International Journal of

Molecular Sciences Article

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

2 3

4

*

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.) Novosibirsk State University, Pirogova str. 1, Novosibirsk 630090, Russia; [email protected] (R.O.A.); [email protected] (O.I.L.) 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.C.); [email protected] (A.L.Z.); [email protected] (E.S.I.); [email protected] (N.S.D.); [email protected] (E.M.M.) 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 enzymes, 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

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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 (Figure 1). 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, Figure 2), 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 (Scheme 1), 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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Figure2.2.Examples Examplesofofestablished establishedTdp1 Tdp1inhibitors inhibitors1–8. 1–8. Figure

2.2.Results Resultsand andDiscussion Discussion 2.1. 2.1.Chemistry Chemistry Sulfonates in in accordance with previously reported methods [27]. [27]. For this Sulfonates10–12 10-12were weresynthesized synthesized accordance with previously reported methods For ◦ purpose, berberine 9 was selectively demethylated at 190 C under vacuum as previously described [29]. this purpose, berberine 9 was selectively demethylated at 190 °C under vacuum as previously After treatment with treatment HBr, berberrubine 13 was isolated 89% yield in (Scheme 1). described [29]. After with HBr,hydrobromide berberrubine hydrobromide 13in was isolated 89% yield Compound 13 was reduced with sodium borohydride in methanol according to a procedure described (Scheme 1). Compound 13 was reduced with sodium borohydride in methanol according to a previously yielding previously tetrahydroberberrubine 14.tetrahydroberberrubine The bromination of compound 14 with a bromineof procedure[30] described [30] yielding 14. The bromination in dioxane solution afforded 12-bromotetrahydroberberrubine 15 in 52% yield. The reaction compound 14 with a bromine in dioxane solution afforded 12-bromotetrahydroberberrubine 15ofin tetrahydroderivatives 14 and with polyfluoroaryl and alkyl15 sulfonylchlorides as welland as with 52% yield. The reaction of 15tetrahydroderivatives 14 and with polyfluoroaryl alkyl tosylchloride in dichloromethane the presenceinofdichloromethane triethylamine produced tetrahydroberberrubine sulfonylchlorides as well as withintosylchloride in the presence of triethylamine 9-O-sulfonates 11a–h (44–84% yields) and 9-O-sulfonates 12-bromotetrahydroberberrubine 9-O-sulfonates produced tetrahydroberberrubine 11a-h (44–84% yields) 12a–h and (49–93% yields). New berberine type 10 derivatives were synthesized by the reaction of berberrubine 12-bromotetrahydroberberrubine 9-O-sulfonates 12a-h (49–93% yields). New berberine type 10 hydrobromide 13 with different by alkyl The reactions were carried out dichloromethane derivatives were synthesized thesulfochlorides. reaction of berberrubine hydrobromide 13 in with different alkyl insulfochlorides. the presence ofThe triethylamine for 5 h at room temperature. Sulfonates 10 were isolated by triethylamine precipitation reactions were carried out in dichloromethane in the presence of from reaction mixtures. for 5the hours at room temperature. Sulfonates 10 were isolated by precipitation from the reaction

mixtures.

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O O

O

N

O

Cl

O

Br

OH

9

N

O OH

OH 14 RSO2Cl NEt3 CH2Cl2

O O

N O S R O

O

Br Br2 dioxane

N

O

13

RSO2Cl NEt3 CH2Cl2

O

O NaBH4 MeOH 0°C

N

O

O

O O

1) 180°C, 20-30 mm Hg 2) HBr, EtOH

O

Br

O

15 RSO2Cl NEt3 CH2Cl2

O O

N O S R O

O S R O 12

O 11

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

10a: R = CH3; 10b: R = C2H5; 10c: R = C3H7; 10d: R = C4H9

O

N

O

O

10

O

Br

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

Scheme Scheme 1. 1. The The synthetic syntheticpathways pathwaysto tosulfonates sulfonates10, 10,11 11and and12. 12.

The structures structures of of the the new new compounds compounds were were confirmed confirmed by by 11H-NMR, The H-NMR,1313C-NMR, C-NMR,IR IRand andHRMS HRMS methods; the the results results are shown in methods; are shown in the the experimental experimental section section and and the the supplementary supplementary information. information.

