Synthesis and biological evaluation of new fluconazole β-lactam conjugates linked via 1,2,3-triazole

Article abstract of DOI:10.1039/c6nj03117j

Novel 1,2,3-triazole-linked β-lactam-fluconazole conjugates 12(a-l) were designed and synthesized. The compounds showed potent antifungal activity against two pathogenic Candida strains; Candida albicans ATCC 24433 and Candida albicans ATCC 10231 with MIC values in the range of 0.0625-2 μg mL^-1. Compounds 12h, 12j and 12k showed promising antifungal activity against all the tested fungal pathogens except C. neoformans ATCC 34554 compared to fluconazole. Compound 12j in which the β-lactam ring was formed using para-anisidine and benzaldehyde was found to be more potent than fluconazole against all the fungal strains with an IC₅₀ value of <0.015 μg mL^-1 for Candida albicans (ATCC 24433). Mechanistic studies for active compounds revealed that the antifungal action was due to ergosterol inhibition. Compounds 12h and 12j at a concentration of 0.125 μg mL^-1 caused 91.5 and 96.8% ergosterol depletion, respectively, compared to fluconazole which at the same concentration caused 49% ergosterol depletion. The molecular docking study revealed that all the fluconazole β-lactam conjugates 12(a-l) could snugly fit into the active site of lanosterol 14α-demethylase (CYP51) with varying degrees of affinities. As anticipated, the binding energy for compound 12j (-58.961 kcal mol^-1) was much smaller than that for fluconazole (-52.92 kcal mol^-1). The synthesized compounds have therapeutic potential for the control of candidemia.

Full text of DOI:10.1039/c6nj03117j

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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Synthesis and biological evaluation of new fluconazole β-lactam
conjugates linked via 1,2,3-triazole
Jaisingh M. Divse,a Santosh B. Mhaske,a Chaitanya R. Charolkar,a Duhita G. Sant,b Santosh G.
Tupe,b Mukund V. Deshpande,b Vijay M. Khedkar,c,d Laxman U. Nawale,c Dhiman Sarkar,c and
Vandana S. Pore*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Novel 1,2,3-triazole-linked β-lactam–fluconazole conjugates 12(a-l) were designed and synthesized. The compounds
showed potent antifungal activity against two pathogenic Candida strains; Candida albicans ATCC 24433 and Candida
albicans ATCC 10231 with MIC values in the range of 0.0625 – 2 μg/mL. Compounds 12h, 12j and 12k showed promising
antifungal activity against all the tested fungal pathogens except C. neoformans ATCC 34554 as compared to fluconazole.
Compound 12j in which β-lactam ring was formed using para-anisidine and benzaldehyde was found to be more potent
than flucanozole against all the fungal strains with an IC50 value of <0.015 µg/mL for Candida albicans (ATCC 24433).
Mechanistiic studies for active compounds revealed that the antifungal action was due to ergosterol inhibition.
Compounds 12h and 12j at 0.125 μg/mL concentration caused 91.5 and 96.8% ergosterol depletion respectively as
compared to fluconazole which at same concentration caused 49% ergosterol depletion. Molecular docking study revealed
that all the fluconazole β-lactam conjugates 12(a-l) could snugly fit into the active site of lanosterol 14 α-demethylase
(CYP51) with varying degree of affinities. As anticipated, binding energy for compound 12j (-58.961 kcal/mol) was much
lesser than fluconazole (-52.92 kal/mol). The synthesized compounds have therapeutic potential for the control of
candidemia.
to its potent activity; excellent safety profile, and favorable
pharmacokinetics characteristics.4 However, fluconazole is not
fungicidal and extensive use of fluconazole has increased the
1. Introduction
number of fluconazole-resistant C. albicans isolates.5 Therefore;
great efforts have been made to modify the chemical structure of
fluconazole, in order to broaden its antifungal spectrum of activity
and to increase its potency.
Fungal infections caused by Candida, Aspergilli and Cryptococcus
have continued to be a major medical problem since past two
decades especially involving immunocompromised patients.1,2 Four
major types of drugs including azoles (e.g. fluconazole), allyamines
(e.g. naftifine), polyene macrolides (e.g. amphotericin B) and
echinocandins (e.g. caspofungin) are most frequently used
antifungal agents for treatment of fungal infections.3 The current
antifungal drugs are either highly toxic (amphotericin B) or are
becoming ineffective due to emergence of resistant strains
(flucytosine and azoles). In spite of significant research on
antifungal agents, the azoles continue to be the mainstay of therapy
for systemic life threatening fungal infections as they can be
administered orally and have broad-spectrum activities against
most of the pathogenic yeasts and filamentous fungi. Fluconazole, a
1,2,4-triazole has shown an exceptional therapeutic performance
against Candida infections. It is a drug of choice for the treatment
of infections by Candida albicans and Cryptococcus neoformans due
Based on the structure of the active site of lanosterol 1,4 α-
demethylase (CYP51) and the extensive investigation of the
structure–activity relationships of azole antifungals, it was found
that 1,2,4-triazole ring and 2,4-difluorophenyl group are essential
for the promising antifungal activity.6 In the course of our ongoing
program for development of new fluconazole analogues, we have
previously reported fluconazole bile acid conjugates,7 new
fluconazole analogues containing one8 and two9 1,2,3-triazole rings
(Figure 1). Some of these molecules showed better activity than
fluconazole and amphotericin B with minimal cytotoxicity. Cu(I)
catalyzed 1,3-dipolar cycloaddition (click chemistry) has been a
method of choice for the synthesis of 1,4-disubstituted-1,2,3-
triazoles.10 The reactions take place under mild conditions, with
high yields and regioselectivity, and uses copper as catalyst that has
no impact on most of the other functional groups. Among the
various reaction conditions, microwave assisted Cu (I) catalyzed
reaction was found to be suitable for this conjugation as the
reaction time is reduced considerably.11
β-Lactam ring is a common structural feature in a number of
broad spectrum antibiotics, including penicillins, cephalosporins,
carbapenems, nocardicins and monobactams, which have been
widely used as chemotherapeutic agents for treating bacterial
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Figure 1. Fluconazole analogues containing 1,2,3triazole ring
infections.12 The compounds containing β-lactam ring also exhibited
many other remarkable biological properties such as cholesterol
absorption inhibition,13 human cytomegalovirus protease
inhibition,14 thrombin inhibition,15 antihyperglycemic,16 anti-
tumour,17 anti-HIV,18 anti-inflammatory, analgesic activities19 and
serine-dependent enzyme inhibition.20
bromoacetate 1 (Scheme 1). Compound 1 on treatment with
sodium azide afforded ethyl azidoacetate 2. Subsequent
saponification of ester functionality of compound 2 with KOH in
methanol gave the potassium salt of azidoacetic acid 3 with 92%
yield. Imines 6 (a-l) were conveniently prepared under microwave
conditions within 5-10 min in excellent yields (90-95%), from the
aldehydes 5 (a-l) and amine 4 (a and b).
