Efficient synthesis of the first betulonic acid-acetylene hybrids and their hepatoprotective and anti-inflammatory activity

Article abstract of DOI:10.1016/j.bmc.2009.05.059

The Sonogashira reaction can be applied for the preparation of acetylenic derivatives of betulonic acid where the triterpenoid moiety can serve as either the halo- or the acetylenic component. This reaction opened access to the first derivatives of betulonic acid containing either the arylethynyl (S{cyrillic}{triple bond, long}S{cyrillic}-Ar(Het) or the ethynyl (S{cyrillic}{triple bond, long}S{cyrillic}N{cyrillic}) moieties. From the fundamental perspective, this work illustrates the possibility of selective Pd-catalyzed cross-coupling at terminal acetylenes in the presence of a terminal alkene. Hepatoprotective and anti-inflammatory properties of selected acetylenic derivatives of betulonic acid were investigated using the CCl₄-induced hepatitis and carrageenan-induced edema models, respectively.

Full text of DOI:10.1016/j.bmc.2009.05.059

Bioorganic & Medicinal Chemistry 17 (2009) 5164–5169
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Efficient synthesis of the first betulonic acid–acetylene hybrids
and their hepatoprotective and anti-inflammatory activity
a
b
b
b
Sergey F. Vasilevsky a, , Anastasiya I. Govdi , El’vira E. Shults , Makhmut M. Shakirov , Irina V. Sorokina ,
*
Tatyana G. Tolstikova b, Dmitry S. Baev b, Genrikh A. Tolstikov b, Igor V. Alabugin c,
*
a Institute of Chemical Kinetics and Combustion, Siberian Branch of the Russian Academy of Sciences, 3 Institutskaya str., 630090 Novosibirsk, Russian Federation
b N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, 9 prosp. Acad. Lavrent’eva, 630090 Novosibirsk, Russian Federation
c Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The Sonogashira reaction can be applied for the preparation of acetylenic derivatives of betulonic acid
where the triterpenoid moiety can serve as either the halo- or the acetylenic component. This reaction
opened access to the first derivatives of betulonic acid containing either the arylethynyl (C„C-Ar(Het)
or the ethynyl (C„CH) moieties. From the fundamental perspective, this work illustrates the possibility
of selective Pd-catalyzed cross-coupling at terminal acetylenes in the presence of a terminal alkene.
Hepatoprotective and anti-inflammatory properties of selected acetylenic derivatives of betulonic acid
were investigated using the CCl4-induced hepatitis and carrageenan-induced edema models, respectively.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 23 June 2008
Revised 18 May 2009
Accepted 22 May 2009
Available online 29 May 2009
Keywords:
Cross-coupling
Arylacetylenes
Betulonic acid
Triterpenoids
Hepatoprotection
Anti-inflammatory activity
1. Introduction
hols of the triterpenoid family (derived from 3-keto-glycyrrhetic
acid)7 has been described in the literature. We were particularly in-
Plant-derived pentacyclic triterpenoids of lupane and oleanane
families provide a convenient structural platform for synthetic
studies aimed at the discovery of new biologically active com-
pounds. A number of semisynthetic derivatives of these ubiquitous
molecules, sometimes obtained through relatively simple transfor-
mations, possess high medical efficiency. In particular, research
published in the last decade has emphasized such pharmacological
properties of oleanane and lupane derivatives as antiviral (HIV-1),
anticancer and immunomodulating activity.1 In the process of fur-
ther development of our research program dedicated to the inves-
tigation of plant metabolites produced by trees and herbs of
Siberia,2 we describe efficient synthesis and selected medicinal
properties of betulonic acid derivatives containing acetylenic
structural units at the C28 position. Introduction of substituents
at this position,3 in particular through the formation of amides,4
provided highly promising results in the search of antitumor
agents in the past.
trigued by the possibility of combining the recent observations of
significant synergistic effect of irradiation on the effect of betulonic
acid derivatives on survival and growth inhibition of several mela-
noma cell lines increases8a with the ability of aryl acetylenes to in-
duce efficient double-stranded DNA photocleavage.8b,c Recently,
the alkyne moiety became one of the most commonly used substit-
uents in the design of new medicinal agents due to the ability of
alkynes to undergo facile cycloaddition reactions useful for the
modular construction of polyfunctional architectures and for label-
ing biomolecules in vitro.9
We envisioned two approaches to such acetylenic derivatives.
Both of them take advantage of the acyl chloride functionality for
the attachment of substituted anilines. In the first approach, the
reaction with amino-substituted phenylacetylene allows prepara-
tion of the key precursor 1—derivative of betulonic acid which pos-
sesses a terminal ethynyl moiety useful for a variety of further
transformations. The second approach is based on the preparation
of an iodo derivative through the condensation with p-iodoaniline
and subsequent Sonogashira coupling of the resulting aryl iodide
with terminal alkynes.
