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European Journal of Medicinal Chemistry 87 (2014) 248e266

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European Journal of Medicinal Chemistry


journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Structureeaffinity/activity relationships of 1,4-dioxa-spiro[4.5]decane


based ligands at a<alpha>1 and 5-HT1A receptors
Silvia Franchini a, Umberto M. Battisti a, Annamaria Baraldi a, Adolfo Prandi a,
Paola Fossa b, Elena Cichero b, Annalisa Tait a, Claudia Sorbi a, Gabriella Marucci c,
Antonio Cilia d, Lorenza Pirona d, Livio Brasili a, *
a  degli Studi di Modena e Reggio Emilia, Via Campi 183, 41125 Modena, Italy
Dipartimento di Scienze della Vita, Universita
b
Dipartimento di Scienze Farmaceutiche, Universita  degli Studi di Genova, Viale Benedetto XV 3, 16132 Genova, Italy
c
Dipartimento di Scienze Chimiche, Universita  degli Studi di Camerino, Via S. Agostino 1, 62032 Camerino, Italy
d
Divisione Ricerca e Sviluppo, Recordati S.p.A., Via Civitali 1, 20148 Milano, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Recently, 1-(1,4-dioxaspiro[4,5]dec-2-ylmethyl)-4-(2-methoxyphenyl)piperazine (1) was reported as a


Received 22 May 2014 highly selective and potent 5-HT1AR ligand. In the present work we adopted an in-parallel synthetic
Received in revised form strategy to rapidly explore a new set of arylpiperazine (7e32) that is structurally related to 1. The
12 September 2014
compounds were tested for binding affinity and functional activity at 5-HT1AR and a<alpha>1-
Accepted 22 September 2014
Available online
adrenoceptor subtypes and SAR studies were drawn. In particular, compounds 9, 27 and 30 emerged
as promising a<alpha>1 receptor antagonists, while compound 10 behaves as the most potent and
efficacious 5-HT1AR agonist. All the compounds were docked into the 5HT1AR theoretical model and the
Keywords:
a<alpha>1 receptor results were in agreement with the biological experimental data. These findings may represent a new
5-HT1AR starting point for developing more selective a<alpha>1 or 5-HT1AR ligands.
1,3-Dioxolane © 2014 Elsevier Masson SAS. All rights reserved.
Arylpiperazine derivatives
Structureeaffinity/activity relationships

1. Introduction tumor growth and progression in human prostate cancer [4,5]. Like
the a<alpha>1-ARs, serotoninergic receptors belong to the G-pro-
a<Alpha>1-adrenoreceptors (a<alpha>1-ARs) have been stud- tein-coupled receptor (GPCRs) superfamily. Among them the 5-
ied over the years as an attractive target for a variety of therapeutic HT1AR subtype has been the most studied as an attractive target
applications. a<alpha>1-ARs can be divided into at least three for the development of novel therapeutic approaches [6]. The 5-
subtypes, namely, a<alpha>1A (a<alpha>1a), a<alpha>1B HT1AR subtype is involved in a wide range of physiological and
(a<alpha>1b) and a<alpha>1D (a<alpha>1d) [1]. Great efforts were physiopathological processes including anxiety [7], depression [8],
dedicated to better define their physiological role(s) and to discover schizophrenia [9], pain perception [10], neuroprotection [11],
new drug candidates. a<Alpha>1B-AR subtypes play an important cognition [12], food intake, sexual function [13], urogenital [14] and
role in cardiac development and/or function as well as in blood cardiovascular response [15]. Their involvement in malignant
pressure response to a<alpha>1-agonists via vasoconstriction [2]. carcinoid syndrome and prostate cancer has recently been
Initially introduced for hypertension management, a<alpha>1 an- confirmed [16]. The main limitation of many 5-HT1AR ligands is
tagonists have become increasingly common for symptomatic their undesired high affinity for other receptor subtypes such as
treatment of benign prostatic hyperplasia (BPH). In particular, a<alpha>1-ARs due to their high degree of homology (approxi-
several uroselective antagonists targeting a<alpha>1A and mately 45%) [17]. Recently, our research group has demonstrated
a<alpha>1D subtypes, predominantly located in the prostate and in that a properly substituted 1,3-dioxolane moiety is a suitable
the human bladder detrusor, have been disclosed [3]. More scaffold for developing selective ligand targeting a<alpha>1-ARs or
recently, a<alpha>1 antagonists have proven to inhibit primary 5-HT1AR [18e20]. In particular, we have identified compound 1,
bearing a 1,4-dioxa-spiro[4.5]decane moiety, endowed with high
affinity and good selectivity for 5-HT1AR over a<alpha>1-ARs
(Fig. 1). Preliminary molecular docking of compound 1, on the
* Corresponding author. recently published theoretical model of the human 5HT1AR,
E-mail addresses: livio.brasili@unimore.it, brasili.livio@unimore.it (L. Brasili).

http://dx.doi.org/10.1016/j.ejmech.2014.09.070
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266 249

Fig. 1. Rationale of the work.

suggested that this compound (the R enantiomer proved to be the molecules. The availability of a large number of carbonyl or sulfonyl
most probable) displayed one salt bridge interaction between the chlorides, which can be either commercially available or easily
piperazine protonated nitrogen atom and D116 side-chain and also prepared in a few steps from commercial starting materials, makes
Van der Waals contacts and pep stacking between the 2-methoxy them very attractive as alkylating agents. As a result, we developed
phenyl ring and C120, T121; and with F361, F362, respectively. a parallel synthetic approach using a Buchi Syncore® Reactor. This
Moreover, compound 1 properly oriented the cyclohexyl portion semi-automatic synthesizer is able to provide up to 24 reactions in
towards a hydrophobic pocket (including A93, Q97, W387, Y390), parallel and to maximize multiple sample work-up efficiency,
thus detecting Van der Waals contacts (Fig. 2). This model showed a saving a huge amount of time. Thus, compound 12 reacted with
useful available space around the spirocyclohexyl ring which could suitable carbonyl and sulfonyl chlorides, using polymer-assisted in-
be modified without affecting the driving interactions of the 2- parallel liquid phase chemistry, to obtain the corresponding amides
methoxyphenylpiperazine moiety. In particular, the C8 position of and sulfonamides. Poly(4-vinylpyridine) or (piperidino methyl)
the 1,4-dioxa-spiro[4.5]decane moiety was initially selected due to polystyrene was employed to scavenge the excess of carbonyl or
its feasible chemistry and because it could be exploited without sulfonyl chlorides, respectively. A benzensulfonic polystyrene resin
adding a second stereogenic centre that would require expensive was added in the last step to scavenge the final product that was
and time-wasting separation of the diastereomers. Thus, a large isolated highly pure without any further purification step. All the
variety of substituents were introduced at position 8: H-bond desired compounds (15e29) were successfully obtained as depic-
donor/acceptor groups that could establish H-bond contacts, an ted in Scheme 3. Finally, compound 12 was further derivatized to
aromatic group that might be beneficial for additional Van der give urea derivatives (30e32) from the corresponding isocyanates
Waals and pep stacking, and bulky groups that could be favorable under basic conditions (Scheme 4).
in detecting hydrophobic contacts. In the present work, we
describe an in-parallel synthetic strategy to rapidly explore this
new set of structural analogues of 1 in order to investigate the effect 2.1.2. The purities of the analogues
on affinity, activity and selectivity at 5-HT1AR and a<alpha>1-ARs. Full assignments of protons and carbons for all the compounds
This approach led to extensive qualitative structureeactivity rela- 7e32 were done by 1H and 13C NMR analysis, through related 1D
tionship studies on compound 1. Furthermore, to help in rational- spectra acquisition and studying the key values obtained from
1
izing the pharmacological results, docking studies on compounds He1H COSY, 1He13C HSQC and HMBC experiments. The final
7e32 were performed to evaluate their interaction with the puta- compounds were converted into oxalate salts using oxalic acid in
tive 5-HT1AR binding site. The computational results obtained, in acetone. The purities of the salts were confirmed by elemental
agreement with the biological experimental data, underlined the analysis and the values obtained are within ±0.4% of the calculated
already proved reliability of the model and gave additional sug- ones. The exact mass of the salts was confirmed by HPLC-QTOF
gestions for the synthesis of new derivatives. measurement.

2. Results and discussion 2.2. Pharmacology

2.1. Chemistry The pharmacological profile of compounds 7e32 was evaluated


by radioligand binding assays using [3H]prazosin to label cloned
2.1.1. Synthesis human a<alpha>1 adrenoceptors expressed in CHO cells and [3H]
The 1,4-dioxaspiro[4.5]decane intermediates 2e4 were ob- 8-OH-DPAT to label cloned human 5-HT1AR expressed in HeLa cells
tained by acetalization of the selected ketones with 3- [22,23]. Functional a<alpha>1 adrenoceptor subtype selectivity
chloropropane-1,2-diol, in acid catalyzed conditions (Scheme 1). was also determined on various isolated tissue types. Blocking ac-
Oxidation of the sulphur atom of compound 2 with 3- tivity was assessed by antagonism of (-)-norepinephrine-induced
chloroperbenzoic acid yielded both the sulfoxide derivative 5 and contraction of rat prostatic vas deferens(a<alpha>1A) [24] or
the sulfone derivative 6 depending on reaction conditions (Scheme thoracic aorta (a<alpha>1D) [25] and by antagonism of (-)-phen-
1). The chloro derivatives 2e6 obtained were subsequently reacted ylephrine-induced contraction of rat spleen (a<alpha>1B) [26].
with 1-(2-methoxyphenyl)piperazine, under KI catalysis, to give Functional characterization of selected compounds, chosen as
final compounds 7e11. Subsequently debenzylation of 9 afforded the best representatives of the different structural classes (7e10, 13,
the free amine 12 which was further employed to synthetize 14, 17, 18, 23, 26, 30 and 31) was performed at the 5-HT1AR ac-
several derivatives. cording to the method of Stanton and Beer [27], using [35S]GTPgS
In particular 12 was reacted with chlorobenzene and 2- binding, in cell membranes from HeLa cells transfected with the
chloropyridine, following the Buchwald-Hartwig amination pro- human cloned 5-HT1AR [28]. Stimulation of [35S]GTPgS binding was
cedure [21], to yield respectively the alkylated compounds 13 and expressed as the percentage increase in binding above the basal
14 (Scheme 2). Moreover, the piperidine nitrogen of 12 unlocks a value; maximal stimulation observed with serotonin was estab-
myriad of new possibilities to fully explore the SAR of this class of lished as 100%.
250 S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266

Fig. 2. Selected docking pose of compound 1 R isomer into the 5HT1AR. The ligand is reported in stick, colored by atom-type (C, cyan). Salt bridge interaction is depicted as yellow
dashed line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Scheme 1. Synthesis of compounds 7e12. Reagents and conditions: i) PTSA, DeaneStark, toluene, 110  C, 22 h; ii) mPCBA, CH2Cl2, 20  C, 45 min.; iii) mPCBA, CH2Cl2, r.t., 24 h; iv) 1-
(2-methoxyphenyl)piperazine, KI, 2-Methoxyethanol, 120  C, 22 h; v) Pd/C, H2, EtOH, 60  C, 5 h.

Scheme 2. Synthesis of 13 and 14. Reagents and conditions: i) Pd2(dba)3/P(i-BuNCH2CH2)3N, t-BuONa, toluene, 110  C, 15 h.

2.3. Structureeaffinity relationship decrease is probably due to the protonation of the nitrogen atom
and a positive charge in this part of the molecule seems to be an
Table 1 shows the affinity constants (pKi) obtained in radio- unfavorable factor for binding. As a consequence, a significant
ligand binding assays. Compound 1 was reported as the reference reversal of selectivity is observed. On the other hand, the benzy-
compound in order to better understand the structureeaffinity lated derivative of 12, compound 9, displays an increased affinity at
relationship. As previously reported [20], 1 binds preferentially to a<alpha>1A and a<alpha>1D subtypes and at 5-HT1AR, although
the 5-HT1AR being 18-fold more selective over the adrenoceptors. protonation of the same nitrogen atom may occur as well. This may
Isosteric substitutions of the carbon atom at position 8 of the 1,4- be explained by the fact that the introduction of a benzyl moiety
dioxaspiro[4.5]decane with sulfur atom, as for 7, or with an oxy- increases lipophilicity which can overcome the negative effect of
gen atom, as for 8, and or with a nitrogen atom, as for compound 12, the positive charge on the nitrogen atom. Similar values were also
was also studied. An increase in the polarity (7 < 8 < 12) led to a obtained for compounds 13 and 14, respectively, the phenyl and the
progressive decrease in affinity at 5-HT1AR, whereas an increase at 2-pyridyl derivatives of 12. Therefore, the introduction of bulky
the a<alpha>1 adrenoceptors, although limited, is observed. substituents at the C-8 position of the 1,4-dioxa-8-azaspiro[4.5]
Oxidation of the sulphur atom of 7 to give the sulfoxide 10 and the decane seems to favor a<alpha>1A and a<alpha>1D binding.
sulfone 11 confirmed this trend of activity as derivative 10 and 11 Differently, all the amidic derivatives synthesized (15e21) showed
show lower affinities for 5-HT1AR with respect to the less polar 7. At lower affinity values both at 5-HT1AR and at a<alpha>1 adreno-
the a<alpha>1 sites the variation is less pronounced. The observed ceptors. Anyway, once more, a preference towards sterically
S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266 251

Scheme 3. Synthesis of compounds 15e29. Reagents and conditions: a) Buchi Syncore® Reactor, in parallel synthesis, CHCl3, r.t, 12 h. *Purification was performed employing
benzensulfonic polystyrene resin.

Scheme 4. Synthesis of compounds 30e32. Reagents and conditions: a) CH2Cl2, 0  C to r.t, 3 h.

hindered aromatic substituents was observed at a<alpha>1A and 2.4. Structureeactivity relationship
a<alpha>1D subtypes with the benzoyl derivative 15, being 100-
and 20-fold more affine towards a<alpha>1A and a<alpha>1D with Antagonist potencies at a<alpha>1 adrenoceptor subtypes are
respect to the trifluoroacetyl derivative 21. The insertion of a sul- listed in Table 2. Compound 1 was employed as reference com-
fonamidic group at position 8 (22e29) of compound 12 resulted pound. With few exceptions, the activity trend parallels the affin-
again in a reverse in selectivity in comparison to compound 1, with ities observed in the binding studies and the selectivity values,
higher affinity at a<alpha>1A and a<alpha>1D. In particular, com- generally toward a<alpha>1D subtype, are larger. These discrep-
pound 27 displays the highest affinities of the series at the ancies are not unusual and may be ascribed to several factors, as
a<alpha>1 adrenoceptor subtypes with a selectivity ratio of 15 previously reported [18]. In particular, the potencies of the tested
(a<alpha>1A/5-HT1A) and 50 (a<alpha>1D/5-HT1A). Of note are the compounds for a<alpha>1A and a<alpha>1B subtypes are higher
affinities toward a<alpha>1 receptors and, among these, the than those of the lead compound 1. On the other hand, the values
preference for the a<alpha>1A and the a<alpha>1D subtypes with obtained for a<alpha>1D subtype are lower, with few exceptions,
respect to the a<alpha>1B subtype. Again, in this respect, the most than the reference compound 1. Thus, a marked loss in selectivity
interesting compound is 27 which shows selectivity ratios of 76 was observed for a large number of molecules. With regard to
(a<alpha>1D/a<alpha>1B) and 33 (a<alpha>1A/a<alpha>1B). a<alpha>1D subtype, an enhancement in antagonistic potency was
Moreover, the position of the substituents on the aromatic ring of observed for compounds 9 and 26. In particular, compound 9 could
the latter derivative seems to be relevant, with the meta position be of great interest for further design of selective a<alpha>1D li-
favored with respect to the para position. Compounds 30e32, gands, since it displays a good antagonist potency (pKb
possessing a more polar ureidic moiety, show a similar trend in a<alpha>1D ¼ 8.60), with a selectivity ratio of 8 and 26 over
affinity at both the 5-HT1AR and at a<alpha>1 adrenoceptors, but a<alpha>1A and a<alpha>1B adrenoceptor subtypes, respectively.
both affinity and selectivity are to a lesser extent. Furthermore, of interest is the fact that several compounds show
preferential antagonist activities for a<alpha>1B subtype. Among
them, the most interesting is compound 30, with a selectivity ratio
of about one order of magnitude. Although this is not a striking
252 S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266

Table 1
Affinity constants (pKi) and selectivities of test and reference compounds for human recombinant a<alpha>1 adrenoceptor subtypes and 5-HT1AR.
H3CO

