Synthesis and anti-HIV-1 activity of new conjugates of 18β- and 18α-glycyrrhizic acids with aspartic acid esters

Article abstract of DOI:10.1007/s10600-012-0217-1

New conjugates of 18β- and 18α-glycyrrhizic acids (GAs) each containing two di- or α-methyl esters of L-aspartic acid in the carbohydrate part of the glycosides were synthesized by the activated ester method using the N-hydroxysuccinimide (HOSu) and N,N'-dicyclohexylcarbodiimide. It was found that the conjugate of 18β-GA with Asp(OMe)(OMe) (4) at a concentration of 250 μg/mL inhibited effectively RT of HIV-1 and the accumulation of virus antigen p24 in MT-4 cell culture (95-97%) and protected cells from the cytopathogenic action of the virus.

Full text of DOI:10.1007/s10600-012-0217-1

Chemistry of Natural Compounds, Vol. 48, No. 2, May, 2012 [Russian original No. 2, March-April, 2012]
SYNTHESIS AND ANTI-HIV-1 ACTIVITY OF NEW
CONJUGATES OF 18ꢀ- AND 18ꢁ-GLYCYRRHIZIC
ACIDS WITH ASPARTIC ACID ESTERS
1
1
1*
L. A. Baltina, Jr., E. S. Chistoedova, L. A. Baltina,
UDC 547.598.458.22+616.98:578.828.6
2
3
R. M. Kondratenko, and O. A. Plyasunova
New conjugates of 18ꢀ- and 18ꢁ-glycyrrhizic acids (GAs) each containing two di- or ꢁ-methyl esters of
L-aspartic acid in the carbohydrate part of the glycosides were synthesized by the activated ester method
using the N-hydroxysuccinimide (HOSu) and N,Nꢂ-dicyclohexylcarbodiimide. It was found that the conjugate
of 18ꢀ-GA with Asp(OMe)(OMe) (4) at a concentration of 250 ꢃg/mL inhibited effectively RT of HIV-1 and
the accumulation of virus antigen p24 in MT-4 cell culture (95–97%) and protected cells from the
cytopathogenic action of the virus.
Keywords: glycyrrhizic acid, conjugates, aspartic acid, anti-HIV-1 activity.
The search for novel immunostimulators and antiviral agents with a new mechanism of action among natural compounds
obtained from renewable plant resources and their modified forms is a burgeoning area of modern bioorganic and medicinal
chemistry. This is explained by the broad distribution of HIV-infections, viral hepatitises B and C, and the emergence of new
viral respiratory infections.
2
9
COOH
COOH
H
2
19
H
1
1
8
O
11
9
22
28
O
1
7
2
5
1
1
5
8
3
5
6
'
27
COOH
COR
1
O
6
a, b
O
+ RNH . HCl
2
O
O
O
2
3
24
4
'
OH
1'
OH
HO
''COOH
HO
COR
6
O
1
O
O
O
OH
1''
OH
3: R
1
=
O
N
O
HO 3''
HO
OH
1, 2
OH
3 - 9
H
H
= Asp(OBu t )(OMe)
: R = Asp(OH)(OMe); 4, 5, 6: 18-ꢀH
1
2
: 18
: 18
4: R
1
= Asp(OMe)(OMe); 5: R
1
6
7
9
1
: R
1
: R
1
= Asp(OMe)(OMe); 8: R
= Asp(OH)(OMe); 7, 8, 9: 18-ꢁH
1
= Asp(OBu t )(OMe)
a. HOSu/DCC, NEM or Et N; b. CF COOH/CH Cl
3
3
2
2
Scheme 1
1
) Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences, 450054, Ufa, fax: +7 (347)
35 60 66, e-mail: baltina@anrb.ru; 2) Bashkir State Medical University, 450000, Ufa, Ul. Lenina, 3, Russia; 3) State Scientific
Center Vector, Kolꢂtsovo, Novosibirsk Oblast, Russia. Translated from Khimiya Prirodnykh Soedinenii, No. 2, March–April,
2
2
012, pp. 235–238. Original article submitted July 29, 2011.
