Synthesis of antitumor-active betulinic acid-derived hydroxypropargylamines by copper-catalyzend mannich reactions

Article abstract of DOI:10.1002/ardp.201200428

Several novel betulin derivates were prepared using Mannich reactions as a key step. Starting from 3-ethynyl-3-hydroxy-lup-20(29)-ene derivatives, copper-catalyzed Mannich reactions yielded hydroxypropargyl ammonium hydrochlorides or their corresponding methiodides. All compounds were screened in a sulforhodamine B assay for their antitumor activity using a panel of 9 human cancer cell lines. Some of these compounds showed significant cytotoxicity; they act by triggering apoptotic cell death as shown by additional acridine orange/propidium iodide assays, Trypan blue tests, DNA laddering experiments, and investigations of the cell cycle. Using copper-catalyzed Mannich reactions as a key step, several novel betulin derivates were prepared. These were screened for their antitumor activity using a panel of 9 human cancer cell lines. Some of the compounds showed significant cytotoxicity by triggering apoptotic cell death.

Full text of DOI:10.1002/ardp.201200428

Arch. Pharm. Chem. Life Sci. 2013, 000, 1–15
1
Full Paper
Synthesis of Antitumor-Active Betulinic Acid-Derived
Hydroxypropargylamines by Copper-Catalyzend Mannich
Reactions
´
Rene Csuk, Christoph Nitsche, Ronny Sczepek, Stefan Schwarz, and Bianka Siewert
¨
Bereich Organische Chemie, Martin-Luther-Universitat Halle-Wittenberg, Saale, Germany
Several novel betulin derivates were prepared using Mannich reactions as a key step. Starting
from 3-ethynyl-3-hydroxy-lup-20(29)-ene derivatives, copper-catalyzed Mannich reactions yielded
hydroxypropargyl ammonium hydrochlorides or their corresponding methiodides. All compounds
were screened in a sulforhodamine B assay for their antitumor activity using a panel of 9 human
cancer cell lines. Some of these compounds showed significant cytotoxicity; they act by triggering
apoptotic cell death as shown by additional acridine orange/propidium iodide assays, Trypan blue
tests, DNA laddering experiments, and investigations of the cell cycle.
Keywords: Antitumor-active compounds / Apoptosis / Betulinic acid / Cell cycle / Mannich reaction
Received: November 14, 2012; Revised: December 10, 2012; Accepted: December 21, 2012
DOI 10.1002/ardp.201200428
Introduction
towards human tumor cell lines compared to parent betulin.
Here, we report our new strategy to combine the higher
cytotoxic activity of alkynyl substituted compounds with
improved water solubility by attaching amino group-bearing
functionalities employing the alkynyl groups in Mannich
reactions.
The natural occurring lupane-type triterpenes betulin and
betulinic acid (BA) (Fig. 1) are widely spread in the plant
kingdom. These compounds gained interest in medicinal
research because of their antiviral [1, 2], antiplasmodial [3],
and anti-inflammatorial [1] activity. Of special interest, how-
ever, is the ability of BA [4] and many of its derivatives [5–7] to
inhibit the growth of human tumor cell lines and in vivo even
of several tumors by triggering apoptosis [8–10]. In an animal
model, BA showed selective cytotoxicity for melanoma cells
with no acute or chronic side effects for nonmalignant cells [4].
BA, however, is only slightly soluble in aqueous solvents.
Therefore, BA derivatives holding polar groups and hence
providing an increased bioavailability are called for. Alkynyl
substituted betulin derivatives [11–17] have scarcely been
prepared so far; even less their synthetic potential has been
explored. Previously, we were able to show [18, 19] that
alkynyl derivatives of betulin show an increased cytotoxicity
The Mannich reaction [20–23] has been known for exactly
100 years [24]; it has become an important tool for the
synthesis of pharmaceutical drugs [23] and more recently
in polymer chemistry [25–27]. Although Mannich reactions
using alkynes [28] have been reported as early as 1933, rather
harsh conditions and moderate yields have limited a wider
application. In general, many reaction conditions have been
described for Mannich reactions employing alkynes, aldehydes,
and amines [29–35]. They differ in solvents, temperatures,
pH-values, additives, and catalysts. A first screening of many
of these conditions resulted in the decision to use a copper-
catalyzed reaction variant that was developed by Reppe [36] as
early as 1955 and refined in 2004 by Bieber and da Silva [37].
Results and discussion
´
Correspondence: Prof. Dr. Rene Csuk, Bereich Organische Chemie,
Martin-Luther-Universitat Halle-Wittenberg, Kurt-Mothes-Strasse 2,
¨
Prerequisites to prepare propargylamines using Mannich
reactions are suitable C,H-acidic alkynes. The synthesis of
BA derivatives bearing an alkynyl group at carbon C3 was
realized by a Grignard reaction using ethynylmagnesium
bromide in THF [18, 19], starting from betulonic acid 1 or
D-06120 Halle, Saale, Germany
E-mail: rene.csuk@chemie.uni-halle.de
Fax: þ49 345 5527030
Abbreviations: AO/PI, acridine orange/propidium iodide; BA, betulinic
acid; SRB, sulforhodamine B.
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R. Csuk et al.
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–15
manner yielding (3 S) configurated compounds with an
axially oriented a-alkynol moiety in ring A of the triterpenoid
skeleton [38]. In diethylether as a solvent, however, invariably
mixtures of the diastereomeric compounds were formed. Due
to the complexation with the solvent THF, the Grignard
reagent is more bulky than in diethylether. The axially ori-
ented methyl group at carbon C4 and the methyl group at
carbon C10 hinder an attack of the organometallic reagent
along the Bu¨rgi–Dunitz-trajectory [39] from the upper side of
the molecule. This parallels quite recent findings of Wagner
and coworkers [40, 41].
The Mannich reactions were performed treating alkynols 3
and 4 in DMSO with several secondary amines, aqueous
formalin, and CuI as the catalyst for one up to several days
(Scheme 2) [37]. During these reactions, the corresponding
copper acetylide complexes and methylene-iminium ions are
formed, since reactions employing alkynes performed with-
out the copper catalyst did not proceed at all. This is in
excellent agreement with early findings of Reppe [36].
Figure 1. Structures of the parent compounds betulinic acid (BA)
and betulin.
its methyl ester 2. This gave alkynic starting materials 3 and 4
(Scheme 1). As shown by exhaustive N.o.e.-NMR experiments,
these Grignard reactions advanced in a diastereoselective
Scheme 1. Synthesis of 3-ethynylbetulinc acid derivatives 3 and 4: (a) HCꢀCMgBr, THF, 258C: 3 77% (from 1); 4 68% (from 2).
Scheme 2. Mannich reactions with alkynol 3 or 4: (a) secondary amine (R2NH), formalin, CuI (cat.) DMSO, 408C, 13–64%.
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Scheme 3. Synthesis of quaternary ammonium iodide salts 20 and 21 from propargylamines 12 and 15: (a) 1. KOH, 2. MeI, Et2O, 258C,
20: 89% (from 12), 21: 82% (from 15).
For the synthesis of the corresponding propargylamines 5–
19, acyclic and cyclic secondary amines where used (Table 1).
All compounds were isolated as their hydrochlorides, and the
copper catalyst was removed by flash chromatography.
The propargylamines 12 and 15 where transformed into
their corresponding methiodides 20 and 21, by reacting the
amines with methyl iodide (Scheme 3) [42, 43].
(Table 1, Fig. 2). Highest activity was found for the N-methyl-
piperazine derivative 14 with IC50-values between 2.5 and
5.8 mM for the different human cancer cell lines. Bulky
and more hydrophobic substituents (as in compounds 13
or 19) resulted in a reduced activity. Compound 18 carrying
a dihexylamine substituent showed significantly lower
activity than all other derivatives. For the methiodides 20
and 21 a lowered activity was found but these compounds
showed a higher selectivity between the different cell lines.
The selectivity index (IC50 tumor cells vs. NiH 3T3 fibroblasts),
however, is low throughout this series of compounds.
Apoptosis is a naturally occurring process by which a cell is
directed to programmed cell death. In this process, cells that
are detrimental to an organism are disposed of in a neat and
orderly manner; this prevents the development of an inflam-
Based on an initial report dealing with the cytotoxic
activity of BA [44] quite a huge number of studies dealt with
the antineoplastic activity of BA and derivatives [45, 46] in vivo
and in vitro. BA is almost insoluble in water but an aqueous
medium is usually the preferred formulation for injection
[47]. Thus, solubility data for several of our compounds have
been obtained in water/DMSO mixtures (95:5, v/v). Whereas
for BA, a solubility of 166 mg/mL was determined, for 5
175 mg/mL was found. The ester 9 showed a reduced solubil-
ity of 82 mg/mL – hence indicating that good solubility is not
an ultimate pre-requisite for achieving high cytotoxicity. The
derivatives 8 (65 mg/mL) and 12 (61 mg/mL) also exhibited a
reduced solubility but still showed high cytotoxicity.
Previous studies concerning modifications performed at
C-28 [18, 48–51] emphasized the need of the presence of a
carboxylic or carbonylic group at this position for obtaining
reasonably high cytotoxicity. Esterification of the C-28 car-
boxylic acid moiety employing lipophilic alcohols did not
result in compounds of improved antitumor activity [45].
Introduction of extra hydrophilic groups in ring A reduced
activity whereas derivatives showing aprotic polar functional
groups showed lower IC50 values [45]. Steric hindrance at
position C-3 seems crucial [52].
Testing of our compounds in colorimetric sulforhodamine
B (SRB) cell assays [53–55] showed a higher cytotoxicity for the
propargylamines compared to their parent alkynic precur-
sors. Propargylamines 5–19 mostly even showed an increased
cytotoxicity than naturally occurring BA. There is only a
limited influence of the secondary amine on the activity
Figure 2. Cytotoxicity of the propargylamine derivatives 3–20
measured in SRB assays using the A549 cancer cell line in
comparison to BA.
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Antitumor-Active Mannich Products
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Figure 3. AO/PI staining of A549 cancer cells after treatment with selected compounds (fluorescence microscopy): (A) BA (30 mM, 24 h);
(B) 5 (15 mM, 48 h); (C) 9 (10 mM, 48 h); (D) 12 (15 mM, 48 h); (E) 14 (10 mM, 48 h); (F) 20 (40 mM, 24 h).
matory response being often associated with necrotic cell
death. BA and most of its derivatives induce apoptosis [4, 47–
49, 56–60]. Many of our Mannich compounds showed good or
moderate antitumor activity. Since SRB tests a priori allow no
conclusion for an apoptotic cell death, selected compounds
were chosen for further studies; these experiments included
dye exclusion tests (acridine orange/propidium iodide (AO/PI)
and Trypan blue), DNA laddering experiments as well as
investigations concerning the cell cycle.
are summarized in Table 2. These results clearly confirm that
our compounds are able to trigger apoptosis.
Caspases are a family of cysteine proteases acting in
concert in a cascade triggered by apoptosis signaling. The
culmination of this cascade is the cleavage of a number of
proteins in the cell, followed by cell disassembly, cell death,
and, ultimately, the phagocytosis and removal of the cell
debris. As a result of the caspase cascade, transcription of
endonucleases is increased during apoptosis leading to a
cutting of intact DNA into smaller fragments of 180 bp
[62]. Experiments using A549 cancer cells and compounds
5, 9, 12, 14, and 20 (BA was used as an internal reference)
revealed the presence of ‘‘DNA ladders’’ (Fig. 4) typical of an
apoptotic cell death.
In contrast to necrosis, membranes of cells undergoing
apoptosis remain intact until a rather late stage of this
process of programmed cell death. The results from the
dying of the cells with AO/PI are shown in Fig. 3 for A549
human adenocarcinomic alveolar basal epithelial cells.
White arrows point out typical characteristics of an apoptotic
process, i.e. blebbing of the membrane and the condensation
of the chromatin. Cells treated with compounds 5, 9, or 12
(and to a lower extent with compound 14) revealed also the
presence of small cell compartments (probably lysosomes or
autophagosomes) exhibiting weak reddish fluorescence. This
parallels recent findings of Fulda and coworkers [61] for B10
(a glycosylated derivative of BA) to induce apoptosis as well as
to initiate macroautophagy.
