Efficient click chemistry towards fatty acids containing 1,2,3-triazole: Design and synthesis as potential antifungal drugs for Candida albicans

Article abstract of DOI:10.1016/j.ejmech.2017.05.001

Candida is an important opportunistic human fungal pathogen. The cis-2-dodecenoic acid (BDSF) showing in vitro activity of against C. albicans growth, germ-tube germination and biofilm formation has been a potential inhibitor for Candida and other fungi.

Full text of DOI:10.1016/j.ejmech.2017.05.001

Accepted Manuscript
Efficient click chemistry towards fatty acids containing 1,2,3-triazole: Design and
synthesis as potential antifungal drugs for Candida albicans
Nina Fu, Suiliang Wang, Yuqian Zhang, Caixia Zhang, Dongliang Yang, Lixing Weng,
Baomin Zhao, Lianhui Wang
PII:
S0223-5234(17)30362-8
10.1016/j.ejmech.2017.05.001
EJMECH 9431
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 February 2017
Revised Date: 10 April 2017
Accepted Date: 1 May 2017
Please cite this article as: N. Fu, S. Wang, Y. Zhang, C. Zhang, D. Yang, L. Weng, B. Zhao, L. Wang,
Efficient click chemistry towards fatty acids containing 1,2,3-triazole: Design and synthesis as potential
antifungal drugs for Candida albicans, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.05.001.
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Graphical Abstract
Efficient Click Chemistry towards Fatty Acids Containing
1,2,3-Triazole: Design and Synthesis as Potential Antifungal Drugs
for Candida albicans
Nina Fu,a Suiliang Wang,a Yuqian Zhang,a Caixia Zhang,a Dongliang Yang,a Lixing Weng,b
Baomin Zhao,a,* Lianhui Wang a,*
a
Key Laboratory for Organic Electronics and Information Displays & Institute of
Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for
Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9
Wenyuan Road, Nanjing 210023, China.
b School of Geography and Biological Information, Nanjing University of Posts &
Telecommunications, 9 Wenyuan Road, Nanjing 210023, China.
Two-steps in “one-pot” synthesis of ATC compounds, BDSF analogues, for efficient application as
anti-fungal agents. The design, synthesis and bioactivity performance were all described.

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Efficient Click Chemistry towards Fatty Acids Containing
1,2,3-Triazole: Design and Synthesis as Potential Antifungal Drugs
for Candida albicans
Nina Fu,a Suiliang Wang,a Yuqian Zhang,a Caixia Zhang,a Dongliang Yang,a Lixing Weng,b
Baomin Zhao,a,* Lianhui Wang a,*
a
Key Laboratory for Organic Electronics and Information Displays & Institute of
Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for
Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9
Wenyuan Road, Nanjing 210023, China.
b School of Geography and Biological Information, Nanjing University of Posts &
Telecommunications, 9 Wenyuan Road, Nanjing 210023, China.
ABSTRACT
Candida is an important opportunistic human fungal pathogen. The
cis-2-dodecenoic acid (BDSF) showing in vitro activity of against C. albicans growth,
germ-tube germination and biofilm formation has been a potential inhibitor for
Candida and other fungi. In this study, facile synthetic strategies toward a novel
family of BDSF analogue, 1-alkyl-1H-1,2,3-triazole-4-carboxylic acids (ATCs) was
developed. The straightforward synthetic method including converting the
commercial available alkyl bromide to alkyl azide, consequently with a typical click
chemistry method, copper(II) sulfate and sodium ascorbate as catalyst in water to
furnish ATCs with mild to good yields. According to antifungal assay,
1-decyl-4,5-dihydro-1H-1,2,3-triazole-4-carboxylic acid（5d） showed antifungal
capability slightly better than BDSF. The 1,2,3-triazole unit played a crucial role for
the bioactivity of ATCs was also confirmed when compared with two alkyl-aromatic
carboxylic acids. Given its simplicity, high antifungal activity, and wide availability of
compounds with halide atoms on the end part of the alkyl chains, the method can be
extended to develop more excellent ATC drugs for accomplishing the challenges in
future antifungal applications.

