Efficient synthesis and biological evaluation of two modafinil analogues

Article abstract of DOI:10.1016/j.bmc.2008.10.027

Non classical bioisosters of modafinil featuring interesting biological profile have been easily produced through replacement of the sulfoxide function with a carbonyl group and modification of the carboxylic acid amide functionality.

Full text of DOI:10.1016/j.bmc.2008.10.027

Bioorganic & Medicinal Chemistry 16 (2008) 9904–9910
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Efficient synthesis and biological evaluation of two modafinil analogues
b
a
b
a
Carmela De Risi a, , Luca Ferraro , Gian P. Pollini , Sergio Tanganelli , Filippo Valente ,
*
Augusto C. Veronese a
a Dipartimento di Scienze Farmaceutiche, Via Fossato di Mortara 19, 44100 Ferrara, Italy
b Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Via Fossato di Mortara 19, 44100 Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Non classical bioisosters of modafinil featuring interesting biological profile have been easily produced
through replacement of the sulfoxide function with a carbonyl group and modification of the carboxylic
acid amide functionality.
Received 11 July 2008
Revised 3 October 2008
Accepted 12 October 2008
Available online 17 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Modafinil
Bioisosters
[3H]5-HT efflux
Rat cerebral cortex slices
1. Introduction
A facile preparation of both racemic modafinil and its achiral
oxidized derivative 2-[(diphenylmethyl)sulfonyl]acetamide has
Modafinil, 2-[(diphenylmethyl)sulfinyl]acetamide [Provigil]
( )-1, Adrafinil, 2-[(diphenylmethyl)sulfinyl]-N-hydroxyacetamide
[Olmifon] ( )-2, the hydroxamic acid derivative of modafinil
(Fig. 1) and Armodafinil [Nuvigil], the R-enantiomer of modafinil,
are among the most widely used and most effective pharmaceuti-
cal agents for the treatment of excessive sleepiness caused by nar-
colepsy, shift work sleep disorder and obstructive sleep apnea.1–3
Modafinil has a complex and still uncertain mode of action and
is theorized to work in a localized manner, utilizing hypocretin,
been recently described.9
Interestingly, owing to the uncomplicated chemistry involved, a
microscale synthesis of racemic modafinil has been described as an
instructive experiment for undergraduate students, keeping them
surely awake!10
The limited data available in the public domain on armodafinil,
with particular regard to chemistry, have been recently enriched
by a paper describing the investigations that led to the develop-
ment of the commercial route providing the chiral sulfoxide.11
The importance of studying the biological activity of each enan-
tiomer stimulated efforts to obtain the two enantiomers of modafi-
nil. Among the reported methods,12–15 the microbial oxidation/
amidation of benzhydrylsulfanyl acetic acid was a very interesting
approach.14 The two operations, namely a highly enantioselective
oxidation of benzhydrylsulfanyl acetic acid to the corresponding
(S)-sulfinyl carboxylic acid and the subsequent amidation were
achieved in very good yield employing the fungus Beauveria bassi-
ana and the bacteria Bacillus subtilis, respectively.
histamine, epinephrine,
c
-aminobutyric acid, and glutamate.2
Moreover, modafinil has a postulated large potential for many uses
in psychiatry and general medicine. Recent work suggests that
modafinil might also be of utility in the treatment of attention def-
icit/hyperactivity disorder (ADHD),4 and in treating opioid-induced
sedation.5
Although modafinil contains an asymmetric sulfoxide func-
tional group, the racemic sulfoxide has been marketed in the Uni-
ted States by Cephalon as Provigil6 since its approval in 1998.
In June 2007, Armodafinil, the R-enantiomer of modafinil, was
approved by US Food and Drug Administration for the same
indications.
O
S
O
S
O
O
Modafinil and its chemical predecessor adrafinil have been
firstly prepared by the procedure patented by L. Lafon7 and later
reported by Mu et al.8
NH2
NHOH
1
2
* Corresponding author. Tel.: +39 0532 455922; fax: +39 0532 455953.
E-mail address: drc@unife.it (C. De Risi).
