Design, synthesis, radiolabeling and in vivo evaluation of potential positron emission tomography (PET) radioligands for brain imaging of the 5-HT7 receptor

Article abstract of DOI:10.1016/j.bmc.2014.01.016

Here we describe the design, synthesis, and pharmacological evaluation of a set of compounds structurally related to the high affinity serotonin 5-HT ₇ receptor agonist N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1- piperazinehexanamide (6, LP-211). Specific structural modifications were performed in order to maintain affinity for the target receptor and to improve the selectivity over 5-HT_1A and adrenergic α₁ receptors. The synthesized compounds have chemical features that could enable labeling with a positron emitter radioisotope (carbon-11 or fluorine-18) and lipophilicity within the range considered optimal for brain penetration and low non-specific binding. 4-[2-(4-Methoxyphenyl)phenyl]-N-(pyridin-4-ylmethyl) piperazinehexanamide (23a) and N-pyridin-4-ylmethyl-3-[4-[2-(4-methoxyphenyl) phenyl]piperazin-1-yl]ethoxy]propanamide (26a) were radiolabeled on the methoxy group with carbon-11. Positron emission tomography (PET) analysis revealed that [¹¹C]-23a and [¹¹C]-26a were P-glycoprotein (P-gp) substrates and rapidly metabolized, resulting in poor brain uptake. These features were not predicted by in vitro tests.

Full text of DOI:10.1016/j.bmc.2014.01.016

Accepted Manuscript
Design, Synthesis, Radiolabeling and In Vivo Evaluation of Potential Positron
Emission Tomography (PET) Radioligands for Brain Imaging of the 5-HT Re-
7
ceptor
Enza Lacivita, Mauro Niso, Hanne D. Hansen, Pantaleo Di Pilato, Matthias M.
Herth, Szabolcs Lehel, Anders Ettrup, Lisa Montenegro, Roberto Perrone,
Francesco Berardi, Nicola A. Colabufo, Marcello Leopoldo, Gitte Moos
Knudsen
PII:
S0968-0896(14)00044-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.01.016
BMC 11347
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
26 September 2013
3 December 2013
14 January 2014
Please cite this article as: Lacivita, E., Niso, M., Hansen, H.D., Pilato, P.D., Herth, M.M., Lehel, S., Ettrup, A.,
Montenegro, L., Perrone, R., Berardi, F., Colabufo, N.A., Leopoldo, M., Knudsen, G.M., Design, Synthesis,
Radiolabeling and In Vivo Evaluation of Potential Positron Emission Tomography (PET) Radioligands for Brain
Imaging of the 5-HT Receptor, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.
7
2
014.01.016
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Design, Synthesis, Radiolabeling and In Vivo Evaluation of Potential Positron Emission
Tomography (PET) Radioligands for Brain Imaging of the 5-HT Receptor
7
a
Enza Lacivita, Mauro Niso, Hanne D. Hansen, Pantaleo Di Pilato, Matthias M. Herth,
a
b,c
a
b,c,d
a
d
b,c
a
a
Szabolcs Lehel, Anders Ettrup, Lisa Montenegro, Roberto Perrone, Francesco Berardi, Nicola
a
A. Colabufo, Marcello Leopoldo* , Gitte Moos Knudsen
a
b,c
Affiliation
a) Dipartimento di Farmacia – Scienze del Farmaco, Università degli Studi di Bari A. Moro, via
Orabona, 4, 70125 Bari, Italy
b) Center for Integrated Molecular Brain Imaging, Rigshospitalet, Blegdamsvej 9, 2100
Copenhagen, Denmark
c) Neurobiology Research Unit, Rigshospitalet and University of Copenhagen, Blegdamsvej 9,
2100 Copenhagen, Denmark
d) PET and Cyclotron Unit, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark
*Corresponding Authors: Marcello Leopoldo, Dipartimento di Farmacia – Scienze del Farmaco,
Università degli Studi di Bari “A. Moro”, via Orabona, 4, 70125, Bari, Italy. E-mail:
marcello.leopoldo@uniba.it; Phone: +39 080 5442798; Fax: +39 080 5442231
1

Abstract
Here we describe the design, synthesis, and pharmacological evaluation of a set of compounds
structurally related to the high affinity serotonin 5-HT
-(2-diphenyl)-1-piperazinehexanamide (6, LP-211). Specific structural modifications were
performed in order to maintain affinity for the target receptor and to improve the selectivity over 5-
HT1A and adrenergic α receptors. The synthesized compounds have chemical features that could
7
receptor agonist N-(4-cyanophenylmethyl)-
4
1
enable labeling with a positron emitter radioisotope (carbon-11 or fluorine-18) and lipophilicity
within the range considered optimal for brain penetration and low non-specific binding. 4-[2-(4-
Methoxyphenyl)phenyl]-N-(pyridin-4-ylmethyl)piperazinehexanamide (23a) and N-pyridin-4-
ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanamide (26a) were
1
radiolabelled on the methoxy group with carbon-11. PET analysis revealed that [ C]23a and
1
1
1
[
C]26a were P-glycoprotein (P-gp) substrates and rapidly metabolized, resulting in poor brain
uptake. These features were not predicted by in vitro tests.
2

1
. Introduction
The fourteen different receptors that are activated by serotonin (5-hydroxytryptamine, 5-HT)
are divided into seven families depending on their pharmacological properties, amino acid
1
sequences, and second-messenger coupling pathways. The 5-HT
7
receptor is the most recently
identified member of the 5-HT receptors family. It was first cloned by sequence homology to other
5
-HT receptors in the mouse. Subsequently, it was cloned from the rat, guinea pig, pig, and human
2
tissue. Important functional roles that have been established for the 5-HT
7
receptor include
3
thermoregulation, circadian rhythm, learning and memory, hippocampal signaling, and sleep. The
5
-HT
7
receptor is possibly also involved in psychiatric and neurological disorders, such as
3
schizophrenia, epilepsy, and migraine. Preclinical studies have shown that 5-HT
7
receptor
antagonists induce an antidepressant-like response, a phenotype that can also be observed in mice
4
lacking the receptor. Furthermore, in clinical trials a new atypical antipsychotic agent with very
5
7
high affinity for the 5-HT receptor, lurasidone, improves depression rating scores in patients.
Moreover, it has been suggested that 5-HT
7
receptor agonist drugs may be beneficial for pain
receptor as a therapeutic target for treatment of
receptors may represent a novel strategy
6
alleviation. Very recent studies indicate the 5-HT
7
neurodevelopmental diseases: selective activation of 5-HT
7
for treatment of Fragile X syndrome, the most common form of inherited intellectual disability and
7
autistic spectrum disorder. Also, the 5-HT
7
receptor plays a key role in stimulating neurite
8
-10
outgrowth from different neurons in vitro, suggesting its involvement in central nervous system
(
CNS) connectivity which may be intimately linked to psychiatric and neurodevelopmental
1
1
disorders.
Positron emission tomography (PET) is a molecular imaging technique that is increasingly
1
2
being used in the drug development process, where PET radioligands enable quantitative
measurement of molecular processes and interactions between a candidate drug and its molecular
targets, making this technique a very valuable tool at all stages from preclinical development to the
1
3
7
clinical trials. Accordingly, imaging of the 5-HT receptor using PET might enable quantification
3

of the distribution expression, and modulation of this receptor under physiological and pathological
conditions.
Various attempts to develop a 5-HT
7
receptor PET radioligand have not been successful so
1
far. Zhang and coworkers have reported a PET study in monkey on [ C]-1 ([ C]-DR4446) (Figure
1
11
1
): although the tracer displayed a regional brain binding compatible with 5-HT
7
receptor brain
14
18
distribution, no further studies were reported. Andries and colleagues developed [ F]-2 (Figure 1)
as a potential 5-HT
specific 5-HT receptor binding. The same research group reported a set of F-labeled
compounds based on the selective 5-HT receptor antagonist 3 (SB-269970) (Figure 2). Among
7
PET radioligand but brain tissue autoradiography failed to give evidence of
1
5
18
7
7
1
these, [ F]-4 ([ F]-2FP3, Figure 1) had a high brain uptake. However, in the absence of a valid
8
18
reference region or an arterial input function it was difficult to fully assess the validity of this PET
1
radioligand. Herth et al. have reported the synthesis, radiolabeling and in vivo evaluation of the
6
1
1
compound [ C]-5 ([ C]-Cimbi-806) (Figure 1). Despite the high brain uptake and relevant
11
1
1
17
distribution pattern, [ C]-5 failed to be displaced by the 5-HT
Thus, to date no fully validated selective 5-HT receptor PET radioligand exists.
Here, we sought to develop a PET radioligand to image the cerebral 5-HT
the years we have identified various high affinity and selective 5-HT
7
receptor selective antagonist 3.
7
7
receptors. Over
1
8-20
7
receptor ligands.
Among
2
1
them, we identified the arylpiperazine derivative 6 (LP-211, Figure 2). However, this compound is
not an ideal potential PET radioligand because the lipophilicity of this compound (calculated
2
2,23
logD7.4 = 4.46) is not considered optimal for brain uptake and low non specific binding,
Thus,
we embarked upon a chemical programme aimed at obtaining analogues of 6 characterized by
lower lipophilicity and endowed with a chemical feature that could be easily radiolabelled with a
1
positron emitting isotope such as C or F.
1
18
2
. Results and discussion
.1. Chemistry
2
4

