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Carbazoles and tetrahydro-beta-carbolines: Synthesis and activity characterizations against Mycobacterium tuberculosis

ProQuest Dissertations and Theses, 2009
Dissertation
Author: Taylor A Choi
Abstract:
Despite the widespread use of chemotherapeutic agents, an estimated one-third of the global population is infected with Mycobacterium tuberculosis and two million lives are consumed annually by this pandemic disease. In order to discover and develop new anti-tuberculosis drugs, two classes of compounds--carbazole and 1,2,3,4-tetrahydro-β-carboline--were synthesized and evaluated. Structure-activity relationships study of the 1,2,3,4-tetrahydro-β-carboline series revealed that substitutions at C-1 and N-2 are crucial for anti-tuberculosis activity. Discovery of two compounds with submicromolar activity and non-toxicity against the two mammalian lines tested invited further study, including spectrums of activity, minimum bactericidal concentration, metabolic stability, pharmacokinetics and in vivo efficacy. Studied revealed that the 1,2,3,4-tetrahydro-β-carboline series is selective towards the Mycobacterium tuberculosis complex. Different oral formulations of ITR406 indicated that PEG400 formulation provided the compound in solution, thereby resulting in greater oral bioavailability. Based on the findings, 1,2,3,4-tetrahydro-β-carboline may be potential anti-tuberculosis lead candidates.

TABLE OF CONTENTS CHAPTER PAGE Acknowledgements..., .v Table of Contents vi List of Tables ix Table of Figures.... .....x List of Symbols and Abbreviations xi Summary .xv Tuberculosis 1 1.1 Tuberculosis 1 1.1.1 Background and Epidemiology 1 1.1.2 Tuberculosis: An Ongoing Challenge 2 1.1.3 Methods to Identify M. tuberculosis Strains 3 1.1.4 Current Treatment 4 1.1.5 Drug Resistance 5 1.1.6 Development of New Anti-TB Drugs 6 1.2 Carbazoles as Potential Leads for the Treatment of Tuberculosis 7 1.3 Tetrahydro-P-carbolines as Potential Leads for the Treatment of Tuberculosis 9 1.4 Scope of the Study 13 1.5 Cited Literature.... 15 2 Materials and Methods..... .20 2.1 Biology 20 2.1.1 General 20 2.1.2 Mycobacterium tuberculosis Culture Conditions 20 2.1.3 Nonreplicating Mycobacterium tuberculosis Culture Conditions......... 20 2.1.4 Microplate Alamar Blue Assay 21 2.1.5 Low Oxygen Recovery Assay 22 2.1.6 Cell Culture 23 2.1.7 Cytotoxicity..... 24 2.1.8 Minimum Bactericidal Concentration Test 24 2.1.9 Macrophage Assay 25 2.1.10 Spectrum of Activity Assay .....26 2.1.11 Aerosol Acute Infection of Animals.... 27 2.1.12 In vivo efficacy in an acute infection mouse model 27 2.1.13 clogP Calculation 28 2.2 Chemistry ...29 2,2.1 General Chemistry .29 2.2.2. Synthesis of Carbazole Analogs 30 2.2.3 Synthesis of Tetrahydro-(3-carboline Analogs 45 2.3 Drug Metabolism and Pharmacokinetics ,. 73 2.3.1 General Information 73 2.3.2 HPLC Conditions .....73 2.3.3 Microsomal Stability Assay 74 VI

