Cytotoxic betulin-derived hydroxypropargylamines trigger apoptosis

Article abstract of DOI:10.1016/j.bmc.2012.11.016

Several novel betulin derivatives were prepared and evaluated for their antitumor activity. Among others, 3-O-acetylbetulinic aldehyde served as an ideal starting material for the synthesis of 28-acetylenic derivatives that were further transformed into Mannich bases. These hydroxypropargylamines were screened for their antitumor activity in a panel of nine human cancer cell lines in a sulforhodamine B (SRB) assay. Several compounds showed a noteworthy antitumor activity. The results from acridine orange/propidium iodide staining and annexinV-FITC assays as well as DNA laddering experiments provided evidence for an apoptotic cell death.

Full text of DOI:10.1016/j.bmc.2012.11.016

Bioorganic & Medicinal Chemistry 21 (2013) 425–435
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cytotoxic betulin-derived hydroxypropargylamines trigger apoptosis
⇑
René Csuk , Ronny Sczepek, Bianka Siewert, Christoph Nitsche
Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 2, D-06120 Halle (Saale), Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Several novel betulin derivatives were prepared and evaluated for their antitumor activity. Among others,
3-O-acetylbetulinic aldehyde served as an ideal starting material for the synthesis of 28-acetylenic deriv-
atives that were further transformed into Mannich bases. These hydroxypropargylamines were screened
for their antitumor activity in a panel of nine human cancer cell lines in a sulforhodamine B (SRB) assay.
Several compounds showed a noteworthy antitumor activity. The results from acridine orange/propidium
iodide staining and annexinV-FITC assays as well as DNA laddering experiments provided evidence for an
apoptotic cell death.
Received 11 September 2012
Revised 7 November 2012
Accepted 12 November 2012
Available online 24 November 2012
Keywords:
Betulin
Hydroxypropargylamines
Mannich reaction
Cancer
Ó 2012 Elsevier Ltd. All rights reserved.
SRB assay
Antitumor
DNA laddering
Annexin V assay
Apoptosis
1. Introduction
solubility by attaching aminogroup-bearing functionalities using
Mannich reactions.
Betulin (1, Fig. 1) is a pentacyclic triterpene showing a lupane
skeleton. It can be obtained in huge amounts from birch bark waste
by extraction. The healing properties of birch bark and birch bark
extracts have been known longtime in folk medicine. Betulin 1
itself is inactive against many tumor cell lines. The opposite is true
for several of its derivatives, especially for betulinic acid (2),
betulonic acid (3) and their derivatives.
Mannich reactions are known for exactly 100 years17 and their
significance for the synthesis of fine chemicals, pharmaceuticals
and polymers is beyond any doubt.17–20 Mannich reactions using
alkynes21 have been in the focus of synthetic organic chemists
since 1933—but moderate yields because of harsh reaction condi-
tions have limited a wider application.22 A breakthrough, however,
was using copper catalysts in these reactions.23
Terpenoids have scarcely been subject of aminomethylation
reactions.24,25 There, the reactive site was usually an alkyl ketone
group. There are even fewer examples for a successful Mannich
reaction of alkyne-modified terpenes so far because Mannich reac-
tions are often accompanied by changes in the substrate structure
due to rearrangement reactions.26–28
Several reports1,2 revealed the great potency of these com-
pounds; especially betulinic acid derivatives have been suggested
for the therapy of human melanoma tumors;3–6 no toxic side ef-
fects were noted in doses up to 500 mg/kg bodyweight, and their
way of action works by an induction of apoptosis.
A major drawback for their application is their low solubility in
water.7,8 Therefore, derivatives of betulin or betulinic acid holding
extra polar groups, and showing an increased solubility are needed.
Alkynyl substituted derivatives9–14 have scarcely been prepared so
far and their synthetic and antitumor potential remains yet to be
explored. Some of these compounds15,16 showed an increased
cytotoxicity towards many human tumor cells compared to parent
betulin. So, we decided to combine the higher cytotoxic activity of
alkynyl substituted betulin derivatives with improved water
⇑
Corresponding author. Tel.: +49 345 55 25660; fax: +49 345 55 27030.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
Figure 1. Structure of important lupane-type triterpenes.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.016

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R. Csuk et al. / Bioorg. Med. Chem. 21 (2013) 425–435
6, from the Grignard reaction of 4 or 5 alkynic 7 and 8 were
obtained.
The Grignard reaction of 5 advanced in THF in a chemoselective
2. Chemistry
Mannich reactions using alkynes, aldehydes and amines (so-
called A3 reactions29) yield propargylamines. Suitable starting
materials for the synthesis of alkynic terpenes can be obtained eas-
ily from naturally occurring betulin (1, Scheme 1). Thus, 1 was
transformed by known procedures into aldehydes 430–36 and
5.33–35,37–46 Reaction of 4 with lithium acetylide15,16 in THF gave
way, and 8 with an intact carbonyl group at C(3) was obtained.
Reactions in diethylether, however, produced a mixture of several
compounds. Compound 8 is characterized in its IR spectrum by a
strong vibration for the carbonyl group at
m
= 1702 cmꢀ1. In the
13C NMR spectrum the carbonyl group was detected at
Scheme 1. Reagents and conditions: Synthesis of 28-acetylenic betulins: (a) [29–31]; (b) [32, 39, 43]; (c) [29–31]; (d) ethynylmagnesium bromide, 25 °C, 10 h, 75%; (e)
ethynylmagnesium bromide, 25 °C, 12 h, 63%.
Scheme 2. Reagents and conditions: (a) Secondary amine, formalin, CuI (cat.), DMSO (or THF), 40 °C, 1–6 d, 40–90%; (b) MeI in Et2O, 5 d (87% for 22; 81% for 23); (c) catalyst,
MeOH, H2 (6 bar), 45%.

R. Csuk et al. / Bioorg. Med. Chem. 21 (2013) 425–435
427
d = 218.0 ppm, the carbons of the alkyne moiety were found at
d = 84.6 and 74.2 ppm, respectively.
Mannich reaction of 6 (Scheme 2) with diisopropylamine and
formalin in DMSO (or THF) in the presence of catalytic amounts
of CuI23 gave the Mannich base 9 in 90% yield. Similarly, from 6
with N-methyl-piperazine compound 13 was obtained. Yields
dropped slightly for the reaction of 7 with several secondary
amines; the products 11–21 were obtained in fair yields.
From the betulone derivative 8 under the same conditions 10
was obtained in 47% yield. For comparison, compounds 11 and
17 were transformed47,48 into their methiodides 22 and 23, respec-
tively. Hydrogenation of 11 in the presence of Lindlar catalyst gave
24 leaving the isopropenyl group of the lupane skeleton
unaffected.
3. Biology
Many structural variations have been applied to the betulin/
betulinic acid skeleton to preserve or to increase antitumor activ-
ity. Of special interest have been the carboxylic group at position
C-28 in ring E and the hydroxy group at position C-3 in ring A.30–
32,49–57
In general, it seems that esterification of C-28 using small to
medium length alcohols (or benzylic alcohol) led to a decrease in
activity as well as introducing hydrophilic carbohydrate-derived
moieties. Introduction of a bidesmosidic moiety, however, in-
creased activity.58,59 Increase in cytotoxic activity has been re-
marked for derivatives varied at C-28 and C-3. Thus, introduction
of an extra N-substituent led to an increase in cytotoxicity.60,61 It
can be assumed that this increase of cytotoxicity is due either to
an increased solubility of these compounds and/or an improved
transport into the tumor cells. The results from in vivo studies sug-
gested that betulinic acid accumulates in regions of lowered pH
values.62,63 Non-sufficient solubility of betulinic acid and many of
its derivatives is a notorious problem in gaining good antitumor
activity. The problem of solubility is crucial for an application be-
cause an aqueous medium is the preferred formulation for injec-
tion. To improve the solubility of triterpenes either the use of
liposomes, microemulsions64 or the addition of organic solvents
might be indicated. Raising of the pH value of the aqueous reaction
medium also increases solubility.7 To get a first insight into this
problem, solubility data for betulinic acid7,8 and several of our
derivatives have been measured in a water/DMSO (95:5, v/v) mix-
ture; for betulinic acid a solubility of 166
lg/ml was found. The
Mannich bases showed better solubility. Thus, for compound 10
182
312
l
l
g/ml, for 19 178
g/ml were determined. For the dicyclohexylamine 12 a rather
g/ml was found, thus giving a possible
lg/ml, for 15 192 lg/ml and for 13 even
low solubility of 100
l
explanation for the rather high IC50 values (IC50 > 30
lines) for this derivative.
lM for all cell
Although betulinic acid and most of its derivatives act by trig-
gering apoptosis, a recent study of Fulda and co-workers using
B10, a glycosylated betulinic acid derivative, pointed out autoph-
agy as an (extra) reason for cell death.65 During autophagy a fusion
of lysosomes and the macrophagosome takes place, therefore,
introducing a proton-accepting group might led to an increased
formation of autophagosomes.
Our compounds were tested for their cytotoxic activity (Table 1,
Fig. 2) in a colorimetric SRB cell assay.66–68 In general, 3-O-acetyl
derivatives showed a higher activity than their parent deacetylated
analogs (cf. Fig. 2); in alkynyl substituted C-28 oxo compounds
activity is retained. This parallels by and large previous findings
of Kim et al.69,70 whereas all derivatives with C-28 decarbonylated
or decarboxylated were shown to be inactive.31,71
The results from the SRB assay also revealed (Table 1, Fig. 2)
that Mannich products having been obtained from small aliphatic

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R. Csuk et al. / Bioorg. Med. Chem. 21 (2013) 425–435
100
45.00
80
60
40
20
0
30.00
15.00
.00
MA
BA
15
22
24
Figure 2. Comparison of IC50 values (in
and standard betulinic acid (BA) for lung carcinoma cell line A549; black bars
lM from SRB assay) for compounds 6–24
programmed cell death
necrosis
indicate inactive compounds.
Figure 3. Summary of the FACS based annexin V/propidium iodide assays for A549
cells and compounds (x-axis) 15 (5 M), 22 (10 M) or 24 (10 M) compared to
standards betulinic acid (BA, 30 M) and maslinic acid (MA, 30 M); percentage (y-
l
l
l
l
l
axis) of cells died by necrosis (in red) or by a programmed cell death (blue) after an
incubation of the cells for a period of 6 h and concentration as indicated.
amines show the highest cytotoxicity. Steric hindrance seems cru-
cial since the dicyclohexyl derivative 12 as well as the dibenzyl
compound 21 displayed decreased activity. Formation of methiod-
ides 22 and 23 gave a 2.5- to 5.8-fold increase in activity compared
to their parent compounds. Hydrogenation of the triple bond in 9
gave 24; the latter compound showed an even better cytotoxicity.