The synthesis of the target compounds 21a-23a, 27a, 35e, 36f, 37e, 38f with a pentamethylene
spacer as compound 6 is reported in Scheme 1. The appropriate arylmethylamine 7-11 was reacted
with 6-bromohexanoyl chloride to give the 6-bromohexanamides 12-16, that were used to alkylate
the appropriate arylpiperazines 17a-f to afford the target compounds.
The target compounds having an ethoxyethyl spacer were synthesized following a different
synthetic pathway (Scheme 1). The arylpiperazines 17a-d were alkylated with 2-bromoethanol to
give alcohols 18a-d. These latters underwent Michael addition to t-butylacrylate to give the esters
19a-d. Acidic hydrolysis of the esters afforded carboxylic acids 20a-d that were subsequently
condensed with the appropriate arylmethylamine to give the target compounds 24a-26a, 28a, 29b,
30c, 31d, 32b, 33c, 34d.
7
2.2. Structure-Activity Relationships for the 5-HT Receptor
Compound 6 was selected as starting point to develop a PET radioligand due to its high affinity for
rat and human cloned 5-HT receptor (K = 0.58 and 15.0 nM, respectively) and for its selectivity
profile over a range of 5-HT receptors. Of importance is the selectivity over 5-HT1A receptors
because there are well known parallels between 5-HT and 5-HT1A receptors in their distributions in
the CNS and in the pharmacological properties. Additionally, a similarity in the putative binding
7
i
7
2
4
2
5
sites of the two receptors has been highlighted. Considering that the affinity value of 6 at 5-HT1A
receptor (K = 379 nM), this compound might appear highly selective (650-fold) if K value at rat
cloned 5-HT receptor is considered or modestly selective (25-fold) if the K value at human cloned
-HT receptor is considered. Indeed, in vitro and in vivo studies have shown that 6 is a 5-HT
i
i
7
i
2
6
5
7
7
-
7
,10
receptor specific agonist. On such basis, in the perspective of developing a PET radioligand, we
targeted compounds showing an in vitro affinity profile comparable to that of 6. The new
compounds are based on the same scaffold of 6 because our previous structure-activity relationship
(
SAR) studies on long-chain arylpiperazine derivatives related to 6 indicated that a) the biphenyl
piperazine moiety is responsible for high affinity for 5-HT
5
7
receptor and selectivity over 5-HT1A

7
receptor; b) the length of the spacer also influences the 5-HT /5-HT1A receptor selectivity: a shorter
2
7
chain might result in a loss of selectivity over 5-HT1A receptor; c) marginal structural
modifications of the terminal amidic fragment can be tolerated. Based on these considerations, we
designed a set of analogs of compound 6 fulfilling the following requirements i) presence of a
1
chemical feature that can be easily radiolabelled with C or F; ii) lipophilicity value within the
1
18
range 2.5 ≤ logP ≤ 3.5 that is considered optimal for adequate brain penetration without an
2
2,23
excessive level of non specific binding
(Table 1).
The first requirement was fulfilled by inserting a methoxy group or a fluoro substituent in a
position of the molecule that could give easy access to the radiolabelled form through conventional
radiochemistry procedures. Thus, we synthesized the compounds 21a-28a because 1-[2-(4-
2
8
methoxyphenyl)phenyl]piperazine is a high-affinity 5-HT
7 i
receptor ligand (K = 2.6 nM). As an
alternative, we also synthesized the compounds 30c and 33c because we found that 1-[3-(2-
28
7 i
methoxyphenyl)phenyl]piperazine shows high 5-HT receptor affinity (K = 8.2 nM). We have also
evaluated compounds displaying the structural feature for radiolabeling on the terminal amidic
fragment. Therefore, we have synthesized N-(4-methoxyphenylmethyl)- amides 32b, 33c, 34d, 37e,
38f and N-(2-fluoro-5-pyridylmethyl)- amides 36f, 35e, 29b, 31d. It is important to note that the
possibility to radiolabel the compounds on the arylpiperazine moiety or on the terminal amide
fragment might be of relevance in view of metabolic degradation in vivo. In fact, it has been
2
0
reported that 6 is metabolized in vivo to the corresponding unsubstituted arylpipiperazine. Thus, if
the new analogues of 6 would undergo similar metabolism, one could expect different radiolabelled
metabolites depending upon which moiety of the molecule was radiolabelled. For brain PET
radioligands metabolism should occur, ideally, outside the brain producing radiometabolites with
poor brain entry and little or no interaction with the target protein, because in PET scans there are
2
3
no means to discriminate between the chemical sources of detected radioactivity. Finally, the
presence of the feature for radiolabelling on the terminal amide fragment permits the study of
arylpiperazine derivatives different from those based on 1-[2-(4-methoxyphenyl)phenyl]piperazine.
6

In fact, compounds 29b, 31d, 32b, 34d were based on the high-affinity 5-HT
7
ligands 1-[2-(4-
2
fluorophenyl)phenyl]- and 1-[2-(4-methylphenyl)phenyl]- piperazine, whereas 35e, 36f, 37e, 38f
8
2
were prepared starting from the newly designed 1-[2-(4-pyridyl)pyridyn-3-yl]- and 1-[3-(4-
9
pyridyl)pyridyn-2-yl]- piperazine derivatives (see below).
The second requirement to be fulfilled by the new molecules is lipophilicity within the logP range
of 2.5-3.5. Lipophilicity can be evaluated in various theoretical and experimental ways. Theoretical
partition coefficients are routinely calculated by various softwares that give values generally well
correlated with the experimental determinations. Since our previous studies on arylpiperazine
2
0,30,31
derivatives substantially confirmed this trend,
for the new molecules.
we have adopted clogD7.4 values for the design
Therefore, we modified the scaffold of compound 6 by replacing one phenyl ring with a pyridyl
ring on one end of the scaffold and/or by replacing the intermediate pentamethylene chain with the
ethoxyethyl spacer. In particular, we have synthesized various N-pyridylmethylamides because our
2
0
7
SAR studies showed that this fragment was not detrimental for 5-HT receptor affinity. Also, we
have evaluated the 1-[3-(4-pyridyl)pyridin-2-yl]piperazine derivatives 35e and 37e, and the 1-[2-(4-
pyridyl)pyridin-3-yl]piperazine derivatives 36f and 38f because we have found these bipyridyl
2
systems as valid substitutes of the biphenyl system. As a result of these modifications, we have
9
obtained a set of compounds showing a wide range of lipophilicity (Table 1).
The target compounds were evaluated for their affinity at human cloned 5-HT
receptors in radioligand binding assay. Moreover, they were evaluated for their adrenergic α
receptor affinity because it has been reported that the 1-(2-biphenyl)piperazinyl scaffold might have
7
and 5-HT1A
1
3
2
affinity for such receptor.
Considering the affinity data for the 5-HT
7
receptor of the target compounds (Table 1), it
emerges that the structural modifications performed on compound 6 were, in general, well tolerated.
In particular, compounds 21a-23a, that have a pentamethylene linker as 6, and the 1-[2-(4-
methoxyphenyl)phenyl]piperazine moiety instead of the 1-(2-biphenyl)piperazine one, displayed
7

almost identical K
i
values as compound 6. The replacement of the pentamethylene spacer in
receptor affinity
compounds 21a-23a with the ethoxyethyl spacer (24a-26a) left the 5-HT
7
unchanged. Interestingly, this modification improved the selectivity over both 5-HT1A and α
1
adrenergic receptors.
Comparing compounds 27a and 28a with 22a and 25a, respectively, the introduction of a
fluoro substituent on the N-pyridylmethyl group led to an increase in affinity. Also in this case, the
compound with the ethoxyethyl spacer was more selective than the compound with the
pentamethylene spacer (28a vs 27a).
Comparing the derivative 28a with the 2-biphenyl analogs 29b and 31d, an increase in 5-
HT
7
receptor affinity was observed, whereas the 3-biphenyl derivative 30c was devoid of affinity
> 1000 nM).
Considering the modification of the terminal amide fragment it can be noted that the N-4-
methoxyphenylmethyl derivatives 32b and 34d displayed lower affinity than the corresponding N-
-fluoro-5-pyridylmethyl derivatives 29b and 31d. 3-Biphenyl derivative 33c was devoid of 5-HT
receptor affinity similar to the analog 30c. When the 2-biphenyl ring in 27a was replaced by a
bipyridyl system (35e and 36f) a marked loss in 5-HT receptor affinity was observed. Also, N-2-
(K
i
2
7
7
fluoro-5-pyridylmethyl derivatives 35e and 36f were almost equipotent as the corresponding N-4-
methoxyphenylmethyl counterparts 37e and 38f.
As noted above, the replacement of the pentamethylene spacer (compounds 21a-23a) with the
ethoxyethyl spacer (24a-26a) resulted in an improvement of selectivity over 5-HT1A and adrenergic
1
α receptors. All the other ethoxyethyl derivatives 29b, 30c, 31d, 32b, 33c, 34d showed 5-HT1A
receptor affinities comparable to those of 24a-26a. Taken together, these data confirmed previous
SAR on the analogs of compound 6.
Considering that, to date, no effective PET 5-HT
7
radiotracer has been identified, a guideline
affinity value does not emerge from the literature. Based on available data we decided to not discard
11
i i
compounds with K values lower than 20 nM at this stage. In fact, for [ C]5 a K value of 8.8 nM
8

17
7 i
has been reported. In another case, four potential 5-HT receptor PET radiotracers showing K =
1
6
1.6-14 nM were evaluated.
Thus, derivatives 26a, 27a, 29b, 37e combined the desired binding affinity profile with
11
11
lipophilicity properties. For the ease of C radiolabelling, we decided to target [ C]-26a for a PET
1
study in vivo and also the corresponding pentamethylene derivative [ C]-23a for comparative
1
purposes.
PET tracers that are not substrates for the P-glycoprotein (P-gp) efflux pump have higher chances
for crossing the blood-brain barrier (BBB). Therefore, we evaluated the efflux ratio (BA/AB)
between basal-to-apical (BA) and apical-to-basal (AB) fluxes in Caco-2 cells monolayer of
compounds 23a and 26a. Generally, a cutoff value of 3 is used to distinguish P-gp substrate from
3
nonsubstrate. The compounds showed an efflux ratio of to 3.33 and 4.48, respectively, which is
3
predictive of a weak interaction with P-gp.
In vitro metabolism of compounds 23a and 26a was evaluated after incubation with rat hepatic S9
3
fraction in the presence of an NADPH-generating system. Compounds 23a and 26a did not
4
undergo significant oxidative metabolism, being the percent of recovery of parent compound after
30 min incubation was 76% and 82%, respectively.
2.3. Radiochemistry
1
1
The radiosynthesis of [ C]-23a and [ C]-26a required the preparation of the corresponding
11
phenolic precursors 39 (Scheme 2) and 48 (Scheme 3), respectively. The phenolic derivative 39 was
obtained by demethylating 23a under mild conditions by means of BBr
3
(Scheme 2).
As for the preparation of the phenolic precursor of [ C]-26a (compound 48), the reaction of
6a with BBr did not afford the desired phenolic derivative. Therefore, an alternative synthetic
1
1
2
3
route was devised (Scheme 3). 4-(2-Bromophenyl)-1-t-butoxycarbonylpiperazine (40) was coupled
with 4-bromophenol under Suzuki conditions to give derivative 41. This latter compound was first
reacted with benzyl bromide to protect the phenolic function (42), then reacted with trifluoroacetic
9