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KEYWORDS Candida albicans, click chemistry, BDSF analogue,
1H-1,2,3-triazole, antifungal agents

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1. Introduction
Fungal infections represent a major problem in human health care and difficult to
treat [1]. Candida albicans (C. albicans) is the most common fungal pathogen,
causing invasive infections that are associated with high mortality [2-4]. Treatment of
C. albicans infections is imperfect because current drugs have significant side effects,
and resistance is developing for these drugs. For example, pathogen resistance to
fluconazole is widespread [2,5,6], and resistance to amphotericin B (AmpB) is on the
rise [7,8]. However, the development of drugs that specifically inhibit fungal growth
without deleterious effects on patients is challenging because both fungi and humans
are eukaryotes. The discovery of natural antimicrobic agents and synthetic analogues
for potential therapeutic application has been the extensively developed strategy in
this area [9].
The ability of C. albicans to cause disease has been linked to dimorphic
switching between yeast to hyphal phase [10], a process that is induced by many
different environmental factors [11,12], and by antifungal agents.
Low-molecular-mass fatty acids, including the diffusible signal factor
cis-2-dodecenoic acid (BDSF) from Burkholderia cenocepacia, have been shown to
regulate the C. albicans dimorphic switch [13,14]. Further analyses have shown that C.
albicans germ tube formation is strongly inhibited by exogenous addition of
physiological levels of BDSF [14,15]. We recently described that BDSF and
trans-BDSF were more effective against biofilm formation than farnesol and the
diffusible signal factor cis-11-methyl-2-dodecenoic acid [16]. Although showing high
antifungal efficiency, the investigation on BDSF and its analogues are still limited by
their drawbacks, such as requirement of sophisticated synthesis and difficulty in
structural optimization. Typically, the synthetic strategy toward BDSF and its
analogues suffers several drawbacks (as shown in Scheme 1) [17,18], including
tedious isolating of the side-products, handling toxic and volatile bromine, using large
amount of moisture-sensitive methyl lithium, moreover, leading to a very low total
yield. In addition, with this synthetic strategy, it is difficult to develop novel BDSF

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analogues with olefin chains containing functional groups which sensitive to strong
base, methyl lithium or bromine. As a result, there is a demand to develop novel
synthetic methodology towards BDSF analogues with efficient medicinal effect.
Scheme 2 synthesis of BDSF and trans-BDSF derivatives
1,2,3-Triazole ring system is a very well-recognized pharmacophore [19,20], which
is derived from Huisgen's 1,3-dipolar cycloaddition of azides and alkynes by
copper-catalyzed click reaction [21,22]. This nitrogen containing heterocycle is
renowned among U.S. FDA approved pharmaceuticals [23]. The moiety is attractive
connecting unit, since it is stable to metabolic degradation, oxidative/reductive
conditions and actively participates in binding to biomolecular targets and improves
its solubility by hydrogen bonding and dipole interactions [24]. There are several
reports available in literature about the biological relevance of 1,2,3-triazoles against
cancer [25], HIV [26], influenza [27], tuberculosis [28], malaria [29], leishmaniasis
[30], inflammation [31], analgesic [32], obesity [33], and also to bacterial infections
[34-38]. Although, 1,2,3-triazole derivatives have gained special attention in the
discovery of new antibiotics used for the treatment of bacterial infections, the
antifungal activity has rarely been documented.
Scheme 2. Design principle of alkyl-1,2,3-triazole-carboxylic acid (ATCs).
As we have found that the double-bond on the α-β position of fatty acid is crucial
for the anti-fungal property of BDSF [13-16]. Inspired by the above research works
on both 1,2,3-triazole moiety and as well as our research exploration on BDSF to find
new and effective antifungal agents, a new design principle toward anti-C. albicans

