Synthesis and evaluation of naphthyl bearing 1,2,3-triazole analogs as antiplasmodial agents, cytotoxicity and docking studies

Article abstract of DOI:10.1016/j.bmc.2016.10.029

Novel series of naphthyl bearing 1,2,3-triazoles (4a–t) were synthesized and evaluated for their in vitro antiplasmodial activity against pyrimethamine (Pyr)-sensitive and resistant strains of Plasmodium falciparum. The synthesized compounds were assessed

Full text of DOI:10.1016/j.bmc.2016.10.029

Accepted Manuscript
Synthesis and evaluation of Naphthyl bearing 1,2,3-Triazole analogs as Anti-
plasmodial agents, Cytotoxicity and Docking Studies
Balabadra Saikrishna, Kotni MeenaKumari, Manga Vijjulatha, Aparna Devi
Allanki, Rajesh Prasad, Puran Singh Sijwali
PII:
S0968-0896(16)30900-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.10.029
BMC 13356
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
5 October 2016
22 October 2016
24 October 2016
Please cite this article as: Saikrishna, B., MeenaKumari, K., Vijjulatha, M., Devi Allanki, A., Prasad, R., Singh
Sijwali, P., Synthesis and evaluation of Naphthyl bearing 1,2,3-Triazole analogs as Antiplasmodial agents,
Cytotoxicity and Docking Studies, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.10.029
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Synthesis and evaluation of Naphthyl bearing 1,2,3-Triazole analogs as
Antiplasmodial agents, Cytotoxicity and Docking Studies
Balabadra Saikrishnaa, Kotni MeenaKumaria, Manga Vijjulathaa,*, Aparna Devi Allankib, Rajesh
Prasadb, Puran Singh Sijwalib
aMolecular Modeling and Medicinal Chemistry Group, Department of Chemistry, Osmania
University, Hyderabad-500007 Telangana, India
bCentre for Cellular and Molecular Biology, Habsiguda, Uppal Road, Hyderabad-500007
Telangana, India
*To whom correspondence should be addressed. Tel: 040-27682337. Fax: 040-27090020
Email: vijjulathamanga@gmail.com
Abstract
Novel series of naphthyl bearing 1,2,3-triazoles (4a-t) were synthesized and evaluated for
their in vitro antiplasmodial activity against pyrimethamine (Pyr)-sensitive and resistant strains
of Plasmodium falciparum. The synthesized compounds were assessed for their cytotoxicity
employing human embryonic kidney cell line (HEK-293), and none of them was found to be
toxic. Among them 4j, 4k, 4l, 4m, 4n, 4t exhibited significant antiplasmodial activity in both
strains, of which compounds 4m, 4n and 4t (~3.0 fold) displayed superior activity to Pyr against
resistant strain. Pyr and selected compounds (4n, 4p and 4t) that repressed parasite development
also inhibited PfDHFR activity of the soluble parasite extract, suggesting that anti-parasitic
activity of these compounds is a result of inhibition of the parasite DHFR. In silico studies
suggest that activity of these compounds might be enhanced due to π-π stacking.
Keywords
Naphthyl 1,2,3-triazoles; Click chemistry; Antiplasmodial activity; Cytotoxicity; Induced fit
docking(IFD)

1. Introduction
Malaria remains as one of the world’s most widespread and dreadful infectious diseases.
Recent estimates indicate approximately 4,38,000 deaths worldwide in 2015, that predominantly
trait children under 5 years of age, pregnant women and patients with HIV [1]. Drug resistance is
a major cause of morbidity and mortality [2], complicates and impedes current efforts in the
treatment of malaria and its control. Antifolates are an important class of antimalarial drugs that
target folate metabolism is essential for survival of malarial parasite. Several compounds with
Quinoline scaffold are found to be effective against the parasite’s Dihydrofolate reductase
(DHFR), a key enzyme in the folate biosynthetic pathway [3].
Point mutations in DHFR have been implicated in drug resistance against antifolates [4].
Dihydrofolate reductase-Thymidylate synthase (DHFR-TS) is a bifunctional enzyme of P.
falciparum remains as one of the popular targets for malaria chemotherapy [5-10]. This enzyme
catalyzes the Nicotinamide Adenine Dinucleotide Phosphate (NADPH) dependent reduction of
Dihydrofolate (DHF) to Tetrahydrofolate (THF) that is essential for DNA synthesis. Inhibition of
DHFR enzyme interrupts DNA synthesis, which ultimately leads to the death of the parasite.
Triazoles scaffold have garnered much attention in recent times due to their wide range of
application in medicinal chemistry. The Huisgen1,3-dipolar cycloaddition reactions of azides and
alkynes is an exceptionally atom-economical, forthright route to yield 1,2,3-triazoles [11]. The
copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) has emerged as an
outstanding procedure for access to 1,4-disubstituted 1,2,3-triazoles, in a regioselective manner
and remains as an attractive scaffold to start with [12-18]. Recently, several naphthyl based
compounds [19] and 1,2,3-triazoles [20-23] have been reported for their antimalarial activity.
Among the several chemical classes that boast the potential to display the antimalarial activity,

prototype structures of few naphthyl and triazoles possessing significant in vitro antimalarial
activity are presented in Fig. 1.
Fig. 1. Bioactive antimalarial agents.
a, b - naphthyl based antimalarials; c,d- triazole based antimalarials
Antimalarial activity of antifolates is suggested to be enhanced by flexibility, aromaticity
and hydrophobic nature [24]. In order to explore the significance of aromatic and hydrophobic
substituents on 1,2,3-triazole moiety, we have bioisoterically replaced the quinoline moiety with
naphthyl and derivatives possessing this substituent’s were synthesized. We investigated the
effect of our novel naphthyl 1,2,3-triazoles on the growth of cultured P. falciparum parasite, as
well as their ability to inhibit parasite DHFR. In silico studies on PfDHFR support us in
understanding the basis of drug resistance and are instrumental in developing novel and effective

inhibitors against resistant DHFR [25-28]. The mode of interactions with the actives is
emphasized by means of docking studies in both wild type and mutant enzymes.
2. Results and Discussion
2.1. Chemistry
Naphthyl bearing 1,2,3-triazoles were synthesized using a convenient approach that
entails linking of naphthyl alkynyl ether (3a/b) with suitable azides (1a-j, 1k) by Click approach,
as outlined in Scheme 3. In connection to this, several azides were synthesized by treating
alkyl/aryl halides with NaN3 in presence of THF:H2O medium (Scheme 1) [29]. A novel (S)-
diethyl-2-(4-azidobenzamido)pentanedioate (1k) was synthesized from its amino derivative
using less hazardous, stable and non-explosive reagents such as t-butyl nitrite (t-BuONO) and
azido trimethyl silane (TMSN3) (Scheme 2). The prepared azides are treated with naphthyl
alkynyl ether (3a/b, that is prepared from naphthols (2a/b) by treating with propargyl bromide,
K2CO3 in DMF), CuSO4.5H2O, sodium ascorbate in 2:1 mixture of t-BuOH and H2O to
capitulate the final products 4a-v. The reactions were carried out under mild conditions and
afforded the final desired products in high yield. The acid compounds 5a and 5b were
synthesized from its ester derivatives on hydrolysis. The detailed synthesis and spectral data are
described in the experimental section.

