Chader Drus 2 Dtscowry D..ign,.ndOevetopm.nr a. lmpo anbe Lipophilicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 ot ... b. Measurcment ol Lipophiticitis ............... _...... . . . . . . . . . . . . . . . . . . . . . 55 c.

Compute zation LogP Values...... of _...................._........__...61 d. I\,4mbrane Lipophiliciiy. ..................62 3. Effscts lonization Lipophtticity O.atBioavaitautity. . . . . . . . . . . . . of on and . 62 4. OlherProperties toflusnce rhat OratBioavaitabitity Abitityto and Cross Bloocl-Brain the Banir................... . . . . . . . . . . . . . . . . . . . . . . . . .6s G. Ouanlilaiive Structure-Activity Rtationships . . . . . . . . . _. . . . . . . . . . . . . . . . . . . . _. . . . . 66 .. . 1. Hislorical. . . . . . . . . . . . . _ . . . . . . . . . . . . _. . . . . . . . . . . . . . 116 2. StecEllectsrTheTafiEquaiionandOtherEquations...._....................67 parameters 3. Methods lJsedto Conelate Physicochemicat with BiologicalAciivity.......... .._....................68 a. Hansch Analysie:A LlnoarMulripte Rogression Anaty$is..............._..68 b. FreandWilsonor d novot\,lethod. . . , , , , _. . . . , , , . , _. . . , , , , , . . . . . . . , , , ,70 . c, Enhancement . - . Factor . . . , , , . . . _. . . . , , , , . . . . 71 d. lLlanual Stepwise Methods: Toptiss Opetionat Schmes Others. . . . 7l and . e. Batch Selection Methodsr Balchwise Toptiss Oprarionat Scheme, Clusrer Analysis, Othe and ....................75

2,3 Gen6lReterencos

..................47

Discovery ll z.r Orug

Drug discoveryis a very time-consuming expensive and pmcess. Estimates ofthe avemae rime requird bringa druglo rhemarker ro range from I 2- | 5 yars an a!emge ar co\r of $600_800 million. For approximatelyevery 10,000compounds lhat areevaluated animal srudies.t0 i; will makeii to human clinicaltrial\ in orderro get t compound the marker. ct,nical on Tfie trials consistofthree phases pdor to drug approval:phaseI (generallya few monrhsto a year anda hal0 evaluates safety,tolerability (dosage the levelsand sideeffects).Dharmacokinetic propenies. pharmacotogicat and effecrs 20-t00 healrhy irl \otunteers: phaie II (abourt_J years)assesses effctiveness the drug, determines the of sideefiectsandother safelvasDects. andclarifies dosing lhe regimen a fes hundred in dLeased pabents: pha.eU (;bour2_6 and rial $irh severa_l padenrs cUojcs hospiral\ eslablrshes lhousand )earsris a larger in and ihat the efficacy of the drug and monitors advene rcactionsftom long-term use. Once the new dlllg application(NiDA) is subminedro rhe Foodand Drug Administration (FDA). ir can b severalmonths io severalyearsbefore it is apFoved for comnercial us.phaseIV studies areconsidered betfieresuhs to found\^irh a druglhalha\ already been owedonrothedrua a market and is in generaluse.Drug candidates rlew chefiical ent|ties,Nct, as they are (or

Di5cov6ry a!$oo 2 I Orug

financial lossesfor the nien called) that fail late in this Focess result in huge,umecovered puchasea alflg is so high. lt is not that it coststhat muchto .ompany.This is why the cost to to that manufacture one drug, but that the profits are needed pay for aI of the dmgs that fail funds havealreadybenexpended. ro makeit to marketafter large sumsof research is wllat is more likely discovered known as a lead In general,drugsare not discovered. a numberof attractivecharacteristics' that has The lead is a prototypecompound compound. such as the desiredbiologiirat or phannacologicalactivity, but mav have other undesirable for rytion difficulties' characteristics, example,high toxicity, otherbiological activities,abso. is modifiedby synproblems.The structureof the leadcompound insotubility. or metabolism Foperties to ihesisio amplify the desiredactivity andto minimize or eliminatethe unwanted pharmacological' worthy ofextensivebiological. cmd lat, a comPound ^druT ^poina\Nherc radyfor clinical tdals' is andaninal studies.is idennfred-therla clinical druq, a ompound to hior to an elaborationof approaches teaddiscoveryandleadmodification'two developd. commondrugsdiscovercdwithout a lead arc discussed

withouta Lead 2.1.A DrugDiscovery a.1 Peniclllln.

In 1928 AlexanderFleming noticed a greenmold growing in a,ciltwe of ,tldp,]rlococc,r rl the arrelr, andwherethe two hadconverged, bacteriawerelysed.L This led to the discovery of penicillin, which was producedby the mold Actually' Fleming was not the first to make rhis obse ationi JohnBudon-sandersonhad done so in 1870,ironica y also at St. Maryt Hospital in London, the sameinstitution where Fleming made the rediscoveryll2lJoseph Lister had teated a woundedpatient with Perirtli,.m, the oryanismlater found to be the producerof penicillin (althoughthe strainsdiscovercdearlier than Fleming's strain did not acid).After Flemingobservd antibiotic,mycophenolic Foclucepenicillin, but,rather,another it he tried many times to rePeatit without success; was his coneague' this Dhenomenon, It Ronald Hare,l3'alwho was able to reproducethe observation. only occurredthe first Dr Hare found time becausea combinationof unlikely eventsall took place simultaneously. initially observed conditions were required to produce the phenomenon that very special by Fleming. The culture dish inoculatedby Fleming must have becomoaccidentallyand lnsteadofplacing thedishin therelrigerator with contaminated themold spore. simultaneously left went on vacaiionasis nomally done,Fleminginadvertendy it on his or incubatorwhenhe the fo owing month,he noticedthe lysedbacteda Ordinarilv' lab bench.Wlen he retumed but pdcillin doesnot lyse thesebacteria;it preventsihem ftom developing, it hasno effet havedeveloped However.while Fleming was on vacation(July if addedafter the bacteria cold, and this providedthe particulartemperature to August) the weatherwas unseasonably to requiredfor the rnold and the staphylococci gtow slowly and Foduce the lysis. Another thepanicularstrainof themold onFleming'sculturewas was circumstance that extraordinary a relativelygoodpenicillin ploducer,althoughmoststrainsofthat mold (Prnictlii"t ) produce no penicillin atall. Themold Fesumablycameftom thelabolatorvjust belowFleming'swhere research molds was going on at that time on that penicillin could be useful as a topical antiseptic,he Although Flerning suggested was not successfulin Foducing penicillin in a form suitable to treat infections. Nothing lhe morewasdoneuntil Sir HowardFlorey at Oxford Univeniry reinvestigated possibility of eucceeded producing penicillin could lhat in torm.In ls40 he in oroducins oenicillin a useful the lull e\lenl oflhe valueof Penicillin ropical5 and \!slemicall).rtlbut Le aanuni,rerea

t0

Chaprr2 DrugDB@ery ltedgn, and Developmenr

for Tlvo until the late 1940s.t61 reasons the delayin the univenal utilization wasnot revealed of of penicillin were the emergence the sulfommide antibacterials(sulfa drugs, 2.1i see Chapter5, Section5.4.B.2,p. 254) in 1935and $e outbreakof World Wd II.
Hr\ (

,--\

'FSOr\'ilR

"*i1'*
produciion,andclinical aPplicarion penicillin of No studiesrelatedto the phafinacology, to ftom havingaccess this wonder werepmitted until after the war to preventthe Germans involvedin chemotherapeutic who wereintenogatingGeman scientists drug.Allied scieniists researh were told that the Gemans thoughtthe iniiial reportof penicillin was madejust for commercialrcasonsto competewith the sulfa drugs.They did not take the rePortseriously gave a reiatively low yield of The original mold was Pdnicillium notatum,a stJain_that which had beencultured ftom a penicillin. It was replacedby Peniciqiun chfsogenrn.LTr mold growing on a gapefiuit in a marketin Peoria,nlnoisl but noi rnunyyean a.lut" *ged regading the actualstructureof penicillin (2.2).131 the in correctstructwewaselucidated 1944with an X-ray crystalstructureby Dorothy Crowfoot differuntil 1949tvl Several was Hodgkin(Oxford); lhesystal structure rot actuallypublished (R ent penicillin analogs goup vaded)wereisolatedearly on; only two of theseearly analogs (2.2, R = PhOCH2,penicillin V; and 2.2, R : CHzPh,penicillin G) are still in usetoday.

s !! *\--a\ -n-

.",
H

ii

^y' '

n-/\cx,
l'coon

v .-penicillin (F - PhocH, peniclllln {R CHrPh) G -

A.2 Llbrium HCI tranquitizerdrug,chlordiazepoxide t7-chloro-2-(methylamino)Thefirst benzodiazepine I0l serendipitously.l 2.3; 4-oxide; Libriuml,wasdiscovereal s-phenyl-3 1,4-benzodiazepine fla Dr Leo Stembach Rochewasinvolvedin a Fogram to synthesize newclassof tranquilizer at I ines a He ly drugs. original setout to prepare serie.of benzhepro\dia/ (2.4r.btrlwhenR wa' CH2NR2and R2 was C6H5,it was found that the actual stmcturewas that of a quinazoline (2.5). 3-oxide
. _ NHCHI HCI

,-J*A'J:

A"
2.3

"2 " t\,"?

2.4

xlw\,'1o

section 2.1 Drug Dl.cdry

11

itY-*"t

CH3NH2

z(o-

a'*<*T '
I

' -lo H

s-

ll

/tH':Cl

a\

r-5,

.-

2.3

Schme 2.1 > Me.hmism for fomation of Librium.

results. The However,none of thesecompoundsgave any interestingpharmacological to programwasabandoned 1955in orderfor Stembach work on a different project tn 1957 in during a generallaboratorycleanupa vial containingwhat was thoughtto be 2.5 (X = ?-Cl' R1 : CHzNHCH:, R2 = C6H5)was found and, as a last effort, was submittedfor pharmasubmitted,this onegavevery Fomising cological testing.Unlike all of the othercompounds results in six different testsused for preliminary screeningof tanquilizers Further investigation revealedthat this compoundwas not a quinazoline 3-oxide, but was instead the reactionof the corle4-oxide, 2.3, presumablyproducedin an unexpected benzodiazepine hloromethyl quin^zoline 3-oxide (2.6) \tith methylanine (Scheme2 1) If this sponding compoundhad not been found in the laboratorycleanup,all of the negativepharmacologand ical resultswould havebeenrcportedfor the quinazoline3-oxide classof compounds, for may not havebeendiscovered many yeals to comebenzodiazepine 4-oxides The examples drug discoverywithout a leadare relatively few in number The typical of is is is occunence thataleadcompound identified,andits structure modifiedto give.eventually, goesto the clinic. the compound that

2.1.8 LeadDiscovery
without a lead. Penicillin v andLibrium are,indeed,two importantdrugsthat werediscovered leadcompounds second-generation for However, oncetheywereidentified,theythenbecame havebeensynthesized a result of the as analogs. myriad of penicillin derivedantibacterials A (2.7,Valium)wassynthesized penicillins. at of Diazepam structure elucidation the earliest RocheevenbeforeLibrium was introducedon to the market;this drug was derivedfrom the leadcornpound Librium and is almost l0 times morepotentthanthe lead.

and Design, DrugDiscoverY, Development

2.2

''l1 - ' ' '' ; Discovefy..., B. Leact ,'13 ,'" : Scronlng . 1. Randorn ,' 14 Scrnlng (orTarcstod Focusd) or 2. NonBndom ,' '14 Sludios .... -,' Metabolism 3. Drug 15 .. - ' -'' obsrvatlons. " 4. Clinlcal ,'16 ,' Discovery to Approaches Lead " 5. Ralional . ... .. ....... .....17 ....... B.

c.
D. E.

