Design, synthesis and preliminary pharmacological evaluation of new analogues of DM232 (unifiram) and DM235 (sunifiram) as cognition modulators

Article abstract of DOI:10.1016/j.bmc.2008.10.017

A series of amides, structurally related to DM232 (unifiram) and DM235 (sunifiram), characterized by a 1,2,3,4-tetrahydropyrazino[2,1-a]isoindol-6(2H)-one, 1,4-diamino-cyclohexane or 1,4-diaminobenzene ring, have been synthesized and tested for cognition-enhancing activity in the mouse passive-avoidance test. Some of the compounds display good antiamnesic and procognitive activity, with higher potency than piracetam, while some cyclohexane derivatives are endowed with amnesia inducing properties.

Full text of DOI:10.1016/j.bmc.2008.10.017

Bioorganic & Medicinal Chemistry 16 (2008) 10034–10042
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and preliminary pharmacological evaluation of new
analogues of DM232 (unifiram) and DM235 (sunifiram) as cognition modulators
Elisabetta Martini a, Monica Norcini b, Carla Ghelardini b, Dina Manetti a, Silvia Dei a, Luca Guandalini a,
Michele Melchiorre a, Simona Pagella a, Serena Scapecchi a, Elisabetta Teodori a, Maria Novella Romanelli a,
*
a Laboratory of Design, Synthesis and Study of Biologically Active Heterocycles (HeteroBioLab), Department of Pharmaceutical Sciences, University of Florence, Via Ugo Schiff 6, 50019
Sesto Fiorentino, Italy
b Department of Preclinical and Clinical Pharmacology, University of Florence Viale Pieraccini 6, 50139 Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of amides, structurally related to DM232 (unifiram) and DM235 (sunifiram), characterized by a
1,2,3,4-tetrahydropyrazino[2,1-a]isoindol-6(2H)-one, 1,4-diamino-cyclohexane or 1,4-diaminobenzene
ring, have been synthesized and tested for cognition-enhancing activity in the mouse passive-avoidance
test. Some of the compounds display good antiamnesic and procognitive activity, with higher potency
than piracetam, while some cyclohexane derivatives are endowed with amnesia inducing properties.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 11 August 2008
Revised 26 September 2008
Accepted 7 October 2008
Available online 11 October 2008
Keywords:
Sunifiram
Cognition-modulators
Cognition-enhancers
Mouse passive-avoidance test
O
1. Introduction
R
N
O
O
HN
CH3
N
Cognition is a complex physiological process involving several
areas of the central nervous system. It has been shown that various
neurotransmitters can modulate, positively or negatively, learning
and memory; thus, their receptors may represent suitable targets
for developing cognition-enhancing drugs.1–5 Substances able to
increase learning and memory may be useful in several kinds of
cognitive dysfunctions, such as age-related memory deficits, neu-
rodegenerative disorders such as Alzheimer’s or Parkinson’s dis-
eases or multiple sclerosis, other neuropsychiatric conditions
such as schizophrenia and attention-deficit hyperactivity disor-
ders.6–10 Despite the efforts, at present the only drugs that have
been approved to treat cognition deficits in Alzheimer’s disease
and other forms of dementia are modulators of the cholinergic
and glutamatergic transmission; other substances targeting these
systems (i.e., other cholinesterase inhibitors, nicotinic agonists, or
AMPA receptor positive allosteric modulators) are now in clinical
trials.
N
N
NH2
N
N
O
O
O2S
O2S
F
2a R = COEt
F
(DM235, Sunifiram)
2b R = COMe
2c R = SO2-iPr
3 MN19
(Sapunifiram)
1 DM232
(Unifiram)
piracetam
2d R = SO2C6H4-p-F
O
N
R1
R1
N
R1
R
N
N
HN
N
N
O
O2S
CH3
CH3
4 (MC68)
R2
HN
R2
R3
19-32
9-18
5-8
R1 = MeCO, EtCO,
R1, R2 = MeCO, EtCO, PhCO,
PrSO2, -F-C6H4SO2
R, R3 = H, CH3
The pyrrolidin-2-one family of cognition enhancers, exempli-
fied by piracetam (Chart 1), has been the subject of studies for al-
most four decades and a few members of the family are in use in
several countries as drugs to control cognition impairment, to af-
ford neuroprotection after stroke and to treat epilepsy.11 The use
i
PrSO2,
p
F-C6H4SO2
i
p
Chart 1.
of this class of substances is controversial, due to the lack of a com-
mon mechanism of action at the molecular level, although some
members of this series (for instance, aniracetam and nefiracetam)
* Corresponding author. Tel.: +39 055 4573691; fax +39 055 4573780.
E-mail address: novella.romanelli@unifi.it (M.N. Romanelli).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.017

E. Martini et al. / Bioorg. Med. Chem. 16 (2008) 10034–10042
10035
R1
R1
have been shown to modulate receptor systems such as the cholin-
N
R1
PhCO
R2
NH2
NH2
HN
ergic and/or glutamatergic ones.12,13
HN
HN
9
EtCO
PhCO
PhCO
b
a
It has previously been reported that DM232 (unifiram, 1) and
DM235 (sunifiram, 2a) show cognition-enhancing properties with
a potency four orders of magnitude greater than piracetam.14,15
These compounds are well tolerated in rodents, but their develop-
ment has been impaired because their mechanism of action has
not been clarified.16 In fact, unifiram and sunifiram did not show
any affinity towards the most important central receptors or trans-
porters.15 These compounds are able to increase acetylcholine re-
lease from rat brain,15 and nitric oxide production in rat
adipocytes, the latter effect being antagonized by nicotinic antago-
nists such as mecamylamine and methyllycaconitine17; there is evi-
dence that AMPA receptors are involved in the cognition-enhancing
effect of these compounds.18 However, a direct interaction of unifi-
ram and sunifiram with nicotinic or AMPA receptors in vitro has
not been proven yet.
10 MeCO
iPrSO2
11
12
13
14
15
PhCO p-FC6H4SO2
MeCO p-FC6H4SO2
EtCO p-FC6H4SO2
NH2
R2
9-15
EtCO
EtCO
R1
R1
NH2
NH2
HN
HN
b
a
N
R1
EtCO
EtCO p-FC6H4SO2
MeCO p-FC6H4SO2
R2
16
17
18
EtCO
NH2
R2
HN
16-18
Scheme 2. Reagents: (a) (R1)2O or R1Cl, AcONa, H2O, dioxane; (b) R2Cl, Na2CO3,
H2O, dioxane.
In order to improve the potency of our compounds, and possibly
to elucidate the mechanism of action, several structural modifica-
tions were performed on the lead compounds 1 and 2a, giving
interesting results. For instance, the extrusion of one of the nitro-
gen atoms of the piperazine ring to give 4-aminopiperidine deriv-
atives, exemplified by MN19 (3, sapunifiram), afforded compounds
with cognition-enhancing properties similar to those of the parent
compound sunifiram.19 Moreover, the replacement of the 4-fluoro-
phenyl moiety of 1 with an isopropyl group gave compound 4
(MC68), endowed with amnesia inducing properties.20
CH3
O2S
BOC
CH3
H3C
N
F
H3C
N
F
h, i
g, c, b
HN SO2
HN SO2
32
38
R
R
R
R
NR1
NR1
NR1
NH
a, c
or
f
e
Asa continuationof thisresearch, wedecidedtomodifythestruc-
ture of our lead compounds following two different approaches: (i)
the flexibility of the benzoylpiperazine moiety of 2 has been reduced
by incorporatingit intoa 1,2,3,4-tetrahydropyrazino[2,1-a]isoindol-
6(2H)-one ring, obtaining compounds 5–8 and (ii) both amide func-
tionshave beenextruded from thesix-memberedring, providingthe
cyclohexane derivatives 9–18 and their aromatic analogues, the 1-4-
diamidobenzenes 19–32. The acyl and sulfonyl groups which deco-
rate the structures were chosen among those giving, in the previous
series, the most interesting compounds.14,15,19 All the designed com-
pounds maintain a diamidic structure, a feature which seems impor-
tant for high nootropic activity.16
b, d
NHR2
NR2
H3C
NO2
NH2
R = H: 19-26
R = CH3: 27, 28
R = H: 33-35
R = CH3: 36, 37
R = H: 29
R = CH3: 30, 31
R1
R2
N
R
R1
N
R
H
H
H
p-FC6H4SO2
PhCO
EtCO
33
34
35
36
37
p-FC6H4SO2 iPrSO2
19
20
21
22
23
24
25
26
27
28
29
30
31
H
H
H
H
H
H
H
H
CH3
CH3
H
CH3
CH3
PhCO
PhCO
EtCO
iPrSO2
p-FC6H4SO2
p-FC6H4SO2
CH3 p-FC6H4SO2
CH3 PhCO
EtCO
iPrSO2
MeCO
MeCO
MeCO
PhCO
iPrSO2
p-FC6H4SO2
2. Chemistry
p-FC6H4SO2 iPrSO2
Compounds 5–8 were obtained by reaction of 1,2,3,4-tetrahyd-
ropyrazino[2,1-a]isoindol-6(2H)-one, prepared according to
Welch,21 with the suitable acyl or sulfonyl chloride (Scheme 1).
