Homopiperazine-rhodamine B adducts of triterpenoic acids are strong mitocans

Article abstract of DOI:10.1016/j.ejmech.2018.06.051

Parent pentacyclic triterpenoic acids such as ursolic-, oleanolic, glycyrrhetinic, betulinic and boswellic acid were converted into their acetylated piperazinyl amides that were coupled with rhodamine B. SRB assays to evaluate their cytotoxicity showed all of these triterpene-homopiperazinyl-rhodamine adducts 16–20 being highly cytotoxic for a panel of human tumor cell lines even in nanomolar concentrations while being significantly less cytotoxic for non-malignant cells. Interestingly enough, these compounds were even more cytotoxic than previously prepared piperazinyl analogs, thus making the homopiperazinyl spacer a very interesting scaffold for the development of biologically active compounds. Extra staining experiments showed that the cytostatic effect of compounds 18 and 20 onto A2780 cancer cells is due to their ability to act as a mitocan.

Full text of DOI:10.1016/j.ejmech.2018.06.051

Accepted Manuscript
Homopiperazine-rhodamine B adducts of triterpenoic acids are strong mitocans
Ratna Kancana Wolfram, Lucie Fischer, Ralph Kluge, Dieter Ströhl, Ahmed Al-
Harrasi, René Csuk
PII:
S0223-5234(18)30542-7
10.1016/j.ejmech.2018.06.051
EJMECH 10521
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 May 2018
Revised Date: 18 June 2018
Accepted Date: 22 June 2018
Please cite this article as: R.K. Wolfram, L. Fischer, R. Kluge, D. Ströhl, A. Al-Harrasi, René. Csuk,
Homopiperazine-rhodamine B adducts of triterpenoic acids are strong mitocans, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.06.051.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Homopiperazine-rhodamine B adducts of triterpenoic acids are strong mitocans
a
a,
a
a
Ratna Kancana Wolfram , Lucie Fischer (née Heller) Ralph Kluge , Dieter Ströhl ,
Ahmed Al-Harrasi b, René Csuk a*
a
Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str. 2, D-
06120 Halle (Saale), Germany
b
University of Nizwa, Chair of Oman’s Medicinal Plants and Marine Natural Products, P.O.
Box 33, PC 616, Birkat Al-Mauz, Nizwa, Sultanate of Oman
Dedicated to Prof. Dr. Andrea T. Vasella, ETH Zurich, on the occasion of his 75th birthday.
Ad multos annos!
Corresponding author:
Prof. Dr. René Csuk
Martin-Luther-University Halle-Wittenberg, Organic Chemistry
Kurt-Mothes-Str. 2,
D-06120 Halle (Saale), Germany
Tel.: +49 345 5525660
Fax: +49 345 27030
Email: rene.csuk@chemie.uni-halle.de
1

