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European Journal of Medicinal Chemistry 105 (2015) 208e219

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Research paper

The synthesis of indolo[2,3-b]quinoline derivatives with a guanidinegroup: Highly selective cytotoxic agents

Katarzyna Sidoryk a, *, Marta �Switalska b, Anna Jaromin c, Piotr Cmoch a, d, Iwona Bujak a,Monika Kaczmarska a, Joanna Wietrzyk b, e, Eddie G. Dominguez f, Robert _Zarnowski f,David R. Andes f, Krzysztof Ba�nkowski a, Marcin Cybulski a, Łukasz Kaczmarek a

a Pharmaceutical Research Institute, 8 Rydygiera St., 01-793 Warsaw, Polandb Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Weigla St., 53-114 Wroclaw, Polandc Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, 14A Joliot-Curie St., 50-383 Wroclaw, Polandd Institute of Organic Chemistry, Polish Academy of Sciences, 44/52 Kasprzaka St., 01-224 Warsaw, Polande Institute of Chemistry Environmental Protection and Biotechnology, Jan Długosz University, 13/15 Armii Krajowej Ave., 42-200 Czestochowa, Polandf Department of Medicine, Section of Infectious Diseases, 4125 Microbial Sciences Building, 1550 Linden Dr., University of Wisconsin-Madison, Madison, WI53706, USA

a r t i c l e i n f o

Article history:Received 23 July 2015Received in revised form5 October 2015Accepted 10 October 2015Available online xxx

Keywords:NeocryptolepineAntiproliferative activityAntifungal activityBiofilmGuanidine groupMechanism of actionApoptosisHemolytic activity

Abbreviations: Boc, tert-butyloxycarbonyl group;DIPEA, N,N-diisopropylethylamine; DMF, dimethythylsulfoxide; DSS, 4,4-dimethyl-4-silapentane-1-sulybenzotriazole monohydrate; HPLC, high performaTBTU, O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-TFA, trifluoroacetic acid.* Corresponding author.

E-mail address: k.sidoryk@ifarm.eu (K. Sidoryk).

http://dx.doi.org/10.1016/j.ejmech.2015.10.0220223-5234/© 2015 Elsevier Masson SAS. All rights re

a b s t r a c t

The synthesis of indolo[2,3-b]quinoline derivatives containing guanidine, amino acid or guanylaminoacid substituents as well as their in vitro evaluation for the cytotoxic and antifungal activity are reported.The influence of the guanidine group on the selective cytotoxic and hemolytic properties of indolo[2,3-b]quinoline was investigated. Most of the compounds displayed a high cytotoxic activity in vitro and two ofthe most promising compounds (3 and 12) exhibited a high selectivity between normal and cancer cell-lines. The cytotoxic activity of compound 3 was about 600-fold lower against normal fibroblasts thanagainst A549 and MCF-7 cancer cell lines. Novel entities acted as the DNA-intercalators when testedusing a DNAemethyl green assay but demonstrated zero or low hemolytic activity in comparison to theirunsubstituted analogs. The mechanism of action was studied for guanidine derivatives 3 and 12 and bothcompounds were found to be very effective inducers of apoptosis.

© 2015 Elsevier Masson SAS. All rights reserved.

1. Introduction

Neocryptolepine is an alkaloid which displays a broad spectrumof biological activities including an antibacterial, antifungal, anti-inflammatory, cytotoxicity, and antimalarial activity [1e6] (Fig. 1).5,11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ, Fig. 1), the syn-thetic analog of neocryptolepine, demonstrates high cytotoxic

BSTU, N,N0-bis-Boc-thiourea;lformamide; DMSO, dime-fonic acid; HOBt, N-hydrox-nce liquid chromatography;uronium tetrafluoroborate;

served.

activity and inhibits the proliferation of mouth cacinoma KB cells ata concentration of 1 mM. Moreover, its activity is similar to thecytotoxic activity of doxorubicin (0.8 mM against KB cells) [7e9].Unfortunately, DiMIQ's high toxicity, lack of selectivity and very lowsolubility in aqueous solutions, especially at neutral pH, seriouslylimit the practical application of this compound in the treatment ofcancer [10]. The high toxicity and low bioavailability of DiMIQ,prompted us to look for new analogs which would conform to thehigh requirements necessary for anticancer drugs: potent and se-lective activity and low side effects. Our recently published resultsreveal that this might be achieved by constructing conjugatescomposed of DiMIQ and amino acids or peptides [11e13]. As it hasbeen proved, the attachment of an amino acid moiety or a shortpeptide chain to DiMIQ significantly improves its physicochemicalproperties, resulting in the auspicious anticancer action in vivowitha relatively low hemolytic effect. Some of the recently reported

Fig. 1. Structures of neocryptolepine and DiMIQ.

K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219 209

neocryptolepine analogs showed promising antifungal and anti-bacterial characteristics which could potentially aid futureanticancer-antimicrobial treatment [13].

However, the cytotoxic activity in vitro of the above mentionedcompounds and other known indolo[2,3-b]quinoline derivativesagainst cancer cells was comparable to their cytotoxic activityagainst normal cell lines [14e17]. The lack of the selectivity of ac-tion of the indolo[2,3-b]quinoline derivatives obtained so farprompted us to search further for more selective antitumor com-pounds with high cytotoxic activity against cancer cells and lowagainst normal cells.

After a detailed literature search, it was assumed that our goalcould be accomplished by the introduction of a guanidine groupinto the indolo[2,3-b]qiunoline conjugates. The guanidine groupwidely exists in various natural products and pharmaceuticallyactive compounds [18,19]. This group has been found inmetabolitesof different living organisms and many naturally occurring sub-stances such as ptilomycalin A and bisguanidine. Ptilomycalin A,which exhibits an antimicrobial, antifungal, antiviral and alsocytotoxic activity, was isolated from the sponges of the Red Sea andthe Caribbean Sea [20e22]. Bisguanidine, TAN-1057, isolated fromFlexibacter sp. PK-74 bacteria, possess a potent activity against b-lactam-resistant, Gram-positive bacteria [23]. The guanidine sub-structure is also present in the active pharmaceutical substancesused to treat influenza A and B viral infections (Zanamivir), as wellas bacterial infections (Chlorhexidine, Sulfaguanidine) (Fig. 2).Furthermore, it seems that the guanidine group plays an importantrole in drug delivery to cancer cells due to its strong basic proper-ties (pKa 12.5). Evidence has also been found for its possible stronginteraction with the phosphate residues of the minor groove of theDNA helix [24]. Taking into consideration all the above facts, it

Fig. 2. Guanidine-containing natural

could be expected that the introduction of the guanyl substituentsinto novel conjugates would increase the DNA interaction and, as aresult, advantageously affect the cytotoxic activity of the novelanalogs. Besides stabilizing the drug-DNA complex, the guanidinegroup might also improve the delivery of the substances insidecancer cells by increasing their hydrophilicity and water solubility,while simultaneously decreasing their toxicity [25e28].

