Download PDF
Synlett
DOI: 10.1055/a-2531-6930
perspectives
Small Molecules in Medicinal Chemistry

Advancements in Isoniazid-Based Heterocyclic Derivatives as Potent Antitubercular Agents: A Comprehensive Review of Synthesis, SAR, and Biological Activity (2017–2023)

Mansi Garg
a   Brawn Laboratories Ltd., Gurugram, Haryana, 122001, India
,
Ekta Verma
b   MIT College of Pharmacy, Moradabad, U.P., 244001, India
,
Shweta Mishra∗
c   SGT College of Pharmacy, SGT University, Gurugram, Haryana, 1225052, India
,
Adarsh Sahu∗
d   Department of Pharmaceutical Sciences, Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar, Madhya Pradesh, India
› Author Affiliations
› Further Information
 
 PDF Download Permissions and Reprints All articles of this category


Abstract

Tuberculosis (TB) continues to be a major health problem worldwide, requiring the development of new and innovative therapeutic agents. Isoniazid (INH) is one of the drugs of choice for treating tuberculosis. It is activated by KatG, which produces nicotinamide adenine dinucleotide (NAD). The resulting metabolites inhibit enoyl-acyl carrier protein (ACP) reductase (InhA), an enzyme involved in the biosynthesis of mycolic acid in Mycobacterium tuberculosis. This inhibition disrupts the production of type II fatty acids, which are essential for mycolic acid synthesis and cell survival. However, INH-resistant mycobacterial strains are becoming more prevalent, primarily due to long-term, widespread use and misuse. Researchers have extensively researched and modified INH, a cornerstone in TB treatment, to improve its efficacy and reduce resistance. Numerous investigations have shown that heterocyclic scaffolds, when coupled with INH, exhibit excellent antitubercular activity by increasing the permeation of the drug into bacterial cells. The review highlights various heterocyclic moieties, including phenylisoxazole, indanyl, indole, and isatin, emphasizing their role in improving pharmacokinetic properties and overcoming drug resistance. Here, we have focused on INH-clubbed heterocyclic derivatives that were investigated from 2018 to 2023 as potential antitubercular agents. This review aims to guide future research and development of INH-based heterocyclic derivatives, offering a valuable resource for researchers in the quest for more effective antitubercular therapies.

1 Introduction

2 Challenges with Current Drug Treatment

3 Literature Reports on INH-Clubbed Heterocyclic Derivatives

4 Conclusion

5 Abbreviations


#

Key words

isoniazid - heterocycles - antituberculosis - drug resistance
1

Introduction

Tuberculosis (TB) is an airborne infectious disease that is the leading cause of death worldwide. It is triggered by the Mycobacterium tuberculosis (Mtb) bacillus.[1] [2] It primarily affects the lungs, however, it can also damage the kidneys, brain, liver, intestines, and other organs.[3] Tuberculosis is a chronic communicable illness that spreads from person to person.[4] Approximately 10.6 million cases of active illness and 1.6 million cases of death were reported globally in 2021 (including 187,000 individuals living with HIV).[5] [6] The End TB Strategy of the World Health Organization (WHO) aims to reduce TB fatalities by 95% and incidence by 90% worldwide by 2035.[6] [7] There are two main health concerns around TB: drug-resistant TB and co-infection with HIV.[8] [9] Resistance to at least two significant antituberculosis drugs, INH and rifampicin, is known as multidrug-resistant tuberculosis (MDR-TB) which is caused by inadequate chemotherapy.[10] [11] Extensive drug resistance (XDR) is an instance of disease caused by Mtb that is resistant to at least INH and rifampicin, any fluoroquinolone, and at least one of three injectable second-line medications (amikacin, capreomycin, or kanamycin).[12] [13] Clinically, TB has been divided into two groups: active TB, in which patients who are typically symptomatic and may be infectious if pulmonary disease is present, while latent TB is an asymptomatic clinical state that is not transmissible and noninfectious, but with varying risk of developing into active TB disease.[14] [15] Longer treatment regimens and rising disease incidence in immunocompromised patients highlight the need for novel medications to broaden the spectrum of efficient TB treatment choices. TB treatment is challenging so it takes at least 6 months in 2 separate phases. In the first phase, INH, rifampin, pyrazinamide, and ethambutol are prescribed whereas the second phase contains INH and rifampin. However, present treatment has many limitations like drug–drug interactions, drug toxicity and intolerance, and inadequate patient adherence as a result of the extended course of treatment.[16]

Antitubercular drugs with their mode of action are listed in Table [1].[17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35]

Table 1 Antitubercular Drugs with Their Mode of Action

Drugs

Mode of action

Year

Structure

Isoniazid

inhibition of mycolic acid production via binding to the NADH-dependent enoyl-ACP reductase (InhA)

1952[17]

Rifampicin

inhibition of bacterial RNA synthesis by binding to the β-subunit of the DNA-dependent RNA polymerase

1963[18]

pyrazinamide

disruption of membrane potential hinders the energy production required for the survival of mycobacteria

1952[19]

Ethambutol

inhibits the activity of arabinosyl transferase, an enzyme that plays a role in the formation of the cell wall

1961[20]

prothionamide

inhibition of mycolic acid synthesis

2019[21]

Ethionamide

inhibition of mycolic acid synthesis by binding to the InhA

1956[22]

Cycloserine

inhibition of peptidoglycan synthesis by blocking the d-alanine racemase enzyme

1955[23]

linezolid

inhibition of protein synthesis

2000[24]

Clofazimine

inhibition of DNA biosynthesis

1986[25]

levofloxacin

inhibition of DNA replication

1996[26]

moxifloxacin

inhibition of DNA biosynthesis

1996[27]

gatifloxacin

inhibition of DNA biosynthesis

1999[28]

amikacin

inhibition of RNA-dependent protein synthesis through binding to 30S ribosomal subunit

1976[29]

capreomycin

inhibition of protein biosynthesis

1963[30]

kanamycin

inhibition of protein synthesis that relies on RNA by attaching to the 30S ribosomal subunit

1957[31]

streptomycin

attaches to the 30S ribosomal subunit and causes misreading of the genetic codes thus preventing protein chain growth

