Effect of novel triazole-amino acid hybrids on growth and virulence of Candida species: in vitro and in vivo studies

Article abstract of DOI:10.1039/c6ob01718e

The increasing incidence of human candidiasis and the tendency of Candida species to become resistant to existing chemotherapies are well-recognized health problems. The present study demonstrates the successful synthesis of novel triazole-amino acid hybrids with potent in vitro and in vivo inhibitory activity against Candida species. Particularly, compounds 68 and 70 showed potent in vitro activity against fluconazole (FLC) resistant as well as sensitive clinical isolates of Candida albicans. Time kill curve analysis of lead inhibitors 68 and 70 showed their fungistatic nature. Secretion of hydrolytic enzymes, mainly proteinases and phospholipases, decreased considerably in the presence of 68 and 70 indicating their interference in fungal virulence. TEM analysis of Candida cells exposed to compounds 68 and 70 clearly showed morphological changes and intracellular damage as their possible mode of action. A preliminary mechanistic study carried out on the two most effective inhibitors (68 and 70) revealed the inhibition of ergosterol biosynthesis thereby causing the cells to lose their integrity and viability. The selected compounds did not show significant cytotoxicity up to a concentration of 200 μg mL^?1 in the HEK293 cell line. An in silico analysis of 68 and 70 binding to a modeled C. albicans CYP51 showed critical H-bonding as well as hydrophobic interactions with the important active site residues indicating the basis of their anti-Candida role. Studies on the larvae of Galleria mellonella showed that the selected inhibitors (68 and 70) were non-toxic, did not provoke an immune response and significantly reduced Candida proliferation in vivo.

Full text of DOI:10.1039/c6ob01718e

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ARTICLE
Effect of novel triazole-amino acid hybrids on growth and
virulence of Candida species: In vitro and in vivo studies
Received 00th January 20xx,
Accepted 00th January 20xx
a,b,‡
a,c,‡
d
d
e
Babita Aneja , Mohammad Irfan , Charu Kapil , Mohamad Aman Jairajpuri , Ronan Maguire ,
e
f
c,†,
b
a,†,
DOI: 10.1039/x0xx00000x
Kevin Kavanagh , M. Moshahid A. Rizvi , Nikhat Manzoor *, Amir Azam , Mohammad Abid *
www.rsc.org/
The increasing incidence of human candidiasis and its tendency to become resistant to existing chemotherapies is a well-
recognized health problem. The present study demonstrates the successful synthesis of novel triazole-amino acid hybrids
with potent in vitro and in vivo inhibitory activity against Candida species. Particularly, compounds 68 and 70 showed
potent in vitro activity against fluconazole (FLC) resistant as well as sensitive clinical isolates of Candida albicans. Time kill
curve analysis of lead inhibitors 68 and 70 showed their fungistatic nature. Secretion of hydrolytic enzymes, mainly
proteinases and phospholipases, decreased considerably in presence of 68 and 70 indicating their interference in fungal
virulence. TEM analysis of Candida cells exposed to compounds 68 and 70 clearly showed morphological changes and
intracellular damage as their possible mode of action. A preliminary mechanistic study carried out on the two most
effective inhibitors (68 and 70) revealed inhibition of ergosterol biosynthesis thereby causing the cells to lose their
integrity and viability. Selected compounds did not show significant cytotoxicity up to a concentration of 200 µg/mL on
HEK293 cell line. An in silico analysis of 68 and 70 binding to a modeled C. albicans CYP51 showed critical H-bond as well as
hydrophobic interactions with the important active site residues indicating the basis of their anti-Candida role. Studies on
the larvae of Galleria mellonella showed that the selected inhibitors (68 and 70) were non-toxic, did not provoke an
immune response and significantly reduced Candida proliferation in vivo.
antibiotics, suppression of immune response during organ and
bone marrow transplantation, cancer treatment and AIDS are
increasing the chances of Candida infections thus, further
Introduction
Over the past three decades, the frequency of fungal
infections in human has increased dramatically worldwide
despite extensive efforts to the discovery of novel antifungal
6
aggravating the condition. Among many Candida species, C.
albicans is a prominent Candida species which is responsible
1
-3
for major cause of candidiasis and isolated from approximate
7
0% cases of candidemia. However, number of infections
agents and the availability of various drugs. Of particular
concern is the severity associated with the ever-increasing
incidences of hospital-acquired systemic mycoses, caused by
various species of Candida, responsible for the crude mortality
8
caused by other non-Candida albicans Candida species (NCAC)
which includes C. glabrata, C. tropicalis and C. parapsilosis has
8
4
also increased.
rate of up to 50% in the United States alone. Adding to this
During both superficial and systemic infection, pathogenicity
of Candida species relies on a number of virulence factors
including the capability to adhere to the surface that assist
them to colonize the host. Adhesion is mainly influenced by
the profile of cell wall proteins and cell surface
disease burden, superficial infections of skin and nails in
humans are affecting ~25% of the general population
5
worldwide. It has been reported that use of broad-spectrum
9
physicochemical properties. In addition to this, they also
adhere to the surface of medical devices and form biofilms
which limit the penetration of substances through the matrix
and decreases the susceptibility of Candida to antifungal
1
0
therapy. Candida also secrete different hydrolytic enzymes
like proteinases, phospholipases and hemolysins and acquire
nutrients and dissemination within the host. They also
modulate host immune response and cause the destruction of
11
host tissues.
Treatment of Candida infections relies on a limited number of
chemotherapeutic agents including polyenes, azoles,
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1
echinocandins, allylamines and flucytosine. Among them, 1,2,3-triazoles bearing phenylalanine (68) and tryptophan tails
2
azoles such as fluconazole (FLC), itraconazole, voriconazole, (70) as potent antifungals against FLC sensitive and resistant
posaconazole, ketoconazole, miconazole and econazole (Fig. 1) clinical isolates of C. albicans. Moreover, compounds with
are widely used for the treatment of both superficial and comparatively lower IC50 values were also subjected to cell
invasive fungal infections due to their favourable proliferation assay on HEK293 (Human Embryonic Kidney) cell
6
pharmacokinetics and safety profile. Azoles target the line and found non-toxic up to 200 µg/mL concentration.
biosynthesis of ergosterol, a major component of fungal Various assays for growth (time kill curve) and virulence
membrane, by inhibiting the cytochrome P450-dependent (proteinase and phospholipase secretion) were performed on
enzyme lanosterol 14α-demethylase (CYP51). The depletion of Candida cells, exposed to lead inhibitors (68 and 70) also
ergosterol and accumulation of toxic methylsterols in showed promising results. A detailed study of lead compounds
membrane results in alteration of membrane fluidity along
(68 and 70) on the larvae of Galleria mellonella further
with disruption of activity of membrane-bound enzymes confirmed their non-toxic behavior and a significant reduction
1
3
leading to the inhibition of fungal growth and replication.
in Candida proliferation in vivo. Because of the importance of
Although azoles have an advantage of oral administration, CYP51 in the antifungal drug studies, molecular docking was
they lack in fungicidal effects as well as in possessing limited performed for the most active compounds against the
selectivity for fungal demethylase enzyme over mammalian predicted three-dimensional model of CYP51 from C. albicans
1
4
cytochrome P450s thus posing serious side effects.
Moreover, the increased use of azole antifungals has modes of the compounds to the target enzyme. Our study
contributed to the emerging resistance in Candida species revealed that the triazole-amino acid hybrids (68 and 70
in order to shed light on the mechanism and plausible binding
)
which is prompting the medicinal chemists worldwide to provide a suitable core to exploit for lead optimization to
develop novel and more effective antifungal agents with a develop potent antifungal agents.
broad spectrum, better pharmacokinetic profile and low
1
5
toxicity.
Results and discussion
Chemistry
The multistep synthetic approach was adopted for the
preparation of the title compounds (65-88) as given in Scheme
1. Substituted benzylamines (17-24) were obtained via two-
step sequence involving oximation of substituted aryl aldehyde
1
1-8) followed by Zn/HCl mediated reduction. Then, in
8
(
another set of reaction azidopropionic acid, 26 was obtained
from 3-bromopropionic acid, 25 by direct azide replacement
using sodium azide in anhyd DMF at 85
azidopropionic acid, 26 with substituted benzylamines 17-24 in
the presence of 1-ethyl-3-(3-dimethylaminopropyl)
°C. Coupling of
carbodiimide (EDC.HCl) and 1-hydroxybenzotriazole (HOBt) in
acetonitrile yielded corresponding azide intermediates 27-34
in moderate yield. In a separate set of reaction, boc-protected
L-amino acids (35-37) were treated with propargyl bromide in
the presence of K
in excellent yield. Finally, the 1,2,3-triazole-amino acid hybrids
41-64) were synthesized via Huisgen 1,3-dipolar cycloaddition
2 3
CO to provide alkyne intermediates (39-40)
Fig. 1 Structure of common azole-based antifungal drugs
(
Previously, it was reported that triazole-amino acids clubbed reaction between alkynes 39-40 and azides 27-34 in the
with indole moiety have potent antifungal effects as it caused presence of copper sulphate and sodium ascorbate in tert-
detrimental effect on sterol biosynthetic pathway of C. butanol:water (1:2) mixture. The key intermediates, 41-64
albicans thus induced cell wall defects leading to cell death in were finally deprotected utilizing p-toluenesulfonic acid in
1
6
combination with currently available azole antifungals. On dichloromethane to yield title compounds 65-88 in high yield
the basis of these findings on triazole-amino acid scaffolds and (Table 1).
in continuation to our efforts for the development of
1
7-19
antimicrobial agents,
we report herein the synthesis of a
series of novel triazole-amino acid hybrids (65-88) and their
screening against three species of Candida (C. albicans, C.
glabrata and C. tropicalis). This leads to the identification of
2
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Br
OH
N
3
OH
c
O
O
26
25
O
O
NOH
H
NH2
H
d
H
a
b
N
N
3
H
Boc
NH
O
R
R
R
1-8
R
9-16
17-24
27-34
f
N
N
N
O
R'
H
N
O
R'
R'
Br
R
OH
41-64
O
BocHN
BocHN
e
O
O
R
R'
g
3
5-37
38-40
1
H
35
O
2
H N
2
3
4-F
N
N
O
R'
4-Cl
2-Cl
4-Me
H
N
N
O
4
5
36
R
N
65-88
6
7
4-OMe
4-CF3
37
N
8
4-OCF3
H
Reagents and conditions (a) NH
2
OH.HCl, Pyridine/ethanol (1:10), reflux; (b) Zn, HCl, ethanol, rt; (c) NaN
3
, DMF, reflux; (d) HOBt, EDC.HCl, NMM, CH
3
CN, rt; (e) Propargyl bromide,
K
2
CO , DMF,rt; (f) CuSO .5H O, sodium ascorbate, tert-butanol:water (1:2), rt; (g) pTSA, DCM, rt
3
4
2
Scheme 1. Synthesis of triazole-amino acid hybrids
Table 1. Structure of triazole-amino acid hybrids
Benzylamine
L-Phenylalanine
L-Proline
L-Tryptophan
O
O
O
NH2
O
O
O
N
H
N
N
N
H
N
N
N
O
O
O
H
N
N
NH2
N
N
N
NH2
HN
NH
NH
6
5
66
67
O
O
O
NH
2
O
O
O
N
H
N
N
H
N
N
N
N
O
O
O
H
O
F
N
N
N
N
N
NH2
F
NH
2
F
F
HN
7
0
6
8
69
O
O
O
NH2
O
O
O
N
N
N
N
N
N
N
N
O
O
H
O
H
Cl
H
N
N
NH2
N
NH2
Cl
N
Cl
NH
HN
Cl
7
1
73
76
7
2
O
O
O
NH2
O
O
N
H
N
N
N
H
N
N
N
N
O
O
O
Cl
H
N
NH2
N
N
Cl
N
HN
Cl
NH
2
NH
Cl
7
4
75
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O
O
O
NH
2
O
O
O
N
N
N
N
H
N
N
N
N
O
O
O
Me
H
H
N
NH2
NH2
NH2
N
N
NH2
Me
N
HN
Me
NH
Me
7
7
78
81
79
O
O
O
O
NH
2
O
O
O
N
N
N
H
N
N
N
O
O
O
H
H
MeO
N
N
N
N
N
MeO
MeO
HN
N
NH2
MeO
F3C
NH
NH
8
0
8
2
O
O
NH
2
O
O
O
N
N
N
H
N
N
N
N
O
H
O
F
3
C
H
O
N
N
N
N
NH
2
F
3
C
N
HN
3
F C
8
3
85
8
8
4
7
O
O
O
NH
2
O
O
O
N
N
N
N
N
N
N
O
O
O
H
F
3
CO
H
H
N
N
NH2
N
NH2
N
F
3
CO
NH
F
3
CO
N
HN
F
3
CO
8
8
8
6
4
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In vitro anti-Candida activity
Candida sp. Fungistatic or fungicidal nature of lead compounds (68
and 70) was determined by time kill curve studies on FLC-sensitive
as well as resistant strains of C. albicans. Both compounds showed
fungistatic effect on the growth of C. albicans with an efficacy much
superior to the conventional standard drug fluconazole. Further, no
significant changes in growth were observed at concentrations
lower than 120 µg/mL of compounds 68 and 70 (Fig. 2).
We generated a library of 24 compounds that features the 1,2,3-
triazole core and an amino acid (Phe/Pro/Trp) as a tail and screened
all the analogues for in vitro anticandidal activity. Preliminary
studies were done on three different Candida species: C. albicans,
C. glabrata and C. tropicalis.to see the effect of these analogues on
the growth of Candida cells in vitro (Table 2). Fluconazole was used
as positive control. The unsubstituted and para-halogen substituted
benzyl compounds with L-phenylalanine tail showed modest activity
(
70.9-179.4 µg/mL) against most pathogenic C. albicans ATCC
0028. Compound 68 with p-fluoro substitution and phenylalanine
9
tail emerged as most potent (IC50 = 70.9 µg/mL) among all in the
series as selective inhibitor of C. albicans ATCC 90028 strain. Chloro
substitution ortho to benzyl group (74) resulted in the decrease of
activity (340.0 µg/mL) in the same strain. Although no clear SAR was
observed in case of analogues with L-proline tail. Moreover,
compound 66 bearing unsubstituted benzyl group and L-proline tail
exhibited IC50 value of 203.5 µg/mL in C. glabrata ATCC 90030
strain. It was found that unsubstituted and para substituted triazole
analogues with tryptophan tail showed best anticandidal activity
against C. albicans ATCC 90028 strain. Compound 70 with p-fluoro
substitution emerged as most potent anticandidal agent (IC50 = 22.4
µg/mL) as compared to 67, with no substitution, having IC50 = 98.8
µg/mL against C. albicans ATCC 90028 strain. It also inhibited the
growth of C. glabrata with IC50 of 163.2 µg/mL. The p-methyl (79,
IC50 = 168.7 µg/mL) and p-methoxy (82, IC50 = 163.9 µg/mL)
substitution were well tolerated as compared to p-trifluoromethyl
(A)
(
85, IC50 = 282.6 µg/mL) and p-trifluoromethoxy (88, IC50 = 1142.7
µg/mL) substitution where the activity was completely lost against
C. albicans ATCC 90028 strain. Based on the results obtained with
standard Candida strains, seven compounds (67, 68, 70, 71, 73, 79
and 82) were selected for further investigation against both FLC-
sensitive and resistant clinical C. albicans strains. Compounds with
p-fluoro substitution with phenylalanine (68) and tryptophan (70)
tails emerged as most potent inhibitor of clinical strains too.
Compound 68 showed moderate inhibitory effect on clinically
isolated C. albicans with IC50 in the range 154.9-188.3 µg/mL.
Similarly, compound 70 also showed inhibition of clinically isolated
Candida cells with IC50 in the range 149.63-176.1 µg/mL. Both
compounds, 68 and 70 showed potent inhibitory activity against
FLC-resistant C. albicans with IC50 values of 671.08 and 634.98
µg/mL, respectively which otherwise is tolerant to FLC (IC50 > 1000
(B)
µg/mL) (Table 3). The results prompted us to further explore the Fig. 2 Time kill curves of A) fluconazole sensitive C. albicans ATCC 90028; B) fluconazole
resistant C. albicans at various concentrations of 68 and 70. The results indicate fungi-
effect of these two lead inhibitors (68 and 70) on the growth of
static nature of these compounds.
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Table 2. In vitro anticandidal activity of compounds 65-88 (IC50 in µg/mL) against standard strains of Candida
1
Comp.
C. alb.
C. gla.
C. tro.
C. alb.
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
179.4
473.8
98.8
506.5
203.5
944.1
1352.0
912.2
163.2
1065.3
267.4
975.5
383.6
247.5
684.3
851.3
326.9
1074.0
1121.1
265.6
427.2
371.1
268.3
272.4
1103.7
1006.4
1081.6
7.8
364.7
741.5
475.7
503.0
678.9
349.0
389.4
927.1
556.4
421.4
852.0
542.3
497.7
684.3
618.3
447.8
571.2
423.6
407.6
468.0
662.3
923.8
971.4
991.0
8.5
377.0
541.3
303.7
335.6
369.3
269.5
314.9
351.8
313.3
303.5
333.8
378.7
300.3
636.5
379.0
292.7
562.3
340.9
310.0
580.3
321.3
747.2
966.0
998.8
n.d.