Finally, some cell cycle experiments were performed by
dyeing the DNA of the A549 cancer cells with DNA-intercalat-
ing PI [63–67] followed by measurement of the living cells by
flow cytometry after having been incubated with the cyto-
Table 2. Results from the Trypan blue staining test of compounds
5, 9, 12, and 20 using A549 cells
Compound Apoptotic cell death (%)
Confidence interval
5
9
12
20
62.8
69.7
72.5
49.1
3.9
3.6
9.3
13.6
Trypan blue staining indicates the integrity of the cyto-
plasmatic membrane, hence allowing to draw some con-
clusions for a quantification of necrotic and apoptotic
cells. The results of these Trypan blue staining experiments
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(indicating the start and progressing of an apoptotic process).
Evaluation of the PI dyeing showed many cells in SubG1
phase (being quite typical of apoptosis according to
Darzynkiewicz et al. [65]). Also, a significant decrease of cells
in G2/M phase is observed; in some experiments an arrest of
cells in the S phase was observed, hence paralleling previous
findings of Santos et al. [62] and Kommera et al. [69] for tumor
cells treated with other triterpenoids. In conclusion, the
Mannich derivatives of BA have been shown to be excellent
inhibitors of cell proliferation leaving only a small number of
cells in G2 phase.
In summary, several Mannich bases from BA were prepared
and tested for their cytotoxic activity. The results from AO/PI
staining and annexinV-FITC assays as well as DNA laddering
experiments provided evidence for an apoptotic cell death.
Some of them are strong regulators of tumor cell prolifer-
ation and induce cell cycle arrest. Thus, evaluation of the PI
dyeing showed many cells in SubG1 phase, but also a signifi-
cant decrease of cells in G2/M phase is observed. The bio-
logical activities make these compounds interesting
candidates for further biological evaluation.
Experimental
General
Melting points are uncorrected (Leica hot stage microscope);
NMR spectra were recorded using the Varian spectrometers
Gemini 200, Gemini 2000 or Unity 500 (d given in ppm, J in
Hz, internal Me4Si); optical rotations were obtained using a
Perkin–Elmer 341 polarimeter (1 cm microcell, 258C), IR spectra
(film or KBr pellet) on a Perkin–Elmer FT-IR spectrometer
Spectrum 1000; MS spectra were taken on an Intectra GmbH
AMD 402 (electron impact, 70 eV) or on a Finnigan MAT TSQ 7000
(electrospray, voltage 4.5 kV, sheath gas nitrogen) instrument.
TLC was performed on silica gel (Merck 5554, detection by UV
absorption). The solvents were dried according to usual pro-
cedures. The purity of the compounds was determined by
HPLC and found to be >98%. Solubility was determined by
HPLC/UV–vis according to [47].
(3b) 3-Ethynyl-3-hydroxylup-20(29)-en-28-acid (3)
Figure 4. DNA ladders in A549 cancer cells that were treated for
24 h with 20 mM of compounds 9 or 14.
To a solution of 1 (5.0 g, 11.0 mmol) in dry THF (150 mL) a
solution of ethynylmagnesium bromide (0.5 M in THF, 50 mL,
25 mmol) was slowly added. The reaction mixture was stirred
under argon for 4 h at 258C, quenched by the addition of meth-
anol and water (5 mL each), and the solvents were evaporated
under reduced pressure. The residue was subjected to chroma-
tography (silica gel, n-hexane/ethyl acetate, 3:1) and 3 (4.06 g,
77%) was obtained as a white solid. Mp 2558C; [a]D ¼ þ12.58
(c ¼ 5.0, MeOH); RF ¼ 0.42 (silica gel, n-hexane/ethyl acetate,
4:1); IR (KBr): n ¼ 3538s, 3074m, 3004s, 2943s, 2869s, 2844s,
1708s, 1644m, 1450s, 1434m, 1392m, 1373m, 1350m, 1332m,
1285m, 1274m, 1238m, 1223m, 1199s, 1167s, 1138m, 1104m,
toxic compounds for 24 h. The results from these investi-
gations are depicted in Fig. 5.
Thus, living A549 cells were treated with dye PI and com-
pounds 5, 9, 12, 14, and 20 for 24 h. The concluding FACS
measurements were performed and evaluated using the pro-
cedures described by Kallioniemi et al. [67] and Dean et al.
[68]. These experiments demonstrate that upon treatment of
the cells with the cytotoxic compounds 5, 12, 14, and 20 a
degradation of the DNA occurs in the living A549 cells
1082m, 1047s, 1008m, 972m cmꢁ1 1H NMR (500 MHz, CD3OD):
;
d ¼ 4.71 (m, 1H, CHa (29)), 4.59 (m, 1H, CHb (29)), 3.02 (brm, 1H,
CH (19)), 2.78 (s, 1H, CH (32)), 2.46 (m, 1H, CHa (16)), 2.32 (m, 1H,
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Figure 5. FACS measurements of A549 cells after treatment with cytotoxic compounds 5 (15 mM), 9 (10 mM), 12 (15 mM), 14 (10 mM) and
20 (40 mM) for 24 h followed by PI staining.
Methyl (3b) 3-ethynyl-3-hydroxylup-20(29)-en-28-oate (4)
CH (13)), 2.23 (m, 1H, CHa (2)), 1.98ꢁ1.87 (m, 3H, CHa (21) þ CHa
(22) þ CHb (2)), 1.76ꢁ1.21 (m, 16H, CH (18) þ CHa (1) þ CHb
To a solution of 2 (4.69 g, 10.0 mmol) in dry THF (150 mL),
(1) þ CHa (12) þ CH (9) þ CHa (7) þ CHb (7) þ CHb (16) þ CHb
solution of ethynylmagnesium bromide (0.5 M in THF, 50 mL,
(21) þ CHb (22) þ CHa (15) þ CHa (11) þ CHb (11) þ CHa
25 mmol) was added dropwise. After stirring at 258C for 4 h
(6) þ CHb (6) þ CH (5)), 1.69 (s, 3H, CH3 (30)), 1.20ꢁ1.14
under argon, the reaction was quenched by the careful addition
of acetic acid (3 mL) and water (400 mL). The mixture was
(m, 1H, CHb (15)), 1.11ꢁ1.02 (m, 1H, CHb (12)), 1.04 (s, 3H, CH3
(24)), 1.00 (s, 3H, CH3 (27)), 0.96 (s, 3H, CH3 (26)), 0.86 (s, 3H, CH3
extracted with ethyl acetate (3 ꢂ 200 mL), the extracts were
(25)), 0.83 (s, 3H, CH3 (23)) ppm; 13C NMR (125 MHz, CD3OD):
washed with an aq. solution of NaHCO3 (150 mL, satd.) and
–
–
d ¼ 178.5 (C28, CO), 150.5 (C20, C CH ), 108.8 (C29, C CH ),
brine (150 mL) and dried with sodium sulfate. The solvents were
evaporated, and the residue subjected to chromatography (silica
gel, n-hexane/ethyl acetate, 4:1) to afford 4 (3.38 g, 68%) as a white
solid. Mp 210–2158C; UV–vis (MeOH): lmax (nm) (log e) ¼ 260
(0.08), 220 (0.94); [a]D ¼ þ1.78 (c ¼ 4.20, CHCl3); RF ¼ 0.63 (silica
gel, n-hexane/ethyl acetate, 4:1); IR (KBr): n ¼ 3550s, 3476s,
3307m, 3264s, 3072m, 2950s, 2870s, 2107w, 1716s, 1641m,
1461m, 1377s, 1351m, 1317m, 1266m, 1226m, 1190s, 1156s,
–
–
2
2
86.8 (C31, C CH), 74.8 (C32, C CH), 73.2 (C3, COH), 54.7
(C17, Cquart.), 53.0 (C5, CH), 50.7 (C18, CH), 49.8 (C9, CH), 49.2
(C19, CH), 42.1 (C14, Cquart.), 41.0 (C4, Cquart.), 40.5 (C8, Cquart.), 38.3
(C13, CH), 37.6 (C1, CH2), 36.9 (C10, Cquart.), 36.8 (C22, CH2),
34.2 (C7, CH2), 33.6 (C2, CH2), 33.4 (C16, CH2), 32.0 (C21, CH2),
29.5 (C15, CH2), 24.8 (C24, CH3), 25.5 (C12, CH2), 20.6 (C11, CH2),
18.2 (C30, CH3), 18.2 (C6, CH2), 16.8 (C23, CH3), 15.7 (C25, CH3),
15.2 (C26, CH3), 13.8 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 959.1
(97% [2M–H]ꢁ), 525.1 (52% [MþHCO2]ꢁ), 479.6 (68% [M–H]ꢁ);
analysis for C32H48O3 (480.72): C, 79.95; H, 10.06; found: C,
79.75; H, 10.21.
1135s, 1108m, 1067m, 1030m, 1008m, 984m, 949m, 914w cmꢁ1
;
1H NMR (500 MHz, CDCl3): d ¼ 4.66 (m, 1H, CHa (29)), 4.53
(m, 1H, CHb (29)), 3.60 (s, 3H, CH3 (31)), 2.92 (ddd, 1H, J ¼ 11.1,
10.8, 4.8 Hz, CH (19)), 2.40 (s, 1H, CH (33)), 2.18ꢁ2.10 (m, 2H,
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Arch. Pharm. Chem. Life Sci. 2013, 000, 1–15
CH (13) þ CHa (16)), 1.89ꢁ1.79 (m, 3H, CHa (2) þ CHa (21) þ CHa
(22)), 1.66ꢁ1.50 (m, 4H, CH (18) þ CHa (1) þ CHa (12) þ CHb (2)),
1.61 (s, 3H, CH3 (30)), 1.44ꢁ1.14 (m, 12H, CH (9) þ CHb (1) þ CHb
(22) þ CHa (7) þ CHb (7) þ CHb (16) þ CHb (21) þ CHa (15) þ CHa
(11) þ CHb (11) þ CHa (6) þ CHb (6)), 1.10ꢁ1.05 (m, 2H, CH
(5) þ CHb (15)), 1.00ꢁ0.95 (m, 1H, CHb (12)), 0.98 (s, 3H, CH3
(24)), 0.92 (s, 3H, CH3 (27)), 0.84 (s, 3H, CH3 (25)), 0.78
(s, 3H, CH3 (23)), 0.76 (s, 3H, CH3 (26)) ppm; 13C NMR (125 MHz,
(C20, C ¼ CH2), 108.7 (C29, C ¼ CH2), 93.2 (C31, C CH), 75.1
(C32, C CH), 73.7 (C3, COH), 56.0 (C17, Cquart.), 53.8 (C5, CH),
53.2 (C34 þ C37, 2ꢂCH2), 50.9 (C18, CH), 49.0 (C9, CH), 47.0
(C19, CH), 43.1 (C33, CH2), 42.2 (C14, Cquart.), 41.3 (C4, Cquart.),
40.5 (C8, Cquart.), 38.2 (C13, CH), 37.8 (C1, CH2), 37.0 (C10, Cquart.),
36.7 (C22, CH2), 34.2 (C7, CH2), 31.9 (C2, CH2), 31.8 (C16, CH2), 30.3
(C21, CH2), 29.4 (C15, CH2), 25.5 (C12, CH2), 25.1 (C24, CH3), 23.1
(C35 þ C36, 2ꢂCH2), 20.6 (C11, CH2), 18.2 (C6, CH2), 18.2 (C30,
CH3), 16.7 (C23, CH3), 15.6 (C25, CH3), 15.2 (C26, CH3), 13.9 (C27,
CH3) ppm; MS (ESI, MeOH): m/z ¼ 564.4 (100% [MþH]þ); analysis
for C37H58ClNO3 (600.31): C, 74.03; H, 9.74; N, 2.33; found: C,
73.89; H, 9.99; N, 2.23.
–
CDCl ): d ¼ 176.6 (C28, CO), 150.5 (C20, C CH ), 109.5 (C29,
–
C CH ), 87.1 (C32, C CH), 75.7 (C33, C CH), 73.5 (C3, COH),
3
2
–
–
2
56.5 (C17, Cquart.), 53.1 (C5, CH), 51.2 (C31, CH3), 50.6 (C18, CH),
49.5 (C9, CH), 46.9 (C19, CH), 42.4 (C14, Cquart.), 41.3 (C4, Cquart.),
40.6 (C8, Cquart.), 38.3 (C13, CH), 37.9 (C1, CH2), 37.2 (C10, Cquart.),
36.9 (C22, CH2), 34.2 (C7, CH2), 32.6 (C2, CH2), 32.2 (C16, CH2), 30.6
(C21, CH2), 29.7 (C15, CH2), 25.6 (C24, CH3), 25.5 (C12, CH2), 20.8
(C11, CH2), 19.4 (C30, CH3), 18.5 (C6, CH2), 17.4 (C23, CH3), 16.4
(C25, CH3), 15.9 (C26, CH3), 14.9 (C27, CH3) ppm; MS (ESI, MeOH):
m/z ¼ 1011.1 (27% [2MþNa]þ), 549.3 (18% [MþNaþMeOH]þ);
analysis for C33H50O3 (494.75): C, 80.11; H, 10.19; found: C,
79.98; H, 10.27.