Herein we report the synthesis of new hybrid compounds withthe indolo[2,3-b]quinoline core [(5,11-dimethyl-5H-indolo[2,3-b]quinoline e DIMIQ or 6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinoline)] and a guanidine group, an amino acidresidue or an N-guanylamino acid. All new conjugates were testedfor their cytotoxic activity against cancer and normal cell lines andagainst fungal biofilms as well as for their DNA interactions. Mostpromising compounds were selected to establish their mechanismof action. Moreover, all novel conjugates were tested for theirability to induce the hemolysis of human erythrocytes, as this test isone of the most important and most frequently studied biocom-patibility measures.

2. Results and discussion

2.1. Synthesis

5,11-dimethyl-5H-indolo[2.3-b]quinolin-9-yl-amine dihydro-chloride (1a) and 6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2.3-b]quinolin-9-yl-amine trihydrochloride (10a) used for the SARstudies were prepared by treating the amino derivatives of 1 and 10with HCl/MeOH. The synthesis of the N-guanidine- and N-guany-lamino acids conjugates of 1 is outlined in Scheme 1. The startingcompound 1 is not commercially available and was synthesized asdescribed previously [14,29]. The guanidinylation of 1 with N,N0-bis-Boc-thiourea in the presence of DIPEA and HgCl2 gave the ex-pected compound 2 with 77% yield. The treatment of the protectedBoc-derivative (2) with the trifluoroacetic acid and then with HCl/MeOH gave N-guanyl-N-(5,11-dimethyl-5H-indolo[2,3-b]chinolin-9-yl)-amine dihydrochloride 3 with an excellent 98% yield. Theguanidinylation of compounds 4 [12] and 7 [12] with BSTU understandard conditions afforded 5 and 8 with a moderate 60% andgood 80% yield, respectively. In the case of Na-bis(tert-butylox-ycarbonyl)guanyl-glycyl-N-(5,11-dimethyl-5H-indolo[2,3-b]

products and pharmaceuticals.

Scheme 1. Reagents and conditions: (a) BSTU, HgCl2, DIPEA, DMF, 24 h, rt; (b) TFA, HCl/MeOH.

K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219210

chinolin-9-yl)-amide 5, the main product was obtained with asmall amount of the impurity 5a. The Boc-removal of compounds 5and 8 with the trifluoroacetic acid and then the treatment of theresidue by hydrogen chloride in methanol gave the hydrochloridederivatives of DiMIQ, compounds 6 and 9, with 85% and 95% yields,respectively. The hydrochlorides of the deprotected derivatives ofDiMIQ were purified by crystallization.

Na-guanyl-N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-amine tetrahydrochloride 12 was obtainedaccording to the above procedure (Scheme 2). The guanidinylationof 10 [17] gave derivative 11 in a moderate 61% yield and the sub-sequent Boc deprotection gave the corresponding derivative 12with a 77% yield. The glycyl and L-prolyl derivatives of 6H-indolo-quinoline 14 and 16 were obtained by reacting 9-amino derivative(10) with the Boc-protected amino acids using the 2-(1H-benzo-triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)method [30]. The coupling reactions were performed in DMF atroom temperature for 2e24 h. The Boc-Na-amino acid derivativesof 10, compounds 13 and 15, were separated by extraction andpurified by column chromatography on the silica gel. Their yieldsafter the purification were 54 and 56%. In the next step the Bocgroups were removed by the trifluoroacetic acid and finally the

trifluoroacetates were converted into the appropriate hydrochlo-rides 14 and 16 by the HCl/MeOH treatment.

The N-guanylamino acid derivatives of 6H-indoloquinoline werealso synthesized (Scheme 3). The synthesis of these derivatives wascarried out analogously to the method used for the N-guanylaminoacid derivatives of 5H-indoloquinoline. The guanidinylation of 14and 16 with BSTU in the presence of DIPEA and HgCl2 afforded theexpected Boc-derivatives 17 and 19 with moderate yields 64% and51%, respectively. The Boc groups were removed by TFA treatmentand the final hydrochlorides 18 and 20 were obtained in goodyields.

The structures of all new compounds were confirmed byextended 1D and 2D NMR experiments (Fig. 3 and Table 1S, Sup-plementaryMaterial), as well as MS (Experimental part). The purityof all final products was verified by the elemental analysis and theC-18 RPHPLC method using acetonitrile-water as the mobile phase.

2.2. Biological studies

2.2.1. Antiproliferative activity in vitroAll synthesized compounds were evaluated for their anti-

proliferative activity in vitro against the following cell lines: A549

Scheme 2. Reagents and conditions: (a) BSTU, HgCl2, DIPEA, DMF, 24 h, rt; (b) TFA, HCl/MeOH; (c) Boc-L-Pro, TBTU, HOBt, DIPEA, DMF; (d) Boc-Gly, TBTU, HOBt, DIPEA, DMF.

Scheme 3. Reagents and conditions: (a) BSTU, HgCl2, DIPEA, DMF, 24 h, rt; (b) TFA, HCl/MeOH.

K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219 211

(non-small cell lung cancer), MCF-7 (breast cancer), LoVo (coloncancer), and KB cells (mouth carcinoma cell). Possible cell toxicityof the indolo[2,3-b]quinoline derivatives was tested by the rate ofviability of the normal murine fibroblasts BALB/3T3. The results ofthe studies on the antiproliferative activity of the guanidine, amino

acid and N-guanylamino acid derivatives of indolo[2,3-b]quinolineare summarized in Table 1. The tested compounds showed a diverseactivity against cancer and normal cells. The amino derivative ofDiMIQ (1a) and 6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2.3-b]quinolin-9-yl-amine (10a) displayed the highest cytotoxic

Fig. 3. The 1H, 13C and 15N NMR signal assignments for analog 16 (in D2O).

K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219212

activity against all cancer cell lines with IC50 values between 0.07and 0.8 mM. Unfortunately, these compounds were also cytotoxicagainst the normal murine fibroblast (BALB/3T3), with IC50 values0.54 and 0.56 mM for 1a and 10a, respectively.

