Synthesis and pharmacological evaluation of bivalent antagonists of the nociceptin opioid receptor

Article abstract of DOI:10.1016/j.ejmech.2011.01.040

Bivalent ligands constituted by two identical pharmacophores structurally related to the Nociceptin Opioid Receptor (NOPr) antagonist JTC-801 were synthesized and their binding affinities for NOPr were evaluated. The novel ligands are formed by two modifi

Full text of DOI:10.1016/j.ejmech.2011.01.040

European Journal of Medicinal Chemistry 46 (2011) 1207e1221
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological evaluation of bivalent antagonists of the
nociceptin opioid receptor
*
Maria Rosaria Del Giudice , Anna Borioni, Giuditta Bastanzio, Maria Sbraccia, Carlo Mustazza,
Isabella Sestili
Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Bivalent ligands constituted by two identical pharmacophores structurally related to the Nociceptin
Opioid Receptor (NOPr) antagonist JTC-801 were synthesized and their binding affinities for NOPr were
evaluated. The novel ligands are formed by two modified JTC-801 units linked by di-iminic and di-aminic
spacers with length ranging from three to ten methylene units. Moreover, the synthesis and the phar-
macological characterization were extended to the corresponding univalent ligands. The latter
compounds consisted in a single modified JTC-801 unit and an alkyl or alkylamino or alkylimino tail. The
purpose of this study is to feature the location and surroundings of the allosteric binding site(s) of
pharmacophores containing the 4-aminoquinoline structure. Most important, the bivalent ligands were
exploited to reveal the eventual occurrence of a supramolecular receptorial architecture of the NOPr.
All the bivalent derivatives 4 and 5 proved to be active in the nanomolar range with no outstanding
dependence on the chain length. They showed potencies from three to ten times higher than the cor-
responding monomers. Consequently, results clearly indicated a positive role of the second pharmaco-
phore in the ligandeprotein interaction. The pharmacological profile of the monomers 7 and 8 clarified
the contribution of the linker chain to NOP receptor affinity and suggested the presence of a lipophilic
acidic site neighbouring the binding site of the JTC-like ligands.
Received 11 October 2010
Received in revised form
17 January 2011
Accepted 25 January 2011
Available online 3 February 2011
Keywords:
Synthesis
Quinoline
Bivalent ligand
NOP receptor antagonism
Selectivity of saturated compounds 5, 7, and 8 was tested by binding experiments on
d
,
k
and
m opioid
receptors. Results indicated a general loss of selectivity as compared to JTC-801. In the [35S]GTP
assay, all the compounds revealed antagonistic properties at the NOP Receptor.
g
S binding
In conclusion the present study set the basis for a systematic investigation on the structural modifi-
cations that can be introduced into novel ligands for NOPr and helped to feature the surrounds of the
allosteric site of NOPr.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
ligands, namely the 4-substituted piperidines and spiropiper-
idines. In the interaction with the NOP receptor, JTC-801 showed
The nociceptin (NOP) receptor [1] is the most recently discov-
ered member of the family of the opioid receptor. The NOP
receptor antagonists currently attract a rapidly growing pharma-
cological interest. Their perspective clinical applications are as
analgesics devoid of tolerance, anorexiants, memory enhancers,
antidepressants and in the treatment of Parkinson’s disease [2].
The first NOPr antagonist used in clinical trials was the analgesic
JTC-801 [3e5], whose molecular structure is based on N-(4-amino-
2-methylquinolin-6-yl)-2-phenoxymethylbenzamide moiety. It
departs from that of the most widely studied classes of NOP
peculiar characteristics associated to structural and conformational
aspects [5]. In Schild analysis [5] it behaved as a non-competitive
antagonist, probably bound to an allosteric site of NOPr. At present,
the location and attributes of JTC-801 binding site are still
undisclosed.
As not much is known about putative NOPr allosteric antago-
nists, we chose JTC-801 as the lead structure to generate new NOPr
ligands in univalent and bivalent form.
The development of bivalent ligands is nowadays a common
practice in medicinal chemistry [6e9]. In particular, bifunctional
ligand design and targeting of dimeric receptors were recently
evidenced as big novel opportunities in the field of opioid receptor
ligands. Bivalent ligands could exhibit distinct pharmacokinetic
and pharmacological profiles compared to the lead univalent
* Corresponding author. Tel./fax: þ39 06 49902516.
E-mail address: mariarosaria.delgiudice@iss.it (M.R. Del Giudice).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.040

1208
M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
O
ligands so far reported were developed as selective agonists for the
O
peripheral nervous system (patent by Clark & Elbing LLP) [13].
These compounds, structurally related to the orthosteric NOP
ligand Ro 646198 [14], showed about 6-fold lower affinity as
compared with the originator (Fig. 1).
N
N
N
N
N
N
Novel compounds 4 and 5 (Fig. 2) are the first bivalent NOPr
ligands based on antagonist pharmacophores. A modification of
JTC-801 structure was accomplished by inserting an iminic eCH]
Nealkyl or an aminic eCH2NHealkyl linker between the two
pharmacophoric units (Fig. 2). Aliphatic linker chains ranged from 3
to 10 methylene units with a corresponding elongation from 4.8 Å
to 13.7 Å. At present, no information on the location of allosteric
sites in nociceptin receptor was available in the literature. There-
fore, this series of bivalent compounds covering a wide range of
distances represent a starting point as probes to the existence of
native dimeric forms of the NOPr.
(Clark & Elbing LLP), IC50 =25.2 nM
O
O
(CH ) N(CH )
NH
N
N
N
N
N
Ro 646198, IC50 = 4.4 nM
(Clark & Elbing LLP), IC50 = 5.5 nM
Fig. 1. Structures of dimeric and monomeric ligands from 1,3,8-triazaspiro[4.5]decan-
4-one pharmacophore.
All the novel compounds, including the univalent congeners 6, 7
and 8 (Fig. 2), were also synthesized and tested to elucidate the
features and surroundings of the binding site(s) location of 4-
aminoquinoline derived pharmacophores.
A structure/activity
compounds, showing unexpected effects on potency and selec-
tivity. The two pharmacophoric moieties of bivalent ligands have
been depicted to interact cooperatively with different units of the
dimeric receptor [10e12].
relationship of bivalent azomethines 4 and univalent azomethines
6 made possible to investigate the influence of configurational
rigidity on the pharmacological properties.
Unsaturated and saturated univalent alkyl derivatives 6, 7
were a helpful tool to factor out the contribution of the aliphatic
chain to the ligandeprotein interaction. Pharmacological profile
of the univalent aminic compounds 8 aided in elucidating the
Moreover, bivalent ligands were used to achieve selectivity
among central and peripheral effects of widely distributed recep-
tors, as they often display different BloodeBrain Barrier permeation
in respect to the monomeric unit. Indeed the only bivalent NOPr
DIMERIC COMPOUNDS
H2N
N
N
O
O
NH
NH2
N
H
O
O
X
4 (a-h): X = -CH=N-(CH2)n-N=CH-
5 (a-h): X= -CH2-NH-(CH2)n-NH-CH2-
REFERENCE COMPOUNDS
MONOMERIC COMPOUNDS
H2N
5'
6'
NH2
4
4'
3'
H
5
N
N
O
3
2
O
N
H
7
O
N
1
CH3
n = 3-10
8
phenoxy
portion
2"
3"
6"
5"
O
4"
R
R
JTC-801 R = -CH2CH3 IC50
=
30 nM
IC50 = 207 nM
R = -CH2NH2 IC50 = 116 nM
6
R = -CH=N-(CH2)n-1-CH3
R = -CH2-NH-(CH2)n-1-CH3
8 R = -CH2-NH-(CH2)n-NH2
3
R = -CHO
7
13
Fig. 2. Structures of NOP receptor ligands and synthesized compounds.