Previously we have reported the synthesis of triazole-linked β-
lactam bile acids conjugates which exhibited good antibacterial and
antifungal activity.21 In present study, we have designed and
synthesized new fluconazole β-lactam conjugates linked via 1,2,3-
triazole. Antibacterial and antifungal activities of the synthesized
compounds were determined and mechanism of action of the lead
compounds was studied. Furthermore, molecular docking was
carried out to gain an insight into the molecular basis of their
interactions with the potential fungal target lanosterol 14α-
demethylase (CYP51).
The mixtures of azides 7 (a-l) and 8 (a-l) were prepared by using
the ketene–imine cycloaddition (Staudinger reaction)22 of
potassium azidoacetate 3 and imines 6 (a-l) in the presence of
triphosgene and triethylamine in anhydrous dichloromethane with
good yields. Compounds 7 (a-l) and 8 (a-l) were obtained as
diastereomeric mixture which was inseparable by crystallization or
column chromatography.21a
Keto compound 10 was synthesized starting from m-
difluorobenzene 9 and further reaction of 10 with propargyl
bromide under Barbier reaction conditions afforded alkyne 11 as a
racemic mixture (Scheme 2).23
2.Result and Discussion
Our next objective was to synthesize 1,2,3-triazole-linked β-
lactam–fluconazole conjugates 12 (a-l).The cycloaddition reaction
of alkyne 11, with a mixture of azides 7 (a-l) and 8 (a-l), in the
presence of Cu(I) catalyst as per our reported procedure7 under
microwave irradiation furnished diastereomeric mixture of novel
conjugates 12 (a-l) in excellent yields (Scheme 3). These
diastereomers were inseparable by flash column chromatography
2.1. Chemistry:
Target molecules 12 (a-l) were synthesized by 1,3-dipolar
cycloaddition reaction of β-lactam containing racemic mixture of
azides 7 (a-l) and 8 (a-l) with fluconazole intermediate 11 having
terminal alkyne, in the presence of Cu(I) catalyst. Briefly, the
synthesis of azides 7 (a-l) and 8 (a-l) was started from ethyl
2 | J. Name., 2012, 00, 1-3
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Scheme 1. Synthesis of mixture of azides 7 (a-l) and 8 (a-l)
OH
O
N
N
N
N
(c) Zn, propargyl bromide,
DMF/THF, 25 oC, 5 h, 95%.
(a) AlCl3, 1,2-dichloroethane,
chloroacetyl chloride, 250C, 7 h
F
F
F
N
N
(b)1,2,4-triazole,NaHCO3,toluene,
reflux, 4 h, (overall 55% in two step)
F
11
F
9
F
10
Scheme 2. Synthesis of alkyne 11
.
N
N
HO
N
F
N
N
OH
O
N3
O
N
N
CuSO4.5H2O
N
R6
R6
Na-Ascorbate
F
F
N
R5
R4
N
N
R5
R4
DMF:H2O
R1
R2
Microwave
F
R1
R2
R3
R3
12 (a-l) as mixture of diastereomers
(dr = 1:1)
11
7 + 8 (a-l)
12a,
R1 = OMe, R2 = R3 = R4 = R5 = R6 = F
12g,
R1 = OMe,R4 = Cl, R2 = R3 = R5 = R6 = H
12b, R1 = OMe, R2 = F, R3 = Cl, R5 = CF3, R4 = R6 = H
12c,
12h, R1 = OMe, R2 = Cl, R3 = R4 = R5 = R6 = H
12i,
R1 = H, R2 = R3 = F, R4 = R5 = R6 = H
12d, R1 = H, R2 = F, R3 = CF3, R4 = R5 = R6 = H
12e,
R1 = OMe, R2 = R4 = Cl, R3 = R5 = R6 = H
12j, R1 = OMe, R2 = R3 = R4 = R5 = R6 = H
12k,
R1 = OMe, R3 = Me, R4 = F, R2 = R5 = R6 = H
12f, R1 = H, R3 = F, R2 = R4 = R5 = R6 = H
R1 = OMe, R4 = F, R2 = R3 = R5 = R6 = H
12l, R1 = H, R4 = F, R2 = R3 = R5 = R6 = H
Scheme 3. Synthesis of 1,2,3-triazole-linked β-lactam fluconazole conjugates 12 (a-l)
and also by crystallization. Their separation by preparative HPLC 2.2. Biological activity
was also not successful. The synthesized molecules, mixture of
2.2.1. Antibacterial activity
azides
7 (a-l) and8 (a-l) and 1,2,3-triazole-linked β-lactam–
fluconazole conjugates 12 (a-l) were characterized by FTIR, 1H NMR,
13C NMR and HR-MS.
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Table 1. Antifungal activity of the 1,2,3-triazole-linked β-lactam fluconazole conjugates 12 (a-l).
C. albicans ATCC
24433
C. albicans
ATCC10231
IC50 MIC
C. neoformans ATCC
34554
A. niger ATCC
10578
A. fumigatus NCIM
908
Compounds
C. glabrata NCYC 388 C. tropicalis ATCC 750
*
IC50
MIC**
IC50
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
MIC
12a
12b
0.28
0.11
0.5
0.25
0.25
0.5
9.2
1.34
3.95
6.18
3.74
2.9
16
4
110.5
41.83
107.2
68.01
102
>256
64
57
128
16
83.45
46.81
14.26
69.79
71.31
8.15
>256
128
32
120.4
83.2
>256
>256
>256
>256
>256
>256
256
63.62
202.9
220.9
118
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
8.68
20.63
32.23
73.6
36.24
18.22
16.97
6.23
8
12c
0.1
8
256
128
>256
>256
128
128
128
32
64
96
12d
0.29
16
8
64
128
>256
16
54.04
41.63
189.8
133.1
64
12e
0.16
0.25
0.125
0.125
0.25
0.5
>256
64
56
12f
0.083
0.086
0.1
4
169
164.4
139.1
52.62
67.11
91.25
112.2
90.43
96.7
12g
0.74
1.93
1.1
2
59.73
45.91
73.79
16.26
66.66
32.34
8.05
32
6.52
32
12h
4
32
17.92
14.67
36.09
15.52
14.2
32
>256
>256
64
12i
0.21
4
16
32
80.9
12j
<0.015
0.041
0.17
0.031
0.0625
0.25
2
0.1
0.5
2
16
64
12.57
90.14
113.7
75.9
12k
0.56
1.7
128
64
17.02
11.93
68.4
32
64
256
12l
4
32
32
256
Fluconazole
0.150
3.29
8
16
128
89.9
>256
128
* IC50: concentration (in μg/mL) causing 50% growth inhibition; **MIC: Minimum Inhibitory Concentration causing >90% growth inhibition.
