The Synthesis of Indolo[2,3-B]Quinoline Derivatives

European Journal of Medicinal Chemistry 105 (2015) 208e219

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European Journal of Medicinal Chemistry

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Research paper The synthesis of indolo[2,3-b]quinoline derivatives with a guanidine group: 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 Bankowski a, Marcin Cybulski a, Łukasz Kaczmarek a a Pharmaceutical Research Institute, 8 Rydygiera St., 01-793 Warsaw, Poland b Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Weigla St., 53-114 Wroclaw, Poland c Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, 14A Joliot-Curie St., 50-383 Wroclaw, Poland d Institute of Organic Chemistry, Polish Academy of Sciences, 44/52 Kasprzaka St., 01-224 Warsaw, Poland e Institute of Chemistry Environmental Protection and Biotechnology, Jan Długosz University, 13/15 Armii Krajowej Ave., 42-200 Cze˛stochowa, Poland f Department of Medicine, Section of Infectious Diseases, 4125 Microbial Sciences Building, 1550 Linden Dr., University of Wisconsin-Madison, Madison, WI 53706, USA article info abstract

Article history: The synthesis of indolo[2,3-b]quinoline derivatives containing guanidine, amino acid or guanylamino Received 23 July 2015 acid substituents as well as their in vitro evaluation for the cytotoxic and antifungal activity are reported. Received in revised form The influence of the guanidine group on the selective cytotoxic and hemolytic properties of indolo[2,3-b] 5 October 2015 quinoline was investigated. Most of the compounds displayed a high cytotoxic activity in vitro and two of Accepted 10 October 2015 the most promising compounds (3 and 12) exhibited a high selectivity between normal and cancer cell- Available online xxx lines. The cytotoxic activity of compound 3 was about 600-fold lower against normal fibroblasts than against A549 and MCF-7 cancer cell lines. Novel entities acted as the DNA-intercalators when tested Keywords: e Neocryptolepine using a DNA methyl green assay but demonstrated zero or low hemolytic activity in comparison to their Antiproliferative activity unsubstituted analogs. The mechanism of action was studied for guanidine derivatives 3 and 12 and both Antifungal activity compounds were found to be very effective inducers of apoptosis. Biofilm © 2015 Elsevier Masson SAS. All rights reserved. Guanidine group Mechanism of action Apoptosis Hemolytic activity

1. Introduction activity and inhibits the proliferation of mouth cacinoma KB cells at a concentration of 1 mM. Moreover, its activity is similar to the Neocryptolepine is an alkaloid which displays a broad spectrum cytotoxic activity of doxorubicin (0.8 mM against KB cells) [7e9]. of biological activities including an antibacterial, antifungal, anti- Unfortunately, DiMIQ's high toxicity, lack of selectivity and very low inflammatory, cytotoxicity, and antimalarial activity [1e6] (Fig. 1). solubility in aqueous solutions, especially at neutral pH, seriously 5,11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ, Fig. 1), the syn- limit the practical application of this compound in the treatment of thetic analog of neocryptolepine, demonstrates high cytotoxic cancer [10]. The high toxicity and low bioavailability of DiMIQ, prompted us to look for new analogs which would conform to the high requirements necessary for anticancer drugs: potent and selective activity and low side effects. Our recently published results 0 Abbreviations: Boc, tert-butyloxycarbonyl group; BSTU, N,N -bis-Boc-thiourea; reveal that this might be achieved by constructing conjugates DIPEA, N,N-diisopropylethylamine; DMF, dimethylformamide; DMSO, dime- composed of DiMIQ and amino acids or peptides [11e13]. As it has thylsulfoxide; DSS, 4,4-dimethyl-4-silapentane-1-sulfonic acid; HOBt, N-hydrox- ybenzotriazole monohydrate; HPLC, high performance liquid chromatography; been proved, the attachment of an amino acid moiety or a short TBTU, O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium tetrafluoroborate; peptide chain to DiMIQ significantly improves its physicochemical TFA, trifluoroacetic acid. properties, resulting in the auspicious anticancer action in vivo with * Corresponding author. a relatively low hemolytic effect. Some of the recently reported E-mail address: [email protected] (K. Sidoryk). http://dx.doi.org/10.1016/j.ejmech.2015.10.022 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved. K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219 209

could be expected that the introduction of the guanyl substituents into novel conjugates would increase the DNA interaction and, as a result, advantageously affect the cytotoxic activity of the novel analogs. Besides stabilizing the drug-DNA complex, the guanidine group might also improve the delivery of the substances inside cancer cells by increasing their hydrophilicity and water solubility, while simultaneously decreasing their toxicity [25e28]. Herein we report the synthesis of new hybrid compounds with Fig. 1. Structures of neocryptolepine and DiMIQ. the 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 acid neocryptolepine analogs showed promising antifungal and anti- residue or an N-guanylamino acid. All new conjugates were tested bacterial characteristics which could potentially aid future for their cytotoxic activity against cancer and normal cell lines and anticancer-antimicrobial treatment [13]. against fungal biofilms as well as for their DNA interactions. Most However, the cytotoxic activity in vitro of the above mentioned promising compounds were selected to establish their mechanism compounds and other known indolo[2,3-b]quinoline derivatives of action. Moreover, all novel conjugates were tested for their against cancer cells was comparable to their cytotoxic activity ability to induce the hemolysis of human erythrocytes, as this test is e against normal cell lines [14 17]. The lack of the selectivity of ac- one of the most important and most frequently studied biocom- tion of the indolo[2,3-b]quinoline derivatives obtained so far patibility measures. prompted us to search further for more selective antitumor compounds with high cytotoxic activity against cancer cells and low against normal cells. 2. Results and discussion After a detailed literature search, it was assumed that our goal could be accomplished by the introduction of a guanidine group 2.1. Synthesis into the indolo[2,3-b]qiunoline conjugates. The guanidine group widely exists in various natural products and pharmaceutically 5,11-dimethyl-5H-indolo[2.3-b]quinolin-9-yl-amine dihydro- active compounds [18,19]. This group has been found in metabolites chloride (1a) and 6-(2-dimethylaminoethyl)-11-methyl-6H-indolo of different living organisms and many naturally occurring sub- [2.3-b]quinolin-9-yl-amine trihydrochloride (10a) used for the SAR stances such as ptilomycalin A and bisguanidine. Ptilomycalin A, studies were prepared by treating the amino derivatives of 1 and 10 which exhibits an antimicrobial, antifungal, antiviral and also with HCl/MeOH. The synthesis of the N-guanidine- and N-guany- cytotoxic activity, was isolated from the sponges of the Red Sea and lamino acids conjugates of 1 is outlined in Scheme 1. The starting the Caribbean Sea [20e22]. Bisguanidine, TAN-1057, isolated from compound 1 is not commercially available and was synthesized as Flexibacter sp. PK-74 bacteria, possess a potent activity against b- described previously [14,29]. The guanidinylation of 1 with N,N0- lactam-resistant, Gram-positive bacteria [23]. The guanidine sub- bis-Boc-thiourea in the presence of DIPEA and HgCl2 gave the ex- structure is also present in the active pharmaceutical substances pected compound 2 with 77% yield. The treatment of the protected used to treat influenza A and B viral infections (Zanamivir), as well Boc-derivative (2) with the trifluoroacetic acid and then with HCl/ as bacterial infections (Chlorhexidine, Sulfaguanidine) (Fig. 2). MeOH gave N-guanyl-N-(5,11-dimethyl-5H-indolo[2,3-b]chinolin- Furthermore, it seems that the guanidine group plays an important 9-yl)-amine dihydrochloride 3 with an excellent 98% yield. The role in drug delivery to cancer cells due to its strong basic proper- guanidinylation of compounds 4 [12] and 7 [12] with BSTU under ties (pKa 12.5). Evidence has also been found for its possible strong standard conditions afforded 5 and 8 with a moderate 60% and a interaction with the phosphate residues of the minor groove of the good 80% yield, respectively. In the case of N -bis(tert-butylox- DNA helix [24]. Taking into consideration all the above facts, it ycarbonyl)guanyl-glycyl-N-(5,11-dimethyl-5H-indolo[2,3-b]

