Introduction

Indoloquinolines isolated from the root of Cryptolepis sanguinolenta, the climbing shrub growing in some African countries, are attracting the constant attention of pharmacologists, since an aqueous macerate or decoction of this root is used in traditional medicine for the treatment of infectious diseases like malaria and various disorders of the body (Alexandra et al., 2000; Cimanga et al., 1996a, b). Among the various isolated products with indoloquinoline structures, cryptolepine i (5-methylindolo[3,2-b]quinoline), and the minor alkaloid neocryptolepine ii (5-methylindolo[2,3-b]quinoline) (Fig. 1) have been intensively studied, which showed broad spectrum of biological activities including antibacterial (Cimanga et al., 1996a, b, 1997, 1998), antifungal (Cimanga et al., 1996a, b, 1997, 1998), and antimalarial (Philippe et al., 1996; Kirby et al., 1995; Wright et al., 1996).

Fig. 1
figure 1

Structures of cryptolepine and neocryptolepine

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Since cryptolepine i and neocryptolepine ii possess linearly arranged tetracyclic planar structures, they behave as a DNA intercalating agent and inhibit topoisomerase II thus revealing a high level of cytotoxicity (Guittat et al., 2003; Jonckers et al., 2002; Bailly et al., 2000). Consequently, SAR studies on these structures as a lead to search for more active drug candidates have been extensively undertaken (Kumar et al., 2008; Lavrado et al., 2010; Parvatkar et al., 2011).

Our previous research on 5-Me-indolo[2,3-b]quinolines revealed that the 11-amino group is important for their activity, especially the 3-aminopropylamino group, which could increase the activity against MV4-11 about 67 times compared to its 11-chloro precursor (Wang et al., 2012). The antiproliferaive test indicated that the 5-methylated derivatives are usually more cytotoxic than their respective 6-methylated congeners. A synergistic effect of the substituents at the C-2 position was also observed. For example, the C-2-Br derivative was 5 times more active compared to the non-halogenated derivative on 11-(3-aminopropylamino)neocryptolepine, and the 2-chloro-11-(4-methyl-1,4-diazepane-1-yl)neocryptolepine was 65 times more active than its corresponding agent with no substituent at C-2.

On the other hand, a recent publication describing effect of substituents on the anticancer activity indicated that the introduction of an ester group to the core structures affects the activity of other plant-derived anticancer agents such taxol (Ojima et al., 1997), camptothecin (Yang et al., 2002), bryostatin (Wender and Hinkle, 2000), and 5-fluorouracil (Xiong et al., 2009). Such an improvement in the anticancer activity is believed to be due to increasing both the lipophilicity and bioavailability of the drug by introducing the ester group into the core structure. Encouraged by these significant information, we examined the introduction of an ester group to the core and checked its influence on the antiproliferative activity. In this study, we report the further investigation of the anticancer potential of the neocryptolepine core. An ester group was introduced at the C-2 position of the A-ring and/or the C-9 position of the D-ring to establish or extend the structure-activity relationship (SAR) study for these regions.

Synthesis and biological evaluation

The preparations of the ester substituted 11-chloro-5-methyl-5H-indolo[2,3-b]quinolines 6 were carried out according to the method we previously described (Wang et al., 2012). Since the dimethyl 1H-indole-3,5-dicarboxylate (2b), a key starting compound, was not commercially available, it was synthesized by installation of an ester group at the C-3 position to the methyl 1H-indole-5-carboxylate 1 using the reaction with trichloroacetyl chloride in the presence of pyridine, followed by successive alkaline treatment with KOH at reflux in MeOH and MeI in DMF (Linton and Kozlowski, 2008). The obtained dimethyl 1H-indole-3,5-dicarboxylate (2b) was combined with N-methylaniline (3b) via chlorination with NCS in the presence of 1,4-dimethylpiperazine, giving 2-(N-methylanilino)indole-3,5-dicarboxylate (4b) in 73 % yield. Heating of 4b at 250 °C in diphenyl ether induced intra-molecular acylation at the C-2 of aniline, forming the tetracyclic indolo[2,3-b]quinolinone 5. The reaction of 5 with POCl3 afforded the 11-chloro-indolo[2,3-b]quinoline 6 as a result of dehydrative chlorination. The amination of 6 via an SNAr reaction smoothly proceeded by heating with amines in THF to give various 11-aminoindolo[2,3-b]quinolines 7–9 as depicted in Scheme 1.

Scheme 1
scheme 1

Preparation of ester substituted 11-amino-neocryptolepines 7–9. Reagents and conditions: (i) a. Pyridine, trichloroacetyl chloride, THF. b. KOH, MeOH, reflux. (ii) a. N-chlorosuccinimide, 1,4-dimethylpiperazine. b. trichloroacetic acid. (iii) diphenyl ether, reflux. (iv) POCl3, toluene, reflux. (v) appropriate amines

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We introduced an ester group to the indole motif at the C-9 position, and varied the C-11 position with different amines (8a–8g). The results of the anti-proliferative evaluation against the human leukemia cell line (MV4-11) are summarized in Table 1. We found that in some cases, their activities were improved compared to those compounds without substituents at C-9. For example, 8a, 8b, 8d, and 8e were shown to be more active compared to compounds 10a, 10b, 10d, and 10e which lack a substitutent at the C-2 or C-9 position. Their IC50 values ranged from 0.044 to 0.811 μM, and the best result was achieved with a 3-aminopropylamino group substituted at C-11 (8b, IC50 = 0.044 μM). Compared with the agent 7, which contained an ester group at the C-2 position on the quinoline subunit, the agent 8b was twice more efficient.

Table 1 Compounds 7–8 as anti-proliferative agents against human leukemia cell line MV4-11
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Furthermore, we have synthesized and evaluated agents 9a–9e, bearing the same aminopropylamino group R2 while varying the ester group at R1 or R2 for the SAR study, and a synergistic effect were detected (Table 2). In general, an ester substituent at the C-9 position can efficiently favor the anticancer activity of the agent while the ester substituent at the C-2 position is not as efficient. Comparing compounds 9b and 9d for instance, the compound 9b was twice more active than 9d, the same trend as observed for the agents 7 and 8b. Compared with 8b, the introduction of a substituent at the C-2 position, compounds 9a, 9b, and 9c for instance, has made the compound less active.

Table 2 The anti-proliferative activity of agents 9 against human leukemia cell line MV4-11
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The solubility of an agent in an aqueous medium can significantly influence its further application, despite its high anticancer activity. In order to improve the water solubility of the agents, we tried to hydrolyze the 9-ester substituted neocryptolepines into a 9-carboxylic acid analog by treating the methyl 11-amino-5H-indolo[2,3-b]quinoline-9-carboxylate 8 with NaOH(aq) in MeOH. The mixture was refluxed overnight to afford the 9-carboxylic acid substituted indolo[2,3-b]quinoline 11 in good yield, as shown in Scheme 2. The evaluation results of their anti-proliferative activity are summarized in Table 3.