Derivatives 9–15 were synthesized according to the method of
Sueess (Scheme 2).22 The commercially-available trans-1,4-cyclo-
hexanediamine was sequentially treated with the suitable anhy-
drides or acyl or sulfonyl chlorides without isolation of the
intermediate monoamide. The same pathway was applied to cis-
1,4-cyclohexanediamine, prepared according to Johnstone,23 pro-
viding compounds 16–18. Compounds 15 and 16 derive from the
double attack of propionic anhydride on both amine groups of,
respectively, trans- and cis-1,4-cyclohexanediamine.
PhCO
PhCO
PhCO
iPrSO2
iPrSO2
iPrSO2
p-FC6H4SO2 iPrSO2
Scheme 3. Reagents: (a) R1Cl, K2CO3, THF; (b) p-F-C6H4SO2Cl, DMAP, pyridine; (c)
H2/Pd/C, EtOAc; (d) Fe, AcOH; (e) R2Cl, pyridine, THF; (f) MeI, tBuOK, THF; (g)
(BOC)2O; (h) HCl, EtOAc; (i) iPrSO2Cl, pyridine, THF.
The synthesis of the 1,4-diaminobenzene analogues 19–32 is
shown in Scheme 3. Commercially-available 4-nitroaniline was
treated with the suitable acyl or sulfonyl chloride and reduced to
the monoamides 33–35, which were then reacted with the desired
acyl or sulfonyl chlorides, to provide compounds 19–26. The same
pathway was applied to the synthesis of compounds 27 and 28,
starting from the commercially-available N-methyl-4-nitro-ani-
line, through the monoamides 36 and 37. Treatment of compounds
27 and 28 with CH3I under basic conditions gave the dimethyl
derivatives 30 and 31. When this reaction was performed on com-
pound 20 only the more acid amide function was methylated,
obtaining compound 29 as the only product. Catalytic hydrogena-
tion of N-BOC-N-methyl-4-nitroaniline,24 obtained under standard
conditions, and subsequent treatment with p-fluorophenylsulfonyl
chloride gave 38, which was deprotected and treated with i-pro-
pylsulfonyl chloride to give compound 32 (Scheme 3).
R1
H
N
N
1
R1-Cl
10b
10a
5 N
O
N
6
CH3CN, NEt3
O
7
5: R1 = CH3CO
6: R1 = C2H5CO
7: R1
=
p
-F-C6H4-SO2
8: R1 = (CH3)2CHSO2
Scheme 1.

10036
E. Martini et al. / Bioorg. Med. Chem. 16 (2008) 10034–10042
As far as the trans-1,4-diaminocyclohexane derivatives are con-
3. Pharmacology
cerned (compounds 9–15, see structures in Scheme 2), none of
them was able to revert scopolamine-induced amnesia. This lack
of activity can be due to the presence of the cyclohexane spacer
The compounds were tested for their ability to revert scopol-
amine-induced amnesia in the mouse passive-avoidance test of Jar-
vik and Kopp,25 slightly modified by us (see Section 5). The
compounds were tested in a 1:10 dilution sequence, up to the dose
of 10 mg/kg; the results are expressed as minimal effective dose
(MED, mg/kg) and are reported in Table 1. Compounds were consid-
ered inactive if they did not show activity up to the dose of 10 mg/kg,
which is four orders of magnitude higher than the MED of the lead
compounds 1 and 2a. The passive-avoidance test was used to evalu-
ate the amnesia inducing or procognitive properties of the com-
pounds; for this reason the compounds were tested without
addition of scopolamine. The results are reported in Table 2. All com-
pounds were dissolved in saline, except compounds 27–31, which
were dissolved in a vehicle consisting of water/dimethylsulfoxide
4:1. Compound 32 proved to be unstable, and it was not tested.
Table 1
Minimal effective dose (MED) of the compounds against scopolamine-induced
amnesia in the mouse passive-avoidance test, in comparison with reference
compounds 1, 2a and 3a
Treatment
Minimal effective
dose (mg/kg)
n
Training
session (s)
Retention
session (s)
D
Saline
Saline/DMSO
4:1
—
—
30 17.2 2.3
19 21.3 2.3
99.5 7.1
96.8 6.1
82.3
75.5
Scopolamine
(S)
1.5
31 16.5 3.3
42.8 8.3
26.3
S + piracetam 30
32 17.6 3.6
37 19.7 5.8
29 20.5 3.4
12 11.3 5.3
19 14.5 3.8
13 15.6 3.2
16 16.3 4.1
18 17.0 3.2
16 17.5 3.1
23 17.8 3.7
12 16.8 3.1
15 19.1 3.5
14 19.1 3.5
21 16.8 3.8
13 20.2 3.5
12 21.6 3.7
14 20.6 3.8
14 20.7 4.3
18 18.8 2.6
108.8 10.4*
104.4 10.6*
91.5 8.0*
119.0 11.2*
90.6 12.5*
74.5 10.3*
85.5 9.4*
26.3 8.9*
16.4 6.3*
106.2 9.5*
89.1 7.9*
104.5 8.6*
76.3 7.9*
88.1 9.7*
103.6 8.5*
75.9 8.9*
66.7 7.1*
91.8 9.9*
70.5 8.3^
91.2
93.8
71.0
107.7
76.1
58.9
69.2
9.3
ꢀ1.1
88.4
72.3
85.4
57.2
61.3
83.4
54.3
46.1
70.6
73.6
S + 1b
S + 2a
S + 2b
0.001
0.001
10
c
c
4. Results and discussion
d
S + 3
0.01
0.1
1.0
10
1.0
1.0
0.1
1.0
1
S + 7
S + 8
The ability of the compounds to revert scopolamine-induced
amnesia is reported in Table 1, expressed as minimal effective dose
(MED). Compounds 7, 8, 16–20, 27–31 show some activity in this
test; the other compounds (5, 6, 9–12, 15, 21–26) did not revert sco-
polamine-induced amnesiaat doses up to 10 mg/kg ip, and therefore
are not shown. On the contrary, compounds 13, 14 were able to
potentiate the effect of scopolamine. Compounds 13, 14, 16–20
and 27–31 were further tested in animals which had not been previ-
ously pre-treated with amnesia inducing drugs (Table 2): this test
confirmed the amnesia inducing properties of compounds 13 and
14, while compounds 16–20 and 27, 28, 30 and 31 show interesting
procognitive activity. Under these conditions, where memory has
not been pharmacologically impaired, some of the compounds show
minimal effective doses one or two orders of magnitude greater than
thoserecorded in scopolamine-treated mice. The procognitive activ-
ity of compounds 1 and 2a was previously revealed by using the so-
cial learning test, performed according to Mondadori.26 These two
compounds exerted beneficial effects on cognitive performance in
the social learning test by prolonging the time normally required
by rats to delete mnemonic information.15,27
S + 13
S + 14
S + 16
S + 17
S + 18
S + 19
S + 20
S + 27
S + 28
S + 29
S + 30
S + 31
10
0.1
0.1
0.01
0.01
0.01
a
All compounds were dissolved in saline, except 27–31, which were dissolved in
saline/dimethylsulfoxide 4:1, and injected ip 20 min before training session. Sco-
polamine (S) was injected immediately after punishment.
b
From Ref. 14.
From Ref. 15.
From Ref. 19.
c
d
^
P < 0.05 in comparison with scopolamine-treated mice.
P < 0.01 in comparison with scopolamine-treated mice.
*
Reduction of the conformational flexibility of the benzoyl group
of DM235 (sunifiram) by freezing it into a tricyclic moiety (see the
structures reported in Scheme 1) gave compound 6, and its lower
homolog 5, which are devoid of activity in the passive-avoidance
test. On the contrary, the replacement of the acetyl or propionyl
group with a sulfonyl moiety gave compounds (7 and 8) endowed
with good nootropic activity. As a matter of fact, they are able to re-
vert amnesia induced by scopolamine with MEDs of 0.1 and 1.0 mg/
kg ip, respectively, both being much more active than piracetam,
although their potency is not as high as that of the reference com-
pounds 1, 2a and 3. The rank order of potency in the tricyclic series
does not correlate with that found in the piperazine series. In fact,
both the N-acetyl and N-propionyl-benzoylpiperazine (2b and 2a,
respectively, see Chart 1) showed cognition-enhancing properties,
although with quite different potency,15 and both the sulfonyl ana-
logues 2c and 2d were devoid of activity.20 The difference between
these two series can be explained by the fact that in in vivo studies
the biological activity is the consequence of both the pharmacoki-
netic and pharmacodynamic properties, which may be differently
affected by structural modifications. Unfortunately, the lack of
knowledge about the mechanism of action makes it impossible to
perform in vitro studies, where pharmacokinetic factors are largely
reduced. Obviously other explanations cannot be ruled out, such as a
different binding mode for the two series, or the interaction with a
different biological target.
