Abstract
1,3-oxazolidin-2-one, an oxygen and nitrogen comprising heterocyclic structure attracts many researchers all over world, evidenced to have unique and potential antibacterial activity. We had designed and synthesized few analogues of 1,3-oxazolidin-2-ones. Their structures were characterized using physical and spectral data. To evaluate their antibacterial potential, Kirby Bauer Agar Diffusion Assay procedure was adopted. Results are very significant and revealed that 1,3-oxazolidin-2-ones have potential antibacterial activity when compared with standard Oxytetracycline. In case of E. coli, unsubstituted parent molecule have better inhibitory effect than the substituted ones while in case of P. vulgaris, substituted analogs have higher effect than the parent one.
Introduction
The pharmaceutical industry faces a huge pressure nowadays to introduce new drugs in market as well as to increase productivity in short time span with minimum investment through R & D process which has increasing attrition rates and escalating costs [1]. Moreover, development of novel drug molecule with improved efficacy, potency and undesirable side effects have always been the single aim of the scientists[2]. Heterocyclic compounds containing five or six membered ring with one or more nitrogen atoms are always of great importance in the pharmaceutical sector as having the bio-isosteric factor[3]. The 1,3-oxazolidin-2-ones are a new class of antimicrobial agents which have a unique structure and good activity against gram-positive pathogenic bacteria. 1,3-Oxazolidin-2-ones are a class of compounds containing 2-oxazolidine in the structure[4,5]. Oxazolidinones inhibit protein synthesis by binding at the P site at the ribosomal 50S subunit. Resistance to other protein synthesis inhibitors does not affect oxazolidinone activity; however rare development of oxazolidinone resistance cases, associated with 23S r-RNA alterations during treatment, has been reported. Linezolid, the first oxazolidinone available, has already taken its place in the clinic for treatment of gram-positive infections. It selectively inhibits bacterial protein synthesis through binding to sites on the bacterial ribosome and prevents the formation of a functional 70S-initiation complex. Specifically, linezolid binds to a site on the bacterial 23S ribosomal RNA of the 50S subunit and prevents the formation of a functional 70S initiation complex, which is an essential component of the bacterial translation process [5,6].
The development of resistance by the antibiotics in the Gram-positive pathogenic bacteria over the last twenty years and continuing today has created a need for new antibiotic classes, which may be unaffected by existing bacterial resistance. The oxazolidin-2-ones not only represent a new class with a novel mechanism of action, but also satisfy the requirement for overcoming the resistance mechanisms. Both linezolid and eperezolid, the first chemical candidates, arose from the piperazine subclass, with the first one being chosen further development because of its enhanced pharmacokinetic properties. The main attractive traits of the oxazolidinone series have encouraged further work in the area, and the patent literature reveals that extensive chemical investigation is currently being made. The unexpected early resistance development emphasizes the need for further exploration of features of the oxazolidinone to eliminate these deficiencies[5]. Keeping this perspective in view, current study involved the synthesis, spectral characterization and antibacterial activity evaluations of some novel 1,3-oxazolidin-2-ones.
Materials & Methods
All melting points were determined in open capillary tubes in Toshniwal Melting point apparatus and are presented without any corrections. The infrared (IR) spectra were recorded on a FTIR-8310 Shimadzu spectrometer using potassium bromide pellets. The proton nuclear magnetic resonance (1H-NMR) spectra were recorded on AMX 400 at 200 MHz using tetramethylsilane (TMS) as the internal standard and DMSO as solvent. Rota evaporator from servewell instruments pvt ltd., Bangalore. UV chamber from Servewell instruments pvt ltd. All reagents were of the highest purity commercially available. The chemical shifts are expressed in part per million (ppm) downfield from the internal standard; and signals are quoted as s (singlet), d (doublet), t (triplet), q (quartet), dd(doublet of doublet) or m (multiplet). The purity of the compounds was checked by Thin Layer Chromatography using Merck Pre-coated silica gel GF aluminium plates and n-hexane: ethylacetate (70:30) as solvent system[7].
