Synthesis and antiviral activity of ureides and carbamates of betulinic acid and its derivatives

Article abstract of DOI:10.1023/B:RUBI.0000008902.36604.97

Ureides and carbamates of betulinic acid and its derivatives were prepared in good yields by interaction of betulinic acid, betulonic acid, and betulonic acid 3-oxime with amines, amino acids, and alcohols. Ureides of betulonic acid containing L-Val and L

Full text of DOI:10.1023/B:RUBI.0000008902.36604.97

Russian Journal of Bioorganic Chemistry, Vol. 29, No. 6, 2003, pp. 594–600. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 6, 2003, pp. 655–661.
Original Russian Text Copyright © 2003 by Flekhter, Boreko, Nigmatullina, Tret’yakova, Pavlova, Baltina, Nikolaeva, Savinova, Galin, Tolstikov.
Synthesis and Antiviral Activity of Ureides and Carbamates
of Betulinic Acid and Its Derivatives
1
O. B. Flekhter*, E. I. Boreko**, L. R. Nigmatullina*, E. V. Tret’yakova*, N. I. Pavlova**,
L. A. Baltina*, S. N. Nikolaeva**, O. V. Savinova**, F. Z. Galin*, and G. A. Tolstikov*
*Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences, pr. Oktyabrya 71, Ufa, 450054 Russia
**State Research Institute of Epidemiology and Microbiology, Minsk, Belarus
Received August 9, 2002; in final form, September 8, 2002
Abstract—Ureides and carbamates of betulinic acid and its derivatives were prepared in good yields by inter-
action of betulinic acid, betulonic acid, and betulonic acid 3-oxime with amines, amino acids, and alcohols.
Ureides of betulonic acid containing L-Val and L-Met residues were found to be effective against herpes sim-
plex type 1 virus.
Key words: antiviral activity; betulinic acid, isocyanates, carbamates, ureides; lupane triterpenoids
1
INTRODUCTION
the resonance to 154–158 ppm. The resonances of the
NHCONH group protons look like broadened singlets
or multiplets at 4.40–9.89 ppm. The aromatic protons
of quinoline and pyridine fragments of ureides (XII)
and (XIII) resonate at a low field (6.85–8.81 ppm) in
their H NMR spectra. In the H NMR spectra of
(XIV)–(XVIII) containing the residues of esters of
amino acids, intensive resonances from three protons of
methoxy group and the H3' proton of amino acid resi-
due were observed at 3.7–3.8 ppm and 4.07–4.74 ppm,
respectively. Additional resonances of the protons of
ëç2 groups of ureides (X) and (XI) are found in the
area of 2.2–3.9 ppm.
We synthesized a new series of nitrogen-containing
derivatives of lupane triterpenoids, ureides and carbam-
ates of betulinic acid (I), betulonic acid (II), and betu-
lonic acid 3-oxime (III), when continuing our research
on chemical modifications of these triterpenoids [1–4].
The syntheses of dihydrobetulinic acid ethylcarbamate
[5] and 3-é-acetylbetulinic acid ureide with the residue
of 10-aminodecanoic acid were previously described in
literature, the latter exhibiting no antiviral activity
against HIV [6].
1
1
RESULTS AND DISCUSSION
Next, 17β-isocyanate of betulonic acid (VIII)
reacted with sodium methylate and sodium benzylate in
boiled benzene, and methyl carbamate (XIX) and ben-
zyl carbamate (XX) were obtained in 91 and 82%
yields, respectively (Scheme 2). The interaction of iso-
cyanate (VIII) with phenol required the presence of tri-
ethylamine. The structures of carbamates (XIX)–(XXI)
were also confirmed by their spectra. The carbon atoms
of NHCO2-group in (XIX)–(XXI) resonate at 155–
156 ppm. The absorption bands of valent oscillations of
NH2-groups (3250–3350 cm–1) and CO groups (1520–
We used isocyanates of lupane acids (VII)–(IX) as
starting compounds for the syntheses. These acids we
prepared from the corresponding acyl chlorides (IV)–
(VI) by the action of activated NaN3 in acetone without
isolation of intermediate compounds in yields of 80–
94% [7]. Isocyanates (VII)–(IX) were further used in
reactions with primary aliphatic amines and heterocy-
clic amines and methyl esters of optically pure L-amino
acids. Ureides (X)–(XVIII) were formed under the
reflux in benzene or chloroform for 2 h in yields of 78–
84% (Scheme 1).
1540 cm–1) are present in the IR spectra.
1
Elemental analysis, ç NMR, and 13C NMR con-
The resonances in the NMR spectra of ureides and
carbamates of lupane acids were assigned on the basis
of literature data for lupane triterpenoids, triterpene
derivatives, and amino acid esters [6, 8, 9].
