Synthesis and In Vitro Antitumor Activity of Novel Bivalent - MDPI

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Oct 15, 2018 - the N9 position, it was found that the antitumor activity of p-methylbenzyl (4C, 28.06 µM) substituent was better than that of the o-methylbenzyl ...

International Journal of

Molecular Sciences Article

Synthesis and In Vitro Antitumor Activity of Novel Bivalent β-Carboline-3-carboxylic Acid Derivatives with DNA as a Potential Target Hongling Gu 1 , Na Li 1 , Jiangkun Dai 1 , Yaxi Xi 1 , Shijun Wang 1 and Junru Wang 1,2, * 1

2

*

College of Chemistry and Pharmacy, Northwest A&F University, No. 22 Xinong Road, Yangling 712100, China; [email protected] (H.G.); [email protected] (N.L.); [email protected] (J.D.); [email protected] (Y.X.); [email protected] (S.W.) State key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China Correspondence: [email protected]; Tel.: +86-29-8709-2662

Received: 22 August 2018; Accepted: 25 September 2018; Published: 15 October 2018

 

Abstract: A series of novel bivalent β-carboline derivatives were designed and synthesized, and in vitro cytotoxicity, cell apoptosis, and DNA-binding affinity were evaluated. The cytotoxic results demonstrated that most bivalent β-carboline derivatives exhibited stronger cytotoxicity than the corresponding monomer against the five selected tumor cell lines (A549, SGC-7901, Hela, SMMC-7721, and MCF-7), indicating that the dimerization at the C3 position could enhance the antitumor activity of β-carbolines. Among the derivatives tested, 4B, 6i, 4D, and 6u displayed considerable cytotoxicity against A549 cell line. Furthermore, 4B, 6i, 4D, and 6u induced cell apoptosis in a dose-dependent manner, and caused cell cycle arrest at the S and G2/M phases. Moreover, the levels of cytochrome C in mitochondria, and the expressions of bcl-2 protein, decreased after treatment with β-carbolines, which indicated that 6i and 6u could induce mitochondria-mediated apoptosis. In addition, the results of UV-visible spectral, thermal denaturation, and molecular docking studies revealed that 4B, 6i, 4D, and 6u could bind to DNA mainly by intercalation. Keywords: bivalent β-carbolines; antitumor; apoptosis; DNA-binding affinity; bcl-2

1. Introduction β-carboline alkaloids, originally isolated from Peganum harmala, are a class of natural and synthetic indole alkaloids with a tricyclic pyrido[3,4-b]indole ring system [1,2]. β-carbolines have multiple biological and pharmacological properties [2–4], of which antitumor activity is the most widely studied [5–8]. Previous studies have shown that β-carbolines exerted their antitumor activity mainly through intercalating into DNA [7,9–13], inducing apoptosis, and inhibiting topoisomerase I and II (Top I and II) [13–15], cyclin-dependent kinases (CDKs) [16,17], mitogen-activated protein kinase (MAPK) [18], and I-Kappa-B kinase (IKK) [19]. Particularly, parent carbolines can be inserted into DNA, which can further cause cell apoptosis [20,21]. The interaction between β-carboline alkaloids and DNA could cause changes in DNA conformation, which further affects DNA replication, transcription, and repair [22,23]. Furthermore, the existing studies have demonstrated that β-carboline could induce HepG2 cells apoptosis by down-regulating bcl-2 expression [24]. DNA-targeted antitumor drugs, such as anthracyclines, acridines, and quinones, which exert antitumor activity through DNA insertion, are considered to be one of the most effective drugs in clinical applications [25]. These drugs exert antitumor activity mainly through non-covalent and covalent interactions with the minor groove, major groove, or base pairs (intercalation) of the DNA double helix. The discovery of antitumor drugs has focused on the development of new Int. J. Mol. Sci. 2018, 19, 3179; doi:10.3390/ijms19103179

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intercalating scaffolds, suchsuch as β-carboline derivatives [7].[7]. Therefore, we DNA-intercalating scaffolds, as β-carboline derivatives Therefore, weexpect expecttoto discover discover and scaffolds with with appropriate appropriate substituents. substituents. develop new antitumor agents by modifying these bioactive scaffolds Previous structure-activity structure-activity relationships relationships(SARs) (SARs) have have shown shown that that the introduction introduction of appropriate Previous the CC11, C C33, and N9 positions of the β-carboline β-carboline scaffolds could enhance enhance antitumor antitumor substituents at the activity and andDNA-binding DNA-binding affinity [5,7,10,26,27]. has also been that reported and activity affinity [5,7,10,26,27]. It hasItalso been reported phenylthat and phenyl heterocyclic 1 3 1 3 heterocyclicatsubstituted the C and C positionsrespectively, of β-carbolines, respectively, have antitumor exhibited substituted the C and Cat positions of β-carbolines, have exhibited potential potential[12,28–30]. antitumor activity [12,28–30]. The introduction of methyl and benzylatsubstituents at the N activity The introduction of methyl and benzyl substituents the N9 position of9 position of β-carbolines could the DNA Further have demonstrated that β-carbolines could enhance theenhance DNA affinity [10]. affinity Further[10]. studies have studies demonstrated that dimerization dimerization of small molecules bylinkers appropriate linkers couldimprove significantly improve the DNA-binding of small molecules by appropriate could significantly the DNA-binding affinity. It has affinity. It has found that compounds the dimerized compounds couldbybind to DNA by a bis-intercalation been found thatbeen the dimerized could bind to DNA a bis-intercalation mode, causing mode, causing pronounced changes in DNA structure Bivalent β-carbolines at the pronounced changes in DNA structure [31,32]. Bivalent[31,32]. β-carbolines linked at the C6linked position or NC96 9 positionhave or N been position have been synthesized, foundanti-Alzheimer to be potentialagents anti-Alzheimer agents position synthesized, and were foundand to bewere potential [33]. In addition, [33].synthesis In addition, synthesis evaluation of bivalent β-carbolines linked by 3–10atmethylene units the and the evaluation of and bivalent β-carbolines linked by 3–10 methylene units the N9 position 9 at antitumor the N position antitumor agents have[34]. also Our beengroup reported [34]. Our group has synthesized 25 as agentsashave also been reported has synthesized 25 bivalent β-carbolines 7 (-OCH 2 1 (-CH9 7 (-OCH3), and N9 positions (-CH3 and -CH2Ph2), and bivalent β-carbolines modified at the C 3 ), C modified at the C1 (-CH ), C ), and N positions (-CH and -CH Ph ), and dimerized at the C 3 3 3 2 2 2 position, and the results showed that the dimerization could significantly increase dimerized at the position, and the Cresults showed that the dimerization could significantly increase antitumor activity antitumor activity of β-carbolines. Moreover, compounds 4A, exhibit 6b, 6d, good and 6e, which activity, exhibit good of β-carbolines. Moreover, compounds 4A, 6b, 6d, and 6e, which antitumor have antitumor activity, have been reported [5,35]. However, the SARs of in thevitro antitumor activity in vitro of been reported [5,35]. However, the SARs of the antitumor activity of bivalent β-carbolines bivalent linked atrarely the C3reported. position were rarely reported. linked at β-carbolines the C3 position were derivatives modified at In this study, we designed designed and andsynthesized synthesizedaaseries seriesofofnovel novelβ-carboline β-carboline derivatives modified 9 position 3 3position, and further investigated the antitumor activity and 9 the N and linked at the C at the N position and linked at the C position, and further investigated the antitumor DNA-binding affinity affinity in in vitro. vitro. We We expected expected to to discover discover novel novel β-carboline β-carboline derivatives with promising DNA-binding activity and and DNA-binding DNA-binding affinity. affinity. antitumor activity 2. Results 2.1. 2.1. Chemistry Chemistry 9 substituted β-carboline-3-carboxylic acid methyl esters Using Using L-tryptophan L-tryptophan as as aa raw raw material, material, N N9 substituted β-carboline-3-carboxylic acid methyl esters (monomers, and 4D) 4D) were were synthesized synthesized mainly mainly through through the the Pictet–Spengler Pictet–Spengler (P–S) (P–S) reaction, reaction, (monomers, 4A, 4A, 4B, 4B, 4C, 4C, and 9 substituted esterification reaction, and 9-substitution reaction, as previously described [5,36]. The N esterification reaction, and 9-substitution reaction, as previously described [5,36]. The N9 substituted β-carboline-3-carboxylic acid methyl methyl esters esters prepared prepared in in the the above β-carboline-3-carboxylic acid above steps steps were were hydrolyzed hydrolyzed and and then then reacted with dibromoalkane to obtain a series of bivalent β-carboline derivatives (dimers, 6a-6f, reacted with dibromoalkane to obtain a series of bivalent β-carboline derivatives (dimers, 6a-6f, 6g6g-6l, 6m-6r, 6s-6x) [37]. synthetic routes reaction conditions of β-carboline monomers 6l, 6m-6r, andand 6s-6x) [37]. TheThe synthetic routes andand reaction conditions of β-carboline monomers (4A, (4A, 4B, 4C, dimers (6a-6f, 6g-6l, 6m-6r, and 6s-6x)are areshown shownininSchemes Schemes 11 and and 2, 2, 4B, 4C, and and 4D) 4D) andand dimers (6a-6f, 6g-6l, 6m-6r, and 6s-6x) respectively. In this study, we synthesized 3 novel monomers and 21 novel dimers, and evaluated their respectively. In this study, we synthesized 3 novel monomers and 21 novel dimers, and evaluated antitumor activity. their antitumor activity.

9 -substituted 9-substituted Scheme β-carboline-3-carboxylic acid methyl esters (4A,(4A, 4B, 4C, β-carboline-3-carboxylic acid methyl esters 4B, and 4C, 4D). and Scheme 1. 1. Synthesis SynthesisofofNN + , HCHO, + Reagents and conditions: (a) H room temperature (r.t.); (b) SOCl , CH OH, reflux; (c) Pd/C, 4D). Reagents and conditions: (a) H , HCHO, room temperature (r.t.); (b)2 SOCl 3 2, CH3OH, reflux; (c) xylene, reflux; reflux; (d) DMF, R1 -Br. Pd/C, xylene, (d)NaH, DMF,r.t., NaH, r.t., R1-Br.

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Scheme 2.2.Synthesis Synthesis of bivalent β-carboline-3-carboxylic acid derivatives (6a-6f, 6g-6l, 6m-6r, of bivalent β-carboline-3-carboxylic acid derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x). and 6s-6x). Reagents and (a) conditions: THF/CH OH–, K r.t.; (b) DMF, Kheat. Reagents and conditions: THF/CH3(a) OH, OH–, r.t.; (b) DMF, 2CO 3, BrR 2Br, 3 OH, 2 CO3 , BrR2 Br, heat.

2.2. In Vitro Vitro Cytotoxicity 2.2. In Cytotoxicity Assay Assay 9 position and linked The antitumor activity ofnovel novelβ-carboline β-carbolinederivatives derivativesmodified modifiedatatthe theNN 9 position The antitumor activity of and linked at 3 position have been evaluated in vitro against A549, Hela, SGC-7901, SMMC-7721, MCF-7, at the C 3 the C position have been evaluated in vitro against A549, Hela, SGC-7901, SMMC-7721, MCF-7, and and MRC5 lines using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) bromide)assay, assay, MRC5 cell cell lines using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium with the results expressed as IC Values. Previous reports have evidenced that β-carbolines substituted 50 IC50 Values. Previous reports have evidenced that β-carbolines with the results expressed as with an alkoxycarbonyl or carboxyl the C3 position, and withand a short or alkyl benzyl the Nat9 substituted with an alkoxycarbonyl oratcarboxyl at the C3 position, withalkyl a short or at benzyl position, exhibited more significant antitumor activitiesactivities against Hela cells [5]. cells In the[5]. present the N9 position, exhibited more significant antitumor against Hela In theresearch present (Table 1), for all the cell lines tested, only the β-carboline monomer substituted with o-fluorobenzyl research (Table 1), for all the cell lines tested, only the β-carboline monomer substituted with o(4D) at N9 position displayed hihest antitumor activity. For A549 cell line, allline, the substituents at fluorobenzyl (4D) at N9 position displayed hihest antitumor activity. For among A549 cell among all the 9 position, it was found that the antitumor activity of p-methylbenzyl (4C, 28.06 µM) substituent the N substituents at the N9 position, it was found that the antitumor activity of p-methylbenzyl (4C, 28.06 was than that the o-methylbenzyl 39.20 µM) substituent. the o-fluorobenzyl side μM) better substituent wasofbetter than that of the(4B, o-methylbenzyl (4B, 39.20 Next, μM) substituent. Next, the ochain with the lowest IC value (4D, 13.94 µM) was considered to be the most beneficial to increase 50 fluorobenzyl side chain with the lowest IC50 value (4D, 13.94 μM) was considered to be the most antitumor activity at the N9 position. Butatthe activities bivalent β-carboline But theofantitumor activities ofderivatives bivalent βbeneficial to increase antitumor activity theantitumor N9 position. (dimer, 6i,derivatives 6.93 µM; 6u, 5.61 µM) apparently of the corresponding monomer carboline (dimer, 6i, were 6.93 μM; 6u, 5.61better μM) than werethat apparently better than that of the (4B, 39.20 µM; 4D, 13.94 µM), indicating that the dimerization was an effective modification corresponding monomer (4B, 39.20 μM; 4D, 13.94 μM), indicating that the dimerization was for an improving the antitumor activity of β-carbolines. The IC values of bivalent β-carboline derivatives 50 effective modification for improving the antitumor activity of β-carbolines. The IC50 values of bivalent were relatively lower when linkers lower were short length and oddshort in carbon numbers (6s,in6.12 µM; β-carboline derivatives werethe relatively wheninthe linkers were in length and odd carbon 9 position and 6u, 5.61 µM). These results indicated that the introduction of o-fluorobenzyl at the N numbers (6s, 6.12 μM; 6u, 5.61 μM). These results indicated that the introduction of o-fluorobenzyl at 3 position could significantly increase the antitumor activity of β-carbolines, the at the the dimerization N9 position and theC dimerization at the C3 position could significantly increase the antitumor and they have better antitumor activity length ofactivity the linkers was to six activity of β-carbolines, and they have when betterthe antitumor when thefour length ofmethylene the linkersunits. was

four to six methylene units.

Int.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 17 Moreover, compounds exhibited low cytotoxicities against the MRC5 cell line. Because Int. J. Mol. Sci. 2018,most 19, x FOR PEER REVIEW 4 of of 17 Moreover, most compounds exhibited low cytotoxicities against the MRC5 cell line. Because Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of of 17 the modest cytotoxicity of 6i (6.93 μM) and 6u (5.61 μM) against the A549 cell line, and in order to Int. J. Mol. Sci. 2018,most 19, x FOR PEER REVIEW 4 of of 17 Moreover, compounds exhibited cytotoxicities against MRC5 cell line. Because the cytotoxicity of 6i (6.93 μM) andlow 6umonomers (5.61 μM) against the the A549 cell4D, line, and in order Int. J.modest Mol. Sci. 19, x FOR PEER REVIEW 4 of to 17 compare the2018, antitumor mechanisms between and against dimers, 4B,MRC5 6i, and 6u,in the A549 Moreover, most compounds exhibited low cytotoxicities the cell line. Because of the modest cytotoxicity of 6i (6.93exhibited μM) andlow 6umonomers (5.61 μM) against the the A549 cell4D, line, and order to Moreover, most compounds cytotoxicities against MRC5 cell line. Because compare the antitumor mechanisms between and dimers, 4B, 6i, and 6u, the A549 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of of 17 cells were selected to do the further investigation. the modest cytotoxicity of 6i (6.93 μM) and 6u (5.61 μM) against the A549 cell line, and in order to Moreover, most compounds exhibited low cytotoxicities against the MRC5 cell line. Because of compare the antitumor between dimers, 4B, 6i, and 6u,inthe A549 Int. J.modest Mol. Sci. 2018, 19, x FOR mechanisms PEER REVIEW 4 of to 17 the cytotoxicity (6.93exhibited μM) and low 6umonomers (5.61 μM) and against the A549 cell4D, line, and order cells were selected do of the6ifurther investigation. Moreover, most compounds cytotoxicities the cell line. Because of Int. J.modest Mol. Sci. 2018, 19, to x FOR PEER REVIEW 4A549 of to 17 compare the antitumor mechanisms between andagainst dimers, 4B,MRC5 6i, and 6u, the the cytotoxicity of (6.93exhibited μM) and low 6umonomers (5.61 μM) against the A549 cell4D, line, and in order cells were selected to do mechanisms the6ifurther investigation. Moreover, most compounds cytotoxicities against the MRC5 cell line. Because of compare the antitumor between monomers and dimers, 4B, 6i, 4D, and 6u, the A549 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 17 the modest cytotoxicity of 6ifurther (6.93 μM) and monomers 6u (5.61 μM) against the 4D) A549 cell line, and in order to Table 1. The IC 50 values (μM) of β-carboline (4A, 4B, 4C, and and bivalent β-carboline cells were selected to do the investigation. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of of 17 compare the antitumor mechanisms between and dimers, 4B,and 6i, 4D, and 6u,in the A549 Moreover, most compounds exhibited cytotoxicities against the MRC5 cell line. Because J.cells Mol. Sci. 2018, 19, the cytotoxicity of 6i (6.93 μM) andlow 6umonomers (5.61 μM) against the A549 cell line, and order to Table 1. The IC50xto3179 values (μM) of exhibited β-carboline monomers (4A, 4B, against 4C, and 4D) bivalent β-carboline were selected do the further investigation. Int. J.modest Mol. Sci. 2018, 19, FOR PEER REVIEW 4A549 of of 17 compare the antitumor mechanisms between monomers and dimers, 4B, 6i, 4D, and 6u,Because the Moreover, most compounds low cytotoxicities the MRC5 cell line. derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x) against A549, SGC-7901, Hela, SMMC-7721, MCF-7, and Int. J.modest Mol. Sci.selected 19, x values FOR PEER REVIEW 4 of to 17 cells were do the further investigation. the cytotoxicity of 6i (6.93 μM) and low 6umonomers (5.61 μM) against the A549 cell line, and order Table 1.2018, The IC50to (μM) of and β-carboline monomers (4A, 4B, against 4C, and 4D) and bivalent β-carboline Moreover, most compounds exhibited cytotoxicities the MRC5 cell line. Because of derivatives (6a-6f, 6g-6l, 6m-6r, 6s-6x) against A549, SGC-7901, Hela, SMMC-7721, MCF-7, and compare the antitumor mechanisms between and dimers, 4B, 6i, 4D, and 6u,in the Int. J.modest Mol. Sci. 2018, 19, PEER REVIEW 4A549 of of 17 cells were selected do the further investigation. MRC5 cell lines. the cytotoxicity of 6i (6.93 μM) andlow 6u (5.61 μM) against the A549 cell line, and in order to Table 1. The IC50xtoFOR values (μM) of and β-carboline monomers (4A, 4B, against 4C, and 4D) and bivalent β-carboline Moreover, most compounds exhibited cytotoxicities the MRC5 cell line. Because derivatives (6a-6f, 6g-6l, 6m-6r, 6s-6x) against A549, SGC-7901, Hela, SMMC-7721, MCF-7, and compare the antitumor mechanisms between monomers and dimers, 4B, 6i, 4D, and 6u, the A549 theJ.modest cytotoxicity 6ifurther (6.93 μM) andlow 6u (5.61 μM) the 4D) A549 cell line, and in order to Int. Mol. Sci.selected 2018, 19, FOR PEER REVIEW 4 of of 17 Table 1. The IC50xtovalues (μM) of exhibited β-carboline monomers (4A,against 4B, against 4C, and and bivalent β-carboline Moreover, most compounds cytotoxicities the MRC5 cell line. Because MRC5 cell lines. cells were do of the investigation.

