Novel tranylcypromine/hydroxylcinnamic acid hybrids as lysine-specific demethylase 1 inhibitors with potent antitumor activity

Article abstract of DOI:10.1248/cpb.c15-00476

Novel tranylcypromine/hydroxylcinnamic acid hybrids 15a, b, and 19a-l were designed and synthesized by connecting tranylcypromine with hydroxylcinnamic acid, and their biological activities were evaluated. The in vitro assay of their inhibitory activities

Full text of DOI:10.1248/cpb.c15-00476

882
Chem. Pharm. Bull. 63, 882–889 (2015)
Vol. 63, No. 11
Regular Article
Novel Tranylcypromine/Hydroxylcinnamic Acid Hybrids as Lysine-
Specific Demethylase 1 Inhibitors with Potent Antitumor Activity
,a
Yan Han,* Chunlei Wu,a Haifeng Lv,b Na Liu,c and Huaying Denga
aꢀDepartmentꢀofꢀOrthopedics,ꢀTheꢀFirstꢀAffiliatedꢀHospital,ꢀWenzhouꢀMedicalꢀUniversity;ꢀWenzhouꢀ325035,ꢀZhejiang,ꢀ
b
P.R. China: ꢀSchoolꢀofꢀPharmacy,ꢀWenzhouꢀMedicalꢀUniversity;ꢀWenzhouꢀ325035,ꢀZhejiang,ꢀP.ꢀR.ꢀChina:ꢀandꢀ
cꢀDepartmentꢀofꢀTraditionalꢀMedicalꢀTraumatologyꢀOrthopedics,ꢀXi’anꢀHonghuiꢀHospital;ꢀXi’anꢀ710054,ꢀShanxi,ꢀP.ꢀR.ꢀ
China.
Received June 9, 2015; accepted August 12, 2015
Novel tranylcypromine/hydroxylcinnamic acid hybrids 15a, b, and 19a–l were designed and synthesized
by connecting tranylcypromine with hydroxylcinnamic acid, and their biological activities were evaluated.
The in vitro assay of their inhibitory activities against lysine-specific demethylase 1 (LSD1) showed that most
of the target compounds displayed high potency with IC50 values ranging from submicromolar to single-digit
micromolar levels. In particular, compound 19l had robust, selective LSD1 inhibitory activity, which was ob-
viously higher than the inhibitory activity against homologues monoamine oxidase-A (MAO-A) and MAO-B,
respectively. Furthermore, the most potent compound 19l selectively inhibited cancer cell but not nontumor
colon cell proliferation in vitro. In addition, compound 19l also dose-dependently increased the expression of
H3K4me2 at the cellular level. Our findings suggest that tranylcypromine/hydroxylcinnamic acid hybrids as
LSD1 inhibitors may hold great promise as therapeutic agents for the treatment of human cancers.
Key wordsꢀ synthesis;ꢀlysine-specificꢀdemethylase;ꢀinhibitoryꢀactivity;ꢀtranylcypromine;ꢀhydroxylcinnamicꢀacid;ꢀ
antitumor agent
Epigeneticꢀ disregulationꢀ oftenꢀ leadsꢀ toꢀ theꢀ aberrantꢀ geneꢀ However, currently PCPA and its derivatives display poor se-
expressionꢀ programsꢀ characteristicꢀ ofꢀ cancer.1,2) Transcrip- lectivity, low potency, or weak antitumor activity, Therefore,
tionalꢀregulationꢀthroughꢀchromatinꢀmodificationꢀisꢀreversibleꢀ identificationꢀofꢀnovelꢀpotentꢀreversibleꢀLSD1ꢀinhibitorsꢀisꢀes-
and dynamic such that enzymes implicated in the disregula- sential to further elucidate LSD1’s role in cancer and identify
tion of chromatin represent a new class of protein targets for whetherꢀorꢀnotꢀeffectiveꢀinhibitionꢀtargetingꢀLSD1ꢀisꢀaꢀviableꢀ
drugꢀ development.ꢀ Lysine-specificꢀ demethylaseꢀ 1ꢀ (LSD1)ꢀ isꢀ therapeutic strategy.
anꢀepigeneticꢀenzymeꢀthatꢀbelongsꢀtoꢀtheꢀamineꢀoxidaseꢀpro-
As part of our ongoing effort to discover novel anticancer
teinꢀ superfamily,ꢀ whichꢀ oxidativelyꢀ cleavesꢀ methylꢀ groupsꢀ ofꢀ agents,ꢀ weꢀ wereꢀ inspiredꢀ byꢀ theꢀ factꢀ thatꢀ hydroxylcinnamicꢀ
histone H3 at lysine 4 (H3K4me1 and H3K4me2) and lysine acids, such as ferulic acid and p-hydroxycinnamicꢀ acid,ꢀ areꢀ
9ꢀ(H3K9me1ꢀandꢀH3K9me2)ꢀthroughꢀflavinꢀadenineꢀdinucleo- known as phenolic compounds occurring in natural plant
tideꢀ (FAD)-dependentꢀ enzymaticꢀ oxidation,ꢀ andꢀ catalyzesꢀ product, and their derivatives displayed selective antiprolifera-
methylatedꢀ lysineꢀ substratesꢀ toꢀ generateꢀ hydrogenꢀ peroxideꢀ tive activity against some types of cancer cells.23–25)ꢀ Onꢀ
andꢀformaldehydeꢀasꢀbyproducts3–5) (Fig. 1).
theꢀ basisꢀ ofꢀ theꢀ aforementionedꢀ information,ꢀ weꢀ conjugatedꢀ
LSD1ꢀ isꢀ alsoꢀ ableꢀ toꢀ demethylateꢀ non-histoneꢀ substrates,ꢀ tranylcypromineꢀ withꢀ hydroxylcinnamicꢀ acidꢀ toꢀ affordꢀ leadꢀ
such as the tumor suppressor p53 and the cell cycle and apo- compounds 15a, b, and different amino acid as the linker con-
ptosis regulator E2F1.6,7)ꢀ Furthermore,ꢀ LSD1ꢀ isꢀ oftenꢀ overex- nectedꢀ withꢀ tranylcypromineꢀ andꢀ hydroxylcinnamicꢀ acidꢀ toꢀ
pressedꢀ inꢀ variousꢀ cancerꢀ cellsꢀ andꢀ tissues:ꢀ neuroblastoma,ꢀ designꢀ novelꢀ hybridsꢀ 19a–l. We hypothesized that the novel
hepatocarcinomas,ꢀcolonꢀcancer,ꢀbreastꢀcancer,ꢀgastricꢀcancer,ꢀ typesꢀ ofꢀ tranylcypromine/hydroxylcinnamicꢀ acidꢀ hybridsꢀ willꢀ
andꢀ bladderꢀ cancerꢀ cells.8–12)ꢀ LSD1ꢀ inhibitionꢀ decreasedꢀ ex- efficaciouslyꢀdevelopꢀinhibitoryꢀeffectꢀofꢀLSD1ꢀandꢀselectivelyꢀ
pression of target genes in these cancers. Therefore, LSD1
hasꢀbeenꢀconsideredꢀanꢀimportantꢀandꢀattractiveꢀtargetꢀforꢀtheꢀ
treatment of cancer.13,14)ꢀ LSD1ꢀ inhibitorsꢀ areꢀ ofꢀ interestꢀ notꢀ
onlyꢀ asꢀ toolsꢀ toꢀ elucidateꢀ theꢀ biologicalꢀ functionsꢀ ofꢀ theꢀ en-
zyme,ꢀbutꢀalsoꢀasꢀpromisingꢀtherapeuticꢀagents.
Toꢀ date,ꢀ aꢀ numberꢀ ofꢀ priorꢀ LSD1ꢀ inhibitorsꢀ haveꢀ beenꢀ re-
portedꢀ includingꢀ monoamineꢀ oxidaseꢀ (MAO)ꢀ inhibitorsꢀ suchꢀ
as tranylcypromine (1, PCPA, Fig. 2), pargyline (2), and de-
rivatives thereof (3–6), polyamines (7), peptides (8), as well as
guanidine derivatives (9).4,15–20)ꢀAmongꢀtheseꢀLSD1ꢀinhibitors,ꢀ
PCPAꢀisꢀaꢀclassicalꢀMAOꢀinhibitorꢀwhichꢀhasꢀbeenꢀwellꢀstud-
ied,ꢀandꢀbiologicalꢀstudiesꢀofꢀPCPAꢀhaveꢀuncoveredꢀtheꢀimpor-
tant roles of LSD1 in several cancer diseases.21)ꢀPCPAꢀinhibitsꢀ
LSD1ꢀ involvingꢀ anꢀ oxidativeꢀ cyclopropylamineꢀ ring-openingꢀ
reaction through a single-electron transfer mechanism.22)
Fig.ꢀ 1.ꢀ ReactionsꢀCatalyzedꢀbyꢀLysine-SpecificꢀDemethylases
*ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀhymed009@163.com
© 2015 The Pharmaceutical Society of Japan

Vol. 63, No. 11 (2015)
Chem. Pharm. Bull.