M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
CHO
CHO
1209
CHO
CHO
OH
e
a
b
c , d
O
O
O
NH2
N
COOH
CONH
COOCH3
1
2
3
O
CH2-NH-(CH2)n-NHCH2
O
CH=N-(CH2)n-N=CH
O
O
f
NH
N
NH
N
O
O
O
O
NH2
NH
NH2
NH
NH2
NH2
N
4 a-h
N
5 a-h
Br
a:
16% aq. NaOH / THF / MeOH 1:1.5:1.5, 80°C; c: (COCl)2, CH2Cl2,
K2CO3 DMF, 25°C; b:
25°C
;
/
COOCH3
(n = 3 - 10)
e:
MeOH, 25°C;
NH2(CH2)nNH2
4,6-Diamino-2-methylquinoline, Pyridine, 25°C;
NaBH4, MeOH, 25°C.
d:
f:
,
Scheme 1. Synthetical route to compounds 4 and 5.
Aldehyde 3
a
-
X
CH=N-(CH2)
CH2-NH-(CH2)
- NH2
CH2-NH-(CH2)
- X
n-1
n
n-1
b
c
(for N-Fmoc derivatives)
O
O
O
O
O
O
NH
NH
NH
NH2
NH2
NH2
N
N
N
6 a-h
X = CH3
X = CH3
, 7 a-h
,
8a-h
X = CH2-NHFmoc,
X = CH2-NHFmoc,
non isolated intermediates
non isolated intermediates
b: NaBH , MeOH, 25°C;
MeOH, 25°C;
a: monoamine or N-Fmocdiamine
(n= 3 - 10),
4
c:
20% piperidine / DMF, 25°C.
Scheme 2. Synthetical route to compounds 6, 7 and 8.

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M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
CH2NH2
CH2NHCOCF3
a
CH2NHCOCF3
CH2NHCOCF3
d, e
b
c
O
O
O
OH
COOH
COOCH3
COOH
9
10
11
CH2NHCOCF3
CH2NH2
f
H2N
H2N
O
O
CONH
N
CONH
12
N
13
Br
a:
Pyridine,
16% aq. NaOH / THF / MeOH 1:1.5:1.5, 90°C; c: TFAA, DIPEA,
K2CO3 DMF, 30°C; b:
/
COOCH3
f: NH
3 / MeOH, 25°C.
15°C;
d:
(COCl) , Toluene, 25°C
;
e:
4,6-Diamino-2-methylquinoline, Pyridine, 25°C;
CH2Cl2 ,
2
Scheme 3. Synthetical route to compound 13.
role played by the nitrogen atoms located at the extremities of
the aliphatic chain. Finally N-(4-amino-2-methylquinolin-6-yl)-
2-[(4-aminomethyl)phenoxymethyl]benzamide 13 (Fig. 2) was
synthesized to evaluate the change of pharmacological proper-
ties of JTC-801 due to the substitution of the 4-ethyl group with
an aminomethyl moiety.
2. Chemistry
The synthetic pathway to the new compounds is outlined in
Schemes 1 and 2.
4-Hydroxybenzaldehyde was allowed to react with methyl 2-
(bromomethyl)benzoate [3] and potassium carbonate to give methyl
Fig. 3. 1H 1D NMR spectra of the synthesized compounds. 4b is representative of the di-iminic derivatives, 5h is representative of the di-aminic derivatives, 6b is representative of
the alkyliminic derivatives and 8f is representative of the aminoalkylaminic derivatives. Typical feature of compounds 4b and 6b is the iminic CH] proton at 8.15 ppm. In the
spectra of 4b and 6b the phenolic H300,H500 merge with the toluic protons at about 7.64 ppm, while in the spectra of 5h and 8f phenolic H300,H500 are shielded to 7.20 ppm.

M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
1211
a
H2C
α
H
N
H
N
CH2
α
azomethine
azomethine
4"
4"
O
O
cis
trans
(CH2)
n
b
X
n
8
5
9
r(Å)
12.2
9.8
N
X
N
X
4f
5c
5g
-CH=
-CH2
-CH2
r (Angstrom)
14.7
O
O
Fig. 4. Panel a: Azomethines 4 cis/trans configuration. Panel b: Distance between carbon atoms substituting the 400 positions of the pharmacophore units in the dimeric compounds
4f, 5c and 5g. The distances determined by Molecular Modelling Chem-X software were calculated for ligands at the minimum energy conformation.
2-(4-formylphenoxymethyl)benzoate 1. The 2-(4-formylphenoxym-
ethyl)benzoic acid 2, obtained by alkaline hydrolysis of 1, was treated
with oxalyl chloride. The obtained acyl chloride and 4,6-diamino-2-
methylquinoline [5] gave 4-amino-6-[[2-(4-formylphenoxymethyl)
benzoyl]amino]-2-methylquinoline 3. The formyl derivative 3 was
the starting material for the preparation of the insaturated
compounds4 (Scheme 1) and6 (Scheme 2) by reactionwithdiamines
and monoalkylamines in methanol. The very slow formation of the
Schiff bases 4 and 6, as well as the previous reaction affording
compound 3 were a typical example of “lazychemistry”. Reduction of
azomethines 4 and6 with sodium borohydride gave the finalbivalent
and univalent derivatives 5 (Scheme 1) and 7 (Scheme 2).
Fig. 5. 1H-1D NMR spectrum of compound 4b. 2D NOESY is inserted in the 1D spectrum. Both spectra were acquired in DMSO-d6. In the NOESY the cross peak between the
azomethine proton and the alpha methylene of the amine chain is evidenced attesting the trans configuration of 4b.

1212
M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
Aminoalkyl monomers 8aeh were prepared by reaction of
prepared from a cell line expressing a tandem fusion protein
between the NOP receptor and the subunit of Go. As previously
reported [16], the forced 1:1 stoichiometry of expression of
receptor and G subunit in such system greatly enhances the GTP
aldehyde 3 and N-Fmoc diamines following the procedure previ-
ously described for the monomers 7 (Scheme 2). Final Fmoc group
cleavage mixture gave the expected compounds 8.
a
a
gS
The synthesis of compound 13 is outlined in Scheme 3. The
starting product, 2,2,2-trifluoro-N-[(4-hydroxyphenyl)methyl]
acetamide [15], was O-alkylated with methyl 2-(bromomethyl)
benzoate to give 9, which in turn, by alkaline hydrolysis to the
aminoacid 10 and reprotection of the amine group with trifluoro-
acetic anhydride, gave 11. Coupling of the acyl chloride derived
from the latter with 4,6-diamino-2-methylquinoline gave 12,
which eventually, by removal of the trifluoroacetyl group with
methanolic ammonia, gave the target compound 13.
The structure of all compounds was confirmed by mono and
bidimensional 1H NMR experiments and MS spectrometry. Some
representative 1H NMR monodimensional spectra have been
reported (Fig. 3).
response and allows detecting even small levels of ligand efficacy.
4. Results and discussion
4.1. Affinity for NOP receptor
Affinity data of all the synthesized compounds were reported in
Tables 1 and 2 and an overview of the affinity e structure rela-
tionship of 4 and 5 bivalent derivatives was depicted in Fig. 6.
Generally, compounds 4 showed a decrease in affinity in respect to
the lead compound JTC-801. This trend was more pronounced in
the case of long chain spacers and may be explained by the pres-
ence of the trans azomethine double bond that conferred a rigid
distended shape to these molecules (Fig. 4a). The presence of the
double bond proved to be less detrimental for binding for the
smaller molecules 4aec. Elongation of the spacer up to 10 methy-
lene units led to a severe loss in affinity in compounds 4deh. The
lowest affinity was reached by 4f (n ¼ 8, IC50 ¼ 113.6 nM), which is
characterized by a distance of 12 Å between the methinic carbons of
the pharmacophoric units (Fig. 4b). Results suggested that the
interaction with the protein could be influenced by the orientation
and flexibility of molecules. In agreement with this hypothesis, an
opposite trend was detected for the more flexible diamines 5
whose affinity increased with chain elongation. Compounds 5
bearing the shorter spacers interacted with the protein more
weakly than the corresponding unsaturated compounds 4.
