Structure-activity relationships and docking studies of synthetic 2-arylindole derivatives determined with aromatase and quinone reductase 1

Article abstract of DOI:10.1016/j.bmcl.2017.11.010

In our ongoing effort of discovering anticancer and chemopreventive agents, a series of 2-arylindole derivatives were synthesized and evaluated toward aromatase and quinone reductase 1 (QR1). Biological evaluation revealed that several compounds (e.g., 2d, IC₅₀ = 1.61 μM; 21, IC₅₀ = 3.05 μM; and 27, IC₅₀ = 3.34 μM) showed aromatase inhibitory activity with half maximal inhibitory concentration (IC₅₀) values in the low micromolar concentrations. With regard to the QR1 induction activity, 11 exhibited the highest QR1 induction ratio (IR) with a low concentration to double activity (CD) value (IR = 8.34, CD = 2.75 μM), while 7 showed the most potent CD value of 1.12 μM. A dual acting compound 24 showed aromatase inhibition (IC₅₀ = 9.00 μM) as well as QR1 induction (CD = 5.76 μM) activities. Computational docking studies using CDOCKER (Discovery Studio 3.5) provided insight in regard to the potential binding modes of 2-arylindoles within the aromatase active site. Predominantly, the 2-arylindoles preferred binding with the 2-aryl group toward a small hydrophobic pocket within the active site. The C-5 electron withdrawing group on indole was predicted to have an important role and formed a hydrogen bond with Ser478 (OH). Alternatively, meta-pyridyl analogs may orient with the pyridyl 3′-nitrogen coordinating with the heme group.

Full text of DOI:10.1016/j.bmcl.2017.11.010

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-activity relationships and docking studies of synthetic
2-arylindole derivatives determined with aromatase and quinone
reductase 1
Allan M. Prior a,c, Xufen Yu a,c, Eun-Jung Park a,b, Tamara P. Kondratyuk a, Yan Lin a, John M. Pezzuto a,b
,
Dianqing Sun a,
⇑
a Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawai’i at Hilo, 34 Rainbow Drive, Hilo, HI 96720, USA
b Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY 11201, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In our ongoing effort of discovering anticancer and chemopreventive agents, a series of 2-arylindole
Received 5 October 2017
Revised 3 November 2017
Accepted 6 November 2017
Available online xxxx
derivatives were synthesized and evaluated toward aromatase and quinone reductase
Biological evaluation revealed that several compounds (e.g., 2d, IC50 = 1.61 M; 21, IC50 = 3.05
27, IC50 = 3.34
1
l
(QR1).
M; and
l
l
M) showed aromatase inhibitory activity with half maximal inhibitory concentration
(IC50) values in the low micromolar concentrations. With regard to the QR1 induction activity, 11 exhib-
ited the highest QR1 induction ratio (IR) with a low concentration to double activity (CD) value (IR = 8.34,
Keywords:
CD = 2.75
aromatase inhibition (IC50 = 9.00
l
M), while 7 showed the most potent CD value of 1.12
l
M. A dual acting compound 24 showed
M) activities. Computational
2-Arylindole
Anticancer
Aromatase
Docking
Estrogen
Quinone reductase 1
l
M) as well as QR1 induction (CD = 5.76
l
docking studies using CDOCKER (Discovery Studio 3.5) provided insight in regard to the potential binding
modes of 2-arylindoles within the aromatase active site. Predominantly, the 2-arylindoles preferred bind-
ing with the 2-aryl group toward a small hydrophobic pocket within the active site. The C-5 electron
withdrawing group on indole was predicted to have an important role and formed a hydrogen bond with
Ser478 (OH). Alternatively, meta-pyridyl analogs may orient with the pyridyl 30-nitrogen coordinating
with the heme group.
Ó 2017 Elsevier Ltd. All rights reserved.