2.2. Effects of the Berberine Berberine Sulfonates Sulfonates on on Tdp1 Tdp1 Activity Activity oligonucleotidereal-time real-time biosensor theofability ofremove Tdp1 fluorophore to remove An oligonucleotide biosensor waswas usedused basedbased on the on ability Tdp1 to fluorophore quenchers from the 3’-end of DNA, described as previously [31]. The hexadecameric quenchers from the 30 -end of DNA, as previously [31]. described The hexadecameric oligonucleotide oligonucleotide carried 5(6)-carboxyfluorescein (FAM) 5’-end andquencher the fluorophore quencher carried 5(6)-carboxyfluorescein (FAM) at the 50 -end andat thethe fluorophore BHQ1 (Black Hole 0 BHQ1 (BlackatHole at the 3’-end. Tdp1 inhibitors prevent removal of thus fluorophore Quencher-1) the 3 Quencher-1) -end. Tdp1 inhibitors prevent removal of fluorophore quenchers, reducing quenchers, thus reducing fluorescence intensity. The results of the Tdp1 assay derivatives 10–12 fluorescence intensity. The results of the Tdp1 assay for derivatives 10–12 andfor cytotoxic effects are Int. Mol. Sci. 2020, 21, FOR PEER REVIEW ofof16 16 Int.J.J.cytotoxic J.Mol. Mol. Sci.2020, 2020, 21, FOR PEERREVIEW REVIEW 16 and effects in Tables 1 and 2. shown inSci. Tables 121, and 2. shown Int. xxxare FOR PEER 555of It is clear from the data in Table 1 that both alkyl sulfonates of berberine and Tdp1 inhibiting (IC —half inhibitory of sulfonate berberine Table 1.1. Table 1.1. Tdp1 inhibiting activity (IC 50 —half maximal inhibitory concentration) of sulfonate berberine Table Tdp1 inhibiting activity (IC 50 —half maximal inhibitory concentration) of sulfonate berberine 50 Table Tdp1 inhibiting activity (IC 50 —half maximal inhibitory concentration) of sulfonate berberine tetrahydroberberine are notactivity very active withmaximal the exception ofconcentration) the tetrahydroberberine derivatives derivatives with aliphatic substituents and their cytotoxicity (CC —half maximal cytotoxic derivatives with aliphatic substituents and their cytotoxicity (CC 50 —half maximal cytotoxic 50 derivatives with aliphatic substituents and their cytotoxicity (CC 50 —half maximal cytotoxic derivatives with aliphatic substituents and their 50—half maximal cytotoxic containing both sufficiently long alkyl substituent andcytotoxicity bromine in(CC a para-position to the sulfonate concentration) in HeLa cells. Furamidine was used as a positive control (IC = 1.2 ± µM). concentration) in HeLa cells. Furamidine was used as a positive control (IC 50 = 1.2 ± 0.3 µM). 50 concentration) in HeLa cells. Furamidine was used as a positive control (IC 50 = 1.2 ± 0.3 µM). concentration) HeLaThe cells. Furamidine was used as a positive control (IC50 = 1.2 ±is0.3 µM). substituent, 12c andin12d. favorable substitution pattern of these derivatives confirmed 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 Structure µM. Bromine substitution is not important for 12e-h activity as comparison with their Structure Structure Structure non-brominated analogues 11e-h shows. 11a-d 11a-d 11a–d 11a-d

10a-d 10a-d 10a-d 10a–d RRRR a;a;Me Me Me a; a; Me b; Et b;Et Et b; b;c;c; Et Pr Pr c; Pr c; Pr d; Bu d; Bu d; Bu d; Bu

IC 5050 µM IC ,µM µM IC 50 ,,, µM 50 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15

CC 5050 CC ,µM µM CC µM CC 50 ,, µM 50 ND* ND* ND* ND * ND ND ND ND ND ND ND ND ND ND ND

>15

ND

IC 5050 ,µM IC , µM µM IC 50 ,, µM 50 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15 >15

CC 5050 CC ,µM ,µM µM CC 50 ,, µM 50 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

*ND—not determined. *ND—not determined. ND >15 *ND—not determined.

IC 50,,,µM IC ,µM µM IC µM IC 50 5050 >15 >15 >15 >15 >15 >15 >15 >15 2.9±1.3 2.9±1.3 2.9±1.3 2.9 ± 1.3 4±1.0 4±1.0 4±1.0 4 ± 1.0

12a-d 12a-d 12a–d 12a-d CC 5050 CC ,µM µM CC ,,µM CC 50 µM 50 ND ND ND ND ND ND ND ND >100 >100 >100 >100 >100 >100 >100 >100

