Abstract
Three esters of rhodamine B (1–3) differing in their alkyl chain lengths as well as several rhodamine B amides (4–9) were synthesized in good yields and tested for their cytotoxicity in SRB assays employing several human tumor cell lines. The rhodamine B esters were unselective but showed cytotoxicity of as low as EC50 = 0.15 ± 0.02 µM. The rhodamine B amides were slightly less cytotoxic but showed good selectivity against MCF-7 and A2780 tumor cell lines. Especially a morpholinyl derivative 4 was ~20 time more cytotoxic for MCF-7 than for nonmalignant NIH 3T3 cells.
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Introduction
Rhodamines are widely used in fluorescence microscopy to stain cell compartments especially mitochondria (Guo et al. 2018; Johnson et al. 1980; Talib et al. 2019; Wang et al. 2018; Zhang et al. 2020). The preferential transport of these xanthylium scaffold based dyes into mitochondria has previously been used to selectively direct cytotoxic compounds into mitochondria (Kahnt et al. 2018; Sommerwerk et al. 2017; Wiemann et al. 2018; Wolfram et al. 2018a, 2018b; Xie et al. 2013). While rhodamine B displays only minor cytotoxicity, di- or triterpenoid conjugates holding an attached rhodamine B moiety with or without a spacer between these parts of the conjugate proved highly cytotoxic. Several of these “mitocanic” conjugates held an even nanomolar activity for human tumor cell lines (Sommerwerk et al. 2017; Wolfram et al. 2018a, 2018b). It has also been shown that these mitocans led to a controlled cell death; some of them could distinguish very well between malignant and nonmalignant cells thus providing a high selectivity for malignant human tumor cell lines (Kahnt et al. 2018; Sommerwerk et al. 2017; Wiemann et al. 2018; Wolfram et al. 2018a, 2018b). As a consequence, mitochondria have emerged as a major drug target inasmuch as they can induce a programmed cell death in human tumor cells (Costantini et al. 2000; Fulda 2010; Galluzzi et al. 2006; Gogvadze et al. 2009a, 2009b; Neuzil et al. 2013). While the exact mechanism still remains unclear it appears that the presence of a lipophilic cation in the conjugates is one of the necessary prerequisites for achieving good cytotoxicity (Neuzil et al. 2013; Sommerwerk et al. 2017). In the case of triterpenoic acids, there are indications that both the choice of the respective triterpenoic scaffold (maslinic acid is better than, e.g., oleanolic acid), the spacer (piperazinyl spacered compounds holding a rhodamine B dye are more cytotoxic than analogs holding an ethylenediamine spacer) and the type of cation (rhodamine B derived compounds are more cytotoxic than, for example, analogs holding a malachite green derived moiety) are of decisive importance (Kahnt et al. 2018; Sommerwerk et al. 2017; Wiemann et al. 2018; Wolfram et al. 2018a, 2018b). Furthermore, it must also be pointed out that the presence of a rhodamine B residue in a molecule does not guarantee the achievement of high cytotoxicity in a conjugate (Wiemann et al. 2018).
Triterpenoic acids are notoriously poorly soluble in water which limits their bioavailability (Csuk and Deigner 2019; Shakurova et al. 2020; Song et al. 2019). The formation of rhodamine B conjugates significantly increases their solubility, but these molecules are nevertheless not perfect according to Lipinski’s “rule of five” rule (Oprea 2002; Walters et al. 1999). This gave rise to the question to what extent the triterpenoid part in these molecules could be avoided at all. Since no “simple” derivatives of rhodamine B have been evaluated for their cytotoxicity against human tumor cell lines we decided to prepare several esters and amides of rhodamine B and to investigate their cytotoxic effects (Fig. 1 and Scheme 1).
Cytotoxicity of compounds 1–9, STA and Rho; 3D bar chart representation (cutoff at 7 µM)
Synthesis of several rhodamine B drug conjugates: a 1. (COCl)2, DMF, CH2Cl2 2. ethanol/1-hexanol/1-eicosanol, NEt3 (2 eq.) 0 °C b 1. (COCl)2, DMF, CH2Cl2 2. morpholine/thiomorpholine/2,6-dimethylmorpholine/N-methylpiperazine/N, N-diethylamine/piperazine, 0 °C, 30 min
Results and discussion
The synthesis of the rhodamine B drug conjugates was straightforward: for the synthesis of the esters (Mai and Allison 1983; Mottram et al. 2012; Rashid and Horobin 1990; Tansil et al. 2011; Wieker et al. 1987; Yu et al. 2001) 1–3, to a solution of rhodamine B acyl chloride either ethanol, hexanol or eicosanol were added in the presence of triethylamine to afford the esters in yields ranging from 48 to 85%. For the synthesis of the amides (Beija et al. 2011; Bui et al. 2014; Dauner et al. 2016; Del Secco et al. 2017; May et al. 2012; Preston et al. 2018; Sodano et al. 2018) 4–9 rhodamine B acyl chloride was allowed to react with an excess of the corresponding amine; thereby the products were obtained in isolated yields ranging from 68 to 92%.
