential of chemicals. Cancer Science, 2003, 94. (65) Prior Publication Data 3-8.* US 2014/O121211 A1 May 1, 2014 Malwal et al. Design, Synthesis, and Evaluation of Thiol-Activated Sources of Sulfur Dioxide (SO2) as Antimycobacterial Agents. Jour nal of Medicinal Chemistry, 2012, 55, 553-557. (including supple (30) Foreign Application Priority Data mental information) (Published Nov.30, 2011).* Gaylord Chemical Company, LLC. Dimethyl Sulfoxide (DMSO) Oct. 31, 2012 (IN) ...... 3162AMUMA2012 Health and Safety Information. Oct. 2007, p. 1-16.* Messeri et al. A Novel Deprotection/Functionalisation Sequence using 2,4-Dintriobenzenesulfonamide: Part I. Tetrahedron Letter, (51) Int. Cl. 1998, 39, 1669-1672.* C07D 295/26 (2006.01) Supporting information of "Malwal et al. Synthesis and CO7D 2II/96 (2006.01) antimycobacterial activity of prodrugs of sulfur dioxide (SO2). A6 IK3I/8 (2006.01) Bioorganic and Medicinal Chemistry Letter 2012, 22,3603-3606”.* A6 IK 45/06 (2006.01) PubMed printout of “Landrigan et al. A historical study of the use of A6 IK3I/222 (2006.01) agar as a delivery vehicle for alcohol or iron to rats. Alcohol. 1989, 6. A6 IK3I/40 (2006.01) 173-178.* A 6LX3/5377 (2006.01) (Continued) A6 IK3I/495 (2006.01) A6 IK3I/4453 (2006.01) CO7D 207/248 (2006.01) Primary Examiner — Michael Barker A 6LX3L/277 (2006.01) Assistant Examiner — Po-Chih Chen C07C3II/I 7 (2006.01) (74) Attorney, Agent, or Firm — Kramer Amado, P.C. C07C3II/8 (2006.01) C07C3II/2 (2006.01) (52) U.S. Cl. (57) ABSTRACT CPC ...... C07D 295/26 (2013.01); A61K3I/18 Disclosed herein are thiol mediated/activated prodrugs of (2013.01); A61 K3I/222 (2013.01); A61 K SO, particularly 2,4-dinitrophenylsulfonamide analogs, 3 1/277 (2013.01); A61K 31/40 (2013.01); having Formula-I or pharmaceutically acceptable salts A61 K3I/4453 (2013.01); A61 K3I/495 thereof exhibiting tunable release profiles of SO, with signifi (2013.01); A61K3I/5377 (2013.01); A61 K cant therapeutic efficacy against bacterial infections. Further, 45/06 (2013.01); C07C311/17 (2013.01); the present invention provides pharmaceutical compositions C07C3II/18 (2013.01); C07C3II/21 comprising compound of Formula I or pharmaceutically (2013.01); C07D 207/48 (2013.01); C07D acceptable salts thereof, along with pharmaceutically accept 2II/96 (2013.01) able carriers/excipients. (58) Field of Classification Search None See application file for complete search history. 15 Claims, 5 Drawing Sheets US 9,079,870 B2 Page 2

(56) References Cited Henderson, Science behind this anti-microbial, anti-oxidant, wine additive. Practical Winery & Vineyard Journal, Jan./Feb. 2009. OTHER PUBLICATIONS http://www.practicalwinery.com/janfeb09/page1.htm, viewed on Nov.30, 2012. Ito et al., A medium-term ratliver bioassay for rapid in vivo detection Fukuyama et al., 2,4-Dinitrobenzenesulfonamides: A Simple and of carcinogenic potential of chemicals. Cancer Science 2003, 94. Practical Method for the Preparation of a Variety of Secondary 3-8.* Chemical Abstract Registr No. 1219349-36-6, indexed in the Regis Amines and Diamines. Tetrahedron Letters, vol. 38, No. 33, pp. try File on STN CAS Online Apr. 15, 2010.* 5831-5834, 1997. CAPLUS printout of “Fukuyama et al., 2,4- Chen et al., Fluorescent and calorimetric probes for detection of dinitrobenzenesulfonamide: a simple and practical method for the thiols. Chem. Soc. Rev., 2010, 39, 2120-2135. preparation ofa variety of secondary amines and diamines, Tetrahe Newton et al., Distribution of Thiols in Microorganisms; Mycothiol dron Letters 1997, 38,5831-5834.”.* Is a Major Thiol in Most Actinomycetes, Journal of Bacteriology, Cardona et al., Extended safety studies of the attenuated live tuber Apr. 1996, p. 1990-1995. culosis vaccine SO2 based on phoP mutant. Vaccine 27 (2009)2499 2505. * cited by examiner U.S. Patent Jul. 14, 2015 Sheet 1 of 5 US 9,079,870 B2

Figure 1(a)

HeM S 9 N-R NO2 R MSH M O R S o ON S-N O2N R 2 6 R GD -R U.S. Patent Jul. 14, 2015 Sheet 2 of 5 US 9,079,870 B2

to. cal U.S. Patent Jul. 14, 2015 Sheet 3 of 5 US 9,079,870 B2

Figure 2

75 o 50 t C 25 0 0 O 20 40 60 80 MIC (M) (b) g 10075 t 50 tee 25 O O O 2O 40 60 80 MIC (M) U.S. Patent Jul. 14, 2015 Sheet 4 of 5 US 9,079,870 B2

Figure 3

O. : 0-0-0 0-0-0-0-0-0 Time (min) U.S. Patent Jul. 14, 2015 Sheet 5 of 5 US 9,079,870 B2

Figure 4

(a) S 100 O O S- 75 O O P E 50 ( ) c 25 (D O 2 4. 6 8 10 (b) pKa

US 9,079,870 B2 1. 2 THOLMEDIATED/ACTIVATED PRODRUGS global health. Although several new promising drug candi OF SULFUR DIOXIDE (SO) HAVING dates are under consideration for clinical use, the increasing ANTI-BACTERAL ACTIVITY incidences of multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis CROSS-REFERENCE TO RELATED (Mtb) necessitates new strategies for targeting this pathogen. APPLICATIONS SO is an environmental pollutant that is toxic to humans at This application claims the benefit of Indian Application elevated levels; chronic exposure to SO, induces oxidative No. 3162/MUM/2012, filed on Oct. 31, 2012, the entire dis stress and asthma-like symptoms. SO2 has diverse docu closures of which is incorporated herein by reference. 10 mented biological effects including damage to biomacromol ecules such as proteins, lipids and DNA. Oxidation of sulfur TECHNICAL FIELD dioxide by metalions produces sulfur trioxide radical, which possibly mediates damage to biomacromolecules. Further The present invention relates to thiol mediated/activated more, sulfite, the anionic form of sulfur dioxide can break prodrugs of SO, particularly 2,4-dinitrophenylsulfonamide 15 disulfide linkages to produce S-Sulfonates. Thus, SO can analogues, having Formula-I or pharmaceutically acceptable participate in oxidative as well as reductive processes under salts thereof exhibiting tunable release profiles of SO with physiological conditions and may perturb redox equilibrium significant therapeutic efficacy against bacterial infections. in cells. Recently, alteration of redox homeostasis in Mtb has Further, the present invention provides pharmaceutical com been proposed as an effective mechanism for targeting this positions comprising compound of Formula I or pharmaceu bacterium. tically acceptable salts thereof, along with pharmaceutically acceptable carriers/excipients. Further Pat Henderson et al. in Practical winery and vine yard Journal 02/2009 discloses broad-spectrum antimicrobial agent, Sulfur dioxide, having an inhibitory effect on a wide Formula-I 25 variety of microorganisms. O R1 Thus, sulfur dioxide may have diverse mechanisms for OES-N-R2 cytotoxicity induction and multiple biological targets. Despite such well-documented deleterious effects, SO, (in NO the form of bisulfite, metabisulfite and sulfite) has also been 30 routinely used as an antibiotic and antioxidant in the food industry, in wineries and is well tolerated in most individuals. Barring certain individual cases of allergies, sulfur dioxide is well-tolerated in humans; in certain meats that are consumed NO on a daily basis, SO, levels can reach up to 450 mg kg'. 35 Thus, it was envisaged that the susceptibility of bacteria to the deleterious effects of SO2 could be exploited to develop new BACKGROUND AND PRIOR ART SO2-based tuberculosis drug candidates. To tap its therapeu tic potential and possibly to avoid undesirable side effects, The bacterial infection is caused due to the Pathogenic controlled delivery of SO2 is necessary. bacteria Such as genus Streptococcus, Staphylococcus, Myco 40 The poor bioavailability of gaseous sulfur dioxide pre bacterium, Helicobacter, Pseudomonas, Enterococcus, cludes its usage for therapeutic purposes and the use of com Escherichia etc. plex inorganic Sulfite mixtures, typically used to generate Among the pathogens S. aureus is the most common spe SO in biological systems, suffers from a lack of control of cies of Staphylococcus to cause Staph infections. The said rate and amount of SO generated. As there were no reliable pathogen is also referred as “golden staph' or “Oro staphira', 45 SO sources available, the present inventors proposed to which is Gram-positive coccal bacterium. Staphylococcus develop organic donors of sulfur dioxide with tunable release aureus is a part of the skin flora found in the nose and on skin. profiles in order to evaluate their efficacy as against bacterial “S. aureus' can cause a range of illnesses from minor skin infections. infections. Such as pimples, impetigo, boils (furuncles), cel lulitis folliculitis, carbuncles, Scalded skin syndrome and 50 It has been reported in Tetrahedron Letters 38, (33), 1997, abscesses, to life-threatening diseases such as pneumonia, 5831-5834 by Fukuyama et al. that amines can be protected meningitis, osteomyelitis, endocarditis, toxic shock syn by the reaction with 2,4-dinitrophenylsulfonyl chloride (DN drome (TSS), bacteremia and septicemia. SCI) as 2,4-dinitrophenylsulfonamides, wherein deprotection Enterococcus faecalis is a nonmotile, gram-positive, of Such amides was carried out by thiols such as 2-mercap spherical commensal bacterium, inhabiting the gastrointesti 55 toacetic acid in basic medium to produce amines in good nal tracts of humans and other mammals. E. faecalis is listed yields (cf below scheme), wherein a byproduct of these as the first to the third leading cause of nosocomial infections deprotection reactions was Sulfur dioxide. which may mostly occur after Surgery of the abdomen or a puncturing trauma, also due to increased use of IV's and SOCI catheters. Further it cause urinary tract infections, bacterimia, 60 endocarditis, meningitis, and wound infections along with NO many other bacteria. Py RNH -- -es One of the bacterial diseases with highest disease burden is or lutidine tuberculosis, caused by the bacterium Mycobacterium tuber 1 culosis. 65 Tuberculosis causes millions of fatalities each year and in NO combination with HIV is proving to be a significant threat to US 9,079,870 B2 3 -continued SONHR Formula-I O R1 NO ROH, PhP DEAD OES-N-R2 Her O NO

