Synthesis and biological evaluation of novel dimiracetam derivatives useful for the treatment of neuropathic pain

Article abstract of DOI:10.1016/j.bmc.2007.12.015

Chemical modifications of dimiracetam, a bicyclic analogue of the nootropic drug piracetam, afforded a small set of novel derivatives that were investigated in in vivo models of neuropathic pain. Compounds 5, 7 and 8 displayed a very promising antihyperalgesic profile in rat models of neuropathic pain induced by both chronic constriction injury of the sciatic nerve and streptozotocin. The compounds completely reverted the reduction of pain threshold evaluated by the paw pressure test. Importantly these derivatives did not induce any behavioural impairment as evaluated by the rotarod test. These results suggest that compounds 5, 7 and 8 might represent novel and well-tolerated therapeutic agents for the relief of neuropathic pain.

Full text of DOI:10.1016/j.bmc.2007.12.015

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3224–3232
Synthesis and biological evaluation of novel dimiracetam
derivatives useful for the treatment of neuropathic pain
Carlo Farina,a Stefania Gagliardi,b Carla Ghelardini,c Marisa Martinelli,b,*
Monica Norcini,c Carlo Parini,b Paola Petrillob and Silvano Ronzonia
aBrane Discovery, Insubrias Biopark, Via Lepetit 34, 21040 Gerenzano, Varese, Italy
bNiKem Research, Medicinal Chemistry, Via Zambeletti 25, 20021 Baranzate, Milano, Italy
`
Dipartimento di Farmacologia e Clinica, Universita di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy
c
Received 19 September 2007; revised 6 December 2007; accepted 11 December 2007
Available online 15 December 2007
Abstract—Chemical modifications of dimiracetam, a bicyclic analogue of the nootropic drug piracetam, afforded a small set of novel
derivatives that were investigated in in vivo models of neuropathic pain. Compounds 5, 7 and 8 displayed a very promising antihy-
peralgesic profile in rat models of neuropathic pain induced by both chronic constriction injury of the sciatic nerve and streptozo-
tocin. The compounds completely reverted the reduction of pain threshold evaluated by the paw pressure test. Importantly these
derivatives did not induce any behavioural impairment as evaluated by the rotarod test. These results suggest that compounds 5,
7 and 8 might represent novel and well-tolerated therapeutic agents for the relief of neuropathic pain.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Me
1. Introduction
H
N
R
N
O
N
O
Neuropathic pain (NeP) is defined as chronic, persistent
pain mostly caused by peripheral or central neural in-
jury.1 Unlike physiological pain it serves no useful pur-
pose and is usually sustained and chronic. The
characteristic symptoms of neuropathic pain are usually
expressed as allodynia, hyperalgesia and spontaneous
pain. The majority of the currently available treatments
for NeP are not adequate: tricyclic antidepressants, anti-
convulsants and opioids are only partially effective and/
or are associated with significant side effects.2 Even
gabapentin, the gold standard for the treatment of
NeP, reduces pain by 30–50% at best in less than 50%
of patients.3 Therefore, safer and more effective treat-
ments for neuropathic pain need to be developed and
significant efforts must be applied to the discovery of no-
vel drug molecules able to alleviate this intractable pain.
N
NH
O
O
O
NH2
O
Me
R = H, Piracetam (Nootropil R
)
Nefiracetam
Dimiracetam
R = Et, Levetiracetam (KeppraR
)
Figure 1. Nootropic drugs.
that some nootropic drugs such as nefiracetam or leveti-
racetam may be useful in treating neuropathic pain both
in animals and in patients.
The validation of nootropic pyrrolidinones in neuro-
pathic pain is noteworthy. For example, nefiracetam, a
Piracetam derivative, featuring a lipophilic anilide moi-
ety, dose dependently reversed the thermal or mechani-
cal hyperalgesia induced by partial sciatic nerve ligation
or streptozotocin treatment in mice.5 Levetiracetam, an
anticonvulsant drug targeting the synaptic vesicle pro-
tein SV2A, was recently shown to be effective in patients
with chronic neuropathic pain, particularly when treat-
ment with other anticonvulsant agents failed.6
Nootropic drugs comprise a series of derivatives of c-
aminobutyric acid (GABA) whose prototype, pirace-
tam, is used in diseases characterized by learning and
memory deficits (Fig. 1).4 Recent studies have shown
Keywords: Neuropathic pain; CCI; Streptozocin; Nootropic drugs.
*
The analgesic action induced by this class of derivatives
was non-opioid in nature, as it was not reversed by the
Corresponding author. Tel.: +39 0 2356947342; fax: +39 0 2356947606;
e-mail: marisa.martinelli@nikemresearch.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.015

C. Farina et al. / Bioorg. Med. Chem. 16 (2008) 3224–3232
3225
Table 1. Chemical and physical characteristics of compounds 2–38
opioid antagonist naloxone. Together, these findings
suggest that nefiracetam, and other cognition enhancers
of the same pyrrolidinone nootropic class, could be
good therapeutic tools against neuropathic pain, having
no liabilities due to possible involvement of the opioid
system, and an exceptional safety profile.7
O
N
N
R
O
Compound
R
H
Mp (ꢁC)
Elemental analysis
12H12N2O2
Thus, taking nefiracetam as the model drug, we contin-
ued the research in this field with the aim of finding com-
pounds endowed with increased potency, efficacy and
with a good safety profile. Dimiracetam,8 a bicyclic pyr-
rolidinone analogue of piracetam, was developed as a
novel cognition enhancer until Phase I, before its devel-
opment was discontinued. This compound, which main-
tains the backbone of piracetam with the acetamide side
chain restricted in a folded conformation, was 10–100
times more potent than piracetam and, as for the other
related derivatives, practically devoid of toxicity.
2
3
185–188
138–139
92–93
C
2-Me
3-Me
C13 H14 N2O2
C13 H14 N2O2
C13 H14 N2O2
C13H11N3O2
C13H11N3O2
C13H11N3O2
C13H11F3N2O2
C13H11F3N2O2
C13H11F3N2O2
C12H11FN2O2
C12H11FN2O2
C12H11ClN2O2
C12H11ClN2O2
C15H18N2O2
C15H18N2O2
C13H14N2O3
C13H14N2O3
C12H12N2O3
C13H14N2O4S
C14H16N2O2
C13H14N2O3
C15H16N2O4
C13H12N2O4
C12H10F2N2O2
C14H16N2O2
C14H16N2O2
C13H13FN2O2
C13H13FN2O2
C13H13FN2O2
C13H13ClN2O2
C13H13FN2O2
C13H13ClN2O2
C13H13FN2O2
C13H13FN2O2
C13H13ClN2O2
C13H13FN2O2
4
5
4-Me
2-CN
117–118
94–95
6
7
3-CN
4-CN
128–130
175–176
95–97
8
9
2-CF3
3-CF3
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
124–125
124–125
148–149
158–159
123–125
159–160
81–82
4-CF3
3-F
4-F
3-Cl
In the present study, a series of variously substituted
dihydro-1H-pyrrolo[1,2-a]imidazole-2,5(3H,6H)-diones
2–38, derivatives of 1, were prepared and their antihy-
peralgesic effect was investigated in several models of
neuropathic pain in rats.