Breast cancer is the most common type of cancer affecting
women worldwide.1 According to the World Health Organization
(WHO), breast cancer caused more than half a million deaths in
2015.2 Postmenopausal woman are most susceptible to develop
breast cancers and these are often associated with increased serum
estrogen levels which are purported to be a prominent driving fac-
tor of tumor growth and proliferation.1,3 Approximately 70–80% of
breast tumors in postmenopausal woman are found to be estrogen
or progesterone dependent.1 In these cases, unusually high levels
of estrogen are found in breast tissue due to the biosynthesis of
estrogen in adipose, breast, and other peripheral tissues by cyto-
chrome P450 aromatase.3 Aromatase is a microsomal enzyme com-
plex that plays a crucial role in the conversion of androgens into
estrogens in peripheral tissues by aromatization of the steroid A
ring.4,5 Specifically, it catalyzes the conversion of androstenedione,
17b-estradiol, and 17b,16a
-estriol, respectively.6 Clinically, estro-
gen lowering aromatase inhibitors are used as first line therapy
to treat estrogen receptor-positive breast cancer in menopausal
woman today.6 Their effectiveness can be attributed to the fact
that aromatase is the only known enzyme in vertebrates that can
catalyze the aromatization of a six-membered ring,6 as such, aro-
matase represents an attractive druggable target to block estrogen
biosynthesis. A representative list of landmark and/or clinically
used aromatase inhibitors is shown in Fig. 1 and several of them
have been approved for the treatment of estrogen-dependent
breast cancer.4,6 Among them, anastrozole, letrozole, and exemes-
tane are clinically used in the US.7 A review article that describes
the history of aromatase inhibitor development was recently
published.6
Although aromatase inhibitors have shown success in the clinic
in treating and/or preventing breast cancer, currently available
drugs exhibit treatment limiting side effects including nausea, fati-
gue, insomnia, headaches and arthralgia.6,8,9 Moreover, mounting
drug resistance is another concern commonly associated
with anti-aromatase drugs.10 Therefore, continued search and
testosterone, and 16a-hydroxytestosterone into estrone,
⇑
Corresponding author.
E-mail address: dianqing@hawaii.edu (D. Sun).
These authors contributed equally to this work.
c
https://doi.org/10.1016/j.bmcl.2017.11.010
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Prior A.M., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.11.010

2
A.M. Prior et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 2. New anti-aromatase and QR1 activating agents as well as design rationale
for 2-arylindoles.
Fig. 1. Landmark and/or clinically used aromatase inhibitors.
development of novel and alternative aromatase inhibitors is highly
warranted.
In addition, activation and/or upregulation of anticarcinogenic
enzymes have also been considered as an alternative strategy to
achieve cancer chemoprevention. Among these anticarcinogenic
enzymes is the cytoprotective enzyme quinone reductase
1
(QR1). QR1 induction was shown to protect cells against chemi-
cal-induced carcinogenesis in animal models.11 Mechanistically,
this detoxification and cytoprotective effect was achieved via the
catalyzing step of transforming excessive quinones into the corre-
sponding hydroquinones by QR1.12,13 In this context, it is therefore
of great importance to develop potent and efficient QR1 inducers
as potential chemopreventive and anticancer agents.
Scheme 1. Synthesis of 1a–f and 2a–d. Reagents and conditions: (i) NaBH3CN,
AcOH, rt, 20 h; (ii) 1. NaH, DMF or DMF/THF (1/2), 0 °C; 2. R1X (X = I, Br, Cl), 0 °C-rt.
For 1e: DMAP, Boc2O, CH3CN. (iii) 1. NH2OHꢀHCl, NaOH, MeOH, rt; 2. Cu(OAc)2 (5
mol%), CH3CN, rt, ultrasound; (iv) R2ONH2ꢀHCl, pyridine, EtOH, rt.
The indole scaffold is ubiquitous in natural products and phar-
maceutical molecules.14 Some indole derivatives such as azolyl-
methyl-1H-indoles,15–18 azolylbenzyl-1H-indoles,15,19,20 5-aryl
(imidazol-1-yl)methyl-1H-indoles,21 6- and 4-aroylindoles,22
derivatizations were achieved by introducing the N-Me functional-
ity (8a, 24a, 31a, and 31c) or reducing the nitro into amino group
(24b and 31b-c). All the synthesized compounds were tested in
aromatase inhibition and QR1 induction assays, and their biologi-
cal activities are shown in Table 1.