Fig. 2. Guanidine-containing natural products and pharmaceuticals. 210 K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219

Scheme 1. Reagents and conditions: (a) BSTU, HgCl2, DIPEA, DMF, 24 h, rt; (b) TFA, HCl/MeOH. chinolin-9-yl)-amide 5, the main product was obtained with a trifluoroacetates were converted into the appropriate hydrochlo- small amount of the impurity 5a. The Boc-removal of compounds 5 rides 14 and 16 by the HCl/MeOH treatment. and 8 with the trifluoroacetic acid and then the treatment of the The N-guanylamino acid derivatives of 6H-indoloquinoline were residue by hydrogen chloride in methanol gave the hydrochloride also synthesized (Scheme 3). The synthesis of these derivatives was derivatives of DiMIQ, compounds 6 and 9, with 85% and 95% yields, carried out analogously to the method used for the N-guanylamino respectively. The hydrochlorides of the deprotected derivatives of acid derivatives of 5H-indoloquinoline. The guanidinylation of 14 DiMIQ were purified by crystallization. and 16 with BSTU in the presence of DIPEA and HgCl2 afforded the a N -guanyl-N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo expected Boc-derivatives 17 and 19 with moderate yields 64% and [2,3-b]quinolin-9-yl]-amine tetrahydrochloride 12 was obtained 51%, respectively. The Boc groups were removed by TFA treatment according to the above procedure (Scheme 2). The guanidinylation and the final hydrochlorides 18 and 20 were obtained in good of 10 [17] gave derivative 11 in a moderate 61% yield and the sub- yields. sequent Boc deprotection gave the corresponding derivative 12 The structures of all new compounds were confirmed by with a 77% yield. The glycyl and L-prolyl derivatives of 6H-indolo- extended 1D and 2D NMR experiments (Fig. 3 and Table 1S, Sup- quinoline 14 and 16 were obtained by reacting 9-amino derivative plementary Material), as well as MS (Experimental part). The purity (10) with the Boc-protected amino acids using the 2-(1H-benzo- of all final products was verified by the elemental analysis and the triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) C-18 RPHPLC method using acetonitrile-water as the mobile phase. method [30]. The coupling reactions were performed in DMF at a room temperature for 2e24 h. The Boc-N -amino acid derivatives 2.2. Biological studies of 10, compounds 13 and 15, were separated by extraction and purified by column chromatography on the silica gel. Their yields 2.2.1. Antiproliferative activity in vitro after the purification were 54 and 56%. In the next step the Boc All synthesized compounds were evaluated for their anti- groups were removed by the trifluoroacetic acid and finally the proliferative activity in vitro against the following cell lines: A549 K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219 211

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.

(non-small cell lung cancer), MCF-7 (breast cancer), LoVo (colon acid and N-guanylamino acid derivatives of indolo[2,3-b]quinoline cancer), and KB cells (mouth carcinoma cell). Possible cell toxicity are summarized in Table 1. The tested compounds showed a diverse of the indolo[2,3-b]quinoline derivatives was tested by the rate of activity against cancer and normal cells. The amino derivative of viability of the normal murine ﬁbroblasts BALB/3T3. The results of DiMIQ (1a) and 6-(2-dimethylaminoethyl)-11-methyl-6H-indolo the studies on the antiproliferative activity of the guanidine, amino [2.3-b]quinolin-9-yl-amine (10a) displayed the highest cytotoxic 212 K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219

the antiproliferative activity of these compounds against the BALB/ 3T3 normal cells (IC50 0.61 and 0.36 mM, respectively) was about 10 times higher than the cytotoxicity of DiMIQ and comparable to the activity against cancer cells. On the other hand, compounds 18 and 20, the N-guanylamino acid derivatives of 6H-indolo[2,3-b]quinoline possessed a lower cytotoxic activity than their amino acid counterparts. For example, IC50 value for compounds 18 and 20 was between 0.82 mM and 8.3 mM against all cancer cell lines. Compounds 3 and 12 were selected for further studies due to their high anticancer activity and speciﬁcity (cancer vs. normal cell- 1 13 15 Fig. 3. The H, C and N NMR signal assignments for analog 16 (in D2O). lines).

