Indium-Catalyzed Annulation of o-Acylanilines with Alkoxyheteroarenes: Synthesis of Heteroaryl [b

18 Pages·2017·1.69 MB·English

molecules
Article
Indium-Catalyzed Annulation of o-Acylanilines
with Alkoxyheteroarenes: Synthesis of
Heteroaryl[b]quinolines and Subsequent
Transformation to Cryptolepine Derivatives
KyoheiYonekura ID,MikaShinoda,YukoYonekuraandTeruhisaTsuchimoto* ID
DepartmentofAppliedChemistry,SchoolofScienceandTechnology,MeijiUniversity,1-1-1Higashimita,
Tama-ku,Kawasaki214-8571,Japan;[email protected](K.Y.);[email protected](M.S.);
[email protected](Y.Y.)
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:AkioBaba (cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:9March2018;Accepted:31March2018;Published:5April2018
Abstract: We disclose herein the first synthetic method that is capable of offering
heteroaryl[b]quinolines(HA[b]Qs)withstructuraldiversity,whichincludetricyclicandtetracyclic
structureswith(benzo)thienyl,(benzo)furanyl,andindolylrings. ThetargetHA[b]Qisaddressedby
theannulationofo-acylanilinesandMeO–heteroareneswiththeaidofanindiumLewisacidthat
effectivelyworkstomaketwodifferenttypesoftheN–CandC–Cbondsinonebatch. Aseriesof
indolo[3,2-b]quinolinespreparedherecanbesubsequentlytransformedtostructurallyunprecedented
cryptolepinederivatives. MechanisticstudiesshowedthattheN–Cbondformationisfollowedby
theC–Cbondformation. Theindium-catalyzedannulationreactionthusstartswiththenucleophilic
attackoftheNH groupofo-acylanilinestotheMeO-connectedcarbonatomoftheheteroarylringin
2
anS Arfashion,andtherebytheN–Cbondisformed. Theresultingintermediatethencyclizesto
N
maketheC–Cbondthroughthenucleophilicattackoftheheteroaryl-ring-basedcarbonatomtothe
carbonylcarbonatom,providingtheHA[b]Qafteraromatizingdehydration.
Keywords: anti-canceractivity;anti-malarialactivity;heteroarenes;indium;Lewisacids;pyridine;
one-pot;quindolines;tandemreaction
1. Introduction
Heteroaryl[b]quinolines (HA[b]Qs), wherein electron-rich heteroaryl rings are fused to the
[b] site of quinoline, are important frameworks found in natural products [1–3] and biologically
active molecules [1,4–6] as well as functional organic materials [7–9]. Due to their significance,
numeroussyntheticapproacheshavebeendevelopedfortheconstructionofsuchstructuralmotifs.
Theseapproachescouldbecategorizedsimplyintothreestrategiesonthebasisofthering-constructing
method(Figure1),whicharetheheteroarylringformation(strategya)[10–22],thecentralpyridyl
ringformation(strategyb)[23–39],andtheformationofbothrings(strategyc)[4,40–53]. Although
thereareadvantagesanddisadvantagestoeachstrategyfromvariousaspects,thestrategybseemsto
bethemostuser-friendlyintermsoftheaccessibilityofthestartingsubstrates.
Ontheotherhand,wehaverecentlyreportedanewC(heteroaryl)–Nbond-formingreactionby
reactingelectron-richmethoxyheteroareneswithaminesviaanucleophilicaromaticsubstitution(S Ar)
N
reaction[54]. Inadditiontothis,wehavealsodevelopedseveralnewC(heteroaryl)–Cbond-forming
reactionsbyreactingalkynes[55–57]orcarbonylcompounds[58–60]withheteroarenes. Allofthese
reactionsareeffectivelycatalyzedbyasaltofanindium(III)Lewisacid,whichhasbeenalsoemployed
Molecules2018,23,838;doi:10.3390/molecules23040838 www.mdpi.com/journal/molecules
Molecules2018,23,838 2of18
forvariousorganictransformationsbyotherresearchgroups[61–71]. Wethereforeenvisagedthat
conductingthetwodifferenttypesofreactionsinatandemfashionwouldbeanewmethodologyof
thestrategybtooffertheHA[b]Qinaneasyway,therebyalsoleadingtothefurtherexpansionof
ourindium-basedtechnology. OurworkinghypothesisisillustratedmoreintelligiblyinScheme1.