TABLE OF CONTENTS (continued) 2.3.4 Gastric Fluid Stability Assay 75 2.3.5 Plasma Protein Binding. 76 2.3.6 Animal Care 76 2.3.7 Chemical Inhibition Assay 76 2.3.8 Maximum Tolerated Dose 77 2.3.9 PK Study ...77 2.3.10 PK Parameters , 78 2.4 Cited Literature 78 3 Synthesis and Anti-Tuberculosis Activity of Carbazole Alkaloids ...80 3.1 Synthesis 80 3.2 Preliminary Structure-Activity Relationships. 81 3.3 Synthesis of Carbazole 15b and its Analogues.... 87 3.4 Structure-Activity Relationships of Carbazole-1,4-quinol Alkaloids 88 3.5.Synthesis of Carbazole 10 Analogues 91 3.6 Structure-Activity Relationships of 9H-Carbazole Alkaloids 92 3.7 Discussion 93 3.8 Cited Literature 95 4.1 Synthesis of Tetrahydro-B-Carbolines 99 4.2 In vitro Studies of Tetrahydro-B-Carbolines 100 4.2.1 In vitro Anti-tuberculosis Activities and Structure-Activity Relationships ,..,.. ....100 4.2.2 Preliminary Assessment of the Pharmacophore 106 4.2.3 Mini Structure-Activity Relationships of Phathalazinone-Containing Tetrahydro-P-Carbolines 106 4.2.4 Minimum Inhibitory Concentrations in the Presence of Serum......... 109 4.2.5 In vitro Activity Against the Erdman Strain , 110 4.2.6 Spectrums of Activity 111 4.2.7 Minimum Bactericidal Concentration 114 4.3 In vivo Studies 114 4.3.1 Maximum Tolerated Dose 114 4.3.2 Efficacy Studies of ITR353 ...114 4.4 Macrophage Assay 118 4.5 Pharmacokinetic Studies 119 4.5.1 General... 119 4.5.2 Plasma Protein Binding 119 4.5.3 Gastric Fluid Stability of ITR406 119 4.5.4 Microsomal Stability of ITR406 120 4.5.5 In vitro Study of ITR406 with Ketoconazole 121 4.5.6 PK Study. , .....123 4.5.7 Formulations 126 4.5.8 PK Study-Revisited 127 4.6 Target Considerations....... 129 4.6.1 Phosphodiesterase 129 VII

TABLE OF CONTENTS (continued) 4.6.2 Target of Isoniazid 130 4.6.3 Phosphopantetheine Adenyl Transferase (PPAT) 131 4.7 Discussion 133 4.8 Cited Literature .....134 Appendix......... 138 Vita............. .........240 VIII

LIST OF TABLES Table 1. In vitro activity against M. tuberculosis H37Rv of compounds derived from the NovaCore library 12 Table 2. Anti-tuberculosis activity, vero cell cytotoxicity, and selectivity indices of the carbazole derivatives 4 84 Table 3. Anti-tuberculosis activity, vero cell cytotoxicity, and selectivity indices of the carbazole derivatives 10-15 86 Table 4. Structure-activity relationships of carbazole-1,4-quinol alkaloids.. 89 Table 5. Structure-activity relationships of carbazole derivatives 94 Table 6. In vitro activities of 31394012 analogues against M. tuberculosis 101 Table 7. In vitro activity of 60871805 analogues against M. tuberculosis.. 103 Table 8. In vitro activity of tetrahydro-(3-carbolines analogues against M. tuberculosis 105 Table 9. In vitro activity of ITR353 analogues against M. tuberculosis 108 Table 10. Minimum inhibitory concentrations in the presence of serum.... 111 Table 11. Spectrums of activity of tetrahydro-p-carboline derivatives 112 Table 12. MICs of ITR367 and ITR353 against a panel of mycobacterium strains 112 Table 13. MICs of ITR368 and ITR369 against a panel of mono-drug resistant isolates 113 Table 14. Evaluation of ITR353 in M. tuberculosis-Infected mice 115 Table 15. Evaluation of ITR406 in M. tuberculosis-infected mice 117 Table 16. In vitro mouse pharmacokinetic profile of ITR353 enantiomers. 122 Table 17. In vitro human pharmacokinetic profile of ITR353 enantiomers 122 Table 18. Inhibition of metabolism of ITR406 by co-administration with ketoconazole 123 Table 19. Concentration of ITR353 in mouse plasma when dosed at 10 mg/kg, formulated in 300 mg/ml HP(3CD with 20% DMSO (v/v) 124 Table 20. Concentration of ITR353 in mouse plasma when dosed at 400 mg/kg, formulated in 5% CMC suspension 126 Table 21. PK profiling of ITR406 128 IX