Table 2
Effect of the compounds in the mouse passive-avoidance test, in comparison with
reference compounds 1, 2a–4a
Treatment
Minimal effective
dose (mg/kg)
n
Training
session (s)
Retention
session (s)
D
Saline
Saline/DMSO 4:1
1
2a
3
—
—
27 19.3 4.1
19 21.3 2.3
18 15.4 3.6
16 18.1 2.7
13 19.1 2.9
18 13.6 3.9
14 19.2 2.5
13 16.9 2.2
13 34.8 4.3
13 25.3 4.5
10 32.0 3.2
10 21.1 3.8
10 22.1 3.3
16 26.2 2.8
20 17.3 4.5
12 19.6 5.7
14 30.0 4.1
97.2 7.5
96.8 6.1
77.9
75.5
116.2
108.6
107.7
47.9
33.6
39.4
130.4
137.6
0.01
0.01
0.1
10
0.01
0.1
10
10
10
10
1.0
1.0
0.1
1.0
1.0
131.6 9.2*
126.7 7.7^
126.8 6.4*
61.5 10.4*
52.8 6.3*
56.3 7.1*
165.2 6.1*
162.9 7.3*
4b
13
14
16
17
18
19
20
27
28
30
31
156.6 14.5* 121.6
125.2 6.7*
116.7 8.3^
104.1
94.6
149.8 12.1* 123.6
121.0 9.0*
113.7
123.6 18.9* 107.0
158.6 10.3* 128.6
a
All compounds were dissolved in saline, except compounds 27, 28, 30 and 31,
which were dissolved in saline/dimethylsulfoxide 4:1, and injected ip 20 min before
training session.
b
From Ref. 20.
^
P < 0.05 in comparison with mice treated with saline or saline/DMSO.
P < 0.01 in comparison with mice treated with saline or saline/DMSO.
*

E. Martini et al. / Bioorg. Med. Chem. 16 (2008) 10034–10042
10037
which increases the distance between the amide functions with re-
spect to piperazine or 4-aminopiperidine derivatives, or to the
introduction of more hydrophilic characteristics. In fact, all com-
pounds have two secondary amide or sulfonamide functions,
which can inhibit the crossing of the blood–brain barrier. However,
the latter hypothesis can be ruled out, since compounds 13 and 14,
carrying also two secondary amide or sulfonamide groups, at doses
of 10 and 1 mg/kg, respectively, were able to increase the amnesic
effect of scopolamine (Table 1). When tested alone (Table 2), they
showed amnesic properties at doses of 0.01 and 0.1 mg/kg, respec-
tively; they were able to reduce the entry latency in the retention
session, at doses one or two orders of magnitude lower than sco-
polamine. This effect, which indicates that the compounds can en-
ter the CNS, was unexpected. However, as stated in the
introduction, we had already experienced a similar behavior in
some of our molecules, such as the 1,4-diazabicyclo[4.3.0]nonan-
9-one derivatives, where the replacement of the p-fluorophenyl
moiety with an i-propyl group switched the activity of the mole-
cules from cognition-enhancing (1) to amnesic (4).20 Here we
found a similar result: the extrusion of the amide function of com-
pound 3 from the 6-membered ring gave compound 13 endowed
with amnesic properties. These findings prompted us to synthesize
the cis analogues of compounds 13 and 14. Compounds 17 and 18,
as well as 16, were initially tested without addition of scopol-
amine, in order to unveil a possible amnesic activity, but on the
contrary, this test highlighted their procognitive effect at the dose
of 10 mg/kg (Table 2). Moreover, at lower doses, compounds 16–18
were able to revert scopolamine-induced amnesia (Table 1), while
compound 15 (the trans isomer of 16) was completely devoid of
activity. The marked influence on activity of structural modifica-
tions such as isomerization suggests that we are modulating a spe-
cific, but still unknown, biological target.
pounds. To test this hypothesis, the mono and dimethyl
analogues of compounds 19 (compounds 27, 31 and 32) and 20
(compounds 28–30) were prepared. It was found that the removal
of the hydrophilic function(s) increases the potency of com-
pounds: indeed, the addition of one or two methyl groups on
the amide moieties of 19 decreases the minimal effective dose
(1.0 mg/kg) by one or two orders of magnitude (compounds 27
and 31, respectively). A similar, even more evident shift can be
seen by comparing the MED of compound 20 (10 mg/kg) with
the N-methyl-derivatives (compounds 28 and 29) or the N,N0-di-
methyl analogue 30. The increase of ClogP values in the series
of compounds 19, 27, 31 and 20, 28–30 (see Tables 3 and 5) is,
as expected, associated to an increase of nootropic potency (Table
1); the absence of a more general correlation between the cogni-
tion-enhancing activity and the calculated lipophilicity values can
be due, as previously suggested, to the different contribution of
pharmacokinetic and pharmacodinamic factors in determining
the potency of the compounds. These results seem to indicate
that the benzene ring is a suitable spacer to maintain potent noo-
tropic activity in this class of substances, since some the com-
pounds (29–31) have MED values in the same range as that of
3 and only one order of magnitude greater than the reference
compounds 1 and 2a.
In conclusion, we have synthesized a series of diastereomeric
1,4-diamidocyclohexanes which show opposing activities in the
mouse passive-avoidance test, suggesting that they are able to
modulate in an opposite way the function of a specific biological
target. In addition, a new class of compounds (1,4-diamidobenz-
enes) has been discovered, which maintain high nootropic activity.
These compounds may give useful information for structure-activ-
ity relationships in the class of nootropic drugs, and possibly to
elucidate their mechanism of action at the molecular level.
In the 1,4-diaminobenzene series, among the amide-sulfona-
mides 19–26, only compounds 19 and 20 were able to revert sco-
polamine-induced amnesia with MED of 1 mg/kg and 10 mg/kg,
respectively (Table 1). Their potency is in the same range as that
of piracetam, but they are three to four orders of magnitude less
potent than the lead compounds 1, 2a and 3. This drop in potency
can be due to the different physico-chemical characteristics of the
compounds, which may affect their ability to cross the blood–
brain barrier. In fact, in 19 and 20 the functional groups are
amides or sulfonamides with H-bond donor properties and a
more hydrophilic character with respect to the endocyclic
amide-sulfonamide functions of compounds 1–3; moreover, com-
pounds 19 and 20 are weak acids while 1–3 are neutral com-
5. Experimental
5.1. Chemistry
All melting points were taken on a Büchi apparatus and are
uncorrected. NMR spectra were recorded on a Brucker Avance
400 spectrometer (400 MHz for 1H NMR, 100 MHz for 13C). Chro-
matographic 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).
When necessary, chromatographic separations were performed
on an Al2O3 column by gravity chromatography (aluminium oxide
Table 3
Experimental details for the synthesis of compounds 5–18 (see general structures in Chart 1) and ClogP values
d
N
R1
R2
Eluenta
Yield (%)
Mp (°C)
ClogP
Anal.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
COCH3
COC2H5
—
—
—
—
EtOAcb
71
38
50
43
22
17
20
6
40
23
15
21
9
130–131
132–134
169–171c
136–138c
286–287
304–305
262–263
224–225
233–234
218–219
219–220
Waxy solid
Waxy solid
Waxy solid
1.48
1.92
2.02
1.20
2.72
2.26
2.34
3.17
1.69
2.16
1.71
1.71
2.16
1.69
C13H14N2O2
C14H16N2O2
C17H15FN2O3S
C14H18N2O3S
C16H22N2O2
C15H20N2O2
C16H24N2O3S
C19H21FN2O3S
C14H19FN2O3S
C15H21FN2O3S
C12H22N2O2
C12H22N2O2
C15H21FN2O3S
C14H19FN2O3S
EtOAcb
SO2C6H4F
SO2CH(CH3)2
COCH2CH3
COCH3
SO2CH(CH3)2
COC6H5
A
B
C
C
C
C
C
C
C
—
C
C
COC6H5
COC6H5
COC6H5
SO2C6H4F
SO2C6H4F
SO2C6H4F
COCH2CH3
COCH2CH3
SO2C6H4F
SO2C6H4F
COCH3
COCH2CH3
COCH2CH3
COCH2CH3
COCH2CH3
COCH3
12
a
A: CH2Cl2/MeOH 98:2; B: CH2Cl2/MeOH/NH4OH 95:5:0.5; C: CH2Cl2/abs.EtOH/NH4OH/petroleum ether 340:65:8:60.
Al2O3 was used as stationary phase.
The compound melts with decomposition.
b
c
d
The ClogP value of the tested compounds has been calculated using the program ‘OSIRIS Property Explorer’ (http://www.organic-chemistry.org/prog/peo/).

