Synthesis and Antiviral Activity of Hydrazides and Substituted Benzalhydrazides of Betulinic Acid and Its Derivatives

Article abstract of DOI:10.1023/A:1023948919315

New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N′-benzalhydrazides, were synthesized. Their antiviral activities toward viruses of influenza A virus, herpes simplex type I virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the highest activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid 3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus. Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under study displayed a low antiviral activity toward enterovirus ECHO6.

Full text of DOI:10.1023/A:1023948919315

Russian Journal of Bioorganic Chemistry, Vol. 29, No. 3, 2003, pp. 296–301. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 3, 2003, pp. 326–332.
Original Russian Text Copyright © 2003 by Flekhter, Boreko, Nigmatullina, Pavlova, Nikolaeva, Savinova, Eremin, Baltina, Galin, Tolstikov.
Synthesis and Antiviral Activity of Hydrazides and Substituted
Benzalhydrazides of Betulinic Acid and Its Derivatives
1
O. B. Flekhter*, E. I. Boreko**, L. R. Nigmatullina*, N. I. Pavlova**, S. N. Nikolaeva**,
O. V. Savinova**, V. F. Eremin**, L. A. Baltina*, F. Z. Galin*, and G. A. Tolstikov*
*Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences,
pr. Oktyabrya 71, Ufa, Bashkortostan, 450054 Russia
**Belarussian Research Institute of Epidemiology and Microbiology, ul. K. Tsetkin 4, Minsk, 220050 Belarus
Received April 2, 2002; in final form, October 1, 2002
Abstract—New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N'-benzalhy-
drazides, were synthesized. Their antiviral activities toward viruses of influenza A virus, herpes simplex type I
virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the high-
est activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid
3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus.
Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under
study displayed a low antiviral activity toward enterovirus ECHO6.
Key words: benzalhydrazides; betulinic acid, hydrazides; lupane triterpenoids, antiviral activity
1
INTRODUCTION
RESULTS AND DISCUSSION
Betulinic acid and its derivatives constitute a new
To continue the studies on the chemical modifica-
tion of lupane triterpenoids [14–16], we have synthe-
sized a new series of nitrogen-containing derivatives of
betulinic acid (I), 3-ketobetulinic (betulonic) acid (II),
and betulonic acid 3-oxime (III) and investigated the
effect of some of these compounds on replication of
influenza A virus (A/FVP/Rostock/34), HSV-1, and
enterovirus ECHO6 in vitro.
2
class of HIV-1 inhibitors. They block the stage of HIV
fusion with cells [1–4]. Chemical modification of betu-
linic acid is a promising approach to design of new
highly effective antiviral agents. For example, ω-ami-
noalkylamides of betulinic acid displayed anti-HIV
activity at nanomolar concentrations [5]. Medium-
effective concentrations in the region from 0.35 to
0.86 nM were characteristic of 3-O-(3',3'-dime-
thyl)succinyl derivatives of betulinic and dihydrobetu-
linic acids [2, 6]. At the same time, the antiviral proper-
ties of betulinic acid itself toward HIV-1 are moderate
(EC50 = 1.4 µM) [3]. Betulin, betulinic acid, and betu-
lonic acid (like other triterpenoids of oleanane, ursane,
and dammarane series) also possess antiviral activity
toward HSV-1 and Epstein–Barr viruses [3, 7–11].
Among lupane triterpenoids, only betulin was studied
for activity against influenza viruses and was found not
to inhibit their replication [9]. The effect of betulinic
acid and its derivatives on other virus types has not been
reported. In the last few years, the unique ability of bet-
ulinic acid to inhibit the growth of human melanoma
and other tumors has also been found [12, 13].
Betulinic acid (I) was prepared by the modified pro-
cedure [17] in two steps, the Jones oxidation of betulin
and subsequent reduction of betulonic acid (II) with
NaBH4 in isopropanol. The 3β/3α epimer ratio was
97 : 3, and the yield reached 95%.
Acid hydrazides and their derivatives are known to
be widely used in medicine as antiinfectious prepara-
tions [18]. Hydrazides of lupane acids and their deriva-
tives have not been described in literature.
We obtained hydrazides (VII)–(IX) by the treat-
ment of betulinic, betulonic, and betulonic acid 3-
oxime chlorides (IV)–(VI) with hydrazine hydrate in
91–95% yields. The starting acid chlorides (IV)–(VI)
were obtained in quantitative yields by the reaction of
the corresponding acids (I)–(III) with oxalyl chloride
in anhydrous benzene at 0°ë. The reflux of hydrazides
(VII)–(IX) with equimolar amount of an aromatic alde-
hyde in ethanol for 6 h resulted in benzalhydrazides
(X)–(XVIII) (87–92% yields). Structures of the com-
pounds were confirmed by IR, UV, and NMR spectros-
copy. Thus, in the 13C NMR spectra, the signals of the
carbon atoms of the C=N bond, the CONH group, and
1
Corresponding author; fax: +7 (3472) 35-6066; e-mail:
obf@anrb.ru
2
Abbreviations: CEF, chicken embryo fibroblast culture; EC
,
50
mean-effective concentration, concentration that causes 50%
inhibition of virus replication; FPV, fowlpox virus; HIV-1, human
immunodeficiency virus type I; HSV-1, type I human herpes sim-
plex virus; MTC, maximum tolerable concentration; PFU,
plague-forming unit; and TCEI , titer of cytopathic effect inhibi-
50
tion by 50%.
