Novel isoniazid derivative as promising antituberculosis agent

Abstract

Background:

A major focus of tuberculosis drug discovery is aimed at the development of novel antibiotics with activity against drug-resistant strains of Mycobacterium tuberculosis.

Results:

We have synthesized ten isoniazid derivatives and investigated for antibacterial activity toward M. tuberculosis H37Rv and isoniazid-resistant strain SRI 1369. It was revealed that only one compound, isonicotinic acid (1-methyl-1H-pyrrol-2-ylmethylene)-hydrazide (1), is active toward isoniazid-resistant strain with minimum inhibitory concentration value of 0.14 μM. This compound is not cytotoxic toward human liver cells (HepG2; IC₅₀ >100 μM), demonstrates good permeability in Caco-2 cells. Accordingly to the results of plasma protein binding assay, unbound fraction of compound 1, which potentially exhibits pharmacologic effects, is 57.9%.

Conclusion:

Therefore, isonicotinic acid (1-methyl-1H-pyrrol-2-ylmethylene)-hydrazide is a promising compound for further preclinical studies.

Isoniazid belongs to the most efficient first-line antituberculosis drug with minimum inhibitory concentration (MIC) value of 0.01–0.2 μg/ml (0.0729–1.458 μM) for actively replicating Mycobacterium tuberculosis [1]. Isoniazid is also bacteriostatic against dormant forms of mycobacteria; therefore, it is also used in clinical medicine for treatment of persistent tuberculosis. However, due to the long-term antimycobacterial therapy, isoniazid-resistant M. tuberculosis strains are becoming more widespread, inducing the development of novel effective antituberculosis agents. A number of attempts have been made to find novel antituberculosis drugs. Unfortunately, the process of antituberculosis drug discovery is very slow. For example, in 2012, for the first time in the 40 years, only one novel antibiotic, bedaquiline, was approved by US FDA for the treatment of tuberculosis. Other antibiotic delamanid was approved in 2014 by the EMA for clinical application in Europe, Japan and South Korea. However, the cases of bedaquiline and delamanid resistance have been already published [2–7]. In 2019, FDA also approved novel antibiotic pretomanid (PA-824) [8] for medical use in the USA. Several experimental antituberculosis drugs from novel chemical classes, such as PNU-100480 [9] and SQ109 [10] are undergoing clinical trials.

Therefore, nowadays, one of the main approaches to develop novel antimycobacterial agents still remains redesigning old drugs with the aim to create analogs effective against resistant M. tuberculosis strains.

Material & methods

Chemical synthesis

A mixture of 1 mmol of isonicotinic acid hydrazide, 1 mmol of corresponding aldehyde, acetophenone or pyruvic acid ethyl ester in 75 ml of water-ethanol (1:1) solution was heated to reflux for 20–60 min to give a precipitate. Then, reaction mixture was cooled and filtered. Resulted compounds were washed twice with water (10 ml) and then were washed twice with ethanol (10 ml). The obtained compounds did not require further purification and recrystallization.

Starting materials and solvents were purchased from commercial suppliers and were used without further purification. ¹H nuclear magnetic resonance (NMR) spectra were recorded on a Varian VXR 400 instrument at 400 MHz with internal standard of tetramethylsilane. Chemical shifts are reported as parts per million (δ) and spin multiplicities are shown as s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet) or m (multiplet).

HPLC-mass spectrometry (MS) analysis was carried out using the Agilent (CA, USA) 1100 LC/MSD SL separations module and Mass Quad G1956B mass detector as described earlier [11].

The purity of compounds was >95%.

Isonicotinic acid (1-methyl-1H-pyrrol-2-ylmethylene)-hydrazide (1)

Yield 72%; m.p. 187°C. MS [m/z]: 229 [M⁺H⁺], R_t = 0.68 min. ¹H NMR (dimethyl sulfoxide [DMSO]-d₆): δ: 3.89 (s, 3H, CH₃), 6.13 (t, 1H, CH, J = 7.3), 6.53 (d, 1H, CH, J = 7.5), 7.00 (s, 1H, CH), 7.80, 7.80 (d, 2H, CH, J = 8.3), 8.37 (s, 1H, CH), 8.70 (d, 2H, CH, J = 8.3), 11.75 (s, 1H, NH).