Despite high biological activity of many natural acetylenic
metabolites,5,6 acetylenic derivatives of betulonic acid remained
previously unknown. Moreover, only a few tertiary acetylenic alco-
In both of these cases, we were interested in determining con-
ditions where the alkene functionality of the isopropylidene moi-
ety characteristic for the lupane family does not interfere with
the Sonogashira cross-coupling. Success of such couplings has
* Corresponding authors.
E-mail addresses: vasilev@kinetics.nsc.ru (S.F. Vasilevsky), alabugin@chem.fsu.
edu (I.V. Alabugin).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.059

S. F. Vasilevsky et al. / Bioorg. Med. Chem. 17 (2009) 5164–5169
5165
not been a priori obvious because the vinyl group is known to react
with aryl halides with the formation of disubstituted olefins (the
Heck reaction). The two reactions proceed under analogous condi-
tions and are catalyzed by the same Pd complexes (such as
PdCl2(PPh3)2 and Pd(OAc)2).10 As a consequence, both the Heck
and the Sonogashira reactions can proceed in parallel for the first
of the above approaches because the substrate can either
alkenylate itself (Heck) or be alkynylated by a terminal alkyne
(Sonogashira). A similar problem could potentially complicate the
cross-coupling reactions of betulonic acid derivatives which con-
tain both the vinyl and the ethynyl residues (approach 1), both of
which can participate in the cross-coupling reactions.
Considering the above, we concentrated our efforts on the first
approach because presence of the terminal alkyne moiety in the
key triterpene alkyne synthetic intermediate 1 (prepared in 55%
yield through the interaction of the acyl chloride 2 with 4-amin-
ophenylacetylene in the presence of triethylamine, Scheme 1)
opens a number of possibilities for further synthetic modifications
which are not limited to the cross-coupling reactions.
the earlier literature observations, the relative cross-coupling reac-
tivity of an alkene and an alkyne moiety from the same molecule,
to the best of our knowledge, has not been studied before.
These results stimulated us to investigate whether the ‘inverse’
version of the Sonogashira reaction with the betulonic acid deriv-
ative as a halogen component would also be possible. Again, the
success of this reaction has not been a priori guaranteed because
the iodobetulonic acid can also act as the olefin component and,
thus, one can expect the mixture of the Heck alkenylation and
Sonogashira alkynylation.
The requisite iodoamide 6 has been synthesized in the 50% yield
through the interaction of 4-iodoaniline with acid chloride 2 in the
presence of triethylamine (Scheme 3). A very interesting feature of
the subsequent coupling step is the direct introduction of terminal
1,3-butadiynyl moiety in Sonogashira reaction. This is not a trivial
task because of the instability of molecules with the terminal
diacetylenic moiety.15
1-Hydroxy-2-methylhexa-3,5-diyne, which serves as the alkyne
component, has been previously synthesized via oxidative cou-
pling of dimethylethynyl carbinol to diacetylenic glycole followed
by its selective fragmentation through the retro Favorsky reaction
in the combined yield of 56%.16 We utilized a significantly im-
proved procedure developed by one of us17 where the key modifi-
cation for increasing the reaction efficiency involved heating of the
diacetylenic glycole with K2CO3 in s-pentaphenyl ether, a high-
boiling non-volatile solvent which minimizes thermal decomposi-
tion. In this procedure, the acetylene is distilled off under the aspi-
rator vacuum without any thermal decomposition whereas m-
pentaphenyl is left in the distillation residue. The yield of twice
distilled diacetylene is 92%.
In particular, interaction with piperidine and morpholine and
paraform under the Mannich reaction conditions (Scheme 2)
yielded 69% and 75% of the propargyl amines 3a, 3b. This com-
pound is potentially interesting considering the data regarding
the activity of 3-arylprop-2-ynyl amines as squalene epoxidase
(squalene monooxygenase) inhibitors in mammals.11
The Cadiot–Chodkiewicz cross-coupling is one of important
synthetic methods traditionally used for the preparation of diacet-
ylene derivatives including
a-diacetylenic alcohols, which were
found to possess high anticancer activity.5 A number of naturally
occurring conjugated butadiynyl carbinols have been prepared by
this method.12 Gratifyingly, the reaction of 1 with dimethyl-3-
bromopropynol in methanol in the presence of CuCl, Et2NH and
Interaction of iodobetylonic acid 6 with the diacetylenic alcohol
proceeded under the cross-coupling conditions analogous to those
NH2OHꢀHCl proceeded without complications and afforded
a
-
utilized for alkyne 1 and a-bromopyridine (PdCl2(PPh3)2, CuI, Et3N,
diacetylenic alcohol 4 in 73% yield.
55 °C) and provided the target 1,3-butadiyne 4 in the 57% yield.
