Regioselective synthesis, characterization and antimicrobial evaluation of S-glycosides and S,N-diglycosides of 1,2-Dihydro-5-(1H-indol-2-yl)-1,2,4- triazole-3-thione

Article abstract of DOI:10.1016/j.ejmech.2013.04.047

Glycosylation of 1,2-Dihydro-5-(1H-indol-2-yl)-1,2,4-triazole-3-thione with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide, 2,3,4,6-tetra-O- acetyl-α-D-galactopyranosyl bromide and 2-acetamido-3,4,6-tri-O-acetyl-2- deoxy-α-D-glucopyranosyl chloride was investigated in the presence of Et₃N and K₂CO₃ as acid scavengers. A regioselective S-glycosides were obtained by using Et₃N whereas, using K₂CO₃ gave a mixtures of two hybrids having two glycosidic bonds. The two products of each mixture were separated and characterized as S,N¹- and S,N²-bis(glycosylated) derivatives. The structures of the newly synthesized compounds were elucidated by ¹H NMR, ¹³C NMR, 2D NMR and mass spectra. The compounds were screened for their antibacterial and antifungal activities. Some compounds exhibited strong inhibition activity compared with the reference drugs (chloramphenicol and baneocin).

Full text of DOI:10.1016/j.ejmech.2013.04.047

Accepted Manuscript
Regioselective synthesis, characterization and antimicrobial evaluation of S-
glycosides and S,N-diglycosides of 1,2-Dihydro-5-(1H-indol-2-yl)-1,2,4-triazole-3-
thione
El Sayed H. El Ashry, El Sayed H. El Tamany, Mohy El Din Abd El Fattah, Ahmed
T.A. Boraei, Heba M. Abd El-Nabi
PII:
S0223-5234(13)00279-1
10.1016/j.ejmech.2013.04.047
EJMECH 6157
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 December 2012
Revised Date: 1 April 2013
Accepted Date: 20 April 2013
Please cite this article as: E.S.H. El Ashry, E.S.H. El Tamany, M.E.D.A. El Fattah, A.T.A. Boraei, H.M.
Abd El-Nabi, Regioselective synthesis, characterization and antimicrobial evaluation of S-glycosides and
S,N-diglycosides of 1,2-Dihydro-5-(1H-indol-2-yl)-1,2,4-triazole-3-thione, European Journal of Medicinal
Chemistry (2013), doi: 10.1016/j.ejmech.2013.04.047.
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ACCEPTED MANUSCRIPT
Highlights
•
•
Unsubstituted 1,2,4-triazole-3-thione was regioselectively glycosylated.
1
13
New structures were confirmed using H NMR, C NMR, 2D NMR and
mass spectra.
1
2
•
•
The differentiation between the S,N - and S,N -diglycosides was done by 2D
HMBC.
Synthesized compounds were tested for some pathogenic bacteria and fungi.

ACCEPTED MANUSCRIPT
Regioselective synthesis, characterization and antimicrobial evaluation of
S-glycosides and S,N-diglycosides of 1,2-Dihydro-5-(1H-indol-2-yl)-
1
,2,4-triazole-3-thione
El Sayed H. El Ashry, El Sayed H. El Tamany, Mohy El Din Abd El Fattah,
Ahmed T.A. Boraei, Heba M. Abd El-Nabi
(AcO)n
H
N
O
N
R
H
N
N
N
O
O
R
N
N
N
O
(
AcO)n
S
S
(AcO)n
(AcO)n
+
R
R
H
N
H
N
O
N
N
H
N
(AcO)n
+
H
N
N
O
N
H
R
LG
S
S
(AcO)n
R
S-Glycosides and S,N-diglycosides of 1,2-Dihydro-5-(1H-indol-2-yl)-1,2,4-triazole-3-
thione were regioselectively synthesized and tested for their antimicrobial activity
against pathogenic human and/or plant fungi and bactria.

ACCEPTED MANUSCRIPT
Regioselective synthesis, characterization and antimicrobial evaluation
of S-glycosides and S,N-diglycosides of 1,2-Dihydro-5-(1H-indol-2-yl)-
1
,2,4-triazole-3-thione
a,b,*
c
c
El Sayed H. El Ashry,
El Sayed H. El Tamany, Mohy El Din Abd El Fattah, Ahmed
a,c
d
T.A. Boraei, Heba M. Abd El-Nabi
a
HEJ Research Institute for Chemistry, International Center for Chemical and Biological
Sciences, Karachi University, Karachi, Pakistan
b
Chemistry Department, Faculty of science, Alexandria University, Alexandria, Egypt
c
Chemistry Department, Faculty of Science, Suez Canal University, Ismailia, Egypt
d
Microbiology Department, Faculty of Agriculture , Suez Canal University, Ismailia, Egypt
-
---------------------------------------------------------------------------------------------------------
A B S T R A C T
---------------------------------------------------------------------------------------------------------
-
Glycosylation of 1,2-Dihydro-5-(1H-indol-2-yl)-1,2,4-triazole-3-thione with 2,3,4,6-tetra-O-
acetyl-α-D-glucopyranosyl bromide, 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide and 2-
acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride was investigated in the
presence of Et N and K CO as acid scavengers. A regioselective S-glycosides were obtained by
3
2
3
using Et N whereas, using K CO gave a mixtures of two hybrids having two glycosidic bonds.
3
2
3
1
2
The two products of each mixture were separated and characterized as S,N - and S,N -
bis(glycosylated) derivatives. The structures of the newly synthesized compounds were
1
13
elucidated by H NMR, C NMR, 2D NMR and mass spectra. The compounds were screened
for their antibacterial and antifungal activities. Some compounds exhibited strong inhibition
activity compared with the reference drugs (chloramphenicol and baneocin).
Keywords:
Indolyltriazoles
1
,2,4-Triazoles
Glycosylation
Bis(glycosides)
Antimicrobial
*
Corresponding author, Tel.: +20 3 4246601: fax: +20 3 427 1360.
E-mail address: eelashry60@hotmail.com (E. S. H. El Ashry)
1

ACCEPTED MANUSCRIPT
1
. Introduction
The pharmacological activity of 1,2,4-triazoles has been extensively explored. Thus,
compounds possessing that ring exhibit anti-HIV, anti-HBV [1], antibacterial [2], antitumor [3],
anti-inflammatory [4], antiviral [5,6], CNS stimulant [7], and antituberculosis [8] activities.
Moreover, 1,2,4-triazole nucleus is found in many drugs such as anastrozole, estazolam,
ribavirin and triazolam (Figure 1). Anastrozole is used for treatment of breast cancer after
surgery and for metastases in post-menopausal women [9,10]. Estazolam and triazolam have
anxiolytic, anticonvulsant, sedative and skeletal muscle relaxant properties [11-13]. Ribavirin is
an anti-viral drug for severe RSV infection (individually), hepatitis C infection and other viral
infections [14]. 1,2,4-Triazole thioglycosides and nucleosides emerge a wide spectrum of
biological activities like antiproliferative [15], antiviral [16, 17] and antitumor [18]. The
carbohydrate moiety is expected to play the role of drug carrier and improve the selectivity of
compounds for cancerous cell lines [19].
Moreover, compounds containing indole nucleus have received considerable attention owing
to their biological importance [20]. Indole derivatives were reported to have antitumor [21],
anticonvulsant, tranquilizing [22], antibacterial, antifungal, antiviral [23], anti-inflammatory
[
24], and immunosuppressive [25] activities. Indole-3-acetic acid is a well known plant growth
hormone [26], tryptamine, homotryptamine, and isotryptamine derivatives have attracted
considerable interest as potent and selective serotonin [27] or melatonin [28] receptor ligands.
(Insert Figure 1)
In continuation of our research on the synthesis of glycosylated heterocycles and their acyclic
analogs [29-31], in this article we intended to study the glycosylation of unsubstituted 1,2,4-
triazole-thiones. Unsubstituted 1,2,4-triazole-thione has four nucleophilic centers which led to its
existence in different tautomeric forms I-V (Figure 2). All these forms are considered in the
literature and even in the electronic database, the actual structure is not proved even with the use
2

