Chemical manipulations on the 1,4-dioxane ring of 5-HT1A receptor agonists lead to antagonists endowed with antitumor activity in prostate cancer cells

Article abstract of DOI:10.1016/j.ejmech.2019.02.056

A series of derivatives obtained by moving the aromatic moiety on the 1,4-dioxane ring of compounds 1–3 from position 6 to position 2 or 3 was prepared and evaluated for the affinity for 5-HT_1A receptor (5-HT_1AR) and α₁-ad

Full text of DOI:10.1016/j.ejmech.2019.02.056

Accepted Manuscript
Chemical manipulations on the 1,4-dioxane ring of 5-HT receptor agonists lead to
1A
antagonists endowed with antitumor activity in prostate cancer cells
Fabio Del Bello, Alessandro Bonifazi, Gianfabio Giorgioni, Wilma Quaglia, Consuelo
Amantini, Maria Beatrice Morelli, Giorgio Santoni, Francisco O. Battiti, Giulio Vistoli,
Antonio Cilia, Alessandro Piergentili
PII:
S0223-5234(19)30170-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.056
EJMECH 11148
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 December 2018
Revised Date: 15 February 2019
Accepted Date: 18 February 2019
Please cite this article as: F. Del Bello, A. Bonifazi, G. Giorgioni, W. Quaglia, C. Amantini, M.B. Morelli,
G. Santoni, F.O. Battiti, G. Vistoli, A. Cilia, A. Piergentili, Chemical manipulations on the 1,4-dioxane ring
of 5-HT receptor agonists lead to antagonists endowed with antitumor activity in prostate cancer cells,
1A
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.056.
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ACCEPTED MANUSCRIPT
Graphical Abstract:

ACCEPTED MANUSCRIPT
Chemical manipulations on the 1,4-dioxane ring of 5-HT1A receptor agonists lead to
antagonists endowed with antitumor activity in prostate cancer cells
Fabio Del Belloa,1, Alessandro Bonifazia,b,1,, Gianfabio Giorgionia, Wilma Quagliaa,*,
Consuelo Amantinic, Maria Beatrice Morellic,d, Giorgio Santonid, Francisco O. Battitib, Giulio
Vistolie, Antonio Ciliaf, Alessandro Piergentilia
aScuola di Scienze del Farmaco e dei Prodotti della Salute, Unità di Chimica Farmaceutica,
Università di Camerino, Via S. Agostino 1, 62032 Camerino, Italy
bMedicinal Chemistry Section, Molecular Targets and Medications Discovery Branch,
National Institute on Drug Abuse – Intramural Research Program, National Institutes of
Health, Baltimore, Maryland, 333 Cassell Drive, Baltimore, Maryland 21224
cScuola di Bioscienze e Medicina Veterinaria, Università di Camerino, Via Madonna delle
Carceri 9, 62032 Camerino, Italy
dScuola di Scienze del Farmaco e dei Prodotti della Salute, Unità di Immunopatologia,
Università di Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy
eDipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli
25, 20133 Milano, Italy
fRecordati S.p.A., Drug Discovery, Via Civitali 1, 20148 Milano, Italy
*Corresponding Author: Phone +390737402237. E-mail wilma.quaglia@unicam.it.
1These authors contributed equally to this work
1

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Abstract
A series of derivatives obtained by moving the aromatic moiety on the 1,4-dioxane ring of
compounds 1-3 from position 6 to position 2 or 3 was prepared and evaluated for the affinity
for 5-HT1A receptor (5-HT1AR) and α1-adrenoceptor (α1-AR) subtypes. Moreover, the flexible
2-ethanolamine linker of the most interesting compounds was replaced by the more
conformationally constrained piperazine ring. In vitro functional studies performed on
derivatives showing the highest affinities for 5-HT1AR highlighted that the shifting of the
diphenyl moiety of derivatives 2 and 13 from position 6 to position 3 of the 1,4-dioxane
nucleus, affording 11 and 16, respectively, modulated the 5-HT1AR functional profile from
agonism to antagonism. Docking simulations, performed on the human 5-HT1AR, further
rationalized the biological results, delving into the features which modulate the shift between
agonist and antagonist activity. Interestingly, compound 11, endowed with mixed 5-
HT1AR/α1d-AR antagonist profile, showed antiproliferative and cytotoxic effects on both PC-3
and DU-145 prostate cancer cell lines higher than those of the α1d-AR antagonist 2 and the 5-
HT1AR antagonist 16. The experiments performed in the presence of the endogenous agonists
norepinephrine and serotonin confirmed the involvement of both receptor systems in the
antitumor activity of 11.
Keywords: 1,4-dioxane compounds; functional profile modulation; 5-HT1A receptor
antagonists; antitumor activity; docking studies.
Abbreviations: 5-HTRs, serotonin receptors, CNS, central nervous system; PNS, peripheral
nervous system; 5-HT, serotonin; GPCRs, G-protein coupled receptors; α1-ARs, α1-
adrenoceptors; SRB, sulforhodamine B; GI50, growth inhibition 50; TGI, total growth
inhibition; LC50, lethal concentration 50; SE, standard error; NE, norepinephrine.
2

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1. Introduction
Serotonin receptors (5-HTRs) are among the most widespread drug targets and are widely
distributed in both the central and peripheral nervous systems (CNS and PNS) [1]. The
heterogeneity of 5-HTRs was pharmacologically hypothesized in 1950’s and evidenced by
radioligand experiments in 1979 [2]. At present, at least seven subfamilies (5-HT1-7) have
been identified and some of them are further subdivided into different subtypes [3]. 5-HTRs
are G-protein coupled receptors (GPCRs), belonging to rhodopsine-like receptor family (class
A), except for 5-HT3R, which is a cation-permeable ion channel. Among the 5-HTRs, 5-HT1A
subtype was the first to be cloned [4] and, though its structure is still unknown, amino acid
residues responsible for ligand binding and G-protein coupling have been identified from
mutagenesis studies [1]. 5-HT1ARs are expressed both pre- and post-synaptically in the CNS
[5], mainly in limbic areas, such as hippocampus, lateral septum, cortical brain regions, as
well as dorsal and medial raphe nuclei. They are also distributed in the PNS, in particular in
the enteric nervous system, in the gut, and in smooth muscle [1]. 5-HT1ARs activation mainly
leads to the inhibition of adenylate cyclase. However, depending on the target cell, 5-HT1AR
stimulation modulates the production of different second messengers and activates or inhibits
different enzymes, channels, and kinases [6,7].
Nearly all physiological and behavioral effects of serotonin (5-HT) are mediated by 5-HT1ARs
[3]. Full and partial 5-HT1AR agonists have been demonstrated to be effective in the treatment
of anxiety, depression, and schizophrenia [8,9], as well as in pain management [10] and, as
levodopa adjuvants, in the treatment of Parkinson’s disease [11]. The potentially therapeutic
applications of 5-HT1AR antagonists have been evaluated in cognitive disorders such as the
cognitive impairment associated with Alzheimer’s disease [12]. Moreover, 5-HT1AR
antagonists co-administered with selective serotonin reuptake inhibitors shortened the onset of
the antidepressant effect [13]. Finally, the growth of PC-3, DU-145, and LNCaP prostate
3