4-Cl
3-i-Pr
4-i-Pr
124–125
94–95
3-OMe
4-OMe
3-OH
210–211
210–211
143–145
98–99
4-SO2Me
4-Et
2. Chemistry
4-CH2OH
4-COOEt
4-COOH
3,5-diF
3,4-Me
3,5-Me
2-F-5-Me
3-F-4-Me
2-Me-3-F
2-Me-3-Cl
2-Me-4-F
2-Me-4-Cl
2-Me-5-F
3-Me-4-F
3-Me-4-Cl
3-Me-5-F
159–161
158–159
252–253
200
Substituted aromatic rings were inserted into the dimi-
racetam structure through a microwave-enhanced Gold-
berg reaction, allowing the synthesis of a small set of 37
compounds (Scheme 1).9
142–143
103–104
96–97
Microwave irradiation greatly accelerates this copper-
catalysed arylation of the secondary amide. Compounds
2–38 (Table 1) were quickly obtained by irradiation of a
mixture of dimiracetam and the corresponding aryl io-
dides or bromides, using NMP as solvent, in the pres-
ence of CuI as catalyst, in a microwave reactor at
150 ꢁC for about 20 min. Notwithstanding the moderate
yields obtained (ranging from 20% to 60%), the de-
scribed procedure is time and energy-saving affording
substantial amounts of the desired derivatives in a short
timeframe. Moreover, after reaction work-up, the final
compounds were obtained without any additional puri-
fication and with purities sufficient for in vivo biological
screening.10
135–137
135–137
142–144
105–107
138–139
147–148
107–109
138–139
111–113
the reaction times were much longer (i.e., 5 h was neces-
sary to get complete conversion into compound 5 vs
20 min of the MW irradiation).
As during the continuation of our study, larger amounts
of selected compounds were required for biological and
toxicological studies, scaling-up of the Goldberg reac-
tion was achieved by using DMF as a solvent and warm-
ing the reactions by conventional heating. The yields
obtained using oil-bath heating were comparable, while
Both enantiomers of the three selected compounds were
synthesized in order to investigate the potential presence
of a more active eutomer (Scheme 2). (S) and (R) methyl
5-oxo-pyrrolidin-2-carboxylate were used as enantio-
pure precursors and were alkylated with tert-butyl-bro-
moacetate to give, respectively, compounds 40a and
40b. Transamidation with ammonia afforded the pri-
mary amides that were readily converted into the corre-
sponding amines by Hofmann-type rearrangement with
the hypervalent iodine compound iodosobenzene–bistri-
fluoroacetate.11 Acidic hydrolysis of tert-butyl esters
and final cyclizations gave the (+)-(7aS) and (À)-(7aR)
dimiracetam (43a, [a]D + 39.9 (c = 0.4, MeOH), and
43b [a]D = À39.2 (c = 0.4, MeOH), respectively).
a
O
O
N
NH
O
N
N
R
2-38
1
O
Scheme 1. (a) ArI or ArBr, CuI, K2CO3, NMP, MW, 150 ꢁC, 20 min.

3226
C. Farina et al. / Bioorg. Med. Chem. 16 (2008) 3224–3232
45 and 60 min). Based on their initial efficacy, the most
b
a
O
O
CONH2
CH2COOtBu
promising compounds were tested further at lower con-
centrations and the results obtained for the minimal
dose giving a statistical significant effect (p < 0.01) are
reported in Table 3. The results represent means SEM
of the mechanical threshold expressed as grams. Com-
pounds were tested in different experiments. In vivo test-
ing facilitates the selection of the compounds with the
most promising clinical properties but does not allow
establishing robust structure–activity relationships be-
cause the resulting activity is a consequence of the over-
all compound properties that can be significantly
modulated even by small structural modifications. Seven
derivatives showed good antihyperalgesic activity. Com-
pounds 4, 5, 8 (10 lg/kg, i.c.v.) and 2, 3, 7 and 16 (30 lg/
kg, i.c.v.) exhibited a dose dependent antihyperalgesic
effect when compared to the animal group treated with
saline. The pain threshold in contralateral non-operated
paw was not modified in all the experiments. Impor-
tantly, at the dose of 10 lg/kg (30–45 min post-dosing),
compounds 4, 5 and 8 did increase the pressure thresh-
old of the lesioned paw to the same level of the contra-
lateral paw and were about 3-fold more effective than
gabapentin or nefiracetam used as reference standards.
COOMe
CH2COOtBu
N
O
N
COOMe
N
H
39a (S)
39b (R)
40a (S)
40b (R)
41a (S)
41b (R)
c
7a
d
.HCl
NH2
e
O
O
N
NH
N
N
O
N
R
CH2COOH
42a (S)
42b (R)
O
O
43a (S)
43b (R)
3S-3R
5S-5R
8S-8R
Scheme 2. (a) NaH, BrCH2COOtBu; (b) 32% NH4OH; (c) PIFA then
TFA, HCl; (d) Ac2O, AcONa and then H+; (e) ArI or ArBr, CuI,
K2CO3, NMP.
Table 2. Chemical and physical characteristics of compounds 3–5 (R
and S pure enantiomers)
7a
O
N
N
R
O
Compound 7a
R
Mp (ꢁC) [a]D in MeOH Elemental
analysis
Since all the tested derivatives were racemic, both enan-
tiomers of 3, 5 and 8 were synthesized. All of these com-
pounds displayed similar efficacy to the racemate in the
animal models of neuropathic pain (data not shown),
suggesting that the enantioselection is not important
for the mechanism of action. We then focused our atten-
tion on compounds 5, 7 and 8 which were tested on
nerve injury-induced hyperalgesia in the rat paw pres-
sure test after po administration (Fig. 2). Compound 5
increased the pressure threshold of the lesioned paw to
the same level of the contralateral paw at a dose of
30 mg/kg po for 30–60 min post-dosing but not at a dose
of 10 mg/kg. Compounds 7 and 8 produced a statisti-
cally significant antihyperalgesic effect at doses of 30
and 50 mg/kg po, respectively. At the highest dose tested
(100 mg/kg po), both compounds completely reverted
hyperalgesia (30–60 min post-treatment). All the com-
pounds tested did not induce any significant effects on
the mechanical threshold of the non-lesioned paw at
all the doses and times tested.
3S
3R
5S
5R
8S
8R
S
2-Me 138–139 À51.3 (c = 0.4)
C13H14N2O2
R
S
138–139 +52.2 (c = 0.4) C13H14N2O2
4-Me 146–148 À134.3 (c = 0.4) C13H14N2O2
144–146 +134.0 (c = 0.4) C13H14N2O2
R
S
4-CN 208–210 À133.2 (c = 0.3) C13H11N3O2
184–186 +133.7 (c = 0.3) C13H11N3O2
R
Finally, compounds 3–5 (R and S pure enantiomers),
whose chemical and physical characteristics are reported
in Table 2, were synthesized under the well-established
experimental condition described before.