O
N
X
O N

Cmps X pKia1Aa pKia1Ba pKia1Da pKia 5HT1A a1D/a1Ab a1D/a1Bb a1B/a1Ab 5HT1A/c a1

1 C 7.04 6.90 <6 8.29 <0.09 <0.12 0.72 17.78

7 S 8.07 6.85 7.83 8.03 0.60 9.50 0.06 0.90

8 O 7.77 6.49 7.60 7.33 0.70 12.9 0.05 0.36

9 8.17 7.78 8.33 7.84 1.4 3.5 0.4 0.32

10 O S n.d. 6.32 7.37 6.93 n.d. 10.5 n.d. 0.36


O
11 S 8.01 6.54 7.29 7.09 0.19 19.0 0.03 0.12
O

12 NH 7.31 7.37 7.43 6.80 1.3 1.1 1.1 0.23

13 N 8.19 6.81 8.09 7.80 0.8 19 0.04 0.4

14 N 7.75 <6 7.64 8.05 0.77 >43 <0.01 2


N

15 7.97 7.17 7.69 7.23 0.52 3.3 0.16 0.18

16 7.99 7.20 7.96 7.30 0.93 5.75 0.16 0.20

17 7.67 6.80 7.55 7.37 0.76 5.62 0.13 0.50

18 7.51 6.96 7.40 7.69 0.77 2.75 0.28 1.51

19 7.93 6.80 7.57 7.44 0.43 5.89 0.07 0.32

20 7.22 <6 7.05 7.19 0.67 >11.22 <0.06 0.93

21 <6 <6 6.38 6.49 >2.40 >2.40 1 1.29

22 8.10 7.07 7.91 7.28 0.64 6.92 0.09 0.15

23 8.34 8.08 7.97 7.57 0.43 0.77 0.55 0.17

24 8.19 7.16 8.15 7.43 0.91 9.77 0.07 0.17

25 8.05 6.83 7.58 7.16 0.34 5.62 0.06 0.13

26 8.28 6.96 8.24 7.61 0.91 19.05 0.05 0.21

27 8.52 7.16 9.04 7.36 3.31 75.86 0.04 0.02

28 8.10 7.07 8.01 7.22 0.81 8.71 0.09 0.13

29 7.73 6.44 7.63 6.84 0.79 15.49 0.05 0.13


S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266 253

Table 1 (continued )

Cmps X pKia1Aa pKia1Ba pKia1Da pKia 5HT1A a1D/a1Ab a1D/a1Bb a1B/a1Ab 5HT1A/c a1

30 7.92 7.09 7.88 7.48 0.91 6.16 0.15 0.36

31 7.70 6.62 7.72 7.10 1.04 12.59 0.08 0.25

32 7.90 6.89 7.45 7.06 0.35 3.63 0.10 0.14

a
Ki values were derived from the ChengePrusoff equation [25] at one or two concentrations and agreed within 10%.
b
Antilog of the difference between the pKi values for a<alpha>1A, a <alpha>1B and a <alpha>1D adrenoceptors.
c
Antilog of the difference between the pKi values for a1 adrenoceptors (higher value) and the 5-HT1AR.

result, especially considering its low affinity for a<alpha>1B adre- pharmacological results. On the basis of our calculations, the newly
noceptor, it is an important value as it is not an easy task to discover synthesized analogues partially share the binding mode of com-
an a<alpha>1B selective antagonist and only a few, at present, are pound 1, as described above. Accordingly, all the compounds dis-
known. Thus, compound 30 may be of interest as a starting point to played one salt bridge interaction between the piperazine
develop a selective antagonist for this subtype. protonated nitrogen atom and D116 side-chain and also Van der
A number of compounds (7e10, 13, 14, 17, 18, 23, 26, 30 and 31) Waals contacts and pp stacking between the 2-methoxy phenyl
were selected for functional characterization studies at 5-HT1AR ring and C120, T121; and with F361, F362, respectively.
based on their affinity as the best representatives of the different Interestingly, when comparing compound 1 and 8, the intro-
structural classes of analogues of 1. The results are reported in duction of an oxygen atom onto the 1,4-dioxa-spiro[4.5]decane
Table 3 and Fig. 3. Contrary to what has already been reported, portion proved to be detrimental for the activity, guiding the
compound 1 behaves as a partial agonist, with a pD2 of 8.61 and agonist towards the polar Q97 side-chain. It could be hypothesized
Emax of 48% [19]. All the derivatives increased the binding of [35S] that the described docking mode probably caused the establish-
GTPgS, with pD2 values between 6.49 and 8.13 and Emax values ment of a weaker salt bridge with the key-residue D116 for 8, in
between 28.3 and 138.8 defining all the compounds, with the comparison with that detected by compound 1. In agreement with
exception of 10, as partial agonists. Compound 10, with a pD2 of 8.13 these data, the sulphoxide 10 (characterized by a bulkier group at
and an Emax value of 138.8, is the most potent and the most effi- C8-position and bearing an H-bond acceptor function), displayed
cacious of the series. A close inspection of the results reported in an additional H-bond with Q97, which could justify its high pD2
Table 3 allows us to make some considerations in terms of struc- values (pD2 ¼ 8.13; the 2-R, 8-R enantiomer proved to be the most
tureeactivity relationships. The isosteric replacement of CH2 with S probable) (Fig. 4).
and O (compound 1 vs compounds 7 and 8) causes a decrease in On the other hand, compounds bearing much more bulky
activity, for both potency (pD2 of 7.48 and 6.82) and efficacy (Emax of groups at C-8 position (9, 13, 14, 17, 18, 23, 26, 30, 31) showed lower
36 and 31), paralleling therefore the decrease in affinity. Comparing pD2 values (pD2 ¼ 6.49e7.65) compared to the potent agonists 1
the activities of the benzyl derivative 9, the phenyl derivative 13 and 10. This is probably due to a weaker salt bridge interaction with
and the 2-pyridyl derivative 14 it is possible to make another point. the key residue D116 while the substituent at 8 position proved to
Compounds 9 and 13 have the same activities indicating that the be oriented towards a much more deep hydrophobic cavity
protonation of the nitrogen atom of the 1,4-dioxa-8-azaspiro[4.5] including F19, Y96, F112 and T188 performing Van der Waals con-
decane core do not affect activity. Furthermore, the difference in tacts and pep stacking interactions (Fig. 5, compound 26 has been
activity between compounds 13 and 14 clearly indicates that the reported). Thus, according to these results, it is reasonable to
nitrogen atom within the aromatic ring (2-pyridine) is a negative conclude that only small substituents at C-8 position of the 1,4-
structural feature, at least for potency (pD2 of 7.14 vs pD2 of 6.49). dioxa-spiro[4.5]decane portion are allowed. On the contrary, the
Compound 10 deserves a specific comment: its potency presence of bulky aromatic substituents improperly switches the
(pD2 ¼ 8.13) is slightly lower than that of compound 1, but it is the compound docking mode.
only one in the series to display an efficacy higher than 100%, with
an Emax value of 139% (Fig. 3). This behavior is at least peculiar, not 2.6. Conclusion
unusual but even not frequently observed. What is the meaning? If
we consider 8-OH-DPAT as full agonist, compound 10 is more than In conclusion, extensive qualitative structureeactivity relation-
that. On the other hand is 8-OH-DPAT actually a true full agonist? ship studies on compound 1 were performed. Some considerations
That is beyond the scope of this paper. But it would be of interest to on structure-affinity and structureeactivity relationships have led
investigate this aspect further. At this stage any hypothesis may to these conclusions:
seem speculative but it might be a working hypothesis. It should be
considered that compound 10 is a mixture of two diastereoisomers a) An increase in the polarity of the substituents, at 8-position
and therefore a mixture of two couples of enantiomers. Therefore of the spirocyclohexyl portion of 1, led to a progressive loss
in order to try to clarify this aspect the separation of the four en- of affinity toward 5-HT1AR.
antiomers is mandatory. A stereoselective synthesis has already b) Even small structural modifications on the spirocyclohexyl
been planned and it will be reported in due course. moiety decrease the activity of the ligands as partial agonists
at 5-HT1AR, the exception being compound 10, which shows
2.5. Molecular modeling a full agonist behavior.
c) The introduction of bulky aromatic substituents, at 8-
Docking studies on the theoretical model of the 5-HT1AR have position of the spirocyclohexyl portion of 1, is well toler-
been performed for compounds 7e32 in order to rationalize the ated, especially in the case of a<alpha>1 adrenoceptor. A
254 S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266

Table 2
Antagonist potency (pKb) and selectivities of test and reference compounds at a<alpha>1 adrenoceptors in isolated rat prostatic vas deferens (a<alpha>1A), spleen
(a<alpha>1B), and thoracic aorta (a<alpha>1D).
H3CO

O
N
X
O N

Cmps X pKba1Aa pKba1Ba pKba1Da a1D/a1Ab a1D/a1Bb a1B/a1Ab

1 6.78 ± 0.15 6.72 ± 0.16 7.88 ± 0.19 12.59 14.45 0.87


7 7.60 ± 0.11 7.63 ± 0.20 6.84 ± 0.05 0.17 0.16 1.07

8 7.55 ± 0.06 7.17 ± 0.05 7.33 ± 0.07 0.60 1.44 0.41


N
9 7.72 ± 0.03 7.18 ± 0.08 8.60 ± 0.19 7.58 26.30 0.29

10 O S n.d. n.d. n.d. n.d. n.d. n.d.

11 7.34 ± 0.20 7.66 ± 0.02 7.20 ± 0.03 0.72 0.34 2.09

12 7.74 ± 0.16 7.78 ± 0.07 6.89 ± 0.05 0.14 0.13 1.09

13 N 7.78 ± 0.15 7.56 ± 0.12 7.43 ± 0.20 0.44 0.74 0.60

14 N 7.63 ± 0.08 7.86 ± 0.10 6.87 ± 0.03 0.17 0.10 1.70


N

15 N 7.71 ± 0.02 8.19 ± 0.02 7.45 ± 0.22 0.55 0.18 3.02

16 7.79 ± 0.02 8.33 ± 0.14 7.29 ± 0.16 0.31 0.09 3.47

17 N 7.43 ± 0.14 7.69 ± 0.06 7.38 ± 0.17 0.89 0.49 1.82


O

18 N 6.95 ± 0.05 7.33 ± 0.13 7.02 ± 0.04 1.17 0.49 2.40

19 N 7.43 ± 0.11 7.86 ± 0.11 6.71 ± 0.01 0.19 0.07 2.69


NH

20 7.17 ± 0.11 7.72 ± 0.05 6.48 ± 0.03 0.20 0.05 3.55

21 6.97 ± 0.11 8.00 ± 0.04 7.50 ± 0.12 3.39 0.31 10.7

O
22 S N 7.48 ± 0.01 7.94 ± 0.19 7.05 ± 0.12 0.37 0.13 2.88
O

23 7.40 ± 0.04 7.91 ± 0.09 6.96 ± 0.04 0.36 0.11 3.23

O
24 Cl S N 7.24 ± 0.08 7.36 ± 0.04 7.37 ± 0.06 1.35 1.02 1.32
O

25 6.98 ± 0.08 7.74 ± 0.21 7.37 ± 0.07 2.45 0.42 5.75

26 7.74 ± 0.08 7.75 ± 0.16 8.02 ± 0.20 1.90 1.86 1.02

27 7.97 ± 0.03 8.14 ± 0.02 7.98 ± 0.16 1.02 0.69 1.48

28 7.46 ± 0.11 8.05 ± 0.07 7.27 ± 0.16 0.64 0.16 3.89


S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266 255

Table 2 (continued )

Cmps X pKba1Aa pKba1Ba pKba1Da a1D/a1Ab a1D/a1Bb a1B/a1Ab

29 7.41 ± 0.20 7.46 ± 0.19 7.18 ± 0.20 0.59 0.52 1.12

30 7.22 ± 0.03 8.18 ± 0.03 7.26 ± 0.18 1.09 0.12 9.12

31 7.38 ± 0.12 7.40 ± 0.12 7.29 ± 0.20 0.81 0.77 1.04

32 7.63 ± 0.04 7.42 ± 0.03 6.53 ± 0.01 0.08 0.13 0.61

a
Potency values were calculated according to van Rossum [26] at one or two concentrations and agreed within 2%.
b
Antilog of the difference between the pKb values for a<alpha>1A, a<alpha>1B, and a<alpha>1D adrenoceptors.

reversal in selectivity was observed, in comparison to the


lead compound 1.
d) In particular, sterically hindered substituents oriented the
a<alpha>1 adrenoceptor selectivity towards a<alpha>1D
and a<alpha>1A subtypes.
Table 3 e) The computational results obtained, in agreement with the
Agonist potency (pD2), relative effectiveness (Emax) in the agonist-induced [35S]
biological experimental data, proved the reliability of the 5-
GTPgS-binding assay at the human 5-HT1AR.
HT1AR model.
H3CO

Among this series, compound 9 showed an interesting affinity


O
N and selectivity toward a<alpha>1A and a<alpha>1D-adrenoceptor
X
N subtypes. Together with compounds 27, it displayed a relevant
O
antagonist activity and selectivity towards a<alpha>1A and
a<alpha>1D adrenoceptors. Furthermore, compound 30 has been
Cmps X pD2 Emaxa
shown to be a new a<alpha>1B selective antagonist notwith-
standing its low affinity for the latter receptor. Therefore, 9, 27 and
30 emerged as promising a<alpha>1 receptors antagonist and they
1 8.61 48 could represent a new starting point for developing more selective
7 7.48 36
ligands for one or the other a<alpha>1 adrenoceptor subtypes.
Research along this line is in progress and will be disclosed in due
8 6.82 31
course.
9 7.21 45

10 8.13 139 3. Experimental


13 N 7.14 42
3.1. General methods
14 N 6.49 41
N
All reagents, solvents and other chemicals were used as pur-
O
chased from SigmaeAldrich without further purification unless
17 N 7.12 33
O

18 7.29 33

23 7.33 28

26 7.65 37

30 7.22 28

O
31 Bun
N N
6.53 30
H
Fig. 3. Stimulation of [35S]GTPgS binding in HeLa cells expressing human recombinant
a
Maximal stimulation expressed as a percentage of the maximal 5-HT response. 5-HT1AR by compounds 1, 10 and the reference full agonist 8-OH-DPAT.
256 S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266

Fig. 4. Selected docking pose of compound 1 and compound 10 (the 2-R, 8-r enantiomer proved to be the most probable) into the 5HT1AR. The ligands are reported in stick, colored
by atom-type (C, cyan; C, green respectively). Salt bridge and H-bond interactions are depicted as yellow and red dashed lines, respectively. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Selected docking pose of compound 1 and compound 26 R isomers into the 5HT1A R. The ligands are reported in stick, colored by atom-type (C, cyan; C, light green
respectively). Salt bridge interaction is depicted as yellow dashed line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)

otherwise specified. Air- or moisture-sensitive reactants and sol- DPX-400 Advance (Bruker) spectrometer operating at 400.13 MHz
vents were employed in reactions carried out under nitrogen at- Chemical shifts are expressed in d (ppm). 1H NMR chemical shifts
mosphere unless otherwise noted. Flash column chromatography are relative to tetramethylsilane (TMS) as internal standard. 13C
purifications (medium pressure liquid chromatography) were car- NMR chemical shifts are relative to TMS at d 0.0 or to the 13C signal
ried out using Merck silica gel 60 (230e400 mesh, ASTM). The of the solvent: CDCl3d 77.04, CD3OD d 49.8, DMSO-d6 d 39.5. NMR
purity of compounds was determined by elemental analysis (C,H,N) data are reported as follows: chemical shift, number of protons/
that was performed on a Carlo Erba 1106 Analyzer in the Micro- carbons, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
analysis Laboratory of the Life Sciences Department of Universit a multiplet; br, broadened), coupling constants (Hz) and assignment
degli Studi di Modena e Reggio Emilia and the results here reported (dotsd ¼ 1,4-dioxa-8-thiaspiro[4.5]decane; tosd ¼ 1,4,8-trioxaspiro
are within ±0.4% of the theoretical values. Melting points were [4.5]decane; doasd ¼ 1,4-dioxa-8-azaspiro[4.5]decane;
determined with a Stuart SMP3 and they are uncorrected. The Bz ¼ benzyl; dotsdo ¼ 1,4-dioxa-8-thiaspiro[4.5]decane 8-oxide;
structures of all isolated compounds were ensured by Nuclear dotsdd ¼ 1,4-dioxa-8-thiaspiro[4.5]decane 8,8-dioxide;
magnetic resonance (NMR) and Mass spectrometry. 1H and 13C piperaz. ¼ piperazine; Phe ¼ 2-methoxyphenyl; arom. ¼ pheny;
NMR (1D and 2D experiments) spectra were recorded on a DPX-200 Pyr. ¼ pyridine; Bzo ¼ benzoyl; Fur ¼ 2-furanyl; Cyc. ¼ Cyclohexyl;
Advance (Bruker) spectrometer operating at 200.13 MHz and on a Pyrro. ¼ Pyrrolidine; Sulf ¼ phenylsulfonyl; Tos ¼ tosyl;
S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266 257

