Recent Advances in Patent Literature

EXPERT OPINION ON THERAPEUTIC PATENTS, 2016 http://dx.doi.org/10.1080/13543776.2017.1253681

REVIEW New antimycobacterial agents in the pre-clinical phase or beyond: recent advances in patent literature (2001–2016) Patricia Bento da Silva, Débora Leite Campos, Camila Maríngolo Ribeiro, Isabel Cristiane da Silva and Fernando Rogério Pavan Faculdade de Ciências Farmacêuticas, UNESP – Univ. Estadual Paulista, Campus Araraquara, Araraquara, São Paulo, Brazil

ABSTRACT ARTICLE HISTORY Introduction: Tuberculosis, an infectious disease, has caused more deaths worldwide than any other Received 14 July 2016 single infectious disease, killing more than 1.5 million people each year; equating to 4,100 deaths a day. Accepted 24 October 2016 In the past 60 years, no new drugs have been added to the first line regimen, in spite of the fact that KEYWORDS thousands of papers have been published on drugs against tuberculosis and hundreds of drugs have Drug patent; Mycobacterium received patents as new potential products. Thus, there is undoubtedly an urgent need for the tuberculosis; tuberculosis deployment of new effective drugs against tuberculosis. Areas covered: This review brings to the reader the opportunity to understand the chemical and biological characteristics of all patented anti-tuberculosis drugs in North America, Europe, Japan, and Russia. The 116 patents discussed here concern new molecules in the early or advanced phase of development in the last 16 years. Expert opinion: Of all 116 patents, only one developed drug, bedaquiline, is used, and then, only in specific cases. Another three drugs are in clinical studies. However, many other compounds, for which there are in vitro and in vivo studies, seem to fulfil the requisite criteria to be a new anti-tuberculosis agent. However, why are they not in use? Why were so many studies interrupted? Why is there no more news for many of these drugs?

1. Tuberculosis capreomycin [CAP], or kanamycin [KAN]) is designated extensively drug-resistant TB (XDR-TB) [6–9]. Tuberculosis (TB) is a chronic, potentially fatal contagious dis- The most effective first-line anti-TB drug, RIF, became avail- ease caused by species in the Mycobacterium tuberculosis able in the 1960s. Regimens for treating new cases of drug- (MTB) complex. The MTB complex comprises tubercle bacilli susceptible TB have an initial phase of 2 months, followed by a of eight distinct subgroups: M. tuberculosis, M. africanum, M. choice of several options for the continuation phase of either canettii, M. bovis, M. caprae, M. pinnipedii, M. microti, and M. 4 or 7 months, representing a total of 6–9 months of treat- mungi [1–4]. TB commonly affects the lungs but may affect ment. For the treatment of active TB, the first-line therapy is a almost any organ system, including the central nervous sys- cocktail of the drugs RIF, INH, ethambutol (ETB), and pyrazina- tem, gastrointestinal tract lymph nodes, genitourinary tract, mide (PZA). After 2 months, the number of drugs is reduced. bones, and liver. The formula used for a non-drug-resistant strain of TB is 2HRZE/4HR, meaning 2 months of INH, RIF, PZA, and ETB, 1.1. Epidemiology followed by 4 months of RIF and INH [10]. Treatment success rates of 85% or higher for new cases are regularly reported to According to a recent report from the World Health WHO. The treatment regimen for MDR-TB is longer and Organization (WHO) [5], TB has caused more deaths than any requires more expensive and more toxic drugs. For most other single infectious disease worldwide, killing more than patients with MDR-TB, the current regimens recommended 1.5 million people each year, equating to 4100 deaths a day. by WHO last 20 months, and treatment success rates are Nearly one-third of the world’s population is infected with much lower than those for drug-susceptible TB [5]. MTB. Each year, more than 9.6 million people develop active Despite the urgent situation, barely 20% of MTB-infected TB, with approximately 480,000 of them affected by multi- people currently receive the recommended treatment, and drug-resistant (MDR) MTB strains. MDR-TB is defined as resis- only about 10% are successfully treated [11]. Therefore, most tance in vitro to at least isoniazid (INH) and rifampicin (RIF); people with MDR-TB can transmit drug-resistant MTB to other MDR-TB with additional resistance to any fluoroquinolone members of their communities because they remain undiag- (such as ofloxacin [OFL] or moxifloxacin [MOX]) and any one nosed or are diagnosed but treated with ineffective first-line of the three second-line injectable agents (amikacin [AMI], drugs [12]. New TB drugs are now emerging from the pipeline,

CONTACT Fernando Rogério Pavan [email protected]; Patricia Bento da Silva [email protected] Tuberculosis Research Laboratory School of Pharmaceutical Science/UNESP Rodovia Araraquara-Jaú, km01, s/n, Campos Ville, Cep.: 14800-903 Araraquara, São Paulo, Brazil © 2016 Informa UK Limited, trading as Taylor & Francis Group 2 P. B. D. SILVA ET AL.

gram-positive bacteria, sharing relatively more orthologous Article highlights genes for energy production and conversion with gram-negative bacteria, in particular Escherichia coli and Pseudomonas ● Tuberculosis is an infectious disease that causes more deaths than any other single infectious disease worldwide, killing more than 1.5 aeruginosa, than with gram-positive bacteria. Thus, MTB is million people each year; neither gram-positive nor gram-negative but is instead ● Despite substantial research and hundreds of promising compounds described as acid-fast, because once it is stained, it resists patented as new potential products, no new drug has been added to the first line regimen in the past 60 years; decolorization with acidified organic solvents [13]. ● The long treatment period and therapeutic side effects of drugs currently in use lead to cessation of treatment and the potential for the selection of resistant mutants; 1.3. Transmission and symptoms ● The main mechanisms by which Mycobaterium tuberculosis (MTB) develops drug resistance are the following: 1. modifying the drug MTB is mainly transmitted by airborne aerosols from people target, 2. changing the pathway that activates or metabolizes the with active pulmonary TB through coughing and, occasionally, drug, 3. reducing permeability (e.g., of efflux pumps), and 4. inactivating enzymes; sneezing, shouting, or speaking. MTB is carried through air- ● This article covers the recent patent literature on tuberculosis drugs borne particles called droplet nuclei. Droplet nuclei are 1–5 in the pre-clinical phase (116 in total), presenting the chemical and μm in size and remain suspended in air for hours [19]. biological characteristics of all patented anti-tuberculosis drugs in North America, Europe, Japan, and Russia in the last 16 years; However, TB is not highly contagious compared to some ● Of all surveyed patents, only one developed drug, bedaquiline, is other infectious diseases. Only about one in three close con- used currently in tuberculosis therapy; however, its use is controver- tacts of a TB patient, and fewer than 15% of more remote sial and highly restricted. Another three substances are in the clinical phase. However, many other compounds, for which there are in vitro contacts, are likely to become infected. The probability of a and in vivo studies, seem to meet the requisite criteria to be con- person exposed to MTB becoming infected depends on the sidered promising new anti-tuberculosis agents. concentration of infectious droplet nuclei in the confined This box summarizes key points contained in the article. space, the time that has passed, air ventilation or recirculation, and general health status [20]. TB usually develops slowly. The symptoms might not begin until months or even years after infection, leading to delays in and combination regimens that include new compounds are seeking care and the transmission of the bacterium to others. – being tested in clinical trials. There are several TB vaccines in People with active TB can infect 10 15 other people through Phase I or Phase II trials. For the time being, however, a close contact over the course of a year. Without adequate treat- vaccine that is effective in preventing TB in adults remains ment, 45% of HIV-negative people with TB, on average, and elusive. practically all HIV-positive people with TB will die. The general symptoms of active TB include lack of appetite and weight loss, fever, night sweats, or fatigue [21]. TB can also cause symptoms that are dependent on the part of the body infected. Most 1.2. Bacteriology infections affect the lungs, which can result in chronic cough MTB is an obligate intracellular, nonmotile pathogen. It is an with blood-containing sputum for more than 3 weeks [20]. aerobic, slim, nonencapsulated, non-spore-forming bacillus Less commonly, TB infections affect other parts of the body, curved rod (0.2–0.4 μm×2–10 μm), with a complex and which is known as extrapulmonary TB. Extrapulmonary sites of thick cell wall rich in lipids such as mycolic acids (long-chain infection commonly include lymph nodes, pleura, and osteoar- fatty acids that surround the bacterial cytoplasmic membrane). ticular areas, although any organ can be involved [22]. The mycolic acids, for which mycobacteria are named, make Extrapulmonary TB is more common in people with a weakened up more than 60% of the total cell wall mass. This pathogen immune system, such as those with HIV coinfections [23]. grows most successfully in tissues with high oxygen content, Moreover, in some cases, the infection causes no symp- such as lungs [13,14]. toms, which is known as latent TB. Latent TB infection (LTBI) The mycobacterial cell wall has a peculiar characteristic: it is is defined as evidence of an immune response to MTB anti- impermeable to a number of compounds, a feature that is gens without evidence of clinically active TB [24,25]. The thought to be advantageous in stressful conditions of osmotic importance of this enormous reservoir of potential disease shock or desiccation as well as to contribute to their resistance and the interactions of MTB with the human host that mediate to many drugs [15]. This characteristic also allows the bacter- clinical latency are largely unknown [13]. ium to grow inside of macrophages, effectively hiding it from the host’s immune system [14,16]. 2. Molecular mechanisms of resistance Despite the fact that mycobacteria are considered gram- positive bacteria structurally, because they do not exhibit a The main mechanisms of MTB drug resistance are the follow- true outer membrane and present a thick layer of peptidogly- ing: (1) modifying the drug target, (2) changing the pathway can, they also share characteristics of gram-negative organ- that activates or metabolizes the drug, (3) reducing perme- isms, such as not retaining Gram stain, having porins in their ability (e.g. of efflux pumps), and (4) inactivating enzymes [26]. outer lipid layer, and displaying a space that may serve a Molecular modification of a target occurs because of similar function as the gram-negative periplasm [17]. A phylo- changes in the bacterial DNA. In the following sections, we genetic study published by Fu and Fu-Liu [18] suggests that describe the mechanism of action and the main mutations MTB is indeed more closely related to gram-negative than to associated with first-line drug resistance. EXPERT OPINION ON THERAPEUTIC PATENTS 3

