Synthesis and biological evaluation of novel delta (δ) opioid receptor ligands with diazatricyclodecane skeletons

Article abstract of DOI:10.1016/j.ejmech.2013.09.014

Considering the interesting pharmacological profile of the delta (δ) selective opioid agonist compound SNC-80, conformationally constrained analogs containing two diazatricyclodecane ring systems in place of dimethylpiperazine core motif were synthesized.

Full text of DOI:10.1016/j.ejmech.2013.09.014

European Journal of Medicinal Chemistry 69 (2013) 413e426
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel delta (
ligands with diazatricyclodecane skeletons
d) opioid receptor
,
b, c,
Giovanni Loriga a , Paolo Lazzari
1, Stefania Ruiu a, Giorgio Marchese a,
*
Ilaria Manca a b, Gian Luca Casu a, Christian Dessì b 2, Gérard Aimè Pinna c,
**
,
,
Battistina Asproni c, Gabriele Murineddu c
,
a C.N.R. Istituto di Farmacologia Traslazionale, UOS Cagliari, Edificio 5, Loc. Piscinamanna, 09010 Pula, CA, Italy
b Neuroscienze PharmaNess S.c.a r.l., Edificio 5, Loc. Piscinamanna, 09010 Pula, CA, Italy
c Dipartimento di Chimica e Farmacia, Università di Sassari, Via F. Muroni 23/A, 07100 Sassari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2013
Received in revised form
3 September 2013
Accepted 5 September 2013
Available online 19 September 2013
Considering the interesting pharmacological profile of the delta (d) selective opioid agonist compound
SNC-80, conformationally constrained analogs containing two diazatricyclodecane ring systems in place
of dimethylpiperazine core motif were synthesized.
The compounds showed subnanomolar or low nanomolar
Depending upon the substituents on the diazatricyclodecane ring, these compounds displayed varying
selectivity for opioid receptor over and receptors.
Amongst the novel compounds, 1Aa showed the more interesting biological profile, with higher
finity and selectivity compared to SNC-80. The receptor agonist profile and antinociceptive activity of
1Aa were confirmed using ex-vivo (isolated mouse vas deferens) and in vivo (tail flick) assays.
d opioid receptor binding affinity.
d
m
k
d
af-
Keywords:
d
d-Agonist SNC-80
d
-Opioid receptor ligands
9,10-Diazatricyclo[4.2.1.12,5]decane
2,7-Diazatricyclo[4.4.0.03,8]decane
Binding assays
Ó 2013 Elsevier Masson SAS. All rights reserved.
Antinociceptive activity
1. Introduction
The
d
selective opioid ligands thus represent for pharmaceutical
industry and academia attractive candidates for a broad range of
pharmacological applications.
The delta (d) receptor is an opioid receptor (d OR) that is broadly
expressed in both the central nervous system and peripheral tis-
sues and organs [1].
Several small-molecule ligands with high affinity and selectivity
d receptors have been recently reported [11,12]. Examples of
for
Although the biology and function of
d
receptors are not
those compounds are typified by agonists such as SNC-80, TAN-67,
and ADL-5859 (Fig. 1) [13e15] and antagonists such as CP-646,777,
naltrindole, and naltriben [16e20].
completely understood [2], modulation of receptors in pre-clinical
d
animal models has shown promising efficacy in conditions such as
analgesia, addiction, Parkinson’s disease, Alzheimer’s disease, and
seizure disorders [3e5]. Moreover, peripheral d-opioid receptors
seem to be involved in cancer [6], cardioprotection [7,8], gastroin-
testinal disorders [9], and weight loss [10].
Amongst the considerable number of reported
[11,12], SNC-80 displayed an interesting pharmacological profile,
with good binding affinity to receptor (nanomolar range) and
remarkable selectivity relative to [13,21]. opioid mediated
d opioid ligands
d
d
m
d
activity of this reference compound was successfully ascertained by
in vivo assays [22e26]. Pre-clinical studies showed in particular
SNC-80 efficacy towards acute pain [27], hypertension [28], Par-
kinson’s disease [4,24], anxiety [22,29], depression [22], motor
dysfunction and neuronal injury after spinal cord ischemia [30].
This unique biopharmacological profile makes it an attractive lead
* Corresponding author. Tel.: þ39 0709242025; fax: þ39 0709242206.
** Corresponding author. Tel.: þ39 079228749; fax: þ39 079228720.
E-mail addresses: giovanni.loriga@ift.cnr.it (G. Loriga), muri@uniss.it
(G. Murineddu).
1
Current address: KemoTech Srl, Edificio 3, Loc. Piscinamanna, 09010 Pula, CA,
Italy.
compound in the search for novel
d opioid receptor ligands.
2
Current address: Dipartimento di Scienze Biomediche, Sezione di Neuroscienze
In this context, our research has been focused on the design of
conformationally restricted analogs of SNC-80 in which the
e Farmacologia Clinica, Università degli Studi di Cagliari, S.S. 554, Bivio per Sestu,
09042 Monserrato, CA, Italy.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.014

414
G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
O
O
N
HO
N
N
O
N
N
HO
O
H
N
N
H
13
14
15
ADL-5859
Agonists:
SNC-80
TAN-67
OH
O
HO
O
N
N
N
N
OH
N
X
16
X = NH: NTI (Naltrindole)17,18
X = O: NTB (Naltriben)19,20
Antagonists:
CP-646,777
Fig. 1. Structures of
d opioid receptor ligands.
dimethylpiperazine core motif was incorporated into diaza-
tricyclodecane ring systems of type 1 and 2 (Fig. 2).
2Bd (see Table 1). In this first stage of the research, the com-
pounds have been obtained without the introduction of specific
steps aimed to the separation of potential stereoisomers. The
novel compounds exhibited good affinity to delta receptors
In this paper, we present the synthesis and preliminary
pharmacological characterization of 9-disubstitutedbenzhydryl-
10-allyl-9,10-diazatricyclo[4.2.1.12,5]decane derivatives 1Aaef,
1Baef and of 2-disubstitutedbenzhydryl-7-allyl-2,7-diazatricyclo
[4.4.0.03,8]decane derivatives 2Aa, 2Ac, 2Ad and 2Ba, 2Bc and
relative to the other opioid receptor subtypes
m and k. Moreover,
interesting profiles were highlighted by ex vivo and in vivo
assays.
Fig. 2. Process of rational drug design based on conformational restriction strategy.

G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
415
Table 1
Binding affinities of benzhydryl derivatives 1Aa, 1Acee, 1Baee, 2Aa, 2Ad, 2Ba, 2Bc, 2Bd and SNC-80 for opioid receptorsa,b (values expressed as Ki).
O
O
R
R
N
N
N
N
OR1
OR1
c
c
2A,B
1A,B
Compd
R
R1
Ki (nM)
Selectivity
d
m
k
m/
d
k/d
1Aa
1Ac
eCH3
eCH3
0.203 ꢀ 0.032
1 ꢀ 0.041
1633 ꢀ 186
600 ꢀ 29
8044
585
2956
1000
Et N
Et
N
585 ꢀ 101
650 ꢀ 96.4
1000 ꢀ 100
706 ꢀ 101
N
1Ad
1Ae
eCH3
eCH3
1.03 ꢀ 0.111
45 ꢀ 3.8
631
44
685
N
H
2000 ꢀ 289
300 ꢀ 58
6.7
1Ba
1Bb
1Bc
eH
eH
eH
0.23 ꢀ 0.03
0.900 ꢀ 0.1
0.208 ꢀ 0.02
20 ꢀ 0.5
173.3 ꢀ 14.5
23.7 ꢀ 2
4.8 ꢀ 0.9
683 ꢀ 9
4.7 ꢀ 0.4
87
193
114
21
759
23
Et N
Et
Me N
Me
N
N
1Bd
1Be
eH
eH
1.3 ꢀ 0.13
160 ꢀ 15.3
36.7 ꢀ 1.7
56 ꢀ 8
123
44
43
N
H
0.833 ꢀ 0.203
3.6 ꢀ 0.5
4.3
2Aa
2Ad
eCH3
eCH3
0.633 ꢀ 0.033
22 ꢀ 4.012
533.3 ꢀ 33.4
293.3 ꢀ 52.13
1267 ꢀ 67
842
13
2002
216
Et N
Et
N
4750 ꢀ 250
(continued on next page)

416
G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
Table 1 (continued )
Compd
R
R1
Ki (nM)
Selectivity
d
m
k
m/
d
k/d
2Ba
2Bc
eH
eH
0.267 ꢀ 0.033
61.6 ꢀ 13.3
n.d.
231
90
e
e
Et N
Et
N
0.700 ꢀ 0.087
63.25 ꢀ 11.9
n.d
N
2Bd
eH
2.5 ꢀ 0.2
170.0 ꢀ 10.01
2383 ꢀ 142
n.d.
68
e
SNC-80
0.71 ꢀ 0.06
2750 ꢀ 250
3356
3873
cAll compounds were prepared as racemates. n.d.: not determined value.
a
Binding assays were carried out in triplicate.
[3H-ADPE], [3H-DAMGO] and [3H-U-69,593] were used for the
d, m and k receptor binding studies, respectively (see Experimental section for full descriptions of the assays).
b
In order to well compare proposed compounds with SNC-80,
hereafter, it is our intention to separate the enantiomers or di-
astereoisomers which constitute, respectively, the racemic or dia-
stereomeric mixtures of the presented products. The separation of
the stereoisomers of defined leads and their structural character-
ization could allow us to evaluate the stereochemical configuration
of enantiomers related to the best receptor profiles. Moreover, this
could allow us to study in deep the relevance of the stereochem-
istry on the biological activity of the compounds. However, this
deepening goes beyond the scope of this first paper which is aimed
to preliminary investigate the pharmacological profile of novel
delta opioid receptor agonists based on conformationally con-
strained compounds containing diazatricyclodecane skeletons.
Thus, to further support our research concerning this novel class of
delta opioid agonists, the separation of the stereoisomers of defined
leads and their structural characterization will be the subject of a
future investigation. Finally, further studies will be performed in
the future to complete the pre-clinical evaluations (i.e. affinity
profiles to various receptors, toxicological data, pharmaco-kinetic
studies) of each enantiomer or diastereoisomer of identified lead
compounds.
1Baef and 2Ba, 2Bc and 2Bd was achieved in two steps by using
alkylation followed by methyl deprotection using BBr3.
3. Biology
The binding affinities of the prepared benzhydryl derivatives 1
and 2 for the m, d, and k opioid receptors were determined on brain
membranes according to a procedure based on that previously
described by Gillan and Kosterlitz [32].
Intrinsic activity of the novel compounds characterized by the
more favorable binding profiles was investigated through isolated
organ assays (mouse vas deferens). In fact, it has been ascertained
that delta opioid receptor agonists are able to reduce the electri-
cally induced contractions of the musculature of the mouse vas
deferens [21]. Moreover, it has been shown that delta antagonist
derivatives are able to block or reduce the inhibition of the elec-
trically evoked contraction amplitude of the vas deferens induced
by delta agonists, with parallel dextral shifts in delta receptor
agonist log concentrationeresponse curves in electrically stimu-
lated tissues.
To evaluate the potential therapeutic activity of the novel
compounds, antinociceptive effect of a selected derivative amongst
those synthesized was assayed in animal model of acute pain (tail
flick test).
2. Chemistry
The general strategy for preparation of the benzhydryl-
diazatricyclodecane opioids 1A,B and 2A,B was to synthesize the
chloro-benzhydryl moiety and then to attach the functionalized
diazatricyclodecane cores (Scheme 1).
4. Results and discussion
The synthesis of 6aef commenced with the conversion of 4-(3-
methoxybenzoyl)benzoic acid (3) [13] into the corresponding am-
ides 4aef using the appropriate amines. Reduction of ketoamides 4
with sodium borohydride in diluted ethanol at room temperature
for 2 h afforded the corresponding alcohols 5aef. Treatment of 5ae
f with 37% HCl provided the corresponding benzhydryl chlorides
6aef in good yields. Alkylation of 9-benzyl-9,10-diazatricyclo
4.1. Radioligand binding assays
Affinities for d, m, and k opioid receptors from mouse brain
membranes were computed by displacement of [3H-DPDPE], [3H-
DAMGO] and [3H-U-69,593], respectively, in equilibrium binding
assays (Table 1). Independent of the used diazatricyclodecane core,
most of the compounds exhibited subnanomolar/low nanomolar
[4.2.1.12,5]decane [31] and 2-benzyl-2,7-diazatricyclo[4.4.0.03,8
]
affinity [Ki (
) ¼ 45 and 22 nM, respectively];
within the 20e2000 nM range. Except 1Ba, 1Bc and 1Be, the other
d
) ¼ 0.20e2.5 nM] except compounds 1Ae and 2Ad [Ki
decane [31] with the appropriate chloride 6, in the presence of
anhydrous potassium carbonate in refluxing acetonitrile, afforded
(d
m-opioid receptor affinities fall
benzyl-benzyhdryl-diazatricyclodecanes 7aef and 8a, c,
d
in
ligands listed in Table 1 showed no significant
affinity.
k
-opioid receptor
moderate to good yields. These intermediates were next subjected
to debenzylation by treatment with H2 on Pd/C to provide 9aef and
10a, c, d. Target ligands 1Aaef and 2Aa, 2Ac and 2Ad were isolated
by alkylation with allyl bromide, whereas completion of compound
Compound 1Aa, the best of this series with a
d opioid receptor
affinity of 0.203 nM and 8044-fold and 2956-fold lower affinity for
and , respectively, serves as a convenient benchmark for all
m
k

