Part 1 Chemistry of NO Donors

31 Pages·2004·0.32 MB·English

Part1 ChemistryofNODonors

3
1
NOandNODonors
TingweiBillCai,PengGeorgeWang,andAlvinA.Holder
Nitricoxide(NO),amagicfreeradicalgasmolecule,hasbeenshowntobeinvolved
in numerous physiological and pathophysiological processes. Among its diverse
functions,NOhasbeenimplicatedintherelaxationofvascularsmoothmuscle,the
inhibitionofplateletaggregation,neurotransmission(Viagrareversesimpotenceby
enhancinganNO-stimulatedpathway),andimmuneregulation[1]. Itwasnamedthe
moleculeoftheyearin1992byScienceandwasthesubjectoftheNobelPrizein1998.
NOhaslimitedsolubilityinwater(2–3mM),anditisunstableinthepresenceof
variousoxidants. Thismakesitdifficulttointroduceassuchintobiologicalsystems
inacontrolledorspecificfashion. Consequently,thedevelopmentofchemicalagents
thatreleaseNOisimportantifwearetotargetitsbioeffectorrolestospecificcelltypes
forbiologicalandpharmacologicalapplications. Basedonourcomprehensivereview
ofNOdonors[2],thischapterfocusesonrecentprogressandcurrenttrendsinNO
donordevelopmentandnovelapplicationswhicharenotcoveredbythefollowing
chapters.
1.1
IntroductiontoNOBiosynthesisandNOdonors
1.1.1
NitricOxideSynthases
EndogenousNOisproducedalmostexclusivelybyl-argininecatabolismtol-citrul-
lineinareactioncatalyzedbyafamilyofnitricoxidesynthases(NOSs)[3]. Inthefirst
step, Arg is hydroxylated to an enzyme-bound intermediate Nù-hydroxy-l-arginine
(NHA), and 1 mol of NADPH (nicotinamide adenine dinucleotide phosphate, re-
ducedform)andO areconsumed. Inthesecondstep,NHAisoxidizedtocitrulline
2
and NO, with consumption of 0.5 mol of NADPH and 1 mol of O (Scheme 1.1).
2
Oxygen activation in both steps is carried out by the enzyme-bound heme, which
derives electrons from NADPH. Mammalian NOS consists of an N-terminal oxy-
NitricOxideDonors
PengGeorgeWang,TingweiBillCai,NaoyukiTaniguchi(Eds.)
Copyright(cid:1)c 2005WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:3-527-31015-0
4 1NOandNODonors
H2N NH2 H2N N OH O NH2
NH 1 NADPH NH 0.5 NADPH NH
N O
O2 H2O O2 H2O
H3N COO H3N COO H2N COO
Arginine Nw-Hydroxyarginine Citrulline Nitric Oxide
Scheme1.1 Endogenoussynthesisofnitricoxide.
genasedomainthatbindsironprotoporphyrinIX(heme),6-(R)-tetrahydrobiopterin
(H B)andArg,andaC-terminalreductasedomainthatbindsFMN(flavinmononu-
4
cleotide),FAD(flavinadeninedinucleotide),andNADPH,withacalmodulinbinding
motiflocatedbetweenthetwodomains. Tobeactive,twoNOSpolypeptidesmust
formahomodimer. ThereductasedomainseachtransferNADPH-derivedelectrons,
throughFADandFMN,tothehemelocatedintheadjacentsubunit. Threedistinct
isoformsofNOShavebeenidentified–neuronal,macrophageandendothelialtypes,
andeachisassociatedwithaparticularphysiologicalprocess(Table1.1). Constitutive
endothelialNOS(eNOSorNOSIII)regulatessmoothmusclerelaxationandblood
pressure;constitutiveneuronalNOS(nNOSorNOSI)isinvolvedinneurotransmis-
sion and long-term potentiation; the NO produced from inducible NOS (iNOS or
NOSII)inactivatedmacrophagecellsactsasacytotoxicagentinnormalimmune
defenseagainstmicroorganismsandtumorcells. Theconstitutiveisoforms(nNOS
andeNOS)requireaddedCa2+ andcalmidulinforactivityandproducearelatively
smallamountofNO,whiletheinducibleisoform(iNOS)hastightlyboundCa2+and
calmodulin,andproducesarelativelylargeamountofNO.
