Synthesis and Antitubercular, Antimicrobial, and Hemolytic Activity of Methyl D-Glucopyranuronate and Its Simplest Derivatives

Article abstract of DOI:10.1134/S1070363217120106

Methyl glucuronate and some of its simplest derivatives have been synthesized, and their antitubercular, antimicrobial, and hemolytic activities have been studied. The simplest derivatives of glucuronic acid have been shown for the first time to exhibit a high antitubercular activity which is comparable with the activity of isoniazid.

Full text of DOI:10.1134/S1070363217120106

ISSN 1070-3632, Russian Journal of General Chemistry, 2017, Vol. 87, No. 12, pp. 2816–2825. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © M.G. Belenok, O.V. Andreeva, B.F. Garifullin, A.S. Strobykina, M.A. Kravchenko, A.D. Voloshina, V.E. Kataev, 2017, published in
Zhurnal Obshchei Khimii, 2017, Vol. 87, No. 12, pp. 1999–2008.
Dedicated to B.I. Buzykin on His 80th Anniversary
Synthesis and Antitubercular, Antimicrobial,
and Hemolytic Activity of Methyl D-Glucopyranuronate
and Its Simplest Derivatives
M. G. Belenoka, O. V. Andreevaa, B. F. Garifullina, A. S. Strobykinaa,
M. A. Kravchenkob, A. D. Voloshinaa, and V. E. Kataeva*
a Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences,
ul. Arbuzova 8, Kazan, 420088 Tatarstan, Russia
*e-mail: kataev@iopc.ru
b Ural Research Institute of Phthisiopulmonology, Ministry of Health Protection of the Russian Federation,
ul. 22 Parts’’ezda 50, Yekaterinburg, 620039 Russia
Received June 8, 2017
Abstract—Methyl glucuronate and some of its simplest derivatives have been synthesized, and their antitubercular,
antimicrobial, and hemolytic activities have been studied. The simplest derivatives of glucuronic acid have
been shown for the first time to exhibit a high antitubercular activity which is comparable with the activity of
isoniazid.
Keywords: glyucuronic acid, methyl D-glucopyranuronate, glucuronides, glucopyranosides, antitubercular activity
DOI: 10.1134/S1070363217120106
Glucuronic acid (1) is the most important parti-
cipant of phase II metabolism of xenobiotics, including
drugs, in human organism. Glucuronic acid is a
structural fragment of uridine diphosphate glucuronic
acid (4, UDPGA), which is a coenzyme ensuring UDP-
glucuronosyl transferase-catalyzed glucuronidation of
xenobiotics oxidized during phase I metabolism; water-
soluble glucuronides 5 thus formed are excreted with
Scheme 1.
O
O
P
O
O
O
P
O
O
OH
OH
O
O
O
R
HO
O
OH
OH
NH
HO
N
HO
HO
OH
O
OH
1 3
OH
O
OH
_
4
O
Xenobiotic
O
R
HO
OH
OH
5
R = OH (1), OCH3 (2), NH2 (3).
2816

SYNTHESIS AND ANTITUBERCULAR, ANTIMICROBIAL, AND HEMOLYTIC ACTIVITY
2817
Scheme 2.
COOH
R4
O
OH
R1
R2
O
O
O
HOOC
HO
O
R3
OH
OH
O
O
OH
OH
OH
HOOC
6_8
9
O
O
O
HO
R1 = OH, R2 = H, R3 =
R4 = OH (7);
R1 = OH, R2 = H, R3 = R4 = OH (6);
HO
OH
OH
O
O
O
HO
2
3
4
R1 =
,
R = OH, R = R = H (8).
OH
HO
OH
urine or are transported with blood from the liver to
other organs [1, 2] (Scheme 1).
other falvonoid glucuronides isolated from higher
plants display biological activity. For example,
scutellarin 8 (4′,5,6-trihydroxyflavone-7-glucuronide)
is a traditional Chinese medicine exhibiting cytotoxic
activity against some human cancer cell lines [6, 7]; it
is also used in clinical practice for the treatment of
strokes [8]. Flavonoid glucuronides isolated from L.
