|Year : 2017 | Volume
| Issue : 6 | Page : 526-534
Synthesis and evaluation of antimicrobial activity of cyclic imides derived from phthalic and succinic anhydrides
Elham Jafari1, Najmeh Taghi jarah-Najafabadi2, Ali Jahanian-Najafabadi3, Safoora Poorirani2, Farshid Hassanzadeh1, Sedighe Sadeghian-Rizi2
1 Department of Medicinal Chemistry and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran
2 Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran
3 Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran
|Date of Web Publication||3-Nov-2017|
Department of Medicinal Chemistry and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan
Source of Support: None, Conflict of Interest: None
Cyclic imides are a group of compounds which have valuable biological properties including cytotoxic, anti-inflammatory, antibacterial and antifungal activities. In this study, succinic and phthalic anhydrides were treated with glycinamide in pyridine to yield the corresponding amic acids. These amic acids underwent ring closure with acetic anhydride and anhydrous sodium acetate to form cyclic imides. In another procedure, succinic and phthalic anhydrides upon reaction with 2-amino-benzylamine in pyridine gave the corresponding cyclic imides. The imides were screened for their antimicrobial activities against three types of bacteria and one type of fungi. Phthalimide derived from benzylamine exhibited remarkable antimicrobial activity against E. coli.
Keywords: Cyclic imides; Glycinamide; 2-Amino-benzylamine; Antimicrobial
|How to cite this article:|
Jafari E, jarah-Najafabadi NT, Jahanian-Najafabadi A, Poorirani S, Hassanzadeh F, Sadeghian-Rizi S. Synthesis and evaluation of antimicrobial activity of cyclic imides derived from phthalic and succinic anhydrides. Res Pharma Sci 2017;12:526-34
|How to cite this URL:|
Jafari E, jarah-Najafabadi NT, Jahanian-Najafabadi A, Poorirani S, Hassanzadeh F, Sadeghian-Rizi S. Synthesis and evaluation of antimicrobial activity of cyclic imides derived from phthalic and succinic anhydrides. Res Pharma Sci [serial online] 2017 [cited 2019 Jul 23];12:526-34. Available from: http://www.rpsjournal.net/text.asp?2017/12/6/526/217433
| Introduction|| |
Cyclic imides and their N-derivatives as an important class of organic compounds, contain bis-amide linkages with a general structure of [-CO-N(R)-CO-]. Their hydrophobicity and neutral structures enable them to easily cross biological membranes ,,,,,. These molecules are reported to exhibit valuable biological effects including antifungal ,,, antibacterial ,,,, anticancer, apoptosis induction ,, anti-inflammatory , androgen receptor antagonists , anxiolytic, and anticonvulsant ,. Structure activity relationship studies of these systems in some literatures revealed that incorporation of heterocyclic moiety in the imide portion or linkage with nitrogen improve their anti-inflammatory and antitumor activities. Antitubercular activity was reported for cyclic imides with a para-sulphonamide group ,. These molecules could be used as building blocks in the synthesis of natural products such as coumarins and atacoumarins and also as core structures in the design and synthesis of peptidomimetics ,. The alkaloid phyllanthimide isolated from leaves of Phyllanthus sellowianus (euphorbiaceae) has been used as a precursor for the synthesis of some analogs of cyclic imides . Some examples with cyclic imide structure are shown in [Figure 1]. Gayoso, et al. have reported antifungal effectiveness of 3,4-dichloro-N-phenyl-alkyle-maleimide derivatives. Compounds 3, 4-dichloro-N-phenyl-methyl-maleimide and 3,4-dichloro-N-phenyl-propilmaleimide displayed antifungal activities with MIC of 100 μg/mL against fungal strains . Sultana, et al. succeeded to synthesize 2-(2-methoxyphenyl)-1H-isoindole- 1, 3(2H)-dione ligand and some of the metal complexes by using a simple method. Synthesized complexes exhibited improved antibacterial effects in comparison to their parent ligand .
Al-Azzawi, et al. synthesized N-(2-acetyl amino-5-substituted -1, 3, 4-oxadiazole -2-yl)- 1,8-naphthalimide derivatives with remarkable antimicrobial activity against E. coli  [Figure 2].
