Novel styryl-indoles as small molecule inhibitors of 25-hydroxyvitamin D-24-hydroxylase (CYP24A1): Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2014.09.035

The synthesis of a series of imidazole styrylindoles and sulfonyl styrylindoles derivatives is described. Evaluation of binding affinity and inhibitory activity against CYP24A1 identified the imidazole styrylindoles as potent inhibitors with activity greater or comparable with the standard ketoconazole.Flexible alignment and docking studies of the inhibitors in the CYP24A1 enzyme active site confirmed that complete occupation of the vitamin D access tunnel is essential to inhibitory activity, allowing exposure to multiple hydrophobic binding interactions and optimal conformation for the interaction of the imidazole nitrogen lone pair and the active site haem.

Full text of DOI:10.1016/j.ejmech.2014.09.035

European Journal of Medicinal Chemistry 87 (2014) 39e51
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel styryl-indoles as small molecule inhibitors of 25-
hydroxyvitamin D-24-hydroxylase (CYP24A1): Synthesis and
biological evaluation
,
Salvatore Ferla a, Mohamed S. Gomaa a 1, Andrea Brancale a, Jinge Zhu b,
,
*
Justin T. Ochalek b, Hector F. DeLuca b, Claire Simons a
a Medicinal Chemistry, School of Pharmacy & Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK
b Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a series of imidazole styrylindoles and sulfonyl styrylindoles derivatives is described.
Evaluation of binding affinity and inhibitory activity against CYP24A1 identified the imidazole styr-
ylindoles as potent inhibitors with activity greater or comparable with the standard ketoconazole.
Flexible alignment and docking studies of the inhibitors in the CYP24A1 enzyme active site confirmed
that complete occupation of the vitamin D access tunnel is essential to inhibitory activity, allowing
exposure to multiple hydrophobic binding interactions and optimal conformation for the interaction of
the imidazole nitrogen lone pair and the active site haem.
Received 23 June 2014
Received in revised form
9 September 2014
Accepted 10 September 2014
Available online 16 September 2014
Keywords:
© 2014 Elsevier Masson SAS. All rights reserved.
Imidazole styrylindoles
Sulfonyl styrylindoles
CYP24A1
Vitamin D
Molecular modelling
1. Introduction
calcitriol and therefore the antiproliferative or prodifferentiation
effects [13e16].
The most biologically active form of vitamin D3, 1
a
,25-
Inhibition of CYP24A1 is therefore an attractive strategy to
enhance endogenous circulating calcitriol and/or increase the half-
life of exogenously administered calcitriol or vitamin D therapeutic.
Studies using the non-specific CYP inhibitors, ketoconazole or
liarozole in combination with calcitriol in prostate and breast
cancer cell lines resulted in increased half-life of calcitriol and
enhanced antiproliferative effect [17,18]. Similarly studies using
CYP24A1 inhibitors, the isoflavone genistein and a novel tetralone
derivative, greatly enhanced the apoptotic and differentiation effect
of calcitriol in prostate cancer cell lines [19,20].
dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol, 1) has an important
role in the regulation of cell proliferation and differentiation [1] in
addition to the key role of calcitriol in the regulation of calcium
homeostasis and bone metabolism [2,3]. Serum levels of calcitriol
have been associated with the prognosis of several cancers, with
higher serum levels associated with improved prognosis [4e6] and
lower levels associated with an increased risk and poorer prognosis
[7e10]. The serum level of calcitriol is tightly regulated in a short
feedback loop by two cytochrome P450 enzymes, the 1a-hydrox-
ylase CYP27B1 that promotes synthesis of calcitriol and the 24-
hydroxylase CYP24A1 that inactivates calcitriol by metabolism
[11,12]. Lower calcitriol levels is directly linked to high expression of
CYP24A1 with increased CYP24A1 levels resulting in increased
metabolism of calcitriol significantly reducing the circulating
Our previous research on non-vitamin D CYP24A1 inhibitors
[20e22] indicated the importance of a hydrophobic group or chain
to allow complete fill of the hydrophobic channel of the CYP24A1
enzyme active site. In the development of the azole inhibitors, a
styryl group was found to be optimal for inhibitory activity [21]
(Fig. 1) resulting in compounds with activity comparable with the
standard inhibitor ketoconazole (Fig. 1). Combining the styryl and
azole haem binding group with an indole group, to mimic the
calcitriol C/D ring unit, resulted in the design of the herein
described indole imidazole series. In addition a series of indole
* Corresponding author.
E-mail address: SimonsC@Cardiff.ac.uk (C. Simons).
URL: http://www.cardiff.ac.uk/phrmy/staffinfo/CS/homepage/CS.html
Present address: Pharmaceutical Chemistry Department, Faculty of Pharmacy,
1
Suez Canal University, Egypt.
http://dx.doi.org/10.1016/j.ejmech.2014.09.035
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

40
S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
Fig. 1. Structures of natural CYP24A1 substrate calcitriol, standard inhibitor for comparison ketoconazole and designed 4- and 5-styrylindole derivatives.
sulphonyl derivatives was designed based on the very the prom-
ising vitamin D sulphonate derivatives [23] (Fig. 1).
and the resulting indole alkylazide derivatives (14 and 15) reduced
via a Staudinger reaction [25] using triphenylphosphine to give the
indole alylamines (16 and 17). The final step to prepare the indole
sulfonamides (18 and 19) involved reaction of the amines (16 and
17) with p-toluenesulfonyl chloride at 0 ꢀC for 30 min in the
presence of triethylamine.
2. Results
2.1. Chemistry
To include a compound for testing with a sulfinate bond, 1-(3-
benzenesulfonyl-propyl)-4-styryl-1H-indole (20) was prepared on
reaction with bromide (4a, n ¼ 3, R ¼ H) with benzene sulfinic acid
sodium salt following described methodology (Scheme 2) [26,27].
The indole sulfonates (23) were prepared in three steps from the
indole styrene (3) with introduction of the lateral alcohol chain
achieved on reaction of 3 with sodium hydride and ethyl-3-
bromopropionate and subsequent reduction of the ester (21) to
the alcohol (22) with LiAlH4. The final sulfonates (23) were readily
prepared by reaction of the alcohols (22) with p-toluenesulfonyl
chloride at room temperature for 24 h in the presence of 4-
dimethylaminopyridine (DMAP) (Scheme 3).
The styrenes (3 and 9) were prepared by Heck reaction of either
styrene (2a) or 1,3-dimethoxy-5-vinylbenzene (2b) with the bro-
moindole (1 or 8) using paladium (II) acetate catalyst, tri(o-tolyl-
phosphine) (TOP) as ligand and triethylamine as basic medium at
110 ꢀC in a sealed tube overnight (Scheme 1) [24]. The indole
bromoalkyl derivatives with either a lateral three carbon chain (4,
10) or lateral four carbon chain (5, 11) were readily obtained after
treatment of the indole (3 or 9) with NaH as base and addition of an
excess of dibromoalkane followed by column chromatography
purification. The imidazole products (6, 7, 12 and 13) were obtained
in good yield on reacting the indole bromoalkyl derivatives (4, 5, 10
and 11) with sodium hydride in DMF followed by reaction with
imidazole at 45 ꢀC overnight (Scheme 1).
2.2. CYP24A1 enzyme inhibition
Preparation of the indole sulfonamides (18 and 19) commenced
from the indole bromoalkyl derivatives (4 and 5) (Scheme 2). The
bromide was displaced with azide on treatment with sodium azide
The CYP24A1 enzymatic assay followed methodology previ-
ously described [23], in short human CYP24A1 with an N-terminal

S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
41
Scheme 1. Reagents and conditions: (i) Pd(OAc)2, ToP, Et3N, 110 ꢀC, 20 h (ii) NaH, DMF, 1,3-dibromopropane or 1,4-dibromobutane, 5 min (iii) NaH, DMF, imidazole, 45 ꢀC, 1 h then r.t.
overnight.
Scheme 2. Reagents and Conditions: (i) NaN3, DMF, r.t., 5 h (ii) (a) PPh3, THF, r.t. 1 h (b) H2O, 60 ꢀC, 2 h (iii) TsCl, Et3N, CH2Cl2, 0 ꢀC, 30 min (iv) C6H5SO2Na, DMF, r.t., 24 h.
fusion to maltose binding protein (MBP) was overexpressed in
Escherichia coli and purified to homogeneity. The hydrolase enzyme
was reconstituted in vitro, and the resulting cell-free assay system
applied in the screening of the compounds to measure Ki and IC50
(Table 1).
activity to the 5-styryl derivates (12 and 13). Substitution of the
styryl aromatic ring (R ¼ H or OCH3) did not affect the inhibitory
activity.
The sulfonyl and sulfinyl derivatives (18, 19, 20 and 23) dis-
played reduced CYP24A1 inhibitory activity compared with the
standard ketoconazole (Table 1). In this series the substitution of
the styryl aromatic ring (R ¼ H or OCH3) did have an effect with the
unsubstituted derivatives (18a, 19a, 20 and 23a) displaying greater
inhibitory activity than the dimethoxy derivatives (18b, 19b and
23b).
The imidazole styryl indole derivatives 6, 7, 12 and 13 were all
potent inhibitors of CYP24A1 (Ki 14e37 nM, IC50 0.19e0.52
with activity comparable with or greater than the standard keto-
conazole (Ki 33 nM, IC50 0.47 M) (Table 1). The 4-styryl derivatives
mM)
m
with the 4-carbon lateral chain (7) were more active than the 3-
carbon chain derivatives (6), which were of equivalent inhibitory

42
S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
Scheme 3. Reagents and conditions: (i) NaH, DMF, ethyl-3-bromopropionate, 0 ꢀC to r.t., 2 h (ii) LiAlH4, THF, 0 ꢀC 1 h then r.t. 4 h (iii) TsCl, DMAP, CH2Cl2, pyridine, r.t., 24 h.
Table 1
IC50 and Ki data for indole imidazole and sulfonyl derivatives vs CYP24A1.
lateral chain and position of the styrene on the indole ring are
important to activity. The 3 carbon derivatives have lower activity,
whereas the introduction of an extra carbon on the lateral chain
results in an increase in inhibitory activity up to 2-fold. Substituting
a 5-indole as the central core instead of the 4-indole results in a
decrease in inhibitory activity.
In order to provide a rational explanation for the observed
enzymatic data, flexible alignment and molecular docking studies
were performed using MOE 2010 [29] and LeadIT2.1.2 [30]. All the
molecular modelling studies were performed using a CYP24A1
homology model built using a rat CYP24A1 crystal structure [31] as
a template and the amino acid sequence of the human isoform
following a previous published homology model technique [32].
After performing a molecular docking of the most promising in-
Compound
IC50
(
m
M)
Ki (mM)
6a
6b
7a
7b
12
13
18a
18b
19a
19b
20
23a
23b
Ketoconazole
0.36
0.44
0.20
0.19
0.52
0.52
4.8
0.026 0.004
0.031 0.004
0.014 0.002
0.014 0.002
0.037 0.004
0.037 0.002
0.34 0.02
0.73 0.18
0.23 0.06
0.74 0.03
0.54 0.07
0.64 0.09
1.19 0.43
0.035
10.4
3.3
10.5
7.6
9.0
16.9
0.47
hibitor (24: IC50 ¼ 0.11
mM; Ki ¼ 7.8 nM; CYP24A1/CYP27B1
Table 2
2.3. CYP27B1 enzyme inhibition and selectivity
IC50 and Ki of indole imidazole derivatives vs CYP24A1 and CYP27B1 and selectivity
data.
Inhibition assay of CYP27B1 was performed in a similar way to
the CYP24A1 assay as previously described [23], and the resulting
cell-free assay system applied in the screening of the compounds to
measure Ki and IC50 (Table 2). Compounds with good binding and
inhibitory activity against CYP24A1 also displayed similar proper-
ties against CYP27B1. A small selectivity was observed for CYP24A1
with the indole imidazole with a lateral four carbon chain (7a)
displaying the best selectivity (selectivity CYP27B1/CYP24A1 ¼1.8)
comparable with the standard, ketoconazole (selectivity CYP27B1/
CYP24A1 ¼ 1.7), although two of the derivatives in this series (6b
and 13) showed a small selectivity for CYP27B1.
Compound
CYP24A1
IC50 Ki (
CYP27B1
IC50 Ki (
Selectivity
m
M)
m
M)
Ki (CYP27B1/
CYP24A1)
(m
M)
(
m
M)
6a
6b
7a
7b
12
13
0.36
0.44
0.20
0.19
0.52
0.52
0.026 0.004 0.21
0.031 0.004 0.14
0.014 0.002 0.15
0.014 0.002 0.10
0.037 0.004 0.28
0.037 0.002 0.15
0.034 0.005 1.3
0.022 0.004 0.71
0.025 0.003 1.8
0.017 0.004 1.2
0.046 0.004 1.2
0.024 0.005 0.65
Ketoconazole 0.47
0.033
0.36
0.058
1.7
selectivity ¼ 3.3) from the previously reported (E)-N-(2-(1H-imi-
dazol-1-yl)-2-phenylethyl)-4-styrylbenzamides [28] in the enzyme
active site (Fig. 2), its active binding conformation pose was kept
rigid while a flexible alignment was executed using a database of
the six indole-imidazole derivatives.
Flexible alignment is a technique which allows determination of
the best alignment in terms of internal strain and overlap of
different molecular features (e.g. aromatic features, H-bond donors/
3. Discussion
The indole-imidazole derivatives were prepared using a 4-step
synthetic pathway. The six compounds (6a, 6b, 7a, 7b, 12 and 13,
Table 1) showed a very interesting activity in the CYP24A1 enzy-
matic assay, which is comparable to our (E)-N-(2-(1H-imidazol-1-
yl)-2-phenylethyl)-4-styrylbenzamides family previously reported
[28]. The inhibition assay would suggest that the length of the

