Synthesis and antiviralactivity of 2,3-seco-derivatives of betulonic acid

Article abstract of DOI:10.1007/s10600-009-9436-5

A lupane 2,3-seco-aldehydoacid that was oxidized further to the 2,3-seco-diacid was synthesized by Beckmann fragmentation of the α-hydroxyoxime of betulonic acid. It was found that both 2,3-seco-derivatives were capable of suppresing reproduction of Herpes type 1 and flu A viruses (EC₅₀ from 1.9 to 21.3 μM).

Full text of DOI:10.1007/s10600-009-9436-5

Chemistry of Natural Compounds, Vol. 45, No. 5, 2009
SYNTHESIS ANDANTIVIRALACTIVITY OF
2
,3-seco-DERIVATIVES OF BETULONIC ACID
I. A. Tolmacheva, V. V. Grishko, E. I. Boreko,2
1
1*
UDC 547.597:542.952.3+578:615.281.8
2
2
O. V. Savinova, and N. I. Pavlova
A lupane 2,3-seco-aldehydoacid that was oxidized further to the 2,3-seco-diacid was synthesized by Beckmann
fragmentation of the α-hydroxyoxime of betulonic acid. It was found that both 2,3-seco-derivatives were
capable of suppresing reproduction of Herpes type 1 and flu A viruses (EC50 from 1.9 to 21.3 μM).
Key words: 2,3-seco-triterpenoids, betulonic acid, Beckmann fragmentation, antiviral activity.
Lupane triterpenoids of plant origin such as betulin and betulinic and betulonic acids exhibit a variety of biological
activity and are interesting as starting materials for chemical and biocatalytic transformations [1, 2]. Semi-synthetic lupane
derivatives include compounds with high antitumor and antiviral activity [1–3]. We have previously proposed a method for
preparing cytotoxic 2,3-seco-derivatives of the lupane and oleanane type that included Beckmann cleavage of α-hydroxyoximes
based on the methyl ester of betulonic acid and allobetulone [4, 5]. Herein we present an analogous approach that was used to
synthesize lupane derivatives with an opened ring A and a free C-28 carboxylic acid.
The starting material for the synthesis of the 2,3-seco-derivatives was betulonic acid (1). During the process
(
Scheme 1) the corresponding hydroxyiminoketone 2 and hydroxyiminoalcohol 3 were prepared. The hydroxyimine substituent
–
1
on C-2 in 2 and 3 was indicated by absorption bands in the IR spectra at 1644 cm and 3248–3400. PMR spectra of 2 and 3
recorded in DMSO-d solutions had a resonance for the hydroxyimino proton at 12.17 ppm (2) or 10.59 (3).
6
COOH
HON
O
HON
HO
a
b
c
1
3
O
1
2
3
COOH
d
NC
HOC
NC
HOOC
4
5
a. i-C H NO /t-C H OH/t-C H OK; b. NaBH /CH OH; c. TsCl/C H N; d. Cr O /H SO /(CH ) CO
6
11
2
4
9
4
9
4
3
5
5
2
3
2
4
3 2
Scheme 1
1
) Institute of Technical Chemistry, Ural Branch, Russian Academy of Sciences, 614013, Russia, Perm′, ul. Akad.
Koroleva, 3, e-mail: grishko@aport.ru; 2) Research Institute of Epidemiology and Microbiology, Ministry of Health, Republic
of Belarus, 220114, Belarus, Minsk, ul. Filimonova, 23, e-mail: bei@mail.by. Translated from Khimiya Prirodnykh Soedinenii,
No. 5, pp. 566–568, September–October 2009. Original article submitted May 25, 2009.
0
009-3130/09/4505-0673 ©2009 Springer Science+Business Media, Inc.
673