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agents was speculated by the replacement of this double bond with 1,2,3-triazole
moiety (as shown in Scheme 2). We considered the replacement of the double bond in
BDSF with the chemically stable heterocyclic 1H-1,2,3-triazole ring would not
compromise their medicinal property, because they were close structural analogues. In
this contribution, a new series of 1,2,3-triazole hybridized BDSF analogues was
designed and synthesized by combination of 1,2,3-triazaole and α, β-unsaturated fatty
acid moieties, named as ATCs (Scheme 3). The efficient and simple one-pot synthetic
method towards ATCs was presented. The inhibiting effects of ATCs on C. albican
yeast and hyphal cell growth were assayed and BDSF was used as control. The results
indicate when the carbon number of the alkyl chains reached 8, the ATCs will show
similar or better medical effects as BDSF. In addition, the 5d displayed better C.
albicans inhibiting performance than BDSF. Considering the conjugation effect of
1,2,3-triazle ring with carboxylic acid group, we evaluated another two alkyl-aromatic
carboxylic acids to elucidate that the double bond in 1H-1,2,3-triazole is different
from that in other aromatic systems.
2. Results and discussion
2.1. Chemistry
Adopting the hybridization approach, a series of ATCs was synthesized using
copper(I)-catalyzed Huisgen’s 1,3-dipolar cycloaddition reaction of alkyl-azides with
a terminal alkyne containing carboxylic acid group. Synthetic route and chemical
structures of the mentioned ATCs were both depicted in Scheme 3. The alkyl-azides
were obtained in the reaction of sodium azide with alkyl-1-bromide compounds in the
present of base in dimethyl sulphoxide (DMSO) in mild condition with high yields
[39]. 1,2,3-Triazole-BDSF hybrids comprising various 4-alkyl-1,2,3-triazole subunits
were subsequently prepared by click chemistry of alkyl-azides and propiolic acid in
the presence of Cu(I), as the catalyst, which was generated in situ by reaction of
copper(II) sulfate and L-ascorbic acid sodium, as reducing agent [40-41]. The
reactions were performed under mild conditions with a “one-pot” fashion to give
ATCs with a good yields ranging from 53~78%. The versatility of target hybrids
could be further extended to the synthesis of more complicated BDSF analogues by

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incorporating other antifungal or antibacterial building blocks. To confirm the
relationship of 1,2,3-triazole with the medical effect of ATCs, we prepared another
two aromatic carboxylic acid containing alkyloxyl-benzene (8) and alkylthienyl group
(9) (see Scheme 3).
Scheme 3. General synthetic route to 5a-d, their structural analogues 8 and 9.
2.2. In vitro biological evaluations and physicochemical profile of ATCs.
2.2.1. Effects of ATCs on C. albicans yeast growth.
To investigate the antifungal activity of ATCs, all ATCs were incubated with C.
albicans yeast cells for 24 hr to study their inhibitory effects on C. albicans yeast
growth. BDSF was also used as positive control. The OD530 of all entries in presence
of drugs with adverse final concentrations of 7.5, 15, 30, 45, 60, 120, 200 and 300 µM
were recorded. As shown in Figure 1, the ATCs with different chemical structure (the
length of alkyl chains) displayed different inhibitory effects at each of the tested
concentrations during C. albicans yeast growth. All ATCs and BDSF tested had no
apparent effect on yeast growth at low concentrations (<15 µM), while the
concentrations were higher than 30 µM, 5c, 5d and BDSF had obvious inhibitory
effect on the yeast growth. When the concentration reached 60 µM, 5c, 5d and BDSF
can inhibit yeast-cell growth significantly by approximately 90%. In contrast, 5a and
5c with shorter alkyl chains can only slightly influence the growth of yeast cells at

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high concentrations (>120 µM). These data suggest that 5d（see Scheme 3）, with the
longest alkyl chain is the most effective inhibitor, followed by 5c, 5b and 5a. Both 5d
and 5c exhibited the similar inhibitory effect-concentration trends as BDSF. Thinking
about their chemical structures, it is found that there are the same atom numbers along
the molecular backbones in 5d and BDSF. These results indicate the atom number in
molecular backbone impart influence on their inhibitory performance.
Fig. 1 Effect of four ATCs (5a-d) and BDSF on C. albicans yeast-cell growth.
The OD 530 values were determined after 24 hr growth at 37 oC with agitation.
2.2.2. ATC drugs inhibit germ-tube formation in C. albicons
As reported previously, the polymorphic C. albicans is harmful in its filamentous
form. Thus, it is very important to understand the antifungal activity of ATC drugs by
treating C. albicans yeast-cells with these compounds, especially their performance in
blocking the dimorphic transition from yeast form to hyphal phase. Four of our ATC
drugs were added to fresh fungal yeast cells and compared their bioactivity
quantatively using medium and growth conditions described previously. Methanol
o
was used as negative control. As shown in figure, after incubation at 37 C for 4
hr, >90% of the C. albicans yeast cells in the control wells formed germ tube and
were in their hyphal phase. As to 5a and 5b, they showed no apparent effect on the
germ tube germination at various concentrations (from 30, 60, 120, 200 to 300 µM),
but the new-born germ tube length is slightly shorter than that of the hyphal cells in
the control wells (images shown in Supporting Information Figure S13-15). In
contrast, both 5d and 5c drugs repressed the yeast-to-hyphal conversion significantly