ACCEPTED MANUSCRIPT
1
3
1
of 1D C NMR and H NMR techniques and hence, the data on the properties of tautomers
partially duplicate each other [32-34].
Reaction of unsubstituted 1,2,4-triazoles with alkyling agents can lead to the attachment
of alky moiety to one or two of the nucleophilic centers as shown by arrows on I-V (Figure 2),
leading to difficulties in assigning the structures of products [32]. The determination of the
nucleophilic center which will be attacked by the glycosyl moiety will solve the problem that
makes most of researchers avoid the use of unsubstituted 1,2,4-triazole-thiones as starting
material.
(Insert Figure 2)
Consequently, many synthetic chemists avoided the use of unsubstituted 1,2,4-triazole-
thiones and instead, they used 4-substituted-1,2,4-triazole-thiones X as a precursor for
glycosylation and other alkylation reactions. Others have limited their alkylation to be on sulfur
only [49-53].
Di-alkylation of unsubstituted 1,2,4-triazole-thione were reported to give a mixture of
4
2
1
S,N - and S,N -bis(alkylated) derivatives [54-55] or yield mainly S,N -bis(alkylated) triazoles
2
[
[
56]. In addition, cyclization of S-alkylated 1,2,4-triazole-thiones can lead to ring closure at N -
4
51-59] or N - [60]. Accordingly, a solution for this problem should be introduced in an
unambiguous manner based on scientific data and reliable interpretation.
In this paper, we aim to investigate the selectivity during glycosylation of indolyl-1,2,4-
triazolethione and studying the effect of the synthesized compounds on some human and/or plant
pathogenic microbes in order to find new leads with potent inhibition activities.
2
. Results and discussion
2
.1 Chemistry
The selected heterocycle 1,2-Dihydro-5-(1H-indol-2-yl)-1,2,4-triazole-3-thione 5 was
previously prepared in 30 % yield by reaction of KSCN with indol-2-carbohydrazide 3 to give
the thiosemicarbazide 4 which was cyclized to 5 by heating in aqueous KOH, followed by
acidification with HCl [46]. In the present work, compound 5 was synthesized in a better yield
3

ACCEPTED MANUSCRIPT
from reaction of carbohydrazide 3 with NH SCN in presence of HCl to yield thiosemicarbazide 4
4
in 88 % yield. An intramolecular heterocyclization of 4 gave 5 in 81 % yield (Scheme 1).
(Insert Scheme 1)
Although, there are four probable nucleophilic centers in the heterocycle 5 that can react
with electrophiles, the sulfur atom is expected to be more nucleophilic and hence has the priority
for reaction [31]. Thus, glycosylation of 5 with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl
bromide 6, 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide 7 and 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-α-D-glucopyranosyl chloride 8 in the presence of Et N gave regioselectively the
3
thioglycosides 9-11, respectively. On the other hand, glycosylation in the presence of K CO
2
3
2
1
1
gave a mixture of S,N - and S,N -diglycosides in overall yield (80-90 %). The H NMR spectra
2
were used to determine their ratio the products, which showed that S-,N -bis(glycosylated)
1
products 12-14 were obtained in higher yields (56-67 %) than the S-,N -bis(glycosylated)
products 15-17 (22-28 %) (Scheme 2). Separation of the bis(glycosylated) mixtures were very
tedious because of the close R values of the isomers. Column chromatography and recycling
f
HPLC were not successful for their separation. Preparative TLC furnished good separation after
running the TLC sheet three times in the appropriate solvent. Moreover, the bis(glycosides) 12-
1
7 were also synthesized in almost the same yields by glycosylation of the corresponding
monoglycosides 9-11 in the presence of K CO .
2
3
1
13
The structures of the synthesized compounds were characterized by H NMR, C NMR,
1
2
1
D NMR and Mass spectra. The H NMR of 5 showed three D O exchangeable signals at 11.77,
2
3.67 and 13.86 ppm for the NH of the indole ring and the two NH of the triazole ring.
1
3
Moreover, its C NMR showed the two triazole carbons at 145.0 and 166.6 ppm which indicated
that the thione form is the most preferable tautomer for 5.
1
In the H NMR spectra of the monoglycosylated products 9-11, the anomeric protons
were observed as doublets at 5.61, 5.02 and 5.51 ppm respectively, with coupling constant values
greater than 8.0 Hz which confirm the β-configuration of these glycosides. The attachment of
4

ACCEPTED MANUSCRIPT
1
3
glycosyl moiety to sulfur clearly evidenced from the C NMR spectra of these compounds
which showed the two triazole carbons below 137.00 ppm, and the absence of C=S signal.
(Insert Scheme 2)
1
Di-glycosylation in compounds 12-17 has been realized from their H NMR spectra. The
spectra showed the disappearance of the two exchangeable proton signals of the triazole ring and
as a result two anomeric protons appeared as doublets with coupling constants around 10.5 and
1
1
1
13
9
.3 Hz, confirming the β-configuration. The 2D NMR spectra ( H- H DQFCOSY, H- C
1
13
HMQC and H- C HMBC) facilitated the spectral assignment of the sugar protons and carbons.
Heteronuclear multiple bond correlation (HMBC) were used for assigning the centers to
which the glycosyl moieties has attached. For example, the HMBC of the glucoside 12 showed
that the S-glucosyl anomeric proton H-1aGlc at 5.50 ppm and the N-glucosyl anomeric proton H-
3
1
7
bGlc at 5.63 ppm have a coupling correlation J with C-3Triazol at 150.67 ppm, while H-3Indol at
C,H
3
.11 ppm has J coupling correlation with C-5Triazol at 157.37 ppm, which strongly suggest that
C,H
2
1
the structure of 12 is S,N -bis(glucosylated) triazole (Figure 3). The H NMR spectrum 15
showed two doublets for the two anomeric protons at 5.46 and 5.71 ppm with J 10.4 and 9.2
1
,2
Hz for the S- and N-linked sugar moieties, respectively. The HMBC of 15 showed that the S-
3
anomeric proton has J coupling correlation with the carbon signal at 158.30 ppm, while the
C,H
3
N-anomeric proton and H-3Indol have J coupling correlation with the triazole carbon at 151.32
C,H
1
ppm indicating that the structure is the S,N -bis(glycosylated) product (Figure 3). Products 12,13
and 16,17 have showed the same coupling correlation as 12 and 15.
(Insert Figure 3)
2
.2 Antimicrobial Activity
P. aeruginosa, B. subtilis (bactria), A. niger, F. oxysporum and R. solani (fungi) were selected
as human and/or plant pathogenic microbes in order to test the inhibition activities of the
synthesized compounds. P. aeruginosa is a highly relevant opportunistic pathogen that causes
disease to animals, humans and plants. It can weaken immune system and can cause infections in
critical body organs, such as the lungs, the urinary tract, kidneys, skin, bones and joints, and is a
frequent nosocomial pathogen that causes a wide range of opportunistic infections [61-63].
5