Thio ¼ thiophenyl). 1He1H correlation spectroscopy (COSY), 1He13C CH2Cl), 50.8 (CH2, C-7/C-9 doasd), 50.9 (CH2, C-7/C-9 doasd), 62.6
heteronuclear multiple quantum coherence (HMQC) and hetero- (CH2, CH2 bn), 67.8 (CH2, C-3 doasd), 74.6 (CH, C-2 doasd), 109.8 (C,
nuclear multiple bond connectivity (HMBC) experiments were C-5 doasd), 125.4 (CH, C-1 Bz), 127.5 (CH, C-3, C-5 Bz), 128.8 (CH, C-
recorded for determination of 1He1H and 1He13C correlations 2, C-6 bn), 129.1 (C, C-4 Bz).
respectively. HR-MS experiments were carried out using an LC-MS
mass spectrometer (6520 Accurate-Mass Q-TOF LC/MS e Agilent 3.2.5. 2-(Chloromethyl)-1,4-dioxa-8l4-thiaspiro[4.5]decan-8-one
Technologies) equipped with an ion spray ionization source (ESI). (5)
MS (þ) spectra were acquired by direct infusion (5 mL/min) of a To a cooled (0  C) solution of compound 2 (0.50 g, 2.4 mmol) in
solution containing the appropriate sample as oxalate salt CH2Cl2 (4.0 mL) was added dropwise mCPBA (0.45 g, 2.64 mmol).
(10 pmol/mL), dissolved in a 0.1% acetic acid solution, with mobile The reaction mixture was allowed to warm to room temperature.
phase methanol/water 50:50, at the optimum ion voltage of After completion the reaction mixture was quenched with satu-
4800 V. The oxalate salts of all tested compounds were used for the rated aqueous sodium bicarbonate. The aqueous layer was extrac-
pharmacological evaluations. ted with CH2Cl2 and the combined organic layers were dried over
anhydrous MgSO4, filtered and concentrated in vacuum to afford 2-
3.2. Synthesis (chloromethyl)-1,4-dioxa-8-thiaspiro[4.5]decane 8-oxide as a
brown oil (0.36 g, 1.52 mmol; yield 63%).
1
3.2.1. General procedure A H NMR (CDCl3, 400 MHz): d ¼ 2.12e2.37 (4H, m, CH2-6, CH2-10
The 1,4-dioxaspiro[4.5]decane derivatives 2e4 were synthe- dotsdo), 3.01e3.38 (4H, m, CH2-7, CH2-9 dotsdo), 3.45e3.64 (2H, m,
sized by reacting the corresponding ketone (1.0 mmol) with 3- CH2Cl), 3.94 (1H, dd, J ¼ 5.0, 8.5 Hz, CHa-3 dotsdo), 4.22 (1H, dd,
chloropropane-1,2-diol (2.0 mmol), in toluene (10 mL) using p- J ¼ 6.1, 8.5 Hz, CHb-3 dotsdo), 4.31e4.58 (1H, m, CH-2 dotsdo); 13C
toluenesulfonic acid (0.05 mmol) as catalyst. The mixture was NMR (CDCl3, 400 MHz): d ¼ 36.9 (CH2, C-6/C-10 dotsdo), 38.5 (CH2,
refluxed and water was removed in a DeaneStark trap until the C-6/C-10 dotsdo), 47.5 (CH2, CH2Cl), 52.9 (CH2, C-7/C-9 dotsdo),
formation of water stopped (20 h). After the reaction is completed 53.2 (CH2, C-7/C-9 dotsdo), 66.8 (CH2, C-3 dotsdo), 74.2 (CH, C-2
the mixture CH2Cl2 and H2O were added. The organic layer was dotsdo), 106.1 (C, C-5 dotsdo); GC-MS (70 eV) m/z ¼ 224 (5) [Mþ],
separated and the aqueous layer was extracted with CH2Cl2. The 196 (10), 156 (100), 139 (95),111 (62), 75 (45).
organic layers were combined, dried over anhydrous Na2SO4,
filtered and concentrated in vacuum to give the title compound. 3.2.6. 2-(chloromethyl)-1,4-dioxa-8l6-thiaspiro[4.5]decane-8,8-
dione (6)
3.2.2. 2-(Chloromethyl)-1,4-dioxa-8-thiaspiro[4.5]decane (2) To a cooled (0  C) of compound 2 (0.47 g, 2.3 mmol) in CH2Cl2
The compound, was obtained as a brown oil from dihydro-2H- (4.0 mL) was added dropwise mCPBA (0.87 g, 5.06 mmol). The re-
thiopyran-4(3H)-one following the general procedure A (1.70 g, action mixture was allowed to warm to room temperature for 24 h.
8.31 mmol, yield 97%). After completion the reaction mixture was quenched with satu-
1
H NMR (CDCl3, 400 MHz): d ¼ 1.76e2.01 (4H, m, CH2-6, CH2-10 rated aqueous sodium bicarbonate. The aqueous layer was extrac-
dotsd), 2.52e2.87 (4H, m, CH2-7, CH2-9 dotsd), 3.30e3.52 (2H, m, ted with CH2Cl2 and the combined organic layers were dried over
CH2Cl), 3.88 (1H, dd, J ¼ 5.2, 8.7 Hz, CHa-3 dotsd), 4.12 (1H, dd, anhydrous MgSO4, filtered and concentrated in vacuum to afford 2-
J ¼ 6.0, 8.7 Hz, CHb-3 dotsd), 4.23e4.38 (1H, m, CH-2 dotsd); 13C (chloromethyl)-1,4-dioxa-8-thiaspiro[4.5]decane 8,8-dioxide
NMR (CDCl3, 400 MHz): d ¼ 26.7 (CH2, C-7/C-9 dotsd), 26.9 (CH2, C- (unassigned diasteromeric mixture 40/60), as a brown oil (0.57 g,
7/C-9 dotsd), 36.4 (CH2, C-6/C-10 dotsd), 37.8 (CH2, C-6/C-10 dotsd), 2.28 mmol, yield 99%).
1
47.3 (CH2, CH2Cl), 65.9 (CH2, C-3 dotsd), 73.2 (CH, C-2 dotsd), 105.9 H NMR (CDCl3, 400 MHz): d ¼ 2.12e2.39 (4H, m, CH2-6, CH2-10
(C, C-5 dotsd). dotsdd), 3.03e3.25 (4H, m, CH2-7, CH2-9 dotsdd), 3.49e3.61 (2H, m,
CH2Cl), 3.92 (1H, dd, J ¼ 5.8, 8.8 Hz, CHa-3 dotsdd), 4.14 (1H, dd,
3.2.3. 2-(Chloromethyl)-1,4,8-trioxaspiro[4.5]decane (3) J ¼ 6.1, 8.8 Hz, CHb-3 dotsdd), 4.30e4.42 (1H, m, CH-2 dotsdd); 13C
The compound, was obtained as a yellow oil from dihydro-2H- NMR (CDCl3, 400 MHz): d ¼ 37.3 (CH2, C-6/C-10 dotsdd), 38.7 (CH2,
pyran-4(3H)-one following the general procedure A (1.60 g, C-6/C-10 dotsdd), 46.5 (CH2, CH2Cl), 52.7 (CH2, C-7/C-9 dotsdd),
8.30 mmol, yield 80%). 53.5 (CH2, C-7/C-9 dotsdd), 66.7 (CH2, C-3 dotsdd), 73.9 (CH, C-2
1
H NMR (CDCl3, 400 MHz): d ¼ 1.61e1.90 (4H, m, CH2-6, CH2-10 dotsdd), 106.3 (C, C-5 dotsdd); GC-MS (70 eV) m/z ¼ 240 (5) [Mþ],
tosd), 3.47 (1H, dd, J ¼ 7.5, 10.9 Hz, CHaCl), 3.61 (1H, dd, J ¼ 4.7, 191 (5), 148 (100), 56 (45).
10.9 Hz, CHbCl), 3.68e3.75 (4H, m, CH2-7, CH2-9 tosd), 3.91 (1H, dd,
J ¼ 5.1, 8.7 Hz, CHa-3 tosd), 4.13 (1H, dd, J ¼ 6.1, 8.7 Hz, CHb-3, tosd), 3.2.7. General procedure B
4.23e4.45 (1H, m, CH-2 tosd); 13C NMR (CDCl3, 400 MHz): d ¼ 35.8 To a solution of 1-phenyl-1,3,8-triazaspiro-[4,5]deacan-4-one
(CH2, C-6/C-10 tosd), 36.9 (CH2, C-6/C-10 tosd), 47.7 (CH2, CH2Cl), (5.0 mmol), KI (0.2 mmol) in anhydrous 2-methoxyethanol
65.3 (CH2, C-3 tosd), 65.6 (CH2, C-7/C-9 tosd), 65.9 (CH2, C-7/C-9 (10 mL) was added the selected halogen derivative 2e5
tosd), 76.3 (CH, C-2 tosd), 106.9 (C, C-5 tosd). (1.0 mmol) portion wise. The resulting mixture was stirred under
reflux for 20 h then, cooled to room temperature and concentrated
3.2.4. 8-Benzyl-2-(chloromethyl)-1,4-dioxa-8-azaspiro[4.5]decane in vacuum. The residue was partitioned between EtOAc and H2O.
(4) The organic layer was separated, and the aqueous layer was
The compound, was obtained as a yellow oil from 1- extracted with EtOAc. The organic layers were combined, washed
benzylpiperidin-4-one following the general procedure A (1.2 g, with H2O, dried over anhydrous Na2SO4, filtered, and concentrated
4.5 mmol, yield 90%). in vacuum. The residue was purified by flash chromatography to
1
H NMR (CDCl3, 400 MHz): d ¼ 1.60e1.91 (m, 4H, CH2-6, CH2-10 yield the desired compound.
doasd), 2.31e2.59 (4H, m, CH2-7, CH2-9 doasd), 3.41e3.52 (2H, m,
CH2Cl), 3.54 (2H, s, CH2 bn), 3.92 (1H, dd, J ¼ 5.0, 8.6 Hz, CHa-3 3.2.8. 1-{1,4-Dioxa-8-thiaspiro[4.5]decan-2-ylmethyl}-4-(2-
doasd), 4.12 (1H, dd, J ¼ 5.3, 8.6 Hz, CHb-3 doasd), 4.29e4.34 (1H, methoxyphenyl)piperazine (7)
m, CH-2 doasd), 7.21e7.43 (5H, m, Bz); 13C NMR (CDCl3, 400 MHz): The compound, was obtained as a dark oil from compound 2
d ¼ 34.7 (CH2, C-6/C10 doasd), 35.6 (CH2, C-6/C10 doasd), 47.4 (CH2, following the general procedure B (0.76 g, 2.09 mmol, yield 86%).
258 S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266

1
H NMR (CDCl3, 400 MHz): d ¼ 1.89e205 (4H, m, CH2-6, CH2-10 doasd), 50.3 (2 CH2, C-3, C-5 piperaz.), 50.7 (CH2, C-7/C-9 doasd),
dotsd), 2.61e2.72 (2H, m, CH2N), 2.74e2.87 (8H, m, CH2-7, CH2-9 50.8 (CH2, C-7/C-9 doasd), 53.9 (2 CH2, C-2, C-6 piperaz.), 55.1 (CH3,
dotsd, CH2-2, CH2-6 piperaz.), 3.02e3.15 (4H, m, CH2-3, CH2-5 OCH3), 61.2 (CH2, CH2eN), 62.2 (CH2, CH2-Bz), 67.9 (CH2, C-3 doasd),
piperaz), 3.67 (1H, dd, J ¼ 7.5, 8.0 Hz, CHa-3 dotsd), 3.87 (3H, s, 73.8 (CH, C-2 doasd), 107.6 (C, C-5 doasd), 110.9 (CH, C-3 Phe), 117.9
OCH3), 4.13 (1H, dd, J ¼ 6.1, 8.0 Hz, CHb-3 dotsd), 4.23e4.40 (1H, m, (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 122.7 (CH, C-4 Phe), 127.0 (C, C-1
CH-2 dotsd), 6.90 (1H, d, J ¼ 7.8 Hz, CH-3 Phe), 6.93e7.02 (2H, m, Bz), 128.0 (2 CH, C-3, C-5 Bz), 128.9 (CH, C-4 Bz), 129.0 (2 CH, C-2, C-
CH-5, CH-6 Phe), 7.04 (1H, ddd, J ¼ 2.5, 7.9, 8.0 Hz, CH-4 Phe); 13C 6 Bz), 141.0 (C, C-1 Phe), 152.0 (C, C-2 Phe).
NMR (CDCl3, 400 MHz): d ¼ 26.6 (CH2, C-7/C-9 dotsd), 26.7 (CH2, C- The free amine (0.18 g, 0.4 mmol) was then converted into the
7/C-9 dotsd), 36.5 (CH2, C-6/C-10 dotsd), 37.9 (CH2, C-6/C-10 dotsd), corresponding hydrogenoxalate from diethyl ether (0.07 g,
50.1 (2 CH2, C-3, C-5 piperaz.), 53.8 (2 CH2, C-2, C-6 piperaz.), 55.1 0.12 mmol, yield 26%).
(CH3, OCH3), 61.1 (CH2, CH2eN), 68.0 (CH2, C-3 dotsd), 73.9 (CH, C-2 mp: 137e139  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.61e1.87 (4H,
dotsd), 107.9 (C, C-5 dotsd), 110.9 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), m, CH2-6, CH2-10 doasd), 2.66e3.15 (14H, m, CH2-7, CH2-9 doasd,
120.7 (CH, C-6 Phe), 122.7 (CH, C-4 Phe), 140.9 (C, C-1 Phe), 152.0 (C, CH2N, 4 CH2 piperaz), 3.67 (1H, dd, J ¼ 7.2, 8.1 Hz, CHa-3 doasd),
C-2 Phe). 3.78 (3H, s, OCH3), 4.02e4.15 (3H, m, CHb-3 doasd, bn-CH2N),
The free amine (0.10 g, 0.27 mmol) was then converted into the 4.41e4.50 (1H, m, CH-2 doasd), 6.83e6.99 (4H, m, Phe), 7.37e7.51
corresponding hydrogenoxalate from diethyl ether (0.12 g, (5H, m, bn); HR-ESI-MS m/z (pos): 438.2752 C26H36N3O3: (calcd.
0.26 mmol, yield 98%). 438.2751); Anal. Calcd. for C30H39N3O11: C, 58.34; H, 6.36; N, 6.80;
mp: 145e147  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.79e1.97 (4H, Found C, 58.42; H, 6.45; N, 6.89.
m, CH2-6, CH2-10 dotsd), 2.60e2.72 (4H, m, CH2-7, CH2-9 dotsd),
2.82e3.10 (10H, m, 4 CH2 piperaz., CH2N), 3.63 (1H, dd, J ¼ 7.8, 3.2.11. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-1,4- dioxa-
8.2 Hz, CHa-3 dotsd), 3.78 (3H, s, OCH3), 4.13 (1H, dd, J ¼ 6.3, 8.2 Hz, 8l4-thiaspiro[4.5]decan-8-one (10)
CHb-3 dotsd), 4.39e4.46 (1H, m, CH-2 dotsd), 6.84e6.95 (4H, m, The compound (unassigned diasteromeric mixture 40/60), was
Phe); HR-ESI-MS m/z (pos): 365.1951 C19H29N2O3S: (calcd. obtained as a brown oil from compound 5 following the general
365.1893); Anal. Calcd. for C21H30N2O7S: C, 55.49; H, 6.65; N, 6.16; procedure B (0.14 g, 0.36 mmol, yield 36%).
1
Found C, 55.67; H, 6.73; N, 6.31. H NMR (CDCl3, 400 MHz): d ¼ 1.71e1.92 (2H, m, CHa-6, CHa-10
dotsdo), 2.42e2.91 (10H, m, CHb-6, CHb-10, CHa-7, CHa-9 dotsdo,
3.2.9. 1-(2-Methoxyphenyl)-4-{1,4,8-trioxaspiro[4.5]decan-2- CH2N, CH2-2, CH2-6 piperaz.), 3.05e3.27 (6H, m, CHb-7, CHb-9
ylmethyl}piperazine (8) dotsdo, CH2-3, CH2-5 piperaz.), 3.67 (0.6H, dd, J ¼ 7.8, 7.9 Hz, CHa-3
The compound, was obtained as a yellow oil from compound 3 dotsdo), 3.74 (0.4H, dd, J ¼ 7.8, 8.2 Hz, CHa-3 dotsdo), 3.87 (3H, s,
following the general procedure B (0.44 g, 1.26 mmol, yield 80%). OCH3), 4.14e27 (1H, m, CHb-3 dotsdo), 4.33e4.40 (0.4H, m, CH-2
1
H NMR (CDCl3, 400 MHz): d ¼ 1.61e1.78 (4H, m, CH2-6, CH2-10 dotsdo), 4.41e4.52 (0.6H, m, CH-2 dotsdo), 6.89 (1H, d, J ¼ 7.9 Hz,
tosd), 2.52e2.89 (6H, m, CH2N CH2-2, CH2-6 piperaz.), 3.01e3.15 CH-3 Phe), 6.93e7.01 (2H, m, CH-5, CH-6 Phe), 7.03 (1H, ddd,
(4H, m, CH2-3, CH2-5 piperaz.), 3.62 (1H, dd, J ¼ 4.5, 8.1 Hz, CHa-3 J ¼ 2.0, 7.9, 8.1 Hz, CH-4 Phe); 13C NMR (CDCl3, 400 MHz): d ¼ 26.5
tosd), 3.73e3.90 (4H, m, CH2-7, CH2-9 tosd), 3.94 (3H, s, OCH3), 4.14 (CH2, C-6/C-10 dotsdo), 27.1 (CH2, C-6/C-10 dotsdo), 45.6 (CH2, C-7/
(1H, dd, J ¼ 6.1, 8.1 Hz, CHa-3 tosd), 4.31e4.45 (1H, m, CH-2 tosd), C-9 dotsdo), 45.7 (CH2, C-7/C-9 dotsdo), 50.5 (2 CH2, C-3, C-5
6.89 (1H, d, J ¼ 7.8 Hz, CH-3 Phe), 6.94e7.02 (2H, m, CH-5, CH-6 piperaz.), 54.0 (2 CH2, C-2, C-6 piperaz.), 55.3 (CH3, OCH3), 60.7
Phe), 7.03 (1H, ddd, J ¼ 2.0, 7.9, 8.1 Hz, CH-4 Phe); 13C NMR (CDCl3, (CH2, CH2eN), 68.4 (CH2, C-3 dotsdo), 74.6 (CH, C-2 dotsdo), 106.6
400 MHz): d ¼ 35.8 (CH2, C-6/C-10 tosd), 37.0 (CH2, C-6/C-10 tosd), (C, C-5 dotsdd), 110.9 (CH, C-3 Phe), 117.8 (CH, C-5 Phe), 120.7 (CH,
50.2 (2 CH2, C-3, C-5 piperaz.), 53.9 (2 CH2, C-2, C-6 piperaz.), 55.1 C-6 Phe), 122.8 (CH, C-4 Phe), 140.9 (C, C-1 Phe), 152.1 (C, C-2 Phe).
(CH3, OCH3), 61.2 (CH2, CH2eN), 65.6 (CH2, C-7/C-9 tosd), 65.7 (CH2, The free amine (0.03 g, 0.08 mmol) was then converted into the
C-7/C-9 tosd), 67.9 (CH2, C-3 tosd), 73.7 (CH, C-2 tosd), 106.7 (C, C-5 corresponding hydrogenoxalate from diethyl ether (0.01 g,
tosd), 110.9 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 0.02 mmol, yield 28%).
122.7 (CH, C-4 Phe), 140.9 (C, C-1 Phe), 152.0 (C, C-2 Phe). mp: 113e114  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.71e1.82 (2H,
The free amine (0.44 g, 1.26 mmol) was then converted into the m, CHa-6, CHa-10 dotsdo), 2.10e2.21 (2H, m, CHb-6, CHb-10
corresponding hydrogenoxalate from diethyl ether (0.150 g, dotsdo), 2.73e3.21 (14H, m, CH2-7, CH2-9 dotsdo, CH2N, 4 CH2
0.35 mmol, yield 26%). piperaz.), 3.61e3.78 (4H, m, CHa-3 dotsdo, OCH3), 4.15 (1H, dd,
mp: 165e167  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.51e1.69 (4H, J ¼ 6.1, 8.2 Hz, CHb-3 dotso), 4.37e4.53 (1H, m, CH-2 dotsdo),
m, CH2-6, CH2-10 tosd), 2.83e3.15 (10H, m, CH2N, 4 CH2 piperaz.), 6.83e7.02 (4H, m, Phe); HR-ESI-MS m/z (pos): 381.1891
3.49e3.71 (5H, m, CHa-3 tosd, CH2-7, CH2-9 tosd), 3.78 (3H, s, C19H29N2O4S: (calcd. 381.1843); Anal. Calcd for C21H30N2O8S: C,
OCH3), 4.10 (1H, dd, J ¼ 6.4, 8.3 Hz, CHb-3 tosd), 4.37e4.51 (1H, m, 53.60; H, 6.43; N, 5.95; Found C, 53.52; H, 6.31; N, 5.62.
CH-2 tosd), 6.84e7.02 (4H, m, Phe); HR-ESI-MS m/z (pos): 349.2118
C19H29N2O4: (calcd. 349.2122); Anal. Calcd. for C21H30N2O8: C, 3.2.12. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-1,4- dioxa-
57.52; H, 6.90; N, 6.39; found C, 57.63; H, 6.95; N, 6.72. 8l6-thiaspiro[4.5]decane-8,8-dione (11)
The compound was obtained as a brown oil from compound 6
3.2.10. 8-Benzyl-2-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}- following the general procedure B (0.32 g, 0.83 mmol, yield 66%).
1
1,4-dioxa-8-azaspiro[4.5]decane (9) H NMR (CDCl3, 400 MHz): d ¼ 2.05e2.30 (4H, m, CH2-6, CH2-10
The compound, was obtained as a brown oil from compound 4 dotsdd), 2.54e2.96 (6H, m, CH2-2, CH2-6 piperaz., CH2N), 2.98e3.19
following the general procedure B (5.60 g, 12.8 mmol, yield 69%). (8H, m, CH2-7, CH2-9 dotsdd, CH2-3, CH2-5 piperaz.), 3.69 (1H, dd,
1
H NMR (CDCl3, 400 MHz): d ¼ 1.72e1.85 (4H, m, CH2-6, CH2-10 J ¼ 7.8, 8.1 Hz, CHa-3 dotsdd), 3.83 (3H, s, OCH3), 4.15 (1H, dd,
doasd), 2.45e2.91 (10H, m, CH2-7, CH2-9 doasd, CH2N, CH2-2, CH2-6 J ¼ 6.0, 8.1 Hz, CH-3 dotsdd), 4.31e4.44 (1H, m, CH-2 dotsdd),
piperaz.), 3.07e3.21 (4H, m, CH2-3, CH2-5 piperaz.), 3.55 (2H, s, bn- 6.86e7.09 (4H, m, Phe); 13C NMR (CDCl3, 400 MHz): d ¼ 32.5 (CH2,
CH2N), 3.65 (1H, dd, J ¼ 7.7, 8.1 Hz, CHa-3 doasd), 3.90 (3H, s, OCH3), C-6/C-10 dotsdd), 33.8 (CH2, C-6/C-10 dotsdd), 48.6 (CH2, C-7/C-9
4.13 (1H, dd, J ¼ 6.1, 8.1 Hz, CHb-3 doasd), 4.31e4.40 (1H, m, CH-2), dotsdd), 48.8 (CH2, C-7/C-9 dotsdd), 50.2 (2 CH2, C-3, C-5 piperaz.),
6.87e7.07 (4H, m, Phe), 7.31e7.52 (5H, m, Bz); 13C NMR (CDCl3, 53.9 (2 CH2, C-2, C-6 piperaz.), 55.1 (CH3, OCH3), 60.6 (CH2, CH2eN),
400 MHz): d ¼ 34.3 (CH2, C-6/C-10 doasd), 35.5 (CH2, C-6/C-10 68.4 (CH2, C-3 dotsdd), 74.8 (CH, C-2 dotsdd), 105.7 (C, C-5 dotsdd),
S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266 259

