molecules

Review The Current Case of Quinolones: Synthetic Approaches and Antibacterial Activity

Abdul Naeem 1, Syed Lal Badshah 1,2,*, Mairman Muska 1, Nasir Ahmad 2 and Khalid Khan 2

1 National Center of Excellence in Physical Chemistry, University of Peshawar, Peshawar, Khyber Pukhtoonkhwa 25120, Pakistan; [email protected] (A.N.); [email protected] (M.M.) 2 Department of Chemistry, Islamia College University Peshawar, Peshawar, Khyber Pukhtoonkhwa 25120, Pakistan; [email protected] (N.A.); [email protected] (K.K.) * Correspondence: [email protected]; Tel.: +92-331-931-6672

Academic Editor: Peter J. Rutledge Received: 23 December 2015 ; Accepted: 15 February 2016 ; Published: 28 March 2016

Abstract: Quinolones are broad-spectrum synthetic antibacterial drugs first obtained during the synthesis of chloroquine. Nalidixic acid, the prototype of quinolones, first became available for clinical consumption in 1962 and was used mainly for urinary tract infections caused by Escherichia coli and other pathogenic Gram-negative bacteria. Recently, significant work has been carried out to synthesize novel quinolone analogues with enhanced activity and potential usage for the treatment of different bacterial diseases. These novel analogues are made by substitution at different sites—the variation at the C-6 and C-8 positions gives more effective drugs. Substitution of a fluorine atom at the C-6 position produces fluroquinolones, which account for a large proportion of the quinolones in clinical use. Among others, substitution of piperazine or methylpiperazine, pyrrolidinyl and piperidinyl rings also yields effective analogues. A total of twenty six analogues are reported in this review. The targets of quinolones are two bacterial enzymes of the class II topoisomerase family, namely gyrase and topoisomerase IV. Quinolones increase the concentration of drug-enzyme-DNA cleavage complexes and convert them into cellular toxins; as a result they are bactericidal. High bioavailability, relative low toxicity and favorable pharmacokinetics have resulted in the clinical success of fluoroquinolones and quinolones. Due to these superior properties, quinolones have been extensively utilized and this increased usage has resulted in some quinolone-resistant bacterial strains. Bacteria become resistant to quinolones by three mechanisms: (1) mutation in the target site (gyrase and/or topoisomerase IV) of quinolones; (2) plasmid-mediated resistance; and (3) chromosome-mediated quinolone resistance. In plasmid-mediated resistance, the efflux of quinolones is increased along with a decrease in the interaction of the drug with gyrase (topoisomerase IV). In the case of chromosome-mediated quinolone resistance, there is a decrease in the influx of the drug into the cell.

Keywords: quinolones; analogues; gyrase; topoisomerase IV; resistance

1. Introduction In the early 20th century, infectious diseases were the most common cause of human illness leading to death, out of which bacterial infections accounted for about one-third of these infections [1]. In addition to bacterial infections, malaria also has an enormous impact on global human health, with over 200 million cases of malaria and over 600,000 deaths each year [2]. While different therapies were used for the treatment of these diseases, the introduction of antibiotic agents opened new avenues for the treatment of bacterial infections. The invention of antibiotics such as sulfonamide, and afterward penicillin by Alexander Fleming in 1928 and synthetic antibiotics such as quinolones were some of the

Molecules 2016, 21, 268; doi:10.3390/molecules21040268 www.mdpi.com/journal/molecules Molecules 2016, 21, 268 2 of 19

Molecules 2016, 21, 268 2 of 18 great milestones of science [3,4]. However, with progress in the development of antibiotics has come increasinglyincreasingly widespreadwidespread antibiotic-resistantantibiotic-resistant pathogens,pathogens, which represent an unprecedentedunprecedented healthhealth issueissue [[5].5]. Here, we focus on an important classclass of synthetic antibiotics known as quinolones, that constitute an importantimportant classclass ofof biologically-activebiologically-active broad-spectrumbroad-spectrum antibacterialantibacterial drugsdrugs [[6].6]. The spectacularspectacular discovery of nalidixic nalidixic acid, acid, which which is is chemically chemically a naphthyridone a naphthyridone and and the the prototype prototype compound compound of the of thequinolones quinolones as a as byproduct a byproduct in inchloroquine chloroquine synthesis synthesis (Figure (Figure 1)1 )by by Lesher Lesher and coworkerscoworkers inin 19621962 initiatedinitiated thethe improvementimprovement ofof quinolonesquinolones [[7,8].7,8]. It is present in a number of biologically-active naturalnatural products. Because Because of of its its valuable valuable me medicinaldicinal activities, activities, quinolones quinolones have have attracted attracted a lot a lot of ofattention attention in inthe the field field of medicinal of medicinal and andsynthetic synthetic chemistry chemistry [9]. In [9 the]. In later the 1960s, later 1960s,nalidixic nalidixic acid came acid into came clinical into clinicaluse for the use cure for theof urinary cure of tract urinary infections tract caused infections by enteric caused bacteria by enteric [10]. bacteria Around [ 10the]. 1970s, Around several the 1970s,new generations several new of quinolones, generations with of quinolones, oxolinic acid with being oxolinic the most acid prominent, being the mosthad been prominent, synthesized had beenand became synthesized available and became for clinical available use for [10–12]. clinical Un usetil [10 1980,–12]. quinolones Until 1980, quinoloneswere a less-used were a less-used class of classantibacterial of antibacterial agents and agents later and a second later a generation second generation of quinolones of quinolones was developed. was developed. In the quest In thefor questpotent for quinolones, potent quinolones, it was discovered it was discovered that alterati thaton alteration with different with differentgroups at groups positions at positions C-6 and C-6C-8 andgives C-8 more gives effective more effectiveantibacterial antibacterial analogues analogues [13]. The [13most]. The significant most significant changes in changes the basic in theskeleton basic skeletonof quinolones of quinolones are the aresubstitution the substitution of fluorine of fluorine at C-6 at C-6and and a key a key ring ring substituent substituent piperazine piperazine or methylpiperazine, pyrrolidinylpyrrolidinyl and and piperidinyl. piperidinyl. Due Due to the to insertionthe insertio of fluorinen of fluorine at C-6, mostat C-6, clinically most usedclinically quinolones used quinolones are fluoroquinolones are fluoroquinolones (FQ) [14–16 (FQ)]. [14–16].

O O

OH

N N

Nalidixic acid

Figure 1. Chemical structure of nalidixic acid.

