Synthesis and antituberculosis activity of the first macrocyclic glycoterpenoids comprising glucosamine and diterpenoid isosteviol

Article abstract of DOI:10.1016/j.carres.2016.05.007

The first macrocyclic glycoterpenoids comprising glucosamine and diterpenoid isosteviol moieties were synthesized and evaluated for inhibition activity against Mycobacterium tuberculosis H37Rv.

Full text of DOI:10.1016/j.carres.2016.05.007

Accepted Manuscript
Synthesis and antituberculosis activity of the first macrocyclic glycoterpenoids
comprising glucosamine and diterpenoid isosteviol
Bulat F. Garifullin, Irina Yu Strobykina, Radmila R. Sharipova, Marionella A.
Kravchenko, Olga V. Andreeva, Olga B. Bazanova, Vladimir E. Kataev
PII:
S0008-6215(16)30157-4
10.1016/j.carres.2016.05.007
CAR 7201
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 18 April 2016
Revised Date: 16 May 2016
Accepted Date: 22 May 2016
Please cite this article as: B.F. Garifullin, I.Y. Strobykina, R.R. Sharipova, M.A. Kravchenko, O.V.
Andreeva, O.B. Bazanova, V.E. Kataev, Synthesis and antituberculosis activity of the first macrocyclic
glycoterpenoids comprising glucosamine and diterpenoid isosteviol, Carbohydrate Research (2016), doi:
10.1016/j.carres.2016.05.007.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and antituberculosis activity of the first macrocyclic
glycoterpenoids comprising glucosamine and diterpenoid isosteviol
Bulat F. Garifullina, Irina Yu. Strobykinaa, Radmila R. Sharipovaa, Marionella A. Kravchenkob,
Olga V. Andreevaa, Olga B. Bazanovaa, and Vladimir E. Kataeva*
a Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy
of Sciences, Arbuzova str., 8, Kazan, 420088, Russia
b Ural Research Institute for Phtisiopulmonology, Ministry of Health Protection of the Russian
Federation, XX Parts’ezda str., 50, Yekaterinburg, 620039, Russia
Keywords: Glucosamine; Isosteviol; Glycoconjugates; Glycoterpenoids; Glycosides; Macrocyclic;
Tuberculosis; Antitubercular.
The first macrocyclic glycoterpenoids comprising glucosamine and diterpenoid isosteviol moieties
were synthesized and evaluated for inhibition activity against M. tuberculosis H37Rv.
1. Introduction
An increasing number of studies have been devoted to the isolation, purification, and structural
elucidation of natural macrocyclic glycosides. Among them a first place is occupied by resin
glycosides (or else fatty acid glycosides) which contain saturated fatty acids mainly jalapinolic acid
(11(S)-hydroxyhexadecanoic acid) and convolvulinolic acid (11(S)-hydroxytetradecanoic acid) as
the aglycon.1 A carbohydrate portion of resin glycosides is typically composed of two1b,c,e to
five1a,b,g,h monosaccharides as
D-glucose, D-fucose, L-rhamnose, and D-quinovose. Many resin
glycosides have cytotoxic,1a-c,e,i antibacterial,1b,i and antifungal1i effects. Perhaps one can single out
from the rank of resin glycosides the macrocyclic glycolipids isolated from the plant Cerastium
glomeratum, named glomerasides,1j and glycolipids isolated from different microorganisms, e.g.
cycloviracins,1i and macroviracins.1d,i Glomerasides were found to be unique 1,6-cyclic esters with
17-, 18- or 19-membered ring formed by
acid.1j Cycloviracins own C2-symmetrical macrodilactone core functionalized with two long side
polymethylene chains having several
-glucopyranose residues.1i Macroviracins are 42- to 46-
D-glucose and 9(R)-, 10(R)- or 11(R)-hydroxydocosanoic
D
membered macrodilactones composed of a glucopyranosyl C22 or C24 fatty acid dimer and a long
side polymethylene chain attached to the core.1d,i Both groups of glycolipids exhibit a powerful
antiviral activity.1d,i The following large group of macrocyclic glycosides contains phenols,1d,2
polyphenols1d or flavonoids1d as the aglycon. Among them cyclic dimers of 4-
(glycosyloxy)benzoates with two and four sugar residues showed inhibitory activity against α- and
β-glucosidases, lipoxygenase, and antioxidant potential.1d,2c Some polyphenols containing
macrocyclic glycosides demonstrated inhibitory activity against HIV-1 enzymes and have shown an
antimicrobial activity against human bacterial pathogens as well.1d The literature has provided
several examples of the macrocyclic glycosides having a terpenoid moiety as the aglycon.1b,i,3
These are glycosides urceolide3a and parkinsenes A-E3b which have some monoterpenoid acids as
the aglycon as well as
aglycon of the macrocyclic glycosides of the syphonoside series is diterpenoid clerodane and the
glycon is
-glucopyranose.3c,d The macrocyclic glycosides lobatosides A-E are 34-membered
macrocycles which are composed of triterpenoid oleanolic acid as the aglycon, and four or five
monosaccharides ( -glucose, -galactose, and
-arabinofuranose).1b,i All above mentioned
D D D D
-glucose,3a, -fucose,3b -quinovose,3b and -apiose3a as the glycon. The
D
D
D
L
macrocyclic terpenoid glycosides have demostrated one or another type of biological activity.
Monoterpene glycosides showed significant analgesis, anti-inflammatory, hepatoprotective, and
hypoglycemic activities.3b Diterpenoid glycoside syphonoside was able to inhibit high density
induced apoptosis.3c Lobatoside E belonging to the macrocyclic triterpenoid glycosides (saponins)
demonstrated a high potency to inhibit the growth of tumor cells.1i

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As far as we had reported on the synthesis of a large range of macrocycles constituted by one,
two or four molecules of diterpenoid steviol or isosteviol,4 the aborementioned publications about
naturally occurring macrocyclic terpenoid glycosides (or else macrocyclic glycoterpenoids3c) gave
us the impetus to synthesize macrocyclic derivatives of diterpenoid isosteviol that would also have
carbohydrate residues. Recently the first synthesis of macrocyclic glycoterpenoids composed of
diterpenoid isosteviol and monosaccharid (α,α’-trehalose or D-glucuronic acid) residues have been
reported.5 In continuation of these studies, herein we describe the first synthesis of macrocyclic
glycoterpenoids comprising glucosamine and isosteviol moieties. Their antituberculosis activities
were also evaluated.
2. Results and Discussion
Diterpenoid isosteviol 1 (16-oxo-ent-beyeran-19-oic acid6) obtained by acid hydrolysis of
commercially available sweetener Sweta7 and commercially available glucosamine hydrochloride 3
were used as starting compounds for the synthesis of target macrocyclic glycoterpenoids. General
strategy for their synthesis consists of four stages and it is shown in Scheme 1. In the first stage, two
isosteviol molecules are coupled with a linker attached to the atoms C16 of ent-beyerane skeletons
and carboxylic groups are functionalized by ω-hydroxypolymethylene chains that afford a terpenoid
precursor 2. In the second stage hydroxyl and amine groups of glucosamine hydrochloride 3 are
protected and anomeric center is brominated that give a carbohydrate precursor 4. In the next stage
terpenoid and carbohydrate precursors are coupled to afford diglycoside 5 which undergoes
macrocyclization in the final stage to provide a target macrocyclic glycoterpenoid 6.
Scheme 1. General strategy for the synthesis of macrocyclic glycoterpenoids. Designations: Ter
means terpenoid (isosteviol moiety), Sug means carbohydrate (glucosamine residue).
2.1.
Chemistry
2.1.1.
Synthesis of starting materials
To obtain terpenoid precursors, initially the 16-oxo group in isosteviol 1 was chemo- and
stereoselectively reduced with sodium borohydride by analogy with described procedure8 to give
dihydroisosteviol 7 (100% de). Then two molecules of dihydroisosteviol 7 were coupled with each
other by the reaction with sebacyl dichloride to afford the binuclear derivative of isosteviol 8.4a,9
This compound was choosen as the intermediate in the pathway towards terpenoid precursors
because it had exhibited the highest antitubercular activity in the series of binucleur isosteviol
derivatives.4f According to the X-ray crystal structure data4f diacid 8 has the maximum folding,
sandwich-like structure. Then diacid 8 was converted to its bis-acyl chloride that was involved in

