Synthesis, PASS predication, in vitro antimicrobial evaluation and pharmacokinetic study of novel n-octyl glucopyranoside esters

Article abstract of DOI:10.1016/j.carres.2019.107812

Octyl β-D-glucopyranoside (OBG), prepared from D-glucose and octan-1-ol employing MW method, was subjected to direct dimolar valeroylation in pyridine at room temperature (25 °C) with valeroyl chloride. This mainly furnished the corresponding 3,6-di-O-valeroate in 57% yield indicating the regioselectivity at C-6 and C-3 positions. For structural elucidation and to get newer glucopyranosides of potential antimicrobial 3,6-di-O-valeroate was further converted into four novel 2,4-di-O-acyl esters reasonably in good yields. Per-O-acetate and per-O-benzoate of OBG were also prepared for SAR study. PASS predication and in vitro antimicrobial studies established them as better antifungal agent than that of antibacterial. SAR study along with AdmetSAR and SwissADME suggested that incorporation of alkanoyl and aromatic ester groups on octyl glucopyranoside core increase antimicrobial potentiality in very low concentration (10 μgmL^?1). Molecular docking revealed that novel 2,4-di-O-tosyl ester and 2,3,4,6-tetra-O-benzoyl ester may act as competitive inhibitors of lanosterol 14-alpha demethylase.

Full text of DOI:10.1016/j.carres.2019.107812

Journal Pre-proof
Synthesis, PASS predication, in vitro antimicrobial evaluation and pharmacokinetic
study of novel n-octyl glucopyranoside esters
Mohammed M. Matin, Sreebash C. Bhattacharjee, Priyanka Chakraborty, Muhammad
S. Alam
PII:
S0008-6215(19)30352-0
DOI:
https://doi.org/10.1016/j.carres.2019.107812
CAR 107812
Reference:
To appear in: Carbohydrate Research
Received Date: 5 June 2019
Revised Date: 30 August 2019
Accepted Date: 8 September 2019
Please cite this article as: M.M. Matin, S.C. Bhattacharjee, P. Chakraborty, M.S. Alam, Synthesis, PASS
predication, in vitro antimicrobial evaluation and pharmacokinetic study of novel n-octyl glucopyranoside
esters, Carbohydrate Research (2019), doi: https://doi.org/10.1016/j.carres.2019.107812.
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Graphical abstract
HO
HO
RO
PASS Predication
Antimicrobial Study
ADMET Prediction
SwissADME
1
O
O
R O
RO
D-glucose
OC H
OC H
8
17
HO
8
17
1
OH
OR
1
2 (OBG)
Molecular Docking
3-10: R = R = various
acyl group(s)
1

Synthesis, PASS Predication, In Vitro Antimicrobial Evaluation and Pharmacokinetic
study of Novel n-Octyl Glucopyranoside Esters
Mohammed M. Matin,a* Sreebash C. Bhattacharjee,a,b Priyanka Chakraborty,a and
Muhammad S. Alamc
aOrganic Research Laboratory, Department of Chemistry, Faculty of Science, University of
Chittagong, Chittagong-4331, Bangladesh.
bChemical Research Division, Bangladesh Council of Scientific & Industrial Research
(BCSIR) Laboratories, Chittagong-4220, Bangladesh.
bMolecular Modeling and Drug Design Laboratory, Bangladesh Council of Scientific &
Industrial Research (BCSIR) Laboratories, Chittagong-4220, Bangladesh.
*Corresponding author. E-mail address: mahbubchem@cu.ac.bd, Tel.: +88 01716 839689.
Abstract
Octyl β-D-glucopyranoside (OBG), prepared from D-glucose and octan-1-ol employing
MW method, was subjected to direct dimolar valeroylation in pyridine at room
temperature (25 °C) with valeroyl chloride. This mainly furnished the corresponding 3,6-
di-O-valeroate in 57% yield indicating the regioselectivity at C-6 and C-3 positions. For
structural elucidation and to get newer glucopyranosides of potential antimicrobial 3,6-di-
O-valeroate was further converted into four novel 2,4-di-O-acyl esters reasonably in good
yields. Per-O-acetate and per-O-benzoate of OBG were also prepared for SAR study.
PASS predication and in vitro antimicrobial studies established them as better antifungal
agent than that of antibacterial. SAR study along with AdmetSAR and SwissADME
suggested that incorporation of alkanoyl and aromatic ester groups on octyl
glucopyranoside core increase antimicrobial potentiality in very low concentration (10
µgmL-1). Molecular docking revealed that novel 2,4-di-O-tosyl ester and 2,3,4,6-tetra-O-
benzoyl ester may act as competitive inhibitors of lanosterol 14-alpha demethylase.
Keywords
n-Octyl β-
D
-glucopyranoside (OBG)
;
Regioselective acylation; PASS predication;
Antimicrobial activities; AdmetSAR, SwissADME; Molecular docking.
1

1. Introduction
Carbohydrate fatty acid (CFA) esters especially selective acyl esters of monosaccharides
containing both hydrophilic group and lipophilic group have synthetic utility as versatile
intermediates in the syntheses of many natural products and their analogues which have a
broad spectrum of applications [1]. These esters are biodegradable, nontoxic, nonallergic,
nonionic surfactants and widely used in processed foods as stabilizing agents or emulsifiers
[2]. However, selective acylation of monosaccharide molecules is a prominent challenge as
monosaccharide molecules contain several hydroxyl groups (2°) of similar reactivity. These
alcoholic groups compete during functionalization (esterification) step leading to a mono-,
di- and polyesters [3]. Various methods have so far been developed and employed
successfully for selective esterification (acylation) [4] such as direct method [5], protection-
deprotection technique [6], organotin technique [7] etc. The latter two methods have many
shortcomings such as increase the number of steps, tedious, expensive and hence generally
decrease the overall yield. Enzyme catalyzed [8] and microwave (MW) assisted acylation
[9] methods were also investigated in the past decade. In the case of enzymatic acylation of
secondary hydroxyls of glycosides, the regioselectivity and yield of the reaction depends on
many expensive factors [10] like the substrate must be able to bind in the active site of the
enzyme [10b]. MW assisted esterification causes burning of sugars and selectivity is
generally poor [9]. So, direct esterification method is preferred for selective acylation of
monosaccharide molecules maintaining proper reaction conditions to reduce the number of
steps [5c].
In recent years, the emergence of multiple antibiotic resistant pathogenic bacteria like
methicillin-resistant Staphylococcus aureus (MRSA) is a global concern [11]. To combat
with these organisms there is urgent need for establishment of new chemotherapeutic agents
with novel mode of action. CFA esters have attracted considerable research interest and
wide range of application in industry and medicine [12] due to their antimicrobial efficacy
comparable to commercially available antimicrobials [13]. For example, in Japan sucrose
esters of fatty acids [14] are used to reduce flat-sour spoilage due to Bacillus and
Clostridium in canned coffee milk drinks [15]. In 2018, Chang et al. [16] reported
regioselective synthesis of several monosaccharide esters for antimicrobial screening against
a panel of bacteria and fungi. They identified 6-O-myristoyl-D-mannopyranose (1) as a lead
compound which was highly active against methicillin resistant Staphylococcus aureus
ATCC 33591 (MRSA) and can be used as an antimicrobial in food processing, preservation,
and for bacterial and fungal diseases in animal and plants [16].
2

OH
O
HO
HO
C13H27OCO
6
O
HO
HO
O
HO
OH
OH
1
2
The antimicrobial property of CFA esters have been studied extensively, although variable
results were reported for various bacterial and fungal species [17]. They can be both
bacteriostatic and bactericidal [18]. Actually, antimicrobial functionality of CFA esters
broadly depends on the carbohydrate core, degree of esterification and number and nature of
fatty acid chain [19]. The sugar moieties present in CFA esters can increase drug water
solubility, decrease toxicity and contribute to the bioactivity of the natural products. It is
believed that CFA esters principally attack the cell membrane of bacteria [20] although
exact mode of action is still unrevealed. Also, relatively a very few studies have examined
the positional role of ester group(s) in the carbohydrate core for antimicrobial activity. Thus,
it is very much reasonable to synthesize novel CFA esters and study their antimicrobial
functionality with positional effect.
In the present study, several novel CFA esters were synthesized using n-octyl β-D-
glucopyranoside (OBG, 2) as the core carbohydrate moiety. Their antimicrobial efficacy
was assessed against seven bacterial and five fungal strains. For structure activity
relationship (SAR) study PASS (Prediction of Activity Spectra for Substances) predication
and pharmacokinetic calculation (AdmetSAR, SwissADME) as well as molecular docking
of these OBG esters were calculated and reported herein.
2. Results and discussion
2.1. Synthesis of n-octyl β-D-glucopyranoside (OBG, 2): Application of MW irradiation
For the microwave assisted preparation of alkyl glycosides, generally, a mixture of sugar
and alcohol is reacted in a high frequency field (2.45 GHz) in the presence of catalytic
amount of an acid and the ratio of the α/β-anomers is influenced by the reaction conditions
and reaction controlling [21]. Thus, we attempted for the synthesis of n-octyl β-
glucopyranoside (OBG, 2) using microwave irradiation (MWI). Thus, MWI of a mixture of
˗glucose in anhydrous 1-octanol with Amberlite IR-120 (H+) resin in a domestic
D-
D
microwave oven for 8 min at 180 watt (Scheme 1) followed by chromatography gave
glycoside 2 along with octanol in good yield. However, to evaporate 1-octanol we used high
temperature which reduces (due to burning) ultimate yield to 15% as solid mp 101-103 °C,
lit. [22] mp 97-103 °C. In its 1H NMR spectrum, additional seventeen protons was observed
for octyl group and H-1 appeared as doublet with high coupling constant (7.8 Hz at δ 4.32
ppm) that requires diaxial relationship with H-2 proton. As H-1 is axially oriented octyloxy
3

