J. Chem. Sci. Vol. 125, No. 6, November 2013, pp. 1573–1594. c Indian Academy of Sciences. Facile green synthesis and potent antimicrobial efficacy of β -aminoheteronapthol via tailored Betti’s protocol and their bis-aryl hydrazone click products K M KHANDARKAR a,b, ∗ , M D SHANTI a , M AHMED c and J S MESHRAM a a Department of Chemistry, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur 440 033, India b Applied Chemistry Department, G H Raisoni Academy of Engineering and Technology, Nagpur 440 016, India c Department of Chemistry, Government Polytechnic Sakoli, Bhandara 441 802, India e-mail: [email protected]MS received 27 March 2013; revised 23 June 2013; accepted 4 July 2013 Abstract. A series of novel Mannich base and their hydrazone derivatives were synthesized by highly resourceful, chic, simple and green technique with exceptionally facile reaction conditions of one-pot, three component reaction with an array of biologically and pharmaceutically active novel heterocycles. The proto- col offers a valuable alternative to known methods and will find applications in the field of green synthesis and antimicrobial study against pathogenic microbes, supporting the development of bioinformatical database of novel and derelict heterocycles. These data indicate their potential to become antifungal agents. The novel products were established by elemental, IR, mass spectroscopic and NMR analysis. The environmental advan- tages of the method include short reaction time, excellent yield, easy work-up, absence of extraction and chromatographic purification steps. Keywords. Green synthesis; zeolite catalyst; click reaction; antimicrobial efficacy. 1. Introduction The incidence of microbial infections in the immune compromised population has significantly increased over the past several years. In particular, Candida species, especially Candida albicans, are often significant pathogens in patients infected with human immunode- ficiency virus (HIV). Another pathogen, Pneumocysist carinii, causes a form of pneumonia (PCP) that is believed to be one of the leading causes of death in patients suffering from AIDS. Furthermore, the treat- ment of infectious diseases is more complicated in immuno-suppressed patients, such as those infected with the HIV, undergoing anticancer treatment and organ transplants. Therefore, there is a vital need for the development of new antimicrobial agents having potent activity against the resistant microorganisms. 1 The rapid development of resistance to existing antibacterial and antifungal drugs posses a major threat to public health which creates a serious challenge to the ∗ For correspondence scientific community. Hence the usage of most of the existing antimicrobials have limited capacity in over- coming the threat of resistance and it is limited not only by the rapidly developing drug resistance, but also the unsatisfactory status of present treatments of bacte- rial and fungal infections and drug side effects. 2 Thus it would be advantageous to provide a pharmacophore species with useful properties that can be used commer- cially against pathogenic and resistant microbes. Mannich bases had abundant commercial appli- cations and it was estimated that at least 35% of Mannich bases related articles are published in phar- maceutical journals. The first synthesis of racemic Mannich-bases of 2-naphthol was achieved by Betti at the turn of the twentieth century. 3 Thereafter numer- ous modifications of this reaction surfaced. 4 Since these compounds have multiple centres for chelation with metal ions, they are likely to be potent inhibitors of metallo-enzymes. 5 Also, these compounds have the potential to be used as scavengers in cases of heavy metal poisoning. 6 They have a broad range of biologi- cal activities including diuretic, anticonvulsant, antipsy- chotic, antimalarial, antiviral, centrally acting muscle relaxant, and anticancer. Also, Mannich-bases of various bioactive compounds have been prepared as 1573
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Facile green synthesis and potent antimicrobial efficacyof β-aminoheteronapthol via tailored Betti’s protocoland their bis-aryl hydrazone click products
K M KHANDARKARa,b,∗, M D SHANTIa, M AHMEDc and J S MESHRAMa
aDepartment of Chemistry, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur 440 033, IndiabApplied Chemistry Department, G H Raisoni Academy of Engineering and Technology,Nagpur 440 016, IndiacDepartment of Chemistry, Government Polytechnic Sakoli, Bhandara 441 802, Indiae-mail: [email protected]
MS received 27 March 2013; revised 23 June 2013; accepted 4 July 2013
Abstract. A series of novel Mannich base and their hydrazone derivatives were synthesized by highlyresourceful, chic, simple and green technique with exceptionally facile reaction conditions of one-pot, threecomponent reaction with an array of biologically and pharmaceutically active novel heterocycles. The proto-col offers a valuable alternative to known methods and will find applications in the field of green synthesisand antimicrobial study against pathogenic microbes, supporting the development of bioinformatical databaseof novel and derelict heterocycles. These data indicate their potential to become antifungal agents. The novelproducts were established by elemental, IR, mass spectroscopic and NMR analysis. The environmental advan-tages of the method include short reaction time, excellent yield, easy work-up, absence of extraction andchromatographic purification steps.
