Synthesis, characterization and antimicrobial evaluation of novel urea, sulfonamide and acetamide 3,4-dihydropyrazino[1,2-a]indol-1(2H)-one derivatives

3.1 Analytical results

A series of four allied candidates (4a–e), (7a–f), (9a–d) and (12a–d) were synthesized substantially through the synthetic route as illustrated in Schemes 1–3. The off structure reaction products were confirmed by means of ¹H NMR and ¹³C NMR spectra, e.g. ¹H NMR spectrum of compound 2 indicated the presence of two triplet signals appeared at δ 4.97–4.93 ppm and δ 3.82–3.76 ppm of adjoining CH₂ protons of bromoethyl group. The ¹³C NMR spectra exhibited highest frequency signal at δ 165.78 ppm assignable to ester carbonyl, where as signals appeared around δ 31.71 and 52.84 ppm were assigned for two CH₂ of bromethyl carbons. Furthermore, the ¹H NMR spectrum of compound (4a–e) showed singlet for methylene proton at δ 4.74 ppm and aromatic protons resonate as multiplets at δ 6.90–7.68 ppm. The ¹³C NMR spectrum is in well agreement with the structure assigned. In the ¹³C NMR spectra, signals appeared around δ 43.18–48.64 ppm are assigned for methylene carbons whereas δ 121.13–136.76 ppm is attributed to aromatic carbons and carbonyl carbon was observed at about δ 159.20 ppm. The key intermediate 5 (Katritzky et al., 2003) was characterized by ¹H NMR spectrum which exhibited lack of the characteristic signal as quartet triplet pattern corresponding to the ethyl ester of compound 2 and the presence of broad singlet at δ 5.18 ppm NH₂ group (D₂O exchangeable). ¹³C NMR spectra exhibited highest frequency signal at δ 158.14 ppm assignable to the pyrazino carbonyl carbon, whereas lowest frequency signal appears between 45.01 and 50.86 ppm for two CH₂ carbons of newly formed 3,4-dihydropyrazino ring in all the spectra. The ¹H NMR spectra of urea derivatives (7a–f) indicated the presence of two broad singlets appeared at around δ 9.54 ppm for NH proton attached to the pyrazino ring whereas other NH proton attached to aryl ring appeared at around δ 8.72 ppm. Moreover, disappearance of broad singlet at δ 5.18 ppm of NH₂ proton of compound 5 clearly confirmed the formation of urea derivatives. In ¹³C NMR spectra of urea derivatives the most characteristic signal appears at δ 160.54 ppm for the carbonyl carbon between two NH groups of urea. Furthermore, the ¹H NMR spectrum of sulfonamide derivatives (9a–d) showed addition of aromatic multiplet and most downfield broad singlet appeared at around δ 10.05–11.02 ppm for sulfonamide NH proton. ¹³C NMR spectra showed highest frequency signal range observed around δ 158.67 ppm assignable to the pyrazino carbonyl carbon. Finally, the ¹H NMR spectrum of acetamide derivatives (12a–d) showed one downfield broad singlet signal appears at around δ 10.24 ppm corresponding to NH protons, whereas sharp singlet of amidic CH₂ protons observed at δ 3.08 ppm. ¹³C NMR revealed the highest frequency signals at δ 168.83 ppm assigned for carbonyl carbon of amide function.

3.2 Antimicrobial activity results

The results of antimicrobial screening of newly prepared compounds were expressed as the MIC values are summarized in Table 1.

Table 1 Results of antibacterial screening of compounds 4a–e, 7a–f, 9a–d and 12a–d.