S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
43
Table 3
acceptors, etc.) [33]. Using this molecular modeling tool the align-
Scoring results from flexible alignment.
ment of the indole-imidazole derivatives with the 24 binding pose
conformation were evaluated and an interesting relation was found
between the flexible alignment results (see Table 3) and the
enzymatic inhibition assay data. The most active compound in the
series, 7b, presents 4 carbons in the lateral chain and the 4-indole
as central core. Fig. 3A, shows the alignment between 7b (yellow)
and 24 (lilac).
Entry
Mol
U (kcal/mol)
F
S
dU
1
2
3
4
5
6
7b
7a
6b
6a
13
12
34.3428
26.3473
32.7516
25.0259
24.4710
23.3696
ꢁ169.6129
ꢁ153.5406
ꢁ148.6960
ꢁ131.2711
ꢁ125.4641
ꢁ119.7692
ꢁ135.2701
ꢁ127.1933
ꢁ115.9444
ꢁ106.2452
ꢁ100.9931
ꢁ96.4266
0.0
0.0
0.0
0.0
0.0
0.0
The imidazole, the 3,5-dimethoxy phenyl ring and the benzyl
portion of the indole core of 7b overlap perfectly with the corre-
sponding groups of 24, with 7b perfectly spatially aligned with our
reference compound. Moreover, as reported in Table 3, this
conformation has the lowest S value (sum of the internal energy of
the ligand [U] and the similarity score [F]) and therefore is the best
in terms of overlapping score. The low S value is due to the good
internal energy of the ligand pose (U value) and the high similarity
score (F value) meaning that the 7b conformation obtained is not
forced in the first case and that the two molecules have a high
shape/functional group similarity in the second case.
Similar observations can be observed for 7a (light blue), a 4-
indole derivative with 4 lateral chain carbons but with no substit-
uent on the aromatic ring. The compound overlaps perfectly with
24 (Fig. 3B) and its S value is the second in the ranking. The absence
of the 3,5-dimethoxy substituents results in a decrease in the
similarity score F with 24 leading to a slightly higher S value.
The 3 carbons derivatives 6b (green) and 6a (white), present a
higher S value as a consequence of the unfavourable visual over-
lapping (Fig. 3C and D respectively).
interaction between its nitrogen lone pair and the haem iron. 7b is
disposed in the active site in the same manner as 24, occupies the
full length of the enzyme channel and forms a H-bond between its
3-methoxy group and Gln82 as found for our reference compound
(Fig. 4).
Docking studies of 6b and 6a, showed the two molecules
orientated in the right position with the imidazole ring perpen-
dicular to the iron, but due to the lack of one carbon on the lateral
chain they are not able to entirely occupy the active site (far from
Gln 82) and their interaction with the CYP24A1 could be less strong
than the 4-carbon lateral chain derivatives influencing their inhi-
bition activity (Fig. 5).
The 5-indole derivatives 13 (green) and 12 (red) showed the
lowest activity among this indole-imidazole series. The flexible
alignment results showed both molecules do not completely
overlap with 24 (Fig. 6) especially in the important imidazole ring
region giving a low S value as a final score.
The docking studies of these two derivatives confirmed our
initial deduction and the possible connection between the spatial
alignment with 24 and the capacity to entirely sit in the enzyme
active site. 13 was not able to occupy the active site due to its
structural flexibility conferred by the presence of the styryl ring in
position 5 instead of 4 (Fig. 7). As previously mentioned, the
inability to accommodate the entire active site can result in a
decrease of CYP24A1 enzymatic inhibition.
The replacement of the imidazole in both the indole-sulfonate
and indole-sulfonamide series resulted in a notable decrease in
inhibitory activity. These results underline the key role of the
imidazole ring in the binding to the CYP24A1 haem group [34,35].
In fact, the higher structural similarity among these three families
Both compounds due to the shorter lateral chain do not
perfectly overlap with 24 especially in the imidazole ring moiety.
The difference in alignment with inhibitor 24 between the 4 carbon
and the 3 carbon derivatives could be a plausible explanation for
the CYP24A1 enzymatic assay results. 7b and 7a overlap better with
24 and this can influence their disposition in the enzyme active site.
The docking studies performed confirmed the importance of the
length of the lateral chain. All the compounds reach the enzyme
active site through the vitamin D access tunnel and are exposed to
multiple hydrophobic residues (Ile131, Trp134, Met246, Phe249,
Thr394, Thr395, Gly499, Tyr500) which have been reported to have
multiple hydrophobic interactions with calcitriol [31]. This dispo-
sition allows the imidazole ring to be optimally positioned for the
Fig. 2. Structure and Docking of 24: the compound occupies the access tunnel with the imidazole ring in an optimal orientation for the interaction with the iron. The hydrogen bond
between the 3-methoxy group and Gln82 stabilises the molecule in a favourable active conformation.

44
S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
Fig. 3. Flexible alignment between 24 (lilac) and (A) 7b (yellow), (B) 7a (light blue), (C) 6b (green), (D) 6a (white). (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
Fig. 4. Docking of 7b: the compound is able to entirely occupy the active site thanks to the 4-carbon lateral chain.
suggests that the decrease in activity in these two last series of
compounds is a consequence of the replacement of the imidazole
by the sulfonate or sulfonamide moiety. The interaction of the
indole-sulfonate and the indole-sulfonamide derivatives with the
haem iron is not strong enough resulting in reduced inhibitory
activity. A rational explanation could be found in the different
availability of the electron lone pair of the imidazole nitrogen in
one case and of the sulfonyl-sulfonamidic oxygen in the other case.
The imidazole lone pair could be more available for the interaction
with the haem group making the imidazole-Fe a strong interaction.
On the other hand, the possibility to share the lone pair between
the two oxygen atoms of the sulfonyl-sulfonamide group makes the
oxygeneFe interaction weaker resulting in the important decrease
of the CYP24A1 inhibitory activity. The use of the more stable sul-
fonamide instead of the sulfonate group gave the desired result
with activity improved by 2e3 fold but not comparable with the
indole-imidazole family.
4. Conclusions
We have developed methods for the preparation of novel styr-
ylindoles (imidazole, sulfonate and sulfonamide series). The thir-
teen new styrylindoles have been assayed for the inhibition of
CYP24A1 and CYP27B1. The imidazole styrylindoles were the most
potent CYP24A1 inhibitors with IC50 values from 0.19 to 0.52
comparable with the CYP inhibitor ketoconazole (IC50 0.47 M). The
sulfonate and sulfonamides displayed only weak CYP24A1 inhibi-
tory activity indicating the importance of the imidazole azole group
in interacting with the active site haem moiety. A four carbon
lateral chain was optimal resulting in complete occupation of the
vitamin D access tunnel and allowing exposure to multiple hy-
drophobic binding interactions and optimal conformation for the
interaction of the imidazole nitrogen lone pair and the active site
haem. A small selectivity for CYP24A1 over CYP27B1 comparable
mM
m
with
ketoconazole
(CYP27B1/CYP24A1
7a
¼
1.8,
ketoconazole ¼ 1.7) was observed.

S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
45
Fig. 5. Docking of 6b and 6a: the compounds are not able to entirely occupy the active site access tunnel as consequence of the shorter lateral chain.
The challenge in the development of CYP24A1 inhibitors is
achieving selectivity between CYP24A1 and CYP27B1. In the
absence of an available crystal structure of CYP27B1 it is essential to
develop a reliable homology model to allow exploitation of any
differences between the active sites of CYP24A1 and CYP27B1,
however with the similarity of the two substrates, 1,25(OH)2D3 and
25(OH) D3 respectively, any differences are likely to be subtle. This
is the current focus of our investigations using the styrylindoles
(e.g. 7a) and styrylbenzamides (e.g. 24) as lead compounds for
further development.
tolylphosphine) (TOP, 0.3 mmol) and palladium (II) acetate
(0.05 mmol) in a sealed glass tube at 110 ꢀC for 5 h. On completion,
water (10 mL) was added. The product was portioned between
CH2Cl2 (100 mL) and water (100 mL), then the organic layer was
dried over MgSO4 and the solvent evaporated under vacuum. The
product was isolated by flash column chromatography (petroleum
ether-EtOAc 100:0 v/v increasing to 90:10 v/v).
5.1.1.1. 4-Styryl-1H-indole (3a, R ¼ H). Obtained in 80% yield as a
green solid. M.p. 126e128 ꢀC. TLC (7:3 Petroleum ether/EtOAc,
Rf ¼ 0.57). 1H NMR (CDCl3):
d
8.30 (bs, 1H, NH), 7.63 (d, J ¼ 8.2 Hz,
2H, Ar), 7.59 (d, J ¼ 16.3 Hz, 1H, alkene), 7.33 (d, J ¼ 7.4 Hz, 2H, Ar),
7.30 (d, J ¼ 16.1 Hz, 1H, alkene), 7.20e7.26 (m, 4H, Ar and indole),
5. Experimental
7.03 (m, 1H, Ar), 6.72 (s, 1H, indole). 13C NMR (CDCl3):
d 136.8, 135.1,
5.1. Materials and methods
128.6, 125.1 (C, Ar), 128.2, 127.5, 126.3, 126.2, 125.4, 121.6, 119.3,
113.6, 109.5, 100.0 (CH, Ar and alkene). Anal. Calcd for C16H13
0.1H2O (221.08692): C, 86.92; H, 6.02; N, 6.34. Found: C, 87.10; H,
5.86; N, 6.40.
N
1,25(OH)2D3 and 25(OH)D3 were purchased from SAFC-Pharma
(Madison, WI). Human MBP-CYP24A1, mouse CYP27B1, bovine
adrenodoxin (Adx), and adrenodoxin reductase (AdR) were purified
as described previously [14]. All solvents used for chromatography
were HPLC grade from Fisher Scientific (UK).
5.1.1.2. 4-(3,5-Dimethoxystyryl)-1H-indole (3b,
R
¼
OCH3).
1H and 13C NMR spectra were recorded with a Bruker Avance
DPX500 spectrometer operating at 500 and 125 MHz, with Me4Si as
internal standard. Mass spectra were determined by the EPSRC
mass spectrometry centre (Swansea, UK). Flash column chroma-
tography was performed with silica gel 60 (230e400mesh) (Merck)
and TLC was carried out on precoated silica plates (kiesel gel 60
Obtained in 65% yield as a green-grey solid. M.p. 96e98 ꢀC. TLC (7:3
Petroleum ether/EtOAc, Rf ¼ 0.50). 1H NMR (CDCl3):
d 8.27 (bs, 1H,
NH), 7.53 (d, J ¼ 16.3 Hz, 1H, alkene), 7.39 (d, J ¼ 7.3 Hz, 1H, Ar), 7.36
(d, J ¼ 8.1 Hz, 1H, Ar), 7.31 (t, J ¼ 2.9 Hz, 1H, indole), 7.23e7.28 (m,
2H, Ar and alkene), 6.87 (m, 1H, indole), 6.78 (d, J ¼ 2.2 Hz, 2H, Ar),
6.45 (t, J ¼ 2.2 Hz, 1H, Ar), 3.88 (s, 6H, 2ꢂ OCH3). 13C NMR (CDCl3):
F254, BDH). Compounds were visualised by illumination under UV
d
161.0 (C),140.0,136.2,129.5,126.3 (C, Ar),129.3,127.9,124.4,122.2,
light (254 nm) or by the use of vanillin stain followed by charring
on a hotplate. Melting points were determined on an electro-
thermal instrument and are uncorrected. All solvents were dried
prior to use and stored over 4 Å molecular sieves, under nitrogen.
All compounds were more than 95% pure.
117.7, 110.6, 104.6, 101.3, 99.7 (CH, Ar and alkene), 55.4 (2ꢂ OCH3).
EI-HRMS (MþH)þ found 280.1332, calculated for C18H18NO2
280.1335.
5.1.1.3. 5-Styryl-1H-indole (9). Obtained in 49% yield as a pale
green solid. M.p. 152e154 ꢀC. TLC (7:3 Petroleum ether/EtOAc,
5.1.1. General method for the preparation of styryl-1H-indoles 3 and
9
Styrene (2a) or 1,3-dimethoxy-5-vinylbenzene (2b) (10 mmol),
4-bromoindole (1) or 5-bromoindole (8) (10 mmol), and triethyl-
amine (20 mmol) were heated in the presence of tri(o-
Rf ¼ 0.62). 1H NMR (CDCl3):
d 8.15 (bs, 1H, NH), 7.80 (s, 1H, Ar), 7.57
(d, J ¼ 7.4 Hz, 2H, Ar), 7.49 (dd, Jx,a ¼ 1.7 Hz, Jx,b ¼ 8.7 Hz, 1H, Arx),
7.37e7.41 (m, 3H, Ar and indole), 7.25e7.31 (m, 2H, Ar and alkene),
7.23 (t, J ¼ 2.8 Hz, 1H, Ar), 7.12 (d, J ¼ 16.3 Hz, 1H, alkene), 6.60 (m,
1H, indole). 13C NMR (CDCl3):
d 138.0, 135.6, 129.6, 128.2 (C, Ar),

