Synthesis and biological evaluation of amino acid-linked 1,2,3-bistriazole conjugates as potential antimicrobial agents

Article abstract of DOI:10.1007/s00044-013-0882-z

A small library of amino acid-linked 1,4-disubstituted 1,2,3-bistriazole conjugates has been synthesized from the N-protected l-Amino acids propargyl esters through one-pot click reaction. All the newly synthesized compounds were tested in vitro for antimicrobial activity against Gram-positive (Staphylococcus aureus, Bacillus subtilis), Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans, Aspergillus niger). The antibacterial evaluation data indicated that compounds 3c, 3h, 5a, 5b, 5c, 5d, 5e, 5g, and 5h exhibited comparable activity to standard ciprofloxacin. However, the bistriazoles 3b, 3c, 3e, 3f, and 3g showed good antifungal activity than others against both fungi. Further, docking simulation of the two most active compounds 5c against S. aureus tyrosyl tRNA synthetase and 5h into E. coli topoisomerase II DNA Gyrase B, respectively, was also performed.

Full text of DOI:10.1007/s00044-013-0882-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2995–3004
DOI 10.1007/s00044-013-0882-z
ORIGINAL RESEARCH
Synthesis and biological evaluation of amino acid-linked
1,2,3-bistriazole conjugates as potential antimicrobial agents
•
•
•
C. P. Kaushik Kashmiri Lal Ashwani Kumar
Sunil Kumar
Received: 24 August 2013 / Accepted: 15 November 2013 / Published online: 4 December 2013
Ó Springer Science+Business Media New York 2013
Abstract A small library of amino acid-linked 1,4-
disubstituted 1,2,3-bistriazole conjugates has been synthe-
sized from the N-protected L-amino acids propargyl esters
through one-pot click reaction. All the newly synthesized
compounds were tested in vitro for antimicrobial activity
against Gram-positive (Staphylococcus aureus, Bacillus
subtilis), Gram-negative (Escherichia coli) bacteria and
fungi (Candida albicans, Aspergillus niger). The antibac-
terial evaluation data indicated that compounds 3c, 3h, 5a,
5b, 5c, 5d, 5e, 5g, and 5h exhibited comparable activity to
standard ciprofloxacin. However, the bistriazoles 3b, 3c,
3e, 3f, and 3g showed good antifungal activity than others
against both fungi. Further, docking simulation of the two
most active compounds 5c against S. aureus tyrosyl tRNA
synthetase and 5h into E. coli topoisomerase II DNA
Gyrase B, respectively, was also performed.
health around the world. This has created a substantial need
for the development of new and more potent antimicrobial
agents with broad spectrum of activity. It requires an
efficient approach to design new agents with novel mech-
anism of action and structural modification to improve their
activity. Tyrosyl-tRNA synthetase from the bacterium
Staphylococcus aureus, largely responsible for hospital-
acquired infections, assist the coupling of tyrosine to
tyrosyl-tRNA (Qiu et al., 2001). Likewise, Type II topoi-
somerase DNA gyrase from the bacterium Escherichia coli,
causing common bacterial infections, like cholecystitis,
urinary tract infection (UTI), diarrhea, pneumonia, creates a
double stranded break and allow the passage of a double-
stranded DNA segment (Kampranis and Maxwel, 1996).
The N-heterocyclic compounds especially 1,2,3-tria-
zoles have received special attention because of their wide
spread use in the synthetic and medicinal chemistry (Kolb
and Sharpless, 2003; Kharb et al., 2011; Thirumurugan
et al., 2013). The 1,2,3-triazoles are considered as inter-
esting heterocycles due to their important pharmacological
activities such as antiprotozoal (Bakunov et al., 2010),
anticonvulsant (Kelley et al., 1995), anti-HIV (Whiting
et al., 2006), antimicrobial (Genin et al., 2000), antiallergic
(Buckle et al., 1986), anticancer (Singh et al., 2012), anti-
inflammatory (Biagi et al., 1986, 1990), antitubercular
(Patpi et al., 2012), etc. The copper(I)-catalyzed azide-
alkyne cycloaddition discovered independently by Meldal
and Sharpless led to significant development of the (Hu-
isgen, 1984) thermal reaction and offers a unique approach
for the regioselective synthesis of 1,4-disubstituted 1,2,3-
triazoles (Tornoe et al., 2002; Rostovtsev et al., 2002). This
cycloaddition has emerged as premier example of click
chemistry because of its high efficiency, versatility, regi-
oselectivity, and excellent functional group compatibility.
The 1,4-disubstituted 1,2,3-triazoles can also be prepared
Keywords 1,4-Disubstituted 1,2,3-bistriazoles Á
Click chemistry Á Antimicrobial activity Á
Docking simulation
Introduction
The growing emergences of bacterial and fungal resistant
to most of the antibiotics are major concern to the public
C. P. Kaushik (&) Á K. Lal
Department of Chemistry, Guru Jambheshwar University
of Science & Technology, Hisar 125001, Haryana, India
e-mail: kaushikcp@gmail.com
A. Kumar Á S. Kumar
Department of Pharmaceutical Sciences, Guru Jambheshwar
University of Science &Technology, Hisar 125001,
Haryana, India
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Med Chem Res (2014) 23:2995–3004
by one-pot reaction of organic halides, sodium azide, and
alkynes in presence of a copper catalyst (Sharpless et al.,
2005; Odlo et al., 2007; Kumar et al., 2011). Moreover,
this method also provides a mean for rapid construction of
compounds or libraries of compounds for drug discovery.
Therefore, in continuation of our interest toward synthesis
of biologically important 1,2,3-triazoles (Lal et al., 2012),
we report herein, one-pot synthesis of a small library of
amino acid-linked 1,4-disubstituted 1,2,3-bistriazole con-
jugates and their biological evaluation for antimicrobial
activities.
L-glutamic acid with propargyl bromide in presence of
potassium carbonate in dry dimethylformamide (DMF)
under nitrogen (Bew and Gipson, 2010). The bisalkynes so
obtained were subjected to one-pot three-component reac-
tion with various benzyl bromides (2a–h), sodium azide,
copper sulfate pentahydrate, and sodium ascorbate in
DMF: water mixture to yield the desired bistriazoles
(3a–h and 5a–h) in good yields (Schemes 1 , 2).
