HETEROCYCLES

Short Paper | Special issue | Vol. 84, No. 2, 2012, pp. 1383-1389
Received, 17th September, 2011, Accepted, 18th October, 2011, Published online, 1st November, 2011.
DOI: 10.3987/COM-11-S(P)99

■ The Synthesis and Antimicrobial Evaluation of Some Spiro-Phthalidyl Benzoxazinones

Caterina Ferraro, István Lengyel, and Ralph Stephani*

Department of Chemistry, St. John's University, 8000 Utopia Parkway, Jamaica, New York 11439, U.S.A.

Abstract

Five N-substituted spiro-phthalidyl benzoxazinones (5a-e) were prepared from anthranilic acid (1) and the appropriate alkyl or aryl halides (2), followed by condensation of the N-substituted anthranilic acids (3) with symmetrical phthaloyl dichloride (4). These compounds were then evaluated for antimicrobial activity against: E. coli, S. aureus, B. subtilis, P. aeruginosa, S. cerevisiae, and A. nidulans. Compound 5a was active against all the microbes tested, especially the A. nidulans. Compound 5c was active against all the bacteria, except the fungus A. nidulans. The N-benzyl (5b) and N-phenyl (5d) derivatives were not active at all. The N-4-fluorophenyl analog 5e showed activity against S. aureus and B. subtilis.

We became interested in N-alkyl and N-aryl-substituted spiro-phthalidyl benzoxazinones (5) as potential antimicrobial agents, in view of their similar structure to Griseofulvin (6), Figure 1.

Griseofulvin a well-known antifungal agent, was first isolated from the fungus Penicillium griseofulvum in 1939.1 It inhibits fungal mitosis by disrupting the mitotic spindle through interaction with polymerized microtubules.2 Griseofulvin is mainly effective against a specific group of fungi known as dermatophytes,3 which cause skin disease in humans and animals.

We chose to determine antimicrobial activity of our benzoxazinones, 5a-e by the method developed by Bauer and Kirby.4 The principle of this method is to measure the zone of inhibition of microbial growth around the antimicrobial agent. The diameter (in mm) of the zone of inhibition around the disk is an indication of the susceptibility or resistance of a microorganism to the microbial agent.


1. Synthesis
Five N-substituted spiro-phthalidyl benzoxazinediones (5a-e) were prepared from anthranilic acid (1) and the appropriate alkyl or aryl halide (2), followed by condensation of the N-substituted anthranilic acids (3) with symmetrical phthaloyl dichloride (4) (Scheme 1).

2. Antimicrobial Testing
Mueller Hinton Agar (MHA) plates were prepared according to DifcoTM. The powder (38 grams) was suspended in 1 liter of purified water and mixed thoroughly, then heated with frequent agitation and boiled for one minute to obtain complete solution. It was autoclaved at 121 oC for 15 minutes, then approximately 25 mL portions of MHA were poured into sterile Petri dishes.
Each test compound was dissolved in DMSO, at four different concentrations (100, 50, 25, and 12.5 mg/mL) and 50 μL was applied to 5 ½ inch sterile paper discs (diameter 12.5 mm). A control disc containing only DMSO was also prepared. The disks were uniformly placed in a circle on the Mueller Hinton Agar plates seeded with the microorganisms. Plates were prepared in triplicate. The plates were then incubated at 37 oC for 24 hours to allow the microbial species to grow. Antibiotic effects of the test compounds were quantified by measuring (millimeters) the average of the three zones of growth-inhibition. N-Phenyl and N-4-fluorophenyl-spiro-[1,2-dihydro-4H-3,1-benzoxazin-2,1’-phthalan]-4,3’-diones were not readily soluble in DMSO. These concentrations were made by suspending the 200 mg of the compound in 2 mL of DMSO and then treated in an ultrasonic homogenizer to ensure a uniform suspension of the compound. Serial dilutions were then prepared at concentrations of 50, 25, and 12.5 mg/mL, and pipetted onto the discs. The diameter of the zone of inhibition of each compound is reported as a measure of growth inhibition.
The results of the antimicrobial screening we performed show (Table 1) that these compounds have potential antimicrobial activity. The N-methyl derivative 5a showed significant activity against all the microbes tested, especially the A. nidulans. The N-methoxymethyl analog 5c showed activity against all the bacteria, as well as strong activity against the yeast, but was inactive against the fungus A. nidulans. The N-4-fluorophenyl derivative 5e showed activity against B. subtilis.
Intended future work to modify these structures by introducing polar groups on the rings, can potentially increase water solubility, and perhaps enhance the antimicrobial activity.

