Structure-activity relationship studies on unifiram (DM232) and sunifiram (DM235), two novel and potent cognition enhancing drugs

Article abstract of DOI:10.1016/j.bmc.2003.10.025

Structure-activity relationships on two novel potent cognition enhancing drugs, unifiram (DM232, 1) and sunifiram (DM235, 2), are reported. Although none of the compounds synthesised reached the potency of the parent drugs, some fairly active compounds have been identified that may represent new leads to develop other cognition enhancing drugs. An interesting result of this research is the identification of two compounds (13 and 14) that are endowed with amnesing activity (the opposite of the activity of the original molecules) and are nearly equipotent to scopolamine. Moreover, two compounds of the series (5 and 6) were found endowed with analgesic activity on a rat model of neuropathic pain at the dose of 1 mg/kg.

Full text of DOI:10.1016/j.bmc.2003.10.025

Bioorganic & Medicinal Chemistry 12 (2004) 71–85
Structure–activity relationship studies on unifiram
(DM232) and sunifiram (DM235), two novel and potent
cognition enhancing drugs
Serena Scapecchi,a,* Elisabetta Martini,a Dina Manetti,a Carla Ghelardini,b
Cecilia Martelli,a Silvia Dei,a Nicoletta Galeotti,b Luca Guandalini,a
Maria Novella Romanellia and Elisabetta Teodoria
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Firenze, Via G. Capponi 9, I-50121 Firenze, Italy
b
`
Dipartimento di Farmacologia Preclinica e Clinica, Universita di Firenze, Viale G. Pieraccini 6, I-50139 Firenze, Italy
Received 5 August 2003; revised 26 September 2003; accepted 14 October 2003
Abstract—Structure–activity relationships on two novel potent cognition enhancing drugs, unifiram (DM232, 1) and sunifiram
(DM235, 2), are reported. Although none of the compounds synthesised reached the potency of the parent drugs, some fairly active
compounds have been identified that may represent new leads to develop other cognition enhancing drugs. An interesting result of
this research is the identification of two compounds (13 and 14) that are endowed with amnesingactivity (the opposite of the
activity of the original molecules) and are nearly equipotent to scopolamine. Moreover, two compounds of the series (5 and 6) were
found endowed with analgesic activity on a rat model of neuropathic pain at the dose of 1 mg/kg.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
same level when the 2-pyrrolidinone ringwas opened to
give the corresponding piperazine derivatives8 (DM 235
(2); (Chart 1), suggesting that in this class of compounds
the 2-oxopyrrolidine moiety is not critical for pharma-
cological action. Both series of compounds were effec-
One of the main symptoms accompanying ageing,
stroke, head injury and neurodegenerative diseases like
Alzheimer’s disease is the decline of cognitive functions.
Cognition enhancers,1 are defined as drugs able to
facilitate attentional abilities and acquisition, storage
and retrieval of information, and to attenuate the
impairment of cognitive functions associated with age
and age-related pathologies.2 By definition, this class of
drugs improves the decline of cognitive functions but
does not change the rate of progression of neurodegen-
eration.3
tive also in other behavioural tests such as social
9,10
learningand Morris water maze.
Further research
confirmed and extended this finding, as it was observed
that the seco derivatives of many classical piracetam-like
drugs behave like the parent compounds.11 Finally,
potent cognition enhancing activity was also found in a
series of closely related 4-aminopiperidines.12
We have continued exploringstructure–activity rela-
tionships in these series of compounds and we now
report the cognition enhancing activity of compounds
where (Chart 2): (i) The 1,4-diazabicyclo[4.3.0]nonan-9-
one moiety has been expanded to a 1,4-diazabicy-
clo[4.4.0]decan-10-one by enlarging the pyrrolidinone
cycle. (ii) The 1,4-diazabicyclo[4.3.0]nonan-9-one, 1,4-
diazabicyclo[4.4.0]decan-10-one and piperazine scaf-
folds have been decorated with other groups, in parti-
cular those present in a series of compounds actingas
allosteric modulators of the AMPA receptor.13ꢀ15 As a
matter of fact, there are indications that AMPA-recep-
Previously, we described a new class of compounds with
a 1,4-diazabicyclo[4.3.0]nonan-9-one structure4 (Chart
1) related to the family of piracetam-like nootropics1,5ꢀ7
by the presence of the 2-pyrrolidinone ring(DM 232 ( 1);
Chart 1). Most of the compounds of this class showed a
very potent cognition enhancing activity in a mouse
passive avoidance assay. Activity was maintained at the
* Correspondingauthor. Tel.: +39-05-5275-7295; fax: +39-05-5240-
776; e-mail: fulvio.gualtieri@unifi.it
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.025

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S. Scapecchi et al. / Bioorg. Med. Chem. 12 (2004) 71–85
Table 1. Chemical and physical characteristics of compounds 3–14
N
n
R2
Mp ꢁC
(Petrol ether)
Purification
eluenta
Analysis
3b
4
5
6
7
8
9
10
11
12
13
14
1
2
2
2
1
2
1
2
1
2
1
2
Hydrogen
Hydrogen
—
Oil
—
II
—
C8H14N2O
c
A
B
C
C
D
D
E
E
F
F
130–133
130–133
166–169
170–172
181–184
86–89
140–142
147–150
114–117
113–116
C15H18N2O2
C14H17FN2O3S
C16H16N4O2
C17H18N4O2
C16H16N2O4
C17H20N2O4
C15H16N2O4
C16H18N2O4
C10H18N2O3S
C11H20N2O3S
Chart 1.
c
III
III
III
I
IV
VI
VI
VI
a See Experimental.
b See ref 4.
c Crystallized directly from abs EtOH/petrol ether.
available methylpyrazine with LDA at ꢀ78 ꢁC.19 The
metallic species obtained, was then reacted with ethyl 2-
bromopropionate to give compound 38. Hydrogenation
of 38 gave 4 directly which was then reacted with the
appropriate acyl or sulfonyl chloride to give compounds
5, 6, 8, 10, 12, 14. A similar pathway allowed the
synthesis of 1,4-diazabicyclo[4.3.0]nonan-9-one 3 with a
new, more efficient, synthetic route19 with respect to our
original synthesis.4 In fact, compound 39 obtained from
metallated methylpyrazine and t-butyl bromoacetate,
was reduced to ester 40 that, when heated at 200 ꢁC,
gave 3. This compound was then reacted with the
appropriate acyl or sulfonyl chloride to give compounds
7, 9, 11, 13.
Chart 2.
tor activation is involved in the antiamnesic effect of
DM232 and DM235.16 (iii) An amino group has been
introduced into the acyl side chain of the piperazine
compound DM 235 (2), mimickingthe basic side chain
of nebracetam.1 Cognition enhancing activity of the
compounds obtained has been evaluated through the
Compounds 15–27, whose chemical and physical char-
acteristics are reported in Table 2, were synthesized as
shown in Scheme 2. 1-Benzoyl-4-benzylpiperazine20 and
41, synthesized from 1-benzylpiperazine and iso-
propylsulfonyl chloride, were hydrogenated with
HCOONH4, Pd/C 10% in MeOH21 to give 2422 and 42.
Commercially available 1-acetylpiperazine, 1-propio-
nylpiperazine (16), 24 and 42,8 were then treated with
commercially available piperonyloyl chloride, iso-
propylsulfonyl chloride, 4-F-benzenesulfonyl chloride
or home made 1,4-benzodioxan-6-carbonyl chloride and
6-quinoxaloyl chloride, to give the desired compounds
15, 17–23, 25–27. 1,4-Benzodioxan-6-carbonyl chloride
was obtained by oxidation of commercially available
passive avoidance test and confirmed with the social
17
learningtest
in the case of one of the most potent
compounds. Moreover, followingthe report of Rashid
et al.18 on the neuropathy-specific analgesic action of
nefiracetam, the compounds have been also screened in
the ligated sciatic nerve model.
23,24
1,4-benzodioxan-6-carboxaldehyde with KMnO4
2. Chemistry
followed by treatment with SOCl2 by standard meth-
ods.25 6-Quinoxaloyl chloride was synthesised from
commercially available 3,4-diaminobenzoic acid with
potassium carbonate and glyoxal sodium bisulfite
hydrate26 followed by reaction with SOCl2.
Compounds 3–14, whose chemical and physical char-
acteristics are reported in Table 1, were synthesized as
reported in Scheme 1. 1,4-Diazabicyclo[4.4.0]decan-10-
one 4 was obtained by metallation of commercially