70.9
326.3
22.4
164.1
420.2
161.0
340.0
376.0
227.1
294.4
478.7
168.7
300.2
554.0
163.9
315.3
526.0
282.6
865.4
985.0
1142.7
15.6
FLC
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1
Abbreviations: C. alb. = C. albicans ATCC 90028; C. gla.= C. glabrata ATCC 90030; C. tro.= C. tropicalis ATCC 750; C. alb.= C. albicans ATCC 1026; n.d. = not done; NA =
not active; FLC = fluconazole
Table 3. In vitro anticandidal activity of selected potential compounds against clinical isolate of C. albicans
†
Compound
*C. alb.
*C. alb.
*C. alb.
C. alb.
6
6
7
7
7
7
8
7
8
0
1
3
9
2
192.86
154.92
149.63
387.90
237.52
563.93
297.62
<7.81
343.63
188.37
153.19
206.48
358.43
249.64
199.52
<7.81
322.05
183.13
176.10
368.69
344.70
258.77
341.76
<7.81
>1000
671.08
634.98
>1000
>1000
>1000
>1000
>1000
FLC
Abbreviations: *C. alb. = C. albicans (fluconazole sensiꢀve); †C. alb. = C. albicans (fluconazole resistant).
Extracellular hydrolytic enzymes (e.g. phospholipases and
proteinases) are potent virulence factors which play an
2
0
important role in the establishment of Candida infection. To
investigate whether the lead compounds (68 and 70) have any
effect on the extracellular secretion of these enzymes, the
inhibition assay was performed using standard as well as
resistant C. albicans strains. Results showed that on treatment
with 120 µg/mL of compound 68, proteinase secretion was
decreased by 29, 22 and 23.5% in standard, FLC-sensitive and
resistant strains of C. albicans, respectively while at the same
concentration, the compound 70 decreased proteinase
secretion by 30, 33 and 17% against the respective strains.
Similar results were obtained at 60 µg/mL concentration of
both compounds, 68 and 70 with no significant differences in
inhibition of proteinase secretion (Fig. 3A). At similar
concentrations, compound 68 caused 40, 38, and 38%
inhibition in phopholipase secretion whereas compound 70
decreased secretions respectively by 36, 27 and 38% in
standard, FLC-sensitive and resistant strains of C. albicans (Fig.
(A)
3B). Our results suggest that both compounds (68 and 70)
significantly reduce the secretion of proteinases and
phospholipases in Candida spp. which are vital to fungal
invasion of host tissues and immunosuppression.
(B)
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Fig. 3 Effect of compounds 68 and 70 on A) Proteinase secretion and; B) Phospholipase
secretion. These hydrolytic enzymes act as potent virulence factor and play an
important role in establishment of Candida infection. Higher Pz value indicates the
lower secretion of enzyme. At 125 µg/mL concentration, compounds 68 and 70
decrease 17 to 38% secretion of these enzymes.
6
0 µg/mL and 48% at 120 µg/mL of 70. However, the decrease
in total ergosterol content for FLC- resistant C. albicans was 7%
at 30 µg/mL, 80% at 60 µg/mL and 91% at 120 µg/mL of 70. In
the presence of 100 µg/mL of FLC, the decrease in ergosterol
content was 25% and 28%, respectively for standard and FLC-
resistant C. albicans (Fig. 5). Our results suggest that the
compounds 68 and 70 inhibit the total ergosterol content
more effectively in comparison to FLC for all the Candida
strains tested here. In conclusion, the lead compounds (68 and
The effect of lead compounds (68 and 70) on the morphology
of C. albicans was monitored by transmission electron
microscopy (TEM) which led to the exploration of the possible
antifungal mechanism of action. Candida cells treated with 120
µg/mL of 68 and 70 and untreated control cells were used for
TEM analysis. It was observed, as shown in Fig. 4, that
untreated cells are normal in shape with uniform cell density
and intact cell wall while treated cells showed several
deformations. On exposure to 68 or 70, significant damage
could be seen in Candida cells wherein the cell shape became
abnormal and the cell wall lost its integrity becoming irregular
and ruptured. The intracellular organelles disappeared
indicating towards necrotic mode of cell death. Thus, we can
conclude from the results of TEM analysis that the lead
compounds (68 and 70) may be interfering with biosynthesis
of cell membrane.
70) caused ergosterol depletion resulting in the disruption of
the cell membrane which is consistent with the results of TEM
analysis.
(A)
Fig. 4 Representative transmission electron micrographs of C. albicans ATCC 90028 cells
exposed to 68 and 70 at their respective MICs. The figure showed significant damage to
Candida cell. The shape of the cell became abnormal and cell wall lost its integrity,
became irregular and ruptured.
It is well known that azole drugs like FLC inhibit ergosterol
biosynthesis destroying both membrane integrity and function
of some membrane-bound proteins and therefore affecting
2
1
fungal cell growth and proliferation. The effect of the lead
compounds (68 and 70) was studied on ergosterol biosynthesis
in C. albicans to further support our suggestion that these
(B)
Fig. 5 UV spectrophotometric sterol profiles of A) fluconazole sensitive standard C.
albicans ATCC 90028 and; B) fluconazole resistant C. albicans. At 120 µg/mL, the mean
compounds interfere with biosynthesis of cell membrane. The decrease in total ergosterol content for standard as well as resistant C. albicans were
found in the range of 48-96%.
ergosterol content of exposed cells was estimated in FLC-
sensitive and resistant Candida cells by a method reported
The cytotoxicity of compounds with comparatively lower IC50 values
2
2
previously by Skaggs et al and FLC was used as a positive
control. The results showed a dose dependent decrease in
ergosterol content when cells were grown in varying
concentrations of 68 and 70 (Fig. 5). The mean decrease in
total ergosterol content for standard strains was found to be
(
65, 67-71, 73-74, 79, 82-83, 85) was checked by MTT assay using
HEK293 cell line (Fig. 6). It is evident from the results that all the
twelve selected compounds (65, 67-71, 73-74, 79, 82-83, 85)
displayed more than 85% cell viability at 50 µg/mL concentration.
Out of 12, eight compounds (67-71, 73, 79, 82-83) displayed more
than 70% cell viability even at a concentration of 200 µg/mL which
indicated non-cytotoxic nature of these compounds. On increasing
the concentration from 400 to 600 µg/mL for all the selected
compounds, the cells were found to be more than 50% viable. The
hemolytic assay was also performed with lead inhibitors 68 and 70
to exclude any possible toxicity on human RBCs. At 125 µg/mL, the
5% at 30 µg/mL, 27% at 60 µg/mL and 73% at 120 µg/mL of
compound 68. The mean decrease in total ergosterol content
for FLC-resistant C. albicans was found to be 2% at 30 µg/mL,
23% at 60 µg/mL and 79% at 120 µg/mL of compound 68.
Similarly, the mean decrease in total ergosterol content for
standard C. albicans was found to be 15% at 30 µg/mL, 37% at
8
| J. Name., 2012, 00, 1-3
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compounds 68 and 70 caused 10% and 15% cell lysis, respectively in cerevisiae. A structure based sequence alignment showed that
comparison to 36% cell lysis caused by FLC at the same CYP51 from S. cerevisiae has 65% sequence homology with CYP51
concentration (Fig. 7). More interestingly, compounds 68 and 70 of C. albicans (Fig. 8). Crystal structure of S. cerevisiae CYP51
were found negligibly toxic up to 500 µg/mL, with only 18% and (PDB.4LXJ) was used as a template to construct a model of C.
2
2% cell lysis, respectively.
albicans CYP51. The model was further refined using energy
minimization and molecular dynamics simulation. We generated 5
model structures for CYP51 from C. albicans and checked for their
Ramachandaran Plot values. Among the 5 models, model 3 showed
nearly 89% Ramachandaran value, careful analysis of the residues in
the disallowed region. Structural superimposition of model 3 with
its template 4LXJ, showed an RMSD value of 0.248 across 501 atom
pairs (Fig. 9A). The mode of binding of lead inhibitors 68 and 70
with modeled C. albicans like CYP51 was identified using molecular
docking with Autodock Vina and Ligplot (Fig. 9B-E). Since both the
lead compounds have azole group flanked by fluorobenzyl and
hydrophobic side chains (Phe in 68 and Trp in 70) we have also
done hydrophobic interaction analysis using LIGPLOT to better
characterize the interactions that leads to in vitro antifungal
activity. The results revealed that proline attached to azole showed
significantly less hydrogen bond and hydrophobic interactions and
the compounds were consequently less effective as inhibitors
(
Tables 1 and 2). Trptophan and phenylalanine based compounds
8 and 70 with a common terminal p-fluorobenzyl group (Table 1)
Fig. 6 Cell viability assay on HEK293 cell line
6
are shown to be more effective inhibitors. The center of the active
site is positioned between I and F helix with heme citing in the
center of the core. Docking studies showed that both 68 and 70
were bound very close to the heme group in the active site pocket
of CYP51 interacting with active site residues with high binding
affinities (68 = -10.1 kcal/mol and 70= -9.2 kcal/mol). A longer side
chain of the compound as compared to the fluconazole appeared to
be accommodated in the active site pocket (Fig. 9B and 9D).
Compound 68 formed H-bond with Ser506 and terminal p-
fluorobenzyl group was very close to the heme, residue S506 in β-
turn connecting β4-1 and β4-2 and interacted with the heme group.
A number of hydrophobic interactions were observed with Leu376,
Phe228, Tyr132, Ile131 and Thr122 (Fig. 9B and 9C). Terminal p-
fluorobenzyl group of the inhibitor was found to be extremely close
to the Tyr132 and Phe228 phenyl ring showing a possible π-π
stacking. Tyr132 has been found to play an important role in
flucanazole susceptibility of CYP51 with a fluconazole resistant
Fig. 7 Hemolytic assay for compounds 68 and 70 on human red blood cells (hRBCs).
2
4
isolate of C. albicans showing mutation in Tyr132. Lead compound
Modeling and in-silico docking
7
0 similarly showed hydrogen bonding with Asp214 and Glu194
Lanosterol 14-α demethylase enzyme (CYP51) is a member of with side chain of tryptophan at the entrance of the substrate
cytochrome P450 superfamily which catalyses the oxidative binding pocket. A number of important hydrophobic interactions
removal of 14-α methyl group from lanosterol in ergosterol were detected with Gln309, Ser222, Tyr221, Ala218, Phe213 and
biosynthesis pathway. Inhibition of CYP51 is shown to directly Ile197, all these residues are part of the helix F, helix I and FG loop
deplete ergosterol resulting in inhibition of fungal growth, making it in the active site pocket of the CYP51 (Fig. 9D and 9E). Several
2
3
the primary target for the azole based antifungal drugs. Our residues that are important in the active site were identified as
results has established that synthesis of ergosterol is inhibited common residues that showed interaction with both 68 and 70 and
which may cause deformation of cell membrane and its leakage interacted laterally and spanned that length of the active site close
indicating classical azole based effect on the fungal CYP51. CYP51 is to the heme group. N-terminal of the helix I together with BC loop
a membrane bound protein that is difficult to purify and structural forms the entrance of the substrate access channel and a strong
assessment is only possible by modelling closely related structures. interaction close to the substrate binding site might affect the
In this study, we have modeled C. albicans like CYP51 from S. architecture of the active site which can distort the specificity of the
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Journal Name
substrate. In the absence of crystal structure of CYP51 from C. based on the active site and optimization of the structure activity
albicans interacting with its natural substrate, the interaction with relationship.
model CYP51 provides a rationale for designing lead inhibitors
Fig. 8 Structure based sequence alignment of CYP51 from S. cerevisiae and CYP51 of C.
(A)
albicans
1
0 | J. Name., 2012, 00, 1-3
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ARTICLE
Binding energy: 68 = -10.1 kcal/mol; 70= -9.2 kcal/mol
Fig. 9 (A) Structural superimposition of model 3 with its template 4LXJ; (B) The plausible mode of binding of 68 in the active site of modeled CYP51; (C) LIGPLOT analysis of binding
of 68; (D) The possible mode of binding of 70 in the active site of modeled CYP51; (E) LIGPLOT analysis of binding of 70
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ARTICLE
In vivo assessment against Galleria mellonella
The insect immune response bears many similarities to the
innate immune response of mammals and as consequence
insects may be used to evaluate the virulence of microbial
pathogens or assess the toxicity of antimicrobial drugs. Larvae
of G. mellonella have been used previously to assess the
toxicity of novel antifungal compounds and results showed a
2
5
strong correlation with those obtained using mammals. In
vivo study on larvae of G. mellonella indicated significant
anticandidal potential of compounds 68 and 70. The viability of
larvae was not affected following administration of the
compounds up to a concentration of 2.5 mg/mL which showed
a non-toxic behaviour towards the larvae (Fig. 10A-C). Again, at
the same concentration no significant alteration in the
haemocyte density of larvae was found which indicated the
lack of an immune response (Fig. 10D). The administration of a
dose of 2.5 mg/mL of compounds 68 and 70 to larvae
previously infected with C. albicans reduced yeast replication
in vivo by 50% and 70%, respectively as measured at 24 h (Fig.
(B)
10E).
(C)
(A)
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1
H NMR chemical shifts (δ) values are reported in parts per
, δ 7.26 and
DMSO-d , δ 2.54). C NMR chemical shifts (δ) are reported in
million (ppm) relative to residual solvent (CDCl
3
1
3
6
3 6
ppm relative to CDCl , δ 77.16 and DMSO-d , δ 39.5 and
coupling constants (J) are expressed in Hertz (Hz). Mass
spectra were recorded on Waters Tandem Quadrupole
Detector (TQD) electron spray ionization mass spectrometer
and on Agilent RRLC MS 6320 Ion Trap spectrometer fitted
with an electrospray ionization (ESI) interface. Melting points
were measured on a digital Buchi melting point apparatus (M-
5
60) and are uncorrected. The specific rotation of 41-64 was
recorded on Anton Paar MCP-100 digital polarimeter at 20
°
C
using sodium D light at a conc. of 1g/100 mL in ethanol.
Purification of the compounds was carried out by silica gel
column chromatography (230-400 mesh size, Merck, Germany)
with the indicated eluent.
(D)
General procedure for the synthesis of oximes (9-16). Example:
Synthesis of 4-fluorobenzaldehyde oxime (10)
4
-Fluorobenzaldehyde oxime was synthesized by reacting 4-
fluorobenzaldehyde (2) (12 mmol, 1.29 mL) with
hydroxylamine hydrochloride (13 mmol, 0.90 g) in ethanol (20
mL) and pyridine (2.0 mL) mixture under reflux for 21-23 h.
After cooling, solvent was evaporated under vacuo. and water
was added to this residue. The mixture was stirred in an ice
bath until the oxime crystallizes. The solid was filtered and
washed with water. The product was crystallized from ethanol.
1
8
Benzaldehyde oxime (9). Colorless oil, yield 80%. R
f
= 0.58
) δ: 8.67 (s,
H), 8.17 (s, 1H), 7.59-7.56 (m, 2H), 7.38 (t, J = 3.3 Hz, 3H). IR
1
(
20% EtOAc in hexane). H NMR (300 MHz, CDCl
3
1
-1
νmax (neat)/cm 3275 (O-H), 1640 (C=N) and 952 (N-O).
4
f
-Fluorobenzaldehyde oxime (10). White solid, yield 84%. R =
(E)
1
0.50 (20% EtOAc in hexane). mp 86-88
°C. H NMR (300 MHz,
Fig. 10 Percentage viability of Galleria mellonella in the presence of (A) 68; (B) 70; (C)
CDCl
m, 2H). IR νmax (neat)/cm 3253 (O-H), 1689 (C=N) and 963 (N-
O).
3
) δ: 8.55 (s, 1H), 8.13 (s, 1H), 7.58-7.53 (m, 2H), 7.10-7.05
DMSO; (D) Haemocyte densities of larvae and (E) fungal load assay
-1
(
4
f
-Chlorobenzaldehyde oxime (11). White solid, yield 89%. R =
Experimental section
1
0.61 (20% EtOAc in hexane). mp 102-104 °C. H NMR (400
Chemistry
MHz, CDCl ) δ: 8.10 (s, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.36 (d, J =
3
-1
All the chemicals and solvents (analytical grade) were
8
.5 Hz, 2H). IR νmax (neat)/cm 3257 (O-H), 1659 (C=N) and 959
N-O).
-Chlorobenzaldehyde oxime (12). White crystalline solid,
purchased
from
Sigma-Aldrich,
USA.
Thin-layer
(
chromatographic analysis was carried out on precoated Merck
silica gel 60 F254 TLC aluminium sheets and spots were
visualized under UV light at 254 nm, iodine- vapor staining or
ninhydrin test. IR spectra were recorded on Agilent Cary 630
2
1
yield 94%. R
NMR (300 MHz, CDCl
.2 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.36-7.26 (m, 2H). IR νmax
f
= 0.58 (20% EtOAc in hexane). mp 72-74
°C. H
3
) δ: 8.88 (s, 1H), 8.59 (s, 1H), 7.84 (d, J =
7
1
13
-1
FT-IR spectrometer. H and C NMR spectra were obtained in
(neat)/cm 3242 (O-H), 1659 (C=N) and 970 (N-O).
