Synthesis and biological evaluation of chiral α-aminoanilides with central antinociceptive activity

Article abstract of DOI:10.1021/jm061078e

Tocainide and related optically active chiral α-aminoanilides were synthesized and tested in vivo via the hot plate test to evaluate their central analgesic action. The aims of the study were to verify if a) the increase in lipophilicity, obtained by the introduction of an alkyl group on the steric center (3f-i), and the replacement of the C=O group with the C=S (10) group as well as the introduction of a methyl or ethyl group on the amidic nitrogen atom (8a-c) would produce an increase in central analgesic efficacy with respect to Tocainide; b) the 2,6-xylidide moiety is crucial for high analgesic activity (3b-e); c) the hydrogen atom bonded to the amidic nitrogen moiety is an essential pharmacophoric element for analgesic activity. Among all the synthesized compounds, 3f showed antinociceptive properties with a good enantioselective index.

Full text of DOI:10.1021/jm061078e

J. Med. Chem. 2007, 50, 1907-1915
1907
Synthesis and Biological Evaluation of Chiral r-Aminoanilides with Central Antinociceptive
Activity
Filomena Corbo,† Carlo Franchini,*,† Giovanni Lentini,† Marilena Muraglia,† Carla Ghelardini,‡ Rosanna Matucci,‡
Nicoletta Galeotti,‡ Elisa Vivoli,‡ and Vincenzo Tortorella†
Department of Pharmaceutical Chemistry, UniVersity of Bari, Via E. Orabona n.4, 70125 Bari, Italy, and Department of Preclinical and
Clinical Pharmacology, UniVersity of Florence, Viale G. Pieraccini, 5/6 - 50139, Florence, Italy
ReceiVed September 13, 2006
Tocainide and related optically active chiral R-aminoanilides were synthesized and tested in ViVo via the
hot plate test to evaluate their central analgesic action. The aims of the study were to verify if a) the increase
in lipophilicity, obtained by the introduction of an alkyl group on the steric center (3f-i), and the replacement
of the CdO group with the CdS (10) group as well as the introduction of a methyl or ethyl group on the
amidic nitrogen atom (8a-c) would produce an increase in central analgesic efficacy with respect to Tocainide;
b) the 2,6-xylidide moiety is crucial for high analgesic activity (3b-e); c) the hydrogen atom bonded to the
amidic nitrogen moiety is an essential pharmacophoric element for analgesic activity. Among all the
synthesized compounds, 3f showed antinociceptive properties with a good enantioselective index.
Introduction
to the effects of procaine on the brain’s superior functions.11
Due to their poor oral bioavailability, lidocaine and procaine
may not be considered as the best tools for the investigation of
alternative systemic mechanisms of antinociception. Therefore,
orally active analogues of lidocaine such as mexiletine (Mex)
and tocainide (Toc) were used. In fact in our previous work12
we demonstrated that (-)-R-Toc is the eutomer responsible for
exerting analgesia. In particular, biological tests have shown
that the antinociceptive effect of the (-)-R enantiomer, such as
the analgesia induced by lidocaine, procaine, and Mex, is due
to a highly stereoselective central presynaptic cholinergic
mechanism of action.13 Thus, in light of these results, the
interpretation of Lindstrom and Lindblom14,15 should be recon-
sidered. They hypothesized that analgesia induced by Toc might
depend on the blocking of Na+ channels of hyperexcited nerve
membranes in the pain-producing foci. This hypothesis prompted
us to synthesize further anilide derivatives, structurally related
to Toc, with the aim of identifying the chemical features
specifically related to the antinociceptive activity and to gain a
better insight to the mechanism involved in their analgesic
action. In particular, the goals of this study were to verify the
following:
a) if the increase in lipophilicity, obtained by replacing the
hydrogen atom, both on the steric center and on the amidic
nitrogen atom, with alkyl groups, and by substituting the CdO
group with a more lipophilic moiety, such as CdS, leads to an
increase in central analgesic efficacy;
b) if the 2,6-xylididic moiety is essential to preserve analgesic
activity;
c) if the hydrogen atom bonded to the amidic nitrogen is a
pharmacophoric element for analgesic action.
Traditional pain treatments (i.e., those based on the use of
NSAIDs and opioid analgesics) have been known to exhibit
several side effects, and this is why there has been a lot interest
in the area of the so-called ‘adjuvant analgesics’, “drugs that
have primary indications other than pain but may be analgesics
in selected circumstances”, in the past 20 years.1 In this
heterogeneous group of alternative drugs for palliative therapy,
local anaesthetics (LAs) have been specifically targeted for the
treatment of neuropathic pain, and the mechanism of their
antinociceptive effect may be due to the blocking of afferent
stimuli. However, the ability of these agents to stabilize
membranes is not limited to the peripheral nerves;2 in fact once
they cross the blood-brain barrier (BBB), LAs exert marked
effects on the central nervous system (CNS). Indeed several
LA-like compounds have exerted various systemic effects both
in laboratory models and humans.3 For example, procaine is
one of foremost LAs investigated for both its systemic effects
and for its neuropsychopharmacological properties. However,
it presents some disadvantages due its rapid metabolism and
relatively high incidence of allergic phenomena. It because of
these reasons that lidocaine (the amide-type substitute of
procaine4) is currently the LA under greatest investigation as a
systemic adjunct in the treatment of pain. Surprisingly, analgesic
plasma lidocaine EC505,6 values are about 100 times lower than
those required to obtain a peripheral blockade of sodium
channels (Na+).7 These findings suggest that the systemic
antinociceptive effects of lidocaine may be ascribed to synergic
mechanisms, implicating targets other than sodium channels.8,9
In agreement with the above hypothesis, we have previously
reported that lidocaine, when given systemically, produces
significant antinociception in mice and rats through the activa-
tion of the cholinergic system via antagonism of the presynaptic
muscarinic receptors.10 It has recently been proposed that
antagonism of the presynaptic muscarinic receptors contributes
Chemistry. The synthesis of compounds 3a-i (Scheme 1)
was carried out using, as starting materials, the commercially
available amino acids alanine, valine, and isoleucine, in their
optically active forms. These R-amino acids were converted into
their N-Boc derivatives 1a-c by a reaction of 2-tert-butoxy-
carbonylimino-2-phenylacetonitrile (Boc-ON)16 and triethy-
lamine (Et3N) in a mixture of dioxane and water. The N-tert-
butoxycarbonyl-R-amino acids were then condensed with
suitably substituted aromatic anilines in the presence of EEDQ,
* To whom correspondence should be addressed. Phone: +39 080
5442743, fax: +39 080 5442050; e-mail: cfranc@farmchim.uniba.it.
† University of Bari.
‡ University of Florence.
10.1021/jm061078e CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/21/2007

1908 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Corbo et al.
Scheme 1a
a Reagents and conditions: (a) Boc-ON, Et3N, dioxane/H2O, rt: (b) EEDQ or IIDQ, Et3N, CHCl3, anilines, ∆, 6 h; (c) HClg, anhyd Et2O.
Scheme 2a
reagent used instead of LR (Lawesson’s reagent) in the presence
of less polar solvents. This reaction was carried out in benzene
at 60 °C, under reflux, for 2 days. The corresponding hydro-
chloride (10) was prepared as described in the Experimental
Section.
Pharmacological Results. The antinociceptive properties of
Toc and its analogues were investigated using the mouse hot
plate test. As shown in Table 1, the compounds 3a (Toc), 3c,
3e, 3f, 3g, and 3h showed antinociceptive properties. In
particular, an increase in the licking latency values was
selectively obtained with the R isomer of 3a, 3f, and 3g.
Conversely, no stereoselective antinociception was detected for
compounds 3c, 3e, and 3h. The increase in the pain threshold
produced by (-)-R-Toc and (-)-R-3f was prevented by pre-
treatment with the muscarinic antagonist atropine, and the
acetylcholine depletor Hemicolinium-3 (HC-3) administered 15
min and 1 h respectively, before the test (Table 2). By contrast,
the antinociception induced by (R)-3c, (S)-3c, (R)-3e, (S)-3e,
(R)-3g, (R)-3h, and (S)-3h was not visibly modified by atropine
pretreatment. None of the other investigated compounds showed
antinociceptive effect. At the highest effective doses, all
antinociceptive compounds did not modify the animals’ behav-
ior. Furthermore, the motor coordination of animals treated with
the above-mentioned compounds was evaluated using the rota-
rod test. As reported in Table 3, 3a (Toc), 3c, 3e, 3f, 3g, and
3h did not produce any impairment of motor coordination in
comparison to the saline-treated group. The number of falls by
control animals progressively decreased at every measurement
since the mice learned how to balance on the rotating rod.
a Reagents and conditions: (a) (CF3CO)2O, anhyd THF, N2; (b) CuCl,
anhyd DMSO, N2; (c) HCl 17%/THF (1:1).
a peptide coupling reagent that avoids racemization,17,18 produc-
ing the desired 2-(tert-butoxycarbonylamino)-N-arylalkanamides
2a-h. Removal of the N-protecting group by gaseous HCl gave
2-amino-N1-arylalkanamide hydrochlorides 3a-h. The com-
pound 2i, N2-(tert-butoxycarbonyl)-N1-(4-chloro-2,6-dimeth-
ylphenyl)valinamide, was synthesized using IIDQ instead of
EEDQ, as previous experimental situations proved that EEDQ
did not give the desired product.