Fluoroquinolones (formally called simply quinolones) are characterized as broad-spectrum Fluoroquinolones (formally called simply quinolones) are characterized as broad-spectrum antibacterial drugs active against both Gram positive and Gram negative bacteria. Besides antibacterial drugs active against both Gram positive and Gram negative bacteria. broad-spectrum activity, the success of fluoroquinolones can be attributed to their properties such as Besidesgood bioavailability broad-spectrum after oral activity, administration, the success relati ofvely fluoroquinolones low toxicity and can favorable be attributed pharmacokinetics to their properties[17]. Although such adverse as good bioavailabilityevents still happen after oralwhile administration, utilizing fluoroquinolones, relatively low these toxicity are and still favorable some of pharmacokineticsthe major antimicrobial [17]. Althoughagents and adverse a lot of events work stillhas happengone into while the structural utilizing fluoroquinolones,evolution in the theseframework are still of somefluoroquinolones of the major to antimicrobial make novel agentsanalogues and with a lot improved of work has potency. gone intoFluoroquinolones the structural evolutionhave been in primarily the framework utilized of for fluoroquinolones the treatment of to urinary make novel tract analoguesinfections with(UTI), improved respiratory potency. tract Fluoroquinolonesinfections, sexually have transmitted been primarily diseases, utilized gastroin fortestinal the treatment and abdominal of urinary infections, tract infections skin and (UTI), soft respiratorytissue infections tract infections,and infections sexually of bone transmitted and joints diseases, [11,18]. gastrointestinalBesides these functions, and abdominal fluoroquinolones infections, skinwith andbroad soft spectrum tissue infections activity have and infectionsbeen widely of boneutilized and in joints veterinary [11,18 ].medicine Besides to these treat functions, bacterial fluoroquinolonesinfections in food-producing with broad animals, spectrum aquaculture activity have and been pets. widely Many utilizedfluoroquinolones in veterinary are available medicine on to treatthe market; bacterial however infections the in use food-producing of these antibacterial animals, agents aquaculture highly and depends pets. Manyon the fluoroquinolones animal species and are availabletheir geographical on the market; distribution however [18]. the They use of have these a antibacterial wide range agentsof medical highly application depends on in the livestock, animal speciespoultry, and fish their and geographical domestic animal distribution in the [treatment18]. They haveand prevention a wide range of ofrespiratory, medical application enteric and in livestock,complex urinary poultry, tract fish andinfections domestic [19]. animal Some inquinolones the treatment such and as 6-chloro-7-methoxy-4(1 prevention of respiratory,H)-quinolones enteric and complexhave good urinary antimalarial tract infections activities, [19 with]. Some good quinolones activity against such as multiple 6-chloro-7-methoxy-4(1 stages of PlasmodiumH)-quinolones infection have[2]. Antimalarial good antimalarial activity activities, was also with shown good in (1 activityH)-quinolone, against multipleand its tetrahydroacridine stages of Plasmodium analogue,infection with [2]. Antimalarialsubstitution of activity various was benzenoid also shown ringin features (1H)-quinolone, and aryl moieties and its tetrahydroacridine[20]. analogue, with substitution of various benzenoid ring features and aryl moieties [20]. 2. Recent Developments in the Synthesis of Quinolones Quinolones are very important in medicinal chemistry as they have broad spectrum antibacterial activity, and they acts as anticancer and antimalarial agents. A lot of work has been carried out on the synthesis of quinolones to develop more effective and economical analogues. A number of different methods are reported for the synthesis of quinolones. In general these, procedures involve

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2. Recent Developments in the Synthesis of Quinolones Quinolones are very important in medicinal chemistry as they have broad spectrum antibacterial activity, and they acts as anticancer and antimalarial agents. A lot of work has been carried out on the synthesis of quinolones to develop more effective and economical analogues. A number of Molecules 2016, 21, 268 3 of 18 differentMolecules 2016 methods, 21, 268 are reported for the synthesis of quinolones. In general these, procedures involve3 of 18 thethe utilization utilization of of a a preactivated substrate, a a multistep strategy strategy and and harsh harsh reaction reaction conditions, which limitlimitthe utilization the the compatibility compatibility of a preactivated and and range range substrate, of of the the functional functional a multistep groups groups strategy involved and harshin in these reaction reactions conditions, [9]. [9]. The The usewhich use of of transitiontransitionlimit the compatibility metal metal catalysts catalysts and has has range also also ofbeen been the utilized utilizedfunctional fo forr groupsthe the synthesis synthesis involved of of quinolones quinolonesin these reactions and and their their [9]. analogues. analogues.The use of Thetransition catalytic metal activation catalysts of hasthe alsoC-H beenbond utilized allows the for synthesis the synthesis of a ofremarkable quinolones variety and their of heterocyclic analogues. compounds,The catalytic including includingactivation quinolones quinolonesof the C-H bond[21]. [21]. Theallows The followin following the synthesisg are are some some of a ofremarkable of the the latest latest varietyreported reported of dataheterocyclic data on on the developmentcompounds, includingof new methods quinolones for the [21]. synthesis The followin of quinolones g are some and theirof the derivatives. latest reported data on the developmentHu etet al. al. developeddevelopedof new methods a a novel novel for method the synthesis in which of quinolonesa broad range and of their 2-arylquinoline-4(1 derivatives. H)-ones were were synthesizedHu et al. byby developed aa metal-freemetal-free a novel oxidative oxidative method intermolecular intermolec in whichular a Mannichbroad Mannich range reaction reaction of 2-arylquinoline-4(1 between between secondary secondaryH)-ones amines amines were and andunmodifiedsynthesized unmodified ketonesby aketones metal-free (Scheme (Scheme 1oxidative). The 1). advantageThe intermolec advantage of thisular of methodth Mannichis method is thatreaction is itthat does itbetween does not requirenot secondary require the use the amines ofuse any of anytransitionand transitionunmodified metal metal catalyst;ketones catalyst; (Scheme and and it occurs it1). occurs The under advantage under mild mild conditions. of conditions.this method This This is method that method it does can can producenot produce require a number athe number use of ofanaloguesany analogues transition in in very metal very high catalyst;high yield yield and [6]. [6]. it occurs under mild conditions. This method can produce a number of analogues in very high yield [6]. O Transition metal free TEMPO (2equiv) O Transition3 metal free3 O R' t O R' Direct sp C-H / sp C-H coupling TEMPOKO Bu (2equiv) (2equiv) 3 3 R R' R BroadDirect substratesp C-H / scopesp C-H coupling DMSOKOtBu (0.05M)(2equiv) R' R N Ar 0 R N Ar Broad substrate scope H DMSO80 C (0.05M), 6hr H Simple and mild condition N Ar 0 N Ar H 80 C , 6hr H ExcellentSimple and yields mild condition 20 examples Excellent yields upto20 examples 98% yield upto 98% yield Scheme 1. Metal-free oxidative intermolecular Mannich reaction for the synthesis of 2-arylquinone- Scheme 1. Metal-free oxidative intermolecular Mannich reaction for the synthesis of 4(1SchemeH)-ones 1. Metal-freefrom secondary oxidative amines intermolecular and ketones Mannich[6]. reaction for the synthesis of 2-arylquinone- 2-arylquinone-4(1H)-ones from secondary amines and ketones [6]. 4(1H)-ones from secondary amines and ketones [6]. Kang and Hong performed alkynylation of 4-quinolones at three different positions for making quinolonesKang andanalogues. Hong performed In the first alkynylation method, they of 4-quinolonesutilized TIPS-EBX at three (an different alkylating positions agent) forto develop making anquinolones efficient and analogues.analogues. site-selective InIn thethe C5 firstfirst alkynylation method, method, they theyof 4- utilizedquinolones utilized TIPS-EBX TIPS-EBX (Scheme (an (an2). alkylating Inalkyla the secondting agent) agent) strategy to developto developa Ru(II) an catalyzedefficientan efficient and C2 and site-selectiveselective site-selective alkynylation C5 C5 alkynylation alkynylation was achieved of of 4-quinolones 4- throughquinolones N-pyrimidyl (Scheme (Scheme2 ).2). group-directed In In the the second second strategy strategycross-couplings aa Ru(II)Ru(II) tocatalyzed obtained C2 useful selectiveselective C2-alkynylat alkynylationalkynylationed was4-quinolones, achievedachieved throughthrough while isoquinolones N-pyrimidylN-pyrimidyl group-directedgroup-directed were developed cross-couplingscross-couplings through C3 alkynylation.to obtainedobtained usefulusefulThese C2-alkynylatedthreeC2-alkynylat differented routes 4-quinolones, of producing while quinolones isoquinolones analogues were are developed site selective through methods C3 andalkynylation. useful for These These obtaining three effectivedifferent derivativesroutes of producing [22]. quinolones analogues are site selectiveselective methods and useful for obtaining effective derivativesderivatives [[22].22]. i Si Pr3 i Si Pr3 O H O TIPS-EBX TIPS-EBX 5 O H O [Ru]TIPS-EBX or [Rh] TIPS-EBX[Rh] 5 [Rh] R=[Ru] Pyrimidyl or [Rh] N R= alkyl N 2 H N R= alkyl R= Pyrimidyl i RN RN 2 H RN Si Pr3 i R R R Si Pr3 Scheme 2. Site selective C-H alkynylation of Quinolones [22]. Scheme 2. Site selective C-H alkynylation of Quinolones [22]. Scheme 2. Site selective C-H alkynylation of Quinolones [22]. Satio et al. synthesized chiral substituted 2,3-dihydro-4-quinolones in high yields using an aza-MichaelSatio et additional. synthesized reaction chiral (Scheme substituted 3). The 2,3-dihydradvantageo-4-quinolones of this method in is high the enantioselectiveyields using an productionaza-MichaelSatio et of addition al. compoundssynthesized reaction and chiral (Schemesome substituted of the3). Theanalogues 2,3-dihydro-4-quinolonesadvantage can beof purifiedthis method in a in singleis high the recrystallization yieldsenantioselective using an step.aza-Michaelproduction The chiral of addition compounds 2,3-dihydro-4-quinolones reaction and (Schemesome of3 the). can Theanalogues be advantageused for can the ofbe preparation thispurified method in of a isothersingle the useful enantioselective recrystallization quinolone analogues.productionstep. The chiral The of compounds2-substituted 2,3-dihydro-4-quinolones and2,3-dihy somedro-4-quinolones of the can analogues be used halt for can mitosis the be preparation purified and are useful in of a singleother antitumor useful recrystallization agents quinolone [23]. step.analogues. The chiral The 2-substituted 2,3-dihydro-4-quinolones 2,3-dihydro-4-quinolones can be used halt for mitosis the preparation and are useful of other antitumor useful agents quinolone [23]. analogues. The 2-substituted 2,3-dihydro-4-quinolones halt mitosis andX are useful antitumor O O agents [23]. X O O O Catalyst O P O benzene / C H OO Catalyst 6 12 N R P OH NH2 R v/v = 1/1 benzene / C6H12 H O OH NH R 0 N R PG free amine2 v/v70 =C 1/1 upto quantitativeH yield X 700C up to 94% ee X PG free amine upto quantitative yield X= C6F5 up to 94% ee X= C6F5 Catalyst Catalyst Scheme 3. Preparation of chiral 2-substituted 2,3-dihydro-4-quinolones through chiral phosphoric acidScheme catalyzed 3. Preparation intra-molecular of chiral aza-Michael 2-substituted addition 2,3-dihy reaction.dro-4-quinolones through chiral phosphoric acid catalyzed intra-molecular aza-Michael addition reaction.