3. Pharmacology
The lack of a well-known mechanism of action for race-
tam derivatives prevents primary in vitro screening and
the selection of active compounds generally relies on
in vivo testing. All the compounds prepared were tested
on the Bennett and Xie12 model of neuropathic pain
after i.c.v. injection. This route of administration was
preferred to obtain a ‘pure’ measurement of efficacy,
avoiding the influence of other parameters that could
prevent an active compound from being detected (e.g.,
poor absorption and/or poor blood brain barrier pene-
tration). The neuropathy was induced by right sciatic
nerve ligation. Fourteen days after the operation a sig-
nificant reduction of the pain threshold was established
in the injured paw. By contrast, in the contralateral paw,
the pain perception remained unchanged. Paw with-
drawal threshold was measured using a Randall and
Selitto apparatus exerting a force that increases at con-
stant rate (32 g/s). Stimulus at which rats withdrew the
paw was recorded before treatment and after drug
administration (100 lg/kg, i.c.v.) at different times (30,
In rodents the administration of the pancreatic toxin
streptozotocin causes both mechanical and thermal
hyperalgesia, possibly by mimicking diabetic neuropa-
thy. Rats were injected ip with 50 mg/kg streptozotocin
and tested for mechanical hyperalgesia in the paw pres-
sure test 21 days after toxin treatment. In this model,
compound 5 produced a statistically significant increase
of the paw mechanical threshold at 50 and 100 mg/kg
after oral administration (Fig. 3. At 100 mg/kg its effi-
cacy was comparable to gabapentin given at the same
dose). In the same model, orally administered com-
pounds 7 and 8 (30–100 mg/kg), increased in a dose
dependent manner the paw pressure threshold reaching
statistical significance at 100 mg/kg with slightly reduced
efficacy compared to that of gabapentin (data not
shown). The compounds did not induce any significant
effect in control animals. Furthermore, in the range of

C. Farina et al. / Bioorg. Med. Chem. 16 (2008) 3224–3232
3227
Table 3. Antihyperalgesic effect of 1–5,7, 8 and 16 in a rat model of neuropathic pain evaluated in paw pressure test
Compound
R
Minimal effective
dose lg/kg (i.c.v.)
Paw
Paw pressure in rats
After analgesic treatment
Before treatment
30 min
45 min
60 min
Vehicle
—
Contra
Les
Les
59.1 3.7
29.3 3.5
24.5 4.7
22.4 + 3.5
53.2 + 3.2
22.1 3.9
23.6 4.1
25.1 2.4
27.6 3.5
24.2 2.8
60.1 3.8
28.6 3.1
28.5 3.5
32.0 3.0
60.7 4.7
29.3 3.2
28.5 2.9
59.6 3.7
30.6 2.9
31.7 2.7
60.7 4.7
29.3 3..2
25.4 2.7
62.6 4.8
27.1 4.2
46.3 4.2*
51.8 6.8*
53.4 + 4.7
23.7 4.4*
46.7 4.5*
41.0 4.5*
51.7 5.2*
46.7 5.1*
57.4 4.7
25.2 2.8
59.3 5.3*
54.0 5.3*
64.5 6.3
28.1 3.5
49.7 5.2*
62.9 5.0
33.5 3.6
59.4 5.8*
64.5 6.3
28.1 3.5
48.5 4.6*
57.3 3.3
25.9 5.1
43.5 6.1*
52.1 4.9*
51.9 + 5.5
27.6 4.5*
49.5 5.2*
42.8 2.5*
52.7 4.6*
55.3 6.3*
62.2 4.7
28.3 3.5
63.4 6.0*
57.4 4.3*
62.9 5.6
31.7 3.9
55.4 4.7*
60.3 4.4
32.2 3.5
55.3 5.3*
62.9 5.6
31.7 3.9
43.7 5.0
59.4 4.4
28.8 3.9
46.1 5.5*
49.3 4.2*
56.4 + 5.1
28.5 4.5*
47.6 5.0*
39.2 2.5*
53.3 3.2*
54.8 3.6*
58.4 4.4
33.2 4.2
51.2 5.3*
45.4 3.6*
60.4 4.8
26.2 4.2
46.8 5.3*
57.8 3.5
35.6 4.1
58.2 6.2*
60.4 4.8
26.2 4.0
42.5 4.6
Gabapentin
Levetiracetam
Vehicle
—
—
30
500
Les
Contra
Les
Les
Nefiracetam
1 (dimiracetam)
30
100
30
Les
Les
2
H
3
Vehicle
2-Me
—
30
Les
Contra
Les
Les
4
3-Me
4-Me
—
10
10
5
Vehicle
Les
Contra
Les
Les
7
Vehicle
3-CN
—
30
10
30
Contra
Les
Les
8
Vehicle
4-CN
—
Contra
Les
Les
16
3-i-Pr
There were 8–14 rats per group. Les, lesioned; Contra, contralateral.
* p < 0.01 versus the saline lesioned dx paw.
antihyperalgesic doses (100 mg/kg po) compounds 5 and
7 did not produce any behavioural impairment as eval-
uated by the rat rotarod test (data not shown).14
(TLC) was performed using Merck silica gel 60 F254
precoated plates. Flash chromatography was performed
using Merck silica gel 60, 230–400 mesh. The reported
yields are unoptimized. Melting points were determined
on a Buchi melting point B545 apparatus and are uncor-
rected. 1H NMR spectra were recorded on a Bruker AC
300. Mass spectra were recorded TSQ 700 instrument
(Thermo-Finnigan), in EI mode with an external probe
(DIS). Elemental analyses were within 0.4% of the theo-
retical value for the indicated elements.
4. Conclusion
Microwave assisted Goldberg reaction allowed rapid
insertion of substituted phenyl rings onto a dimiracetam
scaffold. Compounds 5, 7 and 8 dose dependently re-
verted mechanical hyperalgesia in sciatic nerve-ligated
rats and streptozotocin-treated animals after i.c.v. and
oral administration without inducing any motor impair-
ment. Interestingly the action of these compounds is
neuropathy-specific since no significant activity is de-
tected on the unlesioned contralateral paw in mononeu-
ropathic and in control rats.
The microwave-enhanced Goldberg reactions were per-
formed on a CEM discover microwave apparatus,
equipped with an internal probe that monitors reaction
temperature and maintains the desired temperature by
computer control.
5.1.1. General procedure for dimiracetam arylation with
aryl halides.
All the data suggest that these compounds represent a
novel class of well-tolerated therapeutic agents for the
relief of neuropathic pain. Additional work is in pro-
gress to gain more information on their mechanism of
action and activity profile in other models of neuropath-
ic pain.
5.1.1.1. Method A (MW heating). To a solution of
dimiracetam (1, 0.5 g, 3.5 mmol),8a in N-methylpyrroli-
done (NMP 1 ml), CuI (0.19 g, 1 mmol), K2CO3 (0.5 g,
3.5 mmol) and aryl halide (7 mmol) were added under
stirring. The suspension was heated in a microwave
apparatus at 150 ꢁC for 20 min. Ethyl acetate (50 ml)
and water (5 ml) were added to the suspension and the
mixture was stirred for 30 min the presence of Celite.
The reaction mixture was filtered and the organic phase
was washed with brine, dried over Na2SO4, filtered and
concentrated to dryness. The residue was triturated with
Et2O to give the desired final compounds. CAUTION:
The microwave-enhanced Goldberg reaction should be
performed in an open reaction system in order to enable
5. Experimental
5.1. Chemistry
Reagents obtained from commercial sources were used
without further purification. In order to monitor the
progress of the reaction, thin layer chromatography

3228
C. Farina et al. / Bioorg. Med. Chem. 16 (2008) 3224–3232
Compound 5
Compound 5
Contralateral paw
Lesioned paw
Control
Diabetic
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
80
80
70
60
50
40
30
20
*
70
*
*
60
*
*
*
50
40
30
*
^
*
Before
treatment
Before
treatment
20
30
45
60
75
90
30
45
60
75
90
Before
Before
treatment
20
30
40
50
60
20
30
40
50
60
Time [min]
treatment
Time [min]
Time min
Time min
Compound 7
Compound 7
Contralateral paw
Lesioned paw
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
Control
Diabetic
*
80
70
60
50
40
30
20
80
70
60
50
40
30
20
*
*
*
*
*
*
Before
treatment
Before
treatment
30
45
Time [min]
60
Before
treatment
Before
treatment
20
30
40
50
60
30
45
60
20
30
40
50
60
Time min
Time [min]
Time min
Compound 8
Compound 8
Contralateral paw
Lesioned paw
80
70
60
50
40
30
20
10
0
Control
Diabetic
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
80
70
60
50
40
30
20
*
*
*
*
*
*
Before
treatment
Before
treatment
30
45
60
Before
treatment
Before
treatment
30
45
60
20
30
40
50
60
20
30
40
50
60
Time [min]
Time [[min]
Time min
Time min
vehicle
50 mg/kg
100 mg/kg
vehicle
50 mg/kg
10 mg/kg
30 mg/kg
30 mg/kg
100 mg/kg
Figure 3. Compounds 5, 7 and 8 after po administration in strepto-
zotocin-induced hyperalgesia in the rat paw pressure test. Pain
threshold is reported as the force inducing the first struggling from
Figure 2. Compounds 5, 7 and 8 after po administration in nerve
injury-induced hyperalgesia in the rat paw pressure test. Pain threshold
is reported as the force inducing the first struggling from the rat (g).
the rat (g). Data are means SEM of 9–10 rats. p < 0.05 and *p < 0.01,
*
ˆ
statistically significant from vehicle administration.
