Synthesis of Conjugates of Lupane-Type Pentacyclic Triterpenoids with 2-Aminoethane- and N-Methyl-2-Aminoethanesulfonic Acids. Assessment of in vitro Toxicity

Article abstract of DOI:10.1007/s10600-017-2153-6

Conjugates of betulin and betulinic and betulonic acids with 2-aminoethane- and N-methyl-2-aminoethanesulfonic acids were synthesized for the first time and were interesting as potential biologically active compounds. Experiments in vitro in MDCK cell culture using the MTT assay found that betulin and betulinic-acid derivatives with aminoethanesulfonic acid bound to triterpene C-3 or C-28 through an ester linker were less toxic than the native compounds.

Full text of DOI:10.1007/s10600-017-2153-6

DOI 10.1007/s10600-017-2153-6
Chemistry of Natural Compounds, Vol. 53, No. 5, September, 2017
SYNTHESIS OF CONJUGATES OF LUPANE-TYPE PENTACYCLIC
TRITERPENOIDS WITH 2-AMINOETHANE- AND
N-METHYL-2-AMINOETHANESULFONIC ACIDS.
ASSESSMENT OF in vitro TOXICITY
1*
1
1
N. G. Komissarova, S. N. Dubovitskii, O. V. Shitikova,
1
1
2
E. M. Vyrypaev, L. V. Spirikhin, E. M. Eropkina,
T. G. Lobova, M. Yu. Eropkin, and M. S. Yunusov
2
2
1
Conjugates of betulin and betulinic and betulonic acids with 2-aminoethane- and N-methyl-2-
aminoethanesulfonic acids were synthesized for the first time and were interesting as potential biologically
active compounds. Experiments in vitro in MDCK cell culture using the MTT assay found that betulin and
betulinic-acid derivatives with aminoethanesulfonic acid bound to triterpene C-3 or C-28 through an ester
linker were less toxic than the native compounds.
Keywords: betulin, betulinic acid, betulonic acid, 2-aminoethanesulfonic acid, N-methyl-2-aminoethanesulfonic acid,
toxicity in vitro.
Lupane-type pentacyclic triterpenoids are interesting as platforms for developing new drugs with various biological
activities [1–4]. In continuation of research on the modification of lupane triterpenoids, we developed approached to conjugates
containing aminoalkanesulfonic acids. The endogenous metabolite taurine (2-aminoethanesulfonic acid), its derivatives, and
natural compounds containing taurine possess broad spectra of biological activity [5–7] including antiviral [8–12], antibiotic
[13], and antitumor [14, 15]. Introduction of taurine or its homologs into lupane-type pentacyclic triterpenoids would expand
the spectrum of biologically active compounds in this series and provide new information about the structure–activity (biological)
relationship.
Herein, conjugates containing C-28/C-3 2-aminoethane- or N-methyl-2-aminoethanesulfonic acid (N-methyltaurine)
as the ammonium or sodium salt or the free aminoethanesulfonic acids bound to the triterpene skeleton via an ester linker were
synthesized from betulin (1) chloroacetates, betulonic acid (2) bromoalkyl esters, and betulinic acid (3a) methyl chloroacetates
or benzyl esters. Experiments in vitro assessed the toxicity of the synthesized compounds in MDCK cell culture (canine
kidney cell line).
Starting chloroacetates 4 and 5a,b were obtained from 1 or 3b,c and chloroacetic acid using the carbodiimide method.
Betulonic acid 2-bromoethyl and 3-bromopropyl esters 6a,b were synthesized via O-alkylation of acid 2 with the appropriate
dibromoalkane (DMF, K CO ). Acid 2 was prepared by a modified literature method [16] using sequential oxidation of 1 by
2
3
pyridinium chlorochromate (PCC) (2 h) and treatment of the obtained betulone aldehyde with NaClO –NaH PO in the
2
2
4
presence of 2-methyl-2-butene for 15 min with an aldehyde–NaClO –NaH PO ratio of 1:6:6. The yield of acid 2 after
2
2
4
purification through the potassium salt was 75%. Acid 3a and methyl 3b and benzyl 3c esters were synthesized by the reported
methods [17–19]. N-Methyltaurine was prepared by N-alkylation of methylamine with sodium 2-bromoethanesulfonate in
73% yield [20].
Conjugates of taurine 7 and 8 or N-methyltaurine 9 and 10a,b were synthesized by N-alkylation of the
tetrabutylammonium salt of taurine [21] or N-methyltaurine chloroacetates 4 and 5a or 5b in DMF in the presence of K CO at 95°C.
2
3
1) Ufa Institute of Chemistry, Russian Academy of Sciences, 71 Prosp. Oktyabrya, Ufa, 450054, Russian Federation,
fax: (347) 235 60 66, e-mail: ngkom@anrb.ru; 2) Research Institute of Influenza, Ministry of Health of the Russian Federation,
15/17 Prof. Popova St., St. Petersburg, 197376. Translated from Khimiya Prirodnykh Soedinenii, No. 5, September–October,
2017, pp. 772–778. Original article submitted December 29, 2016.
©
0009-3130/17/5305-0907 2017 Springer Science+Business Media New York
907

29
20
19
13
30
12
21
17
22
11
O
O
25
26
OH
O
R
1
b/c
9
a
28
N
10
15
Cl
3
HO
O
O
27
4
7
5
HO
HO
S
X
1
4
7, 9
23 24
d
O
OH
OR
1
OR
e
a
b/c
O
O
O
O
O
O
HO
Cl
O
3a-c
2
5a,b
8, 10a, b
O
X
O
f
N
R
2
3a
3a
3b
3c
S
h
g
O
O
O
b
Br
N
n
n
O
O
O
O
O
S O
O Na
-
+
6a, b
11a,b
–
+
7: R = H, X = O N Bu ; 9: R = Me, X = OH; 3a: R = H; 3b,5a: R = Me; 3c,5b: R = Bn;
4
–
+
8: R = Me, R = H, X = O N Bu ; 10a: R = R = Me, X = OH; 10b: R = Bn, R = Me, X = OH
1
2
4
1
2
1
2
6a, 11a: n = 1; 6b,11b: n = 2
–
+
a. ClCH CO H, DÑÑ, DMAP, CH Cl ; b. NÍ CH CH SO N Bu , DMF, Ê CO , 95°Ñ, 3 h; c. MeNHCH CH SO H, DMF, Ê CO , 95ꢁC,
2
2
2
2
2
2
2
3
4
2
3
2
2
3
2
3
50 h (for 9), 6 h (for 10à,b); d. 1. PCC, CH Cl , 2 h, 2. NaClO /NaH PO /2-methyl-2-buten, t-BuOH, 15 min; e. NaBH , THF; f. CH N ,
2
2
2
2
4
4
2 2
Et O; g. BnCl, DMF, K CO ; h. BrCH CH Br or BrCH CH CH Br, K CO , DMF, 4 h
2
2
3
2
2
2
2
2
2
3
Betulonic acid derivatives with sodium N-methyl-2-aminoethanesulfonate 11a and 11b were prepared by reacting
bromoalkyl esters 6a and 6b with the sodium salt of N-methyltaurine [22] in the presence of K CO in DMF at 95°C for 50 h.