G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
417
O
O
OH
R
i
H3CO
H3CO
O
O
3
4a-f
ii
O
R
OCH3
X
5a-f, X = OH
6a-f, X = Cl
iii
iv
v
O
O
R
R
Y
N
N
N
Y
N
OCH3
OCH3
8a,c,d, Y = Bn
10a,c,d, Y = H
7a-f, Y = Bn
9a-f, Y = H
vi
vi
vii
vii
O
O
R
R
N
N
N
N
OR1
OR1
1Aa-f, R1 = CH3
1Ba-f, R1 = H
2Aa, 2Ac, 2Ad, R1 = CH3
2Ba, 2Bc, 2Bd, R1 = H
viii
viii
a
b
c
d
e
f
N
N
H
N
H
N
Et
N
Et
Me
N
R
Me
N
Scheme 1. Reagents and conditions: (i) a) SOCl2, toluene, 110 ꢁC, 3 h, b) RH, CHCl3, r.t., 4 h; (ii) NaBH4, EtOH/H2O, r.t., 2 h; (iii) 37% HCl, CHCl3, r.t., 12 h; (iv) 9-benzyl-9,10-
diazatricyclo[4.2.1.12,5]decane [31], K2CO3, CH3CN, 80 ꢁC, 72 h; (v) 2-benzyl-2,7-diazatricyclo[4.4.0.03,8]decane [31], K2CO3, CH3CN, 80 ꢁC, 72 h; (vi) 3 atm H2, 10% Pd/C, EtOH,
60 ꢁC, 6 h; (vii) allyl bromide, K2CO3, acetone, 56 ꢁC, 12 h; (viii) BBr3, CH2Cl2, 0 ꢁC, 1.5 h.
others in terms of presenting structure-binding affinity
binding affinity. Compound 1Ac and 1Ad had a pyrrolidine and a
relationships.
piperidine ring replacements of the diethylamine moiety as
Examination of
diethylcarbamoyl moiety being replaced by various types of car-
bamoyl moieties (1Ace1Ae) reveals a great impact on receptor
d
receptor affinities of those compounds with
compared to 1Aa. Both maintained high
relatively low and receptor affinities. The piperidine derivative
1Ad exhibited higher to selectivity (631-fold) than did
d receptor affinities and
m
k
d
d
m

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G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
pyrrolidine derivative 1Ac (585-fold). Compound 1Ae has a cyclo-
Similar to SNC-80, compound 1Aa showed d agonist behavior in
hexylamine in the carbamoyl moiety and its Ki value indicated that
inhibiting electrically evoked contractions of mouse vas deferens.
Moreover, 1Aa action was antagonized by Naltrindole, but not by
CTAP (Fig. 4).
it had much lower affinity for
d
receptor as compared to 1Aa and no
significant affinity for
m
(Ki > 1000 nM) and (Ki > 300 nM)
k
receptors.
Isolated organ assay results concerning derivative 1Ba reflected
the determined opioid affinity profile of the compound. As for SNC-
80 and 1Aa, d agonist activity was ascertained by the capability of
1Ba to counteract the electrically evoked contractions on mouse vas
deferens (Fig. 5). The effect of 1Ba on mouse vas deferens con-
Compound 1Bae1Be have a phenol OH group in place of the
methoxy moiety. Their receptor affinities were similar to that of
1Aa but their receptor affinities were increased when compared
to that of 1Aa. The diethylamide 1Ba as well as the cyclohexylamide
d
m
1Be displayed significant binding affinities at
m
and
k
receptors.
tractions was antagonized by the selective
However, the action of the novel compound was also partially
counteracted by the selective antagonist CTAP. These results agree
with the ascertained presence of receptors in the mouse vas
d antagonist Naltrindole.
Replacement of the 9,10-diazatricyclo[4.2.1.12,5]decane unit
with a 2,7-diazatricyclo[4.4.0.03,8]decane ring system resulted in
ligands, compounds 2Aa, 2Ac, 2Ad, 2Ba, 2Bc and 2Bd, that had
m
m
interesting but unpredictable effects on
subtype selectivity.
d
binding affinity and
deferens and with the good affinity (Ki ¼ 20 nM) of 1Ba for the
same opioid sub-receptors.
Compound 2Aa with a similar pattern of substitution to 1Aa had
3-fold lower affinity than 1Aa for the opioid receptor. Similarly,
d
4.3. In vivo assays: tail flick (acute pain model)
the methoxy isomer 2Ad displayed 21-fold lower affinity compared
to 1Ad. Compounds 2Ba, 2Bc and 2Bd have a phenol OH group
replacement of the methoxy group. The diethylamide compound
Administration of the compound 1Aa (5 mg/kg, i.p.) induced a
significant increase of % MPE in the test of tail-flick after 30 min
from drug injection (P < 0.05). No significant differences vs. vehicle
treated mice were observed after 60 and 120 min from drug
administration (P > 0.05) (Fig. 6).
2Ba had the highest
d receptor affinity among the three phenol OH
analogs, with a receptor affinity that is comparable to that of 1Aa.
d
All three compounds, 2Ba, 2Bc, and 2Bd, had higher
affinities as compared to 1Aa.
m receptor
Tail-flick test analyses suggest that compound 1Aa may be able
to stimulate delta opioid receptors causing a rapid, but significant,
analgesic effects. To date, the possibility that selective
d opioid
4.2. Isolated organs (mouse vas deferens)
agonists may induce analgesia in absence of receptor stimulation
m
is still debated [21,33,34], partially explaining the moderate anal-
gesic effect induced by the compound 1Aa relative to that detected
with morphine.
In conformity with previously reported data [21], SNC-80
showed agonist profile by inhibiting mouse vas deferens
contraction in isolated organ assays (Fig. 3). This effect was coun-
teracted by the selective antagonist Naltrindole (NTI). Selective
action of the reference compound through the receptor was
further confirmed by the inefficacy of the selective antagonist D-
d
d
d
5. Conclusions
m
Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) to affect SNC-80
activity.
Based on a balance of binding properties, 1Aa and 1Ba were
selected as key compounds for isolated organ assays. The first one
In conclusion, we report here a novel family of d-selective opioid
receptor agonists containing the 9-disubstitutedbenzhydryl-10-
allyl-9,10-diazatricyclo[4.2.1.12,5]decane (compounds 1A,B) and
the 2-disubstitutedbenzhydryl-7-allyl-2,7-diazatricyclo[4.4.0.03,8
]
was in fact the more affine and selective compound for
(Ki ¼ 0.203 nM; Ki /Ki ¼ 8044), with an improved profile relative
to the reference compound SNC-80. Compound 1Ba displayed af-
finity at receptor comparable to 1Aa but with much higher affinity
at the
receptor (Ki ¼ 20 nM).
d
receptors
decane (compounds 2A,B) core structures as conformationally
restricted analogs of the reference delta selective agonist SNC-80.
m
d
d
m
Fig. 3. Concentrationeresponse curves for SNC-80 on the amplitude of twitch con-
tractions elicited by electrical field stimulation of the mouse vas deferens obtained in
the absence and in presence of NTI (50 nM) and CTAP (100 nM). Assays were per-
formed as described under Materials and Methods. Each curve represents the mean
values ꢀ S.E.M. of 10 experiments. Responses are expressed as a percentage of the
amplitude of the twitch response measured before the first addition of SNC-80.
Fig. 4. Concentrationeresponse curves for 1Aa on the amplitude of twitch contrac-
tions elicited by electrical field stimulation of the mouse vas deferens obtained in the
absence and in presence of NTI (50 nM) and CTAP (100 nM). Assays were performed as
described under Materials and Methods. Each curve represents the mean
values ꢀ S.E.M. of 10 experiments. Responses are expressed as a percentage of the
amplitude of the twitch response measured before the first addition of 1Aa.