firmed structures of the resulting compounds. The car-
bon atoms of NCO-group of isocyanates (VII)–(IX)
exhibit chemical shifts of 121.3–121.6 ppm in 13C
NMR spectra and the NCO-groups have the intensive
maximum of valent oscillations at 2260–2270 cm–1 in
IR spectra. The introduction of NHCONH group in
position 17 of lupane acids results in a low-field shift of
The antiviral activity of a number of the synthesized
compounds was examined in the experiments against
the influenza A virus, the herpes simplex type 1 virus,
and the ECHO6 enterovirus on cell cultures. Betulin,
betulinic acid, and betulonic acid were previously
shown to inhibit the virus of herpes simplex [10–12],
1
Corresponding author; fax: (3472) 35-6066; e-mail: obf@anrb.ru
1068-1620/03/2906-0594$25.00 © 2003 MAIK “Nauka /Interperiodica”

SYNTHESIS AND ANTIVIRAL ACTIVITY
595
(IV), (V), (VI)
COR
R2
R1
NCO
R2
R1
(I) R1 + R2 = O, R = OH;
(II) R1 = OH, R2 = H, R = OH;
(III) R1 + R2 = NOH, R = OH;
(IV) R1 + R2 = O, R = Cl;
(V) R1 = OH, R2 = H, R = Cl;
(VI) R1 + R2 = NOH, R = Cl
1 = OH, R2
(VII
) R
= H;
(VIII) R1 + R2 = O;
(IX) R1 + R2 = NOH
R3NH2
O
H2N
OCH3
H
R3
H R3
O
1'
C
OCH3
2'
3'
NHCONHR3
N
H
N
H
O
R2
R1
R2
R1
(X)–(XIII)
(XIV)–(XVIII)
1'
6'
4'
(X) R1 = OH, R2 = H, R3 = CH2(CH2)4CH3;
(XIV) R1 = OH, R2 = H, R3 = CH3;
1'
10'
(XI) R1 + R2 = O, R3 = CH2(CH2)8CH3;
6'CH3
5'
(XV) R1 + R2 = O, R3 = 4'CH CH3 ;
8'
N
(XII) R1 + R2 = O, R3 =
;
6'CH3
4'
7'
(XVI) R1 + R2 = O, R3 = CH2 CH CH3 ;
4'
6'
(XIII) R1 + R2 = NOH, R3 =
(XVII) R1 + R2 = O, R3 = CH2–CH2–S–CH3;
2'
4'
6'
N
(XVIII) R1 + R2 = NOH, R3 = CH2–CH2–S–CH3
Scheme 1.
whereas betulin did not inhibit the influenza viruses (XVII) within the concentration ranges from MIC to
[13]. Ureides of betulonic acid (XV) and (XVII) were 1/32 MIC for (XVII) or 1/8 MIC for (XV). We failed to
found to be the most active inhibitors of the herpes find antiviral properties of the studied compounds
virus among all the compounds studied (table).
against the ECHO6 virus.
Ureide derivative (XVI) exhibited a pronounced
inhibiting activity towards the influenza virus. How-
ever, a decrease in the virus titer in the presence of this
EXPERIMENTAL
2
Silufol plates (Chemapol, Czech Republic) devel-
oped with 20 : 1 chloroform–methanol mixture were
used for TLC. Substances were detected by the treat-
ment with 20% solution of phosphotungstic acid in eth-
anol followed by heating at 100–120°C for 2–3 min.
compound was no higher than 0.9 logPFU/ml, despite
the use of a large range of concentrations. At the same
time, a decrease in the herpes virus titer was from >3 to
1.72 logTCID50/ml in the presence of (VIII) and
2
13ë and H NMR spectra were registered in deutero-
1
PFU is a plaque forming unit.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 6 2003

596
FLEKHTER et al.
;
(XIX) R = CH3
O
1'
CH2
2'
C
N
(XX) R =
;
OR
ROX
X = Na, H
(VIII)
H
1'
(XXI) R =
O
Scheme 2.
chloroform on a Bruker AM-300 spectrometer at the (IV) (0.47 g, 1 mmol) as a white powder; yield 0.38 g
(84%); Rf 0.30; mp 244–246°C; IR (ν, cm–1): 710, 850,
890, 910, 990, 1050, 1100, 1120, 1150, 1190, 1220,
1280, 1380, 1430, 1490, 1510, 1600, 1620, 1700, 2260;
1H NMR (δ, ppm): 0.81, 0.85, 0.88, 0.91, 1.00 (15 H,
5 s, 5 CH3), 1.20–2.00 (21 H, m, CH2, CH), 1.65 (3 H,
s, CH3), 2.38–2.52 (3 H, m, H13, H16), 2.66–2.75 (1 H,
m, H19), 3.05–3.11 (1 H, m, H3), 4.62 and 4.73 (two
broadened 1 H signals, H29); 13C NMR (δ, ppm): 14.8,
15.6, 15.8, 18.3, 19.4, 20.8, 21.2, 24.7, 26.4, 27.5, 29.1,
33.3, 33.4, 33.9, 36.7, 38.9, 39.0, 39.4, 40.5, 41.9, 47.1,
47.9, 49.0, 49.6, 54.8, 71.3 (C17), 78.8 (C3), 110.3
(C29), 121.5 (NCO), 148.6 (C20). Found, %: C 79.53,
H 10.56, N 3.01. Calcd. for C30H47O2N, %: C 79.40, H
10.46, N 3.09.
working frequencies of 75.5 and 300 MHz, respec-
tively; SiMe4 was used as an internal standard. IR spec-
tra were recorded on a Specord M80 spectrometer in
pastes with Vaseline oil. Melting points were deter-
mined on a Boetius hot plate. Betulinic acid (I), betu-
lonic acid (II), and betulonic acid 3-oxime (III) were
prepared according to the procedures described in [14,
15]. Sodium azide was activated before the reaction as
described in [16]. The anhydrous solvents were pre-
pared as described in [17]. Amino acid methyl esters
were prepared as their hydrochlorides according to the
standard procedure [18].
General procedure for preparation of acid chlo-
rides (IV)–(VI). Oxalyl chloride (0.3 ml) was added
dropwise to a stirred solution of acid (I), (II), or (III)
(1 mmol) in anhydrous benzene. The reaction mixture
was stirred for 2 h under nitrogen at 20–22°C. The sol-
vent was evaporated in a vacuum. The yields were 93–
95%. The resulting compounds were used in syntheses
without any further purification; IR spectrum (ν, cm–1):
1820–1810 (COCl).