Scheme 2
scheme 2

Preparation of the 9-caboxylic acid substituted indolo[2,3-b]quinolines 11. Reagents and conditions: MeOH/NaOH(aq)

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Table 3 The anti-proliferative activity of agents 11 against human leukemia cell line MV4-11
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Unfortunately, the anti-proliferative activity of the hydrolyzed derivatives 11 turned out that though the solubility of the agents in an aqueous medium was improved, their anti-proliferative activities dramatically decreased compared to their precursors 8a–8d.

Furthermore, the indoloquinoline derivative compounds bearing an ester substituent (7–9) were also evaluated against other cell lines to evaluate the selective anticancer properties (Table 4). Two kinds of cancer cell lines (human lung cancer cell line A549, human colon cancer cell line HCT116) and a kind of normal cell line (normal mice fibroblast cell line BALB/3T3) were chosen for the tests. Most of the tested compounds were cytotoxic against the cancer cell lines, except for the agent 8c with an IC50 value of more than 5 μM. In general, these tested compounds were selectively cytotoxic against the human colon cancer HCT116 cell line, and had a lower cytotoxicity against the A549 or BALB/3T3 cell lines. Their IC50 values against the HCT 116 cell line were generally 4–5 times lower than those against the A549 or BALB/3T3 cell lines. The agents 7, 8a, 8b, 8g, and the 9 series were shown to be more efficient than the wide spectrum anticancer medicine, doxorubicin, against the HCT116 cells. In addition, the agent 9c revealed a better activity against the A549 cells when compared to doxorubicin.

Table 4 The test of compounds 8 and 9 as anti-proliferative agents against A549, HCT116, and BALB/3T3 cell lines
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Spectroscopic characterization of neocryptolepine derivative 8b interacting with salmon fish sperm DNA

The DNA binding studies of compounds 8b was performed using UV–vis absorption spectroscopy with salmon fish sperm DNA in phosphate buffer of pH 7.0 at 20 °C. The hypochromic effect was observed in the absorption spectra while the DNA solution was gradually added to the solution of the compound 8b. The results in Fig. 2 showed that the absorption band at 298 nm for the 8b decreased while increasing the DNA concentration. It illustrated that there is a strong interaction between 8b and DNA. Then the binding constant of 8b-DNA was calculated as 2.77 × 105 L mol−1 according to double-reciprocal equation.

Fig. 2
figure 2

UV–Vis absorption spectra of compound 8b and 8b-DNA at 20 °C. C 8b  = 50 μmol/L, C DNA = 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μmol/L for curve 1–11 in pH 7.0 phosphate buffer solution; (b) the plot of 1/ΔA vis 1/[DNA] for 8b-DNA

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Conclusion

To summarize, we have synthesized a series of C-2 and/or C-9 ester substituted 11-amino-5-methyl-5H-indolo[2,3-b]quinolines and evaluated their antiproliferative activity for an SAR study. The results indicated that the activities of the agents could be improved by introducing an ester substituent at the C-9 position. Among them, the methyl 11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate (8b) was the most potent with an IC50 value of 0.044 μM against the human leukemia MV4-11 cell line. While compound 9a showed 28 times more selective cytotoxic activity against the human colon cancer cell line than normal mice fibroblast cell line, BALB/3T3.

Experimental

Chemistry

General

The commercially obtained reagents were directly used without further purification. The 1H NMR and 13C NMR spectra were measured on the Varian INOVA-600 or Varian INOVA-400 spectrometer. High resolution mass spectra were obtained on a Bruker micrOTOF II-SKA spectrometer. Melting points were determined on a J-Science RFS-10 hot stage microscope. The scaffolds 7–8 and were prepared by the method we previously mentioned (Wang et al., 2013).

General procedure for the synthesis of Dimethyl 1H-indole-3,5-dicarboxylate 2b

Pyridine (0.30 mL, 3.54 mmol) was added to a suspension of methyl 1H-indole-5-carboxylate (0.48 g, 2.72 mmol) in anhydrous THF (6 mL) at 0 °C. A solution of trichloroacetyl chloride (0.40 mL, 3.54 mmol) in THF (6 mL) was added dropwise via an addition funnel over 1 h. The reaction mixture was then allowed to warm to room temperature to stir over night. The reaction mixture was quenched in 1 M HCl, dried over Na2SO4, and concentrated under vacuum. The resulting solid was then dissolved in MeOH (54 mL), and KOH(s) was added. The reaction mixture was heated to reflux for 5 h, then stirred at ambient temperature for 1 h, followed by concentration under vacuum. The solid was purified by chromatography (SiO2, 25 % EtOAc/Hexane) in 75 % yield.

Dimethyl 1H-indole-3,5-dicarboxylate 2b

White solids; 1H NMR (400 MHz, DMSO-d 6) δH (ppm) 12.28 (s, 1H), 8.69 (d, J = 1.5 Hz, 1H), 8.23 (s, 1H), 7.83 (dd, J = 8.6, 1.7 Hz, 1H), 7.57 (dd, J = 8.6, 0.5 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.34 (s, 2H). 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 167.0, 164.4, 139.0, 134.3, 125.2, 123.3, 122.7, 112.5, 107.4, 51.9, 50.9.

Methyl 5-bromo-1H-indole-3-carboxylate 2c

White solids; 1H NMR (400 MHz, CDCl3) δH (ppm) 8.68 (s, 1H), 8.33 (d, J = 1.9 Hz, 1H), 7.92 (d, J = 3.0 Hz, 1H), 7.37 (dd, J = 8.6, 1.9 Hz, 1H), 7.29 (dd, J = 8.6, 0.4 Hz, 1H), 3.93 (s, 3H). 13C NMR (100 MHz, CDCl3) δC (ppm) 165.1, 134.6, 131.8, 127.4, 126.3, 124.2, 115.7, 112.9, 108.6, 51.3.

Methyl 2-((4-(methoxycarbonyl)phenyl)(methyl)amino)-1H-indole-3-carboxylate 4a

White solids, Mp: 217–219 °C; 1H NMR (600 MHz, DMSO-d 6): δH (ppm) 12.25 (br s, 1 H), 8.00 (d, J = 7.92 Hz, 1 H), 7.81 (d, J = 8.80 Hz, 2 H), 7.39 (d, J = 7.63 Hz, 1 H), 7.29–7.15 (m, 2 H), 6.73 (d, J = 8.80 Hz, 2 H), 3.78 (s, 3 H), 3.66 (s, 3 H), 3.37 (s, 3 H); 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 166.1, 163.5, 151.7, 145.3, 132.6, 130.7, 125.8, 122.7, 121.5, 121.0, 119.0, 112.9, 111.8, 98.4, 51.6, 50.6, 39.1.