Scheme 1. Synthesis of 1a-j.
Reagents and conditions: Aryl/alkyl halide (1 equiv), NaN3 (1.5 equiv), in THF: H2O (1:4),
refluxed at 80-85 oC for 3-4 h. Reported yields are for the isolated Alkyl/Aryl azides.
Scheme 2. Synthesis of novel (S)-diethyl-2-(4-azidobenzamido)pentanedioate (1k).
Reagents and conditions: (S)-diethyl-2-(4-aminobenzamido)pentanedioate (1.0 equiv), t-BuONO
(1.5 equiv), and TMSN3 (1.2 equiv) in acetonitrile at 0 °C to room temperature.

Scheme 3. Synthesis of novel naphthyl 1,2,3-triazole derivatives 4a-v, 5a and 5b.
Reagents and conditions; a) K2CO3 (1.1 equiv), propargyl bromide (1.1 equiv), in DMF refluxed
for 3 h; b) azide 1a-1j,1k (0.75 mmol), alkynes 3a, 3b (1.05 equiv), CuSO4·5H2O (5 mol %), and
Na ascorbate (15 mol %), in t-BuOH:H2O (2:1) (10 mL) at room temp. for 12 h; c) THF:H2O (15
mL), 1N NaOH(7.5 mL) at room temperature for 4 h. Reported yields are for the isolated
products.
2.2. X-ray crystallographic studies
All the synthesized compounds are characterized by spectroscopic (1HNMR, 13CNMR,
LC-MS and HRMS) data and were found to be in good compliance with their depicted
structures. Additionally, the structure of compounds 4c, 4k, 4r, 4s and 4t were further confirmed
by single crystal X-ray diffraction method. Fig. 2 shows a perspective view of these compounds
together with their atomic labeling. Crystallographic data and structure refinement for these
compounds is provided in supporting information. Crystallographic data has been deposited for

compounds 4c, 4k, 4r, 4s and 4t with the Cambridge Crystallographic Data Centre [CCDC
1037001/2/3/4/5 respectively].
Fig. 2. ORTEP drawings (30% probability level) for 4c, 4k, 4r, 4s and 4t.
2.3. In vitro antiplasmodial activity and SAR
The newly synthesized compounds were evaluated for their antiplasmodial activity
against Pyr-sensitive (3D7) and resistant (Dd2) P. falciparum strains. Selected 3D7 high active
compounds (4j, 4k, 4l, 4m, 4n and 4t) were assessed for antiplasmodial activity on Dd2 strain.
Notably, all these compounds inhibited the growth of Dd2 with similar concentrations to that of
wild type strain. The IC50 values of these compounds are summarized in Table 1, and their
distribution was configured graphically as shown in Fig. 3. The compounds 4m (IC50 of 24.0
µM), 4n (IC50 of 31.03 µM) and 4t (IC50 of 13.6 µM) against Pyr resistant Dd2 strain showed
improved antiplasmodial activity than the control drug Pyr (IC50 of 33.95 µM).

Table 1: In vitro antiplasmodial activitya and Cytotoxicityb of the synthesized compounds.
IC50+SEM (µM)
P. falciparum 3D7
IC50 in µM
97.5
P. falciparum Dd2
Compound
ID
HEK-293
IC50 in µM
–
NC
NC
NC
NC
NC
NC
NC
NC
–
4a
4b
54.5
43.45
61.5
46.45
48.6
38.95
80
–
4c
4d
4e
4f
–
–
–
–
4g
4h
4i
–
–
NA
–
4j
39.65
23.55
22.05
21.0
19.6
26.25
21.25
27.3
NA
41.65
34.16
36.93
24.0
31.03
–
NC
NC
NC
NC
NC
NC
NC
NC
–
4k
4l
4m*
4n*
4o
4p
4q
4r
–
–
–

4s
NA
14.7
NA
NA
NA
41.45
0.047
–
–
–
4t*
13.6
NC
4u
–
–
4v
–
–
5a
–
–
5b
–
NC
Pyr
33.95
–
–
3.13 0.01
Doxorubicin
a Pyr was used as a control drug. The IC50 values are average with a standard deviation from two
independent experiments, each done in duplicates. NA: Not Active (Did not inhibit, even at the
highest concentration used), – : not determined. *More potent than Pyr against resistant strain.
b Cell line was treated with different concentrations 1µM to 100µM of compounds for 48 h. Cell
viability was measured employing MTT assay. IC50 values are indicated as the mean +/- SD of
three independent experiments. NC: Non Cytotoxic.
Antiplasmodial activity (IC50 in µM)
100
80
60
40
20
0
Pyr Sensitive 3D7 strain
Pyr Resistant Dd2 strain
Fig. 3. Graphical representation of antiplasmodial activity.

Twenty four naphthyl bearing 1,2,3-triazole derivatives were synthesized using Click
reaction between naphthylalkynylethers and substituted azides. Majority of the synthesized
compounds exhibited fair antiplasmodial activity. Dock pose analysis of the potent molecules
highlighted the presence of lipophilic groups which aid in anchoring the compounds into
hydrophobic pocket. Naphthyl and triazole rings exhibited π-π stacking interactions with Phe58,
and aryl substituent on the N-1 position of the triazole ring interacted with Phe116 of the
enzyme. Rotatable bonds adjacent to the naphthyl and aryl groups allowed the molecules to
maneuver effectively into the active site of both wild type (PDB ID: 1J3I) and mutant (PDB ID:
1J3K) PfDHFR.
Structure activity relationship (SAR) studies of reported inhibitors like Trimethoprim and
Pyr have revealed that the resistance to Pyr arises due to point mutation in PfDHFR, resulting in
the substitution of serine to asparagine (S108N). This mutation is believed to induce steric
clashes between the bulky side chain of N108 and Cl atom of the 5-p-Cl aryl group of Pyr,
reducing the binding affinity between the enzyme and inhibitor. The higher inhibitory potential
of newly synthesized naphthyl-1,2,3-triazole derivatives may be attributed to the absence of such
steric clashes and the hydrophobic naphthyl framework anticipate in contribution of strong π-π
stacking interactions with DHFR.
SAR at the cellular level for these naphthyl 1,2,3-triazole derivatives demonstrated that
the compounds with aromatic and aliphatic hydrophobic groups attached to the either side of
triazole ring showed remarkable antiplasmodial activity. All the compounds in the series 4a–j
have a simple (naphthalen-6-yloxy) methyl group at one end (C-4) of the triazole ring and
hydrophobic groups on the other end, i.e., the N-1 position. These compounds thus exhibited
moderate activity against the wild strain. While replacement of hydrogen with electronegative

Bromine atom in compounds 4k–t and 4v, 5b with (2-bromonaphthalen-6-yloxy) methyl group
at the C-4 position of the triazole ring has shown enhanced activity. Among these, 4k, 4l, 4m,
4n, 4p and 4t compounds showed high antiplasmodial activity on wild and resistant strains. The
compound 4t with a nitro benzyl substituent at the N-1 position of triazole moiety exhibited
higher antiplasmodial activity when compared to all other compounds. The activity has been
observed to increase from compounds 4k (ethyl), 4l (propyl), 4m (isopropyl) to 4n (isobutyl),
and 4p (allyl) has displayed further increase in activity. This is certainly due to branched alkyl
substituent and olefinic character respectively. Compounds 4o and 4q with higher hydrocarbon
chains showed decreased activity towards wild type strain, despite 4r and 4s having 4-
ethylmorpholine and benzyl groups attached respectively to the triazole moiety has contributed
in enhancement of hydrophobic nature but did not exhibit any activity on wild strain. Hence,
moderate hydrobhobicity on the triazole ring (branched alkyl) appears to be optimal for more
potent antiplasmodial activity.
While compounds 4u and 5a with glutamate side chain on the N-1 position and simple
(naphthalen-6-yloxy) methyl on the C-4 position has not shown any activity, where as 5b has
shown activity due the presence of electronegative Br group on the naphthyl ring. This
emphasizes the importance of an electronegative substituent on the naphthyl ring.
2.4. DHFR inhibition Assay
Three compounds that inhibited parasite development (4n, 4p and 4t) were also evaluated
for inhibition of DHFR activity in the soluble parasite extract. Pyr, a standard parasite DHFR
inhibitor, was used as a positive control [30]. Pyr decreased NADPH depletion to nearly 75%
compared to control, indicating that 25% of the total NADPH depletion was by DHFR (Fig. 4).
4n and 4p decreased NADPH depletion to 59% and 87% of the control, indicating 41% and 13%

