Bazedoxifene-scaffold-based mimetics of solomonsterols A and B as novel pregnane x receptor antagonists

Article abstract of DOI:10.1021/jm500351m

Pregnane X receptor (PXR), a member of the NR1I nuclear receptor family, acts as a xenobiotic sensor and a paramount transcriptional regulator of drug-metabolizing enzymes and transporters. The overexpression of PXR in various cancer cells indicates the importance of PXR as a drug target for countering multidrug resistance in anticancer treatments. We describe the discovery of novel bazedoxifene-scaffold-based PXR antagonists inspired by the marine sulfated steroids solomonsterol A and B as natural leads. A luciferase reporter assay on a PXR-transfected HepG2 cell line identified compounds 19-24 as promising PXR antagonists. Further structure-activity relationship studies of the most active PXR antagonist from the series (compound 20, IC₅₀ = 11 μM) revealed the importance of hydroxyl groups as hydrogen-bond donors for PXR antagonistic activity. PXR antagonists 20 and 24 (IC₅₀ = 14 μM), in addition to the downregulation of PXR expression, exhibited inhibition of PXR-induced CYP3A4 expression, which illustrates their potential to suppress PXR-regulated phase-I drug metabolism.

Full text of DOI:10.1021/jm500351m

Subscriber access provided by SCD Université de Rennes1 | SCD de l'Université de Rennes 1
Article
Bazedoxifene scaffold-based mimetics of solomonsterols
A and B as novel pregnane X receptor antagonists
Žiga Hodnik, Lucija Peterlin Maši#, Tihomir Tomaši#, Domen
Smodiš, Claudio D'Amore, Stefano Fiorucci, and Danijel Kikelj
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500351m • Publication Date (Web): 14 May 2014
Downloaded from http://pubs.acs.org on May 18, 2014
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
5x33mm (300 x 300 DPI)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Bazedoxifene scaffoldꢀbased mimetics of
solomonsterols A and B as novel pregnane X
receptor antagonists
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
1
2
Žiga Hodnik, Lucija Peterlin Mašič, Tihomir Tomašić, Domen Smodiš, Claudio D’Amore,
2,
⊥
*,1,⊥
Stefano Fiorucci and Danijel Kikelj
1
University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia
University of Perugia, Dipartimento di Medicina Clinica e Sperimentale, Nuova Facultàdi
2
Medicina e Chirurgia, S. Andrea delle Fratte, 06132 Perugia, Italy
KEYWORDS antagonist, bazedoxifene, mimetic, pregnane X receptor, solomonsterol A,
solomonsterol B
1
ACS Paragon Plus Environment

Page 3 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ABSTRACT
Pregnane X receptor (PXR), a member of the NR1I nuclear receptor family, acts as a
xenobiotic sensor and a paramount transcriptional regulator of drug metabolising enzymes
and transporters. The overexpression of PXR in various cancer cells indicates the importance
of PXR as a drug target for countering multidrug resistance in anticancer treatments. We
describe the discovery of novel, bazedoxifene scaffoldꢀbased PXR antagonists, inspired by the
marine sulphated steroids, solomonsterols A and B, as natural leads. Luciferase reporter assay
on a PXR transfected HepG2 cell line identified compounds 19ꢀ24 as promising PXR
antagonists. Further structureꢀactivity relationship study of the most active PXR antagonist
from the series (compound 20, IC50 = 11 µM) revealed the importance of hydroxy groups as
hydrogen bond donors for PXR antagonistic activity. PXR antagonists 20 and 24 (IC = 14
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
50
µM), in addition to the downꢀregulation of PXR expression, exhibited an inhibition of PXRꢀ
induced CYP3A4 expression, which illustrates their potential to suppress PXRꢀregulated
phase I drug metabolism.
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
INTRODUCTION
Cancer, despite the extent of invested efforts, remains one of the leading fatal diseases
worldwide. By the estimates of the International Agency for Research on Cancer, 14.1 million
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
new cancer cases and 8.2 million cancer deaths were recorded in 2012. Furthermore, the
multidrug resistance (MDR) of cancer cells contributes to the problematic 5% success rate of
2, 3
anticancer agents, and the latest studies point to the pregnane X receptor (PXR) as one of
4
ꢀ7
the key players in the MDR of cancer cells.
PXR, a member of the NR1I nuclear receptor family, is primarily found in the liver, small
intestines and colon, but it is also present in brain, breast, prostate, bone marrow, ovary,
2
placenta and immune cells. It functions as a xenobiotic sensor and as a paramount
8
, 9
transcriptional regulator of drug metabolising enzymes (DMEs) and transporters. Primarily
targeted phase I DMEs include CYP2B6, CYP2C8, CYP2C9, CYP2C19 and CYP3A4,
whereas phase II DMEs regulated by PXR comprise glutathioneꢀSꢀtransferase,
acetyltransferase, methyltransferase, sulfotransferase and uridine 5’ꢀ
8
diphosphoglucuronosyltransferase. PXR also regulates the expression of phase III efflux
ATPꢀbinding cassette (ABC) drug transporters, such as Pꢀglycoprotein (Pgp), breast cancer
9
resistance protein (BCRP) and multiple resistance drug protein (MRP). Considering the
impact of PXR on xenobiotic metabolism and transport, it constitutes a major factor involved
1
0
in drugꢀdrug interactions.
The ability of PXR to initiate the metabolism and efflux of various molecules and its
1
1
4
6
12
13
14
overexpression in breast, prostate, colon, ovarian, osteosarcoma and endometrial
2
cancer cells, makes PXR a promising target for countering MDR in anticancer treatment.
Hence, coꢀtreatment of prostate cancer cell line PCꢀ3 with PXR agonist SR12813 and
anticancer agents paclitaxel or vinblastine resulted in elevated expression of MDR1 and
3
ACS Paragon Plus Environment

Page 5 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4
decreased the efficiency of both chemotherapeutics. On the other hand, the PXR antagonist
sulforaphane increased the chemosensitivity of colorectal cancer cells to irinotecan, which
6
points to the possible use of PXR antagonists to overcome MDR in cancer chemotherapy.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The xenobioticꢀsensing PXR ligand binding domain (LBD) is defined by a spherical and
3
flexible binding pocket with a volume expanding from 1150 to more than 1600 Å , which
15
binds hydrophobic molecules with the ability to form hydrogen bonds. Ligand binding to
PXR is followed by conformational changes of the LBDꢀlocated ligandꢀdependent activation
function 2 (AFꢀ2) and results in dissociation of corepressor proteins. This is followed by the
binding of transcriptional coactivators, which finalises the assembly of proteins that bind to
the promoter regions of its target genes as a heterodimer with retinoid X receptor and initiates
16
the transcription. On the contrary, PXR antagonists disrupt the binding of coactivators to the
16ꢀ18
AFꢀ2 region of the LBD and therefore suppress the transcription of the PXR target genes.
Ecteinascidinꢀ743 (ETꢀ743), which suppresses SR12813ꢀ and paclitaxelꢀinduced PXR
19
activation, stands out as the first reported PXR antagonist. The list of currently known PXR
2
0
antagonists (Figure 1) includes HIV protease inhibitor A792611 and a biguanide antidiabetic
21
agent metformin, both of which act as inhibitors of PXR mediated CYP3A4 expression.
Isothiocyanate sulforaphane inhibits PXR by disrupting the binding of the coactivator to the
AFꢀ2 region of LBD, which also results in PXRꢀcontrolled suppression of CYP3A4
22
transcription. Cytotoxic quinoline alkaloid camptothecin acts as a PXR antagonist by
inhibiting the formation of the PXR complex with the steroid receptor coactivator 1, while
SPB3255 stands out as the most potent PXR antagonist so far, with IC value of 850 nM in
50
16
PXRꢀtransfected HepG2 cells. Undesirable properties of known PXR antagonists, such as
23, 24
25
toxicity,
chemical instability or activities other than inhibition of PXR mediated
2
transcription of genes, are reasons for designing novel and improved PXR antagonists, which
26
is not an easy task due to the promiscuous behaviour of PXR. Hence, in contrast to PXR
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
agonists, the structureꢀbased design of novel PXR antagonists has not been successful so
1
6, 27
far.
Furthermore, recent studies suggest that in addition to the binding pocket, the AFꢀ2
17
helix could serve as a surface for binding the PXR antagonists ketoconazole and
25
coumestrol. The design and discovery of PXR antagonists therefore presents a challenging
task with the demanding combination of fitting a large and flexible binding pocket and/or
binding to a presently unknown part of the AFꢀ2 helix surface.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Structures of currently known PXR antagonists.
We report here the discovery of novel PXR antagonists 20 and 24 resulting from our ligandꢀ
based approach towards the design of PXR modulators using marine sulphated steroids
28
solomonsterols A and B as model compounds. Solomonsterols A and B were isolated from a
marine sponge, Theonella swinhoei, and were shown to act as PXR agonists in a
transactivation experiment on a HepG2 cell line with potency similar to the effective PXR
2
8ꢀ30
agonist rifaximin.
Furthermore, solomonsterol A successfully protected PXRꢀhumanised
mice against the development of clinical signs and symptoms of colitis, apparently by
reducing the generation of TNFα, a paramount cytokine responsible for the development of
31
this disease.
5
ACS Paragon Plus Environment