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As all the synthesized compounds 12 (a-l) had β-lactam The results indicated that the newly synthesized 1,2,3-triazole-
pharmacophore as a one of the functional group, they were tested linked β-lactam fluconazole conjugates exerted their antifungal
for in vitro antibacterial activity at concentrations 0.39-50 µg/mL action through inhibition of lanosterol 14 α-demethylase and
against Escheirchia coli, Pseudomonas aeruginosa and concomitant ergosterol depletion.
Staphylococcus aureus by broth microdilution method.21 None of
the compounds showed antibacterial activity against the tested
24433 when exposed to ½ MIC, MIC and 2X MIC concentration of
strains.
2.2.2. Antifungal activity
Table 2. Depletion of total ergosterol content of C. albicans ATCC
1,2,3-triazole-linked β-lactam fluconazole conjugates
The antifungal activity of the final compounds 12 (a-l) was
Concentration
(µg/mL)
0.0625
0.125
0.25
%Ergosterol
depletion
12.459
91.598
92.459
68.033
92.049
93.238
39.385
59.795
96.885
41.270
67.787
94.344
49.057
67.254
81.475
evaluated by CLSI standard broth microdilution assay against
different fungal pathogens, viz. Candida albicans ATCC 24433, C.
albicans ATCC 10231, Cryptococcus neoformans ATCC 34554,
Candida glabrata NCYC 388, Candida tropicalis ATCC 750,
Aspergillus niger ATCC 10578 and Aspergillus fumigatus NCIM
908.24 Antifungal activity data of 1,2,3-triazole-linked β-lactam
fluconazole conjugates 12 (a-l) is given in Table 1 in terms of IC50
(concentration causing 50% growth inhibition) and MIC(minimum
inhibitory concentration causing >90% growth inhibition).
Fluconazole was used as a standard antifungal drug.
Compound
12h
0.125
0.25
12i
12j
All the compounds showed better antifungal activity than
fluconazole with MIC values ranging from 16 – 0.031 μg/mL against
both C. albicansstrains with an exception of 12a and 12d which
exhibited 2X MIC than fluconazole against C. albicans ATCC 10231.
Compound 12j with β-lactam ring containing para-anisidine and
benzaldehyde was most effective with MIC values 0.031 and 0.5
μg/mL against C. albicans ATCC 24433 and C. albicans ATCC10231,
respectively. It also showed good activity against all other fungal
pathogens tested. Good to moderate activity was observed for the
synthesized compounds 12 (a-l) against Cryptococcus neoformans
ATCC 34554, Candida glabrata NCYC 388 and Candida tropicalis
ATCC 750, whereas they were found least susceptible against A.
niger ATCC 10578and A. fumigatus NCIM 908. Four compounds
12h, 12i, 12j and 12k showed good antifungal activity against C.
tropicalis ATCC 750 (MIC values in the range of 64 – 32 μg/mL)
where fluconazole proved to be ineffective. Based on these results,
12h, 12j and 12k were identified as leads and used for further
mechanistic studies.
0.5
0.03125
0.0625
0.125
0.0625
0.125
0.25
12k
Fluconazole
0.125
0.25
0.5
2.2.4. Molecular Docking
Molecular docking study has become an integral part of drug
discovery research in recent times to identify the potential targets
for different ligands and their associated thermodynamic
interactions with the target enzyme governing the inhibition of the
pathogen. This structure-based drug design approach imparts
knowledge on binding affinities, binding modes and types of
interactions guiding the enzyme–inhibitor complexation which
provides an ample opportunity to medicinal chemists for generating
and analyzing the potential of designed molecules. Thus, with the
aim to rationalize the promising antifungal activity portrayed by the
title compounds 12 (a-l) and to gain an insight into their binding
interactions at the molecular level, a molecular docking study was
carried out with Candida albicans sterol 14α-demethylase (CYP51)
as target enzyme. Molecular docking study revealed that all the
2.2.3. Ergosterol quantification assay
The cellular target of azole antifungals is lanosterol 14 α-
demethylase, an enzyme of ergosterol biosynthesis pathway.
Inhibition of lanosterol 14 α-demethylase results in accumulation of
methylated sterol intermediates and depletion of ergosterol. This
results in membrane instability, loss in cellular integrity and
subsequent death of the fungus.25,26 In spectrophotometric sterol
analysis, ergosterol and late sterol intermediate 24(28)-
dehydroergosterol gives a characteristic four peak curve for a 230-
300 nm scan. For the Candida cells samples treated with 12h, 12i,
12j and 12k, the peak intensity decreased with increase in the
concentration of the compounds (Figure 2) indicating depletion of
ergosterol. Compounds 12h and 12j at 0.125 μg/mL concentration
produced >90% ergosterol depletion, whereas, same concentration
of fluconazole could cause only 49% ergosterol depletion (Table 2). fluconazole β-lactam conjugates 12 (a-l) could snugly fit into the
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12h
Control
12i
Control
0.0625 μg/ml
0.125 μg/ml
0.25 μg/ml
0.5 μg/ml
3
2.5
2
3
0.125 μg/ml
2.5
0.25 μg/ml
2
1.5
1
1.5
1
0.5
0
0.5
0
220
240
260
280
300
220
240
260
280
300
Wavelength (nm)
Wavelength (nm)
12k
Control
Control
12j
0.03125 μg/ml
0.0625 μg/ml
0.125 μg/ml
3
2.5
2
0.0625 μg/ml
0.125 μg/ml
0.25 μg/ml
3
2.5
2
1.5
1
1.5
1
0.5
0
0.5
0
220
240
260
280
300
220
240
260
280
300
Wavelength (nm)
Wavelength (nm)
Fluconazole
Control
3
2.5
2
0.125 μg/ml
0.25 μg/ml
0.5 μg/ml
1.5
1
0.5
0
220
240
260
280
300
Wavelength (nm)
Figure 2. Spectrophotometric analysis of sterol composition of C. albicans ATCC 24433 exposed to
different concentrations of 12h, 12i, 12j, 12k and fluconazole.