In spite of the higher reactivity19 of IIDQ when compared to
EEDQ, the yield of this reaction was lower than that recorded
in the synthesis of previous compounds. The aromatic amine
(4-chloro-2,6-dimethylaniline) used to prepare the compound
2i, was synthesized ex noVo. The synthesis of this substrate was
carried out as reported in Scheme 2: commercially available
4-bromo-2,6-dimethylaniline was converted to N-(4-bromo-2,6-
dimethylphenyl)-2,2,2-trifluoracetamide (4) by reacting it with
trifluoroacetic anhydride in anhydrous THF under a nitrogen
athmosphere. Then, 4 was converted to N-(4-chloro-2,6-dim-
ethylphenyl)-2,2,2-trifluoroacetammide (5) with CuCl as source
of chloride ions, in anhydrous DMSO.20 Finally, removal of
the trifluoroacetyl group to give 6 was carried out using an
equivolumetric mixture of THF and HCl 17%, under reflux,
for 17 h. N-Alkyl-N1-aryl-2-(tert-butoxycarbonylamino)alkana-
mides (7a-c) (Scheme 3) were prepared by reacting 2a and 2f
with CH3I or C2H5I, as alkylant reagents, in DMF/H2O in the
presence of Ba(OH)2. Even under basic conditions, no racem-
ization was detected (as previously reported for the chiral
compounds obtained under the same experimental conditions).21
The corresponding hydrochlorides 8a,b were prepared by
treatment of 7a,b with gaseous HCl, by using anhydrous Et2O
as the solvent. 8c instead was obtained as the tartrate salt by
using L-tartaric acid. 2-tert-Butoxycarbonylamino-N-(2,6-dim-
ethylphenyl)propanethioamide (9) was prepared, as reported in
Scheme 4, by the reaction of 2a with 2,4-bis-(p-pentyloxyphe-
nyl)-1,2,3,4-dithiadiphosphethane 2,4-disulfide,22 a sulfuration
Discussion
In the following discussion one must remember that in vivo
tests, such as the hot plate and rota-rod tests, while obviously
speeding up the selection of the compounds with the most
promising clinical properties, do not always allow sound
structure-activity relationships to be established. In fact, the
resulting activity is the consequence of both pharmacokinetic
and pharmacodynamic properties that may be differently affected
by structural modifications.
All the synthesized compounds can be considered as chiral
analogues of Toc, an antiarrhythmic drug of the IB23 class once
used in treatment of symptomatic life-threatening ventricular
arrhythmias.24 Furthermore, Toc is effective as an analgesic in
the treatment of trigeminal neuralgia in humans, as well as the
nociceptive effect in rats.25 It is noteworthy that many chiral

Chiral R-aminoanilides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1909
Scheme 3a
a Reagents and conditions: (a) DMF/H2O, BaOH2, CH3I or CH3CH2I, rt; (b) HClg, anhyd Et2O, or L-tartaric acid, EtOH.
Scheme 4a
a Reagents and conditions: (a) 2,4-Bis(p-pentyloxyphenyl)-1,2,3,4-dithiadiphosphethane-2,4-disulfide, benzene, 70 °C; (b) HClg, anhyd Et2O.
reported the synthesis of a new series of rigid21,31 or sterically
hindered compounds, in order to evaluate potential central
Table 1. Effect of Toc and Toc Analogues in the Mouse Hot-Plate Test
(52.5 °C)
analgesic action.32 Some compounds showed high stereoselec-
licking latency (s)
tive central analgesic activity,12 probably due to the activation
treatment
before
after
compd
saline
(mg/kg s.c.)
treatment
treatment
of the central cholinergic system. In this experimental protocol
(-)-R-Toc was found to be more active than the (+)-S-
enantiomer. These findings prompted us to plan a new series
of chiral-R-aminoanilides, structurally related to Toc with the
aim of evaluating the effect of some modified structural moieties
(aromatic group, alkylic chain, amidic moiety) on analgesic
activity.
14.3 ( 0.4
13.8 ( 1.1
15.4 ( 1.0
14.3 ( 0.6
13.9 ( 0.8
14.1 ( 1.0
13.8 ( 0.9
14.0 ( 1.3
13.2 ( 1.2
14.9 ( 0.7
14.5 ( 1.0
13.8 ( 0.5
15.6 ( 1.3
15.6 ( 1.4
13.8 ( 0.6
16.2 ( 1.0
15.3 ( 1.4
14.4 ( 1.2
14.0 ( 0.8
14.7 ( 1.0
13.9 ( 1.1
15.8 ( 0.9
13.2 ( 0.8
13.5 ( 1.3
13.5 ( 0.9
14.0 ( 0.8
14.3 ( 0.8
15.1 ( 1.2
26.5 ( 1.7**
14.3 ( 1.5
17.3 ( 2.1
15.8 ( 1.9
23.2 ( 1.8**
25.1 ( 2.0**
17.1 ( 2.1
16.2 ( 2.0
25.5 ( 2.0**
21.1 ( 1.3**
30.3 ( 1.7**
15.8 ( 0.6
27.9 ( 2.1**
19.9 ( 2.0*
20.7 ( 1.3*
21.3 ( 1.7*
17.0 ( 1.4
17.2 ( 1.7
18.2 ( 2.5
16.7 ( 1.9
16.0 ( 1.8
15.1 ( 1.3
13.6 ( 2.1
13.6 ( 1.7
18.8 ( 2.4
18.6 ( 1.9
(R)-3a Toc
(S)-3a Toc
(R)-3b
(S)-3b
(R)-3c
(S)-3c
(R)-3d
(S)-3d
(R)-3e
(S)-3e
(R)-3f
(S)-3f
(R)-3g
(S)-3g
(R)-3h
(S)-3h
(R)-3i
(50)
(50)
(5)
(5)
(30)
(30)
(10)
(15)
(50)
(50)
(10)
(10)
(30)
(40)
(10)
(10)
(50)
(50)
(10)
(10)
(5)
Therefore, we synthesized a first series of compounds, (3a-
e) in which we introduced substituents with different electronic
properties on the aromatic ring. As reported in Table 1, among
all the alanino-anilides, only compounds 3a, 3c, and 3e showed
analgesic activity; in the same series only compound 3a gave
rise to a stereoselective analgesic effect, probably due to the
xylididic moiety. By these preliminary results we deduced that
the 2,6-xylididic moiety was important in central analgesic
action. To verify the influence of steric hindrance on the
stereogenic center, the compounds 3f-i were also synthesized
and tested. First we substituted the methyl group with an
isopropyl moiety on the steric center (3f, 3g, 3i). As reported
in Table 1, all compounds, with the exception of 3i, exhibited
antinociceptive properties. In particular 3f is the most active of
the series and shows both potency and stereoselectivity higher
than tocainide itself.33,34 To confirm the influence of the alkylic
chain on the chiral carbon atom, we synthesized compound 3h,
that possesses the isobutyl chain. Biological results disclose that
the increment of the steric hindrance on this position, such as
in 3h, causes analgesic activity even if it is lower than that
caused by 3f.
(S)-3i
(R)-8a
(S)-8a
(R)-8b
(S)-8b
(R)-8c
(S)-8c
(R)-10
(S)-10
(5)
(20)
(10)
(50)
(50)
a The licking latency values were recorded in correspondence with the
maximum antinociceptive effect of each compound. *P < 0.05, **P <
0.01 in comparison with saline-saline treated mice.
local anaesthetic drugs are used in clinical therapy in their
racemic form. In the last 20 years it was proved, that in all
cases, the enantiomers of these drugs had a different pharma-
cological and pharmacokinetic profile;26 in particular, the
introduction of Mex and Toc in clinical therapy led chemists
to review previous studies on each of the single enantiomers of
local anaesthetic drugs. It was then demonstrated that the
enantiomers of Mex, for example, showed both a different
pharmacokinetic profile27 as well as a diverse inhibitory effect
for the clorgiline resistant monoamino oxidase (CRAO).28 The
R isomer of Toc showed a higher antiarrhythmic activity than
that of the S isomer.29,30 In light of these results, we recently
To complete our investigation, we synthesized and tested
compounds 8a and 8b, that possess an alkylic group (methyl
and ethyl) on the amidic moiety. Furthermore, we obtained 8c
by performing the same substitution on 3f which, as mentioned
above, is the most active among all the tested compounds. The
molecules so obtained (8a, 8b and 8c) did not show any central
analgesic action. To clarify if this loss of activity is dependent
on the increment of lipophilicity, due to an insertion of an alkylic
moiety on the nitrogen amidic atom, or due to the loss of a
hydrogen bond on the receptor site, we synthesized compound

1910 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Corbo et al.