indole-3-carbinols,23
indole-imidazoles,24
and
2-
and
3-aryl(azolyl)methylindoles25 have been previously synthesized
and evaluated as potential anti-aromatase agents. Recently, Cush-
man and coworkers developed a series of resveratrol analogs by
replacing the trans double bond with a thiadiazole moiety and
discovered a promising new compound with dual activity against
aromatase and QR1 (Fig. 2).26–28 Lang and coworkers identified
1-arylpyrrolo[2,3-c]pyridine as a potent aromatase inhibitor with
an IC50 value of 59 nM.29 Due to the structural similarity of these
compounds to 2-arylindole scaffold (Fig. 2), we became interested
in investigating our series of 2-arylindoles as potential dual acting
small molecules that inhibit aromatase while simultaneously
activate QR1. We hypothesized that the more rigid 2-arylindole
skeleton could mimic the resveratrol backbone whilst at the
same time reducing the entropic penalty of binding creating a
more favorable binder.30
Anti-aromatase activity
A brief SAR summary of our 2-arylindole series on aromatase
inhibition is highlighted in Fig. 3. In general, small, polar electron
withdrawing groups (EWGs) on the C-5 indole ring resulted in
favorable anti-aromatase activity and increased in the order of
Br < Cl < NO2 < CN with IC50 values of 39.7, 22.8, 9.0, and 3.3 mM,
respectively. Changing the position of the EWG (CN) from C-5 to
C-3 resulted in a twofold improvement of anti-aromatase activity
(Table 1, entry 11 vs 39) while moving the EWG (Cl) from C-5 to
C-6 resulted in a fivefold decrease in activity (Table 1, entry 38
vs 40). Although the prototype 2-phenylindole (1) showed only
slight activity against aromatase, switching the C-30 carbon to
nitrogen on the 2-phenyl ring resulted in dramatic increase in
activity, providing 21 with an IC50 of 3.05 mM (Table 1, entry 1 vs
31). Subsequent docking studies suggest that the 30-pyridyl
nitrogen can form a favorable N-Fe interaction with the heme of
aromatase which may account for this notable increase in anti-aro-
matase activity. The 2-thiophenyl and 2-naphthyl derivatives, 22
and 23 (Table 1, entries 32–33), did not show encouraging activity.
Attaching various alkyl groups to the nitrogen atom of the indole
motif in general had no improvement in anti-aromatase activity
(Table 1, entries 2–6) except for N-methylation of 24 to give 24a
which effected a twofold increase in activity (Table 1, entry 34 vs
35). The reduced 2-phenylindole variant, indoline 1f did not show
significant activity (entry 7). Similarly, the C-3 oxime derivatives
The 2-arylindole compound library was synthesized according
to Schemes 1 and 2. To start, a preliminary 2-arylindole set was
initially synthesized based on chemical transformations of
commercially available 2-phenylindole (1) and 2-phenylindole-
3-carboxaldehyde (2) to afford N-alkyl derivatives 1a–e, the
reduction product 1f, the oxime derivatives 2a–c, and 3-cyano-2-
phenylindole 2d, respectively (Scheme 1).31 Further modifications
of the 2-arylindole library were done by varying the substituents
R1, R2, and R3 around the 2-arylindole scaffold. Synthesis of these
derivatives was achieved using a one pot process via initial
Sonogashira coupling of 2-iodoanilines with terminal alkynes
followed by base-assisted cyclization (Scheme 2).31
Most of the reactions proceeded smoothly to generate the
expanded 2-arylindole library in good yields. Further chemical
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A.M. Prior et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Scheme 2. Synthesis of 2-arylindole derivatives.
2a-c were largely inactive against aromatase (Table 1, entries 8–
10). A variety of 2-arylindole derivatives bearing electron donating
groups (NMe2, NH2, OMe, alkyl) or electron withdrawing groups (F,
Cl, Br, OCF3) on C-20, C-30 or C-40 position of the 2-aryl ring were
tested against aromatase. However, no significant increase in
anti-aromatase activity was observed for these derivatives com-
pared to 1. In fact, incorporation of 2-aryl substituents on 24 to
give 31 totally destroyed aromatase activity (Table 1, entry 34 vs
43), suggesting that substituents on the 2-aryl moiety are not well
tolerated. On the basis of excellent anti-aromatase activities of 21,
24, and 27, two hybrid derivatives 32 and 33 were subsequently
designed and synthesized in an effort to identify more potent
anti-aromatase agents. Nonetheless, no improvements were
achieved and only 33 showed activity in low micromolar range
(IC50 = 11.5 mM) (Table 1, entries 47 and 48). Moreover, the com-
mercially available 2-phenylbenzimidazole (34) with structural
similarity to 2-phenylindole, was not active (Table 1, entry 49).
