Natural Alkaloids and Heterocycles as G-Quadruplex Ligands and Potential Anticancer Agents

19 Pages·2017·1.75 MB·English

molecules
Review
Natural Alkaloids and Heterocycles as G-Quadruplex
Ligands and Potential Anticancer Agents
TongChe,Yu-QingWang,Zhou-LiHuang,Jia-HengTan,Zhi-ShuHuangandShuo-BinChen* ID
SchoolofPharmaceuticalSciences,SunYat-senUniversity,Guangzhou510006,China;
[email protected](T.C.);[email protected](Y.-Q.W.);
[email protected](Z.-L.H.);[email protected](J.-H.T.);[email protected](Z.-S.H.)
* Correspondence:[email protected];Tel.:[phone]
Received:30December2017;Accepted:20February2018;Published:23February2018
Abstract: G-quadruplexes are four-stranded nucleic acid secondary structures that are formed
in guanine-rich sequences. G-quadruplexes are widely distributed in functional regions of the
humangenomeandtranscriptome,suchashumantelomeres,oncogenepromoterregions,replication
initiationsites,anduntranslatedregions. ManyG-quadruplex-formingsequencesarefoundtobe
associatedwithcancer,andthus,thesenon-canonicalnucleicacidstructuresareconsideredtobe
attractivemoleculartargetsforcancertherapeuticswithnovelmechanismsofaction. Inthismini
review, we summarize recent advances made by our lab in the study of G-quadruplex-targeted
naturalalkaloidsandtheirderivativestowardthedevelopmentofpotentialanticanceragents.
Keywords: naturalalkaloids;anticanceragents;drugdesign;G-quadruplex
1. Introduction
Guanine-rich nucleic acid sequences are able to fold into four-stranded secondary structures
known as G-quadruplexes, which arise from the self-association of guanine bases by Hoogsteen
hydrogenbonding[1]. Putativequadruplexsequences(PQS)arewidespreadthroughoutthehuman
genome,especiallyattelomeresandregulatoryregionsofoncogenes[2–4]. G-quadruplexesarenow
thoughttoplayimportantrolesinseveralbiologicaleventsassociatedwithcancer, particularlyin
telomere maintenance and the expression of oncogenes, and have received much attention[5–8].
The conformations of G-quadruplexes provide recognition sites for small molecules, and thus,
thesestructuresarepromisingtargetsforthedesignofanticancerdrugs[9,10].
Although there remains a long way to go in the development of potent drugs that target
G-quadruplexes,somepromisingleadcompoundshavebeenobtained.Forexample,telomestatin,which
selectivelybindstotelomericG-quadruplexDNA,wasabletocausetelomeredysfunctionbyinhibiting
telomeraseactivityanddisturbingtheshelteringcomplex[11,12]. Pyridostatin(PDS)couldinhibitthe
transcriptionofG-quadruplex-relatedgeneswhileinteractingwithpromoterG-quadruplexes[13].Notably,
theribosomalDNA(rDNA)G-quadruplexligandQuarfloxin(CX-3543),whichwasabletodisrupt
nucleolin/rDNAG-quadruplexcomplexesandblockthebiogenesisofrRNA,hasonceenterthephase
IIclinicaltrials[14]. ThereislargeevidencethatligandswhichcanstabilizeG-quadruplexareableto
interferewiththeG-quadruplexrelatedbiologicaleventsassociatedwithcancer[15].
SomeofthesereportedG-quadruplexligandsarenaturalalkaloids[16,17],whichareanimportant
classofnaturalproductsthathavediverseandsignificantbiologicalactivities,suchasantimalarialand
anticanceractivities. Becausethesecompoundsexhibitchemicaldiversityandstructuralcomplexity
andareavailablefromnaturalsources,naturalalkaloidsandtheirderivatives,suchastelomestatin,
the quindoline derivative CX-3543, the bis-quinoline derivative PDS and the acridine derivative
BRACO-19,havebeeninvestigatedintensivelyfortheirpotentialasG-quadruplexligands[18–20].
Molecules2018,23,493;doi:10.3390/molecules23020493 www.mdpi.com/journal/molecules
Molecules2018,23,493 2of19
Inthispaper,wesummarizethepreviouslypublishedresearchfromourlabonG-quadruplex-targeting
naturalalkaloidsandheterocyclesaspotentialanticanceragents.
Molecules 2018, 23, x 2 of 19
2. G-Quaacrdidruinpe ledxerSivtarutivcetu BreRsACO-19, have been investigated intensively for their potential as G-
quadruplex ligands [18–20]. In this paper, we summarize the previously published research from our
Guanine-rich strands have high propensity to self-associate into unusual structures called
lab on G-quadruplex-targeting natural alkaloids and heterocycles as potential anticancer agents.
G-quadruplexes (Figure 1), which were first reported by Davies and co-workers in 1962 [21].
G-quad2r.u Gp-lQeuxatdorpupolleoxg Sytriuscctulereasr lydifferentfromthatofB-DNAandothertypesofDNA.Thebasic
building block of a G-quadruplex is a structure in which four guanine residues form a ring-like,
Guanine-rich strands have high propensity to self-associate into unusual structures called G-
aromatiqcu,apdlraunpalerxGes- t(eFtirgaudre s1tr),u wcthuirceh vwiaerHe ofirosgt srteepeonrthedy dbryo gDeanviebso nandds cboe-twwoerkeenrst hine N1916,2 N[271,].O G6-andN2
sites. Wquitahdrtuhpeleaxd tvopenoltogoyf iXs -crlaeyarlcyr ydsiftfaerlleongt rfaropmh yth,ant uocf leBa-DrNmAa gannde toicthreer stoypneasn ocfe DsNpeAc. tTrohse cboapsiyc (NMR)
andothbeurilpdoinwg ebrlfouclk toefc ha nGo-lqougaidersu,ptlheex sist rau csttruurcetusroe finm wanhyichG f-oquur agduraunpinlee xreessidhuaevse fobreme na rreinsog-llvikeed, [22,23].
aromatic, planar G-tetrad structure via Hoogsteen hydrogen bonds between the N1, N7, O6 and N2
G-quadruplexstructurescanfoldduetoeitherintra-orintermolecularinteractionsofG-richstrands.