262
0009-3130/12/4802-0262 ©2012 Springer Science+Business Media, Inc.

TABLE 1. Effect of Conjugate 4 on Reproduction of HIV-1 in MT-4 Cell Culture
Inhibition, %
Concentration,
Amount of living
cells, %
Drug
ꢃg/mL
virus reverse transcriptase activity
accumulation of virus antigen
AZT
8ꢀ-GA
8ꢀ-GA
4
10
6.00 ꢅ 2.00
18.20 ꢅ 9.00
–
62.91 ꢅ 5.00
72.53 ꢅ 0.21
98.08 ꢅ 0.11
98.30 ꢅ 0.20
96.90 ꢅ 0.20
97.27 ꢅ 2.68
95.31 ꢅ 3.49
94.17 ꢅ 0.32
98.30 ꢅ 0.82
93.90 ꢅ 0.86
97.08 ꢅ 0.06
95.88 ꢅ 0.19
1
1
500
250
500
250
4
_
_____
Amount of living cells in control: uninfected MT-4 cells, 38.49 ꢅ 3.68%; MT-4 cells infected with HIV-1, 35.49 ꢅ 2.35%.
1
8ꢀ-Glycyrrhizic acid (18ꢀ-GA) (1) and its aglycon 18ꢀ-glycyrrhetic acid (18ꢀ-GLA) are the principal bioactive
components of licorice roots (Glycyrrhiza glabra L. and G. uralensis Fisher) and some of the leading natural compounds of
great value to medicine as scaffolds (bases) for creating new highly effective drugs for treating and preventing viral infections
[
1]. A unique property of GA is a new mechanism of action on HIV. Licorice glycoside is active at early stages of the virus
replication cycle and prevents adsorption of the virus to the cell [2].
Triterpene glycopeptides or conjugates of 18ꢀ-GAwith amino acids and dipeptides are especially interesting derivatives.
We found among them promising immunomodulators, inhibitors of Epstein–Barr virus and SARS-associated coronaviruses,
and anti-HIV-1 agents [3-7].
We synthesized new conjugates of 18ꢀ-GA (1) and its stereoisomer 18ꢁ-GA (2) with esters of L-aspartic acid (Asp)
(
4–9) (Scheme 1) in order to expand the number of N-containing GA derivatives and to study the structure–activity relationship.
These are interesting as inhibitors of aspartate-proteases, which hydrolyze polypeptide substrates of HIV-1 protease.
The amino-acid moieties were introduced into the carbohydrate part of the glycosides as the dimethyl or mixed
ꢁ
-methyl ꢀ-tert-butyl esters of aspartic acid. The COOH of GA was activated using N-hydroxysuccinimide (HOSu) and
N,Nꢂ-dicyclohexylcarbodiimide (DCC) at 0–5°C in THF, dioxane, or DMF using the activated ester method that was described
earlier [4]. The ratio of reagents was GA:HOSu:DCC = 1:5–5.5:2.3–2.8 mmol (Scheme 1). The precipitate of
N,Nꢂ-dicyclohexylurea that was formed during the course of the reaction was filtered off. The resulting activated
hydroxysuccinimide ester (3) was used (in solution) without isolation in the reaction with 2.5–3.0 eq. of L-Asp(OMe)–OMe·HCl
or L-Asp(O-t-Bu)–OMe·HCl at 20–22°C in the presence of an excess (1–2 mmol) of Et N or N-ethylmorpholine (NEM). An
3
excess of the tertiary base had to be used in the reaction for temporary protection of the sterically hindered 30-COOH of the
aglycon. The formation of the CONH bond in activated ester 3 was a bimolecular nucleophilic substitution reaction that
formed the desired product as a result of elimination of the OSu group. The resulting HOSu remained in the aqueous solution
during isolation of the reaction products whereas the target carboxy-substituted GA conjugates were insoluble in H O and
2
precipitated.