All the newly synthesized bistriazoles were well char-
1
acterized by IR, H NMR, 13C NMR and high-resolution
mass (HRMS) spectral techniques. The IR spectrum of a
representative compound 3a showed absorption bands at
3389 and 3144 cm-1,which were attributed to the N–H
stretching and =C–H stretching vibrations of triazole rings,
respectively. Two bands were observed at 1734 and
1682 cm-1 due to C=O stretching of ester and carbamate
groups, respectively. The 1H NMR spectrum of 3a showed
two characteristic singlets at d 7.50 and d 7.57 due to two
triazolyl protons. A multiplet at d 4.61–4.65 integrating for
a single proton was assigned to a proton attached to a-
carbon atom and a broad singlet at d 5.85 was attributable
to the amide proton. Two doublets of doublet signals
observed at d 2.83 and d 3.01 were accounted for protons
Results and discussion
Chemistry
The amino acids L-aspartic acid and L-glutamic acid having
b- and c-carboxylates were chosen as ideal candidates for
generating bistriazoles through click combination of bis-
alkynes with various organic azides. The synthesis of bis-
alkynes
1
commercially available N-Cbz-L-aspartic acid and N-Boc-
and
4
was accomplished by reacting
R
O
O
O
N
N
N
N
Br
n
N
N
n
O
O
O
O
O
O
(i)
O
N
N
O
R
H
H
n
O
R
2a-h
1
3a-h
n = 1,2
=
H
,
R
, NO2
CH
3
Scheme 1 Synthesis of aspartic acid-based 1,4-disubstituted 1,2,3-
bistriazoles (3a–h). Reagents and conditions (i) NaN3, sodium
ascorbate, CuSO4Á5H2O, DMF/H2O, RT. 2a: n = 1, R = H; 2b:
n = 1, R = 2-CH3; 2c: n = 1, R = 3-CH3; 2d: n = 1, R = 4-CH3;
2e: n = 1, R = 2-NO2; 2f: n = 1, R = 3-NO2; 2g: n = 1, R = 4-
NO2; 2h: n = 2, R = H
N
N
R
N
O
O
n
O
O
Br
n
O
O
(i)
O
O
R
n
N
R
N
O
N
O
N
N
H
H
O
O
2a-h
4
5a-h
n = 1,2
R = H
NO
2
CH
,
,
3
Scheme 2 Synthesis of glutamic acid-based 1,4-disubstituted 1,2,3-bistriazoles (5a–h). Reagents and conditions (i) NaN3, sodium ascorbate,
CuSO4.5H2O, DMF/H2O, RT
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Med Chem Res (2014) 23:2995–3004
2997
of b-CH2. Further, all the aromatic protons were observed
in the region d 7.29–7.35. A multiplet at d 5.04–5.09
integrating for four protons was assigned to methylene
protons of protecting group and one of the methylene
attached to ester group. The protons of other methylene
attached to ester group and N-1 of triazole appeared at d
5.21 and d 5.51–5.52, respectively. In 13C NMR spectrum
signals due to carbonyl carbons appeared at d 170.5 (ester)
and d 155.9 (carbamate) and signals for C-5 carbon atoms
of the triazole moiety were observed at d 124.1 and d
123.8. While, both C-4 carbons of the triazole ring reso-
nated at d 142.4 and d 142.6. The signals appeared at
d 36.6 and d 50.3 were assigned to the b-carbon and a-
carbon of the aspartic acid and the signal due to methylene
carbon linked to phenyl (protecting group) appeared at d
67.2. Two signals at d 58.1 and d 54.2 were accounted for
the two methylene carbons attached to the ester linkages
and other two methylene carbons linked to N-1 of triazoles
ring resonated at d 58.9. The high resolution mass
spectrum of this compound exhibited two signals at m/z
610.2402 and m/z 632.2223 corresponding to [M?H]?
and [M?Na]?, which are in agreement with calculated
value.
corresponding to [M?H]? and [M?Na]?, which are in
agreement with calculated value.
Biological activity
Antibacterial activity
In vitro antibacterial activity of newly synthesized amino
acid-linked 1,4-disubstituted 1,2,3-bistriazole conjugates
was studied against two Gram-positive bacteria, S. aureus
(MTCC 3160), Bacillus subtilis (MTCC 121), and one
Gram-negative bacteria, E. coli (MTCC 40), by standard
serial dilution method (Cappucino and Sherman, 1999)
using a stock solution of 50 lg/mL concentration. Cipro-
floxacin was used as standard drug for antibacterial activity
and minimum inhibitory concentrations (MIC; inhibition of
100 % of growth) were determined in terms of lmol/mL
(Table 1).
As evident from the Table 1, most of the tested com-
pounds showed potential antibacterial activity. Some of the
tested compounds are found to possess comparable activity
to the reference (Ciprofloxacin) against all bacterial strains
used. Out of the 16 screened compounds, 5c (MIC,
0.0025 lmol/mL) and 5h (MIC, 0.0025 lmol/mL) dis-
played excellent and almost twofold activity than standard
drug Ciprofloxacin (MIC, 0.0047 lmol/mL) against
S. aureus and E. coli, respectively. In the case of S. aureus,
compounds 3f (MIC, 0.0045 lmol/mL), 5e (MIC,
1
Likewise, the H NMR spectrum of 5a showed charac-
teristic peaks at d 7.54 and 7.56 due to two triazolyl pro-
tons. A singlet corresponding to four protons observed at d
5.51, which was assigned to the methylene protons linked
to N-1 of triazoles ring. While, two singlets observed at d
5.23 and 5.17 were due to the protons of two methylene
groups attached to ester linkages. A multiplet at d
4.28–4.29 integrating for a single proton was assigned to a
proton attached to a-carbon atom and a doublet at d 5.10
with J = 7.6 Hz was attributable to the amide proton. Two
multiplets observed in the region d 1.88–1.93 and d
2.09–2.14 were accounted for protons of b-CH2 and a
multiplet in the region d 2.33–2.38 corresponding to two
protons was due to c-CH2. Further, all the aromatic protons
were observed in the region d 7.27–7.38. In 13C NMR
spectrum, signals due to three carbonyl carbons appeared at
d 172.4, 172.0 (ester), and 155.3 (carbamate). The char-
acteristics signals for C-5 carbons of both the triazole rings
were observed at d 123.7 and 123.8. While, signals due to
C-4 carbon atoms of the two triazole rings appeared at d
143.1 and 142.7. Two signals at d 80.1 and 28.3 were
assigned to the quaternary carbon and three methyl carbons
of tert-butyl group, respectively. Three signals appeared at
d 27.5, 30.0, and 52.8 were assigned to the b-carbon,
c-carbon and a-carbon atoms of the glutamic acid,
respectively. The two methylene carbons linked to N-1 of
triazole rings appeared at d 58.6 and the two methylene
carbons attached to the ester linkages resonated at d 54.3
and 57.9. The high resolution mass spectrum of this com-
pound exhibited two signals at m/z 590.2718, 612.2531
Table 1 In vitro antibacterial activities of the 1,4-disubstituted 1,2,3-
triazoles (3a–h and 5a–h)
Compounds n R
Minimum inhibitory concentration (MIC)a
S. aureusb
B. subtilisc
E. colid
3a
3b
3c
3d
3e
3f
1 H
0.0103
0.0103
0.0098
0.0049
0.0049
0.0045
0.0045
0.0089
0.0049
0.0053
0.0051
0.0051
0.0051
0.0046
0.0046
0.0046
0.0051
0.0047
0.0051
0.0098
0.0049
0.0098
0.0045
0.0089
0.0089
0.0049
0.0053
0.0051
0.0051
0.0051
0.0046
0.0046
0.0046
0.0025
0.0047
1 2-CH3 0.0098
1 3-CH3 0.0049
1 4-CH3 0.0049
1 2-NO2 0.0089
1 3-NO2 0.0045
1 4-NO2 0.0089
3g
3h
5a
5b
5c
5d
5e
5f
2 H
1 H
0.0049
0.0053
1 2-CH3 0.0051
1 3-CH3 0.0025
1 4-CH3 0.0051
1 2-NO2 0.0046
1 3-NO2 0.0092
1 4-NO2 0.0046
5g
5h
2 H
0.0051
0.0047
Ciprofloxacin – –
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Med Chem Res (2014) 23:2995–3004
0.0046 lmol/mL), and 5g (MIC, 0.0046 lmol/mL) displayed
comparable activity to standard. Likewise, compounds 3f
(MIC, 0.0045 lmol/mL), 3f (MIC, 0.0045 lmol/mL), 5e
(MIC, 0.0046 lmol/mL), 5f (MIC, 0.0046 lmol/mL), 5g
(MIC, 0.0046 lmol/mL), and compounds 3e (MIC,
0.0045 lmol/mL), 5e (MIC, 0.0046 lmol/mL), 5f (MIC,
0.0046 lmol/mL), 5g (MIC, 0.0046 lmol/mL) exhibited
very good and comparable activity to Ciprofloxacin against
B. subtilis and E. coli, respectively. Further, many of the
nitro group-containing compounds showed better activity
than that containing methyl group. Moreover, conjugates
derived from N-Boc-L-glutamic propargyl ester were more
potent than compounds derived from N-Cbz-L-aspartic
propargyl ester.