Page 7 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Our strategy to obtain novel PXR modulators from solomonsterols A and B was to replace
their steroid core with a synthetically more easily accessible steroidomimetic scaffold of
32
bazedoxifene, a selective oestrogen receptor modulator, and to evaluate the effect of
sulphated, as well as nonꢀsulphated, bazedoxifeneꢀbased solomonsterols analogues as
potential PXR modulators because, to our knowledge, no studies involving nonꢀsulphated
solomonsterol analogues as PXR modulators were available. Therefore, we have evaluated (i)
the impact of sulphate esters formation, (ii) the effect of the number of hydroxy/sulphate ester
groups attached to the scaffold and (iii) the length of the alkyl linker at position 5 of the
indole moiety on the modulation of PXR (Figure 2).Contrary to our expectations, the
compounds resulting from this study did not display any PXR agonistic activity, but some
bazedoxifene scaffoldꢀbased solomonsterol analogues, unexpectedly, turned out to be low
micromolar PXR antagonists.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OSO Na
3
n
H
NaO SO
3
solomonsterol A (n = 3)
solomonsterol B (n = 2)
H
H
NaO SO
3
H
steroidomimetic design
O
n
O
n
HN
HN
O
R
O
R
O
O
R
R
-
+
R = H or SO Na
3
R
O
n = 0, 2 or 3
bazedoxifene scaffold based analogues
series I)
bazedoxifene scaffold based analogues
(
(series II)
Figure 2. Design of the bazedoxifene scaffold based PXR modulators by a steroidomimetic
approach using solomonsterols A and B as the model compounds.
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
RESULTS AND DISCUSSION
Design. The design of the bazedoxifene scaffold based solomonsterol analogues was
supported by studying their threeꢀdimensional similarity to solomonsterols A and B using the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
33
ROCS (OpenEye Scientific Software, Inc) method of molecular shape and atom type
comparison. The results of the ROCS overlay show that our designed analogues 27a and 29
can adopt similar conformations with sulphate groups overlapping well with those of
solomonsterol B and solomonsterol A (Figure 3).
Figure 3. Overlay of solomonsterol A (left) and solomonsterol B (right) represented by green
sticks with the corresponding shape in grey and its pharmacophoric features coloured red for
Hꢀbond acceptors, blue for Hꢀbond donors, green for rings and yellow for hydrophobic
groups, with the designed bazedoxifene scaffoldꢀbased analogues 29 (in cyan; left) and 27a
33
(in magenta; right), as obtained by ROCS (Openeye Scientific Software, Inc.).
7
ACS Paragon Plus Environment

Page 9 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The design was further supported by molecular docking experiments to explore whether the
designed solomonsterol analogues can form similar interactions with amino acid residues in
the ligandꢀbinding site of PXR to those of solomonsterols A and B. The docking of
solomonsterol A to the PXR ligandꢀbinding pocket had shown that the steroidal nucleus forms
several hydrophobic interactions with the protein, whereas the sulphate groups were predicted
to form ionic interaction with Lys210 (24ꢀOꢀsulfate) and hydrogen bonds with Ser247 (3ꢀOꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
28
34
sulfate) and His407 (2ꢀOꢀsulfate). Our docking experiments using GOLD were able to
28
reproduce the binding pose of solomonsterol A in the PXR ligandꢀbinding site and also
predicted similar interaction patterns for solomonsterol A and its analogue 29 (Figure 4).
Figure 4. Overlay of the GOLDꢀcalculated binding pose of solomonsterol A (cyan lines) and
its bazedoxifene scaffold based analogue 29 (grey sticks) in the PXR ligandꢀbinding site
(green, PDB entry: 1M13). Ionic interaction and hydrogen bonds between the ligand and PXR
residues (magenta) are presented as dashed black lines. Residues forming hydrophobic
35
interactions are presented as green lines. The figure was prepared by PyMOL.
Chemistry. Compounds 11 and 12 are the key intermediates in the synthesis of the envisaged
bazedoxifeneꢀbased solomonsterol A and B mimetics because they enable selective
introduction of various linkers at position 5 of the indole moiety (Schemes 1 and 2).
According to the synthetic plan, orthogonality between the Oꢀprotecting groups of the
8
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hydroxy propiophenones and of 4ꢀaminophenol was required. This was achieved by benzyl
protection of the hydroxy groups of the propiophenones (compounds 5 and 6) and by
methoxymethyl ether (MOM) protecting group, cleavable by mild acidolysis, for the 4ꢀ
aminophenol hydroxy group (compound 4). Therefore, the synthesis of MOMꢀprotected 4ꢀ
aminophenol 3 and its amine hydrochloride 4 was crucial and was completed by a
straightforward synthetic procedure from phenol 1 (Scheme 1a). First, the benzyloxycarbonylꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
6
protected 4ꢀaminophenol 1 was treated with bromomethyl methyl ether, using sodium
hydride as a base to yield totally protected 4ꢀaminophenol 2. Then, the benzyloxycarbonyl
protecting group was cleaved, using a standard catalytic hydrogenation procedure, to yield
intermediate 3, which was transformed to the amine hydrochloride 4 using the acetyl chloride
driven formation of hydrogen chloride in ethanol. Both intermediates 3 and 4 were later used
37
in a twoꢀstep BischlerꢀMöhlau indole synthesis (Scheme 1b).
38
To obtain key intermediates 11 and 12, the benzylꢀprotected 4ꢀhydroxypropiophenones 5
39
and 6 were first αꢀbrominated with bromine in glacial acetic acid to yield compounds 7 and
8. Unfortunately, in the next step, we were not able to complete the oneꢀstep BischlerꢀMöhlau
indole synthesis from intermediates 3 and 7/8 at 120 °C and 150 ºC using DMF as a solvent.
We assume that the failure was due to the instability of the MOM protecting group in acidic
conditions, but we did not manage to isolate any compounds from the complex reaction
mixture that would confirm our assumptions. Furthermore, the addition of triethylamine to the
reaction mixture containing 3 and 7/8 resulted in the formation of small amounts of the
intermediates 9 and 10, but cyclisation to 11 and 12 was again unsuccessful, which could be
37
explained by the reaction mechanism that proposes amine hydrochloride catalysis.
Consequently, our search for the optimal reaction conditions resulted in a twoꢀstep Bischlerꢀ
Möhlau indole synthesis, using freeꢀamine intermediate 3 and triethylamine in ethanol in the
first step to yield compounds 9 and 10. Finally, to complete the indole synthesis and to obtain
9
ACS Paragon Plus Environment

