Synthesis and Evaluation of Anti microbial Activity of Substituted 1, 4-bis (5- phenyl isoxazole-3-yl) benzene derivatives

 

Karri Apparao¹, Tentu Nageswararao¹*, N.Krishnarao¹, S.Cinyya Setty², T.Prsanna Devi³

¹Department of Chemistry, Krishna University, Machilipatnam, Andhra Pradesh, India

²Research and Development Centre, Pharma zel, Visakhapatnam, Andhra Pradesh, India

³Department of Chemistry, Viswabharati College, Visakhapatnam, Andhra Pradesh, India

*Corresponding Author E-mail: tnraochemistry@gmail.com

The preliminary investigation  of  newly  synthesized , evaluated  and the bioevaluation of  1,4-bis (5- phenyl isoxazole-3-yl) benzenewhich  was obtained from the cyclization of 3,3’-(1,4-phenylene) bis (1-phenyl prop-2-en-1-one)  treated with hydroxyl amine hydrochloride in the presence of ethanol. The 3, 3’-(1,4-phenylene) bis (1-phenyl prop-2-en-1-one  can be synthesized from the condensation of  the terepthaldehyde and substituted aryl, alkyl ketones in the presence of potassium tertiarybutaxide. All the newly synthesized compounds were evaluated by advanced spectroscopic analysis (1HNMR, 13CNMR) and also structural determination was calculated by elemental analysis. The titled compounds were screened by the antimicrobial activity.

 

 

 

INTRODUCTION:

Heterocyclic compounds are of the most significance in the organic synthesis which is inseparable from mankind because human are totally dependent on the drug derivatives. These derivatives from heterocyclic rings are present in several compounds, e.g. most of the number of vitamin complexes, antibiotics, chlorophyll, haemine, other plant pigments, amino acids and proteins, drug, dyes stuffs, enzymes, the genetic materials DNA etc.

 

An Isoxazoles are an important five membered heterocyclic ring   which possesses nitrogen and oxygen atoms at 1 and 3 positions. These isoxazole were prepared from chalcones and important intermediate products and they are also contained biological and pharmacological properties¹.

 

Isoxazoles possess an interesting medicinal property and they have industrial applications². Isoxazole steroids show anabolic activity, eg. Denazole³, the CNS active isoxazole, ibotinic. Isoxazole derivatives were used as inhibitors for ulcers, lipoxngenas, acetyl choline esterase⁴. 3-substituted- 5-methyl thioisoxazole were found to exhibit anthelminitic activity ⁵. Spiro isoxazolines and benzofuro isoxazole were used as anti –convergents. 5-amino-3-methyl-4-ureido isoxazoles were found to exhibit anti leukumic activity⁶. Most of the 2-isoxazole derivatives synthesized from α , β – dibromo chalcones showed mild antibacterial activity ⁷.  Some of 4,5-diphenyl Isoxazoles, 3,4-diphenyl-5-trifluoro methyl Isoxazoles and 4,5-diphenyl-3-methyl sulfonamide isoxazole containing a variety of substitutions at the Para position of  one of the phenyl rings were used as analgesic and selective-COX-2 inhibitory and anti inflammatory agents^{8, 9}. Isoxazolines napthaquinines acts as a potential trypanocidal and anti bacterial agents¹⁰.

 

 

METHODS AND MATERIALS:

All the chemicals and synthetic grade reagents were purchased from Merck Chemicals Pvt. Ltd. Melting points of all the synthesized compounds were determined in open capillaries an melting apparatus and are uncorrected. All the reactions were monitored by TLC on precoated silica gel 100 – 150 [mesh]. Spots of the compound were visualized with UV light. Merck silica gel (100 – 200) was used for column chromatography. ¹HNMR (400 MHz) and ¹³CNMR (100M Hz) spectra were recorded on a Bruker AMX 400 MHz. NMR spectra values in CDCl₃ solution using TMS as an internal standard. Molecular weights of the newly synthesized compounds were determined by LCMS spectrometer. All the chemical shifts are reported in δ(ppm) using TMS as an internal standard.

 

EXPERIMENTAL SECTION:

General procedure for the synthesis of 3,3’-(1,4-phenylene) bis (1-phenyl prop-2-en-1-one) :

Synthesis of chalcones by Claisen –Schmidt condensation by reaction of terepthaldehyde and various substituted acetophenones. To the solution of 11gm of potassium hydroxide in 100 ml of water and 61 ml of rectified spirit in 250 ml bolt-head flask provided with mechanical stirrer. Immerse the flask in a bath of crushed ice, pour 0.43 ml of purify substituted acetophenones, start the stirrer and then 0.43mol of pure terepthaldehyde. Keep the temperature of the mixture at about 25⁰c and stir vigoursly until the mixture so thick that stirring is no longer effective (3-4 hrs). Remove the Stirrer and leave the reaction in an refrigerator overnight. Filter the product with suction on Buchner funnel or sintered glass funnel, wash with cold water until the washing are neutral to litmus and than 20 ml if ice cold rectified spirit. The crude chalcones after drying in vacuum pump and recrystalized from ethanol warm to 55⁰c. The final product can be obtained.