Table 2. Effect of Toc and Active Toc Analogues in the Mouse Hot-Plate Test (52.5 °C)a
licking latency (s)
after treatment
treatment
(mg/kg s.c.)
without
pretreatment
pretreatment
with atropine
pretreatment
with HC-3
compd
saline
(R)-3a Toc
(R)-3c
(S)-3c
(R)-3e
(S)-3e
(R)-3f
before treatment
14.3 ( 0.4
13.8 ( 1.1
14.1 ( 1.0
13.8 ( 0.9
14.9 ( 0.7
14.5 ( 1.0
13.8 ( 0.5
15.6 ( 1.4
16.2 ( 1.0
15.3 ( 1.4
15.1 ( 1.2
14.8 ( 1.6
17.5 ( 0.9
18.6 ( 1.3
(50)
(30)
(30)
(50)
(50)
(10)
(30)
(10)
(10)
26.5 ( 1.7**
23.2 ( 1.8**
25.1 ( 2.0**
25.5 ( 2.0**
21.1 ( 1.3**
30.3 ( 1.7**
27.9 ( 2.1**
20.7 ( 1.3*
21.3 ( 1.7*
17.5 ( 1.5
23.7 ( 1.8**
26.6 ( 2.4**
25.0 ( 2.7**
20.5 ( 2.1**
16.9 ( 2.0**
26.4 ( 2.0**
23.9 ( 1.8**
20.7 ( 1.7**
17.5 ( 2.2
(R)-3g
(R)-3h
(S)-3h
a The licking latency values were recorded in correspondence with the maximum antinociceptive effect of each compound. Atropine and hemicolinium-3
(HC-3) at the Dose of 5 mg/kg i.p. and 1 µg per mouse icv, respectively, were administered *P < 0.05, **P < 0.01 in comparison with saline-saline treated
mice.
Table 3. Lack of Effect of Some Compounds on Motor Coordination Expressed as Falls from the Rota-roda
falls from the rota-rod
after treatment
treatment
dose, mg kg-1
before treatment
15 min
30 min
45 min
saline
(R)-3a
(S)-3c
(R)-3e
(R)-3f
(R)-3g
(R)-3h
2.4 ( 0.4
2.9 ( 0.5
2.7 ( 0.4
2.8 ( 0.4
3.1 ( 0.5
2.6 ( 0.4
2.5 ( 0.6
1.6 ( 0.3
1.7 ( 0.4
1.4 ( 0.3
1.5 ( 0.3
2.3 ( 0.4
1.5 ( 0.5
2.2 ( 0.5
1.1 ( 0.3
1.4 ( 0.2
1.2 ( 0.4
1.1 ( 0.3
1.2 ( 0.2
1.3 ( 0.4
1.5 ( 0.5
0.9 ( 0.2
0.9 ( 0.3
1.0 ( 0.2
0.8 ( 0.3
0.9 ( 0.3
0.8 ( 0.3
0.8 ( 0.3
(50)
(30)
(50)
(10)
(30)
(10)
a Each value represents the mean of 6-8 mice. *P < 0.01 in comparison with CMC-treated controls.
10. In this compound the increase in lipophilicity was realized
by replacing the CdO with the CdS group. Here, also we
recorded no antinociceptive action. These results suggest that
the amidic moiety (NH-CO) is crucial in preserving analgesic
activity. In conclusion among all tested compounds only 3a,
3c, 3e, 3f, 3g, and 3h showed analgesic behavior, with ED50
values between 5.7-39.2 mg kg/s.c. (Figure 1). So as to clarify
the mechanism of action of these compounds, we administered
the muscarinic antagonist atropine to antagonize their analgesic
action. The administration of atropine was able to oppose this
effect only in the case of compounds (-)-R-3a and (-)-R-3f.
We can therefore suggest that the antinociception, induced by
these two compounds, is of the cholinergic type. Furthermore,
these compounds produced analgesia without altering their
performance in the rota-rod test. We can therefore suppose that
the observed increase in the pain threshold corresponds to an
actual analgesic effect. This is because the dose of atropine
employed in the present study was able to selectively prevent
muscarinic antinociception without modifying the analgesia
induced by the modulation of other neurotransmitters, such as
opioid and GABA.10,35 The most likely site of analgesic action
is in the central nervous system, since the increase in the pain
threshold induced by (-)-R-3a and (-)-R-3f, is antagonized
by the acethylcholine depletor HC-3, that was administered
directly into the cerebral ventricles. Furthermore, the prevention
of (-)-R-3a and (-)-R-3f antinociception, exerted by HC-3,
suggests that this effect is due to a presynaptic mechanism of
action. This is since a depletion of cerebral acethylcholine is
obtained after HC-3 administration. Concerning the antinoci-
ceptive effect produced by 3c, 3e, 3g, and 3h, no mechanism
of action has so far been explained. Nevertheless, a cholinergic
type of analgesia may be ruled out, since the increase in pain
threshold induced by these compounds was not prevented by
atropine.
Figure 1. ED50 values of molecules possessing an antinociceptive
activity.
As reported in this paper, preliminary exploration of the
mechanism of action of the compounds shows that they present
affinity for the muscarinic receptor. Among the studied com-

Chiral R-aminoanilides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1911
Table 4. Dose-Response Curve of (R)-3f on Mouse Hot-Plate Testa
licking latency in mice (s)
after treatment
treatment
dose, mg kg-1
before treatment
15 min
30 min
45 min
60 min
saline
(R)-3f
(R)-3f
(R)-3f
14.4 ( 1.1
13.8 ( 1.2
15.1 ( 1.4
14.7 ( 0.9
15.6 ( 1.3
16.5 ( 1.6
23.7 ( 1.5*
30.3 ( 1.7*
14.9 ( 1.3
17.2 ( 2.0
20.6 ( 2.3*
23.6 ( 1.7*
15.9 ( 1.8
15.3 ( 1.7
17.1 ( 2.0
18.5 ( 1.2
16.0 ( 1.7
14.5 ( 1.6
14.9 ( 1.5
15.1 ( 1.3
(1)
(5)
(10)
a P < 0.01 in comparison with saline controls. Each value represents the mean of at least 10 mice.
experiments were carried out according to the guidelines of the
European Community Council.
pounds, 3f shows outstanding potency, being active at a dose
of 10 mg/kg via subcutaneous (s.c.) (Table 4). In addition,
analgesia induced by this compound, prevented by HC-3
treatment administered at the dose of 1 µg via intracerebroven-
tricular (i.c.v.), seems to depend on the blocking of the central
presynaptic muscarinic autoreceptors that are physiologically
involved, via a feedback mechanism, in the inhibition of
acethylcholine release. Starting with 3f as the lead compound,
design and synthesis of new analogues are in progress so as to
collect more data on the molecular mechanism of action as well
as on the effect other chemical structural modifications have
on the antinociceptive action.
Hot Plate Test. The method adopted was described by
O’Callaghan and Holzman.36 Mice were placed inside a stainless
steel container, thermostatically set at 52.5 ( 0.1 °C in a precision
water-bath from KW Mechanical Workshop, Siena, Italy. Reaction
times (s) were measured with a stop-watch before and at regular
intervals up to a maximum of 60 min after treatment. The endpoint
used was the licking of the fore or hind paws. Those mice scoring
below 12 and over 18 s in the pretest were rejected (30%). An
arbitrary cutoff time of 45 s was adopted.
The computer program ALLFIT37 was used for the analysis of
the sigmoidal dose-response table (Table 4) obtained in functional
studies; the program uses the constrained four-parameter logistic
model to obtain estimates of half-maximal effective concentration
(EC50) values (Figure 1). All the quoted values are mean ( SEM.
Rota-rod Test. The apparatus consisted of a base platform and
a rotating rod of 3 cm diameter with a nonslippery surface. The
rod was placed at a 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 basis of the number of falls from the rod in 30
s according to Vaught.38 The performance time was measured before
and 15, 30, and 45 min after treatment.