Leucocephala [9] and flowers of Syzygium aromaticum
[10] showed cytotoxic effect against a number of
cancer cell lines, while those isolated from Scutellaria
indica possess anti-inflammatory activity [11] (Scheme 2).
It should be noted that glucuronidation of xeno-
biotics taken by humans endows them with some pharma-
cological activity via transformation into beneficial
compounds (glucuronides). For example, the natural
flavonoid quercetin (6) widely known due to its
biological activity (antioxidant, antitumor, anti-inflam-
matory, antidiabetic [3]), which occurs in vegetables,
fruits, tea, and red wine [4], is metabolized in humans
to quercetin 3-O-β-glucuronide (7), and the latter is
transferred with blood to different tissues, including
brain and muscles, where it performs its useful
functions [3]. The major metabolite of quercetin,
quercetin 3-O-β-glucuronide (7), responsible for its
useful properties, is present as such in fruits, berries,
and some vegetables (lettuce, green bean) [4]. This is
not a single example. Metabolites (glucuronides) of the
natural phenols tyrosol and hydroxytyrosol occurring
in olives protect hemoglobin from oxidation and red
blood cells from morphological change [5]. Many
Natural glucuronides derived from triterpenoids are
also biologically active. The most known is glycyrrhizin
(9), which is a carboxylic acid of the oleanane series
containing two glucuronic acid residues); it was found
to exhibit anti-inflammatory, antiulcer, antiviral, antitumor,
and immunomodulatory activities [12–14]. Biological
activities of tens of other triterpenoid glucuronides
have also been reported (see, e.g., [15–18]). Therefore,
glucuronidation is used to endow natural terpenoids
with new biological properties [19–21].
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2818
BELENOK et al.
Scheme 3.
O
Toxic
drug
Spacer
O
O
HO
HO
OH
OH
Nontoxic prodrug
O
β-Glucuronidase
OH
OH
O
HO
Toxic
drug
Spacer
+
+
HO
OH
Glucuronic acid amide (3, glucuronamide) is a
fragment of glycolipids in some bacteria [22]. Even
more interesting is that amides of glucuronic acid and
some amino acids, primarily L-alanine, constitute O-
polysaccharides in a large number of bacteria, which
act as antigens (see, e.g., [23–25]).
are converted to a highly toxic “cocktail” [33]. If a
protein (usually, of the globulin series) is included in a
prodrug, the latter becomes an immunogen [34]. Since
β-glucuronidase recognizes as substrates only glucuro-
nides (including prodrugs) with the glucopyranuro-
nosyl residue containing free carboxy and hydroxy
groups, glucuronic acid methyl ester 2 or even its 2,3,4-
tri-O-acetyl derivative [27, 28, 36] was used as
carbohydrate fragment instead of glucuronic acid 1
itself to avoid untimely hydrolysis of prodrugs in
blood plasma. It was presumed [36] that it is only in
cancer tissues that carboxylesterase removes acyl
protection from the glucopyranuronate fragment of a
prodrug and hydrolyzes its ester group; next follows
hydrolysis of the prodrug by lysosomal β-glucuro-
nidase, which releases the cytotoxic anticancer agent.