(3b) 3-Hydroxy-3-(3-piperidin-1-yl-prop-1-yn-1-yl)lup-
20(29)-en-28-acid hydrochloride (6)
Compound 6 was prepared as described in the general procedure
from 3 (481 mg, 1.0 mmol), piperidine (0.35 mL, 3.5 mmol),
formalin (37%, 1.2 mL, 14.8 mmol) and copper iodide (4 mg,
0.02 mmol) in DMSO (5 mL) (408C for 5 days), and 6 (180 mg,
30%) was obtained as a colorless solid. Mp 2358C; [a]D ¼ þ8.48
(c ¼ 4.45, MeOH); IR (KBr): n ¼ 3346s, 2948s, 2628s, 2519s, 1688s,
1640s, 1455s, 1376s, 1319m, 1319s, 1241s, 1195s, 1135s, 1078s,
General procedure for the synthesis of acids 5–8
1039s, 984s cmꢁ1
;
1H NMR (500 MHz, CD3OD): d ¼ 4.70
A mixture of alkynol 3 (481 mg, 1.0 mmol), secondary amine
(2.5–4.0 mmol), formalin (37%, 10–15 mmol), copper iodide
(4 mg, 0.02 mmol) and DMSO (5 mL) was stirred at 408C for 1–
5 days. After completion of the reaction (as indicated by TLC),
a solution of ammonium chloride (10 g) and aqueous ammonia
(30%, 5 mL) in water (30 mL) was added. The mixture was
extracted with ethyl acetate (5 ꢂ 10 mL), and the solvents was
evaporated under reduced pressure. The crude residue was dis-
solved in diethylether (50 mL), insoluble material was filtered
off, and at 08C gaseous hydrogen chloride was passed through,
until the precipitation of salts had ceased. After standing for 12 h
at 48C, the product was filtered off and washed with water and
diethylether. Optionally, re-crystallization from methanol (5 mL)
and hydrochloric acid (10%, 5 mL) gave an analytical product.
(m, 1H, CHa (29)), 4.59 (m, 1H, CHb (29)), 4.13 (s, 2H, CH2 (33)),
3.62 (m, 2H, CHa (34) þ CHa (38)), 3.10ꢁ2.96 (m, 3H, CHb
(34) þ CHb (38) þ CH (19)), 2.31 (ddd, 1H, J ¼ 12.9, 11.5,
3.2 Hz, CH (13)), 2.23 (ddd, 1H, J ¼ 12.9, 3.1, 2.7 Hz, CHa (16)),
2.03ꢁ1.76 (m, 7H, CHa (35) þ CHa (37) þ CHb (35) þ CHb
(37) þ CHa (2) þ CHa (21) þ CHa (22)), 1.76ꢁ1.65 (m, 3H, CHa
(1) þ CHa (12) þ CHb (2)), 1.67 (s, 3H, CH3 (30)), 1.62 (dd, 1H,
J ¼ 11.4, 11.4 Hz, CH (18)), 1.58ꢁ1.02 (m, 17H, CH (9) þ CHb
(1) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb (16) þ CHb (21) þ CHa
(15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb (6) þ CHa (36) þ CHb
(36) þ CH (5) þ CHb (15) þ CHb (12)), 1.06 (s, 3H, CH3 (24)), 1.00
(s, 3H, CH3 (27)), 0.97 (s, 3H, CH3 (26)), 0.88 (s, 3H, CH3 (25)), 0.86
(s, 3H, CH3 (23)) ppm; 13C NMR (125 MHz, CD3OD): d ¼ 178.4
–
–
–
(C28, CO), 150.5 (C20, C CH ), 108.7 (C29, C CH ), 94.5 (C31,
–
2
2
C CH), 75.1 (C32, C CH), 72.8 (C3, COH), 56.0 (C17, Cquart.),
53.8 (C5, CH), 53.3 (C34 þ C38, 2ꢂCH2), 51.0 (C18, CH), 49.0
(C9, CH), 47.0 (C19, CH), 45.8 (C33, CH2), 42.2 (C14, Cquart.), 41.3
(C4, Cquart.), 40.5 (C8, Cquart.), 38.2 (C13, CH), 37.8 (C1, CH2), 37.0
(C10, Cquart.), 36.7 (C22, CH2), 34.2 (C7, CH2), 31.9 (C2, CH2), 31.8
(C16, CH2), 30.3 (C21, CH2), 29.4 (C15, CH2), 25.5 (C12, CH2),
25.1 (C24, CH3), 22.8 (C35 þ C37, 2ꢂCH2), 21.2 (C36, CH2), 20.6
(C11, CH2), 18.2 (C6, CH2), 18.2 (C30, CH3), 16.7 (C23, CH3), 15.6
(C25, CH3), 15.2 (C26, CH3), 14.0 (C27, CH3) ppm; MS (ESI, MeOH):
m/z ¼ 578.4 (100% [MþH]þ); analysis for C38H60ClNO3 (614.34): C,
74.29; H, 9.84; N, 2.28; found: C, 74.02; H, 10.04; N, 2.16.
(3b) 3-Hydroxy-3-(3-pyrrolidin-1-yl-prop-1-yn-1-yl)lup-
20(29)-en-28-acid hydrochloride (5)
Compound 5 was prepared as described in the general procedure
from 3 (481 mg, 1.0 mmol), pyrrolidine (0.3 mL, 3.7 mmol),
formalin (37%, 1.2 mL, 14.8 mmol) and copper iodide (4 mg,
0.02 mmol) in DMSO (5 mL) (408C for 5 days), and 5 (262 mg,
44%) was obtained after re-crystallization as a white solid. Mp
2228C; [a]D ¼ þ5.88 (c ¼ 4.30, MeOH); IR (KBr): n ¼ 3395s, 2948s,
2869s, 2585s, 1687s, 1640s, 1455s, 1376s, 1319m, 1241s, 1192s,
1135s, 1167m, 1075m, 1038s, 983m, 948m cmꢁ1 1H NMR
;
(500 MHz, CD3OD): d ¼ 4.71 (m, 1H, CHa (29)), 4.59 (m, 1H, CHb
(29)), 4.22 (s, 2H, CH2 (33)), 3.66 (m, 2H, CHa (34) þ CHa (37)), 3.26
(m, 2H, CHb (34) þ CHb (37)), 3.01 (ddd, 1H, J ¼ 10.6, 10.6,
4.6 Hz, CH (19)), 2.32 (m, 1H, CH (13)), 2.26ꢁ2.14 (m, 3H, CHa
(16) þ CHa (35) þ CHa (36)), 2.07 (m, 2H, CHb (35) þ CHb (36)),
2.00ꢁ1.87 (m, 3H, CHa (2) þ CHa (21) þ CHa (22)), 1.76ꢁ1.64
(m, 3H, CHa (1) þ CHa (12) þ CHb (2)), 1.69 (s, 3H, CH3 (30)),
1.62 (dd, 1H, J ¼ 11.4, 11.4 Hz, CH (18)), 1.58ꢁ1.22 (m, 12H,
CH (9) þ CHb (1) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb
(16) þ CHb (21) þ CHa (15) þ CHa (11) þ CHb (11) þ CHa
(6) þ CHb (6)), 1.19ꢁ1.09 (m, 2H, CH (5) þ CHb (15)), 1.07ꢁ1.01
(m, 1H, CHb (12)), 1.06 (s, 3H, CH3 (24)), 1.00 (s, 3H, CH3 (27)), 0.97
(s, 3H, CH3 (26)), 0.88 (s, 3H, CH3 (25)), 0.85 (s, 3H, CH3 (23)) ppm;
13C NMR (125 MHz, CD3OD): d ¼ 178.5 (C28, CO), 150.5
(3b) 3-(3-Azepan-1-yl-prop-1-yn-1-yl)-3-hydroxylup-
20(29)-en-28-acid hydrochloride (7)
Compound 7 was prepared as described in the general procedure
from 3 (481 mg, 1.0 mmol), azepane (0.3 mL, 2.6 mmol), for-
malin (37%, 1.2 mL, 14.8 mmol) and copper iodide (4 mg,
0.02 mmol) in DMSO (5 mL) (408C for 5 days), and 7 (85 mg,
14%) was obtained after re-crystallization as a colorless solid.
Mp 2318C; [a]D ¼ þ1.08 (c ¼ 5.10, MeOH); IR (KBr): n ¼ 3385m,
2941s, 2867s, 2602m, 1718m, 1638m, 1464m, 1376m, 1168m,
1133m, 1038m, 1011m, 983m, 947w cmꢁ1 1H NMR (500 MHz,
;
CD3OD): d ¼ 4.71 (m, 1H, CHa (29)), 4.59 (m, 1H, CHb (29)), 4.19
(s, 2H, CH2 (33)), 3.59 (m, 2H, CHa (34) þ CHa (39)), 3.11 (m, 2H,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2013, 000, 1–15
Antitumor-Active Mannich Products
9
CHb (34) þ CHb (39)), 3.01 (ddd, 1H, J ¼ 10.8, 10.8, 4.8 Hz, CH (19)),
2.32 (ddd, 1H, J ¼ 12.8, 11.8, 3.5 Hz, CH (13)), 2.24 (ddd, 1H,
J ¼ 12.8, 3.2, 3.2 Hz, CHa (16)), 2.06ꢁ1.85 (m, 5H, CHa
(35) þ CHa (38) þ CHa (2) þ CHa (21) þ CHa (22)), 1.81ꢁ1.66
(m, 5H, CHb (35) þ CHb (38) þ CHa (1) þ CHa (12) þ CHb (2)),
1.69 (s, 3H, CH3 (30)), 1.62 (dd, 1H, J ¼ 11.4, 11.4 Hz, CH (18)),
1.57ꢁ1.00 (m, 19H, CH (9) þ CHb (1) þ CHb (22) þ CHa (7) þ CHb
(7) þ CHb (16) þ CHb (21) þ CHa (15) þ CHa (11) þ CHb
(C25, CH3), 15.2 (C26, CH3), 14.0 (C27, CH3) ppm; MS (ESI,
MeOH): m/z ¼ 594.5 (100% [MþH]þ); analysis for C39H64ClNO3
(630.38): C, 74.31; H, 10.23; N, 2.22; found: C 74.09, H 10.54,
N 2.09.
General procedure for the synthesis of the methyl esters
9–19
A mixture of alkynol 4 (1.0 equiv.), secondary amine (1.05–3.5
equiv.), formalin (37%, 10–15 equiv.), copper iodide (0.01–0.06
equiv.) and DMSO (3–6 mL) was stirred at 408C for 1–5 days. After
the reaction was completed (as indicated by TLC), a solution of
aqueous ammonia (30%, 5 mL) in water (5 mL) was added. The
mixture was extracted with ethyl acetate (5 ꢂ 10 mL), and the
solvents were evaporated under reduced pressure. The crude
residue was dissolved in ethyl acetate (100 mL), the solvent
was stripped off, and the residue re-dissolved in diethylether
(100 mL). Insoluble material was filtered off, and at 08C gaseous
hydrogen chloride was passed through the solution until the
precipitation of salts had ceased. Crystallization was completed
by standing at 48C for 12. The product was collected and washed
with water and diethylether. Optionally, re-crystallization from
methanol (5 mL) and hydrochloric acid (10%, 5 mL) gave an
analytical product.