12

Chaplor DrugDbcov.ry. 2 D*ign..nd D.vetopmnr

ar'f
r-

cHr :, .!)

dlazepam 2.7 The initial diffiulry arisesin the discoveryof the lead compound.Severatapproaches can be taken to identify a lead. The fiIsr requirementfor all of the approaches to have is a meansto assaycompoundsfor a particular biological acrivity, so ihar researchencan tell when a compoundis active. A ,ioaffn) (or sc,,err) a meansof determinins in a is biologicalsystem. relarjlero a conFolcompound. a compound lhe desirea iI ha. acriviry. and, if so, what the ielative potency of rhe compoundis. Note tbe distincrion betweenthe terms activity andpotencj. A.rtl,ry is the particular biological or phamtacologicaleffect (for example,antibacterialactivity or antico$'ulsantactiviry)i pdrc, is the streng$ of that efftSomescreens ln virlo tests,for example,the inhibition of an enzymeor antagonism are of a rcceptor; othersaft ,n yiro tesrs,for example,the abiliry of the compoundto prevent an inducod seizurein a mouse,In genelal, the in litl, tests aft quicker and less eK)en_ sive.Clurcntly, high-throughputscreens(lIS),Irrl very rapid and sinsitive ir vlrro screens initially developedabout 1989-1991,that now can be carried out roboticallv in 1536- or 3456-well riler plales smalltsubmicrogmm) on amounr5 compound ol (di55otu"a .uU-;in croliter volumes)arebecominguniversallyused.With theseulta-high-throughputscreening approaches, is possibleto scren100,000compoundsin a dayl As we will sebelow. it combinatorialchemistry(seeSection2.2.E.5,p. 34) cansupplyhugenumbersof compounds in a short period of time, which, theoretically,shouldprovide an increased numberof ltr, i.e., compounds that elicit a Fedetemined level of activiry in the bioassayand, thercfore, provide more leads.According to Drews,u2l the numberof compounds assayed a large in pharmaceutical companyin the early 1990swas about 200,000a yeari rhat numberroseto 5-6 million during the mid-1990s,and by the end of the 1990sit was >50 milliont However, the increasein the assayrate did not result in a commensurate indease in research productivity, as measured new compoundsentering the market. Of course,it can tale by 12-15 years for a drug to reach the market, so Foducrivity in th early pan of rhe 2lst century should provide a more accurateruler for success drug disoverychanges of made at the end of the 20th ceniury. Cunently, HTS appea$ to have resultedin an increasein the numberof hils, but this may be because mote lipophilic compounds, whih may have more druglike Foperties (se Secrion2.2.R2, p. 53), can be resredby dissolving them in dimethylsulfoxide (DMSO) rather than in water.Nonetheless, is not yet clear if this it increasein hit rate is translaiing into a much greater numbr of leads and development compounds,tl3l An exciting apFoach for scrceningcompounds thar might interact with an enzymein a metabolicpathwaywas demonsrrared Wong, Pompliano,and coworkeff fm rhe discovby ery of lead compounds rhai block bactedalcell wal biosynthesis(as potenrialantibacterial agents).t1al Condidonswerefound to reconstirute six enzymes the cell wall biosyntheiic all in

____'_5 I

Section21 DrugDlscovery

13

for pathwayso that incubationwith the substrate the first enzymeleadsto the formation of product of the last enzyrnein the pathway.Then by screeningcompounds and looking the it is possibleto identify not only compounds that block for the buildup of an intermediate, (and preventthe formation of the bacterial cell wall). but to determinewhich the pathway meansthat the enzymethat actedon that enzymeis blocked (the buildup of an interrnediate intermediate was blocked). Compoundsffeening also can be caffied out by electrosprayionization massspectrofor merylrsl (the technique which JohnFennreceivedthe Nobel prize in 2002)andby NMR complexes compounds of with a macromolecule specromety.tr6l Tightly boundnoncovalent (such as a rcceptor or enzyme)can be observedin $e rnassspctrum.The afnniiy of fte l|Aand (a small moleculethat binds to a receptor)can be measuedby varying the collision This method also can be energyand determiningat what energythe complex dissociates. provided they have ditrerent moleculal used.o screenmixnrles (a libnry) of compounds, so masses andor charges, the l,/z for eachcomplexwith dle biomolulecan be separated in the massspectrometerBy varying the collision energy,it is possiblero detemine which exPloirs in be'r. changee erlher bindto lhebiomolecule The rH \MR merhod lesrmolecute' relaxation ratesor diffrrsion ratesof small moleculeswhen they bind to a macrcmolecule. to This methodalso can be usedto screenmixtures of compounds determinethe onestbat bindbestcan a is Oncethe screen developed, variety of approaches be taten to obtaina lead.As we will seebeloq the typical lead compoundfor a receptoror enzymeis the natural ligand for is for thercceptoror substrate the enzyme.Anothergoodsourceof leadcompounds marketed tfl and rlrugs. llr this case targetwill generallybe well established, $e leadsfucture will be the known to bind well to the targetandto havegoodabsorytionpmperties.The main stumbling lf block to the use of rnarketeddrugs as leadsmay be patentissuesfor commercialization. is the targetmacromolecule not known or ifno new leadshavecomefrom a marketeddrug, otheraooroaches taLen. can

8.1 Random Screening with desiredactivity, a Iandom screen In the absence i{nown drugsand other compounds of all are Rdndomscrceninqinvolvesno inteltectualization; compounds is a valuableapproach. Prior to 1935(the discoveryof sulfa without rcgardto the;r structures. testedin the bioassay the todaythis methodis siill animportantapFoach drugs),this wasessentially only approachi to suchhugenumbers it to discoverdngs or leads.particularlybecause is now possible screen rapidly with HTSS. This is the lead discoverymethodof choicewhennothing of compounds is known aboutthe receptortarget. are The two rhajor classes materialsscreened syntheticchemicalsandnatumlproducts of (microbial, plant, and marine). An exampleof a random screenof synthetic and natunl and wasthe r'war on cancel' declaredby Congress the National CancerInstitute compounds in 1970s.Any new compoundsubmittedwas screened a mousetumor bioassay. in the early but drugsresultedfrom that screen, many known anticancerdrugsalso Few new anticancer did not show activity in the screenused, so a new set of screenswas devisedthat gave by results.In the 1940sand 1950s,a randomscreenof soil samples various morc consistent Howevet in this case, newantibioticswasundertaken. pharmaceutical in of companies search but and leadsuncovefed, two imponantantibiotics.streptomycin the not only werenumerous

14

2 Dl@ery lt rlgn, tnd DovoloPmnl Chaplar Orug

of tetracyclines,werefound. Screening micrcbial brolhs, particular strainsof Strcptomlces, was a cominonftndom scfeenme6odology prior to 1980 Nonrandom (oJ Taryeted or Focu8ed) Screning Nonrandomscreening,alsoc led taryetcd orfocttsenscrcenine,is a morc Darow approach to than is random screening.In this case,compoundshaving a vagueresemblance weakly active compounds uncoverd in a random scrcen, or compoundscontaining differcnt By the firnctional goups thanleads,may be testedselectively. thelate 1970s, NationalCancer scren because ofbudgetaryandmanInstitute'srandomsueenwasmodifiedto a nonrandom powerrcstrictions.Also, the singletumor screenwas changedto a variety of tumor screens it because was rcalizedthat canceris not just a singledisease. 8.2 8.3 DJugMetabollsmStudis gen_ During drug metabolismstudies(seeChapter7) rretdrolites (drugdegradation Products is to isolatedarc screened determineif the activity observed derived er.ted in ,irr) that are the or from the &ug candidate from a metabolit.For examPle, anti-inflannatory dnlg sulin(2.8, Clinoril) is not the activeagent;the metabolicrcductionProduct.2.9, is responsible dac for lhe activity.tl8l

1r
2,4

antihistamineterfenadinehydrochloride (2.10, Seldane)was found to The nonsedating heartrhythm in someuserswho alsoweretal:rngce(aln antiirngal agents, cause abnormal an a tedenadine. This caused builduPof which werefound to block the enzymethat metabolizes led io lhe abnormalheari rhythms.Howevet a metaboliteof tefenadine, which terfenadine, antihistamine, hydrochloride(2.11,Allegra),wasalsofound to be a nonsedating fexofenadin presence antifungalagents. of This, then,is a saferdrug. evenin the but it canbe metabolized for Mtabolitescanbe screend otheractivities aswell.
QHr

HO--H

trf6n.dln. Hcl 2.10

;gD_

21 Drug Dls@ery

15

.\.t:r" t-HoX
P( UoI '

.N--\z
loxoi.ndln. 2.tr HCI

8.4 ClinlcalObservatlon3 during clinical irials will exhibit morethanonepharnacological a Somerimes drugcandidate it may Foduce a side effect.This compound,then,can be usedas a lead (ot a.d!iry; thai is, {rrh luck, as a drug) for the secondaryactivity. In 1947 an a ihistamine,dimenhy'lfnate , 2.12.Dramamine) wastestedatthe allergy clinic at JohnsHopkins Univenity andwasfound furtherstudy who suffered^.from sickne\sl.a car in d].o ro be effecLive relievinga Palient 4rTl thenbecame Iv ic of in lie treatmeDl seas knes\r I andairsicloress pm!ed irseffe(riveness the mosrwidely usddrug for the treatmentof all forrns of motion sickness.