1068-1620/03/29032-0296$25.00 © 2003 MAIK “Nauka /Interperiodica”

SYNTHESIS AND ANTIVIRAL ACTIVITY OF HYDRAZIDES
297
COOH
COR
a, b
R1
R2
R1
R2
(I)–(III)
(IV)–(VI) R3 = Cl
(VII)–(IX) R3 = NHNH2
c
(I), (IV), (VII), (X)–(XII) R1 = H, R2 = OH
(II), (V), (VIII), (XIII)–(XV) R1 + R2 = O
(III), (VI), (IX), (XVI)–(XVIII) R1 + R2 = NOH
1'
2'
CONH CH
Y
R1
R2
Y =
(X) o-, m-OCH3; (XI) p-OC2H5; (XII) p-Cl;
(XIII) o-OCH3; (XIV) p-Br; (XV), (XVII) m-I;
(XVI) H; (XVIII) m-NO2
a: (COCl)2/C6H6; b: NH2NH2 · H2O/(C2H5)2O; c: EtOH, YC6H4CHO
Scheme.
of the aromatic moiety appear at 141–145, 172–175,
and 110–160 ppm, respectively. In the H NMR spec-
Betulonic acid 3-oxime (III) displayed the highest
activity against influenza virus. The virus titer
decreased from 3.52 to 1.98 log PFU/ml in the presence
of (III) at a concentration range from 851.6 (MTC) to
12.8 µM and up to 0.96 log PFU/ml in the presence of
6.4 µM (III) as compared to control. Thus, the EC50
value for (III) is the lowest among those for com-
pounds under study, whereas the MTC/EC50 ratio is the
highest (see table).
1
tra, the signals of the protons of N–CH–Ar and CONH
groups appear in the low-field region at 8–9 ppm as sin-
glets. The IR spectra of all the hydrazides are character-
ized with broad absorption bands of NH2 group at
3500–3000 cm–1, whereas the CONH group absorbs at
1680 and 1550 cm–1. The UV spectra of benzalhy-
drazides exhibit highly intensive absorption maxima at
282–284 and 290–295 nm, typical of the chromophores
of aromatic benzalhydrazides.
Betulinic acid 3-oxime hydrazide (IX) showed less
pronounced activity in the experiments with influenza
virus. The decrease in the virus titer in the presence of
Antiviral activity of betulinic acid derivatives
Influenza A virus
Com-
HSV-1
EC50 (I95), µM
ECHO6 virus
EC50 (I95), µM
pound
EC50 (I95), µM
MTC/EC50
MTC/EC50
MTC/EC50
(II)
5.73 (12.22–2.68)
2.17 (2.47–1.89)
nd
9.58
392.16
nd
2.50 (2.76–2.28)
81.56 (85.69–77.64)
9.96 (11.26–8.79)
>51.60
43.86
1.30
73.32 (77.11–69.69)
4.19 (4.64–3.79)
nd
3.0
6.09
nd
(III)
(VII)
(IX)
21.32
<1
2.04 (2.13–1.98)
25.25
4.69
0.68 (127.14–0.004)
50.73 (60.77–42.34)
406.80 (722.67–229.00)
37.87
6.65
<1
(XII) 71.96 (146.25–35.44)
(XVI) 15.40 (30.13–7.86)
5.16 (5.89–4.54)
7.70 (9.85–6.03)
32.68
181.41
11.34
Note: nd, not determined; I , confidence interval for 95% probability.
95
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 3 2003

298
FLEKHTER et al.
(IX) at concentrations of 51.6 and 25.8 µM was 1.10 solution of acid (I)–(III) in anhydrous benzene (10 ml),
and 1.06 log PFU/ml, respectively, as compared to con- and the mixture was stirred for 2 h at room temperature
trol. Compounds (XII) and (XVI) were practically under nitrogen. Then the solvent was removed in a vac-
inactive toward this virus.
uum. Yield 95–97%.
At the same time, compounds (II), (VII), (XII), and
(XVI) can inhibit the replication of HSV-1. For exam-
ple, betulonic acid (II) was highly effective within the
concentration range from 109.7 (MTC) to 6.6 µM; the
decrease in the virus titer was from 3.0 to 1.59 log
CTIE50 as compared to control. A similar activity was
also observed for (XVI) (the decrease in the virus titer
from 2.00 to 1.36 logTCEI50/ml), but in a wider con-
centration range from 1397.3 (MTC) to 10.5 µM. Com-
pound (XII) appeared to be less active, as in its pres-
ence the HVS-1 titer decreased for 1–2 logTCEI50/ml
in a concentration range from 168.8 (MTC) to 20.2 µM.
Betulonic acid 3-oxime (III) and its hydrazide (IX)
were inactive against this virus.
The derivatives of betulonic acid under study were
little effective against enterovirus ECHO6. Thus, betu-
lonic acid 3-oxime (III) caused the decrease in its titer
for 1.27–1.22 logTCEI50/ml in a concentration range
from 25.5 (MTC) to 6.4 µM. Betulonic acid (II) was
effective in MTC (21.9 µM, titer decrease for 1.93
logTCEI50/ml). Compound (XII) caused the decrease
in the virus titer in a concentration range from 337.5
(MTC) to 42.2 µM, but it was the less pronounced
(0.92–0.4 logCTID50/ml). Compounds (IX) and (XVI)
were inactive.
Acid chloride (IV); amorphous cream-colored pow-
der; IR: 1810 (COCl). Found, %: C 76.00, H 9.72, Cl
7.90. Calcd. for C30H47O2Cl, %: C 75.82, H 9.99, Cl
7.46.
