International Journal of Molecular Sciences

Article The First Berberine-Based Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), an Important DNA Repair

Elizaveta D. Gladkova 1,2, Ivan V. Nechepurenko 1, Roman A. Bredikhin 1, Arina A. Chepanova 3, Alexandra L. Zakharenko 3, Olga A. Luzina 1 , Ekaterina S. Ilina 3, Nadezhda S. Dyrkheeva 3, Evgeniya M. Mamontova 3, Rashid O. Anarbaev 2,3,Jóhannes Reynisson 4 , Konstantin P. Volcho 1,2,* , Nariman F. Salakhutdinov 1,2 and Olga I. Lavrik 2,3

1 N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, 9, Akademika Lavrentieva Ave., Novosibirsk 630090, Russia; [email protected] (E.D.G.); [email protected] (I.V.N.); [email protected] (R.A.B.); [email protected] (O.A.L.); [email protected] (N.F.S.) 2 Novosibirsk State University, Pirogova str. 1, Novosibirsk 630090, Russia; [email protected] (R.O.A.); [email protected] (O.I.L.) 3 Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8, Akademika Lavrentieva Ave., Novosibirsk 630090, Russia; [email protected] (A.A..); [email protected] (A.L.Z.); [email protected] (E.S.I.); [email protected] (N.S.D.); [email protected] (E.M.M.) 4 School of Pharmacy and Bioengineering, Keele University, Hornbeam Building, Staffordshire ST5 5BG, UK; [email protected] * Correspondence: [email protected]

 Received: 31 August 2020; Accepted: 26 September 2020; Published: 28 September 2020 

Abstract: A series of berberine and tetrahydroberberine sulfonate derivatives were prepared and tested against the tyrosyl-DNA phosphodiesterase 1 (Tdp1) DNA-repair enzyme. The berberine derivatives inhibit the Tdp1 enzyme in the low micromolar range; this is the first reported berberine based Tdp1 inhibitor. A structure–activity relationship analysis revealed the importance of bromine substitution in the 12-position on the tetrahydroberberine scaffold. Furthermore, it was shown that the addition of a sulfonate group containing a polyfluoroaromatic moiety at position 9 leads to increased potency, while most of the derivatives containing an alkyl fragment at the same position were not active. According to the molecular modeling, the bromine atom in position 12 forms a hydrogen bond to histidine 493, a key catalytic residue. The cytotoxic effect of topotecan, a clinically important topoisomerase 1 inhibitor, was doubled in the cervical cancer HeLa cell line by derivatives 11g and 12g; both displayed low toxicity without topotecan. Derivatives 11g and 12g can therefore be used for further development to sensitize the action of clinically relevant Topo1 inhibitors.

Keywords: berberine; tetrahydroberberine; Tdp1 inhibitor; cancer; molecular modeling; DNA repair enzyme; SAR

1. Introduction A promising strategy to enhance the efficacy of anticancer therapy is the inhibition of various DNA repair , which counteract the effect of many anticancer drugs [1,2]. This is particularly important where resistance to chemotherapy is observed. An interesting example is the poly (ADP-ribose) polymerase (PARP), which inhibitors were studied both in combination with chemotherapeutic agents and as individual drugs. Now olaparib, rucaparib and niraparib are in clinical use for the treatment

Int. J. Mol. Sci. 2020, 21, 7162; doi:10.3390/ijms21197162 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 16 Int. J. Mol. Sci. 2020, 21, 7162 2 of 16 chemotherapeutic agents and as individual drugs. Now olaparib, rucaparib and niraparib are in clinical use for the treatment of ovarian cancers [3]. Another DNA repair enzyme, tyrosyl-DNA of ovarian cancers [3]. Another DNA repair enzyme, tyrosyl-DNA phosphodiesterase 1 (Tdp1) has phosphodiesterase 1 (Tdp1) has attracted considerable interest in the last few years mainly due to its attracted considerable interest in the last few years mainly due to its ability to repair DNA lesions caused ability to repair DNA lesions caused topoisomerase 1 (Top1) poisons, a well-established class of topoisomerase 1 (Top1) poisons, a well-established class of anticancer drugs [4]. The anticancer activity anticancer drugs [4]. The anticancer activity of Top1 poisons, camptothecin and its clinically of Top1 poisons, camptothecin and its clinically important derivatives, topotecan and irinotecan [5,6], important derivatives, topotecan and irinotecan [5,6], is based on their ability to bind to the covalent is based on their ability to bind to the covalent intermediate complex Top1/DNA and prevent the intermediate complex Top1/DNA and prevent the restoring of DNA integrity. This leads to the restoring of DNA integrity. This leads to the stabilization of the covalent bond between catalytic stabilization of the covalent bond between catalytic tyrosine (Y723) of Top1 and the 3’- end of DNA tyrosine (Y723) of Top1 and the 30- end of DNA (Figure1). The Tdp1 mechanism of action is the (Figure 1). The Tdp1 mechanism of action is the phosphotyrosyl bond hydrolysis [7], resulting in the phosphotyrosyl bond hydrolysis [7], resulting in the resumption of DNA replication and cell division. resumption of DNA replication and cell division.

Figure 1. DNA single-strand cleavage by nucleophilic attack of Tyr723 of Top1, and covalent cleavage Figure 1. DNA single-strand cleavage by nucleophilic attack of Tyr723 of Top1, and covalent complex formation. cleavage complex formation. A few classes of Tdp1 inhibitors are known such as furamidines (compound 1, Figure2), A few classes of Tdp1 inhibitors are known such as furamidines (compound 1, Figure 2), tetracyclines (compound 2), aminoglycosides (compound 3)[8,9]. Also, natural products of various tetracyclines (compound 2), aminoglycosides (compound 3) [8,9]. Also, natural products of various types have been found to inhibit Tdp1, including derivatives of bile acids 4 [10,11], of lichen metabolite types have been found to inhibit Tdp1, including derivatives of bile acids 4 [10,11], of lichen usnic acid 5a,b [12,13] and 6 [14], monoterpenoid derivatives 7 [15–18] and oxinitidine 8 [19] with metabolite usnic acid 5a,b [12,13] and 6 [14], monoterpenoid derivatives 7 [15–18] and oxinitidine 8 inhibitory activity in the micro- or submicromolar range. Importantly, the hydrazinothiazole derivative [19] with inhibitory activity in the micro- or submicromolar range. Importantly, the of usnic acid 5a [13,20] and monoterpene-substituted 4-arylcoumarin 7a [21] significantly increased hydrazinothiazole derivative of usnic acid 5a [13,20] and monoterpene-substituted 4-arylcoumarin topotecan efficacy in vivo. 7a [21] significantly increased topotecan efficacy in vivo. The aim of this study was to establish the potency of a novel structural class of natural The aim of this study was to establish the potency of a novel structural class of natural products, products, the derivatives of berberine 9 (Scheme1), which like their usnic acid counterparts the derivatives of berberine 9 (Scheme 1), which like their usnic acid counterparts are phenolic are phenolic compounds. It is known that the isoquinoline plant alkaloid berberines have many compounds. It is known that the isoquinoline plant alkaloid berberines have many beneficial beneficial physiological effects, e.g., they are hypocholesterolemic, antibacterial, hypoglycemic agents, physiological effects, e.g. they are hypocholesterolemic, antibacterial, hypoglycemic agents, antioxidants and, finally, they suppress tumor growth [22–26]. Interestingly, the sulfonate derivatives antioxidants and, finally, they suppress tumor growth [22–26]. Interestingly, the sulfonate of berberines and tetrahydroberberines have been reported as promising hypocholesterolemic derivatives of berberines and tetrahydroberberines have been reported as promising agents [27,28]. Although berberine derivatives never used as Tdp1 inhibitors, a preliminary molecular hypocholesterolemic agents [27,28]. Although berberine derivatives never used as Tdp1 inhibitors, a modeling study indicated that berberines with 9-sulfonate group would bind to Tdp1. In the present preliminary molecular modeling study indicated that berberines with 9-sulfonate group would bind study, 9-sulfonate-berberine and tetrahydroberberine derivatives 10–12 with aliphatic and aromatic to Tdp1. In the present study, 9-sulfonate-berberine and tetrahydroberberine derivatives 10-12 with substitutes were synthesized and their potency against Tdp1 was tested. To the best of our knowledge, aliphatic and aromatic substitutes were synthesized and their potency against Tdp1 was tested. To this has not been done previously. Using the HeLa cervical cancer cell line, derivatives 11g and 12g the best of our knowledge, this has not been done previously. Using the HeLa cervical cancer cell were found to be nontoxic and sensitized the cancer cells to topotecan. line, derivatives 11g and 12g were found to be nontoxic and sensitized the cancer cells to topotecan.

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FigureFigure 2. 2.Examples Examples of of established established Tdp1 Tdp1 inhibitors inhibitors1 1––88. .