 

 

Characterization of 3, 3’-(1,4-phenylene) bis (1-phenyl prop-2-en-1-one):

 

 

 

The mixture of 3,3’-(1,4-phenylene) bis (1-phenyl prop-2-en-1-one (0.03 mol), and hydroxyl amine hydrochloride (0.045 mol) and sodium acetate in ethanol 35 ml was taken in 100mL RB flask  and subjected to reflux from 6-7 hrs.The reaction mixture was  examined by TLC when the all reactants were consumed. After completion the reaction, the mixture was concentrated by distilling out the solvent under reduced pressure and poured into ice water. The precipitate obtained after filtration, washed and recrystalized from ethanol.

 

 

1)       1,4-bis (5- phenyl isoxazole-3-yl) benzene (5a) :

 

 

 

White solid; yield-85%;

¹HNMR (400MHz, CDCl₃) δ in ppm:8.10(d, J=8.88Hz,1H,Ar-H),8.07(d,8.4Hz,1H,Ar-H), 8.35-8.24 (m,4H,Ar-H),7.51-7.47(m,3H,Ar-H),6.21(s,1H,=CH ,isoxazole). ¹³CNMR (100MHz, CDCl₃) δ in ppm: 166.5, 160.7, 129.4, 129.1, 128.8, 128.2, 127.8, 125.9, 97.3, LCMS (m/z): 354.56. Molecular formula: C₁₂H₁₀N₄. Elemental analysis: calculated: C-79.11, H-4.43, N-7.69, O-8.78. Obtained: C-79.18, H-4.41, N-6.67,O-8.75.

 

 

2)       4,4’-(3,3’-(1,4-phenylene) bis ( isoxazole-5,3-diyl)) bis (2-ethoxy phenol)  (5b)

 

 

 

White solid; yield-87%;

¹HNMR (400MHz, CDCl₃)δ in ppm:9.70 (s,1H,-OH),8.13-8.07 (m,4H,Ar H),6.96(s,1H,Ar-H),6.76(d, J= 8.0Hz,1H,Ar-H), 6.72(d, J= 8.4Hz,1H,Ar H),6.26(s,1H,isoxazole),4.16-3.98(m,2H,CH2)1.33 (t, J=8.4Hz,CH3).  ¹³CNMR (100MHz, CDCl₃) δ in ppm: 168.1,154.8,148.7,146.5,133.2,131.4,127.3,126.9,118.4,115.7,109.2,98.4,63.4,14.6.(fig-1)                        . 

 

LCMS (m/z):488.85. Molecular formula: C₁₄H₁₁N₃ . Elemental analysis:  Calculated: C-68.84, H-5.78, N-5.73,O-19.65. Obtained: C-68.88, H-5.77, N-5.72,O-19.63.

 

 

 

 

 

 

Fig. 1.13C NMR of 4,4’-(3,3’-(1,4-phenylene) bis ( isoxazole-5,3-diyl)) bis (2-ethoxy phenol)  (5b)

 

3)       4,4’-(3,3’-(1,4-phenylene)bis(isoxazole-5.3-diyl) bis (2-methoxy phenol)(5c) :

 

 

 

White solid; yield-90%; m.p – 219⁰c

¹HNMR (400MHz, CDCl₃) δ in ppm:9.63(s,1H,-OH),8.32-8.09(m,4H,Ar-H), 6.82 (s,1H,Ar-H),6.54 (d, J=7.6Hz, 1H, Ar-H), 6.47(d, J=8.0Hz, 1H,Ar-H), 6.27 (s,1H, isoxazole), 3.73(s,3H,-OCH3). ¹³CNMR (100MHz, CDCl₃) δ in ppm:167.8,157.1 , 147.4, 146.3, 134.4,131.4, 128.6,127.6,118.6,11.3,108.4,97.7,55.9(fig-2). LCMS (m/z):460.17. Molecular formula: C₁₂H₉N₃O. Elemental analysis: calculated: C-67.82, H-5.25, N-6.08, 0-20.85. Obtained: C-67.86, H-5.24, N-6.07, O-20.83.