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by approximately 95% at concentrations >200 µM. 5c and 5d, the two closely
structural analogues of BDSF, which can block the yeast-to-hyphal conversion
efficiently, showed the similar antifungal behavior, suggesting they are promising
inhibitors for C. albicans germ tube germination.
Fig. 2 Effects of ATCs on grem tube formation in C. albicans. Cells were grown
in G RPMI medium medium containing 100 ATCs at 37 °C. At least 200 cells were
counted for each sample after every 2 hr of cultivation. Bars represent standard
deviations of three replicates.
Fig. 3 Images for the dosage effects of 5d on morphology of C. albicans at the
concentration of 0 µM (a), 30µM (b), 60 µM (c), 120 µM (d), 200 µM (e), 300 µM (f).

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About 200 yeast cells in each treatment．
2.2.3. ATCs inhibition on C. albicans hyphal growth
Further, inhibitory effects of ATC drugs and BDSF on fungal hyphal growth were
also investigated on commercially available, presterilized, flat-bottomed 96-well
polystyrene microtiter plates. After the fresh yeast cells grew for 4 hr at 37 oC to make
a population of 95% C. albicans cells in their hyphal form, ATC drugs and BDSF
were added to each wells to make a final concentration of 200 μM for each treatment.
Then, microscopic images were captured at 0, 3, 6 and 9 hr. Since 5b showed similar
bioactivity as 5a, only 5a, 5c and 5d were showed in Figure 4. Obviously, the result in
the presence of 5a showed the same trend as the blank (negative control, no drug
added), the length of germ tubes grew from 66.5 ± 12.3 μm to 142.8 ± 21.7μm (at 3
hr), then to 207.0 ± 18.9 μm (at 6 hr) and final to 223.5 ± 34.0 μm (at 9 hr). Note that
the hyphal cell density was increased dramatically after the cells grew for 9 hr. 5c
exhibited an apparent growth inhibiting property that both the germ tube length and
the hyphal cells density were not increased. 5d showed the best performance that the
germ tube length was slightly reduced (from 71.9 ± 12.4 μm to 53.3 ± 9.3 μm), and no
increase of cell density was observed, outperforming the close structural analogue of
BDSF. These results suggest that 5d and 5c can both inhibit the growth of C. albicans
hyphal cells as BDSF, indicating they can be used as anti-C. albicans inhibitors as
BDSF.

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Fig. 4 Images of ATCs (5a, 5c and 5d) inhibition on C. albicans hyphal growth.
o
Images collected after yeast cells were incubated at 37 C for 4 hr in GS medium
supplemented with ATCs (BDSF) at a concentration of 200 μM.
Table 1. Effect of ATCs (5a, 5c and 5d) on hyphae growth of C. albicans. Data
o
collected after yeast cells were incubated at 37 C for 4 hr in GS medium
supplemented with ATCs or BDSF at a concentration of 200 μM.
Drug
Length
Blank
5a
5c
5d
BDSF
/µm
Time/h
70.3 ± 12.3a
149.9 ± 22.3
235.2 ± 40.4
223.9 ± 24.5
66.5 ± 12.3
142.8 ± 21.7
207.8 ± 18.9
223.5 ± 34.1
73.4 ± 15.1
64.4 ± 21.3
54.6 ± 15.3
74.2 ± 14.7
71.8 ± 12.4
59.1 ± 16.5
45.0 ± 11.9
53.3 ± 9.3
65.3 ± 11.5
73.3 ± 13.7
63.5 ± 14.3
64.2 ± 9.0
0
3
6
9
a
In each treatment, about 200 cells were counted. All data were summarized from
five repeating experiments.
2.3. 8 and 9 inhibitory effects on C. albicans germ tube germination.