3-Oxo-28-norlup-20(29)-ene-17b-isocyanate (VIII)
was prepared from betulonic acid chloride (V) (0.47 g,
1 mmol) as a white powder; yield 0.45 g (94%); Rf 0.76;
mp 178–180°C; IR (ν, cm–1): 720, 860, 870, 910, 980,
1060, 1100, 1120, 1160, 1190, 1230, 1290, 1380, 1430,
1490, 1510, 1600, 1620, 1700, 1780, 2270; 1H NMR (δ,
ppm): 0.87, 0.93, 1.01, 1.06, 1.08 (15 H, 5 s, 5 CH3),
1.20–2.00 (21 H, m, CH2, CH), 1.67 (3 H, s, CH3),
2.33–2.57 (4 H, m, H13, H16, H19), 4.62 and 4.73 (two
1 H broadened signals, H29); 13C NMR (δ, ppm): 14.9,
15.6, 15.7, 19.2, 19.5, 20.8, 21.2, 24.9, 26.4, 27.3, 29.1,
33.4, 33.6, 33.9, 36.7, 38.8, 39.0, 39.2, 40.5, 42.0, 47.2,
47.6, 49.0, 49.5, 54.8, 71.2 (C17), 110.3 (C29), 121.6
(NCO), 149.6 (C20), 217.5 (C3). Found, %: C 79.83, H
9.98, N 3.07. Calcd. for C30H45O2N, %: C 79.76, H
General procedure for preparation of isocyan-
ates (VII)–(IX). A solution of acid chloride (IV), (V),
or (VI) (1 mmol) in anhydrous acetone (100 ml) was
treated with activated NaN3 (0.125 g) upon stirring. The
reaction mixture was stirred for 6 h at room tempera-
ture, NaN3 excess was filtered off, and solvent was
evaporated. The residue was chromatographed on a sil-
ica gel column eluted with benzene.
3b-Hydroxy-28-norlup-20(29)-ene-17b-isocyan-
ate (VII) was prepared from betulinic acid chloride 10.06, N 3.10.
Antiviral activity of betulonic acid derivatives
Influenza virus
Herpes simplex type 1 virus
ECHO6 virus
Compound
EC50 (I95), µM
MTC/EC50
EC50 (I95), µM
MTC/EC50
EC50 (I95), µM
MTC/EC50
(VIII)
(XV)
NS
>85.68
NS
<1
>221.36
0.17 (1.92–0.02)
NS
<1
NS
NS
1.22
NS
500.00
NS
4.21(7.14–2.50)
(XVI)
(XVII)
Ӷ5.02
ӷ67
3.0
NS
NS
27.08 (39.54–18.53)
1.46 (2.21–0.96)
444.44
NS
Denotations: MTC, maximal tolerant concentration; EC , average effective concentration; I , confidence interval for 95% probability.
50
95
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 6 2003

SYNTHESIS AND ANTIVIRAL ACTIVITY
597
13C NMR (δ, ppm): 14.0, 14.3 (C10'), 15.7, 15.9, 19.2,
19.6, 20.9, 21.4, 22.6 (C9'), 25.0, 26.6, 26.9 (C2'), 27.2,
29.3 (C6'), 29.4 (C3'), 29.5 (C4'), 29.6 (C5'), 29.7 (C7'),
28-Norlup-20(29)-ene-17b-isocyanate 3-oxime
(IX) was prepared from betulonic acid chloride 3-
oxime (VI) (0.48 g, 1 mmol) as a creme-white powder;
yield 0.37 g (80%); Rf 0.21, mp 212–214°C; IR (ν, cm–1): 29.7, 30.3, 31.8 (C8'), 33.3, 34.0, 35.6, 36.8, 37.7, 39.5
(C1'), 40.4, 40.6, 42.0, 47.2, 47.6, 49.4, 49.6, 54.7, 63.3
(C17), 109.8 (C29), 149.7 (C20), 157.6 (NHCONH),
217.5 (C3). Found, %: C 82.47, H 9.68, N 3.72. Calcd.
for C40H68O2N2, %: C 82.23, H 9.55, N 3.84.
710, 860, 890, 920, 990, 1050, 1110, 1130, 1150, 1200,
1250, 1270, 1380, 1410, 1500, 1610, 1630, 1710, 1780,
1
2260; H NMR (δ, ppm): 0.81, 0.85, 0.88, 0.91, 1.00
(15 H, 5 s, 5 CH3), 1.20–2.00 (21 H, m, CH2, CH), 1.65
(3 H, s, CH3), 2.40–2.53 (4 H, m, H13, H16), 2.65–2.73
(1 H, m, H19), 4.62 and 4.73 (two 1 H broadened sig-
3-Oxo-17b-[N'-(8-aminoquinolyl)ureido]-28-nor-
lup-20(29)-ene (XII) was prepared from (VIII) (0.47 g,
1 mmol) and 8-aminoquinoline (0.19 g) as a yellow
powder; yield 0.48 g (81%); Rf 0.70; mp 252–254°C;
13
nals, H29), 9.48 (1 H, broadened signal, 3-NOH); C
NMR (δ, ppm): 14.6, 15.8, 15.9, 16.1, 17.1, 19.0, 21.1,
25.2, 27.2, 29.6, 30.6, 32.1, 33.9, 36.9, 37.2, 38.3, 38.7,
40.3, 40.7, 42.4, 46.9, 49.4, 50.1, 51.2, 55.6, 71.3
(C17), 110.1 (C29), 121.5 (NCO), 149.6 (C20), 167.3
(C3). Found, %: C 77.32, H 9.84, N 5.92. Calcd. for
C30H46O2N2, %: C 77.19, H 9.95, N 6.00.