* ND—not Based on the data shown in Tables and can be stated that some berberine derivatives Basedon onthe thedata datashown shownin inTables Tables anddetermined. canbe bestated statedthat thatsome someberberine berberinederivatives derivatives Based 111and 222itititcan inhibit Tdp1. was found that the structure of the substituent in the sulfonate affects the inhibitory inhibitTdp1. Tdp1.ItItItwas wasfound foundthat thatthe thestructure structureof ofthe thesubstituent substituentin inthe thesulfonate sulfonateaffects affectsthe theinhibitory inhibitory inhibit activity. Polyfluorinated arylsulfonates 11f-h and 12f-h exhibited inhibitory activity in the low activity. Polyfluorinated arylsulfonates 11f-h and 12f-h exhibited inhibitory activity in thelow low activity. Polyfluorinated arylsulfonates 11f-h and 12f-h exhibited inhibitory activity in the micromolar range, while their non-fluorinated analogs (11e, 12e) were inactive at these micromolar range, range, while while their their non-fluorinated non-fluorinated analogs analogs (11e, (11e, 12e) 12e) were were inactive inactive at at these these micromolar concentrations. In the series of alkylsulfonates, inhibitory activity was found only for compounds concentrations.In Inthe theseries seriesof ofalkylsulfonates, alkylsulfonates,inhibitory inhibitoryactivity activitywas wasfound foundonly onlyfor forcompounds compounds concentrations. with sufficiently long alkyl substituent (propyl-, butyl-) in the sulfonate group and in the presence withaaasufficiently sufficientlylong longalkyl alkylsubstituent substituent(propyl-, (propyl-,butyl-) butyl-)in inthe thesulfonate sulfonategroup groupand andin inthe thepresence presence with of bromine substituent at position 12 (compounds 12c,d). To explain the observed effects, of bromine bromine substituent substituent at at position position 12 12 (compounds (compounds 12c,d). 12c,d). To To explain explain the the observed observed effects, effects, aaa of molecular modeling study was carried out. molecular modeling study was carried out. molecular modeling study was carried out.

Table 2.2.Tdp Tdp inhibiting activity (IC 5050 of sulfonate berberine derivatives with polyfluoroaromatic Table2. Tdp111inhibiting inhibitingactivity activity(IC (IC ofsulfonate sulfonateberberine berberinederivatives derivativeswith withpolyfluoroaromatic polyfluoroaromatic Table 50 )))of

activity. Polyfluorinated Polyfluorinated arylsulfonates arylsulfonates 11f-h 11f-h and and 12f-h 12f-h exhibited exhibited inhibitory inhibitory activity activity in in the the low low activity. activity. Polyfluorinated arylsulfonates 11f-h exhibited inhibitory activity in low activity. Polyfluorinated arylsulfonates 11f-h and and 12f-h 12f-h exhibited inhibitory activity in the the low micromolar range, while their non-fluorinated analogs (11e, 12e) were inactive at these micromolar range, range, while while their their non-fluorinated non-fluorinated analogs analogs (11e, (11e, 12e) were were inactive inactive atat these these micromolar micromolar range, while non-fluorinated analogsactivity (11e, 12e) 12e) found were only inactive at these concentrations. In the the seriestheir of alkylsulfonates, alkylsulfonates, inhibitory was for compounds compounds concentrations. In series of inhibitory activity was found only for concentrations. In series of alkylsulfonates, inhibitory activity was only for compounds concentrations. In the the series alkylsulfonates, inhibitory activity was found found only compounds withaasufficiently sufficiently long alkylof substituent (propyl-, butyl-)in in thesulfonate sulfonate group andfor inthe thepresence presence with long alkyl substituent (propyl-, butyl-) the group and in with a sufficiently long alkyl substituent (propyl-, butyl-) in the sulfonate group and in the presence with a sufficiently long alkyl substituent (propyl-, butyl-) in the sulfonate group and in the presence of bromine substituent at position 12 (compounds 12c,d). To explain the observed effects, a bromine substituent at position position 12 12 (compounds (compounds 12c,d). 12c,d). To To explain explain the the observed observed effects, effects, Int. of J.of Mol. Sci. 2020,substituent 21, 7162 5 of 16 aa bromine at ofmolecular bromine substituent at position 12 (compounds 12c,d). To explain the observed effects, a modelingstudy studywas wascarried carriedout. out. molecularmodeling modeling was Int. J. Mol.molecular Sci. 2020, 21, modeling x FOR PEERstudy REVIEW 5 of 16 molecular study wascarried carriedout. out. TableTable 2. Tdp 1Tdp inhibiting activity (IC50 (IC )(IC of5050sulfonate berberine derivatives withwith polyfluoroaromatic Table Tdp 1 inhibiting inhibiting activity ) of of sulfonate sulfonate berberine derivatives with polyfluoroaromatic 2.2.Tdp activity berberine derivatives polyfluoroaromatic 2.2. 111inhibiting 5050 )))of sulfonate berberine derivatives with polyfluoroaromatic Table 1. Table Tdp1 inhibiting activity (ICactivity 50—half(IC maximal inhibitory concentration) of sulfonate berberine Table Tdptheir inhibiting activity (IC of sulfonate berberine derivatives with polyfluoroaromatic substituents and cytotoxicity (CC ) in HeLa cells. Furamidine was used as a positive control 50 ) in HeLa cells. Furamidine was used as positive control substituents and their cytotoxicity (CC 50 HeLacells. cells. Furamidinewas wasused used asaaapositive positive control substituentsand andtheir theircytotoxicity cytotoxicity(CC (CC50) )ininHeLa substituents derivatives with aliphatic substituents cytotoxicity (CC50—halfwas maximal 50) in HeLa cells.Furamidine Furamidine usedas as cytotoxic a positivecontrol control andµM). their cytotoxicityand (CC50their (IC50substituents = 1.2 0.3 µM). 50 =±1.2 ± 0.3 (IC 1.2±±0.3 0.3µM). µM). (IC50 =1.2 (IC concentration) HeLa 1.2 ± 0.3cells. µM).Furamidine was used as a positive control (IC50 = 1.2 ± 0.3 µM). (IC5050==in

