Antibacterial activity of coumarine derivatives synthesized from 4,7

Behrami Int. J. Res. Chem. Environ. Vol. 4 Issue 3 (58-63) July 2014

International Journal of

Research in Chemistry and Environment Available online at: www.ijrce.org ISSN 2248-9649

Research Paper

Synthesis, Characterization and Antibacterial Studies of 4, 7-Dichloro-chromen2-one derivatives and Its Metal (II, III) Complexes Aziz Behrami Public University of Mitrovica , KOSOVA (Received 01st May 2014, Accepted 18th June 2014)

Abstract: This work reports the synthesese of some new derivatives from 4,7-Dichloro-chromen-2-one and their antibacterial activity. Compounds 4,7-Bis-(2-chloro-ethylamino)-chromen-2-one1a, 4,7-Bis-(2-chloro-ethylamino)3-nitro-chromen-2-one 2a, (4-Dithiocarboxyamino-3-nitro-2-oxo-2H-chromen-7-yl)-dithiocarbamic acid (Complex with Mg II) 3a, [4,7-Bis-(2-chloro-ethylamino)-2H-chromen-3-yl]-phosphonic acid and its metal (III) Complex 4a . All Structures have been synthesized and characterized using melting points , IR spectra , 1H-NMR and 13C-NMR spectra. The purified synthesized compounds 1a,2a,3a,4a at concentrations 2,3,5 mg/ml was subjected to test the antibacterial activity against the bacterial cultures ,Staphylococcus aureus, Escherchia coli and Bacillus cereus. The antibacterial activity of synthesized compounds were compared with antibacterial activity of standard antibiotics cephalexine and streptomycin. The compounds show different bacteriostatic and bacteriocidal activity. Keywords: 4,7-Dichloro-chromen-2-one derivatives ,antibacterial activity © 2014 IJRCE. All rights reserved

Dithiocarboxyamino-3-nitro-2-oxo-2H-chromen-7-yl)dithiocarbamic acid (Complex with Mg II) 3a , [4,7-Bis(2-chloro-ethylamino)-2H-chromen-3-yl]-phosphonic acid and its metal (III) Complex 4a are synthesized.

Introduction Starting from 4,7-Dichloro-chromen-2-one (a), derivatives (1a,2a,3a,4a) are synthesized. Coumarin derivatives are large group of heterocyclic with oxygen as heteroatom. Coumarin is a chemical compound (specifically , a benzo-α-pyrone) found in many plants 1,2,4 notably in high concentration in the tonka bean ( Dipteryx odorata), vanilla grass (Anthoxanthum odoratum) , woodruff (Galium odoratum) , mullein (Verbascum spp), and sweet grass (Hierochloe odorata). Coumarine and their derivatives have shown varius biological activities. Their fame has come mainly from their antithrombic, antiinflammatory, vasodilatory and antiviral activities. Other several coumarin derivatives have antimicrobial properties [5,6,8] with reflux and condensation we have synthesize some new coumarine derivatives and to investigate their antibacterial activity against Staphylococcus aureus, E. coli and Bacillus cereus. The antibacterial activity of synthesized compounds is compared with antibacterial activity of Cefalexine and Streptomycine.

Measurement: The identification of 4,7-Dichlorochromen-2-one derivatives (1a,2a,3a,4a), is made by using melting point , IR , 1H NMR , 13C NMR spectra and elemental analysis. Melting point was determinated on a Electrothermal apparatus (Fisher Scientific 2555) in a open capillary tube and are uncorrected. Infrared spectra were recorded in cm-1 for KBr pellts on a FT-IR Shimadzu 8400S spectrophotometer with resolution 4 cm-1. 1H NMR spectra were recorded on a Bruker UNITY plus-500 ‘NMR 1’ spectrometer using DMSO-d6 as the solvent and TMS as the internal references standard (σ = 0, 00 ppm).Chemical shifts are expressed in δ ppm.Mass spectra were taken on a LKB 9000 mass spectrometer. Element analysze was performed on a Perikin-Elmer 240 BCHN analyzer.The purity of the compounds (synthesized) was routinely checked by TLC using Merck Kieselgel-60 (F-254) and benzene,toluene,glacial acetic acid (80:10:10)as mobile phase. The spots were exposed in iodine vapour for visualization.