In summary, the Mannich products holding a C-28 alkynic skeleton
show an improved cytotoxicity compared to their parent com-
pounds—as long as the terminal amine substituent is small (or a
medium sized cyclic amine containing an extra tertiary amino
group). This parallels previous results of Salvador, who found that
the introduction of N-heterocyclic moieties at C-28 increased cyto-
toxicity. Also, a loss or a decrease of activity for compounds acety-
lated at C-3 is in excellent agreement with previous findings.50–52
Betulinic acid and derivatives act by triggering apopto-
sis63,72,73—a programmed cell death wherein cells that are harmful
to an organism are disposed of in a neat and orderly manner. SRB
tests do not allow conclusions for an apoptotic cell death. Hence,
selected compounds were chosen for further studies. These exper-
iments included a dye exclusion test (acridine orange/propidium
iode, AO/PI) as well as an annexin V assay.
An indicator for an early stage of apoptosis was shown to be the
transfer of phosphatidylserine from the inner membrane to the
outer,74 a finding that can be determined using a FACS based an-
nexin V/propidium iodide assay. Evaluation of the results for com-
pounds 15, 22 and 24 showed that for > 90% of all cells
phosphatidylserine has been translocated to the outer cell mem-
brane (Fig. 3).
An extra AO/PI staining experiment (Fig. 4, left and right part)
using A541 cells (after having been treated with compounds 15,
22 or 24, respectively) showed an almost complete exclusion of
PI by the cells (hence resulting in a green fluorescence during
microscopy because of an incorporation of AO into double stranded
nucleic acids). DNA laddering, that is, the cleavage of DNA by endo-
nucleases into fragments of 180 bp lengths (detected by gel elec-
trophoresis), is regarded as a late stage indicator of apoptosis.
The results from such an experiment are depicted in Figure 4 (mid-
dle), and they clearly show the presence of these fragments and
hence apoptosis.
Besides staining the cells during the AO/PI dyeing experiments,
morphological changes can be found for cells undergoing the pro-
cess of apoptosis. As marked by small white arrows in Figure 4,
blebbing of the cell membrane, condensation of the chromatin as
well as a shrinking of the cells can be noted. In Figure 4 (right part)
a mitotic cell has been marked by a red arrow. This can be inter-
preted as an indicator for a G2/M arrest of the cell induced by treat-
ing the cells with compound 24 hence paralleling previous findings
of Salvador50 and Yang et al.75
In this staining experiment lysosomes or autophagosomes are
colored in red—indicating that maybe besides apoptosis autophagy
is taking place.65
4. Conclusion
In summary, alkynyl Mannich bases of betulin are promising
antitumor agents. The results from acridine orange/propidium io-
dide staining and annexinV-FITC assays as well as DNA laddering
experiments provided evidence for an apoptotic cell death. Some
Figure 4. AO/PI staining of A541 human cancer cells (left: with compound 22; right with compound 24); in the middle: DNA laddering (A541 cells) after having been treated
with compounds 22 or 24.

R. Csuk et al. / Bioorg. Med. Chem. 21 (2013) 425–435
429
of them are strong regulators of tumor cells proliferation and in-
duce a cell cycle arrest. An extra process of autophagy leading to
cell death, however, cannot be ruled out. The biological activities
make these compounds interesting candidates for further biologi-
cal evaluation.
(15) + CHb (2) + CHa (1) + CHa (12)), 1.66 (s, 3H, CH3 (30)), 1.44–
1.09 (m, 10H, CH (9) + CHa (6) + CHb (22) + CHa (7) + CHb (7) + CHb
(16) + CHb (21) + CHa (11) + CHb (11) + CHb (6)), 1.03–0.92 (m, 3H,
CHb (15) + CHb (12) + CHb (1)), 1.02 (s, 3H, CH3 (23)), 0.98 (s, 3H,
CH3 (27)), 0.83–0.81 (m, 9H, CH3 (24) + CH3 (25) + CH3 (26)), 0.77
(d, 1H, J = 9.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3):
d = 171.0 (C31, CO), 150.9 (C20, C@CH2), 109.7 (C29, C@CH2), 84.7
(C33, C„CH), 80.9 (C3, CH), 74.2 (C34, C„CH), 66.0 (C28, CHOH),
55.4 (C5, CH), 50.6 (C17, Cquart.), 50.2 (C9, CH), 48.9 (C18, CH),
48.7 (C19, CH), 43.0 (C14, Cquart.), 40.9 (C8, Cquart.), 38.4 (C1, CH2),
37.8 (C4, Cquart.), 37.3 (C13, CH), 37.1 (C10, Cquart.), 34.3 (C7, CH2),
34.1 (C22, CH2), 33.9 (C21, CH2), 32.4 (C16, CH2), 27.9 (C24, CH3),
27.8 (C15, CH2), 25.1 (C12, CH2), 23.7 (C2, CH2), 21.3 (C32, CH3),
20.8 (C11, CH2), 18.8 (C30, CH3), 18.1 (C6, CH2), 17.5 (C23, CH3),
16.5 (C25, CH3), 16.1 (C26, CH3), 15.1 (C27, CH3) ppm; MS (ESI,
MeOH): m/z = 1039.3 (100%, [2 M+Na]+), 563.2 (25%, [M+Na + Me-
OH]+), 531.5 (6% [M+Na]+), 509.3 (2%, [M+H]+); Anal. for C34H52O3
(508.77): C 80.26; H, 10.30. Found: C 80.04, H 10.56.
5. Experimental
5.1. General
Instrumentation, cell lines and culture conditions, cytotoxicity
assay, dye exclusion tests, the annexin V assay and DNA fragmen-
tation was performed as previously described.76 Solubility was
determined in water/DMSO (95:5, v/v) mixtures (HPLC, UV/vis)
8
according to Ref.
5.2. General procedure for the Mannich reactions
A mixture of alkynol (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 40 °C 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 ꢁ 10 ml), and the sol-
vents were evaporated under reduced pressure. The crude residue
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 0 °C gaseous hydrogen chloride
was passed through the solution until the precipitation of salts
had ceased. Crystallization was completed by standing at 4 °C for
12. The product was collected and washed with water and diethyl-
ether. Optionally, re-crystallization from methanol (5 ml) and
hydrochloric acid (10%, 5 ml) gave an analytical product.
5.7. (28S) 28-Ethynyl-28-hydroxylup-20(29)-en-3-on (8)
Following the procedure given for 6, from the reaction of 23
(3.0 g, 6.46 mmol) with ethynylmagnesium bromide (0.5 M,
20 ml, 10 mmol) for 12 h and chromatography (silica gel, n-hex-
ane/ethyl acetate, 8/2) 8 (1.90 g, 63%) was obtained as a colorless
solid; mp 110–115 °C; ½a D
ane/ethyl acetate 8/2); IR (KBr):
1702s, 1639m, 1458m, 1378m, 1248m, 1111m, 1046m,
961m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 4.87 (m, 1H, CH (28)),
ꢂ
+35.6° (c 5.60, CHCl3); Rf = 0.52 (n-hex-
m
= 3442s, 3308m, 2943s, 2868s,
;
4.68 (m, 1H, CHa (29)), 4.56 (m, 1H, CHb (29)), 2.88 (ddd, 1H,
J = 10.7, 10.7, 6.4 Hz, CH (19)), 2.50–2.43 (m, 1H, CHa (2)), 2.40–
2.34 (m, 1H, CHb (2)), 2.48 (d, 1H, J = 2.1 Hz, CH (32)), 2.11–1.93
(m, 4H, CHa (16) + CHa (21) + CHa (22) + CH (13)), 1.94 (ddd, 1H,
J = 12.6, 5.5, 4.3 Hz, CHa (1)), 1.75 (dd, 1H, J = 11.9, 11.9 Hz, CH
(18)), 1.77–1.65 (m, 2H, CHa (15) + CHa (12)), 1.66 (s, 3H, CH3
(30)), 1.56 (ddd, 1H, J = 13.8, 13.8, 4.1 Hz, CHa (6)), 1.50–1.27 (m,
10H, CH (9) + CHb (22) + CHa (7) + CHb (7) + CHb (16) + CHb
(21) + CHa (11) + CHb (11) + CHb (6) + CHb (1)), 1.23 (d, 1H,
J = 8.6 Hz, CH (5)), 1.16 (ddd, 1H, J = 13.4, 12.4, 4.8 Hz, CHb (15)),
1.02–0.95 (m, 1H, CHb (12)), 1.06 (s, 3H, CH3 (25)), 1.05 (s, 3H,
CH3 (24)), 1.00 (s, 3H, CH3 (23)), 0.99 (s, 3H, CH3 (27)), 0.91 (s,
3H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3): d = 218.0 (C3,
CO), 150.9 (C20, C = CH2), 109.7 (C29, C@CH2), 84.6 (C31, C„CH),
74.2 (C32, C„CH), 65.9 (C28, CHOH), 54.9 (C5, CH), 50.6 (C17,
Cquart.), 49.6 (C9, CH), 48.8 (C18, CH), 48.6 (C19, CH), 47.3 (C4,
Cquart.), 43.1 (C14, Cquart.), 40.8 (C8, Cquart.), 39.5 (C1, CH2), 37.3
(C13, CH), 36.8 (C10, Cquart.), 34.2 (C22, CH2), 34.1 (C2, CH2), 33.9
(C7, CH2), 33.5 (C21, CH2), 32.3 (C16, CH2), 27.8 (C15, CH2), 26.6
(C24, CH3), 25.1 (C12, CH2), 21.3 (C11, CH2), 21.0 (C23, CH3), 19.6
(C6, CH2), 18.8 (C30, CH3), 15.9 (C25, CH3), 15.8 (C26, CH3), 15.0
(C27, CH3) ppm; MS (ESI, MeOH): m/z = 951.3 (35% [2 M+Na]+),
519.1 (56% [M+Na + MeOH]+), 487.5 (5% [M+Na]+), 465.3 (11%
[M+H]+); Anal. for C32H48O2 (464.72): C, 82.70; H, 10.41. Found:
C, 82.56; H, 10.62.
5.3. (3S) 28-Oxolup-20(29)en-3-yl acetate (4)
This compound was prepared according to Refs. 30–32
5.4. 3-Oxolup-20(29)en-28-al (5)
This compound was prepared according to Refs. 33,40,44
5.5. (3S, 28S) 28-Ethynyl-betulin (6)
This compound was prepared according to Refs. 30–32
5.6. (3S, 28S) 3-O-Acetyl-28-ethynyllup-20(29)-en-3,28-diol (7)
A solution of 4 (1.22 g, 2.53 mmol) in THF (50 ml) containing a
solution of ethynylmagnesium bromide (0.5 m, 12 ml, 7.2 mmol)
was stirred at 25 °C for 10 h; the reaction was quenched with
water (10 ml) and extracted with dichloromethane (5 ꢁ 100 ml).