Data are means SEM of 6–11 rats. p < 0.01, statistically significant
from vehicle administration. Test was performed 14 days after sciatic
nerve ligation.
J = 16.7 Hz, 1H), 2.57–2.82 (m, 2H), 2.36–2.54 (m,
1H), 1.92–2.16 (m, 1H). MS: 216 (M)+, 160, 97.
gaseous products to escape, thus avoiding the risk of
explosion.
1
5.1.3. Compound 3. Yield: 45%. H NMR (CDCl3) d:
5.1.1.2. Method B (conventional heating). To a solu-
tion of dimiracetam (1, 0.5 g, 3.5 mmol),8a in DMF
(5 ml), CuI (0.19 g, 1 mmol), K2CO3 (0.5 g, 3.5 mmol)
and aryl halide (7 mmol) were added under stirring.
The suspension was refluxed for 5 h. The solvent was re-
moved under vacuum and the crude was portioned be-
tween ethyl acetate and water. The organic layer was
filtered over Celite and the filtrate was washed with
10% HCl, then with a saturated solution of NaHCO3
and finally with brine. The organic layer was dried over
Na2SO4, filtered and concentrated to dryness. The resi-
due was triturated with Et2O to give the desired final
compounds.
7.20–7.39 (m, 3H), 7.03–7.14 (m, 1H), 5.42–5.75 (m,
1H), 4.44 (d, J = 16.7 Hz, 1H), 3.75 (d, J = 16.4 Hz,
1H), 2.67 (dt, J = 17.0, 9.8 Hz, 1H), 2.46 (dd d,
J = 17.3, 10.1, 3.1 Hz, 1H), 2.33 (dd dd, J = 16.2, 6.5,
6.4, 3.0 Hz, 1H), 2.24 (s, 3H), 1.79–2.09 (m, 1H). MS:
230 (M)+, 143, 118, 97.
1
5.1.4. Compound 4. Yield: 40%. H NMR (CDCl3) d:
7.30 (dd, J = 7.9 Hz, 1H), 7.24 (br s, 1H), 7.13 (dd,
J = 8.2, 1.3 Hz, 1H), 7.06 (d, J = 7.6 Hz, 1H), 5.76–
5.85 (m, 1H), 4.47 (d, J = 16.4 Hz, 1H), 3.73 (d,
J = 16.4 Hz, 1H), 2.54–2.80 (m, 2H), 2.40–2.53 (m,
1H), 2.38 (s, 3H), 1.91–2.13 (m, 1H). MS: 230 (M)+,
174, 118.
1
5.1.2. Compound 2. Yield: 20%. H NMR (CDCl3) d:
7.35–7.50 (m, 4H), 7.18–7.32 (m, 1H), 5.84 (t,
J = 5.8 Hz, 1H), 4.48 (d, J = 16.7 Hz, 1H), 3.74 (d,
1
5.1.5. Compound 5. Yield: 62%. H NMR (CDCl3) d:
77.30 (m, 2H), 7.24 (m, 2H), 5.76–5.87 (m, 1H), 4.48

C. Farina et al. / Bioorg. Med. Chem. 16 (2008) 3224–3232
3229
(d, J = 16.4 Hz, 1H), 3.75 (d, J = 16.4 Hz, 1H), 2.56–
2.83 (m, 2H), 2.41–2.55 (m, 1H), 2.37 (s, 3H), 1.94–
2.12 (m, 1H). 13C NMR (CDCl3) d: 177.3 (CO); 169.1
(CO); 136.6 (Ar); 133.0 (Ar); 130.4 (Ar); 122.1 (Ar),
75.9 (C7a); 47.8 (C3); 30.7 (C6); 28.2 (C7); 21.4 (CH3).
MS: 230 (M)+, 174, 118.
1H), 5.81 (t, J = 5.5 Hz, 1H), 4.49 (d, J = 16.7 Hz,
1H), 3.74 (d, J = 16.7 Hz, 1H), 2.60–2.79 (m, 2H),
2.36–2.56 (m, 1H), 1.84–2.14 (m, 1H). MS: 250 (M)+,
194, 138, 111.
1
5.1.15. Compound 15. Yield: 44%. H NMR (CDCl3) d:
7.37 (m, 4H), 5.80 (t, J = 6.0 Hz, 1H), 4.47 (d,
J = 16.7 Hz, 1H), 3.73 (d, J = 16.7 Hz, 1H), 2.57–2.83
(m, 2H), 2.36–2.54 (m, 1H), 1.89–2.11 (m, 1H). MS:
250 (M)+, 194.
1
5.1.6. Compound 6. Yield: 20%. H NMR (CDCl3) d:
7.79 (dd, J = 7.9, 1.5 Hz, 1H), 7.72 (td, J = 7.9, 1.5 Hz,
1H), 7.45–7.54 (m, J = 7.6, 7.6, 1.2 Hz, 1H), 7.40 (dd,
J = 8.2, 0.9 Hz, 1H), 5.89–6.03 (m, 1H), 4.52 (d,
J = 16.7 Hz, 1H), 3.83 (dt, J = 16.7, 1.0 Hz, 1H), 2.63–
2.82 (m, 1H), 2.40–2.60 (m, 2H), 1.92–2.13 (m, 1H).
MS: 242.22 (MH)+.
1
5.1.16. Compound 16. Yield: 23%. H NMR (CDCl3) d:
7.04–7.43 (m, 4H), 5.83 (t, J = 5.8 Hz, 1H), 4.47 (d,
J = 16.7 Hz, 1H), 3.74 (d, J = 16.7 Hz, 1H), 2.93 (spt,
J = 7.2 Hz, 1H), 2.56–2.80 (m, 2 H), 2.36–2.56 (m,
1H), 1.93–2.18 (m, 1H), 1.26 (d, J = 6.9 Hz, 6H). MS:
259 (MH)+.
1
5.1.7. Compound 7. Yield: 31%. H NMR (CDCl3) d:
7.67–7.80 (m, 2H), 7.47–7.61 (m, 2H), 5.86 (t,
J = 5.7 Hz, 1H), 4.53 (d, J = 17.0 Hz, 1H), 3.77 (d,
J = 17.0 Hz, 1H), 2.67–2.86 (m, 2H), 2.39–2.57 (m,
1H), 1.92–2.14 (m, 1H). MS: 241 (M)+, 185, 129.
5.1.17. Compound 17. Yield: 33%. 1H NMR (CDCl3) d:
7.20–7.36 (m, 4H), 5.80 (t, J = 6.0 Hz, 1H), 4.47 (d,
J = 16.4 Hz, 1H), 3.73 (d, J = 16.4 Hz, 1H), 2.91 (spt,
J = 6.9 Hz, 1H), 2.55–2.81 (m, 2H), 2.37–2.53 (m, 1H),
1.90–2.18 (m, 1H), 1.25 (d, J = 6.9 Hz, 6H). MS: 259.1
(MH)+.