TABLE 1. Antiviral Properties of Compounds 4 and 5
Herpes virus
Flu virus
Compound
No.
MTC (RD/CEF),
ratio
MTC/EC50
MTC/EC90
ratio
MTC/EC50
MTC/EC90
μM
EC50 (I95)*
EC90(I95), μM
EC50 (I95)
EC90(I95), μM
1
.9 (2911.5÷0.001)
14.0
1.5
7.7 (8.8÷6.6)
12.8 (15.0÷11.1)
3.5
2.1
4
5
26.7/26.7
17.9 (28269÷0.01)
2
8
1.3 (186.8÷2.5)
1.8 (716.9÷9.3)
9.7
2.5
63.8 (72.1÷56.4)
101.0 (114.3÷89.3)
1.6
1.0
206.6/103.3
_
*
_____
I95 is the confidence interval.
The structure of 2,3-seco-aldehydonitrile 4 was confirmed by spectral data. The IR spectrum of 4 contained a band
at 2248 cm–1 that corresponded to nitrile vibrations. The PMR spectrum showed the C-3 aldehyde proton as a singlet
1
3
with chemical shift 9.67 ppm. The C NMR spectrum had characteristic resonances for nitrile (117.99 ppm) and aldehyde
206.10 ppm) C atoms. Oxidation of 4 produced diacid 5, the structure of which was confirmed by IR and NMR spectroscopy.
(
The nature of the C-1 methylene resonances in 4 and 5, as described earlier for 2,3-seco-derivatives [4], depended on the
nature of the C-3 substituent. For the aldehyde (4), the C-1 protons resonated as two doublets at 2.23 and 2.59 ppm; for the
carboxylic acid (5), as a singlet at 2.55 ppm.
The antiviral activity of 4 and 5 was studied against flu A virus and Herpes Simplex virus type 1 (Table 1). It was
found that 4 and 5 were capable of suppressing reproduction of Herpes virus type 1 (EC50 = 1.9 and 21.3 μM; ratio
MTC/EC50 = 14.0 and 9.7, respectively). Also, 5 was slightly active against fluAvirus (MTC/EC50 = 1.6) whereas 4 exhibited
moderate antiviral activity (EC50 = 7.7 μM, MTC/EC50 = 3.5).
EXPERIMENTAL
PMR and 13C NMR spectra were recorded in CDCl or DMSO-d solutions on a Varian Mercury+ spectrometer
3
6
(
USA) at operating frequency 300 or 75.5 MHz with HMDS internal standard. IR spectra were recorded in mineral oil on a
Specord M80 spectrophotometer (Germany). Melting points were measured on a PTP instrument for determining melting
points (Russia). Specific optical rotation was determined for CHCl or CHCl :EtOH (4:1) solutions on a Perkin–Elmer
3
3
Model 341 polarimeter (USA) at 589 nm. Elemental analyses (C, H, N) were perfomed using a Leco CHNS-9321P elemental
analyzer (Netherlands) and agreed with those calculated.
TLC was carried out on Sorbfil plates (Russia) using solvents selected individually for each compound. Compounds
were detected using phosphomolybdic acid in EtOH (20%) and heating at 100–120°C for 2–3 min. Column chromatography
was performed over silica gel (Merck, 60–200 μm) with a compound:sorbent ratio of about 1:50. The eluent was selected
individually for each compound. Solvents were purified and dried by literature methods [6]. Betulonic acid [7] was prepared
by oxidation of betulin using Jones reagent [8].
2
-Hydroxyimino-3-oxolup-20(29)-en-28-oicAcid (2). The literature synthesis was used [4]. Purification by column
chromatography (eluent CHCl :EtOAc, 10:1) afforded 2 (2.1 g, 70%), R 0.42 (CHCl :MeOH, 20:1), mp 169–171°C (EtOH),
3
f
3
2
1
[
α] +95.4° (c 0.4, CHCl ), C H NO .
D
3 30 47 4
–
1
IR spectrum (ν, cm ): 3400 (OH), 1710 (COOH), 1698 (C=O), 1644 (C=N).
PMR spectrum (300 MHz, CDCl , δ, ppm, J/Hz): 0.82, 0.96, 0.99, 1.10, 1.15 (5 × 3H, 5s, 5CH ), 1.69 (3H, s,
3
3
CH -30), 2.09, 3.00 (2H, 2d, J = 18.8, 2H-1, AB-system), 3.01 (1H, m, H-19), 4.61, 4.74 (2H, 2s, 2H-29). PMR spectrum
3
AB
(
300 MHz, DMSO-d , δ, ppm): 12.17 (1H, s, NOH).
6
1
3
C NMR spectrum (75.5 MHz, CDCl , δ, ppm): 14.53, 15.30, 16.91, 19.32, 20.21, 21.64, 25.49, 29.03, 29.67,
3
3
1
0.49, 32.04, 32.84, 35.63, 37.03, 38.37, 38.83, 40.40, 41.81, 42.49, 45.86, 46.80, 48.31, 49.09, 52.31, 56.30, 109.84 (C-29),
50.27 (C-20), 153.47 (C-2), 181.76 (C-28), 203.21 (C-3).
674

