Synthesis, characterization and preliminary antimicrobial study of some new phthalimide phenyl hydrazide derivatives

Experiment

Phthalic anhydride was produced by Alpha Chemika India, and para-aminobenzoic acid was supplied by Thomas Baker, Mumbai, India. Glacial acetic acid, 99.98% ethanol, thionyl chloride, and hydrazine 99%, n-hexane, benzene, acetone, and ether were among the solvents and other chemicals that were purchased from commercial sources and employed in the various stages of the synthesis. Thin layer chromatography (TLC) (GF254 merk-Germany) under UV light was applied to ensure that the created substances were pure and to observe reaction progression (254 nm). S1 [(benzene: acetone) (7:3)] and S2 [(ethanol, ethylacetate, n-hexane, and toluene) (3:3:6:4)] were the solvent systems that were used. The melting points were calculated without adjustment using capillary tubes and the Stuart SMP30 apparatus, (Bibby Scientific, UK). The thin film approach acquired FT-IR spectra (υ, cm-1) using an attenuated total reflectance - Fourier transform infrared spectrometer (ATR- FTIR) (IRAffinity-1S, Shimadzu, Japan), at the University of Baghdad’s College of Pharmacy. Part of our study were conducted at the University of Tehran, Iran including Proton nuclear magnetic resonance (1H-NMR) studies, which used a Bruker Ultrashield, (Bruker Ltd, UK) 500 MHz with DMSO as the solvent.

4-(1,3-dioxoisoindolin-2-yl) benzoic acid⁷

Phthalic anhydride and 4-aminobenzoic acid were reacted in an equimolar ratio of 1 mmoL each in 15 ml of glacial acetic acid.

After refluxing the reactants for three hours, the reaction medium was washed with 25 mL of ice-distilled water, filtered, dried, and recrystallized from absolute ethanol to get an intermediate (1).

The yield at 93% was a fluffy white powder and the melting point (m.p.) was 287–290°C. RF (0.74), FTIR: 3100–2553.75 cm^-1 O-H Carboxylic acid stretching; 3067.82 cm^-1. (C-H) stretching of Ar.; 1782.23 and 1766.80 cm^-1 asymmetric and symmetric. Imide (C=O) str.; 1720.50 cm^-1 carboxyl (C=O) str.; 1689.64–1465 cm^-1 Ar.(C=C) str.; 1076 and 705 cm^-1 aromatic (C-H) bend. HNMR interpretation δ = ppm (300MHz, Bruker): 10.50 (1H, s, OH), 7.62–8.12 (8H, m, 2Ar-H).

4-(1,3-dioxoisoindolin-2-yl) benzoyl chloride

An addition of 10 mL of SOCl₂ to the intermediate (1) (10 mmol) in 5 mL dry benzene and three hours of refluxing was done. Once cooled, the overabundance of SOCl2 and benzene were vacuum isolated and then washed with benzene to give intermediate (2).

This yield at 77% was a yellow powder, m.p. 300°C. RF 0.3. FTIR: 1782.23 cm^-1 carboxyl (C=O) str; 1766.80 and 1708.93 cm^-1 asym. and sym. Imide (C=O) str; 1597.06 cm^-1 Ar.(C=C) str.; 1HNMR Interpretation δ= ppm (300MHz, Bruker): 7.62–8.12 (8H, m, 2Ar-H).¹³

4-(1,3-dioxoisoindolin-2-yl) benzo hydrazide (3)¹⁴

The intermediate (2) (10 mmol - 2.67 gm) was treated with 50 mmol of hydrazine hydrate (99%) and dry benzene (15 mL).

After refluxing the mixture for four hours, it was cooled and surplus hydrazine hydrate and solvent were separated under reduced pressure, rinsing the leftover with ether and recrystallizing it with 99% ethanol to get intermediate (3).

The yield was 65% and a white solid, m. p. 270–272°C. RF 0.6.

FTIR: 3421.72 and 3340.71 cm^-1 asym. and sym. primary amide (N-H) str.; 3224.98 cm^-1 amide (N-H) str.; 3066 cm^-1 Ar-(C-H) str.; 1681.93 and 1640.78 cm^-1 asym. and sym. Of imide (C=O) str.; 1631cm^-1 (C=O) amide str.; 1593 cm^-1 (N-H) amide bend. 1573–1442 cm^-1 Ar-(C=C) str.; 1111.86 and 771.53 cm^-1 in and out of a plane (C-H) bend.