ACCEPTED MANUSCRIPT
Bacilli cause an array of infections from ear infections to meningitis and urinary tract infections
to septicemia. Mostly they occur as secondary infections in immunodeficient hosts or otherwise
compromised hosts [64]. A. niger can cause a serious lung disease, aspergillosis if large amounts
of spores are inhaled. Moreover, A. niger is one of the most common causes of otomycosis
(fungal ear infections), which can cause pain, temporary hearing loss, and, in severe cases,
damage to the ear canal and tympanic membrane. In plants, A. niger can cause black mold of
onions and common postharvest disease of onions. The fungus also causes disease in peanuts and
in grapes [65, 66]. F. oxysporum is infamous for causing a condition called Fusarium wilt and as
a result the plant die. Additionally, F. oxysporum can cause disease in nearly every agriculturally
important plant [67]. Rhizoctonia solani can cause various plant diseases such as collar rot, root
rot, damping off and wire stem [68].
Data of the preliminary antimicrobial and the antifungal activities are shown in (Tables 1 and
2
). All the tested compounds exhibited high inhibition activities at 5 mg/mL concentration
against most tested microorganisms. In general, the highest antibacterial activity was displayed
by compounds 2, 11, 13+16, 5, 9 and 3 against P. aeruginosa (Gram negative), while compounds
1
4+17 , 12+15, 12, 4, 1 and 10 displayed good activities. Meanwhile, most of the synthesized
compounds have a strong (highly antibacterial activities against B. subtilis (Gram positive)
especially compounds 3, 4, 5, 9, 10, 14+17, and 1 that gave the same effect like the antibacterial
agent chloramphenicol. The rest of compounds had weak antibacterial activities when the same
concentration was used. Compounds 2, 1, 13+16, 12, 5, 14+17, 10, 3 and 12+15 exhibited good
antifungal activity against F. oxysporum and Rhizoctonia solani. On the other hand, all of the
tested compounds had no significant antifungal activity against Aspergillus niger.
(Insert Table 1)
(Insert Table 2)
3
. Conclusions
6

ACCEPTED MANUSCRIPT
In conclusion, regioselective glycosylation of 1,2-Dihydro-5-(1H-indol-2-yl)-1,2,4-
triazole-3-thione 3 with the α-acetylated-glycosyl halides was done in presence of Et N to give
3
1
2
the S-glycosides. However, in the presence of K CO , a mixture of S,N - and S,N -
2
3
bis(glycosylated)indolyl triazoles were obtained. The antimicrobial activity of the synthesized
compounds showed that some of them have good antifungal and antibacterial activities. The
results exhibit the importance of using these compounds in control of human and plant diseases.
4
. Experimental
4
.1. General
Melting points were determined with a melt-temp apparatus (SMP10) in open capillaries
and are uncorrected. Flash chromatography was performed on silica gel 60 (230-400 mesh
ASTM). TLC was performed on Merck silica gel 60 F254 and spots were visualized by UV light
absorption and/or treatment with a solution of 10 % H SO in aqueous methanol followed by
2
4
1
heating. H NMR spectra were recorded on Avance Bruker NMR spectrometer at 300-500 MHz
1
3
whereas C NMR spectra were done on the same instrument at 75-125 MHz, with TMS as
internal standard. Mass spectra were recorded on mass Finnegan (MAT312) and Jeol
(JMS.600H) for EIMS; HRMS were recorded on Thermo Finnegan (MAT 95XP) and Jeol (JMS
HX110).
4
4
.2. Synthesis of the target heterocycle 5
.2.1. 1-[(1H-Indol-2-yl)-carbonyl]-thiosemicarbazide (4)
A
mixture of indole-2-
carbohydrazide 1 (1.0 mmol) and ammonium thiocyanate NH SCN (2.0 mmol) in distlled water
4
(10 mL) and conc. HCl (1.5 mL) was heated under reflux for 3 h, then filtered while hot and
cooled at room temperature. The formed crystals were collected by filtration then washed with
distilled water and dried to give 2 as white solid in 88 % yield.
o
1
m.p. 272-274 C, R 0.39 (CH Cl /MeOH 9:1); H NMR (DMSO-d , 300 MHz) δ 7.03 (dd, 1 H,
f
2
2
6
J 7.89, J 7.35 Hz, H-5Indol), 7.16-7.21 (m, 2 H, H-3Indol, H-6Indol), 7.43 (d, 1 H, J6,7 8.25 Hz,
4
,5
5,6
H-7Indol), 7.61 (d, 1 H, J4,5 7.89 Hz, H-4Indol), 7.85 (br. s, 2 H, NH ), 9.37 (br. s, 1 H, NH), 10.38
2
1
3
(
br. s, 1 H, NH), 11.64 (br. s, 1 H, NHIndol); C NMR (DMSO-d , 75 MHz) δ 103.89 (C-3Indol),
6
1
12.27 (C-7Indol), 119.83 (C-5Indol), 121.64 (C-4Indol), 123.63 (C-6Indol), 126.89 (C-3aIndol), 129.51
7

ACCEPTED MANUSCRIPT
(
C-2Indol), 136.56 (C-7aIndol), 160.79 (C=O), 182.20 (C=S); LRMS (EI) m/z (Int. %): 59 (24), 63
14), 89 (94), 116 (49), 144 (100), 157 (18), 175 (53), 200 (26), 217 (96), 234 (48); HRMS (EI)
(
calcd for C H N OS (M): 234.0575. Found: 234.0572.
1
0
10
4
4
.2.2. 1,2-Dihydro-5-(1H-indol-2-yl)-1,2,4-triazole-3-thione (5) A solution of 4 (1.0 mmol)
in 4 N aq. KOH (25 mL) was heated under reflux for 3 h. The reaction mixture was cooled and
acidified with conc. HCl to give 3 as white solid in 81 % yield.
4
6
o
1
[
Lit. 294-296] m.p. 295-296 C, R 0.55 (CH Cl /MeOH 9:1); H NMR (DMSO-d , 300 MHz)
f 2 2 6
δ 7.04 (dd, 1 H, J 7.86, J 7.31 Hz, H-5Indol), 7.11 (s, 1 H, H-3Indol), 7.18 (dd, 1 H, J 7.31, J
6,7
4
,5
5,6
5,6
8
.17 Hz, H-6Indol), 7.41 (d, 1 H, J 8.17 Hz, H-7Indol), 7.61 (d, 1 H, J4,5 7.86 Hz, H-4Indol), 11.77
6,7
1
3
(
br. s, 1 H, NHIndol), 13.67 (br. s, 1 H, NHTriazol), 13.86 (br. s, 1 H, NHTriazol); C NMR (DMSO-
d6, 75 MHz) δ 102.98 (C-3Indol), 111.91 (C-7Indol), 119.90 (C-5Indol), 120.96, 123.38, 127.24 (C-
2Indol, C-4Indol, C-6Indol, C-3aIndol), 137.20 (C-7aIndol), 145.25 (C-5Triazol), 166.60 (C-3Triazol); LRMS
(EI) m/z (Int. %): 63 (6), 89 (15), 115 (14), 142 (42), 184 (6), 216 (100); HRMS (EI) calcd for
C H N S (M): 216.0470. Found: 216.0453.
10
8
4
4
.3. Glycosylation of 5 with α-halo-sugar-acetates 6-8 in the presence of TEA
The appropriate glycosyl halide (1.1 mmol) was added portionwise to a well stirred
mixture of 5 (1.0 mmol) and TEA (1.1 mmol), in dry acetone (10 mL) and stirring was continued
overnight. The mixture was filtered off, washed thoroughly with acetone and evaporated in
vacuo. The residues were containing only one product in each reaction (the S-glycosylated
products) which purified by crystallization from ethanol/n-hexane for the glucoside 9 or by flash
column chromatography (ethyl acetate/n-hexane 4:6) for galactoside 10 and (acetone/n-hexane
1
:1) for the N-acetylglucosyl amine 11.
4
.3.1. 3-(2,3,4,6-tetra-O-Acetyl-β-D-glucopyranosylsulfanyl)-5-(1H-indol-2-yl)-2H-1,2,4-
o
triazole (9) Colorless crystals in 70 % yield, m.p. 230-232 C, R 0.43 (ethyl acetate/n-hexane
f
1
6
:4); H NMR (300 MHz, DMSO-d ): δ 1.94, 1.95, 1.99, 2.00 (4s, 12 H, 4 CH CO), 4.01-4.14
6
3
(
m, 3 H, H-5 , H-6 , H-6ꢀ ), 4.96-5.08 (m, 2 H, H-2 , H-4 ), 5.39 (dd, 1 H, J 9.4 J 9.5
Glc Glc Glc Glc Glc 2,3 3,4
Hz, H-3 ), 5.61 (d, 1 H, J 8.7 Hz, H-1 ), 7.01-7.06 (m, 2 H, H-3Indol, H-5Indol), 7.16 (dd, 1H,
Glc
1,2
Glc
J 7.4 J 8.1 Hz, H-6 ), 7.44 (d, 1 H, J 8.1 Hz, H-7Indol), 7.60 (d, 1 H, J4,5 7.7 Hz, H-4Indol),
5
,6
6,7
Indol
6,7
8