 

 

Fig. 2.13C NMR of 4,4’-(3,3’-(1,4-phenylene)bis(isoxazole-5.3-diyl) bis (2-methoxy phenol) (5c)

 

 

1,4-bis (5-(3,5-dimethoxy phenyl) isoxazole-3-yl) benzene (5d) :

 

White solid; yield-88%;

¹HNMR (400MHz, CDCl₃) δ in ppm:8.31-8.25(m,4H,Ar-H),6.92(s,,2H,broad), 6.34(s,1H,Ar-H),6.22(s,1H,isoxazole),3.79(s,6H,-2OCH3). ¹³CNMR (100MHz, CDCl₃) δ in ppm: 167.4, 160.9, 159.4, 132.5, 129.0, 128.5, 128.1, 02.7, 99.3, 54.6(fig-3). LCMS (m/z):484.27. Molecular formula: C₁₂H₁₀N₄. Elemental analysis: calculated: C-69.41, H-4.99, N-5.78, O-19.82. Obtained: C-69.46, H-4.98, N-5.77, O-19.80.

 

 

Fig. 3.13C NMR of 1,4-bis (5-(3,5-dimethoxy phenyl) isoxazole-3-yl) benzene (5d)

 

4)       6,6’ (3,3’-(1,4-phenylene) bis (isoxazole-5,3-diyl) bis(3-( dimethylamino) phenol) (5e) :

 

 

 

White solid; yield-85%;

¹HNMR (400MHz, CDCl₃) δ in ppm: 9.45(s,1H,-OH),7.45(d, J=8.4.Hz,1H,Ar-H), 6.30 (d, J=8.0 Hz,1H,Ar-H), 6.37(s,1H,Ar-H),6.25(s,1H, isoxazole),3.18(s,6H,-2CH3). ¹³CNMR (100MHz, CDCl₃) δ in ppm: 168.0, 161.3, 155.7, 152.6, 129.4, 128.8, 128.5, 128.0, 105.5, 104.4, 98.8, 96.5, 40.5 (fig-4) . LCMS (m/z):427.73. Molecular formula: C₁₂H₉N₃S. Elemental analysis:  calculated: C-69.70, H-5.43, N-11.61,O-13.26.  Obtained: C-69.75, H-5.42, N-11.59,O-13.24.

 

 

Figure. 4.13C NMR of 6,6’ (3,3’-(1,4-phenylene) bis (isoxazole-5,3-diyl) bis(3-( dimethylamino) phenol) (5e)

 

5)       1,4-bis (5-( 4-bromo phenyl) isoxazole-3-yl) benzene (5f):

 

White solid; yield-89%

¹HNMR (400MHz, CDCl₃) δ in ppm : 8.39-8.28 (m,4H,Ar-H),7.69-7.61(m,4H,Ar-H),6.25 (s,1H,isoxazole)   . ¹³CNMR (100MHz, CDCl₃) δ in ppm: 168.4, 160.6, 131.8, 128.3, 127.5, 126.8, 125.2, 122.9, and 98.0 (fig-5). LCMS (m/z):523.68. Molecular formula: C₁₃H₁₀N₄. Elemental analysis:  calculated: C-55.20, H-2.70, Br-30.60, N-5.36, O-6.13. Obtained: C-55.25, H-2.69, Br-30.58, N-5.35, O-6.12.

 

 

Fig.. 5.13C NMR of 1,4-bis (5-( 4-bromo phenyl) isoxazole-3-yl) benzene (5f)

 

6)       4,4’-( 3,3’-(1,4-phenylene) bis( isoxazole-5,3-diyl) dibenzonitrile  (5g):

 

Brown solid; yield-86%;

¹HNMR (400MHz, CDCl₃) δ in ppm: 8.41(m,4H,Ar-H),7.96(d,  J=8.4Hz, 1H,Ar-H), 7.83(d,J=8.0Hz,1H,Ar-H),7.78(d,J=8.0Hz,1H,Ar-H),7.7 (d, J=8.0Hz,1H,Ar-H),6.32(s,1H, isoxazole). ¹³CNMR (100MHz, CDCl₃) δ in ppm: 168.9, 160.8, 132.2, 128.1, 127.7, 126.8, 125.5, 118.7, 112.0, and 97.4(fig-6).  LCMS (m/z):414.49. Molecular formula: C₁₃H₁₀N₄.  Elemental analysis:  calculated: C-73.35, H-3.41, N-13.52. Obtained: C-73.39, H-3.40, N-13.50,O-7.71.