Drugs. The following drugs were used: atropine sulfate (Sigma),
hemicholinium-3 hydrobromide (HC-3) (R.B.I.). Other chemicals
were of the highest quality commercially available. All drugs were
dissolved in isotonic (0.9% NaCl) saline solution. Drug concentra-
tions were prepared in such a way that the necessary dose could
be administered in a volume of 10 mL kg -1 by s.c. route or 5 µL
by i.c.v. route. I.c.v. administration was performed under ether
anaesthesia using isotonic saline as the solvent, according to the
method described by Haley and McCormick.39 Briefly, during
anaesthesia, mice were grasped firmly by the loose skin behind
the head. A 0.4 mm external diameter, hypodermic needle attached
to a 10 µL syringe was inserted perpendicularly through the skull
at a depth of no more than 2 mm into the brain of the mouse where
5 µL were then administered. The injection site was 1.5 mm from
either side of the midline of a line drawn through to the anterior
base of the ears. To ascertain that the drugs were administered
exactly into the cerebral ventricle, some mice were i.c.v. injected
with 5 µL of diluted 1:10 India ink and their brains examined
macroscopically after sectioning.
Chemistry. General. Melting points were determined on a
Gallenkamp melting point apparatus in open glass capillary tubes.
The infrared spectra were recorded on a Perkin-Elmer Spectrum
One FT spectrophotometer, and band positions were given in
reciprocal centimeters (cm-1). 1H NMR routine spectra were
recorded on a Varian XL 390 spectrometer (90 MHz) using CDCl3
as the solvent, unless otherwise indicated, and TMS as the internal
reference. 1H 300-MHz and 13C 75-MHz spectra were recorded on
a Varian Mercury 300 spectrometer. Chemical shifts were reported
in ppm relative to solvent resonance: CDCl3, δ 7.26 (1H NMR)
and δ 77.3 (13C NMR); (CD3)2SO, δ 2.50 (1H NMR); CD3OD, δ
3.31 (1H NMR) and δ 47.8 (13C NMR), unless otherwise indicated.
Amino proton assignments were confirmed by D2O exchange. J
values are given in hertz. EIMS spectra were recorded with a
Hewlett-Packard 6890-5973 MSD gas chromatograph/mass spec-
Conclusions
In conclusion, the results obtained by screening the com-
pounds studied Via the hot plate and rota-rod tests suggests the
following:
(1) The increase in lipophilicity obtained via the methylation
of the nitrogens and the substitution of the CdO group with its
isologous CdS did not result in an augmentation of the analgesic
effect. In fact compounds 8-10 did not exhibit any central
antinociceptive action in the hot-plate test.
(2) The 2,6-xylididic moiety is essential for analgesic activity;
in fact derivative 3b that does not possess a xylididic ring did
not present any analgesic effect, as present in compound 3c
but not in compound 3i. It is important to underline that the
coexistence of the Cl atom in the para position and the methyl
group in the ortho position, as in 3e, restores the analgesic effect,
although it results in the loss of stereoselectivity.
(3) An increase in steric hindrance at the chiral center results
in the formation of the most active compound of the series (3f),
but further lengthening of the alkyl chain, as in 3h, causes a
drastic reduction in activity.
(4) Furthermore, one may observe that compounds 8a-c do
not exhibit any analgesic activity although steric hindrance at
the chiral center is absent and they possess the 2,6-xylididic
moiety. In light of this evidence, one may conclude that the
presence of an intramolecular hydrogen bond between the
amidic and aminic functions is crucial in allowing the molecule
to assume the ideal conformation for receptor binding.
(5) Finally, the biological results suggest that an increase in
the pain threshold is related to the stimulation of the central
cholinergic system without any side effects. In particular,
compound 3f could be a starting point for the discovery of new
compounds potentially useful in analgesia and in the treatment
of age-related degenerative syndromes of the CNS.
Experimental Section
Pharmacology. Pharmacological Methods. Male Swiss albino
mice (23-30 g) from Morini (San Polo d’ Enza, Italy) breeding
farm were used. Fifteen mice were housed per cage. The cages
were placed in the experimental room 24 h before the acclimatiza-
tion test. The animals were kept at 23 ( 1 °C with a 12 h light/
dark cycle, light at 7 a.m., with food and water ad libitum. All

1912 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Corbo et al.
trometer at low resolution; where amine salts are concerned, MS
analyses were performed on the corresponding free base forms
obtained by extraction. Elemental analyses were performed on a
Eurovector Euro EA 3000 elemental analyzer; the data for C, H,
and N were within (0.4 of the theoretical values for all final
compounds except (+)-(S)-3d, (-)-(R)-3h, (+)-(S)-3h, (+)-(S)-8b.
Optical rotations were measured on a Perkin-Elmer Mod 341
spectropolarimeter; concentrations were expressed in g/100 mL and
(+)-(R)-N2-(tert-Butoxycarbonyl)-N1-(4-chloro-2,6-dimeth-
ylphenyl)alaninamide [(+)-(R)-2c]: 20% yield, mp 188-189 °C
(EtOAc/petroleum ether); [R]20 ) +60 (c 1, CHCl3); IR
D
1
(CHCl3): 3430 (NH); 1720 (NHCOO), 1694 (NHCO) cm-1; H
NMR (90 MHz) δ 1.40 (d, overlapping s at 1.43, J ) 9 Hz, 3H,
CH3CH), 1.43 (s overlapping d at 1.40, 9H, CH3C), 2.03 (s, 3H,
CH3Ar), 2.1 (s, 3H, CH3Ar), 4.4 (apparent quintet, J ) 8 Hz, 1H,
CHNH), 5.5 (br d, J ) 8 Hz, 1H; CHNH), 6.8-7.2 (m, 2H, Ar),
8.2 (br s, 1H; NH); MS (70 eV): m/z (%) 326 (M+, 6), 44 (100).
(-)-(S)-N2-(tert-Butoxycarbonyl)-N1-(4-chloro-2,6-dimeth-
ylphenyl)alaninamide [(-)-(S)-2c]: 30% yield, mp 187-188 °C
the cell length was 1 dm. Thus, [R]20 values were given in units
D
of 10-1 deg cm2 g-1. Silica gel chromatographic separations were
performed by flash chromatography with silica gel (Kieselgel 60,
0.040-0.063 mm, Merk) packed in glass columns, using the
technique described by Still et al.40 The weight of the silica gel
was approximately 100 times that of the substance, unless noted
otherwise. The eluting solvent indicated in parentheses for each
purification was determined by TLC, that was performed on
precoated silica gel on aluminum sheets (kieselgel 60, F254, Merck).
TLC plates were visualized with UV light and/or in an iodine
chamber.
(EtOAc/petroleum ether); [R]20 ) -57 (c 1, CHCl3).
D
(+)-(R)-N2-(tert-Butoxycarbonyl)-N1-(2-methylphenyl)alani-
namide [(+)-(R)-2d]: 50% yield, mp 97-99 °C (EtOAc/petroleum
ether); [R]20D ) +53 (c 1.2, MeOH); IR (CHCl3): 3439 (NH), 1700
(CO) cm-1; 1H NMR (300 MHz, CDCl3) δ 1.45 (d, overlapping s
at 1.46, J ) 6.4 Hz, 3H, CH3CH), 1.46 (s, 9H, CH3C), 2.26 (s, 3H,
CH3Ar), 2.1 (s, 3H, CH3Ar), 4.33 (br, apparent quintet, J ) 7.1
Hz, 1H, CHCH3), 4.9 (br s, 1H, CHNH), 7.02-7.10 (m, 1H, Ar
HC-4), 7.14-7.24 (m, 2H, Ar HC-3,5), 7.90 (d, J ) 7.7 Hz, 1H,
Ar HC-6), 8.13 (bs, 1H, ArNH); MS (70 eV): m/z (%): 278 (M+,
16), 44 (100).
Chemicals and Reagents. Boc-ON reagent [2-(tert-butoxycar-
bonylimino)-2-phenylacetonitrile], the enantiomers of alanine, iso-
leucine, and valine, 2,6-dimethylaniline, 4-bromo-2,6-dimethyla-
niline, 2-methylaniline, 4-chloroaniline, 4-chloro-2-methylaniline,
EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline), and IIDQ
(2-isobuthoxy-1-isobuthoxycarbonyl-1,2-dihydroquinoline) were pur-
chased from Aldrich Chemical Co.; 4-bromo-2,6-dimethylaniline
was purchased from Lancaster Synthesis, Inc. in the highest
commercially available quality. Yields refer to purified products.
The structures of the compounds were confirmed by routine
spectrometric analyses. Only spectra for compounds that, to our
knowledge, never have been previously described, are given. Even
though N-tert-butoxycarbonylamino acids are commercialy avail-
able, compounds 1a-c were obtained from the corresponding amino
acids via the N-protection reaction using Boc-ON reagent as
reported above.12,21 (+)-(R) and (-)-(S)-2a, (+)-(R) and (-)-(S)-
2e, (-)-(R) and (+)-(S)-3a, and (-)-(R) and (+)-(S)-3e were
prepared as previously described.12 Solvents were RP grade, unless
otherwise indicated.
The procedures used to prepare the compounds shown in
Schemes 1-4 are as follows.