"^I^Y)>"'
dlm.nhvdrh.le

1?,

BuproThere are other popular examplesof drugs derived fmm clinical observations. (Wellbutrin)' was found to help patients drug pion hydrochtoride(2.13), an antidepressant srop smokng and is now the Iilst dnrg marketedas a smoking cessationaid (Zyban). Ttte for dmg sildenafil citrate (2.14: Viagra) was designed the treatmentof anginaand irnpotence which hydrolyzescy-clicguanohypnensionby blocking the enzymePhosphodiesterase-s' (cdMP), a vasodilatorthat allows increasedblood flow.t2u In 1991 .in. -onopttospttat" sildenafil went into phaseI clinical trials for angina.ln phaseII clinical trials, it was not as I anginaasPfizerhadhoped,soit went backto phase clinical trials to seehow effectiveagainst hish of a dosecould be tolerated.It was during that clinical trial that the volunteersreported inleased erectile function. Given the weak activity againstangina' it was an easydecision as to try to determineits effectiveness the first treatmentfor erectile dysfunction.Sildenafil for works by the mechanism which it was designedas an antianginaldrug, exceptit inhibits insteadof the heart (Figure 2.1). in rhe phosphodiestemse the pnis (phosphodiesterase-5) of release nitnc oxide in the penis causes Sexualstimulation

\')
2.14

16 .-AJs ---lq------.isynoase Nidc onde

Chaptor2 DrugDlscwery, De3ign,and Development

Guantlale cyclas \ cCMP-----__t' sDootb muscle'_'---t_

lsr''r' k '

I I

CTP

+ I
PDE5

GMP

4 inhibirs
I
Flsure 2.1 > Mechanisnoi actionof sildenafl (Viagra)

cyclase' the moleculethatstimulates enzymeguanylate messenger Nitnc oxideis a second to triphosphate cCMP The vasodilatorCGMPrclaxesthe smooth which convertsguanosine musclein the colp r .dttmos ,1, allowing blood to flow into the penis,therebvproducingan vasowhich causes erection.Howvet phosphodiesterase-s CDE 5) hydrolyzesthe CGMP, andtheoutflow of bloodfrom thepenis Sildenafilinhibits thisphosphodiesterase, constriction preventingthe hy&olysis of oGMP andprolonging the vasodilationeffect

8.5 RationalApproachesto Lead Discovery None of the aboveapFoachesto lead discoveryinvolves a major rational component The as lead is just found by soeening techniques, a by-product of dtug metabolismstudies,or Is from clinical investigations. it possibleto detrgn a compoundhavinga pa4icular activity? to Rationalapproaches drug designnow havebecomethe major routesto leaddiscovery The or state Many diseases, at leastthe symptoms for firsi stepis to identiry the cause the disease (either excessor deficiency)of particular chemicalsin arise from an imbalance of disases, the body, from the invasionof a foftign organism,or from aberrantcell gro*th As will be or the discussed later chapters, effects of the imbalancecan be corected by antagonism in particularenzvme(seeChapter5); agonismof a rcceptorGeeChapter3) or by inhibition of a foreign organismenzymeinhibition or interfererce with DNA biosynthesisor function are and arisingftom microorganisms aberrantcell growth to impo(ant approaches treatdiseases 6). GeeChapter then become Oncethe relevantbiochemicalsystemis identified. initial lead compounds the natural recptor ligands or enzyme substntes. For examPle.lead compoundsfor the (+)-norgestrel (2.15, Orral) and l7o-ethynylestradiol(2.16' Activella) were contraceptives (2'17) and 178 estradiol (2.18) WlHeas the steroid the stercidal hormonesprogesterone 2.15and 2.17 and2,18 showweakandshortlasting effects,the oral contmceptives hormones 2.16exert strongFogestationalactivity of long duration.

Sedon 2.2 Lssd Modllicallon: Druq Dlgn and O.velopmenl

17

2 .1 5

2.16

2,18

At Merck it was believedthat serotonin(2.19) was a possiblemediatorof inflammation. Consequently, serotoninwas usedas a lead for anti-inflammatoryagents,andfiom this lead lhe anti-inflarfinatory drug indomethacin(2.20,Indocin) was developed.t22l

1i
2.19

2,20

The rational approaches directedat lead discovery It is not possible,with muchaccuare racy, to foretel toxicity and side effects, anticipatetransportcharacteristics, predict the or metabolic fate of a dnrg. Once a lead is identified, its structurecan be modified unril an effedive drus is obtained.

,.4, -J

2,2 LeadModitlcation: Drug Designand Development

Onceyour lead compoundis in hand,how do you know what to modify in order to improve propefties? thedesiredpharmacological

.,.,22.A ldenlilication ot the Actlve Part:The Pharmacophore

Interactionsof drugs with receptors,known as pidrltuco.bnamics, arc rcry spcific (see Chapter3). Therefore,only a smallpat of the leadcompoundmay be involvedin the appropriate receptorinteractions.The relevantgroupson a molculethat iDtemctwith a receptor and areresponsible the activity are collectively known as rhephamucophrr. The other for atoms in the lead molecule, sometimesrefe[ed to as the awophore. mav be extraneous,

18

Chaptd 2 DrugDls.@ry D.!19n,lnd DercloPrent

to Someof the atoms,of couse, areessentiai maintainthe integrity of the moleculeandhold atoms, groupsin their appropriatepositions.Someof theseextraneous the pharmacophoric and the binding of the pharmacoPhore' thoseatomsned however,mat be interfedry with from the lead compound.Otber atomsin the auxophoremay be dangling in to be exciseal the spacewithin the receptorand arc neitherbinding to the receptornor Preventing phama_ to atomsaPPear b imocuous, it is importantto cophoricatomsfiom binding. Although these thesearethe onesthat canbe modified without loss of krow which atomstheseare,because to potency.As we wilt seelatq thereareotherasPects leadmodificationthat arc asimportant aspdt nacot e/icr (abso4tion, distribuas increasingbinding to the taqet receptot such ard tion, metabolism, excretionor ADME) Modification ofthe atomsthat arenoi intedering with binding could be vr/ impo(ant to solving phamacokineticspIoblems. By determining which ale the pharnacophoric groups and which are the auxophoric and goups on your leadcompound, of the auxophoricgroups'which areinterferingwith lead binding andwhich arenot detrimentalto binding,you will knowwhich groupsmust ompound in One approach leadmodification be excisedandwhich you canretain or modiry asneeded. the is to help makethis determination to cut awaysectionsof ihe leadmoleculeandmeasure how this might effectsof thosemodificationson potency.Considerthis artificial exanple of moryhine(2.21, R = R/ = H)i codeine(2'21' be done.Assumethat the addictiveanalgesics and R : CH3, R/ : H), ard heroin (2.21, R = R/ = COCH3)are the lead compounds, we and want to lorow which groupsare pharmacophoric which areauxophoric'

morphlne(R = R' = H) codeine(R = CH3,R'= H) hroin (R = R' = COCH3) 2.21 binds to the l, opioid recptoN The phamacophore The morphinfamity of analgesics in ;d is shown as the darkenedPsn in 2.21 A decrease potencyon removal of is known an it may have tteenpharmacoPhoric' increasein potencymans a group wil suggestthat no interfering with Properbinding, and essendally changein potency it was auxophorican<t but not interfering with binding. will meanthat it is auxophoric Let's sta( by excisingthe dihydrofuranoxygenatom,which is not in t'\e pharmacophore' ring to the that atom connectsthe cyclohexene This may not seemto b sensiblebecause ring in a charge in lhe conformationof the cyclohexone ring; its removalwill rcsult benzene offreedomofthe molecule Excisionofrhe dihydrofuranoxygen in andanincrease the de$ees the givesnorphinan (2.22,i = H;i23J; hydroxyanalog'levo+hanoll2al(2.22.R = OH, Levobut nore Potentthan momhine as an analgesic, it rctains bromoran). is three to four times ring confomation has not been (note that in 2.22 the cyclohexene ihe addictiveproperties with 2.21: surely,a lower energyconformerwill be favored) for changed easeof comparison

Scion 2.2 Leid Modltlo.llon: Drug lte.lgn and D@loptrent ctt N

t9

(F lcvorphanol oH) 2,22 -

its Possibly,the additionalconfofiBtional mobility alowed the moleculeto approximate (lhe conformationthat most efrectivelybinds to a receptor).Removal bioact e confomation of half of the cyilohexene ring (also not in the phamacoPhor),leaving only methyl sub(223, R : CH3).txl This compoundshowssomseparation stituents,givesbenzomorphan hydtocbloride(2.23,R = CHzCH:C(CH3)2 i of analgesic addictiveeffects;pentazocine and but componentof Tatwin) is less potent than morphine (about as potent as'codeine), has a potencf anddecrease your goalis both to increase muchlower addictionliability. Remember, adverseeffects, such as addi.iive properties.Although this analogis not more poteft than fused morphine,it is less addicting. Cutting away the methylnegmup of the cyclohexane activity in animal tests. ring (2.24) also, surprisingly,haslittte effect on the analgesic

\. xct z..-- z-c[,

I l-'*

)a\

pnlazocln. (F = CHTCH.C(CH3L) HCI 2,24

structure. Removalof all fusedrings, lhe Again, this excisionremoves rigidity of theParent givesan analgesic possessing still t215,_Demerol). for eiample,in rfiecaseof meperidjDe the is of Although porency lower,it cenainly l0-129oof the overal potency morphine.l26l will be much easierto synthesizeanalogsof meperidinethan of molphine. Even acyclic analogsare active. DextoFopoxwhene (2'26, Dafloq again note the side chain is left in a conformationto resemblethe structruof morphine)is one-half to two-thir& aspotnt as bind codeine. Both morphineanddxtsopropoxyphene to the p opioid receptot sotheacdvity a can of dexrropropdxyphene be ascribedto the fact that it can assume confomution related I ro that of the morphinephafinacophore. don't thint any of us, seingdextroProPoxyphene written in a more eneryeticallyfavorableconfonnation,would ever make the connection betweenthis sfucture and that of mor?hine.By cutdng piecesoff of the lead compound,it givesyou new psspertives on possibleactive structues, which shouldopen up comPletely (2'27,Methad$e) Anotheracyclicanalogis methadone to in newscaffolds consider synthesis. potentar analgesic moryhine;the (-)-isomer is usedin the treatmentof opioid as which is as it because is eliminatdfrom the body slower than in abstinence syndroms heroin abusers body to adaptto the falling levelsof drug gradually. morphine,allowing the

20 iH3

Chdler 2 Drug DiscoEry. Deslgn,and ovetopmenr

._Y<o
ocH,cH. |-

2,25

2,26

What if every cut in the Iead producesa compoundwith lower potency?Then, eiiher every excision is removingpart of dre pharmacophor or eachcur causesa conformarional changethat givesa structurel?rr similar ro rhe bioactiveconformation.The lafter possibility is particularly relevantto such a dgid structureas in moryhine.In this case,groupsneedro be addedto the lead structureto ircleare the pharmacophore. example,oripavinederivaFor (2.28, = CH3.R' = CaH?;Immobilon)t tivessuchasetoryhine R whichhasa rwo-carborl bridge and substituent in morphine,is 3200 times morepotent rhanmorphinel2?l not and is usedin veterinarymedicineto immobilize large animals.The relatedanalog,buprenor?hine (2.28,R=cir.-<: , R/ : /-Bu, doublebond reduced;Buprenex)iS 10 20 rimesmore polenr than moryhineand hasa very low level of dependence liability.