2.2. ResultsResults andand DiscussionDiscussion

2.1.2.1. ChemistryChemistry SulfonatesSulfonates10 10–12-12were were synthesized synthesized in in accordance accordance with with previously previously reported reported methods methods [27]. [27]. For this For purpose,this purpose, berberine berberine9 was selectively 9 was selectively demethylated demethylated at 190 ◦C under at 190 vacuum °C under as previously vacuum describedas previously [29]. Afterdescribed treatment [29]. After with treatment HBr, berberrubine with HBr, hydrobromide berberrubine hydrobromide13 was isolated 13 inwas 89% isolated yield in (Scheme 89% yield1). Compound(Scheme 1).13 Compoundwas reduced 13 with was sodium reduced borohydride with sodium in methanol borohydride according in methanol to a procedure according described to a previouslyprocedure [30described] yielding previously tetrahydroberberrubine [30] yielding14 .tetrahydroberberrubine The bromination of compound 14. The14 withbromination a bromine of incompound dioxane solution14 with a a ffbromineorded 12-bromotetrahydroberberrubine in dioxane solution afforded 12-bromotetrahydroberberrubine15 in 52% yield. The reaction 15 of in tetrahydroderivatives52% yield. The reaction14 and of 15tetrahydroderivativeswith polyfluoroaryl and14 and alkyl 15 sulfonylchlorides with polyfluoroaryl as well and as withalkyl tosylchloridesulfonylchlorides in dichloromethane as well as with in tosylchloride the presence in of dichloromethane triethylamine produced in the presence tetrahydroberberrubine of triethylamine 9-producedO-sulfonates tetrahydroberberrubine11a–h (44–84% yields) and9- 12-bromotetrahydroberberrubineO-sulfonates 11a-h (44–84% 9-O -sulfonatesyields) 12aand–h (49–93%12-bromotetrahydroberberrubine yields). New berberine type 9-10O-sulfonatesderivatives 12a were-h synthesized(49–93% yields). by the reactionNew berberine of berberrubine type 10 hydrobromidederivatives were13 with synthesized different by alkyl the sulfochlorides. reaction of berberrubine The reactions hydrobromide were carried out 13 inwith dichloromethane different alkyl insulfochlorides. the presence of The triethylamine reactions were for 5 hcarried at room out temperature. in dichloromethane Sulfonates in10 thewere pr isolatedesence of by triethylamine precipitation fromfor 5 the hours reaction at room mixtures. temperature. Sulfonates 10 were isolated by precipitation from the reaction mixtures.

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O O O O O O O O 1) 180°C, Br 20-30 mm Hg NaBH4 2) HBr, EtOH MeOH Br2 N N 0°C N dioxane N O O O O Cl Br O OH OH OH 91314 15 RSO Cl RSO Cl RSO2Cl 2 2 NEt NEt NEt3 3 3 O CH Cl O CH Cl O CH2Cl2 2 2 2 2 O O O Br

N N N O O O O Br O O O O O S S S R R R O O O 10 11 12

10a: R=CH3; 11a: R=CH3; 12a: R=CH3; 10b: R=C2H5; 11b: R=C2H5; 12b: R=C2H5; 10c: R=C3H7; 11c: R=C3H7; 12c: R=C3H7; 10d: R=C4H9 11d: R=C4H9; 12d: R=C4H9; 11e: R=p-C6H5CH3; 12e: R=p-C6H5CH3; 11f: R=C6F5; 12f: R=C6F5; 11g: R=p-C6F4H; 12g: R=p-C6F4H; 11h: R=p-C6F4CF3 12h: R=p-C6F4CF3

Scheme 1. TheThe synthetic synthetic pathways pathways to to sulfonates sulfonates 1010, ,1111 andand 1212. .

The structures of the newnew compoundscompounds were confirmedconfirmed by 11H-NMR,1313C-NMR,C-NMR, IR IR and and HRMS HRMS methods; the results are shown in the experiment experimentalal section and the supplementary supplementary information. information.

2.2. Effects Effects of the Berberine Sulfonates Sulfonates on on Tdp1 Tdp1 Activity Activity An oligonucleotideoligonucleotide real-time real-time biosensor biosensor was was used used based based on the on ability the ofability Tdp1 toof remove Tdp1 fluorophoreto remove quenchersfluorophore from quenchers the 30-end from of DNA,the 3’-end as previously of DNA, describedas previously [31]. described The hexadecameric [31]. The hexadecameric oligonucleotide carriedoligonucleotide 5(6)-carboxyfluorescein carried 5(6)-carboxyfluorescein (FAM) at the 50-end (FAM) and at the the fluorophore 5’-end and quencher the fluorophore BHQ1 (Black quencher Hole Quencher-1)BHQ1 (Black at Hole the 3 0Quencher-1)-end. Tdp1 inhibitorsat the 3’-end. prevent Td removalp1 inhibitors of fluorophore prevent removal quenchers, of thusfluorophore reducing fluorescencequenchers, thus intensity. reducing The fluorescence results of the intensity. Tdp1 assay The results for derivatives of the Tdp110–12 assayand for cytotoxic derivatives effects 10–12 are shownandInt.Int.Int. J. J. cytotoxic J. Mol.Mol. Mol. in Sci.Sci. TablesSci. 20202020 2020 effects,1, 21, 21 and21,, x,x xare FORFOR2 FOR. shown PEERPEER PEER REVIEWREVIEW REVIEW in Tables 1 and 2. 55 5 ofof of 1616 16 It is clear from the data in Table 1 that both alkyl sulfonates of berberine and Table 1. Tdp1 inhibiting activity (IC —half maximal inhibitory concentration) of sulfonate berberine tetrahydroberberineTableTableTable 1.1. 1. Tdp1Tdp1 Tdp1 inhibitinginhibiting inhibiting are not activity activity activityvery active (IC(IC (IC50505050—half—half —halfwith maximalmaximal maximalthe exception inhibitoryinhibitory inhibitory of concentration)concentration) theconcentration) tetrahydroberberine ofof of sulfonatesulfonate sulfonate berberineberberine derivativesberberine derivatives with aliphatic substituents and their cytotoxicity (CC —half maximal cytotoxic containingderivativesderivativesderivatives both sufficientlywithwithwith aliphaticaliphaticaliphatic long substitusubstitusubstitu alkyl entsentssubstituentents andandand theirtheirtheir and cytotoxicitycytotoxicity cytotoxicitybromine in (CC(CC (CCa 50para505050—half—half—half-position maximalmaximalmaximal to the cytotoxiccytotoxiccytotoxic sulfonate concentration)concentration) in in HeLa HeLa cells. cells. Furamidine Furamidine was was used used as as a a positive positive control control (IC (IC505050= = 1.21.2 ± 0.3 µM).µM). substituent,concentration)concentration) 12c and in in12d HeLa HeLa. The cells. cells. favorable Furamidine Furamidine substitution was was used used aspattern as a apositive positive of these control control derivatives (IC (IC50 = =1.2 1.2 ± is ±0.3 0.3confirmed µM). µM). by the results of the tetrahydroberberine sulfonates with aromatic and polyfluoroaromatic substituents as shown in Table 2. Both sulfonates with para-toluenesulfonyl substituent (11e and 12e) are inactive, but all three fluorinated sulfonates (11f, 11g, 11h) show good inhibitory activity with IC50 values of Code,Code,Code,Code, ~1 µM. Bromine substitution is not important for 12e-h activity as comparison with their StructureStructureStructureStructure non-brominated analogues 11e-h shows. 11a-d11a–d11a-d 10a-d10a–d10a-d10a-d 11a-d 12a-d12a-d12a–d12a-d

RRRR ICICIC50505050,,, µM,µµM µMM CC CC CC CC50505050,, µM,µM µMµM IC IC IC505050,, µM,µM, µMµM CC CC CC505050,, µM,µM ,µMµM IC IC IC505050,, , µM,µM µµMM CC CC CC CC50505050,,, µ µM,µM µMM aa; ;Me Me >15>15 ND*ND* >15 >15 ND ND >15 >15 ND ND aa;; Me Me >>1515 ND* ND * >15>15 ND ND >15>15 ND ND bbb;; Et;Et Et >15>15>15 NDNDND >15 >15 >15 ND ND ND >15 >15 >15 ND ND ND b; Et >15 ND >15 ND >15 ND cc;c; Pr;Pr Pr >15>15>15 NDNDND >15 >15 >15 ND ND ND 2.9±1.32.9±1.32.9±1.3 >100>100>100 cd;d; Pr ;Bu Bu >>15>1515 ND NDND >15> >1515 ND ND ND 2.9 4±1.0 4±1.01.3 > >100100 >100 d; Bu >15 ND >15 ND 4±1.0± >100 d; Bu >15 ND *ND—not*ND—not*ND—not> 15 determined.determined. determined. ND 4 1.0 >100 ± BasedBasedBased on onon the thethe data datadata shown shownshown in inin Tables TablesTables* ND—not 1 11 and andand determined.2 22 it itit can cancan be bebe stated statedstated that thatthat some somesome berberine berberineberberine derivatives derivativesderivatives inhibitinhibitinhibit Tdp1.Tdp1. Tdp1. ItIt It waswas was foundfound found thatthat that thethe the structurestructure structure ofof of thethe the substituentsubstituent substituent inin in thethe the sulfonatesulfonate sulfonate affectsaffects affects thethe the inhibitoryinhibitory inhibitory activity.activity.activity. PolyfluorinatedPolyfluorinatedPolyfluorinated arylsulfonatesarylsulfonatesarylsulfonates 11f-h11f-h11f-h andandand 12f-h12f-h12f-h exhibitedexhibitedexhibited inhibitoryinhibitoryinhibitory activityactivityactivity ininin thethethe lowlowlow micromolarmicromolarmicromolar range,range,range, whilewhilewhile theitheitheirr r non-fluorinatednon-fluorinatednon-fluorinated analogsanalogsanalogs ((11e(11e11e,, , 12e12e12e)) ) werewerewere inactiveinactiveinactive atatat thesethesethese concentrations.concentrations.concentrations. In InIn the thethe series seriesseries of ofof alkylsulfonates, alkylsulfonates,alkylsulfonates, inhibitory inhibitoryinhibitory activity activityactivity was waswas found foundfound only onlyonly for forfor compounds compoundscompounds withwithwith aa a sufficientlysufficiently sufficiently longlong long alkylalkyl alkyl substituentsubstituent substituent (propyl-,(propyl-, (propyl-, butyl-)butyl-) butyl-) inin in thethe the sulfonatesulfonate sulfonate groupgroup group andand and inin in thethe the presencepresence presence ofofof brominebrominebromine substituentsubstituentsubstituent atatat positionpositionposition 121212 (compounds(compounds(compounds 12c,d12c,d12c,d).).). ToToTo explainexplainexplain thethethe observedobservedobserved effects,effects,effects, aaa molecularmolecularmolecular modelingmodeling modeling studystudy study waswas was carriedcarried carried out.out. out.

