Hybridized and isosteric analogues of N1-acetyl-N4-dimethyl-piperazinium iodide (ADMP) and N1-phenyl-N4-dimethyl-piperazinium iodide (DMPP) with central nicotinic action

Article abstract of DOI:10.1016/S0968-0896(98)00259-4

A series of piperazine derivatives, obtained by hybridization of N¹-acetyl-N⁴-dimethyl-piperazinium iodide (1, ADMP) and N¹-phenyl-N⁴-dimethyl-piperazinium iodide (3, DMPP) or of the corresponding tertiary bases (2, 4) with arecoline (5) and arecolone (6) or by isosteric substitution of the phenyl ring of DMPP, has been synthesized. Hybridization afforded compounds that, both as tertiary bases and as iodomethylates, have no affinity for the nicotinic receptor. On the contrary, isosteric substitution gave compounds that maintain affinity for the receptor; among them, two tertiary bases (37, 38), show affinity in the nanomolar range for the nicotinic receptor. The pharmacological profile of these isomeric compounds is quite interesting as they present differences in their peripheral and central effects, suggesting that they interact with different subtypes of the nicotinic receptor. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00259-4

Bioorganic & Medicinal Chemistry 7 (1999) 457±465
Hybridized and Isosteric Analogues of N1-Acetyl-N4-dimethyl-
piperazinium Iodide (ADMP) and N1-Phenyl-N4-dimethyl-
piperazinium Iodide (DMPP) with Central Nicotinic Action
Dina Manetti,a Alessandro Bartolini,b Pier Andrea Borea,c Cristina Bellucci,a
Silvia Dei,a Carla Ghelardini,b Fulvio Gualtieri,a* Maria Novella Romanelli,a
Serena Scapecchi,a Elisabetta Teodoria and Katia Varanic
a
Á
Dipartimento di Scienze Farmaceutiche, Universita di Firenze, via G. Capponi 9, 50121 Firenze, Italy
Á
Á
Dipartimento di Farmacologia, Universita di Ferrara, Via Fossato di Mortara 17±19, 44100 Ferrara, Italy
b
Dipartimento di Farmacologia Preclinica e Clinica, Universita di Firenze, Viale Morgagni 65, 50141 Firenze, Italy
c
Received 15 June 1998; accepted 29 October 1998
AbstractÐA series of piperazine derivatives, obtained by hybridization of N1-acetyl-N4-dimethyl-piperazinium iodide (1, ADMP)
and N1-phenyl-N4-dimethyl-piperazinium iodide (3, DMPP) or of the corresponding tertiary bases (2, 4) with arecoline (5) and
arecolone (6) or by isosteric substitution of the phenyl ring of DMPP, has been synthesized. Hybridization a€orded compounds
that, both as tertiary bases and as iodomethylates, have no anity for the nicotinic receptor. On the contrary, isosteric substitution
gave compounds that maintain anity for the receptor; among them, two tertiary bases (37, 38), show anity in the nanomolar
range for the nicotinic receptor. The pharmacological pro®le of these isomeric compounds is quite interesting as they present dif-
ferences in their peripheral and central e€ects, suggesting that they interact with di€erent subtypes of the nicotinic receptor. # 1999
Elsevier Science Ltd. All rights reserved.
Introduction
As a matter of fact, the number of nicotinic receptors in
patients a€ected by PD or AD is lower than those pre-
sent in the brain of healthy individuals;14 there is an
inverse relationship between smoking and the likelihood
to contract PD or AD;1 treatment with nicotine has
shown positive e€ects on memory, both on humans and
animal models15 and, in contrast to other neuro-
transmitters, the number of nicotinic receptors is
increased after administration of nicotine.16 Finally, the
emerging heterogeneity of nicotinic receptors seems to
provide an opportunity to discover ligands which would
be selective and possibly devoid of nicotine side
e€ects.8,17
Central nicotinic cholinergic receptors (nAChRs) are an
expanding area of research due to their implication in
neurodegenerative disorders such as Parkinson's disease
(PD) and Alzheimer's disease (AD), where cholinergic
transmission seems severely a€ected.1,2
For several years the cholinergic hypothesis of Alzhei-
mer's disease3 has stimulated intense research e€orts,
mainly on muscarinic agonists4±6 while nicotinic ago-
nists were substantially neglected, principally because of
the cardiovascular, gastrointestinal and endocrine side
e€ects of nicotine, as well as its negative connotation
associated with tobacco smoking. In the past few years,
however, thanks also to progress in molecular biology,
physiology and pharmacology of central nicotinic
receptors,7,8 the potential of nicotinic agonists for the
treatment of neurodegenerative disorders has been
recognized and intensive research has been performed
with the synthesis and pharmacological evaluation of
several classes of drugs that possess nicotinic properties
without the undesirable side e€ects of nicotine.9±13
Continuing our long-term research on cholinergic
ligands18,19 we have recently reported the synthesis of a
few nicotinic agonists related to N1-acetyl-N4-dimethyl-
piperazine iodide.20 In the present paper we report the
synthesis and preliminary pharmacological evaluation
of the compounds with general structure A (see Chart
1), obtained by hybridization of N1-acetyl-N4-dimethyl-
piperazinium iodide (1, ADMP)20,21 and the corre-
sponding tertiary base 2,20 N1-phenyl-N4-dimethyl-
piperazinium iodide (3, DMPP)22 and the correspond-
ing tertiary base 4, with arecoline (5),23 arecolone (6)24
and by isosteric substitution of the phenyl ring of
DMPP. Compounds 1, 3, 5 and 6 are endowed with
nicotinic activity, even if they present di€erent degrees
Key words: Piperazine; nicotinic receptor; analgesics; cognition-
enhances.