(R loryhln = CH!;F :CaHt 2,24

Apparendt the additional rigidiq, of the oripavinederivativesincreases appropriate rhe recepiorinieractions(seeChapter3). Theactivity andpotencyofa moleculearcrclatedto rheinteractions thepharmacophoric of groupswith groupson the receptor(seeChapter3, Section3.2.B)_The binding constants of 200drugsandpotentenzymeinhibitors wereusedby Andrewsandcoworkersl28l calcutate to the average binding energiesof common functional groups;theseeneies can be used to determinehow well a new moleculebinds to irs receptor If the tstmoleculehasa measured bindirg energyrhai is lower than rhe calcularedaverage value, it suggesrs the molecule rhat containsgroupsthat do not interacrwilh the rccepror(arenor in the pharmacophore). These groups,then, could be exciseddthout loss of potency,giving a simplified lead for further srucoral modificarion.Tbis Andrlrs dndtrir was carriedour on a highty subsrituted lead compou Iead to a more q nd. Ing srrucrure sa. modified simple anatog thal logivemotecuter rrh rz"r potency Il $e Lesl enhanced compound a bindrng has grearer energy lhanrhecalcutared averagevalue,then the moleculemay bind differently than suspected. leading ro enhanced binding interactions.Tbis indicaresthat manipulationof functional goups is an important lead modilication aDDroach.

seciion 2.2 L.ad Modification:orug Designand Dv.lopment

GroupModification 2.2.8 Functional

by The importanceof functional group nodification is demonstrated 2'29. The antibacterial (2.29,R = NH2), wasfound to havean antidiabeticsideeffect;howeverit agent,carbutamide activity,which couldlead drug because its antibacterial of could not be usedasan antidiabetic (seeChapter5, Section5.2, p. 231). The amino group of carbutanide to bacterialresistance wasreplaced a methyl goup to give tolbutamide(2'29, R : CHI; Orinase)andin so doing by the anlibacterialactivjty waseliminatedfrom the antidiabeticactivity.

R1

,,-\

_TSO,NHCNHCH,CH,CH,CHT

(R lolbut.mid = CH3) 2,29 nedicinal chemistknowswhat functional group will elicit In somecases, experienced an agent that has a a oarticular effect. Chlorothiazide(2.30, Aldocor) is an antihypertensive strongdiuretic effet as well. It was known from sulfanilamidework that the suuonamide udne excretion)aciivity Gee Section2.2.C below). side chain can gjve diuretic (increased drug without as dizzoxide(2.31.Hyperstat)was prepared an antihypertensive Consequently. diuretic activiiy.

aYY"
",,*u{'
2,30
2,31

Obviously, a relationshipexists betweenthe molecular structureof a compoundand its was activity. This phenomenon first rcalized about 135yearsago.

Relatlonships 2.2.C Structure-Activity

that the quatemaryammoniumchancter of suspecting In 1868Crum-Brownard Fraser,t3ol may curare, a potent poison known since the 16tb century that was used on arrowheads, be responsiblefor its muscularparalytic Foprties (it blocks the action of the excitatory blocking acetylcholineon musclere.eptors),examinedthe neuromusaular neurotransmitter alkaloidsin animals ammoniumsaltsandquatemized effecisof a varietyof simplequatemary Fromthesestudiesthey concludedthat the physiologicalactionof a moleculewasa tunction Richardsontsllnotedthat thehypnoticactivity of its chemicalconstitution.Shortly thereafter, are alcoholswas a function of their molecular weight Theseobservations the of aliphatic relarionrldpr (SARo. for future str.&cture basis -aciitiry Drugs can be classifiedas being structurally specific or structurally nonspecific..ttr',/cntrclly specifc dru4s, wbich most drugs are, act at specific sites,such as a receptoror an to in enzyme.Their activity and potencyare very susceptible small changes chemicalstruc_ similar biological activities tend to have common structuralfeatures hrrei moleculeswith d Struct\rallf nonspecific tgshaveno specilic site of actionandusuallyhavelower potency Examplesof thesedrugs Similar biological activitiesmay occur with a variety of structures. and hypnotics,and mary antiseptics disinfectants. sedatives and anesthetics, are gasous

chaprd 2 orug Dl.coery [t.lgn, and Developmenl

with its activity, a multitudeof Eventhoughonly a part of the moleculemay be associated molecularmodificationscould be made.The hallmark of SAR studiesis the synthesisof as many analoSs possibleof the lead andtheir testing to determinethe effect of ssuctureon as are and con" activity (or potency).Onceenoughanalogs prepared sufficientdataaccumulated, Unfortunately, ease syntheof clusionscanbemaderegading structure-activityrelationships. made. is sis,ratherthancogentrationales, often theguiding force behindth choiceof analogs of An excellentexampleof this approachcame ftom the development the sulfonamide antibactenatagents(suua drugs). After a number of analogsof the lead compound sulfanilamide (2,1, R : H; AVC) were preparcd,clinical trials deGrminedthat compounds of this generalstructue efibited diuretic and antidiabeticactivities as well as antimicrocertain bial activity. CompoMdswith eachtype of activity eventuallywere shownto possess structwal featuresin colnmon.On the basisof the biological results of geater than 10,000 Andmicrobial agents havestructue weremade.t32l compounds, sevenl SAR generalizations 2.32(R = SO2NHR/ SO3H). or ,\* rn,-1_/ 2,32 In 2.32. (1) the amino and suronyl goups on the benzenering shouldbe para; (2) rhe (as that anilino aminogroupmay beunsubstituted shown)or mayhavea substituent is removed (3) replacement tie benzenering by other ring systems.or the introduction of of ir rira; the on the additionalsubstituents it, decreases potencyor abolishes activity; (4) R maybeanyof potencyis reduced mostcases; N-monosubstitution (5) in rhealtematives shownbelow.but the

,o+*1.oO*.E*.E* *8-$'.
(R = SO2NHR/)resultsin morepotentcompounds, thepotencyincreases and wilh heteroaromatic substitution;and (6) N-disubstitution (R : SO2NRT),in general,leads to inactive Antidiabetic agentsare compoundswith structure 2,33, where X may be O, S, or N incorpomtdinto a heteroarcmaticstructuresuch as a thiadiazoleor a pFimidine or in an acyclic structuresuch as a urea or thiourea-In the caseof uleas, the N-2 shouldcarry as a substituent chainof at leasttwo carbonatoms,[33] a
H N\

SO'NHC

x\r
2.!l:!

||

-B '

ides (2.34) and Sulfonamidediureticsarc of two generalstructuraltyps:hydrochlorthi^z the high ceiling typet3al(2.35). The forrner compoundshave l,3-disulfamyl groupson the group suchasCl, CF3,or NHR. benzene ring andR2 is an electronegative

Setion 2.2 Le.d Modlllcallon: Drug D.slgn and Dev.lopm.nl

23

R'N!SO,

corH

2.34

ffi
)
Acyloxyl csup Arntirli .tuitr

acyl gNupsMY. impsvul !.(iviry

(TaxoD Figur2.2 > SARfor P&lnaxel Subsbruenl i. Cl. R2 conrainl*ulfamyl-J-carbo\, groups. the hrgh\'eilingcompound' position 2 or 3 andis normally Ph, or PhZ, whereZ may be O, S, CO, or NH andX canbe at NHR. OR.or SR.tssl A more recent exampleof a SAR is that of the natural Foduct anticancerdrug pacli_ raxel (2.36. Ta{ol), which was the first anticancercompoundfound to act by promotingthe assemblyof tubulin into microtubules.therebyblocking mitosis t36lAfter a large number many SAR (actually, structurFpdreno relationship) of modificationswere introduced,l3?l conclusionscould be made(Figure 2.2). A commonway to track the structuralchangesis to annotated w'trhrheuseof molecularadiliry ndpr, structual drawingsof a leadcompound affect activity or potencymeasuedin showwherein the moleculespecificstructuralchanges a single bioassay. o "lHo

"o'\-n ., HO

/:
OB

Chaplor 2 OrugDllcov.ry D6sl9n.and D.velopmonl

For large lead moleculesthat havebenextensivelymodifred,thesemapsconciselysumwith their activities and potencies. Effects marizea hugenumberof facts relating structures toxicity, or largechanges potency. could includethe abolishment an activity,unexpected of in Thesemapsmay depicttheresultsof a longlasting drug discoveryeffort involving nunlercus Their main virtues arethat they canprevent chemists(andthe biologistswho do the sc.eens). your coworkersfrom synthesizing analogsthat havealreadybenmadeandtested,and lhey may dirdct the chemists' qeativify to unexploredregions of the lead compound,yielding novel structual changes. to The atroveexamples Fovide strongevidence supportthe notion lhat a conelation does exist betweenstructureand activity, but doeseach structureinteraci with only one receptor andlead to or y one activity?

Molecules 2.2.O Privileged Structures and Drug-Like

Evans and coworkersfirst introduced the telm p,,lfileged rrruct!/rqr for certain molecular of scaffoldsthat appearto be capable binding to multiple reeptortargets,and,consequendy, with appropdatestructue modifications, could exhibit multiple activities.t38lThe Merck (2.37) as the exampleof this phenomenon, which was earlier $oup usedbenzodiazepines mentionedby Aridns and coworkerswithout referring to them as privileged structures.t3el are A numberof otherprivileged structures lqrown.tl
R

o.- ilo
I I

FNHcoR'

2.3? in by Thecommonalityofmolecular features a vadety ofdrugs wasapparcnt the revelation Likewise, a small number of that only 32 scaffolds describehalf of all known drugs.ta1l The number moietiesaccountfor alargemajority of thesidechainsfound in drugs.la2l average chainsper moleculeis four. If the carbonylsidechainis ignored,then737, of fte side of side chainsin dngs arefrom the top 20 most commonsidechains.On.thebaiis of what is known proposed ihat and aboutpdvilegedstructures commonscaffoldstructure,Ajay andcoworkers possibleinllelnt Foperty of somemolecules,[43] this propertycould and drr,g'Ifendrs is a determinewhich moleculesshould be selectedfor screening.They used a set of one- and in two-dimensionalparameters their computatioirand were able to predict correcdy more laal Medicinal Chemistry(CMC) database in rhan907oof the compounds the Comprehensive using Another computationalapFoachto differentiatedrug-like andnondnrg-likemolecules that a scoringscheme developdt45l wasableto classify correctly 83% of the compounds was and ?7% of the compoundsin the world in the Available chemicals Directory (ACD)ta6J Drug Index (wDD.la7l A vadety of other apFoacheshaveben takento identify drug-like rnolecules,t43l that in Therealsoaremanynonal g-Likemolecules showup asactivecompounds screens, that appearto be active but later are demonstrated befalse posith,es(inactivecompounds to

and DrugOslgn Ov.lopment Seclion22 LeadUodltlcailon:

and to initially appear interactwith a variety of receptors are Thesecompounds in the screen). or (prcrniscuous manyreceptors ifthey antagonize antaSottistu bin,Jers kno'rlnasptumiscuous they arenondruglike because promiscuousinhibitorsifrhey inhibitmanyenzymet However, showlittle relationship lhey act at a site different from that of the naturalligand or substrate, structue and activity, and havepoor selectivity for a specificrcceptor or enzyme. between later to As a result. much time is wastedfollowing up on the activity of thesecompounds, they areinactive.Shoichetandcoworkersshowedthat manyofthese compounds find out that that of aJeactually aggregates molecules,and ii is the aggregate producesthe false-positive theseaggregates dissociate,and the conditionsof the screenare changed, acriviiy.tlgl ff the tobe inactive canbeshown molecules individual