The above transformation also presents an alternative synthesis
of diacetylenyl betulonic acid obtained from the Cadiot–Cho-
dkiewicz reaction (see Scheme 2). Comparison of these two ap-
proaches suggests that both of them can be used for the efficient
preparation of butadiynyl lupane derivatives.
Considering the polyfunctional character of betulonic acid, it
was important to test the possibility of cross-coupling of acetylene
1 with haloarenes. If successful, this will open synthetic access to a
wide variety of betulonic acid derivatives with ethynylaryl and
ethynylhetaryl moieties.
As mentioned above, a possible complication could arise from
the presence of the vinyl and the ethynyl residues in the reacting
molecule. From the literature, it is known that the Heck reaction
can involve not only aryl-, but also alkyl- and cycloalkenes.13 In or-
der to test the possibility of achieving selective alkynylation, we
have chosen 2-bromopyridine as the halogen component. This
choice was based on the observation of R. F. Heck that halogen
derivatives are more reactive towards alkynes than towards
alkenes.14
Structure of the prepared acetylenic derivatives of betulonic
acid has been established from the analysis of spectral data (IR,
1H and 13C NMR). 2D-spectra (COSY) and 1H–13C correlations
(COSY, COLOS) were used for the full assignment of the 13C NMR
signals of compounds 4 and 5. In particular, the unambiguous
assignment of C-39 of the diynyl moiety and C-41 (singlets at
65.27 and 66.65 ppm, respectively) for compound 4 from the
cross-peaks between carbon C-41 and hydrogens of the methyl
0
groups (C42H3 and C42 H3 at 1.54 ppm) in the 1H–13C COLOC spec-
Under standard Sonogashira conditions (PdCl2(PPh3)2, CuI,
Et3N), interaction of ethynyl derivative 1 with 2-bromopyridine
(55 °C, 18 h) provides only the alkynylation product—the disubsti-
tuted acetylene 5 in 62%. Such selectivity is likely to result from
higher reactivity of halides towards Sonogashira reactions than in
the Heck reaction. Although these findings are consistent with
trum. Correlation with the above methyl protons is also observed
for the C-40 carbon (d 86.24 ppm), unlike the C-37 which displays
cross peak with H-33, 35 (7.43 ppm). Assignment of carbons C-37,
38 (s, 88.79 and 88.75) of the alkyne moiety in compound 5 is con-
firmed by the presence of a cross-peak between aromatic hydro-
gens at C-33, 35 (7.50 ppm) and carbon C-37.
29
20
30
19
21
22
32
H
31
N
33
12
26
18
15
Et3N
28
O
11
17
16
13
14
25
10
34
O
H2N
+
37
36
1
4
9
6
Cl
38
2
8
80 °C
35
27
5
3
7
O
O
2
1
23
24
Scheme 1.

5166
S. F. Vasilevsky et al. / Bioorg. Med. Chem. 17 (2009) 5164–5169
H
N
32
31
33
H
N
32
35
31
33
34
O
34
36
37
O
40
41
35
37
36
38
40
41
O
38
39
O
3a
42
39
N
3b
O
N
44
43
40'
41'
i: piperidine, (CH2O)x, Cu(OAc)2,
30% H2SO4, 80 oC, 8 h
ii
i
ii: CH2[N(CH2CH2)O]2, CuCl, 80 oC, 2 h
1
iii: dimethyl-3-bromopropynol, Et2NH,
CuCl, 35 oC, 4 h
iv: 2-bromopyridine, PdCl2(PPh3)2, CuI, PPh3,
Et3N, 80 oC, 18 h
iii
iv
32
H
H
32
31
N
31
N
33
34
33
O
34
O
37
36
37
36
38
35
35
38
39
3'
O
40
41
42
Me
Me42'
2'
O
4'
5'
4
5
N
HO
6'
Scheme 2.
32
35
H
31
N
33
34
O
Et3N
C(OH)Me2
H2N
I
+
4
O
80 oC
36
Cl
PdCl2(PPh3)2,
CuI, PPh3, Et3N
I
O
O
2
6
Scheme 3.
the ALT and AST activity in blood as a result of anticytolytic effect.
Betulonic derivatives 6 and 3a lower the transaminase activity 1.8-
and 1.6-fold whereas dihydroquercetin causes only a 1.4-fold de-
crease in the activity. Compound 3b is inferior to dihydroquercetin
in this regard. None of the compounds display significant antioxi-
dant activity under the above experimental conditions. Neverthe-
less, derivative 3a lowered the TBARS concentration in the blood
1.5 times group whereas dihydroquercetin decreased this parame-
ter only by factor of 1.2. These results suggest that further studies
of analogues of compound 3a in this direction may be warranted.