2.2.2. Activity against fungal biofilms in vitro activity against all cancer cell lines with IC50 values between 0.07 The newly synthesized DiMIQ derivatives were evaluated for and 0.8 mM. Unfortunately, these compounds were also cytotoxic their antifungal activity against Candida albicans biofilms. The ex- against the normal murine fibroblast (BALB/3T3), with IC50 values periments were designed to determine the cell viability of fungal 0.54 and 0.56 mM for 1a and 10a, respectively. biofilms in vitro grown in 96-well ELISA plates after exposure to the The introduction of the guanidine group to the DiMIQ molecule tested compounds [13]. Our previous study demonstrated a strong (compound 3) or to the glycine and L-proline DiMIQ derivatives preferential action of the DiMIQ derivatives substituted with the (compounds 6 and 9) resulted in differences in the cytotoxic ac- amino acid or dipeptide groups against the C. albicans biofilms [13]. tivity. The most advantageous activity profile was obtained for In the current study, the N-guanylamino acid derivative 6 compound 3 when the guanidine moiety was linked directly to the showed the highest antibiofilm activity (ED50 4.2 mM), whereas two DIMIQ core. As it was shown in Table 1, compound 3 demonstrated other molecules, namely the amino group-modified derivative of its highest cytotoxicity against the MCF-7 cell line (IC50 value DiMIQ (1a), and 6-(2-dimethylaminoethyl)-11-methyl-6H-indolo 0.06 mM), the A549 cell line (IC50 value 0.14 mM) and the KB cell line [2.3-b]quinolin-9-yl-amine (10a) displayed a strong antifungal ac- (IC50 value 0.88 mM) but was not active against the LoVo cell line tivity against C. albicans with ED50 values at 41.0 and 34.2 mM, (IC50 value 76.88 mM). In accordance with our proposed hypothesis, respectively. Other 6-(2-dimethylaminoethyl)-11-methyl-6H- the most interesting data were collected when the cytotoxic ac- indolo[2,3-b]quinolone derivatives containing only amino acid tivity of the guanidine DIMIQ (3) against BALB3T3 murine fibro- moieties (14 and 16) were also found active and actually more blasts was tested. It should be pointed out that compound 3 did not efficient than the original natural component (ED50 74.3 and exhibit any antiproliferative activity against the normal cell line 25.5 mM, respectively). (IC50 value 66.43 mM). The cytotoxic activity of compound 3 was Compounds 3 and 12 with the guanidine moiety linked directly about 600-fold lower against the normal cell line than against A549 to the indoquinoline ring did not exhibit any activity against the and MCF-7 cancer cell lines. This very high selective action has C. albicans biofilms. Strikingly, the addition of the guanyl moieties never been observed for any standard anticancer drug. For example, into the DiMIQ structure dramatically reduced their antifungal doxorubicin, widely used in cancer treatment, shows a comparative activity, while maintaining the preferred anticancer properties cytotoxic activity against cancer and normal cell-lines. Although a in vitro (Table 2). high cytotoxic activity was also observed in the case of the N- guanylamino acid derivatives 6 and 9, no significant selectivity of 2.2.3. DNA interactions action was identified. The highest activity against the LoVo cell line It is generally accepted that the cytotoxic activity of the indolo (IC50 0.83 mM) and against the A549 cell line (IC50 1.82 mM) was [2,3-b]quinolone derivatives depends on their ability of action as exhibited by derivative 6, whereas its activity against the KB and the DNA intercalating agents and topoisomerase inhibitors MCF-7 cell lines was lower, with IC50 values 3.88 and 4.75 mM, [6e8,14e17]. Therefore, the novel compounds were evaluated as respectively. Compound 6 exhibited a cytotoxic activity against the potential DNA-interacting agents using a DNA-methyl green assay normal cell line with IC50 value of 4.47 mM. The cytotoxic activity of based upon the displacement of the dye methyl green from the derivative 9 against all cancer cell lines was in the range of DNA-methyl green complex (Table 3). The tested compounds, 7.76e15.57 mM. On the other hand, its cytotoxic activity against the namely 1a, 3, 6, 10a, 12, 14 and 16, showed a pronounced activity in BALB/3T3 normal cell line was lower and was measured at the DNA-methyl green assay, confirming their DNA-intercalating 31.92 mM. properties which are generally associated with cytotoxicity. It was For a more thorough examination of the influence of the guanyl identified that the introduction of any additional substituent into group on the conjugate's cytotoxic activity, guanidine (12), amino the indolo[2,3-b]quinoline core resulted in the increase of the DNA- acid (14 and 16), and N-guanylamino acid (18 and 20) derivatives of interacting properties in comparison to DiMIQ. The strongest 6H-indolo[2,3-b]quinoline were also synthesized. Similarly to the interaction was observed for compounds 6 and 16 with a different data collected for the DIMIQ derivatives, the same influence on the indoloquinoline moiety. It is worth noting that all examined com- antiproliferative activity was observed for the 6H-indolo[2,3-b] pounds showed DNA interacting activity while being cytotoxic at quinolone analogs. The effect was related to the presence and po- different levels during in vitro testing. Although guanidine de- sition of the guanyl group in the molecule. rivatives 3 and 12 possessed a similar but moderate ability to Maximal selective activity was achieved when the guanidine interact with DNA, the discussed assay is a valuable but limited tool group was directly connected with the 6H-indolo[2,3-b]quinoline to predict the cytotoxic selectivity of a particular compound. core (12). The IC50 values for compound 12 were 0.81 and 0.87 mM for MCF-7 and KB cell lines, respectively. Alike compound 3, 12 did 2.2.4. The effect of compounds 3 and 12 on the cell cycle not exhibit cytotoxic activity against normal cell line (IC50 The influence of compounds 3 and 12 on the cell cycle of the KB 30.04 mM). Glycine (14) and L-proline (16) derivatives of 6H-indolo cell line at 1.0, 0.5 and 0.25 mM concentrations (Table 4)was [2,3-b]quinoline acted stronger than DiMIQ or doxorubicin against studied. At the highest concentration (1 mM) compounds 3 and 12 all cancer cell lines with IC50 values ranging from 0.06 to 1.1 mM. But stopped the cells in S phase and decreased the number of cells in K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219 213

Table 1

Comparison 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 ± 0.61 Doxorubicine 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.

G0/G1 phase (similar influence, statistically significant; p 0.05 as significant; p 0.05 as compared to control cells); however, com- compared to control cells). At a concentration of 0.5 mM both de- pound 3 acted stronger on the cell cycle than 12. At the lowest rivatives stopped the cells in G2/M phase and also decreased the concentration (0.25 mM) only compound 3 exerted slight influence number of cells in G0/G1 phase (for 3 results were statistically on the cell cycle (stopped the cells in G2/M phase and decreased the 214 K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219

Table 2 Table 4 The activity of the test derivatives against Cell cycle distribution of the KB cells treated with 3 and 12 derivatives at the con- C. albicans bioﬁlms.a centration of 1.0, 0.5 and 0.25 mM for 72 h. The mean values and standard deviations from the independent experiments. G0/G1 e cells in G1 or G0 phase; S e cells in S m No. ED50 [ M] phase; G2/M cells in G2 or M phase. *indicates statistically signiﬁcant values 1a 41.0 ± 4.9 (p 0.05 as compared to control cells); ManneWhitney U test. 3 197.1 ± 18.7 Compounds Cell cycle distribution Representative histogram 6 4.2 ± 0.2 10a 34.2 ± 4.3 G0/G1 S G2/M 12 287.0 ± 24.1 Control 53.2 ± 5.8 32.1 ± 5.4 14.7 ± 1.1 14 74.3 ± 8.8 16 25.5 ± 3.7

a Mean values ± SD (n ¼ 3).