The introduction of the guanidine group to the DiMIQ molecule(compound 3) or to the glycine and L-proline DiMIQ derivatives(compounds 6 and 9) resulted in differences in the cytotoxic ac-tivity. The most advantageous activity profile was obtained forcompound 3 when the guanidine moiety was linked directly to theDIMIQ core. As it was shown in Table 1, compound 3 demonstratedits highest cytotoxicity against the MCF-7 cell line (IC50 value0.06 mM), the A549 cell line (IC50 value 0.14 mM) and the KB cell line(IC50 value 0.88 mM) but was not active against the LoVo cell line(IC50 value 76.88 mM). In accordance with our proposed hypothesis,the most interesting data were collected when the cytotoxic ac-tivity of the guanidine DIMIQ (3) against BALB3T3 murine fibro-blasts was tested. It should be pointed out that compound 3 did notexhibit any antiproliferative activity against the normal cell line(IC50 value 66.43 mM). The cytotoxic activity of compound 3 wasabout 600-fold lower against the normal cell line than against A549and MCF-7 cancer cell lines. This very high selective action hasnever been observed for any standard anticancer drug. For example,doxorubicin, widely used in cancer treatment, shows a comparativecytotoxic activity against cancer and normal cell-lines. Although ahigh cytotoxic activity was also observed in the case of the N-guanylamino acid derivatives 6 and 9, no significant selectivity ofaction was identified. The highest activity against the LoVo cell line(IC50 0.83 mM) and against the A549 cell line (IC50 1.82 mM) wasexhibited by derivative 6, whereas its activity against the KB andMCF-7 cell lines was lower, with IC50 values 3.88 and 4.75 mM,respectively. Compound 6 exhibited a cytotoxic activity against thenormal cell line with IC50 value of 4.47 mM. The cytotoxic activity ofderivative 9 against all cancer cell lines was in the range of7.76e15.57 mM. On the other hand, its cytotoxic activity against theBALB/3T3 normal cell line was lower and was measured at31.92 mM.

For a more thorough examination of the influence of the guanylgroup on the conjugate's cytotoxic activity, guanidine (12), aminoacid (14 and 16), and N-guanylamino acid (18 and 20) derivatives of6H-indolo[2,3-b]quinoline were also synthesized. Similarly to thedata collected for the DIMIQ derivatives, the same influence on theantiproliferative activity was observed for the 6H-indolo[2,3-b]quinolone analogs. The effect was related to the presence and po-sition of the guanyl group in the molecule.

Maximal selective activity was achieved when the guanidinegroup was directly connected with the 6H-indolo[2,3-b]quinolinecore (12). The IC50 values for compound 12 were 0.81 and 0.87 mMfor MCF-7 and KB cell lines, respectively. Alike compound 3, 12 didnot exhibit cytotoxic activity against normal cell line (IC5030.04 mM). Glycine (14) and L-proline (16) derivatives of 6H-indolo[2,3-b]quinoline acted stronger than DiMIQ or doxorubicin againstall cancer cell lines with IC50 values ranging from 0.06 to 1.1 mM. But

the antiproliferative activity of these compounds against the BALB/3T3 normal cells (IC50 0.61 and 0.36 mM, respectively) was about 10times higher than the cytotoxicity of DiMIQ and comparable to theactivity against cancer cells. On the other hand, compounds 18 and20, the N-guanylamino acid derivatives of 6H-indolo[2,3-b]quino-line possessed a lower cytotoxic activity than their amino acidcounterparts. For example, IC50 value for compounds 18 and 20wasbetween 0.82 mM and 8.3 mM against all cancer cell lines.

Compounds 3 and 12 were selected for further studies due totheir high anticancer activity and specificity (cancer vs. normal cell-lines).

2.2.2. Activity against fungal biofilms in vitroThe newly synthesized DiMIQ derivatives were evaluated for

their antifungal activity against Candida albicans biofilms. The ex-periments were designed to determine the cell viability of fungalbiofilms in vitro grown in 96-well ELISA plates after exposure to thetested compounds [13]. Our previous study demonstrated a strongpreferential action of the DiMIQ derivatives substituted with theamino acid or dipeptide groups against the C. albicans biofilms [13].

In the current study, the N-guanylamino acid derivative 6showed the highest antibiofilm activity (ED50 4.2 mM), whereas twoother molecules, namely the amino group-modified derivative ofDiMIQ (1a), and 6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2.3-b]quinolin-9-yl-amine (10a) displayed a strong antifungal ac-tivity against C. albicans with ED50 values at 41.0 and 34.2 mM,respectively. Other 6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolone derivatives containing only amino acidmoieties (14 and 16) were also found active and actually moreefficient than the original natural component (ED50 74.3 and25.5 mM, respectively).

Compounds 3 and 12 with the guanidine moiety linked directlyto the indoquinoline ring did not exhibit any activity against theC. albicans biofilms. Strikingly, the addition of the guanyl moietiesinto the DiMIQ structure dramatically reduced their antifungalactivity, while maintaining the preferred anticancer propertiesin vitro (Table 2).

2.2.3. DNA interactionsIt is generally accepted that the cytotoxic activity of the indolo

[2,3-b]quinolone derivatives depends on their ability of action asthe DNA intercalating agents and topoisomerase inhibitors[6e8,14e17]. Therefore, the novel compounds were evaluated aspotential DNA-interacting agents using a DNA-methyl green assaybased upon the displacement of the dye methyl green from theDNA-methyl green complex (Table 3). The tested compounds,namely 1a, 3, 6,10a,12,14 and 16, showed a pronounced activity inthe DNA-methyl green assay, confirming their DNA-intercalatingproperties which are generally associated with cytotoxicity. It wasidentified that the introduction of any additional substituent intothe indolo[2,3-b]quinoline core resulted in the increase of the DNA-interacting properties in comparison to DiMIQ. The strongestinteraction was observed for compounds 6 and 16 with a differentindoloquinoline moiety. It is worth noting that all examined com-pounds showed DNA interacting activity while being cytotoxic atdifferent levels during in vitro testing. Although guanidine de-rivatives 3 and 12 possessed a similar but moderate ability tointeract with DNA, the discussed assay is a valuable but limited toolto predict the cytotoxic selectivity of a particular compound.

2.2.4. The effect of compounds 3 and 12 on the cell cycleThe influence of compounds 3 and 12 on the cell cycle of the KB

cell line at 1.0, 0.5 and 0.25 mM concentrations (Table 4) wasstudied. At the highest concentration (1 mM) compounds 3 and 12stopped the cells in S phase and decreased the number of cells in

Table 1Comparison of the antiproliferative activity (IC50) of the indolo[2,3-b]quinoline derivatives.

