Synthesis and antibacterial activity of N1-(carbazol-3-yl)amidrazones incorporating piperazines and related congeners

2.1 Chemistry

The synthesis of the target N′-(carbazol-3-yl)amidrazones 7a–n (shown in Scheme 2) commenced with the preparation of the appropriate N1-(carbazol-3-yl)hydrazonyl chloride 5 (Scheme 1). Precursor 5 is readily accessible via direct coupling of 3-carbazolediazonium chloride 3A (freshly prepared by diazotization of 3-amino-9H-carbazole) with 3-chloropentane-2,4-dione in aqueous ethanol buffered with sodium acetate. The resulting intermediate azo compound 4 undergoes conversion to the corresponding hydrazone structure 5 via loss of an acetyl group (Japp-Klingemann reaction) [19–22]. Unexpectedly, a chlorine atom has been incorporated at carbon-4 of the carbazole moiety in 5 as evidenced by elemental analysis, high-resolution mass spectra-electrospray ion trap (HRMS-ESI), ¹H and ¹³C NMR spectral data (given in the Experimental section). Thus, a DEPT-90 (distortionless enhancement of polarization transfer using a 90° decoupler pulse) experiment for 5 shows the presence of only six aromatic CH signals, whereas the starting 3-aminocarbazole 3 has seven aromatic CH entities. Additionally, the HRMS spectra of 5 display in the monomeric molecular ion region characteristic isotopic chlorine clusters with a percentage relative abundance ratio of 9:6:1 indicative of the presence of two chlorine atoms in the molecule. Under the prevailing reaction conditions, this process of aromatic ring chlorination is hitherto unprecedented, and for which an explanation requires further practical investigations.

Piperazines and the related sec-cyclic amine congeners 6a–n, acting as nitrogen nucleophiles, are expected to add readily onto N′-(carbazol-3-yl)nitrilimine (the reactive 1,3-dipole generated in situ from its hydrazonoyl chloride 5 in the presence of triethylamine) to produce the respective amidrazone adducts 7a–n (Scheme 2). This mode of nucleophile addition of various nucleophiles onto 1,3-dipolar species is well documented in the literature [23–28], and several related amidrazone adducts were obtained from the reaction of amines with hydrazonoyl chlorides [17, 18, 29, 30].

The newly synthesized amidrazones 7a–n were characterized by MS and NMR spectral data, detailed in the Experimental section. As would be expected, these data reveal that the chlorine atom at C-4 in 5 is retained in the adducts 7a–n and is further confirmed by single crystal X-ray structure determination for 7n as a representative of the series (Fig. 3). In essence, the mass spectra display the correct molecular ion peaks for which the high-resolution (HRMS-ESI) data are in good agreement with the calculated values. The monomeric molecular ion regions are dominated by the characteristic isotopic chlorine clusters (with a relative abundance percent ratio of 3:1). DEPT and 2D (correlation spectroscopy, heteronuclear multiple-quantum coherence, heteronuclear multiple-bond correlation) experiments showed correlations that helped in the ¹H and ¹³C signal assignments to the different carbon atoms and their attached and/or neighboring hydrogen atoms.

Fig. 3:

Molecular structure of 7n in the crystal and atom numbering scheme adopted. Displacement ellipsoids are drawn at the 30% probability level, hydrogen atoms as spheres with arbitrary radii.

2.2 X-ray structure determination of 7n

An X-ray crystal structure determination was performed to confirm the structure of 7n (Scheme 2) as a representative example of the new synthetic N1-(4-chloro-9-ethylcarbazol-3-yl)amidrazones 7a–n. A summary of the crystal data and numbers pertinent to data collection and refinement is given in Table 1. The molecular structure of 7n in the crystal is shown in Fig. 3.

2.3 Antibacterial activity

The antimicrobial properties of N1-(4-chloro-9-ethylcarbazol-3-yl)amidrazones 7a–n were evaluated in vitro against an assortment of Gram-positive and Gram-negative bacterial strains, and one fungus Candida albicans. Table 2 shows the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of seven compounds against four Gram-positive bacterial strains. The following three compounds were also highly active against the indicated Gram-negative bacterial strains: 7a against Salmonella typhimurium (ATCC 14028), 7h against Klebsiella pneumoniae (ATCC 700603), and 7f against K. pneumoniae and Shigella sonnei (ATCC 9290), showing MBC/MIC values of 100/12.5, 6.25/3.125, 100/12.5, and 100/12.5, respectively. The MIC values for the compounds against Gram-positive bacteria ranged from 1.56 to 100 µg mL⁻¹ (Table 2), while the MICs against Gram-negative bacteria were in the range between 12.5 and 100 µg mL⁻¹ (vide supra). Among all of the tested pathogens, MRSA was the most susceptible to 7f, and Bacillus cereus to 7b both with an MIC value of 1.56 µg mL⁻¹ with bactericidal activity against MRSA and bacteriostatic (inhibitory) activity against B. cereus. In this regard, special attention should be devoted to compounds 7c, 7f, and 7m showing high activity against MRSA which is considered as a very important pathogen problematic in nosocomial infections. On the other hand, compound 7b and related congeners, showing high activity against B. cereus (known as a pathogen causing food poisoning), could be considered as candidate preservatives in food industry if they are proven to be non-toxic to humans. Furthermore, compounds 7c, 7f, and 7h could be useful lead structures for further development of new antibacterial agents against Gram-positive and Gram-negative pathogens. However, compounds 7a–n did not exhibit any antifungal properties against C. albicans.