Page 11 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
key intermediates 11 and 12, in the second step, amine hydrochloride 4 was reacted with
compounds 9/10 in ethanol in a sealed reactor, which resulted in a oneꢀpot indole synthesis
and MOM protecting group cleavage to give compounds 11 and 12 (Scheme 1b).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The synthesis of the solomonsterol mimetics from intermediates 11 and 12 started with the
introduction of a hydroxyalkyl chain (Scheme 2). With 3ꢀbromoꢀ1ꢀpropanol and potassium
a
b
Scheme 1. Reagents and conditions: (a) bromomethyl methyl ether, NaH, DMF, 0 °C, 3 h; (b)
H
2
2 3
, Pd/C, MeOH, rt, 1.5 h; (c) acetyl chloride, EtOH, 0 °C, 2.5 h; (d) Br , CH COOH, rt, 30
min; (e) 3, Et N, EtOH, 80 °C, 16 h; (f) 4, EtOH, 115 °C, 14 h.
3
carbonate intermediates 13 and 14 were obtained in excellent yields, whereas with 2ꢀ
bromoethanol, the reaction did not yield the desired product. Variations of solvents (acetone,
DMF and acetonitrile), reaction temperature, base (cesium carbonate) as well as the catalysts
(potassium iodide and 18ꢀcrownꢀ6) eventually resulted in the decomposition of reactants 11
and 12. Therefore, to obtain twoꢀcarbon chain analogues, 2ꢀbromoethyl acetate was used
instead of 2ꢀbromoethanol, which in a combination with cesium carbonate as a base in
1
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
acetone gave compounds 15 and 16 in moderate yields. The following ester hydrolysis
yielded compounds 17 and 18. Finally, Oꢀdeprotection of intermediates 11ꢀ14, 17 and 18
using standard catalytic hydrogenation procedure yielded hydroxylated solomonsterol A and
B analogues 19ꢀ24. The latter were then treated with chlorosulfonic acid in anhydrous
pyridine with a following sodium hydroxide driven salt formation to give sulphate ester
analogues 25ꢀ29 (Scheme 2). Due to unknown reasons, although using the same procedure,
we have not been able to prepare the sulphate ester analogue 27a from compound 22.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Reagents and conditions: (a) 3ꢀbromoꢀ1ꢀpropanol, K
2
3
CO , acetone, 60 °C, 90 h;
(
b) 2ꢀbromoethyl acetate, Cs CO , acetone, 60 °C, 20 h; (c) NaOH, dioxane/H
2
3
2
O, rt, 5 h; (d)
H , Pd/C, EtOH/THF, rt, 15 h; (e) chlorosulfonic acid, pyridine, rt, 24 h; then NaOH,
2
pyridine, rt, 24 h.
In an attempt to further explore the structureꢀactivity relationship, Oꢀmethyl and Nꢀalkyl
derivatives of compound 20, the most active antagonist from the first series of solomonsterol
1
1
ACS Paragon Plus Environment

Page 13 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
analogues, were prepared (Figure 5, Scheme 3; compounds 34ꢀ38). The indole hydroxy group
of intermediate 12 was Oꢀbenzylated to produce compound 30, which enabled the selective
introduction of various substituents on the indole nitrogen to give compounds 31, 32 and 33
with Nꢀmethyl, ꢀpropyl and ꢀbenzyl substituents. They were Oꢀdeprotected using a standard
catalytic hydrogenation procedure to give Nꢀsubstituted analogues 34, 35 and 36. To
determine the necessity of hydroxy groups as hydrogen bond donors for PXR antagonism,
compound 20 was Oꢀmethylated with methyl iodide and cesium carbonate as a base to
produce analogue 37. Finally, to explore the effect of the absence of hydrogen bond donors on
PXR antagonism, analogue 38 was synthesised by the Nꢀmethylation of 37, using methyl
iodide and sodium hydride as a base (Scheme 3).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Modification of compound 20, the most active PXR antagonist from the first series
of solomonsterol analogues, for the SAR study demonstrating the importance of Hꢀbond
donors for PXR antagonistic activity.
1
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Reagents and conditions: (a) benzyl bromide, K
compound 31: methyl iodide, NaH, DMF, rt, 1.5 h; for compound 32: 1ꢀbromopropane, NaH,
DMF, rt, 1.5 h; for compound 33: benzyl bromide, NaH, DMF, rt, 1.5 h; (c) H , Pd/C,
, acetone, 60 °C, 2.5 h; (e) methyl iodide,
2 3
CO , acetone, 60 °C, 19 h; (b) for
2
2 3
EtOH/THF, rt, 15 h; (d) methyl iodide, Cs CO
NaH, DMF, rt, 1 h.
Biological evaluation. Because solomonsterols A and B have been shown to act as potent
28
PXR agonists, our bazedoxifene scaffoldꢀbased solomonsterol A and B analogues 19ꢀ29
were first evaluated for PXR agonistic activity in a luciferase reporter assay in a human
hepatocarcinoma cell line (HepG2), transiently transfected with pSG5ꢀPXR, pSG5ꢀRXR,
pCMVꢀβgalactosidase and p(CYP3A4)ꢀTKꢀLuc vectors (c.f. Experimental section). Despite
their similar molecular shape with that of solomonsterols A and B (c.f. Figure 3), compounds
19ꢀ29, at a 10 µM concentration, did not exhibit any significant PXR activation compared to
that of the positive controls solomonsterol A (10 µM) and rifaximin (10 µM), demonstrating
that they are devoid of PXR agonistic activity (Figure 6a). The series of analogues 19ꢀ29 (50
µM) was further tested for PXR antagonism in a luciferase reporter assay on a PXR
transfected HepG2 cell line in the presence of rifaximin (10 µM). The results shown in Figure
6b demonstrate that, whereas the sulphate esters 25ꢀ29 displayed no or only weak inhibition
of rifaximin induced PXR transactivation, the hydroxy analogues 19ꢀ24 exhibited evident
1
3
ACS Paragon Plus Environment

Page 15 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
antagonistic effects, with compound 20 being the most active PXR antagonist among
compounds described here.
A
7
5
4
3
2
.0
.0
.0
.0
×
10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
*
7
7
7
7
×
10
*
×
10
×
10
×
1.0 10
0
NT
R
SOL.A 19
20
21
22
23
24
25
26
27
28
29
37
B
7
×
3.0 10
*
7
7
7
7
6
×
2.5 10
*
2
1
1
5
.0
.5
.0
.0
×
10
×
10
#
#
×
10
#
#
#
#
#
×
10
#
0
NT
R
19
20
21
22
23
24
25
26
27
28
29
37
R
Figure 6. The luciferase reporter assay performed in HepG2 cells transiently transfected with
pSG5ꢀPXR, pSG5ꢀRXR, pCMVꢀβgal and p(cyp3a4)TKLUC vectors and stimulated for 18 h
with (A) rifaximin (R), solomonsterol A (SOLꢀA), 20-29 and 37 (10 ꢁM) or (B) with 10 ꢁM
rifaximin alone (R), or in combination with 20-29 and 37 (50 ꢁM). * p < 0.05 vs. not treated
(NT); # p < 0.05 vs. R.
1
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The sulphate esters 28 and 29 possess a three carbon atom linker and displayed a weak
inhibition of rifaximin induced PXR transactivation compared to sulphate ester 27 with a two
carbon linker and compounds 25 and 26 with sulphate ester groups bound directly to the
bazedoxifene scaffold, which did not exhibit any PXR antagonistic activity. From the
obtained results, a connection between the number of sulphate ester groups and the inhibition
of rifaximin induced PXR transactivation was not evident. In general, the potential low
cellular membrane permeability of sulphated solomonsterol analogues 25ꢀ29 could present the
reason for no or weak PXRꢀmodulatory activities. In the series of hydroxylated analogues 19ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
24 possessing PXR antagonistic activity, preliminary SAR implied that, in the case of
analogues with two hydroxy groups (compounds 19, 21 and 23), the presence and length of
the linker at position 5 of the indole moiety does not impact the affinity towards PXR. These
three analogues displayed similar antagonistic potencies that are, however, weaker compared
to those of the corresponding analogues with three hydroxy groups (compounds 20, 22 and
24). Among the latter, compound 20 stands out as the most active PXR antagonist in the
series.
The antagonistic effect of the two most active PXR antagonists among compounds described
here, 20 and 24, was quantified by concentrationꢀresponse transactivation experiments
performed on HepG2 cells. The concentrationꢀresponse curves revealed the concentrationꢀ
dependent effects of antagonists 20 and 24, with IC50 values of 11 µM and 14 µM,
respectively (Figure 7). Since potency of compound 20 correlated with the potency of PXR
25
antagonist coumestrol (IC = 12 µM), we evaluated the threeꢀdimensional similarity of both
50
compounds, using the ROCS method of molecular shape and atom type comparison (Figure
). Results of overlay revealed better shape similarity (Tanimoto shape score = 0.81) than
8
pharmacophoric feature similarity (Tanimoto colour score = 0.39) of coumestrol and
1
5
ACS Paragon Plus Environment

