Ester and amide derivatives of rhodamine B exert cytotoxic effects on different human tumor cell lines

Article abstract of DOI:10.1007/s00044-020-02591-8

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. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00044-020-02591-8

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02591-8
ORIGINAL RESEARCH
Ester and amide derivatives of rhodamine B exert cytotoxic effects
on different human tumor cell lines
1
Immo Serbian1 Sophie Hoenke1 Oliver Kraft1 René Csuk
●
●
●
Received: 15 April 2020 / Accepted: 11 June 2020
© The Author(s) 2020
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.
Graphical Abstract
●
●
●
●
Keywords Rhodamine B Rhodamine B amide Rhodamine B esters Cytotoxicity SRB assay
Introduction
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 cyto-
toxic. Several of these “mitocanic” conjugates held an even
nanomolar activity for human tumor cell lines (Sommer-
werk 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, mito-
chondria 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
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 pre-
viously been used to selectively direct cytotoxic compounds
into mitochondria (Kahnt et al. 2018; Sommerwerk et al.
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02591-8) contains supplementary
material, which is available to authorized users.
* René Csuk
rene.csuk@chemie.uni-halle.de
1
Martin-Luther-University Halle-Wittenberg, Organic Chemistry,
Kurt-Mothes-Str. 2, D-06120 Halle (Saale), Germany

Medicinal Chemistry Research
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 ethylene-
diamine 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).
7
6
5
4
3
2
1
0
FADU
MCF-7
A375
Rhho
1
2
3
4
5
6
7
8
9
STA
A375
HT29
MCF-7
A2780
FADU
NIH 3T3
Fig. 1 Cytotoxicity of compounds 1–9, STA and Rho; 3D bar chart
representation (cutoff at 7 µM)
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 solu-
bility, 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).
Scheme 1 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,
azine, 0 °C, 30 min
N-diethylamine/piper-
Results and discussion
>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 selec-
tivity (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.
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.
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
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

Medicinal Chemistry Research
Table 1 Cytotoxicity of
Compound
A375
HT29
MCF-7
A2780
FADU
NIH 3T3
compounds 1-9 and rhodamine
B (Rho) (EC50 values in μM
from SRB assays after 72 h of
treatment, the values are
averaged from three independent
experiments performed each in
triplicate, confidence interval CI
= 95%; mean standard mean
error, cutoff 30 µM)
Rho
>30
>30
>30
>30
>30
>30
1
0.38 0.02
0.19 0.01
>20
0.41 0.04
0.19 0.03
>20
0.23 0.03
0.15 0.02
>20
0.21 0.01
0.17 0.01
>20
0.30 0.02
0.15 0.01
>20
0.96 0.05
0.32 0.02
>20
2
3
4
7.09 0.3
1.79 0.1
3.05 0.20
16.05 0.6
1.03 0.03
>30
5.46 0.2
1.54 0.1
1.74 0.04
17.34 0.8
0.54 0.02
>30
1.54 0.3
0.44 0.1
0.49 0.08
3.74 0.3
0.32 0.04
17.8 3.9
0.1 0.01
1.66 0.1
0.52 0.03
0.70 0.14
3.62 0.2
0.27 0.01
26.4 2.1
0.1 0.05
4.53 0.2
1.12 0.1
1.52 0.10
11.78 0.5
0.64 0.03
>30
>30
5
5.09 0.2
7.92 0.30
>30
6
7
8
3.27 0.08
>30
9
STA
0.2 0.02
0.1 0.01
0.008 0.001
0.2 0.02
Human cancer cell lines: A375 (epithelial melanoma), HT29 (colorectal adenocarcinoma), MCF-7 (breast
adenocarcinoma), A2780 (ovarian carcinoma), FaDu (squamous cell carcinoma); nonmalignant: NIH 3T3
(mouse fibroblasts). Staurosporine (STA) was used as a positive standard
Table 2 Selectivity factors of compounds 1–9 with Stumor cell line = EC50
(NIH 3T3) / EC50 (tumor cell line)
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 (Som-
merwerk 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. Further-
more, 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.
Compound
A375
HT29
MCF-7
A2780
FaDu
1
2
4
5
6
7
8
9
2.5
1.7
>4.2
2.8
2.6
>1.9
3.2
–
2.3
1.7
>5.5
3.3
4.6
>1.7
6.1
–
4.2
2.1
4.6
1.9
3.2
2.1
>6.6
4.5
5.2
>2.5
5.1
–
>19.5
11.6
16.2
>8.0
10.2
>1.7
>18.1
9.8
11.3
>8.3
12.1
>1.1
rhodamine B amides were slightly less cytotoxic. Interest-
ingly 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 con-
cerned, 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
Ongoing investigations will provide evidence whether
these compounds are able to trigger permeabilization of the
mitochondrial membrane due to a change in the mito-
chondrial membrane potential or to interfere with the
mitochondrial permeability transition pore.