IR (ν, cm–1): 760, 850, 900, 1200, 1130, 1240, 1320,
1
1450, 1530, 1640, 1670, 1780, 3250–3360; H NMR
(δ, ppm): 0.88, 0.92, 0.96, 1.01, 1.04, (15 H, 5 s, 5
CH3), 1.10–2.00 (21 H, m, CH2, CH), 1.59 (3 H, s,
CH3), 2.44–2.58 (3 H, m, H13, H16), 2.75–2.84 (1 H,
m, H19), 4.52 and 4.79 (two 1 H broadened signals,
H29), 5.13 (1 H, broadened signal, NHCONH), 7.46–
7.61 (3 H, m, H4', H7', H8'), 8.19 (1 H, dd, J 7.1 and
1.2 Hz, H3'), 8.72 (1 H, dd, J 4.1 and 1.2 Hz, H5'), 8.81
(1 H, dd, J 1.2 and 7.7 Hz, H9'), 9.89 (1 H, broadened
signal, NHCONH); 13C NMR (δ, ppm): 14.1, 15.8,
15.9, 19.4, 19.5, 21.0, 21.2, 24.9, 26.4, 27.3, 29.5, 29.9,
33.5, 34.1, 35.5, 36.7, 37.5, 39.5, 40.3, 41.9, 46.4, 47.3,
49.2, 49.6, 54.9, 63.9 (C17), 109.7 (C29), 115.5, 119.2,
121.4, 127.9, 128.2, 136.7, 136.8, 138.2, 147.4, 149.5,
(C20), 154.3 (NHCONH), 218.0 (C3). Found, %: C
78.64, H 8.67, N 7.19. Calcd. for ë39ç53é2N3, %: C
78.40, H 8.88, N 7.04.
General procedure for the synthesis of (X)–
(XIII). Isocyanate (VIII) or (IX) (1 mmol) and amine
(1.6 mmol) were refluxed for 2 h in anhydrous benzene
(10 ml). The reaction mixture was washed with 5% HCl
(15 ml) and water (2 × 10 ml), dried over MgSO4, and
evaporated in a vacuum to dryness. The residue was
chromatographed on a column with alumina eluted
with chloroform.
3b-Hydroxy-17b-[N'-(n-hexyl)ureido]-28-norlup-
20(29)-ene (X) was prepared from isocyanate (VII)
(0.45 g, 1 mmol) and n-hexylamine (0.18 g) as a white
powder; yield 0.44 g (79%); Rf 0.34; mp 194–197°C;
IR (ν, cm–1): 740, 780, 850, 910, 1100, 1120, 1170,
1220, 1260, 1350, 1420, 1480, 1540, 1650, 1690, 1750,
3260–3340; 1H NMR (δ, ppm): 0.80, 0.84, 0.86, 0.88,
0.98 (15 H, 5 s, 5 CH3), 1.10–2.00 (29 H, m, CH2, CH),
1.26 (3 H, s, H6'), 1.66 (3 H, s, CH3), 2.40–2.55 (3 H,
m, H13, H16), 2.65–2.73 (1 H, m, H19), 3.04–3.11 (1
H, m, H3), 3.17–3.25 (2 H, m, H1'), 4.57 and 4.72 (two
broadened 1 H signals, H29), 5.00–5.10 (1 H, m,
NHCONH), 5.20 (1 H, broadened signal, NHCONH);
13C NMR (δ, ppm): 13.9 (C6'), 14.4, 15.3, 16.0, 18.2,
19.2, 20.9, 22.5 (C5'), 25.0, 26.6 (C2'), 27.2, 27.9, 29.8,
29.8 (C3'), 30.3, 31.5 (C4'), 34.1, 35.7, 37.1, 37.5, 38.7,
38.8, 38.8 (C1'), 40.6, 41.9, 47.5, 49.5, 49.7, 50.3, 55.2,
63.3 (C17), 78.8 (C3), 109.7 (C29), 149.8 (C20), 157.8
(NHCONH). Found, %: C 77.85, H 11.27, N 4.92.
Calcd. for C36H62O2N2, %: C 78.04, H 11.12, N 5.06.
17b-[N'-(2-aminopyridyl)ureido]-28-norlup-20(29)-
ene 3-oxime (XIII) was prepared from isocyanate (IX)
(0.47 g, 1 mmol) and 2-aminopyridine (0.14 g) as a
cream-white powder; yield 0.44 g (78%); Rf 0.28; mp
201–203°C; IR (ν, cm–1): 770, 850, 920, 1130, 1180,
1240, 1230, 1340, 1470, 1540, 1660, 1680, 1780,
3250–3350; 1H NMR (δ, ppm): 0.83, 0.90, 0.95, 1.01,
1.12 (15 H, 5 s, 5 CH3), 1.10–2.00 (22 H, m, CH2, CH),
1.70 (3 H, s, CH3), 2.57–2.72 (3 H, m, H13, H16),
2.93–3.04 (1 H, m, H19), 4.61 and 4.73 (two 1 H broad-
ened signals, H29), 5.16 (1 H, broadened signal,
NHëéNç), 6.85 (1 H, broadened signal, H3'), 6.97 (1
H, broadened signal, H4'), 7.55 (1 H, broadened signal,
H2'), 8.16 (1 H, broadened signal, H5'), 9.55 (1 H,
broadened signal, 3-NOH), 9.85 (1 H, broadened sig-
13
3-Oxo-17b-[N'-(n-decyl)ureido]-28-norlup-20(29)-
ene (XI) was prepared from isocyanate (VIII) (0.47 g,
1 mmol) and n-decylamine (0.56 g) as a white powder;
nal, NHCONH); C NMR (δ, ppm): 14.5, 15.8, 17.0,
18.9, 19.3, 21.2, 21.6, 22.9, 25.2, 27.4, 29.6, 30.7, 33.8,
35.2, 37.1, 37.7, 38.1, 38.7, 40.1, 40.7, 42.1, 47.9, 49.6,
yield 0.56 g (83%); Rf 0.53; mp 198–200; IR (ν, cm–1): 50.1, 55.4, 64.3 (C17), 109.7 (C29), 112.1, 116.2,
138.0, 145.4, 154.0, 149.9 (C20), 155.2 (NHCONH),
166.6 (C3). Found, %: C 74.97, H 9.34, N 10.05. Calcd.
for C35H53O2N4, %: C 74.81, H 9.53, N 9.97.
