studies on 4-hydroxy coumarin -...

Studies on 4-Hydroxy Coumarin

17

2.1 Introduction

Coumarin is a freedom gallows among Heterocycles and is known to possess a wide

range of biological activities including antibiotic, anti-malarial, antifungal, anti-viral,

and cytotoxic1-8

. In finicky, the 4-Hydroxycoumarins and its derivatives (3-alkylated)

have stir up a great deal of interest due to their utility as ‘anticoagulant rodenticides as

well as antithrombotic agents’ such as brodifacoum, difethialone, bromadiolone,

coumatetralone, and flocoumafen9 and also as nonpeptide human immunodeficiency

virus (HIV) protease inhibitors10

. The C3 or O-alkylation of 4-Hydroxycoumarin is

undoubtedly one of the most important and challenging reactions in synthetic

chemistry due to its pharmaceutical utility as mentioned above and also can be

diversified to synthesize 3,4-substitued compounds11-14

. In continuation of our interest

in developing novel synthetic methodologies, particularly carbon-carbon, carbon-

heteroatom bond formations to synthesize pharmaceutically relevant heterocycles15

,

we have very recently reported SnO2-catalyzed C3-alkylation of 4-Hydroxycoumarin

with secondary benzyl alcohols and O-alkylation with O-acetyl compounds16

.

O

OH

O

CF3

O

flocoumafen

Moreover, the 4-Hydroxy-3-nitrocoumarins can easily converted to 4-Chloro-3-

nitrocoumarins. Wide range of compounds were synthesized from 4-Chloro-3-

nitrocoumarins have been used for antihistamine17

, neurotropic18

and antimicrobial

agents19

. On the other hand 4-chloro-3-nitrocoumarins may be utilized as a key

intermediate for many other heterocyclic moieties20,21

.


18

2.2 Pharmacological-significance of 4-Hydroxycoumarin

Numerous biological activities have been associated with Coumarins and its

analogues. Among them, antimicrobial, antiviral, anticancer, enzyme inhibition, anti-

inflammatory, antioxidant, anticoagulant and effect on central nervous system are

most prominent. Coumarin nucleus possesses diversified biological activities that can

be briefly summarized as under

Antimicrobial and Molluscicidal 8,22-31

Antiviral 32-36

Anticancer14,37,38

As Enzyme Inhibition 39,40

Antioxidant 41

Anti-inflammatory42,43

Anticoagulant and Cardiovascular44,45

Effect on Central Nervous System 46,47

4-Hydroxycoumarin is a versatile gibbet and is being consistently used as a building

block in organic chemistry as well as in heterocyclic chemistry for the synthesis of

different heterocycles. The synthetic versatility of 4-Hydroxycoumarin has led to the

extensive use of this compound in organic synthesis. 4-Hydroxycoumarin shows

diversified chemical reactivity.

2.2.1 Anti-HIV Activity

In current studies a lot of structurally different Coumarins are found to display potent

anti HIV activity. Various synthetic coumarins seem to indicate that some of them

serve as a potent non-nucleoside reverse transcriptase inhibitors (NNRTIs). Some

derivatives of (A) posses HIV integrase and protease48-63

4-Hydroxycoumarins are typical phenolic compounds and therefore act as a potent

metal chelators and free radical scavengers. They are powerful chain breaking agents.

The report of tetrameric 4-Hydroxycoumarin derived inhibitor provided a lead

example which does not contain catechol moiety. Tetrameric 4-Hydroxycoumarin

derived inhibitors represent a large highly complex yet symmetrical molecule it was

the purpose of the study of this compound to determine the critical component of


19

tetrameric 4-Hydroxycoumarin derived inhibitors and if possible to simplify its

structure while maintaining potency.

These compounds are used as primers. After addition of DDT unit to the 3-terminal

end of the 4-Hydroxycoumarin some primers became very effective inhibitors of RT.

2.2.2 Anticoagulant Activity

Coumarin derivatives possessing a 4-Hydroxy group with a carbon at 3 position (I) of

coumarin based structure possess anticoagulant activity and are referred to as

Hydroxycoumarins which are not present in Coumarin itself.

O O

OH

R

Indicates that groups are important for activity

Superwarfarins have a much longer duration of action than traditional warfarins. After

intentional over ingestion of superwarfarins, patients may be anticoagulated for weeks

to months.64-79

2.2.3 Antibiotic Activity

Compounds like Novobiocin, Coumermycin-A and clorobiocin posses 4-Hydroxy-

coumarin nucleus which inhibit the activity of DNA gyrase acting as antibiotics,80-89

Streptomyces species possess 3-Amino-4-hydroxycoumarin moiety as base.

Moreover, Novobiocin an antibiotic which is primarily active against gram positive

organism was found to be 4-Hydroxycoumarin derivative. The antibacterial spectrum

of this antibiotic corresponds generally to that of penicillin and erythromycin.90-107


20

O

OH

NH

O

O

O

OH

O

OHO

O

H2NO

Coumermycin-A acting as an antibiotic was isolated from Streptomyces rishiriensis

has a two 4-Hydroxycoumarin units in its structure.108-111

O

OHHN

O

O OO

HOO

O

O

NH

HN

O

HO

NH

O OO

O

OHO

O

O

2.3 Synthesis aspect

2.3.1 Literature review for 4-Hydroxycoumarin.

Anschutz' first synthesized 4-Hydroxycoumarin by treating acetylsalicylyl chloride

with the sodium derivative of Malonic ester to form 3-Carboethoxy-4-

hydroxycoumarin on treatment with alkali this compound was decarboxylated to form

4-Hydroxycoumarin112

.


21

Cl

O

OCOCH3

CHCOOEt

COOEt

aq. KOHO O

COOC2H5

OH

Na

O O

OH

+

Zeigler and coworker have cyclised malonic acid diphenyl ester in presence of AlCl3

using Friedal Craft’s alkylation to give 4-Hydroxycoumarin in 85% yield113

.

PhO OPh

O OAlCl3

180-185 oC

O O

OH85%Diphenyl Malonate

Shah et al have evolved a simple process for the synthesis of 4-Hydroxycoumarins in

which a phenol was treated with a malonic acid in the presence of anhydrous Zinc-

-chloride and Phosphorus oxychloride at temperatures 60-75 oC

114.

HO OH

O O O O

OH

OH

Malonic AcidPhenol

ZnCl2

POCl3

64%

+

Selenium catalysed carbonylation of 4-Hydroxyacetophenone in THF containing

PhNO2 under Carbon monoxide atmosphere at 90 oC for 30 hours giving 68%

yield115

.