S. Scapecchi et al. / Bioorg. Med. Chem. 12 (2004) 71–85
73
Scheme 1. (a) LDA, ꢀ78 ꢁC; Br(CH2)2COOC2H5 or BrCH2COOC(CH3)3; (b) H2, Pd/C; (c) 200 ꢁC; (d) CH3CN, Et3N, R1Cl.
Table 2. Chemical and physical characteristics of piperazine derivatives 15–27
N
R1
R2
Mp ꢁC
(Petrol ether)
Purification
eluenta
Analysis
15
16b
17
18
19
20
21
22
23
24
25
26
27
CO–CH3
CO–CH2–CH3
CO–CH2–CH3
CO–CH3
CO–CH2–CH3
CO–CH3
CO–CH2–CH3
CO–CH3
CO–CH2–CH3
CO–C6H5
CO–C6H5
A
Hydrogen
60–61
—
IV
—
IV
IV
V
IV
IV
IV
IV
IV
VI
VI
VI
C15H18N2O4
—
A
B
B
C
C
D
D
65–66
50–51
60–62
55–56
43–44
45–46
60–61
Oil
C16H20N2O4
C14H16N2O4
C15H18N2O4
C15H16N4O2
C16H18N4O2
C9H18N2O3S
C10H20N2O3S
C11H14N2O
C14H20N2O3S
C17H17FN2O3S
C13H19FN2O4S2
Hydrogen
D
CO–C6H5
SO2C6H4-p-F
142–144
105–106
161–163
SO2C6H4-p-F
SO2–CH(CH3)2
a See Experimental.
b See ref 8.
Compounds 28–37, whose chemical and physical char-
acteristics are reported in Table 3, were synthesised as
shown in Scheme 3. The commercially available N-(tert-
butoxycarbonyl)-l-alanine, N-(tert-butoxycarbonyl) gly-
cine and N-(tert-butoxycarbonyl)sarcosine (43), obtained
with standard methods20 7 were activated with N-hydroxy-
succinimide28 and N,N -dicyclohexylcarbodiimide. These
derivatives were then treated with benzylpiperazine28 to

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S. Scapecchi et al. / Bioorg. Med. Chem. 12 (2004) 71–85
Scheme 2. (a) (CH3)2CHSO2Cl, Net3, CH3CN; (b) HCOONH4, Pd/C 10%, MeOH; (c) R2COCl or (CH3)2CHSO2Cl or 4-F-C6H4–SO2Cl, Net3,
CH3CN.
Table 3. Chemical and physical characteristics of compounds 28–37
N
R2
R3
R4
R5
Mp ꢁC (Petrol ether)
Purification eluenta
Analysis
28
29
30
31
32
33
34
35
36
37
A
A
A
A
A
B
B
B
B
B
H
H
H
H
CH3
H
H
H
H
CH3
H
H
CH3
CH3
H
H
H
CH3
CH3
H
H
SO2-CH(CH3)2
H
SO2-CH(CH3)2
72–74
90–91
65–66
80–81
78–79
136–137
190–195
64–65
65–66
131–132
IV
IV
VII
IV
IV
IV
IV
IV
IV
IV
C13H17N3O2
C16H23N3O4S
C14H19N3O2
C17H25N3O4S
C14H19N3O2
C12H16FN3O3S
C15H22FN3O5S2
C13H18FN3O3S
C16H24FN3O5S2
C13H18FN3O3S
H
H
SO2-CH(CH3)2
H
SO2-CH(CH3)2
H
a See Experimental.
give 44–46 that, in turn, were hydrogenated with
HCOONH4, Pd/C 10% in MeOH21 to give 47–49.
Compounds 46 and 49 have already been described in
literature,29,30 but were synthesised with a different
method. Compounds 47–49 were treated with benzoyl
chloride and NEt3 (50–52) and de-protected with HCl to
afford 28, 30, 32. Analogously, the same compounds
were reacted with 4-fluorobenzenesulfonyl chloride (53–
55) and then de-protected to give 33, 35 and 37. Finally,
compounds 29, 31, 34, 36 were synthesised from 28, 30,
33 and 35 with isopropylsulfonyl chloride by the
method already described.
3. Pharmacology
3.1. Cognition enhancing activity
The compounds studied were tested as cognition
enhancers in the mouse passive avoidance test of
Jarvik and Kopp,31 slightly modified by us (see
the Experimental). The same test was used to evalu-
ate the amnesingproperties of compounds
13
and 14. The cognition enhancing properties of com-
pound 22 were also tested in the rat social learning
test.17

S. Scapecchi et al. / Bioorg. Med. Chem. 12 (2004) 71–85
75
Scheme 3. (a) (tBuOCO)2O, NEt3; (b) DCC, N-hydroxysuccinimide, benzylpiperazine; (c) HCOONH4, Pd/C 10%, MeOH; (d) PhCOCl, NEt3;
(e) HCl 2M; (f) 4-F-C6H4-SO2Cl, NEt3; (g) (CH3)2CH–SO2Cl, CH3CN, NEt3.
amnesic activity below the 10 mg/kg minimal effective
dose are reported in Table 4; the remainingcompounds
did not show any activity up to the same dose. It must
be kept in mind that the tests are made in vivo and that
pharmacokinetic factors could play a role on the
potency of the drugs. For instance, the structural modi-
fications introduced to study SARs may affect critical
pharmacokinetic properties such as bioavailability. The
minimal effective doses of piracetam, DM232 and
DM235 are reported in the same table. In general, even
if some molecules show much better potency with
respect to piracetam, none of them reached the potency
of unifiram (DM232, 1) and sunifiram (DM235, 2).
3.2. Analgesic activity
A peripheral mono neuropathy was produced in adult
rats by placingloosely constrictive ligatures around the
common sciatic nerve accordingto the method descri-
bed by Bennett.32 The nociceptive threshold in the rat
was determined with an analgesimeter according to the
paw pressure test.33
4. Results and discussion
4.1. Cognition enhancing activity
To establish structure–activity relationships, all the
synthesised compounds were evaluated in the mouse
passive avoidance test. The compounds showinganti
Cycle enlargement was clearly detrimental for nootropic
activity, as shown by compounds 5 and 6, that are some
hundred times less potent than 1 and 2. However, the