General Procedure for the Synthesis of 2-(tert-Butoxycarbo-
nylamino)-N-arylalkanamides (2a-i). The preparation of (+)-
(R)-N2-(tert-butoxycarbonyl)-N1-(2,6-dimethylphenyl)valinamide [(+)-
(R)-2f] can be taken as the reference procedure for the synthesis
of 2-(tert-butoxycarbonylamino)-N-arylalkanamides (2a-i).
A solution of (+)-(R)-N-(tert-butoxycarbonyl)valine [(+)-(R)-
1f, 0.22 g, 10 mmol], 2,6-dimethylaniline (0.13 g, 1.1 mmol), freshly
recrystallized, EEDQ (0.30 g, 1.2 mmol), and triethylamine (0.15
g, 1.5 mmol) in 50 mL of CHCl3 was refluxed for 6 h. The solvent
was evaporated, and the residue, dissolved in EtOAc, was extracted
with a 10% HCl solution. The organic layer was then washed with
0.1 M NaOH and H2O, dried over anhydrous Na2SO4, and then
evaporated under reduced pressure to give a crude product, that
was purified by recrystallization, producing (+)-(R)-2f as a white
solid (64 mg, 20% yield): mp 203-204 °C; [R]20D ) +56 (c 1.0,
CHCl3); IR (CHCl3): 3678, 3415 (NH), 1692 (CO) cm-1; 1H NMR
(300 MHz, CDCl3) δ 1.02 (d, J ) 6.9 Hz, 3H, CH3CHCH3), 1.07
(d, J ) 6.9 Hz, 3H, CH3CHCH3), 1.45 (s, 9H, CH3C), 2.19 (s, 6H,
CH3Ar), 2.24-2.36 (m, 1H, CH3CHCH3), 4.06 (apparent t, J )
7.8 Hz, 1H, CHNH), 5.15 (br s, 1H; CHNH), 7.02-7.11 (m, 3H,
ArH), 7.50 (br s, 1H; NHAr); MS (70 eV): m/z (%) 320 (M+, 6),
57 (100).
(-)-(S)-N2-(tert-Butoxycarbonyl)-N1-(2-methylphenyl)alani-
namide [(-)-(S)-2d]: 82% yield, mp 97-99 °C (EtOAc/petroleum
ether); [R]20 ) -54 (c 1.2, MeOH).
D
(+)-(R)-N2-(tert-Butoxycarbonyl)-N1-(4-chloro-2-methylphe-
nyl)alaninamide [(+)-(R)-2e]: 20% yield, mp 132-134 °C
(EtOAc/petroleum ether); [R]20 ) +89 (c 0.6, CH2Cl2); IR
D
(CHCl3): 3440 (NH), 1700 (CO) cm-1
;
1H NMR (300 MHz,
CDCl3) δ 1.45 (d, J ) 8.9 Hz, 3H, CH3CH), 1.46 (s, 9H, CH3C),
2.22 (s, 3H, CH3Ar), 4.31 (br, apparent quintet, J ) 7.0 Hz, 1H,
CHCH3), 4.97 (br d, 1H, CHNH), 7.14 (br s overlapping m at,
7.12-7.18, 1H, Ar HC-3), 7.12-7.18 (m overlapping s at 7.14, J
) 6.7 Hz, 1H, Ar HC-5), 7.86 (d, J ) 9.2 Hz, 1H, Ar HC-6), 8.24
(bs, 1H, ArNH).
(-)-(S)-N2-(tert-Butoxycarbonyl)-N1-(4-chloro-2-methylphe-
nyl)alaninamide [(-)-(S)-2e]: 30% yield, mp 133-135 °C (benzene/
hexane); [R]20 ) -86 (c 2.0, CH2Cl2).
D
(-)-(S)-N2-(tert-Butoxycarbonyl)-N1-(4-chloro-2-methylphe-
nyl)alaninamide [(-)-(S)-2f]: 40% yield, mp 200-202 °C (EtOAc/
petroleum ether); [R]20 ) -63 (c 1.0, CHCl3).
D
(+)-(R)-N2-(tert-Butoxycarbonyl)-N1-(4-chloro-2-methylphe-
nyl)valinamide [(+)-(R)-2g]: 35% yield, mp 142-144 °C (EtOAc/
petroleum ether); [R]20D ) +55 (c 1.0, CHCl3); IR (CHCl3): 3436
(NH), 1694 (CO) cm-1; 1H NMR (300 MHz, CDCl3) δ 1.00 (d, J
) 6.9 Hz, 3H, CH3CHCH3), 1.04 (d, J ) 6.9 Hz, 3H, CH3CHCH3),
1.45 (s, 9H, CH3C), 2.22 (s overlapping apparent octet at 2.31,
3H, CH3Ar), 2.31 (apparent octet overlapping s at 2.22, J ) 6.8
Hz, 1H, CH3CHCH3), 4.00 (dd, J ) 8.2, 6.6 Hz, 1H, CHNH), 5.05
(br s, 1H, CHNH), 7.1-7.2 (m, 2H, Ar HC-3,5), 7.76 (d overlapping
br s at 7.81, J ) 9.2 Hz, 1H, Ar HC-6), 7.8 (br s, 1H, ArNH); MS
(70 eV): m/z (%): 340 (M+, 13), 57 (100).
(-)-(S)-N2-(tert-Butoxycarbonyl)-N1-(4-chloro-2-methylphe-
nyl)valinamide [(-)-(S)-2g]: 50% yield, mp 142-144 °C (EtOAc/
petroleum ether); [R]20 ) -57 (c 3.0, MeOH).
D
(+)-(R)-N2-(tert-Butoxycarbonyl)-N1-(2,6-dimethylphenyl)iso-
leucinamide [(+)-(R)-2h]: 40% yield, mp 193-195 °C (EtOAc/
petroleum ether); [R]20D ) +69 (c 1.0, CHCl3); 1H NMR (300 MHz,
CDCl3) δ 0.97 (d overlapping d at 1.00, J ) 6.66 Hz, 3H, CH3-
CHCH3), 1.00 (d overlapping d at 0.97, J ) 6.9 Hz, 3H, CH3-
CHCH3), 1.46 (s, 9H, CH3C), 1.52-1.62 (m, 1H, CH3CHCH3),
1.68-1.88 (m, 2H, CH2CH), 4.25-4.35 (m, 1H, CHNH), 6.00 (br
d, J ) 6.2 Hz, NHCH), 7.00-7.15 (m, 3H, Ar), 7.60 (br s, 1H,
NHAr), 7.8 (br s, 1H, ArNH); MS (70 eV): m/z (%) 334 (M+, 1);
86 (100).
(+)-(R)-N2-(tert-Butoxycarbonyl)-N1-(4-chlorophenyl)alanina-
mide [(+)-(R)-2b]: 72% yield, mp hygroscopic (EtOAc/petroleum
ether); [R]20D ) +44 (c 1.3, MeOH); IR (KBr): 3300 (NH); 1664
(-)-(S)-N2-(tert-Butoxycarbonyl)-N1-(2,6-dimethylphenyl)iso-
leucinamide [(-)-(S)-2h]: 50% yield, mp 193-195 °C (EtOAc/
1
(CO) cm-1; H NMR is reported in the literature41 (MS (70 eV):
m/z (%) 298 (M+, 17), 44 (100).
petroleum ether); [R]20 ) -45 (c 1.0, CHCl3).
D
(-)-(S)-N2-(tert-Butoxycarbonyl)-N1-(4-chlorophenyl)alanina-
(+)-(R)-N2-(tert-Butoxycarbonyl)-N1-(4-chloro-2,6-dimeth-
ylphenyl)valinamide [(+)-(R)-2i]: IIDQ was used as the condens-
ing agent; 20% yield, mp 171-173 °C (EtOAc/petroleum ether);
mide [(-)-(S)-2b]: 77% yield, mp 139-140 °C (EtOAc/petroleum
ether); [R]20 ) -44 (c 1.3, MeOH).
D

Chiral R-aminoanilides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1913
1
[R]20 ) +68 (c 1, CHCl3); IR (CHCl3): 3436 (NH), 1723
[R]20 ) -46.0 (c 1.6, MeOH); H NMR (300 MHz, CD3OD) δ
D
D
(NHCOO), 1694 (NHCO) cm-1; H NMR (300 MHz, CDCl3) δ
1.14 (d, J ) 6.9 Hz, 3H, CH3CHCH3), 1.17 (d, J ) 7.1 Hz, 3H,
CH3CHCH3), 2.29 (s overlapping apparent octet at 2.35, 3H, CH3-
Ar), 2.35 (octet, J ) 6.9 Hz, 1H, CH3CHCH3), 3.99 (d, J ) 5.7
Hz, 1H, CHCO), 7.20 (dd, J ) 8.5, 2.4 Hz, 1H, Ar HC-5), 7.28 (d,
J ) 2.2 Hz 1H, Ar HC-3), 7.40 (d, J ) 8.6, 1H, Ar HC-6); 13C
NMR (75 MHz, CD3OD, δ 46.3) δ 15.3 (1C), 15.6 (1C), 16.4 (1C),
29.0 (1C), 57.3 (1C), 124.7 (1C), 125.5 (1C), 128.8 (1C), 130.2
(1C), 132.1 (1C), 133.5 (1C), 166.0 (1C). Anal. (C12H18Cl2N2O)
C, H, N.