The compounds were subjected to sulforhodamine B assays (SRB) to evaluate their cytotoxicity; the results of these assays are summarized in Table 1.
As a result, compounds 1–9 were cytotoxic for all human tumor cell lines; their EC50 values ranged from excellent 0.15 ± 0.02 µM for compound 2 to very low EC50 values of >20 µM for the eicosyl ester 3. This somewhat surprising result suggests that transport through the membrane(s) is not the limiting factor, since it is known that rhodamine B esters with hydrophobic moieties permeate lipid membranes faster than their hydrophilic analogs (Melikyan et al. 1996; Rokitskaya et al. 2008, 2018).
While the rhodamine B esters are highly active holding EC50 values lower than 1 µM these compounds lack selectivity (Table 2). Although it would make sense to study a homologous series of these esters holding different chain lengths with the aim of finding a “magic” chain length where cytotoxicity is highest, we have refrained from doing so. We justify this by the fact that the selectivity factors of the esters (as compared to those of the amides) are too low, and a sufficiently large differentiation between malignant and nonmalignant cells is not likely.
However, the rhodamine B amides also showed high cytotoxic effects with EC50 values between 0.27 ± 0.01 and 17.34 ± 0.8 µM. Especially compound 8 was very cytotoxic holding EC50 values lower than 1 µM while the other rhodamine B amides were slightly less cytotoxic. Interestingly enough, piperazine derived 9 was significantly less active than N-methyl-piperazine derived compound 7. As far as the selectivity factors of all compounds are concerned, it is of interest to note that the “simple” rhodamine B amides showed a significant selectivity for human tumor cell lines MCF-7 (breast adenocarcinoma) and A2780 (ovarian carcinoma). Especially the morpholinyl derivative 4 exhibited with S = 19.5 the highest selectivity factor for MCF-7 and with S = 18.1 for A2780 tumor cells.
Conclusion
Rhodamine derived dyes are widely used to stain the mitochondria of cells; although rhodamine B is classified as potentially carcinogenic it does not show cytotoxicity up to 30 µM. To get a deeper insight into the cytotoxicity of rhodamine B derived conjugates, three different esters of rhodamine B (1–3) differing in their alkyl chain length as well as six amides (4–9) were prepared in good yields and tested for their cytotoxicity using several human tumor cell lines. Esters and amides of rhodamine B with triterpenoids have previously been shown to be highly cytotoxic for tumor cells holding EC50 values in the nM region (Sommerwerk et al. 2017; Wiemann et al. 2018; Wolfram et al. 2018a). Hence it would be of interest to evaluate whether a triterpenoid scaffold is necessary for cytotoxicity or if simple esters and amides of rhodamine B may also perform as well as in SRB assays. As a result, the ethyl and hexyl ester of rhodamine B showed against MCF-7 tumor cells EC50 values as low as 0.23 and 0.15 µM, respectively. An eicosyl derivative, however, whose lipophilicity is even more close to that of triterpenoids did not show even moderate cytotoxicity although the long nonpolar alkyl chain might be able to interfere with membranes. Furthermore, the rhodamine B esters also lack selectivity.
Surprisingly, the morpholinyl derived rhodamine B amide was not as cytotoxic as the rhodamine B esters (albeit being in a low µM range with EC50 values ranging from 0.44 to 3.76 µM for MCF-7 breast adenocarcinoma cells) but showed good selectivity factors against tumor cells of 8–19.5 for MCF-7 tumor cells. The calculated selectivity factors, however, might even be higher but due to the cutoff limit of the assay the exact values could not be determined.