The dried organic phase (Na2SO4) was evaporated, and the residue
was subjected to chromatography (silica gel, n-hexane/ethyl ace-
tate, 8/2) to yield 7 (0.91 g, 75%) as a colorless solid; mp 201–
5.8. (3S, 28S) 28-[3-(Diisopropylamino)prop-1-yn-1-yl]lup-
20(29)-en-3,28-diol hydrochloride (9)
204 °C; ½a D
tate, 8/2); IR (KBr):
2867s, 2529m, 1708s, 1639m, 1452s, 1377s, 1315m, 1269s,
1194m, 1131w, 1108m, 1035s cmꢀ1 1H NMR (500 MHz, CDCl3):
ꢂ
+15.6° (c 3.80, CHCl3); Rf = 0.63 (n-hexane/ethyl ace-
m
= 3440s, 3298m, 3260s, 3073m, 2939s,
Compound 9 was prepared following the general procedure from
6 (327 mg, 0.7 mmol), diisopropylamine (0.12 ml, 0.85 mmol), for-
malin (37%, 0.3 ml, 3.7 mmol), and catalytical amounts of CuI in
DMSO. The solution was stirred for 20 h at 40 °C to yield 9
(388 mg, 90%) as a colorless solid; mp 235; ½aꢂD +2.6° (c 4.30, MeOH);
;
d = 4.86 (m, 1H, CH (28)), 4.68 (m, 1H, CHa (29)), 4.56 (m, 1H,
CHb (29)), 4.45 (dd, 1H, J = 10.7, 5.8 Hz CH (3)), 2.88 (ddd, 1H,
J = 11.2, 11.2, 6.3 Hz, CH (19)), 2.48 (d, 1H, J = 2.1 Hz, CH (34)),
2.10–1.90 (m, 3H, CHa (22) + CHa (21) + CHa (16)), 2.02 (s, 3H, CH3
(32)), 1.93 (ddd, 1H, J = 12.2, 11.8, 3.6 Hz, CH (13)), 1.74 (dd, 1H,
J = 12.2, 12.2 Hz, CH (18)), 1.71–1.47 (m, 5 H, CHa (2) + CHa
IR (KBr): m = 3419m, 2943s, 2503m, 1640m, 1467m, 1397m, 1046m,
877m cmꢀ1; 1H NMR (500 MHz, CD3OD): d = 4.95 (m, 1H, CH (28)),
4.66 (m, 1H, CHa (29)), 4.56 (m, 1H, CHb (29)), 4.18 (d, 2H,

430
R. Csuk et al. / Bioorg. Med. Chem. 21 (2013) 425–435
J = 1.6 Hz, CH2 (33)), 3.90 (sept., 2H, J = 6.6, 2.6 Hz, 2 ꢁ CH
(34) + (37)), 3.13 (dd, 1H, J = 11.4, 4.9 Hz, CH (3)), 2.96 (m, 1H, CH
(19)), 2.12–1.93 (m, 4H, CHa (22) + CHa (21) + CHa (16) + CH (13)),
1.79 (dd, 1H, J = 11.8, 11.8 Hz, CH (18)), 1.77–1.50 (m, 5 H, CHa
(2) + CHb (2) + CHa (15) + CHa (1) + CHa (12)), 1.67 (s, 3H, CH3 (30)),
1.48–1.14 (m, 11H, CHa (6) + CHb (6) + CH (9) + CHb (12) + CHb
(22) + CHa (7) + CHb (7) + CHb (16) + CHb (21) + CHa (11) + CHb
1.02 mmol), formalin (37%, 0.35 ml, 4.3 mmol) and CuI (4 mg,
0.02 mmol) in DMSO (5 ml). The solution was stirred for 5 d at
40 °C to yield after re-crystallization 11 (297 mg, 53%) as a color-
less solid; mp 205 °C;
= 3319s, 2942s, 2876s, 2636s, 2515s, 1734s, 1639m, 1542m,
1457s, 1376s, 1248s, 1196m, 1108m, 1073m, 1048s, 979s cmꢀ1
½
aꢂD +3.9° (c 4.70, MeOH); IR (KBr):
m
;
1H NMR (500 MHz, CDCl3): d = 4.97 (m, 1H, CH (28)), 4.67 (m, 1H,
CHa (29)), 4.57 (m, 1H, CHb (29)), 4.44 (dd, 1H, J = 11.3, 5.2 Hz,
CH (3)), 4.18 (d, 2H, J = 1.7 Hz, CH2 (35)), 3.20 (m, 4H, 2 ꢁ CHa
(36) + (39) + 2 ꢁ CHb (36) + (39)), 2.95 (m, 1H, CH (19)), 2.09–1.94
(m, 4H, CHa (22) + CHa (21) + CHa (16) + CH (13)), 2.01 (s, 3H, CH3
(32)), 1.83–1.52 (m, 10 H, CH (18) + CHa (2) + CHa (15) + CHb
(2) + CHa (1) + CHa (12) + 2 ꢁ CH2 (37) + (40)), 1.68 (s, 3H, CH3
(30)), 1.50–1.12 (m, 10 H, CH (9) + CHb (22) + CHa (7) + CHb
(7) + CHb (16) + CHb (21) + CHa (11) + CHb (11) + CHa (6) + CHb (6)),
1.09 (s, 3H, CH3 (25)), 1.06–0.99 (m, 12H, CHb (15) + CHb
(12) + CHb (1) + CH3 (27) + 2 ꢁ CH3 (38) + (41)), 0.95 (s, 3H, CH3
(24)), 0.87 (s, 3H, CH3 (23)), 0.75 (s, 3H, CH3 (26)), 0.72 (d, 1H,
J = 10.1 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d = 171.4
(C31, CO), 150.9 (C20, C@CH2), 108.8 (C29, C@CH2), 92.0 (C33,
C„CH), 81.0 (C3, CH), 72.6 (C34, C„CH), 64.6 (C28, CH), 55.3
(C5, CH), 54.8 (C36 + C39, 2 ꢁ CH2), 50.6 (C17, Cquart.), 50.3 (C9,
CH), 48.8 (C18, CH), 48.7 (C19, CH), 42.7 (C14, Cquart.), 42.0 (C35,
CH2), 40.7 (C8, Cquart.), 38.5 (C1, CH2), 37.4 (C4, Cquart.), 37.2 (C13,
CH), 36.8 (C10, Cquart.), 34.2 (C7, CH2), 34.1 (C22, CH2), 34.0 (C21,
CH2), 31.9 (C16, CH2), 27.6 (C15, CH2), 27.2 (C24, CH3), 26.6 (C12,
CH2), 25.0 (C2, CH2), 20.6 (C11, CH2), 19.7 (C32, CH3), 18.0 (C6,
CH2), 17.8 (C30, CH3), 17.3 (C37 + C40, 2 ꢁ CH2), 15.3 (C23, CH3),
15.2 (C25, CH3), 14.7 (C26, CH3), 14.1 (C27, CH3), 9.8 (C38 + C41,
2 ꢁ CH3) ppm; MS (ESI, MeOH): m/z = 622.6 (100%, [MꢀCl]+); Anal.
for C41H68ClNO3 (658.43): C, 74.79; H, 10.41; N, 2.13. Found: C,
74.49; H, 10.67; N, 2.00.
(11)),
1.45
(dd,
12H,
J = 6.6 Hz,
1.8 Hz,
4 ꢁ CH3
(35) + (36) + (38) + (39)), 1.07–0.90 (m, 2H, CHb (15) + CHb (1)),
1.08 (s, 3H, CH3 (23)), 1.04 (s, 3H, CH3 (27)), 0.95 (s, 3H, CH3 (24)),
0.87 (s, 3H, CH3 (26)), 0.75 (s, 3H, CH3 (25)), 0.72 (d, 1H, J = 11.0 Hz,
CH (5)) ppm; 13C NMR (125 MHz, CD3OD): d = 151.9 (C20, C@CH2),
108.8 (C29, C@CH2), 91.3 (C31, C„CH), 78.2 (C3, CHOH), 74.8 (C32,
C„CH), 64.7 (C28, CHOH), 55.3 (C5, CH), 55.0 (C34 + C37, 2 ꢁ CH),
50.6 (C17, Cquart.), 50.3 (C9, CH), 48.9 (C18, CH), 48.7 (C19, CH), 42.7
(C14, Cquart.), 40.7 (C8, Cquart.), 38.6 (C1, CH2), 38.6 (C4, Cquart.), 37.2
(C13, CH), 36.8 (C10, Cquart.), 36.0 (C33, CH2), 34.1 (C7, CH2), 34.0
(C22, CH2), 31.9 (C21, CH2), 30.1 (C16, CH2), 27.6 (C15, CH2), 27.2
(C24, CH3), 26.6 (C2, CH2), 25.1 (C12, CH2), 20.6 (C11, CH2), 18.2
(C30, CH3), 18.0 (C6, CH2), 17.7 (C35 + C36 + C38 + C39, 4 ꢁ CH3),
15.3 (C23, CH3), 15.2 (C25, CH3), 14.7 (C26, CH3), 14.1 (C27, CH3);
MS (ESI, MeOH): m/z = 580.5 (100%, [MꢀCl]+); Anal. for C39H66ClNO2
(616.40): C, 75.99; H, 10.79; N, 2.27. Found: C, 75.88; H, 10.96; N,
2.15.
5.9. (3S, 28S) 28-Hydroxy-28-(3-pyrrolidin-1-yl-prop-1-yn-1-
yl)lup-20(29)-en-3-on hydrochloride (10)
Compound 10 was prepared following the general procedure
from 8 (466 mg, 1.00 mmol), pyrrolidine (0.1 ml, 1.2 mmol), forma-
lin (37%, 0.4 ml, 4.9 mmol) and CuI (4 mg, 0.02 mmol) in DMSO
(4 ml). The solution was stirred 20 h at 40 °C to yield after re-crys-
tallization 10 (215 mg, 37%) as a colorless solid; mp 180 °C; ½a D
+29.8° (c 5.50, MeOH); IR (KBr): = 3374s, 3067s, 2941s, 2584s,
2479s, 1703s, 1639s, 1457s, 1379s, 1201m, 1138s, 1046s, 1021s,
967m cmꢀ1 1H NMR (500 MHz, CDCl3): d = 4.95 (m, 1H, CH (28)),
ꢂ
m
5.11. (3S, 28S) 3-O-Acetyl-28-[3-(dicyclohexylamino)prop-1-yn-
1-yl]lup-20(29)-en-3,28-diol hydrochloride (12)
;
4.67 (m, 1H, CHa (29)), 4.56 (m, 1H, CHb (29)), 4.19 (m, 2H, CH2
(33)), 3.65 (m, 2H, CHa (34) + CHa (37)), 3.24 (m, 2H, CHb
(34) + CHb (37)), 2.96 (ddd, 1H, J = 10.9, 10.9, 5.9 Hz, CH (19)),
2.18 (m, 2H, CHa (35) + CHa (36)), 2.11–1.87 (m, 9H, CHa (2) + CHb
(2) + CHa (16) + CHa (21) + CHa (22) + CH (13) + CHb (35) + CHb
(36) + CHa (1)), 1.82 (dd, 1H, J = 11.9, 11.9 Hz, CH (18)), 1.78–1.63
(m, 2H, CHa (15) + CHa (12)), 1.67 (s, 3H, CH3 (30)), 1.63–1.57 (m,
1H, CHa (6)), 1.55–1.03 (m, 12H, CH (9) + CHb (22) + CHa (7) + CHb
(7) + CHb (16) + CHb (21) + CHa (11) + CHb (11) + CHb (6) + CHb
(1) + CH (5) + CHb (15)), 1.12 (s, 3H, CH3 (25)), 1.02–0.93 (m, 1H,
CHb (12)), 1.06 (m, 6 H, CH3 (24) + CH3 (27)), 1.02 (s, 3H, CH3
(23)), 0.95 (s, 3H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3):