Figure 1. Chemical structures of Modafinil (1) and Adrafinil (2).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.027

C. De Risi et al. / Bioorg. Med. Chem. 16 (2008) 9904–9910
9905
Studies related to the anticonvulsant properties of modafinil
and its sulfone derivative led to discover that the oxidized deriva-
tive of modafinil is also non-toxic and almost as effective as an
anticonvulsant as the parent compound.9
displayed by the parent compound. In fact, it has been reported
that modafinil (0.3–30 lM) increased electrically-evoked, but not
spontaneous, serotonin ([3H]5-HT) efflux from cortical slices in a
concentration-dependent manner.20
These data seem to indicate that the oxidation state at sulphur
atom is not essential, the sulfone moiety being as important as the
sulfoxide. Moreover, modafinil analogs have been prepared intro-
ducing different substituents (e.g. F, Cl, Br, CF3, NO2, NH2, alkyl
and alkyloxy with 1 to 4 carbon atom chain length) into both aro-
matic rings or modifying the primary amide moiety not only as the
hydroxamic acid 2 but also as secondary or tertiary amide function
bearing different alkyl residues.
The known modafinil analogs 1–9 featuring modifications on
both the phenyl groups and the carboxamide function act on CNS
either as stimulating or sedative agents,16 as summarized in the
following Table 1.
2. Results and discussion
2.1. Chemistry
Among the existing methodologies for preparing 3-oxobutano-
ate derivatives we investigated the Blaise reaction21 and the Mel-
drum’s acid (2,2-dimethyl-1,3-dioxane-4,6-dione) acylation22
starting from commercially available materials such as diphenyl-
acetonitrile 3 or diphenylacetic acid 7 as summarized in Scheme 1.
In both cases, the starting materials required a two-carbon
elongation to install the desired carbon chain length.
The protocol based on Blaise reaction involved the reaction of
cyanide 3 with a twofold molar excess of ethyl bromoacetate in
the presence of activated zinc dust in refluxing tetrahydrofuran
to yield the corresponding b-enamino ester 4 in 90% isolated yield.
Subsequent acid hydrolysis afforded the b-ketoester 5 in essen-
tially quantitative yield as a rather unstable oil. This could be also
obtained through a modified protocol for performing the Blaise
reaction,23 involving in situ zinc activation in the presence of cata-
lytic methanesulfonic acid followed by acid hydrolysis of the inter-
mediate b-enamino ester 4.
The alternative approach entailed on the use of the acyl-Mel-
drum derivative 10, which was obtained through triethylamine-
mediated reaction between Meldrum’s acid 9 and diphenylacetic
acid chloride 8.
Alternatively, preparation of the intermediate 10 could be
achieved through a one-step procedure by reaction of 9 with
diphenylacetic acid 7 activated with N,N’-dicyclohexylcarbodiim-
ide (DCC),24,25 providing an enhanced yield and purity of the de-
sired compound.
Our continuous interest in the field,17 prompted us to explore
the effect of replacement of the sulfoxide function in 1 with a car-
bonyl group, which represents a common isosteric replacement.18
This choice favoured a functional group that could be introduced in
a straightforward way with concomitant removal of problems
associated with sulphur chirality.
The bioisosterism represents a successful strategy in rational
drug design,19 useful in the molecular modification and design of
new therapeutically attractive substances of different pharmaco-
logical classes. Nonclassical bioisosteres do not obey the strict ste-
ric and electronic definition of classical isosteres and they do not
have the same number of atoms as the substituent moiety for
which they are used as a replacement. These isosteres are capable
of maintaining similar biological activity by mimicking the spatial
arrangement, electronic properties, or some other physicochemical
property of the molecule or functional group that is critical for the
retention of biological activity. Moreover, replacement of a group
with a bioisostere frequently results in a new compound that re-
tains the activity of the parent one. Thus, this approach is common
in the pharmaceutical industry, since it allows to generate market-
able analogues of a known drug that has a patentable composition
of matter.
Treatment of b-ketoester 5 with methanolic ammonia at 0 °C
resulted in a clean formation of compound 6 in 54% yield, while
reaction of the acyl-Meldrum derivative 10 with the same ammo-
nia source in different experimental conditions (from 0 to 100 °C in
sealed glass tube) proved unsuccessful, mainly decomposition of
the starting material being observed.
The preparation of modafinil analogues as those described in
this paper is lead by the increasing interest in study the pharmaco-
logical behaviour of modafinil isomers and their implicancies in
other diseases.
The formation of a salt between ammonia and the completely
enolized 10 may likely account for the difficulties encountered in
this operation, as previously observed in the literature.26
However, amide formation was eventually achieved in 49%
yield by treatment of a CH3CN solution of 10 with an ammonia
gas-saturated THF solution at 70 °C, according to a reported
protocol.26
In particular, we were interested in the evaluation of the effects
of modafinil analogues on the spontaneous and the electrically-
evoked tritiated serotonin ([3H]5-HT) efflux from rat cortical slices,
in order to preliminary compare their biological activity with that
Table 1
Activity of known modafinil analogs on CNS
In order to overcome this hurdle we envisaged the use of tert-
butyl carbamate as a different ammonia precursor, since the ure-
thane group is less basic than ammonia. Thus, amidation of the
acyl-Meldrum derivative 10 with tert-butyl carbamate following
known directions,25 produced in excellent yield the corresponding
amide 11, that was easily taken to the modafinil analogue 6 in
essentially quantitative yield by action of HCl in EtOAc (Scheme 2).