2-[(Pyridine-4-carbonyl)-hydrazono]-propionic acid ethyl ester (2)

Yield 71%; m.p. 162°C (dec.). MS [m/z]: 236 [M⁺H⁺], R_t = 0.88 min. ¹H NMR (DMSO-d₆): δ: 47 (t, 1H, CH, J = 7.3), 1.25 (t, 3H, CH₂CH₃, J = 7.0), 2.17 (s, 3H, CH₃), 4.32 (q, 2H, CH₂CH₃, J = 7.0), 7.76 (d, 2H, CH, J = 8.2), 8.76 (d, 2H, CH, J = 8.2), 11.16 (s, 1H, NH).

Isonicotinic acid pyridin-2-ylmethylene-hydrazide (3)

Yield 76%; mp 188°C (dec.). MS [m/z]: 227 [M⁺H⁺], R_t = 0.84 min. ¹H NMR (DMSO-d₆): δ: 7.47 (t, 1H, CH, J = 7.3), 7.64 (d, 2H, CH, J = 8.2), 7.73–8.11 (m, 2H, CH), 8.5 (s, 1H, CH), 8.72 (d, 1H, CH, J = 8.0), 8.62 (d, 2H, CH, J = 8.2), 12.25 (s, 1H, NH).

Isonicotinic acid (2-methyl-benzothiazol-6-ylmethylene)-hydrazide (4)

Yield 77%; m.p. 303°C (dec.). MS [m/z]: 297 [M⁺H⁺], R_t = 1.21 min. ¹H NMR (DMSO-d₆) δ, 2.83 (s, 3H, CH₃), 7.82 (d, 2H, CH, J = 8.2), 7.88–7.96 (m, 2H, CH), 8.38 (s, 1H, CH), 8.58 (s, 1H, CH), 8.79 (d, 2H, CH, J = 8.2), 12.06 (s, 1H, NH).

Isonicotinic acid (4-methyl-benzylidene)-hydrazide (5)

Yield 85%; m.p. 166°C. MS [m/z]: 240 [M⁺H⁺], R_t = 1.24 min. ¹H NMR (DMSO-d₆) δ, 2.41 (s, 3H, CH₃), 7.30 (d, 2H, CH, J = 7.8), 7.62 (d, 2H, CH, J = 7.8), 7.78 (d, 2H, CH, J = 8.2), 8.43 (s, 2H, CH), 8.74 (d, 2H, CH, J = 8.2), 12.02 (s, 1H, NH).

Isonicotinic acid {1-[2-hydroxy-3-(2-hydroxy-1,1-dimethyl-ethylamino)-propyl]-1H-indol-3-ylmethylene}-hydrazide (6)

Yield 71%; m.p. 113°C (dec.). MS [m/z]: 410 [M⁺H⁺], R_t = 0.59 min. ¹H NMR (DMSO-d₆) δ, 0.98 (s, 6H, CH₃), 3.21 (s, 4H, CH₂), 3.81 (s, 1H, OH), 4.11 (t, 1H, CH, J = 7.3), 4.91 (s, 1H, OH), 7.19–7.26 (m, 2H, CH), 7.55 (d, 1H, CH, J = 7.8), 7.76 (s, 1CH), 7.88 (d, 2H, CH, J = 8.3), 8.35 (d, 1H, CH, J = 7.8), 8.62 (s, 1H, CH), 8.74 (d, 2H, CH, J = 8.2), 11.56 (s, 1H, NH).