10038
E. Martini et al. / Bioorg. Med. Chem. 16 (2008) 10034–10042
Table of elemental analysis of the new compounds
Calculated %
H
Found %
N
formula
C
N
C
H
N
C13H14N2O2
C14H16N2O2
67.81
68.83
58.95
57.12
70.04
69.20
59.23
60.62
53.49
54.86
63.68
63.68
54.86
53.49
48.37
60.36
61.61
55.89
53.31
70.85
51.54
54.54
49.73
61.42
61.42
62.40
50.98
49.73
54.12
73.56
65.83
55.70
74.31
56.83
6.13
6.60
4.36
6.16
8.08
7.74
7.46
5.62
6.09
6.45
9.80
9.80
6.45
6.09
4.60
5.70
4.08
4.69
6.71
5.55
6.29
4.25
4.96
6.06
6.06
6.40
5.29
4.96
4.16
5.70
7.37
4.67
6.24
5.56
12.17
11.47
8.09
68.11
68.91
59.16
57.31
70.30
69.41
59.42
60.45
53.21
54.99
63.57
63.42
54.98
53.25
48.52
60.15
61.42
55.58
53.24
70.96
51.69
54.63
49.55
61.26
61.65
62.56
50.68
49.36
54.36
73.72
65.99
55.42
74.62
57.06
6.34
6.76
4.50
6.28
8.40
7.58
7.32
5.44
6.34
6.22
9.58
9.96
6.58
6.25
4.35
5.48
4.36
4.42
6.87
5.62
6.56
4.46
5.04
6.42
6.36
6.21
5.63
4.59
4.25
5.85
7.54
4.85
6.42
5.71
12.33
11.65
8.32
5
6
7
8
C17H15FN2O3S
C14H18N2O3S
C16H22N2O2
9.52
9.73
10.21
10.76
8.63
10.07
10.62
8.51
9
C15H20N2O2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
C16H24N2O3S
C19H21FN2O3S
C14H19FN2O3S
C15H21FN2O3S
C12H22N2O2
7.44
8.91
8.53
12.38
12.38
8.53
8.91
7.52
7.19
8.74
8.24
12.15
12.53
8.69
8.75
7.31
C12H22N2O2
C15H21FN2O3S
C14H19FN2O3S
C15H17FN2O4S2
C16H18N2O3S
C19H15FN2O3S
C15H15FN2O3S
C12H18N2O3S
C15H14N2O2
C11H16N2O3S
C14H13FN2O3S
C16H19FN2O4S2
C17H20N2O3S
C17H20N2O3S
C18H22N2O3S
C17H21FN2O4S2
C16H19FN2O4S2
C12H11FN2O2S
C13H12N2O
8.80
7.56
9.07
7.41
8.69
8.65
10.36
11.02
10.93
9.09
10.19
11.35
10.69
9.35
7.25
7.54
8.43
8.24
8.43
8.32
8.09
8.41
6.99
7.32
7.25
6.98
10.52
13.20
17.06
9.99
10.24
13.32
17.21
10.08
12.55
7.52
C9H12N2O
C13H13FN2O2S
C14H14N2O
12.38
7.36
C18H21FN2O4S
90 standardized, Merck). Yields are given after purification, unless
stated otherwise. Where analyses are indicated by symbols, the
analytical results are within 0.4% of the theoretical values. When
reactions were performed under anhydrous conditions, the mix-
tures were maintained under nitrogen.
appearance of the intermediate monoamide (t.l.c.). Solid Na2CO3 (4
eq) and benzoyl chloride or p-fluorobenzenesulfonyl chloride (1.1
eq) were then added, and the mixture heated at 70 °C for 0.5–
3 h. After cooling, the mixture was acidified (HCl 1 N) and
extracted with ethyl acetate; the organic phase was washed with
saturated Na2CO3 and dehydrated (Na2SO4), the solvent was
removed under low pressure to yield a residue which was purified
by flash chromatography. The synthesis of the cis derivatives
16–18 was carried out with the same procedure, except that the
first step was run at RT. Compounds 15 and 16 derive from the
double attack of propionic anhydride on both amine groups. Other
experimental details are reported in Table 3; 1H and 13C NMR spec-
tra are reported in Table 4.
5.1.1. General procedure for the synthesis of compounds 5–8
To
a solution of 1,2,3,4-tetrahydropyrazino[2,1-a]isoindol-
6(2H)-one21 (1.5 mmol) and anhydrous Et3N (2 eq) in CH3CN
(10 mL), cooled at 0 °C, the suitable acyl or sulfonyl chloride (1
eq) was added. After 1 h stirring at room T, the mixture was treated
with saturated NaHCO3 and extracted with CHCl3. Dehydration
(Na2SO4) and removal of the solvent gave a residue which was
purified by column chromatography. Other experimental details
are reported in Table 3; 1H and 13C NMR spectra are reported in Ta-
ble 4. Compounds 5 and 6 are mixtures of A + B rotamers.
5.1.3. N-(4-aminophenyl)-4-fluorobenzenesulfonamide (33)
A mixture of 4-nitroaniline (1 g, 7.24 mmol, 1 eq), dimethylami-
nopyridine (DMAP, 0.1 eq) and p-F-benzenesulfonyl chloride (1 eq)
in anhydrous pyridine (70 mL) was heated at 80 °C for 5 h. After
cooling, the mixture was treated with ether, washed twice with a
saturated solution of CuSO4 and then with brine; dehydration
(Na2SO4) and removal of the solvent gave a yellow solid (mp
142 °C, 90% yield). 1H NMR (DMSO d6, d): 7.32 (d, 2H, J = 9.2 Hz);
7.41–7.47 (m, 2H); 7.91–7.96 (m, 2H); 8.14 (d, 2H, J = 9.2 Hz)
5.1.2. General procedure for the synthesis of cyclohexane
derivatives 9–1822
To a solution of trans-1,4-cyclohexanediamine (2.5 mmol) and
sodium acetate (2.19 eq) in water (15 mL), the suitable anhydride
(acetic or propionic), benzoyl or sulfonyl chloride (1.2 eq) in diox-
ane (10 mL) was added, and the mixture was heated at 70 °C until

E. Martini et al. / Bioorg. Med. Chem. 16 (2008) 10034–10042
10039
Table 4
NMR spectra of compounds 5–18
N
1H NMR (d)
13C NMRa (d)
5b,c
2.19–2.30 (m, 4H, CH3 + 1H, H-1axB); 2.55–2.62 (m, 1H, H-3axA); 2.76–2.82 (m,
1H, H-1axA); 3.09–3.25 (m, 3H, H-3axB + H-4axA + H-4axB); 3.88–3.95 (m, 1H,
H-3eqB); 4.33–4.36 (m, 1H, H-1eqA); 4.43–4.51 (m, 4H, H-10bA,B + H-4eqA,B,);
4.79–4.83 (m, 1H, H-3eqA); 5.29 (dd, 1H, J = 12.8 Hz, 2.9 Hz, H-1eqB); 7.50–7.57
(m, 3H) and 7.89–7.91 (m, 1H) (A+B aromatic protons) ppm
1.03–1.18 (m, 4H, CH3 + 1H, H-1axB); 2.38–2.50 (m, 3H, CH2CH3 + H-3axA); 2.64
(t, 1H, J = 11.2 Hz, H-1axA); 2.94–3.13 (m, 3H, H-3axB + H-4axA,B); 3.90 (d, 1H,
J = 12.8 Hz, H-3eqB); 4.32–4.38 (m, 5H, H-1eqA + H-10bA,B + H-4eqA,B); 4.70 (d,
1H, J = 12.2 Hz, H-3eqA); 5.18 (d, 1H, J = 12.5 Hz, H-1eqB); 7.41–7.43 (m, 2H),
7.48–7.50 (m, 1H) and 7.77 (s, 1H) (aromatic protons) ppm
1.89–1.94 (m, 1H, H-1ax); 2.28 (td, 1H, J = 11.8 Hz, 3.7 Hz, H-3ax); 3.37 (td, 1H,
J = 12.7 Hz, 4.0 Hz, H-4ax); 3.89 (td, 1H, J = 11.6 Hz, 3.8 Hz, H-3eq); 4.36–4.40
(m, 1H, H-1eq); 4.46 (dd, 1H, J = 13.4 Hz, 3.3 Hz, H-4eq); 4.64 (dd, 1H,
J = 10.8 Hz, 4.1 Hz, H-10b); 7.19 (t, J = 8.4 Hz, 2H), 7.50 (t, J = 7.2 Hz, 2H),