1
5.1.8. Compound 8. Yield: 55%. H NMR (CDCl3) d:
7.73 (m, 2H), 7.60 (m, 2H), 5.75–5.98 (m, 1H), 4.54 (d,
J = 16.7 Hz, 1H), 3.77 (d, J = 17.0 Hz, 1H), 2.65–2.91
(m, 2H), 2.41–2.62 (m, 1H), 1.94–2.17 (m, 1H). MS:
242.2 (MH)+.
1
5.1.18. Compound 18. Yield: 63%. H NMR (CDCl3) d:
7.44–7.75 (m, 4H), 5.88 (t, J = 5.5 Hz, 1H), 4.51 (d,
J = 16.7 Hz, 1H), 3.82 (s, 3H), 3.76 (d, J = 16.7 Hz,
1H), 2.61–2.91 (m, 2H), 2.35–2.57 (m, 1H), 1.88–2.16
(m, 1H). MS: 246 (M)+, 190.
1
5.1.9. Compound 9. Yield: 20%. H NMR (CDCl3) d:
7.81 (dd, J = 7.9, 1.6 Hz, 1H), 7.67 (t, J = 7.2 Hz, 1H),
7.47–7.62 (m, 1H), 7.26–7.34 (m, 1H), 5.65 (t,
J = 5.7 Hz, 1H), 4.27–4.56 (m, 1H), 3.64–3.88 (m, 1H),
2.57–2.78 (m, 1H), 2.40–2.57 (m, 1H), 2.19–2.39 (m,
1H), 1.87–2.19 (m, 1H). MS: 284 (M)+, 228; 192, 172,
145.
1
5.1.19. Compound 19. Yield: 25%. H NMR (CDCl3) d:
7.16–7.41 (m, 2H), 6.95 (m, 2H), 5.74 (t, J = 6.0 Hz,
1H), 4.45 (d, J = 16.4 Hz, 1H), 3.81 (s, 3H), 3.73 (d,
J = 16.7 Hz, 1H), 2.35–2.84 (m, 3H), 1.86–2.14 (m,
1H). MS: 246 (M)+, 190, 134.
1
5.1.10. Compound 10. Yield: 35%. H NMR (CDCl3) d:
7.44–7.75 (m, 4H), 5.88 (t, J = 5.5 Hz, 1H), 4.51 (d,
J = 16.7 Hz, 1H), 3.76 (d, J = 16.7 Hz, 1H), 2.61–2.91
(m, 2H), 2.35–2.57 (m, 1H), 1.88–2.16 (m, 1H). MS:
284 (M)+, 228, 172, 145.
5.1.20. Compound 20. Yield: 35%. 1H NMR (CDCl3)
d: 77.16 (t, J = 8.2 Hz, 1H), 6.96 (t, J = 2.2 Hz,
1H), 6.75 (dd, J = 7.9, 1.9 Hz, 1H), 6.69 (dd, 1H),
5.74 (t, J = 5.7 Hz, 1H), 4.38 (d, J = 16.4 Hz, 1H),
3.67 (d, J = 16.4 Hz, 1H), 2.49–2.76 (m, 2H), 2.28–
2.49 (m, 1H), 1.83–2.11 (m, 1H). MS: 232 (M)+,
176.
1
5.1.11. Compound 11. Yield: 51%. H NMR (CDCl3) d:
7.68 (m, 2H), 7.57 (m, 2H), 5.88 (dd, J = 6.0, 5.3 Hz,
1H), 4.51 (d, J = 16.7 Hz, 1H), 3.75 (d, J = 16.7 Hz,
1H), 2.63–2.84 (m, 2H), 2.35–2.59 (m, 1H), 1.92–2.15
(m, 1H). MS: 284 (M)+, 228; 172; 145.
1
5.1.21. Compound 21. Yield: 25%. H NMR (CDCl3) d:
7.98 (d, 2H), 7.65 (d, J = 8.8 Hz, 2H), 5.90 (t, J = 5.2 Hz,
1H), 4.52 (d, J = 17.0 Hz, 1H), 3.77 (d, J = 16.7 Hz, 1H),
3.05 (s, 3H), 2.61–2.91 (m, 2H), 2.31–2.60 (m, 1H), 1.83–
2.16 (m, 1H). MS: 295.1 (MH)+.
1
5.1.12. Compound 12. Yield: 15%. H NMR (CDCl3) d:
7.38 (td, J = 8.2, 6.3 Hz, 1H), 7.29 (dt, J = 10.4, 2.2 Hz,
1H), 7.15 (dd d, J = 8.2, 2.0, 0.8 Hz, 1H), 6.88–7.02 (m,
J = 8.2, 8.2, 2.4, 0.9 Hz, 1H), 5.81 (t, J = 5.5 Hz, 1H),
4.49 (d, J = 16.7 Hz, 1H), 3.74 (d, J = 16.7 Hz, 1H),
2.59–2.87 (m, 2H), 2.33–2.57 (m, 1 H), 1.91–2.16 (m,
1H). MS: 235.1 (MH)+.
1
5.1.22. Compound 22. Yield: 46%. H NMR (CDCl3) d:
7.27 (m, 4H), 5.80 (t, J = 6.0 Hz, 1H), 4.46 (d,
J = 16.4 Hz, 1H), 3.74 (d, J = 16.4 Hz, 1H), 2.65 (q,
J = 7.6 Hz, 2H), 2.55–2.80 (m, 2H), 2.39–2.52 (m, 1H),
1.90–2.14 (m, 1H), 1.11–1.32 (m, 3H). MS: 245.1
(MH)+.
1
5.1.13. Compound 13. Yield: 10%. H NMR (CDCl3) d:
(CDCl3) d: 7.37 (m, 2H), 7.12 (m, 2H), 5.78 (t,
J = 5.7 Hz, 1H), 4.47 (d, J = 16.7 Hz, 1H), 3.74 (d,
J = 16.7 Hz, 1H), 2.55–2.86 (m, 2H), 2.37–2.54 (m,
1H), 1.88–2.10 (m, 1H). MS: 235.1 (MH)+.
1
5.1.23. Compound 23. Yield: 48%. H NMR (CDCl3) d:
7.37–7.50 (m, 4H), 5.81–5.91 (m, 1H), 4.72 (s, 2H), 4.49
(d, J = 16.7 Hz, 1H), 3.76 (d, J = 16.7 Hz, 1H), 2.58–
2.83 (m, 2H), 2.39–2.55 (m, 1H), 1.94–2.13 (m, 1H),
1.56 (br s, 1H). MS: 247.2 (MH)+.
1
5.1.14. Compound 14. Yield: 65%. H NMR (CDCl3) d:
7.44–7.49 (m, 1H), 7.28–7.39 (m, 2H), 7.16–7.25 (m,

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C. Farina et al. / Bioorg. Med. Chem. 16 (2008) 3224–3232
1
5.1.24. Compound 24. Yield: 62%. H NMR (CDCl3) d:
7.96–8.22 (m, 2H), 7.40–7.65 (m, 2H), 5.89 (t,
J = 5.7 Hz, 1H), 4.51 (d, J = 16.7 Hz, 1H), 4.38 (q,
J = 7.2 Hz, 2H), 3.75 (d, J = 16.7 Hz, 1H), 2.63–2.90
(m, 2H), 2.35–2.58 (m, 1H), 1.87–2.16 (m, 1H), 1.39 (t,
J = 7.2 Hz, 3H). MS: 289.4 (MH)+, 218.4.