Code, Code,structure structure Code, structure StructureCode,

Code, Code,structure structure

10a-d R a; Me b; Et c; Pr d; Bu

e;e; e;e; e;

IC50, µM >15 R RR RR >15 >15 >15

11a-d

CC50, µM IC50, µM CC50, µM 11e–h 11e-h 11e-h 11e-h 11e-h ND* >15 ND 50,50 ,µM µM CC 50,,,µM µM IC IC , µM CC5050 µM 50 CC IC 50 IC ND >15CC 50, ,µM µM CC5050, ,µM µM ND IC ND >15 ND ND ND >15 ND ND>15 >15 ND ND

ND >15 >15 >15 *ND—not determined.

12a-d 12e–h IC50, µM CC50, µM 12e-h 12e-h 12e-h >15 12e-h ND IC 50,µM µM CC5050,,µM µM IC50 ,IC µM CC50 , µMCC 50 IC , ,,µM NDCC IC5050>15 µM CC5050, ,µM µM 2.9±1.3 >100 ND ND >15 4±1.0 ND >100 ND >15

>15 >15 >15

ND

Based on the data shown in Tables 1 and 2 it can be stated that some berberine derivatives inhibit Tdp1. It was found that the in the sulfonate affects 1.0structure ±0.20 0.20 of the substituent 11±±2.0 2.0 0.53±±0.01 0.01 the inhibitory 9.9±±4.5 4.5 1.0 11 0.53 9.9 ± 0.20 11 2.0exhibited 0.53 ± 0.01 4.59.9 1.0 ±±±0.20 ±±±2.0 0.53 ±±0.01 ±±4.5 1.01.0 0.20 11 2.0 0.53 0.01 9.9 4.5 activity. Polyfluorinated arylsulfonates 11f-h and11 12f-h inhibitory activity9.9in± the low micromolarf;f;range, while their non-fluorinated analogs (11e, 12e) were inactive at these f;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 position 12 12c,d). To explain observed effects, 1.05±±0.05 0.05(compounds >100 1.3the ±0.30 0.30 95±a±5.0 5.0 1.05 >100 1.3 95 1.05 ±±0.05 >100 1.3 ±±±0.30 1.05 ± 0.05 >100 1.3 ± 0.30 95 ± 5.0 95 0.05 >100 1.3 0.30 95±±5.0 5.0 molecular modeling study was 1.05 carried out. 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). 0.9± ±0.20 0.20 2.6 ±±0.1 0.1 1.4 ±1.4 0.30± 0.30 2.2 ± 1.5 2.2 ± 1.5 0.9 2.6

h;h; Berberine Code, 2.3. structure It isMolecular clear fromModeling the dataofinthe Table 1 that Derivatives both alkyl sulfonates of berberine and tetrahydroberberine are not very active with the exception of were the tetrahydroberberine derivatives containing both Twenty-three berberine derivatives docked into the binding site of Tdp1 (PDB ID: 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.these The favorable pattern of thesethe derivatives isquality confirmed the results keeping crystallinesubstitution water improves prediction of thebydocking scaffold 11e-h molecules 12e-h of the tetrahydroberberine sulfonates with aromatic and polyfluoroaromatic substituents as shown (for section) [13]. R further information CC50, µM IC50, The µM binding predictions CC50, µMof the scoring IC50, µM see the Methodology in Table 2. Both sulfonates para-toluenesulfonyl (11e and 12e) are inactive, but all functions used are givenwith in Table S1, all the ligandssubstituent show reasonable scores. three fluorinated sulfonates (11f,ligand 11g, 11h) goodIC inhibitory activity with ICto values of ~1 µM. 50 the Compound 12f is the withshow the best 50 value and according docking; 12f fits ND ND Bromine substitution is not important for 12e–h activity as comparison with their non-brominated >15 >15 neatly in the catalytic region as shown in Figure 3A. This region contains the catalytic histidine 263 e; analogues 11e–h shows. and 493 amino acid residues thus the ligand blocs any activity of the enzyme. Indeed, 12f forms a BasedH-bond on the data in Tables 1 and 2site it can be stated that some berberine derivatives weak withshown the His493 imidazole group via the bromine substituent as showninhibit in Figure Tdp1. It was found that the structure of the substituent in the sulfonate affects the inhibitory activity. 3B as well as with Asn283’s amide side chain. Finally, the amide group of Asn516 forms an H-bond 1.0 ± 0.20 11 ± 2.0 0.53 ± 0.01 9.9 ± 4.5 Polyfluorinated arylsulfonates 11f–h 12f–h exhibited inhibitory in the low micromolar with one of the oxygen atoms in and the 1.3-benzodioxole moiety of activity the ligand. It is worth mentioning range, while their non-fluorinated analogs (11e, 12e) were inactive at these concentrations. In theif the that the methoxy group on 12f can potentially form a weak H-bond with the thiol on Cys205, f; series of alkylsulfonates, inhibitory activity was found only for compounds with a sufficiently long flexibility of the protein would be accounted for, and the same methoxy group has lipophilic alkylcontacts substituent butyl-)side in the sulfonate groupthe and in the mode. presence of bromine the substituent with(propyl-, Ile285 aliphatic chain, stabilizing binding Interestingly, modeling of at position 12 (compounds 12c,d). To explain the observed effects, a molecular modeling study the 11f ligand, 1.05 which does not contain a bromine group, 1.3 but±also 50 ± value, not give ± 0.05 >100 0.30with a good IC95 5.0 did was carried out. consistent 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