2
3
The yields of conjugates 7–9, 10a,b, and 11a,b were 64–90%. The structures of these compounds were established
13
13
using PMR and C NMR spectroscopy and mass spectrometry. PMR and C NMR spectra of conjugates 8, 9, 10b, and 11a
were fully assigned using 2D experiments (Table 1).
Toxicity in vitro of 1, 2, 3a, 7–9, 10a, and 11a. Assessment of the toxicity of the compounds was an important step
in preclinical studies of their biological activity. The toxicity of synthesized 7–9, 10a, and 11a was screened in vitro using
MDCK cell culture and the MTT assay. The median cytotoxic dose (CTD , dose leading to the death of 50% of the cells) and
50
minimum toxic dose (MTD, dose causing toxic effects to appear in the cells) were determined. The MTD became an independent
important toxicity characteristic if the CTD value exceeded the upper limit of the tested concentrations. According to this
50
criterion, conjugates 7, 9, and 10a were slightly toxic compounds (Table 2). A criterion reflecting the rate of toxicity increase
with increasing concentration (CTD /MTD) served as a guide for comparing the toxicity of compounds with similar CTD
50
50
values, e.g., 8 (CTD 30 ꢀg/mL) and 11a (CTD 37.5 ꢀg/mL). According to this criterion, 11a (CTD /MTD = 1.3; for 8,
50
50
50
4.8) was the most toxic compound.
The toxicity results for 7–9 and 10a showed that adding aminoethanesulfonic acids through ester linkers to triterpene
C-3 or C-28 reduced considerably the toxicity of the conjugates as compared with native precursors 1 and 3a (Table 2,
compounds 1, 7, 9, 3a, 8, 10a). The exception was betulonic acid derivative 11a, the toxicity of which was the same as acid 2
itself (CTD /MTD = 1.5 and 1.6, respectively). An analysis of the toxicities of betulin and betulinic acid derivatives with the
50
same substituents (pairs 7,8 and 9,10a) (Table 2) or derivatives within each series 7,9 and 8,11a with different substituents
showed that the toxicity depended on both the triterpenoid structure and the fine differences in the substituents.
908

13
TABLE 1. C NMR Spectra of 6a,b–9, 10a,b, and 11a,b (ꢂ, ppm)*
C atom
6à
6b
7
8
9
10à
10b
11à
11b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
39.62
34.13
218.01
47.32
54.96
19.63
33.60
40.67
49.89
36.90
21.41
25.52
38.41
42.46
29.65
31.99
56.54
49.34
46.90
150.32
30.57
36.90
26.61
21.03
15.95
15.80
14.63
175.68
109.75
19.39
39.55
34.06
218.03
47.24
54.87
19.56
33.54
40.56
49.82
36.82
21.36
25.44
38.31
42.39
29.51
31.58
56.49
49.25
47.24
150.28
30.49
37.00
26.44
20.95
15.90
16.76
14.55
175.77
109.67
19.29
38.63
26.96
78.75
38.77
55.20
18.21
34.08
40.78
50.26
37.05
20.70
25.09
37.50
42.60
27.31
29.49
46.31
48.71
47.62
150.10
29.60
34.45
27.94
15.34
16.02
15.92
14.68
62.85
109.74
19.03
50.75
172.36
50.66
45.69
38.34
23.64
81.40
37.78
55.40
18.09
34.17
40.62
50.40
37.03
20.83
25.40
38.17
42.32
29.59
32.09
56.49
49.39
46.93
150.50
30.53
36.90
37.99
15.88
16.09
16.45
14.60
175.50
109.55
19.29
50.87
170.50
50.40
45.70
38.27
27.18
76.79
36.69
54.86
17.99
33.78
41.11
49.77
38.53
20.29
24.74
37.09
42.26
26.62
29.12
46.10
48.22
46.99
149.78
28.96
33.99
28.13
15.68
15.93
15.84
14.53
64.96
110.07
18.79
55.98
167.90
46.55
53.07
40.44
37.57
23.38
79.98
37.28
54.55
17.64
33.59
40.09
49.55
36.53
20.36
24.89
37.57
41.89
29.91
31.37
55.79
48.65
46.56
149.97
31.20
36.07
27.63
15.51
15.74
16.43
14.29
175.56
109.70
18.82
57.88
170.18
49.30
52.35
41.47
51.12
38.21
23.92
80.49
37.81
55.06
18.17
34.12
40.36
50.07
37.04
20.88
25.44
38.05
42.42
29.48
31.89
56.31
49.15
47.08
150.47
30.42
36.62
28.16
15.92
16.30
16.97
14.78
175.27
110.24
19.34
58.41
170.68
49.83
52.88
42.00
40.64
35.26
221.43
47.81
56.11
20.76
34.77
41.89
51.21
38.05
22.60
26.58
39.72
43.64
30.87
31.64
57.87
50.27
49.34
151.82
33.02
37.89
27.18
21.41
16.64
16.47
15.08
177.50
110.37
19.60
40.64
35.10
220.08
47.66
56.08
20.71
34.74
41.84
51.15
38.01
22.61
26.84
39.81
43.61
30.81
31.65
57.90
50.49
**
151.72
33.11
37.80
27.10
21.40
16.55
16.44
15.02
177.62
110.35
19.55
30
OÑ(Î)ÑÍ2
OÑ(Î)ÑÍ2
NCH2CH2SO3
NCH2CH2SO3
NCH3
C(O)OCH3
CH2Ph
Ph
48.55
54.24
42.67
48.70
53.67
42.55
51.20
65.57
128.43
128.50
128.83
136.90
C(O)OCH2CH2
C(O)OCH2CH2
29.18
63.37
56.56
62.66
C(O)OCH2CH2CH2
C(O)OCH2CH2CH2
C(O)OCH2CH2CH2
CH2CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CH2CH3
29.60
32.02
61.48
27.41
55.08
63.41
13.64
19.66
23.94
58.69
13.67
19.68
23.97
58.68
_______
*6à, 6b (75.47 MHz, CDCl ); 7, 8 (125.47 MHz, CDCl , TMS); 9, 10b (125.47 MHz, DMSO-d , TMS); 10à (75.47 MHz,
3
3
6
DMSO-d ), 11à (125.47 MHz, CD OD), 11b (75.47 MHz, CD OD); **C-19 resonance overlapped solvent resonances.