LIST OF FIGURES Figure 1. Current first-line tuberculosis chemotherapeutic agents 5 Figure 2. Naturally occurring carbazole alkaloids with anti-TB activity 8 Figure 3. Carbazoles in clinical use 9 Figure 4. Synthesis of carbazoles 4 by iron-mediated or palladium-catalyzed oxidative cyclization 80 Figure 5. (+)-Carquinostatin and (±)-lavaduquinocin 81 Figure 6. Carbazole derivatives 8-15 82 Figure 7. Summary of preliminary findings 86 Figure 8. Synthesis of carbazole 15b and its analogues 87 Figure 9. Summary of structure-activity relationships of carbazole-1,4-quinol alklaoids 90 Figure 10 Synthesis of tricarbonyliron-cyclohexadienylium salt 91 Figure 11. Summary of structure-activity relationships of carbazoles 95 Figure 12. Synthesis of 1,2-disubstituted tetrahydro-3-carbolines with a carbonyl linker 99 Figure 13. Synthesis of 1,2-disubstituted tetrahydro-(3-carbolines with a carbonyl linker.... 100 Figure 14. Representative phathalizone moiety-containing compounds from the NovaCore library 107 Figure 15. In vitro anti-tuberculosis structure-activity relationships of tetrahydro-B-carbolines 110 Figure 16. In vivo efficacy study of ITR353 115 Figure 17. Evaluation of ITR406 in M. tuberculosis-!nfected mice 117 Figure 18. Mouse microsomal stabiliyt of the enantiomers of ITR353 121 Figure 19. Human microsomal stability of the enantiomers of ITR353 122 Figure 20. Concentration of ITR353 in mouse plasma when dosed at 400 mg/kg formulated in 5% CMC suspension 124 Figure 21. Concentration of ITR353 in mouse plasma when dosed at 10 mg/kg formulated in 300 mg/mL HPpCD with 20% DMSO (v/v) 125 Figure 22. Concentration of ITR353 in mouse plasma when dosed at 100 mg/kg bid formulated in NMP-PEG400-EtOH-H2O (20/40/2/38 v/v) 128 Figure 23. Tadalafil 129 Figure 24. Isoniazid and phthalazinone 131 Figure 25. A representative PPAT-targeting compound..... 132 x

LIST OF ABBREVIATIONS AND SYMBOLS APCI ATTC BCG BRL C. albicans cAMP CAN CDCb CPU cGMP CH2CI2 CH3OH CL CLh CLjnt cm2 CMC 13C NMR C02 cuft Cu(OAc)2 d DCE DCM dd DEPT DMEM DMSO DOC DST dt E. coli ED EDC! EHNA EMEM ESI Et20 EtOAc EtOH eV FBS g h atmospheric pressure chemical ionization American type culture collection bacilli Calmette-Guerin biological resources laboratory Candida albicans cyclic adenosine monophosphate eerie ammonium nitrate deuterated chloroform colony forming unit cyclic guanosine monophosphate dichloromethane methanol clearance hepatic clearance intrinsic clearance squared centimeter(s) carboxymethylcellulose carbon-13 nuclear magnetic resonance carbon dioxide cubic feet copper (ii) acetate doublet dichloroethane dichloromethane doublet of a doublet distortionless enhancement by polarization transfer dulbecco's modified eagle's medium dimethyl sulfoxide dissolved oxygen concentration drug susceptibility testing double of a triplet Eschericheria coli erectile dysfunction t-ethyl-3-(3-dimethylaminopropyl)carbodiimide (erthyro-9-(2-hydroxy-3-nonyl)adenine hydrochloride Eagle's minimum essential medium electrospray ionization diethyl ether ethyl acetate ethanol electron volt fetal bovine serum gram(s) hour(s) XI

H2 1HNMR H20 HBSS HCI HIV HOAc HOBt HPLC HP(3CD HRMS HSR Hz IBMX IC50 ICBG INH ip IR IS ITR J K2CO3 KCZ Kg KM KOH L LC LORA M m M. tuberculosis MABA MBC V9 ML pm MM MDR-TB MeCN MeOH mg Mhz MIC hydrogen proton nuclear magnetic resonance water Hank's balanced salt solution hydrochloric acid human immunodeficiency virus acetic acid 1 -hydroxybenzotriazole high pressure liquid chromatography 2-hydroxypropylbetacyclodextrin high resolution mass spectrometry head space ratio hertz 3-isobutyl-1 -methylxanthine concentration that inhibits a response by 50% relative to positive control international cooperative biodiversity group isoniazid intraperitoneal Infrared internal standard institute for tuberculosis research coupling constant(s) potassium carbonate ketoconazole kilogram(s) kanamycin potassium hydroxide liter(s) liquid chromatography low oxygen recovery assay mycobacterium multiplet Mycobacterium tuberculosis microplate Alamar blue assay Minimum bactericidal concentration microgram Microliter(s) micrometer(s) micromolar multiple drug-resistant tuberculosis acetonitrile methanol milligram(s) megahertz minimum inhibitory concentration xii

