Synthesis, antiproliferative and pro-apoptotic activity of 2-phenylindoles

Article abstract of DOI:10.1016/j.bmc.2016.06.050

Breast cancer is the second most common cancer worldwide after lung cancer with the vast majority of early stage breast cancers being hormone-dependent. One of the major therapeutic advances in the clinical treatment of breast cancer has been the introduction of selective estrogen receptor modulators (SERMs). We describe the design and synthesis of novel SERM type ligands based on the 2-arylindole scaffold to selectively target the estrogen receptor in hormone dependent breast cancers. Some of these novel compounds are designed as bisindole type structures, while others are conjugated to a cytotoxic agent based on combretastatin A4 (CA4) which is a potent inhibitor of tubulin polymerisation. The indole compounds synthesised within this project such as 31 and 86 demonstrate estrogen receptor (ER) binding and strong antiproliferative activity in the ER positive MCF-7 breast cancer cell line with IC₅₀values of 2.71?μM and 1.86?μM respectively. These active compounds induce apoptotic activity in MCF-7 cells with minimal effects on normal peripheral blood cells. Their strong anti-cancer effect is likely mediated by the presence of two ER binding ligands for 31 and an ER binding ligand combined with a cytotoxic agent for 86.

Full text of DOI:10.1016/j.bmc.2016.06.050

Accepted Manuscript
Synthesis, antiproliferative and pro-apoptotic activity of 2-Phenylindoles
Patrick M. Kelly, Sandra A. Bright, Darren Fayne, Jade K. Pollock, Daniela M.
Zisterer, D. Clive Williams, Mary J. Meegan
PII:
S0968-0896(16)30473-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.06.050
BMC 13106
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
13 May 2016
24 June 2016
25 June 2016
Please cite this article as: Kelly, P.M., Bright, S.A., Fayne, D., Pollock, J.K., Zisterer, D.M., Clive Williams, D.,
Meegan, M.J., Synthesis, antiproliferative and pro-apoptotic activity of 2-Phenylindoles, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.06.050
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Synthesis, antiproliferative and pro-apoptotic activity of 2-Phenylindoles
1
2
2
2
2
Patrick M. Kelly , Sandra A. Bright *, Darren Fayne , Jade K. Pollock , Daniela M. Zisterer , D.
2
1
Clive Williams , Mary J. Meegan
1
School of Pharmacy & Pharmaceutical Sciences, Trinity Biomedical Sciences Institute,
Trinity College Dublin, Ireland
2
School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity
College Dublin, Ireland
*Corresponding author: Email address: brights@tcd.ie
Graphical abstract:
Abstract
Breast cancer is the second most common cancer worldwide after lung cancer with the vast majority
of early stage breast cancers being hormone-dependent. One of the major therapeutic advances in
the clinical treatment of breast cancer has been the introduction of selective estrogen receptor
modulators (SERMs). We describe the design and synthesis of novel SERM type ligands based on the
2-arylindole scaffold to selectively target the estrogen receptor in hormone dependent breast
cancers. Some of these novel compounds are designed as bisindole type structures, while others are
conjugated to a cytotoxic agent based on combretastatin A4 (CA4) which is a potent inhibitor of
tubulin polymerization. The indole compounds synthesised within this project such as 31 and 86
demonstrate estrogen receptor (ER) binding and strong antiproliferative activity in the ER positive
MCF-7 breast cancer cell line with IC50 values of 2.71 M and 1.86 M respectively. These active
compounds induce apoptotic activity in MCF-7 cells with minimal effects on normal peripheral blood
cells. Their strong anti-cancer effect is likely mediated by the presence of two ER binding ligands for
3
1 and an ER binding ligand combined with a cytotoxic agent for 86.
Introduction
Breast cancer is the foremost cause of cancer death in women. Worldwide, breast cancer
exceeds all other cancers and the incidence rates of breast cancer (more than 1,676,000 new cases
1
diagnosed in 2012) are increasing steadily . The majority of early stage breast cancers are hormone-
dependent and patient prognosis is good. However, when the breast cancer is hormone-
independent then prognosis becomes poor. About 5% of breast cancers, denoted BRCA-1 and BRCA-
2
2
, are considered hereditary .
1

Breast cancer cells have receptors on their surface and in their cytoplasm and nucleus.
Important receptors include hormone receptors such as the estrogen receptor (ER) and the
progesterone receptor (PR). Estrogen receptor-positive (ER+) cancer cells depend on the hormone
estrogen for their growth, so they can be treated with hormone blocking therapies such as
antiestrogens, aromatase inhibitors and selective estrogen receptor modulators (SERMs)
(e.g. tamoxifen). Another important receptor found in breast cancer cells is the human epidermal
growth factor receptor 2 (HER2/neu). Monoclonal antibodies such as Trastuzumab can target this
cell surface receptor which is especially active in some breast cancers. Estrogen receptor-negative
(ER-) and triple negative (ER-, PR- and HER2-) cancers, non responsive to targeted therapies, tend to
be treated with more traditional chemotherapy drugs such as cyclophosphamide and doxorubicin
which target rapidly dividing cells; these cancers are therefore associated with a poorer prognosis
than receptor positive, hormone-dependent tumours.
The hormone estradiol (E2), a member of the estrogen (oestrogen) family of female sex
hormones, plays an important role in the progression and development of breast tumours. Estradiol,
together with the ER complex, can control activation of proto-oncogenes, oncogenes, nuclear
3
, 4-6, 7, 8
proteins, as well as other target genes
. The majority of breast cancers occur during the
postmenopausal period, despite resulting low levels of circulating estrogens, the concentration of
estrogens in breast cancer tissue is several times higher than that found in the plasma or in the area
of the breast considered as normal tissue, suggesting specific tumoral biosynthesis and accumulation
9
, 10
of these hormones
.
11
There are two subtypes of ER, ERα and ERβ, each with several isoforms and splice variants .
1
2
Although ERα and ERβ have similar DNA binding domains (95% sequence homology) , and
moderate to high degree of sequence identity (55-59%) in the ligand binding domains (LBDs), the
overall degree of homology of the receptors is much lower, with different structures and their
1
3, 14
encoding genes on different chromosomes
cancer cells, and ovarian stroma. In contrast, ERβ is mainly found in several non-classic target tissues
kidney, intestinal mucosa, lung parenchyma, bone marrow, bone, brain, endothelial cells, and
. ERα is mostly present in the endometrium, breast-
(
1
5
prostate gland) .
The ER contains two activation function domains, AF1 and AF2 which are located within the
1
6
N-terminal domain and LBDs, and are responsible for regulating transcriptional activity . AF1 is
1
6, 17
known to function independently of estrogen whereas AF2 requires the presence of estrogen
. It
is believed that the two AFs act in synergy to achieve transcription activity. Their activity is also
1
8
believed to be promoter and cell specific . The ER is cytoplasmically based, dimerising when
2

activated by a ligand translocating it to the nucleus where it binds to DNA as a transcription factor to
regulate the activity of different genes. LBD dimerisation is identical for both ER subtypes and
independent of the agonist/antagonist nature of the bound ligand.
The molecular basis of ERα and ERβ agonism and antagonism has been reported in a diverse
1
3, 19
variety of crystal structure determinations
. X-ray crystallography of ligand bound ER has shown
1
3, 14
that the ER LBDs adopt a classical helical sandwich fold, common to all nuclear receptor LBDs
.
The agonist stabilises the receptor conformation so that Helix 12 is aligned across the LBP fold and
the ligand lies within the buried cavity generating the AF2 site and enabling co-activator
2
0
recruitment . For antagonist effects, a hydrophobic core mimicking the steroid nucleus of E2 is
required, together with phenolic groups which form hydrogen bonds with key residues in the
ER(Glu353, Arg394 and His524) and a basic moiety which can interact with Asp351 to anchor the
antagonistic ligands within the LBP. The basic side chains which are too large to be accommodated in
the LBP can cause a displacement of Helix 12. This in turn prevents formation of the AF2 site and
2
0, 21, 22
tissue specific activation mediated by other regions of ER, including AF1
.
Nuclear receptors represent one of the most important classes of drug targets. Selective estrogen
receptor modulator (SERMs) are form of targeted therapies and unlike pure
a
receptor agonists and antagonists, SERMs can act differently in various tissues, selectively inhibiting
or stimulating estrogen-like action. The mechanism of mixed agonism/antagonism depends on the
chemical structure of the SERM, the ratio of coactivators (CoA) to co-repressors (CoR) proteins in
different cell types and the conformation of the ER induced by drug binding. Tamoxifen is an
endocrine therapy standard for ER+ breast cancers in both the neoadjuvant and postoperative
adjuvant settings and a first-line therapy for the treatment of pre/postmenopausal women with
2
3, 24
estrogen-responsive advanced (stage IV) breast cancer
. Since the first clinical use of tamoxifen as
2
3, 25
a SERM in 1971, multiple generations of SERMs have been developed
. There is interest in the
26
development of the tamoxifen metabolite endoxifen .
Figure 1: Estradiol, selected estrogen receptor antagonists and SERMs.
Second and third-generation SERMs were developed to avoid the uterotrophic effects of tamoxifen.
27
28
These include raloxifene , lasofoxifene and the indole containing compounds bazedoxifene and
29
pipendoxifene (Figure 1). Raloxifene functions as an agonist in bone and the cardiovascular system
but acts as a pure antagonist in the breast and uterine tissue. Raloxifene has higher efficacy in
maintaining bone mineral density compared with first generation SERMs, which is likely due to the
unique manner in which it alters the ER structure. Raloxifene was found to decrease the risk of all
3

3
0
ER+ breast cancers by 90% and invasive breast cancers by 72% . The generally positive effects of
raloxifene in the uterus are likely to make it the agent of choice for use as a chemo-preventative in
2
4
postmenopausal women at high risk of breast cancer .
Whilst ER+ cancers are associated with a good prognosis due to range of targeted therapies
directed against the ER, the ER receptor gene is prone to truncations, mutations and deletions as the
3
1-33
cancer cell evolves
as such, a relatively high number of patients with ER+ tumours do not
3
4-36
respond to antiestrogen therapy
. Together with the non-specific targeting effects of traditional
chemotherapy, efforts are currently required to identify new enhanced drugs for the treatment of
breast cancer which overcome these limitations. New breast cancer drugs should also exhibit
2
5,
selectivity towards either ERα or ERβ, known as selective ER subtype modulation (SERSM) activity
3
7, 38
.
We now report the synthesis and evaluation of a novel series of compounds based on the 2-
arylindole scaffold structure (analogous to bazedoxifene and pipendoxifene) with the potential for
treatment of ER+ breast cancers. Many 2-arylindoles have shown pharmacological activity in breast
cancer cells with the 2-arylindole core possessing good ER binding activity. Numerous studies have
described modifications that have demonstrated enhanced binding affinity for the ER including alkyl
substituents at C-3 of the indole nucleus and on the indole nitrogen and demonstrating a very
39-41
sensitive dependency on structural alterations in these molecules . The introduction of a terminal
4
2
amino group on the N-alkyl substituent resulted in estrogen antagonistic activity . Indole
4
3, 44
conjugated dendrimers have also been reported as estrogen receptor ligands
. In the present
study, we now report the synthesis and antiproliferative activity of a novel series of 2-phenylindole
analogues. We will explore the effect of the introduction of extended substitution on the indole
nitrogen on antiproliferative and apoptosis activity. The target indole compounds are designed to
contain a methyl substituent at C-3, a phenolic hydroxyl group at the para position of the C-2 phenyl
ring and a second phenolic group at C-5. These molecular features are recognised as important for
ER binding in nonsteroidal ER ligands, namely, a hydrophobic core with two phenolic groups
4
5
approximately 11 Å apart . Estrogen receptor ligand conjugates containing a variety of cytotoxic
4
6, 47
agents, photodynamic therapeutic agents and radioligands have been reported
to exploit the
high affinity and receptor selectivity of steroidal estrogens and ER ligands to deliver cytotoxic agents
that lack selectivity. We have previously reported that stable conjugates of endoxifen (a tamoxifen
metabolite and ER antagonist) with DNA alkylating agents, aromatase inhibitors and COX2 inhibitors
4
8
demonstrate antiproliferative and ER binding effects .
4