Dimethyl 2-(methyl(phenyl)amino)-1H-indole-3,5-dicarboxylate 4b

White solids, Mp: 168–169 °C; 1H NMR (400 MHz, CDCl3) δH (ppm) 9.09 (s, 1H), 8.80–8.75 (m, 1H), 7.85 (dd, J = 8.5, 1.7 Hz, 1H), 7.25–7.19 (m, 3H), 6.96–6.85 (m, 3H), 3.89 (s, 3H), 3.79 (s, 3H), 3.44 (s, 3H). 13C NMR (100 MHz, CDCl3) δC (ppm) 168.2, 164.3, 149.3, 146.5, 134.9, 129.3, 126.3, 124.0, 123.5(2C), 121.3, 117.1, 110.3, 97.6, 51.9, 51.0, 40.2. ESI-HRMS: m/z Calcd. for C19H17N2O4 [M–H]− 337.1188. Found 337.1166.

Dimethyl 2-((4-(methoxycarbonyl)phenyl)(methyl)amino)-1H-indole-3,5-dicarboxylate 4c

White solids, Mp: 234–236 °C; 1H NMR (400 MHz, CDCl3) δH (ppm) 9.58 (s, 1H), 8.84 (d, J = 1.3 Hz, 1H), 7.96 (dd, J = 8.5, 1.6 Hz, 1H), 7.80–7.73 (m, 2H), 7.38 (d, J = 8.5 Hz, 1H), 6.70–6.63 (m, 2H), 3.93 (s, 3H), 3.82 (d, J = 3.5 Hz, 6H), 3.42 (s, 3H). 13C NMR (100 MHz, CDCl3) δC (ppm) 168.0, 167.3, 163.9, 150.9, 146.7, 135.0, 131.1, 125.7, 124.7, 124.4, 124.0, 120.6, 113.5, 110.9, 100.8, 52.0, 51.8, 51.2, 39.7. ESI-HRMS: m/z Calcd. for C21H20N2O6 [M+Na]+ 419.1219. Found 419.1217.

Dimethyl 2-((4-chlorophenyl)(methyl)amino)-1H-indole-3,5-dicarboxylate 4d

White solids, Mp: 188–189 °C; 1H NMR (600 MHz, CDCl3) δH (ppm) 8.79 (s, 1H), 8.70 (s, 1H), 7.92 (dd, J = 8.5, 1.6 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.21–7.16 (m, 2H), 6.82–6.77 (m, 2H), 3.93 (s, 4H), 3.84 (s, 3H), 3.43 (s, 4H). 13C NMR (100 MHz, CDCl3) δC (ppm) 168.01, 164.1, 148.4, 145.4, 134.8, 129.1, 126.1, 125.9, 124.3, 123.8(2C), 117.5, 110.4, 98.8, 52.08, 51.1, 40.1. ESI-HRMS: m/z Calcd. for C19H18ClN2O4 [M+H]+ 373.0960. Found 373.0965.

Dimethyl 2-((4-bromophenyl)(methyl)amino)-1H-indole-3,5-dicarboxylate 4e

White solids, Mp: 193–194 °C; 1H NMR (400 MHz, CDCl3) δH (ppm) 8.85 (s, 1H), 8.79 (s, 1H), 7.91 (dd, J = 8.5, 1.6 Hz, 1H), 7.34–7.26 (m, 3H), 6.75–6.67 (m, 2H), 3.92 (s, 3H), 3.83 (s, 3H), 3.41 (s, 3H). 13C NMR (100 MHz, CDCl3) δC (ppm) 168.0, 164.0, 148.1, 145.9, 134.7, 132.1, 126.1, 124.4, 123.9(2C), 117.7, 113.2, 110.4, 99.1, 52.0, 51.1, 40.0. ESI-HRMS: m/z Calcd. for C19H18BrN2O4 [M+H]+ 417.0450. Found 417.0452.

Methyl 5-bromo-2-((4-(methoxycarbonyl)phenyl)(methyl)amino)-1H-indole-3-carboxylate 4f

White solids, Mp: 217–218 °C; 1H NMR (600 MHz, CDCl3) δH (ppm) 9.08 (s, 1H), 8.29 (d, J = 1.9 Hz, 1H), 7.81–7.76 (m, 2H), 7.37 (dd, J = 8.5, 2.0 Hz, 1H), 7.22 (d, J = 8.6 Hz, 1H), 6.69–6.64 (m, 2H), 3.84 (s, 3H), 3.81 (s, 3H), 3.42 (s, 3H). 13C NMR (150 MHz, CDCl3) δC (ppm) 167.3, 163.8, 151.0, 146.1, 131.1, 130.7, 127.8, 126.4, 124.4, 120.6, 115.7, 113.5, 112.5, 99.7, 51.9, 51.2, 39.7. ESI-HRMS: m/z Calcd. for C19H18BrN2O4 [M+H]+ 417.0450. Found 417.0457.

Methyl 5-bromo-2-((4-chlorophenyl)(methyl)amino)-1H-indole-3-carboxylate 4g

White solids, Mp: 193–194 °C; 1H NMR (600 MHz, CDCl3) δH (ppm) 8.36 (s, 1H), 8.24 (d, J = 1.9 Hz, 1H), 7.31 (dd, J = 8.5, 1.9 Hz, 1H), 7.23–7.16 (m, 2H), 7.11 (d, J = 8.5 Hz, 1H), 6.79–6.75 (m, 2H), 3.82 (s, 3H), 3.41 (s, 3H). 13C NMR (150 MHz, CDCl3) δC (ppm) 164.0, 147.9, 145.5, 130.4, 129.2, 128.2, 125.9(2C), 124.1, 117.5, 115.5, 112.0, 97.9, 51.1, 40.1. ESI-HRMS: m/z Calcd. for C17H15BrClN2O2 [M+H]+ 393.0005. Found 393.0006.

Methyl 5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-2-carboxylate 5a

Pale gray solids, Mp: >300 °C; 1H NMR (600 MHz, DMSO-d 6): δH (ppm) 12.21 (s, 1H), 8.96 (d, J = 2.05 Hz, 1H), 8.28–8.12 (m, 2H), 7.84 (d, J = 8.80 Hz, 1H), 7.48 (d, J = 7.63 Hz, 1H), 7.36–7.17 (m, 2H), 3.98 (s, 3H), 3.91 (s, 3H); 13C NMR (150 MHz, DMSO-d 6): δC (ppm) 171.0, 165.9, 146.9, 142.0, 134.7, 130.9, 127.8, 124.2, 123.7, 123.2, 122.5, 121.5, 120.3, 115.8, 111.0, 102.9, 52.1, 33.7;

Methyl 5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-9-carboxylate 5b

Pale gray solids, Mp:>300 °C; 1H NMR (400 MHz, DMSO-d 6) δH (ppm) 12.45 (s, 1H), 8.86–8.81 (m, 1H), 8.40 (d, J = 7.8 Hz, 1H), 7.94–7.86 (m, 1H), 7.78 (q, J = 9.1 Hz, 2H), 7.55 (d, J = 8.4 Hz, 1H), 7.42 (t, J = 7.1 Hz, 1H), 3.98 (s, 3H), 3.90 (s, 3H). 13C NMR (100 MHz, DMSO-d 6) δC (ppm) 171.6, 166.9, 147.8, 139.2, 137.9, 131.5, 125.8, 124.7, 124.2, 123.8, 122.5, 122.0, 121.6, 115.5, 110.7, 102.1, 51.9, 33.4. ESI-HRMS: m/z Calcd. for C18H13N2O3 [M−H]− 305.0932. Found 305.0867.