Acid chloride (V); amorphous yellow powder; IR:
1820 (COCl). Found, %: C 76.57, H 9.43, Cl 8.02.
Calcd. for C30H45O2Cl, %: C 76.14, H 9.60, Cl 7.49.
Acid chloride (VI); amorphous gray powder; IR:
1825 (COCl). Found, %: C 74.22, H 9.39, N 2.70, Cl
7.42. Calc. for C30H46O2NCl, %: C 73.80, H 9.52, N
2.87, Cl 7.26.
Synthesis of hydrazides (VII)–(IX). General pro-
cedure. Hydrazine hydrate (100%, 1.5 ml) was added
dropwise under stirring and cooling to 0 ± 5°ë to a
solution of acid chloride (IV), (V), or (VI) (1 mmol) in
diethyl ether (10 ml). The precipitate of hydrazide
formed after cooling to 5–7°ë was filtered, washed
with diethyl ether (2 × 20 ml), and dried.
3b-Hydroxylup-20(29)-en-28-oic acid hydrazide
(VII) was obtained from betulinic acid chloride (IV)
(0.47 g, 1 mmol); yield 0.44 g (93%); Rf 0.31; mp
285°ë; IR: 3470, 3300 (NH2), 1732, 1680, 1560
(CONH), 1478, 1430, 1382, 1368, 1320, 1296, 1254,
1244, 1200, 1112, 1088, 1032, 990, 912, 888, 824, and
760; 1H NMR: 0.76, 0.82, 0.87, 0.92, and 0.97 (15 H, 5
s, 5 CH3), 1.10–2.00 (22 H, m, CH2 and CH), 1.69 (3 H,
s, CH3), 2.27–2.56 (3 H, m, H13 and H16), 3.09 (1 H,
dt, J 3.6 and 11.5, H19), 3.14–3.22 (1 H, m, H3), 4.61
and 4.75 (2 × 1 H, br. s, H29), 6.99 (1 H, br. s, CONH),
and 9.30 (2 H, br. s, NH2); 13ë NMR: 14.7, 15.4, 16.2,
18.3, 19.4, 20.9, 21.5, 22.9, 25.6, 27.4, 28.0, 29.5, 30.8,
33.3, 34.4, 37.2, 37.9, 38.2, 38.9, 40.2, 40.8, 42.4, 46.9,
50.3, 50.6, 55.0, 79.0 (C3), 109.6 (C29), 150.6 (C20),
and 177.2 (CONH). Found, %: C 76.74, H 10.62, N
5.85. Calcd. for C30H50O2N2, %: C 76.53, H 10.73, N
5.95.
Experiments with HIV-1 revealed the pronounced
antiviral activity of (VII) (EC50 calculated from the
decrease of the amount of antigen-containing cells by
the method of indirect immunofluorescence was 0.23
(0.40–0.15) µM; MTC/EC50 = 20).
EXPERIMENTAL
TLC was performed on Silufol (Chemapol, Czech
Republic) using the 20 : 1 chloroform–methanol devel-
oping system. The spots were visualized by spraying
with 20% phosphotungstic acid in ethanol and subse-
quent heating to 100–120°ëfor 2–3 min.
1
3-Ketolup-20(29)-en-28-oic
acid
hydrazide
13ë and H NMR spectra were registered on a (VIII) was obtained from betulonic acid chloride (V)
Bruker AM-300 spectrometer (75.5 and 300 MHz, (0.47 g, 1 mmol). Crystallization of the crude product
respectively) in deuterochloroform with SiMe4 as an from chloroform yielded 0.38 g (80%) of (VIII) as
white powder; IR: 3470 (NH2), 1727, 1696, 1680, 1560
and coupling constants (J, Hz) are given. IR spectra (CONH), 1480, 1434, 1398, 1372, 1330, 1280, 1242,
1214, 1191, 1118, 1078, 1024, 988, 920, 888, 824, and
spectrometers in Vaseline oil. UV spectra [λ, nm (ε, 760; 1H NMR: 0.84, 0.91, 0.97, 1.01, and 1.06 (15 H, 5
s, 5 CH3), 1.10–2.00 (21 H, m, CH2 and CH), 1.68 (3 H,
tometer in 1 : 1 chloroform–methanol. Melting points s, CH3), 2.34–2.52 (2 H, m, H13 and H16), 3.08 (1 H,
internal standard; the values of chemical shifts (δ, ppm)
(ν, cm–1) were recorded on Specord M80 and UR-20
å−1 cm–1] were taken on a Specord M40 spectropho-
were taken on a Boetius hot plate. Betulin was isolated
by the method reported in [19]; betulinic and betulonic
acids and betulonic acid 3-oxime were obtained
according to [17, 20].