6
3
3
909

TABLE 2. Toxicity Screening of 1, 2, 3a, 7–9, 10a, and 11a
Compound
CTD50/MTD
CTD50,* ꢀg/mL
MTD,** ꢀg/mL
> 200
> 200
> 100
37.5
30
40
10
9.4
50
50
100
25
6.3
6.3
6.3
3.1
–
–
–
1.5
4.8
6.3
1.6
3.0
9
7
10à
11à
8
1
2
3à
______
*Median cytotoxic dose from MTT assay; **minimum toxic dose.
Thus, methods for preparing conjugates of betulin and betulinic and betulonic acids with aminoethanesulfonic acids
that were interesting as potential biologically active compounds were developed. They were also intermediates for synthesizing
various sulfonamide and peptide derivatives of lupane-type pentacyclic triterpenoids. The in vitro experiments provided
seminal information on the effects of aminoethanesulfonic acid substituents on the toxicity of these compounds that was
valuable for planning further syntheses of biologically active compounds based on aminoethanesulfonic acids.
EXPERIMENTAL
13
IR spectra were taken in mineral oil on a Shimadzu Prestige-21 IR spectrophotometer. PMR and C NMR spectra
1
were recorded on a Bruker Avance III pulsed spectrometer at operating frequencies 500.13 MHz for H and 125.47 MHz for
13
1
C using a Z-gradient PABBO probe at 298 K or a Bruker AM-300 spectrometer at operating frequencies 300.13 MHz for H
13
13
and 75.47 MHz for C. Chemical shifts in PMR and C NMR spectra were given in ppm relative to solvent (CDCl )
3
resonances (ꢂ 7.27 and ꢂ 77.1) or TMS internal standard. 2D spectra were recorded using standard multi-pulse sequences
H
C
of the Bruker Avance III spectrometer software. Mass spectra were obtained using electrospray ionization (ESI) or chemical
ionization at atmospheric pressure (APCI) on an LCMS-2010EV liquid chromatograph-mass spectrometer (Shimadzu) (sample
injection via syringe, 0.1 mL/min, MeCN–H O eluent, 95:5) in positive- and negative-ion modes at capillary potential 4.5
2
and –3.5 kV. The APCI interface temperature was 250°C, heater 200°C, vaporizer 230°C. The spray-gas (N ) flow rate was
2
1.5 and 2.5 L/min for ESI andAPCI, respectively. Rotation angles were measured on a PerkinElmer 341C instrument. Column
chromatography used SiO (PTSKh-AF-A, Imid, Krasnodar, Russia). Melting points were determined on a Kofler apparatus.
2
13
Betulin (1) was isolated from birch bark by the literature method [23]. Spectral characteristics (PMR and C NMR) of
betulinic acid 3a and its esters 3b and 3c that were prepared according to the literature [17–19] agreed with those reported.
°
Commercial 2-aminoethanesulfonic acid was used. DMF was stored over MgSO and vacuum distilled under Ar from 4A
4
molecular sieves. CH Cl was distilled from CaH . K CO was calcined before the reactions.
2
2
2
2
3
3-Oxolup-20(29)-en-28-oic Acid (betulonic acid) (2). A solution of 1 (5.00 g, 11.29 mmol) in CH Cl (500 mL)
2
2
was treated with PCC (12.17 g, 56.47 mmol), stirred for 2 h, diluted with Et O (500 mL), stirred for 15 min, and filtered
2
through a layer of Al O . The filtrate was concentrated. The residue was purified by flash chromatography over SiO
2
3
2
13
[C H –methyl-tert-butylether (MTBE), 4:1] to afford betulonic aldehyde (4.20 g, 85%) (PMR and C NMR spectra agreed
6
6
with the literature [16]). A solution of the aldehyde (4.20 g, 9.57 mmol) in t-BuOH (200 mL) was treated with 2-methyl-2-
butene (4 mL, 37.75 mmol) and simultaneously dropwise with NaClO (5.20 g, 57.44 mmol) in H O (22 mL) and NaH PO
2
2
2
4
(6.89 g, 57.44 mmol) in H O (21 mL). The reaction mixture was stirred for 15 min, diluted with H O (200 mL), and extracted
2
2
with CHCl . The organic layer was separated, washed with H O, dried over Na SO , and evaporated. The resulting residue
3
2
2
4
was dissolved in C H (150 mL), treated with KOH (0.58 g, 10.36 mmol, 15% solution in H O), and refluxed for 1 h.
6
6
2
The resulting precipitate of the potassium salt of betulonic acid was filtered off and dissolved in a mixture of MTBE (200 mL)
and HCl (10%, 50 mL). The organic layer was separated, washed with H O, dried over Na SO , and evaporated to afford 2
2
2
4
13
(3.26 g, 75%). The PMR and C NMR spectra agreed with the literature data [16].
3ꢃ-Hydroxylup-20(29)-en-28-yl 2-Chloroacetate (4). A solution of 1 (3.00 g, 6.78 mmol) and Et N (2.83 mL,
3
20.33 mmol) in CH Cl (300 mL) was stirred, treated dropwise with a solution of chloroacetic chloride (1.3 mL, 16.26 mmol)
2
2
in CH Cl (50 mL), stirred at 20°C for 30 min, washed with HCl (10%, 100 mL) and H O, dried over Na SO , and evaporated.
