Novel triazole-tetrahydroisoquinoline hybrids as human aromatase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103327

Novel thirteen triazole-tetrahydroisoquinoline derivatives (2a-m) were synthesized and evaluated for their aromatase inhibitory activities. Seven triazoles showed significant aromatase inhibitory activity (IC₅₀ = 0.07–1.9 μM). Interestingly, th

Full text of DOI:10.1016/j.bioorg.2019.103327

Journal Pre-proofs
Novel triazole-tetrahydroisoquinoline hybrids as human aromatase inhibitors
Chanamon Chamduang, Ratchanok Pingaew, Veda Prachayasittikul, Supaluk
Prachayasittikul, Somsak Ruchirawat, Virapong Prachayasittikul
PII:
S0045-2068(19)30632-7
DOI:
https://doi.org/10.1016/j.bioorg.2019.103327
YBIOO 103327
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
23 April 2019
14 August 2019
27 September 2019
Please cite this article as: C. Chamduang, R. Pingaew, V. Prachayasittikul, S. Prachayasittikul, S. Ruchirawat, V.
Prachayasittikul, Novel triazole-tetrahydroisoquinoline hybrids as human aromatase inhibitors, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103327
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1
Novel triazole-tetrahydroisoquinoline hybrids as human
aromatase inhibitors
Chanamon Chamduang a, Ratchanok Pingaew a,*, Veda Prachayasittikul b*, Supaluk
Prachayasittikul b, Somsak Ruchirawat c,d, Virapong Prachayasittikul e
aDepartment of Chemistry, Faculty of Science, Srinakharinwirot University,
Bangkok 10110, Thailand
b Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology,
Mahidol University, Bangkok 10700, Thailand
c Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, and Program in
Chemical Biology, Chulabhorn Graduate Institute, Bangkok 10210,Thailand
d Center of Excellence on Environmental Health and Toxicology, Commission on Higher
Education (CHE), Ministry of Education, Thailand
eDepartment of Clinical Microbiology and Applied Technology, Faculty of Medical
Technology, Mahidol University, Bangkok 10700, Thailand
*Corresponding authors:
E-mail: ratchanok@swu.ac.th (R. Pingaew)
Tel: 662-649-5000 ext 18253; Fax: 662-260-0128
E-mail: veda.pra@mahidol.ac.th (V. Prachayasittikul)
Tel: 662-441-4376; Fax: 662-441-4380

2
Abstract
Novel thirteen triazole-tetrahydroisoquinoline derivatives (2a-m) were synthesized
and evaluated for their aromatase inhibitory activities. Seven triazoles showed significant
aromatase inhibitory activity (IC50 = 0.07-1.9 ꢀM). Interestingly, the analog bearing
naphthalenyloxymethyl substituent at position 4 of the triazole ring (2i) displayed the most
potent aromatase inhibitory activity (IC50 = 70 nM) without significant cytotoxicity to a
normal cell. Molecular docking also suggested that the direct H-bonding interaction with
residue Thr310 may be responsible for a striking inhibitory effect of the most potent
compound 2i.
Keywords: Triazole; Isoquinoline; Sulfonamide; Synthesis; Aromatase inhibitor; Molecular
Docking

3
1. Introduction
High level of estrogen has been noted to promote cancer cell growth, recurrence, and
metastasis of the estrogen-dependent breast cancers. Regardingly, decreasing estrogen level
by inhibiting its biosynthesis is considered to be one of the effective strategies for breast
cancer management [1-7]. Aromatase (CYP19), a member of the cytochrome P450 family, is
a rate-limiting enzyme that catalyzes the biosynthesis of estrogens from androgens.
Therefore, aromatase inhibitors (AIs) have been recognized as one of the widely used drug
classes for management of estrogen-dependent cancer [1-7]. AIs are categorized by their
mechanisms of action into two main types: (i) steroidal AIs (such as formestane and
exemestane) which irreversibly inhibit activity of the aromatase enzyme, and (ii) nonsteroidal
AIs (such as letrozole, anastrozole, and vorozole) which their inhibitory effects are reversible
(Fig. 1) [3-7]. Although available aromatase inhibitors (both steroidal and non-steroidal
types) displayed successful clinical outcomes, long-term use can lead to acquired drug
resistane as well as considerable side effects including musculoskeletal pain, bone loss
(osteoporosis) broken bones, and cardiovascular disease [8-11]. Therefore, development of
novel aromatase inhibitors is still essential to provide alternative drug of choice with more
preferable properties.
Triazoles are core structure regularly found in nonsteroidal AIs [2-3, 5-7]. Among the
classes of AI (Fig. 1), letrozole and anastrozole containing triazole ring were approved by the
United States Food and Drug Administration (FDA) for the treatment of advanced breast
cancer in postmenopausal women [12]. The heterocyclic nitrogen atom of triazole plays an
important role by interacting with the heme iron of the aromatase enzyme [3, 6-7]. Along the
line, the aromatase inhibitory activity of a series of 1,2,3-triazoles bearing sulfonamide has
been reported by our group (Fig. 2) [13]. The study revealed that various tested triazoles
displayed significant inhibitory potency with IC50 ranging from 0.2 to 9.4 μM, where the
tetrahydroisoquinoline-triazole (THIQ-triazole) 1 was shown to be the most potent inhibitor.
The molecular docking results of the triazole 1 against the aromatase disclosed the crucial
interactions namely hydrophobic interaction using THIQ and benzene rings; π-π stacking
interaction using triazole, and coumarinyl rings; and hydrogen bond interaction using
sulfonyl and ether groups. In our previous work, the sulfonamide moiety plays crucial role in
H-bond formation with His480 of the enzyme giving rise to potent aromatase inhibitory

4
effect of the compounds [14]. Therefore, as inspired by the parent compound 1, a series of
novel THIQ-triazole 2 was modified (Fig. 2). Accordingly, THIQ-SO2-benzene-triazole core
of the hit compound 1 was preserved to remain the hydrophobic, π-π stacking, and H-bonding
interactions with the enzyme, but the oxymethylcoumarinyl moiety was replaced with
different hydrophobic groups (R) to attain an optimal interaction with the target.
All of the novel synthesized triazole derivatives were evaluated for their aromatase
inhibitory activities. To provide insights into the important interactions, the molecular
docking study was also performed to investigate possible binding modes governing the
aromatase inhibitory activities.
2. Results and discussion
2.1. Chemistry
Alkynes 5a-k were prepared by alkylation reaction of the corresponding phenols or
carboxylic acids 3a-k with propargyl bromide using K2CO3 as a base in acetone or in DMF,
whereas alkynes 5l and 5m were procured from the commercially available sources. 2-((3-
Azidophenyl)sulfonyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline 6 has been prepared
according to the method reported by our group [13]. The copper(I)-catalyzed alkyne-azide
cycloaddition (CuAAC) of the azide 6 and alkynes 5 was performed to afford the desired
1,2,3-triazole products 2a-m in good yields (70-94%).
Structures of the novel 1,2,3-triazoles 2 were well characterized by their 1H NMR, 13C
NMR, HRMS and IR spectra. 1H NMR of all products showed a singlet peak with down
field chemical shift at ꢁ in the range of 8.10-8.32 ppm, which indicated that the triazole ring
was formed, and HRMS data were in accordance with the expected chemical structures. In
case of derivatives 2j and 2k, their carbonyl carbons (CO) were observed at δ 166.6 and
177.6 ppm, respectively in 13C NMR spectra, and their vibration absorption bands (IR
spectra) of C=O groups appeared at 1717 and 1724 cm-1, respectively.
2.2. Biological activity
2.2.1 Aromatase inhibitory activity and Structure-activity relationships (SAR)
The novel thirteen isoquinoline-sulfonamide-triazole derivatives (2a-m) with different
substituents (R) at position 4 of the triazole core, were assayed for their aromatase inhibitory
activities. The derivatives (2a, 2e-h and 2m) with the inhibition ≤50% were identified as