Compound 5c (n ¼ 5, IC50 ¼ 76.2 nM), for which a distance of about
10 Å (Fig. 4b) was calculated, was the less active compound in this
series (Table 1). Aliphatic chain elongation from 7 to 10 methylene
units, potentially extending to a distance of 16.3 Å, led to a gradual
recovery of affinity (Fig. 6). Affinity reached its best value in
derivative 5g (n ¼ 9, IC50 ¼ 28.1 nM, distance 14.7 Å).
Cis/trans configuration of imines 4 (Fig. 4a) was established by
2D NOESY NMR experiment performed on compound 4b (Fig. 5).
NOE was observed between the azomethinic proton and the N-
a
methylene group of the aminic spacer. Therefore, results sup-
ported the hypothesis of a short spatial distance between these
groups, which is in agreement with a trans configuration. Accord-
ingly, no NOE cross peaks were detected between the N-a methy-
lene group and the phenolic protons that should be spatially close
in the cis configuration.
3. Pharmacology
The binding affinities of the synthesized compounds were
measured by employing [125I]-nociceptin as radiotracer [16]. The
most active compounds were also tested on
estimate their selectivity towards all the opioid receptor subtypes.
In such experiments [3H]-naltrindole for , and [3H]-diprenorphine
for and were used as radioligands [17,18]. The results are shown
d, k and m receptors to
d
k
m
in Table 1 and Table 2.
The biological efficacy of the compounds was evaluated as their
The removal of one pharmacophore unit in compounds 4 caused
a marked decrease in affinity. So the univalent alkylimino
ability to enhance the binding of [35S]GTP
gS in the presence of
a fixed or varied concentration of GDP, using membranes isolated
from cells transfected with NOP receptors. The membranes were
compounds 6 showed IC50 values in a range of about 100e300 nM.
The aliphatic tail played a negative role in the ligandeprotein
Table 1
Binding affinity of dimeric compounds 4 and 5.
Compound
Structure
Compound
Affinity
Selectivity
Linker
IC50(nM) ꢀ SD
n
NOP
k
m
IC50k/IC50NOP
IC50m/IC50NOP
3
4
5
6
7
8
9
10
4a
4b
4c
4d
4e
4f
48.0 ꢀ 11.0
35.6 ꢀ 4.7
50.9 ꢀ 10.7
83.3 ꢀ 6.1
63.1 ꢀ 16.0
113.6 ꢀ 10.3
82.7 ꢀ 2.8
82.1 ꢀ 3.0
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
4g
4h
3
4
5
6
7
8
9
10
5a
5b
5c
5d
5e
5f
76.5 ꢀ 15.9
56.6 ꢀ 4.7
76.2 ꢀ 2.9
71.7 ꢀ 14.5
30.1 ꢀ 1.1
31.6 ꢀ 2.3
28.1 ꢀ 1.9
32.7 ꢀ 7.1
5475 ꢀ 70
1091 ꢀ 15
1496 ꢀ 20
1347 ꢀ 18
1023 ꢀ 12
1269 ꢀ 63
1049 ꢀ 16
900 ꢀ 12
468 ꢀ 10
177
27
28
30
33
37
36
31
15
39
46
31
34
56
40
45
1590 ꢀ 25
2505 ꢀ 55
1390 ꢀ 17
1038 ꢀ 15
1900 ꢀ 59
1160 ꢀ 64
1320 ꢀ 16
5g
5h
JTC-801
30.0 ꢀ 1.3
7512 ꢀ 80
1507 ꢀ 19
250
50
Radioligands: [125I]-nociceptin for NOP, [3H]-naltrindole for
d
and [3H]-diprenorphine for
k
and
m
receptors. Data are given as mean ꢀ SD (n ¼ 3).

M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
1213
Table 2
Binding affinity of monomeric compounds 6, 7, 8 and of compounds 3 and 13.
Compound
Affinity
Selectivity
Structure
Substituent
Compound
IC50(nM) ꢀ SD
NOP
n
k
m
IC50k/IC50NOP
IC50m/IC50NOP
3
4
5
6
7
8
9
10
6a
6b
6c
6d
6e
6f
98.9 ꢀ 5.8
308.9 ꢀ 18.5
186.2 ꢀ 71.4
208.6 ꢀ 26.4
141.9 ꢀ 39.8
146.5 ꢀ 18.2
154.5 ꢀ 2.5
184.5 ꢀ 91.2
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
6g
6h
3
4
5
6
7
8
9
10
7a
7b
7c
7d
7e
7f
83.0 ꢀ 6.0
1115.9 ꢀ 32.6
731.6 ꢀ 32.2
1217.8 ꢀ 82.7
667.5 ꢀ 32.0
482.9 ꢀ 57.8
304.6 ꢀ 28.3
404.7 ꢀ 37.7
3079 ꢀ 87
5924 ꢀ 78
3669 ꢀ 109
1157 ꢀ 63
1850 ꢀ 24
4955 ꢀ 74
2565 ꢀ 58
4348 ꢀ 61
3345 ꢀ 94
5114 ꢀ 83
4453 ꢀ 67
2593 ꢀ 51
1977 ꢀ 25
4339 ꢀ 67
2171 ꢀ 55
2755 ꢀ 62
45
10
13
1.2
6.2
15
13
15
49
8.6
16
2.6
6.6
13
7g
7h
11
9.6
3
4
5
6
7
8
9
10
8a
8b
8c
8d
8e
8f
159.6 ꢀ 2.6
799.8 ꢀ 67.1
206.9 ꢀ 42.9
1106.7 ꢀ 34.8
246.4 ꢀ 34.8
144.0 ꢀ 6.4
211.6 ꢀ 3.4
152.9 ꢀ 17.3
1649 ꢀ 21
4257 ꢀ 67
2155 ꢀ 53
1222 ꢀ 57
998 ꢀ 10
1402 ꢀ 19
5333 ꢀ 76
4885 ꢀ 78
5999 ꢀ 80
9978 ꢀ 143
325 ꢀ 11
14
7.2
18
1.5
5.7
8.6
40
12
9.0
41
7.4
57
3.2
46
13
872 ꢀ 17
8g
8h
6177 ꢀ 72
9103 ꢀ 98
7071 ꢀ 124
1401 ꢀ 18
86
R ¼ CH2NH2
R ¼ CHO
13
3
JTC-801
115.7 ꢀ 10.4
207.3 ꢀ 3.3
30.0 ꢀ 1.3
e
e
e
H N
e
e
e
N
O
O
R ¼ CH2CH3
7512 ꢀ 80
1507 ꢀ 19
250
50
N
H
R
Radioligands: [125I]-nociceptin for NOP, [3H]-naltrindole for
d
and [3H]-diprenorphine for
k
and
m
receptors. Data are given as mean ꢀ SD (n ¼ 3).
interaction and the chain length modulated the effect. The best
affinity was reached in derivative 6a (n ¼ 3, IC50 ¼ 98.9 nM) and the
lowest one in derivative 6b (n ¼ 4, IC50 ¼ 308.9 nM). In the insa-
turated compounds 6 the detrimental effect of the aliphatic chain
was weaker than that recorded in the saturated congeners 7.
Probably, in derivatives 6 the aliphatic substituent was oriented by
the trans iminic function in such a way as to be tolerated by the
receptor region surrounding the recognition site, somehow allow-
ing a moderate interaction.
Binding experiments on univalent derivatives 7, 8 and 13
revealed a general loss in affinity, both in respect to the corre-
sponding dimers 5 and in respect to the lead compound JTC-801.