Taking into account that phase II metabolism of
xenobiotics in human organism consists of their trans-
formation into a water-soluble form for excretion,
many therapeutic agents are subjected to glucuronida-
tion to enhance their bioavailability or detoxification
[26]. Detoxification of therapeutic agents as a result of
their glucuronidation is widely used in the design
drugs for chemotherapy of cancer [26–35]. In order to
avoid damage to healthy cells, highly toxic anticancer
agents (e.g., fluorouracil, anthracycline antibiotics,
etc.) are subjected to glucuronidation [26, 27, 30, 31]
or are covalently bound through a specific spacer to
glucuronic acid [28, 32, 33], its methyl ester [28–30],
or amide [34]. The resulting glucuronide conjugates
(prodrugs) are not toxic, and they do not affect healthy
cells. On the other hand, lysosomal β-glucuronidase
present at a high concentration in cancer cells
(especially in breast cancer cells [35]) hydrolyzes
prodrugs to release cytotoxic anticancer agents directly
at the target site (Scheme 3). Some prodrugs that are
nontoxic due to the presence of glucuroric acid
residues in their molecules include several cytotoxic
anticancer agents [33]. After hydrolysis in cancer
tissues by the action of β-glucuronidase, these prodrugs
It should be noted that one of the pharmacophoric
moieties in all biologically active natural (glucuronides
derived from flavonoids [3–11], terpenoids [12–18],
and polysachharides [23–25]) or synthetic glucuro-
nides [19–21, 26–34, 36] listed above is glucuronic
acid 1 [3–8, 11, 13–17, 26–28, 30–33], or its methyl
ester 2 [9–11, 15, 16, 18, 19, 21, 26–30, 36], or amide
3 [19, 21, 23–25, 34], or even phosphite [21]. However,
there are almost no published data on biological
activity of these pharmacophores. It is only known that
glucuronic acid hydroxamate exhibits antioxidant
activity [37]. In order to fill the gap in the knowledge
of biological activity of the above listed pharma-
cophores, in the present work we synthesized
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SYNTHESIS AND ANTITUBERCULAR, ANTIMICROBIAL, AND HEMOLYTIC ACTIVITY
2819
Scheme 4.
OH
NH
6
NH2
OH
OCH3
OH
OH
OH
CH3ONa,
CH3OH
O
O
O
O
OH
5
4
1
2
5
H
H
4
NNH2
3
3
2
O
HO
HO
1
HO
OH
OH
12
OH
10
2
Ac2O,
HClO4
NH2NH2 H2O,
CH3OH
OCH3
OCH3
OCH3
OAc
OH
NH2NH2 H2O,
CH3OH
O
OH
OH
O
O
O
O
O
AcO
OAc
HO
OH
HO
OH
OAc
11
OH
33% HBr, AcOH
CH2Cl2
OCH3
O
OCH3
Ag2CO3,
acetone,
H2O
OH
Br
O
O
O
AcO
OAc
AcO
OAc
O
O
OAc
13
OAc
14
O
P
Ag2CO3,
CH3OH,
toluene
Cl
THF,
Et3N
O
OCH3
NHNH2
O
OCH3
NH2NH2 H2O,
CH3OH
OCH3
O
P
O_
H
OCH3
OH
O
O
O
O
O
+
Et3NH
HO
OAc
AcO
AcO
OAc
OH
17
OAc
OAc
15
16
glucuronic acid methyl ester 2 and a number of its
simplest derivatives and studied their antitubercular,
antimicrobial, and hemolytic activity.
and compound 2 was isolated as a mixture of α- and β-
1
anomers (Scheme 4). The H NMR spectrum of 2
contained a doublet at δ 5.38 ppm (3J = 3.5 Hz) due to
resonance of anomeric proton of the α-anomer and a
doublet at δ 4.5 ppm (3J = 10.0 Hz) corresponding to
the β-anomer [39]. Acylation of 2 with acetic acid in
the presence of perchloric acid gave tetra-O-acetyl
derivative 11 which was isolated as pure β-anomer; the
Glucuronic acid methyl ester (2) was synthesized
by treatment of D-(+)-glucurono-3,6-lactone 10 with
sodium methoxide by analogy with the procedure
described in [38]. The reaction was not stereoselective,
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 87 No. 12 2017

2820
BELENOK et al.
1
Table 1. Antitubercular activity of compounds 2, 11, 12, 15,
and 17 against Mycobacterium tuberculosis H37Rv
one set of signals in the H NMR spectrum, and the
anomeric proton resonated at δ 5.77 ppm as a doublet
3
of doublets with the coupling constants JHH = 9.1 Hz
Comp. no. MIC, μg/mL
Comp. no. MIC, μg/mL
and 3JHP = 3.4 Hz. The P–H proton signal was observed
at δ 6.87 ppm as a doublet with a direct P–H coupling
constant of 640.4 Hz. The 31P NMR spectrum of 15
contained one singlet at δP 0.90 ppm. These finfings
indicated that phosphonate 15 was isolated as pure
β-anomer.