1
1.0 (d overlapping d at 1.05, J ) 6 Hz, 3H, CH3CHCH3), 1.05 (d
overlapping d at 1.0, J ) 6 Hz, 3H, CH3CHCH3), 1.4 (s, 9H, CH3C),
2.1 (s overlapping m at 2.0-2.3, 6H, CH3Ar), 2.0-2.3 (m, 1H,
CH3CHCH3), 4.1 (apparent t, J ) 9 Hz, 1H, CHNH), 5.4 (br d, J
) 9 Hz, 1H, CHNH), 7.05 (s, 2H, Ar), 7.7 (br s, 1H, ArNH); 13C
NMR (75 MHz, CDCl3) δ 18.4 (1C), 18.8 (1C), 19.9 (2C), 28.5
(3C), 30.0 (1C), 60.8 (1C), 80.3 (1C), 128.1 (2C), 132.5 (1C), 132.6
(1C), 137.4 (2C), 156.4 (1C), 171.0 (1C); MS (70 eV): m/z (%):
354 (M+, 3), 72 (100).
(+)-(S)-N1-(4-Chloro-2-methylphenyl)valinamide hydrochlo-
ride [(+)-(S)-3g]: 98% yield, mp 219-220 °C (MeOH/Et2O);
(-)-(S)-N2-(tert-Butoxycarbonyl)-N1-(4-chloro-2,6-dimeth-
ylphenyl)valinamide [(-)-(S)-2i]: 25% yield, mp 170-171 °C
[R]20 ) +40 (c 1.5, MeOH). Anal. (C12H18Cl2N2O) C, H, N.
D
(EtOAc/petroleum ether); [R]20 ) -71 (c 1, CHCl3).
(-)-(R)-N1-(2,6-Dimethylphenyl)leucinamide hydrochloride
D
General Procedure for the Synthesis of 2-Amino-N1-arylal-
kanamides Hydrochlorides (3a-i). The preparation of (-)-(R)-
N1-(2,6-dimethylphenyl)valinamide hydrochloride [(-)-(R)-3f] will
be described. (+)-(R)-N2-(tert-butoxycarbonyl)-N1-(2,6-dimeth-
ylphenyl)valinamide [(+)-(R)-2f] (0.32 g, 1.0 mmol) in 10 mL of
anhydrous diethyl ether was saturated with gaseous HCl and stirred
at room temperature for 15 min. After removal of the solvent under
reduced pressure, the residue was purified by recrystallization from
[(-)-(R)-3h]: 40% yield, mp 170-171 °C (abs EtOH/Et2O); [R]20
D
) -47 (c 1.6, MeOH); 1H NMR (300 MHz, CD3OD) δ 1.09 (d, J
) 6.0 Hz, 6H, CH3CH), 1.85-1.95 (br m, 3H, CH2CHCH3), 2.23
(s 6H, CH3Ar), 4.05-4.15 (m, 1H, CHCO), 7.88-7.90 (m, 3H,
Ar). Anal. (C14H23ClN2O) C, H, N.
(+)-(S)-N1-(2,6-Dimethylphenyl)leucinamide hydrochloride
[(+)-(S)-3h]: 77% yield, mp 168-170 °C (MeOH/Et2O); [R]20
D
) +48 (c 1.7, MeOH). Anal. (C14H23ClN2O) C, H, N.
(-)-(R)-N1-(4-Chloro-2,6-dimethylphenyl)valinamide hydro-
chloride [(-)-(R)-3i]: 25% yield, mp 248-250 °C (EtOH/Et2O);
[R]20D ) -39 (c 1, MeOH); 1H NMR (300 MHz, CD3OD) δ 1.14
(d, J ) 7.1 Hz, 3H, CH3CH CH3), 1.21 (d, J ) 7.1 Hz, 3H, CH3-
CHCH3), 2.23 (s 6H, CH3Ar), 2.40-2.52 (m, 1H, CH3CHCH3),
4.05 (d, J ) 4.1 Hz, 1H, CHCH), 7.14 (s, 2H, Ar). 13C NMR (75
MHz, CD3OD) δ 16.0 (1C), 17.5 (2C), 18.2 (1C), 30.6 (1C), 58.6
(1C), 127.8 (2C), 132.3 (1C), 132.8 (1C), 137.8 (2C), 167.4 (1C);
MS (70 eV): m/z (%): 254 (M+, 2), 72 (100). Anal. (C13H20-
Cl2N2O‚3/4H2O) C, H, N.
MeOH/Et2O, to give white needle like crystals [(-)-(R)-3f] (80
1
mg, 40%), mp 260-261 °C; [R]20 ) -49.8 (c 1.6, MeOH); H
D
NMR (300 MHz, CD3OD) δ 1.15 (d, J ) 6.9 Hz, 3H, CH3CHCH3),
1.23 (d, J ) 7.1 Hz, 3H, CH3CHCH3), 2.25 (s, 6H, CH3Ar), 2.40-
2.54 (m, 1H, CH3CHCH3), 4.07 (d, J ) 4.2 Hz, 1H, CHCO), 7.08-
7.12 (m, 3H, Ar); 13C NMR (75 MHz, CD3OD) δ 16.1 (1C), 17.6
(2C), 18.2 (1C), 30.6 (1C), 58.6 (1C), 127.6 (2C), 133.4 (1C), 135.6
(2C), 167.3 (1C).
(-)-(R)-N1-(4-Chlorophenyl)alaninamide hydrochloride [(-)-
(R)-3b]: 82% yield, mp 151-152 °C (MeOH/Et2O); [R]20D ) -8.1
1
(+)-(S)-N1-(4-Chloro-2,6-dimethylphenyl)valinamide hydro-
chloride [(+)-(S)-3i]: 30% yield, mp 246-248 °C (EtOH/Et2O);
(c 1.0, MeOH); the H NMR spectrum recorded on the free base
obtained via extraction from an analytical sample of the salt, was
in agreement with that reported in the literature for the (+)-(S)-
3b.42 Anal. (C9H12Cl2N2O) C, H, N.
[R]20 ) +46 (c 1, MeOH). Anal. (C13H20Cl2N2O‚H2O) C, H, N.
D
N-(4-Bromo-2,6-dimethylphenyl)-2,2,2-trifluoracetamide (4).
A solution of 4-bromo-2,6-dimethylaniline (0.50 g, 1.69 mmol) in
10 mL of anhydrous THF was added dropwise to a solution of
trifluoroacetic anhydride (0.79 g, 3.75 mmol) in anhydrous THF
(20 mL) under vigorous stirring, at 0 °C. The reaction mixture was
stirred at room temperature for 14 h and, after removing the solvent
under reduced pressure, taken up with EtOAc. The organic layer
was washed twice with 2 N HCl. The aqueous layers were extracted
twice with EtOAc, and the three organic phases so obtained were
pooled together and dried over Na2SO4. The solvent was then
removed under reduced pressure giving 4 which was then purified
by crystallization (EtOAc/petroleum ether) giving a pale brown solid
(0.35 g, 47%): mp 142-143 °C; IR (CHCl3) 3410, 3239 (NH),
(+)-(S)-N1-(4-Chlorophenyl)alaninamide hydrochloride [(+)-
(S)-3b]: 15% yield, mp hygroscopic (EtOH/Et2O); [R]20D ) +8.3
(c 1.2, MeOH). Anal. (C9H12Cl2N2O‚0.5H2O) C, H, N.
(-)-(R)-N1-(4-Chloro-2,6-dimethylphenyl)alaninamide hy-
drochloride [(-)-(R)-3c]: 41% yield, mp 251-252 °C (EtOH/
Et2O); [R]20D ) -37 (c 2, MeOH). Because of the formation of an
aggregated form in CD3OD solution, the NMR spectra were more
complex than expected. Both xylididic methyls and Ar HC-3,5 were
1
anisochronous. H NMR (300 MHz, CD3OD) δ 1.71 (d, J ) 7.1
Hz, 3H, CH3CH), 2.21 (s, 3H, CH3Ar C-2), 2.27 (s, 3H, CH3Ar
C-6), 4.26 (q, J ) 7.1 Hz, 1H, CH3CH), 7.10 (d, J ) 8.2 Hz, 1H,
Ar HC-3), 7.26 (d, J ) 8.2 Hz, 1H, Ar HC-5); 13C NMR (75 MHz,
CD3OD) δ 16.8 (1C), 17.1 (1C), 23.0 (1C), 49.2 (1C), 128.2 (1C),
128.5 (1C), 132.1 (1C), 133.8 (1C), 134.8 (1C), 168.7 (1C). Anal.