*Corresponding author. Tel.: +1 39 552757295; fax: +1 39 55240776;
e-mail: gualtieri@farm®.scifarm.uni®.it
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(98)00259-4

458
D. Manetti et al. / Bioorg. Med. Chem. 7 (1999) 457±465
candidates for the treatment of neurodegenerative dis-
eases, we have been especially interested in compounds
lacking a permanent charge on the nitrogen atom.
However, a second goal of the research has been to
develop nicotinic agonists useful to characterize nico-
tinic receptors and possibly to draw up a model of
interaction; for this reason we have also synthesized and
studied the corresponding methiodide derivatives that
are unable to cross the BBB, but are known to interact
with high anity with nicotinic receptors.
Chemistry
Synthesis of the compounds designed involved simple
chemistry and the use of standard methods. The syn-
thetic pathways used are described in Schemes 1 and 2
and the compounds obtained are reported in Tables 1±3.
Chart 1.
of potency and selectivity toward this kind of choliner-
gic receptor subtype.
The starting material for the piperazino derivatives of
Scheme 1 were the previously described N,N0-dimethyl-
ethylendiamine,25 N-phenyl-N-methylethylendiamine26
and N-benzyl-N-methylethylendiamine27 that were con-
densed with the commercially available methyl and ethyl
esters of 2,3-dibromopropionic acid, 3,4-dibromobutan-
Since the main objective of our work has been that of
obtaining nicotinic agonists able to cross the blood±
brain barrier (BBB) and therefore be useful as drug
Scheme 1. (a) Anhyd. benzene/N(C2H5)3; (b) CH3l; (c) Pd/C/H2; (d) CH3COCl.

D. Manetti et al. / Bioorg. Med. Chem. 7 (1999) 457±465
459
Scheme 2. (a) 3- or 4-bromopyridine; (b) Pd/C/H2; (c) CH2O/HCOOH; (d) HCl, high temp. (e) CH3l.
2-one28 and 4,5-dibromopentan-3-one,29 respectively.
Among the compounds obtained, 9,30 1131 and 1220,27
have already been reported. Only the series where the
nitrogen 4 of piperazine is unequivocally involved in the
reaction (N1-phenyl and N1-acetyl series) were treated
with methyl iodide to give the quaternary salt. The dis-
appointing pharmacological results obtained, dis-
compounds 35,32 4133 and 4234,35 have already been
described and 39 was obtained in very low yields. Even
in this case, only unequivocally reacting compounds (35
and 39) were quaternized to give 36 and 40.
Pharmacology
couraged
any
further
investigation
on
the
stereochemistry of the compounds which are all racemic
mixtures.
The compounds obtained have been tested in vitro on
rat brain homogenates to evaluate their anities for the
central nicotinic receptors labeled by [3H]-cytisine
(2a43b2 subtype), which represents up >90% of the
high-anity agonist binding sites in rat brain11,36 and
In Scheme 2 the synthetic pathways used to obtain the
compounds reported in Table 3 are reported; of them,
Table 2. Compounds obtained by hybridization of 1, 2, 3, 4 and
arecolone (6)
Table 1. Compounds obtained by hybridization of 1, 2, 3, 4 and
arecoline (5)
N
Y
R1
R2
X
Mp ꢀC
Analysisa [ref]
N
Y
R1
R2
X
Mp ꢀC
Analysisa
7
8
9
H
H
CH3
CH3
CH2C6H5
CH2C6H5
C6H5
C6H5
C6H5
C6H5
Ð
Ð
Ð
Ð
Ð
Ð
Ð
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
Ð
Ð
Ð
Ð
Ð
Ð
Ð
I
Ð
I
Ð
I
b
b
b
b
b
b
C7H14N2O2
C8H16N2O2
[30]
C9H18N2O2
[31]
[20,27]
C13H18N2O2
C14H21IN2O2
C14H20N2O2
C15H23IN2O2
C9H16N2O3
C10H19IN2O3
C10H18N2O3
C11H21IN2O3
21
22
23
24
25
26
27
28
29
30
31
32
33
34
H
H
CH3
CH3
CH2C6H5
CH2C6H5
C6H5
C6H5
C6H5
C6H5
Ð
Ð
Ð
Ð
Ð
Ð
Ð
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
Ð
Ð
Ð
Ð
Ð
Ð
Ð
I
Ð
I
Ð
I
b
b
b
b
b
b
C7H14N2O
C8H16N2O
C8H16N2O
10
11
12
13
14
15
16
17
18
19
20
C9H18N2O
C14H20N2O
C15H22N2O
C13H18N2O
C14H21IN2O
C14H20N2O
C15H23IN2O
C9H16N2O2
C10H19IN2O2
C10H18N2O2
C11H21IN2O2
b
140±142
b
143±145
b
190±192
b
193±195
b
135±137
b
133±135
b
180±182
b
182±184
CH3 CH3
C2H5
CH3 C2H5
CH3
CH3 CH3
C2H5
CH3 C2H5
CH3 CH3
C2H5
CH3 C2H5
CH3
CH3 CH3
C2H5
CH3 C2H5
Ð
Ð
COCH3
COCH3
COCH3
COCH3
Ð
COCH3
COCH3
COCH3
COCH3
Ð
Ð
Ð
I
Ð
Ð
I
aAll compounds have been analyzed for C, H, N and the results
obtained range within ‹0.4 of the theoretical values.
bOil (free base).
aAll compounds have been analyzed for C, H, N and the results
obtained range within ‹0.4 of the theoretical values.
bOil (free base).