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tumor cell lines, as well as the proliferation of PC-3 xenografted subcutaneously in athymic
nude mice have been reported to be inhibited by 5-HT1AR antagonists [14]. 5-HT1ARs share a
high degree of homology with other receptor systems, including α1-adrenoceptors (α1-ARs)
[15]. Indeed, several 5-HT1AR high affinity ligands, belonging to structurally different classes,
including aryloxyalkylamines, arylpiperazines, aminotetralines, indolylalkylamines, ergolines,
and aporphines [16], also show high affinity for α1-ARs. The simultaneous antagonism at 5-
HT1AR and α1-ARs might be potentially useful in the treatment of prostate cancer, as α1-ARs
antagonists have also been demonstrated to inhibit the primary tumor growth and progression
in human prostate cancer [17].
Recently, new classes of ligands, including 2-imidazoline and 1,3-dioxolane derivatives, have
shown high 5-HT1AR affinity [18-20]. The 1,4-dioxane nucleus, differently and properly
substituted in positions 2 and 5 or 6, has also proved to be a versatile scaffold for building
ligands selectively targeting 5-HT1AR [21-23] besides other different receptors [24-31]. In
particular, the effect of the phenyl substitution at position 5 or 6 of the 1,4-dioxane ring,
bearing a 2-phenoxyethylaminomethyl chain with no, one or two methoxy groups, on 5-
HT1AR and α1-ARs affinity has been investigated [21]. The 6,6-diphenyl substitution resulted
to be more favourable than the 5,5-diphenyl, the 6-phenyl and the 5-phenyl substitution.
Among the 6,6-diphenyl-1,4-dioxane ligands, derivatives 1 and 2 showed high affinity for 5-
HT1AR and α1d-AR subtype and selectivity over α1a-AR and α1b-AR. Instead, 3 preferentially
interacted with 5-HT1AR with respect to α1-ARs. Moreover, from functional studies, 1 and 2
behaved as 5-HT1AR partial agonists, while 3 and its corresponding S enantiomer were potent
full agonists, with pD2 values significantly higher than those of the known 5-HT1AR agonists
5-HT and 8-OH-DPAT. Therefore, for these 6,6-diphenyl derivatives, the ortho dimethoxy
substitution seemed to be responsible for preferential 5-HT1AR affinity and full agonism
(compound 3) [21,22].
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To complete the structure-activity relationship (SAR) studies concerning the presence of the
aromatic portion in different positions of the 1,4-dioxane nucleus, the aim of the present study
was to evaluate the effect of the shifting of one or two phenyl rings from position 5 or 6 to
position 2 or 3 on the affinity/activity for 5-HT1AR and α1-AR subtypes. Each modification
performed on the 1,4-dioxane nucleus was associated with no, one or two methoxy groups in
the phenoxy moiety (compounds 4-12) (Fig. 1).
Moreover, based on the observation that arylpiperazines are the most studied classes of 5-
HT1AR ligands, the flexible 2-ethanolamine linker of the 2-methoxyphenoxy derivatives,
which showed the highest affinities for 5-HT1AR (Table 1), was replaced by the more
conformationally constrained piperazine ring, affording derivatives 13-16. Both the presence
of phenyl substituents in position 2 or 3 of the 1,4-dioxane ring and the replacement of the
flexible 2-ethanolamine function with the more conformationally constrained piperazine ring
reduce the possible conformations assumed by the ligand, allowing us to investigate which
conformations are most preferred for interactions with the 5-HT1AR. The preparation of the
cis and trans diastereomers of compounds 7-9 and 15 helped us to evaluate the role played by
the relative configuration on the biological profile. To rationalize the experimental
observations and derive information about receptor-ligand interactions of the reported 1,4-
dioxane ligands, molecular modelling studies were performed.
In addition, based on the observation that both 5-HT1AR and α1d-ARs are expressed in
different prostate tumor cells and are involved in the modulation of cell proliferation
[14,17,32-35], the cytotoxic effects of selected ligands of the present study on PC-3 and DU-
145 cell lines were evaluated.
5

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O
O
O
R1
N
N
NH
O
O
R2
H3CO
13
1: R1 = R2 = H
2: R1 = OCH3; R2 = H
3
: R1 = R2 = OCH3
H3CO
O
R1
O
4
5
6
3
2
4
5
6
3
2
R
R
1
NH
O
1
N
N
O
O
R2
14
: R = 2-C6H5
4
5
: R = 2-C6H5; R1 = R2 = H
15a
: R = 3-C6H5 (trans)
15b: R = 3-C6H5 (cis)
16
: R = 2-C6H5; R1 = OCH3; R2 = H
6: R = 2-C6H5; R1 = R2 = OCH3
7a
7b
: R = 3,3-(C6H5)2
: R = 3-C6H5 (trans); R1 = R2 = H
: R = 3-C6H5 (cis); R1 = R2 = H
8a: R = 3-C6H5 (trans); R1 = OCH3; R2 = H
8b
9a
: R = 3-C6H5 (cis); R1 = OCH3; R2 = H
: R = 3-C6H5 (trans); R1 = R2 = OCH3
9b: R = 3-C6H5 (cis); R1 = R2 = OCH3
10
11
: R = 3,3-(C6H5)2; R1 = R2 = H
: R = 3,3-(C6H5)2; R1 = OCH3; R2 = H
12: R = 3,3-(C6H5)2; R1 = R2 = OCH3
Fig. 1. Chemical structures of compounds 1-16.
2. Results and discussion
2.1. Chemistry
Compounds 4-6, 13 and 14 were prepared according to the procedure reported in Scheme 1.
(3-Bromoprop-1-en-2-yl)benzene (17) [36] was alkylated with ethylene glycol in presence of
Na to give the ether 18, which was subjected to oxymercuration-reduction reaction with
mercury(II) acetate, followed by an aqueous solution of KI and I2 to yield the iododerivative
19. The amination of 19 with the suitable amine (2-phenoxyethanamine, 2-(2-
methoxyphenoxy)ethanamine [37], 2-(2,6-dimethoxyphenoxy)ethanamine [38], or 1-(2-
6

ACCEPTED MANUSCRIPT
methoxyphenyl)piperazine) yielded 4-6 and 14, respectively and that of tosyl derivative 20
[27] with 1-(2-methoxyphenyl)piperazine afforded 13.
Scheme 1a
aReagents: (a) Na, HOCH2CH2OH; (b) Hg(OAc)2, AcOH, H2O; (c) KI, I2, CHCl3; (d) 2-
phenoxyethanamine,
or
2-(2-methoxyphenoxy)ethanamine,
or
2-(2,6-
dimethoxyphenoxy)ethanamine, CH3OCH2CH2OH, ∆; (e) 1-(2-methoxyphenyl)piperazine, 2-
CH3OCH2CH2OH, ∆.
Compounds 7-12, 15 and 16 were prepared according to the procedure reported in Scheme 2.
Esters 21a and 21b [39] were treated with 2 N NaOH to afford the 1,4-dioxane acids 24a and
.
24b. Treatment of oxyrane 22 [40] with 2-bromoethanol in presence of BF3 Et2O yielded 23,
which was cyclized with KOH to the 1,4-dioxane acid 25. Amidation of 24a, 24b or 25 with
the suitable amines 2-phenoxyethanamine, 2-(2-methoxyphenoxy)ethanamine [37], 2-(2,6-
dimethoxyphenoxy)ethanamine [38] or 1-(2-methoxyphenyl)piperazine in the presence of
.
Et3N and EtOCOCl afforded the amides 26-33, whose reduction with BH3 CH3SCH3 complex
or LiAlH4 gave the ethanolamines 7-12 or the piperazines 15 and 16, respectively.
7