The amine function resulted unsuitable for binding and compound
13 showed a diminished affinity (IC50 ¼ 115.7 nM). In compounds 7,
the linker aliphatic chain exerted a further detrimental effect on
affinity. This effect was more pronounced in compounds 7bee
bearing chains from 4 to 8 carbon atoms. Compound 7a (n ¼ 3,
IC50 ¼ 83.0 nM) showed a moderate loss in affinity as compared
with JTC-801, indicating that a short chain could be tolerated in the
interaction with the peculiar site of 4-aminoquinoline derived
pharmacophores. Increase of the lipophilic tail strongly disturbed
interaction, probably addressing the molecule far away from the
pharmacophore site. Further chain elongation led to a partial
recovery of affinity in compounds 7feh. The highest affinity was
displayed by compound 7g (n ¼ 9, IC50 ¼ 304.6 nM). This behaviour
suggested that longer chains could reach the protein in a lipophilic
domain somehow in proximity of the pharmacophore site.
Diamines 8 showed a general improved affinity in respect to
monomers 7, while maintaining the same trend in relation to the
chain length. The effect could be ascribed to the presence of the
primary amine function at the end of the chain. Compounds 8feh
showed the best affinity in the series. This behaviour seemed to
suggest the presence of an acid lipophilic environment to which
aminoalkyl chains of proper length could be docked thus modu-
lating the ligand affinity.
4.2. Affinity for d, k, m receptors
Compounds 5, 7 and 8 were tested to determine their affinity for
the receptors. Data reported in Tables 1 and 2 were compared
with the values detected for JTC-801 taken as a reference. All
compounds showed no affinity for receptor and interacted weakly
with and , without revealing a substantial preference. The
presence of the second pharmacophore unit in the bivalent
d, k, m
d
k
m

1214
M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
4
5
6
7
JTC-801
4
5
1200
1100
1000
900
800
700
600
500
400
300
200
100
0
8
JTC-801
Aldehyde 3
Amine 9
Aldehyde 3
Amine 9
JTC-801
0
1
2
3
4
5
6
7
8
9
10
11
Aliphatic Chain Carbon Atoms
Fig. 6. Structure- NOP Receptor affinity relationship for compounds 4, 5, 6, 7, 8.
derivatives 5 seemed to play a positive role in the interaction,
mainly when the target was the receptor. This was the case of
Exceptions to this trend were represented by compounds 8f and 8h
k
that exhibited a better affinity at
dimers 5f and 5h.
m receptor than the corresponding
ligand 5h that demonstrated eight times higher affinity than JTC-
801 with IC50 ¼ 900 ꢀ 12 nM. Also monomers 7 and 8 compared to
Selectivity of these NOPr ligands towards
reported in Table 1 and Table 2 as /NOP and
As compared to JTC-801, compounds 5 showed a moderate loss
in selectivity vs receptor. That behaviour was not confirmed for
k
and
m receptors was
JTC-801 exhibited a general enhanced affinity for the
k
receptor,
k
m/NOP IC50 ratio.
reaching the best value of IC50 ¼ 872 ꢀ 17 nM in compound 8f.
Difference in affinity for receptor between compounds 5 and their
k
m
k
related 7 and 8 was more pronounced for compounds bringing
aliphatic chain with four, nine and ten methylene units. Thus, 5h
had 5- and 10-fold higher affinity than respectively 7h and 8h.
receptor to which compounds 5 bound more strongly than JTC-801.
The alkyl derivatives 7 and 8 showed the same trend in selectivity
of bivalent congeners and showed a significant decrease in the k/
In the interaction with
m
receptor, a moderate difference in
NOP IC50 ratio. At last the spacer length did not substantially
affinity between bivalent 5 and the corresponding 7 was detected.
All the bifunctional compounds showed somewhat higher affinity
(about 2-fold) than the corresponding univalent congeners. The
only exception was represented by 5a that exhibited a 7-fold higher
affinity than the corresponding monomer 7a. The difference
influence selectivity of all the tested compounds.
4.3. Activity at NOP receptor
All the compounds evaluated for their ability to enhance the
binding of [35S]GTP
gS in the presence of 300 nM GDP, showed
between the affinities of compounds 5 and their related
u-ami-
noalkyl monomers 8 were accentuated in compounds with seven
and nine methylene units. Thus, the derivatives 5e and 5g showed
affinity 10- and 6-fold higher than 8e and 8g respectively.
antagonistic properties maintaining the pharmacological behav-
iour of the originator JTC-801. None of the structural modifications
introduced any agonistic property in the JTC-801 pharmacophore,
despite the wide differences in binding affinities. A more in-depth
study of the compounds with the highest binding affinity (5g,h)
demonstrated that the lack of agonist activity was maintained at all
GDP concentrations (Fig. 7).
0,25
NC
BAS
0,2
5g
5h
5. Conclusions
0,15
Bivalent structures
5 did not exhibit affinity properties
substantially from that of JTC-801. At a first glance their behaviour
indicated that the second pharmacophore unit was not involved in
the interaction. Nevertheless, the study pointed out that the
adverse effect of spacer detected in compounds 7, 8 could be
neutralized in compounds 5 by the second pharmacophore unit.
Recovery of affinity measured in dimers 5 for a chain length ranging
from 10 to 14 Å (5eeh) might be explained by the presence of
a further recognition site in the receptorial architecture. However
on this basis it was not possible to establish whether or not this
second site could belong to the same receptorial entity.
0,1
0,05
0
-11
-10
-9
-8
-7
-6
-5
-4
-3
[GDP] log M
Fig. 7. Effects of compounds 5g and 5h on GTP
gS binding in comparison with noci-
The pharmacological behaviour of our ligands 5, compared with
that exhibited by the spiropiperidine-based bivalent agonists [13],
seemed to substantiate the hypothesis that JTC-801 and its
ceptin NC The binding of [35S]GTP
g
S was measured at increasing GDP concentrations
as indicated on x-axis, in the absence (BAS) or in the presence of the ligands. The
compounds exhibit lack of stimulation at all concentrations of GDP.

M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
1215
structurally related antagonists bound to a different site in respect
6.1.1. Fmoc diamines
to the competitive agonists. It is known that the NOP orthosteric
binding site required very strict structural features of the ligands
[19]. Therefore, the presence of a second pharmacophoric unit in
the Clark compound caused a drop in affinity (Fig. 1). This behav-
iour could be ascribed either to an unsuitable molecular size of the
ligand, if it binds to a monomeric receptor, or to an unsuitable
length of the spacer considering binding to a dimeric receptor. An
opposite behaviour occurred for the antagonists here described.
Bivalent ligands 5 maintain the JTC-801 affinity, despite the adverse
effect of the linker chain. Moreover, they display the highest affinity
for linker chains of at least eight methylene units. This may be
explained hypothesizing either the unlikely presence of an allo-
steric site able to accommodate huge molecules as ligands 5, or
supposing the existence of two separated synergic sites probably
belonging to different units in a receptor dimeric assembly. A final
consideration concerns the increase of affinities in mono-
alkylamino monomers 8 in relation to the linker chain elongation.
This finding indicated that receptor interaction could be assisted by
a lipophilic acidic environment. More experiments are in program
to further elucidate this point.
N-Fmoc-1,3-propane, N-Fmoc-1,4-butane N-Fmoc-1,5-pentane
and N-Fmoc-1,6-hexane diamines were purchased by Fluka as
hydrobromide salts. The other N-Fmoc diamines were prepared as
follows: to a diluted solution of diamine (Aldrich) (7.6 mmol) in
anhydrous tetrahydrofuran (50 mL), a solution of Fmoc chloride
(Sigma) (0.5 g,1.9 mmol) in anhydrous tetrahydrofuran (50 mL) was
slowly added at 0 ꢃC for 3 h. The suspension was allowed to react
overnight at room temperature, and then the solvent was evapo-
rated in vacuo. N-Fmoc diamine was extracted with ethyl ether.
Evaporation of the solvent gave the expected products as viscous
oils. The compounds, whose structures were confirmed by 1H NMR,
were used without further purification (90% purity determined by
microanalysis).