Keywords. Green synthesis; zeolite catalyst; click reaction; antimicrobial efficacy.
1. Introduction
The incidence of microbial infections in the immunecompromised population has significantly increasedover the past several years. In particular, Candida species,especially Candida albicans, are often significantpathogens in patients infected with human immunode-ficiency virus (HIV). Another pathogen, Pneumocysistcarinii, causes a form of pneumonia (PCP) that isbelieved to be one of the leading causes of death inpatients suffering from AIDS. Furthermore, the treat-ment of infectious diseases is more complicated inimmuno-suppressed patients, such as those infectedwith the HIV, undergoing anticancer treatment andorgan transplants. Therefore, there is a vital need forthe development of new antimicrobial agents havingpotent activity against the resistant microorganisms.1
The rapid development of resistance to existingantibacterial and antifungal drugs posses a major threatto public health which creates a serious challenge to the
∗For correspondence
scientific community. Hence the usage of most of theexisting antimicrobials have limited capacity in over-coming the threat of resistance and it is limited notonly by the rapidly developing drug resistance, but alsothe unsatisfactory status of present treatments of bacte-rial and fungal infections and drug side effects.2 Thusit would be advantageous to provide a pharmacophorespecies with useful properties that can be used commer-cially against pathogenic and resistant microbes.
Mannich bases had abundant commercial appli-cations and it was estimated that at least 35% ofMannich bases related articles are published in phar-maceutical journals. The first synthesis of racemicMannich-bases of 2-naphthol was achieved by Betti atthe turn of the twentieth century.3 Thereafter numer-ous modifications of this reaction surfaced.4 Since thesecompounds have multiple centres for chelation withmetal ions, they are likely to be potent inhibitors ofmetallo-enzymes.5 Also, these compounds have thepotential to be used as scavengers in cases of heavymetal poisoning.6 They have a broad range of biologi-cal activities including diuretic, anticonvulsant, antipsy-chotic, antimalarial, antiviral, centrally acting musclerelaxant, and anticancer. Also, Mannich-bases ofvarious bioactive compounds have been prepared as
1573
1574 K M Khandarkar et al.
prodrugs as means of overcoming some of their limi-tations.7 So keeping importance of Mannich-base moi-ety in mind, we had synthesized a series of substitutedcoumarin derivatives via classical Betti’s condensa-tion reaction with their pharmacophores modelling andin vitro microbial activity.8 Despite the importance ofthe compounds, the serious downsides of their com-mercial syntheses include some or all of the following:refluxing for extensive reaction time, large quantities ofmineral or Lewis/Bronsted acids as activators, whichon work-up are hydrolysed with generation of largequantities of corrosive and toxic waste by-products; theuse of stoichiometric quantities of reagents that producemetal salts as waste; poor yields; or production of mix-tures of regioisomers with low selectivity. The enan-tioselective transformations and industrial applicationsare the foremost impediment in the microwave-assistedsynthesis of these compounds.
The aforementioned problems of greener syntheticapproach of Betti’s reaction product are overcome inpresent work by one-pot multicomponent zeolite cata-lysed condensation bioactive Betti’s reaction products.Inspired by the above facts and in continuation of ourongoing research programme in the field of synthe-sis and antimicrobial activity of medicinally impor-tant compounds,9 we report here the click synthesis ofnovel hydrazide-hydrazone/bisaryl hydrazone deriva-tives of bioactive Betti’s product and evaluated them forantimicrobial activity.
Hydrazones are the most important pharmacophoriccores of several antimicrobial, anticonvulsant, anal-gesic, antiinflammatory, antinociceptive, antiplatelet,antitubercular, and antitumoural drugs.10 Hydrazidesand hydrazones are present in many of the bioac-tive heterocyclic compounds that are of wide interestbecause of their diverse biological and clinical applica-tions. This created interest in researchers who have syn-thesized variety of hydrazide derivatives and screenedthem for their various biological activities.11 Hydra-zones have been explored for the reversible conjuga-tion12 and labelling13 of biomolecules. However, theirapplication has been limited, as most hydrazones areprone to hydrolysis and premature cleavage, whereashydrazones (and oximes) that are fully stable underbiological and mildly acidic conditions undergo slowerhydrolysis and are difficult to exchange or cleave.