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S. Scapecchi et al. / Bioorg. Med. Chem. 12 (2004) 71–85
Table 4. Compounds active on mouse passive avoidance test, using
scopolamine (S) as amnesingdrug. All other compounds are inactive
up to 10 mg/kg
Drug(number
of animals)
Minimal
effective dose
mg/kg (ip)
Entry latency
2nd day
1
st day
ꢀ
Saline (13)
Scopolamine(S) (6)
S+1(35)
—
1.5
0.01
0.01
30
0.1
0.1
1
15.0ꢂ5.9
16.6ꢂ4.7
15.2ꢂ3.2
14.9ꢂ3.6
95.6ꢂ8.8
44.5ꢂ8.3a
98.7ꢂ9.1b
95.4ꢂ8.7b
80.6
27.9
83.5
80.5
89.4
88.2
62.3
93.2
83.1
78.6
90.1
84.7
S+2(31)
S+piracetam (56)
S+3 (9)
S+4 (18)
S+5 (16)
S+6 (18)
S+8 (16)
S+10 (20)
S+11 (12)
S+16 (10)
S+17 (12)
S+22 (19)
S+23 (19)
S+24 (10)
S+30 (11)
S+35 (11)
15.9ꢂ2.1 105.3ꢂ7.8b
18.6ꢂ6.0 106.8ꢂ12.5b
15.3ꢂ3.1
77.6ꢂ11.6b
99.4ꢂ11.1b
15.2ꢂ3.3 108.3ꢂ10.6b
1
16.3ꢂ2.7
10
10
1
0.1
1
0.1
1
0.1
1
13.9ꢂ3.2 92.52ꢂ11.5b
15.5ꢂ4.1 105.6ꢂ10.5b
14.6ꢂ3.4
99.3ꢂ8.3b
22.6ꢂ4.5 130.3ꢂ11.2b 107.7
16.4ꢂ3.2 108.2ꢂ9.3b
91.8
73.3
75.2
89.4
70.8
69.6
Figure 1. Effect of compound 22 in a rat social learningtest in com-
parison with piracetam. Each column represents the mean of six rats.
*p<0.01 in comparison with the correspondingsaline-treated rats.
Piracetam and 22 were administered 20 min before the first session.
Vertical lines show SEM.
13.8ꢂ3.6
13.7ꢂ3.9
87.1ꢂ9.6c
88.9ꢂ10.3c
16.3ꢂ4.6 105.7ꢂ9.3b
17.0ꢂ3.5
87.8ꢂ9.1b
1
13.8ꢂ3.2
83.4ꢂ8.4b
a p<0.01 with respect to mice treated with saline.
b p<0.01.
A very interestingresult was obtained when the iso-
propylsulfonyl group was introduced into both 1,4-dia-
zabicyclo[4.3.0]nonan-9-one and 1,4-diazabicyclo[4.4.0]
decan-10-one scaffolding. Indeed, the resulting com-
pounds 13 and 14 are endowed with an opposite action
with respect to the other members of the series and
appear to be potent amnesic drugs, as is shown in Table
5. Their potency and activity seem comparable to those
of scopolamine and their amnesic action was overcome
by compound 1 in a dose-dependent manner that par-
allels that of scopolamine. These results are intriguing
and studies are under way to clarify the mechanism of
action of 13 and 14.
c p<0.05 with respect to mice treated with scopolamine.
unsubstituted 1,4-diazabicyclo[4.4.0]decan-10-one 4 is
active at the dose of 0.1 mg/kg ip which is close to the
potency of the reference compounds. The same happens
for the1,4-diazabicyclo[4.3.0]nonan-9-one analogue 3,
and for the mono substituted piperazines 16 and 24.
Apparently, the N-substitution (or the second N-sub-
stitution in the case of piperazines) while increasingin
some cases nootropic potency, is not critical for high
activity. It is possible that the unsubstituted or mono
substituted compounds are indeed active metabolites of
the parent drugs. Studies are in progress to evaluate this
possibility.
Finally, as regards the results of inserting an amino
group into the acyl chain of the piperazine series, most
of the molecules synthesised (28, 29, 31–34, 36, 37), are
inactive up to the dose of 10 mg/kg. However, two
compounds (30 and 35) show a minimal effective dose
of 1 mg/kg and may represent new leads to develop
other molecular species endowed with cognition enhan-
cingproperties.
Also the substitution of benzoyl (A) and 4-F-benzene-
sulfonyl (B) groups with the acyl groups present in some
ampakines13 such as 6-quinoxaloyl (C), 1,4-benzo-
dioxan-6-carbonyl (D), piperonyloyl (E) was dis-
appointing. As a matter of fact, the compounds
obtained are inactive up to 10 mg/kg dose (7, 9, 12) or
show much lower potency (8, 10, 11) with respect to the
parent compounds, independently of the size of the
cycle. Similar results were observed when the same sub-
stitution was performed on the piperazine scaffoldingof
DM235. Only compound 17 is active at the dose of 1
mg/kg, the other compounds (15, 18–21, 25–27) being
inactive up to the dose of 10 mg/kg. On the contrary,
the insertion of the isopropylsulfonyl group, present in a
new series of AMPA receptor modulators,14 gave better
results as compound 23 is active at 1 mg/kg and 22 at
0.1 mg/kg, close to the potency of the parent drug. The
cognition enhancing properties of this compound were
confirmed by the results of the social learningtest 17 as it
is shown in Figure 1.
4.2. Analgesic properties on neuropathic pain
18
Followingthe interestingreport of Rashid et al.,
all
compounds were tested on the model of neuropatic pain
described by Bennett.32 Experiments were performed on
rats submitted to paw-pressure test 14 days after the
operation since at this time a significant reduction of the
pain threshold of the injured paw (dx) was observed. By
contrast in the controlateral paw the pain perception
remained unchanged. Only two compounds showed
appreciable analgesic activity on this model: 5 (1 mg/kg
ip) and 6 (1 mg/kg ip) exhibited an anti hyperalgic effect
when compared with saline treated group 30, 45 and 60
min after administration. Both compounds did not
modify pain threshold in controlateral, non operated,
paw. Compounds 5 and 6 at the doses of 0.1 mg/kg ip