DMF NO

10 O NO wherein, R1 is independently selected from H. (C1-C8) alkyl, HSCHCO2H 15 aryl, alkylaryl, allyl (C2-C8) alkynyl; or optionally selected EtN R n from the group consisting of Hs N NO O 3 n-PrNH) 4 ON

Though, the release of SO from the deprotection of 2,4- dinitrophenylsulfonamides is known from the above article, the thiol activated 2,4-dinitrophenylsulfonamides analogs as source of SO with tunable release profile which significantly inhibits the growth of bacterial infection is not yet reported. 25 2,4-Dinitrophenylsulfonamides have also been used in thiol detection systems for environmental and biological applications. Xiaoqiang Chen et al. in Chem. Soc. Rev., 2010, (39), 30 2120-2135 focuses on the fluorescent or colorimetric sensors for thiols according to their unique mechanisms between sensors and thiols, including cleavage of Sulfonamide and sulfonate ester by thiols, cleavage of disulfide by thiols, wherein the thiols are selected from cysteine, homocysteine 35 and glutathione. Furthermore, variation of the groups on the amine may provide a handle for modulating SO2 release profiles. Unlike mammalian cells, Mtb does not contain glutathione (GSH) but mycothiol (MSH) as the primary thiol in millimolar con 40 centrations. Gerald L. Newton etal. in Journal of bacteriology, Vol. 178, R2 represents group consisting of allyl or, no. 7 Apr. 1996, 1990-1995 discloses that MSH production is confined to actinomycetes and shows that mycobacteria, 45 including Mycobacterium tuberculosis, produce high levels 21 pi COO(R4) of thiol (MSH). R3-H O 1– MSH is critical for the maintenance of redox homeostasis N R4' and alteration of thiol-levels could induce stress to Mtb. Upon reaction with a 2,4-dinitrophenylsulfonamide, MSH is 50 expected to be arylated to generate sulfur dioxide intracellu wherein, (n) is either 0 or 1: larly. Thus this two-pronged strategy of introduction of SO R3 is independently selected from H. halogen, —CF3, (C1 and a thiol-depleting agent could inhibit Mtb growth. C6) alkoxy, —CN, wherein R4 and R4 are identical or inde Therefore the present inventors have developed organic pendently selected from the group consisting of H. Substi donors/prodrugs of SO2, i.e. 2,4-dinitrobenzenesulfona 55 tuted or unsubstituted (C1-C6) alkyl; and mides analogues with tunable release profiles of SO in order R1 and R2 together form substituted or unsubstituted 5 or 6 to evaluate their therapeutic efficacy against bacterial infec membered heterocyclic ring, optionally containing Substi tions which remains objective of the present invention. tuted or unsubstituted heteroatom such as O or N, wherein 60 Substituents are selected from the group consisting of H, SUMMARY OF THE INVENTION (C1-C6) alkyl, -COO(R5); where R5 is selected from H, (C1-C6) alkyl. In accordance with the objective, the present invention In an aspect, the present invention relates to a pharmaceu provides thiol mediated/activated reliable prodrugs of SO tical composition comprising an effective amount of active having general Formula-I, which exhibits tunable release pro 65 ingredient compound of Formula I or as its salts along with files of SO2 with significant therapeutic efficacy against bac pharmaceutically acceptable excipients for treating bacterial terial infections. infections. US 9,079,870 B2 5 In yet another aspect, the invention provides method of treating bacterial infections in a mammal comprising admin Formula-I istering an effective amount of compound of formula I or its O R1 pharmaceutical salt in association with one or more pharma OES-N-R2 ceutical carriers. NO The bacterial infections according to the invention may be caused due to gram negative, gram positive and Mycobacte rium tuberculosis. In yet another aspect, the invention provides use of com 10 pounds of formula I or its pharmaceutical salt optionally in O association with one or more pharmaceutical carriers for the treatment of bacterial infections in a mammal. wherein, R1 is independently selected from H. (C1-C8) alkyl, aryl, arylalkyl, allyl (C2-C8) alkynyl; or optionally selected 15 from the group consisting of DESCRIPTION OF FIGURES

FIG. 1(a) depicts plausible mechanism for thiol mediated ON decomposition of to generate SO, from compounds such as (1): FIG. 1(b) depicts plausible mechanism for thiol mediated decomposition of to generate SO2 from compound (7).

FIG. 2 depicts Relationship between sulfur dioxide yield 25 generated during cysteine-mediated decomposition of selected 2,4-dinitrophenylsulfonamides prepared in this study and their Mtb inhibitory activity. (a)SO, yield was after 30 min; Spearman rank correlation analysis of SO yield and MIC gave a Spearman correlation coefficient of -0.50 30 (P-value=0.01). (b) SO yield was after 5 min; Spearman rank correlation analysis of SO, yield and MIC gave p=-0.69 (P-value=0.001). FIG.3 depicts kinetics of decomposition of compound (7) 35 to produce sulfur dioxide and 2-hydroxyethylthio-2,4-dini trobenzene (7y), where disappearance of compound (7) and appearance of (7y) was followed by HPLC analysis. Produc tion of sulfite was monitored by ion chromatography analysis. Curve fitting afforded first order rate constant for: appearance 40 of (7y) as 0.011 min', and appearance of sulfite as 0.014 R2 represents group consisting of allyl, or min' FIG. 4 depicts relationship between sulfur dioxide gener ated during cysteine-mediated decomposition of selected 2,4- 45 21 pi COO(R4) dinitrophenylsulfonamides prepared in this study and pK. R3-H O 1– of the amine from which the corresponding Sulfonamide was N R4' prepared. (a) SO, yield was after 5 min: Pearson correlation analysis of SO yield and pK gave a correlation coefficient 50 wherein, (n) is either 0 or 1: p=-0.79 (P-value-0.001) (b) SO, yield was after 30 min: R3 is independently selected from H. halogen, —CF (C1 Pearson correlation analysis of SO yield and pK gave a C6) alkoxy, —CN, correlation coefficient r=-0.71 (P-value-0.0001). wherein R4 and R4' are identical or independently selected from the group consisting of H. Substituted or unsubstituted DETAILED DESCRIPTION OF INVENTION 55 (C1-C6) alkyl; and R1 and R2 together form substituted or unsubstituted 5 or 6 membered heterocyclic ring, optionally containing Substi The invention will now be described in detail in connection tuted or unsubstituted heteroatom such as O or N, wherein with certain preferred and optional embodiments, so that Substituents are selected from the group consisting of H, various aspects thereof may be more fully understood and 60 (C1-C6) alkyl, -COO(R5); where R5 is selected from H, appreciated. (C1-C6) alkyl. The term “alkyl includes all straight chain and branched The present invention provides thiol mediated/activated alkyl selected from the group methyl, ethyl, n-propyl, iso reliable prodrugs of SO having general Formula-I or phar propyl. n-butyl, sec-butyl, iso-butyl, t-butyl, n-penty1 and maceutically acceptable salts thereof, which exhibits tunable 65 n-hexyl and like thereof. release profile of SO2 with significant therapeutic efficacy The term “alkoxy” includes the group selected from meth against bacterial infections. oxy, ethoxy, propoxy, butoxy and like thereof. US 9,079,870 B2 7 8 The terms halogen and halo include fluorine, chlorine, lated derivatives of compounds of Formula I, can be prepared bromine and iodine, and fluoro, chloro, bromo and iodo, by treatment of corresponding alkyl halide with suitable 2,4- respectively. dinitrosulfonamide under suitable condition. The compounds of the present invention may contain one The thiol used according to the instant invention is particu or more asymmetric carbon atoms and may exist in racemic larly cysteine, therefore in preferred embodiment the present and optically active form. invention provides cysteine mediated/activated reliable pro 2,4-dinitrophenylsulfonyl group of Formula I of present drugs of SO having general Formula-I, or pharmaceutically invention, also represented as “DNs' acceptable salts thereof, which exhibit tunable release profile In an embodiment, the present invention relates to synthe of SO with significant therapeutic efficacy against bacterial sis of 2,4-dinitrosulfonamide analogues (Formula-I) which 10 infections. can be prepared by the process known in the art, wherein the A large number of prodrugs of SO having Formula I, process comprises reaction of corresponding amine with 2,4- which are synthesized by the process mentioned hereinabove, dinitrophenylsulfonyl chloride (DNSCl); whereas the alky are disclosed in below Table 1. TABLE 1