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Scheme 2a
COOCH3
O
22
b
O
O
OCH3
Br
O
O
OH
21a
: (trans)
21b: (cis)
COOCH3
23
O
R
OH
O
O
24a
24b
25
(trans): R = H
(cis): R = H
: R = C6H5
e
d
O
N
26a (trans): R = H; R1 = H; R2 = H
26b (cis): R = H; R1 = H; R2 = H
27a (trans): R = H; R1 = OCH3; R2 = H
27b (cis): R = H; R1 = OCH3; R2 = H
28a (trans): R = H; R1 = R2 = OCH3
28b (cis): R = H; R1 = R2 = OCH3
29: R = C6H5; R1 = R2 = H
R
O
N
OCH3
O
O
R
O
O
R1
O
H
32a (trans): R = H
32b (cis): R = H
33: R = C6H5
N
R2
30: R = C6H5; R1 = OCH3; R2 = H
31: R = C6H5; R1 = R2 = OCH3
g
f
7a (trans): R = H; R1 = H; R2 = H
7b (cis): R = H; R1 = H; R2 = H
8a (trans): R = H; R1 = OCH3; R2 = H
8b (cis): R = H; R1 = OCH3; R2 = H
9a (trans): R = H; R1 = R2 = OCH3
9b (cis): R = H; R1 = R2 = OCH3
10: R = C6H5; R1 = R2 = H
O
O
N
O
R
O
R1
O
R
H
N
OCH3
N
15a (trans): R = H
15b (cis): R = H
16: R = C6H5
R2
11: R = C6H5; R1 = OCH3; R2 = H
12: R = C6H5; R1 = R2 = OCH3
.
aReagents: (a) 2 N NaOH; (b) BrCH2CH2OH, BF3 Et2O, CH2Cl2; (c) KOH, MeOH/H2O; (d)
2-phenoxyethanamine,
or
2-(2-methoxyphenoxy)ethanamine,
or
2-(2,6-
dimethoxyphenoxy)ethanamine, Et3N, ClCOOEt, CHCl3; (e) 1-(2-methoxyphenyl)piperazine,
.
Et3N, ClCOOEt, CHCl3 (f) BH3 CH3SCH3, THF (g) LiAlH4, THF.
2.2. Binding experiments
8

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The pharmacological profiles of 4-16 were evaluated by radioligand competition binding
assays with human recombinant α1-AR subtypes expressed in Chinese hamster ovary (CHO)
cells using the labelled ligand [3H]Prazosin and with human recombinant 5-HT1ARs expressed
in HeLa cells using the labelled ligand [3H]8-OH-DPAT, according to previously reported
procedures [23,41,42]. The affinity values, expressed as pKi, are reported in Table 1 together
with those of 1-3, WAY100635, 8-OH-DPAT and BMY-7378, included as reference
compounds.
Moreover, the most interesting compounds 11, 13 and 16 were tested at human recombinant
D2, D3 and D4 receptors stably expressed in HEK293 cells by competition binding
experiments, using [3H]N-methylspiperone as the radioligand, according to previously
reported procedures [41,43]. The results are discussed together with those of 2 [23], included
for useful comparison.
2.3. Functional studies
The functional characterization of compounds 8a, 8b, 10, 11, 13, and 16 showing the highest
affinities for 5-HT1AR, was performed according to previously reported procedures
[23,44,45], using [35S]GTPγS binding, in cell membranes from HeLa cells transfected with
human cloned 5-HT1AR. Stimulation of [35S]GTPγS binding was expressed as the percent
increase in binding above the basal value, considering that the maximal stimulation observed
is with 8-OH-DPAT and was thus taken as 100%. The results are reported in Table 2 together
with those of 1-2 included for useful comparison and those of WAY100635 and 8-OH-DPAT
included as reference compounds.
2.4. SAR and molecular modelling studies
9

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The data reported in Table 1 show that all the novel compounds preferentially bind to 5-
HT1AR with respect to the three α1-AR subtypes, except for compound 6, which doesn’t
interact with any of the studied receptors, compounds 5 and 11, which show similar affinity
values for 5-HT1AR and α1d-AR subtype, and compounds 13 and 15a, which show a
preference for α1d-AR subtype.
Table 1. Affinity constants (pKi) of 1-16, WAY 100635, 8-OH-DPAT, and BMY-7378 for
human recombinant α1-AR subtypes and 5-HT1AR.a
pKi, human cloned receptors
Compound
R
R1
R2
α1a
α1b
α1d
5-HT1A
6,6-(C6H5)2
6,6-(C6H5)2 OCH3
6,6-(C6H5)2 OCH3
H
H
H
OCH3
H
H
OCH3
H
H
H
H
OCH3
OCH3
H
H
OCH3
6.77
7.56
6.47
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
8.58
<6
<6
<6
6.92
7.25
6.49
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
7.17
<6
7.64
<6
<6
8.44
8.94
7.18
<6
7.16
<6
<6
<6
7.02
6.90
<6
9.23
9.18
8.85
6.57
7.12
<6
1
2
3
4
5
6
2-C6H5
2-C6H5
2-C6H5
3-C6H5
3-C6H5
3-C6H5
3-C6H5
3-C6H5
3-C6H5
3,3-(C6H5)2
3,3-(C6H5)2 OCH3
3,3-(C6H5)2 OCH3
6,6-(C6H5)2
2-C6H5
3-C6H5
3-C6H5
H
OCH3
OCH3
H
7a (trans)
7b (cis)
8a (trans)
8b (cis)
9a (trans)
9b (cis)
10
11
12
13
14
15a (trans)
15b (cis)
16
WAY 100635
8-OH-DPAT
7.26
6.67
8.02
7.69
7.01
6.74
7.55
8.40
7.18
8.30
6.76
6.67
7.15
7.92
9.48
8.47
H
OCH3
OCH3
OCH3
OCH3
H
<6
6.50
8.13
<6
8.76
<6
7.27
6.72
7.05
8.34
<6
-
-
-
-
-
-
-
-
-
-
<6
3,3-(C6H5)2
6.45
7.73
<6
6.42
7.18
<6
10

ACCEPTED MANUSCRIPT
BMY 7378
6.42
6.15
8.89
9.43
aAffinity values are reported as pKi = -logKi. Equilibrium dissociation constants (Ki) were derived from IC50
values using the Cheng-Prusoff equation [46]. Each experiment was performed in triplicate. Ki values were from
three experiments, which agreed within ±20%.
11