Depending on their substitution pattern, triterpenoid rhodamine conjugates are quite cytotoxic and several of them are highly selective against tumor cells. The good selectivity of the “simple” morpholinyl derived compounds as in contrast to the complex triterpenoids cannot be explained. One might assume, they are able to interfere with NF-kB, caspase-3/8/9 and or mTOR/PI3K/Akt-pathways which also are known to be altered by triterpenoids.
Ongoing investigations will provide evidence whether these compounds are able to trigger permeabilization of the mitochondrial membrane due to a change in the mitochondrial membrane potential or to interfere with the mitochondrial permeability transition pore.
Experimental
The equipment as well as the details of the cytotoxic evaluation can be found in the supplementary materials file.
Synthesis
Rhodamine B chloride (= N-(9-(2-(chlorocarbonyl)phenyl)-6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminium chloride)
The fluorescent dye rhodamine B (10.0 g, 22.3 mmol) was dissolved in dry CH2Cl2 (250 mL), treated with oxalyl chloride (8.84 mL) at 0 °C. One drop of dry DMF was added, and the solution was allowed to warm up to room temperature. After completion of the reaction, the solvent was removed under reduced pressure. The residue was dissolved in dry CH2Cl2 (50 mL), and the solution was concentrated again to remove excess oxalyl chloride. Yielding rhodamine B chloride (11.0 g, 99%) as a purple solid which is used without further purification
General procedure for the synthesis of the rhodamine B esters 1–3
Rhodamine B acyl chloride (500 mg, 1.0 mmol) was dissolved in dry CH2Cl2 (50 mL), and at 0 °C the corresponding alcohol (1 eq.) and triethylamine (2 mmol, 0.28 mL) were added. The reaction progress was monitored by TLC, and after complete conversion the solvent was removed under reduced pressure. The crude reaction mixture was purified by column chromatography (SiO2, CHCl3/MeOH) to yield the rhodamine B esters (1–3) each as a dark purple solid.
General procedure for the synthesis of the rhodamine B amides 4–9
Rhodamine B acyl chloride (500 mg, 1.0 mmol) was dissolved in dry CH2Cl2 (50 mL). The corresponding amine (4 eq.) was added slowly at 0 °C. The mixture was stirred for 30 min, the solvent was removed under reduced pressure, and the residue was purified by column chromatography (SiO2, CHCl3/MeOH) to yield the rhodamine B amides (4–9) each as a dark purple solid.
3,6-Bis(diethylamino)-9[2-(ethyloxy)carbonyl]-xanthylium chloride (1)
Yield: 433 mg (85%); m.p. 124–127 °C; RF = 0.39 (SiO2, CHCl3/MeOH, 8:2); IR (ATR): ν = 3322w, 3166m, 2981m, 1712s, 1644m, 1589s, 1543s, 1502m, 1463s, 1409s, 1390m, 1366m, 1334s, 1262s, 1240s, 1178s, 1131s, 1077s, 1043m, 1010s, 919m, 828m, 759m, 708m, 665m, 576 m cm−1; 1H NMR (400 MHz, CDCl3): δ = 8.21 (dd, J = 7.9, 1.4 Hz, 1H, 5H), 7.73 (dt, J = 7.5, 1.4 Hz, 1H, 7H), 7.66 (dt, J = 7.7, 1.4 Hz, 1H, 6H), 7.22 (dd, J = 7.5, 1.4 Hz, 1H, 8H), 7.00 (d, J = 9.5 Hz, 2H, 12H + 12′H), 6.84 (dd, J = 9.5, 2.4 Hz, 2H, 13H + 13′H), 6.72 (d, J = 2.4 Hz, 2H, 15H + 15′H), 3.99 (q, J = 7.1 Hz, 2H, 2′H), 3.58 (q, J = 7.2 Hz, 8H, 17H + 17′H + 17′′H + 17′′′H), 1.25 (t, J = 7.1 Hz, 12H, 18H + 18′H + 18′′H + 18′′′H), 0.99 (t, J = 7.1 Hz, 3H, 1H) ppm; 13C NMR (101 MHz, CDCl3): δ = 165.0 (C-3), 158.9 (C-4), 157.7 (C-16 + C-16′), 155.5 (C-14 + C-14′), 133.4 (C-9), 132.9 (C-7), 131.3 (C-12 + C-12′), 131.2 (C-5), 130.3 (C-6), 130.1 (C-8), 130.1 (C-10), 114.2 (C-13 + C-13′), 113.5 (C-11 + C-11′), 96.2 (C-15 + C-15′), 61.5 (C-2), 46.1 (C-17 + C-17′ + C-17′′ + C-17′′′), 13.7 (C-1), 12.6 (C-18 + C-18′ + C-18′′ + C-18′′′) ppm; MS (ESI, MeOH): m/z = 471.4 (100%, [M]+); analysis calcd for C30H35N2O3Cl (507.07): C 71.06, H 6.96, N 5.52; found: C 70.76, H 7.18, N 5.31.