5.1.33. Compound 33. Yield: 20%. 1H NMR (DMSO-d6)
d: 7.32 (dd, J = 8.8, 5.3 Hz, 1H), 7.16 (dd, J = 9.4,
2.8 Hz, 1H), 7.07 (td, J = 8.5, 2.8 Hz, 1H), 5.60–5.77
(m, 1H), 4.15 (d, J = 16.0 Hz, 1H), 3.68–3.86 (m,
J = 16.0, 0.9, 0.9 Hz, 1H), 2.54–2.69 (m, 1H), 2.21–
2.41 (m, 2H), 2.19 (s, 3H), 1.75–2.01 (m, 1H). MS:
249.1 (MH)+.
5.1.25. Compound 25. Yield: 24%. 1H NMR (DMSO-d6)
d: 12.87 (br s, 1H), 7.98 (m, J = 8.8 Hz, 2H), 7.65 (m,
J = 8.8 Hz, 2H), 5.89–6.06 (m, 1H), 4.20 (d,
J = 16.1 Hz, 1H), 3.79 (d, J = 16.1 Hz, 1H), 2.57–2.81
(m, 2H), 2.18–2.36 (m, 1H), 1.79–1.99 (m, 1H). MS:
261.2 (MH)+.
1
5.1.34. Compound 34. Yield: 41%. H NMR (CDCl3) d:
7.33 (d, J = 2.2 Hz, 1H), 7.25 (dd, J = 8.5, 2.2 Hz, 1H),
7.04 (d, J = 8.5 Hz, 1H), 5.64 (br. s., 1H), 4.45 (d,
J = 16.7 Hz, 1H), 3.77 (d, J = 16.7 Hz, 1H), 2.69 (dt,
J = 17.3, 9.8 Hz, 1H), 2.42–2.55 (m, J = 17.3, 10.1,
2.8 Hz, 1H), 2.36 (dd dd, J = 16.2, 6.4, 6.3, 2.8 Hz,
1H), 2.22 (s, 3H), 1.90 (br s, 1H). MS: 264 (M)+, 97.
1
5.1.26. Compound 26. Yield: 15%. H NMR (CDCl3) d:
6.98–7.16 (m, 2H), 6.70 (tt, J = 8.8, 2.2 Hz, 1H), 5.67–
5.84 (m, 1H), 4.51 (d, J = 16.7 Hz, 1H), 3.76 (d,
J = 16.7 Hz, 1H), 2.65–2.86 (m, 2H), 2.40–2.60 (m,
1H), 1.93–2.16 (m, 1H). MS: 253.2 (MH)+.
5.1.35. Compound 35. Yield: 24%. 1H NMR (DMSO-d6)
d: 7.31 (dd, J = 8.2, 6.2 Hz, 1H), 7.04 (td, J = 8.5,
2.7 Hz, 1H), 6.87 (dd, J = 8.8, 2.7 Hz, 1H), 5.66 (br s,
1H), 4.48 (d, J = 16.5 Hz, 1H), 3.72–3.86 (m, J = 16.5,
1.2, 1.2 Hz, 1H), 2.63–2.82 (m, 1H), 2.32–2.58 (m,
2H), 2.22 (s, 3H), 1.81–2.12 (m, 1H). MS: 249.1 (MH)+.
1
5.1.27. Compound 27. Yield: 57%. H NMR (CDCl3) d:
7.18 (d, J = 2.2 Hz, 1H), 7.16 (d, J = 7.9 Hz, 1H), 7.05
(dd, J = 8.2, 2.2 Hz, 1H), 5.77 (t, J = 6.0 Hz, 1H), 4.44
(d, J = 16.4 Hz, 1H), 3.72 (d, J = 16.4 Hz, 1H), 2.63–
2.79 (m, 1H), 2.53–2.63 (m, 1H), 2.37–2.51 (m, 1H),
2.28 (s, 3H), 2.25 (s, 3H), 1.91–2.12 (m, 1H). MS: 245
(MH)+.
1
5.1.36. Compound 36. Yield: 25%. H NMR (CDCl3) d:
7.01–7.22 (m, 3H), 5.70–5.85 (m, 1H), 4.46 (d,
J = 16.4 Hz, 1H), 3.77 (dt, J = 16.4, 1.2 Hz, 1H), 2.61–
2.79 (m, 1H), 2.37–2.54 (m, 2H), 2.36 (s, 3H), 1.89–
2.10 (m, 1H). MS: 249.3 (MH)+.
1
5.1.28. Compound 28. Yield: 54%. H NMR (CDCl3) d:
1
6.98 (s, 2H), 6.89 (s, 1H), 5.79 (t, J = 5.8 Hz, 1H), 4.45
(d, J = 16.4 Hz, 1H), 3.72 (d, J = 16.7 Hz, 1H), 2.71
(dt, J = 16.0, 9.8 Hz, 1H), 2.53–2.65 (m, 1H), 2.37–2.51
(m, 1H), 2.33 (s, 6H), 1.89–2.13 (m, 1H). MS: 244
(M)+, 188, 97.
5.1.37. Compound 37. Yield: 35%. H NMR (CDCl3) d:
7.37 (d, J = 8.5 Hz, 1H), 7.33 (d, J = 2.8 Hz, 1H), 7.13
(dd, J = 8.3, 3.0 Hz, 1H), 5.79 (t, J = 6.0 Hz, 1H), 4.46
(d, J = 16.4 Hz, 1H), 3.73 (d, J = 16.4 Hz, 1H), 2.56–
2.81 (m, 2H), 2.41–2.52 (m, 1H), 2.37–2.41 (m, 3H),
1.91–2.11 (m, 1H). MS: 265 (MH)+.
1
5.1.29. Compound 29. Yield: 10%. H NMR (CDCl3) d:
7.24–7.31 (m, 1H), 7.10–7.17 (m, 1H), 7.07 (dd,
J = 8.8 Hz, 1H), 5.72–5.84 (m, 1H), 4.48 (d,
J = 16.7 Hz, 1H), 3.76 (dt, J = 16.7, 1.0 Hz, 1H), 2.57–
2.81 (m, 2H), 2.43–2.54 (m, 1H), 2.32 (d, J = 2.1 Hz,
3H), 1.93–2.13 (m, 1H). MS: 249.3 (MH)+.
5.1.38. Compound 38. Yield: 43%. 1H NMR (CDCl3)
d: 6.94–7.07 (m, 2H), 6.77 (d, J = 9.1 Hz, 1H), 5.78
(t, J = 5.7 Hz, 1H), 4.47 (d, J = 16.7 Hz, 1H), 3.73
(d, J = 16.4 Hz, 1H), 2.61–2.81 (m, 2H), 2.39–2.53
(m, 1H), 2.37 (s, 3H), 1.93–2.11 (m, 1H). MS: 249
(MH)+.
1
5.1.30. Compound 30. Yield: 46%. H NMR (CDCl3) d:
7.14–7.25 (m, 2H), 7.03 (dd, J = 8.2, 2.2 Hz, 1H), 5.77 (t,
J = 5.8 Hz, 1H), 4.46 (d, J = 16.4 Hz, 1H), 3.72 (d,
J = 16.4 Hz, 1H), 2.59–2.81 (m, 2H), 2.37–2.55 (m,
1H), 2.26 (d, J = 1.6 Hz, 3H), 1.94–2.12 (m, 1H). MS:
249 (MH)+, 192, 97.
