1,3-Dioxane as a scaffold for potent and selective 5-HT1AR agonist with in-vivo anxiolytic, anti-depressant and anti-nociceptive activity

Article abstract of DOI:10.1016/j.ejmech.2019.05.024

A series of compounds generated by ring expansion/opening and molecular elongation/simplification of the 1,3-dioxolane scaffold were prepared and tested for binding affinity at 5-HT_1AR and α₁ adrenoceptors. The compounds with greater affinity were selected for further functional studies. N-((2,2-diphenyl-1,3-dioxan-5-yl)methyl)-2-(2-methoxyphenoxy)ethan-1-ammonium hydrogen oxalate (12) emerged as highly potent full agonist at the 5-HT_1AR (pKi 5-HT_1A = 8.8; pD₂ = 9.22, %E_max = 92). The pharmacokinetic data in rats showed that the orally administered 12 has a high biodistribution in the brain compartment. Thus, 12 was further investigated in-vivo, showing an anxiolytic and antidepressant effect. Moreover, in the formalin test, 12 was able to decrease the late response to the noxious stimulus, indicating a potential use in the treatment of chronic pain.

Full text of DOI:10.1016/j.ejmech.2019.05.024

Accepted Manuscript
1
,3-Dioxane as a scaffold for potent and selective 5-HT R agonist with in-vivo
1A
anxiolytic, anti-depressant and anti-nociceptive activity
Silvia Franchini, Claudia Sorbi, Pasquale Linciano, Gianluca Carnevale, Annalisa Tait,
Simone Ronsisvalle, Michela Buccioni, Fabio Del Bello, Antonio Cilia, Lorenza Pirona,
Nunzio De Nora, Rosa Maria Iacobazzi, Livio Brasili
PII:
S0223-5234(19)30433-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.024
EJMECH 11334
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 December 2018
Revised Date: 27 March 2019
Accepted Date: 6 May 2019
Please cite this article as: S. Franchini, C. Sorbi, P. Linciano, G. Carnevale, A. Tait, S. Ronsisvalle,
M. Buccioni, F. Del Bello, A. Cilia, L. Pirona, N. De Nora, R.M. Iacobazzi, L. Brasili, 1,3-Dioxane as
a scaffold for potent and selective 5-HT R agonist with in-vivo anxiolytic, anti-depressant and anti-
1A
nociceptive activity, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.05.024.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
1
,3-Dioxane as a scaffold for potent and selective 5-HT R agonist with in-vivo anxiolytic,
1
A
anti-depressant and anti-nociceptive activity
*a
a
a
b
a
Silvia Franchini, Claudia Sorbi, Pasquale Linciano, Gianluca Carnevale, Annalisa Tait, Simone
c
d
d
e
e
Ronsisvalle, Michela Buccioni, Fabio Del Bello, Antonio Cilia, Lorenza Pirona, Nunzio De
f
g
a*
Nora, Rosa Maria Iacobazzi, and Livio Brasili
a
Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via
Campi 103, 41125 Modena, Italy.
b
Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con Interesse
Trapiantologico, Oncologico e di Medicina Rigenerativa, Università degli Studi di Modena e
Reggio Emilia, Via del Pozzo 71, 41125 Modena, Italy.
c
Dipartimento di Scienze del Farmaco Sezione di Chimica Farmaceutica e Sezione di Farmacologia e
Tossicologia, Università degli Studi di Catania, Viale Andrea Doria 6, 95125, Catania, Italy
d
Scuola in Scienze del Farmaco e dei Prodotti della Salute, Università degli Studi di
Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy
e
Divisione Ricerca e Sviluppo, Recordati S.p.A., Via Civitali 1, 20148 Milano, Italy
f
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari "Aldo Moro",
Via E. Orabona 4, I-70125 Bari, Italy.
g
IRCCS Istituto tumori "Giovanni Paolo II" of Bari, Italy, Via O. Flacco 65, 70124 Bari, Italy.
1

ACCEPTED MANUSCRIPT
Abstract
A series of compounds generated by ring expansion / opening and molecular elongation /
simplification of the 1,3-dioxolane scaffold were prepared and tested for binding affinity at 5-
HT R and α adrenoceptors. The compounds with greater affinity were selected for further
1
A
1
functional
studies.
N-((2,2-diphenyl-1,3-dioxan-5-yl)methyl)-2-(2-methoxyphenoxy)ethan-1-
ammonium hydrogen oxalate (12) emerged as highly potent full agonist at the 5-HT R (pKi 5-
1
A
HT1A = 8.8; pD = 9.22, %Emax = 92). The pharmacokinetic data in rats showed that the orally
2
administered 12 has a high biodistribution in the brain compartment. Thus, 12 was further
investigated in-vivo, showing an anxiolytic and antidepressant effect. Moreover, in the formalin test,
12 was able to decrease the late response to the noxious stimulus, indicating a potential use in the
treatment of chronic pain.
Keywords
1
,3-dioxane, 5-HT receptor agonist, anxiolytic, anti-depressant, antinociceptive activity
1A
*Corresponding Author: E-mail: livio.brasili@unimore.it
Note: The authors declare no competing financial interest.
2

ACCEPTED MANUSCRIPT
1. Introduction
Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter within the central and peripheral
nervous systems, which exerts its actions through its interaction with seven distinct receptors (5-
HT R). In the 5-HT subfamily, five subtypes have been identified (5-HT , 5-HT , 5-HT , 5-
1
-7
1
1A
1B
1D
ht and 5-HT ), all of which belong to the G-protein-coupled receptors (GPCRs). The 5-HT R is
1
E
1F
1A
widely recognized as a relevant therapeutic target for several psychiatric disorders, such as anxiety,
depression and schizophrenia [1–3] but also for other pathological conditions, including cognitive
deficits [4], neurodegenerative disorders, like Parkinson’s and Alzheimer’s diseases [5–7], ischemic
stroke [8], neuropathic pain [9] and cancer [10].
In the last three decades, a number of 5-HT R ligands have been developed. They range from full
1
A
agonists (8-OH-DPAT, S-14506 and Xaliproden), partial agonists (vilazodone, buspirone,
tandospirone, BMY 7378), inverse agonists (spiperone [11]), biased agonists (F15599 also known
as NLX-101,[2]) to neutral antagonists (WAY 100635).
However, it should be noted that some of these also share affinity for other receptor types (alpha
adrenoceptors, dopamine receptors) and/or 5-HT subtypes or subfamilies (e.g. 8-OH-DPAT for 5-
1
HT ). Therefore, there is still a need to develop potential clinical candidates with a high degree of
7
selectivity and full agonist potency.
3

ACCEPTED MANUSCRIPT
Figure 1: Representative 5-HT R ligands
1
A
Starting from compounds acting at α -adrenoceptors, a series of 5-HT R ligands built on the 1,3-
1
1A
dioxolane scaffold was identified [12]. The 1,3-dioxolane has proved to be a versatile and useful
scaffold for different classes of drugs. We had successfully employed this moiety to improve
potency and selectivity of ligands acting at alpha adrenergic, 5-HT serotoninergic, sigma and
1
1A
TAAR5 receptors.[13][14][15][16]
4