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Abstract
Parent pentacyclic triterpenoic acids such as ursolic-, oleanolic, glycyrrhetinic, betulinic and
boswellic acid were converted into their acetylated piperazinyl amides that were coupled with
rhodamine B. SRB assays to evaluate their cytotoxicity showed all of these triterpene-
homopiperazinyl-rhodamine adducts 16-20 being highly cytotoxic for a panel of human tumor
cell lines even in nanomolar concentrations while being significantly less cytotoxic for non-
malignant cells. Interestingly enough, these compounds were even more cytotoxic than
previously prepared piperazinyl analogs, thus making the homopiperazinyl spacer a very
interesting scaffold for the development of biologically active compounds. Extra staining
experiments showed that the cytostatic effect of compounds 18 and 20 onto A2780 cancer
cells is due to their ability to act as a mitocan.
Keywords: Homopiperazine, rhodamine B, mitocan; triterpenoic acids
1. Introduction
Cancer is still one of the most devastating diseases; approximately 8.9 million deaths in 2016
were caused by cancer.[1] Many therapies have been developed and help to cure patients in
early stages suffering from this disease or to prolong their lifespan for months or even years.
Much progress has been made especially in the development of small-molecule anticancer
drugs.[2, 3] However, many of them kill healthy cells as well as targeted cancer cells. In
search of higher active and more selective drugs, mitochondria have moved in the focus of
scientific interest. Mitochondrial functions of cancer cells differ from those of normal cells.[4]
As a consequence, mitochondria can be regarded as an interesting target for cancer therapy.[5,
6] Thus, the development of cytotoxic agents being able to impair mitochondrial functions
especially in cancer cells seems most rewarding. As a consequence, the development of
mitocans,[7-10] compounds that primarily target mitochondria, was called for.
Several derivatives of pentacyclic triterpenoids [11] showed an increased cytotoxic and a
significant anti-tumor activity both in vitro and in vivo.[11-17] Increased cytotoxicity,
however, was observed for those molecules holding an additional amino group (in)-directly
attached to the carbon skeleton of the triterpenoid.[18-22] Thereby, compounds coupled to a
piperazinyl residue showed significantly better anticancer activity than their parent
compounds.[23-27] Triterpenoids, however, holding both a piperazinyl spacer [28, 29] as well
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as a rhodamine B moiety attached to it were shown to act as mitocanic agents of high
cytotoxicity.[17, 30] Interestingly enough, while SciFinder (as of 2018, May) reports 157
pentacyclic triterpenoids with a piperazinyl moiety,[31-36] there is only one report dealing
with the synthesis and cytotoxic evaluation of ursolic acid derivatives holding a
homopiperazinyl moiety instead of a piperazinyl group.[29] Hence, we became interested in
compounds holding a homopiperazinyl moiety together with a rhodamine B group, and to
investigate their ability to act as mitocanic agents.
2. Results and discussion
2.1. Chemistry
The starting materials (Fig. 1), ursolic acid (1), oleanolic acid (2), glycyrrhetinic acid (3) and
betulinic acid (4) were purchased from commercial suppliers in bulk. 3-O-Acetyl-11-keto-β-
boswellic acid (5) was extracted from the resin of frankincense applying a modification of
Jauch’s procedure.[37] Deacetylation of 5 with an aqueous solution of sodium hydroxide in
EtOH gave 11-keto-β-boswellic acid (10) in almost quantitative yield.
Fig. 1. Structure of parent triterpenoic acids: ursolic acid (1), oleanolic acid (2), glycyrrhetinic
acid (3), betulinic acid (4), 3-O-acetyl-11-keto-β-boswellic acid (5).
Scheme 1 shows the coupling reaction of the triterpenoic acids with homopiperazine. Thus, to
avoid side reactions prior to the coupling of the corresponding triterpenoic acid chloride to
homopiperazine, the HO-C(3) was protected as an acetate. Acetylation of 1-4 with acetic
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anhydride gave compounds 6-9 in good yields (70-90%). Activation of acetylated compounds
5-9 with oxalyl chloride followed by their reactions with homopiperazine gave compounds
11-15 in moderate to good isolated yields (48-95%).
To obtain the desired rhodamine B derivatives, commercially available rhodamine B was
activated by its reaction with oxalyl chloride, and the resulting acid chloride was coupled with
the corresponding triterpenoic acid homopiperazine derivative 11-15 to yield final target
compounds 16-20 in 50-83% isolated yield.
Scheme 1. Synthesis of triterpenoic acid homopiperazine derivatives. Reaction conditions: a)
Ac2O, reflux, 2 h, 70–90%, b) (COCl)2, CH2Cl2, r.t., 3 h, then homopiperazine, NEt3, cat.
DMAP, CH2Cl2, r.t., 24 h, 48–99%, c) Rhd-CO2H, (COCl)2, CH2Cl2, r.t., 3 h, then NEt3, cat.
DMAP, CH2Cl2, r.t., 24 h, 50–83%.
Previous studies have shown that triterpenoic acids holding a rhodamine B moiety directly
attached to the triterpene skeleton are highly cytotoxic but possess a significantly lower
tumor/non-tumor selectivity [17] than derivatives where the triterpene and the rhodamine are
separated by a piperazine spacer.[30] To verify if this holds true also for previously not
investigated 11-keto-β-boswellic acid derivatives, the synthesis of some representative 11-
keto-boswellic acid derived compounds was undertaken. Compound 5 is an ideal starting
material (Scheme 2), and its reaction in dry DMF with methyl iodide or benzyl bromide in the
presence of finely grounded K2CO3 gave the methyl ester 21 and the benzyl ester 22,
respectively.[38]
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Scheme 2. Synthesis of 11-keto-β-boswellic acid derivatives 10, 21-27: a) NaOH, EtOH, r.t.,
12 h; b) MeI, K2CO3, DMF, r.t., 24 h, 92%; c) BnBr, K2CO3, DMF, r.t., 24 h, 57%; d) Rhd-
CO2H, (COCl)2, CH2Cl2, r.t., 3 h, then NEt3, DMAP, CH2Cl2, r.t., 24 h, 25 43%, 26 58%;
Reaction of 5 with oxalyl chloride followed by the addition of benzyl amine (Scheme 3)
furnished the N-benzyl amide 23 in good yield. Deacetylation of 23 with KOH in MeOH at
ambient temperature gave 24. Finally, coupling of 21, 22 or 24 with activated rhodamine B as
described above gave the rhodamine B esters 25-27, respectively.
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Scheme 3: a) (COCl)2, CH2Cl2, r.t., 3 h, then BnNH2, NEt3, DMAP, CH2Cl2, r.t., 24 h, 80%;
b) KOH, MeOH, r.t., 3 d, 76%; c) Rhd-CO2H, (COCl)2, CH2Cl2, r.t., 3 h, then NEt3, DMAP,
CH2Cl2, r.t., 24 h, 57%.
2.2.Biology
The cytotoxicity of the compounds was evaluated in sulforhodamine B assays (SRB), and the
results are summarized in Table 1.
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Table 1. Cytotoxicity of selected compounds (EC50 values in µM from SRB assays after 96 h
of treatment, the values are averaged from three independent experiments performed each in
triplicate, confidence interval CI = 95%, cut-off the assay 30 µM, n.m. not measured; mean ±
standard mean error). Human cancer cell lines: A375 (epithelial melanoma), A2780 (ovarian
carcinoma), HT29 (colorectal adenocarcinoma), MCF7 (breast adenocarcinoma), SW1736
(thyroidea carcinoma); non-malignant: NIH 3T3 (mouse fibroblasts); data for compounds 1-5
and 21 are taken from literature.[30, 39-41] Betulinic acid (BA) was used as a standard.
Compound
A375
n. m.
n. m.
n. m.
A2780
HT29
MCF7
NiH3T3
18.7±1.6
76.4±0.7
18.52±0.93
SW1736
n. m.
11.7±0.6
14.0±2.3
74.57±3.73
10.6±0.7
38.8±3.1
80.09±4.00
12.7±0.1
>60
1
2
3
n. m.
84.70±4.24
76.93±3.85
n. m.
n. m.
8.8±0.9
14.4±2.3
19.4±1.1
10.2±1.2
17.4±1.7
16.1±1.4
26.4±3.0
n. m.
n. m.
4
5
14.4±2.0
3.21±0.17
1.95±0.87
2.30±0.14
18.66±1.63
n. m.
2.85±0.24
2.21±0.06
2.31±0.23
12.0±0.62
1.61±0.08
0.45±0.03
0.02±0.003
0.01±0.001
0.22±0.01
0.04±0.002
n. m.
1.99±0.02
1.88±0.14
1.30±0.04
5.11±1.07
1.47±0.04
0.50±0.07
0.04±0.01
0.04±0.005
0.28±0.01
0.14±0.07
18.4±2.0
2.41±0.20
1.61±0.04
1.50±0.02
10.74±1.00
1.52±0.04
0.39±0.04
0.03±0.004
0.03±0.01
0.22±0.02
0.05±0.02
29.2±3.0
0.90±0.15
1.81±0.21
1.23±0.10
12.30±1.02
1.05±0.06
0.40±0.03
0.14±0.02
0.17±0.07
0.33±0.07
0.36±0.16
>30
n. m.
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
BA
n. m.
3.25±0.15
18.66±1.63
1.84±0.08
n. m.
0.51±0.05
0.04±0.01
0.03±0.01
0.76±0.09
0.05±0.02
n. m.
0.04±0.01
0.03±0.01
0.22±0.04
0.05±0.02
n. m.
24.8±27.7
13.7±1.1
12.8±0.5
0.36±0.06
1.52±0.11
0.59±0.09
17.1±1.7
30.1±57
28.8±100
20.2±1.8
26.4±48.3
9.8±1.2
>30
24.8±27.7
n. m.
10.8±2.2
10.0±0.9
0.30±0.03
1.32±0.12
0.53±0.08
11.0±1.9
28.1±4.9
4.9±1.2
10.7±0.6
7.3±0.9
n. m.
0.29±0.03
1.71±0.15
0.55±0.07
14.4±2.3
0.23±0.03
1.38±0.13
0.49±0.03
14.8±1.9
0.19±0.02
1.01±0.13
0.40±0.04
13.1±1.1
n. m.
n. m.
n. m.
18.3±2.0
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As a result, boswellic acid derived compounds were cytotoxic but their selectivity for tumor
cells (as compared to non-malignant fibroblasts NIH 3T3) was low. The presence of a keto
function at the C-11 position seems to be disadvantageous for gaining good cytotoxicity.
Further investigation of derivatives of 5 (OH at C-3 coupled directly with rhodamine B)
showed that the cytotoxicity is highest for the methyl ester 25. Again, selectivity was low for
all of these compounds.
Fig. 2. Fluorescence microscopic investigations by double staining with Rhodamine 123 and
Hoechst 33342: A2780 ovarian carcinoma cells were analyzed after an incubation for 24
hours with compound 18 (100 nM) and compound 20 (400 nM), respectively. Picture A was
captured with the Zeiss filter set 15, picture B was captured with the Zeiss filter set 09, picture
C was captured with the Zeiss filter set 02 and picture D is an overlay of A, B and C. Scale
bar = 10 µm.
Better results were obtained for compounds derived from parent 1-4. Thus, all of the
acetylated triterpene-homopiperazinyl amides 11-15 were cytotoxic in the µM range while the
triterpene-homopiperazinyl-rhodamine adducts 16-20 showed the highest cytotoxicity for the
human tumor cell lines even in nanomolar concentrations.
Compared to previously reported piperazine-rhodamine compounds,[30] derivatives of ursolic
and oleanolic acid holding each a homopiperazinyl spacer were twice as cytotoxic as their
piperazinyl analogs. Interestingly enough, those analogs derived from glycyrrhetinic acid or
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betulinic acid were less cytotoxic than analogs from parent ursolic or oleanolic acid. Thus, the
glycyrrhetinic acid derived compound 19 exhibited the lowest cytotoxicity of all compounds
of this series while compound 18 displayed the highest. These results parallel previous
findings described for the piperazinyl derivatives.[30]
Furthermore, selectivity of cytotoxicity between tumor cells and nonmalignant fibroblasts
(NIH 3T3) was improved for the new derivatives holding the homopiperazinyl spacer. Thus,
the selectivity factor was S = EC50 (NIH 3T3) / EC50 (2780 cells) = 9 for betulinic acid
derived 20, and this selectivity is even higher (S = 17) for the oleanolic acid derived
compound 18.
Some additional experiments were performed to reveal the mode of action of these
compounds (Figure 2). Major benefit of linking triterpenes to rhodamine B is the relative ease
of localization of the compounds within the (cancer) cells. While staining of the cells with the
bisbenzimidazole dye Hoechst 33342 allows the identification of cell nuclei, Rhodamine 123
is usually applied in living cells to stain the mitochondria. A comparison of subfigures B (Fig.
2) shows an additional orange fluorescence for compound 20 in addition to the green
fluorescence. This effect is due to the higher concentration of the compound 20 (400 nM for
20, 100 nM for 18). However, the fluorescence microscopic studies also revealed that both
compounds 18 and 20 are located not in the nucleus, but in the same area as the Rhodamine
123 dye, i.e. the mitochondria. As described above, cells treated with each of these
compounds showed an inhibited growth as compared to untreated cells (control).
Consequently, it can be assumed that the cytostatic effect of compounds 18 and 20 onto
A2780 cancer cells is due to their ability to act as a mitocan.
3. Conclusion
Parent triterpenoic acids ursolic-, oleanolic, glycyrrhetinic, betulinic and boswellic acid were
acetylated followed by the conversion into their piperazinyl amides that were coupled with
rhodamine B. These compounds were subjected to RB assays to evaluate their cytotoxicity.
All of these triterpene-homopiperazinyl-rhodamine adducts 16-20 were highly cytotoxic for a
panel of human tumor cell lines even in nanomolar concentrations while being significantly
less cytotoxic for non-malignant cells. Several of the homopiperazinyl-rhodamine adducts
were even more cytotoxic than previously prepared piperazinyl analogs, thus making the
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homopiperazinyl spacer a very interesting scaffold for the development of biologically active
compounds. Extra staining experiments showed that the cytostatic effect of compounds 18
and 20 onto A2780 cancer cells is due to their ability to act as a mitocan.
Acknowledgments
We like to thank Mrs. V. Simon for measuring the IR and UV-vis spectra as well as the
optical rotations. The cell lines were kindly provided by Dr. Th. Müller (Dept. of
Haematology/Oncology, Martin-Luther Universität Halle-Wittenberg). Financial support by
the Oman Research Council (ORG/EBR/15/007) and the ScienceCampus Halle WCR
(W13004216) is gratefully acknowledged. The authors declare no conflict of interests.
4. Experimental part
4.1. General
NMR spectra were recorded using the Varian spectrometers Gemini 2000 or Unity 500 (δ
given in ppm, J in Hz; typical experiments: H-H-COSY, HMBC, HSQC, NOESY), MS
spectra were taken on a Finnigan MAT LCQ 7000 (electrospray, voltage 4.1 kV, sheath gas
nitrogen) instrument. The optical rotations were measured on a Perkin-Elmer polarimeter at
20 °C; TLC was performed on silica gel (Merck 5554, detection with cerium molybdate
reagent); melting points are uncorrected (Leica hot stage microscope. IR spectra were
recorded on a Perkin Elmer FT-IR spectrometer Spectrum 1000. The solvents were dried
according to usual procedures. The purity of the compounds was determined by HPLC and
found to be >96%. The parent triterpenoic acids were obtained from Betulinines (Stříbrná
Skalice, Czech Republic) in bulk quantities. Fluorescence microscopic images were recorded
on an Axioskop 20 with an AxioCam MR3 (Carl Zeiss AG). Flow cytometric experiments
were performed on an Attune acoustic focusing cytometer (Life Technologies GmbH). The
SRB assay was performed as previously described.[20, 30, 39, 42]
4.2.Biology
4.2.1. Double-staining experiment using Rhodamine 123 and Hoechst
33342
The Hoechst 33342 dye was used for live-cell fluorescent staining of DNA, and mitochondria
were stained by Rhodamine 123. Approx. 1·106 cells (A2780) were seeded in cell culture
flasks (25 cm2), and the cells were allowed to grow up for 24 h. After removing of the used
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medium, the substance loaded fresh medium was reloaded (or a blank new medium as a
control). After 24 h, the content of the flask was collected and centrifuged (1200 rpm, 4 °C),
the pellet was gently suspended in phosphate-buffered saline (PBS (w/w), 1 ml) and
centrifuged again. The PBS was removed, and the pellet gently suspended in PBS (50 µl)
again. After having mixed the cell suspension with a solution of Hoechst 33342/ Rhodamine
123 (10 µg/ml, 20 µl) the cells were incubated for 30 min at 37°C in the dark and washed
with PBS (w/w, 2x 500 µl). The analysis of the cells was performed with an Axioskop (Zeiss)
epifluorescent microscope after re-suspending the pellet in the supernatant. Different filter
sets were used for the screening: for the compounds the Zeiss filter set 15 (BP 546/12, FT
580, LP 590), for Hoechst 33342 the Zeiss filter set 02 (G 365, FT 395, LP 420) and for
Rhodamine 123 the Zeiss filter set 09 (BP 450-490, FT 510, LP 515), respectively.
4.3.General procedures
4.3.1. General procedure for the acetylation of parent triterpenoic acids
(GP1)
A solution of the triterpenoic acid 1-4 (10.0 g, 22 mmol, 1.0 eq.) in acetic anhydride (160 mL)
was stirred under reflux for 2 h. To the hot reaction mixture, acetic acid (20 mL) was slowly
added. Stirring was continued for another 10 min, then water (40 mL) was slowly added, and
the precipitate was filtered off. The precipitate was washed with water (4×50 mL), cold EtOH
(20 mL) and dried under reduced pressure. The desired products were obtained as off-white
solids that were pure enough (as checked by TLC, NMR, MS) for the next reaction steps.
Analytical samples were obtained either by re-crystallization or column chromatography.
4.3.2. General procedure for the coupling with homopiperazine (GP2)
To an ice-cold solution of acetate 5-9 (0.5 g, 1.0 mmol, 1.0 eq.) in dry CH2Cl2 (10 mL),
oxalyl chloride (0.5 g, 0.3 mL, 4 mmol, 4.0 eq.) was slowly added, and the mixture was
allowed to warm to ambient temperature, and stirring was continued for 3 h. The solvent was
removed under reduced pressure, the residue dissolved in dry THF (10 mL), and the volatiles
were evaporated. To a solution of the residue in dry CH2Cl2 (10 mL), homopiperazine (2.0 g,
2 mmol, 2.0 eq.), NEt3 (0.2 mL, 1.5 mmol, 1.5 eq.) and DMAP (5 mol-%) were added, and
stirring was continued for 2 h. Evaporation of the solvent under reduced pressure and
purification of the residue by column chromatography (silica gel) afforded the desired
products as off-white solids.
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4.3.3. General procedure for the preparation of the rhodamine B
derivatives (GP3)
To an ice-cold solution of rhodamine B (0.3 g, 0.63 mmol, 1 eq.) in dry CH2Cl2 (6 mL),
oxalyl chloride (0.32 g, 0.2 mL, 2.51 mmol, 4 eq.) was slowly added, and the mixture was
allowed to warm to ambient temperature; stirring was continued for 3 h. The solvent was
removed under reduced pressure, the residue dissolved in dry THF (2×10 mL), and the
CH2Cl2 (10 mL) compounds 11-15 (0.3 g, 0.46 mmol, 1 eq.), NEt3 (0.1 mL, 0.69 mmol,
1.5 eq.) and DMAP (5 mol-%) were added, and stirring was continued overnight. Evaporation
of the solvent under reduced pressure and purification of the residue by column
chromatography (silica gel, MeCN/CH2Cl2/H2O 10:1:1) afforded the desired rhodamine B
derivatives as intensively violet solids.
4.4.Syntheses
4.4.1. 3-O-Acetyl-11-keto-β-boswellic acid (5)
[8]
This compound was isolated following a modified Jauch’s procedure
and obtained as a
colorless solid; m.p. 268–270 °C; [α]D = +81.1° (c = 1.0, CHCl3), [lit.: m.p. 271–276 °C, [α]D
= +82.0° (c = 1.25, CHCl3)].[37]
4.4.2. 3-O-Acetyl-ursolic acid (6)
Following GP1 from ursolic acid (1) compound 6 was obtained in 90% yield; m.p. 280–
285 °C, [α]D = +65.6° (c 0.33, CHCl3), [lit.: m.p. 285 °C,[43] [α]D = +69.0° (c = 0.19,
CHCl3)].[39]
4.4.3. 3-O-Acetyl-oleanolic acid (7)
Following GP1 from oleanolic acid (2) compound 7 was obtained in 73% yield; m.p. 266–
269 °C, [α]D = +69.4° (c = 0.30, CHCl3), [lit.: m.p. 265–268 °C, [α]D = +65.0° (c = 0.33,
CHCl3)].[39]
4.4.4. 3-O-Acetyl-18β-glycyrrhetinic acid (8)
Following GP1 from glycyrrhetinic acid (3) compound 8 was obtained in 85% yield; m.p.
316–318 °C, [α]D = +161.9° (c = 0.2, CHCl3) [lit.: m.p. 310–313°C); [α]D = +163.8° (c = 1.0,
CHCl3)]. [44]
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4.4.5. 3-O-Acetyl-betulinic acid (9)
Following GP1 from betulinic acid (4) compound 9 was obtained in 70% yield; m.p. 282–
285 °C, [α]D = +21.0° (c = 0.33, CHCl3), [lit.: m.p. 287–289 °C[45], [α]D = +20.5° (c =
0.58)].[42]
4.4.6. 11-Keto-β-boswellic acid (10)
Compound 5 (10.0 g, 19.5 mmol) was dissolved in ethanol (200 mL), and an aq. solution of
sodium hydroxide (4 M, 100 mL) was added. After stirring at 25 °C for 12 h, the pH was
adjusted to pH 1 by addition with aq. HCl, the crude product was extracted with CHCl3
(5×100 mL) and purified by column chromatography (silica gel, hexane/ethyl acetate, 98:2) to
afford 10 in almost quantitative yield as a colorless solid; m.p. 192–195 °C [α]D = +118.2° (c
= 3.72, CHCl3), [lit.: m.p. 194–195 °C [37], [α]D = +121° (c = 1.11, CHCl3)].[37]
4.4.7. (3α, 4β) 3-Acetyloxy-N-homopiperazinyl-11-oxo-urs-12-en-23-one
(11)
Following GP2 from 5 followed by column chromatography (CHCl3/MeOH, 9:1), 11 was
obtained in 64% yield as an off-white solid; m.p. 170–173 °C; Rf 0.4 (CHCl3/MeOH, 9:1);
[α]D = +29.7° (c = 0.34, CHCl3); UV-Vis (MeOH): λmax (log ε) =278 nm (3.30); IR (KBr):
1
ν = 3442br, 2927m, 1736s, 1654m, 1458m, 1381m, 1249m, 1026m, 755m cm-1; H NMR
(500 MHz, CDCl3): δ = 5.62 (m, 1 H, 3-H), 5.54 (s, 1 H, 12-H), 3.84–3.64 (m, 4 H,
CONCH2), 3.23–2.90 (m, 4 H, HNCH2), 2.54 (ddd, J = 13.5, 13.5, 3.4 Hz, 1 H, 1-Ha), 2.40 (s,
1 H, 9-H), 2.25 (m, 1 H, 6-Ha), 2.11 (s, 3 H, CH3CO), 2.14–2.03 (m, 2 H, CONCH2CH2),
2.02–1.77 (m, 3 H, 2-Ha, 15-Ha, 16-Ha), 1.69–1.56 (m, 3 H, 6-Hb, 2-Hb, 7-Ha), 1.54 (d,
J = 11.2 Hz, 1 H, 18-H), 1.51–1.33 (m, 4 H, 22-Ha, 7-Hb, 21-Ha, 19-H), 1.32 (s, 3 H, 27-H3),
1.32–1.16 (m, 5 H, 15-Hb, 21-Hb, 1-Hb, 22-Hb, 5-H), 1.25 (s, 3 H, 23-H3), 1.24 (s, 3 H,
26-H3), 1.23 (s, 3 H, 25-H3), 1.01 (m, 1 H, 16-Hb), 0.94 (s, 3 H, 30-H3), 0.96–0.92 (m, 1 H,
20-H), 0.82 (s, 3 H, 28-H3), 0.80 (d, J = 6.2 Hz, 3 H, 29-H3) ppm; 13C NMR (125 MHz,
CDCl3): δ = 199.5 (C-11), 174.4 (C-24), 170.9 (CH3CO), 164.7 (C-13), 130.6 (C-12), 71.6
(C-3), 61.8 (C-9), 58.9 (C-18), 54.2 (C-5), 49.6 (C-4), 48.0, 45.8 (4×CONCH2), 45.3 (C-8),
43.7 (C-14), 40.9 (C-22), 39.3 (C-19, C-20), 38.1 (C-10), 34.1 (C-1), 33.9 (C-7), 33.8 (C-17),
30.9 (C-21), 28.8 (C-28), 27.5 (C-16), 27.3 (C-15), 24.5 (C-2), 23.0 (C-23), 21.2 (CH3CO),
21.1 (C-30), 20.8 (CONCH2CH2), 20.6 (C-27), 19.7 (C-6), 18.6 (C-26), 17.4 (C-29), 16.2
13