QR1 activity (Table 1, entry 11 and 39). In general, substituents on
the 2-aryl ring of 1 weakened QR1 activity, except for the
30,50-dimethoxy derivative
7
(CD = 1.12
displayed a 2-fold increase in QR1 induction activity over 1
(CD = 2.65 M, entry 1). The activity of para-substituted 2-arylin-
doles increased in the order of OCF3 < F < Br < Cl with CD values
of 29.10, 17.80, 5.01, and 2.75 M, respectively (Table 1, entries
lM, entry 16) which
l
l
21–22, 24, 26). However, the para substituted analogs 17
(n-pentyl), 18 (amino), and 20 (N,N-diMe) were all inactive as
QR1 inducing agents (Table 1, entries 27, 28 and 30). The meta
derivatives 6 (30-OCH3) and 10 (30-Cl) had comparable activity to
their corresponding para isomers 5 and 11 (Table 1, entries 14,
20 vs 15, 21), however, the meta amino derivative 19 showed
improved activity (CD = 18.40 lM) over its para isomer 18 (Table 1,
entry 28 vs 29). The ortho substituted derivatives 3, 9, and 25 dis-
played minimal QR1 activity with IR values less than 1.5 (Table 1,
entries 12, 19 and 37). The loss of QR1 induction activity is likely
due to steric effects and subsequent conformational change result-
ing from having an ortho substituent on the 2-aryl ring. Notably,
increasing the number of halogen atoms on the 2-aryl ring was
detrimental to the QR1 inductive activity as seen in 8 (30,40-diCl)
and 15 (30,40-diF) that were inactive in comparison to their corre-
sponding mono substituted analogs (Table 1, entries 17, 25 vs 20,
21, and 24). The pyridyl (21), 2-thiophenyl (22), and 2-naphthyl
(23) analogs (Table 1, entries 31–33) were also inactive.
QR1 induction activity
A brief SAR summary for our 2-arylindole library on QR1 induc-
tion is shown in Fig. 4. The prototype 2-phenylindole 1 having a
free indole NH showed very good QR1 induction activity with a
CD value of 2.65 lM (Table 1, entry 1) and was used as a bench-
mark to compare activities of other analogs. N-Alkylation of the
indole nitrogen destroyed QR1 induction activity as seen in deriva-
tives 1a–e (Table 1, entries 2–6). This suggested that the free indole
NH functionality is important for QR1 induction. Relative to 1,
2-phenylindoline 1f only exhibited weak activity (IR = 1.8), indicat-
ing a flat, aromatic indole scaffold is superior to indoline for QR1
induction (Table 1, entry 7). Among the oxime derivatives 2a–c,
only 2a having a free hydroxyl group showed reasonable QR1
Collectively, the SAR studies of this 2-arylindole library from
both aromatase and QR1 assays identified four compounds (24,
26, 29 and 33) that showed dual activity in both assays. The most
promising dual acting compound, 24, exhibited an IC50 value of
9.00
lM and CD value of 5.76 lM for anti-aromatase and QR1
induction, respectively.