We thus expected that the synthesis of HA[b]Qs 4 could be achieved by mixing o-alkynylanilines
1 or o-acylanilines 2 with methoxyheteroarenes 3 in the presence of a catalytic indium Lewis acid
(InX =In). ThefirststageistheS Ar-basedN–Cbond-formingreactionthroughthenucleophilic
3 N
attackoftheaminogroupof1or2toelectrophiliccomplexAtoafford5or6,respectively.Intermediate
5or6successivelycyclizesbyformingtheC–Cbondinanintramolecularfashion,thusgiving7or8,
respectively,viatheactivationmodeofBorC.Theisomerizationordehydrationasthefinalstage
results in the formation of desired HA[b]Q 4. We also expected that combining the two indium
transformations, both of which are compatible with a broad range of substrates, should lead to
the development of the HA[b]Q synthesis with good substrate generality. As stated above, a lot
of studies that synthesize the HA[b]Q have appeared so far in literature, but these studies have
been limited to preparing HA[b]Qs with one to three types of heteroaryl rings, to the best of our
knowledge [72–74]. We report herein that an indium salt effectively catalyzes the N–C and C–C
bond-formingsequencetoaffordarangeofHA[b]Qsincludingtricyclicandtetracyclic[2,3-b]and
[3,2-b]structureswithsulfur-,oxygen-,andnitrogen-basedfive-memberedheteroarylrings. Among
theproducts,indolo[3,2-b]quinolines,whichcanbeeasilyconvertedtocryptolepinederivativesthat
havebeenknowntoexhibitanti-malarialandanti-canceractivities,areincluded[75].
Figure1.Syntheticstrategiesfortheconstructionoftheheteroaryl[b]quinoline(HA[b]Q)structure.
Scheme1.AworkinghypothesisforthesynthesisofHA[b]Qs4.In=InX .
3
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2. ResultsandDiscussion
In order to verify the working hypothesis, we first investigated the possibility of whether
o-ethynylaniline(1a)worksasasubstrateforthesynthesisoftheHA[b]Qunderindiumcatalysis,and
selected3-methoxybenzothiophene(3a)asthesubstratepartner(Table1). Upontreatmentof1aand
3awith5mol%ofIn(NTf ) (Tf=SO CF )inPhClat110◦Cfor24h,wewerepleasedtoobservethat
2 3 2 3
thedesiredannulationproceededtogive11-methyl[1]benzothieno[3,2-b]quinoline(4aa),albeitinlow
yield(entry1). Whilethescreeningofotherindiumsaltsprovidednosignificantimprovementsinthe
yieldof4aa,asmallamountofo-acetylaniline(2a)wasformedalongwith4aawhenusingIn(ONf)
3
(Nf=SO C F )asacatalyst(entries2–6). Inthiscontext,awidevarietyofLewisacids,including
2 4 9
indiumsalts,havebeenknowntoactascatalystsforthehydrationofaC≡Cbondtocreateacarbonyl
functionality [76,77]. A possible explanation for the formation of 2a is thus the indium-catalyzed
hydrationof1awithH O,whichcouldhavebeenpresentinasmallquantityinthereactionmixture.
2
Accordingly,wepresumedthat,asroutesfortheformationof4aa,therewouldbetwopossibilities:
oneisdirectlyfrom1a, andtheotherisindirectlyfrom2aformedinsituafterthehydrationof1a.
Inordertogetaninsightintowhichroutesoperatehere,thefollowingexperimentswereconducted.
Thus,theannulationcarriedoutundertheconditionsofentry3,additionallyincludingfivemolar
equivalentsofH O,resultedinhigheryieldsofboth4aaand2a(entry7). Moreover,theprolonged
2
reactiontimefrom24hto36hraisedtheyieldof4aato61%withthecompleteconsumptionof2a
(entry8). Theseresultssuggestthat4aaislikelytobeformedthroughthegenerationof2abythe
hydrationof1a,whereasthecontributionofthedirectroutefrom1acannotbecompletelyexcluded.
Table1.Indium-catalyzedannulationofo-ethynylanilinewith3-methoxybenzothiophenea.