RIF shows bactericidal effects on both replicating bacteria Bacilli can have a compensatory mutation at promoter region and bacteria with slow metabolism [27]. RIF is an inhibitor of for ahpC in KatG-deficient strains because KatG deficiency leads the β-subunit of the DNA-dependent RNA polymerase; thus, to an imbalance in reactive oxygen species, and this mutation transcription, and consequently protein synthesis, is impaired reduces INH activation. ahpC mutations are less frequent in [28]. RIF resistance is caused by mutations in the rpoB gene phenotypically resistant strains than katG and inhA mutations; that encodes the β-subunit of the RNA polymerase. These however, these mutations can appear together [33–35]. mutations lead to conformational changes in the protein and ETB inhibits the arabinosyl transferase enzyme because ETB is reduce the affinity of the drug to the protein. The main muta- an arabinose analog. Arabinosyl transferase (EmbB) is encoded tions identified are in codon positions 531 and 526: serine by the embB gene, which is in embCAB operon [36,37]. ETB (Ser) is substituted by leucine (Leu), glycine (Gly), or trypto- resistance was elucidated when mutations at position 306 of phan (Trp), and histidine (His) is substituted by tyrosine (Tyr), the embB gene were described. The replacement of Met (ATG) asparagine (Asp), arginine (Arg), proline (Pro), or glutamine at position 306 for Leu (CTG), valine (Val) (GTG), or Ile (ATA, ATT, (Gln). In addition, mutations in positions 533, 522, 518, 516, and ATC) has been observed. These changes alter the drug– 513, and 511 and other sites can cause RIF resistance [29]. protein interaction causing ETB resistance [38]. RIF belongs to the class of rifamycins. Rifabutin, rifapentine, PZA has bactericidal activity against bacteria in a dormant and rifalazil also belong to this class, and they may be used for state inside of phagolysosomes and in other microenviron- the treatment of TB. For strains with mutations at codon 531 ments with an acid pH. Among the first-line drugs, PZA and and 526, cross-resistance for all the rifamycins has been RIF are the two best drugs for sterilization of the infected observed. However, mutations at codon 516 result in resis- lungs [27]. PZA requires activation by the enzyme pyrazina- tance to RIF and rifapentine, but not to rifabutin and rifala- mide (PncA, encoded by pncA), which releases the acid pyr- zin [30]. azinoic (POA) [39]. INH is dependent on activation by the catalase peroxidase The POA is a weak acid that carries a proton in acid pH (pH enzyme (Enzyme KatG, encoded by katG), which eliminates 5.5). Therefore, POA interrupts the membrane potential, which is isonicotinic hidrazyl and isonicotinic acyl radicals. These INH- required for the transport of many nutrients such as amino acids derived radicals form adducts with nicotinamide adenine and nitrogenous bases. Protein synthesis is indirectly reduced dinucleotide (NAD). These adducts (INH-derived radicals linked because of POA action on the plasmatic membrane [40]. to NAD) bind and inhibit the enzyme InhA (encoded by inhA). Mutations in PncA can reduce the activation of PZA in POA, InhA is an NADH-dependent enoyl-acyl carrier protein reduc- which leads to drug resistance. Mutations that have been

tase enzyme that is responsible for mycolic acid synthesis, implicated as responsible for PZA resistance are cysteine(Cys) because it elongates fatty acids. Mycolic acids are the major 138Ser, Gln141Pro, Asp63His, and deletions at the 162 and 288 lipids in the mycobacterial envelope [31]. When the drug INH positions [41]. is metabolized to form adducts and binds to InhA, the mycolic A new PZA target was identified in PZA-resistant clinical acid synthesis is prejudiced; therefore, the bacterial survival isolates without a pncA mutation. POA binds ribosomal pro- and the growth are harmed also. tein S1 (RpsA); PZA inhibits the protein responsible for e The most frequent cause of resistance to INH is katG315 ribosome release after the translation in nonreplicating bacilli. mutations. Mutations in this gene are present in 64% of all PZA/POA has activity against dormant bacteria, especially phenotypically resistant cases. Specifically, a serine codon because of its mechanism of action. RpsA overexpression (AGC) is exchanged for a threonine (Thr; ACC, ACA, ACG, or confers increased PZA resistance, which has been observed ACT). This exchange for Thr is the most frequent substitution, in naturally resistant M. smegmatis [42]. found in 93.4% of isolates, followed by Asp (AAC), isoleucine POA binds to RpsA with hydrogen bonds and hydrophobic (Ile) (ATC), Arg (AGA, CGC, or AGG), or Gly (GGC). Mutations at interactions. These links cause a conformational change that positions 306, 316, 309, 311, 299, 275, 328, 155, and 110 have reduces tmRNA affinity. Mutations in the C-terminus of RpsA also been reported as responsible for INH resistance [32]. (Lysine(Lys)303, Phenylalanine(Phe)307, Phe310, and Arg357) The second most frequent cause of INH resistance an inhA lead to loss of POA activity because this is where POA binds [43]. mutation. These changes can be in the promoter or coding In addition to the aforementioned mutations, mutations regions of the gene. Mutations in the promoter region lead to have been described in the panD gene in PZA-resistant strains overexpression of InhA, while mutations in the coding region without rpsA and pncA mutations. The panD gene encodes the alter InhA so that the drug can no longer bind. Mutations in this aspartate decarboxylase enzyme, which synthesizes β-alanine gene, present in 19% of phenotypically resistant clinical isolates, from aspartate. Overexpression of PanD increases POA resis- can be in positions −15 (most frequent), −8, −47, or −17 (pro- tance. The supposition is that POA blocks PanD and inhibits moter) or in positions 94, 21, 194, 3, 258, or 190 (coding pantothenate or β-alanine synthesis, impairing bacterial meta- region) [32]. bolism. Pantothenate or β-alanine supplementation can res- The ahpC-oxyR intergenic region is the promoter region for cue PZA susceptibility [44,45]. ahpC. Mutations in this region have been reported to be Another prominent mechanism of resistance is altered responsible for INH resistance. ahpC encodes the enzyme alkyl efflux pumps. As mentioned, reducing the permeability of hydroperoxide reductase (AhpC). Mutations in the promoter drugs is a way to reduce their activity. Low permeability may region induce overexpression of AhpC, resulting in resistance. be a result of the high lipophilicity of the mycobacterial cell 4 P. B. D. SILVA ET AL.