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 functions used are Int. J. Mol. Sci. 2020, 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 2.3. Molecular of the Berberine and 493 aminoModeling acid residues thus the Derivatives ligand blocs any activity of the enzyme. Indeed, 12f forms a weakTwenty-three H-bond with berberine the His493derivatives imidazole were site group viainto the bromine substituent as shown docked the binding site of Tdp1 (PDB in ID:Figure 6DIE, 3B as well as with side chain. Finally, the amide821 group Asn516 an H-bond resolution 1.78 Å) Asn283’s [32] withamide three water molecules (HOH814, and of 1078). It isforms established that with one of the oxygen atoms in the 1.3-benzodioxole moiety of the ligand. It is worth mentioning keeping these crystalline water molecules improves the prediction quality of the docking scaffold that methoxy group on potentially form a weak with the thiol on Cys205, if the (for the further information see12f thecan Methodology section) [13]. H-bond The binding predictions of the scoring flexibility of the be all accounted for,show and reasonable the same methoxy functions used areprotein given inwould Table S1, the ligands scores. group has lipophilic contacts with Ile285 aliphatic side chain, stabilizing the binding mode. the12f modeling of Compound 12f is the ligand with the best IC50 value and accordingInterestingly, to the docking; fits neatly the 11f ligand, which does not contain a bromine group, but also with a good IC 50 value, did not give in the catalytic region as shown in Figure 3A. This region contains the catalytic histidine 263 and 493 consistent across theligand scoring functions used,ofi.e., differentIndeed, conformations predicted. amino acidresults residues thus the blocs any activity the enzyme. 12f formswere a weak H-bond This strongly indicates that the bromine group with its weak H-bonds to Asn283 and His493 is with the His493 imidazole site group via the bromine substituent as shown in Figure 3B as well as with essential anchoring the ligand the catalytic site. Asn283’s for amide side chain. Finally,in the amide group of Asn516 forms an H-bond with one of the oxygen Interestingly, derivatives 11e and 12e are essentially inactive (IC50 >15 µM); theymethoxy are structural atoms in the 1.3-benzodioxole moiety of the ligand. It is worth mentioning that the group analogues of the 11f/12f pair with hydrogens on the phenyl ring as well as a para methyl on 12f can potentially form a weak H-bond with the thiol on Cys205, if the flexibility ofsubstitution the protein instead of accounted fluorine groups. According to the modeling, thelipophilic 11e/12e pair has awith different mode would be for, and the same methoxy group has contacts Ile285binding aliphatic side from 11f/12f with the bromine moiety pointing into the aqueous phase for the former pair as shown chain, stabilizing the binding mode. Interestingly, the modeling of the 11f ligand, which does not in Figure S1 in the Supplementary Information. indicates the importance of theacross fluorine contain a bromine group, but also with a good IC50 This value, did not give consistent results the substitution on theused, phenyl the conformations modeling suggests the phenyl is leaning against scoring functions i.e.,rings; different werethat predicted. Thisring strongly indicates that the the carboxyl moiety in the Gly458 backbone, which can form an interaction between the electron bromine group with its weak H-bonds to Asn283 and His493 is essential for anchoring the ligand in deficient fluoride the catalytic site. substituted ring and the lone pairs of the oxygen atom in the carboxylic group as shown in Figure 3B.

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

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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 between as a blue the lineelectron betweendeficient the carboxylic backbone the Gly458 backbone, which can form an interaction fluoride substituted in Gly458 and of thethe centroid (green ball) fluorinated phenyl (3.5 ring group and the lone pairs oxygen atom in of thethe carboxylic group asgroup shown inÅ). Figure 3B.