Material and Methods Experimental Chemistry: Compounds 4,7-Bis-(2chloro-ethylamino)-chromen-2-one 1a, 4,7-Bis-(2-chloroethylamino)-3-nitro-chromen-2-one 2a, (4-

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Behrami Int. J. Res. Chem. Environ. Vol. 4 Issue 3 (58-63) July 2014 H N

Cl

CH2CH2Cl

(Chloro-ethyl)-amine O

Cl

O

ClH2CH2C

C2H5OH, Et3N

N

O

O

H

4,7-Dichloro-chromen-2-one

4,7-Bis-(2-chloro-ethylamino)-chromen-2-one

Figure 1: 4, 7-Bis-(2-chloro-ethylamino)-chromen-2-one (1a) H N

H CH2CH2Cl

N

CH2CH2Cl NO2

HONO2 ClH2CH2C

N

O

O

CH3COOH

ClH2CH2C

H

N

O

O

H

4,7-Bis-(2-chloro-ethylamino)-chromen-2-one

4,7-Bis-(2-chloro-ethylamino)-3-nitro-chromen-2-one

Figure 2: Preparation of 4,7-Bis-(2-chloro-ethylamino)-3-nitro-chromen-2-one (2a)

Cl

Mg

S

H

HS

C

N

Dithiocarbamic acid Mg(NO3)2 Cl

O

O

4,7-Dichloro-chromen-2-one

N

O

O Mg

H

CH3CN,Et3N HONO2

OO N

O

N

O

C S

SH Mg

(4-Dithiocarboxyamino-3-nitro-2-oxo-2H-chromen-7-yl)dithiocarbamic acid (Complex with Mg II)

Figure 3: Preparation of (4-Dithiocarboxyamino-3-nitro-2-oxo-2H-chromen-7-yl)-dithiocarbamic acid (Complex with Mg II) (3a) Preparation of 4,7-Bis-(2-chloro-ethylamino)chromen-2-one (1a): For this synthesis is used 4g 4,7Dichloro-chromen-2-one as substrat in a 100 ml flask mixed 3ml chloro ethylamine , and 6 ml C2H5OH , 1ml Et3N as catalyzer .

C2H5OH gave red crystals product of 70 % yield, meltingpoint, 225 oC. Preparation of (4-Dithiocarboxyamino-3-nitro-2-oxo2H-chromen-7-yl)-dithiocarbamic acid (Complex with Mg II) (3a)

The mixture was refluxed at 90 oC for ca. 120 min. The obtained crystals yellow are filtred and rinsed with ethanol and dried at room temperature. Recrystallization form absolute ethanol gave a yellow product of 80% yield, melting point 217 oC.

In a 100 ml flask were mixed 2.5g of 4,7-Bis-(2chloro-ethylamino)-3-nitro-chromen-2-one , with 6ml CH3CN, 1 ml Et3N as katalyzer, 2ml Dithiocarbomidacid, 2g Mg(NO3)2, 3ml HNO3. The mixture was refkuxed at 100 oC in water bath for ca. 22 h .The flask was placed in an ice bath for 1h until yellow crystalline precipitate was formed.After filtration the product was recrystallized from CH3CN. The recrystallizacion gave a yellow product at 70% yield, meltingpoint, 170 oC.

Preparation of 4, 7-Bis-(2-chloro-ethylamino)-3-nitrochromen-2-one (2a): In a 100 ml flask were mixed 3g 4,7-Bis-(2-chloro-ethylamino)-chromen-2-one with 8ml CH3COOH, 4ml HONO2. The mixture was refluxed at 80 oC for ca. 12h. The obtained red crystals are filtred and dried at room temperature. Recrystallization form

Preparation of [4,7-Bis-(2-chloro-ethylamino)-2Hchromen-3-yl]-phosphonic acid and its metal (III) 59


320 oC in sand bath for ca. 20h .The obtained white crystals are filtred and rinsed with C2H5OH and dried at room temperature.Recrystallization from C2H5OH gave a white product at 70 % yield , melting point 237 oC.

Complex (4a) In a 100 ml flask were mixed 3g 4,7-Bis- (2-chloroethylamino)-2 oxo-2H-Chromen-3-yl]-phosphonic acid, 8ml C2H5OH , 1.5g FeCl2 . The mixture was refluxed at

NHCH2CH2Cl OH P

O

OH O

ClH2CH2CHN

O

[4,7-Bis-(2-chloro-ethylamino)-2-oxo-2H-chromen-3-yl]-phosphonic acid C2H5OH FeCl2 NHCH2CH2Cl P

NHCH2CH2Cl O O

M

O O

ClH2CH2CHN

O

Cl

O

Cl

P O O

O

O NHCH2CH2Cl

[4,7-Bis-(2-chloro-ethylamino)-2H-chromen-3-yl]-phosphonic acid and its metal (III) Complex M=Fe

Figure 4: Preparation of [4,7-Bis-(2-chloro-ethylamino)-2H-chromen-3-yl]-phosphonic acid and its metal (III) Complex (4a) Table 1 characteristics and analytical data of the complexes Comp

Yeld %

MP

M.F.