6.1.2. Methyl 2-(4-formylphenoxymethyl)benzoate (1)
A mixture of 4-hydroxybenzaldehyde (3.7 g, 30 mmol), methyl
2-(bromomethyl)benzoate (6.9 g, 30 mmol) and anhydrous K2CO3
(20.7 g, 150 mmol) in anhydrous DMF (100 mL) was kept under
stirring at 30 ꢃC for 4 h, then ice water was added. The resulting
white precipitate was collected by filtration, washed with water
and dried under vacuum to obtain 1 (6.9 g, 85%); mp 76e78 ꢃC; 1H
In conclusion, the study here reported could open new
perspectives and stimulate further investigation for the synthesis of
ligands for the allosteric site of NOPr in its monomeric or possible
dimeric form.
NMR (DMSO-d6) d
: 9.87 (s, 1H, 40-CHO), 7.92 (d, 1H, H-6), 7.87 (d,
2H, H-30,50), 7.65 (sharp m, 2H, H-3,4), 7.49 (t, 1H, H-5), 7.17 (d, 2H,
H-20,60), 5.53 (s, 2H, OCH2), 3.80 (s, 3H, OCH3). MS (ESI) m/z: 271
(M þ H)þ. Anal. Calcd. for C16H14O4: C, 71.09; H, 5.22. Found: C,
71.10; H, 5.19.
6. Experimental
6.1. Chemistry
6.1.3. 2-(4-Formylphenoxymethyl)benzoic acid (2)
A solution of 1 (5.7 g, 21 mmol) in tetrahydrofuran (18 mL),
methanol (18 mL) and 16% aqueous NaOH (12 mL) was heated at
80 ꢃC under stirring for 45 min. After cooling the solution was
acidified with diluted HCl and the white precipitate 2 was collected
by filtration, washed with water and dried in an oven at 50 ꢃC under
Melting points were determined on a Köfler hot stage apparatus
and are uncorrected.
Column chromatographic separations were accomplished on
Merck aluminium oxide 90. The purity of each compound was
checked on Merck aluminium oxide 60 F254 plates and spots were
located by UV light. Anhydrous sodium sulfate was used to dry
organic solutions. Elemental analyses indicated by the symbols of
the elements were performed by a Carlo Erba EA 1110 instrument
and were within ꢀ0.4% of the theoretical values. The purity of
described compounds determined by microanalysis resulted ꢁ95%.
1H NMR spectra were acquired on a broad band Bruker Avance
400 equipped with temperature controller and z gradient coils.
During acquisition the temperature was maintained at 298 K.
vacuum. Yield 4.1 g, 76%; mp 165e167 ꢃC 1H NMR (DMSO-d6)
d:
9.88 (s, 1H, 40-CHO), 7.96 (d, 1H, H-6), 7.89 (d, 2H, H-30,50), 7.62
(sharp m, 2H, H-3,4), 7.48 (t, 1H, H-5), 7.18 (d, 2H, H-20,60), 5.58 (s,
2H, OCH2). MS (ESI) m/z: 279 (M þ Na)þ. Anal. Calcd. for C15H12O4:
C, 70.29; H, 4.72. Found: C, 70.38; H, 4.79.
6.1.4. 4-Amino-6-[[2-(4-formylphenoxymethyl)benzoyl]amino]-2-
methylquinoline (3)
To a solution of 2 (2.0 g, 7.8 mmol) in anhydrous CH2Cl2 (80 mL),
oxalyl chloride (1.4 mL, 16 mmol) was added with a few drops of
DMF. The resulting mixture was stirred for 48 h at room temper-
ature. Evaporation of the solvent gave 2-(4-formylphenoxymethyl)
benzoyl chloride as a clear syrup which was dissolved in anhydrous
pyridine (100 mL). To the solution cooled in an ice bath 4,6-dia-
mino-2-methylquinoline (1.3 g, 7.5 mmol) was added under stir-
ring. The mixture was allowed to react for weekend at 25 ꢃC, then
the solution was decanted from the undissolved precipitate and
evaporated in vacuo. The residue was suspended in a diluted NaOH
solution and thoroughly extracted with ethyl acetate. The organic
layer was washed with brine and concentrated in vacuo. The
residue was chromatographed on an aluminium oxide column
eluting with ethyl acetate/methanol 9:1 mixture. The obtained
compound 3 was used without further purification. Yield 0.71 g,
22%. An analytical sample was obtained by crystallization from
Chemical shifts were reported in ppm (
tions were used (a apparent; b
d
) and standard abbrevia-
broad; doublet;
¼
¼
d
¼
dd ¼ doublet of doublets; m ¼ multiplet; q ¼ quadruplet;
s ¼ singlet; t ¼ triplet; u ¼ unresolved).
2D TOCSY experiments were collected for 32 scans at
2048 ꢂ 512 data points, for a mixing time of 100 ms, spectra were
processed by sine bell function and zero-filled to 1024 in the t1
dimension.
2D NOESY experiments were collected for 32 scans at
2048 ꢂ 512 data points, for mixing time of 300 ms, 500 ms and 1 s,
spectra were processed by sine bell function and zero-filled to 1024
in the t1 dimension.
2D NOESY NMR experiment aimed to elucidate cis/trans
configuration of imines 4 was established by at mixing times cor-
responding to the T1 relaxation times of the molecular fragments
composing 4.
ethyl acetate: mp 125e128 ꢃC 1H NMR (DMSO-d6)
d: 10.54 (s, 1H,
High Resolution Mass spectra were recorded on a Micromass
(Now Waters) Q-TOF Micro Spectrometer with ESI (þ) source.
Methyl 2-(bromomethyl)benzoate [3] and 4,6-diamino-2-
methylquinoline [5] were obtained according literature methods.
2,2,2-Trifluoro-N-[(4-hydroxyphenyl)methyl]acetamide was prep-
ared as described in the patent literature [15].
NH), 9.82 (s, 1H, 400-CHO), 8.36 (s, 1H, H-5), 7.81 (d, 2H, H-300,500),
7.63 (sharp m, 5H, H-7,8, H-30,40,60), 7.53 (t, 1H, H-50), 7.15 (d, 2H, H-
200,600), 6.47 (bs, 2H, 4-NH2), 6.43 (s,1H, H-3), 5.46 (s, 2H, OCH2), 2.38
(s, 3H, 2-CH3). MS (ESI) m/z: 412 (M þ H)þ. Anal. Calcd. for
C25H21N3O3: C, 72.96; H, 5.15; N, 10.22. Found: C, 72.85; H, 5.11; N,
10.20.

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M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
6.1.5. General procedure for the preparation of bivalent ligands of
N-(4-amino-2-methylquinolin-6-yl)-2-(4-
aminomethylphenoxymethyl)benzamide (5)
(bm, 6H, chain eCH2e). MS (ESI) m/z: 917 (M þ H)þ. Anal. Calcd. for
C57H56N8O4: C, 74.64; H, 6.16; N, 12.22. Found: C, 74.63; H, 6.15; N,
12.20.
(a) Preparation of azomethines 4. To a concentrated solution of 3
(0.3 g, 0.73 mmol) in methanol (3 mL), the appropriate diamine
(0.36 mmol) was added at room temperature. The solution was
stirred and after a while a precipitate separated. The suspension
was kept under stirring for the weekend, then the solid Schiff base 4
was filtered, washed with methanol and ether and dried in vacuo.
(b) Reduction of 4. Compounds 4 (0.1 g) suspended in methanol
(5 mL) at room temperature were reduced by sodium borohydride
(0.5 g) under stirring. After 2 h the resulting solution was concen-
trated in vacuo and the residue treated with water. The solid
compounds 5 that separated were filtered and rinsed thoroughly
with water and ethyl ether.
6.1.5.6. (E,E)-N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
carbonyl]phenyl]methoxy]phenyl]methylene]-1,8-octanediamine (4f).
Yield 42%, mp 118e120 ꢃC 1H NMR (DMSO-d6)
d: 10.50 (s, 2H, NH),
8.36 (s, 2H, H-5), 8.18 (s, 2H, CH]N), 7.62 (m, 12 H, H-8, H-7, H-30, H-
60, H-300,500), 7.53 (t, 2H, H-40), 7.50 (t, 2H, H-50), 7.00 (d, 4H, H-200,600),
6.42 (s, 2H, H-3), 6.38 (s, 4H, 4-NH2), 5.37 (s, 4H, OCH2), 3.46 (t, 4H,
chain NeCH2), 2.37 (s, 6H, 2-CH3),1.56 (m, 4H, chain eCH2e),1.26 (bs,
8H, chain eCH2e). MS (ESI) m/z: 931 (M þ H)þ. Anal. Calcd. for
C58H58N8O4: C, 74.80; H, 6.28; N, 12.04. Found: C, 74.82; H, 6.31; N,
12.07.