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Fig. 5 The dosage effects of 8 and 9 on yeast cell growth of C. albicans at the
concentration of 30，60，120，200 and 300μM. Up row is in presence of 8, and down
row in presence of 9. About 200 yeast cells in each treatment．
The chemical structures of ATCs are different from BDSF by replacing the cis
double bond at the
α−β position with 1,2,3-triazole unit. As is well known, the
aromatic property of triazole ring is stronger than that of the double bond, which will
partially influence the bioactivity of ATCs. Note that 5c and 5d could work as BDSF
and show effectively inhibition on the hyphal development of C. albicans. We were
inspired to imagine that alkyl aromatic carboxylic acids with alkyl chain containing 8
to 10 carbon atoms would either or not display the similar inhibitory effects as ATCs
and BDSF. Thus, 8 and 9, two close analogues of 5c, were synthesized and their
bioactivity on C. albicans yeast cell growth were also examined. In the plate
bioassays, fungal cells grew as budding yeast form in the presence of 30, 60, 120, 300
µM 8 and 9, over 95 % of C. albicans yeast cells switched into the hyphal form after 2
hr incubation. This result shows that 8 and 9 cannot effectively inhibit germ-tube
formation even at high concentration. These results indicate that cis configuration of
the fatty acid in BDSF and 1,2,3-triazole unit in ATCs both play a critical role in the
biological activity. And, the cis-double-bond at the α−β position of BDSF and
1,2,3-triazole unit in ATC drugs cannot be replaced by other aromatic rings like
benzene or thiophene.

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3. Conclusions
In summary, the current study provided a facile approach toward a novel series of
1-alkyl-1H-1,2,3-triazole-4-carboxylic acids (ATCs) based on the structure of BDSF
and trans-BDSF. We identified potent anti-C. albicans agents among them. The most
active compound of 5d was found to display in vitro activity of inhibiting the yeast,
hyphal and germ tube growth more efficient than BDSF. Furthermore, we have
demonstrated that 1,2,3-triazole hybridized structure is crucial for their medicinal
property by comparison with two alkyl-aromatic carboxylic acids. Considering the
wide availability of compounds consisting of alkyl chain with iodo- or bromo- atoms
on the end, the application of the methodology presented by us we be easily
broadened in future integrated other medicinal acitivities, such as anti-bacterials or
anti-cancers. We expect our strategy of synthesis of ATC agents can open new
opportunities for antifungal agents in future biological and medical research and clinic
applications.
4. Experimental Section
4.1. Materials and characterization
Dimethyl sulfoxide (DMSO), ethanol, sodium azide with analytical grade were
purchased from sinopharm chemical reagent Co., Ltd. Silica gel (200~300 mesh) was
purchased from Qingdao Haiyang Company. 1-Bromohexane, 1-bromooctane,
1-ol-6-bromohexane, 1-bromodecane, copper sulfate pentahydrate, and sodium
ascorbate were purchased from Aldrich Chemical. All reagents and chemicals were
used as delivered without further treatment unless mentioned otherwise. Anhydrous
tetrahedronfuran (THF) was freshly distilled from sodium metal in presence of
benzophenone.
The NMR spectra were recorded on a Bruker AV 400 MHz NMR spectrometer
using DMSO-d6 or CDCl3 solution with tetramethylsilane (TMS) as an internal
o
standard at 25 C. Elemental analysis was performed with a Vario EL III Elementar
system (Elementar Analyzen-systeme, Hanau, Germany). Elemental analyses were
the average of three runs. All samples were measured simultaneously for C, H and N.