min mL mM mm mmol MS MTS N2 Na Na2So3 Na2So4 NADPH NaHC03 ND NH4CI NH4OH nm NMP NMR NRP OADC P450 PBS Pd(OAc)2 PDE PE pH PMS po PZA q Q Qp,h r r.t. RED RIF RLU rpm s S. aureus SAR sat SI SM Minute(s) milliliter(s) millimolar millimeter(s) millimole(s) mass spectrometry (3-(4,5-d imethylthiazol-2-y l)-2,5-d iphenyltetrazol i urn bromide, a tetrazole) nitrogen sodium sodium sulfite sodium sulfate P-nicotinamide adenine dinucleotide phosphate sodium bicarbonate not determined ammonium chloride ammonium hydroxide nanometer(s) n-methylpyrrolidone nuclear magnetic resonance non-replicating persistent Oleic acid-albumin-dextrose-catalase cytochrome p450 phosphate-buffered solution palladium (ii) acetate phosphodiesterase petroleum ether power of hydrogen phenylmethasulfazone per os pyrazinamide quadruplet quadrupole hepatic plasma flow resistant room temperature rapid equilibrium dialysis rifampin relative light unit(s) rotations per minute singlet staphylococcus aureus structure-activity relationships saturated selectivity index stremptomycin XIII

t t T TB TEA TFA THBC TiCI4 TLC TOF TsOH UV v/v Vero WHO XDR-TB 5(ppm) A time (day) triplet titre tuberculosis triethylamine trifluoroacetic acid tetrahydro-0-carboline titanium tetrachloride thin layer chromatography time-of-flight p-toluene sulfonic acid ultraviolet volume/volume african green monkey kidney cell line world health organization extensively drug-resistant tuberculosis chemical shift (in parts per million) wavelength XIV

SUMMARY This thesis reports the syntheses and anti-tuberculosis activities of two classes of compounds: carbazole and 1,2,3,4-tetrahydro-|3-carboline. Chapter 1 lays the foundation of this study. It discusses the global impact of tuberculosis and the current need for new anti-tuberculosis drugs. It also introduces carbazole and 1,2,3,4-tetrahydro-p-carboline which may be potential anti-tuberculosis lead candidates. Chapter 2 details the experiment protocols and characterizations of the synthesized compounds. Namely, it describes the assays which were used to determine the inhibitory activity of the compounds against Mycobacterium tuberculosis, cytotoxicity against mammalian cell lines, spectra of activity, metabolic stability, pharmacokinetics, and in vivo efficacy. Chapter 3 describes the synthetic approach taken to synthesize many of the carbazoles. In addition, structure-activity relationships are also discussed. Chapter 4 discusses the synthetic approach and the structure-activity relationships of tetrahydro-(3- carbolines. Discovery of two compounds with submicromolar activity with no toxicity against the two mammalian lines invited further study, including the spectra of activity and metabolic stability. Pharmacokinetic studies were performed at different dosages and in various formulations to optimize the exposure in plasma. In vivo efficacy study was also conducted. Lastly, possible targets are briefly discussed. xv

1. INTRODUCTION 1.1 Tuberculosis 1.1.1 Background and Epidemiology Though it was much more prevalent in the past than it is today, tuberculosis (TB) is still considered a worldwide health threat [1, 2]. Despite the widespread use of chemotherapeutic agents and bacilli Calmette-Guerin (BCG) vaccination, an estimated one-third of the global population is infected with TB and two million lives are consumed annually by this pandemic disease [1, 3]. Mycobacterium tuberculosis, the etiological agent of TB, is an acid-fast bacillus which mainly affects the pulmonary system although extrapulmonary sites may also be involved. The bacteria are transmitted airborne (e.g. coughing and sneezing) and one only needs to inhale a few bacilli to be infected; however, only a small percentage of those infected will become sick. Immunocompromised individuals, particularly HIV-patients and geriatrics, are at higher risk of developing active TB [1]. Mycobacteria are characterized by their thick, lipid-rich cell wall, which serves as a permeability barrier. This renders them less susceptible to many classes of antimicrobial agents. A main component of the lipid is mycolic acid, which is present only in mycobacteria [4]. Hence, they are not identified by Gram stain but rather by acid-fast stain. M. tuberculosis is an obligate aerobic, rod-shaped bacillus of size ranging from 0.2-0.6 x 1-10 urn [5]. Upon encountering hypoxic environments in vivo, a small population of M, tuberculosis terminates growth and shifts down to a - 1 -