770, 840, 910, 1100, 1140, 1190, 1250, 1330, 1440,
1480, 1530, 1650, 1680, 1770, 3240–3350; H NMR
(δ, ppm): 0.86, 0.90, 0.95, 1.02, 1.07 (15 H, 5 s, 5 CH3),
1.20–2.00 (37 H, m, CH2, CH), 1.24 (3 H, s, H10'), 1.67
(3 H, s, CH3), 2.41–2.54 (3 H, m, H13, H16), 2.63–2.72
1
General procedure for the synthesis of (XIV)–
(XVIII). Triethylamine (0.23 ml) was added dropwise
(1 H, m, H19), 3.06–3.17 (2 H, m, H1'), 4.59 and 4.67 to a stirred suspension of amino acid methyl ester
(two 1 H broadened signals, H29), 4.85–4.95 (1 H, m, hydrochloride (1.3 mmol) in anhydrous chloroform
NHCONH), 5.17 (1 H, broadened signal, NHCONH); (5 ml). A solution of isocyanate (VII), (VIII), or (IX)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 6 2003

598
FLEKHTER et al.
in chloroform (5 ml) was added, and the reaction mix- 1.08 (21 H, 7 s, 7 CH3), 1.20–2.00 (24 H, m, CH2, CH),
ture was refluxed for 1 h, kept overnight, washed with
10% HCl (2 × 10 ml), water (2 × 10 ml), dried over
MgSO4, and evaporated in a vacuum. The residue was
chromatographed on an alumina column eluted with
chloroform.
1.70 (3 H, s, CH3), 2.40–2.53 (3 H, m, H13, H16),
2.57–2.64 (1 H, m, H19), 3.74 (3 H, s, OCH3), 4.63 and
4.73 (two broadened signals of 1 H each, H29), 4.68 (1
H, broadened signal, H3'), 5.10 (1H, one broadened
signal, NHCONH), 5.22 (1 H, m,NHëéNç); 13C
NMR (δ, ppm): 14.4, 15.8, 16.0, 19.3, 19.6, 21.0, 21.5,
22.8, 24.9, and 24.9 (C6', C7'), 25.0, 26.7 (C5'), 27.1,
29.7, 29.8, 33.4, 34.1, 35.5, 36.9, 37.9, 39.6, 40.7, 40.8
(C4'), 42.1, 47.3, 47.7, 49.5, 49.8, 51.8 (C3'), 52.1
(C1'), 54.9, 63.8 (C17), 110.1 (C29), 149.5 (C20),
156.6 (NHCONH), 174.8 (C2'), 218.0 (C3). Found, %:
C 74.32, H 10.02, N 4.53. Calcd. for C37H60O4N2, %: C
74.34, H 10.15, N 4.69.
Methyl ester of 3b-hydroxy-28-norlup-20(29)-
en-17b-yl-N-carbamoyl-L-alanine (XIV) was pre-
pared from isocyanate (VII) (0.45 g, 1 mmol) and L-
alanine methyl ester (0.20 g) as a white powder; yield
0.46 g (83%); Rf 0.42; mp 217–219°C; IR (ν, cm–1):
750, 770, 850, 900, 910, 1000, 1100, 1120, 1180, 1210,
1250, 1350, 1400, 1450, 1480, 1540, 1650, 1690, 1760,
3250–3350; 1H NMR (δ, ppm): 0.76, 0.82, 0.95, 0.96,
1.01, (15 H, 5 s, 5 CH3), 1.10–2.00 (22 H, m, CH2, CH),
1.25 (3 H, s, H4'), 1.67 (3 H, s, CH3), 2.39–2.50 (3 H,
m, H13, H16), 2.53–3.61 (1 H, m, H19), 3.14–3.22
(1 H, m, H3), 3.73 (3 H, s, OCH3), 4.09–4.19 (1 H, m,
H3'), 4.60 and 4.69 (two broadened signals of 1 H each,
H29), 5.03 (1 H, m, NHCONH), 5.15 (1 H, broadened
signal, NHCONH); 13C NMR (δ, ppm): 14.4, 15.3,
16.0, 16.1 (C4'), 18.2, 19.0, 19.2, 20.9, 21.6, 21.7, 25.0,
27.2, 28.0, 29.8, 34.1, 35.5, 37.1, 37.6, 38.7, 38.8, 40.7,
42.0, 47.5, 49.0, 49.6, 50.4 (C1'), 55.3, 63.6 (C17), 68.7
(C3'), 78.9 (C3), 109.8 (C29), 149.7 (C20), 156.6
(NHCONH), 173.8 (C2'). Found, %: C 73.19, H 10.04,
N 4.97. Calcd. for ë34ç56é4N2, %: C 73.32, H 10.16,
N 5.03.
