Recent Efforts for the Development of Antitubercular Drug Containing Diazine Ring

There has been considerable interest in the development of new molecule with antibacterial activities particularly against tuberclosis because mycobacterium species have developed resistant against currently used drugs, their toxic effect and long duration of therapy. The diazine (pyridazine, pyrimidine and piperazine) derivatives possess an important class of compound for new drugs research and development. Therefore, many researchers have synthesized these compounds as target structures and evaluated their antitubercular activity. These observations have been guiding for the development of new molecules that possess potent antitubercular activity with minimum side effects or effective against MDR, XDR mycobacterium strains, and also in patient co-infected with HIV/AIDS. *Corresponding author: Mohammad Asif, GRD (PG) Institute of Management and Technolgy, Dehradun, 248009 (UK), India, E-mail: aasif321@gmail.com Received December 11, 2012; Accepted December 27, 2012; Published December 29, 2012 Citation: Asif M (2012) Recent Efforts for the Development of Antitubercular Drug Containing Diazine Ring. Med chem 2: 151-167. doi:10.4172/2161-0444.1000133 Copyright: © 2012 Asif M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Infectious microbial diseases remain a pressing problem worldwide, because microbes have resisted prophylaxis or therapy longer than any other form of life. Infectious diseases have increased dramatically in recent years. In spite of many significant progres in antibacterial therapy, the widespread use and misuse of antibiotics have caused the emergence of resistance to antibiotics, which is a serious threat for the therapy. In particular, the emergence of multidrug resistant (MDR) bacteria has become a serious problem in the treatment of bacterial diseases. Variuos infections are more common because of their causing agents or microbes have a tendency to develop new strains under any circumstances and developing resistance against the available drugs. In addition to the development of new and effective antibacterial agents against MDR bacteria, recently attention has focused on the treatment of tuberculosis (TB) [1][2][3][4]. Although, there is an increasing resistance to antimicrobial drugs [5], to overcome the development of drug resistance necessary to synthesize a new class of drugs that possessing different chemical properties. Therefore, the development of new drugs to deal with bacterial resistant has become one of the most important areas of research today. Therefore, recent efforts have been directed toward exploring new, potent anti-TB agents with low toxicity profiles when compared with currently used anti-TB drugs [6][7][8][9]. Based on this finding, new anti-TB agents having the pyridazine system have been studied. However, some pyridazine or phthalazine compounds have been reported to have anti-TB activity [10,11].
Diazines are (pyridazines, pyrimidine and piperazine) containing two nitrogen atoms at 1,2 position, 1-3 position and 1,4 position in their cyclic structures respectively. The structures of diazines are prepared by replacing two carbon atoms to two nitrogen atoms in the benzene ring at their respective positions. Diazines and its derivatives are noteworthy for their physiological and biological importance [12][13][14]. Recently diazine derivatives have been a subject of intensive research owing to their wide spectrum of pharmacological activities. Differently substituted diazines have been found to have potential antibacterial, antifungal and antiviral including anti-HIV activities, anticancer, analgesic, anti-inflammatory, anticonvulsant, cardio tonic, antiulcer, antihypertensive, and antiasthmatic etc activities. In view of above facts and inspired by the research going on diazine compounds, particularly in relation to microbial infections [15][16][17].
Tuberculosis (TB) is one of the oldest and most pervasive, respiratory transmitted or contagious disease infecting one-third of the world's population and killing between 2 and 3 million people each year. According World Health Organization (WHO) report, TB has spread to every corner of the globe. The increase in TB incidence during recent years is largely due to the prevalence of TB is synergy with Human Immunodeficiency Virus (HIV/AIDs) epidemic, which augments the risk of developing the disease 100-fold where 31% of new TB cases were attributable to HIV co-infection and emergence of MDR-TB and extensively drug resistance (XDR-TB) strains. The treatment of MDR-TB and XDR-TB has become a major concern worldwide. However, the total number of new TB cases is still rising slowly. The occurrence of this disease is linked to dense population, poor nutrition, and poor sanitation. Observed Treatment, short-course (DOTS) strategy, constitutes the cornerstone of the current protocol for control of TB [18][19][20][21][22][23][24][25]. Despite the success of DOTS strategy, the emergence of MDR-TB strains, recurrently isolated from patient's sputum, darken the future. In addition to this, the increase in M. tuberculosis strains resistant to front line anti-TB drugs such as rifampin and INH has further complicated the problem, which clearly indicates the need for more effective drugs for the efficient management of TB. As per WHO reports, approximately 90% of the patients having both TB and HIV died within a few months after clinical symptoms. Therefore, WHO warned the world for ''even greater TB-HIV crisis'' and called for wide availability of free anti-TB drugs to those living with HIV. As per WHO, HIV is spreading rapidly in India with the largest number of TB cases in the world [26,27].