1944[32]

bedaquiline

ATP synthase

2012[33]

delamanid

inhibition of the biosynthesis of cell wall, lipids, and protein

2017[34]

para-aminosalicylic acid

folic acid synthesis inhibition

1946[35]


# 2

Challenges with Current Drug Treatment

The directly observed therapy (DOTS) is an available direct treatment of pulmonary TB. DOTS treatment involves the four key drugs in the regimen INH, rifampicin, pyrazinamide, and ethambutol over a longer (6–9 month) period.[36] In the case of drug resistance (MDR and XDR) first-line drugs and second-line drugs followed by multiple antibiotics are required.[37] These anti-TB drugs also cause hepatotoxicity which minimizes the effectiveness of treatment. Adverse effects are the main cause of nonadherence to current treatment and the emergence of drug resistance.[38] Apart from this, there are several reasons behind the failure of therapy, i.e., lack of time, proper administration of effective drugs, long treatment duration, lower availability of less toxic drugs, late diagnosis, nonadherence to the drug regimen, and evolution of drug-resistant TB strain are the significant challenges towards the TB drug therapy and control programs.[39] The variability in host response, disease progression, and drug-resistant phenotypes complicates the therapy because TB is a disease found in existing infrastructure and resource-constrained countries.[40] It is the biggest challenge in developing the new anti-TB drugs. To achieve this aim, novel entities are being developed based on knowledge of the molecular basis for the evaluation and mechanism of drug resistance of Mtb. Along with this, many scientists are engaged in developing new medicines against sensitive and resistant strains of Mtb from the existing category of medicine.[41]

The prevalence of INH-resistant bacterial strains is increasing primarily due to its widespread and prolonged usage. Therefore, many scientists have prepared new derivatives of the INH for resistant cases. Isoniazid derivatives containing an N-heterocyclic moiety are most prominent in medicinal chemistry due to their biological activities such as antitubercular,[42] antifungal,[43] anticancer,[44] anticonvulsant,[45] etc. Due to their variety of biological activities, INH derivatives have attracted the continuing attention of researchers for new drug developments.[46]

Isoniazid is the most popular anti-TB drug. The enzyme known as the enoyl-ACP reductase (InhA) is the primary target of the INH. It is an enzyme that plays a significant role in the biosynthesis pathway and is necessary for the formation of the outer membrane of Mtb. Isoniazid is a prodrug that needs to be activated by KatG to be used within the realm of combination therapy for the treatment of Mtb. When KatG activates it, INH interacts with the cofactor NADH to produce a covalent adduct.[47] The current review delivers a general overview of TB, INH, and its coupled derivatives against Mycobacterium tuberculosis.


# 3

Literature Reports on INH-Clubbed Heterocyclic Derivatives

To address the increasing incidence of INH-resistant forms of Mtb, Aslan and colleagues (2022) attempted to specifically target INH to overcome this resistance. The authors synthesized 13 new compounds C1–C13 by combining INH with sulfonate esters through a hydrazone linkage (Scheme [1]). All the designed compounds were evaluated for their antitubercular activity against the Mtb strain H37Rv. The results showed that C1, C4, C12, and C13 exhibited MIC values of 0.31 μM against Mtb strain H37Rv, the same as the reference drug INH, and the other compounds in the series are also effective antitubercular agents with an MIC value of 0.62 μM, which was two-fold less activity than that of INH (MIC = 0.31 μM). Compounds C1 (CH3), C2 (t-Bu), and C4 (CH3O) are effective against InhA mutant INH-resistant Mtb at MIC values of 1.56 μM. In addition, C4 is also effective against the INH-resistant Mtb isolate with KatG mutation with an MIC value of 6.25 μM. Structural analysis suggested that introducing iodine (C7), a bulky and lipophilic halogen, can be an essential modification to bind to the enzyme’s active site, resulting in an effective antimycobacterial action. The molecular docking studies revealed that C7 shows the best interaction into the binding pocket of InhA.[48]

Zoom Image
Scheme 1 Synthesis of 3-[(2-isonicotinoylhydrazono)methyl]phenyl 4-substituted benzenesulfonate derivatives
Zoom Image
Figure 1
Zoom Image
Scheme 2 Reagents and conditions: (i) propargyl bromide, K2CO3, DMF, rt, 6 h (92%); (ii) isonicotinohydrazide, DIPEAc, rt, 1 h (90%); (iii) substituted azidobenzenes, CuSO4, Na ascorbate, DMF, rt (80–90%).

Isoniazid and 1,3-triazole are both effective anti-TB medicines that act by inhibiting the synthesis of the cell wall. Combining both of the heterocyclic moieties has the potential to result in the synthesis of a lead molecule that possesses potent antimycobacterial activity. Based on these considerations, a novel series of new INH-1,2,3-triazole conjugates (C14–C24, Figure [1]) was designed and synthesized via click reaction, and they were tested for their antimycobacterial and antimicrobial activity in vitro by Badar et al. (2020). The synthetic scheme was presented in Scheme [2]. The antitubercular evaluation demonstrated that compound C20 is the most potent with a MIC value of 1.56 μg/mL against Mtb strain H37Rv. However, it is less active than INH (0.1 μg/mL). The compounds C15, C16, and C21 showed low toxicity against the RAW 264.7 cell line and also exhibited moderate antitubercular activity with MIC values of 6.25–12.5 μg/mL. Moreover, antimicrobial evaluation resulted in compounds C17, C21, C23, and C24 showing better antibacterial and antifungal activities. To determine a possible method of action, molecular docking research was carried out, and the results revealed that these compounds have a strong binding affinity for enoyl-ACP reductase (InhA) and DNA gyrase.[49]

Based on the antitubercular activity of 1,3-triazole, 16 INH-linked 1,2,3-triazole analogues were designed and synthesized by Patil and researchers (2020) and screened for in vitro antitubercular and antimicrobial activities (Scheme [3]). Compounds C30, C34, and C38 showed more potent antitubercular activity among all the screened compounds. These selected compounds showed low cytotoxicity in MTT assays against RAW 264.7 cells. Moreover, the antimicrobial activity of the analogues against both antibacterial and antifungal pathogens revealed compounds C29–C32 and C34 showed better antimicrobial activity making INH-embedded triazole the most potent class of antituberculosis drugs. Further, molecular docking studies supported in vitro biological results and found that the compound C33 fit into the active site of mycobacterial enoyl-ACP reductase (InhA) with an excellent binding affinity score of –8.395 and binding energy of –52.373 kcal/mol.[50]