inhibition, respectively. However, 4t did not affect NADPH depletion, most likely due to its
rapid precipitation in the assay buffer. Although Pyr is a nanomolar inhibitor of malaria
parasites, it inhibited NADPH depletion by 25%, suggesting that DHFR is responsible for 25%
of the total NADPH depletion activity. Indeed, several other enzymes, such as thioredoxin
reductase and glutathione reductase [31-32], can also utilize NADPH. Alternatively, as Pyr also
precipitated during reaction, its effective concentration might have been lower than that used in
the assay. Similar was the case with 4t, which was most potent on parasites among the three
compounds. Hence, 4n and 4p partially inhibited NADPH depletion activity of the parasite
extract, which was similar to that of Pyr, suggesting that these compounds target parasite DHFR.
Fig. 4. Inhibition of DHFR activity.
Fig. 4. Inhibition of DHFR activity: 500 µg of soluble parasite extract was incubated with
DHFR assay buffer containing 0.05% DMSO (control) or the indicated inhibitors (50 µM Pyr, 25
µM 4n, 25 µM 4p, and 25 µM 4t) for 30 min at room temperature. 60 µM NADPH and 50 µM
DHA were added to the reaction, decrease in NADPH absorbance was monitored for 30 min, and
expressed as the rate of change in absorbance/min. Data shown are percent activity of control
from a representative experiment.

2.5. Cytotoxicity
Cell line used in this study was purchased from the American Type cell Culture.
Human embryonic kidney cell line (HEK-293) was maintained in Eagle’s minimum essential
medium (MEM) containing 10% FBS. Synthesized test compounds were screened on HEK-293
cell line from 1µM - 100µM concentration for their in vitro antiproliferative activity. The MTT
cell proliferation assay was used to estimate cell viability or growth. None of the compounds
inhibited HEK-293 cell growth at maximum concentration checked (100µM) and could be
considered non-toxic, shown in Table 1.
2.6. Docking studies on PfDHFR
Pyrimethamine is known to produce effective antiplasmodial activity by binding to
PfDHFR [33], We selected this compound to compare the performance of the compounds
synthesized in this work by using molecular docking methods. The crystal structures of wild-type
(PDB ID:1J3I) and mutant type (PDB ID:1J3K) PfDHFR-TS were considered for molecular
docking analysis.
Docking simulations (Induced Fit Docking, a computationally intensive docking
protocol) were performed on active compounds (4j, 4k, 4l, 4m, 4n, 4p, 4t) against DHFR to
validate the SAR of triazole derivatives. All these compounds were found to occupy the central
region in the active site similar to Pyr and an overlay of the dock poses clustered at the same
position i.e., naphthyl ring is shown in Fig. 5.

(a)
(b)
Fig. 5. (a) All the docked conformations occupy the same position in the center of the active site
with respect to Pyr (blue in color). (b) Overlay of dock poses of 4t (green), 4n (orange), 4m
(turquoise), 4l (yellow), 4k (plum), and 4j (maroon).
The dock poses of 4t and Pyr in the crystal structure of PfDHFR (PDB ID: 1J3K) are
represented in Fig. 6a and 6b. Docking analysis revealed that amino acids Asn108, Phe58,
Phe116, Arg59 and Arg122, in the binding pocket of mutant enzyme (1J3K) play a vital role in
stabilizing the conformation of 4t, by π-π stacking interactions (Fig. 6a). Triazole and naphthyl
rings of 4m showed two π–π stacking interactions with Phe58. Only one π–π interaction with
Phe58 was observed in 4j, 4k, 4l, 4n and 4p. From the ligand interaction diagram (Fig. 7a and
7b), it can be clearly seen that Br group on naphthyl ring is oriented diametrically opposite to
Asn108 unlike the Pyr, reducing the steric repulsions between bulky side chain of Asn108 and
the Br atom on bromo naphthyl ring of triazole derivatives. Additionally, the bromo naphthyl
ring is projecting into the hydrophobic pocket and exhibits strong π-π interactions with the active
site amino acid Phe58.

(a)
(b)
Fig. 6. (a) Docked conformation of 4t in the active site of 1J3K, showing π-π stacking with
Phe116 and Phe58, -NO2 group of 4t interacting with Arg59 and Arg122. The Br atom on 4t is
away from Asn108. (b) Docked conformation of Pyr in the active site showing π-π stacking with
Phe58, four hydrogen bonds with Ile14, Asp54, and H2O.The Cl atom of Pyr close to Asn108.
(a)
(b)
Fig. 7. (a) Ligand interaction diagram of compound 4t, (b) Ligand interaction diagram of Pyr.

The high inhibitory potential of 4t may be explained on the basis of additional π-π
interaction between benzene ring of p-nitro benzyl group and amino acid Phe116. Dock pose
analysis of 4t also shows polar interactions of –NO2 group with Arg59 and Arg122 of PfDHFR.
The computational parameters like IFD score and Emodel (-9.40 & -83.69) of a potent molecule
4t against mutant PfDHFR (1J3K), surpass that of Pyr (-9.05 & -69.14). While in wild type
PfDHFR (1J3I), IFD score and Emodel (-8.65 & -79.27) of 4t are less compared to Pyr (-10.05
& -84.96) and this is in good agreement with the antiplasmodial activity. Dock pose of 4n in the
active site of wild type PfDHFR (1J3I) showed π-π stacking interaction with Phe58 and IFD
score and Emodel (-9.12 & -80.24), shown in Fig 8.
(a)
(b)
Fig. 8: (a) Docked conformation of compound 4n. (b) Ligand interaction diagram of 4n.
3. Conclusion
In summary, a new series of naphthyl bearing 1,2,3-triazoles were synthesized by [3+2]
cycloaddition of azides with a terminal alkyne in excellent yields. Preparation of triazoles (4u
and 4v) bearing p-aminoglutamate ester substituent adds another dimension to multifaceted Click
chemistry. Most of the synthesized 1,2,3-triazoles exhibited good antiplasmodial activity against
wild type strain, among them few compounds are assessed against the resistant strain of P.

falciparum and exhibited similar potency. Significantly, the compound 4t was found to be more
potent against the resistant strain than the standard antifolate Pyr. The compounds 4n and 4p
inhibited NADPH depletion activity, suggesting that these compounds target parasite DHFR as
the standard antifolate pyrimethamine. The SAR studies of these 1,2,3-triazole derivatives throw
light on structural features responsible for antiplasmodial activity. This work will promote
further studies in synthesis of naphthyl 1,2,3-triazole derivatives for developing new drugs to
combat the widespread drug resistance.
4. Experimental Section
4.1. General information
All chemicals and reagents used in the study were of analytical grade. Reactions progress
was monitored by Merck pre coated plates (60 F254); using UV light (254 nm). Column
chromatography was performed on silica gel (200-300 mesh) using solvent mixtures specified in
the corresponding experiments. Spots were visualized by UV light (254 nm). Melting points
(mp) are reported in degree centigrade, using open capillaries and are uncorrected. Single-crystal
structures of X-ray data were collected on a Bruker SMART APEX CCD X-ray diffractometer;
using graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å). Proton (1H) and carbon (13C)
NMR spectra were recorded on a Bruker (Bio-spin) Ultra shield Arance-III Nano Bay 400MHz
1
NMR spectrometer (400.13 MHz for H; 100.61 MHz for 13C) in CDCl3, and DMSO-d6 as
solvents, at room temperature with TMS as internal standard. Chemical shift values were
reported in δ (ppm). LC-MS were recorded on SHIMADZU 2010A mass spectrometer, HRMS
were recorded on ‘‘High Resolution QSTAR XL hybrid MS/MS system, Applied bio systems’’
under Electron Spray Ionization conditions preparing sample solutions in methanol. The purity
(≥95%) of all compounds evaluated in this work was determined by 1H NMR.