ACCEPTED MANUSCRIPT
In this work, we explored the distance between the basic centre and the 2,2-diphenyl portion (Figure
2a, b), the expansion (Figure 2c, d), the opening and the simplification of the 1,3-dioxolane ring
(
Figure 2e, f), as a way to increase the intrinsic activity and selectivity for 5-HT R. A detailed SAR
1
A
study was carried out, and a potent and selective 5-HT R agonist with in-vivo anxiolytic,
1
A
antidepressant and anti-nociceptive activity was discovered.
Figure 2. A structural modification approach used to design new 5-HT R agonists: a,b) study of
1
A
the distance between the basic center and the diphenyl portion; c,d) ring expansion; e,f) ring
opening and molecular simplification.
2. Results and Discussion
2.1 Chemistry
5

ACCEPTED MANUSCRIPT
All the final compounds 3-16 were tested as oxalate salts, prepared through reaction of the
respective free amines 17-30 with anhydrous oxalic acid, followed by crystallization from dry
diethyl ether (Scheme 1-4). The free amines 17-30 were directly obtained by standard S 2 reaction
N
between the appropriate aliphatic chloride 33-39 and the 2-phenoxyethan-1-amine or 2-(2-
methoxyphenoxy)ethan-1-amine, prepared as previously reported [13]. The reaction was performed
in 2-methoxyethanol at reflux temperature for 18-24 hours, using potassium iodide as a catalyst
(
Scheme 1-4). The two amines 27-28 were synthesized, under the same S 2 conditions, from the
N
chloro-derivative 38 [15] and the appropriate phenoxyethylamine. The de-protection of the
hydroxyl group by treatment of 31-32 with tetra-n-butylammonium fluoride (TBAF) in THF at
room temperature for 24 hours led to amines 27-28 in high yield (Scheme 4). With the sole
exception of 34 [13], which was obtained directly by condensation of benzophenone with 3-chloro-
1,2-propandiol (Scheme 2), the aliphatic chlorides 33 [17], and the brand-new 35-37 were easily
prepared by treatment of the respective alcohols 40-43 with thionyl chloride in dry toluene, under
nitrogen atmosphere, using pyridine as a base (Scheme 1-3). The two diastereomers of the
intermediates 34 and 35 were separated by flash chromatography (Scheme 2). Aliphatic chloride 39
was synthesized as previously reported [15]. Finally, the synthesis of the alcohols 40-43 followed
two different procedures. 40 and 42 were jointly prepared (in stoichiometric ratio 13:1) through the
condensation of benzophenone and 1,2,4-butantriol in refluxing toluene, using p-toluensulfonic acid
(pTSA) as a catalyst and a Dean-Stark trap to remove the water formed, and easily separated by
flash chromatography (Scheme 1 and 3). On the contrary, the same condensation procedure did not
lead to the preparation of alcohols 41 and 43. Therefore, for the synthesis of 41 and 43, the
respective carbonyl starting compounds (benzophenone for 41 and 2,2-diphenylacetaldehyde for
43) were firstly converted into the dimethyl-acetals 44, 45, by refluxing in methanol using pTSA as
a catalyst and trimethyl orthoformate as a water scavenger. The condensation of 44 with 1,2,4-
butantriol and 45 with 2-(hydroxymethyl)propane-1,3-diol in acetonitrile at room temperature, in
6

ACCEPTED MANUSCRIPT
the presence of cobalt chloride (CoCl ) and trimethylsilyl chloride (TMSCl), led to the quantitative
2
preparation of alcohols 41 and 43, respectively (Scheme 2 and 3).
Scheme 1. Reagents and conditions: a) 1,2,4-butantriol (1.7 eq.), pTSA (cat.), dry Toluene, Dean-
Stark trap, N , reflux 65 h, 65% yield; b) thionyl chloride (1.3 eq.), pyridine (2 eq.), dry toluene,
2
N , 0°C to reflux, 45 min, 64% yield; c) 2-phenoxyethan-1-amine or 2-(2-methoxyphenoxy)ethan-
2
1-amine (6.4 eq.), KI (cat.), 2-methoxyethanol, reflux, 24 h, 74% yield (for 17) and 55% yield (for
1
8); d) oxalic acid (1.2 eq.), dry Et O, r.t., 24 h, 63% yield (for 3) and 60% yield (for 4).
2
Scheme 2. Reagents and conditions: a) 1-Chloro-2,3-propandiol (2 eq.), pTSA (cat.), dry Toluene,
Dean-Stark trap, N , reflux 18 h, 5% yield (cis-34) and 42% yield (trans-34); b) 2-phenoxyethan-
2
1
2
-amine or 2-(2-methoxyphenoxy)ethan-1-amine (6.4 eq.), KI (cat.), 2-methoxyethanol, reflux, 18-
4 h, 66% yield (for trans-19), 54% yield (for cis-19), 20% yield (for trans-20), 39% yield (for
7

ACCEPTED MANUSCRIPT
cis-20); c) oxalic acid (1.2 eq.), dry Et O, r.t., 24 h, 37% yield (for trans-5), 57% yield (for cis-5),
2
41% yield (for trans-6), 40% yield (for cis-6), 59% yield (for trans-7), 30% yield (for cis-7), 45%
yield (for trans-8), 62% yield (for cis-8); d) trimethyl orthoformate (10 eq.), pTSA (cat.), MeOH,
reflux, 5 h, 75% yield; e) 1,2,4-butantriol (2 eq.), CoCl (0.6 eq.), TMSCl (1 eq.), ACN, r.t., 17 h,
2
quantitative yield; f) thionyl chloride (1.3 eq.), pyridine (2 eq.), dry toluene, N , 0°C to reflux, 45
2
min, 39% yield (cis-35) and 45% yield (trans-35).
Scheme 3. Reagents and conditions: a) 1,2,4-butantriol (1.7 eq.), pTSA (cat.), dry Toluene, Dean-
Stark trap, N , reflux 65 h, 5% yield; b) thionyl chloride (1.3 eq.), pyridine (2 eq.), dry toluene, N ,
2
2
0
°C to reflux, 45 min, 88.5% yield (36) and 77% yield (37); c) 2-phenoxyethan-1-amine or 2-(2-
methoxyphenoxy)ethan-1-amine (6.4 eq.), KI (cat.), 2-methoxyethanol, reflux, 24 h, 20% yield (for
3), 31% yield (for 24), 20% yield (for 25), 36% yield (for 26); d) oxalic acid (1.2 eq.), dry Et O,
2
2
r.t., 24 h, 26% yield (for 9), 30% yield (for 10), 41% yield (for 11), 57% yield (for 12); e) trimethyl
orthoformate (10 eq.), pTSA (cat.), MeOH, reflux, 5 h, 82% yield; f) 2-(hydroxymethyl)propane-
1
,3-diol (2 eq.), CoCl (0.6 eq.), TMSCl (1 eq.), ACN, r.t., 17 h, quantitative yield.
2
8