S. Scapecchi et al. / Bioorg. Med. Chem. 12 (2004) 71–85
77
Table 5. Amnesia inducingeffect of compounds 13 and 14
ical values. GC/MS was performed with a Perkin-
ElmerAutosystem XLTurbomass.
Drug(number
of animals)
Dose
mg/kg (ip)
Entry latency
2nd day
5.1.1. 4-Pyrazin-2-yl butyric acid ethyl ester (38) and 3-
pyrazin-2-yl propionic acid tert-butyl ester (39). One
equivalent of butyllithium (1.6 M hexane solution) was
added to 1 equiv of diisopropylamine in anhydrous
THF at 0 ꢁC. The mixture was stirred at 0 ꢁC for 30 min,
then cooled at ꢀ78 ꢁC and added with 1 equiv of
methylpyrazine. After 10 min the appropriate bromo-
ester was added and the mixture maintained 1 h at
ꢀ78 ꢁC. Then the reaction was quenched with NH4Cl
and extracted with diethyl ether and the organic phase
dried and evaporated under vacuum. Compound 38 was
obtained after purification by column chromatography
1st day
ꢀ
Saline (13)
Scopolamine (6)
13 (18)
14 (19)
13 (18)
—
1.5
10
10
1
15.5ꢂ3.0
16.6ꢂ4.7
13.7ꢂ3.2
14.3ꢂ3.0
13.6ꢂ3.9
14.5ꢂ4.4
16.0ꢂ4.2
14.1ꢂ3.9
13.8ꢂ3.3
15.3ꢂ4.4
17.2ꢂ3.4
13.8ꢂ4.1
102.6ꢂ8.7
44.5ꢂ8.3b
20.5ꢂ9.4b
22.6ꢂ7.9b
61.5ꢂ10.4b
69.8ꢂ8.5b
36.4ꢂ9.5b
81.2ꢂ9.4c
111.6ꢂ10.3c
41.9ꢂ9.1b
79.8ꢂ9.3c
109.5ꢂ9.6c
87.1
27.9
6.8
8.3
47.9
55.3
20.4
67.1
97.8
20.4
82.6
95.7
14 (19)
1
13a+1 (18)
13a+1 (16)
13a+1 (17)
14a+1 (14)
14a+1 (12)
14a+1 (14)
0.001
0.01
0.1
0.01
0.1
1.0
1
a 10 mg/kg as amnesing drug.
(eluent I) in 15% yield. H NMR (CDCl3) d: 1.26 (t,
b P<0.01 with respect to mice treated with saline.
J=6.0 Hz, 3H,CH2CH3); 2.10–2.20 (m, 2H,
CH2CH2CH2COO); 2.39 (t, J=6.0 Hz, 2H,
CH2CH2CH2COO); 2.88 (t, J=8 Hz, 2H, CH2COO);
4.14 (q, J=8 Hz, 2H, OCH2); 8.41–8.51 (m, 3H, H pyr-
azine protons) ppm. Anal. (C10H14N2O2) C,H,N.
c P<0.01; P<0.05 with respect to mice treated with 13 or 14 (10 mg/
kg).
were devoid of any anti hyperalgic effect. Rats treated
with 5 and 6 at the highest doses showed a normal
behaviour compared to saline control rats. It is inter-
estingthat the two active molecules belongto the 1,4-
diazabicyclo[4.4.0]decan-10-one series and that the cor-
responding1,4-diazabicyclo[4.3.0]nonan-9-one deriva-
tives are inactive in this assay. The mechanism of action
does not seem related to that determiningcongition
enhancement as, in this case the compounds of both
series are active, although with different potencies (see
Table 4 and ref 4). Compounds 5 and 6 represent two
fairly potent analgesic compounds that can be useful
leads to find new drugs for a pathology that is difficult
to control.
Compound 3919 was obtained, in the same way, after
purification by flash chromatography (eluent I) in 20%
yield. 1H NMR (CDCl3) d: 1.42 (s, 9H, C(CH3)3; 2.72 (t,
J=8 Hz, 2H, CH2CH2COO); 3.08 (t, J=8.0 Hz, 2H,
CH2COO); 8.39–8.49 (m, 3H, H pyrazine protons) ppm.
Anal. (C11H16N2O2) C,H,N.
5.1.2. 1,4-Diazabicyclo[4.4.0]decan-10-one (4). Com-
pound 38 was hydrogenated over Pd/C 10% in abs.
EtOH, at 60 psi for 60 h. After filtration, the solvent
was removed under vacuum to give a residue that was
purified by flash chromatography (eluent II). 60% yield.
1H NMR (CDCl3) d: 1.33–1.50 (m, 1H); 1.50–1.73 (m,
1H); 1.73–2.03 (m, 3H, 2CH and NH); 2.25–2.53 (m,
3H); 2.57–2.78 (m, 2H); 2.91–3.08 (m, 2H); 3.20–3.38
(m, 1H); 4.50–4.66 (m, 1H) ppm. 13C NMR (CDCl3) d :
19.60 (t); 27.67 (t); 33.14 (t); 42.40 (t); 46.13 (t); 53.27
(t); 57.28 (d); 169.50 (s). m/z 154 (M+). Anal.
(C8H14N2O) C,H,N.
5. Experimental
5.1. Chemistry
All meltingpoints were taken on a Bu chi apparatus and
are uncorrected. Infrared spectra were recorded with a
Perkin-Elmer 681 spectrophotometer in Nujol mull for
solids and neat for liquids. Unless otherwise stated,
NMR spectra were recorded on a Gemini 200 spectro-
5.1.3. 3-Piperazin-2-yl propionic acid tert-butyl ester
(40). Compound 39 (2 mmol, 0.43 g) was hydrogenated
over Pd/C10% in abs. EtOH at 70 psi for 24 h. After
filtration, the solvent was removed under vacuum to
give compound 40 that was used without purification in
1
1
meter (200 MHz for H NMR, 50.3 MHz for 13C), and
the next step. 93% yield. H NMR (CDCl3) d: 1.2 (s,
chromatographic separations were performed on a silica
gel column by gravity chromatography (Kieselgel 40,
0.063–0.200 mm; Merck) or flash chromatography
(Kieselgel 40, 0.040–0.063 mm; Merck). The composi-
tion of the elutingsystems was the following: I) petro-
leum ether/CH2Cl2/Et2O/EtOH abs. 900:360:360:180;
9H, C(CH3)3; 1.48–1.67 (m, 2H); 2.18–2.43 (m, 5H);
2.57–3.00 (m, 6H) ppm; 13C NMR (CDCl3) d: 28.31 (q);
29.80 (t); 32.00 (t); 46.66 (t); 47.17 (t); 52.33 (t); 55.71
(d); 80.31(s); 172.81 (s). Anal. (C11H22N2O2) C,H,N.
5.1.4. 1,4-Diazabicyclo[4.3.0]nonan-9-one (3). Com-
pound 40 (1.9 mmol, 0.41 g)was heated to 200 ꢁC; after
1 h the reaction was cooled to rt and CHCl3 (100 mL)
was added. The solid residue was eliminated by fil-
tration and the organic phase evaporated under
vacuum. Compound 34,19 was purified by flash chroma-
tography (eluent II) and obtained in 43% yield.
II)
NH4OH/EtOH
abs./CHCl3/petroleum
ether
45:225:600:90; III) NH4OH/EtOH abs./CH2Cl2/petro-
leum ether 4:25:150:50; IV): CHCl3/EtOH abs./petro-
leum ether/NH4OH 340:65:60:8; V): CHCl3/MeOH
90:10; VI): petroleum ether/CH2Cl2/Et2O/EtOH abs./
NH4OH 900:360:360:180:9.9; VII): CH2Cl2/MeOH
95:5. Yields are given after purification, unless differ-
ently stated. Where analyses are indicated by symbols,
the analytical results are within ꢂ0.4% of the theoret-
5.1.5. N-(tert-Butoxycarbonyl)sarcosine (43). NEt3 (2.3
mL; 0.016 mol) and di-tert-butyl dicarbonate (1.23 g;