Page 17 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
antagonist 20. The best overlay can be observed between the phenol rings of both compounds,
indicating the equal distance between hydroxy groups of coumestrol and compound 20.
A
7
6
6
6
1
.0
×
10
*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
110
100
7.5
×
10
#
90
80
70
60
50
40
×
5.0 10
2
0 IC50: 11.39ꢀM
#
2.5
×
10
#
30
2
0
0
0
1
0
NT
R
10
25
50
20 (ꢀM)
0
0ꢀ5
10ꢀ4
1
R
[20], M
B
*
0
0
0
6
6
6
6
×10
1
10
#
100
90
80
70
60
50
40
4×10
#
2
4 IC50: 14 ꢀM
#
2×10
3
2
1
0
0
0
0
0
NT
R
10
25
50
24 (ꢀM)
ꢀ 5
ꢀ 4
0
10
10
R
[24], M
Figure 7. Concentrationꢀresponse curves for compounds 20 (A) and 24 (B). HepG2 cells were
transfected as described above and stimulated with rifaximin (R) alone or in combination with
increasing concentration of 20 or 24 (10, 25 and 50 ꢁM) to determine the IC50 value. * p <
0.05 vs. NT; # p < 0.05 vs. R.
With an aim to study the impact of the indole Nꢀsubstitution for the antagonistic effect of the
most active compound 20 from the first series of solomonsterol analogues, compounds
possessing methyl (34), propyl (35) or benzyl substituents (36) at position 1 of the indole core
have also been biologically evaluated. The necessity of hydrogen bond donors for antagonistic
1
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
activity of the compound 20 was evaluated by testing the Oꢀmethylated analogues 37 and 38
(Figure 5, Scheme 3). A transactivation experiment in the presence of rifaximin on HepG2
cells indicated PXR antagonistic activity of analogues 34ꢀ38, but the morphological analysis
revealed that at a dose of 50 µM, Nꢀalkylated compounds 34, 35, 36 and 38 were cytotoxic.
Cytotoxicity was not observed for antagonist 37, which exhibited weaker potency compared
to its parent compound 20 (Figure 6b). This suggests the necessity of hydroxy groups as
hydrogen bond donors for the low micromolar antagonistic effect of compound 20.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Overlay of coumestrol represented by green sticks with the corresponding shape in
grey and its pharmacophoric features coloured red for Hꢀbond acceptors, blue for Hꢀbond
donors, green for rings and yellow for hydrophobic groups, with the designed bazedoxifene
scaffoldꢀbased analogue 20 (in orange), as obtained by ROCS (Openeye Scientific Software,
3
3
Inc.).
To further support the determined antagonistic activity of the most promising analogues from
the series, 20 and 24, we have evaluated their effects on the rifaximin influenced expression
of PXR and its master target gene CYP3A4 (Figure 9). The realꢀtime PCR analysis, which
was performed on cDNA isolated from HepG2 cells, displayed a significant downꢀregulation
of PXR expression and a strong inhibition of CYP3A4 expression by both analogues. These
results highlight compounds 20 and 24 as promising PXR antagonists with the capacity to
suppress PXRꢀregulated phase I drug metabolism in vitro. To our knowledge, both
1
7
ACS Paragon Plus Environment

Page 19 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
compounds also represent a unique example of PXR antagonists that are capable of downꢀ
regulating PXR expression.
A
B
1
1
.5
.0
3
2
1
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
*
0.5
#
#
#
#
0
.0
NT
Rifaximin
20
24
NT
Rifaximin
20
24
Rifaximin
Rifaximin
Figure 9. RealꢀTime PCR analysis of PXR mRNA relative expression (A) and its target gene
CYP3A4 mRNA relative expression (B) in HepG2 cells treated with rifaximin (10 ꢁM) alone
and in combination with 20 or 24 (50 ꢁM). Values are normalised relatively to GAPDH
mRNA and are expressed relative to those of nonꢀtreated cells (NT), which are arbitrarily set
to 1. Data are the mean of two different experiments. * p < 0.05 vs. NT; # p < 0.05 vs. R.
CONCLUSION
In conclusion, our attempt to obtain novel PXR modulators by replacing a steroid core of
natural leads solomonsterol A and solomonsterol B with a steroidomimetic bazedoxifene
scaffold resulted in the discovery of novel PXR antagonists 19ꢀ24, among which compounds
20 and 24, with IC values of 11 µM and 14 µM, respectively, for the inhibition of rifaximin
50
induced PXR transactivation, represent promising novel PXR antagonists. Although less
16
potent than SPB3255, these compounds stand out as an important new structural class of
PXR antagonists offering good opportunity for further optimization. The SAR study, as well
2
5
as the overlay of compound 20 with previously described PXR antagonist coumestrol,
pointed out the importance of hydroxy groups as hydrogen bond donors for the PXR
antagonistic activity of compound 20. The suppression of PXR master target gene CYP3A4
1
8
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
highlights compounds 20 and 24 as PXR antagonists with the capacity to attenuate PXRꢀ
regulated phase I drug metabolism in vitro. Finally, both compounds represent a unique
example of PXR antagonists that are shown to downꢀregulate the expression of PXR.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
General procedures. All reagents were used as received from commercial sources without
further purification unless otherwise indicated. Analytical TLC was performed on Merck
silica gel (60 F 254) plates (0.25 mm) and components visualized with staining reagents or
ultraviolet light. Column chromatography was carried out on silica gel 60 (particle size 240ꢀ
400 mesh). Reversedꢀphase column chromatography was performed on Biotage Isolera One
system, using Biotage SNAP KPꢀC18ꢀHS cartridge. HPLC analyses were performed on
Agilent Technologies 1100 instrument with G1365B UVꢀVIS detector, G1316A thermostat
and G1313A autosampler using Agilent Eclipse Plus C18 column (5 µm, 4.6 × 150 mm).
Three different methods were used for HPLC analyses: a) Method A: Agilent 5µ C18 column;
mobile phase: 0.1% trifluoroacetic acid in water (A) and methanol (B); gradient: 90% A to
30% A in 20 min, then 5 min 30 % A; flow rate 1.0 mL/min; injection volume: 10 ꢁL; b)
Method B: Agilent 5µ C18 column; mobile phase: 0.1% trifluoroacetic acid in water (A) and
methanol (B); gradient: 90% A to 30% A in 20 min, then 10 min 30 % A; flow rate 1.0
mL/min; injection volume: 10 ꢁL; c) Method C: Agilent 5µ C18 column; mobile phase: 20
mM phosphate buffer (pH 7.0) (A) and methanol (B); gradient: 90% A to 30% A in 15 min,
then 5 min 30 % A; flow rate 1.0 mL/min; injection volume: 10 ꢁL. All tested compounds
1
13
were ≥95% pure by HPLC. H NMR and C NMR spectra were recorded at 400 MHz and
01 MHz, respectively, on a Bruker AVANCE III spectrometer in DMSOꢀd , CD OD or
CDCl solution with TMS as an internal standard at 25 °C. Spectra were assigned using
1
6
3
3
1
9
ACS Paragon Plus Environment