1HNMR Interpretation: 5.1 ppm (2H,s, -NH2), 6.83–8.00 ppm (8H, m, Ar-H), 11.58 ppm (1H, s, -CO-NH).

4-(1, 3-dioxoisoindolin-2-yl) benzohydrazide derivatives (3a-f)^15,16

Using a flask with a round bottom and a magnetic stirrer, each of the aldehydes – listed in further detail in Table 1 – was dissolved in ethanol, adding three drops of glacial acetic acid.

The procedure for carrying out the synthesis of the phthalimide phenyl hydrazide derivatives and the characterization of intermediates and Schiff bases is described as follows:

Table 2. Physical properties of intermediates and schiff bases [1–3f].

No.	Compound structure	Color	Melting points °C	Yield %	RF	Solvent of recrystallization
1		White fluffy powder	287–290°C	94%	0.74	Ethanol
2		Yellow powder	300°C	77%	0.3	-
3		White solid	270–272°C	65%	0.6	Ethanol
3a		Off-white	185°C	85	0.5	Ethanol
3b		Yellow	261°C	80	0.67	Ethanol
3c		Off-white	207°C	82	0.7	Ethanol
3d		Light yellow	213–215°C	60	0.6	Ethanol
3e		Dark green	315–317°C	45	0.72	Ethanol
3f		White	198–200°C	59	0.69	Ethanol

Antibacterial assay

Using a bacterial solution with a concentration of 1.5 × 108 CFU/mL derived from the McFarland turbidity standard, an antibacterial essay using the disc-well diffusion technique was completed (number 0.50). It was used to inoculate the agar by swabbing the top of Mueller Hinton agar (MHA) plates. Under a sterile hood, the surplus liquid was left to air dry. Four wells were formed in each agar plate and were used to study the bacteria, Pseudomonas aeruginosa and Streptococcus pyogenes, and each well received (80 mL) of each synthetic drug. At 37°C, the plates were left in the incubator for 24 hours. Antibacterial activity was assessed based on measuring the diameter of the well’s surrounding inhibitory zone.¹⁷

Disc-well diffusion technique

The disc-well diffusion technique is a widely used method for assessing the antimicrobial activity of compounds against various microorganisms. This technique is particularly valuable in determining the susceptibility of bacteria and fungi to novel antimicrobial agents, such as the newly synthesized phthalimide phenyl hydrazide derivatives in this study.

The procedure involves the preparation of sterile agar plates that serve as a growth medium for the microorganisms. The agar is first inoculated with a standardized suspension of the test organism, ensuring a uniform distribution of the microorganisms across the agar surface. Once the agar solidifies, either sterile filter paper discs or wells are placed on the surface of the agar.¹⁸

Chemistry

The current work focused on developing new compounds incorporating the active moieties (phthalimide and Schiff base) with expected biological activity because both N-substituted phthalimides and Schiff bases are known as biologically active compounds with a wide spectrum of various applications. This target was created using the multistep synthesis shown in Figure 1.

Figure 1. Synthesis of phthalimide and Schiff base) with expected biological effect.

The starting step involved phthalic anhydride used with the primary amine para-amino benzoic acid for synthesizing n-phenyl phthalimide with the assistance of glacial acetic acid as the solvent and reaction catalyst.¹⁸ Intermediate 1 (the resultant of the previous reaction) underwent an SN1 reaction with thionyl chloride to chlorinate the benzoic acid portion of intermediate 1, forming intermediate 2. This reaction occurred in the presence of benzene as a solvent. As the components started to react, hydrogen chloride (HCl) fumes were produced therefore this procedure was performed under a fume hood. After three hours of reflexing, the yellow powder was isolated, composing intermediate 2.¹⁸

For preparing hydrazide derivatives (intermediate 3), we used 50 mmol hydrazine 99% with dry benzene and 10 mmol of intermediate 2.¹⁹ The resultant white powder was recrystallized by absolute ethanol. Six aldehydes were chosen for Schiff base synthesis with intermediate 3 by condensation reaction to give intermediates 3a–f.²⁰

The method we used to create the new Schiff bases involved converting benzoic acid to an active acyl chloride group placed in the para position of a phenyl ring attached to a phthalimide moiety, followed by the nucleophilic replacement of the chloride with hydrazine moiety. This created a position suitable for an amino group, which was then prepared for condensation with the chosen aldehydes and ketones to create the desired Schiff bases.