1
110.9 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 122.8 H NMR (CDCl3, 400 MHz): d ¼ 1.44e1.70 (4H, m, CH2-6, CH2-10
(CH, C-4 Phe), 140.8 (C, C-1 Phe), 152.0 (C, C-2 Phe). doasd), 2.54e2.86 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.),
The free amine (0.32 g, 0.83 mmol) was then converted into the 3.02e3.16 (4H, m, CH2-7, CH2-9 doasd), 3.22e3.36 (4H, m, 2 CH2-3,
corresponding hydrogenoxalate from diethyl ether (0.29 g, CH2-5 piperaz.), 3.69 (1H, dd, J ¼ 6.9, 7.5 Hz, CHa-3 doasd), 3.87 (3H,
0.59 mmol, yield 71%). s, OCH3), 4.13 (1H, dd, J ¼ 7.5, 8.5 Hz, CHb-3 doasd), 4.38e4.50 (1H,
mp: 172e174  C; 1H NMR (DMSO, 200 MHz): d ¼ 2.05e2.19 (4H, m, CH-2 doasd), 6.81e7.06 (7H, m, Phe, CH-2, CH-6, CH-4 arom.),
m, CH2-6, CH2-10 dotsdd), 2.83e3.87 (14H, m, CH2-7, CH2-9 dotsdd, 7.26 (2H, dd, J ¼ 7.3, 8.5 Hz, CH-3, CH-5 arom.); 13C NMR (CDCl3,
CH2N, 4 CH2 piperaz.), 3.61e3.87 (4H, m, CHa-3 dotsdd, OCH3), 4.20 400 MHz): d ¼ 34.4 (CH2, C-6/C-10 doasd), 35.1 (CH2, C-6/C-10
(1H, dd, J ¼ 6.4, 8.2 Hz, CHb-3 dotsdd), 4.41e4.53 (1H, m, CH-2 doasd), 43.1 (2 CH2, C-7, C-9 doasd.), 50.4 (2 CH2, C-3, C-5 piperaz.),
dotsdd), 6.83e7.09 (4H, m, Phe); HR-ESI-MS m/z (pos): 397.1812 54.2 (2 CH2, C-2, C-6 piperaz.), 55.2 (CH3, OCH3), 61.3 (CH2, CH2eN),
C19H29N2O5S: (calcd. 397.1792); Anal. Calcd. for C21H30N2O9S: C, 68.1 (CH2, C-3 doasd), 74.0 (CH, C-2 doasd), 107.9 (C, C-5 doasd),
51.84; H, 6.21; N, 5.76; Found C, 51.97; H, 6.43; N, 5.96. 110.9 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 122.7
(CH, C-4 Phe), 126.5 (2 CH, C-2, C-6 arom.), 128.3 (CH, C-4 arom),
3.2.13. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-1,4-dioxa- 128.9 (2 CH, C-3, C-5 arom.), 140.9 (C, C-1 Phe), 141.2 (C, C-1 arom.),
8-azaspiro[4.5]decane (12) 152 (C, C-2 Phe).
A mixture of 8 (5.20 g, 12.00 mmol) and 5% PdeC (1.0 g) in EtOH The free amine (0.073 g, 0.17 mmol) was then converted into the
(20 mL) was stirred at 60  C under hydrogen atmosphere until the corresponding hydrogenoxalate from diethyl ether (0.02 g,
absorption of hydrogen ceased (8 h) (the hydrogenation was carried 0.05 mmol, yield 28%).
out on an atmospheric pressure hydrogenator). After the PdeC mp: 130e131  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.64e1.97 (4H,
catalyst was filtered off, the solvent was removed by rotary evap- m, CH2-6, CH2-10 doasd), 2.86e3.45 (14H, m, CH2-7, CH2-9 doasd,
oration to yield compound 12 as a yellow oil (4.0 g, 11.75 mmol, CH2eN, 4 CH2 piperaz.), 3.61e3.89 (4H, m, OCH3, CHa-3 doasd),
yield 98%). 4.07e4.16 (1H, m, CHb-3 doasd), 4.43e4.56 (1H, m, CH-2 doasd),
1
H NMR (CDCl3, 400 MHz): d ¼ 1.51e1.72 (4H, m, CH2-6, CH2-10 6.71e6.98 (7H, m, Phe, CH-2, CH-6, CH-4 arom.), 7.17 (2H, dd,
doasd), 2.57e2.81 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.), J ¼ 7.3, 8.4 Hz, CH-3, CH-5 arom.); HR-ESI-MS m/z (pos): 424.2561
2.88e3.02 (4H, m, CH2-7, CH2-9 doasd), 3.05e3.19 (4H, m, CH2-3, C25H34N3O3: (calcd. 424.2595); Anal. Calcd. for C27H35N3O7: C,
CH2-5 piperaz.), 3.65 (1H, dd, J ¼ 7.2, 8.1 Hz, CHa-3 doasd), 3.88 (3H, 63.14; H, 6.87; N, 8.18; found C, 63.25; H, 6.94; N, 8.35.
s, OCH3), 4.12 (1H, dd, J ¼ 6.2, 8.1 Hz, CHb-3 doasd), 4.21 (1H, s
broad, NH), 4.31e4.45 (1H, m, CH-2 doasd), 6.90 (1H, d, J ¼ 7.9 Hz, 3.2.16. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-8-
CH-3 Phe), 6.93e7.01 (2H, m, CH-5, CH-6 Phe), 7.03 (1H, ddd, J ¼ 2.1, (pyridin-2-yl)-1,4-dioxa-8-azaspiro[4.5]decane (14)
7.9, 8.1 Hz, CH-4 Phe); The compound, was obtained as a dark oil from 2-
13
C NMR (CDCl3, 400 MHz): d ¼ 35.3 (CH2, C-6/C-10 doasd), 35.5 chloropyridine following the general procedure C (0.15 g,
(CH2, C-6/C-10 doasd), 42.3 (CH2, C-7/C-9 doasd), 44.8 (CH2, C-7/C- 0.35 mmol, yield 70%).
1
9 doasd), 50.4 (2 CH2, C-3, C-5 piperaz.), 54.2 (2 CH2, C-2, C-6 H NMR (CDCl3, 400 MHz): d ¼ 1.71e1.90 (4H, m, CH2-6, CH2-10
piperaz.), 55.2 (CH3, OCH3), 61.2 (CH2, CH2eN), 68.2 (CH2, C-3 doasd), 2.54e2.91 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.),
doasd), 74.5 (CH, C-2 doasd), 107.9 (C, C-5 doasd), 111.0 (CH, C-3 3.06e3.21 (4H, m, CH2-3, CH2-5 piperaz.), 3.58e3.71 (5H, m, CHa-3,
Phe), 117.8 (CH, C-5 Phe), 120.6 (CH, C-6 Phe), 122.8 (CH, C-4 Phe), CH2-7, CH2-9 doasd), 3.85 (3H, s, OCH3), 4.15 (1H, dd, J ¼ 6.2, 8.1 Hz,
141.1 (C, C-1 Phe), 152.1 (C, C-2 Phe). CHb-3 doasd), 4.32e4.44 (1H, m, CH-2 doasd), 6.58 (1H, dd, J ¼ 5.0,
The free amine (0.20 g, 0.58 mmol) was then converted into the 6.4 Hz, CH-5 Pyr.) 6.68 (1H, d, J ¼ 8.6 Hz, CH-3 Pyr.), 6.84e7.05 (4H,
corresponding hydrogenoxalate from diethyl ether (0.13 g, m, Phe) 7.46 (1H, ddd, J ¼ 1.2, 6.4, 8.6 Hz, CH-4 Pyr.), 8.18 (1H, dd,
0.26 mmol, yield 44%). J ¼ 1.2, 5.0 Hz, CH-6 Pyr.); 13C NMR (CDCl3, 400 MHz): d ¼ 34.1 (CH2,
mp: 149e150  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.75e2.02 (4H, C-6/C-10 doasd), 35.3 (CH2, C-6/C-10 doasd), 43.2 (2 CH2, C-7, C-9
m, CH2-6, CH2-10 doasd), 2.72e3.31 (14H, m, CH2-7, CH2-9 doasd, doasd.), 50.2 (2 CH2, C-3, C-5 piperaz.), 53.8 (2 CH2, C-2, C-6
CH2eN, 4 CH2 piperaz.), 3.63 (1H, dd, J ¼ 7.1, 7.7 Hz, CHa-3 doasd), piperaz.), 55.1 (CH3, OCH3), 61.2 (CH2, CH2eN), 68.0 (CH2, C-3
3.85 (3H, s, OCH3), 4.13 (1H, dd, J ¼ 7.4, 7.7 Hz, CHb-3 doasd), doasd), 73.9 (CH, C-2 doasd), 106.8 (CH, C-3 Pyr), 108.2 (C, C-5
4.42e4.59 (1H, m, CH-2 doasd), 4.82 (2H, s broad, NHþ 2 ), 6.81e6.99 doasd),110.9 (CH, C-3 Phe), 112.5 (CH, C-5 Pyr.), 117.9 (CH, C-5 Phe),
(4H, m, Phe); HR-ESI-MS m/z (pos): 348.2285 C19H30N3O3: (calcd. 120.7 (CH, C-6 Phe), 122.7 (CH, C-4 Phe), 137. 1 (CH, C-4 Pyr.), 140.9
348.2282); Anal. Calcd. for C23H33N3O11: C, 52.37; H, 6.31; N, 7.97; (C, C-1 Phe), 147.2 (CH, C-6 Pyr.), 152 (C, C-2 Phe), 158.7 (C, C-2 Pyr).
Found C, 52.45; H, 6.56; N, 7.83. The free amine (0.15 g, 0.35 mmol) was then converted into the
corresponding hydrogenoxalate from diethyl ether (0.12 g,
3.2.14. General procedure C 0.24 mmol, yield 69%).
Under an atmosphere of nitrogen compound 12, NaOtBu mp: 81e83  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.66e1.90 (4H, m,
(1.4 mmol), and Pd2(dba)3 (0.05 mmol) were solubilized in toluene; CH2-6, CH2-10 doasd), 3.01e3.42 (10H, m, 4 CH2 piperaz., CH2N),
subsequently the selected aryl halide (1.0 mmol) and 2,8,9- 3.55e3.81 (5H, m, CHa-3, CH2-7, CH2-9 doasd) 3.88 (3H, s, OCH3),
triisobutyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane 4.16 (1H, dd, J ¼ 6.4, 8.3 Hz, CHb-3 doasd), 4.48e4.61 (1H, m, CH-2
(0.01 mmol) were added. The reaction mixture was refluxed for doasd), 6.60 (1H, dd, J ¼ 5.2, 6.3 Hz, CH-5 Pyr.), 6.81e7.03 (5H, m,
20 h and was then allowed to cool to room temperature. The CH-3 Pyr., Phe), 7.54 (1H, ddd, J ¼ 1.6, 8.1, 8.6 Hz, CH-4 Pyr.),
mixture was diluted with water then extracted with diethyl ether. 8.03e8.14 (1H, m, CH-6 Pyr.); HR-ESI-MS m/z (pos): 425.2521
The extracts were combined, washed with saturated saline solu- C24H33N4O3: (calcd. 425.2547); Anal. Calcd. for C26H34N4O7: C,
tion, and then dried over MgSO4. The solvent was removed in 60.69; H, 6.66; N, 10.98; Found C, 60.51; H, 6.23; N, 10.65.
vacuum and residue was purified by flash chromatography.
3.2.17. General procedure D
3.2.15. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-8-phenyl- To a solution of compound 11 (1.0 mmol) in CHCl3 was added
1,4-dioxa-8-azaspiro[4.5]decane (13) poly(4-vinylpyridine (0.34 g, 3.0 mmol of functional group) for
The compound, was obtained as a dark oil from chlorobenzene carbonyl chlorides or piperidino methyl(polystyrene) (0.85 g mmol,
following the general procedure C (0.08 g, 0.19 mmol, yield 21%). 3.0 mmol of functional group) for sulfonyl chlorides. Subsequently,
260 S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266