46
S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
Fig. 6. Flexible alignment between 24 (lilac) and 13 (green) and 12 (red): no optimal spatial overlapping was found in the imidazole ring region. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
13.0, 128.6, 126.9, 126.2, 126.1, 124.7, 120.7, 119.5, 111.2, 103.0 (CH, Ar
and alkene). Anal. Calcd for C16H13N 0.1H2O (221.08692): C, 86.92;
H, 6.02; N, 6.34. Found: C, 87.07; H, 6.28; N, 6.44.
108.7, 99.9 (CH, Ar and alkene), 45.6, 32.9, 29.9, 28.8 (CH2). EI-HRMS
(MþH)þ found 354.0857, calculated for C20H21BrN 354.0852.
5.1.2.4. 4-(3,5-Dimethoxystyryl)-1-(4-bromobutyl)-1H-indole
(5b,
5.1.2. General method for the preparation of styryl-1-bromoalkyl-
1H-indoles 4, 5, 10 and 11
R ¼ OCH3). Purified by flash column chromatography eluting with
petroleum ether-EtOAc 100:0 v/v increasing to 90:10 v/v. Obtained
in 69% yield as a thick yellow oil. TLC (3:1 Petroleum ether/EtOAc,
The different 4/5-(3,5-unsubstituted/substituted styryl)-1H-
indole (3 or 9) (7 mmol) and NaH (60% dispersion in mineral oil)
(21 mmol) in dry DMF (10 mL) were cooled to 0 ꢀC using an ice bath
and stirred for 5 min. 1,3-Dibromopropane or 1,4-dibromobutane
(70 mmol) was added and the reaction mixture was stirred for
10 min. On completion, the solvent was evaporated under reduced
pressure and the residue was dissolved in CH2Cl2 (100 mL), washed
with water (2 ꢂ 50 mL) and dried over MgSO4. The organic layer
was then evaporated to dryness and the residue was purified by
flash column chromatography to obtain the pure product.
Rf ¼ 0.55). 1H NMR (CDCl3):
d
7.51 (d, J ¼ 16.4 Hz, 1H, alkene), 7.38
(d, J ¼ 7.3 Hz, 1H, Ar), 7.30 (d, J ¼ 8.0 Hz, 1H, Ar), 7.22e7.31 (m, 2H,
Ar and alkene), 7.18 (d, J ¼ 3.2 Hz, 1H, indole), 6.82 (d, J ¼ 3.2 Hz, 1H,
indole), 6.77 (d, J ¼ 2.2 Hz, 2H, Ar), 6.44 (t, J ¼ 2.2 Hz,1H, Ar), 4.21 (t,
J ¼ 6.8 Hz, 2H, CH2), 3.88 (s, 6H, 2ꢂ OCH3), 3.40 (t, J ¼ 6.6 Hz, 2H,
CH2), 2.06 (m, 2H, CH2), 1.89 (m, 2H, CH2). 13C NMR (CDCl3):
d 161.0
(C), 140.0, 136.4, 129.8, 127.0 (C, Ar), 129.3, 127.9, 127.7, 121.8, 117.3,
108.8, 104.6, 99.8, 99.7 (CH, Ar and alkene), 55.4 (2ꢂ OCH3), 45.6,
32.9, 29.9, 28.8 (CH2). EI-HRMS (M)þ found 413.0990, calculated for
C22H24BrNO2 413.0985.
5.1.2.1. 1-(3-Bromopropyl)-4-styryl-1H-indole (4a,
R
¼
H).
Purified by flash column chromatography eluting with petroleum
ether-diethyl ether 100:0 v/v increasing to 99:1 v/v. Obtained in
50% yield as a thick yellow oil. TLC (3:1 Petroleum ether/EtOAc,
5.1.2.5. 1-(3-Bromopropyl)-5-styryl-1H-indole (10). Purified by
flash column chromatography eluting with petroleum ether-
diethyl ether 100:0 v/v increasing to 99:1 v/v. Obtained in 46%
yield as a white solid. M.p. 78e80 ꢀC. TLC (9:1 Petroleum ether/
Rf ¼ 0.73). 1H NMR (CDCl3):
d
7.61 (d, J ¼ 7.3 Hz, 2H, Ar), 7.55 (d,
J ¼ 16.3 Hz, 1H, alkene), 7.39e7.43 (m, 3H, Ar), 7.26e7.36 (m, 4H, Ar
and alkene), 7.25 (d, J ¼ 3.0 Hz, 1H, indole), 6.83 (d, J ¼ 3.0 Hz, 1H,
indole), 4.40 (t, J ¼ 6.4 Hz, 2H, CH2), 3.34 (t, J ¼ 6.1 Hz, 2H, CH2), 2.39
EtOAc, Rf ¼ 0.50). 1H NMR (CDCl3):
d 7.78 (s, 1H, Ar), 7.56 (d,
J ¼ 7.2 Hz, 2H, Ar), 7.50 (dd, Jx,a ¼ 1.6 Hz, Jx,b ¼ 8.6 Hz, 1H, Arx),
7.37e7.42 (m, 3H, Ar), 7.23e7.30 (m, 2H, Ar and alkene), 7.17 (d,
J ¼ 3.1 Hz, 1H, indole), 7.11 (d, J ¼ 16.3 Hz, 1H, alkene), 6.54 (d,
J ¼ 3.2 Hz, 1H, indole), 4.36 (t, J ¼ 6.4 Hz, 2H, CH2), 3.34 (t, J ¼ 6.1 Hz,
(m, 2H, CH2). 13C NMR (CDCl3):
d 137.9, 136.3, 130.1, 129.9 (C, Ar),
129.5, 128.6, 128.3, 127.4, 127.1, 126.5, 121.9, 117.3, 108.7, 100.1 (CH,
Ar and alkene), 44.1, 32.7, 30.4 (CH2). EI-HRMS (MþH)þ found
341.07795, calculated for C19H19BrN 341.07791.
2H, CH2), 2.39 (m, 2H, CH2). 13C NMR (CDCl3):
d 138.0, 135.7, 129.3,
129.1 (C, Ar), 129.9, 128.6, 128.2, 126.9, 126.2, 126.1, 120.3, 119.8,
109.5, 102.0 (CH, Ar and alkene), 44.1, 32.8, 30.4 (CH2). Anal. Calcd
for C19H18BrN (340.256): C, 67.07; H, 5.33; N, 4.11. Found: C, 66.73;
H, 5.75; N, 4.08.
5.1.2.2. 4-(3,5-Dimethoxystyryl)-1-(3-bromopropyl)-1H-indole (4b,
R ¼ OCH3). Purified by flash column chromatography eluting with
petroleum ether-EtOAc 100:0 v/v increasing to 95:5 v/v. Obtained
in 66% yield as a yellow oil. TLC (3:1 Petroleum ether/EtOAc,
5.1.2.6. 1-(4-Bromobutyl)-5-styryl-1H-indole (11). Purified by flash
column chromatography eluting with petroleum ether-EtOAc
100:0 v/v increasing to 98:2 v/v. Obtained in 84% yield as a yel-
low wax. TLC (9:1 Petroleum ether/EtOAc, Rf ¼ 0.44). 1H NMR
Rf ¼ 0.72). 1H NMR (CDCl3):
d
7.53 (d, J ¼ 16.3 Hz, 1H, alkene), 7.39
(d, J ¼ 7.3 Hz, 1H, Ar), 7.34 (d, J ¼ 8.1 Hz, 1H, Ar), 7.23e7.28 (m, 3H,
Ar, indole and alkene), 6.83 (m, 1H, indole), 6.77 (d, J ¼ 2.3 Hz, 2H,
Ar), 6.44 (t, J ¼ 2.3 Hz, 1H, Ar), 4.38 (t, J ¼ 6.3 Hz, 2H, CH2), 3.88 (s,
(CDCl3):
d
7.78 (s, 1H, Ar), 7.56 (d, J ¼ 7.5 Hz, 2H, Ar), 7.50 (dd,
6H, 2ꢂ OCH3), 3.34 (t, J ¼ 6.1 Hz, 2H, CH2), 2.39 (m, 2H, CH2). 13
C
Jx,a ¼ 1.3 Hz, Jx,b ¼ 8.6 Hz, 1H, Arx), 7.38e7.42 (m, 2H, Ar), 7.34 (d,
J ¼ 8.6 Hz, 1H, Ar) 7.24e7.30 (m, 2H, Ar and alkene), 7.12 (d,
J ¼ 16.3 Hz, 1H, alkene), 7.11 (d, J ¼ 3.2 Hz, 1H, indole), 6.54 (d,
J ¼ 3.2 Hz, 1H, indole), 4.19 (t, J ¼ 6.9 Hz, 2H, CH2), 3.41 (t, J ¼ 6.6 Hz,
2H, CH2), 2.00 (m, 2H, CH2), 1.89 (m, 2H, CH2). 13C NMR (CDCl3):
NMR (CDCl3):
d 161.0 (C), 140.0, 136.3, 129.9, 127.1 (C, Ar), 129.4,
128.3, 127.7, 121.9, 117.4, 108.8, 104.6, 101.1, 99.7 (CH, Ar and alkene),
55.4 (2ꢂ OCH3). EI-HRMS (MþH)þ found 400.0911, calculated for
C
21H23BrNO2 400.0907.
d
138.0, 135.8, 129.1, 129.0 (C, Ar), 130.0, 128.6, 128.2, 126.9, 126.2,
126.0, 120.2, 119.8, 109.5, 101.8 (CH, Ar and alkene), 45.6, 32.9, 29.9,
5.1.2.3. 1-(4-Bromobutyl)-4-styryl-1H-indole
(5a,
R
¼
H).
28.8 (CH2). EI-HRMS (MþH)þ found 354.0849, calculated for
Purified by flash column chromatography eluting with petroleum
ether-EtOAc 100:0 v/v increasing to 90:10 v/v. Obtained in 86%
yield as a thick yellow oil. TLC (3:1 Petroleum ether/EtOAc,
C20H21BrN 354.0852.
Rf ¼ 0.73). 1H NMR (CDCl3):
d
7.64 (d, J ¼ 7.4 Hz, 2H, Ar), 7.58 (d,
5.1.3. General method for the preparation of imidazoles 6, 7, 12 and
13
A suspension of NaH (60% dispersion in mineral oil) (6 mmol) in
dry DMF (15 mL) was stirred and heated at 60 ꢀC for 5 min. Imid-
azole (6 mmol) was added and the reaction mixture was heated at
60 ꢀC for 1 h. The reaction mixture was cooled to room temperature
J ¼ 16.2 Hz, 1H, alkene), 7.42e7.45 (m, 3H, Ar), 7.30e7.37 (m, 4H, Ar
and alkene), 7.19 (d, J ¼ 3.3 Hz, 1H, indole), 6.85 (d, J ¼ 3.3 Hz, 1H,
indole), 4.21 (t, J ¼ 6.7 Hz, 2H, CH2), 3.41 (t, J ¼ 6.5 Hz, 2H, CH2), 2.06
(m, 2H, CH2), 1.89 (m, 2H, CH2). 13C NMR (CDCl3):
d 138.0, 136.4,
130.0,127.4 (C, Ar),129.4,128.7,128.1,127.5,127.0,126.5,121.8,117.2,