Preparation of lupane acid chlorides (IV)–(VI). 38.2, 39.6, 40.7, 42.5, 46.8, 47.3, 50.1, 50.3, 54.9, 55.3,
Oxalyl chloride (0.3 ml) was added under stirring to a 109.5 (C29), 177.2 (CONH), and 218.0 (C3).
dt, J 3.6 and 11.5, H19), 4.60 and 4.73 (2 × 1 H, br. s,
H29), 6.99 (1 H, br. s, CONH), and 9.27 (2 H, br. s,
NH2); 13ë NMR: 14.6, 16.0, 16.1, 19.4, 19.6, 21.0,
21.3, 25.6, 26.6, 29.4, 30.8, 33.2, 33.7, 34.1, 37.0, 37.9,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 3 2003

SYNTHESIS AND ANTIVIRAL ACTIVITY OF HYDRAZIDES
299
Lup-20(29)-en-28-oic acid 3-oxime hydrazide (CONH), 1630 (Ar), 1590, 1550 (CONH), 1484, 1460,
(IX) was prepared from betulonic acid 3-oxime chlo- 1390, 1318, 1296, 1266, 1200, 1178, 1132, 1080, 1054,
1
ride (VI) (0.47 g, 1 mmol); yield 0.46 g (94%); Rf 0.29;
1024, 992, 932, 888, 832, and 756; H NMR: 0.75,
mp >300°ë; IR: 3030 (NH2), 1732, 1680, 1650 (C=N– 0.81, 0.87, 0.91, 0.96, and 0.99 (18 H, 6 s, 6 CH3),
OH), 1555 (CONH), 1478, 1432, 1386, 1360, 1322, 1.10–2.00 (22 H, m, CH2 and CH), 1.66 (3 H, s, CH3),
1278, 1244, 1210, 1199, 1128, 1074, 1020, 992, 912,
2.45–2.68 (3 H, m, H13 and H16), 2.93–3.07 (1 H, m,
888, 824, and 760; 1H NMR: 0.86, 0.92, 0.96, 1.04, and H19), 3.11 (1 H, dd, J 4.7 and 10.7, H3), 4.00–4.14
1.13 (15 H, 5 s, 5 CH3), 1.20–2.00 (21 H, m, CH2 and (2 H, m, éëç2ëç3), 4.61 and 4.76 (2 × 1 H, br. s,
CH), 1.69 (3 H, s, CH3), 2.35–2.56 (3 H, m, H13 and H29), 6.88 (2 H, d, J 7.9, H arom.), 7.64 (2 H, d, J 7.9,
H arom.), 8.16 (1 H, s, N=CH–Ar), and 8.87 (1 H, s,
H16), 3.03–3.17 (1 H, m, H19), 4.62 and 4.75 (2 × 1 H,
br. s, H29), 6.99 (1H, br. s, CONH), 9.15 (2 H, br. s, CONH); C NMR: 14.7, 15.3, 16.1, 16.4, 18.3, 19.5,
13
NH2), and 9.21 (1 H, br. s, NOH); 13C NMR: 14.6, 15.8,
16.1, 19.1, 19.4, 21.2, 21.5, 22.9, 25.6, 27.4, 29.4, 30.8,
33.3, 34.0, 37.2, 37.9, 38.2, 38.7, 40.2, 40.8, 42.5, 46.8,
50.2, 50.3, 55.0, 55.6, 109.6 (C29), 150.5 (C20), 167.3
(C3), and 177.3 (CONH). Found, %: C 74.59, H 10.03,
N 8.80. Calcd. for C30H49O2N3, %: C 74.47, H 10.23, N
8.69.
21.0, 22.3, 25.7, 27.4, 28.0, 29.6, 30.8, 33.4, 34.4, 37.2,
37.5, 38.7, 38.8, 40.8, 40.9, 42.5, 46.5, 50.5, 50.6, 55.3,
55.4, 64.0 (éëH2ëç3), 79.0 (C3), 109.5 (C29), 114.6,
114.6, 126.5, 126.5, 129.0, 160.7 (C arom.), 142.6
(C1'), 150.7 (C20), and 172.6 (CONH). Found, %: C
77.82, H 9.49, N 4.56. Calcd. for C39H58O3N2, %: C
77.68, H 9.71, N 4.65.
Synthesis of benzalhydrazides (X)–(XVIII). Gen-
eral procedure. A mixture of hydrazide (VII), (VIII),
or (IX) (1 mmol) and an aromatic aldehyde (1 mmol) in
ethanol (20 ml) was refluxed for 6 h. The reaction mix-
ture was cooled and poured in 5% HCl (50 ml). The
precipitate was filtered and washed with water to pH
7.0. The target product was purified by column chroma-
tography on alumina. Compounds (XIII)–(XVIII)
were eluted with chloroform; compounds (X)–(XII),
with acetone.
N'-(4-Chlorobenzal)hydrazide of 3b-hydroxy-
lup-20(29)-en-28-oic acid (XII) was obtained from
hydrazide (VII) (0.47 g) and p-chlorobenzaldehyde
(0.14 g); yield 0.53 g (90%); white powder; Rf 0.41; mp
270–273°ë; UV: 290 (log ε 3.94); IR: 1698, 1660
(CONH), 1652, 1605 (Ar), 1555 (CONH), 1517, 1483,
1426, 1394, 1317, 1265, 1244, 1196, 1155, 1116, 1070,
1
1032, 998, 924, 890, 822, and 756; H NMR: 0.70,
0.76, 0.85, 0.93, and 1.02 (15 H, 5 s, 5 CH3), 1.10–2.00
(21 H, m, CH2 and CH), 1.70 (3 H, s, CH3), 2.48–2.65
(3 H, m, H13 and H16), 2.93–3.04 (1 H, m, H19), 3.16
(1 H, dd, J 4.6 and 10.7, H3), 4.55 and 4.68 (2 × 1 H,
br. s, H29), 7.48 (2 H, d, J 8.4, H arom.), 7.67 (1 H, d,
J 8.3, H arom.), 8.28 (1 H, s, N=CH-Ar), and 8.55 (1 H,
N'-(2,3-Dimethoxybenzal)hydrazide
of
3b-
hydroxylup-20(29)-en-28-oic acid (X) was synthe-
sized from hydrazide (VII) (0.47 g) and 2,3-dimethoxy-
benzaldehyde (0.17 g); yield 0.57 g (92%), white pow-
13
der; Rf 0.37; mp 300–303°ë; UV: 295 (log ε 4.61); IR:
1680 (CONH), 1652, 1605 (Ar), 1550 (CONH), 1480,
1440, 1388, 1320, 1295, 1254, 1210, 1185, 1136, 1080,
br. s, CONH); C NMR: 14.4, 15.0, 15.9, 18.5, 19.2,
21.0, 25.7, 27.6, 28.1, 29.2, 30.8, 33.4, 34.1, 37.2, 37.3,
37.3, 38.9, 39.4, 40.7, 40.9, 42.2, 46.2, 50.5, 50.7, 55.0,
55.4, 78.6 (C3), 109.9 (C29), 128.9, 129.0, 129.4,
129.4 (C–Cl), 134.2, 134.6 (C arom.), 144.8 (C1'),
150.9 (C20), and 172.6 (CONH). Found, %: C 75.04, H
8.91, N 4.68, Cl 6.15. Calcd. for C37H53O2N2ël, %: C
74.89, H 9.02, N 4.72, Cl 5.97.
