Resea ch pape
Hi - o-lead op imiza ion o a la ency-associa ed nuclea an igen
inhibi o agains Kaposi’s sa coma-associa ed he pes i us in ec ions
Philine Ki sch
a
,
b
,
c
, Saskia C. S ein
c
,
d
, Aylin Be wange
a
,
b
,
c
, Julia Rinkes
a
,
b
,
c
,
Valen in Jakob
a
,
b
,
c
, Thomas F. Schulz
c
,
d
, Ma in Emp ing
a
,
b
,
c
,
*
a
Depa men o D ug Design and Op imiza ion (DDOP), Helmhol z-Ins i u e o Pha maceu ical Resea ch Saa land (HIPS) - Helmhol z Cen e o In ec ion
Resea ch (HZI), Campus E8.1, 66123, Saa b ücken, Ge many
b
Depa men o Pha macy, Saa land Uni e si y, Campus E8.1, 66123, Saa b ücken, Ge many
c
Ge man Cen e o In ec ion Resea ch (DZIF), Pa ne Si e Hanno e -B aunschweig, 66123, Saa b ücken, Ge many
d
Ins i u e o Vi ology, Hanno e Medical School, Ca l-Neube g-S asse 1, 30625, Hanno e , Ge many
a icle in o
A icle his o y:
Recei ed 11 Feb ua y 2020
Recei ed in e ised o m
25 May 2020
Accep ed 30 May 2020
A ailable online 28 June 2020
Keywo ds:
Hi - o-lead op imiza ion
La ency-associa ed nuclea an igen (LANA)
Kaposi’s sa coma he pes i us (KSHV)
Fluo escence pola iza ion (FP)-Based
in e ac ion inhibi ion assay
Elec opho e ic mobili y shi assay (EMSA)
CuAAC
STD-NMR
abs ac
The La ency-associa ed nuclea an igen (LANA) plays a cen al ole o he la en pe sis ence o he
Kaposi’s Sa coma He pes i us (KSHV) in he human hos and helps o es ablish li elong in ec ions.
He ein, we epo ou e o s owa ds hi - o-lead gene a ion s a ing om a p e iously disco e ed LANA-
DNA inhibi o . By e he ing he i al genome o he hos nucleosomes, LANA ensu es he seg ega ion and
pe sis ence o he i al DNA du ing mi osis. LANA is also equi ed o he eplica ion o he la en i al
episome du ing he S phase o he cell cycle. We aim o inhibi he in e ac ion be ween LANA and he
i al genome o p e en he la en pe sis ence o KSHV in he hos o ganism. Medicinal chemis y-d i en
op imiza ion s udies and s uc u e-ac i i y- ela ionship in es iga ion led o he disco e y o an
imp o ed LANA inhibi o . The unc ional ac i i y o ou compounds was e alua ed using a fluo escence
pola iza ion (FP)-based in e ac ion inhibi ion assay and elec opho e ic mobili y shi assay (EMSA). E en
hough a c ys al s uc u e o he ligand p o ein complex was no a ailable, we success ully conduc ed hi
op imiza ion owa d a low mic omola p o ein-nucleic acid-in e ac ion inhibi o . Addi ionally, we
applied STD-NMR s udies o co obo a e a ge binding and o gain insigh s in o he binding o ien a ion
o ou mos po en inhibi o , p o iding oppo uni ies o u he a ional design o mo e e ficien LANA-
a ge ing an i KSHV agen s in u u e s udies.
©2020 The Au ho (s). Published by Else ie Masson SAS. This is an open access a icle unde he CC BY-
NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
1. In oduc ion
Kaposi’s Sa coma He pes i us (KSHV) is a human gamma
he pes i us and es ablishes a li elong la en in ec ion in B-cells and
endo helial cells [1,2]. The i us was iden ified as he e iological
agen o Kaposi’s Sa coma (KS) and is in ol ed in wo o he
neoplas ic diseases, mul icen ic Cas leman’s disease and pleu al
e usion lymphoma [1,3]. In heal hy indi iduals, KSHV-associa ed
diseases a e a e. Howe e , in immunosupp essed pa ien s, e.g.,
ansplan ecipien s o pa ien s wi h he acqui ed immunodefi-
ciency synd ome (AIDS), KSHV is highly oncogenic [4,5]. Howe e ,
classic KS mainly can also occu in elde ly men especially om
KSHV-endemic a eas and endemic KS in Eas and Cen al A ica [6].
The main key playe o he es ablishmen and main enance o he
la en in ec ion is he la ency-associa ed nuclea an igen (LANA)
[7e9]. I is an o igin-binding p o ein, whose C- e minal domain
binds o he i al genome and whose N- e minal egion in e ac s
simul aneously wi h hos nucleosomes [10e12]. This allows he
seg ega ion o la en i al episomes du ing mi osis and hei pa -
i ioning o daugh e cells [13]. LANA has also addi ional unc ions
like la en i al eplica ion, ansc ip ional con ol and su i al in
he hos cell [14e16]. The C- e minal DNA-binding domain (DBD) o
LANA binds he i al genome in a sequence-specific manne [17].
Loca ed on he e minal epea s (TRs) a e h ee specific LANA
binding si es (LBS), LBS1, LBS2 and LBS3. LBS1 has a hund ed old
highe a fini y o LANA compa ed o LBS2 and LBS3 [17]. In he
majo i y o KSHV-associa ed cance cells he i al genome is p e-
sen and LANA is exp essed [18]. I has been shown, ha he
*Co esponding au ho . Depa men o D ug Design and Op imiza ion (DDOP),
Helmhol z-Ins i u e o Pha maceu ical Resea ch Saa land (HIPS) - Helmhol z
Cen e o In ec ion Resea ch (HZI), Campus E8.1, 66123, Saa b ücken, Ge many..