(C11H16Cl2N2O‚0.5H2O) C, H, N.
1745 (CO) cm-1; H NMR (300 MHz, CD3OD) δ 2.18 (s, 6H,
1
CH3), 7.31 (s, 2H, ArH); 13C (75 MHz, CD3OD) δ 16.6 (2C), 116.5
(q, J ) 287.4 Hz, 1C), 121.4 (1C), 130.9 (2C), 131.4 (1C), 137.9
(2C), 156.4 (q, J ) 37.2 Hz, 1C). MS (70 eV) m/z (%): 295 (M+,
62), 147 (100).
(+)-(S)-N1-(4-Chloro-2,6-dimethylphenyl)alaninamide hydro-
chloride [(+)-(S)-3c]: 78% yield, mp 255-256 °C (EtOH/Et2O);
[R]20 ) +35 (c 2, MeOH). Anal. (C11H16Cl2N2O) C, H, N.
N-(4-Chloro-2,6-dimethylphenyl)trifluoroacetamide (5).20
A
D
(-)-(R)-N1-(2-Methylphenyl)alaninamide hydrochloride [(-)-
(R)-3d]: 47% yield, mp 221-222 °C (EtOH/Et2O); [R]20D ) -27
(c 1, MeOH); 1H NMR (300 MHz, CD3OD) δ 1.66 (d, J ) 7.1 Hz,
3H, CH3CH), 2.27 (s, 3H, CH3Ar C-2), 4.21 (q, J ) 7.1 Hz, 1H,
CH3CH), 7.13-7.21 (m, 1H, Ar HC-3,4), 7.21-7.24 (m, 1H, Ar
HC-5), 7.34 (dd, J ) 7.1 Hz, 2.2 Hz 1H, Ar HC-6); 13C NMR (75
MHz, CD3OD) δ 16.8 (1C), 16.9 (1C), 49.4 (1C), 125.8 (1C), 126.3
(1C), 126.8 (1C), 130.6 (1C), 133.2 (1C), 134.7 (1C), 168.8 (1C).
Anal. (C10H15ClN2O) C, H, N.
mixture of 4 (3.0 g, 10.1 mmol) and CuCl (6.0 g, 61 mmol) in 30
mL of anhydrous DMSO was heated at 188 °C for 6 h; the reaction
mixture was then cooled at room temperature and poured into water.
The resultant crude solid was collected and redissolved with EtOAc.
The organic layer was dried over anhydrous Na2SO4 and concen-
trated under reduced pressure to give 5 (0.63 g, 84%) as a brown
solid: mp 135-137 °C; IR (KBr) 3457, 3240 (NH), 1714 (CO)
1
cm-1; H NMR (300 MHz, CD3Cl3) δ 2.15 (s, 6H, CH3), 7.08 (s,
2H, ArH), 7.71 (br s, 1H, NH); MS (70 eV) m/z (%) 251 (M+, 85),
182 (100).
(+)-(S)-N1-(2-Methylphenyl)alaninamide hydrochloride [(+)-
(S)-3d]: 50% yield, mp 224-225 °C (EtOH/Et2O); [R]20D ) +23
(c 1.2, MeOH). Anal. (C10H15ClN2O‚0.5H2O) C, H, N.
4-Chloro-2,6-dimethylaniline (6).43 A solution of 5 (1.00 g, 4
mmol) in a mixture of HCl 17% (25 mL) and THF (25 mL) was
refluxed for 7 h; water was then added in order to quench the
reaction, and the aqueous phase was extracted three times with Et2O.
The aqueous phase was treated with 4 N NaOH until it reached
pH 14 and was then extracted with Et2O (3 × 50 mL). The
combined organic layers were dried over anhydrous Na2SO4 and
(+)-(S)-N1-(2,6-Dimethylphenyl)valinamide hydrochloride [(+)-
(S)-3f]: 66% yield, mp 256-258 °C (MeOH/Et2O); [R]20D ) +43
(c 1, MeOH). Anal. (C13H21ClN2O) C, H, N.
(-)-(R)-N1-(4-Chloro-2-methylphenyl)valinamide hydrochlo-
ride [(-)-(R)-3g]: 40% yield, mp 230-231 °C (MeOH/Et2O);

1914 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Corbo et al.
concentrated under reduced pressure to give 6 (0.6 g, 98%) as a
yellow oil: 1H NMR (300 MHz, CD3Cl3) δ 2.15 (s, 6H, CH3),
3.54 (br s, 2H, NH2), 6.93 (s, 2H, ArH); MS (70 eV) m/z (%)155
(M+, 100). The corresponding hydrochloride was obtained by
dissolving 0.5 g of 6 in Et2O and then adding few drops of 2 N
HCl. On standing, a gray solid formed: mp 238-239 (lit.: 239.0-
14.0, 7.0 Hz, 1H, CHHCH3), 3.65-3.70 (m, 1H, CH CH3), 3.96
(dt, J ) 14.2, 7.2 Hz, 1H, CHHCH3), 7.08-7.20 (m, 3H, Ar), 8.24
(br s, 3H, NH3). Anal. (C13H21ClN2O), C, H, N.
(+)-(S)-N1-(2,6-Dimethylphenyl)-N1-ethylalaninamide hydro-
chloride [(+)-(S)-8b]: 50% yield, mp 163-164 °C (MeOH/Et2O);
[R]20 ) +60 (c 1.0, MeOH). Anal. (C13H21ClN2O), C, H, N %
D
1
239.5 °C); H NMR (300 MHz, DMSO-d6) δ 2.31 (s, 6H, CH3),
was found to be -0.49 of the theoretical value.
7.17 (s, 2H, ArH), 9.53 (br s, 3H, NH3).
(-)-(R)-N1-(2,6-Dimethylphenyl)-N1-methylvalinamide L-Tar-
trate [(-)-(R)-8c‚C4H6O6]. A solution of (-)-(R)-1-N1-(tert-
butoxycarbonyl)-N1-(2,6-dimethylphenyl)-N1-methylvalinamide [(-)-
(R)-7c, 1 mmol] in EtOAc was treated with 48% HBr under
vigorous stirring at room temperature for 1 h. After this time, the
organic layer was removed under reduced pressure and the aqueous
phase was alkalinized with 2 N NaOH, extracted twice with EtOAc,
and dried over Na2SO4 to give a yellow oil that was converted into
the corresponding tartrate as follows. A solution of 8c (1 mmol) in
EtOH was mixed with a solution of L-tartaric acid (1 mmol) in
EtOH and stirred at room temperature for 10 min; the solvent was
then removed under reduced pressure giving 8c as a white solid
that was recrystallized from acetone to give 8c‚tartrate in the form
of white crystals (0.27 g, 70%). mp 146-148 °C; [R]20D ) -28 (c
1.4, MeOH). In CD3OD solution two forms were present; for the
sake of simplicity, only the predominating NMR spectrum will be
described: 1H NMR (300 MHz) δ 0.76 (d, J ) 7.1 Hz, 3H, CH3-
CHCH3), 0.95 (d, J ) 7.2 Hz, 3H, CH3CHCH3), 1.79-1.86 (m,
1H, CH3CHCH3), 2.25 (s, 3H, CH3Ar), 2.26 (s, 3H, CH3Ar), 3.19
(s, 3H, CH3N), 3.57 (d, J ) 2.7 Hz, 1H, CHCO), 4.40 (s, 2H,
CHOH), 7.20-7.30 (m, 3H, ArH); 13C NMR (75 MHz, CD3OD, δ
46.2) δ 13.5 (1C), 15.1 (1C), 15.3 (1C), 16.8 (1C), 26.0 (1C), 33.4
(1C), 55.3 (1C), 71.4 (2C), 127.5 (1C), 127.9 (2C), 134.0 (1C),
134.5 (1C), 137.3 (1C), 167.3 (1C), 174.1 (2C). Anal. (C18H28N2O7‚
H2O) C, H, N.
General Procedure for the Synthesis of N-Alkyl-N1-Aryl-2-
(tert-butoxycarbonylamino)alkanamides (7a-c). The preparation
of (-)-(R)-N2-tert-butoxycarbonyl-N1-(2,6-dimethylphenyl)-N-me-
thylalaninamide (7a) can be taken as the reference procedure for
the synthesis of N-alkyl-N1-aryl-2-(tert-butoxycarbonylamino)-
alkanamides (7a-c). N2-(tert-Butoxycarbonyl)-N1-(2,6-dimeth-
ylphenyl)alaninamide (2a) (0.298 g, 1 mmol) was dissolved in
DMF/H2O (30 mL)/(10 mL). Ba(OH)2‚8H2O (1.9 g, 6 mmol) and
CH3I (0.85 g, 6 mmol) were added. This suspension was stirred at
room temperature for 12 h. The yellow reaction mixture was then
filtered to remove the excess Ba(OH)2, followed by three extractions
with petroleum ether. The organic layer was then dried over
anhydrous Na2SO4, and the solvent was then removed under reduced
pressure to give a crude yellowish oil (0.28 g, 90%): [R]20
)
D
-47 (c 1, CHCl3); MS (70 eV): m/z (%) 306 (M+, 1), 44 (100).