ACCEPTED MANUSCRIPT
Scheme 4. Reagents and conditions: a) 2-phenoxyethan-1-amine or 2-(2-methoxyphenoxy)ethan-
1-amine (6.4 eq.), KI (cat.), 2-methoxyethanol, reflux, 24 h, 77% yield (for 31), 40% yield (for
32), 41% yield (for 29), 20% yield (for 30); b) TBAF (1.2. eq.), THF, r.t., 24 h, 57% yield (for 27)
and 87% yield (for 28); c) oxalic acid (1.2 eq.), dry Et O, r.t., 24 h, 54% yield (for 13), 45% yield
2
(for 14), 55% yield (for 15), 30% yield (for 16).
2
.2 Structure–affinity and structure-activity relationship studies
Compounds 3-16 were tested for binding affinity (pK ) and activity (pK ) at human α and 5-HT
1A
i
b
1
receptors The most active and selective compounds were chosen for their functional
.
characterization (pD and %Emax).
2
In a previous paper, we showed that compounds 1 and 2 bind to both α and 5-HT R receptors
1
1A
[
12]. Binding studies in human cloned receptors have shown that at all the three α subtypes (α1a,
1
α and α ), the affinities of both compounds are quite similar, with compound 2 showing a small,
1
b
1d
but scarcely significant, preference with respect to 1. In the case of 5-HT R, compound 2 has a
1
A
higher affinity than 1, of about one order of magnitude (9.22 vs 8.45). During the functional studies,
both behaved as antagonists at α adrenoceptors and partial agonists at 5-HT R. Compound 1
1
1A
showed a selective profile towards α1D (more than 100-fold), with respect to the α1A and α1B
subtypes. The functional data for the 5-HT R indicated that the agonist potency decreased by about
1
A
2
8-fold, going from 1 to 2 (pD of 8.8 and 7.36 respectively). This is contrary to the trend observed
2
in the binding experiment, where the affinity of 1 was 6-fold lower than that of compound 2 (pKi
9

ACCEPTED MANUSCRIPT
8.45 and 9.22 respectively). Therefore, the presence of the methoxy group in the ortho position had
a positive effect on binding and a negative one on agonist potency. Furthermore, during the
functional studies at α adrenoceptors the methoxy group increased potency but decreased
1
,
selectivity towards the α1D subtype, while leaving the affinity at the α subtypes in the binding
1
studies almost unchanged.
*
Table 1. Affinity constants (pK or pKi†) for α -adrenoceptors in isolated rat prostatic vas deferens
b
1
(
α ), spleen (α ), and thoracic aorta (α ) and for human cloned α , α α and 5-HT R. Agonist
1A 1B 1D 1a 1b, 1d 1A
‡
35
potency (pD ) and relative effectiveness (% Emax) in the agonist-induced [ S]GTPγS-binding
2
assay at 5-HT . All the compounds were assayed as oxalate salt.
1
A
*
pKb
pKi†
5
-HT1A %Emax
Cmp
Structure
5-
α1A
α1B
α1D
α1a
α1b
α1d
pD2
5-HT1A
HT1A
1
2
6.16
7.53
5.86 8.37 7.43
7.36 8.65 7.71
7.20
7.33
7.94
8.03
8.45
9.22
8.8
24.4
31.6
7.36
3
4
6.34
6.74
6.49 7.04 7.23
6.93 8.17 8.14
7.02
7.83
7.52
8.24
6.14
9.30
6.16
22.1
Trans-5
Cis-5
6.29
6.22
6.78
6.16
5.71
6.14
6.23
6.58
6.26 6.70 6.92
6.83 6.72 6.37
6.82 7.64 7.51
6.75 7.52 6.01
6.66 7.22 7.05
6.43 7.15 6.77
7.13 7.25 7.22
6.91 7.67 7.30
6.99
6.99
7.56
7.43
6.78
6.97
7.51
7.19
7.06
7.53
8.15
8.36
7.02
7.12
7.80
7.78
8.28
7.94
8.70
8.50
7.24
7.22
7.81
7.59
Trans-6
Cis-6
Trans-7
Cis-7
Trans-8
Cis-8
6.24
5.86
53
82.1
9
6.12
6.97 7.55 7.41
7.25
8.30
8.73
7.85
79.5
1
0

ACCEPTED MANUSCRIPT
*
pKb
pKi†
5
-HT1A %Emax
Cmp
Structure
5-
α1A
α1B
α1D
α1a
α1b
α1d
pD2
8.49
5.87
5-HT1A
HT1A
1
0
6.99
7.19 8.68 7.61
6.76 6.76 6.54
7.8
8.99
6.75
9.11
7.63
65.9
11
5.52
5.98
<5
6.55
88.7
1
1
2
3
6.80 7.06 7.47
6.98 7.07 6.85
7.5
7.6
8.79
8.34
9.22
7.51
91.6
69.5
5.64
7.24
1
1
1
4
5
6
6.51
7.65
6.18
7.02 7.46 7.08
7.01 7.73 7.77
7.11 7.75 7.35
7.04
7.60
8.23
8.92
8.81
8.87
8.14
7.53
7.85
7.38
90.2
78.9
77.3
5.52
6.15
8.00
8.89
§
§
¶
§
¶
§
¶
§
§
§
§
BMY-7378
-OH DPAT
6.41
6.82
7.73
8.90
9.27
7.83
n.a.
26
§
§
§
§
8
<6
<6
8.43
100
¶
WAY 100635
*
7.18
8.34
9.48
-
Each experiment was performed in triplicate.
†
The data are expressed as means of 2–3 separate experiments performed in duplicate;
*
†
Standard deviation is within ±10% of the value.
‡
§
According to [18]
See [12];
¶See [19].
In the present study, we wanted to further investigate the importance of other structural elements on
activity, potency and selectivity, such as the distance between the basic center and the lipophilic
diphenyl portion, and the enlargement and opening of the 1,3-dioxolane ring (Figure 2 a-f). The
newly synthesized compounds are reported in Table 1, together with the binding affinities and
activities at both α and 5-HT R. As can be seen, compound 4, having the nitrogen atom moved
1
1A
1
1