G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
419
Based on these profiles,1Aa appears to be a promising candidate
to investigate further for the development of new drugs for the
treatment of
To confirm and validate the adopted synthetic strategies in the
development of novel selective agents, the separation and the
d opioid receptor mediated pathologies.
d
characterization of the enantiomers in the racemic mixtures will be
carried out in the future in order to identify the component with
the best profile.
6. Experimental protocols
6.1. Chemistry. General informations
Melting points were obtained on an Electrothermal IA 9100
digital melting point apparatus or on a Kopfler melting point
apparatus and are uncorrected.
Thin layer chromatography (TLC) was performed with Poly-
gramÒ SIL N-HR/HV254 precoated plastic sheets (0.2 mm).
Flash chromatography (FC) was performed using Merck silica
gel 60 (230e400 mesh ASTM).
Fig. 5. Concentrationeresponse curves for 1Ba on the amplitude of twitch contrac-
tions elicited by electrical field stimulation of the mouse vas deferens obtained in the
absence and in presence of NTI (50 nM) and CTAP (100 nM). Assays were performed as
described under Materials and Methods. Each curve represents the mean
values ꢀ S.E.M. of 10 experiments. Responses are expressed as a percentage of the
amplitude of the twitch response measured before the first addition of 1Ba.
IR spectra were recorded as thin films (for oils) or nujol mulls
(for solids) on NaCl plates with a Jasco FT/IR 460 plus spectropho-
tometer and are expressed in
All NMR spectra were taken on a Varian XL-200 NMR spec-
trometer with 1H and 13C being observed at 200 and 50 MHz
respectively. Chemical shifts for 1H and spectra were reported in
n
(cmꢂ1).
Although all the novel compounds were prepared and evaluated as
racemic mixtures, the biological profile of the evaluated com-
pounds provides support for the design strategies implemented in
the present study. In fact, all of the diazatricyclodecanes displayed
d
or ppm downfield from TMS [(CH3)4Si]. Multiplicities are recor-
ded as s (singlet), br s (broad singlet), d (doublet), t (triplet), dd
(double doublet), dt (double triplet), q (quartet), m (multiplet).
Elemental analyses were performed by Laboratorio di Micro-
analisi, Dipartimento di Chimica e Farmacia, Università di Sassari,
Italy, and are within ꢀ0.4% of the calculated values.
higher affinity for binding at the
the novel compounds, 1Aa displayed the best binding profile, with
Ki values 3-fold lower than that of the reference compound SNC-
80, and Ki /Ki value of 8044 (more than twice the corresponding
d sites than at m or k sites. Amongst
d
All reactions involving air or moisture-sensitive compounds
were performed under argon atmosphere.
m
d
value determined for SNC-80).
As a potential lead compound, 1Aa was evaluated in ex vivo and
in vivo assays and was found to display d selective agonist activity in
Unless otherwise specified, all materials, solvents, and reagents
were obtained from commercial suppliers.
the isolated organ assay (mouse vas deferens) and antinociceptive
activity in acute pain animal model (tail flick test).
6.2. General procedure I: preparation of compounds 4aef
A solution of 4-(3-methoxybenzoyl)benzoic acid 3 [13] (2.24 g,
8.74 mmol) and of SOCl2 (5 mL, 68.92 mmol) in 5 mL of toluene was
allowed to stir at reflux for 3 h under N2. The volatiles were
removed under reduced pressure. The remaining SOCl2 was
coevaporated with toluene (3 ꢃ 5 mL). The acid chloride was dis-
solved in 10 mL of CHCl3, and the resulting solution was added
dropwise to a stirred solution of the appropriate amine or hydra-
zine (1.5 eq) in CHCl3 (13 mL). The biphasic reaction mixture was
allowed to stir at room temperature for 4 h. The organic layer was
separated, washed with water (2 ꢃ 15 mL), and dried (Na2SO4). The
solvent was evaporated under reduced pressure, and the residue
was purified by flash chromatography to afford the desired amide
4aef.
6.2.1. 4-(3-Methoxybenzoyl)-N,N-diethylbenzamide (4a)
General procedure I was used to convert diethylamine into the
title product. Yield 98%; Rf 0.44 (petroleum ether/ethyl acetate 1:1);
IR (nujol): 1650, 1690; 1H NMR (CDCl3)
d 1.00e1.38 (m, 6H), 3.20e
3.38 (m, 2H), 3.50e3.70 (m, 2H), 3.86 (s, 3H), 7.10e7.40 (m, 4H), 7.48
(d, 2H, J ¼ 8.4 Hz), 7.84 (d, 2H, J ¼ 8.4 Hz). Anal. calcd for C19H21NO3:
C, 73.29; H, 6.80; N, 4.50. Found: C, 73.08; H, 6.79; N, 4.49.
Fig. 6. Histogram showing the effects induced by vehicle, morphine and 1Aa (dosages
5 mg/kg, i.p.) in the tail-flick test. Each bar represents mean values ꢀ S.E.M. of % MPE
obtained from 6 to 10 mice at different time points after drug injection. Statistical
analyses have been carried out using Two-Way ANOVA, followed by NewmaneKeuls
post-hoc test (*,**P < 0.05 and P < 0.01 vs. vehicle treated mice).
6.2.2. 4-(3-Methoxybenzoyl)-N,N-dimethylbenzamide (4b)
General procedure I was used to convert dimethylamine into the
title product. Yield 99%; Rf 0.23 (petroleum ether/ethyl acetate 1:1);
IR (nujol): 1680, 1690; 1H NMR (CDCl3)
d 2.90e3.05 (m, 3H), 3.10e

420
G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
3.20 (m, 3H), 3.86 (s, 3H), 6.87e7.60 (m, 6H), 7.80e7.95 (m, 2H).
Anal. calcd for C17H17NO3: C, 72.07; H, 6.05; N, 4.94. Found: C, 71.93;
H, 6.03; N, 4.93.
3H), 5.81 (s, 1H), 6.75e6.95 (m, 3H), 7.10e7.50 (m, 5H). Anal. calcd
for C17H19NO3: C, 71.56; H, 6.71; N, 4.91. Found: C, 71.49; H, 6.70; N,
4.90.
6.2.3. (3-Methoxyphenyl)-[4-(pyrrolidine-1-carbonyl)phenyl]
methanone (4c)
6.3.3. {4-[Hydroxy(3-methoxyphenyl)methyl]phenyl}(pyrrolidin-1-
yl)methanone (5c)
General procedure I was used to convert pyrrolidine into the
title product. Yield 98%; Rf 0.20 (petroleum ether/ethyl acetate 1:1);
General procedure II was used to convert 4c into the title
product. Yield 99%; Rf 0.31 (CHCl3/CH3OH 95:5); IR (nujol): 1680,
IR (nujol): 1690, 1700; 1H NMR (CDCl3)
d
1.90e2.10 (m, 4H), 3.40e
3300; 1H NMR (CDCl3)
d
1.75e2.00 (m, 4H), 2.62 (d, 1H, J ¼ 3.4 Hz),
3.50 (m, 2H), 3.60e3.75 (m, 2H), 3.86 (s, 3H), 7.10e7.45 (m, 4H), 7.61
(dd, 2H, J ¼ 1.6 and 6.6 Hz), 7.83 (dd, 2H, J ¼ 2.0 and 6.6 Hz). Anal.
calcd for C19H19NO3: C, 73.77; H, 6.19; N, 4.53. Found: C, 73.59; H,
6.17; N, 4.52.
3.40 (t, 2H, J ¼ 6.2 Hz), 3.62 (t, 2H, J ¼ 6.2 Hz), 3.78 (s, 3H), 5.81 (d,
1H, J ¼ 3.0 Hz), 6.78e7.00 (m, 3H), 7.10e7.50 (m, 5H). Anal. calcd for
C
19H21NO3: C, 73.29; H, 6.80; N, 4.50. Found: C, 73.20; H, 6.79; N,
4.49.
6.2.4. (3-Methoxyphenyl)-[4-(piperidine-1-carbonyl)phenyl]
methanone (4d)
6.3.4. {4-[Hydroxy(3-methoxyphenyl)methyl]phenyl}(piperidin-1-
yl)methanone (5d)
General procedure I was used to convert piperidine into the title
product. Yield 91%; Rf 0.45 (petroleum ether/ethyl acetate 1:1); IR
General procedure II was used to convert 4d into the title
product. Yield 99%; Rf 0.45 (CHCl3/CH3OH 95:5); IR (nujol): 1690,
(nujol): 1680, 1690; 1H NMR (CDCl3)
d
1.48e1.90 (m, 6H), 3.30e3.40
3300; 1H NMR (CDCl3)
d 1.40e1.75 (m, 6H), 2.85 (bs, 1H), 3.25e3.40
(m, 2H), 3.70e3.80 (m, 2H), 3.86 (s, 3H), 7.10e7.55 (m, 6H), 7.80e
7.90 (m, 2H). Anal. calcd for C20H21NO3: C, 74.28; H, 6.55; N, 4.33.
Found: C, 74.21; H, 6.54; N, 4.32.
(m, 2H), 3.60e3.75 (m, 2H), 3.78 (s, 3H), 5.78 (s, 1H), 6.78e7.00 (m,
3H), 7.20e7.45 (m, 5H). Anal. calcd for C20H23NO3: C, 73.82; H, 7.12;
N, 4.30. Found: C, 73.77; H, 7.10; N, 4.29.
6.2.5. 4-(3-Methoxybenzoyl)-N-cyclohexylbenzamide (4e)
6.3.5. 4-[Hydroxy-(3-methoxyphenyl)methyl]-N-
cyclohexylbenzamide (5e)
General procedure I was used to convert cyclohexylamine into
the title product. Yield 95%; Rf 0.42 (petroleum ether/ethyl acetate
General procedure II was used to convert 4e into the title
product. Yield 85%; Rf 0.30 (CHCl3/CH3OH 9:1); IR (nujol): 1690,
7:3); IR (nujol): 1680, 1690, 3100; 1H NMR (CDCl3)
d 1.00e1.95 (m,
8H), 2.00e2.20 (m, 2H), 3.85 (s, 3H), 3.90e4.10 (m, 1H), 6.05e6.15
(m, 1H), 7.10e7.40 (m, 8H). Anal. calcd for C21H23NO3: C, 74.75; H,
6.87; N, 4.15. Found: C, 74.68; H, 6.85; N, 4.14.
3100, 3300; 1H NMR (CDCl3)
d 1.00e2.10 (m, 11H), 3.78 (s, 3H),
3.80e4.05 (m, 1H), 5.83 (s, 1H), 5.96e6.00 (m, 1H), 6.78e6.95 (m,
2H), 7.20e7.30 (m, 2H), 7.43 (d, 2H, J ¼ 8.2 Hz), 7.70 (d, 2H,
J ¼ 8.2 Hz). Anal. calcd for C21H25NO3: C, 74.31; H, 7.42; N, 4.13.
Found: C, 74.22; H, 7.41; N, 4.11.
6.2.6. 4-(3-Methoxybenzoyl)-N-pyrrolidin-1-yl-benzamide (4f)
General procedure I was used to convert 1-aminopyrrolidine
into the title product. Yield 50%; Rf 0.20 (petroleum ether/ethyl
6.3.6. 4-[Hydroxy-(3-methoxyphenyl)methyl]-N-pyrrolidin-1-yl-
benzamide (5f)
acetate 2:8); IR (nujol): 1680, 1700, 3300; 1H NMR (CDCl3)
d 1.90e
2.00 (m, 4H), 3.00e3.20 (m, 4H), 3.86 (s, 3H), 6.86 (s, 1H), 7.15 (d,
1H, J ¼ 6.0 Hz), 7.30e7.43 (m, 3H), 7.80e7.87 (m, 4H). Anal. calcd for
General procedure II was used to convert 4f into the title
product. Yield 99%; Rf 0.50 (CHCl3/CH3OH 9:1); IR (nujol): 1670,
C
19H20N2O3: C, 70.35; H, 6.21; N, 8.64. Found: C, 70.24; H, 6.20; N,
3100, 3300; 1H NMR (CDCl3)
d 1.90e2.00 (m, 5H), 2.85e3.10 (m,
8.62.
4H), 3.77 (s, 3H), 5.82 (s, 1H), 6.75 (s, 1H), 7.20e7.55 (m, 4H), 7.66 (d,
2H, J ¼ 8.0 Hz), 7.68 (d, 2H, J ¼ 8.0 Hz). Anal. calcd for C19H22N2O3: C,
69.92; H, 6.79; N, 8.58. Found: C, 69.90; H, 6.77; N, 8.56.
6.3. General procedure II: preparation of compounds 5aef
To
a
solution of the appropriate benzophenone 4aef
6.4. General procedure III: preparation of compounds 6aef
(16.58 mmol) in 56 mL of 3:1 ethanol/water mixture was added
portionwise NaBH4 (6.0 eq). The reaction mixture was allowed to
stir at room temperature for 2 h. The volatiles were evaporated
under reduced pressure, and the pH of the solution was adjusted to
5 by dropwise addition of 1 M HCl solution. The obtained solid was
filtered and air dried to afford the pure product 5aef.
A solution of the appropriate alcohol 5aef (37.38 mmol) in
50 mL of CHCl3 was added dropwise to 150 mL of 37% HCl. The
biphasic reaction mixture was allowed to stir at room temperature
overnight. The organic layer was separated, washed with water
(2 ꢃ 25 mL), dried, and evaporated under reduced pressure to give
pure product 6aef.
6.3.1. 4-[Hydroxy-(3-methoxyphenyl)methyl]-N,N-
diethylbenzamide (5a)
6.4.1. 4-[Chloro-(3-methoxyphenyl)methyl]-N,N-diethylbenzamide
(6a)
General procedure II was used to convert 4a into the title
product. Yield 99%; Rf 0.16 (petroleum ether/ethyl acetate 1:1); IR
General procedure III was used to convert 5a into the title
product. Yield 95%; Rf 0.33 (petroleum ether/ethyl acetate 4:6); IR
(nujol): 1680, 3300; 1H NMR (CDCl3)
d 1.00e1.35 (m, 6H), 2.39 (s,
1H), 3.10e3.18 (m, 2H), 3.20e3.30 (m, 2H), 3.79 (s, 3H), 5.82 (d, 1H,
J ¼ 3.2 Hz), 6.75e7.00 (m, 3H), 7.24 (d, 2H, J ¼ 8.2 Hz), 7.25e7.38 (m,
1H), 7.41 (d, 2H, J ¼ 8.2 Hz). Anal. calcd for C19H23NO3: C, 72.82; H,
7.40; N, 4.47. Found: C, 72.67; H, 7.41; N, 4.46.
(nujol): 1680; 1H NMR (CDCl3)
d 1.00e1.78 (m, 6H), 3.08e3.38 (m,
2H), 3.40e3.60 (m, 2H), 3.80 (s, 3H), 6.09 (s, 1H), 6.80e7.00 (m, 4H),
7.35 (d, 2H, J ¼ 8.4 Hz), 7.44 (d, 2H, J ¼ 8.4 Hz). Anal. calcd for
C19H22ClNO2: C, 68.77; H, 6.68; N, 4.22. Found: C, 68.61; H, 6.67; N,
4.21.
6.3.2. 4-[Hydroxy-(3-methoxyphenyl)methyl]-N,N-
dimethylbenzamide (5b)
6.4.2. 4-[Chloro-(3-methoxyphenyl)methyl]-N,N-
dimethylbenzamide (6b)
General procedure III was used to convert 5b into the title
product. Yield 99%; Rf 0.35 (petroleum ether/ethyl acetate 4:6); IR
General procedure II was used to convert 4b into the title
product. Yield 99%; Rf 0.35 (CHCl3/CH3OH 95:5); IR (nujol): 1690,
3300; 1H NMR (CDCl3)
d 2.58 (bs, 1H), 2.90e3.20 (m, 6H), 3.78 (s,