883
Fig.ꢀ 2.ꢀ RepresentativeꢀStructuresꢀofꢀReportedꢀLSD1ꢀInhibitors
Reaction conditions and reagents: a) Di-t-butyl-dicarbonate,ꢀ 1ꢀNꢀ NaOH,ꢀ rt,ꢀ 10ꢀh,ꢀ 56%;ꢀ b)ꢀ MOMCl,ꢀ MeCN,ꢀ NaH,ꢀ rt,ꢀ 3ꢀh,ꢀ 67–71%;ꢀ c)ꢀ EDCI,ꢀ DMAP,ꢀ CH2Cl2, rt, 5–8h,
65–77%;ꢀ d)ꢀ Saturatedꢀ HClꢀ inꢀ dryꢀ AcOEt,ꢀ rt,ꢀ 6–10ꢀh,ꢀ 88–94%;ꢀ e)ꢀ Differentꢀ substitutedꢀ methylꢀ aminoacetate,ꢀ EDCI,ꢀ DMAP,ꢀ CH2Cl2,ꢀ rt,ꢀ 5–10ꢀh,ꢀ 63–72%;ꢀ f)ꢀ 1ꢀNꢀ NaOH,ꢀ
MeOH,ꢀrt,ꢀ1–2ꢀh,ꢀ86–93%.
Chart 1
inhibitꢀ tumorꢀ cellꢀ proliferation.ꢀ Therefore,ꢀ aꢀ totalꢀ ofꢀ twentyꢀ chloromethylꢀmethylꢀetherꢀ(MOM-Cl)ꢀtoꢀgiveꢀ13a and b. Then
target compounds (15a, b, 19a–l) were designed and synthe- compound 11 was respectively reacted with 13a and b in the
sized, and their inꢀ vitro antitumor effects were investigated. presenceꢀofꢀ1-ethyl-(3-(3-dimethylamino)ꢀpropyl)-carbodiimideꢀ
Herein,ꢀtheꢀsynthesisꢀandꢀpreliminaryꢀbiologicalꢀevaluationꢀofꢀ hydrochlorideꢀ(EDCI)ꢀandꢀ4-dimethylaminopyridineꢀ(DMAP)ꢀ
these compounds were reported.
to gain amidated products 14a and b, which was followed
treatedꢀ withꢀ saturatedꢀ HClꢀ inꢀ dryꢀ AcOEtꢀ toꢀ obtainꢀ targetedꢀ
compounds 15a and b. In addition, compounds 13a and b
Results and Discussion
Chemistry The synthetic route to target compounds 15a, wereꢀ alsoꢀ reactedꢀ withꢀ differentꢀ substitutedꢀ methylꢀ aminoac-
b and 19a–l was depicted in Chart 1. Firstly, the alkyl amine etates to offer intermediates 16a–l, which was further hydro-
of 4-(2-aminocyclopropyl)aniline scaffold 10ꢀwasꢀprotectedꢀbyꢀ lyzedꢀwithꢀNaOHꢀsolutionꢀtoꢀgetꢀcompoundsꢀ17a–l. Then tar-
treatment with di-t-butyl-dicarbonateꢀ toꢀ generateꢀ compoundꢀ get compounds 19a–l were prepared from intermediates 17a–l
11,ꢀ andꢀ theꢀ phenolicꢀ hydroxyꢀ ofꢀ 4-hydroxycinnamicꢀ acidꢀ 12a and 11 according to the similar synthetic method of 15a and
or ferulic acid 12bꢀ wasꢀ alsoꢀ protectedꢀ byꢀ etherificationꢀ withꢀ b.ꢀTheꢀfinalꢀproductsꢀ15a, b, and 19a–lꢀwereꢀpurifiedꢀbyꢀcol-

884
Chem. Pharm. Bull.
Vol. 63, No. 11 (2015)
Tableꢀ 1.ꢀ TheꢀStructuresꢀandꢀLSD1ꢀInhibitoryꢀAcivityꢀ(IC50, µM) of 15a, b, and 19a–l
Compd
R
R1
LSD1ꢀinhibitoryꢀactivityꢀ(IC50a), µM)
4
—
H
—
1.8
7.2
15a
15b
19a
19b
19c
19d
19e
—
OCH3
H
—
H
4.9
6.6
H
CH2
>10
3.2
H
CH2Ph
H
CH(CH3)2
CH(CH3)CH2CH3
>10
>10
H
19f
H
2.1
19g
19h
19i
OCH3
OCH3
OCH3
OCH3
OCH3
H
CH2
4.5
7.1
CH2Ph
1.4
19j
19k
CH(CH3)2
CH(CH3)CH2CH3
8.9
>10
19l
OCH3
0.93
a)ꢀDataꢀareꢀexpressedꢀasꢀtheꢀmeanꢀIC50ꢀvalueꢀfromꢀtheꢀdose–responseꢀcurvesꢀofꢀatꢀleastꢀthreeꢀindependentꢀexperiments.
Tableꢀ 2.ꢀ LSD1,ꢀ MAO-Aꢀ andꢀ MAO-Bꢀ Inhibitingꢀ Activityꢀ (IC50 Values,
µM) of Compounds 19f, i, and l
Tableꢀ 3.ꢀ Theꢀ IC50 Values of Active Compounds 19f, i, and l against
Three Human Cancer Cell Lines
a)
IC50 (µM)
Growthꢀinhibitionꢀ(IC50a), µM)
Compd
Compd
LSD1
MAO-A
MAO-B
HGC-27 cells
HCT116 cells
LP-1 cells
4
1.8
2.1
1.4
0.93
2.3
4.1
3.7
3.6
>10
>10
>10
>10
4
12.8
13.5
7.9
15.1
11.8
8.8
16.3
14.5
15.2
13.4
19f
19i
19l
19f
19i
19l
8.1
5.3
a) The data are the mean values of IC50ꢀ fromꢀ atꢀ leastꢀ threeꢀ independentꢀ experi-
ments.
a)ꢀ Theꢀ inhibitoryꢀ effectsꢀ ofꢀ individualꢀ compoundsꢀ onꢀ theꢀ proliferationꢀ ofꢀ cancerꢀ
cellꢀlinesꢀwereꢀdeterminedꢀbyꢀtheꢀMTTꢀassay.ꢀTheꢀdataꢀareꢀtheꢀmeanꢀvaluesꢀofꢀIC50
fromꢀatꢀleastꢀthreeꢀindependentꢀexperiments.
umn chromatography, and their structures were characterized nearly 2–4 fold less than those against LSD1, and also less
byꢀIR,ꢀ1H-NMR,ꢀMS,ꢀandꢀelementalꢀanalyses.ꢀAllꢀcompoundsꢀ potent than the positive control 4ꢀagainstꢀMAO-A.ꢀInꢀaddition,ꢀ
were of >95%ꢀpurityꢀdeterminedꢀbyꢀHPLC.
allꢀ theꢀ compoundsꢀ exhibitedꢀ noꢀ significantꢀ inhibitoryꢀ effectsꢀ
Biological Evaluation All the compounds synthesized in onꢀMAO-Bꢀactivities.
thisꢀstudyꢀwereꢀexaminedꢀinꢀvitroꢀforꢀtheirꢀinhibitoryꢀeffectꢀonꢀ
Next,ꢀ inꢀ orderꢀ toꢀ exploreꢀ theirꢀ antitumorꢀ activity,ꢀ activeꢀ
LSD1ꢀactivityꢀwithꢀaꢀcommercializedꢀassayꢀkit.ꢀLSD1ꢀinhibi- compounds 19f, i, and l were further evaluated their anti-
tor 4ꢀwasꢀusedꢀasꢀaꢀpositiveꢀcontrol.ꢀTheꢀinhibitoryꢀactivityꢀre- proliferative effects against three human cancer cells HGC-27
sultsꢀwereꢀshownꢀinꢀTableꢀ1.ꢀWithꢀtheꢀexceptionꢀofꢀcompoundsꢀ (human gastric cancer cells), HCT-116 (human colon carcino-
19b, d, e, and k,ꢀ mostꢀ ofꢀ theꢀ compoundsꢀ exhibitedꢀ moderateꢀ maꢀcells),ꢀandꢀLP-1ꢀ(humanꢀmyelomaꢀcells)ꢀbyꢀ3-(4,5-dimeth-
to good potency with IC50ꢀ valuesꢀ rangingꢀ fromꢀ submicro- ylthiazol-2-yl)-2,5-diphenyltetrazoliumꢀbromideꢀ(MTT)ꢀassaysꢀ
molar to single-digit micromolar. Among them, compounds inꢀ vitro, and 4 was used as a positive control. The results
19f, i, and l (IC50s=0.93–2.1 µM) were more potent than or (shownꢀ inꢀ Tableꢀ 3)ꢀ illustratedꢀ theꢀ IC50 values of active com-
comparableꢀ toꢀ positiveꢀ controlꢀ 4 (IC50=1.8 µM). Particularly, pounds against each tumor cell line. All the tested compounds
compound 19l showed the most potent activity to LSD1, which showedꢀ significantꢀ antiproliferativeꢀ activities,ꢀ whichꢀ wereꢀ
was nearly 2 times higher than that of 4.
similar or stronger anticancer activities than positive control 4.
Asꢀ LSD1ꢀ belongsꢀ toꢀ theꢀ MAOꢀ family,ꢀ theꢀ highlyꢀ potentꢀ Among these compounds, compound 19l (IC50s=5.3–13.4 µM)
LSD1ꢀinhibitorsꢀ19f, i, and l were chosen to assay its homolo- exhibitedꢀ higherꢀ antiproliferativeꢀ activitiesꢀ thanꢀ theꢀ otherꢀ
giesꢀ MAO-Aꢀ andꢀ MAO-Bꢀ toꢀ assessꢀ theirꢀ selectivityꢀ profilesꢀ target compounds against three tumor cells, and its IC50 value
usingꢀ commerciallyꢀ availableꢀ kits.ꢀ Asꢀ shownꢀ inꢀ Tableꢀ 2,ꢀ theꢀ against HCT-116 cells was nearly 3-fold less than those of 4
inhibitoryꢀeffectsꢀofꢀcompoundsꢀ19f, i, and lꢀonꢀMAO-Aꢀwereꢀ (IC50=15.1 µM).