5
inactive compound (IC50 > 12.5 μM), and the compounds (2b-d and 2i-l) with inhibition
>50% were further evaluated for their IC50 values using ketoconazole and letrozole as the
reference drugs as shown in Table 1.
Seven derivatives (2b-d and 2i-l) displayed the aromatase inhibitory activity better than
the ketoconazole, but lesser than the letrozole. The study indicated that hydrophobic
substituents (R) on the triazole ring play important roles in governing their aromatase
inhibitory activities, and a structure-activity relationship (SAR) of the tested compounds is
discussed. No significant inhibitory effect was observed for triazole 2a bearing
phenoxymethyl (R) group on the triazole ring. However, derivatives 2b-d achieved from an
introduction of lipophilic substituents (methyl, ethyl and propyl moieties) at para position on
the oxyphenyl ring of compound 2a displayed the inhibitory effect with IC50 values in the
range of 0.2-1.7 μM. Apparently, the triazoles 2b with methyl group and 2d with propyl
group had comparable IC50 values with that of the hit compound (1, IC50 = 0.2 μM)
containing 7-coumarinyl substituent on the triazole ring [13]. Unfortunately, triazoles (2e-h)
were shown to be inactive. The results indicated that steric bulky groups (t-butyl, phenyl,
benzyl and bromo groups) substituted on the phenyl ring of R group hindered the crucial
interaction with the target site. Because the volume of the aromatase active site is very small
[15], the bulky group presented in these compounds may lead to loss of ability to reach active
site and loss of the activity. When the phenyl group of compound 2a was replaced with 2-
naphthalenyl ring as found in compound 2i (R= 2-napthalenyloxymethyl), the distinctively
enhanced inhibitory potency was observed. The compound 2i was shown to be the most
active derivative (IC50 = 0.07 μM) with 3-fold more potent than the hit compound 1 (R=
coumarinyloxymethyl) [11].
Inhibitory potency was remarkably improved as noted in ester 2j (IC50 = 0.8 μM)
resulting from the insertion of C=O group between O-Phenyl bond of the analog 2a (IC50 >
12.5 μM). When the phenyl group of compound 2j was replaced with 1-adamantanyl group
leading to ester 2k (IC50 = 1.9 μM), the observed potency was 2-fold reduction. The result
implies that the phenyl ring may be required for π-π stacking and/or hydrophobic interaction
with the target site. Inhibitory potency was observed when the oxymethyl group of compound
2h (IC50 > 12.5 μM) was removed to give the active compound 2l (IC50 = 0.6 μM). This
could suggest that the bromo derivative without oxymethyl group (2l) might be an
appropriate size required for hydrophobic interaction with the target site of action when

6
compared with the bigger molecule containing oxymethyl moiety (2h). At this point, it could
be hypothesized that synthesis of the most active compound 2i without the oxymethyl moiety
is attractive to be further studied. Replacement of the bromo substituent of triazole 2l with
trifluoromethyl (CF3) substituent led to the compound 2m with loss of the activity. It could be
due to the lower lipophilic effect of CF3 group compared with the Br group.
2.2.2 Cytotoxic activity
These triazoles (2a-m) were further evaluated for their cytotoxic effects against the
hormone-dependent breast cancer cell line (T47-D) using MTT assay (Table 1). The results
showed that compounds 2b-f, 2i and 2m exhibited moderate cytotoxic activity (IC50 8.66-
58.85 µM). The compounds 2e, 2f and 2m displayed cytotoxic activity against T47-D cells
whereas they were shown to be inactive for aromatase inhibitory potency (IC50 > 12.5 μM).
These could be anticipated that the potent cytotoxic effect of compounds 2e, 2f and 2m may
be responsible by different mechanisms and/or biological targets [16- 20].
These compounds were also investigated against normal embryonic lung cells (MRC-5)
using MTT assay to determine the safety index (Table 1). It was found that the active analogs
(2b-d and 2k) showed cytotoxicity to non-cancerous cell in the range of 4.66-30.96.
Interestingly, the potent analogs (2i, 2j and 2l) were non-cytotoxic toward the normal cell,
and the most potent triazole 2i had very high safety index with a selectivity index (SI) value
of >1283.
2.3. Molecular docking
Molecular docking of the investigated triazole analogs 2a-m was performed against
the target protein to reveal possible binding modes of the compounds. Crystal structure of
human placental aromatase cytochrome P450 in complex with androstenedione (PDB:
3EQM) was selected as a target protein due to its acceptably high resolution (2.9 Å) and its
androgenic specificity [15]. The target protein underwent preparation processes including
repairing missing side chains and adding essential hydrogen atoms followed by merging non-
polar hydrogen atoms, assigning Gasteiger atomic charges, and specifying atom type of the
protein structures. All investigated compounds were drawn, geometrically optimized, and
prepared by defining rotatable bonds, merging non-polar hydrogen atoms, and assigning
partial atomic charge. Before investigating possible binding modes of the studied compounds,

7
the docking protocol need to be validated to ensure its performance. Redocking is one of the
widely used pose selection validation methods, which is performed by docking a ligand with
known conformation and orientation in the active site of the target, which is typically derived
from a co-crystal protein structure [21]. Success of pose prediction of the docking protocol
was evaluated by comparing the different between original co-crystalized position and redock
position (which is calculated by root mean squared deviation: RMSD value), the docking
protocol providing RMSD less than 2 Å is considered to perform successfully [22]. The
validation of docking protocol was conducted by redocking the co-crystallized ligand
androstenedione (ASD) to the aromatase enzyme. The redocking provided acceptable RMSD
value of 0.705 A° (Fig. 3A). The same docking protocol was subsequently employed to
investigate possible binding modes of the triazole derivatives (2a-m). The molecular docking
results showed that all investigated triazole derivatives could occupy within the active site of
the aromatase enzyme (Fig. 3B). Compound 2i (IC50 = 0.07 µM) was selected as a
representative compound for detailed discussion according to its most potent inhibitory
activity (Fig. 3C). The 2D ligand-protein interaction diagrams (Fig. 4) showed that the
oxygen atoms of oxymethylene linker between naphthalene and triazole rings, and of
methoxyl group substituted on the isoquinoline ring of compound 2i could form hydrogen
bonding interactions with the residues Ser478 and Thr310 of the aromatase enzyme,
respectively (Fig. 4B). The THIQ moiety of compound 2i could also mimic steroidal
backbone of the natural ASD by forming hydrophobic interaction with Ile133 residue (Fig.
4B). Moreover, other hydrophobic parts (naphthalenyl and benzene sulfonyl) of the
compound 2i could form hydrophobic interactions with residues Ser478, His480, Asp309,
Phe221, and Val370 (Fig. 4B). In addition, the same set of enzyme residues (i.e., Ser478,
Asp309, Val370, and Phe221) are involved in the hydrophobic interactions of the second
most potent compound 2d, IC50 = 0.2 µM (Fig. 4C), and the third most potent compound 2b,
IC50 = 0.3 µM (Fig. 4D). Particularly, the Phe221 displayed π-π stacking interactions with the
triazole ring of these active compounds (2i, 2d, and 2b). This Phe221 residue also played role
in forming π-π stacking interaction with the phenyl ring substituted at position 4 of the
triazole moiety of compounds 2d, and 2b as well as the naphthalenyl ring substituted at
position 4 of the triazole moiety of 2i. Such π- π stacking interactions of the Phe221 with the
triazole and with the coumarinyl rings were also reported for the hit compound 1 as shown in
Fig. 2 [13]. In addition, the Ser478 residue establishes a common H-bonding with the