To determine the influence of structural modifications on antiproliferative activity in ER positive
MCF-7 breast cancer cells, the following indole structural types will be investigated:


Group I containing the N-methylaminoalkyl substituent of various hydrocarbon chain lengths
4,5,6,8,12 carbons) located at positions at N-1 of the indole;
Group II are bis-indole type structures having alkyl linking groups of various chain lengths
(
(4,5,6,8,12 carbons);


Group III contain benzyl and hydroxyethyl substituents at N-1 of the indole;
Group IV compounds are conjugate compounds comprising of a 2-phenylindole linking to an
acrylic acid combretastatin CA-4 type ligand, having a tethering hydrocarbon carbon chain
length of 4,5,6,8 or 12 carbons.
Chemistry
The synthesis of the target indole compounds is illustrated in Schemes 1-4. 4-
4
9
Benzyloxypropiophenone 2 was prepared via the benzylation of 4-hydroxypropiophenone 1,
Scheme 1. The stable 4-benzyloxyphenylhydrazine hydrochloride 4 was obtained from 4-
benzyloxyaniline hydrochloride 3 by diazotization with NaNO
2
in concentrated HCl solution at low
temperature and subsequent reduction with SnCl . The ketone 2 was then reacted with 4 in a
2
5
0
Fischer indole synthesis reaction to give the product 5 (84%) , which was then treated with
dibromoalkanes 6-10 of various chain lengths to give the products 11-15, together with the minor
products bis-2-phenylindoles 26-30 and the 2-phenyl-3-bromoalkyl-3H- indoles 36-40 (Scheme 1).
Alkylation of 5 with 1,2-dibromoethane under the same reaction conditions was not successful. The
amination reaction of compounds 11-15 with methylamine, afforded analogues 16-20 which were
then deprotected by hydrogenolysis to afford ER targeting phenolic products 21-25 (Group I
compounds, Scheme 1). The most frequently used procedure for removal of the benzyl protecting
5
1
group required a hydrogenation over Pd/C catalyst which proved unsuccessful with the
quantitative recovery of starting material. Instead, the benzyl protected compounds 16-20 were
treated with 20 % palladium hydroxide (Pearlman's catalyst) to afford the 2-phenylindole analogues
21-25 (Scheme 1). Lower than expected yields of 59-66 % for the above alkylation step, with all of
the starting material 5 having reacted, led to the identification of two series of indole based side
products, bis-indoles 26-30 and rearranged 2-aryl-3H-indoles 36-40 (Scheme 1).
The bis-2-phenylindole compounds 26-30 were also unexpectedly obtained when further reaction of
the initially formed bromoalkylated indole and a second 2-phenyindole molecule occurred in the
reaction conditions to produce the bis-2-phenylindoles (Scheme 1). Compounds 26-30 were
subsequently deprotected by treatment with 20% palladium hydroxide in a hydrogen atmosphere
5

for 30 min, affording the free phenolic products 31-35 in good yields (Group II compounds, Scheme
). These bis-2-phenylindole compounds have an interesting structure as they contain the diphenolic
1
indole structure which is essential for ER binding. Either of the 2-phenylindole termini could
potentially bind in the ER, or alternatively intramolecular or intermolecular bivalent binding may also
52
be possible which could potentially exhibit enhanced ER binding activity . It has been reported that
more complex bis-indole type compounds have demonstrated cytotoxic activity in cultures of PC-
5
3, 54
3(prostate), U251 (CNS), K562 (leukaemia), Hep-2 (Liver), HeLa (cervix), and HCT-15 (colon) cells
.
6

OBn
OH
(a)
BnO
(d)
O
O
OBn
2
(93%)
1
(c)
N
Br
2
+ 4
H
OBn
OBn
(CH2)n
Br
n= 2
n= 3
n= 4
(b)
5
6
7
8
NH2
HN
NH2
9 n= 6
0 n= 10
1
3
4
(92%)
BnO
+
OBn
BnO
N
OBn
OBn
BnO
+
N
CH )
N
(
(CH )
2 n
(CH2)n
2 n
Br 11 n= 2 (63%)
N
Br
1
1
2 n= 3 (65%)
3 n= 4 (66%)
OBn
BnO
(
e) 14 n= 6 (60%)
1
5 n= 10(59%)
26 n= 2 (20%)
27 n= 3 (22%)
28 n= 4 (26%)
29 n= 6 (22%)
30 n= 10(30%)
36 n= 2 (17%)
37 n= 3 (9%)
38 n= 4 (12%)
39 n= 6 (13%)
40 n= 10(11%)
BnO
OBn
N
(f)
(
CH2)n
1
1
1
1
6 n= 2 (92%)
7 n= 3 (93%)
8 n= 4 (95%)
9 n= 6 (91%)
NH
HO
OH
2
0 n= 10(87%)
N
(f)
(CH2)n
N
HO
OH
OH
HO
N
(
CH2)
n
21 n= 2 (80%)
3
3
3
3
3
1
2
3
4
5
n= 2 (67%)
n= 3 (53%)
n= 4 (74%)
n= 6 (61%)
n= 10 (58%)
2
2
2
2
2
3
4
5
n= 3 (71%)
n= 4 (76%)
n= 6 (91%)
n= 10(88%)
NH
Scheme 1. Synthesis of 2-phenylindoles 21-25, bis-indoles 31-35 and 2-aryl-3H-indoles 36-40.
o
Reagents and conditions: (a) C H CH Br, K CO , RT, ACN; (b) HCl, NaNO , SnCl , 0 C; (c) HCl, EtOH, Reflux;
6
5
2
2
3
o
2
2
o
(
d) NaH, THF, 0 C; (e) MeNH , sealed tube, 60 C; (f) Pd(OH) /C(20%), H , RT.
2
2
2
The additional series of minor products isolated in the bromoalkylation reaction of indole 5
was identified as the novel 2-phenyl-3-bromoalkyl-3H–indoles 36-40, Scheme 1. On comparison of
1
the H NMR spectra for 11 and 36 the C-3 methyl group shows a significant change in chemical shift
from 2.23 ppm in compound 11 to 1.58 ppm in compound 36. The CH signals in 36 appear as a
2
7

multiplets centred at 3.15, 2.26, 1.63 and 0.94 ppm. (In compound 11, these multiplets appear at
1
3
4
.07, 3.21, 1.77 and 1.65 ppm). Similarly in the C NMR spectrum for 36, the C-3 methyl group on
the 3H-indole core has moved much more downfield to 24.6 ppm compared with 8.9 ppm for the C-
methyl in compound 11. The signal at 57.6 ppm is assigned to quaternary carbon C-3 on which the
3
methyl and the alkyl chain are located. The C-2 imine carbon on the 3H-indole core appears more
upfield at 179.1 ppm. In the basic reaction conditions, it is proposed that deprotonation of the
indole 5 by sodium hydride generates the indolyl anion, which subsequently reacts through the C-3
position with the dibromoalkane to afford the novel 2-phenyl-3-bromoalkyl-3H–indoles 36-40. This
proposed mechanism is similar to that documented by Ke-Jia et al., for the palladium catalysed C-3
5
5
56
dearomative arylation of indoles and also C-3 benzylation of indoles .
To further investigate the structural-activity relationships of this series of compounds, the
dimethoxyindoles 47-49 were similarly prepared (Scheme 2). The indole scaffold 43, was obtained in
75% yield via the Fischer indole synthesis using 4-methoxyphenylhydrazine hydrochloride 42 and 4-
3
9
methoxypropiophenone 41 . Reaction of the indole 43 with the dibromoalkanes 6-10 with the base
sodium hydride to afford the bromoalkylindole products 44-46 in good yield. Although minor
products of the alkylation reactions were detected by tlc, they were not isolated. Amination of the
alkyl bromides 44-46 was achieved by treatment with methylamine under pressure in a sealed tube
to afford products 47-49 respectively in good yield. Demethylation of the methyl ether 47 with
boron tribromide to afford the phenolic 21 was achieved in very low yield. It was originally intended
to use this route for the preparation of the phenolic indoles. However the required phenolic
products 21-25 were achieved via hydrogenolysis of the benzyl ethers as described above.
8

Br
O
O
(CH2)n
(a)
O
O
Br
6-10
N
H
.
HCl
HN
O
NH2
(b)
43 (75%)
41
42
O
O
(c)
O
O
N
N
n(H C)
2
(H C)
n
2
Br
HN
44 n = 2 (58%)
45 n = 6 (52%)
46 n = 10 (36%)
47 n = 2 (75%)
48 n = 6 (66%)
49 n = 10 (78%)
Scheme 2. Synthesis of 2-phenylindoles 47- 49
Reagents and conditions: (a) HCl, EtOH, Reflux; (b) NaH, THF, 0 C; (c) CH NH , sealed tube, 60 C.
o
o
3
2
A further series of 2-phenyl-3-methylindole compounds containing a variety of alkyl,
hydroxyethyl and benzyl substituents located on the N-1 indole position was prepared for
biochemical evaluation as antiproliferative agents and estrogen receptor ligands. The indole 5 was
reacted with the appropriate alkyl and benzyl bromides 56-61 to afford products 50-54 (Scheme 3).
(
The required bromides 57 and 60 were prepared from the corresponding benzyl alcohols 62 and 64
5
7, 58
respectively using phosphorus tribromide
, while the bromide 59 was obtained from 2-
5
9
benzyloxyethanol 63 on treatment with N-bromosuccinimide ). The expected bis-indole 55 obtained
from the dibromide 61 (63%) was accompanied by the monobrominated product 71(22%). The
deprotection of the benzyl ethers 50-55 was achieved using Pd(OH) /C under an hydrogen
2
atmosphere affording the phenolic compounds 65-70 (Group 3 compounds, Scheme 4). The
synthesis of 51, 52 and 54 was also accompanied by the formation of the corresponding unreported
2-phenyl-3H-indole side products, 72-74 respectively, (Scheme 3), as seen with the previous
alkylation reactions of indole 5 (Scheme 1). Attempted debenzylation of the 2-phenyl-3H-indole
products 72-74 (H
2
/Pd(OH)
2
afforded in each case the 4’,5-dihydroxy-2-phenylindole product 75 in
9