Vol. 63, No. 11 (2015)
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885
Fig.ꢀ 4.ꢀ Immunoblotꢀ Analysisꢀ ofꢀ theꢀ Expressionꢀ ofꢀ H3K4me1/me2/me3ꢀ
and Histone H3 in Vitro
Fig.ꢀ 3.ꢀ Inhibitoryꢀ Effectsꢀ ofꢀ 19l on the Proliferation of HCT116 and
The total levels of histone H3 were used as loading control. HCT116 cells were
incubatedꢀ withꢀ orꢀ withoutꢀ 19l, or 4 at the indicated concentrations for 48h and
theꢀlevelsꢀofꢀproteinꢀexpressionꢀwereꢀdetectedꢀusingꢀH3ꢀmethylationꢀspecificꢀanti-
bodies.ꢀ Dataꢀ shownꢀ areꢀ representativeꢀ imagesꢀ ofꢀ eachꢀ proteinꢀ forꢀ threeꢀ separateꢀ
experiments.
CCD841 Cells
Theseꢀcellsꢀwereꢀincubatedꢀwithꢀtheꢀindicatedꢀconcentrationsꢀofꢀ19l for 48h. Cell
proliferationꢀwasꢀassessedꢀusingꢀtheꢀMTTꢀassay.ꢀDataꢀareꢀmean S.D.ꢀofꢀtheꢀinhibi-
tionꢀ(%)ꢀfromꢀthreeꢀindependentꢀexperiments.
Givenꢀ thatꢀ theseꢀ activeꢀ hybridsꢀ showingꢀ strongꢀ LSD1ꢀ andꢀ Theseꢀhybridꢀmoleculesꢀlinkedꢀwithꢀphenylalanineꢀorꢀproline,ꢀ
antitumorꢀ inhibitoryꢀ effects,ꢀ weꢀ wonderꢀ toꢀ knowꢀ whetherꢀ particularlyꢀ forꢀ proline,ꢀ exhibitedꢀ theꢀ outstandingꢀ inhibitoryꢀ
thoseꢀ compoundsꢀ wereꢀ toxicꢀ toꢀ theꢀ normalꢀ cells.ꢀ Soꢀ theꢀ cy- activitiesꢀtoꢀLSD1ꢀwhileꢀtheꢀhybridsꢀlinkedꢀwithꢀvalineꢀorꢀiso-
totoxicitiesꢀ ofꢀ 19l with the highest anticancer activities were leucine showed relatively weak activities.
further determined for human normal cell CCD841 and
humanꢀ colonꢀ carcinomaꢀ cellsꢀ HCT116ꢀ byꢀ MTTꢀ assay.ꢀ Asꢀ
Conclusion
shown in Fig. 3, treatment with 20µM 19lꢀ promotedꢀ aboveꢀ
Inꢀ summary,ꢀ twoꢀ seriesꢀ ofꢀ novelꢀ hybridꢀ moleculesꢀ 15a, b,
85%ꢀofꢀtheꢀinhibitionꢀinꢀHCT-116ꢀcells,ꢀwhileꢀtheꢀsameꢀtreat- and 19a–lꢀwereꢀdesignedꢀandꢀsynthesizedꢀbyꢀcouplingꢀtheꢀcar-
mentꢀ hadꢀ littleꢀ significantꢀ effectꢀ onꢀ theꢀ survivalꢀ ofꢀ CCD-841ꢀ boxylꢀ groupꢀ ofꢀ hydroxylcinnamicꢀ acidꢀ withꢀ tranylcypromine,ꢀ
cellsꢀ(lessꢀthanꢀ25%ꢀofꢀinhibition).ꢀApparently,ꢀhybridꢀ19l had and their inꢀ vitroꢀ biologicalꢀ activitiesꢀ wereꢀ evaluated.ꢀ Itꢀ wasꢀ
selectiveꢀcytotoxicityꢀtoꢀhumanꢀcancerꢀcellsꢀinꢀvitro.
discovered that most of target compounds showed promising
To get insight into the preliminarily molecular mechanism LSD1ꢀinhibitoryꢀactivities,ꢀparticularlyꢀforꢀ19f, i, and l, which
underlyingꢀtheꢀLSD1ꢀinhibitionꢀonꢀ19l-treated cells, the effects hadꢀ significantꢀ LSD1ꢀ inhibitoryꢀ andꢀ antiproliferativeꢀ activi-
of 19lꢀ onꢀ theꢀ methylationꢀ levelsꢀ ofꢀ LSD1ꢀ substratesꢀ H3K4ꢀ tiesꢀcomparableꢀtoꢀorꢀslightlyꢀstrongerꢀthanꢀpositiveꢀcontrolꢀ4
wereꢀ analyzedꢀ byꢀ Westernꢀ blotꢀ analysisꢀ inꢀ HCT116ꢀ cells.ꢀ against human carcinoma cells. Furthermore, compound 19l
Theꢀ expressionꢀ ofꢀ H3K4ꢀ methylationꢀ andꢀ histoneꢀ H3ꢀ wereꢀ exhibitedꢀsignificantꢀ selectivityꢀforꢀLSD1ꢀoverꢀ itsꢀ homologiesꢀ
examinedꢀ usingꢀ specificꢀ antibodies,ꢀ whichꢀ wereꢀ incubatedꢀ MAO-Aꢀ andꢀ MAO-B.ꢀ Inꢀ addition,ꢀ compoundꢀ 19l also could
withꢀ theꢀ vehicleꢀ aloneꢀ (dimethylꢀ sulfoxideꢀ (DMSO)),ꢀ 19l (1.0 dose-dependently increase the level of H3K4me2 in HCT116
or 5.0µM), or 4 (5.0 µM).ꢀ Asꢀ canꢀ beꢀ seenꢀ inꢀ Fig.ꢀ 4,ꢀ afterꢀ 48ꢀhꢀ cells. Therefore, our novel work could serve as a foundation
treatment with 19l, there was a dose-dependent increase in the forꢀfurtherꢀexplorationꢀofꢀnovelꢀLSD1ꢀinhibitorsꢀforꢀtheꢀtreat-
H3K4me2ꢀ expression,ꢀ whichꢀ wasꢀ slightlyꢀ strongerꢀ thanꢀ thatꢀ ment of cancer.
of 4 at the same concentration of 5.0µM. However, there were
noꢀ significantꢀ reproducibleꢀ changesꢀ inꢀ H3K4me1,ꢀ H3K4me3,ꢀ
Experimental
and histone H3. These results validated that compound 19l
Meltingꢀ pointsꢀ wereꢀ determinedꢀ onꢀ aꢀ RDCSY-Iꢀ capil-
canꢀ specificallyꢀ inhibitꢀ LSD1ꢀ activityꢀ atꢀ theꢀ cellularꢀ levelꢀ inꢀ lary apparatus and were uncorrected. The compounds syn-
HCT116 cells. thesizedꢀ wereꢀ purifiedꢀ byꢀ columnꢀ chromatographyꢀ usingꢀ
Structure–Activity Relationship (SAR) Analysis of silicaꢀ gelꢀ (200–300ꢀ mesh)ꢀ exceptꢀ forꢀ recrystallizationꢀ andꢀ
SARꢀrevealedꢀthatꢀtheꢀinhibitoryꢀactivityꢀofꢀtranylcypromine/ TLC using silica gel 60 F254ꢀ platesꢀ (250ꢀmm;ꢀ Qingdaoꢀ Oceanꢀ
ferulicꢀ acidꢀ hybridsꢀ 19a–f were more potent against LSD1 Chemicalꢀ Company,ꢀ China).ꢀ Infraredꢀ (IR)ꢀ spectraꢀ (KBr)ꢀ
than that of tranylcypromine/p-hydroxycinnamicꢀ acidꢀ hybridsꢀ wereꢀ recordedꢀ onꢀ aꢀ Nicoletꢀ Impactꢀ 410ꢀ instrumentꢀ (KBrꢀ pel-
1
19g–l,ꢀ whichꢀ suggestedꢀ thatꢀ theꢀ electron-donatingꢀ substitu- let). H-NMRꢀ spectraꢀ wereꢀ recordedꢀ withꢀ aꢀ Brukerꢀ Avanceꢀ
tionsꢀ (OCH3)ꢀ onꢀ theꢀ ferulicꢀ acidꢀ derivativesꢀ mayꢀ contributeꢀ 300ꢀMHzꢀ spectrometerꢀ atꢀ 300ꢀK,ꢀ usingꢀ tetramethylsilaneꢀ
toꢀ theirꢀ inhibitoryꢀ activitiesꢀ toꢀ theseꢀ molecules.ꢀ Secondary,ꢀ (TMS)ꢀ asꢀ anꢀ internalꢀ standard.ꢀ MSꢀ spectraꢀ wereꢀ recordedꢀ
hybridsꢀwithꢀaminoꢀacidꢀlinkerꢀsuchꢀasꢀglycine,ꢀphenylalanine,ꢀ onꢀ aꢀ Marinerꢀ Massꢀ Spectrumꢀ (electrosprayꢀ ionizationꢀ (ESI)).ꢀ
orꢀ proline,ꢀ displayedꢀ slightlyꢀ strongerꢀ inhibitoryꢀ effectsꢀ onꢀ Element analysis was performed on an Eager 300 instrument.