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the reactions with 1,4-butandiol and 1,6-hexanediol to afford terpenoid precursors 9 and 105d in
56% and 60% yields.
OH
O
O
O
OH
O
iii, iv
O
O
HO
O
OH
O
9
O
O
O
O
i
ii
OH
O
OH
O
O
O
OH
OH
O
O
O
O
1
7
8
iii, iv
O
OH
O
O
O
10
O
Scheme 2. Reagents and conditions: (i) NaBH4, CH3OH; (ii) ClC(O)(CH2)8C(O)Cl, CH2Cl2,
DMAP; (iii) SOCl2, 50°C; (iv) HO(CH2)nOH, CH2Cl2 (n = 4 or 6).
To prepare carbohydrate precursor 13 (Scheme 3), according to the known procedure,10 the amine
group of glucosamine hydrochloride 3 was protected by 2,2,2-trichloroethoxycarbonyl (Troc) group
to give glucosamine derivative 11 which was per-O-acetylated, and then monosaccharide 12 was
treated with 33% HBr in AcOH in CH2Cl2 to afford glycosyl-donor 13. It is to be noted that exactly
Troc group was chosen for the protection of amine group of glucosamine for two reasons. Firstly,
N-Troc-protected glucosamine derivatives is more reactive than benzylidene or N-phthalyl-
protected derivatives.11 Secondly, the selective removal of the Troc group takes place in a rather
mild conditions under which acetate groups of glucosamine residues and ester groups of linkers are
not affected.12
Scheme 3. Reagents and conditions: (i) NaHCO3, TrocCl, H2O; (ii) Ac2O, Py; (iii) 33% HBr,
AcOH, CH2Cl2.
In the next stage diglycosides 5 (Scheme 1) containing glucosamine residues and isosteviol
moieties were synthesized by the reaction of bromide 13 with both terpenoid precursors 9 and 10.
By analogy with the literature13 bromide 13 was treated with diterpenoid diols 9 and 10 in the
presence of ZnCl2 (Scheme 4). The reactions provided diglycosides 14 and 15 in 20% and 22%
yields, respectively. It is worth noting that both reactions led to the formation of α-glycosides. This
1
was proved by the fact that the anomeric protons in diglycoside 14 resonated in the H NMR
spectrum as a doublet at 4.88 ppm with a vicinal coupling constant of 3.4 Hz, and the anomeric
protons in diglycoside 15 resonated as a doublet at 4.87 ppm with a vicinal coupling constant of 3.5

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Hz. The reason is the amazing property of the Lewis acid ZnCl2 to manifest itself as the
stereoselective activator of the classical Koenigs-Knorr reaction.13 In both reactions glycosides 16
and 17 formed as the byproducts (Scheme 4). Glycoside 16 was detected in the reaction mixture by
MALDI mass spectroscopy which revealed the peaks at 1436.9, [M+Na]+ (calcd. 1436.7 [M+Na]+
for C73H112Cl3NNaO19), and m/z 1452.9, [M+K]+ (calcd. 1452.7 [M+K]+ for C73H112Cl3KNO19).
Glycoside 17 was obtained in 9% yield after flash chromatography. In contrast to diglycosides 14
and 15, the 1H NMR spectrum of glycoside 17 showed methylene protons of the terminal CH2OH
group as a triplet at 3.62 ppm with a coupling constant 6.8 Hz. The MALDI spectrum of glycoside
17 exhibited the peaks at m/z 1493.1, [M+Na]+, and at m/z 1509.1, [M+K]+ corresponding to
molecular formulae C77H120Cl3NNaO19 and C77H120Cl3KNO19.
Scheme 4. Reagents and conditions: (i) bromide 13, ZnCl2, CH2Cl2; (ii) Zn, AcOH; (iii) Ac2O,
Et3N, CH2Cl2
2.1.2.
Synthesis of macrocyclic glycoterpenoids
For closing diglycosides 14 and 15 in target macrocycles it was necessary to remove Troc
groups and then to bind the amine groups by a suitable linker. Previously, during the synthesis of
macrocyclic derivatives of isosteviol by closing diacid 8 it was found that the yield of forming
macrocycle appeared to depend on the length of a linker which have to bind the carboxylic groups
of diacid 8.4f The maximum yield was achieved by using 1,8-octanediol as a linker, that is, the
length of a linker between two ester groups in the formed macrocycle should equal eight methylene
groups.4f Therefore sebacic acid chloroanhydride and 1,6-diisocyanatohexane were chosen as
linkers for macrocyclization of diglycosides 14 and 15. Beforehand we tested macrocyclization of
glucosamine residues with 1,6-diisocyanatohexane in glycoside 21 which was obtained from

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ketoalcohol 205d in 40% yield (Scheme 5). As noted above, since ZnCl2 was used as the catalytic
activator of Koenigs-Knorr reaction, glycoside 21 had α-orientation of the glycoside bond. This
1
followed from the anomeric proton signal in the H NMR spectrum which appeared as a single
doublet at 4.87 ppm with a vicinal coupling constant of 3.6 Hz. The amine group of glycoside 21
was deprotected by zinc dust in acetic acid12 to provide free amine 22 which then was involved in
the reaction with 1,6-diisocyanatohexane according to described procedure14 which afforded
diglycoside 24 in 13% yield, and glycoside 25 in 19% yield (Scheme 5). Following spectral data
confirmed the formation of diglycoside 24. Its MALDI spectrum showed peaks at m/z 1523.8,
[M+H]+, (calc. m/z 1523.9, [M+H]+, C80H123N4O24), m/z 1545.6, [M+Na]+, (calc. m/z 1545.8,
[M+Na]+, C80H122N4NaO24), and m/z 1561.6, [M+K]+, (calc. m/z 1561.8, [M+K]+, C80H122KN4O24).
The IR spectrum of diglycoside 24 demonstrated absorption bands at 1561, 1641, 3367, 3412 cm-1
which are typical to ureic moieties. The 1H NMR spectrun of diglycoside 24 indicated the presence
of ureic protons as a doublet at 4.90 ppm with a coupling constant of 5.7 Hz, and a triplet at 5.24
ppm with a coupling constant of 5.7 Hz. The MALDI spectrum of glycoside 25 showed peaks at
m/z 846.7, [M+H]+, (calc. m/z 846.5, [M+H]+, C44H68N3O13), m/z 868.7, [M+Na]+, (calc. m/z 868.5,
[M+Na]+, C44H67N3NaO13), and m/z 884.7, [M+K]+, (calc. m/z 884.4, [M+K]+, C44H67KN3O13). The
IR and 1H NMR spectra of glycoside 25 were similar to the spectra of diglycoside 24.
Scheme 5. Reagents and conditions (i) SOCl2, 50 °C; (ii) HO(CH2)nOH, CH2Cl2; (iii) bromide 13,
ZnCl2, CH2Cl2; (iv) Zn, AcOH; (v) Ac2O, Et3N, CH2Cl2; (vi) OCN(CH2)6NCO, CH2Cl2.
The tested approach to the macrocyclization of glucosamine residues with 1,6-diisocyanatohexane
linker was then used for the synthesis of target macrocyclic glycoterpenoids. Troc protective groups
in diglycosides 14 and 15 were removed by zinc dust in acetic acid12 to give diamines 26 and 27 in
good yields (96% and 87%). As a rule, free amines formed after removing Troc protective groups
were used without further purification in next steps.15 However, one of the diamines obtained,
namely diamine 27, has been isolated. Its MALDI spectrum showed peaks at m/z 1582.1, [M+H]+,
(calc. m/z 1582.0, [M+H]+, C86H136N2O24), m/z 1604.1, [M+Na]+, (calc. m/z 1603.9, [M+Na]+,
C86H136N2NaO24), and m/z 1620.1, [M+K]+ (calc. m/z 1619.9, [M+K]+, C86H136KN2O24). In the 1H
NMR spectrum of diamine 27 the anomeric protons resonated as a doublet at 4.85 ppm with a
vicinal coupling constant of 3.2 Hz indicating α-orientation of the glycoside bonds. The reactions of
diamines 26 and 27 with 1,6-diisocyanatohexane afforded the target macrocyclic glycoterpenoids