Dimethyl 5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-2,9-dicarboxylate 5c

Pale gray solids, Mp:>300 °C; 1H NMR (400 MHz, DMSO-d 6) δH (ppm) 12.53 (s, 1H), 8.91 (d, J = 2.1 Hz, 1H), 8.78 (d, J = 1.3 Hz, 1H), 8.17 (dd, J = 8.9, 2.2 Hz, 1H), 7.89 (dd, J = 8.4, 1.7 Hz, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 3.95 (s, 3H), 3.91 (d, J = 4.7 Hz, 6H). 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 171.1, 166.9, 165.9, 147.8, 142.0, 137.8, 131.2 127.7, 124.6, 124.1, 123.4, 122.8(2C), 121.8, 116.1, 111.0, 102.7, 52.2, 52.0, 33.8. ESI-HRMS: m/z Calcd. for C20H17N2O5 [M+H]+ 365.1137. Found 365.1140.

Methyl 2-chloro-5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-9-carboxylate 5d

Pale gray solids, Mp: >300 °C; 1H NMR (600 MHz, DMSO-d 6) δH (ppm) 12.54 (s, 1H), 8.81 (s, 1H), 8.29 (s, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 8.2 Hz, 1H), 3.98 (s, 3H), 3.90 (s, 3H).13C NMR (150 MHz, DMSO-d 6) δC (ppm) 170.3, 166.8, 147.9, 138.0(2C), 131.2, 126.9, 125.9, 124.6(2C), 123.6, 122.7, 121.8, 118.1, 111.0, 102.5, 51.9, 33.8. ESI-HRMS: m/z Calcd. for C18H14ClN2O3 [M+H]+ 341.0693. Found 341.0679.

Methyl 2-bromo-5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-9-carboxylate 5e

Pale gray solids, Mp: >300 °C; 1H NMR (600 MHz, DMSO-d 6) δH (ppm) 12.57 (s, 1H), 8.82 (d, J = 1.3 Hz, 1H), 8.44 (d, J = 2.4 Hz, 1H), 7.96–7.87 (m, 2H), 7.81 (d, J = 9.1 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H), 3.90 (s, 3H). 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 170.2, 166.9, 147.9, 138.3, 138.1, 133.9, 127.8, 126.3, 124.6, 123.6, 122.7, 121.8, 118.3, 114.8, 111.0, 102.5, 51.9, 33.7. ESI-HRMS: m/z Calcd. for C18H13BrN2O3 [M+Na]+ 407.0007. Found 407.0009.

Methyl 9-bromo-5-methyl-11-oxo-6,11-dihydro-5H-indolo[2,3-b]quinoline-2-carboxylate 5f

Pale gray solids, Mp: >300 °C; 1H NMR (600 MHz, DMSO-d 6) δH (ppm) 12.34 (s, 1H), 8.88 (d, J = 2.1 Hz, 1H), 8.22 (s, 1H), 8.15 (dd, J = 8.8, 2.1 Hz, 1H), 7.79 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 1.7 Hz, 2H), 3.92 (s, 3H), 3.90 (s, 3H). 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 171.1, 165.8, 147.2, 142.0, 133.6, 131.2, 127.7, 125.5(2C), 123.9, 122.7, 122.2, 115.9, 113.7, 113.0, 102.0, 52.2, 33.7. ESI-HRMS: m/z Calcd. for C18H13BrN2O3 [M+Na]+ 407.0007. Found 407.0007.

9-bromo-2-chloro-5-methyl-5H-indolo[2,3-b]quinolin-11(6H)-one 5g

Pale gray solids, Mp: >300 °C; 1H NMR (600 MHz, DMSO-d 6) δH (ppm) 12.27 (s, 1H), 8.22 (dd, J = 5.2, 1.8 Hz, 2H), 7.77 (d, J = 9.1 Hz, 1H), 7.73 (dd, J = 9.0, 2.6 Hz, 1H), 7.44–7.30 (m, 2H), 3.91 (s, 3H). 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 170.2, 147.3, 137.8, 133.7, 131.0, 126.7, 125.6(2C), 125.4, 124.6, 122.1, 117.8, 113.5, 112.9, 101.7, 33.6. ESI-HRMS: m/z Calcd. for C16H10BrClN2O [M+Na]+ 382.9563. Found 382.9565.

Methyl 11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-2-carboxylate 6a

Orange solids, Mp: 281–283 °C; 1H NMR (600 MHz, CDCl3): δH (ppm) 8.95 (s, 1H), 8.29 (d, J = 8.22 Hz, 2H), 7.74–7.57 (m, 2H), 7.52 (t, J = 7.48 Hz, 1H), 7.22 (t, J = 7.48 Hz, 1H), 4.24 (s, 3H), 4.00 (s, 3H); 13C NMR (150 MHz, CDCl3): δC (ppm) 166.0, 155.1, 154.5, 139.2, 135.7, 131.3, 130.1, 128.2, 125.4, 123.9, 123.8,123.5, 120.9, 118.5, 117.8, 114.3, 52.4, 33.4.

Methyl 11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate 6b

Orange solids, Mp: 248–249 °C; 1H NMR (400 MHz, CDCl3) δH (ppm) 9.01 (s, 1H), 8.45 (d, J = 8.2 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.88 (t, J = 7.7 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 4.43 (d, J = 7.3 Hz, 3H), 3.98 (s, 3H). 13C NMR (150 MHz, CDCl3) δC (ppm) 167.4, 157.6, 156.1, 137.1, 136.5, 131.5, 130.9, 126.0, 125.4, 123.4, 122.9(2C), 121.5, 119.2, 116.7, 114.4, 51.9, 33.4. ESI-HRMS: m/z Calcd. for C18H14ClN2O2 [M+H]+ 325.0744. Found 325.0797.

Dimethyl 11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-2,9-dicarboxylate 6c

Orange solids, Mp: 278–280 °C; 1H NMR (600 MHz, CDCl3) δH (ppm) 9.14 (d, J = 1.8 Hz, 1H), 9.03 (d, J = 1.5 Hz, 1H), 8.51 (dd, J = 8.9, 1.8 Hz, 1H), 8.24 (dd, J = 8.4, 1.6 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 4.55 (s, 3H), 4.06 (s, 3H), 3.99 (s, 3H). 13C NMR (150 MHz, CDCl3) δC (ppm) 167.2, 165.7, 156.4, 155.7, 139.1, 138.0, 132.2, 131.7, 128.6, 125.8, 125.1, 124.4, 123.0, 122.8, 119.2, 117.1, 115.0, 52.7, 52.1, 34.4. ESI-HRMS: m/z Calcd. for C20H16ClN2O4 [M+H]+ 383.0799. Found 383.0799.