group must be present equatorially leading to the formation of β-isomer and assigned as n-
octyl β-
D
-glucopyranoside (OBG, 2).
HO
(a)
O
D-Glucose
HO
HO
O
OH
2
Scheme 1. Reagents and conditions: (a) 1-octanol, IR-120
(H+) resin, MWI, 180 W, 30×16 sec, 15%.
2.2. Regioselective valeroylation of OBG (2): Direct method
OBG in hand, we carried out its dimolar valeroylation (pentanoylation) employing direct
technique without any catalyst. Thus, treatment of OBG (2) with 2.1 equivalent of valeroyl
chloride in dry pyridine at 0 °C for 1 h and 25 °C for 23 h showed the formation of two
products (with little complex mixture, Scheme 2). Initial elution provided faster-moving
component [Rf = 0.58 (n-hexane/EA = 2/1)] as a clear syrup in 11% yield.
HO
HO
C H OCO
C H OCO
4
9
4
9
6
O
O
O
HO
C H OCO
4
9
(a)
OC H
OC H
8 17
HO
OC H
C H OCO
+
4 9
C H OCO
8
17
8
17
4
9
3
OH
OH
OCOC H
4
9
4 (57%)
2 (OBG)
3 (11%)
Scheme 2. Reagents and conditions: (a) C4H9COCl, dry Py, 0 °C-rt, 24 h.
Presence of four carbonyl signals and absence of OH band in its FT-IR spectrum indicated
1
the complete valeroylation of the molecule. In the H NMR spectrum, extra thirty-six
protons appeared in the aliphatic region indicated the attachment of four valeroyloxy groups
in the molecule. Also, H-2, H-3, H-4 and H-6 resonated in the considerable downfield as
compared to its precursor compound 2 which confirmed the attachment of valeroyl groups
at C-2, C-3, C-4 and C-6 positions. This is further supported by its 13C NMR spectrum
where four carbonyl peaks appeared at δ 173.4, 172.9, 172.1 and 172.0. Thus, the compound
was assigned as n-octyl 2,3,4,6-tetra-O-valeroyl-β-D-glucopyranoside (3).
Further elution with n-hexane/EA (8/1) furnished the slower-moving component [Rf = 0.48
(n-hexane/EA = 2/1)] as a clear solid (57%), mp 62-65 °C (Scheme 2). Two ester carbonyl
peaks at 1741 and 1733 cm-1 and a broad hydroxyl band at 3250-3540 cm-1 in its FT-IR
1
spectrum clearly demonstrated the partial valeroylation of this compound. Its H NMR
spectrum exhibited extra eighteen protons at 2.46 (2H), 2.38 (2H), 1.55-1.70 (4H), 1.31-
1.44 (4H) and 0.95 (6H) corresponding to two valeroyl groups. More importantly, H-3 and
H-6 protons deshielded considerably at δ 5.07 (as t) and 4.48-4.42 (as dd), respectively as
compared to its precursor OBG indicating the attachment of valeroyl groups at C-3 and C-6
4

positions. Its 13C NMR spectrum displayed carbonyl peaks at δ 174.4 and 174.2. Other
excess eight characteristic signals also appeared in the CH2 and CH3 region. On the basis of
its spectral and elemental analyses, the compound was assigned as n-octyl 3,6-di-O-
valeroyl-β-D-glucopyranoside (4). It should be noted that, structure of this compound was
also confirmed by its conversion to its 2,4-di-O-acetate, which was well characterized by 1D
and 2D NMR techniques and discussed in the next Section 2.3.
Isolation of 4 in low yield was probably due to the – (i) less selective reaction with
comparatively smaller acylating agent like valeroyl chloride; (ii) less selective nucleophilic
substitution reaction occurs at room temperature and (iii) formation of inseparable complex
mixture of products. However, formation and isolation of 3,6-di-O-valeroate 4 and 2,3,4,6-
tetra-O-valeroate 3 indicated the regiselectivity/reactivity order in OBG (2) under direct
dimolar valeroylation at normal temperature is 6-OH > 3-OH > 4-OH,2-OH.
2.3. Synthesis of 2,4-di-O-acylates of 3,6-di-O-valeroate 4: Direct method
Compound 4 has two free hydroxyl groups at C-2 and C-4 positions. To get newer
glucopyranoside esters of potential activity we exploited these positions for the preparation
of newer CFA esters using four different types of acylating agents. Initially, compound 4
was reacted with dimolar amount of acetic anhydride for 12 h and obtained an oil in
excellent yield (Scheme 3). Its FT-IR spectrum didn’t show any band corresponding to OH
1
group(s) indicating the di-O-acetylation of the molecule. In the H NMR spectrum of this
compound, three three-proton triplets at δ 0.94, 0.91 and 0.88 were assigned for methyl
protons of two CH3(CH2)3CO and one CH3(CH2)7O groups. Whereas, two three-proton
singlets appeared at δ 2.03 and 2.04 were due to two acetyl methyl protons (2×COCH3). The
H-2 and H-4 protons resonated considerable downfield at δ 5.00 (as dd) and 5.09 (as t),
respectively as compared to δ 3.83-3.93 and 3.46-3.59, respectively of its precursor 3,6-di-
O-valeroate 4. These downfield shifts of H-2 and H-4 protons were indicative of the
13
attachment of acetyloxy groups at C-2 and C-4 position, respectively. In its C NMR two
acetyl carbonyl peaks appeared at δ 169.3 and 169.2, also, two acetyl methyl carbons
resonated at δ 20.6 and 20.5. So, the structure of the acetate was assigned as n-octyl 2,4-di-
O-acetyl-3,6˗di-O-valeroyl-β-
D
-glucopyranoside (5).
C H OCO
C H OCO
4
9
4
9
6
O
O
(a)
AcO 4
4 9
HO
O
O
C H OCO
C H OCO
4
9
2OAc
3
OH
5
4
Scheme 3. Reagents and conditions: (a) Ac2O, Py, 0 °C-rt, 12 h, 96%.
5

The assignments of the signals of this compound 5 were established by scanning and
analyzing its DEPT-135, COSY, HSQC and HMBC experiments. In compound 5, the
position of two COCH3 groups at C-2 and C-4 positions and two COC4H9 groups at C-3 and
C-6 were ascertained by analyzing its HMBC experiment (Table 1 and Fig. 1-2).
1
13
1
13
Table 1. H NMR and C NMR shift values of compound 5. H and C assignments
were obtained from COSY, HSQC and HMBC experiments performed on
Bruker DPX-400 spectrometer (CDCl3).
δH (ppm)
(J Hz)
δC (ppm)
(HSQC)
100.8
71.4
Position
HMBC
COSY
1
2
3
4
5
4.50 (d, J = 8.0)
5.00 (dd, J = 9.6 & 8.0)
5.24 (t, J = 9.6)
5.09 (t, J = 9.5)
3.68-3.73 (m)
H: 2, 1’
H: 1,4
-
H: 6
H: 3
H: 4
H: 2
H: 1.3
H: 2,4
H: 3,5
H: 4,6
H: 5
72.5
68.6
71.9
61.9
6a,b
4.25 (dd, J = 12.4 & 4.8, 6a);
4.42 (dd, J = 12.4 & 2.4, 6b)
3.88 (dt, J = 9.6 & 6.4, 1′A);
3.49 [dt, J = 9.6 & 6.9, 1′B)
1.51-1.68 (m)
1′A,B
70.2
H: 1
H: 2’
2′
-
-
H: 1’,3’
2- COCH3
3- COC4H9
4- COCH3
6- COC4H9
2.03 (s)
169.2
173.1
169.3
173.5
H: 2, COCH3
H: 3, CH2
H: 4, COCH3
H: 6a,b, CH2
-
-
-
-
-
2.04 (s)
-
6