In another study, Bhoi et al. (2019) synthesized a series of novel INH-containing 4H-pyrimido[2,1-b]benzothiazole derivatives and screened them for their in vitro antibacterial and antimycobacterial activities. Among them, compound C41 showed the most potent antimycobacterial activity against Mtb strain H37Rv with an MIC value of 6.25 μg/mL which is nearly similar to the standard drug INH (0.20 μg/mL). The SAR correlation envisaged potent activity against Mtb strain H37Rv in the trifluoromethoxy group (C41) at the 8th position, while the dimethyl groups at the 7th and 8th positions showed a remarkable MIC value of 12.5 μg/mL and the greatest percentage inhibition value of 82.11% (Figure [2]). Molecular docking studies showed that C41 is the most docked compound bound to the active site of the Mtb enoyl-acyl carrier protein (ACP) reductase (InhA) enzyme and possessed a binding energy of 9.077 kcal/mol. This study suggested that the synthesized analogue C41 has great potential as a promising candidate for further developing antitubercular agents.[51]

Zoom Image
Scheme 3 Reagents and conditions: (i) propargyl bromide, K2CO3, DMF, rt, 6 h (90%); (ii) isonicotinyl hydrazide, DIPEA, rt, 1 h (92%); (iii) CuSO4, sodium ascorbate, DMF, rt (80–92%).
Zoom Image
Figure 2 SAR study of INH-containing 4H-pyrimido[2,1-b]benzothiazole derivatives

In the continuation of ongoing research for potent antitubercular activity, Borad and coworkers (2019) developed a novel series of INH-spirooxindole hybrids as potential antimycobacterial agents. The compounds were evaluated for their in vitro antimycobacterial activity against the Mtb strain H37Rv with the comparison of first-line drug INH. Among them, compound C42 with chloro substitution (MIC = 12.5 μg/mL and % inhibition = 81.88) was found to be the most potent antimycobacterial agent. The SAR of the synthesized compounds are presented in Figure [3]. Molecular docking studies concluded that compound C42 showed good binding in the binding pocket of INH-resistant enoyl-ACP (CoA) reductase mutant enzyme from Mtb.[52]

Zoom Image
Figure 3 SAR analysis of INH-spirooxindole derivatives

In this study, eight novel phenylisoxazole-linked INH derivatives have been synthesized and evaluated for their antitubercular activity against the Mtb strain H37Rv by Carrasco et al. (2021). The biological evaluation study concluded that compounds C43–C50 exhibited moderate bioactivity (MIC = 0.34–0.41 μM) with respect to the standard drug INH (MIC = 0.91 μM) against the Mtb strain H37Rv done by the TEMA assay. Compound C43 (Figure [4]) is the most active compound against the H37Rv strain with less cytotoxicity (MIC >86.21 μM). Compounds C44 and C45 with MIC values of 12.41 μM and 13.06 μM, respectively, were about two times more cytotoxic compared to the INH against the resistant TB DM97 strain. The SAR study demonstrated that the presence of electron-donating groups (CH3 and OCH3) on the C-4′ position of phenyl ring increases the cytotoxicity. This research shows that the presence of the isoxazole group bound to the phenyl ring with different substituent groups improves the antitubercular activity with respect to the cytotoxicity of the reference drug INH.[53]

Zoom Image
Figure 4

The importance of the bioactivity of hydrazone and hydrazide-based compounds was further explored in INH-acyl hydrazide hybrids by Castelo-Branco et al. (2018). Isoniazid-acyl hydrazide hybrids linked by thiacetazone and gem-difluorinated thiosemicarbazide were screened for antimycobacterial activity using a MABA assay against the Mtb strain H37Rv ATCC 27294 (which is susceptible to all first-line anti-TB drugs). The difluorinated derivatives C46 and C47 showed high potential, with an MIC of 3.59 μM and 6.91 μM, respectively. The SAR study revealed that substituents on the para position of the phenyl ring exert detrimental effects on the activity (Figure [5]). It was found that replacing difluoro substituents with a carbonyl group produces exceptional anti-Mtb activity, nearly twice as potent as INH and RIF, and 30-fold and 1,800-fold more potent than EMB and PZA, respectively. Further compounds with better MIC were evaluated for resistant strains of Mtb and results showed that compound C48 was almost four times more potent than INH against this strain. Similarly, compounds with carbonyl groups showed better activity against Mtb strain SR 2571/0215. Moreover, compound C54 has lower hepatotoxic potential. Compounds C48 and C49 showed almost no mutagenicity only at concentrations ≥ 500 μM.[54]

Ghiano and coworkers (2020) reported the synthesis of N-substituted tosyl N′-acrylhydrazine acrylates via an aza-Michael reaction. The approach entails the aldehyde reacting with tosylhydrazine in anhydrous acetonitrile at room temperature. This is then followed by the addition of methyl propionate, resulting in the synthesis of novel E/Z N′-acrylhydrazone derivatives. The synthesized compounds were screened against Mtb strain H37Rv. Biological evaluation indicates that compounds C50–C51 show most potent antitubercular activity against Mtb strain H37Rv with an MIC value of 1.25 μM. The SAR analysis revealed the role of the regioselectivity of the acrylate on antitubercular activity. It was found that out of 30 synthesized compounds, 19 are less effective and 13 are more potent against the Mtb strain H37Rv. Out of these 13 compounds, most of the potent compounds were E isomers with an MIC below 10 mM. The E isomer of the basic aromatic derivative (1E) containing a phenyl group exhibited significant activity and was one of the most potent derivatives (Figure [6]).[55]

Zoom Image
Figure 5 SAR study of hydrazide-linked INH derivatives
Zoom Image
Figure 6 The SAR study of N-substituted tosyl N′-acryl-hydrazone derivatives