quantitative yield. It is possible that under the hydrogenation conditions, the benzyl substituent at C-
undergoes rearrangement to the N-benzylindole which is subsequently debenzylated to afford the
common product 75, Scheme 3.
3
The use of phenylindole-platinum conjugates to deliver the cytotoxic agents to breast cancer
tumours was developed by Von Angerer et al. It was observed that the binding affinity was only
6
0
slightly decreased using a conjugate when compared to the 2-phenylindole itself . The 2-
phenylindole compounds 21-25 developed in this study contain a secondary amine type basic side
chain similar to that of the tamoxifen metabolite endoxifen, and are utilised in this study for
conjugation to the prototype cytotoxic agent, the acrylic acid combretastatin analogue 76. For the
synthesis of this series of benzyl-protected conjugates 77-81, EDC/HOBt was used as the coupling
reagent, (Scheme 4). The isolated benzyl-protected conjugates 77-81 were then deprotected by
treatment with Pd(OH) under a hydrogen atmosphere to remove the benzyl-protecting group
2
1
13
affording the phenolic amide conjugates 82-86 in high yields (79-94%). Both the H and C NMR
spectra of the conjugates 82-86 displayed a complex signal pattern attributed to the presence of
rotameric centres in these compounds. The presence of these rotameric centre(s) was investigated
10

by variable temperature Nuclear Magnetic Resonance spectroscopy (VT NMR). (See Supplementary
Information). The synthesis of the 5-methoxy-2-phenylindole conjugates with the acrylic acid
combretastatin analogue 76 was also achieved. This coupling reaction of the indoles 47-49 with 76
was carried out as described above for the preparation of 77-81 with DCC used as the coupling
reagent rather than EDC (Scheme 4) to afford the methoxy conjugate products 87-89 in moderate
yields (41-68%). Compounds 87-89 would not be expected to bind in the ER domain as the free
hydroxyl groups essential for binding are not present; however, these compounds were evaluated
for antiproliferative activity as a direct comparison to the free hydroxyl conjugates synthesised.
11

BnO
OBn
OBn
BnO
N
N
OH
(
CH2)n
(a)
1
6 n = 2
7 n = 3
8 n = 4
9 n = 6
(
CH2)n
1
1
1
N
H
N
O
7
7
7
8
8
7 n = 2 (85%)
8 n = 3 (91%)
9 n = 4 (88%)
0 n = 6 (85%)
1 n = 10 (91%)
O
2
0 n = 10
O
COOH
O
O
O
O
(b)
O
O
OH
7
6
HO
N
OH
(CH2)n
N
O
8
8
8
8
8
2 n = 2 (89%)
O
3 n = 3 (79%)
4 n = 4 (83%)
5 n = 6 (94%)
6 n = 10 (90%)
O
O
O
O
O
N
(
CH2)n
4
4
4
7 n = 2
8 n = 6
9 n = 10
N
H
O
O
N
OH
(c)
(CH2)n
N
O
COOH
O
O
8
8
7 n = 2 (41%)
8 n = 6 (68%)
O
O
89 n = 10 (52%)
O
O
O
OH
O
7
6
Scheme 4: Synthesis of 3-arylindole conjugates 82-89
Reagents and conditions: (a) EDC, HOBt, DCM, RT; (b) Pd(OH) /C(20%), H , EtOAc/MeOH, 30 min, RT;
2
2
(c) DDC, HOBt, DCM, RT.
12

Antiproliferative activity
Selected indole containing compounds synthesised via the schemes 1-4 illustrated above, were
evaluated for their ability to reduce the viability of the ER-positive breast cancer cell line, MCF-7.
Tamoxifen was used as a positive control with IC50 value of 4.1 M. The 2-phenylindole-based
analogues 21-25 incorporating the ER-binding indole moiety of the SERM bazedoxifene included
various length alkyl amine positioned at the indole nitrogen. In the present study, the terminal
2
6
methylamine basic substituent was designed to mimic the endoxifen basic side chain structure . It
was hypothesised that by incorporating a basic side chain onto the 2-phenylindole structure that
3
9, 41, 42
high affinity ER ligands with potential antagonist activity may be achieved
.
The 2-phenylindole-based analogues 21-25 (Group I) all showed micromolar IC50 activity
Table 2) with IC50 values in the range 33-49 μM. The ER ligands with an alkyl chain length <6 carbons
(
would be expected to have good oral bioavailability as the CLogP values are less than 4 but the
longer non-polar chain length >4 carbons are predicted be more lipophilic. It is possible that the
moderate antiproliferative activity of these compounds may be due to estrogen receptor
4
2
antagonism as compound 23 had previously been shown to demonstrate ER binding effects . Group
II bis-2-phenylindoles products 31-35 contain two potential ER-binding ligands separated via the
alkyl chain. The bis-2-phenylindole-based analogues 31-35 were significantly more active than the 2-
phenylindole-based analogues and showed low micromolar antiproliferative activity (IC50 values in
the range of 2.75-8.16 μM, comparable with that of tamoxifen, 4.1 M). The increased activity of
these compounds could be explained by bivalent ligand binding where each ER-binding ligand could
potentially bind to an ER LBD, either intramolecularly (binding the same ER LBD monomer) or
5
2
intermolecularly (binding between dimer ER LBDs) . However, the CLogP value increases from 8.00
to 12.23 as the chain length is increased from n=2 (31) to n=10 (35), while the cLogP value for
tamoxifen is calculated to be 6.07.
To investigate the effect on activity of replacement of the N-methylaminoalkyl substituent of various
hydrocarbon chain lengths (4,5,6,8,12 carbons) located at positions at N-1 of the indole by benzyl, n-
butyl and hydroxyethyl substituents, an additional series of indole compounds 65-70 (Group III)
synthesised in this study all containing the N-substituted 2-phenylindole ER type ligand structure
were also evaluated The antiproliferative activity for the compounds tested in this series were
generally in the low to moderate micromolar region (IC50 = 3.5 – 41.0 μM) for compounds with
benzyl, n-butyl and hydroxyethyl substituents, with the exception of 69 which displayed much lower
antiproliferative activity (IC50 = 332 μM). Compounds 65 and 66 were previously reported to show
3
9, 41
estrogen receptor binding effects in calf uterine receptor assay
. From the Group III compounds
13

screened, the bis-indole compound 70, displayed the greatest antiproliferative activity with an IC50
value of 3.5 μM. The novel bis-indole compound 70, similar in structure to the Group II bis-2-
phenylindoles 31-35 above, but containing a more rigid linker system, could potentially bind via one
of the 2-phenylindole within the ER binding site and the other unbound indole has the potential to
interact with Helix-12 and potentially act as an ER antagonist. However this compound also had the
highest CLogP value of any of the tested compounds. The most active of these novel compounds (70)
displayed IC50 value of 3.5 μM which is comparable to that of the control drug tamoxifen with IC50
61
determined as 4.1 M, which is in agreement with reported IC50 values .
The series of novel conjugate scaffold structures 82-87 (Group IV) containing the ER-ligands 21-25
linked to the CA4 analogue 76 were initially evaluated for antiproliferative activity in human breast
cancer MCF-7 cells. This series of conjugates again showed low micromolar antiproliferative activity
(IC50 values in the range 1.86-2.95 μM) with the exception of the methoxy substituted compound 87
which exhibited reduced antiproliferative activity (IC50 = 81 μM) indicating that the phenolic indole
substituents were essential for the enhanced activity of the conjugate compounds. Again however,
this series of compounds are predicted to have poor oral absorption with CLogP greater than 5.
14

b
Compound
21
R
n
MCF-7
IC50 (μM)
40.7
CLogP
a, c
Group I:
-Aryl-3-methylindoles 21-25
2
3
3.66
4.19
4.72
5.78
7.89
2
22
23
24
25
48.97
4
41.68
6
38.90
10
33.0
Group II: bis-2-Phenylindoles
1-35
31
2
3
2.75
4.16
8.16
6.6
8.00
8.53
9.06
3
32
33
34
35
4
6
10.11
12.23
10
5.24
Group III:
-Aryl-3-methylindoles 65-70
65
-C
4
H
9
34.0
38.9
41.0
17.7
332
3.5
5.40
4.91
5.73
2.98
4.88
9.03
2
66
67
68
69
70
-CH (C H )OH
2
6 4
-CH (C H )F
2
6 4
-CH CH OH
2
2
-CH (C H )(OCH )
2
6
2
3 3
-CH (C H )CH -indole
2
6
4
2
Group
IV:
2-Aryl-3-
82
-H
2
3
1.99
2.93
2.34
2.95
1.86
81.28
5.81
6.24
6.87
7.86
9.27
6.91
methylindole- combretastatin
conjugates 82-87
83
84
85
86
87
-H
-H
4
-H
6
-H
10
2
-CH
3
Table 2: Antiproliferative data for 2-phenylindole-based analogues as potential ER-ligands in the
MCF-7 human breast cancer cell line.
a
b
The values represent the mean for three independent experiments performed in triplicate. CLogP
c
values calculated from CS ChemBioDraw 13.0. The IC50 value determined for the control drug
Tamoxifen was 4.1 M.
15

Effects of selective active compounds on cell cycle and cell death.
A small selection [25, 31 and 86] representative of the most active compounds from the
structural groups above which were identified from the cell viability studies, were subjected to cell
cycle analysis. Flow cytometry using propidium iodide stained cells was carried out to analyse the
percentage of cells in each stage of the cell cycle and the number of apoptotic or dead cells. MCF-7
cells were treated for 72 h with 10 µM of each of the selected compounds. Results showed that for
compounds 31 and 86, a significant increase in the levels of apoptosis was observed when compared
to vehicle-treated cells, 54.7 ± 3.5 and 42.1 ± 4.4% apoptosis respectively compared to 5.5 ± 1.3%
apoptosis (Figure 2A). In contrast, the less active compound 25 did not induce a significant increase
in apoptotic levels at this concentration (7.5 ± 4.0% apoptosis). A G2/M arrest, commonly observed
with tubulin-targeting compounds was not observed with any compound treatment (Figure 2C).
Next, the same cohort of compounds (Figure 2B+2D) was tested on peripheral blood
mononuclear cells (PBMCs) isolated from the blood of healthy donors. Cells were treated with 10
µM of each compound for 72 h and subjected to flow cytometry. Results showed that neither 86 or
25 induced apoptosis or a G2/M arrest in healthy PBMCs, whilst compound 31 induced a small
degree of apoptosis, 16.7 ± 0.1% apoptosis, compared to vehicle-treated cells, 1.5 ± 0.6% apoptosis.
The potent activity of 31 and 86, in MCF-7 breast cancer cells and their lack of toxicity in normal
blood cells make them ideal candidates for further anti-cancer studies.
Figure 2: Compounds potently induce apoptosis in MCF-7 cells, but not healthy PBMCs. Cells were
treated for 72 h with a vehicle or the indicated compounds. After the required incubation period,
cells were fixed in 1 mL ethanol and 100 µL PBS and stained with propidium iodide. Cells were
subsequently analysed by flow cytometry to determine the percentage of apoptotic (A) MCF-7 cells
and (B) PBMCs and G2/M-arrested (C) MCF-7 cells and (D) PBMCs. Values represent the mean ±
S.E.M. of three independent experiments.
ER binding assay of selected active compounds
As our results demonstrated a strong ability of indole-based compounds to inhibit the
viability of ER-positive breast cancer MCF-7 cells, it was necessary to test the binding efficiency of
these compounds for both ER isoforms, ERα and ERβ, to determine how effectively the ligand binds
to the receptor and whether the compound is acting as an antagonist or agonist. The most active
compounds from each group of tested SERM type compounds based on the viability results were
selected for the ER binding study (Table 3). The selectivity value is reported as the ratio of RBA
16