(11) þ CHa
(6) þ CHb
(6) þ CHa
(36) þ CHb
(36) þ CHa
(37) þ CHb (37) þ CH (5) þ CHb (15) þ CHb (12)), 1.06
(s, 3H, CH3 (24)), 1.00 (s, 3H, CH3 (27)), 0.98 (s, 3H, CH3 (26)),
0.88 (s, 3H, CH3 (25)), 0.86 (s, 3H, CH3 (23)) ppm; 13C NMR
–
(125 MHz, CD OD): d ¼ 178.4 (C28, CO), 150.5 (C20, C CH ),
–
108.7 (C29, C CH ), 94.0 (C31, C CH), 75.1 (C32, C CH), 73.4
3
–
–
2
2
(C3, COH), 56.0 (C17, Cquart.), 54.4 (C34 þ C39, 2ꢂCH2), 53.9
(C5, CH), 51.0 (C18, CH), 49.0 (C9, CH), 47.0 (C19, CH), 46.8
(C33, CH2), 42.2 (C14, Cquart.), 41.3 (C4, Cquart.), 40.5 (C8, Cquart.),
38.2 (C13, CH), 37.8 (C1, CH2), 37.0 (C10, Cquart.), 36.7 (C22, CH2),
34.2 (C7, CH2), 31.9 (C2, CH2), 31.8 (C16, CH2), 30.3 (C21, CH2), 29.4
(C15, CH2), 25.6 (C35 þ C38, 2ꢂCH2), 25.5 (C12, CH2), 25.1 (C24,
CH3), 23.8 (C36 þ C37, 2ꢂCH2), 20.6 (C11, CH2), 18.2 (C6, CH2),
18.2 (C30, CH3), 16.7 (C23, CH3), 15.6 (C25, CH3), 15.2 (C26, CH3),
14.0 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 592.5 (100% [MþH]þ);
m/z ¼ 1181.1 (100% [2M–H]ꢁ), 636.1 (95% [MþHCO2]ꢁ), 590.5
(14% [M–H]ꢁ); analysis for C39H62ClNO3 (628.37): C, 74.55;
H, 9.95; N, 2.23; found: C, 74.32; H, 10.11; N, 2.03.
Methyl (3b) 3-hydroxy-3-(3-pyrrolidin-1-yl-prop-1-yn-1-
yl)lup-20(29)-en-28-oate hydrochloride (9)
(3b) 3-[3-(Diisopropylamino)prop-1-yn-1-yl]-3-hydroxylup-
20(29)-en-28-acid hydrochloride (8)
Compound 9 was prepared as described in the general procedure
from 4 (495 mg, 1.0 mmol), pyrrolidine (0.2 mL, 2.4 mmol), for-
malin (37%, 0.8 mL, 9.8 mmol) and copper iodide (12 mg,
0.06 mmol) in DMSO (3 mL) (408C for 4 days), and 9 (220 mg,
38%) was obtained after re-crystallization as a colorless solid.
Mp 2008C; [a]D ¼ ꢁ2.68 (c ¼ 5.60, MeOH); UV–vis (MeOH): lmax
(nm) (log e) ¼ 219 (0.51); IR (KBr): n ¼ 3424m, 2949s, 2868m,
2608w, 1126m, 1640w, 1458m, 1377m, 1189m, 1154m, 1137m,
Compound 8 was prepared as described in the general procedure
from
3
(481 mg, 1.0 mmol), diisopropylamine (0.35 mL,
2.5 mmol), formalin (37%, 0.8 mL, 9.9 mmol) and copper iodide
(4 mg, 0.02 mmol) in DMSO (5 mL) (408C for 20 h), and 8
(390 mg, 62%) was obtained after re-crystallization as a colorless
solid. Mp 2408C; [a]D ¼ þ1.98 (c ¼ 4.60, MeOH); IR (KBr):
n ¼ 3172m, 2949s, 2868s, 2539m, 2462m, 1727s, 1644m,
1469m, 1392m, 1376m, 1316m, 1255w, 1167m, 1134s, 1067m,
1039w cmꢁ1
;
1H NMR (500 MHz, CD3OD): d ¼ 4.72 (m, 1H, CHa
(29)), 4.60 (m, 1H, CHb (29)), 4.22 (s, 2H, CH2 (34)), 3.66 (m, 2H, CHa
(35) þ CHa (38)), 3.65 (s, 3H, CH3 (31)), 3.25 (m, 2H, CHb (35) þ CHb
(38)), 2.99 (ddd, 1H, J ¼ 10.8, 10.8, 5.0 Hz, CH (19)), 2.28ꢁ2.15
(m, 4H, CH (13) þ CHa (16) þ CHa (36) þ CHa (37)), 2.07
(m, 2H, CHb (36) þ CHb (37)), 1.97 (ddd, 1H, J ¼ 15.0, 12.6,
3.7 Hz, CHa (2)),1.90ꢁ1.83 (m, 2H, CHa (21) þ CHa (22)),
1.76ꢁ1.65 (m, 3H, CHa (1) þ CHa (12) þ CHb (2)), 1.69
(s, 3H, CH3 (30)), 1.64 (dd, 1H, J ¼ 11.3, 11.3 Hz, CH (18)),
1.55ꢁ1.25 (m, 12H, CH (9) þ CHb (1) þ CHb (22) þ CHa
1037m, 1010m, 982w cmꢁ1
;
1H NMR (500 MHz, CD3OD):
d ¼ 4.71 (m, 1H, CHa (29)), 4.59 (m, 1H, CHb (29)), 4.23
(s, 2H, CH2 (33)), 3.91 (sept, 2H, J ¼ 6.6 Hz, 2ꢂCH (34) þ (37)),
3.01 (ddd, 1H, J ¼ 10.8, 10.8, 4.6 Hz, CH (19)), 2.32 (ddd, 1H,
J ¼ 12.8, 11.8, 3.2 Hz, CH (13)), 2.24 (ddd, 1H, J ¼ 12.8, 3.2,
3.0 Hz, CHa (16)), 2.01ꢁ1.88 (m, 3H, CHa (2) þ CHa (21) þ CHa
(22)), 1.77ꢁ1.66 (m, 3H, CHa (1) þ CHa (12) þ CHb (2)), 1.69
(s, 3H, CH3 (30)), 1.62 (dd, 1H, J ¼ 11.4, 11.4 Hz, CH (18)),
1.58ꢁ1.22 (m, 12H, CH (9) þ CHb (1) þ CHb (22) þ CHa
(7) þ CHb
(7) þ CHb
(16) þ CHb
(21) þ CHa
(15) þ CHa
(11) þ CHb (11) þ CHa (6) þ CHb (6)), 1.19ꢁ1.09 (m, 2H, CH
(5) þ CHb (15)), 1.08ꢁ1.00 (m, 1H, CHb (12)), 1.06 (s, 3H, CH3
(24)), 1.00 (s, 3H, CH3 (27)), 0.94 (s, 3H, CH3 (26)), 0.88
(s, 3H, CH3 (25)), 0.85 (s, 3H, CH3 (23)) ppm; 13C NMR (125 MHz,
(7) þ CHb
(7) þ CHb
(16) þ CHb
(21) þ CHa
(15) þ CHa
(11) þ CHb (11) þ CHa (6) þ CHb (6)), 1.47 (d, 12H, J ¼ 6.6 Hz,
4ꢂCH3 (35) þ (36) þ (38) þ (39)), 1.20ꢁ1.01 (m, 3H, CH
(5) þ CHb (15) þ CHb (12)), 1.05 (s, 3H, CH3 (24)), 1.00
(s, 3H, CH3 (27)), 0.97 (s, 3H, CH3 (26)), 0.88 (s, 3H, CH3 (25)),
0.85 (s, 3H, CH3 (23)) ppm; 13C NMR (125 MHz, CD3OD):
–
CD OD): d ¼ 178.1 (C28, CO), 151.8 (C20, C CH ), 110.3 (C29,
–
C CH ), 94.5 (C32, C CH), 76.4 (C33, C CH), 75.2 (C3, COH),
3
2
–
–
2
–
–
57.9 (C17, Cquart.), 55.1 (C5, CH), 54.5 (C35 þ C38, 2ꢂCH2), 52.3
(C31, CH3), 51.8 (C18, CH), 50.2 (C9, CH), 48.5 (C19, CH), 44.5
(C34, CH2), 43.6 (C14, Cquart.), 42.7 (C4, Cquart.), 41.9 (C8, Cquart.),
39.6 (C13, CH), 39.2 (C1, CH2), 38.4 (C10, Cquart.), 37.8 (C22, CH2),
35.6 (C7, CH2), 33.2 (C2, CH2), 33.1 (C16, CH2), 31.6 (C21, CH2), 30.8
(C15, CH2), 26.8 (C12, CH2), 26.5 (C24, CH3), 24.6 (C36 þ C37,
2ꢂCH2), 22.0 (C11, CH2), 19.6 (C6, CH2), 19.6 (C30, CH3), 18.2
(C23, CH3), 17.0 (C25, CH3), 16.5 (C26, CH3), 15.4 (C27, CH3)
ppm; MS (ESI, MeOH): m/z ¼ 578.4 (100% [MþH]þ); analysis
d ¼ 178.4 (C28, CO), 150.5 (C20, C CH ), 108.7 (C29, C CH ),
–
–
2
2
93.8 (C31, C CH), 75.2 (C32, C CH), 74.3 (C3, COH), 56.0 (C17,
Cquart.), 54.5 (C34 þ C37, 2ꢂCH), 53.9 (C5, CH), 51.0 (C18, CH), 49.0
(C9, CH), 47.4 (C19, CH), 42.2 (C14, Cquart.), 41.4 (C4, Cquart.), 40.5
(C8, Cquart.), 38.2 (C13, CH), 37.8 (C1, CH2), 37.0 (C10, Cquart.), 36.7
(C22, CH2), 35.7 (C33, CH2), 34.2 (C7, CH2), 31.9 (C2, CH2), 31.8
(C16, CH2), 30.3 (C21, CH2), 29.4 (C15, CH2), 25.5 (C12, CH2),
25.2 (C24, CH3), 20.7 (C11, CH2), 18.2 (C6, CH2), 18.2 (C30, CH3),
17.6 (C35 þ C36 þ C38 þ C39, 4ꢂCH3), 16.8 (C23, CH3), 15.6
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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10
R. Csuk et al.
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–15
for C38H60ClNO3 (614.34): C, 74.29; H, 9.84; N, 2.28; found: C,
74.02; H, 9.97; N, 2.12.
(37) þ CHa (38) þ CHb (38) þ CH (5) þ CHb (15) þ CHb (12)), 1.06
(s, 3H, CH3 (24)), 1.00 (s, 3H, CH3 (27)), 0.95 (s, 3H, CH3 (26)), 0.88
(s, 3H, CH3 (25)), 0.86 (s, 3H, CH3 (23)) ppm; 13C NMR (125 MHz,
–
CD OD): d ¼ 176.6 (C28, CO), 150.3 (C20, C CH ), 108.8 (C29,
–
C CH ), 94.0 (C32, C CH), 75.1 (C33, C CH), 73.4 (C3, COH),
3
2
Methyl (3b) 3-hydroxy-3-(3-piperidin-1-yl-prop-1-yn-1-
yl)lup-20(29)-en-28-oate hydrochloride (10)
–
–
2
56.4 (C17, Cquart.), 54.4 (C35 þ C40, 2ꢂCH2), 53.8 (C5, CH), 51.0
(C31, CH3), 50.4 (C18, CH), 49.2 (C9, CH), 47.0 (C19, CH), 46.8 (C34,
CH2), 42.1 (C14, Cquart.), 41.3 (C4, Cquart.), 40.5 (C8, Cquart.), 38.2
(C13, CH), 37.8 (C1, CH2), 37.0 (C10, Cquart.), 36.4 (C22, CH2), 34.2
(C7, CH2), 31.8 (C2, CH2), 31.7 (C16, CH2), 30.2 (C21, CH2), 29.4 (C15,
CH2), 25.6 (C36 þ C39, 2ꢂCH2), 25.5 (C12, CH2), 25.1 (C24, CH3),
23.8 (C37 þ C38, 2ꢂCH2), 20.6 (C11, CH2), 18.2 (C6, CH2), 18.2
(C30, CH3), 16.7 (C23, CH3), 15.6 (C25, CH3), 15.1 (C26, CH3), 14.1
(C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 606.5 (100% [MþH]þ);
analysis for C40H64ClNO3 (642.39): C, 74.79; H, 10.04; N, 2.18;
found: C, 74.59; H, 10.23; N, 2.04.