Isonicotinic acid [1-(3-nitro-phenyl)-ethylidene]-hydrazide (7)

Yield 75%; m.p. 285°C. MS [m/z]: 229 [M⁺H⁺], R_t = 1.15 min. ¹H NMR (DMSO-d₆) ¹H NMR (DMSO-d₆): δ: 2.48 (s, 3H, CH₃), 7.53–7.93 (m, 4H, CH), 8.29 (d, 2H, CH, J = 8.2), 8.67 (s, 1H, CH), 8.77 (d, 2H, CH, J = 8.2), 11.36 (s, 1H, NH).

Isonicotinic acid (2-nitro-benzylidene)-hydrazide (8)

Yield 82%; m.p. 271°C (dec.). MS [m/z]: 229 [M⁺H⁺], R_t = 1.12 min. ¹H NMR (DMSO-d₆) δ, 6.53–6.75 (m, 2H, CH), 7.88 (d, 2H, CH, J = 8.2), 8.10–8.35 (m, 2H, CH), 8.80 (d, 2H, CH, J = 8.2), 8.92 (s, 1H, CH), 12.42 (s, 1H, NH).

Isonicotinic acid [1-(4-nitro-phenyl)-ethylidene]-hydrazide (9)

Yield 79%; m.p. 285°C. MS [m/z]: 229 [M⁺H⁺], R_t = 1.14 min. ¹H NMR (DMSO-d₆) δ, 2.48 (s, 3H, CH₃), 7.58 (d, 2H, CH, J = 8.2), 7.98 (d, 2H, CH, J = 8.2), 8.28 (d, 2H, CH, J = 8.2), 8.65 (s, 1H, CH), 8.77 (d, 2H, CH, J = 8.2), 11.22 (s, 1H, NH).

Isonicotinic acid (2,4-dihydroxy-benzylidene)-hydrazide (10)

Yield 75%; m.p. 287°C (dec.). MS [m/z]: 258 [M⁺H⁺], R_t = 0.69 min. ¹H NMR (DMSO-d₆): δ: 6.23–6.35 (m, 3H, CH), 7.24 (d, 2H, CH, J = 7.8), 7.83 (d, 2H, CH, J = 8.2), 8.49 (s, 1H, CH), 8.74 (d, 2H, CH, J = 8.2), 9.91 (s, 1H, OH), 11.19 (s, 1H, OH), 12.02 (s, 1NH).

MIC determination

Testing of compounds was conducted using 96-well, U-bottom microplates with an assay volume of 0.2 ml/well. At first, the test media, Middlebrook 7H9 broth supplemented with OADC Enrichment (0.1% casitone, 5.6 μg/ml palmitate, 0.5% bovine serum albumin and 4 μg/ml catalase) (BD BioSciences; MD, USA) without compounds, was added (0.1 ml/well) to each well. The test compounds, solubilized in DMSO to make 10 mM solution and diluted in the assay medium to a concentration of 100 μM, were further supplemented to corresponding wells (0.1 ml/well) at twice the planned initial concentration and serially twofold diluted. Then, M. tuberculosis cells at the concentration 1.0 × 10⁶ CFU/ml were added to each well (0.1 ml/well) and incubated during 7 days at 37°C and 90% air humidity. After incubation, the plates were analyzed visually and also each well was read for turbidity, partial or complete clearing. Testing was performed in two independent experiments. As a positive control, isoniazid or rifampicin was used; as a negative control, bacteria in the medium without the addition of compounds, and for sterility control, only the medium was taken. The MIC value was measured as the lowest compound concentration which visually completely inhibits bacterial growth.

Bacteria

MIC screening was conducted for M. tuberculosis H37Rv (SRI 1345), isoniazid-resistant M. tuberculosis (SRI 1369), which is a katG mutant (S315L), rifampin-resistant M. tuberculosis (SRI 1367), which is rpoB mutant (S450L) and ofloxacin-resistant M. tuberculosis (SRI 4000), which is a gyrA mutant (D94N).