values or binding affinities, ERα relative to ERβ, for each of the compounds (see Table 3). For
selectivity values greater than 1, the compounds have a more pronounced affinity for ERα binding
site, while for values less than 1, the compounds have a more pronounced affinity for the ERβ
binding site. The 1-(aminoalkyl)-2-phenylindole 25 which contains the basic secondary amine
substituent exhibited nanomolar activity in ERα and low micromolar activity in ERβ. The bis-indole
compound 31 which lacks the conventional basic side chain required in most ER antagonists such as
tamoxifen and raoxifene interestingly displayed ER binding activity in the low micromolar or
nanomolar regions for both ER isoforms with a preference for ERβ. The combretastatin analogue 86
also displayed nanomolar activity for ER binding with a preference for ERβ. For comparative
purposes, estradiol was also tested as a positive control and displayed a low nanomolar IC50 value for
both ER isoforms.
Compound
ER α
IC50 (nM)
ER β
IC50 (nM)
ERα/ERβ
Selectivity
2
3
8
5
1
6
380
436
574
1.7
1660
41
4.37 ERα
0.09 ERβ
0.27 ERβ
1.76 ERα
155
3
Estradiol
Table 3: ERα and ERβ binding data for the most active novel compounds.
The Lanthascreen® FRET kit contains a fluoromone which is found in the binding site of the receptor,
ERα or ERβ. If the ligand displaces the fluoromone, fluorescence decreases, determining the
efficiency of the ligand to bind to the receptor. The selectivity value is reported as the ratio of RBA
values or binding affinities, ERα relative to ERβ, for each of the compounds. For selectivity values
greater than 1, the compounds have a more pronounced affinity for ERα binding site, while for
values less than 1, the compounds have a more pronounced affinity for the ERβ binding site.
Molecular modelling study of selected indoles
To date, different agonists and antagonists have been co-crystallised with 34 human ERα
1
4, 20-22
isoforms
. Structure-activity relationships for estradiol ER binding affinity have been extensively
1
3, 19
studied
. The phenolic A ring and 17β-hydroxy group of Ring D are essential for estrogenic
activity. Ideally the distance between the oxygen atoms of the phenol at C and the hydroxy at C17 is
3
4
5
approximately 10.3-12.1 Å) . The 2-phenylindoles now reported contain the required
pharmacophore for optimimum binding at the ER. The distance between the 2-phenylindole
hydroxyl groups positioned on the hydrophobic core in selected compounds 25, 31 and 86 is
17

6
2
calculated to be 11.48 Å (MOE 2012.10 ) which is within the optimum distance required for ER
4
5
20
activity as exemplified by raloxifene in the 1ERR X-ray structure (see Supplementary Information).
Therefore the 2-phenylindole combines all the key structural requirements for binding to the ER.
Examples of the most potent in antiproliferative activity of each of the indole containing compounds
synthesised (compounds 25, 31 and 86) were retrospectively modelled in silico in order to gain an
insight into the most probable binding orientation within the human ERα and ERβ isoforms and to
rationalise findings from the biochemistry assays. In brief, multi-conformer databases were created
with OMEGA for compounds synthesised in the current study, which were then docked in 3ERT.pdb
2
2
(
4-Hydroxytamoxifen cocrystallised with hERα) and scored to determine the highest ranking
6
3, 64
docked solution using the software package FRED
. The compounds were included within a
6
5
subset of the DUD ERα antagonist dataset (containing 39 actives and 1125 inactives) for validation
and comparative purposes. Docking ranking accuracy was 89.9% and the compound rankings
detailed below refer to this docking study. The highest ranked docked pose of each compound was
6
6
visualised in the hERα binding site utilising MOE 2015.1001 . An analogous study was undertaken
6
7
for ERβ where the 1NDE X-ray structure with an ER selective 1,3,5-triazine co-crystallised ligand
was utilised, a docking accuracy of 81.9% was achieved for the DUD subset.
For ERα, the interaction of the antagonist side chain with Asp351 residue varies amongst
ligands. 4-Hydroxytamoxifen (4OHT) (ranked 1) displays a tight interaction between the Asp351
2
2
residue and the ligand amine through hydrogen bonding (1.24 Å) 3ERT.pdb (Figure 3). The bis-2-
phenylindole analogue 25 was ranked 6 when docked in hERα and an overlay with 4OHT is shown in
Figure 3A. The two phenols on the 2-phenylindole core form hydrogen bond interactions with
Glu353 directly and to Arg394 via a water bridge interaction, the other phenol is observed to directly
interact with the His524. These three interactions are also seen for 4OHT so it is proposed that 25
should bind within the LBD of the ER similarly. However, 4OHT also interacts with Asp351 which
displaces the Helix-12, but this is not observed with 25. Instead, the long chain of 25 projects
linearily at an angle from the LBP. It is possible that inclusion of an additional basic N-CH substituent
3
at C-7 or C-8 of the aliphatic chain could form an ionic bond with Asp351 when protonated thereby
further increasing ERα binding affinity (Figure 3A). The bis-2-phenylindole analogue 31 was ranked
249 and also demonstrated advantageous interactions within the LBP. However, its ranking was
significantly higher than 25, partly due to shorter chain length and bulkier nature of the phenylindole
at the exit of the pocket. The similarity between both binding poses is illustrated in Figure 3B.
For the docking studies of 25, the X-ray structure of the ERβ (1NDE.pdb) cocrystallised with a
6
7
triazine modulator was used (Figure 3C) . Interestingly, the orientation of the 2-phenylindole core
18

recapitulating the 4OHT LBP interactions is flipped 180° as compared to other docked analogues.
This non-conventional binding mode is reflected in a higher ranking (55) than analogue 31 (ranked
24) and also a significantly lower ERβ binding affinity. However, key phenolic binding interactions
with Glu305, Arg346 and His475 are still maintained. Analogue 31 displays the traditional hydrogen
bonding interactions at the base of the LPB including with Glu305. The distances between the
hydroxyl groups adjacent to Glu305 of triazine (1.49 Å) and that of 25 are 2.46 Å while for 31 they
are 1.88 Å demonstrating that this key hydrogen bonding is possible for all compounds. Interestingly,
the hydroxyl group of the outer pocket located phenylindole is close enough to Asp303 to make
hydrogen bonding interactions with this important amino acid (Figure 3D). These results are in
agreement with the ER binding data suggesting compound 25 has a preference for ERα while 31 has
a preference for ERβ.
Compound 86 is significantly bulkier than any of the other compounds studied. Within the
parameters of FRED docking, a binding pose for 86 was not ranked in the top 1000 molecules, most
likely due to the aliphatic chain linking to the combretastatin analogue clashing with nearby protein
structure so a possible docked pose for discussion is not presented. However, despite this limitation
in computational docking, it is likely the compound does indeed bind in situ as evidenced by the
strong anti-proliferative effects the compound has on breast cancer cells in vitro. This compound
contains the 2-phenylindole binding ERα core as compound 25, so will display the same hydrogen
bonding interactions as described above.
Figure 3: Molecular modelling of compounds 25 and 31 with ERα and β. Overlay of the ERα X-ray
structure of 4OHT (3ERT) on the best ranked docked pose of 2-phenylindoles (A) 25 and (B) 31 and
ERβ X-ray structure of triazine (1NDE) on (C) 25 and (D) 31. The overlays succinctly illustrate the
similarities in positioning of the hydroxyl groups. The distances between the hydroxyl groups of
tamoxifen and that of 25 are 0.89 Å while for 31 they are 1.63 Å, and for the triazine modulator and
that of 25 are 1.97 Å while for 31 it is also 1.97 Å, demonstrating their capacity for involvement in
similar hydrogen bonding networks. 4OHT and Triazine are shaded gray, 25 is green and 31 is
coloured orange. The atoms are coloured by element type, carbon = grey, hydrogen = white, oxygen
=
red, nitrogen = blue, sulphur = yellow. Key amino acid residues are labelled.
19

Conclusions
Many approaches have been reported for the design and evaluation of novel molecules which target
the proliferation of human breast cancer cells. The indole containing ER ligands and the novel
conjugate compounds synthesised in this work were tested for antiproliferative activity in MCF-7 ER-
overexpressing cell line. The novel bis-2-phenylindole ER ligands 31-35, 70 displayed interesting
activity in the low micromolar range with IC50 values of 2.75-8.16 M. The 2-phenylindole-cytotoxic
agent conjugates 82-86 incorporating the acrylic acid derivative of the cytotoxic agent
combretastatin CA4 also displayed good antiproliferative activity with the longest chain linker
(
(CH )12 ) derivative 86 exhibiting an IC50 value of 1.86 μM. Representative compounds 31 and 86 also
2
potently induced apoptosis in breast cancer cells with minimal effects on normal blood cancer cells.
A computational docking study of the interaction of compounds 25 and 31 with ER and ERβ
provides insight into the differential isoform selectivity profile. Both analogues are capable of
o
forming similar interactions within the LBP of both isoforms but 25 is rotated 180 when docked in
ERβ as compared to ERα and is also better ranked in the ERα isoform than in ERβ. These results are
reflected in the in vitro measured binding affinity and ~4.4-fold selectivity of 25 for ERα. Conversely,
analogue 31 is better ranked in ERβ docking studies compared to ERα and also demonstrates the
possibility of forming hydrogen bond donating interactions with ASP308 which is reflected in vitro
with a ~10-fold selectivity for ERβ over ERα. A decrease in cellular anti-cancer efficacy of 25 is likely
to be due to the presence of only one potential ER binding moiety on 25 compared to two ER
binding moieties on 31 and the presence of an ER binding ligand complexed to a cytotoxic agent for
86. In conclusion, the interesting activity of several members of this cohort of compounds in MCF-7
breast cancer cells, ER binding characteristics, induction of apoptosis and their lack of toxicity in
normal blood cells make them potential candidates for further anti-cancer studies.
20