d = 219.4 (C3, CO), 150.9 (C20, C@CH2), 108.8 (C29, C@CH2), 90.7
(C31, C„CH), 74.0 (C32, C„CH), 64.6 (C28, CHOH), 54.6 (C5, CH),
53.2 (C34 + C37, 2 ꢁ CH2), 50.5 (C17, Cquart.), 49.5 (C9, CH), 48.8
(C18, CH), 48.6 (C19, CH), 47.0 (C4, Cquart.), 43.2 (C14, Cquart.), 42.8
(C33, CH2), 40.6 (C8, Cquart.), 39.1 (C1, CH2), 37.3 (C13, CH), 36.6
(C10, Cquart.), 34.1 (C22, CH2), 34.0 (C2, CH2), 34.0 (C7, CH2), 33.3
(C21, CH2), 31.9 (C16, CH2), 27.6 (C15, CH2), 25.7 (C24, CH3), 25.1
(C12, CH2), 23.1 (C35 + C36, 2 ꢁ CH2), 21.1 (C11, CH2), 21.0 (C23,
CH3), 19.3 (C6, CH2), 17.8 (C30, CH3), 15.1 (C25, CH3), 15.0 (C26,
CH3), 14.1 (C27, CH3) ppm; MS (ESI, MeOH): m/z = 548.5 (100%,
[MꢀCl]+); Anal. for C37H58ClNO2 (584.31): C, 76.05; H, 10.00; N,
2.40. Found: C, 75.86; H, 10.24; N, 2.11.
Compound 12 was prepared as described in the general proce-
dure from
7 (204 mg, 0.4 mmol), dicyclohexylamine (0.1 ml,
0.61 mmol), formalin (37%, 0.2 ml, 2.5 mmol) and CuI (2 mg,
0.01 mmol) in DMSO (5 ml). The solution was stirred for three days
at 40 °C to yield after re-crystallization 12 (157 mg, 53%) as a col-
orless solid; mp 220 °C; ½a D
= 3265s, 2938s, 2862s, 2427s, 1737s, 1646m, 1456s, 1372s,
1244s, 1132m, 1108m, 977m cmꢀ1 1H NMR (500 MHz, CDCl3):
ꢂ
+11.2° (c 2.30, MeOH); IR (KBr):
m
;
d = 4.97 (m, 1H, CH (28)), 4.67 (m, 1H, CHa (29)), 4.56 (m, 1H,
CHb (29)), 4.44 (dd, 1H, J = 11.1, 5.2 Hz, CH (3)), 4.20 (m, 2H, CH2
(35)), 3.56 (m, 2H, 2 ꢁ CH (36) + (42)), 2.96 (m, 1H, CH (19)),
2.13–1.90 (m, 4H, CHa (22) + CHa (21) + CHa (16) + CH (13)), 2.02
(s, 3H, CH3 (32)), 1.80 (dd, 1H, J = 11.8, 11.8 Hz, CH (18)), 1.77–
1.52 (m, 15H, CHa (2) + CHa (15) + CHb (2) + CHa (1) + CHa
(12) + 4 ꢁ CHa
(37) + (41) + (43) + (47) + 4 ꢁ CHa
(38) + (40) + (44) + (46) + 2 ꢁ CHa (39) + (45)), 1.68 (s, 3H, CH3
(30)), 1.51–1.15 (m, 20 H, CH (9) + CHb (22) + CHa (7) + CHb
(7) + CHb (16) + CHb (21) + CHa (11) + CHb (11) + CHa (6) + CHb
(6) + 4 ꢁ CHb
(37) + (41) + (43) + (47) + 4 ꢁ CHb
(38) + (40) + (44) + (46) + 2 ꢁ CHb (39) + (45)), 1.10 (s, 3H, CH3
(25)), 1.07–0.97 (m, 3H, CHb (15) + CHb (12) + CHb (1)), 1.06 (s,
3H, CH3 (27)), 0.91 (s, 3H, CH3 (26)), 0.87 (s, 3H, CH3 (23)), 0.86
(s, 3H, CH3 (24)), 0.82 (m, 1H, CH (5)) ppm; 13C NMR (125 MHz,
CDCl3): d = 171.4 (C31, CO), 150.8 (C20, C@CH2), 108.8 (C29,
C@CH2), 92.6 (C33, C„CH), 81.0 (C3, CH), 73.2 (C34, C„CH), 64.6
(C28, CHOH), 61.8 (C36 + C42, 2 ꢁ CH), 55.3 (C5, CH), 50.6 (C17,
Cquart.), 50.2 (C9, CH), 48.9 (C18, CH), 48.7 (C19, CH), 42.8 (C14,
Cquart.), 40.7 (C8, Cquart.), 38.1 (C1, CH2), 37.4 (C4, Cquart.), 37.2
(C13, CH), 36.8 (C10, Cquart.), 36.5 (C35, CH2), 34.1 (C7, CH2), 34.0
(C22, CH2), 31.9 (C21, CH2), 29.1 (C16, CH2), 27.7 (C15, CH2), 27.0
5.10. (3S, 28S) 3-O-Acetyl-28-[3-(dipropylamino)prop-1-yn-1-
yl]lup-20(29)-en-3,28-diol hydrochloride (11)
Compound 11 was prepared as described in the general proce-
dure from
7 (433 mg, 0.85 mmol), dipropylamine (0.14 ml,

R. Csuk et al. / Bioorg. Med. Chem. 21 (2013) 425–435
431
(C24, CH3), 25.0 (C12, CH2), 24.8 (C37 + C41 + C43 + C47, 4 ꢁ CH2),
24.6 (C38 + C40 + C44 + C46, 4 ꢁ CH2), 24.0 (C2, CH2), 23.2
(C39 + C45, 2 ꢁ CH2), 20.6 (C11, CH2), 19.7 (C32, CH3), 19.0 (C30,
CH3), 17.8 (C6, CH2), 15.5 (C23, CH3), 15.3 (C25, CH3), 15.2 (C26,
CH3), 14.1 (C27, CH3) ppm; MS (ESI, MeOH): m/z = 702.5 (100%,
[MꢀCl]+); Anal. for C47H76ClNO3 (738.56): C, 76.43; H, 10.37; N,
1.90. Found: C, 76.19; H, 10.57; N, 1.65.
ppm; 13C NMR (125 MHz, DMSO-d6): d = 171.4 (C31, CO), 150.8
(C20, C = CH2), 108.8 (C29, C = CH2), 91.4 (C33, C„CH), 81.0 (C3,
CH), 73.7 (C34, C„CH), 64.6 (C28, CHOH), 55.3 (C5, CH), 54.4
(C36 + C41, 2 ꢁ CH2), 50.5 (C17, Cquart.), 50.2 (C9, CH), 48.8 (C18,
CH), 48.1 (C19, CH), 47.8 (C14, Cquart.), 46.8 (C35, CH2), 42.7 (C8,
Cquart.), 40.7 (C1, CH2), 38.1 (C10, Cquart.), 37.4 (C4, Cquart.), 37.2
(C13, CH), 36.8 (C22, CH2), 34.0 (C7, CH2), 33.9 (C16, CH2), 31.9
(C21, CH2), 27.6 (C15, CH2), 27.0 (C24, CH3), 25.6 (C37 + C40,
2 ꢁ CH3), 25.0 (C12, CH2), 23.7 (C37 + C39, CH2), 23.2 (C2, CH2),
20.6 (C32, CH3), 19.7 (C11, CH2), 17.8 (C30, CH3), 17.7 (C6, CH2),
15.5 (C25, CH3),, 15.2 (C26 + C23, 2 ꢁ CH3), 14.1 (C27, CH3) ppm;
MS (ESI, MeOH): m/z = 620.7 (100%, [MꢀCl]+); Anal. for
5.12. (3S, 28S) 3-O-Acetyl-28-[3-(dimethylamino)prop-1-yn-1-
yl]lup-20(29)-en-3,28-diol hydrochloride (13)
Compound 13 was prepared as described in the general proce-
dure from 7 (500 mg, 1 mmol), dimethylamine solution (40%,
0.25 ml, 2 mmol), formalin (37%, 0.4 ml, 5 mmol) and CuI (2 mg,
0.01 mmol) in DMSO (5 ml). The solution was stirred for 24 h at
40 °C to yield 13 (318 mg, 53%) as a colorless solid; mp 189 °C;
C
41H66NO3Cl (656.42): C, 75.02; H, 10.13; N, 2.13. Found: C,
74.87; H, 10.02; N, 2.01.