 

 

Fig. 6.13C NMR of  4,4’-( 3,3’-(1,4-phenylene) bis( isoxazole-5,3-diyl) dibenzonitrile  (5g)

 

7)       1,4-bis (5-( 4-nitro phenyl) isoxazole-3-yl) benzene(5h):

 

Brown solid; yield-87%;

¹HNMR (400MHz, CDCl₃) δ in ppm: 8.4.-8.22 (m,6H, Ar-H) ,7.96(d, J=8.0Hz,Ar-H),97.91(d, J=8.4Hz, 1H, Ar-H),6.41(s,1H,isoxazole). ¹³CNMR (100MHz, CDCl₃) δ in ppm: 169.1, 160.8, 145.9 , 132.8, 128.3, 127.1, 124.2, 123.6, 98.2(fig-7).  LCMS (m/z):454.22. Molecular formula: C₁₃H₁₀N₄. Elemental analysis:  calculated: C-63.44, H-3.11, N-12.33, O- 21.13 . Obtained: C-63.49, H-3.10, N-12.31, O-21.11.

 

 

Fig. 7.13C NMR of  1,4-bis (5-( 4-nitro phenyl) isoxazole-3-yl) benzene(5h)

 

 

ANTI BACTERIAL ACTIVITY

The antimicrobial activities of all the newly synthesized compounds (5a-5h) were evaluated against gram negative micro organisms such as E.coli ,  P.aerugenosa and gram positive micro organism such as S.aureus, B.substills strains using Amoxycillin as a reference by the cup plate technique as explained by Hugo and Russe (15).In this technique  the test solutions was placed in contact with agar , which was  already inoculated with test organism. After incubation, zones of inhibition were observed. The test solution may be placed in a small cup sealed to the agar surface in a well cut from the agar with a sterile cork borer or applied in the form of impregnated disc of filter papers.

 

Preparation of Media Petri dishes

Conical flask with medium was cooled to 46⁰c and inoculated with test organism (20 ml of sub culture medium/100 ml of the assay medium) 30 ml of inoculated media distributed into petri dishes. Four cups (8 mm diameter) per plate were made by using a sterile cork borer. The whole operation was carried out under the laminar flow especially. Cups were filled with 0.1 ml of test solution and 0.1 ml of standard solution (100µg/ml, 250µg/ml) and blank (DMSO) were placed in each cups separately under aseptic condition. Then the petri dishes uniform diffusion of drug into the agar medium. All the petri dishes were then incubated at the 37⁰c in one day and zone of inhibition were measured and results are presented in Table 1.

 

ANTI FUGAL ACTIVITY

The newly synthesized compounds (5a-h) were screened for their anti fungal activity against Aspergillus niger, Canadida  albicans and Aspergillus flavus using agar well diffusion assays. Steril molten potato dextrose agar (PDA) medium was inoculated  with 50L of fungal spo suspension and zone of inhibition were measured and results are presented in Table 2.

 

 

 

 

 

RESULT AND DISCUSSION:

Chemistry

The synthetic approach to the titled compounds is given in the scheme. The starting materials,3,3’-(1,4-phenylene) bis (1-phenyl prop-2-en-1-one   (3a-3h) was synthesized by the cyclization of the condensation of terepthaldehyde (1) and reacted  with aryl ketones in the presence of potassium hydroxide in ethanol.               

 

The derivatives of 1, 4-bis (5- phenyl isoxazole-3-yl) benzene can be synthesized from the reaction with hydroxyl amine hydrochloride with3, 3’-(1,4-phenylene) bis (1-phenyl prop-2-en-1-one the presence of potassium hydroxide in ethanol under the reflux conditions. After 2h of reaction the mixture was added to drop wise (20% H₂O₂) 2 hrs. The reaction mixture was cooled to room temperature and transferred to HCl. The precipitated solid product was filtered off, washed with methanol several times dried to get titled compound. The structure of 5a-5h was evaluated by spectral data and elemental analysis.  All the newly synthesized compounds obtained 85%-90% yield.

 

Biological Activity

We observed that the bacterial activity of compound 5a-5h, mostly electron withdrawing group of compound viz; 5g and 5h exhibited  low active potent  while electron donating group of compounds 5b, 5c, 5d,5e exhibited moderate active potent. The compound 5f exhibited good active potential due to halogen group present in the compound.

 

We also observed the Antifungal Activity of compound (5a-5h) exhibited different activity compound 5f showed good activity and rate of the compound showed low to moderate activity.

 

 

 

 

BIOLOGICAL ACTIVITY:

Table 1.  Zone of Inhibition of compound (4a-4h) against tested bacterial activity

 

Table 2. Zone of Inhibition of compound ( 5a-h )against tested fungal strains

 

CONCLUSION:

Here in, we reported the synthesis of bis-isoxazole and its derivatives .We used potassium tertiary but oxide as strong base in this synthesis and reported the new derivatives. The newly synthesized compounds examined antimicrobial activity, compound 5f showed good activity. The entire compound obtained good yield and reaction time is low.

 

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Received on 11.10.2017         Modified on 13.11.2017

Accepted on 20.12.2017         © AJRC All right reserved