Analytically pure samples of carboxy-substituted conjugates 4 and 7 were isolated by column chromatography (CC)
over silica gel (SG) upon elution by a CHCl :MeOH:H O mixture in yields of 52.7 and 49.6%, respectively. The conjugates
3
2
with mixed esters Asp(O-t-Bu)(OMe) 5 and 8 were treated without preliminary chromatographic purification with CF COOH
3
at 20–22°C and chromatographed over SG to afford 6 and 9, each containing two ꢁ-methyl esters of aspartic acid in the
carbohydrate part of the GA molecule, in yields of 48.8 and 44.6%, respectively.
The structures of the prepared compounds were confirmed by spectral methods (IR, UV, PMR, and 13C NMR). Thus,
–
1
the IR spectrum of conjugate 4 contained absorption maxima for OH and NH in the range 3600–3200 cm . The UV spectrum
of this compound in MeOH was characterized by a strong band with ꢄmax 249 nm that was typical for the 11-on-12-ene system
of the aglycon. The PMR spectrum showed four singlets for methoxy protons in the range 3.70–3.77 ppm. The 1 C NMR
spectra of conjugates 4, 6, 7, and 9 had resonances for Asp ester C=O groups in addition to other C=O resonances at weak field
of 169.5–173.5 ppm. The resonance of the free 30-COOH of the aglycon had chemical shift 178.0–180.5 ppm. The amino
acids were also found using ꢁ-CH resonances in the range 52.4–54.6 ppm.
3
The anti-HIV-1 activity of 18ꢀ-GA derivative 4 was studied at Vector State Scientific Center (Novosibirsk Oblast).
The cytotoxicity and antiviral activities of the compounds were studied on passaged human T-lymphocyte (MT-4) culture for
the traditional model of acute HIV-1 infection using strain HIV-1/EVK as before [8, 9]. The antiviral activity was assessed by
the reduction of HIV-1 reverse transcriptase (RT) activity and inhibition of virus antigen accumulation (virus-specific protein
263

p24) on the fourth day of cultivation using an immuno-enzyme analysis method in culture fluid and comparison with a control
without added compound). Furthermore, the fraction of viable cells was determined after staining with trypan blue by
(
counting in a Goryaev chamber. The known anti-HIV drug azidothymidine (AZT) and purified (97%) 18ꢀ-GA were used as
references [10]. The mechanism of action of the former is related to blockage of viral RT and termination of viral DNA
synthesis. Table 1 presents the experimental results.
Compound 4 inhibited effectively HIV-1 RT activity (95–97%) and accumulation of viral antigen p24 (96–97%) at
the studied concentrations. The level of inhibition of virus-specific p24 protein by this compound at a dose of 250 ꢃg was
better than AZT at a dose of 10 ꢃg/mL and comparable with 18ꢀ-GA. In contrast with AZT, 4 was not toxic to the cell culture
and protected MT-4 cells from death as a result of the cytopathogenic action of the virus. The amount of living cells after
addition of the drug (Table 1) was ~10–12 times greater than that for AZT and 3-4 times greater than for 18ꢀ-GA. Thus,
conjugate 4 is interesting for further research as an HIV-1 inhibitor in cell culture.
EXPERIMENTAL
IR spectra were recorded in mineral oil mulls on a Specord M-80 spectrophotometer. UV spectra wee taken in MeOH
or EtOH on a Specord UF-400 spectrometer. PMR and 13C NMR spectra were recorded in CDCl + DMSO-d and CD OD
3
6
3
1
13
with TMS internal standard on a Bruker AM-300 spectrometer at operating frequency 300 ( H) and 75.5 ( C) MHz. Optical
activity was measured in a 1-dm tube at 20–22°C on a Perkin–Elmer 241 MC polarimeter. Melting points were determined on
a Boetius microstage.