8
oxymethylene linker of compounds 2i, 2d, and 2b as well as the hit compound 1 [13].
However, compound 2i is the only one that could form direct H-bond interactions with both
Thr310 and Ser478 residues (Fig. 4B), while that of the Ser478 was only observed for the
others (2d and 2b), Fig. 4C-4D. This suggested that the direct H-bonding interaction with
Thr310 residue may be responsible for a striking inhibitory effect of the most potent
compound 2i. Accordingly, it is suggested that further investigations are necessary to reveal
insight mechanism of inhibition and kinetics of these novel compounds.
Non-steroidal aromatase inhibitors currently used in clinical such as letrozole and
anastrozole are 1,2,4-triazole based compounds. However, the mechanism of aromatase
inhibition of the triazoles is still controversial. Many possible mechanisms of inhibition have
been proposed including competitive, non-competitive (allosteric), or mixed inhibition [23-
25]. Molecular dynamic study revealed that the 1,2,4-triazole-based compounds could elicit
aromatase inhibitory effect via competitive inhibition mechanism, in which a coordination
interaction between nitrogen atom of the 1,2,4-triazole ring and the Fe atom of aromatase
heme is considered a crucial interaction for the inhibitory activity [23].
Our molecular docking finding revealed that the investigated 1,2,3-triazole-based
compounds (2a-m) could competitively bind in active site of the aromatase enzyme to elicit
their inhibitory effects (Fig. 3), and was in agreement with the hypothesized competitive
inhibition mechanism. However, the 2D ligand-protein interaction diagrams (Fig. 4) showed
that these compounds interacted with the target aromatase enzyme via several types of
interactions, except for the direct coordination with Fe atom of the aromatase heme.
Considering the competitive nature as well as limitations caused by long-term used of
available steroidal and non-steroidal aromatase inhibitors [24], current attention has been
given to the discovery of alternative non-competitive/allosteric aromatase inhibitors since an
estrogen receptor modulator namely tamoxifen has been reported to inhibit aromatase
enzyme activity via allosteric mechanism and its three potential allosteric pockets were noted
[25]. This finding has led the searching for the inhibitors with alternative mode of inhibition.
Previous kinetics study indicated that the letrozole, a 1,2,4-triazole containing drug, could act
as a non-competitive or mixed aromatase inhibitor [26]. Allosteric inhibition of the letrozole
was also confirmed by the work of Spinello et al. [27], which investigated the binding mode
of letrozole against allosteric pockets [25] by performing molecular dynamics and free energy
simulation.

9
Aromatase enzyme is a metalloprotein, which contains metal ions in its binding sites.
The binding sites of metalloproteins are highly polarized due to the locating high
coordinated-numbered metal ions. It is widely recognized that the molecular docking of
metalloproteins is challenging due to multiple coordination geometries, lack of sufficiently
accurate force field parameters, and requirement of optimizing metal ion parameters [28].
Most of the current docking methods use force field based fixed electric charges for scoring
electrostatic energy portion of both ligand and protein, in which the determined charges of
metal ions may not appropriate for docking to provide preferable accuracy [29]. To overcome
this limitation, it has been suggested that the protein atoms surrounding the binding sites
together with the nearby metal ions should be included, in addition to the ligand atoms, as a
part of quantum region for quantum mechanics/molecular mechanics (QM/MM) calculations
in scoring function of molecular docking [29]. Several previous works demonstrated that,
despite of extended time of calculation, using QM/MM docking could improve the accuracy
of pose predictions [28, 30-31].
Accordingly, further investigation using in vitro kinetic assay is recommended to
elucidate actual mechanism of inhibition of these novel potential 1,2,3-triazole-based
inhibitors. It is also suggested that further insight investigations using other computational
methods (such as molecular dynamics) and softwares (which are be able for inclusion of
water molecule and metal ion in the docking simulation) are required for overcoming the
limitations of this study to provide more accurate definite binding modalities, interactions,
and mechanisms of action of these compounds.
3. Conclusions
Thirteen tetrahydroisoquinoline-triazole hybrids (2a-m) have been achieved using the
Pictet-Spengler and CuAAC reactions as the key steps. Their aromatase inhibitory activities
and molecular docking were investigated. Seven triazoles exerted the aromatase inhibitory
activity (IC50 = 0.07-1.9 ꢀM) superior than that of the ketoconazole. Particularly, the triazole
2i was shown to be the most potent inhibitor (IC50 = 0.07 ꢀM) without affecting the non-
cancerous cell line. Its inhibition potency was improved 3-fold compared with the hit
compound 1. The molecular docking also suggested that the formation of hydrogen bonding
between the methoxy group of compound 2i and the Thr310 residue of the enzyme may be
essential for its highly potent aromatase inhibitory effect. In summary, a set of