andto 2.2.E SiructureModifications IncleasePotency -i7 fndex theTheraDeutic

How do vou know whal moleculai modifications to nake in order to fine tune the lead compound?The precedingsectionmakesclear that structuremodificationsare the keys to activity and potency manipulations.After ye3rs of structure activity relationship studies. for havebeendeveloped the systematrc molecularmodification apprcaches variousstandard improvement the theldpeuti index (zlso calledthe thetupeuticntio). whi'chis a measue of of rhe safety of a drug as determinedfrom the ratio of the concentrationof a drug that givesun{tesirable effectsto that which gives desirableeffects.The therapeuticindex can b and desirabledrug effecls, but often it undesirable determined any methodthat measures by effect in an animal asthe dose-limitingtoxicity versusthe desirablepharmacological is taken index could be the ratio of the LD5o the (preferablyhumans). example, therapeutic For model (the lethal dosefor 507, of the test animals)to the therapeuticEDso (the effectivedosethat effect in 509. of the test animals);a toxic EDso(the dose produces maximum therapeutic the producestox;city in 50Eoof the test animals) may substitutefor the LD50. The lsrger that lhe therapeuticindex, the greaterthe marginof safetyof the compound.In otherwords. you would like to have to administergram quantitiesof the drug before any undesirableeffects but areobserved. administeronly milligrams of the drug to attain the desirableeffects.There index that must be httainedbefore a drug can is no specificminimum value for a theraPeutic that is being treatedand whetherother therapiesare on be apFovedl it depends the disease suchas canceror alreadyavailable.A low therapeuticindex is tolerablefor lethal diseases, or if no oihertreatment available if the side is evenaslow as I 5), especially AIDS (maybe therapeutic For lesstbreatening diseases, with the treatmentbenefit. effect is rninor compared an example,the therapeuticindex lbr indiceson the order of 10 100Inay be reasonableAs (2.38, is TheMerckIndexis a goodsource Leukeran) 23 tsol chlorambucil agent lheantitumor LDsodatafor drugsin animals. for obtaining
HOOC cl

a\ /-\
chbrambucll 2,36 cl A number of stiictural modification methodologies lbllow

26

Chopler 2

Drug Dlscovery, Deign, and D.veloPnenl

r23456?89101112 C chain Lergth

Flgure 2.3 > General etr@l of ctrbo. chain len8lh o. dtug polency

E.1 HomoloEation A honolosow senes is a group of compoundsthat differ by a constantunit, generally a CH2 group. As will becomemore apparert in Section2-2.F2, p. 53, biological properties For of homologouscompoundsshow regularitiesof increaseand decrease. many seriesof of a satuated ca$on side chain from one (methyl) to frve to nine lengthening compounds, atoms(pntyl to nonyl) producesan incrcasein pharnacologicaleffects;futher lengthening p.55.I show this (Figue 2.3).lnSection 2.2.F2.b, thai decrease potency in in results a sudden ofthe moleculeto permit penetation into phenomenon to lipophilicity codesponds increased through in until its loweredwatersolubility beconesproblernatic its transport cell membmnes anotherFoblem is micelle formation,which aqueous medla.In the caseof aliphaticamineq from Potential interactionwith the the beginsat aboutCt2. This effectivelyremoves comPound examples this potencyversuschainlength of apFopriate receptols.Oneof, if not the,ear_liest phenomenon reportedby Richardson,prlwho wasinvestigatingthe hypnotic activity of was alcohols.The maximumeffectoccunedfor l-hexanol to l-octanol: thenthepotencydeclined on chain lengtheninguntil no aciivity was obse ed for hexadecanol. resorcinolderivativesshowedthat the A studyby Dohme et dlts2l on 4-allryl-substituted pak antibacterialactivity occurred with 4-n-hexylresorcinolGee Table 2.1),_acompound Funckeet dll53l found that in now usedas a topical anesthetic a variety of throat lozenges. estersoccurredwith the t-nonyl ester the peahspasmolyticactivity of a seriesof mandelate (see Table2.1).

E.2 Chain Branching Wten a simple lipophilic rclationship is important, as descnbdabove,then chain branch_ a ing lowers the potencyof a compoundbecause branchedalkyl chain is lesslipophilic than the correspondingsfaight allvl chain as a result of larger molar volumesand shapesof is This phenomenon exemPlifiedby the lower potency of the comhanched compounds. would be the pounds having isoalkyl chains in Table 2.1. In this cas,pharmacokinetics for lower potncywith branching However,anotherexplanation overridingfactor for poteny. chainbranchingmay interferewith receptorbindlng.For exancould be pharmacodynamics; (PhCH2CH2NH2) an excellentsubstrate monoamineoxidase,but for is ple, phenethylamine

Development secrlon2,2 Led Modltlc.llon: DrugDg3lgn.nd TABLE 2,1 > Eflecl of chaln Longth on Pot6ncy. Antibacterial activity of 4-tFalkylresorcinob and spasmolytic gclivity ol mandelat steB

27

liYo' dY
OH

$ir.o,*
% SpasDolltic activiq/

-ol t

methyl ehtyl r-prcpyt

0.3 o.7 ; 22 33 5l 30 0 0 0 0 15.2 23.8 2:7 9.8 28 35 5l 130 190 3',7 22 0.9 8.3 28

,-heptyt

-propyl ' t-htyl t-hexyl

sel to " Relative 3,3.5-trimethylcyclohexdol. at 100%.

(amphetamine) a poor substate.Primary aminesoften arc more is s-methylphenethylamine potent than secondaryamines,which are mor potent ihan tertiary amines.For example, (2.39,Primaquine)is muchmore potentthan its the antimalarialdrug primaquinephosphate secondary tedary aminehomologs. or
cHro

Major phamacological changes occw wilh chain branchingor homologation.Concan the lGaminoaftyphenothiazines (2.40, X : H). w]en R is CH2CH(CHr)N(CH:)z 'ider (promethazineHCI; Phenergan), antispasmodic and antihistaminic activities prdominate. However the straigtt-chain analog2.40 willl R being CH2CH2CHTN(CH3),(promazine) hasgeatly reducedantispasmodic antihistaminicactivities,but sedative and and tranquilizaregreadyeDhanced. the caseof the bnnched chainanalog2.40 with R equal In ing activities

and Dvebpment Chaptr2 Drug oi6coverv'Dellgn'

ff:t#$$lx::'j,,$ilil'l*ffi $ff[{l*Hi:l*n*
ll

#ifiTii*:fl ,:,ffi :31?$$.[#i1,

E.3 Ring_ChainTranslormations

..* ncarron moJ Anorher rhar b:"*^d' '::1,j;TxT::1il:H",1;:#.'::fi" :ljT 240 ' cr Ti'::*::"'i.*1"?ii{'ii::ll.''j:il'.*;;-, il";;;;;,and X "l;li: thebranchedmerhvrgroup
;::1:i;.;,;;.;varenta'('anquiri/er''|nanimarresr'
activity in man

ll'lililil $il",il:'1"",1"';-l;"1"11?'il ::*:tt.*Hr*:nr*xl1l,i'Jli:;:

G"D
I

r-Y\(.^'.,-

ll
2,41

ll
242

"#lnlr:::lrfr l,*#:#i:*n#iltthii]6:+1f * :ffi'll ;'*."tr;,il::"1il##,"'*t;35;;fr

:i5j"x#:ilil:ffi ;;::J:Tffi
'"'S,$.",1* "-'t'::I **un'.^" 1."1.,:*::l::1"'i::Tffi H.:iilill;:iil: n: ;ll nuu"u p,."n''
un' n.' ilTfT:j:I : J:llllr''"lillTi.;;;.,u, .,n.,'.'

S.tr

22 lad ilodlidrbn:

Irrug Dalgn.nd atevelopm.nt

29

TABIE 2.2 >

: L;:ldr d(m

Classlcal lso3telca
dd groups

: *-ddtuc

andS6ups

Tilzlar

:hs

md sroups

I I

1 I

(..3., bonrcDe,diidpdenc) (0.9., bellae, plddine)

In rd rorniring) activity is greadyenhanced. ihis case,howevel an additionalmethylamino !re@ r! also added,which may havean effect in changingthe activify

EJ

Bkisoeterish

or aresubstituents groupsthat havechemicalor Physicalsimiladties,andwhich .lirpr..'pr Bioisosterismis an importantleadmodjtrtioadly sirnilar biological pfopIties.tsal rrece to\icity or to rDodifythe activity a+proach ha\ beenshownio be usefulto artenuale lhal @ ,f r lcad. dd may havea significantrole in the altemtionof pharmakineticsofa lead There isosteres.tsTl 1925Grirnrntsslfdmulated the In and G dlssi'd isosteresl55,s6l nonclassical number law to describesimilaritiesbetweengloupsthathavethe_s^ame displacenent @ later rede_ ."aaoe itectrons, lut may have a different numberof atoms.ErlenmeyerlseJ * in Lld ioneres asatoms,ions, or molecules which the peripherallayersof electronscanbe arc .ffir--d Theserwo definirioDsdescribe.lass/cal irorl?rcrl e).amples ro be idendcal. do bioisosteres not havethe samenumberof atomsanddo 2.2. Nonclassical turiElabl but dr& $e *eric andelectronicnles of theclassicalisosteres, do producesimilar biological also can be in Table 2.3. Ring-chaintratrstonnations are L\amples of Lhese shown EiE tlnt ffirt-Fl Thereare hunabeds examplsof compounds of ro be isostericintercharges. are sho*n in Table2.4. some"',mFles trfu ts ! bioisostericinterchangeJml

Chaptr2 Drug Dlscovery D*19n, and ltevelopment

lsosteres TABLE 2.3 > Nonclassical

fil ))

N cY 'cN

L<t-l
I

.c.
-cH

.{.
lr*o"
"/'\

n- o '.{

E .ll-i_

-cN

f"

Nocl{r

I t9 |
ooooo !-.l_s

I l'o' I

II
Pe

! or

l-os

!
9q

'!"

i",
9

b-on

- c-NH

l"

-t^lJ*

/il-l-*

-i-;-E--

A"t" -t*'*"" --\\

]P H .F

d*o" N-

y\
NJ

{}o"
F

f;__-]

lrl I /'-NH, I

9i
ttlt,cr ,-c.ttg,

./L'cH:-

8?"?