Medicinal Chemistry Research
Experimental
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.
The equipment as well as the details of the cytotoxic eva-
luation 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
3,6-Bis(diethylamino)-9[2-(hexyloxy)carbonyl]-xanthylium
chloride (2)
General procedure for the synthesis of the rhodamine B
esters 1–3
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,
Rhodamine B acyl chloride (500 mg, 1.0 mmol) was dis-
solved 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.
1
824m, 758m, 707m, 681s, 667m, 579 m cm−1; H 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.
General procedure for the synthesis of the rhodamine B
amides 4–9
Rhodamine B acyl chloride (500 mg, 1.0 mmol) was dis-
solved 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 pres-
sure, and the residue was purified by column chromato-
graphy (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;
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,

Medicinal Chemistry Research
1721m, 1645m, 1586s, 1553m, 1528m, 1489m, 1467s,
1433m, 1412s, 1396m, 1381m, 1333s, 1272s, 1249s,
1198m, 1179s, 1160m, 1132s, 1075s, 1040m, 1011m,
3,6-Bis(diethylamino)-9[2-(1-thiomorpholinyl)carbonyl]-
xanthylium chloride (5)
1
974m, 923m, 824m, 708m, 682s, 668 m cm−1; H NMR
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,
(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.
1
665 m cm−1; H 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)
3,6-Bis(diethylamino)-9[2-(1-morpholinyl)carbonyl]-
xanthylium chloride (4)
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,
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,
1
1009m, 975m, 920m, 823m, 682s, 666 m cm−1; H 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
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.

Medicinal Chemistry Research
C34H42ClN3O3 (576.18): C 70.88, H 7.35, N 7.29; found: C
70.59, H 7.53, N 7.01.
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-(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,
3,6-Bis(diethylamino)-9[2-(1-piperazinyl)carbonyl]-
xanthylium chloride (9)
Yield: (724 mg, 67%); m.p. > 250 °C; RF = 0.14 (chloro-
form/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.
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.
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.
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,
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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 +
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons license, and indicate if
changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons license and your intended
use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this license, visit http://creativecommons.
org/licenses/by/4.0/.