EXPERIMENTAL
Melting points are uncorrected and were measured on either a Thomas-Hoover® or Mel-Temp® capillary melting point apparatus. Thin layer chromatography (TLC) was preformed with Analtech® silica gel glass backed plates (250 microns) and Rf values were recorded. Infrared spectra (IR) were recorded using a Perkin Elmer® Fourier Transform (FTIR) Spectrum 1000 Spectrophotometer. NMR Spectra (1H, 13C, and 13C APT) were recorded on a 400 MHz Bruker Spectrometer. NMR spectra are presented using TMS as the internal standard and chemical shifts are given in ppm as δ values. Atlantic Microlab, Inc (Norcross, Georgia) performed elemental analyses. Gas chromatography-mass spectrometry (GC-MS) studies were performed on either a Hewlett Packard® G1800A GCD System or a Shimadzu model QP5050A.
Anthranilic acid, N-methylanthranilic acid, and symmetrical phthaloyl dichloride were purchased from Alfa Aesar (Ward Hill, MA). 4-Fluorophenylanthranilic acid, benzyl bromide, chloromethyl methyl ether, N,N-diisopropylethylamine, and triethylamine were purchased from Aldrich (Milwaukee, WI). N-Phenylanthranilic acid was purchased from Fluka (Milwaukee, WI).
The microorganisms, obtained from Ward’s (Rochester, New York), were Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli MG1655, Saccharomyces cerevisiae W303, and Aspergillus nidulans. Mueller Hinton Agar and Yeast Peptone Dextrose Agar were purchased from Difco. Media used was Tryptic Soy Broth for bacteria, YPD liquid media for yeast, and Yeast Glucose media with Ampicillin for fungus. Antibiotic discs were purchased from VWR (Bristol, CT).

N-Benzylanthranilic acid (3b).
Anthranilic acid, 1, (6.86 g, 60 mmol) and benzyl bromide (10.2 g 60 mmol) were added to an aqueous solution of K2CO3 (3.46 g) (62 mL water) with stirring. The reaction mixture was refluxed for 2.5 h, then allowed to cool to rt, and 50 mL of EtOAc was added. The organic layer was washed with water and dried over MgSO4. The EtOAc was removed under reduced pressure to obtain the crude product, yield 10.4 g, 76%. This was recrystallized from heptane/EtOAc (30/20 mL) to give 9.8 g (72%), mp 171-172 oC (lit.6 mp 174-176 oC); TLC (90:10 hexane/i-PrOH alcohol): Rf = 0.46; IR (CCl4 cm-1): 3540 (OH), 3380 (N-H), 3066 and 3030 (aromatic C-H), 2891 (aliphatic C-H), and 1662 with a shoulder at 1698 cm-1 (aromatic α-electro-negative-substituted COOH); MS: m/z 227 (M+, C14H13NO2); 209 (M – H2O); 208 (C14H10NO); 180 (C13H10N); 152 (C12H8); 132 (C8H4O2); 106 (C7H8N); 91 base peak (C7H7+), 77 (C6H5+), 65 (C5H5+); 51 (C4H3+).