sites. With the advent of X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR) and
Structuralpolymorphismarisesmostlyfromthepropertiesoftheloop,suchasvariationsinstrand
other powerful technologies, the structures of many G-quadruplexes have been resolved [22,23]. G-
stoichioqmuaedtrruyp,lsetxr astnrudctpuroelsa craitny ,foaldn dduge ltyoc eoisthideri cinttorar-s oior nintaenrmgloeleacunldar tihnteersaictteiosnos nof tGh-ericloho sptratnhdast. link to
the guaSntrinucetusrtarla pnodly[m2o4r]p.hMismo raeroisvese rm,othstely sforolumt itohne pcroonpedrittieios nosf ,thseu lcohopa, ssutchhe aps rveasreianticoenso ifn mstreatnadl ions or
ligandss,tooirchmioomleectruyl,a srtrcarnodw pdolianrgity,,m anady gallyscoosiindflicu toernscioent ahnegtleo panodlo tghey soitfesq ouna dthreu lpoolepx tehsa.t Glin-kq utoa tdhreu plexes
guanine strand [24]. Moreover, the solution conditions, such as the presence of metal ions or ligands,
can form via intramolecular folding of a single G-rich sequence or by intermolecular association
or molecular crowding, may also influence the topology of quadruplexes. G-quadruplexes can form
oftwo(dimeric)orfour(tetrameric)separatestrands. Therelativearrangementofstrandpolarity
via intramolecular folding of a single G-rich sequence or by intermolecular association of two
can also give rise to structural polymorphism. For example, the polarities of the four strands in a
(dimeric) or four (tetrameric) separate strands. The relative arrangement of strand polarity can also
G-quadgruivpel erxisec atno bsetraurcrtuanragl epdoalysmfoolrlpohwissm:.a lFlopr aerxaallmelpslet,r atnhed sp;othlarreiteieps aorfa ltlheel sftoruarn dstsraanndds oinn eaa nGt-iparallel
strand;qaudajadcruepnltexp acarna lblee larsrtarnagnedds a;so frolaloltwers:n aallt ipnagrapllealr satlrlaenldsst;r tahnredes p.aTrahlelesle stararrnadns ganemd oennet asnlteipadaratolledl ifferent
conformstartainodn;s a,dnjaacmenetl py,apraallreal lslterla,npdrso; pore allleter,rnaantidnga nptairpaallreal lslteralnqdusa. dThruespel aerxreasng[2em5,e2n6t]s. lTeahde tcoo dnifffoerremnat tionsof
conformations, namely, parallel, propeller, and antiparallel quadruplexes [25,26]. The conformations
G-quadruplexesformedfromthefoldingofdifferentG-richsequencesaredistinct. G-quartetregions,
of G-quadruplexes formed from the folding of different G-rich sequences are distinct. G-quartet
loopregions,groovedimensions,andthenegativeelectrostaticpotentialoftheanionicbackboneand
regions, loop regions, groove dimensions, and the negative electrostatic potential of the anionic
the cenbtraaclkbcohnaen annedl tohef cGen-qtruala cdhraunpnelel xofe sG-aqrueadcrruitpicleaxlese laerme cerintitcsalt helaemt nenetesd thtaot nbeeedc oton bseid coenresiddeirnedo rder to
improvient ohredebri ntod iimnpgrosevlee tchtei vbiitnydionfgd sreulegcticvaitnyd oifd dartuegs c[a1n2d,2id7a–t2e9s ][.12,27–29].

FigurFeig1u.reS t1r. uStcrtuucrteusreos fotfh theeG G--qquuaarrtteett (A(A) a)nadn Gd-Gqu-aqduraudplreuxpesl e(Bxe).s (B).
3. Existence and Distribution of G-Quadruplexes
3. ExistenceandDistributionofG-Quadruplexes
Computational predictions using simple algorithms suggest that over 370,000 sequence motifs
Co(mof puthtea titoypnea lGp≥r3eNd1–i7cGti≥3oNn1s–7Gu≥s3iNn1g–7Gsi≥3m) pinle athleg ohriutmhmans sguengogmese tthhaavte otvheer 3p7o0te,n0t0i0al setoq ufeonrmce motifs
(of theGt-yqpueadrGu≥pl3eNx s1t–r7uGct≥u3reNs 1[2–]7.G Th≥e3sNe P1–Q7SGs ≥ar3e) frienqutehnetlyh fuoumnadn wigtheinn ohmumeanh atevloemtehriec DpNoAte, nrDtiNalA,t o form
transcription start sites, and promoter regions, suggesting that G-quadruplex structures may play a
G-quadruplex structures [2]. These PQSs are frequently found within human telomeric DNA,
pivotal role in the regulation of a variety of cellular processes, including telomere maintenance,
rDNA, transcription start sites, and promoter regions, suggesting that G-quadruplex structures
may pl ay a pivotal role in the regulation of a variety of cellular processes, including telomere
maintenance,ribosomebiogenesis,genereplicationandtranscription[3]. Amethodthatcombines
G-quadruplex-dependentDNApolymerasestallingandnext-generationsequencing(G4-seq)hasbeen
Molecules2018,23,493 3of19
developed and was used to obtain a genome-wide map of G-quadruplex structures in purified,
single-stranded human DNA. G4-seq has identified more than 700,000 G-quadruplexes in the
humangenome,whichismuchhigherthanthevaluespredictedbyearlieralgorithms. Amajority
(70%) of these sequences either comprise G-tracts that are interrupted by non-guanine sequences
and are predicted to form bulges or comprise extra-long loops [4]. Another method, which
couples reverse transcriptase stalling with next-generation sequencing (rG4-seq), was developed
and was used to characterize more than 13,000 G-quadruplexes in the human transcriptome.
TheenrichmentofG-quadruplexesintranscriptsthatmediateRNAprocessingandstabilityreveals
thatRNAG-quadruplexesmayplayrolesintheregulationoftranscriptsviamiRNA-mediatedgene
regulation and alternative polyadenylation [7]. On the other hand, ~10,000 G-quadruplexes were
identifiedinhumanchromatinbyanantibody-basedG-quadruplexchromatinimmunoprecipitation
and high-throughput sequencing approach (G4 ChIP-seq); these G-quadruplexes were identified
predominantlyinregulatory,nucleosome-depletedregions[5]. G-quadruplexstructuresareenriched
inthepromotersand5(cid:48)UTRsofhighlytranscribedgenes,particularlyingenesrelatedtocancer,andin
somaticcopynumberalterations(SCNAs). ThefindingsofG-quadruplex-relatedsequencingsuggest
thatthevarietyandnumberofpotentialG-quadruplexesaregreaterthanoriginallypredicted.
StudiesinvolvingthevisualizationofG-quadruplexesbyantibodiesandsmall-moleculeprobes
have provided crucial evidence of the existence of G-quadruplexes in cells [30]. Schaffitzel et al.,
whowerethefirsttoreportthevisualizationofG-quadruplexformationinabiologicallyrelevant
context, used a G-quadruplex-selective antibody, Sty49, to show that G-quadruplexes form at
telomeresinthemacronucleioftheciliateStylonychialemnae[31]. DNAG-quadruplexeswerethen
observed at the ends of human chromosomes by using the radiolabeled ligand 3H-360A and the
fluorescentligandBMVC[32,33]. Subsequently,DNAG-quadruplexeswerevisualizedinthenuclei
ofhumancellsbyinsitufluorescencelabellingofPDSandimmunofluorescencemicroscopyusing
the G-quadruplex-specific antibody BG4 [13,34]. Notably, the number of BG4 foci increased after
theexposureoflivecellstoG-quadruplexligands,includingPDS,Phen-DC3andTMPyP4,which
demonstrates that these ligands trap the G-quadruplex structures formed in cells. Furthermore,
interactions between small molecules and DNA G-quadruplexes were visualized in live cells by
fluorescence-lifetimeimagingmicroscopyusingDAOTA-M2. Severalfluorescentprobescapableof
discriminatingDNAG-quadruplexesfromsingle-anddouble-strandednucleicacidshavealsobeen
described previously [35–39]. Compared with the detection of DNA quadruplexes, the detection
of cellular RNA quadruplexes was more challenging. RNA quadruplexes were first detected by
immunodetectionusingBG4in2014[40]. Thisfindingprovidessubstantiveevidencefortheexistence
of RNA G-quadruplexes in human cells. On the other hand, this investigation also demonstrates
that the ligand carboxyPDS, which binds to G-quadruplexes invitro, can trap endogenous RNA
G-quadruplexeswhenusedincells. Thisresultcorroboratestheresultsobtainedbyusingstabilizing
ligandsthattargetG-quadruplexesinacellularcontext. RNAG-quadruplexeswerethenobservedin
livecellsbyusingthemultiphotonturn-onfluorescentprobeNaphthoTASQin2015[41].Morerecently,
aG-quadruplex-triggeredfluorogenichybridization(GTFH)probereportedbyourresearchgroup
specificallyvisualizedtheRNAG-quadruplexstructureformedbytheG-richsequencewithinthe
5(cid:48)-UTRofNRASmRNAinthecytoplasmofhumancells[42]. Overall,antibodiesandfluorescent
probescapableofstructure-specificdetectionofG-quadruplexesarepowerfultoolsforbasicbiological
researchandfortheexplorationofG-quadruplexesasapotentialdrugtargets.