We also attempted the preparation of an analogue of 2 using
N,O-di-tert-butoxycarbonyl hydroxylamine27 as the nitrogen
nucleophilic counterpart of the acyl-Meldrum derivative 10
(Scheme 2). We found this reaction gave the b-ketohydroxamic
acid 12 in good yield, but every attempt to cleanly remove the
nitrogen protecting groups failed. As a matter of fact, we obtained
unseparable complex mixtures in which only traces of the desired
N-hydroxy amide were present.
O
S
O
R1
N
X1
R2
X2
Entry
X1
X2
NR1R2
Action on CNS
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
4-Cl
4-F
4-F
H
H
H
H
H
H
H
NHCH3
Stimulating
Stimulating
Stimulating
Sedative
Sedative
Sedative
Stimulating
Stimulating
Stimulating
NHCH(CH3)2
NHC(CH3)3
NHCH2CH3
Piperidine
Morfoline
NH2
4-F
H
NH2
NH2
Furthermore, we were intrigued to apply this chemistry to the
synthesis of modafinil analogues incorporating not only a carbonyl

9906
C. De Risi et al. / Bioorg. Med. Chem. 16 (2008) 9904–9910
O
O
H2N
O
ii
i
CN
OEt
OEt
5
4
3
iii
O
O
O
O
O
OH
O
O
O
vi
9
R
NH2
O
v
O
O
10
6
7 R = OH
8 R = Cl
iv
Scheme 1. Reagents and conditions: (i) Method A: Zn, BrCH2CO2Et, THF, reflux, 30 min; Method B: Zn, MeSO3H cat., BrCH2CO2Et, THF, reflux, 30 min; (ii) 1N HCl, THF, rt, 6 h,
88% (Method A) or 90% (Method B) from 3; (iii) NH3, MeOH, rt, 24 h, 54%; (iv) SOCl2, benzene, reflux, 2 h, 96%; (v) From 7: DCC, Et3N, CH2Cl2, 12 h, 78%; From 8: Et3N, CH2Cl2,
rt, 5 h, 65%; (vi) NH3, THF, CH3CN, 70 °C, 48 h, 49%.
OH
O
O
O
O
i
ii
6
O
NHBoc
O
10
11
iii
iv
O
O
complex mixture
O
N
Boc
Boc
12
Scheme 2. Reagents and conditions: (i) H2N-Boc, CH3CN, reflux, 2 h, 83%; (ii) 3 N HCl in EtOAc, rt, 3 h, 98%; (iii) (Boc)NHO(Boc), benzene, 65 °C, 4 h, 71%; (iv) HCl, EtOAc, rt,
12 h.
, DMAP
O
O
O
O
H2N
OEt
N
H
toluene, reflux, 20 h
92%
5
13
Scheme 3. Synthesis of compound 13.
group instead of the sulfoxide moiety but also a modified amide
functionality. Preliminary studies in this field allowed us to obtain
compound 13, through reaction of the b-ketoester 5 with two
equivalents of N-isopropylamine in refluxing toluene in the pres-
ence of 4-(dimethylamino)pyridine (DMAP) (Scheme 3), basing
on a literature procedure.28

C. De Risi et al. / Bioorg. Med. Chem. 16 (2008) 9904–9910
9907
2.2. Biological activity: effects of modafinil and its analogues on
2.5
2.0
1.5
1.0
0.5
Control
spontaneous and electrically-evoked [3H]5-HT efflux from
cortical slices
modafinil (0.3 μM)
modafinil (1 μM)
modafinil (30 μM)
2.2.1. Spontaneous tritium efflux
In control slices, spontaneous [3H]5-HT efflux at the 45th min
was 0.029 0.003 pmol/min, corresponding to a fractional rate of
3.25 0.06%/5 min, and slowly declined during the collection per-
iod (from the 30th to the 110th min from the onset of the superfu-
*
sion). Neither modafinil (1–30
l
M)20 nor compound
M), added to the superfusion
6 (0.1–
100 M) and compound 13 (0.1–30
l
l
medium after three basal collection periods (45th min of superfu-
sion) affected spontaneous [3H]5-HT efflux from rat cortical slices
(data not shown).