Methyl ester of 3-oxo-28-norlup-20(29)-en-17b-
yl-N-carbamoyl-L-methionine (XVII) was prepared
from isocyanate (VIII) (0.47 g, 1 mmol) and L-
methionine methyl ester (0.26 g) as a white powder;
yield 0.52 g (84%); Rf 0.38; mp 137–140°C; IR (ν, cm–
1): 780, 840, 910, 1000, 1200, 1130, 1190, 1210, 1260,
1340, 1410, 1450, 1470, 1540, 1660, 1690, 1750,
3250–3350; 1H NMR (δ, ppm): 0.85, 0.93, 0.96, 1.03,
1.07 (15 H, 5 s, 5 CH3), 1.20–2.00 (25 H, m, CH2, CH),
1.68 (3 H, s, CH3), 2.13 (1 H, broadened signal, H6'),
2.41–2.53 (3 H, m, H13, H16), 2.55–2.66 (1 H, m,
H19), 3.76 (3 H, s, OCH3), 4.51–4.62 (2 H, m, H29,
H3'), 4.68 (1 H, broadened signal, H29), 5.15–5.25 (1
H, m, NHëéNç), 5.40 (3 H, broadened signal,
NHCONH); 13C NMR (δ, ppm): 14.3, 15.4 (C6'), 15.7,
15.9, 19.2, 19.5, 20.9, 21.3, 25.0, 26.6, 27.2, 29.6, 29.7,
30.1 (C5'), 32.3 (C4'), 33.3, 34.0, 35.5, 36.8, 37.6, 39.5,
40.6, 42.0, 47.3, 47.5, 49.4, 49.6, 49.7 (C3'), 52.2 (C1'),
54.8, 63.6 (C17), 109.9 (C29), 149.6 (C20), 156.7
(NHCONH), 173.8 (C2'), 217.5 (C3). Found, %: C
70.23, H 9.68, N 4.41, S 4.98. Calcd. for C36H58O4N2S,
%: C 70.30, H 9.53, N 4.56, S 5.21.
Methyl ester of 3-oxo-28-norlup-20(29)-en-17b-
yl-N-carbamoyl-L-valine (XV) was prepared from
isocyanate (VIII) (0.47 g, 1 mmol) and L-valine methyl
ester (0.22 g) as a white powder; yield 0.49 g (84%); Rf
0.40; mp 182–184°C; IR (ν, cm–1): 740, 780, 850, 910,
1100, 1120, 1180, 1200, 1250, 1340, 1410, 1460, 1480,
1
1530, 1650, 1690, 1750, 3240–3350; H NMR (δ,
ppm): 0.83, 0.84, 0.91, 0.93, 1.00, 1.04, 1.05 (21 H, 7 s,
7 CH3), 1.20–2.00 (23 H, m, CH2, CH), 1.66 (3 H, s,
CH3), 2.36–2.51 (3 H, m, H13, H16), 2.52–2.63 (1 H,
m, H19), 3.73 (3 H, s, OCH3), 4.40 (1 H, m,
Methyl ester of 28-norlup-20(29)-en-17b-yl-N-
carbamoyl-L-methionine 3-oxime (XVIII) was pre-
NHCONH), 4.57 and 4.64 (two broadened signals of 1 pared from isocyanate (IX) (0.47 g, 1 mmol) and L-
H each, H29), 4.74 (1 H, broadened signal, H3'), 5.53 methionine methyl ester (0.26 g) as a white powder;
(1 H, d, J 7.3 Hz, NHëéNç); 13C NMR (δ, ppm): 14.3, yield 0.50 g (79%); Rf 0.27; mp 189–191°C; IR (ν, cm–1):
15.7, 15.8, 17.7, 19.0 and 19.0 (C5', C6'), 19.2, 20.9,
21.2, 24.9, 26.5, 27.3, 29.7, 29.8, 31.5 (C4'), 33.3, 34.0,
35.4, 36.8, 37.4, 39.5, 40.5, 42.0, 47.3, 47.4, 49.4, 49.7,
51.8 (C1'), 54.9, 57.7 (C3'), 64.3 (C17), 109.7 (C29),
149.7 (C20), 156.8 (NHCONH), 174.3 (C2'), 218.2
(C3). Found, %: C 74.31, H 9.98, N 4.72. Calcd. for
ë36ç59é4N2, %: C 74.17, H 10.05, N 4.81.
710, 770, 860, 900, 920, 1100, 1130, 1190, 1210, 1340,
1410, 1460, 1470, 1530, 1660, 1690, 1750, 3250–3350;
1H NMR (δ, ppm): 0.85, 0.91, 0.96, 1.02, 1.14 (15 H, 5
s, 5 CH3), 1.10–2.00 (26 H, m, CH2, CH), 1.66 (3 H, s,
CH3), 2.07 (3 H, broadened signal, H6'), 2.38–2.54 (3
H, m, H13, H16), 2.95–3.06 (1 H, m, H19), 3.75 (3 H,
s, OCH3), 4.47–4.61 (2 H, m, H29, H3')), 4.66 (1 H,
broadened signal, H29), 5.10 (1 H, broadened signal,
NHCONH), 5.70–5.80 (1 H, m, NHëéNç), 9.50 (1 H,
Methyl ester of 3-oxo-28-norlup-20(29)-en-17b-
yl-N-carbamoyl-L-leucine (XVI) was prepared from
isocyanate (VIII) (0.47 g, 1 mmol) and L-leucine
methyl ester (0.24 g) as a white powder; yield 0.49 g
(82%); Rf 0.41; mp 172–174°C; IR (ν, cm–1): 770, 840,
900, 920, 1100, 1130, 1190, 1220, 1250, 1370, 1410,
1450, 1470, 1520, 1650, 1670, 1760, 3250–3350;
13
broadened signal, 3-NOH); C NMR (δ, ppm): 14.3,
15.4 (C6'), 15.8, 17.1, 18.9, 19.1, 21.2, 21.5, 22.8, 24.9,
27.3, 29.6, 30.1 (C5'), 30.5, 32.1 (C4'), 33.6, 35.4, 37.1,
37.5, 38.2, 38.7, 40.2, 40.6, 41.9, 47.5, 49.4, 49.7, 49.9
(C3'), 52.2 (C1'), 55.5, 63.6 (C17), 109.9 (C29), 149.5
1H NMR (δ, ppm): 0.94, 0.95, 0.96, 0.98, 1.03, 1.07, (C20), 156.8 (NHCONH), 166.8 (C3), 174.3 (C2').