3,6-Bis(diethylamino)-9[2-(hexyloxy)carbonyl]-xanthylium chloride (2)
Yield: 397 mg (70%); m.p. 159–162 °C; RF = 0.41 (SiO2, CHCl3/MeOH, 8:2); IR (ATR): ν = 3063w, 2956w, 2929m, 2858w, 1716m, 1646m, 1584s, 1529m, 1507m, 1481m, 1466s, 1435m, 1411s, 1395m, 1334s, 1272s, 1245s, 1197m, 1177s, 1160s, 1130s, 1072s, 1008m, 977m, 922m, 824m, 758m, 707m, 681s, 667m, 579 m cm−1; 1H NMR (400 MHz, CDCl3): δ = 8.28 (dd, J = 7.8, 1.3 Hz, 1H, 9H), 7.80 (dt, J = 7.5, 1.4 Hz, 1H, 11H), 7.73 (dt, J = 7.7, 1.4 Hz, 1H, 10H), 7.30 (dd, J = 7.5, 1.3 Hz, 1H, 12H), 7.07 (d, J = 9.4 Hz, 2H, 16H + 16′H), 6.90 (dd, J = 9.4, 2.2 Hz, 2H, 17H + 17′H), 6.82 (d, J = 2.3 Hz, 2H, 19H + 19′H), 3.99 (t, J = 6.6 Hz, 2H, 6H), 3.64 (q, J = 7.2 Hz, 8H, 21H + 21′H + 21′′H+21′′′H), 1.43–1.35 (m, 2H, 5H), 1.32 (t, J = 7.1 Hz, 12H, 22H + 22′H + 22′′H + 22′′′H), 1.28–1.07 (m, 6H, 4H + 3H + 2H), 0.82 (t, J = 6.9 Hz, 3H, 1H) ppm; 13C NMR (101 MHz, CDCl3): δ = 165.2 (C-7), 158.9 (C-8), 157.8 (C-20 + C-20′), 155.6 (C-18 + C-18′), 133.4 (C-13), 133.0 (C-11), 131.3 (C-16 + C-16′), 131.3 (C-9), 130.4 (C-10), 130.3 (C-12), 130.2 (C-14), 114.3 (C-17 + C-17′), 113.6 (C-15 + C-15′), 96.4 (C-19 + C-19′), 65.8 (C-6), 46.2 (C-21+C-21′ + C-21′′ + C-21′′′), 31.3 (C-3), 28.3 (C-5), 25.5 (C-4), 22.4 (C-2), 14.0 (C-1), 12.7 (C-22 + C-22′ + C-22′′ + C-22′′′) ppm; MS (ESI, MeOH): m/z = 527.5 (100%, [M]+); analysis calcd for C34H43ClN2O3 (563.18): C 72.51, H 7.70, N 4.97; found: C 72.38, H 7.96, N 4.73.