E-mail add ess: [email p o ec ed] (M. Emp ing).
Con en s lis s a ailable a ScienceDi ec
Eu opean Jou nal o Medicinal Chemis y
jou nal homepage: h p://www.else ie .com/loca e/ejmech
h ps://doi.o g/10.1016/j.ejmech.2020.112525
0223-5234/©2020 The Au ho (s). Published by Else ie Masson SAS. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
Eu opean Jou nal o Medicinal Chemis y 202 (2020) 112525
pe sis ence o i al DNA is a ec ed by dis u bing o influencing
LANA [15]. The inhibi ion o he in e ac ion be ween LANA and i al
DNA could lead o a educ ion o loss o i al genomes in he
in ec ed cells. Today’s ea men o KSHV and KSHV-associa ed
diseases is di ficul and s ill limi ed [19,20]. I is clea , ha he e
is an u gen need o specific d ugs, which in e e e wi h no el
s eps in he KSHV li ecycle. In iew o i s cen al ole du ing la en
i al pe sis ence, LANA is conside ed o be a e y p omising a ge
o he de elopmen o specific an i i al he apeu ics agains KSHV.
In a s udy p e iously published by us in 2019, we desc ibed he
disco e y o fi s inhibi o s, which in e e e wi h he LANA-DNA
in e ac ion [11]. Fu he inhibi o sca olds ha e been iden ified
using a unc ional sc een and an in-house compound lib a y [21].
S a ing wi h a agmen -based d ug disco e y app oach, we suc-
cess ully de eloped a agmen -sized inhibi o Icapable o compe e
wi h he i al DNA (Fig. 1). Fo he e alua ion o unc ional ac i i y
o ou compounds, we used a fluo escence-pola iza ion (FP)-based
assay and elec opho e ic mobili y shi assay (EMSA) expe imen s.
Fo ou mos p omising agmen -sized inhibi o I, we obse ed an
IC
50
alue o 17 ±1
m
M in ou FP-assay using a LANA DNA binding
domain (DBD) mu an and 435 ±6
m
M in he EMSA s udies using
he wild- ype LANA C- e minal domain (CTD).
We confi med a ge binding using mic oscale he mopho esis
(MST) and sa u a ion ans e di e ence (STD)-NMR expe imen s.
Addi ionally, he STD-NMR expe imen s and molecula docking
s udies p o ided impo an in o ma ion on he pu a i e o ien a ion
o Inhibi o Iwhen bound o LANA. Based on he STD-NMR s udies
and docking esul s we sugges ed ha he ni ogen a he py idine
co e ac s as a hyd ogen bond accep o and p o ons 2, 3 and 4 a e no
in di ec in e ac ion wi h he p o ein su ace, hence hese posi ions
should be u he in es iga ed as po en ial g ow h ec o s.
Fu he mo e, wo glu amines a e p esumably in ol ed in hyd ogen-
bond in e ac ions wi h he ca boxyl g oup. Howe e , i was no clea
whe he he ca boxylic acid unc ion is necessa y o binding [11].
Based on hese findings, we emba ked on s uc u e-ac i i y
ela ionship (SAR) s udies and u he medicinal chemis y op i-
miza ion o imp o e he po ency o ou hi compounds. He ein, we
epo ou ecen ad ances in imp o ing ou LANA-DNA-
in e ac ion inhibi o s using compound Ias a s a ing poin . Un-
o una ely, ou e o s in sol ing a co-c ys al s uc u e o inhibi o I
in complex wi h LANA ha e no been success ul o da e. This en-
de s unambiguous expe imen -suppo ed s uc u e-based op imi-
za ion un easible. The e o e, we sys ema ically in es iga ed he
LANA-DNA-in e ac ion inhibi ion o new syn hesized compounds
using FP-based compe i ion assay and EMSA expe imen s as he
SAR d i e s.
2. Design concep
Based on he p e iously applied STD-NMR and docking s udies
we modified Inhibi o Iin a s ep-by-s ep manne . Inhibi o Iwas
di ided in wo egions, he benzoic acid pa Aand he py idine
co e B(Fig. 1). The iazole co e was no ye modified in o de o
exploi he obus and acile Coppe (I)-ca alyzed azide-alkyne
cycloaddi ion (CuAAC) click chemis y. Fi s , egion Awas modified
and a ia ions o he ca boxylic acid we e in oduced. As a second
s ep, we ha e modified he py idine moie y, egion B. F om ou
p e ious esul s we assumed ha he ni ogen a he py idine mo i
is essen ial o binding and unc ions as hyd ogen bond accep o .
STD-NMR da a e ealed ha P o on 1 in e ac s igh ly wi h LANA
while p o on 2 is also in close p oximi y o he p o ein su ace.
Howe e , ou docking s udies sugges ed ha he la e migh be a
leas pa ially sol en exposed. In con as , p o ons a posi ion 3
and 4 did no show di ec con ac wi h he LANA su ace acco ding
o hei weak STD-NMR e ec s. These obse a ions inspi ed us o
in es iga e posi ions 2, 3 and 4 as po en ial g ow h ec o s in he
p esen ed s udy.