General procedure for the synthesis of parent ring (3-(3-fluoro-4-piperazine-1-phenyl)-5-(hydroxymethyl)-1, 3-oxazolidin-2-one)
Step-A The synthesis began by separate SN Ar reactions of 3,4- difluoronitrobenzene with mono piperazine under basic conditions to afford para-substituted nitrobenzene derivatives to good yields. Compound A (0.2mole, 12.91gm) was dissolved in 30 ml of acetone containing few drop of GAA. The compound B (0.2 mole, 23.86gm) was added and reaction mixture was refluxed for 48 hr at 70 ºC with 35ml DMF and 60gm K2CO3. In between, the completion of the reaction was monitored by TLC. After completion of reaction, the solvent was distilled out to get the solid product. The reaction mixture was cooled, poured in crushed ice, filtered, and the separated product compound C was purified by recrystallization from ethanol. Yield 68%; M.Pt. 98-100 oC; Rf 0.62 in gradient(n-hexane: ethylacetate 70:30).
Step-B The compound C and a few amount of Fe dissolve in 50 ml ethanol containing 10ml hydrochloride acid as a catalyst was refluxed for 2-4 hr at 10 ºC. The reaction mixture was stirred for 15 minutes. The mixture was cooled and poured into crushed ice and made basic by 20 % NaOH. The resulting solid compound D was, filtered, and the separated product was purified by recrystallization from acetone. Yield 58%; M.Pt 160-162oC; Rf 0.72 in gradient(n-hexane: ethylacetate 70:30).
Step-C A mixture of compound D, benzylchloroformate (0.02mole), Na2CO3 (30%, 5ml) in 50 ml acetone was refluxed on water bath for 2 hr. In between, the completion of the reaction was monitored by TLC. The mixture was cooled and poured into crushed ice and made acidic by HCl. The resulting solid was, filtered, and the separated product Compound E was purified by recrystallization from ethanol. Yield 70%; M.Pt 190-192oC; Rf 0.83 in gradient(n-hexane: ethylacetate 70:30).
Step-D Compound E was treated with n-BuLi and R-glycidyl butyrate sequentially at 78 ºC in presence of THF to form 1,3-oxazolidin-2-one analogs parent ring (3-(3-fluoro-4-piperazine-1-phenyl)-5-(hyroxymethyl)-1,3-oxazolidin-2-one) 70– 80% yields with defined stereochemistry at the A-ring C-5 position. Yield 75%; M.Pt 196-198oC; Rf 0.76 in gradient(n-hexane: ethylacetate 70:30).
Synthesis of [3-(3-fluoro-4-piperazine-1-phenyl)-5-[hydroxyl(pyrimidin-4-yl)methyl]-1,3 oxazolidin-2-one] (Ox 1.1)
3-(3-fluoro-4-piperazine-1-phenyl)-5-(hyroxymethyl)-1,3-oxazolidin-2-one (0.027M, 8.0 gm) was dissolved in acetone (10ml) and 2-chloropyrazine (0.027 M) was added and refluxed in presence of Ph3P for 8 hr. In between the completion of the reaction was monitored by TLC. Then the solution was poured into crushed ice with stirring and neutralized with glacial acetic acid. Product was filtered, washed with cold water, dried and recrystallised from ethanol.
Synthesis of [3-(3-fluoro-4-piperazine-1-phenyl)-5-pyrazine-1,3 oxazolidin-2-one] (Ox 1.2)
3-(3-fluoro-4-piperazine-1-phenyl)-5-(hyroxymethyl)-1,3-oxazolidin-2-one (0.027M, 8.0 gm) was dissolved in acetone (10ml) and 4-hydroxypyrimidin (0.027 M, 3.09gm) was added and refluxed in presence of a hot mixture of NaH and DMF for 8 hr. In between the completion of the reaction was monitored by TLC. Then the solution was poured into crushed ice with stirring and neutralized with glacial acetic acid. Product was filtered, washed with cold water, dried and recrystallized from ethanol.