activity with an IR value of 3.10 and CD value of 11.70
lM (Table 1,
Computational docking studies
entries 8–10). Introducing a NO2, Cl, or Br function on the indole
moiety at C-5 generally led to a twofold loss in activity relative
to 1 with CD values of 5.76, 5.50, and 6.76 lM, respectively
(Table 1, entries 34, 38, and 41). Interestingly, the introduction of
a cyano group at C-3 or C-5 of the indole moiety totally destroyed
Computational docking has become a powerful virtual screen-
ing tool for discovering novel potential drug leads and advanced
computer based drug design.32 Recently, Del Rio and coworkers
identified highly active aromatase inhibitors with a new core
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Table 1
Aromatase inhibition and QR1 induction activities of 2-arylindole derivatives.a
Entry
Compound
Aromatase
QR1
IRc
% inhibitionb
IC50
(l
M)
CDd
(lM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
1
27.75 2.87
0.00 3.88
0.00 3.58
20.19 1.95
26.29 3.31
11.89 1.81
48.98 2.84
40.36 3.25
24.66 1.17
23.25 1.34
88.00 1.07
27.06 0.45
9.33 3.39
0.00 3.84
48.37 2.57
9.18 3.25
22.65 5.01
20.00 1.78
21.51 2.73
24.15 1.48
15.78 2.93
4.80 0.81
9.27 4.36
21.80 2.67
11.02 3.87
1.86 2.34
0.00 3.22
46.21 1.35
26.74 4.83
0.00 2.63
81.55 2.09
21.98 3.81
0.07 3.06
77.72 2.89
76.99 3.52
41.43 3.41
71.47 0.21
70.53 1.39
82.27 4.98
13.60 2.84
58.08 0.18
32.36 2.32
7.59 3.42
34.04 5.10
21.37 2.89
41.99 2.35
27.90 0.10
93.70 7.00
7.64 7.82
2.30 0.12
0.50 0.63
0.37 0.59
1.90 0.24
0.87 0.81
1.00 0.88
1.80 0.55
3.10 0.29
1.30 1.10
0.50 0.65
0.55 0.64
0.54 1.20
5.20 1.50
1.56 0.53
1.30 0.59
3.75 0.52
0.57 0.63
4.40 1.23
1.40 0.81
5.20 0.79
8.34 1.60
2.80 0.84
1.48 0.59
2.73 0.29
0.58 1.12
2.48 0.63
0.78 0.23
0.56 0.80
2.70 0.71
1.21 0.69
1.14 0.33
0.67 0.15
0.74 0.69
3.92 0.41
0.89 0.49
0.72 0.84
0.78 1.21
5.10 1.03
1.06 0.23
4.25 1.12
5.50 0.98
1.00 0.96
1.07 0.80
1.56 0.90
4.90 1.40
2.14 0.20
1.70 0.20
2.10 0.50
0.86 1.40
2.65 0.51
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
3
4
5
6
7
8
8a
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
24a
24b
25
26
27
28
29
30
31
31a
31b
31c
32
33
34
11.70 3.60
6.40 2.90
1.61 0.20
1.12 0.20
2.10 0.80
2.17 1.80
2.75 0.63
5.10 1.00
17.80 4.60
29.10 7.02
18.40 2.60
3.05 0.61
9.00 0.49
3.88 0.38
5.76 0.80
5.50 0.94
6.87 0.64
22.81 3.39
3.34 0.71
6.80 2.10
6.76 3.10
39.71 1.77
10.33 2.60
46.70 2.05
48
11.50 2.40
31.20 4.00
0.01
49e
Standard controlf
9.95 1.46 (nM)
a
b
c
d
e
f
Concentration for initial testing: 50
Percentage of inhibition in comparison with vehicle-treated controls.
IR: induction ratio at a concentration of 50 M.
CD is the concentration that doubles the activity and is determined for compounds with IR > 2.
2-Phenylbenzimidazole was purchased from Sigma-Aldrich.
IC50 or CD values of known bioactive compounds (letrozole for aromatase and 40-bromoflavone for QR1).
lM.
l

A.M. Prior et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
Fig. 3. Brief SAR of aromatase inhibitory activity based on modifications of 2-arylindole.
with that in the protein bound X-ray cocrystal structure
(Fig. 5a and b). Key hydrogen bond interactions were predicted
between the C-17 carbonyl of androstenedione and the protein at
Met374 (NH, backbone) and Arg115 (NH, side chain). Similarly,
the top scoring pose for letrozole agreed favorably with that in pre-
viously
published
letrozole-aromatase
docking
studies
(Fig. 5b).34,35 In our model, the distance between the triazole func-
tion of letrozole and the heme of aromatase was found to be 2.7 Å.
Both benzonitrile groups of letrozole were predicted to form
hydrogen bonds to the protein within the active site, one to
Met374 (NH) and the other to Ser478 (OH) with measured dis-
tances of 2.0 Å and 2.2 Å, respectively. An additional hydrogen
bond was observed between a benzonitrile group of letrozole and
Arg115 (NH side chain) with a distance of 2.2 Å.
