Synthesis, antimicrobial potency with in silico study of Boc-leucine-1,2,3-triazoles

Article abstract of DOI:10.1016/j.steroids.2020.108675

A library of N-Boc protected Leucine-linked 1,4-disubstituted 1,2,3-triazoles was synthesized and fully characterized, in high yield via copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. In vitro antibacterial activity showed that compound 4h found to be more potent than the reference drug Ciprofloxacin (MIC: 0.0196 μmol/mL) against tested bacterial strains S. entrica, B. subtilis, S. aureus, E. coli and P. auroginosa with MIC: 0.0148, 0.0074, 0.0148, 0.0074, and 0.0074 μmol/mL, respectively and antifungal activity with MIC: 0.0148 μmol/mL as compared to reference drug Fluconazole (MIC: 0.0102 μmol/mL) against A. niger and C. albicans fungal strains. Further, the molecular docking study on 4h and its predecessor alkyne 3 by choosing E. coli topoisomerase II, DNA Gyrase (PDB ID: 1KZN) showed better binding with triazole than alkyne and these results were supported by DFT study using B3LYP/6-311G(d,p) basis set.

Full text of DOI:10.1016/j.steroids.2020.108675

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Synthesis, Antimicrobial Potency with In Silico Study of Boc-Leucine-1,2,3-
Triazoles
Naveen, Ram Kumar Tittal, D. Ghule Vikas, Pinki Yadav, Kashmiri Lal,
Ashwani Kumar
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https://doi.org/10.1016/j.steroids.2020.108675
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Please cite this article as: Naveen, Kumar Tittal, R., Ghule Vikas, D., Yadav, P., Lal, K., Kumar, A., Synthesis,
Antimicrobial Potency with In Silico Study of Boc-Leucine-1,2,3-Triazoles, Steroids (2020), doi: https://doi.org/
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Synthesis, Antimicrobial Potency with In Silico Study of Boc-Leucine-1,2,3-
Triazoles
Naveen,a Ram Kumar Tittal,*,a Ghule Vikas D.,a Pinki Yadav,b Kashmiri Lal,*,b Ashwani
Kumarc
aDepartment of Chemistry, National Institute of Technology Kurukshetra, Haryana-136119, India.
Email: rktittaliitd@nitkkr.ac.in
bDepartment of Chemistry, Guru Jambheshwar university of Science & Technology (GJUS&T), Hisar, Haryana
12500, India. Email: klal_iitd@yahoo.com
cDepartment of Pharmaceutical Sciences, GJUS&T, Hisar, Haryana 12500, India.
_______________________________________________________________________
Abstract
A library of N-Boc protected Leucine-linked 1,4-disubstituted 1,2,3-triazoles was
synthesized and fully characterized, in high yield via copper-catalyzed alkyne-azide cycloaddition
(CuAAC) reaction. In vitro antibacterial activity showed that compound 4h found to be more
potent than the reference drug Ciprofloxacin (MIC: 0.0196 µmol/mL) against tested bacterial
strains S. entrica, B. subtilis, S. aureus, E. coli and P. auroginosa with MIC: 0.0148, 0.0074,
0.0148, 0.0074, and 0.0074 µmol/mL, respectively and antifungal activity with MIC: 0.0148
µmol/mL as compared to reference drug Fluconazole (MIC: 0.0102 µmol/mL) against A. niger
and C. albicans fungal strains. Further, the molecular docking study on 4h and its predecessor
alkyne 3 by choosing E. coli topoisomerase II, DNA Gyrase (PDB ID: 1KZN) showed better
binding with triazole than alkyne and these results were supported by DFT study using B3LYP/6-
311G(d,p) basis set.

Keywords: CuAAC; Boc-Leucine-1,2,3-triazole; Antibacterial activity; Antifungal activity;
Molecular docking and DFT/B3LYP-6113G(d,p).
1. Introduction
The day by day increasing graph of microbial infection is causing a serious threat to public
health all over the world. The continuous and uncontrolled use of chemotherapeutic drugs has
developed multidrug resistance (MDR) to many antibiotics against various bacterial and fungal
strains [1]. MDR severely limits the proper treatment of diseases [2]. Due to this, during last few
years there was a sharp hike in the frequency of microbial diseases which creates the need for
exploration and implementation of second-line antibiotics. So, a multifaceted perspective is
required to modify existing and develop some new novel chemotherapeutic agent with a lesser
side effect, more potency, better selectivity and broad-spectrum pharmacological activity which
can target these MDR-microbes by a different and effective mechanism of action. In search of new
antimicrobials, medicinal chemists generally rely on N-heterocyclic compounds. In this line, 1,2,3-
triazoles have been explored as a significant class of synthetically versatile heterocyclic
compounds. They have attracted paramount attention of organic chemists due to their promising
and fascinating applications in the drug industry, agricultural chemistry, biological chemistry for
the development of antibacterial [3-6], anticancer [7-10], antifungal [11-16], anti-HIV [17-18],
antiallergic [19], antitubercular [20-23], anti-inflammatory [24-27], antiviral [28-31], antimalarial
[32-33], anticonvulsant [34], antitrypanosomal [35], antileishmania, etc. [36-37]. The efficacy of
1,2,3-triazoles in medicinal chemistry and synthesis of various drugs is because of their stability
towards acidic and basic hydrolysis in oxidoreductase environments which resist their metabolic
degradation in a diverse range of pH to confirm their aromatic stabilization. Also, the high dipole
moment, tendency to show dipole-dipole interaction and H- bonding makes 1,2,3-triazoles capable
to effectively bind with different biological targets. Sharpless and Medal have modified the un-
catalyzed 1,3-dipolar cycloaddition reaction (DCR) to a new approach of CuAAC reaction with
regioselective formation of 1,4-disubstituted 1,2,3-triazoles which avoids unnecessary use of high
activation energy of 24-26 kcal mol-1 [38-39]. Subsequently, various other methodologies have
been proposed to show the improvement of process or product for the synthesis of 1,4-disubstituted
1,2,3-triazoles using various copper-based catalyst under different reaction conditions [40].