Piperazine and Pyrazine Derivatives
Pyrazinamide (17) is a synthetic pyrazine analog of nicotinamide. It is active at a MIC of 6-60 μg/mL. Resistance to pyrazinamide develops soon when it is used alone. Its mechanism of action is unknown but it appears to require activation via pyrazinamidases in the organism [39,40]. Since the discovery of pyrazinamide, several derivatives containing pyrazine nucleus have been tested for their activity against Mtb. These pyrazine derivatives substituted with oxadiazole, and oxathiazoline [40]. The derivatized isosteres were expected to be biotransformed by esterases to the active species after penetration of the mycobacterial cell wall. The most active compound of the series 18 exhibited a MIC of 4.5 μg/mL in comparison to 49 μg/mL for pyrazinamide. With the same concept, a series of ring substituted (E)-3-Phenyl-1-(2 pyrazinyl)-2-propen-1-ones were screened for their efficacy against Mtb H37Rv. Among all, compound 19 showed an inhibition of 94% at 12.5 μg/mL [41]. While in a series of pyrazine derivatives, two compound (140a and 20b) have shown equal potency of MIC 6.25 μg/mL against Mtb H37Rv [42]. Compound 20b also showed MIC of <0.25 μg/mL against Mtb H37Ra. On the basis of ethionamide, a series of 5-Alkyl-6-(alkyl/aryl sulfanyl) pyrazine-2-carbothioamide, compound (21) showed 91% inhibition at a MIC <6.25 μg/mL. The activity increased with increasing molecular weight of the alkyl sulfanyl group in the 6-position of the pyrazine ring. Thioamides exhibited higher activity than the corresponding amides [43]. While, S methyl-2-(amino(6-chloropyrazin-2-yl)methylene) hydrazine carbodithioate (22) exhibited moderate potency of MIC 32 μg/mL among simple pyrazine hybrids against Mtb sensitive and wild strains [44].
The new potential anti-TB agents are classified on the basis of their chemical entities. A piperazine derivative BM 212 (28), which arose the interest with its very good in vitro activity of MIC 0.7 μg/mL against Mtb [50].

Pyrimidine Derivatives
The antifolates are much importance as they target the enzyme dihydrofolate reductase (DHFR). Most of the antifolates have selectivity toward the pathogen DHFR rather than the host DHFR, as safe target for the development of anti-infective agents. In this concern, a number of compounds with substituted pyrimidines were evaluated their potency against Mtb H37Rv. Among them, compounds (37a-f) showed in-vitro activity in the range of MIC 25-50 μg/mL [58]. In continuation, a number of trisubstituted pyrimidines, compounds (38) have shown anti-TB potency with a MIC in the range of 12.5-25 μg/mL [59]. To further increase the activity of pyrimidines, synthesized other trisubstituted pyrimidines (39) [60], where the activity profile was remained same as 138. Whereas, chloro-pyrimidines were found to be highly active against Mtb. Compounds (140 a-d) were found to be active at a MIC of 0.78 μg/mL. These compounds were further screened against virulent strain (Mtb H37Rv) and no change in their MIC profile was observed [61]. While, in a series of anilino pyrimidines tested against Mtb H37Ra, the most potent activity was shown by the compound 41 having a MIC of 3.12 μg/mL [62].  (42) has shown promising MIC of 2 μg/mL against Mtb H37Rv on day 14 and 21, which is equal to that of standard amikacin [63]. A series of N-phenyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydro pyrimidine-5-carboxamides were evaluated for their anti-TB activity against Mtb H37Rv. Among all, two compounds with 2,3-dimethylphenyl (43a) and 3,4-dimethyl (43b) carbamoyl side chain, respectively, showed 65% and 63% inhibition [ In this perception, a series of pyrimidine-2,4-diamines, these compounds also carried aryl substituents at the 5-position. Preliminary assay of the abilities of these compounds to inhibit the growth of TB5 Saccharomyces cerevisiae carrying the DHFR genes from Mtb, human and yeast indicated that 5-phenyl-6-((3R,4S)-3,4,5-trihydroxypentyl) pyrimidine-2,4-diamine (44) selectively inhibited Mtb DHFR and had little effect on the human or yeast enzymes [65]. Whereas, in a series of methylene-bis-pyrimidinones and methylene-bis-mercapto-pyrimidines, pyrimidinone derivative (45) shown best potency of 0.1 μg/mL while mercapto-pyrimidines showed moderate activity [66].