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Concerning the 2-ethanolamine derivatives 1-12, the presence of a phenyl ring in position 2 of
the 1,4-dioxane nucleus (compounds 4-6) proves to be unfavourable for the interaction with
both receptor systems. The increased steric bulk in this position might alter the optimal
molecule geometry, interfering with the binding to the corresponding receptor sites.
When the phenyl ring of compounds 4-6 is shifted from position 2 to 3 of the 1,4-dioxane
nucleus, affording the pairs of diastereomers 7-9, the binding profile at α1-ARs is not
significantly affected, while the 5-HT1AR affinities increase (4 vs 7, 5 vs 8 and 6 vs 9).
Among them, the mono-methoxy derivatives 8a and 8b show the highest pKi values at 5-
HT1AR and significant selectivity over α1-ARs. Compared to the 6,6-diphenyl derivative 2,
compounds 8a and 8b show lower 5-HT1AR affinity values but increased 5-HT1AR/α1-ARs
selectivity ratios.
Though the stereochemical relationship between the phenyl ring in position 3 and the side
chain in position 2 doesn’t seem to play a crucial role in the interaction with 5-HT1AR,
however, the affinity values of the trans diastereomers 7a, 8a, and 9a are slightly higher than
those of the corresponding cis diastereomers 7b, 8b, and 9b.
The increase of the steric bulk by inserting a second phenyl ring in position 3 of the 1,4-
dioxane nucleus of 7-9 is tolerated for the interaction with 5-HT1AR. Indeed, derivatives 10-
12 show pKi values for 5-HT1AR even slightly higher than those of the corresponding mono-
phenyl derivatives. Also in this case the presence of one methoxy group in the phenoxy
moiety (11) confers to the ligand the highest 5-HT1AR affinity (pKi = 8.40). However, this
structural modification also causes a significant increase of α1d-AR affinity (pKi = 8.13), with
a consequent reduction of 5-HT1A/α1d-AR selectivity ratio. On the contrary, a good 5-
HT1A/α1d-AR selectivity ratio is maintained by the unsubstituted and the dimethoxy-
substituted derivatives (10 and 12, respectively). Such a selectivity trend is similar to that of
the corresponding 6,6-diphenyl derivatives 1-3.
12

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The SARs observed for the piperazine derivatives 13-16 are different from those of the
corresponding 2-ethanolamine compounds 2, 5, 8, and 11 respectively. The replacement of
the 2-ethanolamine moiety of the lead 2 with a piperazine ring, affording 13, decreases the
affinity for 5-HT1A-R and increases that for α1a-AR. Consequently, a preference for α1a-/α1d-
AR subtypes with respect to 5-HT1AR is observed.
Also in the piperazine series the presence of a phenyl ring in position 2 is detrimental to the
affinity for all the studied receptors (compound 14). The shifting of the phenyl ring from
position 2 to 3, affording 15a and 15b, causes an effect different from that observed for the 2-
ethanolamine derivatives 8a and 8b. Indeed, while both 8a and 8b show a preference for 5-
HT1AR with respect to α1-AR subtypes, only the cis isomer 15b preferentially interacts with
5-HT1AR. On the contrary, the trans diastereomer 15a shows a preference for α1d-AR. The
insertion of a second phenyl ring in position 3 of the 1,4-dioxane nucleus, affording 16,
causes a significant increase of 5-HT1AR affinity and, unlike the 2-ethanolamine derivative
11, a consequent increased 5-HT1AR/α1d-AR selectivity ratio.
Considering the high degree of homology between 5-HT1AR, α1-ARs and dopamine receptors
[15] and based on the observation that arylpiperazine demonstrated to be a suitable scaffold of
dopamine D2-like receptor ligands [47], the most interesting piperazine derivatives 13 and 16
were also evaluated for their binding affinity at cloned human D2, D3 and D4 receptor
subtypes. The corresponding ethanolamines 2 and 11 were included in this study for useful
comparison. Both piperazines 13 (pKi D2 = 6.95; D3 = 6.59; D4 = 5.86) and 16 (pKi D2 = 5.66;
D3 = 5.54; D4 = 5.01) showed affinity values lower than those of the corresponding
ethanolamines 2 (pKi D2 = 7.91; D3 = 7.88; D4 = 8.08) [23] and 11 (pKi D2 = 6.49; D3 = 6.27;
D4 = 6.56), respectively, for all the dopamine D2-like receptor subtypes. Moreover, the 3,3-
diphenyl derivatives 11 and 16 bound D2, D3 and D4 subtypes with affinity values lower than
those of the 6,6-diphenyl compounds 2 and 13, respectively. Interestingly, for all the newly
13