7.55.7.59 (m, 1H), 7.75–7.78 (m, 2H) and 7.83 (d, J = 7.2 Hz, 1H) (aromatic
protons) ppm
1.37 (d, 6H, J = 6.8 Hz, 2CH3);2.55 (dd, 1H, J = 12.4 Hz, 10.8 Hz, H-1ax); 2.91 (td,
1H, J = 24.8 Hz, 3.5 Hz, H-3ax); 3.21–3.36 (m, 2H, CH + H-4ax); 3.92 (dd, 1H,
J = 12.8 Hz, 3.8 Hz, H-3eq); 4.38–4.47 (m, 2H, H-4eq + H-1eq); 4.59 (dd, 1H,
J = 10.7 Hz, 4.2 Hz, H-10b); 7.47–7.60 (m, 3H) and 7.88 (d, J = 7.4 Hz, 1H)
(aromatic protons) ppm
0.98 (t, 3H, CH3CH2, J = 7.2 Hz); 1.20–1.44 (m, 4H, cyclohexane); 1.80–1.89 (m,
4H, cyclohexane); 2.04 (q, 2H, CH2CH3, J = 7.2 Hz); 3.46–3.52 (m, 1H,
cyclohexane); 3.67- 3.79 (m, 1H, cyclohexane); 7.42–7.50 (m, 3H); 7.65 (d, 1H,
NH, J = 7.2 Hz); 7.81–7.83 (m, 2H); 8.21 (d, 1H, NH, J = 7.2 Hz) ppm
1.22–1.25 (m, 2H, cyclohexane); 1.38–1.44 (m, 2H, cyclohexane); 1.77–1.82 (m,
7H, CH3 + 4H cyclohexane); 3.39–3.53 (m, 1H, cyclohexane); 3.68–3.81 (m, 1H,
cyclohexane); 7.44–7.50 (m, 3H); 7.75–7.83 (m, 3H, 2H aromatics + NH); 8.22
(d, 1H, NH, J = 6.4 Hz) ppm
1.22 (d, 6H, 2CH3, J = 6.8 Hz); 1.38–1.50 (m, 4H, cyclohexane); 1.83–1.92 (m, 4H,
cyclohexane); 3.05–3.10 (m, 1H, cyclohexane); 3.14 (sept, 1H, J = 6.8 Hz); 3.60–
3.70 (m, 1H, cyclohexane); 6.99 (d, 1H, NH, J = 8.0 Hz); 7.42–7.52 (m, 3H); 7.81–
7.85 (m, 2H); 8.20 (d, 1H, NH, J = 8.0 Hz) ppm
1.15–1.32 (m, 4H, cyclohexane); 1.67–1.76 (m, 4H, cyclohexane); 2.89–2.91 (m,
1H, cyclohexane); 3.63–3.65 (m, 1H, cyclohexane); 7.41–7.51 (m, 5H); 7.66–
7.79 (m, 3H, 2H aromatics + NH); 7.83–7.88 (m, 2H); 8.13 (d, 1H, NH, J = 7.8 Hz)
ppm
1.05–1.23 (m, 4H, cyclohexane); 1.49–1.68 (m, 4H, cyclohexane); 1.72 (s, 3H,
CH3); 2.55–2.93 (m, 1H, cyclohexane); 3.27–3.43 (m, 1H, cyclohexane); 7.38–
7.45 (m, 2H); 7.62–7.78 (m, 2H, 1H aromatic + NH); 7.81–7.92 (m, 2H, 1H
aromatic + NH) ppm
21.78 (CH3); 39.17 (C-4A); 39.63 (C-4B); 41.37 (C-3A); 46.09 (C-3B); 46.58 (C-1B);
51.44 (C-1A); 57.13 (C-10bB); 57.57 (C-10bA); 122.03 (CHA); 122.42 (CHB);
124.01 (CHB); 124.25 (CHA); 128.93 (CHB); 129.19 (CHA); 131.78 (CHB); 132.44
(CHA); 132.84 (C-10aB); 134.09 (C-10aA); 141.23 (C-6aA); 141.98 (C-6aB); 166.15
(C-11A); 166.42 (C-11B); 169.03 (C-6A); 169.35 (C-6B) ppm
9.38 (CH3); 26.65 (CH2CH3A); 26.83 (CH2CH3B); 39.19 (C-4A); 39.64 (C-4B); 41.46
(C-3A); 45.08 (C-3B); 46.68 (C-1B); 50.42 (C-1A); 57.19 (C-10bB); 57.60 (C-10bA);
122.38, 123.85, 128.81 and 131.69 (aromatic CH); 132.36 (C-10aB); 132.72 (C-
10aA); 141.34 (C-6aA); 142.02 (C-6aB); 166.13 (C-11A); 166.34 (C-11B); 172.40
(C-6A); 172.72 (C-6B) ppm
6b,c
7b
38.66 (C-4); 45.73 (C-3); 50.80 (C-1); 56.88 (C-10b); 116.69 (CH, JC–F = 22.6 Hz);
122.32 (CH); 124.15 (CH); 129.20 (CH); 130.23 (CH, JC–F = 9.0 Hz); 131.80, 132.47
and 141.37 (quat. C); 165.41 (CF, JC–F = 256.1 Hz); 165.99 (C-6) ppm
8b
16.70 (CH3); 39.96 (C-4); 46.08 (C-3); 51.31 (C-1); 54.08 (CHSO2); 57.81 (C-10b);
122.31, 124.12, 129.11 and 131.74 (aromatic CH); 132.57 (quat. C); 141.43 (quat.
C); 166.24 (C-6) ppm
9d
10.47 (CH3); 29.01 (CH2); 31.48 and 31.80 (cyclohexane CH2); 47.45 and 48.23
(CH); 127.69, 128.60 and 131.44 (aromatic CH); 135.23 (quat. C); 165.95 and
172.45 (CO) ppm
10d
11d
12d
13d
14d
23.22 (CH3); 31.46 and 31.78 (cyclohexane CH2); 47.55 and 48.21 (CH); 127.70,
128.60 and 131.44 (aromatic CH); 135.21 (quat. C); 165.95 and 168.69 (CO) ppm
16.91 (CH3); 31.49 and 33.41 (CH2); 48.01 and 52.51 (aliphatic CH); 127.71, 128.61
and 131.46 (aromatic CH), 135.21 (quat. C), 165.93 (CO) ppm
31.19 and 32.50 (CH2); 47.81 and 52.28 (CH); 116.89 (CH, JC–F = 22 Hz); 127.67,
128.60 and 131.44 (benzoyl CH); 129.68 (CH, JC–F = 10 Hz); 135.18 (quat. C);
155.57 (CF, JC–F = 253 Hz); 165.99 (CO) ppm
23.15 (CH3); 31.27 and 32.32 (cyclohexane CH2); 47.05 and 52.09 (cyclohexane
CH); 116.76 (CH, JC–F = 22 Hz); 129.64 (CH, JC–F = 9 Hz); 139.03 (CSO2); 164.37
(CF, JC–F = 248 Hz); 168.66 (CO) ppm
0.93 (t, 3H, CH3CH2, J = 7.6 Hz); 1.01–1.23 (m, 4H, cyclohexane); 1.58–1.68 (m,
4H, cyclohexane); 1.98 (q, 2H, CH2CH3, J = 7.6 Hz); 2.83–2.95 (m, 1H,
cyclohexane); 3.29–3.41 (m,1H, cyclohexane); 7.43 (t, 2H,, J = 8.8 Hz); 7.55 (d,
1H, NH, J = 4.0 Hz); 7.73(d, 1H, NH, J = 4.0 Hz); 7.85–7.88 (m, 2H) ppm
0.96 (t, 6H, J = 7.6 Hz, 2CH3CH2); 1.15–1.21 (m, 4H, cyclohexane); 1.74–1.76 (m,
4H, cyclohexane); 2.02 (q, 4H, 2CH2CH3, J = 7.6 Hz); 3.39–3.47 (m, 2H,
cyclohexane); 7.61 (d, 2H, NH, J = 7.6 Hz) ppm
1.16 (t, 6H, 2CH3CH2, J = 8.0 Hz); 1.51–1.61 (m, 4H, cyclohexane); 1.73–1.82 (m,
4H, cyclohexane); 2.20 (q, 4H, 2CH2CH3, J = 8.0 Hz); 3.86–3.99 (m, 2H,
cyclohexane); 5.42–5.51 (m, 2H, NH) ppm
1.09 (t, 3H, CH3CH2, J = 7.6 Hz); 1.50–1.64 (m, 8H, cyclohexane); 2.17 (q, 2H,
CH2CH3, J = 7.6 Hz); 3.14–3.20 (m, 1H, cyclohexane); 3.61–3.69 (m, 1H,
cyclohexane); 7.26–7.32 (m, 2H); 7.89–7.94 (m, 2H) ppm
1.50–1.63 (m, 8H, cyclohexane); 1.91 (s, 3H, CH3); 3.12–3.20 (m, 1H,
cyclohexane); 3.61–3.70 (m, 1H, cyclohexane); 7.29 (t, 2H, J = 6.8 Hz); 7.90–7.94
(m, 2H) ppm
10.40 (CH3); 28.92 (CH2); 31.29 and 32.34 (cyclohexane CH2); 46.94 and 52.13
(CH); 116.76 (CH, JC–F = 23 Hz); 129.65 (CH, JC–F = 10 Hz); 139.04 (CSO2); 164.37
(CF, JC–F = 249 Hz); 172.41 (CO) ppm
15d
16b
17e
18e
10.46 (CH3); 29.00 (CH2); 31.64 (cyclohexane CH2); 47.37 (CH); 172.42 (CO)
ppm
9.23 (CH3); 26.99, 27.03, 28.77, 28.84 and 28.87 (CH2); 46.15 and 49.45 (CH2);
115.76 (CH, JC–F = 23 Hz); 129.49 (CH, JC–F = 10 Hz); 137.53 (C-SO2), 164.9 (C–F,
JC–F = 251 Hz), 174.99 (CO) ppm
21.23 (CH3); 26.99 and 28.82 (CH2); 46.29 and 49.48 (CH); 115.75 (CH, JC–F =
22 Hz), 129.39 (CH, JC–F = 10 Hz), 137.58, 164.92 (C–F, JC–F = 251 Hz); 171.17
(CO) ppm
a
b
c
APT 13C NMR.
CDCl3.
Mixture of 30:70 A + B rotamers.
DMSO d6.