460
D. Manetti et al. / Bioorg. Med. Chem. 7 (1999) 457±465
Table 3. Compounds obtained by isosteric replacement or substitu-
tion of the phenyl ring of 3 and 4
As a matter of fact, these compounds did not show any
anity for central nicotinic receptors up to a con-
centration of 10 mM and were not investigated any fur-
ther. Better results were obtained with the isosteric
replacement of a carbon atom with a nitrogen in the
phenyl ring of compounds 3 and 4 (Table 3).
Central nicotinic binding, peripheral nicotinic activity
and analgesic activity of active compounds are reported
in Table 4, compared with those of nicotine and of
compounds 1±6. In the evaluation of the results shown
in Table 4, it must be considered that the anity values
for nicotine reported in the literature are fairly di€er-
ent2,24,41,42 and, in our hands, DMPP appears to be
somehow less potent for central nicotinic receptors than
reported in the literature.42
N
Y
R1
X
Mp ꢀC
Analysisa [ref]
35
36
37
38
39
40
41
42
2-pyridyl
2-pyridyl
3-pyridyl
Ð
CH3
Ð
Ð
Ð
CH3
Ð
Ð
Ð
I
Ð
Ð
Ð
I
b
202±204
[32]
C11H18IN3
C10H15N3
C10H15N3
C12H18N2
C13H21IN2
[33]
b
b
b
4-pyridyl
2-CH3-phenyl
2-CH3-phenyl
3-Cl-6-pyridazinyl
2-pyrimidinyl
255±260
113±115
b
Ð
Ð
[34,35]
Compounds 36±38 show anity for the central nicotinic
receptors labeled by cytisine in the nanomolar range. As
a consequence, these compounds were tested also on
ganglionic nicotinic receptors and evaluated in vivo as
analgesics. Finally, compounds 37 and 38, that are ter-
tiary amines and present properties that would make
them potential candidates for the treatment of neuro-
degenerative diseases, were tested also in the passive
avoidance test. In Fig. 1, the e€ect of nicotine and
compounds 37 and 38 on the passive avoidance test is
reported.
aAll compounds have been analyzed for C, H, N and the results
obtained range within ‹0.4 of the theoretical values.
bOil (free base).
on guinea pig ileum (ganglionic nicotinic receptors)37 to
evaluate their activity on peripheral receptors. More-
over, since some nicotinic agonists show analgesic
activity, which is peculiar of their central action and
might be related to their cognitive properties,2,38 those
showing anity for the central nicotinic receptors have
also been tested as analgesics in vivo on mice, using the
hot-plate test.39 Finally, compounds showing both a-
nity for central nicotinic receptor and analgesic activity
were also tested as cognition enhances in the mouse
passive avoidance test.40
Discussion
As reported in the previous section, molecular hybridi-
zation of compounds 1±6, to give compounds 7±34
(Tables 1 and 2), completely abolished the nicotinic
anity of the parent compounds: these compounds do
not show any anity for central nicotinic receptors up
to a concentration of 10 mM. Apparently the introduc-
tion of the carboxylate function of arecoline (5) or of
the ketonic function of arecolone (6) in position 3 of the
Results
Molecular hybridization of compounds 1±6, which gave
origin to compounds 7±34 (Tables 1 and 2) completely
abolished the nicotinic anity of the parent compounds.
Table 4. Nicotinic and analgesic activity of compounds 1±6 and 35±38
N
Nicotinic activity
G.p. ileumb ED50 (mM) ‹SE
Analgesic activityc
Bindinga Ki (mM) ‹SE
MADd (mg)
Ecacye (%)
Nicotine
1 (ADMP)
2
3 (DMPP)
4
5 (Arecoline)
6 (Arecolone)
35
36
37
38
0.0082f
1.07 (0.2)
>100
2.13 (0.15)
3.25 (0.04)
>100
3.43 (0.08)
Ð
0.3 (0.01)
n.d.
Ð
1.25 (0.13)
4.67 (0.11)
>100
0.7
2.5
50
3
Ð
1
n.d.
Ð
71
77
69
67
Ð
99
n.d.
Ð
0.25 (0.03)g
>100
0.224h
0.0058h
>100
0.50 (0.03)
0.09 (0.01)
0.17 (0.01)
100
>300
100
51
Ð
117
aBinding against [3H]-cytisine on rat cerebral cortices.
bContraction and decontraction of guinea pig ileum in presence of hexamethonium.
cHot-plate test on mice.
dMAD=minimum analgesic dose when injected intracerebroventricularly (icv); analgesia was fully reversed by mecamylamine.
eEcacy with respect to morphine (5 mg icv).
fSee ref. 41; Ward,24 Decker2 and Boksa42 report 8.40, 0.8 and 6.7 nM, respectively.
gRef. 42 reports 0.057 mM.
hSee ref. 24.
n.d.=not done.

D. Manetti et al. / Bioorg. Med. Chem. 7 (1999) 457±465
461
piperazine ring of N1-acetyl-N4-dimethyl-piperazinium
iodide (1, ADMP) and N1-phenyl-N4-dimethyl-piper-
azinium iodide (3, DMPP) severely a€ects nicotinic
receptor anity, both for tertiary bases and methiodide
derivatives. The same happens when the substituent at
nitrogen 1 of the piperazine ring is hydrogen, methyl or
benzyl. Interestingly, hybridization has negative con-
sequences also on the muscarinic anity of arecoline, as
compounds 7, 9, 11, 13, 17 do not show any anity for
muscarinic receptors up to a concentration of 10 mM.