Molecules 2016, 21, 268 3 of 18

the utilization of a preactivated substrate, a multistep strategy and harsh reaction conditions, which limit the compatibility and range of the functional groups involved in these reactions [9]. The use of transition metal catalysts has also been utilized for the synthesis of quinolones and their analogues. The catalytic activation of the C-H bond allows the synthesis of a remarkable variety of heterocyclic compounds, including quinolones [21]. The following are some of the latest reported data on the development of new methods for the synthesis of quinolones and their derivatives. Hu et al. developed a novel method in which a broad range of 2-arylquinoline-4(1H)-ones were synthesized by a metal-free oxidative intermolecular Mannich reaction between secondary amines and unmodified ketones (Scheme 1). The advantage of this method is that it does not require the use of any transition metal catalyst; and it occurs under mild conditions. This method can produce a number of analogues in very high yield [6].

O O Transition metal free TEMPO (2equiv) 3 3 R' Direct sp C-H / sp C-H coupling KOtBu (2equiv) R' R R DMSO (0.05M) Broad substrate scope N Ar 0 N Ar H 80 C , 6hr H Simple and mild condition Excellent yields 20 examples upto 98% yield Scheme 1. Metal-free oxidative intermolecular Mannich reaction for the synthesis of 2-arylquinone- 4(1H)-ones from secondary amines and ketones [6].

Kang and Hong performed alkynylation of 4-quinolones at three different positions for making quinolones analogues. In the first method, they utilized TIPS-EBX (an alkylating agent) to develop an efficient and site-selective C5 alkynylation of 4-quinolones (Scheme 2). In the second strategy a Ru(II) catalyzed C2 selective alkynylation was achieved through N-pyrimidyl group-directed cross-couplings to obtained useful C2-alkynylated 4-quinolones, while isoquinolones were developed through C3 alkynylation. These three different routes of producing quinolones analogues are site selective methods and useful for obtaining effective derivatives [22].

i Si Pr3

O H O TIPS-EBX TIPS-EBX 5 [Rh] [Ru] or [Rh] R= Pyrimidyl N R= alkyl N 2 H N i R R R Si Pr3 Scheme 2. Site selective C-H alkynylation of Quinolones [22].

Satio et al. synthesized chiral substituted 2,3-dihydro-4-quinolones in high yields using an aza-Michael addition reaction (Scheme 3). The advantage of this method is the enantioselective production of compounds and some of the analogues can be purified in a single recrystallization Moleculesstep.2016 The, 21 chiral, 268 2,3-dihydro-4-quinolones can be used for the preparation of other useful quinolone4 of 19 analogues. The 2-substituted 2,3-dihydro-4-quinolones halt mitosis and are useful antitumor agents [23].

X O O O O Catalyst P benzene / C6H12 O OH NH R N R 2 v/v = 1/1 H 0 PG free amine 70 C upto quantitative yield X up to 94% ee X= C6F5 Catalyst Scheme 3. Preparation of chiral 2-substituted 2,3-dihydro-4-quinolones through chiral phosphoric Scheme 3. Preparation of chiral 2-substituted 2,3-dihydro-4-quinolones through chiral phosphoric acid acid catalyzed intra-molecular aza-Michael addition reaction. catalyzed intra-molecular aza-Michael addition reaction.

Molecules 2016, 21, 268 4 of 18 Kuppusamy and Kasi synthesized enantiomerically enriched 2-aryl-2,3-dihydroquinolin-4(1H)- Kuppusamy and Kasi synthesized enantiomerically enriched 2-aryl-2,3-dihydroquinolin-4(1H)- ones (inMolecules up to 2016 99%, 21, yield)268 in a one pot synthesis using per-6-ABCD (Scheme4). This synthesis method4 of 18 is ones (in up to 99% yield) in a one pot synthesis using per-6-ABCD (Scheme 4). This synthesis very useful for obtaining enantiomer analogues of quinolones. The yield is very high and it is one-pot methodKuppusamy is very useful and Kasifor obtaining synthesized enantiomer enantiomerica analogueslly enriched of quinolones. 2-aryl-2 The,3-dihydroquinolin-4(1 yield is very high andH)- synthesisonesit is one-pot which(in up synthesis occursto 99% inyield) whic a single hin occurs a stepone in pot reactiona single synthe step withsis reaction using reuse per-6-ABCD with of the reuse expensive of (Scheme the expensive catalyst 4). This catalyst after synthesis separation after frommethodseparation the product. is very from The useful the disadvantage product. for obtaining The disadvantage of enantiomer this method ofanalogues this is that method theof quinolones. low is that temperature the Thelow yieldtemperature and is longvery highand reaction longand time reactionitreaction is conditionsone-pot time synthesis reaction are a whic bitconditions cumbersomeh occurs are in a a bit single [24 cumbersome]. step reaction [24]. with reuse of the expensive catalyst after separation from the product. The disadvantage of this method is that the low temperature and long reaction time reaction conditionsO are a bit cumbersome [24]. O O Per-6-ABCD O + O Ethanol , Water NH2 R N R O 15hrPer-6-ABCD , -50C H + Ethanol , Water 20 examples NH2 R N R 15hr , -50C ee up toH 99% 20 examples R= C6H5- , p-NO2C6H4-, p-CH3OC6H4-, cyclohexyl-, heterocycles-, etc ee up to 99% Scheme 4. Per-6-ABCD catalyzed asymmetric one-pot synthesis of 2-aryl-2,3-dihydro-4-quinolones Scheme 4. Per-6-ABCDR= C catalyzed6H5- , p-NO asymmetric2C6H4-, p-CH one-pot3OC6H4-,synthesis cyclohexyl-, of heterocycles-, 2-aryl-2,3-dihydro-4-quinolones etc using substituted aldehydes [24]. using substituted aldehydes [24]. Scheme 4. Per-6-ABCD catalyzed asymmetric one-pot synthesis of 2-aryl-2,3-dihydro-4-quinolones usingǺkerbladh substituted et al. aldehydessynthesized [24]. functionalized 4-quinolones from 2-iodoanilines and alkynes using Åkerbladhtwo differentet protocols al. synthesized for which functionalized molybdenum 4-quinolones hexacarbonyl from was 2-iodoanilinesused as a solid andsource alkynes of CO using two different(SchemeǺkerbladh 5). protocols In one et al. of synthesized the for methods, which functionalized molybdenum the reaction co 4-quinolonesnditions hexacarbonyl include from was microwave2-iodoanilines used asheating aand solid alkynesof 120 source °C using and of CO (Schemetwothe 5 cyclizeddifferent). In one product protocols of the was methods, for obtained which the molybdenumin reaction 20 min in conditions high hexa yield.carbonyl include The wassecond microwave used procedure as a solid heating involves source of 120theof COuse˝C and the cyclized(Schemeof sensitive product 5). Insubstituents one was of the obtained methods, like nitro- in the 20 and reaction min bromo- in highconditionsgroups, yield. includebut The the second microwave reaction procedure is heating performed involves of 120 at °C room the and use of the cyclized product was obtained in 20 min in high yield. The second procedure involves the use sensitivetemperature. substituents This likesecond nitro- method and bromo-groups,is also one-pot synt buthesis the reactionthat occurs is performedin two reaction at room steps. temperature. In both of thesensitive synthesis substituents methods palladiulike nitro-m is and used bromo- as a catalystgroups, [25]. but the reaction is performed at room This second method is also one-pot synthesis that occurs in two reaction steps. In both of the synthesis temperature. This second method is also one-pot synthesis that occurs in two reaction steps. In both methods palladium is used as a catalyst [25]. Method A of the synthesis methods palladium is used as a catalyst [25]. O I Pd (dba)Method , dppf , AMo(CO) , Et NH R1 + R2 2 3 6 2 R2 0 O H N MW , 120 C , 20min R1 N 2 I H Pd (dba) , dppf , Mo(CO) , Et NH R1 + R2 2 3 6 2 R2 0 13+20 entries H N MW , 120 C , 20min R1 N 2 up Hto 85% yield 13+20 entries Pd(OAc) 2 up to 85% yield {HP(tBu)3}BF4 O Pd(OAc) Et NH Mo(CO)6 , 2Et3N 2 2 {HP(tBu) }BF R rt, 16hr3 4 R1 O rt, 5hr H2N Et NH Mo(CO)6 , Et3N 2 R2 rt, 16hr R1 rt, 5hr HMethod2N B