78
S. Scapecchi et al. / Bioorg. Med. Chem. 12 (2004) 71–85
5.61 mmol), in a minimal amount of THF, were added
to sarcosine (0.5 g; 5.61 mmol) in 1.5 mL of H2O and 15
mL of THF, cooled to 0 ꢁC. After 24 h at rt, the solvent
was removed and the aqueous layer was extracted with
CHCl3. After dryingand evaporation of the solvent, the
product was used as such for the next reaction. 1H
NMR (CDCl3) d: 1.28 (s, 3H, CH3N), 1.34 (s, 9H,
(CH3)3C), 3.83 (s, 2H, CH2N), 9.2 (bs, OH) ppm. Anal.
(C8H15NO4) C,H,N.
3 equiv of NEt3 were added. The mixture was refrig-
erated to 0 ꢁC and then 1 equiv of the proper arylsulfo-
nyl chloride, alkylsulfonyl chloride or acyl chloride was
added. After 24 h at room temperature, the solvent was
removed, the mixture made alkaline with NaHCO3,
extracted with CHCl3 and dried. Removal of the solvent
gave compounds that were used as such for the next step
(41, 50–55), or purified by column chromatography
usingthe eluent indicated in Table 1 (5–14), 2 (15–27)
and 3 (29, 31, 34, 36). Their yields and spectroscopic
characteristics are reported in Table 7 (supplementary
material).
5.2. Activation of protected aminoacids with N-hydroxy-
succinimide
1 equiv of N-(tert-butoxycarbonyl)aminoacid in 5 mL of
CH2Cl2 was cooled to 0 ꢁC and then added with 1 equiv
of N-hydroxysuccinimide and 1.1 equiv of N,N0-diciclo-
hexylcarbodiimide in a minimal amount of CH2Cl2.
After 1 h at 0 ꢁC, the mixture was left at room tem-
perature for 20 h. After filtration, the solution was
cooled to 0 ꢁC and 1 equiv Benzylpiperazine was added.
After 24 h at room temperature, CH2Cl2 was added, the
solution washed with H2O and the dried solvent evapo-
rated under reduced pressure. Compounds 44 and 4629
were purified by flash chromatography with eluent I,
while the derivative 45 was obtained pure. Yields and
spectroscopic characteristics of compounds 44–46 are
reported in Table 7 (supplementary material).
5.5. Deprotection of 4-substituted-1-acyl-piperazine
A solution of HCl 2 M (2 equiv) was added to inter-
mediates 50–55 and the mixture left at room tempera-
ture for 24 h. Then NaHCO3 was added and the
solution extracted with CHCl3 and dried. Removal of
the solvent gave compounds that were purified with
column chromatography using eluent IV (28, 32, 33, 35,
37) or eluent VII for compound 30. Their yields and
spectroscopic characteristics are reported in Table 7
(supplementary material).
6. Pharmacology
6.1. Antiamnesic test: passive-avoidance test
5.3. Removal of the benzyl group
To 1 equiv of 1-benzoyl-4-benzylpiperazine, 41 and 44–
46 in MeOH anhydrous and under N2 of 0.5 equiv of
Pd/C/10% and 5 equiv of HCOONH4 were added. The
mixture was refluxed for 8 h, then filtered and the solu-
tion was evaporated. The residue was made alkaline
with NaHCO3, extracted with CHCl3 and dried. After
removal of the solvent compounds 24, 42, 47, 48 and
4930 were obtained. Their yields and spectroscopic
characteristics are reported in Table 7 (supplementary
material).
The test was performed accordingto the step-through
method described by Jarvik and Kopp.31 The apparatus
consists of a two-compartment acrylic box with a ligh-
ted compartment connected to a darkened one by a
guillotine door. In the original method, mice received a
punishingelectrical shock as soon as they entered the
dark compartment, while in our modified method, after
entry into the dark compartment, mice receive a non-
painful punishment consistingof a fall (from 40 cm)
into a cold water bath (10 ꢁC). For this purpose the dark
chamber was constructed with a pitfall floor. Mice
receive the punishment when enteringthe dark room in
the trainingsession and remember it in the session on
the followingday, unless their memory is impaired by
the amnesic drug. Mice who did not enter after 60 s
latency were excluded from the experiment. For mem-
5.4. Synthesis of acyl or sulfonyl derivatives
To a CH3CN solution of 1 equiv of the suitable piper-
azine derivative (1-benzylpiperazine, 1-acetylpiperazine,
1-propionylpiperazine, 3, 4, 24, 28, 30, 33, 35, 42, 47–49)
Table 6. Antihyperalgic effect of 5 and 6 in a rat model of mononeuropathic dx evaluated in the paw pressure test
Treatment
Dose mg/kg (s.c.)
Paw
Paw pressure in rats
After analgesic treatment.
Before treatment.
30 min
45 min
60 min
Saline
Saline
sn
dx
sn
dx
sn
dx
sn
dx
sn
dx
51.1ꢂ2.5
26.5ꢂ4.3
50.2ꢂ3.9
28.5ꢂ3.5
49.4ꢂ4.8
24.8ꢂ3.5
48.7ꢂ3.9
31.9ꢂ3.1
52.5ꢂ4.5
26.8ꢂ3.3
54.5ꢂ4.6
25.2ꢂ5.1
48.6ꢂ4.3
29.5ꢂ3.6
52.1ꢂ6.0
49.2ꢂ3.1*
51.3ꢂ4.5
32.5ꢂ3.3
54.1ꢂ4.2
48.3ꢂ3.7*
48.2ꢂ5.2
29.7ꢂ4.8
53.1ꢂ3.9
31.3ꢂ5.1
50.8ꢂ4.2
49.7ꢂ3.3*
54.2ꢂ5.1
35.3ꢂ4.5
52.7ꢂ3.7
48.9ꢂ3.5*
53.6ꢂ5.4
30.6ꢂ4.9
51.7ꢂ4.4
28.9ꢂ3.7
47.2ꢂ3.5
51.6ꢂ4.0*
49.6ꢂ3.7
38.4ꢂ3.1
47.3ꢂ5.0
45.1ꢂ4.5*
5
5
5
5
6
6
6
6
0.1
0.1
1.0
1.0
0.1
0.1
1.0
1.0
There were 5–7 rats per group. *p<0.001 versus the saline dx paw.