the appropriate acyl chloride or sulfonyl chloride (1.8 mmol) was The free amine (0.09 g, 0.17 mmol) was then converted into the
added to the reaction mixture. The reaction was stirred at 400 rpm corresponding hydrogenoxalate from diethyl ether (0.09 g,
for 12 h at room temperature After completion of the reaction, the 0.14 mmol, yield 84%).
polymer was filtered, washed successively with CH2Cl2 (2  10 mL) mp: 165e167  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.61e1.91 (4H,
and MeOH (1  10 mL) and dried in vacuo at 20  C for 4 h. The m, CH2-6, CH2-10 doasd), 3.01e3.52 (12H, m, 4 CH2 piperaz., CH2N,
residue was then solubilized in CH2Cl2 and added of benzensulfonic CHa-7, CHa-9, CHa-3 doasd), 3.54e3.97 (6H, m, OCH3, CHa-3, CHb-
resin (1.1 g, 3.0 mmol of functional group). The mixture was stirred 7, CHb-9 doasd), 4.16 (1H, dd, J ¼ 6.1, 7.6 Hz, CHb-3 doasd),
for 12 h at room temperature. Subsequently the resin was filtered, 4.43e4.57 (1H, m, CH-2 doasd), 6.87e7.08 (4H, m, Phe), 7.63 (2H, d,
washed with CH2Cl2 (2 x 10 mL) and treated with NH3/CH3OH 1 N J ¼ 7.8 Hz, CH-3, CH-5 Bzo), 7.80 (2H, d, J ¼ 7.8 Hz, CH-2, CH-6 Bzo);
solution. Evaporation of the solvent afforded the pure desired HR-ESI-MS m/z (pos): 520.2403C27H33F3N3O4: (calcd. 520.2418);
compound. Anal. Calcd. for C29H34F3N3O8: C, 57.14; H, 5.62; N, 6.89; Found C,
57.36; H, 5.87; N, 7.02.
3.2.18. 8-Benzoyl-2-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}-
3.2.20. 8-(Furan-2-carbonyl)-2-{[4-(2-methoxyphenyl)piperazin-
1,4-dioxa-8-azaspiro[4.5]decane (15)
1-yl]methyl}-1,4-dioxa-8-azaspiro[4.5]decane (17)
The compound, was obtained as a dark oil from benzoyl chloride
The compound, was obtained as a yellow oil from furan-2-
following the general procedure D (0.08 g, 0.18 mmol, yield 32%).
1 carbonyl chloride following the general procedure D (0.11 g,
H NMR (CDCl3, 400 MHz): d ¼ 1.65e1.90 (4H, m, CH2-6, CH2-10
0.21 mmol, yield 37%).
doasd), 2.51e2.98 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.), 1
H NMR (CDCl3, 400 MHz): d ¼ d 1.68e1.99 (4H, m, CH2-6, CH2-
3.01e3.23 (8H, m, CH2-7, CH2-9 doasd, CH2-3, CH2-5 piperaz.), 3.66
10 doasd), 2.61e2.92 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.),
(1H, dd, J ¼ 7.4, 8.1 Hz, CHa-3 doasd), 3.86 (3H, s, OCH3), 4.19 (1H,
3.03e3.35 (4H, m, CH2-3, CH2-5 piperaz.), 3.73 (1H, dd, J ¼ 7.5,
dd, J ¼ 6.3, 7.4 Hz, CHb-3 doasd), 4.52e4.71 (1H, m, CH-2 doasd),
7.8 Hz, CHa-3 doasd), 3.78e3.91 (4H, m, CH2-7, CH2-9 doasd), 3.92
6.81e7.03 (4H, m, Phe), 7.37e7.49 (5H, m, Bzo), 13C NMR (CDCl3,
(3H, s, OCH3), 4.14 (1H, dd, J ¼ 6.5, 7.8 Hz, CHb-3 doasd), 4.49e4.79
400 MHz): d ¼ 34.9 (CH2, C-6/C-10 doasd), 35.5 (CH2, C-6/C-10
(1H, m, CH-2 doasd), 6.52 (1H, dd, J ¼ 1.8, 3.6 Hz, CH-4 Fur),
doasd), 40.4 (CH2, C-7/C-9 doasd), 45.3 (CH2, C-7/C-9 doasd), 49.3
6.81e7.02 (5H, m, Phe, CH-3 Fur), 7.52 (1H, dd, J ¼ 0.8, 1.8 Hz, CH-5
(2 CH2, C-3, C-5 piperaz.), 53.8 (2 CH2, C-2, C-6 piperaz.), 55.1 (CH3,
Fur); NMR (CDCl3, 400 MHz): d ¼ 35.6 (CH2, C-6/C-10 doasd), 35.9
OCH3), 60.8 (CH2, CH2eN), 68.1 (CH2, C-3 doasd), 73.9 (CH, C-2
(CH2, C-6/C-10 doasd), 40.2 (CH2, C-7/C-9 doasd), 45.3 (CH2, C-7/C-
doasd), 107.6 (C, C-5 doasd), 110.9 (CH, C-3 Phe), 118.0 (CH, C-5 Phe),
9 doasd), 50.0 (2 CH2, C-3, C-5 piperaz.), 53.9 (2 CH2, C-2, C-6
120.7 (CH, C-6 Phe), 122.9 (CH, C-4 Phe), 126.7 (2 CH, C-3, C-5 Bzo),
piperaz.), 55.2 (CH3, OCH3), 61.1 (CH2, CH2eN), 68.1 (CH2, C-3
128.6 (2 CH, C-2, C-6 Bzo), 130.0 (CH, C-4 Bzo), 136.0 (C, C-1 Bzo),
doasd), 73.9 (CH, C-2 doasd), 108.1 (C, C-5 doasd), 110.8 (CH, C-4
140.9 (C, C-1 Phe), 152.0 (C, C-2 Phe), 169.9 (C, CO).
Fur.), 111.0 (CH, C-3 Phe), 115.8 (CH, C-3 Fur), 118.0 (CH, C-5 Phe),
The free amine (0.08 g, 0.18 mmol) was then converted into the
120.8 (CH, C-6 Phe), 122.9 (CH, C-4 Phe), 140.9 (C, C-1 Phe), 143.6
corresponding hydrogenoxalate from diethyl ether (0.05 g,
(CH, C-5 Fur), 148.1 (C, C-2 Fur), 152.0 (C, C-2 Phe), 169.5 (C, CO).
0.1 mmol, yield 55%).
The free amine (0.11 g, 0.21 mmol) was then converted into the
mp: 162e164  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.51e1.71 (4H,
corresponding hydrogenoxalate from diethyl ether (0.02 g,
m, CH2-6, CH2-10 doasd), 2.90e3.78 (15H, m, 4 CH2 piperaz., CH2N,
0.03 mmol, yield 13%).
CH2-7, CH2-9, CHa-3 doasd), 3.80 (3H, s, OCH3), 4.15 (1H, dd, J ¼ 6.2,
mp: 156e157  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.52e1.82 (4H,
7.2 Hz, CHb-3 doasd), 4.45e4.52 (1H, m, CH-2 doasd), 6.82e703
m, CH2-6, CH2-10 doasd), 2.79e3.18 (10H, m, CH2eN, 4 CH2
(4H, m, Phe), 7.25e7.51 (5H, m, Bzo); HR-ESI-MS m/z (pos):
piperaz.), 3.51e3.77 (5H, m, CHa-3, CH2-7, CH2-9 doasd), 3.78 (3H, s,
452.2531C26H34N3O4: (calcd. 452.2544); Anal. Calcd. for
OCH3), 4.48 (1H, dd, J ¼ 6.2, 8.1 Hz, CHb-3 doasd), 6.61 (1H, s broad,
C28H35N3O8: C, 62.09; H, 6.51; N, 7.76; Found C, 61.91; H, 6.41; N,
CH-4 Fur), 6.83e7.02 (5H, m, Phe, CH-3 Fur), 7.82 (1H, s, CH-5 Fur);
7.53.
HR-ESI-MS m/z (pos): 442.2325C24H32N3O5: (calcd. 442.2336);
Anal. Calcd. for C26H33N3O9: C, 58.75; H, 6.27; N, 7.91; Found C,
3.2.19. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-8-[4- 58.96; H, 6.50; N, 8.13.
(trifluoromethyl)benzoyl]-1,4-dioxa-8-azaspiro[4.5]decane (16)
The compound, was obtained as a yellow oil from 4-(tri- 3.2.21. 8-Cyclohexanecarbonyl-2-{[4-(2-methoxyphenyl)piperazin-
fluoromethyl)benzoyl chloride following the general procedure D 1-yl]methyl}-1,4-dioxa-8-azaspiro[4.5]decane (18)
(0.09 g, 0.17 mmol, yield 31%). The compound, was obtained as a yellow oil from cyclo-
1
H NMR (CDCl3, 400 MHz): d ¼ 1.52e2.08 (4H, m, CH2-6, CH2-10 hexanecarbonyl chloride following the general procedure D (0.09 g,
doasd), 2.52e3.04 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.), 0.20 mmol, yield 35%).
1
3.03e3.31 (4H, m, CH2-3, CH2-5 piperaz.), 3.39e3.63 (2H, m, CH2-7/ H NMR (CDCl3, 400 MHz): d ¼ 1.23e1.93 (m, 14H, CH2-6, CH2-10
CH2-9 doasd), 3.67 (1H, dd, J ¼ 7.2, 8.1 Hz,CHa-3 doasd), 3.87 (3H, s, doasd, CH2-2, CH2-3, CH2-4, CH2-5, CH2-6 Cyc), 2.42e2.59 (1H, m,
OCH3), 3.89e3.95 (2H, m, CH2-7/CH2-9 doasd), 4.16 (1H, dd, J ¼ 6.1, CH-1 Cyc), 2.73e3.24 (10H, m, CH2eN, 4 CH2 piperaz.), 3.47e3.68
7.2 Hz, CHb-3 doasd), 4.38e4.51 (1H, m, CH-2 doasd), 6.89 (1H, d, (5H, m, CHa-3, CH2-7, CH2-9 doasd), 3.84 (3H, s, OCH3), 4.11e4.20
J ¼ 7.9 Hz, CH-3 Phe), 6.89e6.98 (2H, m, CH-5, CH-6 Phe), 7.01e7.11 (1H, m, CHb-3 doasd), 4.51e4.62 (1H, m, CH-2 doasd), 6.89 (1H, d,
(1H, m, CH-4 Phe), 7.55 (2H, d, J ¼ 7.8 Hz, CH-2, CH-6 Bzo), 7.67 (2H, J ¼ 7.8 Hz, CH-3 Phe), 6.89e6.98 (2H, m, CH-5, CH-6 Phe), 7.01e7.11
d, J ¼ 7.8 Hz, CH-3, CH-4 Bzo); 13C NMR (CDCl3, 400 MHz): d ¼ 35.5 (1H, m, CH-4 Phe); 13C NMR (CDCl3, 400 MHz): d ¼ 23.7 (CH2, C-4
(CH2, C-6/C-10 doasd), 35.9 (CH2, C-6/C-10 doasd), 40.1 (CH2, C-7/C- Cyc.) 25.7 (2 CH2, C-3, C-5 Cyc.), 29.4 (2 CH2, C-2, C-6 Cyc.), 34.9
9 doasd), 45.5 (CH2, C-7/C-9 doasd), 50.1 (2 CH2, C-3, C-5 piperaz.), (CH2, C-6/C-10 doasd), 35.5 (CH2, C-6/C-10 doasd), 39.2 (CH2, C-7/C-
54.2 (2 CH2, C-2, C-6 piperaz.), 55.1 (CH3, OCH3), 61.1 (CH2, CH2eN), 9 doasd), 40.1 (CH2, C-7/C-9 doasd), 40.3 (CH, C-1 Cyc.), 42.3 (2 CH2,
68.3 (CH2, C-3 doasd), 74.2 (CH, C-2 doasd), 107.6 (C, C-5 doasd), C-3, C-5 piperaz.), 48.9 (2 CH2, C-2, C-6 piperaz.), 55.5 (CH3, OCH3),
111.0 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), 120.8 (CH, C-6 Phe), 122.2 60.9 (CH2, CH2eN), 68.1 (CH2, C-3 doasd), 74.5 (CH, C-2 doasd),
(C, CF3) 122.9 (CH, C-4 Phe), 125.6 (2 CH, C-3, C-5 Bzo), 127.1 (2 CH, 107.9 (C, C-5 doasd), 111.1 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), 120.9
C-2, C-6 Bzo), 131.6 (C, C-1 Bzo), 138.5 (C, C-4 Bzo), 140.9 (C, C-1 (CH, C-6 Phe), 123.0 (CH, C-4 Phe), 140.8 (C, C-1 Phe), 152.0 (C, C-2
Phe), 152.0 (C, C-2 Phe), 169.8 (C, CO). Phe), 174.2 (C, CO).
S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266 261