Compound 10 was prepared as described in the general pro-
cedure from
4 (371 mg, 0.75 mmol), piperidine (0.1 mL,
1.0 mmol), formalin (37%, 0.3 mL, 3.7 mmol) and copper iodide
(3 mg, 0.016 mmol) in DMSO (4 mL) (408C for 20 h), and 10
(85 mg, 18%) was obtained as a colorless solid. Mp 2158C;
[a]D ¼ ꢁ5.68 (c ¼ 4.35, MeOH); UV–vis (MeOH): lmax (nm)
(log e) ¼ 220 (1.28), 212 (0.37); IR (KBr): n ¼ 3422m, 2947s,
2869m, 2529m, 1727m, 1641w, 1458m, 1376m, 1188m, 1154m,
1136m, 1039m, 883w cmꢁ1; 1H NMR (500 MHz, CD3OD): d ¼ 4.71
(m, 1H, CHa (29)), 4.59 (m, 1H, CHb (29)), 4.13 (s, 2H, CH2 (34)), 3.65
(s, 3H, CH3 (31)), 3.62 (m, 2H, CHa (35) þ CHa (39)), 3.06 (m, 2H, CHb
(35) þ CHb (39)), 2.99 (ddd, 1H, J ¼ 10.8, 10.8, 5.1 Hz, CH (19)),
2.29ꢁ2.21 (m, 2H, CH (13) þ CHa (16)), 2.01ꢁ1.77 (m, 7H, CHa
(36) þ CHa (38) þ CHb (36) þ CHb (38) þ CHa (2) þ CHa
(21) þ CHa (22)), 1.76ꢁ1.66 (m, 3H, CHa (1) þ CHa (12) þ CHb
(2)), 1.69 (s, 3H, CH3 (30)), 1.64 (dd, 1H, J ¼ 11.3, 11.3 Hz, CH
(18)), 1.58ꢁ1.02 (m, 17H, CH (9) þ CHb (1) þ CHb (22) þ CHa
Methyl (3b) 3-hydroxy-3-(3-morpholin-4-yl-prop-1-yn-1-
yl)lup-20(29)-en-28-oate hydrochloride (12)
Compound 12 was prepared as described in the general pro-
cedure from
4 (347 mg, 0.7 mmol), morpholine (0.07 mL,
0.8 mmol), formalin (37%, 0.35 mL, 4.3 mmol) and copper iodide
(4 mg, 0.02 mmol) in DMSO (6 mL) (408C for 2 days), and 12
(260 mg, 59%) was obtained after re-crystallization as a colorless
solid. Mp 2078C; [a]D ¼ ꢁ4.98 (c ¼ 5.75, MeOH); UV–vis (MeOH):
lmax (nm) (log e) ¼ 220 (1.39), 217 (1.43), 214 (0.70); IR (KBr):
n ¼ 3405s, 2947s, 2869s, 2543m, 2451m, 1727s, 1642m, 1453s,
1390s, 1376s, 1352m, 1318m, 1265m, 1238m, 1189s, 1155s, 1134s,
(7) þ CHb
(7) þ CHb
(16) þ CHb
(21) þ CHa
(15) þ CHa
(11) þ CHb (11) þ CHa (6) þ CHb (6) þ CHa (37) þ CHb (37) þ CH
(5) þ CHb (15) þ CHb (12)), 1.06 (s, 3H, CH3 (24)), 0.99 (s, 3H, CH3
(27)), 0.94 (s, 3H, CH3 (26)), 0.88 (s, 3H, CH3 (25)), 0.86 (s, 3H, CH3
(23)) ppm; 13C NMR (125 MHz, CD3OD): d ¼ 178.1 (C28, CO), 151.8
(C20, C ¼ CH2), 110.3 (C29, C ¼ CH2), 95.9 (C32, C CH), 76.6 (C33,
C CH), 74.2 (C3, COH), 57.9 (C17, Cquart.), 55.2 (C5, CH), 53.7
(C35 þ C39, 2ꢂCH2), 52.4 (C31, CH3), 51.8 (C18, CH), 50.7 (C9,
CH), 48.4 (C19, CH), 47.2 (C34, CH2), 43.5 (C14, Cquart.), 42.7 (C4,
Cquart.), 41.9 (C8, Cquart.), 39.7 (C13, CH), 39.2 (C1, CH2), 38.4 (C10,
Cquart.), 37.8 (C22, CH2), 35.6 (C7, CH2), 33.2 (C2, CH2), 33.1 (C16,
CH2), 31.6 (C21, CH2), 30.8 (C15, CH2), 26.8 (C12, CH2), 26.5 (C24,
CH3), 24.3 (C36 þ C38, 2ꢂCH2), 22.6 (C37, CH2), 22.0 (C11, CH2),
19.7 (C6, CH2), 19.6 (C30, CH3), 18.1 (C23, CH3), 17.0 (C25, CH3),
16.5 (C26, CH3), 15.5 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 592.5
(100% [MþH]þ); analysis for C39H62ClNO3 (628.37): C, 74.55; H,
9.95; N, 2.23; found: C, 74.77; H, 10.02; N, 2.14.
1073s, 1038s, 1012m, 983m cmꢁ1 1H NMR (500 MHz, CD3OD):
;
d ¼ 4.71 (m, 1H, CHa (29)), 4.60 (m, 1H, CHb (29)), 4.22 (s, 2H, CH2
(34)), 4.10 (m, 2H, CHa (36) þ CHa (37)), 3.80 (m, 2H, CHb (36) þ CHb
(37)), 3.65 (s, 3H, CH3 (31)), 3.56 (m, 2H, CHa (35) þ CHa (38)), 3.27
(m, 2H CHb (35) þ CHb (38)), 2.99 (ddd, 1H, J ¼ 10.8, 10.8,
5.0 Hz, CH (19)), 2.29ꢁ2.21 (m, 2H, CH (13) þ CHa (16)), 1.98
(ddd, 1H, J ¼ 14.4, 13.1, 3.8 Hz, CHa (2)), 1.90ꢁ1.77 (m, 2H, CHa
(21) þ CHa (22)), 1.76ꢁ1.66 (m, 3H, CHa (1) þ CHa (12) þ CHb (2)),
1.69 (s, 3H, CH3 (30)), 1.64 (dd, 1H, J ¼ 11.4, 11.4 Hz, CH (18)),
1.55ꢁ1.24 (m, 12H, CH (9) þ CHb (1) þ CHb (22) þ CHa (7) þ CHb
(7) þ CHb (16) þ CHb (21) þ CHa (15) þ CHa (11) þ CHb
(11) þ CHa (6) þ CHb (6)), 1.20ꢁ1.08 (m, 2H, CH (5) þ CHb (15)),
1.07ꢁ1.00 (m, 1H, CHb (12)), 1.06 (s, 3H, CH3 (24)), 0.99 (s, 3H, CH3
(27)), 0.94 (s, 3H, CH3 (26)), 0.88 (s, 3H, CH3 (25)), 0.86 (s, 3H, CH3
(23)) ppm; 13C NMR (125 MHz, CD3OD): d ¼ 176.7 (C28, CO), 150.4
Methyl (3b) 3-(3-azepan-1-yl-prop-1-yn-1-yl)-3-
hydroxylup-20(29)-en-28-oate hydrochloride (11)
Compound 11 was prepared as described in the general pro-
cedure from 4 (248 mg, 0.5 mmol), azepane (0.3 mL, 2.6 mmol),
formalin (37%, 1.0 mL, 12.3 mmol) and copper iodide (2 mg,
0.01 mmol) in DMSO (6 mL) (408C for 2 days), and 11 (205 mg,
64%) was obtained after re-crystallization as a colorless solid.
Mp 2148C; [a]D ¼ þ1.28 (c ¼ 5.50, MeOH); IR (KBr): n ¼ 3406m,
3073w, 1946s, 2868s, 2601m, 1726s, 1641m, 1457s, 1377m, 1352m,
–
–
–
(C20, C CH ), 108.8 (C29, C CH ), 95.1 (C32, C CH), 75.2
–
2
2
(C33, C CH), 72.2 (C3, COH), 63.6 (C36 þ C37, 2ꢂCH2), 56.4
(C17, Cquart.), 53.8 (C5, CH), 50.9 (C35 þ C38, 2ꢂCH2), 50.8 (C31,
CH3), 50.4 (C18, CH), 49.2 (C9, CH), 47.0 (C19, CH), 45.9 (C34, CH2),
42.1 (C14, Cquart.), 41.3 (C4, Cquart.), 40.5 (C8, Cquart.), 38.2 (C13, CH),
37.8 (C1, CH2), 37.0 (C10, Cquart.), 36.4 (C22, CH2), 34.1 (C7, CH2),
31.8 (C2, CH2), 31.7 (C16, CH2), 30.2 (C21, CH2), 29.4 (C15, CH2),
25.4 (C12, CH2), 25.1 (C24, CH3), 20.6 (C11, CH2), 18.2 (C6, CH2),
18.1 (C30, CH3), 17.0 (C23, CH3), 15.5 (C25, CH3), 15.1 (C26, CH3),
14.0 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 594.4 (100% [MþH]þ);
analysis for C38H60ClNO4 (630.34): C, 72.41; H, 9.59; N, 2.22;
found: C, 72.39; H, 9.73; N, 2.07.
1317m, 1189m, 1154m, 1136s, 1079w, 1038m, 984m cmꢁ1
;
1H NMR (500 MHz, CD3OD): d ¼ 4.71 (m, 1H, CHa (29)), 4.60
(m, 1H, CHb (29)), 4.18 (s, 2H, CH2 (34)), 3.66 (s, 3H, CH3 (31)),
3.63ꢁ3.45 (brm, 2H, CHa (35) þ CHa (40)), 3.44ꢁ3.28
(brm, 2H, CHb (35) þ CHb (40)), 2.99 (ddd, 1H, J ¼ 10.7, 10.7,
5.1 Hz, CH (19)), 2.28ꢁ2.28 (m, 2H, CH (13) þ CHa (16)),
2.06ꢁ1.84 (m, 5H, CHa (36) þ CHa (39) þ CHa (2) þ CHa
(21) þ CHa (22)), 1.81ꢁ1.66 (m, 5H, CHb (36) þ CHb (39) þ CHa
(1) þ CHa (12) þ CHb (2)), 1.69 (s, 3H, CH3 (30)), 1.64 (dd, 1H,
J ¼ 11.4, 11.4 Hz, CH (18)), 1.55ꢁ1.02 (m, 19H, CH (9) þ CHb
(1) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb (16) þ CHb (21) þ CHa
(15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb (6) þ CHa (37) þ CHb
Methyl (3b) 3-hydroxy-3-(3-thiomorpholin-4-yl-prop-1-yn-
1-yl)lup-20(29)-en-28-oate hydrochloride (13)
Compound 13 was prepared as described in the general pro-
cedure from 4 (248 mg, 0.5 mmol), thiomorpholine (0.06 mL,
0.6 mmol), formalin (37%, 0.2 mL, 2.5 mmol) and copper iodide
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Arch. Pharm. Chem. Life Sci. 2013, 000, 1–15
Antitumor-Active Mannich Products
11
(2 mg, 0.01 mmol) in DMSO (5 mL) (408C for 2 days), and 13
(160 mg, 50%) was obtained after twofold re-crystallization as
a colorless solid. Mp 1988C; [a]D ¼ ꢁ2.38 (c ¼ 3.50, MeOH); UV–vis
(MeOH): lmax (nm) (log e) ¼ 250 (0.15), 208 (1.60); IR (KBr):
n ¼ 3355s, 2946s, 2868s, 2724s, 2656s, 2457s, 1723s, 1641s,
1591s, 1455s, 1390s, 1376s, 1317s, 1266s, 1188s, 1154s, 1135s,
(C19, CH), 46.8 (C34, CH2), 43.6 (C14, Cquart.), 43.3 (C39, CH3), 42.7
(C4, Cquart.), 41.9 (C8, Cquart.), 39.6 (C13, CH), 39.1 (C1, CH2), 38.4
(C10, Cquart.), 37.9 (C22, CH2), 35.4 (C7, CH2), 33.2 (C2, CH2), 33.1
(C16, CH2), 31.6 (C21, CH2), 30.8 (C15, CH2), 26.8 (C12, CH2), 26.6
(C24, CH3), 22.0 (C11, CH2), 19.6 (C6, CH2), 19.6 (C30, CH3),
18.2 (C23, CH3), 17.0 (C25, CH3), 16.5 (C26, CH3), 15.5 (C27,
CH3); MS (ESI, MeOH): m/z ¼ 607.4 (100% [MþH]þ); analysis
for C39H65Cl2N2O3 (680.85): C, 68.80; H, 9.62; N, 4.11; found: C,
68.55; H, 9.83; N, 3.98.