4. G-QuadruplexesasPromisingTargetsforAnticancerDrugDesign
4.1. TelomericG-Quadruplexes
HumantelomerescomprisetandemrepeatsoftheDNAmotifTTAGGGtogetherwithassociated
telomericproteinsandfeaturea50–300-nucleotideprotrusionsofsingle-strandedrepeatsfromthe
3(cid:48) end, termed the G-tail or G-overhang [43]. This G-overhang acts as a substrate for telomerase,
Molecules2018,23,493 4of19
areversetranscriptasewithanRNAcomponentthatisrequiredfortelomereextension. Thestructure
andstabilityoftelomeresarecloselyrelatedtocancer,aging,andgeneticstability. Duetothehigh
frequencyofTTAGGGrepeatsandduetothelackofcompetitionfromacomplementarystrandatthe
3(cid:48) overhang,theformationofaG-quadruplexwouldbefavored.
TheformationofaG-quadruplexcanaffecttelomeresinprofoundways,including(a)inhibiting
telomeraseactivitybyblockingthebindingoftelomerasetothesingle-strandedtelomeresubstrate
to elongate the telomeres. Telomerase is overexpressed in 80–85% of cancer cells and primary
tumors, making it an attractive target for cancer therapeutics [44]; (b) telomere uncapping by
dissociationoftelomere-bindingproteins(e.g.,POT1,TRF1andTRF2)fromtheendofthetelomere.
Telomereuncappingcanleadtotelomericdysfunction,whichischaracterizedbyend-to-endfusion,
inappropriate recombination, anaphase bridges, and G-overhang degradation, and leads to either
apoptosisorsenescence[45];and(c)interferingwithtelomerereplicationbyimpairingreplicationfork
progression. ThesefindingspromptedthedevelopmentofseveralselectiveG-quadruplex-binding
smallmoleculeswiththeaimofdevelopingnoveltelomere-basedanticancertherapies.
Recently, a number of studies showed that the 3(cid:48)-terminal single-stranded overhang could
formseveralconsecutivequadruplexunitsconnectedbyTTAlinkers, calledtelomericmultimeric
G-quadruplexes[46–49]. Thisdistinctivestructureenablesthepossibilityofdesigningsmallmolecules
thatareabletodiscriminatetelomericG-quadruplexesfromalargenumberofotherG-quadruplexes
withvariousbiologicalfunctions[50].Thus,ithasbeensuggestedthatsmallmoleculesthatspecifically
targettelomericmultimericG-quadruplexesmightbemorepromisingasanticanceragentsandmight
havefewersideeffects.
4.2. OncogenePromoterRegionG-Quadruplexes
InadditiontotelomericG-quadruplexes,severalputativeG-quadruplexsequenceshavebeen
identifiedneartranscriptionstartsitesandgenepromoters,indicatingthekeyroleofthesestructures
intheregulationofvariousgenes. Thesegenepromotersincludethepromotersofc-MYC,c-KIT,bcl-2,
K-RAS,VEGF,RB,PDGF-A,hTERT[51–58]. Moreimportantly,alteredexpressionoftheseoncogenes
arerecognizedashallmarksofcancer. Attheturnofthecentury,HanahanandWeinbergproposed
sixvitalcellularandmicroenvironmentalprocessesthatareaberrantlyregulatedduringoncogenic
transformationandmalignancy[59]. Wheneachofthesecategoriesisexamined,oneormorecritical
proteinsarefoundtohaveG-quadruplexesinthecoreorproximalpromoterregions. Transcriptional
repressionofoncogenesbystabilizationofG-quadruplexstructuresorbyinhibitingthebindingof
relatedproteinstothesestructurescouldbeanovelanticancerstrategy.
c-MYC is one of the best-studied oncogenes. Up to 80% of all solid tumors, including
gastrointestinal,ovarianandbreastcancertumors,overexpressc-MYCasdomanynon-Hodgkin’s
lymphoma tumors [60]. The nuclease-hypersensitive element (NHE III ) in the c-MYC promoter
1
regulates85–90%ofc-MYCtranscription. Theregioncontains27guanine-richbases(referredtoas
Pu27herein)thatcanfoldintoaG-quadruplexthatisconsideredatranscriptionrepressorelement.
Several G-quadruplex-binding ligands possess the ability to promote and/or stabilize the c-MYC
G-quadruplex structure, thus repressing c-MYC transcription [51]. Generally, gene transcription
is triggered and regulated by transcription factors. Transcription factors promote or repress gene
transcriptionbyrecognizingandbindingtocertainregionsinpromotersandthenrecruitingother
factors. In most cancer cells, the expression of transcription factors is usually abnormal, which
theoreticallymakestheseproteinspromisingtargetsforthedevelopmentofanticancerdrugs.
4.3. RNAG-Quadruplexes
Thereisemergingevidencethat5(cid:48)-and3(cid:48)-untranslatedregionsaswellasopenreadingframes
(ORFs) contain putative RNA G-quadruplexes. Due to the absence of a complementary strand in
RNA molecules, it has been widely held that G-rich RNA sequences are more susceptible to the
formationofG-quadruplexstructures. UnlikethehighlypolymorphicDNAG-quadruplexstructures
Molecules2018,23,493 5of19
thatdependonthesurroundingconditions,RNAG-quadruplexesalmostexclusivelyadoptasingle
conformation. The presence of a 2(cid:48)-hydroxyl group in the ribose sugar results in additional steric
constraintsontheG-quadruplextopology. These2(cid:48)-hydroxylgroupspreventthebasefromadopting
asyn-conformation,insteadstronglyfavoringtheanti-conformation(byrestrainingtheglycosidic
torsionangle)andimposingadditionalconstraintsonsugarpuckering(ribosehasapreferencefor
C3(cid:48)-endo puckering). As a consequence, the topology of RNA G-quadruplexes is limited to the
parallelconformation,inwhichallfourstrandsareorientedinthesamedirection. Thereisample
evidencethatRNAG-quadruplexesexistintheUTRsandORFsofcancer-relatedgenesthatregulate
transcription, alternative splicing and translation. In addition, the unique chemical properties of
RNAG-quadruplexessuggestthatitispossibletodevelopsmallmoleculesthatselectivelybindto
RNAG-quadruplexesratherthanDNAG-quadruplexes. Thispreferentialbindingisexemplifiedby
thesmallmoleculecarboxyPDS,whichexhibitshighmolecularspecificityforRNAG-quadruplexes
overDNAG-quadruplexes. SelectiveinterventionbytargetingonlyRNAG-quadruplexesmaybea
strategicallyimportanttherapeuticapproachforcancer.