ACCEPTED MANUSCRIPT
(C-25) ppm; MS [ESI, MeOH for C37H58N2O4 (594.88)]: m/z (%) 1189.3 (3) [2M+H]+, 595.3
(100) [M+H]+.
4.4.8. 3β-Acetyloxy-28-(1-homopiperazinyl)-urs-12-en-28-one (12)
Following GP2 from 6 followed by column chromatography (hexane/ethyl acetate, 5:1) 12
was obtained in 84% yield as an off-white solid; m.p. 178–180 °C (lit.: 151–157 °C)[29]; Rf
0.2 (hexane/ethyl acetate, 5:1); [α]D = +9.9° (c = 0.35, CHCl3); UV-Vis (MeOH): λmax (log
ε) = 203 nm (4.04); IR (KBr): ν = 3448br, 2947m, 1734s, 1624s, 1458m, 1371m, 1247s,
1
1028m, 753m cm-1; H NMR (500 MHz, CDCl3): δ = 5.15 (m, 1 H, 12-H), 4.42 (dd, J = 9.1,
7.5 Hz, 1 H, 3-H), 3.96–3.49 (m, 4 H,CONCH2), 3.34–2.97 (m, 4 H, HNCH2), 2.37 (d,
J = 9.9 Hz, 1 H, 18-H), 2.32–2.17 (m, 2 H, 16-H2), 1.99 (s, 3 H, CH3CO), 1.88 (m, 2 H,
11-H2), 1.69 (m, 1 H, 22-Ha), 1.63–1.53 (m, 4 H, 2-H2, 22-Hb, 1-Ha), 1.52–1.38 (m, 4 H, 6-Ha,
21-Ha, 7-Ha, 9-H), 1.39–1.20 (m, 4 H, 6-Hb, 21-Hb, 7-Hb, 19-H), 1.07–0.95 (m, 6 H,
CONCH2CH2, 1-Hb, 20-H, 15-H2), 1.04 (s, 3 H, 27-H3), 0.93 (s, 3 H, 30-H3), 0.90 (s, 3 H,
25-H3), 0.85 (s, 3 H, 29-H3), 0.82 (s, 6 H, 23-H3, 24-H3), 0.77 (d, J = 11.5 Hz, 1 H, 5-H), 0.67
13
(s, 3 H, 26-H3) ppm; C NMR (125 MHz, CDCl3): δ = 176.3 (C-28), 171.0 (CH3CO), 138.4
(C-13), 125.1 (C-12), 80.9 (C-3), 55.3 (C-5), 54.8 (C-18), 49.0 (C-17), 47.5 (C-9), 46.8
(2×CH2N), 46.4 (2×CH2N), 42.2 (C-14, C-8), 39.4 (C-19), 38.7 (C-20), 38.2 (C-1), 37.7
(C-4), 36.9 (C-10), 34.4 (C-22), 32.9 (C-7), 30.5 (C-21), 28.2 (C-23), 28.1 (CONCH2CH2),
27.8 (C-15), 25.6 (C-16), 23.9 (C-27), 23.5 (C-2), 23.3 (C-11), 21.3 (CH3CO), 21.2 (C-30),
18.2 (C-6), 17.4 (C-29), 17.0 (C-26), 16.7 (C-24), 15.5 (C-25) ppm; MS [ESI, MeOH for
C37H60N2O3 (580.88)]: m/z (%) 581.4 (100) [M+H]+.
4.4.9. 3β-Acetyloxy-28-(1-homopiperazinyl)-olean-12-en-28-one (13)
Following GP2 from 7 followed by column chromatography (CHCl3/MeOH, 92:8) 13 was
obtained in 99% yield as an off-white solid; m.p. 187–190 °C; Rf 0.25 (CHCl3/MeOH, 9:1);
[α]D = +16.4° (c = 0.30, CHCl3); UV-Vis (MeOH): λmax (log ε) = 203 nm (4.10); IR (KBr):
ν = 3442br, 2946s, 1733s, 1622s, 1466m, 1371m, 1247s, 1182m, 1028m, 1007m, 986m, 753m
1
cm-1; H NMR (400 MHz, CDCl3): δ = 5.25 (m, 1 H, 12-H), 4.48 (dd, J = 9.6, 7.5 Hz, 1 H,
3-H), 3.78–3.54 (m, 4 H, CONCH2), 3.45 (br s, 1 H, NH), 3.12–2.83 (m, 5 H, HNCH2, 18-H),
2.11 (ddd, J = 14.1, 14.1, 2.6 Hz, 1 H, 16-Ha), 2.03 (s, 3 H, CH3CO), 2.00–1.79 (m, 4 H,
CONCH2CH2, 11-H2), 1.74–1.46 (m, 10 H, 19-Ha, 22-H2, 16-Hb, 2-H2, 15-Ha, 1-Ha, 6-Ha,
9-H), 1.41 (m, 1 H, 6-Hb), 1.37–1.14 (m, 5 H, 7-H2, 21-H2, 19-Hb), 1.13 (s, 3 H, 27-H3), 1.10–
0.97 (m, 2 H, 15-Hb, 1-Hb), 0.93 (s, 3 H, 25-H3), 0.92 (s, 3 H, 30-H3), 0.89 (s, 3 H, 29-H3),
14