Entry InX3 t(h) Yieldof4aa(%)b Yieldof2a(%)b
1 In(NTf ) 24 11 <1
2 3
2 In(OTf) 24 9 <1
3
3 In(ONf) 24 14 2
3
4 InCl 24 2 <1
3
5 InBr 24 3 <1
3
6 InI 24 5 <1
3
7c In(ONf) 24 26 30
3
8c In(ONf)3 36 61(61)d <1
aReagentsandconditions(unlessotherwisespecified):1a(0.250mmol),3a(0.300mmol),InX3(12.5µmol,5mol%),
PhCl(0.20mL),andperformedunderargon(1atm).bDeterminedby1H-NMRusingMeNO2asaninternalstandard.
cPerformedinthepresenceofH2O(1.25mmol,5equiv.).dTheisolatedyieldof4aaisshowninparentheses.
Onthebasisoftheaboveresults,weturnedourattentiontotheannulationwith2ainsteadof
1a(Table2). Asexpected,underthesamereactionconditionsasthoseforentry3ofTable1,4aawas
produced in significantly higher yield of 62% (entry 1). Inspired by this result, we continuously
examinedtheeffectofvariousindiumsaltsotherthanIn(ONf) forthesameannulationreactionof
3
2awith3a. Thus,In(OTf) andIn(NTf ) withthestrongelectron-withdrawingligandsasIn(ONf)
3 2 3 3
alsocatalyzedtheannulation,andtheyieldof4aaincreasedto74%intheuseofIn(NTf ) (entries2
2 3
and3). Amongtheindiumhalidesexamined,InBr andInI werefoundtobehighlyeffective,giving
3 3
4aain92%yieldinboththecases,insharpcontrasttotheinactivityofthefluoridesalt(entries4–7).
However,thecorrespondinghydroxideandacetatesaltsweretotallyinactive(entries8and9). Dueto
theremarkablecatalyticactivityofInBr ,metalbromidesof,forinstance,Sc,Fe,Co,Pd,Cu,Ag,Zn,Pb,
3
andBiweretested,butprovedtobelesseffective(entries10–18). No4aawasformedintheabsence
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ofacatalyst,whichisthusindispensablefortheprogressoftheannulation(entry19). WithInBr as
3
thepromisingcatalyst,acontinuoussurveyofthesolventeffectindicatedthatPhClwouldbethe
mostsuitablesolventofchoicefortheannulation,andthatthereactionrategreatlydecreasesinH O
2
(entries20–27). Whiletheloweringofthecatalystloadingto1mol%accompaniesthedecreaseofthe
reactionrate,thegoodyieldof4aacanbesecuredbyextendingthereactiontimeto96h(entry28).
Favorably,theannulationcanbealsocarriedoutunderanatmosphereofairinsteadofargontoafford
4aain88%yield(entry29).
Table2.Lewisacid-catalyzedannulationofo-acetylanilinewith3-methoxybenzothiophenea.
Entry LewisAcid Solvent Conversionof2a(%)b Yieldof4aa(%)c
1 In(ONf) PhCl 86 62
3
2 In(OTf) PhCl 73 55
3
3 In(NTf ) PhCl 79 74
2 3
4 InF PhCl 4 <1
3
5 InCl PhCl 95 83
3
6 InBr PhCl 97 92
3
7 InI PhCl 97 92
3
8 In(OH) PhCl <1 <1
3
9 In(OAc) PhCl <1 <1
3
10 ScBr PhCl 61 50
3
11 FeBr PhCl 86 62
3
12 CoBr PhCl 31 22
2
13 PdBr PhCl 47 29
2
14 CuBr PhCl 28 18
2
15 AgBr PhCl 3 <1
16 ZnBr PhCl 15 5
2
17 PbBr PhCl <1 <1
2
18 BiBr PhCl 45 33
3
19 None PhCl 5 <1
20 InBr PhMe 91 82
3
21 InBr Bu O 85 74
3 2
22 InBr 1,2-Diethoxyethane 91 80
3
23 InBr 1,4-Dioxane 87 66
3
24 InBr3 MeNO2 88 76d
25 InBr PrCN 82 76
3
26 InBr BuOH 92 73
3
27 InBr H O 48 37
3 2
28e InBr PhCl 89 70
3
29f InBr3 PhCl 97 88
aReagentsandconditions(unlessotherwisespecified):2a(0.250mmol),3a(0.300mmol),Lewisacid(12.5µmol,
5mol%),solvent(0.20mL),andperformedunderargon(1atm). bDeterminedbyGCusingn-dodecaneasan
internalstandard. cDeterminedby1H-NMRusingMeNO2asaninternalstandard. dDeterminedby1H-NMR
usingCH2Br2insteadofMeNO2asaninternalstandard.ePerformedwithInBr3(2.50µmol,1mol%)for96h.f
Performedunderair(1atm).