3e (MIC, 0.0089 lmol/mL), 3f (MIC, 0.0089 lmol/mL),
and 3g (MIC, 0.0089 lmol/mL) showed good antifungal
activity against C. albicans among all the compounds
under study. In case of triazoles derived from aspartic acid
propargyl esters, nitro-substituted (3e–g) were found to be
more active than methyl substituted compounds (3b–
d) against C. albicans. Further, most of the synthesized
compounds showed less potency against fungal strains
compared to bacterial strains.
Docking studies
In order to gain insight into the plausible mechanism of
action of two most potent compounds 5c (S. aureus) and 5h
(E. coli) docking simulations (Hsu et al., 2011) were per-
formed. Compound 5c was docked into active sites of
S. aureus tyrosyl tRNA synthetase (1JIJ) and 5h into E. coli
topoisomerase II DNA Gyrase B complexed with the nat-
ural inhibitor clorobiocin (1kzn). The S. aureus tyrosyl
tRNA synthetase (Xiao et al., 2011) and E. coli topoiso-
merase II DNA Gyrase B (Abdel-Aziz et al., 2011) has
been reported as good targets for studying biological
activity against these bacteria. The least energy binding
mode of compounds has been studied.
Antifungal activity
All the synthesized amino acid-linked 1,2,3-bistriazole
conjugates were screened for antifungal activity against
two fungal strains viz. Aspergillus niger (MTCC 281) and
Candida albicans (MTCC 183) by standard serial dilution
method (Cappucino and Sherman, 1999) using a stock
solution of 50 lg/mL concentration. While carrying out
antifungal activity, fluconazole was used as reference drug
and MIC were determined in terms of lmol/mL (Table 2).
The perusal of antifungal activity indicated that most of
the compounds exhibited good activity. Compounds 3e
(MIC, 0.0089 lmol/mL) and 3f (MIC, 0.0089 lmol/mL)
displayed high activity against A. niger, while, compounds
It was observed from in silico studies of compound 5c
(Fig. 1) that oxygen atom on carbonyl of amide linkage and
N-1 of the triazoles ring forms hydrogen bond with the
Asp-195. The N-1 and N-3 of the second triazole ring
generated hydrogen bonds with Gln-196 and Tyr-170,
respectively. The oxygen atom of the ester linkage form
hydrogen bond with Lys-84. Further, oxygen atom of the
second ester linkage and oxygen atom on carbonyl of ester
linkage are involved in hydrogen bonding interaction with
Asp-40. Secondary structure of tyrosyl tRNA synthetase
showing compound 5c into binding pocket is presented in
Table 2 In vitro antifungal activity of the 1,4-disubstituted 1,2,3-
triazoles (3a–h and 5a–h)
Compounds
n
R
Minimum inhibitory concentration (MIC)a
A. nigerb
C. albicansc
3a
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
2
–
H
0.0103
0.0103
0.0098
0.0098
0.0196
0.0089
0.0089
0.0089
0.0098
0.0212
0.0101
0.0202
0.0101
0.0184
0.0184
0.0184
0.0202
0.0051
3b
2-CH3 0.0098
3-CH3 0.0098
4-CH3 0.0098
2-NO2 0.0089
3-NO2 0.0089
4-NO2 0.0178
3c
3d
3e
3f
3g
3h
H
H
0.0196
0.0106
5a
5b
2-CH3 0.0101
3-CH3 0.0101
4-CH3 0.0202
2-NO2 0.0184
3-NO2 0.0092
4-NO2 0.0092
5c
5d
5e
5f
5g
5h
H
–
0.0202
0.0051
Fig. 1 Docked compound 5c showing hydrogen bonding interactions
with S. aureus tyrosyl tRNA synthetase
Fluconazole
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Med Chem Res (2014) 23:2995–3004
2999
Fig. 2. The docked conformation of 5h (Fig. 3) showed
that the oxygen atom on carbonyl of ester linkage is
involved in hydrogen bonding interaction with Ser-121.
The surface diagram of the docked molecule into the
binding pockets of topoisomerase II is depicted in Fig. 4.
Conclusion
In conclusion, a new series of amino acid-linked 1,2,3-
bistriazole conjugates has been synthesized in good yields
utilizing one-pot click reaction and evaluated for antimi-
crobial activity. It is noteworthy that most of the com-
pounds exhibited comparable antibacterial activity to the
reference drug ciprofloxacin. However, most of the com-
pounds were found to be more potent against bacterial
strains as compared to fungal strains. Further, docking
studies showed that compounds 5c (MIC, 0.0025 lmol/mL)
and 5h (MIC, 0.0025 lmol/mL) inhibits S. aureus tyrosyl
tRNA synthetase and E. coli topoisomerase II DNA Gyrase
B, respectively, through hydrogen bonding interactions.
These findings can be exploited toward development and
testing of triazole-based novel molecules for better anti-
microbial activities.