Methyl 2,11-dichloro-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate 6d

Orange solids, Mp: 283–284 °C; 1H NMR (600 MHz, CDCl3) δH (ppm) 8.90 (d, J = 1.2 Hz, 1H), 8.33 (d, J = 2.3 Hz, 1H), 8.17 (dd, J = 8.4, 1.7 Hz, 1H), 7.73 (dd, J = 9.1, 2.3 Hz, 1H), 7.63 (dd, J = 21.2, 8.7 Hz, 2H), 4.33 (s, 3H), 3.97 (s, 3H). 13C NMR (150 MHz, CDCl3) δC (ppm) 167.3, 157.6, 155.9, 135.9, 135.1, 131.8, 131.6, 129.2, 125.7, 125.3, 124.7, 122.8, 122.3, 120.4, 117.0, 116.2, 52.1, 33.9. ESI-HRMS: m/z Calcd. for C18H13Cl2N2O2 [M+H]+ 359.0354. Found 359.0357.

Methyl 2-bromo-11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate 6e

Orange solids, Mp: 289–290 °C; 1H NMR (600 MHz, CDCl3) δH (ppm) 9.06 (d, J = 1.5 Hz, 1H), 8.60 (d, J = 2.2 Hz, 1H), 8.27 (dd, J = 8.4, 1.7 Hz, 1H), 7.96 (dd, J = 9.1, 2.2 Hz, 1H), 7.74 (dd, J = 20.0, 8.7 Hz, 2H), 4.46 (s, 3H), 3.99 (s, 3H). 13C NMR (150 MHz, CDCl3) δC (ppm) 167.5, 158.5, 156.5, 135.7, 134.4, 131.7, 130.9, 128.8, 128.5, 125.9, 125.2, 123.3, 122.2, 120.8, 117.3, 116.3, 52.1, 33.6. ESI-HRMS: m/z Calcd. for C18H13BrClN2O2 [M+H]+ 402.9849. Found 402.9848.

Methyl 9-bromo-11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-2-carboxylate 6f

Orange solids, Mp: >300 °C; 1H NMR (600 MHz, CDCl3) δH (ppm) 9.14 (d, J = 1.8 Hz, 1H), 8.53 (d, J = 1.9 Hz, 1H), 8.48 (dd, J = 8.9, 1.9 Hz, 1H), 7.84 (d, J = 9.0 Hz, 1H), 7.70–7.60 (m, 2H), 4.43 (s, 3H), 4.04 (d, J = 8.0 Hz, 3H). 13C NMR (150 MHz, CDCl3) δC (ppm) 166.0, 155.4, 153.4, 139.6, 137.9, 137.2, 132.8, 132.0, 128.7, 126.5, 124.7, 124.3, 119.4, 118.7, 114.7, 113.6, 52.6, 33.6. ESI-HRMS: m/z Calcd. for C18H13BrClN2O2 [M+H]+ 402.9849. Found 402.9850.

9-Bromo-2,11-dichloro-5-methyl-5H-indolo[2,3-b]quinoline 6 g

Orange solids, Mp: 288–290 °C; 1H NMR (600 MHz, CDCl3) δH (ppm) 8.45 (d, J = 1.9 Hz, 1H), 8.37 (d, J = 2.3 Hz, 1H), 7.76 (dd, J = 9.1, 2.4 Hz, 1H), 7.66 (d, J = 9.1 Hz, 1H), 7.62 (dd, J = 8.4, 2.0 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 4.30 (s, 3H). 13C NMR (150 MHz, CDCl3) δC (ppm) 155.0, 153.6, 135.6, 135.5, 132.8, 131.8, 128.6, 126.5, 125.4, 125.0, 124.8, 120.08, 119.1, 115.9, 113.1, 33.5. ESI-HRMS: m/z Calcd. for C16H10BrCl2N2 [M+H]+ 378.9404. Found 378.9406.

General procedure for the synthesis of ester substituted 11-amino-5-methyl-5H-indolo[2,3-b]quinolines 7–9

The methyl 11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-2-carboxylate (6) or methyl 11-chloro-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate (7) was heated at reflux with a large excess of an appropriate amine in THF for 3–4 h. The reaction was monitored by TLC. Then the mixture was washed with water and extracted with CH2Cl2. The organic phase was dried over MgSO4 and concentrated under vacuum. The crude product was purified by column chromatography using eluent changed from AcOEt to AcOEt-2 N ammonia in MeOH (10: 1) to give the final product.

Methyl 11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-b]quinoline-2-carboxylate 7

Yellow solids, Mp: 149–150 °C; 1H NMR (400 MHz, DMSO-d 6) δH (ppm) 9.12 (d, J = 1.8 Hz, 1 H), 8.25 (dd, J = 9.0, 1.8 Hz, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.90 (d, J = 9.1 Hz, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.09 (t, J = 7.0 Hz, 1H), 4.17 (s, 3H), 3.99 (t, J = 6.5 Hz, 2H), 3.93 (s, 3H), 2.67 (t, J = 6.3 Hz, 2H), 1.78–1.74 (m, 2H); 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 165.9, 156.2, 151.9, 148.1, 140.2, 130.2, 126.5, 124.5, 124.3, 122.1, 121.3, 118.5, 116.9, 115.3, 115.1, 103.7, 52.2, 47.4, 39.9, 33.1, 32.5. ESI-HRMS: m/z Calcd. for C21H21N4O2 [M−H]− 361.1670. Found 375.1666.

Methyl 11-(3-hydroxypropylamino)-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate 8a

Orange solids, Mp: 223–224 °C; 1H NMR (400 MHz, DMSO-d 6) δH (ppm) 8.60–8.50 (m, 2H), 7.94–7.88 (m, 2H), 7.86–7.81 (m, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.50–7.43 (m, 2H), 4.57 (t, J = 4.7 Hz, 1H), 4.19 (s, 3H), 3.97 (q, J = 6.5 Hz, 2H), 3.87 (s, 3H), 3.47 (dd, J = 10.8, 5.9 Hz, 2H), 1.90 (p, J = 6.4 Hz, 2H); 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 167.4, 158.3, 155.8, 149.2, 137.4, 131.2, 125.7, 124.0(2C), 123.6, 121.3, 118.4, 116.0, 115.6(d), 103.1, 58.4, 51.6, 45.8, 40.0(overlap with DMSO-d6 peaks), 33.6, 32.5. ESI-HRMS: m/z Calcd. for C21H20N3O3[M−H]− 362.1492. Found 362.1483.