4-Methylbenzaldehyde oxime (13). White solid, yield 90%. R =
CDCl /DMSO-d as solvent with tetramethylsilane (TMS) as an
3
6
f
1
internal standard on either a Bruker Spectrospin DPX-300
spectrometer at 300 MHz and 75 MHz, respectively or a Bruker
Avance II 400 spectrometer at 400 MHz. 2D spectra (COSY and
HETCOR) were recorded on Jeol, JNM-EXCP 400 spectrometer.
Splitting patterns are designated as follows: s (singlet), d
0
.56 (20% EtOAc in hexane). mp 74-76
°C. H NMR (400 MHz,
CDCl ) δ: 8.10 (s, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4
3
-1
Hz, 2H), 2.53 (s, 3H). IR νmax (neat)/cm 3260 (O-H), 1659 (C=N)
and 963 (N-O).
4
f
-Methoxybenzaldehyde oxime (14). Yellow oil, yield 90%. R =
1
(doublet), t (triplet), q (quartet), m (multiplet) or brs (broad).
3
0.43 (20% EtOAc in hexane). H NMR (400 MHz, CDCl ) δ: 8.10
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(
s, 1H), 7.52 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 3.83 (s, MHz, CDCl ) δ: 7.95 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 8.3 Hz, 2H),
3
-1
-1
3
H). IR νmax (neat)/cm 3234 (O-H), 1611 (C=N) and 952 (N-O).
-(Trifluoromethyl)benzaldehyde oxime (15). Light yellow General procedure for the synthesis of azides (27-34).
C. Example: Synthesis of N-(4-fluorobenzyl)-3-
) δ: 8.17 (s, 1H), 7.70 (d, J = 8.0 Hz, azidopropanamide (28)
2
4.52 (s, 2H). IR νmax (neat)/cm 3394 (NH ).
4
solid, yield 88%. R = 0.55 (20% EtOAc in hexane). mp 88-90
1
°
f
H NMR (400 MHz, CDCl
3
-1
2
1
4
H), 7.64 (d, J = 8.4 Hz, 2H). IR νmax (neat)/cm 3272 (O-H), To a solution of 3-bromopropionic acid, 25 (19.61 mmol, 3.0 g)
622 (C=N) and 974 (N-O). in anhyd DMF (10 mL) was added sodium azide (39.22 mmol,
-(Trifluoromethoxy)benzaldehyde oxime (16). Colourless 2.55 g). The reaction mixture was heated at 85 C for 3 h. The
= 0.61 (20% EtOAc in hexane). mp 47-49 crude reaction mixture was diluted with ethyl acetate which
C. H NMR (400 MHz, CDCl ) δ: 8.13 (s, 1H), 7.61 (d, J = 8.4 Hz, was then washed with 0.1 N HCl, water and brine. The organic
°
crystals, yield 90%. R
f
1
°
3
-1
2
H), 7.24 (d, J = 8.0 Hz, 2H). IR νmax (neat)/cm 3267 (O-H), layer was dried over anhyd sodium sulphate, filtered and
696 (C=N) and 977 (N-O). concentrated under vacuo to yield 3-azidopropionic acid, 26
1
General procedure for the synthesis of amines (17-24). which was used directly in the next reaction without further
6
2
Example: Synthesis of (4-Fluorophenyl)methanamine (18)
purification. The prepared azido acid (13.03 mmol, 1.5 g) was
C. To this cold
To a solution of 10 (11.51 mmol, 1.6 g) in ethanol (35 mL), a dissolved in acetonitrile (15 mL) and stirred at 0
°
mixture of water (6.0 mL) and conc. HCl (12 mL) was added. solution, HOBt (15.54 mmol, 2.09 g) and EDC.HCl (14.02 mmol,
The reaction mixture was stirred at room temperature for 15 2.69 g) were added. The reaction was stirred for 40 min and
min then it was allowed to cool to 0 °C. Zinc dust (69.04 mmol, then a mixture of NMM (15.54 mmol, 1.71 mL) and 18 (13.03
.38 g) was added slowly to the reaction mixture and it was mmol, 1.28 mL) was added. The reaction mixture was stirred
4
refluxed for 1 h at 80-90 °C. The progress of the reaction was overnight at room temperature. The solid was filtered and the
monitored by TLC. The reaction mixture was filtered and acetonitrile was removed under vacuo. The residue was
concentrated under vacuo to yield hydrochloride salt of amine. dissolved in 50 mL ethyl acetate, washed with saturated citric
The hydrochloride salt was then treated with dried amberlyst acid solution, 5% aq sodium bicarbonate solution and brine.
1
8
A-21 to afford corresponding free amine.
The organic layer was dried over anhyd sodium sulphate,
Phenylmethanamine (17). Light yellow oil, yield 75%. R
1
f
= 0.0 filtered, evaporated and gave crude azide. The crude product
(
40% EtOAc in hexane). H NMR (300 MHz, CDCl ) δ: 7.43-7.24 was purified by column chromatography eluted by a solution
3
-1
27
(
m, 5H), 3.88 (s, 2H). IR νmax (neat)/cm 3367 (NH
2
).
of ethyl acetate:hexane (4:6) to yield pure azide (28).
(
4-Fluorophenyl)methanamine (18). Light yellow oil, yield N-benzyl-3-azidopropanamide (27). Yellow oil, yield 37%. R =
f
1
= 0.0 (40% EtOAc in hexane). H NMR (300 MHz, CDCl
1
8
2%. R
f
3
)
3
0.48 (40% EtOAc in hexane). H NMR (300 MHz, CDCl ) δ: 7.38-
δ: 7.34-7.26 (m, 2H), 7.06-6.99 (m, 2H), 3.84 (s, 2H). IR νmax 7.27 (m, 5H), 6.17 (brs, 1H), 4.45 (d, J = 5.7 Hz, 2H), 3.63 (t, J =
13
-
1
(
neat)/cm 3372 (NH
2
).
3
6.3 Hz, 2H), 2.45 (t, J = 6.4 Hz, 2H). C NMR (75 MHz, CDCl ) δ:
(
4-Chlorophenyl)methanamine (19). Light yellow oil, yield 171.45, 141.63, 128.94, 128.66, 127.32, 45.76, 41.56, 33.82. IR
-1
max (neat)/cm 2093 (azide), 1644 (C=O) and 1544 (NH). MS
1
= 0.0 (40% EtOAc in hexane). H NMR (400 MHz, CDCl
8
6%. R
f
3
)
ν
+
δ: 7.29 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 3.83 (s, 2H). (ESI), m/z: 205.07 [M+H] .
-1
IR νmax (neat)/cm 3365 (NH
2
).
N-(4-fluorobenzyl)-3-azidopropanamide (28). Light yellow
(
2-Chlorophenyl)methanamine (20). Light yellow oil, yield solid, yield 49%. R = 0.45 (40% EtOAc in hexane). mp 36-38
f
°
C.
1
= 0.0 (40% EtOAc in hexane). H NMR (300 MHz, CDCl
1
7
8%. R
f
3
)
H NMR (300 MHz, CDCl
3
) δ: 7.28-7.23 (m, 2H), 7.05-6.99 (m,
δ: 7.39-7.36 (m, 2H), 7.28-7.18 (m, 2H), 3.94 (s, 2H). IR νmax 2H), 6.10 (brs, 1H), 4.42 (d, J = 5.7 Hz, 2H), 3.64 (t, J = 6.3 Hz,
13
-
1
(
neat)/cm 3372 (NH ).
2
2H), 2.45 (t, J = 6.3 Hz, 2H). C NMR (75 MHz, CDCl ) δ: 169.85,
3
(
4-Methylphenyl)methanamine (21). Light yellow oil, yield 160.58, 133.80, 129.50, 115.73, 47.39, 42.99, 35.82. IR νmax
1
= 0.0 (40% EtOAc in hexane). H NMR (400 MHz, CDCl
-1
(neat)/cm 2086 (azide), 1641 (C=O) and 1553 (NH). MS (ESI),
7
3%. R
f
3
)
+
δ: 7.20 (d, J = 7.8 Hz, 2H), 7.15 (d, J = 7.9 Hz, 2H), 3.82 (s, 2H), m/z: 223.1 [M+H] .
-1
2
.34 (s, 3H). IR νmax (neat)/cm 3382 (NH
2
).
N-(4-chlorobenzyl)-3-azidopropanamide (29). Light yellow
= 0.31 (40% EtOAc in hexane). mp 33-35 C.
H NMR (300 MHz, CDCl ) δ: 7.32 (d, J = 8.1 Hz, 2H), 7.23 (d, J =
(4-Methoxyphenyl)methanamine (22). Light yellow oil, yield solid, yield 36%. R
f
°
1
1%. R = 0.0 (40% EtOAc in hexane). H NMR (400 MHz, CDCl )
f 3
1
8
3
δ: 7.22 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 3.79 (brs, 7.2 Hz, 2H), 6.04 (s, 1H), 4.44 (d, J = 5.4 Hz, 2H), 3.66 (t, J = 6.0
13
-
1
5
H). IR νmax (neat)/cm 3367 (NH
2
).
3
Hz, 2H), 2.47 (t, J = 6.1 Hz, 2H). C NMR (75 MHz, CDCl ) δ:
(
4-(Trifluoromethyl)phenyl)methanamine (23). Light yellow 169.98, 136.56, 133.35, 129.04, 128.84, 47.39, 42.96, 35.77. IR
1
= 0.0 (40% EtOAc in hexane). H NMR (400
-1
max (neat)/cm 2095 (azide), 1640 (C=O) and 1544 (NH). MS
oil, yield 67%. R
f
ν
+
) δ: 7.91 (d, J = 8.0 Hz, 2H), 7.69 (d, J = 7.9 Hz, 2H), (ESI), m/z: 239.1 [M+H] .
MHz, CDCl
3
-
1
3
.95 (s, 2H). IR νmax (neat)/cm 3384 (NH
2
).
N-(2-chlorobenzyl)-3-azidopropanamide (30). Light yellow
(
4-(Trifluoromethoxy)phenyl)methanamine (24). Light yellow solid, yield 39%. R
1
f
= 0.48 (40% EtOAc in hexane). mp 38-40
°C.
1
= 0.0 (40% EtOAc in hexane). H NMR (400
oil, yield 71%. R
f
H NMR (300 MHz, CDCl ) δ: 7.39-7.35 (m, 2H), 7.29-7.22 (m,
3
2
H), 6.26 (brs, 1H), 4.55 (d, J = 5.7 Hz, 2H), 3.63 (t, J = 6.4 Hz,
1
4 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6OB01718E
ARTICLE
1
H), 2.46 (t, J = 6.3 Hz, 2H). C NMR (75 MHz, CDCl ) δ: 169.81, CDCl ) δ: 7.34-7.26 (m, 3H), 7.19-7.17 (m, 2H), 4.97 (d, J = 7.5
3
2
1
3
3
3
35.33, 133.63, 130.24, 129.57, 129.08, 127.17, 47.37, 41.65, Hz, 1H), 4.82-4.64 (m, 3H), 3.20-3.06 (m, 2H), 2.53 (s, 1H), 1.43
-
1
13
5.78. IR νmax (neat)/cm 2102 (azide), 1644 (C=O) and 1551 (s, 9H). C NMR (75 MHz, CDCl
3
) δ: 171.16, 155.07, 135.70,
+
(
NH). MS (ESI), m/z: 239.1 [M+H] .
129.41, 128.61, 127.13, 80.06, 75.47, 54.31, 52.65, 38.10,
-1
N-(4-methylbenzyl)-3-azidopropanamide (31). Yellow oil, yield 28.28. IR νmax (neat)/cm 3365 (
≡
CH), 2129 (C≡C), 1762 (C=O,
1
= 0.33 (40% EtOAc in hexane). H NMR (400 MHz, ester), 1687 (C=O, amide) and 1510 (NH). MS (ESI), m/z: 326.2
5
3%. R
f
+
+
CDCl ) δ: 7.19-7.14 (m, 4H), 5.80 (brs, 1H), 4.42 (d, J = 5.6 Hz, [M+Na] , 629.4 [2M+Na] .
3
2
H), 3.66 (t, J = 6.4 Hz, 2H), 2.44 (t, J = 6.4 Hz, 2H), 2.34 (s, 3H). Propargyl ester of boc-L-proline (39). Light yellow oil, yield
1
= 0.64 (20% EtOAc in hexane). H NMR (300 MHz,
1
3
C NMR (75 MHz, CDCl
3
) δ: 169.58, 137.39, 134.88, 129.44, 85%. R
f
-1
127.84, 47.44, 43.56, 35.90, 21.08. IR νmax (neat)/cm 2102 CDCl
3
) δ: 4.78-4.69 (m, 2H), 4.29-4.25 (m, 1H), 3.58-3.40 (m,
(
azide), 1618 (C=O) and 1585 (NH). MS (ESI), m/z: 218.9 2H), 2.48 (s, 1H), 2.26-2.23 (m, 1H), 2.05-1.91 (m, 3H), 1.42 (s,
13
+
M+H] .
[
3
9H). C NMR (75 MHz, CDCl ) δ: 172.42, 153.70, 80.06, 75.11,
-1
N-(4-methoxybenzyl)-3-azidopropanamide (32). Light yellow 58.91, 52.23, 46.30, 30.79, 28.28, 23.61. IR νmax (neat)/cm
solid, yield 41%. R = 0.41 (40% EtOAc in hexane). mp 58-60 C. 3246 ( CH), 2129 (C C), 1754 (C=O, ester) and 1693 (C=O,
H NMR (300 MHz, CDCl ) δ: 7.14 (d, J = 8.4 Hz, 2H), 6.80 (d, J = amide). MS (ESI), m/z: 254.4 [M+H] .
.7 Hz, 2H), 5.77 (brs, 1H), 4.32 (d, J = 5.4 Hz, 2H), 3.73 (s, 3H), Propargyl ester of boc-L-tryptophan (40). Light yellow solid,
f
°
≡
≡
1
+
3
8
3
1
3
1
.58 (t, J = 6.4 Hz, 2H), 2.36 (t, J = 6.4 Hz, 2H). C NMR (75 yield 88%. R = 0.31 (20% EtOAc in hexane). mp 95-96
°
C. H
) δ: 8.17 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H),
7.39, 42.99, 35.82. IR νmax (neat)/cm 2091 (azide), 1637 7.38 (d, J = 7.8 Hz, 1H), 7.21-7.12 (m, 2H), 7.07 (s, 1H), 5.08 (d,
f
3 3
MHz, CDCl ) δ: 169.85, 160.58, 133.80, 129.50, 115.73, 57.04, NMR (300 MHz, CDCl
-1
4
+
(C=O) and 1559 (NH). MS (ESI), m/z: 234.9 [M+H] .
J = 8.1 Hz, 1H), 4.77-4.63 (m, 3H), 3.35-3.33 (m, 2H), 2.50 (s,
1
3
N-(4-(trifluoromethyl)benzyl)-3-azidopropanamide (33). Light 1H), 1.44 (s, 9H). C NMR (75 MHz, CDCl
3
) δ: 171.54, 155.24,
1
= 0.43 (40% EtOAc in hexane). H NMR 136.12, 127.71, 122.95, 122.24, 119.69, 118.76, 111.20,
yellow oil, yield 56%. R
f
(
400 MHz, CDCl ) δ: 7.60 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 7.7 Hz, 109.92, 79.99, 75.37, 54.23, 52.65, 28.32, 27.76. IR ν
3 max
-1
2
2
1
H), 5.96 (brs, 1H), 4.53 (d, J = 5.8 Hz, 2H), 3.68 (t, J = 6.2 Hz, (neat)/cm 3397 (
≡
CH), 2147 (C
≡
C), 1736 (C=O, ester), 1698
1
3
H), 2.48 (t, J = 6.2 Hz, 2H). C NMR (75 MHz, CDCl
+
) δ: 170.01, (C=O, amide) and 1503 (NH). MS (ESI), m/z: 365.09 [M+Na] ,
3
+
42.05, 129.67, 127.87, 125.71, 122.24, 47.38, 43.18, 35.86. IR 707.4 [2M+Na] .
-1
ν
max (neat)/cm 2107 (azide), 1654 (C=O) and 1547 (NH). MS Synthesis of boc-protected triazole-amino acid hybrids (41-
+
-
(ESI), m/z: 272.9 [M+H] , 271.1 [M-H] .
64). Example: Synthesis of (R)-(1-(3-(4-fluorobenzylamino)-3-
(34). oxopropyl)-1H-1,2,3-triazol-4-yl)methyl 2-[(tert-
= 0.27 (40% EtOAc in hexane). H NMR butoxycarbonyl)amino]-3-phenylpropanoate (44)
) δ: 7.32 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.2 Hz, The azide 28 (1.35 mmol, 0.3 g) and alkyne 38 (1.35 mmol,
H), 5.90 (brs, 1H), 4.46 (d, J = 7.1 Hz, 2H), 3.67 (t, J = 6.2 Hz, 0.41 g) were dissolved in tert-butanol (4.0 mL) and H O (8.0
O (0.013 mmol, 0.033 g)
58.72, 134.58, 129.50, 123.53, 115.76, 47.39, 42.99, 35.82. IR and sodium ascorbate (0.067 mmol, 0.013 g) were added and
N-(4-(trifluoromethoxy)benzyl)-3-azidopropanamide
1
Yellow oil, yield 45%. R
400 MHz, CDCl
f
(
3
2
2
1
2
1
3
3 4 2
H), 2.46 (t, J = 6.3 Hz, 2H). C NMR (75 MHz, CDCl ) δ: 169.85, mL). The copper sulphate, CuSO .5H
-1
ν
max (neat)/cm 2102 (azide), 1652 (C=O) and 1547 (NH). MS the reaction mixture was stirred at room temperature. The
+
ESI), m/z: 289.45 [M+H] .