3,6-Bis(diethylamino)-9[2-(eicosyloxy)carbonyl]-xanthylium chloride (3)
Yield: 362 mg (48%); m.p. 144–146 °C; RF = 0.38 (SiO2, CHCl3/MeOH, 9:1); IR (ATR): ν = 2919m, 2850m, 1721m, 1645m, 1586s, 1553m, 1528m, 1489m, 1467s, 1433m, 1412s, 1396m, 1381m, 1333s, 1272s, 1249s, 1198m, 1179s, 1160m, 1132s, 1075s, 1040m, 1011m, 974m, 923m, 824m, 708m, 682s, 668 m cm−1; 1H NMR (500 MHz, CDCl3): δ = 8.29–8.25 (m, 1H, 23H), 7.79 (dt, J = 7.6, 1.4 Hz, 1H, 25H), 7.72 (dt, J = 7.7, 1.3 Hz, 1H, 24H4), 7.30 (dd, J = 7.6, 1.3 Hz, 1H, 26H), 7.06 (d, J = 9.3 Hz, 2H, 30H + 30′H), 6.86 (d, J = 2.5 Hz, 2H, 21H + 21′H), 6.85–6.83 (m, 2H, 33H + 33′H), 3.98 (t, J = 6.7 Hz, 2H, 20H), 3.62 (q, J = 7.4 Hz, 8H, 35H + 35′H + 35′′H + 35′′′H), 1.42–1.37 (m, 2H, 18Ha + 18Hb), 1.31 (t, J = 7.1 Hz, 12H, 36H + 36′H + 36′′H + 36′′′H), 1.29–1.26 (m, 2H, 2Ha + 2Hb), 1.24–1.14 (m, 28H, 3Ha + 3Hb + 4H–17H), 1.13–1.07 (m, 2H, 19Ha + 19Hb), 0.86 (t, J = 6.9 Hz, 3H, 1H) ppm; 13C NMR (126 MHz, CDCl3): δ = 165.2 (C-21), 158.9 (C-22), 157.8 (C-34 + C-34′), 155.6 (C-32 + C-32′), 133.4 (C-27), 133.0 (C-25), 131.3 (C-30 + C-30′), 131.2 (C-23), 130.3 (C-24), 130.3 (C-26), 130.2 (C-28), 114.1 (C-21 + C-21′), 113.6 (C-29 + C-29′), 96.5 (C-33 + C-33′), 65.8 (C-20), 46.1 (C-35 + C-35′ + C-35′′ + C-35′′′), 31.9 (C-3), 29.7–29.2 (C-4–C-17), 28.3 (C-18), 25.8 (C-19), 22.7 (C-2), 14.1 (C-1), 12.6 (C-36+C-36′ + C-36′′ + C-36′′′) ppm; MS (ESI, MeOH): m/z = 723.7 (100%, [M]+); analysis calcd for C48H71ClN2O3 (759.56): C 75.90, H 9.42, N 3.69; found: C 75.75, H 9.61, N 3.55.
3,6-Bis(diethylamino)-9[2-(1-morpholinyl)carbonyl]-xanthylium chloride (4)
Yield: 503 mg (92%); m.p. >250 °C; RF = 0.34 (SiO2, CHCl3/MeOH, 9:1); IR (ATR): ν = 3356w, 3060w, 2976w, 2933w, 2871w, 2795w, 2605w, 2498w, 1644m, 1624m, 1584s, 1528m, 1508m, 1481s, 1465s, 1446s, 1431s, 1411s, 1394s, 1334s, 1271s, 1245s, 1196m, 1178s, 1161s, 1132s, 1094m, 1071s, 1008m, 976m, 921m, 822m, 740s, 682s, 655m, 657 m cm−1; 1H NMR (500 MHz, CDCl3): δ = 7.65–7.62 (m, 2H, 7H + 8H), 7.50–7.47 (m, 1H, 5H), 7.32–7.29 (m, 1H, 6H), 7.19 (d, J = 9.5 Hz, 2H, 12H + 12′H), 6.95 (dd, J = 9.6, 2.4 Hz, 2H, 13H + 13′H), 6.73 (d, J = 2.5 Hz, 2H, 15H + 15′H), 3.60 (q, J = 7.2 Hz, 8H, 17H + 17′H + 17′′H + 17′′′H), 3.45–3.40 (m, 4H, 1H + 1′H), 3.39–3.29 (m, 4H, 2H + 2′H), 1.28 (t, J = 7.1 Hz, 12H, 18H + 18′H + 18′′H + 18′′′H) ppm; 13C NMR (126 MHz, CDCl3): δ = 167.4 (C-3), 157.7 (C-14), 155.7 (C-4), 155.6 (C-16 + C-16′), 135.0 (C-9), 132.0 (C-12 + C-12′), 130.7 (C-10), 130.3 (C-7), 130.2 (C-6), 130.1 (C-8), 127.6 (C-5), 114.2 (C-13 + C-13′), 113.7 (C-11 + C-11′), 96.3 (C-15 + C-15′), 66.6 (C-1), 48.0 (C-2a), 46.2 (C-17 + C-17′ + C-17′′ + C-17′′′), 42.2 (C-2b), 12.6 (C-18 + C-18′ + C-18′′ + C-18′′′) ppm; MS (ESI, MeOH): m/z = 512.3 (100%, [M]+); analysis calcd for C32H38ClN3O3 (548.12): C 70.12, H 6.99, N 7.67; found: C 69.84, H 7.17, N 7.49.