Table 3 3 32.8 ± 8.9* 48.6 ± 8.3* 18.6 ± 4.0 The activity of 1a, 3, 10a, 12 and 16 in the DNA/methyl green 1 mM displacement assay. a e The values represent the concentration (mean ± SD, n ¼ 3e5) required for a 50% decrease in the initial absorbance of the DNA/methyl green solution.

a Compound IC50 [mM] 3 29.2 ± 4.6* 39.8 ± 4.5 31.0 ± 6.5* DiMIQ 367.22 ± 22.3 0.5 mM 1a 150.4 ± 9.8 3 71.48 ± 11.8 6 40.46 ± 22.5 10a 125.1 ± 17.8 12 100.9 ± 29.6 ± ± ± 14 80.3 ± 3.9 3 39.5 3.7 35.3 4.2 25.3 0.6 m 16 42.25 ± 4.3 0.25 M Hoechst 33342 12.11 ± 6.1

number of cells in G0/G1 cycle phase). Moreover, at the concen- 12 33.4 ± 5.9* 46.9 ± 4.1* 19.6 ± 4.7 tration of 1.0 and 0.5 mM cell death was also observed for both 1 mM studied compounds.

2.2.5. The effect of derivatives 3 and 12 on the mitochondrial membrane potential of the KB cells 12 43.8 ± 6.5 31.6 ± 10.5 24.6 ± 9.2 In apoptotic cells the electrochemical gradient across the 0.5 mM mitochondrial membrane breaks down [31]. In our studies we analyzed the inﬂuence of the tested compounds (at concentration of 1.0 and 0.5 mM, after 72 h) on the mitochondrial membrane potential (Jmt) of KB cells (Fig. 4). We observed a slightly but 12 51.6 ± 6.8 31.1 ± 3.3 17.3 ± 3.5 statistically signiﬁcantly decreased Jmt (p 0.05 as compared to 0.25 mM control cells) in 11e17% of the cells independent of the used concentration of the indolo[2,3-b]quinoline derivatives.

2.2.6. Effect of derivatives 3 and 12 on the apoptosis and necrosis of the KB cells The externalization of phosphatidylserine from the cytoplasmic and 12 induced a statistically significant (at p < 0.05) activity of to the extracellular membrane site is observed in apoptotic cells caspase-3/7. Compound 3 at a concentration of 1.0 mMprovedtobe [32]. We studied whether the tested compounds at concentrations a more effective inducer; as the activity of caspase-3/7 was of 1.0 and 0.5 mM are able to induce apoptotic cell death (using observed at a rate 5 times higher than in control cells. Compound annexin V staining) or necrosis (using PI staining) of KB cells after 12 at a concentration of 1.0 mM induced caspase-3/7 activity at the 72 h. The results are summarized in Table 5. Compounds 3 and 12 same degree as in the case of compound 3 at a concentration of increased the number of apoptotic cells (AVþ/PI; about 26e44% of 0.5 mM (however activity was only twice as high in control cells). cells, statistically significant at p < 0.05) as well as necrotic ones (AV±/PIþ; about 6e12% of cells, statistically significant at p < 0.05). 2.2.8. Hemolytic activity 2.2.7. Effect of derivatives 3 and 12 on activity of caspase-3/7 in KB One of the drawbacks of indolo[2,3-b]quinolines described cells earlier is the red blood cell hemolysis [10,12,33]. The hemolytic The activation of the caspase cascades is one of the molecular activity of the most potent neocryptolepine derivatives from this mechanisms involved in apoptotsis. The main effector of caspase is study 1a, 3, 6, 10a, 12, 14 and 16 on human red blood cells was caspase-3/7 [31]. In order to determine the activity of caspase-3/7 assessed to gain further insight into the potential toxic effects of the in KB cells, we used the enzymatic assay as described previously tested compounds. As observed, there was no apparent sign of [32]. Results were normalized to the protein content and reported toxicity to erythrocytes for 1a, 3, 10a, 12 and 16 in the concentration as the relative caspase-3/7 activity in comparison to the untreated range of 10 6e5 10 4 M(Table 6). On the other hand, the gua- control. Results are summarized in Fig. 5. The studied compounds 3 nylglycyl (6) and glycylamide (14) derivatives disrupted K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219 215

J Fig. 4. The mitochondrial membrane potential ( mt) of the KB cells treated with Fig. 5. The activity of caspase-3/7 in the KB cells treated with derivatives 3 and 12 at m derivatives 3 and 12 at a concentration of 1.0 and 0.5 M for 72 h.* indicates statisti- concentrations of 1.0 and 0.5 mM for 72 h.* indicates statistically significant values fi cally signi cant values (p 0.05 as compared to control cells). (p 0.05 as compared to control cells). erythrocyte membranes in a dose dependent manner. The HC50 moiety significantly improves its selective cytotoxic and antifungal m values are 192.4 and 80.3 M for 6 and 14, respectively, whereas for activity. m the reference compound DiMIQ, Jaromin et al. reported 120 M Interestingly, we found that the attachment of the guanidine [33]. It is generally assumed that the molecular weight and the group directly to the DiMIQ or 6H-indolo[2,3-b]quinoline rings (3 balance between hydrophobic and hydrophilic groups are the fac- and 12) resulted in the increased cytotoxic activity against almost tors governing their hemolytic activity. all cancer cell lines, while decreasing this activity against the normal cell line. Moreover, only compounds 3 and 12 exhibited a 3. Conclusion very low antifungal activity contrary to other indolo[2,3-b]quinoline derivatives (1a, 6, 10a, 14, and 16) which displayed a strong A series of indolo[2,3-b]quinoline analogs containing guanidine, antifungal activity against C. albicans with ED50 values ranging from amino acid and the guanylamino acid chains were synthesized with 4.2 to 74.3 mM. The referred results of the biological studies showed the initial objective to increase the selectivity of their action. The that the presence or lack of the guanidine substituent, as well as its influence of the strongly hydrophilic guanidine group on the se- position in the molecule, strongly modulates its biological activity. lective cytotoxic and antifungal activity of new conjugates was Compounds 3 and 12 were chosen for the investigation of the investigated. Our results clearly show that the attachment of the mechanism of action due to their selective antiproliferative activity guanidine or guanylamino acid chain to the indolo[2,3-b]quinoline towards cancer cells and their lack of antifungal properties. These

Table 5 The apoptosis and necrosis of the KB cells treated with derivatives 3 and 12 at a concentration of 1.0 and 0.5 mM for 72 h.* indicates statistically signiﬁcant values (p 0.05 as compared to control cells).