No. Compound IC50 [mM]

BALB/3T3 A549 MCF-7 LoVo KB

DiMIQ 5.77 ± 0.93 2.19 ± 0.48 1.54 ± 0.52 0.20 ± 0.40 1.14 ± 0.61Doxorubicine 1.08 ± 0.03 0.33 ± 0.10 0.44 ± 0.16 0.11 ± 0.03 0.84 ± 0.03

1a 0.54 ± 0.11 0.16 ± 0.26 0.52 ± 0.26 0.07 ± 0.03 0.72 ± 0.08

3. 66.43 ± 12.20 0.14 ± 0.08 0.06 ± 0.01 76.88 ± 16.56 0.88 ± 0.01

6. 4.47 ± 0.38 1.82 ± 0.38 4.75 ± 1.25 0.83 ± 0.01 3.88 ± 1.17

9. 31.92 ± 19.23 11.04 ± 4.73 7.85 ± 3.02 7.76 ± 1.47 15.57 ± 2.88

10a. 0.56 ± 0.05 0.88 ± 0.33 0.33 ± 0.11 0.077 ± 0.004 0.86 ± 0.19

12. 30.04 ± 10.77 3.24 ± 1.05 0.81 ± 0.32 9.38 ± 1.57 0.87 ± 0.20

14. 0.61 ± 0.04 0.74 ± 0.26 0.75 ± 0.30 0.09 ± 0.01 1.13 ± 0.08

16. 0.36 ± 0.07 0.27 ± 0.10 0.32 ± 0.02 0.06 ± 0.01 0.38 ± 0.11

18. 3.55 ± 1.16 1.96 ± 0.79 2.31 ± 0.21 0.82 ± 0.06 1.23 ± 0.44

20. 13.42 ± 3.86 6.22 ± 1.69 2.06 ± 0.59 8.31 ± 1.25 7.37 ± 2.84

IC50 values of the compounds 3 and 12 which showed the selective cytotoxic activity are in bold text.

K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219 213

G0/G1 phase (similar influence, statistically significant; p � 0.05 ascompared to control cells). At a concentration of 0.5 mM both de-rivatives stopped the cells in G2/M phase and also decreased thenumber of cells in G0/G1 phase (for 3 results were statistically

significant; p � 0.05 as compared to control cells); however, com-pound 3 acted stronger on the cell cycle than 12. At the lowestconcentration (0.25 mM) only compound 3 exerted slight influenceon the cell cycle (stopped the cells in G2/M phase and decreased the

Table 2The activity of the test derivatives againstC. albicans biofilms.a

No. ED50 [mM]

1a 41.0 ± 4.93 197.1 ± 18.76 4.2 ± 0.210a 34.2 ± 4.312 287.0 ± 24.114 74.3 ± 8.816 25.5 ± 3.7

a Mean values ± SD (n ¼ 3).

Table 3The activity of 1a, 3, 10a, 12 and 16 in the DNA/methyl greendisplacement assay. a e The values represent the concentra-tion (mean ± SD, n ¼ 3e5) required for a 50% decrease in theinitial absorbance of the DNA/methyl green solution.

Compound IC50a [mM]

DiMIQ 367.22 ± 22.31a 150.4 ± 9.83 71.48 ± 11.86 40.46 ± 22.510a 125.1 ± 17.812 100.9 ± 29.614 80.3 ± 3.916 42.25 ± 4.3Hoechst 33342 12.11 ± 6.1

Table 4Cell cycle distribution of the KB cells treated with 3 and 12 derivatives at the con-centration of 1.0, 0.5 and 0.25 mM for 72 h. The mean values and standard deviationsfrom the independent experiments. G0/G1 e cells in G1 or G0 phase; S e cells in Sphase; G2/M � cells in G2 or M phase. *indicates statistically significant values(p � 0.05 as compared to control cells); ManneWhitney U test.

Compounds Cell cycle distribution Representative histogram

G0/G1 S G2/M

Control 53.2 ± 5.8 32.1 ± 5.4 14.7 ± 1.1

31 mM

32.8 ± 8.9* 48.6 ± 8.3* 18.6 ± 4.0

30.5 mM

29.2 ± 4.6* 39.8 ± 4.5 31.0 ± 6.5*

30.25 mM

39.5 ± 3.7 35.3 ± 4.2 25.3 ± 0.6

121 mM

33.4 ± 5.9* 46.9 ± 4.1* 19.6 ± 4.7

120.5 mM

43.8 ± 6.5 31.6 ± 10.5 24.6 ± 9.2

120.25 mM

51.6 ± 6.8 31.1 ± 3.3 17.3 ± 3.5

K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219214

number of cells in G0/G1 cycle phase). Moreover, at the concen-tration of 1.0 and 0.5 mM cell death was also observed for bothstudied compounds.

2.2.5. The effect of derivatives 3 and 12 on the mitochondrialmembrane potential of the KB cells

In apoptotic cells the electrochemical gradient across themitochondrial membrane breaks down [31]. In our studies weanalyzed the influence of the tested compounds (at concentrationof 1.0 and 0.5 mM, after 72 h) on the mitochondrial membranepotential (Jmt) of KB cells (Fig. 4). We observed a slightly butstatistically significantly decreased Jmt (p � 0.05 as compared tocontrol cells) in 11e17% of the cells independent of the used con-centration of the indolo[2,3-b]quinoline derivatives.

2.2.6. Effect of derivatives 3 and 12 on the apoptosis and necrosis ofthe KB cells

The externalization of phosphatidylserine from the cytoplasmicto the extracellular membrane site is observed in apoptotic cells[32]. We studied whether the tested compounds at concentrationsof 1.0 and 0.5 mM are able to induce apoptotic cell death (usingannexin V staining) or necrosis (using PI staining) of KB cells after72 h. The results are summarized in Table 5. Compounds 3 and 12increased the number of apoptotic cells (AVþ/PI�; about 26e44% ofcells, statistically significant at p < 0.05) as well as necrotic ones(AV±/PIþ; about 6e12% of cells, statistically significant at p < 0.05).

2.2.7. Effect of derivatives 3 and 12 on activity of caspase-3/7 in KBcells

The activation of the caspase cascades is one of the molecularmechanisms involved in apoptotsis. The main effector of caspase iscaspase-3/7 [31]. In order to determine the activity of caspase-3/7in KB cells, we used the enzymatic assay as described previously[32]. Results were normalized to the protein content and reportedas the relative caspase-3/7 activity in comparison to the untreatedcontrol. Results are summarized in Fig. 5. The studied compounds 3

and 12 induced a statistically significant (at p < 0.05) activity ofcaspase-3/7. Compound 3 at a concentration of 1.0 mM proved to bea more effective inducer; as the activity of caspase-3/7 wasobserved at a rate 5 times higher than in control cells. Compound12 at a concentration of 1.0 mM induced caspase-3/7 activity at thesame degree as in the case of compound 3 at a concentration of0.5 mM (however activity was only twice as high in control cells).