wall or specific efflux mechanisms, which can be further antibiotics against mycobacteria or restoring drug susceptibility. divided into those that increase the activity of existing efflux The combination of an EPI anti-TB therapy has been shown to pumps in bacteria or those that increase expression of the be a promising strategy to reduce the emergence of resistant genes that encode them [26,46,47]. strains [50,51]. Efflux pumps are transmembrane proteins that can mediate Molecules with EPI potential include protonophores (carbo- antibiotic resistance. Some classes of efflux pump not only nyl cyanide m-chlorophenyl hydrazone (CCCP), 2,4-dinitrophe- extrude drugs but also have a role in bacterial pathogenicity nol (DNP), and valinomycin); Ca2+ channel blockers (verapamil (colonization and survival of bacteria in a host). The five and its metabolite norverapamil and phenothiazines [chlor- families of efflux pump proteins that are associated with promazine, piperidine and thioridazine]); and plant-derived MDR are the ATP-binding cassette (ABC) superfamily, the efflux pump inhibitors (piperine, reserpine, and berberine). major facilitator superfamily (MFS), the multidrug and toxic Different types of efflux pump inhibitors exhibit different compound extrusion (MATE) family, the small multidrug resis- modes of action that can be either broad or specific to one tance (SMR) family, and the resistance nodulation division class of efflux pumps [50] (RND) family. The difference between families is the substrate used to make the extrusion compound of interest, which may be protons, sodium, or dephosporylated ATP [48]. 3. Patent evaluation In bacteria with increased activity or expression of efflux 3.1. Halogenated agents pumps, the minimum inhibitory concentration (MIC) of antimicrobial agents is typically 2–8 times higher than that in Thioacetazone (also known as thiacetazone, thiosemicarba- susceptible strains. Moreover, with higher activity or expres- zone, benzothiozane, or amithiozone) has been used for TB sion of efflux pumps, mycobacteria may become simulta- treatment since the 1960s. Currently, this drug is not included neously resistant to more than one drug, even of different as a first-line treatment recommended by the WHO; however, classes. However, when resistance to antimicrobials is a result it is used in specific cases in which there is resistance to other of mutations at drug target or at activator enzyme, the MIC is drugs [52]. much higher than that in susceptible strains (100 times higher In 1944, para-aminosalicylic acid was introduced as a treat- e.g.), and resistance may be against only a drug or a specific ment for TB. It is effective against MTB and is currently used to class of drugs [48]. treat MDR-TB [53]. Increased expression of efflux pumps may result from a The University of the Sciences in Philadelphia obtained, in

mutation in a global regulator gene (transcription activator) or 2002, a patent for the synthesis and antimycobacterial activity mutation in the promoter region of the efflux pump [48]. The of halogenated compounds derived from thiocetazone and increased expression of efflux pumps may also be an adaptation para-aminosalicylic acid. The halogenated compounds that for survival of the microorganism, as efflux pump gene expres- characterized this invention are present in one of the two sion can be induced in the presence of an antibiotic [49]. structures (I and IV) presented below (Figure 1) as a pharma- Molecules that inhibit the action of efflux pumps (efflux ceutical salt and showed good anti-TB activity [54]. pump inhibitor [EPI]) have been evaluated for their ability to The literature shows that exchanging the thiosemicarba- reduce the MICs of antibiotics, potentiating the effects of zone group for a semicarbazone, hydrazine, or oxime reduces

X1 H3C H N NN H X2 NH2

Y Structure I

X1 is a halogen, X2 is a halogen or hydrogen, and Y is sulfur or oxygen.

X2 OH

H2N COOH

X1 is a halogen and X2 is a halogen or hydrogen Structure IV

Figure 1. Structures of halogenated derivatives of thioacetazone and para-aminosalycilic acid. EXPERT OPINION ON THERAPEUTIC PATENTS 5

the activity of the compounds, and that replacing the primary 0.2 µM) and M. kansaii, demonstrating a narrow spectrum of amines (R‘-NH2) of the thiosemicarbazone (=N-NH-C(S)-NH2) activity. The mechanism of action is considered similar to that of group with one or two alkyl groups or a sulfur (S) atom with thiacetazone that act against HadABC and, as thiacetazone, PCZ oxygen (O) or nitrogen (N) also gives eliminates activity is a prodrug that needs to be activated by EthA [57]. against the microorganism. The literature also shows that para-aminosalicylic acid loses activity when (1) the primary amine is exchanged for a hydroxyl (HO–), alkoxy (R–O–), ter- 3.2. Thiostrepton tiary amines (R-NR’R’), or amides (R-CO-NR’R”); (2) the hydroxyl In 1955, thiostrepton was described, and, in 1958, it was (HO–) group is cloaked with ester (R’-C(=O)OR”) or ether (R-O- patented. Thiostrepton is a cyclic oligopeptide antibiotic that R’); (3) the hydroxyl (HO–) group is replaced by a thiol (–C–SH is densely packed with thiazoles and other structurally intri- or R–SH) or amino (R-NR’R”) group, or (4) the carboxylic acid guing moieties and is derived from strains of streptomycetes (RC(=O)OH) group is transformed into alkyl esters (R’C=OOR), such as Streptomyces azureus and S. laurentii [58]. amidines (RnE(=NR)NR2), amides (R-CO-NR’R”), or nitrates – Thiostrepton was patented as an anti-MTB agent by (NO )[54]. 3 Vermeulen and Wu in 2004 [59]. According to the authors, a Halogenated derivatives of structures I and/or IV were also mycobacterial infection can be treated or prevented with synthesized, namely 4-acetamido-3-fluorobenzaldehyde thio- thiostrepton. The compound was evaluated against MTB strain semicarbazone (16), 4-acetamido-2-fluorobenzaldehyde thio- H Ra, and it showed growth inhibition with an IC (drug semicarbazone (17), 4-acetamido-3-chlorobenzaldehyde 37 99 concentration inhibiting 99% of microorganism’s activity) in thiosemicarbazone (25), 4-acetamido-3-bromobenzaldehyde the range of 30–100 nM. In an intracellular assay that con- thiosemicarbazone (29), 4-acetamido-3-iodobenzaldehyde sisted of MTB H Ra-infected murine alveolar macrophages thiosemicarbazone (34), 4-acetamido-5-fluorosalicylic acid 37 (MH-S cells), the antibiotic showed an IC (drug concentration methyl ester (11), and 4-amino-5-fluorosalicylic acid (12, 54). 50 inhibiting 50% of microorganism’s activity) value of 30 nM. The derivative 4-acetamido-3-fluorobenzaldehyde thiose- The antibiotic may be administered through different routes: micarbazone (16) showed an MIC against MTB H Rv (ATCC 37 parenteral, intravenous, intrapulmonary, oral, or mucoadhe- 27294) of 0.2 µg/mL and the toxicity (IC )inVerocellswas 50 sion, with doses of 75–750, 150–500, or 250–400 mg/day [59]. >62.5 µg/mL; consequently, the selectivity index (SI) was >312.5, showing that the compound was selective. The in vitro efficacy of compound 16 was tested in a macrophage 3.3. Pharmaceutical compositions