The formation kinetics of the bis-aryl hydrazonebond are 20 to 900 times higher than those of conven-tional hydrazone/oxime bonds between alkyl aldehydes/ketones and alkyl hydrazide or aminoxy probes.14
The hydrazide hydrazones have been demonstrated topossess antibacterial, 15–17 anticonvulsant18–20 and anti-tubercular21 activities. Furthermore, the benefits of
click reactions are well-known, and their applicationshave revolutionized numerous fields including bio-conjugate chemistry, material chemistry and polymersciences.22–25 The non-aldol addition reaction betweena carbonyl and a nucleophilic nitrogen as a clickreaction has key benefits of availability of amines forbioconjugation, rapid incorporation into molecularframeworks, and a simple resultant click product thatminimally impacts the nature of the biomolecules.26
Against this background, and also in the context ofour interest in the synthesis of biologically significantheterocycles via multi-component and click reactions,we report here the green preparation of a library of phar-macologically relevant click fused bisarylhydrazones,containing a Betti’s base fabricated by a process thatcombines the use of zeolite with the synthetic effi-ciency associated with MCRs, providing a convergenceof reaction and environmental economies.27
In this paper, green synthesis of potent Betti’s prod-uct and hydrazone derivatives are exemplified withtheir antimicrobial efficacy. Here, in order to reducethe time period of Betti’s reaction product, enantio-selective transformations and to achieve green chemi-cal synthesis, the reaction was carried out in catalyticmedium of zeolites which enhance reaction rate, sup-port reagents, regioselectivity, entrain by-products andenhance product selectivity.28–30
Furthermore, we have found a novel series of Betti’sproducts and hydrazone derivatives that are effec-tive against resistant microbes, and provide significantactivity advantage over the art. It has been found that thecompounds of this innovation and composition contain-ing these compounds are effective antimicrobial agentsagainst a broad range of pathogenic microorganismswith advantages in low susceptibility to microbial resis-tance, reduced toxicity, and improved pharmacology.
2. Experimental
The solvents and reagents used in the synthetic workwere of analytical grade obtained from Qualigens Indiaand were purified by distillation where necessary. Melt-ing points were determined in open capillaries anduncorrected. Infrared spectra were recorded on BrukerAlfa-T with direct sampling for analysis. 1H NMRspectra were recorded on a Cryo-magnet NMR Spec-trometer 400 MHz (Bruker) instrument using tetra-methylsilane (TMS) as an internal standard with CDCl3
and DMSO as a solvent. Chemical shifts are givenin parts per million (δ scale) and the coupling con-stants are given in Hz. Mass spectra were recordedon a Waters Micromass Q-T of Micro spectrometer.
Tailored Betti’s product and their click hydrazones 1575
The reactions were monitored and the purity of pro-ducts were checked out on Silica gel 60 F254 (Merck)TLC plates with a mixture of petroleum ether (60–80◦C) and ethyl acetate as the eluent and the spots werevisualized under ultraviolet light and iodine chamber.Elemental analyses were performed on a Perkin-Elmer2400 Series II elemental CHNS analyzer. The zeoliticraw mineral was ground and purified via washing withdistilled water by means of a fluidized bed process inorder to remove the non-zeolitic mineral phases. Aftervacuum filtration and drying at 200◦C, zeolites werereutilized for each reaction.
2.1 General procedure for the synthesisof β-amino-quinoline (5)
A mixture of 8-hydroxyquinoline (3) (1 mmol) and car-bonyl compound (1) (1 mmol) was stirred at room tem-perature for 10 min in the presence of zeolite (0.5 g)in ethanol. Then, an equimolar amount of primary orsecondary aromatic/aliphatic amine (2) was added, andresulting reaction mixture was stirred for 1–1.5 h at40◦C. After completion of the reaction (TLC checked),the coloured solid that separated was filtered, washedwith ethanol and purified by recrystallization fromethanol or by filtration through a pad of silica gelusing 9:1 petroleum ether–ethyl acetate to afford purecompounds 5 (compounds 5a–5j).
2.2 General procedure for the synthesisof β-amino-chromene (6)
A mixture of coumarin (4) (1 mmol) and carbonylcompound (1) (1 mmol) in ethanol was stirred at40◦C for 15 min in the presence of zeolite (0.5 g).Then, an equimolar amount of primary or secondaryaromatic/aliphatic amine (2) was added, and result-ing reaction mixture was stirred for 1–2 h. After com-pletion of the reaction (TLC checked), the colouredsolid that separated was filtered, washed with ethanoland purified by recrystallization from ethanol/methanolor by filtration through a pad of silica gel using 9:1petroleum ether–ethyl acetate to afford pure compounds6 (compounds 6a–6g).
2.3 General procedure for the synthesisof N′-substituted arylidenehydrazine derivatives (7)
β-aminoheteronapthol (5/6) (1 mmol) and aldehyde/ketone (1 mmol) were made single phase by dissolvingin minimum quantity of ethanol and then added to petridish containing hydrazine hydrate (80%) (1 mmol).This concoction was then mixed with a glass rod, adry solid product established within 10 s by exother-mic click reaction at room temperature. The solid pro-duct was washed with cold ethanol and recrystallizedby ethanol 7 (compound 7a–7p).