Compounds The apoptosis and necrosis of the KB cells Representative dot-plot

Live cells (AV/PI) Apoptotic (AVþ/PI) Necrotic (AVþ/PIþ)

Control 91.3 ± 3.1 4.8 ± 2.4 3.9 ± 0.8

3 62.1 ± 5.2* 25.7 ± 5.1* 12.2 ± 2.2* 1 mM

3 41.1 ± 12.1* 43.7 ± 12.8* 8.2 ± 1.8* 0.5 mM

12 68.1 ± 4.1* 24.4 ± 3.4* 7.5 ± 2.2* 1 mM

12 61.5 ± 16.2* 32.5 ± 15.8* 5.9 ± 1.4 0.5 mM 216 K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219

® ® Table 6 2996 Pump, Waters 2707Autosampler and Waters 2996 Photo- The hemolytic activity of 1a, 3, 10a, 12 and 16.ae The diode Array Detector. The separation of the analyte from potential HC50 value is the concentration required to lyse 50% of impurities was achieved using a Kromasil C8 column red blood cells after 30 min at 37 C; b e value from a published report [33];ce no activity. Data given as the (150 4.6 mm, 3.5 mm, Kromasil) (6, 9, 10a, 12, 14, 16, 18, 20)ora mean ± SD of three individual determinations. Luna C18 column (250 4.6 mm, 5 mm, Phenomenex) (1a, 3) placed a in a thermostated column heater at 30 C(6, 9, 10a, 12, 14, 16, 18, 20) Compound HC50 [mM] or 25 C(1a, 3). The mobile phases consisting of A (0.1% TFA in ± b DiMIQ 120 10 water), B (0.1% TFA in acetonitrile) were used with the gradient 1a n.a.c fl 3 n.a.c mode at the ow rate of 1 mL/min. The samples were prepared at 6 192.4 ± 10.3 the concentration of about 0.2 mg/mL and they were dissolved in 10a n.a.c methanol (3, 9, 10a, 12, 14, 16) or methanol:water (1:1, v/v) (6)or c 12 n.a. methanol:0.1%TFA in water (1:1, v/v) (1a, 18, 20). The injection 14 80.3 ± 2.3 m 16 n.a.c volume was 10 L. The UV detection at 275 nm was used. All key compounds were proved by an HPLC method to show 95% purity. Compounds 1, 4, 7 and 10 were synthesized earlier [12]. For the biological testing compounds 1 and 10 were obtained as hydro- two compounds seem to be very effective inducers of apoptosis. chloride salts 1a and 10a according to the following procedure. However, derivative 3, whose ability to interact with DNA was stronger than that of compound 12, is also more efficient in the induction of apoptosis. The proved DNA interactions of the tested 4.1.2. General procedure for the synthesis of compounds 1a and 10a compounds probably lead to the inhibition of DNA synthesis and in Compound 1 or 10 was treated with HCl in methanol and the consequence to the arrest of the cells in S cell cycle phase. More- mixture was stirred for 30 min at room temperature. The solution over, an important and promising feature of 3 and 12 is the absence was evaporated to dryness and this procedure was repeated three of hemolysis which is one of the requirements to be fulfilled by a times. The salts 1a or 10a were obtained as orange solids with a candidate compound for intravenous applications. quantitative yield. The summarized promising in vitro results qualify 3 and 12 as anticancer candidates for further in vivo studies and enhanced 4.1.3. 5,11-Dimethyl-5H-indolo[2.3-b]quinolin-9-yl-amine fi investigation of their mechanism of action. For the rst time, the dihydrochloride (1a) problem of the low cytotoxic selectivity of indolo[2,3-b]quinoline 1 H NMR (DMSO-D6): 8.20 (1H, dd, J1 ¼ 1.1 Hz, J2 ¼ 8.4 Hz), derivatives towards normal and cancer cells has been at least 7.82e7.75 (1H, m), 7.50 (1H, m), 7.44e7.41 (2H, m), 7.31 (1H, d, partially solved by synthesizing these guanidine derivatives. The 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 conjugates containing the guanidine group directly linked to the (3H, s), 2.99 (3H, s); 13C NMR (DMSO-D ): 152.8, 141.9, 139.1, 136.3, fi 6 core structure have been con rmed as highly potent and selective 130.1, 125.7, 125.1, 124.8, 120.9, 120.2, 117.0, 115.9, 114.5, 108.8, 48.5, anticancer agents and promising lead compounds for further þ 32.2, 14.7; ESI-MS calcd. for C17H15N3 (261.3) found [MþH] : 262.3; investigations. þ [2MþH] : 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, Cl 4. Experimental section 20.30. HPLC: 95.18%.

4.1. Chemistry 4.1.4. 6-(2-Dimethylaminoethyl)-11-methyl-6H-indolo[2.3-b] quinolin-9-yl-amine trihydrochloride (10a) 4.1.1. General 1H NMR and 13C NMR data, see Table 1S, Supplementary Ma- Melting points (m.p.) were determined using a Mettler Toledo terial; Anal. Calcd. for C20H22N4 3HCl 5H2O [517.87]: C 46.38, H 1 13 15 MP90 apparatus and were uncorrected. The H and C/ N NMR 6.81, N 10.82, Cl 20.54 Found: C 46.67, H 6.55, N 10.85, Cl 20.94. spectra of all compounds studied were measured in CDCl3, DMSO- HPLC: 96.5%. D6 or D2O using Varian-NMR-vnmrs500, Varian-NMR-vnmrs600 and Varian Gemini 200 spectrometers at the temperature of 298 K. 4.1.5. General procedure for the guanidinylation with N,N0-bis-Boc- Standard experimental conditions and standard Varian programs thiourea (2, 6, 8 and 11) were used. To assign the structures under consideration the The amino component (1, 4, 7, 10; 1 eq.) was added to the sus- following 1D and 2D experiments were employed: the 1D selective pension of DIPEA (3 eq.) and BSTU (1.5 eq.) in dry DMF. Next HgCl NOESY, and 2D gradient selected COSY, 1He13C/1He15N HSQC and 2 (1.5 eq.) was added and the mixture was stirred for 2e4 h at room 1He13C/1He15N HMBC. The 1H and 13C NMR chemical shifts relate temperature (TLC monitoring). The solvent was evaporated and the to the TMS (for compounds dissolved in CDCl3 or DMSO-D6) and residue was puriﬁed by column chromatography or preparative TLC DSS (for compounds dissolved in D2O), whereas nitromethane, to afford the title compounds with a moderate 61e80% yield. whose chemical shift of the 15N nucleus is 0.0 ppm, was used as the calibration standard for the nitrogen 15N NMR spectra. The concentration of all solutions used for the measurements was about 4.1.6. N-[bis(tert-butyloxycarbonyl)guanyl]-N-(5,11-dimethyl-5H- 10e20 mg of the compounds in 0.6e0.8 cm3 of the solvent. indolo[2,3-b]quinolin-9-yl)-amine (2) The ESI-MS spectra were recorded on a PE Biosystems Mariner Compound 2 was obtained according to the general procedure mass spectrometer. The progress of the reaction was monitored by of guanidinylation from 1 (200 mg; 0.77 mM), BSTU (334 mg; thin layer chromatography (TLC) with Merck DC-Alufolien Kieselgel 1.15 mM), DIPEA (0.4 mL; 2.31 mM), and HgCl2 (312 mg; 1.15 mM) 60 F254. The chemicals and solvents were purchased from Fluka in DMF (5 mL). The crude product 2 was puriﬁed by preparative TLC Company. Column chromatography was performed on Merck silica (chloroform: methanol 6:1) to afford an orange solid; yield 300 mg gel 60 (230e400 mesh). (77%); m.p. 210 C (decomp.); 1H NMR and 13C NMR data, see ® The chromatographic analysis was performed using a Waters Table 1S, Supplementary Material; HR-MS (ESI) calc. for ® þ Alliance HPLC system (Waters Co. USA, MA) consisting of a Waters C28H34N5O4 [MþH] : 504.2605. Found: 504.2611. K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219 217