H-4
H-1
H-3
H-6
H-2
H-5
C-6
C-1
C4H9OCO
6
O
AcO
4
O
C4H9OCO
2
3
OAc
5
COCH3
COC4H9
Fig. 1. 2D HMBC (CDCl3) spectrum of compound 5.
O
O
6
O
4
O
O
5
2'
O
O
2
3
1
1'
O
O
O
Fig. 2. The HMBC correlations for compound 5.
Encouraged by these successes we used more three acylating agents for derivatization of
valeroate 4. Thus, reaction of valeroate 4 with dimolar decanoyl chloride, benzoyl chloride
and tosyl chloride separately followed by purification furnished 2,4-di-O-decanoate 6, 2,4-
di-O-benzoate 7, and 2,4-di-O-tosylate 8, respectively in good yields (Scheme 4). All these
compounds were characterized by FT-IR, 1H, 13C NMR and elemental analyses.
7

C H OCO
C H OCO
4
9
4
9
6
O
(a)/(b)/(c)
O
HO
RO 4
O
O
C H OCO
C H OCO
4
9
4 9
2OR
3
OH
4
6: R = COC H
9
19
7: R = Bz
8: R = Ts
Scheme 4. Reagents and conditions: (a) C9H19COCl, Py, 0 °C-rt, 11 h, 45
°C, 1h, 76%; (b) C6H5COCl, Py, 0 °C-rt, 14 h, 82%; (c) 4-
CH3.C6H4COCl, Py, 0 °C-rt, 10 h, 78%.
2.4. Synthesis of per-O-acyl esters of OBG (2): Direct method
We have prepared two per-O-acyl esters of OBG (2) to get potential antimicrobial agents as
well as to get idea about structure activity relationship (SAR) during antimicrobial activity
evaluation. Initially, treatment of OBG (2) with excess amount of acetic anhydride in
pyridine gave a faster-moving yellow solid, mp 58-60 °C in excellent yield (92%, Scheme
1
5). In its H NMR spectrum, additional four three-proton singlets at δ 2.10, 2.06, 2.04 and
2.02 indicated the attachment of four acetyl groups in the molecule. The solid was
unambiguously assigned the structure as n-octyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside
(9) on the basis of spectral analyses.
HO
HO
HO
RO
RO 4
RO
6
O
(a)/(b)
O
O
O
2OR
3
OH
2
9: R = Ac
10: R = Bz
Scheme 5. Reagents and conditions: (a) Ac2O, Py, 0 °C-rt, 12 h,
92%; (b) BzCl, dry Py, 0 °C-rt, 12 h, 87%.
Finally, OBG was treated with four equivalent of benzoyl chloride in pyridine and furnished
a white solid, mp 97-100 °C in good yield (Scheme 5). FT-IR spectrum of this compound
showed no signal related to OH group instead exhibited characteristic bands at 1723, 1719,
1
1715, 1710 (CO), 1069 cm-1 (pyranose ring). In the H NMR spectrum, twenty protons
resonated in the aromatic region which indicated the attachment of four benzoyl groups in
the molecule. The solid was unambiguously assigned the structure as n-octyl 2,3,4,6-tetra-
O-benzoyl-β-D-glucopyranoside (10).
Thus, we have successfully synthesized eight acyl esters of OBG using direct acylation
technique with various acylating agents.
8

2.5. Computational antimicrobial activities evaluation: Prediction of Activity Spectra for
Substances (PASS)
The web based application PASS predicts a plethora of pharmacological and toxicological
activities of a compound simultaneously and the programme is designed to anticipate more
than 4000 forms of biological activity including drug and non-drug actions and can be
employed to identify the most probable targets with 90% accuracy [23]. PASS result is
designated as Pa (probability for active compound) and Pi (probability for inactive
compound). Being probabilities, the Pa and Pi values vary from 0.000 to 1.000. Only
activities with Pa>Pi are considered as possible for a particular compound. If Pa>0.7, the
chance to find the activity experimentally is high. When 0.5<Pa<0.7, the chance to find the
activity experimentally is less, but the compound is probably not so similar to known
pharmaceutical agents.
For the biological activity spectrum initially all the drugs was assigned SMILES (simplified
molecular-input line-entry system). With these SMILES, biological activities were obtained
by PASS online version (http://www.pharmaexpert.ru/ PASSonline/index.php). Predicted
biological activities for OBG esters are mentioned in Table 2.
Table 2. Predicted biological activity of synthesized OBG esters using PASS
online software.
Biological Activity
Antibacterial
Pa
Antifungal
Drug
Pi
Pa
Pi
0.533
0.544
0.563
0.567
0.544
0.532
0.404
0.563
0.494
0.014
0.013
0.011
0.011
0.013
0.014
0.029
0.011
0.017
0.685
0.705
0.738
0.725
0.705
0.703
0.537
0.716
0.653
0.010
0.009
0.008
0.009
0.009
0.009
0.025
0.009
0.013
2
3
4
5
6
7
8
9
10
Pa = Probability ‘to be active’; Pi = Probability ‘to be inactive’
Pa values of the OBG esters (3-10) were 0.40 < Pa < 0.56 in antibacterial and 0.53 < Pa <
0.73 in antifungal (Table 2), which indicated that the OBG esters should be more potent
against phytopathogenic fungi as compared to that of bacterial pathogens.
9

2.6. In vitro antimicrobial evaluation of OBG esters
Knowing the possibility of antimicrobial efficacy of OBG esters from PASS predication we
evaluated their activity in vitro against several bacterial and fungal strains.
2.6.1. Effects of OBG esters 3-10 against bacteria
In the present study, we used two Gram-positive and five Gram-negative organisms using
disc diffusion method [24]. Gram-positive organisms were Bacillus subtilis ATCC 6633
and Staphylococcus aureus ATCC 25923. Five Gram-negative organisms were Escherichia
coli ATCC 25922, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 8027,
Pseudomonas aeruginosa ATCC 27853 and Salmonella abony NCTC 6017.
2.6.1.1 Efficacy against Gram-positive organisms
The results of inhibition zone (diameter) of the selected bacteria due to the effect of
synthesized glucoside type CFA esters are presented in Table 3 and Fig. 3. It was found
from Table 3 that these esters were weak to moderate inhibitor against Gram-positive
organisms.
Table 3. Inhibition against Gram-positive bacteria by the OBG esters.
Diameter of zone of inhibition in mm (10 µg dw / disc)
Drug
B. subtilis
2.41 ± 0.82
6.54 ± 0.63
4.62 ± 0.75
*16.45±0.76
NI
S. aureus
NI
2
NI
3
NI
4
NI
5
5.51 ± 0.44
3.55 ± 0.41
2.41 ± 0.82
5.64 ± 0.82
NI
6
5.55 ± 0.41
*14.31 ± 0.82
6.43 ± 0.82
*21.43 ± 0.82
*27.71 ± 0.63
7
8
9
10
Ciprofloxacin**
*35.22 ± 0.39
Data are presented as Mean ± SD. Values are represented for the triplicate of all the experiments.
Significantly inhibition (P < 0.05) values are marked with asterisk (*) sign for test compounds and
double asterisk (**) sign for reference antibiotic ciprofloxacin. dw = Dry weight; NI = No
inhibition. NI was observed for control (DMSO).
10

Fig. 3. Activities against Gram-positive organisms.
2.6.1.2 Efficacy against Gram-negative organisms
The results of inhibition zone (diameter) of the five selected bacteria due to the effect of
synthesized OBG type CFA esters are presented in Table 4 and Fig. 4. It was clear from the
Table 3 and Table 4 that OBG esters were less prone against these Gram-negative
pathogens than Gram-positive strains.
Table 4. Inhibition against Gram-negative bacteria by the OBG esters.
Diameter of zone of inhibition in mm (10 µg dw / disc)
Drug
E. coli
ATCC 25922
NI
E. coli
ATCC 8739
NI
P. aeruginosa P. aeruginosa
S. abony
NCTC 6017
NI
ATCC 8027
ATCC 27853
NI
NI
2
3
4
5
6
7
8
9
8.22 ± 0.48
NI
NI
NI
NI
9.56 ± 0.32
8.06 ± 0.72
NI
12.23 ± 0.66
6.44 ± 0.68
10.50 ± 0.32
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
12.66 ± 0.64
NI
NI
12.56 ± 0.74
7.88 ± 0.56
NI
NI
NI
NI
NI
NI
9.22 ± 0.78
NI
NI
NI
8.22 ± 0.22
NI
10.57 ± 0.82
NI
10
Ciprofloxacin**
13.24 ± 0.42
12.20 ± 0.66
*31.65 ± 0.82
9.49 ± 0.44
14.55 ± 0.74
Data are presented as Mean ± SD. Values are represented for the triplicate of all the experiments.
Significantly inhibition (P < 0.05) values are marked with asterisk (*) sign for test compounds and double
asterisk (**) sign for reference antibiotic ciprofloxacin. dw = Dry weight; NI = No inhibition. NI was
observed for control (DMSO).
11