LSD1ꢀactivityꢀthanꢀhybridsꢀ15a and b without linker. Among 4-(2-Aminocyclopropyl)aniline 10 and compounds 12a and b
these amino acid linkers, different amino acid fragments con- wereꢀcommerciallyꢀavailable.ꢀAllꢀsolventsꢀwereꢀreagentꢀgradeꢀ
nectedꢀ betweenꢀ tranylcypromineꢀ andꢀ hydroxycinnamicꢀ acidsꢀ and,ꢀ whenꢀ necessary,ꢀ wereꢀ purifiedꢀ andꢀ driedꢀ byꢀ standardsꢀ
wereꢀalsoꢀcrucialꢀforꢀtheirꢀLSD1ꢀinhibitoryꢀactivitiesꢀinꢀvitro. methods.ꢀ Solutionsꢀ afterꢀ reactionsꢀ andꢀ extractionsꢀ wereꢀ con-

886
Chem. Pharm. Bull.
Vol. 63, No. 11 (2015)
centrated using a rotary evaporator operating at a reduced N, 7.39. Found: C, 60.08; H, 6.27; N, 7.31.
pressure of ca.ꢀ20ꢀTorr.ꢀOrganicꢀsolutionsꢀwereꢀdriedꢀoverꢀan-
hydrous sodium sulfate.
(E)-2-(3-(4-(Methoxymethoxy)phenyl)acrylamido)acetic
Acid (17a) To a solution of 13a (0.2g, 0.96mmol) in dry
tert-Butyl (2-(4-Aminophenyl)cyclopropyl)carbamate (11) CH2Cl2 (10mL), methyl 2-aminoacetate (0.10g, 1.12mmol),
To a solution of 10 (3.0g, 20mmol) and 1MꢀNaOHꢀ(15ꢀmL)ꢀinꢀ EDCIꢀ (0.27ꢀg,ꢀ 1.44ꢀmmol)ꢀ andꢀ catalyticꢀ amountsꢀ ofꢀ DMAPꢀ
acetoneꢀ(30ꢀmL)ꢀatꢀ0°CꢀwasꢀaddedꢀBoc2Oꢀ(4.4ꢀg,ꢀ20ꢀmmol)ꢀbyꢀ wasꢀ addedꢀ andꢀ theꢀ mixtureꢀ wasꢀ stirredꢀ atꢀ roomꢀ temperatureꢀ
slow dropwise. The solution was allowed to warm to room overnight. Then 20mL of CH2Cl2ꢀwasꢀaddedꢀandꢀtheꢀmixtureꢀ
temperatureꢀ andꢀ stirredꢀ forꢀ 10ꢀh,ꢀ andꢀ thenꢀ extractedꢀ withꢀ was washed with water (30mL×3)ꢀ andꢀ brine.ꢀ Theꢀ organicꢀ
AcOEtꢀ(30×3ꢀmL).ꢀTheꢀorganicꢀextractsꢀwereꢀcombined,ꢀdriedꢀ phaseꢀ wasꢀ driedꢀ overꢀ anhydrousꢀ sodiumꢀ sulfate,ꢀ filteredꢀ andꢀ
withꢀ anhydrousꢀ sodiumꢀ sulfate,ꢀ filtered,ꢀ andꢀ concentratedꢀ toꢀ evaporated inꢀ vacuo,ꢀ andꢀ theꢀ crudeꢀ productꢀ wasꢀ purifiedꢀ byꢀ
afford 2.8g of 11ꢀinꢀ56%ꢀyield.ꢀMSꢀ(ESI)ꢀm/z=249ꢀ[M+H]+.
column chromatography to give 16a, which was then dis-
(E)-3-(4-(Methoxymethoxy)phenyl)acrylic Acid (13a) solved in 5mL methanol containing 1.5mL 2Mꢀ NaOH.ꢀ Theꢀ
4-Hydroxycinnamicꢀ acidꢀ 12a (1.64g, 10mmol) was dissolved reactionꢀwasꢀstirredꢀandꢀrefluxedꢀforꢀ3ꢀh,ꢀandꢀthenꢀcooled.ꢀTheꢀ
in acetonitrile (20mL), and then NaH (0.36g, 15mmol) was solvent was evaporated inꢀ vacuo, the residue was neutralized
addedꢀ atꢀ 0°C.ꢀ Theꢀ mixtureꢀ wasꢀ stirredꢀ forꢀ 10ꢀminꢀ andꢀ thenꢀ to pH=5 with 1Mꢀ HCl.ꢀ Theꢀ precipitateꢀ wasꢀ filtered,ꢀ washedꢀ
addedꢀ MOMClꢀ (0.96ꢀg,ꢀ 12ꢀmmol)ꢀ inꢀ acetonitrileꢀ (5ꢀmL)ꢀ andꢀ with water, and dried in vacuum to afford 17aꢀ inꢀ yieldꢀ 69%.ꢀ
stirred for another 3h. After the reaction was completed, the MSꢀ(ESI)ꢀm/z=266ꢀ[M+H]+.
solvent was removed under reduced pressure. The crude resi-
(S,E)-2-(3-(4-(Methoxymethoxy)phenyl)acrylamido)pro-
due was dissolved in water and washed with ether (10×3mL). panoic Acid (17b)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ obtainedꢀ fromꢀ
Theꢀwaterꢀ layerꢀ wasꢀ acidifiedꢀ withꢀ 1ꢀM HCl to pH 4, and the 13a and (S)-methyl 2-aminopropanoate according to the
precipitationꢀ wasꢀ collectedꢀ byꢀ filtrationꢀ toꢀ giveꢀ 13a, yeild synthetic procedure of 17aꢀ inꢀ yieldꢀ 67%.ꢀ MSꢀ (ESI)ꢀ m/z=280
71%.ꢀMSꢀ(ESI)ꢀm/z=209ꢀ[M+H]+.
(E)-3-(3-Methoxy-4-(methoxymethoxy)phenyl)acrylic
[M+H]+.
(S,E)-2-(3-(4-(Methoxymethoxy)phenyl)acrylamido)-
Acid (13b)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ obtainedꢀ fromꢀ ferulicꢀ 3-phenylpropanoic Acid (17c)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ ob-
acid 12bꢀandꢀMOMClꢀaccordingꢀtoꢀtheꢀsyntheticꢀprocedureꢀofꢀ tained from 13a and (S)-methyl 2-amino-3-phenylpropanoate
13aꢀinꢀyieldꢀ67%.ꢀMSꢀ(ESI)ꢀm/z=239ꢀ[M+H]+.
according to the synthetic procedure of 17aꢀinꢀyieldꢀ66%.ꢀMSꢀ
(E)-N-(4-(2-Aminocyclopropyl)phenyl)-3-(4-hydroxy- (ESI) m/z=356ꢀ[M+H]+.
phenyl)acrylamide Hydrochloride (15a)ꢀ ꢀAꢀ mixtureꢀ ofꢀ
(S,E)-2-(3-(4-(Methoxymethoxy)phenyl)acrylamido)-
compounds 11 (0.25g, 1.01mmol) and 13a (0.2g, 0.96mmol), 3-methylbutanoic Acid (17d)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ ob-
EDCIꢀ(0.27ꢀg,ꢀ1.44ꢀmmol)ꢀandꢀcatalyticꢀamountsꢀofꢀDMAPꢀinꢀ tained from 13a and (S)-methylꢀ 2-amino-3-methylbutanoateꢀ
CH2Cl2 (10mL) was stirred at room temperature overnight. according to the synthetic procedure of 17aꢀinꢀyieldꢀ66%.ꢀMSꢀ
Then 20mL of CH2Cl2ꢀwasꢀaddedꢀandꢀtheꢀmixtureꢀwasꢀwashedꢀ (ESI) m/z=368ꢀ[M+H]+.
with water (30mL×3)ꢀandꢀbrine.ꢀTheꢀorganicꢀphaseꢀwasꢀdriedꢀ
(S,E)-2-(3-(4-(Methoxymethoxy)phenyl)acrylamido)-
overꢀanhydrousꢀsodiumꢀsulfate,ꢀ filteredꢀ andꢀ evaporatedꢀtoꢀaf- 3-methylpentanoic Acid (17e) The title compound was
fordꢀ theꢀ crudeꢀ product,ꢀ whichꢀ wasꢀ thenꢀ purifiedꢀ byꢀ columnꢀ obtainedꢀ fromꢀ 13a and (2S,3R)-methyl 2-amino-3-methylpen-
chromatography to give compound 14a. Then compound 14a tanoate according to the synthetic procedure of 17a in yield
wasꢀdissolvedꢀinꢀ15ꢀmLꢀofꢀsaturatedꢀHClꢀinꢀdryꢀAcOEtꢀandꢀtheꢀ 63%.ꢀMSꢀ(ESI)ꢀm/z=322ꢀ[M+H]+.
reaction was stirred at room temperature for 8h. The solvent
(S,E)-1-(3-(4-(Methoxymethoxy)phenyl)acryloyl)pyrro-
was evaporated inꢀ vacuo to give compound 15a,ꢀ yieldꢀ 61%.ꢀ lidine-2-carboxylic Acid (17f) The title compound was
Analytical data for 15a:ꢀ IRꢀ (KBr,ꢀ cm−1): 3465, 3225, 2926, obtainedꢀ fromꢀ 13a and (S)-methylꢀ pyrrolidine-2-carboxylateꢀ
1
1712, 1629, 1585, 1241, 1192; H-NMRꢀ (DMSO-d6,ꢀ 300ꢀMHz,ꢀ according to the synthetic procedure of 17aꢀinꢀyieldꢀ61%.ꢀMSꢀ
δꢀppm)ꢀ10.29ꢀ(brꢀs,ꢀ1H,ꢀPhNHCO),ꢀ8.34ꢀ(brꢀs,ꢀ3H,ꢀNH3Cl), 7.70 (ESI) m/z=306ꢀ[M+H]+.