ACCEPTED MANUSCRIPT
away by the insertion of a methylene in the lateral chain, with respect to 2, showed a small increase
in binding affinity (pK ) at all α subtypes, while leaving unchanged the affinity at 5-HT R.
i
1
1A
During the functional studies, the antagonist potency of 4 at α subtypes was slightly decreased,
1
while the selectivity towards the α subtype was almost the same (about 10-fold). At 5-HT R, the
1
D
1A
agonist potency was also decreased by about 17-fold. The same modification made on the
desmethoxy derivative 1 to give 3 produced different results. In fact, the binding affinities were
decreased and the decrease was much larger for 5-HT R. During the functional studies at α the
1
A
1,
most significant variation was a complete loss of α1D selectivity, due to a more than 10-fold
decrease in potency at this subtype and a concomitant, although small, increase at the α1A and α1B
subtypes. In compound 6 the diphenyldioxolane was replaced with a benzhydryldioxolane to
increase the distance between the diphenyl moiety and the central amine and two disatereomers
were obtained (t and c). The most important change was in the selectivity 5-HT /α which was
1
A
1,
greatly reduced, as a result of a significant decrease in 5-HT R affinity. Combining the two
1
A
variations, as in 8, was generally negative for both affinity and potency for α and 5-HT receptor
1
1A
systems. The exception was the increase in efficacy at 5-HT R for both diastereomers, which was
1
A
accompanied by a decrease in potency. A similar trend was observed with the desmethoxy
derivatives 5c,t and 7c,t. As far as the stereochemistry is concerned, no clear difference emerged for
the two pairs of diastereomers.
The study of the expansion of the dioxolane ring was achieved in two ways: (i) by the insertion of a
methylene unit adjacent to the oxygen atom in position 1 of the 1,3-dioxolane to give the
asymmetric 1,3-dioxanes 9,10 (Figure 2 c); (ii) by the insertion of the same methylene unit adjacent
to the oxygen atom in position 3 to give the symmetric 1,3-dioxanes 11, 12 (Figure 2 d).
Compound 10 showed a significant enhancement of the affinity at α1b and α1d receptors, with an
α /α selectivity ratio of about 12-fold higher than that of compound 2. The affinity at 5-HT R
1
d
1a
1A
1
2

ACCEPTED MANUSCRIPT
was practically unchanged, with a consequential loss of 5-HT /α selectivity. During the functional
1
A
1
studies, compound 10 showed an increase in α1D selectivity (31-fold) and, at 5-HT R, a significant
1A
increase in agonist potency (about 10-fold) and efficacy (doubled).
With respect to 2, compound 12 showed a decrease, although limited, in affinity at both receptor
systems, the exception being the affinity at the α receptor subtype. Also the antagonist potencies
1
b
at the three α receptor subtypes were decreased. At 5-HT R the agonist potency was enhanced by
1
1A
about 72-fold, while the efficacy was three times the one observed with compound 2.
Compound 14 is the open analogue of 2, obtained by breaking the C2-O1 bond of the 1,3-dioxolane
ring. This molecular variation caused a decrease in affinity at both receptor systems, with the
exception of the affinity at α subtype, showing a significant decrease in 5-HT1A/α selectivity.
1
d
1
These results are in agreement with the antagonist potency trend at the α subtypes. The agonist
1
potency at 5-HT R was retained, while the efficacy was increased by about 3-fold.
1
A
Molecular simplification of 14, by removing the hydroxymethyl moiety, to give 16, interestingly
gave an increase in binding affinity and antagonist potency at the α receptor subtype, the exception
1
being the potency at the α1b subtype. At 5-HT R, the affinity and potency remained unchanged,
1A
with a small variation in efficacy.
Compounds 9, 11, 13 and 15 were synthesized in order to confirm the effects on the activity of the
previously described ortho-methoxy group (compound 2 vs 1). The methoxy group improved, with
some exceptions, the pharmacological parameters at both receptor systems. In particular, the most
significant variation is the potency of compound 12, which shows a pD2 of 9.22, 2240-fold higher
than the desmethoxy derivative 11, showing a pD2 of 5.87.
Overall, the above described structural modifications allowed the identification of compound 12,
which is the most interesting in the series, due to its high potency at 5-HT R. In direct comparison
1
A
with the starting point 2, compound 12 clearly showed enhancement of the pharmacological profile
1
3

ACCEPTED MANUSCRIPT
at 5-HT : the selectivity ratio 5-HT /α was maintained and, despite a limited reduction in
1
A
1A
1
affinity, an increase of about two orders of magnitude in agonist potency and three in efficacy was
observed. These results allowed us to consider 12 as one of the most potent 5-HT R full agonists.
1
A
Therefore, compound 12 was chosen for further pharmacological studies.
2.3. In vitro studies
2
.3.1. Citotoxicity
Firstly, the cytotoxicity (IC ) of 12 was determined by MTT assay, on SH-SY5Y human
5
0
neuroblastoma cell line, across a wide range of concentrations (0.1-100 µM, see Experimental
Section). Cell viability assay was also performed for hydrogen peroxide (H O ), oligomycin A and
2
2
rotenone, to determine their corresponding IC50. The results, reported in Table 2, showed a dose-
dependent cytotoxicity for the above-mentioned compounds at the tested concentrations.
a
Table 2. Cytotoxicity (IC50 µM) of the tested
compounds.
b
1
2
H O
oligomycin A rotenone
2
2
*
*
*
3
1.2 ± 0.6 195 ± 1.7 29 ± 3.4
74.1 ± 4.5
*
According to[18]
a
The IC50 values were determined after 24 h
incubation of the cells SH-SY5Y with the
compounds, by varying concentrations in the range
b
0
.1-100 µM. H O was tested in the range 1-500
2
2
µM.
2.3.2. Neuroprotective capacity
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4

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An assessment of the ability of 12 to prevent the death of human neuroblastoma SH-SY5Y cell lines
caused by H O , oligomycin A and rotenone was carried out in vitro [20]. These neurotoxins were
2
2
used at a concentration equal to their IC . As reported in Table 3, compound 12, at 1 µΜ, showed
5
0
neuroprotective activity against H 0 and oligomycin A damage, while a minimal effect was
2
2
observed against rotenone.
*
Table 3. Neuroprotective effect of 12 on human neuroblastoma cell lines after
the addition of three toxic insults
Compd [µM] H O (195 µΜ) Oligomycin A(30 µΜ) Rotenone (75 µΜ)
2
2
1
1
2 (1 µΜ )
79 ± 5
83 ± 2
69 ± 2
61 ± 4
63 ± 9
2 (0.1 µΜ ) 89 ± 3
*
According to[18]
The data are expressed as percentages of neuroprotection ±SD of three
independent experiments.
2.3.3. Bi-directional transport studies
An evaluation of the ability of 12 to permeate the MDCK-MDR1 monolayers was carried out in
vitro. It is well-known that these cell lines mimic the BBB and express the P-glycoprotein (P-gp),
which is involved in the drug efflux transport [21]. Transport studies were conducted in both
Apical-to-Basolateral and Basolateral-to-Apical directions and the results are shown in Table 4.
Compound 12 was found to have non-significant differences in Papp values between the AP-to-BL
and BL-to-AP directions. The efflux ratio (ER), which was calculated using the equation ER = Papp,
BL-AP / P , AP-BL, was found to be less than 2. This result indicates that 12 is not likely to be
app
considered a suitable substrate for P-gp transport. Therefore, 12 was able to permeate the
monolayer, by passive diffusion, with permeability that was comparable to that of diazepam. The
1
5

ACCEPTED MANUSCRIPT
permeability for the control (Fluorescein isothiocyanate–dextran, FD-4) was within the expected
values.
*
Table 4. Bi-directional Transport Across MDCKII-MDR1
cells of tested compound 12 and reference compounds.
a
Compd
Papp AP(cm/sec) Papp BL(cm/sec) ER PappBL/PappAP
-
5
-5
1
2
2.6*10
1.26*10
1.23*10
2.08*10
0.49
0.84
0.20
-
-
5§
6§
-5§
-7§
§
§
diazepam 1.46*10
FD-4
1.03*10
*
§
According to[22]
See [18]
2.4. Pharmacokinetic studies
Before proceeding to the in vivo studies, preliminary pharmacokinetic analysis was performed in
rats. Compound 12 was administered per os at a dose of 10 mg/Kg and brain and plasma
concentrations were quantified following the previously reported and validated bioanalytical
method [23]. The concentration vs. time curves are reported in Figure 3 and the pharmacokinetic
parameters for brain and plasma are summarized in Table 5.
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6