6.1.5.7. (E,E)-N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
6.1.5.1. (E,E)-N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
carbonyl]phenyl]methoxy]phenyl]methylene]-1,9-nonanediamine (4g).
1
carbonyl]phenyl]methoxy]phenyl]methylene]-1,3-propanediamine (4a).
Yield 58%, mp 138e140 ꢃC H NMR (DMSO-d6)
d: 10.53 (s, 2H, NH),
Yield 50%, mp 165e167 ꢃC 1H NMR (DMSO-d6)
d: 10.51 (s, 2H, NH),
8.39 (s, 2H, H-5), 8.20 (s, 2H, CH]N), 7.64 (m, 12 H, H-8, H-7, H-30, H-
60, H-300,500), 7.54 (t, 2H, H-40), 7.50 (t, 2H, H-50), 7.01 (d, 4H, H-200,600),
6.44 (s, 2H, H-3), 6.43 (s, 4H, 4-NH2), 5.39 (s, 4H, OCH2), 3.47 (t, 4H,
chain NeCH2), 2.39 (s, 6H, 2-CH3),1.54 (m, 4H, chain eCH2e),1.26 (bs,
10H, chain eCH2e). MS (ESI) m/z: 945 (M þ H)þ. Anal. Calcd. for
C59H60N8O4: C, 74.96; H, 6.40; N, 11.86. Found: C, 74.95; H, 6.42; N,
11.88.
8.36 (s, 2H, H-5), 8.20 (s, 2H, CH]N), 7.63 (m, 12 H, H-8, H-7, H-30, H-
60, H-300,500), 7.54 (t, 2H, H-40), 7.49 (t, 2H, H-50), 7.00 (d, 4H, H-200,600),
6.43 (s, 2H, H-3), 6.39 (s, 4H, 4-NH2), 5.38 (s, 4H, OCH2), 3.54 (t, 4H,
chain NeCH2), 2.38 (s, 6H, 2-CH3), 1.87 (quintuplet, 2H, chain central
eCH2e). MS (ESI) m/z: 861 (M þ H)þ. Anal. Calcd. for C53H48N8O4: C,
73.92; H, 5.62; N, 13.02. Found: C, 73.96; H, 5.58; N, 13.12.
6.1.5.2. (E,E)-N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
6.1.5.8. (E,E)-N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
carbonyl]phenyl]methoxy]phenyl]methylene]-1,4-butanediamine (4b).
carbonyl]phenyl]methoxy]phenyl]methylene]-1,10-decanediamine (4h).
Yield 41%, mp 153e155 ꢃC 1H NMR (DMSO-d6)
d: 10.51 (s, 2H, NH),
Yield 57%, mp 127e129 ꢃC 1H NMR (DMSO-d6)
d: 10.53 (s, 2H, NH),
8.36 (s, 2H, H-5), 8.20 (s, 2H, CH]N), 7.62 (m,12 H, H-8, H-7, H-30, H-
60, H-300,500), 7.54 (t, 2H, H-40), 7.51 (t, 2H, H-50), 6.99 (d, 4H, H-200,600),
6.42 (s, 2H, H-3), 6.39 (s, 4H, 4-NH2), 5.37 (s, 4H, OCH2), 3.50 (t, 4H,
chain NeCH2), 2.37 (s, 6H, 2-CH3), 1.59 (m, 4H, chain central
e(CH2)2e). MS (ESI) m/z: 875 (M þ H)þ. Anal. Calcd. for C54H50N8O4:
C, 74.11; H, 5.76; N, 12.81. Found: C, 74.15; H, 5.78; N, 12.84.
8.39 (s, 2H, H-5), 8.20 (s, 2H, CH]N), 7.64 (m, 12 H, H-8, H-7, H-30, H-
60, H-300,500), 7.54 (t, 2H, H-40), 7.50 (t, 2H, H-50), 7.01 (d, 4H, H-200,600),
6.44 (s, 2H, H-3), 6.41 (s, 4H, 4-NH2), 5.39 (s, 4H, OCH2), 3.45 (t, 4H,
chain NeCH2), 2.40 (s, 6H, 2-CH3), 1.47 (m, 4H, chain eCH2e), 1.20
(bm, 12H, chain eCH2e). MS (ESI) m/z: 959 (M þ H)þ. Anal. Calcd. for
C60H62N8O4: C, 75.12; H, 6.52, N. 11.69. Found: C, 75.16; H, 6.57; N,
11.71.
6.1.5.3. (E,E)-N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
carbonyl]phenyl]methoxy]phenyl]methylene]-1,5-pentanediamine (4c).
6.1.5.9. N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
Yield 55%, mp 152e153 ꢃC 1H NMR (DMSO-d6)
d: 10.52 (s, 2H, NH),
carbonyl]phenyl]methoxy]phenyl]methyl]-1,3-propanediamine (5a).
8.36 (s, 2H, H-5), 8.19 (s, 2H, CH]N), 7.63 (m, 12 H, H-8, H-7, H-30, H-
60, H-300,500), 7.54 (t, 2H, H-40), 7.50 (t, 2H, H-50), 6.99 (d, 4H, H-200,600),
6.42 (s, 2H, H-3), 6.39 (s, 4H, 4-NH2), 5.38 (s, 4H, OCH2), 3.50 (t, 4H,
chain NeCH2), 2.37 (s, 6H, 2-CH3), 1.58 (m, 4H, chain eCH2e), 1.31 (m,
2H, chain central eCH2e). MS (ESI) m/z: 889 (M þ H)þ. Anal. Calcd. for
C55H52N8O4: C, 74.29; H, 5.90; N, 12.61. Found: C, 74.31; H, 5.94; N,
12.60.
Yield 92%, mp 143e145 ꢃC 1H NMR (DMSO-d6)
d: 10.51 (s, 2H, NH), 8.37
(s, 2H, H-5), 7.63 (m, 8H, H-8, H-7, H-30, H-60,), 7.50 (two overlapped t,
4H, H-40,H-50), 7.15 (d, 4H, H-300,500), 6.88 (d, 4H, H-200,600), 6.44 (s, 2H, H-
3), 6.40 (s, 4H, 4-NH2), 5.30 (s, 4H, OCH2), 3.53 (s, 4H, 400-CH2N), 2.44 (t,
4H, chain NeCH2), 2.38 (s, 6H, 2-CH3), 1.50 (t, 2H, chain central eCH2).
MS (ESI) m/z: 865 (M þ H)þ. Anal. Calcd. for C53H52N8O4: C, 73.58; H,
6.06; N, 12.96. Found: C, 73.54; H, 6.09; N, 12.94.
6.1.5.4. (E,E)-N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
6.1.5.10. N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
carbonyl]phenyl]methoxy]phenyl]methylene]-1,6-hexanediamine (4d).
carbonyl]phenyl]methoxy]phenyl]methyl]-1,4-butanediamine (5b).
1
Yield 43%, mp 155e157 ꢃC H NMR (DMSO-d6)
d: 10.52 (s, 2H, NH),
Yield 92%, mp 127e129 ꢃC 1H NMR (DMSO-d6)
d: 10.50 (s, 2H, NH),
8.38 (s, 2H, H-5), 8.20 (s, 2H, CH]N), 7.64 (m, 12 H, H-8, H-7, H-30, H-
60, H-300,500), 7.56 (t, 2H, H-40), 7.49 (t, 2H, H-50), 7.01 (d, 4H, H-200,600),
6.44 (s, 2H, H-3), 6.40 (s, 4H, 4-NH2), 5.39 (s, 4H, OCH2), 3.48 (t, 4H,
chain NeCH2), 2.39 (s, 6H, 2-CH3),1.58 (m, 4H, chain eCH2e),1.32 (m,
4H, chain central e(CH2)2e). MS (ESI) m/z: 903 (M þ H)þ. Anal. Calcd.
for C56H54N8O4: C, 74.47; H, 6.03; N, 12.41. Found: C, 74.48; H, 6.05; N,
12.43.