Quinoline and Quinoxaline Derivatives
In this concern, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (60), a protein kinase inhibitor for its anti-TB profile and found to inhibit the growth of two different mycobacterial strains, the slow-growing M. bovis Bacille Calmette Guerin (BCG) and the fast-growing saprophyte M. smegmatis mc2 155, in a dose-dependent manner. While screening for the effect of kinase inhibitors on mycobacterial growth, millimolar concentrations of 60 induced a 40% decrease in the growth of M. bovis BCG. This 60-induced decrease in growth was shown to involve a 2-log fold decrease in the viable counts of M. smegmatis within a 48 h period and a 50% reduction in the number of BCG viable counts within a 10-day period [79].  (61) showed MIC of 6.25 μg/mL against Mtb H37Rv and 0.5 μg/mL against Mtb H37Ra. Which prompted to continue the optimization of quinoxaline 1,4-dioxide [80], a series of quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives were evaluated for their in-vitro anti-TB activity against Mtb H37Rv. Among all, compound 62a exhibited best MIC of 0.78 μM, has a solubility problem, while compound (62b) having MIC of 3.13 μM has a best selectivity index (SI=>40.06). A series of quinoxaline 1,4-di-N-oxide derivatives by varying the 2-position and found that 2-methylquinoxaline 1,4-di-N-oxides (63a and 63b) were most active of the series with a MIC of 0.39, 0.78 μM respectively and also have better selectivity index (8.46, 20.43). The compound 63b is also active against resistant strains of Mtb [81]. In another series, 2-benzyl-3-(methoxycarbonyl) quinoxaline 1,4-dioxide (64) has shown best potency above all, with a MIC=0.10 μg/mL and selectivity index SI=470 [82]. Inspired with the above activity profile of quinolones, a series of Lamivudine prodrugs bearing fluoroquinoles (68) evaluated their efficacy against Mtb H37Rv. All the compounds exhibited an inhibition of 92-100% at a concentration of 6.25 μg/ml [86]. While in ciprofloxacin derivatives, one compound (69) showed in-vivo anti-TB activity by reducing the bacterial load in spleen tissue with 0.76-log10 protections and was considered to be moderately active in reducing bacterial count in spleen [87]. In continuation, Gatifloxacin derivatives and found a more potent compound (70) in comparison to compound 69. In the in vivo animal model 70 decreased the bacterial load in lung and spleen tissues with 3.62-and 3.76-log10 protections, respectively [88]. With this motivation, he was able to find out a most potent molecule (71) which decreased the bacterial load in lung and spleen tissues with 2.42-and 3.66-log10 protections, respectively, at 25 mg/kg body weight [89]. Contrarily, 7-[4-(5-amino-1,3,4 thiadiazole-2-sulfonyl)]-1-piperazinyl fluoroquinolonic derivatives (72a and 72b), showed moderate anti-TB activity at MIC of 10 μg/mL compared to INH standard [90]. The most active compound 73 showed MIC of 0.39 μg/mL against Mtb H37Rv [91]. In another investigation, 6-nitroquinolone (74) was also found to be the most active compound in-vitro with MIC of 0.08 and 0.16 μM against MTB and MDR-TB, respectively. In the in-vivo animal model 74 decreased the bacterial load in lung and spleen tissues with 2.78 and 4.15-log 10 protections, respectively, at the dose of 50 mg/kg body weight [92][93][94]. In the process of investigating novel quinolones as anti-TB agents, many derivatives of quinolones screened for their in vitro efficacy against MTB and MDR-TB. The most potent (in-vitro) compound of the series was screened for in-vivo potency too. Compound 75 exhibited MIC99 of 0.19 μM and 0.09 μM against MTB and MDR-TB, respectively and decreased the bacterial load in lung and spleen tissues with 1.91 and 2.91-log10 protections, respectively, in the in vivo animal model at a dose of 50 mg/kg body weight [95]. In an effort to increase anti-TB potency of quinolones, 1-(cyclo propyl/2,4-difluorophenyl/tert-butyl)-1,4-dihydro-8-methyl-6-nitro-4-oxo-7-(substituted-secondary-amino)quinoline-3-carboxylic acids. The most active compound (76) of the series showed MIC of 0.42 μM and 0.09 μM against MTB and MDR-TB respectively [92].