Fig. 4. Activities against Gram-negative organisms.
2.6.2. Effects of OBG esters 3-10 against fungal pathogens
The results of the percentage inhibitions of mycelial growth [25] due to the effect of the
OBG esters 3-10 against four pathogenic fungi viz. Aspergillus flavus, Aspergillus niger,
Candida albicans and Fusarium solani are presented in Table 5 and Fig. 5. It should be
noted that OBG (2) didn’t show any inhibition whereas introduction of ester groups at
different position increased antifungal activities.
Table 5. Inhibition against fungal pathogens by the OBG esters.
% Inhibition of fungal mycelial growth (10 µg dw / mL PDA)
Drug
A. flavus
NI
A. niger
NI
C. albicans
F. solani
NI
NI
2
32.22 ± 0.44
NI
*65.24 ± 0.42
NI
*68.10 ± 0.82 *73.22 ± 0.64
3
34.74 ± 0.44
53.26 ± 0.44
18.44 ± 0.72
*64.86 ± 0.36
4
35.36 ± 0.76
*65.67 ± 0.76
25.83 ± 0.82
*62.50 ± 0.76
*65.44 ± 0.42
45.83 ± 0.44
*62.50 ± 0.66
51.87 ± 0.68
41.18 ± 0.72
20.36 ± 0.34
51.87 ± 0.82
18.12 ± 0.42
*55.58 ± 0.60
17.11 ± 0.82
5
*68.44 ± 0.82 *70.32 ± 0.58
NI *71.89 ± 0.66
6
7
*68.32 ± 0.76 *70.27 ± 0.68
*68.39 ± 0.68 *73.68 ± 0.64
*70.58 ± 0.60 *65.41 ± 0.68
8
9
10
**Fluconazole
36.44 ± 0.68
*83.78 ± 0.46
Data are presented as Mean ± SD. Values are represented for the triplicate of all the experiments.
Significantly inhibition (P < 0.05) values are marked with asterisk (*) sign for test compounds and double
asterisk (**) sign for reference antibiotic fluconazole. dw = Dry weight; NI = No inhibition. NI was
observed for control (DMSO).
12

Fig. 5. Activities against fungal pathogens.
Most of the OBG esters, except 4, possess antifungal potentiality against the tested
pathogens. Their efficacy, in many cases is better than that of the standard antifungal
fluconazole. OBG ester 3, 6, 8-10 showed excellent inhibition against C. albicans and 3, 5-
10 showed very good efficacy against F. solani.
More interestingly, in vitro results are in complete agreement with PASS predication result
that the OBG esters were more potent against phytopathogenic fungi as compared to that of
bacterial pathogens. Also, higher efficacy of 3, 6 and 8-10 justified that the presence of
aliphatic ester group and aromatic ester group in glucopyranoside molecular framework
increase antimicrobial activities of OBG.
2.7. ADME/T analysis
From AdmetSAR calculation (Table 6), all the glucopyranoside derivatives, except 2,
showed positive result for blood brain barrier (BBB) criteria, predicting that they can pass
through the BBB. They are non-carcinogenic (except 8) and show III category acute oral
toxicity which suggesting relatively harmless for oral administration. All compounds
(except 2) are P-glycoprotein inhibitor where, P-glycoprotein inhibition can interrupt the
absorption, permeability and retention of the chemical species [26]. Amongst the OBG
esters, compound 10 shows highest human intestinal absorption values whereas di-ester
compound (4) showed the lowest value compared to others. Rat acute toxicity of OBG esters
was found below 1.6 (mol/kg) (except 4 and 8) which suggesting lower median lethal dose
(LD50) values. However, all the compounds show weak inhibitory feature for human ether-
a-go-go-related gene (hERG) which can lead to long QT syndrome, so further more study of
this aspect is necessary.
13

Table 6. AdmetSAR calculation of OBG esters.
Human ether –
Rat acute
toxicity
LD50
Human
intestinal
absorption
Acute
oral
toxicity
Blood brain
barrier
a-go-go-
related gene
inhibition
P-glycoprotein
inhibitor
Drug
Carcinogen
(mol/kg)
2
3
- 0.5311
+ 0.9392
+ 0.5816
+ 0.9392
+ 0.9392
+ 0.9319
+ 0.9120
+ 0.9392
+ 0.9312
- 0.5967
+ 0.9106
- 0.6026
+ 0.9106
+ 0.9106
+ 0.9503
+ 0.9449
+ 0.9106
+ 0.9644
NI (0.8709)
I (0.6784)
I (0.7052)
I (0.6784)
I (0.6784)
I (0.8893)
I (0.7723)
I (0.6784)
I (0.7430)
WI (0.8624)
WI (0.8839)
WI (0.9357)
WI (0.8839)
WI (0.8839)
WI (0.8699)
WI (0.8203)
WI (0.8839)
WI (0.8423)
NC (0.9490)
NC (0.8692)
NC (0.9364)
NC (0.8692)
NC (0.8692)
NC (0.8755)
C (0.5550)
III
III
III
III
III
III
III
III
III
1.4828
1.6066
2.0275
1.6066
1.6066
1.7512
2.2737
1.6066
1.5788
4
5
6
7
8
9
NC (0.8692)
NC (0.9149)
10
NI = Non-inhibitor, I = Inhibitor, WI = Weak inhibitor, NC = Non-carcinogenic, C = Carcinogenic
From SwissADME calculation (Table 7), all the glucopyranoside derivatives have good
hydrogen bonds donor and acceptor. According to Lipnski’s rule of five poor absorption or
permeation is more likely when there are more than 5 H-bond donors and 10 H-bond
acceptors [27]. Topological polar surface area (TPSA) data showed the good polarity of the
compounds, where the TPSA value should be less than 200 Ų, More the value more the
polarity. The CYP enzymes, particularly isoforms 1A2, 2C9, 2C19, 2D6, and 3A4, are
responsible for about 90% oxidative metabolic reactions. Inhibition of CYP enzymes will
lead to inductive or inhibitory failure of drug metabolism. Pan-assay interference
compounds (PAINS) are chemical compounds that often give false positive results in high-
throughput screens. PAINS tend to react nonspecifically with numerous biological targets
rather than specifically affecting one desired target. Here PAINS showed no violation with
these compounds.
Table 7. Calculation drug likeliness using SwissADME.
HB
acceptors
HB
donors
CYP1A2 CYP2C19 CYP2C9
CYP2D6
inhibitor
No
CYP3A4
inhibitor
No
No
Yes
Yes
No
Yes
Yes
PAINS
alerts
Drug
TPSA
inhibitor
inhibitor
inhibitor
6
10
8
10
10
10
13
10
10
4
0
2
0
0
0
0
0
0
99.38
No
No
No
0
0
0
0
0
0
0
0
0
2
3
4
5
6
7
8
9
10
123.66
111.52
123.66
123.66
123.66
183.79
123.66
123.66
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
Yes
Yes
No
No
No
No
No
No
No
No
Yes
No
No
No
Yes
No
No
Yes
No
Yes
*HB = Hydrogen bond, TPSA = Topological polar surface area, PAINS = Pan-assay interference compounds
2.8. Molecular Docking and non-bond interaction analysis
In the present study, we tried to find a lanosterol 14-alpha demethylase inhibitor which play
crucial role in ergosterol biosynthesis which ultimately causes rupture of cell membrane
14

[28]. Thus, we validated docking accuracy by redocking of a known ligand- itraconazole
using XP docking methodology. The known co-crystal ligand was first separated and re-
docked using the Glide XP algorithm. Conformation of the complex with the highest
negative docking score was selected from each run. Interestingly, Glide docking with XP
setting of ester 10 produced the docked complex with highest negative docking score, which
denoted that the inhibitory ability of docked ligand was almost similar to that of the crystal,
as shown in Table 8. Ester 10 and 8 showed glide docking score of -13.476 and -12.092
respectively where the known ligand itraconazole showed -12.296. The docked complexes
of these two virtual hits from Glide XP docking were further analyzed for protein–ligand
interaction profiling (Fig. 6). Tetrabenzoate 10 showed three carbon hydrogen bonds with
ILE304 & Gly307, one hydrophobic pi-pi T-shaped with TYR132, one hydrophobic alkyl with
ALA200 and seven hydrophobic pi-alkyl interactions with PHE475, PRO375, ALA476,
LEU376, MET508, ILE131, LYS143 & ILE304.
On the other hand, compound 8 formed a conventional hydrogen bond with CYS470, four
carbon hydrogen bonds with LYS143, GLY307 & HIS468, thirteen hydrophobic alkyl bonds
with LEU204, ILE304, LYS143, LEU300, ILE304, ILE471, LEU121, MET508, PRO375, LEU376,
VAL509 and six hydrophobic pi-alkyl bonds with TYR118, PHE233, PHE475, LEU376 and
CYS470. These all non-bond interactions revealed that novel ester 8 may act as competitive
inhibitors of lanosterol 14-alpha demethylase.
Table 8. Glide docking of OBG ester 3-10.
Glide XP docking
score (kcal/mol)
-13.476
Glide energy
(kcal/mol)
-71.39
Glide emodel
(kcal/mol)
-122.165
-135.642
-44.995
Glide ligand
efficiency
-0.259
Drug
10
-12.296
-12.092
-10.016
-9.22
-8.727
-7.713
-7.024
-7.007
-75.401
-67.215
-57.222
-76.162
-61.135
-61.154
-41.932
-50.074
-0.196
-0.228
-0.313
-0.192
-0.23
-0.175
-0.351
-0.219
itraconazole
8
4
7
5
3
2
9
-81.044
-121.558
-91.42
-100.854
-54.082
-69.298
15