Abb e ia ions
AIDS acqui ed immune deficiency synd ome
CTD C- e minal domain
DCM dichlo ome hane
DMSO dime hylsul oxide
DBD DNA binding domain
DMF dime hyl o mamide
DIPEA diisop opyle hylamine
EE e hyl ace a e
E OH e hanol
EMSA elec opho e ic mobili y shi assay
FA o mic acid
FP fluo escence pola iza ion
HPLC high p essu e liquid ch oma og aphy
HHV-8 human he pes i us 8
KS Kaposi Sa coma
KSHV Kaposi’s sa coma-associa ed he pes i us
LANA la ency-associa ed nuclea an igen
LBS LANA binding si e
LCMS liquid ch oma og aphy mass spec ome e
MeCN ace oni ile
MeOH me hanol
MST mic oscale he mopho esis
PBS phospha e-bu e ed saline
PE pe oleum benzene
STD NMR sa u a ion ans e di e ence nuclea magne ic
esonance
SPR su ace plasmon esonance
TR e minal epea
w wild- ype
Fig. 1. P e iously desc ibed LANA-DNA in e ac ion inhibi o Iand i s p edic ed binding
mode which p o ides he basis o s uc u al op imiza ion by a ional design and
g ow h ec o explo a ion.
P. Ki sch e al. / Eu opean Jou nal o Medicinal Chemis y 202 (2020) 1125252
3. Resul s and discussion
3.1. Chemis y
3.1.1. Modifica ions o egion A
3-azidopy idine 2was gene a ed by a s anda d azida ion
me hod using 3-aminopy idine 1,NaNO
2
and NaN
3
in a mix u e o
E OAc and 6M HCl [11,22]. In a second s ep, as depic ed in Scheme 1,
a ious comme cially a ailable e hynylbenzene de i a i es we e
used in a s anda d coppe -ca alyzed CuAAC eac ion wi h 3-
azidopy idine o p o ide he iazoles 3e5, 8, 10, 11, and 13 [11].
The e hyl es e 6and amide 7analogue we e gene a ed om he
ca boxylic acid 3by hionyl chlo ide-media ed ac i a ion and
subsequen ea men wi h e hanol o aq. ammonia solu ion. The
hyd olysis o 3-chlo o-4-me hyles e in e media e 8wi h NaOH in
e hanol p oduced he co esponding acid 9. The N-ace yl analogue
12 was syn hesized om amine 11 wi h ace yl chlo ide unde basic
condi ions.
3.1.2. Modifica ions o egion B
Compound 16 bea ing an addi ional CH
2
-linke be ween i-
azole and py idine co e was syn hesized s a ing om (b omo-
me hyl)benzene 14, which was con e ed o he azide 15 using
NaN
3
in DMSO [23], ollowed by a click eac ion wi h 4-
e hynylbenzoic acid. Di e en a ylazides 17a-n and 20 deco a ed
wi h a ious subs i u ions we e gene a ed by eac ion o he co -
esponding comme cially a ailable amines 18a-n and 21 wi h
NaNO
2
and NaN
3
in 6 M HCl and E OAc (Scheme 2). The subsequen
CuAAC click eac ion wi h 4-e hynylbenzoic acid p o ided he
a ge molecules 19a-n and 22. The hyd oxypy idine 19o analogue
was gene a ed om he me hoxypy idine 19k by ea ing wi h 48%
aqueous HB solu ion a 80

C.
As depic ed in Scheme 3, he syn heses o a ge compounds
ia Suzuki coupling was achie ed using wo di e en syn he ic
ou es. In ou e 1, Suzuki coupling wi h di e en comme cially
a ailable bo onic acids and halogena ed py idine-3-amines 23,
27 and 28 in p esence o Pd(P(Ph
3
)
4
achie ed phenyl-subs i u ed
py idine amines 24a-b and 29a-b in he fi s s ep. Subsequen ly,
he amines we e con e ed o he co esponding azides 25a-b
and 30a-b ollowed by a CuAAc click eac ion wi h 4-
e hynylbenzoic acid o ob ain he a ge compounds 26a-b and
31a-b. In pa allel, he al e na i e ou e 2 was es ablished o la e
s age modifica ions ia Suzuki coupling. Fi s , halogena ed py i-
dine-3-amines 32a-b we e con e ed o he co esponding azide
33a-b, ollowed by click eac ion wi h 4-e hynylbenzoa e o
ob ain he co esponding iazole in e media es 34a-b.Subse-
quen ly, phenyl-subs i u ed compounds 35a-k we e achie ed ia
Suzuki coupling using co esponding bo onic acids and
Pd(P(Ph
3
)
4
.
Finally, he hyd olysis o he es e s wi h NaOH in me hanol
p oduced he a ge ca boxylic acid compounds 36a-k. As depic ed
in Scheme 4, o he syn hesis o he se ies o py idine-phenoxy
a ge compounds 40a-e, cuppe -ca alyzed Ullmann eac ion was
used in he fi s s ep using 6-b omo-4-me hylpy idin-3-amine 37,
he co esponding phenol de i a i e o hiophenol, Cs
2
CO
3
and CuI
o ob ain he aminopy idine-phenoxy in e media es 38a-e.