S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
47
Fig. 7. Docking of 13: the molecule does not occupy entirely the active site due to its structural flexibility conferred by the styryl ring in position 5 of the central indole core.
and the styryl-1-bromoalkyl-1H-indole (4, 5, 10 or 11) (3 mmol)
was added. The reaction mixture was heated at 60 ꢀC overnight and
then hydrolysed by adding H2O (100 mL). The aqueous layer was
extracted with EtOAc (3 ꢂ 100 mL); the organic layers were
collected and dried over MgSO4. The solvent was then evaporated
to dryness and the residue was purified by flash column chroma-
tography (petroleum ether-EtOAc 50:50 v/v then CH2Cl2eMeOH
100:0 v/v increasing to 98:2 v/v) to obtain the pure desired product.
MeOH, Rf ¼ 0.40). 1H NMR (CDCl3):
7.60 (d, J ¼ 7.5 Hz, 2H, Ar), 7.58
d
(d, J ¼ 16.3 Hz, 1H, alkene), 7.38e7.41 (m, 4H, Ar and imid),
7.23e7.33 (m, 5H, Ar, alkene and imid), 7.13 (d, J ¼ 3.2 Hz, 1H,
indole), 7.06 (s, 1H, imid), 6.82 (d, J ¼ 3.2 Hz, 1H, indole), 4.16 (t,
J ¼ 6.5 Hz, 2H, CH2), 3.85 (t, J ¼ 6.9 Hz, 2H, CH2), 1.85 (m, 2H, CH2),
1.78 (m, 2H, CH2). 13C NMR (CDCl3):
d 137.9, 136.3, 130.1, 127.0 (C,
Ar), 137.0, 129.6, 129.5, 128.6, 127.8, 127.4, 127.1, 126.4, 121.9, 118.6,
117.2, 108.6, 100.1 (CH, Ar and alkene), 46.4, 45.9, 28.6, 27.2 (CH2).
EI-HRMS (MþH)þ found 342.1959, calculated for C23H24N3
342.1965.
5.1.3.1. 1-[3-(1H-imidazol-1-yl)propyl]-4-styryl-1H-indole
(6a,
R ¼ H). Obtained in 72% yield as a yellow glue. TLC (9:1 CH2Cl2/
MeOH, Rf ¼ 0.31). 1H NMR (CDCl3):
7.61 (d, J ¼ 7.4 Hz, 2H, Ar), 7.54
d
5.1.3.4. 1-[4-(1H-imidazol-1-yl)butyl]-4-(3,5-dimethoxystyryl)-1H-
(d, J ¼ 16.1 Hz, 1H, alkene), 7.48 (s, 1H, imid), 7.39e7.42 (m, 3H, Ar),
7.24e7.34 (m, 4H, Ar and alkene), 7.19 (d, J ¼ 8.4 Hz, 1H, Ar), 7.14 (s,
1H, imid), 7.12 (d, J ¼ 3.2 Hz, 1H, indole), 6.93 (s, 1H, imid), 6.86 (d,
J ¼ 3.1 Hz, 1H, indole), 4.17 (t, J ¼ 6.6 Hz, 2H, CH2), 3.91 (t, J ¼ 7.0 Hz,
indole (7b, R ¼ OCH3). Obtained in 75% yield as a thick yellow oil.
TLC (9:1 CH2Cl2/MeOH, Rf ¼ 0.48). 1H NMR (CDCl3):
d 7.50 (d,
J ¼ 16.1 Hz, 1H, alkene), 7.37e7.40 (m, 2H, Ar and imida.), 7.23e7.26
(m, 3H, Ar and alkene), 7.13 (d, J ¼ 3.3 Hz, 1H, indole), 7.06 (s, 1H,
imid), 6.83 (s, 1H, imid), 6.81 (d, J ¼ 3.3 Hz, 1H, indole), 6.76 (d,
J ¼ 2.2 Hz, 2H, Ar), 6.44 (t, J ¼ 2.2 Hz, 1H, Ar), 4.17 (t, J ¼ 6.3 Hz, 2H,
CH2), 3.87 (s, 6H, 2ꢂ OCH3), 3.85 (t, J ¼ 6.7 Hz, 2H, CH2), 1.86 (m, 2H,
2H, CH2), 2.39 (m, 2H, CH2). 13C NMR (CDCl3):
d 137.8, 136.3, 130.2,
127.1 (C, Ar),137.2,130.0,129.6,128.6,127.7,127.5,126.5,122.1,118.5,
117.4, 108.5, 102.4, 100.5 (CH, Ar and alkene), 43.8, 43.0, 31.0 (CH2).
EI-HRMS (MþH)þ found 328.1807, calculated for C22H22N3
328.1808.
CH2), 1.78 (m, 2H, CH2). 13C NMR (CDCl3):
d 161.0 (C), 139.9, 136.3,
129.9,127.9 (C, Ar),137.0,129.6,129.4,127.6,127.0,121.9,118.6,117.4,
108.7, 104.6, 100.1, 99.8 (CH, Ar and alkene), 55.4 (2ꢂ OCH3), 46.4,
45.9, 28.6, 27.2 (CH2). EI-HRMS (MþH)þ found 402.2173, calculated
for C25H28N3O2 402.2176.
5.1.3.2. 1-[3-(1H-imidazol-1-yl)propyl]-4-(3,5-dimethoxystyryl)-1H-
indole (6b, R ¼ OCH3). Obtained in 72% yield as a yelloweorange
glue. TLC (3:1 Petroleum ether/EtOAc, Rf ¼ 0.14). 1H NMR (CDCl3):
d
7.51 (d, J ¼ 16.3 Hz, 1H, alkene), 7.48 (s, 1H, imid), 7.4 0 (d,
J ¼ 7.3 Hz, 1H, Ar), 7.19e7.26 (m, 3H, Ar and alkene), 7.14 (s, 1H,
imid), 7.12 (d, J ¼ 3.1 Hz, 1H, indole), 6.93 (s, 1H, imid), 6.85 (d,
J ¼ 3.1 Hz, 1H, indole), 6.77 (d, J ¼ 2.2 Hz, 2H, Ar), 6.44 (t, J ¼ 2.3 Hz,
1H, Ar), 4.17 (t, J ¼ 6.7 Hz, 2H, CH2), 3.91 (t, J ¼ 6.8 Hz, 2H, CH2), 3.88
5.1.3.5. 1-[3-(1H-imidazol-1-yl)propyl]-5-styryl-1H-indole
(12).
Obtained in 83% yield as a white solid. TLC (9:1 CH2Cl2/MeOH,
Rf ¼ 0.61). M.p. 116e118 ꢀC.1H NMR (CDCl3):
d 7.78 (s, 1H, Ar), 7.56
(d, J ¼ 7.3 Hz, 2H, Ar), 7.45e7.52 (m, 2H, Ar), 7.38e7.40 (m, 2H, Ar),
7.22e7.28 (m, 3H, Ar, imid and alkene), 7.15 (s, 1H, imid), 7.11 (d,
J ¼ 16.3 Hz, 1H, alkene), 7.04 (d, J ¼ 3.1 Hz, 1H, indole), 6.92 (s, 1H,
imid), 6.57 (d, J ¼ 3.1 Hz, 1H, indole), 4.13 (t, J ¼ 6.6 Hz, 2H, CH2),
3.90 (t, J ¼ 6.8 Hz, 2H, CH2), 2.39 (m, 2H, CH2). 13C NMR (CDCl3):
(s, 6H, 2ꢂ OCH3), 2.39 (m, 2H, CH2). 13C NMR (CDCl3):
d 161.0 (C),
139.9, 136.3, 130.0, 127.5 (C, Ar), 137.2, 129.6, 128.2, 127.8, 122.1,
118.5, 117.6, 108.6, 106.2, 104.6, 100.5, 99.8 (CH, Ar and alkene), 55.4
(2ꢂ OCH3), 43.8, 43.0, 31.0 (CH2). EI-HRMS (MþH)þ found
388.2018, calculated for C24H26N3O2 388.2020.
d
137.9, 135.6, 129.5, 129.1 (C, Ar), 137.2, 130.0, 128.6, 128.0, 127.0,
126.3, 126.2, 120.6, 119.9, 118.6, 109.4, 102.4 (CH, Ar and alkene),
43.8, 42.9, 31.0 (CH2). Anal. Calcd for C22H21N3 0.5H2O (336.1814): C,
78.59; H, 6.32; N, 12.49. Found: C, 78.56; H, 6.70; N, 12.48.
5.1.3.3. 1-[4-(1H-imidazol-1-yl)butyl]-4-styryl-1H-indole
R ¼ H). Obtained in 67% yield as a thick yellow oil. TLC (9:1 CH2Cl2/
(7a,