2.2.2. Electrically-evoked tritium efflux
0.0
2.5
The net [3H]5-HT overflow induced by the electrical stimulation
in St1 was (0.036 0.006 pmol/min. Under the present experimen-
tal conditions the St2/St1 ratio remained close to unity
(0.92 0.05). As previously reported,20 the addition to the superfu-
Control
Compound 6 (0.3 μM)
Compound 6 (1 μM)
Compound 6 (30 μM)
Compound 6 (100 μM)
sion medium of modafinil (0.1–30
creased the [3H]5-HT evoked-release in
dependent manner, the threshold concentration being 0.3
The maximal effect (30 M) was in the order of 217% of control val-
l
M), 15 min before St2, in-
concentration-
M.
2.0
1.5
1.0
0.5
a
l
l
*
ues (Fig. 2A). A similar profile of action was displayed by com-
pound 13 (Fig. 2C), while compound 6 increased the [3H]5-HT
evoked-release only at the higher concentration tested (Fig. 2B).
These results suggest that either compound 6 or compound 13
retained the bioactivity of the parent compound in increasing the
electrically-evoked, but not spontaneous, [3H]5-HT efflux. How-
ever, while compound 13 and modafinil display a similar profile
of action, compound 6 possesses a lower potency in affecting cor-
tical serotonergic transmission, at least under the present experi-
mental conditions. Further experiments will be necessary in
order to evaluate the pharmacokinetic profile and the possible
therapeutic prospective of the two compounds.
0.0
2.5
Control
Compound 13 (0.3 μM)
Compound 13 (1 μM)
Compound 13 (30 μM)
3. Conclusion
2.0
1.5
1.0
0.5
In summary, two novel analogues of modafinil 1 have been syn-
thesized through a synthetically simple isosteric replacement of
the sulfoxide function with a carbonyl group. Evaluation of their ef-
fects on the spontaneous and the electrically-evoked tritiated sero-
tonin ([3H]5-HT) efflux from rat cortical slices showed a loss of
biological activity for compound 6, which could be restored modi-
fying the primary amide function into the corresponding N-isopro-
pyl derivative 13. We are currently in the process of synthesizing
several new derivatives in this series.
*
4. Experimental
Melting points were determined on a Büchi-Tottoli apparatus
and were uncorrected.
IR spectra were recorded using a Perkin Elmer Paragon 500 FTIR
spectrophotometer.
Nuclear magnetic resonance (NMR) spectra were taken on a
Varian VXR-200 and a Varian Mercury Plus-400 spectrometers.
Solvents were distilled prior to use and reactions were per-
formed under nitrogen or argon atmosphere.
Silica gel plates were used to monitor synthetic transforma-
tions, visualization being done under UV light and using 2% aque-
ous KMnO4 solution or 2% ethanolic FeCl3 solution. Organic
solutions were dried over anhydrous magnesium sulfate and evap-
orated with a rotary evaporator.
Figure 2. Effects of different concentrations of modafinil (0.1–30
compound 6 (0.1–100 M; Panel B) and compound 13 (0.1–30
electrically-evoked [3H]5-HT efflux from rat brain cortex slices superfused with
Krebs solution containing paroxetine (3 M) from the onset of the experiment. The
slices were electrically stimulated twice at the 45th (St1) and 85th (St2) min from
the onset of superfusion by applying two trains of rectangular pulses of alternate
polarity (40 mA, 2 ms, 3 HzÂ2 min). The drugs were added to the superfusion
medium 15 min before (St2) and maintained till the end of the collection period.
Each histogram represents the mean SEM. of 8–11 experiments. Panel A: *P < 0.05,
l
M; Panel A),
l
lM; Panel C), on the
l
**P < 0.01 significantly different versus control as well as modafinil (0.3
°°P < 0.01 significantly different versus modafinil (1
significantly different versus the other groups; Panel C: *P < 0.05, **P<0.01 signifi-
cantly different vs control as well as compound 13 (0.3
M); °°P < 0.01 significantly
different vs compound 13 (1 M). The statistical analysis was carried out according
to one-way ANOVA followed by the Newman–Keuls test for multiple comparisons.
lM);
l
M); Panel B: *P < 0.005
l
l

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C. De Risi et al. / Bioorg. Med. Chem. 16 (2008) 9904–9910
Chromatographic purifications were carried out using 70–230
4.2. 5-(1-Hydroxy-2,2-diphenylethylidene)-2,2-dimethyl-1,3-
mesh silica gel.