ACCEPTED MANUSCRIPT
1
3
1
2
1.80 (br. s, 1 H, NHIndol) 14.70 (br. s, 1 H, NHTriazol); C NMR (75 MHz, DMSO-d ): δ 20.23,
6
0.32, 20.36 (4 CH CO), 61.72 (C-6 ), 67.84, 69.97 (C-2 , C-4 ), 72.88 (C-3 ), 74.76 (C-
3
Glc
Glc
Glc
Glc
5Glc), 82.70 (C-1 ), 100.80 (C-3Indol), 111.96 (C-7Indol), 119.80 (C-5Indol), 120.78, 122.97,
Glc
1
1
27.59, 136.88 (C-2Indol, C-4Indol, C-6Indol, C-3aIndol, C-7aIndol, C-3Triazol, C-5Triazol), 169.09, 169.21,
69.47, 169.90 (4 CH CO); LRMS (EI) m/z (Int. %): 81 (12), 109 (94), 169 (100), 216 (31), 331
3
(
10), 546 (5); HRMS (EI): m/z Calcd for C H N O S (M): 546.1420. Found: 546.1403.
24 26 4 9
4
.3.2. 3-(2,3,4,6-tetra-O-Acetyl-β-D-galactopyranosylsulfanyl)-5-(1H-indol-2-yl)-2H-1,2,4-
o
triazole (10) Colorless foams in yield 90 %, m.p. 124-125 C, R 0.42 (ethyl acetate/n-hexane
f
1
6
1
:4);; H NMR (600 MHz, CDCl ): δ 1.98, 2.09, 2.11, 2.13 (4s, 12 H, 4 CH CO), 4.02-4.04 (m,
3
3
H, H-5 ) 4.14 (dd, 1H, J 11.7, J 4.8 Hz, H-6 ), 4.24 (dd, 1 H, J 11.71, J 7.48 Hz,H-
Gal
gem
5,6
Gal
gem
5,6ꢀ
6
9
1
ꢀ ), 5.02 (br, 1 H, H-1 ), 5.09 (dd, 1 H, J 9.9 J 3.2 Hz, H-3 ), 5.32 (dd, 1 H, J 10.0 J
Gal Gal 2,3 3,4 Gal 1,2 2,3
.9 Hz, H-2 ), 5.46 (d, 1 H, J 3.2 Hz, H-4 ), 7.10-7.13 (m, 2 H, H-3Indol, H-5Indol), 7.22 (dd,
Gal
3,4
Gal
H, J 7.5 J 8.2 Hz, H-6 ), 7.41 (d, 1 H, J 8.2 Hz, H-7Indol), 7.64 (d, 1 H, J4,5 7.9 Hz, H-
5
,6
6,7
Indol
6,7
1
3
4Indol), 9.17 (br. s, 1 H, NHIndol) 11.67 (br. s, 1 H, NHTriazol); C NMR (75 MHz, CDCl ): δ 20.52,
3
2
8
1
1
0.55, 20.71 (4 CH CO), 61.90 (C-6 ), 67.27 (C-2 , C-4 ), 71.56 (C-3 ), 75.39 (C-5 ),
3 Gal Gal Gal Gal Gal
3.65 (C-1 ), 102.43 (C-3Indol), 111.39 (C-7Indol), 120.48 (C-5Indol), 121.41, 123.59, 128.42,
Gal
36.56 (C-2Indol,C-4Indol, C-6Indol, C-3aIndol, C-7aIndol, C-3Triazol, C-5Triazol), 169.76, 169.92, 170.06,
70.83 (4 CH CO); LRMS (ESI) m/z (Int. %): 272 (4), 331 (100), 547 (5); HRMS (ESI): m/z
3
Calcd for C H N O S (M+1): 547.1493. Found: 547.1745.
2
4
27
4
9
4
.3.3. 3-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosylsulfanyl)-5-(1H-indol-
o
2
-yl)-2H-1,2,4-triazole (11) Colorless crystals in 68 % yield, m.p. 185-186 C, R 0.33
f
1
(
acetone/n-hexane 1:1); H NMR (500 MHz, DMSO-d ): δ 1.80, 1.89, 1.93, 1.97 (4s, 12 H,
6
NHCOCH , 3 OCOCH ), 3.95-4.07 (m, 3 H, H-2GlcNAc, H-5GlcNAc, H-6
Gꢀ lcNAc), 4.17 (dd, 1 H, Jgem
3
3
1
2.2, J 4.9 Hz, H-6
), 4.91 (dd, 1 H, J 9.6, J 9.7 Hz, H-4
), 5.20 (dd, 1 H, J ≈
5
,6
ꢀ
GlcNAc
3,4
4,5
GlcNAc
2,3
J3,4 9.7 Hz, H-3GlcNAc), 5.51 (br, 1 H, H-1GlcNAc), 7.01-7.17 (m, 3 H, H-3Indol, H-5Indol, H-6Indol),
.44 (d, 1 H, J6,7 8.1 Hz, H-7Indol), 7.59 (d, 1 H, J4,5 6.9 Hz, H-4Indol), 8.21 (d, 1 H, J 8.6 Hz,
7
1
3
NHCOCH ),11.73 (br. s, 1 H, NHIndol), 14.29-14.57 (br, 1 H, NHTriazol); C NMR (125 MHz,
3
DMSO-d ): δ 20.29, 20.36, 22.58 (NHCOCH , 3 OCOCH ), 52.13 (C-2GlcNAc), 61.77 (C-6Glc),
6
3
3
6
8.49 (C-4GlcNAc), 73.25 (C-3GlcNAc), 74.95 (C-5GlcNAc), 83.83 (C-1GlcNAc), 101.45 (C-3Indol),
9