1
1018, 998, 920, 888, 824, and 766; H NMR: 0.82,
0.88, 0.91, 0.96, and 1.04 (15 H, 5 s, 5 CH3), 1.10–2.00
(22 H, m, CH2 and CH), 1.69 (3 H, s, CH3), 2.33–2.58
(3 H, m, H13 and H16), 2.88–2.99 (1 H, m, H19), 3.11
(1 H, dd, J 4.7 and 10.7, H3), 3.77 and 3.79 (2 × 3 H, 2
s, 2 éëç3), 4.52 and 4.67 (2 × 1 H, br. s, H29), 6.84
and 6.88 (2 × 1 H, 2 d, J 7.9, 2 H arom.), 6.99 (1 H, t, J
7.9, H arom.), 8.91 (1 H, s, N=CH-Ar), and 9.04 (1 H,
s, CONH); 13C NMR: 14.6, 15.3, 16.1, 18.2, 19.4, 20.9,
25.6, 27.3, 27.9, 29.6, 30.7, 33.1, 34.3, 37.1, 37.4, 38.7,
38.8, 40.7, 40.7, 42.4, 46.3, 50.5, 50.6, 55.1, 55.3, 55.7
(éëç3), 61.5 (éëç3), 78.9 (C3), 109.4 (C29), 113.5,
118.6, 124.3, 127.6, 148.1, 152.4 (C arom.), 141.7
(C1'), 150.8 (C20), and 172.5 (CONH). Found, %: C
75.80, H 9.43, N 4.47. Calcd. for C39H58O4N2, %: C
75.67, H 9.46, N 4.52.
N'-(2-Methoxybenzal)hydrazide of 3-ketolup-
20(29)-en-28-oic acid (XIII) was synthesized from
hydrazide (VIII) (0.47 g) and 2-methoxybenzaldehyde
(0.14 g); yield 0.40 g (69%); cream-colored powder; Rf
0.53; mp 193–195°ë; UV: 282 (log ε 4.47); IR: 1696,
1680 (CONH), 1662, 1600 (Ar), 1555 (CONH), 1518,
1490, 1436, 1320, 1292, 1243, 1206, 1136, 1080, 1040,
1
992, 890, 828, and 766; H NMR: 0.83, 0.88, 0.92,
1.00, and 1.05 (15 H, 5 s, 5 CH3), 1.10–2.00 (21 H, m,
ëç2 and CH), 1.70 (3 H, s, CH3), 2.38–2.52 (3 H, m,
H13 and H16), 2.97–3.08 (1 H, m, H19), 3.88 (3 H, s,
éëç3), 4.60 and 4.75 (2 × 1 H, br. s, H29), 6.78–7.00
(2 H, m, H arom.), 7.28–7.40 (2 H, m, H arom.), 7.60
(1 H, d, J 7.8, H arom.), 7.67 (1 H, d, J 7.8, H arom.),
8.55 (1 H, s, N=CH-Ar), and 9.40 (1 H, br. s, CONH);
13C NMR: 14.6, 15.9, 16.2, 19.4, 19.6, 21.0, 21.5, 25.6,
N'-(4-Ethoxybenzal)hydrazide of 3b-hydroxy-
lup-20(29)-en-28-oic acid (XI) was synthesized from
hydrazide (VII) (0.47 g) and p-ethoxybenzaldehyde
(0.14 ml); yield 0.54 g (89%); white powder; Rf 0.36;
mp 303–305°ë; UV: 290 (log ε 3.92); IR: 1732, 1660
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FLEKHTER et al.
26.6, 29.6, 30.8, 33.1, 33.7, 34.1, 36.9, 37.5, 37.8, 39.6,
40.7, 42.5, 46.3, 47.3, 50.1, 50.3, 55.0, 55.2 (OCH3),
55.6, 109.5 (C29), 110.9, 120.8, 127.6, 131.8, 132.8 (C
arom.), 142.8 (C1'), 150.5 (C20), 157.9 (C arom.),
174.8 (CONH), and 218.1 (C3). Found, %: C 77.93, H
9.18, N 4.90. Calcd. for C38H54O3N2, %: C 77.76, H
9.29, N 4.77.