MeOD d4.
d
e
ppm. The compound was used as such in the next step: it was sol-
ubilized (1.2 g, 1 eq) in hot glacial acetic acid (10 mL) and treated
with iron powder (16 eq). The mixture was heated at 90 °C for
2 h, and then filtered when still hot. Removal of the solvent under
vacuum gave a residue which was purified by flash chromatogra-
phy (CHCl3/petroleum ether/abs. EtOH/NH4OH 340:60:65:8 as elu-
ent) obtaining the title compound as white solid. Mp 141 °C. Yield:
35%. 1H NMR (DMSO d6, d): 4.99 (br s, 2H, NH2); 6.39 (d, 2H,
J = 6.8 Hz); 6.66 (d, 2H, J = 6.8 Hz); 7.34–7.38 (m, 2H); 7.66–7.69
(m, 2H) ppm. Anal. (C12H11FN2O2S) (C, H, N).
5.1.4. General procedure for the synthesis of compounds 34, 35,
37
A mixture of 4-nitroaniline or N-methyl-4-nitroaniline (7 mmol),
the acyl or benzoyl chloride (1.05 eq) and potassium carbonate (2
eq) in anhydrous THF (20 mL) was kept stirring for 12 h at RT. The
mixture was acidified with HCl 10% and extracted with CH2Cl2;
anhydrification (Na2SO4) and removal of the solvent gave a residue,
which was solubilized in ethyl acetate and hydrogenated at 50 psi
over Pd 10%/C for 24 h, then the catalyst was filtered off, and the sol-
vent was removed under vacuum giving the desired compounds.

10040
E. Martini et al. / Bioorg. Med. Chem. 16 (2008) 10034–10042
Table 5
Experimental details for the synthesis of compounds 19–31 (see structures in Scheme 3) and ClogP values
N
R
R1
R2
Eluenta
Yield%
Mp (°C)
ClogPb
Anal.
19
20
21
22
23
24
25
26
27
28
29
30
31
H
H
H
H
H
H
H
H
CH3
CH3
H
CH3
CH3
SO2C6H4F
COC6H5
COC6H5
COCH2CH3
COCH2CH3
COCH3
SO2CH(CH3)2
SO2CH(CH3)2
SO2C6H4F
SO2C6H4F
SO2CH(CH3)2
COC6H5
A
A
A
A
A
A
A
A
B
A
C
D
E
60
17
55
28
34
29
60
65
22
24
29
80
38
227–229
166
250–252
174–175
168
1.96
2.64
3.47
2.45
1.63
2.67
1.17
1.99
2.02
3.07
2.70
3.12
2.07
C15H17FN2O4S2
C16H18N2O3S
C19H15FN2O3S
C19H15FN2O3S
C12H18N2O3S
C15H14N2O2
C11H16N2O3S
C14H13FN2O3S
C16H19FN2O4S2
C17H20N2O3S
C17H20N2O3S
C18H22N2O3S
C17H21FN2O4S2
175
COCH3
COCH3
SO2CH(CH3)2
SO2C6H4F
200–202
180–182
144–145
149–150
135–137
144
SO2C6H4F
COC6H5
COC6H5
COC6H5
SO2C6H4F
SO2CH(CH3)2
SO2CH(CH3)2
SO2CH(CH3)2
SO2CH(CH3)2
SO2CH(CH3)2
135–136
a
A: cyclohexane/ethyl acetate 50:50; B: CH2Cl2/CH3OH/NH4OH 98:2:0.2; C: CH2Cl2/abs.EtOH/NH4OH/petroleum ether/Et2O/toluene 12:6:0.3:29.7:12:40 as eluent; D:
cyclohexane/ethyl acetate 60:40; E: CH2Cl2/abs.EtOH/NH4OH/petroleum ether 340:65:8:60 as eluent.
b
The ClogP value of the tested compounds has been calculated using the program ‘OSIRIS Property Explorer’ (http://www.organic-chemistry.org/prog/peo/).
34: oil, yields: 84% 1H NMR (DMSO d6, d) 6.70 (d, 2H, J = 8.4 Hz);
7.41–7.56 (m, 5H); 7.75 (br s, 1H, NH); 7.86 (d, 2H, J = 8.4 Hz) ppm.
Anal. (C13H12N2O) C, H, N.
5.1.7. Synthesis of compounds 29–31
To a solution of the suitable secondary amide (0.25 mmol) (27 or
28) in anhydrous THF (5 mL), t-BuOK (1 eq) and CH3I (10 eq) were
added, and the mixture was stirred at RT under N2 atmosphere.
When the reaction was performed on compound 20, 0.8 eq of CH3I
was used, and the mixture was heated at 60 °C for 18 h. The mixture
was then treated with 2 N HCl and extracted three times with ethyl
acetate; dehydration (Na2SO4) and removalof the solvent undervac-
uum gave the desired compound. Other experimental details are re-
ported in Table 5; [1H] and [13C] NMR are reported in Table 6.
35: oil, yields: 90% 1H NMR (DMSO d6, d) 1.18 (t, 3H, J = 7.6 Hz);
2.30 (q, 2H, J = 7.6 Hz); 6.58 (d, 2H, J = 8.8 Hz); 7.24 (d, 2H,
J = 8.8 Hz); 7.7 (br s, 1H, NH) ppm. Anal. (C9H12N2O) C, H, N.
37 Mp 122 °C. Yields: 90% after purification with flash chroma-
tography (cyclohexane/ethyl acetate 6:4 as eluent) 1H NMR (DMSO
d6, d) 3.26 (s, 3H); 5.08 (br s, 2H, NH2);6.38 (d, 2H, J = 8.4 Hz); 6.77 (d,
2H, J = 8.4 Hz);7.20–7.22 (m, 5H) ppm. Anal. (C14H14N2O) C, H, N.
5.1.5. N-Methyl-N-(4-aminophenyl)-4-fluorobenzenesulfon-
amide (36)
5.1.8. tert-Butyl 4-(p-fluorophenylsulfonamido)phenyl-
(methyl)carbamate (38)
N-Methyl-4-nitroaniline (2 g, 13.15 mmol, 1 eq) was dissolved in
anhydrous THF (25 mL), then p-F-benzenesulfonyl chloride (1 eq)
and anhydrous pyridine (2 eq) were added and the mixture heated
at 60 °C for 24 h. After cooling, the mixture was acidified with HCl
10% and extracted with ethyl acetate; dehydration (Na2SO4) and re-
moval of the solvent gave a residue, which was purified with flash
chromatography (CH2Cl2/abs.EtOH/NH4OH/petroleum ether/Et2O/
toluene 12.6:3.1:0.2:31.5:12.6:40 as eluent) leaving N-methyl-N-
(4-nitrophenyl)-4-fluorobenzenesulfonamide (mp 144 °C, 65%
yield). 1H NMR (CDCl3, d): 3.25 (s, 3H); 7.15 (t, 2H, J = 8.8 Hz); 7.35
(d, 2H, J = 9.2 Hz); 7.55–7.60 (m, 2H); 8.21 (d, 2H, J = 9.2 Hz) ppm.
This compound (2.65 g, 8.55 mol) was solubilized in methanol
(35 mL), and iron powder (1.39 g) and glacial acetic acid (2.85 mL)
were added. The mixture was left stirring at RT until completion
(t.l.c.), then the solvent was removed under vacuum, the residue
was partitioned between H2O and ethyl acetate, the organic phase
was collected and washed with satd NaHCO3. Dehydration (Na2SO4)
and removal of the solvent gave the title compound (FEB18) as white
solid. Mp 180 °C. Yield: 71%. 1H NMR (CDCl3, d): 3.13 (s, 3H); 6.57 (d,
2H, J = 8.4 Hz); 6.83 (d, 2H, J = 8.4 Hz); 7.11–7.18 (m, 2H); 7.57–7.61
(m, 2H) ppm. Anal. (C13H13FN2O2S) C, H, N.
A solution of (BOC)2O (3.2 g, 14.46 mmol, 1.1 eq) and DMAP
(0.07 g, 0.04 eq) in anhydrous THF (12 mL) was added dropwise
at 0 °C to a solution of N-methyl-4-nitro-aniline (2 g, 13.14 mmol,
1 eq) in anhydrous THF (12 mL). The mixture was allowed to warm
to RT and then stirred for 48 h under N2 atmosphere. After cooling,
the mixture was treated with a 1 M aqueous solution of citric acid
(20 mL) and extracted three times with ethyl acetate; the organic
phase was washed with brine. Dehydration (Na2SO4) and removal
of the solvent gave tert-butyl methyl-(4-nitrophenyl)carbamate24
in 90% yield. 1H NMR (CDCl3) d: 1.46 (s, 9H); 3.30 (s, 3H); 7.42
(d, 2H, J = 9.2 Hz); 8.13 (d, 2H, J = 9.2 Hz) ppm. This compound
(3.3 g, 13.21 mmol) was dissolved in ethyl acetate (65 mL) and
hydrogenated at 50 psi over Pd 10%/C (0.64 g) for 24 h. The mixture
was filtered, the solvent was removed, and the residue was purified
by flash chromatography (CH2Cl2/abs.EtOH/NH4OH/petroleum
ether 340:65:8:60 as eluent) giving tert-butyl 4-aminophe-
nyl(methyl)carbamate in 77% yield. 1H NMR (CDCl3) d: 1.43 (s,
9 H); 3.19 (s, 3H); 3.87 (br s, 2H); 6.67 (d, 2H, J = 8.4 Hz); 6.99 (d,
2H, J = 8.4 Hz) ppm. The compound (2.27 g, 10.22 mmol, 1 eq)
was dissolved in the minimum amount of THF, anhydrous pyridine
(1.65 mL, 2 eq) and p-fluoro-benzensulfonyl chloride (2.2 g, 1.1 eq)
were added, and the mixture was stirred at RT for 12 h and at 60 °C
for 3 h under N2 atmosphere. The mixture was partitioned between
H2O and ethyl acetate; the organic phase was collected and dried
(Na2SO4), Removal of the solvent gave the title compound in 94%
yield. Mp 168 °C. 1H NMR (CDCl3) d: 1.44 (s, 9H); 3.20 (s, 3H);
6.99–7.01 (m, 2H); 7.07–7.12 (m, 4H); 7.76–7.79 (m, 2H) ppm.