The ob ained amines we e ans o med in o he co esponding
azides 39a-e as desc ibed abo e. Las s ep was a CuAAC eac ion o
azides wi h 4-e hynylbenzoic acid o ob ain he a ge compounds
40a-e. The isoquinoline 43 and quinoline 46 analogue we e syn-
hesized s a ing om isoquinoline-4-amin 41 and quinoline-3-
amin 44 by s anda d azida ion o 42 and 45, ollowed by CuAAC
click eac ion wi h 4-e hynylbenzoic acid. Fu he isoquinoline
de i a i es 50a-c we e syn hesized in a 3 s ep p ocedu e (Scheme
5). A di ec ans o ma ion o b omo isoquinolines 47a-c in o he
co esponding azides using NaN
3
, Cu(I) and Na
2
CO
3
a 85

C o e
nigh as desc ibed in li e a u e was no e ficien [24]. LCMS-guided
eac ion moni o ing showed he o ma ion he p ima y amine and
o he side p oduc s. Fo his eason, we ex ended he eac ion ime
un il we de ec ed ull con e sion in o he co esponding p ima y
amine 48a-c wi h he aim o subsequen ly ans o m hese in-
e media es in o he co esponding azides. Indeed, we achie ed
success ul azida ion (in e media es 49a-c) and CuAAC coupling,
espec i ely, using amines 48a-c and he condi ions desc ibed
abo e yielding he desi ed isoquinoline p oduc s 50a-c.
3.2. Func ional e alua ion using LANA-DNA in e ac ion inhibi ion
assays and SAR s udies
The a ge compounds we e es ed o unc ional ac i i y in he
FP-based LANA-DNA in e ac ion inhibi ion assay using LBS2 as he
p obe and an oligome iza ion-deficien LANA DBD mu an [11].
This oligome iza ion-deficien C- e minal LANA mu an (aa1008-
1146) has nine amino acid poin mu a ions: K1055E, K1138S,
K1140D, K1141D, R1039Q, R1040Q, A1121E, K1109A, and D1110A.
Fo his mu an , also in p esence o oligonucleo ides, which
ep esen he i al LANA-binding si es LBS1, LBS2 o LBS3, a high
wa e solubili y was shown [10,11,17,25]. All compounds showing
an IC
50
alues less han 250
m
M we e u he es ed in an o hog-
onal LANA-DNA in e ac ion inhibi ion assay employing EMSA
me hodology, he same LANA DBD mu an and LBS1 as p obe. As
desc ibed abo e, he la e oligo has a highe a fini y o he a ge
ende ing he EMSA expe imen a mo e s ingen ead ou o
compound e ficacy.
Fo he fi s se ies o compounds, we in es iga ed he signifi-
cance o he ca boxylic acid in he Wes e n pa o he molecule
( egion A) by a ying i s posi ion, a aching addi ional g oups o
subs i u ing i by o he pola unc ional g oups capable o
Scheme 1. Modifica ion o egion A.
a
.
a
Reagen s and condi ions: a) NaNO
2
, NaN
3
, E OAc, 6M HCl, 0 C/ , yield 50%, 2 h; b) co esponding e hynylbenzene, CuSO
4
5H
2
0, Na-Asco ba e, DIPEA, MeOH, H
2
O, , 16 h, yield
60e82%; c) 1. SOCl
2
, DMF, 60 C, 1 h, 2. E OH, DIPEA, , 16 h, yield 53%; d) 1. SOCl
2
, DMF, 60 C, 1 h, 2. NH
4
OH, , 16 h, yield 27%; e) 2 M NaOH, MeOH, , 16 h, yield 66%; ) Ac yl
chlo ide, E
3
N, DCM, DMF, , 16 h, yield 14%.
P. Ki sch e al. / Eu opean Jou nal o Medicinal Chemis y 202 (2020) 112525 3
pa icipa ing in hyd ogen bonding. The esul s a e shown in Table 1.
Mo ing he ca boxylic acid om pa a (inhibi o I) ome a po-
si ion (4) dec eases he ac i i y significan ly. Also an addi ional
chlo ine a om a ached in me a posi ion (9) lead o a comple e loss
o ac i i y. The eplacemen o he ca boxylic acid by a me hyl es e
(5), e hyl es e (6) o amide (7) was also de imen al. Fu he mo e,
mo ing om he ca boxylic acid o he me hyl alcohol (10), amine
(11), ace amide (12) o ni ile (13) also esul ed in inac i e com-
pounds. These esul s indica e ha he ca boxylic acid in pa a po-
si ion in egion A is essen ial o inhibi o y ac i i y. The e o e, we
kep he p-ca boxylic acid in egion A fixed o u he op imiza ion
s udies and ocused on he modifica ions a he py idine co e in
egion B. Fi s , we examined he e ec on inse ing a sho linke
be ween he iazole and he py idine co e (16).
This, howe e , esul ed in loss o ac i i y. As desc ibed be o e,
om p e ious STD-NMR and molecula docking expe imen s we
expec ed, ha g owing he agmen -sized Inhibi o Iin di e en
posi ions a he py idine co e ( egion B) would po en ially inc ease
po ency.
To explo e he influence o la ge s uc u al mo i s a he py i-
dine co e in posi ion 4 we in oduced a a ie y o esidues. As lis ed
in Table 2, g owing in his posi ion is accep ed and esul ed in
mode a e o po en inhibi o y e ec s in FP assay anging om IC
50
alues o 86 ±6
m
M(19a) o18±4
m
M(19c). The size o he
in oduced esidue seems o play an impo an ole. While a small
me hyl g oup is no a o able, bu accep ed (19a,IC
50
86 ±6
m
M),
u he inc easing he size om chlo ine (19b) o phenyl (19c)
imp o es IC
50
alues o 29 ±1
m
M
m
M and 18 ±4
m
M, espec i ely.
