Hit-to-lead optimization of a latency-associated nuclear antigen inhibitor against Kaposi's sarcoma-associated herpesvirus infections.

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