Medicinal Chemistry Research
References
synergistic nature of image-based and biochemical approaches.
Histochemistry 94:303–308
Rokitskaya TI, Klishin SS, Severina II, Skulachev VP, Antonenko YN
(2008) Kinetic analysis of permeation of mitochondria-targeted
antioxidants across bilayer lipid membranes. J Membr Biol
224:9–19
Rokitskaya TI, Korshunova GA, Antonenko YN (2018) Effect of alkyl
chain length on translocation of rhodamine B n-alkyl esters across
lipid membranes. Biophys J 115:514–521
Shakurova ER, Pozdnyakova DA, Tretyakova EV, Parfenova LV
(2020) One-pot synthesis of betulin triterpenoid quaternized
pyridine derivatives and their antimicrobial activity. Lett Drug
Des Discov 17:79–84
Sodano F, Gazzano E, Fraix A, Rolando B, Lazzarato L, Russo M,
Blangetti M, Riganti C, Fruttero R, Gasco A, Sortino S (2018) A
molecular hybrid for mitochondria-targeted NO photodelivery.
ChemMedChem 13:87–96
Beija M, Afonso CAM, Farinha JPS, Charreyre M-T, Martinho JMG
(2011) Novel Malachite Green- and Rhodamine B-labeled
cationic chain transfer agents for RAFT polymerization. Poly-
mer 52:5933–5946
Bui DT, Nicolas J, Maksimenko A, Desmaele D, Couvreur P (2014)
Multifunctional squalene-based prodrug nanoparticles for tar-
geted cancer therapy. Chem Commun 50:5336–5338
Costantini P, Jacotot E, Decaudin D, Kroemer G (2000) Mitochon-
drion as a novel target of anticancer chemotherapy. J Natl Cancer
Inst 92:1042–1053
Csuk R, Deigner H-P (2019) The potential of click reactions for the
synthesis of bioactive triterpenes. Bioorg Med Chem Lett 29:949–958
Dauner M, Batroff E, Bachmann V, Hauck CR, Wittmann V (2016)
Synthetic Glycosphingolipids for Live-Cell Labeling. Bioconju-
gate Chem 27:1624–1637
Sommerwerk S, Heller L, Kerzig C, Kramell AE, Csuk R (2017)
Rhodamine B conjugates of triterpenoic acids are cytotoxic
mitocans even at nanomolar concentrations. Eur J Med Chem
127:1–9
Del Secco B, Malachin G, Milli L, Zanna N, Papini E, Cornia A,
Tavano R, Tomasini C (2017) Form matters: stable helical fol-
damers preferentially target human monocytes and granulocytes.
ChemMedChem 12:337–345
Song S, Gao K, Niu R, Yi W, Zhang J, Gao C, Yang B, Liao X (2019)
Binding behavior, water solubility and in vitro cytotoxicity of
inclusion complexes between ursolic acid and amino-appended
β-cyclodextrins. J Mol Liq 296:111993
Talib AJ, Fisher A, Voronine DV, Sinyukov AM, Bustamante Lopez
SC, Ambardar S, Meissner KE, Scully MO, Sokolov AV (2019)
Fluorescence imaging of stained red blood cells with simulta-
neous resonance Raman photostability analysis. Analyst
144:4362–4370
Fulda S (2010) Exploiting mitochondrial apoptosis for the treatment of
cancer. Mitochondrion 10:598–603
Galluzzi L, Larochette N, Zamzami N, Kroemer G (2006) Mito-
chondria as therapeutic targets for cancer chemotherapy. Onco-
gene 25:4812–4830
Gogvadze V, Orrenius S, Zhivotovsky B (2009a) Mitochondria as
targets for cancer chemotherapy. Semin Cancer Biol 19:57–66
Gogvadze V, Orrenius S, Zhivotovsky B (2009b) Mitochondria as
targets for chemotherapy. Apoptosis 14:624–640
Tansil NC, Li Y, Koh LD, Peng TC, Win KY, Liu XY, Han M-Y
(2011) The use of molecular fluorescent markers to monitor
absorption and distribution of xenobiotics in a silkworm model.
Biomaterials 32:9576–9583
Guo R, Yin J, Ma Y, Wang Q, Lin W (2018) A novel mitochondria-
targeted rhodamine analogue for the detection of viscosity