This obse a ion migh hin a a s e ic o ho e ec . The addi ional
bulky phenyl ing s ongly hinde s he o a ion o he bond be-
ween iazole and py idine and, he e o e, migh fix he ni ogen
in he py idine co e in a mo e a o able o ien a ion. In EMSA ex-
pe imen s, 4-subs i u ed compounds 19b (EMSA: 94% inhibi ion @
500
m
M) and 19c (EMSA: 100% inhibi ion @ 500
m
M) showed a
highe e ficiency compa ed o Inhibi o I(EMSA: 83% inhibi ion @
500
m
M) [11]. Addi ionally, we shi ed he ni ogen o he py idine
co e om me a (19a) o pa a posi ion (22) which esul ed in an
inac i e compound. The imp o emen s in he EMSA assay o
compounds 19b-c o e ou ini ial hi compound Iwe e no
pe ec ly mi o ed by he FP IC
50
alues, which p esumably is
oo ed in he usage o di e en DNA p obes (LBS1 s LBS2,
espec i ely).
Ne e heless, he esul s o compounds 19b and 19c we e a
majo s ep owa ds achie ing LANA inhibi o s sui able o cellula
assays and encou aged us o explo e he po en ial o g owing he
hi sca old in his di ec ion e en u he .
In he nex se ies o compounds, Inhibi o Iwas g own in po-
si ion 3 a he py idine co e by in oducing a a ie y o a oma ic
ings. As lis ed in Table 3, a small me hyl esidue in posi ion 3 (19d,
IC
50
o 45 ±5
m
M; EMSA: 78% inhibi ion @ 500
m
M) is ole a ed, bu
he fluo ina ed analogue 19j and mos o he phenyl subs i u ed
compounds 36a-d,36 -i, and 31a showed a comple e loss o only
mode a e ac i i y.
Howe e , compounds wi h an addi ional hyd oxyl unc ion
a ached o he phenyl ing (36e, IC
50
o 153 ±7
m
M, EMSA: 20%
inhibi ion @ 500
m
M) showed mode a e ac i i y.
Mo ing om a phenyl 36a o a smalle and mo e pola u anyl
esidue 36j he po ency was es o ed (IC
50
o 19 ±2
m
M). Un o -
una ely, in EMSA expe imen s we obse ed only a weak e ec (34%
inhibi ion @ 500
m
M) o his compound. In e es ingly, by a aching
an addi ional chlo ine a om in posi ion 4 and ha ing a phenyl in
posi ion 3 (31b) esul ed in a highly po en compound wi h IC
50
o
Scheme 2. Azide syn hesis and CuAAC click eac ion.
a
.
a
Reagen s and condi ions: a) NaN
3
,E
3
N, DMSO, , 16 h, yield 78%; b) 4-e hynylbenzoic acid, CuSO
4
5H
2
0, Na-Asco ba e, DIPEA, MeOH, H
2
O, , 16 h, yield 20e90%; c) NaNO
2
, NaN
3
,
E OAc, 6M HCl, 0 C/ , 2 h, yield 4e98%; d) 48% aq. HB , 80 C, 12 h, yield 86%.
P. Ki sch e al. / Eu opean Jou nal o Medicinal Chemis y 202 (2020) 1125254
38 ±3
m
M and ull inhibi ion in FP and EMSA assays, espec i ely.
These esul s u he co obo a e he no ion o a beneficial o ho
e ec .
To explo e he influence o g owing inhibi o Ia he py idine
co e in posi ion 2, a se o di e en a ge compounds was syn-
hesized (Tables 3 and 4). The di ec a achmen o a ni ile g oup o
he py idine was ole a ed (19e:IC
50
52 ±37
m
M, EMSA: n. i.).
Mo ing o chlo ine, hyd oxy o me hoxy g oup we obse ed a
significan loss in ac i i y (19 :IC
50
>250
m
M; 19o: IC
50
214 ±24
m
M and 19k: IC
50
218 ±192
m
M). Also in EMSA expe i-
men s 19j (75% inhibi ion @ 500
m
M) and 19k (11% inhibi ion @
500
m
M) did no show a significan e ec . An inc ease in ac i i y
was obse ed by in oducing bulkie subs i uen s and an addi ional
me hyl g oup o R
1
. In de ail, an unpola bulky phenyl o p-
Scheme 3. Syn hesis o a ge compounds ia Suzuki coupling using wo di e en ou es.
a
.
a
Reagen s and condi ions: a) co esponding bo onic acid, Na
2
CO
3
, Pd(P(Ph
3
)
4
, 1,4-dioxan, H
2
O, 90 C, 16 h, yield 21e75%; b) NaNO
2
, NaN
3
, E OAc, 6M HCl, 0 C/ , 2 h, yield
86e99%; c) 4-e hynyl benzoic acid, CuSO
4
5H
2
0, Na-Asco ba e, DIPEA, MeOH, H
2
O, , 16 h, yield 36e76%; d) NaNO
2
, NaN
3
, E OAc, 6M HCl, 0 C/ , 2 h, yield 34e80%; e) 4-e hynyl
benzoa e, CuSO
4
5H
2
0, Na-Asco ba e, DIPEA, MeOH, H
2
O, , 16 h, yield 63e80%; ) co esponding bo onic acid, Na
2
CO
3
, Pd(P(Ph
3
)
4
, 1,4-dioxan, H
2
O, 90 C, 16 h, yield 20e90%; g)
2 M NaOH, MeOH, , 16 h, yield 16e83%.
Scheme 4. Syn hesis o a ge compounds ia Ullmann Reac ion.
a
.
a
Reagen s and condi ions: a) co esponding phenol o hiophenol, Cs
2
CO
3
, CuI, DMF, 130 C, 16 h, yield 28e94%; b) NaNO
2
, NaN
3
, E OAc, 6M HCl, 0 C/ , 2 h, yield 70e99%; c) 4-
e hynyl benzoic acid, CuSO
4
5H
2
0, Na-Asco ba e, DIPEA, MeOH, H
2
O, , 16 h, yield 45e93%.