2.2.8. Hemolytic activityOne of the drawbacks of indolo[2,3-b]quinolines described

earlier is the red blood cell hemolysis [10,12,33]. The hemolyticactivity of the most potent neocryptolepine derivatives from thisstudy 1a, 3, 6, 10a, 12, 14 and 16 on human red blood cells wasassessed to gain further insight into the potential toxic effects of thetested compounds. As observed, there was no apparent sign oftoxicity to erythrocytes for 1a, 3,10a,12 and 16 in the concentrationrange of 10�6e5 � 10�4 M (Table 6). On the other hand, the gua-nylglycyl (6) and glycylamide (14) derivatives disrupted

Fig. 4. The mitochondrial membrane potential (Jmt) of the KB cells treated withderivatives 3 and 12 at a concentration of 1.0 and 0.5 mM for 72 h.* indicates statisti-cally significant values (p � 0.05 as compared to control cells).

Fig. 5. The activity of caspase-3/7 in the KB cells treated with derivatives 3 and 12 atconcentrations of 1.0 and 0.5 mM for 72 h.* indicates statistically significant values(p � 0.05 as compared to control cells).

K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219 215

erythrocyte membranes in a dose dependent manner. The HC50values are 192.4 and 80.3 mM for 6 and 14, respectively, whereas forthe reference compound DiMIQ, Jaromin et al. reported 120 mM[33]. It is generally assumed that the molecular weight and thebalance between hydrophobic and hydrophilic groups are the fac-tors governing their hemolytic activity.

3. Conclusion

A series of indolo[2,3-b]quinoline analogs containing guanidine,amino acid and the guanylamino acid chains were synthesizedwiththe initial objective to increase the selectivity of their action. Theinfluence of the strongly hydrophilic guanidine group on the se-lective cytotoxic and antifungal activity of new conjugates wasinvestigated. Our results clearly show that the attachment of theguanidine or guanylamino acid chain to the indolo[2,3-b]quinoline

Table 5The apoptosis and necrosis of the KB cells treated with derivatives 3 and 12 at a concentrcompared to control cells).

Compounds The apoptosis and necrosis of the KB cells

Live cells (AV�/PI�) Apoptotic (AVþ/PI�Control 91.3 ± 3.1 4.8 ± 2.4

31 mM

62.1 ± 5.2* 25.7 ± 5.1*

30.5 mM

41.1 ± 12.1* 43.7 ± 12.8*

121 mM

68.1 ± 4.1* 24.4 ± 3.4*

120.5 mM

61.5 ± 16.2* 32.5 ± 15.8*

moiety significantly improves its selective cytotoxic and antifungalactivity.

Interestingly, we found that the attachment of the guanidinegroup directly to the DiMIQ or 6H-indolo[2,3-b]quinoline rings (3and 12) resulted in the increased cytotoxic activity against almostall cancer cell lines, while decreasing this activity against thenormal cell line. Moreover, only compounds 3 and 12 exhibited avery low antifungal activity contrary to other indolo[2,3-b]quino-line derivatives (1a, 6, 10a, 14, and 16) which displayed a strongantifungal activity against C. albicanswith ED50 values ranging from4.2 to 74.3 mM. The referred results of the biological studies showedthat the presence or lack of the guanidine substituent, as well as itsposition in the molecule, strongly modulates its biological activity.

Compounds 3 and 12 were chosen for the investigation of themechanism of action due to their selective antiproliferative activitytowards cancer cells and their lack of antifungal properties. These

ation of 1.0 and 0.5 mM for 72 h.* indicates statistically significant values (p � 0.05 as

Representative dot-plot

) Necrotic (AVþ/PIþ)

3.9 ± 0.8

12.2 ± 2.2*

8.2 ± 1.8*

7.5 ± 2.2*

5.9 ± 1.4

Table 6The hemolytic activity of 1a, 3, 10a, 12 and 16. a e TheHC50 value is the concentration required to lyse 50% ofred blood cells after 30 min at 37 �C; b e value from apublished report [33]; c e no activity. Data given as themean ± SD of three individual determinations.

Compound HC50a [mM]

DiMIQ 120 ± 10b

1a n.a.c

3 n.a.c

6 192.4 ± 10.310a n.a.c

12 n.a.c

14 80.3 ± 2.316 n.a.c

K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219216

two compounds seem to be very effective inducers of apoptosis.However, derivative 3, whose ability to interact with DNA wasstronger than that of compound 12, is also more efficient in theinduction of apoptosis. The proved DNA interactions of the testedcompounds probably lead to the inhibition of DNA synthesis and inconsequence to the arrest of the cells in S cell cycle phase. More-over, an important and promising feature of 3 and 12 is the absenceof hemolysis which is one of the requirements to be fulfilled by acandidate compound for intravenous applications.

The summarized promising in vitro results qualify 3 and 12 asanticancer candidates for further in vivo studies and enhancedinvestigation of their mechanism of action. For the first time, theproblem of the low cytotoxic selectivity of indolo[2,3-b]quinolinederivatives towards normal and cancer cells has been at leastpartially solved by synthesizing these guanidine derivatives. Theconjugates containing the guanidine group directly linked to thecore structure have been confirmed as highly potent and selectiveanticancer agents and promising lead compounds for furtherinvestigations.

4. Experimental section

4.1. Chemistry

4.1.1. GeneralMelting points (m.p.) were determined using a Mettler Toledo

MP90 apparatus and were uncorrected. The 1H and 13C/15N NMRspectra of all compounds studied were measured in CDCl3, DMSO-D6 or D2O using Varian-NMR-vnmrs500, Varian-NMR-vnmrs600and Varian Gemini 200 spectrometers at the temperature of 298 K.Standard experimental conditions and standard Varian programswere used. To assign the structures under consideration thefollowing 1D and 2D experiments were employed: the 1D selectiveNOESY, and 2D gradient selected COSY, 1He13C/1He15N HSQC and1He13C/1He15N HMBC. The 1H and 13C NMR chemical shifts relateto the TMS (for compounds dissolved in CDCl3 or DMSO-D6) andDSS (for compounds dissolved in D2O), whereas nitromethane,whose chemical shift of the 15N nucleus is 0.0 ppm, was used as thecalibration standard for the nitrogen 15N NMR spectra. The con-centration of all solutions used for the measurements was about10e20 mg of the compounds in 0.6e0.8 cm3 of the solvent.