changes in living cells, zebra fish and living mice. J Mater Chem
B 6:2894–2900
Walters WP, Ajay, Murcko MA (1999) Recognizing molecules with
drug-like properties. Curr Opin Chem Biol 3:384–387
Wang D, Wang Z, Li Y, Song Y, Song Y, Zhang M, Yu H (2018) A
single rhodamine spirolactam probe for localization and pH
monitoring of mitochondrion/lysosome in living cells. N. J Chem
42:11102–11108
Wieker HJ, Kuschmitz D, Hess B (1987) Inhibition of yeast mito-
chondrial F1-ATPase, F0F1-ATPase and submitochondrial par-
ticles by rhodamines and ethidium bromide. Biochim Biophys
Acta Bioenerg 892:108–117
Johnson LV, Walsh ML, Chen LB (1980) Localization of mitochon-
dria in living cells with rhodamine-123. Proc Natl Acad Sci-Biol
77:990–994
Kahnt M, Wiemann J, Fischer L, Sommerwerk S, Csuk R (2018)
Transformation of asiatic acid into a mitocanic, bimodal-acting
rhodamine B conjugate of nanomolar cytotoxicity. Eur J Med
Chem 159:143–148
Mai MS, Allison WS (1983) Inhibition of an oligomycin-sensitive
ATPase by cationic dyes, some of which are atypical uncouplers
of intact mitochondria. Arch Biochem Biophys 221:467–476
May F, Peter M, Huetten A, Prodi L, Mattay J (2012) Synthesis and
characterization of photoswitchable fluorescent SiO2 nano-
particles. Chem Eur J 18:814–821
Wiemann J, Fischer L, Kessler J, Ströhl D, Csuk R (2018) Ugi mul-
ticomponent-reaction:
Syntheses
of
cytotoxic
dehy-
droabietylamine derivatives. Bioorg Chem 81:567–576
Wolfram RK, Fischer L, Kluge R, Ströhl D, Al-Harrasi A, Csuk R
(2018a) Homopiperazine-rhodamine B adducts of triterpenoic
acids are strong mitocans. Eur J Med Chem 155:869–879
Wolfram RK, Heller L, Csuk R (2018b) Targeting mitochondria:
Esters of rhodamine B with triterpenoids are mitocanic triggers of
apoptosis. Eur J Med Chem 152:21–30
Melikyan GB, Dieriy BN, Ok DC, Cohen FS (1996) Voltage-
dependent translocation of R18 and Dil across lipid bilayers leads
to fluorescence changes. Biophys J 71:2680–2691
Mottram LF, Forbes S, Ackley BD, Peterson BR (2012) Hydrophobic
analogues of rhodamine B and rhodamine 101: potent fluorescent
probes of mitochondria in living C. elegans. Beilstein J Org
Chem 8:2156–2165. No. 2243
Neuzil J, Dong L-F, Rohlena J, Truksa J, Ralph SJ (2013) Classifi-
cation of mitocans, anti-cancer drugs acting on mitochondria.
Mitochondrion 13:199–208
Xie C, Chang J, Hao X-D, Yu J-M, Liu H-R, Sun X (2013)
Mitochondrial-targeted prodrug cancer therapy using a rhodamine
B
labeled fluorinated docetaxel. Eur
J Pharm Biopharm
85:541–549
Yu B, Dong C-Y, So PTC, Blankschtein D, Langer R (2001) In vitro
visualization and quantification of oleic acid induced changes in
transdermal transport using two-photon fluorescence microscopy.
J Investig Dermatol 117:16–25
Zhang M, Shen C, Jia T, Qiu J, Zhu H, Gao Y (2020) One-step
synthesis of rhodamine-based Fe3+ fluorescent probes via
Mannich reaction and its application in living cell imaging.
Spectrochim Acta Part A 231:118105
Oprea TI (2002) Virtual screening in lead discovery: a viewpoint.
Molecules 7:51–62
Preston AN, Farr JD, O’Neill BK, Thompson KK, Tsirka SE, Laughlin
ST (2018) Visualizing the brain’s astrocytes with diverse che-
mical scaffolds. ACS Chem Biol 13:1493–1498
Rashid F, Horobin RW (1990) Interaction of molecular probes with
living cells and tissues. Part 2. A structure-activity analysis of
mitochondrial staining by cationic probes, and a discussion of the