The free amine (0.09 g, 0.20 mmol) was then converted into the The free amine (0.23 g, 0.58 mmol) was then converted into the
corresponding hydrogenoxalate from diethyl ether (0.03 g, corresponding hydrogenoxalate from diethyl ether (0.242 g,
0.05 mmol, yield 26%). 0.50 mmol, yield 86%).
mp: 159e161  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.37 (m, 14H, mp: 61e63  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.48e1.72 (4H, m,
CH2-6, CH2-10 doasd, CH2-2, CH2-3, CH2-4, CH2-5, CH2-6 Cyc), CH2-6, CH2-10 doasd), 2.00 (3H, s, COCH3), 3.06e3.33 (10H, m,
2.41e2.58 (1H, m, CH-1 Cyc), 2.92e3.52 (10H, m, CH2eN, 4 CH2 CH2eN, 4 CH2 piperaz.), 3.39e3.62 (4H, m, CH2-7, CH2-9 doasd),
piperaz.), 3.43e3.62 (4H, m, CH2-7, CH2-9 doasd), 3.63e3.72 (1H, m, 3.65e3.77 (1H, m, CHa-3 doasd), 3.78 (3H, s, OCH3), 4.13 (1H, dd,
CHa-3 doasd), 3.78 (3H, s, OCH3), 4.02e4.17 (1H, m, CHb-3 doasd), J ¼ 6.1, 8.2 Hz, CHb-3 doasd), 4.49e4.64 (1H, m, CH-2 doasd),
4.32e4.51 (1H, m, CH-2 doasd), 6.73e7.03 (4H, m, Phe); HR-ESI-MS 6.81e7.02 (4H, m, Phe); HR-ESI-MS m/z (pos): 390.2395
m/z (pos): 458.3041 C26H40N3O4: (calcd. 458.3013); Anal. Calcd. for C21H32N3O4: (calcd. 390.2387); Anal. Calcd. for C23H33N3O8: C,
C28H41N3O8: C, 61.41; H, 7.55; N, 7.67; Found C, 61.74; H, 7.72; N, 57.61; H, 6.94; N, 8.76; Found C, 57.33; H, 6.74; N, 8.58.
7.83.
3.2.24. 2,2,2-Trifluoro-1-(2-{[4-(2-methoxyphenyl)piperazin-1-yl]
3.2.22. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-8- methyl}-1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethan-1-one (21)
(pyrrolidine-2-carbonyl)-1,4-dioxa-8-azaspiro[4.5]decane (19) The compound, was obtained as a yellow oil from 2,2,2-
The compound, was obtained as a yellow oil from pyrrolidine-2- trifluoroacetyl chloride following the general procedure D (0.03 g,
carbonyl chloride following the general procedure D (0.110 g, 0.08 mmol, yield 4%).
1
0.25 mmol, yield 43%). H NMR (CDCl3, 400 MHz): d ¼ 1.63e1.89 (4H, m, CH2-6, CH2-10
1
H NMR (CDCl3, 400 MHz): d ¼ 1.67e1.93 (8H, m, CH2-6, CH2-10 doasd), 2.59e2.88 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.),
doasd, CH2-3, CH2-4 Pyrro.), 2.07 (1H, s broad, NH), 2.56e2.84 (6H, 3.22e3.41 (4H, m, CH2-3, CH2-5 piperaz.), 3.52e3.90 (5H, m, CHa-3,
m, CH2eN, CH2-2, CH2-6 piperaz.), 3.02e3.18 (m, 4H, CH2-3, CH2-5 CH2-7, CH2-9 doasd), 3.92 (3H, s, OCH3), 4.14 (1H, dd, J ¼ 6.1, 7.9 Hz,
piperaz.), 3.25e3.50 (7H, m, CH2-7, CH2-9 doasd, CH-2, CH2-5 CHb-3 doasd), 4.31e4.48 (1H, m, CH-2 doasd), 6.88e7.01 (4H, m,
Pyrro), 3.66 (1H, dd, J ¼ 7.4, 8.0 Hz, CHa-3 doasd), (3H, s, OCH3), 4.13 Phe); 13C NMR (CDCl3, 400 MHz): d ¼ 36.4 (CH2, C-6/C-10 doasd),
(1H, dd, J ¼ 6.4, 10.0 Hz, CHb-3 doasd), 4.31e4.43 (1H, m, CH-2 38.4 (CH2, C-6/C-10 doasd), 41.4 (CH2, C-7/C-9 doasd), 43.3 (CH2, C-
doasd) 6.87 (1H, d, J ¼ 7.9 Hz, CH-3 Phe), 6.89e6.98 (2H, m, CH-5, 7/C-9 doasd), 49.1 (2 CH2, C-3, C-5 piperaz.), 53.8 (2 CH2, C-2, C-6
CH-6 Phe), 7.01 (1H, dd, J ¼ 7.9, 8.1 Hz, CH-4 Phe); 13C NMR piperaz.), 55.5 (CH3, OCH3), 60.8 (CH2, CH2eN), 68.0 (CH2, C-3
(CDCl3, 400 MHz): d ¼ 26.6 (2 CH2, C-3, C-4 Pyrro.), 34.7 (CH2, C-6/ doasd), 74.3 (CH, C-2 doasd), 106.8 (C, C-5 doasd), 110.9 (CH, C-3
C-10 doasd), 36.0 (CH2, C-6/C-10 doasd), 43.6 (CH2, C-7/C-9 doasd), Phe), 116.7 (C, CF3) 117.9 (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 122.9
43.8 (CH2, C-7/C-9 doasd), 47.3 (CH, C-2 Pyrro.), 48.2 (CH2, C-5 (CH, C-4 Phe), 140.8 (C, C-1 Phe), 151.9 (C, C-2 Phe), 160.5 (C, CO).
Pyrro.), 50.3 (2 CH2, C-3, C-5 piperaz.), 53.8 (2 CH2, C-2, C-6 The free amine (0.035 g, 0.08 mmol) was then converted into the
piperaz.), 55.0 (CH3, OCH3), 61.0 (CH2, CH2eN), 68.0 (CH2, C-3 corresponding hydrogenoxalate from diethyl ether (0.03 g,
doasd), 74.0 (CH, C-2 doasd), 107.9 (C, C-5 dotsdd), 110.9 (CH, C-3 0.05 mmol, yield 64%).
Phe), 117.9 (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 122.7 (CH, C-4 Phe), mp: 139e141  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.49e1.73 (4H,
140.9 (C, C-1 Phe), 152.0 (C, C-2 Phe), 162.4 (C, CO). m, CH2-6, CH2-10 doasd), 2.60e3.11 (6H, m, CH2eN, CH2-2, CH2-6
The free amine (0.110 g, 0.25 mmol) was then converted into the piperaz.), 3.29e3.43 (4H, m, CH2-3, CH2-5 piperaz.), 3.48e3.72 (5H,
corresponding hydrogenoxalate from diethyl ether (0.04 g, m, CHa-3, CH2-7, CH2-9 doasd), 3.86 (3H, s, OCH3), 4.14 (1H, dd,
0.06 mmol, yield 26%). J ¼ 6.3, 7.4 Hz, CHb-3 doasd), 4.36e4.53 (1H, m, CH-2 doasd),
mp: 145e147  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.51e1.93 (8H, 6.83e7.02 (4H, m, Phe); HR-ESI-MS m/z (pos): 444.2127
m, CH2-6, CH2-10 doasd, CH2-3, CH2-4 Pyrro.), 2.81e3.47 (17H, m, C21H29F3N3O4: (calcd. 444.2105); Anal. Calcd. for C23H30F3N3O8: C,
CH2eN, 4 CH2 piperaz., CH2-7, CH2-9 doasd, CH-2, CH2-5 Pyrro.), 51.78; H, 5.67; N, 7.88; Found C, 51.89; H, 5.72; N, 7.95.
3.67e3.80 (4H, m, OCH3, CHa-3 doasd), 4.02e4.17 (1H, m, CHb-3
doasd), 4.41e4.59 (1H, m, CH-2 doasd), 6.83e7.09 (4H, m, Phe),
3.2.25. 8-(Benzenesulfonyl)-2-{[4-(2-methoxyphenyl)piperazin-1-
7.12 (1H, s broad, NHþ); HR-ESI-MS m/z (pos): 445.2832
yl]methyl}-1,4-dioxa-8-azaspiro[4.5]decane (22)
C24H37N4O4: (calcd. 445.2809); Anal. Calcd. for C28H40N4O12: C,
The compound, was obtained as a yellow oil from benzene-
53.84; H, 6.45; N, 8.97; Found C, 53.91; H, 6.53; N, 9.11.
sulfonyl chloride following the general procedure D (0.07 g,
0.14 mmol, yield 25%).
1
3.2.23. 1-(2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-1,4- H NMR (CDCl3, 400 MHz): d ¼ 1.57e1.88 (4H, m, CH2-6, CH2-10
dioxa-8-azaspiro[4.5]decan-8-yl)ethan-1-one (20) doasd), 2.59e2.87 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.),
The compound, was obtained as a yellow oil from acetyl chloride 2.92e3.16 (6H, CH2-3, CH2-5 piperaz., CHa-7, CHa-9 doasd),
following the general procedure D (0.23 g, 0.58 mmol, yield 99%). 3.21e3.33 (2H, m, CHb-7, CHb-9 doasd), 3.58 (1H, dd, J ¼ 7.4, 8.1 Hz,
1
H NMR (CDCl3, 400 MHz): d ¼ 1.64e1.86 (4H, m, CH2-6, CH2-10 CHa-3 doasd), 3.87 (3H, s, OCH3), 4.13 (1H, dd, J ¼ 6.1, 8.1 Hz, CHb-3
doasd), 2.16 (3H, s, COCH3), 2.61e2.90 (6H, m, CH2eN, CH2-2, CH2-6 doasd), 4.22e4.37 (1H, m, CH-2 doasd), 6.82e7.02 (4H, m, Phe),
piperaz.), 3.03e3.23 (4H, m, CH2-3, CH2-5 piperaz.), 3.49e3.74 (5H, 7.46e7.66 (5H, m, Sulf) 13C NMR (CDCl3, 400 MHz): d ¼ 29.5 (CH2, C-
m, CHa-3, CH2-7, CH2-9 doasd), 3.91 (3H, s, OCH3), 4.15 (1H, dd, 6/C-10 doasd), 29.8 (CH2, C-6/C-10 doasd), 44.2 (CH2, C-7/C-9
J ¼ 6.4, 7.9 Hz, CHb-3 doasd), 4.37e4.55 (1H, m, CH-2 doasd), 6.90 doasd), 44.3 (CH2, C-7/C-9 doasd), 50.1 (2 CH2, C-3, C-5 piperaz.),
(1H, d, J ¼ 7.9 Hz, CH-3 Phe), 6.93e7.01 (2H, m, CH-5, CH-6 Phe), 54.0 (2 CH2, C-2, C-6 piperaz.), 55.1 (CH3, OCH3), 61.1 (CH2, CH2eN),
7.03 (1H, ddd, J ¼ 2.4, 7.9, 8.1 Hz, CH-4 Phe); 13C NMR (CDCl3, 67.9 (CH2, C-3 doasd), 74.3 (CH, C-2 doasd), 106.3 (C, C-5 doasd),
400 MHz): d ¼ 21.1 (CH3, COCH3), 34.4 (CH2, C-6/C-10 doasd), 35.6 110.9 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 122.7
(CH2, C-6/C-10 doasd), 39.1 (CH2, C-7/C-9 doasd), 44.0 (CH2, C-7/C-9 (CH, C-4 Phe), 127.3 (2 CH, C-2, C-6 Sulf), 129.5 (2 CH, C-3, C-5 Sulf),
doasd), 50.2 (2 CH2, C-3, C-5 piperaz.), 53.8 (2 CH2, C-2, C-6 133.2 (C, C-1 Tos), 141.0 (C, C-1 Phe), 145.4 (C, C-4 Sulf), 152.0 (C, C-2
piperaz.), 55.1 (CH3, OCH3), 61.0 (CH2, CH2eN), 68.0 (CH2, C-3 Phe).
doasd), 74.3 (CH, C-2 doasd), 107.6 (C, C-5 doasd), 110.9 (CH, C-3 The free amine (0.07 g, 0.14 mmol) was then converted into the
Phe), 118.0 (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 122.9 (CH, C-4 Phe), corresponding hydrogenoxalate from diethyl ether (0.05 g,
140.9 (C, C-1 Phe), 152.0 (C, C-2 Phe), 168.5 (C, CO). 0.09 mmol, yield 62%).
262 S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266

mp: 186e188  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.50e1.78 (4H, 134.7 (C, C-4 Sulf), 139.0 (C, C-1 Sulf), 140.9 (C, C-1 Phe), 152.0 (C, C-
m, CH2-6, CH2-10 doasd), 2.63e3.42 (14H, m, CH2eN, 4 CH2 piperaz, 2 Phe).
CH2-7, CH2-9 doasd), 3.41e3.58 (1H, m, CHa-3 doasd), 3.79 (3H, s, The free amine (0.07 g, 0.13 mmol) was then converted into the
OCH3), 3.91e4.12 (1H, m, CHb-3 doasd), 4.37e4.52 (1H, m, CH-2 corresponding hydrogenoxalate from diethyl ether (0.05 g,
doasd), 6.82e7.06 (4H, m, Phe), 7.52e7.88 (5H, m, Sulf); HR-ESI- 0.08 mmol, yield 59%).
MS m/z (pos): 488.2219C25H34N3O5S: (calcd. 488.2214); Anal. mp: 173e175  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.52e1.79 (4H,
Calcd. for C27H35N3O9S: C, 56.14; H, 6.11; N, 7.27; Found C, 56.23; H, m, CH2-6, CH2-10 doasd), 2.66e3.45 (14H, m, CH2eN, 4 CH2 piperaz,
6.32; N, 7.38. CH2-7, CH2-9 doasd), 3.58 (1H, dd, J ¼ 6.1, 7.9 Hz, CHa-3 doasd), 3.74
(3H, s, OCH3), 4.10 (1H, dd, J ¼ 6.3, 7.9 Hz, CHb-3 doasd), 4.31e4.49
(1H, m, CH-2 doasd) 6.84e7.02 (4H, m, Phe), 7.58 (2H, d, J ¼ 8.6 Hz,
3.2.26. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-8-(4-
CH-3, CH-5 Sulf), 7.67 (2H, d, J ¼ 8.6 Hz, CH-2, CH-6 Sulf); HR-ESI-
methylbenzenesulfonyl)-1,4-dioxa-8-azaspiro[4.5]decane (23)
MS m/z (pos): 522.1845 C25H33ClN3O5S: (calcd. 522.1824); Anal.
The compound, was obtained as a yellow oil from 4-
Calcd. for C27H34ClN3O9S: C, 52.98; H, 5.60; N, 6.87; Found C, 53.09;
methylbenzene-1-sulfonyl chloride following the general proce-
H, 5.51; N, 6.97.
dure D (0.13 g, 0.26 mmol, yield 44%).
1
H NMR (CDCl3, 400 MHz): d ¼ 1.74e1.96 (4H, m, CH2-6, CH2-10
3.2.28. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-8-
doasd), 2.46 (3H, s, CH3), 2.54e2.89 (6H, m, CH2eN, CH2-2, CH2-6
[4(trifluoromethoxy)benzenesulfonyl]-1,4-dioxa-8-azaspiro[4.5]
piperaz.), 2.94e3.20 (6H, CH2-3, CH2-5 piperaz., CHa-7, CHa-9
decane (25)
doasd), 3.22e3.37 (2H, m, CHb-7, CHb-9 doasd), 3.62 (1H, dd,
The compound, was obtained as a yellow oil from 4-(tri-
J ¼ 7.5, 7.9 Hz, CHa-3 doasd), 3.89 (3H, s, OCH3), 4.07 (1H, dd, J ¼ 6.1,
fluoromethoxy)benzene-1-sulfonyl chloride following the general
7.9 Hz, CHb-3 doasd), 4.25e4.38 (1H, m, CH-2 doasd), 6.90 (1H, d,
procedure D (0.11 g, 0.19 mmol, yield 34%).
J ¼ 7.7 Hz, CH-3 Phe), 6.93e7.01 (2H, m, CH-5, CH-6 Phe), 6.99e7.08 1
H NMR (CDCl3, 400 MHz): d ¼ 1.73e1.99 (4H, m, CH2-6, CH2-10
(1H, m, CH-4 Phe), 7.35 (2H, d, J ¼ 8.0 Hz, CH-3, CH-5 Tos), 7.68 (2H,
doasd), 2.54e2.90 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.),
d, J ¼ 8.0 Hz, CH-2, CH-6 Tos); 13C NMR (CDCl3, 400 MHz): d ¼ 21.1
3.01e3.28 (6H, CH2-3, CH2-5 piperaz., CHa-7, CHa-9 doasd),
(CH3, CH3), 29.4 (CH2, C-6/C-10 doasd), 29.9 (CH2, C-6/C-10 doasd),
3.31e3.42 (2H, m, CHb-7, CHb-9 doasd), 3.64 (1H, dd, J ¼ 7.5, 8.0 Hz,
44.1 (CH2, C-7/C-9 doasd), 44.2 (CH2, C-7/C-9 doasd), 50.2 (2 CH2, C-
CHa-3 doasd), 3.91 (3H, s, OCH3), 4.10 (1H, dd, J ¼ 6.2, 8.0 Hz, CHb-3
3, C-5 piperaz.), 53.9 (2 CH2, C-2, C-6 piperaz.), 55.1 (CH3, OCH3),
doasd), 4.30e4.41 (1H, m, CH-2 doasd), 6.89 (1H, d, J ¼ 8.0 Hz, CH-3
61.0 (CH2, CH2eN), 68.0 (CH2, C-3 doasd), 74.0 (CH, C-2 doasd),
Phe), 6.94e7.01 (2H, m, CH-5, CH-6 Phe), 7.03e7.12 (1H, m, CH-4
106.5 (C, C-5 doasd), 110.9 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), 120.7
Phe), 7.39 (2H, d, J ¼ 8.2 Hz, CH-3, CH-5 Sulf), 7.84 (2H, d,
(CH, C-6 Phe), 122.7 (CH, C-4 Phe), 127.3 (2 CH, C-2, C-6 Tos), 129.4
J ¼ 8.3 Hz, CH-2, CH-6 Sulf); 13C NMR (CDCl3, 400 MHz): d ¼ 34.2
(2 CH, C-3, C-5 Tos), 133.3 (C, C-1 Tos), 141.0 (C, C-1 Phe), 143.2 (C, C-
(CH2, C-6/C-10 doasd), 35.4 (CH2, C-6/C-10 doasd), 44.1 (CH2, C-7/C-
4 Tos), 152.0 (C, C-2 Phe).
9 doasd), 44.2 (CH2, C-7/C-9 doasd), 50.0 (2 CH2, C-3, C-5 piperaz.),
The free amine (0.13 g, 0.26 mmol) was then converted into the
53.8 (2 CH2, C-2, C-6 piperaz.), 55.1 (CH3, OCH3), 60.9 (CH2, CH2eN),
corresponding hydrogenoxalate from diethyl ether (0.12 g,
68.0 (CH2, C-3 doasd), 73.9 (CH, C-2 doasd), 106.5 (C, C-5 doasd),
0.20 mmol, yield 75%).
110.9 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), 120.5 (2 CH, C-3, C-5 Sulf)
mp: 182e184  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.52e1.79 (4H,
120.7 (CH, C-6 Phe), 122.7 (CH, C-4 Phe), 127.1 (C, OCF3), 129.4 (2 CH,
m, CH2-6, CH2-10 doasd), 2.35 (3H, s, CH3), 2.66e3.45 (14H, m,
C-2, C-6 Sulf), 134.9 (C, C-4 Sulf), 140.9 (C, C-1 Phe), 151.9 (C, C-4
CH2eN, 4 CH2 piperaz, CH2-7, CH2-9 doasd), 3.58 (1H, dd, J ¼ 6.7,
Sulf), 152.0 (C, C-2 Phe).
8.2 Hz, CHa-3 doasd), 3.74 (3H, s, OCH3), 4.10 (1H, dd, J ¼ 6.5, 8.2 Hz,
The free amine (0.10 g, 0.19 mmol) was then converted into the
CHb-3 doasd), 4.31e4.49 (1H, m, CH-2 doasd), 6.83e7.03 (4H, m,
corresponding hydrogenoxalate from diethyl ether (0.05 g,
Phe), 7.40 (2H, d, J ¼ 8.2 Hz, CH-3, CH-5 Tos), 7.65 (2H, d, J ¼ 8.2 Hz,
0.08 mmol, yield 42%).
CH-2, CH-6 Tos); HR-ESI-MS m/z (pos): 502.2362 C26H36N3O5S:
mp: 174e176  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.62e1.91 (4H,
(calcd. 502.2370); Anal. Calcd. for C28H37N3O9S: C, 56.84; H, 6.30; N,
m, CH2-6, CH2-10 doasd), 2.75e3.38 (14H, m, CH2eN, 4 CH2 piperaz,
7.10; Found C, 56.98; H, 6.54; N, 7.42.
CH2-7, CH2-9 doasd), 3.56e3.72 (1H, m, CHa-3 doasd), 3.80 (3H, s,
OCH3), 4.00e4.18 (1H, m, CHb-3 doasd), 4.31e4.51 (1H, m, CH-2
3.2.27. 8-(4-Chlorobenzenesulfonyl)-2-{[4-(2-methoxyphenyl) doasd), 6.78e7.08 (4H, m, Phe) 7.68 (2H, d, J ¼ 7.6 Hz, CH-3, CH-5
piperazin-1-yl]methyl}-1,4-dioxa-8-azaspiro[4.5]decane (24) Sulf), 7.95 (2H, d, J ¼ 7.6 Hz, CH-2, CH-6 Sulf); HR-ESI-MS m/z (pos):
The compound, was obtained as a yellow oil from 4- 572.2015 C26H33F3N3O6S: (calcd. 572.2037); Anal. Calcd. for
chlorobenzene-1-sulfonyl chloride following the general proce- C28H34F3N3O10S: C, 50.83; H, 5.18; N, 6.35; Found C, 50.65; H, 5.02;
dure D (0.07 g, 0.13 mmol, yield 23%). N, 6.14.
1
H NMR (CDCl3, 400 MHz): d ¼ 1.72e1.96 (4H, m, CH2-6, CH2-10
doasd), 2.51e2.92 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.), 3.2.29. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-8-(4-
2.98e3.26 (6H, CH2-3, CH2-5 piperaz., CHa-7, CHa-9 doasd), nitrobenzenesulfonyl)-1,4-dioxa-8-azaspiro[4.5]decane (26)
3.30e3.42 (2H, m, CHb-7, CHb-9 doasd), 3.64 (1H, dd, J ¼ 7.5, 7.8 Hz, The compound, was obtained as a yellow oil from 4-
CHa-3 doasd), 3.90 (3H, s, OCH3), 4.09 (1H, dd, J ¼ 6.1, 7.8 Hz, CHb-3 nitrobenzene-1-sulfonyl chloride following the general procedure
doasd), 4.28e4.42 (1H, m, CH-2 doasd), 6.90 (1H, d, J ¼ 7.8 Hz, CH-3 D (0.110 g, 0.21 mmol, yield 36%).
1
Phe), 6.93e7.01 (2H, m, CH-5, CH-6 Phe), 7.02e7.12 (1H, m, CH-4 H NMR (CDCl3, 400 MHz): d ¼ 1.74e2.01 (4H, m, CH2-6, CH2-10
Phe), 7.54 (2H, d, J ¼ 8.5 Hz, CH-3, CH-5 Sulf), 7.74 (2H, d, doasd), 2.47e2.88 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.),
J ¼ 7.5 Hz, CH-2, CH-6 Sulf); 13C NMR (CDCl3, 400 MHz): d ¼ 34.1 2.97e3.22 (6H, CH2-3, CH2-5 piperaz., CHa-7, CHa-9 doasd),
(CH2, C-6/C-10 doasd), 35.3 (CH2, C-6/C-10 doasd), 44.1 (CH2, C-7/C- 3.32e3.46 (2H, m, CHb-7, CHb-9 doasd), 3.63 (1H, dd, J ¼ 7.2, 7.3 Hz,
9 doasd), 44.2 (CH2, C-7/C-9 doasd), 50.1 (2 CH2, C-3, C-5 piperaz.), CHa-3 doasd), 3.89 (3H, s, OCH3), 4.08 (1H, dd, J ¼ 6.2, 7.3 Hz, CHb-3
53.8 (2 CH2, C-2, C-6 piperaz.), 55.1 (CH3, OCH3), 61.0 (CH2, CH2eN), doasd), 4.27e4.41 (1H, m, CH-2 doasd), 6.91 (1H, d, J ¼ 7.9 Hz, CH-3
68.1 (CH2, C-3 doasd), 73.8 (CH, C-2 doasd), 106.4 (C, C-5 doasd), Phe), 6.94e7.03 (2H, m, CH-5, CH-6 Phe), 6.99e7.08 (1H, m, CH-4
110.9 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 122.7 Phe), 8.00 (2H, d, J ¼ 8.6 Hz, CH-3, CH-5 Sulf), 8.41 (2H, d,
(CH, C-4 Phe), 128.7 (2 CH, C-3, C-5 Sulf), 129.1 (2 CH, C-2, C-6 Sulf), J ¼ 8.6 Hz, CH-2, CH-6 Sulf); 13C NMR (CDCl3, 400 MHz): d ¼ 34.2
S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266 263