5.14. (3S, 28S) 3-O-Acetyl-28-[3-morpholin-4-yl-prop-1-yn-1-
½
a D
ꢂ
+11.3° (c 4.9, MeOH); IR (KBr):
2604s, 2462s, 1734s, 1640m, 1456s, 1375s, 1317m, 1248s,
1132m, 1108m, 1024s, 979s cmꢀ1 1H NMR (500 MHz, DMSO-d6):
m
= 3374s, 2944s, 2873s,
yl]lup-20(29)-en-3,28-diol hydrochloride (15)
;
Compound 15 was prepared as described in the general proce-
d = 10.9 (br, 1H, NH), 4.78 (s, 1H, CH (28)), 4.61 (d, 1H, J = 2.1 Hz,
CHa (29)), 4.51 (s, 1H, CHb (29)), 4.35 (dd, 1H, J = 4.6, 11.5 Hz, CH
(3)), 4.07 (s, 2H, CH2 (35)), 2.94–2.86 (m, 1H, CH (19)), 2.72 (s,
3H, CH3 (36)), 2.51 (s, 3H, CH3 (37)), 2.38–2.33 (m, 1H, CHa (22)),
2.10–2.01 (m, 1H, CHa (21)), 1.97 (s, 3H, CH3 (32)), 1.95–1.85 (m,
2H, CHa (16) + CH (13)), 1.70–1.63 (m, 1H, CH (18)), 1.61 (s, 1H,
CH3 (30)), 1.60–1.04 (m, 16 H, CH2 (12) + CH2 (11) + CH2 (6) + CH2
(15) + CH2 (7) + CHa (1) + CH (9) + CH2 (2) + CHb (22) + CHb (21)),
0.99 (s, 3H, CH3 (27)), 0.96 (s, 3H, CH3 (25)), 0.93–0.84 (m, 2H,
CHb (1) + CHb (16)), 0.80 (s, 3H, CH3 (26)), 0.77 (m, 6 H, 2 ꢁ CH3
(24 + 23)), 0.65 (d, 1H, J = 5.8 Hz, CH (5)) ppm; 13C NMR
(125 MHz, DMSO-d6): d = 170.1 (C31, CO), 150.8 (C20, C@CH2),
109.4 (C29, C@CH2), 91.6 (C33, C„CH), 79.8 (C3, CH), 73.7 (C34,
C„CH), 63.8 (C28, CHOH), 54.5 (C5, CH), 50.1 (C17, Cquart.), 49.4
(C9, CH), 48.4 (C18, CH), 48.1 (C19, CH), 45.8 (C14, Cquart.), 42.5
(C8, Cquart.), 41.7 (C35, CH3); 40.4 (C36 + C37, 2 ꢁ CH3), 38.9 (C13,
CH), 37.7 (C1, CH2), 37.4 (C4, Cquart.), 36.5 (C10, Cquart.), 36.5 (C35,
CH2), 34.0 (C7, CH2), 33.8 (C22, CH2), 31.7 (C21, CH2), 29.1 (C16,
CH2), 27.7 (C15, CH2), 27.0 (C24, CH3), 24.7 (C12, CH2), 23.3 (C2,
CH2), 20.9 (C32, CH3), 20.4 (C11, CH2), 18.6 (C30, CH3), 17.7 (C6,
CH2), 16.4 (C25 + C23, 2 ꢁ CH3), 15.8 (C26, CH3), 14.7 (C27, CH3)
ppm; MS (ESI, MeOH): m/z = 566 (100%, [MꢀCl]+); Anal. for
dure from 7 (500 mg, 1 mmol), morpholine (100 ll, 1.15 mmol),
formalin (37%, 0.4 ml, 5 mmol) and CuI (2 mg, 0.01 mmol) in DMSO
(5 ml). The solution was stirred for 24 h at 40 °C to yield after re-
crystallization 15 (238 mg, 37%) as a colorless solid; mp 206 °C;
½
a D
ꢂ
+5.0° (c 6.5, MeOH); IR (KBr):
2450m, 1734s, 1640m, 1455s, 1375s, 1247s, 1196m, 1125s,
1075m, 1028s, 978s cmꢀ1 1H NMR (500 MHz, DMSO-d6):
m = 3346s, 2943s, 2871s,
;
d = 11.80 (s, 1H, NH), 4.76 (s, 1H, CH (28)), 4.59 (d, 1H, J = 2.1 Hz,
CHa (29)), 4.49 (m, 1H, CHb (29)), 4.33 (dd, 1H, J = 4.8, 11.8 Hz,
CH (3)), 4.10 (s, 2H, CH2 (35)), 4.00–3.70 (m, 6 H, CH2 (36) + CH2
(39) + CHa (37) + CHa (38)), 3.13–2.96 (m, 2H, CHb (37) + CHb
(38)), 2.93–2.83 (m, 1H, CH (19)), 2.00–1.96 (m, 2H, CHa
(22) + CHa (21), 1.95 (s, 3H, CH3 (32)), 1.93–1.83 (m, 2H, CHa
(16) + CH (13)), 1.68–1.61 (m, 1H, CH (18)), 1.60 (s, 3H, CH3 (30)),
1.58–1.08 (m, 16 H, CH2 (12) + CH2 (11) + CH2 (6) + CH2 (15) + CH2
(7) + CHa (1) + CH (9) + CH2 (2) + CHb (22) + CHb (21)), 0.97 (s, 3H,
CH3 (27)), 0.94 (s, 3H, CH3 (25)), 0.92–0.85 (m, 2H, CHb (1)+ CHb
(16)), 0.79 (s, 3H, CH3 (26)), 0.76 (m, 6 H, 2 ꢁ CH3 (24 + 23)),
0.72–0.70 (m, 1H, CH (5)) ppm; 13C NMR (125 MHz, DMSO-d6):
d = 169.7 (C31, CO), 150.5 (C20, C@CH2), 109.1 (C29, C@CH2), 91.9
(C33, C„CH), 79.7 (C3, CH), 73.1 (C34, C„CH), 64.7 (C36 + 39,
2 ꢁ CH2), 63.7 (C28, CHOH), 62.9 (C37 + 38, 2 ꢁ CH2), 54.5 (C5,
CH), 50.0 (C17, Cquart.), 49.3 (C9, CH), 48.4 (C18, CH), 48.0 (C19,
CH), 44.9 (C35, CH2), 44.2 (C14, Cquart.), 42.5 (C8, Cquart.), 38.9
(C13, CH), 37.7 (C1, CH2), 37.3 (C4, Cquart.), 36.5 (C10, Cquart.) 34.0
(C7, CH2), 33.7 (C22, CH2), 33.6 (C16, CH2), 31.7 (C21, CH2), 27.6
(C15, CH2), 27.4 (C24, CH3), 24.7 (C12, CH2), 23.3 (C2, CH2), 20.9
(C32, CH3), 20.4 (C11, CH2), 18.5 (C30, CH3), 17.6 (C6, CH2), 15.8
(C25 + C23, 2 ꢁ CH3), 15.1 (C26, CH3), 14.7 (C27, CH3) ppm; MS
(ESI, MeOH): m/z = 608.5 (100%, [MꢀCl]+); Anal. for C39H62NO4Cl
(644.37): C, 72.69; H, 9.70; N, 2.17. Found: C, 72.51; H, 9.93; N,
2.11.
C37H60NO3Cl (602.33): C, 73.78; H, 10.04; N, 2.33. Found: C,
73.59; H, 10.16; N, 2.20.
5.13. (3S, 28S) 3-O-Acetyl-28-[3-azepan-prop-1-yn-1-yl]lup-
20(29)-en-3,28-diol hydrochloride (14)
Compound 14 was prepared as described in the general proce-
dure from 7 (500 mg, 1 mmol), azepane (113 ll, 1.15 mmol), for-
malin (37%, 0.4 ml, 5 mmol) and CuI (2 mg, 0.01 mmol) in DMSO
(5 ml). The solution was stirred for 24 h at 40 °C to yield after re-
crystallization 14 (366 mg, 56%) as a colorless solid; mp 210 °C;
½
a D
ꢂ
+21.5° (c 6.3, CHCl3); IR (KBr):
2450m, 1734s, 1640m, 1455s, 1375s, 1247s, 1196m, 1125s,
1075m, 1028s, 978s cmꢀ1 1H NMR (500 MHz, DMSO-d6): d = 4.96
m
= 3346s, 2943s, 2871s,
5.15. (3S, 28S) 3-O-Acetyl-28-[3-thiomorpholin-4-yl-prop-1-yn-
1-yl]lup-20(29)-en-3,28-diol hydrochloride (16)
;
(s, 1H, CH (28)), 4.67 (d, 1H, J = 1.8 Hz, CHa (29)), 4.56 (m, 1H,
CHb (29)), 4.44 (dd, 1H, J = 5.1, 11.2 Hz, CH (3)), 4.15 (s, 2H, CH2
(35)), 3.62–3.53 (m, 2H, CHa (36) + CHa (41)), 3.31–3.29 (m, 2H,
CHb (36) + CHb (41)), 2.99–2.92 (m, 1H, CH (19)), 2.10–203 (m,
3H, CHa (22) + CHa (21) + CHa (16)), 2.01 (s, 3H, CH3 (32)), 2.00–
1.86 (m, 5 H, + CH2 (37) + CH2 (40) + CHa (22)), 1.84–1.69 (m, 8H,
+CHa (1) CH2 (38) + CH2 (39) + CH2 (12) + CH (13)), 1.68 (s, 3H,
CH3 (30)), 1.65–1.15 (m, 13H, CH2 (11) + CH2 (6) + CH2 (15) + CH2
(7) + CH (9) + CH2 (2) + CHb (21) + CH (18)), 1.10 (s, 3H, CH3 (27)),
1.05 (s, 3H, CH3 (25)), 1.03–0.96 (m, 2H, CHb (1) + CHb (16)), 0.91
(s, 3H, CH3 (26)), 0.87–0.82 (m, 7H, 2 ꢁ CH3 (24 + 23) + CH (5))
Compound 16 was prepared as described in the general proce-
dure from
7
(500 mg, 1 mmol), thiomorpholine (109 ll,
1.15 mmol), formalin (37%, 0.4 ml, 5 mmol) and CuI (2 mg,
0.01 mmol) in DMSO (5 ml). The solution was stirred for 24 h at
40 °C to yield after re-crystallization 16 (284 mg, 43%) as a color-
less solid; mp 208–211 °C; ½a D
= 3385s, 3068m, 2943s, 2873s, 2363m, 1732s, 1639m, 1454s,
1375s, 1318m, 1248s, 1133m, 1108m, 1028s, 980s cmꢀ1 1H NMR
ꢂ
+6.0° (c 5.5, MeOH); IR (KBr):
m
;
(500 MHz, DMSO-d6): d = 4.78 (s, 1H, CH (28)), 4.61 (d, 1H,
J = 2.0 Hz, CHa (29)), 4.51 (m, 1H, CHb (29)), 4.35 (dd, 1H, J = 4.5,
11.5 Hz, CH (3)), 4.14 (s, 2H, CH2 (35)), 3.75–3.65 (m, 2H, CHa

432
R. Csuk et al. / Bioorg. Med. Chem. 21 (2013) 425–435
(36) + CHa (39)), 3.20–3.05 (m, 4H, CHb (36) + CHb (39) + CHa
(37) + CHa (38)), 2.95–2.80 (m, 3H, CH (19) + CHb (37) + CHb (38)),
2.10–2.03 (m, 1H, CHa (22)), 2.00–1.85 (m, 5 H, CH3 (32) + CHa
(21) + CHa (16)), 1.75–1.63 (m, 2H, CH (13) + CH (18)), 1.61 (s,
3H, CH3 (30)), 1.59–1.01 (m, 16 H, CH2 (12) + CH2 (11) + CH2
(6) + CH2 (15) + CH2 (7) + CHa (1) + CH (9) + CH2 (2) + CHb
(22) + CHb (21)), 0.99 (s, 3H, CH3 (27)), 0.96 (s, 3H, CH3 (25)),
0.94–0.89 (m, 2H, CHb (1)+ CHb (16)), 0.81 (s, 3H, CH3 (26)), 0.78
(m, 6 H, 2 ꢁ CH3 (24 + 23)), 0.67–0.64 (m, 1H, CH (5)) ppm; 13C
NMR (125 MHz, DMSO-d6): d = 170.1 (C31, CO), 150.8 (C20,
C@CH2), 109.4 (C29, C@CH2), 91.7 (C33, C„CH), 79.9 (C3, CH),
73.3 (C34, C„CH), 63.8 (C28, CHOH), 54.5 (C5, CH), 52.2
(C36 + C39, 2 ꢁ CH2), 50.1 (C17, Cquart.), 49.4 (C9, CH), 48.5 (C18,
CH), 48.1 (C19, CH), 45.7 (C14, Cquart.), 42.5 (C8, Cquart.), 40.1 (C36,
CH3), 38.9 (C13, CH), 37.7 (C1, CH2), 37.3 (C4, Cquart.), 36.5 (C10,
Cquart.), 36.4 (C35, CH2), 34.1 (C7, CH2), 33.8 (C22, CH2), 33.6 (C16,
CH2), 31.7 (C21, CH2), 27.6 (C15, CH2), 27.4 (C24, CH3), 24.7 (C12,
CH2), 23.9 (C37 + C38, CH2), 22.3 (C2, CH2), 20.9 (C32, CH3), 20.4
(C11, CH2), 18.5 (C30, CH3), 17.7 (C6, CH2), 16.4 (C25 + C23,
2 ꢁ CH3), 15.8 (C26, CH3), 14.7 (C27, CH3) ppm; MS (ESI, MeOH):
m/z = 624.6 (100%, [MꢀCl]+); Anal. for C39H62NO3SCl (660.43): C,
70.93; H, 9.46; N, 2.12; S, 4.86. Found: C, 70.76; H, 9.64; N, 2.04;
S, 4.63.