Synthesis of [3-(4acetylpiperazine-1-phenyl)-5-hydroxymethyl-1,3 oxazolidin-2-one] (Ox 1.3)
3-(3-fluoro-4-piperazine-1-phenyl)-5-(hyroxymethyl)-1,3-oxazolidin-2-one (0.027M, 8.0 gm) was dissolved in cold pyridine (8ml) at 4ºC.To this clear solution acetic anhydride (10ml) and THF (2.5ml) was added gradually with stirring at room temp. The mixture was heated under refluxed for1-2hr. The mixture was cooled and poured into crushed ice. The resulting solid was, filtered, and the separated product was purified by recrystallization from ethanol.
Synthesis of [3-(3-fluoro-4-piperazine-1-phenyl)-5-carbaldehyde-1,3 oxazolidin-2-one] (Ox 1.4)
3-(3-fluoro-4-piperazine-1-phenyl)-5-(hyroxymethyl)-1,3-oxazolidin-2-one (0.027M, 8.0 gm) was taken in 250 ml RBF. Potassium dichromate (0.027M, 7.94 gm) was added in acidic medium. Then all were refluxed at 1200 C for 45 minutes. Then filtered the solution and recrystallized with acetone. After recrystallization, melting point was determined. The progress of reaction evaluated by TLC.
Synthesis of [3-(3-fluoro-4-piperazine-1-phenyl)-5-oxime-1,3 oxazolidin-2-one] (Ox 1.5)
3-(3-fluoro-4-piperazine-1-phenyl)-5-(hyroxymethyl)-1,3-oxazolidin-2-one (0.027M, 8.0 gm) was taken in 250 ml RBF. Hydroxyl amine (0.027M,1.0 gm) was added at low temperature. After 30min, all were refluxed at 600 C for 90 minutes.Then filtered the solution and recrystalized with acetone. After recrysalization, melting point was determined. The progress of reaction evaluated by TLC.
Kirby Bauer Agar Diffusion Assay
Nutrient agar media was taken in a pre-sterilized Petri-dish. After that the microorganisms were spreaded over the cooled nutrient agar media with the help of L- shaped glass rod. With the help of sterilized borer, wells were formed in the seeded agar plate and filled with different concentrations of 1,3-oxazolidin-2-one derivatives(250, 500 & 1000 mg/ml). In each seeded plate, reference compound oxytetracyclin (1000mg/ml) was also placed & incubated at 370C for 24 hr. The diameters of zone of inhibition (mm) were recorded and compared with standard drug oxytetracyclin[8-18].
Results & Discussion
Table 1 Derivatives of 1,3-Oxazolidin-2-one
See Illustration 1
3(3-fluoro-4-piperazine-1-phenyl)-5-(hydroxymethyl)-1,3-oxazolidin-2-one (Parent):
C14H18FN3O3; Mol wt. 295.30; M.P.( ºC) 196-198; white solid; Rf 0.76; Freely soluble in acetone and soluble in Ethanol; % yield (w/w) 75%; IR(KBr Disc, cm-1): 2919.36(Aromatic-C-H str ), 1064.74(C-N str ), 3014.38(aliphatic C-H str) , 3158.08(N-H str), 1285.60(C-O str of oxazolidin-2-one ), 1146.61(aromatic C-C str), 1512.14(C=O str ), 1149.41 ( C-F str); 1H-NMR(ppm): 1.3-1.329(t,2H, CH2OH), 2.048(s, 1H, -CH2OH), 3.199-3.233(d, 2H, CH2-oxazolidinone), 1.432-1.461(s, 1H, NH- piperazine), 6.89-6.949(dd, 8H, piperazine), 7.064-8.011(m, 3H, Ar-H), 4.392-4.401(s, 1H, CH-methine).
3(3-fluoro-4-piperazine-1-phenyl)-5-[hydroxyl(pyrimidin-4-yl)methyl]-1,3-oxazolidi n-2-one (Ox 1.1):
C18H20FN5O3; Mol. Wt. 373.38; M.P.( ºC) 205-207°C; White cream solid; Rf 0.72; Freely soluble in acetone and soluble in Ethanol; % yield (w/w) 72%; IR(KBr Disc, cm-1): 3194.12(Aromatic-C-H str ), 1385.43(C-N str ), 3036.06(aliphatic C-H str), 3446.91(N-H str ), 1680.94(C-O str of oxazolidin-2-one), 1149.97(aromatic C-C str), 1733.10(C=O str), 1598.32 (C=N str pyrimidine), 1149.21 ( C-F str); 1H-NMR(ppm): 1.311-1.346(t, 2H, -CH2O), 3.209-3.253(dd, 8H, piperazine), 3.697-3.711(dd, 2H, -CH2-oxazolidinone), 1.410(s, 1H, NH-piperazine), 4.246-4.255(s, 1H, CH-methine), 6.891-8.256(m, 6H, Ar-H).