N-Methoxymethylanthranilic acid (3c).
Diisopropylethylamine (25.0 g, 194 mmol) was slowly added to a mixture of anthranilic acid, 1, (10.97 g, 80 mmol) and 1,2-dichloroethane (125 mL) with stirring. Chloromethyl methyl ether (7.08 g, 88 mmol) was added to the reaction mixture and stirred for 30 min. at rt, then washed with water and the organic layer was dried over MgSO4. After filtration the 1,2-dichloroethane was removed under reduced pressure yielding 24.5 g of solid crude product which was crystallized upon treatment with aqueous MeOH; Recrystallization from MeOH gave 1.6 g (11%) of pure N-methoxymethyl anthranilic acid, mp 89-91 oC; TLC (90/10 hexane/i-PrOH): Rf = 0.52; IR (CCl4, cm-1): 3365 (N-H) and 1690 (aromatic carboxylic acid); 1H-NMR (CDCl3): δ = a 3.51 (s, 3H), b 4.81 (t, 1H), c 5.42 (s, 2H), d 6.67 (t, 1H), e 6.86 (d, 1H), f 7.40 (t, 1H), g 7.97 (d, 1H), and h 8.27 (bs, 1H) ppm. .13C-NMR (CDCl3): δ = C1: 57.73, C2: 90.48, C3: 110.52, C4:111.85, C5: 115.98, C6: 132.01, C7: 135.24, C8: 150.26, C9: 168.04 ppm; MS: 181 (M+, C9H11NO3); 150 (M – OCH3)+; 132 base peak (C8H6NO)+, 120 (C7H4O2)+; 105 (C7H5O)+; 92 (C6H6N)+; and 65 (C5H5)+. Anal. Calcd for C9H11NO3: C 59.67; H 10.26; N 12.97. Found: C59.99; H 10.25; N 12.917.

N-Methyl-spiro-[1,2-dihydro-4H-3,1-benzoxazin-2,1’-phthalan]-4,3’-dione (5a). The method used by Butula and Otting5 was modified as follows.
Symmetrical phthaloyl dichloride (2.03 g, 10 mmol) was dissolved in 105 mL of 1,2-dimethoxyethane. The solution was stirred at rt. Triethylamine (2.02 g, 20 mmol) was added slowly as the flask was immersed in an ice-bath. A solution of N-methylanthranilic acid, 1.51 g, 10 mmol, in 40 mL of dimethoxyethane was added dropwise while stirring was continued at rt. The precipitate (Et3NH+Cl-) was filtered, and the solvent was removed under reduced pressure. The solid residue was recrystallized from aqueous MeOH to yield 2.2 g (78%) of colorless crystals, mp 212-214 oC (lit.5 mp 210-212 oC); TLC (90/10 hexane/i-ProH); Rf = 0.30; IR, cm-1 (NaCl plate): 1770 (γ-lactone); 1759 (δ-lactone); 1H-NMR (CDCl3): δ = 2.77 (s, 3H); 6.97 (d, 1H); 7.12; 7.74 (d, 1H); 7.76 (t, 1H); 7.86 (t, 1H); 7.95 (d, 1H); 8.08 (d, 1H) ppm.
13C-NMR (CDCl3): δ = C1: 33.04, C2: 112.24, C3: 113.90, C4: 114.80, C5: 121.63, C6: 123.80, C7: 126.36, C8: 127.74, C9: 130.96, C10: 132.87, C11: 135.83, C12: 137.06, C13: 142.89; C14: 146.54; C15: 160.02; C16: 166.31 ppm; MS: m/z 281 (M+, C16H11NO4); 236 (M-45, M-CH3NO, C15H8O3); 152 (C12H8); 133 (C8H7NO); 132 (C8H6NO); 105 base peak, (C7H7N); 104 (C7H7O12); 77 (C6H5)+and 50 (C4H2); 76 (C6H4).