2
2
2
2
4
910

The residue was chromatographed over SiO (C H –MTBE, 8:1) to afford chloroacetate 4 (2.18 g, 63%), mp 152–154ꢁÑ
2
6 6
–1
1
(lit. [24]: 154–156ꢁC). IR spectrum (ꢄ, cm ): 1660, 1740, 3325. Í NMR spectrum (300 MHz, CDCl , ꢂ, ppm, J/Hz): 0.67
3
(1H, d, J = 8.9, H-5), 0.76 (3Í, s, ÑH -24), 0.82 (3Í, s, ÑH -25), 0.96 (3H, s, CH -23), 0.98 (3H, s, CH -26), 1.03 (3H, s,
3
3
3
3
CH -27), 1.68 (3H, s, CH -30), 2.43 (1H, td, J = 10.5, 5.5, H-19), 3.18 (1H, dd, J = 11.0, 5.3, H-3), 3.86 (2H, s, ÎÑ(Î)ÑÍ ), 3.94,
3
3
2
13
4.37 (each 1H, d, J = 11.0, H-28), 4.60, 4.70 (each1H, br.s, H-29) (only characteristic resonances are given). The C NMR
spectrum agreed with the literature data [24].
Preparation of 5a and 5b. A solution of ester 3b or 3c (1.06 mmol) in CH Cl (60 mL) was treated under Ar with
2
2
chloroacetic chloride (2.13 mmol) and DMAP (2.13 mmol), stirred for 5 min, treated with DCC (2.13 mmol), and stirred for 20 min.
The precipitate of N,N-dicyclohexylurea was filtered off. The organic layer was washed with HCl (10%, 20 mL) and H O,
2
dried over Na SO , and evaporated. The residue was purified by flash chromatography over SiO (C H –MTBE, 4:1).
2
4
2
6 6
20
Methyl Ester of 3ꢃ-(2-Chloroacetoxy)lup-20(29)-en-28-oic Acid (5a). Yield 0.54 g (94%), amorph., [ꢅ] +4ꢁ
(c 0.15, CHCl ). IR spectrum (ꢄ, cm ): 1650, 1734. Í NMR spectrum (300 MHz, CDCl , ꢂ, ppm, J/Hz): 0.68 (1Í, d,
D
–1
1
3
3
J = 10.0, Í-5), 0.84 (3H, s, CH -24), 0.85 (3H, s, CH -25), 0.86 (3H, s, ÑH -23), 0.91 (3H, s, ÑÍ -27), 0.95 (3Í, s, ÑÍ -26),
3
3
3
3
3
1.68 (3H, s, ÑÍ -30), 1.87 (2H, m, H-21, 22), 2.19 (1H, m, H-13), 2.22 (1H, m, H-16), 3.00 (1Í, td, J = 11.0, 4.5, Í-19), 3.66
3
13
(3H, s, Ñ(Î)OCH ), 4.05 (2Í, s, ÑÍ Cl), 4.56 (1H, dd, J = 10.0, 6.0, Í-3), 4.60, 4.73 (each 1H, s, H-29). C NMR spectrum
3
2
(75.47 MHz, CDCl , ꢂ, ppm): 14.64 (Ñ-27), 16.13 (C-25), 16.40 (C-26), 15.92 (C-24), 18.10 (C-6), 19.31 (C-30), 20.86
3
(C-11), 23.52 (C-2), 25.41 (C-12), 27.88 (C-23), 29.63 (C-15), 30.55 (C-21), 32.12 (C-16), 34.17 (C-7), 36.92 (C-22), 37.06
(C-10), 37.97 (C-4), 38.18 (C-13), 38.28 (C-1), 40.65 (C-8), 41.23 (CH Cl), 42.37 (C-14), 49.41 (C-19), 51.26 (Ñ(Î)OCH ),
2
3
46.95 (C-18), 50.40 (C-9), 55.35 (C-5), 56.51 (C-17), 83.35 (Ñ-3), 109.62 (C-29), 150.52 (C-20), 167.14 (C=O), 176.64
+
(C-28). Mass spectrum (ESI), m/z 453 [M+H-ClCH CO H] (calcd for C H ClO , 546.5).
2
2
33 51
4
20
Benzyl Ester of 3ꢃ-(2-Chloroacetoxy)lup-20(29)-en-28-oic Acid (5b). Yield 0.61 g (92%), mp 57–59ꢁÑ, [ꢅ] –9ꢁ
D
–1
1
(c 0.22, CHCl ). IR spectrum (ꢄ, cm ): 700, 1650, 1730, 1762. Í NMR spectrum (300 MHz, CDCl , ꢂ, ppm, J/Hz): 0.76
3
3
(3H, s, ÑÍ -24), 0.83 (3H, s, CH -25), 0.84 (3Í, s, ÑÍ -26), 0.85 (3H, s, CH -23), 0.94 (3H, s, CH -27), 1.68 (3H, s,
3
3
3
3
3
ÑÍ -30), 1.88 (2H, m, H-21, 22), 2.18 (1H, td, J = 11.0, 5.4, H-13), 2.28 (1H, d, J = 11.8, H-16), 3.02 (1H, td, J = 10.5, 4.2,
3
H-19), 4.02, 4.05 (each1H, d, J = 18.5, CH Ñl), 4.55 (1H, dd, J = 10.5, 4.3, H-3), 4.60, 4.72 (each 1H, s, H-29), 5.08, 5.18
2
13
(each 1H, d, J = 19.6, CH Ph), 7.36 (5H, m, Ph). C NMR spectrum (75.47 MHz, CDCl , ꢂ, ppm): 14.64 (Ñ-27), 15.83
2
3
(C-25), 16.19 (C-26), 16.43 (C-24), 18.14 (C-6), 19.35 (C-30), 20.90 (C-11), 23.56 (C-2), 25.46 (C-12), 27.94 (C-23), 29.55
(C-15), 30.55 (C-21), 32.10 (C-16), 34.18 (C-7), 36.94 (C-22), 37.07 (C-10), 38.00 (C-4), 38.15 (C-13), 38.31 (C-1), 40.65
(C-8), 41.28 (CH Cl), 42.40 (C-14), 46.96 (C-19), 49.41 (C-18), 50.43 (C-9), 55.37 (C-5), 56.53 (C-17), 65.74 (CH Ph),
2
2
83.35 (C-3), 109.68 (C-29), 128.08, 128.26, 128.50 (Ph), 136.50 (Ph: C-1), 150.55 (C-20), 167.17 (C=O), 175.83 (C-28).