þ 4.1.7. N-guanyl-N-(5,11-dimethyl-5H-indolo[2,3-b]qinolin-9-yl)- [2MþNa] 823.5; Anal. Calcd. for C23H24N6O 4H2O 3HCl amine dihydrochloride (3) [581.92]: C 47.47, H 6.06, N 14.44, Cl 18.28. Found: C 47.52, H 5.99, N Boc-derivative 2 (219 mg, 0.43 mM) was treated with TFA (5 mL) 14.52, Cl 18.60; HPLC: 95.8%. and stirred for 24 h (TLC monitoring). The solution was evaporated to dryness, next HCl/CH3OH was added and evaporated to dryness. 4.1.13. N-[bis(tert-butyloxycarbonyl)guanyl]-N-[6-(2- This procedure was repeated three times. The residue was crys- dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]- tallized from ethyl acetate to afford a yellow solid; yield 160 mg amine (11) (98%); m.p. 260 C(decomp.); 1H NMR and 13C NMR data, see Compound 11 was obtained according to the general procedure Table 1S, Supplementary Material; ESI-MS calcd. for C18H17N5 from 10 (100 mg; 0.31 mM), BSTU (137 mg; 0.47 mM), DIPEA þ þ (303.3) found [MþH] : 304.4; [2MþH] : 607.3; Anal. Calcd. for (0.163 mL; 0.93 mM), and HgCl2 (128 mg; 0.47 mM) in DMF (4 mL). C18H17N5 3H2O 2HCl [430.32]: C 50.24, H 5.86, N 16.27, Cl 16.48 The crude product 11 was puriﬁed by column chromatography Found: C 50.28, H 5.64, N 16.35, Cl 16.75; HPLC: 99.7%. (dichloromethane: ethanol 3:1) and crystallized from ethyl acetate to afford an orange solid; yield 105 mg (61%); m.p. 220 C a 1 4.1.8. N -[bis(tert-butyloxycarbonyl)guanyl]-glycyl-N-(5,11- (decomp.); H NMR (DMSO-D6): 11.51 (1H, s, NH), 10.12 (1H, s, NH), dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-amide (5) 8.75 (1H, br s), 8.39e8.38 (1H, m), 8.03e8.02 (1H, m), 7.81e7.76 Compound 5 was obtained according to the general procedure (2H, m), 7.73e7.71 (1H, m), 7.56e7.53 (1H, m), 3.58 (2H, m), 3.18 from 4 (84 mg; 0.26 mM), BSTU (116 mg; 0.4 mM), DIPEA (0.14 mL; (3H, s, 11-CH3), 2.92 (5H, br s), 2.89 (1H, s), 2.73 (1H, s), 2.31 (1H, s), 13 0.78 mM), and HgCl2 (109 mg; 0.4 mM) in DMF (3 mL). The crude 1.55 (9H, s, CH3, Boc), 1.40 (9H, s, CH3, Boc); C NMR (DMSO-D6): product 5 was puriﬁed by preparative TLC (chloroform: methanol 162.7 (CO, Boc), 162.2 (CO, Boc), 153.5, 152.1, 151.6, 145.6, 139.6, 6:1) to afford an orange solid; yield 90 mg (62%); m.p. 140e142 C; 138.4, 129.9, 129.0, 127.5, 124.6, 123.9, 123.8, 123.0, 120.7, 119.1, 1H NMR and 13C NMR data, see Table 1S, Supplementary Material; 115.7, 109.2, 83.3, 78.5, 54.6, 42.7, 38.7, 36.5, 35.7, 30.7, 27.8, 27.6, þ þ HR-MS (ESI) calc. for C30H37N6O5 [MþH] : 561.2820. Found: 14.5, 11.2; HR-MS (ESI) calc. for C31H41N6O4 [MþH] : 561.3179. 561.2825. Found: 561.3189.