N-Benzyl-spiro-[1,2-dihydro-4H-3,1-benzoxazin-2,1’-phthalan]-4,3’-dione (5b).
Symmetrical phthaloyl chloride (1.48 g, 7.27 mmol) was stirred at rt in 20 mL CHCl3. Diisopropylethylamine (2.07 g, 16.0 mmol) was added slowly as the flask was cooled in an ice-bath. A suspension of N-benzylanthranilic acid (1.5 g, 6.61 mmol) in 30 mL CHCl3 was then added dropwise while stirring at rt. The CHCl3-layer was washed with water and dried over MgSO4. The solvent was removed under reduced pressure. The solid residue crystallized upon treatment with MeOH. It was recrystallized from aqueous MeOH to give 2.1 g (89%) of pure product, mp 180-183 oC; TLC (90/10 hexane/i-ProH): Rf = 0.51; IR (CCl4): 1797 cm-1 (γ-lactone); 1766 cm-1 (δ-lactone); 1H-NMR (CDCl3): δ = a: 4.31 (d, 1H), b: 4.44 (d, 1H), c: 6.75 (d, 1H), d: 7.08 (t, 1H), e: 7.21-7.33 (m, 5H), f 7.47 (d, 1H), g: 7.65-7.71 (m, 3H), h: 7.91 (d, 1H), i: 8.10 (d, 1H) ppm. 13C-NMR (CDCl3): δ = C1: 50.26, C2: 112.28, C3: 114.63, C4: 114.87, C5: 121.52, C6: 123.35, C7: 125.89, C8: 126.18, C9: 127.71, C10: 127.91, C11: 129.22, C12: 130.89, C13: 132.74; C14: 135.49; C15: 135.55; C16: 136.67; C17: 142.31; C18: 145.66; C19: 159.92; and C20: 165.95 ppm; MS: 357 (M+, C22H15NO4); 209 (C14H11NO); 180 base peak (C13H10N); 152 (C12H8); 105 (C7H5O); 91 (C7H7+); and 77 (C6H5+); Anal. Calcd for C22H15NO4: C 73.94; H 4.23; N 3.92. Found: C 73.99; H 4.25; N 3.97.


N-Methoxymethyl-spiro-[1,2-dihydro-4H-3,1-benzoxazin-2,1’-phthalan]-4,3’-dione (5c).
A mixture of symmetrical phthaloyl dichloride (6.91 g, 34 mmol) and 1,2-dimethoxyethane (75 mL) was stirred at rt. N,N-Diisopropylethylamine (9.68 g, 75 mmol) was added. N-Methoxymethyl anthranilic acid (3c, 5.6 g, 31 mmol) in of 1,2-dimethoxyethane, 25 mL was added dropwise to the flask while stirring at rt. Stirring was continued for 48 h after the addition was completed. The white precipitate, consisting of N,N-diisopropylethylamine hydrochloride, was collected. The filtrate was evaporated to dryness to give an oil, 8.29 g, 86%; TLC (90/10 hexane/i-PrOH): Rf = 0.33; IR (cm-1 NaCl plate): 1811 (γ-lactone); 1725 (δ-lactone); 1H-NMR (CDCl3): δ = a: 3.43 (s, 3H); b: 5.35 (s, 2H); c: 7.09 (d, 1H); d: 7.26 (t, 1H); e: 7.55 (t, 1H); f: 7.78 (t, 1H); g: 7.86 (t, 1H); h: 7.96 (d, 1H); i: 8.06 (d, 1H); j: 8.13 (d, 1H); 13C-NMR (CDCl3): δ = C1: 57.77, C2: 91.06, C3: 121.34, C4: 122.62, C5: 124.10, C6: 124.83, C7: 125.59, C8: 128.48, C9: 130.28, C10: 131.28, C11: 133.39, C12: 133.69, C13: 135.67; C14: 146.56; C15: 148.66; C16: 164.43; and C17: 165.25 ppm; MS: m/z 311 (M+ C17H13NO5); 280 (M-OCH3)+; 250 (M-61, unassigned); 223 (M-2CO2, C15H13NO); 179 (C9H9NO3); 163 (C9H9NO2); 148 (base peak, C8H4O3); 120 (C7H4O); 104 (C7H4O); 76 (C6H4)+; 45 (CH3OCH2)+. Anal. Calcd for C17H13NO5: C 65.65; H 4.21; N 4.52. Found: C 65.49; H 4.29; N 4.58.