In the carrageenan-induced paw edema model, only compound
3a displayed a statistically significant effect by lowering the edema
index 1.4 times relatively to the control group (Table 2). Compound
3b showed only a tendency to the anti-inflammatory effect
whereas derivative 6 did not exhibit an appreciable effect. It is
2. Biological activity: hepatoprotective and anti-inflammatory
properties of acetylenic derivatives of betulonic acid
The hepatoprotective and anti-inflammatory properties of com-
pounds described in the previous section were investigated using
the CCl4-induced hepatitis and carrageenan-induced edema mod-
els, respectively. These models were described in detail in the
experimental part.
The results of biochemical analysis of blood serum of animals
with toxic hepatitis (Table 1) show that all compounds decrease
the alkaline phosphatase activity, which confirms their antichole-
static properties. Compound 6 displayed the most apparent effect
comparable with that of dihydroquercetin. Compound 3a has a
moderate effect whereas compound 3b shows weak anticholestatic
properties. It is found that two of the studied compounds decrease
Table 1
Effect of betulonic derivatives on biochemical parameters in the blood serum of mice with CCl4-hepatisis
Agent
Dose (mg/kg)
ALP,
l
cat/L
ALT,
lcat/L
AST,
l
cat/L
TBARS, lmol/l
Control
3b
6
3a
—
18.27 1.42
14.28 1.50
10.27 0.64***
12.80 1.53*
11.31 1.13**
5.04 0.72
3.48 0.45
2.75 0.42*
3.08 0.34*
3.63 0.59
5.23 0.76
5.25 0.66
3.09 0.53*
3.25 0.38*
3.88 0.70
1.63 0.24
3.21 0.62*
2.03 0.31
1.06 0.14
1.42 0.29
50
20
50
100
Dihydroquercetin
*p < 0.05; **p < 0.01, ***p < 0.001—differences with control are significant.

S. F. Vasilevsky et al. / Bioorg. Med. Chem. 17 (2009) 5164–5169
5167
lonic acid.19 Chemical shifts were given in (d in ppm) relative to the
residual signals of CHCl3 (dy 7.24 ppm and dc 76.90 ppm). Coupling
constants (J in Hz) were accurate to 0.2 Hz. The multiplicity of sig-
nals in the 13C NMR spectra was determined using J-modulation
(JMOD). Due to the complexity of signals in the 1H NMR spectra
for the betulonic acid derivatives, only the characteristic signals
are assigned. Most protons of the triterpenoid skeleton resonate
between 0.8 and 2.7 ppm. Assignment of the alkyne carbons in
the 13C NMR was carried out through comparison with the respec-
tive chemical shifts of alkynes described earlier.20
Table 2
Effect of betulonic derivatives on the index of paw edema indexes in carrageenan-
induced edema model
Control
3b
6
3a
Indomethacin
51.9 3.9***
75.5 3.1
65.8 3.6#
77.2 3.4###
56.1 4.5**
**p < 0.01, ***p < 0.001—differences with control are significant.
#p < 0.05, ###p < 0.001—differences with indomethacin are significant.
noteworthy that compound 3a has anti-inflammatory activity
which is comparable to that of indomethacin.
3.1. N-(3-Oxolup-20(29)en-28-oyl)-4-ethynylaniline 1
In conclusion, we have shown that the Sonogashira reaction is
applicable for the preparation of acetylenic derivatives of betulonic
acid and that the triterpenoid moiety can serve as either the halo-
or the acetylenic component. This reaction has opened access to
first derivatives of betulonic acid containing either the arylethynyl
(C„C-Ar(Het) or the ethynyl (C„CH) moieties. These highly reac-
tive triterpenoid derivatives open broad possibilities for the prep-
aration of new families of promising compounds for the
development of biologically active molecules for medicinal
applications.18
A fundamentally important conclusion from the present work is
the possibility of selective Pd-catalyzed cross-coupling at the ter-
minal acetylenic carbon of the ethynyl derivative of betulonic acid
in the presence of a terminal alkene substituent which is also reac-
tive towards haloarenes and hetarenes under these conditions.
This is a useful finding because efficient chemical processes for
practical applications should involve the minimum number of pro-
tecting group manipulations.
Similar selectivity was found during the reaction of the iodo
derivative with diacetylenic alcohol—only alkynylation product of
the Sonogashira reaction is isolated, no alkenylation product of
the Heck reaction is observed even though the starting material
has both the halo- and the olefin components for the Heck cross-
coupling. Hepatoprotective and anti-inflammatory properties of
selected acetylenic derivatives of betulonic acid were investigated
using the CCl4-induced hepatitis and carrageenan-induced edema
models. These studies established that compound 3a has signifi-
cant hepatoprotective, anti-inflammatory effects and a potential
antioxidant effect. Compound 6 displayed significant hepatopro-
tective properties, whereas compound 3b showed no significant
hepatoprotective and anti-inflammatory activities. These observa-
tions indicate that biological properties of this family of com-
pounds are sensitive to the nature of substituents and, thus,
justify synthetic studies aimed at the preparation of this promising
class of medicinally interesting compounds.