model infected with MTB Erdman (ATCC 35801), and an effective concentration was shown to reduce in the col- An invention patented by Stockel in 2006 [60] consisted of

ony-forming units by 90% (EC90)and99%(EC99)in7days, metathesis and acid–base reactions in which the bioactive which is equal to a 1.7-fold and >3.2-fold reduction, respec- complex formed is composed of two active drugs, one cation tively. The sample was tested against MTB strains resistant and other anion. Metathesis reactions occur between a stan- to INH (ATCC 35822), RIF (ATCC 35838), ETB, KAN, ciproflox- dard amino drug and a sodium-halogenated molecule in an acin (CIP), and thiocetazone (THI), and the compound aqueous or alcoholic aqueous medium. The bioactive complex showed excellent activity, with MIC values of ≤0.1, ≤0.1, may also be formed by an acid–base reaction that takes place 0.2, ≤0.1, ≤0.1, and ≤0.1 µg/mL, respectively. No cross-resis- after protonation of the reagents. tance was observed between the compound and INH, RIF, The cationic antimycobacterial portion of the bioactive ETB, KAN, or CIP. By Microplate Alamar Blue assay (MABA), complexes may be guanidine, quinoline, pyridinol, amidine, the MIC of compound 16 showed that it was 20 times more imidazoline, bisbenzimidazoles, or N,Nʹ-substituted biguanides active against MTB than THI and that it is an anti-TB derived from hydroxylamines. The anionic portion may be agent [54]. phenolates, mercaptides, carboxylates, sulfonates, phosphates, Other halogenated derivatives of THI (17, 25, 29, and 34) phosphonates, phosphinates, 2-hydroxyl-1,4-naphthoqui- also showed good anti-TB activity and are considered good nones, bisphosphonates, or similar complexes [60]. new antimycobacterial agents. Of the derivatives of para-ami- Bioactive complexes are formed by acid–base reactions nosalicylic acid, only compound 12 was active against a sus- between a standard amino drug and a drug that donates ceptible MTB strain, and others tests were only conducted on protons, e.g. carboxylic acids, alkyl or aryl sulfonic acids, or this sample. The results showed that compound 12 is a possi- alkyl or aryl phosphoric, phosphorous, phosphonic, bispho- ble anti-TB agent. The halogenated derivatives described in sphonic, or phosphinic acids [60]. this patent may be used orally [54]. The complexes synthesized were chlorhexidine distearate, iso- In a reaction between 4-pyridine aldehyde (excess) with niazid dodecyl benzene sulfonate, N-cocoylamine-L-arginine ethyl thiosemicarbazide in water–ethanol solutions of 48–55% and ester triclosanate, chlorhexidine dithymol, chlorhexidine di-2-mer- perchloric acid at 80–85ºC, a new thiosemicarbazone was captobenzthiazole, chlorhexidine dilaurate, chlorhexidine di [4- obtained that was patented in 2004 by JSC Pharmasyntez and amino-1-hydroxybutylidene] bis-phosphonate, chlorhexidine named 4-thioureido-iminomethylpyridinium per-chlorate beta cyclodextrin sulfobutyl ether, chlorhexidine di-ortho phenyl (Perchlozone®,PCZ)[55,56]. A thiosemicarbazone compound, phenol, poly (hexamethylene) biguanide stearate, and chlorhex- perchlozone, was approved for MDR-TB treatment in 2012 in idine distearate. The MIC values of the complexes were deter-

Russia. Its activity against MTB has been recognized since the mined against MTB H37Rv by the microdilution technique, and the 1950s. In addition, its activity is directed only against MTB (MIC50 resulting values ranged between 6.25 and 0.78 µg/mL [60]. 6 P. B. D. SILVA ET AL.

3.4. Benzothiazin derivatives infections. These derivatives were patented in 2012 by Ecole Polytechnique Federale de Lausanne [63]. Benzothiazin is a bicyclic heterocycle containing a benzene In 2013, the University of Queensland St. Lucia patented ring fused with a thiazine ring. This class of compounds has eight other novel benzothiazinone derivatives as anti-TB generated several patents because of its high antimycobacter- agents. The most effective compounds in this class were 2- ial activity. The Leib Niz Institute for Natural Product Research [(2S)-2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8-yl]-8-nitro-6-(tri- and Infection Biology E.V. Hans-Knöll-Institut patented eleven fluoromethyl)-4H-1,3-benzothia-zin-4-one (BTZ 043) and 2-[4- formula (I) novel benzothiazin derivatives in 2007 (Figure 2) for cyclohexylmethyl)piperazin-1-yl]-8-nitro-6-(trifluoromethyl)- use as antibacterial agents and especially for TB. Wherein R1 4 H-1,3-benzothiazin-4-one (PBTZ 169) (patented by and R2 are, independently from each other, NO , CN, CONR7R8, 2 Innovative Medicines for TB, Lausanne, Switzerland). BTZ 043 COOR9, CHO, halogen, NR7R8,SONR7R8,SR9, OCF , or mono-, 2 3 was used to generate PBTZ 169, which is simpler to synthe- di, or trifluoromethyl; R3 and R4 are, independently each from size because it does not have chiral centers [64]. other, H, a saturated or unsaturated, linear or branched ali- To inhibit the enzyme DprE1, BTZ 043 and its analog PBTZ phatic radical having 1–7 chain members, a cycloalkyl having 169, as prodrugs, need to be activated by bioreduction of the 3–6 carbon atoms, benzyl, SR9,orOR9;orR5 and R6 are, nitro group by mycobacterial enzymes. Because of this, they independently from each other, a saturated or unsaturated, present safety concerns for human use. DprE1 plays an impor- halogenated or unhalogenated, linear or branched aliphatic tant role in the biosynthesis of arabinan, a precursor to the cell radical having 1–8 chain members, a cycloalkyl having 3–6 wall. Preclinical studies show that BTZ is a bactericidal com- carbon atoms, phenyl, or R5 and R6 together representing a pound that has exceptional activity against replicating MTB bivalent radical-(CR9 )m-, or R5 and R6 together representing 2 H Rv at a concentration of 1 ng/mL and against latent TB at bivalent radicals of formula (II) or (III) (Figure 2), wherein m is 37 <10 ng/mL, in addition to its activity against MDR-TB. 1–4 or represents bivalent radicals of saturated or unsaturated Resistance is attributed to mutations in the Cys387 residue mono- or polyheterocycles with heteroatoms N, S, or O and codon [65–67]. substituted by (R10)x, wherein x is 1–4[61]. In 2009, the same applicant patented 10 new compounds, also derived from benzothiazin: 2-[4-(4-chlorophenyl)piperazin-1-yl]-6,8-dinitro-4H-1,3-benzothiazin-4-one, 2- 3.5. Avermectins and milbemycins as antimycobacterial [4-(5-chloro-2-methylphenyl)piperazin-1-yl]-8-nitro-6-(trifluo agents romethyl)-4H-1,3-benzothiazin-4-one, 8-nitro-6-(trifluorom