with dry HClg at 0 °C for 1 h. Precipitation was completed by stand-
ing for 12 h at 4 °C; the product was filtered off, washed with water
and diethyl ether and dried to yield 18 (143 mg, 91%) as a colorless
solid; mp 226–228 °C; ½a D
ꢂ
+16.6° (c 4.05, CHCl3); IR (KBr):
= 3301m, 2944s, 2477m, 1732s, 1643w, 1455m, 1370m, 1247s,
m
1132w, 1027m cmꢀ1
;
1H NMR (500 MHz, DMSO-d6): d = 11.70
(br, 1H, NH), 6.05–5.95 (m, 2H, 2 ꢁ CH (37)), 5.60–5.48 (m, 4H,
2 ꢁ CH2 (38)), 4.80 (br, 1H, CH (28)), 4.62 (d, 1H, J = 1.8 Hz, CHa
(29)), 4.52 (s, 1H, CHb (29)), 4.36 (dd, 1H, J = 4.6, 11.5 Hz, CH (3)),
3.97 (s br, 2H, CH2 (35)), 3.80–3.65 (m, 4H, 2 ꢁ CH2 (36)), 2.95–
2.85 (m, 1H, CH (19)), 2.05–2.00 (m, 1H, CHa (22)), 1.98 (s, 3H,
CH3 (32)), 1.93–1.81 (m, 3H, CHa (21) + CH (13) + CHa (16)), 1.70–
1.65 (m, 1H, CH (18)), 1.63 (s, 3H, CH3 (30)), 1.69–1.08 (m, 15H,
CH2 (11) + CH2 (6) + CH2 (15) + CH2 (7) + CHa (1) + CH (9) + CH2
(2) + CHb (22) + CHb (21) + CHb (16)), 1.02–0.89 (m, 8H, CH2
(12) + 2 ꢁ CH3 (27) + (25)), 0.86–0.83 (m, 1H, CHb (1)), 0.82–0.75
(m, 9H, 3 ꢁ CH3 (26) + (24) + (23)), 0.65 (d, 1H, J = 8.5 Hz, CH (5))
ppm; 13C NMR (125 MHz, DMSO-d6): d = 170.0 (C31, C@O), 150.8
(C20, C@CH2), 125.0 (C37, CH2@CH), 117.0 (C38, CH@CH2), 109.4
(C29, CH2@C), 91.9 (C33, C„C), 79.8 (C3, CH), 74.3 (C34, C„C),
66.7 (C28, CH), 55.5 (C5, CH), 50.3 (C17, Cquart.), 50.6 (C35, CH2),
50.2 (C9, CH), 49.2 (C18, CH), 48.8 (C19, CH), 43.1 (C14, Cquart.),
41.0 (C8, Cquart.), 40.9 (C36, 2 ꢁ CH2), 38.5 (C4, Cquart.), 37.9 (C1,
CH2), 37.5 (C13, CH), 37.2 (C10, Cquart.), 34.7 (C7, CH2), 34.3 (C21,
CH2), 34.1 (C16, CH2), 32.4 (C22, CH2), 28.0 (C23, CH3), 27.8 (C15,
CH2), 25.2 (C12, CH2), 23.8 (C2, CH2), 21.6 (C32, CH3), 20.9 (C11,
CH2), 18.8 (C30, CH3), 18.2 (C6, CH2), 16.5 (C24, CH3), 16.2
(C26 + C25, 2 ꢁ CH3), 15.1 (C27, CH3) ppm; MS (ESI, MeOH): m/
z = 618.8 (100%, [M+H]+); Anal. for C41H64NO3Cl (654.40): C,
75.25; H, 9.86; N, 2.14. Found: C, 75.13; H, 9.99; N, 1.97.
5.16. (3S, 28S) 3-O-Acetyl-28-[(diallylamino)-1-yl-prop-1-yn-1-
yl]lup-20(29)-en-3,28-diol (17)
Compound 17 was prepared as described in the general proce-
dure from 7 (500 mg, 1 mmol), diallylamine (109 ll, 1.15 mmol),
formalin (37%, 0.4 ml, 5 mmol) and CuI (2 mg, 0.01 mmol) in THF
(5 ml). The crude residue was purified by column chromatography
(silica gel, n-hexane/ethyl acetate, 8/2) to yield 17 (487 mg, 79%) as
5.18. (3S, 28S) 3-O-Acetyl-28-[piperidine-1-yl-prop-1-yn-1-
a colorless solid; mp 75–80 °C ½a D
(n-hexane/ethyl acetate, 8/2); IR (KBr):
1735s, 1642s, 1454s, 1376s, 1326s, 1245s, 1028s, 1008s cmꢀ1
ꢂ
+142.9° (c 0.6, CHCl3); Rf = 0.26
= 3457br, 3075m, 2943s,
1H
yl]lup-20(29)-en-3,28-diol hydrochloride (19)
m
;
Compound 19 was prepared as described in the general proce-
NMR (400 MHz, CHCl3): d = 5.84–5.70 (m, 2H, 2 ꢁ CH (37)), 5.21–
5.05 (m, 4H, 2 ꢁ CH2 (38)), 4.84 (s, 1H, CH (28)), 4.64 (s, 1H, CHa
(29)), 4.51 (s, 1H, CHb (29)), 4.40 (dd, 1H, J = 6.4, 9.7 Hz, CH (3)),
3.35 (s, 2H, CH2 (35)), 3.10–3.00 (m, 4H, 2 ꢁ CH2 (36)), 2.91–2.78
(m, 1H, CH (19)), 2.21–2.10 (m, 2H, CHa (22) + CHa (16)), 1.97 (s,
3H, CH3 (32)), 1.96–1.85 (m, 2H, CHa (21) + CH (13)), 1.74–1.65
(m, 3H, CH (18) + CHa (1) + CHa (12)), 1.62 (s, 3H, CH3 (30)), 1.69–
1.08 (m, 14H, CH2 (11) + CH2 (6) + CH2 (15) + CH2 (7) + CH
(9) + CH2 (2) + CHb (22) + CHb (21) + CHb (16)), 0.98 (s, 3H, CH3
(27)), 0.93 (s, 3H, CH3 (25)), 0.92–0.81 (m, 2H, CHb (12) + CHb
(1)), 0.80–0.75 (m, 9H, 3 ꢁ CH3 (26) + (24) + (23)), 0.73 (d, 1H,
J = 10.5 Hz, CH (5)) ppm; 13C NMR (125 MHz, CDCl3): d = 171.0
(C31, C@O), 151.0 (C20, C@CH2), 135.0 (C38, 2 ꢁ CH2@CH), 118.2
(C37, 2 ꢁ CH@CH2), 109.6 (C29, CH2@C), 85.9 (C33, C„C), 80.9
(C3, CH), 80.8 (C34, C„C), 66.1 (C28, CH), 56.3 (C35, CH2), 55.4
(C5, CH), 50.8 (C17, Cquart.), 50.2 (C9, CH), 49.0 (C18, CH), 48.7
(C19, CH), 43.0 (C14, Cquart.), 41.7 (C36, 2 ꢁ CH2), 40.9 (C8, Cquart.),
38.4 (C4, Cquart.), 37.8 (C1, CH2), 37.2 (C13, CH), 37.0 (C10, Cquart.),
34.7 (C7, CH2), 34.1 (C21, CH2), 34.0 (C16, CH2), 32.4 (C22, CH2),
28.0 (C23, CH3), 27.8 (C15, CH2), 25.1 (C12, CH2), 23.7 (C2, CH2),
21.3 (C32, CH3), 20.9 (C11, CH2), 18.8 (C30, CH3), 18.1 (C6, CH2),
16.5 (C24 + C26, 2 ꢁ CH3), 16.1 (C25, CH3), 15.0 (C27, CH3) ppm;
MS (ESI, MeOH): m/z = 618.6 (100%, [M+H]+); Anal. for C41H63NO3
(617.94): C, 79.69; H, 10.28; N, 2.27. Found: C, 79.52; H, 10.48;
N, 2.14.
dure from 7 (300 mg, 0.59 mmol), piperidine (114
formalin (37%, 0.4 ml, 5 mmol) and CuI (20 mg, 0.1 mmol) in THF
(5 ml); 19 (225 mg, 67%) was obtained as colorless solid
(255 mg, 67%); mp 212–215 °C; ½a D +74.0° (c 0.6, CHCl3); IR
(KBr) = 3405br, 2943s, 2870s, 2641m, 2534m, 1733s, 1638m,
1456s, 1384s, 1247s, 1197w, 1133w, 1107w, 1031w cmꢀ1 1H
ll, 1.15 mmol),
a
ꢂ
m
;
NMR (400 MHz, CD3OD): d = 4.90–4.85 (m, 1H, CH (28)), 4.57 (d,
1H, J = 1.8 Hz, CHa (29)), 4.49–4.45 (m, 1H, CHb (29)), 4.34 (dd,
1H, J = 5.4, 10.8 Hz, CH (3)), 4.00 (s br, 2H, CH2 (35)), 3.62–3.42
(m, 2H, CHa (36) + CHa (40)), 3.09–2.80 (m, 4H, CH (19) + CHb
(36) + CHb (40)), 2.22–1.84 (m, 9H, CHa (22) + CHa (16) + CH3
(32) + CHa (21) + CH (13) + CHa (37) + CHa (39)), 1.74–1.05 (m,
24H, CHb (37) + CHb (39) + CH2 (38) + CH (18) + CHa (1) + CH2
(12) + CH3 (30) + CH2 (11) + CH2 (6) + CHa (15) + CH2 (7) + CH
(9) + CH2 (2) + CHb (22) + CHb (21) + CHb (16)), 1.01 (s, 3H, CH3
(27)), 0.96 (s, 3H, CH3 (25)), 0.94–0.88 (m, 2H, CHb (15) + CHb
(1)), 0.86 (s, 3H, CH3 (26)), 0.81 (s, 3H, CH3 (24)), 0.77 (s, 3H, CH3
(23)), 0.75–0.71 (m, 1H, CH (5)) ppm; 13C NMR (100 MHz, CD3OD):
d = 171.4 (C31, C@O), 150.9 (C20, C@CH2), 108.8 (C29, CH2@C), 92.0
(C33, C„C), 81.0 (C3, CH), 73.1 (C34, C„C), 64.7 (C28, CH), 55.4
(C5, CH), 52.3 (C36 + C40, 2 ꢁ CH2), 50.5 (C17, Cquart.), 50.3 (C9,
CH), 48.9 (C18, CH),48.7 (C19, CH), 45.9 (C35, CH2), 42.8 (C14,
Cquart.), 40.7 (C8, Cquart.), 38.6 (C4, Cquart.), 38.5 (C1, CH2), 37.4
(C10, Cquart.), 37.2 (C13, CH), 36.9 (C16, CH2), 36.8 (C22, CH2),
34.1 (C7, CH2), 31.9 (C21, CH2), 27.6 (C15, CH2), 27.0 (C23, CH3),
25.0 (C12, CH2), 23.3 (C2, CH2), 22.8 (C37 + C39, 2 ꢁ CH2), 21.1
(C38, CH2), 20.6 (C11, CH2), 19.7 (C32, CH3), 18.0 (C6, CH2), 17.7
(C30, CH3), 15.5 (C24, CH3), 15.3 (C26, CH3), 15.2 (C25, CH3), 14.2
(C27, CH3) ppm; MS (ESI, MeOH): m/z = 606.7 (100%, [M+H]+);
Anal. for C39H63N2O3Cl (643.38): C, 72.81; H, 9.87; N, 4.35. Found:
C, 72.69; H, 10.03; N, 4.18.