Experimental section
General Chemical Synthesis and Analysis: All reagents were commercially available and were used
without further purification unless otherwise indicated. Tetrahydrofuran (THF) was distilled
immediately prior to use from Na/benzophenone under a slight positive pressure of nitrogen,
toluene was dried by distillation from sodium and stored on activated molecular sieves (4 Å) and
dichloromethane was dried by distillation from calcium hydride prior to use. IR spectra were
recorded as thin films on NaCl plates or as KBr discs on a Perkin- Elmer Paragon 100 FT-IR
1
13
spectrometer. H and C NMR spectra were obtained on a Bruker Avance DPX 400 instrument at 20
1
13
°
C, 400.13 MHz for H spectra, 100.61 MHz for C spectra, in CDCl (internal standard
3
tetramethylsilane) by Dr. John O’Brien and Dr. Manuel Ruether in the School of Chemistry, Trinity
College Dublin. Low resolution mass spectra were run on a Hewlett–Packard 5973 MSD GC–MS
system in an electron impact mode, while high resolution accurate mass determinations for all final
target compounds were obtained on a Micromass Time of Flight mass spectrometer (TOF) equipped
with electrospray ionisation (ES) interface operated in the positive ion mode at the High Resolution
Mass Spectrometry Laboratory by Dr. Martin Feeney in the School of Chemistry, Trinity College
Dublin. Elemental analysis was carried out in the microanalytical laboratory, University College
Dublin, Belfield, Dublin 4. Thin layer chromatography was performed using Merck Silica Gel 60 TLC
aluminium sheets with fluorescent indicator visualising with UV light at 254 nm. Flash
chromatography was carried out using standard Silica Gel 60 (230–400 mesh) obtained from Merck.
All products isolated were homogenous on TLC. Analytical high-performance liquid chromatography
(HPLC) to determine the purity of the final compounds was performed using a Waters 2487 Dual
Wavelength Absorbance detector, a Waters 1525 binary HPLC pump, a Waters In-Line Degasser AF
and a Waters 717plus Autosampler. The column used was a Varian Pursuit XRs C18 reverse phase
150 x 4.6 mm chromatography column. Samples were detected using a wavelength of 254 nm. All
samples were analysed using acetonitrile (70%): water (30%) over 10 min and a flow rate of 1
mL/min. Unless otherwise indicated, the purity of the final products was ≥95%. Chromatograpic
separations were also carried out on Biotage SP4 instrument. Microwave experiments were carried
out with the Biotage Initiator and Discover CEM microwave synthesisers. Full spectroscopic
4
9
39
60
42
68
50
42
39
41
characterisation is now provided for compounds 2 , 43 , 44 , 45 , 46 , 5 , 23 , 65 , 66 .
5
8
59
57
69
Compounds 57 , 59 , 60 and 76 were synthesised as previously reported.
21

1
-(4-(Benzyloxy)phenyl)propan-1-one (2)
-Hydroxypropiophenone (1) (5.0 g, 33 mmol) was dissolved in acetonitrile (50 mL) under N
solution was added K CO (3.0 g, 21.6 mmol) and benzyl bromide (5.7 g, 4.0 mL, 33 mmol). The
4
2
, to the
2
3
mixture was stirred at RT for 16 h. The mixture was filtered and the solvent was evaporated under
reduced pressure. The resulting oil was dissolved in DCM (100 mL) and the mixture was extracted
with 1M HCL (50 mL), water (2 × 50 mL), brine (50 mL) and dried over Na
2
SO . The solvent was
4
evaporated under reduced pressure to obtain a pale yellow resin. The material was purified via flash
chromatography on silica gel (DCM) to afford the product as a white solid. (7.5 g, 93%), Mp 100-102
4
9
-1
1
°
C . IR : νmax (KBr) cm : 2938, 1680 (C=O), 1599, 1224, 1012. H NMR (CDCl
3
, 400 MHz) δ 7.97 (d, J =
.35 Hz, 2H, Ar-H), 7.34 - 7.51 (m, 5H, Ar-H), 7.04 (d, J = 9.35 Hz, 2H, Ar-H), 5.15 (s, 2H, CH ), 2.98 (q,
, 100 MHz) δ 199.0 (C=O), 162.0
9
2
1
3
J = 7.02 Hz, 2H, CH
2
), 1.24 (t, J = 7.31 Hz, 3H, CH
3
). C NMR (CDCl
3
(ArC), 135.8 (ArC), 129.8 (ArC), 129.8 (ArC), 128.3 (ArC), 127.8 (ArC), 127.1 (ArC), 114.1 (ArC), 69.7
+
(CH
2
), 31.0 (CH
2
), 8.0 (CH
3
). HRMS (EI): Found 263.1052 (M+Na) , C16
H
16
2
O Na requires 263.1048.
1-(4-(Benzyloxy)phenyl)hydrazine hydrochloride (4)
Note: The reaction mixture and all added solutions were maintained at 0 °C during this procedure. 4-
Benzyloxyaniline hydrochloride (3) (3.0 g, 12.5 mmol) was added to conc. aq. HCl (25 mL) and stirred
for 10 min at 0 °C, followed by dropwise addition of NaNO
over the course of 15 min. The mixture was stirred for additional 15 min and then a solution of SnCl
6.4 g, 33.1 mmol) in conc. aq. HCl (7.5 mL) was added dropwise. The reaction mixture was stirred
for 1 h and filtered to yield an off-white precipitate, which was washed with water and triturated
2
(852 mg, 12.3 mmol) in water (6 mL)
2
(
-
1
with Et
2
O, to afford an off-white solid (2.9 g, 92%); Mp 179-181 °C. IR : νmax (KBr) cm : 3232, 2907
1
(
br), 1638, 1618, 1511, 1246, 1178. H NMR (DMSO-d
6
, 400 MHz) δ 10.14 (br s, 3H, NH-NH
2
), 7.35 -
1
3
7.48 (m, 4H, Ar-H), 7.27 - 7.35 (m, 1H, Ar-H), 6.92 - 7.05 (m, 4H, Ar-H), 5.06 (s, 2H, CH
2
). C NMR
(
(
(
DMSO-d , 100 MHz) δ 153.7 (ArC-O), 139.1 (ArC-N), 137.3 (ArC), 128.5 (ArC), 128.4 (ArC), 128.3
6
ArC), 127.9 (ArC), 127.7 (ArC), 117.1 (ArC), 116.9 (ArC), 115.5 (ArC), 115.3 (ArC), 69.5 (CH ). HRMS
2
+
EI): Found 215.1180 (M+H) , C13
H
15
2
N O requires 215.1184.
5-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-3-methyl-1H-indole (5)
1-(4-(Benzyloxy)phenyl)propan-1-one (2) (1.9 g, 8.0 mmol) and 1-(4-(benzyloxy)phenyl)hydrazine
hydrochloride (4) (2.0 g, 8.0 mmol) were dissolved in EtOH (50 mL) and 5 drops of conc. HCl was
added and the mixture was refluxed for 2 h. The reaction mixture was then allowed to cool and was
stirred for 1 h. The mixture was filtered to afford an pale pink solid. The solid was washed with cold
5
0
-1
EtOH (10 mL) followed by n-hexane (20 mL). (2.8 g, 84%). Mp: 149-152 °C . IR: νmax (KBr) cm : 3470
1
(
N-H), 2880, 2820, 1620 (C=C). H NMR (CDCl
3
, 400 MHz) δ 7.86 (br s, 1H, NH), 7.48 - 7.58 (m, 6H, Ar-
H), 7.33 - 7.47 (m, 6H, Ar-H), 7.25 - 7.31 (m, 1H, Ar-H), 7.14 - 7.18 (m, 1H, Ar-H), 7.07 - 7.13 (m, 2H,
22

1
3
Ar-H), 6.97 (d, J = 7.53 Hz, 1H, Ar-H), 5.18 (s, 2H, CH
2
), 5.15 (s, 2H CH
, 100 MHz) δ 157.7 (BnO-C), 157.3 (BnO-C), 143.6 (Ar-C-CH ), 137.3 (C
29.9 (ArC), 128.5 (ArC), 128.2 (ArC), 128.2 (ArC), 127.7 (ArC), 127.6 (Ar-C), 127.1 (ArC), 120.4 (ArC),
2
), 2.43 (s, 3H, CH
3
) C NMR
(CDCl
3
2
q
), 136.3 (C ), 136.0 (C
q
q
),
1
1
6
14.7 (ArC), 114.6 (ArC), 114.3 (ArC), 112.2 (C
9.6 (CH ), 9.3 (CH
q
), 110.8 (C=C), 108.8 (ArC), 102.0 (ArC), 70.0 (CH ),
2
+
2
3
). HRMS (EI): Found 442.1797 (M+Na) , C29
H
25NNaO requires 442.1783.
2
General method for alkylation of 5-(benzyloxy)-2-(4-(benzyloxy)phenyl)-3-methyl-1H-
indole (5).
The indole (5) dissolved in THF (10 mL) was added dropwise to a stirring mixture of NaH in THF (10
mL) at 0 °C under N
dibromoalkane (6)-(10) was added and the mixture was stirred for 30 min at 0 °C. The reaction was
refluxed for a further 30 min under N atmosphere, the mixture was allowed to cool at which point
2
atmosphere. The mixture was stirred for 10 min, at which point the appropriate
2
water was added dropwise until the solution became clear. The solvent was then evaporated under
reduced pressure to obtain a brown oil. The resin was dissolved in DCM (50 mL) and washed with
water (3 × 50 mL), brine (50 mL) and dried over Na
2
SO . The solvent was evaporated under reduced
4
pressure to obtain a brown oil. The material was purified via flash chromatography over silica gel to
afford the desired product.
5-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-1-(4-bromobutyl)-3-methyl-1H-indole (11)
As per general method above, indole (5) (800 mg, 1.90 mmol), NaH (5 eq, 228 mg, 9.50 mmol) and
,4-dibromobutane (6) (1 eq, 1.90 mmol, 411 mg, 0.2 mL) were reacted. The material was purified
via flash chromatography over silica gel (n-Hexane:DCM, gradient 4:1 to 2:1) to afford the product as
1
-
1
a white solid (658 mg, 63%); Mp 101-102 °C. IR : νmax (KBr) cm : 3062.1, 3232.52, 3086.0, 2927. 50,
1
1
613.8, 1506.0, 1478.9, 1454.2, 1242.7, 1223.6, 1026.0, 736.1, 696.7. H NMR (CDCl
3
, 400 MHz) δ
7.50 - 7.57 (m, 4H, Ar-H), 7.39 - 7.50 (m, 5H, Ar-H), 7.36 - 7.39 (m, 1H, Ar-H), 7.33 (d, J = 8.18 Hz, 2H,
Ar-H), 7.27 (d, J = 9.35 Hz, 1H, Ar-H), 7.17 (d, J = 2.34 Hz, 1H, Ar-H), 7.10 - 7.15 (m, 2H, Ar-H), 7.01
(
dd, J = 2.34, 8.77 Hz, 1H, Ar-H), 5.19 (s, 2H, CH
t, J = 6.43 Hz, 2H, CH ), 2.23 (s, 3H, CH ), 1.77 (quin, J = 7.02 Hz, 2H, CH
, 100 MHz) δ 158.1 (BnO-C), 152.7 (BnO-C), 137.5 (Ar-C-CH
), 131.3 (ArC), 131.2 (ArC), 128.2 (ArC), 128.1 (ArC), 127.7 (ArC), 127.3 (ArC), 127.2 (ArC), 124.4
2
), 5.17 (s, 2H, CH
2
), 4.07 (t, J = 7.02 Hz, 2H, CH
), 1.65 (quin, J = 6.40 Hz, 2H,
), 137.4 (C ), 136.3
2
), 3.21
(
2
3
2
1
3
CH
2
). C NMR (CDCl
3
2
q
(
(
(
C
q
C=C), 115.3 (C
q
), 114.4 (ArC), 111.8 (ArC), 109.8 (ArC), 108.0 (C=C), 102.0 (ArC), 69.7 (CH
), 32.6 (CH ), 29.3 (CH ), 28.1 (CH ), 8.9 (CH
requires 554.1689.
2
), 43.2
+
CH
2
), 42.4 (CH
2
2
2
2
3
). HRMS (EI): Found 554.1688 (M+H) ,
7
9
C
33
H
33 BrNO
2
23