Table 7. Supplementary material to ‘Structure–activity relationship studies in a class of potent cognition enhancing drugs’
Sigla
N
Yield (%)
35
IR n (cmꢀ1
)
1H NMR (CDCl3) d
13C NMR (CDCl3) d
EM107
15
—
2.12 (s, 3H, CH3CO); 3.42–3.77
(m, 8H, CH2 piperazine);
4.27 (s, 4H, CH2O); 6.81–6.98
(m, 3H, aromatics) ppm.
21.79 (q); 41.84 (t); 46.55 (t); 64.65 (t); 64.82 (t);
117.09 (d); 117.68 (d); 121.03 (d); 128.35 (s); 143.73 (s);
145.56 (s); 169.50 (s); 170.43 (s).
MN69
17
49
—
0.98 (t, J=7.2 Hz, 3H, CH3CH2); 2.18
(q, J=7.2 Hz, 2H, CH2CH3); 3.18–3.36
(m, 8H, CH2 piperazine); 4.05 (s, 4H,
CH2O); 6.63–6.80 (m, 3H,
9.79 (q); 26.89 (t); 43.45 (t); 45.66 (t); 64.63
(t); 64.81 (t); 117.08 (d); 117.66 (d); 121.01 (d); 128.38 (s);
143.73 (s); 145.54 (s); 170.41 (s); 172.80 (s).
aromatics) ppm.
EM105
MN62
18
19
20
21
74
61
48
37
—
—
—
—
2.11 (s, 3H, CH3CO); 3.48–3.66
(m, 8H, CH2 piperazine);
6.00 (s, 2H, CH2O); 6.83–6.89
(m, 3H, aromatics) ppm.
21.77 (q); 41.76 (t); 46.48 (t); 101.89 (t);
108.30 (d), 108.57 (d); 121.99 (d); 128.89 (s); 148.00 (s);
149.40 (s); 169.46 (s); 170.36 (s).
1.15 (t, J=7.3 Hz, 3H, CH3CH2); 2.36
(q, J=7.3 Hz, 2H, CH2CH3); 3.44–3.76 (m,
8H, CH2 piperazine); 6.00 (s, 2H, CH2O);
6.78–6.96 (m, 3H, aromatics) ppm.
10.97 (q); 26.92 (t); 43.45 (t); 43.60 (t); 102.05
(t); 108.35 (d); 108.83 (d); 122.05 (d); 129.02 (s); 148.07 (s);
149.45 (s); 170.45 (s); 172.83 (s).
EM108
EM109
2.10 (s, 3H, CH3CO); 3.55–3.66 (m, 8H,
CH2 piperazine); 7.78 (d, J=8.8 Hz,
1H, aromatic); 8.10–8.18 (m, 2H, aromatics);
8.88 (s, 2H, aromatics) ppm.
21.81(q); 41.76 (t); 46.50 (t); 128.44 (d); 128.89
(d); 130.77 (d); 136.87 (s); 142.59 (s); 143.53 (s); 146.32 (d);
146.43 (d); 169.32 (s); 169. 41 (s).
1.15 (t, J=7.3 Hz, 3H, CH3CH2); 2.37 (q, J=7.3 Hz,
2H, CH2CH3); 3.49–3.70 (m, 8H, CH2
piperazine); 7.81 (dd, J=8.4 Hz, 1.8 Hz,
1H, aromatic); 8.12 (d, J=1.8 Hz,
9.70 (q); 26.78 (t); 41.80 (t); 42.69 (t); 45.48 (t); 47.90 (t);
128.37 (d); 128.84 (d); 130.64 (d); 136.87 (s); 142.50 (s); 143.41 (s);
146.27 (d); 146.37 (d); 169.21 (s); 172.63 (s).
1H, aromatic); 8.19 (d, J=8.8 Hz,
1H, aromatic); 8.90 (s, 2H, aromatic) ppm.
EM106
22
66
53
—
—
1.33 (d, J=7.0 Hz, 6H,
(CH3)2CH);2.10 (s, 3H, CH3CO);
3.11–3.27 (m, 1H, CH(CH3)2); 3.29–3.42 (m, 4H,
CH2 piperazine); 3.48–3.53 (m, 2H, CH2 piperazine);
3.62–3.68 (m, 2H, CH2 piperazine) ppm.
17.11 (q); 21.79 (q); 42.16 (t); 46.50 (t); 46.77 (t);
47.17 (t); 53.89 (d); 169.34 (s).
DM360 23
1.16 (t, J=7.3 Hz, 3H, CH3CH2); 1.35 (d, J=7.0 Hz, 6H,
(CH3)2CH); 2.36 (q, J=7.3 Hz, 2H, CH2CH3);
3.13–3.26 (m, 1H, CH(CH3)2); 3.34–3.41
(m, 4H, CH2 piperazine); 3.48–3.53
9.80 (q); 17.12 (q); 26.90 (t); 46.28 (t); 46.57 (t); 46.81 (t);
47.21 (t); 53.87 (d); 172.65 (s).
(m, 2H, CH2 piperazine); 3.67–3.73
(m, 2H, CH2 piperazine) ppm.
MN32
24
71
—
1.76 (bs, 1H, NH); 2.82–3.03 (m, 4H, CH2 piperazine);
3.38–3.53 (m, 2H, CH2 piperazine);
3.62–3.84 (m, 2H, CH2 piperazine);
–
7.32–7.46 (m, 5H, aromatics) ppm.
(Continued on next page.)

Table 7 (continued)
Sigla
N
Yield (%)
95
IR n (cmꢀ1
)
1H NMR (CDCl3) d
13C NMR (CDCl3) d
DM366 25
DM368 26
DM369 27
—
1.36 (d, J=7.0 Hz, 6H, (CH3)2CH);
3.17–3.28 (m, 1H, CH(CH3)2);
3.33–3.42 (m, 8H, CH2 piperazine);
7.39–7.47 (m, 5H, aromatics) ppm.
25.37 (q); 54.48 (t); 55.04 (t); 62.17 (d); 135.63 (d); 137.23 (d);
138.69 (d); 143.57 (s); 179.10 (s).
26
35
—
—
2.98–3.18 (m, 4H, CH2 piperazine);
3.53–3.91 (m, 4H, CH2 piperazine);
7.30–7.44 (m, 5H+2H, aromatics);
7.74–7.81 (m, 2H, aromatics) ppm.
45.93 (t); 46.46 (t); 117.03 (d, JCꢀF=22.9 Hz); 127.46 (d);
129.00 (d); 130.64 (d); 130.75 (d,
JCꢀF=11.0 Hz); 131.64 (d, JCꢀF=2.8 Hz);
135.01 (s); 165.73 (d, JCꢀF=256.4 Hz); 170.74 (s).
1.32 (d, J=7.0 Hz, 6H,
(CH3)2CH); 3.05–3.12 (m, 4H,
CH2 piperazine); 3.10–3.19 (m, 1H,
CH(CH3)2); 3.46–3.51 (m, 4H,
CH2 piperazine); 7.21–7.29
(m, 2H, aromatics); 7.73–7.80
(m, 2H, aromatics) ppm.
17.09 (q); 46.15 (t); 46.92 (t); 54.20 (d); 117.05
(d, JCꢀF=22.8 Hz); 130.75 (d, JCꢀF=9.1 Hz); 131.64
(d, JCꢀF=2.7 Hz); 165.76 (d, JCꢀF=255.5 Hz).
MN45
EM112
MN58
EM123
MN40
MN44
28
29
30
31
32
33
43
24
42
20
67
52
3300–3380 (NH2); 1670 (CO); 1630 (CO).
1.72 (s, 2H, NH2); 3.18–3.43 (m, 4H,
CH2 piperazine); 3.42 (s, 2H,
CH2CO); 3.47–3.72 (m, 4H, CH2 piperazine);
7.33–7.41 (m, 5H, aromatics) ppm.
42.38 (t); 43.59 (t); 44.46 (t); 127.30 (d); 129.00 (d);
130.60 (d); 135.20 (s); 170.91 (s, CO);
171.93 (s, CO)ppm.
—
1.37 (d, J=6.6 Hz, 6H, (CH3)2CH); 3.09–3.24
(m, 1H, CH(CH3)2); 3.40–3.73 (m, 8H,
CH2 piperazine); 3.97 (s, 2H, CH2CO); 5.40 (bs,
1H, NH); 7.27–7.45 (m, 5H, aromatics) ppm.
16.98 (q); 44.65 (t); 44.75 (t); 44.93 (t); 54.18 (d),
127.40 (d); 129.04 (d); 130.57 (d); 135.19 (s);
166.99 (s, CO); 170.92 (s, CO).
3340 (NH); 1670 (CO); 1620 (CO).
2.00 (bs, 1H, NH); 2.42 (s, 3H,
CH3N); 3.39 (s, 2H, CH2CO);
3.45–3.67 (m, 8H, CH2 piperazine);
7.27–7.41 (m, 5H, aromatics) ppm.
37.00 (q); 42.43 (t); 44.99 (t); 52.83 (t);
127.40 (d), 129.00 (d); 130.50 (d); 135.30 (s);
170.10 (s, CO); 170.90 (s, CO) ppm.
—
1.40 (d, J=6.9 Hz, 6H, (CH3)2CH);
3.03 (s, 3H, CH3N); 3.28–3.35 (m,
1H, CH(CH3)2); 4.15 (s, 2H, CH2CO);
7.40–7.47 (m, 5H, aromatics) ppm.
17.23 (q); 37.14 (q); 42.38 (t); 45.22 (t); 51.98 (t); 58.84 (d);
127.40 (d); 129.04 (d); 130.55 (d); 135.27 (s);
166.88 (s, CO); 170.94 (s, CO).
3300–3380 (NH); 1650 (CO); 1640 (CO).
3340–3400 (NH2); 1660 (CO).
1.12 (d, J=6.6 Hz, 3H, CH3); 1.79 (bs,
2H, NH2); 3.18–3.60 (m, 8H, CH2
piperazine); 3.61–3.64 (m, 1H, CHCH3);
7.19–7.30 (m, 5H, aromatics) ppm.
22.07 (q); 42.45 (t); 45.21 (t); 47.18 (d); 127.30 (d);
128.90 (d); 130.41 (d); 135.29 (s); 170.70
(s,CO); 175.00 (s,CO) ppm.
1.53 (bs, 2H, NH2); 2.98
41.64 (t); 43.64 (t); 44.04 (t); 46.25 (t); 46.34 (t); 116.90
(dd, JCꢀF=21.9 Hz); 130.70 (d, JCꢀF=9.2 Hz); 131.60
(d, JCꢀF=3.6 Hz); 165.70 (d, JCꢀF=254.0 Hz);
171.90 (s, CO) ppm.
(t, J=5.1 Hz, 4H, CH2 piperazine); 3.37
(s, 2H, CH2CO); 3.44–3.48 (m, 2H,
CH2 piperazine); 3.61–3.71 (m, 2H, CH2
piperazine); 7.16–7.27 (m, 2H, aromatics);
7.69–7.79 (m, 2H, aromatics) ppm.
(Continued on next page.)