- 2 - dormant (or non-replicating) state [6, 7]. M. tuberculosis is slow-growing—its population doubling time is approximately 24 hours [8]. Its unique cell wall structure allows M. tuberculosis to lie dormant as a latent infection. In addition, it can readily grow inside macrophages, hiding from the host's immune system [9, 10]. In the case of latent TB, patients are asymptomatic until the host immune system has been compromised [11]. At an opportune time, M. tuberculosis revives and initiates the production of lesions and progressive TB [7]. 1.1.2 Tuberculosis: An Ongoing Challenge Several factors aggrandize the challenge of the TB therapy. One problem in TB control is the persistence of the bacilli, despite prolonged chemotherapy. A small fraction of the bacterial population, commonly known as the persisters, is genetically sensitive [6] yet does not respond to the standard anti-mycobacterial agents [7, 8]. A positive tuberculin skin test is usually the only evidence of latent TB infection. However, a negative test does not exclude TB. An estimated 25% of apparently immunocompetent persons will have a negative tuberculin skin test at the time of diagnosis of TB [13]. Another problem in eradication of the disease is the inaccuracy and a delay in detection of TB. Furthermore, multiple drug therapy is essential in treating TB as monotherapy increases the selection of drug-resistant mutants [14]. The TB patients must follow the multiple drug therapy for prolonged periods of time, which often leads to patient non compliance, thereby contributing to the antibiotic resistance of M. tuberculosis

- 3 - [9]. Phenotypic resistance in nonreplicating persisters and genetic mutation both pose a threat to the current TB therapy [2, 6]. In addition, the bacilli reside both intracellular^ and extracellularly. The intracellular location of the bacilli could render some drugs ineffective. Though many drugs can penetrate the necrotic tissues, they fail to effectively eradicate all bacilli in the lesions [2]. In addition, the BCG vaccine has shown inconsistent efficacy in clinical trials, further challenging the eradication of the disease [15]. Effective TB control is complicated by the spread of HIV, a virus which weakens the host immune system, thereby allowing latent TB to reactivate and/or making the patient more susceptible to TB infection [2, 16, 17]. Drug susceptibility testing (DST) is not routinely done in many under-developed nations, or it is performed only when there is a treatment failure or suspicion of multi-drug resistant M. tuberculosis (MDR-TB). DST of MDR-TB is not standardized; hence, reproducibility and validity of the data are often questionable [18]. 1.1.3 Methods to Identify M. tuberculosis Strains Three acid-fast smear methods are available to identify mycobacterial species from nonmycobacterial species: Kinyoun, Ziehl-Neelsen, and fluorochrome acid- fast smears [19]. The method of choice is Ziehl-Neelsen staining [5]. Although the acid-fast smear is an important tool in identifying mycobacteria, it alone is not adequate to distinguish M. tuberculosis from other mycobacterial species and must be followed by culture [19]. Conventional biochemical tests such as nitrate reductase and niacin production, growth rate, temperature of growth and

- 4 - morphology of colonies can all help in identifying M. tuberculosis strains [21]. M. tuberculosis is slow-growing and forms nonpigmented, rough colonies on an agar plate. All strains of M. tuberculosis are positive for the niacin production, although some strains of M. chelonae and M. simiae also give positive results. All strains of M. tuberculosis (and some strains of M. kansasii) reduce nitrate. An acid fast isolate which tests positive in both the niacin production and the nitrate reductions test may be classified as M. tuberculosis [19]. 1.1.4 Current Treatment The conventional drug regimen begins with two months of rifampin, isoniazid, pyrazinamide, and ethambutol (or streptomycin)—this is known as the intensive phase. The structures of the first-line tuberculosis chemotherapeutic agents are listed in Figure 1. This phase is followed by a continuation phase during which rifampin and isoniazid are taken for four months to target the bacteria that reside in the host, as well as to prevent relapse of the disease by the persisting bacilli [3, 15]. This can be highly effective if the drug regimen is adhered to appropriately. However, this complex and lengthy treatment makes patient compliance difficult, further contributing to the emergence of multiple drug- resistant (MDR) and extensively drug-resistant (XDR) TB which may require more than two years of therapy, thereby further increasing the costs and side effects [21, 22].