5
-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-1-(5-bromopentyl)-3-methyl-1H-indole (12)
As per general method above, indole (5) (800 mg, 1.90 mmol), NaH (5 eq, 228 mg, 9.50 mmol) and
,5-dibromopentane (7) (1 eq, 1.9 mmol, 0.44 g, 0.3 mL) were reacted. The material was purified via
flash chromatography over silica gel (n-Hexane:DCM, gradient 4:1 to 2:1) to afford the product as a
1
-
1
white solid (701 mg, 65%). Mp 98-101 °C. IR : νmax (KBr) cm : 3031.0, 2947.5, 2911.3, 2866.2, 1610.6,
1
1
480.6, 1455.0, 1378.5, m1230.7, 1020.6, 704.6.. H NMR (CDCl
3
, 400 MHz) δ 7.50 - 7.58 (m, 4H, Ar-
H), 7.36 - 7.50 (m, 6H, Ar-H), 7.33 (d, J = 8.77 Hz, 2H, Ar-H), 7.24 - 7.28 (m, 1H, Ar-H), 7.11 - 7.19 (m,
H, Ar-H), 7.01 (dd, J = 2.34, 8.77 Hz, 1H, Ar-H), 5.19 (s, 2H, CH ), 5.18 (s, 2H, CH ), 4.04 (t, J = 7.31
Hz, 2H, CH ), 3.28 (t, J = 6.72 Hz, 2H, CH ), 2.23 (s, 3H, CH ), 1.58 - 1.75 (m, 4H, 2 × CH ), 1.27 (quin, J
, 100 MHz) δ 158.1 (BnO-C), 152.7 (BnO-C), 137.5 (Ar-C-CH ),
), 131.3 (ArC), 131.2 (ArC), 128.4 (ArC), 128.2 (ArC), 128.1 (ArC), 127.7 (ArC),
27.3 (ArC), 127.2 (ArC), 124.5 (C=C), 114.3 (ArC), 111.7 (ArC), 109.8 (ArC), 107.8 (C=C), 102.0 (ArC),
0.6 (CH ), 69.7 (CH ), 43.1 (CH ), 33.0 (CH ), 31.7 (CH ), 28.7 (CH ), 24.9 (CH ), 8.9 (CH ). HRMS (EI):
requires 568.1848.
3
2
2
2
2
3
2
1
3
=
1
1
7
7.60 Hz, 2H, CH
2
). C NMR (CDCl
3
2
37.4 (C ), 136.4 (C
q
q
2
2
2
2
2
2
2
3
+
79
Found 568.1848 (M+H) , C34
H
35 BrNO
2
5
-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-1-(6-bromohexanoyl)-3-methyl-1H-indole (13)
As per general method above, Indole (5) (800 mg, 1.90 mmol), NaH (5 eq, 228 mg, 9.5 mmol) and
,6-dibromohexaneane (8) (1 eq, 1.90 mmol, 460 mg, 0.3 mL) were reacted. The material was
purified via flash chromatography on silica gel (n-Hexane:DCM, gradient 4:1 to 2:1) to afford the
1
-
1
product as a white solid (730 mg, 66%); Mp 101-102 °C. IR : νmax (KBr) cm : 3063.1, 3032.7, 1932.3,
1
1
931.4, 1612.9, 1506.0, 1479.6, 1454.9, 1238.2, 1174.5, 1026.18, 735.94, 696.79. H NMR (CDCl
3
,
400 MHz) δ 7.51 - 7.58 (m, 4H, Ar-H), 7.40 - 7.50 (m, 5H, Ar-H), 7.36 - 7.39 (m, 1H, Ar-H), 7.30 - 7.35
(
m, 2H, Ar-H), 7.24 - 7.30 (m, 1H, Ar-H), 7.10 - 7.18 (m, 3H, Ar-H), 7.01 (dd, J = 2.01, 8.53 Hz, 1H, Ar-
H), 5.19 (s, 2H, CH ), 5.18 (s, 2H, CH ), 4.03 (t, J = 7.28 Hz, 2H, CH ), 3.33 (t, J = 6.78 Hz, 2H, CH ), 2.23
s, 3H, CH ), 1.74 (quin, J = 7.15 Hz, 2H, CH ), 1.56 - 1.67 (m, 2H, CH ), 1.24 - 1.35 (m, 2H, CH ), 1.10 -
.21 (m, 2H, CH , 100 MHz) δ 158.1 (BnO-C), 152.6 (BnO-C), 137.5 (Ar-C-CH ), 137.4
), 136.4 (C ), 131.3 (ArC), 131.2 (ArC), 128.4 (ArC), 128.2 (ArC), 128.1 (ArC), 127.7 (ArC), 127.3
ArC), 127.2 (ArC), 124.6 (C=C), 114.3 (ArC), 111.7 (ArC), 109.8 (ArC), 107.7 (C=C), 102.0 (ArC), 70.6
CH ), 69.7 (CH ), 43.2 (CH ), 33.3 (CH ), 32.1 (CH ), 29.3 (CH ), 27.2 (CH ), 25.4 (CH ), 8.9 (CH ). HRMS
EI): Found 582.1999 (M+H) , C35 requires 582.2002.
2
2
2
2
(
3
2
2
2
1
3
1
2
). C NMR (CDCl
3
2
(
(
(
(
C
q
q
2
2
2
2
2
2
2
2
3
+
79
H
37 BrNO
2
5
-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-1-(8-bromooctyl)-3-methyl-1H-indole (14)
As per general method above, indole (5) (800 mg, 1.90 mmol), NaH (5 eq, 228 mg, 9.50 mmol) and
,8- dibromooctane (9) (1 eq, 1.90 mmol, 516 mg, 0.4 mL) were reacted. The material was purified
via flash chromatography on silica gel (n-Hexane:DCM, gradient 4:1 to 2:1) to afford the product as a
1
-
1
white solid (700 mg, 60%); Mp 104-106 °C. IR : νmax (KBr) cm : 3062.9, 3032.5, 2929.4, 1612.8,
24

1
1
505.9, 1476.9, 1454.1, 1287.4, 1241.7, 1025.8, 735.6, 696.3. H NMR (CDCl
3
, 400 MHz) δ 7.50 - 7.59
(
m, 4H, Ar-H), 7.39 - 7.50 (m, 5H, Ar-H), 7.35 - 7.39 (m, 1H, Ar-H), 7.31 - 7.35 (m, 2H, Ar-H), 7.24 -
.28 (m, 1H, Ar-H), 7.15 - 7.19 (m, 1H, Ar-H), 7.10 - 7.15 (m, 2H, Ar-H), 7.00 (dd, J = 2.26, 8.78 Hz, 1H,
Ar-H), 5.18 (s, 2H, CH ), 5.17 (s, 2H, CH ), 4.00 (t, J = 7.53 Hz, 2H, CH ), 3.40 (t, J = 7.03 Hz, 2H, CH ),
.23 (s, 3H, CH ), 1.82 (quin, J = 7.15 Hz, 2H, CH ), 1.56 - 1.66 (m, 2H, CH ), 1.36 (quin, J = 7.15 Hz, 2H,
CH ), 1.09 - 1.26 (m, 6H, 3 × CH ). C NMR (CDCl , 100 MHz) δ 158.0 (BnO-C), 152.6 (BnO-C), 137.6
Ar-C-CH ), 137.4 (C ), 136.4 (C ), 131.3 (ArC), 131.2 (ArC), 128.4 (ArC), 128.2 (ArC), 128.1 (ArC),
27.7 (ArC), 127.3 (ArC), 127.2 (ArC), 124.6 (C=C), 114.2 (ArC), 111.6 (ArC), 109.9 (ArC), 107.6 (C=C),
7
2
2
2
2
2
3
2
2
1
3
2
2
3
(
2
q
q
1
1
01.9 (ArC), 70.6 (CH
), 27.5 (CH ), 26.2 (CH
10.2321.
2
), 69.7 (CH
2
), 43.4 (CH
2
), 33.6 (CH
2
), 32.3 (CH
2
), 29.5 (CH
2
), 28.4 (CH ), 28.1
2
+
79
(CH
2
2
2
), 8.9 (CH
3
). HRMS (EI): Found 610.2301 (M+H) , C37
H
41 BrNO
2
requires
6
5
-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-1-(12-bromododecyl)-3-methyl-1H-indole (15)
As per general method above, indole (5) (800 mg, 1.90 mmol), NaH (5 eq, 228 mg, 9.50 mmol) and
,12- dibromododecane (10) (1 eq, 1.9 mmol, 623 mg) were reacted. The material was purified via
flash chromatography on silica gel (n-Hexane:DCM, gradient 4:1 to 2:1) to afford the product as a
1
-
1
white solid (750 mg, 59%); Mp 71-73 °C. IR : νmax (KBr) cm : 3062.8, 3032.8, 2922.7, 2853.7, 1613.0,
1
1
505.9, 1479.6, 1287.2, 1241.7, 1025.9, 735.8, 696.12. H NMR (CDCl
3
, 400 MHz) δ 7.50 - 7.57 (m,
4
H, Ar-H), 7.30 - 7.49 (m, 8H, Ar-H), 7.24 - 7.28 (m, 1H, Ar-H), 7.14 - 7.17 (m, 1H, Ar-H), 7.12 (s, 2H,
), 5.16 (s, 2H, CH ), 3.99 (t, J = 7.53 Hz,
), 1.59 (m, 4H, 2 × CH ), 1.39 - 1.51 (m, 8H, 4 ×
, 100 MHz) δ 158.0 (BnO-C), 152.6 (BnO-C), 137.6
), 131.3 (ArC), 128.3 (Ar-C), 128.2 (ArC), 128.1 (ArC), 127.7 (ArC),
27.3 (ArC), 127.1 (ArC), 124.6 (C=C), 114.2 (ArC), 111.6 (ArC), 109.9 (ArC), 107.6 (C=C), 101.9 (ArC),
0.6 (CH ), 69.6 (CH ), 43.5 (CH ), 33.7 (CH ), 32.4 (CH ), 29.6 (CH ), 29.3 (CH ), 29.04 (CH ), 29.01
), 28.7 (CH ), 28.3 (CH ), 27.7 (CH ), 26.3 (CH ), 8.9 (CH ). HRMS (EI):
requires 666.2947.
Ar-H), 6.99 (dd, J = 2.51, 9.03 Hz, 1H, Ar-H), 5.18 (s, 2H, CH
2
2
2H, CH
2
), 3.44 (t, J = 7.03 Hz, 2H, CH
2
), 2.22 (s, 3H, CH
3
2
1
3
CH
2
), 1.09 - 1.37 (m, 8H, 4 × CH
2
). C NMR (CDCl
3
(Ar-C-CH
2
), 137.4 (C
q
), 136.4 (C
q
1
7
2
2
2
2
2
2
2
2
(CH
2
2
), 28.5 (CH
2
), 28.4 (CH
2
2
2
2
3
+
79
Found 666.2941 (M+H) , C41
H
49 BrNO
2
General method for amination of 1-bromoalkyl-2-phenylindoles (11-15)
The appropriate 1-bromoalkyl-2-phenylindole (11-15) was dissolved in dry THF (10 mL) and
transferred to a pressure tube. To this solution was added a solution of methylamine (2 M in THF)
and the mixture was flushed with N before sealing the tube. The tube was left stir at 65 °C for 48 h.
2
The tube was then allowed to cool to reduce the internal pressure, then opened carefully and the
solvent was evaporated under reduced pressure to obtain a brown oil. The oil was dissolved in DCM
(30 mL) and was washed with a pH 10 aq. solution (30 mL). The pH 10 aq. solution was extracted
with DCM (2 × 30 mL). The organic layers were combined and washed with water (50 mL), brine (50
25