Method B Scheme 5. Synthesis of 4-quinolones via palladium catalyzed carbonylative Sonogashira cross-coupling Scheme 5. Synthesis of 4-quinolones via palladium catalyzed carbonylative Sonogashira cross-coupling reaction [25]. reactionScheme [25]. 5. Synthesis of 4-quinolones via palladium catalyzed carbonylative Sonogashira cross-coupling reactionMonastyrskyi [25]. et al. developed a protocol for the synthesis of 3-aryl-4(1H)-quinolones from ethyl acetoacetate (EAA) using hypervalent diaryliodonium salts under mild and metal-free conditions (SchemeMonastyrskyi 6). This method et al. developed has also a beenprotocol applied for the for synthesis the synthesis of 3-aryl-4(1 of theH antimalarial)-quinolones compoundfrom ethyl acetoacetateELQ-300. This (EAA) compound using hypervalentis active against diaryliodonium Plasmodium falciparumsalts under and mild Plasmodium and metal-free vivax at conditions all stages (Schemeof its lifecycle 6). This and method is in the has preclinical also been testing applied stage. for The the toxicity synthesis of thisof theproduct antimalarial is not yet compound reported, ELQ-300.but the preclinical This compound data provides is active important against Plasmodium information falciparum concerning and efficacy. Plasmodium The vivaxusefulness at all ofstages this ofsynthetic its lifecycle method and isis itsin productthe preclinical that is testing highly stage.desirable, The toxicityas malaria of thisis one product of the is deadliest not yet reported,diseases, butaffecting the preclinical millions of data people provides in Asia important and Africa informat [26]. ion concerning efficacy. The usefulness of this synthetic method is its product that is highly desirable, as malaria is one of the deadliest diseases, affecting millions of people in Asia and Africa [26].

Molecules 2016, 21, 268 5 of 19

Monastyrskyi et al. developed a protocol for the synthesis of 3-aryl-4(1H)-quinolones from ethyl acetoacetate (EAA) using hypervalent diaryliodonium salts under mild and metal-free conditions (Scheme6). This method has also been applied for the synthesis of the antimalarial compound ELQ-300. This compound is active against Plasmodium falciparum and Plasmodium vivax at all stages of its lifecycle and is in the preclinical testing stage. The toxicity of this product is not yet reported, but the preclinical data provides important information concerning efficacy. The usefulness of this synthetic method is its product that is highly desirable, as malaria is one of the deadliest diseases, affecting millions of people

Moleculesin Asia 2016 and, Africa21, 268 [26]. 5 of 18 Molecules 2016, 21, 268 5 of 18 O O 1 O O COOEt R Ar IX O 1 O 2 COOEt R Base,Ar2IX DMF Conrad-Limphac COOEt R1 N Base, DMF Conrad-Limphac R2 H COOEt R1 N EAA R3-aryl-4(1H)-quinolones2 H EAA 3-aryl-4(1H)-quinolones Scheme 6. Metal free arylation of ethylacetoacetate with hypervalent diaryliodonium for the Scheme 6. Metal free arylation of ethylacetoacetate with hypervalent diaryliodonium for the synthesis synthesisScheme 6.of 3-aryl-4(1Metal freeH )-quinolonesarylation of [26]. ethylacetoacetate with hypervalent diaryliodonium for the of 3-aryl-4(1H)-quinolones [26]. synthesis of 3-aryl-4(1H)-quinolones [26]. Wang et al. synthesized a wide variety of quinolones in a high yield in one step process using a mild WangN-H etactivation/Heck al. synthesizedsynthesized reactiona a wide wide variety varietymethod of of quinolones quinolones(Scheme 7). in in aThis ahigh high process yield yield in in inone onevolves step step processthe process palladium using using a catalyzed-oxidativeamild mild N-H N-H activation/Heck activation/Heck annulation reaction of reaction acrylamide method method with (Sch (Scheme strainedeme 77). ).arynes. This This This process technique involvesinvolves was the themade palladiumpalladium possible forcatalyzed-oxidative the first time as previousannulation attempts of acrylamide by other with researchers strained resulted arynes. This in lower technique yields was [27]. made possible for the firstfirst time as previousprevious attemptsattempts byby otherother researchersresearchers resultedresulted inin lowerlower yieldsyields [[27].27]. TfO 3 TfO R TMS R3 TMS