Recently,ahigh-throughputmethodwasdevelopedthatidentifiesRNAG-quadruplexregionson
the basis of the propensity of the RNA to stall reverse transcriptase in a K+-dependent manner.
By applying this method to RNA from mammalian cell lines and yeast, >10,000 endogenous
regions that form RNA G-quadruplexes invitro were identified, thereby expanding the catalog
ofendogenousregionswithexperimentallyconfirmedpropensitytofoldintoRNAG-quadruplex
structuresbyafactorof>100. TodeterminethefoldingstateoftheseRNAG-quadruplexregions
invitroandincells,dimethylsulfate(DMS)treatmentwasperformedbeforemappingthereverse
transcriptasestops. Theresultsshowedthat,incontrasttopreviousassumptions,regionsthatfolded
intoRNAG-quadruplexstructuresinvitrowereoverwhelminglyunfoldedineukaryoticcells[61].
Takentogether,variousindependentstudiesonRNAG-quadruplexesrevealpromisingregulatory
functionsofthesestructuresinmultiplebiologicalprocesses. However,thesechallengingpuzzles
haveonlybeenpartiallysolved,andcomprehensivestudiesshouldbeundertakentounderstandthe
behaviorofRNAG-quadruplexes,suchasthefoldingdynamics,stabilityandbiologicalrelevance
invivoandthedetailedmechanismofRNAG-quadruplexesinvolvedinregulatoryprocesses.
5. NaturalAlkaloidsandTheirDerivativesasG-QuadruplexLigands
5.1. MethodstoDiscoverG-QuadruplexLigands
Asmentionedabove,G-quadruplexesplayimportantrolesinseveralbiologicaleventsassociated
with cancer. Selective intervention by targeting G-quadruplexes may be a strategically important
therapeutic approach for cancer. Thus, the discovery of potential G-quadruplex was important.
Generally,agoodG4ligandbindsG-quadruplexwithstrongpotencyandselectivityoverduplexand
othernucleicacidstructuresinvitro. Whileinvivo,theyarepotenttointerfereG4-involvedbiological
events, suchasthetelomeremaintenanceandgeneexpression. Variousmethods, includingthose
invitro,incellsandinsilicon,havebeendevelopedandappliedinthescreenofG-quadruplexligands.
SeveralfrequentlyusedG-quadruplexligandscreeningmethodsaresummarizedinTable1. Besides,
optimizationbasedontheseknownG-quadruplexinteractivemoietiesisemergingasaneffective
approachtodevelopnewG-quadruplexligands,whichwillbeelaboratedinthefollowingparts.
Table1.MethodsforthediscoveryofG-quadruplexligands.
Methods ScreeningIndex Ref.
3D-QSARPharmacophore Chemicalfeatureofactivecompounds [62]
Moleculardocking EstimatedbindingaffinitytoG-quadruplex [63]
Fluorescencedisplacement(FID) CompetedbindingtowardsG-quadruplex [64]
NMR BindingsitewithG-quadruplex [65]
Fluorescenceresonanceenergytransfer(FRET) StabilityeffectofcompoundsonG-quadruplex [66]
Surfaceplasmonresonance(SPR) BindingaffinitytoG-quadruplex [67]
G-quadruplexonOligoAffinitySupport(G4-OAS) BindingaffinitytoG-quadruplex [68]
Molecules2018,23,493 6of19
Molecules 2018, 23, x 6 of 19
5.2. QuindolineDerivatives
5.2. Quindoline Derivatives
Quindoline(1,Figure2)isanaturallyoccurringindolo[3,2-b]quinolinealkaloid. Quindolinewas
originallyQsuyinntdhoelisnize e(1d, Finigu19re0 26) aisn ad nhaatusrasillnyc oecbcuererninpg rinepdoalroe[d3,2fr-bo]mquicnroylpinteo laelkpailnoeid(.2 Q).uQinudoinlidnoe lwinaes was
firsteoxrtirgaicntaeldly fsryonmthtehseizAedf riinc a1n90p6l aanntdC hraysp stionlecpei sbeseann gpurienpoalreendta fr(Pomer icprlyopctaocleepaien)ei n(21).9 Q78u.inTdhoilsinceo mwapso und
hasanfitrismt eixctrroabctieadl, faronmti mthael Aarfriaicla,na nptlai-nint Cflraymptmoleaptios srayn,gaunindoalennttiah (yPeproiplilpocidaceemaei)c ianc 1t9iv78it. iTehs.isI cnom20p0o0u,nNde idle
has antimicrobial, antimalarial, anti-inflammatory, and antihypolipidemic activities. In 2000, Neidle
etal. firstreportedthatthe2,10-disubstitutedquindolinederivative3couldbindtoG-quadruplex
et al. first reported that the 2,10-disubstituted quindoline derivative 3 could bind to G-quadruplex
structuresinhumantelomeres,showingittohavemodestcytotoxicityagainstseveralcancercells
structures in human telomeres, showing it to have modest cytotoxicity against several cancer cells as
aswellasinhibitoryactivityagainsttelomerase, withanIC (telIC )of16µM[69]. Theyfurther
well as inhibitory activity against telomerase, with an IC50 5(0telIC50) 5o0f 16 μM [69]. They further
determinedthatthe2,7-disubstitutedquindolinederivative4showedmorepotentactivitythan3,
determined that the 2,7-disubstituted quindoline derivative 4 showed more potent activity than 3,
withawtietlhI Ca 5te0lIvCa50l uvaeluoef 6o.f3 6.µ3M μM[7 [07]0.].

FiguFreig2u.reS t2r.u Sctrtuucrteusroesf oqfu qinuidnodloinliened deerrivivaattiivveess aanndd bbininddiningg mmodoed weiwthi tGh-qGu-aqduraudprleuxp. lex.