ACCEPTED MANUSCRIPT
1
3
11.93 (C-7Indol), 119.71 (C-5Indol), 120.70, 122.88, 127.66, 136.86 (C-2Indol, C-4Indol, C-6Indol, C-
aIndol, C-7aIndol, C-3Triazol, C-5Triazol), 169.26, 169.50, 169.57, 169.91 (4 CH CO); LRMS (ESI)
3
m/z (Int. %): 150 (10), 210 (5), 330 (100), 546 (4); (HRMS (ESI): m/z Calcd for C H N O S
2
4
28
5
8
(M+1): 546.1653. Found: 546.1924.
4
.4. Synthesis of the bis-glycosides 12-17
Method A The appropriate glycosyl halide (2.2 mmol) was added portion wise to a well stirred
mixture of 5 (1.0 mmol) and K CO (2.5 mmol), in dry acetone (10 mL) and stirring was
2
3
continued overnight. The mixture was filtered off, washed thoroughly with acetone and
1
2
evaporated in vacuo. The residues were containing both S-, N - and S-, N -glycosides that
purified by crystallization form ethanol in case of glucoside 12 to get it as colorless crystals and
preparative TLC for purifying the remaining glycosides using (ethyl acetate/n-hexane 4:6) as the
eluent for glucosides and galactosides and (CH Cl /MeOH 9.9:0.1) for the N-acetylglucosyl
2
2
amines.
Method B The appropriate glycosyl halide (1.1 mmol) was added portion wise to a well stirred
mixture of the monoglycosylated compounds 9-11 (1.0 mmol) and K CO (1.1 mmol), in dry
2
3
acetone (10 mL) and stirring was continued overnight. The workup was performed as mentioned
2
in glycosylation of 5 in the presence of K CO to get the S-,N -bis(glycosides) 12-14 (35-60 %
2
3
1
yield) and S-,N -bis(glycosides) 15-17 (20-32 % yield).
.4.1. 2-(2,3,4,6-tetra-O-Acetyl-β-D-glucopyranosyl)-3-(2,3,4,6-tetra-O-acetyl-β-D-
4
glucopyranosylsulfanyl)-5-(1H-indol-2-yl)-2H-1,2,4-triazole (12) Colorless crystals in a yield
o
6
7 % (Method A) and 65 % (Method B), m.p. 170-171 C, R 0.40 (ethyl acetate/n-hexane 6:4);
f
1
H NMR (300 MHz, CDCl ): δ 1.84, 1.97, 2.00, 2.018, 2.024, 2.05, 2.08, 2.09 (8s, 24 H, 8
3
CH CO), 3.96-3.99 (m, 2 H, H-5a , H-5b ), 4.10 (dd, 1 H J 12.6, J5a,6a 2.4 Hz, H-6aGlc),
gem
3
Glc
Glc
4
.15 (dd, 1 H Jgem 12.9, J5b,6b 2.1 Hz, H-6b ), 4.24-4.34 (m, 2 H, H-6
ꢀ
H-6 ꢀb ), 5.09 (dd, 1
Glc, Glc
Glc
H, J3a,4a 9.6 J4a,5a 9.9 Hz, H-4a ), 5.16 (dd, 1 H, J
10.5 J2a,3a 9.9 Hz, H-2a ), 5.25-5.40 (m, 3
Glc
Glc
1a,2a
H, H-3a , H-3b , H-4b ), 5.50 (d, 1 H, J
10.5 Hz, H-1a ), 5.63 (d, 1 H, J
9.3 Hz, H-
Glc
Glc
Glc
1a,2a
Glc
1b,2b
1
bGlc), 5.81 (dd, 1 H, J1b,2b 9.3 J2b,3b 9.0 Hz, H-2b ), 7.08-7.25 (m, 3 H, H-3Indol, H-5Indol, H-
Glc
6Indol), 7.41 (d, 1 H, J6,7 8.1 Hz, H-7Indol), 7.64 (d, 1 H, J4,5 7.8 Hz, H-4Indol), 8.98 (br. s, 1 H,
1
0

ACCEPTED MANUSCRIPT
1
3
NHIndol); C NMR (75 MHz, CDCl ): δ 20.30, 20.53, 20.64, 20.68 (8 CH CO), 61.49, 61.97 (C-
3
3
6
3
a , C-6b ), 67.57, 68.22 (C-4a , C-4b ), 69.79 (C-2b ), 70.01 (C-2a ), 73.18, 73.67 (C-
Glc Glc Glc Glc Glc Glc
a , C-3b ), 74.88, 76.52 (C-5a , C-5b ), 83.64 (C-1b ), 84.44 (C-1a ), 102.99 (C-
Glc
Glc
Glc
Glc
Glc
Glc
3Indol), 111.30 (C-7Indol), 120.36 (C-5Indol), 121.41 (C-4Indol), 123.54 (C-6Indol), 127.91, 128.143
C-2Indol, C-3aIndol), 136.56 (C-7aIndol), 150.67 (C-3Triazol), 157.37 C-5Triazol), 168.43, 169.22,
(
1
1
69.38, 169.49, 169.98, 170.19, 170.57, 170.61 (8 CH CO); LRMS (ESI) m/z (Int. %): 109 (20),
3
69 (100), 211 (13), 271 (30), 331 (54), 547 (17), 877 (18); HRMS (ESI): m/z Calcd for
C H N O S (M+1): 877.2495. Found: 877.2664.
3
8
45
4
18
4
.4.2.
2-(2,3,4,6-tetra-O-Acetyl-β-D-galactopyranosyl)-3-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosylsulfanyl)-5-(1H-indol-2-yl)-2H-1,2,4-triazole (13) White solid in a yield 59
o
1
%
(Method A) and 58 % (Method B), m.p. 119-121 C, R 0.41 (ethyl acetate/n-hexane 6:4); H
f
NMR (300 MHz, CDCl ): δ 1.86, 1.98, 2.00, 2.01, 2.04, 2.16, 2.22 (7 s, 24 H, 8 CH CO), 4.09-
3
3
4
4
3
2
.38 (m, 6 H, H-5a , H-5b , H-6a , H-6b , H-6
ꢀ
H-6 ꢀb ), 5.16-5.20 (m, 2 H, H-4a , H-
Gal
Gal
Gal
Gal
Gal, Gal Gal
bGal), 5.38 (dd, 1H, dd, 1 H, J1b,2b 9.2, J2b,3b 10.2 Hz, H-2b ), 5.48-5.57 (m, 3 H, H-1a , H-
Gal
Gal
a , H-3b ,), 5.67 (d, 1 H, J
9.2 Hz, H-1b ), 6.13 (dd, 1 H, J 9.9, J2a,3a 9.5 Hz, H-
1a,2a
Gal
Gal
1b,2b
Gal
aGal), 7.01 (s, 1 H, H-3Indol), 7.18 (dd, 1 H, J 8.0, J 7.4 Hz, H-5 ), 7.32 (dd, 1 H, J 7.4,
4,5
5,6
Indol
5,6
J 8.2 Hz, H-6 ), 7.48 (d, 1 H, J 8.2 Hz, H-7Indol), 7.67 (d, 1 H, J4,5 8.0 Hz, H-4Indol), 9.38
6
,7
Indol
6,7
1
3
(
br. s, 1 H, NHIndol); C NMR (100 MHz, CDCl ): δ 20.33, 20.51, 20.59, 20.63, 20.70 (8
3
CH CO), 61.11 (C-6a , C-6b ), 66.76, 67.14 (C-4a , C-4b ), 67.29, 68.37 (C-2a , C-
3
Gal
Gal
Gal
Gal
Gal
2
1
1
1
1
b ,), 71.17, 71.70 (C-3a , C-3b ), 73.77, 75.00 (C-5a , C-5b ), 84.76, 84.90 (C-1a , C-
Gal Gal Gal Gal Gal Gal
bGal), 102.76 (C-3Indol), 111.30 (C-7Indol), 120.33 (C-5Indol), 121.36 (C-4Indol), 123.45 (C-6Indol),
28.03, 128.47 (C-2Indol, C-3aIndol), 136.53 (C-7aIndol), 151.33 (C-3Triazol), 157.06 C-5Triazol),
67.72, 168.37, 169.80, 169.96, 170.09, 170.25, 170.60 (8 CH CO); LRMS (ESI) m/z (Int. %):
3
09 (11), 169 (85), 211 (15), 271 (5), 331 (100), 547 (17), 877 (8); HRMS (ESI): m/z Calcd for
C H N O S (M+1): 877.2444. Found: 877.2699.
3
8
45
4
18
4
.4.3.
2-(2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-3-(2-
acetamido-2-deoxy-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosylsulfanyl)-5-(1H-indol-2-
yl)-1H-1,2,4-triazole (14) White solid in a yield 56 % (Method A) and 57 % (Method B), m.p.
o
1
2
24-226 C, R 0.60 (CH Cl /MeOH 9:1); H NMR (500 MHz, DMSO-d ): δ 1.59, 1.79, 1.83,
f 2 2 6
1
1