The synthesis of a novel series of (E)-N′-(5-X-salicylidene) isonicotinohydrazide derivatives (IL) and its copper (CuL) and cobalt (CoL) complexes was reported by Dueke-Eze et al. (2020). Scheme [4] shows the condensation reaction of INH with the matching 5-substituted 2-hydroxybenzaldehyde to form Schiff bases, which were then reacted with Cu(II) and Co(II) chloride in ethanol to get the appropriate metal complexes (Figure [7]). All the synthesized compounds were evaluated for their antitubercular activity against the Mtb strain H37Rv. The antitubercular activity order for metal ligands and metal complexes was as follows: IL < CuL < CoL, and for each ligand, the order of activity was L4 < L1 < L3 < L2. However, compound C57 displayed excellent antitubercular activity but having high LC50 values so it is not used for further modification. Based on the analysis of results, it was found that the ligand having a nitro group (C62) has better antitubercular activity compared to INH.[56]

Zoom Image
Scheme 4 Synthesis of N′-(5-X-salicylidene) isonicotinohydrazide derivatives
Zoom Image
Figure 7

A new series of isatin nicotinohydrazide derivatives against TB was designed and synthesized by Elsayed et al. (2021). Their antitubercular evaluation against the Mtb strain ATCC 27294 revealed that the majority of the compounds exhibited moderate to good antitubercular activity, with MIC values ranging from 0.24 μg/mL to 7.81 μg/mL. Additionally, compounds C60–C62 showed the highest potency and equipotency as antitubercular INH drugs (MIC = 0.24 mg/mL). According to the findings of the SAR analysis, the antitubercular activity of all the compounds was affected by two main factors: the substitution of nitrogen and the inclusion of halogen at position 5 of the oxindole ring as mentioned in Figure [8]. Based on N-substitution, compounds containing isobutyl groups (C62–C64) showed significant activity with MIC values of 1.95 μg/mL, 0.48 μg/mL, and 0.24 μg/mL. Likewise, compound C60 with an N-benzyl moiety showed strong action (MIC = 0.24 μg/mL). Whereas compounds with the N-ethylcarboxylate group showed the lowest activity (MIC = 7.81 μg/mL). Molecular docking confirmed that the compounds C61 and C62 fit exactly in the active region of the DprE1 enzyme with binding energy values of –9.7 kcal/mol and –10.1 kcal/mol. The N-isobutyl group of C61 also interacted with nonpolar residues bordering a hydrophobic pocket.[57]

Zoom Image
Figure 8 The SAR study of isatin nicotinohydrazide based derivatives

Vosátka and colleagues (2018) designed and synthesized the novel series of N-alkyl-2-isonicotinoylhydrazine-1-carboxamides, and their cyclic analogues were evaluated against Mtb strain H37Ra and two strains of nontuberculous mycobacteria (M. avium, M. kansasii). The synthetic scheme is presented in Scheme [5]. The results revealed that the introduction of small alkyl groups such as methyl, ethyl, and propyl in the carboxamides group enhanced the antitubercular activity (MIC ≤ 2 μM). Interestingly, utilizing pentyl and butyl groups improved anti-Mtb activity (MIC = 8 μM). N-Compounds containing the decyl group showed the highest Mtb cell suppression compared to derivatives with long aliphatic chains. The carboxamides that are most effective against M. avium are those that replace C11 to C16 alkyls (MICs ≤125 μM), with C12 particularly more effective. Moreover, isonicotinoyl-N-propylhydrazine-1-carboxamide was shown to be the most effective drug against both M. kansasii strains, having MIC values of 4–16 μM. Overall, compound C65 was the most potent derivative, inhibiting the growth of both susceptible and drug-resistant Mtb strains with uniform MIC values of 4–8 μM and no cross-resistance to antitubercular medications such as INH. These compounds were also nontoxic for HepG2.[58]

Zoom Image
Scheme 5 Synthesis of N-alkyl-2-isonicotinoylhydrazine-1-carboxamides derivatives

In 2021, de Faria et al. discovered two series of alkyl hydrazides and hydrazones-based INH derivatives with promising in silico properties, including membrane permeability and spontaneous radical formation, which resulted in improved efficacy against the most common resistant Mtb strain S315T compared to INH. The in silico predictions concerning the extremely high reactivity of alkyl hydrazides were validated by the assessments of kinetics, cytotoxicity, and biological activity. The first series comprises the isonicotinoyl hydrazones that originated from aliphatic aldehydes, known as the hydrazone series. Another set in this series includes the reduced counterparts, referred to as the N′-alkyl hydrazides or alkyl hydrazide series. The alkyl hydrazides are likely too reactive, which appears to impede their performance. Compounds from the hydrazone series appear to be just slightly less reactive than INH, resulting in a high rate of hydrolysis of the hydrazone compounds and contributing primarily to INH compounds. The observed MIC values against H37Rv for C66 (4 μM) and C67 (2 μM) are approximately greater than those determined for INH. As a result, increasing the amount of alkyl chains boosts antitubercular activity more than INH. The MIC values for the two studied hydrazone compounds C66 and C67 (Figure [9]) are comparable to those of INH.[59]

Zoom Image
Figure 9

In 2020, Gaonkar and coworkers reported a novel series of INH hydrazone derivatives to improve the therapeutic efficacy of the INH. The synthetic scheme is presented in Scheme [6]. All the synthesized compounds were evaluated against the Mtb strain H37Rv, results found that most of the INH derivatives exhibited moderate to good activity. SAR studies suggested that the compounds with an aryl, heteroaryl, or alkyl group (C68; Figure [10]) substitution on aryl/heteroaryl groups (C69; Figure [10]) exhibited inhibitory activity against the Mtb strain H37Rv. Moreover, the presence of nitro, chloro, hydroxy, and methoxy substituents on aryl/heteroaryl moieties decreases the antimycobacterial activity. The stability studies revealed that the synthesized INH derivatives were more stable than the standard drug INH under basic pH conditions.[60]

Zoom Image
Scheme 6 Synthesis of INH-hydrazone derivatives
Zoom Image
Figure 10

Shah and coworkers (2020) prepared a novel series of N-([2-aryl-5-methyl-1,3-oxazole-4-yl]methylene)isonicotino/nicotino hydrazides derivatives that were designed utilizing molecular hybridization of a biologically active scaffold, INH-oxazole, into a single molecular framework. Biological evaluation studies revealed that C70 and C71 derivatives showed the highest in vitro antitubercular activity against the Mtb strain H37Rv with a MIC value of 1.56 μg/mL. The majority of the compounds exhibited moderate to fair activity in comparison to other anti-TB drugs, such as INH (MIC = 0.1 μg/mL) and rifampicin (MIC = 0.2 μg/mL). Based on the SAR investigations, it was determined that the activity of the compound is influenced by the presence of methyl and methoxy groups on the phenyl ring connected to the oxazole moiety, as shown in Figure [11]. Molecular docking studies revealed that compounds with methoxy substitutions and compounds with methyl substitutions on the phenyl ring possessed greater inhibitory activity than other analogues, as compounds had a higher docking score than other hybrid compounds.[61]