Sr. No. Compound R1 R2 R3 R4 1. 1 benzy 2. 2 benzy 4-methoxy 3. 3 benzy 4-chloro 4. 4 benzy 4-CF 5. 5 benzy 2- CF 6. 6 pheny 7. 7 pheny 4-methoxy 8. 8 pheny 2-methoxy 9. 9 pheny 3-fluoro O. 10 pheny 4-fluoro 1. 11 pheny 4-CN 2. 12 (S) N (1-phenylethyl)- 3 13 (R) N (1-phenylethyl)- 4. 14 2-phenylethyl 5. 15 Methyl benzy 16 n-propyl 17 Phenyl 6. 18 Methyl pheny 7. 19 Methyl 2-phenylethyl 2O benzyl

8 21 H COO(R4) R4 and R4 = methyl

19. 22 COR5

R1 N-DNs DNS-N-R2 Formula -I R5 = methyl 20. 23 r N1 DNS X - X = O

24 DNS r N Y X - X = CH 25 r N1 DNS X - X = N-Methyl US 9,079,870 B2

TABLE 1-continued

Sr. No. Compound R1 R2 R3

21. 32 R1 = allyl R2 = allyl 22. 33 R1 = propynyl or R2 = benzyl propargyl

23. 34 O N benzyl

O 2AWe ON S O

24. 35 O N phenyl 2-methoxy

O2 2\S2Y. C N

25. 36 ON phenyl 3-methoxy

& sa' 2\ C N

26. 37 ON phenyl 4-methoxy

29 & S CO Y. US 9,079,870 B2 11 12 TABLE 1-continued

Sr. No. Compound R4

27. 38 ON2 phenyl

S 29 o212 V N

28. 39 ON phenyl 2-Fluoro

NO 4° O2 V N

29. 40 benzyl NO

2 O S 2 O2V C N

30. 41 NO2 benzyl

O sa21 oav C N US 9,079,870 B2 13 14 TABLE 1-continued

Sr. No. Compound R1 R2 31. 42 ON benzyl

In accordance with Table 1, the thiol activated prodrugs of XXX. N-benzyl-N-(3-(N-(3-methoxyphenyl)-2,4-dinitrophe SO having Formula I of instant invention, encompasses the nylsulfonamido)propyl)-2,4-dinitrobenzenesulfonamide following compounds; (36): i. N-benzyl-2,4-dinitrophenylsulfonamide (1): XXXi. N-benzyl-N-(3-(N-(4-methoxyphenyl)-2,4-dinitrophe ii. N-(4-methoxybenzyl)2,4-dinitrophenylsulfonamide (2): nylsulfonamido)propyl)-2,4-dinitrobenzenesulfonamide iii. N-(4-chlorobenzyl)2,4-dinitrophenylsulfonamide (3): 25 (37): iv. N-(4-trifluoromethyl benzyl) 2,4-dinitrophenylsulfona XXXii. N-benzyl-N-(3-(2,4-dinitro-(N-phenyl) phenylsul mide (4): fonamido)propyl)-2,4 dinitrobenzene sulfonamide (38); V. N-(2-trifluoromethyl benzyl) 2,4-dinitrophenylsulfona XXXiii. N-benzyl-N-(3-(N-(2-fluorophenyl)-2,4-dinitrophe mide (5): nylsulfonamido)propyl)-2,4-dinitro vi. N-phenyl, 2,4-dinitrophenylsulfonamide (6): 30 XXXiv. benzenesulfonamide (39); vii. N-(4-methoxyphenyl) 2,4-dinitrophenylsulfonamide (7): XXXV. N-benzyl-N-(3-(N-benzyl-2-nitrophenylsulfonamido) viii. N-(2-methoxyphenyl) 2,4-dinitrophenylsulfonamide propyl)-2,4-dinitrobenzenesulfon-amide (40); (8): XXXVi. N-benzyl-N-(3-(N-benzyl-3-nitrophenylsulfona ix. N-(3-fluorophenyl) 2,4-dinitrophenylsulfonamide (9); mido)propyl)-2,4-dinitrobenzenesulfon-amide (41); x. N-(4-fluorophenyl) 2,4-dinitrophenylsulfonamide (10); as XXXvii. N-benzyl-N-(3-(N-benzyl-4-nitrophenylsulfona Xi. N-(4-cyanophenyl) 2,4-dinitrophenylsulfonamide (11); mido)propyl)-2,4-dinitrobenzenesulfon-amide (42); xii. (S) N-(1-phenylethyl)-2,4-dinitrobenzenesulfonamide In an another embodiment, the compounds of the present (12); invention are characterized by spectral analysis such as NMR, xiii. (R) N-(1-phenylethyl)-2,4-dinitrobenzenesulfonamide CNMR, IR, Mass and UV, elemental analysis and melting (13): point etc. xiv. N-(2-phenylethyl) 2,4-dinitrophenylsulfonamide (14); 40 Also the 2,4-dinitrophenylsulfonamide analogues of For XV. (N-benzyl, N-methyl) 2,4-dinitrophenylsulfonamide mula I, are well characterized by the known chromatographic (15): techniques like HPLC, GC, TLC etc. xvi. (N-benzyl, N-propyl) 2,4-dinitrophenylsulfonamide According to another preferred embodiment, the com (16): pounds of formula I are tested for its antibacterial activity by xvii. (N-benzyl, N-phenyl) 2,4-dinitrophenylsulfonamide 45 reacting with thiols. (17); The antibacterial activity of compounds of Formula I xviii. (N-methyl, N-phenyl) 2,4-dinitrophenylsulfonamide against the growth of gram positive bacteria Such as S. aureus (18); and E. feacalis is represented in (Table 2a). xix. N-methyl, N-(2-phenylethyl) 2,4-dinitrophenylsulfona One such study of the invention includes Mtb inhibitory mide (19); 50 activity. In the instant study, the generation of SO, particu XX. N-benzyl, N-(2-phenylethyl) 2,4-dinitrophenylsulfona larly the yield of SO2 after commencement of the reaction mide (20): with cysteine with compounds of 2,4-dinitrophenylsulfona XXi. Methyl 2-(2,4-dinitrophenyl)sulfonyl mide analogues (Formula I) and their antimycobacterial aminopropanoate (21); activity, (Mtb inhibitory activity) is given below in Table 2b. xxii. Methyl 1(2,4-dinitrophenyl)sulfonylpyrrolidine-2- The SO, analysis during thiol mediated decomposition and carboxylate (22); 55 antimycobacterial activity of 2,4 dinitrosulfonamides and xxiii. 4-(2,4-dinitrophenyl)sulfonylmorpholine (23); related analogues is demonstrated below in Table 3. xxiv. 1-(2,4-dinitrophenyl)sulfonylpiperidine (24); In yet another embodiment the invention provides com XXV. 1-(2,4-dinitrophenyl)sulfonyl-4-methylpiperazine pounds of Formula I, which comprises more than one SO (25); moiety to generate high percentage release of SO/mol after xxvi. N,N-diallyl-2,4-dinitrobenzenesulfonamide (32); 60 commencement of the reaction with thiol and thereby XXvii. N-benzyl-2,4-dinitro-N-prop-2-yn-1-ylbenzene enhance the antibacterial activity of the said compounds. The sulfonamide (33): release profile of compounds having more than one SO xxviii. N,N'-(propane-1,3-diyl)bis(N-benzyl-2,4-dinitroben moiety is given below in Table 5. Zenesulfonamide) (34); Further the present invention discloses that among the 2.4 xxix. N-benzyl-N-(3-(N-(2-methoxyphenyl)-2,4-dinitrophe 65 dinitrobenzylsulfonamide analogue compounds of Formula nylsulfonamido)propyl)-2,4-dinitrobenzenesulfonamide I, the compound (1) i.e. N-benzyl-2-4-dinitrophenylsulfona (35); mide produces nearly 100% yield of SO in 30 min after US 9,079,870 B2 15 16 commencement of the reaction of compound (1) with cys fied cellulose such as microcrystalline cellulose, hydroxypro teine, which strongly inhibits Mtb growth (MIC about 0.05 pyl cellulose (HPC): xylitol, sorbitol or maltitol; gelatin: ug/ml) (cf Table 2b). polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), lubricants such as magnesium Stearate or talc and/or agents In another embodiment, cysteine-mediated decomposition for achieving a depot effect such as carboxypolymethylene, of compounds of the Formula I or its salts generated elevated 5 carboxymethyl-cellulose, cellulose acetate phthalate or poly levels of SO in 30 minutes (75%) with MICs in the range of vinyl acetate, coatings layer Such as polyvinylpyrrolidone or 0.05 to >100 g/ml. shellac, gum arabic, talc, titanium oxide or Sugar. In another embodiment, the present invention provides a Solutions or Suspensions containing the compounds of the pharmaceutical composition comprising of the active ingre general formula I according to the invention can additionally dient of Formula I or its pharmaceutically acceptable salts, 10 comprise taste-enhancing agents such as Saccharin, cycla along with pharmaceutically acceptable excipients or carri mate or Sugar and other flavourings. They can moreover com ers, for the treatment of antibacterial infections in a mammal. prise Suspending excipients or preservatives. Generally, the quantity of active compound will range In yet another embodiment, the invention provides method between 0.5% to 90% by weight of the composition. Nor of treating or inhibiting the growth of bacterial infections in a mally, the effective amount of dosage of antibacterial active 15 Subject comprising administering an effective amount of component will be in the range of about 0.1 to about 100 compound of formula I or its pharmaceutical salt in associa mg/kg, more preferably about 3.0 mg to about 50 mg/kg of tion with one or more pharmaceutical carriers. body weight/day. The bacterial infections according to the invention may be The quantity of the compound of formula I used in phar caused due to gram negative, gram positive such as S. aureus, maceutical compositions of the present invention will vary E. feacalis and Mycobacterium tuberculosis. depending upon the body weight of the patient and the mode In a specific embodiment, the invention provides methods of administration and can be of any effective amount to of inhibiting the growth of Mycobacterium tuberculosis achieve the desired therapeutic effect. The compound of the HR strain which comprises administering an effective present invention can also be administered optionally with amount of compound of Formula I or its pharmaceutical salt other antibacterial actives depending on the disease condi in association with one or more pharmaceutical carriers. tions. The additional antibacterial active compound may be 25 In yet another embodiment, the invention provides use of selected from the group consisting of Fluoroquinolones, compounds of formula I or its pharmaceutical salt optionally Cephalosporins, Penicillins, Carbepenems, Aminoglyco in association with one or more pharmaceutical carriers for sides, Tetracyclines etc. the treatment of bacterial infections in a subject. Further the synergistic antimycobacterial effect of instant Accordingly, the invention provides the use of the com pharmaceutical composition can be achieved in combination 30 pounds of Formula I to inhibit the growth of bacterial infec with additional known anti-tuberculosis drugs selected from tions caused due to pathogens selected from the group con the group consisting of rifampin, isoniazide, ethambutol, sisting of Staphylococcus, Enterococcus and Mycobacterium pyrazinamide etc. The pharmaceutical composition accord particularly Mycobacterium tuberculosis (Mtb) HR strain ing to the invention can be in the form of a solid such as, pills, (Table 2a, 2b and 3). powders, granules, tablets, capsules, pellets, beads etc. or can 35 The subject as described in above embodiment is a mam be present in the liquid form Such as Solutions, emulsions, mal. Suspensions, syrup etc. or can be used in the form of inhalants The following examples, which include preferred embodi or Parenteral injection. ments, will serve to illustrate the practice of this invention, it According to the invention, the pharmaceutical composi being understood that the particulars shown are by way of tions containing compounds of Formula-I may be adminis examples and for purpose of illustrative discussion of pre tered using any effective amount, any form of pharmaceutical 40 ferred embodiments of the invention only and are not limiting composition and any route of administration effective for the the scope of the invention. treatment of tuberculosis. After formulation with an appro priate pharmaceutically acceptable carrier in a desired dos Experimental age, as known by those of skill in the art, the pharmaceutical compositions of present invention can be administered by any 45 The preliminary results of instant invention indicated that means that delivers the active pharmaceutical ingredient(s) to the rate of SO generation affected inhibitory activity of 2,4- the site of the body whereby it can exerta therapeutic effect on dinitrophenylsulfonamide related compounds. the patient. The antibacterial activity of thiol mediated/activated pro The excipients or carriers are selected from inert diluents drugs of SO2 i.e. 2,4-dinitrophenylsulfonamide related com Such as dextrose, Sugar, Sorbitol, mannitol, polyvinylpyrroli 50 pounds against the gram positive pathogenic bacteria selected done, disintegrants such as Starch, Crosslinked polymers, from the group S. aureus, E. feacalis is given herein below binders such as Sucrose, lactose; starches, cellulose or modi Table 2a. TABLE 2a

MIC (ig/mL) MIC (ig/mL) S. airetts E. feacais Sr. No Compound Structure ATCC 29213 ATCC 29212

NO >16 >16

1 S N O|| HCO NO US 9,079,870 B2 17 18 TABLE 2a-continued MIC (ig/mL) MIC (ig/mL) S. airetts E. feacais Sr. No Compound Structure ATCC 292.13 ATCC 29212