CsF O CsF O Pd(OAc) (5 mol%) 1 2 R O OMe R1 O OMe N Pd(OAc) (5 mol%) 1 Cu(OAc)22 (2equiv) N R OMe+ R3 R1 OMe 2 HN Cu(OAc)2 (2equiv) 2 N R H R3 DMSO/Dioxane = 1/9 R H + R2 H DMSO/Dioxane = 1/9 R2 R3 3 Scheme 7. Palladium catalyzed-oxidative annulation of acrylamide with strained arynesR for the synthesisScheme 7.of Palladiumquinolones catalyzed-oxidative[27]. annulation of acrylamide with strained arynes for the Scheme 7. Palladium catalyzed-oxidative annulation of acrylamide with strained arynes for the synthesis of quinolones [27]. synthesis of quinolones [27]. Manikandan and Jeganmohan prepared 2-quinolones by Ru-catalyzed cyclization of anilides with propiolatesManikandan or acrylates and Jeganmohan with further prepar conversioned 2-quinolones into 3-halo-2-quinolones by Ru-catalyzed cyclization and 2-chloro-quinolones of anilides with (SchemepropiolatesManikandan 8). Theor acrylates 2-quinolones and Jeganmohan with producedfurther prepared conversion through 2-quinolones this into mechanism 3-halo-2-quinolones by Ru-catalyzed are very cyclizationuseful and 2-chloro-quinolonesfor the of anilidessynthesis with of otherpropiolates(Scheme quinolone 8). orThe acrylates 2-quinolonesderivatives, with and furtherproduced are prepared conversion through in this intoexcellent mechanism 3-halo-2-quinolones yield. Theare very mechanisms useful and 2-chloro-quinolones for proposed the synthesis for the of above(Schemeother quinolonereactions8). The 2-quinoloneswerederivatives, strongly and produced supported are prepared through by experi in this excellentmental mechanism evidence yield. areThe and very mechanisms deuterium useful for proposedlabeling the synthesis studies, for the of suggestingotherabove quinolonereactions easy were derivatives,isolation strongly of andre supportedaction are prepared intermediates by experi in excellentmental during evidence yield. synt Thehesis. and mechanisms deuteriumThe halogenated proposedlabeling products studies, for the madeabovesuggesting through reactions easy such wereisolation synthesis strongly of methodsre supportedaction stillintermediates by need experimental to be duringassayed evidence syntfor theirhesis. and toxicity deuteriumThe halogenated in mouse labeling models products studies, for theirsuggestingmade future through easymedicinal such isolation synthesis value of reaction[28]. methods intermediates still need to during be assayed synthesis. for their The toxicity halogenated in mouse products models made for throughtheir future such medicinal synthesis value methods [28]. still need to be assayed for their toxicity in mouse models for their future medicinal value [281-]. Ru(II)AgSbF6 H Cu(OAc) , H O H 1- Ru(II)A2gSbF2 6 H N O N Me N O 0 H DCE,Cu(OAc) 1102,C, H2 12hrO H Ru(II)AgSbF6 H N O N O Me N O 0 DCE, 110 C, 012hr 1 RCOOH,Ru(II)AgSbF i-PrOH6 H 1 2- 30% HCl, 130 C, 10hr R R1 R O 0 2 04 1 RCOOH,130 C, i-PrOH 24hr R H 1 2- 30% HCl, 130ORC, 10hr R R1 R 2 0 3 2 4 R 130 C, COOR24hr R O OR R2 COOR3 O H N O H NBS or NCS N O AIBN 1 X NBS or NCS R X= Br, Cl R2 AIBN 1 X R X= Br, Cl R2

N Cl POCl3 N Cl POCl R1 3 R2 1 R 2 Scheme 8. Synthesis of 2-quinolones through rutheniumR catalyzed cyclization of anilides with substitutedScheme 8. propiolatesSynthesis of or 2-quinolonesacrylates [28]. through ruthenium catalyzed cyclization of anilides with substituted propiolates or acrylates [28].

Molecules 2016, 21, 268 5 of 18

O O 1 O COOEt R Ar2IX Base, DMF Conrad-Limphac COOEt R1 N R2 H EAA 3-aryl-4(1H)-quinolones Scheme 6. Metal free arylation of ethylacetoacetate with hypervalent diaryliodonium for the synthesis of 3-aryl-4(1H)-quinolones [26].

Wang et al. synthesized a wide variety of quinolones in a high yield in one step process using a mild N-H activation/Heck reaction method (Scheme 7). This process involves the palladium catalyzed-oxidative annulation of acrylamide with strained arynes. This technique was made possible for the first time as previous attempts by other researchers resulted in lower yields [27].

TfO R3 TMS

CsF O O Pd(OAc)2 (5 mol%) R1 OMe R1 OMe N Cu(OAc)2 (2equiv) N R3 H + R2 H DMSO/Dioxane = 1/9 R2

R3 Scheme 7. Palladium catalyzed-oxidative annulation of acrylamide with strained arynes for the synthesis of quinolones [27].

Manikandan and Jeganmohan prepared 2-quinolones by Ru-catalyzed cyclization of anilides with propiolates or acrylates with further conversion into 3-halo-2-quinolones and 2-chloro-quinolones (Scheme 8). The 2-quinolones produced through this mechanism are very useful for the synthesis of other quinolone derivatives, and are prepared in excellent yield. The mechanisms proposed for the above reactions were strongly supported by experimental evidence and deuterium labeling studies, suggesting easy isolation of reaction intermediates during synthesis. The halogenated products Molecules 2016, 21, 268 6 of 19 made through such synthesis methods still need to be assayed for their toxicity in mouse models for their future medicinal value [28].

1- Ru(II)AgSbF6 H Cu(OAc)2, H2O H H N O N Me N O DCE, 1100C, 12hr Ru(II)AgSbF6 O 2- 30% HCl, 1300C, 10hr 1 RCOOH, i-PrOH 1 H R1 R R 1300C, 24hr R2 OR4 R2 COOR3 O H N O NBS or NCS AIBN 1 X R X= Br, Cl R2

N Cl POCl3

R1 R2 Scheme 8. Synthesis of 2-quinolones through ruthenium catalyzed cyclization of anilides with Scheme 8.substitutedSynthesis propiolates of 2-quinolones or acrylates [28]. through ruthenium catalyzed cyclization of anilides with substituted propiolates or acrylates [28].

Suet et al. synthesized and evaluated bisaryl quinolones for biological activities against P. falciparum [29]. Pateland his coworkers synthesized a series of novel pyridoquinolone analogues by introducing sulfonamide, urea, thiourea, amine, amide, piperazine and β-lactam moieties in the pyridoquinolone molecule. These quinolones on the one hand inhibit gyrase and topoisomerase IV of bacteria while the other attached groups are also inhibitors of specific metabolic pathways. These medicinal molecules have antibacterial and antifungal activities and some of them are even active against drug resistant Mycobacterium tuberculosis strains. However there is a need to test these novel quinolones in mammals for their potential toxicity [30–36]. Komarnicka et al. made phosphine derivatives of sparfloxacin in high yield by treating sparfloxacin with methoxy(diphenyl)phosphine. The oxide derivative was made by treating the aminomethyl(diphenyl)phosphine analog of sparfloxacin with 35% hydrogen peroxide in chloroform at room temperature with continuous stirring for 3 h. The aminomethyl(diphenyl)phosphine analog of sparfloxacin showed antibacterial activity of similar nature to sparfloxacin, while the oxide form had high antitumor activity against cancer cell lines [37]. One of the beautiful modifications in the existing quinolone is the production of a series of 1,2,4-triazole-ciprofloxacin hybrid compounds that have potential antibacterial potency against both Gram-positive as well as Gram-negative bacteria. These 1,2,4-triazole-ciprofloxacin hybrid compounds were more active than ciprofloxacin against both vulnerable and quinolone resistant bacteria. Such promising hybrid triazole analogues of other quinolone compounds should also be prepared and tested against both resistant and non-resistant bacterial strains that cause different diseases. In addition to determine their action against pathogenic bacteria, they should also be tested for viral infections, especially HIV [38]. A number of new natural antibiotics are being discovered that can target fluoroquinolone resistant bacteria (FQR). For example deoxynymbomycins can target the mutant bacterial DNA gyrase and can be used against FQR bacteria but the chances of resistance to this new drug are still there [39]. Similarly, the FQR Neisseria gonorrhoeae type II topoisomerase can be inhibited by a novel compound called spiropyrimidinetrione AZD0914 [40] so on one hand we have the continuously evolving resistant strains to quinolones and other antibacterial agents, but on the other hand scientific community is working hard to design and synthesize new drugs for the resistant bacterial strains.