Fig. 2 Secondary structure view of docked compound 5c with
S. aureus tyrosyl tRNA synthetase
Experimental
Chemistry
Melting points (°C) were recorded in open capillaries and
are uncorrected. The IR spectra were observed on SHI-
MAZDU IR AFFINITY-I FTIR spectrophotometer using
potassium bromide (KBr) and values are given in cm-1
.
The 1H NMR and 13C NMR spectra were reported on
Bruker Avance III 400 nano bay spectrophotometer at 400
and 100 MHz, respectively, in deuterated chloroform
(CDCl3) using tetramethysilane as an internal standard
(chemical shift in d, ppm). Coupling constant (J) values are
in Hertz (Hz). HRMS were recorded with LCMS-QTOF
Module No. G6540 A (UHD) instrument. The completion
of reactions and the purity of the compounds was analyzed
by thin layer chromatography (TLC) using readymade
silica gel plates (SIL G/UV254, ALUGRAM) in ethyl
acetate hexane mixture. Starting materials were procured
from Aldrich and were used without further purification.
Fig. 3 Docked pose of 5h showing hydrogen bonding with active site
residues of E. coli topoisomerase II DNA Gyrase
General method for the preparation of 1,4-disubtituted
1,2,3-bistriazoles (3a–h and 5a–h)
To a solution of substituted benzyl bromide 2a–
h (1 mmol), sodium azide (3.0 mmol), alkyne 1 and 4
(0.5 mmol) in DMF/water (9:1) was added copper sulfate
Fig. 4 Surface diagram showing docked molecules 5h with E. coli
topoisomerase II DNA Gyrase
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Med Chem Res (2014) 23:2995–3004
(S)-Bis((1-(3-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)2-
(benzyloxycarbonylamino) succinate (3c) White semi-
solid, Yield: 77 %; IR (KBr): 3382 (N–H str), 3140 (=C–H
str triazole), 3036 (=C–H str aromatic), 2959 (C–H str
CH2), 1738 (C=O str ester), 1680 (C=O str) cm-1. 1H NMR
(400 MHz, CDCl3): d 2.28–2.34 (m, 6H, CH3), 2.81 (dd,
1H, bCH, J = 17.2 Hz, J = 4.1 Hz), 2.96 (dd, 1H, bCH,
J = 17.1 Hz, J = 4.0 Hz), 4.61–4.65 (m, 1H, aCH),
5.05–5.14 (m, 4H, PhCH2 ? OCH2), 5.29 (s, 2H, OCH2),
5.45 (brs, 4H, NCH2), 5.85 (brs, 1H, NH), 7.01–7.39 (m,
13H, Ar–H), 7.46 (s, 1H, triazole), 7.56 (s, 1H, triazole).
13C NMR (100 MHz, CDCl3): d 21.6 (CH3), 22.3 (CH3),
36.4 (bCH2), 50.5 (aCH), 54.3 (OCH2), 58.0 (OCH2), 58.7
(NCH2), 67.5 (OCH2; carbamate), 123.9 (C-5 triazole),
124.0 (C-5 triazole), 127.7, 128.4, 128.5, 128.7, 129.2,
129.4, 134.1, 136.5, 139.2 (aromatic carbons), 142.3 (C-4
triazole), 142.4 (C-4 triazole), 155.7 (C=O), 170.3 (C=O
ester). HRMS: m/z (M?) Cacld. for C34H35N7O6: 637.2649,
found: 638.2725(M?H)?, 660.2540 (M?Na)?.
(5 mol%) and sodium ascorbate (10 mol%). The reaction
mixture was stirred overnight at ambient temperature and
the progress of the reaction was monitored by TLC. After
completion of the reaction, the reaction mixture was
diluted with ice-cold water (30 mL) and the product was
extracted with ethyl acetate (3 9 20 mL). Combined
organic layer was washed with aqueous ammonia solution
followed by water and dried over anhydrous Na2SO4.
After filtration, the solvent was evaporated under vacuum
and the crude product obtained was purified by column
chromatography (hexane/ethyl acetate gradient) to afford
the pure desired product.
(S)-Bis((1-benzyl-1H-1,2,3-triazol-4-yl)methyl) 2-(benzyl-
oxycarbonylamino)succinate
(3a) White
Yield: 85 %; IR (KBr): 3389 (N–H str), 3144 (=C–H str
semisolid,
triazole), 3034 (=C–H str aromatic), 2957 (C–H str CH2),
1734 (C=O str ester), 1682 (C=O str) cm-1 1H NMR
.
(400 MHz, DMSO-d6): d 2.83 (dd, 1H, bCH, J = 16.8 Hz,
J = 4.0 Hz), 3.01 (dd, 1H, bCH, J = 16.8 Hz, J =
4.0 Hz), 4.61–4.65 (m, 1H, aCH), 5.04–5.09 (m, 4H,
PhCH2 ? OCH2), 5.21 (brs, 2H, OCH2), 5.51–5.52 (brs,
4H, NCH2), 5.85 (brs, 1H, NH), 7.29 (s, 5H, Ar–H), 7.35
(brs, 10H, Ar–H), 7.50 (s, 1H, triazole), 7.57 (s, 1H, tria-
zole). 13C NMR (100 MHz, CDCl3): d 36.6 (bCH2), 50.3
(aCH), 54.2 (OCH2), 58.1 (OCH2), 58.9 (NCH2), 67.2
(OCH2; carbamate), 123.8 (C-5 triazole), 124.1 (C-5 tria-
zole), 128.1, 128.2, 128.3, 128.6, 128.9, 129.2, 134.5,
136.1 (aromatic carbons), 142.4 (C-4 triazole), 142.6 (C-4
triazole), 155.9 (C=O carbamate), 170.5 (C=O ester).
HRMS: m/z (M?) Cacld. for C32H31N7O6: 609.2336,
found: 610.2402 (M?H)?, 632.2223 (M?Na)?.
(S)-Bis((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)
2-(benzyloxycarbonylamino) succinate (3d) White semi-
solid, Yield: 71 %; IR (KBr): 3383 (N–H str), 3142
(=C–H str triazole), 3032 (=C–H str aromatic), 2949 (C–H
1
str CH2), 1736 (C=O str ester), 1685 (C=O str) cm-1. H
NMR (400 MHz, CDCl3): d 2.29–2.35 (m, 6H), 2.80 (dd,
1H, bCH, J = 17.0 Hz, J = 4.2 Hz), 2.95 (dd, 1H, bCH,
J = 17.0 Hz, J = 4.2.0 Hz), 4.64–4.67 (m, 1H, aCH),
5.03–5.11 (m, 4H, PhCH2 ? OCH2), 5.22 (s, 2H, OCH2),
5.47 (brs, 4H, OCH2), 5.87 (s, 1H, NH), 7.19–7.28 (m,
13H, Ar–H), 7.50 (s, 1H, triazole), 7.54 (s, 1H, triazole).