The ESI-MS spectra were recorded on a PE Biosystems Marinermass spectrometer. The progress of the reaction was monitored bythin layer chromatography (TLC) withMerck DC-Alufolien Kieselgel60 F254. The chemicals and solvents were purchased from FlukaCompany. Column chromatography was performed on Merck silicagel 60 (230e400 mesh).

The chromatographic analysis was performed using a Waters®

Alliance HPLC system (Waters Co. USA, MA) consisting of a Waters®

2996 Pump, Waters® 2707Autosampler and Waters® 2996 Photo-diode Array Detector. The separation of the analyte from potentialimpurities was achieved using a Kromasil C8 column(150 � 4.6 mm, 3.5 mm, Kromasil) (6, 9, 10a, 12, 14, 16, 18, 20) or aLuna C18 column (250� 4.6mm, 5 mm, Phenomenex) (1a, 3) placedin a thermostated column heater at 30 �C (6, 9,10a,12,14,16,18, 20)or 25 �C (1a, 3). The mobile phases consisting of A (0.1% TFA inwater), B (0.1% TFA in acetonitrile) were used with the gradientmode at the flow rate of 1 mL/min. The samples were prepared atthe concentration of about 0.2 mg/mL and they were dissolved inmethanol (3, 9, 10a, 12, 14, 16) or methanol:water (1:1, v/v) (6) ormethanol:0.1%TFA in water (1:1, v/v) (1a, 18, 20). The injectionvolume was 10 mL. The UV detection at 275 nm was used. All keycompounds were proved by an HPLC method to show �95% purity.

Compounds 1, 4, 7 and 10 were synthesized earlier [12]. For thebiological testing compounds 1 and 10 were obtained as hydro-chloride salts 1a and 10a according to the following procedure.

4.1.2. General procedure for the synthesis of compounds 1a and 10aCompound 1 or 10 was treated with HCl in methanol and the

mixture was stirred for 30 min at room temperature. The solutionwas evaporated to dryness and this procedure was repeated threetimes. The salts 1a or 10a were obtained as orange solids with aquantitative yield.

4.1.3. 5,11-Dimethyl-5H-indolo[2.3-b]quinolin-9-yl-aminedihydrochloride (1a)

1H NMR (DMSO-D6): 8.20 (1H, dd, J1 ¼ 1.1 Hz, J2 ¼ 8.4 Hz),7.82e7.75 (1H, m), 7.50 (1H, m), 7.44e7.41 (2H, m), 7.31 (1H, d,J ¼ 8.1 Hz), 6.8 (1H, dd, J1 ¼ 2.2 Hz, J2 ¼ 8.1 Hz), 4.85 (2H, br s), 4.18(3H, s), 2.99 (3H, s); 13C NMR (DMSO-D6): 152.8, 141.9, 139.1, 136.3,130.1, 125.7, 125.1, 124.8, 120.9, 120.2, 117.0, 115.9, 114.5, 108.8, 48.5,32.2, 14.7; ESI-MS calcd. for C17H15N3 (261.3) found [MþH]þ: 262.3;[2MþH]þ: 523.4; Anal. Calcd. for C17H15N3 � 2HCl � H2O [352.25]:C 57.96, H 5.44, N 11.93, Cl 20.13 Found: C 58.32, H 5.40, N 12.17, Cl20.30. HPLC: 95.18%.

4.1.4. 6-(2-Dimethylaminoethyl)-11-methyl-6H-indolo[2.3-b]quinolin-9-yl-amine trihydrochloride (10a)

1H NMR and 13C NMR data, see Table 1S, Supplementary Ma-terial; Anal. Calcd. for C20H22N4 � 3HCl � 5H2O [517.87]: C 46.38, H6.81, N 10.82, Cl 20.54 Found: C 46.67, H 6.55, N 10.85, Cl 20.94.HPLC: 96.5%.

4.1.5. General procedure for the guanidinylation with N,N0-bis-Boc-thiourea (2, 6, 8 and 11)

The amino component (1, 4, 7, 10; 1 eq.) was added to the sus-pension of DIPEA (3 eq.) and BSTU (1.5 eq.) in dry DMF. Next HgCl2(1.5 eq.) was added and the mixture was stirred for 2e4 h at roomtemperature (TLC monitoring). The solvent was evaporated and theresiduewas purified by column chromatography or preparative TLCto afford the title compounds with a moderate 61e80% yield.

4.1.6. N-[bis(tert-butyloxycarbonyl)guanyl]-N-(5,11-dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-amine (2)

Compound 2 was obtained according to the general procedureof guanidinylation from 1 (200 mg; 0.77 mM), BSTU (334 mg;1.15 mM), DIPEA (0.4 mL; 2.31 mM), and HgCl2 (312 mg; 1.15 mM)in DMF (5 mL). The crude product 2was purified by preparative TLC(chloroform: methanol 6:1) to afford an orange solid; yield 300 mg(77%); m.p. 210 �C (decomp.); 1H NMR and 13C NMR data, seeTable 1S, Supplementary Material; HR-MS (ESI) calc. forC28H34N5O4 [MþH]þ: 504.2605. Found: 504.2611.

K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219 217

4.1.7. N-guanyl-N-(5,11-dimethyl-5H-indolo[2,3-b]qinolin-9-yl)-amine dihydrochloride (3)

Boc-derivative 2 (219mg, 0.43 mM) was treatedwith TFA (5 mL)and stirred for 24 h (TLC monitoring). The solution was evaporatedto dryness, next HCl/CH3OH was added and evaporated to dryness.This procedure was repeated three times. The residue was crys-tallized from ethyl acetate to afford a yellow solid; yield 160 mg(98%); m.p. 260 �C(decomp.); 1H NMR and 13C NMR data, seeTable 1S, Supplementary Material; ESI-MS calcd. for C18H17N5(303.3) found [MþH]þ: 304.4; [2MþH]þ: 607.3; Anal. Calcd. forC18H17N5 � 3H2O� 2HCl [430.32]: C 50.24, H 5.86, N 16.27, Cl 16.48Found: C 50.28, H 5.64, N 16.35, Cl 16.75; HPLC: 99.7%.

4.1.8. Na-[bis(tert-butyloxycarbonyl)guanyl]-glycyl-N-(5,11-dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-amide (5)

Compound 5 was obtained according to the general procedurefrom 4 (84 mg; 0.26 mM), BSTU (116 mg; 0.4 mM), DIPEA (0.14 mL;0.78 mM), and HgCl2 (109 mg; 0.4 mM) in DMF (3 mL). The crudeproduct 5 was purified by preparative TLC (chloroform: methanol6:1) to afford an orange solid; yield 90 mg (62%); m.p. 140e142 �C;1H NMR and 13C NMR data, see Table 1S, Supplementary Material;HR-MS (ESI) calc. for C30H37N6O5 [MþH]þ: 561.2820. Found:561.2825.