Tab. 1.1: PropertiesofNOSisoforms.
NOS Locations Characteristics MajorBiologicalFunctions
nNOS(NOS-I) Brain,spinalcord,peripheral Constitutive,Ca2+dependent Neuromediator
iNOS(NOS-II) Macrophages,othertissues Inducible,Ca2+independent Hostdefender,cytotoxic
eNOS(NOS-III) Endothelium Constitutive,Ca2+dependent Vasodilatortonemodulator
The first step of an NOS catalyzed reaction is a “classical” P450-dependent N-
hydroxylationofaguanidine,exceptfortheinvolvementofH B.AsshowninScheme
4
1.2,Fe(III)heme1firstacceptsoneelectrontogiveFe(II)heme2,whichbindsO to
2
produceferrous-dioxyheme3. ThesecondelectronfromH Breduces3toperoxy-
4
iron 4. Arg donates a proton to 4 to facilitate O–O bond cleavage to generate an
oxo-iron (IV) cation radical species 5, which then rapidly hydroxylates the neutral
guanidiniumtoNHA[4].
ThesecondstepofNOSoxidationisagreaterchallengetoenzymologistssince
thereisnodirectanalogyinothersystems. Avarietyofproposedreactionstepscanbe
1.1IntroductiontoNOBiosynthesisandNOdonors 5
FeIIFIMNH2 FMNHF.eII O2 OF2eII H4B H4B+. R NH N+OH_N2HH R NH NH_ON2HH R NH NHN2 OH
O O
S1 S2 S3 SFeII4 SFeIV+. 5 SFeII1I
Scheme1.2 ThefirststepofNOSreaction.
roughlysummarizedinthreemechanisms(Scheme1.3). ThepopularMechanism
I was proposed by Marletta and modified by Ingold and others [5, 6], a superoxo-
iron(III)hemeintermediate6abstractsthehydrogenatomoftheNHAtofurnishan
iminoxyradical8,whichuponnucleophilicattackbythehydroperoxoiron(III)heme
7onitscarbongeneratesNOandcitrulline. Thismechanism,however,appearsnot
tobesupportedbythecrystalstructureanalysisoftheNOS-NHAcomplex[7–9]orby
arecentspectralstudy[10]. ThesecondmechanismwasproposedbyGroves(Mecha-
nismII),wheretheNOS-catalyzedaerobicoxidationofNHAoccursviaaradical-type
auto-oxidationprocess[11,12],i.e.,NHAisoxidizedbytheFe(III)hemetogenerate
animinoxylradical8,whichtautomerizestotheá-nitrosoradical12. Insertionofa
dioxygenmoleculebetween12andFe(II)hemeformsanenergeticá-nitrosoperoxy
Fe(III)hemeintermediatethatdecomposestogenerateNO[13,14]. However,direct
ligationofNHAtohemeironhasbeenprecludedbytheX-raycrystallographicdata
[7–9]. Thethirdmechanism,proposedbySilvermanandothers[15–18],mainlyin-
Mechanism I
NH2 NH2 NH2 8 H2N NH2
R NH N OH O2 , e- R NH ON OH R NH ON OH R NH ON O H R NH O + NO
O O O OH
FeIII FeIII 6 FeIII 7 FeIII 9 FeIII 10
Mechanism II
NH
NH 2
R NH N2 OH -H+R NH ON RN NHN2 O RN NHN2 O O2 RH2NN N O R N NHO2+ NO
H H H O H
FeIII FeIII 11 8 FeII 12 O OFeIV 14
FeIII 13
Mechanism III
R NH NHNH2 OHO2 , e- R NHONHHON2 OH R NH NOHOH2N 1O5H-H+R NH-ONHON2 1O7- RHNH2NOON O- R NHON-HO2 + NO
FeIII FeIII 6 FeIII 16 FeIII 18 FeIII 19 FeIII 20
Scheme1.3 ThesecondstepofNOSreaction.