There are some procedures available for cyclic imides synthesis, such as the dehydrative condensation of a cyclic anhydride and amine , the use of expensive catalysts , and microwave synthesis . In this study, the first method, with some modifications was used to prepare the compounds.
| Materials and Methods|| |
Melting points were determined in open capillaries using electrothermal 9200 melting point apparatus (UK) (England) and are uncorrected. IR (KBr discs) was recorded with a WQF-510 FT-IR spectrophotometer (China). 1H-NMR spectra were recorded on Bruker 400 MHz spectrometers (Germany) using TMS as an internal standard and either DMSO-d6 or CDCl3 as solvents. Mass spectra were recorded on Finnigan TSQ-70 Mass spectrometer (United States). All chemicals were purchased from Merck Company (Germany).
Amic acids synthesis procedures from (2-amino acetamide or glycinamide) (A1G, A2G, A3G)
Phthalic, succinic and maleic anhydride (0.01 mol) and glycinamide (0.01 mol) were transferred separately to round bottom flasks and then freshly distilled and dried pyridine was added slowly while shaking. The mixture was heated under refluxed for 5 h. Excess of pyridine was distilled off under reduced pressure, and then methanol was added to residue to obtain crystalline solids [Figure 3].
|Figure 3: Synthesis of cyclic imides of (2-amino acetamide or glycinamide).|
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Preparation of phthalimide
Obtained amic acid of phthalic anhydride (0.01 mol) in 20 mL acetic anhydride and 0.001 mol anhydrous sodium acetate was refluxed with stirring for 4 h ,,. The resulted homogenous solution was cooled to room temperature and then poured into excess cold water with vigorous stirring. The obtained precipitate was filtered, washed with distilled water, dried and finally purified by column chromatography on silica gel using CHCl3- MeOH (49:1) as eluent to give FA1G and FA1GAc [Figure 3].
Preparation of succinimide
Obtained amic acid of succinic anhydride (0.01 mol) was dissolved in acetic anhydride (20 mL) and anhydrous sodium acetate (0.001 mol) and refluxed with stirring for 4 h ,,. The solvent was removed by distillation under reduced pressure.
The obtained residue was dissolved in chloroform, then solvent was removed by distillation under reduced pressure. The obtained product was purified by column chromatography on silica gel using CHCl3- MeOH (49:1) as eluent to afford FA3G [Figure 3].
Preparation of cyclic imides of (2-amino benzylamine)
Preparation of phthalimide
Phthalic anhydride (0.01 mol) and 2-aminobenzylamine (0.01 mol) were placed in a round bottom flask and freshly distilled and dried pyridine was then added slowly while shaking. The mixture was heated under reflux for 5 h. The solvent was distilled off under reduced pressure. Obtained residue was dissolved in ethyl acetate and purified by column chromatography to afford A1B [Figure 4].
Preparation of succinimide
Succinic anhydride (0.01 mol) and 2-aminobenzylamine (0.01 mol) were put in a round bottom flask and freshly distilled and dried pyridine was then added slowly while shaking. The mixture was heated under reflux for 5 h. Excess of pyridine was distilled off under reduced pressure; then ethanol and water were added to the residue to obtain light brown precipitant A3B [Figure 4].
Microorganisms were obtained from Persian Type Culture Collection (PTCC). Staphylococcus aureus PTCC1337 as Gram-positive, Escherichia More Details coli, PTCC 1338 and Pseudomonas aeruginosa, PTCC1074, as Gram-negative bacteria and Candida albicans PTCC, 5027 as fungus. Mueller Hinton Agar, Mueller Hinton Broth and Sabouraud Dextrose Agar were purchased from Merck (Germany) Roswell Park Memorial Institute (RPMI)-1640 culture medium was procured from Gibco, USA.