4.1.9. Na-[(tert-butyloxycarbonyl)carbamoil]-glycyl-N-(5,11-dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-amide (5a)

1H NMR and 13C NMR data, see Table 1S, Supplementary Ma-terial; HR-MS (ESI) calc. for C25H28N5O4 [MþH]þ: 462.2130. Found:462.2141.

4.1.10. Na-guanyl-glycyl-N-(5,11-dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-amide trihydrochloride (6)

Product 5 (85 mg, 0.15 mM) was treated with TFA (5 mL) andstirred for 24 h (TLC monitoring). The solution was evaporated todryness, next HCl/CH3OH was added to the residue and evaporatedto dryness. The procedure was repeated three times. The crudeproduct was crystallized from ethyl acetate to afford an yellowsolid; yield 60 mg (85%); m.p. 250 �C (decomp.); 1H NMR and 13CNMR data, see Table 1S, Supplementary Material; ESI-MS calcd. forC20H20N6O (360.4) found [MþH]þ: 361.4; [2MþH]þ: 721.5; Anal.Calcd. for C20H20N6O � 5H2O � 3HCl [559.87]: C 42.91, H 5.94, N15.01, Cl 19.01 Found: C 42.66, H 5.47, N 14.82, Cl 19.20; HPLC:97.7%.

4.1.11. Na-[bis(tert-butyloxycarbonyl)guanyl]-L-prolyl-N-(5,11-dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-amide (8)

Compound 8 was obtained according to the general procedurefrom 7 (155 mg; 0.43 mM), BSTU (188 mg; 0.65 mM), DIPEA(0.24 mL; 1.72 mM), and HgCl2 (176 mg; 0.65 mM) in DMF (6 mL).The crude product 8 was purified by column chromatography(dichloromethane: methanol 10:1) to afford an orange solid; yield208 mg (80%); m.p. 240 �C (decomp.); 1H NMR and 13C NMR data,see Table 1S, Supplementary Material; HR-MS (ESI) calc. forC33H41N6O5 [MþH]þ: 601.3133. Found: 601.3138.

4.1.12. Na-guanyl-L-prolyl-N-(5,11-dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-amide trihydrochloride (9)

Product 8 (80 mg, 0.133 mM) was treated with TFA (6 mL) andstirred for 24 h (TLC monitoring). The solution was evaporated todryness, next HCl/CH3OH was added and evaporated to dryness.The procedure was repeated three times. The residue was crystal-lized from ethyl acetate to afford a yellow solid; yield 73 mg (95%);m.p. 210 �C (decomp.); 1H NMR and 13C NMR data, see Table 1S,Supplementary Material; ESI-MS calcd. for C23H24N6O (400.4)found [MþH]þ: 401.4; [MþNa]þ: 423.4; [2MþH]þ: 801.5;

[2MþNa]þ 823.5; Anal. Calcd. for C23H24N6O � 4H2O � 3HCl[581.92]: C 47.47, H 6.06, N 14.44, Cl 18.28. Found: C 47.52, H 5.99, N14.52, Cl 18.60; HPLC: 95.8%.

4.1.13. N-[bis(tert-butyloxycarbonyl)guanyl]-N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-amine (11)

Compound 11 was obtained according to the general procedurefrom 10 (100 mg; 0.31 mM), BSTU (137 mg; 0.47 mM), DIPEA(0.163 mL; 0.93 mM), and HgCl2 (128 mg; 0.47 mM) in DMF (4 mL).The crude product 11 was purified by column chromatography(dichloromethane: ethanol 3:1) and crystallized from ethyl acetateto afford an orange solid; yield 105 mg (61%); m.p. 220 �C(decomp.); 1H NMR (DMSO-D6): 11.51 (1H, s, NH), 10.12 (1H, s, NH),8.75 (1H, br s), 8.39e8.38 (1H, m), 8.03e8.02 (1H, m), 7.81e7.76(2H, m), 7.73e7.71 (1H, m), 7.56e7.53 (1H, m), 3.58 (2H, m), 3.18(3H, s, 11-CH3), 2.92 (5H, br s), 2.89 (1H, s), 2.73 (1H, s), 2.31 (1H, s),1.55 (9H, s, CH3, Boc), 1.40 (9H, s, CH3, Boc); 13C NMR (DMSO-D6):162.7 (CO, Boc), 162.2 (CO, Boc), 153.5, 152.1, 151.6, 145.6, 139.6,138.4, 129.9, 129.0, 127.5, 124.6, 123.9, 123.8, 123.0, 120.7, 119.1,115.7, 109.2, 83.3, 78.5, 54.6, 42.7, 38.7, 36.5, 35.7, 30.7, 27.8, 27.6,14.5, 11.2; HR-MS (ESI) calc. for C31H41N6O4 [MþH]þ: 561.3179.Found: 561.3189.

4.1.14. N-guanyl-N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-amine tetrahydrochloride (12)

Product 11 (200 mg, 0.357 mM) was treated with TFA (10 mL)and stirred for 24 h (TLC monitoring). The solution was evaporatedto dryness, next HCl/CH3OH was added and evaporated to dryness.The procedure was repeated three times. The residue was crystal-lized from ethyl acetate to afford a yellow solid; yield 140mg (77%);m.p. 200e202 �C; 1H NMR and 13C NMR data, see Table 1S, Sup-plementary Material; ESI-MS calcd. for C21H24N6 (360.4) found[MþH]þ: 361.4; Anal. Calcd. for C21H24N6 � 5H2O � 4HCl [596.37]:C 42.29, H 6.42, N 14.09, Cl 23.78 Found: C 42.20, H 6.59, N 14.00, Cl24.06; HPLC: 96.5%.

4.1.15. Na-[tert-butyloxycarbonyl]-N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-glycylamide (13)

TBTU (452.6 mg, 1.41 mM), HOBt (215.7 mg, 1.41 mM), andDIPEA (0.4 mL, 2.82 mM) was added to the solution of Boc-Gly(247.6 mg, 1.41 mM) in DMF (3 mL), and the mixture was stirredfor 15 min at room temperature. Then the solution of 6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2.3-b]quinolin-9-ylamine (10) (300 mg, 0.94 mM) in 2 mL DMF was added and thereaction mixture was stirred at room temperature for 24 h (TLCmonitoring). After the reaction had been completed, the solventwas evaporated under reduced pressure at ca. 40 �C. The resultingoil was treated with water and CHCl3, the organic layer was sepa-rated and washed successively with the NaHCO3 aq solution andNaCl aq solution. The extract was dried over anhydrous MgSO4,filtered and evaporated to dryness. The column chromatography ofthe residue (dichloromethane e methanol, 10:1 / 3:1) affordedthe title compound (249 mg, 56%) as a yellow solid. m.p.190e192 �C; 1H NMR and 13C NMR data, see Table 1S, Supple-mentary Material; HR-MS (ESI) calc. for C27H34N5O3 [MþH]þ:476.2656. Found: 476.2662.