acid to give the deprotected arylpiperazine 43. This latter was alkylated with 2-bromoethanol to
give the alcohol 44 that underwent Michael addition to t-butylacrylate to give the ester 45.
Debenzylation of 45 by hydrogenolysis gave the phenol 46, that, after acidic hydrolysis of the ester
function, afforded carboxylic acid 47. Condensation of 47 with 4-pyridylmethylamine (9) gave the
desired phenolic derivative 48.
1
1
11
11
The preparation of [ C]23a and [ C]26a succeeded applying standard C-labelling conditions
Schemes 2 and 3). The precursor eluted ~ 300 sec prior to the radiolabelled product as indicated by
the UV-absorption chromatogram (see Supplementary Data). The radiosynthesis including HPLC
(
1
1
11
purification and formulation generated an injectable solution of [ C]23a and [ C]26a
radiochemical purity > 98%) within 70-80 min. Typically, 300–900 MBq (RCY > 40%) of the
final product was isolated with a specific activity (A ) of 50–200 GBq/µmol at end of synthesis. A
at the time of injection was 196 ± 133 GBq/µmol (average ± S.D.), n=3) for [ C]23a and 87 ± 61
(
s
s
1
1
1
GBq/µmol (average ± S.D.), n= 2) for [ C]26a.
1
1
.4. PET imaging of [ C]23a and [ C]26a in pigs.
1
11
2
1
1
The ability of [ C]23a and [ C]26a to image brain 5-HT
11
7
receptors in vivo was tested with PET in
1
pigs. After intravenous injection of [ C]23a and [ C]26a the radioactivity uptake was very limited
1
11
1
1
11
suggesting that neither [ C]23a nor [ C]26a freely crossed the BBB. This could be due to the
lipophilicity of the compounds or to the interaction of the compounds with the BBB efflux
transporter P-gp. To test the latter hypothesis, we pre-administered cyclosporine A (CsA), which is
an inhibitor of P-gp, before a second injection of the respective radioligand. This resulted in
11
11
increased brain uptake of [ C]23a and [ C]26a, as shown by the summed PET images (Figure 3).
However, the uptake only reached a standardized uptake value of ~1.0, indicating a uniform uptake
1
of radioactivity after intravenous injection of both [ C]23a and [ C]26a in the pig.
1
11
The time activity curves (TACs) revealed that CsA pre-treatment tripled the brain radioactivity
1
uptake of [ C]23a, whereas the effect of CsA on [ C]26a uptake was more modest (Figure 4). The
1
11
1
0

11
11
limited brain uptake of radioactivity after intravenous injection of [ C]23a and [ C]26a with an
increased uptake following CsA administration suggest that these two radioligands are substrates of
1
P-gp. CsA did not alter the arterial input of [ C]23a and [ C]26a (data not shown). It cannot be
1
11
1
1
35
ruled out that [ C]26a is also a substrate for other efflux transporters or it simply poorly diffuses
through the BBB as the brain uptake was only moderately improved by CsA pre-administration.
The cerebral kinetics of both radioligands were rather slow with limited wash-out detected within
1
1
the acquisition time (90 min). After i.v. injection, the fraction of [ C]23a in the plasma declined
rapidly, however with some inter-pig variability, as in one pig ~85% of the total radioactivity in the
1
plasma was attributed to [ C]23a after 5 min, whereas in the second pig this percentage was only
1
1
0% (Figure 5A). The metabolism of [ C]26a was very fast, with only ~30% of the parent fraction
1
5
remaining after 5 min (Figure 5B).
1
1
11
We could not determine the nature of the metabolites formed from [ C]23a and [ C]26a or if the
formed metabolites crossed the BBB. It can be speculated, however, that a N-dealkylation
1
metabolism observed for compound 6 also takes place for [ C]23a and [ C]26a, producing the
1
11
1
same metabolite [ C]-1-[2-(4-methoxyphenyl)phenyl]piperazine. A previous study reported that
1
1
1
[
C]-1-[2-(4-methoxyphenyl)phenyl]piperazine readily crossed the BBB in the non-human
3
primates. We cannot exclude that the observed brain radioactivity after intravenous injection of
6
1
1
11
C]23a and [ C]26a was due to the presence of the parent radioligands as well, but it is rather
[
unlikely given the rapid metabolism of the radioligands and the rather high brain uptake reported
1
for [ C]-1-[2-(4-methoxyphenyl)phenyl]piperazine in non-human primates and in the rat.
1
37
The two radioligands tested in this study did not show sufficient brain uptake to warrant further
investigation, particularly since CsA only moderately enhanced brain uptake.
3
. Conclusions
Here, we tested two compounds for their suitability as PET radioligands for imaging of 5-HT
receptors in the CNS. Starting from our high-affinity ligand 6, and guided by previous SAR studies
7
1
1

from our laboratory, we designed a set of compounds possessing chemical features amenable to
carbon-11 or fluorine-18 labeling and calculated logP values within the range that is considered
optimal for a radioligand. Affinity screening of compounds at the 5-HT
7
receptor as well as at
selected off-target receptors (serotonin 5-HT1A and adrenergic α ) guided our selection of N-
1
pyridin-4-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanamide (26a)
and its analog 4-[2-(4-methoxyphenyl)phenyl]-N-(pyridin-4-ylmethyl)piperazinehexanamide (23a).
The in vitro data on the microsomal stability and interaction with P-gp of 23a and 26a were in
1
agreement with the accepted criteria for a PET radioligand candidate, therefore [ C]23a and
1
1
1
[
C]26a were synthesized in yields sufficient for in vivo evaluation starting from phenolic
11
11
precursors 39 and 48. Unfortunately, initial PET investigations of [ C]23a and [ C]26a
1
1
11
11
unexpectedly revealed that [ C]23a and [ C]26a were substrates for the P-gp. Moreover, [ C]23a
1
and, especially, [ C]26a were very rapidly metabolized. When P-gp efflux was inhibited with CsA,
1
brain uptake of both radioligands increased, however not to a degree that warranted further
1
receptor in vivo. The main weakness of [ C]23a and
1
investigation of their specificity to the 5-HT
7
1
1
[
C]26a seems to be the poor in vivo pharmacokinetic profile, that was not predicted by the in vitro
tests. The poor predictive power of the tests could be due to interspecies variability that, however,
cannot be completely eliminated. Further optimization of the structure of compounds 23a and 26a
will thus be required in order to improve their overall pharmacokinetic properties in vivo.
1
2

4. Experimental section
Chemicals were purchased from Acros, Fluka, Sigma, Tocris, or Merck. Unless otherwise stated, all
chemicals were used without further purification. Thin layer chromatography (TLC) was performed
using plates from Merck (silica gel 60 F254 and aluminium oxide 60 F254). Column chromatography
was performed with 1:30 Merck silica gel 60A (63-200 µm) as the stationary phase. Reactions were
1
performed under microwave irradiation with a Biotage Initiator+ synthesizer. H NMR spectra were
recorded at 300 MHz on a Varian Mercury-VX spectrometer. All spectra were recorded on free
bases. All chemical shift values are reported in ppm (δ). Recording of mass spectra was done on an
HP6890-5973 MSD gas chromatograph/mass spectrometer; only significant m/z peaks, with their
percentage of relative intensity in parentheses, are reported. All spectra were in accordance with the
assigned structures. HRMS-ESI analyses were performed on a Bruker Daltonics MicrOTOF-Q II
mass spectrometer, mass range 50-800 m/z, electrospray ion source in positive ion mode. Elemental
analyses (C,H,N) of the tartrate salts of the target compounds were performed on a Eurovector Euro
EA 3000 analyzer. Analyses indicated by the symbols of the elements were within ± 0.4 % of the
theoretical values. The purity of the target compounds listed in Table 1 was assessed by RP-HPLC
and combustion analysis. All compounds showed ≥ 95% purity. RP-HPLC analysis was performed
on a Perkin-Elmer series 200 LC instrument using a Phenomenex Prodigy ODS-3 RP-18 column,
(
250 × 4.6 mm, 5 µm particle size) and equipped with a Perkin-Elmer 785A UV/VIS detector
setting λ= 254 nm. All target compounds (Table 1) were eluted with CH OH/H O/Et N, 7:3:0.01 at
3
3
2
3
8
27
28
39
a flow rate of 1 mL/min. Compounds 11, 17a-d, 17e,f, and 40 were prepared according to
literature methods. The Human Embryonic Kidney 293 cell line (HEK-293 cells) was kindly gifted
by Prof. Susanna Cotecchia, Dip. Bioscienze, Biotecnologie e Scienze Farmacologiche, Università
degli Studi di Bari “Aldo Moro”. Cell culture reagents were purchased from EuroClone (Milan,
Italy). 5-HT7A cDNA was purchased from Missouri S&T cDNA Resource Center (Rolla, MO
65409). G418 (geneticin), 5-HT, phentolamine hydrochloride, were obtained from Sigma-Aldrich
1
3