In view of emerging importance of green synthesis andour general interest in solid acid catalysed chemicalreactions, we envision expedited Betti’s reactions usingzeolites as catalysts. Both natural and synthetic zeo-lites are used as a catalyst to extend the Betti’s reactiontowards the greener protocol.
Initially we focused on the synthesis of β-aminoheteronapthol from aromatic carbonyl com-pound, amine and 8-hydroxy quinoline or coumarin.
Most of the earlier proposed procedures for the pro-duction of Mannich-base involve various organic andmineral acid catalysed reaction conditions. The synthe-sis of these compounds was reported by performingthe reaction in the presence of Lewis acid,31 Bronstedacid catalysts,32 Lewis base,33 transition metal salt34
catalysts condition and even uncatalysed one-pot, three-component synthesis of Betti’s base in water.35 How-ever, all of these chemical processes required reflux-ing for extensive reaction time, large quantities ofmineral or Lewis acids as activators which on work-up are hydrolysed with generation of large quanti-ties of corrosive and toxic waste by-products, longreaction time (days) along with purification steps andthe uncatalysed Betti’s reaction involves only basic2-pyridinyl, 2-pyrazinyl and 2-pyrimidinyl amine asamine moiety which act as a catalyst.
Here, we wish to disclose our results, studying theapplication of natural and synthetic zeolite as a catalystin synthesis of some novel and bioactive Betti’s reac-tion product and their hydrazone (arylidenehydrazine)derivatives by, catalyst-free most placid reaction condi-tion of click synthesis in least amount of solvent.
3.1 Chemistry
The Betti’s classical procedure, a Mannich-typeaminoalkylation condensation of 2-naphthol and aro-matic aldehyde in the presence of ammonia, wasmodified by condensing carbonyl compound (1), aro-matic amine (2) and heteronapthol [here, 8-hydroxyquinolone (3) or 7-hydroxy-4-methyl-coumarin (4)]in the presence of zeolite to prepare the β-aminoheteronapthol (5/6) (scheme 1).
The natural zeolite applied here are scolecite(CaAl2Si3O.
103H2O), stilbite (NaCa4 (Si27Al9)O.72 28(H2O)),
fluorapophyllite (KCa4Si8O20(F,OH)·8(H2O)) and meso-lite (Na2Ca2(Al2Si3O10)3 ·8H2O) and synthetic zeoliteare crystalline molecular sieves which can be repre-sented by general formula −M2/nO[(AI2O3).(SiO2)X].yH2O, where M represents alkali metals with valency-n.These aluminosilicates are 3A, 4A, 5A and 13X withpore size 3 Å, 4 Å, 5 Å and 10 Å, respectively. Thealkali metal in aluminosilicate 3A and 4A is potassium,5A is calcium and 13A is sodium. A detailed study oncatalyst standardization is obtained from the chemicaloxide-form composition of the zeolitic raw minerals asshown in table 1.
In present work of modified Betti’s reaction productsynthesis, use of zeolite catalyst reduces the time periodof reaction with the yield of 92%–98% and the catalystwas recovered completely which was reused in threeconsecutive reactions having more than 90% yield. Theproducts of modified Betti’s reaction are specified intable 2.
The catalyst was cleanly separated after completionof the reaction (monitored by TLC) by simple filtrationin hot condition. The obtained solid condensation pro-duct was further purified by recrystallization in ethanol.As the granular size of the zeolite is used, 100%recovery of the catalyst is possible even after threeconsecutive runs. And it was found that the recovered
Table 1. Oxide-form chemical composition of the raw mineral, natural zeolites and molecularsieves in weight %.
Tailored Betti’s product and their click hydrazones 1583
Table 2. Tailored Betti’s reaction between carbonyl compound, amine and heteronapthol (here, quinoline/chromene)a.
N
Ar2 NH2
O
CH3
O
Ar1COR NOH
Ar1
NHAr2Ar1 HN Ar2
OHO O Zeolite / EtOH Zeolite / EtOH
Ar3OHAr4OH
Ar3 =Ar4 =
R
R
R = H, Cl, Aryl
(5)(6)
(2)(4)
(1)
(3)
Reaction Yieldb
Entry Ar2NH2 Ar1COR time (min) (%) Product/Compound
1O
C2H5ONH2 CH3
OHC O
OH
60 96
C2H5O
H3C
ONH
O
OH
NHO
5a
2O
NH2
H3C
CH3OHC O
OH90 93
H3C
CH3
NHO
OHO
O
NH
5b
3C2H5O
NH2
O
OHC
HO
60 97
C2H5O
ONH
OH
NHO
5c
4C2H5O
NH2
O N
OHC
Cl
105 92
C2H5O
O
NH
N Cl
N
OH 5d
5NH2
O
NN
O CHO 120 95O
NN
HN
O
N
HO
5e
6O
C2H5ONH2
O CHO 90 98
NHO
O
NH
O
C2H5O 5f
7O
C2H5ONH2 NO2
Cl
O
120 97
C2H5O
O2N
O
NH
Cl
N
HO
5g
1584 K M Khandarkar et al.
Table 2. (Continued).