6 1NOandNODonors
volvestheoxidationofthenitrogenontheprotonatedN-hydroxyguanidinomoiety
(MechanismIII).ItwassuggestedthattheinitialN–Hbondcleavagebysuperoxo-
iron(III)heme6generatesaradicalcationintermediate15,which,uponheterolysis
oftheO–Hbond, givestheiminoxyradical17. Thenucleophilicattackofperoxo-
iron(III)heme18on17givesanintermediatesimilarto13,whichdecomposestoNO
andcitrulline. Morerecently,StuehrhasemphasizedtheinvolvementofH Binthe
4
secondstepoftheNOSreaction[19–21].
1.1.2
ChemistryofReactiveNitrogenSpecies
OneofNO’smajorbiologicalactionsistoactivateguanylatecyclasedirectlytogener-
atecyclicguanosinemonophosphate(cGMP)asanintracellularsecondmessenger,
followedbykinase-mediatedsignaltransduction. Inanotherpathway,NOundergoes
oxidationorreductioninbiologicalsystemstoconverttomanydifferentreactivenitro-
−•
genspecies(RNS).Itcanreactwithmolecularoxygen(O ),superoxideanion(O )
2 2
− −
ortransitionmetals(M)toproduceRNSsuchasN O ,NO ,NO ,NO ,peroxyni-
2 3 2 2 3
−
trite(OONO ),andmetal-nitrosyladducts(RouteA,Scheme1.4)[22,23]. Among
theseRNS,peroxynitritestandsoutasanimportantspecies[24, 25]. Thereaction
−•
betweenNOandO producesperoxynitriteatadiffusioncontrolledrate[26–28].
2
Peroxynitriteisastrongoxidizingandnitratingspeciesthatcausesmoleculardam-
ageleadingtodisease-causingcellulardysfunction[29,30]. NOcanalsoberapidly
oxidizedbyoxygen,superoxideortransitionmetalstonitrosonium(NO+)whichre-
actswithnucleophiliccenterssuchasROH,RSHandRR′NHtoproduceRO–NO,
RS–NOorRR′N–NO,respectively(RouteB,Scheme1.4)[31,32]. Theseproductssub-
sequentlyundergootherreactionstoexhibittheirbiologicaleffects. Inaddition,NO
−
alsoundergoesaone-electronreductiontoproducenitroxyl(NO )(RouteC,Scheme
1.4). Thereducingpotentialofthisreductionisapproximately+0.25V[33]. Nitroxyl
convertsrapidlytoN Ounderphysiologicalconditions. Othercompetingreactions
2
RONO RSNO RR'-NO L-Arg NH2OH
ROH RSH RR'NH NOS RSH
oxidation reduction
NO+ NO NO- NO
2
B C
H2O H2O2 M M A O2- O2 NO O2 M
NO2- ONOO- M-NO M-NO ONOO- NO2 / N2O3 NO2- ONOO- M-NO
M O2- O2 H2O
NO2- NO - RS-NO NO2- / NO3-
3
( NO -)
2
Scheme1.4 Oxidationandreductionofreactivenitrogenspecies.
1.2ClassificationofNODonors 7
−
ofnitroxylincludeadditiontothiolgroups(singletNO )togenerateNH OH,and
2
− −
reactionwithoxygen(tripletNO )toformperoxynitrite(OONO ). Nitroxylhasalso
beenproventoexhibitmanybiologicalfunctions[34],suchasvasodilatation[35–37]
andcytotoxicity[38–40].
1.2
ClassificationofNODonors
IntensiveresearchonthebiologicalfunctionsofNOandotherreactivenitrogenoxide
speciesdemandsexogenoussourcesofNOdonorsasresearchtoolsandpharmaceu-
ticals. Sincethemid-1980s,thedevelopmentofnewNOdonorshasofferedseveral
advantagesoverthepreviousNOdonors,suchasspontaneousreleaseofNO,dona-
tionofNOundercontrolledrates,andeventhetargetingofNOtocertaintissues.