TableTableTable 2. 2.2. Tdp TdpTdp 11 1 inhibitinginhibiting inhibiting activityactivity activity (IC(IC (IC505050)) )ofof of sulfonatesulfonate sulfonate berberineberberine berberine derivativesderivatives derivatives withwith with polyfluoroaromaticpolyfluoroaromatic polyfluoroaromatic

substituentssubstituentssubstituents and andand their theirtheir cytotoxicitycytotoxicity cytotoxicity (CC (CC(CC505050)) )in inin HeLaHeLa HeLa cells.cells. cells. Furamidine FuramidineFuramidine was waswas usedused used as asas a a a positive positivepositive controlcontrol control

(IC(IC(IC505050 == = 1.21.2 1.2 ±± ± 0.30.3 0.3 µM).µM). µM).

Code,Code,Code, structurestructure structure

11e-h11e-h11e-h 12e-h12e-h12e-h RRR ICICIC505050,, ,µMµM µM CC CC CC505050,, ,µMµM µM IC IC IC505050,, ,µMµM µM CC CC CC505050,, ,µMµM µM

NDND NDND >15>15 ND >15>15 ND e;e;e; >15 >15

1.01.01.0 ±± ± 0.200.20 0.20 11 11 11 ±± ± 2.02.0 2.0 0.53 0.53 0.53 ±± ± 0.010.01 0.01 9.9 9.9 9.9 ±± ± 4.54.5 4.5

f;f;f;

1.051.051.05 ±± ± 0.050.05 0.05 >100 >100 >100 1.3 1.3 1.3 ±± ± 0.300.30 0.30 95 95 95 ±± ± 5.05.0 5.0

g;g;g;

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50 TableTable 1. 1. Tdp1 Tdp1 inhibiting inhibiting activity activity (IC (IC50—half—half maximal maximal inhibitory inhibitory concentration) concentration) of of sulfonate sulfonate berberine berberine TableTable 1. 1. Tdp1 Tdp1 inhibiting inhibiting activity activity (IC (IC5050—half—half maximal maximal inhibitory inhibitory concentration) concentration) of of sulfonate sulfonate berberine berberine 50 derivativesderivatives withwith aliphaticaliphatic substitusubstituentsents andand theirtheir cytotoxicitycytotoxicity (CC(CC50—half—half maximalmaximal cytotoxiccytotoxic derivativesderivatives withwith aliphaticaliphatic substitusubstituentsents andand theirtheir cytotoxicitycytotoxicity (CC(CC5050—half—half maximalmaximal cytotoxiccytotoxic 50 concentration)concentration) in in HeLa HeLa cells. cells. Furamidine Furamidine was was used used as as a a positive positive control control (IC (IC50 = = 1.2 1.2 ± ± 0.3 0.3 µM). µM). concentration)concentration) in in HeLa HeLa cells. cells. Furamidine Furamidine was was used used as as a a positive positive control control (IC (IC5050 = = 1.2 1.2 ± ± 0.3 0.3 µM). µM).

Code,Code, Code,Code, StructureStructure StructureStructure 10a-d 11a-d11a-d 10a-d10a-d 11a-d 12a-d12a-d 10a-d 11a-d 12a-d12a-d 50 50 50 50 50 50 RR ICIC50, ,µM µM CC CC50, ,µM µM IC IC50, ,µM µM CC CC50, ,µM µM IC IC50, ,µM µM CC CC50, ,µM µM RR ICIC5050, ,µM µM CC CC5050, ,µM µM IC IC5050, ,µM µM CC CC5050, ,µM µM IC IC5050, ,µM µM CC CC5050, ,µM µM aa; ;Me Me >15>15 ND*ND* >15 >15 ND ND >15 >15 ND ND aa; ;Me Me >15>15 ND*ND* >15 >15 ND ND >15 >15 ND ND bb; ;Et Et >15>15 NDND >15 >15 ND ND >15 >15 ND ND bb; ;Et Et >15>15 NDND >15 >15 ND ND >15 >15 ND ND cc; ;Pr Pr >15>15 NDND >15 >15 ND ND 2.9±1.32.9±1.3 >100>100 cc; ;Pr Pr >15>15 NDND >15 >15 ND ND 2.9±1.32.9±1.3 >100>100 dd; ;Bu Bu >15>15 NDND >15 >15 ND ND 4±1.0 4±1.0 >100 >100 dd; ;Bu Bu >15>15 NDND >15 >15 ND ND 4±1.0 4±1.0 >100 >100 *ND—not*ND—not determined. determined. *ND—not*ND—not determined. determined. BasedBased onon thethe datadata shownshown inin TablesTables 11 andand 22 itit cancan bebe statedstated thatthat somesome berberineberberine derivativesderivatives BasedBased onon thethe datadata shownshown inin TablesTables 11 andand 22 itit cancan bebe statedstated thatthat somesome berberineberberine derivativesderivatives inhibitinhibit Tdp1.Tdp1. It It waswas foundfound thatthat thethe structurestructure ofof thethe substituentsubstituent inin thethe sulfonatesulfonate affectsaffects thethe inhibitoryinhibitory inhibitinhibit Tdp1. Tdp1. It It was was found found that that the the structure structure of of the the substituent substituent in in the the sulfonate sulfonate affects affects the the inhibitory inhibitory activity.activity. PolyfluorinatedPolyfluorinated arylsulfonatesarylsulfonates 11f-h11f-h andand 12f-h12f-h exhibitedexhibited inhibitoryinhibitory activityactivity inin thethe lowlow activity.activity. PolyfluorinatedPolyfluorinated arylsulfonatesarylsulfonates 11f-h11f-h andand 12f-h12f-h exhibitedexhibited inhibitoryinhibitory activityactivity inin thethe lowlow micromolarmicromolar range,range, whilewhile theitheirr non-fluorinatednon-fluorinated analogsanalogs ((11e11e, , 12e12e)) werewere inactiveinactive atat thesethese micromolarmicromolar range,range, whilewhile theitheirr non-fluorinatednon-fluorinated analogsanalogs ((11e11e, , 12e12e)) werewere inactiveinactive atat thesethese concentrations.concentrations. InIn thethe seriesseries ofof alkylsulfonates,alkylsulfonates, inhibitoryinhibitory activityactivity waswas foundfound onlyonly forfor compoundscompounds concentrations.concentrations. InIn thethe seriesseries ofof alkylsulfonates,alkylsulfonates, inhibitoryinhibitory activityactivity waswas foundfound onlyonly forfor compoundscompounds withwith a a sufficiently sufficiently long long alkyl alkyl substituent substituent (propyl-, (propyl-, butyl-) butyl-) in in the the sulfonate sulfonate group group and and in in the the presence presence withwith a a sufficiently sufficiently long long alkyl alkyl substituent substituent (propyl-, (propyl-, butyl-) butyl-) in in the the sulfonate sulfonate group group and and in in the the presence presence ofof brominebromine substituentsubstituent atat positionposition 1212 (compounds(compounds 12c,d12c,d).). ToTo explainexplain thethe observedobserved effects,effects, aa Int.of J.of Mol. brominebromine Sci. 2020 ,substituent21substituent, 7162 atat positionposition 1212 (compounds(compounds 12c,d12c,d).). ToTo explainexplain thethe observedobserved effects,effects,5 of 16 aa molecularmolecular modeling modeling study study was was carried carried out. out. Int. J. Mol.molecular molecularSci. 2020, 21 ,modeling modelingx FOR PEER study studyREVIEW was was carried carried out. out. 5 of 16

Table 2. Tdp 1 inhibiting activity (IC ) of50 sulfonate berberine derivatives with polyfluoroaromatic TableTable 2.2. TdpTdp 11 inhibitinginhibiting activityactivity50 (IC(IC50)) ofof sulfonatesulfonate berberineberberine derivativesderivatives withwith polyfluoroaromaticpolyfluoroaromatic Table 1. TableTdp1Table inhibiting 2.2. TdpTdp 11 inhibitingactivityinhibiting (IC activityactivity50—half (IC (ICmaximal5050)) ofof sulfonatesulfonate inhibitory berberineberberine concentration) derivativesderivatives of sulfonate withwith polyfluoroaromaticpolyfluoroaromatic berberine substituents and their cytotoxicity (CC ) in50 HeLa cells. Furamidine was used as a positive control substituentssubstituents andand theirtheir cytotoxicitycytotoxicity 50 (CC(CC50)) inin HeLaHeLa cells.cells. FuramidineFuramidine waswas usedused asas aa positivepositive controlcontrol derivativessubstituentssubstituents with aliphatic andand theirtheir substitu cytotoxicitycytotoxicityents and (CC(CC 50their50)) inin HeLaHeLacytotoxicity cells.cells. FuramidineFuramidine (CC50—half waswas maximal usedused asas acytotoxica positivepositive controlcontrol (IC 1.250 0.3 µM). 50 (IC= 50 = 1.2 ± 0.3 µM). (IC50 = ±1.2 ± 0.3 µM). concentration)(IC(IC50 = = in1.2 1.2 HeLa ± ± 0.3 0.3 cells.µM). µM). Furamidine was used as a positive control (IC50 = 1.2 ± 0.3 µM).