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Microscopy images were collected by utilizing an OLYMPUS IX71 Inverted
fluorescence microscope, equipped with a CCD. Cell concentration was determined
with a BioTech PowerWave XS2 Microplate spectrophotometer. The clean medium
and culture media were sterilized with a TOMY SX-500 High Pressure Sterilization
pot.
4.2. Synthesis
Typical procedure for the synthesis of ATC compounds. In a 100 mL two-necked
round-bottom flask (RBF), sodium azide (0.975g, 15 mmol), KI (0.083g， 0.5mmol)
and 1-bromohexane (1.64g，10mmol) were mixed in 30 mL of DMSO, and the
o
solution was stirred at 60 C under argon atmosphere for 16 hr. After the reaction
mixture was cooled to room temperature, a solution of sodium ascorbate (67 mg, 0.33
mmol) in ethanol (3 mL) was added in one portion. CuSO4•5H2O (62.5 mg， 0.25
mmol) in H2O (3 mL) was added dropwise. Then，propiolic acid (0.42g，6 mmol) was
o
added at three portions. The reaction was kept stirring under N2 at 65 C for another
10 hr. After the reaction was cooled to room temperature, the solution was neutralized
with 0.5 M HCl before being extracted with ethyl acetate (EA). Pure products were
obtained by silica gel chromatography with eluent of petroleum/EA (6:1, v/v).
Syntheis of 1-hexyl-1H-1,2,3-triazole-4-carboxylic acid (5a). White solid. Total
o
1
yield, 78%; mp. 171-173 C. H NMR (400 MHz, DMSO-d6) δ (ppm): 8.68 (s, 1H),
13
4.45 – 4.31 (t, 2H), 1.83– 1.79 (m, 2H), 1.24 (m, 6H), 0.82 (t, 3H). C NMR (100
MHz, DMSO-d6) δ (ppm): 162.22, 140.06, 129.19, 50.07, 30.97, 29.88, 25.83, 22.33,
14.26. Anal. calcd (%) for C9H15N3O2: C, 54.81; H, 7.67; N, 21.30, Found (%): C,
54.83; H, 7.59; N, 21.34.
Syntheis of 1-(6-hexanol)-1H-1,2,3-triazole-4-carboxylic acid (5b). White solid.
Total yield, 53%; mp. 116-117 oC. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.67 (s,
13
1H), 4.39 (m, 2H), 1.83 (m, 2H), 1.40 (m, 2H), 1.27−1.18 (m, 6H). C NMR (100
MHz, DMSO-d6) δ (ppm): 162.23, 140.13, 129.17, 61.00, 50.07, 29.98, 26.09, 25.31.
Anal. calcd (%) for C9H15N3O3: C, 50.69; H, 7.09; N, 19.71, Found (%): C, 50.73; H,
7.07; N, 19.73.
Syntheis of 1-octyl-1H-1,2,3-triazole-4-carboxylic acid (5c). White solid. Total
o
1
yield, 65%; 138-139 C. H NMR (400 MHz, DMSO-d6) δ (ppm): 8.67 (s, 1H), 4.37

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(t, 2H), 1.85 – 1.79 (m, 2H), 1.26 – 1.17 (m, 10H), 0.83 (t, 3H). 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 162.21, 140.06, 129.19, 50.07, 31.60, 29.92, 28.93, 28.74, 26.17,
22.49, 14.35. Anal. calcd (%) for C11H19N3O2: C, 58.64; H, 8.50; N,18.65, Found (%):
C, 58.73; H, 8.41; N, 18.63.
Syntheis of 1-decyl-1H-1,2,3-triazole-4-carboxylic acid (5d). White solid. Total
o
1
yield, 59%; mp. 171-172 C. H NMR (400 MHz, DMSO-d6) δ (ppm): 8.67 (s, 1H),
4.39 (t, 2H), 1.83 (m, 2H), 1.25 (d, 14H), 0.83 (t, 3H). 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 162.21, 140.06, 129.20, 50.07, 31.73, 29.91, 29.32, 29.27, 29.10,
28.76, 26.16, 22.54, 14.39. Anal. calcd (%) for C10H23N3O2: C, 61.63; H, 9.15; N,
16.59, Found (%): C, 61.67; H, 9.11; N, 16.61.
Syntheis of 4-octyloxylphenyl-carboxylic acid (8). In a freshly dried Schlenck tube
(100 mL), 4-bromo-1-octyloxylbezene (5.70 g, 20 mmol) was dissolved in dry THF
o
(40 mL). The solution was cooled to −78 C in a dry ice/acetone bath. Then, n-BuLi
(1.6 M in hexane, 15 mL) was added dropwise. After the lithium exchange reaction
finished, dry CO2 was bubbled into the solution via a long needle for 2 hr. Later, the
extraction with ethyl acetate was conducted before neutralized with 1 M HCl. Pure
product was isolated by silica gel chromatography with eluent of petroleum/EA (8:1,
o
1
v/v). White solid. Total yield, 75%; mp. 112-113 C. H NMR (400 MHz, CDCl3) δ
(ppm): 8.07 (d, 2H), 6.94 (d, 2H), 4.04 (t, 2H), 1.84 (m, 2H), 1.48 (m, 2H), 1.35 (m,
8H), 0.91 (t, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm): 172.16, 163.72, 132.35,
121.40, 114.20, 68.30, 31.82, 29.34, 29.24, 29.10, 26.00, 22.67, 14.11. Anal. calcd (%)
for C15H22O2: C, 71.97; H, 8.86, Found (%): C, 72.08; H, 8.84.
Syntheis of 5-octylthienyl-carboxylic acid (9). 9 was synthesized using a similar
o
1
procedure as 8. White solid. Total yield, 82%; mp. 112-113 C. H NMR (400 MHz,
CDCl3) δ 7.72 (d, J = 1.5 Hz, 1H), 7.25 – 7.23 (m, 1H), 2.67 – 2.56 (m, 2H), 1.70 –
1.54 (m, 2H), 1.35 – 1.22 (m, 12H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 167.95, 144.51, 135.97, 132.31, 129.24, 31.87, 30.42, 30.24, 29.48, 29.24,
29.19, 22.68, 14.12. Anal. calcd (%) for C9H15N3O2: C, 64.96; H, 8.39, Found (%): C,
65.06; H, 8.36.
4.3. C. albicans assay
4.3.1. Strains, media and culture conditions
Wild type C. albicans strain SC5314 was used in this study. SC5314 was routinely
o
grown at 37 C in YPD agar (1% yeast extract, 2% peptone, 2% dextrose, 2% agar).