The 11-substituted quindoline derivatives designed and synthesized by our research group
The 11-substituted quindoline derivatives designed and synthesized by our research group
exhibited significantly improved ability to stabilize telomeric G-quadruplexes [71,72]. The
exhibiintetrdosdiugcntiiofinc aonf tellyecimtropnr odvoendatainbgi lgitryoutopss taatb pioliszietiotenl o1m1 eenrhicanGc-eqsu tahde rbuapsilceixtye sof[ 7th1e,7 n2i]t.roTgheeni anttormod iunc tion
ofeletchtero pnydriodninaet inrigngg,r othuupss eanthpaonsciitnigo nth1e1 eelnechtaronscteasticth ientbearascictiiotyn obfettwheeenni trthoeg edneraitvoamtiviens tahnedp tyhrei dine
ring,tnheugsateivneh ealneccitnrogsttahteic ecleenctterro sotfa tthice iGnt-qeruaacdtriounplbexe.t wTheee nmtohset adcetirvive adteivrievsaatinvde, t5h (eSnYeUgIaQt-i5v)e, sehleocwtreods tatic
centerimopfrtohveeGd -inqhuiabditriounp loefx t.elTohmeermasoes tacatcivtiivtye (dteleICri5v0 a=t 0iv.4e4, μ5M(S)Y aUndIQ in-d5)u,csehdo aw medarkimedp creosvseadtioinn hinib cietlilo nof
telomgerroawsetha acntidv ait yce(lltuellIaCr sen=e0sc.4en4cµe Mph)eannodtypined inu cheudmaanm leaurkkeemdicae Kss5a6t2i ocenllisn acnedl lcoglroonw ctahncaenr dSWa6c2e0ll ular
50
cells [73]. The growth cessation was accompanied by a shortening of telomere length and
senescencephenotypeinhumanleukemiaK562cellsandcoloncancerSW620cells[73]. Thegrowth
upregulation of the expression of the cyclin-dependent kinase inhibitors p16, p21 and p27.
cessationwasaccompaniedbyashorteningoftelomerelengthandupregulationoftheexpressionof
Further studies on SYUIQ-5 indicated that the 11-alkylamino group on quindoline could lead to
thecyclin-dependentkinaseinhibitorsp16,p21andp27.
in situ protonation of the 5-N atom [74]. Molecular modeling studies revealed that the crescent-
FurtherstudiesonSYUIQ-5indicatedthatthe11-alkylaminogrouponquindolinecouldleadto
shaped aromatic core was stacked on two guanine residues of the G-quartet, and the 5-N
insituelpecrtortoopnoastiitoivne ocfenthteer 5o-Nveraltaopmpe[d7 4w].itMh othleec ucalatironm cohdaenlinnegl ostfu tdhiee squreavderualpeldext.h Hatotwheevcerre, stcheen tp-Kshaa ped
aromavtailcuceos roef wthaes 5s-Nta caktoemdso onf tthweo 1g1-uaamniinnoeqrueisniddoulienseos fwtehree G8.-2q–u8.a4r atentd, athnuds tchoeu5ld-N bee eleacsitlryo ipnoflsuiteinvceedce nter
overlabpyp tehde cwointdhittihoencs aitni othnec hsoalnuntieolno. fAtnh ealqteurandartiuvpe lteox .thHe oinwtreovdeur,ctthioenp oKf aa vpaolsuiteisveo fchthaerg5e- Nviaa tionm sistuo fthe
11-ampirnootqonuaintidono lwinaess Nw-emreet8h.y2l–a8ti.o4na natd ththeu 5s-pcoosuiltdionb.e Beaasseidly oinn flthuiesn hcyepdobthyesthise, ac osnedriietsi oonf s5i-nN-tmheetshoylul tion.
Analtqeurinnadtoivlienet odtehreiviantitvroesd uwcetrioe ndoesfiagnpeods iftoivr esccrheaernginegv ilaigianndsist uwpitrho tboenttaetri obninwdainsgN a-bmileittyh yalnadti othnaat tthe
selectively recognized telomeric G-quadruplexes. The results showed that the 5-N-methyl quindoline
5-position. Basedonthishypothesis,aseriesof5-N-methylquindolinederivativesweredesignedfor
derivatives not only exhibited significantly higher binding affinity for G-quadruplexes
screeningligandswithbetterbindingabilityandthatselectivelyrecognizedtelomericG-quadruplexes.
(approximately 5-fold higher than the non-methylated derivatives) but also exhibited selectivity
Theresultsshowedthatthe5-N-methylquindolinederivativesnotonlyexhibitedsignificantlyhigher
toward antiparallel telomeric quadruplexes. Derivative 6 markedly inhibited telomerase activity in a
bindingaffinityforG-quadruplexes(approximately5-foldhigherthanthenon-methylatedderivatives)

but also exhibited selectivity toward antiparallel telomeric quadruplexes. Derivative 6 markedly
Molecules2018,23,493 7of19
inhibitedtelomeraseactivityinacell-freesystem(telIC =0.31µM).Thedeterminationofshort-term
50
cellviabilitybyatwo-daycytotoxicityassay(MTTassay)showedthat6hadapotentinhibitoryeffect
onleukemiaHL60cellsandlymphomaCA46cells. Long-termexposureofHL60andCA46cellsto6
inducedmarkedcessationofpopulationgrowth,acellularsenescencephenotype,andshorteningof
telomerelength.
QuindolinederivativeswerecapableofinteractingwiththetelomericG-quadruplexstructureand
exhibitedinhibitionoftelomeraseactivity. However,studiesshowedthatcryptolepinecouldinteract
with the CC sites of the DNA fragment d(CCTAGG) in a base-stacking intercalation mode.Thus,
2
furtherstudieswereundertakentoimprovetheselectivityofthesecompounds.Ithasbeenreportedthat
acombinationofpeptidesthatselectivelybindtoDNAscaffoldswouldgreatlyincreasetheselectivity
ofthesepeptidesforG-quadruplexesversusduplexDNA.Aseriesofpeptidyl-benzofuroquinoline
conjugatesweredesignedandsynthesized.Thepositivechargeandalkyl-chainlengthofthedipeptidyl
side chain proved to be important for the interaction of the side chain with the G-quadruplex.
Derivative7,witha-Lys-Argdipeptidefragment,exhibitedthemostpotenttelomeraseinhibition
(telIC =5.5µM)andhad50timeshigherselectivityfortelomericG-quadruplexthanfortheduplex.
50
Additionally,cellularexperimentsalsoshowedamarkedcessationinpopulationgrowthandacellular
senescencephenotypeaswellasshorteningofthetelomerelength[75].
In addition to telomeric single-stranded overhangs, G-quadruplexes are also present in the
promoterorregulatoryregionsofimportantoncogenessuchasc-MYC,BCL-2andc-KIT.c-MYCis
animportantproto-oncogene,andtheaberrantoverexpressionofthisgeneisalwaysassociatedwith
avarietyofmalignanttumors. FurtherstudieshaveshownthatSYUIQ-5canalsointeractwiththe
c-MYCG-quadruplexandinhibittheexpressionofthisgene. APCRstopassayshowedthatinthe
presenceofSYUIQ-5,thePu22c-MYColigomerwasinducedtoformaG-quadruplex,whichprevented
hybridizationwithacomplementarystrandthatoverlappedwiththelastGrepeat. Theexpression
ofc-MYCwasseentobemarkedlyinhibitedwhenthemRNAandproteinlevelsinHL-60andK562
cellsweremeasured. Derivative8,withashortsidechain,showedmorepotentialstabilizationofthe
c-MYCG-quadruplex. Furtherstudiesshowedthat8couldinhibittheexpressionofc-MYCinthe
hepatocellularcarcinomacelllineHepG2. Thebindingofderivative8withc-MYCG-quadruplex
wasexploredbyYangetal.[76]. StructuralNMRresultsshowedquindolinecouldstackeffectively
onbothsidesofG-quadruplex,andunexpectedly,thestructureshowedthatthequindoline-induced
reorientationoftheflankingsequencesledtotheformationofanewbindingpocket(Figure2).