Fig. 6. Non-bond interactions of (a) compound 10 and (b) compound 8 with lanosterol 14-alpha
demethylase.
3. Structure activity relationship (SAR)
It is essential to understand the mechanisms of antimicrobial action for the design and
improvement of antimicrobial agents. Unlike antibiotics, CFA esters have diverse modes of
action that appear to be nonspecific. Thus, we attempted to derive SAR of the OBG esters
on the basis of our results. It was evident from Table 3 (Fig. 3), Table 4 (Fig. 4) and Table 5
(Fig. 5) that incorporation of valeroyl, decanoyl, benzoyl and tosyl groups, increased the
antimicrobial potentiality of OBG (2).
Incorporation of valeroyl and other acyl group(s) gradually increased hydrophobicity of the
OBG esters 4, 3, 5-10. Previously we observed that the hydrophobicity of materials is an
important parameter with respect to bioactivity such as toxicity or alteration of membrane
integrity, because it is directly related to membrane permeation [29]. Hunt [30] also
proposed that the potency of aliphatic alcohols is directly related to their lipid solubility
through the hydrophobic interaction between alkyl chains from alcohols and lipid regions in
the membrane. We believe that a similar hydrophobic interaction might occur between the
acyl chains of glucopyranosides accumulated in the lipid like nature of the bacteria
membranes. As a consequence of their hydrophobic interaction, bacteria lose their
membrane permeability, ultimately causing death of the bacteria [30].
It was evident that, synthesized OBG esters 3-10 were more active against Gram-positive
pathogen than that of Gram-negative bacterial organisms. More importantly, OBG esters 3-
10 were highly prone against fungal pathogens than that of tested bacterial organisms.
4. Conclusion
Thus, dimolar valeroylation of OBG (2) at room temperature exhibited regioselectivity
mainly at C-6 OH and C-3 OH positions confirming its reactivity order as 6OH > 3OH >
4OH,2OH. 3,6-Di-O-valeroylglucopyranoside, thus obtained, was then converted into four
newer 2,4-di-O-acylates with other acylating agents. Also, per-O-acetate and per-O-
16

benzoates of OBG were prepared. Both PASS predication and in vitro antimicrobial activity
test established that these OBG esters possess excellent antifungal activity as compared to
antibacterial. SAR study demonstrated that for better antimicrobial activities aromatic
moieties (phenyl ester) should be in combination with aliphatic acyl moieties.
Parmacokinetic study such as AdmetSAR and SwissADME along with molecular docking
of 8 and 10 are also discussed in connection with their drug likeliness. Overall, a very
simple and effective direct acylation method is used to prepare several novel OBG esters (3-
8) which have basically antifungal potentiality at a very low concentration (10 µgmL-1).
5. Experimental
5.1. General methods
Evaporations were carried out under reduced pressure using a Buchi rotary evaporator (R-
100, Switzerland) with a bath temperature below 40 °C. Melting points were determined on
an electro-thermal melting point apparatus (England) and are uncorrected. Column
chromatography was performed with silica gel G60. Thin layer chromatography (TLC) was
performed on Kieselgel GF254 and the spots were detected by spraying the plates with 1%
H2SO4 and heating at 150–200 °C until coloration took place. The solvent system employed
for the TLC analyses was chloroform/methanol and/or n-hexane/ethyl acetate in different
proportions. FT-IR spectra were recorded on a FT-IR spectrophotometer (Shimadzu, IR
1
Prestige-21) in KBr disc. H (400 MHz, Bruker DPX-400 spectrometer, Switzerland) and
13C (100 MHz) NMR spectra were recorded in CDCl3 solution using tunable multinuclear
probe. Chemical shifts were reported in δ unit (ppm) with reference to TMS as an internal
standard and J values are shown in Hz. Elemental analyses were carried out with C,H-
analyzer.
5.2. Synthesis
5.2.1. Octyl
solution of
β
-D
-glucopyranoside (2). Amberlite IR-120 (H+) resin (2.0 g) was added to a
-glucose (1.0 g, 5.551 mmol) in anhydrous 1-octanol (20 mL) and the mixture
D
was placed in a domestic microwave oven. The mixture was irradiated 30 sec span for
sixteen times (i.e. 8 min) at 180 watt when TLC indicated completion of reaction with the
formation of a faster-moving product. The resin was filtered off and the filtrate concentrated
under reduced pressure. The resulting syrup was purified by passage through a silica gel
column (with ethyl acetate/methanol, 8/1, v/v as eluant) to give the desired glycoside 2.
However, some octanol (bp ~195 °C) was present with glucoside 2, which took our tedious
effort (heating at 90-100 °C) to remove octanol and we obtained a little amount of
17

compound 2 (0.244 g, 15%) as a solid. Recrystallization from absolute ethanol gave the
glucoside 2 as needles, mp 101-103 °C, lit. [22] mp 97-103 °C.
Rf = 0.51 (ethyl acetate/methanol = 2/1, v/v); FT-IR (KBr): ν 3200-3560 (OH), 1080 cm-1
1
(pyranose ring); H NMR (400 MHz, CDCl3): δH 4.32 (d, 1H, J1,2 7.8 Hz, H-1), 3.78-3.91
[br m, 7H, H-6, CH3(CH2)6CHAHBO and 4×OH], 3.63 (t, 1H, J2,3;3,4 = 9.2 Hz, H-3), 3.50-
3.57 [m, 2H, H-5 and CH3(CH2)6CHAHBO], 3.39 (dd, 1H, H-2), 3.32 (t, 1H, J4,5 9.3 Hz, H-
4), 1.61-1.68 [m, 2H, CH3(CH2)5CH2CH2O], 1.22-1.37 [m, 10H, CH3(CH2)5CH2CH2O],
0.90 [t, 3H, J 6.6 Hz, CH3(CH2)7O]; 13C NMR (100 MHz, CDCl3): δC 102.8 (C-1), 76.4 (C-
3), 75.5 (C-2), 73.4 (C-5), 70.6 [CH3(CH2)6CH2O], 69.5 (C-4), 61.4 (C-6), 31.8, 29.6, 29.5,
29.3, 25.9, 22.7 [CH3(CH2)6CH2O], 14.1 [CH3(CH2)7O]; Anal. Calcd for C14H28O6: C,
57.51; H, 9.65. Found: C, 57.62; H, 9.63.
5.2.2. Octyl 2,3,4,6-tetra-O-valeroyl-
β
-
D
-glucopyranoside (3) and octyl 3,6-di-O-valeroyl-
-glucopyranoside (2, 0.5 g, 1.71 mmol) in
β
-D
-glucopyranoside (4). A solution of octyl β-D
pyridine (2 mL) was cooled to 0 °C whereupon valeroyl chloride (0.433 g, 3.591 mmol) was
added to this mixture. The mixture was stirred at the same temperature for 1 h and then
stirred 23 h at room temperature. TLC (n-hexane/EA, 1/2, v/v) indicated the formation of
two products, the slower-moving component being the major one. Usual work-up followed
by concentration gave a syrupy residue. During column chromatography, initial elution
provided faster-moving component as 2,3,4,6-tetra-O-valeroate 3 (0.118 g, 11%) as a clear
syrup.
Rf = 0.58 (n-hexane/EA = 2/1); FT-IR (KBr): ν 1756, 1752, 1750, 1733 (CO), 1090 cm-1
(pyranose ring); 1H NMR (400 MHz, CDCl3): δH 5.25 (t, 1H, J3,4 9.4 Hz, H-3), 5.11 (t, 1H,
J
4,5 9.6 Hz, H-4), 5.01 (1H, dd, J2,3 9.6 Hz, H-2), 4.50 (d, 1H, J1,2 8.0 Hz, H-1), 4.21 (dd, 1H,
J6a,6b 12.1, J5,6a 4.8 Hz, H-6a), 4.18 (dd, 1H, J5,6b 2.0 Hz, H-6b), 3.87 [dt, 1H, J 9.6 and 6.4
Hz, CH3(CH2)6CHAHBO], 3.66-3.72 (m, 1H, H-5), 3.47 [dt, 1H, J 9.6 and 6.8 Hz,
CH3(CH2)6CHAHBO], 2.36 [t, 2H, J 7.6 Hz, CH3(CH2)2CH2CO], 2.21-2.31 [m, 6H,
3×CH3(CH2)2CH2CO],
4×CH3CH2CH2CH2CO],
1.48-1.67
1.26-1.40
[m,
[m,
10H,
18H,
CH3(CH2)5CH2CH2O
CH3(CH2)5(CH2)2O
and
and
13
4×CH3CH2(CH2)2CO], 0.88-0.96 [m, 15H, CH3(CH2)7O and 4×CH3(CH2)3CO]; C NMR
(100 MHz, CDCl3): δC 173.4, 172.9, 172.1, 172.0 (CO), 100.9 (C-1), 72.5 (C-3), 72.0 (C-5),
71.2 (C-2), 70.1 [CH3(CH2)6CH2O], 68.4 (C-4), 62.0 (C-6), 33.8(2), 33.7(2)
[4×CH3(CH2)2CH2CO], 31.8, 29.5, 29.3, 29.2, 27.0, 26.9, 26.8(2), 25.9, 22.6, 22.2(3), 22.1
[CH3(CH2)6CH2O and 4×CH3(CH2)2CH2CO], 14.0 [CH3(CH2)7O], 13.7, 13.6(2), 13.5
18