N-Phenyl-spiro-[1,2-dihydro-4H-3,1-benzoxazin-2,1’-phthalan]-4,3’-dione (5d).
A mixture of symmetrical phthaloyl dichloride (5.24 g, 26 mmol), CHCl3 (75 mL), and N,N-diisopropylethylamine (7.33 g, 57 mmol) was stirred at rt. N-Phenylanthranilic acid (5.0 g, 23 mmol) was suspended in CHCl3 (75 mL) and added dropwise to the flask. After 15 h of stirring at rt, the mixture was washed with water and the organic layer was dried over MgSO4. The CHCl3 was removed on a rotary evaporator, giving 6.1 g of crude product. This was triturated with EtOAc and the resulting crystals were collected on a Büchner funnel, and recrystallized from aqueous MeOH to give pure 5d, 4.8 g (61%), mp 241-242 oC; TLC (90/10 EtOAc/MeOH): Rf = 0.65; IR (cm-1 NaCl plate): 1769 (γ-lactone); 1758 (δ-lactone); 1H-NMR (DMSO): δ = a: 6.65-6.80 (m, 1H); b: 6.95-7.10 (m, 2H); c: 7.12-7.30 (m,6H); d: 7.31-7.40 (m, 3H); e: 7.80-7.96 (dd, 1H) ppm; 13C-NMR (DMSO-d6): δ = C1: 125.71, C2: 126.39, C3: 126.46, C4: 127.03, C5: 127.13, C6: 127.61, C7: 127.83, C8: 128.07, C9: 128.21, C10: 128.94, C11: 129.88, C12: 130.37, C13: 130.83; C14: 131.18; C15: 132.21; C16: 132.93; C17: 136.48; C18: 142.03; C19: 143.65; C20: 167.23; and C21: 168.92 ppm; MS: m/z 343 (M+ C21H13NO4); 298 (unassigned); 195 (base peak, C13H9NO); 167 (C12H9N); 152 (C12H8); 119 (C6H5N=C=O) and 77 (C6H5+). Anal. Calcd for C21H13NO4: C 73.53; H 3.81; N 4.10. Found: C 73.49; H 3.92; N 4.08.

N-4-Fluorophenyl-spiro-[1,2-dihydro-4H-3,1-benzoxazin-2,1’-phthalan]-4,3’-dione (5e).
A mixture of symmetrical phthaloyl dichloride (4.83 g, 24 mmol), CHCl3 (75 mL), and N,N-diisopropylethylamine (6.76 g, 52 mmol) was stirred at rt. N-4-Fluorophenylanthranilic acid (5.0 g, 22 mmol) was suspended in CHCl3 (75 mL) and added dropwise to the flask. After 15 h of stirring at rt, the reaction mixture was extracted with water and the organic layer was dried over MgSO4. The filtrate was evaporated to dryness on a rotary evaporator, giving 5.9 g of a solid residue. This was triturated with EtOAc until it turned crystalline, and collected on a Büchner funnel. The product was recrystallized from aqueous MeOH affording 4.4 g (55%) of pure 5e, mp 205-207 oC; TLC (90/10 EtOAc/MeOH): IR (NaCl plate): 1769 cm-1 (γ-lactone); 1758 cm-1 (δ-lactone); 1H-NMR (DMSO-d6): δ = a: 6.99 (m, 1H); b: 7.07 (t, 2H); c: 7.10 (d, 1H); d: 7.16 (t, 1H); e: 7.18 (t, 1H); f: 7.44 (d, 1H); g: 7.47 (t, 2H); h: 7.54 (t, 2H); i: 7.95 (d, 1H) ppm; 13C-NMR (DMSO-d6): δ = C1: 116.27, C2: 116.49, C3: 123.66, C4: 127.72, C5: 127.99, C6: 128.81, C7: 129.45, C8: 129.66, C9: 130.27, C10: 130.98, C11: 131.51, C12: 132.12, C13: 132.83; C14: 133.55; C15: 136.71; C16: 140.46; C17: 142.24; C18: 159.50; C19: 161.92; C20:. 167.71; and C21: 169.36 ppm; MS: m/z 361 (M+ C21H12NO4F); 316 (M-45); 213 (base peak, C13H8NOF); 185 (C12H8NF); 119 (C6H5NCO); 92 (C6H6N); 76 (C6H4); and 50 (C4H2). Anal. Calcd for C21H12NO4F: C .69.86; H 3.82; N 4.45. Found: C 69.59; H 3.69; N 4.58.

ACKNOWLEDGEMENTS
We would like to acknowledge the assistance of Ms Meropi Aravantinou with the antimicrobial testing.

References

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