+
Mass spectrum (ESI), m/z 529 [M + H – ClÑH CO H] (calcd for C H ClO , 622).
2
2
39 55
4
Preparation of 6a and 6b. Asuspension of acid 3 (4.40 mmol) and K CO (4.40 mmol) in DMF (18 mL) was treated
2
3
dropwise with 1,2-dibromoethane or 1,3-dibromopropane (4.40 mmol) in DMF (2 mL) and stirred for 4 h. The K CO was
2
3
filtered off. The filtrate was evaporated. The residue was filtered through a layer of Al O . The filtrate was evaporated.
2
3
20
2-Bromoethyl Ester of 3-Oxolup-20(29)-en-28-oic Acid (6a). Yield 1.9 g (77%), amorph., [ꢅ] +4ꢁ (ñ 0.25,
ÑHCl ). IR spectrum (ꢄ, cm ): 1650, 1726. Í NMR spectrum (300 MHz, CDCl , ꢂ, ppm, J/Hz): 0.92 (3H, s, CH -25), 0.97
D
–1
1
3
3
3
(3H, s, CH -26), 0.98 (3H, s, CH -27), 1.02 (3H, s, CH -24), 1.07 (3H, s, CH -23), 1.69 (5H, s, CH -30, m, H-15, H-18), 1.91
3
3
3
3
3
(3H, m, H-1, 21, 22), 2.24 (1H, td, J = 12.0, 4.8, H-13), 2.28 (1H, m, H-16), 2.44 (Í, m, H-2), 3.02 (1Í, td, J = 11.5, 5.3,
H-19), 3.54 (2Í, t, J = 6.0, CH Br), 4.40, 4.42 (each 1Í, dt, J = 12.0, 6.0, C(O)OCH CH ), 4.61, 4.74 (each 1H, s, H-29).
2
2
2
13
+
C NMR spectrum (Table 1). Mass spectrum (APCI), m/z 561 [M + H] (calcd for C H BrO , 560).
32 49
3
20
3-Bromopropyl Ester of 3-Oxolup-20(29)-en-28-oic Acid (6b). Yield 1.9 g (76%), mp 80–83ꢁÑ, [ꢅ] –6ꢁ (c 0.40,
D
–1
1
CHCl ). IR spectrum (ꢄ, cm ): 1630, 1730. Í NMR spectrum (300 MHz, CDCl , ꢂ, ppm, J/Hz): 0.92 (3H, s, CH -25), 0.96
3
3
3
(3H, s, CH -26), 0.97 (3H, s, CH -27), 1.01 (3H, s, CH -24), 1.06 (3H, s, CH -23), 1.68 (5H, s, CH -30, m, H -15, H-18),
3
3
3
3
3
b
1.88 (3H, m, H-1, 21, 22), 2.20 (2H, m, H-13, 16), 2.43 (2Í, m, H-2), 3.00 (1Í, m, H-19), 3.48 (2Í, t, J = 6.5, CH Br), 4.21
2
13
(2Í, m, C(O)OCH CH ), 4.64, 4.73 (each 1H, s, H-29) (only characteristic resonances are given). Table 1 lists the C NMR
2
2
+
spectrum. Mass spectrum (APCI), m/z 575 [M + H] (calcd for C H BrO , 574).
33 51
3
Tetrabutylammonium 2-{2-[3ꢃ-Hydroxylup-20(29)-en-28-yloxy]-2-oxoethylamino}ethanesulfonate (7).
–
+
A solution of NH CH CH SO Bu N (0.16 g, 0.44 mmol) in DMF (4 mL) was treated with K CO (0.06 g, 0.44 mmol),
2
2
2
3
4
2
3
stirred for 10 min, treated dropwise with betulin chloroacetate (4, 0.23 g, 0.44 mmol) in DMF (5 mL), and stirred at 95°C for
3 h. The K CO was filtered off. The filtrate was evaporated. The residue was chromatographed over SiO (C H –MTBE,
2
3
2
6 6
–1
1
4:1; CHCl –MeOH, 10:1) to afford 7 (0.25 g, 68%), amorph. IR spectrum (ꢄ, cm ): 1035, 1192, 1640, 1740, 3378. Í NMR
3
911

spectrum (500 MHz, CDCl , ꢂ, ppm, J/Hz): 0.67 (1Í, d, J = 9.1, H-5), 0.76 (3H, s, CH -24), 0.82 (3H, s, CH -25), 0.96 (6H,
3
3
3
s, CH -23, 27), 1.01 (12H, t, J = 7.3, butyl: CH CH CH CH ), 1.03 (3H, s, CH -26), 1.45 (10H, m, butyl: CH CH CH CH ,
3
2
2
2
3
3
2
2
2
3
2H-2), 1.67 (8H, m, butyl: CH CH CH CH ), 1.69 (3H, s, CH -30), 1.76 (1H, dd, J = 12.2, 8.8, H-22), 1.83 (1H, d, J = 12.4,
2
2
2
3
3
H-16), 1.95 (1H, m, H-21), 2.42 (1H, td, J = 10.5, 5.5, H-19), 3.03 (2H, t, J = 6.3, NCH CH SO ), 3.13 (2H, t, J = 6.3,
2
2
3
NCH CH SO ), 3.18 (1H, dd, J = 11.2, 4.5, H-3), 3.32 (8H, t, J = 6.5, butyl: CH CH CH CH ), 3.48 (2H, s, OC(O)CH ),
2
2
3
2
2
2
3
2
13
3.88, 4.29 (each 1H, d, J = 11.0, H-28), 4.58, 4.68 (1H, s, H-29). Table 1 lists the C NMR spectrum. Mass spectrum (ESI),
m/z 606 [M – N Bu ] (calcd for C H N O S, 848).