To investigate the binding stability of the ligands molecular dynamic (MD) runs were conducted Tothe investigate the binding of stability the12eligands molecular (MD) were using docked conformations 11f, 12fofand for 10 ps at 1000 K.dynamic In general, the runs ligands are conducted using docked conformations of 11f, andstable 12e for 10 ps at 1000 K. In general, the stable within the the binding pocket and not ejected; the12f most intramolecular bond being between ligands are stable within binding pocket and not ejected; theInmost stablethe intramolecular bond the fluorinated phenyl ringthe and the Gly458 carboxyl group for 12f. contrast, phenyl group in 12e being between the fluorinated phenyl ring and the Gly458 carboxyl group for 12f. In contrast, the is very mobile. The other H-bonding interactions predicted are often broken to be reestablished during phenyl the MDgroup 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]. Top1 are in used anticancer drugs for the treatment forpoisons, variousthe oncological diseases Since Tdp1poisons is involved the as removal of DNA damage caused by Top1 activity of Tdp1 can [33–35]. Since Tdp1 is involved the removal ofThus, DNA itdamage caused Top1 poisons, can the enhance activity lead to the development of druginresistance [36]. is believed thatby Tdp1 inhibition of Tdp1 can lead to the development of drug resistance [36]. Thus, is believed that intrinsic Tdp1 inhibition the efficacy of Top1 poisons [37]. Tdp1 inhibitors should have theitlowest possible toxicity can enhance the efficacy of Top1 poisons [37]. Tdp1 inhibitors should have the lowest possible to minimize potential side effects. Therefore, we studied the intrinsic cytotoxicity of the compounds intrinsic toxicity to minimize sideEZ4U effects. Therefore, we studied the intrinsic cytotoxicity of against HeLa cells (cervical potential carcinoma). cell proliferation and cytotoxicity assay results are the compounds against HeLa cells (cervical carcinoma). EZ4U cell proliferation and cytotoxicity shown in Figure 4 for the ligands with Tdp1 inhibitory activity. assay results are shown in Figure 4 for the ligands with Tdp1 inhibitory activity.

Figure Figure 4. 4. The Theberberine berberine derivatives’ derivatives’ cytotoxicity cytotoxicity according according to to the the EZ4U EZ4U test. test. Error Error bars bars show show standard standarddeviations. deviations.

Theresults resultsshow showthat that9-O-sulfonates 9-O-sulfonatesof of12-bromotetrahydoberberine 12-bromotetrahydoberberinewith withaliphatic aliphaticsubstituents substituents The 12c and 12d, (Table 1 and Figure 4, black and red traces) are non-toxic up to 100 µM. The toxicityof of 12c and 12d, (Table 1 and Figure 4, black and red traces) are non-toxic up to 100 µM. The toxicity 9-O-sulfonates strongly strongly depends on the structure 9-O-sulfonates structure of of the the polyfluorinated polyfluorinatedfragment. fragment.Compounds Compoundswith witha were thethe most toxic; CCCC of 2.6 µMµM andand 2.2 2.2 µMµM for 11h andand 12h, aCF CF 3-groupin inthe thepara-position para-position were most toxic; 50 values of 2.6 for 11h 3 -group 50 values respectively (Table 2 and Figure 4, green and brown traces). The replacement of the trifluoromethyl 12h, respectively (Table 2 and Figure 4, green and brown traces). The replacement of the group with a fluorine the para-position reduced toxicity with CC50toxicity values with of 10 CC µM50for 11f and trifluoromethyl groupatom with in a fluorine atom in the para-position reduced values of 12f (Table 2 and Figure 4, blue and magenta traces, respectively). The compounds with a hydrogen 10 µM for 11f and 12f (Table 2 and Figure 4, blue and magenta traces, respectively). The compounds atomain the para-position were non-toxic (11g, 2 and(11g, Figure 4, violet or4, moderately toxic with hydrogen atom in the para-position wereTable non-toxic Table 2 andtrace) Figure violet trace) or (12g, Table 2toxic and (12g, Figure 4, orange moderately Table 2 and trace). Figure 4, orange trace). 2.5.Sensitizing SensitizingEffects Effects 2.5. The sensitizing of the inhibitors on topotecan’s cytotoxic potential investigated. The sensitizingeffect effect of berberine the berberine inhibitors on topotecan’s cytotoxicwas potential was In order to determine the optimal concentration for the inhibitors to provide the maximum sensitizing investigated. In order to determine the optimal concentration for the inhibitors to provide the effect, but remaining non-toxic, theirremaining concentrations were varied topotecan concentration of 2with µM, maximum sensitizing effect, but non-toxic, theirwith concentrations were varied its CC50 for HeLa cells. Topotecan significantly increased cytotoxicity of compounds 11g, 12g, topotecan concentration of 2 µM, its CC50 for HeLa cells.the Topotecan significantly increased the

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and 11f, the reliability was confirmed by the Mann–Whitney U-test, p = 0.05 and the results are shown cytotoxicity of compounds 11f, the in Figure 5. The original data11g, are 12g, givenand in Table S2. 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.