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28 and 29 (Scheme 6) in 40% and 15% yields. The MALDI spectrum of macrocycle 28 showed
peaks at m/z 1694.3, [M+H]+, (calc. m/z 1694.0, [M+H]+, C90H141N4O26), m/z 1716.3, [M+Na]+,
(calc. m/z 1716.0, [M+Na]+, C90H140N4NaO26), and m/z 1732.2, [M+K]+, (calc. m/z 1731.9,
[M+K]+, C90H140KN4O26). The MALDI spectrum of macrocycle 29 showed peaks at m/z 1750.7,
[M+H]+, (calc. m/z 1751.0, [M+H]+, C94H149N4O26), and m/z 1772.7, [M+Na]+, (calc. m/z 1773.0,
1
[M+Na]+, C94H148N4NaO26). The H NMR spectra of macrocycles 28 and 29 displaed the
methylene protons of the ureidic moieties NHC(O)NHCH2 as multiplets at 3.02-3.24 ppm, and
ureidic moieties own protons appeared as dublets at 4.92 ppm (3J = 10 Hz) and 4.96 ppm (3J = 10
Hz), as well as multiplets at 5.40-5.47 ppm and 5.21-5.30 ppm. Diglycoside 27 was also involved in
the reaction with sebacic acid chloroanhydride to give macrocyclic glycoterpenoid 30 in 23% yield
(Scheme 6). The MALDI spectrum of macrocycle 30 showed peaks at m/z 1771.1, [M+Na]+, (calc.
m/z 1771.0, [M+Na]+, C96H150N2NaO26), and 1787.1, [M+K]+, (calc. m/z 1787.0, [M+K]+,
C96H150KN2O26). The 1H NMR spectrum of macrocycle 30 showed the amidic protons as a doublet
at 5.82 ppm with a vicinal constant of 9.3 Hz. The anomeric protons of macrocyclic
glycoterpenoids 28-30 resonated as dublets at 4.83, 4.84, 4.83 ppm with coupling constants 3.4,
3.5, 3.6 Hz, respectively, that confirmed α-orientation of the glycoside bonds. It is to be noted that
diacid 31 was formed simultaneously with macrocycle 30 and was isolated in 20% yield after
column chromatography. The MALDI spectrum of glycoterpenoid 31 showed peaks at m/z 1973.2,
[M+Na]+, (calc. m/z 1973.2, [M+Na]+, C106H168N2O30Na), and m/z 1989.3 [M+K]+, (calc. m/z
1989.1, C106H168N2O30K).
2.2. Antituberculosis activity
Since both glucosamine16 and isosteviol17 included in the target macrocyclic glycoterpenoids
28-30 are biologically active molecules, there is no doubt that macrocycles 28-30 should have also
a broad spectrum of biological activity. As we have previously studied the ability of some
glycoterpenoids based on isosteviol and trehalose 32-34,5d as well as glucuronic acid 35, 365e (Fig.
1) to inhibit the in vitro growth of M. tuberculosis H37Rv,5d,e the target macrocyclic
glycoterpenoids 28-30 as well as some of their precursors 19, 23, 24, and byproduct 31 were also
screened against M. tuberculosis H37Rv. The obtained values of minimum inhibitory concentration
(MIC) are presented in Table 1. To analyse the data obtained, MIC values found for isosteviol 1,9
diacid 8,9 glycoconjugates of isosteviol and trehalose 32-34,5d glycocnjugates of isosteviol and
glucuronic acid 35, 36,5e 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-α- -glucopyranose 37,21 as
D
well as antitubercular drugs isoniazid and pyrazinamide are also presented in Table 1. The
comparison of MIC values indicates that antituberculosis activity of target macrocycles 28-30, their
precursors 19, 24, and diglycoside 31 is almost the same. Macrocyclic glycoterpenoids 345d and
36,5e as well as their precursors 325d and 355e showed a similar activity. One can see that
antituberculosis activities of isosteviol glycoconjugates 19, 24, 28-32, 34-36 are 19-13-fold lower
than the activity of antitubercular drug isoniazid in control experiments, but, at the same time, these
compounds appeared to be 8-17-fold better inhibitors than antitubercular drug pyrazinamide whose
MIC equals 101.5 µM.18 Several important conclusions can be drawn from an examination of
Table 1. Firstly, the binding of two molecules of isosteviol 1 (MIC 157 µM) with 1,8-octanedioate
linker increases its antituberculosis activity by 2.5 times (MIC for diacid 8 equals 62 µM). The
functionalization of isosteviol 1 with 3,4,6-tri-O-acetyl-glucosamine moiety increases its
antituberculosis activity by 9 times (MIC for glycoside 23 equals 17.4 µM). The functionalization
of isosteviol 1 with several 3,4,6-tri-O-acetyl-glucosamine moieties increases its antituberculosis
activity already by 22 times. This is another confirmation of the known fact that glycosylation of
bioactive compounds enhances their activity.19 By the way, the antituberculosis activity of
glucosamine itself, or rather, its derivative 37 (MIC 16.2 µM), was not changed after it had been
coupled with isosteviol to afford glycoside 23 (MIC 17.4 µM). Secondly, the antituberculosis
activity of macrocyclic glycoterpenoids investigated does not depend on the presence (or absence)
of nitrogen-containig functional groups. Thus MIC values for nitrogen-containig macrocyclic

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AcO
AcO
O
OAc
4s
OAc
OAc
6s
5s
OAc
3s
O
1s
2s H
N
O
O
HN
H2N
20
O
17
11
1
5
O
O
19
13
3
9
7
16
NH
O
O
O
O
O
O
15
O
18
2s'
H2N
O
OAc
OAc
OAc
OAc
4s'
HN
3s'
5s'
i
O
1s'
O
14
OAc
OAc
O
6s'
ii
O
O
O
O
18'
5'
19'
15'
13'
7'
9'
16'
O
O
O
3'
O
1'
11'
20'
26
28
17'
AcO
O
AcO
O
OAc
OAc
OAc
OAc
O
O
H
HN
H2N
N
O
O
O
O
O
NH
O
O
OAc
OAc
OAc
O
H2N
OAc
OAc
OAc
HN
O
i
O
O
O
15
O
O
O
O
ii
O
O
O
O
O
O
29
27
iii
AcO
AcO
O
OAc
OAc
OAc
OAc
O
O
OH
O
O
O
OH
HN
HN
O
O
O
O
O
O
O
O
O
O
OAc
OAc
OAc
OAc
OAc
OAc
HN
HN
O
O
O
O
O
+
O
O
O
O
O
O
O
O
O
30
31
Scheme 6. Reagents and conditions (i) Zn, AcOH; (ii) OCN(CH2)6NCO, CH2Cl2
(iii) ClOC(CH2)8COCl, CH2Cl2.

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O
O
O
O
O
O
O
O
O
O
O
O
O
AcO
AcO
AcO
O
O
O
AcO
AcO
AcO
O
O
AcO
OAc
OAc
O
AcO
O
O
O
O
O
OAc
OAc
34
O
O
O
O
O
HO
O
32
33
O
O
AcO
O
O
OAc
H2N
AcO
AcO
O
O
OAc
NH
AcO
O
O
HO
O
O
HO
O
OH
O
O
O
O
O
35
NH2
HN
O
O
O
O
O
36
O
O
OH
O
OH
O
O
HO
O
O
H
H
OH
N
OAc
O
N
HN
HO
NH
O
HO
OH
O
AcO
AcO
O
37
HO
OH
O
O
NH
OAc
Fig. 1. Glycoterpenoids which have already been synthesized and have been subjected to biological
evaluation as antituberculosis agents.5d,e
Table 1
In vitro inhibitory activities against the growth of M. tuberculosis H37Rv
Compound
MIC (µM)
157.09
62.09
Compound
MIC (µM)
1
31
6.4
12.75d
0.35d
8.35d
10.55e
9.25e
16.2
8
32
19
23
24
28
29
30
33
9.1
17.4
8.2
7.4
7.1
7.2
34
35
36
37
101.510
pyrazinamide
isoniazid
0.7
glycoterpenoids 28-30, 36 and the MIC value for macrocyclic glycoterpenoid 34 which has no
nitrogen atoms are almost the same. The striking conclusion that has been already marked5d is that