4.2. General procedure for the synthesis of azides (1a-j) [29]
To a stirred solution of the corresponding aryl/alkyl halide (1.0 equiv) in a 50 mL
water/THF mixture (1:4), NaN3 (1.5 equiv) was added. The resulting suspension was refluxed at
o
80-85 C for 3-4 h. The aqueous layer was extracted with DCM (3 x 10 mL) and the combined
organic solvent was dried over anhydrous Na2SO4. The solvent was removed under reduced
pressure, leaving the azide, which was sufficiently pure and used for further step without
purification.
4.3. Typical procedure for the synthesis of (S)-diethyl 2-(4-azidobenzamido)pentanedioate (1k)
(S)-diethyl 2-(4-aminobenzamido)pentanedioate (1 g, 2.87 mmol) was dissolved in
acetonitrile (20 mL) in a 50 mL round bottomed flask and cooled to 0°C in an ice bath. To this
stirred mixture t-BuONO (444 mg, 512 µL, 4.31 mmol) was added followed by TMSN3 (397
mg, 457 µL, 3.44 mmol) in a dropwise manner. The mixture was warmed to room temperature
and stirred for 2 h. The resulting mixture was concentrated under vacuum and the crude product
was purified by column chromatography on silica gel, eluted with absolute hexane to afford 1k
as light yellow solid (Yield:92%); mp: 42-44 ºC ; 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 8.8
Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 4.79-4.75 (m, 1H), 4.27-4.23 (m, 2H), 4.14-4.10 (m, 2H),
2.54-2.41 (m, 2H), 2.32-2.29 (m, 1H), 2.20-2.12 (m, 1H), 1.32 (t, J = 7.2 Hz, 3H), 1.24 (t, J =
7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 173.5, 171.9, 166.1, 143.7, 130.1, 128.9, 119.0,
61.8, 60.9, 52.5, 30.5, 27.1, 14.2, 14.1; ESI-MS: m/z 349 (M+H)+. HRMS (ESI): m/z calcd for
1
1
C16H21O5N4 [M+H]+ 349.15065, found 349.15157. [α]25D +25.680 deg cm3 g− dm− (c 0.0016 g
3
cm− in Chloroform).
4.4. General procedure for the synthesis of alkynes 3a and 3b [34]

To a suspension of K2CO3 (10.5 g, 76.3 mmol for 2a ; 6.8 g, 49.3 mmol for 2b) in DMF
(15 mL) was added a solution of appropriate 2-naphthol (10 g, 69.4 mmol of 2a ; 10 g, 44.8
mmol of 2 b) in DMF (15 mL). After the reaction was heated to 60oC for 30 min, a solution of 3-
bromoprop-1-yne (9 g, 6.8 mL for 2a ; 5.9 g, 4.4 mL for 2b) in DMF (10 mL) was added in a
dropwise manner and the mixture was refluxed for 3 h. (monitored by TLC; Hexane:EtOAc-9:1).
The mixture was neutralized with dil. aqueous HCl, then extracted with EtOAc(3x30 mL), dried
over anhydrous Na2SO4 and concentrated under vacuum, purified by column chromatography
(silica gel, hexane:EtOAc-9:1 as developing solvent) to obtain the desired product.
4.4.1. 2-(Porp-2-yn-1-yloxy)naphthalene (3a)
1
Light orange solid (Yield:95%); mp: 48-50 ºC ; H NMR (400 MHz, CDCl3) δ 7.79-7.75 (m,
3H), 7.45 (td, J = 5.6, 1.2 Hz, 1H), 7.36 (td, J = 5.6, 1.2 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.19
(dd, J = 6.4, 2.4 Hz, 1H), 4.81 (d, J = 2.4 Hz, 2H), 2.55 (t, J = 2.4 Hz, 1H) ; 13C NMR (100
MHz, CDCl3) δ 155.5, 134.3, 129.6, 129.3, 127.7, 126.9, 126.5, 124.0, 118.7, 107.5, 78.5, 75.7,
55.9.
4.4.2. 2-Bromo-6-(porp-2-yn-1-yloxy)naphthalene (3b)
Color less solid (Yield:96%); mp: 70-72 ºC ; 1H NMR (400 MHz, CDCl3) δ 7.93 (d, J = 2.0 Hz,
1H), 7.64 (m, 2H), 7.51 (dd, J = 6.4, 2.0 Hz, 1H), 7.22-7.19 (m, 2H), 4.80 (d, J = 2.4 Hz, 2H),
2.56 (t, J = 2.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 155.7, 132.8, 130.4, 129.8, 129.7, 128.7,
128.6, 119.8, 117.5, 107.5, 78.2, 75.9, 55. 9.
4.5. General procedure for the synthesis of compounds (4a-v)
In a round bottomed flask, Aryl/ Alkyl azide 1a-j,1k (0.75 mmol) and naphthalen-2-
ylprop-2-yn-1-ylether (3a) or 6-bromo naphthalen-2-ylprop-2-yn-1-ylether (3b) (1.05 equiv)
were added to a mixture of copper(II) sulfate pentahydrate (9.4 mg, 0.05 equiv),and sodium