3,6-Bis(diethylamino)-9[2-(1-thiomorpholinyl)carbonyl]-xanthylium chloride (5)
Yield: 471 mg (83%); m.p. 233 °C; RF = 0.35 (SiO2, CHCl3/MeOH, 9:1); IR (ATR): ν = 3311w, 3060w, 2972w, 2906w, 1628m, 1585s, 1527m, 1508m, 1481m, 1467s, 1411s, 1394m, 1335s, 1305m, 1273s, 1245s, 1178s, 1131s, 1093m, 1072s, 1049m, 1036m, 1008m, 951m, 920m, 742m, 682s, 665 m cm−1; 1H NMR (500 MHz, CDCl3): δ = 7.64–7.60 (m, 2H, 7H7 + 8H), 7.48–7.45 (m, 1H, 5H), 7.29–7.27 (m, 1H, 6H), 7.17 (d, J = 9.5 Hz, 2H, 12H + 12′H), 6.93 (dd, J = 9.5, 2.4 Hz, 2H, 13H + 13′H), 6.72 (d, J = 2.5 Hz, 2H, 15H + 15′H), 3.59 (qd, J = 7.3, 2.1 Hz, 8H, 17H + 17′H + 17′′H + 17′′′H), 3.58–3.46 (m, 4H, 2H + 2′H), 2.47–2.37 (m, 4H, 1H + 1′H), 1.27 (t, J = 7.1 Hz, 12H, 18H + 18′H + 18′′H + 18′′′H) ppm; 13C NMR (126 MHz, CDCl3): δ = 167.7 (C-3), 157.7 (C-14), 155.6 (C-16 + C-16′), 155.6 (C-4), 135.3 (C-9), 132.1 (C-12 + C-12′), 130.5 (C-10), 130.3 (C-7), 130.2 (C-6), 130.0 (C-8), 127.3 (C-5), 114.2 (C-13 + C-13′), 113.7 (C-11 + C-11′), 96.3 (C-15 + C-15′), 50.0 (C-2a), 46.2 (C-17 + C-17′ + C-17′′ + C-17′′′), 44.1 (C-2b), 27.6 (C-1), 12.6 (C-18 + C-18′ + C-18′′ + C-18′′′) ppm; MS (ESI, MeOH): m/z = 528.4 (100%, [M]+); analysis calcd for C32H38ClN3O2S (564.19): C 68.13, H 6.79, N 7.45, S 5.68; found: C 67.90, H 6.82, N 7.29, S 5.56.
3,6-Bis(diethylamino)-9[2-(1-(2,6-dimethylmorpholinyl)carbonyl)]-xanthylium chloride (6)
Yield: 483 mg (84%); m.p. 179–181 °C; RF = 0.38 (SiO2, CHCl3/MeOH, 9:1); IR (ATR): ν = 2974w, 2933w, 2871w, 1644m, 1629m, 1585s, 1528m, 1507m, 1481m, 1467m, 1455m, 1431m, 1411s, 1394m, 1331s, 1272s, 1245s, 1196m, 1178s, 1159s, 1131s, 1094m, 1071s, 1039m, 1009m, 975m, 920m, 823m, 682s, 666 m cm−1; 1H NMR (500 MHz, CDCl3): δ = 7.69–7.65 (m, 2H, 7H + 8H), 7.51–7.48 (m, 1H, 5H), 7.36–7.33 (m, 1H, 6H), 7.26–7.19 (m, 2H, 12H + 12′H), 6.96–6.87 (m, 2H, 13H + 13′H), 6.83–6.79 (m, 2H, 15H + 15′H), 4.12 (d, J = 13.2 Hz, 1H, 2Ha), 3.66–3.57 (m, 8H, 17H + 17′H + 17′′H + 17′′′H), 3.42 (d, J = 12.7 Hz, 1H, 2′Ha), 3.22–3.12 (m, 2H, 1H), 3.09–3.00 (m, 2H, 1′H), 2.58 (t, J = 11.9 Hz, 1H, 2H′b), 2.21 (t, J = 11.6 Hz, 1H, 2Hb), 1.30 (t, J = 7.1 Hz, 12H, 18H + 18′H+18′′H + 18′′′H), 1.08–0.98 (m, 6H, 19H + 19′H) ppm; 13C NMR (126 MHz, CDCl3): δ = 167.3 (C-3), 157.7 (C-14), 155.8 (C-16), 155.6 (C-16′), 155.5 (C-4), 135.2 (C-9), 132.4 (C-12), 131.9 (C-12′), 130.3 (C-10), 130.3 (C-7), 130.3 (C-6), 130.0 (C-8), 127.6 (C-5), 114.1 (C-13), 113.9 (C-13′), 113.7 (C-11), 113.6 (C-11′), 96.5 (C-15 + C-15′), 71.9 (C-1), 71.7 (C-1′), 52.9 (C-2), 47.1 (C-2′), 46.1 (C-17 + C-17′ + C-17′′ + C-17′′′), 18.6 (C-19), 18.4 (C-19′), 12.60 (C-18 + C-18′ + C-18′′ + C-18′′′) ppm; MS (ESI, MeOH): m/z = 540.4 (100%, [M]+); analysis calcd for C34H42ClN3O3 (576.18): C 70.88, H 7.35, N 7.29; found: C 70.59, H 7.53, N 7.01.