The macrocyclic lactone derivatives of avermectins (ivermec- ethyl)-2-{4-[3-trifluoromethyl)phenyl]piperazin-1-yl}-4H-1, 3 tin, selamectin, and doramectin) and milbemycins (moxidec- -benzothiazin-4-one, 2-[benzyl(ethyl)amino]-8-nitro-6-(triflu tin), products obtained from Streptomyces fermentation, oromethyl)-4H-1,3-benzothiazin-4-one, 2-[benzyl(methyl)am began to be used as antihelmintics in the 1980s. These aver- ino]-8-nitro-6-(trifluoromethyl)-4H-1, 3-benzothiazin-4-one, mectins and milbemycins are derived from a 16-membered 2-[4-(2-fluorophenyl)piperazin-1-yl]-8-nitro-6-(trifluorometh macrocyclic lactone, and the structures of the compounds yl)-4H-1,3-benzothiazin-4-one, 2-(4-benzylpiperazin-1-yl)-8- from these two families are very similar; however, avermectin nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one, 2-(ben- has a substituent at position 13, the bisoleandrosyloxy, while zylamino)-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4- milbemycin has no substituent [68]. one, 2-[methyl[1 R)-1-phenylethyl]amino}-8-nitro-6-(trifluor- Thompson, Ramon-Garcia, and Lim patented new therapeutic omethyl)-4H-1,3-benzothiazin-4-one, and 2-[benzyl(methyl) regimens for mycobacterial infections in 2014, using the antihel- amino-6-chloro-8-nitro-4H-1,3-benzothiazin-4-one [62]. minthic agents avermectins and milbemycins, not only for sensi- Two new 2-piperazin-1-yl-4H-1,3-benzothiazin-4-one deri- tive strains but also for MDR, extensively drug-resistant (XDR), and vatives were synthesized and used to treat mammalian MTB totally drug-resistant (TDR) strains [69]. Mycobacterial infections, as described in the patent, may be caused by slowly or rapidly grow- ing mycobacteria and sensitive or resistant strains of MTB, includ- 5 R ing MDR, XDR-, and TDR-TB. Infections may be caused by M. bovis, O M.africanum,M.canetti,M.microti,M.leprae,M.avium,M.avium- R1 R6 O intracellulare, M. kansasii, M. fortuitum, M. chelonae, M. marinum, M. R3 S N ulcerans,andM. leprae. The subject to be treated may be any (I) mammal other than a human, e.g. M. avium infections in cattle N causing Johne’sdisease[69]. Ivermectin, selamectin, and moxidectin were evaluated R2 R4 O against a panel of 27 clinical isolates, for sensitive, MDR, and XDR strains from different locations and with different resistance profiles for first-line and second-line drugs. Except for three MDR (CI15072, CI12081, and BC-MDR2) and two susceptible (BC-DS4 10 and BC-DS5) strains that were less sensitive to ivermectin, with R m (II) O (III) MIC90 >24μg/mL, the other strains showed MIC50 s<8µg/mL. With the aim of evaluating whether ivermectin, selamectin, and Figure 2. General formulae for benzothiazinone derivatives. moxidectin are bactericidal or bacteriostatic, the authors EXPERT OPINION ON THERAPEUTIC PATENTS 7

performed a time-kill kinetics experiment using MTB strain H37Rv by nitrogen and whose basic structure is C9H7 N. The diaryl- and drugs at different concentrations, and they constructed 21- quinoline TMC 207 was developed and patented by Janssen day kill curves. In this experiment, the avermectins were Pharmaceutical in 2004 [70], and it stood out from a library of observed to be bactericidal, thus reduced the initial bacterial 229 novel compounds of the general formula (Figure 3). viability in six adjustments of greatness. An independent experi- R1 is bromo, p = 1, R2 is alkyloxy, R3 is optionally substituted ment was conducted in similar, but not identical conditions to naphthyl or phenyl, q = 1, R4, and R5 are each a hydrogen, 6 that of the M. tuberculosis H37Rv and mc25857 (resistant) strains methyl, or ethyl, R is hydrogen, r is equal to 0 or 1, R7 is with the avermectins at a concentration of 20 µg/mL, and bac- hydrogen, R8 is hydrogen or alkyl, R9 is oxo; or R8 and R9 tericidal activity was observed for both strains [69]. together form the radical =N-CH=CH-. For TMC 207, the gen- Furthermore, in vitro pharmacodynamic parameters were eval- eral formula is Ia, where, R1 is bromo, R2 is alkyloxy, R3 is uated by the construction of dose–response curves. The kill naphthyl, R4 and R5 are methyls, R6 is hydrogen, and r and p kinetics of the avermectins and milbemycins were observed to are equal to 1. The IUPAC name is (1R,2S)-1-(6-bromo-2-meth- be dependent on the time of exposure to MTB, and the bactericidal oxyquinolin-3-yl)-4-(dimethylamino)-2-(naphthalen-1-yl)-1- effects of avermectins and milbemycins were found to be compar- phenylbutan-2-ol. Its structure has two stereogenic centers (R, able or better than RIF, a standard first-line drug. A pharmacoki- S), and it was prepared by successive steps. netics analysis and in vivo assays in an acute TB mouse model In 2005, 2006, and 2008, more patents were issued for TMC- showed that after nine consecutive days of oral treatment, MTB 207, promoting its use as a compound to treat drug-resistant was reduced by 1 log10 CFU in the lungs when compared to that of mycobacterial disease and latent TB, in addition to the prepara- the control [69]. tion of an enantiomer and fumarate salt of bedaquiline [71–74]. The avermectin that showed the highest activity against The compound, approved in 2012, inhibits ATP synthase, MTB H37Rv, CDC 1551, and Erdman 107 in this study was depleting ATP in cells and therefore acting on dormant MTB. It selemectin. Selemectin showed in vivo activity against MTB shows low MIC values of 0.03 and 0.12 µg/mL in sensitive and H37Rv at doses of 10 and 50 mg/kg and in vitro activity against resistant isolates, respectively. Known resistance mechanisms M. avium, M. chelonae, M. gordonae, M. intracellulare, M. micro- against this compound result from mutations in the atpE gene, tti, and M. phlei. Avermectin may be administered orally [69]. which modify the binding site or efflux pumps [67,75]. Bedaquiline, when combined with verapamil, showed enhanced bactericidal activity in a mouse model. In addition, 3.6. Quinoline derivatives combination with verapamil prevented the selection of beda- – Bedaquiline (TMC 207) is a quinoline derivative that belongs to quiline-resistant mutants and reduced by 8 16-fold the MICs a class of organic compounds characterized by two fused of clofazimine and bedaquiline in clinical isolates [51,76]. An hexagonal rings, wherein one carbon in position 1 is replaced adverse effect of bedaquiline is prolongation of the QT interval

6 (R )p

1 7 R4 (R )p R OH N (CH2)q (Ia) R5 NR2 R3

6 (R )p

1 7 R4 (R )p R OH N (CH2)q (Ib) R5 R3 N R9

R8 R1 is bromo, p = 1, R2 is alkyloxy, R3 is optionally substituted naphthyl or phenyl, q = 1, R4, and R5 are each a hydrogen, methyl, or ethyl, R6 is hydrogen, r is equal to 0 or 1, R7 is hydrogen, R8 is hydrogen or alkyl, R9 is oxo; or R8 and R9 together form the radical = N-CH=CH-.