The long time use of antibiotics leads to loss of appetite and consequently loss of muscles and
health due to unbalanced secretion of digestive juices or suppression in protein biosynthesis. It is
known that Leucine, an essential amino acid is important for the protein biosynthesis. It cannot be
synthesized in human body. During metabolism the end product of Leucine are acetoacetate,
acetyl-CoA and a minor metabolite -hydroxy -methylbutyric acid which is responsible for
pharmacological action and protein biosynthesis [41]. This year, Hang Wu, Buchang Zhang and
co-workers have reported the importance of Leucine-responsive regulatory proteins (Lrps) for
various biological processes in bacteria which stimulate transcription of some structural, resistance
and regulatory genes [42]. Also, the important role of Leucine was reported recently by Zhang and
co-workers as a Leucine-rich repeat and sterile alpha motif containing 1 (LRSAM1) for different
cellular activities like antibacterial autophagy and cell adhesion [43]. Considering the importance
of Leucine metabolism, current need for the development of advanced antimicrobial, biological
potential of 1,2,3-triazoles and our interest in biological active triazoles, we thought to synthesize
N-Boc protected Leucine-linked 1,4-disubstituted 1,2,3-triazoles as shown in Figure 1. Herein,
we report the synthesis, in vitro antimicrobial activity and in silico study of N-Boc protected
leucine linked 1,4-disubstituted 1,2,3-triazoles.
R
O
O
N
N
O
NH
N
O
Figure 1. Design of N-Boc protected Leucine linked 1,4-disubstituted 1,2,3-triazole.
2. Materials and methods
2.1. General Chemistry
All the commercially available chemicals purchased from Sigma Aldrich. The starting
chemicals including solvents were used purification as per the reported protocols. The melting
points of all compounds were recorded in open capillaries using LABCO melting point apparatus.
The 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded using Avance III 400
MHz Bruker spectrometer. Tetramethylsilane (TMS) was used as an internal standard (chemical
shift (δ) in ppm, coupling constant (J) in Hertz (Hz)). Infrared spectra of compounds were recorded

on SHIMAZDU IR AFFINITY-I FT-IR spectrophotometer. The high- resolution mass spectra
(HRMS) were recorded using SCIEX-QTOF spectrometer. Readymade silica gel plates (SIL
G/UV254, ALU-GRAM) were used to run thin-layer chromatography (TLC) and visualized in
ultraviolet lamp box.
2.2. General procedure for the synthesis of Boc-Leucine linked triazoles (4a-j) [3].
Differently substituted benzyl bromide, NaN3 & alkyne 3 in THF:water (9:1 v/v) in 1.0
mmol, 3.0 mmol & 1.0 mmol, respectively were taken in a round bottom flask, then 10 mol %
CuSO4.5H2O and 20 mol % Na-ascorbate were added to it. This reaction solution was allowed to
stir for 2-4 hrs at room temperature (i.e. 30˚C). The progression of reaction was studied by using
thin layer column chromatography (TLC). The reaction was quenched with the addition of ice-
cold water in the reaction mixture, after the completion of reaction. Now, the reaction product was
diluted with ethyl acetate and extracted with brine solution followed by washing with aq. NH4Cl:
NH3 (9:1 v/v) solution. The organic layer of ethyl acetate was evaporated by rotary evaporator
under vacuum to obtain the desired triazoles in considerable to good yields.
2.2.1 (1-benzyl-1H-1,2,3-triazol-4-yl)methyl (tert-butoxycarbonyl)-L-leucinate (4a)
◦
1
Appearance: off white solid, Yield: 95 %, m.p: 63-65 C. H
NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.38 (d, J = 5.8 Hz, 3H),
7.28 (d, J = 5.9 Hz, 2H), 5.53 (s, 2H), 5.26 (s, 2H), 4.92 (s, 1H),
4.28 (t, J = 11.2 Hz, 1H), 1.64 (dd, J = 13.5, 7.0 Hz, 2H), 1.54
N
N
O
N
O
O
N
H
O
(dt, J = 10.2, 5.8 Hz, 1H), 1.41 (s, 9H), 0.89 (dd, J = 6.4, 3.1 Hz, 6H). 13C NMR (101 MHz, CDCl3)
δ 173.33(s), 155.44(s), 142.98(s), 134.34(s), 129.15(s), 128.85(s), 128.09(s), 123.68(s), 79.91(s),
75.22(s), 58.25(s), 54.25(s), 52.17(s), 41.36(s), 28.27(s), 24.72(s), 22.77(s), 21.76(s). FT IR (KBr):
vmax 3387(s), 3142(s), 2955(m), 2872(m), 1749(s), 1688(s), 1504(m), 1391(m). HRMS [M+H]+:
m/z for C21H31N4O4: calculated 403.2345 and found 403.2175
2.2.2 (1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl (tert-butoxycarbonyl)-L-leucinate (4b)
◦
1
Appearance: off white solid, Yield: 84%, m.p: 74-76 C. H
NMR (400 MHz, CDCl3) δ 7.64 (s, 1H), 7.37 (dd, J = 13.5, 7.5
Hz, 1H), 7.29 (t, J = 7.2 Hz, 1H), 7.14 (dt, J = 14.6, 8.2 Hz,
2H), 5.59 (s, 2H), 5.27 (s, 2H), 4.91 (d, J = 8.3 Hz, 1H), 4.29 (t,
N
N
O
N
F
O
O
N
H
O

J = 11.2 Hz, 1H), 1.66 (dd, J = 14.2, 6.7 Hz, 2H), 1.57-1.49 (m, 1H), 1.42 (s, 9H), 0.89 (dd, J =
13
6.4, 3.4 Hz, 6H). C NMR (101 MHz, CDCl3) δ 173.34(s), 161.77(s), 159.31(s), 155.45(s),
142.99(s), 130.84 (d, J = 44.9 Hz), 124.87 (d, J = 3.7 Hz), 123.85(s), 115.88 (d, J = 21.1 Hz),
79.91(s), 58.23(s), 52.17(s), 47.75 (d, J = 4.3 Hz), 41.38(s), 28.26(s), 24.72(s), 22.75(s), 21.76 (s).
FT IR (KBr): vmax 3389(s), 3146(m), 2957(s), 2872(m), 1751(s), 1686(s), 1506(w), 1368(m).
HRMS [M+H]+: m/z for C21H30FN4O4: calculated 421.2251 and found 421.2082.
2.2.3. (1-(2-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl (tert-butoxycarbonyl)-L-leucinate (4c)
Appearance: off white solid, Yield: 80%, m.p: 83-85 ◦C.1H NMR
N
(400 MHz, CDCl3) δ 7.66 (s, 1H), 7.32 (t, J = 7.4 Hz, 1H), 7.23
(t, J = 7.6 Hz, 2H), 7.17 (d, J = 7.3 Hz, 1H), 5.66 (s, 2H), 5.27 (s,
2H), 4.92 (s, 1H), 4.29 (t, J = 10.7 Hz, 1H), 1.64 (dd, J = 13.2, 6.6
N
O
N
Br
O
O
N
H
O
Hz, 2H), 1.53 – 1.43 (m, 1H), 1.41 (s, 9H), 0.89 (dd, J = 6.2, 3.0 Hz, 6H); 13C NMR (101 MHz,
CDCl3) δ 173.34(s), 155.45(s), 142.88(s), 133.91(s), 133.27(s), 130.46 (d, J = 9.8 Hz), 128.26(s),
124.07(s), 123.51(s), 79.90(s), 58.24(s), 53.88(s), 52.17(s), 41.40(s), 28.28(s), 24.73(s), 22.78(s),
21.77(s). FT IR (KBr): vmax 3383(s), 3149(m), 2959(s), 2870(w), 1747(s), 1691(s), 1519(s),
1367(m)cm-1. HRMS [M+H]+: m/z for C21H30BrN4O4: calculated 481.1450 and found 481.1246
2.2.4. (1-(2-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl (tert-butoxycarbonyl)-L-leucinate (4d)
◦
Appearance: off white solid, Yield: 93%, m.p: 80-82 C.1H
N
N
O
NMR (400 MHz, CDCl3) δ 8.18 (dd, J = 8.1, 1.3 Hz, 1H),
7.80 (s, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 8.4 Hz, 1H),
7.28 (s, 1H), 5.96 (s, 2H), 5.33 (s, 2H), 4.86 (d, J = 10.0 Hz,
N
O
NO2
O
N
H
O
1H), 4.33-4.29 (m, 1H), 1.68 (dd, J = 13.5, 7.2 Hz, 2H), 1.51 (dt, J = 5.8, 2.8 Hz, 1H), 1.43 (s,
9H), 0.93 (dd, J = 6.5, 2.5 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 173.34(s), 155.51(s), 142.82(s),
133.91(s), 133.27(s), 130.50 (d, J = 17.4 Hz), 128.26(s), 124.07(s), 123.51(s), 79.95(s), 58.24(s),
53.88(s), 52.26(s), 41.31(s), 28.28(s), 24.73(s), 22.84(s), 21.71(s). FT IR (KBr): vmax 3385(s),
3134(m), 3088(m), 2961(s), 1732(s), 1691(s),1522(s), 1368(s). HRMS [M+H]+: m/z for
C21H30N5O6: calculated 448.2196 and found 448.2001.
2.2.5. (1-(3-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl (tert-butoxycarbonyl)-L-leucinate (4e)