Zoom Image
Figure 11 SAR investigation of INH-oxazole derivatives

In 2021, Santoso et al. designed and synthesized a series of INH-isatin hydrazone derivatives and evaluated them against the Mtb strain H37Rv (Scheme [7]). The biological evaluation concluded that compound C74 was the most potent antitubercular agent (MIC = 0.017 μM) in comparison with rifampicin (MIC = 0.048 μM). Molecular docking studies validated the in vitro results and showed the interaction of INH derivatives with their active site.[62]

Zoom Image
Scheme 7 Synthesis of INH-isatin hydrazone derivatives. Reagents and conditions: (a) NaH (2 equiv), DMF (0.02 M), rt, 1 h, BnBr or 4-Br-BnBr (1 equiv); (b) INH (2 equiv), EtOH (0.003 M), H2SO4 (cat.), reflux, 4 h.

Considering the therapeutic efficacy of INH against Mtb, in 2020 Różycka and researchers designed and synthesized a novel series of INH-carborane derivatives and tested them against the Mtb strain H37Rv and mutant (ΔkatG). Compound C75 (Figure [12]) showed the highest activity against the wildtype Mtb strain, and compound C76 exhibited an increase in activity against Mtb DkatG as compared to INH. Selected compounds containing closo-carborane showed substantial action, while one modified with nido-carborane showed high activity against in vitro Mtb, comparable to INH. All INH-carborane hybrids could inhibit the growth of the ΔkatG mutant in lower concentrations than INH.[63]

Zoom Image
Figure 12

In 2020, Volynets et al. discovered the novel 10 INH derivatives and evaluated them for activity against the Mtb strain H37Rv and INH-resistant strain SRI 1369. Compound C77 (Figure [13]) showed activity against INH-resistant strain SRI 1369 with an MIC value of 0.14 μM. Additionally, it was less resistant to rifampicin than INH. Compound C77 exhibited no cytotoxicity against human liver cells (HepG2; IC50 >100 μM) and demonstrated favorable permeability in Caco-2 cells. The author noted that C77 was active against INH, rifampicin, and fluoroquinolone-resistant strains. It also had good ADME properties and low cytotoxicity against human liver cells (HepG2).[64]

Zoom Image
Figure 13

Keeping in mind the therapeutic significance of the INH against Mtb in addition to the importance of pyrazole as anti-TB agent, Koli et al. planned a one-pot multicomponent synthesis of 5-amino-1-isonicotinoyl-3-(substituted phenyl)-1H-pyrazole-4-carbonitrile derivatives from various substituted aromatic aldehydes, INH, and malononitrile in aqueous EtOH. All 9 synthesized compounds were tested for antibacterial, antioxidant, and antitubercular activities. Most of the compounds showed significant antibacterial and similar antifungal potential within the micromolar range, while nearly all of the synthesized compounds showed potent antioxidant activity. The research findings indicate that compound C78 (Figure [14]) has significant activity against Mtb (MIC = 0.125 μg/mL)[65].

Zoom Image
Figure 14

By following a structure-based molecular hybridization approach, Nazim et al. (2020) prepared the novel series of 1-indanyl INH derivatives (Scheme [8]) and evaluated them against Mtb on 3 mycobacterial strains ATCC H37Rv, known as INH-sensitive (INH-S) and INH-resistant strains (INH-R). In comparison to the positive reference drug INH, compound C79 (1-indanyl-INH derivative, Figure [15]) was more potent than INH and had high antimycobacterial activity against both INH-S and INH-R strains of Mtb.[66]

Zoom Image
Scheme 8 Synthesis of 1-indanyl hydrazide Schiff base derivatives
Zoom Image
Figure 15

To develop novel potent antitubercular agents, Bakale et al. (2023) designed and synthesized the novel series of INH-triazole derivatives using click chemistry as discussed in Scheme [9]. The in vitro antitubercular activity against the Mtb strain H37Rv was examined. Nine compounds were found to be the most active among all of the synthesized INH-triazole derivatives. The MIC value of these compounds was found to be 0.78 μg/mL as compared to the standard drugs ciprofloxacin (MIC = 1.56 μg/mL) and ethambutol (MIC = 3.12 μg/mL). The most active compound of the series was compound C80 (Figure [16]) which exhibited better MIC values in antitubercular activity and efficacy against tuberculosis. The biological results concluded that INH-triazole derivatives having electron-donating and withdrawing substituents showed excellent activities. All the biological active derivatives showed low cytotoxicity against the cell line RAW 264.7. The molecular dynamic study discloses that the simulated protein–ligand complex of 4BFT-144 136 with compound C80 showed the highest stability with minimum deviation.[67]

Mardianingrum et al. (2019) discovered the novel 3 isonicotinohydrazide derivatives N′-(4-fluorobenzoyl)isonicotinohydrazide (C81, Figure [17]), N′-(3-chlorobenzoyl)isonicotinohydrazide (C82), and N′-(3-bromobenzoyl) isonicotinodyrazide (C83), which were evaluated for their antimycobacterial and antibacterial activity. The antibacterial activity revealed that C82 showed the lowest MIC value (0.26 ppm) against Staphylococcus aureus. While C83 exhibited MIC values of 0.30 ppm and 0.24 ppm against Bacillus subtilis and Escherichia coli. As compared to INH, all the tested compounds were found to be less active against the strain H37Rv. The most active compound was C83 against the Escherichia coli (MIC = 0.24 ± 2.20 ppm).[68]

Zoom Image
Scheme 9 Synthesis of INH-triazole derivatives. Reagents and conditions: (a) propargyl bromide, K2CO3, DMF, rt, 6 h (92%); (b) substituted azido benzenes, CuSO4, sodium ascorbate, PEG 400, rt (88–96%); (c) isonicotinohydrazide, AcOH, MeOH, rt, (80–95%).
Zoom Image
Figure 16
Zoom Image
Figure 17