ACCEPTED MANUSCRIPT
1
5
2
.946, 1.95, 1.97, 200, 2.01 (8s, 24 H, 8 CH CO), 3.90-4.28 (m, 7 H, H-2aGlcNAc, H-5aGlcNAc, H-
3
bGlcNAc, H-6aGlcNAc, H-6bGlcNAc, H-6
ꢀ
H-6
ꢀ
GlcNAc), 4.39 (dd, 1 H, J1b,2b ≈ J2b,3b 9.4 Hz, H-
GlcNAc,
bGlcNAc), 4.91 (dd, 1 H, J3a,4a 9.7 J4a,5a 9.6 Hz, H-4aGlcNAc), 5.00 (dd, 1 H, J3b,4b 9.9 J4b,5b 9.7 Hz,
H-4bGlcNAc), 5.21 (dd, 1 H, J2a,3a ≈ J3a,4a 9.7 Hz, H-3aGlcNAc), 5.48 (dd, 1 H, J2b,3b ≈ J3b,4b 9.9 Hz,
H-3bGlcNAc), 5.65 (d, 1 H, J1a,2a 10.8 Hz, H-1aGlcNAc), 5.94 (d, 1 H, J1b,2b 9.4 Hz, H-1bGlcNAc), 6.98
(d, 1 H, J 1.2 Hz, H-3Indol), 7.02 (dd, 1 H, J4,5 7.9, J5,6 7.4 Hz, H-5Indol), 7.14 (dd, 1 H, J5,6 7.4
Hz, J 8.2, H-6 ), 7.45 (d, 1 H, J 8.2 Hz, H-7Indol), 7.58 (d, 1 H, J4,5 7.9 Hz, H-4Indol), 8.05
6
,7
Indol
6,7
1
3
(
d, 1 H, J 8.5 Hz, NH Ac), 8.29 (d, 1 H, J 9.4 Hz, NH Ac), 11.62 (br. s, 1 H, NH ); C NMR
b a Indol
(
125 MHz, DMSO-d ): δ 20.08, 20.26, 20.31, 22.46, 22.37, 22.50 (8 CH CO), 51.82 (C-
6
3
2
4
1
aGlcNAc), 52.88 (C-2bGlcNAc), 61.53, 61.70 (C-6aGlcNAc, C-6bGlcNAc), 67.93 (C-4bGlcNAc), 68.43 (C-
aGlcNAc), 71.90 (C-3bGlcNAc), 73.15 (C-3aGlcNAc), 73.33, 75.21 (C-5aGlcNAc, C-5bGlcNAc), 82.57 (C-
bGlcNAc), 84.86 (C-1aGlcNAc), 101.56 (C-3Indol), 111.87 (C-7Indol), 119.52 (C-5Indol), 120.56 (C-
4Indol), 122.46 (C-6Indol), 127.63, 128.44 (C-2Indol, C-3aIndol), 136.94 (C-7aIndol), 151.19 (C-3Triazol),
56.63 (C-5Triazol), 169.21, 169.25, 169.42, 169.47, 169.51, 169.55, 169.82, 169.92 (8 CH CO);
1
3
LRMS (EI) m/z (Int. %): 43 (100), 84 (39), 108 (24), 125 (60), 184 (14), 216 (99), 330 (31), 365
(
7), 485 (5), 545 (13), 874 (2); HRMS (ESI): m/z Calcd for C H N O S (M+1): 875.2763.
38 47 6 16
Found: 875.3068.
4
.4.4.
1-(2,3,4,6-tetra-O-Acetyl-β-D-glucopyranosyl)-3-(2,3,4,6-tetra-O-acetyl-β-D-
glucopyranosylsulfanyl)-5-(1H-indol-2-yl)-1H-1,2,4-triazole (15) White solid in a yield 22 %
o
1
(
Method A) and 24 % (Method B), m.p. 103-105 C, R 0.44 (ethyl acetate/n-hexane 6:4); H
f
NMR (600 MHz, CDCl ): δ 1.89, 1.99, 2.00, 2.041, 2.042, 2.045, 2.06, 2.08 (8s, 24 H, 8
3
CH CO), 3.99-4.39 (m, 6 H, H-5a , H-5b , H-6a , H-6b , H-6
ꢀ
H-6 ꢀb ), 5.15-5.19 (m,
Glc, Glc
3
Glc
Glc
Glc
Glc
2
H, H-2aGlc, H-4a ), 5.25 (dd, 1 H, J
≈ J4b,5b 10.2 Hz, H-4b ), 5.33 (dd, 1 H, dd, 1 H,
3b,4b Glc
Glc
J2a,3a ≈ J3a,4a 9.6 Hz, H-3a ), 5.38 (dd, 1 H, J
9.6, J3b,4b 10.2 Hz, H-3b ), 5.46 (d, 1 H,
Glc
Glc
2b,3b
J1a,2a 10.2 Hz, H-1a ), 5.70 (d, 1 H, J
9.0 Hz, H-1b ), 6.02 (dd, 1 H, J
≈ J2b,3b 9.6 Hz,
Glc
1b,2b
Glc
1b,2b
H-2b ), 6.91 (d, 1 H, J 1.2 Hz, H-3 ), 7.19 (dd, 1 H, J 7.8 J 7.2 Hz, H-5Indol), 7.33 (dd,
Glc
Indol
4,5
5,6
1
H, J 7.2 J 7.8 Hz, H-6 ), 7.47 (d, 1 H, J 7.8 Hz, H-7Indol), 7.67 (d, 1 H, J4,5 7.8 Hz, H-
5,6 6,7 Indol 6,7
1
3
4Indol), 9.38 (br. s, 1 H, NHIndol); C NMR (150 MHz, CDCl ): δ 20.57, 20.63, 20.68, 20.70,
3
2
6
0.72, 20.76, 20.86 (8 CH CO), 62.21, 62.27 (C-6a , C-6b ), 67.94 (C-4b ), 68.33 (C-4a ),
3 Glc Glc Glc Glc
8.99 (C-2b ), 70.04 (C-2a ), 73.35 (C-3b ), 73.92 (C-3a ), 74.42 (C-5b ), 75.76 (C-
Glc
Glc
Glc
Glc
Glc
1
2