G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
421
(nujol): 1690; 1H NMR (CDCl3)
6.53 (s, 1H), 6.70e7.60 (m, 8H). Anal. calcd for C17H18ClNO2: C,
d
2.90e3.20 (m, 6H), 3.79 (s, 3H),
6.5.2. 4-[(10-Benzyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]-N,N-dimethylbenzamide (7b)
67.21; H, 5.97; N, 4.61. Found: C, 67.15; H, 5.95; N, 4.61.
General procedure IV was used to convert 9-benzyl-9,10-
diazatricyclo[4.2.1.12,5]decane and 6b into the title product. Yield
50%; Rf 0.45 (petroleum ether/ethyl acetate 1:1); IR (nujol): 1700;
6.4.3. {4-[Chloro(3-methoxyphenyl)methyl]phenyl}(pyrrolidin-1-
yl)methanone (6c)
1H NMR (CDCl3)
d 1.60e2.30 (m, 8H), 2.70e2.85 (m, 4H), 2.90e3.15
General procedure III was used to convert 5c into the title
product. Yield 99%; Rf 0.21 (petroleum ether/ethyl acetate 8:2); IR
(m, 6H), 3.36 (s, 2H), 3.79 (s, 3H), 4.29 (s, 1H), 6.65e6.75 (m, 1H),
7.10e7.40 (m, 10H), 7.55 (d, 2H, J ¼ 8.4 Hz). Anal. calcd for
(nujol): 1680; 1H NMR (CDCl3)
d
1.90e2.00 (m, 4H), 3.42 (t, 2H,
C32H37N3O2: C, 77.54; H, 7.52; N, 8.48. Found: C, 77.37; H, 7.50; N,
J ¼ 6.4 Hz), 3.64 (t, 2H, J ¼ 6.4 Hz), 3.79 (s, 3H), 6.09 (s, 1H), 6.90e
7.00 (m, 3H), 7.20e7.60 (m, 5H). Anal. calcd for C19H20ClNO2: C,
69.19; H, 6.11; N, 4.25. Found: C, 69.07; H, 6.09; N, 4.24.
8.49.
6.5.3. {4-[(10-Benzyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]phenyl}pyrrolidin-1-yl-methanone (7c)
General procedure IV was used to convert 9-benzyl-9,10-
diazatricyclo[4.2.1.12,5]decane and 6c into the title product. Yield
44%; Rf 0.42 (petroleum ether/ethyl acetate 1:1); IR (nujol): 1680;
6.4.4. {4-[Chloro(3-methoxyphenyl)methyl]phenyl}(piperidin-1-yl)
methanone (6d)
General procedure III was used to convert 5d into the title
product. Yield 99%; Rf 0.50 (petroleum ether/ethyl acetate 8:2); IR
1H NMR (CDCl3)
d 1.50e2.25 (m, 12H), 2.70e2.90 (m, 4H), 3.30e
(nujol): 1690; 1H NMR (CDCl3)
d
1.40e1.80 (m, 6H), 3.30e3.45 (m,
3.45 (m, 4H), 3.55e3.65 (m, 2H), 3.79 (s, 3H), 4.29 (s, 1H), 6.65e6.75
(m, 1H), 7.00e7.50 (m, 10H), 7.55 (d, 2H, J ¼ 8.4 Hz). Anal. calcd for
2H), 3.60e3.78 (m, 2H), 3.80 (s, 3H), 6.09 (s, 1H), 6.80e7.00 (m, 3H),
7.20e7.50 (m, 5H). Anal. calcd for C20H22ClNO2: C, 69.86; H, 6.45; N,
4.07. Found: C, 69.70; H, 6.44; N, 4.06.
C34H39N3O2: C, 78.28; H, 7.54; N, 8.05. Found: C, 78.22; H, 7.55; N,
8.04.
6.4.5. 4-[Chloro-(3-methoxyphenyl)methyl]-N-
cyclohexylbenzamide (6e)
6.5.4. {4-[(10-Benzyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]phenyl}piperidin-1-yl-methanone (7d)
General procedure IV was used to convert 9-benzyl-9,10-
diazatricyclo[4.2.1.12,5]decane and 6d into the title product. Yield
85%; Rf 0.27 (petroleum ether/ethyl acetate 7:3); IR (nujol): 1650;
General procedure III was used to convert 5e into the title
product. Yield 97%; Rf 0.31 (petroleum ether/ethyl acetate 8:2); IR
(nujol): 1690, 3100; 1H NMR (CDCl3)
d 1.00e1.80 (m, 8H), 1.95e2.10
(m, 2H), 3.78 (s, 3H), 3.85e4.05 (m, 1H), 5.90e6.00 (m, 1H), 6.09 (s,
1H), 6.80e7.00 (m, 3H), 7.20e7.30 (m, 1H), 7.46 (d, 2H, J ¼ 8.4 Hz),
7.72 (d, 2H, J ¼ 8.4 Hz). Anal. calcd for C21H24ClNO2: C, 70.48; H,
6.76; N, 3.91. Found: C, 70.27; H, 6.74; N, 3.90.
1H NMR (CDCl3)
d 1.40e1.85 (m, 10H), 1.90e2.00 (m, 2H), 2.15e
2.25 (m, 2H), 2.70e2.85 (m, 4H), 3.20e3.35 (m, 2H), 3.36 (s, 2H),
3.60e3.75 (m, 2H), 3.79 (s, 3H), 4.28 (s, 1H), 6.65e6.75 (m, 1H),
7.05e7.40 (m, 10H), 7.55 (d, 2H, J ¼ 8.0 Hz). Anal. calcd for
C35H41N3O2: C, 78.47; H, 7.71; N, 7.84. Found: C, 78.35; H, 7.69; N,
6.4.6. 4-[Chloro-(3-methoxyphenyl)methyl]-N-pyrrolidin-1-yl-
benzamide (6f)
7.81.
General procedure III was used to convert 5f into the title
product. Yield 95%; Rf 0.50 (CHCl3/CH3OH 9:1); IR (nujol): 1680,
6.5.5. 4-[(10-Benzyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]-N-cyclohexylbenzamide (7e)
3300; 1H NMR (CDCl3)
d
1.70e2.10 (m, 4H), 2.90e3.15 (m, 4H), 3.79
General procedure IV was used to convert 9-benzyl-9,10-
diazatricyclo[4.2.1.12,5]decane and 6e into the title product. Yield
42%; Rf 0.45 (petroleum ether/ethyl acetate 8:2); IR (nujol): 1720,
(s, 3H), 6.09 (s, 1H), 6.75 (s, 1H), 6.77e7.00 (m, 2H), 7.20e7.35 (m,
2H), 7.69 (d, 2H, J ¼ 8.0 Hz), 7.71 (d, 2H, J ¼ 8.0 Hz). Anal. calcd for
C
19H21ClN2O2: C, 66.18; H, 6.14; N, 8.12. Found: C, 66.04; H, 6.13; N,
3100; 1H NMR (CDCl3)
d 1.00e1.55 (m, 5H),1.58e1.85 (m, 6H),1.90e
8.10.
2.10 (m, 4H), 2.15e2.25 (m, 2H), 2.70e2.90 (m, 4H), 3.36 (s, 2H),
3.78 (s, 3H), 4.31 (s, 1H), 5.85 (d, 2H, J ¼ 7.4 Hz), 6.65e6.75 (m, 1H),
7.00e7.40 (m, 8H), 7.55e7.65 (m, 4H), 8.10 (d, 1H, J ¼ 7.2 Hz). Anal.
calcd for C36H43N3O2: C, 78.65; H, 7.88; N, 7.64. Found: C, 78.59; H,
7.87; N, 7.63.
6.5. General procedure IV: preparation of compounds 7aef and 8a,
c, d
A mixture of the appropriate chloride 6aef (1.5 eq), 9-benzyl-
6.5.6. 4-[(10-Benzyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]-N-pyrrolidin-1-yl-benzamide (7f)
9,10-diazatricyclo[4.2.1.12,5]decane
[31]
or
2-benzyl-2,7-
diazatricyclo[4.4.0.03,8]decane [31] (0.5 g, 2.19 mmol), and anhy-
drous K2CO3 (3 eq) in 20 mL of anhydrous acetonitrile was allowed
to stir at reflux for 72 h, under Ar. The solution was filtered, and the
volatiles were evaporated under reduced pressure. The residue was
purified by flash chromatography to afford the desired compounds
7aef and 8a, c, d.
General procedure IV was used to convert 9-benzyl-9,10-
diazatricyclo[4.2.1.12,5]decane and 6f into the title product. Yield
87%; Rf 0.39 (petroleum ether/ethyl acetate 2:8); IR (nujol): 1680,
3100; 1H NMR (CDCl3)
d 1.20e2.00 (m, 8H), 2.10e2.40 (m, 4H),
2.70e2.90 (m, 4H), 2.95e3.05 (m, 4H), 3.36 (s, 2H), 3.76 (s, 3H), 4.15
(s, 1H), 5.85 (s, 1H), 6.70e7.70 (m, 11H), 8.00e8.20 (m, 2H). Anal.
calcd for C34H40N4O2: C, 76.09; H, 7.51; N, 10.44. Found: C, 75.98; H,
7.50; N, 10.41.
6.5.1. 4-[(10-Benzyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]-N,N-diethylbenzamide (7a)
General procedure IV was used to convert 9-benzyl-9,10-
diazatricyclo[4.2.1.12,5]decane and 6a into the title product. Yield
52%; Rf 0.40 (petroleum ether/ethyl acetate 7:3); IR (nujol): 1680;
6.5.7. 4-[(7-Benzyl-2,7-diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-
methoxyphenyl)methyl]-N,N-diethylbenzamide (8a)
General procedure IV was used to convert 2-benzyl-2,7-
diazatricyclo[4.4.0.03,8]decane and 6a into the title product. Yield
65%; Rf 0.21 (petroleum ether/ethyl acetate 1:1); IR (nujol): 1680;
1H NMR (CDCl3)
d 1.00e1.30 (m, 6H), 1.60e2.20 (m, 8H), 2.75e2.85
(m, 4H), 3.20e3.36 (m, 2H), 3.37 (s, 2H), 3.40e3.60 (m, 2H), 3.80 (s,
3H), 4.28 (s, 1H), 6.70e6.80 (m, 1H), 7.00e7.40 (m, 10H), 7.55 (d, 2H,
J ¼ 8.4 Hz). Anal. calcd for C34H41N3O2: C, 77.98; H, 7.89; N, 8.02.
Found: C, 77.71; H, 7.87; N, 8.00.
1H NMR (CDCl3)
d 1.00e1.95 (m, 14H), 2.70e2.90 (m, 4H), 3.15e3.35
(m, 2H), 3.40e3.60 (m, 2H), 3.78 (s, 3H), 3.80e4.00 (m, 2H), 5.07 (s,
1H), 6.65e6.75 (m, 1H), 7.00e7.40 (m, 10H), 7.52 (d, 2H, J ¼ 8.2 Hz).