Code, StructureCode,Code, Code, structure structure structure Code,Code, structure structure 10a-d 11a-d 12a-d

12e–h R IC50, µM CC50, µM IC50, µM CC50, µM IC50, µM CC50, µM 11e-h11e-h11e–h 12e-h12e-h a; Me >15 ND* 11e-h11e-h >15 ND >15 12e-h12e-h ND RRR ICICIC5050,50 ,µM µM, µM CC CC CC5050, ,,µM µMµM IC50, IC ICµM5050, ,µM µM CC50, µM CC CC5050, ,µM µM b; Et RR >15 ICICND5050, ,µM µM >15 CC CC5050, ,µM µM ND IC IC5050 >15, ,µM µM ND CC CC5050, ,µM µM c; Pr >15 ND >15 ND 2.9±1.3 >100 >15NDND >15 ND NDND d; Bu >15 ND>15 >15 NDND ND 4±1.0>15 >100 NDND e;e; >15>15 >15>15 e;e;e; >15 *ND—not determined. >15 Based on the data shown in Tables 1 and 2 it can be stated that some berberine derivatives inhibit Tdp1. It was found that the1.0 structure ± 0.20 of the substituent 11 ± 2.0 in the sulfonate 0.53 ± affects0.01 the inhibitory 9.9 ± 4.5 1.01.01.0 ± ± 0.20 0.200.20 11 11 11 ± ± 2.0 2.02.0 0.53 0.53 0.530.01 ± ± 0.01 0.01 9.9 4.5 9.9 9.9 ± ± 4.5 4.5 activity. Polyfluorinated arylsulfonates1.0 ± ±0.20 11f-h and11 12f-h ±± 2.0 exhibited inhibitory 0.53± ± 0.01 activity in± the9.9 low ± 4.5 micromolarf; f; range, while their non-fluorinated analogs (11e, 12e) were inactive at these f;f; concentrations. In the series of alkylsulfonates, inhibitory activity was found only for compounds with a sufficiently long alkyl substituent (propyl-, butyl-) in the sulfonate group and in the presence of bromine substituent at position1.05 ± 120.05 (compounds >100 12c,d ). To explain 1.3 the ± 0.30 observed effects, 95 a± 5.0 1.051.051.05 ± ± 0.05 0.050.05 >100 >100>100 1.3 1.3 1.30.30 ± ± 0.30 0.30 95 5.0 95 95 ± ± 5.0 5.0 molecular modeling study was 1.05carried ± ±0.05 out. >100 ± 1.3 ± 0.30 ± 95 ± 5.0 Int. g;J.g; Mol. Sci. 2020, 21 , x FOR PEER REVIEW 6 of 16 g;g;g; Table 2. Tdp 1 inhibiting activity (IC50) of sulfonate berberine derivatives with polyfluoroaromatic

substituents and their cytotoxicity (CC50) in HeLa cells. Furamidine was used as a positive control

(IC50 = 1.2 ± 0.3 µM). 0.90.9 ± 0.200.20 2.6 2.6 ± 0.10.1 1.4 1.40.30 ± 0.30 2.2 1.5 2.2 ± 1.5 ± ± ± ±

h;h;

Code, 2.3.structureIt is Molecular clear from Modeling the data of in the Table Berberine1 that bothDerivatives alkyl sulfonates of berberine and tetrahydroberberine are not veryTwenty-three active with berberine the exception derivatives of were the tetrahydroberberine docked into the binding derivatives site of Tdp1 containing (PDB ID: both 6DIE, sufficiently long alkyl substituent and bromine in a para-position to the sulfonate substituent, resolution 1.78 Å) [32] with three water molecules (HOH814, 821 and 1078). It is established that 12c and 12d. The favorable substitution pattern of these derivatives is confirmed by the results keeping these crystalline water11e-h molecules improves the prediction12e-h quality of the scaffold of the(for tetrahydroberberine further information sulfonates see the Methodology with aromatic sectio andn) polyfluoroaromatic [13]. The binding substituentspredictions of as the shown scoring R IC50, µM CC50, µM IC50, µM CC50, µM in Tablefunctions2. Both used sulfonates are given with in Tablepara-toluenesulfonyl S1, all the ligands substituent show reasonable (11e and scores.12e) are inactive, but all three fluorinatedCompound sulfonates 12f is the (11f ligand, 11g, 11h with) show the best good IC inhibitory50 value and activity according with IC to50 thevalues docking; of ~1 µ12fM. fits ND ND Bromine substitution is not important for 12e–h activity as comparison with their non-brominated e; neatly in the catalytic>15 region as shown in Figure 3A. This region>15 contains the catalytic histidine 263 analoguesand 49311e–h aminoshows. acid residues thus the ligand blocs any activity of the enzyme. Indeed, 12f forms a weakBased H-bond on the datawith shown the His493 in Tables imidazole1 and2 itsite can group be stated via the that bromine some berberine substituent derivatives as shown inhibit in Figure Tdp1.3B Itas was well found as with that Asn283’s the structure amide of side the substituentchain. Finally, in thethe sulfonateamide group affects of theAsn516 inhibitory forms activity.an H-bond 1.0 ± 0.20 11 ± 2.0 0.53 ± 0.01 9.9 ± 4.5 Polyfluorinatedwith one of the arylsulfonates oxygen atoms11f–h in andthe 1.3-benzodioxo12f–h exhibitedleinhibitory moiety of activitythe ligand. in the It is low worth micromolar mentioning f; range,that while the methoxy their non-fluorinated group on 12f analogscan potentially (11e, 12e form) were a weak inactive H-bond at these with concentrations. the thiol on Cys205, In the if the seriesflexibility of alkylsulfonates, of the inhibitory would activity be accounted was found for, only and for the compounds same methoxy with agroup sufficiently has lipophilic long alkylcontacts substituent with (propyl-,Ile285 aliphatic butyl-) side in the chain, sulfonate stabilizing group the and binding in the mode. presence Interestingly, of bromine the substituent modeling of at positionthe 11f 12ligand, (compounds 1.05which ± 0.05 does12c ,notd). contain To explain >100 a bromine the observed group, e 1.3butffects, ± also 0.30 a molecularwith a good modeling IC 9550 ±value, 5.0 study did wasnot give carriedconsistent out. results across the scoring functions used, i.e., different conformations were predicted. g; This strongly indicates that the bromine group with its weak H-bonds to Asn283 and His493 is essential for anchoring the ligand in the catalytic site. Interestingly, derivatives 11e and 12e are essentially inactive (IC50 >15 µM); they are structural analogues of the 11f/12f pair with hydrogens on the phenyl ring as well as a para methyl substitution instead of fluorine groups. According to the modeling, the 11e/12e pair has a different binding mode from 11f/12f with the bromine moiety pointing into the aqueous phase for the former pair as shown in Figure S1 in the Supplementary Information. This indicates the importance of the fluorine substitution on the phenyl rings; the modeling suggests that the phenyl ring is leaning against the carboxyl moiety in the Gly458 backbone, which can form an interaction between the electron deficient fluoride substituted ring and the lone pairs of the oxygen atom in the carboxylic group as shown in Figure 3B.