Figure Figure 5. 5. The The influence influence of of 22 µM µM topotecan topotecan on on Tdp1 Tdp1 inhibitors’ inhibitors’ cytotoxicity. cytotoxicity. The The unshaded unshaded histogram histogram bars denote cell viability in the presence of a Tdp1 inhibitor. The hatched histogram bars denote cell viability in the presence of a Tdp1 inhibitor. The hatched histogram bars bars indicate indicate cell cell viability in the presence of a combination of a Tdp1 inhibitor with 2 µM of topotecan. The concentration viability in the presence of a combination of a Tdp1 inhibitor with 2 µM of topotecan. The of the Tdp1 inhibitor in µMinhibitor is given in under each pairunder of bars. Error show standard deviations. concentration of the Tdp1 µM is given each pairbars of bars. Error bars show 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 (CC50 ). Topotecan weakly, concentration. In the presence of topotecan, a significant decrease in cell survival is observed (~30%) but significantly reduces this value12g to 62 µM. Compound 11f is inherently toxic, 90% of the cells die at all concentrations. Compound has a low toxicity potential of 95 µM (CCand 50). Topotecan weakly, at 20 µM. At lower concentrations, the effect of topotecan is significant. CC value for90% 11fof decreases 50 but significantly reduces this value to 62 µM. Compound 11f is inherently toxic, and 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 of topotecan was negligible or unreliable. decreases three fold, from 11 to 3.7 µM, in the presence of topotecan. For other ligands (11h, 12c, d, f, Non-toxic concentrations of the berberine derivatives (5 µM) were then tested at different h), the effect of topotecan was negligible or unreliable. concentrations topotecan. Theof most compound 11f caused 20% cell death this concentration; Non-toxicofconcentrations thetoxic berberine derivatives (5 µM) were thenat tested at different the rest of the compounds were not toxic. In general, our Tdp1 inhibitors doubled the cytotoxic concentrations of topotecan. The most toxic compound 11f caused 20% cell death at this potential of topotecan as can be seen in Figure 6 and Table 3. concentration; the rest of the compounds were not toxic. In 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

6.8 ± 1.1

Tpc 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

120

Tpc + DMSO Tpc + 11g Tpc + 12g Tpc + 11f

HeLa cell viability, %

100

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HeLa cell viability, %

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60 40 120

Tpc + DMSO Tpc + 11g Tpc + 12g Tpc + 11f

20 100 800 60

0

2

4

6

8

10

Topotecan, μM 40

Figure 6. The influence of the Tdp1 inhibitors at 5 µM on topotecan cytotoxicity. Error bars show standard deviations. 20 Table 3. The influence0of 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 0 2 4 6 8 10 Tpc 6.8 ± 1.1 Topotecan, μM Tpc + 11g 3.5 ± 0.6 2.3 ± 0.5 Figure6. 6. The The influence of ofthe theTdp1 Tdp1 inhibitors µMon ontopotecan topotecancytotoxicity. cytotoxicity. Errorbars barsshow show Figure influence Tpc + 12g 2.9inhibitors ± 0.4 atat55µM 3.3 ± 1.1 Error standard deviations. standard deviations. Tpc + 11f 1.7 ± 0.3 not determined For comparison, the of was to 11f Table 3. The influence of the Tdp1 inhibitors at 5 µM on theincreased cytotoxic of Compound topotecan (Tpc). For comparison, theconcentration concentration ofthe theinhibitors inhibitors was increasedpotential to20 20µM. µM. Compound 11fwas was not used due to its high toxicity. Again, compounds 12g and 11g had a significant effect, p < 0.05, not used Compounds due to its highCC toxicity. Again, compounds 12g CC and50,11g significant effect, p < 0.05, 50, µM - 5 µM Tdp1 inhibitor µMhad - 20 aµM Tdp1 inhibitor confirmed the Table 3). ItIt isis interesting to note confirmed by byTpc theMann–Whitney Mann–Whitney U-test U-test (Figure (Figure 77and and Table 3). interesting to note that that the the 6.8 ± 1.1 sensitizing effect of Tdp1 inhibitors was practically the same at 5 and 20 µM. sensitizingTpc effect of Tdp1 inhibitors was practically the same at 5 and 20 µM. + 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

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.

Figure Influenceofof Tdp1 inhibitors at µM 20 on µMtopotecan on topotecan cytotoxicity. Error bars show Figure 7. 7. Influence Tdp1 inhibitors at 20 cytotoxicity. Error bars show standard standard deviations. deviations.

