Novel hybrid-pyrrole derivatives: their synthesis, antitubercular evaluation and docking studies

Abstract

Using novel hybrid molecules for the treatment of tuberculosis is one of the latest approaches. Keeping this concept in mind, thirty two hybrid compounds were synthesized, with pyrrole as one of the moieties, clubbed to coumarin, ibuprofen and isoniazid. The compounds were evaluated against Mycobacterium tuberculosis H₃₇Rv strain. Compounds 7e and 8e exhibited MIC of 3.7 and 5.10 μg mL⁻¹ and growth inhibition of 95% and 92%, respectively. These compounds were also active against single drug resistant bacterial strains. The compounds were devoid of cytotoxicity when tested against Vero African green monkey kidney cell line. Docking study was carried out on enoyl acyl carrier protein enzyme to provide some understanding into the mechanism of action of these compounds.

---

1. Introduction

Despite the enormous efforts that have been made in the quest of new drugs, tuberculosis (TB) still is one of the rapidly emerging and deadly infections caused by the pathogen Mycobacterium tuberculosis (Mtb). In the year 2013, 9 million new cases were reported with 1.4 million fatal incidents.¹ The rampant bacterial resistances viz. multi-drug resistant (MDR) TB and extensively drug-resistant (XDR) TB to existing anti-TB agents have become a significant concern for the effective treatment of TB.^2,3

1.1. Antitubercular agents

Antitubercular agents can be divided into two classes: first-line drugs: isoniazid (INH), pyrazinamide (PZA), ethambutol (EMB), rifampin (RIF), and streptomycin given for 6 months. If the treatment fails due to bacterial drug resistance, second line drugs such as p-amino salicylic acid (PAS), kanamycin, fluoroquinolones, capreomycin, ethionamide, and cycloserine are used. These are mostly either less effective or more toxic with serious side-effects. Hence, it is quite essential to develop safe and cost-effective new antitubercular agents.

1.2. Molecular hybridization

Molecular hybridization of a potential pharmacophore scaffold is being used as a valuable approach by medicinal chemists to design new prototypes. These molecules effectively address resistance problem and have increased efficacy. Hybrid molecules are generated by the covalent addition of two active subunits either by (a) a spacer or (b) fusion or (c) merging of compounds with desired activities.⁴

Various hybrid derivatives have been synthesized and evaluated for antitubercular activity, for example isoniazid-pyrazinamide,⁵ isoniazid-fluoroquinolone⁶ and pyrazole-quinazoline⁷ (Fig. 1) and a synergistic activity profile has been observed.

Fig. 1 Structures of different hybrid drugs showing antitubercular activity.

Pyrrole derivatives have been found to possess a wide spectrum of activities^8,9 of which anti-TB is one of the prominent ones.^10–12 Moreover, coumarin containing compounds have been widely explored for different activities such as antibacterial, anticancer and antitubercular.^13–15 Calanolide A, a natural product with coumarin nucleus is reported to have antitubercular action at MIC of 3.13 μg mL⁻¹.¹⁶ Similarly, ibuprofen (MIC 75 μg mL⁻¹), naproxen (MIC 90 μg mL⁻¹) and other known anti-inflammatory agents are reported to possess good antitubercular activity against Mtb H₃₇Rv.¹⁷ Because INH contains a pyridine nucleus, large numbers of scientists have developed novel agents bearing a pyridine moiety for the treatment of TB.¹⁸

Keeping in view the hybrid concept along with the antitubercular potential of different moieties it was planned to synthesize hybrid compounds (Fig. 2) that comprise the pyrrole nucleus with the aforementioned fragments and their evaluation of anti-TB activity.

Fig. 2 Compounds represented with antitubercular activity and portions highlighted have been used to design new hybrid compounds.

All the compounds were synthesized by conventional, as well as microwave methods. Microwave reactions obtained higher yields with reduced reaction times. Entire cell assay of these compounds was performed by microplate Alamar blue assay (MABA) against drug sensitive and resistant strains. Compounds with more than 60% growth inhibitory property were further selected for cytotoxicity studies to determine whether they are non-toxic or toxic.

These compounds are thought to act by inhibiting enoyl acyl carrier protein enzyme (InhA target of INH). This was supported by docking studies of the synthesized pyrrole-hybrids using the GLIDE module of Schrödinger. Because N-(3,5-dichlorophenyl)-5-oxo-1-phenylpyrrolidine-3-carboxamide is a direct inhibitor of InhA, which forms a hydrogen bond with Tyr158, we focused on docking the most potent compound in a similar manner.

2. Results and discussion

2.1. Chemistry

The concept of hybridization is being widely used to synthesize novel agents to target different diseases, especially related to the development of resistance to existing drugs. A similar approach has been used to develop novel antitubercular agents containing different moieties linked together directly or through linkers. In this paper we report hybrids of a pyrrole moiety with different moieties, such as isobutyl benzene, pyridyl, naphthyl and coumarin, to explore their potential as antitubercular agents. Thirty two different hybrid compounds (7a–p and 8a–p) reported herein were prepared following Scheme 1. Step 1a describes the preparation of intermediate hydrazides (3a–o) from different acids (1a–o). Primarily, corresponding aromatic acid (1a–o) and catalytic amount of concentrated HCl was refluxed in methanol to produce the corresponding ester (2a–o). The hydrazides (3a–o) were synthesized by refluxing ester (2a–o) with 99% hydrazine hydrate according to the reported procedure.¹⁹ Step 1b involves the preparation of coumarin based hydrazides (3p), which obtained from 7-hydroxy-4-methyl-2H-chromen-2-one by refluxing with ethyl 2-bromoacetate in the presence of potassium carbonate as base. The starting material phenacyl bromide (4) was readily prepared from acetophenone by treating with bromine at room temperature in diethyl ether. 1,4-Diphenylbutane-1,4-dione (5) was synthesized by refluxing bromo acetophenone 4 with zinc and a pinch of iodine suspension in dry tetrahydrofuran. 1-Phenylpentane-1,4-dione (6) was purchased from Sigma-Aldrich. Finally, the title compounds (7a–p and 8a–p) were produced by the nucleophilic attack of the most basic nitrogen of hydrazides (3a–p) to the carbonyl group of the γ-diketone (5 and 6) following Paal–Knorr cyclocondensation. The Paal–Knorr condensation was carried out by conventional heating, as well as under microwave irradiation; however, carrying the reaction by conventional method needs a catalyst (p-toluene sulfonic acid), and took a longer time with lesser yields. The neat reagents were irradiated for 7–15 minutes in the range of 100–130 °C, water droplets appeared as the reaction proceeded. The details are provided in the ESI.†

Scheme 1 Synthetic route of hybrid-pyrrole analogues 7a–p and 8a–p.

The structures of all the synthesized compounds (7a–p and 8a–p) were established by analytical data. In general, IR peaks at 3268–3182 cm⁻¹ for NH and 1700–1640 cm⁻¹ for C=O confirmed that all the derivatives contain an amide bond. The analysis of ¹H NMR spectra (7a–p and 8a–p) showed the signals of the corresponding protons, which were confirmed on the basis of their chemical shifts, multiplicities and coupling constants. These spectra showed two characteristic signals for the CONH proton between 8.0 to 11.0 ppm and CH=CH pyrrole protons at 6.2–6.8 ppm singlet type in case of 7a–p. However, CH=CH pyrrole protons of the second series (8a–p) appeared as doublet with different chemical shift values. The NH protons of all the compounds were confirmed by D₂O exchange. In ¹³C NMR, peaks were observed at 164–169 (C=O), 104–110 (pyrrole ring), further confirming the desired structures. Remaining resonances were also observed at their expected values. In MS (ESI) m/z was found at M + H showing a 100% base peak, which corresponds with the actual molecular weight of the compounds. The elemental analysis confirmed the purity of the compounds, experimental values were found within ±0.4% of the theoretical values. The purification of all the compounds (7a–p and 8a–p) was achieved by chromatography using a gradient elution method (hexane and 0–30% ethyl acetate mixture), and was confirmed by HPLC coupled with mass spectrometry. All the compounds are more than 90% pure.