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tested compounds 11, 13 and 16 the affinity for 5-HT1AR was significantly higher than those
for D2-like subtypes, with a consequent high selectivity for 5-HT1AR over D2-like receptors.
More and more interesting results emerge from the functional studies performed with
compounds 8a, 8b, 10, 11, 13 and 16, selected on the basis of their highest 5-HT1AR
affinities. The data reported in Table 2 show that the stereochemical relationship between the
phenyl ring in position 3 and the side chain in position 2 of the 1,4-dioxane nucleus
significantly affects the functional activity at 5-HT1AR. Indeed, while the cis diastereomer 8b
behaves as a 5-HT1AR partial agonist (pD2 = 7.13, Emax = 24.3), the corresponding trans
isomer is a 5-HT1AR antagonist. The presence of two phenyl ring in position 3 makes the
phenoxy derivative 10 a weak 5-HT1AR partial agonist (pD2 = 6.71, Emax = 32.9) and the 2-
methoxyphenoxy derivative 11 a potent 5-HT1AR antagonist, with a pKB value of 8.50. The
crucial role of the diphenyl moiety in position 3 of the 1,4-dioxane nucleus and of one ortho
methoxy group in the phenoxy moiety in conferring an antagonism behaviour to the ligand is
confirmed by the functional results of the piperazine compound 16, which also behaves as a
potent 5-HT1AR antagonist (pKB = 8.14). Therefore, the most interesting result of the present
paper is that a modulation of the 5-HT1AR functional profile from agonism to antagonism is
obtained by shifting the diphenyl moiety of the 2-methoxy agonists 2 and 13 from position 6
to position 3 of the 1,4-dioxane nucleus, affording the novel antagonists 11 and 16,
respectively. Moreover, the intriguing mixed 5-HT1AR/α1d-AR profile of compound 11 might
be therapeutically beneficial in the treatment of prostate cancer.
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Table 2. Antagonist Potency (pKB), Agonist potency (pD2) and relative efficacy (Emax%) at
the human recombinant 5-HT1AR.
5-HT1A Binding [35S]GTPγS
Compound
R
R1
R2
pKi
apD2 (bpKB) cEmax%
6,6-(C6H5)2
6,6-(C6H5)2 OCH3
3-C6H5
3-C6H5
3,3-(C6H5)2
3,3-(C6H5)2 OCH3
6,6-(C6H5)2
3,3-(C6H5)2
H
H
H
H
H
H
H
-
9.23
9.18
8.02
7.69
7.55
8.40
8.30
7.92
9.48
8.47
9.11
9.40
(8.20)
7.13
6.71
(8.50)
7.01
(8.14)
(9.33)
7.60
77.1
81.5
-
24.3
32.9
-
39.3
-
-
1
2
8a (trans)
8b (cis)
10
11
13
16
OCH3
OCH3
H
-
-
-
WAY 100635
8-OH-DPAT
100
apD2 values are the negative logarithm of the agonist concentration required to obtain 50% of the
maximal stimulation of [35S]GTPγS binding and were calculated from three experiments, which
agreed within ±20%.bpKB values were evaluated according to the equation pKB = log10 ([B]/(dr - 1)),
where [B] is the antagonist concentration and the dose-ratio (dr) is the ratio between the
concentrations of agonist required to produce half-maximal response in the presence and the absence
c
of the antagonist. Maximal stimulation is expressed as a percentage of the maximal 8-OH-DPAT
response.
With a view to further rationalizing the biological results and to delving into the features
which modulate the shift between agonist and antagonist activity, docking simulations were
performed on the human 5-HT1AR homology model by using PLANTS [48] and by applying
the same previously reported computational procedures [23]. Since the study comprises both
agonists and antagonists, docking simulations involved only the 5-HT1AR homology model in
its open inactive state which was generated by using the resolved 5-HT1B structure in complex
with ergotamine (PDB Id = 4IAR) and should be able to conveniently accommodate all
simulated ligands.
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In detail, the following discussion will be focused on the comparison of three pairs of close
analogues (i.e. 8a vs. 8b, 10 vs. 11 and 13 vs. 16) which differ for their intrinsic activity and
allow a precise evaluation of the role played by the configurational aspects of the dioxane ring
(8a vs. 8b) as well as by the substituents on the phenoxy ring (10 vs. 11) and on the dioxane
nucleus (13 vs. 16). Thus, Fig. 4A compares the best putative complexes as computed for 8a
and 8b within the binding cavity of the inactive open 5-HT1AR model and reveals significant
differences involving both the phenoxy ring and the 3-phenyl dioxane moiety. The key
stabilizing interactions and the observed differences can be summarized as follows: (1) the
protonated amino group stabilizes the expected key contacts with Asp116 and Tyr390; (2) the
dioxane ring elicits H-bonds with Gln97 and Asn386; these contacts appear to be stronger in
the putative complex of 8a; (3) in both complexes the phenoxy ring approaches Phe361 and
Phe362 even with different arrangements which impact on its closeness to Ala203 (see
below); (4) only the methoxy group of 8a is able to elicit clear H-bonds with Ser199 and
Thr200, while the same function in 8b at most approaches Ala365; (5) the 3-phenyl ring
assumes slightly different poses in the two ligands: in both cases, it contacts Phe112 and
Tyr96 but only 8b is able to also approach Trp102 and the disulphide bridge Cys109-Cys187.
On these grounds, one may suppose that the greater affinity and potency of 8a can be
explained by considering the stronger H-bonds elicited by both the methoxy group and the
dioxane ring, while the shift between agonist and antagonist activity can be ascribed to the
different arrangements of the aromatic rings which, though stabilizing comparable π-π
stacking interactions in the two ligands, contact different residues. In detail, attention is here
focused on the resulting different distance between the phenoxy ring and Ala203 since a
recent study revealed that the activity shift as seen between methysergide (antagonist) and its
demethylated metabolite (methylergonovine, agonist) in 5-HT2BR can be rationalized by
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considering the steric clashes between the side chain of Ala225 and the N-methyl group of the
former [49].
Fig. 4. Comparison of the key interactions stabilizing the putative complexes of (A) 8a vs. 8b,
(B) 10 vs. 11 and (C) 13 vs. 16. In all Figures, the antagonist is colored by atom and the
agonist is colored in blue for easy identification. The inlet in Fig. 4A shows the alignment of
the TM5 helix of 5-HT1AR and 5-HT2BR focusing on the region around Ala203.
As indicated in the inlet in Fig. 4A, sequence alignment reveals that Ala225 of 5-HT2BR
corresponds to Gly202 in 5-HT1AR and thus the residue which seems to have a comparable
role in 5-HT1AR is Ala203 which indeed correspond to a conserved Ala226 in 5-HT1BR. As
anticipated above, the different pose of the phenoxy ring results in a different closeness with
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Ala203. In detail, while the agonist 8b remains at 5 Å from Ala203, the antagonist 8a is
seen to approach Ala203 at 3 Å, a significantly reduced distance which can induce steric
clashes especially when considering that the arrangement of the phenoxy ring of 8a is here
markedly tethered by the H-bonds elicited by the methoxy group.
The role of the different arrangement of the phenoxy ring is further confirmed by the second
comparison between 10 and 11. Fig. 4B compares the two computed poses and reveals a
satisfactory superimposition of the two ligands apart from the phenoxy ring which also here
shows different arrangements and the ring of 11 appears to be closer to Ala203 than that of 10
(3.5 Å vs. 4.5 Å) and also in this case the arrangement of the phenoxy ring of the antagonist
11 is rigidified by the H-bonds elicited by the methoxy group with Ser199 and Thr200 which
are clearly missed in the unsubstituted analogue 10 and can also explain the greater affinity
observed for 11.
The last comparison (13 vs 16) further confirms the role of Ala203 and offers additional
insights concerning the potential role of the residues belonging to the extracellular loops. Fig.
4C compares the two corresponding complexes and reveals that the phenoxy rings, the
dioxane nucleus and the connected phenyl rings are differently accommodated. With regard to
the phenoxy moiety, the two ligands similarly approach Ala203 (at 3.5 Å) even though the
potential clashes involve the phenyl ring in the antagonist 16 and the methoxy group in the
agonist 13. One may suppose that the methoxy group of 13, which is not involved in clear H-
bonds, should have flexibility enough to minimize the steric clashes, while the phenyl ring of
16, which stabilizes π−π stacking with Phe361 and Phe362, should remain fixed against
Ala203. Concerning the phenyl rings connected to dioxane and similarly to what was
described in the first comparison, both ligands approach Phe112 and Tyr96 but only agonist
13 is able to contact Trp102 and the disulphide bridge Cys109-Cys187. As an aside and for
the sterically less hindered ligands, docking results also generated overturned poses in which
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the dioxane moiety approach Ser199 and Thr200 and the phenoxy ring contacts Phe112 and
Tyr96. Although these poses are clearly understandable by considering the rather symmetric
structure of these ligands, the here reported complexes can be preferred when considering the
frequency of the two binding modes, the computed docking scores and the offered structure-