Minimum Inhibitory Concentration (MIC) for bacteria (μg/mL)
Compound numbers	Gram-negative		Gram-positive
	E.C. MTCC 443	P.A. MTCC 1688	S.A. MTCC 96	S.P. MTCC 442
4a	200	200	500	250
4b	100	100	250	100
4c	125	125	500	250
4d	500	250	500	500
4e	62.5	50	125	100
7a	200	200	500	200
7b	200	100	250	200
7c	125	200	250	500
7d	200	250	500	250
7e	200	125	500	500
7f	100	100	500	500
9a	100	200	125	250
9b	200	125	500	500
9c	500	200	500	125
9d	100	100	250	100
12a	250	125	500	125
12b	62.5	50	125	50
12c	500	250	500	500
12d	500	500	500	250
Ampi.	100	100	250	100

S.A., Staphylococcus aureus; S.P., Streptococcus pyogenes; E.C., Escherichia coli; P.A., Pseudomonas aeruginosa; MTCC, Microbial Type Culture Collection; Ampi., Ampicillin.

Many of newly synthesized compounds are found to exhibit good to excellent antimicrobial activity. From antimicrobial activity data (Tables 1 and 2), it is observed that compounds 4b (R = 4-Cl–C₆H₄), 7b (R = Cl), 9d (R = 3,4-di-Cl–C₆H₃), 4e (R = morpholine) and 12b (R₁, R₂ = morpholine) are most active compounds. Data of antibacterial activity reveal that, compounds 4b (R = 4-Cl–C₆H₅), 7f (R = –COOMe), 9a (quinoline-8-sulfonamide) and 9d (R = 3,4-di-Cl–C₆H₃), are considered to be good active against Escherichia coli. When we have taken the morpholine group as substitution in compounds 4e and 12b, it shows excellent activity against E. coli. Compounds 4b (R = 4-Cl–C₆H₄), 7b (R = Cl), 7f (R = COOMe) and 9d (R = 3,4-di-Cl–C₆H₃) are considered as good active against Pseudomonas aeruginosa, while compounds 4e (R = morpholine) and 12b (R₁, R₂ = morpholine) are considered as very good active against P. aeruginosa. Compounds 4b (R = 4-Cl–C₆H₄), 7b (R = Cl), 7c (R = OMe), and 9d (R = 3,4-di-Cl–C₆H₃) are considered as good active against Staphylococcus aureus, while compounds 4e (R = morpholine), 12b (R₁, R₂ = morpholine) and 9a (quinoline-8-sulfonamide) are considered as very good active against S. aureus. Compounds 4b (R = 4-Cl–C₆H₄), 4e (R = morpholine) and 9d (R = 3,4-di-Cl–C₆H₃) are considered to be good active against Streptococcus pyogenes, while compound 12b (R₁, R₂ = morpholine) is considered as very good active against S. pyogenes. For the antifungal activity, we have screened the same compounds which are used for antibacterial activity. Compounds 4b (R = 4-Cl–C₆H₄), 4e (R = morpholine), 9a (quinoline-8-sulfonamide), 9b (R = 4-OCF₃–C₆H₄) and 12a (4-methyl piperazine) are considered as good active against Candida albicans, while compounds 4c (R = 4-OCH₃–C₆H₅), 7b (R = Cl), 7c (R = OCH₃), 7d (R = CN), 9d (R = 3,4-di-Cl–C₆H₃) and 12b (R₁, R₂ = morpholine) are considered as excellent active against C. albicans. Compounds 4e and 12b are considered as good active against Aspergillus niger. Only compound 12b is good active against Aspergillus clavatus among the entire screened compounds. We have discussed and compared antibacterial and antifungal activities based on standard drugs ampicillin and griseofulvin, respectively.

3.3 Antimicrobial testing method

The MICs of synthesized compounds were carried out by broth microdilution method using DMSO as diluents to get desired concentration of compounds to test upon standard bacterial strains. Serial dilutions were prepared in primary and secondary screening. The control tube containing no antibiotic was immediately subcultured (before inoculation) by spreading a loopful evenly over a quarter of plate of medium suitable for the growth of the test organism and put for incubation at 37 °C overnight. The tubes were then incubated overnight. The MIC of the control organism was read to check the accuracy of the compound concentrations. The MIC was defined as the lowest concentration of the antibiotic or test sample allowing no visible growth. All the tubes showing no visible growth (same as control tube) were subcultured and incubated overnight at 37 °C. The amount of growth from the control tube before incubation (which represents the original inoculum) was compared. Subcultures might show similar number of colonies indicating bacteriostatic, a reduced number of colonies indicating a partial or slow bactericidal activity and no growth if the whole inoculum has been killed. The test must include a second set of the same dilutions inoculated with an organism of known sensitivity. Each synthesized compound was diluted obtaining 2000 μg/mL concentration as a stock solution. In primary screening 500, 250 and 200 μg/mL concentrations of the synthesized compounds were taken. The active synthesized compounds found in this primary screening were further tested in second set of dilution against all microorganisms. The compounds found active in primary screening were similarly diluted to obtain 100, 62.5, 50 and 25 μg/mL concentrations. The highest dilution showing at least 99% inhibition is taken as MIC.