3,6-Bis(diethylamino)-9[2-(1-(4-methylpiperazinyl)carbonyl)]-xanthylium chloride (7)
Yield: 384 mg (68%); m.p. >250 °C; RF = 0.29 (SiO2, CHCl3/MeOH, 9:1); IR (ATR): ν = 3061w, 2975w, 2899w, 2869w, 2717w, 2468w, 1629m, 1583s, 1528m, 1508m, 1481s, 1466s, 1411s, 1394s, 1334s, 1299m, 1272s, 1245s, 1196m, 1177s, 1159s, 1131s, 1113s, 1093m, 1071s, 1009s, 976m, 921m, 822m, 739s, 681s, 656 m cm−1; 1H NMR (500 MHz, CDCl3): δ = 7.74–7.70 (m, 1H, 5H), 7.64 (dt, J = 7.6, 1.4 Hz, 1H, 7H), 7.60 (dt, J = 7.5, 1.4 Hz, 1H, 8H), 7.26 (dd, J = 7.5, 1.4 Hz, 1H, 6H), 7.14 (d, J = 9.5 Hz, 2H, 12H + 12′H), 6.89 (dd, J = 9.6, 2.4 Hz, 2H, 13H + 13′H), 6.69 (d, J = 2.4 Hz, 2H, 15H + 15′H), 3.65–3.50 (m, 8H, 17H + 17′H + 17′′H + 17′′′H), 3.51–3.43 (m, 4H, 2H + 2′H), 2.71–2.54 (m, 4H, 1H + 1′H), 2.52–2.43 (m, 3H, 19H), 1.26 (t, J = 7.2 Hz, 12H, 18H + 18′H + 18′′H + 18′′′H) ppm; 13C NMR (126 MHz, CDCl3): δ = 167.3 (C-3), 157.7 (C-14), 156.0 (C-4), 155.6 (C-16 + C-16′), 134.9 (C-9), 131.9 (C-12 + C-12′), 130.6 (C-10), 130.3 (C-7), 130.1 (C-6), 129.9 (C-8), 128.1 (C-5), 114.1 (C-13 + C-13′), 113.7 (C-11 + C-11′), 96.2 (C-15 + C-15′), 53.4 (C-1 + C-1′), 46.1 (C-17 + C-17′ + C-17′′ + C-17′′′), 44.8 (C-19), 40.5 (C-2 + C-2′), 12.6 (C-18 + C-18′ + C-18′′ + C-18′′′) ppm; MS (ESI, MeOH): m/z = 525.5 (100%, [M]+); analysis calcd for C33H41ClN4O2 (561.17): C 70.63, H 7.36, N 9.98; found: C 70.44, H 7.56, N 9.63.