+
–
4
50 92 2 6
Tetrabutylammonium 2-{2-[17ꢃ-Methoxycarbonyllup-20(29)-en-3ꢃ-yloxy]-2-oxoethylamino}ethanesulfonate
20
–1
(8) was prepared analogously to 7. Yield 0.30 g (82%), mp 78–80ꢁÑ, [ꢅ] –8.5ꢁ (ñ 0.43, CHCl ). IR spectrum (ꢄ, cm ):
D
3
1
1034, 1166, 1190, 1200, 1640, 1729, 3420. Í NMR spectrum (500 MHz, CDCl , ꢂ, ppm, J/Hz): 0.76 (1Í, d, J = 9.5, Í-5),
3
0.82 (3H, s, CH -25), 0.83 (3H, s, CH -26), 0.91 (3H, s, CH -24), 0.82 (3H, s, CH -23), 0.95 (3H, s, CH -27), 0.96 (1H, m,
3
3
3
3
3
H-1), 1.01 (12H, t, J = 7.3, butyl: CH CH CH CH ), 1.14 (1H, m, H-15), 1.28 (1H, m, H-11), 1.36 (4H, m, 1H-6, 2H-7,
2
2
2
3
1H-21), 1.45 (4H, m, 1H-11, 15, 16, 22), 1.46 (2H, m, H-2), 1.47 (8H, sextet, J = 7.3, butyl: CH CH CH CH ), 1.49 (1H, m,
2
2
2
3
H-6), 1.58 (1H, t, J = 11.3, H-18), 1.65 (9H, m, butyl: CH CH CH CH , H-1), 1.65 (8H, m, butyl: CH CH CH CH ), 1.68
2
2
2
3
2
2
2
3
(3H, s, CH -30), 1.88 (2Í, m, H-21, 22), 2.21 (1Í, td, J = 12.0, 5.5, H-13), 2.23 (1Í, dd, J = 10.0, 3.3, H-16), 2.90 (1H, br.s,
3
NH), 3.00 (1H, m, H-19), 3.01 (2H, t, J = 6.2, NCH CH SO ), 3.12 (2H, t, J = 6.2, NCH CH SO ), 3.31 (8H, t, J = 6.5, butyl:
2
2
3
2
2
3
CH CH CH CH ), 3.45 (2H, s, OC(O)CH ), 3.67 (3H, s, C(O)OCH ), 4.50 (1H, dd, J = 11.0, 5.5, H-3), 4.60, 4.73 (each 1H,
2
2
2
3
2
3
13
+
–
s, H-29). Table 1 lists the C NMR spectrum. Mass spectrum (ESI), m/z 634 [M – N Bu ] (calcd for C H N O S, 876).
4
51 92 2 7
Preparation of 9, 10a, and 10b. General Method. A suspension of N-methyltaurine (0.19 mmol) in DMF (3 mL)
was treated with K CO (0.19 mmol), stirred for 15 min, treated dropwise with chloroacetate 4, 5a, or 5b (0.19 mmol) in DMF
2
3
(2 mL), and stirred at 95°C for 50 h (for 9) or 6 h (for 10a and 10b). The K CO was filtered off. The filtrate was evaporated
2
3
to dryness. The resulting oily residue was triturated with hexane. The resulting solid was filtered off. Solid compound 9 was
rinsed with MTBE and dried in vacuo (KOH, 80°C). Compounds 10a and 10b that were obtained after hexane work-up were
chromatographed over SiO (C H –MTBE, 4:1; CHCl –MeOH, 4:1).
2
6
6
3
2-({2-[3ꢃ-Hydroxylup-20(29)-en-28-yloxy]-2-oxoethyl}(methyl)amino)ethanesulfonic Acid (9). Yield 0.11 g
20
–1
1
(90%), amorph., [ꢅ] +9.5ꢁ (ñ 0.64, CH OH). IR spectrum (ꢄ, cm ): 1038, 1192, 1213, 1637, 1653, 1748, 3420. Í NMR
D
3
spectrum (500 MHz, DMSO-d , ꢂ, ppm, J/Hz): 0.62 (1Í, d, J = 9.8, Í-5), 0.65 (3H, s, CH -24), 0.76 (3H, s, CH -25), 0.83 (1H, m,
6
3
3
H -1), 0.87 (3H, s, CH -23), 0.94 (3H, s, CH -27), 0.99 (5H, s, CH -26, m, H -12, 15), 1.09 (1H, m, H -22), 1.20 (1H, m,
a
3
3
3
a
a
H -11), 1.26 (2Í, m, H-9, H -16), 1.34 (5Í, m, H -6, 21, 2H-7, H -11), 1.44 (3H, m, 2H-2, H -6), 1.54 (1H, m, H -1), 1.57
a
a
a
b
b
b
(1H, m, H-18), 1.60 (2H, m, H-13, H -12), 1.65 (4H, s, CH -30, m, H -15), 1.72 (1H, m, H -22), 1.74 (1H, m, H -16), 1.91
b
3
b
b
b
(1H, m, H -21), 2.48 (1H, m, H-19), 2.66 (3H, s, NCH ), 2.84 (2Í, t, J = 7.5, NCH CH SO ), 2.96 (1Í, dd, J = 10.0, 5.3,
b
3
2
2
3
H-3), 3.21 (2H, m, NCH CH SO ), 3.28 (1Í, d, J = 10.8, H -28), 3.98 (2H, s, OC(O)CH ), 4.37 (1H, d, J = 10.8, H -28), 4.57,
2
2
3
a
2
b
13
–
4.71 (each 1H, s, H-29), 8.73 (1H, br.s, SO H). Table 1 lists the C NMR spectrum. Mass spectrum (ESI), m/z 620 [M – H]
3
(calcd for C H NO S, 621).
35 59
6
2-({2-[17ꢃ-Methoxycarbonyllup-20(29)-en-3ꢃ-yloxy]-2-oxoethyl}(methyl)amino)ethanesulfonicAcid (10a). Yield
20
–1
0.08 g (66%), amorph., [ꢅ] –8ꢁ (ñ 0.65, CH OH). IR spectrum (ꢄ, cm ): 1045, 1174, 1188, 1201, 1640, 1729, 3446.
D
3
1
Í NMR spectrum (300 MHz, DMSO-d , ꢂ, ppm, J/Hz): 0.79 (10H, m, H-5, s, ÑH -23, 24, 25), 0.83 (3H, s, CH -26), 0.94
6
3
3
(3H, s, CH -27), 1.64 (3H, s, CH -30), 2.12 (1H, m, H-16), 2.24 (3H, s, NCH ), 2.63 (2H, m, NCH CH SO ), 2.75 (2H, m,
3
3
3
2
2
3
NCH CH SO ), 2.90 (1H, m, H-19), 3.23 (2H, s, OC(O)CH ), 3.58 (3H, s, C(O)OCH ), 4.40 (1H, dd, J = 10.5, 5.3, H-3),
2
2
3
2
3
13
–
4.56, 4.69 (each 1H, s, H-29). Table 1 lists the C NMR spectrum. Mass spectrum (ESI), m/z 648 [M – H] (calcd for
C H NO S, 649).