◦
Appearance: off white solid, Yield: 85%, m.p: 69-71 C.1H
N
N
O
NMR (400 MHz, CDCl3) δ 7.60 (s, 1H), 7.36 (dd, J = 14.4, 7.4
Hz, 1H), 7.06 (d, J = 7.2 Hz, 2H), 6.96 (d, J = 9.1 Hz, 1H), 5.53
(s, 2H), 5.28 (s, 2H), 4.91 (s, 1H), 4.32 – 4.26 (m, 1H), 1.64
N
O
O
N
H
O
F
(dd, J = 13.5, 6.6 Hz, 2H), 1.57-1.53 (m, 1H), 1.41 (s, 9H), 0.90 (dd, J = 5.7, 2.0 Hz, 6H). 13C
NMR (101 MHz, CDCl3) δ 173.38(s), 164.23(s), 161.76(s), 155.47(s), 143.24(s), 136.74 (d, J =
7.1 Hz), 130.82 (d, J = 8.3 Hz), 123.69 (d, J = 20.9 Hz), 115.88 (d, J = 21.0 Hz), 115.02 (d, J =
22.3 Hz), 79.95(s), 58.23(s), 53.57(s), 52.20(s), 41.31(s), 28.25(s), 24.73(s), 22.75(s), 21.74(s). FT
IR (KBr): vmax 3387(s), 3142(m), 2959(s), 2872(m), 1745(s), 1686(s), 1593(m), 1391(m). HRMS
[M+H]+: m/z for C21H30FN4O4: calculated 421.2251 and found 421.2082
2.2.6. (1-(3-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl (tert-butoxycarbonyl)-L-leucinate (4f)
Appearance: off white solid, Yield: 91%, m.p: 78-80 C.1H
◦
N
N
O
NMR (400 MHz, CDCl3) δ 7.60 (s, 1H), 7.50 (d, J = 7.8 Hz,
1H), 7.43 (s, 1H), 7.26 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 7.8 Hz,
1H), 5.50 (s, 2H), 5.28 (s, 2H), 4.89 (s, 1H), 4.29 (t, J = 11.3
N
O
O
N
H
O
Br
Hz, 1H), 1.65 (dd, J = 13.5, 7.0 Hz, 1H), 1.50 – 1.44 (m, 2H), 1.41 (s, 9H), 0.90 (dd, J = 6.4, 3.7
Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 173.36(s), 155.46(s), 143.28(s), 136.57(s), 132.01(s),
130.87 (d, J = 31.3 Hz), 126.58(s), 123.75(s), 123.12(s), 79.93(s), 58.26(s), 53.43(s), 52.19(s),
41.34(s), 28.27(s), 24.74(s), 22.78(s), 21.76(s). FT IR (KBr): vmax 3402(s), 3152(m), 2957(s),
1738(w), 1688(w), 1579(m), 1506(s), 1365(s). HRMS [M+H]+: m/z for C21H30BrN4O4: calculated
481.1450 and found 481.1246
2.2.7 (1-(3-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl (tert-butoxycarbonyl)-L-leucinate (4g)
Appearance: off white solid, Yield: 86%, m.p: 68-70 C.1H
◦
N
N
O
NMR (400 MHz, CDCl3) δ 8.18 (dd, J = 8.1, 1.3 Hz, 1H), 7.80
(s, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.56 (t, J = 7.2 Hz, 1H), 7.28
(s, 1H), 5.96 (s, 2H), 5.32 (s, 2H), 4.88 (d, J = 8.2 Hz, 1H), 4.33
N
O
O
N
H
O
NO2
– 4.30 (m, 1H), 1.67 (dd, J = 12.6, 6.4 Hz, 2H), 1.53-1.47 (m, 1H), 1.42 (s, 9H), 0.92 (dd, J = 6.5,
2.2 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 173.37(s), 155.43(s), 147.49(s), 143.20(s), 134.41(s),
130.38(s), 129.75(s), 125.43(s), 124.83(s), 79.96(s), 58.22(s), 52.18(s), 50.89(s), 41.40(s),