13C NMR (100 MHz, CDCl3): d 21.7 (CH3), 36.5 (bCH2),
50.3 (aCH), 53.6 (OCH2), 58.4 (OCH2), 64.3 (NCH2), 66.9
(OCH2; carbamate), 124.1 (C-5 triazole), 124.5 (C-5 tria-
zole), 127.8, 128.5, 128.6, 129.8, 129.5, 133.6, 136.6,
136.8 (aromatic carbons), 142.1 (C-4 triazole), 142.5 (C-4
triazole), 156.3 (C=O), 170.2 (C=O ester). HRMS:
m/z (M?) Cacld. for C34H35N7O6: 637.2649, found:
638.2726 (M?H)?, 660.2544 (M?Na)?.
(S)-Bis((1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)2-
(benzyloxycarbonylamino) succinate (3b) White semi-
solid, Yield: 73 %; IR (KBr): 3386 (N–H str), 3144 (=C–H
str triazole), 3034 (=C–H str aromatic), 2958 (C–H str
CH2), 1736 (C=O str ester), 1697 (C=O str) cm-1. 1H NMR
(400 MHz, CDCl3): d 2.27 (s, 6H), 2.82 (dd, 1H, bCH,
J = 17.2 Hz, J = 4.0 Hz), 2.98 (dd, 1H, bCH, J =
17.2 Hz, J = 4.0 Hz), 4.59–4.63 (m, 1H, aCH), 5.02–5.06
(m, 4H, PhCH2 ? OCH2), 5.18 (brs, 2H, OCH2), 5.46 (brs,
4H, NCH2), 5.84 (brs, 1H, NH), 7.05–7.32 (m, 13H,
Ar–H), 7.48 (s, 1H, triazole), 7.54 (s, 1H, triazole). 13C
NMR (100 MHz, CDCl3): d 21.3 (CH3), 22.7 (CH3), 36.4
(bCH2), 50.1 (aCH), 54.3 (OCH2), 58.2 (OCH2), 58.9
(NCH2), 67.2 (OCH2; carbamate), 123.8 (C-5 triazole),
124.2 (C-5 triazole), 127.9, 128.1, 128.3, 128.6, 128.9,
129.0, 129.6, 134.4, 136.1, 139.0 (aromatic carbons), 142.3
(C-4 triazole), 142.7 (C-4 triazole), 155.8 (C=O), 170.2
(C=O ester). HRMS: m/z (M?) Cacld. for C34H35N7O6:
637.2649, found: 638.2721 (M?H)?, 660.2542 (M?Na)?.
(S)-Bis((1-(2-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)2-
(benzyloxycarbonylamino) succinate (3e) Yellowish semi-
solid, Yield: 71 %; IR (KBr): 3380 (N–H str), 3144 (=C–H
str triazole), 3034 (=C–H str aromatic), 2957 (C–H str
CH2), 1734 (C=O str ester), 1682 (C=O str), 1533 (N=O
str), 1350 (N=O str) cm-1. 1H NMR (400 MHz, CDCl3): d
2.90 (dd, 1H, bCH, J = 17.2 Hz, J = 4.4.0 Hz), 3.08 (dd,
1H, bCH, J = 17.4 Hz, J = 4.4 Hz), 4.65–4.69 (m, 1H,
aCH), 5.10–5.14 (m, 4H, PhCH2 ? OCH2), 5.24 (s, 2H,
OCH2), 5.28 (brs, 1H, NH), 5.96 (brs, 4H, NCH2), 7.07–
7.15 (m, 2H, Ar–H), 7.33 (brs, 5H, Ar–H), 7.50–7.67 (m,
4H, Ar–H), 7.79 (s, 1H, triazole), 7.84 (s, 1H, triazole),
123

Med Chem Res (2014) 23:2995–3004
3001
8.14–8.19 (m, 2H, Ar–H). 13C NMR (100 MHz, CDCl3): d
36.6 (bCH2), 50.9 (aCH), 58.7 (OCH2), 58.8 (NCH2), 67.2
(OCH2; carbamate), 123.3 (C-5 triazole), 124.9 (C-5 tria-
zole), 127.4 127.8, 128.3, 129.6, 130.7, 134.7, 136.1, 136.9
(aromatic carbons), 142.5 (C-4 triazole), 142.7 (C-4 tria-
zole), 147.4, 147.5 (aromatic carbons), 156.5 (C=O), 170.5
(C=O ester). HRMS: m/z (M?) Cacld. for C32H29N9O10:
699.2037, found: 700.2116 (M?H)?, 722.1933 (M?Na)?.
str triazole), 3037 (=C–H str triazole), 2952 (C–H str CH2),
1737 (C=O str ester), 1680 (C=O str) cm-1 1H NMR
.
(400 MHz, CDCl3): d 2.83 (dd, 1H, bCH, J = 17.6 Hz,
J = 4.4 Hz), 3.00 (dd, 1H, bCH, J = 17.2 Hz, J =
4.4 Hz), 3.20 (t, 4H, J = 7.2 Hz, PhCH2CH2), 4.59 (t, 4H,
J = 7.2 Hz, NCH2), 4.62–4.64 (m, 1H, aCH), 5.09–5.15
(brs, 4H, PhCH2 ? OCH2), 5.21 (s, 2H, OCH2), 5.80 (brs,
1H, NH), 7.09–7.11 (m, 5H, Ar–H), 7.23–7.34 (m, 10H,
Ar–H), 7.35 (s, 1H, triazole), 7.40 (s, 1H, triazole). 13C
NMR (100 MHz, CDCl3): d 36.6 (PhCH2), 36.7(bCH2),
51.7 (aCH), 58.1 (OCH2), 58.9 (NCH2), 67.2(OCH2; car-
bamate), 124.2 (C-5 triazole), 124.5 (C-5 triazole), 127.2,
128.2, 128.3, 128.6, 128.7, 128.9, 136.0, 136.8, 136.9
(aromatic carbons), 141.8 (C-4 triazole), 142.0 (C-4 tria-
zole), 155.9 (C=O), 170.5 (C=O ester). HRMS: m/z (M?)
Cacld. for C34H35N7O6: 637.2649, found: 638.2731
(M?H)?, 660.2549 (M?Na)?.
(S)-Bis((1-(3-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)2-
(benzyloxycarbonylamino) succinate (3f) Yellowish
semisolid, Yield: 71 %; IR (KBr): 3379 (N–H str), 3144
(=C–H str triazole), 3034 (=C–H str aromatic), 2957 (C–H
str CH2), 1734 (C=O str ester), 1682 (C=O str), 1530 (N=O
str), 1344 (N=O str) cm-1. 1H NMR (400 MHz, CDCl3): d
2.89 (dd, 1H, bCH, J = 17.2 Hz, J = 4.1 Hz), 2.99 (dd,
1H, bCH, J = 17.4 Hz, J = 4.0 Hz), 4.63–4.66 (m, 1H,
aCH), 5.09–5.17 (m, 4H, PhCH2 ? OCH2), 5.26 (s, 2H,
OCH2), 5.38 (brs, 1H, NH), 5.92 (brs, 4H, NCH2), 7.01–
7.12 (m, 2H, Ar–H), 7.33–7.67 (m, 9H, Ar–H), 7.69 (s, 1H,
triazole), 7.80 (s, 1H, triazole), 8.19–8.24 (m, 2H, Ar–H).