ascorbate (23 mg, 0.15 equiv) dissolved in 2:1 of t-BuOH and H2O (10 mL) at room temperature.
The reaction mixture was stirred for 12 h at room temperature and was poured into ice cold water
(20 mL). The aqueous layer was extracted with CH2Cl2 (10 mL) thrice. The combined organic
layer was concentrated in vacuo. The residue was purified by short column chromatography on
silica gel (70−230 mesh) eluted with hexane/ EtOAc (4/1) to afford the desired products 4a-v.
4.5.1. 1-Ethyl-4-((naphthalen-6-yloxy)methyl)-1H-1,2,3-triazole (4a)
1
Light yellow solid (Yield:95%); mp: 97-99 ºC ; H NMR (400 MHz, CDCl3) δ 7.78-7.74 (m,
3H), 7.65 (s, 1H), 7.45 (td, J = 5.6, 1.2 Hz, 1H), 7.35 (td, J = 5.6, 1.2 Hz, 1H), 7.28 (d, J = 2.4
Hz, 1H), 7.19 (dd, J = 6.4, 2.4 Hz, 1H), 5.342 (s, 2H), 4.43 (q, J = 7.2 Hz, 2H), 1.57 (t, J = 7.2
Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 156.2, 144.1, 134.4, 129. 6, 129.2, 127.6, 126.9, 126.5,
123.9, 122.1, 118.8, 107.2, 62.1, 45.4, 15.5; LC-MS (ESI), m/z 254.15 (M+H)+, HRMS (ESI):
m/z calcd for C15H16ON3 [M+H]+ 254.12879, found 254.12841.
4.5.2. 4-((Naphthalen-6-yloxy)methyl)-1-propyl-1H-1,2,3-triazole (4b)
1
Light yellow solid (Yield:94%); mp: 86-88 ºC; H NMR (400 MHz, CDCl3) δ 7.78-7.74 (m,
3H), 7.62 (s, 1H), 7.44 (td, J = 5.6, 1.2 Hz, 1H), 7.34 (td, J = 5.6, 1.2 Hz, 1H), 7.27 (d, J =
2.4Hz, 1H), 7.19 (dd, J = 6.4, 2.4 Hz, 1H), 5.342 (s, 2H), 4.33 (t, J = 7.2 Hz, 2H), 1.99-1.90
13
(sextet, J = 7.2 Hz, 2H), 0.96 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl3) δ 156.2, 144.0,
134.4, 129.6, 129.2, 127.6, 126.9, 126.5, 123.9, 122.6, 118.8, 107.2, 62.2, 52.0, 23.7, 11.1; LC-
MS (ESI), m/z 268.15 (M+H)+, HRMS (ESI): m/z calcd for C16H18ON3 [M+H]+ 268.14444,
found 268.14399.
4.5.3. 1-Isopropyl-4-((naphthalen-6-yloxy)methyl)-1H-1,2,3-triazole (4c)
1
Light red crystals (Yield:91%); mp: 110-112 ºC; H NMR (400 MHz, CDCl3) δ 7.78-7.75 (m,
3H), 7.67 (s, 1H), 7.45 (td, J = 5.6, 1.2 Hz, 1H), 7.35 (td, J = 5.6, 1.2 Hz, 1H), 7.29 (d, J = 2.4

Hz, 1H), 7.20 (dd, J = 6.4, 2.4 Hz, 1H), 5.33 (s, 2H), 4.85 (septet, J = 6.4 Hz, 1H), 1.60 (d, J =
13
6.4 Hz, 6H); C NMR (100 MHz, CDCl3) δ 156.3, 143.8, 134.5, 129.5, 129.2, 127.6, 126.9,
126.5, 123.9, 120.2, 118.8, 107.2, 62.3, 51.1, 23.0; LC-MS (ESI), m/z 266 (M-H)+, HRMS
(ESI): m/z calcd for C16H18ON3 [M+H]+ 268.14444, found 268.14396.
4.5.4. 1-Isobutyl-4-((naphthalen-6-yloxy)methyl)-1H-1,2,3-triazole (4d)
1
Colorless solid (Yield:86%); mp: 82-84 ºC; H NMR (400 MHz, CDCl3) δ 7.78-7.73 (m, 3H),
7.59 (s, 1H), 7.44 (td, J = 5.6, 1.2 Hz, 1H), 7.34 (td, J = 5.6, 1.2 Hz, 1H), 7.27 (d, J = 2.4 Hz,
1H), 7.19 (dd, J = 6.4, 2.4 Hz, 1H), 5.35 (s, 2H), 4.17 (d, J = 7.2 Hz, 2H), 2.28-2.17 (septet, J =
6.8 Hz, 1H), 0.95 (d, J = 6.4 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 156.2, 143.9, 134.4, 129.5,
129.2, 127.6, 126.9, 126.5, 123.9, 122.9, 118.8, 107.3, 62.2, 57.7, 29.7, 19.9, 10.4; LC-MS
(ESI), m/z 282.40 (M+H)+, HRMS (ESI): m/z calcd for C17H20ON3 [M+H]+ 282.16009, found
282.15952.
4.5.5. 4-((Naphthalen-6-yloxy)methyl)-1-pentyl-1H-1,2,3-triazole (4e)
1
Colorless solid (Yield:95%); mp: 76-78 ºC; H NMR (400 MHz, CDCl3) δ 7.78-7.74 (m, 3H),
7.62 (s, 1H), 7.45 (td, J = 5.6, 1.2 Hz, 1H), 7.35 (td, J = 5.6, 1.2 Hz, 1H), 7.27 (d, J = 2.4 Hz,
1H), 7.19 (dd, J = 6.4, 2.4 Hz, 1H), 5.34 (s, 2H), 4.35 (t, J = 7.2 Hz, 2H), 1.95-1.88 (quintet,
13
2H), 1.40-1.27 (m, 4H), 0.89 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl3) δ 156.2, 143.9,
134.5, 129.5, 129.2, 127.6, 126.9, 126.5, 123.9, 122.6, 118.8, 107.3, 62.1, 50.4, 29.9, 28.6, 22.1,
13.8; LC-MS (ESI), m/z 296.20 (M+H)+, HRMS (ESI): m/z calcd for C18H22ON3 [M+H]+
296.17574, found 296.17503.
4.5.6. 1-Allyl-4-((naphthalen-6-yloxy)methyl)-1H-1,2,3-triazole (4f)
Light yellow solid (Yield:90%); mp: 79-80 ºC; 1H NMR (400 MHz, CDCl3) δ 7.77-7.74 (m, 3H),
7.64 (s, 1H), 7.44 (td, J = 6.4, 1.2 Hz, 1H), 7.35 (td, J = 6.4, 1.2 Hz, 1H), 7.27 (d, J = 2.4 Hz,

1H), 7.18 (dd, J = 6.4, 2.4 Hz, 1H), 6.07-5.97 (m, 1H), 5.37-5.29 (tq, J=6.8/1.2/0.8Hz, 2H),
5.34 (s, 2H), 4.98 (dt, J = 3.6, 1.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 156.2, 144.4, 134.4,
131.1, 129.6, 129.2, 127.6, 126.9, 126.5, 123.9, 122.6, 120.4, 118.8, 107.3, 62.1, 52.8; LC-MS
(ESI), m/z 266.20 (M+H)+, HRMS (ESI): m/z calcd for C16H16ON3 [M+H]+ 266.12879, found
266.12831.
4.5.7. 1-Cyclopentyl-4-((naphthalen-6-yloxy)methyl)-1H-1,2,3-triazole (4g)
Colorless solid (Yield:93%); mp: 108-110 ºC; 1H NMR (400 MHz, CDCl3) δ 7.78-7.75(m, 3H),
7.65 (s, 1H), 7.45 (td, J = 6.4, 1.2 Hz, 1H), 7.35 (td, J = 6.4, 1.2 Hz, 1H), 7.29 (d, J = 2.4 Hz,
1H), 7.20 (dd, J = 6.4, 2.4 Hz, 1H), 5.33 (s, 2H), 4.98-4.91 (quintet, 1H), 2.32-2.23 (m, 2H),
2.11-2.02 (m, 2H), 1.95-1.86 (m, 2H), 1.82-1.73 (m, 2H); 13C NMR (100 MHz, CDCl3) δ
156.3, 143.8, 134.5, 129.5, 129.2, 127.6, 126.9, 126.5, 123.9, 121.2, 118.8, 107.2, 62.2, 61.9,
33.4, 24.1; LC-MS (ESI), m/z 294.25 (M+H)+, HRMS (ESI): m/z calcd for C18H19ON3Na
[M+Na]+ 316.14203, found 316.14122.
4.5.8. 4-(2-(4-((Naphthalen-6-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)morpholine (4h)
Light yellow solid (Yield:92%); mp: 94-96 ºC; 1H NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.77-
7.73 (m, 3H), 7.45 (td, J = 6.4, 1.2 Hz, 1H), 7.35 (td, J = 6.4, 1.2 Hz, 1H), 7.27 (brs, 1H), 7.19
(dd, J = 6.4, 2.4 Hz, 1H), 5.36 (s, 2H), 4.47 (t, J = 6.0 Hz, 2H), 3.62 (t, J = 4.8 Hz, 4H), 2.81 (t, J
13
= 6.0 Hz, 2H), 2. 46 (t, J =4.8 Hz, 4H); C NMR (100 MHz, CDCl3) δ 156.1, 144.0, 134.4,
129.6, 129.2, 127.6, 126.9, 126.5, 123.9, 123.3, 118.8, 107.3, 66.7, 62.1, 57.8, 53.4, 47.5; LC-
MS (ESI), m/z 339.25 (M+H)+ , HRMS (ESI): m/z calcd for C19H23O2N4 [M+H]+ 339.18155,
found 339.18114.
4.5.9. 1-Benzyl-4-((naphthalen-6-yloxy)methyl)-1H-1,2,3-triazole (4i)