48
S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
5.1.3.6. 1-[4-(1H-imidazol-1-yl)butyl]-5-styryl-1H-indole
Obtained in 67% yield as a white solid. TLC (9:1 CH2Cl2/MeOH,
(13).
indole), 6.77 (d, J ¼ 2.2 Hz, 2H, Ar), 6.44 (t, J ¼ 2.2 Hz,1H, Ar), 4.21 (t,
J ¼ 6.8 Hz, 2H, CH2), 3.88 (s, 6H, 2ꢂ OCH3), 3.30 (t, J ¼ 6.7 Hz, 2H,
Rf ¼ 0.50). M.p. 114e116 ꢀC. 1H NMR (CDCl3):
d
7.77 (s, 1H, Ar), 7.55
CH2), 1.97 (m, 2H, CH2), 1.62 (m, 2H, CH2). 13C NMR (CDCl3):
d 161.0
(d, J ¼ 7.5 Hz, 2H, Ar), 7.48 (dd, Jx,a ¼ 1.5 Hz, Jx,b ¼ 8.6 Hz, 1H, Arx),
7.36e7.42 (m, 3H, Ar and alkene), 7.30 (d, J ¼ 8.6 Hz, 1H, Ar),
7.24e7.28 (m, 2H, Ar and imid), 7.10 (d, J ¼ 16.3 Hz, 1H, alkene), 7.07
(s, 1H, imid), 7.05 (d, J ¼ 3.1 Hz, 1H, indole), 6.84 (s, 1H, imid), 6.53
(d, J ¼ 3.1 Hz, 1H, indole), 4.15 (t, J ¼ 6.4 Hz, 2H, CH2), 3.86 (t,
J ¼ 6.9 Hz, 2H, CH2), 1.86 (m, 2H, CH2), 1.79 (m, 2H, CH2). 13C NMR
(C), 140.0, 136.4, 129.8, 127.0 (C, Ar), 129.3, 127.9, 121.8, 117.3, 108.8,
104.6, 99.9, 99.7 (CH, Ar and alkene), 55.4 (2ꢂ OCH3), 51.0, 46.0,
27.5, 26.4 (CH2). EI-HRMS (MþH)þ found 377.1975, calculated for
C22H25N4O2 377.1972.
5.1.5. General method for the preparation of amines 16 and 17
To a solution of azide-1H-indole (14 or 15) (2 mmol) in dry THF
(6 mL) was added triphenylphosphine (2.4 mmols) and the reaction
stirred at room temperature until evolution of nitrogen ceased
(about 1 h). H2O (0.4 mL, 22 mmols) was added to the reaction
mixture which was heated at 60 ꢀC for 2 h. The reaction mixture
was concentrated under reduced pressure. The residue was stirred
for 20 min with aqueous 2 M HCl (20 mL) and then extracted with
CH2Cl2 (2 ꢂ 20 mL). To the aqueous layer was added aqueous 1 M
NaOH (50 mL) until basic pH. The aqueous solution was then
extracted with EtOAc (2 ꢂ 100 mL), dried (MgSO4) and the solvent
removed under reduced pressure to afford the pure product as a
glue.
(CDCl3): d 138.0, 135.7, 129.2, 129.0 (C, Ar), 137.0, 129.9, 129.7, 128.6,
128.1, 126.9, 126.2, 126.1, 120.3, 119.9, 118.6, 109.4, 102.0 (CH, Ar and
alkene), 46.4, 45.8, 28.6, 27.2 (CH2). Anal. Calcd for C23H23N3 0.1H2O
(342.99077): C, 80.54; H, 6.81; N, 12.25. Found: C, 80.39; H, 7.39; N,
12.20.
5.1.4. General method for the preparation of azides 14 and 15
Sodium azide (4.5 mmol) was added to a solution of styryl-1-
bromoalkyl-1H-indole (4 or 5) (3 mmol) in DMF (3 mL). The
resulting green reaction mixture was stirred at room temperature
for 5 h then H2O (20 mL) was added and the aqueous layer
extracted with EtOAc (3 ꢂ 20 mL). The combined organic phase was
washed with brine (30 mL), dried over MgSO4 and evaporated in
vacuo to give the pure product as a glue.
5.1.5.1. 3-(4-Styryl-indol-1-yl)propylamine
(16a,
R
¼
H).
Obtained in 77% yield as a yellow glue. TLC (CH2Cl2, Rf ¼ 0.16). 1H
5.1.4.1. 1-(3-Azido-propyl)-4-styryl-1H-indole (14a,
R
¼
H).
NMR (CDCl3):
d
7.61 (d, J ¼ 7.4 Hz, 2H, Ar), 7.55 (d, J ¼ 16.3 Hz, 1H,
Obtained in 82% yield as an orange glue. TLC (4:1 Petroleum ether/
alkene), 7.37e7.43 (m, 3H, Ar), 7.23e7.35 (m, 4H, Ar and alkene),
7.21 (d, J ¼ 3.0 Hz, 1H, indole), 6.82 (d, J ¼ 3.0 Hz, 1H, indole), 4.27 (t,
J ¼ 6.8 Hz, 2H, CH2), 2.75 (t, J ¼ 6.9 Hz, 2H, CH2), 2.02 (m, 2H, CH2),
EtOAc, Rf ¼ 0.50). 1H NMR (CDCl3):
7.62 (d, J ¼ 7.3 Hz, 2H, Ar), 7.55
d
(d, J ¼ 16.4 Hz, 1H, alkene), 7.39e7.43 (m, 3H, Ar), 7.26e7.35 (m, 4H,
Ar and alkene), 7.19 (d, J ¼ 3.1 Hz, 1H, indole), 6.84 (d, J ¼ 3.0 Hz, 1H,
indole), 4.29 (t, J ¼ 6.6 Hz, 2H, CH2), 3.29 (t, J ¼ 6.4 Hz, 2H, CH2), 2.12
1.67 (bs, 2H, eCH2NH2). 13C NMR (CDCl3):
d 138.0, 136.4, 129.9,
126.9 (C, Ar),129.3,128.5,128.1,127.3,127.3,126.4,121.7,117.1,108.8,
99.7 (CH, Ar and alkene), 43.9, 39.3, 33.7 (CH2). EI-HRMS (MþH)þ
found 277.1701, calculated for C19H21N2 277.1699.
(m, 2H, CH2). 13C NMR (CDCl3):
d 137.9, 136.3, 130.1, 127.0 (C, Ar),
129.5, 128.1, 127.4, 127.1, 126.5, 122.0, 117.3, 108.6, 100.2 (CH, Ar and
alkene), 48.3, 43.2, 29.3 (CH2). EI-HRMS (MþH)þ found 303.1608,
calculated for C19H19N4 303.1604.
5.1.5.2. 3-[4-(3,5-Dimethoxystyryl)-indol-1-yl]propylamine
(16b,
R ¼ OCH3). Obtained in 72% yield as a yellow glue. TLC (1:1 Pe-
5.1.4.2. 1-(3-Azido-propyl)-4-(3,5-dimethoxystyryl)-1H-indole (14b,
R ¼ OCH3). Obtained in 72% yield as an orange glue. TLC (4:1 Pe-
troleum ether/EtOAc Rf ¼ 0.15). 1H NMR (CDCl3):
d 7.50 (d,
J ¼ 16.2 Hz, 1H, alkene), 7.36 (d, J ¼ 7.2 Hz, 1H, Ar), 7.21e7.29 (m, 3H,
Ar, and alkene), 7.16 (d, J ¼ 3.2 Hz, 1H, indole), 6.81 (dd, Jx,a ¼ 0.8 Hz,
Jx,b ¼ 2.6 Hz, 1H, indole) 6.74 (d, J ¼ 2.3 Hz, 2H, Ar), 6.42 (d,
J ¼ 2.3 Hz,1H, Ar), 4.29 (t, J ¼ 6.7 Hz, 2H, CH2), 3.88 (s, 6H, 2ꢂ OCH3),
3.87 (bs, 2H, eCH2NH2), 3.29 (t, J ¼ 6.3 Hz, 2H, CH2), 2.11 (m, 2H,
troleum ether/EtOAc, Rf ¼ 0.50). 1H NMR (CDCl3):
d 7.50 (d,
J ¼ 16.2 Hz, 1H, alkene), 7.36 (d, J ¼ 7.2 Hz, 1H, Ar), 7.21e7.29 (m, 3H,
Ar, and alkene), 7.16 (d, J ¼ 3.2 Hz, 1H, indole), 6.81 (dd, Jx,a ¼ 0.8 Hz,
Jx,b ¼ 2.6 Hz, 1H, indole) 6.75 (d, J ¼ 2.2 Hz, 2H, Ar), 6.42 (t,
J ¼ 2.2 Hz,1H, Ar), 4.23 (t, J ¼ 6.7 Hz, 2H, CH2), 3.85 (s, 6H, 2ꢂ OCH3),
3.24 (t, J ¼ 6.2 Hz, 2H, CH2), 2.07 (m, 2H, CH2). 13C NMR (CDCl3):
CH2). 13C NMR (CDCl3):
d 161.0 (C), 140.0, 136.3, 129.8, 127.0 (C, Ar),
129.4, 128.1, 127.7, 121.9, 117.4, 108.7, 104.6, 100.2, 99.7 (CH, Ar and
alkene), 55.4 (2ꢂ OCH3), 48.3, 43.2, 29.3 (CH2). EI-HRMS (MþH)þ
found 337.1911, calculated for C21H25N2O2 337.1911.
d
161.0 (C), 139.9, 136.3, 129.7, 127.0 (C, Ar), 129.3, 128.2, 127.6, 121.9,
117.4, 108.8, 104.6, 100.4, 99.7 (CH, Ar and alkene), 55.3 (2ꢂ OCH3),
48.3, 43.1, 29.3 (CH2). EI-HRMS (MþH)þ found 363.1817, calculated
for C21H23BrN4O2 363.1816.
5.1.5.3. 4-(4-Styryl-indol-1-yl)butylamine
(17a,
R
¼
H).
Obtained in 60% yield as a yellow glue. TLC (CH2Cl2, Rf ¼ 0.16). 1H
5.1.4.3. 1-(4-Azido-butyl)-4-styryl-1H-indole (15a,
R
¼
H).
NMR (CDCl3):
d
7.61 (d, J ¼ 7.3 Hz, 2H, Ar), 7.44e7.58 (m, 4H, Ar and
Obtained in 86% yield as a yellow glue. TLC (4:1 Petroleum ether/
alkene), 7.26e7.35 (m, 4H, Ar and alkene), 7.19 (d, J ¼ 3.1 Hz, 1H,
indole), 6.81 (t, J ¼ 3.1 Hz, 1H, indole), 4.18 (t, J ¼ 6.9 Hz, 2H, CH2),
2.72 (t, J ¼ 7.0 Hz, 2H, CH2), 1.88e1.94 (m, 4H, CH2, eCH2NH2), 1.49
EtOAc, Rf ¼ 0.57). 1H NMR (CDCl3):
7.63 (d, J ¼ 7.4 Hz, 2H, Ar), 7.58
d
(d, J ¼ 16.2 Hz, 1H, alkene), 7.41e7.44 (m, 3H, Ar), 7.35 (d,
J ¼ 16.2 Hz, 1H, alkene), 7.26e7.33 (m, 3H, Ar), 7.10 (d, J ¼ 3.1 Hz, 1H,
indole), 6.85 (d, J ¼ 3.1 Hz, 1H, indole), 4.18 (t, J ¼ 6.97 Hz, 2H, CH2),
(m, 2H, CH2). 13C NMR (CDCl3):
d 138.0, 136.4, 129.9, 127.3 (C, Ar),
129.2,128.7, 128.4, 128.0,127.0, 126.4,121.6, 117.1, 108.8, 99.6 (CH, Ar
and alkene), 46.4, 41.7, 30.9, 27.2 (CH2). EI-HRMS (MþH)þ found
291.1857, calculated for C20H23N2 291.1856.
3.28 (t, J ¼ 6.8 Hz, 2H, CH2), 1.95 (m, 2H, CH2), 1.61 (m, 2H, CH2). 13
C
NMR (CDCl3):
d 138.0, 136.4, 130.0, 127.4 (C, Ar), 129.3, 128.7, 128.1,
127.5, 127.0, 126.5, 121.5, 117.2, 108.8, 99.3 (CH, Ar and alkene), 45.6,
32.9, 29.9, 28.8 (CH2). EI-HRMS (MþH)þ found 317.1764, calculated
for C20H21N4 317.1761.
5.1.5.4. 4-[4-(3,5-Dimethoxystyryl)-indol-1-yl]butylamine
(17b,
R ¼ OCH3). Obtained in 79% yield as a yellow glue. TLC (1:1 Pe-
troleum ether/EtOAc Rf ¼ 0.16). 1H NMR (CDCl3):
d 7.51 (d,
5.1.4.4. 1-(4-Azido-butyl)-4-(3,5-dimethoxystyryl)-1H-indole (15b,
R ¼ OCH3). Obtained in 57% yield as an orange glue. TLC (4:1 Pe-
J ¼ 16.2 Hz, 1H, alkene), 7.38 (d, J ¼ 7.1 Hz,1H, Ar), 7.23e7.30 (m, 3H,
Ar and alkene), 7.17 (d, J ¼ 3.3 Hz, 1H, indole), 6.76 (d, J ¼ 3.1 Hz, 1H,
indole), 6.74 (d, J ¼ 2.2 Hz, 2H, Ar), 6.42 (d, J ¼ 2.3 Hz, 1H, Ar), 4.24
(m, 2H, CH2), 3.86 (s, 6H, 2ꢂ OCH3), 3.36 (bs, 4H, CH2, eCH2NH2),
troleum ether/EtOAc, Rf ¼ 0.34). 1H NMR (CDCl3):
d 7.51 (d,
J ¼ 16.2 Hz,1H, alkene), 7.38 (d, J ¼ 7.1 Hz, 1H, Ar), 7.23e7.30 (m, 3H,
Ar and alkene), 7.18 (d, J ¼ 3.3 Hz, 1H, indole), 6.82 (d, J ¼ 3.1 Hz, 1H,
1.90 (m, 2H, CH2), 1.70 (m, 2H, CH2). 13C NMR (CDCl3):
d 161.0 (C),