Derivatives Derivatives11g 11gand and12g 12gcan canbe beconsidered consideredto tobe bethe thelead leadcompounds compoundssince sincethey theyare, are,unlike unlike11f, 11f, non-toxic non-toxic(11g) (11g)or ormoderately moderatelytoxic toxic(12g) (12g)and andhave haveaapronounced pronouncedsensitizing sensitizingeffect effecton ontopotecan. 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. RB (rotatable bonds)) are given in Table S3. The MW of the ligands lies between 325.4 and 684.4 g mol−1 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.

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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 Gaussian 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)

KDI2b = IMW × Ilog P × IHD × IHA × IRB × IPSA

(2)

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 CH2 Cl2 -MeOH. The 1 H and 13 C 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].

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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% Spectra NMR 1 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, OCH2 O), 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, H-8). Spectra NMR 13 C (100 MHz, DMSO-d6 , δ, ppm): 26.18 (C-5), 40.02 (SO2 CH3 ), 55.70 (C-6), 57.44 (OCH3 ), 102.23 (OCH2 O), 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 (C-2), 150.17 (C-3), 151.89 (C-10). Spectra IR (cm−1 ): 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 C20 H18 NO6 S+ , 400.085 found: 400.081. 3.1.4. 9-Ethanesulfonyloxy-10-methoxy-2,3-methylenedioxyprotoberberine chloride (10b) Yield: 25% Spectra NMR 1 H (400 MHz, DMSO-d6 , δ, ppm, J/Hz): 1.52 (3H, t, J = 7.32 Hz, CH2 CH3 ), 3.22 (2H, t, J = 6.05 Hz, H-5), 3.89 (2H, q, J1 = 3.50 Hz, J2 = 10.96 Hz, CH2 CH3 ), 4.12 (3H, s, OCH3 ), 5.01 (2H, t, J = 5.91 Hz, H-6), 6.18 (2H, s, OCH2 O), 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 13 C (100 MHz, DMSO-d6 , δ, ppm): 8.18 (CH2 CH3 ), 26.19 (C-5), 47.00 (SCH2 ), 55.83 (C–6), 57.44 (OCH3 ), 102.20 (OCH2 O), 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), 151.71 (C-10). Spectra IR (cm−1 ): 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 C21 H20 NO6 S+ , 414.101 found: 414.096. 3.1.5. 10-Methoxy-2,3-methylenedioxy-9-((propane-1-sulfonyl)oxy)protoberberine chloride (10c) Yield: 34% Spectra NMR 1 H (400 MHz, DMSO-d6 , δ, ppm, J/Hz): 1.11 (3H, t, J = 7.45 Hz, CH2 CH3 ), 1.94–2.03 (2H, m, CH2 CH3 ), 3.22 (2H, t, J = 5.72 Hz, H-5), 3.87 (2H, t, J = 7.58 Hz, SCH2 CH2 ), 4.12 (3H, s, OCH3 ), 5.01 (2H, t, J = 6.02 Hz, H-6), 6.19 (2H, s, OCH2 O), 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-d , δ, ppm): 12.49 (CH CH ), 17.17 (CH CH CH ), 26.16 (C-5), 53.27 (SCH ), 6 2 2 3 2 3 2 55.80 (C-6), 57.46 (OCH3 ), 102.18 (OCH2 O), 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), 143.91 (C-8), 147.70 (C-2), 150.12 (C-3), 151.69 (C-10). Spectra IR (cm−1 ): 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 C22 H22 NO6 S+ , 428.116 found: 428.121.

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3.1.6. 9-(Butane-1-sulfonyl)oxy-10-methoxy-2,3-methylenedioxy protoberberine chloride (10d) Yield: 50% Spectra NMR 1 H (400 MHz, DMSO-d6 , δ, ppm, J/Hz): 0.97 (3H, t, J = 7.34 Hz, CH2 CH3 ), 1.48–1.58 (2H, m, CH2 CH3 ), 1.89–1.97 (2H, m, SCH2 CH2 ), 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, OCH2 O), 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). Spectra NMR 13 C (100 MHz, DMSO-d6 , δ, ppm): 13.44 (CH3 CH2 ), 20.74 (CH3 CH2 ), 25.29 (SCH2 CH2 ), 26.19 (C-5), 51.75 (SCH2 ), 55.80 (C-6), 57.46 (OCH3 ), 102.20 (OCH2 O), 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 (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−1 ): 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 C23 H24 NO6 S+ , 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 crystal structure of TDP1 Tdp1 (PDB ID: 6DIE, resolution 1.78 Å) [32] which was obtained from the Protein Data Bank (PDB) [40,41]. The Scigress version

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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 PARP

Tyrosyl-DNA phosphodiesterase 1 Poly (ADP-ribose) polymerase

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International Journal of Molecular Sciences Article The First Berberine-Based Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), an Important DNA...
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