(
Milano, Italy). 5CT was purchased from Tocris Bioscience (Bristol, UK). FuGENE HD
Transfection Reagent was purchased from Promega (Milano, Italy); Opti-MEM was obtained from
3
3
3
Invitrogen. [ H]-5CT, [ H]-8-OH-DPAT, [ H]-prazosin, and human recombinant serotonin 5-HT1A
receptors expressed in HEK-293-EBNA cells, were obtained from PerkinElmer Life and Analytical
Sciences (Boston, MA, USA). Wistar Hannover rats (250-300 g) were obtained from Harlan (Italy).
MultiScreen plates with Glass fiber filters were purchased from Merck Millipore (Billerica, MA,
USA). Rat liver S9 fraction was purchased from from BD Bioscience (Milan, Italy). Analytical high
performance liquid chromatography (HPLC) measurements in the radiochemistry laboratory were
performed on a Dionex system consisting of a P680A pump, a UVD 170U detector and a Scansys
1
1
radiodetector. Chemical purity was checked either by HPLC or by GC. [ C]Methane was produced
1
4
11
14
2 2
via the N(p,α) C reaction by bombardment of an [ N]N containing 10% H target with a 17
MeV proton beam in a Scanditronix MC32NI cyclotron. Radioactive synthesis were carried out on
an automated Scansys module.
4.1. Chemical Synthesis
4.1.1. General Procedure for the Preparation of Compounds 12-16.
A cooled solution of amine 7-11 (1.4 mmol) in CH
2 2
Cl (20 mL) was stirred vigorously with 2%
aqueous NaOH (5.6 mL, 1.7 mmol) while 6-bromohexanoyl chloride (0.36 g, 1.7 mmol) in CH
2
Cl
2
(
10 mL) was added dropwise. The same NaOH solution was then used to maintain pH at 9, and at
2
constant pH the layers were separated. The organic phase was washed with 3N HCl, with H O, and
then dried over Na
2
SO
4
and evaporated under reduced pressure. The crude residue was purified by
/AcOEt, 1:1, as eluent) to give pure desired compound.
chromatographic column (CHCl
3
4.1.1.1. 6-Bromo-N-(pyridin-2-ylmethyl)hexanamide (12)
1
4

1
Amber oil, 87% yield. H NMR: δ 1.43-1.53 (m, 2H), 1.71 (quintet, 2H, J= 7.6 Hz), 1.83-1.92 (m,
2
H), 2.30 (t, 2H, J= 7.4 Hz), 3.40 (t, 2H, J= 6.7 Hz), 4.55 (d, 2H, J= 4.7 Hz), 6.77 (br t, 1H, D
2
O
+
exchanged), 7.17-7.24 (m, 2H), 7.63-7.69 (m, 1H), 8.52-8.54 (m, 1H). ESI/MS m/z 307 (M+Na) .
ESI-MS-MS m/z: 227 (31), 206 (17), 205 (100).
4.1.1.2. 6-Bromo-N-(pyridin-3-ylmethyl)hexanamide (13)
1
Lightly yellow oil, 53% yield. H NMR: δ 1.44-1.52 (m, 2H), 1.69 (quintet, 2H, J= 7.6 Hz), 1.87
(
quintet, 2H, J= 7.2 Hz ), 2.24 (t, 2H, J= 7.4 Hz), 3.40 (t, 2H, J= 6.8 Hz), 4.45 (d, 2H, J= 6.1 Hz),
5.94 (br s, 1H, D
2
O exchanged), 7.23-7.26 (m, 1H), 7.62-7.63 (m, 1H), 8.50-8.52 (m, 2H). GC/MS
+
m/z: 286 (M +2, 2), 284 (M , 2), 150 (75), 135 (90), 92 (100).
+
4.1.1.3. 6-Bromo-N-(pyridin-4-ylmethyl)hexanamide (14)
1
Colourless oil, 71% yield. H NMR and GC/MS data are consistent with those previously reported.
4.1.1.4. 6-Bromo-N-(2-fluoro-pyridin-5-ylmethyl)hexanamide (15)
1
Brown-red oil, 49% yield. H NMR (CDCl
3
): δ 1.44-1.52 (m, 2H), 1.64-1.74 (m, 2H), 1.82-1.90 (m,
2
H), 2.24 (t, 2H, J= 7.5 Hz), 3.40 (t, 2H, J= 6.6 Hz), 4.43 (d, 2H, J= 6.1 Hz), 5.88 (br s, 1H), 6.90
(
dd, 1H, J= 2.8, 8.5 Hz), 7.76 (dt, 1H, J= 2.5, 7.7 Hz), 8.12 (dd, 1H, J= 0.8, 1.4 Hz). ESI/MS m/z
+
04 (M+H) . ESI-MS/MS m/z 245 (20), 223 (100).
3
4.1.1.5. N-(4-Methoxybenzyl)-6-bromohexanamide (16)
1
White solid, 75% yield . H NMR (CDCl
3
): δ 1.42-1.52 (m, 2H), 1.61-1.78 (m, 2H), 1.81-1.92 (m,
2
H), 2.21 (t, 2H, J= 7.4 Hz), 3.40 (t, 2H, J= 6.8 Hz), 3.80 (s, 3H), 4.37 (d, 2H, J= 5.5 Hz), 5.65 (br
+
2
t, 1H, D O exchanged), 6.86 (d, 2H, J= 8.5 Hz), 7.20 (d, 2H, J= 8.5 Hz). ESI/MS m/z 336 (M+Na) .
ESI-MS/MS m/z 256 (14), 235 (16), 234 (100), 121 (58).
1
5

4
.1.2. General Procedure for the Preparation of Compounds 18a-d.
A mixture of arylpiperazine 17a-d (5.5 mmol), 2-bromoethanol (6.6 mmol), K
KI (2.0 mmol) in CH CN (20 mL) was stirred under reflux overnight (compound 18a) or was
2
3
CO (6.6 mmol) and
3
heated at 150 °C for 10 minutes by microwave irradiation (compounds 18b-d). After cooling, the
solvent was removed under reduced pressure and the residue was partitioned between AcOEt (20
mL) and H
with AcOEt (20 mL). The combined organic layers were dried over Na
reduced pressure. The crude residue was purified by chromatographic column (CHCl
as solvent) to afford pure target compound as oil.
2
O (20 mL). The organic layer was separated and the aqueous phase was extracted twice
SO and evaporated under
/MeOH, 19:1,
2
4
3
4.1.2.1. 2-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]ethanol (18a)
1
Brown oil, 65% yield. H NMR (CDCl
3
): δ 2.09 (br s, 1H, D
2
O exchanged), 2.61 (t, 2H, J= 5.5
Hz), 2.69 (app t, 4H), 3.25 (app t, 4H), 3.66 (t, 2H, J= 5.5 Hz), 3.84 (s, 3H), 6.94 (d, 2H, J= 8.8
Hz), 7.00 (d, 1H, J= 8.0 Hz), 7.10-7.15 (m, 1H), 7.22-7.30 (m, 2H), 7.46 (d, 2H, J= 8.8 Hz).
+
GC/MS m/z 313 (M +1, 11), 312 (M , 50), 281 (100), 238 (27), 168 (23).
+
4.1.2.2. 2-[4-[2-(2-Methylphenyl)phenyl]piperazin-1-yl]ethanol (18b)
1
Brown oil, 82% yield. H NMR (CDCl
3
): δ 2.09 (br s, 1H, D
2
O exchanged), 2.18 (s, 3H), 2.31 (br s,
4
H), 2.44-2.48 (m, 2H), 2.75-2.80 (m, 2H), 2.87-2.94 (m, 2H), 3.55 (t, 2H, J= 5.3 Hz), 7.02-7.19
+
m, 5H), 7.28-7.34 (m, 3H). GC/MS m/z 297 (M +1, 6), 296 (M , 29), 265 (100), 194 (23).
+
(
4.1.2.3. 2-[4-[3-(2-Methoxyphenyl)phenyl]piperazin-1-yl]ethanol (18c)
1
Brown oil, 65% yield. H NMR (CDCl
3
): δ 2.09 (br s, 1H, D
2
O exchanged), 2.61 (t, 2H, J= 5.5
Hz), 2.69 (app t, 4H), 3.25 (app t, 4H), 3.66 (t, 2H, J= 5.5 Hz), 3.81 (s, 3H), 6.90 (dd, 1H, J= 1.9,
+
.2 Hz), 6.96-7.09 (m, 4H), 7.28-7.34 (m, 3H). GC/MS m/z 313 (M +1, 11), 312 (M , 50), 281
+
8
(
100), 238 (27), 168 (23).
1
6

4.1.2.4. 2-[4-[2-(4-Fluorophenyl)phenyl]piperazin-1-yl]ethanol (18d)
1
Yellow oil, 82% yield . H NMR (CDCl
3
): δ 2.09 (br s, 1H, D
2
O exchanged), 2.43 (br s, 4H), 2.53
(
t, 2H, J= 5.5 Hz), 2.84-2.87 (m, 4H), 3.59 (t, 2H, J= 5.5 Hz), 7.02-7.08 (m, 4H), 7.20-7.23 (m,
+
H), 7.57-7.62 (m, 2H). GC/MS m/z 300 (M , 4), 269 (100), 207 (43), 198 (41).
2
4.1.3. General Procedure for the Preparation of Compounds 19a-d.
To a cooled suspension of NaH (0.07 g, 3.0 mmol) in anhydrous THF (5 mL) was added, dropwise
and under nitrogen, a solution of alcohol 18a-d (3.0 mmol) in the same solvent (10 mL). The
reaction mixture was stirred at room temperature for 8-10 hours. Then, a solution of t-butyl acrylate
(
0.4 mL, 2.71 mmol) in anhydrous THF (10 mL) was added dropwise and the reaction mixture was
stirred at room temperature for 20-22 hours. Then, the solvent was removed under reduced pressure
and the residue was cautiously taken up with brine and extracted with AcOEt (2 × 30 mL). The
combined organic layers were washed with H
pressure. The crude residue was purified by chromatographic column (CHCl
eluent) to afford pure compound as wax.
2
O, dried over Na
2
SO
4
and evaporated under reduced
3
/AcOEt, 1:1, as
4.1.3.1. t-Butyl-3-[2-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanoate (19a)
1
Orange wax, 26% yield. H NMR (CDCl
3
): δ 1.43 (s, 9H), 2.42 (br s, 4H), 2.47 (t, 2H, J= 6.5 Hz ),
2.54 (t, 2H, J= 6.3 Hz), 2.86 (app t, 4H), 3.56 (t, 2H, J= 5.8 Hz), 3.66 (t, 2H, J= 6.5 Hz), 3.85 (s,
3
H), 6.90-6.95 (m, 2H), 7.00-7.07 (m, 2H), 7.19-7.28 (m, 2H), 7.55-7.60 (m, 2H). ESI/MS m/z 463
+
M+Na) , ESI-MS/MS m/z 407 (100).
(
4.1.3.2. t-Butyl-3-[2-[4-[2-(2-methylphenyl)phenyl]piperazin-1-yl]ethoxy]propanoate (19b)
1
7