4.1.16. N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-glycylamide trihydrochloride (14)

Product 13 (200 mg, 0.42 mM) was treated with TFA (5 mL) andstirred for 2 h (TLC monitoring). The solution was evaporated todryness, next HCl/CH3OH was added and evaporated to dryness.This procedure was repeated three times. The residue was crys-tallized from ethyl acetate to afford an orange solid; yield 200 mg

K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219218

(98%); m.p. 250 �C (decomp.); 1H NMR and 13C NMR data, seeTable 1S, Supplementary Material; ESI-MS calcd. for C22H25N5O(375.3) found [MþH]þ: 376.3; Anal. Calcd. forC22H25N5O � 3H2O � 3HCl [538.89]: C 49.03, H 6.36, N 13.00, Cl19.74 Found: C 48.85, H 6.25, N 12.92, Cl 19.99; HPLC: 95.2%.

4.1.17. Na-[tert-butyloxycarbonyl]-N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-L-prolylamide (15)

TBTU (303.3 mg, 0.945 mM), HOBt (144.6 mg, 0.945 mM), andDIPEA (0.26 mL, 1.85 mM) was added to the solution of Boc-L-Pro(349.8 mg, 0.945 mM) in DMF (4 mL), and the mixture was stirredfor 20 min at room temperature. Then the solution of 6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2.3-b]quinolin-9-ylamine (10) (200 mg, 0.63 mM) in 2 mL DMF was added and thereaction mixture was stirred at room temperature for 24 h (TLCmonitoring). After the reaction had been completed, the solventwas evaporated under reduced pressure at ca. 40 �C. The resultingoil was treated with water and CHCl3, the organic layer wasseparated and washed successively with the NaHCO3 aq. solutionand NaCl aq. solution. The extract was dried over anhydrousMgSO4, filtered and evaporated to dryness. The column chroma-tography of the residue (dichloromethane e methanol,10:1/8:1) afforded the title compound (174 mg, 54%) as a yellowfoam. 1H NMR and 13C NMR data, see Table 1S, SupplementaryMaterial; HR-MS (ESI) calc. for C30H38N5O3 [MþH]þ: 516.2965.Found: 516.2975.

4.1.18. N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-L-prolylamide trihydrochloride (16)

Product 15 (134mg, 0.259mM)was treatedwith TFA and stirredfor 2 h (TLC monitoring). The solution was evaporated to dryness,next HCl/CH3OH was added and evaporated to dryness. The pro-cedure was repeated three times. The residue was crystallized fromethyl acetate to afford an orange solid; yield 95 mg (70%); m.p.270 �C (decomp.); 1H NMR and 13C NMR data, see Fig. 3; ESI-MScalcd. for C25H29N5O (415.5) found [MþH]þ: 416.5; Anal. Calcd.for C25H29N5O � 3H2O � 3HCl [578.95]: C 51.86, H 6.62, N 12.10, Cl18.37. Found: C 52.09, H 6.46, N 12.26, Cl 18.47; HPLC: 98.7%.

4.1.19. Na-[bis(tert-butyloxycarbonyl)guanyl]-glycyl-N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-amide (17)

Compound 17 was obtained according to the general procedurefrom 14 (110 mg; 0.29 mM), BSTU (128 mg; 0.44 mM), DIPEA(0.15 mL; 0.87 mM), and HgCl2 (119 mg; 0.44 mM) in DMF (4 mL).The crude product 17 was purified by preparative TLC (chloroform:methanol 10:1) to afford a yellow foam; yield 115 mg (64%); 1HNMR and 13C NMR data, see Table 1S, Supplementary Material; HR-MS (ESI) calc. for C33H44N7O5 [MþH]þ: 618.3401. Found: 618.3404.

4.1.20. Na-guanyl-glycyl-N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-amide trihydrochloride (18)

Product 17 (30 mg, 0.048 mM) was treated with TFA (3 mL) andstirred for 3 h (TLC monitoring). The solution was evaporated todryness, next HCl/CH3OH was added and evaporated to dryness.The procedure was repeated three times. The residue was crystal-lized from ethyl acetate to afford a yellow solid; yield 24 mg (86%);m.p. 212 �C (decomp.); 1H NMR and 13C NMR data, see Table 1S,Supplementary Material; ESI-MS calcd. for C23H27N7O (417.51)found [MþH]þ: 418.24; Anal. Calcd. for C23H27N7O � 3H2O � 3HCl[579.94]: C 49.70, H 6.43, N 14.49, Cl 18.34. Found: C 49.96, H 6.47, N14.78, Cl 18.29; HPLC: 96.15%.

4.1.21. Na-[bis(tert-butyloxycarbonyl)guanyl]-L-prolyl-N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-amide (19)

Compound 19 was obtained according to the general procedurefrom 16 (110 mg; 0.26 mM), BSTU (115 mg; 0.39 mM), DIPEA(0.14 mL; 0.79 mM), and HgCl2 (107 mg; 0.39 mM) in DMF (3 mL).The crude product 19 was purified by column chromatography(dichloromethane: methanol 5:1) to afford a yellow foam; yield90 mg (51%); 1H NMR and 13C NMR data, see Table 1S, Supple-mentary Material; HR-MS (ESI) calc. for C36H48N7O5 [MþH]þ:658.3705. Found: 658.3717.

4.1.22. Na-guanyl-L-prolyl-N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-amide trihydrochloride (20)

Product 19 (70 mg, 0.11 mM) was treated with TFA (4 mL) andstirred for 24 h (TLC monitoring). The solution was evaporated todryness, next HCl/CH3OH was added and evaporated to dryness.The procedure was repeated three times. The residue was crystal-lized from ethyl acetate to afford a yellow solid; yield 60 mg (96%);m.p. 210 �C (decomp.); 1H NMR and 13C NMR data, see Table 1S,Supplementary Material; ESI-MS calcd. for C26H31N7O (457.57)found [MþH]þ: 458.27; Anal. Calcd. for C26H31N7O � 4H2O � 3HCl[639.01]: C 48.87, H 6.62, N 15.34, Cl 16.64. Found: C 48.97, H 6.60, N15.05, Cl 16.92; HPLC: 96.77%.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2015.10.022.

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