8O
C2H5ONH2
O
O
120 91C2H5O
O
NH
O
N
HO
5h
9 NH2
OH
O
105 93 N
OHHO
HN 5i
10
N
NNH2
N N
OCH3
OH
CHO
90 94
CH3
N
OHN
N
HN
N
N
O
OH
5j
11O
C2H5ONH2
CHO
OCH3H3C
OH
O90 95
H3C
H3C
O
HONH
OHO
O
O
HO
O
6a
12HN O O CHO 90 87
N
O
O
OH
O
O
6b
13HN O
OHC OCH3 105 90OHO O
OCH3
N
O
6c
14 NH2
O
C2H5O
N
OHC
Cl
105 96
O
C2H5ONH
N Cl
O
OH
O
6d
Tailored Betti’s product and their click hydrazones 1585
Table 2. (Continued).
15O
C2H5ONH2
OOHC
O
120 90O
HO
O
OO
O
C2H5O
NH
6e
16 NH2
C2H5O
O
O
150 92 O
HO
ONH
C2H5O
O
6f
17O
NH
O CHO 120 95O
HO
O
O
N
O
6g
aAll reactions were performed in EtOH at 40◦C in the presence of natural/synthetic zeolite as a catalyst. The solid obtainedfrom the reactions was purified by washing with cold EtOH and by recrystallization in MeOH/EtOHbOf pure and isolated product
catalyst shows almost same yield with three successivereactions (table 3). The recovered catalyst was washedwith ethyl acetate, then dried at 70◦C prior to usefor next run in model reaction and activated at 350◦Cafter three consecutive runs. The activity of the catalystdecreases after fourth run plausibly due to impedimentof pores of the zeolite.
Interestingly, the addition of 0.5 g catalyst was foundto be sufficient to provide excellent yields of pro-ducts (<90%) after moderately short reaction time (typi-cally between 1 and 3 h). Lower catalyst loading andreaction time (less than 30 min) at room temperatureturned out to be inefficient and resulted in significantlyreduced yields (85% and below). The protocol was alsoamenable to a range of substrates, including differentaldehydes as well as more challenging substrates, suchas ketone and benzoyl chloride (table 2). The products
aReaction condition: carbonyl compound: amine:quinoline/chromene, 1:1:1 (mol ratio), natural zeolite =stilbite (0.5 g crystalline), temperature = 40◦C
were obtained in excellent yields for all the investigatedsubstrates (85–97%), regardless of the nature and posi-tion of the substituents and/or hetero aromatic natureof the carbonyl compound. In any case, reactions takea longer time to reach completion for these more chal-lenging substrates (2–2.5 h) as compared to aldehydes,which provide quantitative conversion to products in0.5–2 h of reaction. A series of optimization experi-ments pointed to a remarkable acceleration of reactionrates and therefore yields, at slightly increased reactiontemperatures. Temperatures as low as 40◦C providedalmost quantitative yields of products at reduced reac-tion times (ranging from 1 to 2 h; table 2) by employingzeolite as catalyst. Yields were somewhat reduced forsubstrates with larger chains, but in any case the devisedprotocol was suitable for both aromatic and aliphatic(cyclic and acyclic) substituents. The time period ofreaction decreases with further increase in temperature(from 50 to 70◦C) but did not have much influence onthe yields of products as compared to those at 40◦C(table 4). But when compared to other catalysts, zeolitesafford a much reduced reaction period with promis-ingly greater yield and a complete green sustainabletechnique.
3.1a Bis-aryl hydrazones: Synthesis of thesehydrazides, hydrazones and their derivatives involvesstrong acid catalysed reaction condition of multiplesteps of hours or overnights reflux in abundant sol-vent.36 These harsh reaction conditions and hazardous
1586 K M Khandarkar et al.
Table 4. The effect of reaction temperature on the activityof zeolite for the Betti’s condensation of carbonyl compound,amine and quinoline/chromene (5/6).