1
Yellowish wax, 33% yield. H NMR (CDCl
3
): δ 1.43 (s, 9H), 2.17 (s, 3H), 2.29 (br s, 4H), 2.43-
2.50 (m, 4H), 2.74-2.79 (m, 2H), 2.86-2.92 (m, 2H), 3.52 (t, 2H, J= 5.8 Hz), 3.64 (t, 2H, J= 6.6
+
Hz), 7.02-7.19 (m, 5H), 7.28-7.34 (m, 3H). ESI/MS m/z 447 (M+Na) , ESI-MS/MS m/z 391 (100).
4.1.3.3. t-Butyl-3-[2-[4-[3-(2-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanoate (19c)
1
Brownish wax, 38% yield. H NMR (CDCl
3
): δ 1.45 (s, 9H), 2.48 (t, 2H, J= 6.4 Hz), 2.52-2.69 (m,
6
5
H), 3.24 (app t, 4H), 3.63 (t, 2H, J= 5.8 Hz), 3.69 (t, 2H, J= 6.3 Hz), 3.80 (s, 3H), 6.88-7.10 (m,
+
H), 7.29-7.33 (m, 3H). ESI/MS m/z 463 (M+Na) , ESI-MS/MS m/z 407 (100).
4.1.3.4. t-Butyl-3-[2-[4-[2-(4-fluorophenyl)phenyl]piperazin-1-yl]ethoxy]propanoate (19d)
1
Orange wax, 43% yield. H NMR (CDCl
3
): δ 1.43 (s, 9Η), 2.41 (br s, 4H), 2.45-2.49 (t, 2H, J= 6.3
Hz), 2.52-2.56 (t, 2H, J= 5.8 Hz), 2.84 (app t, 4H), 3.54 -3.58 (t, 2H, J= 5.8 Hz), 3.63-3.68 (t, 2H,
+
J= 6.3 Hz), 7.02-7.10 (m, 4H), 7.18-7.31 (m, 2H), 7.57-7.62 (m, 2H). ESI/MS m/z 451 (M+Na) ,
ESI-MS/MS m/z 395 (100).
4.1.4. General Procedure for the Preparation of Compounds 20a-d.
To a solution of ester 19a-d (1.14 mmol) in dioxane (5 mL) was added 3N HCl (5 mL). The
reaction mixture was stirred at room temperature for 48 h, then dioxane was evaporated in vacuo.
The acidic solution was neutralized with aqueous NaOH (5%, w/v) and extracted with AcOEt (3 ×
2 4
20 mL). The combined organic layers were washed with brine, dried over Na SO and evaporated
under reduced pressure to yield the desired compound that was used without further purification.
4.1.4.1. 3-[2-[4-[2-(4-Methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanoic acid (20a)
1
Quantitative yield. Pale brown semisolid. H NMR (CDCl
3
): δ 2.48 (t, 2H, J= 5.2 Hz), 2.63 (br s,
O exchanged), 3.62 (t, 2H, J= 5.2
Hz), 3.69 (t, 2H, J= 5.8 Hz), 3.85 (s, 3H), 6.92 (d, 2H, J= 8.8 Hz), 7.04 (q, 2H, J= 7.6 Hz), 7.21 (t,
4
H), 2.70 (t, 2H, J= 5.2 Hz), 2.94 (app t, 4H), 3.06 (br s, 1H, D
2
1
8

+
H, J= 7.4 Hz), 7.52 (d, 2H, J= 8.8 Hz). ESI/MS m/z 385 (M+H) , ESI-MS/MS m/z 295 (100), 266
2
(
31), 238 (32).
4
4
4
.1.4.2. 3-[2-[4-[2-(2-Methylphenyl)phenyl]piperazin-1-yl]ethoxy]propanoic acid (20b)
1
4% Yield. Pale brown semisolid. H NMR (CDCl
3
): δ 2.17 (s, 3H), 2.30 (br s, 4H), 2.43-2.50 (m,
H), 2.74-2.79 (m, 2H), 2.86-2.92 (m, 2H), 3.06 (br s, 1H, D
2
O exchanged), 3.52 (t, 2H, J= 5.8
+
Hz), 3.64 (t, 2H, J= 6.6 Hz), 7.02-7.19 (m, 5H), 7.28-7.34 (m, 3H). ESI/MS m/z 369 (M+H) , ESI-
MS/MS m/z 279 (54), 250 (100), 222 (33).
4.1.4.3. 3-[2-[4-[3-(2-Methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanoic acid (20c)
1
0% Yield. Pale brown semisolid. H NMR (CDCl
6
3
): δ 2.52 (app t, 2H), 2.71-2.83 (m, 6H), 3.06
(
2
br s, 1H, D O exchanged), 3.28 (br s, 4H), 3.64-3.73 (m, 4H), 3.79 (s, 3H), 6.85-6.98 (m, 1H),
+
.01-7.08 (m, 4H), 7.27-7.33 (m, 3H). ESI/MS m/z 385 (M+H) , ESI-MS/MS m/z 295 (100), 266
7
(
31), 238 (32).
4.1.4.4. 3-[2-[4-[2-(4-Fluorophenyl)phenyl]piperazin-1-yl]ethoxy]propanoic acid (20d)
1
0% Yield. Pale brown semisolid. H NMR (CDCl
9
3
): δ 2.5 (t, 2H, J= 5.9 Hz), 2.64 (br s, 4H), 2.71
O exchanged), 3.63 (t, 2H, J= 5.4 Hz), 3.70
(
(
t, 2H, J= 5.2 Hz), 2.94 (app t, 4H), 3.06 (br s, 1H, D
2
t, 2H, J= 5.9 Hz), 7.02-7.10 (m, 4H), 7.17-7.20 (m, 1H), 7.25-7.31 (m, 1H), 7.52-7.58 (m, 2H).
-
ESI/MS m/z 371 (M-H) , ESI-MS/MS m/z 299 (73), 281 (10), 186 (100).
4
.1.5. General Procedure for the Preparation of Final Compounds 21a-23a, 35e, 36f, 37e, 38f.
A stirred mixture of alkyl bromide 12-16 (0.7 mmol), the appropriate arylpiperazine (0.6 mmol) and
CO (0.7 mmol) in acetonitrile (20 mL) was refluxed overnight. After cooling, the mixture was
evaporated to dryness and H O (20 mL) was added to the residue. The aqueous phase was extracted
with AcOEt (2 × 30 mL). The collected organic layers were dried over Na SO and evaporated
K
2
3
2
2
4
1
9

under reduced pressure. The crude residue was purified by chromatographic column
CHCl /MeOH, 19:1, as solvent) to afford pure target compound.
(
3
4.1.5.1. 4-[2-(4-Methoxyphenyl)phenyl]-N-(pyridin-2-ylmethyl)piperazinehexanamide (21a)
1
Pale yellow oil, 93% yield. H NMR (CDCl
3
): δ 1.33 (quintet, 2H, J= 7.4 Hz), 1.48 (quintet, 2H, J=
.5 Hz), 1.63-1.71 (m, 2H), 2.28 (app q, 4H), 2.35 (br s, 4H), 2.85 (app t, 4H), 3.85 (s, 3H), 4.55 (d,
H, J= 4.7 Hz ), 6.73 (br t, 1H, D O exchanged), 6.93 (d, 2H, J= 8.8 Hz), 7.03 (q, 2H, J= 6.7 Hz),
7
2
7
2
.17-7.24 (m, 4H), 7.20 (d, 2H, J= 8.5 Hz), 7.66 (dt, 1H, J= 1.9, 7.7 Hz), 8.52 (d, 1H, J= 4.8 Hz).
+
ESI/MS m/z 473 (M+H) . ESI-MS/MS m/z 473 (11), 365 (92), 266 (32), 205 (100). Anal.Calcd for
29 36 4 2 4 6 6
C H N O •C H O ; C, 63.65; H, 6.80; N, 9.00. Found: C, 63.55; H, 6.78; N, 8.87.
4.1.5.2. 4-[2-(4-Methoxyphenyl)phenyl]-N-(pyridin-3-ylmethyl)piperazinehexanamide (22a).
1
Brown oil, 46% yield. H NMR (CDCl
3
): δ 1.32 (quintet, 2H, J= 7.6 Hz), 1.48 (quintet, 2H, J= 7.4),
1
.64-1.72 (m, 2H), 2.22 (t, 2H, J= 7.6 Hz), 2.30 (t, 2H, J= 7.7 Hz), 2.36 (br s, 4H), 2.85 (br s, 4H),
.85 (s, 3H), 4.45 (d, 2H J= 6.1 Hz), 5.84 (br t, 1H, D O exchanged), 6.93 (d, 2H, J= 8.8 Hz), 7.03
3
2
(
(
q, 2H, J= 7.4 Hz) 7.18-7.24 (m, 4H), 7.57 (d, 2H, J= 8.8 Hz), 8.53 (br s, 2 H). GC/MS m/z: 472
+
M ), 281 (21), 234 (100). Anal.Calcd for C29
H
36
N
4
O
2
•C
4
H
6
O
6
; C, 63.65; H, 6.80; N, 9.00. Found:
C, 63.85; H, 6.92; N, 8.75.
4.1.5.3. 4-[2-(4-Methoxyphenyl)phenyl]-N-(pyridin-4-ylmethyl)piperazinehexanamide (23a)
1
Orange oil 38% yield. H NMR (CDCl
3
): δ 1.34 (quintet, 2H, J= 7.4 Hz), 1.52 (quintet, 2H, J= 7.4
Hz), 1.69 (quintet, 2H, J= 7.5 Hz), 2.26 (t, 2H, J= 7.4 Hz), 2.35 (t, 2H, J= 7.6 Hz), 2.40 (br s, 4H),
2
8
1
2
.87 (br s, 4H), 3.85 (s, 3H), 4.45 (d, 2H, 6.0 Hz), 6.05 (br t, 1H, D O exchanged), 6.93 (d, 2H, J=
.8 Hz), 7.04 (q, 2H, J= 7.4 Hz), 7.16-7.28 (m, 4H), 7.56 (d, 2H, J= 8.8 Hz), 8.54 (dd, 2H, J= 4.7,
+
.7 Hz). ESI/MS m/z 473 (M+H) . ESI-MS/MS m/z 473 (21), 365 (15), 269 (20), 266 (23), 205
2
0