Minimal bactericidal concentration determination

Minimal bactericidal concentration (MBC) was determined subsequent to MIC testing by subculturing diluted aliquots from wells that fail to exhibit macroscopic growth. A total of 100 μl aliquots were removed from the wells and inoculated onto Middlebrook 7H10 agar plates (one plate per well) and then incubated for 16–21 days at 37°C. Once growth was apparent, the bacterial colonies were enumerated. The MBC was defined as the lowest concentration of compound exhibiting 99.9% kill over the same time period used to determine the MIC (18–24 h).

Intracellular drug activity

The purpose of this assay is to assess the inhibition of M. tuberculosis H37Rv phagocytized by J774.A1 macrophages when exposed to a series of test compounds. J774.A1 is an adherent macrophage cell line originating in mice routinely used for the determination of inhibition of intracellular pathogens. The cells were grown in RPMI 1640 medium containing L-glutamine and fetal bovine serum (FBS). J774.A1 cells were maintained in tissue culture flasks at 37°C in the air with 5% CO₂. For infection experiments, macrophage cells were added to 12-well tissue culture chambers at a density 2.0 × 10⁵ (1 ml/well) in the medium with 10% FBS and cultivated overnight. Then, the medium was replaced with fresh medium supplemented with 1% FBS to prevent further macrophage proliferation. After incubation for 24 h the number of cells per well was calculated using ocular micrometer. The medium was changed with fresh medium supplemented with 1% FBS and inoculated with M. tuberculosis at a multiplicity of infection of five Mycobacteria/macrophage. The macrophage cells were infected during 4 h and then nonphagocytosed Mycobacteria were removed by washing and fresh medium was added. The compounds were added at three concentrations and infection proceeded for 7 days [12].

At the end of the compound exposure period, assay medium containing test compound was removed and each well was washed three times with phosphate-buffered saline (PBS). At 0 and 7 days, the macrophages were lysed by replacing the growth media with 1 ml of filter sterilized 0.25% (w/v) sodium dodecyl sulfate in PBS. A total of 100 μl of lysate was removed from each well, diluted in sterile PBS and then plated on Middlebrook 7H10 agar. The 7H10 agar plates were incubated at 37°C for 16–21 days to determine the cell number or CFU. In order to determine the percent reduction of intracellular mycobacteria, the number of colonies resulting from the test wells was divided by the number of colonies resulting from the negative control wells and the results were multiplied by 100.

The compounds in each concentration were tested in two independent experiments. Rifampin was taken as a positive control. Also, in parallel, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed in order to confirm that the compounds are not cytotoxic toward uninfected macrophages.

Intracellular drug-screening assays were conducted using only M. tuberculosis H37Rv (SRI 1345).

Absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties investigation

Plasma protein-binding, Caco-2 permeability and HepG2 cytotoxicity assays were performed as described previously [13].

Cytochrome P450 inhibition assay

Compound 1 was tested for inhibition of six cytochrome P450 enzyme isoforms – CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 [14–16]. Compounds were prepared as a 7-point dilution series in acetonitrile (Fisher Scientific, NJ, USA): DMSO (Fisher Scientific, 9:1). The final DMSO content in the reaction mixture was equal in all solutions used within an assay and was <0.2%. Samples were run in duplicate. Compounds were incubated with human liver microsomes in sample buffer containing 2 mM NADPH (Sigma, MO, USA) and probe substrate in a 200 μl assay final volume. Reactions were incubated at 37°C for the optimal time (10–60 min) and terminated by addition of methanol containing internal standard (propranolol) for analytical quantification. Samples were incubated at 4°C for 10 min and centrifuged at 4°C for 10 min. The supernatant was removed and the probe substrate metabolite was analyzed by LC–MS/MS using an Agilent 6410 mass spectrometer coupled with an Agilent 1200HPLC and a CTC PAL chilled autosampler, all controlled by MassHunter software (Agilent). After separation on a C18 reverse phase HPLC column (Agilent Zorbax StableBond 3.5 μm, 2.1 × 30 mm) using an acetonitrile (acetonitrile with 0.1% formic acid) – water (H₂O with 0.1% formic acid) gradient system, peaks were analyzed by MS using electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. A decrease in the formation of the metabolite compared with vehicle control was used to calculate an IC₅₀ value (the test concentration that produces 50% inhibition).