/"t H:c-i\

u*-c."n,

I
r*J'o-

,-c-ol

93
nt-S-o.

i-!i-cHr

*,

9^",

lFr

;i

Ld Moditicatlon:DrugDesignand D.velopment

31

EE

23 >

Coiti {e,l

o
-/i\NRR'
7'c'N-R

r-d
IN

.^d
F'

IN "-d

,/-d

Ia\>-,

R'

.-'t

\
DR

N-s\

,-\
OR

ll ,Ji

tr)

N'Nr

.2--n

,N

Ztlo*

-\'IlcN

:I

jo qo ""n ,,"-Y)
x =O.N R

cr,

cr

N(CN),

C(CN)r

,!:

,,o'.

x
*t""

CN ,N-

*1"n,
CN

N 'II'-N H ,

*-SozNHu

,[.r.

32 IABLE 2,3 > Co tiaued

Chaptr2 Drug Dbcovery D.5lgn, rnd tt EloPmnl

l("rl Y
|Fl l,
v *,

.'fl
R

NOr

Y
'NR:

I l l .t t"I "r"t *"o

aS

r l^ .. ,
\N'/

rt.',) T-tr r ./ |
L-i/ \N

T-o.. r-o .,
LZ UN

n-or r(r.
LNI

LN2

|r3l

*--;/-o^^ C-o-'" f1__l li

R

-'

*- ,i"'

"to \2

o ' " ' ""

"'1Ao *A*'A *,

F6;
EF

-{-)-

in Bioisosterism,however,alsd can lead to changes activity or potency For example'if or phenothiazine neurolepticdrugs (2.40) is replacedbv {H=CHthe sulfur atom of the drugs (2'43) result A change antidepressant then dibenzazepine {H2CH2- bioisosteres, in enzyme affinity also can be observed.For examPle,when the thi^zolone ring in- a isozymeover the selectivefor the cyctooxygenase-2 series;f anti-inflammatorycompounds by,an 5 5. lseeChapler Section 5 8 2 b p 280rqas (ubslituled I isoryme cycloorygenasefor by O r.theseleclivity lhelwo isoz)me' te!er.ed '" ' as ri orazolone ng(i.e..theS ra replaced

R 2,43

It is actually quite surprising that bioisosierismshould b such a successfulapproach to lead modification. Perusal of Table 2.2, and esPeciallyof Table 23' makes it clear will that in making a bioisosteric rcplacement,ore or morc of the following Parameters

LadModfil@rbn: OrugOellgn.nd It votopmsnt

33

Il

TABLE 2.4 >

|.

Exampt$

ol Blobogteric

Anatogs

Neumleplis (dtipsychodc)

o
X= ,/C\ d CHCIi

2.

Alli.inflrnD

ory rgeDas

x
o
= NHOH ,

--{

N_N

fi

)\,

Y =C H r O Y =F

Z-Ct z =S C H rG u l i n d a c )

X = NH,O,CH,

-< ) c=D
H \H

lhtrge: size, shape,electronic distribution, lipid solubiltty. water solubility, p1<., chemical rwtivity, and hydrogenbonding.Because drug must get to the site of action. then interacr a rtrh ii (seChapter3), bioisosteic modificationsmadeto a moleculemav haveone or more dt' rhe following effects: | . .Sru ral. Il rhemoietytharis reptaced a bioiso\tere a snuctural in holdine cr by has role otherlundionalirjes a panicular in geomerry. size.shape. hydrogen then and bondin; will be important.

34

chapier2 orugDlsco{oryitellgn' tnd oovelopmenr

with a 2. Receptorintemctions lf the moiety replacedis involvedin a specifrcinteraction solubility will be exceptlipid andwater tft"" all of theparameters ."""p- o. "*V.", important. tranrpon and for is 3, PharnatoLinerirsH the moiei) rePtaced necessar) ab'orption pra' and hydrogen excretion of the compound, &en liPophilicity, hyftophiticitv' bonding will be important. metabo]ism'then 4. Metabolism.ll the moiefyreplacedis involvedin blocking or aiding &e chemicalreactivity will be imPortant This approachallows of It is because these subdechangesthat bioisosterismis effective with oirly someof the panmeten in order to algment tbe the medicinal chemist to tinker ma) lorcir) Mul!iplealteralrons be and of rlectivrty. andduralion action Io reduce Dorenct, in!olved ol for effecls. examplerl modificalion a lunclionaliry 1."...i* . --i.rU"r-ce inLinairuat,oaect.u'e.rhelipophjlicrrvofInemoleculelherebyreducingitsabilit)ropenat a different site the etrateceli walls and crossother membmnes, moleculecanbe modified replacement' bioieo'teric can But absorprion where these wi$ a lipophilicgotrp lo increase rho'egrouPs (seclion2 2 A p 17' couldhavidenrified studv * maaeli ptrarinacopnore rthescrssrons InaI bindrng wilhoutan effecton receptor on lhe lead;ar couldbe modified $at caDbe qafel)modinedMod-ifiarelhe posjtron' in led to lillle change porency)Those and sbape result dDolher the of carions rhis.;n. h;wever.may change o\erall molecular 'n

uyr4.

E.5 CombinaiorialChemiltry a. GenralAsPecla of'n?"ical /i'wi?srafamjly" or tnvohe'theslnthesis biosvnlhesis Conbinatunatchenistry o' <tructurer molecules,tol of havinga cenajnbasechemical of comoounds It.plTott "'' or leadmodihcalion llprcarD rnese for panicularty leaddi\covery biologiiatscreerung. uay b) {ovalentassemoiy and in librariesare prepared a sysremaric repetilive chemical that build up pafis of the overa structure) bloc*s oi tuiaine"Au".r" (v^tiou; reactantmolecules the urruy of moleculeswith a common rcaf''d (the Parentstructurein u a ni* is that it is canied out on a solid of me advantage this metbodology *rno*"aO i"tiifr can of of "i ,rtari'olati;n andpurincarion lhe producr eachreaction t,ioo*. i"ir"i".ri.i ($ilh a latiety o[ solvent')of the pol]menc dt .i.pr. nlrauon andwa'hing ri" ol "LJ.*.4uhich the buildingblockshavebeenanached of Becau'e lhe insolubrl'ly the to suooon whichailow\ theuseofexcerr Lo uttac-hed thepolymeri' removed *iiln.r. ."."tft;ne ".t or reactionsThe d;sadvanrases rhis methodolo$ are !h d;". i'h. synrheLic ;;;i;; of op tl" reactionsandthe sluggishness reactions'An altemativestrategy ai6"uftv in reagenr' ""nfing \ in it ro car4 out rhereacrions solution ith ercess scauenei,ie,ndlo8)'r rcovateh comPlereL afterlhereaclionis scavenger i" J." *"i*g"a *ith a polymenc*upponed lo atlached tnescavenger "ft"i reagent es filttatjonremo! lhe excess ln thisapproach. {Fisure2.4). to usepolymer-supported .11""-"t f"""." ,ft"'"i"auct in solutiont63lAnother appmachis polvmerreactions *lr, sitrrion ,.a.rion' To avoidproblem ol hererogeneous I*i."ii

..iiti. oort.,r'vr.". erv.ol -are approacbe' lhe solid rlrpair.t.i... u.ong the \atious combinatonai A;;" rrmmob i'ed or.rn the oon used.lhe method'for a'sembling buildingblocks the state entitier)in shrcn $e (a fraclionof lhe lotal Ubmryor individual .otunonr,nd numbers

{6al polvmen beused can

r
+l:LT 2 2 Lead Modillcallon: Drug D*lgn and ltevelopmoni

35

oo t//
-b6*.HCt

G'-.:.

-Jl
Figure 2,4 > Use of polymq-bomd retgents 10scavengeexce$ taciants in a re&lio.

o[ are an.l I'b de. are screened. rhe mannerin which rfie struclures acnle compounds determined. in Tlre nurnberof possibledifferent compounds a library (N) is detemined by the numbr usedin each step(r) and the numberof synthetic steps(r). If an equal of building blocks nunber of building blocks is used in each synthetic step, then Equation 2.1 holds. ff the (e.9.. c, andd for a three-step ,, synthesis), number buildingblocksin eachstepis varied of drenEquation2.2 is relevant.

(2.rt
(2.2' that -{ combinatoriallihary of all of the pentapeptides comprisethe 20 commonly encoded = 20r or 3.2 million different peptides.Combinationalchemistry amino aidswould be /r' originally was used to make peptide libranes, but now is most cornmonly employed for of rhe synthesisof large arraysof diversenonpeptidicsmall molecules.lt is only because combinatorialchemistrywas ableto that of rbe discoveryanddevelopment HTS techniques generated a library in a in rhrive.Unlesstherewerea methodto test3 milljon newcompounds periodof time, thercwouldbe no advantage beingableto makethatmanycompounds. to short possibledruglike moleculeswith concludethat thereare up to 10130 Theo;edcalestirnates the molecularweightsbelow 800, but this amountexceeds massof the universe.The much even if would stitt tate over 1o5l yearsto synthesize, moleculesl6sl lower estimateof 1060 Therefore,even combinatorialmethodsare a million compoundsa day were preparedll66l rclative to the total number of compoundspossible, but the belief is that inconsequential quicker than by conventionalsyntheiic ir can approachthe theoreticalvalue of compounds that Howevet thediversityof moleules canbe attainedby combinatorialchemistry methods.

and De6lgn. Developnnl chapler? DrugDlscovory.