10
tetrahydroisoquinoline-triazole hybrids was highlighted as promising compounds to be
further developed for managing estrogen-dependent diseases and cancers. The molecular
docking results are also useful for guiding the design and synthesis of new aromatase
inhibitors with improved potency and properties.
4. Experimental
4.1 General
Column chromatography was carried out using silica gel 60 (70–230 mesh ASTM).
Analytical thin-layer chromatography (TLC) was performed with silica gel 60 F254 aluminum
1
sheets. H- and13C-NMR spectra were recorded on a Bruker AVANCE 300 and a Bruker
1
13
AscendTM 400 NMR spectrometers. H NMR and C NMR chemical shifts are reported in
ppm using tetramethylsilane (TMS) or residual nondeuterated solvent peak as an internal
standard. The following standard abbreviations were used for signal multiplicities: singlet (s),
doublet (d), triplet (t), quartet (q) and multiplet (m). FTIR spectra were obtained using a
universal attenuated total reflectance attached on a Perkin–Elmer Spectrum One
spectrometer. Mass spectra were recorded on a Bruker Daltonics (microTOF). Melting points
were determined using a Griffin melting point apparatus and were uncorrected.
Compound 6 was prepared according to literature procedures [13]. All chemicals were
used without further purification and purchased from commercial sources. A recombinant
human aromatase (CYP19) and O-benzyl fluorescein benzyl ester (DBF) were supplied with
the BD GentestTM kit from BD Biosciences-Discovery Labware (Woburn, USA). Reagents
using in cell culture and assay were obtained from the following sources: DMEM
(Dulbecco’s Modified Eagle’s Medium), RPMI-1640 (Rosewell Park Memorial Institute
medium), FBS (fetal bovine serum) from Hyclone laboratories (Logan, UT, USA); MTT
(3(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide), L-glutamine, penicillin-
streptomycin, insulin and glucose from Sigma-Aldrich (St. Louis, MO, USA).
4.2 General procedure for the synthesis of alkynes (5a-i)
A propargyl bromide (12 mmol) was added to a suspension of an appropiate phenol 3a-i (10
mmol) and potassium carbonate (15 mmol) in acetone (15 mL). The suspension was heated to
reflux until completion of reaction (monitored by TLC). The reaction was allowed to cool
and then concentrated under reduced pressure. Water (30 mL) was added and extracted with

11
EtOAc (3 × 30 mL). The organic extracts were combined and washed with water (20 mL)
and brine (20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and
concentrated. The crude product was purified by column chromatography.
1H NMR spectra of (prop-2-yn-1-yloxy)benzene (5a) [32], 1-methyl-4-(prop-2-yn-1-
yloxy)benzene (5b) [32], 1-ethyl-4-(prop-2-yn-1-yloxy)benzene (5c) [33], 1-(tert-butyl)-4-
(prop-2-yn-1-yloxy)benzene (5e) [33], 4-(prop-2-yn-1-yloxy)-1,1'-biphenyl (5f) [34], 1-
bromo-4-(prop-2-yn-1-yloxy)benzene (5h) [32], and 2-(prop-2-yn-1-yloxy)naphthalene (5i)
[35] were consistent with those of the published result.
1-(prop-2-yn-1-yloxy)-4-propylbenzene (5d)
Pale yellow oil. 90% yield. 1H NMR (300 MHz, CDCl3) δ 0.91 (t, J = 7.3 Hz, 3H, CH3), 1.59
(sext, J = 7.6 Hz, 2H, CH2CH3), 2.49 (t, J = 2.6 Hz, 1H, HCꢂC), 2.52 (t, J = 7.4 Hz, 2H,
CH2Ar), 4.65 (s, 2H, CH2O), 6.88 (d, J = 8.6 Hz, 2H, ArH), 7.09 (d, J = 8.6 Hz, 2H, ArH). 13C
NMR (75 MHz, CDCl3) δ 13.8, 24.7, 37.2, 55.9, 75.3, 78.9, 114.7, 129.3, 135.8, 155.7.
1-benzyl-4-(prop-2-yn-1-yloxy)benzene (5g)
1
Pale yellow oil. 93% yield. H NMR (300 MHz, CDCl3) δ 2.52 (t, J = 2.5 Hz, 1H, HCꢂC),
3.95 (s, 2H, CH2Ar), 4.67 (s, 2H, CH2O), 6.92 (d, J = 8.6 Hz, 2H, ArH), 7.13 (d, J = 8.6 Hz,
13
2H, ArH), 7.16-7.34 (m, 5H, ArH). C NMR (75 MHz, CDCl3) δ 41.1, 55.9, 75.4, 78.8,
115.0, 126.0, 128.4, 128.9, 129.9, 134.3, 141.4, 156.0.
4.3 General procedure for the synthesis of alkynes (5j-k)
A propargyl bromide (12 mmol) was added to a suspension of an appropiate carboxylic acid
3j-k (10 mmol) and potassium carbonate (15 mmol) in dimethylformamide (10 mL). The
suspension was stirred at room temperature until completion of reaction (monitored by TLC).
The reaction was allowed to cool and then concentrated under reduced pressure. Water (30
mL) was added and extracted with EtOAc (3 × 30 mL). The organic extracts were combined
and washed with water (20 mL) and brine (20 mL). The organic layer was dried over
anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by
column chromatography.
1H NMR spectra of prop-2-yn-1-yl benzoate (5j) was consistent with that reported in the
literature [36].
prop-2-yn-1-yl adamantane-1-carboxylate (5k)

12
Pale yellow oil. 80% yield. 1H NMR (300 MHz, CDCl3) δ 1.69 (s, 6H, 3 × CH2), 1.90 (s, 6H,
3 × CH2), 2.00 (s, 3H, 3 × CH), 2.42 (t, J = 2.5 Hz, 1H, HCꢂC), 4.62 (s, 2H, CH2O). 13C
NMR (75 MHz, CDCl3) δ 27.9, 36.6, 38.7, 40.7, 51.7, 74.4, 78.1, 176.8.
4.4 General procedure for the synthesis of triazoles (2a-m)
To a stirred solution of azide 6 (0.2 mmol) and the corresponding alkyne 5 (0.2 mmol)
in t-BuOH:H2O (3:3 mL), CuSO445H2O (0.2 mmol) and ascorbic acid (0.5 mmol) were
added. The reaction mixture was stirred at room temperature for 2-12 h (monitored by TLC),
then concentrated under reduced pressure. The residue was added water (10 mL) and
extracted with dichloromethane (3 × 20 mL). The combined organic phases were washed
with water (20 mL), dried over anh. Na2SO4 and evaporated to dryness. The crude product
was purified using silica gel column chromatography and eluted with
methanol:dichloromethane (1:50).
4.4.1 6,7-dimethoxy-2-((3-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)phenyl)sulfonyl)-1,2,3,4-
tetrahydroisoquinoline (2a)
o
White solid. 78% yield. mp 153-154 C. IR (UATR) cm-1: 1598, 1518, 1347, 1226, 1157,
1117. 1H NMR (300 MHz, CDCl3) δ 2.85 (t, J = 5.7 Hz, 2H, C4-H), 3.45 (t, J = 5.7 Hz, 2H,
C3-H), 3.82 (s, 6H, 2 × OCH3), 4.28 (s, 2H, C1-H), 5.32 (s, 2H, CH2O), 6.53, 6.54 (2s, 2H,
ArH), 6.97-7.07 (m, 3H, ArH), 7.33 (t, J = 7.4 Hz, 2H, ArH), 7.71 (t, J = 8.0 Hz, 1H, ArH),
7.90 (d, J = 7.9 Hz, 1H, ArH), 8.02 (d, J = 8.0 Hz, 1H, ArH), 8.13 (s, 1H, CHN), 8.20 (s, 1H,
13
ArH). C NMR (75 MHz, CDCl3) δ 28.2, 43.9, 47.2, 55.9, 56.0, 61.9, 109.0, 111.4, 114.8,
119.3, 120.8, 121.5, 122.9, 124.4, 124.8, 127.5, 129.7, 130.8, 137.5, 139.2, 145.8, 147.9,
148.1, 158.1. HRMS-TOF: m/z [M + Na]+ 529.1513 (Calcd for C26H26N4NaO5S: 529.1516).
4.4.2
6,7-dimethoxy-2-((3-(4-((p-tolyloxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)sulfonyl)-
1,2,3,4-tetrahydroisoquinoline (2b)
o
White solid. 93% yield. mp 129-130 C. IR (UATR) cm-1: 1612, 1509, 1347, 1226, 1157,
1117. 1H NMR (300 MHz, CDCl3) δ 2.31 (s, 3H, CH3), 2.85 (t, J = 5.7 Hz, 2H, C4-H), 3.45
(t, J = 5.7 Hz, 2H, C3-H), 3.82 (s, 6H, 2 × OCH3), 4.28 (s, 2H, C1-H), 5.29 (s, 2H, CH2O),
6.53, 6.55 (2s, 2H, ArH), 6.93 (d, J = 8.6 Hz, 2H, ArH), 7.12 (d, J = 8.4 Hz, 2H, ArH), 7.71
(t, J = 8.0 Hz, 1H, ArH), 7.89 (d, J = 8.0 Hz, 1H, ArH), 8.02 (dd, J = 8.0, 1.2 Hz, 1H, ArH),
13
8.12 (s, 1H, CHN), 8.20 (t, J = 1.7 Hz, 1H, ArH). C NMR (75 MHz, CDCl3) δ 20.5, 28.2,
43.9, 47.2, 55.9, 56.0, 62.0, 108.9, 111.4, 114.6, 119.3, 120.8, 122.9, 124.4, 124.8, 127.4,