8.35 (s, 2H, H-5), 7.63 (m, 8H, H-8, H-7, H-30, H-60,), 7.50 (two
partially overlapped t, 4H, H-40, H-50), 7.16 (d, 4H, H-300,500), 6.87 (d,
4H, H-200,600), 6.44 (s, 2H, H-3), 6.40 (s, 4H, 4-NH2), 5.30 (s, 4H,
OCH2), 3.54 (s, 4H, 400-CH2N), 2.45 (t, 4H, chain NeCH2), 2.38 (s,
6H, 2-CH3), 1.51 (m, 4H, chain central e(CH2)2e). MS (ESI) m/z:
879 (M þ H)þ. Anal. Calcd. for C54H54N8O4: C, 73.77; H, 6.20; N,
12.75. Found: C, 73.81; H, 6.25; N, 12.73.
6.1.5.5. (E,E)-N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
6.1.5.11. N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
carbonyl]phenyl]methoxy]phenyl]methylene]-1,7-heptanediamine (4e).
carbonyl]phenyl]methoxy]phenyl]methyl]-1,5-pentanediamine (5c).
1
Yield 38%, mp 120e122 ꢃC H NMR (DMSO-d6)
d: 10.55 (s, 2H, NH),
Yield 95%, mp 128e130 ꢃC 1H NMR (DMSO-d6)
d: 10.49 (s, 2H, NH),
8.39 (s, 2H, H-5), 8.20 (s, 2H, CH]N), 7.64 (m, 12 H, H-8, H-7, H-30, H-
60, H-300,500), 7.54 (t, 2H, H-40), 7.50 (t, 2H, H-50), 7.01 (d, 4H, H-200,600),
6.44 (s, 2H, H-3), 6.41 (s, 4H, 4-NH2), 5.39 (s, 4H, OCH2), 3.49 (t, 4H,
chain NeCH2), 2.39 (s, 6H, 2-CH3), 1.57 (m, 4H, chain eCH2e), 1.29
8.36 (s, 2H, H-5), 7.63 (m, 8H, H-8, H-7, H-30, H-60,), 7.53 (t, 2H, H-40),
7.49 (t, 2H, H-50), 7.15 (d, 4H, H-300,500), 6.87 (d, 4H, H-200,600), 6.43
(s, 2H, H-3), 6.38 (s, 4H, 4-NH2), 5.30 (s, 4H, OCH2), 3.54 (s, 4H, 400-
CH2N), 2.38 (bs, 10H, 2-CH3, chain NeCH2), 1.34 (bt, 4H, chain CH2),

M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
1217
1.23 (um, 2H, chain central eCH2). MS (ESI) m/z: 893 (M þ H)þ.
Anal. Calcd. for C55H56N8O4: C, 73.95; H, 6.32; N, 12.55. Found: C,
73.97; H, 6.34; N, 12.50.
The residue was treated thoroughly with water, filtered and dried
in vacuo.
(b) Reduction of 6. Compounds 6 (0.1 g) dissolved in methanol
(5 mL) at room temperature were reduced by sodium borohydride
(0.5 g) under stirring. After 2 h the resulting solution was concen-
trated in vacuo and the residue treated with water. The solid
compounds 7 that separated were filtered, rinsed thoroughly with
water and dried in vacuo.
6.1.5.12. N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
carbonyl]phenyl]methoxy]phenyl]methyl]-1,6-hexanediamine (5d).
Yield 95%, mp 133e135 ꢃC 1H NMR (DMSO-d6)
d: 10.48 (s, 2H, NH),
8.36 (s, 2H, H-5), 7.63 (m, 8H, H-8, H-7, H-30, H-60,), 7.52 (t, 2H, H-40),
7.47 (t, 2H, H-50), 7.13 (d, 4H, H-300,500), 6.85 (d, 4H, H-200,600), 6.42 (s,
2H, H-3), 6.37 (s, 4H, 4-NH2), 5.29 (s, 4H, OCH2), 3.52 (s, 4H, 400-
CH2N), 2.37 (bs, 10H, 2-CH3, chain NeCH2), 1.34 (bt, 4H, chain CH2),
1.22 (um, 4H, chain central e(CH2)2e). MS (ESI) m/z: 907 (M þ H)þ.
Anal. Calcd. for C56H58N8O4: C, 74.13; H, 6.45; N, 12.36. Found: C,
74.17; H, 6.49; N, 12.31.
6.1.6.1. (E)-N-(4-amino-2-methylquinolin-6-yl)-2-[4-(propylimino-
methyl)phenoxymethyl]benzamide (6a). Yield 75%, mp 125e127 ꢃC
1H NMR (DMSO-d6)
d: 10.50 (s, 1H, NH), 8.36 (s, 1H, H-5), 8.18 (s,
1H, CH]N), 7.62 (m, 6 H, H-8, H-7, H-30, H-60, H-300,500), 7.54 (t, 1H,
H-40), 7.50 (t, 1H, H-50), 6.99 (d, 2H, H-200,600), 6.42 (s, 1H, H-3), 6.38
(s, 2H, 4-NH2), 5.37 (s, 2H, OCH2), 3.44 (t, 2H, chain NeCH2), 2.38
(s, 3H, 2-CH3), 1.56 (m, 2H, chain CH2), 1.29 (t, 3H, chain CH3). MS
(ESI) m/z: 453 (M þ H)þ. Anal. Calcd. for C28H28N4O2: C, 74.30; H,
6.24; N, 12.39. Found: C, 74.29; H, 6.21; N, 12.38.
6.1.5.13. N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
carbonyl]phenyl]methoxy]phenyl]methyl]-1,7-heptanediamine (5e).
Yield 92%, mp 128e130 ꢃC 1H NMR (DMSO-d6)
d: 10.51 (s, 2H, NH),
8.38 (s, 2H, H-5), 7.64 (m, 8H, H-8, H-7, H-30, H-60,), 7.52 (m, 4H, H-
40, H-50), 7.17 (d, 4H, H-300,500), 6.88 (d, 4H, H-200,600), 6.44 (s, 2H, H-3),
6.38 (s, 4H, 4-NH2), 5.31 (s, 4H, OCH2), 3.54 (s, 4H, 400-CH2N), 2.39
(bs, 10H, 2-CH3, chain NeCH2), 1.36 (bt, 4H, chain CH2), 1.21 (um,
6H, chain central e(CH2)3e). MS (ESI) m/z: 921 (M þ H)þ. Anal.
Calcd. for C57H60N8O4: C, 74.31; H, 6.57; N, 12.17. Found: C, 74.29; H,
6.55; N, 12.15.
6.1.6.2. (E)-N-(4-amino-2-methylquinolin-6-yl)-2-[4-(butylimino-
methyl)phenoxymethyl]benzamide (6b). Yield 72%, mp 120e123 ꢃC.
1H NMR (DMSO-d6)
d: 10.51 (s, 1H, NH), 8.37 (s, 1H, H-5), 8.19 (s, 1H,
CH]N), 7.63 (m, 6 H, H-8, H-7, H-30, H-60, H-300,500), 7.56 (t,1H, H-40),
7.49 (t, 1H, H-50), 7.00 (d, 2H, H-200,600), 6.40 (s, 3H, H-3, 4-NH2), 5.38
(s, 2H, OCH2), 3.49 (t, 2H, chain NeCH2), 2.38 (s, 3H, 2-CH3), 1.54 (m,
2H, chain CH2), 1.29 (m, 2H, chain CH2), 0.88 (t, 3H, chain CH3). MS
(ESI) m/z: 467 (M þ H)þ. Anal. Calcd. for C29H30N4O2: C, 74.64; H,
6.48; N, 12.01. Found: C, 74.67; H, 6.51; N, 12.06.