dioxane-4,6-dione (10)
4.1. Ethyl 3-Oxo-4,4-diphenylbutanoate (5)
4.2.1. Method A
To a cooled (0 °C) solution of 9 (3.40 g, 23.60 mmol) in CH2Cl2
(15 mL), Et3N (8.51 mL, 59.00 mmol) was added over a period of
20 min and the solution stirred at the same temperature for
15 min. A solution of 8 (5.44 g, 23.60 mmol) in CH2Cl2 (20 mL)
was successively added dropwise over a period of 2 h and stirring
was continued for 5 h with gradual warming to ambient tempera-
ture. The reaction mixture was diluted with CH2Cl2 (20 mL), cooled
at 0 °C and treated with 2 N HCl (20 mL). After being stirred at 0 °C
for 15 min, the aqueous phase was separated from the organic
layer and extracted with CH2Cl2 (2 Â 10 mL), the combined organic
phases were dried and concentrated to dryness. The yellow solid
thus obtained was recrystallized from EtOAc/petroleum ether
1:10 and filtered to give 10 (5.20 g, 65%) as a white solid, mp 49-
4.1.1. Method A
Ethyl bromoacetate (0.25 mL, 2.25 mmol) was added dropwise
to
a refluxing suspension of activated zinc dust (0.70 g,
10.71 mmol) in THF (5 mL) until it turned green. Compound 3
(1.00 g, 5.17 mmol) was added and successively ethyl bromoace-
tate (1 mL, 9.02 mmol) was dropped over a period of 30 min. Heat-
ing was continued for 30 min, then the reaction mixture was
cooled to room temperature, saturated aqueous NaHCO3 solution
(10 mL) was added and the solution stirred for about 15 min. After
being filtered through a bed of Celite, the solution was extracted
with Et2O (3 Â 15 mL), the combined extracts were washed with
brine (3 Â 15 mL), dried and concentrated in vacuo. The crude
product was purified by flash chromatography (Et2O/petroleum
ether 4:6) to give enamino ester 4 (1.30 g, 90%) as a white solid,
51 °C. IR (KBr): 1729, 1668, 1569, 1415 cmÀ1 1H NMR (200 MHz,
;
CDCl3): d (ppm) 1.71 (s, 6H), 5.06 (s, 1H), 7.25-7.37 (m, 10H); 13C
NMR (50 MHz, CDCl3): d (ppm) 27.8, 57.0, 92.0, 105.1, 127.5,
128.7, 129.3, 137.9, 171.1, 178.3, 195.8. Anal. Calcd for C20H18O5:
C, 70.99; H, 5.36. Found: C, 71.00; H, 5.34.
mp 58 °C. IR (KBr): 3460, 3310, 1663, 1608, 1554, 1165 cmÀ1 1H
;
NMR (200 MHz, CDCl3): d (ppm) 1.24 (t, J = 7.0 Hz, 3H), 4.10 (q,
J = 7.0 Hz, 2H), 4.56 (s, 1H), 4.92 (s, 1H), 7.23–7.36 (m, 10H),
7.00–9.00 (br, 2H); 13C NMR (50 MHz, CDCl3): d (ppm) 14.5, 57.1,
58.8, 86.6, 127.2, 128.7, 129.2, 139.8, 163.7, 170.4. Anal. Calcd for
C18H19NO2: C, 76.84; H, 6.81; N, 4.98. Found: C, 76.70; H, 6.82;
N, 4.97.
4.2.2. Method B
Et3N (0.30 mL, 2.10 mmol) was added to a 0 °C cold solution of 7
(0.42 g, 2.00 mmol) in CH2Cl2 (15 mL). After being stirred at the
same temperature for 10 min, a solution of 9 (0.28 g, 2.00 mmol)
in CH2Cl2 (10 mL) and N,N’-dicyclohexylcarbodiimide (0.45 g,
2.20 mmol) were successively added. The mixture was stirred at
room temperature for 20 h, then it was diluted with CH2Cl2
(20 mL) and filtered. Brine (20 mL) was added to the filtrate and
the phases were separated. The aqueous layer was extracted with
CH2Cl2 (3 Â 10 mL) and the organic fractions were collected, dried,
and evaporated. The crude solid residue was purified by flash cro-
matography (EtOAc/petroleum ether/AcOH 1:9:0.1) to give 10
(0.52 g, 78%), showing the same analytical and spectroscopic data
as the compound obtained by Method A.