The enzyme/substrate pairs, incubation time and control inhibitors for each isoform are listed in Table 1.

Table 1. . The enzyme/substrate pairs, incubation time and control inhibitors for each isoform of cytochrome P450.

P450 isoform	Substrate	Substrate concentration (μM)	Human liver microsome concentration (mg/ml)	Incubation time (min)	Positive control
CYP2B6	Bupropion	100	0.25	10	Ticlopidine
CYP2C8	Amodiaquine	5	0.25	10	Montelukast
CYP2C9	Tolbutamide	100	0.5	15	Sulphenazole
CYP2C19	Mephenytoin	100	0.25	60	Tranylcypromine
CYP2D6	Dextromethorphan	5	0.5	10	Quinidine
CYP3A4	Midazolam	2.5	0.25	10	Ketoconazole
CYP3A4	Testosterone	50	0.25	10	Ketoconazole

In vitro microsomal stability assay

Compound was tested for microsomal stability using pooled human liver S9 microsomes (Celsis, MD, USA) [17,18]. Compounds were incubated with human liver microsomes at 37°C in duplicate. Each reaction contained 0.3 mg/ml human microsomal protein in assay buffer (2 mM NADPH (Sigma), 3 mM MgCl₂ (Sigma), 100 mM potassium phosphate buffer pH 7.4 (BD Gentest, MA, USA)). Samples were removed at 0, 5, 15, 30 and 45 min, mixed with an equal volume of stop solution (ice cold methanol containing propranolol as an internal standard) and incubated for >10 min at -20°C. An additional volume of water was added, samples were centrifuged to remove precipitated protein and the supernatants were analyzed by LC–MS/MS to quantitate the remaining parent compound. A control reaction omitting NADPH (control buffer) was performed for compound to detect NADPH-free degradation. Verapamil (Sigma) and dextromethorphan (Sigma) were included as control compounds.

Determination of compound microsomal half-life stability and intrinsic clearence (CL_int). Data were converted to % compound remaining compared with the time zero. Data were fitted to a first order decay model to determine the compound half-life. CL_intwas calculated from the half-life and the protein concentrations:

$$\begin{array}{l}{\text{CL}}_{int}=ln(2)/({\text{T}}_{1/2} [\text{microsomal} \text{protein}])\hfill \\ {\text{T}}_{1/2}=0.693/-\text{k}\hfill \\ {\text{CL}}_{int}=\text{intrinsic} \text{clearance}; {\text{T}}_{1/2}=\text{half}-\text{life}; \text{k}=\text{slope}\hfill \end{array}$$

Results

In order to find novel compounds active toward isoniazid-resistant isolates we have synthesized and investigated ten isoniazid derivatives. The activity of compounds was determined toward H37Rv and isoniazid-resistant strain under aerobic conditions. As it can be seen from Table 2, the only one compound – isonicotinic acid (1-methyl-1H-pyrrol-2-ylmethylene)-hydrazide (1) was active toward isoniazid-resistant strain SRI 1369 with MIC value of 0.14 μM.

Table 2. . Structures and antituberculosis activity of isonicotinic acid hydrazide derivatives (IC₅₀ [μM], IC₉₀ [μM] and minimum inhibitory concentration [μM]) toward Mycobacterium tuberculosis H37Rv strain under aerobic conditions and minimum inhibitory concentration toward isoniazid-resistant strain SRI 1369 (INH-R) (μM).