5.1.39. Compound 40a. To a solution of (S)-5-oxo-pyr-
rolidine-2-carboxylic acid methyl ester (30 g, 0.19 mol)
in CH3CN (500 ml), 55% NaH (9.12 g, 0.21 mol) was
added portionwise over 30 min at a temperature ranging
between 0 ꢁC and 5 ꢁC. The mixture was stirred for
additional 90 min and then a solution of bromoacetic
acid tert-butyl ester (33.6 ml, 0.21 mol) in CH3CN
(35 ml) was added dropwise over 15 min. The reaction
mixture was warmed to room temperature and stirred
for 3 h. The solvent was evaporated and the solid crude
was dissolved in water and EtOAc (100/300 ml). The or-
ganic layers were separated and washed with a saturated
solution of NaHCO3, then with water and finally dried
over Na2SO4, filtered and evaporated to get a gummy
mass, which was triturated with hexane to afford the title
compound as a white powder (39 g, 75%). Mp 70–71 ꢁC,
[a]D = À41 (c = 5, DCM). Compound 40b was prepared
following the same procedure of 40a. Mp 68–69 ꢁC,
[a]D = +39.9 (c = 5, DCM).
1
5.1.31. Compound 31. Yield: 35%. H NMR (CDCl3) d:
7.18–7.28 (m, 1H), 7.08 (t, J = 8.3 Hz, 1H), 6.92 (d,
J = 7.9 Hz, 1H), 5.73 (br s, 1H), 4.46 (d, J = 16.7 Hz,
1H), 3.78 (d, J = 16.4 Hz, 1H), 2.70 (dt, J = 17.3,
9.4 Hz, 1H), 2.48 (dd d, J = 17.0, 10.1, 2.8 Hz, 1H),
2.29–2.43 (m, 1H), 2.14 (s, 3H), 1.75–2.01 (m, 1H).
MS: 248 (M)+, 136, 109.
5.1.32. Compound 32. Yield: 25%. 1H NMR (DMSO-d6)
d: 7.42–7.52 (m, 1H), 7.28–7.34 (m, 2H), 5.75 (br. s.,
1H), 4.17 (d, J = 16.1 Hz, 1H), 3.79 (d, J = 15.8 Hz,
1H), 2.54–2.69 (m, 1H), 2.22–2.41 (m, 2H), 2.21 (s,
3H), 1.89 (br. s., 1H). MS: 265.3 (MH)+.

C. Farina et al. / Bioorg. Med. Chem. 16 (2008) 3224–3232
3231
1
5.1.40. Compound 41a. Compound 40a (36.5 g, 0.13 mol)
was added portionwise to a solution of 32% NH4OH
(365 ml). The resulting suspension was stirred for
90 min at room temperature. The aqueous phases were
extracted with EtOAc (6 · 200 ml). The combined or-
ganic layer was dried and evaporated to get a crude solid
which on trituration with AcOEt afforded the desired
compound as a white solid (21.2 g, 65%). Mp 128–
129 ꢁC, [a]D = À31.3 (c = 5, DCM). Compound 41b
was prepared following the same procedure as 41a.
Mp 132–135 ꢁC, [a]D = +31.4 (c = 5, DCM).
5.1.44. Compound 3R. Yield: 44%. H NMR (CDCl3) d:
1H NMR (CDCl3) d: 7.20–7.39 (m, 3H), 7.03–7.14 (m,
1H), 5.42–5.75 (m, 1H), 4.44 (d, J = 16.7 Hz, 1H), 3.75
(d, J = 16.4 Hz, 1H), 2.67 (dt, J = 17.0, 9.8 Hz, 1H),
2.46 (ddd, J = 17.3, 10.1, 3.1 Hz, 1H), 2.33 (dd dd,
J = 16.2, 6.5, 6.4, 3.0 Hz, 1H), 2.24 (s, 3H), 1.79–2.09
(m, 1H). MS: 230 (M)+, 143, 118, 97.
1
5.1.45. Compound 5S. Yield: 60%. H NMR (CDCl3) d:
77.30 (m, 2H), 7.24 (m, 2H), 5.76–5.87 (m, 1H), 4.48 (d,
J = 16.4 Hz, 1H), 3.75 (d, J = 16.4 Hz, 1H), 2.56–2.83
(m, 2H), 2.41–2.55 (m, 1H), 2.37 (s, 3H), 1.94–2.12 (m,
1H). 13C NMR (CDCl3) d: 177.3 (CO); 169.1 (CO);
136.6 (Ar); 133.0 (Ar); 130.4 (Ar); 122.1 (Ar), 75.9
(C7a); 47.8 (C3); 30.7 (C6); 28.2 (C7); 21.4 (CH3). MS:
230 (M)+, 174, 118.
5.1.41. Compound 42a. Compound 41a (2 g, 8.2 mmol)
was added to a solution of iodosobenzene–bistrifluoro-
acetate (3.5 g, 8.2 mmol) in CH3CN (27 ml) and water
(14 ml). The reaction mixture was stirred for 4 h. The or-
ganic solvent was evaporated under vacuum and the
aqueous phase was washed with diethylether, basified
with solid Na2CO3 and extracted with DCM
(5 · 80 ml). The combined organic layers were dried
and evaporated to give (S)-(2-amino-5-oxo-pyrrolidin-
1-yl)-acetic acid tert-butyl ester as a yellow oil (1.5 g,
Yield: 86%). One gram of this compound (4.7 mmol)
was dissolved in DCM (1 ml) and then TFA (5 ml)
was added dropwise maintaining the temperature in
the range of 15–20 ꢁC. The solution was stirred for 3 h
and then was evaporated under vacuum. The reaction
crude was redissolved in 2 N HCl (4.7 ml, 9.4 mmol)
and after few minutes the solution was evaporated to
get a gummy solid that after trituration with diethyl-
ether afforded the desired compound as a white solid
(0.65 g, 71%). Mp 175–177 ꢁC, [a]D = +1.9 (c = 1,
H2O). Compound 42b was prepared following the same
procedure as 42a. Mp 174–176 ꢁC, [a]D = À1.1 (c = 1,
H2O).
1
5.1.46. Compound 5R. Yield: 54%. H NMR (CDCl3) d:
77.30 (m, 2H), 7.24 (m, 2H), 5.76–5.87 (m, 1H), 4.48 (d,
J = 16.4 Hz, 1H), 3.75 (d, J = 16.4 Hz, 1H), 2.56–2.83
(m, 2H), 2.41–2.55 (m, 1H), 2.37 (s, 3H), 1.94–2.12 (m,
1H). 13C NMR (CDCl3) d: 177.3 (CO); 169.1 (CO);
136.6 (Ar); 133.0 (Ar); 130.4 (Ar); 122.1 (Ar), 75.9
(C7a); 47.8 (C3); 30.7 (C6); 28.2 (C7); 21.4 (CH3). MS:
230 (M)+, 174, 118.
1
5.1.47. Compound 8S. Yield: 58%. H NMR (CDCl3) d:
7.73 (m, 2H), 7.60 (m, 2H), 5.75–5.98 (m, 1H), 4.54 (d,
J = 16.7 Hz, 1H), 3.77 (d, J = 17.0 Hz, 1H), 2.65–2.91
(m, 2H), 2.41–2.62 (m, 1H), 1.94–2.17 (m, 1H). MS:
242.2 (MH)+.
1
5.1.48. Compound 8R. Yield: 53%. H NMR (CDCl3) d:
7.73 (m, 2H), 7.60 (m, 2H), 5.75–5.98 (m, 1H), 4.54 (d,
J = 16.7 Hz, 1H), 3.77 (d, J = 17.0 Hz, 1H), 2.65–2.91
(m, 2H), 2.41–2.62 (m, 1H), 1.94–2.17 (m, 1H). MS:
242.2 (MH)+.