2
1.5
0.8
3.1
15
17
0.8
0.3
0.1
11
12
Isoniazid
1
anomeric proton of 11 resonated in the H NMR
spectrum as a doublet at δ 5.77 ppm (3J = 7.7 Hz).
By analogy with the procedure given in [44], The
Koenigs–Knorr reaction of 13 with methanol in the
presence of silver carbonate afforded glucopyranuro-
nate 16 which was isolated as the β-anomer, as
followed from the presence of a doublet signal of the
The reaction of 11 with hydrazine hydrate in
methanol led to not only removal of the acetyl
protecting groups but also hydrazinolysis of the ester
group. Removal of the acetyl protection has made both
ester and aldehyde (open-chain isomer) groups
accessible for the reaction with hydrazine. As a result,
hydrazono hydrazide 12 was obtained in 55% yield
(Scheme 4). Compound 12 showed the molecular ion
peak m/z 245.1 [M + Na]+ in the ESI mass spectrum.
Its IR spectrum contained absorption bands due to
stretching vibrations of the hydrazide and hydrazone
groups at 3300 (OH, NH2), 1616 (amide I), 1535
(amide II), and 1349 cm–1 (amide III), as well as
anomeric proton at δ 4.48 ppm (3J = 7.7 Hz) in the H
1
NMR spectrum. In the final stage, compound 16 was
treated with hydrazine hydrate to obtain 73% of
hydrazide 17. The IR spectrum of 17 contained
absorption bands typical of stretching vibrations of the
hydrazide moiety, whereas no acetyl proton signals
were present in the H NMR spectrum. The anomeric
proton of 17 appeared in the H NMR spectrum as a
doublet at δ 4.05 ppm with a vicinal coupling constant
of 7.8 Hz, which indicated conservation of the β-
orientation of the glycoside bond.
1
1
1
carbonyl stretching band at 1665 cm–1. The H NMR
spectrum of 12 lacked signals assignable to acetyl and
methoxy protons, and the anomeric proton (1-H)
resonated as a doublet at δ 7.35 ppm (3J = 6.2 Hz). The
hydrazide NH proton signal was observed as a
broadened singlet at δ 8.97 ppm, when the spectrum
was recorded from a solution in DMSO-d6. In the 13C
NMR spectrum of 12, the C1 signal (hydrazone
moiety) characteristically appeared at δC 147.06 ppm.
Two set of signals were observed in the 13C NMR
spectrum of 12, indicating the presence of two isomers.
The given spectral data are consistent with those
reported in [40, 41].
The synthesized compounds were tested for
antimycobacterial activity against Mycobacterium
tuberculosis H37Rv. The results are given in Table 1.
The antitubercular (tuberculostatic) effects of methyl
tetraacetylglucuronate 11, phosphonate 15, and 1-O-
methyl-β-D-glucopyranuronohydrazide 17 were com-
parable with the activity of the antitubercular drug
isoniazid used as reference. The activity of methyl
glucuronate 2 and 1-hydrazono-D-glucuronohydrazide
12 was an order of magnitude lower, but it never-
theless exceeded the activity of the second-line anti-
tubercular drug pyrazinamide by a factor of 4–8 (MIC
12.5 μg/mL [45]).
By reaction of 11 with 33% HBr in AcOH by
analogy with [42] we obtained bromide 13. The
We also examined hemolytic activity of the syn-
thesized glucuronic acid derivatives (Table 2). Com-
pounds 2, 11, 12, 15, and 17 caused almost no damage
to human erythrocytes (their hemolysis did not exceed
0.5%) at a concentration at which they showed
antitubercular activity. Moreover, these compounds
exhibited no hemolytic activity at concentrations 1000
times higher than their minimum inhibitory concentra-
tions with respect to Mycobacterium tuberculosis H37Rv.