O O

OH

OHSe , THF

PhNO2

CO

O

+


22

Intramolecular Claisen Condensation of methyl acetylsalicylate with NaOMe in liquid

paraffin at 160-260 oC for 5 hours gave 20% of 4-Hydroxycoumarin

116.

OAc

OMe

O

O O

OH

NaOMe

HCl/H2O

methyl 2-acetoxybenzoate20%

Substituted 4-Hydroxycoumarin was synthesized via new Baker-Venkatraman

rearrangement117

.

AcClBuLi, THF

ZnCl2

O O

OH

OCONEt2OCONEt2

Ac

1. NaH, THF

2. TFA, PhMe+

One-pot synthesis of 4-Hydroxycoumarin by reacting 2-Hydroxyacetophenone with

acylating agents in the presence of base was reported118

.

OH O O

OH

Et2CO3

NaH, PhMe

O

4,7-Dihydroxycoumarin was synthesized from resorcinol and Malonic acid in

Borontrifluoride-diethyl etherate complex (BF3•Et2O) at 90 oC

119.

HO OH

OO O O

OH

HOOHHO BF3 Et2O

90 oC+


23

A simple and efficient procedure has been developed for 4-Hydroxycoumarin in

which the Meldrum’s acid reacts with phenol in the presence of Eaton’s reagent as

cyclization reagent120

.

O

O

OHO

O

O O

OH

Eaton's reagent+

2.3.2 Synthesis aspect for 3-Morphonyle Methyl Derivatives of

4-Hydroxycoumarin

On extending one step for 4-Hydroxycoumarin by using the Formaldehyde and

secondary amine like Morpholine, it is known as Mannich type reaction was reported

by Stanciu et al121

.

One step synthesis of the compound by scheme 10 also reported as antimicrobial

agent has been reported by Badran M. M. et al122

.

O O

OH

O O

OH

N

ONH

O

H O

Ghose and et. al have synthesised the Benzylaminocoumarine by using the various

derivative of Benzaldehyde with Nano crystals of the Zinc oxide (ZnO) and water as

solvent123

.


24

O O

OH

O O

OH

N

ONH

O

CHO

Nano ZnOR

R

Where R= -CH3, -NO2, -OCH3, -X etc

2.4 Aim of current work

The Aim of present work to prepare of 3rd

substituted 4-Hydroxycoumarin

derivatives by using 4-Hydroxycoumarin, 2° amine and formaldehyde, such as

mannich reaction, to find out optimum reaction condition and characterization

of products.

2.5 Reaction schemes

2.5.1 Step-I: Synthesis of 4-Hydroxycoumarin-(4-Hydroxy-2H-chromen-2-

one).

OH

OH

O

OH

OO O

OH

POCl3

Anhy. ZnCl2

R

R

2.5.2 Step-II: 4-Hydroxy-3-(morpholinomethyl)-2H-chromen-2-one.

O O

OH

R

O O

OH

RN

ONH

O

H O


25

2.6 Plausible Reaction Mechanism

OEt

O

OH

O

Cl

O

Cl

O

Cl

O

Cl

O

OH

R OH OCl

HH

OH

O

OCl

O

O

R

RR

Step I

POCl3

O

OToutomarization

O O

OH

R

O NH HCHO

-H2O

O NOO

OH

OO

OHN

OStep II

R

R

2.7 Experimental

2.7.1 Materials and methods

Melting points were determined in open capillary tubes and are uncorrected.

Formation of the products was routinely checked by TLC on silica gel-G plates of 0.5

mm thickness and spots were located by iodine and UV. IR spectra were recorded on

Shimadzu FT-IR-8400 instrument using KBr method. Mass spectra were recorded

on Shimadzu GC-MS-QP-2010 model using Direct Injection Probe technique and

Turbo Spray model using Chemical ionization technique. 1H NMR was determined

in CDCl3/DMSO solution on a Bruker Ac 400 MHz spectrometer.


26

2.7.2 General procedure for the Synthesis of

4- Hydroxycoumarins.

Step-I

A mixture of Phenols (0.5 mol), Malonic acid (0.5 mol), anhydrous Zinc chloride (1.5

mol) and Phosphorus oxychloride (1.5 mol) was placed in a 1 L round-bottom flask and

heated at 60-65 oC for 24 hrs., cooled and decomposed with crushed ice and allowed to

stand overnight. The resulting crude solid was collected by filtration. The solid was then

extracted with 10% sodium bicarbonate with stirring. The resultant mixture was filtered

and filtrate was acidified with dilute hydrochloric acid to obtain solid, it was collected

by filtration. Similarly other members have been prepared.

2.7.3 General procedure for the Synthesis of 4-Hydroxy-3-

(morpholinomethyl) -2H-chromen-2-one

Step-II

In a 50 ml single neck round bottom flask 15 ml IPA was charged and then, to this

5,8-Dimethyl-4-hydroxycoumarin (0.0026 mol), secondary amine (0.0026 mol) and

40% aq. solution of formaldehyde (0.00312 mol) were added. Add 1 ml conc. HCl to

the reaction mass and refluxed for 8-10 hrs. Reaction mass wash, cooled to room

temperature, poured on to crushed ice and neutralized with aq.NaHCO3 solution.

Obtained solid was filtered and wash with methanol to give pure product.

2.8 Physical data

Physical data of 4-Hydroxy-3-(substituted)-2H-chromen-2-one

O O

OHR R1


27

Table 1

Sr.

No. Code R R1 Molecular

Formula

M.W.

Gm/mole

M.P.

°C Rf

1 KSP-101 5,8

diMe

N

C17H21NO3 287 240-242 0.46

2 KSP-102 5,8

diMe

N N CH3

C17H22N2O3 302 438-240 0.41

3 KSP-103 5,8

diMe

N N

H3C

C18H24N2O3

316 240-242 0.40

4 KSP-104 5,8

diMe

O N

C16H19NO4

289 248-250 0.42

5 KSP-105 5,8

diMe

N

CH3

C18H23NO3 301 216-218 0.48

6 KSP-106 5,8

diMe

N

C16H15NO3

269 252-254 0.44

7 KSP-107 5,8

diMe

NHN

C16H20N2O3

288 236-238 0.34


28

Sr.