28.27(s), 24.76(s), 22.80(s), 21.78(s). FT IR (KBr): vmax 3385(s), 3132(m), 3088(m), 2961(s),
1732(s), 1695(s), 1522(s), 1368(m). HRMS [M+H]+: m/z for C21H30N5O6: calculated 448.2196
and found 448.2001.
2.2.8. (1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl (tert-butoxycarbonyl)-L-leucinate (4h)
Appearance: off white solid, Yield: 90%, m.p: 77-79 ◦C.1H NMR
N
O
N
N
(400 MHz, CDCl3) δ 7.56 (s, 1H), 7.28 (t, J = 6.8 Hz, 2H), 7.08
(t, J = 8.5 Hz, 2H), 5.50 (s, 2H), 5.27 (s, 2H), 4.87 (s, 1H), 4.28
(dd, J = 13.3, 8.3 Hz, 1H), 1.69 (d, J = 11.0 Hz, 2H), 1.55 (dt, J
O
O
N
H
O
F
= 14.3, 7.4 Hz, 1H), 1.42 (s, 9H), 0.90 (dd, J = 6.4, 3.5 Hz, 6H) 13C NMR (101 MHz, CDCl3) δ
173.35(s), 164.14(s), 161.67(s), 155.43(s), 143.20(s), 129.99 (d, J = 8.4 Hz), 123.54(s), 116.18 (d,
J = 21.8 Hz), 79.91(s), 58.28(s), 53.47(s), 52.18(s), 41.37(s), 28.26(s), 24.73(s), 22.77(s), 21.76(s).
FT IR (KBr): vmax 3393(s), 3154(s), 2978(s), 2873(), 1734(s), 1688(s), 1512(s), 1367(m). HRMS
[M+H]+: m/z for C21H30FN4O4: calculated 421.2251 and found 421.2082.
2.2.9. (1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl (tert-butoxycarbonyl)-L-leucinate (4i)
Appearance: off white solid, Yield: 94%, m.p: 98-100 ◦C.1H NMR
N
O
N
N
O
(400 MHz, CDCl3) δ 7.57 (s, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.16 (d,
J = 8.3 Hz, 2H), 5.49 (s, 2H), 5.28 (s, 2H), 4.86 (s, 1H), 4.29 (t, J
= 11.1 Hz, 1H), 1.67 (dd, J = 13.3, 5.8 Hz, 2H), 1.50-1.44 (m, 1H),
O
N
H
O
Br
1.42 (s, 9H), 0.91 (dd, J = 6.4, 3.4 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 173.35(s), 155.43(s),
143.28(s), 133.41(s), 132.35(s), 129.69(s), 123.63(s), 123.05(s), 79.92(s), 58.26(s), 53.53(s),
52.19(s), 41.36(s), 28.26(s), 24.74(s), 22.79(s), 21.77(s). FT IR (KBr): vmax 3406(s), 3134(m),
2953(s), 2872(m), 1736(s), 1691(s), 1506(s), 1369(m). HRMS [M+H]+: m/z for C21H30BrN4O4:
calculated 481.1450 and found 481.1246
2.2.10. (1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl (tert-butoxycarbonyl)-L-leucinate (4j)
Appearance: off white solid, Yield: 87%, m.p: 88-90 C.1H NMR
◦
N
O
N
N
O
(400 MHz, CDCl3) δ 7.57 (s, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.16 (d,
J = 8.3 Hz, 2H), 5.49 (s, 2H), 5.28 (s, 2H), 4.86 (s, 1H), 4.29 (t, J =
11.1 Hz, 1H), 1.67 (dd, J = 13.3, 5.8 Hz, 2H), 1.50 – 1.44 (m, 1H),
O
N
H
O
NO2

1.42 (s, 9H), 0.91 (dd, J = 6.4, 3.4 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 173.39(s), 155.47(s),
143.28(s), 133.46(s), 132.35(s), 129.69(s), 123.71(s), 123.05(s), 79.98(s), 58.26(s), 53.53(s),
52.14(s), 41.36(s), 28.26(s), 24.74(s), 22.79(s), 21.77(s). FT IR (KBr): vmax 3350(w), 3144(m),
2961(s), 2872(m), 1744(s), 1705(s), 1526(s), 1348(s). HRMS [M+H]+: m/z for C21H30N5O6:
calculated 448.2196 and found 448.2001.
3. Results and discussion
3.1. Synthesis of Boc-Leucine linked triazoles 4a-j, [3, 44-45].
We have explored the click reaction of C-terminal propargyl esters of N-protected L-Leucine with
various organic azides. Initially, N-Boc protected Leucine 2, as shown in Scheme 1, was prepared
by treating Leucine with Boc2O in aqueous sodium hydroxide (NaOH) solution with the help of
reported procedure [44]. The synthesis of alkyne precursor 3 was accomplished by reacting N-
Boc-Leucine 2, propargyl bromide, and anhydrous potassium carbonate (K2CO3) in dry
dimethylformamide (DMF). This solution was stirred for 20 hrs at ambient temperature under inert
N2 atmosphere [45]. The so prepared alkyne 3 was allowed to react in presence of variously
substituted organic azides formed in situ via reaction of benzyl bromides with NaN3 in presence
of CuSO4.5H2O and Na-ascorbate using various combinations of solvent systems under different
set of reaction conditions as described in Table 1. However, better reaction condition for the form-
NH2
O
O
(i)
(ii)
HO
OH
O
O
N
O
N
H
H
O
O
O
2
3
1
Br
Conditions (i)
: NaOH, Boc2O, stirred overnight
(iii)
(ii)
(rt);
propargyl bromide, K2CO3, DMF, stirred
R
(iii)
at ambient temp., 20h;
CuSO4.5H2O, sodum
,
°
ascorbate, NaN3 THF: H2O (9:1 v/v), 2-4 h, 30 C
HN
O
O
N
N
N
4a 4f
4g 4h 4i 4j
m-Br; : m-NO2; : p-F; : p-Br; : p-NO2
4b
4c
4d
4e
R: : H; :o-F; : o-Br; :o-NO2; : m-F; :
O O
R
4a-j
Scheme 1: Synthesis of Boc-Leucine linked-1,2,3 triazoles (4a-j).

Table 1. Reaction of 3 with in situ generated PhCH2N3 from PhCH2Br and NaN3 using
CuSO4.5H2O/sodium ascorbate in different solvent systems for the formation of triazole 4a.a
Sr. No.
1
Solvent
t-BuOH:H2O
t-BuOH:H2O
t-BuOH:H2O
t-BuOH:H2O
t-BuOH
Composition Temp(
̊
Time(h)
4.0
3.5
3.0
3.0
24
4a Yield(%)b
1:1
1:1
30
40
30
40
77
82
88
86
c
2
3
9:1
4
9:1
5
100%
1:1
30-40
30
6
DMF:H2O
DMF:H2O
DMF:H2O
DMF:H2O
DMF
3.5
3.0
2.5
2.5
24
79
82
85
82
c
7
1:1
40
8
9:1
30
9
9:1
40
10
11
12
13
14
15
16
17
18
19
20
100%
1:1
30-40
30
THF:H2O
THF:H2O
THF:H2O
THF:H2O
THF
3.0
2.5
2.0
2.0
24
87
88
95
92
c
1:1
40
9:1
30
9:1
40
100%
1:1
30-40
30
DMSO:H2O
DMSO:H2O
DMSO:H2O
DMSO:H2O
DMSO
3.5
2.5
2.0
2.0
24
82
85
88
82
c
1:1
40
9:1
30
9:1
40
100%
30-40
aReaction Condition: Benzyl azide and alkyne 3 (1 mmol each); CuSO4.5H2O (10 mol%) and sodium ascorbate (20
mol%), rt, 2-4 h for S. No. 10,15 & 20 time: 24h. bYield refers to purification by recrystallization. cNo reaction.
-ation of (1-benzyl-1H-1,2,3-triazol-4-yl) methyl (tert-butoxycarbonyl)-L-Leucinate 4a in high
yield of 95% from alkyne 3 required tetrahydrofuran (THF): H2O (9:1 v/v) mixture as solvent
system wherein reaction was completed in 2h after stirring reaction mixture at ambient temperature
(30
̊
IR spectrum of the representative compound 4a showed a characteristic band in the region 3142
cm-1 due to =C−H stretching of 1,2,3-triazole ring. The N−H stretching band appeared at 3387 cm-
1. The band observed at 1749 cm-1 was due to the C=O stretching of the ester group. The 1H NMR
spectrum exhibited a characteristic peak at δ 7.56 ppm appeared as singlet equivalent to one proton