36 59
7
2-({2-[17ꢃ-Benzyloxycarbonyllup-20(29)-en-3ꢃ-yloxy]-2-oxoethyl}(methyl)amino)ethanesulfonic Acid (10b).
20
–1
Yield 0.09 g (64%), amorph., [ꢅ] +11ꢁꢆ(c 0.38, ÑH OH). IR spectrum (ꢄ, cm ): 1041, 1129, 1149, 1191, 1231, 1640, 1719,
1724, 3300. Í NMR spectrum (500 MHz, DMSO-d , ꢂ, ppm, J/Hz): 0.69 (3H, s, CH -24), 0.78 (3H, s, CH -25), 0.79 (7H, m,
D
3
1
6
3
3
H-5, s, CH -23, 26), 0.92 (4H, m, H -1, s, CH -27), 0.97 (1H, m, H -12), 1.03 (1H, m, H -15), 1.12 (1H, m, H -11), 1.22 (2H,
3
a
3
a
a
a
m, H -7, H -15), 1.27 (2H, m, H -6, H-9), 1.32 (3H, m, H -7, 11, H -21), 1.42 (2H, m, H -6, H -16), 1.47 (1H, m, H -22), 1.55
a
b
a
b
a
b
a
a
(1H, m, H -2), 1.57 (2H, m, H -2, H-18), 1.60 (2H, m, H -1, 12), 1.64 (3H, s, CH -30), 1.75 (1H, m, H -21), 1.80 (1H, m,
a
b
b
3
b
H -22), 2.11 (1H, td, J = 11.0, 4.0, H-13), 2.17 (1H, d, J = 13.0, H -16), 2.26 (3H, s, NCH ), 2.62 (2H, m, NCH CH SO ), 2.78
b
b
3
2
2
3
(2H, m, NCH CH SO ), 2.94 (1H, td, J = 11.0, 5.5, H-19), 3.22, 3.26 (each 1H, d, J = 17.3, OC(O)CH ), 4.41 (1H, dd,
2
2
3
2
J = 10.5, 5.3, H-3), 4.56, 4.68 (each 1H, s, H-29), 5.08, 5.12 (each 1H, d, J = 12.3, CH Ph), 7.35 (5H, s, Ph). Table 1 lists the
2
13
–
C NMR spectrum. Mass spectrum (ESI), m/z 724 [M – H] (calcd for C H NO S 725).
42 63
7
912

Preparation of 11a and 11b. A suspension of the Na-salt of N-methyltaurine (0.14 g, 0.89 mmol) and K CO
2
3
(0.12 g, 0.89 mmol) in DMF (8 mL) was stirred under Ar for 15 min, treated dropwise with the appropriate bromoalkyl ester
6a or 6b (0.50 g, 0.89 mmol) in DMF (12 mL), and stirred for 50 h at 95°C. The K CO was filtered off. The filtrate was
2
3
evaporated. The oil residue was triturated with hexane. The solution was decanted. The solid was rinsed with MTBE (TLC
monitoring) and dried (KOH, 80°C).
Sodium 2-({2-[3-Oxolup-20(29)-en-17ꢃ-carbonyloxy]ethyl}(methyl)amino)ethanesulfonate (11a). Yield 0.42 g
20
–1
1
(74%), mp 170–172ꢁÑ, [ꢅ] +15.3ꢁ (ñ 0.45, CH OH). IR spectrum (ꢄ, cm ): 1200, 1720, 3420. Í NMR spectrum
D
3
(500 MHz, CD OD, ꢂ, ppm, J/Hz): 0.93 (3H, s, CH -25), 0.98 (3H, s, CH -26), 1.01 (6H, s, CH -24, 27), 1.05 (3H, s, CH -23),
3
3
3
3
3
1.10 (1H, qd, J = 12.5, 5.0, H-12), 1.20 (1H, m, H -15), 1.30 (1H, dt, J = 12.5, 5.0, H -11), 1.40 (1H, m, H-5), 1.46 (10H, m,
a
a
1H-1, 6, 16, 21, 22, 2H-7, H-9, 1H-11, 15), 1.50 (1H, m, H-6), 1.66 (1H, t, J = 12.5, H-18), 1.68 (3H, s, CH -30), 1.73 (1Í, m,
3
H-12), 1.91 (2Í, m, H-1, 22), 2.28 (2H, m, H-16, 21), 2.30 (1Í, m, H-13), 2.33 (3H, s, NCH ), 2.48 (2H, m, H-2), 2.70 (2H,
3
m, C(O)OCH CH )), 2.93 (2Í, m, NCH CH SO ), 3.00 (1Í, m, H-19), 3.03 (2Í, m, NCH CH SO ), 4.18, 4.24 (each 1Í, dt,
2
2
2
2
3
2
2
3
13
J = 11.5, 5.3, C(O)OCH CH ), 4.59, 4.71 (each 1H, s, H-29). Table 1 lists the C NMR spectrum. Mass spectrum (ESI),
m/z 618 [M – Na] (calcd for Ñ H NNaO S, 641).
2
2
–
35 56
6
Sodium 2-({2-[3-Oxolup-20(29)-en-17ꢃ-carbonyloxy]propyl}(methyl)amino)ethanesulfonate (11b). Yield 0.40 g
20
1
(69%), mp 162–164ꢁÑ, [ꢅ] +15.5ꢁ (c 0.54, CH ÎÍ). Í NMR spectrum (300 MHz, CD OD, ꢂ, ppm, J/Hz): 0.95 (3H, s,
D
3
3
CH -25), 0.99 (3H, s, CH -26), 1.02 (3H, s, CH -24), 1.03 (3H, s, CH -27), 1.06 (3H, s, CH -23), 1.70 (5H, s, CH -30, m,
3
3
3
3
3
3
1H-12, H-18), 1.88 (4Í, m, 1H-1, 22, C(O)OCH CH CH ), 2.29 (3Í, m, H-13, 1H-16, 21), 2.38 (3H, s, NCH ), 2.51 (4Í, m,
2
2
2
3
2H-2, C(O)OCH CH CH ), 2.92 (2Í, m, NCH CH SO ), 3.00 (3Í, m, NCH CH SO , H-19), 4.12 (2Í, m, C(O)OCH CH CH ),
2
2
2
2
2
3
2
2
3
2
2
2
13
4.61, 4.73 (each 1H, s, H-29) (only characteristic resonances are given). Table 1 lists the C NMR spectrum. Mass spectrum
(ESI), m/z 632 [M – Na] (calcd for Ñ H NNaO S, 655).