(
progress of the reaction was monitored by TLC. After
General procedure for the synthesis of propargyl ester of boc- completion of the reaction, the content of the reaction was
protected alkynes (38-40). Example: Synthesis of propargyl washed with brine and the compound was extracted with ethyl
ester of boc-L-phenylalanine (38)
acetate, dried over anhyd sodium sulphate and concentrated.
Boc-protected L-phenylalanine (11.31 mmol, 3.0 g) was The product was purified by column chromatography eluted
dissolved in anhyd DMF (10 mL) and the solution was allowed with ethyl acetate: hexane (3:7) to yield pure product in
2
9
to cool to 0
g) was added to this cold solution and stirred for 15 min. (R)-(1-(3-(benzylamino)-3-oxopropyl)-1H-1,2,3-triazol-4-
Propargyl bromide (11.31 mmol, 1.26 mL, 80 wt. % in toluene) yl)methyl 2-[(tert-butoxycarbonyl)amino]-3-
was added dropwise to the mixture and stirred at 0 C for 1 h. phenylpropanoate (41). Off white solid, yield 86%. R = 0.76
The reaction mixture was allowed to attain room temperature. (10% MeOH in DCM). mp 93-95
°C. Anhyd potassium carbonate (16.96 mmol, 2.34 quantitative yield.
°
f
1
°
C. [ꢀ] ꢂꢁ ꢃ
ꢄ= -28.00. H NMR
After removal of DMF under vacuo, the residue was dissolved (300 MHz, CDCl ) δ: 7.50 (s, 1H), 7.26-7.15 (m, 7H), 7.10 (d, J =
3
in ethyl acetate. The organic layer was washed with saturated 7.5 Hz, 2H), 6.99-6.97 (m, 1H), 5.91 (brs, 1H), 5.14 (s, 2H), 4.88
citric acid solution, water and brine which was dried over (d, J = 7.2 Hz, 1H), 4.62 (t, J = 5.6 Hz, 2H), 4.47-4.45 (m, 1H),
anhyd sodium sulphate and concentrated. The crude product 4.32 (d, J = 5.7 Hz, 2H), 3.02-2.95 (m, 2H), 2.78 (t, J = 6.3 Hz,
1
3
was then purified by column chromatography eluted by ethyl 2H), 1.33 (s, 9H). C NMR (75 MHz, CDCl ) δ: 171.65, 168.99,
3
2
8
acetate: hexane (2:8) to yield pure alkyne (38).
155.08, 142.07, 137.79, 135.82, 129.33, 128.69, 128.50,
Propargyl ester of boc-L-phenylalanine (38). Light yellow oil, 127.69, 127.59, 126.99, 125.09, 80.03, 58.23, 54.40, 46.25,
1
yield 87%. R = 0.52 (20% EtOAc in hexane). H NMR (300 MHz,
f
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DOI: 10.1039/C6OB01718E
ARTICLE
Journal Name
-
1
4
3.67, 38.05, 36.32, 28.28. IR νmax (neat)/cm 3078 (CH), 1598 (R)-(1-(3-(4-fluorobenzylamino)-3-oxopropyl)-1H-1,2,3-
+
(
C=C) and 1460 (N=N). MS (ESI), m/z: 508.3 [M+H] .
R)-(1-(3-(benzylamino)-3-oxopropyl)-1H-1,2,3-triazol-4-
yl)methyl 1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylate 0.71 (10% MeOH in DCM). mp 62-64
42). Light yellow oil, yield 84%. R = 0.84 (10% MeOH in NMR (300 MHz, CDCl ) δ: 8.88 (s, 1H, NH), 7.53 (d, J = 7.5 Hz,
triazol-4-yl)methyl
2-[(tert-butoxycarbonyl)amino]-3-(1H-
(
indol-3-yl)propanoate (46). Light yellow solid, yield 90%. R =
f
1
°
C. [ꢀ] ꢂꢁ ꢃ
ꢄ= -20.00. H
(
f
3
DCM). [ꢀ] ꢂꢁ ꢃ = -48.00. H NMR (300 MHz, CDCl
.31-7.19 (m, 5H), 6.04 (brs, 1H), 5.26-5.21 (m, 2H), 4.73-4.71 H), 6.96 (t, J = 8.6 Hz, 2H, Ar-H), 6.56-6.52 (m, 2H, Ar-H, NH),
m, 2H), 4.41 (s, 2H), 4.25-4.16 (m, 1H), 3.53-3.39 (m, 2H), 5.25-5.21 (m, 1H, NH), 5.14-5.05 (m, 2H, OCH ), 4.65-4.49 (m,
), 3.23 (brs, 2H, CH ), 2.94-
H). C NMR (75 MHz, CDCl ) δ: 173.02, 168.97, 154.44, 2.82 (m, 2H, CH ), 1.43 (s, 9H, Boc). C NMR (75 MHz, CDCl3)
1
) δ: 7.71 (s, 1H), 1H, Ar-H), 7.35 (d, J = 8.1 Hz, 1H, Ar-H), 7.17-7.05 (m, 5H, Ar-
3
7
(
2
2
9
1
4
.89-2.84 (m, 2H), 2.21-2.15 (m, 1H), 1.94-1.86 (m, 3H), 1.45 (s, 3H, CH, CH
2
), 4.33-4.32 (m, 2H, CH
2
2
1
3
13
3
2
42.91, 137.75, 128.70, 127.69, 124.98, 79.94, 58.99, 57.94, δ: 172.11, 169.45, 163.82, 155.26, 142.52, 136.09, 133.43,
6.63, 43.66, 36.58, 30.79, 29.79, 28.41, 24.33. IR νmax 129.31, 127.74, 125.01, 123.22, 121.97, 119.49, 118.76,
-1
(
neat)/cm 3067 (CH), 1549 (C=C) and 1454 (N=N). MS (ESI), 115.69, 111.29, 109.48, 79.98, 58.21, 54.51, 46.08, 43.01,
-1
35.59, 28.33, 27.97. IR νmax (neat)/cm 3080 (CH), 1512 (C=C)
+
+
m/z: 358.3 [M+H-Boc] , 458.4 [M+H] .
+
and 1460 (N=N). MS (ESI), m/z: 565.3 [M+H] .
(
R)-(1-(3-(benzylamino)-3-oxopropyl)-1H-1,2,3-triazol-4-
yl)methyl
2-[(tert-butoxycarbonyl)amino]-3-(1H-indol-3- (R)-(1-(3-(4-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
= 0.67 triazol-4-yl)methyl 2-[(tert-butoxycarbonyl)amino]-3-
= -20.00. H NMR phenylpropanoate (47). White solid, yield 88%. R = 0.72 (10%
yl)propanoate (43). Light yellow solid, yield 92%. R
f
1
(
(
(
10% MeOH in DCM). mp 68-70
°
C. [ꢀ] ꢂꢁ ꢃ
ꢄ
f
1
300 MHz, CDCl ) δ: 8.74 (s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.30 MeOH in DCM). mp 98-100
°
C. [ꢀ] ꢂꢁ ꢃ
) δ: 7.56 (s, 1H), 7.27-7.22 (m, 5H), 7.10-7.04 (m,
H), 6.13 (s, 1H), 5.23-5.16 (m, 1H), 5.04-5.00 (m, 2H), 4.60- 4H), 6.60 (brs, 1H), 5.18 (s, 2H), 4.98 (d, J = 7.8 Hz, 1H), 4.68-
.44 (m, 3H), 4.32 (t, J = 6.0 Hz, 2H), 3.16-3.14 (m, 2H), 2.90- 4.63 (m, 2H), 4.50-4.46 (m, 1H), 4.34 (d, J = 5.7 Hz, 2H), 3.08-
ꢄ= -16.00. H NMR (300
3
d, J = 7.8 Hz, 1H), 7.26-7.19 (m, 4H), 7.12-6.99 (m, 4H), 6.46 (s, MHz, CDCl
3
1
4
2
1
1
1
1
3
13
.77 (m, 2H), 1.37 (s, 9H). C NMR (75 MHz, CDCl
3
) δ: 172.12, 2.98 (m, 2H), 2.87 (t, J = 6.3 Hz, 2H), 1.39 (s, 9H). C NMR (75
69.48, 155.24, 142.43, 137.63, 136.09, 128.74, 127.61, MHz, CDCl
3
) δ: 171.66, 169.09, 155.13, 142.17, 136.45, 135.79,
27.51, 124.99, 123.29, 121.91, 119.45, 118.75, 111.34, 133.32, 129.31, 129.04, 128.78, 128.52, 127.02, 125.07, 80.06,
09.41, 79.97, 58.23, 54.52, 46.11, 43.71, 35.64, 28.35, 28.06. 58.20, 54.43, 46.25, 42.93, 38.01, 36.26, 28.27. IR νmax
-1
-1
IR νmax (neat)/cm 3060 (CH), 1525 (C=C) and 1458 (N=N). MS (neat)/cm 3067 (CH), 1521 (C=C) and 1462 (N=N). MS (ESI),
+
ESI), m/z: 547.3 [M+H] .
+
m/z: 542.5 [M+H] .
(
(R)-(1-(3-(4-fluorobenzylamino)-3-oxopropyl)-1H-1,2,3-
(R)-(1-(3-(4-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
triazol-4-yl)methyl
2-[(tert-butoxycarbonyl)amino]-3- triazol-4-yl)methyl
1-(tert-butoxycarbonyl)pyrrolidine-2-
= 0.77 carboxylate (48). Colorless oil, yield 87%. R = 0.67 (10% MeOH
= -44.00. H NMR (300 MHz, CDCl ) δ: 7.62 (s,
phenylpropanoate (44). Creamish solid, yield 89%. R
1
f
f
C. [ꢀ] ꢂꢁ ꢃ
ꢄ
= -4.00. H NMR (300 in DCM). [ꢀ] ꢁꢂ ꢃ
ꢄ
1
(10% MeOH in DCM). mp 88-90
°
3
3
MHz, CDCl ) δ: 7.57 (s, 1H), 7.22 (m, 3H), 7.14 (brs, 2H), 7.06 1H), 7.19 (d, J = 9.0 Hz, 2H), 7.05-6.98 (m, 2H), 6.59 (brs, 1H),
(brs, 2H), 7.00-6.95 (m, 2H), 6.36 (brs, 1H), 5.19 (s, 2H), 4.96 5.17-5.10 (m, 2H), 4.68-4.51 (m, 2H), 4.29-4.24 (m, 2H), 4.20-
(
brs, 1H), 4.67 (brs, 2H), 4.51 (m, 1H), 4.35 (brs, 2H), 3.04 (m, 4.16 (m, 1H), 3.41-3.27 (m, 2H), 2.84-2.74 (m, 2H), 2.11-1.98
3
1
13
2
1
1
5
3
3
H), 2.86 (brs, 2H), 1.39 (s, 9H). C NMR (75 MHz, CDCl ) δ: (m, 2H), 1.86-1.77 (m, 2H), 1.35-1.26 (m, 9H). C NMR (75
71.66, 168.97, 160.77, 155.33, 142.18, 140.27, 135.81, MHz, CDCl ) δ: 173.02, 169.15, 154.46, 143.01, 136.57, 133.31,
3
33.66, 129.47, 128.52, 127.01, 125.03, 115.39, 80.07, 58.23, 129.05, 128.76, 125.02, 79.97, 58.99, 58.06, 46.67, 42.89,
-1
36.59, 30.78, 29.78, 28.41, 24.34. IR νmax (neat)/cm 3089
-1
4.44, 46.23, 42.96, 38.05, 36.30, 28.27. IR νmax (neat)/cm
057 (CH), 1514 (C=C) and 1458 (N=N). MS (ESI), m/z: 526.1 (CH), 1547 (C=C) and 1495 (N=N). MS (ESI), m/z: 392.5 [M+H-
+
+
+
+
Boc] , 492.4 [M+H] .
[M+H] , 548.1 [M+Na] .
(R)-(1-(3-(4-fluorobenzylamino)-3-oxopropyl)-1H-1,2,3-
(R)-(1-(3-(4-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
triazol-4-yl)methyl
1-(tert-butoxycarbonyl)pyrrolidine-2- triazol-4-yl)methyl
2-[(tert-butoxycarbonyl)amino]-3-(1H-
carboxylate (45). Light yellow oil, yield 80%. R
MeOH in DCM). [ꢀ] ꢁꢂ ꢃ
f
= 0.70 (10% indol-3-yl)propanoate (49). Light yellow solid, yield 87%. R =
f
1
= -44.00. H NMR (300 MHz, CDCl
C. [ꢀ] ꢂꢁ ꢃ
.73 (d, J = 11.1 Hz, 1H), 7.20-7.12 (m, 2H), 7.04-6.97 (m, 2H), NMR (300 MHz, CDCl ) δ: 8.88 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H),
1
= -20.00. H
ꢄ
3
) δ: 0.73 (10% MeOH in DCM). mp 70-72
°
ꢄ
7
3
6.55 (brs, 1H), 5.33-5.22 (m, 2H), 4.79-4.66 (m, 2H), 4.39-4.37 7.34 (d, J = 8.1 Hz, 1H), 7.26-7.22 (m, 2H), 7.18-7.13 (m, 2H),
(
m, 2H), 4.26-4.23 (m, 1H), 3.52-3.39 (m, 2H), 2.92-2.85 (m, 7.10-7.03 (m, 3H), 6.65 (brs, 1H), 6.53 (s, 1H), 5.25-5.20 (m,
1
H), 2.21-1.84 (m, 4H), 1.44 (s, 9H). C NMR (75 MHz, CDCl
3
2
3
)
1H), 5.09-5.04 (m, 2H), 4.64-4.48 (m, 3H), 4.31 (d, J = 6.0 Hz,
1
δ: 172.99, 169.24, 153.67, 142.68, 138.75, 134.33, 129.39, 2H), 3.23-3.21 (m, 2H), 2.94-2.82 (m, 2H), 1.43 (s, 9H). C NMR
3
1
2
1
4
24.95, 115.37, 79.83, 58.92, 46.63, 46.28, 42.59, 35.96, 30.76, (75 MHz, CDCl
3
) δ: 172.12, 169.56, 155.26, 142.51, 136.26,
-
1
8.39, 23.59. IR νmax (neat)/cm 3075 (CH), 1512 (C=C) and 136.08, 133.33, 128.86, 128.80, 127.72, 125.04, 123.24,
+
452 (N=N). MS (ESI), m/z: 376.05 [M+H-Boc] , 476.04 [M+H] , 121.96, 119.49, 118.75, 111.31, 109.45, 79.99, 58.19, 54.50,
+
+
98.2 [M+Na] .
-1
46.10, 42.99, 35.55, 28.33, 27.97. IR νmax (neat)/cm 3083
1
6 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6OB01718E
ARTICLE
-
CH), 1514 (C=C) and 1462 (N=N). MS (ESI), m/z: 579.2 [M-H] , (R)-(1-(3-(4-methylbenzylamino)-3-oxopropyl)-1H-1,2,3-
(
+
5
81.1 [M+H] .
R)-(1-(3-(2-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
triazol-4-yl)methyl
2-[(tert-butoxycarbonyl)amino]-3- in DCM). [ꢀ] ꢁꢂ ꢃ
phenylpropanoate (50). White solid, yield 84%. R = 0.73 (10% 1H), 7.12-7.03 (m, 4H), 6.45 (brs, 1H), 5.19 (m, 2H), 4.69-4.64
triazol-4-yl)methyl
1-(tert-butoxycarbonyl)pyrrolidine-2-
= 0.67 (10% MeOH
= -48.00. H NMR (300 MHz, CDCl ) δ: 7.70 (s,
(
carboxylate (54). Colorless oil, yield 83%. R
f
1
ꢄ
3
f
1
MeOH in DCM). mp 118-120
° ꢄ= -16.00. H NMR (300 (m, 2H), 4.34-4.32 (m, 2H), 4.23-4.19 (m, 1H), 3.51-3.37 (m,
C. [ꢀ] ꢂꢁ ꢃ
MHz, CDCl ) δ: 7.48 (s, 1H), 7.29-7.13 (m, 7H), 6.99-6.97 (m, 2H), 2.87-2.79 (m, 2H), 2.31 (s, 3H), 2.19-2.12 (m, 2H), 1.95-
3
1
H), 6.05 (brs, 1H), 5.13 (s, 2H), 4.89 (d, J = 7.5 Hz, 1H), 4.61 (t, 1.81 (m, 3H), 1.42 (s, 9H). C NMR (75 MHz, CDCl
3
2
3
) δ: 172.99,
J = 5.8 Hz, 2H), 4.48-4.47 (m, 1H), 4.41 (d, J = 5.7 Hz, 2H), 3.05- 168.94, 154.42, 142.83, 137.26, 134.86, 129.32, 127.70,
1
3
.90 (m, 2H), 2.78 (t, J = 6.2 Hz, 2H), 1.33 (s, 9H). C NMR (75 124.97, 79.92, 58.98, 58.08, 46.62, 43.38, 36.52, 30.78, 29.78,
2
-1
3
MHz, CDCl ) δ: 171.60, 168.97, 155.28, 143.42, 137.44, 135.82, 28.40, 24.33, 21.05. IR νmax (neat)/cm 3089 (CH), 1547 (C=C)
+
33.62, 130.05, 129.52, 129.29, 129.07, 128.45, 127.05, and 1454 (N=N). MS (ESI), m/z: 358.3 [M+H-Boc] , 471.99
1
1
2
+
26.94, 125.33, 79.98, 58.20, 54.31, 46.10, 41.60, 37.98, 36.24, [M+H] , 494.13 [M+Na] .