(d, J=6.9Hz, 2H, ArH), 7.59 (d, 2H, J=6.9Hz, Ar-H), 7.40
(E)-2-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)acryl-
(d, 1H, J=16.2Hz, ArCH=), 7.13–7.18 (m, 4H, Ar-H), 6.55 (d, amido)acetic Acid (17g)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ obtainedꢀ
1H, J=16.2Hz, CH=CH-Ar), 2.78 (m, 1H, CHN), 2.27 (m, from 13b and methyl 2-aminoacetate according to the synthet-
1H, PhCH), 1.35 (m, 1H, CHH cyclopropane), 1.23 (m, 1H, ic procedure of 17aꢀinꢀyieldꢀ65%.ꢀMSꢀ(ESI)ꢀm/z=296ꢀ[M+H]+.
CHHꢀcyclopropane);ꢀMSꢀ(ESI)ꢀm/z=330ꢀ[M]+. Anal. Calcd for
(S,E)-2-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)acryl-
C18H19ClN2O2: C, 65.35; H, 5.79; N, 8.47. Found: C, 65.28; H, amido)propanoic Acid (17h)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ ob-
5.92; N, 8.41. tained from 13b and (S)-methyl 2-aminopropanoate according
(E)-N-(4-(2-Aminocyclopropyl)phenyl)-3-(4-hydroxy- to the synthetic procedure of 17aꢀ inꢀ yieldꢀ 67%.ꢀ MSꢀ (ESI)ꢀ
3-methoxyphenyl)acrylamide Hydrochloride (15b) The m/z=310ꢀ[M+H]+.
titleꢀcompoundꢀwasꢀobtainedꢀfromꢀcompoundsꢀ11 and 13b ac-
(S,E)-2-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)-
cording to the synthetic procedure of 15aꢀinꢀyieldꢀ57%.ꢀAna- acrylamido)-3-phenylpropanoic Acid (17i) The title com-
lytical data for 15b:ꢀIRꢀ(KBr,ꢀcm−1): 3448, 3220, 2933, 1710, poundꢀ wasꢀ obtainedꢀ fromꢀ 13b and (S)-methyl 2-amino-
1618, 1570, 1245, 1183; 1H-NMRꢀ(DMSO-d6,ꢀ300ꢀMHz,ꢀδ ppm) 3-phenylpropanoate according to the synthetic procedure of
10.21ꢀ(brꢀs,ꢀ1H,ꢀPhNHCO),ꢀ8.31ꢀ(brꢀs,ꢀ3H,ꢀNH3Cl), 7.68 (d, 2H, 17aꢀinꢀyieldꢀ63%.ꢀMSꢀ(ESI)ꢀm/z=386ꢀ[M+H]+.
J=6.9Hz, ArH), 7.41 (d, 1H, J=16.2Hz, ArCH=), 7.13–7.22
(S,E)-2-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)-
(m, 5H, Ar-H), 6.61 (d, 1H, J=16.2Hz, CH=CH-Ar), 2.75 (m, acrylamido)-3-methylbutanoic Acid (17j) The title com-
1H, CHN), 2.28 (m, 1H, PhCH), 1.33 (m, 1H, CHH cyclopro- poundꢀ wasꢀ obtainedꢀ fromꢀ 13b and (S)-methyl 2-amino-
pane), 1.19 (m, 1H, CHHꢀ cyclopropane);ꢀ MSꢀ (ESI)ꢀ m/z=360 3-methylbutanoateꢀaccordingꢀtoꢀtheꢀsyntheticꢀprocedureꢀofꢀ17a
[M]+. Anal. Calcd for C19H21ClN2O3·H2O:ꢀ C,ꢀ 60.24;ꢀ H,ꢀ 6.12;ꢀ inꢀyieldꢀ60%.ꢀMSꢀ(ESI)ꢀm/z=338ꢀ[M+H]+.

Vol. 63, No. 11 (2015)
Chem. Pharm. Bull.
887
(S,E)-2-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)- 3446, 3234, 2938, 1724, 1617, 1575, 1252, 1181; 1H-NMRꢀ
acrylamido)-3-methylpentanoic Acid (17k) The title com- (DMSO-d6,ꢀ 300ꢀMHz,ꢀ δꢀ ppm)ꢀ 10.15ꢀ (brꢀs,ꢀ 1H,ꢀ NHCO),ꢀ 8.19ꢀ
poundꢀ wasꢀ obtainedꢀ fromꢀ 13b and (2S,3R)-methyl 2-amino- (brꢀs,ꢀ 3H,ꢀ NH3Cl), 7.66 (d, 2H, J=6.9Hz, ArH), 7.57 (m, 2H,
3-methylpentanoate according to the synthetic procedure of Ar-H), 7.39 (d, 1H, J=16.2Hz, ArCH=), 7.13–7.17 (m, 4H,
17aꢀinꢀyieldꢀ62%.ꢀMSꢀ(ESI)ꢀm/z=352ꢀ[M+H]+.
Ar-H), 6.58 (d, 1H, J=16.2Hz, CH=CH-Ar), 4.65 (m, 1H,
(S,E)-1-(3-(3-Methoxy-4-(methoxymethoxy)phenyl)ac- NCHCO),ꢀ 2.73ꢀ (m,ꢀ 1H,ꢀ CHN), 2.66 (m, 1H, NCHCH), 2.27
ryloyl)pyrrolidine-2-carboxylic Acid (17l) The title com- (m, 1H, PhCH), 1.35 (m, 1H, CHH cyclopropane), 1.17 (m, 1H,
poundꢀ wasꢀ obtainedꢀ fromꢀ 13b and (S)-methyl pyrrolidine- CHH cyclopropane), 0.93 (d, 6H, J=7.5Hz, 2×CH3);ꢀMSꢀ(ESI)ꢀ
2-carboxylateꢀ accordingꢀ toꢀ theꢀ syntheticꢀ procedureꢀ ofꢀ 17a in m/z=429ꢀ [M]+. Anal. Calcd for C23H28ClN3O3: C, 64.25; H,
yieldꢀ57%.ꢀMSꢀ(ESI)ꢀm/z=336ꢀ[M+H]+.
(E)-N-(2-((4-(2-Aminocyclopropyl)phenyl)amino)-
6.56; N, 9.77. Found: C, 64.06; H, 6.68; N, 9.62.
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-2-((E)-3-(4-
2-oxoethyl)-3-(4-hydroxyphenyl)acrylamide Hydrochloride hydroxyphenyl)acrylamido)-3-methylpentanamide Hydro-
(19a)ꢀ ꢀTheꢀtitleꢀcompoundꢀwasꢀobtainedꢀfromꢀcompoundsꢀ11 chloride (19e)ꢀ ꢀTheꢀtitleꢀcompoundꢀwasꢀobtainedꢀfromꢀcom-
and 17a according to the synthetic procedure of 15a in yield pounds 11 and 17e according to the synthetic procedure of 15a
59%.ꢀ Analyticalꢀ dataꢀ forꢀ 19a:ꢀ IRꢀ (KBr,ꢀ cm−1): 3432, 2941, inꢀ yieldꢀ 53%.ꢀ Analyticalꢀ dataꢀ forꢀ 19e:ꢀ IRꢀ (KBr,ꢀ cm−1): 3471,
1
1
1725, 1620, 1577, 1265, 1187; H-NMRꢀ(DMSO-d6,ꢀ300ꢀMHz,ꢀ 3242, 2940, 1728, 1621, 1569, 1250, 1186; H-NMRꢀ (DMSO-
δꢀ ppm)ꢀ 10.31ꢀ (brꢀs,ꢀ 1H,ꢀ PhNHCO),ꢀ 10.16ꢀ (brꢀs,ꢀ 1H,ꢀ NHCO),ꢀ d6,ꢀ 300ꢀMHz,ꢀ δꢀ ppm)ꢀ 10.25ꢀ (brꢀs,ꢀ 1H,ꢀ NHCO),ꢀ 8.28ꢀ (brꢀs,ꢀ 3H,ꢀ
8.33ꢀ (brꢀs,ꢀ 3H,ꢀ NH3Cl), 7.71 (d, J=6.9Hz, 2H, ArH), 7.59 NH3Cl), 7.70 (d, J=6.9Hz, 2H, ArH), 7.59 (m, 2H, Ar-H), 7.40
(d, 2H, J=6.9Hz, Ar-H), 7.41 (d, 1H, J=16.2Hz, ArCH=), (d, 1H, J=16.2Hz, ArCH=), 7.13–7.18 (m, 4H, Ar-H), 6.56 (d,
7.15–7.19 (m, 4H, Ar-H), 6.57 (d, 1H, J=16.2Hz, CH=CH- 1H, J=16.2Hz, CH=CH-Ar),ꢀ4.55ꢀ(m,ꢀ1H,ꢀNCHCO),ꢀ2.76ꢀ(m,ꢀ
Ar), 3.73 (m, 2H, NCH2), 2.78 (m, 1H, CHN), 2.29 (m, 1H, 1H, CHN), 2.28 (m, 1H, PhCH), 2.17 (m, 1H, NCHCH), 1.36
PhCH), 1.36 (m, 1H, CHH cyclopropane), 1.22 (m, 1H, CHH (m, 1H, CHH cyclopropane), 1.15–1.21 (m, 4H, CHH cyclo-
cyclopropane);ꢀ MSꢀ (ESI)ꢀ m/z=387ꢀ [M]+. Anal. Calcd for propane, CH3), 0.92 (m, 3H, CH3);ꢀ MSꢀ (ESI)ꢀ m/z=443ꢀ [M]+.
C20H22ClN3O3: C, 61.93; H, 5.72; N, 10.83. Found: C, 61.76; H, Anal. Calcd for C24H30ClN3O3·0.5H2O:ꢀ C,ꢀ 63.64;ꢀ H,ꢀ 6.90;ꢀ N,ꢀ
5.85; N, 10.69.