2.2. Antitubercular activity

Preliminary anti-TB screening of all the synthesized compounds (7a–p and 8a–p) was performed against a drug sensitive strain of Mtb H₃₇Rv (ATCC 27294) by microplate Alamar blue assay (MABA) as reported by Collins and Franzblau.²⁰ This methodology is simple, non-toxic, uses a thermally stable reagent and shows good correlations with the proportional and BACTEC methods.²¹ The minimum inhibitory concentration (MIC), i.e. concentration of compounds required to completely inhibit Mtb growth, were recorded. The MIC was calculated from a dose response curve. The compounds with more than 90% inhibition of initial primary screening were further assayed for the determination of MIC against different Mtb clinical isolates (drug sensitive and resistant). The pyrrole scaffold was used as the basis for the development of structural analogues, which yielded compounds 7a–p and 8a–p and their activity data are presented in Table 1. The activity results indicate that thirteen compounds (7a, 7e, 7j, 7l, 7n, 7p, 8a, 8e, 8h, 8j, 8l, 8n and 8p), which have different fragments show moderate to good activity in the range of 60% to 95% inhibition at 50 μg mL⁻¹ concentrations, while rest of the compounds show either a decreased inhibition or are devoid of inhibition. The MIC values of the 2,5-di phenyl pyrrole analogs (7a–p) were found to be better as compared to their 2-methyl-5-phenyl analogues (8a–p) as the overall hydrophobicity increased. Gratifyingly, compounds 7e and 8e with 4-pyridoyl fragments were found to be most active with MIC value of 3.7 and 5.10 μg mL⁻¹ and 95% and 92% growth inhibition, respectively, comparable or better than other existing drugs, e.g. pyrazinamide 50 μg mL⁻¹; cycloserine 50 μg.²²

Table 1 Preliminary antitubercular activity of 7a–p and 8a–p against Mtb H₃₇Rv

ID	R	MIC^a^,^b	GI^c	% Cell viability^d	Docking score	ID	R	MIC^a^,^b	GI^c	% Cell viability^d	Docking score
a Minimum inhibitory concentrations (MIC) are the lowest concentration which prevents the complete growth of inoculum.b μg mL^−1.c Growth inhibition (GI) of Mtb H37Rv at 50 μg mL^−1.d Percent viability of cells at 100 μg mL^−1; docking score calculated as Kcal mol^−1; ND, not determined; NS, not significant; PC, N-(3,5-dichlorophenyl)-5-oxo-1-phenylpyrrolidine-3-carboxamide which is used as reference molecule in the docking study.
7a		25	70	100	−5.017	8a		50	70	100	−6.412
7b		>100	26	ND	−4.585	8b		>100	26	ND	−5.956
7c		>100	NS	ND	−5.393	8c		>100	NS	ND	−6.496
7d		>100	22	ND	−7.164	8d		>100	22	ND	−6.045
7e		3.70	95	100	−5.873	8e		5.10	92	100	−6.832
7f		>100	NS	ND	−5.741	8f		>100	NS	ND	−6.150
7g		62.5	52	ND	−6.164	8g		>100	42	ND	−6.337
7h		>100	NS	ND	−5.469	8h		12.5	62	95	−6.463
7i		>100	28	ND	−6.697	8i		>100	28	ND	−6.305
7j		12.5	75	90	−6.102	8j		25	65	100	−7.034
7k		>100	NS	ND	−4.653	8k		>100	NS	ND	−4.369
7l		6.25	70	100	−4.528	8l		12.5	60	90	−4.565
7m		>100	40	ND	−4.355	8m		>100	40	ND	−5.181
7n		12.5	80	97	−6.502	8n		25	75	100
7o		>100	NS	ND	−3.058	8o		>100	NS	ND	−3.840
7p		6.25	80	97	−6.076	8p		6.25	85	100	−6.097
RIF		0.06	98	ND	ND	INH		0.4	94	ND	ND
PYZ		50	85	ND	ND	PC		∼125	—	ND	−8.907

---

The compounds 7e and 8e have also shown good activity against rifampin (RMP-R1 and RMP-R2) and fluoroquinolone resistant (FQ-R) Mtb strains; whereas, they exhibited moderate activity against isoniazid resistant (INH-R) strains with MIC value of >67.8 μg mL⁻¹ (Table 2). As 7e and 8e both show activity against the resistant strain, these molecules can serve as leads for the further generation of more active compounds. The intracellular (macrophage) drug screening assay evaluates intracellular drug effectiveness. The assay results depict that reduction in cell viability exhibited by compound 7e is 2.4 log reduction of CFU at 3.7 μg mL⁻¹ concentration, which is comparable with the control drug INH. This is important because Mtb can survive inside macrophages, which contributes to treatment failure and disease relapse.

Table 2 Cytotoxicity, minimum bactericidal concentration (MBC), drug sensitive and single drug-resistant (SDR) antitubercular activities of 7e and 8e^a

	TC50	Strain	MIC	IC50	IC90	MBC*
a All concentrations are in μg mL^−1; selectivity index (SI) = TC50/MIC; * MBC of rifampin is 0.78 μg mL^−1.
7e	>100	H37Rv	3.7	2.71	3.72	4.4
		INH-R1	>67.8	>67.8	>67.8
		INH-R2	>67.8	>67.8	>67.8
		RMP-R1	1.25	0.78	1.12
		RMP-R2	2.44	2.84	4.06
		FQ-R1	2.40	1.62	2.61
		LORA	>67.8	>67.8	>67.8
		Intracellular macrophage (3.7 and 37.0 μg mL^−1)	2.4 (log reduction of CFU)
8e	>100	H37Rv	5.10	4.92	5.16	7.8
		INH-R1	>55.4	>55.4	>55.4
		INH-R2	>55.4	>55.4	>55.4
		RMP-R1	14.9	14.4	19.9
		RMP-R2	30.4	21.32	30.4
		FQ-R1	28.5	20.77	27.7
		LORA	1.94	0.86	1.27
		Intracellular macrophage (5.10 and 51.0 μg mL^−1)	2.4 (log reduction of CFU)
INH		H37Rv	0.4–0.6	0.12	0.21
		INH-R1	>200	110	130
		INH-R2	>200	95	110

---

The cytotoxicity of compounds was assessed in Vero African green monkey kidney cell line. Selectivity index is defined as TC₅₀/MIC. The compounds 7e and 8e are non-toxic with high selectivity index (>27 and >19, respectively). The MBC is determined subsequent to MIC testing by sub-culturing diluted aliquots from wells that fail to exhibit macroscopic growth. Because the MBC value is close to MIC, the compound 7e and 8e is bactericidal in nature (Table 1 and Fig. 3).

Fig. 3 Dose response plot for minimum bactericidal concentration (MBC) determination.

It is believed that compounds with an MIC ≤ 6.25 μg mL⁻¹ and an SI > 10 are interesting compounds, and are considered as excellent leads.²³ These features make 7e and 8e very promising anti-TB agents.

2.3. Structure activity relationship (SAR)

Based on the above observation the following structure activity relationship could be established.

(1) Replacement of one of the phenyl groups with a methyl group at 5-position in the pyrrole ring resulted in decrease in activity as can be seen from the differences in MIC of 7a–p and 8a–p, e.g. MIC of 7e is 3.7 μg mL⁻¹, whereas 8e is 5.10 μg mL⁻¹.

(2) In general, the group with electropositive character attached to an amide link resulted in an increase in activity change of phenyl group to pyridyl group in 7e and 8e obtained the most active compounds with low MIC, GI of 95% and 92% and cell viability 100%. On the other hand, when the phenyl group is attached with a steric (+π) group (7b–d and 8b–d), the growth inhibitory activity decreases. Moreover, introduction of an OCH₃ group in the phenyl ring (+σ and −π) favors the inhibitory property.

(3) Introduction of bulky groups such as p-isobutyl benzene, or coumarin or naphthyl group also increased the activity as indicated by MIC of 6.25 μg mL⁻¹ for 7l, 7p and 8p, 12.5 μg mL⁻¹ for 7j, 7n and 8l.

(4) Moreover, when tested against a panel of single-drug resistant Mtb strains, derivatives 7e and 8e maintained the activity as for the wild type, indicating that these derivatives may act with a different mechanism of action when compared to the existing drugs.

2.4. Docking study

Because the most potent molecules share fragments of INH, it was assumed that the active compounds may act via a mechanism similar to isoniazid but directly inhibit the enzyme; therefore, docking studies of all the synthesized molecules were carried out on InhA. The crystal structure of enoyl acyl carrier protein (2H7M) was used for docking studies to get a preliminary idea about the interaction of ligands with their target. It was observed that some of the compounds having docking scores more than −6.0 Kcal mol⁻¹ were not good in in vitro assay, which may be attributed to their high log P value. These results suggest that these inhibitors do not yet have optimal membrane permeability or are actively pumped out of the bacterial cells by efflux pumps.²⁴ Visual inspection of the target ligand interactions made it clear that the hydrogen bonding interaction with Tyr158 and NAD⁺ is important for activity. As supported by the in vitro results, compounds (7f, 7h, 7o and 8o) are inactive because they do not show this interaction. Additionally, extra interaction with Phe149 has proven to be important for potency. Compounds lacking π stacking with Phe149 were found less active in in vitro studies compared to active ones. There may be some other factor for their reduced activity in addition to this.

A complete overview of receptor–inhibitor binding interactions of 7e is illustrated in Fig. 4. The inhibitor fits the binding pocket of InhA in the same manner as co-crystalized ligand. The most active compound 7e tightly binds with the enzyme to form a complex. The oxygen of the amide group and the pyridyl nitrogen are connected through hydrogen bond to the 2′-hydroxyl moiety of the nicotinamide ribose and the hydroxyl group of Tyr158, one of the catalytic residues in the InhA active site. This hydrogen-bonding network is the most important feature among all the InhA-inhibitors identified to date. The hydrogen bonds formed between Tyr158 and compound 7e clearly support that this compound might be acting through this mechanism. 7e also shows van der Waals interactions with the hydrophobic residues Gly96, Met103, Phe149, Met155, Pro156, Ala157, Met161, Pro193, Ala198, Ile215, and Leu218.

Fig. 4 (A) Reference ligand binding pose; (B) surface of the binding pose between reference ligand and InhA; (C) docked conformation of the most potent inhibitor 7e with the crystal structure of a cofactor NAD⁺ and Tyr158 in InhA; (D) surface of the binding pose which shows the interactions between InhA site and compound 7e.