Methyl 11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate 8b

Yellow solids, Mp: 170–171 °C; 1H NMR (600 MHz, DMSO-d 6) δH (ppm) 8.63 (d, J = 1.4 Hz, 1H), 8.52 (d, J = 7.7 Hz, 1H), 7.93–7.87 (m, 2H), 7.83 (t, J = 7.7 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.4 Hz, 1H), 4.19 (s, 3H), 4.00 (t, J = 6.6 Hz, 2H), 3.86 (s, 3H), 2.71 (t, J = 6.3 Hz, 2H), 1.84–1.78 (m, 2H); 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 167.4, 158.3, 155.7, 149.1, 137.5, 131.1, 125.6, 124.1(2C), 123.6, 121.2, 118.3, 116.0, 115.5(2C), 102.6, 51.6, 47.2, 33.1, 32.5. ESI-HRMS: m/z Calcd. for C21H21N4O2[M−H]− 361.1670. Found 361.1639.

Methyl 5-methyl-11-morpholino-5H-indolo[2,3-b]quinoline-9-carboxylate 8c

Orange solids, Mp: 213–215 °C; 1H NMR (400 MHz, CDCl3) δH (ppm) 9.01 (d, J = 1.6 Hz, 1H), 8.50 (d, J = 8.2 Hz, 1H), 8.20 (dd, J = 8.4, 1.7 Hz, 1H), 7.83 (d, J = 4.1 Hz, 2H), 7.74 (d, J = 8.4 Hz, 1H), 7.57–7.47 (m, 1H), 4.41 (s, 3H), 4.18–4.09 (m, 4H), 3.98 (s, 3H), 3.75–3.66 (m, 4H); 13C NMR (100 MHz, CDCl3) δC (ppm) 167.8, 158.4, 156.7, 151.3, 138.1, 131.0, 129.7, 126.5, 126.0, 122.4, 122.0, 121.0(2C), 120.7, 116.5, 115.1, 67.6, 51.9, 51.0, 33.7. ESI-HRMS: m/z Calcd. for C22H22N3O3 [M+H]+ 376.1661. Found 376.1662.

Methyl 5-methyl-11-(4-methyl-1,4-diazepan-1-yl)-5H-indolo[2,3-b]quinoline-9-carboxylate 8d

Orange solids, Mp: 179–180 °C; 1H NMR (400 MHz, CDCl3) δH (ppm) 8.93 (s, 1H), 8.47 (d, J = 8.2 Hz, 1H), 8.26–8.18 (m, 1H), 7.80 (dd, J = 3.7, 1.7 Hz, 2H), 7.71 (d, J = 8.4 Hz, 1H), 7.50 (ddd, J = 8.1, 4.2, 1.7 Hz, 1H), 4.37 (d, J = 0.8 Hz, 3H), 3.97 (d, J = 0.9 Hz, 3H), 3.93–3.87 (m, 2H), 3.78 (t, J = 6.1 Hz, 2H), 3.14 (d, J = 4.5 Hz, 2H), 3.05–2.90 (m, 2H), 2.63 (s, 3H), 2.39–2.23 (m, 2H); 13C NMR (100 MHz, CDCl3) δC (ppm) 168.1, 159.5, 157.8, 152.3, 138.5, 130.7, 129.7, 126.8, 125.7, 122.8, 122.0, 121.5, 121.0, 120.8, 116.8, 115.0, 61.3, 57.9, 52.9, 52.2, 51.9, 47.2, 33.3, 29.2. ESI-HRMS: m/z Calcd. for C24H27N4O2 [M+H]+ 403.2134. Found 403.2135.

Methyl 11-(4-aminobutylamino)-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate 8e

Yellow oil; 1H NMR (600 MHz, DMSO-d 6) δH (ppm) 8.62 (t, J = 6.1 Hz, 1H), 8.53 (s, 1H), 7.92 (dd, J = 8.3, 7.0, 2H), 7.85 (t, J = 7.7 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1 H), 4.20 (s, 3H), 3.90 (t, J = 7.1 Hz, 2H), 3.87 (s, 3H), 2.67 (t, J = 7.4 Hz, 2H), 1.85–1.76 (m, 2H), 1.48 (d, J = 6.4 Hz, 2H); 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 167.4, 158.4, 155.9, 149.0, 137.4, 131.2, 125.8, 124.0 (2C), 123.7, 121.4, 118.4, 116.1, 115.7, 115.5, 103.0, 51.7, 47.3, 38.6, 32.5, 27.6, 24.9. ESI-HRMS: m/z Calcd. for C22H25N4O2 [M + H]+ 377.1978. Found 377.1978.

Methyl 11-(2-(dimethylamino)ethylamino)-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate 8f

Yellow solids, Mp: 172–173 °C; 1H NMR (400 MHz, DMSO-d 6) δH (ppm) = 8.70 (s, 1H), 8.54 (d, J = 8.3 Hz, 1H), 7.97–7.91 (m, 2H), 7.86 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.26 (t, J = 4.5 Hz, 1H), 4.21 (s, 3H), 4.00 (dd, J = 10.8, 5.5 Hz, 2H), 3.87 (s, 3H), 2.65 (t, J = 6.1 Hz, 2H), 2.27 (s, 6H); 13C NMR (150 MHz, CDCl3) δC (ppm) = 168.2, 157.4, 155.6, 149.3, 138.3, 130.5, 127.0, 125.3, 123.8, 122.5, 120.6, 119.7, 116.1, 115.6, 114.8, 106.0, 58.2, 51.5, 45.2, 44.7, 32.6. ESI-HRMS: m/z Calcd. for C22H23N4O2 [M−H]− 375.1826. Found 375.1806.

Methyl 11-(3-(dimethylamino)propylamino)-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate 8g

Yellow solids, Mp: 105–106 °C; 1H NMR (600 MHz, CDCl3) δH (ppm) = 8.65 (d, J = 1.4 Hz, 2H), 8.07 (dd, J = 8.4, 1.5 Hz, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.70 (dd, J = 11.9, 4.4 Hz, 2H), 7.65 (d, J = 8.6 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 4.23 (d, J = 1.2 Hz, 3H), 4.20–4.10 (m, 2H), 3.94 (s, 3H), 2.72–2.64 (m, 2H), 2.39 (s, 6H), 1.95–1.73 (m, 2H); 13C NMR (150 MHz, CDCl3) δC (ppm) = 168.4, 158.3, 155.5, 149.0, 137.5, 130.3, 126.2, 124.1, 123.6, 123.4, 120.7, 119.1, 115.9, 115.8, 114.5, 103.0, 59.5, 51.5, 50.1, 45.4, 32.5, 26.3. ESI-HRMS: m/z Calcd. for C23H25N4O2[M−H]− 389.1983. Found 389.1949.

Dimethyl 11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-b]quinoline-2,9-dicarboxylate 9a

Yellow solids, Mp: 75–76 °C; 1H NMR (400 MHz, DMSO-d 6) δH (ppm) 9.17 (s, 1H), 8.63 (s, 1H), 8.28 (d, J = 8.1 Hz, 1H), 7.97–7.89 (m, 2H), 7.55 (d, J = 8.4 Hz, 1H), 4.20 (s, 3H), 4.02 (t, J = 6.6 Hz, 2H), 3.94 (s, 3H), 3.87 (s, 3H), 2.75 (t, J = 6.3 Hz, 2H), 1.87–1.82 (m, 2H). 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 167.4, 165.9, 158.2, 155.4, 148.9, 140.2, 130.7, 126.6, 125.7, 124.1, 123.7, 122.0, 119.0, 116.4, 115.8, 115.2, 102.5, 52.3, 51.6(2C), 47.1, 32.8, 32.5. ESI-HRMS: m/z Calcd. for C23H25N4O4 [M+H]+ 421.1876. Found 421.1877.