4.1 Materials and methods

All reactions were monitored by thin layer chromatography (TLC), carried out on 0.2 mm silica gel 60 F254 (Merck) plates using UV light (254 and 366 nm) for detection. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker spectrometer operating at 300 and 75 MHz for ¹H and ¹³C respectively using either CDCl₃ or DMSO-d₆ as the solvent. Chemical shifts, δ, are reported in parts per million (ppm) relative to solvent resonance: CDCl₃, δ 7.26 (¹H NMR), and 77.3 (¹³C NMR); DMSO-d₆, δ 2.50 (¹H NMR), and 40.2 (¹³C NMR). Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Coupling constants, J, are reported in Hertz. All melting points have been determined on a manually operated Veego (VMP-1) melting point apparatus and are reported uncorrected.

4.2 Synthesis of ethyl 1-(2-bromoethyl)-1H-indole-2-carboxylate (2)

A mixture of 1,2-dibromo ethane (52.0 mmol, 4.5 mL), potassium carbonate (79.3 mmol, 10.9 g) and ethyl 1H-indole-2-carboxylate 1 (26.4 mmol, 5.0 g) in of acetonitrile (50 mL) was at reflux for 72 h. After cooling the reaction mixture the salt was filtered out and washed with (20 mL) acetonitrile. The residue was concentrated and purified by column chromatography with petroleum ether/ethyl acetate (6:1 to 3:1) as an eluent to give as viscous oil 2: Yield 65%; m.p. 155–158 °C. ¹H NMR (300 MHz, DMSO-d₆) δ: 7.70–7.66 (t, 2H, J = 7.5 Hz, ArH), 7.39–7.35 (m, 1H, ArH), 7.32 (s, 1H, indole), 7.17–7.12 (t, 1H, J = 7.8 Hz, ArH), 4.97–4.93 (t, 2H, J = 13.2 Hz), 4.36–4.29 (q, 2H, J = 6.8 Hz, ester), 3.82–3.76 (t, 2H, J = 6.6 Hz, CH₂Br), 1.36–1.31 (t, 3H, J = 3.6 Hz, ester) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ: 13.83, 31.71, 52.84, 61.34, 103.80, 117.27, 123.12, 125.52, 127.60, 131.71, 148.34, 147.20, 165.78 ppm.

4.3 General procedure for the synthesis of 2-(substituted)-3,4-dihydropyrazino[1,2-a]indol-1(2H)-one 4a–e

To a solution of ethyl 1-(2-bromoethyl)-1H-indole-2-carboxylate 2 (0.100 g, 0.169 mmol) in acetonitrile (10 mL) at room temperature appropriate amines (3a–e) (0.186 mmol) was added. The mixture was heated at 80 °C for 20 min. After cooling the reaction mixture was quenched with cold water. The formed solid was filtered and washed with water to give pure compound 4a–e respectively.