ThestructuraldissimilaritiesofthediverseNOdonorshaveledtoremarkablyvaried
chemicalreactivitiesandNO-releasemechanisms. GenerallyNOdonorsreleaseNO
through three kinds of mechanisms. The first route is that donating NO sponta-
neously, whichreleasesNOthroughthermalorphotochemicalself-decomposition
ofe.g. S-nitrosothiols,diazeniumdiolates,oximes. Thesecondrouteisthatreleasing
NObychemicalreactionswithacid,alkali,metalandthiol. Organicnitrates,nitrites
andsyndnoniminesgiveNOthoughthismechanism. Thethirdrouteisenzymatic
oxidationwhereNOdonors,forexample,N-hydroxyguanidines,needmetabolicac-
tivationbyNOsynthasesoroxidasesforNOrelease. SomeNOdonorsreleaseNO
by more than one route, e.g. organic nitrates can also generate NO by enzymatic
catalysis. Classification of all NO donors could be confusing, since all nitrogen–
oxygen-bondedcompoundshavethepotentialtodecompose,beoxidized,orbere-
duced to produce reactive nitrogen species. However, similar chemical structures
usuallyhaveasimilarNO-releasingmechanism,soallcurrentNOdonorsandtheir
pathwaysofNOgenerationaresummarizedinTable1.2accordingtotheirchemical
classification.
ManymedicinesmayworkbyanNO-dependentmechanism. Recentstudieshave
shownthatangiotensin-convertingenzyme(ACE)inhibitors(i.e. Enalapril,Captro-
pril,Cilazapril)improveendothelium-dependentvasodilatorresponsiveness[41–43].
ACEinhibitorsinhibitthedegradationofbradykinin,therebyaugmentingNOpro-
duction. Anothercalciumchannelblocker,amlodipine,alsoreleasesNOfromblood
vessels, and kinins mediate the generation of NO [44]. These new findings give a
goodexplanationforthecardioprotectiveeffectsofthesedrugs. Furthermore,estro-
gen,statins(HMG-CoAreductaseinhibitor)andessentialfattyacidshavetheability
toaugmentNOsynthesis[45,46]. Alloftheabovemoleculesdonothavestructural
moietieswhichcanreleaseNOdirectly, sotheycanbecalledNOstimulators, and
theyarenotdiscussedinthisbook. CurrentlyusedNOdonorswillbeintroducedin
thefollowingchapters.
8 1NOandNODonors
Tab. 1.2: CurrentmajorclassesofNOdonors.
Chemical RepresentativeCompounds PathwayofNOGeneration
Class
Non-enzymatic Enzymatic
ONO2
Organic O2NO Thiols Cyt-P450,GST,etc
nitrate ONO2
H3C
O
Organic H3C NO Hydrolysis,trans- Xanthineoxidase,
nitrite H3C nitrosation,thiols, etc
light,heat
Metal-NO Na2[Fe(CN)5(NO)]•2H2O Light,thiols,re- A membrane-
complex ductants,nucleo- boundEnzyme
HO philes
NO
N-Nitro- N OH−,light Cyt-P450related
samine Me enzyme
OH - +
N-Hydroxyl N O NH4 Light,heat Peroxidases
nitrosamine N O
Me
+N
Nitrosimine _ N Thiols,light ?
N O
N
O
AcHN N
Nitrosothiol S O Spontaneous, Unknown en-
CO2H enhancedbythiols, zymes
light,metalions
C-Nitroso O2N N O Light,heat ?
compound
R1 + O-
R2 N
Diazetine Spontaneous, ?
dioxide R3R4 N+ _O thiols
R R
Furoxan + _ Thiols Unknownenzyme
&benzo- N N O
O
furoxan
Ar +
N N
Oxatriazole- _ Thiols ?