–
36 58
6
Screening for Toxicity in vitro. The toxicity of the compounds was studied using a continuous culture of Madin-
Darby canine kidney cells (MDCK) that was obtained initially from the Centers for Disease Control and Prevention (CDC,
Atlanta, USA). Toxicity in vitro was assessed using the MTT assay, i.e., reduction of the dye thiazolyl blue tetrazolium
bromide (MTT) by cells in culture. The experiments with the cell culture were conducted as follows. A weighed portion
(5 mg) of compound in a sterile tube was diluted with DMSO to a concentration of 1 mg/mL (stock solution). Then, six
sequential two-fold dilutions (100, 50, 25, 12.5, 6, and 3 ꢀg/mL) with MDCK growth medium (Biolot, St. Petersburg) produced
the solutions used to determine the toxicity. The test was performed in triplicate for each concentration. A 1-d culture of
MDCK cells grown in 96-well plates was inspected visually using an inverted microscope for the integrity of the monolayer.
The plates were rinsed twice with medium without serum. The test compound (100 ꢀL per well) of the appropriate concentration
was placed into each well. The plates were incubated for 72 h at 37°C with 5% CO . Test results were recorded visually by
2
evaluating the integrity of the cell monolayer as compared with a control.
The MTT assay used 96-well plates and the standard protocol [25]. Optical density was recorded at 550 nm on
a Varioscan microplate reader (Thermo Fisher). The median cytotoxic dose (CTD ) was determined by linear regression of
50
2
the photometric data using the Excel 2010 program. The regression equations were valid because R > 0.9.
ACKNOWLEDGMENT
The work was supported financially by a grant from the Russian Science Foundation (Project No. 14-13-01307).
REFERENCES
1.
2.
3.
4.
P. A. Krasutsky, Nat. Prod. Rep., 23, 919 (2006).
R. Csuk, Expert Opin. Ther. Pat., 24 (8), 913 (2014).
P. Yogeeswari and D. Sriram, Curr. Med. Chem., 12 (6), 657 (2005).
H. Wang, R. Xu, Y. Shi, L. Si, P. Jiao, Z. Fan, X. Han, X. Wu, X. Zhou, F. Yu, Y. Zhang, D. Zhou, and S. Xiao,
Eur. J. Med. Chem., 110, 376 (2016).
5.
6.
H. Ripps and W. Shen, Mol. Vision, 18, 2673 (2012).
N. Chen and J. Xu, Tetrahedron, 68 (11), 2513 (2012).
913

7.
8.
9.
10.
11.
R. C. Gupta, T. Win, and S. Bittner, Curr. Med. Chem., 12 (17), 2021 (2005).
A. A. Shtro, A. V. Slita, L. A. Karpinskaya, A. V. Galochkina, and V. V. Zarubaev, Lechashchii Vrach, 10, 52 (2012).
S. Q. Yang, M. Froeyen, E. Lescrinier, P. Marliere, and P. Herdewijn, Org. Biomol. Chem., 9 (1), 111 (2011).
M. G. Mutchnick, M. N. Ehrinpreis, J. L. Kinzie, and R. R. Peleman, Antiviral Res., 24 (2–3), 245 (1994).
J. Yoon, A. Jekle, R. Najafi, F. Ruado, M. Zuck, B. Khosrovi, B. Memarzadeh, D. Debabov, L. Wang, and M. Anderson,
Antiviral Res., 92 (3), 470 (2011).
12.
T. P. Shiau, E. Low, B. Kim, E. D. Turtle, C. Francavilla, D. J. R. O’Mahony, L. Friedman, L. D’Lima, A. Jekle,
D. Debabov, M. Zuck, N. J. Alvarez, M. Anderson, R. Najafi, and R. K. Jain, Bioorg. Med. Chem. Lett.,
23 (20), 5650 (2013).
13.
14.
F. Kong, N. Zhao, M. M. Siegel, K. Janota, J. S. Ashcroft, F. E. Koehn, D. B. Borders, and G. T. Carter,
J. Am. Chem. Soc., 120 (51), 13301 (1998).
Y. Takahashi, J.-R. Nakijiquinones, M. Ushio, T. Kubota, S. Yamamoto, J. Fromont, and J. Kobayashi, J. Nat. Prod.,
73 (3), 467 (2010).
15.
16.
17.
18.
A. Aiello, E. Fattorusso, P. Luciano, A. Macho, M. Menna, and E. Munoz, J. Med. Chem., 48 (9), 3410 (2005).
A. Barthel, S. Stark, and R. Csuk, Tetrahedron, 64 (39), 9225 (2008).
D. S. H. L. Kim, Z. Chen, T. Nguyen, J. M. Pezzuto, S. Qiu, and Z.-Z. Lu, Synth. Commun., 27 (9), 1607 (1997).
N. V. Uzenkova, N. I. Petrenko, M. M. Shakirova, E. E. Shul ts, and G. A. Tolstikov, Chem. Nat. Compd.,
41, 692 (2005).
19.
20.
21.
22.
23.
X. Y. Wang, S. Y. Zhang, J. Li, H. N. Liu, X. Xie, and F. J. Nan, Acta Pharm. Sin., 35, 1463 (2014).
J. W. Schick and E. F. Degering, Ind. Eng. Chem., 39 (7), 906 (1947).
D. G. I. Kingston and Z.-Y. Zhao, EP Pat. 0473326 A1, Mar. 4, 1992.
X. Fang, J. Ling, F. Liu, and Y. Chen, CN Pat. 102675160A, Sept. 19, 2012.
M. S. Yunusov, N. G. Komissarova, and N. G. Belenkova, RU Pat. No. 2,270,201, Feb. 20, 2006; Byull. Izobret.,
No. 5, 1 (2006).
24.
25.
H. Kommera, G. N. Kaluderovic, J. Kalbitz, and R. Paschke, Arch. Pharm. Chem. Life Sci., 343 (8), 449 (2010).
E. Borenfreund, H. Babich, and N. Martin-Alguacil, Toxicol. in Vitro, 2 (1), 1 (1988).
914