ACCEPTED MANUSCRIPT
5
1
1
1
a ), 83.33 (C-1a ), 83.72 (C-1b ), 104.93, (C-3Indol), 111.83 (C-7Indol), 121.23 (C-5Indol),
Glc
Glc
Glc
21.62 (C-4Indol), 122.71 (C-2Indol), 125.13 (C-6Indol), 128.02 (C-3aIndol), 136.77 (C-7aIndol),
51.32 (C-5Triazol), 158.30 (C-3Triazol), 168.65, 169.37, 169.43, 169.58, 170.23, 170.31, 170.55,
70.94 (8 CH CO); LRMS (ESI) m/z (Int. %): 109 (22), 123 (100), 169 (21), 331 (10), 877 (20);
3
HRMS (EI): m/z Calcd for C H N O S (M): 876.2371. Found: 876.2328.
3
8
44
4
18
4
.4.5.
1-(2,3,4,6-tetra-O-Acetyl-β-D-galactopyranosyl)-3-(2,3,4,6-tetra-O-acetyl-β-D-
galactopyranosylsulfanyl)-5-(1H-indol-2-yl)-1H-1,2,4-triazole (16) White solid in a yield 27
o
%
(Method A) and 28 % (Method B), Colorless crystals, m.p. 111-113 C, R 0.45 (ethyl
f
1
acetate/n-hexane 6:4); H NMR (400 MHz, CDCl ): δ 1.86, 1.98, 2.00, 1.98, 2.00, 2.01, 2.04,
3
2
.16, 2.22 (7 s, 24 H, 8 CH CO), 4.15-4.25 (m, 6 H, H-5a , H-5b , H-6a , H-6b , H-6ꢀa
3 Gal Gal Gal Gal Gal,
H-6
ꢀ
b ), 5.17-5.20 (m, 2 H, H-3a , H-3b ), 5.38 (dd, 1 H, J
10.3, J2a,3a 10.0 Hz, H-2aGal),
Gal
Gal
Gal
1a,2a
5
.48 (d, 1 H, J3a,4a 3.1 Hz, H-4a ), 5.51 (d, 1 H, J
10.3 Hz, H-1a ), 5.56 (d, 1 H, J
2.7
Gal
1a,2a
Gal
3b,4b
Hz, H-4bGal), 5.67 (d, 1 H, J1b,2b 9.2 Hz, H-1b ), 6.13 (dd, 1 H, J
9.2 J2b,3b 9.7 Hz, H-2bGal),
Gal
1b,2b
7
.18 (dd, 1 H, J4,5 8.1, J 7.2 Hz, H-5Indol), 7.24 (s, 1 H, H-3Indol), 7.32 (dd, 1 H, J 7.2, J 8.7
5,6 5,6 6.7
Hz, H-6Indol), 7.48 (d, 1 H, J 8.7 Hz, H-7Indol), 7.67 (d, 1 H, J4,5 8.2 Hz, H-4Indol), 9.48 (br. s, 1
6,7
1
3
H, NHIndol); C NMR (100 MHz, CDCl ): δ 20.50, 20.57, 20.65, 20.70 (8 CH CO), 61.12, 61.90
3
3
(C-6a , C-6b ), 66.43, 67.11, 67.32, 67.42 (C-2a , C-2b , C-4a , C-4b ), 71.61, 71.99
Gal Gal Gal Gal Gal Gal
(
C-3a , C-3b ), 73.75, 74.52 (C-5a , C-5b ), 83.93 (C-1a ), 84.82 (C-1b ), 105.14 (C-
Gal
Gal
Gal
Gal
Gal
Gal
3Indol), 111.78 (C-7Indol), 121.12 (C-5Indol), 121.61 (C-4Indol), 122.97 (C-2Indol), 124.98 (C-6Indol),
1
1
1
28.09 (C-3aIndol), 136.82 (C-7aIndol), 151.17 (C-5Triazol), 158.25 (C-3Triazol), 168.43, 169.50,
69.93, 170.00, 170.15, 170.28, 170.35, 170.52 (8 CH CO); LRMS (EI) m/z (Int. %): 69 (11),
3
09 (41), 115 (12), 127 (24), 139 (15), 144 (20), 169 (100), 186 (22), 546 (6), 876 (16); HRMS
(
ESI): m/z Calcd for C H N O S (M+1): 877.2444. Found: 877.2699.
38 45 4 18
4
.4.6.
1-(2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-3-(2-
acetamido-2-deoxy-3,4,6-tri-O-acetyl-2-deoxy-β-D-5-(1H-indol-2-yl)-
glucopyranosylsulfanyl)-2H-1,2,4-triazole (17) White solid in a yield 24 % (Method A) and
o
1
2
5 % (Method B), m.p. 92-94 C, R 0.54 (CH Cl /MeOH 9:1); H NMR (500 MHz, DMSO-d ):
f
2
2
6
δ 1.67, 1.79, 1.94, 1.95, 1.96, 1.99, 2.03 (8s, 24 H, 8 CH CO), 3.97 (dd, 1 H, J
10.3, J2a,3a 9.7
3
1a,2a
Hz, H-4aGlcNAc), 4.02-4.22 (m, 5 H, H-5aGlcNAc, H-6aGlcNAc, H-6bGlcNAc, H-6
ꢀ
H-6 ꢀb GlcNAc),
GlcNAc,
1
3

ACCEPTED MANUSCRIPT
4
9
.23 (m, 1 H, H-5bGlcNAc), 4.67 (dd, 1 H, J1b,2b ≈ J2b,3b 9.5 Hz, H-2bGlcNAc), 4.92 (dd, 1 H, J3a,4a
.7, J4a,5a 9.8 Hz, H-4aGlcNAc), 5.02 (dd, 1 H, J3b,4b ≈ J4b,5b 9.8 Hz, H-4bGlcNAc), 5.19 (d, 1 H, J2a,3a
≈
J3a,4a 9.7 Hz, H-3aGlcNAc), 5.41-5.46 (m, 2 H, H-1aGlcNAc, H-3bGlcNAc), 6.18 (d, 1 H, J1b,2b 9.5 Hz,
H-1bGlcNAc), 7.08 (s, 1 H, H-3Indol), 7.11 (dd, 1 H, J4,5 7.5, J5,6 7.7 Hz, H-5Indol), 7.24 (dd, 1 H,
J5,6 7.5 Hz, J6,7 8.2, H-6Indol), 7.49 (d, 1 H, J6,7 8.2 Hz, H-7Indol), 7.68 (d, 1 H, J4,5 7.8 Hz, H-
4Indol), 7.99 (d, 1 H, J 8.6 Hz, NH Ac), 8.16 (d, 1 H, J 9.2 Hz, NH Ac), 11.85 (br. s, 1 H,
b
a
1
3
NHIndol); C NMR (125 MHz, DMSO-d ): δ 20.30, 20.35, 20.39, 22.50, 22.60 (8 CH CO), 51.45
6
3
(C-2bGlcNAc), 52.18 (C-2aGlcNAc,), 61.75, 61.79 (C-6aGlcNAc, C-6bGlcNAc), 68.02 (C-4bGlcNAc), 68.51
(C-4aGlcNAc), 72.56 (C-3bGlcNAc), 73.13 (C-3aGlcNAc,), 73.21, 74.79 (C-5aGlcNAc, C-5bGlcNAc), 82.56
(C-1bGlcNAc), 84.05 (C-1aGlcNAc), 104.41 (C-3Indol), 112.07 (C-7Indol), 120.08 (C-5Indol), 121.20 (C-
4Indol), 123.78 (C-6Indol), 127.67, 128.58 (C-2Indol, C-3aIndol), 136.96 (C-7aIndol), 150.79 (C-5Triazol),
57.28 (C-3Triazol), 169.22, 169.28, 169.34, 169.54, 169.59, 169.86, 169.91 (8 CH CO); LRMS
1
3
(ESI) m/z (Int. %): 150 (5), 210 (3), 330 (100), 546 (60), 875 (5); HRMS (ESI): m/z Calcd for
C H N O S (M+1): 875.2763. Found: 875.3068.
3
8
47
6
16
4
4
.5 Antimicrobial activity
.5.1 Bacteria Strains
Two isolates bacteria Pseudomonas auroginosa (gram negative) and Bacillus subtilis
(positive bacteria) were used to evaluate the antibacterial activity of the synthesized compounds.
4
.5.2 Fungal Strains
Isolates of plant pathogenic fungi Fusarium oxysporum, Rhizoctonia solani and
Aspergillus niger were used to study the antifungal activity.
4
.5.3 Tested Compounds
Tested compounds (5.0 mg) were dissolved in 1.0 mL dimethylsulphoxide (DMSO) to
make the test concentration.
4
.5.4 Determination of antibacterial and antifungal activities
1
4