of methine of triazole ring to confirm triazole formation. The singlet corresponding to 2H observed
at δ 5.26 ppm was assigned to the methylene protons linked to N-1 of triazole ring whereas the
singlet observed at δ 5.53 ppm was due to two protons of methylene group attached to ester linkage.
A singlet at δ 1.41 ppm integrating for nine protons was assigned to methyl protons of protecting
group. Further, a broad singlet at δ 4.92 ppm was attributed to -NH proton. However, the 13C NMR
spectrum showed signals due to carbonyl carbon at δ 173.33 ppm (ester) and δ 155.44 ppm
(carbamate) and signals at δ 142.98 and δ 134.34 ppm attributed to 4 and 5 carbon atom positions
in the triazole ring. Also, two signals at δ 75.22 and δ 58.25 ppm were accounted for the methylene
carbons attached to the ester linkage and other methylene carbons linked to N-1 of triazoles,
respectively. Finally, the HRMS spectrum of the compound 4a displayed m/z signal due to (M+H)+
at 403.2175 which is in good agreement with the calculated value for C21H31N4O4: 403.2345.
On the basis of information derived from the standardization of reaction condition for the
formation of representative triazole 4a, other derivatives of N-Boc protected Leucine linked 1,4-
disubstituted 1,2,3-triazoles 4b-j were synthesized under these reaction condition. The complete
detail for the formation of N-Boc protected L-Leucine linked 1,4-disubstituted-1,2,3-triazoles (4a-
j) is presented in the Table 2. The formation of alkyne 3 and all synthesized triazoles 4a-j were
Table 2. The reaction of N-Boc protected L-Leucine linked alkyne (3) for the formation of
corresponding 1,4-disubstituted 1,2,3-triazoles (4a-j) using CuAAC reaction.a
Sr. No.
Compound
R
H
Time(h)
Yield (%)b
2.0
1
2
4a
4b
4c
4d
4e
4f
95
84
80
93
85
91
86
90
94
87
2.5
3.0
3.0
4.0
3.5
4.0
2.0
2.5
2.0
o-F
3
o-Br
o-NO2
m-F
4
5
6
m-Br
m-NO2
p-F
7
4g
4h
4i
8
9
p-Br
10
4j
p-NO2
aReaction Condition: Benzyl azide 4a-j and alkyne 3 (1 mmol each); CuSO4.5H2O(10 mol%) and sodium ascorbate
(20 mol%), rt, 2-4 h. bYield refers to purification by recrystallization

structurally confirmed on the basis of FT IR, 1H NMR, and 13C NMR data. However, the structures
of all new N-Boc protected Leucine linked 1,4-disubstituted 1,2,3-triazoles (4a-j) were finally
established by HRMS data.
3.2. Biological Activities
3.2.1. Antibacterial activity
All the newly synthesized N-Boc protected Leucine linked 1,4-disubstituted 1,2,3-triazole
(4a-j) including alkyne 3 were screened in vitro for their antibacterial activity against S. aureus,
S. entrica, B. subtilis (gram positive bacteria) and E. coli, P. auroginosa (gram negative bacteria)
by a standard method of serial dilution. In this method, a 100 µg/mL concentration stock solution
was prepared using DMSO and commonly used antibacterial drug Ciprofloxacin was used as
solvent and reference drug, respectively [46]. The dilution of the initial stock solution of 50 to 3.12
µg/mL concentration was made. Each of these diluted lot was incubated at 37 ̊C for 24 hours after
inoculating with 100 µg/mL suspension of the chosen bacterial strain in sterile saline. A blank
experiment under similar conditions with a nutrient medium was also performed to observe the
solvent effect on the bacterial growth. A minimum inhibitory concentration, MIC in µmol/mL for
alkyne 3, all the 1,2,3-triazoles 4a-j are tabulated in Table 3.
Table 3. In vitro antibacterial activity of N-Boc protected L-Leucine linked 1,2,3-triazoles (4a-j,
MIC in µmol/mL).
Sr. No Compound
R
---
S. entrica B. subtilis S. aureus
E. coli P. auroginosa
1
2
3
0.0464
0.0155
0.0074
0.0260
0.0139
0.0074
0.0130
0.0279
0.0148
0.0130
0.0279
0.0196
0.0929
0.0310
0.0148
0.0130
0.0558
0.0148
0.0260
0.0558
0.0074
0.0260
0.0279
0.0196
0.0464
0.0310
0.0148
0.0130
0.0279
0.0297
0.0260
0.0279
0.0148
0.0260
0.0279
0.0196
0.0929
0.0310
0.0297
0.0130
0.0558
0.0297
0.0260
0.0279
0.0074
0.0130
0.0558
0.0196
0.0464
0.0155
0.0148
0.0130
0.0279
0.0074
0.0130
0.0558
0.0074
0.0260
0.0279
0.0196
4a
4b
4c
4d
4e
4f
H
3
o-F
4
o-Br
o-NO2
m-F
5
6
7
m-Br
m-NO2
p-F
8
4g
4h
4i
9
10
11
12
p-Br
p-NO2
4j
Ciprofloxacin