No. Code

R R1 Molecular

Formula

M.W.

gm/mole M.P.(°C) Rf

8 KSP-

108 6Me N

C16H19NO3 273 182-184 0.45

9 KSP-

109 6Me N N CH3

C16H20N2O3 288 202-204 0.43

10 KSP-

110 6Me NN

C21H22N2O3 350 218-220 0.46

11 KSP-

111 6Me

NN

C22H24N2O3 364 206-208 0.42

12 KSP-

112 6Me NN

H3C

C17H22N2O3 302 192-194 0.44

13 KSP-

113 6Me O N

C15H17NO4 275 198-200 0.38

14 KSP-

114 6Me NHN

C15H18N2O3 274 214-218 0.40

Rf value was determined using solvent system = Ethyl Acetate: Hexane (5:5)

2.9 Spectral discussion

2.9.1 IR spectra

IR spectra of the synthesized compounds were recorded on Shimadzu FT-IR 8400

using KBr pallet method. Various functional groups present were identified by

characteristic frequency obtained for them.

The characteristic bands of Hydroxy groups were obtained for stretching at 3650-

3400 cm-1

and those for bending were obtained at 1250-1050 cm-1

. It gives aromatic

C-H stretching frequencies between 3200-3000 cm-1

and bending vibration near 1500-

1300 cm-1

respectively. C-H stretching frequencies for methyl and methylene group

were obtained near 2950 cm-1

to 2850 cm-1

. The Characteristic frequency of C=C

(Vinyl) stretching showed near 1000-900 cm-1

.


29

2.9.2 Mass spectra

Mass spectra of the synthesized compounds were recorded on Shimadzu GC-MS-

QP-2010 using Direct Injection Probe technique and Turbo Spray using Chemical

ionization technique. The molecular ion peak (M+1 and M-1) was found in

agreement with molecular weight of the respective compound. Characteristic M+2

ion

peaks with one-third intensity of molecular ion peak were observed in case of

compounds having chlorine atom. Fragmentation pattern can be observed to be

particular for these compounds and the characteristic peaks obtained for each

compound.

2.9.3 1HNMR spectra

1HNMR spectra of the synthesized compounds were recorded on Bruker Avance II

400 spectrometer by making a solution of samples in CDCl3 and DMSO-d6 solvent

using tetramethylsilane (TMS) as the internal standard unless otherwise mentioned.

Numbers of protons and carbons identified from NMR spectrum and their chemical

shift (δ ppm) were in the agreement of the structure of the molecule. J values were

calculated to identify o, m and p coupling and it gives geometrical isomer. In some

cases, aromatic protons were obtained as multiplet. 1H spectral interpretation can be

discussed as under.

2.10 Analytical data

4-Hydroxy-2H-chromen-2-one

O O

OH

Yield: 60%; mp 252-254 ºC; IR (cm-1

): 3473 and 3377 (-O-H stretching of hydroxy

group), 3064 (-C-H stretching of aromatic ring), 1701 (-C=O stretching of coumarin),

1554, 1502 and 1442 (-C=C- stretching of aromatic ring), 1398 (-C-H asymmetrical

deformation of -CH3 group), 1317 (-C-H symmetrical deformation of -CH3 group),


30

1050 (-C-O-C- stretching); MS: m/z 162; Anal. Calcd. for C9H6O3: C, 66.67; H, 3.73;

O, 29.60; Found: C, 66.52; H, 3.42; O, 29.54%.

2.10.1 4-Hydroxy-5,8-dimethyl-3-((piperidin-1-yl)methyl)-2H-

chromen-2-one (KSP-101)

OO

OHN

Yield: 62%; mp 218-222 ºC; IR (cm-1


group), 3060 (-C-H stretching of aromatic ring), 2970 (-C-H asymmetrical stretching

of -CH3 group), 2831 (-C-H symmetrical stretching of -CH3 group), 1689 (-C=O

stretching of coumarin), 1523, 1500 and 1422 (-C=C stretching of aromatic ring),

1387 (-C-H asymmetrical deformation of -CH3 group), 1320 (-C-H symmetrical

deformation of -CH3 group), 1050 (-C-O-C stretching); MS: m/z 287; Anal. Calcd. for

C17H21NO3: C, 71.06; H, 7.37; N, 4.87; O, 16.70; Found: C, 71.01; H, 7.27; N, 4.67; O,

16.60%.

2.10.2 4-Hydroxy-5,8-dimethyl-3-((4-methylpiperazin-1-

yl)methyl)2H-chromen- 2-one (KSP-102)

OO

OHN

N

Yield: 67%; mp 210-215 ºC; IR (cm-1



of -CH3 group), 2807 (-C-H symmetrical stretching of -CH3 group), 1700 (C=O


31



deformation of -CH3 group), 1050 (-C-O-C stretching); MS: m/z 302; Anal. Calcd. for

C17H22N2O3: C, 67.53; H, 7.33; N, 9.26; O, 15.87; Found: C, 67.03; H, 7.03; N, 9.06; O,

15.27%.

2.10.3 3-((4-Ethylpiperazin-1-yl)methyl)-4-hydroxy-5,8-dimethyl-

2H-chromen-2-one (KSP-103)

OO

OHN

N

Yield: 67%; mp 240-242 ºC; IR (cm-1






deformation of -CH3 group),1050 (-C-O-C stretching); 1HNMR (DMSO-d6) δ ppm:

0.96 (t, 3H), 1.97 (s, 3H), 2.14 (s, 3H), 2.36 (m, 4H), 2.43 (t, 2H), 3.02 (t, 2H), 3.22

(t, 2H), 4.06 (s, 2H), 6.75(d, 1H) and 7.08 (d, 1H), 10.20 (s, 1H) MS: m/z 316; Anal.

Calcd. For C18H24N2O3: C, 68.33; H, 7.65; N, 8.85; O, 15.17; Found: C, 68.13; H, 7.55;

N, 8.80; O, 15.10%.


32

2.10.4 4-Hydroxy-5,8-dimethyl-3-(morpholinomethyl)-2H-


OO

OHN

O

Yield: 72%; mp 231-235 ºC; IR (cm-1






deformation of -CH3 group), 1050 (-C-O-C stretching); 1HNMR (DMSO-d6) δ ppm:

1.97 (s, 3H), 2.11 (s, 3H), 2.21 (t, 2H), 2.88 (t, 2H), 3.79 (t, 4H), 4.02 (s, 2H),

6.80(d, 1H)and 7.11 (d, 1H), 10.00 (s, 1H) MS: m/z 289; Anal. Calcd. C16H19NO4: C,

66.42; H, 6.62; N, 4.84; O, 22.12; Found: C, 66.32; H, 6.12; N, 4.24; O, 22.00%.