S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
49
133.8, 133.6, 132.0,131.9 (C, Ar),129.2,128.1, 127.7, 121.7,117.3, 108.9,
104.6, 99.8, 99.7 (CH, Ar and alkene), 55.4 (2ꢂ OCH3), 45.9, 30.3,
28.9, 23.7 (CH2). EI-HRMS (MþH)þ found 351.2070, calculated for
5.1.6.4. N-(4-{4-[2-(3,5-dimethoxy-phenyl)-vinyl]-indol-1yl}-butyl)-
4-methyl-benzenesulfonamide (19b, R ¼ OCH3). A yellow glue was
obtained in 20% yield after purification by flash column chroma-
tography (dichloromethane). TLC (dichloromethane) Rf 0.24. 1H
C
22H27N2O2 351.2067.
NMR (CDCl3):
d
7.71 (d, J ¼ 8.3 Hz, 2H, alkene), 7.50 (d, J ¼ 16.3 Hz,
1H, alkene), 7.36 (m, 1H, Ar), 7.22e7.29 (m, 5H, Ar and alkene), 7.11
(d, J ¼ 3.3 Hz, 1H, indole), 6.78 (d, J ¼ 3.3 Hz, 1H, indole), 6.76 (d,
J ¼ 2.1 Hz, 2H, Ar), 6.44 (t, J ¼ 2.1 Hz, 1H, Ar), 4.47 (t, J ¼ 6.4 Hz, 1H,
eCH2NHeSO2PhCH3), 4.12 (t, J ¼ 6.6 Hz, 2H, CH2), 3.87 (s, 6H,
2ꢂ OCH3), 2.92 (dd, Jx,a ¼ 6.6 Hz, Jx,b ¼ 6.8 Hz, 2H, CH2), 2.42 (s, 1H,
5.1.6. General method for the preparation of benzenesulfonamides
18 and 19
Toluenesulfonyl chloride (1 mmol) and indole amine (16 or 17)
(1.1 mmol) were dissolved in dry CH2Cl2 (10 mL) under nitrogen
atmosphere. The mixture was treated dropwise with triethylamine
(2.2 mmol) under ice-cooling and then stirred for 30 min at 0 ꢀC. On
completion, the reaction mixture was washed with aqueous 2 M
HCl (2 ꢂ 30 mL) and with brine (25 mL). Evaporation of the organic
solvent after drying over MgSO4 gave the crude compound. The
product was isolated by flash column chromatography or recrys-
tallisation giving the desired compound.
CH3), 1.86 (m, 2H, CH2), 1.47 (m, 2H, CH2). 13C NMR (CDCl3):
d 161.0
(C), 143.4, 140.0, 136.8, 136.3, 129.8, 126.9 (C, Ar), 129.7, 129.4, 128.0,
127.7, 127.0, 121.7, 117.3, 108.8, 104.6, 99.8, 99.7 (CH, Ar and alkene),
55.4 (2ꢂ OCH3), 45.8, 42.6, 27.1, 27.0 (CH2), 21.5 (CH3). EI-HRMS
(MþH)þ found 505.2150, calculated for C29H33N2O4S 505.2156.
5.1.7. Synthesis of (E)-3-(4-styryl-1H-indol-1-yl)propyl
benzenesulfinate (20)
5.1.6.1. 4-Methyl-N-[3-(4-styryl-indol-1-yl)propyl]benzenesulfona-
mide (18a, R ¼ H). Obtained in 39% yield as a white solid after
recrystallisation from ethyl acetate/hexane. TLC (CH2Cl2, Rf ¼ 0.30).
To a solution of 1-(3-bromopropyl)-4-styryl-1H-indole (4a)
(2.9 mmol) in DMF (10 mL) was added benzene sulfinic acid sodium
salt (2.9 mmol) and the reaction stirred at room temperature for
24 h. The reaction mixture was then evaporated in vacuo and the
residue was dissolved in CH2Cl2 (100 mL), extracted with H2O
(2 ꢂ 50 mL) and dried over MgSO4. The organic layer was reduced
in vacuo and the crude compound was purified by column chro-
matography (petroleum ether-EtOAc 100:0 v/v increasing to
70:30 v/v) giving a yellow oil in 53% yield. TLC (petroleum ether e
M.p. 130e132 ꢀC. 1H NMR (CDCl3):
d
7.69 (d, J ¼ 8.2 Hz, 2H, Ar), 7.60
(d, J ¼ 7.4 Hz, 2H, Ar), 7.53 (d, J ¼ 16.3 Hz, 1H, alkene), 7.38e7.42 (m,
3H, Ar), 7.28e7.33 (m, 4H, Ar and alkene), 7.21e7.24 (m, 2H, Ar), 7.15
(d, J ¼ 3.1 Hz, 1H, indole), 6.79 (d, J ¼ 3.1 Hz, 1H, indole), 4.45 (t,
J ¼ 6.5 Hz, 1H, eCH2NHeSO2PhCH3), 4.23 (t, J ¼ 6.5 Hz, 2H, CH2),
2.92 (q, J ¼ 6.5 Hz, 2H, CH2), 2.43 (s, 1H, CH3), 2.04 (m, 2H, CH2). 13
C
NMR (CDCl3):
d
143.6, 137.9, 136.4, 136.2, 130.1, 127.6 (C, Ar), 129.8,
EtOAc 7:3 v/v) Rf 0.29. 1H NMR (CDCl3):
d
7.87 (d, J ¼ 8.2 Hz, 2H, Ar),
129.4, 128.6, 128.1, 127.4, 127.1, 127.0, 126.5, 121.9, 117.2, 108.6, 100.1
(CH, Ar and alkene), 43.3, 40.5, 30.0 (CH2), 21.5 (CH3). EI-HRMS
(MþH) þ found 431.1783, calculated for C26H27N2O2S 431.1788.
7.63 (m, 1H, Ar), 7.60 (d, J ¼ 7.5 Hz, 2H, Ar), 7.51e7.55 (m, 3H, Ar and
alkene), 7.38e7.41 (m, 3H, Ar), 7.27e7.32 (m, 2H, Ar and alkene),
7.22 (m, 2H, Ar), 7.15 (d, J ¼ 3.2 Hz, 1H, indole), 6.81 (d, J ¼ 3.2 Hz,
1H, indole), 4.32 (t, J ¼ 6.7 Hz, 2H, CH2), 3.01 (t, J ¼ 6.9 Hz, 2H, CH2),
2.32 (m, 2H, CH2). 13C NMR (CDCl3):
d 138.9, 137.8, 136.3, 130.1,
5.1.6.2. N-(3-{4-[2-(3,5-dimethoxy-phenyl)-vinyl]-indol-1yl}-pro-
pyl)-4-methyl-benzenesulfonamide (18b, R ¼ OCH3). A white solid
was obtained in 45% yield after purification by flash column chro-
matography (dichloromethane). TLC (CH2Cl2, Rf ¼ 0.40). M.p.
129.3, 127.0 (C, Ar), 133.8, 129.5, 128.6, 127.9, 127.7, 127.4, 127.1,
126.5, 122.1, 117.3, 108.6, 100.5 (CH, Ar and alkene), 60.3, 53.0, 44.3
(CH2). EI-HRMS (MþH)þ found 402.1523, calculated for C25H24NO2S
402.1522.
142e144 ꢀC. 1H NMR (CDCl3):
d
7.68 (d, J ¼ 8.3 Hz, 2H, Ar), 7.50 (d,
J ¼ 16.4 Hz, 1H, alkene), 7.37 (m, 1H, Ar), 7.28 (d, J ¼ 8.3 Hz, 2H, Ar),
7.24 (d, J ¼ 16.4 Hz, 1H, alkene) 7.22 (d, J ¼ 4.4 Hz, 2H, Ar), 7.10 (d,
J ¼ 3.1 Hz, 1H, indole), 6.79 (d, J ¼ 3.3 Hz, 1H, indole), 6.76 (d,
J ¼ 2.2 Hz, 2H, Ar), 6.44 (t, J ¼ 2.2 Hz, 1H, Ar), 4.44 (t, J ¼ 6.4 Hz, 1H,
eCH2NHeSO2PhCH3), 4.23 (t, J ¼ 6.6 Hz, 2H, CH2), 3.88 (s, 6H,
2ꢂ OCH3), 2.92 (q, J ¼ 6.5 Hz, 2H, CH2), 2.43 (s, 1H, CH3), 2.04 (m,
5.1.8. General method for the preparation of the ethyl esters (21)
The styryl indole derivatives (3) (4.3 mmol) and NaH (60%
dispersion in mineral oil) (12.9 mmol) in dry DMF (20 mL) were
cooled to 0 ꢀC using an ice bath and stirred for 5 min. Ethyl-3-
bromopropionate (12.9 mmol) was added and the reaction
mixture was stirred for 2 h at room temperature. On completion,
the solvent was evaporated under reduced pressure and the residue
was dissolved in CH2Cl2 (100 mL), washed with H2O (2 ꢂ 50 mL)
and dried over MgSO4. The organic layer was then evaporated to
dryness.
2H, CH2). 13C NMR (CDCl3):
d 161.0 (C), 143.6, 139.9, 136.4, 136.2,
129.8,127.6 (C, Ar),129.8,129.4,128.1,127.6,127.0,121.9,117.4,108.7,
104.6, 100.1, 99.8 (CH, Ar and alkene), 55.4 (2ꢂ OCH3), 43.3, 40.5,
30.0 (CH2), 2.5 (CH3). EI-HRMS (MþH)þ found 491.1989, calculated
for C28H31N2O4S 491.1999.
5.1.8.1. 3-(4-Styryl-indol-1-yl)-propionic acid ethyl ester (21a,
R ¼ H). A yellow oil was obtained in 60% yield after purification by
flash column chromatography (petroleum ether then CH2Cl2). TLC
5.1.6.3. 4-Methyl-N-[4-(4-styryl-indol-1-yl)butyl]benzenesulfona-
mide (19a, R ¼ H). A yellow glue was obtained in 33% yield after
purification by flash column chromatography (petroleum ether e
ethyl acetate 100:0 v/v increasing to 80:20 v/v). TLC (dichloro-
(CH2Cl2) Rf 0.83. 1H NMR (CDCl3):
d
7.64 (d, J ¼ 7.7 Hz, 2H, Ar), 7.59
(d, J ¼ 16.2 Hz, 1H, alkene), 7.42e7.45 (m, 3H, Ar), 7.29e7.37 (m, 4H,
Ar and alkene), 7.26 (d, J ¼ 3.2 Hz, 1H, indole), 6.85 (d, J ¼ 3.2 Hz,1H,
indole), 4.59 (t, J ¼ 6.9 Hz, 2H, CH2), 4.18 (q, J ¼ 7.2 Hz, 2H, CH2), 3.34
(t, J ¼ 6.1 Hz, 2H, CH2), 2.87 (t, J ¼ 6.9 Hz, 2H, CH2), 1.26 (t, J ¼ 7.2 Hz,
methane) Rf 0.40. 1H NMR (CDCl3):
d
7.71 (d, J ¼ 8.2 Hz, 2H, Ar), 7.61
(d, J ¼ 7.4 Hz, 2H, Ar), 7.53 (d, J ¼ 16.3 Hz, 1H, alkene), 7.36e7.42 (m,
3H, Ar), 7.28e7.33 (m, 4H, Ar and alkene), 7.23 (d, J ¼ 4.4 Hz, 2H, Ar),
7.11 (d, J ¼ 3.1 Hz, 1H, indole), 6.79 (d, J ¼ 3.2 Hz, 1H, indole), 4.43 (t,
J ¼ 6.3 Hz, 1H, eCH2NHeSO2PhCH3), 4.13 (t, J ¼ 6.5 Hz, 2H, CH2),
2.92 (q, J ¼ 6.7 Hz, 2H, CH2), 2.43 (s, 1H, CH3), 1.87 (m, 2H, CH2), 1.47
3H, CH3). 13C NMR (CDCl3):
d 171.1 (C), 137.9, 136.1, 130.0, 127.1 (C,
Ar), 129.4, 128.6, 128.2, 127.4, 127.1, 126.5, 121.9, 117.5, 117.3, 108.5,
101.3, 100.2 (CH, Ar and alkene), 60.9, 42.0, 35.0 (CH2), 14.1 (CH3).
EI-HRMS (MþH)þ found 320.1648, calculated for C21H22NO2
320.1645.
(m, 2H, CH2). 13C NMR (CDCl3):
d 143.5, 137.9, 136.8, 136.3, 129.9,
126.9 (C, Ar), 129.7, 129.3, 128.6, 127.9, 127.4, 127.2, 127.0, 126.4,
121.8, 117.1, 108.7, 99.8 (CH, Ar and alkene), 45.8, 42.6, 27.1, 27.1
(CH2), 21.5 (CH3). EI- HRMS (MþH)þ found 445.1944, calculated for
5.1.8.2. 3-{4-[2-(3,5-Dimethoxy-phenyl)-vinyl]-indol-1-yl}-propionic
acid ethyl ester (21b, R ¼ OCH3). A yellow oil was obtained in 53%
C
27H29N2O2S 445.1944.