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G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
Anal. calcd for C34H41N3O2: C, 77.98; H, 7.89; N, 8.02. Found: C,
77.80; H, 7.88; N, 8.00.
3100; 1H NMR (CDCl3)
d 1.40e1.90 (m, 13H), 2.20e2.40 (m, 2H),
2.75e2.95 (m, 2H), 3.06 (s, 2H), 3.22e3.45 (m, 2H), 3.60e3.75 (m,
2H), 3.80 (s, 3H), 4.22 (s, 1H), 6.70e6.80 (m, 1H), 7.00e7.40 (m, 5H),
7.55 (d, 2H, J ¼ 8.0 Hz). Anal. calcd for C28H35N3O2: C, 75.47; H, 7.92;
N, 9.43. Found: C, 75.23; H, 7.90; N, 9.42.
6.5.8. {4-[(7-Benzyl-2,7-diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-
methoxyphenyl)methyl]phenyl}pyrrolidin-1-yl-methanone (8c)
General procedure IV was used to convert 2-benzyl-2,7-
diazatricyclo[4.4.0.03,8]decane and 6c into the title product. Yield
44%; Rf 0.20 (petroleum ether/ethyl acetate 1:1); IR (nujol): 1680;
6.6.5. 4-[(9,10-Diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-methoxyphenyl)
methyl]-N-cyclohexylbenzamide (9e)
1H NMR (CDCl3)
d
1.40e2.00 (m,12H), 2.75e3.10 (m, 4H), 3.30e3.50
General procedure V was used to convert 7e into the title
product. Yield 80%; Rf 0.32 (CHCl3/CH3OH 9:1); IR (nujol): 1680,
(m, 2H), 3.55e3.75 (m, 2H), 3.78 (s, 3H), 4.00e4.20 (m, 2H), 5.07 (s,
1H), 6.90e7.60 (m, 13H). Anal. calcd for C34H39N3O2: C, 78.28; H,
7.54; N, 8.05. Found: C, 78.20; H, 7.53; N, 8.03.
3100, 3300; 1H NMR (CDCl3)
d 1.05e1.45 (m, 6H), 1.50e2.00 (m,
6H), 2.00e2.35 (m, 7H), 2.40e2.55 (m, 2H), 3.60e3.77 (m, 3H), 3.78
(s, 3H), 4.33 (s, 1H), 6.74 (d, 1H, J ¼ 6.8 Hz), 7.08 (d, 1H, J ¼ 11.6 Hz),
7.10e7.25 (m, 3H), 7.57 (d, 2H, J ¼ 8.0 Hz), 7.75 (d, 2H, J ¼ 8.0 Hz).
Anal. calcd for C29H37N3O2: C, 75.78; H, 8.11; N, 9.14. Found: C,
75.70; H, 8.09; N, 9.13.
6.5.9. {4-[(7-Benzyl-2,7-diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-
methoxyphenyl)methyl]phenyl}piperidin-1-yl-methanone (8d)
General procedure IV was used to convert 2-benzyl-2,7-
diazatricyclo[4.4.0.03,8]decane and 6d into the title product. Yield
90%; Rf 0.36 (petroleum ether/ethyl acetate 6:4); IR (nujol): 1680;
6.6.6. 4-[(9,10-Diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-methoxyphenyl)
methyl]-N-pyrrolidin-1-yl-benzamide (9f)
1H NMR (CDCl3)
d 1.30e2.10 (m,14H), 2.75e2.90 (m, 4H), 3.20e3.45
(m, 2H), 3.55e3.75 (m, 2H), 3.77 (s, 3H), 3.79 (s, 2H), 5.10 (s, 1H),
6.65e7.40 (m, 11H), 8.00e8.20 (m, 2H). Anal. calcd for C35H41N3O2:
C, 78.47; H, 7.71; N, 7.84. Found: C, 78.40; H, 7.70; N, 7.82.
General procedure V was used to convert 7f into the title
product. Yield 75%; Rf 0.27 (CHCl3/CH3OH 8:2); IR (nujol): 1680,
3200; 1H NMR (CDCl3)
d 1.20e1.90 (m, 8H), 1.92e2.30 (m, 8H),
2.95e3.00 (m, 1H), 3.05e3.20 (m, 4H), 3.76 (s, 3H), 3.98 (s, 1H), 4.80
(bs, 1H), 6.65e6.80 (m, 2H), 7.00e7.30 (m, 4H), 7.90e8.05 (m, 2H).
Anal. calcd for C27H34N4O2: C, 72.62; H, 7.67; N, 12.55. Found: C,
72.48; H, 7.66; N, 12.53.
6.6. General procedure V: preparation of compounds 9aef and 10a,
c, d
A solution of the appropriate benzyl diazatricyclodecane 7 or 8
(0.38 mmol) in 5 mL of ethanol was hydrogenated over 10% Pd/C
(0.1 eq) in a Parr shaker under a hydrogen pressure of 3 atm at 60 ꢁC
for 6 h. The mixture was filtered through Celite and the catalyst
washed with several portions of ethanol. The solution was evapo-
rated to afford the pure product 9aef or 10a, c, d.
6.6.7. 4-[(2,7-Diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-methoxyphenyl)
methyl]-N,N-diethylbenzamide (10a)
General procedure V was used to convert 8a into the title
product. Yield 90%; Rf 0.27 (CHCl3/CH3OH 9:1); IR (nujol): 1670,
3100; 1H NMR (CDCl3)
d 1.00e1.40 (m, 6H), 1.90e2.50 (m, 10H),
2.90e3.00 (m, 2H), 3.10e3.30 (m, 2H), 3.40e3.60 (m, 3H), 3.96 (s,
3H), 4.95 (s, 1H), 6.65e6.85 (m, 1H), 7.00e7.40 (m, 5H), 7.48 (d, 2H,
J ¼ 6.0 Hz). Anal. calcd for C27H35N3O2: C, 74.79; H, 8.14; N, 9.69.
Found: C, 74.70; H, 8.13; N, 9.67.
6.6.1. 4-[(9,10-Diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-methoxyphenyl)
methyl]-N,N-diethylbenzamide (9a)
General procedure V was used to convert 7a into the title
product. Yield 99%; Rf 0.40 (CHCl3/CH3OH 9:1); IR (nujol): 1630,
3200; 1H NMR (CDCl3)
d
1.00e1.45 (m, 6H), 1.90e2.43 (m, 10H),
6.6.8. {4-[(2,7-Diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-
2.90e3.10 (m, 2H), 3.10e3.30 (m, 2H), 3.40e3.60 (m, 2H), 3.66 (bs,
1H), 3.79 (s, 3H), 4.26 (s, 1H), 6.75e6.80 (m, 1H), 7.03e7.38 (m, 5H),
7.60 (d, 2H, J ¼ 8.0 Hz). Anal. calcd for C27H35N3O2: C, 74.79; H, 8.14;
N, 9.69. Found: C, 74.61; H, 8.12; N, 9.67.
methoxyphenyl)methyl]phenyl}pyrrolidin-1-yl-methanone (10c)
General procedure V was used to convert 8c into the title
product. Yield 90%; Rf 0.27 (CHCl3/CH3OH 9:1); IR (nujol): 1680,
3100; 1H NMR (CDCl3)
d 1.10e2.20 (m, 11H), 2.25e2.35 (m, 2H),
2.95e3.05 (m, 2H), 3.30e3.40 (m, 3H), 3.55e3.75 (m, 3H), 3.78 (s,
3H), 4.96 (s, 1H), 7.00e7.09 (m, 2H), 7.15e7.30 (m, 2H), 7.40e7.55
(m, 4H). Anal. calcd for C27H33N3O2: C, 75.14; H, 7.71; N, 9.74.
Found: C, 74.97; H, 7.70; N, 9.73.
6.6.2. 4-[(9,10-Diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-methoxyphenyl)
methyl]-N,N-dimethylbenzamide (9b)
General procedure V was used to convert 7b into the title product.
Yield 99%; Rf 0.35 (CHCl3/CH3OH 9:1); IR (nujol): 1620, 3100; 1H NMR
(CDCl3)
d
2.00e2.50 (m, 10H), 2.90e3.10 (m, 9H), 3.78 (s, 3H), 4.34 (s,
6.6.9. {4-[(2,7-Diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-
1H), 6.70e6.85(m,1H), 7.00e7.60(m, 7H). Anal. calcdforC25H31N3O2:
C, 74.04; H, 7.70; N, 10.36. Found: C, 73.88; H, 7.68; N, 10.33.
methoxyphenyl)methyl]phenyl}piperidin-1-yl-methanone (10d)
General procedure V was used to convert 8d into the title
product. Yield 99%; Rf 0.40 (CHCl3/CH3OH 9:1); IR (nujol): 1680,
6.6.3. {4-[(9,10-Diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
3100; 1H NMR (CDCl3)
d 1.40e2.10 (m, 13H), 2.15e2.30 (m, 1H),
methoxyphenyl)methyl]phenyl}pyrrolidin-1-yl-methanone (9c)
General procedure V was used to convert 7c into the title
product. Yield 99%; Rf 0.23 (CHCl3/CH3OH 9:1); IR (nujol): 1650,
2.80e3.00 (m, 2H), 3.15e3.45 (m, 2H), 3.50e3.75 (m, 2H), 3.78 (s,
3H), 5.00e5.25 (m, 3H), 5.70e5.90 (m, 1H), 6.65e7.40 (m, 6H),
7.95e8.15 (m, 2H). Anal. calcd for C28H35N3O2: C, 75.47; H, 7.92; N,
9.43. Found: C, 75.35; H, 7.91; N, 9.40.
3300; 1H NMR (CDCl3)
d 1.80e2.10 (m, 11H), 2.30e2.40 (m, 2H),
2.80e3.00 (m, 2H), 3.30e3.45 (m, 4H), 3.55e3.70 (m, 2H), 3.79 (s,
3H), 4.29 (s, 1H), 6.70e6.80 (m, 1H), 7.00e7.30 (m, 3H), 7.43 (d, 2H,
J ¼ 8.0 Hz), 7.53 (d, 2H, J ¼ 8.0 Hz). Anal. calcd for C27H33N3O2: C,
75.14; H, 7.71; N, 9.74. Found: C, 75.02; H, 7.70; N, 9.72.
6.7. General procedure VI: preparation of compounds 1Aaef and
2Aa, c, d
A mixture of the appropriate diazatricyclodecane 9 or 10
(0.98 mmol), allyl bromide (1.5 eq), and anhydrous K2CO3 (3 eq) in
acetone (25 mL) was stirred at reflux overnight. The inorganic salt
was filtered off, and the solvent was then evaporated to give pure
product 1A or 2A.
6.6.4. {4-[(9,10-Diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]phenyl}piperidin-1-yl-methanone (9d)
General procedure V was used to convert 7d into the title
product. Yield 99%; Rf 0.40 (CHCl3/CH3OH 9:1); IR (nujol): 1680,