Microplate alamar blue assay (MABA) for antimicrobial evaluation
The inocula of bacterial and fungal strains (1.5 × 108 CFU/mL) were prepared from Mueller Hinton Agar and Sabouraud Dextrose Agar cultures respectively. Prepared suspensions of bacteria were adjusted to 0.5 Mc Farland standard turbidity and the fungal suspensions turbidity was measured spectrometrically at 580 nm. Finally, prepared inoculum density for bacterial and fungal strains were equal to 1.0 × 105 CFU/mL and 1.0 × 106 CFU/mL respectively. The synthesized compounds were dissolved in DMSO (0.5 mL) and diluted with water up to 1 mL to obtain concentration of 5120 μg/mL as the stock solution. The stock solution was serially diluted to give concentrations of 2560 to 80 μg/mL.
Mueller Hinton Broth was used as medium for bacterial growth and RPMI 1640 was used as medium for fungal growth. 20 μL of bacterial suspension was distributed in all 96 wells of microplate.
Then 20 μL of each concentration of the compounds were added to wells with the exception of those wells acting as positive control (containing standard antibiotic) and growth control (containing culture media without testing materials). After adding alamar blue (20 μL) to all of 96 wells the total volume in each well was adjusted to 200 μL using culture medium.
The final concentrations of the compounds in the wells were 512, 256, 32, 16, and 8 μg/mL. After incubation, the MIC was defined as the lowest concentration, which prevented a color change from blue to pink. The test was carried out in triplicates.
Following the MIC test, the content of each well that showed no growth was removed and spread onto a plate containing appropriate medium for bacteria and fungi to determine MBC (minimum bactericidal concentration) and MFC (minimum fungicidal concentration) results.
Pharmacokinetic parameters estimation using Swiss ADME
Absorption and Lipinski's “rule of five” parameters of the compound A1B which exhibited remarkable antimicrobial activity were calculated using online Swiss ADME. Lipinski's “rule of five” including molecular weight (MW), number of H-bond donors (NHBD), number of H-bond acceptors (NHBA) and log P are widely used as a filter for drug-like properties .
| Results|| |
4-(2-amino-2-oxoethylamino)-4-oxobut-2- enoic acid (A2G)
Brown powder, yield: 40%, m.p.: 244-246 °C, MS (m/z, %): 172 (100) for C6H8N2O4, M.W.: 172.14, IR (KBr, cm-1), 3420, 3324 (NH2), 3197 (NH), 1702,1 657 (C=O). 1H-NMR δH (400 MHz; DMSO), 8.70 (1H, t, J = 4Hz, NH), 7.4, 7.04 (2H, s, NH2), 6.98 (1H, d, J = 15.2 Hz, OH-CO-CH=CH-CONH-CH2-CO-NH2), 6.50 (1H, d, J = 15.6 Hz, OH- CO- CH=CH-CONH-CH2-CO-NH2), 3.73 (2H, d, J = 4 Hz, OH-CO-CH=CH-CONH-CH2-CO- NH2).
4-(2-amino-2-oxoethylamino)-4-oxobutanoic acid (A3G)
White powder, yield: 50%. m.p.: 155-157 °C, MS (m/z, %): 174 (100) for C6H10N2O4, M.W.: 174.15, IR (KBr, cm-1), 3422, 3332 (NH2), 1718, 1651 (C=O).1H-NMR (400 MHz, DMSO) δ: 12.1 (1H, OH), 8.11 (1H, t, J = 4 Hz, NH), 7.1, 7.03 (2H, s ,NH2), 3.6 (2H, d, J = 4 Hz, OH-CO-CH2-CH2-CONH-CH2-CO- NH2), 2.43 (2H, t, J = 8 Hz, OH-CO-CH2-CH2-CONH-CH2-CO-NH2), 2.37 (2H, t, J = 8 Hz, OH-CO-CH2-CH2-CONH-CH2-CO-NH2).
2-(1, 3-dioxoisoindolin-2-yl) acetamide (FA1G)
White powder, yield: 40% , m.p.: 255-256 °C (lit. 255-257 °C), MS (m/z, %): 204  for C10H8N2O3, M.W.: 204.18, IR (KBr, cm-1), 3413, 3321 (NH2), 3066 (CH, Ar), 1770, 1682 (C=O) 1H-NMR (400 MHz, DMSO) δ: 7.88 (2H, dd, J = 6 Hz, J = 2.8 Hz, CO-C=CH-CH=CH-CH=C-CO), 7.86 (2H, dt, J = 6 Hz, J = 2.8 Hz CO-C=CH-CH=CH-CH=C-CO), 4.13 (2H, s, CH2), 7.68, 7.23 (NH2, s).