Table 7 (continued)
Sigla
N
Yield (%)
56
IR n (cmꢀ1
)
1H NMR (CDCl3) d
13C NMR (CDCl3) d
EM111
34
—
1.15 (d, J=6.9 Hz, 6H, (CH3)2CH);
2.82–2.93 (m, 4H, CH2 piperazine);
3.06–3.11 (m, 1H, CH(CH3)2);
15.74 (q); 42.86 (t); 43.37 (t); 45.22 (t); 51.49 (d);
116.23 (d, JCꢀF=22.9 Hz); 130.15 (d, JCꢀF=9.1 Hz); 130.48
(d, JCꢀF=3.6 Hz); 164.21 (d, JCꢀF=251.9 Hz); 166.44 (s, CO).
3.42–3.52 (m, 4H, CH2 piperazine); 3.81 (s, 2H,
CH2CO); 5.04 (bs, 1H, NH); 7.43–7.52
(m, 2H, aromatics); 7.75–7.82
(m, 2H, aromatics) ppm.
MN57
35
46
3350 (NH); 1650 (CO).
2.08 (bs, 1H, NH); 2.35 (s, 3H, CH3);
2.83–3.01 (m, 4H, CH2
36.89 (q); 41.32 (t); 44.37 (t); 52.71 (t); 116.90
(dd, JCꢀF=22.7 Hz); 130.70 (d, JCꢀF=9.1 Hz); 131.60 (s);
165.70 (d, JCꢀF=254.0 Hz); 169.70 (s, CO) ppm.
piperazine); 3.29 (s, 2H, CH2CO);
3.47–3.58 (m, 2H, CH2 piperazine); 3.62–3.71
(m, 2H, CH2 piperazine); 7.16–7.27
(m, 2H, aromatics); 7.69–7.77 (m, 2H,
aromatics) ppm.
EM122
36
30
—
1.34 (d, J=6.9 Hz, 6H, (CH3)2CH); 2.93
(s, 3H, CH3N); 2.97–3.03
(m, 4H, CH2 piperazine); 3.18–3.29
(m, 1H, CH(CH3)2); 3.55–3.60
(m, 2H, CH2 piperazine);
18.45 (q); 37.11 (q); 41.25 (t); 44.03 (t); 46.10 (t); 54.35 (d);
117.04 (d, JCꢀF=22.9 Hz); 130.78 (d, JCꢀF=7.3 Hz); 131.61
(s); 165.76 (d, JCꢀF=255.5 Hz); 166.61 (s, CO).
3.62–3.69 (m, 2H, CH2 piperazine); 4.05
(s, 2H, CH2CO); 7.20–7.28
(m, 2H, aromatics); 7.73–7.80
(m, 2H, aromatics) ppm.
MN36
37
41
42
60
91
74
3450 (NH2); 1650 (CO).
1.12 (d, J=6.9 Hz, 3H, CH3); 2.01
(bs, 2H, NH2); 2.79–3.08 (m, 4H,
CH2 piperazine); 3.48–3.59
22.15 (q); 41.63 (t); 44.83 (t); 46.23 (t);
46.46 (t); 47.29 (d); 116.90
(dd, JCꢀF=22.7 Hz); 130.70 (d, JCꢀF=9.2 Hz); 131.5
(d, JCꢀF=2.7 Hz); 165.7 (d, JCꢀF=254.0 Hz);
174.8 (s, CO) ppm.
(m, 4H, CH2 piperazine); 3.66 (q, J=6.8 Hz,
1H, CHCH3); 7.15–7.23 (m, 2H,
aromatics); 7.68–7.74 (m, 2H, aromatics) ppm.
—
—
1.36 (d, J=6.6 Hz, 6H, (CH3)2CH);
2.42–2.56 (m, 4H, CH2
piperazine); 3.09–3.22 (m, 1H,
CH(CH3)2); 3.35–3.42 (m, 4H,
CH2 piperazine); 3.57 (s, 2H, CH2Ph);
7.27–7.37 (m, 5H, aromatics) ppm.
—
—
1.34 (d, J=7.0 Hz, 6H, (CH3)2CH); 1.68 (bs, 1H, NH);
2.87–2.93 (m, 4H,
CH2 piperazine); 3.12–3.22 (m, 1H,
CH(CH3)2); 3.31–3.35 (m, 4H, CH2
piperazine) ppm.
(Continued on next page.)