TLC was performed on Sorbfil plates (ZAO Sorbpolimer) using CHCl :MeOH:H O (45:10:1). Spots were detected
3
2
by H SO solution (5%) in EtOH with subsequent heating at 110–120°C for 2–3 min. Column chromatography used KSK
2
4
silica gel (50–150, dry classification, ZAO Sorbpolimer). HPLC was carried out on a DuPont chromatograph with an absorbance
detector at 254 nm over a Jupiter 5ꢃ C18 reversed-phase column with mobile phase (MP) MeOH:H O:HOAc (60:35:5, v/v)
2
(
for 18ꢀ-GA) and over a Chromsil CN column with MP phosphate buffer (pH 5.17):CH CN:dioxane (210:15:10) (for
3
1
1
8ꢁ-GA) at flow rate 1 mL/min (sample concentration 2 mg/mL).
Purified 18ꢀ-GA (87 ± 0.8%) was obtained from G. uralensis roots from the Siberian population as before [11];
8ꢁ-GA (85 ± 0.1%), by the literature method [12].
We used DCC and HOSu (Aldrich) and L-aspartic acid and its ꢀ-tert-butyl ester (hydrochloride) (Reanal, Hungary).
The dimethyl ester of L-aspartic acid was prepared as the hydrochloride by the literature method [13]: mp 115–116°C,
2
0
[
ꢁ] +30° (c 0.08, MeOH), lit. [13] mp 116–117°C.
D
Et N and N-ethylmorpholine were stored for 1 d over KOH and distilled. DMF was distilled from BaO and stored
over 4-A molecular sieves. Other solvents were purified by standard methods [14]. Solvents were evaporated in vacuo at 40–
3
°
4
5°C. Molecular sieves were calcined for 3 h at 300–350°C.
Preparation of Mixed ꢁ-Methyl ꢀ-tert-Butyl Ester of L-aspartic Acid. A solution of the ꢀ-tert-butyl ester of
L-aspartic acid (5 g) in MeOH (100 mL) at 0–5°C was treated with an Et O solution of diazomethane until a stable yellow
2
color developed. The mixture was stirred for 30 min with cooling in an ice bath, held at 20–22°C for 5–6 h, treated with
several drops of glacial HOAc to destroy the excess of diazomethane, and evaporated to dryness in vacuo. The solid was
dissolved in EtOAc (50 mL) and washed with NaHCO solution (3 ꢆ 50 mL) and H O. The organic phase was dried over
3
2
2
0
MgSO and evaporated. The solid was recrystallized from EtOAc:hexane. Yield 5.4 g (85.3%), mp 157–159°C, [ꢁ] +16°
c 0.02, EtOH), lit. [13] mp 158–160°C, [ꢁ] +18.3° (2%, MeOH).
4
D
2
0
(
D
General Method for Synthesizing Protected Conjugates 4, 5, 7, and 8. A solution of 18ꢀ-GA or 18ꢁ-GA (1 mmol)
in THF, dioxane, or DMF (25–30 mL) at 0–5°C was treated with HOSu (5–5.5 mmol) and DCC (2.3–2.8 mmol), stirred with
cooling for 2–3 h, and stored in a refrigerator overnight. The solid N,Nꢂ-dicyclohexylurea was filtered off. The cold filtrate
was treated with L-aspartic acid ester hydrochloride (2.5–3.0 mmol) and Et N or NEM (5–6 mmol), stirred with cooling for
3
1
h, held for 22–24 h at 20–22°C, and treated with cold H O acidified with citric acid to pH 4–5. The precipitate was filtered
2
off, washed with H O, and dried.
2
General Method for Removing the tert-Butyl Ester Group of the Conjugates 5 and 8. A solution of protected
conjugate (5 or 8, 0.5 mmol) in CF COOH (5 mL) was held at room temperature for 30–40 min and evaporated. The solid was
3
chromatographed over SG with elution by CHCl :MeOH:H O (300:10:1ꢇ30:10:1, stepwise gradient, v/v). Fractions that
3
2
were homogeneous according to TLC were combined and evaporated.
