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Synthesis and Structural Activity Relationship Study of Antitubercular Carboxamides

The unusual structure and chemical composition of the mycobacterial cell wall, the tedious duration of therapy, and resistance developed by the microorganism have made the recurrence of the disease multidrug resistance and extensive or extreme drug resistance. The prevalence of tuberculosis in synergy with HIV/AIDS epidemic augments the risk of developing the disease by 100-fold. The need to synthesize new drugs that will shorten the total duration of effective treatment and/or significantly reduce the dosage taken under DOTS supervision, improve on the treatment of multidrug-resistant tuberculosis which defies the treatment with isoniazid and rifampicin, and provide effective treatment for latent TB infections which is essential for eliminating tuberculosis prompted this review. In this review, we considered the synthesis and structure activity relationship study of carboxamide derivatives with antitubercular potential.


Introduction
Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, which most commonly affects the lungs. The M. tuberculosis complex is a set of evolutionary closely related slow growing mycobacterial species, all containing the mobile insertion sequence IS6610 in their genome and causing TB disease in humans and other mammals. It is transmitted from person to person via droplets from the throat and lungs of people with the active respiratory disease. In healthy people, infections with Mycobacterium tuberculosis often cause no symptoms since the person's immune system acts to "wall off " the bacteria. The symptoms of active TB of the lung are coughing, sometimes with sputum or blood, chest pains, weakness, weight loss, fever, and night sweats [1]. TB is a worldwide pandemic [2] and still remains one of the foremost among infectious diseases in the world causing the maximum number of deaths due to the spread of single microorganisms [3]. Of the new TB cases reported, 95% occur in developing countries every year. Currently, among the infected individuals, approximately eight million develop active TB, and almost two million die from the diseases [4]. The World Health Organization has expressed concern over the emergence of virulent drug-resistant strains of TB and is calling for measures to be strengthened and implemented to prevent the global spread of these deadly TB strains. The unusual structure and chemical composition of the mycobacterium cell wall and effective TB treatment is difficult, which makes many antibiotics ineffective and hinders the entry of drugs. Multidrug resistance tuberculosis (MDR-TB), defined as resistance to at least isoniazid and rifampicin [5], is a serious threat to tuberculosis control and prevention. Isoniazid blocks the biosynthesis of mycolic acids, the essential components of mycobacterial cell wall, and is believed to be oxidized by catalysed peroxidase (Kat G) to the active form [6]. Mutations in the Kat G and the Inh A genes are associated with 70-80% of INH-resistant M. tuberculosis isolate [7]. Resistance to rifampicin has been associated with mutations in the 81 bp core region of the rpoB gene encoding the -subunit of RNA polymerase [8,9] in over 90% cases. The development of resistance by M. tuberculosis to the commonly used antitubercular drugs necessitates a longer duration of therapy. The emergence of multidrug resistance has forced the development of new structural classes of antitubercular agents, with several of them showing promising activity against M. tuberculosis [10]. The tedious duration of therapy and resistance developed by the microorganism has made the recurrence of the disease especially as MDR-TB and XDR-TB a global challenge in tuberculosis chemotherapy [11]. XDR-TB is extensive or extreme drug resistance is MDR-TB that is also resistant to three or more of the six classes of second line drugs. The increase in TB incidence during recent years is largely due to the prevalence of TB in synergy with human immunodeficiency virus (HIV/AIDS) epidemic, which augments the risk of developing the disease by 100-fold, where 31% of new TB cases were attributed to HIV coinfection and emergence of MDR-TB and XDR-TB strains. The treatment of MDR-TB and XDR-TB has become a major concern worldwide. The occurrence of TB is linked to dense population, poor nutrition, and poor sanitation. Observed treatment short-course (DOTS) strategy constitutes the cornerstone of the current protocol for the control of TB [12][13][14][15][16][17][18][19]. Currently, the recommended standard chemotherapeutic regimen for TB treatment is prescribed under DOTS. The chemotherapeutic regimen consists of an initial 2-month phase of treatment with isoniazid (INH), rifampicin (RIF), pyrazinamide (PYR), and ethambutol (ETH) followed by a continuation phase of treatment lasting for 4 months with isoniazid and rifampicin. Poor patience compliance can promote the emergence of drug resistance, and this is particularly true in TB chemotherapy [20]. In the last forty years, only a few drugs have been approved by the Food and Drug Administration (FDA) to treat TB, reflecting the inherent difficulties in discovery and clinical testing of new agents and lack of pharmaceutical industry research in the area. There is unequivocal need for new drugs that should show improvement over the existing regimens in the following areas: (a) shortening the total duration of effective treatment and/or significantly reducing the total number of doses needed to be taken under DOTS supervision; (b) improving the treatment of MDR-TB, which cannot be treated with INH and RIF; and/or (c) providing more effective treatment of latent/dormant TB infection, which is essential for eliminating tuberculosis [21]. Recently, bedaquiline formerly known as TMC 207 was approved by United State Food and Drug Administration for the treatment of adult with pulmonary multidrug resistance tuberculosis when an effective treatment regimen cannot otherwise be provided [22]. It has no cross resistance to the available tuberculosis agents. Bedaquiline is marketed as SIRTURO and chemically known as (1R,2S)-1-(6-bromo-2-methoxy-3-quinolinyl)-4-(dimethylamino)-2-(1-naphthalenyl)-1-phenyl-2-butanol. Though this drug has good antitubercular activity, it needs 24 weeks of treatment thereby encouraging the development of resistance specie arising from noncompliance to prescription and it has also been shown to cause adverse effects like hemoptysis and anorexia [23]. Delamanid marketed as Deltyba is also indicated for use as part of an appropriate combination regimen for pulmonary MDR-TB in adult patients when an effective treatment regimen cannot otherwise be composed for reasons of resistance or tolerability. It must be administered as directly observed therapy (DOT) because of its adverse effect and it lasts for 24 weeks. There has been no established safe dosage for patients with renal or hepatic impairment and children or adolescents [24].

Synthesis of Pyrazine Derived Carboxamides
Pyrazine carboxamide is an important component in the intensive phase of short-course treatment of TB owing to its sterilizing effect, ability to act in acidic environments, and excellent synergy with rifampicin. Martin et al. [48] synthesized binuclear analogues with the -CONH-bridge connecting the pyrazine and benzene rings with antimycobacterial activity. They proposed the formation of centrosymmetric dimer pairs with the peptidic carboxamido group of some peptides needed for binding to the receptor site possibly by hydrogen bond formation. The target compounds were synthesized by microwave assisted coupling reaction of methyl ester of substituted pyrazine carboxylic acids (2) with ring substituted benzylamines (3) which yielded series of substituted N-benzyl pyrazine-2-carboxamides (4a-j) (Scheme 1). They used hydrophobic electron withdrawing (halogens), alkyl substituents on the pyrazine (methyl, t-butyl), and their combination of substituents (alkyl, alkoxy, acetyl, OH, and halogens) on benzene part.
The antimycobacterial evaluation of the compounds showed no improvement in comparison with pyrazinamide. The most active compound in this series is compound 4c (MIC, 25 g/mL) against pyrazinamide (MIC 6.25 g/mL).

Synthesis of N-[(2 I -Substituted Phenyl)-1,3 I -thiazol-5-one]-naphtho[2,1-b]furan-2carboxamide Derivatives
Murugan et al. [52] reported the synthesis of N- (12)(13)(14)(15). A mixture of 2-hydroxy-1-naphthaldehyde (8), ethyl bromoacetate, and anhydrous potassium carbonate was heated under reflux for 24.35 h. The reaction mixture was filtered and potassium carbonate was washed with acetone which was evaporated to get carboxylate (9). To this point, hydrazine hydrate and ethanol were added and refluxed for 18.3 h. The excess ethanol was distilled off to get the respective carbohydrazide (10). The carbohydrazide 10 was mixed with a solution of various substituted aromatic aldehydes (11) in ethanol in DMF. The reaction mixture was refluxed for 8.2 h, cooled to room temperature, and poured into crushed ice to yield carboxamide (12). To the carboxamide 12 in 1,4-dioxane, mercaptoacetic acid and catalytic amount of anhydrous zinc chloride were added. The mixture was refluxed for 4.4 h, cooled, and poured into sodium bicarbonate solution to remove unreacted mercaptoacetic acid which was filtered to get the final products (14-17) (Scheme 3). The antitubercular activities of the compounds were assessed against M. tuberculosis using microplate AlamarBlue assay (MABA). They reported four (14-17) (Figure 2) of the tested compounds to be active at concentrations of 50 and 100 g/mL (Table 1).