Reaction conditions: 1 mmol quinoline/chromene, 1 mmolsubstrate amine and carbonyl compound, 0.5 g zeolite, withstirring
catalyst, solvent and byproducts were utterly removedhere by carrying out the reaction in limited amountof solvent without catalyst using principle of clickreaction which is wide scope, high yielding and stereo-specific with high atom economy.22–25 The hydrazonemoiety was selected for its high stability at physiologi-cal pH, and liability under strongly acidic and basicconditions as demonstrated by drug delivery agents intumour targeting.37,38 To our knowledge, such catalyst-and solvent-free Betti’s bis-arylhydrazone click producthas not yet been explored.
In the present work, we mixed equimolar quantitiesof hydrazine hydrate 80%, β-aminoheteronapthol (5/6)(solubilized in minimum ethanol) and aromatic aldehy-des together in a petri dish at room temperature withthe help of a glass rod. Surprisingly, the reaction startedimmediately after mixing and a shiny immense hydra-zone product appeared within a few seconds with gen-tle heat production, scheme 2. The progress of the reac-tion and by-products was monitored by thin-layer chro-matography and then the reaction mixture was allowedto stand for 1–2 min to obtain hydrazone product (7) ina complete quantitative yield.
Excited by the above result, a range of β-aminoheteronapthol hydrazone derivatives were syn-thesized by the simple mixing of aromatic aldehyde andhydrazine hydrate (80%) with β-aminoheteronaptholjust solubilized in ethanol for homogenous reactionat room temperature; the results are summarized intable 5. All the products were obtained in quantitativeyield (>85%) in analytically pure form, recrystallizedby ethanol/methanol without any further purification.
A plausible mechanism is proposed for the acidcatalysed condensation of carbonyl compound, amineand heteronapthol to give β-amino-phenol using zeo-lite (scheme 3). The carbonyl compound (1) combineswith the amine (2) in mild acidic medium provided byzeolite to give (A) which on dehydrolysis provides sta-ble electrophile iminium ion (B). The heteronapthol (C)functions as a very good carbon nucleophile, giving theintermediate represented by the resonance structures,which subsequently loses a proton to furnish β-amino-phenol. It is easy to identify from this mechanism thatacid catalysis aid the reaction and indeed increased rateswith greener approach have been observed using zeolitecatalysis.
3.2 In vitro antimicrobial screening
A chemical library of the compounds from the series5a–5j, 6a–6g and 7a–7p were tested for antimicrobialand antifungal activity. The fungal and bacterial cul-tures were kindly provided by Rajiv Gandhi Biotech-nology Centre, Nagpur. Agar well diffusion methodwas used to determine the antibacterial activity of thesecompounds, with amoxicillin and gentamicin as thestandard antimicrobial agents and nystatin and flucana-zol as the standard antifungal agents. For the diffusionwell method, the solvent used was dimethylsulphoxide(DMSO) and its antimicrobial activity against all theproposed pathogenic microbial cultures was found toonegligible to be considered as zero. The concentrationsof the compounds taken were 1, 0.5, 0.25, 0.1, 0.01 and0.001 mg/ml in DMSO with 20 μl solution in each well(5 mm diameter hole cut in agar gel).
3.2a Test-microbes: The antibacterial activity ofcompound was assessed against pathogenic strains bothGram-positive and Gram-negative bacteria, the patho-genic Gram-positive bacteria for testing were Staphy-lococcus aureus, Streptococcus mutants, Bacillus sub-tilis and Gram-negative bacteria were Escherichia coli,Pseudomonas aeruginosa, Salmonella typhi, Klebsiella
Ar H2N NH2R2R1
O
R
ON
NH
OR2
R1
N NR2
R1 R
ii R= CH3
(5/6)
i R= OC 2H 5
Ar
Ar2 H2O
C2H5OH H2O
Scheme 2. Bis-aryl hydrazone derivative via click reaction.
Tailored Betti’s product and their click hydrazones 1587
Table 5. Bis-aryl hydrazones of β-aminoheteronapthol by click reaction.a
Ar H2N NH2R2R1
O
R
ON
NH
OR2
R1
N NR2
R1 R
ii R = CH3
(5/6)
i R = O
C 2H 5
Ar
Ar2 H2O
C2H5OH H2O
Reaction Yieldb
Entry Compound R1COR2 time (s) (%) Product
1 5a O
F
6 90
O
NH
O
OH
NHO
N NH
F
H3C
7a
2 5b
O
OCH3
7 93
NHO
OHO
NHN N
H3C
H3CH3CO
7b
3 5c O
H3C
9 87
CH3
O
NH
OH
NHON NH
7c
4 5d O CHO 10 94
O
NH
N
Cl
NHO
N NH
O
7d
5 5e
CHO
OCH3
OH 15 86
H3C
N N
NN
HN
O
N
HO
OOH
7e
6 5f
O
Cl 5 97
NNH
N
OH
O
NH
OCl
7f
1588 K M Khandarkar et al.
Table 5. (Continued).