ACCEPTED MANUSCRIPT
1
1
1
1
80
70
60
50
Brain (in nmol/g)
Plasma (in nmol/mL)
1
.0
.5
.0
0
0
30 60 120
240
Time (min)
480
Figure 3. Cerebral (blue circles) and plasmatic (red squares) concentrations (nmol/g and
nmol/mL, respectively) of 12 after an oral dose of 10 mg/kg.
Table 5. Pharmacokinetic parameters in rat brain and plasma after an oral dose of 12 (10mg/kg),
calculated using “PK Solutions” software.[24]
tmax
min)
AUC(0-t)
AUC(0-
∞
)
t ½
Brain/Plasma
(B/P)
[a]
8
Cmax
(
(nmol/g·min)
(min)
(
nmol/g·min)
*
Plasma
0.095
157.9
30
60
17.4
18.1
102.6
6
1
.2 (15-45 min)
≈420
#
Brain
7167.6
7173.4
42 (45-480 min)
[
a]
concentrations are expressed as nmol/mL in the plasma and in nmol/g in the brain.
Comparing brain and plasma curves, it is possible to observe a different profile of 12 in the two
compartments. The areas under the brain or plasma concentration vs. time (AUC0-t) were 7167 and
17 nmol/g·min, respectively. This difference is reflected in the high brain/plasma ratio (B/P), which
was calculated as the ratio between the brain and the plasma AUC(0-t). Compound 12 showed a B/P
1
7

ACCEPTED MANUSCRIPT
ratio value of 420, demonstrating its elevate capability to permeate the blood-brain barrier (BBB).
These data were supported by in silico BBB-passage prediction for non-active transport. Predicted
brain/blood partition coefficient (QPlogBB) and predicted apparent MDCK cell permeability
(
QPPMDCK) were calculated with QiKProp [25]. Compound 12 showed a QPlogBB of 0.467 (for
CNS penetration -3< QPlogBB <1.2) and a QPPMDCK of 1250 (for CNS penetration QPPMDCK
500), suggesting that is able to cross the BBB by passive diffusion. Moreover, the predicted
>
QPPMDCK is in accordance with the bi-directional transport studies on MDCK-MDR1
monolayers.
As shown in Table 5, the concentration of 12 in the rat brain increased rapidly, reaching a maximum
of 157.9 ± 8.7 nmol/g (Cmax) in the first 60 minutes (tmax). The effect of 12 in the behavioral studies
was evaluated at this time point. The initial increased concentrations of 12 in the brain occurred
during stable plasma levels, as observed for (R)-8-OH-DPAT, dipropylaminotetraline (DPAT)
derivatives and (S)-UH-301. The concentration then decreased, following a biphasic trend: rapidly
during the first phase (60-120 minutes) and more slowly during the second phase (120-480 min)
with two half-lives (t ) of 6.16 and 142 min, respectively. This trend follows the one reported for
½
(R)-8-OH-DPAT [26]. The rapid decrease of 12 in the brain does not seems to be linked to a drug
efflux transport by P-gp, as suggested by the in vitro transport studies.
In the plasma, the concentration of 12 was lower than in the brain, with a Cmax of 0.095 nmol/mL
during the first 30 minutes, but with a monophasic and slow elimination rate, resulting in a t1/2 of
102.6 min, 3-times higher than the reference drug 8-OH-DPAT (t1/2 = 27 min) [26].
According to the high bio-distribution of 12 in brain, this compound was tested in vivo for its
activity on the central nervous system.
2.5. In vivo behavioural studies
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Compound 12 was assessed in adult male Sprague-Dawley rats for anxiolytic, locomotor and anti-
depressant activity.
2.5.1. Anxiolytic effect
The anxiolytic effect of 12 was evaluated in rats using the Elevated Plus Maze test (EPM) [27].
Compound 12 was administered per os at three different concentrations (5, 10 and 20 mg/Kg). 8-
OH-DPAT (0.5 mg/kg, i.p.) was used as a positive control. The percentage of time spent by the rat
between the open arms of the maze (Figure 4A) and the number of entries (Figure 4B) were used as
a measure of the anxiolytic effect of the compound. The administration of 12 at the dosages of 10
and 20 mg/kg significantly increased the percentage of the time spent by the rat in the open-arm
section (20% and 18% with a P<0.001 and P<0.01 respectively) with an effect that is comparable to
8-OH-DPAT (19% at 0.5 mg/kg). At the same time, rats administered with 12 spent less time in the
closed arms with respect to the rat administered with the vehicle. In addition, 12, at 10 and 20
mg/kg, was able to increase (P<0.05) the number of the open arm entries, whereas no differences
were observed between 8-OH-DPAT and the control group. Overall, these data show that 12
exhibits anxiolytic-like activity in the EPM test.
A
B
1
00
10
8
Closed arms
Open arms
Closed arms
Open arms
8
6
4
2
0
0
0
0
0
##
#
#
##
#
##
6
*
*
4
*
**
*
*
*
*
2
0
(
mg/Kg)
8-OH-DPAT
12
(mg/Kg)
8-OH-DPAT
12
Vehicle
-
0.5
5
-
10
-
20
-
Vehicle
-
0.5
+
5
-
10
-
20
-
+
-
-
+
+
+
-
-
+
+
+
Figure 4. Elevated plus maze test for the evaluation of the anxiolytic effect of 12 in rats
administered per os with 5, 10 and 20 mg/Kg. (A) Percentage of time spent by the rat in the open
1
9

ACCEPTED MANUSCRIPT
and closed arms of the maze. (B) Number of entries in the open and closed arms of the maze, in
the 300 sec test session. 8-OH-DPAT, (0.5 mg/Kg, i.p.) was used as a positive control. *P<0.05,
##
###
*
*P<0.01, ***P<0.001, P<0.01,
P<0.001 vs. vehicle treated rats (Anova followed by
Dunnett’s test).
2.5.2. Locomotor activity and anxiolytic effect
The potentiality of compound 12 to possess excitatory activity was evaluated using the Open Field
test [27]. The test measures the total distance tracked by the rats as an index of the locomotory
activity following CNS excitation. Compound 12 was administered per os at 10 mg/Kg. 8-OH-
DPAT (at 0.5 mg/Kg) was used as a reference. As reported in Figure 5A, the rats administered both
12 and 8-OH-DPAT ran a comparable total distance, with the respect to the untreated group. This
lack of significant variation in the locomotor activity revealed an absence of excitatory effect for
compound 12. In addition, the Open Field test confirmed the anxiolytic effect of 12 observed in the
Elevated Pluz Maze test, by measuring the attitude of the rats to explore the open field area of the
maze (time spent and number of entries). Due to their nature, anxious rats avoid bright and open
spaces, preferring to stay close to the walls of the field (thigmotaxis). On the contrary, a decreased
level of anxiety in the animals leads to increased exploratory behavior. The administration of
compound 12 at the dose of 10 mg/kg induced an anti-thigmotactic effect, as indicated by a
significant increase in the percentage of the time spent and number of entries into the central area of
the open field (Figure 5B-C), with respect to the group of untreated and control animals.
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0