G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
423
6.7.1. 4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]-N,N-diethylbenzamide (1Aa)
2, 129.4, 133.7, 137.4, 144.9, 147.4, 159.6, 166.5. Anal. calcd for
32H41N3O2: C, 76.92; H, 8.27; N, 8.41. Found: C, 76.77; H, 8.25; N,
C
General procedure VI was used to convert 9a into the title
product. Yield 96%; Rf 0.27 (petroleum ether/ethyl acetate 1:1); mp
8.40.
99e101 ꢁC; IR (nujol): 1680; 1H NMR (CDCl3)
d
1.00e1.30 (m, 6H),
6.7.6. 4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]-N-pyrrolidin-1-yl-benzamide (1Af)
General procedure VI was used to convert 9f into the title
product. Yield 80%; Rf 0.35 (CHCl3/CH3OH 95:5); mp 70e72 ꢁC; IR
1.50e2.20 (m, 8H), 2.70e2.90 (m, 6H), 3.20e3.35 (m, 2H), 3.40e
3.55 (m, 2H), 3.80 (s, 3H), 4.28 (s, 1H), 4.98e5.15 (m, 2H), 5.70e5.90
(m, 1H), 6.65e6.80 (m, 1H), 7.00e7.20 (m, 5H), 7.54 (d, 2H,
J ¼ 8.0 Hz); 13C NMR (CDCl3)
d
12.9, 14.2, 25.5ꢃ 2, 25.6, 25.7, 39.1,
(nujol): 1680, 3200; 1H NMR (CDCl3)
d 1.20e1.80 (m, 8H), 2.00e
43.2, 55.2, 55.7, 63.2, 63.3, 65.1, 65.3, 69.7, 112.0, 113.5, 115.6, 120.3,
126.5ꢃ 2, 127.8ꢃ 2, 129.3, 135.7, 137.4, 145.0, 145.2, 159.5, 171.2.
Anal. calcd for C30H39N3O2: C, 76.07; H, 8.30; N, 8.87. Found: C,
75.88; H, 8.28; N, 8.84.
2.55 (m, 8H), 3.40e3.60 (m, 2H), 3.75 (s, 3H), 3.95 (s, 1H), 4.25e4.40
(m, 2H), 4.60 (d, 2H, J ¼ 6.8 Hz), 5.40e5.55 (m, 2H), 5.95e6.15 (m,
1H), 6.65e6.80 (m, 2H), 7.10e7.25 (m, 4H), 7.89 (d, 2H, J ¼ 8.0 Hz),
8.02 (s, 1H); 13C NMR (CDCl3)
d 10.8, 13.9, 19.1, 21.6, 23.6, 28.8, 29.6,
30.2, 38.6, 41.7, 55.0, 63.7, 64.2, 68.0, 71.7, 111.3, 114.5, 121.2, 124.5,
127.7, 127.8, 128.3, 128.6, 128.7, 129.2, 130.8, 142.3, 142.9, 159.5,
169.6. Anal. calcd for C30H38N4O2: C, 74.04; H, 7.87; N, 11.51. Found:
C, 73.93; H, 7.86; N, 11.48.
6.7.2. 4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]-N,N-dimethylbenzamide (1Ab)
General procedure VI was used to convert 9b into the title
product. Yield 88%; Rf 0.41 (petroleum ether/ethyl acetate 1:1); mp
100e103 ꢁC; IR (nujol): 1670; 1H NMR (CDCl3)
d
1.50e2.10 (m, 8H),
6.7.7. 4-[(7-Allyl-2,7-diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-
methoxyphenyl)methyl]-N,N-diethylbenzamide (2Aa)
2.70e3.10 (m, 12H), 3.79 (s, 3H), 4.29 (s, 1H), 4.95e5.15 (m, 2H),
5.70e5.95 (m, 1H), 6.60e6.68 (m, 1H), 7.00e7.40 (m, 5H), 7.56 (d,
General procedure VI was used to convert 10a into the title
2H, J ¼ 7.4 Hz); 13C NMR (CDCl3)
25.5ꢃ 2, 25.6ꢃ 2, 29.6, 35.3, 39.6,
d
product. Yield 80%; Rf 0.22 (CH2Cl2/Acetone 1:1); mp 217e220 ꢁC
55.1, 55.6, 63.2, 65.2, 65.3, 69.6,112.0,113.4,115.7,120.2,127.2,127.3,
127.7, 128.7, 129.3, 134.7, 137.2, 145.1, 145.4, 159.5, 171.5. Anal. calcd
for C28H35N3O2: C, 75.47; H, 7.92; N, 9.43. Found: C, 75.30; H, 7.91;
N, 9.41.
(hydrochloride); IR (nujol): 1680; 1H NMR (CDCl3)
d 1.00e1.95 (m,
16H), 2.77e2.89 (m, 2H), 3.15e3.30 (m, 2H), 3.32e3.40 (m, 2H),
3.40e3.60 (m, 2H), 3.79 (s, 3H), 5.00e5.25 (m, 3H), 5.70e5.90 (m,
1H), 6.65e6.75 (m, 1H), 7.00e7.30 (m, 5H), 7.53 (dd, 2H, J ¼ 2.0 and
8.0 Hz); 13C NMR (CDCl3)
d
12.8, 14.2, 22.1ꢃ 2, 22.4ꢃ 2, 29.7, 39.2,
6.7.3. {4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]phenyl}-pyrrolidin-1-yl-methanone (1Ac)
General procedure VI was used to convert 9c into the title
product. Yield 94%; Rf 0.17 (petroleum ether/ethyl acetate 1:1); mp
43.3, 53.7, 55.1, 55.3, 56.0ꢃ 2, 70.3, 111.7, 112.6, 113.4, 113.5, 119.9,
120.3, 126.4, 126.5, 126.6, 127.2, 127.4, 127.8, 129.2, 129.3, 159.5.
Anal. calcd for C30H39N3O2: C, 76.07; H, 8.30; N, 8.87. Found: C,
75.84; H, 8.28; N, 8.85.
46e48 ꢁC; IR (nujol): 1680; 1H NMR (CDCl3)
d 1.60e2.30 (m, 12H),
2.70e2.95 (m, 6H), 3.30e3.45 (m, 2H), 3.50e3.70 (m, 2H), 3.79 (s,
3H), 4.28 (s, 1H), 4.95e5.15 (m, 2H), 5.70e5.95 (m, 1H), 6.65e6.75
(m, 1H), 7.00e7.15 (m, 3H), 7.41 (d, 2H, J ¼ 8.0 Hz), 7.55 (d, 2H,
6.7.8. {4-[(7-Allyl-2,7-diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-
methoxyphenyl)methyl]phenyl}pyrrolidin-1-yl-methanone (2Ac)
General procedure VI was used to convert 10c into the title
product. Yield 99%; Rf 0.25 (CH2Cl2/Acetone 1:1); mp 126e128 ꢁC;
J ¼ 8.0 Hz); 13C NMR (CDCl3)
d
24.4, 25.5ꢃ 2, 25.6ꢃ 2, 26.3, 46.1,
49.6, 55.1, 55.7, 63.2ꢃ 2, 65.2, 66.6, 69.7, 112.0, 113.5, 115.6, 120.3,
127.2ꢃ 2, 127.6ꢃ 2, 129.3, 135.7, 137.5, 145.1, 145.7, 159.5,169.5. Anal.
calcd for C30H37N3O2: C, 76.40; H, 7.91; N, 8.91. Found: C, 76.24; H,
7.89; N, 8.89.
IR (nujol): 1680; 1H NMR (CDCl3)
d 1.10e2.00 (m, 12H), 2.70e3.00
(m, 4H), 3.20e3.50 (m, 4H), 3.50e3.70 (m, 2H), 3.77 (s, 3H), 5.00e
5.25 (m, 3H), 5.70e5.90 (m, 1H), 6.65e6.80 (m, 1H), 7.00e7.35 (m,
3H), 7.40 (d, 2H, J ¼ 8.0 Hz), 7.53 (d, 2H, J ¼ 8.0 Hz); 13C NMR (CDCl3)
d
19.0, 22.0ꢃ 2, 22.3ꢃ 2, 24.3, 26.2, 46.0, 49.5, 53.4, 53.6, 55.0, 55.2,
6.7.4. {4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]phenyl}-piperidin-1-yl-methanone (1Ad)
General procedure VI was used to convert 9d into the title
product. Yield 99%; Rf 0.40 (petroleum ether/ethyl acetate 4:6); mp
55.9, 70.2, 111.7, 112.1, 113.2, 113.4, 115.5, 119.7, 120.1, 127.1ꢃ 2, 127.2,
127.5, 129.2,137.5, 159.5,169.5. Anal. calcd for C30H37N3O2: C, 76.40;
H, 7.91; N, 8.91. Found: C, 76.28; H, 7.88; N, 8.90.
134e136 ꢁC; IR (nujol): 1650; 1H NMR (CDCl3)
d
1.40e1.90 (m, 10H),
6.7.9. {4-[(7-Allyl-2,7-diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-
methoxyphenyl)methyl]phenyl}piperidin-1-yl-methanone (2Ad)
General procedure VI was used to convert 10d into the title
product. Yield 90%; Rf 0.27 (petroleum ether/ethyl acetate 7:3); mp
1.91e2.00 (m, 2H), 2.10e2.25 (m, 2H), 2.70e2.95 (m, 6H), 3.20e
3.45 (m, 2H), 3.55e3.75 (m, 2H), 3.80 (s, 3H), 4.28 (s,1H), 5.00e5.20
(m, 2H), 5.70e5.90 (m, 1H), 6.70e6.80 (m, 1H), 7.00e7.40 (m, 5H),
7.55 (d, 2H, J ¼ 8.0 Hz); 13C NMR (CDCl3)
d
19.1, 24.5, 25.6ꢃ 2, 25.7ꢃ
94e96 ꢁC (hydrochloride); IR (nujol): 1680; 1H NMR (CDCl3)
d 1.15e
2, 29.2, 30.9, 53.8, 55.2, 55.7, 63.2, 63.3, 65.2, 65.3, 69.7, 112.0, 113.5,
115.6, 120.3, 127.0, 127.8, 128.8, 129.3, 130.9, 134.9, 137.4, 145.2,
145.3, 159.5, 170.2. Anal. calcd for C31H39N3O2: C, 76.67; H, 8.09; N,
8.65. Found: C, 76.51; H, 8.06; N, 8.64.
2.00 (m, 18H), 2.75e2.85 (m, 2H), 3.20e3.40 (m, 2H), 3.60e3.75 (m,
2H), 3.78 (s, 3H), 5.00e5.25 (m, 3H), 5.70e5.90 (m, 1H), 6.65e7.40
(m, 6H), 7.95e8.15 (m, 2H); 13C NMR (CDCl3)
d
9.9, 13.0, 18.1ꢃ 2,
21.8, 22.6, 26.6, 27.8, 28.5, 28.8, 29.2, 37.6, 54.2, 66.8, 70.5, 111.9,
118.0, 125.9, 126.0, 127.6ꢃ 2, 127.7, 128.9, 129.2, 129.9ꢃ 2, 131.2,
131.3, 166.3, 166.4. Anal. calcd for C31H39N3O2: C, 76.67; H, 8.09; N,
8.65. Found: C, 76.56; H, 8.08; N, 8.64.
6.7.5. 4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
methoxyphenyl)methyl]-N-cyclohexylbenzamide (1Ae)
General procedure VI was used to convert 9e into the title
product. Yield 99%; Rf 0.31 (petroleum ether/ethyl acetate 8:2); mp
6.8. General procedure VII: preparation of compounds 1Baef and
2Ba, c, d
58e60 ꢁC; IR (nujol): 1680, 3100; 1H NMR (CDCl3)
d 1.10e1.80 (m,
14H), 1.85e2.10 (m, 4H), 2.15e2.20 (m, 2H), 2.70e2.95 (m, 5H), 3.78
(s, 3H), 4.31 (s, 1H), 5.00e5.20 (m, 2H), 5.70e5.95 (m, 2H), 6.65e
6.75 (m, 1H), 7.00e7.25 (m, 3H), 7.50e7.70 (m, 4H); 13C NMR
To a solution of the appropriate methoxy derivative 1A or 2A
(0.50 mmol) in CH2Cl2 (10 mL), cooled in an ice bath, BBr3 (2 eq)
was added dropwise, and the mixture was stirred at 0 ꢁC for 1.5 h.
The cold bath was removed, the reaction was quenched with 1%
(CDCl3)
d
24.8ꢃ 2, 25.5ꢃ 2, 25.6, 25.7ꢃ 2, 33.2ꢃ 2, 48.5, 55.1, 55.7,
63.2, 63.3, 65.2, 65.3, 69.6,112.1, 113.4, 115.6, 120.2,126.9ꢃ 2, 127.9ꢃ