a 4.1.9. N -[(tert-butyloxycarbonyl)carbamoil]-glycyl-N-(5,11- 4.1.14. N-guanyl-N-[6-(2-dimethylaminoethyl)-11-methyl-6H- dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-amide (5a) indolo[2,3-b]quinolin-9-yl]-amine tetrahydrochloride (12) 1H NMR and 13C NMR data, see Table 1S, Supplementary Ma- Product 11 (200 mg, 0.357 mM) was treated with TFA (10 mL) þ terial; HR-MS (ESI) calc. for C25H28N5O4 [MþH] : 462.2130. Found: and stirred for 24 h (TLC monitoring). The solution was evaporated 462.2141. to dryness, next HCl/CH3OH was added and evaporated to dryness. The procedure was repeated three times. The residue was crystal- a 4.1.10. N -guanyl-glycyl-N-(5,11-dimethyl-5H-indolo[2,3-b] lized from ethyl acetate to afford a yellow solid; yield 140 mg (77%); quinolin-9-yl)-amide trihydrochloride (6) m.p. 200e202 C; 1H NMR and 13C NMR data, see Table 1S, Sup- Product 5 (85 mg, 0.15 mM) was treated with TFA (5 mL) and plementary Material; ESI-MS calcd. for C21H24N6 (360.4) found þ stirred for 24 h (TLC monitoring). The solution was evaporated to [MþH] : 361.4; Anal. Calcd. for C21H24N6 5H2O 4HCl [596.37]: dryness, next HCl/CH3OH was added to the residue and evaporated C 42.29, H 6.42, N 14.09, Cl 23.78 Found: C 42.20, H 6.59, N 14.00, Cl to dryness. The procedure was repeated three times. The crude 24.06; HPLC: 96.5%. product was crystallized from ethyl acetate to afford an yellow a solid; yield 60 mg (85%); m.p. 250 C (decomp.); 1H NMR and 13C 4.1.15. N -[tert-butyloxycarbonyl]-N-[6-(2-dimethylaminoethyl)- NMR data, see Table 1S, Supplementary Material; ESI-MS calcd. for 11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-glycylamide (13) þ þ C20H20N6O (360.4) found [MþH] : 361.4; [2MþH] : 721.5; Anal. TBTU (452.6 mg, 1.41 mM), HOBt (215.7 mg, 1.41 mM), and Calcd. for C20H20N6O 5H2O 3HCl [559.87]: C 42.91, H 5.94, N DIPEA (0.4 mL, 2.82 mM) was added to the solution of Boc-Gly 15.01, Cl 19.01 Found: C 42.66, H 5.47, N 14.82, Cl 19.20; HPLC: (247.6 mg, 1.41 mM) in DMF (3 mL), and the mixture was stirred 97.7%. for 15 min at room temperature. Then the solution of 6-(2- dimethylaminoethyl)-11-methyl-6H-indolo[2.3-b]quinolin-9- a 4.1.11. N -[bis(tert-butyloxycarbonyl)guanyl]-L-prolyl-N-(5,11- ylamine (10) (300 mg, 0.94 mM) in 2 mL DMF was added and the dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-amide (8) reaction mixture was stirred at room temperature for 24 h (TLC Compound 8 was obtained according to the general procedure monitoring). After the reaction had been completed, the solvent from 7 (155 mg; 0.43 mM), BSTU (188 mg; 0.65 mM), DIPEA was evaporated under reduced pressure at ca. 40 C. The resulting (0.24 mL; 1.72 mM), and HgCl2 (176 mg; 0.65 mM) in DMF (6 mL). oil was treated with water and CHCl3, the organic layer was sepa- The crude product 8 was puriﬁed by column chromatography rated and washed successively with the NaHCO3 aq solution and (dichloromethane: methanol 10:1) to afford an orange solid; yield NaCl aq solution. The extract was dried over anhydrous MgSO4, 208 mg (80%); m.p. 240 C (decomp.); 1H NMR and 13C NMR data, ﬁltered and evaporated to dryness. The column chromatography of see Table 1S, Supplementary Material; HR-MS (ESI) calc. for the residue (dichloromethane e methanol, 10:1 / 3:1) afforded þ C33H41N6O5 [MþH] : 601.3133. Found: 601.3138. the title compound (249 mg, 56%) as a yellow solid. m.p. 190e192 C; 1H NMR and 13C NMR data, see Table 1S, Supple- a þ 4.1.12. N -guanyl-L-prolyl-N-(5,11-dimethyl-5H-indolo[2,3-b] mentary Material; HR-MS (ESI) calc. for C27H34N5O3 [MþH] : quinolin-9-yl)-amide trihydrochloride (9) 476.2656. Found: 476.2662. Product 8 (80 mg, 0.133 mM) was treated with TFA (6 mL) and stirred for 24 h (TLC monitoring). The solution was evaporated to 4.1.16. N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b] dryness, next HCl/CH3OH was added and evaporated to dryness. quinolin-9-yl]-glycylamide trihydrochloride (14) The procedure was repeated three times. The residue was crystal- Product 13 (200 mg, 0.42 mM) was treated with TFA (5 mL) and lized from ethyl acetate to afford a yellow solid; yield 73 mg (95%); stirred for 2 h (TLC monitoring). The solution was evaporated to 1 13 m.p. 210 C (decomp.); H NMR and C NMR data, see Table 1S, dryness, next HCl/CH3OH was added and evaporated to dryness. Supplementary Material; ESI-MS calcd. for C23H24N6O (400.4) This procedure was repeated three times. The residue was crys- þ þ þ found [MþH] : 401.4; [MþNa] : 423.4; [2MþH] : 801.5; tallized from ethyl acetate to afford an orange solid; yield 200 mg 218 K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219