+
-1
8.23. IR νmax (neat)/cm 3062 (CH), 1521 (C=C) and 1445 (R)-(1-(3-(4-methylbenzylamino)-3-oxopropyl)-1H-1,2,3-
+
(
N=N). MS (ESI), m/z: 542.1 [M+H] .
triazol-4-yl)methyl
2-[(tert-butoxycarbonyl)amino]-3-(1H-
(
R)-(1-(3-(2-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
indol-3-yl)propanoate (55). Light yellow solid, yield 83%. R =
f
C. ꢄ[ꢀ] ꢂꢁ ꢃ
f
= 0.77 (10% MeOH NMR (400 MHz, CDCl ) δ: 8.75 (s, 1H), 7.55 (d, J = 7.7 Hz, 1H),
ꢄ= -16.00. H
1
triazol-4-yl)methyl
1-(tert-butoxycarbonyl)pyrrolidine-2- 0.67 (10% MeOH in DCM). mp 68-70
°
carboxylate (51). Colorless oil, yield 83%. R
in DCM). [ꢀ] ꢂꢁ ꢃ
3
1
= -48.00. H NMR (300 MHz, CDCl ) δ: 7.69 (d, J 7.38 (d, J = 7.6 Hz, 1H), 7.19- 6.99 (m, 7H), 6.56 (s, 1H), 5.98
ꢄ
7.5 Hz, 1H), 7.39-7.23 (m, 4H), 6.43 (brs, 1H), 5.26-5.20 (m, (brs, 1H), 5.29-5.26 (m, 1H), 5.13-5.09 (m, 2H), 4.66-4.56 (m,
3
=
2
1
1
1
1
2
H), 4.78-4.63 (m, 2H), 4.50 (d, J = 5.4 Hz, 2H), 4.26-4.23 (m, 3H), 4.40-4.34 (m, 2H), 3.25-3.19 (m, 2H), 2.95-2.84 (m, 2H),
1
H), 3.55-3.39 (m, 2H), 2.91-2.83 (m, 2H), 2.21-2.10 (m, 1H), 2.33 (s, 3H), 1.44 (s, 9H). C NMR (75 MHz, CDCl ) δ: 172.13,
3
3
1
3
3
.95-1.84 (m, 3H), 1.45 (s, 9H). C NMR (75 MHz, CDCl ) δ: 169.44, 155.24, 142.42, 141.22, 137.35, 136.09, 134.58,
73.21, 167.98, 154.44, 142.91, 137.75, 133.45, 128.70, 129.39, 127.75, 125.01, 123.32, 121.90, 119.44, 118.76,
26.98, 124.98, 79.94, 58.10, 57.99, 46.44, 43.66, 36.58, 30.79, 111.37, 109.36, 79.97, 58.23, 54.54, 46.11, 43.49, 35.63, 28.35,
-1
-1
9.79, 28.41, 23.60. IR νmax (neat)/cm 3073 (CH), 1544 (C=C) 28.06, 21.07. IR νmax (neat)/cm 3089 (CH), 1514 (C=C) and
+
-
1462 (N=N). MS (ESI), m/z: 559.15 [M-H] , 561.65 [M+H] .
+
and 1447 (N=N). MS (ESI), m/z: 492.3 [M+H] .
R)-(1-(3-(2-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
(
(R)-(1-(3-(4-methoxybenzylamino)-3-oxopropyl)-1H-1,2,3-
triazol-4-yl)methyl
2-[(tert-butoxycarbonyl)amino]-3-(1H- triazol-4-yl)methyl
2-[(tert-butoxycarbonyl)amino]-3-
indol-3-yl)propanoate (52). Light yellow solid, yield 88%. R
f
1
=
phenylpropanoate (56). Creamish solid, yield 95%. R = 0.76
f
C.ꢄ[ꢀ] ꢂꢁ ꢃ
ꢄ
= -16.00. H (10% MeOH in DCM). mp 108-110
°
C. ꢄ[ꢀ] ꢂꢁ ꢃ
) δ: 7.60 (s, 1H), 7.24-7.22 (m, 3H), 7.10 (d, J =
.41-7.35 (m, 3H), 7.29-7.08 (m, 5H), 6.60 (s, 1H), 6.36 (brs, 8.5 Hz, 2H), 7.06-7.04 (m, 2H), 6.84 (d, J = 8.5 Hz, 2H), 5.79
ꢄ= -16.00. H NMR
1
0
.41 (10% MeOH in DCM). mp 58-62
°
NMR (300 MHz, CDCl
3
) δ: 8.83 (s, 1H), 7.56 (d, J = 7.8 Hz, 1H), (400 MHz, CDCl
3
7
1
4
9
1
1
1
2
H), 5.29-5.25 (m, 1H), 5.14-5.09 (m, 2H), 4.68-4.54 (m, 3H), (brs, 1H), 5.22 (s, 2H), 4.95 (d, J = 7.8 Hz, 1H), 4.74-4.67 (m,
.49 (brs, 2H), 3.26-3.19 (m, 2H), 2.95-2.88 (m, 2H), 1.46 (s, 2H), 4.55-4.53 (m, 1H), 4.31 (d, J = 5.6 Hz, 2H), 3.78 (s, 3H),
1
3
13
3
H). C NMR (75 MHz, CDCl ) δ: 172.12, 169.54, 155.24, 3.11-2.99 (m, 2H), 2.83 (t, J = 6.2 Hz, 2H), 1.40 (s, 9H). C NMR
42.43, 136.07, 134.96, 133.48, 129.73, 129.59, 129.09, (75 MHz, CDCl ) δ: 171.64, 168.82, 159.09, 155.09, 142.08,
3
27.75, 127.11, 125.02, 123.28, 121.92, 119.46, 118.78, 135.83, 129.82, 129.34, 129.11, 128.50, 126.99, 125.06,
11.32, 109.42, 79.96, 58.24, 54.48, 46.02, 41.70, 35.53, 28.35, 114.09, 80.02, 58.26, 55.29, 54.39, 46.23, 43.19, 38.04, 36.36,
-1
8.02. IR νmax (neat)/cm 3067 (CH), 1514 (C=C) and 1447 28.27. IR νmax (neat)/cm 3070 (CH), 1514 (C=C) and 1458
-1
+
+
(N=N). MS (ESI), m/z: 538.1 [M+H] .
(N=N). MS (ESI), m/z: 581.3 [M+H] .
R)-(1-(3-(4-methylbenzylamino)-3-oxopropyl)-1H-1,2,3-
(
(R)-(1-(3-(4-methoxybenzylamino)-3-oxopropyl)-1H-1,2,3-
triazol-4-yl)methyl
2-[(tert-butoxycarbonyl)amino]-3- triazol-4-yl)methyl
1-(tert-butoxycarbonyl)pyrrolidine-2-
= 0.63 carboxylate (57). Light yellow oil, yield 86%. R = 0.71 (10%
= -44.00. H NMR (400 MHz, CDCl ) δ:
phenylpropanoate (53). Creamish solid, yield 90%. R
f
f
C.ꢄ[ꢀ] ꢂꢁ ꢃ
ꢄ
= -16.00. H NMR MeOH in DCM). [ꢀ] ꢁꢂ ꢃ
1
ꢄ
1
(
(
(
10% MeOH in DCM). mp 105-107
°
3
300 MHz, CDCl ) δ: 7.57 (s, 1H), 7.26-7.21 (m, 3H), 7.11-7.04 7.69 (d, J = 4.5 Hz, 1H), 7.16-7.08 (m, 2H), 6.86-6.83 (m, 2H),
3
m, 6H), 6.34 (brs, 1H), 5.21 (s, 2H), 4.99 (d, J = 7.8 Hz, 1H), 6.10 (brs, 1H), 5.26-5.21 (m, 2H), 4.73-4.66 (m, 2H), 4.33-4.32
4.65 (t, J = 6.3 Hz, 2H), 4.56-4.50 (m, 1H), 4.33 (d, J = 5.7 Hz, (m, 2H), 4.24-4.12 (m, 1H), 3.79 (s, 3H), 3.52-3.45 (m, 2H),
2
H), 3.11-2.96 (m, 2H), 2.83 (t, J = 6.3 Hz, 2H), 2.31 (s, 3H), 1.40 2.86-2.78 (m, 2H), 1.96-1.82 (m, 2H), 1.70 (m, 2H), 1.43 (s, 9H).
13
1
s, 9H). C NMR (75 MHz, CDCl
3
(
3
) δ: 171.64, 168.88, 155.09,
3
C NMR (75 MHz, CDCl ) δ: 173.03, 168.89, 159.10, 154.42,
1
1
2
1
42.05, 137.32, 135.83, 134.74, 129.36, 128.49, 127.72, 142.92, 129.95, 129.11, 124.97, 114.08, 79.92, 58.99, 58.12,
26.99, 125.08, 80.02, 58.24, 54.40, 46.23, 43.45, 38.05, 36.35, 55.29, 46.63, 43.17, 36.59, 30.79, 29.78, 28.41, 24.34. IR νmax
-
1
-1
8.27, 21.07. IR νmax (neat)/cm 3067 (CH), 1521 (C=C) and (neat)/cm 3095 (CH), 1514 (C=C) and 1462 (N=N). MS (ESI),
+
462 (N=N). MS (ESI), m/z: 522.2 [M+H] .
+
m/z: 388.2 [M+H-Boc] , 488.08 [M+H] .
+
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 17
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Organic & Biomolecular Chemistry
Page 18 of 26
View Article Online
DOI: 10.1039/C6OB01718E
ARTICLE
Journal Name
-
1
(
R)-(1-(3-(4-methoxybenzylamino)-3-oxopropyl)-1H-1,2,3-
triazol-4-yl)methyl 2-[(tert-butoxycarbonyl)amino]-3-(1H- and 1460 (N=N). MS (ESI), m/z: 613.27 [M-H] , 615.13 [M+H] .
indol-3-yl)propanoate (58). Light yellow solid, yield 92%. R (R)-(1-(3-(4-trifluoromethoxybenzylamino)-3-oxopropyl)-1H-
= -20.00. H 1,2,3-triazol-4-yl)methyl 2-[(tert-butoxycarbonyl)amino]-3-
) δ: 8.84 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H), phenylpropanoate (62). Light yellow solid, yield 89%. R = 0.69
36.54, 30.72, 28.37. IR νmax (neat)/cm 3091 (CH), 1510 (C=C)
-
+
f
1
=
0
.67 (10% MeOH in DCM). mp 72-74
° ꢄ
C. [ꢀ] ꢂꢁ ꢃ
NMR (400 MHz, CDCl
3
f
° ꢄ= -24.00. H NMR
C. [ꢀ] ꢂꢁ ꢃ
1
7
.38 (d, J = 8.0 Hz, 1H), 7.19-7.15 (m, 2H), 7.10-7.07 (m, 3H), (10% MeOH in DCM). mp 110-113
6.83 (d, J = 8.6 Hz, 2H), 6.52 (s, 1H), 6.02 (brs, 1H), 5.29-5.26 (300 MHz, CDCl ) δ: 7.49 (s, 1H), 7.28-7.21 (m, 5H), 7.09-6.89
3
(m, 1H), 5.12-5.09 (m, 2H), 4.67-4.52 (m, 3H), 4.34-4.30 (m, (m, 4H), 5.91 (brs, 1H), 5.23 (s, 2H), 4.67 (d, J = 7.4 Hz, 1H),
2
9
1
1
5
H), 3.78 (s, 3H), 3.26-3.20 (m, 2H), 2.96-2.83 (m, 2H), 1.44 (s, 4.71 (t, J = 5.8 Hz, 2H), 4.48-4.46 (m, 1H), 4.41 (d, J = 5.4 Hz,
13
1
H). C NMR (75 MHz, CDCl ) δ: 172.12, 169.36, 159.07, 2H), 3.12-3.01 (m, 2H), 2.82 (t, J = 6.8 Hz, 2H), 1.40 (s, 9H).
3
C
3
55.24, 142.43, 136.09, 129.66, 128.93, 127.76, 125.00, NMR (75 MHz, CDCl
23.31, 121.91, 119.44, 118.76, 114.10, 111.35, 109.39, 79.95, 137.89, 137.09, 135.86, 128.54, 127.61, 127.32, 124.78,
8.23, 55.29, 54.54, 46.10, 43.23, 35.61, 28.35, 28.02. IR ν 123.29, 121.56, 115.45, 79.32, 58.51, 54.71, 46.31, 43.71,
3
) δ: 171.82, 169.78, 156.24, 142.63,
max
-1
-1
(neat)/cm 3067 (CH), 1514 (C=C) and 1460 (N=N). MS (ESI), 38.67, 35.55, 28.35. IR νmax (neat)/cm 3067 (CH), 1533 (C=C)
+
and 1447 (N=N). MS (ESI), m/z: 592.3 [M+H] .
-
m/z: 575.37 [M-H] , 577.10 [M+H] .
+
(R)-(1-(3-(4-trifluoromethylbenzylamino)-3-oxopropyl)-1H-
(R)-(1-(3-(4-trifluoromethoxybenzylamino)-3-oxopropyl)-1H-
1
,2,3-triazol-4-yl)methyl
2-[(tert-butoxycarbonyl)amino]-3- 1,2,3-triazol-4-yl)methyl 1-(tert-butoxycarbonyl)pyrrolidine-
= 0.53 (10% 2-carboxylate (63). Light yellow oil, yield 89%. R = 0.65 (10%
= -44.00. H NMR (300 MHz, CDCl ) δ:
phenylpropanoate (59). White solid, yield 94%. R
f
f
° ꢄ ꢄ
C. ꢄ[ꢀ] ꢂꢁ ꢃ = -20.00. H NMR (400 MeOH in DCM).ꢄꢄ[ꢀ] ꢂꢁ ꢃ
1
1
MeOH in DCM). mp 103-105
MHz, CDCl ) δ: 7.57-7.55 (m, 3H), 7.33-7.22 (m, 4H), 7.05-7.04 7.71 (d, J = 9.7 Hz, 1H), 7.24-7.16 (m, 2H), 7.02-6.95 (m, 2H),
m, 2H), 6.19 (brs, 1H), 5.22 (s, 2H), 4.94 (d, J = 7.2 Hz, 1H), 6.43 (brs, 1H), 5.41-5.28 (m, 2H), 4.75-4.68 (m, 2H), 4.42-4.39
3
3
(
4
.75-4.68 (m, 2H), 4.49-4.47 (m, 1H), 4.43 (d, J = 5.7 Hz, 2H), (m, 2H), 4.28-4.25 (m, 1H), 3.67-3.47 (m, 2H), 2.81-2.76 (m,
1
3
13
3
.09-2.98 (m, 2H), 2.89 (brs, 2H), 1.39 (s, 9H). C NMR (75 2H), 2.12-1.89 (m, 4H), 1.39-1.32 (m, 9H). C NMR (75 MHz,
) δ: 171.68, 169.22, 155.14, 142.24, 141.96, 135.76, CDCl ) δ: 173.12, 168.12, 158.23, 154.30, 142.73, 139.32,
29.30, 128.53, 127.82, 127.04, 125.62, 125.57, 125.09, 80.09, 135.03, 129.02, 124.82, 121.67, 114.23, 79.78, 58.23, 46.85,
MHz, CDCl
3
3
1
5
-1
8.19, 54.43, 46.23, 43.11, 37.96, 36.27, 28.26. IR νmax 46.30, 42.80, 35.67, 30.56, 28.23, 23.70. IR νmax (neat)/cm
-1
(
neat)/cm 3032 (CH), 1529 (C=C) and 1447 (N=N). MS (ESI), 3056 (CH), 1544 (C=C) and 1460 (N=N). MS (ESI), m/z: 542.3
+
[M+H] .
+
m/z: 576.1 [M+H] .
(R)-(1-(3-(4-trifluoromethylbenzylamino)-3-oxopropyl)-1H-
(R)-(1-(3-(4-trifluoromethoxybenzylamino)-3-oxopropyl)-1H-
1,2,3-triazol-4-yl)methyl 1-(tert-butoxycarbonyl)pyrrolidine- 1,2,3-triazol-4-yl)methyl 2-(tert-butoxycarbonyl)-3-(1H-indol-
2
-carboxylate (60). Colorless oil, yield 94%. R = 0.48 (10% 3-yl)propanoate (64). Light yellow solid, yield 89%. R = 0.62
f
f
1
MeOH in DCM). [ꢀ] ꢂꢁ ꢃ = -40.00. H NMR (300 MHz, CDCl
1
) δ: (10% MeOH in DCM). mp 70-72
.52 (d, J = 7.8 Hz, 1H), 7.35-7.07 (m, 4H), 6.76 (brs, 1H), 5.29- (300 MHz, CDCl ) δ: 8.92 (s, 1H), 7.56 (d, J = 7.0 Hz, 1H), 7.32-
° ꢄ= -24.00. H NMR
C. ꢄ[ꢀ] ꢂꢁ ꢃ
3
7
5
4
1
3
.05 (m, 2H), 4.61-4.56 (m, 2H), 4.53 (d, J = 6.0 Hz, 2H), 4.25- 7.28 (m, 4H), 7.14-7.02 (m, 5H), 6.43 (s, 1H), 6.21 (brs, 1H),
.16 (m, 1H), 3.21 (brs, 2H), 3.03-2.86 (m, 2H), 2.21-2.15 (m, 5.46-5.41 (m, 1H), 5.14-5.03 (m, 2H), 4.65-4.51 (m, 3H), 4.23
13
1
H), 1.94-1.86 (m, 3H), 1.42 (s, 9H). C NMR (75 MHz, CDCl
3
3
)
(m, 2H), 3.19-3.12 (m, 2H), 2.87-2.72 (m, 2H), 1.43 (s, 9H).