Page 21 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
gradient COSY, HSQC and DEPT experiments. IR spectra were recorded on a Thermo
Nicolet Nexus 470 ESP FTꢀIR spectrometer. Mass spectra were obtained using a
VGAnalytical Autospec Q mass spectrometer. All reported yields are yields of purified
products.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Benzyl (4ꢀhydroxyphenyl)carbamate (
1). To the solution of 4ꢀaminophenol (4.37 g, 40.0
mmol) in a mixture of water (50 mL) and THF (50 mL) was added Na CO (8.48 g, 80.0
2
3
mmol) and the mixture was cooled to 0 °C. Benzyl chloroformate (5.90 mL, 41.6 mmol) in
THF (20 mL) was added dropwise during 1 h. The brown suspension was then stirred for 30
min at room temperature and the THF was removed in vacuo. Brown solution was diluted
with water to 100 mL and extracted with ethyl acetate (1 x 100 mL), which was then washed
with brine (2 x 80 mL) and dried over Na SO . The solvent was removed under reduced
2
4
pressure and the crude product was recrystallized from ethyl acetate to yield 1 as an offꢀwhite
crystals. Yield: 8.71 g (90%); IR (ATR) ν 3302, 1700, 1534, 1516, 1383, 1308, 1234, 1063,
ꢀ1 1
8
27, 800, 746, 734 cm ; H NMR (400 MHz, DMSOꢀd
6 2
) δ 5.12 (s, 2H, CH ), 6.66ꢀ7.00 (m,
2H, 2 x ArꢀH), 7.24 (d, 2H, J = 8.1 Hz, 2 x ArꢀH), 7.31ꢀ7.47 (m, 5H, 5 x ArꢀH), 9.12 (s, 1H,
361
OH), 9.44 (br s, 1H, NH) ppm (lit. H NMR (400 MHz, DMSOꢀd ) 5.11 (s, 2H), 6.66ꢀ6.68
6
13
(d, 2H), 7.22 (d, 2H), 7.35ꢀ7.49 (m, 5H), 9.11 (s, 2H), 9.43 (s, 2H) ppm); C NMR (101
6
MHz, DMSOꢀd ) δ 65.41, 115.12 (2C), 120.00 (2C), 127.92, 127.98 (2C), 128.39 (2C),
+
+
130.50, 136.83, 152.81, 153.51 ppm. HRMS m/z for C14
found 244.0975.
3
H13NO ([M+H ] ): calcd 244.0974;
Benzyl (4ꢀ(methoxymethoxy)phenyl)carbamate (
mg, 27.5 mmol) in dry DMF (50 mL) at 0 °C was added benzyl (4ꢀhydroxyphenyl)carbamate
1) (6.08 g, 25.0 mmol) under argon atmosphere. The mixture was stirred for 30 min at 0 °C
2). To a suspension of sodium hydride (695
(
and then for 30 min at room temperature, upon which the hydrogen gas evolvement had
stopped. The reaction mixture was again cooled to 0 °C, bromomethyl methyl ether (2.50 mL,
2
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
27.5 mmol) was added and the mixture was stirred for 2 h at room temperature. The red
solution was concentrated in vacuo and the residue was suspended in ethyl acetate (150 mL).
Suspension was washed with 10% citric acid (4 x 80 mL), 1M NaOH(aq) (2 x 80 mL), brine (1
2 4
x 100 mL) and dried over Na SO . The solvent was removed under reduced pressure and the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
crude product was recrystallized from ethanol to yield 2 as white crystals. Yield: 4.20 g
ꢀ
(59%); IR (ATR) ν 3328, 1700, 1542, 1509, 1413, 1228, 1150, 1064, 995, 844, 763, 741 cm
1
1
;
H NMR (400 MHz, CDCl ) δ 3.50 (s, 3H, CH ), 5.16 (s, 2H, CH ), 5.22 (s, 2H, OꢀCH ꢀO),
3
3
2
2
13
6
.62 (br s, 1H, NH), 6.99ꢀ7.04 (m, 2H, 2 x ArꢀH), 7.29ꢀ7.45 (m, 7H, 7 x ArꢀH) ppm; C
3
NMR (101 MHz, CDCl ) δ 55.96, 67.00, 94.82, 116.93 (2C), 120.47, 128.34 (2C), 128.36
(2C), 128.64 (2C), 131.99, 136.13, 153.56, 153.64 ppm. HRMS m/z for C16H17NO
4
+
+
(
[M+H ] ): calcd 288.1236; found 288.1228.
4ꢀ(Methoxymethoxy)aniline (3).Benzyl (4ꢀ(methoxymethoxy)phenyl)carbamate (2) (4.20 g,
14.6 mmol) was dissolved in methanol (200 mL), 10% Pd/C (630 mg) was added and the
reaction mixture was stirred under hydrogen atmosphere for 3 h. The catalyst was filtered off
and the solvent was removed under reduced pressure. Brown oil was purified by column
chromatography with ethyl acetate/hexane (1:1) as an eluent to afford 3 as orange oil. Yield:
1.88 g (84%); IR (ATR) ν 3355, 2953, 1626, 1507, 1227, 1193, 1148, 1072, 989, 916, 825
ꢀ1 1
cm ; H NMR (400 MHz, CDCl
3 3 2 2
) δ 3.42ꢀ3.55 (m, 5H, CH , NH ), 5.10 (s, 2H, CH ), 6.63ꢀ
13
6.68 (m, 2H, 2 x ArꢀH), 6.87ꢀ6.92 (m, 2H, 2 x ArꢀH) ppm; C NMR (101 MHz, CDCl
3
) δ
8
55.83, 95.51, 116.23 (2C), 117.87 (2C), 141.21, 150.22 ppm. HRMS m/z for C H11NO
2
+ +
(
[M+H ] ): calcd 154.0868; found 154.0871.
4ꢀ(Methoxymethoxy)benzenaminium chloride (4). Acetyl chloride (286 µl, 4.0 mmol) was
added into the absolute ethanol (10 mL) under argon atmosphere. Reaction mixture was
stirred for 1 h at room temperature. The colourless solution was then cooled to 0 °C and the
solution of 4ꢀ(methoxymethoxy)aniline (3) (612 mg, 4.0 mmol) in absolute ethanol (15 mL)
2
1
ACS Paragon Plus Environment