2.10.5 4-Hydroxy-5,8-dimethyl-3-((2-methylpiperidin-1-yl)methyl)-


OO

OHN

Yield: 70%; mp 230-235 ºC; IR (cm-1







33

deformation of -CH3 group), 1050 (-C-O-C stretching); MS: m/z 301; Anal. Calcd.


15.90%.

2.10.6 4-Hydroxy-5,8-dimethyl-3-((2-methylpiperidin-1-yl)methyl)-


OO

OHN

Yield: 64%; mp 232-235 ºC; IR (cm-1








17.72%.

2.10.7 4-Hydroxy-5,8-dimethyl-3-((piperazin-1-yl)methyl)-2H-


OO

OHN

HN

Yield:55%; mp 250-255 ºC; IR (cm-1






34




16.55%.

2.10.8 4-Hydroxy-6-methyl-3-((piperidin-1-yl)methyl)-2H-


OO

OHN

Yield: 57%; mp 210-220 ºC; IR (cm-1



of -CH3 group), 2861(-C-H symmetrical stretching of -CH3 group), 1710 (C=O





17.52%.

2.10.9 4-Hydroxy-6-methyl-3-((4-methylpiperazin-1-yl)methyl)-2H-


OO

OHN

N

Yield: 59%; mp 215-225 ºC; IR (cm-1




35






16.64%.

2.10.10 4-Hydroxy-6-methyl-3-((4-phenylpiperazin-1-yl)methyl)-2H-


OO

OHN

N

Yield: 51%; mp 203-207 ºC; IR (cm-1








13.61%.


36

2.10.11 3-((4-Benzylpiperazin-1-yl)methyl)-4-hydroxy-6-methyl-2H-

chromen-2- one (KSP-111)

OO

OHN

N

Yield: 54%; mp 203-207 ºC; IR (cm-1








13.61%.

2.10.12 3-((4-Ethylpiperazin-1-yl)methyl)-4-hydroxy-6-methyl-2H-


OO

OHN

N

Yield: 50%; mp 220-223 ºC; IR (cm-1








37


15.67%.

2.10.13 4-Hydroxy-6-methyl-3-(morpholinomethyl)-2H-chromen-2-

one (KSP-113)

OO

OHN

O

Yield: 52%; mp 200-205 ºC; IR (cm-1








23.05%.

2.10.14 4-Hydroxy-6-methyl-3-((piperazin-1-yl)methyl)-2H-

chromen-2-one (KSP -114)

OO

OHN

HN

Yield: 70%; mp 224-230 ºC; IR (cm-1






38



C15H18N2O3: C, 65.68; H, 6.61; N, 10.21; O, 17.50; Found: C, 65.38; H, 6.41; N, 10.01;

O, 17.40%.

2.11 Results and discussion

Various 3-substituted 4-Hydroxycoumarin derivatives were prepared by

reaction of different secondary Amines & the formaldehyde. The compounds

prepared in this chapter possess Chromene nucleus and has substitution at C3

position.

2.12 Spectra of some compounds

2.12.1 Mass spectra

Mass spectra of 3-((4-Ethylpiperazin-1-yl)methyl)-4-hydroxy-5,8-dimethyl-2H-



39

Mass spectra of 4-Hydroxy-5,8-dimethyl-3-(morpholinomethyl)-2H-chromen-2-

one (KSP-104)

2.12.2 IR spectra

IR spectra of 3-((4-Ethylpiperazin-1-yl)methyl)-4-hydroxy-5,8-dimethyl-2H-



40

IR spectra of 4-Hydroxy-5,8-dimethyl-3-(morpholinomethyl)-2H-chromen-2-one

(KSP-104)

2.12.3 1HNMR spectra

1HNMR spectra of 3-((4-Ethylpiperazin-1-yl)methyl)-4-hydroxy-5,8-dimethyl-



41

1HNMR spectra of 4-Hydroxy-5,8-dimethyl-3-(morpholinomethyl)-2H-chromen-

2-one (KSP-104)


42

2.13 Biological evaluation

2.13.1 Antimicrobial evaluation

All of the synthesized compounds (KSP- 101 to 114) were tested for their

antibacterial and antifungal activity (MIC) in vitro by broth dilution method 124-126

with two Gram-positive bacteria Staphylococcus aureus MTCC-96, Streptococcus

pyogenes MTCC 443, two Gram-negative bacteria Escherichia coli MTCC 442,

Pseudomonas aeruginosa MTCC 441 and three fungal strains Candida albicans

MTCC 227, Aspergillus Niger MTCC 282, Aspergillus clavatus MTCC 1323 taking

Gentamycin, Ampicillin, Chloramphenicol, Ciprofloxacin, Norfloxacin, Nystatin and

Greseofulvin as standard drugs. The standard strains were procured from the

Microbial Type Culture Collection (MTCC), Institute of Microbial Technology,

Chandigarh, India.

The minimal inhibitory concentration (MIC) values for all the newly synthesized

compounds, defined as the lowest concentration of the compound preventing the

visible growth, were determined by using micro dilution broth method according to

NCCLS standards 124

.

Minimal Inhibition Concentration [MIC]:-

The main advantage of the ‘Broth Dilution Method’ for MIC determination lies in the

fact that it can readily be converted to determine the MIC as well.

Serial dilutions were prepared in primary and secondary screening.

The control tube containing no antibiotic is immediately subcultured (before

inoculation) by spreading a loopful evenly over a quarter of plate of medium

suitable for the growth of the test organism and put for incubation at 37 0

C

overnight.

The MIC of the control organism is read to check the accuracy of the drug

concentrations.

The lowest concentration inhibiting growth of the organism is recorded as the

MIC.

The amount of growth from the control tube before incubation (which

represents the original inoculums) is compared.


43

Methods used for primary and secondary screening: -

Each synthesized drug was diluted obtaining 2000 μg mL-1

concentration, as a stock

solution. Inoculum size for test strain was adjusted to 108

cfu (colony forming unit)

per milliliter by comparing the turbidity.

Primary screen: - In primary screening 1000 μg mL-1

, 500 μg mL-1

and 250 μg mL-1

concentrations of the synthesized drugs were taken. The active synthesized drugs

found in this primary screening were further tested in a second set of dilution against

all microorganisms.

Secondary screen: - The drugs found active in primary screening were similarly

diluted to obtain 200 μg mL-1

, 100 μg mL-1

, 50 μg mL-1

, 25 μg mL-1

, 12.5 μg mL-1

,

and 6.250 μg mL-1

concentrations.