C
δ: 172.12, 169.72, 155.27, 142.58, 136.06, 127.65, 125.61, NMR (75 MHz, CDCl ) δ: 171.88, 169.57, 156.12, 142.87,
3
1
23.18, 119.50, 111.29, 80.01, 58.16, 54.48, 46.09, 43.14, 137.51, 136.24, 128.54, 127.78, 127.21, 124.73, 122.89,
-1
3
5.51, 29.70, 28.32, 27.91. IR νmax (neat)/cm 3082 (CH), 1546 121.72, 119.03, 118.56, 111.47, 109.25, 79.89, 58.21, 54.76,
+
C=C) and 1452 (N=N). MS (ESI), m/z: 426.05 [M+H-Boc] , 46.23, 43.53, 36.03, 28.42. IR νmax (neat)/cm 3078 (CH), 1510
-1
(
+
26.04 [M+H] .
+
(C=C) and 1460 (N=N). MS (ESI), m/z: 630.1 [M+H] .
5
(
R)-(1-(3-(4-trifluoromethylbenzylamino)-3-oxopropyl)-1H-
,2,3-triazol-4-yl)methyl
1H-indol-3-yl)propanoate (61). Light yellow solid, yield 76%. fluorobenzylamino)-3-oxopropyl)-1H-1,2,3-triazol-4-yl)methyl
Deprotection of boc-protected triazole-amino acid hybrids
1
2-[(tert-butoxycarbonyl)amino]-3- (65-88).
Example:
Synthesis
of
(R)-(1-(3-(4-
(
° ꢄ
C. [ꢀ] ꢂꢁ ꢃ
= -20.00. H 2-amino-3-phenylpropanoate (68) The compound 44 (0.57
1
R = 0.47 (10% MeOH in DCM).ꢄ mp 55-60
NMR (400 MHz, CDCl ) δ: 8.71 (s, 1H), 7.54 (t, J = 8.0 Hz, 3H), mmol, 0.3 g) was dissolved in dichloromethane (10 mL). To this
f
3
7
.35 (d, J = 8.1 Hz, 1H), 7.24-7.14 (m, 3H), 7.08 (t, J = 7.4 Hz, solution p-toluene sulphonic acid (pTSA) (1.71 mmol, 0.32 g)
2
H), 6.50 (s, 1H), 6.28 (brs, 1H), 5.29-5.26 (m, 1H), 5.14-5.09 was added. The reaction was stirred for 3 h at room
(
m, 2H), 4.63-4.52 (m, 3H), 4.43 (s, 2H), 3.23 (s, 2H), 2.99-2.88 temperature. After completion of the reaction, it was
1
m, 2H), 1.43 (s, 9H). C NMR (75 MHz, CDCl
3
(
3
) δ: 172.99, neutralized by sat. sodium bicarbonate solution and the
1
1
1
69.43, 154.47, 153.76, 143.02, 142.26, 137.93, 136.29, compound was extracted by ethyl acetate, washed with water
27.79, 126.04, 125.47, 125.08, 124.50, 121.87, 119.23, and brine to get final compound (68).
18.45, 111.21, 109.34, 79.98, 58.96, 57.99, 46.66, 42.99, (R)-(1-(3-(benzylamino)-3-oxopropyl)-1H-1,2,3-triazol-4-
yl)methyl 2-amino-3-phenylpropanoate (65). Yellow solid,
1
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 19 of 26
Journal Name
yield 85%. R = 0.69 (10% MeOH in DCM). mp 165-167
Orga Pn l ie ca s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
View Article Online
DOI: 10.1039/C6OB01718E
ARTICLE
1
C. H (neat)/cm 3290 (NH ) and 3070 (CH). MS (ESI), m/z: 376.0
-1
°
f
2
+
NMR (400 MHz, DMSO-d
6
) δ: 7.86 (s, 1H), 7.29-7.15 (m, 10H), [M+H] .
.68 (brs, 1H), 5.17 (s, 2H), 4.57 (t, J = 6.7 Hz, 2H), 4.49 (s, 2H), (R)-(1-(3-(4-fluorobenzylamino)-3-oxopropyl)-1H-1,2,3-
.25 (d, J = 5.6 Hz, 2H), 3.63-3.60 (m, 1H), 3.17-3.12 (m, 2H), triazol-4-yl)methyl 2-amino-3-(1H-indol-3-yl)propanoate (70).
6
4
2
1
1
4
1
3
1
.79 (t, J = 7.3 Hz, 2H). C NMR (75 MHz, CDCl
69.13, 162.49, 142.56, 137.85, 133.70, 129.54, 128.68, (300 MHz, CDCl
27.69, 127.31, 126.85, 123.08, 57.28, 55.91, 46.19, 43.63, (d, J = 8.1 Hz, 1H), 7.11-6.98 (m, 5H), 6.87 (t, J = 8.2 Hz, 2H),
3
) δ: 173.89, Yellow oil, yield 90%. R
f
= 0.41 (10% MeOH in DCM). H NMR
3
) δ: 8.81 (s, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.27
-1
0.65, 36.42. IR νmax (neat)/cm 3290 (NH
+
2
) and 3070 (CH). MS 6.64-6.58 (m, 2H), 5.14-4.96 (m, 2H), 4.54-4.42 (m, 2H), 4.22
(ESI), m/z: 408.2 [M+H] .
(d, J = 5.7 Hz, 2H), 3.74 (t, J = 6.0 Hz, 1H), 3.11-3.00 (m, 2H),
1
3
(
R)-(1-(3-(benzylamino)-3-oxopropyl)-1H-1,2,3-triazol-4-
3
2.84-2.77 (m, 2H). C NMR (75 MHz, CDCl ) δ: 175.13, 169.42,
yl)methyl pyrrolidine-2-carboxylate (66). Yellow oil, yield 89%. 142.56, 136.19, 133.53, 129.27, 127.64, 124.88, 123.23,
1
= 0.33 (10% MeOH in DCM). H NMR (300 MHz, DMSO-d
R
f
6
) δ: 121.94, 119.41, 118.76, 115.66, 111.32, 110.33, 57.85, 55.23,
-1
8
4
3
2
.51 (brs, 1H), 7.87 (s, 1H), 7.32-7.15 (m, 5H), 5.14 (brs, 1H), 46.09, 42.92, 35.73, 30.53. IR νmax (neat)/cm 3272 (NH
2
) and
-
.58 (t, J = 6.9 Hz, 2H), 4.50 (s, 2H), 4.26 (d, J = 5.7 Hz, 2H), 3074 (CH). MS (ESI), m/z: 463.0 [M-H] , 465.2 [M+H] .
+
.64-3.59 (m, 1H), 3.23-3.17 (m, 2H), 2.78 (t, J = 6.6 Hz, 2H), (R)-(1-(3-(4-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
3
.01-1.91 (m, 2H), 1.72-1.65 (m, 2H). C NMR (75 MHz, CDCl
1
3
)
triazol-4-yl)methyl
2-amino-3-phenylpropanoate
(71).
δ: 173.24, 169.35, 148.04, 138.37, 128.39, 127.59, 127.11, Creamish solid, yield 88%. R = 0.43 (10% MeOH in DCM). mp
f
1
1
6
22.66, 60.03, 56.07, 46.12, 43.20, 36.20, 30.78, 29.54, 24.32. 120-122 °C. H NMR (400 MHz, DMSO-d ) δ: 7.97 (s, 1H), 7.34
-1
IR νmax (neat)/cm 3290 (NH
+
58.6 [M+H] .
2
) and 3070 (CH). MS (ESI), m/z: (d, J = 8.2 Hz, 2H), 7.28-7.12 (m, 7H), 6.92-6.88 (m, 1H), 5.18-
3
5.10 (m, 2H), 4.61-4.55 (m, 2H), 4.49 (s, 2H), 4.24-4.23 (m, 2H),
3
3.61-3.58 (m, 1H), 2.88-2.77 (m, 4H). C NMR (75 MHz, CDCl )
3
1
(R)-(1-(3-(benzylamino)-3-oxopropyl)-1H-1,2,3-triazol-4-
yl)methyl 2-amino-3-(1H-indol-3-yl)propanoate (67). Yellow δ: 173.66, 169.09, 157.13, 142.17, 136.45, 133.32, 129.31,
1
oil, yield 88%. R = 0.41 (10% MeOH in DCM). H NMR (300 128.78, 128.52, 127.01, 125.07, 124.24, 58.20, 54.43, 46.25,
f
-1
3 2
MHz, CDCl ) δ: 8.96 (s, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.28 (d, J = 42.93, 40.92, 36.26. IR νmax (neat)/cm 3294 (NH ) and 3070
+
.8 Hz, 1H),7.19-6.96 (m, 8H), 6.74 (brs, 1H), 6.66 (s, 1H), 5.13- (CH). MS (ESI), m/z: 442.0 [M+H] .
7
4
.97 (m, 2H), 4.59-4.46 (m, 2H), 4.27 (d, J = 5.4 Hz, 2H), 3.75 (t, (R)-(1-(3-(4-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
1
J = 6.0 Hz, 1H), 3.07-3.04 (m, 1H), 2.78-2.70 (m, 3H). C NMR triazol-4-yl)methyl pyrrolidine-2-carboxylate (72). Yellow oil,
3
1
(
75 MHz, CDCl
3
) δ: 174.98, 169.45, 142.46, 137.78, 136.22, yield 90%. R
33.67, 128.67, 127.52, 124.81, 123.38, 121.88, 119.35, CDCl ) δ: 8.55 (s, 1H), 7.86 (s, 1H), 7.35 (d, J = 8.4 Hz, 2H), 7.17
18.70, 111.37, 110.13, 57.94, 55.16, 46.14, 43.62, 35.83, (d, J = 8.4 Hz, 2H), 5.16 (brs, 1H), 4.57 (t, J = 6.8 Hz, 2H), 4.49 (s,
f
= 0.37 (10% MeOH in DCM). H NMR (300 MHz,
1
1
3
3
-1
0.41. IR νmax (neat)/cm 3391 (NH
-
2
) and 3068 (CH). MS (ESI), 2H), 4.24 (d, J = 6.0 Hz, 2H), 3.65-3.60 (m, 2H), 3.04-2.98 (m,
1H), 2.78 (t, J = 6.8 Hz, 2H), 2.01-1.89 (m, 2H), 1.78-1.67 (m,
+
m/z: 445.0 [M-H] , 447.0 [M+H] .
1
3
(
R)-(1-(3-(4-fluorobenzylamino)-3-oxopropyl)-1H-1,2,3-
triazol-4-yl)methyl 2-amino-3-phenylpropanoate
Creamish solid, yield 85%. R = 0.67 (10% MeOH in DCM). mp 42.84, 36.28, 31.83, 29.56, 24.56. IR νmax (neat)/cm 3284
3
2H). C NMR (75 MHz, CDCl ) δ: 172.24, 169.18, 148.20,
(68). 138.78, 128.92, 127.67, 127.42, 122.02, 61.24, 56.29, 46.23,
-1
f
1
+
184-186
°C. H NMR (300 MHz, CDCl ) δ: 7.51 (s, 1H), 7.21-7.16 (NH ) and 3078 (CH). MS (ESI), m/z: 392.0 [M+H] .
3 2
(m, 3H), 7.08-7.03 (m, 4H), 6.89 (t, J = 8.7 Hz, 2H), 6.52 (brs, (R)-(1-(3-(4-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
1
H), 5.09 (s, 2H), 4.57 (t, J = 5.6 Hz, 2H), 4.26 (d, J = 5.4 Hz, 2H), triazol-4-yl)methyl 2-amino-3-(1H-indol-3-yl)propanoate (73).
1
3
1
3
.66-3.62 (m, 1H), 2.99-2.93 (m, 1H), 2.81-2.72 (m, 3H).
) δ: 174.59, 169.07, 160.52, 142.28, (400 MHz, CDCl
36.92, 133.70, 129.41, 129.30, 128.54, 126.85, 125.03, (d, J = 7.9 Hz, 1H),7.28 (brs, 2H), 7.19-7.11 (m, 2H), 7.15-7.07
15.65, 57.84, 55.66, 46.19, 42.91, 40.89, 36.16. IR ν (m, 3H), 6.68 (s, 1H), 5.95 (brs, 1H), 5.29-5.10 (m, 2H), 4.63-
) and 3074 (CH). MS (ESI), m/z: 426.2 4.58 (m, 2H), 4.35-4.34 (m, 2H), 3.87 (t, J = 6.1 Hz, 1H), 3.17-
C
Yellow oil, yield 87%. R = 0.67 (10% MeOH in DCM). H NMR
f
NMR (75 MHz, CDCl
3
3
) δ: 8.57 (s, 1H), 7.59 (d, J = 7.3 Hz, 1H), 7.37
1
1
max
-1
(neat)/cm 3283 (NH
2
+
13
[M+H] .
3.15 (m, 2H), 2.95-2.85 (m, 2H). C NMR (75 MHz, CDCl
3
) δ:
(R)-(1-(3-(4-fluorobenzylamino)-3-oxopropyl)-1H-1,2,3-
174.39, 169.56, 142.32, 136.78, 133.54, 128.74, 128.51,
triazol-4-yl)methyl pyrrolidine-2-carboxylate (69). Light 127.72, 125.31, 123.24, 121.63, 119.39, 118.43, 111.33,
yellow solid, yield 89%. R = 0.33 (10% MeOH in DCM). mp = 109.45, 58.01, 54.23, 46.57, 42.81, 35.76, 30.42. IR νmax
f
1
-1
7
0-76
°C. H NMR (400 MHz, CDCl ) δ: 7.60 (s, 1H), 7.16-7.13 (neat)/cm 3264 (NH ) and 3063 (CH). MS (ESI), m/z: 479.0 [M-
3 2
-
m, 2H), 7.02-6.98 (m, 2H), 5.94 (brs, 1H), 4.75 (s, 2H), 4.70 (t, J H] , 481.2 [M+H] .
+
(
=
3
1
1
1
6.1 Hz, 2H), 4.36 (d, J = 5.8 Hz, 2H), 3.62-3.57 (m, 1H), 3.12- (R)-(1-(3-(2-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
.03 (m, 2H), 2.86 (t, J = 6.2 Hz, 2H), 2.23-2.07 (m, 2H), 1.86- triazol-4-yl)methyl 2-amino-3-phenylpropanoate (74). Yellow
1
3
1
.74 (m, 2H). C NMR (75 MHz, CDCl
60.25, 148.02, 134.24, 129.31, 127.74, 126.89, 122.70, MHz, CDCl
15.31, 56.09, 46.12, 42.49, 36.21, 29.53, 24.46. IR νmax 4H), 6.58 (brs, 1H), 5.20 (s, 2H), 4.58 (t, J = 6.2 Hz, 2H), 4.38 (s,
3
) δ: 173.98, 169.40, oil, yield 84%. R
f
= 0.53 (10% MeOH in DCM). H NMR (300
3
) δ: 7.49 (s, 1H), 7.24-7.19 (m, 5H), 7.04-6.99 (m,
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DOI: 10.1039/C6OB01718E
ARTICLE
Journal Name
1
H), 4.20-4.16 (m, 2H), 3.72-3.68 (m, 1H), 3.01-2.76 (m, 4H). Yellow oil, yield 85%. R = 0.50 (10% MeOH in DCM). H NMR
2
f
1
3
3 3
C NMR (75 MHz, CDCl ) δ: 174.32, 169.40, 161.23, 147.45, (300 MHz, CDCl ) δ: 8.63 (s, 1H), 7.52 (brs, 1H), 7.32-6.97 (m,
1
35.18, 133.49, 129.87, 129.52, 128.99, 128.26, 127.06, 8H), 6.64 (s, 1H), 5.99-5.91 (m, 1H), 5.19-5.00 (m, 2H), 4.65-
25.91, 123.05, 56.09, 54.23, 46.14, 43.24, 41.56, 36.29. IR 4.51 (m, 2H), 4.27-4.25 (m, 2H), 3.79 (brs, 1H), 3.09-3.08 (m,
1
-1
13
ν
2 3
max (neat)/cm 3279 (NH ) and 3067 (CH). MS (ESI), m/z: 1H), 2.79-2.74 (m, 3H), 2.25 (s, 3H). C NMR (75 MHz, CDCl )
+
42.2 [M+H] ,.
4
δ: 174.89, 169.76, 142.41, 137.54, 136.38, 133.45, 129.54,
(R)-(1-(3-(2-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
128.32, 127.43, 125.21, 123.67, 121.83, 119.75, 118.32,
triazol-4-yl)methyl pyrrolidine-2-carboxylate (75). Yellow oil, 111.63, 109.23, 58.42, 54.61, 46.21, 43.68, 35.42, 28.35, 21.07.