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compare the antitumor mechanisms between and dimers, 4B, 6i, 4D, and 6u,in the A549 derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x) against A549, SGC-7901, Hela, SMMC-7721, MCF-7, and the modest cytotoxicity of 6i (6.93 μM) andlow 6umonomers (5.61 μM) against the A549 cell line, and order to Moreover, most compounds cytotoxicities against the MRC5 cell line. Because of Table 1. The ICSubstituents 50 (μM) of exhibited β-carboline monomers (4A, 4B, 4C, and bivalent β-carboline MRC5 cell lines. a cells werethe selected tovalues do the further investigation. IC 50 (μM) Mean ±and SD 4D) derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x) against A549, SGC-7901, Hela, SMMC-7721, MCF-7, and compare antitumor mechanisms between monomers and dimers, 4B, 6i, 4D, and 6u,in the A549 the modest cytotoxicity of 6i (6.93 μM) andlow 6u (5.61 μM) against the A549 cell line, and order to Moreover, most compounds cytotoxicities against the MRC5 cell line. Because of Table 1. The IC50tovalues of exhibited β-carboline monomers (4A, 4B, 4C, and 4D) and bivalent β-carboline Moreover, most compounds exhibited low cytotoxicities against the MRC5 cell line. Because of MRC5 cell lines. cells werethe selected do the(μM) further investigation. aA549 compare antitumor mechanisms between monomers and dimers, 4B, 6i, 4D, and 6u, the A549 the modest cytotoxicity of 6i (6.93 μM) and 6u (5.61 against cell line, and in order to Substituents ICμM) 50 (μM) Mean ±the SD the derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x) against A549, SGC-7901, Hela, SMMC-7721, MCF-7, and Moreover, most compounds exhibited low cytotoxicities against MRC5 cell line. Because of MRC5 cell lines. Table 1. The ICSubstituents 50 (μM) of β-carboline monomers (4A, 4B, 4C, 4D) and bivalent β-carboline cellsmodest werethe selected tovalues do the further investigation. compare antitumor mechanisms between monomers and dimers, 4B, 6i, 4D, and 6u,inthe A549 aA549 Compounds SGCSMMCthe cytotoxicity of 6i (6.93 μM) and against 6u (5.61 against the cell line, and order to derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x) A549, SGC-7901, Hela, SMMC-7721, MCF-7, and ICμM) 50 (μM) Mean ±and SD MRC5 cell lines. theCompounds modest cytotoxicity of 6i (6.93 µM) and 6u (5.61 µM) against the A549 cell line, and in order to cells were selected to do the further investigation. compare the antitumor mechanisms between monomers and dimers, 4B, 6i, 4D, and 6u, the A549 b b b Table 1.cytotoxicity The(6a-6f, 50 values (μM) of and β-carboline monomers (4A, 4B, 4C, ±and 4D) and bivalent β-carboline Hela MCF-7 RIC 1 Substituents R 2(6.93 A549 MRC5 a SMMC-7721, SGCSMMCthe modest of 6i μM) and against 6umonomers (5.61 μM) against the A549 cell line, and order to IC 50 (μM) Mean SD derivatives 6g-6l, 6m-6r, 6s-6x) A549, SGC-7901, Hela, MCF-7, and cells werethe selected to do the further investigation. MRC5 lines. compare antitumor mechanisms between and dimers, 6i, 4D, and 6u,inthe A549 b b and Table 1.cell The 50 values (μM) of β-carboline 4B, 4D) bivalent b monomers b4C, bβ-carboline a 4B, 7901 7721 Hela MCF-7 RIC 1 Substituents Rfurther 2 A549 MRC5 Compounds IC50(4A, (μM) Mean ±and SD SGCSMMCcells were selected to do the investigation. derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x) against A549, SGC-7901, Hela, SMMC-7721, MCF-7, and compare antitumor mechanisms between monomers and dimers, 6i,bivalent 4D, and 6u, 4D, the A549 compare thethe antitumor mechanisms between and dimers, 4B, 6i, and 6u, the A549 cells Table 1. The 50 values (μM) of β-carboline monomers 4B, 4D) bmonomers b and MRC5 cell lines. a 4B, b b4C, bβ-carboline 7901 7721 IC50(4A, (μM) Mean ±and SD Hela MCF-7 RIC 1 Substituents Rfurther 2 A549 MRC5 Compounds SGCSMMCcells were selected to do the investigation. derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x) against A549, SGC-7901, Hela, SMMC-7721, MCF-7, and Table 1. The IC 50 values (μM) of β-carboline (4A, 4B, and 4D) a bIC50 b and bivalent MRC5 cell lines. b monomers b4C, ± bβ-carboline (μM) Mean SD 7901 7721 Compounds SGCSMMCHela MCF-7 MCF-7, RIC 1 Substituents R 2 A549 MRC5 derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x) against A549, SGC-7901, Hela, SMMC-7721, and cells were selected tovalues do the further investigation. The 50 (μM) 4B, 4C, and 4D) were selected tolines. do the further investigation. MRC51.cell b - of β-carboline >80 b monomers >80 b (4A, >80 >80 band bivalent >80 bβ-carboline 35.60 ± 0.30 4ATable a Compounds Hela MCF-7 MCF-7, RIC 1 Substituents R2 of and A549 MRC5 SGCSMMC7901A549, 7721 derivatives 6g-6l,(μM) 6m-6r, 6s-6x) against SGC-7901, Hela, SMMC-7721, and IC50(4A, (μM) Mean SD Table 1. The(6a-6f, 50 values β-carboline monomers 4B, 4C, ±and 4D) and bivalent β-carboline b b MRC51.cell lines. b b (6a-6f, 6g-6l,(μM) 6m-6r, 6s-6x) SGC-7901, Hela, SMMC-7721, and >80 b against >80A549, >80 >80 >80 MCF-7, 35.60 ± 0.30 4Aderivatives Compounds SGCSMMC7901 7721 Hela MCF-7 RIC 1 Substituents R-2 of and A549 MRC5 a Table The 50 values β-carboline monomers 4B, 4C, ±and 4D) and bivalent β-carboline IC50(4A, (μM) Mean SD MRC5 cell lines. b against b b b b derivatives (6a-6f, 6g-6l,(μM) 6m-6r, and 6s-6x) A549, SGC-7901, Hela, SMMC-7721, MCF-7, and Hela MCF-7 R 1 50 values R 2 A549 MRC5 7901 7721 >80 >80 >80 >80 >80 35.60 ± 0.30 4AMRC5 a Compounds SGCSMMCTable 1. The IC of β-carboline monomers (4A, 4B, 4C, and 4D) and bivalent β-carboline Substituents IC 50 (μM) Mean ± SD cell lines. b b 39.20 ± 42.31 ± 31.28 ± derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x) against A549, SGC-7901, Hela, SMMC-7721, MCF-7, and 7901 7721 b b b a -2 >80 >80 IC >80 >80 >80 4D) and 35.60 ±bivalent 0.30 4A Hela RIC 1 SubstituentsR A549 MRC5 Compounds 50 (μM) Mean ±(4A, SD SGCSMMCTable 1. cell Thelines. values of39.20 β-carboline monomers 4B, 4C,MCF-7 and β-carboline >80 >80 MCF-7, 51.78 0.10 - (µM) 4BMRC5 50 6g-6l, derivatives (6a-6f, 6m-6r, and 6s-6x) SGC-7901, SMMC-7721, and 42.31 ±IC 31.28 ±b b b a >80 ±b against >80A549, >80 >80 >80 35.60 ±± 0.30 4A b 7901 7721 50 (μM) Mean ± Hela, SD Compounds Hela MCF-7 R1 Substituents R- 2 A549 MRC5 SGCSMMCMRC5 cell lines. 1.18 2.13 2.33 a >80 >80 b 51.78 ± 0.30 0.10 4B 39.20 ±b 42.31 31.28 ±b SubstituentsR- 2 (μM) Mean ± SD >80and >80 b±IC50against >80 35.60 4A b 7901 7721 Compounds Hela MCF-7 R 1 A549 MRC5 SGCSMMCMRC5 cell lines. derivatives (6a-6f, 6g-6l, 6m-6r, 6s-6x) A549, SGC-7901, Hela, SMMC-7721, MCF-7, a 1.18 2.13 2.33 Mean >80 >80 51.78 ±± 0.10 4B >80 ±b >80 bIC >80 >80 >80 b 35.60 0.30 4A 39.20 42.31 ± 50 (μM) 31.28 ± ± SD b b Compounds SGCSMMC7901 7721 Hela MCF-7 R1 SubstituentsR--2 A549 MRC5 a 28.06 47.98 42.55 SubstituentsR--2 Mean ± SD 1.18 2.13 2.33 Compounds 39.20 ±±b 42.31 ±b±IC50 (μM) 31.28 ±±b SGCSMMC>80 >80 51.78 ± 0.10 4B b b >80 >80 >80 >80 >80 35.60 ± 0.30 4A Hela MCF-7 R 1 A549 MRC5 7901 7721 and lines. 4C >80 >80 b 35.35 0.30 a b 28.06 47.98 42.55 ±b Compounds SGCSMMCMean ± SD >80 >80 51.78 ±±± 0.10 4B MRC5 cell 39.20 42.31 31.28 Hela MCF-7 R1 SubstituentsR---2 A549 MRC5 1.18 2.13 2.33 7901 7721 >80 ±b >80 bb±IC50 (μM) >80 >80 b >80 35.60 0.30 4A 0.13 0.09 0.98 b 4C >80 b >80 b 35.35 ± 0.30 Compounds SGCSMMCHela MCF-7 R1 R-- 2 A549 MRC5 28.06 47.98 42.55 39.20 42.31 31.28 1.18 2.13 2.33 51.78 4B 7901 7721 >80 ±±b >80 ±b± >80 ±b± >80 b >80 b 35.60 ± 0.10 0.30 4A 0.13 0.09 0.98 Hela R1 A549 MRC5 7901 7721 4C >80 >80 35.35 ±±± 0.30 >80 >80 51.78 0.10 4B Compounds 1.18 2.13 2.33 28.06 47.98 42.55 SGCSMMCa 39.20 42.31 31.28 ±± >80 ±±±b >80 b±±± >80IC >80 >80 35.60 0.30 4A SubstituentsR---2 (µM) Mean ± MCF-7 SD 13.94 17.84 26.36 53.92 7901 7721 0.13 0.09 0.98 1.18 2.13 2.33 28.06 ±± 47.98 ±± 42.55 ±±±b 50 4C >80 b >80 ±b 35.35 ± 0.30 0.30 Hela MCF-7 R1 R--2 A549 MRC5 51.78 0.10 4B 39.20 42.31 31.28 >80 >80 >80 >80 >80 35.60 ± 4A 4D 35.61 ± 1.33 35.38 ± 0.01 b b Compounds 13.94 17.84 26.36 53.92 ± 4C >80 >80 35.35 ± 0.30 -28.06 ± 47.98 ± 42.55 ± 0.13 0.09 0.98 7901 7721 b b b b >80 >80 >80 35.60 4A 1.18 2.13 2.33 >80 >80 51.78 ± 0.10 4B 39.20 ± 42.31 ± 31.28 ± R R MRC5 A549 SGC-7901 Hela SMMC-7721 MCF-7 b 1 0.07 0.66 1.33 2.02 4D 35.61 ± 1.33 35.38 0.01 13.94 17.84 26.36 53.92 28.06 47.98 42.55 0.13 0.09 0.98 >80 ±±± >80 ±±± >80 ±±± 35.60 ±± 0.30 4A 4C >80 >80 35.35 -- 2 1.18 2.13 2.33 >80 >80 ± 51.78 0.10 4B 39.20 42.31 31.28 0.07 0.66 1.33 2.02 4D 35.61 ± 1.33 35.38 ± 0.01 >80 >80 >80 >80 >80 35.60 0.30 4A 4C 35.35 ± 0.13 0.09 0.98 13.94 ± 17.84 ± 26.36 ± 53.92 ± 28.06 ± 47.98 ± 42.55 ± 39.20 42.31 31.28 1.18 ± 2.13 ± 2.33 ± >80 >80 ± 51.78 ± 0.10 4B 73.45 24.27 17.76 17.89 0.07 0.66 1.33 2.02 0.13 0.09 0.98 13.94 ±± 17.84 26.36 53.92 4D 35.61 ± 1.33 35.38 ±± 0.30 0.01 >80 39.20 42.31 31.28 >80>80 >80 ±± >80 >80 ±± 35.60 4A 4A >80 35.60 ± 0.30 4C >80 >80 ± >80 32.57 35.35 -28.06 47.98 42.55 51.78 0.10 4B 1.18 2.13 2.33 0.10 25.73 0.32 6a -(CH 73.45 24.27 17.76 17.89 4D -- 2)335.61 ±± 1.33 35.38 39.20 42.31 31.28 13.94 17.84 26.36 53.92 0.07 ±± 0.66 ±± 1.33 ±± 2.02 >80 >80 ± 51.78 ±±± 0.01 0.10 4B 0.13 0.09 0.98 4C >80 >80 35.35 0.30 1.18 2.13 2.33 28.06 47.98 42.55 0.88 0.12 0.17 0.03 32.57 25.73 ± 0.32 6a -(CH 73.45 24.27 17.76 17.89 39.20 42.31 31.28 13.94 17.84 26.36 53.92 0.07 0.66 1.33 2.02 4D -- 2)335.61>80 1.33 35.38 0.01 >80 >80 ±± 51.78 ±± 0.10 4B 1.18 ±±± 2.13 ±±± 2.33 ±±± 0.13 0.09 0.98 4C >80 35.35 0.30 28.06 47.98 42.55 0.88 0.12 0.17 0.03 25.73 ±±±0.32 6a -(CH 35.61 1.33 35.38 ±±± 0.10 0.01 >80 >80 >80 51.78 --- 2)34B 0.07 0.66 1.33 2.02 1.18 ±±± 1.1847.98 2.1342.31 2.33 73.45 24.27 17.76 17.89 39.20 42.31 31.28 39.20 ± 2.13±± 31.28 2.33 53.92 51.78 ± 0.10 4B4D 13.94 17.84 26.36 28.06 ±± 42.55 0.13 0.09 0.98 4C >80 >80 ±±± >80 32.57 35.35 0.30 70.88 12.26 15.48 18.22 0.88 0.12 0.17 0.03 0.07 ±±± 0.66 ±±± 1.33 ±±± 2.02 1.18 2.13 2.33 73.45 24.27 17.76 17.89 32.57 ±± 0.30 25.73 0.32 6a -(CH 28.06 47.98 42.55 >80 >80 ±± 51.78 0.10 4B 4D 35.61 ±± 0.31 1.33 35.38 0.01 13.94 17.84 26.36 53.92 4C >80 >80 35.35 -- 22))43-0.13 0.09 0.98 6b 64.44 ± 0.09 66.53 ± -(CH 70.88 ± 12.26 ± 15.48 ± 18.22 ± 32.57 ±± 0.10 25.73 ±± 0.32 6a -(CH 28.06 47.98 42.55 73.45 24.27 17.76 17.89 0.88 0.12 ± 0.17 ± 0.03 ± 4C >80 >80 ± 35.35 0.30 -- 2)31.18 2.13 2.33 0.07 0.66 1.33 2.02 4D 35.61 1.33 35.38 0.01 0.13 0.09 0.98 13.94 17.84 26.36 53.92 2.33 0.04 0.32 0.52 6b 66.53 ±±0.31 -(CH 2)412.26 ±±± 18.22 28.06 47.98 42.55 73.45 24.27 17.76 17.89 0.88 0.12 0.17 0.03 4C >80 ±±± >80 64.44 35.35 ±± 0.09 0.30 >80 32.57 0.10 25.73 6a 4C 28.06 ± 0.09±±± 42.55 0.98 70.88 35.35 ± 0.30 0.13 0.09 0.98 0.07 ±±±± 0.1315.48 0.6647.98 1.33 2.02 4D -- 2 3 35.61>80 ± 0.32 1.33 35.38 0.01 13.94 17.84 26.36 53.92 2.33 0.04 0.32 0.52 6b 64.44 66.53 ±±± 0.31 -(CH 4C >80 >80 ±± 35.35 0.30 -- 22))43-32.57 ±±± 0.09 0.10 25.73 0.32 6a -(CH 0.13 ±± 0.09 ±± 0.98 ±± 0.88 0.12 0.17 0.03 70.88 12.26 15.48 18.22 28.06 47.98 42.55 73.45 24.27 17.76 17.89 13.94 17.84 26.36 53.92 0.07 0.66 1.33 2.02 4D 35.61 1.33 35.38 0.01 17.65 ± 18.72 ± 18.15 ± 76.85 ± 2.33 0.04 0.32 0.52 0.13 0.09 0.98 0.88 0.12 0.17 0.03 70.88 ± 12.26 ± 15.48 ± 18.22 ± 6b 64.44 0.09 66.53 ± 0.31 -(CH 2 ) 4 13.94 17.84 26.36 53.92 4C >80 >80 35.35 ± 0.30 32.57 0.10 25.73 0.32 6a -(CH 73.45 24.27 17.76 17.89 4D - 2)335.61 ± 0.03 1.33 35.38 ± 0.11 0.01 0.07 ±± 0.66 ±± 1.33 ±± 2.02 ±± 6c -(CH 55.15 39.34 17.65 18.15 6b 64.44 66.53 -(CH 70.88 12.26 15.48 18.22 13.94 17.84 26.36 53.92 2.33 0.04 ±± 0.0718.72 0.3217.84 0.52 4D -- 222)))453--35.61 ±±±±0.31 1.33 35.38 ±±± 0.09 0.01 0.13 0.09 0.98 4D 13.94 ± 0.66± 26.36 1.33 76.85 35.61 53.92 ± 2.02 35.38 ± 0.01 0.88 0.12 0.17 0.03 32.57 0.10 25.73 0.32 6a -(CH 0.07 0.66 1.33 2.02 73.45 ± ± 1.33 24.27 17.76 ± 17.89 0.09 0.07 0.03 3.01 6c -(CH 55.15 39.34 17.65 18.72 18.15 76.85 70.88 12.26 15.48 18.22 13.94 17.84 26.36 53.92 2.33 0.04 0.32 0.52 4D - 222))543-35.61 ±± 0.03 1.33 35.38 ±± 0.11 0.01 6b 64.44 0.09 66.53 0.31 0.07 ±±± 0.66 ±±± 1.33 ±±± 2.02 ±±± 0.88 0.12 0.17 0.03 32.57 0.10 25.73 0.32 6a -(CH 73.45 24.27 17.76 17.89 0.09 0.07 0.03 3.01 6c -(CH 22))54-55.15 ± 0.03 39.34 ± 0.11 4D 35.61 1.33 35.38 0.01 6b 64.44 ± 0.09 66.53 ± 0.31 -(CH 0.07 0.66 1.33 2.02 2.33 0.04 0.32 0.52 17.65 ± 18.72 ± 18.15 ± 76.85 ± 13.94 17.84 26.36 53.92 70.88 ± 12.26 ± 15.48 ± 18.22 ± 73.45 24.27 17.76 17.89 0.88 ± 0.12 ± 0.17 ± 0.03 ± 32.57 ± 0.10 25.73 ± 0.32 6a -(CH2)375.47 40.03 58.41 32.36 0.09 0.07 0.03 3.01 0.07 0.66 1.33 2.02 2.33 0.04 ±±± 0.1218.72 0.3217.76 0.52 17.65 ±± 18.15 ±± ± 0.32 6c -(CH 55.15 ±±±0.31 0.03 39.34 ±± 0.09 0.11 73.45 24.27 17.76 17.89 - 22)))435-- 35.61 1.33 35.38 0.01 6b 64.44 66.53 -(CH 70.88 12.26 15.48 18.22 32.57 0.10 25.73 0.32 6a 0.88 0.12 0.17 0.03 6a4D -(CH 24.27 ± 0.17±± 17.89 0.03 76.85 25.73 73.45 ± 0.88 32.57 ± 0.10 66.99 ± 0.10 33.49 ± 0.01 6d -(CH 22)63 75.47 40.03 58.41 32.36 6c -(CH 55.15 39.34 73.45 24.27 17.76 17.89 17.65 18.72 18.15 76.85 0.09 ±± 0.07 ±± 0.03 ±± 3.01 ±± 32.57 0.10 25.73 0.32 6a 0.07 0.66 1.33 2.02 2.33 0.04 0.32 0.52 6b 64.44 ±± 0.11 0.09 66.53 ±± 0.03 0.31 -(CH22))534-0.88 0.12 0.17 0.03 70.88 12.26 15.48 18.22 2.32 0.15 0.72 0.02 66.99 33.49 6d -(CH222)65375.47 40.03 58.41 32.36 73.45 24.27 17.76 17.89 17.65 18.72 18.15 76.85 0.09 0.07 0.03 3.01 6c 55.15 0.03 39.34 0.11 32.57 ±± 0.10 25.73 ±± 0.01 0.32 6a 0.88 ±±± 0.12 ±±± 0.17 ±±± 2.33 0.04 0.32 0.52 ±±± 6b 64.44 0.09 66.53 