Reading Result: - The highest dilution showing at least 99 % inhibition zone is taken

as MIC. The result of this is much affected by the size of the inoculums. The test

mixture should contain 108

organism/ml.

The results obtained from antimicrobial susceptibility testing are depicted in Table 1.


44

Table-1:- In vitro Antimicrobial Screening Results for KSP-101 to 114

Code Minimal inhibition concentration (µg mL

-1 )

Gram-positive Gram-negative Fungal species

Staphylo

-coccus

aureus

Streptoco

ccus

pyogenes

Escheric

-hia coli

Pseudomon

-as

aeruginosa

Candida

albicans

Asperg

illus

niger

Aspergil

-lus

clavatus

KSP-101 500 500 500 500 250 1000 500

KSP-102 500 1000 1000 1000 >1000 >1000 >1000

KSP-103 100 100 250 200 1000 500 500

KSP-104 1000 500 1000 1000 1000 500 1000

KSP-105 200 100 100 200 250 1000 1000

KSP-106 1000 1000 500 500 250 1000 1000

KSP-107 500 500 250 250 250 1000 1000

KSP-108 100 100 200 250 1000 500 1000

KSP-109 62.5 1000 200 1000 500 >1000 1000

KSP-110 150 250 100 150 500 500 1000

KSP-111 1000 500 62.5 62.5 >1000 >1000 >1000

KSP-112 200 200 100 100 >1000 1000 500

KSP-113 500 1000 500 500 500 >1000 >1000

KSP-114 150 250 100 150 500 500 500

Gentamycin 0.25 0.5 0.05 1 - - -

Ampicillin 250 100 100 100 - - -

Chloramphenicol 50 50 50 50 - - -

Iprofloxacin 50 50 25 25 - - -

Norfloxacin 10 10 10 10 - - -

Nystatin - - - - 100 100 100

Greseofulvin - - - - 500 100 100


45

2.14 References

1. Murray R.D.H., Mendez J., Brown S.A., Wiley, New York., NY., 1982.

2. Naser-Hijazi B., Stolze B., Zanker K.S., Springer, Berlin., 1994.

3. Spino C., Dodier M., Sotheeswaran S., Bioorg Med Chem Lett. 1998., 8.,

3475-3478.

4. Murakami A., Gao G., Omura M., Yano M., Ito C., Furukawa H., Bioorg Med

Chem Lett. 2000, 10, 59-62.

5. Xia Y., Yang., Z.Y., Xia P., Hackl T., Hamel E., J Med Chem. 2001, 44, 3932-

3936.

6. Itoigawa M., Ito C., Tan H.T.-W., Kuchide M., Tokuda H., Nishino H.,

Cancer Lett. 2001, 169,15-19.

7. Yamaguchi T., Fukuda T., Ishibashi F., Iwao M., Tetrahedron Lett., 2006., 47,

3755-3757.

8. Yamamoto Y., Kurazono M., Bioorg Med Chem Lett. 2007., 17,1626-1628.

9. Manolov I., Danchev N.D., Archiv der Pharmzie. 2003., 336, 83-94.

10. Ivezic Z., Trkovnik M., PCT Int Appl., 2003., 41 pp. WO 2003,029237.

11. Estevez-Braun A., Gonzalez A.G., Nat Prod Rep., 1997., 14,465-475.

12. Clerici A., Porta O., Synthesis., 1993, 99-102.

13. Mizuno T., Nishiguchi I., Hirashima T., Ogawa A., Kambe N., Sonoda N.,

Synthesis., 1988., 257-258.

14. Wang S., Milne G.W.A., Yan X., Posey I.J., Nicklaus M.C., Graham L., J

Med Chem., 1996., 39, 2047-2054.

15. Narayana K.R., Varala R., Zubaidha P.K., J Org Chem., 2012., 2, 3A.

16. (a) Narayana V.R., Zubaidha P.K., Ravi V., Tetrahedron Lett., 2012 . (b)

Figueira V.B.C., Esqué A.G., Varala R., González-Bello C., Prabhakar S.,

Lobo A.M., Tetrahedron Lett. 2010., 51,2029-2031. (c) Varala R., Ramu E.,

Adapa. S.R., Monatsh. Chemie., 2008.,139,1369-1372. (d) Enugala R.,

Nuvvula S., Kotra V., Varala R., Adapa S.R., 2008., 75, 2523-2533. (e) Ramu

E., Varala R., Sreelatha N., Adapa S.R., Tetrahedron Lett., 2007.,4,7184-7190.

17. Buckle D.R., Outred D.J., Ross J.W., Smith H., Smith R.J., Spicer B.A. J.

Med. Chem., 1979., 22., 158.


46

18. Savel’ev V.L., Samsonova O.L., Lezina V.P., Troitskaya V.S., Kozlovskii I.

I., Beshimov A., Kozlovskaya M.M., Pharmaceutical Chemistry Journal.,

2003., 37., 476.

19. Biljana D., Vidoslav D., Niko R., Rastko V., Radosav P. Chemical Papers.,

2010., 64., 354.

20. Zeeshan M., Iaroshenko V.O., Dudkin S., Volochnyuk D.M., Langer P.,

Tetrahedron Lett., 2010., 51., 3897.

21. Savel'ev V.L., Artamonova O.S., Makarov A.V., Troitskaya V.S., Antonova

A.V., Vinokurov V.G. Khim. Geterotsikl. Soedin., 1989., 25.,1208.

22. Oduszek B and Uher M., Synth. Commun., 2000., 30., 1749.

23. Nishizono N., Oda K., Ohno K., Minami M and Machida M., Heterocycles.,

2001., 55., 1897.

24. Ito K., Higuchi Y., Tame C and Hariya J., Heterocycles., 1993., 35., 937.

25. Hagen V., Frings S., Wiesner S and Kaupp B., J. Chem. Bio. Chem., 2003., 4.,

434.

26. Rao L. and Mukerjee K., Ind. J. Chem., 1994., 55., 14777.

27. Rahman M and Gray I., Phytochemistry., 2002., 59., 73.

28. Schinkovitz A., Gibbons S., Stavri M., Cocksedge J and Bucar F., Plant Med.,

2003., 69., 369.

29. Chowdhury R., Hasan., M. and Rashid A., Fitoterapia., 2003., 74., 155.

30. Kawase M., Tanaka T., Sohara Y., Tani S. and Sakagami H., In vivo., 2003.,

17., 509.