These results were quite unexpected but, though nega-
tive, could provide useful information for the develop-
ment of a model of the central nicotinic receptor.
Among the isosteres of DMPP, compound 36 maintains
anity for central nicotinic receptors comparable to
that of DMPP. Much like the tertiary base of the parent
compound (4), the corresponding tertiary base (35)
lacks anity for the nicotinic receptor up to a con-
centration of 100 mM. On the contrary, 37 and 38, which
are tertiary bases, show relevant anity for the central
nicotinic receptors (90 and 170 nM, respectively).
1
Figure 1. Prevention by compounds 37 (100 mg kg ip) and 38
(30 mg kg 1 ip) of amnesia induced by scopolamine (3 mg kg 1 ip) and
mecamylamine (20mg kg 1ip) in comparison with nicotine (1.5mg kg
1
sc) in mouse passive-avoidance test. Compounds 37 and 38 and nico-
tine were injected 20 min before training session while scopolamine
and mecamylamine were administered immediately after the training
session. Each column represents the mean of at least 10 mice. *P<0.01
in comparison with mecamylamine-treated mice.
However, the two compounds show di€erent pharma-
cological pro®les. Compound 37 is active on ganglionic
nicotinic receptors (ED50=4.67 mM) and lacks analgesic
activity; compound 38, although slightly less potent, has
no activity on neuronal ganglia but possess de®nite
analgesic activity in mouse hot-plate test, with a mini-
mum analgesic dose (MAD) of 100 mg, when injected
intracerebroventricularly (icv) and higher ecacy
(117%) with respect to morphine (5 mg, icv). The
analgesic e€ect was fully reversed by mecamylamine.
rings of 36±38 with a second nitrogen atom is detri-
mental for anity (41, 42), while introduction of a
methyl group in position 2 of the phenyl ring of DMPP
completely abolishes nicotinic activity (39, 40). This
result can be very informative toward the development
of a model of interaction, studies which are in progress
and about which we will refer in due time.
Experimental
Chemistry
Both 37 and 38, at doses of 100 and 30 mg/kg, respec-
tively, completely prevent the amnesic action of meca-
mylamine but not that of scopolamine in mouse passive
avoidance test, when injected intraperitoneal (ip), thus
showing that, as expected, they can cross the blood±
brain barrier, they are able to produce the same e€ects
as nicotine on central nicotinic receptors and they do
not interact with the muscarinic ones (Fig. 1).
All melting points were taken on a Buchi apparatus and
are uncorrected. Infrared spectra were recorded with a
Perkin±Elmer 681 spectrophotometer in a Nujol mull
for solids and neat for liquids. NMR spectra were
recorded on a Gemini 200 spectrometer. Chromato-
graphic separations were performed on a silica gel col-
umn by gravity chromatography (Kieselgel 40, 0.063±
0.200 mm, Merck) or ¯ash chromatography (Kieselgel
40, 0.040±0.063 mm, Merck). Yields are given after
puri®cation, unless otherwise stated. Where analyses are
indicated by symbols, the analytical results are within
‹0.4% of the theoretical values. Since the hydro-
chlorides of tertiary bases are often mixtures of mono-
and bis-hydrochlorides (or bis- and tris-hydrochlorides)
with variable amounts of water, which makes dicult to
obtain good analytical data, ®nal compounds have
usually been analyzed as free bases, after column chro-
matography puri®cation and accurate removal of the
solvent.
These results suggest that both 37 and 38 interact with
the nicotinic receptor subtypes responsible for cognitive
e€ects of nicotine, while only 38 is able to activate the so
far unidenti®ed central nicotinic subtype(s) mediating
analgesic activity. Compounds that have high anity
for central nicotinic receptors, but do not show analge-
sic activity are known43 and the most obvious explana-
tion is that analgesic activity is mediated by nicotinic
subtype(s) di€erent from those which predominate in
the CNS, like the a4b2 and a7 subtypes, even if other
explanations cannot be ruled out.43
Whatever the case, compounds 37 and 38 look very
promising as leads for the development of new nicotinic
ligands that can be useful to characterize central nico-
tinic receptors, develop models for nicotinic receptor
binding sites and design drugs useful in the treatment
of neurodegenerative diseases. In this respect, pre-
liminary structure±activity relationship studies have
shown that further isosteric substitution of the phenyl
1-Methyl-4-phenylpiperazine hydrochloride (3). Commer-
cially available N-phenylpiperazine (0.2 g, 1.23 mmol)
was dissolved in 5 mL of abs EtOH and 0.97 g of formic
acid (21 mmol) and 0.47 g of formaldehyde (40% in
H2O, 6.2 mmol) were added to the solution. The solu-
tion was re¯uxed for 3 h, then evaporated, alkalinized

462
D. Manetti et al. / Bioorg. Med. Chem. 7 (1999) 457±465
1
with NaHCO3 and extracted with CHCl3. Evaporation
of the dried solution gave 0.2 g of an oil (93% yield) that
was transformed into the hydrochloride with HCl/abs
IR and H NMR spectra are consistent with the pro-
posed structure. Its chemical and physical character-
istics are reported in Table 1.