Figure 3. The docked configuration of 12f in the binding site of Tdp1 as predicted by the ChemPLP scoring function. (A) The protein surface is rendered; blue depicts a hydrophilic region with a partial positive charge on the surface; red depicts hydrophobic region with a partial negative charge and

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 16

0.9 ± 0.20 2.6 ± 0.1 1.4 ± 0.30 2.2 ± 1.5

h;

2.3. Molecular Modeling of the Berberine Derivatives Twenty-three berberine derivatives were docked into the binding site of Tdp1 (PDB ID: 6DIE, resolution 1.78 Å) [32] with three water molecules (HOH814, 821 and 1078). It is established that keeping these crystalline water molecules improves the prediction quality of the docking scaffold (for further information see the Methodology section) [13]. The binding predictions of the scoring functionsInt. J. Mol. Sci.used2020 are, 21 ,given 7162 in Table S1, all the ligands show reasonable scores. 6 of 16 Compound 12f is the ligand with the best IC50 value and according to the docking; 12f fits neatly in the catalytic region as shown in Figure 3A. This region contains the catalytic histidine 263 and2.3. Molecular493 amino Modeling acid residues of the Berberinethus the Derivativesligand blocs any activity of the enzyme. Indeed, 12f forms a weakTwenty-three H-bond with berberinethe His493 derivatives imidazole weresite group docked via intothe bromine the binding substituent site of Tdp1 as shown (PDB in ID: Figure 6DIE, 3Bresolution as well as 1.78 with Å) Asn283’s [32] with amide three waterside chain. molecules Finally, (HOH814, the amide 821 group and of 1078). Asn516 It isforms established an H-bond that withkeeping one theseof the crystalline oxygen atoms water in moleculesthe 1.3-benzodioxo improvesle the moiety prediction of the qualityligand. ofIt is the worth docking mentioning scaffold that(for the further methoxy information group on see 12f the can Methodology potentially form section) a weak [13]. H-bond The binding with the predictions thiol on Cys205, of the scoring if the flexibilityfunctions usedof the are protein given inwould Table S1,be allaccounted the ligands for, show and reasonablethe same methoxy scores. group has lipophilic contacts with Ile285 aliphatic side chain, stabilizing the binding mode. Interestingly, the modeling of Compound 12f is the ligand with the best IC50 value and according to the docking; 12f fits neatly thein the 11f catalytic ligand, which region does as shown not contain in Figure a bromine3A. This group, region but contains also with the a catalytic good IC histidine50 value, did 263 not and give 493 consistentamino acid results residues across thus thethe ligand scoring blocs functions any activity used, of i.e., the enzyme.different Indeed,conformations12f forms were a weak predicted. H-bond Thiswith strongly the His493 indicates imidazole that site the group bromine via the group bromine with substituent its weak asH-bonds shown into Figure Asn2833B asand well His493 as with is essentialAsn283’s for amide anchoring side chain. the ligand Finally, in the the amide catalytic group site. of Asn516 forms an H-bond with one of the oxygen atomsInterestingly, in the 1.3-benzodioxole derivatives 11e moiety and of12e the are ligand. essentially It is worthinactive mentioning (IC50 >15 µM); that thethey methoxy are structural group analogueson 12f can of potentially the 11f/12f form pair awith weak hydrogens H-bond withon the the phenyl thiol onring Cys205, as well if as the a para flexibility methyl of substitution the protein insteadwould beof accountedfluorine groups. for, and According the same methoxyto the modeling, group has the lipophilic 11e/12e pair contacts has a with different Ile285 binding aliphatic mode side fromchain, 11f stabilizing/12f with the bromine binding mode.moiety Interestingly,pointing into the aqueous modeling phase of the for11f theligand, former which pair as does shown not in Figure S1 in the Supplementary Information. This indicates the importance of the fluorine contain a bromine group, but also with a good IC50 value, did not give consistent results across the substitutionscoring functions on the used, phenyl i.e., rings; different the conformationsmodeling sugg wereests that predicted. the phenyl This ring strongly is leaning indicates against that the the carboxylbromine groupmoiety with in the its weakGly458 H-bonds backbone, to Asn283 which and can His493 form isan essential interaction for anchoringbetween the ligandelectron in deficientthe catalytic fluoride site. substituted ring and the lone pairs of the oxygen atom in the carboxylic group as shown in Figure 3B.

FigureFigure 3. TheThe docked docked configuration configuration of of 12f12f inin the the binding binding site site of of Tdp1 Tdp1 as as predicted predicted by by the the ChemPLP ChemPLP scoringscoring function. function. ( (AA)) The The protein protein surface surface is is rendered; rendered; blue blue depicts depicts a a hydrophilic hydrophilic region region with with a a partial partial positivepositive charge charge on on the the surface; red red depicts hydrop hydrophobichobic region region with with a a partial negative negative charge charge and and

grey shows neutral areas. The ligand occupies the catalytic pocket blocking access to it. (B) H-bonds are shown as green lines between 12f and the amino acids Asn283, His493 and Asn516 side chains. A potential lone pair-π stacking interaction is shown as a blue line between the carboxylic backbone group in Gly458 and the centroid (green ) of the fluorinated phenyl group (3.5 Å).

Interestingly, derivatives 11e and 12e are essentially inactive (IC50 >15 µM); they are structural analogues of the 11f/12f pair with hydrogens on the phenyl ring as well as a para methyl substitution instead of fluorine groups. According to the modeling, the 11e/12e pair has a different binding mode from 11f/12f with the bromine moiety pointing into the aqueous phase for the former pair as shown in Figure S1 in the Supplementary Information. This indicates the importance of the fluorine substitution on the phenyl rings; the modeling suggests that the phenyl ring is leaning against the carboxyl moiety in Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 16

Int. J. Mol. Sci. 2020, 21, 7162 7 of 16 grey shows neutral areas. The ligand occupies the catalytic pocket blocking access to it. (B) H-bonds are shown as green lines between 12f and the amino acids Asn283, His493 and Asn516 side chains. A the Gly458potential backbone, lone pair- whichπ stacking can forminteraction an interaction is shown betweenas a blue thelineelectron between deficientthe carboxylic fluoride backbone substituted ringgroup and the in Gly458 lone pairs and ofthe the centroid oxygen (green atom ball) in of the the carboxylic fluorinated group phenyl as group shown (3.5 in Å). Figure 3B. To investigate the binding stability of the ligands molecular dynamic (MD) runs were conducted usingTo the investigate docked conformations the binding ofstability11f, 12f ofand the12e ligandsfor 10 psmolecular at 1000 K.dynamic In general, (MD) the runs ligands were are conductedstable within using the the binding docked pocket conformations and not ejected; of 11f the, 12f most and stable 12e for intramolecular 10 ps at 1000 bond K. In being general, between the ligandsthe fluorinated are stable phenyl within ring the and binding the Gly458 pocket carboxyl and no groupt ejected; for 12f the. In most contrast, stable the intramolecular phenyl group inbond12e beingis very between mobile. the The fluorinated other H-bonding phenyl interactions ring and the predicted Gly458 are carboxyl often broken group to for be 12f reestablished. In contrast, during the phenylthe MD group run. in 12e is very mobile. The other H-bonding interactions predicted are often broken to be reestablished during the MD run. 2.4. Cytotoxicity 2.4. Cytotoxicity Top1 poisons are used as anticancer drugs for the treatment for various oncological diseases [33–35]. SinceTop1 Tdp1 poisons is involved are inused the as removal anticancer of DNA drugs damage for the caused treatment by Top1 for poisons, various the oncological activity of Tdp1diseases can [33–35].lead to theSince development Tdp1 is involved of drug in resistance the removal [36 ].of Thus,DNA itdamage is believed caused that by Tdp1 Top1 inhibition poisons, canthe enhanceactivity ofthe Tdp1 efficacy can lead of Top1 to the poisons development [37]. Tdp1 of drug inhibitors resistan shouldce [36]. have Thus, the it lowestis believed possible that intrinsicTdp1 inhibition toxicity canto minimize enhance potentialthe efficacy side of e ffTop1ects. Therefore,poisons [37]. we studiedTdp1 inhibitors the intrinsic should cytotoxicity have the of lowest the compounds possible intrinsicagainst HeLatoxicity cells to minimize (cervical potential carcinoma). side EZ4U effects. cell Therefore, proliferation we studied and cytotoxicity the intrinsic assay cytotoxicity results areof theshown compounds in Figure 4against for the HeLa ligands cells with (cervical Tdp1 inhibitory carcinoma). activity. EZ4U cell proliferation and cytotoxicity assay results are shown in Figure 4 for the ligands with Tdp1 inhibitory activity.

FigureFigure 4. 4. TheThe berberine berberine derivatives’ derivatives’ cytotoxicity cytotoxicity acco accordingrding to to the the EZ4U EZ4U test. Error Error bars bars show show standardstandard deviations. deviations.

TheThe results results show show that that 9-O-sulfonates 9-O-sulfonates of of 12-bromo 12-bromotetrahydoberberinetetrahydoberberine with with aliphatic aliphatic substituents substituents 12c12c andand 12d12d,, (Table (Table 11 andand FigureFigure4 4,, black black and and red red traces) traces) are are non-toxic non-toxic upup toto 100100 µM.µM. The The toxicity toxicity of of 9-O-sulfonates9-O-sulfonates strongly strongly depends on the structure ofof thethe polyfluorinatedpolyfluorinated fragment.fragment. CompoundsCompounds with with a aCF CF3-group3-group in in the thepara para-position-position were were the the most most toxic; toxic; CC CC50 values50 values of 2.6of 2.6µM µM and and 2.2 2.2µM µM for 11hfor 11hand and12h, 12h,respectively respectively (Table (Table2 and Figure2 and4 , greenFigure and 4, browngreen traces).and brown The replacementtraces). The of replacement the trifluoromethyl of the trifluoromethylgroup with a fluorine group atomwith ina fluorine the para-position atom in the reduced para-position toxicity withreduced CC50 toxicityvalues with of 10 CCµM50 for values11f and of 1012f µM(Table for 211f and and Figure 12f (Table4, blue 2 andand magentaFigure 4, traces,blue and respectively). magenta traces, The respectively). compounds with The acompounds hydrogen withatom a inhydrogen the para-position atom in the were para non-toxic-position ( 11gwere, Table non-toxic2 and ( Figure11g, Table4, violet 2 and trace) Figure or moderately4, violet trace) toxic or moderately(12g, Table2 toxic and Figure(12g, Table4, orange 2 and trace). Figure 4, orange trace).