0.31 -(CH2)470.88 12.26 15.48 18.22 2.32 0.15 0.72 0.02 66.99 33.49 -(CH 6c -(CH222))))6345--- 55.15 ±±±±0.01 0.03 39.34 ±±± 0.10 0.11 32.57 25.73 0.32 6a 0.09 0.07 0.03 3.01 0.88 0.12 ±±± 0.0418.72 0.1715.48 75.47 40.03 58.41 32.36 73.45 24.27 17.76 17.89 17.65 ±± 18.15 ±± ± 0.31 70.88 12.26 15.48 18.22 2.33 0.04 0.32 0.52 6b 64.44 0.09 66.53 0.31 -(CH -(CH 12.26 ± 0.32±±± 18.22 0.52 76.85 66.53 70.88 ± 2.33 64.44 ± 0.09 6b6d 50.32 69.97 53.97 2.32 0.15 0.72 0.02 0.09 ±± 0.07 ±±± 3.01 ±± 0.88 0.12 0.17 0.03 75.47 40.03 58.41 32.36 66.99 ±± 0.11 33.49 ±± 0.03 0.01 6d -(CH222))54364-70.88 12.26 15.48 18.22 32.57 0.10 25.73 0.32 6a 6c -(CH 55.15 39.34 17.65 ± 18.72 18.15 76.85 6b 64.44 0.09 66.53 0.31 2.33 0.04 0.32 0.52 ±±± 6e 75.36 ± 0.89 >80 >100 -(CH 2 ) 8 50.32 ± 69.97 ± 53.97 66.99 33.49 6d -(CH 70.88 12.26 15.48 18.22 75.47 40.03 58.41 32.36 2.32 ±± 0.15 ± 0.72 ± 0.02 ± 6b 64.44 ±± 0.10 0.09 66.53 ±± 0.01 0.31 0.88 0.12 0.17 0.09 0.07 0.03 3.01 6c -(CH22))654-55.15 0.03 39.34 0.11 2.33 0.04 0.32 0.52 17.65 18.72 18.15 76.85 3.15 ±± 0.13 ±± 0.56 ±± 6e 75.36 >80 ± >100 -(CH222)86450.32 53.97 12.26 15.48 18.22 75.47 40.03 58.41 32.36 2.32 0.15 0.72 0.02 6b 64.44 0.09 66.53 ±±±0.89 0.31 66.99 ±± 0.10 33.49 0.01 2.33 0.04 0.32 0.52 0.09 ±± 0.0969.97 0.0718.72 0.03 3.01 6c -(CH22))55- 55.15 0.03 39.34 0.11 17.65 18.72 ± 18.15 76.85 ± ± 0.03 6c6d -(CH 17.65 ± 0.07± 18.15 0.03 70.88 55.15 76.85 ± 3.01 39.34 ± 0.11 3.15 0.13 0.56 6e 75.36 >80 ± >100 -(CH 6b 64.44 ±± 0.09 66.53 0.31 66.99 0.10 33.49 ±±± 0.89 0.01 6d -(CH222)))8456--2.33 0.04 ±± 0.32 ±± 0.52 ±± 2.32 0.15 0.72 0.02 50.32 69.97 53.97 70.88 12.26 15.48 18.22 75.47 40.03 58.41 32.36 17.65 18.72 18.15 76.85 0.09 0.07 0.03 3.01 6c -(CH 55.15 0.03 39.34 0.11 29.04 ± 51.24 ± 66.3 ± 2-C6H4-CH 3.15 0.13 0.56 2.33 0.04 0.32 0.52 2.32 0.15 0.72 0.02 50.32 ± 69.97 ± 53.97 ± 6e 75.36 ± 0.89 >80 >100 -(CH 2 ) 8 17.65 18.72 18.15 76.85 6b 64.44 ± 0.10 0.09 66.53 ± 0.01 0.31 66.99 33.49 6d -(CH2)65475.47 ±± 40.03 ± 58.41 ± 32.36 ± 6c 55.15 0.03 39.34 0.11 0.09 0.07 0.03 3.01 6f 57.35 ± 1.13 >80 >100 29.04 51.24 66.3 ±± 2-C26)H -CH 6e 75.36 >80 >100 -(CH 85- 450.32 ±± 53.97 17.65 18.72 18.15 76.85 3.15 ±±± 0.1569.97 0.1358.41 0.56 6c 55.15 ±±±0.89 0.03 39.34 0.11 2.33 0.04 0.32 0.52 2.32 0.15 0.72 0.02 66.99 ±± 0.10 33.49 0.01 -(CH 22))6 0.09 0.07 0.03 3.01 ±± ± 0.01 40.03 58.41 32.36 ± -(CH 40.03 ± 0.72 32.36 0.02 75.47 33.49 75.47 ± 2.32 66.99 ± 0.10 6d6d 6- -H 0.06 3.22 1.02 CH 6f 57.35 >80 >100 29.04 ±±± 51.24 ±±± 66.3 2-C2226) -CH 50.32 69.97 53.97 17.65 18.72 18.15 76.85 ±± 3.15 0.13 0.56 6c 55.15 ±± 1.13 0.03 39.34 0.11 6e 75.36 0.89 -(CH 85- 40.09 0.07 0.03±±± 3.01 2.32 0.15 0.72 0.02 66.99 ±± 0.10 33.49 0.01 6d -(CH 2)675.47 40.03 58.41 32.36 22-6)H 0.06 3.22 1.02 CH 6f 57.35 ± 1.13 >80 >100 6c -(CH 58- 455.15 0.03 39.34 ± 6e 75.36 ± 0.89 >80 >100 0.09 0.07 0.03 3.01 3.15 0.13 0.56 29.04 ± 51.24 ± 66.3 ± 2-C -CH 17.65 18.72 18.15 76.85 ± 50.32 ± 69.97 ± 53.97 ± 75.47 40.03 58.41 32.36 2.32 ±± 0.15 ± 0.72 ± 0.02 ± 66.99 ± 0.11 0.10 33.49 ± 0.01 6d -(CH2)653.14 18.33 29.42 17.99 0.06 3.22 1.02 CH 0.09 0.07 0.03 3.01 3.15 ±±± 3.1551.24 0.1369.97 0.56 29.04 ±± 66.3 ±±± 2-C2226-)) H 4-CH 6f 57.35 ±±±0.89 1.13 >80 >100 ± ± 0.89 40.03 58.41 32.36 6c 55.15 0.03 39.34 0.11 >80 6e 75.36 >80 >100 -(CH 8 6e6g -(CH 50.32 ± 0.13 53.97 0.56 75.47 75.36 >100 50.32 69.97 53.97 66.99 ±± 0.10 0.10 33.49 0.01 6d 65- 2.32 0.15 0.72 0.02 32.61 73.49 ± 0.01 8.06 -(CH 22)38 - 453.14 18.33 29.42 17.99 6f 57.35 >80 >100 75.47 40.03 58.41 32.36 29.04 51.24 66.3 2-C226)-H -CH 0.06 ±± 3.22 ±± 1.02±±± CH 66.99 ±± 0.10 33.49 6d -(CH 0.09 0.07 0.03 3.01 3.15 0.13 0.56 6e 75.36 ±± 1.13 0.89 >80 ± >100 -(CH 2)6 82.32 0.15 0.72 0.02 50.32 69.97 53.97 2.28 0.28 1.03 0.15 32.61 ± 0.10 73.49 8.06 6g -(CH )63H 53.14 ± 18.33 ±±± 29.42 ±±± 17.99 75.47 40.03 58.41 32.36 29.04 51.24 66.3 ±±± 2-C -CH 2220.06 3.22 1.02 CH 6f 57.35 ± 1.13 >80 >100 66.99 33.49 0.01 6d 6- 42.32 0.15 0.72 0.02 3.15 0.13 0.56 6e 75.36 ± 0.89 >80 >100 -(CH 2 ) 8 50.32 69.97 53.97 -CH H - 18.33 2 -C 2.28 0.28 1.03 0.15 32.61 ± 0.10 >80 73.49 ±±± 8.06 -(CH 22) 6f 57.35 1.13 >80 >100 66.99 33.49 0.01 6d -6368-- 4-4 0.06 3.22 1.02 CH 2.32 0.15 ±±± 0.0669.97 0.7251.24 0.02 53.14 29.42 17.99 75.47 ± ± 1.13 40.03 58.41 32.36 29.04 ± 3.22 66.3 ± 1.02 76.73 57.35 >100 6f6g 29.04 51.24 66.3 ±±± 2-C -CH 50.32 ±± 53.97 3.15 0.13 0.56 6e 75.36 0.89 >80 >100 -(CH 26)H ± 8.09 17.75 45.75 CH 2.28 0.28 1.03 0.15 --863-- 40.06±±± 3.22 ±±± 1.02±±± CH 2.32 0.15 0.72 0.02 53.14 18.33 29.42 17.99 32.61 73.49 ±± 1.13 8.06 6g 50.32 69.97 53.97 66.99 ± 0.10 33.49 0.01 6d -(CH 6f 57.35 >80 ± >100 29.04 51.24 66.3 2-C2226))H -CH 6e 75.36 0.89 -(CH 3.15 0.13 0.56 6h 50.78 0.09 24.71 ± 0.5 -(CH 2 ) 4 76.73 ± 8.09 ± 17.75 ± 45.75 ± 32.61 ±± 0.10 73.49 6g -(CH 2)3850.32 69.97 53.97 53.14 18.33 29.42 17.99 2.28 0.28 ±± 1.03 ± 0.15± 6e 75.36 ±± 8.06 0.89 >80 >100 2.32 0.15 0.72 0.02 0.06 3.22 1.02 CH 6f 57.35 1.13 >80 >100 3.15 0.13 0.56 29.04 51.24 66.3 2-C26-H 4-CH 5.0 0.25 0.93 ±± 0.55 50.78 0.09 24.71±± ± 0.89 0.5 -(CH 2 -±± ± 8.06 8.09 45.75 50.32 69.97 53.97 53.14 18.33 29.42 17.99 2.28 0.28 1.03 0.15 6e 75.36 >80 >100 2)4 32.61 ± 0.10 73.49 6g 22 383 6g6h -(CH 18.33 ± 1.03 17.99 0.15 76.73 73.49 53.14 ± 2.28 32.61 ± 0.10 3.15 0.13 0.56 26-)H 0.06±±±± 0.2817.75 3.2229.42 1.02 CH 6f 57.35 ± 8.06 1.13 >80 >100 29.04 51.24 ± 66.3 ±±± 2-C 4-CH 5.0 0.25 0.93 0.55 6h 50.78 ±± 0.09 24.71 ±±± 0.89 0.5 -(CH 22))483-6e 75.36 >80 >100 32.61 0.10 73.49 8.06 6g -(CH 3.15±± 0.13 ±± 0.56±±± 2.28 0.28 1.03 0.15 76.73 8.09 17.75 45.75 50.32 69.97 53.97 53.14 18.33 29.42 17.99 29.04 51.24 66.3 2-C26-H4-CH 0.06 3.22 1.02 CH 6f 57.35 1.13 >80 ±±± >100 6.93 ± 7.18 ± 9.88 ± 59.29 5.0 0.25 0.93 0.55 3.15 0.13 0.56 2.28 0.28 1.03 0.15 76.73 ± 8.09 ± 17.75 ± 45.75 ± 6h 50.78 ± 0.09 24.71 ± 0.5 -(CH 2 ) 4 29.04 51.24 66.3 2-C226) H -CH 6e 75.36 ±± 1.13 0.89 >80 ± >100 8 432.61 ± 0.10 0.10 73.49 8.06 6g -(CH 53.14 18.33 29.42 17.99 6f 57.35 >80 >100 0.06±±± 3.22±± 1.02±±± CH 6i -(CH )-453--- -414.22 37.99 6.93 7.18 9.88 ± ± 0.5 50.78 ±±± 0.09 24.71±±±± ±0.28 0.5 -(CH 222) 76.73 8.09 ±± 45.75 29.04 51.24 66.3 2-C 6)H -CH 5.0 0.25± 0.9317.75 0.55 6h6h -(CH 8.09 ± 0.93±± 45.75 0.55 59.29 24.71 76.73 ± 5.0 50.78 ± 0.09 6f 57.35 1.13 >80 >100 3.15 0.13 0.56 2.28 0.28 1.03 0.15 32.61 0.10 73.49 8.06 6g -(CH 2) -340.06 3.22 1.02 CH 53.14 ± 18.33 ±± 0.2517.75 29.42 17.99 0.33 0.01 0.04 5.86 6i -(CH 5 14.22 37.99 ± 0.10 6.93 ±±±± 7.18 9.88 59.29 76.73 8.09 17.75 45.75 29.04 51.24 66.3 2-C2 H -CH 5.0 ±±± 0.25 0.93 0.55 6f 57.35 1.13 >80 >100 6h 50.78 24.71±±±0.28 0.5 26) 4- 40.06 3.22±±± 1.02 CH 2.28 0.28 1.03 0.15±±± 32.61 ± 0.09 0.10 73.49 8.06 6g -(CH 2-)353.14 18.33 29.42 17.99 0.33 0.01 0.04 5.86 6i -(CH 22)6-)5H 14.22 ± 0.28 37.99 ± 0.10 6f 57.35 1.13 >80 >100 6h 50.78 ± 0.09 24.71 ± 0.5 -(CH 4-- 40.06 3.22 1.02 CH 5.0 0.25 0.93 0.55 6.93 ± 7.18 ± 9.88 59.29 ± 29.04 ± 51.24 ± 66.3 ± 2 -C -CH 76.73 8.09 17.75 45.75 Int. J.6gMol. Sci. 2018, 19, x FOR PEER of 17 53.14 18.33 29.42 17.99 2.28 ±± 0.28±±± 1.03 ±± 0.15 ±± 32.61 ±5 0.10 73.49 ± 8.06 -(CHREVIEW 2)364.49 12.18 0.33 0.01 0.04 5.86 22 0.06 3.22 1.02 CHREVIEW 5.0 0.25±±± 0.93±±±7.18 ±18.05 0.55±±± Int.6iJ.6h 5± 0.10 of 17 ± 5.86 6.93 7.18 9.88 ±± ± 0.28 6iMol. Sci. 2018, 19, x FOR PEER -(CH 14.22 0.28 37.99 0.10 -(CH 6.93 0.01 9.88 0.04 59.29 14.22 59.29 37.99 ± 0.10 53.14 18.33 ± 0.3326.26 29.42 17.99 6f 57.35 1.13 >80 >100 50.78 0.09 24.71± 0.5 -(CH 2)))4355--- 76.73 8.09 17.75 45.75 32.61 73.49 ±±±0.03 8.06 2.28 0.28 1.03 0.15 Int. J.6g REVIEW of 17 41.0 ±±±50.07 16.68 -(CH 6jMol. Sci. 2018, 19, x FOR PEER 64.49 12.18 26.26 18.05 6i -(CH 222))-56 14.22 ±±±0.28 37.99 0.10 53.14 18.33 29.42 17.99 6.93 7.18 9.88 59.29 ±± 0.33±±± 0.01±±± 0.04±±± 5.86 32.61 73.49 8.06 6g 3-0.06 3.22 1.02 CHREVIEW 5.0 0.25 0.93 0.55 Int. J. Mol. Sci. 2018, 19, x FOR PEER 5 of 17 6h 50.78 ± 0.09 24.71 0.5 -(CH 2)4 2.28 0.28 1.03 0.15 76.73 8.09 17.75 45.75 3.31 ±± 0.06±± 1.03±± 1.03±± 41.0 ± 0.07 16.68 0.03 -(CHREVIEW 6j 2 64.49 12.18 26.26 18.05 53.14 18.33 29.42 17.99 6.93 7.18 9.88 59.29 0.33 0.01 0.04 5.86 6iMol. Sci. 2018, 19, x FOR PEER 22)6 5314.22 ± 0.28 37.99 ± 0.10 32.61 73.49 8.06 6g 2.28 0.28 1.03 0.15 Int. 6h J. 5 of 17 5.0 ± 0.25±± 0.93 ± 0.55 ± 50.78 ± 0.09 24.71 ± 0.5 -(CH2)476.73 8.09 17.75 45.75 28.43 19.84 18.41 17.95 3.31 0.06 1.03 1.03 41.0 ±±50.07 16.68 0.03 -(CH 6j 6iMol. Sci. 2018, 19, x FOR PEER -(CH2222)))63456-- 14.22 0.28 37.99 32.61 0.10 73.49 8.06 6g 0.33 0.01 0.04 5.86 2.28 0.28±±±± 0.0618.41 0.15 64.49 12.18 26.26 18.05 -(CH 12.18 ± 1.03 18.05 1.03 28.43 16.68 64.49 41.0 ± 0.07 53.14 18.33 29.42 17.99 ±±± ± 0.03 19.84 ±±± 17.95 6.93 7.18 9.88 ±±±± 59.29 Int.6j6m J. of 17 ± 3.31 76.73 8.09 17.75 45.75 5.0 0.25 0.93±26.26 0.55 6h 50.78 0.09 24.71±±±± ±0.03 0.5 49.05 ± 0.12 33.32 -(CHREVIEW 2)344.93 37.36 35.00 36.69 28.43 19.84 ± 18.41 17.95 3.31 0.06 1.03 1.03 0.33± 0.01±±±± 0.04±±±± 5.86 2.28 0.28 0.15 64.49 ±±± 12.18 26.26 18.05 41.0 ±±±50.07 16.68 0.03 6jMol. Sci. 2018, 19, x FOR PEER 6 76.73 8.09 17.75 45.75 32.61 0.10 73.49 8.06 6g -(CHREVIEW 49.05 0.12 33.32 -(CH 222)))35 6i 14.22 0.28 37.99 6.93 ±±± 7.18 9.88 59.29 6h 50.78 0.09 24.71±±±±0.03 0.5 -(CH 43--5.0 0.25 0.93 0.55 28.43 19.84 18.41 17.95 Int.6m J. of 17 0.28 0.04 0.31 0.02 65.85 0.07 6k 33.93 0.52 8 49.05 0.12 33.32 0.03 -(CH 6m 2 ) 3 44.93 37.36 35.00 36.69 41.0 ±±±±0.07 16.68 -(CH 6j 2)6476.73 8.09 17.75 45.75 64.49 ±± 12.18 26.26 18.05 3.31 0.06±±±± 1.03±±± 6h 50.78 0.09 24.71±±±0.03 0.5 2.28 0.28 1.03±±± 0.15 0.28 0.04 0.31 0.02 0.33 0.01 0.04 5.86 28.43 19.84 18.41 17.95 6i 5 14.22 0.28 37.99 0.10 5.0 0.25 0.93 0.55 6.93 7.18 9.88 59.29 49.05 0.12 33.32 0.03 -(CH 6m 2 ) 3 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW of 17 ± 2.99 2.99 2.08 2.09 2.02 0.28 0.04 0.31 0.02 65.85 0.07 6k 33.93 0.52 2 6k6m -(CH 37.36 ± 2.09 36.69 2.02 44.93 33.93 44.93 65.85 ± 0.07 ±± ± 0.52 37.36 ±± 36.69 76.73 8.09 17.75 45.75 64.49 12.18 ±± 2.0835.00 26.26 18.05 3.31 0.06 1.03 1.03 6h 50.78 0.09 24.71±±± ±0.03 0.5 2))8 41.0 ±±±50.07 16.68 -(CH 6j 22 64- 5.0 0.25 0.93±35.00 0.55 49.05 0.12 33.32 0.03 0.33±±± 0.01 0.04 5.86 6iMol. Sci. 2018, 19, x FOR PEER -(CHREVIEW 22)538 14.22 0.28 37.99 0.10 6.93 7.18 9.88 ±±±± 59.29 0.28 0.04 0.31 0.02 28.43 19.84 18.41 17.95 49.06 ± 13.36 ± 37.79 ± 27.96 Int. J. of 17 2.99 2.08 2.09 2.02 65.85 0.07 6k 33.93 -(CH 6h 50.78 0.09 24.71±±±0.52 0.5 41.0 ±±50.07 16.68 0.03 -(CH222))84356-6j 5.0 ±±± 0.25 0.93 0.55 3.31 0.06 1.03 1.03 44.93 37.36 35.00 36.69 76.73 8.09 17.75 45.75 49.06 13.36 37.79 27.96 64.49 12.18 26.26 18.05 0.28 0.04±±±±±± 0.31±±±±± 0.02±±±±± 6.93 7.18 9.88 59.29 0.33 0.01 0.04 5.86 6i 14.22 0.28 37.99 0.10 49.05 0.12 33.32 0.03 6n 26.39 ±5 0.01 42.72 ± 2.18 -(CH 26)H 4- 4Int. 6m J. Mol. Sci. 2018, 19, x FOR PEER REVIEW of 17 28.43 19.84 18.41 17.95 30.13 39.26 59.68 70.76 2-C -CH 49.06 ± 13.36 37.79 27.96 2.99 2.08 2.09 2.02 5.0 0.25 0.93 0.55 3.31 0.06 1.03 1.03 44.93 ±± 37.36 ±± 36.69 65.85 0.07 6k 33.93 0.52 -(CH 86.93 7.18 9.88 ±±± 59.29 6h 50.78 0.09 24.71±±±2.18 0.5 6n 26.39 0.01 42.72 -(CH 222)))46 -CH 41.0 ±±±±±0.07 16.68 0.03 -(CH 6j 6 64.49 12.18 26.26 18.05 6i 543---H4 14.22 0.28 37.99 0.10 0.33±±±±± 0.3735.00 0.01±39.26 0.04 5.86 2 -C 49.06 13.36 37.79 27.96 0.28 0.04 0.31 0.02 2.23 0.35 1.02 0.99 49.05 0.12 33.32 ±±±±0.52 0.03 -(CH 6l 69.66 0.30 37.66 0.13 6n 26.39 0.01 42.72 2.18 28.43 19.84 18.41 17.95 6l6m 30.13 ± 1.22 59.68 1.52 44.93 69.66 70.76 ± 2.02 37.66 ± 0.13 30.13 39.26 59.68 70.76 2-C2226))H -CH 65.85 0.07 6k 33.93 -(CH 845- 4±± ± 0.30 37.36 35.00 36.69 6.93 7.18 9.88 ±±± 59.29 2.99 2.08±±± 2.09±±±± 2.02 6i 14.22 0.28 37.99 0.10 5.0 0.25 0.93 0.55 2.23 0.35 1.02 0.99 3.31 0.06 1.03 1.03 49.06 13.36 37.79 27.96 41.0 ±±±0.07 16.68 0.03 6j 60.33 0.01 0.04 5.86 64.49 12.18 26.26 18.05 6n 26.39 0.01 42.72 2.18 -(CH 2)4 CH 0.28 ±±± 0.04±± 0.31±± 0.02±±± -)H 0.37 1.22 1.52 2.02 CH 2.23 0.35 1.02 0.99 49.05 0.12 33.32 ±± 0.52 0.03 -(CH 6m 28.43 19.84 18.41 17.95 6l 69.66 0.30 37.66 ±± 0.07 0.13 30.13 39.26 59.68 70.76 2-C2222 6) -CH 44.93 37.36 35.00 36.69 6.93 7.18 9.88 59.29 2.99 2.08 2.09 2.02 6i 5--414.22 0.28 37.99 0.10 65.85 6k 33.93 -(CH 83 0.33 0.01 0.04 5.86 6n 26.39 0.01 42.72 2.18 3.31 0.06 1.03 1.03 41.0 ± 0.07 16.68 ± 0.03 -(CH 6j 22)6464.49 12.18 26.26 18.05 2.23 0.35 1.02 0.99 49.06 13.36 37.79 27.96 11.23 ± 17.18 ± 18.76 ± 49.37 ± 28.43 ± 19.84 ± 18.41 ± 17.95 ± 0.28 0.04 0.31 0.02 49.05 0.12 33.32 0.03 -(CH 6m 358- 422-) 0.37 1.22 1.52 2.02 CH 6l 69.66 ± 0.30 37.66 ± 0.13 6i 14.22 0.28 37.99 0.10 65.85 ± 0.07 6k 33.93 ± 0.52 0.33 0.01 0.04 5.86 2.99 2.08 2.09 2.02 30.13 ± 39.26 ± 59.68 ± 70.76 2-C 6H -CH 6.93 ± 7.18 ± 9.88 ± 59.29 ± 11.23 ± 17.18 ± 18.76 ± 49.37 ± 44.93 37.36 35.00 36.69 2.23 ±± 0.35 ± 1.02 ±± 0.99 ±± 64.49 12.18 26.26 18.05 3.31 0.06 1.03 1.03 41.0 ±±±0.07 16.68 ±± 0.12 0.03 6j 66n 26.39 0.01 42.72 2.18 6o -(CH 222))534 36.62 31.28±0.07 49.06 13.36 37.79 27.96 11.23 17.18 18.76 49.37 49.05 0.12 33.32 0.03 -(CH 6m -- 4-0.28 0.04 0.31 0.02 6m -(CH 19.84 ± 0.31±± 17.95 0.02 70.76 33.32 28.43 ± 0.28 49.05 ± 0.12 0.37 1.22 1.52 2.02 CH 0.33 0.01 0.04 5.86 2.99 2.08 ±±±± 0.0439.26 2.0918.41 2.02 30.13 ±± 59.68 ±± ± 0.03 2-C 6-)) H -CH 6l 69.66 ±±±±0.12 0.30 37.66 0.13 64.49 12.18 26.26 18.05 6i -(CH 14.22 0.28 37.99 0.10 6o -(CH 222 585-3 36.62 31.28±0.07 