5.17. (3S, 28S) 3-O-Acetyl-28-[(diallylamino)-1-yl-prop-1-yn-1-
yl]lup-20(29)-en-3,28-diol hydrochloride (18)
Compound 18 was prepared by dissolving compound 17
(150 mg, 0.24 mmol) in diethylether and treating this solution

R. Csuk et al. / Bioorg. Med. Chem. 21 (2013) 425–435
433
5.19. (3S, 28S) 3-O-Acetyl-28-[3-pyrrolidine-1-yl-prop-1-yn-1-
yl]-lup-20(29)-en-3,28-diol hydrochloride (20)
(C15, CH2), 25.0 (C12, CH2), 23.7 (C2, CH2), 21.3 (C32, CH3), 20.8
(C11, CH2), 18.8 (C30, CH3), 18.1 (C6, CH2), 16.5 (C24, CH3), 16.3
(C26, CH3), 16.1 (C25, CH3), 15.0 (C27, CH3) ppm; MS (ESI, MeOH):
m/z = 718.6 (100%, [M+H]+); Anal. for C49H68NO3Cl (754.52): C,
78.00; H, 9.08; N, 1.86. Found: C, 77.87; H, 9.17; N, 1.64.
Compound 20 was prepared as described in the general proce-
dure from 7 (200 mg, 0.39 mmol), pyrrolidine (94 ll, 1.15 mmol),
formalin (37%, 0.4 ml, 5 mmol) and CuI (20 mg, 0.1 mmol) in THF
(5 ml); 20 (164 mg, 67%) was obtained as a colorless solid; mp
5.21. (3S, 28S) 3-O-Acetyl-28-[3-(methyldipropylaminium)-
206–207 °C; ½a D
ꢂ
+21.1° (c 2.49, CHCl3); IR (KBr):
m
;
= 3442br,
prop-1-in-1-yl]lup-20(29)-en-3,28-diol iodide (22)
2943s, 1705m, 1638s, 1442m, 1384s, 1047w cmꢀ1
1H NMR
(400 MHz, CD3OD): d = 4.96–4.91 (m, 1H, CH (28)), 4.66 (d, 1H,
J = 2.2 Hz, CHa (29)), 4.55 (dd, 1H, J = 2.4, 1.3 Hz, CHb (29)), 4.43
(dd, 1H, J = 5.4, 10.9 Hz, CH (3)), 4.09–4.02 (s br, 2H, CH2 (35)),
3.35–3.25 (m, 4H, 2 ꢁ CH2 (36)), 2.99–2.90 (m, 1H, CH (19)), 2.10–
1.90 (m, 11H, CHa (22) + CHa (16) + CH3 (32) + CHa (21) + CH
(13) + 2 ꢁ CH2 (37)), 1.82–1.68 (m, 3H, CH (18) + CHa (1) + CHa
(15)), 1.67 (s, 3H, CH3 (30)), 1.66–1.13 (m, 14H, CH2 (12) + CH2
(11) + CH2 (6) + CH2 (7) + CH (9) + CH2 (2) + CHb (22) + CHb
(21) + CHb (16)), 1.08 (s, 3H, CH3 (25)), 1.04 (s, 3H, CH3 (27)), 1.02–
0.93 (m, 2H, CHb (15) + CHb (1)), 0.90 (s, 3H, CH3 (26)), 0.86 (s, 3H,
CH3 (24)), 0.85 (s, 3H, CH3 (23)), 0.84–0.77 (m, 1H, CH (5)) ppm;
13C NMR (100 MHz, CD3OD): d = 173.2 (C31, C@O), 152.5 (C20,
C@CH2), 110.3 (C29, CH2@C), 91.3 (C33, C„C), 82.7 (C3, CH), 76.6
(C34, C„C), 66.2 (C28, CH), 56.9 (C5, CH), 54.6 (C36, 2 ꢁ CH2), 52.1
(C17, Cquart.), 51.8 (C9, CH), 50.4 (C18, CH), 50.2 (C19, CH), 44.7
(C35, CH2), 44.3 (C14, Cquart.), 42.3 (C8, Cquart.), 39.7 (C4, Cquart.),
39.0 (C1, CH2), 38.7 (C13, CH), 38.4 (C10, Cquart.), 35.7 (C16, CH2),
35.5 (C22, CH2), 35.0 (C7, CH2), 33.5 (C21, CH2), 29.2 (C15, CH2),
28.6 (C23, CH3), 24.8 (C12, CH2), 24.7 (C37, 2 ꢁ CH2), 24.6 (C2,
CH2), 22.2 (C11, CH2), 21.3 (C32, CH3), 19.4 (C6, CH2), 19.3 (C30,
CH3), 17.1 (C24, CH3), 16.8 (C26, CH3), 16.7 (C25, CH3), 15.7 (C27,
CH3) ppm; MS (ESI, MeOH): m/z = 592.6 (100%, [M+H]+); Anal. for
A solution of compound 11 (200 mg, 0.3 mmol) in methanol
(5 ml) was treated with an aq. solution of KOH (satd., 0.5 ml)
for 10 min. After extraction with ethyl acetate (3 ꢁ 100 ml), the
organic layers were dried (Na2SO4), the solvents were evaporated,
and the remaining solid was treated in diethylether (10 ml) with
methyl iodide (2 ml, 32.0 mmol) for 5 days under argon. The pre-
cipitate was washed with ether to yield 22 (202 mg, 87%) as an
off-white solid; mp 170 °C; ½a D
= 3275s, 3078m, 2965s, 2877s, 1732s, 1648m, 1534m, 1456s,
1392s, 1371s, 1318m, 1246s, 1155m, 1119s, 1077m, 1024s,
977s cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.00 (m, 1H, CH (28)),
ꢂ
+9.7° (c 4.55, MeOH); IR (KBr):
m
;
4.67 (m, 1H, CHa (29)), 4.57 (m, 1H, CHb (29)), 4.44 (dd, 1H,
J = 11.1, 5.2 Hz, CH (3)), 4.36 (d, 2H, J = 1.7 Hz, CH2 (35)), 3.37
(m, 4H, 2 ꢁ CHa (36) + (39) + 2 ꢁ CHb (36) + (39)), 3.13 (s, 3H,
CH3 (42)), 2.96 (m, 1H, CH (19)), 2.10–1.91 (m, 4H, CHa
(22) + CHa (21) + CHa (16) + CH (13)), 2.02 (s, 3H, CH3 (32)),
1.85–1.52 (m, 6 H, CH (18) + CHa (2) + CHa (15) + CHb (2) + CHa
(1) + CHa (12)), 1.81 (q, 4H, J = 7.3 Hz, 2 ꢁ CH2 (37) + (40)), 1.68
(s, 3H, CH3 (30)), 1.51–1.14 (m, 10H, CHa (6) + CHb (6) + CH
(9) + CHb (22) + CHa (7) + CHb (7) + CHb (16) + CHb (21) + CHa
(11) + CHb (11)), 1.10 (s, 3H, CH3 (25)), 1.07–1.00 (m, 3H, CHb
(15) + CHb (12) + CHb (1)), 1.06 (s, 3H, CH3 (27)), 1.03 (t, 6 H,
J = 7.3 Hz, 2 ꢁ CH3 (38) + (41)), 0.91 (s, 3H, CH3 (24)), 0.87 (s,
3H, CH3 (23)), 0.86 (s, 3H, CH3 (26)), 0.83 (d, 1H, J = 11.6 Hz, CH
(5)) ppm; 13C NMR (125 MHz, CDCl3): d = 172.8 (C31, CO), 152.2
(C20, C@CH2), 110.3 (C29, C@CH2), 94.9 (C33, C„CH), 82.4 (C3,
CH), 73.7 (C34, C„CH), 66.1 (C28, CHOH), 64.6 (C36 + C39,
2 ꢁ CH2), 56.7 (C5, CH), 53.3 (C17, Cquart.), 52.0 (C35, CH2), 51.6
(C9, CH), 50.3 (C18, CH), 50.1 (C19, CH), 48.9 (C42, CH3), 44.2
(C14, Cquart.), 42.1 (C8, Cquart.), 39.5 (C1, CH2), 38.8 (C4, Cquart.),
38.6 (C13, CH), 38.2 (C10, Cquart.), 35.6 (C7, CH2), 35.4 (C22,
CH2), 35.3 (C21, CH2), 33.3 (C16, CH2), 29.0 (C15, CH2), 28.4
(C24, CH3), 27.0 (C12, CH2), 26.4 (C2, CH2), 22.0 (C11, CH2), 21.1
(C32, CH3), 19.2 (C6, CH2), 19.2 (C30, CH3), 17.0 (C37 + C40,
2 ꢁ CH2), 16.7 (C23, CH3), 16.6 (C25, CH3), 15.6 (C26, CH3), 15.5
(C27, CH3), 10.9 (C38 + C41, 2 ꢁ CH3) ppm; MS (ESI, MeOH): m/
z = 636.5 (100%, [MꢀCl]+); Anal. for C42H70INO3 (763.91): C,
66.03; H, 9.24; N, 1.83. Found: C 65.86, H 9.43, N 1.72.
C39H62NO3Cl (628.37): C, 74.55; H, 9.95; N, 2.23. Found: C, 74.33;
H, 10.12; N, 2.03.