1.6 g (3.4 mmol) of the chloroanhydride 2, 0.39 g (3.4 mmol) of
p-aminophenylacetylene and 3 mL of triethylamine were added to
a flask with 15 mL of dry benzene under the argon atmosphere. The
flask was fitted with a reflux condenser and kept at 70–75 °C for
18 h. The Et3NꢀHCl precipitate was filtered off and washed with
benzene (3 ꢁ 10 mL). Solvent was removed in vacuo. The residue
was dissolved in benzene and washed with diluted HCl (1:4), dried
over anhydrous Na2SO4 and concentrated. The concentrated solu-
tion was filtered through alumina (2 ꢁ 2.5 cm) and washed with
benzene. Solvent was removed in vacuo to afford 1.0 g (55%) of
compound 1, mp 159–160 °C (benzene).
HRMS, found: m/z 553.3918 [M]+. C38H51NO2. Calcd: M =
553.3919. 1H NMR (CDCl3, 300.13 MHz) d 0.90 (3H, s, Me-25),
0.95 (3H, s, Me-24), 0.98 (6H, s, Me-26, 27), 1.03 (3H, s, Me-23),
1.67 (3H, s, Me-30), 3.14 (1H, dt, 19, J1 = 4 Hz, J2 = 11 Hz), 4.59
(1H, s, 29), 4.73 (1H, s, 29), 3.01 (1H, s, 38), 7.39 (4H, m, 32, 36,
33, 35); 13C NMR d 14.37 (C-27), 15.77 (C-26), 15.82 (C-25),
19.32 (C-30), 19.43 (C-6), 20.82 (C-24), 21.27 (C-11), 25.45 (C-
12), 26.38 (C-23), 29.41 (C-21), 30.60 (C-15), 33.53 (C-16), 33.63
(C-7), 33.93 (C-2), 36.76 (C-22), 37.49 (C-13), 37.85 (C-10), 39.46
(C-1), 40.55 (C-8), 42.43 (C-14), 46.27 (C-19), 47.13 (C-4), 49.86
(C-18), 50.07 (C-9), 54.92 (C-5), 56.45 (C-17), 62.41 (C-37), 86.76
(C-38), 109.42 (C-29), 116.29 (C-34), 121.75 (C-32, 36), 137.66
(C-33, 35), 137.89 (C-31), 150.30 (C-20), 174.31 (C-28), 217.73
(C-3); IR, cmꢂ1
, m: 1696 (C@O), 2108 (C„C), 3387 (C„C-H); Anal.
Calcd for C38H51NO2: C, 82.32; H, 8.72; N, 2.38. Found: C, 82.41;
H, 9.28; N, 2.53.
3.2. N-(3-Oxolup-20(29)en-28-oyl)-4-(N-piperidinepropargyl-
1)aniline 3a
A mixture of 25 mg (0.85 mmol) of paraform and 72 mg
(0.85 mmol) of piperidine in to 5 mL of dioxane was stirred under
argon. After the reaction was kept at 55 °C for 30 min, 8 mg
(0.047 mmol) of Cu(OAc)2, 0.2 mL of 30% H2SO4 and 221 mg
(0.4 mmol) of compound 1 were added and stirred at 80 °C for
8 h. After the reaction was complete, the reaction mixture was
transferred to a separatory funnel and washed with aqueous
ammonia to remove the copper salts. The organic layer was sepa-
rated, dried over Na2SO4 and filtered. The solvent was removed in
vacuo. Hexane was added and the resulting precipitate was filtered
off to afford 180 mg (69%) of the Mannich base, mp 137–139 °C
(benzene).
3. Experimental
Melting points were determined with a Kofler apparatus. Col-
umn chromatography was performed on Al2O3 (‘Aldrich’) and the
Silufol UV-254 plates were used for TLC analysis. The IR-spectra
were recorded in KBr pellets on a ‘Bruker IFS 66’ instrument.
PdCl2(PPh3)2, methyl-3-butyn-2-ol,, Et3N, CuCl (‘Aldrich’) were
commercially available reactants. All the organic solvents were of
analytical quality. Mass spectra (HRMS) were measured on a ‘Finn-
igan MAT’, model 8200, 70 eV. Combustion analysis was performed
with CHN-analyzer (Model 1106, ‘Carlo Erba’, Italy).