Sampiron and coworkers (2019) developed 8 novel hydrazones, benzohydrazones, and INH-acylhydrazones as potential antituberculosis agents. Biological evaluation studies disclose that INH-acylhydrazone derivatives showed significant activity against the standard Mtb strain, with excellent results. Compounds C84 and C85 (Figure [18]) showed the lowest MIC values (0.12 μg/mL) among all the compounds, while C86 also presented good activity (0.24 μg/mL) against Mtb. INH-acylhydrazone C85 presented significant activity against NTM, with a significant MIC value of 3.9 μg/mL against Mycobacterium smegmatis. The author concluded that INH-acylhydrazone derivatives such as C84, especially C85, and C86 had surprising MIC values, better than those given by first-line drugs used for TB treatment, such as EMB (1–5 μg/mL), and close to RIF (0.05–0.50 μg/mL) and INH (0.02–0.20 μg/mL).[69]

Zoom Image
Figure 18

Oliveria and coworkers (2017) synthesized a series of INH derivatives tethered phenolic or heteroaromatic derivatives by mechanochemical technique and evaluated them in vitro for Mtb strain H37Rv. Compounds containing isonicotinoyl derivatives showed good antimycobacterial action with MIC values of 0.0125 μg/mL or 0.125 μg/mL. Compound C87 (Figure [19]) was significantly inhibiting InhA enzyme (79% at 50 μM). Comparing isonicotinoyl derivatives derived from indoles, it can be concluded that compounds with an indol-3-yl frame exhibit similar activity, with inhibition values ranging from 32% to 43%. Compound C88 with a nitro substituent increases activity against Mtb multidrug-resistant clinical isolates. On cytotoxicity evaluation of all the compounds, it was found that derivatives with N-heterocyclic INH showed a good SI value on MRC5 human fibroblast cells.[70]

Zoom Image
Figure 19

The same research group designed the 24 heterocyclic amine-azachalcones compounds with antituberculosis potential against the Mtb strain H37Rv. Compounds were synthesized via Claisen–Schmidt condensation. Fifteen of the synthetic derivatives were shown to be effective against Mtb. Analogues C89 and C90 (Figure [20]) were shown to be less cytotoxic (SI = 1.39 and 3.49, respectively), and displayed a stronger potential for antituberculosis activity (MIC = 6.62 μM and 4.85 μM, respectively). The SAR study revealed that morpholine derivatives showed better MIC than thiomorpholine derivatives. Furthermore, the presence of a six-membered heteroaromatic ring that bonds directly to the cyclic amine group, as well as hybrid pyrrolidine-substituted derivatives showed no significant effect on antitubercular activity. The antitubercular activity of compound C91 was the best out of all the synthesized derivatives, with a MIC of 9.54 μM and SI of 9.33.[71]

Zoom Image
Figure 20 SAR study of the designed amine-azachalcones-based compounds

By considering the various structural aspects of INH in tuberculosis treatment, Atta et al. (2018) designed the novel hybrids of pyridine-INH derivatives and evaluated them for their antitubercular activity against Mtb strain H37Rv. After in vitro antimycobacterial evaluation, compounds C92–C95 (MIC = 7.30–8.74 μM, Figure [22]) were found to be the most potent analogues having the least cytotoxicity effect on the HEK cell line as compared to INH. The SAR analysis is discussed in Figure [21]. These compounds showed good fitting scores into the InhA active site in the docking studies better than the standard antitubercular drug INH. In addition, in silico drug-likeness prediction revealed that most of the studied compounds are safe and possess promising pharmacokinetic features.[72]

Zoom Image
Figure 21 SAR discussion of INH derivatives
Zoom Image
Figure 22

As quinoline was found in bedaquiline, a recently developed anti-TB drug, Rani and colleagues (2020) used the molecular hybridization approach to develop INH-quinoline compounds. These quinoline and INH scaffolds were linked through an isoindoline-1,3-dione linkage. Further, the compounds were screened for antimycobacterial activities against the avirulent Mtb strain mc26230 and cytotoxicity against mammalian Vero cells. Most of the synthesized compounds showed promising antimycobacterial activity (MIC = 5.1–11.9 μM) and noncytotoxicity against mammalian Vero cells. The SAR investigations showed that introducing INH at C-5 of the isoindoline ring increased the antimycobacterial activity while also reducing the cytotoxicity. The hybrid of quinidine and INH that included an octyl spacer maintained its noncytotoxic (IC50 ≥ 167 μM) properties while maintaining its antimycobacterial potency (MIC99 = 5.1 μM; Figure [23]). Molecular docking studies validated that the synthesized compounds bind in the same way within the active site of InhA.[73]

Zoom Image
Figure 23 SAR analysis of quinazoline-INH derivatives
Zoom Image
Figure 24

Kumar et al. (2018) have designed novel reversed INH via structural modification of various efflux pump inhibitor (EPI) cores with covalently linking INH to reduce the emergence of drug resistance mediated by EPI. The authors synthesized the novel 14 reversed INH agents and 2 benzhydrazide-based compounds and further evaluated for antimycobacterial activity against sensitive, INH monoresistant, and MDR clinical isolates of Mtb. The biological activity results concluded that compounds C96–C98 (Figure [24]) had comparable activity as INH. From the SAR study of the synthesized analogues, it was concluded that chloro-substituted phenothiazine (C96 and C97) and thioxanthene (C98) based compounds exhibited higher activity compared to their unsubstituted derivatives. However, introducing piperidine and piperazine moieties to EPI structural motifs improved efficacy against Mtb isolates that were drug sensitive (H37Rv), INH monoresistant (R5401), and MDR (X_61 and X_60). The presence of the N-atom in the pyridyl moiety of RINH drugs was responsible for their antimycobacterial action. It was found that substituting the INH moiety with a benzhydrazide group resulted in a significant decrease in the potency of the antimycobacterial effect. Additionally, compounds C96, C97, C99 and C100 (MIC90 ≤ 13.42 μM) exhibit >90% inhibition of macrophage activity, and these compounds also demonstrate encouraging inhibition of EB efflux.[74]