264

3
-O-{2-O-[N-(ꢀ-D-Glucopyranosyluronoyl)-L-aspartic Acid Dimethyl Ester]-N-(ꢀ-D-glucopyranosyluronoyl)-
L-aspartic Acid Dimethyl Ester}-(3ꢀ,20ꢀ)-11-oxo-18ꢀ-olean-12-en-30-oic Acid (4). GA (0.82 g, 1 mmol), HOSu (0.6 g,
.2 mmol), DCC (0.6 g, 2.8 mmol), and L-Asp(OMe)-OMe·HCl (0.6 g, 3 mmol) in THF (20 mL) in the presence of NEM
0.6 mL, 5 mmol) afforded after purification by CC over SG as described above 4 (0.58 g, 52.7%) as an amorphous powder,
5
(
[
1
2
0
–1
ꢁ] +60° (c 0.02, MeOH), C H O N . IR spectrum (ꢈ, cm ): 3600–3200 (OH, NH), 1740 (COOR), 1710 (COOH),
D 54 80 22 2
11
650 (C =O), 1520 (CONH). UV spectrum (MeOH, ꢄmax, nm, log ꢉ): 249 (4.1).
PMR spectrum (CD OD, ꢊ, ppm): 0.82, 0.85, 1.06, 1.14, 1.14, 1.27, 1.41 (3H each, s, CH ), 2.07 (1H, s, H-2), 2.42
3
3
(
1H, s, H-9), 2.55 (1H, t, H-18), 2.65-2.93 (m, CH , CH), 3.70, 3.72, 3.74, 3.77 (3H each, s, COOCH ), 5.62 (1H, s, H-12).
2
3
1
3
C NMR spectrum (CD OD, ꢊ, ppm): 40.41 (C-1), 27.62 (C-2), 90.78 (C-3), 40.66 (C-4), 56.42 (C-5), 18.47 (C-6),
3
3
3.82 (C-7), 46.77 (C-8), 63.17 (C-9), 38.09 (C-10), 202.70 (C-11), 129.11 (C-12), 171.44 (C-13), 44.59 (C-14), 27.21
(
1
(
C-15), 27.38 (C-16), 32.98 (C-17), 48.19 (C-18), 42.46 (C-19), 44.94 (C-20), 32.16 (C-21), 38.56 (C-22), 28.42 (C-23),
7.03 (C-24), 17.19 (C-25), 19.37 (C-26), 23.85 (C-27), 28.91 (C-28), 29.78 (C-29), 180.50 (C-30), 104.88 (C-1ꢂ), 81.40
C-2ꢂ), 75.92 (C-3ꢂ), 73.43 (C-4ꢂ), 77.68 (C-5ꢂ), 171.26 (C-6ꢂ), 104.88 (C-1ꢂꢂ), 75.42 (C-2ꢂꢂ), 76.09 (C-3ꢂꢂ), 73.49 (C-4ꢂꢂ),
7
1
7.19 (C-5ꢂꢂ), 171.44 (C-6ꢂꢂ). Additional resonances: 51.58, 50.94 (2ꢁ-CH, 4COOCH Asp), 36.84, 36.72 (CH Asp each),
3 2
68.91, 168.75 (COOCH3).
-O-{2-O-[N-(ꢀ-D-Glucopyranosyluronoyl)-L-aspartic Acid ꢁ-Methyl Ester]-N-(ꢀ-D-glucopyranosyluronoyl)-
L-aspartic Acid ꢁ-Methyl Ester}-(3ꢀ,20ꢀ)-11-oxo-18ꢀ-olean-12-en-30-oic Acid (6). 18ꢀ-GA (0.82 g, 1 mmol), HOSu
3
(
(
(
0.6 g, 5.2 mmol), DCC (0.6 g, 2.8 mmol), L-Asp(O-t-Bu)-OMe·HCl (0.6 g, 3 mmol), and Et N (0.7 mL, 5.1 mmol) in dioxane
3
–
1
30 mL) afforded protected conjugate 5 (1.1 g) after reprecipitation from MeOH by Et O. IR spectrum (ꢈ, cm ): 3600–3200
2
11
OH, NH), 1750 (COOR), 1665 (C =O), 1550 (CONH), R 0.58.