N'-(4-Bromobenzal)hydrazide of 3-ketolup-
20(29)-en-28-oic acid (XIV) was synthesized from
hydrazide (VIII) (0.47 g) and p-bromobenzaldehyde
(0.20 g); yield 0.32 g (51%); amorphous cream-colored
(log ε 4.44); IR: 1726, 1675 (CONH), 1650 (C=N–
OH), 1600 (Ar), 1560 (CONH), 1512, 1484, 1460,
1441, 1394, 1330, 1297, 1263, 1205, 1165, 1138, 1090,
1
1042, 992, 920, 886, 818, and 760; H NMR: 0.87,
0.90, 0.94, 0.99, and 1.01 (15 H, 5 s, 5 CH3), 1.20–2.00
(21 H, m, CH2 and CH), 1.70 (3 H, s, CH3), 2.31–2.67
(3 H, m, H13 and H16), 2.85–3.05 (1 H, m, H19), 4.59
and 4.73 (2 × 1 H, br. s, H29), 7.25–7.37 and 7.58–7.70
(5 H, 2 m, H arom.), 8.38 (1 H, br. s, N=CH-Ph), 8.52
(1 H, br. s, CONH), and 9.50 (1 H, br. s, =N–OH); 13C
NMR: 14.0, 15.1, 15.8, 16.2, 18.5, 18.7, 21.1, 22.4,
25.5, 27.0, 29.6, 30.5, 32.2, 33.7, 36.9, 37.1, 37.4, 38.4,
39.5, 40.6, 42.1, 46.3, 50.1, 54.4, 55.0, 55.4, 108.9
(C29), 126.8, 128.4, 128.4, 129.4, 129.4, 134.9 (C
arom.), 145.8 (C1'), 150.8 (C20), 163.9 (C3), and 172.7
(CONH). Found, %: C 77.97, H 9.53, N 7.22. Calcd. for
C37H53O2N3, %: C 77.70, H 9.36, N 7.35.
substance; Rf 0.45; UV: 294 (log ε 4.33); IR: 1726,
1700, 1660 (CONH), 1625 (Ar), 1550 (CONH), 1520,
1488, 1460, 1415, 1392, 1318, 1296, 1260, 1215, 1162,
1
1140, 1098, 1028, 990, 916, 888, 830, and 766; H
NMR: 0.91, 0.94, 0.95, 0.99, and 1.01 (15 H, 5 s, 5
CH3), 1.10–2.00 (21 H, m, CH2 and CH), 1.69 (3 H, s,
CH3), 2.43–2.51 (3 H, m, H13 and H16), 3.00–3.14 (1
H, m, H19), 4.58 and 4.72 (2 × 1 H, br. s, H29), 7.50–
7.62 (2 H, m, H arom.), 7.28–7.40 (2 H, m, H arom.),
7.60 (1 H, d, J 7.8, H arom.), 7.67 (1 H, d, J 7.8, H
arom.), 8.55 (1 H, s, N=CH–Ar), and 9.40 (1 H, br. s,
N'-(3-Iodobenzal)hydrazide of lup-20(29)-en-28-
oic acid 3-oxime (XVII) was obtained from hydrazide
(IX) (0.48 g) and m-iodobenzaldehyde (0.24 g); yield
0.61 g (87%); white powder; Rf 0.36; mp 198–202°ë;
UV: 294 (log ε 4.38); IR: 1680 (CONH), 1660 (C=N–
OH), 1605 (Ar), 1565 (CONH), 1488, 1461, 1392,
1317, 1288, 1244, 1206, 1195, 1139, 1080, 1038, 988,
13
CONH); C NMR: 14.4, 16.2, 16.5, 19.2, 19.7, 20.9,
21.6, 25.5, 26.9, 29.2, 30.5, 33.3, 34.0, 34.3, 37.0, 37.7,
38.5, 39.0, 40.5, 42.5, 46.7, 47.9, 49.9, 50.0, 54.7, 55.3,
109.5 (C29), 124.0 (C-Br), 130.9, 131.0, 131.4, 131.4,
134.6 (C arom.), 142.1 (C1'), 149.9 (C20), 174.3
(CONH), and 218.3 (C3). Found, %: C 69.81, H 7.94,
N 4.28, Br 12.76. Calcd. for C37H51O2N2Br, %: C
69.89, H 8.10, N 4.41, Br 12.57.
1
920, 890, 820, and 768; H NMR: 0.86, 0.88, 0.92,
0.98, and 1.03 (15 H, 5 s, 5 CH3), 1.10–2.00 (22 H, m,
CH2 and CH), 1.70 (3 H, s, CH3), 2.45–2.72 (3 H, m,
H13 and H16), 2.92–3.04 (1 H, m, H19), 4.62 and 4.76
(2 × 1 H, br. s, H29), 7.12 (1 H, t, J 7.4, H arom.), 7.62
(1 H, d, J 7.4, H arom.), 7.70 (1 H, d, J 7.4, H arom.),
8.15 (1 H, br. s, N=CH-Ar), 8.80 (1 H, br. s, CONH),
N'-(3-Iodobenzal)hydrazide of 3-ketolup-20(29)-
en-28-oic acid (XV) was prepared from hydrazide
(VIII) (0.47 g) and m-iodobenzaldehyde (0.24 g); yield
0.39 g (57%); white powder; Rf 0.48; mp 178–180°ë;
13
and 9.55 (1 H, br. s, =N–OH); C NMR: 14.3, 15.6,
15.9, 16.9, 18.9, 19.2, 21.1, 22.7, 25.5, 27.1, 29.5, 30.5,
32.5, 33.8, 37.0, 37.2, 38.5, 39.9, 40.6, 42.2, 46.1, 49.8,
50.1, 55.0, 55.3, 55.5, 94.1 (C1), 109.3 (C29), 126.4,
130.1, 136.0, 136.2, 138.5 (C arom.), 144.7 (C1'), 150.6
(C20), 164.5 (C3), and 173.5 (CONH). Found, %: C
63.81, H 7.46, N 5.92, I 17.85. Calcd. for C37H52O2N3I,
%: C 63.68, H 7.53, N 6.02, I 18.19.