A solution of 4 (0.45 g, 1.60 mmol) in THF (6 mL) was treated
with 1 N HCl (3 mL) and the solution stirred at room temperature
for 3 h. Saturated aqueous NaHCO3 solution (10 mL) was added,
and the mixture was extracted with EtOAc (3 Â 10 mL). The organ-
ic phases were combined and washed with brine (2 Â 100 mL). The
organic layer was dried and evaporated to dryness. The oily residue
was purified by flash chromatography (EtOAc/petroleum ether 4:6)
to furnish 5 (0.44 g, 98%) as a yellow oil. IR (neat): 1741, 1714,
1497, 1028 cmÀ1 1H NMR (200 MHz, CDCl3): d (ppm) 1.25 (t,
;
J = 7.0 Hz, 3H), 3.54 (s, 2H), 4.16 (q, J = 7.0 Hz, 2H), 5.36 (s, 1H),
7.23-7.36 (m, 10H); 13C NMR (50 MHz, CDCl3): d (ppm) 14.0,
48.6, 61.3, 64.0, 127.4, 127.6, 128.7, 137.3, 167.1, 200.7. Anal. Calcd
for C18H18O3: C, 76.57; H, 6.43. Found: C, 76.65; H, 6.41.
4.2.3. tert-Butyl (3-Oxo-4,4-diphenylbutanoyl)carbamate (11)
A solution of 10 (0.34 g, 1.00 mmol) and tert-butyl carbamate
(0.12 g, 1.00 mmol) in CH3CN (20 mL) was heated at reflux for
2 h. Evaporation of the solvent, followed by flash cromatography
of the residue (EtOAc/petroleum ether 1:2) gave 11 (0.29 g, 83%)
4.1.2. Method B
A stirred suspension of zinc dust (0.68 g, 10.40 mmol) in THF
(5 mL) was treated with MeSO3H (25
at reflux for 10 min. Ethyl bromoacetate (0.25 mL, 2.25 mmol)
was added dropwise until it turned green, then (1.00 g,
lL, 0.38 mmol) and heated
as
a white solid, mp 147 °C. IR (KBr): 3236, 1753, 1722,
3
1690 cmÀ1 1H NMR (200 MHz, CDCl3): d (ppm) 1.46 (s, 9H), 3.89
.
5.18 mmol) was added. Ethyl bromoacetate (1 mL, 9.02 mmol)
was successively added over a period of 30 min, then the reaction
mixture was heated for 30 min, cooled to 0 °C and treated with 3 N
HCl (5 mL). The solution was stirred for 3 h at room temperature,
then the solvent was concentrated in vacuo. The residue was ex-
tracted with EtOAc (3 Â 15 mL), the combined organic phases were
washed with brine (2 Â 100 mL) and dried. The solvent was evap-
orated, and the residue was purified by flash chromatography
(EtOAc/petroleum ether 4:6) to afford 5 (1.32 g, 90%), showing
the same analytical and spectroscopic data as the compound ob-
tained by Method A.
(s, 2H), 5.31 (s, 1H), 7.20–7.38 (m, 10H), 7.60 (br, 1H); 13C NMR
(50 MHz, CDCl3): d (ppm) 28.0, 50.4, 64.6, 83.1, 127.5, 128.8,
129.1, 137.3, 150.5, 172.2, 208.3. Anal. Calcd for C21H23NO4: C,
71.37; H, 6.56; N, 3.96. Found: C, 71.45; H, 6.55; N, 3.97.
4.3. 3-Oxo-4,4-diphenylbutanamide (6)
4.3.1. Method A
A solution of 5 (0.25 g, 0.88 mmol) in saturated methanolic
ammonia (5 mL) was stirred at room temperature for 24 h, then
the solvent was evaporated. Flash chromatography of the residue
(EtOAc/petroleum ether 1:2 increasing to 2:1) gave a colorless
oil, which was triturated with EtOAc/petroleum ether 1:10. After
some time, a solid started separating out. The obtained suspension
was stirred for about 30 min, then it was filtered to afford 6 (0.12 g,
54%) as a white solid, mp 85 °C. IR (KBr): 3418, 3180, 1715, 1668,
4.1.3. Diphenylacetyl chloride (8)
A solution of 7 (5.00 g, 23.60 mmol) in benzene (50 mL) was
treated with SOCl2 (6 mL, 82.25 mmol) and heated at reflux for
2 h. Evaporation of the solvent gave 8 (5.20 g, 96%) as a colorless
oil, which solidified on standing, mp 56–59 °C. IR (KBr): 3029,
1379cmÀ1 1H NMR (200 MHz, CDCl3): d (ppm) 3.52 (s, 2H), 5.23
;
1774, 1495, 1453 cmÀ1
.