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A
B
C
Vehicle
8
-OH-DPAT at 0.5 mg/Kg
8
6
4
2
000
000
000
000
0
15
8
*
1
2 at 10 mg/Kg
6
*
1
0
5
0
4
2
0
Figure 5. Open Field Test for the evaluation of the locomotor activity and anxiolytic effect of 12
10 mg/Kg per os) in rats. 8-OH-DPAT (0.5 mg/Kg, i.p.) was used as a positive control. (A) Total
(
distance tracked (in cm) by the treated and untreated rats. (B) Percentage of time spent in the central
open field. (C) Number of entries in the central open field. *P<0.05 vs. vehicle treated rats (Anova
followed by Dunnett’s test). Moving traces of untreated (D) and treated (E) mice in the open field
test.
2.5.3. Anti-depressant activity
To assay the anti-depressant activity of compound 12, the Forced Swim test (Porsolt) was used in
rats [28–30]. The administration of 12 at the doses of 10 and 20 mg/kg was able to significantly
reduce the time that the rats spent immobile and to increase the time spent swimming, with an effect
comparable to that of 8-OH-DPAT (Figures 6A and B, respectively).
2
1

ACCEPTED MANUSCRIPT
A
B
C
300
200
100
0
150
100
50
60
Vehicle
8
1
1
-OH-DPAT (0.5 mg/Kg)
2 (5 mg/Kg)
***
***
2 (10 mg/Kg)
**
40
20
0
12 (20 mg/Kg)
**
***
***
0
Figure 6. Porsolt’s Test for the evaluation of the antidepressant activity of 12 (5, 10 and 20 mg/Kg
per os) in rats. (A) time spent immobile (in sec), (B) time spent swimming (sec) and (C) time spent
climbing (sec) of treated and untreated rats. 8-OH-DPAT (0.5 mg/Kg, i.p.) was used as a positive
control. ***P<0.001 and **P<0.01 vs. vehicle treated rats (Anova followed by Dunnett’s test)
In contrast, the administration of 12 at all doses did not significantly influence the time spent
climbing (Figure 6C). The same results were obtained using the reference drug 8-OH-DPAT. Taken
together, these data are in accordance with the effect of 5-HT R agonists, whereas
1
A
catecholaminergic agents cause a decrease in the time spent immobile, together with an increase in
the time spent climbing [31,32]. This behaviour indicates that the anti-depressant action of 12 is
strictly due to its interaction with the serotonergic system. These data are in agreement with the
higher affinity of 12 for the 5-HT R rather than for the α-adrenergic receptor (5-HT1a/α1D
1
A
selectivity = 16), as shown by the binding data.
2.5.4. Anti-nociceptive activity
The formalin test was used to assess the potential analgesic activity of compound 12 in vivo.[33]
Indeed, at the dose of 10mg/kg, i.p., 12 was able to significantly decrease perception of the II phase
2
2

ACCEPTED MANUSCRIPT
of the noxious stimulus. This effect was reverted by WAY-100635 at 3mg/kg i.p. This is a
confirmation that the nociceptive effect of 12 is mediated by the stimulation of 5-HT R (Figure 3).
1
A
1
50
00
Vehicle
*
12 (10mg/Kg i.p.)
WAY-100635 (3mg/Kg i.p.) + 12
1
(10mg/Kg i.p.)
*
50
0
Phase I (0-5 min)
Phase II (15-30 min)
Figure 7: Formalin test to study the effect of intraperitoneal (i.p.) injection of 12 (10 mg/kg) or
vehicle on the early (0-5 min) and late (15-30 min) phase of the noxious stimulus. Data are means ±
S.E.M. of 8 and 10 mice per group. *p < 0.05 vs. mice treated with vehicle.
3. Conclusions
Starting from Leads 1 and 2, a new class of 1,3-dioxane-based 5-HT R ligands was discovered.
1
A
Several compounds acted as potent 5-HT Rs agonists, among which 12 was the most potent, with
1
A
a maximal activity of 92% compared to 8-OH-DPAT. In vitro, compound 12 proved to penetrate the
BBB. This result was confirmed by the pharmacokinetic analysis of 12 in rat brain and plasma,
which showed a preferential distribution in the brain compartment. The behavioral tests in rats
treated orally with 12 at a dose of 10 mg/Kg demonstrated an anxiolytic and anti-depressant effect.
Compound 12 showed also a good anti-nociceptive activity that was reverted by the co-
administration of the 5-HT R antagonist WAY-100635
1
A
2
3

ACCEPTED MANUSCRIPT
4
. Experimental Section
4.1. Chemistry
All the reagents and solvents were commercially available from Sigma-Aldrich. The moisture-
sensitive reactions were performed under an inert atmosphere of argon. Each reaction was
monitored by TLC on Merck 60G F254 plates and detected at 254 nm. All the compounds were
purified by flash column chromatography using silica gel 60 (230-400 mesh, ASTM) supplied by
Merck, unless otherwise specified. The purity of the final compounds was assessed by elemental
analysis (C,H,N) on a Carlo Erba 1106 analyzer and the results obtained are within ±0.4% of the
theoretical values. The melting points were determined with Stuart SMP3 apparatus and are
1
13
uncorrected. The structure elucidation was confirmed by H and C NMR (1D and 2D) on a DPX-
2
6
00 Avance (Bruker) spectrometer at 200 MHz or on a DPX-600 Avance (Bruker) spectrometer at
00 MHz. The chemical shifts are expressed in δ (ppm) using tetramethylsilane (TMS) as internal
1
3
1
standard or the C signal of the solvent (CDCl δ 77.04, CD OD δ 49.8, DMSO-d δ 39.5). H
3
3
6
NMR peak patterns are as follows: s (singlet), d (doublet), t (triplet), dd (double doublet), ddd
double dd), m (multiplet), br (broad singlet). The assignment of cis-trans configuration was done
(
by NOESY experiments. Low resolution MS analysis was performed on a 6310A Ion Trap (Agilent
Technologies) whereas high resolution mass spectra were recorded on a hybrid QTOF mass
spectrometer (PE SCIEX-QSTAR), both equipped with an electrospray ionization source (ESI).
4.1.1. General procedure for the synthesis of the oxalate salts (3-16)
To a solution of the appropriate amine 17-30 (1 eq.) in 5 mL of dry Et O at room temperature and
2
under nitrogen atmosphere, anhydrous oxalic acid (1.2 eq.) was added. The suspension was stirred
for 30 min and left to settle down for 24 h. The precipitate was collected by filtration, washed with
dry Et O and dried to afford the title compound.
2
2
4