While in the series of Tetracycline incorporated with quinolones, compound 77 was found to be the most active against MTB with a MIC of 0.2 μg/mL and also nontoxic to the CEM cells until 200 μM [96]. Thus, developing quinolones as anti-TB agents is a worthy approach.
Compound 78 showed best potency of MIC 0.5 μg/mL against Mtb H37Rv and 0.5-2 μg/mL against resistant strains. However, the in vivo testing in a mouse model of TB infection did not show significant anti-TB activity, probably because of its poor bioavailability [97]. In continuation, 5-nitrofuran, 5-nitrothiophene and arylfuran coupled benzothiadiazines on evaluation, these compounds exhibited moderate anti-TB activity. The most active compound (79) displayed a MIC of 1 μg/mL against Mtb H37Rv [98]. Clofazimine (81) is a fat-soluble riminophenazine dye used in combination with RIF and dapsone as multidrug therapy (MDT) for the treatment of leprosy. It has been used investigationally in combination with other anti-TB drugs to treat M. avium infections in AIDS patients and M. avium para-TB infection in Crohn's disease patients. On this basis and to minimize the side-effects and to improve the anti-TB activity of Clofazimine [100], 3-(2,4-dichloroanilino)-10-(2,4-dichlorophenyl)-2,10-dihydro-2-(2,2,6,6tetramethyl piperid-4-ylimino) phenazine (B4128) (82) posses a similar mode of action of Clofazimine [101]. With the same motivation, a series of phthalimido-and naphthalimido-linked phenazines were found two compounds (83a and 84b) with a potency of MIC 1 μg/mL against Mtb H37Rv. These compounds also exhibited potency against resistant strains of Mycobacterium [102]. Whereas in a series of phenazine carboxamides, compounds 85a and 85b showed excellent activity against Mtb H37Rv with a MIC of 0.19 μg/L and also against DR strains of Mtb. Most interestingly, this series was found to be nontoxic [103], validating them as future anti-TB drugs. While a macrolactone (86) derived from benzo[a]phenazine exhibited best potency against Mtb H37Rv with a MIC 0.62 μg/mL, which is better than that of RIF [104]. With the same motivation, a series of pyrrolo[l,2-a] quinoxaline-2-or -4-carboxylic acid hydrazides and one compound (241) showed an interesting activity at 6.25 μg/mL against Mtb H37Rv, with a 100 percentage inhibition [105].

Future Prospectives
Development of new anti-TB drugs is the need to control TB. In the last fourty years no new anti-TB drug has been brought to the market. However, in recent years there is an enhanced activity in the research and development of new anti-TB drugs. Some compounds are presently in clinical development, while others are being investigated pre-clinically in an attempt to explore new anti-TB molecules. This review provides an overview of the pyridazines against M. tuberclosis [1][2][3][4][5]30,31].