Fig. 4. SAR for QR1 induction of 2-phenylindole derivatives.
through high-throughput docking protocol.33 The currently
accepted binding modes of non-steroidal aromatase inhibitors
(e.g., letrozole and anastrozole) to aromatase have been learned
from computational34,35 and site directed mutagenesis studies35,36
because the X-ray cocrystal structures of these compounds bound
to aromatase are lacking. In 2011, Wood et al.34 and Hong et al.35
reported the proposed binding modes of letrozole with aromatase
using computational docking. Both studies agreed that the triazole
moiety of letrozole binds to the heme prosthetic group of aro-
matase. In the model reported by the Chen group, the distance
between the triazole of letrozole and iron of heme was reported
to be 3.7 Å.35 The Potter group reported more clearly the role of
the benzonitrile groups of letrozole.34 One of the nitrile groups
forms a hydrogen bond to Met374 (NH), the same residue that
androstenedione hydrogen bonds with, while the other nitrile
group forms a hydrogen bond with Ser478 (OH).34 This is sup-
ported by earlier mutagenesis experimental studies that demon-
strated a S478A mutation destroys letrozole activity.36
To gain further insight into the potential binding modes of our
active 2-phenylindole inhibitors with human aromatase, a similar
computational docking approach was undertaken using CDOCKER
(Discovery Studio 3.5).37 The protein X-ray crystal structure of
human aromatase (PDB code: 3EQM) was recovered from the pro-
tein data bank (PDB) and the bound natural substrate ligand
(androstenedione) was removed prior to docking. All ligands for
docking were initially constructed in ChemDraw and exported into
Discovery Studio (DS). The ligands were prepared for docking using
the prepare ligands protocol in DS followed by energy minimiza-
tion. Before docking our 2-arylindole library, the reliability of our
docking protocol (CDOCKER) was verified by docking androstene-
dione and letrozole back into the active site of aromatase. The
CDOCKER energy scoring function (ꢁCDOCKER energy) that takes
into account ligand-receptor interaction energy and internal ligand
strain energy was used to rank docked poses. Encouragingly, the
top scoring pose for docked androstenedione overlaid favorably
After validation of the docking protocol, the binding poses for
our 2-arylindole scaffolds were predicted using analogous docking
experiments. The –CDOCKER energy scores for the docked library
ranged from ꢁ9.5 to 26 where a more positive value means more
favorable binding prediction. Compound 21 was the best scoring
ligand and returned a –CDOCKER energy score of 26. This was
the second most active aromatase inhibitor in our series. Com-
pound 2d, the most active aromatase inhibitor in our series
returned a favorable –CDOCKER energy score of 17. Overall, aro-
matase inhibitors gave favorable –CDOCKER energy scores with
values greater than 10. In general, compounds that were bulky
and/or had substituents on the 2-aryl ring, especially on the para
C-40 position, scored poorly and is consistent with SAR data. The
lowest scoring ligand was 31a which showed a –CDOCKER energy
score of ꢁ9.5 and was not active in the aromatase assay. The dock-
ing results were intriguing as more than one favorable pose were
predicted depending on the substitution pattern around the 2-
arylindole scaffold. Generally, the 2-arylindole scaffold preferred
to orient within the active site in such that the aromatic 2-aryl
group positioned towards a well-known hydrophobic pocket com-
prising residues Phe134, Val370, Leu372, Val373, Met374, and
Leu477 (Fig. 5c and d). In 2d (Fig. 5c), the 2-aryl group made close
contact to Phe134 (2.7 Å), Leu372 (3.0 Å), Met374 (3.1 Å), and
Leu477 (3.1 Å) whereas in Fig. 5d, 27 made closest contact with
Phe134 (2.9 Å), Leu372 (3.4 Å), Val373 (3.2 Å), Met374 (3.5 Å),
and Leu477 (3.5 Å). The indole NH of 2d (Fig. 5c) oriented away
from the heme and toward residues Leu477, Phe221, and Ser478.