Colorless solid (Yield:90%); mp: 140-144 ºC; 1H NMR (400 MHz, CDCl3) δ 7.77-7.73 (m, 3H),
7.56 (s, 1H), 7.45 (t, J = 6.4 Hz, 1H), 7.40-7.33 (m, 4H), 7.29-7.27 (m, 2H), 7.25 (d, J = 2.4 Hz,
13
1H), 7.16 (dd, J = 6.4, 2.4 Hz, 1H), 5.54 (s, 2H), 5.31 (s, 2H); C NMR (100 MHz, CDCl3) δ
156.1, 144.6, 134.4, 134.4, 129.5, 129.2, 128.8, 128.1, 127.6, 126.9, 126.5, 123.9, 122.6, 118.8,
107.3, 62.2, 54.3; LC-MS (ESI), m/z 313.95 (M-H)+, HRMS (ESI): m/z calcd for C20H18ON3
[M+H]+ 316.14444, found 316.14393.
4.5.10. 1-(4-Nitrobenzyl)-4-((naphthalen-6-yloxy)methyl)-1H-1,2,3-triazole (4j)
1
Yellowish solid (Yield:92%); mp: 102-104 ºC; H NMR (400 MHz, CDCl3) δ 8.19 (d, J = 8.8
Hz, 2H), 7.78-7.72 (m, 3H), 7.63 (s, 1H), 7.45 (td, J = 6.4, 1.2 Hz, 1H), 7.41-7.34 (m, 3H), 7.24
13
(d, J = 2.4 Hz, 1H), 7.16 (dd, J = 6.4, 2.4 Hz, 1H), 5.64 (s, 2H), 5.35 (s, 2H); C NMR (100
MHz, CDCl3) δ 155.9, 148.1, 145.2, 141.5, 134.3, 129.6, 129.2, 128.6, 127.7, 126.9, 126.6,
124.3, 124.0, 122.9, 118.7, 107.4, 62.1, 53.2; LC-MS (ESI), m/z 360.95 (M+H)+, HRMS (ESI):
m/z calcd for C20H17O3N4 [M+H]+ 361.12952, found 361.12906.
4.5.11. 4-((2-Bromonaphthalen-6-yloxy)methyl)-1-ethyl-1H-1,2,3-triazole (4k)
Colorless needles (Yield:94%); mp: 118-120 ºC; 1H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 2.0
Hz, 1H), 7.67-7.61 (m, 3H), 7.51 (dd, J = 6.8, 2.0 Hz, 1H), 7.25 (d, J = 2.4 Hz, 1H), 7.20 (dd, J
13
= 6.4, 2.4 Hz, 1H), 5.32 (s, 2H), 4.43 (q, J = 7.2 Hz, 2H), 1.58 (t, J = 7.2 Hz, 3H); C NMR
(100 MHz, CDCl3) δ 156.5, 143.9, 132.9, 130.2, 129.7, 129.6, 128.6, 128.6, 122.0, 119.9, 117.4,
107.2, 62.2, 45.4, 15.5; LC-MS (ESI), m/z 330.80 (M-H)+, HRMS (ESI): m/z calcd for
C15H15ON3Br [M+H]+ 332.03930, found 332.04021.
4.5.12. 4-((2-Bromonaphthalen-6-yloxy)methyl)-1-propyl-1H-1,2,3-triazole (4l)
Colorless solid (Yield:92%); mp: 100-102 ºC; 1H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 2.0 Hz,
1H), 7.67-7.61 (m, 3H), 7.50 (dd, J = 6.8, 2.0 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.20 (dd, J =

6.4, 2.4 Hz, 1H), 5.32 (s, 2H), 4.33 (t, J = 7.2 Hz, 2H), 2.0-1.91 (m, 2H), 0.96 (t, J = 7.2 Hz,
3H); 13C NMR (100 MHz, CDCl3) δ 156.5, 143.8, 132.9, 130.2, 129.7, 129.6, 128.6, 128.6,
122.5, 119.9, 117.4, 107.2, 62.2, 52.1, 23.7, 11.1; LC-MS (ESI), m/z 347.25 (M+H)+, HRMS
(ESI): m/z calcd for C16H17ON3Br [M+H]+ 346.05495, found 346.05586.
4.5.13. 4-((2-Bromonaphthalen-6-yloxy)methyl)-1-isopropyl-1H-1,2,3-triazole (4m)
Colorless solid (Yield:90%); mp: 122-124 ºC; 1H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 2.0Hz,
1H), 7.67-7.62 (m, 3H), 7.51 (dd, J = 6.4, 2.0 Hz, 1H), 7.30 (brs, 1H), 7.21(dd, J = 6.4, 2.4 Hz,
1H), 5.31 (s, 2H), 4.90-4.80 (m, 1H), 1.60 (d, J = 6.8 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ
156.5, 143.6, 132.9, 130.2, 129.7, 129.6, 128.6, 128.6, 120.2, 119.9, 117.4, 107.2, 62.3, 53.2,
23.0; LC-MS (ESI), m/z 347.30 (M+H)+, HRMS (ESI): m/z calcd for C16H17ON3Br [M+H]+
346.05495, found 346.05596.
4.5.14. 4-((2-Bromonaphthalen-6-yloxy)methyl)-1-isobutyl-1H-1,2,3-triazole (4n)
1
Colorless solid (Yield:88%); mp: 88-90 ºC; H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 2.0 Hz,
1H), 7.67-7.60 (m, 3H), 7.51 (dd, J = 6.4, 2.0 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.20 (dd, J = 6.4,
2.4 Hz, 1H), 5.33 (s, 2H), 4.18 (d, J = 7.2 Hz, 2H), 2.30-2.16 (m, 1H), 0.95 (d, J = 6.8 Hz, 6H);
13C NMR (100 MHz, CDCl3) δ 156.5, 143.7, 132.9, 130.2, 129.7, 129.6, 128.6, 128.6, 123.0,
119.9, 117.4, 107.3, 62.236, 57.7, 29.7, 19.9; ESI-MS: m/z 360(M)+, 362 (M+2)+, HRMS (ESI):
m/z calcd for C17H19ON3Br [M+H]+ 360.07060, found 360.07169.
4.5.15. 4-((2-Bromonaphthalen-6-yloxy)methyl)-1-pentyl-1H-1,2,3-triazole (4o)
1
Colorless solid (Yield:93%); mp: 96-98 ºC; H NMR (400 MHz, CDCl3) δ 7.94(d, J = 2.0 Hz,
1H), 7.69-7.63 (m, 3H), 7.52 (dd, J = 6.4, 2.0 Hz, 1H), 7.27 (d, J = 2.4 Hz, 1H), 7.22 (dd, J = 6.4,
2.4 Hz, 1H), 5.34 (s, 2H), 4.37 (t, J = 7.2 Hz, 2H), 1.95-1.92 (quintet, 2H), 1.37-1.27 (m, 4H),
13
0.91 (t, J = 6.8 Hz, 3H); C NMR (100 MHz, CDCl3) δ 156.5, 143.85, 132.95, 130.25, 129.75,