NMR spectra were recorded on a ‘Bruker Avance 300’ spectrom-
eter at (300.13 (1H) and 75.47 MHz (13C) and on a ‘Bruker AM-400’
400.13 (1H) and 100.61 MHz (13C) at 25 °C. Structural assignments
in the NMR spectra were carried out by means of proton–proton
and carbon–proton correlation spectroscopy (COSY, COLOC) using
Bruker DRX-500 (500.13 (1H) b 125.76 MUw (13C)) for compounds
4 and 5, taking into account the available 13C assignments for betu-
HRMS, Found: m/z 650.4821 [M]+. C44H62N2O2. Calcd: M =
650.4806. 1H NMR (CDCl3, 400.13 MHz) d 0.88 (3H, s, Me-25),
0.91 (3H, s, Me-24), 0.96 (6H, s, Me-26, 27), 1.04 (3H, s, Me-23),
1.66 (3H, s, Me-30), 2.55 (4H, m, 40, 44), 3.13 (1H, dt, 19,
J1 = 4 Hz, J2 = 11 Hz), 3.44 (2H, s, CH2-39), 4.60 (1H, s, 29), 4.75
(1H, s, 29), 7.40 (4H, m, 32, 36, 33, 35); 13C NMR d 14.42 (C-27),
15.78 (C-26), 15.83 (C-25), 19.36 (C-30), 19.47 (C-6), 20.89 (C-
24), 21.30 (C-11), 23.75 (C-41, 43), 25.46 (C-12), 25.71 (C-42),
26.41 (C-23), 29.44 (C-21), 30.61 (C-15), 33.54 (C-16), 33.69
(C-7), 34.02 (C-2), 36.79 (C-22), 37.48 (C-13), 37.95 (C-10), 39.50

5168
S. F. Vasilevsky et al. / Bioorg. Med. Chem. 17 (2009) 5164–5169
(C-1), 40.56 (C-8), 42.46 (C-14), 46.29 (C-19), 47.21 (C-4), 48.31
(C-39), 49.88 (C-9), 50.06 (C-18), 54.91 (C-5), 53.30 (C-40, 44),
56.45 (C-17), 84.24 (C-38), 84.59 (C-37), 109.47 (C-29), 118.49
(C-34), 119.39 (C-32, 36), 132.27 (C-33, 35), 137.89 (C-31),
54.59 (C-5), 56.23 (C-17), 65.27 (C-39), 66.65 (C-41), 72.30 (C-
38), 78.20 (C-37), 86.24 (C-40), 109.22 (C-29), 116.16 (C-34),
119.14 (C-32, 36), 132.92 (C-33, 35), 138.73 (C-31), 150.03 (C-
20), 174.10 (C-28), 217.88 (C-3); IR, cmꢂ1
, m: 1692 (C@O), 2145
150.42 (C-20), 174.28 (C-28), 218.01 (C-3). IR, cmꢂ1
,
m: 1710
and 2232 (C„C). Anal. Calcd for C43H57NO3: C, 81.17; H, 10.10;
(C@O); 2227 (C„C). Anal. Calcd for C44H62N2O2: C, 81.18; H,
N, 2.01. Found: C, 81.21; H, 9.03; N, 2.20.
9.60; N, 4.30. Found: C, 80.85; H, 9.91; N, 4.15.
3.5. N-(3-Oxolup-20(29)en-28-oyl)-4-(2-ethynylpyridyl)aniline
5
3.3. N-(3-Oxolup-20(29)en-28-oyl)-4-(N-morpholinopropargyl-
1)aniline 3b
Upon stirring, 78 mg (0.49 mmol) of
a-bromopyridine, 7 mg
(0.04 mmol) of CuI, 7 mg (0.01 mmol) of PdCl2(PPh3)2, 4 mg
(0.02 mmol) of PPh3, 300 mg (0.542 mmol) of acetylene 1 and
3 mL of triethylamine were added under argon to 10 mL of toluene
The mixture was heated to 55 °C with reflux condenser for 18 h.