ACCEPTED MANUSCRIPT
13
0.85 (s, 3 H, 23-H3), 0.84 (s, 3 H, 24-H3), 0.82 (m, 1 H, 5-H), 0.74 (s, 3 H, 26-H3) ppm; C
NMR (100 MHz, CDCl3): δ = 175.9 (C-28), 171.0 (CH3CO), 144.7 (C-13), 121.4 (C-12),
80.9 (C-3), 55.4 (C-5), 48.4 (2×CONCH2), 47.8 (C-17), 47.7 (C-9), 47.3 (CONCH2), 46.5
(C-19), 46.4 (CONCH2), 43.7 (C-18), 41.9 (C-14), 39.2 (C-8), 38.1 (C-1), 37.7 (C-4), 37.0
(C-10), 34.1 (C-21), 33.0 (C-29), 32.8 (C-7), 30.4 (C-20), 30.1 (C-22), 28.8 (CONCH2CH2),
28.1 (C-23), 28.0 (C-15), 25.9 (C-27), 24.0 (C-30), 23.5 (C-2), 23.4 (C-11), 22.8 (C-16), 21.3
(CH3CO), 18.2 (C-6), 17.1 (C-26), 16.7 (C-24), 15.4 (C-25) ppm; MS [ESI, MeOH for
C37H60N2O3 (580.88)]: m/z (%) 581.3 (100) [M+H]+.
4.4.10. (3β, 18β)-3-Acetyloxy-30-(1-homopiperazinyl)-olean-12-en-11,30-
dione (14)
Following GP2 from 8 followed by column chromatography (CHCl3/MeOH, 92:8) 14 was
obtained in 48% yield as an off-white solid; m.p. 260–264 °C; Rf 0.2 (hexane/ethyl acetate,
2:1); [α]D = +109.8° (c = 0.38, CHCl3); UV-Vis (MeOH): λmax (log ε) = 203 nm (3.92),
262 nm (3.93); IR (KBr): ν = 3441br, 2951s, 1730s, 1657m, 1633m, 1465m, 1385m, 1248s,
1
1163m, 1029m, 987m, 540m cm-1; H NMR (400 MHz, CDCl3): δ = 6.78 (br s, 1 H, NH),
5.67 (s, 1 H, 12-H), 4.50 (dd, J = 11.6, 4.8 Hz, 1 H, 3-H), 3.90–3.66 (m, 4 H, CONCH2),
3.30–3.12 (m, 4 H, HNCH2), 2.77 (ddd, J = 13.6, 13.6, 3.5 Hz, 1 H, 1-Ha), 2.34 (s, 1 H, 9-H),
2.27 (m, 1 H, 18-H), 2.23 (m, 2 H, CONCH2CH2), 2.09–1.97 (m, 4 H, 21-H2, 16-Ha, 19-Ha),
2.03 (s, 3 H, CH3CO), 1.82 (ddd, J = 13.6, 13.6, 3.8 Hz, 1 H, 15-Ha), 1.75–1.53 (m, 5 H, 2-H2,
7-Ha, 19-Hb, 6-Ha), 1.47–1.30 (m, 4 H, 6-Hb, 7-Hb, 22-H2), 1.34 (s, 3 H, 27-H3), 1.22 (s, 3 H,
29-H3), 1.21–1.15 (m, 1 H, 15-Hb), 1.14 (s, 3 H, 25-H3), 1.10 (s, 3 H, 26-H3), 1.08–0.97 (m,
2 H, 1-Hb, 16-Hb), 0.86 (s, 6 H, 23-H3, 24-H3), 0.80 (s, 3 H, 28-H3), 0.78 (m, 1 H, 5-H) ppm;
13C NMR (100 MHz, CDCl3): δ = 199.9 (C-11), 175.2 (C-30), 171.0 (CH3CO), 169.3 (C-13),
128.5 (C-12), 80.6 (C-3), 61.7 (C-9), 55.0 (C-5), 48.0 (C-18), 46.7 (2×CONCH2), 45.3 (C-8),
45.0 (CONCH2), 44.6 (CONCH2), 44.4 (C-20), 43.9 (C-19), 43.3 (C-14), 38.8 (C-1), 38.0
(C-4), 37.7 (C-22), 36.9 (C-10), 32.9 (C-21), 32.7 (C-7), 31.8 (C-17), 28.4 (C-28), 28.0
(C-23), 26.9 (C-29), 26.8 (C-16), 26.3 (C-15), 26.1 (CONCH2CH2), 23.5 (C-2), 23.0 (C-27),
21.3 (CH3CO), 18.7 (C-26), 17.4 (C-6), 16.7 (C-24), 16.4 (C-25) ppm; MS [ESI, MeOH for
C37H58N2O4 (594.87)]: m/z (%) 595.5 (100) [M+H]+.
4.4.11. 3β-Acetyloxy-28-(1-homopiperazinyl)-lup-20(29)en-28-one (15)
Following GP2 from 9 followed by column chromatography (CHCl3/MeOH, 9:1) 15 was
obtained in 86% yield as an off-white solid; m.p. 196–199 °C; Rf 0.24 (CHCl3/MeOH, 9:1);
15

ACCEPTED MANUSCRIPT
[α]D = -6.5° (c = 0.34, CHCl3); UV-Vis (MeOH): λmax (log ε) = 201 nm (4.09); IR (KBr):
ν = 3445br, 2945s, 2869m, 1734s, 1628s, 1456m, 1374m, 1247s, 1185m, 1029m, 979m,
881m, 754m cm-1; NMR (400 MHz, CDCl3): δ = 4.68 (m, 1 H, 29-Ha), 4.55 (m, 1 H, 29-Hb),
4.40 (dd, J = 9.6, 8.0 Hz, 1 H, 3-H), 3.67–3.50 (m, 4 H, CONCH2), 3.04–2.89 (m, 5 H,
HNCH2, 19-H), 2.84 (m, 1 H, 13-H), 2.08 (m, 1 H, 16-Ha), 1.98 (s, 3 H, CH3CO), 1.96–1.87
(m, 3 H, CONCH2CH2, 22-Ha), 1.78 (ddd, J = 10.5, 10.5, 7.5 Hz, 1 H, 21-Ha), 1.65 (s, 3 H,
30-H3), 1.66–1.60 (m, 2 H, 12-Ha, 1-Ha), 1.57 (m, 2 H, 2-H2), 1.53–1.47 (m, 2 H, 16-Hb,
18-H), 1.48–1.42 (m, 1 H, 6-Ha), 1.42–1.39 (m, 7 H, 6-Hb, 11-Ha, 15-Ha, 21-Hb, 7-H2, 22-Hb),
1.25–1.18 (m, 2 H, 9-H, 11-Hb), 1.12 (m, 1 H, 15-Hb), 0.97–0.88 (m, 2 H, 12-Hb, 1-Hb), 0.92
(s, 3 H, 27-H3), 0.90 (s, 3 H, 26-H3), 0.82 (s, 3 H, 25-H3), 0.79 (s, 6 H, 24-H3, 23-H3), 0.73 (d,
13
J = 8.9 Hz, 1 H, 5-H) ppm; C NMR (100 MHz, CDCl3): δ = 174.5 (C-28), 171.0 (CH3CO),
151.2 (C-20), 109.2 (C-29), 81.0 (C-3), 55.5 (C-5), 55.0 (C-17), 53.0 (C-18), 50.8 (C-9), 46.9
(4×CONCH2), 45.7 (C-19), 42.0 (C-14), 40.7 (C-8), 38.4 (C-1), 37.8 (C-4), 37.1 (C-10), 36.9
(C-13), 36.1 (C-22), 34.3 (C-7), 32.3 (C-16), 31.4 (C-21), 29.9 (C-15), 29.0 (CONCH2CH2),
27.9 (C-23), 25.6 (C-12), 23.7 (C-2), 21.3 (CH3CO), 21.2 (C-11), 19.7 (C-30), 18.2 (C-6),
16.5 (C-24), 16.2 (C-25), 16.1 (C-26), 14.7 (C-27) ppm; MS [ESI, MeOH for C37H60N2O3
(580.88)]: m/z (%) 581.3 (100) [M+H]+.
4.4.12. 9-[2-[[4-[(3α,4β)-3-Acetyloxy-11,24-dioxo-urs-12-en-24-yl]-1-
homopiperazinyl]carbonyl]-phenyl]-3,6-bis[diethylamino]-
xanthylium chloride (16)
Following GP3 from rhodamine B and oxalyl chloride rhodamine B chloride was obtained.
To a solution of rhodamine B chloride in dry CH2Cl2 (10 mL), 11 (0.22 g, 0.46 mmol, 1 eq.),
NEt3 (70 mg, 0.10 mL, 0.69 mmol, 1.5 eq.) and DMAP (5 mol-%) were added. Work-up as
described and purification by column chromatography afforded 16 in 83% yield; m.p. 213–
215 °C; Rf 0.5 (MeCN/CH2Cl2/H2O, 10:1:1); UV-Vis (MeOH): λmax (log ε) = 274 nm (4.40),
647 nm (4.96); IR (KBr): ν = 3442br, 1732m, 1634m, 1590m, 1470m, 1413m, 1338m, 1247m,
1181m, 1074m, 684m cm-1; 1H NMR (500 MHz, CDCl3): δ = 7.71–7.59 (m, 2 H, 3″-H, 5″-H),
7.50–7.44 (m, 1 H, 4″-H), 7.35–7.20 (m, 3 H, 6″-H, 1′-H, 8′-H), 6.96–6.75 (m, 4 H, 2′-H,
7′-H, 4′-H, 5′-H), 5.55 (s, 1 H, 12-H), 5.51 (m, 1 H, 3-H), 3.95–3.21 (m, 16 H, 4×CONCH2,
N′(CH2CH3)2, N″(CH2CH3)2,), 2.51 (ddd, J = 13.8, 13.8, 3.0 Hz, 1 H, 1-Ha), 2.38 (s, 1 H,
9-H), 2.18 (m, 1 H, 6-Ha), 2.11 (s, 3 H, CH3CO), 2.09 (m, 1 H, 16-Ha), 1.98–1.76 (m, 5 H,
2-H2, 15-Ha, CONCH2CH2), 1.65 (dddd, J = 15.6, 15.6, 7.1, 5.6 Hz, 1 H, 6-Hb), 1.55 (d,
J = 11.4 Hz, 1 H, 18-H), 1.53–1.37 (m, 6 H, 22-H2, 7-Ha, 21-Ha, 19-H, 5-H), 1.34 (s, 3 H,
16

ACCEPTED MANUSCRIPT
27-H3), 1.33 (m, 12 H, N′(CH2CH3)2, N″(CH2CH3)2), 1.31–1.22 (m, 4 H, 21-Hb, 7-Hb, 1-Hb,
15-Hb), 1.25 (s, 3 H, 26-H3), 1.23 (s, 3 H, 23-H3), 1.22 (s, 3 H, 25-H3), 1.02 (m, 1 H, 16-Hb),
13
0.96–0.90 (m, 4 H, 20-H, 30-H3), 0.82 (s, 3 H, 28-H3), 0.80 (s, 3 H, 29-H3) ppm; C NMR
(125 MHz, CDCl3): δ = 199.5 (C-11), 174.4 (C-24), 170.8 (CH3CO), 168.4 (CO-Rhd), 164.8
(C-13), 158.8 (C-9′), 157.7 (C-4a′, C-10a′), 155.7 (C-3′, C-6′), 134.0 (C-1″), 132.3 (C-1′,
C-8′), 131.3 (C-2″), 130.5 (C-12), 130.0 (C-6″), 129.8 (C-3″), 129.4 (C-5″), 127.1 (C-4″),
114.7 (C-2′, C-7′), 113. 5 (C-8a′, C-9a′), 96.4 (C-4′, C-5′), 72.3 (C-3), 61.6 (C-9), 59.1
(C-18), 54.0 (C-5), 49.5 (C-4), 47.7 (4×CONCH2), 46.3, 46.2 (N′(CH2CH3)2, N″(CH2CH3)2),
45.2 (C-8), 43.7 (C-14), 40.8 (C-22), 39.3 (C-19, C-20), 38.0 (C-10), 34.3 (C-1), 33.9 (C-7),
33.8 (C-17), 31.0 (C-21), 29.0 (CONCH2CH2), 28.7 (C-28), 27.9 (C-16), 27.4 (C-15), 23.3
(C-2), 22.7 (C-23), 21.3 (CH3CO), 21.1 (C-30), 20.8 (C-6), 20.6 (C-27), 18.5 (C-26), 17.4
(C-29), 16.0 (C-25), 12.7 (N′(CH2CH3)2, N″(CH2CH3)2) ppm; MS [ESI, MeOH for
C65H89ClN4O5 (1055.88)]: m/z (%) 1019.7 (100) [M-Cl]+.
4.4.13. 9-[2-[[4-[(3β)-3-acetyloxy-28-oxours-12-en-28-yl]-1-
homopiperazinyl]carbonyl]phenyl]-3,6-bis(diethylamino)-
xanthylium chloride (17)
As described above for the synthesis of 16, to a solution of rhodamine B chloride in dry
CH2Cl2 (10 mL), 12 (0.21 g, 0.46 mmol, 1 eq.), NEt3 (70 mg, 0.10 mL, 0.69 mmol, 1.5 eq.)
and DMAP (5 mol-%) were added. Work up as described and purification by column
chromatography afforded 17 in 74% yield; m.p. 238–245 °C; Rf 0.5 (MeCN/CH2Cl2/H2O,
10:1:1); UV-Vis (MeOH): λmax (log ε) = 275 nm (4.21), 647 nm (4.74;R (KBr): ν = 3440br,
2926m, 1729m, 1590s, 1528m, 1468m, 1413m, 1338m, 1275m, 1247m, 1181m, 1133m,
1074m, 1010m, 921m, 684m cm-1; 1H NMR (500 MHz, CDCl3): δ = 7.68–7.56 (m, 2 H, 3″-H,
5″-H), 7.47–7.39 (m, 1 H, 4″-H), 7.31–7.27 (m, 1 H, 6″-H), 7.25–7.15 (m 2 H, 1′-H, 8′-H),
6.90–6.73 (m, 4 H, 2′-H, 7′-H, 4′-H, 5′-H), 5.17 (m, 1 H, 12-H), 4.46 (dd, J = 9.5, 7.8 Hz,
1 H, 3-H), 4.06–3.04 (m, 16 H, 4×CONCH2, N″(CH2CH3)2, N′(CH2CH3)2), 2.40 (m, 1 H,
18-H), 2.18–2.04 (m, 2 H, 16-H2), 2.02 (s, 3 H, CH3CO), 1.88 (m, 2 H, 11-H2), 1.77–1.54 (m,
3 H, 2-H2, 1-Ha), 1.54–1.41 (m, 5 H, 6-Ha, 21-Ha, 7-Ha, 9-H, 22-Ha), 1.40–1.18 (m, 5 H, 6-Hb,
21-Hb, 7-Hb, 19-H, 22-Hb), 1.32 (t, J = 7.2 Hz, 6 H, N′(CH2CH3)2), 1.31 (t, J = 7.0 Hz, 6 H,
N″(CH2CH3)2), 1.08–0.84 (m, 6 H, 1-Hb, 20-H, 15-H2, CONCH2CH2), 1.02 (s, 3 H, 27-H3),
17