1039s, 1003s, 981s, 920s cmꢁ1 1H NMR (500 MHz, CD3OD):
;
d ¼ 4.71 (m, 1H, CHa (29)), 4.59 (m, 1H, CHb (29)), 4.21
(s, 2H, CH2 (34)), 3.95ꢁ2.80 (br m 8H, CHa (36) þ CHa (37) þ CHb
(36) þ CHb (37) þ CHa (35) þ CHa (38) þ CHb (35) þ CHb (38)),
3.65 (s, 3H, CH3 (31)), 2.99 (ddd, 1H, J ¼ 11.0, 11.0, 5.1 Hz, CH
(19)), 2.29ꢁ2.20 (m, 2H, CH (13) þ CHa (16)), 1.97 (m, 1H, CHa (2)),
1.89ꢁ1.83 (m, 2H, CHa (21) þ CHa (22)), 1.75ꢁ1.68 (m, 3H, CHa
(1) þ CHa (12) þ CHb (2)), 1.69 (s, 3H, CH3 (30)), 1.64 (dd, 1H,
J ¼ 11.4, 11.4 Hz, CH (18)), 1.55ꢁ1.23 (m, 12H, CH (9) þ CHb
(1) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb (16) þ CHb (21) þ CHa
(15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb (6)), 1.18ꢁ1.03
(m, 3H, CH (5) þ CHb (15) þ CHb (12)), 1.07 (s, 3H, CH3 (24)),
1.00 (s, 3H, CH3i(27)), 0.94 (s, 3H, CH3 (26)), 0.88 (s, 3H, CH3
(25)), 0.86 (s, 3H, CH3 (23)) ppm; 13C NMR (125 MHz, CD3OD):
Methyl (3b) 3-[3-(dimethylamino)prop-1-yn-1-yl]-3-
hydroxylup-20(29)-en-28-oate hydrochloride (15)
Compound 15 was prepared as described in the general pro-
cedure by using 4 (297 mg, 0.6 mmol), dimethylamine solution
(7.9 M in H2O, 0.15 mL, 1.2 mmol), formalin (37%, 0.25 mL,
3.1 mmol) and copper iodide (4 mg, 0.02 mmol) in DMSO
(6 mL) at 408C for 20 h to obtain 15 after recrystallization as a
colorless solid (120 mg, 34%). Mp 2158C; [a]D ¼ ꢁ4.38 (c ¼ 5.25,
MeOH); UV–vis (MeOH): lmax (nm) (log e) ¼ 221 (1.44), 218 (1.28),
215 (0.64), 213 (0.22); IR (KBr): n ¼ 3384s, 2947s, 2869s, 2605m,
1726s, 1641m, 1464s, 1376m, 1189m, 1154s, 1135s, 1038m,
–
–
d ¼ 176.7 (C28, CO), 150.3 (C20, C CH ), 108.9 (C29, C CH ), 95.2
–
–
2
2
(C32, C CH), 75.2 (C33, C CH), 72.2 (C3, COH), 56.4 (C17, Cquart.),
53.9 (C5, CH), 53.2 (C35 þ C38, 2ꢂCH2), 50.9 (C31, CH3), 50.4 (C18,
CH), 49.2 (C9, CH), 47.0 (C19, CH), 46.6 (C34, CH2), 42.2 (C14,
Cquart.), 41.3 (C4, Cquart.), 40.5 (C8, Cquart.), 38.2 (C13, CH), 37.9
(C1, CH2), 37.0 (C10, Cquart.), 36.4 (C22, CH2), 34.1 (C7, CH2), 31.8
(C2, CH2), 31.7 (C16, CH2), 30.2 (C21, CH2), 29.4 (C15, CH2),
25.4 (C12, CH2), 25.1 (C24, CH3), 24.6 (C36 þ C37, 2ꢂCH2), 20.6
(C11, CH2), 18.2 (C6, CH2), 18.2 (C30, CH3), 16.7 (C23, CH3), 15.6
(C25, CH3), 15.1 (C26, CH3), 14.1 (C27, CH3) ppm; MS (ESI, MeOH):
m/z ¼ 610.4 (100% [MþH]þ); analysis for C38H60ClNO3S (646.41):
C, 70.61; H, 9.36; N, 2.17; found: C, 70.55; H, 9.42; N, 2.07.
1012m, 983m cmꢁ1
;
1H NMR (500 MHz, CD3OD): d ¼ 4.71
(m, 1H, CHa (29)), 4.59 (m, 1H, CHb (29)), 4.16 (s, 2H, CH2 (34)),
3.65 (s, 3H, CH3 (31)), 3.02ꢁ2.95 (m, 7H, CH (19) þ 2ꢂCH3
(35) þ (36)), 2.29ꢁ2.21 (m, 2H, CH (13) þ CHa (16)), 1.97 (ddd,
1H, J ¼ 14.3, 13.1, 3.7 Hz, CHa (2)), 1.90ꢁ1.82 (m, 2H, CHa
(21) þ CHa (22)), 1.75ꢁ1.65 (m, 3H, CHa (1) þ CHb (2) þ CHa
(12)), 1.69 (s, 3H, CH3 (30)), 1.63 (dd, 1H, J ¼ 11.3, 11.3 Hz, CH
(18)), 1.55ꢁ1.25 (m, 12H, CH (9) þ CHb (1) þ CHb (22) þ CHa
(7) þ CHb
(7) þ CHb
(16) þ CHb
(21) þ CHai(15) þ CHa
(11) þ CHb (11) þ CHa (6) þ CHb (6)), 1.17ꢁ1.09 (m, 2H, CH
(5) þ CHb (15)), 1.09ꢁ1.03 (m, 1H, CHb (12)), 1.06 (s, 3H, CH3
(24)), 0.99 (s, 3H, CH3 (27)), 0.94 (s, 3H, CH3 (26)), 0.88
(s, 3H, CH3 (25)), 0.85 (s, 3H, CH3 (23)) ppm; 13C NMR (125 MHz,
Methyl (3b) 3-hydroxy-3-[3-(4-methylpiperazin-1-yl)prop-
1-yn-1-yl]lup-20(29)-en-28-oate hydrochloride (14)
–
CD OD): d ¼ 178.0 (C28, CO), 151.8 (C20, C CH ), 110.3 (C29,
–
C CH ), 95.9 (C32, C CH), 76.5 (C33, C CH), 74.4 (C3, COH),
3
2
–
–
2
Compound 14 was prepared as described in the general pro-
cedure from
57.9 (C17, Cquart.), 55.2 (C5, CH), 52.2 (C31, CH3), 51.8 (C18, CH),
50.6 (C9, CH), 48.5 (C19, CH), 48.0 (C34, CH2), 43.5 (C14, Cquart.),
42.8 (C35 þ C36, 2ꢂCH3), 42.7 (C4, Cquart.), 41.9 (C8, Cquart.), 39.6
(C13, CH), 39.2 (C1, CH2), 38.4 (C10, Cquart.), 37.8 (C22, CH2),
35.2 (C7, CH2), 33.2 (C2, CH2), 33.1 (C16, CH2), 31.6 (C21, CH2),
30.8 (C15, CH2), 26.8 (C12, CH2), 26.5 (C24, CH3), 22.0 (C11, CH2),
19.6 (C6, CH2), 19.5 (C30, CH3), 18.2 (C23, CH3), 17.0 (C25, CH3),
16.5 (C26, CH3), 15.2 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 552.4
(100% [MþH]þ); analysis for C36H58ClNO3 (588.30): C, 73.50;
H, 9.94; N, 2.38; found: C, 73.22; H, 10.12; N, 2.23.
4
(248 mg, 0.5 mmol), N-methylpiperazine
(0.09 mL, 0.8 mmol), formalin (37%, 0.3 mL, 3.7 mmol) and cop-
per iodide (2 mg, 0.01 mmol) in DMSO (6 mL) (408C for 2 days),
and 14 (218 mg, 64%) was obtained after re-crystallization as a
colorless solid. Mp 2388C; [a]D ¼ þ1.98 (c ¼ 4.40, MeOH); UV–vis
(MeOH): lmax (nm) (log e) ¼ 270 (0.10), 210 (1.50); IR (KBr):
n ¼ 3406s, 2943s, 2869s, 2404s, 1715s, 1639m, 1451s, 1377s,
1318m, 1190s cmꢁ1
;
1H NMR (500 MHz, CD3OD): d ¼ 4.71
(m, 1H, CHa (29)), 4.59 (m, 1H, CHb (29)), 4.29 (s, 2H, CH2 (34)),
3.91ꢁ3.51 (m, br 8H, CHa (36) þ CHa (37) þ CHb (36) þ CHb
(37) þ CHa (35) þ CHa (38) þ CHb (35) þ CHb (38)), 3.65
(s, 3H, CH3 (31)), 3.05ꢁ2.93 (m, 4H, CH3 (39) þ CH (19)),
2.29ꢁ2.18 (m, 2H, CH (13) þ CHa (16)), 1.96 (m, 1H, CHa (2)),
1.90ꢁ1.81 (m, 2H, CHa (21) þ CHa (22)), 1.78ꢁ1.65 (m, 3H, CHa
(1) þ CHa (12) þ CHb (2)), 1.69 (s, 3H, CH3 (30)), 1.64 (dd, 1H,
J ¼ 11.3, 11.3 Hz, CH (18)), 1.54ꢁ1.21 (m, 12H, CH (9) þ CHb
(1) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb (16) þ CHb (21) þ CHa
(15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb (6)), 1.20ꢁ1.00
(m, 3H, CH (5) þ CHb (15) þ CHb (12)), 1.07 (s, 3H, CH3 (24)),
1.01 (s, 3H, CH3 (27)), 0.93 (s, 3H, CH3 (26)), 0.87 (s, 3H, CH3
(25)), 0.85 (s, 3H, CH3 (23)) ppm; 13C NMR (125 MHz, CD3OD):
Methyl (3b) 3-[3-(diethylamino)prop-1-yn-1-yl]-3-
hydroxylup-20(29)-en-28-oate hydrochloride (16)
Compound 16 was prepared as described in the general pro-
cedure from 4 (247 mg, 0.5 mmol), diethylamine (0.06 mL,
0.6 mmol), formalin (37%, 0.2 mL, 2.5 mmol) and copper iodide
(2 mg, 0.01 mmol) in DMSO (5 mL) (408C for 2 days), and 16
(80 mg, 13%) was obtained after re-crystallization as a colorless
solid. Mp 2318C; [a]D ¼ ꢁ5.98 (c ¼ 4.80, MeOH); IR (KBr):
n ¼ 3384s, 2947s, 2869s, 2602m, 1726s, 1635m, 1464s, 1378m,
1189m, 1164m, 1136m, 1037m, 1039w, 982w cmꢁ1 1H NMR
;
–
–
d (ppm) ¼ 178.2 (C28, CO), 151.8 (C20, C CH ), 110.3 (C29, C
(500 MHz, CD3OD): d ¼ 4.71 (m, 1H, CHa (29)), 4.59 (m, 1H, CHb
(29)), 4.22 (s, 2H, CH2 (34)), 3.65 (s, 3H, CH3 (31)), 3.30 (br m, 4H,
2ꢂCH2 (35) þ (37)), 2.99 (m, 1H, CH (19)), 2.29ꢁ2.19 (m, 2H, CH
(13) þ CHa (16)), 1.97 (m, 1H, CHa (2)), 1.90ꢁ1.80 (m, 2H, CHa
–
–
2
CH2), 96.8 (C32, C CH), 76.7 (C33, C CH), 73.7 (C3, COH), 57.9
(C17, Cquart.), 54.9 (C5, CH), 52.0 (C31, CH3), 51.8 (C18, CH), 51.5
(C36 þ C37, 2ꢂCH2), 50.6 (C9, CH), 48.9 (C35 þ C38, 2ꢂCH2), 48.5
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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12