The unremitting and steady rise in tuberculosis together with the emergence of resistance against traditional antitubercular drug regimen and the pathogenic synergy with HIV has put enormous pressure on public health systems to introduce new treatments. In drug resistant tuberculosis it is important to understand how the resistance emerges. Consequently, great efforts have been made in the area of Mtb genomics, proteomics and target identification via advanced technologies and therefore several welcome developments comes in the light having novel target with newer mode of action. In this concern, some new class of drugs antibiotics is under study and was approved for the treatment of MDR-TB. Remarkably, the mechanisms of action of these new arrivals are well-understood with new and novel target. Also, in the field of clinical research, well-established classes of compounds and molecular targets are still interesting, however, in some of the cases when similar target molecules are present in humans; future development has to ensure a high degree of selectivity [106][107][108][109]. Further investment in developing fundamental genetic systems and more accurate models of human disease would significantly facilitate TB drug discovery efforts in the long term, in particular enabling robust validation of novel targets. However, all these possibilities therefore, there is a demand in continuing research in this direction to achieve the goal of eradicating Mtb from the world in coming years.

Disscussion
Tuberculosis (TB) is a chronic infectious disease caused by Mtb. The term MDR-TB is used to describe strains that are resistant to two or more of the five first-line anti-TB drugs. Treatment regimen of tuberculosis comprises five first line antiTB drugs namely INH, RIF, pyrazinamide, streptomycin and ethambutol followed by second line antiTB drugs namely fluoroquinolones and one of the injectable aminoglycosides. Besides the traditional antitubercular drugs available commercially, several new heterocycles were synthesized in recent past. The new potential antitubercular agents have been classified according to their chemical entities. In an effort to developed new and more effective therapies, molecules that can also effective against MTB and MDR-TB. Natural products play a major role in drug discovery, as a unique source of original structures, which can provide models for future drug design. In the field of antitubercular agents, the lichen dibenzofuran derived secondary metabolite: usnic acid has been shown to display an interesting activity, but its weak potency did not permit its further development as an antimycobacterial drug [26,37,92,110].
In view of the persistent drug-resistant TB problem of currently used anti-TB agents, it is important that new anti-TB molecules or drugs should address different targets, as those of currently used drugs including the shortening of TB therapy. The unique structure of the mycobacterial cell wall makes it a useful target for drug development and studies can be directed to specific sites like cell wall biosynthetic pathways [111,112]. Although one possible long term solution to the problem is a better vaccine, in the short term, the major reliance will be on chemotherapy requiring the development of novel, effective and non-toxic anti-TB agents [113,114]. The identification of novel target sites will also be needed to circumvent the problems associated with the increasing occurrence of MDR-TB and XDR-TB strains. One of these attractive targets for the rational design of new anti-TB agents are the mycolic acids, the major components of the cell wall of Mtb [26,115,116].

Conclusion
The difficulty in managing TB includes the prolonged duration of the treatment, the emergence of drug resistance, and coinfection with HIV/ AIDS. Tuberculosis control programme requires new drugs that act on novel drug targets to help in combating resistant forms of Mtb and reduce the treatment duration. The availability of the various chemotherapeutic agents, TB remains a leading killer worldwide. This is mainly due to the lack of new drugs, particularly for effective treatment against MDR-TB, XDR-TB, and patients co-infected with HIV/AIDS. Therefore, there is an urgent need for the development of new and effective anti-TB drugs particularly against resistant strains with lesser side-effects [117][118][119]. More importantly, the newly developed drugs are required to reduce the duration of treatment. The newer anti-TB compounds need to be developed on the understanding of the molecular mechanisms of drug action and drug resistance. Focusing on the existing anti-TB targets for drug development may be of limited value because chances of resistance by mutation in the protein target may render the drugs ineffective. Precisely, because of this observed drug-resistance by the bacterium, it is imperative to develop smart new drugs that inhibit novel targets that are structurally and functionally different from those currently known [120]. Medicinal chemists will be interested to working on pyridazine molecules due to their wide range of biological activities pertcularly agaist microbes. In view of above facts and inspired by the research going on pyridazine derivatives, particularly against mycobacterium. Different new pyridazines will be synthesized in the future for development of new anti-TB molecules.