Page 23 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
was added dropwise during 15 min. Brown reaction mixture was then stirred at 0 °C for 75
min and the solvent was evaporated under reduced pressure to give 4 as brown solid. Yield:
745 mg (98%); IR (ATR) ν 2922, 2591, 1662, 1610, 1504, 1455, 1354, 1274, 1217, 1171,
ꢀ1 1
1
107, 826 cm ; H NMR (400 MHz, DMSOꢀd
6 2
) δ 3.37 (s, 3H, signal overlapped with H O,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CH
3
), 5.20 (s, 2H, CH ), 7.08ꢀ7.13 (m, 2H, 2 x ArꢀH), 7.27ꢀ7.32 (m, 2H, 2 x ArꢀH), 10.09 (br
2
+
ꢀ
13
s, 3H, NH Cl ) ppm; C NMR (101 MHz, DMSOꢀd ) 55.60, 93.86, 117.00 (2C), 124.50
3
6
(2C), 125.15, 156.14 δ ppm.
Synthesis of compounds
5
and
6. To the solution of 4’ꢀhydroxypropiophenone (2.70 g, 18
mmol) or 3’,4’ꢀdihydroxypropiophenone (2.99 g, 18 mmol) in acetone (50 mL) was added
CO (4.98 g, 36.0 mmol) and the mixture was stirred for 30 min at room temperature.
K
2
3
Benzyl bromide (19.8 mmol or 39.6 mmol) was added and the mixture was refluxed
overnight. Solvent was removed in vacuo and the residue was suspended in diethyl ether (200
mL). The suspension was washed with water (1 x 100 mL), brine (1 x 100 mL) and dried over
Na
2 4
SO . Removal of the solvent under reduced pressure gave yellow crystals that were
suspended in petroleum ether (50 mL) and filtered to yield compound 5 or 6.
1
ꢀ(4ꢀ(Benzyloxy)phenyl)propanꢀ1ꢀone (
677, 1597, 1509, 1386, 1257, 1224, 117, 1011, 950, 844, 798, 756, 700 cm ; H NMR (400
) δ 1.24 (t, 3H, J = 7.3 Hz, CH ), 2.98 (q, 2H, J = 7.3 Hz, CH ), 5.16 (s, 2H, Arꢀ
), 7.01ꢀ7.06 (m, 2H, 2 x ArꢀH), 7.35ꢀ7.49 (m, 5H, 5 x ArꢀH), 7.95ꢀ8.00 (m, 2H, 2 x ArꢀH)
5). Yield: 4.02 g (93%); white crystals; IR (ATR) ν
ꢀ1
1
1
MHz, CDCl
CH
ppm (lit. H NMR (300 MHz, CDCl
3
3
2
2
38 1
3
) δ 1.24 (t, 3H, J = 7.3 Hz), 2.97 (q, 2H, J = 7.3 Hz),
13
5.15 (s, 2H), 7.03 (d, 2H, J = 8.9 Hz), 7.31ꢀ7.48 (m, 5H), 7.97 (d, 2H, J = 8.9 Hz) ppm); C
NMR (101 MHz, CDCl ) δ 8.46, 31.45, 70.12, 114.54 (2C), 127.51 (2C), 128.26, 128.72
3
+
+
(2C), 130.23, 130.26 (2C), 136.24, 162.45, 199.52 ppm. HRMS m/z for C16
16 2
H O ([M+H ] ):
calcd 241.1229; found 241.1233.
2
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1ꢀ(3,4ꢀbis(Benzyloxy)phenyl)propanꢀ1ꢀone (
ν 1675, 1594, 1580, 1515, 1453, 1424, 1266, 1178, 1146, 1022, 878, 786, 732 cm ; H NMR
400 MHz, CDCl ) δ 1.21 (t, 3H, J = 7.3 Hz, CH ), 2.93 (q, 2H, J = 7.3 Hz, CH ), 5.24 (s, 2H,
ArꢀCH ), 5.26 (s, 2H, ArꢀCH ), 6.96 (d, 1H, J = 8.4 Hz, ArꢀH), 7.31ꢀ7.52 (m, 10H, 10 x Arꢀ
6).Yield: 5.54 g (89%); white crystals; IR (ATR)
ꢀ1 1
(
3
3
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
39 1
H), 7.57 (dd, 1H, J = 2.1, 8.4 Hz, ArꢀH), 7.65 (d, 1H, J = 2.0 Hz, ArꢀH) ppm (lit. H NMR
(CDCl ) δ 1.20 (t, 3H, J = 7 Hz), 2.92 (q, 2H, J = 7 Hz), 5.23 (s, 2H), 5.25 (s, 2H), 6.97 (d,
3
1
3
1
1
1
H, J = 8 Hz), 7.42 (m, 12H) ppm); C NMR (101 MHz, CDCl ) δ 8.51, 31.34, 70.80, 71.14,
3
12.93, 113.69, 122.86, 127.11 (2C), 127.42 (2C), 127.97, 128.04, 128.57 (2C), 128.64 (2C),
+
+
22 3
30.43, 136.54, 136.85, 148.56, 152.94, 199.47 ppm. HRMS m/z for C23H O ([M+H ] ):
calcd 347.1647; found 347.1641.
Synthesis of compounds
7 and 8. To the stirring solution of 1ꢀ(4ꢀ(benzyloxy)phenyl)propanꢀ1ꢀ
one (5) (0.96 g, 4.0 mmol) or 1ꢀ(3,4ꢀbis(benzyloxy)phenyl)propanꢀ1ꢀone (6) (1.38 g, 4.0
mmol) in glacial acetic acid (14 mL) was added bromine (640 mg, 4.0 mmol) in glacial acetic
acid (14 mL) dropwise, during 30 min at room temperature. Concentration of the yellow
solution under reduced pressure and recrystallization of the crude product from the mixture of
diethyl ether and hexane yielded compound 7 or 8.
1
ꢀ(4ꢀ(Benzyloxy)phenyl)ꢀ2ꢀbromopropanꢀ1ꢀone (
ATR) ν 1664, 1599, 1566, 1506, 1335, 1238, 1180, 986, 922, 849, 751, 729 cm ; H NMR
(400 MHz, CDCl ) δ 1.92 (d, 3H, J = 6.6 Hz, CH ), 5.17 (s, 2H, CH ), 5.28 (q, 1H, J = 6.6
7). Yield: 1.05 g (82%); white crystals; IR
ꢀ1 1
(
3
3
2
Hz, CH), 7.03ꢀ7.08 (m, 2H, 2 x ArꢀH), 7.35ꢀ7.48 (m, 5H, 5 x ArꢀH), 8.01ꢀ8.06 (m, 2H, 2 x
38 1
ArꢀH) ppm; (lit. H NMR (300 MHz, CDCl ) δ 1.91 (d, 3H, J = 6.6 Hz), 5.17 (s, 2H), 5.27
3
(q, 1H, J = 6.6 Hz), 7.05 (d, 2H, J = 9.0 Hz), 7.31ꢀ7.50 (m, 5H), 8.03 (d, 2H, J = 9.0 Hz)
1
3
ppm); C NMR (101 MHz, CDCl
3
) δ 20.26, 41.45, 70.24, 114.81 (2C), 127.03, 127.53 (2C),
128.35, 128.76 (2C), 131.36 (2C), 136.02, 163.10, 192.00 ppm. HRMS m/z for C16
H
15BrO
2
+
+
(
[M+H ] ): calcd 319.0334; found 319.0336.
2
3
ACS Paragon Plus Environment

Page 25 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1ꢀ(3,4ꢀBis(benzyloxy)phenyl)ꢀ2ꢀbromopropanꢀ1ꢀone (8). Yield: 1.21 g (71%); white crystals;
ꢀ1
IR (ATR) ν 1672, 1579, 1507, 1425, 1381, 1260, 1198, 1137, 996, 917, 875, 752, 725 cm ;
1
H NMR (400 MHz, CDCl
3
3 2
) δ 1.88 (d, 3H, J = 6.6 Hz, CH ), 5.19ꢀ5.26 (m, 3H, CH , CH),
5.28 (s, 2H, CH
2
), 6.98 (d, 1H, J = 8.5 Hz, ArꢀH), 7.32ꢀ7.44 (m, 6H, 6 x ArꢀH), 7.45ꢀ7.53 (m,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
9
4
H, 4 x ArꢀH), 7.64 (dd, 1H, J = 2.1, 8.5 Hz, ArꢀH), 7.69 (d, 1H, J = 2.1 Hz, ArꢀH) ppm (lit.
1
H NMR (CDCl ) δ 1.87 (d, 3H, J = 7 Hz),5.23 (m, 5H), 6.98 (d, 1H, J = 8 Hz), 7.43 (m,
3
13
1
1
1
2H) ppm); C NMR (101 MHz, CDCl ) δ 20.33, 41.26, 70.81, 71.20, 112.80, 114.60,
3
23.82, 127.09 (2C), 127.17, 127.42 (2C), 128.04, 128.12, 128.62 (2C), 128.69 (2C), 136.31,
+
+
3
36.67, 148.71, 153.67, 191.98 ppm. HRMS m/z for C23H21BrO ([M+H ] ): calcd 425.0752;
found 425.0747.
Synthesis of compounds
9 and 10. To the solution of 4ꢀ(methoxymethoxy)aniline (3) (1.06 g,
6
.9 mmol) and triethylamine (1.75 mL, 12.6 mmol) in absolute ethanol (50 mL) was added 1ꢀ
4ꢀ(benzyloxy)phenyl)ꢀ2ꢀbromopropanꢀ1ꢀone (7) (2.01 g, 6.3 mmol) or 1ꢀ(3,4ꢀ
(
bis(benzyloxy)phenyl)ꢀ2ꢀbromopropanꢀ1ꢀone (8) (2.68 g, 6.3 mmol) and the mixture was
purged with argon for 10 min. The reaction mixture was then refluxed overnight under argon
atmosphere. The mixture was then concentrated under reduced pressure and suspended in
ethyl acetate (150 mL). The white precipitate was filtered off and the filtrate was washed with
water (2 x 80 mL), 1% citric acid (2 x 100 mL) and brine (1 x 100 mL). The organic phase
2 4
was dried over Na SO and concentrated in vacuo to yield compound 9 or 10.
1ꢀ(4ꢀ(Benzyloxy)phenyl)ꢀ2ꢀ((4ꢀ(methoxymethoxy)phenyl)amino)propanꢀ1ꢀone (
9). Yield: 2.41
g (98%); red solid; IR (ATR) ν 3394, 1670, 1596, 1512, 1254, 1221, 1149, 1078, 972, 921,
ꢀ1
1
8
39, 819, 747 cm ; H NMR (400 MHz, CDCl ) δ 1.48 (d, 3H, J = 6.9 Hz, CHꢀCH ), 3.49 (s,
3
3
3
H, OꢀCH
3
), 4.49 (br s, 1H, NH), 5.04 (q, 1H, J = 6.9 Hz, CH), 5.09 (s, 2H,OꢀCH
2
ꢀO), 5.18
2
(s, 2H, PhꢀCH ), 6.62ꢀ6.67 (m, 2H, 2 x ArꢀH), 6.89ꢀ6.94 (m, 2H, 2 x ArꢀH), 7.05ꢀ7.10 (m, 2H,
1
3
2
x ArꢀH), 7.36ꢀ7.49 (m, 5H, 5 x ArꢀH), 8.00ꢀ8.05 (m, 2H, 2 x ArꢀH) ppm; C NMR (101
2
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MHz, CDCl ) δ 19.85, 53.90, 55.82, 70.24, 95.52, 114.89 (4C), 117.99 (2C), 127.53 (2C),
3
127.83, 128.35, 128.77 (2C), 130.80 (2C), 136.04, 141.96, 149.85, 163.06, 199.48 ppm.
+ +
4
HRMS m/z for C24H25NO ([M+H ] ): calcd 392.1862; found 392.1865.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1ꢀ(3,4ꢀBis(benzyloxy)phenyl)ꢀ2ꢀ((4ꢀ(methoxymethoxy)phenyl)amino)propanꢀ1ꢀone (10).
Yield: 3.05 g (97%); red solid; IR (ATR) ν 2931, 1671, 1597, 1508, 1426, 1262, 1226, 1133,
ꢀ1 1
1078, 988, 919, 822, 734 cm ; H NMR (400 MHz, CDCl ) δ 1.41 (d, 3H, J = 6.9 Hz, CHꢀ
3
CH ), 3.49 (s, 3H, OꢀCH ), 4.49 (br s, 1H, NH), 4.95 (q, 1H, J = 6.9 Hz, CH), 5.08 (s, 2H, Oꢀ
3
3
CH
2
2 2
ꢀO), 5.24 (s, 2H, PhꢀCH ), 5.28 (s, 2H, PhꢀCH ), 6.58ꢀ6.63 (m, 2H, 2 x ArꢀH), 6.88ꢀ6.93
(m, 2H, 2 x ArꢀH), 6.97ꢀ7.01 (m, 1H, ArꢀH), 7.32ꢀ7.51 (m, 10H, 10 x ArꢀH), 7.61ꢀ7.65 (m,
13
2H, 2 x ArꢀH) ppm; C NMR (101 MHz, CDCl
3
) δ 19.79, 54.14, 55.83, 70.84, 71.24, 95.46,
112.97, 114.24, 115.24 (2C), 117.94 (2C), 123.24, 127.11 (2C), 127.40 (2C), 127.91, 128.06,
128.13, 128.63 (2C), 128.70 (2C), 136.32, 136.70, 141.46, 148.72, 150.14, 153.65, 199.31
+ +
5
ppm. HRMS m/z for C31H31NO ([M+H ] ): calcd 498.2280; found 498.2285.
Synthesis of compounds 11 and 12. To the solution of 1ꢀ(4ꢀ(benzyloxy)phenyl)ꢀ2ꢀ((4ꢀ
(methoxymethoxy)phenyl)amino)propanꢀ1ꢀone (9) (2.19 g, 5.6 mmol) or 1ꢀ(3,4ꢀ
bis(benzyloxy)phenyl)ꢀ2ꢀ((4ꢀ(methoxymethoxy)phenyl)amino)propanꢀ1ꢀone (10) (2.80 g, 5.6
mmol) in absolute ethanol (50 mL) was added 4ꢀ(methoxymethoxy)benzenaminium chloride
(4) (534 mg, 2.8 mmol). The mixture was purged with argon for 10 min and stirred overnight
in a sealed reactor, at 115 °C. Reaction mixture was then concentrated in vacuo and
suspended in ethyl acetate (100 mL). Suspension was washed with water (1 x 100 mL), 10%
citric acid (1 x 100 mL) and brine (1 x 100 mL). Organic phase was dried over Na SO and
2
4
concentrated under reduced pressure. Crude product was then purified by column
chromatography with ethyl acetate/hexane (1:2) as an eluent to afford compound 11 or 12.
2
5
ACS Paragon Plus Environment