Figure 3. Binding mode of 12j into the active site of lanosterol 14α-demethylase (CYP51) (the π-π stacking interaction)
6 | J. Name., 2012, 00, 1-3
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Table 3. Quantitative estimates of the per-residue interaction analysis of the fluconazole β-lactam conjugates 12 (a-l)
with lanosterol 14α-demethylase (CYP51).
Antifungal activity in μg/mL
Compound
MIC
IC50
Docking Score
Binding Energy (kcal/mol)
12a
12b
12c
12d
0.50
0.25
0.25
0.50
0.28
0.11
0.1
-7.46
-7.61
-7.58
-7.40
-53.38
-55.12
-55.54
-53.12
0.29
12e
0.25
0.16
-7.54
-55.10
12f
12g
12h
12i
0.125
0.125
0.25
0.5
0.083
0.086
0.1
-7.95
-7.91
-7.59
-7.45
-56.27
-56.79
-55.99
-53.95
0.21
12j
12k
12l
0.031
0.0625
0.25
<0.015
0.041
0.17
-8.50
-8.28
-7.69
-58.96
-57.89
-55.82
active site of CYP51 with varying degree of affinities adopting a very per-residual interactions revealed that the van der Waals
similar orientation and at co-ordinates close to that of the native interactions are more prevalent over the electrostatic contribution
ligand - fluconazole in the crystal structure (Figure 3).
in its overall binding affinity towards CYP51. An extensive network
of favorable van der Waals interactions with Val359 (-1.47
kcal/mol), Leu356 (-2.74 kcal/mol), Ala291 (-1.79 kcal/mol),Tyr116
(-4.21 kcal/mol), Phe110 (-2.13 kcal/mol) through the Fluconazole
component of the β-lactam conjugate stabilized the compound into
the active site while the para-methoxy subsititued aromatic ring
attached to the β-lactam was engaged in similar interactions with
Met460 (-3.75 kcal/mol), Leu208 (-3.48 kcal/mol), Met106 (-2.03
kcal/mol), Ile105 (-2.40 kcal/mol) and Tyr103 (-2.95 kcal/mol)
residues. Furthermore the second aromatic ring attached to β-
lactam was also involved in significant van der Waals interactions
with Val461 (-2.59 kcal/mol), Thr459 (-2.28 kcal/mol), His294 (-2.84
kcal/mol), Phe290 (-4.15 kcal/mol) and Glu205 (-2.54 kcal/mol)
residues lining the active site stabilizing the enzyme-inhibitor
complex. The compound was engaged in a set of relatively few but
significant electrostatic interactions as well with Ala291 (-1.36
kcal/mol), Phe290 (-1.41 kcal/mol), Glu205 (-2.51 kcal/mol), Tyr116
(-1.94 kcal/mol) and Tyr103 (-1.23 kcal/mol) residues. The
enhanced binding affinity of 12j can also be attributed to a very
strong van der Waals (-8.82 kcal/mol) as well as electrostatic (-
5.734 kcal/mol) interaction with Heme moiety present in the active
site of CYP51. This is a very important observation considering the
fact that even Fluconazole is coordinated with the iron metal in the
active site of CYP51 indicating that these compounds may as well
share the same inhibition mechanism of action as Fluconazole. A
very prominent pi-pi (π-π) stacking interaction was also observed
between the aromatic ring attached to the β-lactam and His294
residue in the active site. While 12j displayed only single pi-pi
stacking interaction, the other molecules in the dataset were
engaged in such interaction with Tyr116, Tyr103, Phe110, Phe290
residues as well. Such pi-pi stacking interactions not only serve the
function of an "anchor" guiding the 3D orientation of the ligand in
its active site but also facilitate the steric and electrostatic
interactions adding to the stability of the enzyme-inhibitor complex.
The per-residue ligand interaction analysis revealed that the
primary driving forces for mechanical interlocking was the steric
complementarity between these ligands and the active site residues
Their complexation was stabilized through
a
network of
significant steric and electrostatic interactions. The minimum
energy for the formation of complex between ligand and receptor
(Glide energy) for each of these analogues was observed to be
negative ranging from -58.961 kcal/mol to -53.125 kcal/mol with an
average docking score of -7.74 while the docking score for
Fluconazole was -7.34 with binding energy of -52.92 kcal/mol. This
was in harmony with the experimental finding where we found the
MIC of these compounds better than Fluconazole. Also a significant
correlation was observed between the docking score and the
antifungal activity- the active compounds having higher scores
while those with relatively low activity were also predicted to have
a lower score (Table 3). A higher negative value for docking score
and the binding energy signifies a good binding affinity of the ligand
towards the target and vice versa. The binding energy signifies the
energy required for a ligand to cover the entire enzyme surface and
its putative interactions at the active site. A detailed quantitative
analysis of the per-residue interaction between the compounds 12
(a-l) and the residues lining the active site of the enzyme was
carried out to identify the most significantly interacting residues
and the type of interactions (bonded and non-bonded interactions)
that govern the binding of these molecules to CYP51. However to
maintain the brevity of text, this analysis is elucidated in the section
only for the most active compound 12j while the results for the
remaining compounds are summarized in Table 3 (and Table 2S)
and their binding modes are provided in the supplementary
material as Figures (4S-14S).
The lowest energy docked conformation of the most active 12j
into the active site of CYP51 showed that the inhibitor binds at the
same co-ordinates as the native ligand with a significantly higher
binding affinity (Figure 3). The compound binds with the highest
affinity with a docking score of -8.501 and binding energy -58.961
kcal/mol. The higher binding affinity can be attributed to the
specific bonded and non-bonded per residue interactions with the
residues lining the active site of CYP51. A detailed analysis of the
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Journal Name
of CYP51 which is reflected in the relatively higher number of added. The reaction mixture was placed in a microwave reactor and
favorable van der Waals interactions than the other components irradiated for 5-8 min. at 540 watt. It was then cooled to room
contributing to the overall binding affinity. Overall, it is evident temperature, quenched with crushed ice and extracted with ethyl
from the docking simulations and more specifically from the per- acetate. The extract was washed with water and brine, dried over
residue interaction energy distribution that these molecules have anhydrous sodium sulphate. Solvent was evaporated under reduced
excellent affinity for the lanosterol 14α-demethylase (CYP51) pressure and crude product was purified by column
enzyme making them pertinent starting points for structure-based
lead optimization.
chromatography to obtain β-lactam-bile acid conjugates 12a to 12l
with 88-93% yields.