9.28. Found: C, 63.78; H, 7.02; N, 9.36.
(E)-N-((2S)-1-((4-(2-Aminocyclopropyl)phenyl)amino)-
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-1-((E)-3-(4-hy-
1-oxopropan-2-yl)-3-(4-hydroxyphenyl)acrylamide Hydro- droxyphenyl)acryloyl)pyrrolidine-2-carboxamide Hydro-
chloride (19b)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ obtainedꢀ fromꢀ chloride (19f)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ obtainedꢀ fromꢀ
compounds 11 and 17b according to the synthetic procedure compounds 11 and 17f according to the synthetic procedure
of 15aꢀinꢀyieldꢀ55%.ꢀAnalyticalꢀdataꢀforꢀ19b:ꢀIRꢀ(KBr,ꢀcm−1): of 15aꢀinꢀyieldꢀ48%.ꢀAnalyticalꢀdataꢀforꢀ19f:ꢀIRꢀ(KBr,ꢀcm−1):
3440, 3236, 2942, 1718, 1620, 1573, 1258, 1176; 1H-NMRꢀ 3457, 3234, 2918, 1719, 1621, 1568, 1255, 1178; 1H-NMRꢀ
(DMSO-d6,ꢀ 300ꢀMHz,ꢀ δꢀ ppm)ꢀ 10.27ꢀ (brꢀs,ꢀ 1H,ꢀ PhNHCO),ꢀ (DMSO-d6,ꢀ300ꢀMHz,ꢀδꢀppm)ꢀ10.36ꢀ(brꢀs,ꢀ1H,ꢀPhNHCO),ꢀ8.37ꢀ
10.11ꢀ (brꢀs,ꢀ 1H,ꢀ PhNHCO),ꢀ 8.36ꢀ (brꢀs,ꢀ 3H,ꢀ NH3Cl), 7.71 (d, (brꢀs,ꢀ 3H,ꢀ NH3Cl), 7.72 (d, J=6.9Hz, 2H, ArH), 7.61 (d, 2H,
J=6.9Hz, 2H, ArH), 7.61 (d, 2H, J=6.9Hz, Ar-H), 7.41 (d, J=6.9Hz, Ar-H), 7.42 (d, 1H, J=16.2Hz, ArCH=), 7.17–7.19
1H, J=16.2Hz, ArCH=), 7.15-7.19 (m, 4H, Ar-H), 6.56 (d, 1H, (m, 4H, Ar-H), 6.56 (d, 1H, J=16.2Hz, CH=CH-Ar), 4.46
J=16.2Hz, CH=CH-Ar),ꢀ4.75ꢀ(m,ꢀ1H,ꢀNCHCO),ꢀ2.80ꢀ(m,ꢀ1H,ꢀ (m,ꢀ 1H,ꢀ NCHCO),ꢀ 3.48ꢀ (m,ꢀ 2H,ꢀ NCH2), 2.81 (m, 1H, CHN),
CHN), 2.28 (m, 1H, PhCH), 1.35-1.38 (m, 4H, CHH cyclopro- 2.30 (m, 1H, PhCH), 2.03 (m, 2H, NCHCH2), 1.68 (m, 2H,
pane, NCHCH3), 1.23 (m, 1H, CHHꢀcyclopropane);ꢀMSꢀ(ESI)ꢀ NCH2CH2), 1.38 (m, 1H, CHH cyclopropane), 1.24 (m, 1H,
m/z=401ꢀ [M]+. Anal. Calcd for C21H24ClN3O3: C, 62.76; H, CHHꢀcyclopropane);ꢀMSꢀ(ESI)ꢀm/z=427ꢀ[M]+. Anal. Calcd for
6.02; N, 10.46. Found: C, 62.58; H, 6.23; N, 10.35.
C23H26ClN3O3: C, 64.55; H, 6.12; N, 9.82. Found: C, 64.46; H,
(E)-N-((2S)-1-((4-(2-Aminocyclopropyl)phenyl)amino)- 6.29; N, 9.71.
1-oxo-3-phenylpropan-2-yl)-3-(4-hydroxyphenyl)acryl-
(E)-N-(2-((4-(2-Aminocyclopropyl)phenyl)amino)-
amide Hydrochloride (19c) The title compound was 2-oxoethyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide Hy-
obtainedꢀ fromꢀ compoundsꢀ 11 and 17c according to the drochloride (19g)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ obtainedꢀ fromꢀ
synthetic procedure of 15aꢀ inꢀ yieldꢀ 51%.ꢀ Analyticalꢀ dataꢀ forꢀ compounds 11 and 17g according to the synthetic procedure
19c:ꢀ IRꢀ (KBr,ꢀ cm−1): 3455, 3228, 1726, 1622, 1573, 1246, of 15aꢀinꢀyieldꢀ56%.ꢀAnalyticalꢀdataꢀforꢀ19g:ꢀIRꢀ(KBr,ꢀcm−1):
1182; 1H-NMRꢀ (DMSO-d6,ꢀ 300ꢀMHz,ꢀ δꢀ ppm)ꢀ 10.13ꢀ (brꢀs,ꢀ 3477, 3248, 2935, 1726, 1615, 1568, 1250, 1172; 1H-NMRꢀ
1H, NHCO),ꢀ 8.36ꢀ (brꢀs,ꢀ 3H,ꢀ NH3Cl), 7.69 (d, 2H, J=6.9Hz, (DMSO-d6,ꢀ 300ꢀMHz,ꢀ δꢀ ppm)ꢀ 10.35ꢀ (brꢀs,ꢀ 1H,ꢀ PhNHCO),ꢀ
ArH), 7.58 (d, 2H, J=6.9Hz, Ar-H), 7.41 (d, 1H, J=16.2Hz, 10.17ꢀ (brꢀs,ꢀ 1H,ꢀ NHCO),ꢀ 8.39ꢀ (brꢀs,ꢀ 3H,ꢀ NH3Cl), 7.77 (d, 2H,
ArCH=), 7.22–7.28 (m, 3H, Ar-H), 7.15–7.18 (m, 6H, Ar-H), J=6.9Hz, ArH), 7.43 (d, 1H, J=16.2Hz, ArCH=), 7.13–7.22
6.60 (d, 1H, J=16.2Hz, CH=CH-Ar),ꢀ 5.11ꢀ (m,ꢀ 1H,ꢀ NCHCO),ꢀ (m, 5H, Ar-H), 6.65 (d, 1H, J=16.2Hz, CH=CH-Ar), 3.76 (m,
3.09 (d, 2H, J=6.0Hz, PhCH2), 2.77 (m, 1H, CHN), 2.29 (m, 2H, NCH2), 2.83 (m, 1H, CHN), 2.32 (m, 1H, PhCH), 1.38 (m,
1H, PhCH), 1.36 (m, 1H, CHH cyclopropane), 1.21 (m, 1H, 1H, CHH cyclopropane), 1.25 (m, 1H, CHH cyclopropane);
CHHꢀ cyclopropane);ꢀ MSꢀ (ESI)ꢀ m/z=477ꢀ [M]+. Anal. Calcd MSꢀ (ESI)ꢀ m/z=417ꢀ [M]+. Anal. Calcd for C21H24ClN3O4: C,
for C27H28ClN3O3·3H2O:ꢀC,ꢀ60.95;ꢀH,ꢀ6.44;ꢀN,ꢀ7.90.ꢀFound:ꢀC,ꢀ 60.36; H, 5.79; N, 10.06. Found: C, 60.17; H, 5.93; N, 9.92.
60.78; H, 6.58; N, 7.79.
(E)-N-((2S)-1-((4-(2-Aminocyclopropyl)phenyl)amino)-
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-2-((E)-3-(4-hy- 1-oxopropan-2-yl)-3-(4-hydroxyphenyl)acrylamide Hydro-
droxyphenyl)acrylamido)-3-methylbutanamide Hydrochlo- chloride (19h)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ obtainedꢀ fromꢀ
ride (19d)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ obtainedꢀ fromꢀ com- compounds 11 and 17h according to the synthetic procedure
pounds 11 and 17d according to the synthetic procedure of of 15aꢀinꢀyieldꢀ51%.ꢀAnalyticalꢀdataꢀforꢀ19h:ꢀIRꢀ(KBr,ꢀcm−1):
1
15aꢀ inꢀ yieldꢀ 53%.ꢀ Analyticalꢀ dataꢀ forꢀ 19d:ꢀ IRꢀ (KBr,ꢀ cm−1): 3468, 3232, 1715, 1622, 1565, 1253, 1184; H-NMRꢀ (DMSO-

888
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Vol. 63, No. 11 (2015)
d6,ꢀ 300ꢀMHz,ꢀ δꢀ ppm)ꢀ 10.14ꢀ (brꢀs,ꢀ 1H,ꢀ NHCO),ꢀ 8.34ꢀ (brꢀs,ꢀ 3H,ꢀ J=6.9Hz, ArH), 7.41 (d, 1H, J=16.2Hz, ArCH=), 7.13–7.22
NH3Cl), 7.72 (d, J=6.9Hz, 2H, ArH), 7.60 (m, 2H, Ar-H), 7.41 (m, 5H, Ar-H), 6.63 (d, 1H, J=16.2Hz, CH=CH-Ar), 4.45
(d, 1H, J=16.2Hz, ArCH=), 7.11–7.18 (m, 5H, Ar-H), 6.58 (d, (m,ꢀ 1H,ꢀ NCHCO),ꢀ 3.46ꢀ (m,ꢀ 2H,ꢀ NCH2), 2.80 (m, 1H, CHN),
1H, J=16.2Hz, CH=CH-Ar),ꢀ4.77ꢀ(m,ꢀ1H,ꢀNCHCO),ꢀ2.83ꢀ(m,ꢀ 2.31 (m, 1H, PhCH), 2.04 (m, 2H, NCHCH2), 1.68 (m, 2H,
1H, CHN), 2.29 (m, 1H, PhCH), 1.36–1.42 (m, 4H, CHH cy- NCH2CH2), 1.38 (m, 1H, CHH cyclopropane), 1.23 (m, 1H,
clopropane, NCHCH3), 1.23 (m, 1H, CHHꢀcyclopropane);ꢀMSꢀ CHHꢀcyclopropane);ꢀMSꢀ(ESI)ꢀm/z=457ꢀ[M]+. Anal. Calcd for
(ESI) m/z=431ꢀ [M]+. Anal. Calcd for C22H26ClN3O4·0.5H2O:ꢀ C24H28ClN3O4: C, 62.95; H, 6.16; N, 9.18. Found: C, 63.06; H,
C, 59.93; H, 6.17; N, 9.53. Found: C, 60.04; H, 6.36; N, 9.60.