(
100). Anal.Calcd for C29
H
36
N
4
O
2
•C
4
H
6
O
6
; C, 63.65; H, 6.80; N, 9.00. Found: C, 63.54; H, 6.56; N,
+
(M+H) : 473.2911, found: 473.2905.
9.10. HRMS (ESI+) m/z calcd for C29
36
H N
4
O
2
4.1.5.4. N-(2-Fluoropyridin-5-ylmethyl)-4-[2-(4-methoxy)phenyl]-1-piperazinehexanamide
(
27a)
Orange wax, 81% yield. H NMR (CDCl
1
3
): δ 1.31 (quintet, 2H, J= 7.5 Hz), 1.49 (quintet, 2H, J=
.4 Hz ), 1.66 (quintet, 2H, J= 7.6 Hz ), 2.21 (t, 2H, J= 7.6 Hz), 2.30 (t, 2H, J= 7.5 Hz ), 2.36 (br s,
H), 2.86 (app t, 4H), 3.85 (s, 3H), 4.42 (d, 2H, J= 6.1 Hz), 5.91 (br t, 1H, D O exchanged), 6.87-
7
4
6
2
2
.95 (m, 3H), 7.00-7.08 (m, 2H), 7.19-7.27 (m, 2H), 7.56 (d, 2H, J= 8.8 Hz), 7.75 (dt, 1H, J= 8.0,
+
.5 Hz), 8.11-8.12 (m, 1H). ESI/MS m/z 513 (M+Na) . ESI-MS/MS m/z 491 (42), 269 (19), 266
(
4 2 4 6 6
31), 223 (100). Anal.Calcd for C29H35FN O •C H O ; C, 61.86; H, 6.45; N, 8.74. Found: C, 61.54;
H, 6.56; N, 9.10.
4
.1.5.5. N-(2-Fluoropyridin-5-ylmethyl)-4-[2-(4-pyridyl)pyridyl]-1-piperazinehexanamide
35e)
Brown oil 76% yield. H NMR (CDCl
(
1
3
): δ 1.27-1.35 (m, 2H), 1.42-1.52 (m, 2H), 1.60-1.70 (m,
H), 2.20 (t, 2H, J= 7.4 Hz), 2.30 (t, 2H, J= 7.6 Hz), 2.37 (br t, 4H), 3.11 (br t, 4H), 4.41 (d, 2H, J=
.1 Hz), 6.11 (br t, 1H, D O exchanged), 6.86-6.95 (m, 2H), 7.46 (dd, 1H, J= 7.4, 1.9 Hz), 7.53 (dd,
2
6
2
6
2
6
2
H, J= 6.1, 2.8 Hz), 7.75 (dt, 1H, J= 8.00, 2.5 Hz), 8.09 (app d, 1H, J= 1.4 Hz), 8.27 (dd, 1H, J=
-
.6, 1.9 Hz), 8.64 (dd, 2H, J= 6.1, 1.7 Hz). ESI/MS m/z 461 (M-H) . ESI-MS/MS m/z 441 (100),
90 (33), 239 (44), 221 (29), 196 (13), 170 (58). Anal.Calcd for C26
.09; N, 13.72. Found: C, 58.74; H, 6.20; N, 13.54.
6 4 6 6
H31FN O•C H O ; C, 58.51; H,
4.1.5.6. N-(2-Fluoropyridin-5-ylmethyl)-(4-(2-(pyridin-4-yl)pyridin-3-yl)piperazin-1-
yl)hexanamide (36f)
2
1

1
Orange oil, 46% yield. H NMR (CDCl
3
): δ 1.35-1.42 (m, 2H), 1.44-1.58 (m, 2H), 1.61-1.78 (m,
H), 2.19 (q, 2H, J= 7.4 Hz), 2.32 (t, 2H, J= 7.4 Hz), 2.42 (br s, 2H), 2.64 (br s, 2H), 2.89 (br s,
H), 3.11 (br s, 2H), 4.42-4.45 (m, 2H), 5.89 (br t, 1H, D O exchanged), 6.88-6.91 (m, 1H), 7.17-
2
2
7
8
1
5
2
.41 (m, 2H), 7.73-7.78 (m, 1H), 7.90-7.93 (m, 2H), 8.04-8.06 (m, 1H), 8.36-8.38 (m, 1H), 8.66-
-
.68 (m, 2H). ESI/MS m/z 461 (M-H) . ESI-MS/MS m/z 441 (100), 290 (49), 264 (28), 239(29),
96(26), 170 (51). Anal.Calcd for C26
8.34; H, 6.25; N, 13.65.
6 4 6 6
H31FN O•C H O ; C, 58.51; H, 6.09; N, 13.72. Found: C,
4.1.5.7. N-4-Methoxybenzyl-(4-(3-(pyridin-4-yl)pyridin-2-yl)piperazin-1-yl)hexanamide (37e)
1
Orange oil, 66% yield. H NMR (CDCl
3
): δ 1.26-1.38 (m, 2H), 1.43-1.58 (m, 2H), 1.60-1.68 (m,
2
4
7
8
H), 2.18 (t, 2H, J= 7.4 Hz), 2.32 (t, 2H, J= 7.4 Hz), 2.37 (br t, 4H), 3.12 (br t, 4H), 3.79 (s, 3H),
.35 (d, 2H, J= 5.5 Hz), 5.89 (br t, 1H, D O exchanged), 6.85 (dd, 2H, J= 8.5 Hz), 6.93 (dd, 1H, J=
2
.4, 5.0 Hz), 7.20 (d, 2H, J= 8.5 Hz), 7.46 (dd, 1H, J= 7.4, 1.7 Hz), 7.53 (dd, 2H, J= 1.7, 6.1 Hz),
-
.27 (dd, 2H, J= 1.9, 5.0 Hz), 8.65 (dd, 2H, J= 1.7, 4.4 Hz). ESI/MS m/z 472 (M-H) . ESI-MS/MS
m/z 366 (33), 326 (34), 293 (31), 275 (43), 239(72), 196 (31), 184 (34), 170 (100). Anal.Calcd for
•C ; C, 61.62; H, 6.63; N, 11.23. Found: C, 61.40; H, 6.45; N, 11.55.
C
28
H
35
N
5
O
2
4 6 6
H O
4.1.5.8. N-4-Methoxybenzyl-(4-(2-(pyridin-4-yl)pyridin-3-yl)piperazin-1-yl)hexanamide (38f)
1
Yellow oil, 52% yield. H-NMR (CDCl
3
): δ 1.30-1.39 (m, 2H), 1.44-1.57 (m, 2H),1.64-1.69 (m,
2
H), 2.16-2.23 (q, 2H, J= 7.3 Hz), 2.41 (t, 2H, J= 7.4 Hz), 2.43 (br t, 2H), 2.63 (br s, 2H), 2.88
app t, 2H), 3.09 (br s, 2H), 3.79 (s, 3H), 4.35-4.38 (m, 2H), 5.67 (br t, 1H, D O exchanged), 6.83-
.87 (m, 2H), 7.16-7.28 (m, 4H), 7.90-7.92 (m, 2H), 8.36 (dd, 1H, J= 1.4, 4.7 Hz), 8.67 (dd, 2H, J=
(
2
6
1
-
.7, 4.7 Hz). ESI/MS m/z 472 (M-H) . ESI-MS/MS m/z 301(18), 275 (28), 239 (29), 196 (52), 170
(
100). Anal.Calcd for C28
H
35
N
5
O
2
4 6 6
•C H O ; C, 61.62; H, 6.63; N, 11.23. Found: C, 61.30; H, 6.65;
N, 11.52.
2
2

4
.1.6. General Procedure for the Preparation of Final Compounds 24a-26a, 28a, 29b, 30c, 31d,
2d, 33c, 34d.
3
A mixture of the acid 20a-d (0.32 mmol) and 1,1’-carbonyldiimidazole (0.06 g; 0.38 mmol) in
anhydrous THF (10 mL) was stirred overnight. Then, a solution of the amine 7-11 (0.32 mmol) in
anhydrous THF (10 mL) was added dropwise and the mixture was stirred until the acid disappeared
(
TLC). The solvent was evaporated in vacuum and the residue was partitioned between AcOEt (20
mL) and H O (20 mL). The separated organic layer was washed with H O (20 mL), dried (Na SO
and concentrated in vacuum. The crude residue was purified by chromatographic column by
CHCl /MeOH, 19:1, as solvent) to give pure target compound.
2
2
2
4
)
(
3
4.1.6.1. N-Pyridin-2-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-
yl]ethoxy]propanamide (24a)
1
Pale yellow wax, 55% yield. H NMR (CDCl
3
): δ 2.39 (br s,4H), 2-55 (dt, 4H, J= 7.0, 2.5 Hz), 2.79
(
app t, 4H), 3.59 (t, 2H, J= 5.8 Hz), 3.74 (t, 2H, J= 5.8 Hz), 3.84 (s, 3H), 4.57 (d, 2H, J= 5.2 Hz),
2
6.90-6.96 (m, 3H), 7.04 (dt, 1H, J= 8.0, 1.1 Hz), 7.15-7.27 (m, 4H), 7.50 (br t, 1H sc D O), 7.53-
+
.57 (m, 2H), 7.63 (dt, 1H, J= 1.9, 8.7 Hz), 8.51 (app dd, 1H). ESI/MS m/z 475 (M+H) , ESI-
7
34 4 3 4 6 6
MS/MS m/z 367 (10), 313 (100), 295 (48), 266 (31), 224 (12). Anal.Calcd for C28H N O •C H O ;
C, 61.53; H, 6.45; N, 8.97. Found: C, 61.40; H, 6.75; N, 9.05.
4.1.6.2. N-Pyridin-3-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-
yl]ethoxy]propanamide (25a)
1
Pale yellow oil, 60% yield. H NMR (CDCl
3
): δ 2.36 (br s, 4H), 2.47-2.54 (m, 4H), 2.76 (br t, 4H),
3
3
2
.56 (t, 2H, J= 5.5 Hz), 3.71 (t, 2H, J= 5.5 Hz), 3.84 (s, 3H), 4.45 (d, 2H, J= 6.1 Hz), 6.90-6.93 (m,
2
H), 7.03-7.08 (m, 1H), 7.19-7.29 (m, 4H), 7.52-7.56 (m, 2H), 7.60 (br t, 1H sc D O),8.48-8.51 (m,
+
H). ESI/MS m/z 475 (M+H) , ESI-MS/MS m/z 314 (12), 313 (100), 295 (34), 266 (31), 224 (13).
34 4 3 4 6 6
Anal.Calcd for C28H N O •C H O ; C, 61.53; H, 6.45; N, 8.97. Found: C, 61.37; H, 6.35; N, 9.16.
2
3