4.2.1. 3-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-
yl)propyl)-1H-1,2,3-triazol-1-yl)-1-(4-methoxyphenyl)-4-
(perfluorophenyl)azetidin-2-one;12a.Brown colored solid; Yield:
2.2.5. Cytotoxicity testing of the compounds 12 (a-l)
Compounds 12 (a-l) were checked for cytotoxicity by MTT assay
using MCF-7, A-431 and HUVEC (Human umbilical vein endothelial
cells) cell line27 and hemolysis assay.28 All the compounds at tested
concentration (up to 100 μg/mL) were found to be non-toxic to the
HUVEC cells (Table 1S). For hemolysis assay, the concentrations
tested were in the range of 2–256 μg/mL. None of the compounds
showed hemolysis at these concentrations except for 12b. Even for
12b, the hemolysis observed was negligible and at much higher
concentrations than MIC i.e. 1.94% and 1.21% hemolysis at 256 and
128 μg/mL, respectively (Table 1S).
1
90%; IR (CHCl3, cm-1) 3415, 1761; H NMR (CDCl3, 400 MHz) δ 8.05-
8.09 (m, 1H), 7.85 (d, J = 7.3 Hz, 1H), 7.71 (d, J = 9.5 Hz, 1H), 7.29 (d,
J = 8.0 Hz, 2H, Ar-H), 7.31-7.40 (m, 1H, Ar-H), 6.89 (d, J = 9.0 Hz, 2H),
6.70-6.80 (m, 2H), 6.26-6.33 (m, 1H), 5.91-5.93 (m, 1H), 5.21 (bs,
1H), 4.70-4.76 (m, 1H), 4.28-4.50 (m, 1H), 3.81 (s, 3H), 3.37-3.49 (m,
1H), 3.09-3.23 (m, 1H); 13C NMR (CDCl3, 50 MHz) δ 163.9, 157.5,
156.5, 146.5, 142.4, 142.3, 129.7, 129.3, 124.4, 123.2, 118.3, 114.8,
111.6, 111.3, 105.6, 104.5, 104.2, 104.0, 74.9, 66.5, 56.5, 55.5, 52.9,
52.8, 33.9, 33.7, 29.7, 14.1; ESI-MS (m/z): 648.15 [M+H]+; HRMS
(ESI-qTOF): calcd for C29H21F7N7O3 [M+H]+, 648.1589: found:
648.1583.
4.2.2. 4-(3-chloro-2-fluoro-5-(trifluoromethyl)phenyl)-3-(4-(2-(2,4-
difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-
1,2,3-triazol-1-yl)-1-(4-methoxyphenyl)azetidin-2-one; 12b. Brown
colored solid; Yield: 89%; IR (CHCl3, cm-1) 3415, 1761; 1H NMR
(CDCl3, 500 MHz) δ 8.02-8.06 (m, 2H), 7.81 (s, 1H), 7.47-7.51 (m, 1H,
Ar-H), 7.28-7.36 (m, 3H, Ar-H), 7.23-7.26 (m, 1H, Ar-H), 6.89 (t, J =
8.54 Hz, 2H), 6.67-6.78 (m, 2H), 6.25-6.39 (m, 1H), 5.83-5.84 (m,
1H), 5.18 (bs, 1H), 4.63-4.69 (m, 1H), 4.05-4.19 (m, 1H), 3.81 (s, 3H),
3.18-3.27 (m, 1H), 3.03-3.11 (m, 1H); 13C NMR (CDCl3, 50 MHz) δ
162.6, 157.4, 157.1, 142.8, 142.7, 129.8, 129.1, 124.5, 123.6, 123.0,
118.6, 114.8, 111.6, 111.4, 104.3, 104.1, 103.9, 74.7, 67.1, 56.5,
55.8, 55.5, 55.3, 36.5, 33.8, 33.6, 31.4, 29.7, 29.1, 14.1; ESI-MS
(m/z): 678.14 [M+H]+; HRMS (ESI-qTOF): calcd for C30H23ClF6N7O3
[M+H]+, 678.1450: found: 678.1454.
3. Conclusions
In conclusion, 12 new fluconazole β-lactam conjugates linked via
1,2,3-triazole were synthesized and evaluated for antifungal
potential. Compounds 12h, 12i, 12j and 12k were found to be most
potent compounds and exhibited better antifungal activity than
fluconazole against pathogenic strains of C. albicans and C.
tropicalis. Mode of action studies revealed that the antifungal
action of the compounds was through inhibition of lanosterol 14 α-
demethylase enzyme of ergosterol biosynthesis pathway. Results of
spectrophotometric sterol analysis corroborated with that of the
molecular docking studies confirming the mode of action of the
lead compounds. The compounds were non-hemolytic and non-
cytotoxic. The synthesized fluconazole β-lactam conjugates hold
promise for the control of Candidemia. Further optimization of lead
compounds and pre-clinical evaluation of the series will help in
discovery of potential antifungal drug candidate.
4.2.3. 4-(2,3-difluorophenyl)-3-(4-(2-(2,4-difluorophenyl)-2-
hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,3-triazol-1-yl)-1-
phenylazetidin-2-one;12c. Yellow colored solid; Yield: 91%; IR
1
(CHCl3, cm-1) 3415, 1761; H NMR (CDCl3, 400 MHz) δ 8.03-8.07 (m,
1H), 7.82 (s, 1H), 7.33-7.40 (m, 5H, Ar-H), 7.28-7.31 (m, 1H, Ar-H),
7.18-7.23 (m, 1H, Ar-H), 6.85-6.98 (m, 3H), 6.68-6.76 (m, 2H), 6.25-
6.34 (m, 1H), 5.86-5.89 (m, 1H), 5.18 (bs, 1H), 4.60-4.65 (m, 1H),
4.01-4.22 (m, 1H), 3.20-3.29 (m, 1H), 2.99-3.12 (m, 1H); 13C NMR
(CDCl3, 50 MHz) δ 165.2, 158.2, 158.0, 151.2, 142.5, 136.1, 129.8,
129.5, 125.6, 123.0, 117.3, 111.4, 104.1, 98.0, 67.0, 56.6, 56.3, 55.5,
49.6, 33.9, 33.6, 29.7, 14.1; ESI-MS (m/z): 564.14 [M+H]+; HRMS
(ESI-qTOF): calcd for C28H22F4N7O2 [M+H]+, 564.1766: found:
564.1769.