- 5 - Figure 1. Current first-line tuberculosis chemotherapeutic agents [2] O O CrV* fir* NH2 N H CO"*!^! V ^ V ^ r'N H Isoniazid Pyrazinamide CH3 HO- 0 H C -.A,HO. H HO OH Ethambutol UHO, A HQ J H N ~ H2N Streptomycin 1.1.5 Drug Resistance MDR-TB is defined as being resistant to at least isoniazid and rifampin, both of which are first-line anti-tuberculosis drugs. XDR-TB is defined as MDR plus resistance to any fluoroquinolone along with one of three injectable drugs— amikacin, kanamycin, or capreomycin—which are second-line anti-tuberculosis agents [15, 16]. Drug resistance can be broadly categorized into 4 types: spontaneous, acquired, amplified, and primary drug resistance. Mutations conferring resistance to anti-TB drugs occur spontaneously, albeit the frequency of intrinsic mutation is fairly low [23]. Such spontaneous mutations are escalated by inadequate treatment (such as insufficient dosage of the drugs, or not taking the drugs for the required duration of the treatment). Acquired drug resistance

- 6 - occurs when the spontaneously emergent M. tuberculosis mutants are selected for during treatment [24]. This commonly occurs as a result of inadequate chemotherapy. Amplified drug resistance occurs when drug-resistant strains develop resistance to other drugs during treatment. Primary drug resistance occurs when drug-resistant strains are transmitted to previously uninfected individuals [16]. However, one must keep in mind that these textbook classifications are not readily applicable in clinical settings as it is extremely difficult to accurately assess the level of resistance [24]. 1.1.6 Development of New Anti-TB Drugs No new anti-TB drug has been introduced into the market in approximately the past 40 years [2, 16]. It is imperative that new, efficacious drugs are introduced to shorten and/or to simplify the treatment as well as to combat the emerging drug-resistant strains of M. tuberculosis [11, 27]. Otherwise, MDR- and XDR-TB will comprise an increasing fraction of TB cases and eradication of the disease will be so much more challenging [3, 16]. Moreover, human immunodeficiency virus (HIV) increases the risk of reactivation of latent M. tuberculosis [25, 26]. Studies have shown that TB is a common cause of death in HIV-positive patients, especially in the developing countries where both HIV and TB are highly prevalent. Two classes of antiretroviral drugs have clinically relevant adverse drug interactions with rifampin (and its structural analogues), one of the most potent first line anti-TB drugs. Mainly, rifampin is a well known CYP3A inducer and many antiretroviral drugs are metabolized by the CYP3A enzymes [12, 26].

- 7 - Considering the MDR-TB and co-infection with AIDS, new drugs may have to be introduced in pairs or more. New drugs should be unrelated mechanistically and structurally to existing drugs [8]. 1.2 Carbazoles as Potential Leads for the Treatment of Tuberculosis As a part of our drug discovery program at the Institute for Tuberculosis Research (ITR), we routinely screen synthetic and natural product-derived compounds for anti-TB activity. Synthetic compounds are either purchased or prepared by the chemists in our group. Isolated natural products from plants, fungi, and bacteria are obtained through collaborations with various institutions as well as within our university. A collaborative effort within the framework of an International Cooperative Biodiversity Group (ICBG) [28, 29] led to the isolation of six anti-TB active carbazole alkaloids from the stem bark of Micromelum hirsutum Oliv. (Rutaceae). Of six isolated compounds, one was determined to be a novel structure. The novel structure, micromeline, was found to have a minimum inhibitory concentration (MIC) of 31.5 ug/mL against M. tuberculosis H37Rv [29]. Of major interest was the lack of mammalian cell cytotoxicity among the series, suggesting some degree of selective cytotoxicity. This prompted a literature search on the effect(s) of other known carbazole alkaloids on M, tuberculosis. Clausine K or clauszoline J (Figure 2), a natural product isolated independently from several sources [30, 31], was reported to have weak antituberculosis activity (MIC of 100 ug/mL against the h^Ra strain) [31].