N-[2-(1, 3-dioxoisoindolin-2-yl) acetyl]- acetamide (FA1GAc)
White powder, yield: 10%, m.p.: 263-265 °C, C12H10N2O4, M.W.: 246, IR (KBr, cm-1), 3237 (NH), 3097 (CH, Ar), 1783, 1640, 1690 (C=O), 1H-NMR (400 MHz, CDCl3) δ: 8.35, (NH, s) , 7.89 (2H, dd, J = 5.2 Hz, J = 3.2 Hz, CO-C=CH-CH=CH-CH=C-CO), 7.75 (2H, dt, J = 5.2 Hz, J = 3.2 Hz CO-C=CH-CH= CH-CH=C-CO), 4.81 (2H, s, CH2), 2.27 (3H,s, CH3).
2-(2, 5-dioxopyrrolidin-1-yl) actamide (FA3G)
White powder, yield: 23 %, m.p.: 165-167.5 °C, MS (m/z, %), 156 (6) for C6H8N2O3, M.W.: 156, IR (KBr, cm-1), 3413, 3321 (NH2), 1770, 1682 (C=O) 1H-NMR (400 MHz, CDCl3) δ: 5.51 (2H, s, NH2), 4.14 (2H, s, CH2), 2.75 (4H, s, O=C -CH2-CH2-C=O).
Isoindolo [1,2-b]quinazolin-12(10H)-one (A1B)
Yellow powder, yield: 30% , m.p.: 174-176 °C (lit. 175-177 °C), MS (m/z, %): 234 (6) for C15H10N2O, M.W.: 234, IR (KBr, cm-1), 1650 (C=N), 1601 (C=C), 1H-NMR (400 MHz, CDCl3) δ: 8.08 (1H, d, J = 7.2 Hz, N-CO-C=CH-CH=CH-CH=C-C=N), 7.92 (1H, d, J = 8Hz, N-CO-C=CH-CH=CH-CH=C-C=N), 7.67-7.74 (2H, t, J = 8 Hz, N-CO-C=CH-CH=CH-CH=C-C=N), 7.51 (1H, d, J = 8 HZ, N-C=CH-CH=CH-CH =C-CH2), 7.34 (1H, t, J = 8 Hz, N-C=CH-CH=CH-CH =C-CH2), 7.25 (1H, t, J = 8 Hz, N-C=CH-CH=CH-CH=C-CH2), 7.21 (1H, d, J = 8 Hz, N-C=CH- CH=CH-CH =C-CH2), 5.01 (2H, s, CH2).
2, 3-dihydropyrrolo [2, 1-b]quinazoline-1 (9H)-one (A3B)
Light brown powder, yield: 30% , m.p.: 165-166 °C (lit. 185-187 °C), C11H10N2O,
M.W.: 186, IR (KBr, cm-1), 3026, 3066 (CH, Ar), 1685 (C=N), 1662 (C=O), 1H-NMR (400 MHz, CDCl3) δ: 7.23-7.06 (4H, m, Ar-H), 4.85 (2H, s, CH2), 2.94 (2H, t, J = 4 Hz CH2- C=O), 2.68 (2H, t, J = 4 Hz, CH2-C=N).
Results of online Swiss ADME software
Absorption and Lipinski's “rule of five” properties of the compound A1B are shown in [Table 1].
The MICs of all tested compounds were evaluated at concentrations 8 to 512 μg/mL. Compound A1B showed significant inhibition at 16 μg/mL concentration against E. coli as a gram-negative bacteria.