4. Experimental
4.1 General
All the reagents were purchased from Merck and Spectrochem and
used without further purification. Melting points of all the
synthesized compounds were determined in an open capillary tube
and are uncorrected. Infrared (IR) spectra were recorded on a
Shimadzu FT-IR spectrometer using KBr pellets. 1H NMR spectra
were recorded on a Bruker DRX-400 MHz NMR spectrometer. The
13C NMR spectra were recorded on a Bruker DRX-100 MHz NMR in
CDCl3 using tetramethylsilane (TMS) as an internal standard and the
chemical shifts recorded in δ ppm. High-resolution mass spectra
(HR-MS) were recorded on an Agilent 6520 (QTOF) ESI-HRMS
instrument and LC-MS spectrometer. The purity of each of the
compounds was checked by thin-layer chromatography (TLC) using
silica gel (60F254), and iodine/ultraviolet light for visualization.
4.2.4. 3-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-
yl)propyl)-1H-1,2,3-triazol-1-yl)-4-(2-fluoro-3-
(trifluoromethyl)phenyl)-1-phenylazetidin-2-one;12d. Wine red
colored solid; Yield: 90%; IR (CHCl3, cm-1) 3415, 1761; 1H NMR
(CDCl3, 400 MHz) δ 8.02-8.07 (m, 3H), 7.80 (s, 1H), 7.33-7.41 (m, 6H,
Ar-H), 7.20-7.24 (m, 1H, Ar-H), 7.00-7.21 (m, 1H, Ar-H), 6.67-6.74
(m, 2H), 6.25-6.33 (m, 1H), 5.90-5.93 (m, 1H), 5.21 (bs, 1H), 4.60-
4.65 (m, 1H), 3.99-4.17 (m, 1H), 3.17-3.25 (m, 1H), 3.02-3.10 (m,
1H); 13C NMR (CDCl3, 50 MHz) δ 162.5, 158.0, 157.8, 151.3, 142.7,
136.0, 132.16, 129.6, 125.7, 123.0, 117.3, 111.5, 104.1, 74.8, 67.1,
56.5, 56.3, 55.2, 36.5, 33.7, 31.4, 29.7; ESI-MS (m/z): 614.18 [M+H]+;
HRMS (ESI-qTOF): calcd for C29H22F6N7O2 [M+H]+, 614.1734: found:
614.1727.
4.2 General procedure for synthesis of fluconazole β-lactam
conjugates 12 (a-l).
The alkyne of fluconazole 11 (1 equiv) and the azides 7/8 (a-l)
(1 equiv) were dissolved in DMF:H2O (7:3). To this solution
CuSO4·5H2O (0.05 equiv) and sodium ascorbate (0.5 equiv) were
8 | J. Name., 2012, 00, 1-3
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4.2.5. 3-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol- MS (m/z): 626.14 [M+H]+; HRMS (ESI-qTOF): calcd for
1-yl)propyl)-1H-1,2,3-triazol-1-yl)-4-(4-fluoro-3-methylphenyl)-1-
C29H24Cl2F2N7O3 [M+H]+, 626.1280: found: 626.1269.
(4-methoxyphenyl)azetidin-2-one; 12e. cream colored solid; Yield: 4.2.10. 3-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
88%; IR (CHCl3, cm-1) 3415, 1761; 1H NMR (CDCl3, 400 MHz) δ 7.99- 1-yl)propyl)-1H-1,2,3-triazol-1-yl)-1-(4-methoxyphenyl)-4-
8.06 (m, 1H), 7.15-7.40 (m, 6H), 6.67-7.02 (m, 6H), 6.29 (d, J = 10.9 phenylazetidin-2-one; 12j. Yellow colored solid; Yield: 90%; IR
1
Hz, 1H), 5.33-5.53 (m, 1H), 4.13-4.84 (m, 4H), 3.76 (m, 3H), 3.11- (CHCl3, cm-1) 3415, 1761; H NMR (CDCl3, 500 MHz) δ 7.96-8.03 (m,
3.46 (m, 2H), 2.26 (s, 1H), 2.05-2.10 (m, 3H); 13C NMR (CDCl3, 50 1H), 7.79 (s, 1H), 7.41-7.52 (m, 1H), 7.33-7.35 (m, 2H, Ar-H), 7.22-
MHz) δ 169.7, 158.2, 157.0, 141.9, 130.0, 129.7, 126.5, 125.6, 7.26 (m, 1H, Ar-H), 7.07-7.16 (m, 4H), 6.80-6.87 (m, 2H), 6.66-6.74
119.1, 118.8, 115.4 114.5, 67.5, 60.6, 55.4, 33.7, 14.6, 14.4; HRMS (m, 2H), 6.28-6.34 (m, 1H), 5.58-5.61 (m, 1H), 5.07 (bs, 1H), 4.49-
(ESI-qTOF): calcd for C30H27F3N7O3 [M+H]+,590.2122: found: 4.58 (m, 1H), 4.0-4.02 (m, 1H), 3.78 (s, 3H), 2.97-3.19 (m, 2H); 13C
590.2108.