The Et3NꢀHBr precipitate was filtered off and washed with toluene
(3 ꢁ 10 mL). Organic fractions were combined and solvent was
evaporated in vacuo. The product was precipitated upon tritiration
with hexane and filtered to afford 193 mg (62%) of compound 5,
mp 181–182 °C (benzene). 1H NMR (CDCl3, 300.13 MHz) d 0.89
(3H, s, Me-25), 0.94 (3H, s, Me-24), 0.97 (6H s, Me-26, 27), 1.06
(3H, s, Me-23), 1.66 (3H, s, Me-30), 3.13 (1H, dt, 19, J1 = 4 Hz,
J2 = 11 Hz), 4.62 (1H, s, 29), 4.76 (1H, s, 29), 7.50 (4H, m, 32, 33,
35, 36), 7.46 (1H, d, 30, J = 3 Hz), 7.64 (1H, t, 40, J1 = 1.7 Hz,
J = 7.7 Hz), 7.20 (1H, m, 50), 8.58 (1H, d, 60, J = 4.9 Hz); 13C NMR d
14.10 (C-27), 15.47 (C-26), 15.55 (C-25), 19.05 (C-30), 19.15 (C-
6), 20.58 (C-24), 21.00 (C-11), 25.15 (C-12), 26.10 (C-23), 29.14
(C-21), 30.29 (C-15), 33.22 (C-16), 33.31 (C-7), 33.71 (C-2), 36.47
(C-22), 37.15 (C-13), 37.61 (C-10), 39.19 (C-1), 40.25 (C-8), 42.14
(C-14), 45.97 (C-19), 46.90 (C-4), 49.57 (C-18), 49.75 (C-9), 54.60
(C-5), 56.20 (C-17), 87.75 (C-38), 88.79 (C-37), 109.17 (C-29),
116.93 (C-34), 119.18 (C-32, 36), 122.18 (C-60), 126.58 (C-40),
132.39 (C-33, 35), 135.71 (C-50), 138.58 (C-31), 143.10 (C-20),
149.58 (C-30), 150.09 (C-20), 174.14 (C-28), 217.72 (C-3); IR,
5.22 g (60 mmol) of morpholine was added to 1.8 g (60 mmol)
of paraform in 15 mL of dioxane at room temperature. The mixture
was heated to 70 °C with reflux condenser for 2–2.5 h until full dis-
solution of paraform. The reaction mixture was then filtered. Evap-
oration of solvent in vacuo provided 5 g (89.6%) of di(N-
morpholino)methane, bp 97–109°/2 mm, n2D0 1.4792 (lit. bp 87–
92°/1 mm, n2D0 1.4790).21
To the mixture of 260 mg (0.47 mmol) of acetylene 1, 93 mg
(0.5 mmol) of di(N-morpholino)methane in 10 mL of dioxane and
13 mg (0.13 mmol) of CuCl were added under argon atmosphere.
The reaction mixture was kept at 80 °C for 2 h, diluted with
15 mL of dichloromethane and then washed with aqueous ammo-
nia. The organic phase was dried over Na2SO4, filtered and concen-
trated in vacuo. The product was precipitated with hexane to
afford 230 mg (74.9%) of compound 3b, mp 151–152.5 °C (ben-
zene). HRMS, Found: m/z 652.4578 [M]+. C43H60N2O3. Calcd:
M = 652.4599. 1H NMR (CDCl3, 400.13 MHz) d 0.90 (3H, s, Me-
25), 0.94 (3H, s, Me-24), 0.98 (6H, s, Me-26, 27), 1.03 (3H, s, Me-
23), 1.67 (3H, s, Me-30), 2.61 (4H, m, 40, 400), 3.13 (1H, dt, 19,
J1 = 4 Hz, J2 = 11 Hz), 3.44 (2H, s, CH2-39), 3.75 (4H, m, 41, 410),
4.59 (1H, s, 29), 4.73 (1H, s, 29), 7.37 (4H, m, 32, 36, 33, 35); 13C
NMR d 14.43 (C-27), 15.79 (C-26), 15.83 (C-25), 19.36 (C-30),
19.47 (C-6), 20.89 (C-24), 21.31 (C-11), 25.47 (C-12), 26.41 (C-
23), 29.45 (C-21), 30.61 (C-15), 33.54 (C-16), 33.69 (C-7), 34.02
(C-2), 36.79 (C-22), 37.48 (C-13), 37.95 (C-10), 39.51 (C-1), 40.56
(C-8), 42.46 (C-14), 46.29 (C-19), 47.22 (C-4), 47.96 (C-39), 49.88
(C-9), 50.06 (C-18), 54.91 (C-5), 52.31 (C-40, 400), 56.47 (C-17),
66.74 (41, 410), 83.33 (C-38), 85.12 (C-37), 109.49 (C-29), 118.14
(C-34), 119.42 (C-32, 36), 132.29 (C-33, 35), 138.08 (C-31),
cmꢂ1
, m: 1703 (C@O), 2219 (C„C). Anal. Calcd for C43H54N2O2: C,
81.80; H, 8.43; N, 4.38. Found: C, 81.80; H, 8.62; N, 4.44.
3.6. N-(3-Oxolup-20(29)en-28-oyl)-4-iodoaniline 6
0.92 g (4.20 mmol) of p-iodoaniline, 4 mL of triethylamine and
2.0 g (4.2 mmol) of chloroanhydride 2 were added consecutively
to 15 mL of dry benzene upon stirring. Reaction was then heated
to 80 °C for 16 h. The reaction mixture was filtered through alu-
mina (2 ꢁ 2.5 cm). Evaporation of the solvent in vacuo provided
1.4 g (50%) of the product, mp 163–164 °C (benzene). 1H NMR
(CDCl3, 300.13 MHz) d 0.89 (3H, s, Me-25), 0,94 (3H, s, Me-24),
0.98 (6H, s, Me-26, 27), 1.03 (3H, s, Me-23), 1.67 (3H, s, Me-30),
3.13 (1H, dt, 19, J1 = 4 Hz, J2 = 11 Hz), 4.58 (1H, s, 29), 4.72 (1H, s,
29), 7.26 (2H, m, 32, 36), 7.58 (2H, m, 33, 35); 13C NMR d 14.38
(C-27), 15.68 (C-26), 15.77 (C-25), 19.32 (C-30), 19.43 (C-6),
20.82 (C-24), 21.27 (C-11), 25.45 (C-12), 26.38 (C-23), 29.39 (C-
21), 30.59 (C-15), 33.53 (C-16), 33.63 (C-7), 33.93 (C-2), 36.76 (C-
22), 37.49 (C-13), 37.84 (C-10), 39.46 (C-1), 40.55 (C-8), 42.43 (C-
14), 46.27 (C-19), 47.13 (C-4), 49.86 (C-18), 50.07 (C-9), 54.92 (C-
5), 56.45 (C-17), 86.76 (C-34), 109.42 (C-29), 121.75 (C-32, 36),
137.66 (C-33, 35), 137.89 (C-31), 150.30 (C-20), 174.31 (C-28),
150.41 (C-20), 174.33 (C-28), 218.07 (C-3). IR, cmꢂ1
, m: 1703
(C@O); 2225 (C„C).