P. Ki sch e al. / Eu opean Jou nal o Medicinal Chemis y 202 (2020) 112525 5

chlo ophenyl was accep ed in posi ion 2 and we obse ed IC
50
alues o 36 ±5
m
M o 36k and 58 ±7
m
M o 36l and mode a e
inhibi ion in EMSA. Analogues 26a and 26b wi h pola hyd oxyl
g oups a ached a he phenyl showed good po ency wi h IC
50
Scheme 5. Syn hesis o isoquinoline de i a i es.
a
.
a
Reagen s and condi ions: a) NaNO
2
, NaN
3
, E OAc, 6M HCl, 0 C/ , 2 h, yield 68e91%; b) 4-e hynyl benzoic acid, CuSO
4
5H
2
0, Na-Asco ba e, DIPEA, MeOH, H
2
O, , 16 h, yield
25e90%; c) NaN
3
,Na
2
CO
3,
CuSO
4
5H
2
0, Na-Asco ba e, L-p oline, DMF, H
2
O, 85 C, 24 h, yield 88e97%.
Table 1
Inhibi ion ac i i ies o compounds wi h modifica ion in egion A.
Cpd R FP Assay (LBS2)
a
Cpd R FP Assay (LBS2)
IC
50
IC
50
Inhibi o I 17 ±1
m
M9n.i.
4>250
m
M10 n.i.
5n.i.
b
11 >250
m
M
6n.i. 12 n.i.
7n.i. 13 >250
m
M
a
Fluo escence-pola iza ion assay using LBS2 as p obe, da a ep esen ing a e age
o duplica es ±s anda d de ia ion.
b
No inhibi ion a 500
m
M.
Table 2
Inhibi o y ac i i ies o analogues modified in posi ion 4 eobse ing highe e fi-
ciency o 4-subs i u ed compounds.
Cpd R FP Assay (LBS2)
a
EMSA (LBS1)
b
IC
50
inhibi ion @ 500
m
M
Inhibi o I 17 ±1
m
M 82%
16 >250
m
M n.d.
d
19a 86 ±6
m
M 39%
19b 29 ±1
m
M 94%
19c 18 ±4
m
M 100%
22 n.i.
c
n.d.
a
Fluo escence-pola iza ion assay using LBS2 as p obe, da a ep esen ing a e age
o duplica es ±s anda d de ia ion.
b
Elec opho e ic mobili y shi assay using LBS1 as p obe.
c
No inhibi ion a 500
m
M.
d
No de e mined.
P. Ki sch e al. / Eu opean Jou nal o Medicinal Chemis y 202 (2020) 1125256
alues o 21 ±3
m
M and 25 ±1
m
M, espec i ely. Fu he mo e, he
e ficiency o hese wo analogues in ou EMSA s udies was high
wi h a ull inhibi ion @ 500
m
M. By a aching me hylamine (19g),
isop opylamine (19h) and anilino (19i) a posi ion 2 we obse ed an
inc ease in ac i i y om small o bigge size, whe eby he
me hylamine compound 19g was comple ely inac i e and he
anilino analogue 19i showed a mode a e ac i i y o IC
50
o 110
m
M
and 29% inhibi ion in EMSA.
Addi ionally, a se ies o compounds was syn hesized wi h a
mo e flexible and bulky phenoxy g oup in posi ion 2 (Table 5). The
phenoxy analogue 19l, simila o he aminophenyl compound 19i,
was inac i e, indica ing ha an amino linke be ween py idine and
phenyl is mo e sui able o ac i i y compa ed o he oxygen linke .
By a aching an addi ional me hylg oup in posi ion 4 (R
1
) a he
py idine co e 40a an inc ease in ac i i y compa ed o 19l was
obse ed leading o a mode a e IC
50
o 198 ±8
m
M. The fluo ina ed
analogues 40b-d also showed mode a e ac i i ies like compound
40a while he o-fluo o analogue 40b possessed he bes IC
50
o
64 ±2
m
M. Fo he m-(40c) and p-fluo o (40d) de i a i es IC
50
so
122 ±3
m
M and 134 ±2
m
M we e obse ed, espec i ely. Un o -
una ely, all hese compounds showed no e ec in EMSA
expe imen s.
Exchanging he oxygen linke by a sul u (40e,IC
50
175 ±10
m
M)
was ole a ed (compa e wi h 40a,IC
50
198 ±8
m
M). As expec ed, by
emo ing he ni ogen in he py idine co e esul ed in an inac i e
compound (19m). As onishingly, mo ing he ni ogen o he phe-
noxy esidue (19n) yielded a highly po en compound wi h an IC
50
o 19 ±1
m
M showing also ull inhibi ion in he EMSA expe imen s.
In igued by he no ion ha agmen g owing in posi ion 3 was
possible in combina ion wi h o ho-subs i uen s, we ocused ou
e o s on u he explo ing hese wo posi ions by ins alling a
connec ed s uc u al mo i . To his end, we designed and syn he-
sized isochinoline analogues (Table 6). In gene al, isoquinoline
analogues we e pleasingly e ec i e. The unsubs i u ed isochinoline
Table 3
Inhibi o y ac i i ies o analogues modified in posi ion 3. Mos de i a i es
subs i u ed in his posi ion (R
2
) showed a significan dec ease in ac i i y.
cpd R
1
R
2
FP Assay (LBS2) EMSA (LBS1)
IC
50
Inhibi ion @ 500
m
M
Inhibi o I HH 17±1
m
M 82%
19d HMe 45±5
m
M 78%
19j H F n. i. n. d.