33 o Pi—/N-S I NO ON

11 NO

Nt ng

O NO NC

10 NO

N ng

O NO F 7 NO NH O O NO MeO

8 NO OMe Nt ng

10. 24 O US 9,079,870 B2 19 20 In accordance with Table 2a the Minimum inhibitory con ment of the reaction with cysteine (10 equiv). SO was deter centration (MIC) of prodrugs of mined as Sulfite in pH 7.4 using an ion chromatograph SO having Formula I to inhibit growth of bacterial infec equipped with a conductivity-based detector. Under Such tions caused due to pathogens like S. aureus and E. feacalis reaction conditions, the benzylamine derivative (1) produced were found >16 g/mL. 5 100% SO, in 30 min (Table 2b, entry 1). Presence of an The inventors demonstrated that small modifications to the electron withdrawing or electron donating group did not sig structure of the amine resulted in a significant change in the nificantly affect the yield of SO, in comparison with (1) half-lives of SO2 generation (t1/2=2 to 63 min) Thus, varying (Table 2b, entries 2-5). The sulfanilide (6) produced 55 uM the amine structure could potentially help modulate anti (55% yield) SO, under comparable reaction conditions mycobacterial activity. In order to study the effects of varying 10 (Table 2b, entry 6). Amongst the aniline derivatives, inventors sterics and/or electronics on chemical and biological proper found a strong electronic effect on the yield of sulfur dioxide ties, analogues with comparable clogPs to compound (1) during decomposition of aniline derivatives, wherein the were synthesized by process known in the art. presence of an electron donating group enhanced the yield of The calculated partition coefficients (clogP) of thiol-medi SO, in comparison with aniline (Table 2b, entries 7 and 8). ated prodrugs of sulfur dioxide (Formula I), yield of SO and 15 Conversely, when an electron withdrawing group is present anti-mycobacterial activities of specific compounds of For on the aromatic ring of aniline, the yield of SO decreased in mula I is represented in Table 2b. comparison with aniline (Table 2b, entries 9-11). The effect TABLE 2b SO yield, SO2 yield, Mol. Yield C 30 min 5 min Entry Compound W. (%) logP' (M) (IM) pKaBI MIC (ig/mL) MIC (IM) 1 1 337.OS 41 2.87 100 83 9.51 O.OS O.15 2 2 367.33 33 2.78 84 74 9.SO O.25 O.68 3 3 371.75 54 3.58 96 77 9.44 0.4 1.07 4 4 405.31 43 3.75 75 79 9.45 0.4 O.98 5 5 40531 S4 3.75 84 81 9.23 1.56 3.84 6 6 323.28 30 2.76 55 37 4.64 3.13 9.69 7 7 353.30 66 2.69 8O 39 S.11 1.56 4.4 8 8 353.30 70 2.69 86 57 4.42 3.13 8.85 9 9 341.27 70 2.93 24 5 3.22 25f 73 10 10 341.27 48 2.92 55 18 3.8O 6.25 18.31 11 11 34829 47 2.24 5 6 1.63 >50 >100 12 12 351.33 47 3.17 97 8O 9.73 1.56 4.44 13 13 351.33 38 3.17 94 78 9.73 1.56 4.44 14 14 351.33 79 3.19 100 76 9.79 0.4 1.13 15 15 351.33 86 2.41 100 79 9.70 0.4 1.10 16 16 379.38 73 3.47 96 68 9.90 3.13 8.25 17 17 413.40 47 3.87 94 44 3.92 1.56 4.44 18 18 337.31 81 2.10 94 89 5.64 O.78 2.31 19 19 365.56 95 2.74 84 79 10.13 O.78 2.13 2O 2O 441.46 80 4...SO 75 37 9.88 3.13 7.09 21 Isoniazide 137.13 -0.66 O.OS 0.37 22 Ethambutol 204.31 — 2.08 1.56 7.63 23 Pyrizinamide 123.11 -0.67 6.25 SO.8 Yield is for reaction of the amine with 2,4-dinitrophenylsulfonyl chloride or alkylation of 2,4-dinitrophenylsulfonamide. Calculated using Chembiodraw Ultra. Sulfur dioxide as sulfite was quantified using an ion chromatograph equipped with a conductivity detector: yields are 5 min and 30 min after treatment of compound (1001M) with 10 equiv of cysteine in pH 7.4 phosphate buffer, values are for the corresponding amine and were calculated using Marvinsketch 5.7.1. Minimum inhibitory concentration (MIC) is the minimum concentration of the compound required to inhibit 99% ofbacterial growth and was found against Mycobacterium tuberculosis H37Rv strain. Accordingly the inventors were prepared the benzylamine was particularly strong in the presence of a 4-cyano group analogues 2-5 (Table 2b, entries 2-5) with electron donating 50 (11), whose sulfur dioxide yield (30 min) was 5% (Table 2b, and electron withdrawing groups using a previously reported entry 11). In the presence of an additional alkyl, aryl, or method of reaction of the corresponding amine with 2,4- methylene group, either comparable or slightly diminished dinitrophenylsulfonyl chloride (DNSCI). yield of SO2 in comparison with compound (1) was observed Similarly, aniline derivatives 6-11 were prepared using the (Table 2b, entries 12-20). aforementioned procedure with substituents capable of per 55 turbing the electronics of the amine (Table 2b, entries 6-11). The ability of compounds of Formula I to inhibit Myco In order to study the effect of changing sterics around the bacterium tuberculosis (HR) growth was evaluated using a nitrogen bearing the DNS group, compound 12 and 13 with an reported protocol. The inventors found minimum inhibitory alpha-methyl substituent were prepared (Table 2b, entries concentrations (MICs) ranged from 0.05 to >100 mg/mL 12-13). The 1-phenyl-2-aminoethyl derivative 14 was pre 60 (Table 2b). While compound (1) was still most potent Mtb pared by treatment of the corresponding amine with DNsCl inhibitor in series of compounds of Formula I (Table 2b), (Table 2b, entry 14), whereas the alkylated derivatives 15-20 several derivatives (2-4), (14), (15), (18) and (19) had potent were prepared by treating the corresponding alkylhalide with Mtb inhibitory activities with MICs.<1 mg/mL (Table 2b, a suitable 2,4-dinitrosulfonamide (Table 2b, entries 15-20). entries 2-4, 14,15, 18 and 19); Further the present inventors were evaluated cysteine-me 65 The N-benzyl-2,4-dinitrophenylsulfonamide (1), (Table diated SO2 yields from the library of said compounds (Table 2b) with a MIC of 0.05 g/mL (0.15uM), is better than those 2b) by recording the yield of SO 30 min after commence of clinically used tuberculosis drugs such as isoniazid, etham US 9,079,870 B2 21 butoland pyrazinamide, which were evaluated under similar assay conditions (Table 2b, entries 21, 22 and 23). equV-1 In accordance with FIG. 2, the data analysis revealed that pH 7.4 pH 7.4 2 the most potent Mtb inhibitors generated elevated levels of SO HSO SO SO in 30 min (75% yield). Spearman rank correlation 5 analysis of MICs with sulfur dioxide yields (FIG. 2a) The present inventors were used an ion chromatograph revealed a moderate negative correlation value of Spearman (IC) equipped with a conductivity detector for quantitative rank correlation coefficient p=-0.50 (P-value=0.02) between SO analysis as Sulfite. Further cysteine-mediated decompo MICs and SO2 yields. Similar data collected for SO2 yields 10 sition of compounds of (Table 3 entries 1-15) in pH 7.4 buffer after 5 min (Table 2b), however, correlated well with MICs was carried out, where all 2,4-dinitrosulfonamides (100 uM) (FIG. 2b) p=-0.69 (P-value=0.001). Such results indicate a tested were found to generate sulfite (24-100 uM) in 30 min. role for efficiency of SO2 generation, which in turn is related to reactivity of the