- 8 - Figure 2. Naturally occurring carbazole alkaloids with anti-TB activity. H H micromeline clausine K (clauszoline J) Many carbazole alkaloids have been isolated from plants and microorganisms and tested for their biological effects. Their structural heterogeneity provides a great diversity of physicochemical and biological properties [32]. The literature search also revealed several synthetically derived carbazoles in clinical use, predominantly as anti-cancer and anti-viral agents (Figure 3) [32-35]. In addition, carprofen is a non-steroidal anti-inflammatory (NSAID) drug once used clinically but now restricted to use in geriatric dogs to relieve arthritic symptoms. Studies have shown a low frequency of adverse gastrointestinal effects in dogs [36]. Carazolol is a beta blocker of the B-adrenergic receptor [37] and carvedilol is a non-selective beta blocker used in the treatment of congestive heart failure [38, 39]. With the exception of the carbazole natural products published by Ma et al. [29], little information was available on the antimicrobial activities of this class of compounds. As this class of compounds represented a novel class of antimicrobial agents, various carbazoles were acquired from commercial and

- 9 - Figure 3. Carbazoles in clinical use. Carprofen Carazolol Carvedilol academic sources to gather as much SAR information as possible. Readily available starting materials and ease of synthesis enabled the quick establishment of a carbazole library. 1.3 Tetrahvdro-B-carbolines as Potential Leads for the Treatment of Tuberculosis A NovaCore library of 50K compounds, purchased from ChemBridge, was tested against the M. tuberculosis H37Rv strain. All compounds are compliant to Lipinski's "Rule of 5" which serves as a general rule of thumb to evaluate the drug-likeness of a chemical compound. The rule states that, in general, an orally active drug has no more than one violation of the following criteria: 1) molecular weight less than 500 daltons, 2) <5 hydrogen bond donors, 3) £10 hydrogen bond acceptors, and 4) octanol-water partition coefficient log P <5 [40]. Of nearly 50 different templates, one of the series of compounds—possessing a tetrahydro-P-carboline (THBC) framework—showed promising anti-TB activity (Table 1). A few interesting trends common to this class were observed: from a

- 10- biological activity viewpoint, no or minimal toxicity against a mammalian cell line was observed among those tested. This is a unique and promising feature as toxicity is a common deterrent in the progression of a compound along the development pipeline. From a chemistry viewpoint, all compounds had a chiral center at the C-1 position. Moreover, all of the compounds had substituents only at C-1 and C-2 positions. The majority of functional groups at C-1 were either a substituted aryl or an unsubstituted hexyl, whereas various substituents at C-2 were connected to the skeleton by either an alkyl or a carbonyl linker. Most of the compounds were highly lipophilic (clogP = >3). Due to a wide range of anti-TB activity (MIC values ranged from 8 to >30 uM), substituent diversity, and insufficient number of compounds with closely related substituents, only a crude generalization about the structure-activity relationships (SAR) was deduced. Three compounds—31394012, 60871805, and 73732723—had MICs below 30 uM. It seemed that N-2 showed greater sensitivity towards type of substitution than C-1. Compounds containing both pyridazinone (15427623) and dihydropyridazinone (29779099) moieties linked to the THBC nucleus were inactive at the highest concentration tested, regardless of the substituent at C-1. On the other hand, 31394012, which has a fused phenyl ring, was active. Though mechanism of action is only speculative since the target is not yet known, it is possible that (if a binding pocket exists) biaryl-aromaticity and/or hydrophobicity is preferred and that phthalazinone constitutes a pharmacophore. The length of linker chain may also influence potency (27609257). It can be