By observing antibacterial screening results, it was found that in case of S. entrica
compounds 4b and 4e each having F-moiety on benzene ring with MIC value 0.0074 µmol/mL
showed more than two fold better antibacterial activity than the reference drug Ciprofloxacin with
MIC: 0.0196 µmol/mL. Similarly, in case of B. subtilis among all the synthesized compounds,
compound 4h again having F-moiety on benzene ring with MIC: 0.0074 µmol/mL showed two
folds better antibacterial activity than the reference drug Ciprofloxacin with MIC: 0.0196
µmol/mL. Otherwise, all N-Boc protected L-Leucine linked 1,4-disubstituted-1,2,3-triazoles
containing F-moiety at ortho, meta and para position of the benzene ring i.e. 4b, 4e and 4h are
more potent than reference drug Ciprofloxacin (MIC: 0.0196 µmol/mL) for all tested bacterial
strains with MIC value either 0.0074 or 0.0148 µmol/mL except 4b and 4e showed little higher
MIC: 0.0297 µmol/mL for E. coli and S. aureus. Compounds containing o-Br, m-Br and p-Br are
also next more potent than F-moiety containing triazoles i.e. o-Br containing compound 4c for B.
subtilis, S. aureus, E. coli, and P. auroginosa; m-Br containing compound 4f for S. entrica and P.
auroginosa and p-Br containing compound 4i for S. entrica and E. coli with MIC: 0.0130
µmol/mL, each are also more potent than reference drug Ciprofloxacin with MIC: 0.0196
µmol/mL. Moreover, compound 4c and 4h with MIC: 0.0130 µmol/mL and 0.0148 µmol/mL,
respectively were found to be more potent among all triazoles than reference drug Ciprofloxacin
against S. aureus. Compound 4h with MIC: 0.0074 µmol/mL was found more potent among all
triazoles than reference drug Ciprofloxacin with MIC value 0.0196 µmol/mL against E. coli. The
activity data revealed that in the case of P. auroginosa the compound 4e and 4h both with MIC:
0.0074 µmol/mL are more potent than Ciprofloxacin. However, alkyne 3 is least active to all
bacterial strains with MIC values ranging from 0.0464 to 0.0929 µmol/mL, as shown in Figure 2.
The SAR study which is abbreviated as structure-activity relationship shows that all N-
Boc-protected-L-Leucine linked 1,2,3-triazoles showed better efficacy than alkyne which
confirms that incorporation of 1,2,3-triazole ring improves the antibacterial activity to a large
extent. It was also observed that compound containing o, m, or p-F-group in benzyl moiety shows
better antibacterial activity than unsubstituted benzyl, o, m, or p-Br- and o, m, or p-NO2-group
substituted benzyl group containing triazoles.

Figure 2. Antibacterial activities of synthesized Leucine linked alkyne 3 & triazoles 4a-j.
3.2.2. Antifungal activity
All the synthesized N-Boc protected L-Leucine linked 1,2,3-triazoles 4a-j including
predecessor alkyne 3 were also screened for in vitro antifungal activity by the same standard
dilution method (Cappucim and Sherman,1999) against A. niger and C. albicans fungal strains.
The commonly used antifungal drug Fluconazole was used as a reference. The DMSO and
Sabouraud dextrose broth were used, respectively as solvent and culture media. The suspension of
Aspire was prepared in sterile saline from 24 hours old culture of fungus developed on Sabouraud
dextrose broth (SDB, Hi-Media, Mumbai). The samples were incubated at 37
̊
after 2 days for A. niger. In case of the C. albicans samples were incubated at 25
̊
MIC of all the tested compounds were recorded in µmol/mL as shown in Table 4.
The observation of antifungal activity data as shown in Figure 3, revealed that all N-Boc
protected L-Leucine linked 1,2,3-triazoles showed better activity with MIC value in the range of
0.013 to 0.0311 µmol/mL than N-Boc protected L-Leucine linked alkyne 3 with MIC: 0.1859 and
0.0929 µmol/Ml for A. niger and C. albicans, respectively as compared to the Fluconazole
(reference drug) with MIC value of 0.0102 µmol/mL [47]. In case of both fungal strains A. niger
and C. albicans, compounds 4c and 4h with MIC 0.013 and 0.0148 µmol/mL, respectively
exhibited almost comparable activity with the reference drug Fluconazole. Similarly, in the case
of C. albicans compound, 4c and 4i both having MIC values 0.013 µmol/mL are found to have a
considerable activity as compared to the reference drug Fluconazole.

Table 4. Antifungal in vitro activity results of N-Boc protected L-Leucine linked alkyne 3 and
corresponding 1,4-disubstituted 1,2,3-triazoles (4a-j) in MIC: µmol/mL.
Sr. No
Compound
R
---
A. niger
0.1859
0.0311
0.0297
0.0130
0.0279
0.0297
0.0260
0.0279
0.0148
0.0260
0.0279
0.0102
C. albicans
0.0929
0.0155
0.0148
0.0130
0.0139
0.0148
0.0130
0.0279
0.0148
0.0130
0.0279
0.0102
1
2
3
4a
4b
4c
4d
4e
4f
H
3
o-F
4
o-Br
o-NO2
m-F
5
6
7
m-Br
m-NO2
p-F
8
4g
4h
4i
9
10
11
12
p-Br
p-NO2
4j
Fluconazole
Figure 3. Antifungal activities of synthesized Leucine linked alkyne 3 & triazoles 4a-j.

3.3. Pharmacokinetics properties for drug-likeness
The drug like property of compounds depends on their absorption, distribution,
metabolism, excretion (ADME) in human body. All these processes correlate well with
pharmacokinetic properties such as molecular weight, TPSA, permeability, octanol-water
coefficient (logP) etc. In the present study, the pharmacokinetic property of N-Boc protected L-
Leucine linked alkyne 3 and its 1,2,3-triazole derivatives 4a-j were calculated using web tool of
Swiss ADME and are presented in Table 5 [48].
Table 5. Physicochemical data for drug-likeness score for synthesized N-Boc-L-Leucine linked
alkyne 3 and its 1,2,3-triazoles 4a-j calculated using a web tool of Swiss ADME.
Compound
MWa
HAb
19
29
30
30
32
30
30
32
30
30
32
RBc
9
HBAd HBDe
MRf
TPSAg MLogPh
WSi
GIAj
LVk
0
3
269.34
402.49
420.48
481.38
447.48
420.48
481.38
447.48
420.48
481.38
447.48
4
6
7
6
8
7
6
8
7
6
8
1
1
1
1
1
1
1
1
1
1
1
73.37
64.63
95.34
95.34
95.34
2.14
2.26
2.64
2.86
1.43
2.64
2.86
1.43
2.64
2.86
1.43
-2.61
86.70
4a
4b
4c
4d
4e
4f
12
12
12
13
12
12
13
12
12
13
109.41
109.37
117.11
-4.00 76.108
-4.17 76.108
-4.92 76.108
-4.08 60.300
-4.17 76.108
-4.92 76.108
-4.08 60.300
-4.17 76.108
-4.92 76.108
-4.08 60.300
0
0
0
118.23 141.16
1
109.37
117.11
95.34
95.34
0
0
4g
4h
4i
118.23 141.16
1
109.37
117.11
95.34
95.34
0
0
4j
118.23 141.16
1
amolecular weight, bheavy atoms, crotable bonds, dhydrogen bond acceptors, ehydrogen bond donors, fmolar
refractivity, gtotal polar surface area, hoctanol-water partition coefficient, iwater solubility, jgastrointestinal
absorbance, kLipinski's violations
The calculated physicochemical data of synthesized N-Boc protected L-Leucine linked alkyne 3
and its 1,4-disubstituted 1,2,3-triazoles 4a-j as presented in Table 5, showed that the low
molecular weight of drugs is the primary requirement of any drug to get absorbed in human body.
Compounds with higher molecular weights have fewer tendencies to cross the cell membrane and
are less likely to be absorbed in the body. Thus, they have fewer tendencies to reach the biological
target. Therefore, molecular weights of drugs should be less than equal to 500 (500). The
distribution, absorption and excretion of compounds depend upon their lipophilicity. The logP
value of a molecule is a log value of its partition coefficient between n-C8H17OH and H2O
log(coctanol/cwater). It is a measure of lipophilicity of any chemical. The smaller the value of logP