13C NMR (100 MHz, CDCl3): d 37.0 (bCH2), 51.4 (aCH),
58.3 (OCH2), 58.9 (NCH2), 67.4 (OCH2; carbamate), 123.7
(C-5 triazole), 124.5 (C-5 triazole), 127.0, 127.4, 128.6,
129.2, 130.5, 133.2, 136.9 (aromatic carbons), 142.6 (C-4
triazole), 142.9 (C-4 triazole), 147.9 (aromatic carbon),
155.9 (C=O), 170.4 (C=O ester). HRMS: m/z (M?) Cacld.
for C32H29N9O10: 699.2037, found: 700.2120 (M?H)?,
722.1929 (M?Na)?.
(S)-Bis((1-benzyl-1H-1,2,3-triazol-4-yl)methyl) 2-(tert-but-
oxycarbonylamino)pentanedioate (5a) White semisolid,
Yield: 87 %; IR (KBr): 3379 (N–H str), 3131 (=C–H str
triazole), 3074 (=C–H str aromatic), 2978 (C–H str CH2),
1730 (C=O str ester), 1688 (C=O str) cm-1 1H NMR
.
(400 MHz, CDCl3): d 1.39 (s, 9H, tBu-CH3), 1.86–1.93 (m,
1H, bCH), 2.09–2.14 (m, 1H, bCH), 2.33–2.38 (m, 2H,
cCH2), 4.28–4.29 (m, 1H, aCH), 5.10 (d, 1H, J = 7.6 Hz,
NH), 5.17 (s, 2H, OCH2), 5.23 (s, 2H, OCH2), 5.51 (s, 4H,
NCH2), 7.27 (s, 5H, Ar–H), 7.36–7.38 (brs, 5H, Ar–H),
7.54 (s, 1H, triazole), 7.56 (s, 1H, triazole). 13C NMR
(100 MHz, CDCl3): d 27.5 (tBu-CH3), 28.3 (bCH2), 30.0
(cCH2), 52.8 (aCH), 54.3 (OCH2), 57.9 (OCH2), 58.6
(NCH2), 80.1 (tBu-C), 123.7 (C-5 triazole), 123.8 (C-5
triazole), 128.2, 128.3, 128.9, 129.2, 134.4 (aromatic car-
bons), 142.7 (C-4 triazole), 143.1 (C-4 triazole), 155.3 (C=
O), 172.0 (C=O ester), 172.4 (C=O ester). HRMS: m/z
(M?) Cacld. for C30H35N7O6: 589.2649, found: 590.2718
(M?H)?, 612.2531 (M?Na)?.
(S)-Bis((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)2-
(benzyloxycarbonylamino) succinate (3g) Yellowish
semisolid, Yield: 71 %; IR (KBr): 3384 (N–H str), 3140
(=C–H str triazole), 3030 (=C–H str aromatic), 2956 (C–H
str CH2), 1733 (C=O str ester), 1679 (C=O str), 1525 (N=O
str), 1340 (N=O str) cm-1. 1H NMR (400 MHz, CDCl3): d
2.82 (dd, 1H, bCH, J = 17.2 Hz, J = 4.2 Hz), 2.97 (dd,
1H, bCH, J = 17.2 Hz, J = 4.1 Hz), 4.67–4.69 (m, 1H,
aCH), 5.03–5.15 (m, 4H, PhCH2 ? OCH2), 5.21 (s, 2H,
OCH2), 5.32 (brs, 1H, NH), 5.90 (brs, 4H, NCH2), 7.05–
7.12 (m, 2H, Ar–H), 7.36–7.43 (m, 7H, Ar–H), 7.49 (s, 1H,
triazole), 7.57 (s, 1H, triazole), 8.21–8.23 (m, 4H, Ar–H).
13C NMR (100 MHz, CDCl3): d 36.8 (bCH2), 51.8 (aCH),
57.9 (OCH2), 58.6 (NCH2), 67.1 (OCH2; carbamate), 123.9
(C-5 triazole), 124.6 (C-5 triazole), 127.5, 128.3 (aromatic
carbons), 141.8 (C-4 triazole), 142.2 (C-4 triazole), 144.3,
147.5 (aromatic carbons), 156.3 (C=O), 170.0 (C=O).
HRMS: m/z (M?) Cacld. for C32H29N9O10: 699.2037,
found: 700.2119 (M?H)?, 722.1935 (M?Na)?.
(S)-Bis((1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)
2-(tert-butoxycarbonylamino) pentanedioate (5b) White
semisolid, Yield: 87 %; IR (KBr): 3375 (N–H str), 3130
(=C–H str triazole), 3076 (=C–H str aromatic), 2972 (C–H
1
str CH2), 1728 (C=O str ester), 1678 (C=O str) cm-1. H
NMR (400 MHz, CDCl3): d 1.39 (s, 9H, tBu-CH3), 1.96–2.
02 (m, 2H, bCH2), 2.30 (s, 6H, CH3), 2.67–2.80 (m, 2H, cCH2),
4.32–4.37 (m, 1H, aCH), 5.14 (d, 1H, J = 7.1 Hz, NH),
5.19 (s, 2H, OCH2), 5.26 (s, 2H, OCH2), 5.51 (s, 4H, NCH2),
7.19–7.44 (m, 8H, Ar–H), 7.48 (s, 1H, triazole),
7.53 (s, 1H, triazole). 13C NMR (100 MHz, CDCl3): d 20.7
(CH3), 26.2 (tBu-CH3), 27.7 (bCH2), 29.6 (cCH2), 53.1
(aCH), 57.3 (OCH2), 59.3 (OCH2), 64.4 (NCH2), 66.7
(NCH2), 80.3 (tBu-C), 122.5 (C-5 triazole), 122.9 (C-5
triazole), 126.3, 128.5, 130.0, 136.1, 136.6, 138.5 (aromatic
(S)-Bis((1-phenethyl-1H-1,2,3-triazol-4-yl)methyl) 2-(ben-
zyloxycarbonylamino)succinate (3h) Yellowish semi-
solid, Yield: 71 %; IR (KBr): 3388 (N–H str), 3141 (=C–H
123

3002
Med Chem Res (2014) 23:2995–3004
carbons), 143.1 (C-4 triazole), 143.6 (C-4 triazole), 155.8
(C=O), 171.4 (C=O ester), 172.1 (C=O ester). HRMS:
m/z (M?) Cacld. for C32H39N7O6: 617.2962, found:
618.3042 (M?H)?, 640.2855 (M?Na)?.