The quindoline derivatives were seen to interact with the G-quadruplex in the c-MYC gene
withacertaindegreeofselectivity,andthespecificitieswerefurtherimproved. Theheteroaromatic
1,4-substituted 1,2,3-triazole ring system has attracted extensive interest in drug design owing to
thewideapplicabilityofthissystemasasyntheticbuildingblockendowedwithpharmacological
potential.Aseriesofnoveltriazole-containingbenzofuroquinolinederivatives(T-BFQs)weredesigned
and synthesized by 1,3-dipolar cycloaddition of a series of alkyne and azide building blocks [77].
TheselectivityofthesenovelT-BFQstowardc-MYCG-quadruplexDNAwassignificantlyimproved,
andadistinctincreaseinbindingaffinitywasobserved. Furthercellularexperimentsshowedthat
derivative 9 specifically downregulated c-MYC gene transcription and expression in RAJI cells.
Derivative9exhibitedinhibitionoftumorcellproliferationbuthadaweakeffectonprimarycultured
mousemesangialcells(inwhichproliferationdoesnotdependonc-MYCexpression). Theseresults
indicatedthatinhibitionby9presumablyoccursviathedownregulationoftranscriptionofthec-MYC
gene. Tumor-bearingmicetreatedwithderivative9hadanaveragetumorvolumeof<400mm3and
exhibitedaninhibitionrateof38.1%(Table2).
Recently, four series of 7,11-disubstituted quindoline derivatives were also synthesized by
introducing a second cationic amino side chain and a 5-N-methyl group as ligands to bind to the
c-MYCpromoterG-quadruplex. Invitroevaluationsshowedthatthederivatesexhibitedincreased
stabilities and binding affinities, and most of the derivatives had better selectivity for the c-MYC
G-quadruplex(overduplexDNA)comparedtoSYUIQ-5. Moreover,thenewderivativesprevented
Molecules2018,23,493 8of19
NM23-H2, a transcription factor, from effectively binding to the c-MYC G-quadruplex. Further
studies showed that derivative 10 downregulated c-MYC transcription by targeting the promoter
G-quadruplexanddisruptingtheNM23-H2/c-MYCinteractioninRAJIcells. Derivative10could
inhibitBMuolrekcuiletst ’2s01l8y, 2m3, xp h omacellproliferationthroughcellcyclearrestandapoptosisan8d ofs 1u9 ppress
tumorgrowthinahumanBurkitt’slymphomaxenograftwithaninhibitionrateof27.4%(Table2)[78].
G-quadruplex and disrupting the NM23-H2/c-MYC interaction in RAJI cells. Derivative 10 could
inhibit Burkitt’s lymphoma cell proliferation through cell cycle arrest and apoptosis and suppress
Table2.Anti-tumoractivityofalkaloidderivativesinvivo.
tumor growth in a human Burkitt’s lymphoma xenograft with an inhibition rate of 27.4% (Table 2)
[78].
Dosage TumorGrowth DaystoComplete
Derivatives XenograftModel (mg·kg−1) Inhibition Response Ref.
Table 2. Anti-tumor activity of alkaloid derivatives in vivo.
9 A549humanlungcancer 6.25;i.p. 38.1% 12 [77]
Dosage Tumor Growth Days to Complete
D10erivatives RAJIXheunmoagnraBfut rMkiottd’selly mphoma 30;i.p. 27.4% 14 Ref.[ 78]
(mg·kg−1) Inhibition Response
15 20;i.p. 35.2% 20 [79]
9 A549 human lung cancer 6.25; i.p. 38.1% 12 [77]
16 SiHahumancervicalsquamouscancer 5;i.p. 64.8% 21 [79]
RAJI human Burkitt’s
21 10 30; i.p. 1;i.p. 27.4% 49.3% 14 20 [78] [79]
lymphoma
24 MCF-7humanbreastcancer 7.5;i.p. 60.1% 20 [79]
15 20; i.p. 35.2% 20 [79]
SiHa human cervical
16 5; i.p. 64.8% 21 [79]
squamous cancer
5.3. Isaindigot2o1n eDerivatives 1; i.p. 49.3% 20 [79]
MCF-7 human breast
24 7.5; i.p. 60.1% 20 [79]
Isaindigotone(11,Figucraenc3e)r isanalkaloidisolatedfromtherootofthetraditionalChineseherb
IsatisindigoticaFort[80]. Thiscompoundexhibitsexcellenteffectsagainstinfluenza,epidemichepatitis,
5.3. Isaindigotone Derivatives
andepidemicencephalitis.Thestructureofisaindigotonecomprisesapyrrolo[2,1-b]quinazolinemoiety
conjugatedwIsiatihndaigboetnoznye l(i1d1e, nFieggurreo u3)p i.s Oanu arlkgarloouidp isfiorlsattesdy fnrothme sthizee rdooat soef rtihees torafdiistaioinnadl iCghoitnoensee hdeerrbi vatives
toexplorIseattihs einedfifgeoctticsao Ffotrht e[d80e]r. ivTahtisiv ceosmopnouGn-dq ueaxdhirbuitpsl eexxcsetlalebnitl iteyffeacntsd asgealeincstti viintfylu[e8n1z–a8, 3e]p.iTdehmeiuc nfused
hepatitis, and epidemic encephalitis. The structure of isaindigotone comprises a pyrrolo[2,1-
aromaticringsandtetheredsidechainsofthesederivativesseemedtoallowthecompoundstoadopt
b]quinazoline moiety conjugated with a benzylidene group. Our group first synthesized a series of
flexibleandadaptiveconformations,whichhelpintherecognitionoftelomericG-quadruplexcores
isaindigotone derivatives to explore the effects of the derivatives on G-quadruplex stability and
andgroosevleecstiivnitay [48:11–b83in]. dTihneg umnfuosdeed. aDroemriavtiact rivinegs1 2ansdi gtentihfiecreadn tslidyei nchdauincse odf ctheellses ednereisvcaetinvcese saenemdetde lomere
shortenitno galolofwH thLe- 6c0omceplolusnadnsd toC aAdo4p6t fcleexllisblaet amndi cardoamptiovlea crocnofonrcmeanttiroantsi,o wnhsi.chA hTelRp AinP th-Le IrGecoagsnsiatiyons howed
thatderoivf atteilvomee1r2ici nGh-qibuiatderduptelelxo mcoerersa saenda cgtrivooitvyesw init ha a4n:1 tbeliInCdingv maloudee.o Df7er.i8vaµtMive. T12o sbiegnttiefircainntvlye stigate
50
induced cell senescence and telomere shortening of HL-60 cells and CA46 cells at micromolar
howtheplanarityofanunfusedaromaticligandimpactsthequadruplex-bindingpropertiesofthe
concentrations. A TRAP-LIG assay showed that derivative 12 inhibited telomerase activity with an
ligand, another series of isaindigotone derivatives were designed and were reported to stabilize
telIC50 value of 7.8 μM. To better investigate how the planarity of an unfused aromatic ligand impacts
the c-KITG-quadruplex. The bestderivative, 13, reducedthe transcriptionof c-KITby 52.5%and
the quadruplex-binding properties of the ligand, another series of isaindigotone derivatives were
exhibiteddessiiggnneidfi acnadn wtceyret oretpooxritceidty toi nstaabigliazset trhoei nc-tKeIsTt iGn-aqlusatdrroumplaelx.t Tuhme obresct edlelrliivnaeti.vSe,A 13R, raenduaclyedsi tshes howed
thatexptarannssicorniptoiofnt hoef ca-rKoImT bayti c52s.y5%st eamnd veixahitbhiteedin stirgondifuiccantito cnytooftoaxibcietyn zine na egraisntrgo,inatebsteinnazly sltirdomenael group
and twotucmatoiro cneillc lianme. iSnAoRs aindaelycshisa sihnoswiendt othatht eexpqaunisnioanz oolfo thnee amromoiaettiyc snysottemo nvliya tmhea ixnitrmodizuectsiotnh eof sat acking
benzene ring, a benzylidene group and two cationic amino side chains into the quinazolone moiety
interactions of the derivatives with the G-quartet but also incorporates features of flexibility that
not only maximizes the stacking interactions of the derivatives with the G-quartet but also
preventthederivativefromintercalatingwithduplexDNA.