1
anomeric proton of 13 resonated in the H NMR
spectrum as a doublet at δ 6.64 ppm (3J = 4.1 Hz),
indicating that the α-anomer was formed. Following
the procedure described in [43], bromide 13 was
converted to methyl-2,3,4-tri-O-acetyl-D-glucopyra-
nuronate 14 which was reacted with 2-chloro-1,3,2-
benzodioxaphosphinin-4-one to produce phosphonate
15 in 64% yield (Scheme 4). The 1H NMR spectrum of
14 displayed doublets at δ 5.53 (3J = 3.6 Hz) and
4.80 ppm (3J = 7.6 Hz) corresponding to the α- and
β-anomers, respectively. Phosphonate 15 showed only
Furthermore, the synthesized compounds were tested
for antimicrobial activity against gram positive
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SYNTHESIS AND ANTITUBERCULAR, ANTIMICROBIAL, AND HEMOLYTIC ACTIVITY
2821
Table 2. Hemolytic activity of compounds 2, 11, 12, 15, and
17
Staphylococcus aureus ATCC 209p and Bacillus
cereus ATCC 8035, gram negative Escherichia coli
CDCF-50 and Pseudomonas aeruginosa ATCC 9027,
and fungi Aspergillus niger BKMF-1119, Trichophyton
mentagrophytes var. gypseum 1773, and Candida
albicans 855-653 (Table 3). Compound 12 showed a
weak bacteriostatic activity against S. aureus and weak
fungistatic activity against C. albicans. Compounds 2
and 11 also showed a weak activity against C.
albicans, whereas hydrazide 17, which was the most
active antitubercular agent (Table 1), displayed no
antimicrobial activity at all.
Comp. no.
Concentration, μg/mL Hemolysis, %
2
100
10
1
0.3
0
0
0.1
100
10
1
0
11
0.6
0.3
0
In summary, we were the first to demonstrate that
the simplest glucuronic acid derivatives exhibit high
antitubercular activity. Methyl 1,2,3,4-tetra-O-acetyl-β-
D-glucopyranuronate 11, triethylammonium (methyl
2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) phosphonate
15, and methyl β-D-glucopyranosiduronohydrazide 17
were are comparable in antitubercular activity to the
first-line antitubercular drug isoniazid. The activity of
methyl β-D-glucopyranuronate 2 and 1-hydrazono-D-
glucuronohydrazide 12 exceeds the activity of the
second-line antitubercular drug pyrazinamide (MIC
12.5 μg/mL) by a factor of 4–8.
0.1
100
10
1
0
12
15
17
0.3
0.3
0.2
0
0.1
100
10
1
1.69
0.5
0.5
0
0.1
100
10
1
EXPERIMENTAL
0.5
0.5
0.5
0.2
The IR spectra (400–4000 cm–1) were recorded on a
Bruker Vector 22 spectrometer (Germany) with Fourier
transform; samples were examined as films or Nujol
mulls. The 1H, 13C, and 31P NMR spectra were measured
on Bruker Avance-400 and Avance-600 spectrometers
(Germany). The mass spectra (electrospray ionization)
were obtained on a Bruker Daltonik AmazonX
instrument (Bremen, Germany); positive ion detection,
a.m.u. range 100–1500 Da, capillary voltage 4500 V,
0.1
nebulizing gas nitrogen (200°C, flow rate 8 L/min);
samples were dissolved in methanol or water to a
concentration of 10–5 M; the data were processed using
DataAnalysis 4.0 (Bruker). The optical rotations were
Table 3. Antimicrobial (bacteriostatic and fungistatic) activity of compounds 2, 11, 12, and 17
Minimum inhibitory concentration (MIC), μg/mL
Compound no.