3(3-fluoro-4-piperazine-1-phenyl)-5-pyrazine-1,3-oxazolidin-2-one (Ox 1.2):
C18H20FN5O3; Mol. Wt. 373.38; M.P.( ºC) 188-190°C; White cream solid; Rf 0.65; Freely soluble in acetone and soluble in Ethanol; % yield (w/w) 73%; IR(KBr Disc, cm-1): 3043.49(Aromatic C-H str ), 1153.43(C-N str ), 2872.03(aliphatic C-H str) , 3211.95(N-H str ), 1693.12(C-O str of oxazolidin-2-one) , 1324.21(aromatic C-C str), 1592.29(C=O str ), 1069.21 ( C-F str); 1H-NMR(ppm): 1.289(t, 2H, -CH2O), 3.719-3.927(dd, 8H, piperazine), 2.097(s, 1H, NH-piperazine), 4.497(s, 1H, CH-methine), 6.559-7.035(m, 6H, Ar-H).
3(4-acetylpiperazine-1-phenyl)-5-hydroxymethyl-1,3-oxazolidin-2-one (Ox 1.3):
C16H20FN3O4; Mol. Wt. 337.34; M.P.( ºC) 200-203°C; white solid; Rf 0.82; Freely soluble in acetone and soluble in Ethanol; % yield (w/w) 80%; IR(KBr Disc, cm-1): 3045.45(Aromatic-C-H str ), 833.65(C-N str ), 2837.64(aliphatic C-H str), 3163.27(N-H str ), 1226.77(C-O str of oxazolidin-2-one), 974.73(aromatic C-C str), 1556.31(C=O str ), 1163.21 ( C-F str), 1062.31 ( O-H str); 1H-NMR(ppm): 7.068-7.178(dd, 8H, piperazine), 2.178(s, 1H, NH-piperazine ring), 4.618(s, 1H, CH-methine), 6.967-6.964(m, 6H, Ar-H), 3.133-3.184(5H, -C2H5).
3(3-fluoro-4-piperazine-1-phenyl)-5-carbaldehyde-1,3-oxazolidin-2-one (Ox 1.4):
C14H16FN3O3; Mol. Wt. 293.29; M.P.( ºC) 195-198°C; white solid; Rf 0.84; Freely soluble in acetone and soluble in Ethanol; % yield (w/w) 66%; IR(KBr Disc, cm-1): 2837.78(Aromatic-C-H str ), 1152.20 (C-N str ), 1955.95(aliphatic C-H str), 2931.20(N-H str ), 1233.52(C-O str of oxazolidin-2-one), 1014.84(aromatic C-Cstr), 1664.06 (C=O str ), 1064.21 ( C-F str); 1H-NMR(ppm): 7.317-7.407(dd, 8H, piperazine), 2.097(s, 1H, NH-piperazine ring), 6.231(s, 1H, CH-methine), 6.577-6.729(m, 6H, Ar-H).
3(3-fluoro-4-piperazine-1-phenyl)-5-oxime-1,3-oxazolidin-2-one (Ox 1.5):
C14H17FN4O3; Mol. Wt. 308.30; M.P.( ºC) 185-188; white solid; Rf 0.75; Freely soluble in acetone and soluble in Ethanol; % yield (w/w) 64%; IR(KBr Disc, cm-1): 2848.38(Aromatic-C-H str ), 1056.88(C-N str ), 2916.40(aliphatic C-H str), 3198.42(N-H str ), 1665.64 (C=O str ), 1273.06(C-O str of oxazolidin-2-one), 3406.32 (OH str of oxime), 1604.21 ( C=N str of oxime), 975.35 (N-O str of oxime), 1001.31 (C-F str); 1H-NMR(ppm): 7.317-7.407(dd, 8H, piperazine), 2.097(s, 1H, NH-piperazine ring), 6.231(s, 1H, CH-methine), 6.577-6.729(m, 6H, Ar-H).