In another study by Patel et al. (2020) pyridine has been combined with INH using the pharmacophore hybrid approach to increase the biological response to Mtb and/or avoid resistance events. Compounds C101 and C102 (MIC = 7.8 μM and 4.5 μM; IC50 = 3.2 μM and 1.5 μM) showed the most promising antimycobacterial action among all the synthesized derivatives. According to the SAR analysis of antimycobacterial activity, compounds with ethyl, furyl, methyl, and cyclopropyl ring substitution at position 2 of the quinazoline ring were responsible for increased antitubercular activity. The presence of a methyl group as an electron-donating substituent in Schiff bases derived from INH showed significant effects on the antimycobacterial activity, as compared to the electron-withdrawing group at position 5 of the pyridine ring. It is interesting to note that, when compounds evaluated for antimycobacterial activity in hypoxic conditions, C101 and C102 (Figure [25]) demonstrated reasonable low oxygen recovery activity (LORA) as compared to rifampicin.[75]

Zoom Image
Figure 25

Kaur et al. (2020) designed INH and pyrimidine hybrid analogues through a molecular modeling study for antitubercular activity against the Mtb strain H37Rv. The schematic path for the synthesis of the designed compounds is presented in Scheme [10]. The compounds C103 and C104 (Figure [26]) containing a phenyl group at position C-6 of the pyrimidine scaffold, exhibited the highest level of activity (MIC99 = 10 μM) and were the least cytotoxic components of the series. The most active compounds C103 and C104 were found to interact with bovine lactoperoxidase (PDB ID: 3I6N) as well as a cytochrome C peroxidase (PDB ID: 2V2E) mutant N184R Y36A.[76]

Zoom Image
Scheme 10 Synthesis of pyrimidine-INH derivatives. Reagents and conditions: (a) HCl, NH4Cl, PPE; (b) HNO3, 0 °C; (c) POCl3, 105 °C; (d) K2CO3/DMF.
Zoom Image
Figure 26

In the search of an improved pharmacokinetic profile of antitubercular drugs, Dragostin et al. (2019) discovered the novel INH derivatives with the aim of high potency and efficacy against resistance of Mtb. The designed compounds were synthesized using Scheme [11]. All the tested compounds showed lox cytotoxicity against fibroblast cell line. Moreover, all the compounds were found to be on the Mtb strain ATCC 25177. In conclusion, all the synthesized INH derivatives showed a good biocompatibility on cell morphology analysis.[77]

Zoom Image
Scheme 11 Synthesis of INH-linked derivatives

Pflégr et al. (2021) adopted a novel approach to designing compounds known as ‘mee-too’. This technique combines INH with aniline, using pyruvic acid as a bridge. As per the synthetic scheme, a total of 22 compounds were synthesized (Scheme [12]). All the synthesized derivatives were screened for susceptible Mtb strain H37Rv and nontuberculous mycobacteria, and it was found that most of the compounds displayed a significant effect against Mtb, M. kansasii, as well as MDR-TB. Compounds having CF3 and OCF3 groups on the position 4 of the aniline ring were the most potent derivatives against MDR-TB). In the mechanistic study, it was found that these compounds inhibit the enoyl-ACP reductase (InhA) in mycobacteria. In addition, none of them show any cytotoxic and cytostatic activity for HepG2 cells.[78]

Zoom Image
Scheme 12 Synthesis of 2-(2-isonicotinoylhydrazineylidene)propanamide derivatives. Reagents and conditions: (a) pyruvic acid reflux, 2 h; (b) EDC·HCl, HOBT, DMF, 0 °C.

Shtyrlin and coworkers (2021) designed and synthesized the novel series of isonicotinoyl hydrazones based on pyridoxine, evaluated for their antimycobacterial activity against Mtb strain H37Rv. Biological evaluation studies revealed that compound C108 (MIC = 0.62 μg/mL) was the most potent against the Mtb strain H37Rv. Likewise, cytotoxicity studies demonstrate that compound C108 (Figure [27]) was also less cytotoxic as compared to INH moxifloxacin and ethambutol. Interestingly, compound C108 showed weak complexation with Fe3+ ions, low acute toxicity (LD50 > 2000 mg/kg per one mice), and equally efficacious as reported in INH, ethambutol, and moxifloxacin in the mouse model of drug-sensitive TB.[79]

Zoom Image
Figure 27

Pflégr and coworkers (2022) developed a novel series of 23 N-(cyclo)alkyl-2-(2-isonicotinoylhydrazineylidene)propenamide derivatives. All the synthesized compounds were evaluated against the Mtb strain H37Rv. The synthetic strategy was based upon condensation of INH and pyruvic acid, followed by carbodiimide-mediated coupling. The biological result revealed that most of the derivatives demonstrated MIC against Mtb ranging from ≤ 0.125 μM to 2 μM. The most active molecules C109 were substituted by a longer n-alkyl chain (n = 8–14; Figure [28]). These compounds showed comparable or even several times lower MIC values than the parent isonicotinohydrazide.[80]

Zoom Image
Figure 28

A novel series of indole and INH-based derivatives were designed and synthesized by Rathod et al. (2020). All the synthesized compounds were screened in vitro for antitubercular activity against the Mtb strain H37Rv. Results revealed that compounds C110 and C111 (Figure [29]) were found to be most potent against the Mtb strain H37Rv (MIC = 3.12 μg/mL and 6.25 μg/mL). The other compounds were moderately active (MIC = 12–50 μg/mL) against the Mtb strain H37Rv in comparison to the standard antitubercular drugs INH (0.625 μg/mL). The SAR studies revealed that electron-withdrawing groups preferentially increase the antitubercular activity. Molecular docking study of these compounds showed that C110 and C111 revealed similar binding modes and binding interactions to those observed for standard drugs such as INH, PZA, STM, and CPF.[81]

Zoom Image
Figure 29

# 4

Conclusion

TB is still the most common infectious illness in the world due to limited availability of chemotherapeutic medications for MDR, XDR, and HIV/AIDS patients. The present review summarizes the rationale for molecular hybridization of isoniazid with various heterocyclic scaffolds to improve the properties of INH that are effective against MDR and XDR tuberculosis. Despite the enormous number of INH-based derivatives synthesized and analyzed, only a few compounds in each study stood out as having superior activity to INH. Based on the results of all the studies discussed in this review, it can be concluded that INH, when combined with other heterocyclic or cyclic moieties, may demonstrate improved activity against drug-resistant Mycobacterium tuberculosis.