65.85 0.07 6k 33.93 0.52 22) 44.93 37.36 35.00 36.69 41.0 ±±±0.07 16.68 0.03 -(CH 6j 3.31 0.06 1.03 1.03 11.23 17.18 18.76 49.37 2.23 0.35 1.02 0.99 0.61 0.03 0.46 1.65 6n 26.39 0.01 42.72 ±± 0.30 2.18 2)564-6o -(CH 36.62 0.12 31.28±0.07 0.28 0.04 0.31 0.02 49.06 13.36 37.79 27.96 6l 69.66 37.66 0.13 64.49 12.18 26.26 18.05 30.13 39.26 59.68 70.76 2-C2226) -CH 0.37 ±±± 1.22 ±±± 1.52 ±±± 2.02 ±±± CH 41.0 ±±±±0.07 16.68 0.03 -(CH 6j )-H 6- 40.33 0.01 0.04 5.86 0.61 0.03 0.46 1.65 2.99 2.08 2.09 2.02 11.23 17.18 18.76 49.37 65.85 0.07 6k 33.93 0.52 8 3.31 0.06 1.03 1.03 44.93 37.36 35.00 36.69 6o -(CH 2) 5 36.62 ± 0.12 31.28±0.07 2.23 0.35 1.02 0.99 0.61 0.03 0.46 1.65 6n 26.39 ±0.07 0.01 42.72 ±± 0.30 2.18 -(CH 2-6) )H 4- 449.06 ±± 13.36 ±± 37.79 ±± 27.96 ±± 64.49 12.18 26.26 18.05 30.13 39.26 59.68 70.76 2-C -CH 22 0.37 1.22 1.52 2.02 CH 6l 69.66 37.66 ± 0.13 41.0 ± 16.68 0.03 -(CH 6j 6 3.31 0.06 1.03 1.03 6o 36.62 ± 0.52 0.12 31.28±0.07 2.99 2.08 2.09 2.02 65.85 ± 0.01 0.07 6k 33.93 -(CH222))485-44.93 37.36 35.00 36.69 0.61 0.03 0.46 1.65 11.23 17.18 18.76 49.37 55.04 ± 19.28 ±± 17.84 ± 15.66 49.06 13.36 37.79 27.96 2.23 0.35 1.02 0.99 6n 26.39 42.72 -(CH 6n6p -(CH 13.36 ± 1.02±±±±± 27.96 0.99 55.04 42.72 49.06 ± 2.23 26.39 ± 0.01 6l 69.66 ±±±2.18 0.30 37.66 0.13 41.0 ±±0.07 16.68 0.03 -(CH 6j 64 - -4226-))H 0.37 1.22 1.52 2.02 CH 3.31 0.06 1.03 1.03 64.49 12.18 26.26 18.05 ±±±± ± 2.18 19.28 ±±±± 15.66 30.13 39.26 59.68 70.76 2-C -CH 0.61±±±± 0.3517.84 0.0337.79 0.46 1.65 44.93 37.36 35.00 36.69 2.99 2.08 2.09 2.02 65.85 0.07 6k 33.93 0.52 6o 36.62 0.12 31.28±0.07 47.60 ± 0.02 26.75 ± 0.51 -(CH 222) 11.23 17.18 18.76 49.37 55.04 19.28 17.84 15.66 6n 26.39 0.01 42.72 2.18 -(CH )-664586---2.23 0.35 1.02 0.99 0.37 ±± 1.22 ±±± 1.52 ±±± 2.02 ±±± CH 3.31 0.06 1.03 1.03 44.93 37.36 35.00 36.69 41.0 ±±±±0.07 16.68 ±±±± 0.51 0.03 -(CH 6j 47.60 0.02 26.75 6p -(CH 222)6)) 6l 69.66 0.30 37.66 0.13 30.13 39.26 59.68 70.76 2-C H -CH 65.85 0.07 6k 33.93 0.52 -(CH 8- 42.99 2.08 2.09 2.02 55.04 19.28 ±± 17.84 15.66 0.61 0.03 0.46 1.65 2.22 0.02 0.22 0.33 6o 36.62 0.12 31.28±0.07 47.60 ± 0.02 26.75 ± 0.51 6p -(CH 22)652.23 0.35 1.02 0.99 11.23 17.18 ± 18.76 ± 49.37 ± 44.93 37.36 35.00 36.69 65.85 ± 0.02 0.07 6k 33.93 ± 0.51 0.52 -(CH 226) )-H 8- 43.31 ±± 0.06 ±± 1.03 ±± 1.03 ±± 2.22 0.02 0.22 0.33 0.37 1.22 1.52 2.02 CH 55.04 19.28 17.84 15.66 6l 69.66 0.30 37.66 0.13 2.99 2.08 2.09 2.02 30.13 39.26 59.68 70.76 2-C2 -CH 47.60 ± 26.75 ± 6p -(CH 6 0.61 ±± 0.6117.18 0.0317.18 0.46 1.65 2.22 0.02 0.22 0.33 6o -(CH 2 36.62 ± 0.12 31.28±0.07 11.23 ± 18.76 ± ± 0.12 6o6k -(CH 11.23 ± 0.03± 18.76 0.46 49.37 36.62 49.37 ± 1.65 31.28±0.07 44.93 37.36 35.00 36.69 65.85 0.07 33.93 0.52 -(CH 22) ))5865----42.99 2.08 2.09 2.02 47.60 0.02 26.75 ±±±±0.12 0.51 6p -(CH 22 0.37 1.22 1.52 2.02 CH 6l 69.66 0.30 37.66 ±± 0.13 30.13 39.26 59.68 70.76 2-C 6-)H -CH 2.22 0.02 0.22 0.33 55.04 19.28 17.84 15.66 43.05 23.65 35.78 45.21 11.23 17.18 18.76 49.37 0.61±±±± 0.03±±±± 0.46±±±± 1.65±±±± 6o -(CH 22))58-36.62 31.28±0.07 65.85 ± 0.07 6k 33.93 0.52 -(CH 2.99 2.08 2.09 2.02 44.93 37.36 35.00 36.69 43.05 23.65 35.78 45.21 2.22 ±± 0.02 ±± 0.22 ±± 0.33 ±± 30.13 39.26 59.68 70.76 2-C 6-H -CH 0.37 1.22 1.52 2.02 CH 6l 69.66 ±±± 0.93 0.30 37.66 0.13 47.60 0.02 26.75 0.51 6p 6q 42.06 ± 0.09 29.82 -(CH 222) 856-- 455.04 19.28 17.84 15.66 43.05 23.65 35.78 45.21 6o -(CH ) 36.62 0.12 31.28±0.07 0.61 0.03 0.46 1.65 2.99 2.08 2.09 2.02 30.13 39.26 59.68 ± 70.76 2-C -CH 65.85 ±±± 0.09 0.07 6k 33.93 ±±± 0.93 0.52 -(CH )8H 8-- 46q 42.06 29.82 -(CH 222)66l 69.66 0.30 37.66 0.13 0.37 ±± 1.22 ±±± 1.52 2.02 CH ±±± ± 0.51 23.65 35.78 45.21 2.22 0.02 0.22 0.33 2.01 0.05 0.82 1.66 47.60 ±± 0.09 0.02 26.75 ±±±0.93 0.51 6p 2 86- 42.06 29.82 -(CH 6p6q -(CH 19.28 ± 0.22±±± 15.66 0.33 43.05 26.75 55.04 ± 2.22 47.60 ± 0.02 0.61 0.03 0.46 1.65 55.04 19.28 15.66 30.13 39.26 ± 59.68 70.76 ± 2-C226))H -CH 6l 69.66 0.30 37.66 0.13 2.99 2.08 ±±±± 0.0217.84 2.0917.84 2.02 2.01 0.05 0.82 1.66 43.05 23.65 35.78 45.21 -866-- 40.37 1.22 1.52 2.02 CH 6q 42.06 ±± 0.09 29.82 ±± 0.93 -(CH 2) 2.22 ±±± 0.02 ±± 0.22 ±±± 0.33 ±±± 2.01 0.05 0.82 1.66 47.60 0.02 26.75 0.51 6p -(CH 55.04 19.28 17.84 15.66 30.13 39.26 59.68 70.76 2-C226-)H 4-CH 6l 69.66 ± 0.30 37.66 ± 0.13 0.37 1.22 1.52 2.02 CH 6q 42.06 ± 0.09 29.82 ± 0.93 -(CH 2 ) 8 2.01 0.05 0.82 1.66 43.05 ± 23.65 ± 35.78 ± 45.21 ± 46.61 65.93 58.25 68.26 2-C6H4-CH 55.04 ± 19.28 ± 17.84 ± 15.66 ± 2.22 0.02 0.22 0.33 47.60 ± 0.02 26.75 ± 0.51 6p -(CH 226)-H 6- 46l 69.66 0.30 37.66 0.13 0.37 1.22 1.52 2.02 CH 30.13 ± 39.26 ± 59.68 ± 70.76 ± 2 -C -CH 46.61 ±± 58.25 ±± ± 0.93 2-C6H4-CH 2.01 0.05±±± 0.0565.93 0.8235.78 1.66 6q 42.06 ±± 0.01 0.09 29.82 0.93 -(CH 6r >80 56.11 6q6p -(CH 23.65 ± 0.82±± 45.21 1.66 68.26 29.82 43.05 ± 2.01 42.06 ± 0.09 43.05 23.65 35.78 45.21 46.61 65.93 58.25 68.26 2-C22 -CH 47.60 0.02 26.75 ±±±±0.51 -(CH 226)))H 688 -- -42.22 0.02 0.22 0.33 0.37 1.22 1.52 2.02 CH 6l 69.66 0.30 37.66 ±± 0.01 0.13 6r >80 56.11 46.61 65.93 58.25 68.26 2-C -CH 2.01 ±± 0.05 ±± 0.82 ±± 1.66 ±± 226-)H 0.03 1.18 2.98 1.25 CH 6q 42.06 ± 0.01 0.09 29.82 ± 0.93 -(CH 8- 46r >80 56.11 2.22 0.02 0.22 0.33 43.05 23.65 35.78 45.21 0.37 ± 1.22 ± 1.52 ± 2.02 ± CH 22-6-H40.03 1.18 2.98 1.25 CH 46.61 65.93 58.25 68.26 2-C -CH 6r >80 56.11 ± 0.01 2.01 ± 0.05 ± 0.82 ± 1.66 ± -CH 2-)6 0.03 1.18 2.98 1.25 CH 6q 42.06 ± 0.09 29.82 ± 0.93 -(CH 8-H4 43.05 23.65 35.78 45.21 2 -C 6r 56.11 ±± 0.09 0.01 26-H40.03 1.18 2.98 1.25 CH 6r6q 46.61 ± 1.18±± 58.25 2.98 25.20 68.26 ± 1.25 56.11 ± 0.01 46.61 65.93 58.25 68.26 2-C -CH 6.12 8.06 13.77 43.05 23.65 ±± 45.21 2.01±±± >80 42.06 0.05±±±±± 0.0335.78 0.82±±65.93 1.66 29.82>80 ±±0.93 -(CH 2)8CH 25.20 6.12 8.06 13.77 2)-30.03±± 1.18±± 2.98±± 1.25 ± CH 6r >80 56.11 ±± 0.02 0.01 22.82 14.71 ±± 0.08 6s -(CH 222 46.61 65.93 58.25 68.26 2-C 6H -CH 25.20 6.12 8.06 13.77 6q 42.06 0.09 29.82 0.93 -(CH ) 8- 42.01 0.05 0.82 1.66 22.82 ± 0.02 14.71>80 ± 0.08 6s -(CH 2)325.20 ± 6.12 ± 8.06 ± 13.77 ± 2)-31.18 2.98 1.25 CH 1.12 0.03 0.08 0.11 6r 56.11 0.01 22.82 ± 0.02 14.71 ± 0.08 6s -(CH 2 2.01 ± 0.05 ± 0.82 ± 1.66 ± 46.61 65.93 58.25 68.26 -CH2-C6H41.12 0.03 0.08 0.11 25.20 6.12 8.06 13.77 22.82 14.71>80 ±±0.08 -(CH 2)3- 6s6s -(CH 6.12 0.08 13.77 0.11 68.26 14.71 25.20 ± 1.12 22.82 ± 0.02 1.18±±8.06 ±58.25 2.98 ±± 1.25 CH 3 41.12 0.03±± 0.08 0.11 6r 56.11 ±± 0.02 0.01 46.61 ± 0.0365.93 ±± ± 0.08 2-C26-) H -CH 22.82 ±± 0.01 0.02 14.71 ± 0.08 6s -(CH 2)31.12 0.03 0.08 0.11 25.20 6.12 8.06 13.77 25.69 12.39 9.01 15.20 46.61 65.93 58.25 68.26 2-C26-H4-CH 0.03±±±± 1.18±±± 2.98±±± 1.25±±± CH 6r >80 56.11 25.69 12.39 9.01 15.20 1.12 ± 0.03±± 0.08± 0.11 ± 22.82 ± 0.01 0.02 14.71 0.08 6s 6t >100 18.26 ± 0.18 -(CH 22)4325.20 6.12 8.06 13.77 25.69 12.39 9.01 15.20 6r >80 56.11 ± 20.03 1.18 2.98 1.25 CH 6t >100 18.26 ± 0.18 -(CH2)4±± ± 0.18 12.39 1.12 0.03±±± 0.599.01 0.08±±9.01 ±15.20 0.11 ±± 1.21 0.59 0.19 0.32 22.82 ± 0.02 14.71 ±±0.18 0.08 6s >100 18.26 -(CH 0.03 1.18 2.98 1.25 CH222)-)434- 25.20 6.12 8.06 13.77 6t6t -(CH 12.39 0.19 15.20 0.32 25.69 18.26 25.69 ± 1.21 >100 1.21 0.59 0.19 0.32 25.69 12.39 9.01 15.20 6t >100 18.26 -(CH 1.12 ±± 0.03±± 0.08±± 0.11 ±± 1.21 0.59 0.19 0.32 22.82 ± 0.02 14.71 ±± 0.18 0.08 6s -(CH22))43-25.20 6.12 8.06 13.77 6t >100 18.26 ± 0.18 -(CH 2)41.21 0.59 0.19 0.32 25.69 12.39 9.01 15.20 36.03 ±± 5.61 ±±± 5.86 ±± 14.63 ±± 25.20 6.12 8.06 13.77 1.12 0.03 0.08 0.11 22.82 ± 0.02 14.71 ± 0.08 6s -(CH2)336.03 5.61 5.86 14.63 1.21±± 0.59±±± 0.19±± 0.32±± 6t >100 18.26 ±± 0.11 0.18 6u 39.89 ± 0.05 16.68 -(CH 25.69 12.39 9.01 15.20 5.61 14.63 22.82 0.02 14.71 0.08 6s -(CH2222))))5534--- 1.12 0.03 0.08 0.11 39.89 ±± 0.05 16.68 ± 0.11 -(CH 6u6u -(CH 5.61 0.04 5.86 0.08 14.63 0.36 36.03 16.68 36.03 ± 2.99 39.89 ± 0.05 36.03 ±± ± 0.11 5.61 5.86 1.21 0.59±± 0.19±±5.86 ±14.63 0.32 ±± 2.99 0.04 0.08 0.36 6t >100 18.26 ±±0.11 0.18 6u 39.89 ± 0.05 16.68 -(CH222)5451.12 0.03 0.08 0.11 25.69 12.39 ± 9.01 15.20 2.99 0.04 0.08 0.36 36.03 ± 5.61 ± 5.86 ± 14.63 ± 6u 39.89 ± 0.05 16.68 ± 0.11 -(CH 2)51.21 ± 0.59 ± 0.19± 0.32 ± 2.99 0.04 0.08 0.36 6t >100 18.26 ± 0.18 -(CH2)425.69 12.39 9.01 15.20 6u 39.89 ± 0.05 16.68 ±± 0.18 0.11 -(CH22))45-2.99 0.04 0.08 0.36 36.03 5.61 5.86 14.63 7.95 8.60 22.85 42.41 25.69 12.39 9.01 15.20 1.21±±± 0.59±±± 0.19±±± 0.32±±± 6t >100 18.26 -(CH 7.95 2.99 0.04±± 0.06 8.60 0.08±8.60 ±22.85 0.36 ± 6u 39.89 0.05 16.68 ±±±0.06 0.11 -(CH 24.65 67.75 ± 0.11 6v6v -(CH 7.95 0.13 22.85 1.36 42.41 24.65 42.41 ± 2.08 67.75 ± 0.11 36.03 5.61 5.86 14.63 7.95 8.60 22.85 42.41 ± ± 0.06 6t >100 18.26 0.18 -(CH22222))))6645-6-- 1.21 0.59 0.19 0.32 6v -(CH 24.65 ±± 0.06 67.75 ±± 0.11 7.95 ± 8.60 ± 22.85 ± 42.41 ± 2.99 0.04 0.08 0.36 0.06 0.13 1.36 2.08 6u 39.89 0.05 16.68 0.11 -(CH 2 ) 5 6v 2 6 24.65 0.06 67.75 0.11 1.21 ± 0.59± 0.19± 0.32 ± 36.03 5.61 5.86 14.63 0.06 0.13 1.36 2.08 7.95 8.60 22.85 42.41 6v -(CH 24.65 67.75 2.99 ±± 0.04±± 0.08±± 0.36 ±± 0.06 0.13 1.36 2.08 6u 39.89 ±± 0.11 0.05 16.68 ±± 0.06 0.11 -(CH22))65-36.03 5.61 5.86 14.63 6v -(CH22))56-24.65 ±± 0.11 0.06 67.75 ±± 0.05 0.11 0.06 0.13 1.36 2.08 7.95 8.60 22.85 42.41 76.32 16.54 29.98 31.34 36.03 5.61 5.86 14.63 2.99±±± 0.04±±±± 0.08±±±± 0.36±±± 6u 39.89 16.68 -(CH 76.32 16.54 29.98 31.34 0.06±± 0.13±± 1.36 ± 2.08 ± 6v 24.65 ±± 0.02 0.06 67.75 ±± 0.16 0.11 60.78 44.57 -(CH 6w 7.95 8.60 22.85 42.41 76.32 16.54 29.98 31.34 6u 39.89 0.05 16.68 0.11 -(CH2222)))8856--2.99 0.04 0.08 0.36 60.78 ± 0.16 44.57 ± 0.02 -(CH 6w 76.32 ± 16.54 ± 29.98 ± 31.34 ± 0.06 0.13 1.36 2.08 3.36 0.33 0.07 1.88 6v 24.65 ± 0.02 0.06 67.75 ± 0.16 0.11 60.78 44.57 -(CH22)866w 2.99 ± 0.04± 0.08± 0.36 ± 7.95 8.60 22.85 42.41 3.36 0.33 0.07 1.88 76.32 16.54 29.98 31.34 60.78 44.57 -(CH 6w 0.06±± 0.13±± 1.36 ±± 2.08 ±± 3.36 0.33 0.07 1.88 6v -(CH22))86-24.65 ±± 0.02 0.06 67.75 ±± 0.16 0.11 7.95 8.60 22.85 42.41 60.78 ±± 0.11 0.16 44.57 ±± 0.06 0.02 -(CH 6w 8-43.36 0.33 0.07 1.88 76.32 ± 16.54 29.98 31.34 2-C26)H 34.27 ± 45.00 ± 39.61 ±± -CH 7.95 ± 8.60 ± 22.85 42.41 ± 0.06 0.13 1.36 2.08 6v -(CH 2)624.65 67.75 2-C6H434.27 45.00 39.61 -CH 3.36 ± 0.33±± 0.07±± 1.88±± 60.78 ±± 0.10 0.16 44.57 0.02 -(CH 6w 6x >80 >80 63.28 76.32 16.54 29.98 31.34 2-C226))H 34.27 45.00 39.61 -CH 6v -(CH 68-- 424.65 ±± 0.06 67.75 0.11 0.06 0.13 1.36 2.08 6x >80 >80 63.28 ± 0.10 2-C 34.27 45.00 39.61 -CH 3.36 0.33 ±± 0.07 ±± 1.88 ±± CH 226-)H 0.05 0.16 2.59 60.78 ± 0.10 0.16 44.57 ± 0.02 -(CH 6w 8- 46x >80 >80 ± 63.28 0.06 0.13 1.36 2.08 76.32 16.54 29.98 31.34 CH 2-6H40.05 0.16 2.59 2-C 34.27 ± 45.00 ± 39.61 ± -CH 6x >80 >80 63.28 ± 0.10 3.36 ± 0.33 ± 0.07 ± 1.88 ± CH2-)80.05 0.16 2.59 60.78 ± 0.16 44.57 ± 0.02 -(CH 6w 76.32 16.54 29.98 31.34 6x >80 >80 63.28 ± 0.10 CH 26-H40.05 0.16 2.59 2-C 34.27 45.00 ± 39.61 ± -CH 15.39 ± 57.69 ± 76.32 ± 16.54 ± 29.98 ± 31.34 ± 3.36 0.33 0.07 1.88 60.78 ± 0.16 44.57 ± 0.02 -(CH2)86w c 15.39 57.69 -8- 40.05±± 0.16 ± 2.59 ± 6x c >80 ±± 63.28 ±± 0.30 0.10 5-FU C4H3FN 2CH O2-C 2 22) >80 >80 52.32 ± 2.06 16.33 6H 34.27 45.00 39.61 -CH 15.39 57.69 60.78 0.16 44.57>80 0.02 6w 3.36 0.33 0.07 1.88 5-FU C4H3FN-(CH 2O2 >80 >80 52.32 ±± 2.06 16.33 15.39 57.69 0.05 ±± 0.16 2.59 0.09 1.39 6x c >80 ± 63.28 ±± 0.30 0.10 5-FU C4H3FN 2CH O2-C 2 26-H4>80 ± >80 ± 52.32>80 ± 2.06 16.33 0.30 3.36 0.33 0.07 1.88 34.27 45.00 39.61 -CH 0.09 1.39 c 15.39 57.69 5-FU C4H3FN 2CH O2-C 2 2>80 ± >80 ± 52.32>80 ± 2.06 16.33 0.05 ±± 0.16 2.59 0.09 1.39 6xa c >80 ± 63.28 ±± 0.30 0.10 6 H4 34.27 45.00 39.61 -CH 5-FU C 4H3FN2O2 when cell >80 >80 52.32 ± 2.06 16.33 ± 0.30 0.09 1.39 15.39 ± 57.69 ± IC 50 was the concentration viability was reduced by 50% after treatment with β-carbolines 2-C26-H445.00 ± 39.61 ± -CHCH 0.05 ± 0.16 2.59 6xa IC >80 >80 β-carbolines 63.28 ± 0.10 cell34.27 viability was by 50% after treatment with c 50 was the concentration 0.09 1.39 a IC 5-FU C4H3FN2CH O2 when >80reduced >80 52.32 ± 2.06 16.33 ±± 0.10 0.30 15.39 ±of three 57.69 50 was theall concentration viability was reduced 2.59 by 50% after treatment with 6xfor >80 >80 63.28 b A549: 2when - meancell 0.05 0.16 h, and data were the values independent experiments. lung±β-carbolines carcinoma, a IC c 48 50 was theall concentration cell viability was reduced 2.59 by after b A549:with 0.09 1.39 5-FU C4Hwere 3FN2CH O 2 when >80 >8050% 52.32treatment ± 2.06 16.33 ± 0.30 for 48 h, and data the independent experiments. lung±β-carbolines carcinoma, 2- mean values 0.05 0.16 15.39 ±of three 57.69 a b IC 50 was the concentration when cell viability was reduced by 50% after treatment with β-carbolines for 48 h, and all data were the mean values of three independent experiments. A549: lung carcinoma, c 0.09 1.39 b SGC-7901: gastric carcinoma, Hela: cervical carcinoma, SMMC-7721: liver carcinoma, MCF-7: breast 5-FU C4Hwere 3FN2O 2 >80 >80 experiments. 52.32 ± 2.06A549: 16.33 ± 0.30 15.39 ±of three 57.69 ± carcinoma, for h, and all data the mean values independent lung a IC48 SGC-7901: gastric Hela:cell cervical carcinoma, SMMC-7721: livertreatment carcinoma, MCF-7: breast concentration when viability was reduced by 50% after with c 50 was the b A549: 15.39 57.69 ±β-carbolines 1.39 5-FU Ccarcinoma, 4Hwere 3FN2Othe 2 >80 >80 52.32 2.06 16.33 ± 0.30 for 48 h, and all data mean three independent experiments. lung carcinoma, SGC-7901: gastric carcinoma, Hela: values cervical SMMC-7721: liver± carcinoma, MCF-7: breast c 0.09 ± ofcarcinoma,