incorporates features of flexibility that prevent the derivative from intercalating with duplex DNA.

Figure 3. Structures of isaindigotone derivatives and binding mode with G-quadruplex.
Figure3.StructuresofisaindigotonederivativesandbindingmodewithG-quadruplex.
Molecules2018,23,493 9of19
Currently, a number of small molecule ligands that stabilize G-quadruplexes have been
developed[84,85]. However,selectivelytargetingaspecificG-quadruplexisstilladifficulttask[86,87].
Typically, the interaction between protein and DNA is highly specific and stable. By targeting
the interaction of a G-quadruplex-related protein with the G-quadruplex, new opportunities are
emergingforthedevelopmentofsmallmoleculesthattargetG-quadruplexes. Intheprocessofc-MYC
transcription, one of the transcription factors, NM23-H2, has an important role in transcriptional
reactivation.StudieshaverevealedthatNM23-H2canrecognizeandbindtothec-MYCNHEIII region
1
andactasanunwindingproteinontheG-quadruplex[88]. Therefore,theinteractionofNM23-H2
withthec-MYCG-quadruplexmightbeakeyprocessinthetranscriptionalregulationofthec-MYC
gene,andderivativesthatcaninterferewiththebindingofNM23-H2tothec-MYCG-quadruplex
areexpectedtorepressc-MYCtranscription. Anin-housesmall-moleculelibraryscreeningbySPR
identifiedtheisaindigotonederivative14(SYSU-ID-01),whichsignificantlyinterferedwiththebinding
ofNM23-H2tothec-MYCpromoterDNA[79]. Furtheranalysesofthederivative-proteininteraction
andtheprotein-DNAinteractionprovidedinsightintothemodeofactionofSYSU-ID-01. Resultsof
cellularevaluationshowedthatSYSU-ID-01couldabrogatethebindingofNM23-H2tothec-MYC
promoter,resultingindownregulationofc-MYCtranscriptionanddramaticallysuppressedHeLacell
growth. However,inprimaryculturedmousemesangialcells,inwhichproliferationdoesnotdepend
onc-MYCexpression,insteadofproliferationarrest,SYSU-ID-01promotedcellproliferation.
Theseresultsinspiredfurthermodification,andaseriesofnewisaindigotonederivativeswere
designedandsynthesized[89,90]. TheabilitiesofthesederivativestointeractwithG-quadruplexes
orwithNM23-H2andtodisrupttheG-quadruplex-NM23-H2interactionwereevaluated. Among
thesederivatives,derivative15showedmarkeddisruptionoftheG-quadruplex-NM23-H2interaction.
This compound exhibited significant effects on c-MYC-related processes in SiHa cells, including
inhibition of transcription and translation, inhibition of cell proliferation, induction of apoptosis,
and regulation of the cell cycle. In contrast, derivative 15 promoted cellular growth instead of
proliferation arrest in primary cultured mouse mesangial cells. A SiHa xenograft mouse model
of human cervical squamous cancer showed that after 3 weeks of treatment, compared with the
vehiclegroup,treatmentwithderivative15at20mg/kgresultedinastatisticallysignificantreduction
in tumor weight, with a tumor growth inhibition rate of 35.2% (Table 2). Furthermore, to design
andsynthesizehighlyspecificNM23-H2-bindingligands,theG-quadruplex-ligandfeaturesofthe
derivatives were removed, and only one positive amino side chain remained at the 6-position.
A series of novel isaindigotone derivatives with a variety of substituents, such as alkyl, hydroxyl,
alkoxy, alkamino, fluoro, and trifluoromethyl groups, at the 4(cid:48)-position of the styrene ring were
designed and synthesized and screened for NM23-H2-selective binding of ligands. Among these
derivatives,derivative16exhibitedsignificantbindingaffinitytowardNM23-H2anddisruptedthe
NM23-H2/c-MYCG-quadruplexinteractioninvitroandincells. Inhibitionofthetranscriptionof
theoncogenec-MYCby16inducedcell-cycleG0/G1-phasearrest,cellularapoptosis,proliferation
inhibition, and migration inhibition. In addition, 16 exhibited potent antiproliferation activity in
severalcancercelllinesandexhibitedgoodantitumoractivitybyinhibitingthegrowthofcervical
squamouscancerinBALB/C-nu/numicewithaSiHaxenograft,withatumorgrowthinhibitionrate
of64.8%(Table2).
5.4. BerberineDerivatives
Berberine (17) is an alkaloid isolated from Chinese herbs that has long been used as an
antimicrobialagent,andthiscompoundhasattractedalotofattentionforitsantineoplasticeffects.
Berberine appears to suppress the growth of a variety of tumor cells, including breast cancer,
leukemia, prostate carcinoma, and gastric carcinoma cells. In 1999, Tsuruo and coworkers were
thefirsttoreportthatberberineinhibitstelomeraseactivity,thusinterruptingtelomereelongation[91].
The13-substitutedberberinederivative,18,thenshowsweakbindingwithtelomericG-quadruplex
DNAandpoortelomeraseinhibition,while9-O-subtitutedberberinederivatives,byintroducinga
Molecules2018,23,493 10of19
Molecules 2018, 23, x 10 of 19
sidechainwithterminalaminogroupoutsideofthecrescent-shapedringsystem,showsignificantly
improved ability to stabilize telomeric G-quadruplexes [92–94]. A recent X-ray structure of provided
improvedabilitytostabilizetelomericG-quadruplexes[92–94].ArecentX-raystructureofprovidedan
an important clue for interpreting data regarding the binding of berberine with telomeric G-
importantclueforinterpretingdataregardingthebindingofberberinewithtelomericG-quadruplex.
quadruplex. The result showed that two berberine molecules could simultaneously interacting with
Theresultshowedthattwoberberinemoleculescouldsimultaneouslyinteractingwitheachexternal
eGa-cthet reaxdtervniaal πG--stteatcrkaidn gv,iaw πh-islteactkhiengc,r ewscheilnet -tshhea pcreedscreinntg-shoafpbeedr brienrgin eof sbtaecrkberoivneer sttwacok govuearn itnweos
guanines (Figure 4) [95].