UV: 292 (log ε 4.40); IR: 1727, 1696, 1672, 1660
(CONH), 1610 (Ar), 1540 (CONH), 1514, 1480, 1464,
1418, 1393, 1320, 1300, 1263, 1210, 1158, 1131, 1089,
1
1044, 992, 918, 890, 824, and 766; H NMR: 0.83,
0.85, 0.89, 0.91, and 1.01 (15 H, 5 s, 5 CH3), 1.10–2.00
(21 H, m, CH2 and CH), 1.70 (3 H, s, CH3), 2.54–2.70
(3 H, m, H13 and H16), 3.12–3.23 (1 H, m, H19), 4.61
and 4.75 (2 × 1 H, br. s, H29), 7.20 (1 H, t, J 7.8, H
arom.), 7.28 (1 H, s, H arom.), 7.60 (1 H, d, J 7.8, H
arom.), 7.67 (1 H, d, J 7.8, H arom.), 8.55 (1 H, s,
N'-(2-Nitrobenzal)hydrazide of lup-20(29)-en-
28-oic acid 3-oxime (XVIII) was obtained from
hydrazide (IX) (0.48 g) and o-nitrobenzaldehyde
(0.15 g); yield 0.53 g (88%); white powder; Rf 0.39; mp
13
216–218°ë; UV: 284 (log ε 4.20); IR: 1727, 1680
(CONH), 1655 (C=N–OH), 1610 (Ar), 1555 (CONH),
1480, 1426, 1386, 1372, 1323, 1280, 1255, 1240, 1195,
N=CH–Ar), and 9.40 (1 H, br. s, CONH); C NMR:
14.6, 15.9, 16.3, 19.4, 19.6, 21.0, 21.5, 25.6, 26.6, 29.6,
30.7, 33.1, 33.7, 34.1, 36.9, 37.5, 38.4, 39.6, 40.7, 42.5,
46.2, 47.3, 50.0, 50.1, 54.9, 55.0, 94.0 (C-I), 109.5
(C29), 128.0, 130.4, 136.1, 136.9, 138.8 (C arom.),
145.1 (C1'), 150.9 (C20), 173.3 (CONH), and 218.0
(C3). Found, %: C 64.94, H 7.75, N 3.97, I 18.53.
Calcd. for C37H51O2N2I, %: C 65.08, H 7.54, N 4.10, I
18.59.
1
1128, 1090, 1028, 988, 920, 890, 824, and 756; H
NMR: 0.83, 0.84, 0.89, 0.95, and 1.01 (15 H, 5 s, 5
CH3), 1.20–2.00 (21 H, m, CH2 and CH), 1.70 (3 H, s,
CH3), 2.36–2.52 (3 H, m, H13 and H16), 2.94–3.05 (1
H, m, H19), 4.60 and 4.74 (2 × 1 H, br. s, H29), 7.29–
7.38 (1 H, m, H arom.), 7.71, 8.13, and 8.23 (3 × 1 H,
N'-(Benzal)hydrazide of lup-20(29)-en-28-oic br. s, H arom.), 8.51 (1 H, br. s, N=CH–Ar), 9.45 (1 H,
br. s, CONH), and 9.53 (1 H, br. s, =N–OH); 13C NMR:
13.9, 15.7, 16.3, 18.6, 18.9, 21.1, 22.4, 22.7, 25.5, 27.0,
29.6, 30.5, 32.4, 33.7, 37.0, 37.1, 38.0, 38.4, 39.5, 40.6,
acid 3-oxime (XVI) was obtained from hydrazide (IX)
(0.48 g) and benzaldehyde (0.15 ml); yield 0.52 g
(91%); white powder; Rf 0.42; mp 264–267°ë; UV: 292
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 3 2003

SYNTHESIS AND ANTIVIRAL ACTIVITY OF HYDRAZIDES
301
6. Lee, K.-H., Kashiwada, Y., Hashimoto, F., Cosen-
tino, L.M., and Manak, M., US Patent 5 679 828, 1997.
7. Kashiwada, Y., Nagao, T., Hashimoto, A., Ikeshiro, Y.,
Okabe, H., Cosentino, L.M., and Lee, K.H., J. Nat.
Prod., 2000, vol. 63, pp. 1619–1622.
8. Carlson, R.M., Krasutsky, P.A., and Karim, M.R.U., US
Patent 5 750 578, 1998.
9. Platonov, V.G., Zorina, A.D., Gordon, M.A.,
Chizhov, N.P., Balykina, L.V., Mikhailov, Yu.D.,
Ivanen, D.R., Tran Kim Kvi, and Shavva, A.G., Khim.–
Farm. Zh., 1995, vol. 29, no. 2, pp. 42–46.
10. Kurokawa, M., Basnet, P., Ohsugi, M., Hozumi, T.,
Kadot, S., Namba, T., Kawana, and Shiraki, K.,
J. Pharm. Exp. Ther., 1999, vol. 289, pp. 72–78.
11. Ryu, S.Y., Lee, C.-K., Lee, C.O., Kim, H.S., and
Zee, O.P., Arch. Pharm. Res. (Seoul), 1992, vol. 15,
no. 3, pp. 242–245. Cited by Hwang, S-M., US Patent
6214350, 2001.