Figure 3. General formula (Ia) or general formula (Ib) for novel substituted quinoline derivatives. 8 P. B. D. SILVA ET AL. in the heart’s electrical cycle. The QT interval, a measure of the A Phase III clinical trial (NCT00685360) with delamanid, in time from onset of the Q wave to the end of the T wave, association with the optimized background regimen (OBR), represents the electrical depolarization and repolarization of was tested against pulmonary MDR-TB and showed good the left and right ventricles, respectively, and can be measured results in sputum culture conversion at 2 months: 45.4% of in an echocardiogram [52]. If verapamil is administered with the total patients treated with 100 mg of delamanid twice a bedaquiline, this adverse effect is reduced because verapamil day with the OBR showed sputum culture conversion, com- increases the PR interval in the cardiac conduction system. pared to only 29.6% of patients treated with placebo and the Thus, in addition to improving the activity of several drugs, OBR [85,86]. verapamil may also have a cardioprotective effect against A 477-compound library was prepared and evaluated bedaquiline [77,78]. against several bacteria, including MTB, but the (6-chloro-2- There are various Phase III clinical trials expected to be ethyl-N-(4-(4-(4-(trifluoromethoxy)phenyl)piperidin-1-yl)ben- implemented in coming years, and bedaquiline is one of the zyl) imidazo[1,2-a]pyridine-3-carboxamide) compound, Q203, drugs to be tested. These trials will be performed to identify showed the highest activity against TB. This compound was better therapeutic strategies for MDR and XDR-TB. Some of patented in May 2011 with other molecules that have a gen- these trials will test various compounds in conjunction with eral formula, and different structures could be formed, bedaquiline, including linezolid, pretomanid, moxifloxacin, depending on the substituent [87]. Q203 showed a lower and levofloxacin. Its effect in association with delamanid has MIC value (2.7 nM) than bedaquiline, based on a test by not been tested and is not recommended by WHO or its Qurient Therapeutics (Gyeonggi-do, Korea). This imidazopiri- manufacturers, but there is an ongoing US National Institutes dine amide inhibits the growth of MTB by targeting the of Health-sponsored study (ACTG 5343) that will answer some respiratory cytochrome bc1 complex – specifically cytochrome questions about this association [79]. b subunit (QcrB) – showing potential against latent, intracellular, and multidrug-resistant TB [67]. In 2012, AstraZeneca and the Global Alliance for TB Drug Development synthesized and evaluated the antimycobacter- 3.7. Imidazole derivatives ial activity of more than 1000 analogs of PA-824 with the Imidazole (Figure 4) is a heterocyclic aromatic compound that objective of increasing the aqueous solubility and maintain may have various substituents and that is one building block stability and efficiency of the compound by substituting one of histidine and histamine. It was first synthesized by Heinrich or both phenyl groups with a pyridine, pyridazine, pyrazine, or

Debus in 1858, but other imidazole derivatives were discov- pyrimidine. In the study, (6S)-2-nitro-6-[[6-[4-(trifluoro- ered in the 1840s [80]. methoxy)phenyl]pyridine-3-yl]methoxy]6,7-dihydro-5H-imida- Delamanid,or(2R)-2-methyl-6-nitro-2-[(4-{4-[4-(trifluoro- zol[2,1-b][1,3]oxazine (TBA-354) was the most promising of methoxy)phenoxy]piperidin-1- yl}phenoxy)methyl]-2,3dihy- the compounds, with activity against both replicating and droimidazo[2,1-b]oxazole, formerly known as OPC-67683, is a latent MTB and showing MIC values of 0.006 and 0.27 µM, nitroimidazo-oxazole developed by Otsuka Japanese respectively. In addition, it acts against MDR-TB [84,88]. Laboratory, patented in 2004, and published in 2006 [81,82]. Another nitroimidazole molecule studied by AstraZeneca in Compound PA-824 (1997) belongs to a new generation of 2015 was N-(2-hydroxyethyl)-1-((6-methoxy-5-methylpyrimi- nitroimidazoles, with its ring fused with a furan heterocycle din-4-yl)methyl)-6-methyl-1H-pyrrolo[3,2-b]pyridine-3-carboxa- replacing the -pyran. The C-2 stereogenic center has an R mide, TBA-7371, which had a new mechanism of action configuration. This carbon, bound to a set of rings, confers a similar to that of delamanid, of inhibiting the DprE1 enzyme lipophilic character to the molecule. This configuration, that is important to cell wall biosynthesis [89]. TBA-7371 although different from PA-824 (whose stereogenic center is showed excellent in vitro and in vivo efficacy against MTB, S), has a similar geometry. By fixing the core nitroimidazole in with an MIC and minimum bactericidal concentration (MBC) the same position in both molecules, the lipophilic moiety is of 1.56–3.12 and 0.78–1.56 µM, respectively, besides being positioned in the same direction relative to the plane. Its determined safe [90]. mechanism of action is similar to PA-824: it blocks mycolic acid biosynthesis by inhibition of DprE1, but present resis- 3.8. Ethylenediamine derivatives tance mechanisms are caused by mutations in the Rv3547 gene that reduce its production. PA-824 has shown low MIC Lederle Laboratories, in 1950, developed (2S)-2-[(2-{[(2S)-1- values of 0.002 and 0.7 µM in replicating and nonreplicating hydroxybutan-2-yl]amino}ethyl)amino]butan-1-ol (ethambutol, TB, respectively [67,83,84]. ETB), the first derivative of ethylenediamine drug used to treat TB, had an MIC of 5 µg/mL, but could cause optic neuropathy. Protopova prepared a library in collaboration with Clif Barry

4 N 3 (NIH/NIAID) of 63,238 compounds containing a 1,2-ethylenediamine pharmacophore (Figure 5) of ETB. In 2003, 119 of 5 2 these were patented by Sequella, Inc. These compounds N 1 showed good activity against MTB [91], but N-[(2E)-3,7- ′ H dimethyl-2,6-octadienyl]-N -tricyclo[3.3.1.13,7]dec-2-yl-1,2- ethanediamine) (SQ-109), with one ethylenediamine linked to Figure 4. General structure of imidazole. a geranial and adamantine group, showed the lowest MICs, in EXPERT OPINION ON THERAPEUTIC PATENTS 9

H2N use that is used to treat both gram-positive and gram-negative NH 2 bacterial infections; however, it has severe adverse effects such as myelosuppression and optical and peripheral neuropathy, Figure 5. Chemical structure of the 1,2-ethylenediamine pharmacophore. and it entails a long chemotherapeutic regimen. Linezolid, in combination with bedaquiline and pretomanid, is in Phase IIa the range of 0.7–1.56 µM, both in sensitive strains or strains clinical trials. This treatment is considered a rescue therapy for resistant to first-line drugs. This result showed activity 4 times XDR-TB and is expected to be developed in South Africa [79]. greater than that of ETB and that was comparable to INH. In With the aim of maintaining the activity and reducing the vivo tests of SQ-109 showed that this compound was able to adverse effects of linezolid, in 1995, the American cure induced TB in mice at a concentration 100 times lower Pharmaceutical Company Upjohn (now Pfizer) patented a than that of ETB, with an adequate level of toxicity and good compound specifically as an antimycobacterial [101]. Based pharmacokinetics [92]. Its mechanism of action, although not on the chemical structure of linezolid, the laboratory devel- yet fully elucidated, appears to involve inhibition of cell wall oped N-[[(5S)-3-[3-fluoro-4-(4-thiomorpholinyl)phenyl]-2-oxo- biosynthesis, as it has high antimycobacterial activity even 5-oxazolidinyl] methyl]- or N-({(5S)-3-[3-fluoro-4-(thiomorpho- against ETB-resistant strains. It is believed that SQ-109 has a lin-4-yl)phenyl]-2-oxooxazolidin-5-yl}methyl) acetamide, or different mechanism of action than other drugs [67]. sutezolid (PNU-100480), by replacing the nitrogen atom of Protopova et al. also synthesized and evaluated the anti- the morpholine ring with a sulfur atom. This compound mycobacterial activity of a 10,358 compound library contain- showed better potential that of linezolid against susceptible ing as dipiperidine pharmacophore that patented by Sequella, (MIC = 0.71–0.9 µM) and resistant (MIC = 0.18 µM) MTB. In Inc., in 2009 [93]. The compound adamantane containing March 2010, Pfizer, Inc., and Johns Hopkins University hydroxydipiperidine designated 1-[[1-(1-adamantylmethyl) obtained another patent for sutezolid as a compound to piperidin-4-yl]methyl]piperidin-4-ol, SQ-609, was the best of treat TB, including MDR and latent TB, in addition to other this family [94]. This molecule acts blocking Mmpl3 membrane compounds such as PA-824, delamanid, bedaquiline, and SQ- transporter, very important to the synthesis of cell wall. 109 [102]. In 2013, Pfizer sold this compound to Sequella, Besides of that, some investigations demonstrated others Inc [103]. mechanisms that can explain his potent activity against latent Other compounds of this class are (5R)-3-[4-[1-[(2S)-2,3- MTB and MDR-TB strains [67,95,96]. dihydroxypropanoyl]-3,6-dihydro-2H-pyridin-4-yl]-3,5- difluoro-phenyl]-5- (isoxazol-3-yloxymethyl)oxazolidin-2-one;