ACCEPTED MANUSCRIPT
4.1.1.1.
2-(2,2-diphenyl-1,3-dioxolan-4-yl)-N-(2-phenoxyethyl)ethan-1-ammonium
hydrogen
oxalate (3)
1
White solid (60% yield). H NMR (600 MHz, DMSO-d ) δ 1.92-2.00 (m, 2H), 3.08 -3.12 (m, 1H),
6
3
8
.16 – 3.26 (m, 1H), 3.35 (td, J = 2.7, 5.1 Hz, 2H), 3.72 (dd, J = 6.5, 8.2 Hz, 1H), 4.08 (dd, J = 6.6,
13
.2 Hz, 1H), 4.23 (m, 3H), 6.91 – 7.06 (m, 3H), 7.29 – 7.39 (m, 8H), 7.41 – 7.47 (m, 4H). C NMR
(151 MHz, DMSO) δ 30.12, 44.68, 46.45, 63.89, 69.35, 74.26, 109.48, 115.06, 121.70, 126.15,
126.18, 128.47, 128.49, 128.54, 128.70, 130.05, 142.85, 142.91, 158.21, 164.23. M.p. [202-204°C].
+
+
HRMS m/z [M+H] Calcd. for C H NO : 390.2064. Found: 390.2065. El. Anal. Calcd. for
2
5
28
3
C H NO : C 67.63, H 6.10, N 2.92. Found: C 67.60, H 6.10, N 2.90.
27
29
7
4.1.1.2.
2-(2,2-diphenyl-1,3-dioxolan-4-yl)-N-(2-(2-methoxyphenoxy)ethyl)ethan-1-ammonium
hydrogen oxalate (4)
1
White solid (59% yield). H NMR (600 MHz, DMSO-d ) δ 1.94-1.97 (m, 2H), 3.13-3.14 (m, 1H),
6
3
4
=
.23-3.24 (m, 1H), 3.33-3.35 (m, 2H), 3.63 – 3.83 (m, 4H), 4.09 (dd, J = 6.6, 8.2 Hz, 1H), 4.15 –
.29 (m, 3H), 6.91 (td, J = 1.7, 7.6 Hz, 1H), 6.94 – 7.10 (m, 3H), 7.22 – 7.40 (m, 6H), 7.44 (ddd, J
13
1.4, 8.3, 11.2 Hz, 4H). C NMR (151 MHz, DMSO) δ 30.27, 44.93, 46.59, 55.90, 65.67, 69.39,
7
1
4.33, 109.49, 112.81, 115.36, 121.19, 122.74, 126.16, 126.18, 128.48, 128.49, 128.53, 128.70,
+
42.85, 142.92, 147.57, 149.90, 164.66. M.p. [178-180°C]. HRMS m/z [M+H] Calcd. for
+
C H NO : 420.2169. Found: 420.2170. El. Anal. Calcd. for C H NO : C 66.00, H 6.13, N 2.75.
26
30
4
28 31
8
Found: C 66.05, H 6.10, N 2.75.
4.1.1.3. Trans-N-((-2-benzhydryl-1,3-dioxolan-4-yl)methyl)-2-phenoxyethan-1-ammonium hydrogen
oxalate (trans-5)
1
White solid (51% yield). H NMR (600 MHz, DMSO-d ) δ 3.10-3.18 (m, 2H), 3.29-3.31 (m, 2H),
6
3.61 (dd, J = 5.8, 8.6 Hz, 1H), 4.01 (dd, J = 6.4, 8.6 Hz, 1H), 4.14 – 4.25 (m, 3H), 4.31-4.34 (m,
2
5

ACCEPTED MANUSCRIPT
1
3
1
H), 5.77 (d, J = 5.9 Hz, 1H), 6.96-7.00 (m, 3H), 7.20 (t, J = 7.3 Hz, 2H), 7.24 – 7.43 (m, 10H). C
NMR (151 MHz, DMSO) δ 47.11, 49.34, 54.89, 64.12, 67.85, 72.73, 105.45, 115.04, 121.61,
26.88, 126.90, 128.62, 128.68, 129.28, 129.32, 130.03, 141.15, 158.28, 164.42. M.p. [187-192°C].
1
+
+
HRMS m/z [M+H] Calcd. for C H NO : 390.2064. Found: 390.2065. El. Anal. Calcd. for
2
5
28
3
C H NO : C 67.63, H 6.10, N 2.92. Found: C 67.65, H 6.10, N 2.90.
27
29
7
4.1.1.4. Cis-N-((-2-benzhydryl-1,3-dioxolan-4-yl)methyl)-2-phenoxyethan-1-ammonium hydrogen
oxalate (cis-5)
1
White solid (57% yield). H NMR (600 MHz, DMSO-d ) δ 2.74 (dd, J = 4.8, 13.2 Hz, 1H), 3.01
6
(dd, J = 4.3, 13.2 Hz, 1H), 3.18-3.23 (m, 2H), 3.70 (dd, J = 4.8, 8.6 Hz, 1H), 3.94 (dd, J = 6.8, 8.6
Hz, 1H), 4.11 – 4.18 (m, 2H), 4.24 (d, J = 5.8 Hz, 1H), 4.31 – 4.42 (m, 1H), 5.62 (d, J = 5.8 Hz,
1
3
1
H), 6.99-7.00 (m, 3H), 7.20 (td, J = 1.6, 7.3 Hz, 2H), 7.24 – 7.46 (m, 10H). C NMR (151 MHz,
DMSO) δ 47.23, 50.33, 55.06, 64.23, 67.95, 72.89, 106.16, 115.03, 121.61, 126.94, 128.60, 128.62,
+
129.35, 129.37, 130.04, 140.99, 141.00, 158.28, 164.31. M.p. [201-203°C]. HRMS m/z [M+H]
+
3
Calcd. for C H NO : 390.2064. Found: 390.2060. El. Anal. Calcd. for C H NO : C 67.63, H
2
5
28
27 29
7
6.10, N 2.92. Found: C 67.60, H 6.15, N 2.91.
4.1.1.5.
Trans-N-((-2-benzhydryl-1,3-dioxolan-4-yl)methyl)-2-(2-methoxyphenoxy)ethan-1-
ammonium hydrogen oxalate (trans-6)
1
White solid (49% yield). H NMR (600 MHz, DMSO-d ) δ 3.15-3.16 (m, 1H), 3.19-3.20 (m, 1H),
6
3
.27-3.29 (m, 2H), 3.61 (dd, J = 5.8, 8.6 Hz, 1H), 3.75 (s, 3H), 4.02 (dd, J = 6.4, 8.5 Hz, 1H), 4.17
t, J = 5.4 Hz, 2H), 4.21 (d, J = 6.0 Hz, 1H), 4.32-4.34 (m, 1H), 5.78 (d, J = 6.0 Hz, 1H), 6.91 (dd, J
1.7, 7.7 Hz, 1H), 6.94 – 7.05 (m, 3H), 7.20 (td, J = 1.4, 7.2 Hz, 2H), 7.27-7.30 (m, 4H), 7.33 –
(
=
1
3
7
1
.41 (m, 4H). C NMR (151 MHz, DMSO) δ 47.02, 49.00, 54.83, 55.74, 64.39, 67.38, 71.96,
05.49, 112.43, 113.94, 115.57, 121.73, 122.87, 127.22, 127.24, 128.78, 128.88, 130.09, 140.59,
2
6