NMR (CDCl3, 50 MHz) δ 163.6, 158.1, 157.9, 157.0, 142.2, 131.1,
129.8, 129.0, 128.6, 126.6, 126.5, 126.1, 119.0, 118.8, 114.5, 114.4,
4.2.6. 3-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol- 111.5, 111.3, 104.0, 103.8, 74.6, 67.5, 61.04, 56.5, 55.9, 55.4, 34.2,
1-yl)propyl)-1H-1,2,3-triazol-1-yl)-4-(3-fluorophenyl)-1-
29.6; ESI-MS (m/z): 558.18 [M+H]+; HRMS (ESI-qTOF): calcd for
phenylazetidin-2-one; 12f. Brown colored solid; Yield: 91%; semi- C29H26F2N7O3 [M+H]+, 558.2060: found: 558.2057.
solid; IR (CHCl3, cm-1) 3415, 1761; 1H NMR (CDCl3, 400 MHz) δ 7.89- 4.2.11. 3-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
8.01 (m, 1H), 7.73 (d, J = 7.82 Hz, 1H), 7.30-7.37 (m, 2H, Ar-H), 7.0- 1-yl)propyl)-1H-1,2,3-triazol-1-yl)-4-(4-fluorophenyl)-1-(4-
7.24 (m, 7H, Ar-H), 6.84-6.87 (m, 1H, Ar-H), 6.63-6.78 (m, 3H), 6.20- methoxyphenyl)azetidin-2-one; 12k. White colored solid; Yield:
6.29 (m, 1H), 5.53-5.56 (m, 1H), 5.26 (bs, 1H), 4.47-4.72 (m, 2H), 93%; semi-solid; IR (CHCl3, cm-1) 3415, 1761; 1H NMR (CDCl3, 500
3.02-3.43 (m, 2H); 13C NMR (CDCl3, 50 MHz) δ 163.7, 161.2, 158.5, MHz) δ 7.96-8.0 (m, 1H), 7.79-7.81 (m, 1H), 7.35-7.38 (m, 1H), 7.29-
158.4, 154.3, 151.4, 144.2, 142.4, 136.2, 130.4, 129.8, 129.5, 125.6, 7.33 (m, 2H, Ar-H), 7.22-7.26 (m, 2H, Ar-H), 7.09-7.13 (m, 2H), 6.80-
123.2, 117.4, 116.3, 113.8, 111.5, 104.1, 75.1, 74.8, 67.4, 60.4, 56.5, 6.88 (m, 4H), 6.67-6.78 (m, 2H), 6.22-6.29 (m, 1H), 5.55-5.59 (m,
534.1, 33.9, 29.6, 21.0, 14.1; ESI-MS (m/z): 546.14 [M+H]+; HRMS 1H), 5.11 (bs, 1H), 4.56-4.61 (m, 1H), 3.87-4.20 (m, 1H), 3.79 (s, 3H),
(ESI-qTOF): calcd for C28H23F3N7O3 [M+H]+, 546.1860: found: 3.19-3.26 (m, 1H), 2.99-3.13 (m, 1H); 13C NMR (CDCl3, 50 MHz) δ
546.1860.
4.2.7. 4-(4-chlorophenyl)-3-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3- 122.9, 119.0, 118.8, 115.9, 115.7, 114.6, 111.5, 104.0, 74.9, 74.8,
163.7, 163.6, 161.6, 157.8, 151.5, 142.4, 129.6, 128.5, 126.9, 123.2,
(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,3-triazol-1-yl)-1-(4-
67.5, 62.5, 60.4, 56.4, 34.0, 29.6, 14.1; ESI-MS (m/z): 576.19 [M+H]+;
methoxyphenyl)azetidin-2-one; 12g. Wine red colored semi-solid; HRMS (ESI-qTOF): calcd for C29H25F3N7O3 [M+H]+, 576.1965: found:
Yield: 90%; IR (CHCl3, cm-1) 3415, 1761; 1H NMR (CDCl3, 500 MHz) δ 576.1968.
7.99-8.03 (m, 1H), 7.80 (s, 1H), 7.31-7.37 (m, 3H, Ar-H), 7.23-7.26 4.2.12. 3-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
(m, 1H, Ar-H), 7.07-7.15 (m, 1H), 6.81-6.93 (m, 5H), 6.68-6.74 (m, 1-yl)propyl)-1H-1,2,3-triazol-1-yl)-4-(4-fluorophenyl)-1-
2H), 6.26-6.34 (m, 1H), 5.56-5.59 (m, 1H), 5.15 (bs, 1H), 4.56-4.63 phenylazetidin-2-one; 12l. Dark brown semi-solid; Yield: 90%; semi-
(m, 1H), 3.87-4.16 (m, 1H), 3.80 (s, 3H), 2.99-3.25 (m, 2H); 13C NMR solid; IR (CHCl3, cm-1) 3415, 1761; 1H NMR (CDCl3, 400 MHz) δ 7.96-
(CDCl3, 50 MHz) δ 163.5, 161.5, 157.8, 157.1, 151.4, 142.4, 133.8, 8.0 (m, 1H), 7.79-7.83 (m, 1H), 7.32-7.4 0 (m, 5H, Ar-H), 7.20-7.25
130.4, 129.7, 129.5, 123.2, 122.4, 118.7, 114.6, 113.8, 113.7, 111.5, (m, 2H, Ar-H), 7.09-7.18 (m, 2H, Ar-H), 6.78-6.87 (m, 2H), 6.69-6.73
111.4, 104.0, 74.6, 67.45, 60.5, 66.5, 55.4, 34.0, 33.5, 29.6; ESI-MS (m, 2H), 6.24-6.31 (m, 1H), 5.59-5.63 (m, 1H), 5.11-5.22 (m, 1H),
(m/z): 592.14 [M+H]+; HRMS (ESI-qTOF): calcd for C29H25ClF2N7O3 4.54-4.61 (m, 1H), 3.88-4.20 (m, 1H), 3.03-3.26 (m, 2H) ; 13C NMR
[M+H]+, 592.1670: found: 592.1663.
4.2.8. 4-(2-chlorophenyl)-3-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3- 142.4, 136.2, 129.7, 129.4, 128.4, 125.5, 122.9, 117.4, 116.0, 115.7,
(CDCl3, 50 MHz) δ 164.0, 163.9, 161.4, 158.6, 158.4, 151.4, 144.2,
(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,3-triazol-1-yl)-1-(4-
111.5, 104.3, 104.0, 74.92, 74.8, 74.7, 67.4, 60.4, 56.5, 34.0, 33.6,
methoxyphenyl)azetidin-2-one; 12h. Cream colored solid; Yield: 29.3. ESI-MS (m/z): 546.18 [M+H]+; HRMS (ESI-qTOF): calcd for
92%; IR (CHCl3, cm-1) 3415, 1761; 1H NMR (CDCl3, 500 MHz) δ 7.99- C28H23F3N7O2 [M+H]+, 546.1860: found: 546.1865.