(5) more will be the absorption of drug and higher value of logP indicates their less metabolism
and fast excretion from the body. All our synthesized triazoles have logP value <5. The TPSA
which is abbreviated as topological polar surface area is another factor that correlates well with
properties like blood brain barrier penetration and intestinal absorption. If the total area of all the
polar atoms exposed to the surface of a molecule is less than 160 Å2, then the molecule is likely to
pass easily through the cell membrane. In our study, TPSA value of all synthesized compounds
lies between 64.63-141.16, which shows their permeation and good absorption (% ABS= 66.2998-
76.1077). The % absorption (% ABS) can be calculated by % ABS = 109-(0.345 × TPSA) formula.
The water solubility (WS) of any drug also determines its absorption and distribution properties.
Almost 80% of the explored drugs highlighted logS value more than -5. Molecules under study
showed WS in a range of -2.61 to -4.92. The number of H-bond donor and acceptor of a good drug
must have cut off value 5 and 10, respectively. All the synthesized compounds (3 and 4a-j)
showed good permeation and absorption, as indicated by unity value of H-bond donors for all
compounds and H-bond acceptors value for alkyne is 4 and for triazoles it lies between 6-8 which
are less than their cut off value i.e. 5 and 10, respectively. It was observed that the majority of
orally active drugs (almost 90%) obeys Rule-of-five proposed by Lipinski [49]. The parameters of
all the tested compounds (3 and 4a-j) were found within the limits of the rule. According to this
rule, the MW of any ideal drug must be less than and equal to 500 (i.e.  500), the octanol-water
partition coefficient (LogP) must be  5, H-donors  5 and H-acceptors 10 and number of
violations to this rule should be 1. All the synthesized molecules fairly obey this rule-of-five and
these bioactive molecules have good drug like property.
As a general rule, if the bioactivity score of any drug is > -0.5 then the compound is active
and if the bioactivity score is < -0.5 then the compound is inactive. The bioactivity score of N-Boc
protected Leucine linked alkyne 3 and its 1,2,3-triazoles 4a-j as per the data given in the Table 6,
showed that the synthesized compounds are active with bioactivity score > -0.5.

Table 6. Bioactivity score of N-Boc protected Leucine linked alkyne 3 and 1,2,3-triazoles 4a-j by
Molinspiration Cheminformatics software [50].
Compound
R
----
GPCRa
0.29
0.28
0.28
0.12
0.16
0.28
0.18
0.12
0.28
0.19
0.13
ICMb
0.43
0.26
0.21
0.14
0.21
0.24
0.17
0.17
0.23
0.18
0.18
KIc
NRLd
0.14
PIe
EIf
3
-0.45
-0.31
-0.30
-0.39
-0.40
-0.27
-0.36
-0.40
-0.28
-0.34
-0.40
0.66
0.55
0.52
0.38
0.38
0.53
0.41
0.37
0.51
0.43
0.37
0.40
0.32
0.29
0.24
0.18
0.30
0.24
0.18
0.29
0.25
0.19
4a
4b
4c
4d
4e
4f
H
-0.12
-0.11
-0.19
-0.23
-0.09
-0.23
-0.20
-0.10
-0.21
-0.20
o-F
o-Br
o-NO2
m-F
m-Br
m-NO2
p-F
4g
4h
4i
p-Br
4j
p-NO2
aG-protein coupled receptor; bion channel modulator; ckinase inhibitor; dnuclear receptor ligand; eprotease inhibitor;
fenzyme inhibitor
3.4. Molecular docking studies
The docking studies of heterocycles like triazoles were performed and studied by choosing
bacterial enzyme DNA gyrase of E. coli (topoisomerase II, PDB ID: 1KZN). The present results,
considered the docking studies of N-Boc-L-Leucine linked alkyne 3 and its most active triazole 4h
on the suitably active sites of DNA gyrase enzyme of E. coli bacteria by making use of Autodock
Vina [51]. The three-dimensional (3D) structures of alkyne 3 and most active triazole 4h were
generated by Marvin sketch [52]. The binding affinity of alkyne 3 and its most active triazole 4h
showing very good conformations as shown in Figure 5 and 6. The ≡CH of alkyne 3 was involved
in p-alkyl interaction with ILE A:78 as highlighted by the pink line in dotted form in Figure 5a.
The alkyl group of leucine in alkyne 3 was also involved in alkyl interaction with ILE A:78. The
carbon of 3 was found to be involved in carbon-hydrogen interaction with ASP A:73. The carbonyl
oxygen of ester in alkyne 3 showed prevalent H-bond interaction with GLY A:77. But, the
carbonyl oxygen of carbamate in 3 exhibited prevalent H-bond association with ARG A:136. The
ether oxygen of carbamate in alkyne 3 exhibited prevalent H-bond interaction with ARG A:76.
The highest stable conformation of alkyne 3 anchoring with interacting residues is shown in
Figure 5b. However, the ether oxygen of ester of leucine moiety of compound 4h showed Van
der Waals interaction with PRO A:79. The alkyl group of leucine moiety of 4h showed alkyl

interaction with ILE A:90. The triazole ring of 4h showed pi-anion interaction with GLU A:50
and pi-alkyl interaction with ILE A:78. In triazole 4h benzene ring exhibited p-alkyl association
with ALA A:47 also pi-sigma association with THR A:165. The CH2 of benzyl moiety of 4h
exhibited C and H-bond interaction with ASN A:46. The F-atom substitution on benzyl moiety of
the triazole 4h exhibited conventional H-bond interaction with VAL A:167 and halogen interaction
with VAL A:71. The Boc-Leucine linked 1,2,3-triazole 4h demonstrated a high binding energy of
-8.2 kcal/mol as compared to its alkyne precursor 3 (-6.6 kcal/mol). It was therefore revealed that
the incorporation of a triazole moiety into its corresponding alkyne improves the antibacterial
activity of the resulted hybrid compound. The most stable conformation of N-Boc protected
Figure 5a-b. Binding interactions of Boc-Leucine linked alkyne 3 docked with E. coli bacterial
enzyme (DNA gyrase, topoisomerase II, PDB ID: 1KZN).

leucine linked 1,4-disubstituted 1,2,3-triazole 4 anchoring with interacting residues is shown in
Figure 6b and both the structures in Figure 5b and 6b were generated by making use of discovery
studio visualizer [53].
Figure 6a-b. Binding interactions of Boc-Leucine linked 1,2,3-triazole 4h docked with E. coli
bacterial enzyme (DNA gyrase, topoisomerase II, PDB ID: 1KZN).