mL) and dried over Na
2
SO . The solvent was evaporated under reduced pressure to obtain a brown
4
oil. The material was purified via flash chromatography over silica gel (DCM : MeOH) to afford the
desired product.
4-(5-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-3-methyl-1H-indol-1-yl)-N-methylbutan-1-
amine (16)
As per general method above, (11) (440 mg, 0.80 mmol) and methylamine (20 eq, 15.9 mmol, 8.0
mL) were reacted. The material was purified via flash chromatography on silica gel (DCM:EtOAc,
gradient 4:1 to 2:1) to afford the product as a brown resin (365 mg, 92 %); Mp 79-82 °C. IR : νmax
-
1
(
KBr) cm : 3333.3, 3032.7, 2928.0, 2860.2, 2790.8, 1612.4, 1505.8, 1476.9, 1454.3, 1287.0, 1241.2,
1
1
025.8, 736.2, 696.7. H NMR (CDCl
3
, 400 MHz) δ 7.49 - 7.58 (m, 4H, Ar-H), 7.38 - 7.49 (m, 5H, Ar-H),
7
.35 - 7.38 (m, 1H, Ar-H), 7.30 - 7.34 (m, 2H, Ar-H), 7.26 (d, J = 8.53 Hz, 1H, Ar-H), 7.08 - 7.18 (m, 3H,
), 5.16 (s, 2H, CH ), 4.03 (t, J = 7.28 Hz,
), 2.21 (s, 3H, CH ), 1.57 - 1.69 (m, 2H, CH
, 100 MHz) δ 158.1 (BnO-C), 152.6 (BnO-C), 137.5 (Ar-C-CH
Ar-H), 6.99 (dd, J = 2.26, 8.78 Hz, 1H, Ar-H), 5.17 (s, 2H, CH
2
2
2
1
1
1
5
H, CH
.31 - 1.42 (m, 2H, CH
37.4 (C ), 136.4 (C
24.5 (C=C), 114.3 (ArC), 111.7 (ArC), 109.8 (ArC), 107.9 (C=C), 102.0 (ArC), 70.6 (CH
2
), 2.39-2.51 (m, 3H, N-H, CH
2
), 2.36 (s, 3H, CH
3
3
2
),
),
13
2
). C NMR (CDCl
3
2
q
q
), 131.3 (ArC), 128.2 (ArC), 128.1 (ArC), 127.7 (ArC), 127.3 (ArC), 127.2 (ArC),
), 69.7 (CH ),
). HRMS (EI): Found 505.2852
2
2
0.5 (CH
2
), 43.1 (CH
2
), 35.2 (CH
3
), 27.2 (CH
2
), 25.8 (CH
2
), 8.9 (CH
3
+
(
M+H) , C34
H
37
N
2
O
2
requires 505.2855.
5-(5-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-3-methyl-1H-indol-1-yl)-N-methylpentan-1-
amine (17)
As per general method above, (12) (450 mg, 0.80 mmol) and methylamine (20 eq, 15.9 mmol, 8.0
mL) were reacted. The material was purified via flash chromatography on silica gel (DCM:EtOAc,
gradient 4:1 to 2:1) to afford the product as a brown resin (381 mg, 93%); Mp 81-83 °C. IR : νmax (KBr)
-
1
cm : 3327.0, 3032.7, 2930.2, 2859.8, 2790.5, 1612.6, 1505.9, 1476.9, 1454.3, 1287.58, 1243.51,
1
1
174.9, 1025.9, 736.25, 696.8. H NMR (CDCl
3
, 400 MHz) δ 7.49 - 7.57 (m, 4H, Ar-H), 7.38 - 7.49 (m,
5
H, Ar-H), 7.34 - 7.38 (m, 1H, Ar-H), 7.29 - 7.34 (m, 2H, Ar-H), 7.23 - 7.27 (m, 1H, Ar-H), 7.08 - 7.17
), 5.16 (s, 2H, CH ), 4.01 (t, J =
), 2.21 (s, 4H, N-H, CH ), 1.60 (quin, J =
(m, 3H, Ar-H), 6.99 (dd, J = 2.26, 8.78 Hz, 1H, Ar-H), 5.17 (s, 2H, CH
2
2
7.28 Hz, 2H, CH
7.30 Hz, 2H, CH
100 MHz) δ 158.0 (BnO-C), 152.6 (BnO-C), 137.5 (Ar-C-CH
2
), 2.45 - 2.54 (m, 2H, CH
2
), 2.41 (s, 3H, CH
), 1.16 (quin, J = 7.40 Hz, 2H, CH
), 137.4 (C ), 136.4 (C ), 131.3 (ArC), 128.4
3
3
1
3
2
), 1.40 (quin, J = 7.28 Hz, 2H, CH
2
2
). C NMR (CDCl ,
3
2
q
q
(ArC), 128.2 (ArC), 128.1 (ArC), 127.7 (ArC), 127.3 (ArC), 127.2 (ArC), 124.5 (C=C), 114.3 (ArC), 111.7
(ArC), 109.8 (ArC), 107.8 (C=C), 102.0 (ArC), 70.6 (CH
2
), 69.7 (CH
2
), 50.9 (CH
2
), 50.3 (CH
2
), 43.3 (CH
2
),
+
35.4 (CH
3
), 29.4 (CH
2
), 28.3 (CH
2
), 8.9 (CH
3
). HRMS (EI): Found 519.3033 (M+H) , C35
H
39
N
2
O
2
requires
519.3012.
26

6-(5-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-3-methyl-1H-indol-1-yl)-N-methylhexan-1-
amine (18)
As per general method above, (13) (540 mg, 0.90 mmol) and methylamine (20 eq, 18.5 mmol, 10.0
mL) were reacted. The material was purified via flash chromatography on silica gel (DCM:EtOAc,
gradient 4:1 to 2:1) to afford the product as a brown resin (470 mg, 95%); Mp 79-82 °C. IR : νmax (KBr)
-
1
cm : 3324.6, 3033.3, 2929.5, 2858.1, 2790.9, 1613.49, 1515.8, 1479.3, 1454.23, 1380.7, 1287.2,
1
1
7
-
241.3, 1174.7, 1025.9, 735.5, 696.8. H NMR (CDCl
3
, 400 MHz) δ 7.49 - 7.58 (m, 4H, Ar-H), 7.39 -
.49 (m, 5H, Ar-H), 7.35 - 7.39 (m, 1H, Ar-H), 7.30 - 7.35 (m, 2H, Ar-H), 7.23 - 7.28 (m, 1H, Ar-H), 7.09
7.18 (m, 3H, Ar-H), 7.00 (dd, J = 2.01, 8.53 Hz, 1H, Ar-H), 5.18 (s, 2H, CH ), 5.16 (s, 2H, CH ), 4.00 (t, J
7.28 Hz, 2H, CH ), 2.61 (br s, 1H, N-H) 2.49 - 2.58 (m, 2H, CH ), 2.45 (s, 3H, CH ), 2.22 (s, 3H, CH ),
.55 - 1.67 (m, 2H, CH ), 1.38 - 1.49 (m, 2H, CH ), 1.09 - 1.25 (m, 4H, 2 × CH , 100
), 137.4 (C ), 136.4 (C ), 131.3 (ArC), 131.2
ArC), 128.4 (ArC), 128.2 (ArC), 128.1 (ArC), 127.7 (ArC), 127.3 (ArC), 127.2 (ArC), 124.6 (C=C), 114.2
ArC), 111.6 (ArC), 109.9 (ArC), 107.7 (C=C), 101.9 (ArC), 70.6 (CH ), 69.7 (CH ), 51.0 (CH ), 43.3 (CH ),
), 8.9 (CH ). HRMS (EI): Found 533.3162
2
2
=
1
2
2
3
3
1
3
2
2
2
). C NMR (CDCl
3
MHz) δ 158.0 (BnO-C), 152.6 (BnO-C), 137.6 (Ar-C-CH
2
q
q
(
(
2
2
2
2
35.4 (CH
3
), 29.5 (CH
2
), 28.6 (CH
2
), 26.3 (CH
2
), 26.2 (CH
2
3
+
(
M+H) , C36
H
41
N
2
O
2
requires 533.3168.
8-(5-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-3-methyl-1H-indol-1-yl)-N-methyloctan-1-
amine (19)
As per general method above, (14) (400 mg, 0.70 mmol) and methylamine (20 eq, 13.7 mmol, 7.0
mL) were reacted. The material was purified via flash chromatography on silica gel (DCM:EtOAc,
gradient 4:1 to 2:1) to afford the product as a brown resin (351 mg, 91%); Mp 81-84 °C. IR : νmax (KBr)
-
1
cm : 3327.5, 3062.7, 3032.9, 2927.5, 2855.2, 2790.0, 1612.9, 1506.1, 1479.3, 1454.3, 1380.6,
1
1
287.5, 1241.8, 1174.3, 1025.9, 735.9, 696.6. H NMR (CDCl
3
, 400 MHz) δ 7.49 - 7.58 (m, 4H, Ar-H),
7
.39 - 7.49 (m, 5H, Ar-H), 7.35 - 7.38 (m, 1H, Ar-H), 7.30 - 7.34 (m, 2H, Ar-H), 7.23 - 7.28 (m, 1H, Ar-
), 5.16 (s, 2H, CH ), 3.99 (t,
), 2.22 (s, 3H, CH ), 2.09 (br s, 1H,
), 1.07 - 1.30 (m, 8H, 4 × CH
, 100 MHz) δ 158.0 (BnO-C), 152.6 (BnO-C), 137.6 (Ar-C-CH ), 137.4 (C ), 136.4 (C
ArC), 128.3 (ArC), 128.2 (ArC), 128.1 (ArC), 127.7 (ArC), 127.3 (ArC), 127.2 (ArC), 124.6 (C=C), 114.2
ArC), 111.6 (ArC), 109.9 (ArC), 107.6 (C=C), 101.9 (ArC), 70.6 (CH ), 69.7 (CH ), 51.3 (CH ), 43.5 (CH ),
), 26.3 (CH ), 8.9 (CH ). HRMS (EI): Found
H), 7.09 - 7.17 (m, 3H, Ar-H), 6.99 (d, J = 8.53 Hz, 1H, Ar-H), 5.18 (s, 2H, CH
J = 7.28 Hz, 2H, CH ), 2.58 (t, J = 7.03 Hz, 2H, CH ), 2.45 (s, 3H, CH
N-H), 1.61 (d, J = 6.53 Hz, 2H, CH ), 1.43 - 1.52 (m, 2H, CH
2
2
2
2
3
3
1
3
2
2
2
). C NMR
), 131.3
(
(
(
CDCl
3
2
q
q
2
2
2
2
35.6 (CH
3
), 29.6 (CH
2
), 28.8 (CH
2
), 28.6 (CH
2
), 26.7 (CH
2
2
3
+
5
61.3469 (M+H) , C38
H
45
N
2
O
2
requires 561.3481.
27