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 6 2003

SYNTHESIS AND ANTIVIRAL ACTIVITY
599
Found, %: C 68.61, H 9.39, N 6.58, S 5.16. Calcd. for chloroform. The substance was obtained as a yellow
C36H60O4N3S, %: C 68.50, H 9.51, N 6.66, S 5.08.
powder; yield 0.27 g (50%); Rf 0.38; mp 204–206°C;
IR (ν, cm–1): 770, 850, 920, 1100, 1160, 1260, 1330,
General procedure for the synthesis of (XIX) and
(XX). Isocyanate derivative of betulonic acid (VIII)
(0.47 g, 1 mmol) and sodium methylate or benzylate
(2 mmol) were refluxed in anhydrous benzene (20 ml),
with the course of reaction being monitored by TLC.
The reaction mixture was washed with 5% HCl (15 ml),
water (2 × 10 ml), dried over MgSO4, and evaporated in
a vacuum.
1
1440, 1470, 1520, 1670, 1690, 1780, 3250–3340; H
NMR (δ, ppm): 0.88, 0.92, 0.95, 0.99, 1.01 (15 H, 5 s,
5 CH3), 1.10–2.00 (21 H, m, CH2, CH), 1.70 (3 H, s,
CH3), 2.33–2.45 (4 H, m, H13, H16, H19), 4.58 and
4.66 (two broadened signals of 1 H each, H29), 4.76
(1 H, broadened signal, NHCO), 7.48–7.62 (5 H, m,
aromatic protons); 13C NMR (δ, ppm): 14.8, 15.3, 16.2,
18.9, 19.3, 20.9, 21.4, 25.0, 26.5, 27.0, 29.2, 29.9, 33.3,
34.0, 35.3, 36.9, 38.0, 39.2, 40.3, 42.0, 47.4, 47.6, 49.4,
49.6, 54.5, 63.6 (C17), 110.2 (C29), 123.4 and 123.4
(C2', C6'), 128.9 (C4'), 131.3 and 131.4 (C3', C5'),
148.0 (C1'), 149.7 (C20), 156.4 (NHCO2), 218.0 (C3).
Found, %: C 79.33, H 9.31, N 2.63. Calcd. for
C36H51O3N, %: C 79.20, H 9.44, N 2.57.
3-Oxo-17b-methoxycarbonylamino-28-norlup-
20(29)-ene (XIX) was prepared after reflux for 25 h as
a white powder; yield 0.44 g (91%); Rf 0.71; mp 175–
176°C; IR (ν, cm–1): 770, 840, 900, 1100, 1140, 1180,
1250, 1340, 1440, 1480, 1540, 1660, 1690, 1770,
3250–3350; 1H NMR (δ, ppm): 0.92, 0.95, 1.01, 1.03,
1.06 (15 H, 5 s, 5 CH3), 1.10–2.00 (21 H, m, CH2, CH),
1.67 (3 H, s, CH3), 2.37–2.52 (4 H, m, H13, H16 H19),
3.62 (3 H, s, OCH3), 4.52 (1 H, broadened signal,
NHCO), 4.60 and 4.70 (two broadened signals of 1 H
each, H29); 13C NMR (δ, ppm): 14.3, 15.8, 15.9, 19.2,
19.6, 21.0, 21.4, 24.9, 26.6, 27.1, 29.1, 29.5, 33.4, 34.1,
35.0, 36.9, 37.8, 38.5, 39.5, 40.6, 42.1, 47.6, 49.0, 49.7,
51.5 (OCH3), 54.9, 63.6 (C17), 110.2 (C29), 149.2
(C20), 155.2 (NHCO2), 217.8 (C3). Found, %: C 77.07,
H 10.45, N 2.78. Calcd. for C31H49O3N, %: C 76.95, H
10.23, N 2.90.
Antiviral properties were determined in the exper-
iments on cell cultures of the herpes simplex type 1
virus (VGP-1), virus of the A/FPV/Rostok/34 (H7N1)
flue, and the ECHO6 enterovirus. The experiments
were performed by the method of plaque reduction on
the culture of primary fibroblasts of chicken embryos
(FCE) with FPV, by the method of estimation of the
cytoplasmic effect on the culture of the human rab-
domyosarcoma (RD) with VGP-1, and by the method
[19] with the ECHO6 virus. The compounds under
study were preliminarily dissolved in 10% aqueous eth-
anol and, then, the solutions were diluted by the culture
medium to the desired concentrations. The antiviral
effect was determined as a decrease in the virus titer in
the presence of the examined compounds in compari-
son with the control. The concentrations of 50% inhibi-
tion of the virus propagation (an average effective con-
centration EC50) and the ratio of the maximally tolerant
concentration (MTC) to EC50 were also calculated. The
MTC values of the compounds for unidentified cell cul-
tures were determined after 72 h of incubation.
3-Oxo-17b-benzyloxycarbonylamino-28-norlup-
20(29)-ene (XX) was prepared after reflux for 10 h and
purification by chromatography on an alumina column
eluted with benzene as a yellow powder; yield 0.46 g
(82%); Rf 0.43; mp 225–226°C; IR (ν, cm–1): 700, 830,
910, 1120, 1140, 1250, 1350, 1440, 1490, 1540, 1660,
1690, 1770, 3250–3340; 1H NMR (δ, ppm): 0.76, 0.78,
0.85, 0.91, 0.96 (15 H, 5 s, 5 CH3), 1.10–2.00 (21 H, m,
CH2, CH), 1.70 (3 H, s, CH3), 2.35–2.50 (4 H, m, H13,
H16, H19), 4.52 and 4.63 (two broadened signals of 1
H each, H29), 4.68 (2 H, broadened signal, H1'), 4.70
(1 H, broadened signal, NHCO), 7.15 (1 H, m, H5'),
ACKNOWLEDGMENTS
13
7.32–7.46 (4 H, m, H3', H4', H6', H7'); C NMR (δ,
This study was supported by the Russian Founda-
tion for Basic Research and Byelorussian Foundation
for Basic Research, project nos. 00-03-81174, 01-03-
33131, 02-03-81007, B99R-031, and B02R-016.