13
130.1, 130.8, 130.9, 137.5, 139.2, 146.0, 147.9, 148.1, 156.0. HRMS-TOF: m/z [M + Na]+
543.1662 (Calcd for C27H28N4NaO5S: 543.1673).
4.4.3
2-((3-(4-((4-ethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)sulfonyl)-6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline (2c)
o
White solid. 76% yield. mp 135-136 C. IR (UATR) cm-1: 1611, 1509, 1347, 1226, 1157,
1117. 1H NMR (300 MHz, CDCl3) δ 1.22 (t, J = 7.6 Hz, 3H, CH3), 2.61 (q, J = 7.6 Hz, 2H,
CH2), 2.85 (t, J = 5.8 Hz, 2H, C4-H), 3.45 (t, J = 5.9 Hz, 2H, C3-H), 3.82 (s, 6H, 2 × OCH3),
4.28 (s, 2H, C1-H), 5.30 (s, 2H, CH2O), 6.53, 6.54 (2s, 2H, ArH), 6.95 (d, J = 8.6 Hz, 2H,
ArH), 7.15 (d, J = 8.5 Hz, 2H, ArH), 7.71 (t, J = 8.0 Hz, 1H, ArH), 7.89 (d, J = 7.9 Hz, 1H,
13
ArH), 8.02 (d, J = 8.0 Hz, 1H, ArH), 8.12 (s, 1H, CHN), 8.20 (s, 1H, ArH). C NMR (75
MHz, CDCl3) δ 15.8, 28.0, 28.2, 43.9, 47.2, 55.9, 56.0, 62.1, 109.0, 111.5, 114.7, 119.3,
120.7, 123.0, 124.3, 124.8, 127.4, 128.9, 130.8, 137.4, 137.5, 139.2, 146.0, 147.9, 148.1,
156.1. HRMS-TOF: m/z [M + Na]+ 557.1813 (Calcd for C28H30N4NaO5S: 557.1829).
4.4.4
6,7-dimethoxy-2-((3-(4-((4-propylphenoxy)methyl)-1H-1,2,3-triazol-1-
yl)phenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (2d)
o
White solid. 93% yield. mp 139-140 C. IR (UATR) cm-1: 1611, 1509, 1347, 1226, 1157,
1117. 1H NMR (300 MHz, CDCl3) δ 0.91 (t, J = 7.3 Hz, 3H, CH3), 1.50-1.68 (m, 2H, CH2),
2.52 (t, J = 7.6 Hz, 2H, CH2), 2.83 (t, J = 5.7 Hz, 2H, C4-H), 3.42 (t, J = 5.9 Hz, 2H, C3-H),
3.79 (s, 6H, 2 × OCH3), 4.26 (s, 2H, C1-H), 5.27 (s, 2H, CH2O), 6.51, 6.52 (2s, 2H, ArH),
6.93 (d, J = 8.6 Hz, 2H, ArH), 7.10 (d, J = 8.5 Hz, 2H, ArH), 7.68 (t, J = 8.0 Hz, 1H, ArH),
7.87 (d, J = 7.9 Hz, 1H, ArH), 7.99 (dd, J = 8.0, 1.1 Hz, 1H, ArH), 8.10 (s, 1H, CHN), 8.17
(s, 1H, ArH). 13C NMR (75 MHz, CDCl3) δ 13.7, 24.7, 28.2, 37.1, 43.9, 47.2, 55.9, 56.0,
62.0, 109.0, 111.5, 114.6, 119.3, 120.8, 123.0, 124.3, 124.8, 127.4, 129.5, 130.8, 135.8,
137.5, 139.2, 146.0, 147.9, 148.1, 156.2. HRMS-TOF: m/z [M + Na]+ 571.1989 (Calcd for
C29H32N4NaO5S: 571.1986).
4.4.5
2-((3-(4-((4-(tert-butyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)sulfonyl)-6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline (2e)
White solid. 78% yield. mp 81-82 oC. IR (UATR) cm-1: 1610, 1514, 1348, 1238, 1158, 1118.
1H NMR (300 MHz, CDCl3) δ 1.31 (s, 9H, 3 × CH3), 2.85 (br t, 2H, C4-H), 3.45 (br t, J = 5.4
Hz, 2H, C3-H), 3.82 (s, 6H, 2 × OCH3), 4.28 (s, 2H, C1-H), 5.31 (s, 2H, CH2O), 6.53, 6.55
(2s, 2H, ArH), 6.97 (d, J = 8.8 Hz, 2H, ArH), 7.34 (d, J = 8.7 Hz, 2H, ArH), 7.71 (t, J = 8.0
Hz, 1H, ArH), 7.89 (d, J = 7.8 Hz, 1H, ArH), 8.02 (d, J = 8.1 Hz, 1H, ArH), 8.13 (s, 1H,