ACCEPTED MANUSCRIPT
0.90 (br s, 9 H, 29-H3, 30-H3, 25-H3), 0.83 (s, 3 H, 23-H3), 0.82 (s, 3 H, 24-H3), 0.79 (m, 1 H,
5-H), 0.68 (s, 3 H, 26-H3) ppm; 13C NMR (125 MHz, CDCl3): δ = 176.6 (C-28), 171.0
(CH3CO), 168.6 (CO-Rhd), 168.0 (C-9′), 157.7, 157.6 (C-4a′, C-10a′), 155.7, 155.6 (C-3′,
C-6′), 138.5 (C-13), 135.9 (C-1″), 132.5 (C-1′, C-8′), 130.1 (C-2″), 130.0 (C-6″), 129.6 (C-3″,
C-5″), 126.8 (C-4″), 125.0 (C-12), 113.8, 113.6 (C-2′, C-7′), 113.5 (C-8a′, C-9a′), 96.4, 96.2
(C-4′, C-5′), 80.9 (C-3), 55.3 (C-5), 55.2 (C-18), 48.9 (C-17), 47.5 (C-9), 46.2, 46.1
(N′(CH2CH3)2, N″(CH2CH3)2, 4×CONCH2), 42.3 (C-14), 39.3 (C-8), 38.7 (C-19, C-20), 38.2
(C-1), 37.6 (C-4), 36.9 (C-10), 34.8 (C-22), 33.7 (CONCH2CH2), 32.9 (C-7), 30.4 (C-21),
28.0 (C-23), 27.9 (C-15), 23.5 (C-2), 23.4 (C-27), 23.3 (C-11), 21.3 (CH3CO), 21.4 (C-16),
21.2 (C-30), 18.1 (C-6), 17.5 (C-29), 16.9 (C-26), 16.7 (C-24), 15.5 (C-25), 12.7, 12.6
(N′(CH2CH3)2, N″(CH2CH3)2) ppm; MS [ESI, MeOH for C65H89ClN4O5 (1041.90)]: m/z (%)
1005.9 (100) [M-Cl]+.
4.4.14. 9-[2-[[4-[(3β)-3-Acetyloxy-28-oxo-olean-12-en-28-yl]1-
homopiperazinyl]carbonyl]phenyl]-3,6-bis(diethylamino)-
xanthylium chloride (18)
As described above for 16 to a solution of rhodamine B chloride in dry CH2Cl2 (10 mL), 13
(0.27 g, 0.46 mmol, 1 eq.), NEt3 (70 mg, 0.10 mL, 0.69 mmol, 1.5 eq.) and DMAP (5 mol-%)
were added. Work up as described and purification by column chromatography afforded 18 in
82% yield; m.p. 245–248 °C; Rf 0.5 (MeCN/CH2Cl2/H2O, 10:1:1); UV-Vis (MeOH): λmax
(log ε) = 277 nm (4.20), 647 nm (4.76); IR (KBr): ν = 3442br, 2926m, 1629m, 1590s, 1528m,
1468m, 1413m, 1338m, 1275m, 1247m, 1181m, 1133m, 1074m, 1010m, 921m, 684m cm-1; 1H
NMR (500 MHz, CDCl3): δ = 7.67–7.55 (m, 2 H, 3″-H, 5″-H), 7.43 (m, 1 H, 4″-H), 7.32–7.28
(m, 1 H, 6″-H), 7.27–7.18 (m 2 H, 1′-H, 8′-H), 6.85–6.73 (m, 4 H, 2′-H, 7′-H, 4′-H, 5′-H),
5.22 (m, 1 H, 12-H), 4.46 (dd, J = 9.5, 7.6 Hz, 1 H, 3-H), 4.02–3.40 (m, 12 H, 2×CONCH2,
N′(CH2CH3)2, N″(CH2CH3)2), 3.38–2.95 (m, 5 H, 2×CONCH2, 18-H), 2.16–2.04 (m, 1 H,
16-Ha), 2.02 (s, 3 H, CH3CO), 1.96–1.78 (m, 4 H, CONCH2CH2, 11-H2), 1.77–1.36 (m, 9 H,
2-H2, 6-Ha, 15-Ha, 16-Hb, 7-Ha, 1-Ha ,19-Ha, 9-H), 1.35–1.14 (m, 6 H, 6-Hb, 7-Hb, 22-Ha,
19-Hb, 21-H2), 1.31 (m, 12 H, N′(CH2CH3)2, N″(CH2CH3)2), 1.09 (s, 3 H, 27-H3), 1.13–0.96
(m, 3 H, 22-Hb, 15-Hb, 1-Hb), 0.93 (s, 3 H, 30-H3), 0.88 (s, 6 H, 29-H3, 25-H3), 0.83 (s, 3 H,
23-H3), 0.82 (s, 3 H, 24-H3), 0.79 (m, 1 H, 5-H), 0.67 (s, 3 H, 26-H3) ppm; 13C NMR
(125 MHz, CDCl3): δ = 176.2 (C-28), 171.0 (CH3CO), 168.7 (CO-Rhd), 165.7 (C-9′), 157.7
(C-4a′, C-10a′), 155.6 (C-3′, C-6′), 144.7 (C-13), 136.3 (C-1″), 132.5 (C-1′, C-8′), 130.1
18

ACCEPTED MANUSCRIPT
(C-2″), 130.0 (C-6″), 129.6 (C-3″, C-5″), 126.7 (C-4″), 121.4 (C-12), 114.8 (C-2′, C-7′),
113.6 (C-8a′, C-9a′), 96.4, 96.2 (C-4′, C-5′), 80.9 (C-3), 55.3 (C-5), 47.7 (C-17), 47.6 (C-9),
46.3 (C-19), 46.2, 46.0 (N′(CH2CH3)2, N″(CH2CH3)2, 4×CONCH2), 43.6 (C-18), 42.0 (C-14),
39.0 (C-8), 38.0 (C-1), 37.7 (C-4), 37.0 (C-10), 34.0 (C-21), 33.8 (C-22), 33.0 (C-29), 32.8
(C-7), 30.3 (C-20), 28.0 (C-23), 27.9 (C-16), 27.8 (C-15), 25.7 (C-27), 24.0 (C-30), 23.7
(CONCH2CH2), 23.5 (C-2), 23.3 (C-11), 21.3 (CH3CO), 18.2 (C-6), 16.9 (C-26), 16.6 (C-24),
15.4 (C-25), 12.7 (N′(CH2CH3)2, N″(CH2CH3)2) ppm; MS [ESI, MeOH for C65H89ClN4O5
(1041.90)]: m/z (%) 1005.9 (100) [M-Cl]+.
4.4.15. 9-[2-[[4-[(3β, 18β)-3-Acetyloxy-11,30-dioxo-urs-12-en-30-yl]1-
homopiperazinyl]carbonyl]-phenyl]-3,6-bis(diethylamino)-
xanthylium chloride (19)
As described above for 16 to a solution of rhodamine B chloride in dry CH2Cl2 (10 mL), 14
(0.21 g, 0.46 mmol, 1 eq.), NEt3 (70 mg, 0.10 mL, 0.69 mmol, 1.5 eq.) and DMAP (5 mol-%)
were added. Work up as described and purification by column chromatography afforded 19 in
50% yield; m.p. 165–170 °C; Rf 0.5 (MeCN/CH2Cl2/H2O, 10:1:1); UV-Vis (MeOH): λmax
(log ε) = 271 nm (4.47), 644 nm (4.83); IR (KBr): ν = 3441br, 2971m, 1725m, 1590s, 1467m,
1
1414m, 1384m, 1338m, 1248m, 1181m, 1133m, 1074m, 684m cm-1; H NMR (500 MHz,
CDCl3): δ = 7.69–7.59 (m, 2 H, 3″-H, 5″-H), 7.51–7.42 (m, 1 H, 4″-H), 7.33–7.27 (m, 1 H,
6″-H), 7.25–7.18 (m 2 H, 1′-H, 8′-H), 7.00–6.86 (m, 2 H, 2′-H, 7′-H), 6.84–6.64 (m, 2 H,
4′-H, 5′-H), 5.66 (s, 1 H, 12-H), 4.50 (m, 1 H, 3-H), 3.78–3.31 (m, 12 H, CONCH2,
N′(CH2CH3)2, N″(CH2CH3)2), 3.14–3.08 (m, 4 H, RhdCONCH2), 2.77 (m, 1 H, 1-Ha), 2.34
(m, 1 H, 9-H), 2.23 (m, 1 H, 18-H), 2.04 (s, 3 H, CH3CO), 2.07–1.97 (m, 5 H, 16-Ha, 19-Ha,
21-Ha, CONCH2CH2), 1.81 (m, 1 H, 15-Ha), 1.74–1.53 (m, 5 H, 2-H2, 6-Ha, 21-Hb, 19-Hb),
1.49–1.34 (m, 5 H, 6-Hb, 7-H2, 22-H2), 1.33 (s, 3 H, 27-H3), 1.31 (m, 12 H, N′(CH2CH3)2,
N″(CH2CH3)2), 1.19 (s, 3 H, 29-H3), 1.18–1.14 (m, 1 H, 15-Hb), 1.15 (s, 3 H, 25-H3), 1.10 (s,
3 H, 26-H3), 1.06–0.95 (m, 2 H, 1-Hb, 16-Hb), 0.87 (br s, 6 H, 23-H3, 24-H3), 0.79 (m, 1 H,
5-H), 0.76 (s, 3 H, 28-H3) ppm; 13C NMR (125 MHz, CDCl3): δ = 199.9 (C-11), 175.2
(C-30), 171.0 (CH3CO), 169.8 (CO-Rhd), 169.5 (C-13), 158.7 (C-9′), 157.7 (C-4a′, C-10a′),
155.7 (C-3′, C-6′), 134.0 (C-1″), 133.8 (C-5″), 132.0 (C-1′, C-8′), 130.9 (C-2″), 130.0 (C-6″),
129.9 (C-3″), 128.4 (C-12), 126.9 (C-4″), 114.5, 114.3 (C-2′, C-7′), 113.5 (C-8a′, C-9a′),
96.4, 96.3 (C-4′, C-5′), 80.6 (C-3), 61.7 (C-9), 55.0 (C-5), 48.1 (C-18), 46.1 (N′(CH2CH3)2,
N″(CH2CH3)2), 45.9 (4×CONCH2), 45.3 (C-8), 44.4 (C-20), 44.3 (C-19), 43.3 (C-14), 38.8
19