2.5.2.5. Sensitizing Sensitizing Effects Effects TheThe sensitizingsensitizing e ffeffectect of theof berberinethe berberine inhibitors inhibitors on topotecan’s on topotecan’s cytotoxic potentialcytotoxic waspotential investigated. was investigated.In order to determine In order the to optimaldetermine concentration the optimal for concentration the inhibitors tofor provide the inhibitors the maximum to provide sensitizing the maximumeffect, but remainingsensitizing non-toxic, effect, but their remaining concentrations non-toxic, were varied their withconcentrations topotecan concentration were varied of 2withµM, its CC for HeLa cells. Topotecan significantly increased the cytotoxicity of compounds 11g, 12g, topotecan50 concentration of 2 µM, its CC50 for HeLa cells. Topotecan significantly increased the

Int. J. Mol. Sci. 2020, 21, 7162 8 of 16

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 16 and 11f, the reliability was confirmed by the Mann–Whitney U-test, p = 0.05 and the results are shown incytotoxicity Figure5. The of compounds original data 11g are, given12g, and in Table 11f, S2.the reliability was confirmed by the Mann–Whitney U-test, p = 0.05 and the results are shown in Figure 5. The original data are given in Table S2.

FigureFigure 5.5. TheThe influenceinfluence ofof 22 µµMM topotecantopotecan onon Tdp1Tdp1 inhibitors’inhibitors’ cytotoxicity.cytotoxicity. TheThe unshadedunshaded histogramhistogram barsbars denotedenote cellcell viabilityviability inin thethe presencepresence ofof aa Tdp1Tdp1 inhibitor.inhibitor. TheThe hatchedhatched histogramhistogram barsbars indicateindicate cellcell viabilityviability inin the the presence presence of a combinationof a combination of a Tdp1 of inhibitora Tdp1 withinhibitor 2 µM with of topotecan. 2 µM of The topotecan. concentration The µ ofconcentration the Tdp1 inhibitor of the Tdp1 in M inhibitor is given in under µM is each given pair under of bars. each Error pair bars of bars. show Error standard bars show deviations. standard deviations. 11g is non-toxic in the concentration range used; 100% of living cells with a 100 µM maximum concentration. In the presence of topotecan, a significant decrease in cell survival is observed (~30%) at 11g is non-toxic in the concentration range used; 100% of living cells with a 100 µM maximum all concentrations. Compound 12g has a low toxicity potential of 95 µM (CC ). Topotecan weakly, concentration. In the presence of topotecan, a significant decrease in cell survival50 is observed (~30%) but significantly reduces this value to 62 µM. Compound 11f is inherently toxic, and 90% of the cells die at all concentrations. Compound 12g has a low toxicity potential of 95 µM (CC50). Topotecan weakly, µ 11f atbut 20 significantlyM. At lower reduces concentrations, this value theto 62 eff µM.ect of Compound topotecan is11f significant. is inherently CC toxic,50 value and for 90% ofdecreases the cells three fold, from 11 to 3.7 µM, in the presence of topotecan. For other ligands (11h, 12c, d, f, h), the effect die at 20 µM. At lower concentrations, the effect of topotecan is significant. CC50 value for 11f ofdecreases topotecan three was fold, negligible from 11 or to unreliable. 3.7 µM, in the presence of topotecan. For other ligands (11h, 12c, d, f, µ h), theNon-toxic effect of topotecan concentrations was negligible of the berberine or unreliable. derivatives (5 M) were then tested at different 11f concentrationsNon-toxic of concentrations topotecan. The of most the toxicberberine compound derivativescaused (5 µM) 20% cellwere death then at tested this concentration; at different theconcentrations rest of the compoundsof topotecan. were The not most toxic. toxic In general, compound our Tdp111f inhibitorscaused 20% doubled cell death the cytotoxic at this potentialconcentration; of topotecan the rest as of canthe becompounds seen in Figure were6 notand to Tablexic. In3. general, our Tdp1 inhibitors doubled the cytotoxic potential of topotecan as can be seen in Figure 6 and Table 3. Table 3. The influence of the Tdp1 inhibitors at 5 µM on the cytotoxic potential of topotecan (Tpc).

Compounds CC50, µM–5 µM Tdp1 inhibitor CC50, µM–20 µM Tdp1 inhibitor Tpc 6.8 1.1 ± Tpc + 11g 3.5 0.6 2.3 0.5 ± ± Tpc + 12g 2.9 0.4 3.3 1.1 ± ± Tpc + 11f 1.7 0.3 not determined ±

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 16

120 Tpc + DMSO 100 Tpc + 11g Tpc + 12g Int. J. Mol. Sci. 2020, 21, 7162 80 Tpc + 11f 9 of 16 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 16 60

12040 Tpc + DMSO

HeLa cell viability, % viability, HeLacell 10020 Tpc + 11g Tpc + 12g 800 Tpc + 11f

0246810 60 Topotecan, μM 40 Figure 6. The influence of the Tdp1 inhibitors at 5 µM on topotecan cytotoxicity. Error bars show

standard deviations.% viability, HeLacell 20

Table 3. The influence0 of the Tdp1 inhibitors at 5 µM on the cytotoxic potential of topotecan (Tpc).

Compounds CC50, µM - 5 µM Tdp1 inhibitor CC50, µM - 20 µM Tdp1 inhibitor 0246810 Tpc 6.8 ± 1.1 Topotecan, μM Tpc + 11g 3.5 ± 0.6 2.3 ± 0.5 FigureFigure 6.Tpc6. TheThe + 12g influenceinfluence ofof thethe Tdp1Tdp12.9 inhibitorsinhibitors ± 0.4 atat 55µ µMM on on topotecan topotecan cytotoxicity. cytotoxicity. 3.3 ± 1.1 ErrorError barsbars showshow standardstandardTpc deviations. deviations. + 11f 1.7 ± 0.3 not determined

µ 11f ForForTable comparison, comparison, 3. The influence the the concentration concentration of the Tdp1 inhibitors of of the the inhibitorsinhibitors at 5 µM on was was the increased cytotoxicincreased potential to to 20 20 µM.M. of Compoundtopotecan Compound (Tpc). 11f was was not used due to its high toxicity. Again, compounds 12g and 11g had a significant effect, p < 0.05, not used Compoundsdue to its high CC toxicity.50, µM -Again, 5 µM Tdp1compounds inhibitor 12g CCand50 ,11g µM had - 20 aµM significant Tdp1 inhibitor effect, p < 0.05, confirmedconfirmed byby thethe Mann–WhitneyMann–Whitney U-testU-test (Figure(Figure7 7and and Table Table3). 3). It It is is interesting interesting to to note note that that the the sensitizing effectTpc of Tdp1 inhibitors was practically the 6.8 same ± 1.1 at 5 and 20 µM. sensitizingTpc effect + 11g of Tdp1 inhibitors 3.5was ± practically0.6 the same at 5 and 20 2.3 µM. ± 0.5 Tpc + 12g 2.9 ± 0.4 3.3 ± 1.1 Tpc + 11f 1.7 ± 0.3 not determined

For comparison, the concentration of the inhibitors was increased to 20 µM. Compound 11f was not used due to its high toxicity. Again, compounds 12g and 11g had a significant effect, p < 0.05, confirmed by the Mann–Whitney U-test (Figure 7 and Table 3). It is interesting to note that the sensitizing effect of Tdp1 inhibitors was practically the same at 5 and 20 µM.

FigureFigure 7.7. InfluenceInfluence of of Tdp1 Tdp1 inhibitors inhibitors at 20 at µM 20 onµM to onpotecan topotecan cytotoxicity. cytotoxicity. Error bars Error show bars standard show standarddeviations. deviations.

DerivativesDerivatives11g 11gand and12g 12gcan canbe beconsidered consideredto tobe bethe thelead leadcompounds compounds sincesince theythey are,are, unlikeunlike 11f11f,, non-toxicnon-toxic ( 11g(11g)) or or moderately moderately toxic toxic ( 12g(12g)) and and have have a a pronounced pronounced sensitizing sensitizing e ffeffectect on on topotecan. topotecan. 2.6. Chemical Space

The calculated molecular descriptors MW (molecular weight), log P (water-octanol partition Figure 7. Influence of Tdp1 inhibitors at 20 µM on topotecan cytotoxicity. Error bars show standard coefficient), HD (hydrogen bond donors), HA (hydrogen bond acceptors), PSA (polar surface area) and deviations. 1 RB (rotatable bonds)) are given in Table S3. The MW of the ligands lies between 325.4 and 684.4 g mol− and falls into drug-like and Known Drug Space (KDS) regions. Log P spans from 1.6 to 5.4, i.e., over the Derivatives 11g and 12g can be considered to be the lead compounds since they are, unlike 11f, non-toxic (11g) or moderately toxic (12g) and have a pronounced sensitizing effect on topotecan.