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glycoconjugate of trehalose and isosteviol 33 which has no nitrogen atoms showed the same
antituberculosis activity as antitubercular drug isoniazid in control experiment. This is all the more
surprising that both isosteviol 1,9 and trehalose20 are poor inhibitors of the growth of M.
tuberculosis H37Rv.9,20 Of course, this unexpected finding requires a careful study.
3. Conclusions
We have proposed for the first time a synthetic approach to macrocyclic glycoterpenoids
containing glucosamine and isosteviol moieties. The target macrocycles 28-30 as well as some of
their precursors 19, 23, 24 were examined for the ability to inhibit the in vitro growth of M.
tuberculosis H37Rv. All compounds tested showed moderate tuberculostatic activity. MIC values
for them were within range of 6.4-17.4 µM while MIC value for antitubercular drug isoniazid
(reference compound in experiment) was 0.7 µM.
4. Experimental
4.1. Chemistry
4.1.1. General
NMR experiments were carried out with Avance-400 or Avance-500 (Bruker) spectrometers
in CDCl3 at 400 or 500 MHz at 30 ºС. MALDI mass spectra were measured on DYNAMO
MALDI TOF instrument (Thermo BioAnalysis, Santa Fe, New Mexico). Samples were prepared as
0.1% solutions of compounds in an appropriate solvent. The matrix was p-nitroaniline (Acros). IR
spectra of the compounds 24, 25 were recorded with Bruker Vector-22 Fourier spectrometer in the
wavenumber range from 400 to 4000 cm-1. Melting points of substances were determined on a
BOETIUS compact heating table. Optical rotations were determined on a Perkin-Elmer 341
polarimeter (concentration c is given as g/100 mL) (PerkinElmer, Inc, USA) at 20 °C, λ = 589 nm.
The completeness of the reactions and the purity of the compounds were monitored by TLC on
Sorbfil plates (Sorbfil, Russia). Spots were detected by treatment with the 5% solution of sulfuric
acid, followed by heating up to 120 ºС. The isolation of individual substances 19, 21, 23-25, 28,
29–31 was performed with a flash chromatography on Silicagel KSKG (< 0.063 mm, Crom-Lab
Ltd, Russia). All solvents were dried according to standard protocols. Isosteviol 1,7
dihydroisosteviol 7,8 diacid 8,4a,9 diols 9, 10,5d compounds 13,10 20,5d and 3721 were prepared
according to the literature.4a,5d,7,8,9,10,21 The physicochemical properties of these compounds agreed
with those published. Sweetener Sweta was obtained from Stevian Biotechnology Corporation Sdn
Bhd (Malaysia), glucosamine hydrochloride 3 were purchased from abcr GmbH&Co.
4.1.2. Synthesis of compounds
4.1.2.1. General procedure for the synthesis of diglycosides (14-17)
ZnCl2 (2 equiv) was added to a solution of diol 9 or 10 (1 equiv) and bromide 13 (2.2-2.5
equiv) in CH2Cl2 under argon. The reaction mixture was stirred for 15 h at room temperature, then
was diluted with CH2Cl2, washed with 5% NaHCO3, water and brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by silica gel flash chromatography
(hexane/EtOAc = 5/1).
4.1.2.1.1. Bis{19-[3’,4’,6’-tri-O-acetyl-2’-deoxy-2’-(2”,2”,2”-trichloroethoxycarbonyl)amino-α-D-
glucopyranosyl-oxybutyl-4-oxycarbonyl]-ent-beyeran-16-yl}-1,8-octanedioat (14)
20
This compound was prepared as a white foam in 20% yield (0.37 g). [α]D +22.2 (с 1.40,
CH2Cl2); Anal. Calcd for C88H130Cl6N2O28: C, 56.32; H, 6.98; Cl, 11.33; N, 1.49. Found: C,
1
56.44; H, 6.97; Cl, 11.37; N, 1.49; Н NMR (400 MHz, СDСI3): δ 0.70 (s, 6Н, C20H3, C20’H3),
0.90 (s, 6Н, C17H3, C17’H3), 1.18 (s, 6Н, C18H3, C18’H3), 0.83–1.89 (m, 58Н, ent-beyerane skeleton,
two (СН2)2 linkers and (СН2)6 linker), 2.00 (s, 6Н, CH3CO, C’H3CO), 2.03 (s, 6Н, CH3CO,

ACCEPTED MANUSCRIPT
C’H3CO), 2.09 (s, 6Н, CH3CO, C’H3CO), 2.16 (d, 2Н, J = 13.0 Hz, C3Нeq, C3’Нeq), 2.30 (t, 4Н, J =
7.4 Hz, C16OC(O)СН2, C16’OC(O)СН2), 3.44–3.52 (m, 2H, C1sOCH2, C1s’OCH2), 3.70–3.78 (m,
2H, C1sOCH2, C1s’OCH2), 3.93–4.01 (m, 4H, H-5s, H-5s’, H-2s, H-2s’), 4.03–4.17 (m, 6H, H-6s, H-
6s’, C19(O)ОСН2, C19’(O)OСН2), 4.27 (dd, 2Н, J = 12.4, 4.6 Hz, H-6s, H-6s’), 4.64 (d, 2H, J =
12.1 Hz, 2 OCH2CCl3), 4.72 (dd, 2H, J = 10.5, 4.2 Hz, C16H, C16’H), 4.81 (d, 2H, J = 12.1 Hz, 2
OCH2CCl3), 4.89 (d, 2H, J = 3.4 Hz, H-1s, H-1s’), 5.10 (t, 2H, J = 9.9 Hz, H-4s, H-4s’), 5.24 (t,
2H, J = 9.9 Hz, H-3s, H-3s’), 5.29 (d, 2H, J = 9.8 Hz, 2 NH) ppm; MALDI-TOF MS m/z calcd for
C88H130Cl6N2O28Na [M+Na]+ 1899.7, found 1899.5; calcd for C88H130Cl6N2O28K [M+K]+ 1915.6,
found 1915.6.
4.1.2.1.2. Bis{19-[3’,4’,6’-tri-O-acetyl-2’-deoxy-2’-(2”,2”,2”-trichloroethoxycarbonyl)amino-α-D-
glucopyranosyl-oxyhexyl-6-oxycarbonyl]-ent-beyeran-16-yl}-1,8-octanedioat (15)
20
This compound was prepared as a white foam in 22% yield (0.52 g). [α]D +17.7 (с 2.05,
CH2Cl2); Anal. Calcd for C92H138Cl6N2O28: C, 57.17; H, 7.20; Cl, 11.01; N, 1.45. Found: C,
1
57.14; H, 7.19; Cl, 11.03; N, 1.44; Н NMR (400 MHz, СDСI3): δ 0.70 (s, 6Н, C20H3, C20’H3),
0.89 (s, 6Н, C17H3, C17’H3), 1.16 (s, 6Н, C18H3, C18’H3), 0.81–1.89 (m, 66 Н, ent-beyerane skeleton,
two (СН2)4 linkers and (СН2)6 linker), 1.99 (s, 6Н, CH3CO, C’H3CO), 2.02 (s, 6Н, CH3CO,
C’H3CO), 2.09 (s, 6Н, CH3CO, C’H3CO), 2.15 (d, 2Н, J = 12.8 Hz, C3Нeq, C3’Нeq), 2.30 (t, 4Н, J =
7.4 Hz, C16OC(O)СН2, C16’OC(O)СН2), 3.41–3.49 (m, 2H, C1sOCH2, C1s’OCH2), 3.65–3.73 (m,
2H, C1sOCH2, C1s’OCH2), 3.90–3.99 (m, 4H, H-5s, H-5s’, H-2s, H-2s’), 4.00–4.15 (m, 6H, H-6s,
H-6s’, C19(O)ОСН2, C19’(O)OСН2), 4.26 (dd, 2Н, J = 12.3, 4.6 Hz, H-6s, H-6s’), 4.64 (d, 2H, J =
12.1 Hz, 2 OCH2CCl3), 4.70 (dd, 2H, J = 10.5, 4.2 Hz, C16H, C16’H), 4.79 (d, 2H, J = 12.1 Hz, 2
OCH2CCl3), 4.87 (d, 2H, J = 3.5 Hz, H-1s, H-1s’), 5.09 (t, 2H, J = 9.8 Hz, H-4s, H-4s’), 5.24 (t,
2H, J = 9.8 Hz, H-3s, H-3s’), 5.37 (d, 2H, J = 9.9 Hz, 2 NH) ppm; MALDI-TOF MS m/z calcd for
C92H138Cl6N2O28Na [M+Na]+ 1955.7, found 1955.7; calcd for C92H138Cl6N2O28K [M+K]+ 1971.7,
found 1971.7.
4.1.2.1.3. O-{19-[3’,4’,6’-tri-O-acetyl-2’-deoxy-2’-(2”,2”,2”-trichloroethoxycarbonyl)amino-
glucopyranosyl-oxyhexyl-6-oxycarbonyl]-ent-beyeran-16-yl}-O’-[19-(6’-
α-D-
hydroxyhexyloxycarbonyl)-ent-beyeran-16-yl]-1,8-octanedioat (17)
20
This compouns was prepared as a white foam in 9% yield (0.16 g). [α]D -2.3 (с 0.75,
CH2Cl2); Anal. Calcd for C77H120Cl3NO19: C, 62.91; H, 8.23; Cl, 7.23; N, 0.95. Found: C, 62.95;
1
H, 8.21; Cl, 7.25; N, 0.94; Н NMR (400 MHz, СDСI3): δ 0.70 (s, 6Н, C20H3, C20’H3), 0.89 (s,
3Н, C17H3), 0.90 (s, 3H, C17’H3), 1.15 (s, 3H, C18H3), 1.16 (s, 3Н, C18’H3), 0.82–1.90 (m, 66Н, ent-
beyerane skeleton, two (СН2)4 linkers, and (СН2)6 linker), 2.00 (s, 3Н, CH3CO), 2.03 (s, 3Н,
CH3CO), 2.09 (s, 3Н, CH3CO), 2.16 (d, 2Н, J = 13.1 Hz, C3Нeq, C3’Нeq), 2.30 (t, 4Н, J = 7.4 Hz,
C16OC(O)СН2, C16’OC(O)СН2), 3.42–3.49 (m, 1H, C1sOCH2), 3.62 (t, 2H, J = 6.8 Hz,
C19OC(O)(CH2)5CH2OH), 3.66–3.74 (m, 1H, C1sOCH2), 3.91–4.15 (m, 7H, H-2s, H-5s, H-6s,
C19(O)ОСН2, C19’(O)OСН2), 4.26 (dd, 1Н, J = 12.4, 4.5 Hz, H-6s), 4.62–4.73 (m, 3H, OCH2CCl3,
C16H, C16’H), 4.80 (d, 1H, J = 12.2 Hz, OCH2CCl3), 4.88 (d, 1H, J = 3.6 Hz, H-1s), 5.10 (t, 1H, J
= 9.6 Hz, H-4s), 5.25 (t, 1H, J = 9.9 Hz, H-3s), 5.39 (d, 1 H, J = 9.7 Hz, NH) ppm; MALDI-TOF
MS m/z calcd for C77H120Cl3NO19Na [M+Na]+ 1492.7, found 1493.1; calcd. for C77H120Cl3NO19K
[M+K]+ 1508.7, found 1509.1.
4.1.2.2. O-{19-[3’,4’,6’-tri-O-acetyl-2’-deoxy-2’-acetamido-α-D-glucopyranosyl-oxyhexyl-6-
oxycarbonyl]-ent-beyeran-16-yl}-O’-[19-(6’-acetoxyhexyloxycarbonyl)-ent-beyeran-16-yl]-1,8-
octanedioat (19)
To a solution of monoglycoside 17 (0.12 g, 0.08 mmol) in AcOH (6 mL) Zn powdered (0.048
g, 0.8 mmol) was added under argon. The reaction mixture was stirred for 3 h at room temperature,
then was concentrated under reduced pressure. The residue diluted with CH2Cl2, washed with 5%
NaHCO3 and water and brine, dried over Na2SO4, and concentrated under reduced pressure to give
glicoside 18 (0.1 g, 95 %) which was used without further purification in the next step.