Anal. (C18H21FN2O4S) C, H, N.
5.1.6. General procedure for the synthesis of benzene deriva-
tives 19–28
To a solution of the suitable aniline (33–37 or commercially-
available N-(4-aminophenyl)acetamide, 2.5 mmol) and anhydrous
pyridine (2 eq) in anhydrous THF (15 mL), the suitable acyl or sul-
fonyl chloride (1 eq) was added at 0 °C. The mixture was allowed to
warm to RT and heated under reflux for 8 h under stirring. After
cooling, the mixture was treated with 10% HCl and extracted with
ethyl acetate; the organic phase was washed with H2O, dehydrated
(Na2SO4) and then the solvent was removed under vacuum. Flash
chromatography gave the desired compound. Other experimental
details are reported in Table 5; 1H and 13C NMR spectra are re-
ported in Table 6.
5.1.9. 4-Fluoro-N-(4-(N-methylpropan-2-ylsulfonamido)-
phenyl)-benzenesulfonamide (32)
A mixture of compound 38 (3.65 g, 9.60 mmol, 1 eq), dissolved
in ethyl acetate (15 mL), and 2 N HCl (10.48 mL, 1 eq) was left stir-
ring at RT until disappearance of the starting material (t.l.c.). Water
was added, the aqueous phase was washed with ethyl acetate, then

E. Martini et al. / Bioorg. Med. Chem. 16 (2008) 10034–10042
10041
Table 6
NMR spectra of compounds 19–31
N
1H NMR (d)
13C NMRa (d)
19b
1.18 (d, J = 8.0 Hz, 6H, 2CH3); 3.13 (sept, 1H, J = 8.0 Hz, CH); 7.07 (d, 2H,
J = 6.8 Hz); 7.10 (d, 2H, J = 6.8 Hz); 7.35–7.40 (m, 2H); 7.74–7.78 (m, 2H); 9.64
(br s, 1H, NH); 10.14 (br s, 1H, NH) ppm
1.24 (d, J = 8.0 Hz, 6H, 2CH3); 3.19 (sept, 1H, J = 8.0 Hz, CH); 7.21 (t, 2H,
J = 8.0 Hz); 7.51–7.60 (m, 3H); 7.70–7.73 (m, 2H); 7.94 (d, J = 8.0 Hz, 2H) ppm
7.05 (d, J = 8.0 Hz, 2H); 7.39 (t, J = 8.8 Hz, 2H); 7.49–7.64 (m, 5H); 7.76–7.82 (m,
2H); 7.90 (d, J = 8.4 Hz, 2H) ppm
16.58 (CH3); 51.95 (CH); 116.8 (CH, JC–F = 23 Hz); 121.11 (CH); 122.69 (CH);
130.20 (CH, JC–F = 9 Hz); 133.59, 135.74 and 136.25 (quat. C); 164.73 (C–F,
JC–F = 250 Hz) ppm
16.11 (CH3); 52.14 (CH); 120.28, 121.35, 127.55, 128.32 and 131.47 (aromatic
CH) 133.83, 134.07 and 135.23 quat. C); 165.32 (CO) ppm
116.89 (CH, JC–F = 22 Hz); 121.54 (CH); 121.64 (CH); 121.96 (CH); 128.03 (CH);
128.86 (CH); 130.25 (CH, JC–F = 9 Hz); 133.17, 135.19, 135.24 and 136.45 (quat.
C); 163.75 (CH, JC–F = 250 Hz); 165.91 (CO) ppm
10.1 (CH3); 29.87 (CH2); 116.80 (CH, JC–F = 23 Hz), 120.17, 122.31, 130.18 and
132.50 (JC–F = 9 Hz) (aromatic CH); 136.22, 136.25 and 136.83 (quat. C); 164.69
(CF, JC–F = 250 Hz); 172.26 (CO) ppm
20b
21b
22b
23b
1.10 (t, J = 8.0 Hz, 3H, CH3CH2); 2.26 (q, J = 8.0 Hz, 2H, CH2CH3); 6.98 (d,
J = 8.0 Hz, 2H); 7.35–7.45 (m, 4H); 7.73–7.76 (m, 2H); 9.79 (br s, 1H, NH); 10.06
(br s, 1H, NH) ppm
1.07 (t, J = 7.6 Hz, 3H, CH3CH2); 1.22 (d, J = 6.8 Hz, 6H, 2CH3); 2.28 (q, J = 7.6 Hz,
2H, CH2CH3); 3.15 (sept, J = 6.8 Hz, 1H, CH); 7.14 (d, J = 8.0 Hz, 2H); 7.51 (d,
J = 8.0 Hz, 2H) 9.56 (br s, 1H, NH); 9.81 (br s, 1H, NH) ppm
2.03 (s, 3H, CH3); 7.50–7.58 (m, 5H); 7.68 (d, 2H, J = 8.0 Hz); 7.94 (d, 2H,
J = 8.0 Hz); 9.91 (br s, 1H, NH); 10.18 (br s, 1H, NH) ppm
1.22 (d, 6H, 2CH3, J = 6.8 Hz); 2.01 (s, 3H, CH3); 3.15 (sept, 1H, CH, J = 6.8 Hz,);
7.11–7.16 (m, 2H); 7.48–7.51 (m, 2H); 9.55 (br s, 1H, NH); 9.89 (br s, 1H, NH)
ppm
10.15 (CH3); 16.60 (CH3); 29.87 (CH2); 51.49 (CH); 120.42 and 121.11 (aromatic
CH); 133.79 and 136.09 (quat. C) 172.21 (CO) ppm
24b
25b
24.38 (CH3); 119.65, 121.28, 128.03, 128.83 and 131.90 (aromatic CH); 134.79,
135.46 and 135.70 (quat. C); 165.69 and 168.46 (CO) ppm
16.61 (CH3); 24.31 (CH3); 51.51 (CH); 120.4 and 121.12 (aromatic CH); 133.7
and 136.1 (quat. C); 168.5 (CO) ppm
26b
27c
1.98 (s, 3H, CH3); 6.97 (d, 2H, J = 8.0 Hz); 7.35–7.43 (m, 4H); 7.73–7.76 (m, 2H);
9.85 (br s, 1H, NH); 10.06 (br s, 1H, NH) ppm.
24.31 (CH3); 116.80 (JC–F = 22 Hz), 120.16, 122.36 and 130.17 (JC–F = 9 Hz) (aromatic
CH); 132.62, 136.25 and 136.75 (quat. C); 164.69 (CF, JC–F = 250.0 Hz); 168.55 (CO)
ppm.
16.50 (CH3); 38.20 (CH3); 53.02 (CH); 116.16 (JC–F = 23 Hz), 120.14, 127.87 and
130.49 (JC–F = 10 Hz) (aromatic CH); 132.45, 136.80 and 137.51 (quat. C); 165.27
(CF, JC–F = 254 Hz) ppm
16.58 (CH3); 35.45 (CH3); 52.25 (CH); 120.14, 128.13, 128.52, 128.59 and 129.74
(aromatic CH) 136.77, 137.34 and 140.46 (quat. C); 169.96 (CO) ppm
15.72, 43.61, 50.40, 121.31, 126.64, 126.96, 127.23, 128.25, 131.58, 152.05 ppm
1.39 (d, 6H, 2CH3, J = 6.8 Hz); 3.14 (s, 3H, CH3); 3.31 (sept, 1H, CH, J = 6.8 Hz);
7.03 (d, 2H, J = 8.8 Hz); 7.12–7.21 (m, 4H); 7.38 (br s, 1H); 7.55–7.59 (m, 2H)
ppm
1.18 (d, 6H, 2CH3, J = 6.4 Hz); 3.16 (sept, 1H, CH, J = 6.4 Hz); 3.33 (s, 3H, CH3);
7.09–7.12 (m, 4H); 7.18–7.30 (m, 5H); 9.75 (br s, 1H, NH) ppm
1.36 (d, 6H, J = 7.2 Hz); 3.38 (s, 3H); 3.42 (sept, 1H, J = 7.2 Hz); 7.44–7.49 (m,
2H); 7.52–7.55 (m, 2H); 7.59–7.63 (m, 1H); 7.76 (d, 2H, J = 8.8 Hz); 7.95 (d, 2H,
J = 8.8 Hz) ppm
1.15 (d, 6H, J = 6.8 Hz); 3.23 (s, 3H), 3.33 (s, 3H); 3.37 (sept, 1H, J = 6.8 Hz); 7.17–
7.34 (m, 9H) ppm
1.36 (d, 6H, J = 6.8 Hz); 3.16 (s, 3H); 3.31 (sept, 1H, J = 6.8 Hz); 3.38 (s, 3H);
7.09–7.17 (m, 4H); 7.35 (d, 2H, J = 9.2 Hz); 7.56–7.58 (m, 2H) ppm
28b
29d
30b
31c
16.99, 38.22, 38.69, 52.56, 126.02, 127.95, 128.17, 128.64, 129.89, 136.62, 140.38,
140.76, 169.93 ppm
16.77, 38.04, 39.15, 53.02, 116.17 (d, J = 23.0 Hz), 126.96, 127.26, 130.46 (d, J =
10.0 Hz), 132.90, 139.38, 140.95, 165.28 (d, J = 256 Hz) ppm
a
b
c
APT 13C NMR.