They tested all compounds against M. tuberculosis H 37 Rv strain at the concentration of 6.25 g/mL using DMSO as a solubilizing agent. The antitubercular activity result indicated that the substitution of 4,5-dichloroimidazole ring at 4-position of 1,4-DHP affects the antitubercular activity when 3,5-diester group in classic DHP structure was replaced by carboxamide moiety. On comparison, the most active compound is 20d with 3-chlorophenyl group at 3,5dicarboxamide position. 3-Nitrophenyl and 4-nitrophenyl substituted compounds were also relatively active, but other substitutions did not show good activity. Although none of the new compounds had antibacterial activity comparable with rifampicin, the results serve as valuable probes to study the structure function relationship for antitubercular activity.

Synthesis of Novel Thiadiazolyl Pyrrolidine Carboxamides
A new direction in the synthesis of antitubercular agents is directed on the design of molecules acting as enzyme inhibitors. The target enzyme should play a vital role in any phase of the life cycle of the pathogen and should be absent in the host. Enoyl-acyl carrier protein reductase is a FAS II enzyme involved in the bacterial fatty acid biosynthetic pathway in the mycobacterium and other bacteria [61]. These enzymes are involved in fatty acid elongation in the cell wall synthesis. The prime TB drug isoniazid is reported to be a potent enoyl-ACP reductase inhibitor but requires initial activation by Kat G, a catalase peroxidase enzyme [62]. This activation step necessitated the search for new antitubercular agents which can act as direct enoyl ACP reductase inhibitors. This prompted Boyne et al. [63] to synthesize thiadiazolyl pyrrolidine carboxamides (26a-e) and tested their enoyl ACP reductase inhibition activity. In their synthesis, 5-oxo-1-phenylpyrrolidine-3-carboxylic acid 23 was synthesized by refluxing a mixture of itaconic acid [21], aniline [22], and water for 1 h or until the odour of aniline becomes faint after which the reaction was chilled for 1 h. The synthesis of 2-amino-5-(4-substituted)phenyl aryl-1,3,4-thiadiazole 25 was achieved by dissolving aromatic aldehyde and thiosemicarbazide, respectively, in warm alcohol and warm water and mixing the two solutions slowly with stirring. The target compounds were synthesized by dissolving compounds 23 and 25 in dry DMF. HBTU and  Virsodia et al. [69] carried out the synthesis of the target compounds utilizing various substituted acetoacetanilides (29a-n). Compounds (29a-n) were synthesized by reacting substituted amines and ethyl acetoacetate in toluene with a catalytic amount of NaOH or KOH (Scheme 6). The reaction mixture was heated at 120 ∘ C for 10-15 h. Fourteen different acetoacetanilides were synthesized bearing various electron withdrawing and electron donating groups like 2,3-diCH 3 , 3,4-diCH 3 , 4-CH 3 , H, 2,5-diCH 3 ; 2,4-diCH 3 ; 3-Cl-4-F; 4-F; 4-Cl; 2-F; 4-OCH 3 , 2,5-diCl, and 3-NO 2 on the phenyl ring. Acetoacetanilides, thus obtained, were used as 1,3-diketone adducts for the multicomponent Biginelli reaction.
The acetoacetanilides (29a-n) were reacted with substituted aldehydes and urea in methanol using concentrated HCl in catalytic amount to obtain the title compounds (31a-v, 32a-g) as depicted in Scheme 6.