1
-1
yield 92%. R
CDCl ) δ: 8.55 (brs, 1H), 7.87 (s, 1H), 7.44-7.41 (m, 1H), 7.31- 459.2 [M-H] , 461.2 [M+H] , 483.2 [M+Na] .
.27 (m, 2H), 7.17-7.14 (m, 1H), 5.14 (brs, 1H), 4.58 (t, J = 6.8 (R)-(1-(3-(4-methoxybenzylamino)-3-oxopropyl)-1H-1,2,3-
Hz, 2H), 4.50 (s, 2H), 4.31 (d, J = 6.6 Hz, 2H), 3.78-3.62 (m, 1H), triazol-4-yl)methyl 2-amino-3-phenylpropanoate (80). Light
f 2
= 0.35 (10% MeOH in DCM). H NMR (300 MHz, IR νmax (neat)/cm 3279 (NH ) and 3059 (CH). MS (ESI), m/z:
-
+
+
3
7
3
1
1
1
.21-3.14 (m, 2H), 2.83 (t, J = 6.6 Hz, 2H), 2.11-2.02 (m, 1H), yellow solid, yield 87%. R
3
f
= 0.57 (10% MeOH in DCM). mp 168-
1
1
C. H NMR (400 MHz, DMSO-d ) δ: 7.85 (s, 1H), 7.30-7.18
.95-1.84 (m, 3H). C NMR (75 MHz, CDCl
3
) δ: 173.42, 169.44, 170
°
47.49, 135.22, 133.46, 129.81, 129.50, 128.96, 128.43, (m, 4H), 7.14-7.08 (m, 3H), 6.86-6.83 (m, 2H), 6.68-6.65 (m,
6
27.05, 123.02, 60.23, 56.09, 46.16, 41.53, 36.29, 29.69, 24.56. 1H), 5.09 (s, 2H), 4.60-4.54 (m, 2H), 4.49 (s, 2H), 4.18 (d, J = 5.6
13
-1
IR νmax (neat)/cm 3279 (NH ) and 3074 (CH). MS (ESI), m/z: Hz, 2H), 3.72 (s, 3H), 3.58-3.56 (m, 1H), 2.85-2.73 (m, 4H).
2
C
+
92.0 [M+H] .
3
3
NMR (75 MHz, CDCl ) δ: 173.65, 168.78, 158.43, 142.38,
(
R)-(1-(3-(2-chlorobenzylamino)-3-oxopropyl)-1H-1,2,3-
137.61, 133.83, 129.11, 128.64, 126.90, 125.11, 122.93, 56.35,
-1
triazol-4-yl)methyl 2-amino-3-(1H-indol-3-yl)propanoate (76). 55.30, 52.39, 46.22, 43.24, 40.43, 36.36. IR νmax (neat)/cm
1
= 0.50 (10% MeOH in DCM). H NMR 3286 (NH
-
) and 3070 (CH). MS (ESI), m/z: 436.5 [M-H] , 438.1
Yellow oil, yield 82%. R
f
2
+
) δ: 8.83 (s, 1H), 7.47 (brs, 1H), 7.29-6.96 (m, [M+H] .
(300 MHz, CDCl
3
8
4
1
1
1
1
H), 6.68 (s, 1H), 6.59 (s, 1H), 5.16-4.97 (m, 2H), 4.58 (s, 2H), (R)-(1-(3-(4-methoxybenzylamino)-3-oxopropyl)-1H-1,2,3-
.53 (t, J = 6.3 Hz, 2H), 4.36 (d, J = 5.4 Hz, 2H), 3.08-3.06 (m, triazol-4-yl)methylpyrrolidine-2-carboxylate (81). Yellow solid,
3
1
1
3 f
H), 2.80-2.72 (m, 3H). C NMR (75 MHz, CDCl ) δ: 174.65, yield 90%. R = 0.14 (10% MeOH in DCM). H NMR (300 MHz,
69.52, 147.43, 142.39, 135.14, 133.49, 129.89, 129.53, CDCl ) δ: 8.46 (brs, 1H), 7.86 (s, 1H), 7.09 (d, J = 8.4 Hz, 2H),
3
29.02, 127.06, 124.89, 123.03, 121.95, 119.44, 118.68, 6.85 (d, J = 8.4 Hz, 2H), 4.57 (t, J = 6.8 Hz, 2H), 4.50 (s, 2H), 4.18
11.36, 109.95, 57.96, 56.04, 46.12, 41.58, 36.25, 29.68. IR (d, J = 5.7 Hz, 2H), 3.72 (s, 3H), 3.69-3.64 (m, 1H), 3.42-3.37 (m,
-1
ν
2
max (neat)/cm 3260 (NH ) and 3063 (CH). MS (ESI), m/z: 2H), 2.76 (t, J = 7.0 Hz, 2H), 1.96-1.91 (m, 2H), 1.78-1.65 (m,
-
79.0 [M-H] , 481.2 [M+H] .
+
13
2H). C NMR (75 MHz, CDCl
4
3
) δ: 173.65, 169.36, 148.26,
(
R)-(1-(3-(4-methylbenzylamino)-3-oxopropyl)-1H-1,2,3-
138.37, 131.56, 128.98, 123.26, 114.21, 61.19, 57.23, 55.51,
-1
triazol-4-yl)methyl 2-amino-3-phenylpropanoate (77). Light 46.15, 42.00, 36.01, 30.63, 29.37, 24.31. IR νmax (neat)/cm
+
yellow solid, yield 89%. R
f
= 0.50 (10% MeOH in DCM). mp 168- 3290 (NH
2
) and 3074 (CH). MS (ESI), m/z: 388.2 [M+H] .
1
C. H NMR (300 MHz, CDCl ) δ: 7.52 (d, J = 10.8 Hz, 1H), (R)-(1-(3-(4-methoxybenzylamino)-3-oxopropyl)-1H-1,2,3-
1
7
2
2
1
1
3
70
°
.19-6.98 (m, 9H), 6.54-6.36 (m, 1H), 5.09 (s, 2H), 4.58-4.54 (m, triazol-4-yl)methyl 2-amino-3-(1H-indol-3-yl)propanoate (82).
3
H), 4.24 (s, 2H), 3.66 (brs, 1H), 2.81-2.74 (m, 2H), 2.47 (brs, Yellow solid, yield 89%. R = 0.53 (10% MeOH in DCM). mp 150-
f
1
3
1
3 3
H), 2.23 (s, 3H). C NMR (75 MHz, CDCl ) δ: 174.38, 169.17, 154 °C. H NMR (400 MHz, CDCl ) δ: 8.41 (s, 1H), 7.91 (s, 1H),
47.49, 142.19, 137.28, 134.79, 129.33, 128.56, 127.70, 7.48 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 7.9 Hz, 1H),7.09-7.06 (m,
26.88, 125.06, 123.03, 57.94, 55.59, 46.21, 43.43, 40.66, 5H), 6.96 (t, J = 7.4 Hz, 1H), 6.89 (m, 2H), 5.06 (s, 2H), 4.57 (t, J
-
1
6.43, 21.07. IR νmax (neat)/cm 3298 (NH
+
2
) and 3067 (CH). MS = 6.4 Hz, 2H), 4.17 (d, J = 5.6 Hz, 2H), 3.71 (s, 3H), 3.66-3.63 (m,
13
1H), 3.03-2.89 (m, 2H), 2.76 (t, J = 6.5 Hz, 2H). C NMR (75
(ESI), m/z: 422.2 [M+H] .
(R)-(1-(3-(4-methylbenzylamino)-3-oxopropyl)-1H-1,2,3-
MHz, CDCl ) δ: 173.12, 169.45, 142.79, 136.32, 133.59, 129.54,
3
triazol-4-yl)methyl pyrrolidine-2-carboxylate (78). Light 128.43, 127.61, 125.51, 123.21, 121.87, 119.37, 118.63,
yellow solid, yield 83%. R = 0.26 (10% MeOH in DCM). mp 109- 114.21, 111.53, 109.25, 58.53, 55.21, 54.76, 46.62, 43.38,
f
1
-1
1
7
=
5
11
°C. H NMR (300 MHz, CDCl ) δ: 8.43 (brs, 1H), 7.85 (s, 1H), 35.54, 30.56. IR νmax (neat)/cm 3279 (NH ) and 3082 (CH). MS
3 2
-
.11-7.04 (m, 4H), 4.57 (t, J = 6.8 Hz, 2H), 4.50 (s, 2H), 4.21 (d, J (ESI), m/z: 475.2 [M-H] , 477.2 [M+H] .
+
5.7 Hz, 2H), 3.68-3.63 (m, 1H), 3.19-2.94 (m, 2H), 2.77 (t, J = (R)-(1-(3-(4-(trifluoromethyl)benzylamino)-3-oxopropyl)-1H-
.7 Hz, 2H), 2.29 (s, 3H), 1.99-1.90 (m, 2H), 1.71-1.64 (m, 2H). 1,2,3-triazol-4-yl)methyl 2-amino-3-phenylpropanoate (83).
1
3
C NMR (75 MHz, CDCl
3
) δ: 172.10, 169.42, 148.27, 136.58, Light yellow solid, yield 88%. R
f
= 0.57 (10% MeOH in DCM). mp
C. H NMR (400 MHz, DMSO-d ) δ: 7.87 (s, 1H), 7.65
2.92, 35.99, 30.65, 29.38, 21.11. IR νmax (neat)/cm 3294 (d, J = 8.0 Hz, 2H), 7.36 (t, J = 8.4 Hz, 2H), 7.30-7.18 (m, 4H),
1
1
4
36.26, 129.25, 128.56, 127.63, 123.25, 59.61, 55.51, 46.14, 158-160
°
6
-1
+
(NH
2
) and 3074 (CH). MS (ESI), m/z: 372.2 [M+H] .
7.12 (d, J = 6.5 Hz, 1H), 6.68-6.66 (m, 1H), 5.09 (m, 2H), 4.58 (t,
J = 7.7 Hz, 2H), 4.49 (s, 2H), 4.34 (d, J = 5.6 Hz, 2H), 3.60-3.57
(
R)-(1-(3-(4-methylbenzylamino)-3-oxopropyl)-1H-1,2,3-
1
3
3
triazol-4-yl)methyl 2-amino-3-(1H-indol-3-yl)propanoate (79). (m, 1H), 2.84-2.76 (m, 4H). C NMR (75 MHz, CDCl ) δ: 174.15,
2
0 | J. Name., 2012, 00, 1-3
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Journal Name
Orga Pn l ie ca s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
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DOI: 10.1039/C6OB01718E
ARTICLE
1
69.49, 146.34, 142.24, 141.93, 129.30, 128.89, 128.53, in DCM). H NMR (300 MHz, CDCl ) δ: 8.73 (s, 1H), 7.65 (d, J =
1
1
4
3
27.76, 127.03, 125.61, 125.09, 123.15, 56.06, 52.65, 46.18, 7.8 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H),7.36-7.25 (m, 5H), 7.12-6.99
-
1
3.10, 36.28. IR νmax (neat)/cm 3298 (NH
2
) and 3070 (CH). MS (m, 3H), 6.46 (s, 1H), 5.23-5.14 (m, 2H), 4.60-4.53 (m, 2H), 4.32
-
ESI), m/z: 474.8 [M-H] , 476.3 [M+H] .
+
(
(d, J = 6.0 Hz, 2H), 3.87-3.76 (m, 1H), 3.25-3.11(m, 2H), 2.90-
1
3
(
R)-(1-(3-(4-trifluoromethylbenzylamino)-3-oxopropyl)-1H-
,2,3-triazol-4-yl)methyl pyrrolidine-2-carboxylate (84). 156.24, 143.43, 137.89, 135.23, 128.56, 127.51, 127.32,
Yellow oil, yield 89%. R = 0.35 (10% MeOH in DCM). H NMR 124.43, 123.29, 122.56, 121.76, 119.32, 118.67, 111.48,
3
2.77 (m, 2H). C NMR (75 MHz, CDCl ) δ: 174.24, 169.48,
1
1
f
(
300 MHz, CDCl
3
) δ: 8.43 (brs, 1H), 7.56 (s, 1H), 7.40-7.21 (m, 109.62, 58.45, 54.76, 46.23, 43.54, 35.72, 30.35. IR νmax
-1
4
H), 5.23 (brs, 1H), 4.64 (m, 2H), 4.47 (s, 2H), 4.34 (d, J = 6.5 (neat)/cm 3294 (NH ) and 3066 (CH). MS (ESI), m/z: 531.3
2
+
Hz, 2H), 3.78-3.64 (m, 1H), 3.32-3.21 (m, 2H), 2.87 (t, J = 8.8 [M+H] .
1
3
Hz, 2H), 2.31-2.21 (m, 1H), 2.08-1.77 (m, 3H). C NMR (75
MHz, CDCl ) δ: 174.12, 169.72, 149.58, 137.26, 129.65, 127.78,
26.92, 125.61, 123.18, 58.16, 54.48, 46.09, 43.14, 35.51, In vitro anti-Candida activity. The anticandidal assessment of
Biology
3
1
3
-
1
1.03, 29.70, 24.32. IR νmax (neat)/cm 3298 (NH
+
2
) and 3095 title compounds (65-88) was performed by broth dilution
30
technique according to standard protocol of NCCLS, 2012. In
(CH). MS (ESI), m/z: 426.05 [M+H] .
(R)-(1-(3-(4-(trifluoromethyl)benzylamino)-3-oxopropyl)-1H-
this study, C. albicans ATCC 90028, C. albicans 10261, C.
1,2,3-triazol-4-yl)methyl
2-amino-3-(1H-indol-3- glabrata ATCC 90030, C. tropicalis ATCC 750 and FLC-
yl)propanoate (85). Yellow oil, yield 85%. R
f
= 0.64 (10% MeOH sensitive/resistant clinical isolates of C. albicans were used for
) δ: 8.96-8.94 (m, 1H), 8.15 anticandidal activity assessment. Fluconazole was used as
d, J = 8.0 Hz, 1H), 8.06- 8.00 (m, 3H), 7.64 (t, J = 7.2 Hz, 1H), positive control. Varying concentrations (1000 to 7.8 µg/mL) of
1
in DCM). H NMR (400 MHz, CDCl
3
(
7
2
2
1
1
1
2
.57-7.51 (m, 4H), 7.15 (t, J = 7.1 Hz, 1H), 6.99 (s, 1H), 4.97 (s, test compounds were dispensed into 96-well plate in
H), 4.65-4.59 (m, 2H), 4.43-4.38 (m, 2H), 3.51-3.48 (m, 1H), Sabouraud dextrose broth medium in a final volume of 100 µL.
1
.96 (brs, 2H), 2.88 (brs, 2H). C NMR (75 MHz, CDCl ) δ: Candida cells were harvested and their turbidity was assessed
3
3
75.40, 169.53, 147.34, 141.88, 136.45, 133.46, 129.99, according to McFarland standards. Then, 100 µL of cells
3
28.87, 127.76, 125.61, 124.54, 123.16, 121.78, 119.64, (approximate 2.5×10 cells/mL) were dispensed into the 96
18.74, 110.98, 109.12, 58.67, 55.89, 46.20, 43.12, 36.23, wells and incubated at 37 °C for 24 h. After incubation, the
-
1
9.68. IR νmax (neat)/cm 3284 (NH
+
2
) and 3063 (CH). MS (ESI), growth was measured turbidometrically at 600 nm using
Thermo Multiskan spectrophotometer. IC50 was determined as
m/z: 515.3 [M+H] .
(R)-(1-(3-(4-trifluoromethoxybenzylamino)-3-oxopropyl)-1H-
50% inhibition of Candida growth and calculated by plotting a
1
,2,3-triazol-4-yl)methyl 2-amino-3-phenylpropanoate (86). graph between concentration (log10) and % inhibition.
1
Yellow oil, yield 89 %. R
f
= 0.57 (10% MeOH in DCM). H NMR Time kill curve study. Fungicidal or fungistatic effect of lead
(
300 MHz, CDCl
H), 5.14 (s, 2H), 4.61 (t, J = 5.8 Hz, 2H), 4.51 (s, 2H), 4.33-4.25 according to the method reported by Jiang et al.
m, 2H), 3.65-3.52 (m, 1H), 3.12-3.01 (m, 2H), 2.82 (t, J = 6.8 standard as well as resistant strains of C. albicans, the cells
3
) δ: 7.57 (s, 1H), 7.32-7.11 (m, 9H), 6.54 (brs, inhibitors 68 and 70 was determined by time-kill curve assay
3
1
1
For
(
1
3
Hz, 2H). C NMR (75 MHz, CDCl
3
) δ: 172.87, 168.71, 157.26, were exposed to 60 and 120 µg/mL concentrations of 68 and
42.77, 137.56, 134.09, 130.45, 128.92, 127.87, 127.45, 70. Fluconazole (125 and 1000 µg/mL) was used as positive
26.09, 123.29, 121.56, 58.51, 54.71, 46.31, 43.71, 40.83, control against standard and FLC-resistant C. albicans,
1
1
3
-
1
5.55. IR νmax (neat)/cm 3287 (NH
+
2
) and 3056 (CH). MS (ESI), respectively. Untreated cells were used as negative control. All
time-kill curve experiments were conducted in triplicates and
m/z: 492.3 [M+H] .