31. Zaha A. and Hazem A., New Microbio., 2002., 25., 213.

32. Gleye C., Lewin G., Laurens A., Jullian C and Loiseau C., J. Nat. Prod.,

2003., 66., 323.

33. Clercq E. De., Med. Res. Rev., 2000., 20., 323.

34. Makhija T. and Kulkarni M., J. Comput. Aid. Mol. Des., 2001., 15., 961.

35. Bourinbaiar S., Tan X. and Nagorny R., Acta Virol., 1993., 37., 241.

36. Zhao H., Neamati N., Pommier Y and Burke R., Jr. Heterocycles., 1997., 45.,

2277.

37. (a)Vlientick J., Bruyne., T. De., Apers S and Pieters A., Plant Med., 1998.,

64., 97. (b) Valenti P., Fitoterapia., 1996., 68., 115. (c) Rosskopf F., Kraus J

and Franz G., Pharmazie., 1992., 47., 139. (d) Finn J., Creaven B. and Egan

A., Melanoma Res., 2001., 11., 461.

http://www.springerlink.com/content/?Author=V.+L.+Savel%27ev

http://www.springerlink.com/content/?Author=O.+S.+Artamonova

http://www.springerlink.com/content/?Author=A.+V.+Makarov

http://www.springerlink.com/content/?Author=V.+S.+Troitskaya

http://www.springerlink.com/content/?Author=A.+V.+Antonova

http://www.springerlink.com/content/?Author=V.+G.+Vinokurov


47

38. (a) Kawaii S., Tomono Y., Ogawa K., Sugiura M., Yano M., Yoshizawa Y.,

Ito C. and Furukawa H., Anticancer Res., 2001., 21., 1905. (b) Wang J., Hsieh

J., Lin L and Tseng H., Cancer Lett., 2002., 183., 163. (c) Finn J., Creaven S

and Egan A., Eur. J. Pharmacol., 2003., 481., 159. (d) Edenharder R and Tang

X., Food Chem. Toxicol., 1997., 35., 357. (e) Ahmed S., James K., Owen P.,

Patel K., Bioorg. & Med. Chem. Lett., 2002., 12., 1343.

39. (a) Ho T., Purohit A., Vicker N., Newman P., Robinson J., Leese P.,

Ganeshapillai D., Woo L., Potter L and Reed J., Biochem. Biophys. Res.

Commun., 2003., 305., 909.(b) Bruhimann C., Ooms F., Carrupt A., Testa B.,

Catto M., Leonetti F., Altomare C and Carotti A., J. Med. Chem., 2001., 44.,

3195. (c) Jo., S., Gyibg L., Bae K., Lee K and Jun., H., Plant Med., 2002, 68,

84.

40. (a) Wang H., Ternai B and Polya G., Phytochemistry., 1997., 44., 787.(b)

Sardari S., Nishibe S., Horita K., Nikaido T. and Daneshtalab., M.,

Pharmazie., 1999., 54., 554.

41. (a) Yang B., Zhao B., Zhang K. and Mack P., Biochem. Biophys. Res.

Commun., 1999, 260, 682.(b) Wang X. and Ng B., Plant Med., 2001., 67.,

669. (c) Costantino., L. Rastelli., G and Albasini., A., Pharmazie., 1996., 51.,

994. (d) Kaneko T. Baba N. and Matsuo M., Cytotechnology., 2001., 35., 43

(e) Paya M., Halliwell B and Hoult S., Biochem. Pharmacol., 1992., 44., 205.

42. (a) Fernandez-Puntero B., Barroso I., Idlesias I and Benedi J., Bio. Pharm.

Bull., 2001, 24., 777. (b) Lazarova G., Kostova I and Neychev H., Fitoterapia

1993., 64., 134 Maddi. V., Raghu S. and Rao A., J. Pharm. Sci., 1992., 81.,

964. (c) Nicolaides N., Fylaktakidou C., Litinas E and Hadlipavlou-Litina D.,

Eur. J. Med. Chem., 1998., 33., 715.

43. (a) Delgado G., Olivares S., Chavez M.I., Ramirez-Apan T., Linares E and

Bye R., J. Nat. Prod., 2001., 64., 861. (b) Ghate M., Manoher D., Kulkarni V.,

Shosbha R. and Kattimani S., Eur. J. Med. Chem., 2003., 38., 297.

44. (a) Hadlipavlou-Litina D., J. Arzneim-Forsch./Drug Res., 2000., 50., 631(b)

Ferrer M., Leiton J. and Zaton L.J., Protein Chem., 1998., 17., 115.

45. (a) Carotti A., Bioorg. & Med. Chem., 2003, 11, 123. (b) Chiou F., Huang L.,

Chen F and Chen C., Planta Med., 2001., 67., 282.


48

46. (a) Santana L., Uriarte E., Fall Y., Teijeira M., Teran C., Garcia-Martinez E.

and R. Tolf., Eur. J. Med. Chem., 2002., 37., 503. (b) Gonzalez-Gomez. M.,

Santana L., Uriarte E., Brea J., Villlazon M., Loza I., De Luca M., Rivas E.,

Montegero Y and Fontela A., Bioorg. & Med. Chem. Lett., 2003, 13, 175.

47. (a) Darke P.L., Huff J.R., Advances in Pharmacology 1994, 25, 399., August

T.J., Ander M. W., Murad F., Eds Academic Press, San Diego., 1994., 25,

399-454.(b) Thaisrivongs., S. HIV Protease Inhibitors. Annu. Rep. Med.

Chem., 1994, 17, 133-144.

48. Redshaw S., Inhibitors of HIV Proteinase. Exp. Opin. Invest. Drugs., 1994, 3,

273-286.

49. West M.L., Fairlie D.P., Trends Pharm. Sci., 1995, 16, 67-75.

50. Thaisrivongs S.T., Tomich P.K., Watenpaugh K.D., Chong K.T., Howe W.J.,

Yang C.P., Strohbach J.W., Turner S.R., McGrath J.P., Bohanon M.J., Lynn J.

C., Mulichak A.M., Spinelli P.A., Hinshaw R.R., Pagano P.J., Moon J.B.,

Ruwart M.J., Wilkinson K.F., Rush B.D., Zipp G.L., Dalga., R.J., Schwende

F.J., Howard G.M., Padbury G.E., Toth L.N., Zhao Z., Koeplinger K.A.,

Kakuk T.J., Cole S.L., Zaya R.M., Piper R.C., Jeffrey P., J. Med. Chem.,

1994., 37., 3200-3204.

51. Tummino P.J., Ferguson D., Hupe D., Biochem. Biophys. Res. Commun.,

1994, 201, 290-294.