The antitubercular activities of the compounds were tested against M. tuberculosis H 37 Rv strain. Percentage inhibition data of compounds (31a-v, 32a-g) are reported in Table  3. Compounds 31c and 32f, with dimethyl phenyl and 3,4dimethylcarbamoyl side chain, respectively, showed 65% and 63% inhibition. Thus, methyl group at these positions showed higher potency. But substitutions on 4-phenyl ring also alter the activity of compound. Compound 31m was having 3,4dimethylphenyl carbamoyl side chain as in compound 32f, but NO 2 group is at meta-position in compound 31m which leads to a decrease in % inhibition from 63% to 13%. Thus, compounds with methyl substitution on phenyl carbamoyl side chain with -OPh or -NO 2 substitution at meta-position of 4-phenyl ring were more potent than the same substitution NaOH/KOH,
To this homogenous mixture, ethanol and appropriate aldehyde 30 were added. The reaction was stirred below 25 ∘ C for 6 h. The solid that precipitated was filtered and washed with cold ethanol and dried to afford 34a-d.
They synthesized the carboxamides 35(a-d)-50(a-d), by mixing appropriate carboxylic acid 34a-d and DCC in dichloromethane, and stirred them for 10 min at 0 ∘ C. To this mixture, appropriate primary or secondary amine was added and stirred for 8 h. The solid urea separated was filtered off and the organic layer was washed with water and dried over sodium sulphate and distilled under reduced pressure to yield the desired product (Scheme 7).
The compounds were screened for their in vitro antimycobacterial activity against M. tuberculosis (MTB) and M. smegmatis ATCC 14468 (MC2) by agar dilution method for the determination of MIC in duplicate. The result of the MIC is as given in Table 4. The structural core is presented in Figure 3.
The synthesis was achieved as reported by Sharma et al. [76] as follows: the starting material, phenothiazine 51 with 1-bromo-3-chloropropane underwent a nucleophilic substitution reaction yielding 10- The results of the antitubercular activities are summarized in Table 5. All the compounds 51, 52, 53a-s, 54a-s, and 55a-s were screened for their antitubercular activity against M. tuberculosis (H37Rv strain). The investigation of antimicrobial data revealed that compounds 56c, 56d, 56e, 56f, 56h, 56i, and 56j displayed high activity, compounds 55h, 55j, 56b, 56g, and 56q showed moderate activity, and the other compounds showed less activity compared with standard drugs.
The compounds exhibited a structure activity relationship (SAR) because the activity of compounds varies with substitution. The nitrogroup-containing compounds 56h, 56i, and 56j showed higher activity than the chloro-group-(56c and 56d) or the bromo-group-containing compounds (56e and 56f). In addition, the chloro-and bromo-derivatives also had a higher activity than the other tested compounds. Based on the SAR, it could be concluded that the activity of compounds depended on the electron withdrawing nature of the substituent groups. The sequence of the activity is the following: International Journal of Medicinal Chemistry 9

Synthesis of Tetrahydropyrazolopyrimidine Carboxamides
To identify a new starting point in the development of new TB drugs, the Novartis internal small molecule chemical library was screened for activity against Mycobacterium bovis BCG as a surrogate of M. tuberculosis by measuring ATP levels using the BacTiter-Glo assay as described in literature [77]. Subsequent hit confirmation with M. tuberculosis H37Rv led to the identification of tetrahydropyrazolo[1,5-a]pyrimidine scaffold as one of the hit series. Two other groups have also independently reported this scaffold as a hit from their own phenotypic high-throughput screening campaigns against TB [78][79][80]. Yokokawa et al. [81] described the synthesis of tetrahydropyrimidine carboxamides exploring the structure activity relationship (SAR) and structure-property relationship (SPR) of this class and the results of in vivo pharmacokinetics and pharmacological evaluation of selected compounds in mice. Their initial SAR study identified the key pharmacophore required for anti-TB activity as summarized in Figure 4. The NH of the tetrahydropyrimidine ring, the secondary amide linker, and