Table 7 (continued)
Sigla
N
Yield (%)
70
IR n (cmꢀ1
)
1H NMR (CDCl3) d
13C NMR (CDCl3) d
44
—
1.37 (s, 9H, (CH3)3C); 2.33–2.38
(m, 4H, CH2 piperazine);
—
3.31–3.38 (m, 2H, CH2 piperazine);
3.44 (s, 2H, CH2Ph); 3.54–3.66 (m, 2H, CH2
piperazine); 3.86
(s, 2H, CH2CO); 5.61 (bs, 1H,
NH); 7.21–7.27 (m, 5H, aromatics) ppm.
45
99
—
1.39 (s, 9H, (CH3)3C); 2.37–2.42
(m, 4H, CH2 piperazine);
2.57 (s, 2H, CH2CO); 2.79
—
(s, 3H, CH3N); 3.28–3.41
(m, 2H, CH2 piperazine); 3.43 (s, 2H, CH2Ph); 3.52–3.62
(m, 2H, CH2 piperazine); 7.17–7.31 (m, 5H,
aromatics) ppm.
46
97
—
1.22 (d, J=6.4 Hz, 3H, CH3CH);
1.37 (s, 9H, (CH3)3C);
—
2.41 (bs, 1H, NH); 2.20–2.58
(m, 4H, CH2 piperazine);
3.25–3.66 (m, 4H, CH2 piperazine);
3.5 (s, 2H, CH2Ph); 4.51–4.60
(m, 1H, CHCH3); 7.20–7.27
(m, 5H, aromatics) ppm.
47
48
49
50
75
63
46
99
—
—
—
—
1.38 (s, 9H, (CH3)3C); 2.75–2.81 (m, 5H, CH2+NH
piperazine); 3.27–3.32 (m, 2H, CH2 piperazine);
3.50–3.55 (m, 2H, CH2 piperazine); 3.88 (d, J=4.4. Hz,
2H, CH2NH); 5.56 (bs, 1H, NHCO) ppm.
—
—
—
—
1.32 (s, 9H, (CH3)3C); 2.31 (bs, 1H, NH); 2.63–2.78
(m, 4H, CH2 piperazine); 2.74 (s, 3H, CH3N);
3.12–3.23 (m, 2H, CH2 piperazine); 3.38–3.43
(m, 2H, CH2 piperazine); 3.92 (s, 2H, CH2N) ppm.
1.20 (d, J=6.6 Hz, 3H, CH3CH); 1.34 (s, 9H, (CH3)3C);
2.20 (bs, 1H, NH); 2.73–2.82 (m, 4H, CH2 piperazine);
3.38–3.61 (m, 4H, CH2 piperazine); 4.45–4.58 (m, 1H,
CHCH3); 5.65 (bs, 1H, NHCO) ppm.
1.38 (s, 9H, (CH3)3C); 3.21–3.41
(m, 4H, CH2 piperazine);
3.42–3.63 (m, 4H, CH2 piperazine);
3.85 (s, 2H, CH2CO); 5.51 (bs, 1H, NH);
7.31–7.40 (m, 5H, aromatics)ppm.
51
99
—
1.42 (s, 9H, (CH3)3C); 2.88 (s, 3H, CH3N);
3.28–3.71 (m, 8H, CH2 piperazine);
4.04 (s, 2H, CH2CO); 7.27–7.43 (m, 5H, aromatics) ppm.
—
(Continued on next page.)

Table 7 (continued)
Sigla
N
Yield (%)
64
IR n (cmꢀ1
)
1H NMR (CDCl3) d
13C NMR (CDCl3) d
52
—
1.22 (d, J=7.3 Hz, 3H, CH3CH); 1.35 (s, 9H,
(CH3)3C); 3.28–3.73 (m, 8H, CH2 piperazine);
4.45–4.61 (m, 1H, CHCH3); 5.58 (d, J=9.2 Hz,
1H, NH); 7.33–7.35 (m,
—
5H, aromatics) ppm.
53
99
—
1.38 (s, 9H, (CH3)3C); 2.90–2.98
(m, 4H, CH2 piperazine);
3.45–3.47 (m, 2H, CH2
—
piperazine); 3.66–3.70
(m, 2H, CH2 piperazine); 3.86
(d, J=4.4 Hz, 2H, CH2NH); 5.39
(bs, 1H, NH); 7.16–7.27 (m, 2H,
aromatics); 7.68–7.76
(m, 2H, aromatics) ppm.
54
99
—
1.37 (s, 9H, (CH3)3C); 2.81 (s, 3H, CH3N); 2.94–2.97
(m, 4H, CH2 piperazine); 3.41–3.52
(m, 2H, CH2 piperazine);
—
3.59–3.68 (m, 2H, CH2 piperazine); 3.92
(s, 2H, CH2CO); 7.15–7.25
(m,2H, aromatics); 7.67–7.72 (m,
2H, aromatics) ppm.
55
99
—
1.19 (d, J=6.6 Hz, 3H, CH3CH);
1.34 (s, 9H, (CH3)3C);
—
2.79–2.95 (m, 2H, CH2 piperazine); 3.11–3.21
(m, 2H, CH2 piperazine); 3.35–3.58 (m, 2H, CH2
piperazine); 3.61–3.76 (m, 1H,
CH2 piperazine); 3.86.3.97
(m, 1H, CH2 piperazine); 4.42–4.56 (m, 1H,
CH–CH3); 5.36 (d, J=8.1 Hz,
1H, NH); 7.09–7.32 (m, 2H, aromatics);
7.68–7.82 (m, 2H, aromatics) ppm.

84
S. Scapecchi et al. / Bioorg. Med. Chem. 12 (2004) 71–85
ory disruption, mice were ip injected with the amnesic
drugs (scopolamine, 13 or 14). All investigated drugs
were given, ip, 20 min before the training session, while
amnesic drugs were injected immediately after termina-
tion of the trainingsession. The maximum entry latency
allowed in the retention session was 120 s. The degree of
received punishment memory (fall into cold water) was
expressed as the increase in seconds between training
and retention latencies. Piracetam, DM232 (1), and
DM235 (2) were used as the reference drugs.
taken to tie the ligatures such that the diameter of the
nerve was seen to be just barely constricted when viewed
with 40ꢃ magnification. In every animal, an identical
dissection was performed on the opposite side except
that the sciatic nerve was not ligated. The left paw was
untouched.
6.4. Paw pressure test
The nociceptive threshold in the rat was determined
with an analgesimeter (Ugo Basile, Varese, Italy),
33
All compounds elicited their antiamnesic effect without
changing either gross behaviour or motor coordination,
as revealed by the rota-rod test (data not shown). None
of the drugs, at the active doses, increased the number
of falls from the rotatingrod in comparison with saline-
treated mice. The number of falls in the rota-rod test
progressively decreased since mice learned how to bal-
ance on the rotatingrod. The spontaneous motility and
inspection activity of mice was unmodified by the
administration of the studied compounds as revealed by
the hole-board test in comparison with saline-treated
mice (data not shown).
accordingto the method described by Leighton et al.
Rats scoringbelow 40 gor over 75 gduringthe test
before drugadministration (25%) were rejected. An
arbitrary cut-off value of 250 gwas adopted.
Acknowledgements
This research was financed with funds from Italian
Ministry of University and Research (MIUR).
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