3.4. N-(3-Oxolup-20(29)en-28-oyl)-4-(5-hydroxy-5-
methylhexa-1,3-diynyl)aniline 4
188 mg (0.339 mmol) of compound I, 0.2 mL of diethylamine,
3.4 mg (0.05 mmol) of hydroxylamine hydrochloride and 1 mg
(0.01 mmol) of CuCl were added under argon to 2 mL of methanol
upon stirring. After formation of the bright yellow precipitate of
copper acetylide, 62 mg (0.38 mmol) of 1-bromo-3-methylbut-1-
yn-3-ol was added. The reaction mixture was kept at 30–35 °C
for 4 h, diluted with 20 mL of toluene and washed with aqueous
ammonia. The organic phase was dried over Na2SO4, filtered and
concentrated in vacuo. The product was precipitated with hexane
to afford 156 mg (73%) of compound 4, mp 183–184 °C (benzene).
1H NMR (CDCl3, 300.13 MHz) d 0.89 (3H, s, Me-25), 0.94 (6H, s, Me-
26,27), 0.98 (3H, s, Me-24), 1.03 (3H, s, Me-23), 1.66 (3H, s, Me-30),
1.54 (6H, s, 42, 43), 3.12 (1H, dt, 19, J1 = 4 Hz, J2 = 11 Hz), 4.58 (1H,
s, 29), 4.72 (1H, s, 29), 7.42 (4H, m, 32, 33, 35, 36); 13C NMR d 14.10
(C-27), 15.48 (C-26), 15.52 (C-25), 19.04 (C-30), 19.15 (C-6), 20.58
(C-24), 20.99 (C-11), 25.13 (C-12), 26.10 (C-23), 29.12 (C-21), 30.26
(C-15), 30.68 (C-42, 420), 33.21 (C-16), 33.32 (C-7), 33.71 (C-2),
36.47 (C-22), 37.15 (C-13), 37.59 (C-10), 39.19 (C-1), 40.24 (C-8),
42.14 (C-14), 45.95 (C-19), 46.91 (C-4), 49.55 (C-18), 49.71 (C-9),
217.73 (C-3); IR, cmꢂ1
, m: 1694 (C@O), 3443 (NH); Anal. Calcd for
C36H50INO2: C, 66.61; H, 8.09; N, 1.91. Found: C, 65.98; H, 7.69;
N, 2.14.
3.7. N-(3-Oxolup-20(29)en-28-oyl)-4-(5-hydroxy-5-
methylhexa-1,3-diynyl)aniline 4
130 mg (0.198 mmol) of iodide 6, 6 mg (0.03 mmol) of CuI,
10 mg (0.01 mmol) of PdCl2(PPh3)2, 2 mL of triethyl amine and
33 mg (0.27 mmol) of diacetylene alcohol were added to 8 mL of

S. F. Vasilevsky et al. / Bioorg. Med. Chem. 17 (2009) 5164–5169
5169
toluene under argon and upon stirring. The reaction mixture was
kept at 55 °C for 12 h. At the end of the reaction, 10 mL of toluene
was added. The reaction mixture was washed with aqueous
ammonia. The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. The product was precipitated by hexane
to afford 72 mg (57%) of compound 4, mp 183–184 °C (benzene).
Physical and spectral properties of this compound are identical to
those for the compound produced in the Cadiot–Chodkiewicz reac-
tion (see above, mp 183–184 °C).
No. 07-03-00048a, Grant 5.9.3. of the Russian Academy of Sciences
(2009–2011) and the Chemical Service Centre of SB RAS. Work at
FSU is supported by supported by the National Science Foundation
(CHE-0848686).
References and notes
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3.8. Biochemical experiments
The experiments were performed on outbred male mice, weigh-
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This work was supported by the Interdisciplinary Grant No. 93
of SB of the Russian Academy of Sciences (2009-2011), Grant RFBR