1H NMR (200 MHz, CDCl3): d (ppm) 5.48
(s, 1H), 5.49 (br, 1H), 6.84 (br, 1H), 7.18-7.40 (m, 10H); 13C NMR
(50 MHz, CDCl3): d (ppm) 48.4, 65.1, 127.8, 129.0, 129.1, 136.9,
167.6, 204.5. Anal. Calcd for C16H15NO2: C, 75.87; H, 5.97; N,
5.53. Found: C, 76.05; H, 5.95; N, 5.54.
(s, 1H), 7.33–7.38 (m, 10H); 13C NMR (50 MHz, CDCl3): d (ppm)
68.7, 128.5, 128.7, 129.0, 136.2, 173.5. Anal. Calcd for C14H11ClO:
C, 72.89; H, 4.81. Found: C, 72.70; H, 4.82.

C. De Risi et al. / Bioorg. Med. Chem. 16 (2008) 9904–9910
9909
4.3.2. Method B
5.2. Preparation and superfusion of cortical slices
An ammonia gas-saturated THF solution (2 mL) was added to a
solution of 10 (0.20 g, 0.59 mmol) in CH3CN (10 mL). The reaction
mixture was vigorously stirred at 70 °C for 48 h, then the solvent
was evaporated. The residue was dissolved in EtOAc (30 mL) and
2 N HCl (20 mL) was added. The biphasic mixture was stirred at
room temperature for 15 min, the organic phase separated, washed
with brine (2 Â 10 mL) and evaporated. Flash chromatography of
the residue (EtOAc/petroleum ether 1:2 increasing to 2:1) gave a
colorless oil, which was processed as described in Method A to af-
ford 6 (73 mg, 49%), showing the same analytical and spectroscopic
data as the compound obtained by Method A.
Experiments were performed as previously described.20 Briefly,
the animals were killed, their brains promptly isolated and 400-
lm-thick slices (25–30 mg each) were obtained from both the left
andrightfrontalcorticesbyusingafreshtissuevibratome-likeappa-
ratus. The tissue was then allowed to equilibrate for 15 min at room
temperature in a Krebs solution (mM composition: NaCl 118.5, KCl
4.7, CaCl2 1.8, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 10) con-
tinuously bubbled with a mixture of 95% O2 plus 5% CO2. Thereafter,
the slices were incubated at 37 °C for 30 min in Krebs solution with
the addition of ascorbic acid 0.05 mM and disodium EDTA 0.03 mM
and containing 50 nM [3H]5-HT (specific activity 27.8 Ci/mmol, Du-
Pont NEN, Boston, MA). The slices were then rinsed, transferred into
oxygenatedsuperfusion chambers(0.45 mlvolume each;twoslices/
chamber) and superfused at a flow rate of 0.25 ml/min with Krebs
4.3.3. Method C
A solution of 11 (0.29 g, 0.82 mmol) in 3 N HCl solution in AcOEt
(10 mL) was stirred at room temperature for 3 h. The mixture was
kept to pH 9–10 by careful addition of saturated aqueous NaHCO3
solution and then extracted with EtOAc (3 Â 10 mL). The combined
organic layers were washed with brine (20 mL), dried and evapo-
rated. The resulting oil was treated as described in Method A to
give 6 (0.20 g, 98%), showing the same analytical and spectroscopic
data as the compound obtained by Method A.
solution containing the 5-HT uptake inhibitor paroxetine (3 lM).
After 30 min of superfusion, the release experiment started by col-
lecting superfused samples every 5 min from each chamber.
5.3. Experimental protocols: spontaneous and electrically-
evoked [3H]5-HT efflux
4.3.4. N,O-Bis-(tert-butoxycarbonyl)-N-hydroxy-3-oxo-4,4-
diphenylbutanamide (12)
Spontaneous [3H]5-HT efflux was determined in successive 5-
min fractions from the 30th to the 110th min of superfusion. After
the collection of three basal samples, modafinil was added to the
superfusion medium and maintained until the end of the
experiment.