compound with thiols, in the observed Mtb The derivatives 26 and 27, which had one nitro group on the inhibitory activity of series of compounds of Formula I. 15 aryl ring, did not produce any detectable levels of SO, even In accordance with (Chart 1) 2,4-dinitrophenylsulfona after 4 h and neither did the analogues 28 and 29 (Table 3, mides 21-25 and 1 of primary and secondary amines were entries 12-15). prepared by known process. To study the effect of increasing Further the compounds of (Table 3, entries 1 to 15) were sterics on the nitrogen bearing the Sulfonamide on its reactiv screened for their antimycobacterial activity against Myco ity toward thiols, N-methyl (15) and N-propyl (16) deriva bacterium tuberculosis H.R. and minimum inhibitory con tives were prepared by alkylation of (1), whereas to study the centrations (MICs) were determined. effect of electronic modulation of the aromatic ring attached In accordance with Table 3, eight compounds were found to the DNs group on the rate of SO2 generation, aniline derivatives (6), (7), (9) were prepared by the reaction of the 25 to have MICs.<10 ugmL-1 (Table3, entries 1.3.4.6 to 10). The corresponding amine with DNSC1. Additionally, to study the compounds 21-25, 1, 15, 16, 6, 7 and 9 which all produced effect of removal of one or both the aryl nitro groups and SO upon reaction with cysteine, were found to have greater replacement of the aryl ring with a methyl group, compounds inhibitory activity (MICs25 ugmL-1) than compound 26 to 26 to 29 were prepared by the known method in the art. 29 that were unreactive to cysteine (MICD-50 ugmL-1, Table 30 3, entries 12-15). The results as disclosed in Table 3 clearly indicate a correlation between the analogue's ability to Chart: 1 release sulfur dioxide within 30 min and its Mtb inhibitory potency. COMe COMe ONs 35 The efficient Mtb inhibitor in the series of compounds of Formula I was the benzylamine derivative i.e compound (1) Me -N t N-DNS kur with MIC of 0.05ug mL (0.15uM), which was better than DNS 23; X = O the MIC of isoniazid (0.05 ug mL (0.37 uM) determined 21 22 24; X = CH2 40 under similar conditions. Hence, further studies conducted 25; X = N-Me were focused on understanding the mechanisms of efficacy of H Y DNS R N compound (1). Upon reaction with a number of biologically N YONs relevant nucleophiles such as amino acids and nucleosides in R 45 pH 7.4 buffer, a nearly quantitative recovery of compound (1) R was observed (HPLC analysis), which was selectively reac s 6: R, R = H ; K = Me 7; R = OMe, R = H tive to thiols under physiological pH. 16; R = n-propyl 9; R = HR = F The present inventors also showed the thiol-selectivity of Ph O X compounds of Formula-I under biologically relevant condi ls | Ph O 50 tions. Accordingly the decomposition of 1 in the presence of N1iH ls N1TYR thiols generated Sulfur dioxide, benzylamine, and the arylated H thiol. Benzylamine, a decomposition product of (1) did not Y 28; R = Ph show a significant inhibition of growth of Mtb at 100 ugmL-1 26; X = NO2, Y = H 29: R = Me 55 27; X = H, Y = NO2 s (Table 3, entry 16); a similar observation for N-methylben ON Zylamine was recorded (Table 3, entry 17), and sodium sulfite was inactive against Mtb at 100 ug mL. To understand if combinations of decomposition products were responsible X NO 60 for Mtb inhibitory activity, a mixture of cysteine and com pound (1) in pH 7.4 buffer approximately 2hpost mixing was 30; X = Cl 31; X = BnNH tested at 2 Lig mL'; the said mixture was found to be inactive against Mtb. SO from environmental and biological samples is typi- 65 In view of above it was demonstrated that sulfur dioxide cally quantified by estimation of sulfite, the anionic form of (SO) formation (and not sulfite) intracellularly contributes to SO in basic media (eq. 1). the Mtb inhibitory activity of compound (1). US 9,079,870 B2 23 24 TABLE 3 TABLE 4 SO2 analysis during Thiol-Mediated Decomposition, Max and Antimycobacterial Activity of 2,4-dinitrosulfonamides and SO2 Related Analogues (compounds of chart 1 5 K yield SO2 SO yield, 30 min MIC MIC Entry Compoundp -cloggP" P (min') to of() (IM)'d pKifKaz, Entry Compound source (IM) (Lig/mL) (IM) 1 1 2.87 f 2g 1OO 9.34 2 15 2.41 f 4g 1OO 9.58 1 21 93 6.25 18.7 2 5 83 12.5 35 3 16 3.47 0.1517 4.6 96 9.68 3 23 : 96 6.25 19.6 10 4 6 2.76 O.O273 25 94 4.64 4 24 yes 91 O.78 2.5 5 7 2.69 0.0575 12 97 S.29 5 25 yes 88 12.5 38 6 9 2.93 O.O106 63 86 3.38 6 1 yes 100 O.OS O.15 7 15 yes 100 0.4 1.1 Calculated using Chembiodraw Ultra. 8 16 yes 96 3.13 8.25 Rate analysis of sulfite release from the compound (100 uM) in the presence of cysteine (10 9 6 yes 55 3.13 9.7 15 equiv) in pH 7.4 phosphate buffer (20 mM). 10 7 yes 79.5 1.56 4.4 Half-life was estimated from rate constants, 11 9 yes 24 25 73 “Maximum amount of sulfur dioxide generated the during the reaction with no further 12 26 O O >50 >100 increase in total sulfur dioxide; values reported are a sum of sulfite and sulfate (minor, see Supporting Information), 13 27 O O >50 >100 HPLC analysis showed complete disappearance of 2,4-dinitrophenylsulfonamide, 14 28 O O >50 >100 ev,alues are for the amine without a DNs group. 15 29 O O >50 >100 16 BnNH O >100 >250 20 faccurate rate constant could not be determined. 17 BNHMe no >100 >250 gApproximate half-life estimated based on yields of sulfur dioxide. 18 31 O O >100 >250 19 isoniazide no O.OS 0.37 In accordance with Table 4, the compounds tested were 2 Sulfur dioxide was detected using a pararosaniline-based colorimetric assay, found to be excellent sources of SO with maximum yields of Sulfur dioxide as sulfite was quantified using an ion chromatograph equipped with a 25 SO, rang1ng from 86 to 100 uM. For compound 1 and 1 5, conductivity detector; yields are 30 min after treatment of compound (100LM) with 10 equiv under the assay conditions, accurate determination of rate of cysteine in pH 7.4 phosphate buffer, Minimum inhibitory concentration (MIC) is the minimum concentration of the compound constant (k) for SO2 formation was not possible; the t, for ligibit % ofbacterial growth and was found against Mycobacterium tubercu- compound 1 was estimated to be 2 m1n, and for compound 1 5, t was 4 min SO2 release during cysteine-mediated decom ints S. ta, E. shows t pists of s (). so position of 16, 6, 7 and 9 followed pseudo-first-order kinetics generationinhibit Mtb on growth. the Mtb Further, inhibitory to study activity, the effect the time of rate courses o of2 with half-lives ranging from 4.6 to 63 min. cysteine-mediated SO generation from compounds 1, 15, 16. The compounds having more than one SO, moiety wherein 6, 7 and 9 which have comparable estimated cell permeability the percentage of SO2 release (after 30 mints) is more than the (-clogP. Table 4), were determined and compared with their single SO2 moiety compound, per mole of compound as antimycobacterial activities. shown Table 5. TABLE 5 Compound Structure % SO2 (30 min) 34 ON 152%