5-imine N NH.HCl
O
NH
Syndonimine O N N Spontaneous, Prodrugsrequire
+
_N O enhancedbylight, enzymatichydro-
oxidants,pH>5 lysis
R2 R1
NOH
Oxime Spontaneous, Cyt-P450
O2N CONH2 O2/FeIII-porphyrin
1.3NewClassesofNODonorsunderDevelopment 9
Tab. 1.2 (continued)
Chemical RepresentativeCompounds PathwayofNOGeneration
Class
Non-enzymatic Enzymatic
H
Hydroxy- N OH Anto-oxidation Catalase/H2O2
amine H enhancedbymetal
ions
NH
N-Hydroxy- HO CO2H Oxidants NOS,Cyt-P450
N N
guanidine& H H
guanidine NH2
O
Hydroxy- H2N NH H2O2/CuZn-SOD Peroxidase
urea orceruloplasimin,
OH
H2O2/Cu2+,
heme-containing
proteins
O
Hydroxamic OH ? Guanylatecyclase
N
acid H
1.3
NewClassesofNODonorsunderDevelopment
DifferenttypesofNOdonorswillbediscussedintheotherchaptersexceptforthe
followingtwoclasses.
1.3.1
Nitroarene
6-Nitrobenzo[á]pyrene(6-nitroBaP)wasfoundtoreleaseNOundervisible-lightirra-
diation,whilenosuchphotodegradationwasobservedwithothernitratedBaPs,such
as1-and3-nitroBaPs[47]. ItcaninduceDNAstrandbreaksuponphotoirradiation.
NOisgeneratedfrom6-nitroBaPvia6-nitriteBaP,whichisproducedfrom6-nitroBaP
byanintramolecularrearrangementmechanism(Scheme1.5). Thisfindingmaybe
usefulforthedevelopmentofanewtypeofphotochemicallytriggeredNOdonors.
NO
hν
NO2 NO2 O
6-nitroBaP 6-nitriteBaP 6-Oxy-BaP radical
Scheme1.5 Photochemicalreactionof6-nitroBap.
10 1NOandNODonors
1.3.2
HydroxamicAcids
Hydroxamicacids[generalformulaRC(O)N(R′)OH]havebeenusedasinhibitorsof
peroxidases[48],ureases[49]andmatrixmetalloproteinases,andasanti-hypertensive,
anti-cancer,anti-tuberculousandantifungalagents[50,51]. Althoughsomeofthese
bioactivities are attributed to the chelating ability of the hydroxamate group, the
hypotensive effects are due to their ability to release NO [52]. Experiments have
shownthathydroxamicacidscantransferNOtoruthenium(III)andcausevascular
relaxationinrataortabyactivationoftheiron-containingguanylatecyclaseenzyme.
Ofthehydroxamicacidsinvestigated,benzohydroxamicacid(Fig.1.1)showedhigher
NOreleasingabilitythanaceto-,salicyl-,andanthranilichydroxamicacids.
O
OH
N
H Fig.1.1 Benzohydroxamicacid.
1.4
DevelopmentofNO-DrugHybridMolecules
An innovative approach to harnessing the beneficial properties of NO is to attach
anNO-releasingmoietytoanexistingdrug(Fig.1.2). Differenthybridcompounds
canoffervariousdrugactionswithsynergisticeffects,withreducedtoxicityandside
effects. Severalpharmaceuticalcompaniesareactivelyengagedinthisresearcharea.
AseriesofcompoundsarecurrentlyinthePhase-IorPhase-IIclinicalstudy. Forex-
ample,NicoxinFrance(www.nicox.com)hasdevelopedtheNO-releasingderivative
ofacetylsalicyclicacid,NCX-4016,whichisclaimedtobeabletoovercomethemajor
drawbackassociatedwiththeuseofaspirinasapainreliever[53]. NCX-4016also
showsabroadermechanismthanaspirinandcaninhibitadditionalinflammatory
mediators[54]. NitroMedinBoston(www.nitromed.com)hasreportedthatnitrosy-
latedá-adrenoreceptorantagonistsmoxisylate(S-NO-moxisylate)hadlowertoxicity
andfeweradversesideeffectsinthetreatmentoferectiledysfunction[55].
General:
Drug NO
CH3
OAc ONO2 CH3 O CSHN3O
O N O CH3
H3C
O
H3C CH3
NCX-4016 S-NO-Moxisylyte
Fig.1.2 NO-drugconjugates.