46.61 ± 65.93 ± 58.25 ± 68.26 ± 46.61 ± 65.93 ± 58.25 ± 68.26 ± >80 56.11 ± 0.01 >80 56.11 ± 0.01 0.03 1.18 2.98 1.25 0.03 1.18 2.98 1.25 25.20 ± 6.12 ± 8.06 ± 13.77 ± 25.20 ± 6.12 ± 8.06 ± 13.77 ± 22.82 ± 0.02 14.71 ± 0.08 6s -(CH2)322.82 ± 0.02 14.71 ± 0.08 6s -(CH2)31.12 0.03 0.08 0.11 1.12 0.03 0.08 0.11 25.69 ± 12.39 ± 9.01 ± 15.20 ± Int. J. Mol. 25.69 ± 12.39 ± 9.01 ± 15.20 ± 6t Sci. 2018, 19, 3179 -(CH2)4>100 18.26 ± 0.18 6t >100 18.26 ± 0.18 -(CH2)41.21 0.59 0.19 0.32 1.21 0.59 0.19 0.32 36.03 ± 5.61 ± 5.86 ± 14.63 ± 36.03 ± 5.61 ± 5.86 ± 14.63 ± 6u 39.89 ± 0.05 16.68 ± 0.11 -(CH2)56u 39.89 ± 0.05 -(CH2)51. Cont.16.68 ± 0.11 2.99 0.04 0.08Table 0.36 2.99 0.04 0.08 0.36 7.95 ± 8.60 ± 22.85 ± 42.41a ± 7.95 ± 8.60 ± 22.85IC ± 50 (µM) 42.41 ± Substituents 6v -(CH2)624.65 Mean ± 0.06 ± SD 67.75 ± 0.11 6v -(CH2)624.65 ± 0.06 67.75 ± 0.11 0.06 0.13 1.36 2.08 Compounds b b b b 0.06 0.13 1.36 2.08 R1 R2 A549 SGC-7901 Hela SMMC-7721 MCF-7 b 76.32 ± 16.54 ± 29.98 ± 31.34 ± 76.32 ± 16.54 ± 29.98 ± 31.34 ± 60.78 ± 0.16 44.57 ± 0.02 -(CH2)86w 60.78 ± 0.16 44.57 ±±0.02 -(CH22))88- 6w 6w -(CH 16.54 0.07 31.34 1.88 44.57 ± 0.02 76.32 ± 3.36 3.36 0.33 ± 0.33 0.0729.98 ±1.88 3.36 0.33 0.07 1.88 434.27 ± 45.00 ± 39.61 ± -CH2-C -CH -C66H H 4-4 34.27 ±± 0.0545.00 45.00 ± 39.61 -CH22-C 6H >80 >80 >80 63.28 ± 0.10 >80 34.27 ± 0.16± 39.61 ± 2.59 6x6x 6x >80 >80 63.28 ± 0.10 CH CH22-0.05 0.16 2.59 CH 20.05 0.16 2.59 5-FU c C4 H3 FN2 O2 15.39 ± 0.09 >80 >80 52.32 ± 2.06 57.69 ± 1.39 15.39 ± 57.69 ± 15.39 ± 57.69 ± c C4H3FN2O2 >80 >80 52.32 ± 2.06 16.33 ± 0.30 a5-FU c IC50 was the concentration by 50% after treatment with± β-carbolines 5-FU C4H3FN2O2 when cell >80 was reduced >80 52.32 ± 2.06 16.33 0.30 0.09 viability 1.39 0.09 1.39 b 6r 6r