Page 27 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2ꢀ(4ꢀ(Benzyloxy)phenyl)ꢀ3ꢀmethylꢀ1Hꢀindolꢀ5ꢀol (11). Yield: 793 mg (43%); yellow crystals;
IR (ATR) ν 3423, 1609, 1508, 1486, 1455, 1384, 1239, 1194, 1177, 1020, 939, 830, 800, 733
ꢀ1
1
cm ; H NMR (400 MHz, DMSOꢀd
2.3, 8.5 Hz, ArꢀH), 6.78 (d, 1H, J = 2.3, ArꢀH), 7.09ꢀ7.17 (m, 3H, 3 x ArꢀH), 7.32ꢀ7.38 (m,
H, ArꢀH), 7.39ꢀ7.45 (m, 2H, 2 x ArꢀH), 7.47ꢀ7.51 (m, 2H, 2 x ArꢀH), 7.53ꢀ7.58 (m, 2H, 2 x
6 3 2
) δ 2.29 (s, 3H, CH ), 5.17 (s, 2H, CH ), 6.59 (dd, 1H, J
=
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
13
ArꢀH), 8.63 (s, 1H, ArꢀOH), 10.71 (s, 1H, NH) ppm; C NMR (101 MHz, DMSOꢀd ) δ 9.87,
6
69.21, 102.05, 104.80, 111.16, 111.35, 114.96 (2C), 126.05, 127.71 (2C), 127.84, 128.44
(2C), 128.54 (2C), 130.17, 130.22, 134.22, 137.03, 150.33, 157.26 ppm. HRMS m/z for
+
+
C
22 2
H19NO ([M+H ] ): calcd 330.1494; found 330.1495.
2ꢀ(3,4ꢀBis(benzyloxy)phenyl)ꢀ3ꢀmethylꢀ1Hꢀindolꢀ5ꢀol (12).Yield: 1030 mg (42%); offꢀwhite
ꢀ1 1
solid; IR (ATR) ν 3383, 1506, 1452, 1371, 1240, 1199, 1133, 1001, 942, 797, 733 cm ; H
NMR (400 MHz, DMSOꢀd ) δ 2.22 (s, 3H, CH ), 5.20 (s, 2H, CH ), 5.24 (s, 2H, CH ), 6.60
6
3
2
2
(
dd, 1H, J = 2.3, 8.6 Hz, ArꢀH), 6.77 (d, 1H, J = 2.2, ArꢀH), 7.10ꢀ7.20 (m, 3H, 3 x ArꢀH), 7.29
d, 1H, J = 1.8 Hz, ArꢀH), 7.31ꢀ7.44 (m, 6H, 6 x ArꢀH), 7.47ꢀ7.52 (m, 4H, 4 x ArꢀH), 8.63 (s,
(
1
3
1H, ArꢀOH), 10.71 (s, 1H, NH) ppm; C NMR (101 MHz, DMSOꢀd
6
) δ 9.86, 70.03, 70.11,
02.07, 105.04, 111.13, 111.49, 113.71, 114.51, 120.17, 126.51, 127.43 (2C), 127.55 (2C),
27.77 (2C), 128.40 (2C), 128.43 (2C), 130.17 (2C), 134.16, 137.27 (2C), 147.56, 148.09,
1
1
1
+
+
3
50.34 ppm. HRMS m/z for C29H25NO ([M+H ] ): calcd 436.1913; found 436.1903.
Synthesis of compounds 13 and 14.To the solution of 2ꢀ(4ꢀ(benzyloxy)phenyl)ꢀ3ꢀmethylꢀ1Hꢀ
indolꢀ5ꢀol (11) (82 mg, 0.25 mmol) or 2ꢀ(3,4ꢀbis(benzyloxy)phenyl)ꢀ3ꢀmethylꢀ1Hꢀindolꢀ5ꢀol
(12) (108 mg, 0.25 mmol) in acetone (6 mL) were added potassium carbonate (172 mg, 1.25
mmol) and 3ꢀbromoꢀ1ꢀpropanol (34 µl, 0.38 mmol). The mixture was stirred at 60 °C for 90
h. Yellow suspension was filtered and the filtrate concentrated under reduced pressure. Crude
product was then purified by column chromatography with dichloromethane/methanol as an
eluent to afford compound 13 or 14.
2
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3ꢀ((2ꢀ(4ꢀ(Benzyloxy)phenyl)ꢀ3ꢀmethylꢀ1Hꢀindolꢀ5ꢀyl)oxy)propanꢀ1ꢀol (13).Yield: 80 mg
(83%); offꢀwhite crystals; IR (ATR) ν 3419, 1506, 1466, 1452, 1381, 1287, 1241, 1208, 1180,
ꢀ1
1
1
064, 1024, 964, 830, 807, 741 cm ; H NMR (400 MHz, DMSOꢀd
), 2.36 (s, 3H, CH ), 3.60 (q, 2H,J = 6.3 Hz, CH ), 4.05 (t, 2H, J = 6.4 Hz, CH
t, 1H, J = 5.2 Hz, OH), 5.18 (s, 2H, ArꢀCH ), 6.72 (dd, 1H, J = 2.4, 8.7 Hz, ArꢀH), 6.97 (d,
1H, J = 2.4 Hz, ArꢀH), 7.13ꢀ7.18 (m, 2H, 2 x ArꢀH), 7.20 (d, 1H, J = 8.9 Hz, ArꢀH), 7.33ꢀ7.39
6
) δ 1.89 (p, 2H, J = 6.4
Hz, CH
2
3
2
2
), 4.55
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2
(m, 1H, ArꢀH), 7.40ꢀ7.45 (m, 2H, 2 x ArꢀH), 7.47ꢀ7.52 (m, 2H, 2 x ArꢀH), 7.55ꢀ7.60 (m, 2H,
13
2
6
x ArꢀH), 10.87 (s, 1H, NH) ppm; C NMR (101 MHz, DMSOꢀd
6
) δ 9.87, 32.43, 57.54,
5.01, 69.22, 101.00, 105.47, 111.43, 111.63, 115.00 (2C), 125.91, 127.72 (2C), 127.85,
128.44 (2C), 128.59 (2C), 128.79, 130.81, 134.39, 137.01, 152.43, 157.33 ppm. HRMS m/z
+
+
for C H NO ([M+H ] ): calcd 388.1913; found 388.1919.
25
25
3
3ꢀ((2ꢀ(3,4ꢀBis(benzyloxy)phenyl)ꢀ3ꢀmethylꢀ1Hꢀindolꢀ5ꢀyl)oxy)propanꢀ1ꢀol (14).Yield: 97 mg
(79%); yellow crystals; IR (ATR) ν 3520, 3292, 1540, 1482, 1455, 1379, 1250, 1211, 1052,
ꢀ1 1
9
92, 824, 804, 742 cm ; H NMR (400 MHz, DMSOꢀd
6
) δ 1.89 (p, 2H, J = 6.4 Hz, CH
2
),
2.71 (s, 3H, CH
3
), 3.60 (q, 2H,J = 6.2 Hz, CH
2
), 4.05 (t, 2H,J = 6.4 Hz, CH ), 4.55 (t, 1H,J =
2
5
.1 Hz, OH), 5.21 (s, 2H, ArꢀCH ), 5.25 (s, 2H, ArꢀCH ), 6.72 (dd, 1H, J = 2.4, 8.7 Hz, Arꢀ
2
2
H), 6.97 (d, 1H, J = 2.3 Hz, ArꢀH), 7.15ꢀ7.23 (m, 3H, 3 x ArꢀH), 7.30ꢀ7.37 (m, 3H, 3 x ArꢀH),
1
3
7
.38ꢀ7.44 (m, 4H, 4 x ArꢀH), 7.48ꢀ7.52 (m, 4H, ArꢀH), 10.86 (s, 1H, NH) ppm; C NMR
(101 MHz, DMSOꢀd ) δ 9.86, 32.44, 57.53, 64.99, 70.03, 70.13, 100.98, 105.71, 111.40,
11.76, 113.73, 114.52, 120.20, 126.36, 127.43 (2C), 127.56 (2C), 127.78 (2C), 128.40 (2C),
28.43 (2C), 129.80, 130.75, 134.32, 137.26 (2C), 147.33, 148.11, 152.45 ppm. HRMS m/z
6
1
1
+
+
for C H NO ([M+H ] ): calcd 494.2331; found 494.2336.
32
31
4
Synthesis of compounds 15 and 16. To the solution of 2ꢀ(4ꢀ(benzyloxy)phenyl)ꢀ3ꢀmethylꢀ1Hꢀ
indolꢀ5ꢀol (11) (132 mg, 0.4 mmol) or 2ꢀ(3,4ꢀbis(benzyloxy)phenyl)ꢀ3ꢀmethylꢀ1Hꢀindolꢀ5ꢀol
(12) (192 mg, 0.4 mmol) in acetone (20 mL) were added cesium carbonate (391 mg, 1.2
2
7
ACS Paragon Plus Environment