ACCEPTED MANUSCRIPT
(C-1), 38.0 (C-4), 37.7 (C-22), 36.9 (C-10), 32.8 (C-21, C-7), 31.8 (C-17), 29.7
(CONCH2CH2), 28.5 (C-28), 28.0 (C-23), 27.3 (C-29), 26.7 (C-16), 26.3 (C-15), 23.5 (C-2),
23.0 (C-27), 21.3 (CH3CO), 18.7 (C-26), 17.4 (C-6), 16.7 (C-24), 16.4 (C-25), 12.6
(N′(CH2CH3)2, N″(CH2CH3)2) ppm; MS [ESI, MeOH for C65H87ClN4O6 (1055.88)]: m/z (%)
1019.7 (100) [M-Cl]+.
4.4.16. 9-[2-[[4-[(3b)-3-Acetyloxy-20(29)en-28-oxo-lup-28-yl]-1-
homopiperazinyl]carbonyl]-phenyl]-3,6-bis(diethylamino)-
xanthylium chloride (20)
As described above for 16 to a solution of rhodamine B chloride in dry CH2Cl2 (10 mL), 15
(0.26 g, 0.46 mmol, 1 eq.), NEt3 (70 mg, 0.10 mL, 0.69 mmol, 1.5 eq.) and DMAP (5 mol-%)
were added. Work-up as described and purification by column chromatography afforded 20 in
58% yield; m.p. 256–260 °C; Rf 0.5 (MeCN/CH2Cl2/H2O, 10:1:1; UV-Vis (MeOH): λmax (log
ε) = 275 nm (4.32), 646 nm (4.84); IR (KBr): ν = 3448br, 2940m, 1590s, 1467m, 1413m,
1
1338m, 1247m, 1181m, 1133m, 1074m, 684m cm-1; H NMR (500 MHz, CDCl3): δ = 7.68–
7.57 (m, 2 H, 3″-H, 5″-H), 7.47–7.42 (m, 1 H, 4″-H), 7.33–7.18 (m, 3 H, 6″-H, 1′-H, 8′-H),
7.10–6.87 (m, 2 H, 2′-H, 7′-H), 6.86–6.70(m, 2 H, 4′-H, 5′-H), 4.71 (m, 1 H, 29-Ha), 4.55 (m,
1 H, 29-Hb), 4.44 (m, 1 H, 3-H), 3.80–3.26 (m, 16 H, 4×CONCH2, N′(CH2CH3)2,
N″(CH2CH3)2), 2.99 (m, 1 H, 19-H), 2.84 (m, 1 H, 13-H), 2.10 (m, 1 H, 16-Ha), 2.02 (s, 3 H,
CH3CO), 1.91 (m, 1 H, 22-Ha), 1.84–1.72 (m, 3 H, CONCH2CH2, 21-Ha), 1.71–1.54 (m, 4 H,
12-Ha, 1-Ha, 2-H2), 1.65 (s, 3 H, 30-H3), 1.52 (m, 1 H, 18-H), 1.49–1.16 (m, 9 H, 6-H2, 11-H2,
15-Ha, 21-Hb, 22-Hb, 7-H2, 9-H, 16-Hb), 1.12 (m, 1 H, 15-Hb), 1.31 (m, 12 H, CONCH2CH2,
N′(CH2CH3)2, N″(CH2CH3)2), 0.98–0.87 (m, 2 H, 1-Hb, 12-Hb), 0.90 (s, 6 H, 27-H3, 26-H3),
13
0.81 (s, 9 H, 25-H3, 24-H3, 23-H3), 0.76 (m, 1 H, 5-H ppm; C NMR (125 MHz, CDCl3):
δ = 174.5 (C-28), 171.0 (CH3CO), 168.6 (CO-Rhd), 168.1 (C-9′), 157.7 (C-4a′, C-10a′),
155.7, 155.6 (C-3′, C-6′), 151.4 (C-20), 133.8 (C-1″), 132.5 (C-1′, C-8′), 130.2 (C-2″), 130.0
(C-6″), 129.9 (C-5″), 129.5 (C-3″), 126.7 (C-4″), 114.0, 113.7 (C-2′, C-7′), 113.5 (C-8a′,
C-9a′), 109.2 (C-29), 96.4, 96.3 (C-4′, C-5′), 81.0 (C-3), 55.5 (C-5), 54.9 (C-17), 52.7 (C-18),
50.8 (C-9), 46.2, 46.1 (N′(CH2CH3)2, N″(CH2CH3)2), 45.9 (C-19), 42.8 (4×CONCH2), 42.0
(C-14), 40.7 (C-8), 38.4 (C-1), 38.3 (CONCH2CH2), 37.8 (C-4), 37.1 (C-10), 36.8 (C-13),
36.1 (C-22), 34.3 (C-7), 32.3 (C-16), 31.5 (C-21), 29.9 (C-15), 27.9 (C-23), 25.5 (C-12), 23.7
(C-2), 21.3 (CH3CO), 21.2 (C-11), 19.4 (C-30), 18.2 (C-6), 16.5 (C-24), 16.3 (C-25), 16.2
20

ACCEPTED MANUSCRIPT
(C-26), 14.6 (C-27), 12.7 (N′(CH2CH3)2, N″(CH2CH3)2) ppm; MS [ESI, MeOH for
C65H89ClN4O5 (1041.90)]: m/z (%) 1005.8 (100) [M-Cl]+.
4.4.17. 11-Keto-β-boswellic acid methyl ester (21)
To a solution of 10 (6.07 g, 13 mmol, 1.0 eq.) in DMF (100 mL) finely grounded potassium
carbonate (2.0 g, 14 mmol, 1.1 eq.) was added. After stirring at ambient temperature for
30 min, methyl iodide (2.2 g, 1.0 mL, 15 mmol, 1.2 eq.) was added, and stirring was
continued for 24 h. The mixture was slowly added to 5% hydrochloric acid (300 mL) and a
precipitate was formed. The crude product was filtered off and washed with water
(2×100 mL). After purification by column chromatography (silica gel, [hexane/ethyl
acetate/HOAc, 7:3:0.5%]:CHCl3, 1:1) 21 (5.73 g, 92%) was obtained as a white solid; m.p.
226–228 °C [α]D = +111.2° (c = 4.34, CHCl3) [lit. m.p. 220–225 °C, [α]D = +111.2° (c = 4.34,
CHCl3)][16]
.
4.4.18. 11-Keto-β-boswellic acid benzyl ester (22)
As described for the synthesis of 21, from 10 (10.0 g, 22 mmol, 1.0 eq.) and benzyl bromide
(4.1 g, 2.9 mL, 24 mmol, 1.1 eq.) followed by aqueous workup and re-crystallisation from
MeOH, compound 22 was obtained as a white solid in 57% yield; m.p. 202–204 °C; Rf 0.23
(hexane/ethyl acetate, 5:1); [α]D = +111.0° (c = 0.37, CHCl3); UV-Vis (MeOH): λmax (log
ε) = 264 nm (3.97); IR (KBr): ν = 3448br, 2928m, 1726s, 1644s, 1456m, 1385m, 1214m,
1169m, 1107m, 1056m, 968m, 754m, 698m cm-1; 1H NMR (500 MHz, CDCl3): δ = 7.37–7.29
(m, 5 H, Ph), 5.53 (s, 1 H, 12-H), 5.15 (d, J = 12.3 Hz, 1 H, CHaPh), 5.09 (d, J = 12.3 Hz,
1 H, CHbPh), 4.13 (m, 1 H, 3-H), 2.49 (ddd, J = 13.4, 13.4, 3.3 Hz, 1 H, 1-Ha), 2.41 (s, 1 H, 9-
H), 2.30 (dddd, J = 14.8, 14.8, 4.3, 2.6 Hz, 1 H, 2-Ha), 2.09 (ddd, J = 13.7, 13.7, 4.8 Hz, 1 H,
16-Ha), 1.87 (ddd, J = 13.7, 13.7, 5.2 Hz, 1 H, 15-Ha), 1.82 (m, 1 H, 6-Ha), 1.72 (m, 1 H, 6-
Hb), 1.65 (ddd, J = 13.0, 13.0, 3.8 Hz, 1 H, 7-Ha), 1.58–1.51 (m, 2 H, 2-Hb, 18-H), 1.51–1.38
(m, 5 H, 22-Ha, 5-H, 21-Ha, 7-Hb, 19-H), 1.36–1.24 (m, 3 H, 22-Hb, 1-Hb, 21-Hb), 1.32 (s,
3 H, 23-H3), 1.31 (s, 3 H, 27-H3), 1.20 (m, 1 H, 15-Hb), 1.12 (s, 3 H, 26-H3), 1.00 (m, 1 H, 16-
Hb), 0.97 (s, 3 H, 25-H3), 0.95 (s, 3 H, 30-H3), 0.94 (m, 1 H, 20-H), 0.82 (s, 3 H, 28-H3), 0.80
13
(d, J = 6.6 Hz, 3 H, 29-H3) ppm; C NMR (125 MHz, CDCl3): δ = 199.5 (C-11), 176.4 (C-
24), 164.9 (C-13), 135.7 (Ci-Ph), 130.5 (C-12), 128.5 (Cm-Ph), 128.4 (Co-Ph), 128.2 (Cp-Ph),
70.7 (C-3), 66.2 (CH2Ph), 60.4 (C-9), 59.0 (C-18), 48.9 (C-5), 47.5 (C-4), 45.1 (C-8), 43.8 (C-
14), 40.9 (C-22), 39.3 (C-19, C-20), 37.4 (C-10), 34.0 (C-1, C-17), 32.9 (C-7), 30.9 (C-21),
28.8 (C-28), 27.5 (C-16), 27.2 (C-15), 26.3 (C-2), 24.2 (C-23), 21.1 (C-30), 20.5 (C-27), 18.9
21