Table 2 Antibacterial activities of 1,3-oxazolidin-2-one derivatives
See Illustration 2
Parentoxazolidinonei.e. 3-(3-fluoro-4-piperazine-1-phenyl)-5-(hyroxymethyl)-1,3-oxazolidin-2-one were synthesized by the four step nucleophillic substitution reactions as explained above. Five derivatives of oxazolidinones (Ox 1.1 – Ox 1.5) were prepared from the parent oxazolidin-2-one by single step nucleophillic attack(Table 1). Structure of all the compounds was characterized by physical data as well as spectral analysis. To evaluate the test compounds for possible antibacterial activity, they were tested with the strains of E. coli & P. vulgaris at three different concentrations( 250, 500 & 1000 µg/ml) and compared it with the standard oxytetracycline(1000 µg/ml)(Table 2). Kirby Bauer Agar Diffusion Assay(KBADA) procedure was adopted. As can be seen, standard oxytetracycline have similar effects on E. coli & P. vulgaris, while all the 1,3-oxazolidin-2-one analogues have better growth inhibitory effect on E. coli than that on P. vulgaris. Ox 1.1, Ox 1.2, Ox 1.4 & Ox 1.5 have lesser growth inhibition potential than the unsubstituted parent 1,3-oxazolidin-2-one compound. So a brief SAR can be established for the analysis of anti-E. coli activity of 1,3-oxazolidin-2-ones. According to the SAR, the hydroxymethyl group at the 5th position on the parent 1,3-oxazolidin-2-one is responsible for its growth inhibitory effect on E. coli while the piperazinyl substitution at 4th position with acetyl group doesn’t increase or decrease the activity. While surprisingly in case of P. vulgaris, situation is completely opposite. All the derivatives of parent 1,3-oxazolidin-2-one have higher activity than the parent one. Oxidizing of the 5th hydroxyl methyl doesn’t change the anti-P. vulgaris spectrum, while the acetylation at the 4th position of piperazine rings improve the inhibitory effect.
Acknowledgement
Authors would like to express their gratitude towards the management of Jaipur National University for providing facility to conduct this research. Author Rajeev K Singla is grateful to Department of Science & Technology, Ministry of Science & Technology, Government of India for providing young scientist fellowship(SR/FT/LS-149/2011).
References
1. Sharma O P, Kotnala A, Shrivastava B, Singla R K, High-Throughput Screening: Effective Tool in Drug Discovery, Pharmacologyonline, 2(2011) 134-150.
2. Bhatia R, Sharma V, Shrivastava B, Singla R K, A Review on Bioisosterism: A Rationale approach for Drug Design and Molecular Modification, Pharmacologyonline, 1(2011) 272-279.
3. Sharma O P, Singla R K, Shrivastava B, Scientific Assessment of Antioxidant Potential of Pyrimidine-2,4(1H, 3H)-diones, Indo Global Journal of Pharmaceutical Sciences, 2(2012) 142-146.
4. Prasad J V, New Oxazolidinones, Current opinion in Microbiology, 10(2007) 454-460.
5. Pandit N, Singla R K, Shrivastava B, Current updates on Oxazolidinone and its Significance, International Journal of Medicinal Chemistry, 2012(2012) 24 Pages, Article id159285, DOI 10.1155/2012/159285 .
6. Stevens D L, Dotter B, Madaras-Kelly K, A review of linezolid: the first oxazolidinone antibiotic, Expert Review of Anti-infective Therapy, 2(2004) 51-59.
7. Joseph A, Pai A, Srinivasan K K, Kedar T, Thomas A T, Jessy E M, Singla R K, Synthesis and Anticancer Activity of Some Novel 3(1,3,4-thiadiazol-2-yl)-quinazolin-4-(3H)-ones, Orbital- The Electronic Journal of Chemistry, 2(2010) 158-167.