12-(5-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-3-methyl-1H-indol-1-yl)-N-methyldodecan-
1-amine (20)
As per general method above, (15) (357 mg, 0.50 mmol) and methylamine (20 eq, 13.7 mmol, 7.0
mL) were reacted. The material was purified via flash chromatography on silica gel (DCM:EtOAc,
gradient 4:1 to 2:1) to afford the product as a brown resin (287 mg, 87%); Mp 66-69 °C. IR : νmax (KBr)
-
1
cm : 3326.9, 3063.3, 3033.2, 2927.5, 2853.4, 2790.1, 1613.5, 1506.1, 1479.5, 1454.7, 1287.2,
1
1
7
-
242.0, 1174.2, 1026.4, 735.2, 696.4. H NMR (CDCl
3
, 400 MHz) δ 7.49 - 7.58 (m, 4H, Ar-H), 7.39 -
.49 (m, 5H, Ar-H), 7.35 - 7.38 (m, 1H, Ar-H), 7.30 - 7.34 (m, 2H, Ar-H), 7.24 - 7.28 (m, 1H, Ar-H), 7.08
7.18 (m, 3H, Ar-H), 6.99 (dd, J = 2.01, 8.53 Hz, 1H, Ar-H), 5.18 (s, 2H, CH ), 5.16 (s, 2H, CH ), 3.99 (t, J
7.28 Hz, 2H, CH ), 2.60 (t, J = 7.03 Hz, 2H, CH ), 2.46 (s, 3H, CH ), 2.22 (s, 3H, CH ), 2.02 (br. s., 1H,
), 1.51 (s, 2H, CH ), 1.08 - 1.36 (m, 16H, 8 × CH
MHz) δ 158.4 (BnO-C), 153.0 (BnO-C), 138.0 (Ar-C-CH ), 137.8 (C ), 136.8(C
ArC), 128.7 (ArC), 128.6 (ArC), 128.5 (ArC), 128.1 (ArC), 127.7 (ArC), 127.6 (ArC), 125.0 (C=C), 114.6
2
2
=
2
2
3
3
1
3
N-H), 1.56 - 1.66 (m, 2H, CH
2
2
2
). C NMR (CDCl
3
, 100
2
q
q
), 131.7 (ArC), 131.6
(
(
ArC), 112.0 (ArC), 110.3 (ArC), 108.0 (C=C), 102.3 (ArC), 71.0 (CH
2
2
), 70.0 (CH
2
2
), 51.9 (CH
2
2
), 43.9 (CH
2
2
),
),
36.1 (CH
3
), 30.1 (CH
), 26.6 (CH
2
), 29.5 (CH
2
), 29.4 (CH
2
), 29.1 (CH
2
), 28.8 (CH
), 28.6 (CH
), 27.6 (CH
), 27.3 (CH
+
26.8 (CH
2
2
), 9.3 (CH
3
). HRMS (EI): Found 617.4100 (M+H) , C42
H
53
N
2
2
O requires 617.4107.
General method for debenzylation of indoles (16)-(20)
The protected aminated indole (16)-(20), (0.3 mmol) was dissolved in MeOH:EtOAc (1:4) (20 mL)
and transferred to a 3-neck flask. To the flask was added Pd(OH) /C (20%, 20 mg) and the mixture
was placed under a H atmosphere. The hydrogenation reaction was monitored via TLC until the
2
2
starting material had disappeared (10 – 60 min). The reaction mixture was filtered through celite and
the solvent was evaporated under reduced pressure to obtain a brown oil. The material was purified
via flash chromatography to affoed the desired product.
2
-(4-Hydroxyphenyl)-3-methyl-1-(4-(methylamino)butyl)-1H-indol-5-ol (21)
As per general method above, the protected aminated indole (16) (150 mg, 0.30 mmol) and
Pd(OH) /C (20 mg, 20%) were reacted under a hydrogenation atmosphere for 30 min. The material
was purified via flash chromatography on silica gel (DCM:MeOH, gradient 40:1 to 30:1) to afford the
2
-
1
product as an off white solid (77 mg, 80%); Mp 61-63 °C. IR : νmax (KBr) cm : 3433.5 (br), 3246.3,
1
2
931.6, 2852.2, 1613.1, 1508.2, 1462.8, 1361.2, 1262.7, 1268.0, 1098.0, 841.4. H NMR (CD
3
OD, 400
MHz) δ 7.20 - 7.27 (m, 3H, Ar-H), 6.91 - 6.97 (m, 2H, Ar-H), 6.89 (s, 1H, Ar-H), 6.74 (dd, J = 2.51, 8.53
Hz, 1H, Ar-H), 4.12 (t, J = 6.53 Hz, 2H, CH ), 2.70 (t, J = 7.53 Hz, 2H, CH ), 2.54 (s, 3H, NCH ), 2.13 (s,
H, CH ), 1.54 - 1.65 (m, 2H, CH ), 1.29 - 1.40 (m, 2H, CH ), (N-H and OH - not observed). C NMR
CD OD, 100 MHz) δ 156.7 (C-OH), 149.7 (C-OH), 137.6 (C ), 131.0 (C ), 130.9 (ArC), 130.8 (ArC),
29.1 (C ), 122.8 (C=C), 114.6 (ArC), 110.3 (ArC), 109.2 (ArC), 106.8 (C=C), 102.0 (ArC), 48.0 (CH
2
2
3
1
3
3
3
2
2
(
3
q
q
1
q
2
)
28

+
41.9 (CH
2
), 31.5 (CH
3
), 26.0 (CH
2
), 22.6 (CH
2
), 7.6 (CH
3
). HRMS (EI): Found 325.1916 (M+H) ,
C
20
H
25
N
2
O
2
requires 325.1916.
2-(4-Hydroxyphenyl)-3-methyl-1-(5-(methylamino)pentyl)-1H-indol-5-ol (22)
As per general method above, the protected aminated indole (17) (150 mg, 0.30 mmol) and
Pd(OH) /C (20 mg, 20%) were reacted under a hydrogen atmosphere for 30 min. The material was
purified via flash chromatography over silica gel (DCM:MeOH, gradient 40:1 to 30:1) to afford the
2
-
1
product as an off white solid (69 mg, 71%); Mp 63-65 °C. IR : νmax (KBr) cm : 3433.5 (br.), 2925.5,
1
2
7
2
852.2, 1615.6, 1507.5, 1461.9, 1365.9, 1199.8, 1695.5, 838.29. H NMR (CD
3
OD, 400 MHz) δ 7.17 -
),
), 1.41 - 1.51
OD, 100 MHz) δ 156.6
), 123.0 (C=C), 114.5 (ArC), 110.1 (ArC), 109.1 (ArC),
), 28.9 (CH ), 28.5 (CH ), 24.8 (CH ), 22.6 (CH ), 7.6
requires 339.2073.
.26 (m, 3H, Ar-H), 6.85 - 6.97 (m, 3H, Ar-H), 6.69 - 6.75 (m, 1H, Ar-H), 4.06 (t, J = 7.00 Hz, 2H, CH
.79 (t, J = 7.00 Hz, 2H, CH ), 2.61 (s, 3H, NCH ), 2.13 (s, 3H, CH ), 1.52 - 1.62 (m, 2H, CH
), 1.09 - 1.17 (m, 2H, CH ), (N-H and O-H - observed). C NMR (CD
C-OH), 149.6 (C-OH), 137.6, 131.0 (ArC), 128.9(C
2
2
3
3
2
1
3
(m, 2H, CH
2
2
3
(
q
106.5 (C=C), 102.0 (ArC), 65.0 (CH
2
), 31.6 (CH
3
2
2
2
2
+
(CH
3
). HRMS (EI): Found 339.2069 (M+H) , C21
H
27
N
2
O
2
2-(4-Hydroxyphenyl)-3-methyl-1-(6-(methylamino)-n-hexanoyl)-1H-indol-5-ol (23)
As per general method above, the protected aminated indole (18) (150 mg, 0.30 mmol) and Pd(OH)
C (20%, 20 mg) were reacted under a hydrogen atmosphere for 30 min. The material was purified
via flash chromatography over silica gel (DCM:MeOH, gradient 40:1 to 30:1) to afford the product as
2
/
4
2
-1
an off white solid (75 mg, 76%); Mp 75-76 °C . IR : νmax (KBr) cm : 3433.5 (br), 2927.1, 2852.2,
1
1
7
6
2
613.8, 1560.0, 1508.1, 1461.4, 1366.7, 1168.8, 1100.4, 840.8. H NMR (CD
3
OD, 400 MHz) δ 7.13 -
.22 (m, 3H, Ar-H), 6.89 - 6.96 (m, 2H, Ar-H), 6.87 (s, 1H, Ar-H), 6.67 - 6.73 (m, 1H, Ar-H), 4.03 (t, J =
.78 Hz, 2H, CH ), 2.79 (t, J = 7.53 Hz, 2H, CH ), 2.62 (s, 3H, CH ), 2.11 (s, 3H, CH ), 1.42 - 1.58 (m, 4H,
× CH ), 1.01 - 1.21 (m, 4H, 2 × CH ). C NMR (CD OD, 100 MHz) δ 158.6 (C-OH), 151.5 (C-OH), 139.7
), 133.0 (ArC), 130.9 (C ), 125.1 (C=C), 116.4 (ArC), 112.1 (ArC), 111.3 (ArC), 108.4 (C=C), 104.0
ArC), 50.4 (CH ), 44.4 (CH
2
2
3
3
1
3
2
2
3
(
(
(
C
q
q
2
2
), 33.7 (CH
3
2
), 30.7 (CH
2
), 27.2 (CH
2
), 27.1 (CH
2
), 27.0 (CH
2
), 9.6 (CH
3
). HRMS
+
EI): Found 353.3327 (M+H) , C22
H
29
N
O
2
requires 353.2229.
2
-(4-Hydroxyphenyl)-3-methyl-1-(8-(methylamino)octyl)-1H-indol-5-ol (24)
As per general method above, the protected aminated indole (19) (150 mg, 0.30 mmol) and
Pd(OH) /C (20 mg, 20%) were reacted under a hydrogen atmosphere for 30 min. The material was
purified via flash chromatography over silica gel (DCM:MeOH, gradient 40:1 to 30:1) to afford the
2
-
1
product as an off white solid (92 mg, 91%); Mp 71-72 °C. IR : νmax (KBr) cm : 3428.6 (br.), 3241.4,
1
2
931.0, 2852.2, 1614.2, 1508.4, 1462.8, 1380.6, 1260.2, 1147.4, 1093.0, 839.0. H NMR (CD
3
OD, 400
MHz) δ 7.12 - 7.24 (m, 3H, Ar-H), 6.90 - 6.98 (m, 2H, Ar-H), 6.89 (s, 1H, Ar-H), 6.68 - 6.75 (m, 1H, Ar-
29