36a H>250
m
Mn.d.
36 Hn.i. n. d.
36g H>250
m
Mn.d.
36b Hn. i. n. d.
36c H110 ±32
m
Mn.i.
36d Hn. i. n. d.
36i Hn. i. n. .d
36e H153 ±7
m
M 20%
31a H>250
m
Mn.d.
36h H>250
m
Mn.d.
36j H19 ±2
m
M 34%
31b Cl 38 ±3
m
M 100%
a
Fluo escence-pola iza ion assay using LBS2 as p obe, da a ep esen ing a e age o
duplica es ±s anda d de ia ion.
b
Elec opho e ic mobili y shi assay using LBS1 as p obe.
c
No inhibi ion a 500
m
M.
d
No de e mined.
Table 4
Inhibi o y ac i i ies o analogues modified in posi ion 2. A aching pola hyd oxyl
benzene g oups inc eases inhibi o y ac i i y.
cpd R
1
R
2
FP Assay (LBS2) EMSA (LBS1)
IC
50
Inhibi ion @ 500
m
M
Inhibi o I HH 17±1
m
M 82%
19e HCN 52±37
m
M n.i.
19 Me Cl >250
m
M n.d.
19o H OH 214 ±24
m
M 75%
19k H OMe 218 ±192
m
M 11%
36k Me 36 ±5
m
M 36%
36l Me 58 ±7
m
M 30%
26a Me 21 ±3
m
M 100%
26b Me 25 ±1
m
M 100%
19g Hn.i. n.d.
19h H>250
m
M n.d.
19i H110 ±20
m
M 29%
a
Fluo escence-pola iza ion assay using LBS2 as p obe, da a ep esen ing a e age o
duplica es ±s anda d de ia ion.
b
Elec opho e ic mobili y shi assay using LBS1 as p obe.
c
No inhibi ion a 500
m
M.
d
No de e mined.
P. Ki sch e al. / Eu opean Jou nal o Medicinal Chemis y 202 (2020) 112525 7
43 showed an FP IC
50
alue o 33 ±1
m
M and 96% inhibi ion in EMSA
expe imen s. Mo ing om isoquinoline o quinoline 46 esul ed in
a sligh loss in ac i i y compa ed o 43 (46, IC
50
70 ±34
m
M, 61%
inhibi ion in EMSA).
A aching an addi ional me hyl (50a) o chlo ine (50b) a he
isoquinoline mo i was beneficial o he inhibi o y e ec . No e-
wo hy, 50a showed he lowes FP IC
50
o 8 ±1
m
M epo ed o da e,
while 50b also possessed a decen IC
50
o 17 ±1
m
M. Fu he mo e,
in EMSA expe imen s 100% inhibi ion was de ec ed o bo h com-
pounds a 500
m
M. Finally, an isoquinoline me hyles e analogue
50c was inac i e, howe e .
3.3. Fu he cha ac e iza ion and EMSA s udies using wild- ype
LANA
Fo u he e alua ion and cha ac e iza ion he mos p omising
compounds we e selec ed. On he bases o ou esul s, we chose
compounds 19c,31b,26a-b,19n,50a, and 50b, which possessed
he bes IC
50
alues in he FP-based assay and showed s ong
inhibi o y e ec s a 500
m
M in EMSA using he oligome iza ion-
defizien LANA DBD mu an (aa1008-1146).
Fi s , hese compounds we e ini ially es ed o inhibi ion a
250
m
M using LANA DBD mu an (Fig. 2, A) o see i hey a e also
able o dis u b he LANA-DNA in e ac ion a a lowe concen a ion
in EMSA and compa ed hese esul s also wi h inhibi o I. Inhibi o I
and he compounds 19c,31b, and 26b showed no inhibi o y e ec
on he LANA
mu
-DNA in e ac ion a 250
m
M. Howe e , s ong
inhibi o y e ec s we e obse ed a his concen a ion o com-
pounds 26a,19n,50a, and 50b as obse ed by he disappea ance o
he bands o he LANA
mu
-DNA complex (uppe band, Fig. 2A).
We also de e mined he inhibi o y ac i i y o ou bes com-
pounds agains he in e ac ion be ween wild- ype LANA CTD
(aa934-1162) and i al LBS1 (Fig. 2, B) in EMSA. Ou wild- ype LANA
CTD cons uc is longe compa ed o he LANA DBD mu an and has
no mu a ions and s ill shows a su ficien solubili y in aqueous
medium also in p esence o i al LBS1. The compounds we e also
es ed a 250
m
M. Un o una ely, no inhibi o y e ec was obse ed
o compounds I,19c,31b,26a-b,19n, and 50b. Howe e , Com-
pound 50a showed a significan e ec and was able o inhibi he
in e ac ion be ween wild- ype LANA CTD and LBS1.
Fu he mo e, we i a ed he compounds showing an e ec i e
inhibi ion in EMSA using he LANA DBD mu an (Fig. 2, A), in EMSA
expe imen s using he LANA DBD mu an and LBS1 as a p obe o
de e mine he IC
50
alues. The esul s a e lis ed in Table 7 and
de ailed in o ma ion can be ound in he suppo ing in o ma ion.