[4×CH3(CH2)3CO]. Anal. Calcd for C34H60O10: C, 64.94; H, 9.62. Found: C, 64.87; H, 9.65.
Further elution with n-hexane/EA (8/1) furnished the 3,6-di-O˗valeroate 4 (0.449 g, 57%) as
a clear solid, mp 62-65 °C. Rf = 0.48 (n-hexane/EA = 2/1); FT-IR (KBr): ν 3250-3540 (OH),
1
1741, 1733 (CO), 1088 cm-1 (pyranose ring); H NMR (400 MHz, CDCl3): δH 5.07 (t, 1H,
J2,3;3,4 9.6, H-3), 4.48 (dd, 1H, J6a,6b 12.1, J5,6a 4.5 Hz, H-6a), 4.42 (dd, 1H, J5,6b 2.0 Hz, H-
6b), 4.34 (d, 1H, J1,2 8.2 Hz, H-1), 3.83-3.93 [m, 2H, H-2 and CH3(CH2)6CHAHBO], 3.46-
3.59 [m, 3H, H-4, H-5 and CH3(CH2)6CHAHBO], 2.46 [t, 2H, J 7.7 Hz, CH3(CH2)2CH2CO],
2.38 [t, 2H, J 7.6 Hz, CH3(CH2)2CH2CO], 1.55-1.70 [m, 6H, CH3(CH2)5CH2CH2O and
2×CH3CH2CH2CH2CO],
1.31-1.44
[m,
14H,
CH3(CH2)5CH2CH2O
and
2×CH3CH2(CH2)2CO], 1.21-1.28 (br s, 2H, 2×OH), 0.95 [t, 6H, J 7.6 Hz, 2×CH3(CH2)3CO],
13
0.89 [t, 3H, J 7.2 Hz, CH3(CH2)7O]; C NMR (100 MHz, CDCl3): δC 174.4, 174.2 (CO),
101.0 (C-1), 74.3 (C-3), 74.2 (C-5), 71.1 (C-2), 70.4 [CH3(CH2)6CH2O), 69.4 (C-4), 63.0
(C-6), 34.1, 33.9 [2×CH3(CH2)2CH2CO], 31.8, 29.6, 29.3, 29.2, 27.0, 26.9, 25.9, 22.6, 22.2,
22.1 [CH3(CH2)6CH2O and 2×CH3(CH2)2CH2CO], 14.0 [CH3(CH2)7O], 13.6, 13.5
[2×CH3(CH2)3CO]; Anal. Calcd for C24H44O8: C, 62.58; H, 9.63. Found: C, 62.64; H, 9.61.
5.2.3. General procedure for 2,4-di-O-acylation of compound 4 and 2,3,4,6-tetra-O-
acylation of 2 by direct method. To a solution of the 4 or 2 (0.1 g) in dry pyridine (1 mL)
corresponding acyl halide (2.2 or 4.4 eq.) was added slowly at 0 ºC followed by addition of
catalytic amount of DMAP. The reaction mixture was allowed to attain room temperature
and stirring was continued for 11-18 h. For compounds 6 reaction mixture was stirred
additional 1-2 h at 45 °C. A few pieces of ice was added to the reaction mixture to
decompose excess acyl halide and extracted with DCM (5×3 mL). The DCM layer was
washed successively with 5% hydrochloric acid, saturated aqueous sodium hydrogen
carbonate solution and brine. The DCM layer was dried and concentrated under reduced
pressure. The residue thus obtained on column chromatography (elution with n-hexane/ethyl
acetate) gave the corresponding OBG esters.
5.2.4. Octyl 2,4-di-O-acetyl-3,6-di-O-valeroyl-β-D-glucopyranoside (5): Oil, yield 96%; Rf
= 0.57 (n-hexane/EA = 2/1); FT-IR (KBr): ν 1761, 1760, 1733, 1729 (CO), 1091 cm-1
(pyranose ring); 1H NMR (400 MHz, CDCl3): δH 5.24 (t, 1H, J3,4 9.6 Hz, H-3), 5.09 (t, 1H,
J4,5 9.5 Hz, H-4), 5.00 (dd, 1H, J2,3 9.6 Hz, H-2), 4.50 (d, 1H, J1,2 8.0 Hz, H-1), 4.25 (dd, 1H,
J6a,6b 12.4, J5,6a 4.8 Hz, H-6a), 4.42 (dd, 1H, J5,6b 2.4 Hz, H-6b), 3.88 [dt, 1H, J 9.6 and 6.4
Hz, CH3(CH2)6CHAHBO], 3.68-3.73 (m, 1H, H-5), 3.49 [dt, 1H, J 9.6 and 6.9 Hz,
CH3(CH2)6CHAHBO], 2.36 [t, 2H, J 7.6 Hz, CH3(CH2)2CH2CO], 2.27 [t, 2H, J 7.5 Hz,
19