424
G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
KOH, and the mixture extracted with dichloromethane. The extract
was dried (Na2SO4) and concentrated to give pure product 1B or 2B.
32.2ꢃ 2, 48.1ꢃ 2, 53.9, 60.5, 60.6, 66.7, 67.7, 113.8, 114.4, 117.7, 124.5,
126.8ꢃ 2, 126.9, 127.2ꢃ 2, 129.2, 134.2, 141.9, 144.2, 157.1, 165.8.
Anal. calcd for C31H39N3O2: C, 76.67; H, 8.09; N, 8.65. Found: C,
76.61; H, 8.07; N, 8.64.
6.8.1. 4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
hydroxyphenyl)methyl]-N,N-diethylbenzamide (1Ba)
General procedure VII was used to convert 1Aa into the title
product. Yield 99%; Rf 0.25 (petroleum ether/ethyl acetate 3:7); mp
112e115 ꢁC (CH3CN/water); IR (nujol): 1680, 3400; 1H NMR (CDCl3)
6.8.6. 4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
hydroxyphenyl)methyl]-N-pyrrolidin-1-yl-benzamide (1Bf)
General procedure VII was used to convert 1Af into the title
product. Yield 95%; Rf 0.40 (CHCl3/CH3OH 9:1); mp 76e78 ꢁC; IR
d
1.00e1.35 (m, 6H), 1.60e2.30 (m, 8H), 2.70e3.10 (m, 6H), 3.20e
3.35 (m, 2H), 3.45e3.75 (m, 2H), 4.26 (s, 1H), 5.30e5.50 (m, 2H),
6.40e6.60 (m, 2H), 6.65e6.75 (m, 1H), 6.90e7.40 (m, 5H), 7.51 (d,
(nujol): 1680, 3200, 3400; 1H NMR (CDCl3)
d 1.20e2.05 (m, 10H),
2.10e2.40 (m, 6H), 3.80e4.10 (m, 2H), 4.81 (d, 2H, J ¼ 7.0 Hz), 4.82e
5.05 (m, 4H), 5.50e5.70 (m, 2H), 5.90e6.15 (m, 1H), 6.60e6.85 (m,
2H), 7.00e7.40 (m, 4H), 7.90e8.10 (m, 2H), 11.60 (bs, 1H); 13C NMR
2H, J ¼ 8.0 Hz); 13C NMR (CDCl3)
d
12.8, 14.2, 24.6ꢃ 2, 25.0, 29.7,
39.5, 43.5, 54.9, 61.2ꢃ 2, 67.2, 67.5, 68.3, 114.5, 115.1, 118.9, 124.9,
126.8, 127.8, 129.9, 136.3, 142.5, 143.2, 157.3, 171.1. Anal. calcd for
(CDCl3) d 10.3, 13.5, 18.5, 20.7, 22.3, 23.1, 27.1, 29.0, 29.7, 41.2, 64.6,
C
9.12.
29H37N3O2: C, 75.78; H, 8.11; N, 9.14. Found: C, 75.63; H, 8.09; N,
65.4, 67.4, 71.1, 113.1, 115.4, 119.3, 124.8, 127.6, 127.7, 127.9, 128.2,
128.5, 128.9, 130.4, 140.7, 146.8, 156.9, 166.1. Anal. calcd for
C
29H36N4O2: C, 73.70; H, 7.68; N, 11.85. Found: C, 73.59; H, 7.66; N,
6.8.2. 4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
hydroxyphenyl)methyl]-N,N-dimethylbenzamide (1Bb)
11.82.
General procedure VII was used to convert 1Ab into the title
product. Yield 90%; Rf 0.25 (petroleum ether/ethyl acetate 3:7); mp
6.8.7. 4-[(7-Allyl-2,7-diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-
hydroxyphenyl)methyl]-N,N-diethylbenzamide (2Ba)
99e101 ꢁC; IR (nujol): 1650, 3300; 1H NMR (CDCl3)
d
1.50e2.10 (m,
General procedure VII was used to convert 2Aa into the title
8H), 2.50e3.10 (m, 12H), 4.30 (s, 1H), 5.30e5.55 (m, 2H), 6.40e6.60
product. Yield 75%; Rf 0.32 (CHCl3/CH3OH 9:1); mp 147e150 ꢁC; IR
(m, 1H), 6.65e6.80 (m, 1H), 7.00e7.80 (m, 7H), 8.70 (bs, 1H); 13C
(nujol): 1685, 3330; 1H NMR (CDCl3)
d
1.10e2.30 (m, 16H), 2.85e
4.00 (m, 8H), 4.85 (s, 1H), 5.30e5.60 (m, 2H), 6.40e7.60 (m, 9H),
9.55 (bs, 1H); 13C NMR (CDCl3)
NMR (CDCl3)
d
24.6, 25.1, 29.6ꢃ 2, 38.6, 54.9, 55.2, 61.3, 67.3, 68.0,
68.3, 71.7, 112.4, 113.8, 119.8, 124.9, 127.2, 127.6, 127.7, 127.8, 128.7,
130.8, 135.6, 142.7, 143.1, 159.8, 171.0. Anal. calcd for C27H33N3O2: C,
75.14; H, 7.71; N, 9.74. Found: C, 75.07; H, 7.70; N, 9.71.
d
12.8, 14.2, 16.8, 22.6, 29.7ꢃ 2, 39.8,
43.8, 50.7, 51.9, 54.9, 58.6, 58.8, 69.4, 114.15, 114.4, 115.1, 115.4, 118.4,
118.9, 125.1, 126.8, 127.3, 127.9, 128.3, 129.8, 157.4, 157.5, 171.4. Anal.
calcd for C29H37N3O2: C, 75.78; H, 8.11; N, 9.14. Found: C, 75.68; H,
8.08; N, 9.11.
6.8.3. {4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
hydroxyphenyl)methyl]phenyl}-pyrrolidin-1-yl-methanone (1Bc)
General procedure VII was used to convert 1Ac into the title
product. Yield 93%; Rf 0.45 (CHCl3/CH3OH 9:1); mp 156e158 ꢁC; IR
6.8.8. {4-[(7-Allyl-2,7-diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-
hydroxyphenyl)methyl]phenyl}pyrrolidin-1-yl-methanone (2Bc)
General procedure VII was used to convert 2Ac into the title
product. Yield 99%; Rf 0.33 (CHCl3/CH3OH 9:1); mp 140e142 ꢁC; IR
(nujol): 1650, 3300; 1H NMR (CDCl3)
d 1.60e2.20 (m, 12H), 2.50e
3.10 (m, 8H), 3.55e3.80 (m, 3H), 4.28 (s, 1H), 5.20e5.60 (m, 2H),
6.40e6.60 (m, 1H), 6.85e7.00 (m, 1H), 7.20e7.60 (m, 6H), 8.53 (bs,
(nujol): 1670, 3300; 1H NMR (CDCl3)
d 1.30e2.40 (m, 12H), 2.80e
1H); 13C NMR (CDCl3)
d
19.1, 24.4, 24.6, 25.2, 26.3, 29.7, 46.4, 49.9,
3.15 (m, 2H), 3.20e4.00 (m, 8H), 4.90 (s, 1H), 5.35e5.60 (m, 2H),
6.30e6.55 (m, 1H), 6.70e6.80 (m, 1H), 6.85e6.95 (m, 1H), 7.00e7.15
(m, 1H), 7.20e7.40 (m, 3H), 7.50e7.60 (m, 2H), 9.20 (bs, 1H); 13C
61.2, 61.3, 67.4, 67.5, 68.1, 68.3, 114.6, 115.2, 118.9, 125.1, 124.4,
127.6ꢃ 2, 128.7, 129.9, 130.8, 136.1, 144.0, 157.4, 169.4. Anal. calcd for
C
29H35N3O2: C, 76.12; H, 7.71; N, 9.18. Found: C, 75.88; H, 7.69; N,
NMR (CDCl3)
d
19.0ꢃ 2, 22.8, 23.6, 24.2, 27.6, 46.5, 49.8, 53.4, 54.8,
9.17.
58.6, 59.9, 68.0, 71.7, 114.6, 115.3, 118.1, 118.8, 125.1, 127.5, 127.7,
128.7ꢃ 2, 129.7, 130.8ꢃ 2, 142.7, 157.6, 169.5. Anal. calcd for
6.8.4. {4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
hydroxyphenyl)methyl]phenyl}-piperidin-1-yl-methanone (1Bd)
General procedure VII was used to convert 1Ad into the title
product. Yield 99%; Rf 0.20 (petroleum ether/ethyl acetate 7:3); mp
C29H35N3O2: C, 76.12; H, 7.71; N, 9.18. Found: C, 76.02; H, 7.70; N,
9.17.
6.8.9. {4-[(7-Allyl-2,7-diazatricyclo[4.4.0.03,8]dec-2-yl)-(3-
hydroxyphenyl)methyl]phenyl}piperidin-1-yl-methanone (2Bd)
General procedure VII was used to convert 2Ad into the title
product. Yield 91%; Rf 0.38 (CHCl3/CH3OH 9:1); mp 70e73 ꢁC; IR
133e135 ꢁC; IR (nujol): 1680, 3300; 1H NMR (CDCl3)
d 1.40e1.90 (m,
10H), 1.95e2.00 (m, 4H), 2.55e2.75 (m, 4H), 3.00e3.20 (m, 2H),
3.22e3.80 (m, 4H), 4.35 (s, 1H), 5.35e5.60 (m, 2H), 6.45e6.70 (m,
1H), 6.75e6.85 (m, 1H), 7.00e7.40 (m, 5H), 7.53 (d, 2H, J ¼ 8.2 Hz),
(nujol): 1680, 3400; 1H NMR (CDCl3)
d
1.20e1.90 (m, 20H), 3.15e
3.40 (m, 2H), 3.55e3.80 (m, 2H), 5.20e5.45 (m, 3H), 6.10e6.20 (m,
1H), 6.60e7.30 (m, 6H), 7.80e8.00 (m, 3H); 13C NMR (CDCl3)
10.9,
8.95 (bs, 1H); 13C NMR (CDCl3)
d
19.1, 24.4ꢃ 2, 24.7ꢃ 2, 25.2ꢃ 2,
54.9, 55.3ꢃ 2, 61.4ꢃ 2, 67.2, 67.3, 68.4, 112.4, 113.9, 119.9, 124.8,
127.6, 127.7, 127.9, 128.8, 130.0, 130.9135.4, 142.8, 143.1, 159.9, 169.8.
Anal. calcd for C30H37N3O2: C, 76.40; H, 7.91; N, 8.91. Found: C,
76.26; H, 7.89; N, 8.89.
d
14.0, 19.1ꢃ 2, 22.9, 23.7, 24.3, 27.7, 28.9, 29.7, 30.3, 38.7, 50.2, 68.1,
71.8, 114.5, 115.5, 120.1, 120.7, 127.0, 128.6, 128.7, 128.8, 128.9, 130.9,
140.5,141.0,142.5,156.8,168.0. Anal. calcd for C30H37N3O2: C, 76.40;
H, 7.91; N, 8.91. Found: C, 76.30; H, 7.89; N, 8.90.
6.8.5. 4-[(10-Allyl-9,10-diazatricyclo[4.2.1.12,5]dec-9-yl)-(3-
hydroxyphenyl)methyl]-N-cyclohexylbenzamide (1Be)
General procedure VII was used to convert 1Ae into the title
6.9. Chemicals and drugs for in vitro and in vivo assays
product. Yield 80%; Rf 0.29 (CHCl3/CH3OH 95:5); mp 68e70 ꢁC; IR
(nujol): 1690, 3300; 1H NMR (CDCl3)
d
1.00e1.50 (m, 10H), 1.55e
[3H]-DAMGO, [3H]-DPDPE, and [3H]-U 69,593 were purchased
from Perkin Elmer Italia. Naltrindole, Naloxone, CTAP, and U 69,593
were obtained from Tocris Cookson Ltd (Bristol, UK). Where not
differently specified, all the other chemicals were purchased from
Sigma Aldrich (Milan, Italy).
2.05 (m, 9H), 2.08e2.25 (m, 2H), 2.40e2.80 (m, 4H), 4.31 (s, 1H),
5.40e5.60 (m, 2H), 6.20e6.45 (m, 1H), 6.70 (d, 1H, J ¼ 7.8 Hz), 6.90e
7.25 (m, 4H), 7.56 (d, 2H, J ¼ 8.0 Hz), 7.74 (d, 2H, J ¼ 8.0 Hz), 8.40e
8.50 (bs, 1H); 13C NMR (CDCl3)
d
23.7ꢃ 2, 24.3ꢃ 2, 24.4ꢃ 2, 24.8,