14
13
CHN), 8.20 (s, 1H, ArH). C NMR (75 MHz, CDCl3) δ 28.2, 31.5, 34.1, 43.9, 47.2, 55.9,
56.0, 62.0, 109.0, 111.4, 114.2, 119.3, 120.8, 122.9, 124.4, 124.8, 126.4, 127.4, 130.8, 137.5,
139.2, 144.3, 146.0, 147.9, 148.1, 155.8. HRMS-TOF: m/z [M + Na]+ 585.2127 (Calcd for
C30H34N4NaO5S: 585.2142).
4.4.6 2-((3-(4-(([1,1'-biphenyl]-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)sulfonyl)-6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline (2f)
o
White solid. 86% yield. mp 160-161 C. IR (UATR) cm-1: 1609, 1519, 1348, 1239, 1158,
1117. 1H NMR (300 MHz, CDCl3) δ 2.85 (t, J = 5.7 Hz, 2H, C4-H), 3.45 (t, J = 5.7 Hz, 2H,
C3-H), 3.82 (s, 6H, 2 × OCH3), 4.29 (s, 2H, C1-H), 5.37 (s, 2H, CH2O), 6.53, 6.54 (2s, 2H,
ArH), 7.11 (d, J = 8.8 Hz, 2H, ArH), 7.32 (t, J = 7.4 Hz, 2H, ArH), 7.43 (t, J = 7.5 Hz, 1H,
ArH), 7.52-7.60 (m, 4H, ArH), 7.72 (t, J = 8.0 Hz, 1H, ArH), 7.90 (d, J = 7.9 Hz, 1H, ArH),
13
8.03 (d, J = 8.0 Hz, 1H, ArH), 8.15 (s, 1H, CHN), 8.21 (s, 1H, ArH). C NMR (75 MHz,
CDCl3) δ 28.2, 43.9, 47.2, 55.9, 56.0, 62.0, 109.0, 111.4, 115.1, 119.3, 120.9, 122.9, 124.4,
124.8, 126.8, 126.9, 127.5, 128.3, 128.8, 130.8, 134.7, 137.5, 139.3, 140.6, 145.7, 147.9,
148.1, 157.6. HRMS-TOF: m/z [M + Na]+ 605.1834 (Calcd for C32H30N4NaO5S: 605.1829).
4.4.7
2-((3-(4-((4-benzylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)sulfonyl)-6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline (2g)
o
White solid. 87% yield. mp 137-138 C. IR (UATR) cm-1: 1611, 1509, 1348, 1226, 1157,
1117. 1H NMR (300 MHz, CDCl3) δ 2.85 (t, J = 5.8 Hz, 2H, C4-H), 3.45 (t, J = 5.9 Hz, 2H,
C3-H), 3.82 (s, 6H, 2 × OCH3), 3.94 (s, 2H, CH2Ph), 4.28 (s, 2H, C1-H), 5.29 (s, 2H, CH2O),
6.53, 6.55 (2s, 2H, ArH), 6.96 (d, J = 8.6 Hz, 2H, ArH), 7.10-7.32 (m, 7H, ArH), 7.71 (t, J =
8.0 Hz, 1H, ArH), 7.89 (d, J = 8.0 Hz, 1H, ArH), 8.02 (d, J = 8.0 Hz, 1H, ArH), 8.11 (s, 1H,
13
CHN), 8.20 (s, 1H, ArH). C NMR (75 MHz, CDCl3) δ 28.2, 41.1, 43.8, 47.2, 55.9, 56.0,
62.0, 109.0, 111.5, 114.8, 119.3, 120.8, 122.9, 124.3, 124.8, 126.1, 127.4, 128.5, 128.8,
130.0, 130.8, 134.3, 137.5, 139.2, 141.3, 145.9, 147.9, 148.1, 156.5. HRMS-TOF: m/z [M +
H]+ 597.2162 (Calcd for C33H33N4O5S: 597.2166).
4.4.8
2-((3-(4-((4-bromophenoxy)methyl)-1H-1,2,3-triazol-1-yl)phenyl)sulfonyl)-6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline (2h)
White solid. 91% yield. mp 73-74 oC. IR (UATR) cm-1: 1600, 1489, 1344, 1244, 1157, 1119.
1H NMR (300 MHz, CDCl3) δ 2.85 (t, J = 5.8 Hz, 2H, C4-H), 3.45 (t, J = 5.8 Hz, 2H, C3-H),
3.82 (s, 6H, 2 × OCH3), 4.29 (s, 2H, C1-H), 5.28 (s, 2H, CH2O), 6.53, 6.54 (2s, 2H, ArH),
6.93 (d, J = 8.9 Hz, 2H, ArH), 7.42 (d, J = 8.9 Hz, 1H, ArH), 7.71 (t, J = 8.0 Hz, 1H, ArH),

15
7.90 (d, J = 7.9 Hz, 1H, ArH), 8.02 (d, J = 8.0 Hz, 1H, ArH), 8.10 (s, 1H, CHN), 8.19 (s, 1H,
13
ArH). C NMR (75 MHz, CDCl3) δ 28.2, 43.8, 47.2, 55.9, 56.0, 62.1, 109.0, 111.5, 113.8,
116.6, 119.3, 120.9, 122.9, 124.4, 124.8, 127.5, 130.8, 132.5, 137.4, 139.4, 145.3, 147.9.
148.1, 157.2. HRMS-TOF: m/z [M + Na]+ 607.0613 (Calcd for C26H25BrN4NaO5S:
607.0621).
4.4.9
6,7-dimethoxy-2-((3-(4-((naphthalen-2-yloxy)methyl)-1H-1,2,3-triazol-1-
yl)phenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (2i)
o
White solid. 94% yield. mp 150-151 C. IR (UATR) cm-1: 1599, 1519, 1348, 1257, 1158,
1118. 1H NMR (300 MHz, CDCl3) δ 2.84 (t, J = 5.8 Hz, 2H, C4-H), 3.45 (t, J = 5.9 Hz, 2H,
C3-H), 3.81 (s, 6H, 2 × OCH3), 4.29 (s, 2H, C1-H), 5.44 (s, 2H, CH2O), 6.53, 6.54 (2s, 2H,
ArH), 7.20-7.50 (m, 4H, ArH), 7.70 (t, J = 8.0 Hz, 1H, ArH), 7.74-7.81 (m, 3H, ArH), 7.89
(d, J = 7.9 Hz, 1H, ArH), 8.02 (d, J = 8.1 Hz, 1H, ArH), 8.16 (s, 1H, CHN), 8.21 (s, 1H,
13
ArH). C NMR (75 MHz, CDCl3) δ 28.2, 43.8, 47.2, 55.9, 56.0, 62.0, 107.4, 109.0, 111.5,
118.6, 119.4, 120.8, 123.0, 124.1, 124.3, 124.8, 126.6, 126.9, 127.4, 127.7, 129.3, 129.7,
130.8, 134.4, 137.5, 139.3, 145.7, 148.0, 148.1, 156.0. HRMS-TOF: m/z [M + Na]+ 579.1670
(Calcd for C30H28N4NaO5S: 579.1673).
4.4.10
(1-(3-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)phenyl)-1H-1,2,3-
triazol-4-yl)methyl benzoate (2j)
White solid. 87% yield. mp 75-76 oC. IR (UATR) cm-1: 1717, 1600, 1519, 1451, 1348, 1268,
1157, 1116. 1H NMR (400 MHz, CDCl3) δ 2.85 (t, J = 5.6 Hz, 2H, C4-H), 3.44 (t, J = 5.7 Hz,
2H, C3-H), 3.82 (s, 6H, 2 × OCH3), 4.27 (s, 2H, C1-H), 5.58 (s, 2H, CH2O), 6.53, 6.54 (2s,
2H, ArH), 7.45 (t, J = 7.6 Hz, 2H, ArH), 7.58 (t, J = 7.3 Hz, 1H, ArH), 7.71 (t, J = 8.0 Hz,
1H, ArH), 7.90 (d, J = 7.8 Hz, 1H, ArH), 8.02 (d, J = 8.0 Hz, 1H, ArH), 8.08 (d, J = 7.6 Hz,
2H, ArH), 8.21 (s, 1H, ArH), 8.22 (s, 1H, ArH, CHN). 13C NMR (100 MHz, CDCl3) δ 28.2,
43.9, 47.2, 55.9, 57.9, 108.8, 111.3, 119.4, 122.3, 122.9, 124.4, 124.7, 127.6, 128.5, 129.5,
129.8, 130.8, 133.4, 137.4, 139.0, 144.3, 147.8, 148.0, 166.6. HRMS-TOF: m/z [M + H]+
535.1636 (Calcd for C27H27N4O6S: 535.1646).
4.4.11
(1-(3-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)phenyl)-1H-1,2,3-
triazol-4-yl)methyl adamantane-1-carboxylate (2k)
White solid. 79% yield. mp 76-77 oC. IR (UATR) cm-1: 1724, 1599, 1519, 1348, 1226, 1158,
1117. 1H NMR (400 MHz, CDCl3) δ 1.60-1.90 (m, 12H, 6 × CH2), 1.99 (s, 3H, 3 × CH), 2.84
(t, J = 5.8 Hz, 2H, C4-H), 3.42 (t, J = 5.9 Hz, 2H, C3-H), 3.80 (s, 6H, 2 × OCH3), 4.26 (s, 2H,