129.65, 128.65, 128.7, 122.5, 119.9, 117.4, 107.3, 62.23, 50.5, 29.9, 28.6, 22.1, 13.8; LC-MS
(ESI), m/z 375.30 (M+H)+, HRMS (ESI): m/z calcd for C18H21ON3Br [M+H]+ 374.08625, found
374.08750.
4. 5.16. 4-((2-Bromonaphthalen-6-yloxy)methyl)-1-allyl-1H-1,2,3-triazole (4p)
Light yellow solid (Yield:90%); mp: 92-94ºC; 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 2.0 Hz,
1H), 7.66-7.60 (m, 3H), 7.50 (dd, J = 6.4, 2.0 Hz, 1H), 7.23 (d, J = 2.4 Hz, 1H), 7.19 (dd, J =
6.4, 2.4 Hz, 1H), 6.07-5.97 (m, 1H), 5.38-5.29 (m, 2H), 5.31 (s, 2H), 4.99 (dt, J = 3.6, 1.2 Hz,
2H); 13C NMR (100 MHz, CDCl3) δ 156.4, 144.1, 132.9, 131.1, 130.2, 129.7, 129.6, 128.6,
128.6, 122.6, 120.5, 119.9, 117.4, 107.2, 62.1, 52.8; LC-MS (ESI), m/z 345.15 (M+H)+, HRMS
(ESI): m/z calcd for C16H15ON3Br [M+H]+ 344.03930, found 344.04036.
4.5.17. 4-((2-Bromonaphthalen-6-yloxy)methyl)-1-cyclopentyl-1H-1,2,3-triazole (4q)
Colorless solid (Yield:89%); mp: 112-114 ºC; 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 2.0 Hz,
1H), 7.67-7.61 (m, 3H), 7.50 (dd, J = 6.8, 2.0 Hz, 1H), 7.25 (s, 1H), 7.21 (dd, J = 6.4, 2.4Hz,
1H), 5.30 (s, 2H), 4.97-4.90 (m, 1H), 2.32-2.23 (m, 2H), 2.10-2.02 (m, 2H), 1.96-1.86 (m, 2H),
1.81-1.74 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 156.5, 143.6, 132.9, 130.2, 129.7, 129.6,
128.6, 128.5, 121.2, 119.9, 117.4, 107.2, 62.2, 61.9, 33.4, 24.1; LC-MS (ESI), m/z 373.20
(M+H)+, HRMS (ESI): m/z calcd for C18H19ON3Br [M+H]+ 372.07060, found 372.07173.
4.5.18.
4-(2-(4-((2-Bromonaphthalen-6-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)morpholine
(4r)
1
Light yellow needles (Yield:90%); mp: 152-154 ºC; H NMR (400 MHz, CDCl3) δ 7.92 (d, J =
2.0 Hz, 1H), 7.78 (s, 1H), 7.67-7.60 (m, 2H), 7.50 (dd, J = 6.8, 2.0 Hz, 1H), 7.24 (d, J = 2.4 Hz,
1H), 7.21 (dd, J = 6.4, 2.4 Hz, 1H), 5.34 (s, 2H), 4.47 (t, J = 6.0 Hz, 2H), 3.63 (t, J = 4.8 Hz, 4H),
13
2.82 (t, J = 6.0 Hz, 2H), 2.46 (t, J = 4.8 Hz, 4H); C NMR (100 MHz, CDCl3) δ 156.4, 143.8,

132.9, 130.2, 129.8, 129.6, 128.6, 128.5, 123.3, 119.9, 117.4, 107.3, 66.9, 62.1, 57.8, 53.5, 47.5;
LC-MS (ESI), m/z 417.45 (M-H)+, HRMS (ESI): m/z calcd for C19H22O2N4Br [M+H]+
417.09207, found 417.09338.
4.5.19. 4-((2-Bromonaphthalen-6-yloxy)methyl)-1-benzyl-1H-1,2,3-triazole (4s)
White needles (Yield:89%); mp: 134-136 ºC; 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 2.0 Hz,
1H), 7.65-7.59 (m, 2H), 7.56 (1H, s), 7.50 (dd, J = 6.8, 2.0 Hz, 1H), 7.38-7.36 (m, 3H), 7.29-
7.27 (m, 2H), 7.22 (d, J = 2.4 Hz, 1H), 7.17 (dd, J = 6.4, 2.4 Hz, 1H), 5.54 (s, 2H), 5.29 (s, 2H);
13C NMR (100 MHz, CDCl3) δ 156.4, 144.3, 134.4, 132.9, 130.2, 129.7, 129.6, 129.2, 128.9,
128.6, 128.5, 128.1, 122.6, 119.8, 117.4, 107.2, 62.2, 54.3; LC-MS (ESI), m/z 393.25 (M-H)+,
HRMS (ESI): m/z calcd for C20H17ON3Br [M+H]+ 394.05495, found 394.05625.
4.5.20. 4-((2-Bromonaphthalen-6-yloxy)methyl)-1-(4-nitrobenzyl)-1H-1,2,3-triazole (4t)
Yellow needles (Yield:93%); mp: 118-120 ºC; 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 8.8 Hz,
2H), 7.91 (d, J = 2.0 Hz, 1H), 7.66-7.59 (m, 3H), 7.51 (dd, J = 6.8, 2.0 Hz, 1H), 7.40 (d, J = 8.8
Hz, 2H), 7.22 (d, J = 2.4 Hz, 1H), 7.17 (dd, J = 6.4, 2.4 Hz, 1H), 5.66 (s, 2H), 5.33 (s, 2H); 13C
NMR (100 MHz, CDCl3) δ 156.3, 148.1, 144.9, 141.4, 132.8, 130.2, 129.8, 129.7, 128.7, 128.6,
128.5, 124.4, 122.9, 119.8, 117.5, 107.3, 62.0, 53.2; LC-MS (ESI), m/z 440 (M+H)+, HRMS
(ESI): m/z calcd for C20H16O3N4Br [M+H]+ 439.04003, found 439.04152.
4.5.21.
(S)-Diethyl2-(-4-(4-((naphthalen-6-yloxy)methyl)-1H-1,2,3-triazol-1-
yl)benzamido)pentane dioate (4u)
Colorless solid (Yield:91%); mp: 146-148 ºC; 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 8.01
(d, J = 8.8 Hz, 2H), 7.87 (d, J = 8.8 Hz, 2H), 7.79-7.76 (m, 3H), 7.46 (td, J = 6.4, 1.2 Hz, 1H),
7.36 (td, J = 6.4, 1.2 Hz, 1H), 7.31 (d, J = 2.4 Hz, 1H), 7.30 (brs, 1H), 7.22 (dd, J = 6.4, 2.4 Hz,
1H), 5.44 (s, 2H), 4.81-4.76 (m, 1H), 4.29-4.22 (m, 2H), 4.17-4.09 (m, 2H), 2.59-2.43 (m, 2H),