ACCEPTED MANUSCRIPT
Glycoside 18 0.10 g (0.08 mmol) was dissolved in CH2Cl2 (6 mL), then Et3N (0.068 mL, 0.49
mmol) and Ac2O (1 mL, 7.7 mmol) were added. The reaction mixture was stirred for 2 h at room
temperature, then diluted with CH2Cl2, washed with sat. NaHCO3 and 1.0 M HCl, dried over
Na2SO4, and concentrated under reduced pressure. Silica gel flash chromatography (eluent –
hexane/EtOAc = 5/1) of the residue afforded compound 19 in 57% yield (0.06 g) as a colourless oil.
20
[α]D +10.2 (с 1.00, CH2Cl2); Anal. Calcd for C78H123NO19: C, 67.95; H, 8.99; N, 1.02. Found:
1
C, 67.91; H, 8.96; N, 1.03; Н NMR (400 MHz, СDСI3): δ 0.70 (s, 6Н, C20H3, C20’H3), 0.90 (s,
6Н, C17H3, C17’H3), 1.15, 1.16 (2s, 6Н, C18H3, C18’H3), 0.78 - 1.90 (m, 66 Н, ent-beyerane skeleton,
two (СН2)4 linkers, and (СН2)6 linker), 1.95 (s, 3H, CH3CO, C’H3CO), 2.01 (s, 3Н, CH3CO,
C’H3CO), 2.03 (s, 3Н, CH3CO, C’H3CO), 2.04 (s, 3Н, CH3CO, C’H3CO), 2.09 (s, 3Н, CH3CO,
C’H3CO), 2.15 (d, 2Н, J = 13.1 Hz, C3Нeq, C3’Нeq), 2.30 (td, J = 7.4, 2.4 Hz, 4Н, C16OC(O)СН2,
C16’OC(O)СН2), 3.39–3.49 (m, 2H, C1sOCH2, C1s’OCH2), 3.61–3.71 (m, 2H, C1sOCH2, C1s’OCH2),
3.91–4.14 (m, 6H, H-5s, H-6s, C19(O)ОСН2, C19’(O)OСН2), 4.24 (dd, 1H, J = 12.3, 4.5 Hz, H-6s),
4.32–4.37 (m, 1H, H-2s), 4.71 (td, 2H, J = 9.8, 4.4 Hz, C16H, C16’H), 4.83 (d, 1H, J = 3.6 Hz, H-
1s), 5.11 (t, 1H, J = 9.5 Hz, H-4s), 5.21 (t, 1H, J = 9.5 Hz, H-3s), 5.78 (d, 1H, J = 9.5 Hz, NH)
ppm; MALDI-TOF MS m/z calcd for C78H123NO19Na [M+Na]+ 1400.9, found 1400.9; calcd for
C78H123NO19K [M+K]+ 1416.8, found 1416.9.
4.1.2.3. 4-[3’,4’,6’-tri-O-acetyl-2’-deoxy-2’-(2”,2”,2”-trichloroethoxycarbonyl)amino-α-D-
glucopyranosyl]-butyl-16-oxo-ent-beyeran-19-oat (21)
To a solution of ketoalcohol 20 (0.40 g, 1.02 mmol) and bromide 13 (0.71 g, 1.30 mmol) in
CH2Cl2 (15 mL) ZnCl2 (0.21 g, 1.54 mmol) was added under argon. The reaction mixture was
stirred for 30 h at room temperature, then was diluted with CH2Cl2, washed with 5% NaHCO3,
water and brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was
purified by silica gel flash chromatography (hexane/EtOAc = 3/1) to give glycoside 21 in 58%
20
yield (0,47 g) as a colourless oil. [α]D +1.0 (с 0.47, CH2Cl2); Anal. Calcd for C39H56Cl3NO13: C,
54.90; Cl, 12.47; H, 6.62; N, 1.64. Found: C, 55.11; Cl, 12.52; H, 6.59; N, 1.63; 1Н NMR (400
MHz, СDСI3): δ 0.69 (s, 3Н, C20H3), 0.94 (s, 3Н, C17H3), 1.18 (s, 3Н, C18H3), 0.80–1.90 (m, 22Н,
ent-beyerane skeleton and (СН2)2 linker), 1.97 (s, 3Н, CH3CO), 2.00 (s, 3Н, CH3CO), 2.06 (s, 3Н,
CH3CO), 2.16 (d, 1Н, C3Нeq, J = 13.3 Hz), 2.59 (dd, 1Н, C15 Hα, J = 18.6, 3.7 Hz), 3.42–3.49 (m,
1H, C1sOCH2), 3.68–3.75 (m, 1H, C1sOCH2), 3.89-3.95 (m, 1H, H-5s), 3.99 – 4.10 (m, 4H, H-6s,
H-2s, C19(O)ОСН2), 4.24 (dd, 1H, H-6s, J = 12.3, 4.5 Hz), 4.63 (d, 1Н, OCН2CCl3, J = 12.1 Hz),
4.76 (d, 1Н, OCН2CCl3, J = 12.1 Hz), 4.87 (d, 1H, H-1s, J = 3.6 Hz), 5.07 (t, 1H, H-4s, J = 9.8 Hz),
5.21 (t, 1H, H-3s, J = 9.7 Hz), 5.27 (d, 1H, NH, J = 9.7 Hz) ppm; MALDI-TOF MS m/z calcd for
C39H56Cl3NO13Na [M+Na]+ 876.3, found 876.4; calcd for C39H56Cl3NO13K [M+K]+ 892.2, found
892.4.
4.1.2.4. 4-(3’,4’,6’-tri-O-acetyl-2’-deoxy-2’-amino-α-D-glucopyranosyl)-butyl-16-oxo-ent-beyeran-
19-oat (22)
To a solution of glycoside 21 (0.47 g, 0.55 mmol) in AcOH (12 mL) Zn powdered (1.2 g, 18
mmol) was added under argon. The reaction mixture was stirred for 1 h at room temperature, then
was concentrated under reduced pressure. The residue diluted with CH2Cl2, washed with saturated
solution NaHCO3, dried over Na2SO4, and concentrated under reduced pressure to give glycoside
22 in 92% yield (0.34 g) as a colourless oil. Anal. Calcd for C36H55NO11: C. 63.79; H, 8.18; N,
2.07. Found: C, 63.44; H, 8.21; N, 2.05. 1Н NMR (400 MHz, СDСI3): δ 0.71 (s, 3Н, C20H3), 0.97
(s, 3Н, C17H3), 1.20 (s, 3Н, C18H3), 0.83–1.96 (m, 22Н, ent-beyerane skeleton and (СН2)2 linker),
2.01 (s, 3Н, CH3CO), 2.07 (s, 3Н, CH3CO), 2.08 (s, 3Н, CH3CO), 2.19 (d, 1Н, C3Нeq, J = 13.2 Hz),
2.61 (dd, 1Н, C15Hα, J = 18.5, 3.7 Hz), 2.93 (dd, 1Н, H-2s, J = 10.2, 3.6 Hz), 3.43–3.52 (m, 1H,
C1sOCH2), 3.71–3.79 (m, 1H, C1sOCH2), 3.92–3.99 (m, 1H, H-5s), 4.00–4.13 (m, 3H, H-6s,
C19(O)ОСН2), 4.28 (dd, 1H, H-6s, J = 12.2, 4.6 Hz), 4.86 (d, 1H, H-1s, J = 3.6 Hz), 4.95 (t, 1H, H-
4s, J = 9.8 Hz,), 5.11 (t, 1H, H-3s, J = 9.8 Hz) ppm; MALDI-TOF MS m/z calcd for
C36H56NO11 [M+H]+ 678.4; found 678.4; calcd for C36H55NO11Na [M+Na]+ 700.4; found 700.4.