activity relationships. For example, when considering the five best score poses as generated
for the four possible stereoisomers of the compounds 8a and 8b, 16 out of the 20 monitored
complexes assume the here discussed binding mode which represents the majority in all the
four considered isomers and always corresponds to the best generated pose.
Taken together, the reported docking results suggest that the receptor activation is influenced
by both the steric hindrance elicited around Ala203 and the capacity to approach key residues
on the extracellular loops such as the disulphide bridge. The first factor appears to be more
relevant because it is present in all considered comparisons and received a compelling
confirmation by recent experimental studies. Nevertheless, an optimal involvement of some
key residues of the ECL segments also seems to have a non-negligible role, especially for
sterically hindered ligands as seen in the last comparison, where the different poses of the
diphenyl system seem to play a pivotal role as also confirmed by experimental studies which
investigated the role of ECL segments in receptor binding and activation [50].
2.5. Cell growth inhibition studies
Based on the observation that both 5-HT1AR and α1d-ARs are expressed in different prostate
tumor cells and are involved in the modulation of cell proliferation [14,17, 32-35], the in vitro
antiproliferative effects of the potent α1d-AR antagonist 2 [21], the mixed 5-HT1AR/α1d-AR
antagonist 11 and the preferential 5-HT1AR antagonist 16 were evaluated on PC-3 and DU-
145 prostate cancer cell lines, as well as on normal human fibroblast cells as control [51,52],
by the sulforhodamine B (SRB) assay, according to the National Cancer Institute protocol
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[53]. PC-3 and DU-145 cells were selected as it is well known that they both are androgen
independent, tumorigenic and with potential metastatic abilities. Therefore, they represent a
good in vitro model of hormonal refractory cells, useful to evaluate therapies alternative to the
conventional ones. Moreover, evidences indicate that both 5-HT1AR and α1d-AR antagonists
are able to inhibit the proliferation of these cell lines [14, 17, 32-35].
The data reported in Fig. 2 show that all the compounds demonstrated to reduce dose-
dependently the growth of both cell lines after 48 h of culture.
Fig. 2. In vitro antitumor activity of 2, 11 and 16 in human PC-3 and DU-145 cells was
carried out using SRB assay. Growth Inhibition 50 (GI50) represents the drug concentration
(µM) required to inhibit 50% net of cell growth. Total Growth Inhibition (TGI) represents the
drug concentration (µM) required to inhibit 100% of cell growth. Lethal Concentration 50
(LC50) represents the drug concentration (µM) required to kill 50% of the initial cell number.
Each quoted value represents the mean of quadruplicate determinations ± standard error (SE).
*p<0.01 vs untreated cells.
In particular, compound 11 is able to inhibit the growth of the PC-3 and DU-145 cells with
GI50, TGI and LC50 values lower than those of the α1d-AR antagonist 2 and the 5-HT1AR
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antagonist 16 in both cell lines. Interestingly, this result suggests that the combination of 5-
HT1AR and α1d-AR antagonism might be responsible for the best antitumor activity displayed
by 11. Moreover, all the compounds proved to be more effective on PC-3 and DU-145 cells
respect to normal human fibroblasts (2: GI50 = 38.4 ± 2.6 µM, TGI = 121.6 ± 4.7 µM, LC50 >
300 µM; 11: GI50 = 30.5 ± 2.3 µM, TGI = 50.2 ± 1.9 µM, LC50 = 99.6 ± 3.9 µM; 16: GI50 >
300 µM, TGI> 300 µM, LC50 > 300 µM).
To confirm the involvement of both HT1AR and α1d-AR in the antitumor activity of compound
11, the cell viability was evaluated in PC-3 and DU-145 cells stimulated with the endogenous
agonists norepinephrine (NE, 100 µM) or 5-HT (100 µM), alone or in combination with sub-
optimal doses of 11 (5 or 10 µM). As shown in Fig. 3, NE and 5-HT significantly increased
proliferation in both cancer cell lines. Interestingly, the mixed 5-HT1AR/α1d-AR antagonist 11,
at the doses of 5 and 10 µM, markedly reduces the proliferation induced by both the
endogenous agonists, demonstrating that both receptor systems are involved in its antitumor
activity.
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Fig. 3. Cell viability was evaluated by SRB assay in PC-3 and DU-145 cells treated with NE
or 5-HT in combination or not with sub-optimal doses (5 or 10 µM) of compound 11. Each
*
quoted value represents the mean of quadruplicate determinations ± SE. p<0.01 vs medium
(control); °p<0.01 vs NE or 5-HT treated cells.
3. Conclusion
In conclusion, the shifting of the diphenyl moiety from position 6 to position 3 of the 1,4-
dioxane nucleus of the potent 5-HT1AR agonist 2, affording 11, caused a modulation of the 5-
HT1AR profile from agonism to antagonism. This result was confirmed by compounds 13 and
16, in which the flexible 2-ethanolamine function of 2 and 11, respectively, was replaced by
the more conformationally constrained piperazine ring. Indeed, unlike the 6,6-diphenyl
derivative 13, which partially activates the 5-HT1AR, the 3,3-diphenyl compound 16 behaves
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as a potent antagonist. Docking simulations, performed on the human 5-HT1AR, further
rationalized the biological results and suggested that the receptor activation is influenced by
both the steric hindrance elicited around Ala203 and the capacity to approach key residues on
the extracellular loops. Interestingly, compound 11, endowed with mixed 5-HT1AR/α1d-AR
antagonist profile, shows antiproliferative and cytotoxic effects on both PC-3 and DU-145
prostate cancer cell lines higher than those of the α1d-AR antagonist 2 and the 5-HT1AR
antagonist 16. This result, suggesting the involvement of both receptor systems, was
confirmed by the experiments performed in the presence of the endogenous agonists NE and
5-HT. Due to its interesting biological profile, 11 may represent a lead compound for the
development of novel antitumor agents for the treatment of prostate cancer.
4. Experimental section
4.1. Chemistry
4.1.1. General
Melting points were taken in glass capillary tubes on a Büchi SMP-20 apparatus and are
uncorrected. IR and NMR and NOE spectra were recorded on Perkin-Elmer 297 and Varian
Mercury AS400 instruments, respectively. Chemical shifts are reported in parts per million
(ppm) relative to tetramethylsilane (TMS), and spin multiplicities are given as s (singlet), d
(doublet), dd (double doublet), t (triplet), or m (multiplet). IR spectral data (not shown
because of the lack of unusual features) were obtained for all compounds reported and are
consistent with the assigned structures. Mass spectra were obtained using a Hewlett Packard
1100 MSD instrument utilizing electron-spray ionization (ESI). The microanalyses were
recorded on FLASH 2000 instrument (ThermoFisher Scientific). The elemental composition
of the compounds agreed to within ±0.4% of the calculated value. When the elemental
analysis was not included, crude compounds were used in the next step without further
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purification. Chromatographic separations were performed on silica gel columns (Kieselgel
40, 0.040-0.063 mm, Merck) by flash chromatography. The term “dried” refers to the use of
anhydrous sodium sulfate. Chemical names were generated according to IUPAC
Nomenclature of Organic Chemistry (http://www.acdlabs.com/iupac/nomenclature). The
purity of the novel compounds was determined by combustion analysis and was ≥95%.
4.1.2. General procedure for the synthesis of compounds 4-6, 13 and 14.
A solution of iodide 19 or tosylate 20 (1.65 mmol) and the suitable amine (2-
phenoxyethanamine,
2-(2-methoxyphenoxy)ethanamine
[37],
2-(2,6-
dimethoxyphenoxy)ethanamine [38], or 1-(2-methoxyphenyl)piperazine) (3.30 mmol) in 2-
methoxyethanol (10 mL) was refluxed for 4 h. After evaporation of the solvent, the residue
was dissolved in 2 N NaOH and extracted with CHCl3, which was dried over Na2SO4. After
evaporation of the solvent the residue was purified by column chromatography eluting with
EtOAc/MeOH (95:5).
4.1.2.1. 2-Phenoxy-N-((2-phenyl-1,4-dioxan-2-yl)methyl)ethanamine (4). This
compound was prepared starting from 19 and 2-phenoxyethanamine: an oil was obtained
(53% yield). 1H NMR (CDCl3) δ 1.50 (br s, 1H, exchangeable with D2O), 2.90 (m, 4H), 3.06
(d, 1H, J = 12.1 Hz), 3.58-4.01 (m, 6H), 4.25 (d, 1H, J = 12.1 Hz), 6.79-7.32 (m, 10H). The
free base was transformed into the oxalate salt that was crystallized from EtOH: mp 200-201
°C. ESI/MS: m/z 314.2 [M + H]+. Anal. Calcd for C19H23NO3·H2C2O4: C, 62.52%; H, 6.25%;
N, 3.47%. Found: C, 62.73%; H, 6.41%; N, 3.36%.
4.1.2.2. 2-(2-Methoxyphenoxy)-N-((2-phenyl-1,4-dioxan-2-yl)methyl)ethanamine (5).
This compound was prepared starting from 19 and 2-(2-methoxyphenoxy)ethanamine [37]: an
1
oil was obtained (33% yield). H NMR (CDCl3) δ 1.72 (br s, 1H, exchangeable with D2O),
2.95 (m, 3H), 3.09 (d, 1H, J = 12.1 Hz), 3.60-4.08 (m, 7H), 3.78 (s, 3H), 4.27 (d, 1H, J = 12.1
Hz), 6.82-7.52 (m, 9H). The free base was transformed into the oxalate salt that was
24