Methyl 11-(3-aminopropylamino)-2-chloro-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate 9b

Yellow solids, Mp: 185–186 °C; 1H NMR (600 MHz, DMSO-d 6) δH (ppm) 8.59 (d, J = 2.0 Hz, 1H), 8.54 (s, 1H), 7.89 (dd, J = 8.4, 1.4 Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H), 7.79 (dd, J = 9.1, 2.1 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 4.12 (s, 3H), 3.93 (t, J = 6.7 Hz, 2H), 3.86 (s, 3H), 2.67 (t, J = 6.4 Hz, 2H), 1.86–1.77 (m, 2H). 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 167.4, 158.1, 155.8, 147.9, 136.1, 130.7, 125.8(2C), 123.9, 123.2, 118.6, 117.5, 116.7, 116.2, 104.6, 103.1, 51.8, 46.8, 39.9(overlap with DMSO-d6 peaks), 33.3, 32.6. ESI-HRMS: m/z Calcd. for C21H22ClN4O2 [M+H]+ 397.1431. Found 397.1432.

Methyl 11-(3-aminopropylamino)-2-bromo-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate 9c

Yellow solids, Mp: 109–112 °C; 1H NMR (400 MHz, DMSO-d 6) δH (ppm) 8.76 (s, 1H), 8.57 (s, 1H), 7.97–7.88 (m, 2H), 7.84 (d, J = 9.2 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 4.16 (s, 3H), 3.95 (t, J = 6.7 Hz, 2H), 3.86 (s, 3H), 2.67 (t, J = 6.4 Hz, 2H), 1.82 (t, J = 6.5 Hz, 2H). 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 167.4, 158.1, 155.8, 147.9, 136.4, 133.4, 126.1, 125.9, 123.9(2C), 118.6, 117.7, 117.3, 116.2, 113.5 103.1, 51.7, 46.6, 39.3, 33.1, 32.6, 32.3. ESI-HRMS: m/z Calcd. for C21H22BrN4O2 [M+H]+ 441.0926. Found 441.0927.

Methyl 11-(3-aminopropylamino)-9-bromo-5-methyl-5H-indolo[2,3-b]quinoline-2-carboxylate 9d

Yellow solids, Mp: 162–163 °C; 1H NMR (600 MHz, DMSO-d 6) δH (ppm) 9.10 (d, J = 1.6 Hz, 1H), 8.24 (dd, J = 8.9, 1.7 Hz, 1H), 8.10 (d, J = 1.9 Hz, 1H), 7.88 (d, J = 9.0 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.39 (dd, J = 8.4, 1.9 Hz, 1H), 4.14 (s, 3H), 3.97 (t, J = 6.6 Hz, 2H), 3.92 (s, 3H), 2.71 (t, J = 6.2 Hz, 2H), 1.83–1.77 (m, 2H). 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 165.8, 156.5, 150.5, 149.0, 140.4, 130.7, 126.7(2C), 126.2, 124.0, 121.5, 118.5, 115.6, 114.9, 110.4, 102.3, 52.2, 47.5, 39.9(overlap with DMSO-d6 peaks), 32.9, 32.6. ESI-HRMS: m/z Calcd. for C21H22BrN4O2 [M+H]+ 441.0926. Found 441.0927.

N-(3-aminopropyl)-9-bromo-2-chloro-5-methyl-5H-indolo[2,3-b]quinolin-11-amine 9e

Yellow solids, Mp: 152–154 °C; 1H NMR (600 MHz, DMSO-d 6) δH (ppm) 8.59 (d, J = 1.8 Hz, 1H), 8.03 (d, J = 1.7 Hz, 1H), 7.86 (d, J = 9.2 Hz, 1H), 7.82 (dd, J = 9.1, 2.0 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.39 (dd, J = 8.4, 1.9 Hz, 1H), 4.12 (s, 3H), 3.91 (t, J = 6.6 Hz, 2H), 2.63 (t, J = 6.3 Hz, 2H), 1.77 (p, J = 6.5 Hz, 2H). 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 156.6, 150.9, 148.1, 136.3, 130.7, 126.9, 126.1, 125.2, 124.2, 123.3, 118.3, 117.3, 116.5, 109.9, 102.9, 46.9, 39.5(overlap with DMSO-d6 peaks), 33.3, 32.5. ESI-HRMS: m/z Calcd. for C19H19BrClN4 [M+H]+ 417.0482. Found 417.0480.

General procedure for the synthesis of 11-amino-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylic acid 11

Methyl 11-amino-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylate was dissolved in MeOH and refluxed with 20 % NaOH (aq.) over night. The reaction was monitored by TLC. Then the mixture was neutralized with 1 N HCl (aq.) and concentrated to remove the MeOH under vacuum. The crude product was purified by reverse-phase chromatography using eluent changed from H2O to H2O/CH3CN (50:1) to give carboxylic acid.

11-(3-aminopropylamino)-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylic acid 11a

White solids, Mp: >300 °C; 1H NMR (600 MHz, D2O) δH (ppm) 8.10 (d, J = 8.2 Hz, 1H), 7.90 (t, J = 7.5 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.58 (dd, J = 9.9, 4.9 Hz, 2H), 7.53 (s, 1H), 7.17 (dd, J = 7.9, 4.8 Hz, 1H), 3.69–3.57 (m,5H), 2.93–2.83 (m, 2H), 2.06–1.91 (m, 2H). 13C NMR (150 MHz, D2O) δC (ppm) 168.9, 151.6, 146.9, 139.0, 135.8, 133.7, 127.4, 125.1, 123.7, 123.3, 122.4, 118.8, 116.2, 115.4, 111.0, 97.9, 44.8, 36.4, 34.3, 27.9. ESI-HRMS: m/z Calcd. for C20H19N4O2 [M−H]− 347.1513. Found 347.1505.

11-(2-(dimethylamino)ethylamino)-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylic acid 11b

White solids, Mp: 276–280 °C; 1H NMR (400 MHz, DMSO-d 6) δH (ppm) = 9.03 (d, J = 8.0 Hz, 2H), 8.57 (s, 1H), 8.15 (dd, J = 19.0, 8.6 Hz, 2H), 8.04 (t, J = 7.8 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H), 4.37 (dd, J = 11.4, 5.7 Hz, 2H), 4.30 (s, 3H), 3.55 (t, J = 5.5 Hz, 2H), 2.78 (s, 6H). 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 167.6, 152.5, 149.3, 140.71, 136.7, 133.3, 127.7, 125.2, 124.9, 124.5 (2C), 120.6, 116.9, 112.2, 99.5, 56.0, 43.1, 42.8, 35.7, 34.4. ESI-HRMS: m/z Calcd. for C21H21N4O2 [M−H]− 361.1670. Found 361.1653.