-CH2-C6H4-CH2-C6H4CH2CH2-

5 of 16

MRC5 60.78 ± 0.16 63.28 ± 0.10 16.33 ± 0.30

for 48 h, anda all data were the mean values of three independent experiments. A549: lung carcinoma, SGC-7901: gastric IC 50 was the concentration when cell viability was reduced by 50% after treatment with β-carbolines a IC50 was the concentration when cell viability was reduced by carcinoma, 50% after treatment with β-carbolines carcinoma, Hela: cervical carcinoma, SMMC-7721: liver MCF-7: breast carcinoma, MRC5: normal for 48 h, cand all data were the mean values of three independent experiments. bb A549: lung carcinoma, lung 5-FU (5-fluorouracil, could into DNA, interfering DNA synthesis) was used as a forcell. 48 h, and all data were the meanwhich values of threeinsert independent experiments. A549:with lung carcinoma, SGC-7901: gastric carcinoma, Hela: cervical carcinoma, SMMC-7721: liver carcinoma, MCF-7: breast positive control. SGC-7901: gastric carcinoma, Hela: cervical carcinoma, SMMC-7721: liver carcinoma, MCF-7: breast carcinoma, MRC5: normal lung cell. cc 5-FU (5-fluorouracil, which could insert into DNA, interfering carcinoma, MRC5: normal lung cell. 5-FU (5-fluorouracil, which could insert into DNA, interfering with DNA synthesis) was used as a positive control. with DNA synthesis) was used as a positive control.

2.3. The Morphological Observation of Cell Apoptosis

2.3. The Morphological Observation of Cell Apoptosis 2.3. The Morphological Observation of Cell Apoptosis Apoptotic morphology is one of the most intuitive and reliable ways to determine whether cells Apoptotic morphology is one of the most intuitive and reliable ways to determine whether cells Apoptotic morphology is one offeatures the most intuitive and reliable ways tomembrane determine whether cells cell contraction, are are apoptotic [38]. The typical of apoptosis include blebbing, apoptotic [38]. The typical features of apoptosis include membrane blebbing, cell contraction, are apoptotic [38]. The typical features of apoptosis include membrane blebbing, cell contraction, chromatin formation of apoptotic [39]. identify the chromatincondensation, condensation, andand formation of apoptotic bodies [39].bodies To identify theTo typical features of typical features of chromatin condensation, and formation of apoptotic bodies [39]. To identify the typical features of apoptosis, we performed Hoechst 33342/propidium iodide (PI) dual staining assay. As shown in apoptosis, Hoechst 33342/propidium (PI) dual assay. As shown in apoptosis,we we performed performed Hoechst 33342/propidium iodide (PI) iodide dual staining assay. staining As shown in Figure 1, A549 cells in the control group were evenly dyed weak blue with clear edge. However, after Figure A549 cells in the group were evenly dyedevenly weak blue with clear edge. However, Figure 1,1,A549 cells in control the control group were dyed weak blue with after clear edge. However, incubation with 6i, 4D, and 6u for 48 h, the cell density decreased significantly, and the cells were incubation with 6i, 4D, 6i, and4D, 6u for 48 h,6u thefor cell48 density decreased significantly, and the significantly, cells were after incubation with and h,fluorescence, the cell density decreased and the cells stained with strong blue fluorescence and strong red and shrinkage, condensation, or stained with strong blue fluorescence and strong red fluorescence, and shrinkage, condensation, or fragmentation wasstrong observed (Figure 1). These results second series and of dimer (6i) were stained with blue fluorescence andindicated strong that red the fluorescence, shrinkage, condensation, fragmentation was observed (Figure 1). These results indicated that the second series of dimer (6i) and the fourth series fluorine atom) monomer (4D) and dimer (6u) could cellseries of dimer (6i) or fragmentation was(containing observed (Figure 1).of results indicated that theinduce second and the fourth series (containing fluorine atom) ofThese monomer (4D) and dimer (6u) could induce cell apoptosis and further cell necrosis, while the second series of monomer (4B) could only induce cell and further necrosis, while the second series monomer (4B) could only induce(6u) cell could induce cell andapoptosis the fourth series cell (containing fluorine atom) ofofmonomer (4D) and dimer apoptosis in a dose-dependent manner. apoptosis in a dose-dependent manner.

apoptosis and further cell necrosis, while the second series of monomer (4B) could only induce cell apoptosis in a2018, dose-dependent manner. Int. J. Mol. Sci. 19, x FOR PEER REVIEW 6 of 17

Figure1.1.Morphological Morphologicalobservation observationwas was performed performed by by Hoechst Hoechst 33342/propidium 33342/propidium iodide Figure iodide(PI) (PI)dual dual staining.A549 A549 cells cells were were treated treated with 4B, 6i, 4D, staining. 4D, and and 6u 6u at at 88 μM µM for for 48 48 h, h, and andthen thenstained stainedwith with Hoechst33342 33342 inconditions. dark conditions. The cells stained were under observed under a Hoechst andand PI atPI37at◦ C37in °C dark The stained werecells observed a fluorescence fluorescence microscope (Leica Nussloch, DM6B, Leica, Nussloch, Germany). 33342 was used to stain microscope (Leica DM6B, Leica, Germany). Hoechst 33342Hoechst was used to stain the living and the livingnuclei, and apoptotic nuclei,blue andfluorescence emitted blue (upper). fluorescence (upper). used stainnuclei the dead apoptotic and emitted PI was usedPItowas stain thetodead and nuclei and emitted red fluorescence (lower). emitted red fluorescence (lower).

2.4. 2.4.Cell CellCycle CycleAssay Assay InInorder thecell cell cycle arrest induced by β-carbolines, we performed propidium orderto to determine determine the cycle arrest induced by β-carbolines, we performed propidium iodide iodide (PI) staining flow cytometry. As shown in Figure 2 and2,Table when the concentration (PI) staining using using flow cytometry. As shown in Figure 2 and Table when2,the concentration of 4B, 6i,of 4B, 6i,and 4D,6u and 6u4 was µM, the percentages in thephase G2/M phase increased 46.96%,35.46%, 42.40%, 4D, was μM, 4the percentages of cellsof in cells the G2/M increased to 46.96%,to42.40%, 35.46%, and 40.34% group respectively, 12.25%), respectively, 4B, 6i, and 6u could and 40.34% (control (control group 12.25%), indicating indicating that 4B, 6i, that 4D, and 6u 4D, could induce cell induce cell cycle the G2/M At 8 µM, the percentages of cells in and the S cycle arrest at thearrest G2/Matphase in A549phase cells. in At A549 8 μM,cells. the percentages of cells in the S phase of 4B 6i increased to 45.81% and 53.30% (control group 27.6%), respectively, indicating that 4B andindicating 6i could phase of 4B and 6i increased to 45.81% and 53.30% (control group 27.6%), respectively, exert activity by causing S and G2/M phase arrest. At 8 μM of 4D phase and 6u,arrest. the percentages that 4Bantitumor and 6i could exert antitumor activity by causing S and G2/M At 8 µMofof cells in the G1 phase increased to 88.01% and 81.62% (control group 60.15%), respectively, indicating that they could exert antitumor activity by causing G1 and G2/M phase arrest. Moreover, at 8 μM, the percentages of cells in sub-G1 by 4B, 6i, 4D, and 6u increased to 4.15%, 21.32%, 46.29%, and 44.92% (control group 1.78%), respectively, indicating that they could also induce apoptosis.