Staphylococcus
aureus
Bacillus
cereus
Escherichia Pseudomonas Aspergillus
Trichophyton
mentagrophytes albicans
Candida
coli
>500
>500
>500
>500
1.5
aeruginosa
>500
>500
>500
>500
3.0
niger
>500
>500
>500
>500
2
>500
>500
250
>500
2.4
>500
>500
>500
>500
7.8
>500
>500
>500
>500
250
250
11
12
250
17
>500
Norfloxacine
Ketoconazole
–
–
–
–
–
3.9
3.9
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2822
BELENOK et al.
measured on a Perkin Elmer-341 polarimeter (USA).
The progress of reactions and the purity of products
were monitored by thin-layer chromatography on
Sorbfil PTSKh-AF-A plates (Krasnodar, Russia); spots
were visualized by treatment with a 5% solution of
H2SO4 or 5% solution of H3PO4·12MoO3·H2O, followed
by heating. The products were isolated by column
chromatography on silica gel (0.06–0.20 mm, Acros).
NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 3.07–
4.69 m, 6.91 d (1H, 1-H, J = 6.7 Hz), 8.97 br.s (1H,
NH). 13C NMR spectrum (100 MHz, D2O), δC, ppm:
70.47, 70.76, 71.16, 71.25, 71.34, 71.80, 75.42, 76.19,
91.70, 147.06 (C=N), 169.54, 172.79 (C=O). Mass
spectrum (ESI): m/z 245.1 [M + Na]+. Found, %: C
32.67; H 6.73; N 25.38. C6H14N4O5. Calculated, %: C
32.43; H 6.35; N 25.21.
Methyl α/β-D-glucopyranuronate (2). Metallic
sodium, 0.057 g (2.48 mmol), was dissolved in 30 mL
of anhydrous methanol, and the solution was added to
a solution of 5 g (28.4 mmol) of D-(+)-glucurono-3,6-
lactone 10 in 20 mL of anhydrous methanol. The
mixture was stirred for 5 h at room temperature, the
solvent was removed under reduced pressure, and the
residue was subjected to dry column chromatography
using chloroform–methanol (5:1 to 3:1). Yield 2.91 g
(49%), light yellow oil, [α]D20 = 42.8° (c = 1.0,
CH3OH). 1H NMR spectrum (600 MHz, D2O), δ, ppm:
3.38–3.45 m (1H, 2β-H), 3.61–3.74 m (5H, 2α-H,
3α-H, 3β-H, 4α-H, 4β-H), 3.83–3.88 m (1H, 5α-H),
3.93 s (3H, CH3O), 3.93 s (3H, CH3O), 4.18 d (1H, 5β-H,
J = 10.0 Hz), 4.50 d (1H, 1β-H, J = 10.0 Hz), 5.38 d
(1H, 1α-H, J = 3.5 Hz). 13C NMR spectrum (100 MHz,
D2O), δC, ppm: 55.60 and 55.63 (OCH3), 73.23, 73.59,
73.79, 73.99, 74.84, 76.23, 77.19, 77.72 (C2α, C2β, C3α,
C3β, C4α, C4β, C5α, C5β), 94.98 (C1α), 98.74 (C1β),
173.55 and 174.50 (C=O). Mass spectrum (ESI), m/z:
231.1 [M + Na]+, 247.0 [M + K]+. Found, %: C 40.01;
H 5.92. C7H12O7. Calculated, %: C 40.39; H 5.81.
Methyl 2,3,4-tri-O-acetyl-α-D-glucopyranosyluro-
nate bromide (13) was synthesized as described in
[42]. Yield 94%, mp 105–106°C (from EtOH);
published data [46]: mp 106–107°C (from EtOH).
Methyl 2,3,4-tri-O-acetyl-α/β-D-glucopyranuro-
nate (14) was synthesized as described in [43]. Yield
91%, mp 83°C; published data [43]: mp 81°C.