f
A solution of protected conjugate 5 (1.1 g) was treated with CF COOH (5 mL) at 20-22°C as described above. The
3
2
0
solid was chromatographed over SG as described above. Yield 0.52 g (48.8%), R 0.47, [ꢁ] +50° (c 0.05, MeOH),
C H O N . IR spectrum (ꢈ, cm ): 3600–3200 (OH, NH), 1740 (COOMe), 1660 (C =O), 1545 (CONH).
f
D
–
1
11
5
2 76 22 2
PMR spectrum (CD OD, ꢊ, ppm): 0.80, 0.90, 0.95, 1.02, 1.02, 1.04, 1.30 (3H each, s, CH ), 2.44 (1H, s, H-9), 3.05
3
3
(
(
3
(
1H, s, H-1), 3.20 (3H, s, OCH ), 3.35 (1H, s, H-3), 2.74, 2.84, 3.54, 3.62 (all s, CH, CH Asp), 5.45 (1H, s, H-12).
3
2
1
3
C NMR spectrum (CD OD, ꢊ, ppm): 40.4 (C-1), 27.4 (C-2), 90.9 (C-3), 40.7 (C-4), 56.5 (C-5), 18.5 (C-6), 33.8
3
C-7), 46.8 (C-8), 63.2 (C-9), 38.1 (C-10), 202.8 (C-11), 128.9 (C-12), 171.5 (C-13), 44.6 (C-14), 27.6 (C-15), 28.4 (C-16),
3.0 (C-17), 48.1 (C-18), 42.4 (C-19), 45.0 (C-20), 32.1 (C-21), 39.1 (C-22), 28.8 (C-23), 17.1 (C-24, C-25), 19.4 (C-26), 23.9
C-27), 29.3 (C-28), 30.0 (C-29), 180.5 (C-30), 105.5 (C-1ꢂꢂ), 104.9 (C-1ꢂ), 76.0 (C-3ꢂ), 75.0 (C-3ꢂꢂ), 73.5 (C-4ꢂꢂ), 72.4 (C-4ꢂ),
7
7.3, 77.3 (C-5ꢂꢂ, C-5ꢂ), 172.5, 171.9 (C-6ꢂ, C-6ꢂꢂ). Additional resonances: 173.1, 173.0, 171.5, 170.8 (2 COOH, 2COOCH Asp),
3
5
3.5, 52.5, 51.5, 51.2 (2ꢁ-CH, 2COOCH Asp).
3
3
-O-{2-O-[N-(ꢀ-D-Glucopyranosyluronoyl)-L-aspartic Acid Dimethyl Ester]-N-(ꢀ-D-glucopyranosyluronoyl)-
L-aspartic Acid Dimethyl Ester}-(3ꢀ,20ꢀ)-11-oxo-18ꢁ-olean-12-en-30-oic Acid (7). 18ꢁ-GA (0.82 g, 1 mmol), HOSu
0.6 g, 5.2 mmol), DCC (0.5 g, 2.3 mmol), and L-Asp(OMe)-OMe·HCl (0.5 g, 2.5 mmol) in DMF (10 mL) in the presence of
NEM (0.6 mL, 5 mmol) afforded a product that was chromatographed over a column of SG as described above. Yield 0.55 g
(
2
0
–1
(
(
49.6%), [ꢁ] +26° (c 0.02, MeOH), C H O N . IR spectrum (ꢈ, cm ): 3600–3200 (OH, NH), 1740 (COOMe), 1720
D 54 80 22 2
11
COOH), 1660 (C =O), 1540 (CONH).