Withtheproperreactionconditionsinhand, wenextexaminedthescopeoftheo-acylaniline
substrate to 3a (Table 3). Similar to o-acetylaniline (2a), its derivatives with the OH, OMe, or
methylenedioxygroupsuccessfullyparticipatedintheannulation(4aa–4da). Theformationof4ba
in such high yield shows that the OH group does not interfere with the progress of the desired
annulationbyactingasthenucleophilicsite,astheNH groupdoes. Noundesiredringfragmentation
2
oftheacetalmoietyin4dawasobserved,evenundertheLewisacidicconditions[78]. Thebulkier
Molecules2018,23,838 5of18
isopropylgrouponthecarbonylcarbonatomdoesnotaffecttheefficiencyoftheannulation,giving
4eain97%yield. ACF group,theC–Fbondofwhichisknowntoincreasemetabolicstabilityand
3
membranepermeability,thusleadingtoimprovementinbioavailability[79],canbealsoinstalledonto
theC11-positionofthebenzothieno[3,2-b]quinolinestructure(4faand4ga). Acommerciallyavailable
hydrochloride–hydrateadductofo-acylaniline2gcanbeusedasasubstratewithoutneutralizingand
drying. Ourprotocolisapplicableaswelltoo-acylanilineswithaseriesofarylgroupswithdifferent
electronicandstericnatures,inwhichthesimplephenylgroupfor4haand4ia,p-MeOC H for4ja,
6 4
p-FC H for4ka,o-MeC H for4la,ando-fused-aroylC H for4maareincluded. Theatmosphereof
6 4 6 4 6 4
airwasagainconfirmedtobeavailableonthesynthesisof4ha. Inthereactionofaminoanthraquinone
2mwithtwocarbonylmoieties,onlytheoneadjacenttotheNH groupworkedasareactionsiteto
2
providehexacyclic-fusedringsystem4mainoneshot. OfimportancetonoteisthattheMeO,Cl,andF
groupsonthearylringareknowntobehaveasleavinggroupsinthegeneralS Arreaction,butwere
N
foundtobecompatiblewiththereactionconditions,thuscontributingtothehigh-yieldformationof
thetargetmolecules(4ca,4ga,4ia,4ja,and4ka)[80].
Besidesthebenzothieno[3,2-b]quinoline,ourmethodisapplicabletopreparingarangeofHA[b]Qs
byusingothersulfur-andoxygen-basedmethoxyheteroarenes(Table4). Thereplacementof3awith
2-methoxybenzothiophene(3b)enablestheswitchofthefused-ringorientationfromthe[3,2-b]to
the [2,3-b], and products 4ab and 4bb were obtained in high yields. However, in contrast to the
successfulconstructionofthieno[2,3-b]quinoline4ac,4hc,and4ad,changingthefused-ringorientation
to the [3,2-b] in this case resulted in low yield of 4he. In the reaction of 3-methoxythiophene (3e),
aself-condensationreaction,inwhichtwomoleculesof2hreactwitheachothertoformcyclicdiimine9,
occurredasamajorsidereaction(Figure2). Thisresultislikelytoberelated,atleastinpart,tothe
relativelylowreactionrateofthedesiredS Arprocessbetween2hand3e,and,infact,70%of3e
N
loadedforthereactionremainedunconsumed. Inthiscontext,wehavepreviouslyconfirmedthatthe
S Araminationreactionof3-methoxythiophene(3e)requiresahigherloadingofanindiumcatalyst
N
as well as higher temperature compared to those for the reaction of 2-methoxythiophene (3c) [54].