Compound	R1	R2	IC50	IC90	MIC	INH-R, MIC
1	1-Methyl-1H-pyrrol	H	<0.2†	0.6	0.4	0.14
2	Propionic acid ethyl ester	CH3	0.5	0.6	0.6	>200
3	Pyridine	H	1.2	1.4	1.3	>200
4	2-Methyl-benzothiazole	H	1.0	1.7	1.4	>200
5	Toluene	H	1.1	1.8	1.4	>200
6	1-(2-Hydroxy-3-(2-hydroxy-1,1-dimethyl-ethylamino)-propyl-1H-indol	H	2.4	4.9	4.3	>200
7	3-Nitro-phenyl	CH3	4.6	7.8	5.8	>200
8	2-Nitro-phenyl	H	3.5	8.2	6.1	>200
9	4-Nitro-phenyl	CH3	5.8	10	8	>200
10	2,4-Dihydroxy-phenyl	H	36	51	42	>200

^†Compound 1 at the concentration of 0.2 μM inhibits growth of M. tuberculosis.

MIC: Minimum inhibitory concentration.

Therefore, the compound 1 was taken for extensive biological investigations.

This compound was tested toward three M. tuberculosis resistant strains. MIC values of compound 1 toward isoniazid-resistant (SRI 1369), rifampicin-resistant (SRI 1367) and ofloxacin-resistant M. tuberculosis strains (SRI 4000) are presented in Table 3. As it can be seen from the table, compound 1 is active toward all investigated resistant M. tuberculosis strains with MIC values in the submicromolar range.

Table 3. . The activity of compound 1 toward isoniazid-resistant (INH-R) (SRI 1369), rifampicin-resistant (RIF-R) (SRI 1367) and ofloxacin-resistant (OFX-R) (SRI 4000) M. tuberculosis strains (MIC, μM).

Compound	H37Rv	INH-R	RIF-R	OFX-R
1	0.4	0.14	0.13	0.14
Rifampicin	0.016	0.016	3.6	0.018
Isoniazid	0.44	>100	0.32	NA

NA: Not applicable; compound not used in assay.

In order to establish whether compound 1 is bacteriostatic or bactericidal we have used MBC assay. MBC values were not determined, since there were too many colonies on agar plates. Therefore, the compound 1 lacks bactericidal activity and can be considered as a bacteriostatic agent.

The compound 1 was investigated for intracellular antimycobacterial activity in macrophages. Intracellular killing activity is shown as log reduction values calculated as decrease in M. tuberculosis concentration from zero hour to 7 days after infection. The concentrations of compound 1 were chosen based on the results of MIC determination. The mid concentration displays MIC value and the lower concentration is in ten-times below the MIC and the higher concentration is in ten-times above the MIC. Compound cytotoxicity is represented as a percent of viable cells. These data are shown in Table 4.

Table 4. . Macrophage and MTT results for compound 1.

Compound	Macrophage log reduction (low concentration)	Macrophage log reduction (mid concentration)	Macrophage log reduction (high concentration)	MTT % viability (low concentration)	MTT % viability (mid concentration)	MTT % viability (high concentration)
1	2.02	2.26	2.43	100	96	83
Rifampin (positive control)	1.06	2.39	2.86	95	85	73

Plasma protein binding for compound 1 was determined by equilibrium dialysis [19]. Compound 1 was tested using a semipermeable membrane which separates two compartments containing protein (human plasma) and buffer. Molecules can penetrate freely, but proteins cannot pass through the membrane. Accordingly to the results of this experiment (Table 5), unbound fraction of compound 1, which potentially exhibits pharmacologic effects is 57.9%.

Table 5. . Plasma protein binding for compound 1.

Compound	Mean plasma fraction unbound (%)	Mean plasma fraction bound (%)	Recovery (%)
1	57.9	42.1	77.5
Propranolol	24.4	75.6	98.4
Warfarin	0.62	99.4	106

The permeability of compound 1 was assessed using a Caco-2 cell monolayer in both directions – apical (A) and basal (B) [20,21]. The control compounds such as atenolol (low permeability), propranolol (high permeability) and talinolol (P-gp efflux control) were included in each experiment. As it can be seen from Table 6, compound 1 exhibits good apparent permeability Caco-2 cells.