R 2 saO o2 Y N O n-1N1 N 2 NO %Z S

NO 35 ON 161%

NO US 9,079,870 B2 25 26 TABLE 5-continued

Compound Structure % SO2 (30 min)

36 O2N 1.84%

O O N 2AWe ON S ca Y. N nu-1N1 N/ NO

OaS

37 ON o1 1839/o

NO 2 ca Y. N n-1N1 Na NO

OMS

38 ON 160%

K 2 o2V N-1a-N 2 NO %2

39 ON 141%

N-1S-1 N2° NO; C / NO US 9,079,870 B2 27 28 TABLE 5-continued Compound Structure % SO2 (30 min) 40 88%

oav2 N-1-N/O NO C % NO

41 NO 99%

sa' 2\ N-N-Nz' NO C % NO

42 ON 98%

4\29 N-1-Nz' NO C % NO

In accordance with Table 5, the '% of SO2 release of com niline, and 3-fluoroaniline, with the k for their corresponding pounds comprising more than one SO, moiety, after com DNs derivative (Table 4, entries 1-6). mencement of the reaction with thiol (30 mints) were found 50 With regard to the proposed mechanism (FIG.1a), a com 88%. parison ofk and pK revealed that an increased amine pK. The plausible mechanism of decomposition of compounds resulted in higher rates of SO release. A comparison of of Formula I, (compound 1) is represented in FIG. 1a, estimated half-life of SO, generation (t) and MICs showed wherein the thiol attack on the 2,4-dinitrophenylsulfonamide that the fluoro derivative 9, which was the longest among the produced intermediate I, which was converted to intermediate 55 compounds tested, was also the least potent of the 2,4-dini II by a proton transfer that Subsequently gave SO. It was trophenylsulfonamides that evaluated in the instant study observed that the rate constant for SO generation (k) was (Table 3). Further analysis of data for, compounds 1, 15, 16, depend on the stability of the transition state leading to the 6, 7 and 9 which have comparable-clogP values, showed that formation of the protonated amine intermediate II. Hence, the a higher rate of SO2 generation (k) correlated well with stronger the basicity (or weaker the conjugate acid), the 60 improved inhibitory activity. greater would be the tendency of nitrogen bearing the 2,4- Therefore the instant study provides a rational basis for dinitrophenylsulfonyl group to get protonated and leave as further design of analogues of compound (1) with anti-myco the amine resulting in a higher k. bacterial activity. However, without data pertinent to drug Further the step of protonation is rate determining step in uptake of 2,4-dinitrophenylsulfonamide compounds in Mtb, the said mechanism. As basicity data for 2,4-dinitrosulfona 65 it is not possible to attribute differences in MICs of com mides was unavailable, the inventors compared the pKa of pounds 1, 15, 16, 6, 7 and 9 only to differences in rates of benzylamine, N-alkylbenzylamines, aniline, 4-methoxya cysteine-mediated SO generation from said compounds. US 9,079,870 B2 29 30 Further to test the selectivity of SO inhibitory activity The invention will now be illustrated with help of toward Mtb, a cell viability assay was conducted using human examples. The aforementioned embodiments and below embryonic kidney 293 cells (HEK) cell lines and the IC50 for mentioned examples are for illustrative purpose and are not compound (1) was determined as 7 uM. Based on the MIC meant to limit the scope of the invention. Various modifica and IC50, compound (1) was nearly 50-fold selective in 5 tions of aforementioned embodiments and below mentioned inhibitory activity (SI-47) toward Mtb over human embry examples are readily apparent to a person skilled in the art. All onic kidney cells. such modifications may be construed to fall within the scope In addition to generation of SO2, the reaction of compound and limit of this invention as defined by the appended claims. (1) with thiols could also affect cellular redox balance by mycothiol depletion in Mtb. The possible role of thiol-deple 10 EXAMPLES tion in the observed efficacy of compound (1) was determined by testing the analogue of compound (1) without a Sulfonyl Example 1 group 31 (Chart 1), which is unreactive with thiols showed no Mtb inhibitory activity (Table 3, entry 19). These results General Procedure for the Synthesis of Support a role for thiol depletion and perhaps protein S-ary 15 2,4-Dinitrophenylsulfonamides lation as a contributor to the observed antibacterial activity of compound (1). A solution of the amine in DCM containing triethylamine In view of above, the inventors provide evidence for the or pyridine was treated with a solution of DNsCl in DCM at utility of masked sources of Sulfur dioxide as antimycobac 0 or -40°C. The reaction mixture was warmed to RT. Workup terial agents and whose Mtb inhibitory activity in part included extraction with DCM or EtOAc followed by silica depended on the rate of thiol-mediated sulfur dioxide genera gel chromatography using mixtures of PE/EtOAc or DCM as tion. Consequently, a major impediment to development of the eluent to produce the desired compound as a yellow solid new drugs for infectious diseases is the cost of multistep (unless otherwise stated). synthesis to prepare potential drug candidates. The com 25 pounds prepared in this study can be obtained in one step from Example 2 relatively inexpensive commercial sources, making them especially attractive for further development as potential drug General Procedure for the Synthesis of alkylated candidates. derivatives of 2,4-Dinitrophenylsulfonamides Alternatively, a detailed kinetic analysis of thiol-mediated 30 decomposition and Sulfite generation from compound (7) is To a solution of the 2,4-dinitrophenylsulfonamide in DMF, represented in FIG. 1b, wherein compound (7) decomposed KCO (2 eq.) and alkyl halide (2 eq.) were added at RT and to simultaneously produce Sulfur dioxide, and by-products reaction mixture was stirred for 2 h. The reaction was like 4-methoxyaniline (7x) and 2-hydroxyethylthio-2,4-dini quenched with water and extracted with EtOAc. The organic trobenzene (7y). Further the rate determining step in the said 35 layer was dried on NaSO and concentrated under reduced mechanism was the step of protonation (either through proton pressure to get crude product, which was purified by silica gel transfer or addition of proton). column chromatography using mixtures of petroleum ether/ Further the inventors found a good positive correlation EtOAc or DCM as the eluant to get desired compound as a r=0.79 (P-values0.0001) between pKa of the amine and yellow solid (unless otherwise stated). SO2 yields (5 min) and r=0.71 (P-value-0.001) for SO2 40 yields (30 min) during cysteine-mediated decomposition of Example 3 2,4-dinitrosulfonamides Supporting that the proposed mecha nism of formation of protonated intermediate as the rate Cysteine-Activated Sulfur Dioxide Release determining step could be a general mechanism of thiol mediated SO2 generation from 2,4-dinitrophenylsulfona 45 P-Rosaniline assay for sulfur dioxide: The dye was pre mides (FIG. 4). pared using a literature procedure. Assay conditions: 900 uL As a majority of the potent Mtb inhibitors in the instant of p-rosaniline-based dye was mixed with 50 uL of satd study were completely decomposed in 30 min, it is perhaps HgCl, solution and 50 