ACCEPTED MANUSCRIPT
Agar-diffusion technique was used for the determination of the preliminary antibacterial
[
69]. A suspension of organisms were added to sterile nutrient agar media at 45⁰ C and the
mixture was transferred to sterile Petri dishes and allowed to solidify. Holes of 10 mm in
diameter were made using a cork borer. An amount of 100 µL of the synthesized compounds was
poured inside the holes. A hole filed with DMSO was used as a control. The plates were then
incubated at 37⁰ C for 24 h. and observed for the bacterial activity. Antifungal activity of each
compound (5 mg/ mL) was evaluated by the paper disc diffusion method [70].
All the fungal cultures were inoculated onto PDA (potato dextrose agar) plates. Sterile filter
paper discs (10 mm for fungi) were placed on these cultures and impregnated with DMSO were
placed on the culture plates. Chloramphenicol and baneocin were used as reference drugs for
bacteria (gram positive and gram negative) and fungi, respectively. The results were recorded
for each tested compound as average diameter of inhibition zones of bacterial or fungal around
the disks in mm. Two replicate were conducted against each organism.
Acknowledgements
The support from the Higher Education Commission (HEC) Pakistan (Project No. 20-
6
97/R&D/06/38) is highly appreciated.
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2
0

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Captions
Table 1. In vitro antifungal activity of the tested compounds.
Table 2. In-vitro Antibacterial activity for tested compounds.
Figure 1. Selected 1,2,4-triazole drugs
Figure 2. Expected sites of Alkylation of S, N-Unsubstituted 1,2,4-triazole Tautomers.
Figure 3. HMBC representation showing the correlations in the glucosides 12 and 15.
Scheme 1. Synthesis of 1,2-Dihydro-5-(1H-indol-2-yl)-1,2,4-triazole-3-thione 5.
Scheme 2. Synthesis of S- and bis-glycosides.
2
1

ACCEPTED MANUSCRIPT
N
N
N
N
N
N
N
Cl
CN
CN
H C
CH3
3
CH3
CH3
Anastrozole
Estazolam
O
N
N
NH2
N
N
N
HO
N
N
Cl
O
Cl
OH OH
Ribavirin
Triazolam
Figure 1. Selected 1,2,4-triazole drugs
Figure 2. Expected sites of Alkylation of S,N-unsubstituted 1,2,4-triazole Tautomers.

ACCEPTED MANUSCRIPT
OAc
AcO
AcO
7
.11
H
O
OAc
2
S
HN
7
N
.
O
6
.
OAc
H 5.46
1
N
5
1
AcO
AcO
0
5
S
1
N
H
N
N
OAc
N
N
O
AcO
H
6.91
H 5.50
O
AcO
OAc H 5.70
OAc H 5.63
AcO
AcO
AcO
AcO
S,N(1)
S,N(2)
Figure 3. HMBC representation showing the correlations in the S,N-bis(glucosides)
2 and 15.
1
O
O
O
O
PTSA / Toluene
N
N
H
1
NH NH H O
2 2 2
N
H
2
HN NH2
O
N
3
H
H
N
HN NH
NH SCN
4
NH
1. aq. KOH
S
2. Conc. HCl
N
N
S
N
O H N
H
2
5
H
4

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of 1,2-Dihydro-5-(1H-indol-2-yl)-1,2,4-triazole-3-thione 5.
R2
OAc
R1
N
H
AcO
R2
OAc
N
H
O
N
N
O
R
R1
AcO
N
R
N
+
N
S
O
N
OAc
R2
AcO
R2
O
S
R
1
R
R
AcO
1
R AcO
15-17
12-14
6-8
6
-8
K CO ,
2
3
R2
OAc
K CO ,
2
3
Acetone
N
H
O
R
Acetone
R1
AcO
H
S
N
N
N
H
6-8
X
N
OAc
R2
N
Et N, Acetone
N
N
O
3
S
1
2
6
: X= Br, R= OAc, R = OAc, R = H
7: X= Br, R= OAc, R = H, R = OAc
: X= Cl, R= NHAc, R = OAc, R = H
H
H
1
1
2
R
R
AcO
1
2
5
8
9
-11
1
2
9
1
1
, 12, 15: R= OAc, R = OAc, R = H
1
2
0, 13, 16: R= OAc, R = H, R = OAc
1
2
1: 14, 17: R= NHAc, R = OAc, R = H
Scheme 2. Synthesis of S- and S,N-bis(glycosides).

ACCEPTED MANUSCRIPT
Table 1: The in-vitro antibacterial activity of tested compounds against Bacillus
subtilis and Pseudomans aeruginosa.
.
Compounds numbers
Diameter of zone of
inhibition in mm
Bacteria
Gram+
Gram-
Bacillus subtilis
Pseudomans aeruginosa
1
2
3
4
5
9
12
5.6
12
2.65
4.85
3.00
2.5
12
12
3.5
12
3.25
0.5
1
1
1
0
1
2
12
5.1
6.5
5.2
4.87
12
3.90
2.5
1
1
1
2+15
3+16
4+17
2.75
3.55
2.85
Zero
7.9
CONTROL with solvent
CONTROL with anti-
bacterial
Zero
12
(Chloramphenicol)

ACCEPTED MANUSCRIPT
Table 2: The in vitro antifungal activity of the tested compounds against Fusarium
oxysporum, Rhizoctonia solani and Aspegil lus niger.
Compound
Number
Diameter of zone
of inhibition in
mm
Fungi
F. oxysporum
R. solani
5mg/mL
1.95
2.7
A. niger
5mg/mL
1.85
5
mg/mL
2.3
1
2
3
4
5
9
1.9
1.3
1.3
0.85
1.05
0.8
ZERO
ZERO
ZERO
ZERO
ZERO
ZERO
ZERO
ZERO
ZERO
ZERO
ZERO
1.0
1.7
ZERO
2.1
0.6
1
1
1
0
1
2
0.75
1.75
1.15
0.3
ZERO
1.95
1
1
1
2+15
3+16
4+17
ZERO
1.6
2.3
0.8
0.75
ZERO
CONTROL with
solvent
ZERO
CONTROL with
anti-fungal
2.00
2.00
2.00
(Baneocin)

ACCEPTED MANUSCRIPT
Electronic Supplementary material
Regioselective synthesis, characterization and antimicrobial
evaluation of S-glycosides and S,N-diglycosides of 1,2-Dihydro-
5
-(1H-indol-2-yl)-1,2,4-triazole-3-thione
a,b,*
c
c
El Sayed H. El Ashry,
El Sayed H. El Tamany, Mohy El Din Abd El Fattah,
a,c
d
Ahmed T.A. Boraei, Heba M. Abd El-Nabi
a
HEJ Research Institute for Chemistry, International Center for Chemical and
Biological Sciences, Karachi University, Karachi, Pakistan
b
Chemistry Department, Faculty of science, Alexandria University, Alexandria, Egypt
c
Chemistry Department, Faculty of Science, Suez Canal University, Ismailia, Egypt
d
Microbiology Department, Faculty of Agriculture , Suez Canal University, Ismailia,
Egypt