2.5. In silico pharmacokinetic property

Many drugs, at a late stage of development, as well as lead compounds fail due to adverse pharmacokinetic properties. Therefore, it is important to incorporate ADME (adsorption, distribution, metabolism and excretion) properties of the above derivatives to continue the further research. Most of the existing anti-TB drugs are hydrophilic molecules, and hence their low cellular penetration may be contributing to the development of Mtb drug resistance.²⁵ Therefore, the development of hydrophobic molecules could increase the cellular penetration of target tissues as it was hypothesized that they are likely to pass easily through Mtb's cell envelope.²⁶ In addition to their promising in vitro bactericidal activity against Mtb, all the fragment based pyrrole derivatives have good physicochemical properties that indicate great potential of these agents as orally available compounds. They fulfill at least four of the five physicochemical properties defined by the Lipinski ‘rule-of-five,’ which predicts aqueous solubility and intestinal permeability. All the compounds of both the series have <10 hydrogen bond acceptors, <5 hydrogen bond donors and molecular weights <500 gmol⁻¹. Details are provided in the ESI.† In terms of lipophilicity, compounds 7e, 8a–k and 8m–p show miLog P value of <5, while the rest of the compounds display miLog P values just above 5. The ease of synthesis coupled with the promising physicochemical properties signify that these compounds can be attractive leads for further development as novel anti-TB agents.

3. Conclusions

The hybrid design of compounds to develop potent antitubercular agents was successfully achieved. Thirty two compounds were synthesized and screened for anti-TB activity. Two compounds 7e and 8e were active against Mtb at low MIC and also against rifampin and fluoroquinolone resistance strains. The biological evaluation of the synthesized compounds helped in identifying a hybrid of 4-pyridyl, with a pyrrole moiety, as a lead molecule.

4. Experimental

4.1. Analysis and instruments

All the chemicals were purchased from Sigma-Aldrich, Spectrochem Pvt. Ltd. and S.D. Fine Chemicals (India) and were used without further purification. Tetrahydrofuran was dried over sodium/benzophenone prior to use. Anhydrous reactions were performed under a positive pressure of inert nitrogen gas. Melting points were determined on a digital melting point apparatus by the open tube capillary method and are uncorrected. Thin layer chromatography (TLC) plates (silica gel G) were used to check the purity of commercial reagents used and desired product purity and to monitor the reaction progress. Various solvent systems (ethyl acetate–hexane (3 : 7) and methanol–chloroform (1 : 9)) were used to run the TLC and spots were located under iodine vapors/Uv light. An infrared spectrum (IR) was recorded on a Bruker FTIR spectrometer using KBr pellet. Elemental analyses were carried out on a Perkin-Elmer 2400 analyzer (USA) and were found within ±0.4% of the theoretical values. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker spectrometer.

HPLC and MS analysis. The purity of the compounds was measured by reversed-phase liquid chromatography and a mass spectrometer (Agilent 1100 LC/MSD) with a UV detector at λ = 214 and 254 nm and an electron spray ionization (ESI) source. MS data were recorded using an Agilent 1100LC/MSD VL system (Phenomenex Gemini C18 column, 3 × 50 mm; 0.5 mL min⁻¹ flow rate, acetonitrile–water binary solvent (95 : 5)). UV detection was monitored at 214 and 254 nm. MS data were acquired in positive mode scanning over the mass range of 50–1000 (attached in ESI†).

Microwave irradiation experiments. A mono mode CEM-Discover microwave reactor (CEM Corporation, P.O. Box 200, Matthews, NC 28105) was used in the standard configuration, including proprietary software. All the experiments were carried out in sealed microwave process vials (10 mL) at the maximum power and designated temperature (100–130 °C). After the completion of the reaction, the vial was cooled to 50 °C via air-jet cooling before it was opened.
Step 1a.
General procedure for the preparation of substituted hydrazide (3a–o). To a solution of appropriate acids in ethanol, 2 drops of concentrated sulfuric acid was added and refluxed at 80 °C for 8–14 h. The reaction progress was monitored by TLC. After the completion of ester formation, hydrazine hydrate (1.5 times, 90%) was added and refluxed for 4–5 h. The residual solvent was evaporated under reduced pressure; solid was filtered and washed with ice-cold water. The crude mass was purified by recrystallization from methanol. The purity was checked by TLC. After recrystallization by methanol, the desired compounds 3a–o, were isolated as solid (60–80%).
Step 1b.
Typical procedure for the preparation of 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide (3p). 7-hydroxy-4-methyl-2H-chromen-2-one or 7-hydroxy-2H-chromen-2-one (10 mmol) was dissolved in 100 mL dry acetone. To this, anhydrous potassium carbonate (15 mmol), followed by ethyl 2-bromoacetate (15 mmol) were added and refluxed for 12 h. After cooling, the organic layer was filtered and concentrated in vacuo. The mixture was diluted with water (40 mL), extracted with CHCl₃ (15 mL × 3), dried over anhydrous Na₂SO₄, concentrated under reduced pressure and crystallized from methanol to obtain 2p.

To the solution of ester (2p) in absolute ethanol (100 mL), hydrazine hydrate (15 mmol, 90%) was added and refluxed for 8–10 h. On cooling, the precipitate obtained was filtered and washed with ice-cold water, dried and recrystallized from methanol as solid crystal 3p (70% yield).