CH3(CH2)2CH2CO], 2.04 (s, 3H, CH3CO), 2.03 (s, 3H, CH3CO), 1.51-1.68 [m, 6H,
CH3(CH2)5CH2CH2O and 2×CH3CH2CH2CH2CO], 1.27-1.42 [m, 14H,
CH3(CH2)5CH2CH2O and 2×CH3CH2(CH2)2CO], 0.94 [t, 3H, J 7.6 Hz, CH3(CH2)3CO], 0.91
[t, 3H, J 7.6 Hz, CH3(CH2)3CO], 0.88 [t, 3H, J 7.2 Hz, CH3(CH2)7O]; 13C NMR (100 MHz,
CDCl3): δC 173.5, 173.1, 169.3, 169.2 (CO), 100.8 (C-1), 72.5 (C-3), 71.9 (C-5), 71.4 (C-2),
70.2 [CH3(CH2)6CH2O], 68.6 (C-4), 61.9 (C-6), 33.8, 33.7 [2×CH3(CH2)2CH2CO], 31.8,
29.4, 29.3, 29.2, 26.9, 26.8, 25.8, 22.6, 22.2, 22.1 [CH3(CH2)6CH2O and
2×CH3(CH2)2CH2CO], 20.6, 20.5 (CH3CO), 14.0 [CH3(CH2)7O], 13.7, 13.5
[2×CH3(CH2)3CO]; Anal. Calcd for C28H48O10: C, 61.74; H, 8.88. Found: C, 61.79; H, 8.86.
The assignments of the signals of this compound 5 were established by scanning and
analyzing its DEPT-135, COSY, HSQC and HMBC experiments.
5.2.5. Octyl 2,4-di-O-decanoyl-3,6-di-O-valeroyl-β-D-glucopyranoside (6): Oil, yield 76%;
Rf = 0.65 (n-hexane/EA = 2/1); FT-IR (KBr): ν 1756, 1749, 1742, 1738 (CO), 1063 cm-1
(pyranose ring); 1H NMR (400 MHz, CDCl3): δH 5.23 (t, 1H, J3,4 9.6 Hz, H-3), 5.09 (t, 1H,
J4,5 9.6 Hz, H-4), 5.00 (dd, 1H, J2,3 9.6 Hz, H-2), 4.48 (d, 1H, J1,2 8.0 Hz, H-1), 4.20 (dd, 1H,
J6a,6b 12.2, J5,6a 4.8 Hz, H-6a), 4.16 (dd, 1H, J5,6b 2.0 Hz, H-6b), 3.85 [dt, 1H, J 9.6 and 6.4
Hz, CH3(CH2)6CHAHBO], 3.66-3.71 (m, 1H, H-5), 3.45 [dt, 1H, J 9.6 and 6.8 Hz,
CH3(CH2)6CHAHBO], 2.42 [t, 2H, J 7.6 Hz, CH3(CH2)2CH2CO], 2.20-2.36 [m, 6H,
CH3(CH2)2CH2CO and 2×CH3(CH2)7CH2CO], 1.49-1.65 [m, 10H, CH3(CH2)5CH2CH2O,
2×CH3CH2CH2CH2CO and 2×CH3(CH2)6CH2CH2CO], 1.21-1.39 [br m, 38H,
CH3(CH2)5(CH2)2O, 2×CH3CH2(CH2)2CO and 2×CH3(CH2)6(CH2)2CO], 0.87-0.95 [m, 15H,
CH3(CH2)7O, 2×CH3(CH2)3CO and 2×CH3(CH2)8CO]; 13C NMR (100 MHz, CDCl3): δC
173.4, 172.9, 172.1, 172.0 (CO), 100.9 (C-1), 72.5 (C-3), 72.0 (C-5), 71.1 (C-2), 70.1
[CH3(CH2)6CH2O], 68.4 (C-4), 62.0 (C-6), 34.0(2), 33.8, 33.7 [2×CH3(CH2)2CH2CO and
2×CH3(CH2)7CH2CO], 31.8 (3) [CH3(CH2)5CH2CH2O and 2×CH3(CH2)6CH2CH2CO], 30.0,
29.7, 29.4, 29.3(2), 29.2(3), 29.1, 29.0, 28.9, 28.7, 26.8(2), 25.9, 24.8, 24.7, 22.6(2), 22.2(2)
[CH3(CH2)5CH2CH2O, 2×CH3(CH2)2CH2CO and 2×CH3(CH2)6(CH2)2CO], 14.0(3), 13.6(2)
[CH3(CH2)7O, 2×CH3(CH2)3CO and 2×CH3(CH2)8CO]; Anal. Calcd for C44H80O10: C,
68.71; H, 10.48. Found: C, 68.69; H, 10.46.
5.2.6. Octyl 2,4-di-O-benzoyl-3,6-di-O-valeroyl-β-D-glucopyranoside (7): White solid, mp
62-65 °C, yield 83%; Rf = 0.60 (n-hexane/EA = 2/1); FT-IR (KBr): ν 1756, 1747, 1731,
1
1726 (CO), 1086 cm-1 (pyranose ring); H NMR (400 MHz, CDCl3): δH 8.03 (d, 2H, J 7.6
Hz, Ar-H), 7.99 (d, 2H, J 7.2 Hz, Ar-H), 7.56-7.64 (m, 2H, Ar-H), 7.45 (t, 4H, J 7.8 Hz, Ar-
20

H), 5.65 (t, 1H, J3,4 9.6 Hz, H-3), 5.43 (t, 1H, J4,5 9.6 Hz, H-4), 5.35 (dd, 1H, J2,3 9.6 Hz, H-
2), 4.72 (d, 1H, J1,2 8.0 Hz, H-1), 4.27-4.32 (m, 2H, H-6), 3.88-3.97 (m, 2H, H-5 and
CH3(CH2)6CHAHBO], 3.48-3.55 [m, 1H, CH3(CH2)6CHAHBO], 2.30 [t, 2H, J 7.2 Hz,
CH3(CH2)2CH2CO], 2.09 [t, 2H, J 7.6 Hz, CH3(CH2)2CH2CO], 1.50-1.63 [m, 6H,
CH3(CH2)5CH2CH2O
and
2×CH3CH2CH2CH2CO],
1.08-1.26
[br
m,
14H,
CH3(CH2)5CH2CH2O and 2×CH3CH2(CH2)2CO], 0.92 [t, 6H, J 7.2 Hz, 2×CH3(CH2)3CO],
0.85 [t, 3H, J 7.2 Hz, CH3(CH2)7O]; 13C NMR (100 MHz, CDCl3): δC 173.4, 173.0
(C4H9CO), 165.1, 165.0 (PhCO), 133.5, 133.2, 129.9, 129.8, 129.5, 129.0, 128.5, 128.4 (Ar-
C), 101.2 (C-1), 72.1 (C-3), 72.0 (C-5), 71.8 (C-2), 70.3 [CH3(CH2)6CH2O], 69.7 (C-4),
62.5 (C-6), 33.8, 33.7 [2×CH3(CH2)2CH2CO], 31.7 [CH3(CH2)5CH2CH2O], 29.4, 29.2, 29.1
[CH3(CH2)2(CH2)3(CH2)2O],
26.9,
26.8
[2×CH3CH2CH2CH2CO],
25.8
[CH3CH2CH2(CH2)5O], 22.6 [CH3CH2(CH2)6O], 22.2, 21.9 [2×CH3CH2(CH2)2CO], 14.0
[CH3(CH2)7O], 13.7, 13.3 [2×CH3(CH2)3CO]; Anal. Calcd for C38H52O10: C, 68.24; H, 7.84.
Found: C, 68.31; H, 7.87.
5.2.7. Octyl 2,4-di-O-tosyl-3,6-di-O-valeroyl-β-D-glucopyranoside (8): Thick syrup, yield
78%; Rf = 0.51 (n-hexane/EA = 2/1); FT-IR (KBr): ν 1755, 1749 (CO), 1400 (br, SO2), 1096
cm-1 (pyranose ring); 1H NMR (400 MHz, CDCl3): δH 7.82 (4H, d, J = 8.0 Hz, Ar-H), 7.31-
7.39 (4H, m, Ar-H), 5.10-5.16 (m, 2H, H-3 and H-4), 4.82 (dd, 1H, J2,3 9.6 Hz, H-2), 4.34
(d, 1H, J1,2 8.0 Hz, H-1), 4.21 (dd, 1H, J6a,6b 12.4, J5,6a 5.2 Hz, H-6a), 4.17 (dd, 1H, J5,6b 2.0
Hz, H-6b), 3.87-3.92 [m, 1H, CH3(CH2)6CHAHBO], 3.63-3.69 (m, 1H, H-5), 3.50-3.59 [m,
1H, CH3(CH2)6CHAHBO], 2.52 (s, 3H, CH3-C6H4), 2.48 (s, 3H, CH3-C6H4), 2.28-2.47 [m,
4H,
2×CH3(CH2)2CH2CO],
1.55-1.63
[br
[m,
m,
6H,
CH3(CH2)5CH2CH2O
CH3(CH2)5CH2CH2O
and
and
2×CH3CH2CH2CH2CO],
1.27-1.39
14H,
2×CH3CH2(CH2)2CO], 0.88-0.95 [m, 9H, CH3(CH2)7O and 2×CH3(CH2)3CO]; Anal. Calcd
for C38H56O12S2: C, 59.35; H, 7.34. Found: C, 59.34; H, 7.36.
5.2.8. Octyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (9): Yellow solid, mp 58-60 °C,
yield 92%; Rf = 0.56 (n-hexane/EA = 2/1); FT-IR (KBr): ν 1745, 1744, 1742, 1741 (CO),
1
1093 cm-1 (pyranose ring); H NMR (400 MHz, CDCl3): δH 5.22 (t, 1H, J3,4 9.5 Hz, H-3),
5.10 (t, 1H, J4,5 9.6 Hz, H-4), 5.00 (dd, 1H, J2,3 9.6 Hz, H-2), 4.50 (d, 1H, J1,2 8.0 Hz, H-1),
4.28 (dd, 1H, J6a,6b 12.4, J5,6a 4.8 Hz, H-6a), 4.15 (dd, 1H, J5,6b 2.0 Hz, H-6b), 3.88 [dt, 1H, J
9.6 and 6.4 Hz, CH3(CH2)6CHAHBO], 3.67-3.72 (m, 1H, H-5), 3.49 [dt, 1H, J 9.6 and 6.8
Hz, CH3(CH2)6CHAHBO], 2.10 (s, 3H, CH3CO), 2.06 (s, 3H, CH3CO), 2.04 (s, 3H, CH3CO),
2.02 (s, 3H, CH3CO), 1.53-1.62 [m, 2H, CH3(CH2)5CH2CH2O], 1.22-1.36 [m, 10H,
13
CH3(CH2)5CH2CH2O], 0.90 [t, 3H, J 6.8 Hz, CH3(CH2)7O]; C NMR (100 MHz, CDCl3):
21