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To prepare a standard cell suspension, a single colony was inoculated into YPD
o
medium and incubated for 18 hr at 37 C with agitation. The fungal cells were
harvested by centrifugation, washed twice with PBS (pH 7.2) buffer solution, and
resuspended at a cell concentration of 1×107 cells/mL. RPMI medium containing
34.53 g/L morpholinepropanesulfonic acid (MOPS) were used for hyphal induction at
o
37 C. Stock solution was prepared by dissolution in distilled methanol and kept at
o
−20 C until use. When incubated with C. albicans, the final concentration of
methanol was 1% in all assays. GS medium (pH 7.0) containing L-glucose (5 g/L),
Na2HPO4·12H2O (0.26 g/L), KH2PO4 (0.66 g/L), MgSO4·7H2O (0.88 g/L), NH4Cl
(0.33g/L), biotin (16 µg/L) and 1% (v/v) bovine serum.
4.3.2. In vitro susceptibility C. albicans yeast to ATCs assay
The antibacterial susceptibility testing was performed using the standard broth
microdilution method (BMM) and interpreted as per CLSI guideline. A standard cell
suspension of C. albicans was diluted (at 1×105 cells/mL) in YPD medium added
methanol or various concentrations (300、200、120、60、45、30、20、15、7.5 µM)
of ATC compounds or BDSF. The mixture (150 µL, 1×105 cells/mL) was transferred
to flat-bottomed 96-well polystyrene microtiter plates and cultured at 37 oC for 24 hr.
Then, the optical density of the supernatant was measured at 530 nm with a
microtiter plate reader (OD 530 nm measurement).
4.3.3. ATCs inhibition on C. albicans hyphal growth assay
Inhibitory effects of ATC compounds and BDSF on fungal hyphal growth were
investigated on commercially available, presterilized, flat-bottomed 96-well
polystyrene microtiter plates. A standard cell suspension of C. albicans was diluted (at
1×105 cells/mL) in GS medium supplemented with methanol or 100 µM of ATC
compounds, then 150 µL cells suspension was transferred to the wells. BDSF was
used as a positive control. To investigate the effect of ATCs on germ tube generation,
o
cells were grown at 37 C for 4 hr, then every 2 hr took a record, 95% of candida
cells in GS medium with methanol were switched into their mycelium phase.