a (98%); m.p. 250 C (decomp.); 1H NMR and 13C NMR data, see 4.1.21. N -[bis(tert-butyloxycarbonyl)guanyl]-L-prolyl-N-[6-(2- Table 1S, Supplementary Material; ESI-MS calcd. for C22H25N5O dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]- þ (375.3) found [MþH] : 376.3; Anal. Calcd. for amide (19) C22H25N5O 3H2O 3HCl [538.89]: C 49.03, H 6.36, N 13.00, Cl Compound 19 was obtained according to the general procedure 19.74 Found: C 48.85, H 6.25, N 12.92, Cl 19.99; HPLC: 95.2%. from 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). fi a The crude product 19 was puri ed by column chromatography 4.1.17. N -[tert-butyloxycarbonyl]-N-[6-(2-dimethylaminoethyl)- (dichloromethane: methanol 5:1) to afford a yellow foam; yield 15 11-methyl-6H-indolo[2,3-b]quinolin-9-yl]-L-prolylamide ( ) 90 mg (51%); 1H NMR and 13C NMR data, see Table 1S, Supple- TBTU (303.3 mg, 0.945 mM), HOBt (144.6 mg, 0.945 mM), and þ mentary Material; HR-MS (ESI) calc. for C36H48N7O5 [MþH] : DIPEA (0.26 mL, 1.85 mM) was added to the solution of Boc-L-Pro 658.3705. Found: 658.3717. (349.8 mg, 0.945 mM) in DMF (4 mL), and the mixture was stirred a for 20 min at room temperature. Then the solution of 6-(2- 4.1.22. N -guanyl-L-prolyl-N-[6-(2-dimethylaminoethyl)-11- dimethylaminoethyl)-11-methyl-6H-indolo[2.3-b]quinolin-9- methyl-6H-indolo[2,3-b]quinolin-9-yl]-amide trihydrochloride (20) ylamine (10) (200 mg, 0.63 mM) in 2 mL DMF was added and the Product 19 (70 mg, 0.11 mM) was treated with TFA (4 mL) and reaction mixture was stirred at room temperature for 24 h (TLC stirred for 24 h (TLC monitoring). The solution was evaporated to monitoring). After the reaction had been completed, the solvent dryness, next HCl/CH3OH was added and evaporated to dryness. was evaporated under reduced pressure at ca. 40 C. The resulting The procedure was repeated three times. The residue was crystal- oil was treated with water and CHCl3, the organic layer was lized from ethyl acetate to afford a yellow solid; yield 60 mg (96%); separated and washed successively with the NaHCO3 aq. solution m.p. 210 C (decomp.); 1H NMR and 13C NMR data, see Table 1S, and NaCl aq. solution. The extract was dried over anhydrous Supplementary Material; ESI-MS calcd. for C26H31N7O (457.57) fi þ MgSO4, ltered and evaporated to dryness. The column chroma- found [MþH] : 458.27; Anal. Calcd. for C H N O 4H O 3HCl e 26 31 7 2 tography of the residue (dichloromethane methanol, [639.01]: C 48.87, H 6.62, N 15.34, Cl 16.64. Found: C 48.97, H 6.60, N / 10:1 8:1) afforded the title compound (174 mg, 54%) as a yellow 15.05, Cl 16.92; HPLC: 96.77%. foam. 1H NMR and 13C NMR data, see Table 1S, Supplementary þ Material; HR-MS (ESI) calc. for C H N O [MþH] : 516.2965. 30 38 5 3 Appendix A. Supplementary data Found: 516.2975. Supplementary data related to this article can be found at http:// 4.1.18. N-[6-(2-dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b] dx.doi.org/10.1016/j.ejmech.2015.10.022. quinolin-9-yl]-L-prolylamide trihydrochloride (16) Product 15 (134 mg, 0.259 mM) was treated with TFA and stirred References for 2 h (TLC monitoring). The solution was evaporated to dryness, [1] Z.W. Mei, L. Wang, W.J. Lu, C.Q. Pang, T. Maeda, W. Peng, M. Kaiser, I.E. Sayed, next HCl/CH3OH was added and evaporated to dryness. The pro- T. Inokuchi, Synthesis and in vitro antimalarial testing of neocryptolepines: cedure was repeated three times. The residue was crystallized from SAR study for improved activity by introduction and modification of side ethyl acetate to afford an orange solid; yield 95 mg (70%); m.p. chains at C2 and C11 on indoloquinolines, J. Med. Chem. 56 (4) (2013) 1431e1442. 270 C (decomp.); 1H NMR and 13C NMR data, see Fig. 3; ESI-MS þ [2] E. Shaban, K.J. Wicht, N. Wang, Z.W. Mei, I. Hayashi, T.E. Sayed, T.J. Egan, calcd. for C25H29N5O (415.5) found [MþH] : 416.5; Anal. Calcd. T. Inokuchi, Synthesis and antimalarial activity of some neocryptolepine an- for C25H29N5O 3H2O 3HCl [578.95]: C 51.86, H 6.62, N 12.10, Cl alogues carrying a multifunctional linear and branched carbon-side chains, e 18.37. Found: C 52.09, H 6.46, N 12.26, Cl 18.47; HPLC: 98.7%. Heterocycles 89 (4) (2014) 1055 1064. [3] L. Wang, M. Switalska, Z.W. Mei, W.J. Lu, Y. Takahara, X.W. Feng, T.E. Sayed, J. Wietrzyk, T. Inokuchi, Synthesis and in vitro antiproliferative activity of new 11-aminoalkylamino-substituted 5H- and 6H-indolo[2,3-b]quinolines; a 4.1.19. N -[bis(tert-butyloxycarbonyl)guanyl]-glycyl-N-[6-(2- structure-activity relationship of neocryptolepines and 6-methyl congeners, e dimethylaminoethyl)-11-methyl-6H-indolo[2,3-b]quinolin-9-yl]- Bioorg. Med. Chem. 20 (2012) 4820 4829. 17 [4] W. Peng, M. Switalska, L. Wang, Z.W. Mei, Y. Edazawa, C.Q. Pang, T.E. Sayed, amide ( ) J. Wietrzyk, T. Inokuchi, Synthesis and in vitro antiproliferative activity of new Compound 17 was obtained according to the general procedure 11-aminoalkylamino-substituted chromeno[2,3-b]indoles, Eur. J. Med. Chem. e from 14 (110 mg; 0.29 mM), BSTU (128 mg; 0.44 mM), DIPEA 58 (2012) 441 451. [5] W.J. Lu, M. Switalska, L. Wang, M. Yonezawa, T.E. Sayed, J. Wietrzyk, (0.15 mL; 0.87 mM), and HgCl2 (119 mg; 0.44 mM) in DMF (4 mL). T. Inokuchi, In vitro antiproliferative activity of new 11-aminoalkylamino- The crude product 17 was purified by preparative TLC (chloroform: substituted 5H-indolo[2,3-b]quinolines; improving activity of neo- methanol 10:1) to afford a yellow foam; yield 115 mg (64%); 1H cryptolepines by installation of ester substituent, Med. Chem. Res. 22 (2013) 4492e4504. NMR and 13C NMR data, see Table 1S, Supplementary Material; HR- þ [6] E.L. Larghi, A.B.J. Bracca, A.A. Arroyo Aguilar, D.A. Heredia, J.L. Pergomet, MS (ESI) calc. for C33H44N7O5 [MþH] : 618.3401. Found: 618.3404. S.O. Simonetti, T.S. Kaufman, Neocryptolepine: a promising Indoloisoquinoline alkaloid with interesting biological activity. Evaluation of the drug and its most relevant analogs, Curr. Top. Med. Chem. 15 (17) (2015) 1683e1707. a [7] W. Peczynska-Czoch, F. Pognan, L. Kaczmarek, J. Boratynski, Synthesis and 4.1.20. N -guanyl-glycyl-N-[6-(2-dimethylaminoethyl)-11-methyl- structures-activity relationship of methyl-substituted indolo[2,3-b]quino- 6H-indolo[2,3-b]quinolin-9-yl]-amide trihydrochloride (18) lines: novel cytotoxic, DNA topoisomerase II inhibitors, J. Med. Chem. 37 Product 17 (30 mg, 0.048 mM) was treated with TFA (3 mL) and (1994) 3503e3510. stirred for 3 h (TLC monitoring). The solution was evaporated to [8] L. Kaczmarek, W. Peczynska-Czoch, J. Osiadacz, M. Mordarski, A. Sokalski, J. Boratynski, E. Marcinkowska, H. Kusnierczyk-Glazman, C. Radzikowski, dryness, next HCl/CH3OH was added and evaporated to dryness. Synthesis, and cytotoxic activity of some novel indolo[2,3-b]quinoline de- The procedure was repeated three times. The residue was crystal- rivatives: DNA topoisomerase II inhibitors, Bioorg. Med. Chem. 7 (1999) e lized from ethyl acetate to afford a yellow solid; yield 24 mg (86%); 2457 2464. 1 13 [9] J. Osiadacz, L. Kaczmarek, A. Opolski, J. Wietrzyk, E. Marcinkowska, m.p. 212 C (decomp.); H NMR and C NMR data, see Table 1S, K. Biernacka, C. Radzikowski, M. Jon, W. Peczynska-Czoch, Microbial conver- Supplementary Material; ESI-MS calcd. for C23H27N7O(417.51) sion of methyl and methoxy-substituted derivatives of 5H-indolo[2,3-b] þ quinoline as a method of developing novel cytotoxic agents, Anticancer Res. found [MþH] : 418.24; Anal. Calcd. for C23H27N7O 3H2O 3HCl 19 (1999) 3333e3342. [579.94]: C 49.70, H 6.43, N 14.49, Cl 18.34. Found: C 49.96, H 6.47, N [10] A. Jaromin, A. Kozubek, K. Suchoszek-Lukaniuk, M. Malicka-Blaszkiewicz, 14.78, Cl 18.29; HPLC: 96.15%. W. Peczynska-Czoch, L. Kaczmarek, Liposomal formulation of DIMIQ, potential K. Sidoryk et al. / European Journal of Medicinal Chemistry 105 (2015) 208e219 219

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