Table 6. . Caco-2 permeability for compound 1.

Compound	Assay duration (h)	Mean A→B Papp† (10-6 cm/s)	Mean B→A Papp† (10-6 cm/s)	Efflux ratio‡
1	1 2	26.6 20.3	24.7 16.0	0.93 0.79
Atenolol	1 2	0.036 0.31	0.31 0.42	8.7 1.4
Propranolol	1 2	17.3 25.4	43.3 21.1	2.5 0.83
Talinolol	1 2	0.017 0.072	5.04 6.3	298 87.6

^†P_app is the apparent permeability rate coefficient = (dQ/dt)/(C₀A).

^‡Efflux ratio (Re) is P_app (B→A)/P_app (A→B).

An Re >2 indicates a potential substrate for P-gp or other active transporters.

Compound 1 was tested for inhibition of six cytochrome P450 enzyme isoforms – CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. It was revealed that compound 1 inhibits only one cytochrome P450 enzyme isoform – CYP2C19 with IC₅₀ value of 15 μM.

Compound was tested for microsomal stability using pooled human liver S9 microsomes. Microsomes were incubated with the test compound in the presence of the cofactor NADPH. It was established that compound 1 has NADPH-dependent metabolism (Table 7).

Table 7. . Microsomal stability for compound 1.

Compound	Concentration (μM)	Test species	NADPH-dependent CLint† (μl/min/mg)	NADPH-dependent T1/2‡ (min)	NADPH-free CLint† (μl/min/mg)	NADPH-free T1/2‡ (min)
1	1	Human	15.5	149	<12.8	>180
Verapamil	1	Human	135	17.1	<12.8	>180
Dextromethorphan	1	Human	24.2	95.3	<12.8	>180

^†Microsomal intrinsic clearance = ln(2)/(T1/2 [microsomal protein]).

^‡Half-life = 0.693/-k.

The cytotoxicity of compounds toward eukaryotic cells was determined using the human liver cells (HepG2). The IC₅₀ value for compound 1 was >100 μM, while IC₅₀ for staurosporine, which was used as positive control was 0.045 μM. Hence, the compound 1 is not cytotoxic.

Discussion

Isoniazid is the frontline antibiotic for the treatment of latent and active tuberculosis infections. The aim of this study was to synthesize and investigate isoniazid derivatives for antituberculosis activity and particularly, toward resistant M. tuberculosis strains.

As it can be seen from the Table 2, all synthesized isonicotinic acid hydrazide derivatives possess antibacterial activity toward wild-type M. tuberculosis H37Rv strain under aerobic conditions with MIC values in the range from 0.4 to 42 μM.

We have investigated the influence of substituents R1 and R2 of isoniazid derivatives on the antimycobacterial activity. It was revealed that in a series of derivatives with R2 = H, the presence of 1-methyl-1H-pyrrol (compound 1) in the position R1 is the most favored for antimycobacterial activity (MIC = 0.4 μM). The substituents pyridine, 2-methyl-benzothiazole and toluene have almost equal impact on the antimycobacterial activity (MIC = 1.3, 1.4 and 1.4 μM, correspondingly), which is lower in about 3.5-times than in the case of 1-methyl-1H-pyrrol. The introduction of substituents – 1-(2-Hydroxy-3-(2-hydroxy-1,1-dimethyl-ethylamino)-propyl-1H-indol, 2-nitro-phenyl and 2,4-dihydroxy-phenyl at the position R1 of isonicotinic acid hydrazide leads to decrease in antimycobacterial activity in comparison with 1-methyl-1H-pyrrol in more than 10-, 15- and 100-times, respectively. Therefore, the order of potency for R1 substituents of investigated isoniazid derivatives can be proposed as following: 1-methyl-1H-pyrrol >pyridine >2-methyl-benzothiazole = toluene >1-(2-Hydroxy-3-(2-hydroxy-1,1-dimethyl-ethylamino)-propyl-1H-indol >2-nitro-phenyl >2,4-dihydroxy-phenyl.