2.4. Cell Cycle Assay In order to determine the cell cycle arrest induced by β-carbolines, we performed propidium iodide (PI) staining using flow cytometry. As shown in Figure 2 and Table 2, when the concentration of 4B, 6i, 4D, and 6u was 4 μM, the percentages of cells in the G2/M phase increased to 46.96%, 42.40%, 35.46%, Int.and J. Mol. Sci. 2018, 19, 3179group 12.25%), respectively, indicating that 4B, 6i, 4D, and 6u could induce cell 6 of 16 40.34% (control cycle arrest at the G2/M phase in A549 cells. At 8 μM, the percentages of cells in the S phase of 4B and 6i increased to 45.81% and 53.30% (control group 27.6%), respectively, indicating that 4B and 6i could 4D and 6u, the percentages of cells in the G1 phase increased to 88.01% and 81.62% (control group exert antitumor activity by causing S and G2/M phase arrest. At 8 μM of 4D and 6u, the percentages of 60.15%), respectively, indicating that they could exert antitumor activity by causing G1 and G2/M cells in the G1 phase increased to 88.01% and 81.62% (control group 60.15%), respectively, indicating phase Moreover, at 8 µM, activity the percentages ofG1 cells sub-G1 by 4B, 6i, Moreover, 4D, and 6uatincreased that arrest. they could exert antitumor by causing andinG2/M phase arrest. 8 μM, the to 4.15%, 21.32%, 46.29%, and 44.92% (control group 1.78%), respectively, indicating that they also percentages of cells in sub-G1 by 4B, 6i, 4D, and 6u increased to 4.15%, 21.32%, 46.29%, andcould 44.92% induce apoptosis. (control group 1.78%), respectively, indicating that they could also induce apoptosis.

Figure 2. 2.The wasdetected detected flow cytometry PI single staining. A549 were Figure Thecell cell cycle cycle was byby flow cytometry usingusing PI single staining. A549 cells werecells treated ◦ treated with 4B, 6i, 4D, and 6u at 2, 4, and 8 µM for 48 h, and then stained with PI for 30 min at 37 with 4B, 6i, 4D, and 6u at 2, 4, and 8 μM for 48 h, and then stained with PI for 30 min at 37 °C. The C. The proportions of cells containing different DNA content were analyzed by FCS Express 5.0 (De Novo Software, Thornhill, Canada). Table 2. The percentage of cell cycles of A549 cells at indicated compound concentrations (4B, 6i, 4D, and 6u at 2, 4, and 8 µM). Compounds

Concentration (µM)

4B

Percentage of Cell Cycles (%) SubG1

G1

S

G2/M

2 4 8

6.56 10.49 4.15

59.38 43.06 52.09

25.16 9.98 45.81

15.46 46.96 2.1

6i

2 4 8

6.86 26.33 21.32

57.38 45.19 46.64

32.08 12.41 53.30

10.54 42.40 0.06

4D

2 4 8

6.41 39.03 46.29

56.89 47.21 88.01

24.17 17.33 11.69

18.94 35.46 0.3

6u

2 4 8

6.84 46.25 44.92

56.24 47.06 81.62

23.79 12.60 0.08

19.97 40.34 18.30

Control

1% DMSO

1.78

60.15

27.6

12.25

2.5. Cell Apoptosis Assay To better understand the apoptosis induced by β-carboline derivatives, we further performed Annexin V-FITC/PI dual staining assay using flow cytometry. The transfer of phosphatidylserine from the cell inner membrane to the cell outer membrane is considered as a potential marker of cell apoptosis.

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Moreover, Annexin V is a cellular protein which selectively binds phosphatidylserine [40,41]. As shown in Figure 3 and Table 3, after treatment with 8 µM of 4B, 6i, 4D, and 6u for 48 h, the percentages of early apoptosis were 8.40%, 4.24%, 5.28%, and 7.65%, respectively. And the percentages of late apoptosis or necrosis were 69.65%, 83.24%, 77.09%, and 83.52%, respectively, indicating that 4B, 6i, 4D, and 6u could induce significant apoptosis compared with the control group (early apoptosis: 4.22%; late apoptosis or necrosis: 7.44%). To sum up, the results of cell cycle and apoptosis experiments demonstrated that 4B, 6i, 4D, and 6u could exert antitumor activity by inducing cell cycle arrest at the Int.G2/M J. Mol. Sci. 2018, and 19, x FOR PEER REVIEW 8 of 17 S or phase significant cell apoptosis.

Figure 3. 3. The cell apoptosis usingAnnexin AnnexinV-FITC/PI V-FITC/PI dual staining. Figure The cell apoptosiswas wasdetected detectedby by flow flow cytometry cytometry using dual staining. A549 cells were treated with 4B, 6i, 4D, and 6u at 2, 4, and 8 µM for 48 h, and then stained with Annexin A549 cells were treated with 4B, 6i, 4D, and 6u at 2, 4, and 8 μM for 48 h, and then stained with ◦ C. The proportions of the living cells, the early apoptosis cells, and the V-FITC and V-FITC PI for 15and min 3715 Annexin PIatfor min at 37 °C. The proportions of the living cells, the early apoptosis latecells, apoptotic analyzed by FCS Express and thecells late were apoptotic cells were analyzed by5.0. FCS Express 5.0. Table 3. The percentage of cell apoptosis of A549 cells at indicated compound concentrations (4B, 6i, Table 3. The percentage of cell apoptosis of A549 cells at indicated compound concentrations (4B, 6i, 4D, and 6u at 2, 4, and 8 µM). 4D, and 6u at 2, 4, and 8 μΜ). Comp. Comp.

Concentration Concentration (µM) (μM)

2 4 2 8

4B 4B

4

6i

2 4 8 8

4D

2 4 4 8

2

6i

8

2 4 2 8

6u Control 4D

6u

Control

4 1% DMSO

Percentage of Cell Apoptosis (%)

Percentage of Cell Apoptosis (%)

Normal Living Cell Normal Living Cell

75.93 48.59 75.93 21.95

Early Apoptosis Early Apoptosis

3.18

Late Apoptosis and Necrosis Late Apoptosis and Necrosis

20.89 36.06 69.65

15.35 3.18

20.89

48.59

15.35

6.85 12.65 8.40 4.24

36.06

21.03 69.65 42.41 83.24

72.12

6.85

21.03

72.12 44.94 21.95 12.52

8.40

73.94 47.93 44.94 17.63

7.27 6.80 12.65 5.28

12.52

4.24

66.05 32.53 73.94 8.83

12.14 7.276.54 7.65

42.41 83.24

18.78 45.26 77.09

18.78

21.81 60.93 83.52 7.44

47.93 88.34

6.804.22

45.26

8

17.63

5.28

77.09

2

66.05

12.14

21.81

4

32.53

6.54

60.93

8

8.83

7.65

83.52

1% DMSO

88.34

4.22

7.44

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2.6. The Detection of Apoptosis-Related Protein (Cytochrome C, bcl-2) 2.6. ofofApoptosis-Related Protein (Cytochrome C, 2.6. The The Detection Apoptosis-Related Protein (Cytochrome C, bcl-2) bcl-2) A Detection large number of studies have found that the release of cytochrome C (Cyt C) from mitochondria to cytoplasm, and the decrease expression of bcl-2ofprotein, areC) important features A largenumber number studies have found that the of release cytochrome C (Cyt C) fromof A large of of studies have found thatin the release cytochrome C (Cyt from mitochondria apoptosis [42,43]. As shown in Figure 4, the levels of Cyt C (in mitochondria) and the expression mitochondria to cytoplasm, and decreaseofinbcl-2 expression protein, are important features ofof to cytoplasm, and the decrease inthe expression protein, of arebcl-2 important features of apoptosis [42,43]. bcl-2 protein decreased in a dose-dependent manner after treatment with 6i and 6u. The results apoptosis As4, shown in of Figure themitochondria) levels of Cyt and C (in mitochondria) and the expression of As shown [42,43]. in Figure the levels Cyt C4,(in the expression of bcl-2 protein decreased demonstrated that 6i and 6u could induce mitochondria-mediated apoptosis. bcl-2 protein decreased in a dose-dependent manner after treatment with 6i and 6u. The results in a dose-dependent manner after treatment with 6i and 6u. The results demonstrated that 6i and 6u demonstrated that 6i and 6u could induce mitochondria-mediated apoptosis. could induce mitochondria-mediated apoptosis.

Figure 4. Western blot analysis of cytochrome C and bcl-2 proteins. A549 cells were treated with with Figure 4. Western blot8analysis analysis of cytochrome cytochrome C and and bcl-2 proteins. A549 cellswere weretreated treatedwith withwith with 6i and4. 6uWestern at 2, 4, and μM for of 48 h. β-actin was usedbcl-2 as an internalA549 control. Figure blot C proteins. cells 6i and 6u at 2, 4, and 8 µM for 48 h. β-actin was used as an internal control. 6i and 6u at 2, 4, and 8 μM for 48 h. β-actin was used as an internal control.

2.7. DNA Binding Studies 2.7. DNA Binding Studies 2.7. DNA The Binding bindingStudies mode and binding strength of β-carboline derivatives (4B, 6i, 4D, and 6u) to DNA The binding mode and binding strength of β-carboline derivatives (4B, 6i, 4D, and 6u) to DNA wereThe studied through spectral, thermal denaturation, and molecular docking binding modeUV-visible and binding strength of β-carboline derivatives (4B, 6i, 4D, and studies. 6u) to DNA were studied through UV-visible spectral, thermal denaturation, and molecular docking studies. were studied through UV-visible spectral, thermal denaturation, and molecular docking studies. 2.7.1. UV-Visible Spectral Study 2.7.1. UV-Visible Spectral Study 2.7.1. UV-Visible Study In order toSpectral reveal the binding mode of β-carboline derivatives to DNA, the UV absorption In order to reveal the binding mode of β-carboline derivatives to DNA, the UV absorption spectra of the interactions of β-carbolines (4B, 6i, 4D, and 6u) with calf to thymus DNA (CT absorption DNA) were In order reveal theofbinding mode of 6i, β-carboline derivatives DNA,DNA the (CT UV spectra of the to interactions β-carbolines (4B, 4D, and 6u) with calf thymus DNA) were investigated (Figure 5). The absorbance at 3256i, nm of 4B, 6i, 4D, and 6uthymus was gradually decreased with spectra of the (Figure interactions of β-carbolines 4D, 6u)6i, with (CT DNA) were investigated 5). The absorbance(4B, at 325 nm and of 4B, 4D,calf and 6u wasDNA gradually decreased red shift (4B-15 nm,5). 6i-18 nm, and 6u-20 nm), indicating that and 4B, 6u 6i, was and gradually 6u could bind to DNA by investigated (Figure The absorbance at 325 nm of 4B, 6i, 4D, decreased with with red shift (4B-15 nm, 6i-18 nm, and 6u-20 nm), indicating that 4B, 6i, and 6u could bind to DNA intercalation. Among them, the intercalation ability of 4D was very weak (may be a reasonable red shift (4B-15 nm, 6i-18 them, nm, and nm), indicating andweak 6u could DNA by by intercalation. Among the6u-20 intercalation ability ofthat 4D4B, was6i,very (maybind be ato reasonable experimentalAmong error), and it may interact with DNA through other intermolecular forces. intercalation. them, the intercalation ability of 4D was very weak (may be a reasonable experimental error), and it may interact with DNA through other intermolecular forces. experimental error), and it may interact with DNA through other intermolecular forces.

Figure 5. UV-visible spectroscopy study of β-carbolines with calf thymus (CT) DNA. UV-visible Figure 5. UV-visible spectroscopy study 200 of β-carbolines calf thymus DNA. UV-visible absorption titrations were done by adding nM CT DNAwith solution each time(CT) to the quartz cuvette absorption titrations were done by adding 200 nM CT DNA solution each time to the quartz Figure 5. UV-visible spectroscopy study of β-carbolines with calf thymus (CT) DNA. UV-visible containing 10 µM 4B, 6i, 4D, and 6u. The absorption spectra were recorded from 200 nm to 350cuvette nm. containingtitrations 10 μM 4B, 6i, 4D, and The absorption were recorded from nm to 350 nm. absorption were done by6u. adding 200 nM CTspectra DNA solution each time to 200 the quartz cuvette

containing 10 μM 4B, 6i, 4D, and 6u. The absorption spectra were recorded from 200 nm to 350 nm.

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2.7.2. Thermal Denaturation Study 2.7.2. Thermal Denaturation Study The binding affinity of β-carboline derivatives (4B, 6i, 4D, and 6u) to DNA were investigated The affinity of β-carboline derivatives (4B,molecules 6i, 4D, andinteract 6u) to DNA throughbinding thermal denaturation experiments. Small with were DNAinvestigated through the through thermal denaturation experiments. Small molecules interact with DNA through the intercalation mode, which makes the DNA double helix more stable, thereby increasing the melting intercalation mode, makes the DNA double helix more stable, thereby increasing melting temperature (Tm) which [44]. Therefore, the thermal denaturation experiment provides athe simple and temperature (T ) [44]. Therefore, the thermal denaturation experiment provides a simple and effective m effective method for detecting DNA-binding affinity. As shown in Figure 6, the ΔTm values of 4B, 6i, method for 6u detecting DNA-binding affinity. shown in Figure 6, the ∆T of 4B,bind 6i, 4D, 6uby m values 4D, and were 7.5, 8.5, 3.5, and 9.0 °C,Asrespectively, indicating that they could to and DNA ◦ were 7.5, 8.5, 3.5, and 9.0binding C, respectively, that DNA by intercalation, intercalation, and the strength ofindicating 4B, 6i, and 6uthey was could greaterbind thantothat of 4D. Thus, thermal and the binding strength of 4B, 6i, and 6u was greater than that of 4D. Thus, thermal denaturation denaturation results indicated that 4B, 6i, 4D, and 6u could exhibit significant DNA-binding affinity. results indicated that 4B, 6i, 4D, and 6u could exhibit significant DNA-binding affinity.

Figure 6. Thermal denaturation study of the complexes of β-carbolines (4B, 6i, 4D, and 6u) and Figure 6. Thermal denaturation study of the complexes of β-carbolines 4D,, and 6u) and CTCT-DNA. Melting temperatures were measured in PBS-EDTA buffer (1 mM(4B, Na26i, HPO 4 0.1 mM EDTA, HPO4, 0.1 EDTA, Melting temperatures measured in PBS-EDTA Na24B,6i,4D, or 6umM + DNA PHDNA. 7.4) with a β-carboline (4B, were 6i, 4D, and 6u)/DNA ratio of buffer 0.5. ∆T(1m mM = (∆T − m PH 7.4) with a β-carboline (4B, 6i, 4D, and 6u)/DNA ratio of 0.5. ΔT m = (ΔTm4B,6i,4D, or 6u + DNA − ΔTmDNA). DNA ∆Tm ).

2.7.3. Molecular Docking Study 2.7.3. Molecular Docking Study InIn order to to further predict thethe binding mode of β-carboline to DNA, the Surflex-Dock program in order further predict binding mode of β-carboline to DNA, the Surflex-Dock program theinSybyl-X 2.0 package (Tripos, Princeton, NJ, USA) used to perform docking studies the Sybyl-X 2.0 package (Tripos, Princeton, NJ,was USA) was used to molecular perform molecular docking between derivatives and DNA,and to explore of antitumor activity. studies β-carboline between β-carboline derivatives DNA, topossible exploremechanisms possible mechanisms of antitumor The structures docking scores of the DNA-β-carboline complex arecomplex shown in 7. in The total 7. activity. The and structures and docking scores of the DNA-β-carboline areFigure shown Figure scores of 4B, 6i, 4D, and 6u were 7.5936, 8.9892, 7.6925, and 8.6512, respectively, indicating that 4B, 6i, The total scores of 4B, 6i, 4D, and 6u were 7.5936, 8.9892, 7.6925, and 8.6512, respectively, indicating 4D, and4B, 6u6i, could into well the DNA. the scores of dimers and 6u) were higher than that 4D,well andinsert 6u could insertMoreover, into the DNA. Moreover, the(6i scores of dimers (6i and 6u) that of monomers (4Bthat andof4D), indicating that the4D), dimers have stronger DNA-binding thanDNAthe were higher than monomers (4B and indicating that the dimers haveability stronger corresponding monomers, exerting monomers, better antitumor activity. In addition, the planar activity. structureIn binding ability than thethereby corresponding thereby exerting better antitumor of addition, β-carbolines 6i, structure and 4D) could interact with through π-π stacking, the carbon-hydrogen the (4B, planar of β-carbolines (4B,DNA 6i, and 4D) could interact with DNA through π-π bond (non-classical hydrogen bond),bond and the π-alkyl bondhydrogen (hydrophobic force). presence stacking, the carbon-hydrogen (non-classical bond), andThethe π-alkylof the bond fluorine bond between and 6u might support its bond higherbetween antitumor activity. (hydrophobic force). DNA The presence of the fluorine DNA and 6u might support its higher antitumor activity.

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Figure 7. Three-dimensional conformations of the complexes of β-carbolines (4B, 6i, 4D, and 6u) Figure 7. Three-dimensional conformations of the complexes of β-carbolines (4B, 6i, 4D, and 6u) docked with DNA. docked with DNA.