(5R)-3- [4- [1- [(2S)-2,3- dihydroxy-1-oxopropyl]-1,2,3,6-tetrahy- 3.9. Oxazolidinone derivatives dro-4-pyridinyl] −3,5- difluorophenyl]-5- [(3-isoxazolyloxy) methyl]-2-oxazolidinone; and (5 R)-3-(4-{1-[(2S)-2,3-dihydroxy- Oxazolidinones are a class of compounds containing 2-oxazo- propanoyl]-1,2,3,6-tetrahydro-4-pyridyl}-3,5-difluorophenyl)-5- lidone in the structure, Figure 6, which is a five-membered (1,2-oxazol-3-yloxymethyl)-1,3-oxazolidin-2-one) (AZD-5847), cyclic compound comprising a nitrogen atom, an oxygen which was discovered in 1999, but was patented in 2001, atom, and a carbonyl group [97]; the first was synthesized by 2002, and 2010 by AstraZeneca UK Limited, including the DuPont in 1970. These compounds can inhibit the protein chemical process and intermediate compounds generated, synthesis when they bind to the 50 s ribosomal subunit, the phosphorylation and purification process beyond their specifically in the 23S rRNA. intermediates, and its use as a new drug for TB treatment, N-[[(5S)-3-[3-fluoro-4-(4-morpholinyl) phenyl] −2-oxo-5-oxa- respectively [104–109]. This compound differs from linezolid zolidinyl] methyl] acetamide, linezolid, was the first oxazolidi- and sutezolid by the presence of a 3-isoxazol group instead of none compound of its class to be used for the treatment of TB. the acetamide group, inclusion of a fluor atom in the aromatic Patents for the crystal form of this compound were issued in ring, and substitution of the group (S)-1-(5,6-dihydropyridin-1 September 2002. Specifically, Pharmacia & Upjohn Company (2H)-yl)-2,3-dihydroxypropan-1-one for a morpholine or thio- patented crystal form II of linezolid, which differs from form I morpholine. Although the 5-carbon stereochemical configura- in its infrared and X-ray spectra and melting point [98]. Symed tion is R in sutezolid and linezolid and S in AZD-5847, by Labs Limited, in April 2009 and May 2010, obtained patents for maintaining the position of the oxazolidinone group, the the preparation of linezolid and its intermediate compounds group linked to C-5 in each, it is oriented toward the same other than crystalline form III of linezolid, respectively [99,100]. face relative to the plane. AZD-5847 was found to be more Linezolid is an antibiotic approved for broad-spectrum clinical potent than linezolid in in vitro and in vivo assays. In in vitro tests, the MIC is about 40% lower than that of linezolid and O the MBC of AZD-5847 was nearly 3 times lower. In vivo, the minimum effective dose (MED) of AZD was 2 times lower than R ON1 that shown by linezolid. This compound shows bactericidal effects against MTB in macrophages and in murine models of acute and chronic infection and, at this time is on hold in R2 R3 Phase II [67,110–112]. In 2013, Dong-A Pharmaceutical Company (Yongin, S. R1 = alkyl or aryl; R2 = H or aryl and R3 = OH or NH2 Korea) and Rhee et al. patented the synthesis process and Figure 6. General structure of 2-oxazolidinone derivatives. antibacterial activity for gram-positive bacteria such as 10 P. B. D. SILVA ET AL.

10 staphylococci, enterococci, and streptococci, and anaerobic 1 N 9 microorganisms such as Bacteroides and Clostridia, yet acid- 2 8 resistant microorganisms such as M. tuberculosis and M. avium 3 7 of 75 novel derivatives of oxazolidinone, including highlighted N 4 6 the Tedizolid ((R)-3-(4-(2-(2-methyltetrazol-5-yl)-pyridin-5-yl)-3- 5 fluorophenyl)-5-hydroxymethyloxazolidin-2-one (DA-7157)) Figure 7. General structure of phenazine. and its corresponding prodrug Tedizolid phosphate ((R)-3-(4- (2-(2-methyltetrazol-5-yl)-pyridin-5-yl)-3-fluorophenyl)-2-oxo- 5-oxazolidinyl) methyl disodium phosphate (DA-7218)), this accumulation in fat and promotes skin discoloration. This last molecule distinguishes from linezolid by replacing the analog was selected from othersbecauseofitsdesirable acetamide for a hydroxymethyl group [103,113] In 2016, Dr. properties, showing MIC90 values in H37Rv of 0.016 µg/mL Reddy’s Laboratories Limited patented the process to synthe- and in drug-resistant clinical isolates of 0.08 µg/mL. In size and purify tedizolid phosphate [114]. Tedizolid phosphate addition, TBI-166 acts against intracellular and nonreplicat- is a prodrug that, after activation in vivo, inhibits protein ing MTB. Its mechanism of action is blocking DNA synth- synthesis by binding to subunit 23S in the ribosome initiation esis, and because it is less lipophilic than CFM, it shows complex. This drug demonstrates promising intracellular less accumulation in the skin [123,125]. effects, decreasing the number of mycobacteria inside of THP-1 macrophages in 1.3 log after 72 h of exposure [115]. Das et al. used the structure of eperezolid oxazolidinone to 3.11. Spectinomycin derivatives synthesize others compounds. The authors substituted the Spectinomycin is a bacteriostatic antibiotic, a product piperazinyl-phenyl-oxazolidinone core with five-membered secreted by Streptomyces spectabilis,withastructurecon- heterocycles with a methylene linker or replaced a piperazine sisting of a fused tricyclic. The diaminocyclitol moiety acti- ring with other diamino-heterocycles to obtain 27 compounds namine is fused to a single sugar component [116]. Among the compounds synthesized and patented by actinospectose through a β-glycosidic and hemiketal linkage Mehta et al. in 2002 and 2003, N-[[(5S)-3-[3-fluoro-4-[4-[(5- to form the other ring (Figure 8), inhibits protein synthesis, nitrofuran-2-yl)methyl]piperazin-1-yl]phenyl]-2-oxo-1,3-oxazo- but shows little antimycobacterial activity and is used to lidin-5-yl]methyl]acetamide, or ranbezolid (rabnbaxy or RBX- treat gonorrheal infections. Lee et al. used the base struc- 7644), which has a 5-nitrofuryl ring linked by a methylene ture of spectinomycin to synthesize semisynthetic spectino-