The distance between the indole NH and Ser478 side chain hydro-
xyl was measured to be 4.6 Å, suggesting a hydrogen bond interac-
tion is unlikely. A slight variation in binding was predicted for
compounds having a C-5 EWG such as 25, 26, 27, 29, and 33. In
these cases, the 2-aryl group occupied the same hydrophobic
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6
A.M. Prior et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 5. Docking models of natural substrate androstenedione, known aromatase inhibitor letrozole, 2d, 21, and 27 with aromatase (3EQM). a) An overview of superimposed
binding poses of androstenedione (pink), letrozole (yellow), and 2d (orange) with aromatase as well as the zoom-in view of binding site. Crystal structure position of bound
androstenedione is shown in blue. b) Superimposed binding poses of androstenedione (pink) and letrozole (yellow) to aromatase active site with the crystal structure of
bound androstenedione in blue showing hydrogen bonding interactions. c) Predicted binding pose of 2d with aromatase and representative binding pose for 2-arylindoles. d)
Predicted binding mode for C-5 substituted 2-arylindoles 25, 26, 27, 29, and 33. For clarity, only 27 is shown as a representative. e) Alternative binding mode for 30-pyridyl
derivative 21 showing a predicted N-Fe interaction of 2.7 Å. Phe134, Val370, Leu372 and Val373 are removed from Fig. 5b–e for clarity.
pocket as 2d, however the orientation of the indole moiety was
flipped in such that the indole NH pointed toward the back of
the pocket and Trp224 (Fig. 5d). The flipped docking pose of these
derivatives is not surprising given the relatively symmetrical
nature of the 2-arylindole scaffold. This may also highlight the
limitation associated with molecular modelling. Additionally the
Please cite this article in press as: Prior A.M., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.11.010

A.M. Prior et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
7
2-arylindole scaffold was rotated by approx. 35° relative to 2d,
which brought the C-5 indole substituents in close proximity
(<2.5 Å) to Ser478 side chain hydroxyl group (Fig. 5d). This sug-
gested a favorable hydrogen bond may be possible in these cases.
Notably, the six membered indole ring of C-5 substituted analogs
(e.g., 27) was positioned toward a small gap in-between Thr310
and Ser478, the same space normally occupied by one of the ben-
zonitrile groups of letrozole. The six membered indole ring of 2d
oriented towards residues Ala306 and Thr310 with close contacts
of 3.6 Å and 3.1 Å, respectively. The C-2 nitrile group of 2d posi-
tioned towards the side chain of Arg115 and had a close contact
of 3.2 Å. Very interestingly, 21 having a 30-pyridyl nitrogen in the
2-aryl ring, docked in a completely opposite fashion to the other
2-arylindoles. Instead, the 30-pyridyl nitrogen preferred to coordi-
nate to the iron of heme in all top 10 scoring poses with a mea-
sured nitrogen-heme distance of approximately 2.4 Å (Fig. 5e).
The 2-aryl substituent oriented towards Ala306 and Thr310 while
the indole backbone formed close contacts with Phe134, Val370,
Leu372, Met374, and Leu477 all within a distance of 4.1 Å.
Collectively, the predicted binding modes for 2-arylindoles
within the active site of aromatase provided some valuable
insights that may help rationalize SAR data for these compounds.
The active site of aromatase is relatively small being less than
400 Å3.35 The 2-aryl ring predominantly docked into a small
hydrophobic pocket. This suggests that substituents on the 2-aryl
ring are not generally well tolerated which is consistent with activ-
ity data. Active compounds 25, 26, 27, 29, and 33 docked in an ori-
entation that put the C-5 EWG close to Ser478 (OH), strongly
suggesting favorable hydrogen bonding or dipole-dipole interac-
tions may be possible in these cases. Given the mostly hydrophobic
nature of the active site of aromatase, the 2-fold increase in activity
of N-methyl derivative 24a over free NH derivative 24 may be a
result of favorable van der Waals (VDW) interaction between the
small hydrophobic methyl group and the active site. Moreover,
the free indole NH function was not found to play a significant role
in any of our predicted binding models.
leads for further optimization of chemical synthesis and expand
potential applications for the development of new chemopreven-
tive and chemotherapeutic agents.
Acknowledgments
This work was supported in part by the National Institutes of
Health grant P20GM103466 and UH Hilo DKICP RTRF fund.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2017.11.010.
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In summary, a series of 2-arylindole derivatives were designed,
synthesized, and evaluated for aromatase inhibition and QR1
induction. Biological evaluation revealed that 2d was the most
potent anti-aromatase inhibitor (IC50 = 1.60
also displayed good aromatase inhibitory activity with IC50 values
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l
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Please cite this article in press as: Prior A.M., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.11.010