(+)-(S)-N2-tert-Butoxycarbonyl-N1-(2,6-dimethylphenyl)-N1-
methylalaninamide [(+)-(S)-7a]: 80% yield, oil; [R]20 ) +49
D
(c 1, CHCl3).
(-)-(R)-N2-tert-Butoxycarbonyl-N1-(2,6-dimethylphenyl)-N1-
ethylalaninamide [(-)-(R)-7b]: 50% yield, oil; [R]20 ) -28 (c
D
1, CHCl3); IR (CHCl3): 3433 (NH), 1705 (NHCOO), 1650
(NHCO), cm-1; H NMR (200 MHz, CDCl3) δ 1.04 (d, J ) 6.72
1
Hz, 3H, CH3CH), 1.12 (t, J ) 7.3 Hz, 3H, CH3 CH2), 1.39 (s, 9H,
CH3 C), 2.21 (s, 3H, CH3Ar), 2.22 (s, 3H, CH3Ar), 3.27 (dt, J )
13.4, 7.2 Hz, 1H, CHHCH3), 3.9-4.1 (m, 2H; CHHCH3
+
(+)-(S)-N1-(2,6-Dimethylphenyl)-N1-1-methylvalinamide L-
CHCH3), 5.42 (br d, J ) 8.4, 1H, NHCO), 7.0-7.2 (m, 3H, ArH);
tartrate [(+)-(S)-8c‚C4H6O6]: 80% yield, mp 160-163 °C; [R]20
) +41 (c 1.8, MeOH). Anal. (C18H28N2O7‚H2O) C, H, N.
D
MS (70 eV): m/z (%) 320 (M+, 1), 44 (100).
(+)-(S)-N2-tert-Butoxycarbonyl-N1-(2,6-dimethylphenyl)-N1-
(+)-(R)-2-tert-Butoxycarbonylamino-N-(2,6-dimethylphenyl)-
propanethioamide [(+)-(R)-9]. A solution of 2a (0.29 g, 1 mmol)
and 2,4-bis-(p-pentyloxyphenyl)-1,2,3,4-dithiadiphosphethane 2,4-
disulfide (0.52 g, 1mmol) in benzene was refluxed for 2 days. After
this time, the reaction mixture was washed with Na2CO3 5% (pH
12), and the alkaline layer was extracted three times with Et2O.
The combined organic layers were dried over Na2SO4 and
concentrated under reduced pressure, giving a yellowish oil that
was purified by silica gel chromatography (petroleum ether/EtOAc
ethylalaninamide [(+)-(S)-7b]: 65% yield, oil; [R]20 ) +30 (c
D
1, CHCl3).
(-)-(R)-N2-tert-Butoxycarbonyl-N1-(2,6-dimethylphenyl)-N1-
methylvalinamide [(-)-(R)-7c]: 60% yield, oil; [R]20 ) -21 (c
D
1, CHCl3); IR (CHCl3): 1707 (CO, carbamate), 1649 (CO, amide)
cm-1; H NMR (300 MHz, CDCl3) δ 0.67 (d, J ) 6.9 Hz, 3H,
1
CH3CHCH3), 0.80 (d, J ) 6.9 Hz, 3H, CH3CHCH3), 1.39 (s, 9H,
CH3C), 1.62-1.76 (m, 1H, CH3CHCH3), 2.17 (s, 3H, CH3Ar), 2.23
(s, 3H, CH3Ar), 3.14 (s, 3H, CH3N), 3.94 (dd, J ) 6.8, 4.4 Hz,
1H, CHNH), 5.25 (br d, J ) 9.6 Hz, 1H, NHCO), 7.05-7.22 (m,
3H, ArH); MS (70 eV); m/z (%) 334 (M+, 1), 57 (100).
2:1) to produce 9 (0.15 g, 50%) as a white solid: mp 150-152
1
°C; [R]20 ) +34.2 (c 2, CHCl3); H NMR (300 MHz, CDCl3) δ
D
1.36 (s, 9H, CH3C), 1.61 (d, J ) 6.8 Hz, 3H, CH3CH), 2.21 (s,
6H, CH3Ar), 4.80 (apparent quintet, J ) 6.8 Hz, 1H, CH), 5.60 (br
d, J ) 6.8 Hz, 1H, NHCOO), 7.08-7.11 (m, 2H, Ar HC-3,5),
7.15-7.20 (m, 1H, Ar HC-4), 9.82 (br s, 1H, NHAr); MS (70 eV)
m/z (%) 308 (M+, 22), 293 (100).
(+)-(S)-N2-tert-Butoxycarbonyl-N1-(2,6-dimethylphenyl)-N1-
methylvalinamide [(+)-(S)-7c]: 65% yield, oil; [R]20 ) +31 (c
D
1, CHCl3).
(-)-(R)-N1-(2,6-Dimethylphenyl)-N1-methylalaninamide hy-
drochloride [(-)-(R)-8a]:44 50% yield, mp 190-205 °C (abs
EtOH/Et2O); [R]20D ) -25 (c 1.75, MeOH); IR (KBr): 1651 (CO)
cm-1; two forms were present in CD3OD solution; for the sake of
simplicity, only the predominating one will be described; 1H NMR
(300 MHz) δ 1.18 (d, J ) 6.9 Hz, 3H, CH3CH), 2.26 (s, 3H, CH3
Ar), 2.28 (s, 3H, CH3 Ar), 3,19 (s, 3H, CH3N), 3.63 (q, J ) 7.0,
1H, CH), 7.22-7.31 (m, 3H, ArH); 13C NMR (75 MHz, CD3OD)
δ 15.1 (1C), 16.6 (1C), 16.9 (1C), 34.8 (1C), 35.9 (1C), 129.3 (1C),
129.5 (1C), 129.6 (1C), 135.7 (1C), 136.0 (1C), 138.8 (1C), 169.6
(1C); MS (70 eV) m/z (%) 206 (M+, 1), 44 (100). Anal. (C12H19-
ClN2O) C, H, N.
(-)-(S)-2-tert-Butoxycarbonylamino-N-(2,6-dimethylphenyl)-
propanethioamide [(-)-(S)-9]: 60% yield, mp 148-150 °C; [R]20
) -16.9 (c 2, CHCl3).
D
(-)-(R)-2-Amino-N-(2,6-dimethylphenyl)propanethioamide Hy-
drochloride [(-)-(R)-10]. Obtained in a 30% yield via the method
reported above for its oxygenated isologous (3a): mp 230 °C dec
1
(EtOH/Et2O); [R]20 ) -59 (c 2, MeOH); H NMR (300 MHz,
D
CDCl3 + (CD3)2SO) δ 1.65 (br d, J ) 6.2, 3H, CH3CH), 2.04 (s,
3H, CH3Ar), 2.12 (s, 3H, CH3Ar), 2.72 (br s, 3H, NH3), 4.80-
4.82 (m, 1H, CH), 6.94-7.00 (m, 3H, ArH), 8.39 (br s, 1H, NHCS);
Anal. (C11H17ClN2S) C, H, N.
(+)-(S)-N1-(2,6-Dimethylphenyl)-N1-methylalaninamide hy-
drochloride [(+)-(S)-8a]: 70% yield, mp 191-193 °C (MeOH/
Et2O); [R]20D ) +40 (c 1.75, MeOH). Anal. (C12H19ClN2O) C, H,
N.
(+)-(S)-2-Amino-N-(2,6-dimethylphenyl)propanethioamide hy-
drochloride [(+)-(S)-10]: 60% yield, mp 230 °C dec (EtOH/Et2O);
[R]20 ) +58 (c 2, MeOH). Anal. (C11H17ClN2S‚1/3‚H2O) C, H,
D
(-)-(R)-N1-(2,6-Dimethylphenyl)-N1-ethylalaninamide hydro-
N.
chloride [(-)-(R)-8b]: 40% yield, mp 162-163 °C (MeOH/Et2O);
1
[R]20 ) -90 (c 1.5, MeOH); H NMR (300 MHz, DMSO-d6) δ
Supporting Information Available: Elemental analysis of the
compounds submitted for pharmacological evaluation. This material
is available free of charge via the Internet at http://pubs.acs.org.
D
0.99 (d, J ) 6.9 Hz, 3H, CH3-CH), 1.04 (t, J ) 7.3 Hz, 3H, CH3
CH2), 2.18 (s, 3H, CH3Ar), 2.22 (s, 3H, CH3Ar), 3.19 (dt, J )

Chiral R-aminoanilides
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JM061078E