Int. J. Mol. Sci. 2020, 21, 7162 10 of 16 three defined volumes in chemical space, with most in lead-like chemical space and only one ligand 12h in KDS. The HD, HA, RB and PSA values are within the lead- and drug-like definitions (for the definition of lead-like, drug-like and Known Drug Space regions see ref. [38] and Table S4). The Known Drug Indexes (KDIs) for the ligands were calculated to gauge the balance of the molecular descriptors (MW, log P, HD, HA, PSA and RB). This method is based on the analysis of drugs in clinical use, i.e., the statistical distribution of each descriptor is fitted to a function and normalized to 1 resulting in a weighted index. Both the summation of the indexes (KDI2a—Equation (1)) and multiplication (KDI2b—Equation (2)) methods were used [39]. The numerical results are given in Table S3 in the supplementary information.

KDI2a = IMW + Ilog P + IHD+ IHA + IRB + IPSA (1)

KDI = I I I I I I (2) 2b MW × log P × HD× HA × RB × PSA The KDI2a values range from 4.35 to 5.70 with a theoretical maximum of 6 and the average of 4.08 for known drugs. KDI2b range from 0.06 to 0.73, with a theoretical maximum of 1 and with KDS average of 0.18. The berberine ligands can be considered reasonably well balanced in terms of their molecular descriptors and therefore biocompatible. The most active compound 12f, has a KDI2a value of 4.58 and KDI2b of 0.12; the relatively low KDI2b value can be explained by its high MW, which is compensated by the favorable values for the other descriptors resulting in a good KDI2a value. The KDI2b index is sensitive to any outliers since multiplication of small numbers leads to small numbers.

3. Materials and Methods

3.1. Chemistry Berberine chloride was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Methanesulfochloride and ethanesulfochloride were purchased from Acros Organics (Belgium), 1-propanesulfochloride and 1-buthanesulfochloride were purchased from Alfa Aesar (Karlsruhe, Germany). 48% aqueous HBr solution was purchased from Acros Organics (the Netherlands). All solvents used in the reactions were purified and dried. Bromine, sodium borohydride and triethylamine were purchased from Sigma-Aldrich. Column chromatography was carried out on neutral alumina LL40/250. All reactions were monitored by TLC analysis using Merck Aluminium oxide 60 F254 plastic sheets (Darmstadt, Germany), eluent CH2Cl2-MeOH. The 1H and 13C NMR spectra were recorded on a Bruker AM-400 spectrometer (400.13 and 100.61 MHz) for 5–10% solutions of the compounds in CDCl3 or DMSO-d6 using the signal of the solvent CDCl3 as the standard (δ 7.24 for 1H and δ 76.90 for 13C). The IR spectra were measured on a Vector 22 FTIR spectrometer in KBr pellets. High-resolution electrospray ionization (HRESI) mass spectra were carried out using a time-of-flight high-resolution mass spectrometer micrOTOF-Q (Bruker Daltonics, Germany) with an Agilent 1200 liquid chromatograph (Agilent Technologies, USA/Germany). Positive ion scanning in the range m/z = 100–3000. Drying gas (nitrogen) flow rate of 4 L/min; temperature—190 °C; sprayer pressure—1.0 bar. The spectroscopic and analytical measurements were carried out at the Multi-access Chemical Service Center of the Siberian Branch of the Russian Academy of Sciences.

3.1.1. General Synthesis of Compounds 11a–g and 12a–g Desired compounds 11a–d, f, g and 12a–g were synthesized according to the general procedure described previously [27], compound 11e was also synthesized according to the procedure described previously [30]. Int. J. Mol. Sci. 2020, 21, 7162 11 of 16

3.1.2. General Procedure for the Synthesis of Sulfonates 10a–d 2.5 mmol of triethylamine was added to the suspension of 1 mmol of berberrubine hydrobromide in 25 mL of methylene chloride. 1.5 mmol of sulfochloride in 3 mL of methylene chloride was added under stirring at room temperature. After 5 hours the precipitate was filtered, washed with methylene chloride and the pure product 10a–d was isolated.

3.1.3. 10-Methoxy-9-methanesulfonyloxy-2,3-methylenedioxyprotoberberine chloride (10a)

Yield: 7%

1 Spectra NMR H (400 MHz, DMSO-d6, δ, ppm, J/Hz): 3.22 (2H, t, J = 6.07 Hz, H-5), 3.73 (3H, s, SCH3), 4.12 (3H, s, OCH3), 5.00 (2H, t, J = 5.72 Hz, H-6), 6.19 (2H, s, OCH2O), 7.11 (1H, s, H-4), 7.82 (1H, s, H-1), 8.27 (1H, d, J = 9.06 Hz, H-12), 8.34 (1H, d, J = 9.06 Hz, H-11), 9.08 (1H, s, H-13), 9.72 (1H, s, 13 H-8). Spectra NMR C (100 MHz, DMSO-d6, δ, ppm): 26.18 (C-5), 40.02 (SO2CH3), 55.70 (C-6), 57.44 (OCH3), 102.23 (OCH2O), 105.60 (C-1), 108.49 (C-4), 120.20 (C-13b), 120.86 (C-13), 121.79 (C-8a), 126.47 (C-12), 128.03 (C-11), 131.05 (C-4a), 131.44 (C-12a), 133.31 (C-13a), 138.59 (C-9), 144.04 (C-8), 147.77 1 (C-2), 150.17 (C-3), 151.89 (C-10). Spectra IR (cm− ): 804.21, 925.71, 1033.71, 1097.35, 1224.63, 1263.20, + + 1365.42, 1506.20, 1610.34, 1623.84, 3039.40, 3320.97, 3635.33. MS (ESI): m/z (M ) calcd for C20H18NO6S , 400.085 found: 400.081.

3.1.4. 9-Ethanesulfonyloxy-10-methoxy-2,3-methylenedioxyprotoberberine chloride (10b)

Yield: 25%

1 Spectra NMR H (400 MHz, DMSO-d6, δ, ppm, J/Hz): 1.52 (3H, t, J = 7.32 Hz, CH2CH3), 3.22 (2H, t, J = 6.05 Hz, H-5), 3.89 (2H, q, J1 = 3.50 Hz, J2 = 10.96 Hz, CH2CH3), 4.12 (3H, s, OCH3), 5.01 (2H, t, J = 5.91 Hz, H-6), 6.18 (2H, s, OCH2O), 7.11 (1H, s, H-4), 7.82 (1H, s, H–1), 8.27 (1H, d, J = 9.48 Hz, H-12), 8.34 (1H, d, J = 9.48 Hz, H-11), 9.10 (1H, s, H-13), 9.64 (1H, s, H–8). Spectra NMR 13C (100 MHz, DMSO-d6, δ, ppm): 8.18 (CH2CH3), 26.19 (C-5), 47.00 (SCH2), 55.83 (C–6), 57.44 (OCH3), 102.20 (OCH2O), 105.61 (C-1), 108.45 (C–4), 120.18 (C-13b), 120.90 (C-13), 121.98 (C-8a), 126.42 (C-12), 128.02 (C-11), 131.02 (C-4a), 131.27 (C-12a), 133.36 (C-13a), 138.63 (C-9), 143.91 (C-8), 147.75 (C-2), 150.17 (C-3), 1 151.71 (C-10). Spectra IR (cm− ): 806.14, 923.78, 1033.71, 1097.35, 1164.85, 1222.70, 1278.2063, 1363.49, + + 1506.20, 1608.42, 1621.92, 3023.98, 3423.19, 3629.54. MS (ESI): m/z (M ) calcd for C21H20NO6S , 414.101 found: 414.096.

3.1.5. 10-Methoxy-2,3-methylenedioxy-9-((propane-1-sulfonyl)oxy)protoberberine chloride (10c)

Yield: 34%

1 Spectra NMR H (400 MHz, DMSO-d6, δ, ppm, J/Hz): 1.11 (3H, t, J = 7.45 Hz, CH2CH3), 1.94–2.03 (2H, m, CH2CH3), 3.22 (2H, t, J = 5.72 Hz, H-5), 3.87 (2H, t, J = 7.58 Hz, SCH2CH2), 4.12 (3H, s, OCH3), 5.01 (2H, t, J = 6.02 Hz, H-6), 6.19 (2H, s, OCH2O), 7.11 (1H, s, H-4), 7.82 (1H, s, H-1), 8.27 (1H, d, J = 9.33 Hz, H-12), 8.34 (1H, d, J = 9.33 Hz, H-11), 9.10 (1H, s, H-13), 9.64 (1H, s, H-8). Spectra NMR 13 C (100 MHz, DMSO-d6, δ, ppm): 12.49 (CH2CH3), 17.17 (CH2CH2CH3), 26.16 (C-5), 53.27 (SCH2), 55.80 (C-6), 57.46 (OCH3), 102.18 (OCH2O), 105.60 (C-1), 108.42 (C-4), 120.14 (C-13b), 120.86 (C-13), 121.93 (C-8a), 126.37 (C-12), 127.98 (C-11), 130.96 (C-4a), 131.23 (C-12a), 133.33 (C-13a), 138.55 (C-9), 1 143.91 (C-8), 147.70 (C-2), 150.12 (C-3), 151.69 (C-10). Spectra IR (cm− ): 817.71, 925.71, 1037.56, 1095.42, 1162.92, 1222.70, 1263.20, 1363.49, 1506.20, 1610.34, 1621.92, 3023.98, 3427.04. MS (ESI): m/z (M+) calcd + for C22H22NO6S , 428.116 found: 428.121. Int. J. Mol. Sci. 2020, 21, 7162 12 of 16