5.20. (3S, 28S) 3-O-Acetyl-28-[dibenzylamine-1-yl-prop-1-yn-1-
yl]-lup-20(29)-en-3,28-diol hydrochloride (21)
Compound 21 was prepared as described in the general proce-
dure from
7
(500 mg, 1 mmol), dibenzylamine (227 ll,
1.15 mmol), formalin (37%, 0.4 ml, 5 mmol) and CuI (20 mg,
0.1 mmol) in THF (5 ml); compound 21 (291 mg, 38%) was ob-
tained as a colorless solid; mp 161–163 °C; ½a D
CHCl3); IR (KBr): = 3287br, 2943s, 2452br, 1736s, 1641w,
1499w, 1457s, 1376m, 1248s, 1132w, 1028m cmꢀ1 1H NMR
ꢂ
+7.5° (c 4.1,
m
;
(400 MHz, CDCl3): d = 12.80–12.75 (m, 1H, NH), 7.75–7.60 (m,
4H, CH (38)), 7.40–7.30 (m, 6 H, CH3 (39) + (40)), 5.02–4.96 (br,
1H, CH (28)), 4.63 (d, 1H, J = 1.9 Hz, CHa (29)), 4.54–4.50 (m,
1H, CHb (29)), 4.40 (dd, 1H, J = 5.7, 10.3 Hz, CH (3)), 4.37–4.09
(m, 4H, 2 ꢁ CH2 (36)), 3.65–3.55 (m, 2H, CH2 (35)), 2.94–2.85
(m, 1H, CH (19)), 2.15–2.00 (m, 2H, CHa (16) + CHa (21)), 1.97
(s, 3H, CH3 (32)), 1.95–1.86 (m, 2H, CHa (22) + CH (13)), 1.77–
1.64 (m, 3H, CH (18) + CHa (1) + CHa (12)), 1.63 (s, 3H, CH3
(30)), 1.60–1.00 (m, 14H, CH2 (11) + CH2 (6) + CHa (15) + CH2
(7) + CH (9) + CH2 (2) + CHb (22) + CHb (21) + CHb (16) + CHb (12)),
0.98 (s, 3H, CH3 (27)), 0.98–0.96 (m, 1H, CHb (15)), 0.95 (s, 3H,
CH3 (25)), 0.94–0.93 (m, 1H, CHb (1)), 0.78 (s, 3H, CH3 (26)),
0.77 (s, 3H, CH3 (24)), 0.69 (s, 3H, CH3 (23)), 0.62 (d, 1H,
J = 9.0 Hz, CH (5)) ppm; 13C NMR (100 MHz, CDCl3): d = 171.0
(C31, C@O), 150.7 (C20, C@CH2), 131.5 (C39, 4 ꢁ CH), 130.1
(C40, 2 ꢁ CH), 129.2 (C38, 4 ꢁ CH), 128.6 (C37, 2 ꢁ CH), 109.8
(C29, CH2@C), 91.8 (C33, C„C), 80.8 (C3, CH), 73.4 (C34, C„C),
65.7 (C28, CH), 56.5 (C36, 2 ꢁ CH2), 55.3 (C5, CH), 51.6 (C17,
Cquart.), 50.2 (C9, CH), 48.9 (C18, CH), 48.7 (C19, CH), 43.0 (C14,
Cquart.), 40.9 (C35, CH2), 40.8 (C8, Cquart.), 38.8 (C4, Cquart.), 38.3
(C1, CH2), 37.7 (C10, Cquart.), 37.3 (C13, CH), 34.5 (C16 + C22,
2 ꢁ CH2), 34.2 (C7, CH2), 32.3 (C21, CH2), 27.9 (C23, CH3), 27.9
5.22. (3S, 28S) 3-O-Acetyl-28-[diallylmethylaminium-prop-1-yn-
1-yl]lup-20(29)-en-3,28-diol iodide (23)
Compound 23 was obtained by treating compound 17 (200 mg,
0.23 mmol) with methyl iodide (19 ll, 0.3 mmol) in abs. diethyl
ether (10 ml) for 5 days as described above. Compound 23 (141 -
mg, 81%) was obtained as an off-white solid; mp 170–173 °C;
½
aꢂD
ꢀ42.4° (c 3.15, MeOH); IR (KBr):
m
= 3405br, 2943s, 1732m,
1H
1639w, 1456m, 1376s, 1384s, 1248s, 1113w, 1029m cmꢀ1
;
NMR (400 MHz, CD3OD): d = 6.08–5.92 (m, 2H, CH (38 + 39)), 5.7-
4–5.63 (m, 4H, 2 ꢁ CH2 (41 + 40)), 4.92 (br, 1H, CH (28)), 4.58 (d,
1H, J = 1.9 Hz, CHa (29)), 4.50–4.45 (m, 1H, CHb (29)), 4.35 (dd, 1-
H, J = 5.4, 10.8 Hz, CH (3)), 4.20 (s, 2H, CH2 (35)), 4.00–3.93 (m, 4-
H, 2 ꢁ CH2 (36 + 37)), 3.00 (s, 3H, CH3 (42)), 2.93–2.88 (m, 1H, CH
(19)), 2.05–1.94 (m, 1H, CHa (22) + CHa (21)), 1.92 (s, 3H, CH3
(32)), 1.91–1.82 (m, 2H, CHa (16) + CH (13)), 1.76–1.61 (m, 3H,
CH (18) + CHa (12) + CHa (1)), 1.59 (s, 3H, CH3 (30)), 1.58–1.08
(m, 14H, CH2 (11) + CH2 (6) + CHa (15) + CH2 (7) + CH (9) + CH2

434
R. Csuk et al. / Bioorg. Med. Chem. 21 (2013) 425–435
(2) + CHb (22) + CHb (12) + CHb (16) + CHb (21)), 1.01 (s, 3H, CH3
(27)), 0.97 (s, 3H, CH3 (25)), 0.95–0.89 (m, 2H, CHb (1) + CHb (15)-
), 0.82 (s, 3H, CH3 (26)), 0.79–0.75 (m, 6 H, 2 ꢁ CH3 (24) + (23)),
0.74–0.72 (m, 1H, CH (5)) ppm; 13C NMR (100 MHz, CD3OD):
d = 171.5 (C31, C@O), 150.8 (C20, C@CH2), 128.6 (C40 + C41,
CH2@CH), 124.3 (C38 + C39, CH@CH2), 108.9 (C29, CH2@C), 94.1
(C33, C„C), 81.0 (C3, CH), 72.4 (C34, C„C), 64.7 (C28, CH), 63.6
(C36 + C37, 2 ꢁ CH2), 55.3 (C5, CH), 51.2 (C17, Cquart.), 50.5 (C35,
CH2), 50.2 (C9, CH), 48.9 (C18, CH), 48.7 (C19, CH), 46.5 (C42,
CH3), 42.8 (C14, Cquart.), 40.7 (C8, Cquart.), 38.1 (C4, Cquart.), 37.4
(C1, CH2), 37.2 (C13, CH), 36.8 (C10, Cquart.), 34.1 (C7, CH2), 34.0
(C21, CH2), 33.9 (C16, CH2), 31.9 (C22, CH2), 27.6 (C15, CH2), 27.0
(C23, CH3), 25.0 (C12, CH2), 23.2 (C2, CH2), 20.6 (C11, CH2), 19.7
(C32, CH3), 17.8 (C6, CH2), 17.6 (C30, CH3), 15.5 (C24, CH3), 15.3
(C26, CH3), 15.2 (C25, CH3), 14.1 (C27, CH3) ppm; MS (ESI, MeOH):
m/z = 632.7 (100%, [M]+), 127.1 (100%, [I]-) ; Anal. for C42H66NO3I
(759.88): C, 66.39; H, 8.75; N, 1.84. Found: C, 66.23; H, 8.95; N,
1.77.
References and notes
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5.23. (3S, 28S)-28-[3-(Diisopropylamino)propyl]lup-20(29)-en-
3,28-diol hydro-chloride (24)
15. Csuk, R.; Barthel, A.; Kluge, R.; Ströhl, D. Bioorg. Med. Chem. 2010, 18, 7252.
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Hydrogenation (6 bar) of 9 (300 mg, 0.49 mmol) in MeOH (5 ml)
in the presence of Lindlar’s catalyst (1.1 g, 0.5 mmol, 5% Pd auf
CaCO3 treated with Pb) followed by usual work-up gave 24
(135 mg, 45%) as a colorless solid; mp 245 °C; ½a D
MeOH); IR (KBr): = 3417br, 2942s, 2870s, 2660m, 1638m,
1465m, 1386m, 1172w, 1134m, 1089m, 1045m, 982m cmꢀ1 1H
ꢂ
+6.7° (c 4.15,
m
;
NMR (500 MHz, CD3OD): d = 4.68 (m, 1H, CHa (29)), 4.56 (m, 1H,
CHb (29)), 4.05 (d, 1H, J = 10.0 Hz, CH (28)), 3.74 (sept., 2H, J = 6.5,
2.6 Hz, 2 ꢁ CH (34) + (37)), 3.30 (m, 3H, CH2 (33) + CH (3)), 2.97
(ddd, 1H, J = 11.1, 11.1, 6.1 Hz, CH (19)), 2.16 (ddd, 1H, J = 12.3,
12.3, 3.3 Hz, CH (13)), 2.11–1.91 (m, 3H, CHa (22) + CHa
(21) + CHa (16)), 1.87–1.51 (m, 10 H, CH (18) + CHa (2) + CHb
(2) + CHa (15) + CHa (1) + CHa (12) + CHa (7) + CHb (7) + CH2 (32)),
1.68 (s, 3H, CH3 (30)), 1.49–1.13 (m, 11H, CH (9) + CHa (31) + CHb
(31) + CHb (12) + CHb (22) + CHb (16) + CHb (21) + CHa (11) + CHb
(11) + CHa (6) + CHb (6)), 1.40 (dd, 12H, J = 6.5 Hz, 1.7 Hz, 4 ꢁ CH3
(35) + (36) + (38) + (39)), 1.11–0.84 (m, 2H, CHb (15) + CHb (1)),
1.10 (s, 3H, CH3 (23)), 1.04 (s, 3H, CH3 (27)), 0.95 (s, 3H, CH3
(24)), 0.87 (s, 3H, CH3 (26)), 0.75 (s, 3H, CH3 (25)), 0.71 (d, 1H,
J = 10.4 Hz, CH (5)) ppm; 13C NMR (125 MHz, CD3OD): d = 151.3
(C20, C@CH2), 108.5 (C29, C@CH2), 78.2 (C3, CH), 70.8 (C28, CHOH),
55.4 (C5, CH), 54.9 (C34 + C37, 2 ꢁ CH), 50.4 (C9, CH), 50.0 (C17,
Cquart.), 47.0 (C33, CH2), 49.9 (C18, CH), 48.7 (C19, CH), 42.6 (C14,
Cquart.), 40.9 (C8, Cquart.), 38.6 (C1, CH2), 38.6 (C4, Cquart.), 36.9
(C10, Cquart.), 36.6 (C13, CH), 34.1 (C7, CH2), 33.0 (C22, CH2), 32.8
(C21, CH2), 32.2 (C31, CH2), 28.7 (C16, CH2), 27.6 (C15, CH2), 27.2
(C24, CH3), 26.6 (C2, CH2), 25.2 (C12, CH2), 24.9 (C32 CH2),
20.7 (C11, CH2), 19.0 (C30, CH3), 18.0 (C6, CH2), 17.5
(C35 + C36 + C38 + C39, 4 ꢁ CH3), 16.0 (C23, CH3), 15.4 (C25, CH3),
14.7 (C26, CH3), 14.3 (C27, CH3) ppm; MS (ESI, MeOH):
m/z = 584.5 (100% [MꢀCl]+); Anal. for C39H70ClNO2 (620.43): C,
75.50; H, 11.37; N, 2.26. Found: C, 75.32, H, 11.58, N, 2.03.
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