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crystallized from EtOH: mp 194-195 °C. 13C NMR DMSO δ (ppm): 164.6, 149.7, 147.6,
139.5, 129.1, 128.4, 126.7, 122.4, 121.2, 114.6, 112.7, 74.9, 70.4, 66.3, 65.7, 61.2, 55.9, 52.4,
47.6. ESI/MS: m/z 344.2 [M + H]+. Anal. Calcd for C20H25NO4·H2C2O4: C, 60.96%; H,
6.28%; N, 3.23%. Found: C, 60.80%; H, 6.43%; N, 3.11%.
4.1.2.3. 2-(2,6-Dimethoxyphenoxy)-N-((2-phenyl-1,4-dioxan-2-yl)methyl)ethanamine
(6). This compound was prepared starting from 19 and 2-(2,6-dimethoxyphenoxy)ethanamine
[38]: an oil was obtained (30% yield). 1H NMR (CDCl3) δ 1.65 (br s, 1H, exchangeable with
D2O), 2.78-2.98 (m, 4H), 3.60-4.13 (m, 7H), 3.75 (s, 6H), 4.29 (d, 1H, J = 12.1 Hz), 6.52-7.55
(m, 8H). The free base was transformed into the oxalate salt that was crystallized from 2-
PrOH: mp 139-140 °C. ESI/MS: m/z 374.2 [M + H]+. Anal. Calcd for C21H27NO5·H2C2O4:
C, 59.60%; H, 6.31%; N, 3.02%. Found: C, 59.43%; H, 6.12%; N, 3.12%.
4.1.2.4. 1-((6,6-diphenyl-1,4-dioxan-2-yl)methyl)-4-(2-methoxyphenyl)piperazine (13).
This compound was prepared starting from 20 and 1-(2-methoxyphenyl)piperazine: a solid
1
was obtained (33% yield; mp 54-55 °C). H NMR (CDCl3) δ 2.42-3.18 (m, 10H), 3.40-3.88
(m, 4H), 3.84 (s, 3H), 4.61 (d, 1H, J = 12.3 Hz), 6.81-7.60 (m, 14H). The free base was
13
transformed into the oxalate salt that was crystallized from EtOH: mp 134-136 °C. C NMR
DMSO δ (ppm): 163.6, 152.4, 144.2, 142.0, 140.5, 128.9, 128.5, 128.5, 127.9, 127.5, 125.7,
123.5, 121.3, 118.7, 112.4, 79.1, 71.5, 68.6, 66.3, 57.3, 55.9, 53.0, 48.3. ESI/MS: m/z 445.2
[M + H]+. Anal. Calcd for C28H32N2O3·H2C2O4·H2O: C, 65.20%; H, 6.57%; N, 5.07%.
Found: C, 65.03%; H, 6.34%; N, 4.93%.
4.1.2.5. 1-(2-methoxyphenyl)-4-((2-phenyl-1,4-dioxan-2-yl)methyl)piperazine (14).
This compound was prepared starting from 19 and 1-(2-methoxyphenyl)piperazine: an oil was
1
obtained (51% yield). H NMR (CDCl3) δ 2.28-3.01 (m, 10H), 3.45-3.81 (m, 4H), 3.82 (s,
3H), 3.99 (d, 1H, J = 12.1 Hz), 4.43 (d, 1H, J = 12.1 Hz), 6.79-7.55 (m, 9H). The free base
was transformed into the oxalate salt that was crystallized from 2-PrOH: mp 140-141 °C. 13C
25