3.1.6. 9-(Butane-1-sulfonyl)oxy-10-methoxy-2,3-methylenedioxy protoberberine chloride (10d)

Yield: 50%

1 Spectra NMR H (400 MHz, DMSO-d6, δ, ppm, J/Hz): 0.97 (3H, t, J = 7.34 Hz, CH2CH3), 1.48–1.58 (2H, m, CH2CH3), 1.89–1.97 (2H, m, SCH2CH2), 3.22 (2H, t, J = 5.96 Hz, H-5), 3.91 (2H, t, J = 7.68 Hz, SCH2), 4.11 (3H, s, OCH3), 5.02 (2H, t, J = 6.04 Hz), 6.18 (2H, c, OCH2O), 7.10 (1H, s, H-4), 7.82 (1H, s, H-1), 8.27 (1H, d, J = 9.21 Hz, H-12), 8.34 (1H, d, J = 9.21 Hz, H-11), 9.13 (1H, s, H-13), 9.67 (1H, s, H-8). 13 Spectra NMR C (100 MHz, DMSO-d6, δ, ppm): 13.44 (CH3CH2), 20.74 (CH3CH2), 25.29 (SCH2CH2), 26.19 (C-5), 51.75 (SCH2), 55.80 (C-6), 57.46 (OCH3), 102.20 (OCH2O), 105.62 (C-1), 108.45 (C-4), 120.19 (C-13b), 120.91 (C-13), 121.98 (C-8a), 126.42 (C-12), 128.01 (C-11), 131.02 (C-4a), 131.30 (C-12a), 133.36 1 (C-13a), 138.62 (C-9), 143.97 (C-8), 147.75 (C-2), 150.16 (C-3), 151.72 (C-10). Spectra IR (cm− ): 811.92, 925.71, 1035.63, 1099.28, 1222.70, 1265.13, 1365.42, 1504.27, 1610.34, 1619.99, 2960.33, 3425.11. MS (ESI): + + m/z (M ) calcd for C23H24NO6S , 442.132 found: 442.133.

3.2. Biology

3.2.1. Detection of Tdp1 Activity The methodology has been reported in our previous work [30] and consists of fluorescence intensity measurement in a reaction of quencher removal from a fluorophore quencher-coupled DNA oligonucleotide catalyzed by Tdp1. The reaction was carried out at different concentrations of inhibitors (the control samples contained 1% of DMSO, Sigma, St. Louis, MO, USA). The reaction mixtures contained Tdp1 buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, and 7 mM β-mercaptoethanol), 50 nM biosensor, and an inhibitor being tested. Purified Tdp1 (1.5 nM) triggered the reaction. The biosensor (50-[FAM] AAC GTC AGGGTC TTC C [BHQ]-30) was synthesized in the Laboratory of Biomedical Chemistry at the Institute of Chemical Biology and Fundamental Medicine (Novosibirsk, Russia). The reactions were incubated on a POLARstar OPTIMA fluorimeter (BMG LABTECH, GmbH, Ortenberg, Germany) to measure fluorescence every 55 s (ex. 485/em. 520 nm) during the linear phase (here, data from minute 0 to minute 8). The values of IC50 were determined using a six-point concentration response curve in minimum three independent experiments and were calculated using MARS Data Analysis 2.0 (BMG LABTECH, GmbH, Ortenberg, Germany).

3.2.2. Cytotoxicity Assays Cytotoxicity of the compounds to HeLa (human cervical cancer) cell line was examined using the EZ4U Cell Proliferation and Cytotoxicity Assay (Biomedica, Vienna, Austria), according to the manufacturer’s protocols. The cells were grown in Iscove’s modified Dulbecco’s medium (IMDM) with 40 µg/mL gentamicin, 50 IU/mL penicillin, 50 µg/mL streptomycin (MP Biomedicals, Santa Ana, CA, USA), and 10% of fetal bovine serum (Biolot, St. Petersburg, Russia) in a 5% CO2 atmosphere. After formation of a 30–50%-monolayer, the tested compounds were added to the medium. The volume of the added reagents was 1/100 of the total volume of the culture medium, and the amount of DMSO (Sigma, St. Louis, MO, USA) was 1% of the final volume. Control cells were grown in the presence of 1% DMSO. The cell culture was monitored for 3 days. To assess the influence of the inhibitors on the cytotoxic effect of topotecan (ACTAVIS GROUP PTC ehf., Bucharest, Romania), 50% cytotoxic concentrations of topotecan and of each inhibitor were determined to attain a defined single-agent effect. Then, minimum two independent tests were performed with each inhibitor in combination with topotecan. When using a combination of drugs, Tdp1 inhibitors were first added, then topotecan was added immediately (within 10–15 min).

3.3. Molecular Modeling The compounds were docked against the structure of TDP1 Tdp1 (PDB ID: 6DIE, resolution 1.78 Å) [32] which was obtained from the (PDB) [40,41]. The Scigress version Int. J. Mol. Sci. 2020, 21, 7162 13 of 16

FJ 2.6 program [42] was used to prepare the crystal structure for docking, i.e., the hydrogen atoms were added, the co-crystallized ligand benzene-1,2,4-tricarboxylic acid was removed as well as crystallographic water molecules except HOH 814, 821 and 1078. The waters were set on toggle—bound or displaced by the ligand during docking—and spin—automatic optimization of the orientation of the hydrogen atoms. The Scigress software suite was also used to build the inhibitors and the MM2 [43] force field was used to optimize the structures. Furthermore, Scigress was used for the 10 ps MD runs at 1000 K; the MM2 force filed was used, 5 Å radius was defined around the ligand and allowed to be flexible whereas the rest of the protein structure was held ridged (locked). First the binding pocket with the ligand was structurally optimized followed by the MD run. The docking center was defined as the position of a carbon on the ring of the co-crystallized benzene-1, 2, 4-tricarboxylic acid (x = 6.052, − y = 14.428, z = 33.998) with 10 Å radius. Fifty docking runs were allowed for each ligand with − default search efficiency (100%). The basic amino acids lysine and arginine were defined as protonated. Furthermore, aspartic and glutamic acids were assumed deprotonated. The GoldScore (GS) [44] and ChemScore (CS) [45,46] ChemPLP (Piecewise Linear Potential) [47] and ASP (Astex Statistical Potential) [48] scoring functions were implemented to predict the binding modes and relative energies of the ligands using the GOLD v5.4.1 software suite. The QikProp 3.2 [49] software package was used to calculate the molecular descriptors of the molecules. The reliability of it QikProp established for the calculated descriptors. [50] The Known Drug Indexes (KDI) were calculated from the molecular descriptors as described by Eurtivong and Reynisson [40]. Five of the compounds (9 and 10a–d) carry a positive charge and QikProp does not compute charged molecules. The MW, Log P, HD and HA were derived using the Scigress version FJ 2.6 program [42] software, PSA and RB are not available in this software suite.

4. Conclusions The berberine and tetrahydroberberine sulfonates and their brominated analogues were tested to evaluate their Tdp1 inhibitory activity. To our knowledge, this is the first report that documents berberine-based compounds as inhibitors of this enzyme. The IC50 values are in the 0.53 to 4 µM range. Of the alkyl sulfonates, only derivatives with bromine substitution on site 12 of tetrahydroberberrubine are active. While both tetrahydroberberrubine and 12-bromtetrahydroberberrubine derivatives containing polyfluoroaromatic substituents all have inhibitory activity with IC50 values of ~1 µM. According to the inhibitory activity, toxicity data and ability to sensitize topotecan against the HeLa cancer cell line the two most promising derivatives were identified—11g and 12g. These results indicate that sulfonates of tetrahydroberberine have potential to be developed as new agents for anticancer therapy due to their inhibitory activity and lack of toxicity.

Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/19/ 7162/s1. Author Contributions: Chemistry investigation, E.D.G., I.V.N., R.A.B. and O.A.L.; in vitro investigation, A.L.Z., A.A.C., E.S.I., N.S.D., E.M.M. and R.O.A.; modeling, J.R.; methodology, N.F.S. and O.I.L.; project administration, K.P.V.; supervision, K.P.V.; writing—original draft, E.D.G., A.L.Z. and O.A.L.; writing—review and editing, K.P.V., J.R., N.F.S. and O.I.L. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by the Russian Science Foundation grant № 19-13-00040. Acknowledgments: Authors would like to acknowledge the Multi-Access Chemical Research Center SB RAS for spectral and analytical measurements. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

Tdp1 Tyrosyl-DNA phosphodiesterase 1 PARP Poly (ADP-ribose) polymerase Int. J. Mol. Sci. 2020, 21, 7162 14 of 16

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