EtOH: mp 190±192 ꢀC; H NMR (free base, CDCl3) d
1
2.35 (s, 3H, CH3N), 2.55±2.60 (m, 4H, CH2), 3.19±3.24
(m, 4H, CH2), 6.85±6.95 (m, 3H, aromatics), 7.23±7.31
(m, 2H, aromatics) ppm; anal. (C11H16N2 2HCl H2O)
C, H, N, Cl.
1-Acetyl-4-methyl-2-carboxymethylpiperazine (17). 0.316 g
of 7 (2 mmol) were dissolved in CHCl3 (5 mL), cooled at
0 ꢀC and added to 0.28 mL of NEt3 (2 mmol) and
0.17 mL of acetylchloride (2 mmol). After 2 h at 0 ꢀC,
10 mL of CHCl3 were added, the solution washed with
H2O, dried and evaporated to give an oil that was pur-
i®ed by column chromatography using CHCl3/abs
.
.
1,4-Dimethyl-2-carboxyethylpiperazine (10). N,N-Dime-
thylethylendiamine25 (5 mmol) and NEt3 (9 mmol) dis-
solved in 20 mL of anhyd benzene were added to a
solution of commercially available ethyl 2,3-dibromo-
propionate (5 mmol), dissolved in anhyd benzene (20 mL),
kept at 40 ꢀC. The solution was re¯uxed for 3 h and the
solid formed was ®ltered after cooling. The benzene
solution was then washed with H2O, dried and evapo-
rated at reduced pressure. The oily residue was puri®ed
by column chromatography, using CHCl3/MeOH (9/1)
EtOH/petr. ether/NH4OH (600/225/90/30) as eluent:
1
Yield 40%; IR (neat) n 1680 (CON), 1720 (COO) cm
;
1H NMR (CDCl3) d 2.07 (s, 3H, CH3CO), 2.19 (s, 3H,
CH3N), 2.21±2.30 (m, 1H, CHN), 2.58±2.85 (m, 2H,
CH2), 3.21±3.59 (m, 4H, CH2), 3.66 (s, 3H, CH3N); 13C
NMR (CDCl3) d 23.30 (q), 45.72 (q), 48.36 (t), 54.16
(q), 56.41 (t), 57.65 (t), 59.15 (d), 172.66 (s), 172.71 (s)
ppm; anal. (C9H16N2O3) C, H, N.
1
as eluent: Yield 25%; IR (neat) n 1720 (CO) cm 1; H
NMR (CDCl3) d 1.27 (t, 3H, CH3CH2); 2.35 (s, 3H,
CH3N); 2.36 (s, 3H, CH3N); 2.38±2.53 (m, 3H, CHN);
2.72±3.01 (m, 3H, CHN); 3.06±3.18 (m, 1H, CHN); 4.22
(q, 2H, CH2CH3); 13C NMR (CDCl3) d 16.11 (q), 45.45
(q), 47.79 (q), 55.35 (t), 56.45 (t), 59.36 (t), 62.69 (t),
68.17 (d), 173.44 (s) ppm; anal. (C9H18N2O2) C, H, N.
Similarly, starting from 8, 19 was obtained, whose IR
1
and H NMR spectra are consistent with the proposed
structure. Its chemical and physical characteristics are
reported in Table 1.
1-Phenyl-4,4-dimethyl-2-carboxymethylpiperazinium iod-
ide (14). An excess of methyl iodide (2 mL) was added
to a solution of 13 (1 mmol) in anhyd ether and the
solution kept in the dark at room temperature over-
night. The solid obtained was ®ltered and recrystallized
from anhyd EtOH/anhyd ether: mp 140±142; Yield
In the same way, starting from N-methyl-N-pheny-
lethylendiamine26 and the appropriate 2,3-dibromopro-
pionate, the following compounds were obtained:
1
compound 13; its hydrochloride was obtained treating
the free base with HCl/abs EtOH: mp 75±77 ꢀC from
anhyd EtOH/anhyd ether. Analytical data indicate that
the salt is the bis-hydrochloride with a small amount of
mono-hydrochloride and water;
90%; IR (nujol) n 1720 (CO) cm 1; H NMR (D2O) d
3.21 (s, 6H, CH3N), 3.40±3.59 (m, 2H, CH2N), 3.67 (s,
3H, CH3O), 3.63±3.80 (m, 4H, CH2N), 4.49±4.52 (m,
1H, CHCO), 6.83±6.87 (m, 2H, arom.), 7.20±7.29 (m,
3H, arom.); anal. (C14H21IN2O2) C, H, N.
compound 15; its hydrochloride was obtained treating
the free base with HCl/abs EtOH: mp 74±76 ꢀC from
anhyd EtOH/anhyd ether. Analytical data indicate that
the salt is the bis-hydrochloride with a small amount of
mono-hydrochloride and water.
In the same way, starting from 15, 17 and 19, 16, 18 and
1
20 were obtained, respectively, whose IR and H NMR
spectra are consistent with the proposed structure. Their
chemical and physical characteristics are reported in
Table 1.
1
IR and H NMR spectra of 13 and 15 are consistent
with the proposed structures and their chemical and
physical characteristics are reported in Table 1.
2-Acetyl-1,4-dimethylpiperazine (23). Following the pro-
cedure described for 10, starting from N,N0-dimethyl-
ethylendiamine25 and 3,4-dibromo-2-butanone28 com-
pound 23 was obtained as an oil in 25% yield, after
column chromatography puri®cation (CHCl3/MeOH,
4-Methyl-2-carboxymethylpiperazine (7). 1-Benzyl-4-
methyl-2-carboxymethylpiperazine31 (11, 0.28 g) was
dissolved in abs EtOH (20 mL) and hydrogenated at a
pressure of 55 psi in presence of 10% Pd/C for 18 h.