(m, 2H, cCH2), 4.14–4.21 (m, 1H, aCH), 5.22 (d, 1H, J =
7.0 Hz, NH), 5.29 (s, 2H, OCH2), 5.36 (s, 2H, OCH2), 5.55
(s, 4H, NCH2), 7.22–7.44 (m, 6H, Ar–H), 7.64 (s, 1H,
triazole), 7.70 (s, 1H, triazole), 8.23–8.35 (m, 2H, Ar–H).
13C NMR (100 MHz, CDCl3): d 26.2 (tBu-CH3), 28.2
(bCH2), 29.9 (cCH2), 50.8 (aCH), 53.5 (OCH2), 57.4
(OCH2), 59.4 (NCH2), 78.8 (tBu-C), 122.6 (C-5 triazole),
123.1 (C-5 triazole), 126.4, 130.8, 133.1, 133.9 (aromatic
carbons), 142.5 (C-4 triazole), 143.1 (C-4 triazole), 155.1
(C=O), 171.8 (C=O ester), 172.7 (C=O ester). HRMS:
m/z (M?) Cacld. for C30H33N9O10: 679.2350, found: 680.
2439 (M?H)?, 702.2240 (M?Na)?.
(S)-Bis((1-(3-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)
2-(tert-butoxycarbonylamino) pentanedioate (5c) White
semisolid, Yield: 87 %; IR (KBr): 3381 (N–H str), 3129
(=C–H str triazole), 3079 (=C–H str aromatic), 2973 (C–H str
CH2), 1727 (C=O str ester), 1680 (C=O str) cm-1. 1H NMR
(400 MHz, CDCl3): d 1.40 (s, 9H, tBu-CH3), 1.95–2.00 (m,
2H, bCH2), 2.28 (s, 6H, CH3), 2.65–2.84 (m, 2H, cCH2),
4.29–4.33 (m, 1H, aCH), 5.20 (d, 1H, J = 7.2 Hz, NH),
5.25 (s, 2H, OCH2), 5.30 (s, 2H, OCH2), 5.48 (s, 4H,
NCH2), 7.06–7.31 (m, 8H, Ar–H), 7.45 (s, 1H, triazole),
7.57 (s, 1H, triazole). 13C NMR (100 MHz, CDCl3): d 20.5
(CH3), 26.1(tBu-CH3), 27.8 (bCH2), 29.5 (cCH2), 53.3
(aCH), 57.4 (OCH2), 59.5 (OCH2), 64.3 (NCH2), 66.9
(NCH2), 80.5 (tBu-C), 122.4 (C-5 triazole), 123.0 (C-5
triazole), 126.5, 128.0, 130.2, 136.2, 136.8, 138.4 (aromatic
carbons), 143.2 (C-4 triazole), 143.9 (C-4 triazole), 155.9
(C=O), 171.6 (C=O ester), 172.0 (C=O ester). HRMS:
m/z (M?) Cacld. for C32H39N7O6: 617.2962, found:
618.3045 (M?H)?, 640.2857 (M?Na)?.
(S)-Bis((1-(3-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)2-
(tert-butoxycarbonylamino) pentanedioate (5f) Yellow-
ish semisolid, Yield: 87 %; IR (KBr): 3383 (N–H str), 3125
(=C–H str triazole), 3080 (=C–H str triazole), 2980 (C–H
str CH2), 1729 (C=O str ester), 1684 (C=O str), 1535 (N=O
str), 1345 (N=O str) cm-1. 1H NMR (300 MHz, CDCl3): d
1.46 (s, 9H, tBu-CH3), 1.80–1.90 (m, 2H, bCH), 2.73–2.86
(m, 2H, cCH2), 4.20–4.27 (m, 1H, aCH), 5.27 (d, 1H, J =
7.2 Hz, NH), 5.33 (s, 2H, OCH2), 5.40 (s, 2H, OCH2), 5.60
(s, 4H, NCH2), 7.28–7.43 (m, 6H, Ar–H), 7.62 (s, 1H,
triazole), 7.75 (s, 1H, triazole), 8.20–8.31 (m, 2H, Ar–H).
13C NMR (100 MHz, CDCl3): d 26.3 (tBu-CH3), 28.1
(bCH2), 29.6 (cCH2), 50.5 (aCH), 53.2 (OCH2), 57.6
(OCH2), 59.6 (NCH2), 78.9 (tBu-C), 122.5 (C-5 triazole),
123.2 (C-5 triazole), 125.3, 130.3, 133.4, 133.9 (aromatic
carbons), 142.7 (C-4 triazole), 143.4 (C-4 triazole), 146.8,
155.4 (C=O), 171.5 (C=O ester), 172.6 (C=O ester).
HRMS: m/z (M?) Cacld. for C30H33N9O10: 679.2350,
found: 680.2436 (M?H)?, 702.2239 (M?Na)?.
(S)-Bis((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)
2-(tert-butoxycarbonylamino) pentanedioate (5d) White
semisolid, Yield: 87 %; IR (KBr): 3384 (N–H str), 3134
(=C–H str triazole), 3075 (=C–H str aromatic), 2977 (C–H str
CH2), 1728 (C=O str ester), 1687 (C=O str) cm-1. 1H NMR
(400 MHz, CDCl3): d 1.44 (s, 9H, tBu-CH3), 1.83–1.99 (m,
1H, bCH), 2.09–2.20 (m, 1H, bCH2), 2.37–2.40 (m, 8H,
cCH2 ? CH3), 4.30–4.31 (m, 1H, aCH), 5.12 (d, 1H, J =
7.6 Hz, NH), 5.19 (s, 2H, OCH2), 5.24 (s, 2H, OCH2), 5.49
(s, 4H, NCH2), 7.20 (s, 8H, Ar–H), 7.54 (s, 1H, triazole),
7.56 (s, 1H, triazole). 13C NMR (100 MHz, CDCl3): d 21.2
(CH3), 27.5 (tBu-CH3), 28.3 (bCH2), 30.0 (cCH2), 52.8
(aCH), 54.1 (OCH2), 57.9 (OCH2), 58.6 (NCH2), 80.1
(tBu-C), 123.3 (C-5 triazole), 123.7 (C-5 triazole), 128.2,
128.3, 129.8, 129.9, 131.3, 138.8 (aromatic carbons), 142.9
(C-4 triazole), 143.5 (C-4 triazole), 155.3 (C=O), 172.0
(C=O ester), 172.4 (C=O ester). HRMS: m/z (M?) Cacld.
for C32H39N7O6: 617.2962, found: 618.3039 (M?H)?,
640.2851 (M?Na)?.
(S)-Bis((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)2-
(tert-butoxycarbonylamino) pentanedioate (5g) Yel-
lowish semisolid, Yield: 87 %; IR (KBr): 3381 (N–H str),
3129 (=C–H str triazole), 3077 (=C–H str triazole), 2969
(C–H str CH2), 1727 (C=O str ester), 1679 (C=O str), 1541
(N=O str), 1342 (N=O str) cm-1 1H NMR (400 MHz,
.