# 5

Abbreviations

Table 3 Abbreviations

acyl carrier protein

ACP

absorption, distribution, metabolism and excretion

ADME

coenzyme A

CoA

ciprofloxacin

CPF

directly observed therapy

DOTS

ethidium bromide

EB

ethambutol

EMB

efflux pump

EP

efflux pump inhibitor

EPI

human embryonic kidney

HEK

human immunodeficiency virus/ acquired immunodeficiency syndrome

HIV/AIDS

enoyl-ACP reductase

InhA

isoniazid, isonicotinic acid hydrazide

INH

INH-resistant

INH-R

INH-sensitive

INH-S

low oxygen recovery activity

LORA

multidrug-resistant

MDR

multidrug-resistant tuberculosis

MDR-TB

minimal inhibitory concentrations

MIC

Mycobacterium tuberculosis

Mtb

nicotinamide adenine dinucleotide

NAD

non-tuberculous mycobacteri

NTM

protein data bank ID

PDB (PDB ID)

pyrazinamide

PZA

rifampicin

RIF

reversed isoniazids

RINH

structure–activity relationship

SAR

selectivity index

SI

streptomycin

STM

tuberculosis

TB

tetrazolium microplate assay

TEMA

World Health Organization

WHO

extensively drug-resistant

XDR

#
#

Conflict of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The corresponding author Shweta Mishra acknowledge the Shree Guru Gobind Singh Tricentenary University, Gurugram, Haryana for providing research facilities.


Corresponding Authors

Shweta Mishra
SGT College of Pharmacy, SGT University
Gurugram, Haryana, 1225052
India   

Adarsh Sahu
Department of Pharmaceutical Sciences, Dr. Harisingh Gour Central University
Sagar, 470003
India   

Publication History

Received: 30 November 2024

Accepted after revision: 03 February 2025

Accepted Manuscript online:
03 February 2025

Article published online:
16 May 2025

© 2025. Thieme. All rights reserved

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany


   Permissions and Reprints All articles of this category
Zoom Image
Scheme 1 Synthesis of 3-[(2-isonicotinoylhydrazono)methyl]phenyl 4-substituted benzenesulfonate derivatives
Zoom Image
Figure 1
Zoom Image
Scheme 2 Reagents and conditions: (i) propargyl bromide, K2CO3, DMF, rt, 6 h (92%); (ii) isonicotinohydrazide, DIPEAc, rt, 1 h (90%); (iii) substituted azidobenzenes, CuSO4, Na ascorbate, DMF, rt (80–90%).
Zoom Image
Scheme 3 Reagents and conditions: (i) propargyl bromide, K2CO3, DMF, rt, 6 h (90%); (ii) isonicotinyl hydrazide, DIPEA, rt, 1 h (92%); (iii) CuSO4, sodium ascorbate, DMF, rt (80–92%).
Zoom Image
Figure 2 SAR study of INH-containing 4H-pyrimido[2,1-b]benzothiazole derivatives
Zoom Image
Figure 3 SAR analysis of INH-spirooxindole derivatives
Zoom Image
Figure 4
Zoom Image
Figure 5 SAR study of hydrazide-linked INH derivatives
Zoom Image
Figure 6 The SAR study of N-substituted tosyl N′-acryl-hydrazone derivatives
Zoom Image
Scheme 4 Synthesis of N′-(5-X-salicylidene) isonicotinohydrazide derivatives
Zoom Image
Figure 7
Zoom Image
Figure 8 The SAR study of isatin nicotinohydrazide based derivatives
Zoom Image
Scheme 5 Synthesis of N-alkyl-2-isonicotinoylhydrazine-1-carboxamides derivatives
Zoom Image
Figure 9
Zoom Image
Scheme 6 Synthesis of INH-hydrazone derivatives
Zoom Image
Figure 10
Zoom Image
Figure 11 SAR investigation of INH-oxazole derivatives
Zoom Image
Scheme 7 Synthesis of INH-isatin hydrazone derivatives. Reagents and conditions: (a) NaH (2 equiv), DMF (0.02 M), rt, 1 h, BnBr or 4-Br-BnBr (1 equiv); (b) INH (2 equiv), EtOH (0.003 M), H2SO4 (cat.), reflux, 4 h.
Zoom Image
Figure 12
Zoom Image
Figure 13
Zoom Image
Figure 14
Zoom Image
Scheme 8 Synthesis of 1-indanyl hydrazide Schiff base derivatives
Zoom Image
Figure 15
Zoom Image
Scheme 9 Synthesis of INH-triazole derivatives. Reagents and conditions: (a) propargyl bromide, K2CO3, DMF, rt, 6 h (92%); (b) substituted azido benzenes, CuSO4, sodium ascorbate, PEG 400, rt (88–96%); (c) isonicotinohydrazide, AcOH, MeOH, rt, (80–95%).
Zoom Image
Figure 16
Zoom Image
Figure 17
Zoom Image
Figure 18
Zoom Image
Figure 19
Zoom Image
Figure 20 SAR study of the designed amine-azachalcones-based compounds
Zoom Image
Figure 21 SAR discussion of INH derivatives
Zoom Image
Figure 22
Zoom Image
Figure 23 SAR analysis of quinazoline-INH derivatives
Zoom Image
Figure 24
Zoom Image
Figure 25
Zoom Image
Scheme 10 Synthesis of pyrimidine-INH derivatives. Reagents and conditions: (a) HCl, NH4Cl, PPE; (b) HNO3, 0 °C; (c) POCl3, 105 °C; (d) K2CO3/DMF.
Zoom Image
Figure 26
Zoom Image
Scheme 11 Synthesis of INH-linked derivatives
Zoom Image
Scheme 12 Synthesis of 2-(2-isonicotinoylhydrazineylidene)propanamide derivatives. Reagents and conditions: (a) pyruvic acid reflux, 2 h; (b) EDC·HCl, HOBT, DMF, 0 °C.
Zoom Image
Figure 27
Zoom Image
Figure 28
Zoom Image
Figure 29