3. Analogues of Quinolones The first member of the quinolone family was nalidixic acid, which was synthesized as a byproduct in the synthesis of chloroquine. Later, it was modified in order to enhance its activities. One modification was the substitution of fluorine at the C-6 position, which was found to provide good antibacterial activities. Moreover, substitution of piperazine or methyl piperazine, pyrrolidinyl and piperidinyl moieties was also determined to result in increased antibacterial activities. Following are the analogues of quinolones available in the common literature (Figure2)[15,16,41–48]. Molecules 2016, 21, 268 6 of 18

Suet et al. synthesized and evaluated bisaryl quinolones for biological activities against P. falciparum [29]. Pateland his coworkers synthesized a series of novel pyridoquinolone analogues by introducing sulfonamide, urea, thiourea, amine, amide, piperazine and β-lactam moieties in the pyridoquinolone molecule. These quinolones on the one hand inhibit gyrase and topoisomerase IV of bacteria while the other attached groups are also inhibitors of specific metabolic pathways. These medicinal molecules have antibacterial and antifungal activities and some of them are even active against drug resistant Mycobacterium tuberculosis strains. However there is a need to test these novel quinolones in mammals for their potential toxicity [30–36]. Komarnicka et al. made phosphine derivatives of sparfloxacin in high yield by treating sparfloxacin with methoxy(diphenyl)phosphine. The oxide derivative was made by treating the aminomethyl(diphenyl)phosphine analog of sparfloxacin with 35% hydrogen peroxide in chloroform at room temperature with continuous stirring for 3 h. The aminomethyl(diphenyl)phosphine analog of sparfloxacin showed antibacterial activity of similar nature to sparfloxacin, while the oxide form had high antitumor activity against cancer cell lines [37]. One of the beautiful modifications in the existing quinolone is the production of a series of 1,2,4-triazole-ciprofloxacin hybrid compounds that have potential antibacterial potency against both Gram-positive as well as Gram-negative bacteria. These 1,2,4-triazole-ciprofloxacin hybrid compounds were more active than ciprofloxacin against both vulnerable and quinolone resistant bacteria. Such promising hybrid triazole analogues of other quinolone compounds should also be prepared and tested against both resistant and non-resistant bacterial strains that cause different diseases. In addition to determine their action against pathogenic bacteria, they should also be tested for viral infections, especially HIV [38]. A number of new natural antibiotics are being discovered that can target fluoroquinolone resistant bacteria (FQR). For example deoxynymbomycins can target the mutant bacterial DNA gyrase and can be used against FQR bacteria but the chances of resistance to this new drug are still there [39]. Similarly, the FQR Neisseria gonorrhoeae type II topoisomerase can be inhibited by a novel compound called spiropyrimidinetrione AZD0914 [40] so on one hand we have the continuously evolving resistant strains to quinolones and other antibacterial agents, but on the other hand scientific community is working hard to design and synthesize new drugs for the resistant bacterial strains.

3. Analogues of Quinolones The first member of the quinolone family was nalidixic acid, which was synthesized as a byproduct in the synthesis of chloroquine. Later, it was modified in order to enhance its activities. One modification was the substitution of fluorine at the C-6 position, which was found to provide good antibacterial activities. Moreover, substitution of piperazine or methyl piperazine, pyrrolidinyl Molecules 2016 21 and ,piperidinyl, 268 moieties was also determined to result in increased antibacterial activities. Following 7 of 19 are the analogues of quinolones available in the common literature (Figure 2) [15,16,41–48].

O O O O O O F OH O OH OH

N N O N N N HN

Nalidixic acid Oxolinic acid Norfloxacin O O O O F OH F OH N N HN N N N O

Molecules 2016, 21, 268Ci profloxacin Ofloxacin 7 of 18 O O O O F F OH H OH N N N N N O O N H H Levofloxacin Moxifloxacin O O NH2 O O F F OH OH H N 2 N N N N O O HN F N

Sparfloxacin 8-methoxyfloroquinolone O O O O F F OH OH HN N N N HN N N N SN O O 6-Fluoro-7-(3-methoxy-4-methylamino-pyrrolidin-1-yl) -4-oxo-1-phenyl-1,4-dihydro-[1,8]naphthyridine-3- Voreloxin carboxylic acid O O

F OH F O O HN N N F O OH O N N 5-Fluoro-6-(3-methoxy-4-methylamino- HN F pyrrolidin-1-yl)-3-oxo-3H-7-oxa-11b-aza- benzo[de]anthracene-2-carboxylic acid Orbifloxacin O O NH2 O O F F OH OH

N N N N HN F HN O

Lomefloxacin Gatifloxacin O O O O F OH OH O N N N N N HN O H2N CHF2 Garenofloxacin Gemifloxacin

Figure 2. Cont.

Molecules 2016, 21, 268 8 of 19

Molecules 2016, 21, 268 8 of 18

O O F OH O O F H N N N OH F H N N N 2 H HN F Trovafloxacin Enoxacin O O O O F OH F OH H2N N N N N

Enrofloxacin Pazufloxacin O O O O F F OH OH O N N O N N S Cl O N H2N H Prulifloxacin Besifloxacin NH2 O O O O O O F F F OH OH OH HN N N N N N N N O Cl O H2N F Balofloxacin Antofloxacin Sitafloxacin O O O O O O F F F OH OH OH

N N N N N N N

Enrofloxacin Danofloxacin Flumequine O O O O

F OH F OH NH2 O O N N N N F OH HN N

N N N O N F F Marbofloxacin Sarafloxacin Difloxacin Figure 2. Common analogues of quinolones used for the treatment of different diseases. Figure 2. Common analogues of quinolones used for the treatment of different diseases. 4. Clinical Use 4. Clinical Use There are a number of quinolone analogues used in clinical practice all over the world due to theirThere increased are a number antibacterial of quinolone range and favorable analogues pharmacokinetics, used in clinical while practice the search all over for new the hybrid world due to theirnext increasedgeneration quinolones antibacterial is ongoing. range Among and favorable the most suitable pharmacokinetics, quinolones candidates while the that search are highly for new hybridprescribed next generation are gatifloxacin, quinolones levofloxacin, is ongoing. ciprofloxa Amongcin. There the most are several suitable quinolones quinolones that candidateshave more that toxicity for the patients than usefulness, like halogenated quinolones, but such toxic analogues are highly prescribed are gatifloxacin, levofloxacin, ciprofloxacin. There are several quinolones that have more toxicity for the patients than usefulness, like halogenated quinolones, but such toxic analogues should be used when no other option is available and the toxic damage is less Molecules 2016, 21, 268 9 of 19 as compared to the pathogen harm [49]. The quinolones are also suitable choice for lung infections like cystic fibrosis. Clinafloxacin is cytotoxic, but it can be used against multi-drug resistant Burkholderia cepacia [49]. Pseudomonas aeruginosa-related respiratory diseases can also be treated best with anionic fluroquinolones. In P. aeruginosa based diseases like cystic fibrosis where they form a thick biofilm of anionic molecules such as rhamnolipids and alginate, it is difficult for most antibiotics to penetrate and inhibit bacterial growth. However the negatively charged fluoroquinolones have the ability to cross the mucus and thus have better pharmacodynamics due to their negatively charged nature [50]. Some quinolones like ciprofloxacin, levofloxacin, moxifloxacin, ofloxacin, and gatifloxacin are also approved by Federal Drug Administration (FDA) for pediatric use for different diseases like conjunctivitis, otitis, sinusitis, respiratory tract infections, pneumonia, UTI and gastrointestinal diseases [51]. Fluoroquinolones can be used to cure tuberculosis (TB) as other active agents used for treatment take six months or more, while FQ can shorter this time span [52]. The development of multi-drug resistance in Mycobacterium tuberculosis strains has already alarmed the medical community as these strains are highly resistant to antibiotics. Quinolones also suffer the same fate as antibiotics as several strains of M. tuberculosis generate mutations in their DNA gyrase enzyme encoded by GyrA and GyrB genes [53–55]. Mutations in the amino acids Ala90 and Asp94 of GyrA of several strains of M. tuberculosis resulted in high levels of resistance to levofloxacin [56]. The level of resistance to various analogues of quinolones is different in different countries [54,57–61]. The quinolones can also be used to treat diseases that occur as a result of excessive production of aldosterone. Some quinolone analogues can inhibit a group of mitochondrial cytochrome P450 (CYP) enzymes that are involved in aldosterone biosynthesis. Thus they can be used for the treatment of heart related diseases like hypertension, primary aldosteronism, congestive heart failure, cardiac fibrosis, ventricular remodelling, and diabetic nephropathy that occurs due to high level of aldosterone production [62]. Beside the antibacterial role of quinolone, there are certain quinolonyl diketo acid analogues that are active against human immunodeficiency virus (HIV). They act against multiple viral enzymes that include reverse transcriptase, integrase and ribonuclease. These agents bind in the active site of integrase, ribonuclease and reverse transcriptase with the help of magnesium ion with acidic residues in their catalytic site and in that way they halt the function of these enzymes [63–67]. These quinolonyl diketo acid analogues are synthesized by introduction of various alkylating group at the nitrogen atom of the quinolinone ring [63]. Similarly the mono and bifunctional quinolinonyl diketo acids are also potent inhibitors of strand transfer function of HIV integrase, which are synthesized by treating aniline with ethyl orthoformate and ethyl acetoacetate [65–67]. The antitumor activities of quinolones were also investigated by different research groups. A Mannich base can be prepared from ciprofloxacin and kojic acid that possesses antitumor activities. This Mannich base and its complex with copper are toxic to cancerous cells in different ways that include inhibition of replication, disruption of polarization of mitochondrial membrane resulting into loss of ATP activity of cancerous cell [68]. It is also reported that quinolones acts against other types of cancer where its mode of action is suggested to be inhibition of topoisomerase II. In veterinary medicine the quinolones are also prescribed due to their favorable pharmacodynamics and pharmacokinetics, especially for various types of diseases and wounds in dogs and cats. The commonly prescribed fluoroquinolones for different dog and cat diseases includes ciprofloxacin, difloxacin, enrofloxacin, marbofloxacin, orbifloxacin and pradofloxacin [69,70].