Step 2.
Synthesis of phenacyl bromide. Phenacyl bromide was prepared according to the reported procedure and used for the preparation of 1,4-diphenylbutane-1,4-dione.²⁷
Synthesis of 1,4-diphenylbutane-1,4-dione (5). To a solution of phenacyl bromide (3 g, 15 mmol) in dry tetrahydrofuran (30 mL), zinc (1.04 g, 16.5 mmol) and pinch of iodine were added and refluxed at 80 °C for 3 h. After the completion of the reaction, zinc was removed by filtration through Celite. The organic filtrate was extracted from the water layer, dried over anhydrous sodium sulfate, concentrated under reduced pressure and purified by column chromatography. Yield 40%, FTIR (KBr pellet) cm⁻¹: 1674 (C=O str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.03 (d, 4H, J = 7.6 Hz, aromatic-H), 7.57 (t, 2H, J = 6.8 Hz, aromatic-H), 7.47 (d, 4H, J = 7.6 Hz, aromatic-H), 3.46 (s, 4H, CH₂).
Step 3.
General procedure for the preparation of pyrrole derivatives (7a–p and 8a–p). Method I (conventional method). An equimolar mixture of 1,4-diphenylbutane-1,4-dione (5, 10 mmol) or 1-phenylpentane-1,4-dione (6, 10 mmol), catalytic amount of p-toluene sulfonic acid and hydrazide derivatives (10 mmol) (3a–p) in absolute ethanol (30 mL) were refluxed for 10–24 h. After the completion of the reaction, the residual ethanol was evaporated to dryness, and then purified by column chromatography to yield 3a–p.
Method II (microwave irradiation method). To a dry 10 mL microwave vial equipped with a magnetic stir bar, 1,4-diphenylbutane-1,4-dione (5, 2 mmol) or 1-phenylpentane-1,4-dione (6, 2 mmol) and hydrazide derivative (3a–p) (2 mmol) were added and the vial was capped. The vial was shaken to mix the contents, and then heated in a microwave at 100–130 °C for 7–15 min (CEM Discover reactor). Silica gel column chromatography (10 cm × 2 cm; hexanes–ethyl acetate, 0–30%) obtained 7a–p and 8a–p as solids in satisfactory yield.
Spectral data.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)benzamide (7a). FTIR (KBr pellet) cm⁻¹: 3200 (N–H str.), 1648 (C=O str.), 1483 (C–O–N str.), 1294 (C–N str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.61 (bs, 1H, NH, D₂O exchangeable), 8.49 (d, 2H, J = 7.2 Hz, aromatic-H), 7.96–8.01 (m, 3H, aromatic-H), 7.72–7.77 (m, 2H, aromatic-H), 7.45–7.47 (m, 4H, J = 7.2 Hz, aromatic-H), 7.37–7.42 (m, 4H, aromatic-H), 6.34 (s, 2H, CH=CH–pyrrole); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 164.80 ([double bond splayed left]C=O), 138.86, 135.26, 131.25, 129.94, 128.64, 128.07, 127.97, 127.50, 126.12, 126.66, 126.45, 107.35 (C̲H=C̲H–pyrrole); MS-ESI: m/z 339.2 (M + 1); anal. calcd for C₂₃H₁₈N₂O: C, 81.63; H, 5.36; N, 8.29, found C, 81.65; H, 5.40; N, 8.29. N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-methylbenzamide (7b). FTIR (KBr pellet) cm⁻¹: 3208 (amide N–H), 1688 (C=O str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.01 (bs, 1H, NH, D₂O exchangeable), 7.92 (d, 1H, J = 7.2 Hz, aromatic-H), 7.65–768 (m, 6H, aromatic-H), 7.35–7.39 (m, 5H, aromatic-H), 7.20–7.22 (m, 2H, aromatic-H), 6.42 (s, 2H, CH=CH–pyrrole), 2.36 (s, 2H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 165.61 ([double bond splayed left]C=O), 138.26, 135.27, 131.25, 129.64, 128.64, 128.07, 127.47, 127.05, 126.03, 126.69, 126.47, 107.48 (C̲H=C̲H–pyrrole), 20.02 (CH₃); MS-ESI: m/z 353.2 (M + 1); anal. calcd for C₂₄H₂₀N₂O: C, 81.79; H, 5.72, found C, 81.80, H, 5.75; N, 7.95.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-3-methylbenzamide (7c). FTIR (KBr pellet) cm⁻¹: 3209 (amide N–H), 1656 (C=O str.), 1584 (C–N str.), 1477 (C–O–N str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.36 (bs, 1H, NH, D₂O exchangeable), 7.51–7.55 (m, 6H, aromatic-H), 7.31–7.35 (m, 6H, aromatic-H), 7.19–7.22 (m, 2H, aromatic-H), 6.43 (s, 2H, CH=CH–pyrrole), 2.28 (s, 2H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 165.90 ([double bond splayed left]C=O), 138.08, 135.36, 132.81, 131.79, 131.64, 128.54, 128.38, 127.85, 127.15, 126.84, 124.25, 107.46 (C̲H=C̲H–pyrrole), 20.84 (CH₃); MS-ESI: m/z 353.2 (M + 1); anal. calcd for C₂₄H₂₀N₂O: C, 81.79; H, 5.72; N, 7.96, found C, 81.82, H, 5.77; N, 7.96.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-4-methylbenzamide (7d). FTIR (KBr pellet) cm⁻¹: 3209 (aromatic C–H str.), 1656 (C=O str.), 1584 (C–N str.), 1477 (C–O–N str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 8.89 (bs, 1H, NH, D₂O exchangeable), 7.82 (d, 2H, J = 7.2 Hz, aromatic-H), 7.59–7.51 (m, 6H, aromatic-H), 7.33 (d, 2H, J = 7.2 Hz, aromatic-H), 6.95 (d, 4H, J = 6.8 Hz, aromatic-H), 6.43 (s, 2H, CH=CH–pyrrole), 2.36 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 166.08 ([double bond splayed left]C=O), 140.14, 135.26, 131.25, 129.64, 128.64, 128.07, 127.47, 127.05, 126.03, 126.69, 126.47, 107.38 (C̲H=C̲H–pyrrole), 21.2 (CH₃); MS-ESI: m/z 353.2 (M + 1); anal. calcd for C₂₄H₂₀N₂O: C, 81.79; H, 5.72; N, 7.76, found C, 81.82, H, 5.87; N, 7.96.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)isonicotinamide (7e). FTIR (KBr pellet) cm⁻¹: 3254 (amide N–H), 1675 (C=O str.), 1287 (C–N str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.75 (bs, 1H, NH, D₂O exchangeable), 8.58 (d, 2H, J = 2.8 Hz, aromatic-H), 7.99–8.07 (m, 1H, aromatic-CH), 7.51 (d, 2H, J = 2.8 Hz, aromatic-CH), 7.45 (d, 4H, J = 7.2 Hz, aromatic-CH), 7.24 (t, 3H, J = 7.2 Hz, aromatic-H), 7.14 (t, 2H, J = 7.2 Hz, aromatic-CH), 6.33 (s, 2H, CH=CH–pyrrole); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 164.68 ([double bond splayed left]C=O), 149.94, 138.86, 135.26, 131.25, 129.64, 128.64, 128.07, 127.47, 127.05, 126.03, 126.69, 126.47, 120.93, 107.38 (C̲H=C̲H–pyrrole); MS-ESI: m/z 340.2 (M + 1); anal. calcd for C₂₂H₁₇N₃O: C, 77.86; H, 5.05; N, 12.38, found C, 77.84; H, 5.45; N, 12.40.
2-Bromo-N-(2,5-diphenyl-1H-pyrrol-1-yl)benzamide (7f). FTIR (KBr pellet) cm⁻¹: 2947 (amide N–H), 1675 (C=O str.), 1511 (C–N str.), 1291 (C–O–N str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.42 (bs, 1H, NH, D₂O exchangeable), 7.56 (d, 4H, J = 7.6 Hz, aromatic-H), 7.50 (t, 1H, J = 7.6 Hz, aromatic-H), 7.35 (t, 4H, J = 7.6 Hz, aromatic-H), 7.22–7.27 (m, 4H, aromatic-H), 6.92 (t, 1H, J = 7.6 Hz, aromatic-H), 6.33 (s, 2H, CH=CH–pyrrole); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 166.19 ([double bond splayed left]C=O), 135.71, 133.00, 131.49, 131.26, 128.71, 128.01, 127.83, 126.81, 119.42 (C–Br), 107.19 (C̲H=C̲H–pyrrole); MS-ESI: m/z 417.2 (M + 1), 419.1 (M + 2); anal. calcd for C₂₃H₁₇BrN₂O: C, 66.20; H, 4.11; N, 6.71, found C, 66.26, H, 4.17; N, 6.77.
2-Chloro-N-(2,5-diphenyl-1H-pyrrol-1-yl)benzamide (7g). FTIR (KBr pellet) cm⁻¹: 3208 (amide N–H), 1698 (C=O str.), 1618 (C–N str.), 1392 (C–O–N str.), 1020 (C–Cl str.); ¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 8.23 (bs, 1H, NH, D₂O exchangeable), 7.58 (d, 4H, J = 6.9 Hz, aromatic-H), 7.38–7.44 (m, 6H, aromatic-H), 7.35 (d, 2H, J = 3 Hz, aromatic-H), 7.20 (d, 2H, J = 3 Hz, aromatic-H), 6.45 (s, 2H, CH=CH–pyrrole); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 167.08 ([double bond splayed left]C=O), 134.16 (C–Cl), 133.76, 131.25, 129.64, 128.27, 127.41, 127.22, 126.03, 125.99, 125.47, 107.18 (C̲H=C̲H–pyrrole); MS-ESI: m/z 373.1 (M + 1); anal. calcd for C₂₃H₁₇ClN₂O: C, 74.30; H, 4.62; N, 7.51, found C, 74.80; H, 4.71; N, 7.53.
2,4-Dichloro-N-(2,5-diphenyl-1H-pyrrol-1-yl)benzamide (7h). FTIR (KBr pellet) cm⁻¹: 3208 (amide N–H), 1688 (C=O str.), 1628 (C–N str.), 1392 (C–O–N str.), 1025 (C–Cl str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 8.68 (bs, 1H, NH, D₂O exchangeable), 7.86–7.88 (m, 4H, aromatic-H), 7.44–7.47 (m, 6H, aromatic-H), 7.41–7.42 (m, 2H, aromatic-H), 6.60 (s, 2H, CH=CH–pyrrole); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 168.23 ([double bond splayed left]C=O), 135.74 (C–Cl), 131.25, 129.04, 128.64, 128.55, 127.42, 127.00, 108.21 (C̲H=C̲H–pyrrole); MS-ESI: m/z 406.1 (M + 1); anal. calcd for C₂₇H₂₀N₂O₄: C, 67.83; H, 3.96; N, 6.88, found C, 67.89, H, 3.96; N, 6.89.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-phenoxyacetamide (7i). FTIR (KBr pellet) cm⁻¹: 3254 (amide N–H), 1693 (C=O str.), 1598 (C–N str.), 1491 (C–O–N str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 8.78 (bs, 1H, NH, D₂O exchangeable), 7.46 (d, 4H, J = 8.0 Hz, aromatic-H), 7.32 (t, 4H, J = 8.0 Hz, aromatic-H), 7.25–7.29 (m, 2H, aromatic-H), 7.20 (t, 2H, J = 8.0 Hz, aromatic-H), 6.96–7.00 (m, 1H, aromatic-H), 6.70 (d, 2H, J = 8.0 Hz, aromatic-H), 6.40 (s, 2H, CH=CH–pyrrole), 4.45 (s, 2H, OCH₂); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 167.88 ([double bond splayed left]C=O), 156.38, 133.62, 131.15, 129.64, 128.64, 128.07, 127.47, 127.05, 126.03, 126.69, 126.27, 122.83, 106.99 (C̲H=C̲H–pyrrole), 66.72 (OC̲H₂); MS-ESI: m/z 369.2 (M + 1); anal. calcd for C₂₄H₂₀N₂O₂: C, 78.24; H, 5.47; N, 7.60, found C, 78.26; H, 5.71; N, 7.61.