52. Tummino P.J., Ferguson D., Hupe L, Hupe D., Competitive Biochem.

Biophys. Res. Commun., 1994., 200., 1658-1664.

53. Vara Prasa J.V.N., Para K.S., Lunney E.A., Ortwine D.F., Dunbar J.B., Jr.,

Ferguson D., Tummino P.J., Hupe D., Tait., B.D., Domagala J.M., Humblet

C., Bhat T.N., Liu B., Guerin D.M.A., Baldwin E.T., Erickson J.W., Sawyer

T.K., J. Am. Chem. SOC., 1994., 116., 6989-6990.

54. Lunney E.A., Hagen S.E., Domagala J.M., Humblet C., Kosinski J., Tait B.

D., Warmus J.S., Wilson M., Ferguson D., Hupe D., Tummino P.J., Baldwin

E.T., Bhat T.N., Liu B., Erickson J.W., J. Med. Chem., 1994., 37., 2664-2677.

55. Vara Prada J.V.N., Para K.S., Tummino P.J., Ferguson D., McQuade T.J.,

Lunney E.A., Rapundalo S.T., Batley B.L., Hingorani G., Domagala J.M.,

Gracheck S.J., Bhat T.N., Liu. B., Baldwin E.T., Erickson J.W., Sawyer T. K.,

J. Med. Chem., 1995., 38., 898-905.


49

56. Thaisrivongs S., Watenpaugh K.D., Howe W.J., Tomich P.K., Dolak L.A.,

Chong K.T., Turner S.R., Strohbach J.W., Mulichak A.M., Janakiraman M.N.,

Moon J.B., Lynn J.C., Horng M.M., Hinshaw R.., Pagano P.J., J. Med. Chem.,

1995., 38., 3624-3637.

57. Mohamadi F., Richard N.G.J., Guida W.C., Liskamp R., Lipton M., Caufield

C., Chang G., Hendrickson T., Sill W.C., J. Comput. Chem., 1990., 11.,440-

467.

58. Romines K.R., Watenpaugh K.D., Tomich P.K., Howe W.J., Morris J.K.,

Lovasz K.D., Mulichak A.M., Finzel B.C., Lynn J.C., Horng M.M., Schwende

F.J., Ruwart M.J., Zipp G.L., Chong K.T., Dolak L.A., Toth L.N., Howard G.

M., Rush B.D., Wilkinson K.F., Possert P.L., Dalga R.J., Hinshaw R.R., J.

Med. Chem., 1995., 38., 1884-1891.

59. Romines K.R., Watenpaugh K.D., Howe W.J., Tomich P.K., Lovasz K.D.,

Morris J.K., Janakiraman M.N., Lynn J.C., Horng M.M., Chong K.T.,

Hinshaw R.R., Dolak L.A., J. Med. Chem. ., 1995.

60. Schechter I., Berger A., I. Papain. Biochem. Biophys. Res. Commun., 1967.,

27., 157-162.38., 4463-4473.

61. Casley-Smith J.R., Window J. Microvasc Res., 1976., 11, 279-305.

62. Casley-Smith J.R., Foldi-Borcsok E., Foldi M., Br J Exp Pathol., 1974., 55,

89-93.

63. Casley-Smith J.R., Gaffney R.M., J. Pathol., 1981., 133, 69-74.

64. Paya M., Halliwell B., Hoult J.R.S., Biochem Pharmacol., 1992., 44, 205-214.

65. Paya M., Goodwin P.A., de las Heras B., Hoult J.R.S., Biochem Pharmacol.,

1994, 48, 445-451.

66. Hoult J.R.S., Forder R.A., De las Heras B., Lobo I.B., Paya M., Agents

Actions., 1994., 42, 44-49.

67. Pasanen M., Rannala Z., Tooming A., Sotaniemi E.A., Pelkonen O., Rautio

A., Toxicology., 1997., 123, 177-184.

68. Hardt T.J., Ritshel W.A., Arzneim-Forsch/Drug Res., 1983., 33(II), 1662-

1666.

69. Foldi-Borcsok V.E., Bedall F.K., Rahlfs V.W., Arzneim-Forsch/Drug Res.,

1971., 21, 2025-2030.

70. Leal L. K.A.M., Matos M.E., Ribeiro R.A., Ferreira F.V., Viana G.S.B.,

Phytomedicine., 1997., 4, 221-227., Chem Abstrs., 1998., 128., 39440e.


50

71. Shimizu M., et al. Chem Pharm Bull., 1990., 38, 2283-2284.

72. Lino C.S., Taveira M.L., Viana G.S.B., Matos F.J.A., Phytotherapy Research.,

1997., 11, 211-215.

73. Sekiya K., Okuda H., Arichi S., Acta (BBA)-Lipids and Lipid Metabolism .,

1982., 713, 68-72.

74. Lee R.E., Bykadi G., Ritschel W.A., Arzneim-Forsch/Drug Res., 1981., 31,

640-642.

75. Hoult J.R.S., Paya M., Gen Pharmac., 1996., 27, 713-722.

76. Alami I., Jouy N., Clerivet A., J. Phytopathology., 1999., 147, 515-519.

77. Celia H., Hoermann L., Schultz P., Lebeau L., Mallouh V.., Wigley D.B.,

Wang J.C., Mioskowski C., Oudet P., J. Mol. Biol., 1994., 236., 618.,628.

78. Maxwell Mol. Microbiol., 1993., 9., 681-686.

79. Maxwell A., Trends Microbiol, 1997., 5., 102.,109.

80. Lewis R.J., Singh O.M.P., Smith C.V., Skarzynski T., Maxwell A., Wonacott

A.J., Wigley D.B., EMBO J., 1996., 15., 1412.,1420.

81. Shen L.L., Quinolone DNA interaction., in, D.C. Hooper., J.S. Wolfson

(Eds.)., Quinolone Antimicrobial Agents., American Society for

Microbiology., Washington., DC., 1993., 77., 95.

82. Sugino N.P., Higgins P.O., Brown C.L., Peebles N.R., Cozzarelli Proc. Natl.

Acad. Sci. USA., 1978., 75., 4838.,4842.

83. Tsai F.T.F., Singh O.M., Skarzynski T., Wonacott A.J., Weston S.., Tucker A.,

Pauptit R.A., Breeze A.L., Poyser J.P., O'Brien R., Ladbury J.E., Wigley D.B.,

clorobiocin., Proteins., 1997., 28., 41.,52.