-11 - postulated that more active (MIC <1uM) compounds can be discovered by manipulating the substituent at C-1, while retaining the phthalazinone moiety. More closely related analogues were made to better deduce the influence of the C-1 substitutions. Some SAR can be drawn from oxo-chromene-carboxamide substituted compounds. Comparing 60871805 and 51582835, which share the same C-1 substituent, diaryl-aromaticity and/or hydrophobicity is again preferred and phenyl ring-fused chromenone may serve as yet another pharmacophore. The carbonyl linker may be essential as the alkyl linked chromeneone-substituted compounds were inactive (60871805 vs. 11762087). Perhaps of equal or greater importance is the position of the linker to the nucleus. It cannot be excluded that if the linker were appended at the 3-position of the chromen-4-one as opposed to the 2-position, then the compound may not exhibit anti-TB activity. A literature search revealed an abundance of both naturally and synthetically-derived compounds possessing the THBC skeleton. This framework is commonly found in biological tissues and fluids [41, 42], in fruit- and meat-derived products such as juices, jams, and sausages, suggestive of the relative safety of this class. These compounds arise from condensation of tryptophan and/or tryptamine and aldehydes during production, processing, and storage of food [40]. Moreover, this chemical entity exhibits broad and potent biological activities [5], including tadalafil, the active compound in Cialis® [43]. Using 31394012, 60871805, and 73732723 as a template, the substituent at N-2 of each compound was retained, and substituents at C-1 position were varied.

- 12- Table 1. In vitro activity against M. tuberculosis H37Rv of compounds derived from the NovaCore library. Cmpd 21109618 22338747 31394012 29779099 15427623 27609257 60871805 51582835 35217042 11762087 73732723 R1 jb J> J6 ^ ^O Fxx Fxi "6 i T 0 6 R2 0 O 1 Ni'NT° O N-V° 0 H ..rxr 0 0 0 -A 0 0 o -XH) MABA (MM) >30 >30 8.0 >30 >30 >30 4.2 >30 >30 >30 6.2 ClogP KOWWIN 4.32 5.71 3.78 3.80 3.28 4.12 4.52 4.12 5.38 4.82 4.04

- 13- Such analogues were synthesized and tested for in vitro activity. Active compounds with significant potency and little to no cytotoxicity were evaluated further to determine their potential as new antituberculosis leads. 1.4 Scope of the Study This thesis reports the syntheses and anti-tuberculosis activities of two classes of compounds: carbazole and 1,2,3,4-tetrahydro-(3-carboline. Carbazole derivatives were synthesized using one of two methods. In one method, tricarbonyliron- cyclohexadienylium salts and the arylamines underwent an electrophilic aromatic substitution to afford functionalized tricarbonyliron complexes, which upon oxidative cyclization, fully substituted carbazole derivatives were furnished. In another approach, the palladium(0)-catalyzed arnination of aryl halides by arylamines resulted in the formation of A/,A/-diarylamines, which upon palladium(ll)-catalyzed oxidative cyclization, fully functionalized carbazoles were provided [44]. For the synthesis of tetrahydro-P-carboline derivatives, the Pictet- Spengler reaction [45-47] was utilized, followed by amide bond formation [47, 48]. A systematic approach of chemical modification was made for each class of compounds in order to better comprehend the structure-activity relationships (SAR). All synthesized compounds were tested for MICs against the replicating H37Rv M. tuberculosis strain using the Microplate Alamar Blue Assay (MABA) [50] and those with reasonable in vitro potency (MIC < 30 uM) were subsequently evaluated for their activity against the non-replicating persistent (NRP)-/W.

Full document contains 256 pages
Abstract: Despite the widespread use of chemotherapeutic agents, an estimated one-third of the global population is infected with Mycobacterium tuberculosis and two million lives are consumed annually by this pandemic disease. In order to discover and develop new anti-tuberculosis drugs, two classes of compounds--carbazole and 1,2,3,4-tetrahydro-β-carboline--were synthesized and evaluated. Structure-activity relationships study of the 1,2,3,4-tetrahydro-β-carboline series revealed that substitutions at C-1 and N-2 are crucial for anti-tuberculosis activity. Discovery of two compounds with submicromolar activity and non-toxicity against the two mammalian lines tested invited further study, including spectrums of activity, minimum bactericidal concentration, metabolic stability, pharmacokinetics and in vivo efficacy. Studied revealed that the 1,2,3,4-tetrahydro-β-carboline series is selective towards the Mycobacterium tuberculosis complex. Different oral formulations of ITR406 indicated that PEG400 formulation provided the compound in solution, thereby resulting in greater oral bioavailability. Based on the findings, 1,2,3,4-tetrahydro-β-carboline may be potential anti-tuberculosis lead candidates.