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-4-methoxybenzamide (7j). FTIR (KBr pellet) cm⁻¹: 3182 (amide N–H), 1678 (C=O str.), 1300 (C–N str.); ¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 8.27 (bs, 1H, NH, D₂O exchangeable), 7.82 (d, 2H, J = 8.7 Hz, aromatic-H), 7.59–7.51 (m, 6H, aromatic-H), 7.33 (t, 4H, J = 7.5 Hz, aromatic-H), 6.95 (d, 2H, J = 8.7 Hz, aromatic-H), 6.42 (s, 2H, CH=CH–pyrrole), 3.80 (s, 3H, OCH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 167.68 ([double bond splayed left]C=O), 133.98, 131.25, 129.64, 128.64, 128.07, 127.47, 127.05, 126.03, 126.89, 126.38, 116.83, 107.22 (C̲H=C̲H–pyrrole), 58.7; MS-ESI: m/z 369.2 (M + 1); anal. calcd for C₂₄H₂₀N₂O₂: C, 78.24; H, 5.47; N, 7.60, found C, 78.46; H, 5.77; N, 7.60.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-phenylacetamide (7k). FTIR (KBr pellet) cm⁻¹: 3271 (amide N–H), 1676 (C=O str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 8.17 (bs, 1H, NH, D₂O exchangeable), 7.89–7.90 (m, 2H, aromatic-H), 7.92–7.94 (m, 5H, aromatic-H), 7.56–7.52 (m, 4H, aromatic-H), 7.28–7.31 (m, 4H, aromatic-H), 6.47 (s, 2H, CH=CH–pyrrole), 3.52 (s, 2H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 171.21 ([double bond splayed left]C=O), 136.22, 133.62, 131.15, 129.64, 128.64, 128.07, 127.55, 127.05, 126.61, 107.57 (C̲H=C̲H–pyrrole), 41.07 (OC̲H₃); MS-ESI: m/z 353.2 (M + 1); anal. calcd for C₂₈H₂₂N₂O₂: C, 81.79; H, 5.72; N, 7.95, found C, 81.76; H, 5.78; N, 7.96.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-(4-isobutylphenyl)propanamide (7l). FTIR (KBr pellet) cm⁻¹: 3233 (aromatic C–H str.), 1669 (C=O str.), 1601 (C–N str.), 1354 (C–O–N str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 7.49 (bs, 1H, NH, D₂O exchangeable), 7.29 (d, 4H, J = 2.7 Hz, aromatic-H), 7.17–7.24 (m, 6H, aromatic-H), 6.82 (d, 2H, J = 7.8 Hz, aromatic-H), 6.71 (d, 2H, J = 7.8 Hz, aromatic-H), 6.25 (s, 2H, CH=CH–pyrrole), 3.35 (q, 1H, J = 7.2 Hz, CH̲–CH₃), 2.36 (d, 2H, J = 6.9 Hz, CH̲₂–CH), 1.77 (sep, 1H, J = 6.9 Hz, CH–(CH₃)₂), 1.28 (d, 3H, J = 7.2 Hz, CH–CH₃), 0.82 (d, 6H, J = 6.9 Hz, CH–(CH₃)₂); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 171.68 ([double bond splayed left]C=O), 140.12, 134.41, 132.51, 129.42, 128.64, 127.47, 126.03, 126.47, 125.19, 107.8 (C̲H=C̲H–pyrrole), 45.41 (C̲H–CH₃), 44.84 (C̲H₂–CH), 29.05 {C̲H–(CH₃)₂}, 22.18 {CH–(C̲H₃)₂}, 15.65 (CH–C̲H₃); MS-ESI: m/z 423.2 (M + 1); anal. calcd for C₂₉H₃₀N₂O: C, 82.43; H, 7.16; N, 6.63, found C, 82.49; H, 7.23; N, 6.63.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-(naphthalen-1-yl)acetamide (7m). FTIR (KBr pellet) cm⁻¹: 3218 (amide N–H), 1686 (C=O str.), 1628 (C–N str.), 1390 (C–O–N str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.96 (bs, 1H, NH, D₂O exchangeable), 8.16 (m, 2H, aromatic-H), 7.92 (m, 5H, aromatic-H), 7.83–7.87 (m, 5H, aromatic-H), 7.54–7.48 (m, 4H, aromatic-H), 7.18 (d, 1H, aromatic-H), 6.38 (s, 2H, CH=CH–pyrrole), 4.13 (s, 2H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 168.81 ([double bond splayed left]C=O), 134.86, 133.15, 129.14, 128.34, 127.47, 126.11, 126.29, 125.22, 124.89, 107.10 (C̲H=C̲H–pyrrole), 41.56 (OC̲H₂); MS-ESI: m/z 403.1 (M + 1); anal. calcd for C₂₈H₂₂N₂O: C, 83.56; H, 5.51; N, 6.96, found C, 83.59; H, 5.59; N, 6.97.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-(naphthalen-2-yloxy)acetamide (7n). FTIR (KBr pellet) cm⁻¹: 3228 (amide N–H), 1690 (C=O str.), 1612 (C–N str.), 1389 (C–O–N str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.82 (bs, 1H, NH, D₂O exchangeable), 8.16 (m, 3H, aromatic-H), 7.92 (m, 4H, aromatic-H), 7.83 (m, 3H, aromatic-H), 7.57–7.44 (m, 5H, aromatic-H), 7.36 (m, 2H, aromatic-H), 6.40 (s, 2H, CH=CH–pyrrole), 4.65 (s, 2H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 168.08 ([double bond splayed left]C=O), 157.10, 133.26, 131.25, 132.04, 129.64, 128.64, 128.07, 127.47, 127.05, 126.03, 126.69, 126.47, 120.93, 118.29, 107.68 (C̲H=C̲H–pyrrole), 105.11, 67.23 (OC̲H₂); MS-ESI: m/z 419.2 (M + 1); anal. calcd for C₂₈H₂₂N₂O₂: C, 80.36; H, 5.30; N, 6.69, found C, 80.45; H, 5.76, N, 6.69.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-(o-tolyl)acetamide (7o). FTIR (KBr pellet) cm⁻¹: 3260 (amide N–H), 1675 (C=O str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.27 (bs, 1H, NH, D₂O exchangeable), 7.46 (d, 4H, J = 7.2 Hz, aromatic-H), 7.33–7.35 (m, 4H, aromatic-H), 7.06–7.16 (m, 5H, aromatic-H), 6.95–6.99 (m, 1H, aromatic-H), 6.34 (s, 2H, CH=CH–pyrrole), 3.45 (s, 2H, CH₂), 2.25 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 169.07 ([double bond splayed left]C=O), 138.86, 135.26, 133.25, 129.64, 128.64, 128.07, 127.47, 127.05, 126.03, 126.69, 126.47, 106.48 (C̲H=CH̲–pyrrole), 39.5 (C̲H₂), 18.94 (C̲H₃); MS-ESI: m/z 367.2 (M + 1); anal. calcd for C₂₅H₂₂N₂O: C, 81.94; H, 6.05; N, 7.64, found C, 81.95; H, 6.11; N, 7.66.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetamide (7p). FTIR (KBr pellet) cm⁻¹: 3216 (amide N–H), 1727 (C=O str.), 1689 (C=O str.), 1613 (C–N str.), 1391 (C–O–N str.); ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.54 (bs, 1H, NH, D₂O exchangeable), 7.57 (d, 4H, J = 8.4 Hz, coumarin–CH), 7.47 (d, 4H, J = 6.8 Hz, aromatic-H), 7.30 (t, 4H, J = 6.8 Hz, aromatic-H), 7.21 (d, 2H, J = 6.8 Hz, aromatic-H), 6.83 (d, 1H, J = 8.4 Hz, coumarin–CH), 6.75 (s, 1H, coumarin–CH), 6.37 (s, 2H, CH=CH–pyrrole), 6.21 (s, 1H, coumarin–CH), 4.62 (s, 2H, OCH₂), 2.37 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 166.77 ([double bond splayed left]C=O), 160.22 ([double bond splayed left]C=O), 154.35, 153.19, 135.22, 131.33, 128.86, 126.78, 126.38, 113.85, 112.26, 111.59, 107.51 (C̲H=C̲H–pyrrole), 101.61, 66.32 (OC̲H₂), 18.09 (coumarin C̲H₃); MS-ESI: m/z 451.2 (M + 1); anal. calcd for C₂₇H₂₀N₂O₄: C, 74.30; H, 4.62; N, 6.22, found C, 74.36; H, 4.70; N, 6.23.
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8a). FTIR (KBr pellet) cm⁻¹: 3284 (amide N–H), 1662 (C=O str.), 1600 (C–N str.), 1278 (C–O–N str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 11.12 (bs, 1H, NH), 7.86 (d, 2H, J = 7.2 Hz, aromatic-H), 7.48 (t, 1H, J = 7.6 Hz, aromatic-H), 7.37–7.42 (m, 4H, aromatic-H), 7.22 (t, 1H, J = 7.6 Hz, aromatic-H), 7.11 (t, 1H, J = 7.2 Hz, aromatic-H), 6.18 (d, 1H, J = 3.6 Hz, pyrrole–H), 5.93 (d, 1H, J = 3.6 Hz, pyrrole–H), 2.14 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 166.50 ([double bond splayed left]C=O), 132.28, 131.94, 131.72, 130.73, 128.13, 127.95, 127.31, 126.62, 125.94, 105.86, 104.88, 102.99, 10.80; MS-ESI: m/z 277.2 (M + 1); anal. calcd for C₁₈H₁₆N₂O: C, 78.24; H, 5.84; N, 10.14, found C, 78.26; H, 5.94; N, 10.15.
2-Methyl-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8b). FTIR (KBr pellet) cm⁻¹: 3246 (amide N–H), 1658 (C=O str.), 1597 (C–N str.), 1392 (C–O–N str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 9.10 (bs, 1H, NH), 8.18–8.20 (m, 2H, aromatic-H), 7.87–7.89 (m, 3H, aromatic-H), 7.23–7.27 (m, 2H, aromatic-H), 7.15–7.16 (m, 2H, aromatic-H), 6.23 (d, 1H, J = 3.6 Hz, pyrrole–H), 5.97 (d, 1H, J = 3.6 Hz, pyrrole–H), 2.38 (s, 3H, CH₃), 2.16 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 166.61 ([double bond splayed left]C=O), 142.67, 132.71, 131.61, 131.0, 129.04, 128.31, 127.94, 127.08, 126.94, 126.17, 106.26, 105.13, 21.09, 10.81; MS-ESI: m/z 291.2 (M + 1); anal. calcd for C₁₉H₁₈N₂O: C, 78.59; H, 6.25; N, 9.65, found C, 78.62; H, 6.29; N, 9.64.
3-Methyl-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8c). FTIR (KBr pellet) cm⁻¹: 3246 (amide N–H), 1657 (C=O str.), 1599 (C–N str.), 1285 (C–O–N str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.72 (bs, 1H, NH), 7.89–7.90 (m, 2H, aromatic-H), 7.83 (s, 1H, aromatic-H), 7.74–7.79 (m, 4H, aromatic-H), 7.20–7.21 (m, 2H, aromatic-H), 6.23 (d, 1H, J = 3.6 Hz, pyrrole–H), 5.97 (d, 1H, J = 3.6 Hz, pyrrole–H), 2.38 (s, 3H, CH₃), 2.14 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 166.80 ([double bond splayed left]C=O), 140.66, 133.21, 131.13, 130.9, 129.00, 128.01, 127.99, 127.00, 126.49, 126.17, 106.60, 105.30, 21.07, 10.79; MS-ESI: m/z 291.14 (M + 1); anal. calcd for C₁₉H₁₈N₂O: C, 78.59; H, 6.25; N, 9.65, found C, 78.54; H, 6.28; N, 9.64.
4-Methyl-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8d). FTIR (KBr pellet) cm⁻¹: 3236 (amide N–H), 1660 (C=O str.), 1605 (C–N str.), 1297 (C–O–N str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.72 (bs, 1H, NH), 7.58 (d, J = 8.0 Hz, 2H, aromatic-H), 7.36 (d, 2H, J = 8.0 Hz, aromatic-H), 7.23–7.27 (m, 2H, aromatic-H), 7.16–7.20 (m, 3H, aromatic-H), 6.23 (d, 1H, J = 3.6 Hz, pyrrole–H), 5.97 (d, 1H, J = 3.6 Hz, pyrrole–H), 2.37 (s, 3H, CH₃), 2.12 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 166.58 ([double bond splayed left]C=O), 142.67, 132.71, 131.61, 131.0, 129.04, 128.31, 127.94, 127.08, 126.94, 126.17, 106.26, 105.13, 21.09, 10.81; MS-ESI: m/z 291.2 (M + 1); anal. calcd for C₁₉H₁₈N₂O: C, 78.59; H, 6.25; N, 9.65, found C, 78.55; H, 6.27; N, 9.65.
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)isonicotinamide (8e). FTIR (KBr pellet) cm⁻¹: 3226 (amide N–H), 1665 (C=O str.), 1590 (C–N str.), 1276 (C–O–N str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 10.25 (bs, 1H, NH), 8.44 (2H, d, J = 5.6 Hz, aromatic-H), 7.40 (2H, d, J = 5.6 Hz, aromatic-H), 6.14 (d, 1H, J = 3.2 Hz, pyrrole–H), 5.94 (d, 1H, J = 3.2 Hz, pyrrole–H), 2.02 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 165.12 ([double bond splayed left]C=O), 149.92, 138.54, 132.52, 131.34, 130.46, 128.03, 126.79, 126.41, 120.80, 106.60, 105.55, 10.68; MS-ESI: m/z 278.2 (M + 1); anal. calcd for C₁₇H₁₅N₃O: C, 73.63; H, 5.45; N, 15.15, found C, 73.66; H, 5.65; N, 15.20.