7 5f N
OHC Cl
8 91
NNH
N
OH
O
NH
ON Cl
7g
8 5f O CHO9 94
NNH
N
OH
O
NH
OO
7h
9 5g
H3C
NH2
O
12 89
CH3
NO2
H2N
O
NH
HN
Cl
N OH
N
7i
10 5h
O
10 88
O
HN
NH
O
N
OHN
7j
11 6aOOH
6 91N
HN
O
HN
O
OH
O
O
OH
O
OH
CH3 CH3
7k
12 6bO
5 92 NN
O
HO
O
ON
7l
13 6c
OOH
7 90
O
OH
O
NN
NOH
OCH3
7m
Tailored Betti’s product and their click hydrazones 1589
Table 5. (Continued).
14 6d
O
NO2
8 86
O2N O
NH
NH
N
O
HO
ON
Cl
7n
15 6e O 7 87O
HO
O
OO
O
NH
NHN
7o
16 6f
OCH3
O
8 83 H3CO
O
OH
O
NH
O
NHN
7p
aReaction condition‘ Mixing of reactants with a spatula in a petri dish for 10–15 s followed by allowing the mixture to standfor up to 15–30 s if necessarybOf pure and isolated product
pneumonia. The pathogenic fungi referred for antifun-gal activity were Aspergillus niger, Phythophthora sp.,Aspergillus flavus, Candida albicans, Rhizopus orazyaeand Fusarium oxysporium. All the microbes were sub-culture in sterilized nutrient broth.
3.2b Agar well diffusion method: For the antibacte-rial efficacy, Muller Hinton agar medium (33.9 g) wastaken in distilled water (1 L) which was autoclavedat 15 lbs pressure at 121◦C for 15 min and pouredonto 100 mm petriplates (25–30 ml/plate) in moltenstate. The antimicrobial efficacy of compounds wereassessed by the mortification of mycelia growth of fun-gus observed as a zone of inhibition near the wellscratch on Potato Dextrose agar medium. The micro-bial inoculum (300 μl) was uniformly spread using ster-ile cotton swab on a sterile Petri dish agar. The 20 μlsolution of compounds of concentration 1, 0.5, 0.25,0.1, 0.01 and 0.001 mg/ml in DMSO was taken in eachwell cut in agar gel plate. The systems were incubatedunder aerobic conditions for 24 h at 36◦C ± 1◦C forbacterial colony and at 25◦C ± 1◦C for fungal colonialgrowth. After incubation, confluent microbial growthwas observed. Inhibition of the microbial growth wasassayed by measuring the diameter of inhibition zone
(mm) formed around the well (NCCLS, 1993). Refer-ence commercial antibacterials used were amoxicillinand gentamicin and antifungal used were nystatin andflucanazol. Tests were performed in duplicates andresults were presented in tables 6 and 7.
The compounds 5d, 5f, 5j, 7a, 7d and 7h pro-vide better antibacterial properties against most ofthe pathogenic bacteria compared to standard antibac-terial, Gentamicin and it has also been found thatthese Betti’s reaction product and bisarylhydrazoneare active against Methicillin-resistant Staphylococ-cus aureus bacterium and certain quinolone resistantpathogens like S. pyogenes, E. coli, P. aeuruginosa. Thecompounds 5a, 5d, 5f, 5j, 6d and 6g demonstrated verygood antifungal results against all the pathogenic fungiunder consideration. The pathogenic Phytophthora sp,which was not successfully medicated, was also foundto be overawed by these compounds. These potentproducts also have the MIC at very low concentration.
3.2c Minimum inhibitory concentration (MIC) deter-mination: Certain compounds of the subject have beenfound to have MIC values (μg/ml) that are up to about500 times lower than amoxicillin and ciprofloxacin
1590 K M Khandarkar et al.
R'
NH
Ar2
iminium ion
(2)
Zeoite
(1)
(A)
(B)
OAr
H
SiO Al
Proton transfer and -amino hydroxy compound formation
R
C N
R'
Ar2
Ar1OH
Ar
Iminium ion formation
R
CO N
R'
Ar2
Ar1
H R
C N
R'
Ar2
Ar1
R
CO N
R'
Ar2
Ar1H
H R
CO N
R'
Ar2
Ar1H
H Resonance R
C N
R'
Ar2
Ar1
Ar1
C
R
OSi
OAl
Zeoite
HAr1
C
R
OH
Ar1
C
R
OH
H
SiO
Al
O
H
R
C N
R'
Ar2
Ar1
O
HSi
OAl
H
O
Ar
O
R
C N
R'
Ar2
Ar1
Ar
SiO
Al
HO
Ar RC N
R'
Ar2
Ar1 O
Ar RC N
R'
Ar2
Ar1
H
SiO Al
O
Ar
O
Ar
O
ArH
Enolizable carbonylcompound(C)
Scheme 3. Zeolite catalysed mechanism for β-aminoheteronaphthol formation.
and comparable enough to replace gentamicin. More-over, invented compound inhibits the antifungal activityagainst widespread pathogenic fungi having MIC about10 times reduced amount of known antifungal agentslike flucanazole. The MIC of the compounds is given intables 8 and 9.