1a

80

217 oC

C13H14Cl2N2H2

2a

70

225 oC

C13H13Cl2N3H4

3a

70

170 oC

Mg3C17H7N4O6S4

4a

70

237 oC

FeC26H28Cl2N4O1 0P2

Elemental analysis. Calculatet :Found (calc) % C 51.84 51.60 45.11 45.09 26.83 26.80 81.07 80.00

P

S

26 26 8 8

Mg ___

Fe __

___

__

14.81 14.80 ___

__

Cl 23.54 23.51 20.48 20.46 ___

7.4 7.3

37.0 36.98

H 4.69 4.66 3.79 3.75 1.43 1.41 8.0 7.09

Table 2 Antibacterial activity- Staphylococcus aureus Inhibition zone (mm) Compound 2mg/ml 3mg /ml 5mg/ml 1a 10 15 17 2a 10 16 18 3a 11 16 18 4a 12 17 19 Cefalexine 9 9 9 10 μg Streptomycine 20 20 20 10 μg

60

N 9.30 9.29 12.14 12.12 11.38 11.37 14.70 14.68

O 10.62 10.60 18.49 18.47 19.50 19.49 42.00 41.80


Figure 1: Stokes disc: showing inhibition zones of control and test organism against s. aureus organism Table 3: Antibacterial activity – E. Coli Inhibition zone (mm) Compound 2mg/ml 3mg /ml 5mg/ml 1a 10 12 14 2a 11 12 15 3a 12 13 16 4a 11 13 17 Cephalexine 9 9 9 10 μg Streptomycine 23 23 23 10 μg

Figure 2: Stokes disc: showing inhibition zones of control and test organism against E. coli organism Table 4: Antibacterial activity – Bacillus cereus Inhibition zone (mm) Compound 2mg/ml 3mg /ml 5mg/ml 1a 12 16 23 2a 10 15 21 3a 13 19 24 4a 11 18 21 Cephalexine 9 9 9 10 μg Streptomycine 23 23 23 10 μg

Figure 3: Stokes disc: showing inhibition zones of control and test organism against B. cereus organism 61


For (3a) IR bands (KBr,cm -1) 2980cm-1(C-H, stretch.), 3200cm-1(C-NH,stretch.), 2565cm-1 (S-H) ,1720cm-1(C=O,tretch.),1600cm-1(C=C,stretch),1280cm1 (N-O),1523cm-1 (N=O2) ,1050cm-1(C-O),1240 cm-1 -1 (C=S) ,750cm (C-S ) 740cm-1 (C-H) , 1030cm-1 (CS),650cm-1 (Me-O) , 600cm-1 (Me –S)

Antibacterial activity: The purified synthesized compounds (1a,2a,3a,4a) was subjected to test in vitro its antibacterial activity against three bacterial cultures , Staphylococcus aureus, E. Coli and B. cereus. Antibacterial activity of compounds was investigated applying the Kirby-Bayer method or disc method (d=5.5 mm max. capacity 10 μg)

1

H NMR (DMSO-d6) δppm 2.0ppm (H,NH) , 4.0ppm (H,NH), 6.4ppm s(H,SH), 6.40ppm,6.42,7,38 ppm (3H aromatic)

Results and Discussion By reacting equimolar amounts of 4,7-Dichlorochromen-2-one and correspomding reagents (according scheme 1) under reflux reaction condictions product 1a is synthesized in 80 % yield.

13

CNMR (DMSO) δppm 126.9,123.8,122,0,118.1ppm (4 C aromatic) 162ppm (C,C=O)194.7ppm (C,C=S), 196ppm (C,C=S) , 166ppm (C,C-N) 139.0ppm (C,C-N), 151.1ppm (C-O), 88.9ppm (C,C=C)

By reacting equimolar amounts of 4,7-Bis-(2chloro-ethylamino)-chromen-2-one and corresponding reagents (according scheme 2) under reflux reaction condictions product 2a is synthesized in 70 % yield.