(R)-(1-(3-(4-trifluoromethoxybenzylamino)-3-oxopropyl)-1H-
mean colony count data (log10 CFU/mL) were plotted as a
1
,2,3-triazol-4-yl)methyl pyrrolidine-2-carboxylate (87). Light function of time for each strain.
1
yellow oil, yield 89%. R = 0.38 (10% MeOH in DCM). H NMR Proteinase and phospholipase assay. Previously identified
f
(
300 MHz, CDCl
3
) δ: 8.54 (brs, 1H), 7.89 (s, 1H), 7.67-7.43 (m, proteinase positive standard C. albicans ATCC 90028 along
H), 7.31-7.23 (m, 2H), 5.20 (brs, 1H), 4.64 (t, J = 7.2 Hz, 2H), with FLC-sensitive and resistant clinical isolates of C. albicans
2
4
3
1
1
5
.54 (s, 2H), 4.29 (d, J = 6.7 Hz, 2H), 3.78-3.62 (m, 1H), 3.28- were used for this study. 1.5 mL of the 24 h old Candida cells
.21 (m, 2H), 2.89 (t, J = 7.6 Hz, 2H), 2.22-2.04 (m, 2H), 1.89- were transferred into micro centrifuge tube and centrifuged at
1
3
.72 (m, 2H). C NMR (75 MHz, CDCl
3
) δ: 173.45, 169.52, 3000 rpm for 5 min. The pellets obtained were washed in
49.23, 138.45, 129.34, 128.43, 127.89, 127.34, 122.89, 60.56, phosphate buffer saline (PBS) twice and centrifuged to remove
6.45, 46.58, 43.76, 36.52, 30.93, 29.81, 24.56. IR νmax the residual medium. After standardizing the suspensions
-
1
(
neat)/cm 3290 (NH
2
) and 3065 (CH). MS (ESI), m/z: 442.3 (MacFarland standard), cells were exposed to 60 and 120
µg/mL of lead inhibitors (68 and 70) for 1 h. Then, 1 µL from
+
M+H] .
[
(R)-(1-(3-(4-trifluoromethoxybenzylamino)-3-oxopropyl)-1H-
each cell suspension treated with test compound plated at
1
,2,3-triazol-4-yl)methyl
2-amino-3-(1H-indol-3- equidistant points on proteinase and phospholipase agar
3
C for 3-4 days. Formation of
2
yl)propanoate (88). Yellow oil, yield 89%. R
f
= 0.62 (10% MeOH media and incubated at 37
°
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ARTICLE
Journal Name
transparent halo around the Candida colonies indicated the here, %24(28)DHE = [(A230/518)×F]/pellet weight, A281.5 and
effect of test compounds on extracellular enzyme secretion. 230 absorbance at 281.5 nm and 230 nm, respectively and F is
Enzyme activity was measured by dividing the diameter of the factor for dilution in ethanol.
A
3
3
colony by the diameter of colony plus zone of clearance. The Cell proliferation assay. MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyl
experiment was performed in triplicate. tetrazolium bromide), Dulbecco’s modified Eagle’s medium
Transmission electron microscopy (TEM) analysis. The (DMEM), 0.25% trypsin, 0.02% EDTA mixture were purchased from
morphology of Candida cells was analyzed using TEM following Hi Media (Mumbai, India). Fetal bovine serum (FBS) was obtained
3
4
the standard protocol. Mid-log phase cells were harvested, from Gibco (Grand Island, NY). Human embryonic kidney (HEK293)
standardized (A600 ≈ 0.1) and exposed to 120 µg/mL cell line was procured from National Centre for Cell Sciences
concentration of test inhibitors 68 and 70 for 1 h. Then, cells (NCCS), Pune, India. The cells were cultured and maintained as a
were washed thrice with PBS to remove residual medium and monolayer in DMEM supplemented with 10% FBS and antibiotics
fixed
phosphate/magnesium buffer (40 mM K
.5 mM MgCl ). Cells were washed twice for 15 min in 0.1 M cultured twice in a week. Cell count of approximate 2 × 10
overnight
in
2.5%
glutaraldehyde
in (100 units/mL penicillin and 100 µg/mL streptomycin) at 37 °C in
2
HPO /KH PO , pH 6.5, humified atmosphere of 5% CO
4
2
4
2
in T-25 flasks. The cells were sub-
4
0
2
sodium phosphate buffer (pH 6.0) and post-fixed for 2 h in 2% cells/well were seeded in 96-well plate (150 µL/well) and incubated
osmium tetroxide. Again, cells were washed twice for 15 min for 24 h before treatment. The cells were then treated with varying
in distilled water and then en bloc stained with 1% uranyl concentrations (10-600 µg/mL) of the test compounds. After 48 h of
acetate (aqueous) for 30 min. After two further washes, cells incubation at 37 °C, the exhausted serum supplemented medium
were dehydrated in 95% and 100% ethanol. Cells were was removed and serum free media (50 µL) was added in each well.
exposed to propylene oxide for 2×10 min and infiltrated for 1 h After that, 20 µL/well of MTT at a concentration of 5 mg/mL in PBS
in 1:1 propylene/epoxy embedding material (Epon) mixture was added to each well and the plates were incubated for 4 h at 37
and then overnight in fresh Epon. After polymerization for 48 h °C. Formazan crystals, the metabolized MTT product, were
at 60
°C, ultrathin sections were cut using a microtome solubilized in DMSO (150 µL/well) and was quantified by reading
LeicaEM UC6) and transferred to a copper grid. Samples were the absorbance at 570 nm after incubation of 10 min on iMark
(
stained with uranyl acetate (saturated solution of uranyl Microplate Reader (Bio-Rad, Hercules, CA). All assays were
acetate in 50% alcohol) followed by lead citrate. Samples were performed in triplicate. Percent viability was taken as the relative
3
5
washed three times in Milli-Q (MQ) water and dried by absorbance of treated versus untreated control cells.
touching with Whatman filter paper. Sections were examined
Hemolytic assay. The hemolytic activities of the test inhibitors
and 70 and conventional antifungal drug FLC were
with
a Jeol (Japan) JEM-2100F transmission electron
6
8
microscope at 120 KV.
3
6
determined on human red bood cells (hRBCs). Human
erythrocytes from healthy individuals were collected in tubes
containing EDTA as anti-coagulant. The erythrocytes were
Ergosterol estimation assay. The total intracellular sterols
2
were extracted as reported earlier with slight modifications.
2
Three separate conical flasks containing 120, 60 and 30 µg/mL
of compounds 68 and 70 in Sabouraud dextrose broth
inoculated with fresh cells of C. albicans ATCC 90028
harvested by centrifugation for 10 min at 2000 rpm and 20 °C,
and washed three times in PBS. To the pellet, PBS was added
to yield a 10% (v/v) erythrocytes/PBS suspension. The 10%
suspension of erythrocytes was then further diluted with PBS
at 1:10 ratio. 100 µL of final diluted erythrocytes was added to
(standard) and FLC-resistant C. albicans. Fluconazole (40
µg/mL) was used as positive control while untreated cells as
negative control for comparison. All the conical flasks were
1
00 µL of PBS having previously determined concentration
incubated at 35 °C for 16 h. After incubation, the cells were
gradient (1000 to 7.8 µg/mL) of test compounds in micro-
centrifuge tubes. Total hemolysis was achieved with 1% Triton
harvested at their stationary phase and the weight of pellet
was determined. The pellet was treated with 25% alcoholic
potassium hydroxide (KOH) solution followed by incubation at
X-100. The tubes were incubated for 1 h at 37 °C and then
centrifuged for 10 min at 2000 rpm and at room temperature.
From the supernatant fluid, 150 µL was transferred to a flat-
bottomed microtiter plate (Tarson), and the absorbance was
measured spectrophotometrically at 450 nm by Thermo
Multiskan spectrophotometer. The hemolysis percentage was
calculated by following equation:
85 °C for 1 h. After incubation, sterol was extracted by the
addition of n-heptane:distilled water (1:3) mixture. The
heptane layers were transferred in fresh test tube, diluted five-
fold in 100% ethanol and scanned spectrophotometrically
between 240 and 300 nm. The presence of ergosterol and the
late sterol intermediate 24(28)DHE in the extracted sample
resulted in a characteristic four peak curve. The absence of
detectable ergosterol in extract was indicated as a flat line.
Ergosterol content was calculated as percentage wet weight of
the cell by the following equation:
%Hemolysis = [(A450 of test compound treated sample – A450 of
buffer treated sample)/(A450 of 1% Triton X-100 treated sample
450 of buffer treated sample)]×100%.
–
A
Where A450 is absorbance at 450 nm.
Modeling CYP51 like protein structure. Primary protein
sequence of CYP51 was extracted from protein database at
%Ergosterol + %24(28)DHE = [(A281.5/290)×F]/pellet weight
2
2 | J. Name., 2012, 00, 1-3
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3
NCBI and its homologous sequence from PDB data bank with no response was observed, the larvae were considered to be
7
3
PDB ID-4LXJ. Modeller 9.15 was used to generate 5 models dead.
8
39
of Candida CYP51 protein using S. cervasiae CYP51 protein Three larvae were inoculated with 20 μL of the compounds 68
(
PDB ID-4LXJ) as template. Structure validation of these and 70 solutions at concentration of 1.25 or 2.5 mg/mL. Larvae
4
0
41
models was performed using Procheck , verify 3D and were then incubated at 30
°
C, in the dark, for 24 h. Haemocyte
density in larvae was ascertained by piercing the backs of the
In-silico docking. 3D structures of the two shortlisted anterior end (‘head’) of three larvae with a sterile needle and
4
2
superimposed in Chimera for structure comparison.
4
3
compounds, 68 and 70 were drawn in Chem Draw. We used collecting the yellow haemolymph (‘blood’), ensuring no white
model 3 for further in-silico analysis. Computational docking flocular material was removed – this is the fat body and will
method predicts the ligand-macromolecule bounded states impede counting. Haemolymph was diluted 1 in 10 in cold PBS
with their binding affinities using scoring function. We have containing 0.37% (v/v) 2-mercaptoethanol to reduce clotting
4
4
used AUTODOCK VINA 1.1.2 in the present study that uses and melanisation. The solution was mixed gently by pipetting.
Iterated Local Search Global Optimization algorithm for local Haemocytes were counted on a haemocytometer (0.0025
4
5,46
2
We performed in silico blind docking of the mm , BlauBrand, Germany) and the density in the original
minima search.
complete protein molecule of model 3 against the two larvae calculated.
shortlisted compounds 68 and 70. We used ADT tools to Five larvae of G. mellonella were inoculated with C. albicans at
5
process the protein and ligand PDBs into ADT desired format a density of 5×10 / 20µl and incubated at 30
°C in the dark. 1 h
PDBQT). Protein processing includes removal of water post inoculation, larvae were inoculated with compounds 68
(
molecules, addition of hydrogen and charges to the protein and 70 both at concentrations of 2.5 mg suspended in PBS
and likewise ligand was processed (68 and 70) for ADT format. supplemented with 12.5% DMSO (v/v). Three larvae were
Grid dimensions of X, Y and Z covering the complete model 3 selected and placed in a sterile pestle with 3 mL of PBS and
of CYP51 protein molecule was 64, 60 and 52 with 1 Å spacing. ground to a pulp with a mortar. The resulting homogenate was
Dimensions of the centre grid box were 23.27, 15.94 and 19.74 diluted with PBS and 100 μL of sample was plated onto three
in case of whole CYP51 molecule blind docking. Complex with YEPD-erythromycin plates (to prevent bacterial growth). The
the minimum binding energy and involving more number of homogenate was diluted 1/10 in PBS prior to plating. Plates
interactions were chosen as the basis for further interface were incubated at 30 °C for 24 h and colony forming units
analysis. We mapped and visualized these interacting partners were enumerated. The fungal load in the larvae was calculated
4
7
in PyMol. Binding affinities of both the lead compounds (68 by multiplying this figure by the relevant dilution factors.
and 70) with modeled CYP51 were quantitated and are
represented as kcal/mol. Images were prepared in LIGPLOT
4
8
Conclusions
visualizing program and polar and hydrophobic contacts
between them were noted down.
In summary a novel series of 1,2,3-triazole-amino acid hybrids
was prepared in an attempt to search for potent antifungal
agents that can be effective against FLC-sensitive and resistant
In vivo assessment of efficacy of lead inhibitors (68 and 70)
against Galleria mellonella larvae. Larvae of the sixth
developmental stage of G. mellonella were obtained from the
Meal Worm Company (Sheffield, England). Larvae of G.
strains of Candida species. Out of 24, twelve compounds (65
,
6
7-71, 73-74, 79, 82-83, 85) showed comparatively lower IC50
mellonella were stored in wood shavings in the dark at 15 °C
values against various standard Candida strains and were
prior to use. Larvae that were chosen for experiments weighed
on 0.21 g and were used within 3–4 weeks of receipt. Ten
healthy larvae were placed in sterile 9 cm petri dishes with
Whatman filter paper inserted inside. A culture of C. albicans
found to be non-cytotoxic on HEK293 cell line upto a
concentration of 200 µg/mL. Seven compounds (67-68, 70-71,
79 82) were selected for their inhibitory potential against
7
3,
,
FLC sensitive and resistant clinical C. albicans isolates and
identified two lead compounds 68 and 70 with most potent
antifungal activity against all the strains used. Time kill testing
assay confirmed the fungistatic nature of the lead compounds
8
was grown to the stationary phase (1–2×10 /mL) in YEPD broth
at 30 °C and 200 rpm. Cells were harvested by centrifugation
2,056×g for 5 minutes on a Beckmann GS-6 bench centrifuge)
(
5
washed in PBS and re-suspended in PBS at a cell density 5×10
(
68 and 70). They also effectively inhibited the extracellular
per 20 µL. Larvae were inoculated by injecting 20 µL through
the last left pro-leg into the haemocoel using a Myjector
secretion of the enzymes in proteinase and phopholipase
assays against FLC sensitive and resistant clinical C. albicans
isolates. These enzymes are responsible for the fungal invasion
of host tissues and immune suppression. TEM analysis showed
that compounds 68 and 70 caused significant damage to C.
albicans cells. Alterations in cell wall structure suggests that
these antifungal compounds may cause cell death resulting
from inhibiting the synthetic route of fungal cell wall
formation. The effect of 68 and 70 on intracellular ergosterol
syringe (Terumo Europe) and placed at 30 °C in dark. One h
post inoculation, larvae were inoculated with compounds 68
and 70, both at concentrations of 2.5 mg suspended in PBS,
supplemented with 12.5% DMSO (v/v), through the last right
pro-leg. Larvae injected with 20 µL of PBS supplemented with
12.5% DMSO (v/v) were used as controls. For assessment of
larval viability, larvae were gently probed with a needle and if
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ARTICLE
Journal Name
content in FLC-sensitive and resistant C. albicans clinical strains 11 W. L. Chaffin, J. L. López-Ribot, M. Casanova, D. Gozalbo and
were also examined which revealed inhibition of ergosterol J. P. Martínez, Microbiol. Mol. Biol. Rev., 1998, 62, 130-180.
biosynthesis thereby producing anti-Candida effect. Over all 12 S. Wang, Y. Wang, W. Liu, N. Liu, Y. Zhang, G. Dong, Y. Liu, Z.
understanding of the interaction of the lead inhibitors 68 and Li, X. He, Z. Miao, J. Yao, J. Li, W. Zhang and C. Sheng, ACS
with the modeled Candida like CYP51 protein will help in Med. Chem. Lett., 2014, , 506-511.
knowledge based design of better leads with more potent 13 R. Di Santo, A. Tafi, R. Costi, M. Botta, M. Artico, F. Corelli, M.
70
5
antifungal activity. The compounds 68 and 70 not only proved
most potent anti-Candida agents in in vitro assays but also
Forte, F. Caporuscio, L. Angiolella and A. T. Palamara, J. Med.
Chem., 2005, 48, 5140-5153.
displayed promising in vivo efficacy in G. mellonella larvae 14 M. A. Ator, S. J. Schmidt, J. L. Adams, R. E. Dolle, L. I. Kruse, C.
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was also observed which indicated the lack of an immune
Miao, Q. Jiang, J. Yao, Y. Zhou, J. Zhu and J. Lü, J. Med.
Chem., 2006, 49, 2512-2525.
response. Compound 68 caused about 50% inhibition to larvae 16 K. Pawar, A. Yadav, P. Prasher, S. Mishra, B. Singh, P. Singh
infected with C. albicans while 70 reduced 70% yeast and S. S. Komath, Med. Chem. Comm., 2015, , 1352-1359.
replication in vivo as measured at 24 hrs. Our results strongly 17 M. Irfan, B. Aneja, U. Yadava, S. I. Khan, N. Manzoor, C. G.
support that the lead inhibitors (68 and 70) provide a good Daniliuc and M. Abid, Eur. J. Med. Chem., 2015, 93, 246-254.
starting point for lead optimization and efficacy for clinical 18 B. Aneja, M. Irfan, M. I. Hassan, P. Amresh, U. Yadava, C. G.
6
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Daniliuc, M. Zafaryab, M. M. A. Rizvi, A. Azam, M. Abid, J.
Enzyme Inhib. Med. Chem., 2015, 1-19.
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9 M. M. Masood, V. K. Pillalamarri, M. Irfan, B. Aneja, M. A.
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Mohammad Abid gratefully acknowledges UGC, Govt. of India
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for Raman Postdoctoral Fellowship in USA (F. No. 5-123/2016
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