Results of MIC, MBC and MFC are depicted in [Table 2] and [Table 3].
|Table 2: Minimum inhibitory concentrations of synthesized compounds against bacteria and fungi.|
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|Table 3: Minimum fungicidal and minimum bactericidal concentrations of synthesized compounds against bacteria and fungi.|
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| Discussion|| |
In the first step to produce cyclic imides, the reaction was proceed via nucleophilic attack of amino group (NH2-CH2) of glycinamide on one carbonyl group in cyclic anhydrides to yield corresponding amic acids. Treatment of amic acids with acetic anhydride gave the cyclic imides through dehydrative cyclization mechanism [Figure 5]. In another procedure, benzylic amine of 2-amino-benzylamine acted as a nucleophile and attacked the carbonyl group of the cyclic anhydride which resulted in the ring opening and subsequently forming the cyclic imides. NH2-ph group of 2-amino-benzylamin attacked the carbonyl group of cyclic imide and the second ring closure was achieved simultaneously to produce cyclic imide [Figure 6]. Reaction between maleic anhydride and the same amines could not produced cyclic imides by these methods.
IR spectra of the prepared imides (FA1G and FA3G) showed disappearance of absorption bands which belong to (O-H) carboxylic and (N-H). This can be clear proof for the synthesis of cyclic imides.
According to the antimicrobial evaluations, phthalimide derived from benzylamine (A1B) exhibited remarkable antimicrobial activity against E.coli . Phthalimide, isoindoline-1,3- dione have shown a wide array of pharmacological activities. Schiff base, azetidinone and acetyl oxadiazole derivatives of this cyclic imide with high antibacterial activities were synthesized and reported by Azzawi, et al. .
Literature survey revealed that naphthalimides, one type of cyclic imides with strong hydrophobicity, have remarkable antimicrobial activities ,,,. Relative similarity of this compound (A1B) with naphthalimides, can be responsible for its antibacterial activity. Sortino, et al. demonstrated that N-phenyl- and N- phenylalkylmaleimides can display antifungal activities with their intact maleimide ring and opening of the maleimide ring would lead to the loss of antifungal activity ,. In our study, cyclic imides derived from succinic anhydride, A3G (opened derivative) or FA3G (intact ring), did not show significant activity against tested microorganisms.
Lipinski's “rule of five” explains four physicochemical properties for orally active drugs. In this regard , most molecules with acceptable membrane permeability, exhibit hydrogen bond donors (OH, NH) of less than 5, hydrogen bond acceptor of less than 10, molecular weight under 500 g/mol and partition coefficient (log P) less than 5. Any value greater than these ranges is considered as a violation. The number of violations less than 4 is acceptable for drug-like molecule. Compound A1B can, therefore, be considered as a drug-like molecule according to Lipinski's “rule of five”.
| Conclusion|| |
Compound A1B with significant efficacy against E. coli at 16 μg/mL concentration and acceptable Lipinski's parameters can be regarded as a drug likeness molecule. The activity of this compound can be associated with its stability as well as relatively lipophilic structure. Other compounds showed weak activities against tested microorganisms.
| Acknowledgements|| |
The content of this paper is extracted from the Pharm. D thesis (No. 292243) which was financially supported by the Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
| References|| |
Marulasiddaiah R, Kalkhambkar RG, Kulkarni MV. Synthesis and Biological Evaluation of Cyclic Imides with Coumarins and Azacoumarins. Open J Med Chem. 2012,2:89-97.
PATIL M.M. RAJPUT S.S. Succinimides: synthesis, reaction, and biological activity. Int J Pharm Pharm Sci. 2014,6(11): 8-14.
Dhivare RS, Rajput SS. Synthesis and antimicrobial activity of five membered cyclic imide derivatives of mono, di and tri substituted aromatic amines and napthyl amine. World J Pharm Sci. 2015;4(6):1650-1658.
Al-Majidi SMH, Ahmad MR, Kareem Khan A. Synthesis and characterization of novel 1,8-Naphthalimide derivatives containing 1,3-oxazoles, 1,3-thiazoles, 1,2,4-triazoles as antimicrobial agents. J Al-Nahrain Uni. 2013;16(4):55-66.
Al-Azzawi AA, Hassan AS. Synthesis and preliminary evaluation of antimicrobial activity of new sulfonamido and acetamido cyclic imides linked to benzothiazole moiety K. J Pharm Sci. 2011,2:59-80.
Tozato Prado SR, Cechinel-Filho V, Campos-Buzzi F, Correa R, Correia Suter Cadena SM, Martinelli de Oliveira MB. Biological evaluation of some selected cyclic imides: Mitochondrial effects and in vitro
cytotoxicity. Z Naturforsch. 2004;59c: 663D672.