3
β-Hydroxy-2-hydroxyiminolup-20(29)-en-28-oic Acid (3). The literature synthesis was used [4]. Purification
using column chromatography (eluent CHCl :EtOAc, 10:1) afforded 3 (1.16 g, 64%), R 0.3 (CHCl :MeOH, 20:1), mp 240–
3
f
3
2
1
2
42°C (CHCl :EtOAc), [α] +23.9° (c 0.4, CHCl :EtOH, 4:1), C H NO .
3 3 30 47 4
D
–
1
IR spectrum (ν, cm ): 3380, br. 3248 (OH), 1690 (COOH), 1644 (C=N).
PMR spectrum (300 MHz, CDCl , δ, ppm, J/Hz): 0.70, 0.74, 0.90, 0.98, 1.09 (5 × 3H, 5s, 5CH ), 1.68 (3H, s,
3
3
CH -30), 2.20, 3.40 (2H, 2d, J = 12.9, 2H-1, AB-system), 3.00 (1H, m, H-19), 3.79 (1H, s, H-3), 4.59, 4.73 (2H, 2s, 2H-29).
3
AB
PMR spectrum (300 MHz, DMSO-d , δ, ppm): 10.59 (1H, s, NOH).
C NMR spectrum (75.5 MHz, CDCl , δ, ppm): 14.69, 15.57, 15.61, 16.55, 18.22, 19.35, 21.23, 25.36, 28.35,
6
1
3
3
2
1
9.70, 30.56, 32.12, 34.00, 36.93, 38.01, 38.21, 40.99, 41.18, 42.45, 42.94, 46.90, 49.46, 49.87, 54.92, 56.55, 78.53 (C-3),
09.68 (C-29), 150.43 (C-20), 158.43 (C-2), 176.71 (C-28).
2
,3-seco-1-Cyanolup-20(29)-en-3-al-28-oic Acid (4). The literature synthesis was used [4]. Purification using
column chromatography (eluent hexane:EtOAc, 5:1) afforded 4 (0.82 g, 57%), R 0.46 (hexane:EtOAc, 7:3), mp 169–171°C
f
1
–1
(
hexane:EtOAc), [α]2 +30.6° (c 0.5, CHCl ), C H NO . IR spectrum (ν, cm ): 2248 (C≡N), 1722 (COOH), 1688 (CHO).
D
3 30 45 3
PMR spectrum (300 MHz, CDCl , δ, ppm, J/Hz): 0.89, 0.94, 1.04, 1.08, 1.14 (5 × 3H, 5s, 5CH ), 1.68 (3H, s,
3
3
CH -30), 2.23, 2.59 (2H, 2d, JAB = 18.3, 2H-1, AB-system), 3.00 (1H, td, J = 10.5, 5.7, H-19), 4.61, 4.72 (2H, 2s, 2H-29), 9.67
3
(
1H, s, H-3).
1
3
C NMR spectrum (75.5 MHz, CDCl , δ, ppm): 14.59, 15.72, 18.74, 19.17, 19.50, 20.08, 21.78, 22.78, 23.50,
3
2
1
5.37, 29.63, 30.40, 31.87, 33.19, 36.93, 38.37, 40.57, 42.21, 42.83, 44.52, 46.83, 48.93, 49.05, 50.75, 56.33, 110.00 (C-29),
17.99 (C-2), 150.00 (C-20), 182.08 (C-28), 206.10 (C-3).
2
,3-seco-1-Cyanolup-20(29)-en-3,28-dioicAcid (5). The literature synthesis was used [4]. Purification using column
chromatography (eluent CHCl :EtOAc, 10:1) afforded 5 (0.43 g, 62%), R 0.2 (CHCl :EtOAc, 10:1), mp 72–74°C
3
f
3
1
(
hexane:EtOAc), [α]2 +18.1° (c 0.2, CHCl ), C H NO .
D
3 30 45 4
–
1
IR spectrum (ν, cm ): 2244 (C≡N), 1710, 1690 (COOH), .
PMR spectrum (300 MHz, CDCl , δ, ppm, J/Hz): 0.92, 0.98, 1.03, 1.27, 1.35 (5 × 3H, 5s, 5CH ), 1.68 (3H, s,
3
3
CH -30), 2.55 (2H, s, 2H-1), 3.00 (1H, m, H-19), 4.61, 4.72 (2H, 2s, CH -29).
3
2
1
3
C NMR spectrum (75.5 MHz, CDCl , δ, ppm): 14.59, 15.84, 18.35, 19.22, 21.01, 21.68, 25.18, 25.31, 26.95,
3
2
1
9.15, 29.73, 30.40, 31.93, 33.24, 36.80, 38.32, 40.51, 42.33, 42.73, 44.89, 46.45, 46.87, 48.95, 50.94, 56.46, 109.94 (C-29),
18.29 (C-2), 150.06 (C-20), 182.84 (C-28), 184.49 (C-3).
Antiviral Properties of 4 and 5. Antiviral activity was determined using cell cultures with Herpes Simplex virus
type 1 (HSV, strain 1C) and flu A/FPV/Rostock/34 (H7N1). Compounds were dissolved beforehand in EtOH (10%, stock
solution concentration 5 mg/mL) and then in medium to the required concentrations. Antiviral activity was studied by estimating
the cytopathic effect on a transfer culture of human rhabdomyosarcoma (RD) cells with HSV-1 and by the reduction of plaque
in cell culture of primary chicken embryo fibroblasts (CEF) with FPV [9]. The criterion for antiviral activity was a decrease
of virus titre in the presence of the tested compounds compared with a control. The concentrations of compounds suppressing
virus multiplication by 50 and 90% (mean-effective concentration, EC , and concentration for 90% reduction of virus titre,
5
0
EC ) were determined using the Fung computer program [10] based on probit analysis and weighted linear regression. The
9
0
ratios of maximum tolerated concentration (MTC) of the compounds to EC50 and EC90 were also calculated. MTC of the
compounds were determined from the maximum concentration of a compound that did not affect the morphology of uninfected
cell culture after incubation for 72 h.
ACKNOWLEDGMENT
The work was supported financially by RFBR Grant No. 08-03-00265-a.
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