G. Loriga et al. / European Journal of Medicinal Chemistry 69 (2013) 413e426
425
In vivo assays were carried out by solubilizing compounds in
saline solution containing CremophorÒEL (Basf GmbH) and Ethanol
(both at 5 wt%). All animal experiments were performed according
to the UE guidelines for the care and use of experimental animals
(CEE Nꢁ 86/609). Male CD-1 mice (Charles River, Calco, LC, Italy),
weighing 20e30 g, were housed in the animal care quarters;
temperatures were maintained at 22 ꢀ 2 ꢁC (humidity 55 ꢀ 5%) on a
12 h light/dark cycle. Food and water were available ad libitum
before to start with the experimental procedures.
the registration of the contractions induced by electric stimuli. The
samples were then equilibrated for 60 min, changing the Krebs
solution every 15 min. The vas deferens segments were then sub-
jected to treatment cycles wherein the electrical stimulation lasted
3 min, with an interval of 15 min before the following stimulation.
The isometric contractions were evoked with sequences of 3 pulses
(sequence frequency 0.1 Hz; pulse duration 2 ms) by means of
platinum electrodes placed at the sides of the samples. The elec-
trical stimuli were generated by a Grass S88K stimulator and
amplified (multiplexing pulse booster 316S; Ugo Basile, Comerio,
Italy). The musculature contractions of the vas deferens were
monitored by a computer, recorded, and analysed by analysis sys-
tem (PowerLab 400). The compounds to be tested were added to
the vas deferens in cumulative doses, in the absence or in the
presence of Naltrindole or CTAP, delta and mu reference antago-
nists, respectively.
The effect of the tested compounds on the contractions of vas
deferens musculature was expressed in percentage. Values were
calculated by referring the amplitude of the contractions induced
by the electrical stimulations after each addition of the compound
to the amplitude of the contractions obtained in the absence of the
same compound (100%). The reported results are an average of the
data obtained in seven different experiments.
6.10. Radioligand binding methods
Ligand binding assays were carried out according to previously
reported procedures [32]. Improvements were assured by slight
modifications. Briefly, whole brain minus cerebellum was homog-
enized with Polytron in 50 volumes (w/v) of 50 mM TriseHCl (pH
7.4), centrifuged at 48,000ꢃ g for 20 min at 4 ꢁC, re-suspended in
50 volumes of the same buffer solution, and incubated at 37 ꢁC for
45 min. After a further centrifugation step at 48,000ꢃ g for
20 min at 4 ꢁC, the final pellet was re-suspended in the same buffer
solution. Brain membranes were incubated with the appropriate
concentration of [3H]-DAMGO or [3H]-DPDPE or [3H]-U 69,593 in
TriseHCl buffer at 25 ꢁC for 60 min in the absence or presence of
naloxone (1 mM) (for mu and delta receptors) or U 69,593 (10 mM)
(kappa receptors). The binding reaction was stopped by rapid
filtration under vacuum through glass-fiber filters (Whatman GF/B)
using a Brandell 36-sample harvester (Gaithesburg, MD, USA) and
thereafter the filters were washed with 4 ꢃ 5 mL ice-cold 50 mM
TriseHCl buffer (pH 7.4). Filter-bound radioactivity was counted in
a liquid scintillation counter (Tricarb 2900; PerkinElmer Life Sci-
ences, Boston, MA, USA) using 4 mL of scintillation fluid (Packard
Ultima Gold MV, Packard, USA). Displacement curves were carried
6.12. In vivo assays: antinociceptive activity
Tail-flick test was adopted to assess antinociception in mice.
Compounds to be tested were administered by intraperitoneal (i.p.)
injection.
A tail-flick meter (Ugo Basile) equipped with an irradiant heat
source that focused 2.5 cm of the distal tip of the tail was used. A
15 s cut-off time for heat exposure was used to avoid cutaneous
damage and the intensity of the thermal source was adjusted to
produce a 3- to 5-sec latency in vehicle-treated mice.
Each animal was tested before drug administration to determine
control latency and the animals were used only in the determina-
tion of one time point. Data were transformed to the %MPE by the
following equation [37]:
out using serial dilutions ranging from 100 mM to 0.1 nM of the
unlabeled compound. Drugs were dissolved in DMSO. To avoid
possible undesired effects on radioligand binding, DMSO concen-
tration in the different assays never exceeded 0.1% (v/v). Protein
determination was performed by means of Bradford protein assay
using bovine serum albumine (BSA, SigmaeAldrich, Milan, Italy) as
a standard, according to the protocol of the supplier (Bio-Rad,
Milan, Italy) [35].
All receptor binding experiments were performed in triplicate
and results were confirmed in at least four independent experi-
ments. Data from radioligand inhibition experiments were
analyzed by nonlinear regression analysis of a Sigmoid Curve using
GraphPad Prism program (GraphPad Software, Inc., San Diego, CA,
USA). Ki values were calculated from the obtained IC50 values by
means of the equation of Cheng and Prusoff [36]. Values of 0.92 nM,
1.34 nM and 0.68 nM for the dissociation constants of [3H]-
DAMGO, [3H]-DPDPE and [3H]-U 69,593 were used respectively.
% MPE ¼ ½ðtest latency ꢂ control latencyÞ=
ꢃ ðcut ꢂ off ꢂ basal latencyÞꢄ ꢃ 100
where the latencies were expressed in the seconds and the cut-off
was of 15 s.
All the assays were carried out with groups of ten animals. Mice
were tested after 0.5, 1, and 2 h from i.p. administration of opioids
(5 mg/kg) or corresponding vehicles. Statistical analysis was carried
out using two-way ANOVA followed by NewmaneKeuls post hoc
test.
6.11. Isolated organs
Acknowledgments
Assays were performed according to the method described by
Bilsky et al. on vas deferent segments taken from mice immediately
after sacrifice [21]. After the explant, the vas deferent was trans-
ferred in a Petri dish containing a Krebs oxygenated solution (NaCl
118.2 mM, KCl 4.75 mM, KH2PO4 1.19 mM, NaHCO3 25.0 mM,
glucose 11.0 mM, and CaCl2 2.54 mM) in order to proceed to the
cleaning of the tissue and its subsequent dissection in segments
having 1.0e1.2 cm length. Vas deferent segments were placed in
tubs and they were immersed in 10 mL of an oxygenated Krebs
solution (95% O2 and 5% CO2) and kept at 37 ꢁC. One end of the vas
deferens segment was attached to a fixed support, at the tub bot-
tom, while the other end was connected to an isometric force
transducer (WPI Fort10, Biological Instruments, Besozzo, Italy) for
This study was supported in part by grant from Regione
Autonoma della Sardegna (Pacchetti Integrati di agevolazione PIA,
Industria, Artigianato e Servizi, Bando 2007).
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