13
2.37-2.29 (m, 1H), 2.23-2.14 (m, 1H), 1.32 (t, J = 7.2 Hz, 3H), 1.24 (t, J = 7.2 Hz, 3H); C
NMR (100 MHz, CDCl3) δ 173.6, 171.8, 165.7, 156.0, 145.4, 139.2, 134.4, 133.9, 129.7, 129.2,
128.9, 127.7, 126.9, 126.6, 124.0, 120.7, 120.2, 118.7, 107.2, 62.0, 61.9, 61.0, 52.7, 30.5, 26.9,
14.2, 14.1; LC-MS (ESI), m/z 530.35 (M-H)+, HRMS (ESI): m/z calcd for C29H31O6N4 [M+H]+
531.22381, found 531.22505.
4.5.22.
(S)-Diethyl2-(4-(4-(((6-bromonaphthalen-2-yl)oxy)methyl)-1H-1,2,3-triazol-1-
yl)benzamido)pentane dioate (4v)
1
Light yellow solid (Yield:90%); mp: 166-168 ºC; H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H),
8.01 (d, J = 8.8 Hz, 2H), 7.93 (d, J = 2.0 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.69-7.63 (m, 2H),
7.52 (dd, J = 6.8, 2.0 Hz, 1H), 7.32 (d, J = 7.2 Hz, 1H), 7.28 (ds, 1H), 7.24 (dd, J = 6.4, 2.4 Hz,
1H), 5.42 (s, 2H), 4.81-4.76 (m, 1H), 4.29-4.23 (m, 2H), 4.16-4.10 (m, 2H), 2.59-2.43 (m, 2H),
13
2.37-2.29 (m, 1H), 2.23-2.14 (m, 1H), 1.32 (t, J = 7.2 Hz, 3H), 1.24 (t, J = 7.2 Hz, 3H); C
NMR (100 MHz, CDCl3) δ 173.6, 171.8, 165.6, 156.3, 145.1, 139.1, 133.9, 132.9, 130.3, 129.8,
129.7, 128.9, 128.7, 128.6, 120.8, 120.2, 119.8, 117.5, 107.3, 62.0, 61.9, 61.0, 52.8, 30.6, 26.9,
14.2, 14.1; LC-MS (ESI), m/z 609.25 (M-H)+, HRMS (ESI): m/z of C29H29O6N4Br [M+2]+
found 611.13506.
4.6. General procedure for the synthesis of 5a and 5b [35]
A solution of corresponding ester (4u/v) (0.5 g) in H2O:THF (1:1, 15 mL) was treated
with 1N aqueous NaOH solution (7.5 mL) at room temperature for 4 h. THF was evaporated
under reduced pressure and 1N HCl solution was added drop wise to the residue. The white
crystalline precipitate was collected by filtration, washed with water, and dried over CaCl2 under
vacuum to give 5a/b.

4.6.1.(S)-2-(4-(4-((Naphthalene-2-yloxy)methyl)-1H-1,2,3-triazol-1-yl)benzamido)pentanedioic
acid (5a)
White solid (Yield:82%); mp:172-174ºC; 1H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 8.81 (d,
J = 7.6 Hz, 1H), 8.10 (q, J = 8.8, 6.0 Hz, 4H), 7.87-7.84 (m, 3H), 7.57 (d, J = 2.4 Hz, 1H), 7.49
(td, J = 6.4, 1.2 Hz, 1H), 7.37 (td, J = 6.4, 1.2 Hz, 1H), 7.24 (dd, J = 6.4, 2.4 Hz, 1H), 5.38 (s,
13
2H), 4.46-4.40 (m, 1H), 2.38 (t, J = 7.2 Hz, 2H), 2.16-2.08 (m, 1H), 2.02-1.93 (m, 1H); C
NMR (100 MHz, DMSO-d6) δ 174.2, 165.2, 156.3, 144.5, 138.8, 134.6, 129.9, 129.6, 129.1,
128.0, 127.3, 126.9, 124.3, 123.5, 120.1, 119.1, 112.2, 107.7, 104.7, 61.6, 53.0, 32.1, 27.5; LC-
MS (ESI), m/z 475.25 (M+H)+, HRMS (ESI): m/z calcd for C25H23O6N4 [M+H]+ 475.16121,
found 475.16324.
4.6.2.
(S)-2-(4-(4-(((6-Bromonaphthalen-2-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)benzamido)
pentanedioicacid (5b)
Colorless solid (Yield:80%); mp:180-182ºC; 1H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 8.80
(d, J = 7.6 Hz, 1H), 8.14 (d, J = 2.0 Hz, 1H), 8.10 (q, J = 8.8, 6.8 Hz, 4H), 7.88-7.81 (m, 2H),
7.61-7.59 (m, 2H), 7.30 (dd, J = 6.4, 2.4 Hz, 1H), 5.38 (s, 2H), 4.46-4.40 (m, 1H), 2.38 (t, J = 7.2
Hz, 2H), 2.16-2.07 (m, 1H), 2.02-1.92 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 174.7, 165.4,
156.8, 144.3, 138.8, 134.6, 133.3, 130.3, 129.9, 129.8, 129.6, 129.5, 129.2, 123.6, 120.3, 120.1,
117.0, 107.8, 61.6, 53.0, 31.8, 27.2; LC-MS (ESI), m/z 553 (M)+, HRMS (ESI): m/z of
C25H21O6N4Br [M+2]+ found 555.07176.
4.7. Crystallographic studies
Single-crystal structures of X-ray data were collected for 4c, 4k, 4r, 4s, and 4t on a
Bruker SMART APEX CCD X-ray diffractometer, using graphite-monochromated Mo-Kα
radiation (λ = 0.71073 Å). Data were reduced using SAINTPLUS [36] and a multi-scan

absorption correction using SADABS [37] was performed. The structure was solved and refined
against F2 using SHELX-97 [38]. All ring hydrogen atoms were assigned on the basis of
geometrical considerations and were allowed to ride upon the respective carbon atoms. All
hydrogen atoms were assigned fixed Uiso values, equal to 1.2Ueq. of the parent atom. The
molecular structures of 4c, 4k, 4r, 4s, and 4t are illustrated in Fig. 2 with 30% probability
displacement ellipsoid.
4.8. Antiplasmodial activity, DHFR inhibition assay and Cytotoxicity
4.8.1. Antiplasmodial activity
P. falciparum 3D7 and Dd2 strains were obtained from the Malaria Research and
Reference Reagent Resource Centre (MR4), cell culture reagents and SYBR Green 1 were from
Invitrogen, Pyr and DMSO were from Sigma, human serum was from Millipore. Both the strains
were grown in RPMI 1640 medium (containing 2 g/litre sodium bicarbonate, 2 g/litre glucose, 25
µg/ml gentamicin, 300 mg/litre glutamine, 10 % human serum, and human erythrocytes at 2%
haematocrit [39]) at 37°C in the presence of a gas mixture (5% O2, 5% CO2, 90% N2). Parasites
were synchronized by treatment with 5% D-sorbitol at ring stage [40]. Stocks of all the
compounds and Pyr were prepared in DMSO at 20-50 mM concentrations, and the stocks were
serially diluted 2-fold in 50 µl culture medium across rows of a 96 well tissue culture plate.
DMSO (0.05%) or chloroquine (500 nM) was added to the control wells. 50 µl parasite
suspension (1% ring-infected erythrocytes at 4% haematocrit) was added to each well. The plate
was incubated in a modular incubator chamber (Billups-Rothenberg, Inc.) with the gas mixture at
37°C for 50 hours. At the end of incubation, 100 µl lysis buffer (20 mM Tris-Cl, 5 mM EDTA,
0.008% saponin, 0.08% Triton X-100, pH 7.5) with SYBR Green 1 (at the manufacturer’s
recommended dilution) was added to each well, the plate was incubated at 37°C for 30 min, and