84. Hooper D.C., Wolfson J.S., McHugh G.L., Winters M.B., Swartz M.N.,

Agents Chemother., 1982., 22., 662.,671.

85. Gormley N.A., Orphanides G., Meyer A., Cullis P.M., Maxwell A.,

Biochemistry., 1996.,35., 5083.,5092.

86. Birch A.J., Holloway P.W., Richards R.W., Biochim. Biophys. Acta 1962., 57.,

143-145.

87. Bunton C.A., Kenner G.W., Robinson J.T., Webster B.R., Tetrahedron.,

1963., 19., 1001.,1010.

88. Calvert R.T., Spring M.S., Stoker J.R, J. Pharm. Pharmacol., 1972., 24., 972-

978.


51

89. Ste¡ensky M., Muhlenweg A., Wang Z.X., Li S. M., Heide L., Antimicrob.

Agents Chemother., 2000., 44., 1214., 1222.

90. Ste¡ensky M., Li S.M., Heide L., J. Biol. Chem., 2000., 275., 21754-21760.

91. Marahiel M.A., Stachelhaus T., Mootz H.D., Chem. Rev., 1997., 97.,

2651,2673.

92. Stachelhaus T., Mootz H.D., Marahiel M.A., Chem. Biol., 1999., 6., 493.,505.

93. Challis G.L., Ravel J., Townsend C.A., Chem. Biol., 2000., 7., 211-244.

94. Wang Z.X., Li S.M., Heide L., Agents Chemother., 2000., 44., 3040-3048.

95. van Wageningen A.M., Kirkpatrick P.N., Williams D.H., Harris B.R.,

Kershaw J.K., Lennard N.J.., Jones M.., Jones S.J., Solenberg P.J., Sequencing

Chem. Biol., 1998., 5., 155-162.

96. Lauer R., Russwurm C., Bormann Eur. J. Biochem., 2000., 267., 1698-1706.

97. Quadri L.E.N., Weinreb P.H., Lei M., Nakano M.M., Zuber P., Walsh C.T,

Biochemistry., 1998., 1585-1595.

98. Lambalot R.H., Gehring A.M., Flugel R.S., Zuber P., LaCelle M., Marahiel

M.A., Reid R., Khosla C., Walsh C.T., Chem. Biol., 1996., 3, 923-936.

99. Keating T.A., Miller D.A., Walsh C.T., Biochemistry., 2000., 39., 4729-4739.

100. Seebach E., Juaristi., Miller D.D., David D., Schikli C., Weber T., Helv. Chim.

Acta., 1987., 70., 237.

101. Herbert R.B., Wilkinson B., Ellames G. J., Kunec E. K., J. Chem. Soc. Chem.

Commun., 1993., 205-206.

102. Gokhale R.S., Hunziker D., Cane D.E., Khosla C., Chem. Biol., 1999., 6., 117-

125.

103. Steller S., Vollenbroich D., Leenders F., Stein T., Conrad B., Hofemeister J.,

Jacques P., Thonart P., Vater J., Chem. Biol., 1999., 6., 31-41.

104. Leuthner B., Heider J., J. Bacteriol., 2000., 18., 272-277.

105. Bolhofer W.A., J. Chem. Soc., 1954., 76., 1322-1323.

106. Barkovich R.J., Shtanko A., Shepherd J.A., Lee P.T., Myles D.C., Tzagologg

A., Clarke C. F., J. Biol. Chem., 1997., 272., 9128-9128.

107. Lee P.T., Hsu A.Y., Ha H.T., Clarke C.F., J. Bacteriol. 1997., 179., 1748-

1754.

108. Khalameyzer V., Fischer I., Bornscheuer U.T., Altenbuchner J.., Appl.

Environ. Microbio.,. 1999., 65., 477-482.


52

109. Atta-Asafo-Adjei., Lawton M.P., Philpot R.M., J. Biol. Chem., 1993., 268.,

9681-9689. 312 Chemistry & Biology., 2001., 84.,301-312.

110. Dannhardt G., Kiefer W. Cyclooxygenase inhibitors-current status and future

prospects. Eur J Med Chem., 2001., 36, 109-126.

111. Casley-Smith J.R., Casley-Smith J.R., Modern treatment of lymphoedema II.

The benzopyrones. Austral J Dermatol 1992., 33., 69-74.

112. Anschutz R., Ber. 1903., 36., 465., Ann., 1909., 867., 169.

113. Ziegler E., Junek H., Monatshefte fuer Chemie., 1955., 86., 29-38.

114. Shah V.R., Bose J.L., Shah R.C., J. Org. Chem., 1960., 25., 677.

115. Ogawa A., Kondo K., Murai S., Sonoda N., J. Chem. Soc., Chem.

Commun.,

1982., 21., 1283. Tetrahedron., 1985., 41., 4813.

116. Ye Dingyue., Zhou Yushen., Su Qiang Chinese Patent., CN 1101045.

117. Kalinin A.V., Da Silva Alcides J.M., Lopes C.C., Lopes., Rosangela S.C.,

Snieckus V., Tetrahedron Lett., 1998., 39., 4995.

118. Jung J.C., Jung Y.J., Park O.S. Synth. Commun., 2001., 31., 1195.

119. Pisani L., Muncipinto G., Fabiola M.T., Nicolotti O., Leonetti F., Catto M.,

Caccia C., Salvati P., Soto-Otero R., Mendez-Alvarez E., Passeleu C., Carotti

A. J. Med. Chem., 2009., 52., 6685.

120. Gao W.T., Hou W.D., Zheng M.R., Tang L.J., Synth. Commun., 2010, 40.,

732.

121. Stanciu., Magdalena Cristina and Nicolaescu., Tatiana From Analele

Stiintifice ale Universitatii "Al. I.Cuza" din Iasi., Chimie., 2005, 13., 13-18.

122. Badran M.M. et al From Revue Roumaine de Chimie., 1990, 35(6)., 777-83.

123. Ghosh., Partha, Pratim and Das., Asish R., Tetrahedron Letters., 2012., 53

(25)., 3140- 3143.

124. National Committee for Clinical and Laboratory Standards., Method for

Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically

Approved Standard., fourth ed. NCCLS., Villanova., Italy., 1997., Document

M 100-S7. S100-S157.

125. Isenberg D.H., Essential Procedure for Clinical Microbiology., American

Society for Microbiology., Washington., 1998.

126. Zgoda J.R., Porter J.R. Pharm. Biol., 2001., 39., 221.

studies on 4-hydroxy coumarin -...

Documents