In a series of studied isonicotinic acid hydrazide derivatives with R2 = CH₃, the order of efficiency for R1 substituent is following: propionic acid ethyl ester >3-nitro-phenyl >4-nitro-phenyl (corresponding MIC values are 0.6, 5.8 and 8 μM).

The established structure–activity relationships of isoniazid derivatives can be useful for further chemical optimization in order to improve antituberculosis activity.

Among ten investigated compounds only one compound, isonicotinic acid (1-methyl-1H-pyrrol-2-ylmethylene)-hydrazide (1), possesses antibacterial activity toward isoniazid-resistant strain SRI 1369. We hypothesize that antimycobacterial activity of this compound is due to combination of isoniazid and pyrrole action, since both heterocycles posses antituberculosis activity. The literature data demonstrates that pyrrole compounds posses promising antituberculosis activity [22–29]. For example, pyrrole derivatives such as pyrrolnitrin analogs 1, BM212 and LL3858 have antibacterial activity toward both drug-sensitive and multidrug-resistant M. tuberculosis [21–23]. In addition, pyrrole compound, derived from the hybrids of BM212 and SQ109, is effective toward multidrug-resistant M. tuberculosis [26]. It should be noted, that isoniazid-pyrrole hybrid (LL-3858), which was developed by Lupin Limited currently is in initial stages of Phase II clinical trial for the treatment of tuberculosis [30–33].

Since M. tuberculosis can survive inside macrophages, it is very important to establish intracellular killing activity for investigated compounds. It was found that the compound 1 possess intramacrophage antibacterial activity against M. tuberculosis comparable to that of rifampin. Moreover, this compound is not cytotoxic toward macrophage cells (Table 4).

The compound 1 possess good ADMET properties. The compound 1 has high permeability through Caco-2 cell line, which is used as a model of human intestinal absorption of drugs and demonstrates suitability of compounds for oral dosing. The compound 1 has good metabolic stability in human liver microsomes and medium CL_int value.

According to the data of cytochrome P450 inhibition assay, among six investigated P450 enzyme isoforms - CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, compound 1 similarly to isoniazid inhibits CYP2C19, which is involved in the metabolism of a large number of medicines [34], indicating that compound 1 might contribute to drug interactions. The compound is not cytotoxic toward human hepatoma HepG2 cell line, which is usually used in drug metabolism and hepatotoxicity studies.

Conclusion

Therefore, the compound isonicotinic acid (1-methyl-1H-pyrrol-2-ylmethylene)-hydrazide (1) demonstrates high antibacterial activity toward M. tuberculosis H37Rv under aerobic conditions. This compound is active against isoniazid-, rifampicin- and fluoroquinolone-resistant strains and also has good ADME properties and low cytotoxicity toward human liver cells (HepG2). These obtained data indicate that the compound 1 is valuable candidate for further preclinical studies.

Summary points.

• Ten isoniazid derivatives were synthesized and studied for antimycobacterial activity.

• All tested compounds inhibit growth of H37Rv strain under aerobic conditions with minimum inhibitory concentration (MIC) values in the range from 0.4 to 42 μM.

• Only one compound from ten investigated – isonicotinic acid (1-methyl-1H-pyrrol-2-ylmethylene)-hydrazide (1) was active toward isoniazid-resistant strain (MIC = 0.14 μM).

• The compound 1 also inhibits growth of rifampicin-resistant and ofloxacin-resistant M. tuberculosis strains with MIC values of 0.13 and 0.14 μM, respectively.

• The compound 1 possesses good ADME properties.

• The compound 1 is not cytotoxic toward human liver cells (HepG2; IC₅₀ >100 μM).