ACCEPTED MANUSCRIPT
4.1.2.5. 4-[3’,4’,6’-tri-O-acetyl-2’-deoxy-2’-acetamido-α-D-glucopyranosyl]-butyl-16-oxo-ent-
beyeran-19-oat (23)
The mixture of Et3N (0.16 mL, 1.16 mmol) and Ac2O (2.4 mL, 18.29 mmol) was added to a
solution of amine 22 (0.13 g, 0.19 mmol) in CH2Cl2 (5 mL) under argon. The reaction mixture was
stirred for 2 h at room temperature, then diluted with CH2Cl2, washed with sat. NaHCO3 and 1.0 M
HCl, dried over Na2SO4, and concentrated under reduced pressure. Silica gel flash chromatography
(hexane/EtOAc = 1/1) of the residue afforded glycoside 23 in 73% yield (0.10 g) as a colorless oil.
20
[α]D +16.4 (с 0.65, CH2Cl2); Anal. Calcd for C38H57NO12: C, 63.40; H, 7.98; N, 1.95. Found: C,
1
63.54; H, 7.95; N, 1.96.01; Н NMR (400 MHz, СDСI3) : δ 0.72 (s, 3Н, C20H3), 0.97 (s, 3Н,
C17H3), 1.21 (s, 3Н, C18H3), 0.86–1.93 (m, 22Н, ent-beyerane skeleton and (СН2)2 linker), 1.96 (s,
3Н, CH3CO), 2.01 (s, 3Н, CH3CO), 2.02 (s, 3Н, CH3CO), 2.09 (s, 3Н, CH3CO), 2.19 (d, 1Н, C3Нeq,
J = 13.2 Hz), 2.63 (dd, 1Н, C15Hα, J = 18.5, 3.7 Hz), 3.41–3.49 (m, 1H, C1sOCH2), 3.70–3.78 (m,
1H, C1sOCH2), 3.88–3.94 (m, 1H, H-5s), 4.01–4.15 (m, 3H, H-6s, C19(O)ОСН2), 4.24 (dd, 1H, H-
6s, J = 12.3, 4.5 Hz), 4.31–4.38 (m, 1H, H-2s), 4.84 (d, 1H, H-1s, J = 3.6 Hz), 5.11 (t, 1H, H-4s, J
= 9.7 Hz,), 5.19 (t, 1H, H=3s, J = 9.8 Hz), 5.83 (d, 1H, NH, J = 9.4 Hz) ppm; MALDI-TOF MS m/z
calcd for C38H57NO12Na [M+Na]+ 742.4, found 742.3; calcd for C38H57NO12K [M+K]+ 758.4, found
758.3.
4.1.2.6. The synthesis of compounds (24) and (25)
A solution of 1,6-diisocyanatohexane (0.024 g, 0.14 mmol) in CH2Cl2 (1 mL) was added in
portions during 1 h to a solution of amine 22 (0.21 g, 0.29 mmol) in CH2Cl2 (10 mL) under argon.
The reaction mixture was stirred for 40 h at room temperature, then washed with water, and dried
over Na2SO4. The residue was purified by silica gel flash chromatography (hexane/EtOAc = 5/1) to
give diglycoside 24 and glycoside 25.
4.1.2.6.1. 1,6-Bis[2’-(16”-oxo-ent-beyeran-19”-carboxybutyl-6”’-oxy)-4’,5’-diacetoxy-6’-
methylacetoxy-tetrahydropyran-3’-ureidyl]hexane (24)
20
This compound was prepared in 13% yield (0.06 g) as a colorless oil. [α]D +27.7 (с 1.95,
CH2Cl2); Anal. Calcd for C80H122N4O24: C, 63.06; H, 8.07; N, 3.68. Found: C, 63.27; H, 8.05; N,
3.70; IR (film); ν 1561, 1641 [NHC(O)], 1726 [С(О)O], 1746 [CH3C(O)O], 3367, 3412 см-1 (NH);
1Н NMR (400 MHz, СDСI3) : δ 0.72 (s, 6Н, C20H3, C20’H3), 0.97 (s, 6Н, C17H3, C17’H3), 1.21 (s,
6Н, C18H3, C18’H3), 0.87–1.90 [m, 52Н, ent-beyerane skeleton, two (СН2)2 linkers, and (СН2)4
linker), 1.99 (s, 6Н, CH3CO, C’H3CO), 2.00 (s, 6Н, CH3CO, C’H3CO), 2.08 (s, 6Н, CH3CO,
C’H3CO), 2.16 (d, 2H, C3Нeq, C3’Нeq, J = 13.1 Hz), 2.64 (dd, 2H, C15 Hα, C15’ Hα, J = 18.5, 3.5 Hz),
3.00–3.21 (m, 4H, 2C(O)NHCH2), 3.36–3.45 (m, 2H, C1sOCH2, C1s’OCH2), 3.73–3.82 (m, 2H,
C1sOCH2, C1s’OCH2), 3.87–3.95 (m, 2H, H-2s, H-2s’), 4.04–4.26 (m, 10H, 2H-6s, 2H-6s’, H-5s,
H-5s’, C19(O)ОСН2, C19’(O)OСН2), 4.82 (d, 2H H-1s, H-1s’, J = 3.5 Hz), 4.90 (d, 2H 2 NHC(O), J
= 9.9 Hz), 5.05–5.19 (m, 4H, H-4s, H-4s’, H-3s, H-3s’), 5.24 (t, 2H, 2 C(O)NHCH2 J = 5.7 Hz)
ppm; MALDI-TOF MS m/z calcd for C80H123N4O24 [M+H]+ 1523.9, found 1523.8; calcd for
C80H122N4O24Na [M+Na]+ 1545.8, found 1545.6; calcd for C80H122N4O24K [M+K]+ 1561.8, found
1561.6.
4.1.2.6.2. 4’-[3”,4”,6”-tri-O-acetyl-2”-deoxy-2”-(ureidyl-6”’-isocyanatohexane)-α-D-
glucopyranosyl]-butyl-16-oxo-ent-beyeran-19-oat (25)
This compound was prepared in 19% yield (0.005 g) as a colorless oil. Anal. Calcd for
C44H67N3O13: C, 62.47; H, 7.98; N, 4.97. Found: C, 62.51; H, 8.01; N, 4.95; IR (film); ν 1561,
1
1641 ]NHC(O)], 1726 [С(О)O], 1746 [CH3C(O)O], 2272 (NCO), 3364, 3408 см-1 (NH); Н NMR
(400 MHz, СDСI3) : δ 0.74 (s, 3Н, C20H3), 0.98 (s, 3Н, C17H3), 1.23 (s, 3Н, C18H3), 0.87–1.90 (m,
30 Н, ent-beyerane skeleton, (СН2)2 linker, and (СН2)4 linker), 2.01 (s, 3Н, CH3CO), 2.04 (s, 3Н,
CH3CO), 2.09 (s, 3Н, CH3CO), 2.18 (d, 1H, C3Нeq, J = 13.5 Hz), 2.65 (dd, 1H, C15 Hα, J = 18.5, 3.7
Hz), 3,03–3.24 (m, 2H, C(O)NHCH2), 3.28 (t, 2H, CH2NCO, J = 6.6), 3.36–3.43 (m, 1H,