5.1.42. Compound 43a. A mixture of compound 42a (4 g,
20.6 mmol) and anhydrous NaOAc (1.69 g, 20.6 mmol)
in acetic anhydride (60 ml) was refluxed for 4 h under
nitrogen. The solvent was then removed under vacuum
and the crude was partitioned between EtOAc (200 ml)
and water (50 ml), the organic layer was separated and
washed with a saturated solution of NaHCO3 (50 ml)
and water (50 ml), dried over Na2SO4, filtered and evap-
orated to get 1-acetyl-tetrahydro-pyrrolo[1,2-a]imidaz-
5.2. Pharmacology
Male Sprague–Dawley rats weighting 150–200 g were
used in all experiments (Harlan, Italy). Four rats were
housed per cage. The cages were placed in the experimen-
tal room 24 h before the test for acclimatization. The ani-
mals were fed a standard laboratory diet and tap water ad
libitum and kept at 23 1 ꢁC with a 12 h light/dark cycle,
light at 7 a.m. All experiments were carried out in accor-
dance with the NIH Guide for the Care and Use of Labo-
ratory animals. All efforts were made to minimize animal
suffering, and to reduce the number of animals used.
ole-2,5-dione as
a brown oil (2.4 g, 64%). The
protected compound (2.2 g, 12 mmol) was dissolved in
water (44 ml) and then Amberlite IR 120 (2.2 g, 16–45
mesh, H+ form) was added. The reaction mixture was
filtered and the water was evaporated under vacuum.
The obtained solid was triturated with isopropanol to
afford the desired compound (0.9 g, 54%). Mp 197–
200 ꢁC [a]D = +39.9 (c = 0.4, MeOH). Compound 43b
was prepared following the same procedure as 43a.
Mp 200–201 ꢁC, [a]D = À39.2 (c = 0.4, MeOH).
Results are given as means SEM; analysis of variance
(ANOVA), followed by Fisher’s PLSD procedure for
post hoc comparison, was used to verify the significance
between two means. P values less than 0.05 were consid-
ered significant. Data were analysed with the StatView
for the Macintosh computer program
1
5.1.43. Compound 3S. Yield: 48%. H NMR (CDCl3) d:
7.20–7.39 (m, 3H), 7.03–7.14 (m, 1H), 5.42–5.75 (m,
1H), 4.44 (d, J = 16.7 Hz, 1H), 3.75 (d, J = 16.4 Hz,
1H), 2.67 (dt, J = 17.0, 9.8 Hz, 1H), 2.46 (dd d,
J = 17.3, 10.1, 3.1 Hz, 1H), 2.33 (dd dd, J = 16.2, 6.5,
6.4, 3.0 Hz, 1H), 2.24 (s, 3H), 1.79–2.09 (m, 1H). MS:
230 (M)+, 143, 118, 97.
5.2.1. Chronic constriction injury model. A peripheral
mononeuropathy was produced in adult rats by placing
loosely constrictive ligatures around the common sciatic
nerve according to the method described by Bennett and
Xie.12

3232
C. Farina et al. / Bioorg. Med. Chem. 16 (2008) 3224–3232
Rats were anaesthetized with chloral hydrate. The com-
mon sciatic nerve was exposed at the level of the middle
of the thigh by blunt dissection through the biceps femo-
ris. Proximal to sciatica’s trifurcation, about 1 cm of the
nerve was freed of adhering tissue and four ligatures (3/0
silk tread) were tied loosely around it with about 1 mm
spacing. The length of the nerve thus affected was 1 cm
long. Great care was taken to tie the ligatures such that
the diameter of the nerve was seen to be just barely con-
stricted when viewed with 40x magnification. The left
paw was untouched. Animals were tested for neuropath-
ic pain 14 days after nerve injury.
long) was divided into 5 equal sections by 6 discs. Thus,
up to 5 rats were tested simultaneously on the appara-
tus, with a rod-rotation of 16 rpm.
References and notes
1. (a) Merskey, H.; Bogduk, N. Classification of Chronic
Pain. 2nd ed.; IASP Press: Seattle, 1994, p 394; (b) Butera,
J. A. J. Med. Chem. 2007, 50, 2543–2546; (c) Kennedy, J.
D. J. Med. Chem. 2007, 50, 2547–2556.
2. Sindrup, S. H.; Jensen, T. S. Pain 1999, 83, 389–400.
3. Dworkin, R. H. Clin. J. Pain 2002, 18, 343–349.
4. (a) Gouliaev, A. H.; Senning, A. Brain Res. Rev. 1994,
19, 180; (b) Croisile, B.; Trillet, M.; Fondarai, J.;
Laurent, B.; Mauguiere, F.; Billardon, M. Neurology
1993, 43, 301.
5. Rashid, H.; Ueda, H. J. Pharm. Exp. Ther. 2002, 303, 226.
6. (a) Price, M. J. Clin. J. Pain 2004, 20, 33; (b) Arroyo, S.;
Crawford, P. Epileptic Disord. 2003, S57.
7. Gualtieri, F.; Manetti, D.; Romanelli, M. N.; Ghelardini,
C. Curr. Pharm. Des. 2002, 8, 125.
8. (a) Pinza, M.; Farina, C.; Cerri, A.; Pfeiffer, U.; Ricca-
boni, M. T.; Banfi, S.; Biagetti, R.; Pozzi, O.; Magnani,
M.; Dorigotti, L. J. Med. Chem. 1993, 36, 4214; (b) Pinza,
M.; Farina, C.; Banfi, S.; Pfeifer, U. US Patent 4,719,222;
(c) Pinza, M.; Farina, C.; Banfi, S.; Pfeifer, U. US Patent
4,703,0954.
9. Lange, J. H. M.; Hofmeyer, L. J. F.; Hout, F. A. S.;
Osnabrug, S. J. M.; Verveer, P. C.; Kruse, C. G.; Feenstra,
R. W. Tetrahedron Lett. 2002, 43, 1101–1104.
5.2.2. Streptozotocin treatment. Rats were injected intra-
peritoneally with streptozotocin (50 mg/kg). Animals
were tested for mechanical hyperalgesia 21-day after
toxin treatment in the paw pressure test.
5.2.3. Paw pressure test. The nociceptive threshold in the
rat was determined with an analgesimeter (Ugo Basile,
Varese, Italy), according to the method described by
Leighton et al.13 The stimulus at which rats withdrawn
the paw was evaluated before and at different times after
drug treatment. Rats scoring below 40 g or over 75 g
during the test before drug administration (25%) were
rejected. Results represent the mean of mechanical
thresholds expressed as grams. To avoid any possible
damage to the animal paw the maximum applied force
was fixed at 240 g.
10. PCT application WO2004/085438, published on October
7th, 2004.
5.2.4. Rat rotarod test. Potential motor incoordination/
ataxia caused by compounds’ injection was evaluated
using a rotarod apparatus in which animals were re-
quired to walk against the motion of rotating drum over
30 s.14 The time taken to fall off the rotarod was re-
corded as the number of falls in 30 s. The rod (30 cm
11. Prakash, O.; Batra, H.; Kaur, H.; Sharma, P. K.; Sharma,
V.; Singh, S. P.; Moriarty, R. M. Synthesis 2001, 541–543.
12. Bennett, G. J.; Xie, Y. K. Pain 1988, 33, 87–107.
13. Leighton, G. E.; Rodriguez, R. E.; Hill, R. G.; Hughes, J.
Br. J. Pharmacol. 1988, 93, 553–560.
14. Vaugh, J. L. Neuropharmacology 1985, 24, 211–216.