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NMR DMSO δ (ppm): 163.3, 152.3, 141.1, 140.9, 128.8, 127.9, 126.9, 123.2, 121.3, 118.4,
112.3, 76.2, 70.9, 66.5, 62.5, 61.2, 55.7, 54.6, 48.9. ESI/MS: m/z 369.2 [M + H]+. Anal. Calcd
for C22H28N2O3·H2C2O4: C, 62.87%; H, 6.60%; N, 6.11%. Found: C, 62.79%; H, 6.72%; N,
5.98%.
4.1.3. General procedure for the synthesis of compounds 7-12.
.
A solution of 10 M BH3 CH3SCH3 (1.7 mL) in dry THF (10 mL) was added dropwise at rt to
a stirred solution of the suitable amide 26-31 (3.10 mmol) in dry THF (50 mL) under a stream
of dry nitrogen with exclusion of moisture. When the addition was completed, the reaction
mixture was heated at reflux for 8 h. After the mixture was cooled to 0 °C, excess borane was
destroyed by cautious dropwise addition of EtOH (13 mL). The resulting mixture was left to
stand overnight at rt, cooled to 0 °C, acidified with concentrated HCl, and then heated to 60
°C for 1 h. Removal of the solvent under reduced pressure gave a residue, which was
dissolved in H2O. The aqueous solution was basified with 2 N NaOH and extracted with
CHCl3. Removal of dried solvents gave a residue, which was purified by column
chromatography, eluting with EtOAc.
4.1.3.1. trans-2-Phenoxy-N-((3-phenyl-1,4-dioxan-2-yl)methyl)ethanamine (7a). This
1
compound was prepared starting from 26a: an oil was obtained (78% yield). H NMR
(CDCl3) δ 1.82 (br s, 1H, exchangeable with D2O), 2.42-2.92 (m, 4H), 3.57-4.02 (m, 7H),
4.38 (d, 1H, J = 9.0 Hz), 6.84-7.42 (m, 10H). The free base was transformed into the oxalate
salt that was crystallized from MeOH: mp 213-214 °C. ESI/MS: m/z 314.2 [M + H]+. Anal.
Calcd for C19H23NO3·H2C2O4: C, 62.52%; H, 6.25%; N, 3.47%. Found: C, 62.39%; H,
6.29%; N, 3.47%.
4.1.3.2. cis-2-Phenoxy-N-((3-phenyl-1,4-dioxan-2-yl)methyl)ethanamine (7b). This
1
compound was prepared starting from 26b: an oil was obtained (70% yield). H NMR
(CDCl3) δ 2.12 (br s, 1H, exchangeable with D2O), 2.24 (dd, 1H, J = 12.7, 3.4 Hz), 2.93 (t,
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2H, J = 5.5 Hz), 3.20 (dd, 1H, J = 12.7, 10.8 Hz), 3.60-4.15 (m, 7H), 4.90 (d, 1H, J = 3.5 Hz),
6.82-7.39 (m, 10H). The free base was transformed into the oxalate salt that was crystallized
from MeOH: p.f. 222-223°C. ESI/MS: m/z 314.2 [M + H]+.
Anal. Calcd for
C19H23NO3·H2C2O4: C, 62.52%; H, 6.25%; N, 3.47%. Found: C, 62.63%; H, 6.31%; N,
3.48%.
4.1.3.3. trans-2-(2-Methoxyphenoxy)-N-((3-phenyl-1,4-dioxan-2-yl)methyl)ethanamine
1
(8a). This compound was prepared starting from 27a: an oil was obtained (30% yield). H
NMR (CDCl3) δ 1.81 (br s, 1H, exchangeable with D2O), 2.42-2.96 (m, 4H), 3.61-4.08 (m,
7H), 3.90 (s, 3H), 4.36 (d, 1H, J = 9.0 Hz), 6.83-7.41 (m, 9H). The free base was transformed
13
into the oxalate salt that was crystallized from EtOH: mp 176–177 °C. C NMR DMSO δ
(ppm): 165.0, 149.9, 147.5, 137.9, 129.1, 129.0, 128.0, 122.6, 121.2, 115.3, 112.8, 80.2, 76.1,
66.6, 66.5, 65.6, 56.0, 47.9, 46.8. ESI/MS: m/z 344.2 [M + H]+. Anal. Calcd for
C20H25NO4·H2C2O4: C, 60.96%; H, 6.28%; N, 3.23%. Found: C, 60.73%; H, 6.27%; N,
3.18%.
4.1.3.4. cis-2-(2-Methoxyphenoxy)-N-((3-phenyl-1,4-dioxan-2-yl)methyl)ethanamine
1
(8b). This compound was prepared starting from 27b: an oil was obtained (45% yield). H
NMR (CDCl3) δ 1.72 (br s, 1H, exchangeable with D2O), 2.21 (dd, 1H, J = 12.7, 3.6 Hz), 2.93
(t, 2H, J = 5.5 Hz), 3.21 (dd, 1H, J = 12.7, 10.6 Hz), 3.58-4.16 (m, 7H), 3.85 (s, 3H), 4.88 (d,
1H, J = 3.4 Hz), 6.85-7.39 (m, 9H). The free base was transformed into the oxalate salt that
was crystallized from EtOH: mp 181-182 °C. 13C NMR DMSO δ (ppm): 164.5, 149.8, 147.5,
137.7, 128.9, 128.2, 126.1, 122.6, 121.1, 115.1, 112.7, 77.2, 71.8, 66.9, 65.5, 59.5, 55.9, 46.5,
43.0. ESI/MS: m/z 344.2 [M + H]+. Anal. Calcd for C20H25NO4·H2C2O4·H2O: C, 58.52%; H,
6.47%; N, 3.10%. Found: C, 58.88%; H, 6.32%; N, 3.09%.
4.1.3.5.
trans-2-(2,6-Dimethoxyphenoxy)-N-((3-phenyl-1,4-dioxan-2-
yl)methyl)ethanamine (9a). This compound was prepared starting from 28a: an oil was
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obtained (88% yield). 1H NMR (CDCl3) δ 2.46-2.87 (m, 4H), 3.81 (s, 6H), 3.82-4.20 (m, 8H),
4.30 (d, 1H, J = 9.0 Hz), 6.52-7.40 (m, 8H). The free base was transformed into the oxalate
salt that was crystallized from 2-PrOH/Et2O: mp 138-140°C. ESI/MS: m/z 374.2 [M + H]+.
Anal. Calcd for C21H27NO5·H2C2O4: C, 59.60%; H, 6.31%; N, 3.02%. Found: C, 59.79%; H,
6.49%; N, 2.91%.
4.1.3.6.
cis-2-(2,6-Dimethoxyphenoxy)-N-((3-phenyl-1,4-dioxan-2-
yl)methyl)ethanamine (9b). This compound was prepared starting from 28b: an oil was
obtained (57% yield). 1H NMR (CDCl3) δ 2.24 (dd, 1H, J = 12.7, 3.3 Hz), 2.90 (m, 2H), 3.31
(dd, 1H, J = 12.7, 11.1 Hz), 3.60-4.27 (m, 7H), 3.82 (s, 6H), 4.50 (br s, 1H, exchangeable
with D2O), 4.90 (d, 1H, J = 3.6 Hz), 6.53-7.38 (m, 8H). The free base was transformed into
the oxalate salt that was crystallized from EtOH: mp 123-125°C. ESI/MS: m/z 374.2 [M +
H]+. Anal. Calcd for C21H27NO5·H2C2O4·2H2O: C, 55.30%; H, 6.66%; N, 2.80%. Found: C,
55.47%; H, 6.50%; N, 3.00%.
4.1.3.7. N-((3,3-diphenyl-1,4-dioxan-2-yl)methyl)-2-phenoxyethanamine (10). This
compound was prepared starting from 29: an oil was obtained (81% yield): 1H NMR (CDCl3)
δ 1.62 (br s, 1H, exchangeable with D2O), 2.32 (m, 1H), 2.91 (m, 2H), 3.28 (dd, 1H, J = 12.6,
10.0 Hz), 3.58-4.05 (m, 6H), 4.49 (dd, 1H, J = 10.0, 3.5 Hz), 6.81-7.51 (m, 15H). The free
base was transformed into the oxalate salt that was crystallized from MeOH: mp 231-233°C.
ESI/MS: m/z 390.2 [M + H]+. Anal. Calcd for C25H27NO3·H2C2O4: C, 67.63%; H, 6.10%; N,
2.92%. Found: C, 67.77%; H, 6.13%; N, 3.00%.
4.1.3.8. N-((3,3-Diphenyl-1,4-dioxan-2-yl)methyl)-2-(2-methoxyphenoxy)ethanamine
1
(11). This compound was prepared starting from 30: an oil was obtained (99% yield). H
NMR (CDCl3) δ 2.34 (dd, 1H, J = 12.9, 3.5 Hz), 2.75 (br s, 1H, exchangeable with D2O),
2.98-4.17 (m, 9H), 3.87 (s, 3H), 4.61 (dd, 1H, J = 10.7, 3.5 Hz), 6.85-7.52 (m, 14H). The free
base was transformed into the oxalate salt that was crystallized from EtOH: mp 200-202 °C.
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