50
S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
yield after purification by flash column chromatography (petro-
leum ether then CH2Cl2). TLC (CH2Cl2) Rf 0.71. 1H NMR (CDCl3):
5.1.10.1. Toluene-4-sulfonic acid 3-(4-styryl-indol-1-yl)-propyl ester
(23a, R ¼ H). A yellow glue was obtained in 53% yield after puri-
fication by flash column chromatography (petroleum ether e ethyl
acetate 100:0 v/v increasing to 90:10 v/v). TLC (petroleum ether e
d
7.52 (d, J ¼ 16.3 Hz, 1H, alkene), 7.39 (d, J ¼ 7.2 Hz, 1H, Ar),
7.30e7.36 (m, 2H, Ar and alkene), 7.27 (d, J ¼ 7.2 Hz, 1H, Ar), 7.24 (d,
J ¼ 3.2 Hz, 1H, indole), 6.81 (d, J ¼ 3.1 Hz, 1H, indole), 6.78 (d,
J ¼ 2.0 Hz, 2H, Ar), 6.45 (t, J ¼ 2.0 Hz, 1H, Ar), 4.50 (t, J ¼ 6.9 Hz, 2H,
CH2), 4.15 (q, J ¼ 7.2 Hz, 2H, CH2), 3.88 (s, 6H, 2ꢂ OCH3), 2.86 (t,
J ¼ 6.9 Hz, 2H, CH2), 1.24 (t, J ¼ 7.2 Hz, 3H, CH3). 13C NMR (CDCl3):
ethyl acetate 8:2 v/v) Rf 0.48. 1H NMR (CDCl3):
d
7.78 (d, J ¼ 8.2 Hz,
2H, Ar), 7.60 (d, J ¼ 7.1 Hz, 2H, Ar), 7.52 (d, J ¼ 16.4 Hz, 1H, alkene),
7.29e7.42 (m, 7H, Ar and alkene), 7.20e7.24 (m, 3H, Ar), 7.04 (d,
J ¼ 3.2 Hz, 1H, indole), 6.74 (d, J ¼ 3.2 Hz, 1H, indole), 4.26 (t,
J ¼ 6.5 Hz, 2H, CH2), 3.99 (t, J ¼ 5.5 Hz, 2H, CH2), 2.47 (s, 3H, CH3),
d
171.1 (C), 161.0 (C), 140.0, 136.1, 129.8, 127.1 (C, Ar), 129.3, 128.3,
127.7, 121.9, 117.4, 108.7, 104.6, 100.1, 99.7 (CH, Ar and alkene), 55.4
(2ꢂ OCH3), 60.9, 42.0, 35.0 (CH2), 14.1 (CH3). EI-HRMS (MþH)þ
found 380.1859, calculated for C23H26NO4 380.1856.
2.20 (m, 2H, CH2). 13C NMR (CDCl3):
d 145.0, 139.9, 136.1, 132.7,
130.0, 127.0 (C, Ar), 129.9, 129.4, 128.6, 128.1, 127.9, 127.4, 127.1,
126.4, 121.9, 117.2, 108.5, 100.1 (CH, Ar and alkene), 67.0, 42.2, 29.4
(CH2), 21.6 (CH3). EI-HRMS (MþH)þ found 432.1627, calculated for
C26H26NO3S 432.1628.
5.1.9. General method for the preparation of 3- styryl indole
propanol derivatives (22)
5.1.10.2. Toluene-4-sulfonic acid 3-{4-[2-(3,5-dimethoxy-phenyl)-vi-
nyl]-indol-1-yl}-propyl ester (23b, R ¼ OCH3). A yelloweorange glue
was obtained in 50% yield after purification by flash column chro-
matography (petroleum ether e ethyl acetate 100:0 v/v increasing
to 80:20 v/v). TLC (petroleum ether e ethyl acetate 1:1 v/v) Rf 0.57.
A solution of 3-ethyl styryl indole propionate derivative (21)
(2.1 mmol) in dry THF (12 mL) under nitrogen atmosphere was
cooled to 0 ꢀC. LiAlH4 (1 M solution in THF) (8.4 mmol) was added
dropwise via syringe. The yellow reaction mixture was stirred at
0
ꢀC for 1 h, then at room temperature for 4 h. The reaction was
1H NMR (CDCl3):
d
7.78 (d, J ¼ 8.2 Hz, 2H, Ar), 7.48 (d, J ¼ 16.5 Hz,1H,
quenched by the addition of EtOAc (70 mL), the organic layer was
washed with H2O (3 ꢂ 50 mL), dried over MgSO4, and evaporated to
dryness.
alkene), 7.33e7.37 (m, 3H, Ar), 7.21e7.24 (m, 3H, Ar and alkene),
7.04 (d, J ¼ 3.1 Hz, 1H, indole), 6.76 (d, J ¼ 2.2 Hz, 2H, Ar), 6.74 (d,
J ¼ 3.1 Hz, 1H, indole), 6.44 (t, J ¼ 2.2 Hz, 1H, Ar), 4.26 (t, J ¼ 6.6 Hz,
2H, CH2), 3.99 (t, J ¼ 5.7 Hz, 2H, CH2), 3.88 (s, 6H, 2ꢂ OCH3), 2.47 (s,
5.1.9.1. 3-(4-Styryl-indol-1-yl)-propan-1-ol (22a, R ¼ H). A yellow-
eorange wax was obtained in 20% after purification by flash col-
umn chromatography (CH2Cl2). TLC (CH2Cl2) Rf 0.28. 1H NMR
3H, CH3), 2.04 (m, 2H, CH2). 13C NMR (CDCl3):
d 161.0 (C), 145.0,
139.9, 136.1, 132.7, 129.8, 127.6 (C, Ar), 129.9, 129.4, 128.1, 27.9, 127.1,
121.9, 117.4, 108.6, 104.6, 100.1, 99.7 (CH, Ar and alkene), 55.4
(2ꢂ OCH3), 67.0, 42.2, 29.4 (CH2), 21.6 (CH3). EI-HRMS (MþH)þ
found 492.1831, calculated for C28H30NO5S 492.1839.
(CDCl3):
d
7.61 (d, J ¼ 7.3 Hz, 2H, Ar), 7.56 (d, J ¼ 16.5 Hz,1H, alkene),
7.39e7.42 (m, 3H, Ar), 7.35 (d, J ¼ 7.4 Hz, 2H, Ar), 7.24e7.31 (m, 3H,
Ar and alkene), 7.23 (d, J ¼ 3.2 Hz,1H, indole), 6.83 (d, J ¼ 3.2 Hz,1H,
indole), 4.33 (t, J ¼ 6.8 Hz, 2H, CH2), 3.64 (t, J ¼ 5.8 Hz, 2H, CH2), 2.10
5.2. CYP24A1 and CYP27B1 inhibition assay
(m, 2H, CH2),1.37 (bs,1H, eCH2OH). 13C NMR (CDCl3):
d 138.0,136.5,
129.9,127.0 (C, Ar),129.3,128.6,128.2,127.3,127.2,126.4,121.7,117.1,
108.8, 99.8 (CH, Ar and alkene), 59.5, 42.8, 32.6 (CH2). EI-HRMS
(MþH)þ found 278.1539, calculated for C19H20NO 278.1539.
Inhibition of CYP24A1 was carried out as described previously
[23]. Briefly, reaction mixture containing 0.1
mM each of Adx and
AdR, 0.075 M MBP-CYP24A1, 2.5 M 1,25(OH)2D3, varying con-
m
m
centrations of inhibitors, and 0.5 mM NADPH was incubated at
37 ꢀC for 25 min in a buffer of 20 mM Tris (pH 7.5) and 125 mM
NaCl. All inhibitors were dissolved in ethanol (>10 mM) or DMSO
(>50 mM) and further diluted in ethanol to make working stock
(<1 mM). The reaction was extracted with CH2Cl2 and analysed by
HPLC. The IC50 values were determined by fitting the relative ac-
tivity (V/V0) against the inhibitor concentration [I] using the
equation V/V0 ¼ IC50/(IC50 þ [I]), where V and V0 are the reaction
rates in the presence and absence of inhibitors. The assay for each
compound was performed in at least duplicate and in triplicate for
compounds with good inhibitory properties.
5.1.9.2. 3-{4-[2-(3,5-Dimethoxy-phenyl)-vinyl]-indol-1-yl}-propan-
1-ol (22b, R ¼ OCH3). A brownish wax was obtained in 48% yield
after purification by flash column chromatography (CH2Cl2eMeOH
100:0 v/v increasing to 98:2 v/v). TLC (CH2Cl2) Rf 0.20. 1H NMR
(CDCl3):
d
7.52 (d, J ¼ 16.4 Hz, 1H, alkene), 7.34e7.38 (m, 2H, Ar),
7.22e7.27 (m, 3H, Ar, alkene and indole), 6.81 (d, J ¼ 3.1 Hz, 1H,
indole), 6.77 (d, J ¼ 2.1 Hz, 2H, Ar), 6.44 (t, J ¼ 2.1 Hz, 1H, Ar), 4.33 (t,
J ¼ 6.7 Hz, 2H, CH2), 3.88 (s, 6H, 2ꢂ OCH3), 3.63 (t, J ¼ 5.9 Hz, 2H,
CH2), 2.10 (m, 2H, CH2), 1.65 (bs, 1H, eCH2OH). 13C NMR (CDCl3):
d
161.0 (C), 140.0, 136.5, 129.7, 126.9 (C, Ar), 129.2, 128.3, 127.7, 121.7,
117.3, 108.9, 104.6, 99.7, 99.6 (CH, Ar and alkene), 55.4 (2ꢂ OCH3),
59.4, 42.8, 35.6 (CH2). EI-HRMS (MþH)þ found 338.1755, calculated
for C21H24NO3 338.1751.
The Ki values were calculated using equation Ki ¼ IC50/(1 þ [S]/
KM), where [S] is the substrate concentration and KM ¼ 0.19
mM [14].
Inhibition assay of CYP27B1 was performed in a similar way to
that of CYP24A1 as previously described [23]. The concentration of
5.1.10. General method for the synthesis of toluene-sulfonic acid 3-
styryl indole propyl ester derivatives (23)
substrate 25(OH)D3 was 2.5 mM and KM for CYP27B1 was 0.48 mM.
To a cooled (0 ꢀC) solution of 3-styryl indole propanol derivative
(22) (0.9 mmol) and 4-dimethylaminopyridine (0.2 mmol) in dry
CH2Cl2 (7 mL) and pyridine (0.7 mL) under nitrogen atmosphere
was added 4-toluenesulfonyl chloride (2 mmol) portion-wise. The
reaction was stirred at 0 ꢀC for 10 min then stirred for 24 h at room
temperature. On the completion, the reaction mixture was washed
with aqueous saturated NaHCO3 (50 mL) and the organic layer was
separated. The aqueous layer was extracted with CH2Cl2 (50 mL)
and both organic layers were washed with aqueous 1 M HCl
(50 mL). The organic layer was washed with aqueous saturated
NaHCO3 (50 mL) and then dried over MgSO4. The solvent was
evaporated under vacuum.
5.3. Molecular modelling
Docking studies were performed using LeadIT2.1.2 docking
program by BioSolve.IT [29]. The important amino acid residues of
the active pocket (Gln82, Ile131, Trp134, Met246, Ala326, Glu329,
Thr330, Val391, Phe393, Thr394, Ser498, Gly499, Tyr500) [32] were
selected and then the selection was extended to 12 Å in order to
include in the docking site the haem iron region and the access
tunnel to the catalytic site. A ligands database in mol2 format,
prepared using MOE [30], was used as input for the docking cal-
culations. The iron atom of the catalytic site was set as essential
pharmacophoric feature. Ligand docking was performed using the