3. Discussion 3. Discussion β-carboline alkaloids are a class of natural and synthetic indole alkaloids with a wide range of β-carboline a classantitumor, of naturalantiviral, and synthetic indole alkaloids with a wide range of pharmacological alkaloids activities, are including antiparasitic, and antibacterial activities [5,6]. pharmacological activities, antitumor, antiviral, antiparasitic, antibacterial Particularly, previous studiesincluding have shown that β-carboline alkaloids exertedand antitumor activityactivities through [5,6]. Particularly, previous have shown that β-carboline alkaloids antitumor activityI insertion into DNA [10–12],studies inducing apoptosis [13,45], inhibiting CDKs exerted [16], and topoisomerase 3 9 through insertion into DNA [10–12], inducing apoptosis [13,45], inhibiting CDKs [16], and and II activity [13,15]. The substitution at the C and N positions of β-carbolines could significantly 3 9 topoisomerase I andinsertion II activity [13,15]. The substitution at thestudies C andhave N positions ofdimerization β-carbolines increase their DNA ability [7]. Moreover, the existing found that could significantly increase their DNA insertion ability [7]. Moreover, the existing studies have found of small molecules could significantly increase [26]. that dimerization small molecules could significantly increase [26]. DNA bindingofaffinity [31,32]. Therefore, designing and synthesizing DNA-targeted β-carboline DNA binding affinity [31,32]. Therefore, designing andantitumor synthesizing DNA-targeted β-carbolinea derivatives is an important way to find potentially effective drugs. Here, we synthesized 9 derivatives is an important way to find potentially effective antitumor drugs. Here, we synthesized series of bivalent β-carboline derivatives modified at the N position and dimerized at the C3 position, aand series of bivalent β-carbolineactivity derivatives modified at the N9 position dimerized evaluated their antitumor and DNA binding affinity in vitro. and The IC of the 4B, C 6i,3 50 valuesat position, and evaluated activity binding affinity in vitro. The IC50 values of 4D, and 6u against A549their wereantitumor 39.20, 6.39, 13.94, and DNA 5.61 µM, respectively. 4B, 6i, 4D, and 6u against A549 were 39.20, 6.39, 13.94, and 5.61 μM, respectively. Previous studies have shown that the antitumor activity of β-carboline could be significantly 9 Previous shown at that activity of β-carboline could be[5,35]. significantly improved withstudies benzylhave substituted thethe N antitumor position and dimerized at the C3 position In this 9 position and dimerized at the C3 position [5,35]. In this improved with benzyl substituted at the N study, the monomers exhibited good-to-strong antitumor activity with the tendency of o-fluorobenzyl > study, the monomers exhibited> benzyl. good-to-strong antitumor activityof with the tendency of op-methylbenzyl > o-methylbenzyl Furthermore, for the dimers o-methylbenzyl substituted fluorobenzyl > p-methylbenzyl o-methylbenzyl > benzyl. for the dimers of oat the N9 position, most dimers>were generally more active Furthermore, than the corresponding monomers, 9 position, most dimers were generally more active than the methylbenzyl substituted at the N following the tendency of 5 > 4 > 6 > 3 > 8 methylene units. For the dimers of o-fluorobenzyl corresponding monomers, following tendency 5 > 4 > 6 > more 3 > 8 methylene For the dimers substituted at the N9 position, mostthe dimers wereofgenerally active thanunits. the corresponding of o-fluorobenzyl substituted at theofN59 position, dimers were generally more active thanthat the monomers, following the tendency > 3 > 6 > 4most > 8 methylene units. These results indicated 9 3 corresponding monomers, following the tendency of 5 > 3 > 6 > 4 > 8 methylene units. These results N -benzyl substitution and C -dimerizition with 5–6 methylene units were favorable for increasing indicated N9-benzyl andwith C3-dimerizition 5–6 methylene antitumorthat activity, which substitution was consistent the results ofwith previous studies. units were favorable for increasing antitumor activity, which was consistent with the results of previous studies. Previous studies have shown that cell cycle arrest and cell apoptosis are the main mechanisms studiesderivatives have shown cell cycle arrest and cell the main usedPrevious by β-carboline asthat antitumor drugs [4,46]. Ourapoptosis previousare studies havemechanisms shown that used by β-carboline derivatives as Our studiesand have shown that the the introduction of substituents (aantitumor methyl or drugs benzyl[4,46]. group) at previous the N9 position the dimerization 9 position and the dimerization of βintroduction of substituents (a methyl or benzyl group) at the N of β-carbolines could significantly increase the antitumor activity, and could cause cell cycle arrest carbolines increaseapoptosis the antitumor activity, and could cause[36,37]. cell cycle at the at the S or could G2/Msignificantly phase and induce in a dose-dependent manner In arrest the present S or G2/M phase and induce apoptosis in a dose-dependent manner [36,37]. In the present study, we study, we found that the cell cycle arrested at the S and G2/M phases with sub G1 peaks, and induced found that the cell cycle arrested at the S and G2/M phases with sub G1 peaks, and induced apoptosis after treatment with 4B, 6i, 4D, and 6u. Furthermore, the reduction of cytochrome C (Cyt C) in

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apoptosis after treatment with 4B, 6i, 4D, and 6u. Furthermore, the reduction of cytochrome C (Cyt C) in mitochondria, and the decrease in expression of bcl-2 protein, are the typical features of mitochondria-mediated apoptosis [47]. Additionally, the results of western blot showed that the levels of Cyt C (in mitochondria) and the expression of bcl-2 protein decreased in a dose-dependent manner after incubation with 6i and 6u. Taken together, these results revealed that 6i and 6u could induce cell apoptosis through a mitochondria-mediated pathway. To date, DNA intercalators have been effective antitumor drugs in clinical application. After the drugs are inserted into DNA, they can cause conformational changes of the double helix, which disrupts DNA replication, transcription, and repair [23]. The antitumor activity of a large number of natural and synthetic β-carboline derivatives that could interact with DNA by intercalation have been discovered and evaluated. Furthermore, the interactions between these derivatives (4B, 6i, 4D, and 6u) and DNA were investigated by UV-visible spectroscopy, thermal denaturation, and molecular modeling studies. The absorbance of 4B, 6i, 4D, and 6u decreased with red shift, and the ∆Tm values of 4B, 6i, 4D, and 6u were 7.5, 8.5, 3.5, and 9.0 ◦ C, respectively, indicating that 4B, 6i, and 6u could stably bind to DNA by intercalation, which is in agreement with the results of molecular docking studies. In conclusion, a series of novel bivalent β-carboline-3-carboxylic acid derivatives were synthesized, and antitumor activity and DNA-binding affinity were preliminarily evaluated in vitro. The SARs studies revealed that the introduction of appropriate substituents at the N9 position and dimerization at the C3 position of β-carbolines could significantly increase the antitumor activity against A549, Hela, SGC-7901, SMMC-7721, and MCF-7 cell lines. Furthermore, 4B, 6i, 4D, and 6u could further induce tumor cell cycle arrest and apoptosis, and could interact with DNA through intercalation. In summary, further studies of 4B, 6i, 4D, and 6u could be performed to develop potential DNA-targeted agents in clinical applications. 4. Experimental Section 4.1. Materials and Methods All chemicals and solvents used in the synthesis were purchased from suppliers. They were dried and purified when needed. Melting points were determined on a capillary melting apparatus and were uncorrected (SGW X-4, Shanghai Shenguang Instrument Co., Ltd., Shanghai, China). The 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker Avance III at 500 MHz using tetra methyl silane (TMS) as the internal standard. The column chromatography and analytical thin layer chromatography (TLC) were performed with silica gel (100–200 mesh) and silica gel GF254 (Qingdao Marine Chemical Company, Qingdao, China). All cell lines (A549, SGC-7901, Hela, SMMC-7721, and MCF-7) were purchased from the American Type Culture Collection (Manassas, VA, USA), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HycloneLaboratories Inc., Logan, Utah, USA) supplemented with 10% fetal bovine serum (FBS, Gibco&Invitrogen, Carlsbad, CA, USA) at 37 ◦ C and 5% CO2 . 4.2. Chemistry 4.2.1. General Synthesis Procedure for N9 Substituted β-Carboline-3-carboxylic Acid Methyl Esters (4A, 4B, 4C, and 4D) Using L-tryptophan as the raw material, β-carboline monomers (4A, 4B, 4C, and 4D) were synthesized through the Pictet–Spengler reaction (P–S reaction), esterification reaction, oxidation reaction, and N9 substitution reaction, as previously described [5,26,36]. Reaction routes and conditions are shown in Scheme 1.

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4.2.2. General Reaction of Synthesis of Bivalent β-Carboline Derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x) The N9 substituted β-carboline-3-carboxylic acid methyl esters (4A, 4B, 4C, and 4D) prepared in the above steps were hydrolyzed and reacted with dibromoalkane to obtain a series of bivalent β-carboline-3-carboxylic acid derivatives (6a-6f, 6g-6l, 6m-6r, and 6s-6x), as previously described [34,35,37,48]. Reaction routes and conditions are shown in Scheme 2. 4.3. MTT Assay The cytotoxic activities of β-carboline-3-carboxylic acid derivatives were investigated by the MTT method, as previously described [49]. Cells (1 × 104 cells per well) seeded into 96-well plates (JET, Guangzhou, China) were incubated with DMEM containing various concentrations (2.5, 5, 10, 20, 40, and 80 µM) of β-carbolines (4A, 4B, 4C, 4D, 6a-6f, 6g-6l, 6m-6r, and 6s-6x) for 48 h at 37 ◦ C and 5% CO2 . Then, 10 µL of 5 mg/mL MTT reagent dissolved in PBS (Phosphate buffered saline) was added per well, and plates were incubated at 37 ◦ C for 4 h. The medium was carefully removed and 100 µL dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance was measured at 490 nm with a microplate reader (ThermoFisher Scientific Inc., Waltham, MA, USA). The β-carbolines were dissolved in DMSO as stock solutions (10 mM) at −20 ◦ C, and diluted in the final experimental solution using DMEM before use. A549 (lung carcinoma), SGC-7901 (gastric carcinoma), Hela (cervical carcinoma), SMMC-7721 (liver carcinoma), and MCF-7 (breast carcinoma) cell lines were used for the antitumor activity screening. The IC50 values were the mean values of three independent experiments. 4.4. Hoechst 33342/PI Dual Staining Assay The morphological observation of apoptosis was performed by Hoechst 33342 and propidium iodide (PI) dual staining according to the manufacturer’s instructions. A549 cells (2 × 104 cells per well) seeded into 24-well plates (JET, Guangzhou, China) were incubated with DMEM containing 4 µM of β-carbolines (4B, 6i, 4D, and 6u) for 48 h at 37 ◦ C and 5% CO2 . Then, the cells were stained by Hoechst 33342 and PI at 37 ◦ C for 30 min in dark conditions. Finally, cells were observed and photographed with a fluorescence microscope (LeicaDM6B, Leica, Nussloch, Germany). 4.5. Cell Cycle Assay The cell cycle was determined by PI single staining. A549 cells (1 × 106 cells per well) seeded into 6-well plates (JET, Guangzhou, China) were incubated with DMEM containing various concentrations (2, 4, and 8 µM) of β-carbolines (4B, 6i, 4D, and 6u) for 48 h at 37 ◦ C and 5% CO2 . The cells were harvested, washed with pre-cooled PBS (4 ◦ C), and fixed with pre-cooled 90% ethanol (4 ◦ C). Then, the cells were incubated with 20 µL RNAse (30 µg/mL) and 20 µL PI (50 µg/mL) (Sigma Aldrich, Saint Louis, MO, USA) for 30 min at 37 ◦ C. The samples were analyzed using flow cytometry (BD FACSCalibur, San Jose, CA, USA). 4.6. Cell Apoptosis Assay The cell apoptosis was evaluated using Annexin V-FITC/PI dual staining (Beyotime, China). A549 cells (1 × 106 cells per well) seeded into 6-well plates (JET, Guangzhou, China) were incubated with DMEM containing various concentrations (2, 4, and 8 µM) of β-carbolines (4B, 6i, 4D, and 6u) for 48 h at 37 ◦ C and 5% CO2 . The cells in the supernatant and adherent were collected and washed with PBS, and then the cells were stained with Annexin V-FITC and PI for 20 min at r.t. The samples were analyzed using flow cytometry (Becton Dickinson, San Jose, CA, USA).

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4.7. Western Blot Assay The level of cytochrome C (Cyt C) in mitochondria, and the expression of bcl-2 protein, were evaluated by western blot. A549 cells (1 × 106 cells per well) seeded into 6-well plates (JET, Guangzhou, China) were incubated with DMEM containing various concentrations (2, 4, and 8 µM) of β-carbolines (6i and 6u) for 48 h at 37 ◦ C and 5% CO2 . Then, the cells were lysed with RIPA lysis buffer for 30 min on ice (containing 1% PMSF), centrifuged at 12,000× g for 5 min at 4 ◦ C, and then the supernatants were transferred to 1.5 mL EP tubes. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA), as previously described [50]. The membranes were blocked for 2 h at r.t. with 5% non-fat milk in TBST buffer (20 mM Tris-HCl,150 mM NaCl and 0.05% Tween-20, pH7.4), and then incubated with the mouse anti-bcl-2 monoclonal antibody (Sigma, Saint Louis, MO, USA), anti-cytochrome C monoclonal antibody (Beyotime, Haimen, China), and anti-β-actin antibody (1:4000 dilution; Abcam, Cambridge, MA, USA) overnight at 4 ◦ C, and finally incubated with the peroxidase conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at 37 ◦ C for 1 h. Immunostained proteins were visualized using ECL reagent according to the manufacturer’s instructions (Pierce, Rockford IL, CA, USA). β-actin was used as an internal control. 4.8. UV-Visible Spectral Study UV-visible spectroscopy was carried out to determine the binding mode of β-carboline derivatives to DNA (CT DNA) using UV-2500 spectrophotometer (Shimadzu, Kyoto, Japan) at 25 ◦ C. Stock solutions of 10 mM β-carboline derivatives were dissolved in DMSO, and 10 µM CT DNA was prepared in 100 mM KBPES buffer (30 mM Potassium Phosphate with 100 mM KCl, pH 7.0) [13]. UV-visible absorption titrations were performed by adding 200 nM CT DNA solution to the quartz cuvette containing approximately 10 µM derivative solutions. UV-Visible absorption spectra were recorded from 200 nm to 350 nm. 4.9. Thermal Denaturation Study The DNA-binding modes of the β-carboline derivatives (4B, 6i, 4D, and 6u) with CT DNA were examined by DNA thermal denaturation experiments, as previously described [10]. The solutions containing complexes of CT DNA and 4B, 6i, 4D, or 6u were heated using a thermostatic water bath, and the absorbance was measured in 1 ◦ C steps from 30 ◦ C to 95 ◦ C at 260 nm (At ) by a UV-Vis spectrophotometer (A is the absorbance of the solution at 260 nm at different temperatures, t is the temperature). The experiments were performed in PE buffer (pH = 7.4) with a β-carboline/DNA ratio of 0.5. The ∆Tm value of DNA alone is 56.8 ◦ C. ∆Tm = ∆Tm (DNA + 4B, 6i, 4D, or 6u) − ∆Tm (DNA alone) . 4.10. Molecular Modeling Study Molecular docking software (Sybyl-X 2.0 Tripos, Princeton, NJ, USA) and Discovery Studio 2017 client (BIOVIA, San Diego, CA, USA) was used to investigate the interaction of 4B, 6i, 4D, and 6u with DNA. The DNA was retrieved from the Protein Data Bank (PDB entry code: 1Z3F). The crystal structure of the DNA molecule was prepared by adding all the hydrogen atoms, and the charge was added using the AMBER7 FF99 method. The structures of 4B, 6i, 4D, and 6u were drawn in the Sybyl-X 2.0 package (Tripos, Princeton, NJ, USA). Hydrogen atoms were added, and then the energy was optimized using the Tripos force field and the Gasteiger–Hückel method. The Surflex-Dock scoring function is a weighted sum of non-linear functions based on the binding affinities of DNA-ligand complexes and their crystallographic structures. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/10/ 3179/s1.

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Author Contributions: J.W. conceived and designed the experiments; H.G., N.L., J.D., Y.X. performed the experiments; H.G. analyzed data and drafted the manuscript; S.W. edited and revised the manuscript. All authors approved the final version of the paper. Funding: This work was financially supported by the National Natural Science Foundation of China (Grant No. 81773603), the Open Foundation of Key Laboratory of Synthetic and National Foundation Molecule Chemistry of the Ministry of Education (Northwest University, China), the State Key Laboratory of Drug Research (Grant No. SIMM1705KF-09), the National Training Program on Undergraduate Innovation and Entrepreneurship (Grant No. 201803018, Northwest A&F University, China). Acknowledgments: Chengbao Wang, from the college of veterinary medicine in the university provided some technical support and materials in the biochemical experiments. H.W., an undergraduate from the innovative experimental college in the university participated in some experiments within the present research. Conflicts of Interest: The authors declare no conflict of interest.

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