ppm): 14.2, 15.5, 16.2, 18.5, 19.5, 21.1, 21.7, 25.1,
26.9, 27.2, 29.0, 29.7, 31.8, 33.5, 34.2, 36.5, 37.8, 39.2,
40.3, 42.2, 46.9, 48.4, 49.0, 49.6, 51.5, 52.9, 63.7
(C17), 110.2 (C29), 127.9 and 128.1 (C3' and C7'),
128.8 and 128.9 (C4' and C6'), 130.2 (C5'), 140.4 (C2'),
150.5 (C20), 156.5 (NHCO2), 218.3 (C3). Found, %: C
79.64, H 9.52, N 2.35. Calcd. for C37H53O3N, %: C
79.36, H 9.56, N 2.50.
REFERENCES
1. Flekhter, O.B., Karachurina, L.T., Poroikov, V.V., Nig-
matullina, L.R., Baltina, L.A., Zarudii, F.S., Davy-
dova, V.A., Spirikhin, L.V., Baikova, I.P., Galin, F.Z.,
and Tolstikov, G.A., Bioorg. Khim., 2000, vol. 26,
pp. 215–223.
2. Flekhter, O.B., Karachurina, L.T., Nigmatullina, L.R.,
Baltina, L.A., Zarudii, F.S., Davydova, V.A., Spi-
rikhin, L.V., Baikova, I.P., Galin, F.Z., and Tol-
stikov, G.A., Khim.–Farm. Zh., 2000, vol. 34, pp. 3–5.
3-Oxo-17b-phenyloxycarbonylamino-28-norlup-
20(29)-ene (XXI). Phenol (0.18 g, 2 mmol) and several
drops of triethylamine were added to a solution of iso-
cyanate (VIII) (0.47 g, 1 mmol) in anhydrous benzene
(50 ml). The reaction mixture was refluxed for 25 h,
washed with water (3 × 15 ml), dried over MgSO4, and
evaporated in a vacuum. The residue was purified by
chromatography on a silica gel column eluted with
3. Flekhter, O.B., Nigmatullina, L.R., Karachurina, L.T.,
Baltina, L.A., Zarudii, F.S., Davydova, V.A., Galin, F.Z.,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 6 2003

600
FLEKHTER et al.
and Tolstikov, G.A., Khim.–Farm. Zh., 2000, vol. 34, 11. Kurokawa, M., Basnet, P., Ohsugi, M., Hozumi, T.,
pp. 17–20.
Kadota, S., Namba, T., Kawana, T., and Shiraki, K.,
J. Pharm. Exp. Ther., 1999, vol. 289, pp. 72–78.
4. Flekhter, O.B., Karachurina, L.T., Plyasunova, O.A.,
Nigmatullina, L.R., Baltina, L.A., Pokrovskii, A.G.,
Davydova, V.A., Zarudii, F.S., Galin, F.Z., Shul’ts, E.E.,
and Tolstikov, G.A, RF Patent 2 174 982, 2001.
5. Protiva, J. and Vystrcil, A., Collect. Czech. Chem. Com-
mun., 1976, vol. 41, no. 4, pp. 1200–1207.
6. Evers, M., Poujade, C., Soler, F., Ribeill, Y., James, C.,
Lelievre, Y., Gueguen, J.-C., Reisdorf, D., Morize, I.,
Pauwels, R., De Clercq, E., Henin, Y., Bousseau, A.,
Mayaux, J.-F., Pecq, J.-B.L., and Dereu, N., J. Med.
Chem., 1996, vol. 39, pp. 1056–1068.
12. Ryu, S.Y., Lee, C.-K., Lee, C.O., Ki, H.S., and Zee, O.P.,
Arch. Pharm. Res. (Seoul), 1992, vol. 15, pp. 242–245.
13. Platonov, V.G., Zorina, A.D., Gordon, M.A., Chi-
zhov, N.P., Balykina, L.V., Mikhailov, Yu.D., Ivanen, D.R.,
Tran Kim Qui, and Shavva A.G., Khim.–Farm. Zh.,
1995, vol. 29, pp. 42–46.
14. Pezzuto, J.M. and Kim, D.S.H.L., US Patent 5 804 575,
1998.
15. Valterova, E., Klinot, J., and Vystrcil, A., Collect. Czech.
Chem. Commun., 1983, vol. 48, pp. 649–661.
7. Drefahl, G. and Huneck, S., Chem. Ber., 1961, no. 8,
16. Nelles, J., Ber. Deutsch. Chem. Ges., 1933, vol. 66,
pp. 193–196.
pp. 1345–1347.
8. Peng, C., Bodenhausen, G., Qiu, S., Fong, H.H.S., Farn-
sworth, N.R., Yuan, S., and Zheng, C., Magn. Res.
Chem., 1998, vol. 36, pp. 267–278.
17. Gordon, A.J. and Ford, R.A., The Chemist’s Companion:
A Handbook of Practical Data, Techniques, and Refer-
ences, New York: Wiley, 1972, pp. 437–444.
9. Baltina, L.A., Kondratenko, R.M., and Tolstikov, G.A.,
Zh. Obshch. Khim., 1984, vol. 54, no. 11, pp. 2573–
2579.
10. Carlson, R.M., Krasutsky, P.A., and Karim, M.R.U., US
Patent 5 750 578, 1998.
18. Baltina, L.A. and Tolstikov, G.A., Zh. Obshch. Khim.,
1991, vol. 61, no. 5, pp. 1227–1233.
19. Boreko, E.I., Pavlova, N.I., Zaitseva, G.V., and
Mikhailopulo, I.A., Vopr. Virusol., 2001, no. 5, pp. 40–
42.
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