ACCEPTED MANUSCRIPT
C1sOCH2), 3.76–3.84 (m, 1H, C1sOCH2), 3.87–3.93 (m, 1H, H-2s), 4.03–4.26 (m, 5H, 2 H-6s, H-
5s, C19(O)ОСН2), 4.83 (d, 1H H-1s, J = 3.6 Hz), 4.87 (d, 1H, NHC(O), J = 9.8 Hz), 5.07–5.17 (m,
2H, H-3s, H-4s), 5.18–5.22 [m, 1H, C(O)NHCH2] ppm; MALDI-TOF MS m/z calcd for
C44H68N3O13 [M+H]+ 846.5, found 846.7; calcd for C44H67N3O13Na [M+Na]+ 868.5, found 868.7;
calcd for C44H67N3O13K [M+K]+ 884.4, found 884.7.
4.1.2.7. The general procedure for the synthesis of diamines (26, 27)
To a solution of diglycoside 14 or 15 (1 equiv) in AcOH was added Zn powdered (100 equiv)
under argon. The reaction mixture was stirred for 3 h at room temperature, then was concentrated
under reduced pressure. The residue was diluted with CH2Cl2, washed with 5% NaHCO3 and water
and brine, dried over Na2SO4, and concentrated under reduced pressure.
4.1.2.7.1. Bis{19-[3’,4’,6’-tri-O-acetyl-2’-deoxy-2’-amino-α-D-glucopyranosyl-oxybutyl-4-
oxycarbonyl]-ent-beyeran-16-yl}-1,8-octanedioat (26)
This compound was prepared as a white foam in 96% yield (0.11 g) and was used without
further purification in the next step.
4.1.2.7.2. Bis{19-[3’,4’,6’-tri-O-acetyl-2’-deoxy-2’-amino-α-D-glucopyranosyl-oxyhexyl-6-
oxycarbonyl]-ent-beyeran-16-yl}-1,8-octanedioat (27)
This compound was prepared as a white foam in 87% yield (0.13 g). Anal. Calcd for
1
C86H136N2O24: C, 65.29; H, 8.66; N, 1.77. Found: C, 65.53; H, 8.69; N, 1.78; Н NMR (400 MHz,
СDСI3) : δ 0.70 (s, 6Н, C20H3, C20’H3), 0.90 (s, 6Н, C17H3, C17’H3), 1.16 (s, 6Н, C18H3, C18’H3),
0.81–1.89 [m, 66 Н, ent-beyerane skeleton, two (СН2)4 linkers, and (СН2)6 linker), 2.02 (s, 6Н,
CH3CO, C’H3CO), 2.07 (s, 6Н, CH3CO, C’H3CO), 2.08 (s, 6Н, CH3CO, C’H3CO), 2.15 (d, 2Н, J =
13.5 Hz, C3Нeq, C3’Нeq), 2.30 (t, 4Н, J = 7.4 Hz, C16OC(O)СН2, C16’OC(O)СН2), 2.93 (dd, 2H, J =
10.2, 3.3 Hz, H-2s, H-2s’), 3.40–3.49 (m, 2H, C1sOCH2), C1s’OCH2), 3.66–3.75 (m, 2H, C1sOCH2,
C1s’OCH2), 3.89–4.13 (m, 8H, H-6s, H-6s’, C19(O)ОСН2, C19’(O)OСН2, H-5s, H-5s’), 4.28 (dd, 2H,
J = 12.3, 4.7 Hz, H-6s, H-6s’), 4.72 (dd, 2H, J = 10.5, 4.2 Hz, C16H, C16’H), 4.85 (d, 2H, J = 3.2
Hz, H-1s, H-1s’), 4.95 (t, 2H, J = 9.8 Hz, H-4s, H-4s’), 5.12 (t, 2H, J = 9.7 Hz, H-3s, H-3s’) ppm;
MALDI-TOF MS m/z calcd for C86H137N2O24 [M+H]+ 1582.0, found 1582.1; calcd for
C86H136N2O24Na [M+Na]+ 1603.9, found 1604.1; calcd for C86H136N2O24K [M+K]+ 1619.9, found
1620.1.
4.1.2.8. General procedure for the synthesis of macrocycles (28, 29)
1,6-Diisocyanatohexane (1 equiv) was added to a solution of diglycoside 26 or 27 (1 equiv) in
CH2Cl2 under argon. The reaction mixture was stirred for 40 h at room temperature, then was
concentrated under reduced pressure. The residue was purified by silica gel flash chromatography
(hexane/EtOAc = 5/1).
4.1.2.8.1. 2,13,16,20,35,40-Hexaoxa-22,24,31,33-tetraaza-1,14(16
α,4α)di(19-nor-ent-beyeran)-
21,34(1,2)di(3’,4’,6’-tri-O-acetyl-2’-deoxy-
α-D-glucopyranosyl)cyclohentetracontaphan-
3,12,15,23,32,41-hexaon (28)
This compound was prepared in 39.5% yield (0.05 g) as a white powder. Mp 106-108 °C
20
(hexane/EtOAc); [α]D +17.5 (с 0.83, CH2Cl2); Anal. Calcd for C90H140N4O26: C, 63.81; H, 8.33;
1
N, 3.31. Found: C, 63.77; H, 8.35; N, 3.32; Н NMR (400 MHz, СDСI3) : δ 0.69 (s, 6Н, C20H3,
C20’H3), 0.90, 0.92 (2s, 6Н, C17H3, C17’H3), 1.20 (s, 6Н, C18H3, C18’H3), 0.81–1.90 (m, 58Н, ent-
beyerane skeleton, two (СН2)2 linkers, and (СН2)6 linker), 1.99 (s, 6Н, CH3CO, C’H3CO), 2.00 (s,
6Н, CH3CO, C’H3CO), 2.09 (s, 6Н, CH3CO, C’H3CO), 2.13–2.19 (m, 2Н, C3Нeq, C3’Нeq), 2.27–
2.36 (m, 4Н, C16OC(O)СН2, C16’OC(O)СН2), 3.02–3.24 (m, 4H, 2C(O)NHCH2,), 3.36–3.44 (m,
2H, C1sOCH2, C1s’OCH2), 3.74–3.82 (m, 2H, C1sOCH2, C1s’OCH2), 3.87–3.95 (m, 2H, H-2s, H-2s’),
4.03–4.27 (m, 10 H, 2 H-6s, 2 H-6s’, C19(O)ОСН2, C19’(O)OСН2, H-5s, H-5s’), 4.66 (dd, 2H, J =
10.6, 4.4 Hz, C16H, C16’H), 4.83 (d, 2H, J = 3.4 Hz, H-1s, H-1s’), 4.92 [d, 2H, J = 10.0 Hz, 2