5. Targets of Quinolones The main targets of quinolones are DNA topoisomerases. These enzymes are essential for DNA replication, transcription, recombination and condensed DNA remodeling, and function by carrying out transient single- and double-strand breaks. The topoisomerases modulate the supercoiling of DNA to enable proper function and interaction with proteins. These enzymes also have a fundamental role in most nucleic acid processes, like helping to control levels of DNA under- and over-winding, as Molecules 2016, 21, 268 10 of 19 well as removing knots and tangles from bacterial chromosomes [71–73]. The enzymes that cleave just one strand of the DNA are called type I topoisomerases, and they are further divided into type IA, based on the protein covalent linkage to 5’-phosphate, or type IB if the linkage is to 3’ phosphate of the DNA strand. The type II topoisomerases cleave both strands of the double stranded DNA concurrently. The binding and subsequent hydrolysis of ATP with type II topoisomerase results in a conformational changes in DNA. In most of the bacteria, two different but structurally similar type II topoisomerases are present, namely: gyrase and topoisomerase-IV. Both gyrase and topoisomerase-IV are heterotetrameric subunits. The gyrase has two each GyrB and GyrA subunits while the topoisomerase-IV contains two subunits each of ParE and ParC subunits in Gram-positive bacteria. On the other hand GrlA and GrlB are the subunits of topoisomerase-IV in the case of Gram-positive species [71,74–77]. Schoeffler and Berger have reviewed the structure and function of the type II topoisomerase mechanism in great detail, as well as the role of their ATP-dependent domains [76,78,79]. The processes of ligation and DNA cleavage, which make up the central part of enzyme function, use a non-canonical two-metal ion mechanism [80,81]. Gyrase and topoisomerase-IV make staggered cuts in DNA that are four base pairs apart and on opposite strands, leaving a 5’ overhang. In this process, a covalent bond is formed between active site tyrosine residues and the newly generated 5’ DNA termini, in order to maintain a genomic integrity [16,71,74,75,77]. It is noteworthy that humans also have type II enzymes; topoisomerase IIα and topoisomerase IIβ which have significant amino acid sequence similarity to bacterial enzymes, but in humans, the genes that code for subunits A and B are fused, forming a single polypeptide chain. Thus, the human type II enzymes function as homodimers. Due to this difference, the clinically-relevant quinolones can discriminate between the human and bacterial type II topoisomerases [16,71,82].

6. Mode of Action of Quinolones It was first recognized in 1977 that the bacterial enzyme gyrase is the primary target of quinolones. Later, with the discovery of topoisomerases, it was suggested that this enzyme could also be a target of quinolones. Analysis of Escherichia coli strains carrying drug-resistance mutations in these enzymes showed that quinolones inhibit gyrase and topoisomerase-IV as primary and secondary targets, respectively [8]. There are several high-resolution structures of topoisomerase in different catalytic modes with DNA, which have shown how the topoisomerases modify the topology of DNA [83–85]. In a recent investigation, the interaction of quinolones and type II topoisomerase of human and bacteria are mediated by different types of interactions. It was shown that the quinolones target the gyrase and topoisomerase-IV enzymes and inhibit bacterial growth [86]. These enzymes have the potential to fragment the cell genome, which is essential for cell survival and reproduction. Quinolones inhibit cell growth and division by increasing the concentration of enzyme-DNA cleavage complexes [16,73]. Thus quinolones are referred to as topoisomerase poisons as they convert gyrase and topoisomerase IV into cellular toxins [87].

7. Interaction of Quinolones with Topoisomerases The interaction of drugs with proteins plays an important role in stabilizing the complexes. In the case of Gram-negative bacteria, quinolones bind to gyrase, while in Gram-positive bacteria, the drugs preferentially bind to topoisomerase-IV [88,89]. The amino acids that are usually associated with quinolone-resistance are GyrA-Serine 83 and GyrA-aspartate 87 in E. coli [90–94]. Mutation of these two amino acids to alanine results in resistance to quinolones, which showed the importance of these two amino acids in drug binding [95]. Consequently, it is has been assumed that these two amino acids play a crucial role in mediating quinolone-enzyme interactions. A large number of crystallographic studies have been performed to explain the quinolone-enzyme interactions. These structures showed how the drugs interact with both the enzyme and DNA and how these drugs halt the normal process of DNA cleavage and religation. There are differences in the interaction of drugs with the enzyme among the resolved structures as they vary with the species and the drug used. However they are Molecules 2016, 21, 268 11 of 19 helpful in designing more potent analogues that can bind more tightly with the enzyme and DNA. In addition, molecular docking and simulation of such structures and different quinolone analogues Molecules 2016, 21, 268 11 of 18 will be helpful in modelling the interactions to help design new drugs [83,84,96–100]. Osheroff and coworkers providedprovided most most of of the the data data about about the interaction the interaction of quinolones of quinolones and topoisomerases and topoisomerases [16,45,73, 77[16,45,73,77,81,82,86,101–109].,81,82,86,101–109]. In a recent In study, a recent the study, quinolone the quinolone structure wasstructure placed was near placed the serine near andthe serine acidic residues,and acidic but residues, it was foundbut it was that found the amino that acidsthe amino were acids not close were enough not close to mediateenough to a directmediate binding a direct to 2+ drugs.binding On to thedrugs. other On hand, the a other structure hand, of aa quinolonestructure complexof a quinolone was found complex having was a non-catalytic found having Mg a 2+ chelatednon-catalytic by the Mg C3/C42+ chelated keto acid by the of the C3/C4 drugs keto [86 ].acid The of Mg the drugswas in [86]. coordination The Mg2+ with was four in coordination molecules of water,with four two ofmolecules which were of water, located two close of enough which towere the serinelocated and close acidic enough residues to favorablethe serine for and hydrogen acidic bondingresidues favorable (Figure3). for The hydrogen above studies bonding suggested (Figure 3) that. The the above interaction studies of suggested water to that metal the bridged interaction the quinoloneof water to to metal the enzyme bridged [ 83the,84 quinolone,86,93,97,99 to]. the It wasenzyme also observed[83,84,86,93,97,99]. that a mutation It was inalso the observed place of serinethat a ormutation nearby in another the place amino of serine residue or limitsnearby the another variety amino of metal residue ions thatlimits support the variety the drug of metal activity ions [ 101that]. 2+ Thussupport mutation the drug resulted activity in decrease[101]. Thus in affinitymutation of theresu enzymelted in fordecrease quinolones in affinity at the of noncatalytic the enzyme Mg for site.quinolones Additionally, at the noncatalytic these mutations Mg2+ decrease site. Additionally, the affinity these of gyrase mutations and topoisomerase-IV decrease the affinity for quinolones of gyrase and intopoisomerase-IV the case of mutation for quinolones in both residues, and in therethe case is a of complete mutation loss in ofboth relevant residues, quinolones there is a to complete stabilize cleavageloss of relevant complexes quinolones [16,86,103 to stabilize]. cleavage complexes [16,86,103].