2-Bromo-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8f). FTIR (KBr pellet) cm⁻¹: 3218 (amide N–H), 1672 (C=O str.), 1596 (C–N str.), 1292 (C–O–N str.), 748 (C–Br str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.19 (bs, 1H, NH), 7.56–7.59 (m, 1H, aromatic-H), 7.38 (d, 2H, J = 7.2 Hz, aromatic-H), 7.24–7.35 (m, 6H, aromatic-H), 6.19 (d, 1H, J = 4.0 Hz, pyrrole–H), 5.99 (d, 1H, J = 3.2 Hz, pyrrole–H), 2.28 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 166.2 ([double bond splayed left]C=O), 134.41, 133.22, 131.69, 131.42, 130.89, 129.14, 127.96, 127.72, 127.11, 126.59, 119.25, 106.5, 105.39, 10.2; MS-ESI: m/z 355.2, 357 (M + 1, M + 2); anal. calcd for C₁₈H₁₅BrN₂O: C, 60.86; H, 4.26; N, 7.89, found C, 60.88; H, 4.31; N, 7.89.
2-Chloro-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8g). FTIR (KBr pellet) cm⁻¹: 3210 (amide N–H), 1651 (C=O str.), 1603 (C–N str.), 1282 (C–O–N str.), 1027 (C–Cl str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 11.01 (bs, 1H, NH), 7.86 (d, J = 7.2 Hz, 2H, aromatic-H), 7.42 (d, 2H, J = 6.4 Hz, aromatic-H), 7.22 (s, 2H, aromatic-H), 7.12 (d, 1H, J = 6.0 Hz, aromatic-H), 6.88 (d, 2H, J = 7.2 Hz, aromatic-H), 6.17 (s, 1H, pyrrole–H), 5.92 (s, 1H, pyrrole–H), 2.12 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 165.92 ([double bond splayed left]C=O), 162.23, 132.27, 131.99, 130.83, 129.26, 127.95, 126.56, 125.87, 123.85, 113.35, 105.78, 104.80, 10.98; MS-ESI: m/z 311 (M + 1), 312 (M + 2); anal. calcd for C₁₉H₁₈N₂O: C, 69.57; H, 4.86; N, 9.01, found C, 69.58; H, 4.84; N, 9.00.
2,4-Dichloro-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8h). FTIR (KBr pellet) cm⁻¹: 3216 (amide N–H), 1672 (C=O str.), 1584 (C–N str.), 1290 (C–O–N str.), 1048 (C–Cl str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.47 (s, 1H, NH), 7.30–7.35 (s, 1H, aromatic-H), 7.20–7.25 (m, 2H, J = 8.0 Hz, aromatic-H), 6.19 (d, 1H, J = 3.6 Hz, pyrrole–H), 5.88 (d, 1H, J = 3.6 Hz, pyrrole–H), 2.20 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 164.58 ([double bond splayed left]C=O), 137.30, 132.87, 131.62, 131.39, 130.63, 130.46, 130.40, 129.79, 127.88, 127.50, 127.09, 126.54, 106.53, 105.45, 11.02; MS-ESI: m/z 345.0, 346 (M + 1, M + 2); anal. calcd for C₁₈H₁₄Cl₂N₂O: C, 62.62; H, 4.09; N, 8.11, found C, 62.71; H, 4.10; N, 8.11.
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)-2-phenoxyacetamide (8i). FTIR (KBr pellet) cm⁻¹: 3187 (amide N–H), 1685 (C=O str.), 1596 (C–N str.), 1233 (C–O–N str.), 1079 (C–O–C str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.85 (bs, 1H, NH), 7.25–7.36 (m, 7H, aromatic-H), 7.04 (t, 1H, J = 7.2 Hz, aromatic-H), 6.85 (d, 2H, J = 8.0 Hz, aromatic-H), 6.24 (d, 1H, J = 3.6 Hz, pyrrole–H), 6.02 (d, 1H, J = 3.6 Hz, pyrrole–H), 4.60 (s, 2H, OCH₂), 2.14 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 167.23 ([double bond splayed left]C=O), 156.37, 132.81, 131.31, 130.56, 129.41, 128.05, 127.09, 126.46, 122.06, 114.16, 106.68, 105.51, 66.51, 10.88; MS-ESI: m/z 306.2 (M + 1); anal. calcd for C₁₉H₁₈N₂O₂: C, 74.49; H, 5.92; N, 9.14, found C, 74.59; H, 5.97; N, 9.15.
4-Methoxy-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8j). FTIR (KBr pellet) cm⁻¹: 3211 (amide N–H), 1652 (C=O str.), 1604 (C–N str.), 1283 (C–O–N str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 10.91 (bs, 1H, NH), 7.84 (d, 2H, J = 8.4 Hz, aromatic-H), 7.39 (d, 2H, J = 7.2 Hz, aromatic-H), 7.19 (t, 2H, J = 7.2 Hz, aromatic-H), 7.08 (d, 1H, J = 7.2 Hz, aromatic-H), 6.85 (d, 2H, J = 8.4 Hz, aromatic-H), 6.15 (d, 1H, J = 3.2 Hz, pyrrole–H), 5.90 (d, 1H, J = 3.2 Hz, pyrrole–H), 3.76 (s, 3H, OCH₃), 2.11 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 160.38 ([double bond splayed left]C=O), 157.21, 127.31, 126.39, 125.86, 124.25, 122.92, 121.60, 120.85, 118.89, 108.32, 100.79, 99.80, 49.98, 5.97; MS-ESI: m/z 307.2 (M + 1); anal. calcd for C₁₉H₁₈N₂O₂: C, 74.49; H, 5.92; N, 9.14, found C, 74.49; H, 5.98; N, 9.15.
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)-2-phenylacetamide (8k). FTIR (KBr pellet) cm⁻¹: 3244 (amide N–H), 1670 (C=O str.), 1600 (C–N str.), 1348 (C–O–N str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.17 (bs, 1H, NH), 7.24–7.38 (m, 6H, aromatic-H), 7.12–7.19 (m, 4H, aromatic-H), 6.14 (d, 1H, J = 3.6 Hz, pyrrole–H), 5.89 (d, 1H, J = 3.6 Hz, pyrrole–H), 3.52 (s, 2H, CH₂), 2.01 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 169.97 ([double bond splayed left]C=O), 132.99, 130.47, 128.92, 128.85, 127.86, 126.13, 126.5, 106.79, 105.6, 41.0, 10.1; MS-ESI: m/z 291.1 (M + 1); anal. calcd for C₁₉H₁₈N₂O: C, 78.59; H, 5.92; N, 9.65, found C, 78.62; H, 5.92; N, 9.65.
2-(4-Isobutylphenyl)-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)propanamide (8l). FTIR (KBr pellet) cm⁻¹: 3227 (amide N–H), 1666 (C=O str.), 1591 (C–N str.), 1281 (C–O–N str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.61 (bs, 1H, NH), 7.83–7.84 (m, 2H, aromatic-H), 7.34–7.36 (m, 3H, aromatic-H), 7.28 (d, 2H, J = 8.0 Hz, aromatic-H), 7.11 (d, 2H, J = 8.0 Hz, aromatic-H), 6.05 (d, 1H, J = 3.6 Hz, pyrrole–H), 5.90 (d, 1H, J = 3.6 Hz, pyrrole–H), 3.58 (q, 1H, J = 6.0 Hz, CH–CH₃), 2.44 (d, 2H, J = 6.8 Hz, CH–CH₂–Ph), 2.12 (s, 3H, CH₃), 1.85 (d, 1H, J = 6.0 Hz, CH), 1.48 (d, 3H, J = 9.6 Hz, CH₃), 0.90 (d, 6H, J = 6.0 Hz, CH–(CH₃)₂); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 176.11 ([double bond splayed left]C=O), 141.71, 134.69, 132.3, 131.22, 130.16, 129.60, 127.08, 107.13 (CH near the Ph of pyrrole), 106.72 (CH near the methyl of pyrrole), 45.40 (CH–CH₃), 39.98 (C̲H₂–CH), 29.15 [C̲H–(CH₃)₂], 22.12 [CH–(C̲H₃)₂], 17.41 (CH–C̲H₃), 10.72 (CH₃); MS-ESI: m/z 360.3 (M + 1); anal. calcd for C₂₄H₂₈N₂O: C, 79.96; H, 7.83; N, 7.77, found C, 79.98, H, 7.87; N, 7.78.
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)-2-(naphthalen-1-yl)acetamide (8m). FTIR (KBr pellet) cm⁻¹: 3211 (amide N–H), 1665 (C=O str.), 1596 (C–N str.), 1317 (C–O–N str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.19 (bs, 1H, NH), 7.57–7.45 (m, 4H, aromatic-H), 7.31–7.32 (m, 2H, aromatic-H), 7.06–7.20 (m, 6H, aromatic-H), 6.13 (d, 1H, J = 3.6 Hz, pyrrole–H), 5.90 (d, 1H, J = 3.6 Hz, pyrrole–H), 4.11 (s, 2H, CH₂), 2.11 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 169.12 ([double bond splayed left]C=O), 136.76, 132.69, 131.22, 130.6, 130.3, 129.16, 127.53, 126.84, 106.09, 105.21, 41.5, 10.8; MS-ESI: m/z 341.2 (M + 1); anal. calcd for C₂₃H₂₀N₂O: C, 81.15; H, 5.92; N, 8.23, found C, 81.19, H, 5.96; N, 8.23.
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)-2-(naphthalen-2-yloxy)acetamide (8n). FTIR (KBr pellet) cm⁻¹: 3245 (amide N–H), 1692 (C=O str.), 1628 (C–N str.), 1394 (C–O–N str.), 1063 (C–O–C str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 11.02 (bs, 1H, NH), 7.29–7.36 (m, 6H, aromatic-H), 7.24–7.25 (m, 4H, aromatic-H), 7.03–7.08 (m, 2H, aromatic-H), 6.45 (d, 1H, J = 3.6 Hz, pyrrole–H), 5.88 (d, 1H, J = 3.6 Hz, pyrrole–H), 4.66 (s, 2H, OCH₂), 2.12 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 170.11 ([double bond splayed left]C=O), 156.09, 136.12, 131.22, 130.43, 129.29, 127.13, 126.64, 118.19, 106.3 (CH near the methyl of phenyl), 105.12 (CH near the methyl of pyrrole), 70.01 (OC̲H₂), 10.31 (C̲H₃); MS-ESI: m/z 357.2 (M + 1); anal. calcd for C₂₃H₂₀N₂O₂: C, 77.51; H, 5.66; N, 7.86, found C, 77.55, H, 5.68; N, 7.85.
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)-2-(o-tolyl)acetamide (8o). FTIR (KBr pellet) cm⁻¹: 3249 (amide N–H), 1674 (C=O str.), 1601 (C–N str.), 1348 (C–O–N str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 7.54 (bs, 1H, NH), 7.06–7.36 (m, 7H, aromatic-H), 7.02 (t, 2H, J = 8.0 Hz, aromatic-H), 6.14 (d, 1H, J = 3.6 Hz, pyrrole–H), 5.92 (d, 1H, J = 3.6 Hz, pyrrole–H), 3.66 (s, 2H, OCH₂), 3.16 (s, 3H, CH₃), 2.09 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 169.07 ([double bond splayed left]C=O), 136.76, 132.69, 131.3, 131.22, 130.61, 130.37, 129.94, 129.61, 127.3, 126.41, 106.34, 105.2, 39.5 (C̲H₂), 18.94 (C̲H₃), 10.78 (C̲H₃); MS-ESI: m/z 305.2 (M + 1); anal. calcd for C₂₀H₂₀N₂O: C, 78.92; H, 6.62; N, 9.20, found C, 78.95; H, 6.68; N, 9.20.
2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)acetamide (8p). FTIR (KBr pellet) cm⁻¹: 3208 (amide N–H), 1711 (C=O str.), 1682 (C=O str.), 1612 (C–N str.), 1391 (C–O–N str.); ¹H NMR (400 MHz, CDCl₃): δ (ppm) 8.87 (bs, 1H, NH), 7.76 (J = 8.4 Hz, aromatic-H), 7.06–7.36 (m, 7H, aromatic-H), 7.02 (t, 2H, J = 8.4 Hz, aromatic-H), 6.99 (1H, dd, J = 8.4 Hz, J = 2.0 Hz, aromatic-H), 6.08 (d, 1H, J = 3.6 Hz, pyrrole–H), 6.08 (s, 1H, coumarin–H), 5.93 (d, 1H, J = 3.6 Hz, pyrrole–H), 4.70 (s, 2H, OCH₂), 2.28 (s, 3H, CH₃), 2.10 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ (ppm) 170.21 ([double bond splayed left]C=O), 160.89 (C=O of coumarin), 159.09 (C–O), 131.76, 130.46, 129.09, 127.78, 117.44, 108.03 (CH near the Ph of pyrrole), 106.21 (CH near the methyl of pyrrole), 104.73, 68.20 (OC̲H₂), 19.53 (coumarin C̲H₃), 10.18 (pyrrole C̲H₃); MS-ESI: m/z 389.2 (M + 1); anal. calcd for C₂₃H₂₀N₂O₄: C, 71.12; H, 5.19; N, 7.21, found C, 71.16; H, 5.25; N, 7.22.