4.1 (E)-N′-(4-Chloro-9-ethyl-9H-carbazol-3-yl)-2-oxopropane-hydrazonoyl chloride (5)

This compound was prepared by the following two-step procedure. Step (i) 9-Ethyl-9H-carbazol-3-amine (3, 2.1 g, 10 mmol) was dissolved in 6 n aqueous hydrochloric acid (20 mL). To this solution, cooled at −5 to 0°C, was dropwise added a solution of sodium nitrite (1.2 g, 17 mmol) in water (2 mL) with efficient stirring. Stirring was continued at 0–4°C for 20–30 min. Step (ii) The fresh diazonium chloride solution, prepared in step (i), was added to a cold solution (−5 to 0°C) of 3-chloropenta-2,4-dione (1.35 g, 10 mmol) and sodium acetate (25 g, 0.3 mol) in 50% aqueous ethanol (20 mL) with vigorous stirring, which was continued at 0–4°C for 20 min. Thereafter, cold water (100 mL) was added to the reaction mixture and stirred for additional 20 min. The resulting precipitate was collected by suction filtration, dried, and purified by column chromatography (using silica as adsorbent and chloroform-hexane (1:1, v/v) as eluent). The title compound was obtained as a yellowish solid. Yield: 1.2 g (34%); m.p. 178–180°C. – IR (KBr): ν_max = 3417, 1680, 1523 cm⁻¹. – HRMS ((+)-ESI): m/z = 370.04879 (calcd. 370.04844 for C₁₇H₁₅³⁵Cl₂N₃ONa, [M+Na]⁺), 372.04586 (calcd. 372.04549 for C₁₇H₁₅³⁵Cl³⁷ClN₃ONa, [M+2+Na]⁺), 374.04289 (calcd. 374.04254 for C₁₇H₁₅³⁷Cl₂N₃ONa, [M+4+Na]⁺). – ¹H NMR (500 MHz, CDCl₃): δ = 1.46 (t, J = 7.2 Hz, 3H, NCH₂CH₃), 2.61 (s, 3H, COCH₃), 4.40 (q, J = 7.2 Hz, 2H, NCH₂CH₃), 7.30 (pseudo-t, J = 7.5 Hz, 1H, 6-H), 7.36 (d, J = 8.8 Hz, 1H, 1-H), 7.43 (d, J = 8.2 Hz, 8-H), 7.55 (pseudo-t, J = 7.6 Hz, 1H, 7-H), 7.70 (d, J = 8.8 Hz, 1H, 2-H), 8.59 (d, J = 7.9 Hz, 1H, 5-H), 9.05 (s, 1H, N–H) ppm. – ¹³C NMR (125 MHz, CDCl₃): δ = 13.8 (NCH₂CH₃), 25.3 (COCH₃), 37.8 (NCH₂CH₃), 108.0 (C-1), 108.6 (C-8), 113.5 (C-2), 114.1 (C-4), 119.3 (C-6), 120.1 (C-4a), 121.7 (C-4b), 123.1 (C-5), 125.8 (N–C=N), 126.6 (C-7), 130.3 (C-3), 137.0 (C-9a), 140.4 (C-8a), 188.2 (COCH₃) ppm. – C₁₇H₁₅Cl₂N₃O (348.23): calcd. C 58.63, H 4.34, Cl 20.36, N 12.07; found C 58.45, H 4.19, Cl 20.16, N 11.88.

4.2 General procedure for the preparation of amidrazones 7a–n

To a stirred cold suspension (−5 to 0°C) of the hydrazonoyl chloride (5) (0.20 g, 0.57 mmol) in ethanol (20 mL) in a dark flask was added triethylamine (2 mL), followed by the addition of the particular cyclic secondary amine 6a–n (2.8 mmol). Stirring was continued at −5 to 0°C for 2–4 h, and then at ambient temperature for 10–12 h. Thereafter, the reaction mixture was diluted with water (100 mL) and stirred for 10 min. The resulting crude solid product was collected by suction filtration, washed with cold water, and dried.

4.3 Antibacterial activity

The antibacterial activity of compounds 7a–c, 7f, 7h, and 7k–m was determined by the standard broth dilution assay [32] using 96-well microtiter plates. Stock solutions of the compounds in 80% methanol were serially diluted in 100 mL volumes of Mueller-Hinton broth (Oxoid) to obtain known concentrations of the compound per milliliter. To each well was added 10 µL of a 1/100 dilution of the standardized broth culture (0.5 McFarland standards). Control wells contained broth and bacteria without the compound, broth with 80% methanol, and broth plus the compound without bacteria. The plates were incubated aerobically at 35°C for 24 h. MIC was taken as the lowest concentration of the compound that resulted in complete inhibition (visible turbidity) of bacterial growth. A loopful of fluid from those wells showing no growth was subcultured onto Mueller-Hinton plates for aerobic incubation to test for viability, and the lowest concentration of the compound that demonstrated no viability represented the MBC of that compound.

4.4 X-ray diffraction structure determination of 7n

Yellow block crystals were grown slowly during 1 week from a dilute solution of 7n in ethanol. A suitable crystal with approximate dimensions of 0.12 × 0.08 × 0.02 mm³ was mounted with epoxy glue on a glass fiber. Data were collected at room temperature (293 K) using an Oxford Xcalibur diffractometer. Data were acquired and processed using CrysAlisPro software [33]. Cell parameters were determined and refined using CrysAlisPro [33]. A multiscan absorption collection was applied with maximum and minimum transmission factors of 1.000 and 0.797, respectively. The structure was solved by Direct Methods and refined by full-matrix least squares on F² using all unique data using Shelxtl [34–36]. All nonhydrogen atoms were refined anisotropically with the hydrogen atoms placed on the calculated positions using a riding model. A summary of the crystallographic data is given in Table 1.

CCDC 1453328 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.