1
3
C NMR spectrum (CDCl + DMSO-d , ꢊ, ppm): 41.8 (C-1), 26.9 (C-2), 88.5 (C-3), 41.9 (C-4), 54.5 (C-5), 18.4
3
6
(
C-6), 47.6 (C-7), 47.2 (C-8), 60.0 (C-9), 36.8 (C-10), 198.6 (C-11), 123.7 (C-12), 168.9 (C-13), 44.2 (C-14), 26.8 (C-15),
3
2
5.5 (C-16), 35.4 (C-17), 43.5, 41.5 (C-18, C-20), 33.6, 31.9 (C-19, C-21), 28.0 (C-23), 15.7 (C-24), 16.0 (C-25), 17.5 (C-26),
0.2 (C-27), 28.8 (C-28, C-29), 178.0 (C-30), 103.5 (C-1ꢂ), 102.5 (C-1ꢂꢂ), 81.5 (C-2ꢂ), 75.3, 71.6 (C-3ꢂ, C-3ꢂꢂ), 76.0, 75.5
(C-5ꢂ, C-5ꢂꢂ), 69.6, 69.2 (C-4ꢂ, C-4ꢂꢂ), 170.0, 169.5 (C-6ꢂ, C-6ꢂꢂ). Additional resonances: 170.5, 170.3, 169.2, 168.8 (4 COOCH3),
5
4.6, 54.2, 51.7, 51.4, 51.2, 50.9 (2ꢁ-CH, 4 COOCH Asp).
3
3
-O-{2-O-[N-(ꢀ-D-Glucopyranosyluronoyl)-L-aspartic Acid ꢁ-Methyl Ester]-N-(ꢀ-D-glucopyranosyluronoyl)-
L-aspartic Acid ꢁ-Methyl Ester}-(3ꢀ,20ꢀ)-11-oxo-18ꢁ-olean-12-en-30-oic Acid (9). 18ꢁ-GA (0.82 g, 1 mmol), HOSu
(
(
0.6 g, 5.2 mmol), DCC (0.5 g, 2.3 mmol), L-Asp(O-t-Bu)-OMe (0.6 g, 3 mmol), and NEM (0.6 mL, 5.0 mmol) in DMF
20 mL) afforded protected conjugate 8 (0.85 g) that was reacted without further purification with CF COOH (5 mL) as
3
2
0
described above and purified by chromatography over SG to afford 9 (0.45 g, 44.6%), R 0.5, [ꢁ] +25° (c 0.04, MeOH),
C H O N . IR spectrum (ꢈ, cm ): 3600-3200 (OH, NH), 1744 (COOMe), 1712 (COOH), 1640 (C =O), 1540 (CONH).
f
D
–
1
11
5
2
76 22
2
1
3
C NMR spectrum (CDCl + DMSO-d , ꢊ, ppm): 41.5 (C-1), 26.8 (C-2), 88.5 (C-3), 41.7 (C-4), 54.5 (C-5), 17.4
3
6
(
C-6), 31.8 (C-7), 45.2 (C-8), 60.2 (C-9), 36.8 (C-10), 199.5 (C-11), 123.7 (C-12), 166.5 (C-13), 42.5 (C-14), 27.6 (C-15),
265

2
8.2 (C-16), 32.0 (C-17), 42.2 (C-19), 43.9 (C-20), 38.0 (C-22), 16.5, 15.7 (C-24, C-25), 18.4 (C-26), 20.5 (C-27), 178.5
(
(
C-30), 107.0 (C-1ꢂꢂ), 105.5 (C-1ꢂ), 75.5 (C-3ꢂ), 75.0 (C-3ꢂꢂ), 71.5, 69.5 (C-4ꢂꢂ, C-4ꢂ), 77.3, 77.2 (C-5ꢂꢂ, C-5ꢂ), 170.0, 169.5
C-6ꢂ, C-6ꢂꢂ). Additional resonances: 173.5, 173.0, 172.5, 172.0 (2 COOH and 2COOCH Asp), 54.2, 54.0, 51.7, 51.4 (2ꢁ-CH,
3
2
COOCH Asp).
3
ACKNOWLEDGMENT
The work was supported financially by the Russian Ministry of Science (SC 14.740.11.0367) and NSh-3756.2010.3.
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