Page 29 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mmol) and 2ꢀbromoethyl acetate (66 µl, 0.6 mmol). The mixture was stirred at 60 °C for 20 h,
concentrated in vacuo and suspended in ethyl acetate (50 mL). Suspension was washed with
water (1 x 50 mL), 10% citric acid (1 x 50 mL), saturated aqueous NaHCO
3
solution (1 x 50
mL) and brine (1 x 50 mL). Organic phase was dried over Na SO and concentrated under
2
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
reduced pressure. Crude product was then purified by column chromatography with ethyl
acetate/hexane (1:2) as an eluent to afford compound 15 or 16.
2ꢀ((2ꢀ(4ꢀ(Benzyloxy)phenyl)ꢀ3ꢀmethylꢀ1Hꢀindolꢀ5ꢀyl)oxy)ethyl acetate (15). Yield: 76 mg
(46%); offꢀwhite crystals; IR (ATR) ν 3387, 1736, 1609, 1480, 1446, 1379, 1232, 1209, 1054,
ꢀ
1 1
9
69, 833, 802, 741 cm ; H NMR (400 MHz, DMSOꢀd
), 4.18ꢀ4.22 (m, 2H, CH ), 4.34ꢀ4.37 (m, 2H, CH
(dd, 1H, J = 2.4, 8.7 Hz, ArꢀH), 7.02 (d, 1H, J = 2.4 Hz, ArꢀH), 7.13ꢀ7.18 (m, 2H, 2 x ArꢀH),
6
) δ 2.07 (s, 3H, CH
3
ꢀC=O), 2.35 (s,
3H, ArꢀCH
3
2
2
), 5.18 (s, 2H, ArꢀCH
2
), 6.74
7.22 (d, 1H, J = 8.8 Hz, ArꢀH), 7.33ꢀ7.38 (m, 1H, ArꢀH), 7.40ꢀ7.45 (m, 2H, 2 x ArꢀH), 7.47ꢀ
13
7
.52 (m, 2H, 2 x ArꢀH), 7.56ꢀ7.61 (m, 2H, 2 x ArꢀH), 10.91 (s, 1H, NH) ppm; C NMR (101
MHz, DMSOꢀd
6
) δ 9.87, 20.71, 62.81, 66.20, 69.22, 101.33, 105.56, 111.51, 111.57, 115.01
(2C), 125.85, 127.72 (2C), 127.85, 128.45 (2C), 128.61 (2C), 129.78, 131.03, 134.55, 137.01,
+
+
1
51.91, 157.37, 170.42 ppm. HRMS m/z for C H NO ([M+H ] ): calcd 416.1862; found
26 25 4
416.1851.
2ꢀ((2ꢀ(3,4ꢀbis(Benzyloxy)phenyl)ꢀ3ꢀmethylꢀ1Hꢀindolꢀ5ꢀyl)oxy)ethyl acetate (16). Yield: 88 mg
(42%); offꢀwhite crystals; IR (ATR) ν 3380, 2868, 1732, 1510, 1482, 1448, 1377, 1259, 1215,
ꢀ1 1
1134, 1019, 958, 815, 800, 733 cm ; H NMR (400 MHz, CDCl
3 3
) δ 2.15 (s, 3H, CH ꢀC=O),
2
.29 (s, 3H, ArꢀCH ), 4.24ꢀ4.31 (m, 2H, CH ), 4.45ꢀ4.52 (m, 2H, CH ), 5.22ꢀ5.29 (m, 4H, 2 x
3
2
2
ArꢀCH ), 6.86ꢀ6.92 (m, 1H, ArꢀH), 7.01ꢀ7.11 (m, 3H, 3 x ArꢀH), 7.14 (s, 1H, ArꢀH), 7.24 (d,
2
1H, J = 8.7 Hz, ArꢀH), 7.32ꢀ7.45 (m, 6H, 6 x ArꢀH), 7.47ꢀ7.55 (m, 4H, 4 x ArꢀH), 7.82 (s, 1H,
13
NH) ppm; C NMR (101 MHz, CDCl
3
) δ 9.58, 21.03, 63.27, 67.06, 71.28, 71.45, 102.41,
107.80, 111.36, 112.63, 114.95, 115.04, 120.84, 126.76, 127.29 (2C), 127.32 (2C), 127.93
2
8
ACS Paragon Plus Environment