R. Csuk et al.
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–15
(21) þ CHa (22)), 1.75ꢁ1.59 (m, 4H, CHa (1) þ CHb (2) þ CHa
(12) þ CH (18)), 1.68 (s, 3H, CH3 (30)), 1.48ꢁ1.01 (m, 21H, CH
(9) þ CHb (1) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb (16) þ CHb
(21) þ CHa (15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb (6) þ CH
(5) þ CHb (15) þ CHb (12) þ 2ꢂCH3 (36) þ (38)), 1.06 (s, 3H, CH3
(24)), 0.99 (s, 3H, CH3 (27)), 0.94 (s, 3H, CH3 (26)), 0.88 (s, 3H, CH3
(25)), 0.86 (s, 3H, CH3 (23)) ppm; 13C NMR (125 MHz, CD3OD):
(2 mg, 0.01 mmol) and DMSO (4 mL) was stirred at 408C for
20 h. After the reaction was completed, aqueous ammonia
(30%, 5 mL) in water (5 mL) was added. The mixture was
extracted with ethyl acetate (5 ꢂ 10 mL), and the solvents were
evaporated under reduced pressure. The residue was subjected
to chromatography (silica gel, n-hexane/ethyl acetate, 3:1) to
afford a pale yellow oil which was dissolved in diethylether
(10 mL). At 08C gaseous hydrogen chloride was passed through
until the precipitation of salts had ceased. Crystallization was
completed by standing at 48C for 12 h. The product was filtered
off and dried. Compound 17 (54 mg, 15%) was obtained as a
colorless solid. Mp 209–2118C; [a]D ¼ ꢁ6.78 (c ¼ 3.60, MeOH);
UV–vis (MeOH): lmax (nm) (log e) ¼ 217 (0.56), 212 (0.44);
IR (KBr): n ¼ 3313m, 2937s, 2869s, 2528m, 1725s, 1640w,
1456m, 1377m, 1317m, 1189m, 1155m, 1134m, 1039m, 982w,
–
–
d ¼ 176.7 (C28, CO), 150.3 (C20, C CH ), 108.8 (C29, C CH ), 94.4
–
–
2
2
(C32, C CH), 75.1 (C33, C CH), 72.2 (C3, COH), 56.4 (C17, Cquart.),
53.9 (C5, CH), 51.0 (C31, CH3), 50.4 (C18, CH), 49.2 (C9, CH), 48.1
(C36 þ C38, 2ꢂCH2), 47.0 (C19, CH), 42.1 (C14, Cquart.), 41.3 (C4,
Cquart.), 40.6 (C34, CH2), 40.5 (C8, Cquart.), 38.2 (C13, CH), 37.9 (C1,
CH2), 37.0 (C10, Cquart.), 36.4 (C22, CH2), 34.1 (C7, CH2), 31.8 (C2,
CH2), 31.7 (C16, CH2), 30.2 (C21, CH2), 29.3 (C15, CH2), 25.4 (C12,
CH2), 25.1 (C24, CH3), 20.6 (C11, CH2), 18.2 (C6, CH2), 18.1 (C30,
CH3), 16.7 (C23, CH3), 15.5 (C25, CH3), 15.1 (C26, CH3), 14.1 (C27,
CH3), 8.3 (C36 þ C38, 2ꢂCH3) ppm; MS (ESI, MeOH): m/z ¼ 580.4
(100% [MþH]þ); analysis for C38H62ClNO3 (616.36): C, 74.05;
H, 10.14; N, 2.27; found: C, 73.87; H, 10.33; N, 2.11.
882m cmꢁ1
;
1H NMR (500 MHz, CD3OD): d ¼ 4.71 (m, 1H, CHa
(29)), 4.59 (m, 1H, CHb (29)), 4.21 (s, 2H, CH2 (34)), 3.65 (s, 3H, CH3
(31)), 3.23 (m, 4H, 2ꢂCHa (35) þ (41) þ 2ꢂCHb (35) þ (41)), 2.99
(ddd, 1H, J ¼ 10.8, 10.8, 4.7 Hz, CH (19)), 2.30ꢁ2.21 (m, 2H, CH
(13) þ CHa (16)), 1.98 (ddd, 1H, J ¼ 14.3, 13.3, 3.9 Hz, CHa (2)),
1.90ꢁ1.83 (m, 2H, CHa (21) þ CHa (22)), 1.80ꢁ1.67 (m, 7H, þ CHa
(1) þ CHa (12) þ CHb (2) þ 2ꢂCH2 (36) þ (42)), 1.69 (s, 3H, CH3
(30)), 1.63 (dd, 1H, J ¼ 11.5, 11.5 Hz, CH (18)), 1.55ꢁ1.02
(m, 27H, CH (9) þ CHb (1) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb
(16) þ CHb (21) þ CHa (15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb
Methyl (3b) 3-[3-(diisopropylamino)prop-1-yn-1-yl]-3-
hydroxylup-20(29)-en-28-oate hydrochloride (17)
Compound 17 was prepared as described in the general pro-
cedure from 4 (297 mg, 0.6 mmol), diisopropylamine (0.09 mL,
0.64 mmol), formalin (37%, 0.25 mL, 2.5 mmol) and copper
iodide (2 mg, 0.01 mmol) in DMSO (6 mL) (408C for 20 h), and
17 (155 mg, 48%) was obtained as a colorless solid. Mp 2158C;
[a]D ¼ ꢁ7.78 (c ¼ 6.15, MeOH); UV–vis (MeOH): lmax (nm)
(log e) ¼ 220 (1.49); IR (KBr): n ¼ 3252m, 2946s, 2472m, 1724m,
1642w, 1465m, 1389m, 1318w, 1165m, 1136m, 1038m, 1039w,
(6),
þ 2ꢂCH2 (37) þ (43) þ 2ꢂCH2 (38) þ (44) þ 2ꢂCH2
(39) þ (45) þ CH (5) þ CHb (15) þ CHb (12)), 1.05 (s, 3H, CH3
(24)), 1.00 (s, 3H, CH3 (27)), 0.95 (s, 3H, CH3 (26)), 0.93 (t, 6H,
J ¼ 7.2 Hz, 2ꢂCH3 (40) þ (46)), 0.89 (s, 3H, CH3 (25)), 0.86
(s, 3H, CH3 (23)) ppm; 13C NMR (125 MHz, CD3OD): d ¼ 178.0
–
–
–
(C28, CO), 151.7 (C20, C CH ), 110.4 (C29, C CH ), 95.9 (C32,
–
2
2
982w cmꢁ1
;
1H NMR (500 MHz, CD3OD): d ¼ 4.71 (m, 1H, CHa
C CH), 76.5 (C33, C CH), 73.6 (C3, COH), 57.9 (C17, Cquart.),
55.4 (C5, CH), 55.0 (C35 þ C41, 2ꢂCH2), 52.4 (C31, CH3), 51.8
(C18, CH), 50.7 (C9, CH), 48.5 (C19, CH), 43.6 (C14, Cquart.), 42.8
(C34, CH2), 42.8 (C4, Cquart.), 41.9 (C8, Cquart.), 39.6 (C13, CH), 39.4
(C1, CH2), 38.5 (C10, Cquart.), 37.9 (C22, CH2), 35.6 (C7, CH2),
33.3 (C2, CH2), 33.1 (C16, CH2), 32.4 (C38 þ C44, 2ꢂCH2), 31.6
(C21, CH2), 30.8 (C15, CH2), 27.5 (C36 þ C42, 2ꢂCH2), 26.9 (C12,
CH2), 26.6 (C24, CH3), 25.3 (C37 þ C43, 2ꢂCH2), 23.5 (C38 þ C44,
2ꢂCH2), 22.0 (C11, CH2), 19.6 (C6, CH2), 19.6 (C30, CH3), 18.1
(C23, CH3), 17.0 (C25, CH3), 16.5 (C26, CH3), 15.6 (C27, CH3),
14.3 (C40 þ C46, 2ꢂCH3) ppm; MS (ESI, MeOH): m/z ¼ 692.5
(100% [MþH]þ); analysis for C46H78ClNO3 (728.57): C, 75.83;
H, 10.79; N, 1.92; found: C, 75.67; H, 10.88; N, 1.78.
(29)), 4.60 (m, 1H, CHb (29)), 4.21 (s, 2H, CH2 (34)), 3.90 (sept. 2H,
J ¼ 6.7 Hz, 2ꢂCH (35) þ (38)), 3.65 (s, 3H, CH3 (31)), 2.99 (ddd, 1H,
J ¼ 10.6, 10.6, 5.1 Hz, CH (19)), 2.29ꢁ2.21 (m, 2H, CH (13) þ CHa
(16)), 1.96 (m, 1H, CHa (2)), 1.90ꢁ1.82 (m, 2H, CHa (21) þ CHa (22)),
1.76ꢁ1.65 (m, 3H, CHa (1) þ CHa (12) þ CHb (2)), 1.69 (s, 3H, CH3
(30)), 1.64 (dd, 1H, J ¼ 11.4, 11.4 Hz, CH (18)), 1.54ꢁ1.21
(m, 12H, CH (9) þ CHb (1) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb
(16) þ CHb (21) þ CHa (15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb
(6)), 1.47 (d, 12H, J ¼ 6.7 Hz, 4ꢂCH3 (36) þ (37) þ (39) þ (40)),
1.20ꢁ1.14 (m, 2H, CH (5) þ CHb (15)), 1.10ꢁ1.01 (m, 1H, CHb
(12)), 1.05 (s, 3H, CH3 (24)), 0.99 (s, 3H, CH3 (27)), 0.94 (s, 3H, CH3
(26)), 0.88 (s, 3H, CH3 (25)), 0.85 (s, 3H, CH3 (23)) ppm; 13C NMR
–
(125 MHz, CD OD): d ¼ 178.1 (C28, CO), 151.8 (C20, C CH ), 110.3
–
(C29, C CH ), 94.9 (C32, C CH), 76.6 (C33, C CH), 74.4 (C3, COH),
3
2
–
–
2
57.9 (C17, Cquart.), 55.8 (C35 þ C38, 2ꢂCH), 55.3 (C5, CH), 52.3
(C31, CH3), 51.8 (C18, CH), 50.7 (C9, CH), 48.4 (C19, CH), 43.6 (C14,
Cquart.), 42.9 (C4, Cquart.), 41.9 (C8, Cquart.), 39.6 (C13, CH), 39.2
(C1, CH2), 38.4 (C10, Cquart.), 37.8 (C22, CH2), 37.1 (C34, CH2), 35.6
(C7, CH2), 33.2 (C2, CH2), 33.1 (C16, CH2), 31.6 (C21, CH2), 30.8
(C15, CH2), 26.9 (C12, CH2), 26.6 (C24, CH3), 22.0 (C11, CH2),
19.6 (C6, CH2), 19.6 (C30, CH3), 18.2 (C23, CH3), 18.2
(C36 þ C37 þ C39 þ C40, 4ꢂCH3), 17.0 (C25, CH3), 16.5 (C26,
CH3), 15.4 (C27, CH3) ppm; MS (ESI, MeOH): m/z ¼ 608.5 (100%
[MþH]þ); analysis for C40H66ClNO3 (644.41): C, 74.55; H, 10.32;
N, 2.17; found: C, 74.36; H, 10.56; N, 2.11.
Methyl (3b) 3-[3-(dicyclohexylamino)prop-1-yn-1-yl]-3-
hydroxylup-20(29)-en-28-oate hydrochloride (19)
Compound 19 was prepared as described in the general pro-
cedure from
4
(247 mg, 0.5 mmol), dicyclohexylamine
(0.13 mL, 0.79 mmol), formalin (37%, 0.2 mL, 2.5 mmol) and
copper iodide (4 mg, 0.02 mmol) in DMSO (5 mL) (408C for 3
days), and 19 (153 mg, 42%) was obtained after re-crystallization
as a colorless solid. Mp 2208C; [a]D ¼ þ0.88 (c ¼ 5.10, MeOH); UV–
vis (MeOH): lmax (nm) (log e) ¼ 218 (0.59); IR (KBr): n ¼ 3423m,
2938m, 2862m, 2423m, 1723m, 1639w, 1458m, 1376w, 1136w,
1037w, 1012m, 880w cmꢁ1
;
1H NMR (500 MHz, CD3OD):
d ¼ 4.71 (m, 1H, CHa (29)), 4.60 (m, 1H, CHb (29)), 4.26
(s, 2H, CH2 (34)), 3.66 (s, 3H, CH3 (31)), 3.59 (m, 2H, 2ꢂCH
(35) þ (41)), 2.99 (ddd, 1H, J ¼ 10.8, 10.8, 4.9 Hz, CH (19)),
2.29ꢁ2.21 (m, 2H, CH (13) þ CHa (16)), 2.20ꢁ2.04 (m, 4H,
4ꢂCHa (36) þ (40) þ (42) þ (46)), 2.01–1.93 (m, 5H, 4ꢂCHa
Methyl (3b) 3-[3-(dihexylamino)prop-1-yn-1-yl]-3-
hydroxylup-20(29)-en-28-oate hydrochloride (18)
A mixture of 4 (248 mg, 0.5 mmol), dihexylamine (0.14 mL,
0.6 mmol), formalin (37%, 0.2 mL, 2.5 mmol), copper iodide
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 000, 1–15
Antitumor-Active Mannich Products
13
(37) þ (39) þ (43) þ (45) þ CHa (2)), 1.91ꢁ.83 (m, 2H, CHa
(21) þ CHa (22)), 1.77ꢁ1.62 (m, 6H, CHa (1) þ CHa (12) þ CHb
(2) þ CH (18) þ 2ꢂCHa (38) þ (44)), 1.69 (s, 3H, CH3 (30)),
1.55ꢁ1.03 (m, 25H 4ꢂCHb (36) þ (40) þ (42) þ (46) þ 4ꢂCHb
(34) þ (39) þ (43) þ (45) þ 2ꢂCHb (38) þ (44) þ CH (9) þ CHb
(1) þ CHb (22) þ CHa (7) þ CHb (7) þ CHb (16) þ CHb (21) þ CHa
(15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb (6) þ CH (5) þ CHb
(15) þ CHb (12)), 1.06 (s, 3H, CH3 (24)), 1.01 (s, 3H, CH3 (27)),
0.95 (s, 3H, CH3 (26)), 0.89 (s, 3H, CH3 (25)), 0.86 (s, 3H, CH3 (23))
ppm; 13C NMR (125 MHz, CD3OD): d ¼ 177.8 (C28, CO), 151.5
19.4 (C30, CH3), 17.9 (C23, CH3), 16.8 (C25, CH3), 16.4 (C26, CH3),
15.2 (C27, CH3) ppm; MS (ESI, MeOH): 608.5 (100% [M–Cl]þ);
analysis for C39H62INO4 (735.82): C, 63.66, H, 8.49; N, 1.90,
I, 17.25; found: C 63.60, H 8.62, N 2.07.