Inadditiontothesulfur-containingHA[b]Qs,thetetracyclicandtricyclicoxygen-containinganalogues
canbeaddressedbyourmethodinmoderatetogoodyields(4af,4hf,and4ag). Whenpreparing4ag,
InI workedasacatalystmoreefficientlythanInBr . Unfortunately,noannulationreactionof2awith
3 3
2-methoxy-1-phenylpyrrole(3h)forthesynthesisofpyrrolo[2,3-b]quinoline4ahproceeded,dueto
someundesiredsidereactions, includingN-methylationof2abytheMeOgroupof3hactingasa
sourceofamethylgroup.
AscollectedseparatelyinTable5,wesuccessivelypresenttheresultofconstructingtheframework
oftheindolo[3,2-b]quinoline,whichisalternativelynamedquindoline,havingbeenknowntoshow
cytotoxic activity against human cancer cell lines [81]. As in our preceding S Ar amination [54],
N
commercially unavailable 3-methoxyindole was not required, but rather commercially available
3-acetyloxyindole(3i)canbeusedhereagainasasubstrate. Thus,mixing2a,3i,andInBr (5mol
3
%)inPhCl,andthenheatingthemixtureat110◦Cfor24hgave4aiin55%yield. Otherquindoline
derivatives 4di, 4fi, and 4gi could also be synthesized by our method. Unlike the annulation of
2g–HCl–H Owith3-methoxybenzothiophene(3a)(see4gainTable3),thepre-removalofHCland
2
H O from 2g–HCl–H O as a commercial source is required here to obtain 4gi in reasonable yield.
2 2
With 2g–HCl–H O instead, the formation of 4gi resulted in only 1% NMR yield. These results
2
inspiredustoaddresscryptolepinederivatives, duetotheirpotentialitiesasanti-malarialand/or
anti-cancerdrugs.
Molecules2018,23,838 6of18
Table3.Indium-catalyzedannulationofo-acylanilineswith3-methoxybenzothiophenea.
aReagentsandconditions(unlessotherwisespecified):2(0.250mmol),3a(0.300mmol),InBr3(12.5µmol,5mol%),
PhCl(0.20mL),andperformedunderargon(1atm).Yieldsofisolated4basedon2areshownhere.bYieldswhen
performedunderair(1atm).cPerformedinPhCl(0.40mL)for36h.dPerformedwithaHCl–H2Oadductof2g.
ePerformedinPhCl(0.50mL)at130◦C.
Molecules2018,23,838 7of18
Table4.Indium-catalyzedannulationofo-acylanilineswithmethoxyheteroarenesa.
aReagentsandconditions(unlessotherwisespecified):2(0.250mmol),3(0.300mmol),InBr3(12.5µmol,5mol%),
PhCl(0.20mL),andperformedunderargon(1atm).Yieldsofisolated4basedon2areshownhere.bPerformed
witha1.2:1mixtureof2b(0.300mmol)and3b(0.250mmol).cPerformedwith3(0.500mmol,2equiv.).dPerformed
withInBr3(37.5µmol,15mol%).ePerformedfor3h.fPerformedwithInI3insteadofInBr3.
Figure2.Amajorbyproductformedinthereactionof2hwith3e.
Molecules2018,23,838 8of18
Table5.Indium-catalyzedannulationofo-acylanilineswith3-acetyloxyindolea.
aReagentsandconditions(unlessotherwisespecified):2(0.250mmol),3i(0.625mmol),InBr3(12.5µmol,5mol%),
PhCloro-C6H4Cl2asasolvent,andperformedunderargon(1atm).Yieldsofisolated4basedon2areshownhere.
bPerformedonthefollowinglargerscaleinthepresenceofH2O:2a(2.20mmol),3i(5.50mmol),H2O(11.0mmol),
InBr3(110µmol,5mol%),PhCl(1.7mL).cPerformedinPhCl(0.30mL).dPerformedino-C6H4Cl2(0.20mL).e
Performedwith2g–HCl–H2Oasacommercialsourceinsteadof2g.