12. Pisha, E., Chai, H., Lee, L.-S., Chagwedera, T.E., Farn-
sworth, N.R., Cordel, G.A., Beecher, C.W.W.,
Fong, H.H.S., Kinghorn, A.D., Brown, D.M., Wani,
M.C., Wall, M.E., Hicken, T.J., Das Gupta, T.K., and
Pezzuto, J.M., Nat. Med., 1995, vol. 1, pp. 1046–1051.
13. Fulda, S., Jeremias, L, Steiner, H.H., Pietsch, T., and
Debatin, K.-M., Int. J. Cancer, 1999, vol. 82, pp. 435–
441.
42.2, 46.3, 50.0, 50.1, 55.2, 55.4, 108.9 (C29), 121.0,
123.6, 129.9, 132.5, 137.1 (C arom.), 148.6 (ë–NO2),
144.9 (C1'), 150.8 (C20), 163.9 (C3), and 172.5
(CONH). Found, %: C 74.14, H 8.61, N 9.25. Calcd. for
C37H52O3N4, %: C 73.95, H 8.74, N 9.33.
Antiviral activity towards HSV-1, influenza
A/FVP/Rostock/34 (H7N1) virus, HIV-1 (strain zmb),
and enterovirus ECHO6 was estimated in the experi-
ments with cell cultures. The studies were performed
using the methods of plague reduction in a primary
CEF culture bearing FVP and inhibition of cytopatho-
genic activity of HSV-1 and ECHO6 using human rhab-
domyosarcoma cell culture [21]. Tests for anti-HIV-1
activity were carried out using Molt4/8 cell culture
(MTT test and detection of infectiousness) with regis-
tration of antigen-containing cells by the method of
indirect immunofluorescence [22]). The compounds
under study were preliminarily dissolved in 10% etha-
nol and then in supporting medium to obtain the neces-
sary concentrations. A decrease in the virus titer in the
presence of the compounds under study as compared to
that in control sample was used as a criterion of the
antiviral activity. The value of EC50 and the EC50/MTC
ratio were also calculated. The MTC values of the com-
pounds under study for noninfected cells were deter-
mined after 72-h incubation.
14. 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.
ACKNOWLEDGMENTS
15. 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.
16. Flekhter, O.B., Nigmatullina, L.R., Karachurina, L.T.,
L.R., Baltina, L.A., Zarudii, F.S., Davydova, V.A.,
Galin, F.Z., and Tolstikov, G.A., Khim.–Farm. Zh.,
2000, vol. 34, pp. 17–20.
The work was supported by the Russian Foundation
for Basic Research, project nos. 00-03-81174, 01-03-
33131, and 02-03-81007, and by the Belarussian–Rus-
sian Foundation for Basic Research, project nos. B99R-
031 and B02R-016.
REFERENCES
17. Pezzuto, J.M. and Kim, D.S.H.L., US Patent. 5 804 575,
1998.
1. Mayaux, J.F., Bousseau, A., Pauwels, R., Huet, T.,
Henin, Y., Dereu, N., Evers, M., Soler, F., Poujade, C.,
De Clercq, E., and Le Pecq, J.-B., Proc. Natl. Acad. Sci.
USA, 1994, vol. 91, pp. 3564–3568.
18. Mashkovskii, M.D., Lekarstvennye sredstva (Medicinal
Preparations), Moscow: Meditsyna, 1993, vol. II, p. 612.
19. Flekhter, O.B., Karachurina, L.T., Nigmatullina, L.R.,
Sapozhnikova, T.A., Baltina, L.A., Zarudii, F.S.,
Galin, F.Z., Spirikhin, L.V., Tolstikov, G.A., Plya-
sunova, O.A., and Pokrovskii, A.G., Bioorg. Khim.,
2002, vol. 28, pp. 543–550.
20. Valterova, E., Klinot, J., and Vystrcil, A., Collect. Czech.
Chem. Commun., 1983, vol. 48, pp. 649–661.
2. Hashimoto, F., Kashiwada, Y., Cosentino, L.M.,
Chen, C.H., Garret, P.E., and Lee, K.H., Bioorg. Med.
Chem., 1997, vol. 5, pp. 2133–2143.
3. Fujioka, T., Kashiwada, Y., Kilkuskie, R.E., Cosen-
tino, L.M., Ballas, L.M., Jiang, J.B., Janzen, W.P.,
Chen, L.S., and Lee, K.H., J. Nat. Prod., 1994, vol. 57,
pp. 243–247.
21. Boreko, E.I., Pavlova, N.I., Zaitseva, G.V., and
4. De Clercq, E., Clin. Microbiol. Rev., 1995, vol. 8,
Mikhailopulo, I.A., Vopr. Virusol., 2001, no. 5, pp. 40–
pp. 200–239.
42.
5. Soler, F., Poujade, C., Evers, M., Carry, J-C., Henin, Y., 22. Pauwels, R., Development of New Agents Against the
Bousseau, A., Huet, T., Pauwels, R., De Clercq, E., May-
aux, J.F., Pecq, J.-B.L, and Dereu, N., J. Med. Chem.,
1996, vol. 39, pp. 1069–1083.
Human Immunodeficiency Virus (Evaluation Methods,
Structure–Activity Relationships, and Mechanism of
Action), Leuven: Katolique Univ., 1990.
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