ACCEPTED MANUSCRIPT
+
+
1
4
6
40.64, 146.69, 148.56, 168.61. M.p. [201-203°C]. HRMS m/z [M+H] Calcd. for C H NO :
26 30 4
20.5285. Found: 420.5280. El. Anal. Calcd. for C H NO : C 66.00, H 6.13, N 2.75. Found: C
28
31
8
6.02, H 6.15, N 2.70.
4.1.1.6. Cis-N-((-2-benzhydryl-1,3-dioxolan-4-yl)methyl)-2-(2-methoxyphenoxy)ethan-1-ammonium
hydrogen oxalate (cis-6)
1
White solid (32% yield). H NMR (600 MHz, DMSO-d ) δ 2.74-2.76 (m, 1H), 3.03-3.09 (m, 1H),
6
3
4
1
.16-3.21 (m, 2H), 3.69 (dd, J = 4.8, 8.6 Hz, 1H), 3.75 (s, 3H), 3.94 (dd, J = 6.8, 8.6 Hz, 1H), 4.10-
.15 (m, 2H), 4.23 (d, J = 5.8 Hz, 1H), 4.34-4.39 (m, 1H), 5.62 (d, J = 5.8 Hz, 1H), 6.87 – 6.94 (m,
1
3
H), 6.95 – 7.05 (m, 3H), 7.13 – 7.23 (m, 2H), 7.26-7.30 (m, 4H), 7.33 – 7.42 (m, 4H). C NMR
(151 MHz, DMSO) δ 47.11, 50.12, 54.89, 55.73, 64.29, 67.52, 71.83, 106.38, 112.45, 114.09,
1
1
4
2
15.57, 120.30, 121.71, 122.93, 127.27, 128.82, 128.92, 129.00, 130.10, 140.40, 146.68, 148.66,
+
+
68.61. M.p. [203-205°C]. HRMS m/z [M+H] Calcd. for C H NO : 420.5285. Found:
26
30
4
20.5283. El. Anal. Calcd. for C H NO : C 66.00, H 6.13, N 2.75. Found: C 66.00, H 6.10, N
2
8
31
8
.73.
4.1.1.7. Trans-2-(2-benzhydryl-1,3-dioxolan-4-yl)-N-(2-phenoxyethyl)ethan-1-ammonium hydrogen
oxalate (trans-7)
1
White solid (56% yield). H NMR (600 MHz, DMSO-d ) δ 1.70 – 1.87 (m, 2H), 2.90-2.95 (m, 2H),
6
3
1
2
6
1
.22-3.24 (m, 2H), 3.40 (dd, J = 6.2, 8.1 Hz, 1H), 3.91 (dd, J = 6.0, 8.1 Hz, 1H), 3.95 – 4.00 (m,
H), 4.02 – 4.15 (m, 3H), 5.61 (d, J = 6.0 Hz, 1H), 6.86 – 6.94 (m, 3H), 7.10 (td, J = 1.5, 7.2 Hz,
1
3
H), 7.14 – 7.32 (m, 10H). C NMR (151 MHz, DMSO) δ 29.67, 44.75, 46.53, 55.18, 64.05,
9.56, 73.63, 105.08, 115.05, 121.67, 126.82, 128.60, 128.63, 129.23, 129.28, 130.05, 141.34,
+
+
58.23, 164.81. M.p. [172-174°C]. HRMS m/z [M+H] Calcd. for C H NO : 404.2220. Found:
2
6
30
3
2
7

ACCEPTED MANUSCRIPT
4
04.2220. El. Anal. Calcd. for C H NO : C 68.14, H 6.33, N 2.84. Found: C 68.14, H 6.30, N
2
8
31
7
2.85.
4.1.1.8. Cis-2-(2-benzhydryl-1,3-dioxolan-4-yl)-N-(2-phenoxyethyl)ethan-1- ammonium hydrogen
oxalate (cis-7)
1
White solid (30% yield). H NMR (600 MHz, DMSO-d ) δ 1.68-1.74 (m, 1H), 1.80-1.84 (m, 1H),
6
2
1
7
1
1
4
2
.85 – 2.96 (m, 2H), 3.24 – 3.30 (m, 2H), 3.46 (dd, J = 5.8, 8.1 Hz, 1H), 3.91 (dd, J = 6.6, 8.0 Hz,
H), 3.90-4.20 (m, 4H), 5.56 (d, J = 5.9 Hz, 1H), 6.97 – 7.02 (m, 3H), 7.18 – 7.22 (m, 2H), 7.28 -
13
.35 (m, 10H). C NMR (151 MHz, DMSO) δ 29.99, 44.48, 46.58, 55.37, 64.05, 69.00, 73.79,
05.58, 115.05, 121.70, 126.87, 128.58, 128.60, 129.28, 129.32, 130.06, 141.18, 141.21, 158.23,
+
+
64.73. M.p. [172-174°C]. HRMS m/z [M+H] Calcd. for C H NO : 404.2220. Found:
26
30
3
04.2223. El. Anal. Calcd. for C H NO : C 68.14, H 6.33, N 2.84. Found: C 68.15, H 6.35, N
2
8
31
7
.80.
4.1.1.9.
Trans-2-(2-benzhydryl-1,3-dioxolan-4-yl)-N-(2-(2-methoxyphenoxy)ethyl)ethan-1-
ammonium hydrogen oxalate (trans-8)
1
White solid (45% yield). H NMR (600 MHz, DMSO-d ) δ 1.88-1.92 (m, 2H), 3.07-3.10 (m, 2H),
6
3
4
7
.30-3.32 (m, 2H), 3.50 (dd, J = 6.2, 8.1 Hz, 1H), 3.74 (s, 3H), 4.00 (dd, J = 6.0, 8.1 Hz, 1H), 4.03 –
.09 (m, 1H), 4.17-4.20 (m, 3H), 5.71 (d, J = 6.0 Hz, 1H), 6.91 (dd, J = 1.7, 7.6 Hz, 1H), 6.95 –
1
3
.10 (m, 3H), 7.17 – 7.24 (m, 2H), 7.26-7.29 (m, 4H), 7.35-7.37 (m, 4H). C NMR (151 MHz,
DMSO) δ 29.65, 44.80, 46.51, 55.16, 55.91, 65.65, 69.54, 73.65, 105.10, 112.82, 115.40, 121.19,
22.74, 126.82, 128.57, 128.60, 128.63, 129.24, 129.29, 129.33, 141.33, 147.57, 149.91, 164.81.
1
+
+
M.p. [185-187°C]. HRMS m/z [M+H] Calcd. for C H NO : 434.2326. Found: 434.2325. El.
2
7
32
4
Anal. Calcd. for C H NO : C 66.53, H 6.35, N 2.68. Found: C 66.50, H 6.35, N 2.65.
2
9
33
8
2
8