4.4 Synthesis of 2-amino-3,4-dihydropyrazino[1,2-a]indol-1(2H)-one (5)

Mixture of hydrazine monohydrate (1.69 mmol, 0.084 g) and compound 2 (0.169 mmol, 0.050 g) was heated at 80 °C for 30 min (TLC check). After cooling the reaction mixture to room temperature, the solid precipitated was filtered and washed with excess of water to get colorless solid; Yield, 0.042 g, 75%; m.p. 234–238 °C. ¹H NMR (300 MHz, DMSO-d₆) δ: 7.68–7.65 (d, 1H, J = 8.1 Hz, ArH), 7.54–7.51 (d, 1H, J = 8.1 Hz, ArH), 7.32–7.27 (m, 1H, ArH),7.31–7.08 (m, 1H, ArH), 7.04 (s, 1H, indole CH), 5.18 (br.s, 2H, NH₂, D₂O-exchangable), 4.40–4.38 (t, 2H, J = 6.0 Hz, NCH₂CH₂N), 3.91–3.87 (t, 2H, J = 6.3 Hz, NCH₂CH₂N) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ: 49.74, 104.46, 111.05, 120.77, 122.38, 124.35, 127.39, 129.87, 136.63, 158.64 ppm. LC–MS: (202.2, 94.95%).

4.5 General procedure for the synthesis of 1-(aryl)-3-(1-Oxo-3,4-dihydropyrazino[1,2-a]indol-1(2H)-yl)urea 7a–f

To a solution of compound 5 (0.100 g, 0.248 mmol) in dry dichloromethane (10 mL) at room temperature was added the substituted aromatic isocyanates (6a–f) (0.273 mmol). The reaction mixture was stirred at room temperature overnight. The obtained solid was filtered and washed with 10 mL of chloroform to get pure product.

4.6 General procedure for the synthesis of substituted-N-(1-oxo-3,4-dihydropyrazino[1,2-a]indol-1(2H)-yl)aryl sulfonamide 9a–d

To a solution of compound 5 (0.100 g, 0.248 mmol) in dichloromethane (10 mL) at room temperature was added appropriate aromatic sulfonyl chloride 8a–d (0.248 mmol) and pyridine (0.497 mmol). The reaction mixture was stirred at room temperature for 4–5 h and then diluted with water (25 mL). The product was extracted with dichloromethane (3 × 20 mL); the extract was washed with dilute citric acid (20 mL, pH = 7.8). The organic layer was dried over sodium sulfate and evaporated under reduced pressure to obtain pure compound.

4.7 Synthesis of 2-chloro-N-(3,4-dihydro-1-oxopyrazino[1,2-a]indol-1(2H)-yl)acetamide (10)

In compound 5 (0.994 mmol, 0.200 g) chloroacetic acid (1.044 mmol, 0.099 g) and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.193 mmol, 0.299 g) were added in (10 mL) of dichloromethane and the reaction mixture was stirred for 10–15 min. The solid was filtered and washed with water, dried and recrystallized in ethyl acetate to get white solid, Yield, 0.198 g, 72%; m.p. 256–258 °C. ¹H NMR (300 MHz, DMSO-d₆) δ: 10.83 (br.s, 1H, pyrazino NH), 7.72–7.69 (d, 1H, J = 8.1 Hz, ArH), 7.58–7.55 (d, 1H, J = 8.1 Hz, ArH), 7.37–7.32 (t, 1H, J = 7.8 Hz, ArH), 7.18 (s, 1H, indole NH), 7.14–7.12 (d, 1H, J = 7.2 Hz, ArH), 4.49–4.45 (t, 2H, J = 5.7 Hz, NCH₂CH₂N), 4.24 (s, 2H, CH₂Cl), 4.01–3.97 (t, 2H, J = 5.4 Hz, NCH₂CH₂N) ppm. ¹³C NMR (75 MHz, DMSO-d₆) δ: 49.88, 106.34, 111.35, 121.07, 122.66, 125.05, 127.12, 129.12, 136.79, 158.85, 165.68 ppm. LC–MS: (278.1, 98.04%).

4.8 General procedure for the synthesis of N-(3,4-dihydro-1-oxopyrazino[1,2-a]indol-1(2H)-yl)-2-(substituted)acetamide (12a–d)

To a solution of compound 10 (0.180 mmol) in acetonitrile (5 mL) respective amines 11a–d (0.190 mmol) was added and the solution stirred for 1 h in the seal vial at 80 °C. Water was added in the mixture and the solid residue was filtered and dried to give pure 12a–d respectively.