Filtration and evaporation of the solvent gave an oil
that was puri®ed by column chromatography using
CHCl3/MeOH (9/1) as eluent: Yield 92%; IR (neat) n
3200±3400 (NH), 1720 (CO) cm 1; 1H NMR (CDCl3) d
2.30 (s, 3H, CH3N); 2.32 (bs, NH); 2.52±2.68 (m, 1H,
CHN); 2.73±3.12 (m, 5H, CHN); 3.58 (dd, 1H, CHCO);
3.73 (s, 3H, CH3O); 13C NMR (CDCl3) d 46.16 (t),
48.26 (q), 53.93 (q), 57.14 (t), 59.04 (t), 68.74 (d), 174.35
(s); anal. (C7H14N2O2) C, H, N.
1
9/1 as eluent): IR (neat) n 1725 (CO) cm 1; H NMR
(CDCl3) d 2.12±2.29 (m, 1H, CHN), 2.20 (s, 3H, CH3C),
2.23 (s, 3H, CH3N), 2.38 (s, 3H, CH3N), 2.43±2.54 (m,
2H, CHN), 2.80±2.93 (m, 3H, CHN), 3.13 (dd, 1H,
CHN); 13C NMR (CDCl3) d 28.93 (q), 45.62 (q), 47.86
(q), 56.05 (t), 56.45 (t), 58.35 (t), 75.57 (d), 210.68 (s);
anal. (C8H16N2O) C, H, N.
The following compounds were obtained in the same way:
compound 24, starting from N,N0-dimethylethylendia-
mine and 4,5-dibromo-3-pentanone;29
In the same way, starting from 1-benzyl-4-methyl-2-
carboxyethylpiperazine20,27 (12), 8 was obtained, whose
compound 25, starting from N-benzyl-N0-methylethyl-
endiamine27 and 3,4-dibromo-2-butanone;

D. Manetti et al. / Bioorg. Med. Chem. 7 (1999) 457±465
463
compound 26, starting from N-benzyl-N0-methylethyl-
endiamine and 4,5-dibromo-3-pentanone;
The following compounds were obtained similarly: 30
(starting from 29), 32 (starting from 31) and 34 (starting
from 33). Their IR and H NMR spectra are consistent
with the proposed structure. Chemical and physical
characteristics are reported in Table 2.
1
compound 27, starting from N-phenyl-N0-methylethy-
lendiamine and 3,4-dibromo-2-butanone; its hydro-
chloride was obtained treating the free base with HCl/
abs EtOH: mp 60±62 ꢀC from anhyd EtOH/anhyd
ether. Analytical data indicate that the salt is the bis-
hydrochloride with a small amount of mono-hydro-
chloride and water.
1-Benzyl-4-(30-pyridinyl)-piperazine (43). Commercially
available 3-bromopyridine (1 g, 6 mmol), N-benzylpi-
perazine (4.5 g, 25 mmol) and CuSO4 (0.130 g) were
added to 5 mL of H2O and heated at 140 ꢀC in a steel
bomb for 24 h. Ether extraction of the cooled mixture
gave an oily product that was puri®ed by column chro-
matography (using CHCl3/abs EtOH/petr. ether/
NH4OH, 340/65/60/8 as eluent) to give 0.55 g of the title
compound with a 35% yield. The compound was used
Compound 29, starting from N-phenyl-N0-methylethy-
lendiamine and 4,5-dibromo-3-pentanone; its hydro-
chloride was obtained treating the free base with HCl/
abs EtOH: mp 65±67 ꢀC from anhyd EtOH/anhyd
ether. Analytical data indicate that the salt is the bis-
hydrochloride with a small amount of mono-hydro-
chloride and water.
1
as such in the following reaction. H NMR (CDCl3) d
2.62±2.68 (m, 4H, CH2N), 3.10±3.17 (m, 4H, CH2N),
3.57 (s, 2H, NCH2Ph), 7.12±7.15 (m, 2H, CH), 7.25±
7.35 (m, 5H, arom), 8.08±8.12 (m, 1H, CH), 8.40±8.44
(m, 1H, CH).
1
IR and H NMR spectra of 24, 25, 26, 27 and 29 are
consistent with the proposed structure. Chemical and
physical characteristics are reported in Table 2.
In the same way, starting from 4-bromopyridine, com-
1
pound 44 was obtained with a yield of 98%, whose H
NMR spectrum is consistent with the proposed structure.
4-Methyl-2-acetylpiperazine (21). Following the proce-
dure described for 7 and starting from 25, the title
compound was obtained as an oil in 98% yield: IR
1-(30-Pyridinyl)-piperazine (45). 0.25 g of Pd/C 10% and
0.62 g (10 mmol) of ammonium formiate were added to
a solution of 43 (0.55 g, 2 mmol) in MeOH (15 mL) and
re¯uxed for 3 h. The cooled reaction mixture was ®l-
tered, the solvent evaporated, and the residue alkali-
nized with NaHCO3; CHCl3 extraction gave 0.22 g of
title compound with a 68% yield. The compound was
used as such in the following reaction. 1H NMR
(CDCl3) d 2.98±3.06 (m, 4H, CH2N), 3.12±3.19 (m, 4H,
CH2N), 7.10±7.14 (m, 2H, CH), 8.02±8.12 (m, 1H, CH),
8.41±8.43 (m, 1H, CH).