N,O-Bis-(tert-butoxycarbonyl)hydroxylamine
(0.35 g,
1.50 mmol) was added to a solution of 10 (0.51 g, 1.50 mmol) in
benzene (10 mL) and the mixture was heated at 65 °C for 4 h. Evap-
oration of the solvent and flash chromatography of the residue
(EtOAc/hexanes 1:19) gave 12 (0.50 g, 71%) as a yellow oil. IR
For the experiment on electrically-stimulated slices, [3H]5-HT
overflow was evoked by electrical field stimulations (3 Hz,
40 mA, 2 msec, for 2 min) at the 45th (St1) and 85th (St2) min from
the onset of superfusion. The effects of modafinil, compound 6 and
compound 13 were evaluated by adding the drugs to the superfu-
sion medium 15 min before St2. At the end of the experiment, the
slices were solubilized in 1 ml of Soluene 350 (Packard Instru-
ments, Downers Grove, IL). The radioactivity of the 5-min samples
and slices was determined by liquid scintillation spectrometry.
;
(KBr): 1785, 1711, 1629 cmÀ1 1H NMR (200 MHz, CDCl3): d
(ppm) 1.49 (s, 9H), 1.52 (s, 9H), 3.95 (part of a AB system,
J = 16.0 Hz, 1H), 4.13 (part of a AB system, J = 16.0 Hz, 1H), 5.33
(s, 1H), 7.23–7.34 (m, 10H); 13C NMR (50 MHz, CDCl3): d (ppm)
27.5, 27.8, 27.9, 51.3, 64.1, 86.0, 86.4, 127.4, 128.6, 129.05, 137.3,
149.7, 150.8, 163.4, 200.2. Anal. Calcd for C26H31NO7: C, 66.51; H,
6.65; N, 2.98. Found: C, 66.60; H, 6.64; N, 2.97.
4.3.5. 3-Oxo-4,4-diphenyl-N-(propan-2-yl)-butanamide (13)
A solution of 5 (0.50 g, 1.77 mmol) in toluene (20 mL) contain-
ing isopropylamine (0.30 mL, 3.55 mmol) and 4-(dimethyl-
amino)pyridine (65 mg, 0.53 mmol) was heated at reflux for 20 h,
then the solvent was evaporated. The crude residue was purified
by flash chromatography (EtOAc/petroleum ether 1:2 increasing
to 2:1) to give a colorless oil, which was triturated with EtOAc/
petroleum ether 1:10. After some time, a solid started separating
out. The suspension was stirred for about 30 min, then it was fil-
tered to afford 13 (0.48 g, 92%) as white needles, mp 101 °C. IR
5.4. Data presentation and statistical analysis
The amount of tritium released per 5-min sample was calculated
either as pmol/min or as fraction of the total tissue tritium content at
thebeginningof therespective collectionperiod (fractionalrate). For
calculation of the net tritium overflow evoked by the electrical stim-
ulation, the estimated spontaneous [3H]-efflux (the latter was as-
sumed to decline linearly from the 5-min period before to that 15–
20 min after the onset of stimulation) was subtracted from the total
[3H]-efflux during stimulation and the subsequent 13 min; this dif-
ference wascalculatedas a percentageof the totaltissue tritiumcon-
tent at the onset of stimulation.20 The [3H]-overflow resulting from
the first stimulation (St1) has been used as an internal control for
the [3H]-overflow produced by the second stimulation (St2). The ef-
fectsof thedrugtested werequantifiedby evaluatingthechangesin-
ducedintheSt2/St1 ratioincomparisonwithcontrolslicesassayedin
parallel. The data were presented as mean SEM. The term [3H]5-HT
efflux was used as an index of 5-HT (and its metabolites) release.
(KBr): 3285, 1734, 1640, 1570, 1406 cmÀ1 1H NMR (200 MHz,
;
CDCl3): d (ppm) 1.11 (d, J = 6.6 Hz, 6H), 3.44 (s, 2H), 4.03 (m, 1H),
5.25 (s, 1H), 6.64 (br, 1H), 7.19–7.33 (m, 10H); 13C NMR (50 MHz,
CDCl3): d (ppm) 22.6, 40.0, 41.6, 64.9, 127.7, 129.0, 129.1, 137.1,
164.3, 205.1. Anal. Calcd for C19H21NO2: C, 77.26; H, 7.17; N,
4.74. Found: C, 77.35; H, 7.18; N, 4.76.
5. Biological experiments
5.1. Animals
Acknowledgments
Authors are grateful to MIUR (COFIN 2006) and Fondazione Cas-
sa di Risparmio di Ferrara for financial support.
Male adult Sprague–Dawley rats with a body weight of 300–
350 g were housed in cages in groups of five animals at a constant
room temperature (20 °C) and exposed to a 12 h:12 h light–dark
cycle (lights on at 06.00 a.m.). Food and water were provided ad
libitum. Following delivery, the animals were allowed to adapt to
the environment for at least 1 week before the experiment started.
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