4.1.6.3. N-Pyridin-4-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-
yl]ethoxy]propanamide (26a)
1
Colourless oil, 39% yield. H NMR (CDCl
3
): δ 2.36 (br s,4H), 2.50-2.56 (app q, 4H), 2.73 (br t,
4
6
H), 3.59 (t, 2H, J= 5.5 Hz), 3.73 (t, 2H, J= 5.5 Hz), 3.84 (s, 3H), 4.46 (d, 2H, J= 6.3 Hz), 6.85-
.93 (m, 3H), 7.02-7.08 (m, 1H), 7.16-7.25 (m, 4H), 7.43 (br t, 1H, D
2
O exchanged), 7.54 (dd, 2H,
+
J= 1.9, 8.8 Hz), 8.52 (dd, 2H, J= 1.5, 6.1 Hz). ESI/MS m/z 497 (M+Na) , ESI-MS/MS m/z 497
(
100), 336 (9), 335 (94). Anal.Calcd for C28
H
34
N
4
O
3
•C
1.67; H, 6.35; N, 8.85. HRMS (ESI+) m/z calcd for C28
74.2706.
4
H
6
O
6
; C, 61.53; H, 6.45; N, 8.97. Found: C,
+
(M+H) : 474.2704, found:
6
4
H
34
N
4
O
3
4.1.6.4. N-(2-Fluoropyridin-5-ylmethyl)-3-[2-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-
yl]ethoxy]propanamide (29a)
1
Amber oil, 95% yield. H NMR (CDCl
3
): δ 2.37 (br s, 4H), 2.50 (app t, 4H), 2.78 (br t, 4H), 3.56 (t,
2
8
8
2
5
H, J= 5.5 Hz), 3.70 (t, 2H, J= 5.5 Hz), 3.84 (s, 3H), 4.43 (d, 2H, J= 6.3 Hz), 6.86 (dd, 1H, J= 2.9,
.4 Hz), 6.94 (d, 2H, J= 8.8 Hz), 7.06 (dt, 1H, J= 1.1, 7.7 Hz), 7.19-7.25 (m, 4H), 7.55 (d, 2H, J=
.8 Hz), 7.72 (dt, 1H, J= 2.5, 8.0 Hz), 8.10 (br s, 1H). ESI/MS m/z 491 (M-H). ESI-MS/MS m/z
98 (18), 297 (100). Anal.Calcd for C28
9.97; H, 6.25; N, 8.85.
4 3 4 6 6
H33FN O •C H O ; C, 59.80; H, 6.12; N, 8.72. Found: C,
4.1.6.5. N-(2-Fluoropyridin-5-ylmethyl)-3-[2-[4-[2-(2-methylphenyl)phenyl]piperazin-1-
yl]ethoxy]propanamide (29b)
1
Pale yellow oil, 87% yield. H NMR (CDCl
3
): δ 2.16 (s, 3H), 2.24 (br s, 4H), 2.42-2.45 (m, 2H),
2
.48 (t, 2H, J= 5.5 Hz), 2.67-2.71 (m, 2H), 2.80-2.85 (m, 2H), 3.52 (t, 2H, J= 5.5 Hz), 3.68 (t, 2H,
J= 5.5 Hz), 4.43 (d, 2H, J= 6.1 Hz), 6.86 (dd, 1H, J= 3.0, 8.5 Hz), 6.94 (d, 1H, J= 8.3 Hz), 7.01-
.13 (m, 3H), 7.21-7.33 (m, 4H), 7.72 (dt, 1H, J= 2.5, 8.0 Hz), 8.10 (br s, 1H). ESI/MS m/z 475
7
2
4

-
M-H) . ESI-MS/MS m/z 197 (76), 182 (38), 179 (100), 167 (85). Anal.Calcd for
(
C
28
H33FN
4
O
2
4 6 6
•C H O ; C, 61.33; H, 6.27; N, 8.94. Found: C, 61.55; H, 6.35; N, 8.95.
4.1.6.6. N-(2-Fluoropyridin-5-ylmethyl)-3-[2-[4-[3-(2-methoxyphenyl)phenyl]piperazin-1-
yl]ethoxy]propanamide (30c)
1
Pale yellow oil, 38% yield. H NMR (CDCl
3
): δ 2.53 (t, 2H, J= 5.6 Hz), 2.62 (app q, 4H), 2.69 (t,
2
H, J= 5.1 Hz), 3.17 (app t, 4H), 3.64 (t, 2H, J= 5.5 Hz), 3.74 (t, 2H, J= 5.6 Hz), 3.80 (s, 3H), 4.45
(
d, 2H, J= 6.1 Hz), 6.84 (dt, 1H, J= 8.3, 2.7 Hz), 6.96-7.04 (m, 4H), 7.27-7.30 (m, 5H), 7.71 (dt,
+
H, J= 8.3, 2.5 Hz), 8.12 (s, 1H). ESI/MS m/z 515 (M+Na) . ESI-MS/MS m/z 516 (48), 515 (100),
1
3
6
4 3 4 6 6
35 (83). Anal.Calcd for C28H33FN O •C H O ; C, 59.80; H, 6.12; N, 8.72. Found: C, 59.68; H,
.25; N, 8.95.
4.1.6.7. 3-[2-[4-[2-(4-Fluorophenyl)phenyl]piperazin-1-yl]ethoxy]-N-(2-fluoropyridin-5-
1
ylmethyl)-propanamide (31d). Pale yellow oil, 43% yield. H NMR (CDCl
3
): δ 2.38 (br s, 4H),
2
6
1
.48-2.54 (m, 4H), 2.78 (br t, 4H), 3.57 (t, 2H, J= 5.5 Hz), 3.70 (t, 2H, J= 5.5 Hz), 4.43 (d, 2H, J=
.1 Hz), 6.86 (dd, 1H, J= 2.7, 8.3 Hz), 6.95 (d, 1H, J= 7.4 Hz), 7.04- 7.10 (m, 3H), 7.28 (dd, 1H, J=
.7, 7.4 Hz), 7.28- 7.31 (m, 2H), 7.54- 7.59 (m, 2H), 7.73 (dt, 1H, J= 2.5, 8 Hz), 8.10 (s, 1H).
+
ESI/MS m/z 503 (M+Na) . ESI-MS/MS m/z 503 (100), 323 (85). Anal.Calcd for
27 30 2 4 2 4 6 6
C H F N O •C H O ; C, 59.04; H, 5.75; N, 8.88. Found: C, 59.18; H, 5.98; N, 8.85.
4.1.6.8. N-(4-Methoxybenzyl)-3-[2-[4-[2-(2-methylphenyl)phenyl]piperazin-1-
yl]ethoxy]propanamide (32b)
1
Brown oil, 96% yield. H NMR (CDCl
3
): δ 2.15 (s, 3H), 2.22 (br s, 4H), 2.45 (br s, 2H), 2.48 (t, 2H,
J= 5.6 Hz), 2.70 (br s, 2H), 2.80 (br s, 2H), 2.53 (br t, 2H), 3.69 (t, 2H, J= 5.6 Hz), 3.76 (s, 3H),
.37 (d, 2H, J= 5.8 Hz), 6.81-6.84 (m, 2H), 7.06-7.12 (m, 2H), 7.18-7.30 (m, 9H). ESI/MS m/z 486
4
2
5

-
M-H) . ESI-MS/MS m/z 208 (33), 190 (21), 182 (22), 178 (100). ). Anal.Calcd for
(
C
30
H
37
N
3
O
3
4 6 6
•C H O ; C, 64.03; H, 6.80; N, 6.59. Found: C, 64.32; H, 6.98; N, 6.85.
4.1.6.9. N-(4-Methoxybenzyl)-3-[2-[4-[3-(2-methoxyphenyl)phenyl]piperazin-
1
yl]ethoxy]propanamide (33c)
1
Pale orange oil, 28% yield. H NMR (CDCl
3
): δ 2.51 (t, 2H, J= 5.5 Hz), 2.57-2.61 (m, 6H), 3.15 (br
t, 4H), 3.63 (t, 2H, J= 5.5 Hz), 3.73 (s, 3H), 3.74 (t, 2H, J= 5.5 Hz), 3.80 (s, 3H), 4.39 (d, 2H, J= 5.8
Hz), 6.80-6.87 (m, 4H), 6.96-7.04 (m, 4H), 7.18 (d, 2H, J= 8.5 Hz), 7.29-7.34 (m, 3H). ESI/MS m/z
-
02 (M-H) . ESI-MS/MS m/z 297 (100). Anal.Calcd for C30
5
H
37
N
3
O
4
•C
4
H
6
O
6
; C, 62.47; H, 6.63; N,
6.43. Found: C, 62.32; H, 6.88; N, 6.45.
4.1.6.10. N-(4-Methoxybenzyl)-3-[2-[4-[2-(4-fluorophenyl)phenyl]piperazin-1-
yl]ethoxy]propanamide (34d)
1
Pale orange oil, 31% yield. H NMR (CDCl
3
): δ 2.34 (br s, 4H), 2.48 (app t, 4H), 2.75 (br t, 4H),
3
3
4
.55 (t, 2H, J= 5.5 Hz), 3.70 (t, 2H, J= 5.5 Hz), 3.76 (s, 3H), 4.37 (d, 2H, J= 5.8 Hz), 6.81-6.84 (m,
H), 6.95 (d, 1H, J= 7.4 Hz), 7.04-7.10 (m, 3H), 7.16-7.21 (m, 4H), 7.55-7.60 (m, 2H). ESI/MS m/z
-
90 (M-H) . ESI-MS/MS m/z 208 (34), 190 (27), 186 (42), 178 (100). Anal.Calcd for
29 3 3 4 6 6
C H34FN O •C H O ; C, 61.77; H, 6.28; N, 6.55. Found: C, 61.62; H, 6.38; N, 6.87.
4
.1.7. 4-[2-(4-Hydroxyphenyl)phenyl]-N-(pyridin-4-ylmethyl)piperazinehexanamide (39)
Boron tribromide (1M in CH Cl , 2.4 mL, 2.4 mmol) was added dropwise to an ice cooled solution
of 23a (0.35 g, 0.75 mmol) in anhydrous CH Cl (10 mL). The mixture was stirred overnight at
room temperature. Then, the reaction mixture was poured on ice, neutralized with conc. NH OH
and extracted with AcOEt (3 × 20 mL). The combined organic layers was washed with brine, dried
2
2
2
2
4
over Na
2
SO , and concentrated under reduced pressure. The crude residue was chromatographed
4
1
/MeOH, 9:1, as eluent) to give compound 39 as white powder (0.11 g, 43% yield). H NMR
(
CHCl
3
2
6