8.01 (m, 1H), 7.79 (s, 1H), 7.28-7.35 (m, 3H, Ar-H), 7.23-7.24 (m, 1H,
4.3 Antifungal susceptibility assay
Ar-H), 7.02-7.17 (m, 4H), 6.87-6.89 (m, 2H), 6.67-6.72 (m, 2H), 6.27-
6.36 (m, 1H), 5.90-5.93 (m, 1H), 5.13 (bs, 1H), 4.52-4.59 (m, 1H),
3.91-4.0 (m, 1H), 3.80 (s, 3H), 3.01-3.22 (m, 2H); 13C NMR (CDCl3, 50
MHz) δ 163.5, 161.8, 161.5, 158.0, 157.8, 157.1, 151.4, 142.4,
132.8, 130.0, 129.7, 129.5, 128.4, 123.2, 122.4, 118.7, 114.6, 113.8,
113.7, 111.5, 111.4, 104.0, 74.6, 74.8, 67.45, 59.0, 56.4, 56.2, 36.4,
33.8, 14.4; ESI-MS (m/z): 592.14 [M+H]+; HRMS (ESI-qTOF): calcd for
C29H25ClF2N7O3 [M+H]+, 592.1670: found: 592.1663.
Antifungal activities of the synthesized compounds (in terms of IC50
and MIC) against C. albicans ATCC 24433, C. albicans ATCC 10231, C.
glabrata NCYC 388, C. neoformans ATCC 34664, C. tropicalis ATCC
750 (CLSI - Clinical Laboratory Standards Institute document M27-
A3)24 and A. fumigatus NCIM 908, A. niger ATCC 10578 (CLSI M38-
A2)24 were determined by CLSI broth microdilution assay.25
4.2.9.4-(2,4-dichlorophenyl)-3-(4-(2-(2,4-difluorophenyl)-2-
hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,3-triazol-1-yl)-1-(4-
methoxyphenyl)azetidin-2-one; 12i. Brown colored solid; Yield:
4.4 Effect of triazoles on sterol profile of C. albicans ATCC 24433
1
90%; IR (CHCl3, cm-1) 3415, 1761; H NMR (CDCl3, 400 MHz) δ 8.01
Inhibition of lanosterol 14 α-demethylase by azoles was analyzed by
spectrophotometric quantification of ergosterol depletion as
described previously.25,26 The concentrations of 12h, 12i, 12j, 12k
and fluconazole used in the assay were 1/2 MIC, MIC and 2X MIC
concentrations.
(s, 1H), 7.83 (s, 1H), 7.30-7.36 (m, 4H, Ar-H), 7.21-7.25 (m, 1H, Ar-
H), 7.05-7.14 (m, 2H), 6.86-6.90 (m, 2H), 6.72-6.76 (m, 2H), 6.23-
6.33 (m, 1H), 5.83-5.89 (m, 1H), 5.14 (bs, 1H), 4.58-4.65 (m, 1H),
3.95-3.99 (m, 1H), 3.80 (s, 3H), 3.07-3.26 (m, 2H); 13C NMR (CDCl3,
50 MHz) δ 163.8, 161.3, 161.2, 157.8, 157.6, 157.1, 142.2, 165.5,
135.4, 129.7, 129.6, 127.7, 127.2, 118.7, 114.6, 111.6, 111.4, 104.3,
103.7, 74.9, 71.0, 67.0, 60.3, 58.6, 58.5, 55.4, 34.1, 33.8, 29.6; ESI-
4.5 Cytotoxicity assays
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ARTICLE
Journal Name
The active compounds 12 (a-l) were studied for cytotoxicity (MTT 8. (a) N. G. Aher, V. S. Pore, N. Mishra, A. Kumar, P. K. Shukla, A.
assay)27against two human cancer cell lines (MCF-7 and A431) and
were found to be non-cytotoxic. The cytotoxic effect of lead
compounds 12 (a-l) was checked at the concentrations of 100, 50,
25, 12.5, 6.25, 3.125, 1.5625 and 0.7813 μg/mL to determine the
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,
,
GI50 and GI90 (Table1S). These compounds 12 (a-l) were further 9. V. S. Pore, S. G. Agalave, P. Singh, P. K. Shukla, V. Kumar and M.
evaluated against human primary cells (HUVECs) to check the I. Siddiqi, Org. Biomol. Chem., 2015, 13, 6551.
selectivity of these compounds (Table1s). The GI90 (>100 µg/mL) 10. (a) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless,
values of the compounds 12 (a-l) indicate that the compounds are
potent and specific inhibitors against Candida strains. Compound
12b has exhibited highest cytotoxicity (GI50 <25; >32 µg/mL)
against MCF-7 and A431 cells.
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4.6 Hemolysis study
All the compounds were tested for RBC hemolysis as described
previously.28 The concentrations tested were in the range of 4–512
μg/mL.
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Acknowledgments
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The authors thank CSIR, New Delhi for financial support under
ORIGIN (CSC-0108) and OSDD (HCP-0001). JMD thank University
Grant Commission, New Delhi for a senior research fellowship (20-
06/2010 (i)-EU-IV). Authors are also thankful to Schrödinger Inc. for
providing the Demo license of Schrödinger Molecular Modeling
Suite and especially Craig Coel for his valuable support that has
significantly helped in this study. HPLC analysis of diastereomers by
Mrs. S. S. Kunte is gratefully acknowledged.
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Supplementary data
Supplementary data (experimental details, Proton and Carbon
NMRs of new compounds and Tables of biological activity)
associated with this article can be found, in the online version, at
http:// dx.doi.org/10.1016/
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10 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6NJ03117J
Synthesis and biological evaluation of new fluconazole βꢀlactam conjugates
linked via 1,2,3ꢀtriazole
Jaisingh M. Divse,a Chaitanya R. Charolkar,a Santosh B. Mhaske,a Laxman U. Nawale,b Dhiman
Sarkar,b Duhita G. Sant,c Santosh G. Tupe,c Mukund V. Deshpande,c Vijay M. Khedkar,b,d and
Vandana S. Pore*a
aOrganic Chemistry Division, CSIR-National Chemical Laboratory, Pune 411008, India
bCombichem-Bioresource Center, Organic Chemistry Division, CSIR-National Chemical
Laboratory, Pune 411008, India
cBiochemical Sciences Division, CSIR-National Chemical Laboratory, Pune 411008, India
dSchool of Health Sciences, University of KwaZulu Natal, Westville Campus, Durban 4000,
South Africa.
Graphical abstract –
Novel fluconazole conjugates with potent antifungal activity are reported here. They were also
found to be non hemolytic and non cytotoxic.