8. Singla R K, Kumar A, Khan S, Shrivastava R, Bhat V G & Jagani H, Evaluation of Antimicrobial Activity of 3-(4-1H-Indol-3-yl)-(2,3-dihydro-1H-benzo[b]diazepin-2-yl)- 2H-chromen-2-one, Indo Global Journal of Pharmaceutical Sciences, 1(2011) 127-133.
9. Johnson D B, Shringi B N, Patidar D K, Chalichem N S S & Javvadi A K, Screening of antimicrobial activity of alcoholic & aqueous extract of some indigenous plants, Indo-Global Journal of Pharmaceutical Sciences, 1(2011) 186-193.
10. Reddy V B, Singla R K, Bhat V G & Shenoy G G, Synthesis and antimicrobial activities of some novel benzimidazole derivatives, Asian Journal of Research in Chemistry, 2(2009) 162-167.
11. Pandya K, Solanki B, Shah B, Parekh D, Soni H, Maniar K, Gurav N & Patel P, Phyto-chemical screening & evaluation of antibacterial activity of polyherbal formulation, Indo Global Journal of Pharmaceutical Sciences, 1(2011) 206-218.
12. Sharma O P, Shrivastava B, Singla R K & Bhat VG, Synthesis & antimicrobial activity of some novel pyrimidine-2,4(1H,3H)-diones, Indo Global Journal of Pharmaceutical Sciences, 1(2011) 252-257.
13. Mudgal M M, Singla R K, Bhat V G & Shenoy G G, Synthesis and antimicrobial activity of some novel [1,2,4]triazolo[3,4-B][1,3,4]thiadiazole derivatives, Pharmacologyonline Newsletter, 2(2009) 188-193.
14. Sharma O P, Singla R K, Shrivastava B, Bhat V G, Shenoy G G, Jayashree B S & Sreenivasan K K, Synthesis, spectral characterization & antimicrobial evaluation of some novel pyrimidine-2,4(1H, 3H)-diones, Indo Global Journal of Pharmaceutical Sciences, 2(2012) 70-75.
15. Jawaharmal, Lamba H S, Narwal S, Singh G, Saini D R, Kaur A & Narwal S, Synthesis of novel imidazole compounds and evaluation of their antimicrobial activity, Indo Global Journal of Pharmaceutical Sciences, 2(2012) 147-156.
16. Gupta R, Gupta A K, Singla R K & Aiswarya G, Preliminary investigation of antimicrobial property of acacia leucophloea leaves extract, International Journal of Phytomedicine, 3(2011) 308-311.
17. Singla R K, Jaiswal N, Bhat V G & Jagani H, Antioxidant and antimicrobial activities of Cocos nucifera Linn. (Arecaceae) endocarp extracts, Indo Global Journal of Pharmaceutical Sciences, 1(2011) 354-361.
18. Yadav V, Jayalakshmi S, Singla R K, Patra A, Evaluation of Antibacterial Activity of Callicarpa macrophylla Vahl. Stem Extracts, WebmedCentral Ayurvedic Medicine, 3(2012) WMC003651.
Source(s) of Funding
This project was funded by Jaipur National University, Jaipur, India. Author R K Singla receiving Young Scientist Fellowship from Department of Science & Technology(Ministry of Science & Technology), Government of India(SR/FT/LS-149/2011).
Competing Interests
Authors declare no competing interests.
Disclaimer
This article has been downloaded from WebmedCentral. With our unique author driven post publication peer
review, contents posted on this web portal do not undergo any prepublication peer or editorial review. It is
completely the responsibility of the authors to ensure not only scientific and ethical standards of the manuscript
but also its grammatical accuracy. Authors must ensure that they obtain all the necessary permissions before
submitting any information that requires obtaining a consent or approval from a third party. Authors should also
ensure not to submit any information which they do not have the copyright of or of which they have transferred
the copyrights to a third party.
Contents on WebmedCentral are purely for biomedical researchers and scientists. They are not meant to cater to
the needs of an individual patient. The web portal or any content(s) therein is neither designed to support, nor
replace, the relationship that exists between a patient/site visitor and his/her physician. Your use of the
WebmedCentral site and its contents is entirely at your own risk. We do not take any responsibility for any harm
that you may suffer or inflict on a third person by following the contents of this website.