CDCl3): d 1.50 (s, 9H, tBu-CH3), 1.83–1.92 (m, 2H, bCH),
2.77–2.85 (m, 2H, cCH2), 4.11–4.13 (m, 1H, aCH), 5.23 (d,
1H, J = 7.7 Hz, NH), 5.38 (s, 2H, OCH2), 5.43 (s, 2H,
OCH2), 5.66 (s, 4H, NCH2), 7.43–7.65 (m, 4H, Ar–H), 7.68
(s, 1H, triazole), 7.80 (s, 1H, triazole), 8.25 (brs, 4H, Ar–
H). 13C NMR (100 MHz, CDCl3): d 26.0 (tBu-CH3), 28.3
(bCH2), 29.7 (cCH2), 50.9 (aCH), 53.3 (OCH2), 57.3
(OCH2), 59.5 (NCH2), 78.6 (tBu-C), 122.9 (C-5 triazole),
123.7 (C-5 triazole), 129.6 (aromatic carbons), 142.6 (C-4
triazole), 143.1 (C-4 triazole), 144.6 (aromatic carbons),
155.1 (C=O), 171.8 (C=O ester), 172.7 (C=O ester).
HRMS: m/z (M?) Cacld. for C30H33N9O10: 679.2350,
found: 680.2442 (M?H)?, 702.2244 (M?Na)?.
(S)-Bis((1-(2-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)2-
(tert-butoxycarbonylamino) pentanedioate (5e) Yellow-
ish semisolid, Yield: 87 %; IR (KBr): 3378 (N–H str), 3129
(=C–H str triazole), 3078 (=C–H str aromatic), 2978 (C–H
str CH2), 1727 (C=O str ester), 1682 (C=O str), 1530 (N=O
str), 1342 (N=O str) cm-1. 1H NMR (300 MHz, CDCl3): d
1.40 (s, 9H, tBu-CH3), 1.84–1.99 (m, 2H, bCH2), 2.68–2.90
123

Med Chem Res (2014) 23:2995–3004
3003
(S)-Bis((1-phenethyl-1H-1,2,3-triazol-4-yl)methyl)2-(tert-
butoxycarbonylamino) pentanedioate (5h) White semi-
solid, Yield: 87 %; IR (KBr): 3385 (N–H str), 3132 (=C–H
str triazole), 3078 (=C–H str aromatic), 2975 (C–H str
CH2), 1730 (C=O str ester), 1684 (C=O str) cm-1. 1H NMR
(300 MHz, CDCl3): d 1.46 (s, 9H, tBu-CH3), 1.81–1.88 (m,
2H, bCH), 2.70–2.77 (m, 2H, cCH2), 3.17 (t, 2H, J =
7.5 Hz, PhCH2), 4.27–4.31 (brs, 1H, aCH), 4.52 (t, 4H,
J = 7.5 Hz, NCH2), 5.07–5.19 (m, 4H, OCH2), 5.29 (d,
1H, J = 7.6 Hz, NH), 7.24–7.31 (m, 10H, Ar–H), 7.45 (s,
1H, triazole), 7.54 (s, 1H, triazole). 13C NMR (100 MHz,
CDCl3): d 26.1 (tBu-CH3), 28.2 (bCH2), 29.9 (PhCH2),
34.3 (cCH2), 50.8 (aCH), 53.5 (OCH2), 57.6 (OCH2), 59.7
(NCH2), 78.4 (tBu-C), 122.7 (C-5 triazole), 123.5 (C-5
triazole), 125.6, 127.2, 128.7, 136.8 (aromatic carbons),
142.1 (C-4 triazole), 142.8 (C-4 triazole), 155.5 (C=O),
171.6 (C=O ester), 172.4 (C=O ester). HRMS: m/z (M?)
Cacld. for C32H39N7O6: 617.2962, found: 618.3040
(M?H)?, 640.2849 (M?Na)?.
at 25 1 °C for 120 h in case of A. niger. Antifungal
activity was determined by measuring MIC. Fluconazole
was also assessed under similar conditions for comparison
with the synthesized compounds.
Computational details
The crystal structure of tyrosyl tRNA synthetase and topoi-
somerase II DNA Gyrase B was obtained from the Brook-
haven Protein Data Bank http://www.rcsb.org/pdb (PDB
entry: 1J1J and 1kzn). To carry out docking studies, the 2D
structures of various ligands were drawn and these were
converted to 3D and their energy was minimized (Marvin
Sketch, 1998–2008). Ligand files were prepared in pdb
format with explicit hydrogen addition. Co-crystallized
ligand was removed from pdb files and protein molecule was
prepared by deleting solvent molecules and non-complex
ions using Chimera (UCSF Chimera, 2000–2011). Incom-
plete side chains were replaced using Dun Brack Rotamer
library (Dunbrack, 2002). Hydrogens were added and ga-
steiger charges were calculated using Antechamber (Wang
et al., 2006). The prepared file was saved as pdb format and is
used for further studies. All pdb files were transformed
into pdbqt format. Docking studies were carried out by
using Auto Dock Vina 1.1.2. Grid center was placed on the
active site. The sizes and center of grid box were
center_x = –10.9089597492, center_y = 14.432242632,
center_z = 86.4200507331, size_x = 25.0, size_y = 25.0,
size_z = 25.0 and center_x = 19.5975431717, center_y =
30.2651505494, center_z = 35.2957776043, size_x =
25.0, size_y = 23.0014700724, size_z = 23.651263131 for
1JIJ and 1kzn, respectively. Exhaustiveness of the global
search algorithm was set to be 8. Then, finally docking results
were viewed using pdb and pdbqt files (Discovery Studio
Visualizer, 2005–2009; PyMol, 2008).
Antibacterial activity
All the newly synthesized conjugates were tested for their
in vitro antibacterial activity against Gram-positive bacte-
ria viz. S. aureus (MTCC 3160), B. subtilis (MTCC 441),
and Gram-negative bacteria E. coli (MTCC 7443) by
standard serial dilution method using a stock solution of
50 lg/mL concentration. Double-strength nutrient broth
was used as culture media and dimethylsulfoxide was used
as solvent control. The stock solutions of the test com-
pounds were serially diluted in test tubes containing 1 mL
of sterile medium to get the concentration of 25_1.56 lg/
mL and then inoculated with 100 lL of suspension of
respective microorganism in sterile saline. The inoculated
test tubes were incubated at 37 1 °C for 24 h. Cipro-
floxacin was used as standard drug and was also tested
under similar conditions for comparison with the synthe-
sized compounds.
Acknowledgments Financial assistance from University Grants
Commission, New Delhi for major research project to C.P.K. is highly
acknowledged.
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