3,6-Bis(diethylamino)-9[2-(diethylamino)carbonyl]-xanthylium chloride (8)
Yield: 472 mg (88%); m.p. 139–141 °C; RF = 0.34 (SiO2, CHCl3/MeOH, 9:1); IR (ATR): ν = 3356w, 3061w, 2971m, 2932w, 2873w, 1646m, 1615s, 1586s, 1553m, 1527m, 1509m, 1481m, 1465s, 1450m, 1430s, 1412s, 1395s, 1379s, 1335s, 1274s, 1247s, 1196m, 1178s, 1131s, 1076s, 1009s, 978m, 959m, 921m, 855m, 821m, 789m, 770m, 761m, 682s, 666m, 626m, 618 m cm−1; 1H NMR (400 MHz, CDCl3): δ = 7.58–7.53 (m, 2H, 5H + 6H), 7.44–7.40 (m, 1H, 7H), 7.27–7.24 (m, 1H, 8H), 7.15 (d, J = 9.5 Hz, 2H, 12H + 12′H), 6.86 (dd, J = 9.6, 2.5 Hz, 2H, 13H + 13′H), 6.66 (d, J = 2.5 Hz, 2H, 15H + 15′H), 3.55 (q, J = 7.2 Hz, 8H, 17H + 17′H + 17′′H + 17′′′H), 3.06 (dq, J = 14.4, 7.0 Hz, 4H, 2H + 2′H), 1.21 (t, J = 7.1 Hz, 12H, 18H + 18′H + 18′′H + 18′′′H), 0.99 (t, J = 7.0 Hz, 3H, 1H), 0.52 (t, J = 7.0 Hz, 3H, 1′H) ppm; 13C NMR (101 MHz, CDCl3): δ = 167.8 (C-3), 157.6 (C-4), 155.9 (C-16 + C-16′), 155.5 (C-14 + C-14′), 136.6 (C-9), 132.1 (C-7), 130.2 (C-12+C-12′), 130.0 (C-5), 129.9 (C-6), 129.4 (C-8), 126.5 (C-10), 113.9 (C-13 + C-13′), 113.6 (C-11 + C-11′), 96.1 (C-15 + C-15′), 46.1 (C-17 + C-17′ + C-17′′ + C-17′′′), 43.4 (C-2), 38.3 (C-2′), 14.0 (C-1), 12.6 (C-18 + C-18′ + C-18′′ + C-18′′′), 11.6 (C-1′) ppm; MS (ESI, MeOH): m/z = 498.4 (100%, [M]+); analysis calcd for C32H40ClN3O2 (534.14): C 71.96, H 7.55, N 7.87; found: C 71.70, H 7.78, N 7.69.
3,6-Bis(diethylamino)-9[2-(1-piperazinyl)carbonyl]-xanthylium chloride (9)
Yield: (724 mg, 67%); m.p. > 250 °C; RF = 0.14 (chloroform/methanol, 8:2); IR (ATR) v = 3401br, 1589m, 1529w, 1411s, 1328s, 1275s, 1246m, 1180s, 1132m, 1074m, 1011w, 977m, 922m, 820m, 683m; 1H NMR (500 MHz, CD3OD): δ = 7.79–7.74 (m, 3H, 3H + 4H + 5H), 7.52 (m, 1H, 6H), 7.28–7.25 (d, 1H, 10H), 7.10–7.09 (m, 1H, 11H), 6.98–6.97 (d, 1H, 13H), 3.72–3.59 (m, 6H, 15Ha + 15Hb + 17Ha+17Hb+20Ha+20Hb), 3.08–3.05 (t, 4H, 18Ha + 18Hb + 19Ha + 19Hb), 1.33–1.30 (t, 3H, 16Ha + 16Hb + 16Hc) ppm; 13C NMR (126 MHz, CD3OD): δ = 169.53 (C-1), 159.2 (C-8), 157.3 (C-12), 156.7 (C-14), 135.7 (C-7), 133.0 (C-10), 132.3 (C-2), 131.8 (C-6), 131.5 (C-5), 131.4 (C-4), 128.9 (C-3), 115.4 (C-11), 114.8 (C-9), 97.4 (C-13), 46.9 (C-15), 46.8 (C-17+C-20), 44.5 (C-18+C-19), 12.8 (C-16) ppm; MS (ESI, MeOH): m/z = 256.2 (24%, [M + H]2+), 511.4 (100%, [M]+); analysis calcd for C32H39ClN4O2 (547.14): C 70.25, H 7.18, N 10.24; found: C 70.01, H 7.34, N 10.02.
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Acknowledgements
We like to thank Dr. D. Ströhl and his team for the NMR spectra and the late Dr. R. Kluge for measuring the MS spectra. Many thanks are also due to V. Simon and M. Schneider for taking the IR spectra. The microanalyses have been determined by S. Ludwig and M. Schneider. The cell lines have been provided by the Department of Oncology (Martin-Luther-University Halle Wittenberg). Open Access funding provided by Projekt DEAL.
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Serbian, I., Hoenke, S., Kraft, O. et al. Ester and amide derivatives of rhodamine B exert cytotoxic effects on different human tumor cell lines. Med Chem Res 29, 1655–1661 (2020). https://doi.org/10.1007/s00044-020-02591-8
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DOI: https://doi.org/10.1007/s00044-020-02591-8