3.5. DFT study
The Density Functional Theory (DFT) calculations on alkyne derivative of N-Boc
protected L-Leucine i.e. 2-tert-butoxycarbonylamino-4-methyl-pentanoic acid prop-2-ynyl ester 3
and N-Boc protected Leucine-linked 1,4-disubstituted 1,2,3-triazoles (4a-j) were performed with
the help of B3LYP at 6-311G(d,p) level of theory using a Gaussian 09 package [54]. For
convenience, HOMO which is abbreviated as highest occupied molecular orbital and its counter
lowest unoccupied molecular orbital (LUMO) of alkyne 3 along with representative and most
active triazole 4a and 4h, respectively for easy reference are presented in Figure 7. However,
complete reference to the HOMO and LUMO diagrams of alkyne 3 along with its respective N-
Boc protected Leucine-linked 1,2,3-triazoles (4a-j) are given in Figure, SI-1. It is known that
frontier orbitals are the important parameter of any chemical compound to design and ascertain
their reactivity including pharmacological properties [55-56]. The smaller value of the energy gap
(ΔELUMO-HOMO) indicated easier electron transfer between them and the higher ΔELUMO-HOMO
revealed better electron-donating and accepting nature, respectively. The calculated energy gap
ΔELUMO-HOMO valuesfor alkyne 3 and all triazole compounds are presented in Table 7. The HOMO
and the LUMO energies of N-Boc-Leucine-linked alkyne 3 and its triazoles (4a-j) were compared
to explore the pharmacological activity of triazoles with respect to alkyne, whereby it was observed
that the band gap energy of all triazoles 4a-j (3.95-6.03 eV) is less than alkyne 3 (7.07 eV). The
HOMO (3)
LUMO (3)

HOMO (4a)
LUMO (4a)
HOMO (4h)
LUMO (4h)
Figure. 7 HOMOs and LUMOs of alkynes 3 and triazoles 4a & 4h.
lower value of triazoles than alkyne showed that incorporation of a triazole unit on to the alkyne
lowers the band gap energy (ΔELUMO-HOMO) between respective frontier molecular orbitals (FMOs)
and enhances the chemical reactivity and thus enhances the biological activity of the triazoles as
compared to the respective alkyne 3 [57]. Also, with the help of chemical reactivity parameters i.e.
electronic chemical hardness (η), chemical potential (μ) and electrophilicity (ω), the correlation of
pharmacological properties was explored. Wherein, it was observed that all triazoles 4a-j with
lesser chemical hardness values (1.98-3.08 eV) than alkyne 3 (3.54 eV) are softer, hence more
reactive. Likewise, the electronic chemical potential (μ) which can be described as the -ive of the
electronegativity showed that electronic potential of all the triazoles ranges from -3.92 to -5.01 eV
and that of alkyne is -3.61 eV [58]. However, the electrophilicity index (ω) of all triazoles showed
values from 2.50-6.34 eV, which is higher than value of alkyne (1.84 eV). Overall, it highlighted
that the introduction of 1,2,3-triazole moiety in alkyne lowers the ΔELUMO-HOMO along with lower
chemical hardness (η) and higher chemical potential (μ) and electrophilicity index (ω) of a
molecule.

Table 7. Energy of HOMO and LUMO, energy gap ΔELUMO-HOMO, chemical hardness (η), chemical
potential (μ), and electrophilicity index (ω) of alkyne 3 and its 1,2,3-triazoles 4a-j.
ELUMO
Compound EHOMO (eV)
ΔELUMO-HOMO (eV)
η (eV)
μ (eV)
ω (eV)
(eV)
-0.07
-0.84
-1.16
-1.23
-3.03
-1.06
-1.16
-2.86
-1.01
-1.16
-2.69
3
-7.15
-7.00
-6.96
-6.99
-6.98
-7.05
-7.06
-7.15
-7.04
-7.05
-7.13
7.07
6.15
5.81
5.76
3.95
5.99
5.90
4.28
6.03
5.90
4.44
3.54
3.08
2.90
2.88
1.98
3.00
2.95
2.14
3.02
2.95
2.22
-3.61
-3.92
-4.06
-4.11
-5.01
-4.05
-4.11
-5.00
-4.02
-4.10
-4.91
1.84
2.50
2.84
2.93
6.34
2.74
2.86
5.85
2.69
2.86
5.42
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4. Conclusion
A library of N-Boc L-Leucine-linked 1,2,3-triazoles was synthesized in high yield via
CuAAC reaction and all were characterized by FT IR, 1H NMR, 13C NMR, and MS data. The in
vitro antibacterial and antifungal activity screening results of Boc protected Leucine-linked alkyne
3 and its 1,2,3-triazoles 4a-j showed that incorporation of triazole unit enhances the biological
activity of the alkynes and among all N-Boc-Leucine linked 1,2,3-triazoles 4a-j, compound 4h
found to be more potent than the reference antibacterial drug Ciprofloxacin against all tested
bacterial strains S. entrica, B. subtilis, S. aureus, E. coli and P. auroginosa. Also, antifungal
activity was found comparable with MIC: 0.0148 µmol/mL to normally used antifungal drug
Fluconazole (MIC: 0.0102 µmol/mL) against A.niger and C.albicans fungal strains. The molecular
docking study of alkyne 3 and its most active 1,2,3-triazole 4h on DNA Gyrase of E. coli
(topoisomerase II, PDB ID: 1KZN) revealed better binding with triazole 4h than precursor alkyne
3 which was supported by DFT study using B3LYP at 6-311G(d,p) basis set.

Conflict of Interest
The authors declare no competing financial interest.
Acknowledgement
Authors thank Dr. Anil Kumar (Deptt. Bio & Nano Tech., GJUS&T, Hisar, Haryana, India) for
extending facilities for pharmacological studies.
Appendix A. Supplementary data
The supplementary material of this article can be found online, at:
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