linker to the piperazin-1-yl-3-phenyloxazolidin-2-one core mycin analogs with antimycobacterial activity. The authors [117,118]. Ranbezolid showed modest in vitro activity against synthesized over 140 substituted spectinamide analogs and susceptible and drug-resistant MTB strains, with MIC values of evaluated their antitubercular potency through in vitro and ~2 and 4 µg/mL, respectively. In another test, this compound in vivo assays [126]. demonstrated activity against MDR-TB strains, with an MIC 90 Among the analogs of spectinomycin evaluated in recent value of 16.0 µg/mL; against strains monoresistant to INH and years, Spectinamide 1599 stands apart. Spectinamide was RIF, an MIC value of 4.0 µg/mL; and to other isolates, an 90 obtained by semisynthesis, and the aim of the structural MIC value of 8.0 µg/mL. It showed good activity at a con- 90 modifications was to increase its target affinity and prevent centration of 8.0 µg/mL against intracellular MTB, with a its efflux, therefore improving its activity. This compound greater than 200-fold decrease in the number of organisms showed MIC values ranging 0.8–1.6 µg/mL in susceptible when compared to that of the control. In addition, it showed strains and 0.3–1.9 µg/mL in MDR-TB and latent TB. This good activity against gram-positive, fastidious gram-negative, molecule inhibits protein synthesis by binding to the 16S and anaerobic microorganisms, and no cytotoxicity at bacterial ribosomal subunit and blocking ribosome translo- 100.0 µg/mL, when tested against macrophages [119–121]. cation. It showed potent in vivo effects in mice in multiple TB infection models, and good in vitro safety profile besides, pharmacokinetics, and pharmacodynamic performance in 3.10. Phenazine derivatives mice [67,126]. The patent for this compound was obtained by University of Tennessee Research Foundation, USA, 2014 Phenazines include a group of nitrogen-containing heterocyc- [127,128]. lic compounds that differ in their chemical and physical properties, based on the type and position of functional groups present (Figure 7). CFM is a phenazine with bactericidal prop- H3CH N H O erties against M. leprae that has been in clinical use for leprosy HO for more than 40 years [122]. O O The riminophenazine TBI-166 (3-(4-methoxycyclohexyl) H imino-N-(2-methoxypyridin-3-yl)-5-[4-(trifluoromethoxy) H3C NH OOCH3 phenyl]phenazin-2-amine) wasdesignedasananalogof HH CFM in a 2012 study of more than 500 anti-MTB com- O pounds by Lu et al. and the Global Alliance for TB Drug H Development [123,124]. It was designed because of the high lipophilicity of CFM that results in extensive Figure 8. General structure of spectinomycin. EXPERT OPINION ON THERAPEUTIC PATENTS 11

3.12. Other chemical classes pharmaceutical industry. It would be fortunate if a single new molecule addressed each of the criteria above; however, in Caprazamycins (CPZs) are novel lipo-nucleoside antibiotics combination with existing antibiotics or others that might be that are produced by Streptomyces sp. MK730-62F2 and introduced, a new antibiotic could meet all of these needs as well belong to the lipouridyl antibiotic family. Capuramycin (CM) as the current therapy. Here, we surveyed 116 patents in loca- is a naturally occurring nucleoside antibiotic produced by S. tions with substantial pharmaceutical research: North America, griseus. Both of these antibiotics inhibit bacterial translocase I, Europe, Japan, and Russia. Of all of the patented compounds, an enzyme essential to peptidoglycan biosynthesis in all bac- only one developed drug, bedaquiline, is in current application, teria [129]. though in a controversial and highly restricted use. Another Caprazamycin was first described in 2003 by the Microbial three are currently being evaluated by clinical studies. Chemistry Research Foundation (MCRF) and Meiji Seika Kaisa, However, many others, with results from in vitro and in vivo Ltd., of Japan. A patent in 2004 described the production of testing, seem to fulfill the needed characteristics for a new anti- novel antibiotics such as CPZEN-45 [130], based on the capra- TB agent. Why are they not in use? Why have so many studies zamycin structure. CPZEN-45 showed an MIC value against been interrupted? Why is there no news on many of these H Rv of 1.56 µg/mL and against MDR-TB of 6.25 µg/mL, 37 agents? Patenting a product, in this case a molecule, means, or demonstrated activity against XDR-TB in mice, and acts should mean, that it has value, and that, in the future, it might be against latent TB [67,96,131,132]. sold or transferred to an interested party such a pharmaceutical Based on a library of over 7000 CM analogs created by company. In fact, we should understand that the pharmaceutical Daiichi-Sankyo, in 2004, Sequella, Inc., licensed a novel class industry has a commercial and not a social calling: the pharma- of antibiotics. SQ-641 shows excellent activity against MTB ceutical industry will only invest in something that will provide a and M. avium complex, with MIC values equal to 1.0 and financial return. This alert should motivate the researcher to think 0.016–16 µg/mL, respectively. However, the compound early about the development strategy. The researcher must ask shows poor hydrosolubility and intracellular activity, after the before beginning a study: does the synthesis of this molecule or completion of preclinical trials [133,134]. other means of production have viable routes, high yield, and low cost? Otherwise, we will continue to produce numerous papers and patents, even as we enter the postantibiotic era. 4. Expert opinion TB is one of the oldest diseases known to man. A drug against Funding

TB, streptomycin, created by Alexander Fleming, was pivotal in the development of antibiotics. Years later, in the mid-1950s, a This paper has been funded by grant#2013/14957-5 São Paulo Research new series of antibiotics were discovered that became part of Foundation (FAPESP), by Coordination for the Improvement of Higher an anti-TB multidrug therapy with a high success rate, which Education Personnel (CAPES) whith the Post-doctoral National Program (PNPD) and Programa de Apoio ao Desenvolvimento Científico (PADC) of remains the main TB treatment to this day. This therapy was so School of Pharmaceutical Sciences/UNESP, Brazil. effective that eradication of the disease was once believed possible. This did not materialize; worse, the therapy is expected to be ineffective in the near future. Declaration of interest Globalization has expanded the range of the disease from the The authors have no relevant affiliations or financial involvement with any Southern hemisphere to the Northern, with a high rate of resis- organization or entity with a financial interest in or financial conflict with tant bacteria. The lack of low-cost, accurate diagnostics, which the subject matter or materials discussed in the manuscript. This includes has promoted the use of empirical therapies, has increased employment, consultancies, honoraria, stock ownership or options, expert selective pressure on these microorganisms. The long treatment testimony, grants or patents received or pending, or royalties. period and side effects to therapy cause many patients to stop treatment, which further increases selection of resistant mutants. References Finally, no effective vaccine exists to control the disease. New therapies for TB are urgently needed. However, a new antibiotic Papers of special note have been highlighted as either of interest (•)orof •• must overcome specific barriers presented by this bacterium: (1) considerable interest ( ) to readers. 1. Alexander KA, Laver PN, Michel AL, et al. Novel Mycobacterium it must surpass the highly lipid barrier that is the mycolic acid cell tuberculosis complex pathogen, M. mungi. Emerg Infect Dis. wall; (2) in the absence of lipophilic properties, the molecule 2010;16(8):1296–1299. must be small enough to enter through a few porins; (3) once 2. Brosch R, Gordon SV, Marmiesse M, et al. A new evolutionary inside of the bacteria, it needs to escape bacterial efflux pumps; scenario for the Mycobacterium tuberculosis complex. Proc Natl – (4) if the bacteria are located within macrophages, the new Acad Sci USA. 2002;99(6):3684 3689. 3. Huard RC, Fabre M, De Haas P, et al. Novel genetic polymorphisms antibiotic must be able to act within these cells or activate that further delineate the phylogeny of the Mycobacterium tuber- them; (5) to prevent recurrence, the antibiotic should also act culosis complex. J Bacteriol. 2006;188(12):4271–4287. on dormant bacteria. The current treatment regimen meets 4. Van Soolingen D, De Haas PE, Haagsma J, et al. Use of various these criteria. Therefore, the new antibiotic must have features genetic markers in differentiation of Mycobacterium bovis strains more desirable than those of the current antibiotics, such as: (6) from animals and humans and for studying epidemiology of bovine tuberculosis. J Clin Microbiol. 1994;32(10):2425–2433. reduced time of treatment; (7) action against resistant bacteria; 5. Global Tuberculosis Report. WHO – World Health Organization. and (8) fewer side effects. In addition, the new antibiotic should Geneva. 2015 [cited 2015 Dec 12]. Available from: http://www. overcome the major hurdle of attracting interest from the who.int/tb/publications/global_report/en/ 12 P. B. D. SILVA ET AL.

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