ACCEPTED MANUSCRIPT
(C-6), 18.2 (C-26), 17.4 (C-29), 13.3 (C-25) ppm; MS [ESI, MeOH for C37H52O4 (560.82)]:
m/z (%) 1143.3 (100) [2M+Na]+, 1121.2 (29) [2M+H]+, 583.3 (11) [M+Na]+, 561.4 (22)
[M+H]+.
4.4.19. 3-O-Acetyl-11-keto-β-boswellic acid benzylamide (23)
To an ice-cold solution of 5 (2.0 g, 4.0 mmol, 1.0 eq.) in dry CH2Cl2 (20 mL), oxalyl chloride
(2.0 g, 1.4 mL, 16 mmol, 4.0 eq.) was slowly added, and the mixture was allowed to warm to
ambient temperature, and stirring was continued for 3 h. The solvent was removed under
reduced pressure, the residue dissolved in dry THF (10 mL), and the volatiles were again
evaporated. To a solution of the residue in dry CH2Cl2 (15 mL), benzyl amine (1.3 g, 1.3 mL,
12 mmol, 3.0 eq.), NEt3 (0.6 g, 0.3 mL, 5.8 mmol, 1.5 eq.) and DMAP (5 mol-%) were added,
and stirring was continued overnight. Evaporation of the solvent under reduced pressure and
purification of the residue by chromatography (silica gel, hexane/ethyl acetate, 5:1) afforded
23 as a white solid in 80% yield; m.p 173–178 °C; Rf 0.4 (CHCl3/MeOH, 9:1); [α]D = +52.2°
(c = 0.36, CHCl3); UV-Vis (MeOH): λmax (log ε) = 280 nm (3.35); IR (KBr): ν = 3442br,
1
2927m, 1737m, 1654m, 1522m, 1455m, 1372m, 1247m, 1027m, 699m cm-1; H NMR
(500 MHz, CDCl3): δ = 7.38–7.27 (m, 5 H, Ph), 5.74 (dd, J = 5.5, 5.1 Hz, 1 H, NH), 5.56 (s,
1 H, 12-H), 5.33 (m, 1 H, 3-H), 4.47 (dd, J = 14.4, 5.7 Hz, 1 H, CHaPh), 4.37 (dd, J = 14.4,
4.9 Hz, 1 H, CHbPh), 2.55 (ddd, J = 13.2, 13.2, 3.5 Hz, 1 H, 1-Ha), 2.41 (s, 1 H, 9-H), 2.31
(dddd, J = 14.6, 14.6, 4.2, 2.5 Hz, 1 H, 2-Ha), 2.10 (m, 1 H, 16-Ha), 2.09 (s, 3 H, CH3CO),
1.89 (m, 1 H, 15-Ha), 1.81–1.60 (m, 4 H, 6-H2, 7-Ha, 2-Hb), 1.55 (d, J = 11.4 Hz, 1 H, 18-H),
1.53–1.44 (m, 3 H, 22-Ha, 7-Hb, 21-Ha), 1.42 (m, 1 H, 5-H), 1.40 (m, 1 H, 19-H), 1.35 (s, 3 H,
27-H3), 1.39–1.19 (m, 4 H, 22-Hb, 21-Hb, 15-Hb, 1-Hb,), 1.18 (s, 3 H, 23-H3), 1.17 (s, 3 H, 26-
H3), 1.11 (s, 3 H, 25-H3), 1.02 (m, 1 H, 16-Hb), 0.96 (s, 3 H, 30-H3), 0.88 (m, 1 H, 20-H), 0.83
(s, 3 H, 28-H3), 0.81 (d, J = 6.4 Hz, 3 H, 29-H3) ppm; 13C NMR (125 MHz, CDCl3): δ = 199.0
(C-11), 174.9 (C-24), 170.1 (CH3CO), 164.6 (C-13), 138.1 (Ci-Ph), 130.6 (C-12), 128.8 (Cm-
Ph), 128.0 (Co-Ph), 127.6 (Cp-Ph), 73.5 (C-3), 60.3 (C-9), 59.0 (C-18), 50.5 (C-5), 46.7 (C-4),
45.0 (C-8), 43.8 (CH2Ph), 43.7 (C-14), 40.9 (C-22), 39.3 (C-19), 39.2 (C-20), 37.4 (C-10),
34.9 (C-1), 34.0 (C-17), 33.1 (C-7), 30.9 (C-21), 28.8 (C-28), 27.5 (C-16), 27.2 (C-15), 24.7
(C-23), 24.0 (C-2), 21.3 (CH3CO), 21.1 (C-30), 20.5 (C-27), 19.5 (C-6), 18.3 (C-26), 17.4 (C-
29), 13.5 (C-25) ppm; MS [ESI, MeOH for C39H55NO4 (601.41)]: m/z (%) 1225.3 (54)
[2M+Na]+, 922.4 (8) [3M+Ca]+, 624.4 (67) [M+Na]+, 602.3 (100) [M+H]+.
4.4.20. 11-Keto-β-boswellic acid benzylamide (24)
22

ACCEPTED MANUSCRIPT
To a solution of 23 (1.8 g, 3.1 mmol, 1 eq.) in MeOH (50 mL), KOH (0.9 g, 15.7 mmol, 5 eq.)
was added, and the mixture was stirred at ambient temperature for 3 d. The mixture was
slowly added to hydrochloric acid (1 M, 15 mL). The aqueous phase was extracted with
CH2Cl2 (4×15 mL), the combined organic phases were washed with water (20 mL), brine
(20 mL) and dried over MgSO4. Evaporation of the solvent under reduced pressure and
purification of the residue by column chromatography (silica gel, hexane/ethyl acetate, 5:1)
afforded 24 as a white solid in 76% yield; m.p. 216–218 °C; Rf 0.13 (hexane/ethyl acetate,
2:1); [α]D = +118.0° (c = 0.35, CHCl3); UV-Vis (MeOH): λmax (log ε) = 266 nm (3.83); IR
(KBr): ν = 3423br, 2925m, 2869m, 1728m, 1655s, 1511m, 1455m, 1382m, 1234m, 1200m,
1057m, 966m, 700m cm-1; 1H NMR (500 MHz, CDCl3): δ = 7.35–7.25 (m, 5 H, Ph), 5.75 (dd,
J = 5.5, 5.2 Hz, 1 H, NH), 5.55 (s, 1 H, 12-H), 4.48 (dd, J = 14.6, 5.5 Hz, 1 H, CHaPh), 4.37
(dd, J = 14.6, 5.2 Hz, 1 H, CHbPh), 4.13 (m, 1 H, 3-H), 2.50 (ddd, J = 13.7, 13.7, 3.5 Hz, 1 H,
1-Ha), 2.43 (s, 1 H, 9-H), 2.42 (dddd, J = 14.4, 14.4, 4.5, 2.5 Hz, 1 H, 2-Ha), 2.09 (ddd,
J = 13.7, 13.7, 4.7 Hz, 1 H, 16-Ha), 1.86 (ddd, J = 13.7, 13.7, 4.7 Hz, 1 H, 15-Ha), 1.75–1.66
(m, 3 H, 6-H2, 7-Ha), 1.56 (m, 1 H, 2-Hb), 1.53 (d, J = 11.0 Hz, 1 H, 18-H), 1.51–1.42 (m,
4 H, 5-H, 22-Ha, 7-Hb, 21-Ha), 1.41–1.30 (m, 4 H, 22-Hb, 19-H, 1-Hb, 21-Hb), 1.31 (s, 3 H,
27-H3), 1.29 (s, 3 H, 23-H3), 1.19 (m, 1 H, 15-Hb), 1.15 (s, 3 H, 26-H3), 1.09 (s, 3 H, 25-H3),
1.00 (m, 1 H, 16-Hb), 0.95 (s, 3 H, 30-H3), 0.94 (m, 1 H, 20-H), 0.82 (s, 3 H, 28-H3), 0.79 (d,
13
J = 6.5 Hz, 3 H, 29-H3) ppm; C NMR (125 MHz, CDCl3): δ = 199.2 (C-11), 176.2 (C-24),
164.7 (C-13), 138.3 (Ci-Ph), 130.5 (C-12), 128.7 (Cm-Ph), 127.9 (Co-Ph), 127.5 (Cp-Ph), 70.8
(C-3), 60.4 (C-9), 59.0 (C-18), 48.8 (C-5), 47.4 (C-4), 45.0 (C-8), 43.8 (C-14), 43.7 (CH2Ph),
40.9 (C-22), 39.3 (C-19), 39.2 (C-20), 37.6 (C-10), 34.2 (C-1), 34.0 (C-17), 33.2 (C-7), 30.9
(C-21), 28.8 (C-28), 27.5 (C-16), 27.2 (C-15), 26.6 (C-2), 25.1 (C-23), 21.1 (C-30), 20.5 (C-
27), 19.7 (C-6), 18.3 (C-26), 17.4 (C-29), 13.5 (C-25) ppm; MS [ESI, MeOH for C37H53NO3
(559.84)]: m/z (%) 1141.4 (87) [2M+Na]+, 1119.2 (48) [2M+H]+, 859.5 (8) [3M+Ca]+, 582.3
(6) [M+Na]+, 560.3 (100) [M+H]+, 542.3 (11) [M+H-H2O]+.
4.4.21. 3,6-Bis(diethylamino)-9-[2-[3α,4β)-[24-methoxy-11,24-dioxours-12-
en-3-oxycarbonyl]-phenyl]-xanthylium chloride (25)
As described above for 16 to a solution of rhodamine B chloride in dry CH2Cl2 (10 mL), 21
(0.44 g, 0.91 mmol, 1 eq.), NEt3 (0.14 g, 0.15 mL, 1.38 mmol, 1.5 eq.) and DMAP (5 mol-%)
were added. Work up as described and purification by column chromatography afforded 25 in
43% yield; m.p. 165–170 °C; Rf 0.5 (MeCN/ CH2Cl2/H2O, 10:1:1); UV-Vis (MeOH): λmax
(log ε) = 270 nm (4.40), 641 nm (4.97); IR (KBr): δ = 3441br, 2973m, 1723m, 1648m, 1590s,
23

ACCEPTED MANUSCRIPT
1
1467m, 1413m, 1338m, 1274m, 1181m, 1133m, 1075m, 683m cm-1; H NMR (500 MHz,
CDCl3): δ = 8.18 (dd, J = 7.8, 0.9 Hz, 1 H, 3″-H), 7.87 (dt, J = 7.6, 1.2 Hz, 1 H, 5″-H), 7.78
(dt, J = 7.8, 1.2 Hz, 1 H, 4″-H), 7.38 (dd, J = 7.6, 1.0 Hz, 1 H, 6″-H), 7.10, 7.09 (2×d,
J = 9.5 Hz, 2 H, 1′-H, 8′-H), 6.97, 6.84 (2×dd, J = 9.5, 2.4 Hz, 2 H, 2′-H, 7′-H), 6.78, 6.77
(2×d, J = 2.4 Hz, 2 H, 4′-H, 5′-H), 5.57 (s, 1 H, 12-H), 5.29 (m, 1 H, 3-H), 3.62 (s, 3 H,
OMe), 3.63 (m, 8 H, N(CH2CH3)2, N′(CH2CH3)2), 2.59 (ddd, J = 13.3, 13.3, 3.3 Hz, 1 H,
1-Ha), 2.47 (s, 1 H, 9-H), 2.20–2.09 (m, 2 H, 2-Ha, 16-Ha), 1.92 (ddd, J = 13.2, 13.2, 4.7 Hz,
1 H, 15-Ha), 1.85–1.71 (m, 3 H, 6-H2, 7-Ha), 1.56 (d, J = 11.0 Hz, 1 H, 18-H), 1.54–1.43 (m,
6 H, 7-Hb, 22-Ha, 21-Ha, 5-H, 19-H, 2-Hb), 1.42 (s, 3 H, 27-H3), 1.37–1.22 (m, 4 H, 22-Hb,
21-Hb, 1-Hb, 15-Hb), 1.32 (t, J = 7.2 Hz, 12 H, N(CH2CH3)2, N′(CH2CH3)2), 1.20 (s, 3 H,
26-H3), 1.14 (s, 3 H, 23-H3), 1.06 (m, 1 H, 16-Hb), 1.03 (s, 3 H, 25-H3), 0.97 (s, 3 H, 30-H3),
13
0.96 (m, 1 H, 20-H), 0.84 (s, 3 H, 28-H3), 0.83 (d, J = 6.0 Hz, 3 H, 29-H3) ppm; C NMR
(125 MHz, CDCl3): δ = 199.1 (C-11), 175.7 (C-24), 165.4 (C-13), 164.2 (CO-Rhd), 158.9
(C-9′), 157.8, 157.7 (C-4a′, C-10a′), 155.7, 155.4 (C-3′, C-6′), 133.9 (C-1″), 133.3 (C-5″),
131.2, 131.1 (C-1′, C-8′), 130.6 (C-6″, C-2″), 130.5 (C-4″), 130.4 (C-12), 130.2 (C-3″), 114.5,
113.9 (C-2′, C-7′), 113.6, 113.5 (C-8a′, C-9a′), 96.4, 96.3 (C-4′, C-5′), 75.0 (C-3), 60.5 (C-9),
59.1 (C-18), 51.6 (OMe), 51.1 (C-5), 46.8 (C-4), 46.2, 46.1 (N(CH2CH3)2, N′(CH2CH3)2),
45.1 (C-8), 43.8 (C-14), 40.9 (C-22), 39.3 (C-19, C-20), 37.2 (C-10), 35.0 (C-1), 34.0 (C-17),
32.9 (C-7), 30.9 (C-21), 28.9 (C-28), 27.5 (C-16), 27.2 (C-15), 24.0 (C-23), 23.6 (C-2), 21.1
(C-30), 20.5 (C-27), 18.8 (C-6), 18.4 (C-26), 17.5 (C-29), 13.1 (C-25), 12.6 (N(CH2CH3)2,
N′(CH2CH3)2) ppm; MS [ESI, MeOH for C59H77N2O6Cl (945.72)]: m/z (%) 909.5 (100) [M-
Cl]+.
4.4.22. 3,6-Bis(diethylamino)-9-[2-[(3α,4β)-[24-benzyloxy-11,24-dioxours-
12-en-3-oxycarbonyl]-phenyl]-xanthylium chloride (26)
As described above for 16 to a solution of rhodamine B chloride in dry CH2Cl2 (10 mL), 22
(0.32 g, 0.57 mmol, 1 eq.), NEt3 (87 mg, 0.18 mL, 0.86 mmol, 1.5 eq.) and DMAP (5 mol-%)
were added. Work-up as described and purification by column chromatography afforded 26 in
58% yield; m.p. 175–180 °C; Rf 0.5 (MeCN/ CH2Cl2/H2O, 10:1:1); UV-Vis (MeOH): λmax
(log ε) = 273 nm (4.42), 641 nm (4.99); IR (KBr): ν = 3442br, 2974m, 1720m, 1648m, 1590s,
1
1467m, 1413m, 1338m, 1274m, 1181m, 1133m, 1075m, 683m cm-1; H NMR (500 MHz,
CDCl3): δ = 8.17 (dd, J = 7.9, 0.8 Hz, 1 H, 3″-H), 7.86 (dt, J = 7.6, 1.2 Hz, 1 H, 5″-H), 7.76
(dt, J = 7.8, 1.1 Hz, 1 H, 4″-H), 7.36 (dd, J = 7.6, 1.0 Hz, 1 H, 6″-H), 7.32–7.25 (m, 5 H, Ph),
24