4.2. Antitubercular activity

All the synthesized compounds were tested in vitro against Mycobacterium tuberculosis H₃₇Rv (Mtb H₃₇Rv) in a high throughput screen using a procedure adapted from the microplate Alamar blue assay. The experiment was carried out in 96-well microplates containing 7H9-Tween-OADC medium by measuring bacterial growth after 5 days in the presence of test compounds. Test compounds were prepared as ten-fold serial dilutions in DMSO. Compounds 7a–p and 8a–p and reference drugs were dissolved in DMSO at a concentration of 5 mg mL⁻¹ and refrigerated until used. Plates were inoculated with Mtb H₃₇Rv ATCC 27294, covered and sealed with Parafilm and incubated at 37 °C for five days. Testing was conducted in duplicate and the following controls were taken (i) medium only (sterility control); (ii) organism in medium (negative control); and (iii) reference compounds (positive control). The growth was measured by OD₅₉₀ and fluorescence (Ex 560/Em 590) using a BioTek™ Synergy 4 plate reader. To calculate the MIC, the 10-point dose response curve was plotted as % growth and fitted to the Gompertz model using GraphPad Prism 5. The MIC was defined as the minimum concentration at which growth was completely inhibited and was calculated from the dose response curve at zero growth.

% Inhibition = [1 − (growth index of test sample/growth index of control)] × 100.

Intracellular activity assay. Murine J774 macrophages were infected with a luminescent strain of H₃₇Rv (which constitutively expresses luxABCDE) at a multiplicity of infection of 1. After 18 h, extracellular bacteria were removed by washing, and subsequently test and positive control (INH) were added. Infected macrophages were incubated in the presence of compound for 4 days at 1 and 10 times MIC (as determined in aerobic culture in liquid medium). Bacteria were harvested from macrophages by lysis with 0.1% SDS, inoculated into growth media and allowed to grow aerobically for 5 days, in which the amount of bacteria present was determined by luminescence. All assays were conducted in triplicate; each assay included a positive control (INH) and a negative control (DMSO). The baseline of infection was determined by harvesting bacteria from macrophages at 0 days before compound addition and plating for CFU determination in triplicate.