DMSO d6,8
.
CDCl3.
CD3OD.
d
it was made alkaline with 10% NaOH and extracted three times
with ethyl acetate. Dehydration (Na2SO4) and removal of the sol-
vent gave p-fluoro-N-(4-(methylamino)phenyl)benzenesulfon-
amide in 40% yield. 1H NMR (CDCl3) d: 2.75 (s, 3H); 3.53–3.82
(m, 1 H); 6.43 (d, 2H, J = 8.8 Hz); 6.85 (d, 2H, J = 8.8 Hz); 7.05 (t,
3H, J = 8.4 Hz); 7.68–7.72 (m, 2H) ppm. This compound was used
as such for the following step: it was dissolved (1.08 g, 3.86 mmol,
1 eq) in the minimum amount of anhydrous THF, treated with any-
drous pyridine (0.63 mL, 2 eq) and i-propylsulfonyl chloride (1.5
eq, 0.64 mL), and the mixture was left stirring at RT for 12 h. The
same work-up reported for compounds 19–28 gave a residue
which was purified by flash chromatography (CH2Cl2/abs.EtOH/
NH4OH/petroleum ether/Et2O 360:180:9.9:900:360 as eluent).
The title compound was further crystallized from i-propanol, yield-
ing a white solid mp 52–54 °C (19% yield). 1H NMR (CDCl3) d: 1.35
(d, 6H, J = 7.2 Hz); 3.29 (sept, 1H, J = 7.2 Hz); 3.31 (s, 3H); 7.07–7.16
(m, 4H); 7.27–7.30 (m, 2H); 7.47 (br s, 1H); 7.81–7.85 (m, 2H) ppm.
13C NMR (CDCl3) d: 16.76, 39.35, 52.95, 116.44 (d, J = 23 Hz),
122.31, 127.01, 130.02 (d, J = 10 Hz), 155.05 (d, J = 251 Hz) ppm.
Anal. (C16H19FN2O4S2) C, H, N. This compound was unstable: after
some time, the compound transformed into a tarry solid.
received a punishing electrical 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
consisting of 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 entering the dark room
in the training session and remember it in the session on the fol-
lowing day, unless their memory is impaired by the amnesic drug.
Mice who did not enter after 60 s latency in the training session
were excluded from the experiment; about 20–30% of mice was
excluded from each group. For memory disruption, mice were in-
jected with the amnesic drugs (scopolamine, 13 or 14). All investi-
gated drugs were injected ip, in a 1:10 dilution sequence, 20 min
before the training session; amnesic drugs were injected immedi-
ately after termination of the training session. Saline and saline/
DMSO treated mice received an additional injection of saline
immediately after the training session as control of scopolamine
injection. The maximum entry latency allowed in the retention
session was 180 s. The degree of received punishment memory
(fall into cold water) was expressed as the difference in seconds
between training and retention latencies. Piracetam and com-
pounds 1, 2a and 3 were used as the reference drugs.
5.2. Pharmacology
All compounds were dissolved in saline, except compounds 27–
31, which were dissolved into a mixture of water/DMSO 4:1.
All compounds elicited their effect without changing either
gross behavior or motor coordination, as revealed by the rota-rod
test (data not shown). None of the drugs, at the active doses, in-
creased the number of falls from the rotating rod in comparison
with saline-treated mice. The number of falls in the rota-rod test
5.2.1. Passive-avoidance test
The test was performed according to the step-through method
described by Jarvik and Kopp.25 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

10042
E. Martini et al. / Bioorg. Med. Chem. 16 (2008) 10034–10042
progressively decreased since mice learned how to balance on the
rotating rod. The spontaneous motility and inspection activity of
mice was unmodified by the administration of the studied com-
pounds as revealed by the hole-board test in comparison with sal-
ine-treated mice (data not shown).
References and notes
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2. Schmitt, J. A.; Wingen, M.; Ramaekers, J. G.; Evers, E. A.; Riedel, W. J. Curr.
Pharm. Des. 2006, 12, 2473.
3. Robbins, T. W.; Murphy, E. R. Trends Pharmacol. Sci. 2006, 27, 141.
4. Chebib, M.; Hanrahan, J. R.; Mewett, K. N.; Duke, R. K.; Johnston, G. A. R. Annu.
Rev. Med. Chem. 2004, 39, 13.
5. Passani, M. B.; Bacciottini, L.; Mannaioni, P. F.; Blandina, P. Neurosci. Biobehav.
Rev. 2000, 24, 107.
6. Rogers, J. M.; Panegyres, P. K. J. Clin. Neurosci. 2007, 14, 919.
7. Wilens, T. E.; Decker, M. W. Biochem. Pharmacol. 2007, 74, 1212.
8. Gualtieri, F.; Guandalini, L.; Manetti, D.; Martini, E.; Romanelli, M. N. Med.
Chem. Rev. Online 2005, 2, 471.
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Expert Opin. Invest. Drugs 2007, 16, 59.
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2002, 8, 125.
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Fukunaga, K.; Narahashi, T. Mol. Pharmacol. 2007, 71, 580.
13. Zhao, X.; Kuryatov, A.; Lindstrom, J. M.; Yeh, J. Z.; Narahashi, T. Mol. Pharmacol.
2001, 59, 674.
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18. Galeotti, N.; Ghelardini, C.; Pittaluga, A.; Pugliese, A. M.; Bartolini, A.; Manetti,
D.; Romanelli, M. N.; Gualtieri, F. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2003,
368, 538.
5.2.2. Rota-rod test
The apparatus consisted of a base platform and a rotating rod with
a diameterof3 cmanda non-slipperysurface. The rodwas placedata
height of 15 cm from the base. The rod, 30 cm in length, was divided
into five equal sections by six disks. Thus, up to five mice were tested
simultaneously on the apparatus, with a rod-rotating speed of
16 rpm. The integrity of motor coordination was assessed on the ba-
sis of the number of falls from the rod in 30 s according to Vaught
et al.28 Those mice scoring less than 3 and more than 6 falls in the pre-
test were rejected (20%). The performance time was measured before
(pretest) and 15, 30 and 45 min after the beginning of the test.
5.2.3. Hole-board test
The hole-board test consisted of a 40 cm square plane with 16
flush mounted cylindrical holes (3 cm diameter) distributed 4 by
4 in an equidistant, grid-like manner. Mice were placed on the cen-
ter of the board one by one and allowed to move about freely for a
period of 10 min each. Two electric eyes, crossing the plane from
mid-point to mid-point of opposite sides, thus dividing the plane
into four equal quadrants, automatically signaled the movement
of the animal (counts in 5 min) on the surface of the plane (spon-
taneous motility). Miniature photoelectric cells, in each of the 16
holes, recorded (counts in 5 min) the exploration of the holes
(exploratory activity) by the mice.
19. Manetti, D.; Martini, E.; Ghelardini, C.; Dei, S.; Galeotti, N.; Guandalini, L.;
Romanelli, M. N.; Scapecchi, S.; Teodori, E.; Gualtieri, F. Bioorg. Med. Chem. Lett.
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Galeotti, N.; Guandalini, L.; Romanelli, M. N.; Teodori, E. Bioorg. Med. Chem.
2004, 12, 71.
5.2.4. Statistical analysis
All experimental results are given as means SEM. Analysis of
variance (ANOVA), followed by Fisher’s protected least significant
difference (PLSD) procedure for post hoc comparison, was used
to verify significance between two means. Data were analysed with
the StatView software for the Macintosh (1992). P values of less
than 0.05 were considered significant.
21. Welch, W. M. J. Org. Chem. 1982, 47, 886.
22. Sueess, H.; Hesse, M. Helv. Chim. Acta 1979, 62, 1040.
23. Johnston, T. P.; McCaleb, G. S.; Clayton, S. D.; Frye, J. L.; Krauth, C. A.;
Montgomery, J. A. J. Med. Chem. 1977, 20, 279.
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Zavala, M. Tetrahedron Lett. 2007, 48, 17.
25. Jarvik, M. E.; Kopp, R. Psychol. Rep. 1967, 21, 221.
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27. Ghelardini, C.; Galeotti, N.; Gualtieri, F.; Manetti, D.; Bucherelli, C.; Baldi, E.;
Bartolini, A. Drug Dev. Res. 2002, 56, 23.
Acknowledgements
28. Vaught, J.; Pelley, K.; Costa, L. G.; Sether, P.; Enna, S. J. Neuropharmacology 1985,
24, 211.
This work was supported by grants from MUR (PRIN 2006) and
the University of Florence (ex 60%).