ACCEPTED MANUSCRIPT
7.08, 7.07 (2×d, J = 9.7 Hz, 2 H, 1′-H, 8′-H), 6.95, 6.82 (2×dd, J = 9.7, 2.4 Hz, 2 H, 2′-H,
7′-H), 6.77, 6.76 (2×d, J = 2.4 Hz, 2 H, 4′-H, 5′-H), 5.54 (s, 1 H, 12-H), 5.28 (m, 1 H, 3-H),
5.09 (d, J = 12.2 Hz, 1 H, CHaPh), 5.00 (d, J = 12.2 Hz, 1 H, CHbPh), 3.62 (q, J = 7.2 Hz,
4 H, NCH2CH3), 3.61 (q, J = 7.2 Hz, 4 H, N′CH2CH3), 2.56 (ddd, J = 13.1, 13.1, 3.2 Hz, 1 H,
1-Ha), 2.45 (s, 1 H, 9-H), 2.15 (m, 1 H, 2-Ha), 2.12 (ddd, J = 13.7, 13.7, 4.7 Hz, 1 H, 16-Ha),
1.89 (ddd, J = 13.7, 13.7, 4.8 Hz, 1 H, 15-Ha), 1.79 (m, 2 H, 6-H2), 1.73 (ddd, J = 12.7, 12.7,
3.7 Hz, 1 H, 7-Ha), 1.55 (d, J = 11.2 Hz, 1 H, 18-H), 1.52–1.41 (m, 6 H, 22-Ha, 5-H, 7-Hb,
21-Ha, 19-H, 2-Hb), 1.40 (s, 3 H, 27-H3), 1.38–1.20 (m, 4 H, 22-Hb, 21-Hb, 1-Hb, 15-Hb), 1.31
(t, J = 7.0 Hz, 12 H, N(CH2CH3)2, N′(CH2CH3)2), 1.15 (s, 3 H, 23-H3), 1.13 (s, 3 H, 26-H3),
1.04 (m, 1 H, 16-Hb), 0.96 (s, 3 H, 30-H3), 0.95 (m, 1 H, 20-H), 0.94 (s, 3 H, 25-H3), 0.82 (br
13
s, 6 H, 28-H3, 29-H3) ppm; C NMR (125 MHz, CDCl3): δ = 199.1 (C-11), 174.9 (C-24),
165.4 (C-13), 164.1 (CO-Rhd), 158.8 (C-9′), 157.8, 157.7 (C-4a′, C-10a′), 155.6, 155.4 (C-3′,
C-6′), 135.1 (Ci-Ph), 133.9 (C-1″), 133.3 (C-5″), 131.2, 131.1 (C-1′, C-8′), 130.6 (C-6″),
130.5 (C-2″, C-4″), 130.4 (C-12), 130.2 (C-3″), 128.6 (Cm-Ph), 128.5 (Co-Ph), 128.4 (Cp-Ph),
114.5, 113.9 (C-2′, C-7′), 113.6, 113.5 (C-8a′, C-9a′), 96.4, 96.3 (C-4′, C-5′), 75.0 (C-3), 66.7
(CH2Ph), 60.4 (C-9), 59.1 (C-18), 51.2 (C-5), 46.9 (C-4), 46.2, 46.1 (N(CH2CH3)2,
N′(CH2CH3)2), 45.0 (C-8), 43.7 (C-14), 40.9 (C-22), 39.3 (C-19, C-20), 37.3 (C-10), 35.0
(C-1), 34.0 (C-17), 32.9 (C-7), 30.8 (C-21), 28.8 (C-28), 27.5 (C-16), 27.2 (C-15), 24.0
(C-23), 23.6 (C-2), 21.1 (C-30), 20.5 (C-27), 18.8 (C-6), 18.2 (C-26), 17.5 (C-29), 13.3
(C-25), 12.6 (N(CH2CH3)2, N′(CH2CH3)2) ppm; MS [ESI, MeOH for C65H81N2O6Cl
(1021.82)]: m/z (%) 985.6 (100) [M-Cl]+.
4.4.23. 3,6-Bis(diethylamino)-9-[2-[(3α,4β)-[24-benzylamino-11,24-
dioxours-12-en-3-oxycarbonyl]-phenyl]-xanthylium chloride (27)
As described above for 16 to a solution of rhodamine B chloride in dry CH2Cl2 (10 mL), 24
(0.20 g, 0.29 mmol, 1 eq.), NEt3 (44 mg, 0.06 mL, 0.44 mmol, 1.5 eq.) and DMAP (5 mol-%)
were added. Work-up as described and purification by column chromatography afforded 27 in
57% yields; m.p. 195–205 °C; Rf 0.5 (MeCN/CH2Cl2/H2O, 10:1:1); UV-Vis (MeOH): λmax
(log ε) = 270 nm (4.21), 640 nm (4.76); IR (KBr): δ = 3442br, 2926m, 1718m, 1648m, 1590s,
1
1466m, 1413m, 1338m, 1247m, 1181m, 1075m, 923m, 683m cm-1; H NMR (500 MHz,
CDCl3): δ = 8.16 (dd, J = 7.9, 0.9 Hz, 1 H, 3″-H), 7.82 (dt, J = 7.5, 1.0 Hz, 1 H, 5″-H), 7.75
(dt, J = 7.9, 1.0 Hz, 1 H, 4″-H), 7.34 (dd, J = 7.9, 1.0 Hz, 1 H, 6″-H), 7.30–7.16 (m, 5 H, Ph),
7.10, 7.07 (2×d, J = 9.8 Hz, 2 H, 1′-H, 8′-H), 6.97, 6.85 (2×dd, J = 9.6, 2.4 Hz, 2 H, 2′-H,
25

ACCEPTED MANUSCRIPT
7′-H), 6.76, 6.74 (2×d, J = 2.4 Hz, 2 H, 4′-H, 5′-H), 6.06 (m, 1 H, NH), 5.54 (s, 1 H, 12-H),
5.30 (m, 1 H, 3-H), 4.37–4.32 (m, 2 H, CH2Ph), 3.62 (m, 8 H, N′CH2CH3, N″CH2CH3), 2.56
(ddd, J = 13.0, 13.0, 3.5 Hz, 1 H, 1-Ha), 2.44 (s, 1 H, 9-H), 2.34 (m, 1 H, 2-Ha), 2.11 (ddd,
J = 13.6, 13.6, 4.6 Hz, 1 H, 16-Ha), 1.89–1.82 (m, 1 H, 15-Ha), 1.77 (m, 1 H, 6-Ha), 1.61–1.34
(m, 11 H, 6-Hb, 2-Hb, 21-Ha, 7-H2, 1-Hb, 22-H2, 18-H, 5-H, 19-H), 1.39 (s, 3 H, 27-H3), 1.31
(t, J = 7.0 Hz, 12 H, N′(CH2CH3)2, N″(CH2CH3)2), 1.28–1.18 (m, 2 H, 21-Hb, 1-Hb), 1.12 (s,
3 H, 26-H3), 1.11 (s, 3 H, 23-H3), 1.05–0.84 (m, 3 H, 15-Hb, 16-Hb, 20-H), 0.99 (s, 3 H,
13
25-H3), 0.95 (s, 3 H, 30-H3), 0.82 (d, J = 6.2 Hz, 3 H, 29-H3), 0.80 (s, 3 H, 29-H3) ppm; C
NMR (125 MHz, CDCl3): δ = 199.1 (C-11), 175.2 (C-24), 165.2 (C-13), 164.0 (CO-Rhd),
159.0 (C-9′), 157.8 (C-4a′, C-10a′), 155.4 (C-3′, C-6′), 138.3 (Ci-Ph), 133.7 (C-2″), 132.8
(C-5″), 131.3, 131.2 (C-1′, C-8′), 130.9 (C-1″), 130.5 (C-4″), 130.4 (C-12), 130.3 (C-6″,
C-3″), 129.0 (Cp-Ph), 128.5 (Co-Ph), 128.0 (Cm-Ph), 114.6, 114.4 (C-2′, C-7′), 113.8, 113.5
(C-8a′, C-9a′), 96.2 (C-4′, C-5′), 75.7 (C-3), 60.6 (C-9), 59.0 (C-18), 51.0 (C-5), 47.2 (C-4),
46.8, 46.2 (N′(CH2CH3)2, N″(CH2CH3)2), 45.0 (C-8), 43.7 (CH2Ph), 43.6 (C-14), 40.1 (C-22),
39.3 (C-19, C-20), 37.4 (C-10), 35.4 (C-1), 33.8 (C-17), 33.1 (C-7), 31.1 (C-21), 28.7 (C-28),
27.5 (C-16), 27.2 (C-15), 24.5 (C-23), 23.7 (C-2), 21.1 (C-30), 20.4 (C-6), 20.4 (C-27), 18.2
(C-26), 17.5 (C-29), 13.3 (C-25), 12.5 (N′(CH2CH3)2, N″(CH2CH3)2) ppm; MS [ESI, MeOH
for C65H82N3O5Cl (1020.84)]: m/z (%) 984.5 (100) [M-Cl]+.
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