uL of 0.2 mM sulfite. The obtained less likely that they would stay unreacted, especially in the solution was covered with aluminum foil for 15 min until presence of (estimated) millimolar concentration of mycothi 50 violet color developed and the absorbance was measured. A ols in Mtb. similar protocol was used for compounds which were treated It is found that anti-mycobacterial activity of closely with 10 equiv. cysteine in pH 7.4 phosphate buffer containing related structural analogues of compound of Formula I of 1-5% DMSO (or ethanol). present invention with comparable clogPs correlated well Ion chromatography analysis: An ion chromatograph with the analogue's ability to generate Sulfur dioxide upon 55 attached with a conductivity detector was used for sulfite treatment with cysteine. analysis. One mMNaHCO/3.2 mMNaCO was the eluant, In view of above, the inventors provide evidence for the and the flow rate was 0.7 mL/min Using stock solutions of utility of masked sources of Sulfur dioxide as antimycobac sulfite, a calibration curve was generated (R2=0.9999). To 3 terial agents and whose Mtb inhibitory activity in part mL of 1 mM stock solution of compound in acetonitrile, 24 depended on the rate of thiol-mediated sulfur dioxide genera 60 mL of phosphate buffer (pH-7.4, 20 mM) was added and tion. Consequently, a major impediment to development of vortexed for 20s. To this mixture, 3 mL of 10 mM cysteine new drugs for infectious diseases is the cost of multistep solution (pH 7.4) was added, and the reaction mixture was synthesis to prepare potential drug candidates. The com stirred at RT under inert atmosphere. Aliquots at appropriate pounds prepared in this study can be obtained in one step from time intervals were analyzed by IC. Maximum sulfur dioxide relatively inexpensive commercial sources, making them 65 yield was calculated based on completion of the reaction with especially attractive for further development as potential drug no further increase in Sulfite formation. In all such cases, candidates. when sulfur dioxide reached a maximum, HPLC analysis of US 9,079,870 B2 31 32 the reaction mixture showed complete disappearance of the NMR spectrometer in DMSO-d solvents. Further the mass 2,4-dinitrophenylsulfonamide. spectra are taken on high resolution mass spectroscopy HRMS (ESI-TOF). Example 4 7.1 N-benzyl-2,4-dinitrophenylsulfonamide (1) Antimycobacterial Activity Assay H NMR (DMSO-d6): 89.06-9.04 (t, J=5.8 Hz, 1H), 8.86 Ten-fold serial dilutions of each test compound/drug were 8.85 (d. J=2.4 Hz, 1H), 8.52-8.50 (dd, J–8.8, 2.4 Hz, 1H), prepared and incorporated into Middlebrook 7H11 agar 8.12-8.10 (d. J=8.4 Hz, 1H), 7.26-7.20 (m, 5H), 4.21-4.19 (d. medium with OADC growth supplement. Inoculum of Myco 10 J=6.0, 2H); 13C NMR (DMSO-d6): 8 149.4, 147.7, 139.6, bacterium tuberculosis H.R. were prepared from fresh 135.1, 132.5, 128.7, 128.3, 128.0, 126.7, 120.4, 48.0; FTIR Middlebrook 7H11 agar slants with OADC growth supple (KBr, cm-1): 3379, 3098, 2345, 1537, 1352, 1161; mp 150 ment adjusted to 1 mg ml-1 (wet weight) in Tween 80 151° C.; HRMS (ESI-TOF) CHNOS IM+Nat: (0.05%) saline diluted to 10° to give a concentration of 360.0266. Found: 360.0275. approximately 107 cfu mL. Five uL ofbacterial suspension 15 was spotted into 7H11 agar tubes containing 10-fold serial dilutions of drugs per mL. The tubes were incubated at 37°C., 7.2 N-(4-chlorobenzyl) and final readings were recorded after 28 days. The minimum 2,4-dinitrophenylsulfonamide (3) inhibitory concentration (MIC) is defined as the minimum concentration of compound required to completely inhibit the H NMR (400 MHz, DMSO-d): 8 9.06 (s, 1H), 8.87-8.86 bacterial growth. (d. J=1.8 Hz, 1H), 8.54–8.52 (dd, J=6.9, 1.8 Hz, 1H), 8.13 8.11 (d. J=6.9 Hz, 1H), 7.33-7.31 (d.J=6.7 Hz, 2H), 7.26-7.25 Example 5 (d. J=6.7 Hz, 2H), 4.20 (s.2H); 'CNMR (100 MHz, DMSO d): & 149.7, 147.6, 138.4, 136.2, 132.6, 132.0, 130.0, 128.7, Safety data of the byproducts like amino and nitro 25 127.5, 120.4, 40.0; FTIR (KBr, cm): 3385, 3100, 1540, compounds 1346, 1165; mp 161-162° C.; HRMS (ESI-TOF): Calculated CHCINOS M+Nat: 393.9877. Found M+Na": The LD50 of amine byproduct in an Oral Rat model was 393.9879. found as 552 mg/kg and therefore, the amine byproducts are non-toxic in humans. 30 7.3 N-(4-trifluoromethylbenzyl) The thiolated-2,4-dinitrophenyl compound as a byproduct 2,4-dinitrophenylsulfonamide (4) has been tested in an animal model and found to be relatively non-toxic and hence the same are non-toxic in humans. "H NMR (400 MHz, DMSO-d): 8 9.15 (s, 1H), 8.85 (s, Example 6 1H),8.50-8.49 (d.J=6.9 Hz, 1H), 8.11-8.10 (d. J=6.8 Hz, 1H), 35 7.62-7.60 (d. J=6.4 Hz, 2H), 747-7.42 (d. J=6.3 Hz, 2H), 4.32 Pharmaceutical Composition (s. 2H); 'C NMR (100 MHz, DMSO-d): & 149.7, 1474, 1419, 138.4,128.9, 128.6, 128.3, 127.4,125.8, 125.5, 121.1, 120.4, 120.3, 46.2: FTIR (KBr, cm): 3389, 1854, 1354, 40 1322, 1170, 1120; 176-177° C.; HRMS (ESI-TOF): Calcu Compound of formula 1 10.0% wiw lated CHFNOSM+Na":428.0140. Found M+Na": Color Amaranth 0.3% wiw 428O136. Raspberry Flavor 0.7% wiw Magnesium Stearate 2.0% wiw Mannitol q.S. to 100.0% w/w 7.4 N-(2-trifluoromethylbenzyl) 2,4-dinitrophenylsulfonamide (5) 45 General Procedure for Preparation of Pharmaceutical Com position: H NMR (400 MHz, DMSO-d): 8 9.23 (s, 1H), 8.91-8.90 The mannitol was dissolved in water, Subsequently phar (d. J=2.2 Hz, 1H), 8.60-8.58 (dd, J=8.7, 2.2 Hz, 1H), 8.18 maceutically acceptable colour and flavour were added to the 8.16 (d. J=8.6 Hz, 1H), 7.71-7.69 (d.J=7.8 Hz, 1H), 7.65-7.60 maniitol wherein the water was evaporated to adsorb color 50 (m. 2H), 7.49-7.45 (dd, J=7.8, 2.2 Hz, 1H), 4.39 (s. 2H); 'C and flavor on mannitol. Further compound of Formula I and NMR (100 MHz, DMSO-d): & 150.2, 1479, 138.1, 135.8, diluent i.e. magnesium Stearate were mixed together to derive 133.2, 131.7, 129.9, 128.5, 127.8, 127.7, 126.8, 126.5, 126.4, at Suitable dosage form Such as tablet, capsule, powder. 126.3, 126.2, 126.0, 120.6, 43.6; FTIR (KBr, cm): 3385, Suitable colours, flavours and pharmaceutical excipients 1556, 1540, 1422, 1350, 1313, 1171, 1121, 1104, 1038; mp may be selected from the ones known in the art to prepare 55 157-158° C.; HRMS (ESI-TOF): Calculated Suitable dosage forms for conventional modes of administra CHFNOS M+Na": 428.0140. Found M+Na": tions. 428.01.41. Example 7 7.5 N-(4-methoxyphenyl) 60 2,4-dinitrophenylsulfonamide (7) Spectral Data and Characterization of the Array of 2,4-dinitrophenylsulfonamide Analogous H NMR (DMSO-d6) & 10.70 (s, 1H), 8.88-8.87 (d. J–2.0 (Compounds of Formula I) HZ, 1H), 8.60-8.57 (dd, J=8.8, 2.0 Hz, 1H), 8.13-8.11 (d. J=8.8 Hz, 1H), 7.05-7.04, (d. J=7.2 Hz, 2H), 6.87-6.85 (d. The characterization of the compounds of Formula I is 65 J=7.2 Hz, 2H), 3.69 (s.3H); 13C NMR (DMSO-d6) & 157.9, performed by using FT-IR spectrometer and melting point 150.4, 148.3, 136.8, 132.1, 128.5, 127.6, 125.1, 120.7, 115.1, apparatus; whereas spectral analysis include "H and 'C 56.7: FTIR (KBr, cm-1): 3345,3098, 2343, 1539, 1357, 1246,