chemi\lr' The Innj apProache' beg ng' of combinatorial synlhetic conventional eoual ma\ noL '' in peptide nthe'i\b) cevsenandcosrorKers areat(ibuiedlo ft'rkal6/l with applrcalioDs ') <ynmesrs Dy were librarvsvnrhesis Iollowed in inidaleffods Peplide These andHoughlen.loel 701 bv nonpe ide libraries motecute ana u"' andcoworker' Ji o.l,.ia. u, z**.r 'mit andcoqorkenI-2 andTenett il rl'i' *a .i*-1""'' Libraris Peptide b. SplltSynih63ls: pool' ot t}re known asa ryiit ryntlerir (alsoc alledmix and split' split and The initiat approach, general discovery,gf{:"th f:l lead cornmon is merhod'. rhemosr at ,a". *rJi, *-.0n" ed rhat assa) ar librarymixtures Ineresurr ' makinslareelibrarie'tl0al0bcompounds, aJe aini each conr ns onelibrarymemberi e ' bead I. *rlecdon of polvmer ilfiti.'" ii-" ' building ol"every combinalion "il rhe librarvconiain'e!ery passible ilra, i,". ..tr*d." ""i block'Theseriou'linuution\arelha|ltisaPplicableonIytolhes}nlhe'Isollequenceao|e sr wh onb aboutIo0 500pmolof Producr' ich makes ruclure u.^a .ii"or." "arrie, mir\ \peclronehc memos "u.f, -a difficulror impo.siblc For rimple compounds derlerminarion or ?al $ousandc mrrrrons lhe be used. bul lhjc i' not aPplicable.if tibraryconlain'man) may libnry membels'In that case' of membersthat may not be pure or are isomeric with other lo (see melhods nerl seclionrneed be utiliTed encodins " -i"f.".;f, Lo.a'rnall wouldbe apPtied .-".tt. ot hou the split ')'n$esis approach acids This method can be -ri*"ty .i possibletripeptidesof thee amino Cl--".t"i mixture of a of thc tripeptides of His' tihary. A homogeneous to *v .i"" "fl uootut"a 2 in scheme 2 a. "*t on u"i"ir"i s* ij' I zl ."uld be svnrherizid a Merrifieldresin "hoqn buildsro the N lernunusThe and fl."-,f,", ftl..in.fa v"the'is siartsat rheC rerminus of " stepis very imponani to ensurethat eachtube containsthe samemixtue hornogenization comPounos. resin-bound lo pepride binding a panrcularrcceptof fo' aclive the Wlat itvou wantlo delernune mosL of lrbrary morelhan)2 nxllronL'rl a and Houehten cowor*ers prepaled combinaLorial anlago st of lhe /'/ opioid-recepto iienLitl rtrebesrherapeplide ""J"1-lr.*".""4.t1"twnich morphine endorphin'bindr' The prme's \houn rn scneme and ro ror f*e recepror me$)lbenrhydrylamrne conraining tubes wilh 20 'eparale 2.2 wa. cani;d our.bul 'lafiing pePlide amide' $!en peflide' aJe re-.inrTlu' resinproduces , rtlsHAi ;f n'n t"n. amrno ", 'rre |he conlaining 2u slandald libnry of penlapeplidet fiom'ir.i A combinalorial cleaved tuDes r'o rnto lu then'eparaled on acidsqa" conrlructed lhe MBttA re\rn homogeniTed rn acrd so thaL eacn $as addeda differentN-acetylamino eachof the 20 differenltube\ havresin-linkedN-acetylhexapeptides tibrary of aI Possible fi" tfr"." *^ startjng with " "o-tinutorial tube;ontained alt of the N-acetylhexapeptides each ir"-tfr" .r-" N ,".ti.*, 2 acidIFrgure 5Ar' Forerample lhe fir't lubemayconlainall i i"*r"a,""no (lnr' resrn al "'irf."* ftat haveN-acetylalanine the N lermrnus N resin-tinted -aceqlhexaPeptide' fromlheC termjnu'backto lhe rhe tf,. V."ifi;ld s)'ilhesisburld< peptide .""irt .it. lhat ^\'i'rt *-"a tubeco;ld conLain rerin-linked N-acetvlhexapeptides eran all tr'. iili'-;"".r. ltot 6gresin can lhe N-acetylpeptides becleave6 and sith N-aceivlcvsleine, so forth.All of and amide' An aliquorfrom eachofthe 20 tubeci' removed r-"i.(rm*p.orde i. "i". fft" mosrporenr which aminoacid is be't ar lhc N Frminustin indicales in-$e ne\t e'(cept ".'?"J. N-acetylArg "tiquol bebe't' Thenlhieproce'crcrepealed wasfoundIo Lhi,ca.e is tetrapeptides made'is split into 20 tubes' rihary of MBHA-bound i position'.theneach " ""-ti"""la"r "*i. aim"rent umino ucla it coupiedin eachtube at the next-to-N-terminal a that was shownin the previousassay tube is N-terminal cappdwith N-acetylArg' because

secrion 2.2 L@d Modlncallon:D.ug D8lgnand DevloPmenr

37

lne-Kdl " I :n!!!l!!l i

., d

and split) inro 3 equal

ttl

ul
l

orbinq

hoiogqte,

and didde d

l
Itr

ill

ut ut
I

"t
lihra4 ol
edPepbda gdP.Plids

9 tiPPttds

thcn cl@v .mbine and honogtuq lbnrv oL d b giw a snbhdonal

of librarvof aUPosibletripeplides hntidine of synthesis s combinatorial Schgmo2.2 > Solid-ph4e

from the resin' is to be best (Figure 2.5B). Again, an aliquot from eachnrbe.after cleavage and assayed, the best amino acid at the penultimateposition is determined.This processis and lherapeptide. thebesr only each containing oneN -acer) are unlilrhere 20rube\. repeared fol lhe by oi$e 20 is derermined ar'ay.Follo$inglhisptrcedure. mo't polenlanragonisr t}|e opioid receptorfound up to thattime wasidentified(Ac-Arg-Phe-Met-Try-Met-Thr-NH2). ,. to The entireprocesstook about2 monthsl Can you imaginehow long this would ta,ke carry out onecompoundat a time'? the This methodologysoundsfoolproof, but it is not. Sometimes, most potent aliquot is less potent than what was observedin the assaysfrom the prcvious iteration. How is that possible,because previousiteration had to havecontainedall of the peptidesin the next the multiple compounds with assaying This is a cornmonphenomenon ireration(andthensome)? interactions ln the particularly peptides. Oneexplanationis PePtide-peptide simultaneously, iteration there are fewer peptidesin the tube ftan in the previousassay:the necessary next Possiblythe activecomponentis peptide-peptideinteractionmay be lost in the later assay. or more intemcting peptidesand one is removedin the next itelation. Or maybe really two

38
Esin H '-AC-AITXXXXX-N-MBHA 2. lr'-Ac-CyrXX)C(X-N-MBHA H 3. ai-Ao-Ai8.XX)O(X-N-MBHA H Esin Esin

Chapler 2 Drug Ol.coEry, D.slgn,.nd Dovglopmenl

Eachtlbe onlains a mbi.atonat lbEry of 3.2x ld diffeFnl MBHA resin-bound lv-aelylhexapeprid* rll havingrhesme miro acidar theN teminus,

20.,V-A.-ThrX)C(XX-N-MBHA H

Esin

(B) l, /V-AC-Aig-AIa-XXXX-N_MBHA csin g 2. X Ac ArfCysIntGN-MBHA $in 3. ,-Ac-ArrArs XXXX-N-MBHA esin

IH
I

I
20.tr'-Ac-Ars-I1tr-XXXX-N-MBHA Esin H Flgu6 2.s > Conbinatorialsynlhesis .ll of the lr' acetylhexapeptides ihe 20 comonly encoded of of

Eacblube contaiN a conbindorisl librEJyol MBHA ein-bound t-celyhexipeprides all with A. Ary al th N-leminus, oneol the 20 ahino sids al tnepe.utinai. N-leminus. dd a mdon mixtuE of all po$ible adino

thereis a confornational differencein the activepeptideasthe numberofpeptidesdiminishes. generally not makeusefuldrugs, discussed Section Peptides do as p.47. in 2.2.E.7, c. EncodlngComblnatorlalLlbrarles Before tuming our attention to the more important nonpeptidelibnries, let's consider an altemativeapproach the identificationof the most potentanalogin a combinatoriallibrary to oiher than repeateditentions until one compoundremains.A more rapid approachwould be to test the entire library at once and identify the activecomponentof the library directly. As mentionedabove,with large libraries of complex moleculesii is not readily possibleto determine structureof theactivecomponent. thatcase, the In encoding methods needed.lT3l are This is similar to the way in whih Foteins areoften sequenced biology; the protein is not in but the sequenced, the genethat encodes protein is.l79lAlthough .thestructureof tlrc actual compoundmay not be direcdy elucidated,certain tag moleculesthat encodethe structure may be determined.lsol importantapproach involvesthe attachment uniquearmys One thai of of readily analyzable, chemically inen, small moleculetags to eachbadin a split synthesis was reportedby Still and coworkers.l8'lIdeal encodingtags must surviveorganic sy hesis assays, readily decoded be conditions,not inlerfere with screening without ambiguity.encode large numbersof compounds, ard the test compoundand the encodingtag must be able to be packedinto a very small volume. In the Still method, groups of tags arc attachedto a bead at eachcombinatorialstepin a split synthesis. The tags createa record of the buildinB in that stp. At the end of the synthesis,the tags arc rcmoved and analyzed, blocks used which decodes structureof the compoundattachedto that bead.As dpictedin Scheme the 2.3, one or more readily cleavabletag molecules(Tagsx) are attachedto about l% of the polymer bead sites (about I pnoybead), and theseencodebuilding block I (BB1). Then

I
I

sdion 2.2 !..d lllodllicattonl Drug ltetign tnd D'rclopment

39

(Berd l-BBr-BB2 -BB3

',1" i,o"'**
|
6A* -,/ Sctrem. 2.3 > * *e,: BBi

r,fi" i

'*x

f.s,x , rassl + rr*"'' .-

"**"*^" secbo@c'pb,

I i'd N r'\ md 'o@lek b di' bLildnsblocltt

Still nethodolo$r fd encoding conbimtdial

PePti'le libidies on a Polvmq b3d

buiidingblock2 (BB2) lo lags one or moreothercleavable (TagsYraranached encode aod so foflh Althougha differenttag could Deused BBl. followedbv Tassz to encode ol du-\tures rv of k, for eachdildin; bloc it is moreefficientuousemixtures tagsbecause with just l0 iags 1024(2'") synlheses synlhese\l 2d represent different tagsca-n different canbe Derformed. -scaie Ttvo on ftre'tue, ne"a ro U. cherniially inen andretiably analyzed lhe femtomolar and thiny 2'44 tagsare pbolocleavable' th-lo(y tags exarnoles-ot are 2.44and2145The tags nitraE)cleavedThe released areanalyzed 2.45iaesareoiidativetyrcericammonium all deteclionl ol thetagshavedlEeF caDture usingelecnon gas by capJtary cbomarography HPLCIs2land are Ortrersensitivedeiectionmerhods fluorescence-based unL.etenLioritimes. lE3l cC rnassspectronetrY

O3lgn, Dddopnnr Chaflor2 DrugDllcovry. rnd

how will youknow which of thelargenumberof Ifyou areusingthesplit synftesismethod, polymerbeads anactivecompound? approachtsal to chemicallyattachacomnercial is has One The assay run with th library still attached eachpolymer bead. is to dyeto the ta4et receptor. If a compoundbinds to ihe dyelabeled receptor,then the bead to which the compoundis attachedwill take on the color of the dye, and the colored beadscan be removedmanually of The intensityof the color in the beadis an indication of the tightness binding anddecoded. of &e compoundto the receptor which may makethis process clearer(or maybenoo. A com Lefs go throughan example, of binatoriallibary of all of thehexapeptides seine, leucine,lysine,isoleucine,andglutamate (56- or 15,625-nenber libffy) is Fepaftd with the appropdatetag moleculesat eachitration. To do this you ned6 x 3 (3-bit binary code) or 18 different tag molecules.Tags I 3 building block, tags?-9 are only usedto definebuilding block 1, tagsH arefor the second for the d rd, tags 10 12 for the fourth, tags 13-15 for the fifth, and tags 16-18 for the sixth building block. Arbitrarily assigna 3-bit binary code to eachamino acid building block, for = ;t example,001 Ser,010: Lu,011 = Lys, 100= Ile, and 110: Glu. Ifa tag is used, peptidesyntheses are represents binary bit 1; if no tag is used.it meansbinary bit 0. Because and then coupling typically done on a resin to which the C-teminal amino acid is attached, occursback to the N terminus,tags 16-18 encodethe N-terminal amino acid, and tags 1 3 encodethe C-teminal amino acid. LeCs say an active bead is identified. and you want to structureis. The tagsarerernoved from the bead(by photolysisor krow what the hexapeptide linker was used).the carboxylic acid producedby cleavage is oxidation.depending which on morevolatile, andtheelectroncaptureGC is run. In lhis example, trimethylsilylatedto makeit These tagscanb decoded to let'ssaythattags1,2, 5, 6, 8, 9, 12. 14,and16 weredetected. as shownin Figure 2.6. identify the hexapeptide the Another encodingprocesssegregates test compoundfiom the coding tag molecule by attachingthe test compoundto the extedor of the bead and the coding tag molecule to This Fevents the tag rnoleculesfrom interfering with binding of the intenor of the bead.l85l the test compoundto the target receptor.A polymer bead systemwas developdin which to only the surface the beadis exposd an organicsolventthat containsthe organic-soluble of derivatizing reagentithis allows a nonpeptidictest compoundto be constructedwhile the interior of the beadremainsin water without denvatizingreagent.The codin8 moleculeis a

'fas l, Trg 2. 1t 0

Tag 8.Tag9, 0 tt ott

Tag12, Tag1,1 Tagt6 001

noT,gl0

010

100

I
noTrgT

I I
nolagll

lt
n o 'r 1 g 7

100- 010 001 0ll

0ll

l l 0 = Ile-llu-Ser

L)'s Lys CIU

(lromlhl 3-blrbln.ry cod$ iniiiatty a$tgned whenmakhglhe library) Flgu.e 2.6 > the ofa bouid to a polymer An example encoding structure pepride of bead the Slill by

i4.