16
C1-H), 5.27 (s, 2H, CH2O), 6.52, 6.53 (2s, 2H, ArH), 7.70 (t, J = 8.0 Hz, 1H, ArH), 7.87 (d,
J = 7.9 Hz, 1H, ArH), 7.99 (dd, J = 8.0, 1.2 Hz, 1H, ArH), 8.19 (t, J = 1.7 Hz, 1H, ArH), 8.20
13
(s, 1H, ArH, CHN). C NMR (100 MHz, CDCl3) δ 27.8, 28.2, 36.4, 38.7, 40.7, 43.9, 47.2,
55.9, 56.0, 57.2, 108.8, 111.3, 119.4, 121.8, 122.8, 124.4, 124.7, 127.5, 130.8, 137.4, 139.0,
144.6, 147.8, 148.0, 177.6. HRMS-TOF: m/z [M + Na]+ 615.2252 (Calcd for
C31H36N4NaO6S: 615.2248).
4.4.12
2-((3-(4-(4-bromophenyl)-1H-1,2,3-triazol-1-yl)phenyl)sulfonyl)-6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinoline (2l)
o
White solid. 71% yield. mp 204-205 C. IR (UATR) cm-1: 1599, 1517, 1475, 1344, 1227,
1157, 1119. 1H NMR (300 MHz, CDCl3) δ 2.85 (t, J = 5.7 Hz, 2H, C4-H), 3.48 (t, J = 5.9 Hz,
2H, C3-H), 3.82 (s, 6H, 2 × OCH3), 4.32 (s, 2H, C1-H), 6.53, 6.55 (2s, 2H, ArH), 7.62 (d, J =
8.5 Hz, 2H, ArH), 7.74 (t, J = 8.0 Hz, 1H, ArH), 7.80 (d, J = 8.5 Hz, 2H, ArH), 7.92 (d, J =
7.9 Hz, 1H, ArH), 8.08 (d, J = 8.1 Hz, 1H, ArH), 8.21 (s, 1H, ArH), 8.23 (s, 1H, CHN). 13C
NMR (75 MHz, CDCl3) δ 28.1, 43.8. 47.3, 55.9, 56.0, 109.0, 111.5, 117.5, 119.2, 122.8,
122.9, 124.4, 124.8, 127.4, 128.7, 130.9, 132.2, 137.4, 138.6, 143.9, 147.9, 148.1 HRMS-
TOF: m/z [M + Na]+ 577.0513 (Calcd for C25H23BrN4NaO4S: 577.0516).
4.4.13
6,7-dimethoxy-2-((3-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-
yl)phenyl)sulfonyl)-1,2,3,4-tetrahydroisoquinoline (2m)
o
White solid. 70% yield. mp 159-160 C. IR (UATR) cm-1: 1622, 1516, 1484, 1324, 1228,
1158, 1120. 1H NMR (300 MHz, CDCl3) δ 2.84 (t, J = 5.7 Hz, 2H, C4-H), 3.49 (t, J = 5.9 Hz,
2H, C3-H), 3.81, 3.82 (2s, 6H, 2 × OCH3), 4.33 (s, 2H, C1-H), 6.54 (s, 2H, ArH), 7.70-7.80
(m, 3H, ArH), 7.94 (d, J = 7.9 Hz, 1H, ArH), 8.02-8.13 (m, 3H, ArH), 8.24 (s, 1H, ArH),
13
8.32 (s, 1H, CHN). C NMR (75 MHz, CDCl3) δ 28.1, 43.8, 47.3, 55.9, 56.0, 108.4, 111.5,
118.3, 119.2, 122.9, 124.0 (q, 1JCF = 271), 124.4, 124.8, 126.0, 126.1, 127.6, 130.2 (q, 2JCF
=
33), 130.9, 133.2, 137.4, 139.5, 147.6, 147.9, 148.1. HRMS-TOF: m/z [M + Na]+ 567.1290
(Calcd for C26H23F3N4NaO4S: 567.1284).
4.5 Aromatase inhibition assay
Aromatase inhibitory effect was performed using the method reported by Stressor et
al. [37] with minor modification [13, 38]. This method was carried out according to the
Gentest kit using CYP19 enzyme and DBF as a fluorometric substrate. DBF was dealkylated
by aromatase and then hydrolyzed to give the fluorescein product.