the pyrazole ring were all found to be essential to retain low micromolar values of MIC (minimal inhibitory concentration, defined as the concentration that prevents 50% of bacterial growth at 5 days postinhibitor exposure). Significant differential anti-TB activity of the stereoisomers at the C-5 and C-7 positions was observed, and the absolute stereochemistry of the active enantiomer was confirmed to be 5R, 7S with X-ray crystal analysis. The corresponding (5S,7R) isomers were proved to be inactive (MIC > 20 M). This result indicates that the (5R,7S) form of this scaffold may interact appropriately with some pocket of the yet unknown biological target inside the TB bacteria. Next, they focused on the exploration of SAR and SPR for the phenyl left-hand side (LHS), benzyl righthand side (RHS), and trifluoromethyl at the C-7 position to optimize the balance of potency and physicochemical properties.     Compound 65 exhibited the best potency against MTB H37Rv in the whole cell assay (MIC 0.15 M); however, it is highly lipophilic (log = 6.3) and shows high plasma protein binding (>99.0%) and low aqueous solubility (<4 M at pH 6.8), which are in general unfavorable drug-like properties. To reduce the lipophilicity of the scaffold, replacement of the LHS phenyl with 2-pyridyl and 2-furyl groups led to compounds 66 and 67, which were tolerated and reduced log significantly (by 0.8−1.9). Introduction of polar substituents at the para-position of the LHS phenyl afforded compounds 68, 69, and 70, which also reduced log and achieved anti-TB activity comparable with compound 65 ( Table 6). Introduction of the 2-and 3-pyridyl rings on the RHS reduced log without affecting the potency (compounds 71 and 72). However, all of these modifications had little effect on solubility and plasma protein binding. Replacement of the core 7-trifluoromethyl substituent with difluoromethyl in 66 afforded compound 73, which interestingly also increased aqueous solubility. The combination of the LHS pyridyl with RHS pyridyl generated compound 74, which led to a significant decrease in log  MDR-TB isolates suggesting a novel mechanism of action. Studies to elucidate a mechanism of action of this series will be discussed elsewhere [82]. The in vivo pharmacokinetics (PK) of compounds 65, 66, 70, and 73 were evaluated in mice by oral (po) and intravenous (iv) routes at doses of 25 and 5 mg/kg, respectively. All four compounds displayed low to moderate total systemic clearance and volume of distribution with elimination half-lives ranging from 1.3 to 4 h. These compounds showed good oral bioavailability (45−100%) and good oral exposure in systemic circulation. In addition, these compounds exhibited no significant CYP inhibition (based on reversible inhibition assays using midazolam for CYP 3A4/5, bufuralol for CYP2D6, and diclofenac for CYP 2C9 as markers) and induction.

Conclusion
This work has reviewed the synthesis and antitubercular activities of over two hundred carboxamide derivatives. In most of the synthesis reported, there was almost always a comparison between the antitubercular activities of the novel compounds with isoniazid, rifampicin, or pyrazinamide. The review reveals the following compounds as being more active than isoniazid, rifampicin, or pyrazinamide. From the work of Dolež al et al. [49], it was shown that, from the antitubercular activity carried out at Czech Republic, compounds 7a, 7e, 7i, and 7l were more potent against M. tuberculosis than pyrazinamide but only 7l was found to be more active than pyrazinamide when the IC 90 was carried out at TAACF USA. The work of Sriram et al. [70] also revealed that compounds 39a, 37b, 40b, 41b, 43b, 45b, 49b, 50b, 36d, 39d, 46d, 47d, and 50d were more active than isoniazid whereas compounds 43b, 47d, and 50d were found to be more active than rifampicin. Since rifampicin (MIC 0.23 M) is more active than isoniazid (MIC 0.66 M), it can be said categorically that only three of the one hundred and thirtyfour new derivatives of carboxamide reviewed were found to be more active than the existing antitubercular agents. The most active compound is 43b (MIC 0.12 M). Yokokawa et al. [81] also revealed compound 65 with MIC 0.15 M and 67 with 0.13 M.