As epo ed ea lie by us, inhibi o Ishowed an IC
50
in FP
assay (LBS2) o 17 ±1
m
MandanIC
50
in EMSA o 426 ±2
m
M
using LANA DBD mu an [11]. The obse ed IC
50
alues using
LBS2 o he mos p omising inhibi o s we e basically in he same
ange. Addi ionally, we also es ed he mos p omising inhibi o s
Table 5
Inhibi o y ac i i ies o Phenoxy analogues. Shi ing ni ogen o he phenoxy esidue
imp o es inhibi o y e ficiency.
cpd R
2
FP Assay (LBS2) EMSA (LBS1)
IC
50
inhibi ion @ 500
m
M
Inhibi o I 17 ±1
m
M 82%
19l n. i. n. d.
40a 198 ±8
m
Mn.i.
40b 64 ±2
m
Mn.i.
40c 122 ±3
m
Mn.i.
40d 134 ±2
m
Mn.i.
40e 175 ±10
m
Mn.i.
19m n.i. n. d.
19n 19 ±1
m
M 100%
a
Fluo escence-pola iza ion assay using LBS2 as p obe, da a ep esen ing a e age o
duplica es ±s anda d de ia ion.
b
Elec opho e ic mobili y shi assay using LBS1 as p obe.
c
No inhibi ion a 500
m
M.
d
No de e mined.
Table 6
Inhibi o y ac i i ies o Isoquinoline de i a i es. Adding an annula ed ing s uc u e
in di ec ion o iden ified g ow h ec o esul s in he mos e ficien inhibi o s o
da e.
Cpd R FP Assay (LBS2) EMSA (LBS1)
IC
50
inhibi ion @ 500
m
M
Inhibi o I 17 ±1
m
M 82%
43 33 ±1
m
M 96%
46 70 ±34
m
M 61%
50a 8±1
m
M 100%
50b 17 ±1
m
M 100%
50c >250
m
Mn.d.
a
Fluo escence-pola iza ion assay using LBS2 as p obe, da a ep esen ing a e age o
duplica es ±s anda d de ia ion;
b
Elec opho e ic mobili y shi assay using LBS1 as
p obe;
c
No inhibi ion a 500
m
M;
d
No de e mined.
P. Ki sch e al. / Eu opean Jou nal o Medicinal Chemis y 202 (2020) 1125258
in FP assay using LBS1 and LBS3, espec i ely. Compound 50a
showed a 2- old be e IC
50
o 8 9
m
Magains allLBScompa ed
o I. Fu he mo e, we could inc ease he inhibi o y ac i i y in
EMSA expe imen s using LBS1 by 7- old. As a consequence
compounds 19n and 50a a e he mos po en LANA-LBS1-
inhibi o s epo ed so a (IC
50
alues o 64 ±12
m
Mand
53 ±3
m
M). In e es ingly, compounds showing inc eased IC
50
alues o 50e60
m
M agains LBS1 and LBS3 in FP assay we e also
no e ec i e in EMSA a 250
m
M. Excep ed is howe e inhibi o I,
which showed also IC
50
alues a ound 20
m
M agains LBS1 and
LBS3, bu no e ec a 250
m
MinEMSA.
As desc ibed abo e, only compound 50a showed an inhibi o y
e ec a a concen a ion o 250
m
M in EMSA using wild- ype LANA
CTD (Fig. 3 A). A dose- esponse EMSA expe imen wi h wild- ype
LANA CTD yielded an IC
50
alue o 60 ±4
m
M(Fig. 3 B).
These esul s indica e ha compound 50a is equally po en
agains wild- ype LANA CTD and he oligome iza ion-deficien
LANA DBD mu an . In compa ison o inhibi o I(IC
50
o
Fig. 2. EMSA gels wi h inhibi o I,19c,31b,26a-b,19n,50a-b. Compounds we e es ed a a final concen a ion o 250
m
M and LBS1 was used as p obe. (A) Using an oligome iza ion-
deficien LANA DBD mu an , a s ong inhibi o y e ec (disappea ance o LANA-DNA complex band) was obse ed o compounds 26a,19n and 50a-b (B) Using wild- ype LANA CTD,
a significan inhibi o y e ec o compound 50a was obse ed.
Table 7
Compa ison o mos e ficien LANA-DNA inhibi o s.
Cpd S uc u e FP-Assay
a
IC
50
(LBS2) (LANA
mu
) FP-Assay IC
50
(LBS1) (LANA
mu
) FP-Assay IC
50
(LBS3) (LANA
mu
)EMSA
b
IC
50
(LBS1) (LANA
mu
)
Inhibi o I 17 ±1
m
M20±3
m
M19±3
m
M 426 ±2
m
M
19c 18 ±4
m
M52±2
m
M42±3
m
M n.i. a 250
m
M
c
31b 38 ±3
m
M55±7
m
M45±4
m
M n.i. a 250
m
M
26a 21 ±3
m
M30±2
m
M34±3
m
M 156 ±27
m
M
26b 25 ±1
m
M64±1
m
M63±8
m
M n.i. a 250
m
M
19n 19 ±1
m
M15±1
m
M25±1
m
M64±12
m
M
50a 8±1
m
M9±2
m
M8±1
m
M53±43
m
M
50b 17 ±1
m
M14±1
m
M15±1
m
M93±8
m
M
a
Fluo escence-pola iza ion assay using LBS1, LBS2 and LBS3 as p obe, da a ep esen ing a e age o duplica es ±s anda d de ia ion.
b
Elec opho e ic mobili y shi assay using LBS1 as p obe.
c
No inhibi ion a 250
m
M.
P. Ki sch e al. / Eu opean Jou nal o Medicinal Chemis y 202 (2020) 112525 9