17
Briefly, 100 µL of cofactor, containing 78.4 µL of 50 mM phosphate buffer (pH 7.4);
20 µL of 20x NADPH-generating system (26 mM NADP+, 66 mM glucose-6-phosphate, and
66 mM MgCl2); and 1.6 µL of 100 U/mL glucose-6-phosphate dehydrogenase, was pipetted
o
into a 96-well plate and preincubated in 37 C (water bath) for 10 min. The reaction was
initiated by addition of 100 µL of enzyme/substrate mixture containing 77.3 µL of 50 mM
phosphate buffer (pH 7.4); 12.5 µL of 16 pmol/mL CYP19; 0.2 µL of 0.2 mM DBF, and 10
µL of 0.25 mM diluted tested sample or 10% DMSO as a negative control or letrozole as a
positive control. Fluorescence signal was recorded using an excitation wavelength of 490 nm
and emission wavelength of 530 nm with cutoff 515 nm. Percentage of inhibition (%
inhibition) was calculated as shown in Equation (1). Samples with % inhibition greater than
50 were further diluted and assayed in triplicate. Finally, IC50 values were determined by plot
of concentrations versus % inhibition.
(1)
4.6 Cytotoxicity assay
The cytotoxic activity of compounds (2a-m) was tested using hormone-dependent
breast cancer cell line (T47-D) and normal embryonic lung cell line (MRC-5). T47-D cells
were grown in RPMI-1640 medium supplemented with 2 mM L-gluthamine, 100 U/mL
penicillin-streptomycin, 0.2 U/mL insulin, 4.5 g/L glucose and 10% FBS whereas MRC-5
cells were grown in DMEM medium supplemented with 100 U/mL penicillin streptomycin
and 10% FBS. Briefly, the cells suspended in the corresponding culture medium were
inoculated in 96-well microtiter plates (Corning Inc., NY, USA) at a density of 10,000-
o
20,000 cells per well, and then incubated at 37 C under a humidified atmosphere with 95%
air and 5% CO2 for 24 h. An equal volume of additional medium containing either the serial
dilutions of the tested compounds, positive control (etoposide and/or doxorubicin) or negative
control (DMSO) was added to the desired final concentrations. The microtiter plates were
further incubated for 48 h. Cell viability in each well was determined by staining with MTT
assay [39-41]. The MTT solution (10 mL/100 mL medium) was added to all wells of the
assay, and the plates were incubated for 2-4 h. Subsequently, DMSO was added to dissolve
the resulting formazan by sonication. The plates were read on a microplate reader (Molecular
Devices, USA) using a test wavelength of 550 nm and a reference wavelength of 650 nm.

18
The IC50 value was determined as the compound concentration that inhibited cell growth by
50%. The compounds exhibited IC50 > 50 µg/mL were considered as noncytotoxic.
4.7 Molecular docking
Molecular docking was performed to investigate possible binding modalities of the
compounds (2a-m) toward the aromatase enzyme. Crystal structure of the target protein,
human placental aromatase protein co-crystallized with natural substrate (ASD), was
retrieved from RSCB protein data bank (PDB ID: 3EQM, http://www.rcsb.org/), and was
prepared by adding essential hydrogen atoms and repairing missing side chains using the
WHAT IF web server version 10.1 [42]. Subsequently, non-polar hydrogen atoms were
merged, Gasteiger atomic charges were assigned, and atom type of the protein structures
were specified using AutoDock Tools version 1.5.6. [43, 44]. All investigated triazole
analogs (2a-m) were drawn using Marvin Sketch version 6.1.4 [45], and were geometrically
optimized by Gaussian 09 [46] using Becke’s three-parameter hybrid method with the Lee-
Yang-Parr correlation functional (B3LYP) together with the 6-31g(d) basis set. The
structurally optimized compounds were prepared by merging non-polar hydrogen atoms,
defining rotatable bonds and assigning partial atomic charge using AutoDock Tools version
1.5.6 [43, 44]. AutoDock Vina, as a part of the PyRx 0.8 software [47], was used to perform
the molecular docking simulation. Grid boxes size of 62.06 × 71.95 × 51.46 A° were
generated and the center of binding cavity was allocated using x, y and z coordinates of
83.4375, 50.1006, and 46.3803, respectively. The co-crystallized ligand, ASD, was re-docked
to the aromatase proteins as to validate docking protocol. The re-docking results were
evaluated by calculation of root mean standard deviation (RMSD) using Chimera software
[48]. Docking poses of the investigated compounds were visualized using PyMOL [49], and
two-dimensional ligand-protein interaction diagrams were generated using PoseView
(http://proteins.plus) [50].
Acknowledgments
This project is financially supported by Srinakharinwirot University (grant no.
012/2562). We gratefully acknowledge the support from the annual budget grant (BE 2562-
2563) of Mahidol University. We are also indebted to Chulabhorn Research Institute for
recording mass spectra and bioactivity testing.

19
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Figure Legends
Fig. 1. Representative aromatase inhibitors.
Fig. 2. Aromatase inhibitors containing triazole; hit compound (1) and modified compounds
(2).
Scheme 1. Synthesis of triazole-tetrahydroisoquinoline hybrids 2a-m through the Click
reaction.
Fig. 3. Possible binding modalities of the investigated triazoles (heme and Fe atom of the
aromatase enzyme active site are shown in orange and red, respectively, and key protein
residues are presented as colored dotted); (A) Redocking of the natural substrate ASD
providing RMSD = 0.705 A°. Docking pose of the original ASD is shown in yellow while
those of the redocked ASD is shown in green; (B) Docking poses of thirteen investigated
triazoles in the active site of aromatase enzyme; (C) The most potent compound 2i occupying
the enzyme active site.
Fig. 4. The 2D ligand-protein interactions of natural substrate ASD* (A), compound 2i (B),
compound 2d (C), and compound 2b (D).*Additional hydrogen bond between Asp309 and
carbonyl oxygen at C-3 of ASD has been reported (data not shown) [15].

24
ꢀ Thirteen tetrahydroisoquinolines bearing triazole were synthesized.
ꢀ Seven triazoles displayed aromatase inhibitory activity (IC50 = 0.07-1.9 ꢀM).
ꢀ The naphthalenyl analog 2i was shown to be the most potent compound.
ꢀ Molecular docking suggested the direct H-bonding interaction between the methoxy group
of 2i and the aromatase.

25
Table 1 Aromatase inhibitory activity and cytotoxic activity (IC50, μM) of triazoles (2a-m).
aromatase
inhibitory
activity
cytotoxic activity
selectivity
compound
R
indexa
(SI)
T47-D
MRC-5
>12.5
Non-
cytotoxic
Non-
cytotoxic
–
2a
0.3±0.1
22.26±0.579 30.96±4.978
103.21
2b
2c
2d
2e
1.7±0.4
0.2±0.0
>12.5
14.85±2.474 4.66±0.473 2.74
26.81±3.415 6.40±0.586 31.99
8.66±0.424 6.43±0.848
–
>12.5
14.78±1.209 77.68±0.572 –
2f

26
>12.5
>12.5
Non-
cytotoxic
75.26±2.298 –
2g
2h
Non-
cytotoxic
Non-
–
cytotoxic
58.85±6.208 Non-
cytotoxic
0.070±0.025
0.8±0.4
>1283.23
>116.91
2i
2j
Non-
Non-
cytotoxic
cytotoxic
Non-
cytotoxic
1.9±0.5
0.6±0.4
13.28±0.360 6.99
2k
Non-
Non-
>150.03
2l
cytotoxic
cytotoxic
>12.5
34.78±1.032 67.10±4.186 –
2m
2.6±0.7
–
–
–
–
–
Ketoconazoleb
Letrozoleb
0.0019±0.0002 –
–
–
Etoposideb
–
13.35±0.374
Doxorubicinb
–
0.88±0.021 2.19±0.37
T47-D = hormone-dependent breast cancer cell line; MRC-5 = normal embryonic lung cell
line.

27
Non-cytotoxic= IC50> 50 µg/mL.
aSI = IC50 for MRC-5/ IC50 for aromatase.
bKetoconazole, letrozole, etoposide and doxorubicin were used as reference drugs.

28
Fig. 1. Representative aromatase inhibitors.

29
Fig. 2. Aromatase inhibitors containing triazole; hit compound (1) and modified compounds
(2).