1
(neat) n 3200±3350 (NH), 1720 (CO) cm 1; H NMR
(CDCl3) d 1.00 (d, 3H, CH3CO), 1.83 (t, 1H, CHN),
2.02±2.15 (m, 1H, CHN), 2.10 (bs, NH), 2.24 (s, 3H,
CH3N), 2.37±2.69 (m, 2H, CHN), 2.73±2.86 (m, 2H,
CHN), 3.03±3.12 (m, 1H, CHN); 13C NMR (CDCl3) d
23.59 (q), 34.50 (t), 45.30 (t), 47.61 (q), 53.40 (t), 72.70
(d), 210.22 (s); anal. (C7H14N2O) C, H, N.
In the same way, starting from 26, 22 was obtained
1
whose IR and H NMR spectra are consistent with the
proposed structure. Its chemical and physical char-
acteristics are reported in Table 2.
In the same way, starting from 44, compound 46 was
1
obtained, with a yield of 67%, whose H NMR spec-
trum is consistent with the proposed structure.
1,2-Diacetyl-4-methylpiperazine (31). Following the pro-
cedure described for 17 and starting from 21, the title
compound was obtained as an oil in 14% yield: IR (neat)
1-Methyl-4-(30-pyridinyl)piperazine (37). Following the
procedure described for 3 and starting from 45, the title
compound was obtained as an oil in 46% yield after
puri®cation by column chromatography, using CHCl3/
abs EtOH/petr. ether/NH4OH (340/65/60/8) as eluent.
1H NMR (CDCl3) d 2.46 (s, 3H, CH3N), 2.71±2.76 (m,
4H, CH2N), 3.30±3.35 (m, 4H, CH2N), 7.16±7.18 (m,
2H, CH), 8.11±8.12 (m, 1H, CH), 8.29±8.31 (m, 1H,
CH); 13C NMR (CDCl3) d 48.12 (q), 50.22 (t), 56.70 (t),
124.29 (d), 125.45 (d), 140.31 (d), 142.42 (d), 148.84 (s);
anal. (C10H15N3) C, H, N.
1
n 1680 (CON), 1720 (COO) cm 1; H NMR (CDCl3) d
1.17 (d, 3H, CH3CO), 2.05 (s, 3H, CH3 CON), 2.22 (s,
3H, CH3N), 2.75±2.96 (m, 2H, CH), 3.11±3.22 (m, 1H,
CH), 3.46±3.77 (m, 2H, CH), 3.98±4.20 (m, 1H, CH),
4.31±4.47 (m, 1H, CH); 13C NMR (CDCl3) d 23.92 (q),
24.01 (q), 45.60 (q), 47.83 (t), 55.73 (t), 56.42 (t), 72.71 (d),
171.40 (s), 171.55 (s); anal. (C9H16N2O2) C, H, N.
Similarly, starting from 22, 33 was obtained whose IR
1
and H NMR spectra are consistent with the proposed
structure. Its chemical and physical characteristics are
reported in Table 2.
The hydrochloride was obtained treating the free base
with HCl/abs EtOH: mp >260 ꢀC from anhyd EtOH/
.
.
1-Phenyl-4,4-dimethyl-2-acetylpiperazinium iodide (28).
Following the procedure described for 14 and starting
from 27, the title compound was obtained in 92% yield:
mp 135±137 ꢀC; IR (nujol) n 1720 (CO) cm 1; 1H NMR
(D2O) d 2.06 (s, 3H, CH3CO), 3.23 (s, 6H, CH3N),
3.47±3.53 (m, 2H, CH2N), 3.68±3.82 (m, 4H, CH2N),
4.32±4.37 (m, 1H, CHCO), 6.83±6.90 (m, 3H, arom.),
7.21±7.29 (m, 2H, arom.); anal. (C14H21IN2O) C, H, N.
anhyd ether; anal. (C10H15N3 2HCl H2O) C, H, N.
Cl% calcd 26.44; found 27.02.
The following compounds were obtained in the same
way:
compound 35, with a yield of 79%, starting from the
commercially available 1-(20-pyridinyl)-piperazine; its

464
D. Manetti et al. / Bioorg. Med. Chem. 7 (1999) 457±465
hydrochloride was obtained treating the free base with
HCl/abs EtOH. mp 228±230 ꢀC from anhyd EtOH/
anhyd ether. Analytical data indicate that the salt is the
bis-hydrochloride with a small amount of tris-hydro-
chloride and water;
England Nuclear, Boston, MA; 39.7 Ci/mmol) were
performed in 250 mL of bu€er which contained 2 nM
[3H]-cytisine, membranes from 15 mg (wet weight) of
tissue and the compound to be tested. After 75 min of
incubation at 2 ꢀC, separation of bound from free radi-
oligand was performed by rapid ®ltration through
Whatman GF/C glass ®ber ®lter, which were washed
three times with ice-cold bu€er, dried and counted in
5 mL of Aquassure (Packard, Downers Grove, USA).
Binding in the presence of 10 mM ( )-nicotine bitar-
trate was de®ned unspeci®c and was, routinely, about
10% of the total binding. Ki values were calculated from
the Cheng±Pruso€ equation45 using 1.5 nM as the Kd
for [3H]-cytisine, determined by previous saturation
experiments.
compound 38, with a yield of 81%, starting from 46; its
hydrochloride was obtained treating the free base with
HCl/abs EtOH. mp 210±212 ꢀC from anhyd EtOH/
.
.
anhyd ether (hygroscopic). Anal. (C10H15N3 2HCl H2O)
C, H, N; Cl% calcd 26.44; found 26.92;
compound 42, which has already been described.34,35
1H NMR spectra of 35 and 38 are consistent with the
proposed structures; their chemical and physical char-
acteristics are reported in Table 3.