(
CDCl
3
): δ 1.34 (quintet, 2H, J= 7.5 Hz), 1.52 (quintet, 2H, J= 7.5 Hz), 1.68 (quintet, 2H, J= 7.5
O exchanged), 2.27 (t, 2H, J= 7.3 Hz), 2.35 (t, 2H, J= 7.7 Hz), 2.41 (br s, 4H),
.86 (br t, 4H), 4.45 (d, 2H, J= 6.2 Hz), 6.20 (br t, 1H, D O exchanged), 6.88 (d, 2H, J= 8.4 Hz),
Hz), 2.17 (s, 1H, D
2
2
6
4
2
.97-7.07 (m, 2H), 7.19-7.23 (m, 4H), 7.48 (d, 2H, J= 8.4 Hz), 8.54 (d, 2H, J= 6.2 Hz). ESI/MS m/z
-
57 (M-H) . ESI-MS/MS m/z 367 (72), 350 (18), 349 (100). HRMS (ESI+) m/z calcd for
+
(M+H) : 459.2755, found: 459.2757.
C
28
H
34
N
4
O
2
4
.1.8. 4-[2-(4-Hydroxyphenyl)phenyl]-1-t-butoxycarbonylpiperazine (41)
To a degassed mixture of 4-(2-bromophenyl)-1-t-butoxycarbonylpiperazine (40) (1.20 g, 3.6
mmol), 4-hydroxyboronic acid (0.75 g, 5.4 mmol) and 2M aqueous NaHCO (5.4 mL) in dioxane
10 mL) was added tetrakis(triphenylphosphino)palladium(0) (13 mg, 0.11 mmol) under argon
atmosphere. The mixture was refluxed for 12 h. After cooling, the solvent was distilled off in vacuo
and the residue was taken up with Et O (30 mL) and washed first with H O and then with brine.
The organic layer was separated, dried over anhydrous Na SO and concentrated. The crude residue
3
(
2
2
2
4
was chromatographed on a silica gel column (petroleum ether/AcOEt, 9:1) to give pure 41 as pale
1
yellow oil (0.86 g, 67% yield). H NMR (CDCl
3
): δ 1.45 (s, 9H), 2.78 (br t, 4H), 3.33 (br t, 4H),
6.86 (d, 2H, J= 7.7 Hz), 6.98 (d, 2H, J= 8.0 Hz), 7.05-7.10 (m, 1H), 7.21-7.24 (m, 2H), 7.50 (d, 2H,
-
J= 7.7 Hz). ESI/MS m/z 353 (M-H) . ESI-MS/MS m/z 353 (12), 297 (21), 253 (100).
4
.1.9. 4-[2-(4-Benzyloxyphenyl)phenyl]-1-t-butoxycarbonylpiperazine (42)
A mixture of phenolic derivative 41 (0.23 g, 0.65 mmol), benzyl bromide (0.12 g, 0.72 mmol) and
CO (0.10 g, 0.72 mmol) in acetone (30 mL) was refluxed overnight. After cooling, the solvent
was evaporated under reduced pressure and the residue taken up with H O (20 mL) and extracted
with AcOEt (3 × 20 mL). The collected organic layers were washed with H O (20 mL), dried
Na SO ) and evaporated under reduced pressure to give a crude residue. After purification through
a silica gel column (petroleum ether/AcOEt, 9:1, as eluent) pure 42 was obtained (0.15 g, 52%
K
2
3
2
2
(
2
4
2
7

1
yield) as a colourless wax. H NMR (CDCl
3
): δ 1.44 (s, 9H), 2.78 (br s, 4H), 3.32 (br s, 4H), 5.11
(
(
s, 2H), 7.00 (dd, 2H, J= 2.0, 6.7 Hz), 7.08 (dd, 2H, J= 1.1, 7.4 Hz), 7.22-7.29 (m, 2H), 7.38-7.48
+
m, 5H), 7.57 (dd, 2H, J= 2.0, 6.7 Hz). ESI/MS m/z 467 (M+Na) . ESI-MS/MS m/z 367 (100).
4
.1.10. 1-[2-(4-Benzyloxyphenyl)phenyl]piperazine (43)
Trifluoroacetic acid (5 mL) was added to a solution of 42 (0.15 g, 0.34 mmol) in CH
The reaction mixture was stirred at room temperature for 5 h, basified with aqueous NaOH (40%,
w/v), and extracted with CH Cl (3 × 20 mL). The combined organic layers were dried (Na SO
and concentrated in vacuo to give pure 43 as brown wax (0.12 g, quantitative yield). H NMR
2
2
Cl (20 mL).
2
2
2
4
)
1
(
(
CDCl
3
): δ 2.85 (br s, 9H), 5.11 (s, 2H), 7.01 (d, 2H, J= 8.5 Hz), 7.08 (d, 2H, J= 7.4 Hz), 7.22-7.29
+
m, 2H), 7.33-7.48 (m, 5H), 7.60 (d, 2H, J= 8.8 Hz). GC/MS m/z 345 (M +1, 21), 344 (M , 83),
+
303 (23), 302 (100), 211 (18), 210 (40), 196 (15), 182 (15), 167 (14).
4.1.11. 2-[4-[2-(4-Benzyloxyphenyl)phenyl]piperazin-1-yl]ethanol (44)
A mixture of 43 (0.61 g, 1.8 mmol), 2-bromoethanol (0.15 mL, 2.1 mmol), K
mmol) and KI (0.08 g, 0.5 mmol) in CH CN (20 mL) was refluxed overnight under stirring. After
cooling, the solvent was removed under reduced pressure and the residue was partitioned between
AcOEt (20 mL) and H O (20 mL). The organic layer was separated and the aqueous phase was
extracted twice with AcOEt (20 mL). The combined organic layers were dried over Na SO and
2 3
CO (0.29 g, 2.1
3
2
2
4
evaporated under reduced pressure. The crude residue was purified by column chromatography
1
/MeOH, 19:1, as solvent) to afford pure 44 as an orange oil (0.49 g, 71% yield). H NMR
(
(
CHCl
3
CDCl
3
): δ 2.42 (br s, 1H, D
2
O exchanged), 2.43 (br s, 4H), 2.52 (t, 2H, J= 5.5 Hz), 2.86 (br t, 4H),
3
2
.59 (t, 2H, J= 5.5 Hz), 5.11 (s, 2H), 7.00 (d, 2H, J= 8.8 Hz), 7.07 (d, 2H, J= 6.3 Hz), 7.21-7.27 (m,
+
H), 7.34-7.48 (m, 5H), 7.57 (d, 2H, J= 8.8 Hz). GC-MS m/z 389 (M +1, 8), 388 (M , 31), 358
+
(
29), 357 (100), 342 (12).
2
8

4.1.12. t-Butyl-3-[2-[4-[2-(4-benzyloxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanoate (45)
To a cooled suspension of NaH (0.03 g, 1.2 mmol) in anhydrous THF (5 mL) was added, dropwise
and under nitrogen, a solution of alcohol 44 (0.48 g, 1.2 mmol) in the same solvent (10 mL). The
reaction mixture was stirred at room temperature for 8 hours. Then, a solution of t-butyl acrylate
(
0.16 mL, 1.11 mmol) in anhydrous THF (10 mL) was added dropwise and the reaction mixture
was stirred at room temperature for 20-24 hours. Then, the solvent was removed under reduced
pressure and the residue was cautiously taken up with brine and extracted with AcOEt (2 × 30 mL).
The combined organic layers were washed with H
2
O, dried over Na
/AcOEt, 1:1, as eluent) to afford
): δ 1.43 (s, 9H), 2.41 (br s, 4H),
.48 (t, 2H, J= 6.5 Hz), 2.54 (t, 2H, J= 5.8 Hz), 2.86 (br t, 4H), 3.56 (t, 2H, J= 5.8 Hz), 3.66 (t, 2H,
2 4
SO and evaporated under
reduced pressure. The crude residue was chromatographed (CHCl
3
1
pure 45 as a pale yellow wax (0.19 g, 29% yield). H NMR (CDCl
3
2
J= 6.5 Hz), 5.11 (s, 2H), 6.99-7.07 (m, 4H), 7.20-7.28 (m, 2H), 7.29-7.49 (m, 5H), 7.57 (dd, 2H, J=
+
.9, 6.9 Hz). ESI/MS m/z 539 (M+Na) . ESI-MS/MS m/z 461 (100).
1
4.1.13. t-Butyl-3-[2-[4-[2-(4-hydroxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanoate (46)
A methanolic solution of 45 (0.20 g, 0.38 mmol) was hydrogenated at normal pressure and room
temperature in the presence of 10% palladium on charcoal (0.2 g), until the uptake ceased. The
catalyst was removed by fitration through Celite and the solvent was evaporated in vacuo. The
crude residue was chromatographed on a silica gel column (CHCl
3
/AcOEt, 1:1, as eluent) to afford
1
the desired compound 16 as a pale yellow wax (0.07 g, 45% yield)., 45% yield. H NMR (CDCl
3
):
δ 1.43 (s, 9H), 2.45-2.50 (m, 6H), 2.59 (t, 2H, J= 5.7 Hz), 2.86 (br t, 4H), 3.58 (t, 2H, J= 5.7 Hz),
3.66 (t, 2H, J= 6.5 Hz), 6.85 (d, 2H, J= 8.8 Hz), 6.99-7.04 (m, 2H), 7.19-7.24 (m, 3H), 7.50 (d, 2H,
+
J= 8.8 Hz). ESI/MS m/z 449 (M+Na) . ESI-MS/MS m/z 371 (100).
4.1.14. 3-[2-[4-[2-(4-Hydroxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanoic acid (47)
2
9