FTIR spectrum of intermediate 1 as shown in (Table 2) compound 1 shows strong absorption bands at 1782.23 and 1766.80 cm^-1 due to asymmetric. and symmetric. Imide (C=O) str. Other absorption bands appeared at 3100–2553.75cm^-1 and at 1720.50 cm^-1 and belong to O-H carboxylic acid str. and carboxyl (C=O) str., respectively.²¹

FTIR spectra of intermediate of compound 2 showed O-H carboxylic acid str vanishing. The absorption bands demonstrated the success of the dehydration reaction.

Finally, the FTIR spectrum of intermediate compound 3 showed two strong absorption bands at 3421.72 cm-1 and 3224.98 cm^-1, respectively, corresponding to the (-NH₂-NH) group, which is evidence for the attainment of hydrazine intermediate. While FTIR spectra of the prepared Schiff bases (3a–f) displayed the vanishing of these two distinctive absorption bands at 3421.72 and 3224.98 cm^-1, which belong to the (-NH₂-NH) group in hydrazine intermediate 3 and formation of the new distinct absorption band at 1654cm^-1 due to (C=N) imine. These two facts indicate the existence of the Schiff base formation. All the underlying data for FTIR results are shown in Alassadi (2023).²²

Aside from the FTIR spectrum of the Schiff bases [3a–f], distinct absorption bands at 1620 and 1611 cm^-1 were seen, attributed to asymmetric and symmetric stretching of imide (C=O) str. Other absorptions appeared at 1716.65 cm^-1 for (C=O) amide str., 1654 cm^-1 for (N=C) str. of the Schiff base, and 3224.98 cm^-1 for (N-H) str of amide.

Moreover, the FTIR spectra of Schiff bases 3a, 3c revealed distinct absorption bands at 2916 and 2877 cm^-1 asymmetric and symmetric stretching. The appearance of aliphatic (CH₃) str., while 3b demonstrated two absorptions, 3267.41 cm^-1 and 1519 cm^-1 str for phenolic (O-H) asymm. Tables 3 and 4 include complete details of the FTIR spectrum records for compounds [1–3] [3a–3f].

Furthermore, 1HNMR spectra of the produced compounds displayed distinctive unambiguous signs of intermediate 1, phthalimidobenzoic acid at σ = 7.62–8.12 ppm about aromatic protons. At σ = 10.50 ppm, a distinct signal of (OH) carboxylic proton occurred. The 1HNMR spectra of intermediate 2 revealed the elimination of the (OH) carboxyl proton peak and the emergence of multiplet peaks exclusively at σ = 7.62–8.12 ppm pertaining to protons of two aromatic rings. Intermediate 3 displayed signals at σ = 5.1 ppm and 11.58 ppm corresponding to the NH₂ and NH of the hydrazine part, respectively, whereas aromatic protons emerged at σ = 6.83–8.00 ppm.

The 1HNMR spectra of Schiff base 3a revealed a signal at σ = 2.7 ppm that belonged to CH₃ protons, multiplet signals at σ = 7.45–7.97 ppm that related to aromatic, and singlet signals at σ = 8.5 and 11.58 ppm that belonged to the CH of imide and NH of amide, respectively. Schiff base 1HNMR spectrum [3b] Singlet signals at σ = 11.25 ppm of phenolic OH and multiplet peaks at σ = 7.88–8.12 ppm of aromatic protons were seen.

Antibacterial evaluation

Antimicrobial activities for the resultant Schiff bases against two types of bacteria and one type of fungus were evaluated, and the results are listed in Table 4. The results indicated that Schiff bases 3d and 3f showed very high activity against Pseudomonas aeruginosa, Streptococcus pyogenes and the fungus Candida albicans.

Pseudomonas aeruginosa compounds and 3e and 3f showed high activity against Streptococcus pyogenes. While compounds 3a–3f were only slightly active against E. coli and there was no activity against Staphylococcus aureus and Candida albicans.