Triethylammonium (methyl 2,3,4-tri-O-acetyl-β-
D-glucopyranosyluronate) phosphonate (15). Gluco-
pyranuronate 14, 1 g (3.0 mmol), was dissolved in
8 mL of anhydrous THF, and 3 mL of anhydrous
triethylamine and 0.67 g (3.3 mmol) of 2-chloro-1,3,2-
benzodioxaphosphinin-4-one were added. The mixture
was stirred for 24 h at room temperature, treated with
3 mL of water, and left to stand for 1 h. The solvent
was removed under reduced pressure, and the product
was isolated by column chromatography on silica gel
(CH2Cl2–MeOH–Et3N, 50:1:0.5 to 50:2:0.5). Yield
0.95 g (64%), light yellow oil, [α]D20 = 33.5° (c = 1.0,
1
CHCl3). H NMR spectrum (400 MHz, CDCl3), δ,
ppm: 1.30 t [9H, N(CH2CH3)3, J = 7.4 Hz], 1.93 s (3H,
CH3CO), 1.94 s (3H, CH3CO), 1.96 s (3H, CH3CO),
3.02 q [6H, N(CH2CH3)3, J = 7.5 Hz], 3.63 s (3H,
CH3O), 4.55 d (1H, 5-H, J = 10.2 Hz), 4.89–4.94 m
(1H, 2-H), 5.09 t (1H, 4-H, J = 9.6 Hz), 5.52 t (1H,
3-H, J = 9.7 Hz), 5.77 d.d (1H, 1-H, J = 9.1, 3.4 Hz),
6.87 d (1H, PH, J = 640.4 Hz). 13C NMR spectrum
(100 MHz, CDCl3), δC, ppm: 8.41 [N(CH2CH3)3],
20.27 (CH3CO), 20.43 (2C, CH3CO), 45.64
[N(CH2CH3)3], 52.48 (CH3O), 68.89 (C3), 69.08 (C5),
69.42 (C4), 70.07 d (C2, JCP = 5.5 Hz), 90.79 d (C1,
JCP = 4.8 Hz), 167.92 (COOCH3), 169.37 (CH3CO),
169.66 (2C, CH3CO). 31P NMR spectrum (100 MHz,
CDCl3): δP 0.90 ppm. Mass spectrum (ESI): m/z 397.1
(Irel 100%) [M]–. Found, %: C 45.98; H 6.93; N 2.88; P
6.29. C19H34NO12P. Calculated, %: C 45.69; H 6.86; N
2.80; P 6.20.
Methyl 1,2,3,4-tetra-O-acetyl-β-D-glucopyranuro-
nate (11) was synthesized according to the procedure
described in [38]. Yield 32%, mp 177°C (from
MeOH), [α]D20 = 8.1° (c = 1.1, CHCl3); published data:
mp 176°C [38], 177–178°C (from EtOH) [46], [α]D25
7.4° (c = 2.0, CHCl3) [46].
=
(2S,3S,4R,5R)-6-Hydrazinylidene-2,3,4,5-tetra-
hydroxyhexanohydrazide (12). Hydrazine hydrate,
4 mL (80.0 mmol), was added to a solution of 0.6 g
(1.6 mmol) of glucopyranuronate 11 in 20 mL of
anhydrous methanol. The mixture was kept for 24 h at
room temperature, the precipitate was filtered off, and
the solvent and excess hydrazine hydrate were
removed under reduced pressure. Yield 0.18 g (54%),
[α]D20 = –8.7° (c = 0.5, H2O). IR spectrum, ν, cm–1:
3300 (OH, NH2), 1665 (C=O), 1616 (amide I), 1535
1
Methyl (methyl 2,3,4-tri-O-acetyl-β-D-gluco-
pyranosid)uronate (16) was synthesized as described
in [44]. Yield 75%, mp 154–155°C (from EtOH);
published data [44]: mp 154.1–154.5°C (from i-PrOH).
(amide II), 1349 (amide III), 1080 (OH). H NMR
spectrum (600 MHz, D2O), δ, ppm: 3.40–3.45 m, 3.58–
3.70 m, 3.87–3.99 m, 4.21 d (J = 9.1 Hz), 4.31 d (J =
7.1 Hz), 4.37–4.41 m, 7.35 d (1H, 1-H, J = 6.2 Hz). 1H
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SYNTHESIS AND ANTITUBERCULAR, ANTIMICROBIAL, AND HEMOLYTIC ACTIVITY
2823
Methyl β-D-glucopyranosiduronohydrazide (17).