(CH2, C-6/C-10 doasd), 35.3 (CH2, C-6/C-10 doasd), 44.2 (CH2, C-7/C- 3.03e3.25 (6H, CH2-3, CH2-5 piperaz., CHa-7, CHa-9 doasd),
9 doasd), 44.3 (CH2, C-7/C-9 doasd), 50.2 (2 CH2, C-3, C-5 piperaz.), 3.30e3.49 (2H, m, CHb-7, CHb-9 doasd), 3.64 (1H, dd, J ¼ 7.5, 8.0 Hz,
53.9 (2 CH2, C-2, C-6 piperaz.), 55.1 (CH3, OCH3), 61.0 (CH2, CH2eN), CHa-3 doasd), 3.90 (3H, s, OCH3), 4.11 (1H, dd, J ¼ 6.5, 8.0 Hz, CHb-3
68.1 (CH2, C-3 doasd), 74.0 (CH, C-2 doasd), 106.2 (C, C-5 doasd), doasd), 4.33e4.45 (1H, m, CH-2 doasd), 6.91e7.05 (4H, m, Phe), 7.12
110.9 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 122.7 (1H, dd, J ¼ 3.5, 5.0 Hz, CH-4 Thio), 7.52 (1H, d, J ¼ 3.5 Hz CH-3 Thio),
(CH, C-4 Phe), 124.1 (2 CH, C-2, C-6 Sulf), 128.4 (2 CH, C-3, C-5 Sulf), 7.59 (1H, d, J ¼ 5.0 Hz, CH-5 Thio); 13C NMR (CDCl3, 400 MHz):
140.9 (C, C-1 Phe), 142.5 (C, C-1 Sulf), 149.9 (C, C-4 Sulf), 152.0 (C, C- d ¼ 34.0 (CH2, C-6/C-10 doasd), 35.2 (CH2, C-6/C-10 doasd), 44.2
2 Phe). (CH2, C-7/C-9 doasd), 44.3 (CH2, C-7/C-9 doasd), 50.0 (2 CH2, C-3, C-
The free amine (0.110 g, 0.21 mmol) was then converted into the 5 piperaz.), 53.8 (2 CH2, C-2, C-6 piperaz.), 55.1 (CH3, OCH3), 60.9
corresponding hydrogenoxalate from diethyl ether (0.07 g, (CH2, CH2eN), 68.0 (CH2, C-3 doasd), 73.8 (CH, C-2 doasd), 106.6 (C,
0.11 mmol, yield 53%). C-5 doasd), 110.9 (CH, C-3 Phe), 118.0 (CH, C-5 Phe), 120.7 (CH, C-6
mp: 126e127  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.58e1.92 (4H, Phe), 122.8 (CH, C-4 Phe), 127.3 (CH, C-4 Thio), 131.7 (CH, C-5 Thio),
m, CH2-6, CH2-10 doasd), 2.73e3.41 (14H, m, CH2eN, 4 CH2 piperaz, 132.0 (CH, C-3 Thio), 136.7 (C, C-2 Thio), 140.8 (C, C-1 Phe), 151.9 (C,
CH2-7, CH2-9 doasd), 3.51e3.67 (1H, m, CHa-3 doasd), 3.89 (3H, s, C-2 Phe).
OCH3), 3.97e4.12 (1H, m, CHb-3 doasd), 4.34e4.51 (1H, m, CH-2 The free amine (0.127 g, 0.25 mmol) was then converted into the
doasd), 6.75e7.07 (4H, m, Phe) 7.89 (2H, d, J ¼ 8.6 Hz, CH-3, CH-5 corresponding hydrogenoxalate from diethyl ether (0.126 g,
Sulf), 8.53 (2H, d, J ¼ 8.6 Hz, CH-2, CH-6 Sulf); HR-ESI-MS m/z (pos): 0.22 mmol, yield 86%).
533.2082 C25H33N4O7S: (calcd. 533.2064); Anal. Calcd. for mp: 166e168  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.60e1.77 (4H,
C27H34N4O11S: C, 52.08; H, 5.50; N, 9.00; Found C, 52.31; H, 5.72; N, m, CH2-6, CH2-10 doasd), 2.75e3.28 (14H, m, CH2eN, CH2-2, CH2-6,
9.31. CH2-3, CH2-5 piperaz., CH2-7, CH2-9 doasd), 3.66 (1H, dd, J ¼ 7.0,
7.6 Hz, CHa-3 doasd), 3.79 (3H, s, OCH3), 4.07 (1H, dd, J ¼ 6.3, 7.6 Hz,
3.2.30. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-8-(3- CHb-3 doasd), 4.37e4.53 (1H, m, CH-2 doasd), 6.83e7.01 (4H, m,
nitrobenzenesulfonyl)-1,4-dioxa-8-azaspiro[4.5]decane (27) Phe), 7.28 (1H, dd, J ¼ 3.7, 5.0 Hz, CH-4 Thio), 7.65 (1H, d, J ¼ 3.7 Hz
The compound, was obtained as a yellow oil from 3- CH-3 Thio), 8.04 (1H, d, J ¼ 5.0 Hz, CH-5 Thio); HR-ESI-MS m/z
nitrobenzene-1-sulfonyl chloride following the general procedure (pos): 494.1791 C23H32N3O5S2: (calcd. 494.1778); Anal. Calcd. for
D (0.133 g, 0.25 mmol, yield 44%). C25H33N3O9S2: C, 51.44; H, 5.70; N, 7.20; Found C, 51.23; H, 5.61; N,
1
H NMR (CDCl3, 400 MHz): d ¼ 1.75e2.00 (4H, m, CH2-6, CH2-10 7.02.
doasd), 2.48e2.89 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.),
2.95e3.24 (6H, CH2-3, CH2-5 piperaz., CHa-7, CHa-9 doasd), 3.2.32. 8-Methanesulfonyl-2-{[4-(2-methoxyphenyl)piperazin-1-
3.32e3.50 (2H, m, CHb-7, CHb-9 doasd), 3.64 (1H, dd, J ¼ 7.2, 7.8 Hz, yl]methyl}-1,4-dioxa-8-azaspiro[4.5]decane (29)
CHa-3 doasd), 3.89 (3H, s, OCH3), 4.09 (1H, dd, J ¼ 6.2, 7.8 Hz, CHb-3 The compound, was obtained as a yellow oil from meth-
doasd), 4.26e4.42 (1H, m, CH-2 doasd), 6.90 (1H, d, J ¼ 7.9 Hz, CH-3 anesulfonyl chloride following the general procedure D (0.13 g,
Phe), 6.95e7.01 (2H, m, CH-5, CH-6 Phe), 7.02e7.11 (1H, m, CH-4 0.31 mmol, yield 53%).
1
Phe), 7.80 (1H, t, J ¼ 7.9 Hz, CH-5 Sulf.), 8.12 (1H, d, J ¼ 7.9 Hz, H NMR (CDCl3, 400 MHz): d ¼ 1.77e1.91 (4H, m, CH2-6, CH2-10
CH-6 Sulf.), 8.43 (1H, d, J ¼ 7.9 Hz, CH-4 Sulf.), 8.57 (1H, s, CH-2 doasd), 2.83 (3H, s, SO2CH3), 2.93e3.15 (6H, m, CH2eN, CH2-2, CH2-
Sulf.); 13C NMR (CDCl3, 400 MHz): d ¼ 34.1 (CH2, C-6/C-10 doasd), 6 piperaz.), 3.16e3.35 (6H, m, CH2-3, CH2-5 piperaz., CHa-7, CHa-9
35.3 (CH2, C-6/C-10 doasd), 44.2 (CH2, C-7/C-9 doasd), 44.4 (CH2, C- doasd), 3.40e3.54 (2H, m, CHb-7, CHb-9 doasd), 3.71 (1H, dd,
7/C-9 doasd), 50.1 (2 CH2, C-3, C-5 piperaz.), 53.8 (2 CH2, C-2, C-6 J ¼ 7.5, 8.2 Hz, CHa-3 daosd), 3.89 (3H, s, OCH3), 4.23 (1H, dd, J ¼ 6.3,
piperaz.), 55.0 (CH3, OCH3), 60.9 (CH2, CH2eN), 68.0 (CH2, C-3 8.2 Hz, CHb-3 doasd), 4.53e4.65 (1H, m, CH-2 doasd), 6.91 (1H, d,
doasd), 74.0 (CH, C-2 doasd), 106.2 (C, C-5 doasd), 110.9 (CH, C-3 J ¼ 7.7 Hz, CH-3 Phe), 6.95e7.01 (2H, m, CH-5, CH-6 Phe), 7.01e7.12
Phe), 117.9 (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 122.3 (CH, C-2 Sulf.), (1H, m, CH-4 Phe); 13C NMR (CDCl3, 400 MHz): d ¼ 34.3 (CH2, C-6/C-
122.8 (CH, C-4 Phe), 126.9 (CH, C-4 Sulf), 130.2 (CH, C-5 Sulf.), 132.7 10 doasd), 34.9 (CH3, SO2CH3), 35.5 (CH2, C-6/C-10 doasd), 43.8
(CH, C-6 Sulf.), 138.9 (C, C-3 Sulf.); 140.8 (C, C-1 Phe), 148.1 (C, C-1 (CH2, C-7/C-9 doasd), 43.9 (CH2, C-7/C-9 doasd), 49.3 (2 CH2, C-3, C-
Sulf.), 151.9 (C, C-2 Phe). 5 piperaz.), 53.8 (2 CH2, C-2, C-6 piperaz.), 55.2 (CH3, OCH3), 60.7
The free amine (0.133 g, 0.25 mmol)was then converted into the (CH2, CH2eN), 67.8 (CH2, C-3 doasd), 73.1 (CH, C-2 doasd), 107.2 (C,
corresponding hydrogenoxalate from diethyl ether (0.096 g, C-5 doasd), 111.0 (CH, C-3 Phe), 118.1 (CH, C-5 Phe), 120.8 (CH, C-6
0.15 mmol, yield 77%). Phe), 123.2 (CH, C-4 Phe), 140.1 (C, C-1 Phe), 151.9 (C, C-2 Phe).
mp: 134e136  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.58e1.92 (4H, The free amine (0.13 g, 0.31 mmol) was then converted into the
m, CH2-6, CH2-10 doasd), 2.67e3.21 (12H, m, CH2eN, CH2-2, CH2-6, corresponding hydrogenoxalate from diethyl ether (0.03 g,
CH2-3, CH2-5 piperaz., CHa-7, CHa-9 doasd.), 3.23e3.44 (2H, m, 0.05 mmol, yield 16%).
CHb-7, CHb-9 doasd), 3.42e3.64 (1H, m, CHa-3 doasd), 3.77 (3H, s, mp: 158e160  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.26e1.91 (4H,
OCH3), 3.97e4.14 (1H, m, CHb-3 doasd), 4.33e4.49 (1H, m, CH-2 m, CH2-6, CH2-10 doasd), 2.78e3.41 (17H, m, CH3, CH2eN, CH2-2,
doasd), 6.97 (4H, m, Phe), 7.98 (1H, t, J ¼ 7.8 Hz CH-5 Sulf), 8.18 CH2-6, CH2-3, CH2-5 piperaz., CH2-7, CH2-9 doasd), 3.62e3.87 (1H,
(1H, d, J ¼ 7.8 Hz, CH-6 Sulf), 8.37 (1H, d, J ¼ 7.8 Hz CH-4 Sulf), 8.57 m, CHa-3 doasd), 3.89 (3H, s, OCH3), 4.07-4.22 (1H, m, CHb-3
(1H, s, CH-2 Sulf); HR-ESI-MS m/z (pos): 533.2062 C25H33N4O7S: doasd), 4.35e4.51 (1H, m, CH-2 doasd), 6.85e7.01 (4H, m, Phe);
(calcd. 533.2064); Anal. Calcd. for C27H34N4O11S: C, 52.08; H, 5.50; HR-ESI-MS m/z (pos): 426.2043 C20H32N3O5S: (calcd. 426.2057);
N, 9.00; Found C, 52.12; H, 5.69; N, 9.23. Anal. Calcd. for C22H33N3O9S: C, 51.25; H, 6.45; N, 8.15; Found C,
51.56; H, 6.77; N, 8.41.
3.2.31. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-8-
(thiophene-2-sulfonyl)-1,4-dioxa-8-azaspiro[4.5]decane (28) 3.2.33. General procedure E
The compound, was obtained as a yellow oil from thiophene-2- To a solution of compound 12 (1.0 mmol) in CH2Cl2, under ni-
sulfonyl chloride following the general procedure D (0.127 g, trogen was added Et3N (1.1 mmol). The mixture was cooled to 0  C,
0.25 mmol, yield 44%). and selected isocyanate (1.1 mmol) was added slowly. The reaction
1
H NMR (CDCl3, 400 MHz): d ¼ 1.78e2.01 (4H, m, CH2-6, CH2-10 was allowed to warm to room temperature and stirred for 3 h. Then
doasd), 2.42e2.92 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.), after addition of water the solvent was removed in vacuum and
264 S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266