δC 170.6, 170.3, 169.4, 169.2 (CO), 100.8 (C-1), 72.9 (C-3), 71.8 (C-5), 71.4 (C-2), 70.2
[CH3(CH2)6CH2O], 68.6 (C-4), 62.1 (C-6), 31.8, 29.4, 29.3, 29.2, 25.8, 22.6
[CH3(CH2)6CH2O], 20.7, 20.6(3) (CH3CO), 14.0 [CH3(CH2)7O]; Anal. Calcd for C22H36O10:
C, 57.38; H, 7.88. Found: C, 57.45; H, 7.92.
5.2.9. Octyl 2,3,4,6-tetra-O-benzoyl-β-D-glucopyranoside (10): White solid, mp 97-100 °C,
yield 87%; Rf = 0.67 (n-hexane/EA = 2/1); FT-IR (KBr): ν 1723, 1719, 1715, 1710 (CO),
1
1069 cm-1 (pyranose ring); H NMR (400 MHz, CDCl3): δH 8.04 (d, 2H, J 7.2 Hz, Ar-H),
7.98 (d, 2H, J 7.2 Hz, Ar-H), 7.92 (d, 2H, J 7.2 Hz, Ar-H), 7.84 (d, 2H, J 7.2 Hz, Ar-H),
7.51-7.58 (m, 3H, Ar-H), 7.28-7.49 (m, 9H, Ar-H), 5.93 (t, 1H, J3,4 9.6 Hz, H-3), 5.69 (t,
1H, J4,5 9.6 Hz, H-4), 5.54 (dd, 1H, J2,3 9.8 Hz, H-2), 4.85 (d, 1H, J1,2 8.0 Hz, H-1), 4.66 (dd,
1H, J6a,6b 12.0, J5,6a 3.2 Hz, H-6a), 4.53 (dd, 1H, J5,6b 5.2 Hz, H-6b), 4.17 (ddd, 1H, H-5),
3.94 [dt, 1H, J 9.6 and 6.0 Hz, CH3(CH2)6CHAHBO], 3.56 [dt, 1H, J 9.6 and 6.5 Hz,
CH3(CH2)6CHAHBO], 1.49-1.58 [m, 2H, CH3(CH2)5CH2CH2O], 1.04-1.25 [br m, 10H,
13
CH3(CH2)5CH2CH2O], 0.85 [t, 3H, J 6.8 Hz, CH3(CH2)7O]; C NMR (100 MHz, CDCl3):
δC 166.2, 165.9, 165.2, 165.1 (CO), 133.4, 133.2, 133.1, 133.0, 129.9, 129.8, 129.7(2),
129.6, 129.5, 128.9(2), 128.4, 128.3(3) (Ar-C), 101.3 (C-1), 73.0 (C-3), 72.2 (C-5), 72.0 (C-
2), 70.4 [CH3(CH2)6CH2O], 70.0 (C-4), 63.3 (C-6), 31.7, 29.4, 29.2, 29.1, 25.8, 22.6
[CH3(CH2)6CH2O], 14.0 [CH3(CH2)7O]; Anal. Calcd for C42H44O10: C, 71.17; H, 6.26.
Found: C, 71.25; H, 6.28.
5.3. Evaluation of in vitro antimicrobial activities
In the present study, five human pathogenic bacteria were used as test organisms, to detect
the antibacterial activities of different OBG esters as shown in Table 9. Among these human
pathogens two was Gram-positive and five were Gram-negative. In vitro antifungal
activities of the synthesized OBG derivatives were investigated against four human
pathogenic fungi. The test tube cultures of the bacterial and fungal pathogens were collected
from the Microbiology Laboratory, Department of Microbiology, University of Chittagong,
Bangladesh.
22

Table 9. Name of the microorganisms.
Sl.
Strain
Reference
Source
Gram-positive bacteria
(i)
(ii)
Bacillus subtilis
Staphylococcus aureus
ATCC 6633
ATCC 25923
Gastrointestinal tract
Clinical strains
Gram-negative bacteria
(iii)
(iv)
(v)
(vi)
(vii)
Escherichia coli
Escherichia coli
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Salmonella abony
ATCC 25922
ATCC 8739
ATCC 8027
ATCC 27853
Clinical isolate
Lower gut of animals
Animal skin
Animal skin
Natural sugars
NCTC 6017
Name of the fungi
Type of organism
Human pathogenic
Human pathogenic
Human pathogenic
Human pathogenic
(i)
Aspergillus flavus
(ii)
(iii)
(iv)
Aspergillus niger
Candida albicans
Fusarium solani
5.3.1. Screening of antibacterial activity
For antibacterial activities test, the disc diffusion method [24] was followed. Dimethyl
sulfoxide (DMSO) was used as a solvent for test chemicals and a 2% solution of the
compound was used in the investigation. The plates were incubated at 37 °C for 48 h. Proper
control was maintained with DMSO without chemicals. Mueller-Hinton (agar and broth)
medium was used for culture of bacteria. Each experiment was carried out three times. All
the results were compared with the standard antibacterial antibiotic ciprofloxacin (10 µg
dw/disc, brand name Ciprocin, Square Pharmaceuticals Ltd., Bangladesh).
5.3.2. Screening of mycelial growth
The antifungal efficacy of the newly synthesized OBG esters (3-10) was investigated based
on food poisoning technique [25]. Sabouraud (agar and broth, PDA) medium was used for
culture of fungi. Linear mycelial growth of fungus was measured after 3~5 days of
incubation. The percentage inhibition of radial mycelial growth of the test fungus was
(
C −T
)






I =
×100
calculated as
C
where, I = percentage of inhibition, C = diameter of the fungal colony in control (DMSO), T
= diameter of the fungal colony in treatment. The results were compared with standard
antifungal antibiotic fluconazole (10 µg dw/mL medium, brand name Flugal® 50, Square
Pharmaceuticals Ltd., Bangladesh).
23

5.4. ADME/T Analysis
We have analyzed ADME/T of all the compounds by using computational approaches. For
these calculations, at first, we have drawn all the structures of OBG esters in ChemDraw
18.0 to collect InChI Key, isomeric SMILES and SD file format. Absorption, distribution,
metabolism, excretion and toxicity (ADMET) of all the OBG esters were predicted by using
AdmetSAR [31] and SwissADME [32] free web tools. SMILES (simplified molecular-input
line-entry system) strings were used throughout the process. InChI Key were used to find
these compounds in different databases, we found only 9 and 10 in PubChem database [33]
as CID 9847074 and 101430141, respectively.
5.5. Molecular Docking and non-bond interaction analysis
Lanosterol 14-alpha demethylase catalyzes C14-demethylation of lanosterol which is
important for ergosterol biosynthesis [28]. Inhibition of this enzyme stopped ergosterol
synthesis, which further facilitates cell membrane to rapture in microorganisms. Azoles are
known as inhibitors of lanosterol 14-alpha demethylase and the crystal structures of their
complexes with the bacterial or fungal enzyme were determined previously [34]. Looking
for the structural understanding we have docked these new synthetic compounds with
lanosterol 14-alpha demethylase and compare docking score and non-bond interaction with
bounded itraconazole.
5.5.1. Ligand preparation
After drawing the entire synthetic compounds in ChemDraw 18.0 we have saved all the
compounds as SDF format. Further, all the SDF prepared for dataset by using Schrödinger
Suite 2013 [35] software. Here, all ligands were minimized in Ligprep2.5 wizard, by
applying OPLS 2005 force field. During the minimization, the module Epik 2.2 [36] was
utilized to fix the ionization state of the ligand at pH 7.0 ± 2.0. From this analysis, up to 32
possible stereoisomers of each compound were generated and we selected the best
conformer with lowest energy for structural understanding.
5.5.2. Protein Preparation
Protein structures for molecular docking were prepared using the protein preparation
module of Schrödinger Suite 2013 [35] software, in which the crystal structure is initially
assigned proper hydrogen, charges and bond orders. As the main target of the molecular
docking was lanosterol 14-alpha demethylase, its crystal structure was retrieved from the
RCSB protein data bank (PDB id: 5V5Z; organism: Candida albicans). At neutral pH, all
hydrogen bonds in the structure were optimized, deleting unnecessary water. Afterwards,
24

the minimization process was run with the OPLS 2005 force field, considering structural
changes of not more than 0.30 Å of RMSD. The active site of the protein was fixed for
docking simulation by generating a grid box at the reference ligand binding the protein. Grid
generation parameters were kept at default, with a box size of 18 Å × 18 Å × 18 Å, and the
OPLS 2005 force field utilized for post minimization. The charge cutoff and van der Waals
scaling factor were set to 0.25 and 1.00, respectively [37].
5.5.3. Molecular docking
We carried out extra precision (XP) flexible docking using the Glide module of
Schrödinger-Maestro v9.4 [38] which is more sophisticated than SP/HTVS in scoring
function [38]. Here, all ligands were treated flexibly, considering the partial charge and van
der Waals factor of 0.15 and 0.80, respectively. Minimization was performed to the docked
complex after docking using the OPLS 2005 force field. The best-docked pose with lowest
Glide score value was recorded for each ligand [37].
Acknowledgements
This work was supported by the Ministry of Education, Bangladesh and BANBEIS [grant
no. PS 201660, 2016-17]. One of the authors (P.C.) is indebted to Ministry of Science and
Technology for NST fellowship.
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