Gayoso CW, Lima EO, Souza EL, Filho VC, Trajano VN, Pereira FO, Lima IO. Antimicrobial effectiveness of maleimides on fungal strains isolated from onychomycosis. Brazilian Archives of Bio & Tech. 2006, 49(4): 661-664.
Sortino M, Filho VC, Corre R, Zacchino S. N-Phenyl and N-phenylalkyl-maleimides acting against Candida spp.: Time-to-kill, stability, interaction with maleamic acids. Bioorg Med Chem. 2008;16: 560-568.
Dhivare RS, Rajput SS. Microwave assisted solvent free synthesis and antifungal evaluation of 3, 5-bis-(4- hydroxy-3-methoxybenzylidene)-nphenyl-piperidine-2, 6-dione derived from N-phenyl glutarimides. Int J Chem Tech Res. 2016,9(3):325-331.
Guri D, QingPeng W, HuiZhen Z, YiYi Z, Song LVJ, ChengHe Z. A series of naphthalimide azoles: design, synthesis and bioactive evaluation as potential antimicrobial agents. Sci China Chem. 2013;56(7):952-969.
Khalil AEG, Berghot MA, Gouda MA. Synthesis and study of some new N-substituted imide derivatives as potential antibacterial agents. Chem paper. 2010;64(5):637-644.
AL- Azzawi AM, Al-Obiadi KKH. Synthesis and antimicrobial screening of new BisSchiff bases and their acetyl oxadiazole azetidinone derivatives from pyromellitic diimid. Int J Res Pharm & Chem. 2016,6(1):1-8.
Dhivare RS, Rajput SS. Synthesis and antimicrobial evaluation of some novel bis-heterocyclic chalcones from cyclic imides under microwave irradiation. Chem Sci Rev Lett. 2015,4(15):937-944.
Sultana K, Khan NH, Shahid K. Synthesis, characterization and In Vitro a
ntibacterial evaluation of Sn, Sb, and Zn coordination complexes of 2-(2- methoxyphenyl)-1H-isoindole-1, 3(2h)-dione. Int J Pharm Sci Rev Res. 2014;28(2):1-5.
Yunesa JA, Cardosob AA, Yunesc RA, Corread R, Campos-Buzzid F, Cechinel Filhod V. Antiproliferative effects of a series of cyclic imides on primary endothelial cells and a leukemia cell line. Z Naturforsch. 2008;63c:675-680.
Campos-Buzzid F, Corread R, Souzaa MM, Yunes RA, Nunes RJ,Cechinel-Filho V. Studies on new cyclic imides obtained from aminophenazone with analgesic properties. Arzneim.-Forsch./Drug Res. 2002;52,6:455-461.
Al-Azzawi AM. Synthesis, characterization and evaluation of antibacterial activity of several new 3,4-dimethyl maleimides and 1,8-naphthalimides containing 1,3,4-xadiazole ring. J Al-Nahrain Uni Sci. 2011;14(1):15-29.
Abdel-Aziz AAM. Novel and versatile methodology for synthesis of cyclic imides and evaluation of their cytotoxic, DNA binding, apoptotic inducing activities and molecular modeling study. Eur J Med Chem: 2007;42:614-626.
Bakhta MF, Shahar Yar M, Abdel-Hamid SG, Qasoumi SIA, Samad A. Molecular properties prediction, synthesis and antimicrobial activity of some newer oxadiazole derivatives. Eur J Med Chem. 2010;45:5862-5869.
Al-Azzawi AM, Hamd AS. Synthesis and antimicrobial screening of new naphthalimides linled to oxadiazole, thiadiazole and triazole cycles. Int J Res Pharm & Chem. 2014,4(2):283-290.
Shaki H, Khosravi A, Gharanjig K, Mahboubi A. Synthesis and biological properties of novel cationic fluorescent dye. Int J Tec Res & App. 2015;29: 103-106.
Kumari G, Singh RK. Green synthesis, antibacterial activity, and SAR of some novel naphthalimides and allylidenes. Med Chem Res. 2015;24:171-181.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]