6.24; N, 9.01.
(E)-N-((2S)-1-((4-(2-Aminocyclopropyl)phenyl)amino)-
Enzyme Assayꢀ ꢀLSD1ꢀ inhibition.ꢀ Theꢀ LSD1ꢀ screeningꢀ
1-oxo-3-phenylpropan-2-yl)-3-(4-hydroxyphenyl)acryl- biochemicalꢀassayꢀkitꢀwasꢀpurchasedꢀfromꢀCaymanꢀChemicalꢀ
amide Hydrochloride (19i) The title compound was (700120). Test compounds were diluted to 20× the desired
obtainedꢀ fromꢀ compoundsꢀ 11 and 17i according to the testꢀ concentrationꢀ inꢀ 100%ꢀ DMSOꢀ andꢀ 2.5ꢀµL of the diluted
synthetic procedure of 15aꢀ inꢀ yieldꢀ 52%.ꢀ Analyticalꢀ dataꢀ drugꢀ sampleꢀ wasꢀ addedꢀ toꢀ aꢀ blackꢀ 96-wellꢀ plateꢀ (Corning,ꢀ
for 19i:ꢀ IRꢀ (KBr,ꢀ cm−1): 3435, 3218, 1732, 1618, 1576, 1252, cat. 3693). The LSD1 enzyme stock was diluted with assay
1
1171; H-NMRꢀ (DMSO-d6,ꢀ 300ꢀMHz,ꢀ δꢀ ppm)ꢀ 10.11ꢀ (brꢀs,ꢀ 1H,ꢀ buffer,ꢀ andꢀ 40ꢀµL of the diluted LSD1 enzyme was added to
NHCO),ꢀ8.30ꢀ(brꢀs,ꢀ3H,ꢀNH3Cl), 7.70 (d, 2H, J=6.9Hz, ArH), theꢀappropriateꢀwells.ꢀSubstrate,ꢀconsistingꢀofꢀhorseradishꢀper-
7.39 (d, 1H, J=16.2Hz, ArCH=), 7.23–7.28 (m, 3H, Ar-H), oxidase,ꢀH3K4me2ꢀpeptideꢀcorrespondingꢀtoꢀtheꢀfirstꢀ21ꢀaminoꢀ
7.12–7.19 (m, 7H, Ar-H), 6.59 (d, 1H, J=16.2Hz, CH=CH- acids of the N-terminal tail of histone H3, and 10-acetyl-3,7-
Ar),ꢀ 5.09ꢀ (m,ꢀ 1H,ꢀ NCHCO),ꢀ 3.10ꢀ (d,ꢀ 2H,ꢀ J=6.0Hz, PhCH2), dihydroxyphenoxazineꢀ wasꢀ thenꢀ addedꢀ toꢀ wells.ꢀ Resorufinꢀ
2.73 (m, 1H, CHN), 2.27 (m, 1H, PhCH), 1.34 (m, 1H, CHH wasꢀ analyzedꢀ onꢀ anꢀ Envisionꢀ plateꢀ readerꢀ withꢀ anꢀ excitationꢀ
cyclopropane), 1.15 (m, 1H, CHHꢀ cyclopropane);ꢀ MSꢀ (ESI)ꢀ wavelength of 530nm and an emission wavelength of 595nm
m/z=507ꢀ [M]+. Anal. Calcd for C28H30ClN3O4: C, 66.20; H, inꢀorderꢀtoꢀevaluateꢀtheꢀinhibitionꢀrateꢀofꢀtheꢀtestedꢀcompound.ꢀ
5.95; N, 8.27. Found: C, 66.08; H, 6.04; N, 8.11.
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-2-((E)-3-(4-hy- 4.0 (GraphPad Software, San Diego, CA, U.S.A.).
droxyphenyl)acrylamido)-3-methylbutanamide Hydrochlo- MAO-Aꢀ andꢀ MAO-Bꢀ inhibition.ꢀ MAO-Aꢀ andꢀ MAO-Bꢀ
The IC50 values were calculated using GraphPad Prism version
ride (19j)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ obtainedꢀ fromꢀ com- wereꢀ purchasedꢀ fromꢀ Sigmaꢀ (catalogueꢀ numberꢀ Mꢀ 7316ꢀ andꢀ
pounds 11 and 17j according to the synthetic procedure of Mꢀ7441).ꢀTheꢀMAOꢀGloꢀAssayꢀkitꢀ(Promega,ꢀcatalogueꢀnum-
15aꢀ inꢀ yieldꢀ 55%.ꢀ Analyticalꢀ dataꢀ forꢀ 19j:ꢀ IRꢀ (KBr,ꢀ cm−1): berꢀ V1402)ꢀ wasꢀ usedꢀ toꢀ measureꢀ theꢀ effectꢀ ofꢀ inhibitorsꢀ onꢀ
3459, 3228, 1716, 1621, 1568, 1246, 1178; 1H-NMRꢀ(DMSO-d6, MAO-Aꢀ andꢀ MAO-Bꢀ activity.ꢀ Theꢀ assayꢀ wasꢀ performedꢀ atꢀ
300ꢀMHz,ꢀ δꢀ ppm)ꢀ 10.28ꢀ (brꢀs,ꢀ 1H,ꢀ PhNHCO),ꢀ 8.33ꢀ (brꢀs,ꢀ 3H,ꢀ room temperature in 50µL (25µL reaction solution+25µL de-
NH3Cl), 7.70 (d, J=6.9Hz, 2H, ArH), 7.59 (d, 2H, J=6.9Hz, tection reagent) in 96 well half area white plates (Corning, cat.
Ar-H), 7.39 (d, 1H, J=16.2Hz, ArCH=), 7.12–7.18 (m, 5H, 3693)ꢀonꢀaꢀTecanꢀFreedomꢀEVOꢀliquidꢀhandlerꢀ(TecanꢀGroupꢀ
Ar-H), 6.56 (d, 1H, J=16.2Hz, CH=CH-Ar), 4.67 (m, 1H, Ltd.). 50nMꢀ MAO-Aꢀ orꢀ 125ꢀnMꢀ MAO-Bꢀ wereꢀ incubatedꢀ withꢀ
NCHCO),ꢀ 2.81ꢀ (m,ꢀ 1H,ꢀ CHN), 2.69 (m, 1H, NCHCH), 2.30 differentꢀinhibitorꢀconcentrationsꢀforꢀ15ꢀminꢀatꢀRTꢀinꢀMAO-Aꢀ
(m, 1H, PhCH), 1.36 (m, 1H, CHH cyclopropane), 1.21 (m, 1H, orꢀMAO-BꢀBufferꢀ(MAOꢀGloꢀAssayꢀkit).ꢀReactionꢀwasꢀstartedꢀ
CHH cyclopropane), 0.95 (d, 6H, J=7.5Hz, 2×CH3);ꢀMSꢀ(ESI)ꢀ with the addition of 40mMꢀ MAOꢀ substrateꢀ forꢀ MAO-Aꢀ orꢀ
m/z=459ꢀ [M]+. Anal. Calcd for C24H30ClN3O4: C, 62.67; H, 14 mMꢀ forꢀ MAO-B.ꢀ Theꢀ reactionꢀ wasꢀ stoppedꢀ afterꢀ 30ꢀminꢀ
6.57; N, 9.14. Found: C, 62.53; H, 6.75; N, 9.04.
with the detection reagent. Luminescence was measured after
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-2-((E)-3-(4- 20ꢀminꢀ incubationꢀ inꢀ theꢀ darkꢀ usingꢀ aꢀ microplateꢀ reader.ꢀ Allꢀ
hydroxyphenyl)acrylamido)-3-methylpentanamide Hydro- compounds were tested twice in duplicate and IC50 values
chloride (19k)ꢀ ꢀTheꢀ titleꢀ compoundꢀ wasꢀ obtainedꢀ fromꢀ were calculated using GraphPad Prism version 4.0 (GraphPad
compounds 11 and 17k according to the synthetic procedure Software).