Functional studies. 2 cm segments of the terminal por-
tion of the guinea pig ileum were suspended, under 1 g
tension, in PSS with the following composition (mM):
NaCl (137), KCl (2.7), CaCl2 (1.8), MgCl2 (1.05),
NaH2PO4 (0.42), NaHCO3 (11.9), glucose (5.6). The
solution was aerated with a gas mixture of 95% O2, 5%
CO2 and maintained at 34 ꢀC. Tension changes were
recorded isotonically. After 1 h equilibration, the tissues
were exposed to nicotine or to the drug to be tested.
Concentration response curves were constructed non-
cumulatively. Tissues were exposed to drugs for 30 s with
at least 10 min rest between exposures. E€ects of the
antagonist hexamethonium (30 mM) were investigated
after 30 min equilibration.
1-(20-Methyl-phenyl)-4-methylpiperazine (39). o-Tolui-
dine (4 g, 38 mmol) and N-methyl-bis-ethanolamine
(4.16 g, 38 mmol) were mixed, cooled with ice, and neu-
tralized with 6.6 mL of 37% HCl. The mixture was then
heated at 220 ꢀC for 16 h leaving H2O to distill o€. The
cooled mixture was alkalinized with 10% NaOH,
extracted with CHCl3 and the residue ¯ash-chromato-
graphed using pet. ether/CH2Cl2/diethyl ether/abs
EtOH/NH4OH (900/360/360/180/9.9) as eluent, to give
0.2 g (3% yield) of the title compound. 1H NMR
(CDCl3) d 2.30 (s, 3H, CH3C), 2.36 (s, 3H, CH3N),
2.56±2.62 (m, 4H, CH2N), 2.92±2.97 (m, 4H, CH2N),
6.97±7.05 (m, 2H, aromatics), 7.13±7.20 (m, 2H, aro-
matics); anal. (C12H18N2) C, H, N.
Antinociceptive activity. Analgesic activity was eval-
uated on mice with the hot-plate test39 and the results
are given as minimum analgesic dose (MAD) when
injected intracerebroventricularly (icv) and ecacy
(E%) with respect to the analgesic activity of 5 mg
of morphine icv. More details on the protocols used
can be found in previous publications.46,47 The nicotinic
origin of analgesia was checked by its reversal by
mecamylamine.
The hydrochloride was obtained treating the free base
with HCl/abs EtOH. Mp 252±255 ꢀC from anhyd
EtOH/anhyd ether. Analytical data indicate that the
salt is the bis-hydrochloride with a small amount of
mono-hydrochloride and water.
1,1-Dimethyl-4-(20-pyridinyl)piperazinium iodide (36).
Following the procedure described for 14, the title
compound was obtained starting from 35, in 80% yield:
mp 202±204 ꢀC from anhyd EtOH/anhyd ether; 1H
NMR (CDCl3) d 3.62 (s, 6H, CH3N), 3.79±3.89 (m, 4H,
CH2N); 3.91±4.00 (m, 4H, CH2N); 6.72±6.84 (m, 2H,
CH pyr), 7.58±7.66 (m, 1H, CH pyr), 8.19±8.25 (m, 1H,
CH pyr); anal. (C11H18IN3) C, H, N.
Antiamnesic test (passive-avoidance test). The test was
performed according to the step-through method
described by Jarvik and Kopp,40 as we modi®ed it for
testing drugs endowed with analgesic properties. The
apparatus consists of a two-compartment acrylic box
with a lighted compartment connected to a darkened
one by a guillotine door. In the original method, mice
received a punishing electrical shock as soon as they
entered the dark compartment, while in our modi®ed
method, after entry into the dark compartment, mice
receive a non-painful punishment consisting of a fall
into a cold water bath (10 ꢀC). For this purpose, the
dark chamber was constructed with a pitfall ¯oor. The
latency times for entering the dark compartment were
measured in the training test and after 24 h in the
retention test. For memory disruption, mice were ip
injected with the amnesic drugs scopolamine or meca-
mylamine. All investigated drugs were injected 20 min
before the training session, while scopolamine and
mecamylamine were injected immediately after termi-
nation of the training session. The maximum entry
latency allowed in the retention session was 120 s. The
In the same way, starting from 39, compound 40 was
obtained with a yield of 98%, whose 1H NMR spectrum
is consistent with the proposed structure. Its chemical
and physical characteristics are reported in Table 3.
Pharmacology
Binding studies. Cerebral cortices of male Wistar rats
(150±200 g) were dissected on ice. The tissue was
homogenized in 50 mM Tris±HCl bu€er (pH 7.4 at 2 ꢀC)
containing 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, and
2.5 mM CaCl2. The homogenate was centrifuged at
40,000 g for 10 min; the pellet was re-suspended in ice-
cold bu€er, recentrifuged and re-suspended again in
bu€er. Binding experiments44 with [3H]-cytisine (New

D. Manetti et al. / Bioorg. Med. Chem. 7 (1999) 457±465
465
memory of received punishment (fall into cold water)
was expressed as the increase in seconds between train-
ing and retention latencies.
24. Ward, J. S.; Merritt, L.; Bymaster, F. P.; Calligaro, D. O.
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27. Jucker, E.; Rissi, E. Helv. Chim. Acta 1962, 45, 2383.
28. Buchman, E. R.; Sargent, E. J. Am. Chem. Soc. 1945, 67,
400.
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