Methyl 3-[(3b) 3-hydroxy-28-methoxy-28-oxolup-
20(29)en-3-yl]-N,N,N-trimethylprop-2-yn-1-aminium iodide
(21)
Following the procedure given for 20, compound 21 (180 mg,
82%) from 15 (174 mg, 0.32 mmol) and methyl iodide (2 mL,
32.0 mmol) as an off-white solid. Mp 2168C, [a]D ¼ þ1.18
(c ¼ 5.80, MeOH), IR (KBr): n ¼ 3385m, 2947s, 2869m, 1726s,
1642m, 1458m, 1377m, 1318m, 1189m, 1154m, 1137m, 1075w,
1038m, 1009m, 984m cmꢁ1; 1H NMR (500 MHz, CD3OD): d ¼ 4.71
(m, 1H, CHa (29)), 4.59 (m, 1H, CHb (29)), 4.46 (d, 2H,
JN,H ¼ 2.3 Hz, CH2 (34)), 3.66 (s, 3H, CH3 (31)), 3.26 (s, 9H,
3ꢂCH3 (35) þ (36) þ (37)), 2.99 (ddd, 1H, J ¼ 10.8, 10.8,
5.0 Hz, CH (19)), 2.28ꢁ2.21 (m, 2H, CH (13) þ CHa (16)), 1.99
(ddd, 1H, J ¼ 14.3, 13.2, 3.8 Hz, CHa (2)), 1.90ꢁ1.83 (m, 2H, CHa
(21) þ CHa (22)), 1.75ꢁ1.68 (m, 3H, CHa (1) þ CHb (2) þ CHa (12)),
1.69 (s, 3H, CH3 (30)), 1.63 (dd, 1H, J ¼ 11.4, 11.4 Hz, CH (18)),
1.55ꢁ1.23 (m, 12H, CH (9) þ CHb (1) þ CHb (22) þ CHa (7) þ CHb
(7) þ CHb (16) þ CHb (21) þ CHa (15) þ CHa (11) þ CHb
(11) þ CHa (6) þ CHb (6)), 1.18ꢁ1.04 (m, 3H, CH (5) þ CHb
(15) þ CHb (12)), 1.08 (s, 3H, CH3 (24)), 0.98 (s, 3H, CH3 (27)),
0.94 (s, 3H, CH3 (26)), 0.89 (s, 3H, CH3 (25)), 0.87 (s, 3H, CH3 (23))
ppm; 13C NMR (125 MHz, CD3OD): d ¼ 178.1 (C28, CO), 151.7
–
–
–
(C20, C CH ), 109.9 (C29, C CH ), 95.2 (C32, C CH), 76.5 (C33,
–
2
2
C CH), 75.6 (C3, COH), 62.6 (C35 þ C41, 2ꢂCH), 57.6 (C17, Cquart.),
55.2 (C5, CH), 52.3 (C31, CH3), 52.0 (C18, CH), 50.5 (C9, CH),
48.2 (C19, CH), 43.4 (C14, Cquart.), 42.7 (C4, Cquart.), 41.8 (C8,
Cquart.), 39.5 (C13, CH), 39.2 (C1, CH2), 38.3 (C34, CH2), 37.6
(C10, Cquart.), 37.6 (C22, CH2), 35.5 (C7, CH2), 33.1 (C2, CH2),
32.9 (C16, CH2), 31.5 (C21, CH2), 30.7 (C15, CH2), 26.8 (C12,
CH2), 26.4 (C24, CH3), 26.2 (C36 þ C40 þ C42 þ C46, 4ꢂCH2),
25.8 (C37 þ C39 þ C43 þ C45, 4ꢂCH2), 25.3 (C38 þ C44,
2ꢂCH2), 21.9 (C11, CH2), 19.5 (C6, CH2), 19.5 (C30, CH3), 18.0
(C23, CH3), 16.8 (C25, CH3), 16.3 (C26, CH3), 15.6 (C27, CH3)
ppm; MS (ESI, MeOH): m/z ¼ 688.6 (100% [MþH]þ); analysis
for C46H74ClNO3 (724.54): C, 76.25; H, 10.29; N, 1.93; found: C,
76.11; H, 10.45; N, 1.74.
Methyl (3b) 3-[3-(4-methylmorpholin-4-ium-4-yl)prop-1-yn-
1-yl]lup20(29)-en-28 oate iodide (20)
–
–
–
(C20, C CH ), 110.3 (C29, C CH ), 97.6 (C32, C CH), 76.6
Compound 20 was obtained by treating a methanolic solution of
12 (200 mg, 0.32 mmol) in methanol with KOH (satd. aq.), fol-
lowed by extraction with ethyl acetate (3 ꢂ 100 mL). The organic
layers were dried over Na2SO4, the solvent was evaporated, and
the remaining solid was treated under argon with diethylether
and methyl iodide (2 mL, 32.0 mmol) for 7 days. The product was
filtered off and washed with ether to yield 20 (210 mg, 89%) as an
off-white solid. Mp 1808C, [a]D ¼ ꢁ2.58 (c ¼ 5.10, MeOH), IR (KBr):
n ¼ 3385s, 2947s, 2870s, 2361w, 1727s, 1640m, 1450s, 1377s,
1318m, 1189s, 1154s, 1136s, 1066m, 1037s, 983m, 948m, 896m
cmꢁ1; 1H NMR (500 MHz, CD3OD): d ¼ 4.71 (m, 1H, CHa (29)), 4.61
(d, 2H, JN,H ¼ 2.7 Hz, CH2 (34)), 4.59 (m, 1H, CHb (29)), 4.09ꢁ4.01
(m, 4H CHa (36) þ CHa (37) þ CHb (36) þ CHb (37)), 3.68ꢁ3.63
(m, 2H CHa (35) þ CHa (38)), 3.65 (s, 3H, CH3 (31)), 3.58ꢁ3.52
(m, 2H CHb (35) þ CHb (38)), 3.35 (s, 3H, CH3 (39)), 2.99 (ddd, 1H,
J ¼ 10.8, 10.8, 5.2 Hz, CH (19)), 2.29ꢁ2.21 (m, 2H, CH (13) þ CHa
(16)), 1.98 (ddd, 1H, J ¼ 14.3, 13.2, 3.8 Hz, CHa (2)), 1.90ꢁ1.83
(m, 2H, CHa (21) þ CHa (22)), 1.75ꢁ1.67 (m, 3H, CHa (1) þ CHa
(12) þ CHb (2)), 1.69 (s, 3H, CH3 (30)), 1.64 (dd, 1H, J ¼ 11.4,
11.4 Hz, CH (18)), 1.55ꢁ1.22 (m, 12H, CH (9) þ CHb (1) þ CHb
–
2
2
(C33, C CH), 74.0 (C3, COH), 57.9 (C17, Cquart.), 57.7 (C34, CH2),
55.4 (C5, CH), 53.4 (C35 þ C36 þ C37, 3ꢂCH3), 52.4 (C18, CH),
51.8 (C31, CH3), 50.6 (C9, CH), 48.5 (C19, CH), 43.5 (C14, Cquart.),
42.8 (C4, Cquart.), 41.9 (C8, Cquart.), 39.6 (C13, CH), 39.3 (C1, CH2),
38.4 (C10, Cquart.), 37.8 (C22, CH2), 35.6 (C7, CH2), 33.2 (C2, CH2),
33.1 (C16, CH2), 31.6 (C21, CH2), 30.7 (C15, CH2), 26.8 (C12, CH2),
26.6 (C24, CH3), 22.0 (C11, CH2), 19.6 (C6, CH2), 19.6 (C30, CH3),
18.1 (C23, CH3), 17.0 (C25, CH3), 16.5 (C26, CH3), 15.3 (C27, CH3)
ppm; MS (ESI, MeOH): 566.5 (100% [M–Cl]þ); analysis for
C37H60INO3 (693.78): C, 64.05; H, 8.72; N, 2.02; found: C 64.00,
H 8.89, N 1.85.
Cell lines and culture conditions
The cultures of the cell were maintained as monolayer in RPMI
1640 (PAA Laboratories, Pasching, Germany) supplemented with
10% heat inactivated fetal bovine serum (Sigma AG, Germany)
and penicillin/streptomycin (PAA Laboratories) at 378C in a
humidified atmosphere of 5% CO2/95% air.
(22) þ CHa
(7) þ CHb
(7) þ CHb
(16) þ CHb
(21) þ CHa
Biological testing
(15) þ CHa (11) þ CHb (11) þ CHa (6) þ CHb (6)), 1.20ꢁ1.14
(m, 2H, CH (5) þ CHb (15)), 1.12ꢁ1.02 (m, 1H, CHb (12)), 1.07
(s, 3H, CH3 (24)), 0.99 (s, 3H, CH3 (27)), 0.94 (s, 3H, CH3 (26)),
0.88 (s, 3H, CH3 (25)), 0.87 (s, 3H, CH3 (23)) ppm; 13C NMR
The cytotoxicity assay (SRB), the AO/PI test, the Trypan blue
counting, the DNA laddering and the cell cycle analysis exper-
iments were performed as previously described [56, 70, 71].
–
(125 MHz, CD OD): d ¼ 177.9 (C28, CO), 151.6 (C20, C CH ),
–
110.1 (C29, C CH ), 98.2 (C32, C CH), 76.5 (C33, C CH), 72.8
3
2
Many thanks are due to Dr. R. Kluge for the measurement of the MS
spectra and to Dr. D. Stro¨hl for recording the NMR spectra, and to
Mr. E. Sorge for his help with the cell cycle investigations. Support by
the ‘‘Gru¨nderwerkstatt – Biowissenschaften’’ is gratefully acknowledged.
The cell lines were kindly provided by Dr. T. Mu¨ller (Dept. of Haematology/
Oncology, Univ. Halle).
–
–
2
(C3, COH), 61.5 (C36 þ C37, 2ꢂCH2), 60.2 (C35 þ C38, 2ꢂCH2),
57.7 (C17, Cquart.), 56.1 (C34, CH2), 55.3 (C5, CH), 52.2 (C18, CH),
51.7 (C31, CH3), 50.5 (C9, CH), 48.8 (C19, CH), 48.3 (C39, CH3), 43.4
(C14, C Cquart.), 42.6 (C4, Cquart.), 41.7 (C8, Cquart.), 39.4 (C13, CH),
39.2 (C1, CH2), 38.2 (C10, Cquart.), 37.7 (C22, CH2), 35.4 (C7, CH2),
33.0 (C2, CH2), 32.9 (C16, CH2), 31.5 (C21, CH2), 30.6 (C15, CH2),
26.6 (C12, CH2), 26.5 (C24, CH3), 21.9 (C11, CH2), 19.5 (C6, CH2),
The authors have declared no conflict of interest.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

14
R. Csuk et al.
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–15
[25] M. Laus, A. A. Sante, M. Tramontini, P. Ferruti, Polym.
Commun. 1984, 25, 281–284.
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