of 15aꢀinꢀyieldꢀ50%.ꢀAnalyticalꢀdataꢀforꢀ19k:ꢀIRꢀ(KBr,ꢀcm−1):
Cell Culture HGC-27, HCT-116, LP-1, or human normal
3472, 3244, 2934, 1720, 1616, 1570, 1245, 1181; 1H-NMRꢀ cellꢀ lineꢀ CCD841ꢀ cellsꢀ wereꢀ maintainedꢀ inꢀ 10%ꢀ fetalꢀ bovineꢀ
(DMSO-d6,ꢀ300ꢀMHz,ꢀδꢀppm)ꢀ10.20ꢀ(brꢀs,ꢀ1H,ꢀPhNHCO),ꢀ8.28ꢀ serumꢀ (FBS)ꢀ Dulbecco’sꢀ modifiedꢀ Eagle’sꢀ mediumꢀ (DMEM)ꢀ
(brꢀs,ꢀ 3H,ꢀ NH3Cl), 7.67 (d, 2H, J=6.9Hz, ArH), 7.38 (d, 1H, mediumꢀ (Gibco,ꢀ Invitrogen),ꢀ whichꢀ wereꢀ supplementedꢀ withꢀ
J=16.2Hz, ArCH=), 7.10–7.19 (m, 5H, Ar-H), 6.58 (m, 1H, 10%ꢀ fetalꢀ calfꢀ serumꢀ (PAA,ꢀ Austria)ꢀ andꢀ antibioticsꢀ [100ꢀIU/
CH=CH-Ar), 2.71 (m, 1H, CHN),ꢀ4.53ꢀ(m,ꢀ1H,ꢀNCHCO),ꢀ2.26ꢀ mL penicillin and 100IU/mL streptomycin (Amresco)]. All of
(m, 1H, PhCH), 2.15 (m, 1H, NCHCH), 1.35 (m, 1H, CHH the cell lines were purchased from the Shanghai Institute of
cyclopropane), 1.13–1.18 (m, 4H, CHH cyclopropane, CH3), CellꢀBiologyꢀ(Shanghai,ꢀChina)ꢀandꢀwereꢀgrownꢀatꢀ37°Cꢀinꢀaꢀ
0.91 (m, 3H, CH3);ꢀMSꢀ(ESI)ꢀm/z=473ꢀ [M]+. Anal. Calcd for 5%ꢀCO2 atmosphere with medium changes every 2d.
C25H32ClN3O4·2H2O:ꢀ C,ꢀ 58.87;ꢀ H,ꢀ 7.11;ꢀ N,ꢀ 8.24.ꢀ Found:ꢀ C,ꢀ
MTT Assayꢀ ꢀTheꢀ inhibitoryꢀ effectsꢀ onꢀ cellꢀ proliferationꢀ
58.68; H, 7.23; N, 8.15.
ofꢀ testꢀ compoundsꢀ wereꢀ investigatedꢀ byꢀ theꢀ MTTꢀ method.ꢀ
(2S)-N-(4-(2-Aminocyclopropyl)phenyl)-1-((E)-3-(4- HGC-27,ꢀ HCT-116,ꢀ LP-1,ꢀ orꢀ CCD841ꢀ cellsꢀ atꢀ aꢀ finalꢀ densityꢀ
hydroxy-3-methoxyphenyl)acryloyl)pyrrolidine-2-carbox- of 1.0×104 cells/well were placed in 96-well cell plates over-
amide Hydrochloride (19l) The title compound was night and treated with or without different concentrations of
obtainedꢀ fromꢀ compoundsꢀ 11 and 17l according to the test compounds for various periods of time. During the last
synthetic procedure of 15aꢀ inꢀ yieldꢀ 47%.ꢀ Analyticalꢀ dataꢀ 4ꢀhꢀ culture,ꢀ theꢀ cellsꢀ wereꢀ exposedꢀ toꢀ MTTꢀ (5ꢀmg/mL),ꢀ andꢀ
for 19l:ꢀ IRꢀ (KBr,ꢀ cm−1): 3465, 3246, 2911, 1716, 1625, 1560, the resulting formazan crystals were dissolved in 150µL of
1252, 1172; 1H-NMRꢀ (DMSO-d6,ꢀ 300ꢀMHz,ꢀ δ ppm) 10.24 DMSOꢀ andꢀ measuredꢀ usingꢀ aꢀ spectrophotometerꢀ (Tecan)ꢀ atꢀ
(brꢀs,ꢀ 1H,ꢀ PhNHCO),ꢀ 8.35ꢀ (brꢀs,ꢀ 3H,ꢀ NH3Cl), 7.76 (d, 2H, aꢀ testꢀ wavelengthꢀ ofꢀ 570ꢀnm.ꢀ Experimentsꢀ wereꢀ conductedꢀ inꢀ

Vol. 63, No. 11 (2015)
Chem. Pharm. Bull.
889
Metzgerꢀ E.,ꢀ Schuleꢀ R.,ꢀ Eggertꢀ A.,ꢀ Buettnerꢀ R.,ꢀ Kirfelꢀ J.,ꢀ Cancer
Res., 69, 2065–2071 (2009).
ꢀ 9)ꢀ ZhaoꢀZ.ꢀK.,ꢀDongꢀP.,ꢀGuꢀJ.,ꢀChenꢀL.,ꢀZhuangꢀM.,ꢀLuꢀW.ꢀJ.,ꢀWangꢀD.ꢀ
R.,ꢀLiuꢀY.ꢀB.,ꢀTumour Biol., 34, 173–180 (2013).
triplicate.ꢀ Inhibitionꢀ rateꢀ (%)=[(Acontrol−Atreated)/Acontrol]×100%.ꢀ
The IC50 values were calculated using GraphPad Prism version
4.0 (GraphPad Software).
Western Blot Assayꢀ ꢀTheꢀ inhibitoryꢀ activityꢀ ofꢀ LSD1ꢀ
wasꢀ determinedꢀ byꢀ Westernꢀ blotꢀ assayꢀ withꢀ theꢀ totalꢀ lysatesꢀ
byꢀ radioꢀ immunoprecipitationꢀ assayꢀ (RIPA)ꢀ bufferꢀ (Sigmaꢀ
R0278)ꢀorꢀhistoneꢀpurifiedꢀwithꢀkitꢀfromꢀEpigentekꢀ(OP-0006).ꢀ
After harvested and lyzed, the cell lysates (50µg/lane) were
separatedꢀbyꢀsodiumꢀdodecylꢀsulfate-polyacrylamideꢀgelꢀelec-
trophoresisꢀ (SDS-PAGE)ꢀ (12%ꢀ Bis-trisꢀ gel)ꢀ andꢀ transferredꢀ
ontoꢀ nitrocelluloseꢀ membranes.ꢀ Concentrationꢀ ofꢀ wholeꢀ cellꢀ
lysatesꢀ andꢀ histoneꢀ extractsꢀ wereꢀ determinedꢀ usingꢀ aꢀ Microꢀ
BCAꢀ Proteinꢀ Assayꢀ Kitꢀ (Thermoꢀ Scientificꢀ 23235).ꢀ Weꢀ usedꢀ
5%ꢀ bovineꢀ serumꢀ albuminꢀ (BSA)ꢀ toꢀ blockꢀ theꢀ membraneꢀ inꢀ
theꢀcaseꢀofꢀH3K4me1/me2/me3ꢀantibodyꢀandꢀ5%ꢀfat-freeꢀmilkꢀ
forꢀ histoneꢀ H3ꢀ antibody.ꢀ Theꢀ targetꢀ proteinsꢀ wereꢀ probedꢀ
with anti-H3K4me1, anti-H3K4me2, anti-H3K4me3, and
anti-H3ꢀantibodiesꢀ(Biovision,ꢀ6864-25,ꢀ6865-25,ꢀ6866-25,ꢀandꢀ
6806-50),ꢀrespectively.ꢀTheꢀboundꢀantibodiesꢀwereꢀdetectedꢀbyꢀ
horseradishꢀ peroxidaseꢀ (HRP)-conjugatedꢀ secondꢀ antibodiesꢀ
and visualized using the enhanced chemiluminescent reagent.
10)ꢀ Wissmannꢀ M.,ꢀ Yinꢀ N.,ꢀ Müllerꢀ J.ꢀ M.,ꢀ Greschikꢀ H.,ꢀ Fodorꢀ B.ꢀ D.,ꢀ
Jenuweinꢀ T.,ꢀ Voglerꢀ C.,ꢀ Schneiderꢀ R.,ꢀ Güntherꢀ T.,ꢀ Buettnerꢀ R.,ꢀ
MetzgerꢀE.,ꢀSchüleꢀR.,ꢀNat. Cell Biol., 9, 347–353 (2007).
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Kirfel J., Carcinogenesis, 31, 512–520 (2010).
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13)ꢀ Liuꢀ K.,ꢀ Liuꢀ Y.,ꢀ Lauꢀ J.ꢀ L.,ꢀ Minꢀ J.,ꢀ Pharmacol. Ther., 151, 121–140
(2015).
14)ꢀ MaꢀL.ꢀY.,ꢀZhengꢀY.,ꢀWangꢀS.,ꢀWangꢀB.,ꢀWangꢀZ.,ꢀPangꢀL.,ꢀZhangꢀ
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(2015).
15)ꢀ MimasuꢀS.,ꢀUmezawaꢀN.,ꢀSatoꢀS.,ꢀHiguchiꢀT.,ꢀUmeharaꢀT.,ꢀYokoya-
ma S., Biochemistry, 49, 6494–6503 (2010).
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132, 6827–6833 (2010).
17)ꢀ UedaꢀR.,ꢀSuzukiꢀT.,ꢀMinoꢀK.,ꢀTsumotoꢀH.,ꢀNakagawaꢀH.,ꢀHasegawaꢀ
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Conflict of Interest Theꢀ authorsꢀ declareꢀ noꢀ conflictꢀ ofꢀ
interest.
18)ꢀ Sharmaꢀ S.ꢀ K.,ꢀ Wuꢀ Y.,ꢀ Steinbergsꢀ N.,ꢀ Crowleyꢀ M.ꢀ L.,ꢀ Hansonꢀ A.ꢀ
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22)ꢀ YangꢀM.,ꢀCulhaneꢀJ.ꢀC.,ꢀSzewczukꢀL.ꢀM.,ꢀJaliliꢀP.,ꢀBallꢀH.ꢀL.,ꢀMa-
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