The intracellular activity = [log CFU Day 4 compound] − [log CFU Day 4 DMSO]

Activity against resistant isolates. The activity of the compounds against five resistant isolates of Mtb strains under aerobic conditions was assessed by determining the minimum inhibitory concentration (MIC) of the compounds. Strains tested were two isoniazid resistant strains (INH-R1 and INH-R2), two rifampicin resistant strains (RIF-R1 and RIF-R2) and a fluoroquinolone resistant strain (FQ-R1). The assay is based on the measurement of growth in liquid medium of each strain in which the readout is optical density (OD).

INH-R1 was derived from H₃₇Rv and is a katG mutant (Y155* = truncation). INH-R2 is strain ATCC35822. RIF-R1 was derived from H₃₇Rv and is an rpoB mutant (S522L). RIF-R2 is strain ATCC35828. FQ-R1 is a fluoroquinolone-resistant strain derived from H₃₇Rv and has an unidentified mutation.

The MIC of compounds was determined by measuring bacterial growth after 5 days in the presence of the compounds. The compounds were prepared as 10-point two-fold serial dilutions in DMSO and diluted into 7H9-Tween-OADC medium in 96-well plates with a final DMSO concentration. Plates were inoculated with Mtb and incubated for 5 days; growth was measured by OD₅₉₀. To calculate the MIC, the 10-point dose response curve was plotted as % growth and fitted to the Gompertz model using GraphPad Prism 5. In addition dose response curves were generated using the Levenberg–Marquardt algorithm and the concentrations that resulted in 50% and 90% inhibition of growth were determined (IC₅₀ and IC₉₀, respectively).

4.3. Evaluation of cytotoxicity

A drug cytotoxicity control plate assay (MTT cell proliferation) was conducted using Vero African green monkey kidney cell line to confirm that concentrations utilized for testing were not toxic to that cell line. The cells were plated in flat-bottom 96-well plates cultured for 72 h in a controlled atmosphere and non-adherent cells were washed by gentle flushing with RPMI 1640 supplemented with 10% fetal bovine serum. Compounds were prepared as 10-point three-fold serial dilutions in DMSO. The highest concentration of compound tested was 100 μg mL⁻¹, in which compounds were soluble in DMSO. The plates were incubated for an additional 2 days after the addition of test compounds and MTT reagent was added. Cell viability was measured on the basis of absorbance in each well and using positive control drug and medium as blank. Each plate included staurosporine as a control.

4.4. Computational work

The protein crystal structure of Mtb ENR (InhA) (PDB ID 2H7M) with co-crystallized ligand was downloaded from a protein data bank. The protein was prepared using the “protein preparation tool” of Schrödinger. After adding hydrogen atoms, bond orders and formal charges were checked, following which minimization was performed with the root-mean-square-displacement (RMSD) cutoff of 0.30 Å. A grid file was created surrounding the area in the cavity that contains co-crystallized ligand [N-(3,5-dichlorophenyl)-5-oxo-1-phenylpyrrolidine-3-carboxamide] and the grid was defined as an enclosing box with 15 Å in all three dimensions. Three dimensional coordinates of all the ligands (7a–p and 8a–p), and their isomeric, ionization and tautomeric states were generated using the LigPrep module of Maestro 9.4 (LigPrep with default setting, Schrödinger). The poses were selected based on hydrophobic, hydrogen bond and charge–charge interactions. Special care was also taken to identify poses that may involve binding modes of the molecules in the hydrophobic cavity. The cutoff distance for hydrogen bond, van der Waals interaction, and electrostatic interaction is 3.20 Å (angle >90–180°), 4.20 Å, and 4.00 Å, respectively.

Prediction of pharmacokinetic property (ADME). Pharmacokinetic properties of the title compounds (7a–p and 8a–p) were calculated using Molinspiration online property calculation program. Polar surface area (TPSA), miLog P, number of rotatable bonds (n-ROTB), molecular volume (MV), number of hydrogen donor (n-OH and NH) and acceptor atoms (n-O and N atoms) and violations of Lipinski's rule of five of the titled compounds (7a–p and 8a–p) are presented in ESI.†

Software used

(a) Schrödinger Suite 2013 Protein Preparation Wizard; LLC, New York, NY, Glide 5.9 Schrödinger, LLC; New York, NY, 2013, LigPrep, version 2.6, Schrödinger, LLC, New York, NY, 2013. (b) Molinspiration property calculation program.

Conflict of interest

The authors report no declarations of conflict of interest.

Acknowledgements

This work was financially supported by University Grant Commission, New Delhi, India to Rikta Saha. We gratefully acknowledge the TAACF (Tuberculosis Antimicrobial Acquisition Coordinating Facility, National Institute of Allergy and Infectious Diseases (Contract no. HHSN272201100009I), USA for carrying out the biological activity.

Notes and references

1. D. J. Payne, M. N. Gwynn, D. J. Holmes and D. L. Pompliano, Nat. Rev. Drug Discovery, 2007, 6, 29–40.
2. M. Berry and O. M. Kon, Eur. Respir. Rev., 2009, 18, 195–197.
3. J. C. Palomino and A. Martin, J. Antimicrob. Chemother., 2013, 68, 275–283.
4. F. W. Muregi and A. Ishih, Drug Dev. Res., 2010, 71, 20–32.
5. A. Imramovsky, S. Polanc, J. Vinsova, M. Kocevar, J. Jampilek, Z. Reckova and J. Kaustova, Bioorg. Med. Chem., 2007, 15, 2551–2559.
6. A. V. Shindikar and C. L. Viswanathan, Bioorg. Med. Chem. Lett., 2005, 15, 1803–1806.
7. U. Pandit and A. Dodiya, Med. Chem. Res., 2013, 22, 3364–3371.
8. V. Padmavathi, C. Prema Kumari, B. C. Venkatesh and A. Padmaja, Eur. J. Med. Chem., 2011, 46, 5317–5326.
9. K. M. Hilmy, M. M. Khalifa, M. A. Hawata, R. M. Keshk and A. A. el-Torgman, Eur. J. Med. Chem., 2010, 45, 5243–5250.
10. M. Biava, G. C. Porretta, G. Poce, A. De Logu, R. Meleddu, E. De Rossi, F. Manetti and M. Botta, Eur. J. Med. Chem., 2009, 44, 4734–4738.
11. S. D. Joshi, H. M. Vagdevi, V. P. Vaidya and G. S. Gadaginamath, Eur. J. Med. Chem., 2008, 43, 1989–1996.
12. A. Kamal, S. Prabhakar, M. Janaki Ramaiah, P. Venkat Reddy, C. Ratna Reddy, A. Mallareddy, N. Shankaraiah, T. Lakshmi Narayan Reddy, S. N. Pushpavalli and M. Pal-Bhadra, Eur. J. Med. Chem., 2011, 46, 3820–3831.
13. X. M. Peng, G. L. Damu and C. Zhou, Curr. Pharm. Des., 2013, 19, 3884–3930.
14. H. Li, X. Wang, G. Xu, L. Zeng, K. Cheng, P. Gao, Q. Sun, W. Liao and J. Zhang, Bioorg. Med. Chem. Lett., 2014, 24, 5274–5278.
15. A. H. Rezayan, P. Azerang, S. Sardari and A. Sarvary, Chem. Biol. Drug Des., 2012, 80, 929–936.
16. Z. Q. Xu, W. W. Barrow, W. J. Suling, L. Westbrook, E. Barrow, Y. M. Lin and M. T. Flavin, Bioorg. Med. Chem., 2004, 12, 1199–1207.
17. J. D. Guzman, D. Evangelopoulos, A. Gupta, K. Birchall, S. Mwaigwisya, B. Saxty, T. D. McHugh, S. Gibbons, J. Malkinson and S. Bhakta, BMJ, 2013, 3, e002672.
18. T. J. de Faria, M. Roman, N. M. de Souza, R. De Vecchi, J. V. de Assis, A. L. dos Santos, I. H. Bechtold, N. Winter, M. J. Soares, L. P. Silva, M. V. De Almeida and A. Bafica, Antimicrob. Agents Chemother., 2012, 56, 2259–2267.
19. H. L. Yale, K. Losee, J. Martins, M. Holsing, F. M. Perry and J. Bernstein, J. Am. Chem. Soc., 1953, 75, 1933–1942.
20. L. Collins and S. G. Franzblau, Antimicrob. Agents Chemother., 1997, 41, 1004–1009.
21. R. S. Reis, I. Neves Jr, S. L. Lourenco, L. S. Fonseca and M. C. Lourenco, J. Clin. Microbiol., 2004, 42, 2247–2248.
22. Global Alliance for TB Drug Development (TB Alliance), Tuberculosis, 2008, 88, 85–169.
23. I. Orme, Antimicrob. Agents Chemother., 2001, 45, 1943–1946.
24. X. He, A. Alian, R. Stroud and P. R. Ortiz de Montellano, J. Med. Chem., 2006, 49, 6308–6323.
25. E. Nuermberger and J. Grosset, Eur. J. Clin. Microbiol. Infect. Dis., 2004, 23, 243–255.
26. A. Kumar, G. Patel and S. K. Menon, Chem. Biol. Drug Des., 2009, 73, 553–557.
27. R. M. Cowper and L. H. Davidson, Org. Synth., 1939, 19, 24.

---

Footnote

† Electronic supplementary information (ESI) available: Physicochemical properties, calculated ADMET properties, ¹H, ¹³C-NMR and purity data of the compounds can be found online. See DOI: 10.1039/c4ra14440f

---