Hydrazine hydrate, 3 mL (64.6 mmol), was added to a
solution of 0.45 g (1.3 mmol) of glucopyranuronate 16
in 25 mL of anhydrous methanol, and the mixture was
stirred for 30 min at room temperature. The precipitate
was filtered off, washed with methanol, and dried
under reduced pressure. Yield 0.21 g (73%), white
powder, mp 233–234°C, [α]D20 = –45° (c = 0.5, H2O).
IR spectrum, ν, cm–1: 3417, 3350, 3332, 3284 (OH,
NH, NH2), 1672 (C=O), 1670 (amide I), 1557 (amide
inhibition of growth and proliferation of test cultures.
The following test cultures were used: gram positive:
Staphylococcus aureus ATCC 209p, Bacillus cereus
ATCC 8035; gram positive Escherichia coli CDCF-50,
Pseudomonas aeruginosa ATCC 9027; funfi:
Aspergillus
niger
BKMF-1119,
Trichophyton
mentagrophytes var. gypseum 1773, Candida albicans
855-653. Norfloxacin and ketoconazole were used as
reference drugs.
Hemolytic activity. Compounds 2, 11, 12, and 17
were tested for hemolytic activity against human
erythrocytes by comparing the optical density of
solutions of these compounds and dispersed human
erythrocytes in saline with the optical density of blood
after 100% hemolysis according to the procedure
described in [48]. In keeping with GOST R ISO 10993
4-99, a compound is considered cytotoxic with respect
to human erythrocytes if the hemolysis percentage
exceeds 2%.
1
II), 1370 (amide III), 1029 (OH). H NMR spectrum
(600 MHz, DMSO-d6), δ, ppm: 2.95–3.02 m (1H, 2-
H), 3.10–3.17 m (1H, 3-H), 3.17–3.29 m (1H, 4-H),
3.30 s (3H, CH3O), 3.38–3.42 m (1H, 5-H), 4.05 d
(1H, 1-H, J = 7.8 Hz), 4.26 br.s (2H, NH2), 4.95–5.16
m (3H, OH), 9.21 br.s (1H, NH). 13C NMR spectrum
(100 MHz, D2O), δC, ppm: 57.47 (OCH3), 71.20,
72.76, 74.45, 75.33, (C2, C3, C4, C5), 103.55 (C1),
169.01 (C=O). Mass spectrum (ESI), m/z: 245.2 [M +
Na]+. Found, %: C 37.98; H 6.52; N 12.87. C7H14N2O6.
Calculated, %: C 37.84; H 6.35; N 12.61.
ACKNOWLEDGMENTS
Antitubercular activity. Compounds 2, 11, 12, 15,
and 17 were tested for antitubercular activity against
Mycobacterium tuberculosis H37Rv laboratory strain
(hereinafter MBT) by the vertical diffusion method on
a Novaya solid nutrient medium. The nutrient medium
was placed in 5-mL test tubes and inoculated with an
MBT suspension (0.1 mL) diluted to a turbidity value
of 10 GKI units, and the test tubes were incubated for
24 h. The test tubes were then set in a vertical position,
and 0.3 mL of a solution of compounds to be tested in
aqueous ethanol at concentrations of 12.5, 6.2, 3.1, 1.5,
0.8, 0.3, and 0.1 μg/mL was added on the free edge of
the tube. The test tubes were than placed in a
thermostat and incubated for 10 days at 37°C under
sterile conditions. The growth of MBT was assessed by
the standard procedure according to which bacterial
growth inhibition by 10 mm and more indicated
tuberculostatic activity. The MBT growth inhibition
zone (mm) is proportional to the tuberculostatic
activity. A growth inhibition zone of 100 mm and more
was assumed as complete inhibition. The anti-
tubercular drug isoniazid was used as reference; it
inhibited the growth of MBT with a MIC value of
0.1 μg/mL.
This study was performed under financial support
by the Russian Science Foundation (project no. 14-50-
00014).
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