S. Ferla et al. / European Journal of Medicinal Chemistry 87 (2014) 39e51
51
[11] J.A. Omdahl, E.V. Bobrovnikova, A. Annalora, P. Chen, R. Serda, Expression,
structure-function, and molecular modeling of vitamin D P450s, J. Cell. Bio-
chem. 88 (2003) 356e362.
[12] G. Jones, D.E. Prosser, M. Kaufman, 25-Hydroxy vitamin D-24-hydroxylase
(CYP24A1): its important role in the degradation of vitamin D, Arch. Bio-
chem. Biophys. 523 (2012) 9e18.
default values and no water molecules were considered. Ten output
solutions were obtained from each compound and visual inspec-
tion in MOE was used to identify the interaction between ligand
and protein.
The flexible alignment studies were performed using MOE 2010.
The MOE flexible alignment tool generates different possible con-
formations for each of the different six molecules present in the
input mol2 format database that could overlap with the assigned
template. The quality of the alignment is evaluated by a score which
is a sum of the internal strain of the obtained conformation (the
smaller, the better) and the overlap of molecular features (aromatic
regions, donors/acceptors). MOE, for each alignment performed,
evaluates the average internal energy of the ligands U, the similarity
score F (the lower value is the better two structures overlap) and
the value S (sum of U and F values obtained for each alignment). A
good alignment should present a dU value (the average strain en-
ergy of the molecules in the alignment in kcal/mol) lower than
1 kcal/mol meaning that the obtained conformation are not ener-
getically disadvantaged. In our case, we kept our template rigid and
the flexible alignment of the six compounds was run. The obtained
data with a dU of 0.0 (no energy penalty) were kept and ranked
according to the lowest S value.
[13] M.G. Anderson, M. Nakane, X. Ruan, P.E. Kroeger, J.R. Wu-Wong, Expression of
VDR and CYP24A1 mRNA in human tumors, Cancer Chemother. Pharmacol. 57
(2006) 234e240.
[14] K. Mimori, Y. Tanaka, K. Yoshinaga, T. Masuda, K. Yamashita, M. Okamoto,
H. Inoue, M. Mori, Clinical significance of the overexpression of the candidate
oncogene CYP24 in esophageal cancer, Ann. Oncol. 15 (2004) 236e241.
[15] H.C. Horvath, P. Lakatos, J.P. Kosa, K. Bacsi, K. Borka, G. Bises, T. Nittke,
P.A. Hershberger, G. Speer, E. Kallay, The candidate oncogene CYP24A1: a
potential biomarker for colorectal tumorigenesis, J. Histochem. Cytochem. 58
(2010) 277e285.
[16] G. Chen, S.H. Kim, A.N. King, L. Zhao, R.U. Simpson, P.J. Christensen,
Z.W. Wang, D.G. Thomas, T.J. Giordano, L. Lin, D.E. Brenner, D.G. Beer,
N. Ramnath, CYP24A1 is an independent prognostic marker of survival in
patients with lung adenocarcinoma, Clin. Cancer Res. 17 (2011) 817e826.
[17] J. Zhao, B.K. Tan, S. Marcelis, A. Verstuyf, R. Bouillon, Enhancement of anti-
proliferative activity of 1a, 25-dihydroxyvitamin D-3 (analogs) by cytochrome
P450 enzyme inhibitors is compound- and cell-type specific, J. Steroid Bio-
chem. Mol. Biol. 57 (1996) 197e202.
[18] J.R. Muindi, W.D. Yu, Y. Ma, K.L. Engler, R.X. Kong, D.L. Trump, C.S. Johnson,
CYP24A1 inhibition enhances the antitumor activity of calcitriol, Endocri-
nology 151 (2010) 4301e4312.
[19] A.V. Krishnan, S. Swami, J. Moreno, R.B. Bhattacharyya, D.M. Peehl,
D. Feldman, Potentiation of the growth-inhibitory effects of vitamin D in
prostate cancer by genistein, Nutr. Rev. 65 (2007) S121eS123.
[20] S.W. Yee, M.J. Campbell, C. Simons, Inhibition of Vitamin D3 metabolism en-
hances VDR signalling in androgen-independent prostate cancer cells,
J. Steroid Biochem. Mol. Biol. 98 (2006) 228e235.
Acknowledgements
We acknowledge Cancer Research UK for funding (SF, Grant
Ref. C7735/A10649), the Embassy of the Arab Republic of Egypt
(MSG, PhD scholarship) and the EPSRC Mass Spectrometry Centre,
Swansea, UK for mass spectroscopy data.
[21] A.S. Aboraia, S.W. Yee, M.S. Gomaa, N. Shah, A.C. Robotham, B. Makowski,
D. Prosser, A. Brancale, G. Jones, C. Simons, Synthesis and CYP24A1 inhibitory
activity of N-(2-(1H-imidazol-1-yl)-2-phenylethyl)arylamides, Bioorg. Med.
Chem. 18 (2010) 4939e4946.
[22] A.S. Aboraia, B. Makowski, A. Bahja, D. Prosser, A. Brancalea, G. Jones,
C. Simons, Synthesis and CYP24A1 inhibitory activity of (E)-2-(2-substituted
benzylidene)- and 2-(2-substituted benzyl)-6-methoxy-tetralones, Eur. J.
Med. Chem. 45 (2010) 4427e4434.
References
[23] J. Zhu, R. Barycki, G. Chiellini, H.F. DeLuca, Screening of selective inhibitors of
[1] K.K. Deeb, D.L. Trump, C.S. Johnson, Vitamin D signalling pathways in cancer:
potential for anticancer therapeutics, Nat. Rev. Cancer 7 (2007) 684e700.
[2] H.A. Morris, Vitamin D: a hormone for all seasonsdhow much is enough?
Clin. Biochem. Rev. 26 (2005) 21e32.
[3] H.F. DeLuca, Overview of general physiologic features and functions of vitamin
D, Am. J. Clin. Nutr. 80 (2004) 1689Se1696S.
[4] T.E. Robsahm, S. Tretli, A. Dahlback, J. Moan, Vitamin D3 from sunlight may
improve the prognosis of breast-, colon- and prostate cancer (Norway), Cancer
Causes Control 15 (2004) 149e158.
[5] W. Zhou, R.S. Heist, G. Liu, K. Asomaning, D.S. Neuberg, B.W. Hollis, J.C. Wain,
T.J. Lynch, E. Giovannucci, L. Su, D.C. Christiani, Circulating 25-hydroxyvitamin
D levels predict survival in early- stage non-small-cell lung cancer patients,
J. Clin. Oncol. 25 (2007) 479e485.
[6] S. Swaimi, A.V. Krishnan, D. Feldman, Vitamin D metabolism and action in the
prostate: implications for health and disease, Mol. Cell. Endocrinol. 347 (2011)
61e69.
[7] L. Lipworth, M. Rossi, J.K. McLaughlin, E. Negri, R. Talamini, F. Levi,
S. Franceschi, C. La Vecchia, Dietary vitamin D and cancers of the oral cavity
and esophagus, Ann. Oncol. 20 (2009) 1576e1581.
[8] J. Wactawski-Wende, J.M. Kotchen, G.L. Anderson, A.R. Assaf, R.L. Brunner,
M.J. O'Sullivan, K.L. Margol, J.K. Ockene, L. Phillips, L. Pottern, R.L. Prentice,
J. Robbins, T.E. Rohan, G.E. Sarto, S. Sharma, M.L. Stefanick, L. Van Horn,
R.B. Wallace, E. Whitlock, T. Bassford, S.A.A. Beresford, H.R. Black, D.E. Bonds,
R.G. Brzyski, B. Caan, R.T. Chlebowski, B. Cochrane, C. Garland, M. Gass, J. Hays,
G. Heiss, S.L. Hendrix, B.V. Howard, J. Hsia, F.A. Hubbell, R.D. Jackson,
K.C. Johnson, H. Judd, C.L. Kooperberg, L.H. Kuller, A.Z. LaCroix, D.S. Lane,
R.D. Langer, N.L. Lasser, C.E. Lewis, M.C. Limacher, J.E. Manson, Calcium plus
vitamin D supplementation and the risk of colorectal cancer, N. Engl. J. Med.
354 (2006) 684e696.
1a,25-dihydroxyvitamin D3 24-hydroxylase using recombinant human
enzyme expressed in Escherichia coli, Biochemistry 49 (2010) 10403e10411.
[24] B.A. Patel, C.B. Ziegler, N.A. Cortese, J.E. Plevyak, T.C. Zebovitz, M. Terpko,
R.F. Heck, Palladium-catalyzed vinylic substitution reactions with carboxylic
acid derivatives, J. Org. Chem. 42 (1977) 3903e3907.
[25] F.L. Lin, H.M. Hoyt, H. van Halbeek, R.G. Bergman, C.R. Bertozzi, Mechanistic
investigation of the Staudinger ligation, J. Am. Chem. Soc. 127 (2005)
2686e2695.
[26] L. Munoz, E. Rosa, M.P. Bosch, A. Guerrero, A new practical and efficient
sulfone-mediated synthesis of trifluoromethyl ketones from alkyl and alkenyl
bromides, Tetrahedron Lett. 46 (2005) 3311e3313.
[27] C. Billaud, J.P. Goddard, T.L. Gall, C. Mioskowski, Preparation of alcohols from
sulfones and trialkylboranes, Tetrahedron Lett. 44 (2003) 4451e4454.
[28] S. Ferla, A. Brancale, H.F. DeLuca, J. Zhu, C. Simons, Small moleculae inhibitors
of CYP24A1for treatment of various cancer, Abs. Pap. Am. Chem. Soc. 245
(2013) 73 (MEDI).
[29] http://www.biosolveit.de/.
[30] http://www.chemcomp.com/MOE-Molecular_Operating_Environment.htm.
[31] A.J. Annalora, D.B. Goodin, W.H. Hong, Q. Zhang, E.F. Johnson, C.D. Stout,
Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in
vitamin D metabolism, J. Mol. Biol. 396 (2010) 441e451.
[32] M.S. Gomaa, A. Brancale, C. Simons, Homology model of
1a,25-
dihydroxyvitamin D3 24-hydroxylase cytochrome P450 24A1 9CYP24A1):
active site architecture and ligand binding, J. Steroid Biochem. Mol. Biol. 104
(2007) 53e60.
[33] P. Labute, C. Williams, Flexible alignment of small molecules, J. Med. Chem. 44
(2001) 1483e1490.
[34] I. Schuster, H. Egger, P. Nussbaumer, R.T. Kroemer, Inhibitors of vitamin D
hydroxylase: structure-activity relationship, J. Cell. Biochem. 88 (2003)
372e380.
[35] I. Schuster, H. Egger, D. Bikle, G. Herzig, G.S. Reddy, A. Stuetz, G. Stuetz,
G. Vorisek, Selective inhibitors of vitamin D hydroxylase in human keratino-
cytes, Steroids 66 (2001) 409e422.
[9] M. Rossi, J.K. McLaughlin, P. Lagiou, C. Bosetti, R. Talamini, L. Lipworth,
A. Giacosa, M. Montella, S. Franceschi, E. Negri, C. La Vecchia, Vitamin D intake
and breast cancer risk: a case-control study in Italy, Ann. Oncol. 20 (2009)
374e378.
[10] H. Orell-Kotikangas, U. Schwab, P. Osterlund, K. Saarilahti, O. Makitie,
A.A. Makitie, High prevalence of vitamin D insufficiency in patients with head
and neck cancer at diagnosis, Head Neck 34 (2012) 1450e1455.