ACCEPTED MANUSCRIPT
NHC(O)], 5.06–5.18 (m, 4H, H-4s, H-4s’, H-3s, H-3s’), 5.40–5.47 (m, 2H, 2 C(O)NHCH2) ppm;
MALDI-TOF MS m/z calcd for C90H141N4O26 [M+H]+ 1694.0, found 1694.3; calcd for
C90H140N4O26Na [M+Na]+ 1716.0, found 1716.3; calcd for C90H140N4O26K [M+K]+ 1731.9, found
1732.2.
4.1.2.8.2. 2,13,16,23,38,45-Tetraoxa-25,27,34,36-tetraaza-1,14(16
α,4α)di(19-nor-ent-beyeran)-
24,37(1,2)di(3’,4’,6’-tri-O-acetyl-2’-deoxy-
α-D-glucopyranosyl)cyclohexatetracontaphan-
3,12,15,26,35,46-hexaon (29)
This compound was prepared in 14.5% yield (0.03 g) as a white powder. Mp 110-111 °C
20
(hexane/EtOAc); [α]D +16.7 (с 1.05, CH2Cl2); Anal. Calcd for C94H148N4O26: C, 64.51; H, 8.52;
1
N, 3.20. Found: C, 64.55; H, 8.49; N, 3.19; Н NMR (500 MHz, СDСI3) : δ 0.69 (s, 6Н, C20H3,
C20’H3), 0.90 (s, 6Н, C17H3, C17’H3), 1.16 (s, 6Н, C18H3, C18’H3), 0.84–1.88 (m, 66 Н, ent-beyerane
skeleton, two (СН2)4 linkers, and (СН2)6 linker), 1.99 (s, 6Н, CH3CO, C’H3CO), 2.00 (s, 6Н,
CH3CO, C’H3CO), 2.08 (s, 6Н, CH3CO, C’H3CO), 2.15 (d, 2Н, J = 13.5 Hz, C3Нeq, C3’Нeq), 2.27–
2.35 (m, 4Н, C16OC(O)СН2, C16’OC(O)СН2), 3.04–3.20 (m, 4H, 2C(O)NHCH2), 3.37–3.44 (m, 2H,
C1sOCH2, C1s’OCH2), 3.60–3.68 (m, 2H, C1sOCH2, C1s’OCH2), 3.89–4.03 (m, 4H, H-2s, H-2s’, H-
5s, H-5s’), 4.07–4.24 (m, 8H, 2 H-6s, 2 H-6s’, C19(O)ОСН2, C19’(O)OСН2), 4.66 (dd, 2H, J = 10.3,
4.1 Hz, C16H, C16’H), 4.84 (d, 2H, J = 3.5 Hz, H-1s, H-1s’), 4.96 [d, 2H, J = 10.0 Hz, 2 NHC(O)],
5.08 (t, 2H, J = 9.7 Hz, H-4s, H-4s’), 5.16 (t, 2H, J = 9.7 Hz, H-3s, H-3s’), 5.21–5.30 (m, 2
C(O)NHCH2) ppm; MALDI-TOF MS m/z calcd for C94H149N4O26 [M+H]+ 1751.0, found 1750.7;
calcd for C94H148N4O26Na [M+Na]+ 1773.0, found 1772.7.
4.1.2.9. Synthesis of macrocycle (30) and diglycoside (31)
A solution of sebacyl dichloride (0. 012 g, 0.05 mmol) in CH2Cl2 (3 mL) was added to a
solution of diglycoside 27 (0.08 g, 0.05 mmol) and pyridine (0.008 g, 0.1 mmol) in CH2Cl2 (5 mL)
under argon. The reaction mixture was stirred for 8 h at room temperature, then diluted with
CH2Cl2, washed with 0.1 M HCl and water, dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by flash chromatography (hexane/EtOAc = 1/1) to give
macrocycle 30 and diglycoside 31.
4.1.2.9.1. 2,13,16,23,38,45-Hexaoxa-25,36-diaza-1,14(16α,4α)di(19-nor-ent-beyeran)-
24,37(1,2)di(3’,4’,6’-tri-O-acetyl-2’-deoxy-
α
-D-glucopyranosyl)cyclohexatetracontaphan-
3,12,15,26,35,46-hexaon (30)
20
This compound was prepared in 23% yield (0.02 g) as a white foam. [α]D +13.8 (с 0.53,
CH2Cl2); Anal. Calcd for C96H150N2O26: C, 65.95; H, 8.65; N, 1.60. Found: C, 66.01; H, 8.62; N,
1
1.60; Н NMR (500 MHz, СDСI3) : δ 0.70 (s, 6Н, C20H3, C20’H3), 0.91 (s, 6Н, C17H3, C17’H3), 1.16
(s, 6Н, C18H3, C18’H3), 0.80–1.89 [m, 78Н, ent-beyerane skeleton, two (СН2)4 linkers, and two
(СН2)6 linkers), 2.00 (s, 6Н, CH3CO, C’H3CO), 2.03 (s, 6Н, CH3CO, C’H3CO), 2.09 (s, 6Н,
CH3CO, C’H3CO), 2.10–2.19 (m, 6H, C3Нeq, C3’Нeq, 2 NHC(O)CH2),
2.28–2.33 (m, 4Н,
C16OC(O)СН2, C16’OC(O)СН2,), 3.39–3.47 (m, 2Н, C1sOCH2, C1s’OCH2), 3.65–3.71 (m, 2H,
C1sOCH2, C1s’OCH2), 3.91–4.12 (m, 8Н, C19(O)ОСН2, C19’(O)ОСН2, H-6s, H-6s’, H-5s, H-5s’),
4.24 (dd, 2Н, J = 12.4, 4.5 Hz, H-6s, H-6s’), 4.32–4.38 (m, 2Н, H-2s, H-2s’), 4.68 (dd, 2Н, J =
10.2, 4.4 Hz, C16H, C16’H), 4.83 (d, 2Н, J = 3.6 Hz, H-1s, H-1s’), 5.11 (t, 2H, J = 9.9 Hz, H-4s,
H-4s’), 5.21 (t, 2H, J = 10.0 Hz, H-3s, H-3s’), 5.82 (d, 2H, J = 9.3 Hz, 2 NH) ppm; MALDI-TOF
MS m/z calcd for C96H150N2O26Na [M+Na]+ 1771.0, found 1771.1; calcd for C96H150N2O26
K
1787.0, found 1787.1.
4.1.2.9.2. Bis{19-[8-carboxy-2-octylacetamido-3,4,6-tri-O-acetyl-2-deoxy-
α
-D-glucopyranosyl-
oxyhexyl-6-oxycarbonyl]-ent-beyeran-16-yl}-1,8-octanedioat (31)
20
This compound was prepared in 20% yield (0.01 g) as a white foam. [α]D +4.4 (с 1.06,
CH2Cl2/MeOH = 3/1); Anal. Calcd for C106H168N2O30: C, 65.27; H, 8.68; N, 1.44. Found: C,
65.05; H, 8.70; N, 1.45; 1Н NMR (400 MHz, СDСI3) : δ 0.70 (s, 6Н, C20H3, C20’H3), 0.90 (s, 6Н,

ACCEPTED MANUSCRIPT
C17H3, C17’H3), 1.17 (s, 6Н, C18H3, C18’H3), 0.81–1.90 [m, 90Н, ent-beyerane skeleton, two (СН2)4
linkers and three (СН2)6 linkers), 2.01 (s, 6Н, CH3CO, C’H3CO), 2.03 (s, 6Н, CH3CO, C’H3CO),
2.09 (s, 6Н, CH3CO, C’H3CO), 2.11–2.20 (m, 6H, C3Нeq, C3’Нeq, 2 NHC(O)CH2), 2.28–2.37 (m,
8Н, C16OC(O)СН2, C16’OC(O)СН2, 2 CH2COOH), 3.38–3.47 (m, 2Н, C1sOCH2, C1s’OCH2), 3.64–
3.72 (m, 2H, C1sOCH2, C1s’OCH2), 3.90–4.12 (m, 8Н, C19(O)ОСН2, C19’(O)ОСН2, H-6s, H-6s’, H-
5s, H-5s’), 4.24 (dd, 2Н, J = 12.3, 4.1 Hz, H-6s, H-6s’), 4.33–4.39 (m, 2Н, H-2s, H-2s’), 4.70 (dd,
2Н, J = 10.5, 4.2 Hz, C16H, C16’H), 4.83 (d, 2Н, J = 3.4 Hz, H-1s, H-1s’), 5.11 (t, 2H, J = 9.8
Hz, H-4s, H-4s’), 5.21 (t, 2H, J = 9.5 Hz, H-3s, H-3s’), 5.81 (d, 2H, J = 9.1 Hz, 2 NH) ppm;
MALDI-TOF MS m/z calcd for C106H168N2O30Na [M+Na]+ 1973.2, found 1973.2; calcd for
C106H168N2O30K [M+K]+ 1989.1, found 1989.3.
4.2. In vitro assays
Compounds 19, 23, 24, 28-31 were tested for antituberculosis activity by the vertical diffusion
method on a Novaya solid nutrient medium using H37Rv laboratory strain as a test culture. The
nutrient medium was placed in 5 mL test tubes and inoculated with 0.1 mL of test culture diluted to
a turbidity of 10 units, and the test tubes were incubated for 24 h to grow M. tuberculosis. The test
tubes were then set vertically, and 0.3 mL of a solution of compounds 19, 23, 24, 28-31 in aqueous
EtOH with a concentration of 12.5, 6.2, 3.1, 1.5, 0.7, 0.35, or 0.1 ꢀg/mL was added dropwise (test
solutions were prepared by serial decimal dilutions of the initial solution of 100 µg of compounds
tested in the mixture of 5 mL of 96% ethanol and 5 mL of sterile distilled water). The test tubes
were incubated for 10 days at 37 °C under sterile conditions, and the zone of bacterial growth
inhibition was measured. An inhibition zone of longer than 10 mm indicated tuberculostatic
activity. Antitubercular drug isoniazid which was used as a control inhibited the in vitro growth of
M. tuberculosis at MIC 0.1 ꢀg/mL (0.7 µM).
Acknowledgments
The microbiological assay was performed under financial support of the Russian Science
Foundation (grant № 14-50-00014).
Appendix: Supplementary information
Supplementary information to this article can be found online at …
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The first macrocyclic glycoterpenoids comprising glucosamine and diterpenoid isosteviol moieties
were synthesized and evaluated for inhibition activity against M. tuberculosis H37Rv.