(Figure4)[95].

FFiigguurree 44.. SSttrruuccttuurreess ooff bbeerrbbeerriinnee ddeerriivvaattiivveess aanndd bbiinnddiinngg mmooddee wwiitthh GG--qquuaaddrruupplleexx..
AAmmoonngg tthheessee ddeerriivvaattiivveess,, ddeerriivvaattiivvee 1199 mmaarrkkeeddllyy iinnhhiibbiitteedd tteelloommeerraassee aaccttiivviittyy,, wwiitthh aat etleIlICC50
50
value of 16 μM. Introduction of a positively charged terminal aza-aromatic group may enhance the
valueof16µM.Introductionofapositivelychargedterminalaza-aromaticgroupmayenhancethe
electrostatic interaction of the derivative with the phosphate backbone and improve the interaction
electrostaticinteractionofthederivativewiththephosphatebackboneandimprovetheinteractionwith
with the loop region via π-π stacking. Derivative 20, which had an aza-aromatic group, could
theloopregionviaπ-πstacking. Derivative20,whichhadanaza-aromaticgroup,couldselectively
sbeinledcttivoetleyl obminedr itco Gte-lqoumaedrriuc pGle-qxuesadorvueprledxuepsl eoxveDr NduAp.lDexe rDivNaAtiv. eDe2r0ivhaatdivea 2te0lI hCad vaa tleluICe5o0 fv1al4uµe Mof,
50
w14h μicMh,i swmhuicchh ilso wmeurcthh alonwthear tthofanb etrhbaetr ionfe b(eterlbICerin=e 7(t0elIµCM50 )=. R70ec μenMtl)y. ,Raenceewntllyy,s ya nntehwesliyz esdynbtehrebseirziende
50
berberine derivative, 21 (Ber8), with a chlorohexyl side chain at the 9-position, was seen to strongly
derivative,21(Ber8),withachlorohexylsidechainatthe9-position,wasseentostronglyinteractwith
interact with telomeric G-quadruplexes and could effectively induce acute cell growth arrest in cancer
telomericG-quadruplexesandcouldeffectivelyinduceacutecellgrowtharrestincancercells.
cells.
The inhibition of cancer cell growth by Ber8 was associated with apparent cell cycle arrest,
The inhibition of cancer cell growth by Ber8 was associated with apparent cell cycle arrest, cell
cellsenescence,andprofoundDNAdamageattelomericregions. Furthermechanicalstudiesrevealed
senescence, and profound DNA damage at telomeric regions. Further mechanical studies revealed
thatBer8couldnotonlyincreasetheformationofendogenoustelomericG-quadruplexesincancer
that Ber8 could not only increase the formation of endogenous telomeric G-quadruplexes in cancer
cellsbutalsodelocalizeTRF1andPOT1fromthetelomereandinducetelomereuncapping.Invivo
cells but also delocalize TRF1 and POT1 from the telomere and induce telomere uncapping. In vivo
experimentsshowedthat21exhibitedtumor-inhibitionactivityinSiHaxenografttumors(Table2)[96,97].
experiments showed that 21 exhibited tumor-inhibition activity in SiHa xenograft tumors (Table 2)
[59.56.,9O7]t.h erAlkaloidDerivatives
55..55.. 1O.tQhueri nAalzkoalloinide DDeerrivivaatitvivese s
Thedesignofderivatives22–25isderivedfromthe11-substitutedquindolinederivativeSYUIQ-5
5.5.1. Quinazoline Derivatives
(Figure 5) [98–100]. By retaining the 11-substituted amino side chain of SYUIQ-5 and opening
the fiTvhee- mdeesmigbne oref ddeirnidvaotlieversin 2g2,–2a5 niso dne-croivpelda nfraormfl ethxeib 1l1e-ssutrbusctittuurteedw qausinodbotaliinnee dd.eriTvhatiisvem SoYleUcIuQle-
5h a(sFiagnur“ei m5)i t[a9t8iv–1e0”0t]e. tBrayc ryectlaicinainrogm thaeti c11s-yssutbesmtitfuotremd eadmviniao isnitdrea mchoalienc uolfa SrYhUydIQro-5g eanndb oonpdesn,inwgh tihche
feinvaeb-mleesmthbeeraeddo pintidoonleo frimngo,d ae rnaotenl-ycotwpliasnteadr aflnedxibnleea rs-tprulacntuarrec ownafso rombtaatiinoends.o Tfhthise maroyllecguroleu phsasa nadn
“ailmlowitastitvhee” ltiegtarnacdytcolics taarcokmwateilcl soynsttehme Gfo-rqmueadrt evti.a Tinhteraremfoorlee,cuthlaers ehydderroivgaetniv beosnedffse, cwtivheiclhy ebninadbletos
ttheleo amdeorpictiGon-q ouf amdorudperleaxteelsyb tuwtisntoetdt aonddu npelaerx-epsl.aTnahre cmonofsotremffaetcitoinves odfe trhivea atrivyel ,g2ro2,uhpass aandK allvoawluse thoef
D
l3i.g1a4n×d 1to0 −s7taMck. NwMellR oann dthme oGl-eqcuualartredt.o Tckhienrgefaonrea,l ythseessein ddeicriavtaettihvaets heyffdercotigveenlyb obninddi ntgo, tπe-lπomstearcikc inGg-
qinutaedrarcutpiolenxse,sa nbdute nleoctt rtoos dtautipcleinxteesr.a Tchtieo nmsomsta eyffaeccctoivuen tdfeorrivtahteivoev, e2r2a,l lhianst ear aKcDt ivoanl.uDe eorfi v3a.1t4iv ×e 1202−7a lMso.
NinMduRc eadnddi smtionlcetccuellalrs ednoecskcienngc eananaldysteelso minedriecastheo trhteant ihnygdinroHgeLn-6 b0ocnedllisnagt, mπi-cπro smtaoclkairncgo nincteenrtarcattiioonnss,.
Aannde fefleeccttivroesNta-t(i2c- (iqnuteinraacztoiolinns-2 -myla)yp haeccnoyul)nbte nfozra mthide eo(vQePraBl)l dienrtievraatcitvieo,n2.3 D,weraisvsautibvsee q2u2e anltslyo oinbtdauinceedd.
Tdhisetisnectd ecreilvl asteinveesschenacvee asntrdo ntegloimndeurec tsiohnoratebniliintige si,n leHaLd-i6n0g cteollsth aet cmonicvreormsioolnaro cfohnycbenritdratteiolonms. eArinc
eGf-fqecutaivderu Np-l(e2x-e(qsutoinpaazroallilne-l2G-y-ql)u pahdernuypll)ebxeensz.aSmAiRdea n(QalPysBi)s dinedriivcaatteivset,h 2a3t,t wheaps rseusbesnecqeuoefnatnlya odbdtiatiinonedal.
These derivatives have strong induction abilities, leading to the conversion of hybrid telomeric G-
quadruplexes to parallel G-quadruplexes. SAR analysis indicates that the presence of an additional