As previously demonstrated, the HOTf adduct of the 11-methylated cryptolepine (11-Me-10)
shows higher anti-malarial and antitrypanosomal activities than that of the original cryptolepine
(10)(Figure3). SincetheN-methylationofthepyridineringof4aiwithmethyltriflate(MeOTf)has
been already reported [82], we targeted the synthesis of analogues thereof from other quindoline
derivatives4di,4fi,and4gi(Table6). TheN-methylationinaccordancewiththemodifiedliterature
proceduresuccessfullydelivered10di,10fi,and10gi,whicharenewcompoundsunreportedinthe
literature[82]. Especially,10fi,whichhasthe11-CF groupinsteadofthe11-CH groupin11-Me-10,
3 3
mightbeexpectedtobepromisinginviewofanti-malarialandantitrypanosomalactivities,dueto
thepossiblehigherbioavailability. Moreover,sincetheacid-freecryptolepinederivativeshavebeen
thefocusofexamininganti-canceractivity(11and11-Me-11inFigure3),thereshouldbeademand
fortheacid-freeform. Accordingly,weconfirmedthattheneutralizationof,forinstance,10fiwitha
Na CO aqueoussolutionprovides11fiwithnoTfOHinquantitativeyield(Scheme2).
2 3
In order to get insight into the reaction pathway of the present annulation reaction,
someexperimentswereperformed(Scheme3). Atfirst,upontreating2ewith3aatroomtemperature
ratherthanthestandardheatingtemperature,onlytheS Ar-basedintermolecularN–Cbond-forming
N
reactionproceededtofurnish6eain53%yieldwith60%conversionof2e,thusbeingnotcontaminated
by 12ea derived from the C–C bond formation as a possible alternative first stage, and by final
annulation product 4ea [Equation (1) in Scheme 3]. Subsequently, 6ea isolated from the reaction
ofEquation(1)washeatedunderthestandardreactionconditions, andthereby4eawasobtained
highlyefficientlyviatheintramolecularC–Cbond-formingannulation[Equation(2)inScheme3].
Ontheotherhand, Me -2e, whereinthenitrogenatomisdimethylatedandwouldthusnolonger
2
act as a nucleophilic site, did not participate in making a C–C bond with 3a, leading possibly to
Me -12ea. Asaresult,Me -2ewasrecoveredquantitatively,evenunderthestandardheatingreaction
2 2
conditions[Equation(3)inScheme3]. Accordingly,theseresultsstronglysuggestthattheannulation
reactionproceedsintheorderoftheS Ar-basedintermolecularN–Cbondformation,followedbythe
N
S Ar-basedintramolecularC–Cbondformation. ExperimentalproceduresforEquations(1)and(2)as
E
wellasspectralandanalyticaldata(meltingpoint,NMR,andHRMS),andNMRchartsforproducts
6eaand4eaareprovidedinSupplementaryMaterials.
Molecules2018,23,838 9of18
Figure 3. Anti-malarial, antitrypanosomal and anti-cancer activities of cryptolepine,
11-methylcryptolepine,andtheirHOTfadducts[82].
Table6.N-Methylationofindolo[3,2-b]quinolineswithMeOTfa.
aReagents:4(0.100mmol),MeOTf(0.190mmol),solvent[CH2Cl2(1.2mL)ortoluene(0.60mL)].Yieldsofisolated
10basedon4areshownhere.
Scheme2.Neutralizationof10fi.
Molecules2018,23,838 10of18
Scheme3.Controlexperimentsformechanisticstudies.
Onthebasisoftheaboveexperimentalresultsaswellasthepreviousones,aproposedreaction
mechanism is illustrated in Scheme 4 that exemplifies the reaction of 2e with 3a. First up is the
S Ar-basedintermolecularaminationof3abythenucleophilicattackofthenitrogenatomof2evia
N
previouslyproposedtransitionstateA[54],followedbythereleaseoftheindiumcatalyst(In)and
MeOHtogiveintermediate6ea. Nextisthenucleophilicattackofthethienylringtothecarbonyl
moietyactivatedbyInasshownintransitionstateB,herebyproviding8ea,andthendesiredstructure
4eaafteraromatizingdehydration.Thering-closingC–Cbond-formingprocessmightbeacceleratedby
theelectronflowfromthelonepaironthenitrogenatom. However,duetothefactthat6eaistheonly
intermediateconfirmedduringtheannulationprocess[Equation(1)inScheme3],therate-determining
stepislikelytobepresentattheintramolecularC–Cbond-formingstage.