US 9,079,870 B2 39 40 4. The pharmaceutical composition for the treatment of an Formula II infection by at least one pathogen of the genus Staphylococ cus, Enterococcus or Mycobacterium according to claim 1, ON NO further comprising a pharmaceutically acceptable excipient, O a pharmaceutically acceptable vehicle, or a mixture thereof. | 5. A pharmaceutical composition for the treatment of an S-N infection by at least one pathogen of the genus Staphylococ O cus, Enterococcus or Mycobacterium, comprising an effec tive amount of a thiolactivated prodrug of SO, selected from 10 the group consisting of i. N-(4-chlorobenzyl) 2,4-dinitrophenylsulfonamide (3): Formula III ii. N-(4-trifluoromethylbenzyl) 2,4-dinitrophenylsulfona ON mide (4): Xs iii. N-(2-trifluoromethyl benzyl) 2,4-dinitrophenylsul 15 fonamide (5): Oly, iv. N-(2-methoxyphenyl) 2,4-dinitrophenylsulfonamide (8): V. N-(3-fluorophenyl) 2,4-dinitrophenylsulfonamide (9); I vi. N-(4-cyanophenyl) 2,4-dinitrophenylsulfonamide (11): vii. (S)N-(1-phenylethyl)-2,4-dinitrobenzenesulfonamide (12); viii. (R)N-(1-phenylethyl)-2,4-dinitrobenzenesulfona mide (13): R2 is selected from the group consisting of allyl, a group ix. N-(2-phenylethyl) 2,4-dinitrophenylsulfonamide (14); of Formula IV and a group of Formula V: 25 X. (N-benzyl, N-propyl) 2,4-dinitrophenylsulfonamide (16): xi. (N-benzyl, N-phenyl) 2,4-dinitrophenylsulfonamide Formula IV (17); xii. N-methyl, N-(2-phenylethyl) 2,4-dinitrophenylsul 1N1 i. 30 fonamide (19); R3 xiii. N-benzyl, N-(2-phenylethyl) 2,4-dinitrophenylsul 21 fonamide (20): Formula V xiv. Methyl 2-(2,4-dinitrophenyl)sulfonyl COO(R4) aminopropanoate (21); 35 XV. methyl 1-(2,4-dinitrophenyl)sulfonylpyrrolidine-2- carboxylate (22); Xvi. 1-(2,4-dinitrophenyl)sulfonyl-4-methylpiperazine (25); xvii. N,N-diallyl-2,4-dinitrobenzenesulfonamide (32); wherein n is either 0 or 1; R3 is independently selected 40 xviii, N-benzyl-2,4-dinitro-N-prop-2-yn-1-ylbenzene from the group consisting of H, halogen, —CF3, (C1 sulfonamide (33): C6) alkoxy, and —CN; and R4 and R4' are indepen xix. N.N'-(propane-1,3-diyl)bis(N-benzyl-2,4-dinitroben dently selected from the group consisting of and Sub Zenesulfonamide) (34); stituted or unsubstituted (C1-C6) alkyl; or XX. N-benzyl-N-(3-(N-(2-methoxyphenyl)-2,4-dinitro b) R1 and R2, together with the nitrogen atom to which 45 phenylsulfonamido)propyl)-2,4-dinitrobenzene R1 and R2 are attached, form a 5 or 6 membered sulfonamide (35); heterocyclic ring, optionally containing an additional xxi. N-benzyl-N-(3-(N-(3-methoxyphenyl)-2,4-dinitro substituted or unsubstituted heteroatom selected from phenylsulfonamido)propyl)-2,4 dinitrobenzenesulfona the group consisting of O and N, said 5 or 6 membered mide (36): heterocyclic ring being optionally Substituted with a 50 xxii. N-benzyl-N-(3-(N-(4-methoxyphenyl)-2,4-dinitro Substituent selected from the group consisting of H, phenylsulfonamido)propyl)-2,4-dinitrobenzene (C1-C6) alkyl, and COO(R5): sulfonamide (37); wherein R5 is H or (C1-C6) alkyl: xxiii. N-benzyl-N-(3-(2,4-dinitro-(N-phenyl) phenylsul further comprising an active ingredient selected from fonamido)propyl)-2,4 dinitro benzene Sulfonamide the group consisting of anti-tuberculosis drugs 55 (38); Selected from the group consisting of rifampin, iso xxiv. N-benzyl-N-(3-(N-(2-fluorophenyl)-2,4-dinitrophe niazide, ethambutol, and pyrazinamide, Fluoroqui nylsulfonamido)propyl)-2,4-dinitrobenzenesulfona nolones; Cephalosporins; Penicillins; Carbepen mide (39); ems; Aminoglycosides; Tetracyclines; and XXV. N-benzyl-N-(3-(N-benzyl-2-nitrophenylsulfona mixtures thereof. 60 mido)propyl)-2,4-dinitrobenzenesulfon-amide (40); 2. The pharmaceutical composition according to claim 1, xxvi. N-benzyl-N-(3-(N-benzyl-3-nitrophenylsulfona wherein the infection is caused by Mycobacterial tuberculo mido)propyl)-2,4-dinitrobenzenesulfon-amide (41); S1S. and 3. The pharmaceutical composition according to claim 1, xxvii. N-benzyl-N-(3-(N-benzyl-4-nitrophenylsulfona wherein the infection is caused by a pathogen selected from 65 mido)propyl)-2,4-dinitrobenzenesulfonamide (42); the group consisting of Staphylococcus aureus and Entervo said pharmaceutical composition further comprising an COccus faecalis. active ingredient selected from the group consisting of US 9,079,870 B2 41 42 anti-tuberculosis drugs selected from the group consist 10. The method of treating or inhibiting growth of an ing of rifampin, isoniazids, ethambutol, and pyraZina infection according to claim 8, wherein, the infection is mide: Fluroquinolones; Cephalosporins; Penicillins; caused by Mycobacterial tuberculosis. Carbepenems; Aminoglycosides; Tetracyclines; and 11. The method according to claim 7, wherein the subject 5 is a mammal. mixtures thereof. 12. A method of treating or inhibiting growth of an infec 6. A pharmaceutical composition according to claim 5. tion by at least one pathogen of the genus Staphylococcus, further comprising a pharmaceutically acceptable excipient, Enterococcus or Mycobacterium in a subject comprising a pharmaceutically acceptable vehicle, or a mixture thereof. administering to the Subject a pharmaceutical composition 7. A method of treating or inhibiting growth of an infection 10 according to claim 5, together with a pharmaceutically by at least one pathogen of the genus Staphylococcus, Entero acceptable excipient, a pharmaceutically acceptable vehicle, coccus or Mycobacterium in a subject comprising adminis or a mixture thereof. tering to the Subject a pharmaceutical composition according 13. The method of treating or inhibiting growth of an infection according to claim 12, wherein the infection is to claim 1, said pharmaceutical composition further compris caused by a pathogen selected from the group consisting of ing a pharmaceutically acceptable excipient, a pharmaceuti 15 Staphylococcus aureus and Enterococcus faecalis. cally acceptable vehicle, or a mixture thereof. 14. The method of treating or inhibiting growth of an 8. The method of treating or inhibiting growth of an infec infection according to claim 12, wherein the infection is tion according to claim 7, further comprising administering at caused by Mycobacterial tuberculosis. least one additional active ingredient. 15. The method of treating or inhibiting growth of a bac 9. The method of treating or inhibiting growth of an infec terial infection according to claim 12, wherein the Subject is a tion according to claim 7, wherein, the infection is caused by mammal. Mycobacterial tuberculosis.