Glu/Asp

O O

H O Ser

2 O O F OH N N HN

Figure 3. Quinolone-enzyme interaction [86]. Figure 3. Quinolone-enzyme interaction [86]. Thus the existence of a metal ion-water complex verifies that serine and acidic residues act as anchorThus points the existencethat coordinate of a metal the bridge ion-water to the complex enzyme verifies and that that the serine water-metal and acidic ion residues bridge actis the as anchorprimary points interaction that coordinate between the the drug bridge and to bacterial the enzyme type andII enzymes that the [104]. water-metal It is noteworthy ion bridge that is the primarytype II topoisomerase interaction between of humans the druglacks andserine bacterial and acidic type residues II enzymes to affix [104 the]. It water-metal is noteworthy ion that bridge the typeand is II therefore topoisomerase not capable of humans of utilizing lacks serine this critical and acidic mechanism residues to to interact affix the with water-metal quinolones. ion That bridge is andwhy isthe therefore toxicity not of capablequinolones of utilizing is also thisminor critical for humans mechanism [104]. to These interact differences with quinolones. in topoisomerases That is why thestructure toxicity provide of quinolones a base is alsoby which minor forquinolones humans [104differentiate]. These differences between inthe topoisomerases bacterial and structure human providetopoisomerases a base by and which opens quinolones beneficial differentiateavenues for this between class of the drugs. bacterial and human topoisomerases and opens beneficial avenues for this class of drugs. 8. Bacterial Resistance to Quinolones 8. Bacterial Resistance to Quinolones Like other antibiotics, quinolones face resistance from infectious bacteria due to their widespread Like other antibiotics, quinolones face resistance from infectious bacteria due to their widespread use and this resistance is a major clinical issue. Bacterial resistance to quinolones is increasing and it use and this resistance is a major clinical issue. Bacterial resistance to quinolones is increasing and is common reported throughout the world. Most vancomycin-resistant enterococcus (VRE) and it is common reported throughout the world. Most vancomycin-resistant enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) are also cross-resistant to fluoroquinolones [109,110]. methicillin-resistant Staphylococcus aureus (MRSA) are also cross-resistant to fluoroquinolones [109,110]. Bacterial resistance to quinolones can be classified into three categories [16]. Bacterial resistance to quinolones can be classified into three categories [16]. 9. Target-Mediated Quinolones Resistance 9. Target-Mediated Quinolones Resistance Mutation in the target site of quinolone: gyrase (GyrE subunit) and topoisomerase-IV (ParC Mutation in the target site of quinolone: gyrase (GyrE subunit) and topoisomerase-IV (ParC subunit) of bacteria are the most common and they result in resistance to quinolones. The serine and subunit) of bacteria are the most common and they result in resistance to quinolones. The serine acidic residue of amino acids that bind the water-metal on bridges undergo mutation frequently and acidic residue of amino acids that bind the water-metal on bridges undergo mutation frequently results in ten times less binding affinity for quinolones [95,104]. Most probably, the disturbance in the water-metal ion bridge leads to the quinolone resistance. Normally, mutations in serine comprise above 90% of the mutant pool in both clinical and laboratory isolates, while changes at the acidic residues comprising the bulk of other mutations [92,93]. In most of the resistant strains of bacteria, serine is the most common residue that is mutated: to leucine in MRSA and to isoleucine, arginine or tyrosine in GyrA subunit in VRE. After the mutation in gyrase and topoisomerase-IV, wild type

Molecules 2016, 21, 268 12 of 19 results in ten times less binding affinity for quinolones [95,104]. Most probably, the disturbance in the water-metal ion bridge leads to the quinolone resistance. Normally, mutations in serine comprise above 90% of the mutant pool in both clinical and laboratory isolates, while changes at the acidic residues comprising the bulk of other mutations [92,93]. In most of the resistant strains of bacteria, serine is the most common residue that is mutated: to leucine in MRSA and to isoleucine, arginine or tyrosine in GyrA subunit in VRE. After the mutation in gyrase and topoisomerase-IV, wild type DNA cleavage does not stop in the absence of drugs, however quinolones have little ability to enhance the cleavage of enzyme mediated DNA [16,102,111–113]. In addition, the binding capability of quinolones is largely reduced and it loses much of their ability to inhibit DNA ligation or to make enzyme-DNA-drug complex [16,104].

10. Plasmid-Mediated Quinolone Resistance The second method of bacterial resistance to quinolones are plasmids that carry quinolone resistance genes that are also a rising clinical issue, commonly causing low level resistance (less than 10-fold) [114–116] though a 250-fold resistance has also been reported [117,118]. The transmittance of plasmids mediated quinolone resistance from one bacterial generation to another can be done horizontally via conjugation as well as vertically (target mediated resistance is transmitted only vertically). The plasmids responsible for quinolones bear additional genes which offer resistance to other drug classes [115,119,120]. Plasmid mediated quinolone resistance is due to three group of genes. These genes are qnr genes, AAC (6)-Ib-cr and the third group comprised of efflux pumps. The qnr genes offer resistance by decreasing the binding of DNA to gyrase and topoisomerase-IV; as a result there is decrease in the target site of chromosomes. The efflux pump is also a plasmid encoded protein that causes resistance to quinolones [121,122]. The second gene associated with quinolone resistance is an alternative protein of an aminoglycoside acetyltransferase having a mutation at two specific points. The enzymes decrease the activity of the quinolones by acylation of unsubstituted nitrogens of the C7 piperazine ring in norfloxacin and ciprofloxacin [123].

11. Chromosome-Mediated Quinolone Resistance The concentration of quinolones inside the cell is regulated by the diffusion-mediated uptake of drugs and pump-mediated efflux. For a drug to enter into cell of Gram-negative bacteria, it must pass through extra barriers. Therefore the influx of drug into Gram-negative species is facilitated by protein channels called porins. The down regulation of porins expression leads to low level quinolone resistance [109,118,119]. Along with the plasmid encoded efflux pump, if the expression of chromosomes encoded efflux pumps are enhanced, it will also offer low level resistance. Usually the mutations in regulatory proteins cause the up-regulation of these efflux pumps [109,124]. The resistance that arises due to lowering the concentration of quinolones within the cell may not be a big clinical issue but it provides a favorite environment to other kind of resistance to antibacterial agents [124,125].

12. Conclusions Quinolones are utilized worldwide as broad spectrum antibacterial agents, effective against both Gram-positive as well as Gram-negative bacteria. There are several quinolones derivatives that also work as antimalarial, antiviral and anticancer agents. After their initial synthesis as a byproduct of chloroquine, quinolones evolved as a separate class of synthetic drugs. Currently different synthetic methods are explored to synthesize more effective novel analogues of quinolones. Due to the overuse of these drugs, certain bacteria produce resistance to them and these strains of bacteria are now a common threat worldwide. Several mechanisms for resistance have been explained among which mutation at the target site is common. There is a need to explore new synthetic routes for novel quinolone analogues that are cost effective, less cytotoxic and have better pharmacokinetic and pharmacodynamic properties. This latest information about quinolones can help scientists to develop such analogues which can avoid resistance to quinolones and can extend their clinical utilization in the future. Molecules 2016, 21, 268 13 of 19

Acknowledgments: We thank Kiera Reifschneider for her suggestions and support in writing of this manuscript. A.N. acknowledges financial support from National Center of Excellence in Physical Chemistry, University of Peshawar, Pakistan for covering the costs to publish in open access. Author Contributions: A.N.; S.B. and M.M. provided the concept and designed the manuscript. N.A. and K.K. participated in the discussion during preparation and writing of the manuscript. All authors read and approved the final manuscript of this review. Conflicts of Interest: All the authors declare that they have no competing interests.

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