residue was purified by flash chromatography to yield the desired (4H, m, CH2-6, CH2-10 doasd), 2.92e3.53 (16H, m, CH2eN, CH2-2,
compound. CH2-6, CH2-3, CH2-5 piperaz., NCH2CH2CH2CH2, CH2-7, CH2-9
doasd), 3.67 (1H, dd, J ¼ 7.1, 7.8 Hz, CHa-3 doasd), 3.78 (3H, s, OCH3),
3.2.34. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-N-phenyl- 4.15 (1H, dd, J ¼ 6.4, 7.8 Hz, CHb-3 doasd), 4.38-4.51 (1H, m, CH-2
1,4-dioxa-8-azaspiro[4.5]decane-8-carboxamide (30) doasd), 6.50 (1H, s broad, NH), 6.87e7.16 (4H, m, Phe); HR-ESI-
The compound, was obtained as a brown oil from phenyl iso- MS m/z (pos): 447.2962 C24H39N4O4: (calcd. 447.2966); Anal.
cyanate following the general procedure E (0.214 g, 0.46 mmol, Calcd. for C26H40N4O8: C, 58.19; H, 7.51; N, 10.44; Found C, 57.92; H,
yield 79%). 7.22; N, 10.31.
1
H NMR (CDCl3, 400 MHz): d ¼ 1.70e1.89 (4H, m, CH2-6, CH2-10
doasd), 2.51e2.87 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.), 3.2.36. N-tert-butyl-2-{[4-(2-methoxyphenyl)piperazin-1-yl]
3.06e3.25 (4H, m, CH2-3, CH2-5 piperaz.), 3.48e3.67 (4H, CH2-7, methyl}-1,4-dioxa-8-azaspiro[4.5]decane-8-carboxamide (32)
CH2-9 doasd), 3.70 (1H, dd, J ¼ 7.5, 7.8 Hz, CHa-3 doasd), 3.90 (3H, s, The compound, was obtained as a brown oil from tert-butyl
OCH3), 4.13 (1H, dd, J ¼ 6.4, 7.8 Hz, CHb-3 doasd) 4.35-4.45 (1H, m, isocyanate following the general procedure E (0.250 g,
CH-2 doasd), 6.11 (1H, s broad, NHCO), 6.90 (1H, d, J ¼ 7.0 Hz, CH-3 0.55 mmol, yield 90%).
1
Phe), 6.92e7.01 (2H, m, CH-5, CH-6 Phe), 7.01e7.101 (2H, m, CH-4 H NMR (CDCl3, 400 MHz): d ¼ 1.39 (9H, s, 3 CH3), 1.63e1.85 (4H,
Phe, CH-4 Arom.), 7.30 (2H, dd, J ¼ 7.2, 8.2 Hz, CH-3, CH-5 Arom.), m, CH2-6, CH2-10 doasd), 2.57e2.98 (6H, m, CH2eN, CH2-2, CH2-6
7.38 (2H, d, J ¼ 8.2 Hz, CH-2, CH-6 Arom.); 13C NMR (CDCl3, piperaz.), 3.02e3.27 (4H, m, CH2-3, CH2-5 piperaz.), 3.30e3.57 (4H,
400 MHz): d ¼ 34.7 (CH2, C-6/C-10 doasd), 35.8 (CH2, C-6/C-10 CH2-7, CH2-9 doasd), 3.70 (1H, dd, J ¼ 7.2, 7.9 Hz, CHa-3 doasd), 3.91
doasd), 42.1 (CH2, C-7/C-9 doasd), 42.2 (CH2, C-7/C-9 doasd), 50.3 (2 (3H, s, OCH3), 4.17 (1H, dd, J ¼ 6.1, 7.9 Hz, CHb-3 doasd), 4.31-4.52
CH2, C-3, C-5 piperaz.), 53.9 (2 CH2, C-2, C-6 piperaz.), 55.1 (CH3, (1H, m, CH-2 doasd), 6.17 (1H, s broad, NHCO), 6.91 (1H, d,
OCH3), 61.1 (CH2, CH2eN), 68.1 (CH2, C-3 doasd), 74.1 (CH, C-2 J ¼ 7.8 Hz, CH-3 Phe), 6.93-7.03 (2H, m, CH-5, CH-6 Phe), 7.01-7.11
doasd), 107.5 (C, C-5 doasd), 110.9 (CH, C-3 Phe), 117.9 (CH, C-5 Phe), (1H, m, CH-4 Phe); 13C NMR (CDCl3, 400 MHz): d ¼ 29.2 (3 CH3,
119.8 (2 CH, C-2, C-6 Arom.), 120.7 (CH, C-6 Phe), 122.6 (CH, C-4 CH3), 34.8 (CH2, C-6/C-10 doasd), 35.7 (CH2, C-6/C-10 doasd), 41.9
Phe), 122.7 (CH, C-4 Arom.), 128.5 (2 CH, C-3, C-5 Arom.), 138.9 (C, (CH2, C-7/C-9 doasd), 42.0 (CH2, C-7/C-9 doasd), 50.0 (2 CH2, C-3, C-
C-1 Arom.), 141.0 (C, C-1 Phe), 152.0 (C, C-2 Phe), 154.6 (C, NCON). 5 piperaz.), 50.5 (C, C(CH3)3) 53.8 (2 CH2, C-2, C-6 piperaz.), 55.1
The free amine (0.21 g, 0.46 mmol) was then converted into the (CH3, OCH3), 61.0 (CH2, CH2eN), 68.0 (CH2, C-3 doasd), 73.8 (CH, C-2
corresponding hydrogenoxalate from diethyl ether (0.20 g, doasd), 107.8 (C, C-5 doasd), 110.9 (CH, C-3 Phe), 118.0 (CH, C-5 Phe),
0.36 mmol, yield 78%). 120.7 (CH, C-6 Phe), 122.8 (CH, C-4 Phe), 140.8 (C, C-1 Phe), 152.0 (C,
mp: 62e64  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.62e1.94 (4H, C-2 Phe), 156.5 (C, NCON).
m, CH2-6, CH2-10 doasd), 2.40e2.79 (6H, m, CH2eN, CH2-2, CH2-6 The free amine (0.25 g, 0.55 mmol) was then converted into the
piperaz.), 2.93e3.21 (4H, m, CH2-3, CH2-5 piperaz.), 3.36e3.65 (5H, corresponding hydrogenoxalate from diethyl ether (0.153 g,
CHa-3, CH2-7, CH2-9 doasd), 3.78 (3H, s, OCH3), 4.17 (1H, dd, J ¼ 6.2, 0.28 mmol, yield 47%).
8.0 Hz, CHb-3 doasd), 4.24e4.51 (1H, m, CH-2 doasd), 6.56 (1H, s mp: 58e60  C; 1H NMR (DMSO, 200 MHz): d ¼ 1.18 (9H, s, 3
broad. NH), 6.88e7.15 (5H, m, Phe, CH-4 Arom.) 7.21e7.41 (4H, m, CH3), 1.37e1.74 (4H, m, CH2-6, CH2-10 doasd), 2.70e3.50 (14H, m,
CH-3, CH-5, CH-2, CH-6 Arom.); HR-ESI-MS m/z (pos): 467.2371 CH2eN, CH2-2, CH2-6, CH2-3, CH2-5 piperaz., CH2-7, CH2-9 doasd),
C26H35N4O4:(calcd. 467.2353); Anal. Calcd. for C28H36N4O8: C, 3.42e3.61 (1H, m, CHa-3 doasd), 3.74 (3H, s, OCH3), 4.11e4.23 (1H,
60.42; H, 6.52; N, 10.07; Found C, 60.75; H, 6.83; N, 10.33. m, CHb-3 doasd), 4.41-4.61 (1H, m, CH-2 doasd), 6.83 (1H, s broad,
NH), 6.91e7.12 (4H, m, Phe); HRESIMS m/z (pos): 447.2971
3.2.35. N-butyl-2-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}- C24H39N4O4: (calcd. 447.2966); Anal. Calcd. for C26H40N4O8: C,
1,4-dioxa-8-azaspiro[4.5]decane-8-carboxamide (31) 58.19; H, 7.51; N, 10.44; Found C, 58.23; H, 7.67; N, 10.52.
The compound, was obtained as a brown oil from n-butyl iso-
cyanate following the general procedure E (0.125 g, 0.27 mmol, 3.3. Pharmacology
yield 48%).
1
H NMR (CDCl3, 400 MHz): d ¼ 0.92 (3H, t, J ¼ 7.3 Hz, CH3), 1.21- 3.3.1. Functional antagonism in isolated tissues
1.52 (4H, m, CH2CH3, CH2CH2CH3), 1.61e1.97 (4H, m, CH2-6, CH2-10 Male Wistar rats (275e300 g) were killed by cervicaldislocation,
doasd), 2.57e2.94 (6H, m, CH2eN, CH2-2, CH2-6 piperaz.), and the organs required were isolated, freed fromadhering con-
3.02e3.35 (6H, m, CH2-3, CH2-5 piperaz., NCH2CH2CH2CH2), nective tissues, and set up rapidly under asuitable resting tension in
3.41e3.62 (4H, CH2-7, CH2-9 doasd), 3.70 (1H, dd, J ¼ 7.1, 8.1 Hz, 20 mL of organ baths containingphysiological salt solution kept at
CHa-3 doasd), 3.91 (3H, s, OCH3), 4.17 (1H, dd, J ¼ 6.4, 8.1 Hz, CHb-3 37  C and aerated with 5%CO2e95% O2 at pH 7.4. Concentration-
doasd), 4.38e4.54 (1H, m, CH-2 doasd), 6.14 (1H, s broad, NHCO), response curves wereconstructed by cumulative addition of
6.91 (1H, d, J ¼ 7.7 Hz, CH-3 Phe), 6.93e7.01 (2H, m, CH-5, CH-6 agonist. The concentrationof agonist in the organ bath was
Phe), 7.01e7.10 (1H, m, CH-4 Phe); 13C NMR (CDCl3, 400 MHz): increased approximately3-fold at each step, with each addition
d ¼ 13.5 (CH3, CH2CH3), 19.8 (CH2, CH2CH3), 32.1 (CH2, being made only afterthe response to the previous addition had
NCH2CH2CH3), 34.5 (CH2, C-6/C-10 doasd), 35.7 (CH2, C-6/C-10 attained a maximallevel and remained steady. Contractions were
doasd), 40.5 (CH2, CH2CH2CH2CH3), 41.9 (CH2, C-7/C-9 doasd), 42.1 recorded bymeans of a force displacement transducer connected to
(CH2, C-7/C-9 doasd), 50.0 (2 CH2, C-3, C-5 piperaz.), 53.8 (2 CH2, C- the MacLab system PowerLab/800 and to a polygraph channel
2, C-6 piperaz.), 55.1 (CH3, OCH3), 61.0 (CH2, CH2eN), 68.0 (CH2, C-3 recorder(Gemini). In addition, parallel experiments in which tis-
doasd), 73.9 (CH, C-2 doasd), 107.7 (C, C-5 doasd), 110.9 (CH, C-3 suesdid not receive any antagonist were run in order to check
Phe), 118.0 (CH, C-5 Phe), 120.7 (CH, C-6 Phe), 123.4 (CH, C-4 Phe), anyvariation in sensitivity.
140.7 (C, C-1 Phe), 152.0 (C, C-2 Phe), 157.2 (C, NCON).
The free amine (0.121 g, 0.27 mmol) was then converted into the 3.3.2. Vas deferens prostatic portion
corresponding hydrogenoxalate from diethyl ether (0.069 g, This tissue was used to assess the antagonism toward
0.13 mmol, yield 48%). a<alpha>1A adrenoceptors [24]. Prostatic portions of 2 cm length
mp: 128e130  C; 1H NMR (DMSO, 200 MHz): d ¼ 0.86 (3H, t, were mounted under 0.5 g of tension at 37  C in tyrode solution of
J ¼ 7.0 Hz, CH3), 1.11-1.42 (4H, m, CH2CH3, CH2CH2CH3), 1.51e1.73 the following composition (mM): NaCl, 130; KCl, 1; CaCl2, 1.8;
S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266 265

MgCl2, 0.89; NaH2PO4, 0.42; NaHCO3, 25; glucose, 5.6. Cocaine and immediately frozen and stored at 70  C until use. On the day of
hydrochloride (0.1 mM) was added to the tyrode to prevent the experiment, cell membranes (80e90 mg of protein) were resus-
neuronal uptake of (-)-noradrenaline. The preparations were pended in binding buffer (50 mM Tris, 2.5 mM MgCl2, and 10 mM
equilibrated for 60 min with washing every 15 min. After the pargiline, pH 7.4). Membranes were incubated in a final volume of
equilibration period, tissues were primed twice by addition of 0.32 mL for 30 min at 30  C with 1 nM [3H]8-OH-DPAT, in the
10 mM noradrenaline. After another washing and equilibration absence or presence of various concentrations of the competing
period of 60 min, a noradrenaline concentrationeresponse curve drugs (1pMe1 mM); each experimental condition was performed in
was constructed (basal response). The antagonist was equilibrated triplicate. Nonspecific binding was determined in the presence of
for 30 min before construction of a new concentrationeresponse 10 mM 5-HT. Binding to recombinant human a<alpha>1 adreno-
curve to the agonist. (-)-Noradrenaline solutions contained 0.05% ceptor subtypes was performed in membranes from Chinese
Na2S2O5 to prevent oxidation. hamster ovary (CHO) cells transfected by electroporation with DNA
expressing the gene encoding each a<alpha>1 adrenoceptor sub-
3.3.3. Spleen type. Cloning and stable expression of the human a<alpha>1
This tissue was used to assess the antagonism toward adrenoceptor genes were performed as described [22]. CHO cell
a<alpha>1B adrenoceptors [25]. The spleen was removed and membranes (70 mg of protein) were incubated in 50 mM Tris (pH
bisected longitudinally into two strips, which were suspended in 7.4) with 0.1e0.4 nM [3H]prazosin, in a final volume of 0.32 mL for
tissue baths containing Krebs solution of the following composition 30 min at 25  C, in the absence or presence of competing drugs (1
(mM): NaCl, 120; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.5; KH2PO4, 1.2; pMe1 mM). Nonspecific binding was determined in the presence of
NaHCO3, glucose, 11; K2EDTA, 0.01. Propranolol hydrochloride 10 mM Tamsulosin. The incubation was stopped by addition of ice-
(4 mM) was added to block b-adrenoceptors. The spleen strips were cold Tris buffer and rapid filtration through Unifilter B filters (Per-
placed under 1 g of resting tension and equilibrated for 2 h. The kineElmer) using a Filtermate cell harvester (Packard), and the
cumulative concentrationeresponse curves to phenylephrine were radioactivity retained on the filters was determined by TopCount,
measured isometrically and obtained at 30 min intervals, the first PerkineElmer liquid scintillation counting at 90% efficiency.
one being discarded and the second taken as a control. The
antagonist was allowed to equilibrate for 30 min before con-
3.3.6. [35S]GTPgS binding assay
structing a new concentrationeresponse curve to the agonist.
The effects of the various compounds tested on [35S]GTPgS
binding in HeLa cells expressing the recombinant human 5-HT1A
3.3.4. Aorta
receptor were evaluated according to the method of Stanton and
This tissue was used to assess the antagonism toward
Beer [27] with minor modifications [29]. Stimulation experiments:
a<alpha>1D adrenoceptors [26]. Thoracic aorta was cleaned from
Cell membranes (50e70 mg of protein) were resuspended in buffer
extraneous connective tissues and placed in Krebs solution of the
containing 20 mM HEPES, 3 mM MgSO4, and 120 mM NaCl (pH 7.4).
following composition (mM): NaCl, 118.4; KCl, 4.7; CaCl2, 1.9;
The membranes were incubated with 30 mM GDP, and various
MgSO4, 1.2; NaH2PO4, 1.2; NaHCO3, 25; glucose, 11.7; K2EDTA, 0.01.
concentrations (from 0.01 nM to 10 mM) of test drugs or 8-OH-DPAT
Cocaine hydrochloride (0.1 mM) and propranolol hydrochloride
(reference curve) for 20 min at 30  C in a final volume of 0.5 mL.
(4 mM) were added to prevent the neuronal uptake of
Samples were transferred to ice, [35S]GTPgS (200 pM) was added,
(-)-noradrenaline and to block b-adrenoceptors, respectively. Two
and samples were incubated for another 30 min at 30  C. The pre-
helicoidal strips (15 mm  3 mm) were cut from each aorta,
incubation with both agonist and antagonist, before initiating the
beginning from the end that is most proximal to the heart. The
[35S]GTPgS binding, ensures that agonist and antagonist are at
endothelium was removed by rubbing with filter paper. The
equilibrium. Nonspecific binding was determined in the presence
absence of acetylcholine (100 mM) induced relaxation in prepara-
of 10 mM GTPgS. Incubation was stopped by the addition of ice-cold
tions contracted with (-)-noradrenaline (1 mM) was taken as an
HEPES buffer and rapid filtration on Unifilter B filters (Perkin Elmer)
indicator that the vessels were denuded successfully. Vascular
using a Filtermate cell harvester (Packard). The filters were washed
strips were then tied with surgical thread and suspended in a jac-
with ice-cold Hepes buffer, and the radioactivity retained on the
keted tissue bath containing tyrode solution. Strip contractions
filters was determined by TopCount, Perkin Elmer liquid scintilla-
were measured isometrically. After at least a 2 h equilibration
tion counting at 90% efficiency.
period under an optimal tension of 1 g, cumulative (-)-noradren-
aline concentrationeresponse curves were recorded at 1 h in-
tervals, the first two being discarded and the third one taken as 3.3.7. Data analysis
control. The antagonist was equilibrated with the tissue for 30 min Binding data were analyzed using the nonlinear curve-fitting
before the generation of the fourth cumulative concen- program GraphPad (Prism for windows, version 5.04). Scatchard
trationeresponse curve to (-)-noradrenaline. (-)-Norad-renaline plots were linear for all preparations. None of the pseudo-Hill co-
solutions contained 0.05% K2EDTA in 0.9% NaCl to prevent efficients (nH) were significantly different from unity (p > 0.05).
oxidation. Equilibrium dissociation constants (Ki) were derived from the
ChengePrusoff equation Ki ¼ IC50/(L/Kd), where L and Kd are the
3.3.5. Radioligand binding assay at human recombinant 5-HT1AR concentration and the equilibrium dissociation constant of the
and a<alpha>1 adrenoceptor subtypes radioligand. pKi values are the mean of 2e3 separate experiments
A human cell line (HeLa) stably transfected with genomic clone performed in duplicate [30]. Stimulation of [35S]GTPgS binding
G-21 coding for the human 5-HT1A serotoninergic receptor was induced by the compounds tested was expressed as the percent
used. Cells were grown as monolayers in Dulbecco's modified Ea- increase in binding above basal value, with the maximal stimula-
gle's medium supplemented with 10% fetal calf serum and genta- tion observed with 8-OH-DPAT taken as 100%. The concen-
mycin (100 mg/mL) under 5% CO2 at 37  C. Cells were detached from trationeresponse curves of the agonistic activity were analyzed by
the growth flask at 95% confluence by a cell scraper and were lysed GraphPad as reported above [28]. The maximum percentage of
in ice-cold Tris (5 mM) and EDTA buffer (5 mM, pH 7.4). Homoge- stimulation of [35S]GTPgS binding (Emax) achieved for each drug,
nates were centrifuged for 20 min at 40000 g, and pellets were and the concentration required to obtain 50% of Emax
resuspended in a small volume of ice-cold Tris/EDTA buffer (above) (pD2 ¼ log10 [EC50]), were evaluated.
266 S. Franchini et al. / European Journal of Medicinal Chemistry 87 (2014) 248e266

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