The percentage of mycelial growth inhibition (P) forpathogenic bacteria and fungi was calculated by the for-mula P = (C − T) / C × 100, where C is the diameter ofthe control colony and T that of the treated ones (stan-dard drug).38 The growth inhibitions of compoundscompared with standard drugs against pathogenic bac-teria are shown in figure 1 and against pathogenic fungiare shown in figure 2.
3.2d Structure activity relationship (SAR): Inquinoline-based Betti’s product (5a–5j), the furan(5d), 2-chloroquinolin (5f) and purine (5j) substitu-tion increases antimicrobial activity tremendously as
compared to other substituted benzene and pyrazolring. Aliphatic amine (5i) substitution decreases theantimicrobial activity of Betti’s product. 4-oxo-4H-chromene and 2-chloroquinoline ring in coumarinbased Betti’s product (6a–6g) also reduces the antibac-terial activity of the Betti’s product. Presence of bothmorpholine and furan ring raise the antibacterial acti-vity in coumarin based Betti’s compound (6g). Thederivatization (7a–7p) of Betti’s base overall decreasesthe antibacterial activity.
Antifungal activity of Betti’s product and their hydra-zone derivatives are comparable with nystatin but thequinoline based Betti’s products having 2-chloroqui-noline (5d) and furan (5f) ring substitution has theactivity over and much above the nystatin. In coumarin-based Betti’s product, 4-oxo-4H-chromene with benzo-caine (6e) and furan with morpholine (6g) has effec-tive antifungal activity. Syringaldehyde coumarin-basedBetti’s product has better antifungal activity comparedto hydrazone derivative of the Betti’s products with
Tailored Betti’s product and their click hydrazones 1591
Table 6. Antibacterial efficacy of the compounds (0.25 mg/ml) against pathogenic bacteria.
Zone of inhibitionGram-positive bacteria Gram-negative bacteria
A = Gentamicin; B = Amoxicillin; a = the diameters of zone of inhibition were in mm
respect to nystatin but its own salicylaldehyde hydra-zone derivative (7k) has least antifungal activity. Theeffective antimicrobial activity of the synthesized com-pounds is mainly due to the presence of vicinal aminohydroxy group in the structure of the compounds.
4. Conclusion
An array of biologically and pharmaceutically activenovel heterocycles were engendered by tailored Betti’sreaction with green fundamentals. Natural as well assynthetic zeolite was demonstrated in general to be anefficient and versatile catalyst for the three-component
Betti’s condensation reaction. A range of carbonyl com-pounds and amines could be efficiently reacted withquinoline/coumarin to give the corresponding substi-tuted β-aminoheteronaphthols. Furthermore, we havedeveloped a general, highly efficient, catalyst-free, leastsolvent consuming, high yield, step-economic, and eco-friendly method for the synthesis of bis-aryl hydra-zones derivatives via the one-pot, three componentcoupling click reaction of β-aminoheteronaphthols,hydrazine hydrate and aldehydes/ketones acetate underlenient conditions. The antibacterial and antifungalproperties were characterized by zone of inhibitionand compared with standard drugs. The compoundswere better antimicrobial agent compared to standards
1592 K M Khandarkar et al.
Table 7. Antifungal efficacy of the compounds (0.25 mg/ml) against pathogenic fungi.a
Zone of inhibitionAspergillus Phythophthora Aspergillus Candida Rhizopus Fusarium
Figure 1. Growth inhibition of compounds with respect to gentamicin against pathogenic bacteria.
Figure 2. Growth inhibition of compounds with respect to nystatin against pathogenic fungi.
amoxicillin and flucanazol. The compounds 5d, 5f and5j were showing very good antibacterial activity againstmaximum pathogenic bacteria with lower MIC thangentamicin and compounds 5d, 5f, 6d and 6g were
having better antifungal activity amongst all and MICvalue analogous to nystatin. Thus, these green tech-nologically synthesized compounds could be used asputative more potent antimicrobial compounds.
1594 K M Khandarkar et al.
Supporting information
Theelectronic supporting information can be seen inwww.ias.ac.in/chemsci.
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