2 2 L6.d ilodlllcrtlon: Drugtt dgn .nd Dcveloprn.nl

41

mediun of the intedor of lhe bead.Once in --Er:ie. whrchcan be synthesized the aqueous the r r' e beadis identifiedby a colorimetricassay,l86l tagpeptidemoleculein the interior :e or which encodes is sequenced Edmandegradation by massspectrometry,[37] by :: nr bead to the testcompoundattached dle exterior of the bead, :E iruc$re of to orher intercstingaltematives moleculartag encodingare encodingby radiGftquency Encodingmethods a polymedc matrix havingunusualshapes.t8el anrlencodingwith --,-:li:l --t sorneday be displacedby massspectralanalyses,such as imaging time-of-flight sec(TOF-SIMS).[9o] in Mary huidleds of beads be assayed a can :.iir! ion massspectrometry 10 beads/sec, althoughthereis a problem with ftagat :rngle measurement the rate of about leading to a complicatedanalysis.Mass accuracyis about +0.01 of compounds, -nrarion the 3.2 x 106-member ftom 20 amino acids,all peptides, library of pentapptides :-u. so in (samemolecular weight), can be sepamted. i\.epl rhosecontainingleucine and isoleucine B] incorpomtionof a ')N label into either leucineor isoleucine,eventhosepeptidescanbe

d. NonpptideLlbr.rlca do later in Section2.2.8.7,p. 47, peptides not makevery usefuldmgs,espcially .t sdiscussed describedabovefor the s],nthesis of if an orally activdrug is sought.TI| sametechniques pepridelibrariescould be utilized to preparenonpeptide libaries; however, thereis ar impornamely,reactivity. unr differencebetweenthe chemistrywith pepiidesvqsus nonpeptides, In a rypical peptide coupling reaction the carMiimide-activated N-protectedamino acids are all about the samein reactivity with the different amino acids in the growing peptide methodworks well. However,with nonaminoacid of chajn.Because that, the split synthesis reagents, such as different acid eblorides,the structureof th acid chlo.ide wil affect the That could lead to mixores in which someof rarsof reactionwith differcnt nucleophiles. havereactedandothershavenot. For eachreactionthe conditionshaveto be rhecomponents sorked out to be surecompletereactionhasoccurred, that analogsare Over th yealsit hasbeenrecognized when largenumbersof nonpeptide manyfaLsenegatiws (z\ activecompoundthat doesnot prcduce screened simuttaneoudy, level of activity in the assay)and/ake a l7't, i.e.. a compoundthat showsa predetermined portiv?s (an inactivecompound givesa hit) areobserved. falsepositivemay arisefrom that A morc thanonecompound. an impurity in th sampletestedor asa rcsult of a complexbetvreen Falsepositivesarc a wasteof time, but false negativesmeanthat potentialdrugs (or at least conPaniesto cany are lead compounds) being overlooked.It is typical for pharmaceutical our single entity screensto avoid theseploblems. Becauseof dis, individual compounds, synthesison a solid supportalows the Nonetheless, rarherthan mixtures, are synthesized. are npidly androbotically.Thereactions sFftesis of largenumt'rsof individual compounds miqotubes containingthepolymericsupport.This method caniedout individually in separate bcause library of the is referredto aspdrall"J s)rtherir ratherthan combinatorialsynahesis in compounds(in the range of 5G-ld compoundsin amouns of 1-5omg) is synthesized parallel without combining any of lhe tubs.One strategythat can b usedfor potentially more effective libraries is to selectprivileged structuresas lhe scatrold.Another strategyis io designa scaffoldbasedon an importantmolecularrcognitionmotif in the taryetreceptor. Th libraries shouldincorporatedifferent setsof (commerciallyavailable)building blocks to proyide a large numberof diversesuuctures,and they shouldcontain as much functionality Molecular diversity, however,is difficult to detetuine; as possibleas recognitionelements. with Dixon andVillar havefoundthai aproteir canbind a setof structurallydivene molecules

Dign'and Dwlopme chapler2 OrugDl5cov6ry

ScheBe 2.4 >

Sotid-phasesynthesis of a nonpeptide librdy of privil'ged sitrctwes

can compoundr exhibit to clo\el) relaled lhese bul ,rmilarDoenlbindingaFfinttier' analog\ all Parallel $an canDe compounoc man) more cangenerale very \ ;at binding. 'ynlhesis is dnd rradilionally. theco't percompound muchlo$erlo" 'rnlhesized ";;;;;;;r;;;t.pridelibra4otapri;ilesedstrucruIeIbenrodrazepinesri''hosnin drrecll'to l2'41ir notalrached ofthe ben/odraleprne Nolefiat rhefrIslprece scheme 2.4.iqrl to give 2'48 in a to 2'49)' but is attached 2'46 instead tfr" f"tt".t",fttbolystirene. direclly ppr'.ia;etl1 tate ""i"*". ta-zodicarboxy or DEAD) lf 2'47$ereartached vi l'"'"i,.o"or;n" t ma) re\ult' reacrron ro b) hindlance fie potvmer rhefiIstchemicdr i;;;;;;l;;;;*. ro or In aryt'rannane lhepterrnce palladrum to acrdchtoride rtre inliii'. .",,pir"g wnlcn "l problema "n polymer to groupis typ'call' arLdched the '=., 2.50.To a'oid lhar ene 'pacer rs -rnores 6rsrreactant awayfrom lhe pollmer 'o lhal slerichindrance nor a proDrem the ttt" spu"e', *trictt *;tt r'' removedat the end of the svnthesis ln * b"-p"".a ijf ""-"" R' R) andRr'TheSrrlle be divert^ elPn?n8can varied: sr Lhi'solid-oha,e nrle.i". Ulrce I ro a' man) acidchrorides areavairabre var) Rr I Each .;r wiLhas :;;d';; J;;;. ;;"; aminoacidfluondein rhe'ameFmoc-prorecred .an be coupledio amino "iii.'I-"o".,iijJo, Fmoc-protected $irh different can 2.50proJucrs betreated *.rt ol Lrte ,rr. Acericacid cau'e' "."iI"por * ,r'r, , can be incorpordted R2 groups lariei) of nr"';ai', "ide wilh bd'eandlhe \amearKyr ".iJ 2 ol aurocvclrration 2.51Io 2.52 Again.each 52 canbe lrealed

n"irJi iiitii.**i

of to alkvl with z.s2canie treated a different halide sivea library r2 "rspacer polymer resingive' (helblar) of benTodiazepine'54r' and lrom 2.53icteavaqe lhe

$a' oflhoseproducls in lf 25 ditre;t acidchlodde.qere used lhe Slill couplingdndeach ol lheneach tho\ecompounos acidfluoride\' amrno with 25differenlI moc protecled rrealed 15'625 z! aitr-*t alkvl halides,therewould be a library of 25 x 25 x 25 or "r-t"LiJ*i* building blocks' using only ?5 different difierent benzodiazepines

lrs

2.2 LesdModlticatlon: tuug ltellgn and Ovolopment

Deipire the potential of combinatorial library (or paralel) synthesis,natual products le.r !o provide greater structual diversity than standardcombinatodal chemistry About !.i of ihe chemicalscaffoldsin the Dictionary of Natural Productsandthe BioactiveNatu&l (a havenot beensynthesized. compounds) a Pirducts Database total ofmore than100,000 generallyhavedrug-like activein assays F:Ih.rmore. naturalFoducts that are biologically and metabolizedle5lIsolatronof compounds :-.rFrues. i.e., are capableof being absorbed however, shouldbecomplementary' and -::! naturalsources from combinatorialapproaches, bioactivecompounds ftom nattime taLento isolateandchancterize Sif,.rally the excessive rewardmay be greatrmoleular of :-r1 prcductextraclsis a disadvantage themethod,but the opportunity to identify a variety of scaffoldsfor screen::lelsrly, which gives the greatest for computational ng. To attain a wide diversity of chemicalstructures screening Pulposes, rh:mists often reject compoundsthat arc similar in structure;believirg that similar comaJuds would havesimilar biological activities. ln general,structurally similar compounds lr;\e similar biological activiiy. However,the biological sinilarity rllay not be very srong; to considered be 8590sEuctually similar to :i hasbenshownihat only 30% of compounds have the sameactivity.s] Addingjust one methylene r activecompoundwil themselves recepiL of changed froma poorbinder thechemokine ruup ro a 4-h)drorypiperidine-analog simj do binde "'l This maybebecause lar compounds nol necessarjly r. :. r CCRI into a polenl iin{l ro the targeireceptorthe sameway. Constructionof chemical libraries basedon natual product hits is a sensiblecompromethodfor For example,Nicolaou and coworken developeda solid-phase ris approach. '-:r prepararion largenaturdl producFlike libraries based lie pri! ileged on combinarorial of producr\. Seneral .ullcrurc2.2-dimerhylbevoplran. a scaflold The "61 loundin manynatural scaffold 2.5. In this examplethe 2,2-dimethylbenzopymn rerhodology is shownin Scheme by 1i7) is generated reactionof the startingo-Fenyl phenol(2.55)with a polystyrerc-based .elenenyl bromide resin. A variety of reactionsare possibleto elaboratethe side chainsto iunher enbancethe library (2.56). The 6nal products can be releasedfrom the polymeric ruppon by oxidation/eliminationto 2.57. A library of more than 10,000analogswas readily prepared this aPproach. by

O-**

-'Y+ '
*-''z\--""'
.

synthesis a natu.alprodDct-like of combinatorial library scheme 2.5 > Solid-pbase