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Aliquots of fungal cells were visualized by differential interference contrast (DIC)
microscopy, photographs and calculation of yeast-form number in each sample were
recorded.
Acknowledgement
This work was financially supported by the National Natural Science Foundation of
China (21204038, 51103074, 51203077), the National Synergistic Innovation Center
for Advanced Materials (SICAM), the Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD) and Program for Changjiang Scholars
and Innovative Research Team in University (IRT-15R37).
Supplementary data
The spectra of synthesized compounds are given in supplementary material for this
article. Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.XXX.
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CAPTIONS LIST
Scheme 1 Typical synthetic routes of BDSF and trans-BDSF derivatives.
Scheme 2. Design principle of alkyl 1,2,3-triazole carboxylic acids (ATCs).
Scheme 3. General synthetic route to ATCs, 8 and 9.
Fig. 1 Effect of four ATCs (5a-d) and BDSF on C. albicans yeast-cell growth. The
OD 530 values were determined after 24 hr growth at 37 oC with agitation.
Fig. 2 Effects of ATCs on grem tube formation in C. albicans. Cells were grown in G
RPMI medium medium containing 100 ATCs at 37 °C. At least 200 cells were
counted for each sample after every 2 hr of cultivation. Bars represent standard
deviations of three replicates.
Fig. 3 Images for the dosage effects of 5d on morphology of C. albicans at the
concentration of 0 µM (a), 30µM (b), 60 µM (c), 120 µM (d), 200 µM (e), 300 µM (f).
about 200 yeast cells in each treatment．
Fig. 4 Images of ATCs inhibition on C. albicans hyphal growth. Images collected
o
after yeast cells were incubated at 37 C for 4 hr in GS medium supplemented with
ATCs (BDSF) at a concentration of 200 μM.
Fig. 5 The dosage effects of 8 and 9 on yeast cell growth of C. albicans at the
concentration of 30，60，120，200 and 300μM. Up row is in presence of 6, and down
row in presence of 7. About 200 yeast cells in each treatment．

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Table 1. Effect of ATCs on hyphae growth of C. albicans. Data collected after yeast
o
cells were incubated at 37 C for 4 hr in GS medium supplemented with ATCs or
BDSF at a concentration of 200 μM.

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1) A series of 1-alkyl 1H-1,2,3-triazole-4-carboxylic acids (ATCs) have been explored as novel
anti-fungal inhibitors for treatment of C. albicans.
2) This two-step in one-pot strategy can be extended to multiple function anti-fungal drugs.
3) 5c and 5d both showed advanced medicinal activity in vitro.
4) Medicinal effects and structural relationship of 1,2,3-triazole moiety has been identified.

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Supporting Information for :
Efficient Click Chemistry towards Anti-Fungal fatty acids
Containing 1,2,3-Triazole Subunit for Candida albicans
Nina Fu,a Suiliang Wang,a Yuqian Zhang,a Caixia Zhang,a Dongliang Yang,a Lixing Weng,b
Baomin Zhao,a,* Lianhui Wang a,*
a
Key Laboratory for Organic Electronics and Information Displays & Institute of
Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for
Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9
Wenyuan Road, Nanjing 210023, China.
b School of Geography and Biological Information, Nanjing University of Posts &
Telecommunications, 9 Wenyuan Road, Nanjing 210023, China.
E-mail addresses: iambmzhao@njupt.edu.cn，iamlhwang@njtech.edu.cn

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Table of Contents
1. NMR Spectra of ATC compounds
1.1 Spectroscopic data of 5a
1.2 Spectroscopic data of 5b
1.3 Spectroscopic data of 5c
1.4 Spectroscopic data of 5d
1.5 Spectroscopic data of 6
1.6 Spectroscopic data of 7
2. Effects of 5a，5b and 5c on C. albicans yeast growth
2.1 Effects of 5a on C. albicans yeast growth
2.2 Effects of 5b on C. albicans yeast growth
2.3 Effects of 5c on C. albicans yeast growth

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1. Spectra
1.1 Spectroscopic data of 5a
Figure S1. 1H NMR spectrum of 5a in DMSO-d6.
Figure S2. 13C NMR spectrum of 5a in DMSO-d6.

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1.2 Spectroscopic data of 5b
Figure S3. 1H NMR spectrum of 5b in DMSO-d6.
Figure S4. 13C NMR spectrum of 5b in DMSO-d6.

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1.3 Spectroscopic data of 5c
Figure S5. 1H NMR spectrum of 5c in DMSO-d6.
Figure S6. 13C NMR spectrum of 5c in DMSO-d6.

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1.4 Spectroscopic data of 5d
Figure S7. 1H NMR spectrum of 5d in DMSO-d6.
Figure S8. 13C NMR spectrum of 5d in DMSO-d6.

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1.5 Spectroscopic data of 8
Figure S9. 1H NMR spectrum of 8 in CDCl3.
Figure S10. 13C NMR spectrum of 8 in CDCl3.
1.6 Spectroscopic data of 9