For (4a) IR bands (KBr,cm -1) 3000cm-1 (C-H stretch.), 1720 cm-1 (C=O) , 1600 (C=C stretch.) ,1350cm-1 (C-P), 1110cm-1 (P=O) ,1250 cm-1 (N-H), 750 cm-1 (C-H bend.) 600 cm-1 (C-Cl stretch.) 600cm-1 (Me –O) .

By reacting equimolar amounts of 4,7-Bis-(2chloro-ethylamino)-3-nitro-chromen-2-one and corresponding reagents (according scheme 3) under reflux reaction condictions product 3a is synthesized in 70% yield.

1

HNMR (DMSO-d6) δppm, 2.0ppm (H,NH), 2.93ppm t(4H , CH2CH2Cl) , 3,34ppm t(4H, CH2CH2Cl),4.0ppm s(H,NH) , 5.93,5.95,6.95,ppm (3H, aromatic) 13

CNMR (DMSO) δppm, 46.5ppm (C,C-L), 47.3ppm (C,C-Cl) 49.7ppm (C,C-N), 54.4ppm (C,C-N) , 97.8,104.5.108.9,128.0ppm (4C , aromatic) 103ppm (CP) , 151.1ppm (C,C-O) , 162.0 ppm (C,C=O)

By reacting equimolar amounts 4,7-Bis- (2chloro-ethylamino)-2 oxo-2H-Chromen-3-yl]-phosphonic acid 4a is synthesized in 70% yield. The structure of 4,7-Dichloro-chromen-2-one derivative (1a,2a,3a,4a)were determined fromtheir IR, 1H NMR, 13C NMR spectar and their melting points as follows.

Conclusion From the results the following conclusion were drawn: The study provides the first evidence that compounds (1a,2a,3a,4a,) obviously inhibit the growth of S. auerus , E. coli and B. cereus.

For (1a), IR bands (KBr, cm-1) 3000cm-1 (C-H stretch.), 1720 cm-1 (C=O), 1600 (C=C stretch.), 1250 cm-1 (N-H), 750 cm-1 (C-H bend.) 600 cm-1 (C-Cl stretch.)

The compounds (1a,2a,3a,4a) compared with the antibacterial activity of Streptomycine in S. aureus, E. coli and B. cereus. This study provided the first evidence that these compounds 1a,2a,3a,4a showed a significant antibacterial effect against S. aureus, E. coli and B. Cereus. The chemical structures of synthesize compounds were determined according to extensive NMR experiments and published data.

1

H NMR (DMSO-d6) δppm , 2.0ppm (H,NH), 2.93ppm d(2H,CH2NH),3.64ppm t(4H,CH2CH2Cl)3.34ppm d(3H,CH2NH),3.61ppmt(4H,CH2CH2Cl)5.19ppm,6.37pp m,6.39ppm,7.41ppm q(4H aromatic) 13

C NMR (DMSO) δppm , 47.3ppm(C,C-Cl), 49.4ppm (C,C-N), 54.4ppm (C,C-N), 46.5ppm (C,C-Cl), 109.0,105.1,127,116.2,88.9ppm (5C aromatic)166.6 ppm (C,C-N), 162.0ppm (C,C=O)

Acknowledgement The authors thank Prof. Branko Stanovnik, University of Ljubljana and its laboratory staff for 1H NMR spectrum and elemental analyses.

For (2a) IR bands (KBr,cm -1) 3000cm-1 (C-H stretch.), 1720 cm-1 (C=O) , 1600 (C=C stretch.) ,1523cm-1 (N=O2),1250 cm-1 (N-H), 852 cm-1 (C-N), 750 cm-1 (C-H bend.) 600 cm-1 (C-Cl stretch.)

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1

H NMR (DMSO-d6) δppm 2.0ppm (H,NH), 2.93ppm d(2H,CH2NH), 3.64ppm t(4H,CH2CH2Cl) 3.34ppm d(3H,CH2NH), 3.61ppm t (4H ,CH2CH2Cl), 4.0ppm s(H,NH) 6.37ppm, 6.39ppm, 7.41ppm q(3H aromatic) 13

C NMR (DMSO) δppm 47.3ppm(C,C-Cl), 48.4ppm (C,C-N), 46.5 (C,CH2Cl), 54.4ppm (C,CH2NH), 46.5ppm (C,C-Cl), 108ppm (C-NO2), 109.0,127,116.2ppm (3C aromatic)165.7 ppm (C,C-N), 162.0ppm (C,C=O)

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