11-(3-(Dimethylamino)propylamino)-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylic acid 11c

Pale Yellow solids, Mp: 280–282 °C; 1H NMR (400 MHz, DMSO-d 6) δH (ppm) 8.97 (s, 1H), 8.89 (d, J = 8.5 Hz, 1H), 8.52 (s, 1H), 8.12 (dd, J = 16.6, 8.6 Hz, 2H), 8.05–7.98 (m, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.70 (t, J = 7.7 Hz, 1H), 4.26 (s, 3H), 4.01 (dd, J = 12.9, 6.5 Hz, 2H), 3.15–3.04 (m, 2H), 2.70 (s, 6H), 2.31 (dt, J = 14.0, 7.2 Hz, 2H); 13C NMR (150 MHz, DMSO-d 6) δC (ppm)167.7, 152.4, 149.3, 136.8, 133.2, 127.3, 124.9, 124.5 (2C), 124.2, 120.8, 116.9, 116.7, 112.2, 98.6, 53.7, 45.3, 42.1, 35.6, 25.1. ESI-HRMS: m/z Calcd. for C22H23N4O2 [M−H]− 375.1826. Found 375.1828.

11-(3-Hydroxypropylamino)-5-methyl-5H-indolo[2,3-b]quinoline-9-carboxylic acid 11d

White solids, Mp: >300 °C; 1H NMR (600 MHz, DMSO-d 6) δH (ppm) 8.86–8.72 (m, 2H), 8.59 (s, 1H), 8.10 (d, J = 8.5, 1H), 8.05 (d, J = 8.3, 1H), 7.99 (t, J = 7.6, 1H), 7.76 (d, J = 8.3, 1H), 7.66 (t, J = 7.5, 1H), 4.24 (s, 3H), 4.04 (dd, J = 11.8, 6.0, 2H), 3.46 (t, J = 5.6, 2H), 2.07–1.82 (m, 2H); 13C NMR (150 MHz, DMSO-d 6) δC (ppm) 167.4, 152.4, 148.7, 140.2, 136.7, 132.9, 127.0, 124.8, 124.4, 124.1, 123.9, 120.6, 116.8, 116.5, 111.9, 98.2, 58.0, 46.1, 35.6, 32.8. ESI-HRMS: m/z Calcd. for C20H18N3O3 [M–H]− 348.1354. Found 348.1327.

Antitumor screening test

Cell lines

Established in vitro, human cell line: MV4-11 (leukemia), A549 (lung cancer), HCT116 (colon cancer), and normal mice fibroblast (Balb/3T3) were used. These lines were obtained from American Type Culture Collection (Rockville, Maryland, USA) and were being maintained at the Institute of Immunology and Experimental Therapy, Wroclaw, Poland.

MV4-11 cells were cultured in the RPMI 1640 supplemented with 2 mM l-glutamine, 1.0 mM sodium pyruvate and 10 % fetal bovine serum (all from Sigma-Aldrich Chemie GmbH, Steinheim, Germany), HCT 116, and A549 cells were cultured in the RPMI 1640 + OptiMEM (50:50) medium (Gibco, Scotland, UK) supplemented with 2 mM l-glutamine and 5 % fetal bovine serum (all from Sigma-Aldrich Chemie GmbH, Steinheim, Germany), BALB/3T3 cells were cultured in Dulbecco medium (IIET) supplemented with 2 mM l-glutamine and 1.0 mM sodium pyruvate, 10 % fetal bovine serum (all from Sigma-Aldrich Chemie GmbH, Steinheim, Germany). All culture medium was supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin (both from Polfa, Tarchomin S.A., Poland). All cell lines were grown at 37 °C with 5 % CO2 humidified atmosphere.

Antiproliferative assay in vitro

Test solutions of the compounds tested (1 mg/ml) were prepared by dissolving the substances in 100 μl of DMSO completed with 900 μl of tissue culture medium. Afterward, the tested compounds were diluted in culture medium to reach the final concentrations of 10, 1, 0.1, 0.01, and 0.001 μg/ml.

Twenty-four hours before the addition of the tested compounds, the cells were plated in 96-well plates (Sarstedt, Germany) at a density of 1 × 104 cells per well. The assay was performed after 72 h of exposure to varying concentrations of the tested agents. The in vitro cytotoxic effect of all agents was examined using the MTT (MV4-11) or SRB (A549, HCT116 and Balb/3T3) assay.

The results were calculated as an IC50 (inhibitory concentration 50)—the dose of tested agent which inhibits proliferation of 50 % of the cancer cell population. IC values were calculated for each experiment separately and mean values ± SD are presented in the Table 1, 2, 3 and 4. Each compound in each concentration was tested in triplicate in a single experiment, which was repeated 3–5 times.

MTT assay

This technique was applied for the cytotoxicity screening against leukemia cells growing in suspension culture. An assay was performed after 72 h exposure to varying concentrations (from 0.001 to 10 μg/ml) of the tested agents. For the last 3–4 h of incubation 20 μl of MTT solution were added to each well (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; stock solution: 5 mg/ml, Sigma-Aldrich, Germany). The mitochondria of viable cells reduce the pale yellow MTT to a navy blue formazan: the more viable cells are present in well, the more MTT will be reduced to formazan. When incubation time was completed, 80 μl of the lysing mixture were added to each well (lysing mixture: 225 ml dimethylformamide, POCh, Gliwice, Poland, 67.5 g sodium dodecyl sulfate, Sigma-Aldrich, Germany, and 275 ml of distilled water). After 24 h, when formazan crystals had been dissolved, the optical densities of the samples were read on an Multiskan RC photometer (Labsystems, Helsinki, Finland) at 570 nm wavelength. Each compound in given concentration was tested in triplicates in each experiment, which was repeated 3–5 times.

SRB assay

This technique was applied for the cytotoxicity screening against cells growing in adherent culture. The details of this technique were described by Skehan (Skehan et al., 1990). The cytotoxicity assay was performed after 72-h exposure of the cultured cells to varying concentrations (from 0.01 to 10 μg/ml) of the tested agents. The cells attached to the plastic were fixed by gently layering cold 50 % TCA (trichloroacetic acid, Aldrich-Chemie, Germany) on the top of the culture medium in each well. The plates were incubated at 4 °C for 1 h and then washed five times with tap water. The background optical density was measured in the wells filled with culture medium, without the cells. The cellular material fixed with TCA was stained with 0.4 % sulforhodamine B (SRB, Sigma, Germany) dissolved in 1 % acetic acid (POCh, Gliwice, Poland) for 30 min. Unbound dye was removed by rinsing (4x) with 1 % acetic acid. The protein-bound dye was extracted with 10 mM unbuffered Tris base (Sigma, Germany) for determination of optical density (at 540 nm) in a computer-interfaced, 96-well microtiter plate reader Multiskan RC photometer (Labsystems, Helsinki, Finland).