6.1.5.14. N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
carbonyl]phenyl]methoxy]phenyl]methyl]-1,8-octanediamine (5f).
Yield 93%, mp 140e142 ꢃC 1H NMR (DMSO-d6)
d: 10.51 (s, 2H, NH),
8.38 (s, 2H, H-5), 7.64 (m, 8H, H-8, H-7, H-30, H-60,), 7.52 (m, 4H, H-
40, H-50), 7.16 (d, 4H, H-300,500), 6.75 (d, 4H, H-200,600), 6.44 (s, 2H, H-3),
6.38 (s, 4H, 4-NH2), 5.31 (s, 4H, OCH2), 3.54 (s, 4H, 400-CH2N), 2.39
(bs, 10H, 2-CH3, chain NeCH2), 1.35 (bt, 4H, chain CH2), 1.21 (um,
8H, chain central e(CH2)4e). MS (ESI) m/z: 935(M þ H)þ. Anal.
Calcd. for C58H62N8O4: C, 74.48; H, 6.69; N, 11.99. Found: C, 74.50; H,
6.71; N, 11.95.
6.1.6.3. (E)-N-(4-amino-2-methylquinolin-6-yl)-2-[4-(pentylimi-
nomethyl)phenoxymethyl]benzamide (6c). Yield 77%, mp 88e90 ꢃC
1H NMR (DMSO-d6)
d: 10.50 (s, 1H, NH), 8.37 (s, 1H, H-5), 8.19 (s, 1H,
CH]N), 7.63 (m, 6 H, H-8, H-7, H-30, H-60, H-300,500), 7.56 (m, 2H, H-40,
H-50), 7.00 (d, 2H, H-200,600), 6.43 (s,1H, H-3), 6.39 (s, 2H, 4-NH2), 5.38
(s, 2H, OCH2), 3.47 (t, 2H, chain NeCH2), 2.38 (s, 3H, 2-CH3), 1.56 (m,
2H, chain CH2),1.25 (m, 4H, chain e(CH2)2e), 0.85 (t, 3H, chain CH3).
MS (ESI) m/z: 481 (M þ H)þ. Anal. Calcd. for C30H32N4O2: C, 74.96; H,
6.72; N, 11.66. Found: C, 74.93; H, 6.70; N, 11.62.
6.1.5.15. N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
carbonyl]phenyl]methoxy]phenyl]methyl]-1,9-nonanediamine (5g).
Yield 96%, mp 135e137 ꢃC 1H NMR (DMSO-d6)
d
: 10.46 (s, 2H, NH),
6.1.6.4. (E)-N-(4-amino-2-methylquinolin-6-yl)-2-[4-(hexylimino-
8.36 (s, 2H, H-5), 7.63 (m, 8H, H-8, H-7, H-30, H-60,), 7.54 (t, 2H, H-40),
7.48 (t, 2H, H-50), 7.16 (d, 4H, H-300,500), 6.88 (d, 4H, H-200,600), 6.45 (s,
2H, H-3), 6.33 (s, 4H, 4-NH2), 5.31 (s, 4H, OCH2), 3.55 (s, 4H, 400-
CH2N), 2.39 (bs, 10H, 2-CH3, chain NeCH2), 1.36 (bt, 4H, chain CH2),
1.21 (um,10H, chain central e(CH2)5e). MS (ESI) m/z: 949 (M þ H)þ.
Anal. Calcd. for C59H64N8O4: C, 74.64; H, 6.80; N. 11.81. Found: C,
74.67; H, 6.83; N, 11.80.
methyl)phenoxymethyl]benzamide (6d). Yield 73%, mp 90e92 ꢃC 1H
NMR (DMSO-d6)d:10.52(s,1H, NH), 8.38(s,1H,H-5), 8.21(s,1H,CH]
N), 7.64 (m, 6 H, H-8, H-7, H-30, H-60, H-300,500), 7.54 (m, 2H, H-40, H-50),
7.01 (d, 2H, H-200,600), 6.44 (s,1H, H-3), 6.39 (s, 2H, 4-NH2), 5.39 (s, 2H,
OCH2), 3.49 (t, 2H, chain NeCH2), 2.40 (s, 3H, 2-CH3), 1.57 (m, 2H,
chain CH2), 1.27 (m, 6H, chain e(CH2)3e), 0.85 (t, 3H, chain CH3). MS
(ESI)m/z:495 (Mþ H)þ. Anal. Calcd. for C31H34N4O2: C, 75.26; H, 6.93;
N, 11.33. Found: C, 75.20; H, 6.91; N, 11.34.
6.1.5.16. N,N0-bis[[4-[[2-[[(4-amino-2-methylquinolin-6-yl)amino]
carbonyl]phenyl]methoxy]phenyl]methyl]-1,10-decanediamine (5h).
6.1.6.5. (E)-N-(4-amino-2-methylquinolin-6-yl)-2-[4-(heptylimi-
Yield 96%, mp 125e127 ꢃC 1H NMR (DMSO-d6)
d
: 10.46 (s, 2H, NH),
nomethyl)phenoxymethyl]benzamide (6e). Yield 75%, mp 87e90 ꢃC.
8.36 (s, 2H, H-5), 7.63 (m, 8H, H-8, H-7, H-30, H-60,), 7.54 (t, 2H, H-40),
7.48 (t, 2H, H-50), 7.16 (d, 4H, H-300,500), 6.88 (d, 4H, H-200,600), 6.45 (s,
2H, H-3), 6.33 (s, 4H, 4-NH2), 5.31 (s, 4H, OCH2), 3.55 (s, 4H, 400-
CH2N), 2.39 (bs, 10H, 2-CH3, chain NeCH2), 1.35 (bt, 4H, chain CH2),
1.21 (um,12H, chain central e(CH2)6e). MS (ESI) m/z: 963 (M þ H)þ.
Anal. Calcd. for C60H66N8O4: C, 74.80; H, 6.91; N, 11.64. Found: C,
74.87; H, 6.95; N, 11.68.
1H NMR (DMSO-d6)
d: 10.53 (s,1H, NH), 8.38 (s,1H, H-5), 8.20 (s,1H,
CH]N), 7.64 (m, 6 H, H-8, H-7, H-30, H-60, H-300,500), 7.53 (m, 2H, H-
40, H-50), 7.01 (d, 2H, H-200,600), 6.44 (s, 1H, H-3), 6.41 (s, 2H, 4-NH2),
5.39 (s, 2H, OCH2), 3.48 (t, 2H, chain NeCH2), 2.39 (s, 3H, 2-CH3),
1.58 (m, 2H, chain CH2), 1.26 (m, 8H, chain e(CH2)4e), 0.85 (t, 3H,
chain CH3). MS (ESI) m/z: 509 (M þ H)þ. Anal. Calcd. for
C32H36N4O2: C, 75.55; H, 7.14; N, 11.02. Found: C, 75.57; H, 7.18; N,
11.05.
6.1.6. General procedure for the preparation of N-(4-amino-2-methyl
quinolin-6-yl)-2-[4-(alkylaminomethyl)phenoxymethyl]benzamide (7)
(a) Preparation of Azomethines 6. To a solution of 3 (0.3 g,
0.73 mmol) in methanol (5 mL) an excess of the appropriate
alkylamine (1.50 mmol) was added at room temperature. The
solution was stirred overnight then the solvent was evaporated.
6.1.6.6. (E)-N-(4-amino-2-methylquinolin-6-yl)-2-[4-(octylimino-
methyl)phenoxymethyl]benzamide (6f). Yield 79%, mp 74e75 ꢃC 1H
NMR (DMSO-d6) d: 10.50 (s, 1H, NH), 8.36 (s, 1H, H-5), 8.19 (s, 1H,
CH]N), 7.63 (m, 6 H, H-8, H-7, H-30, H-60, H-300,500), 7.52 (m, 2H, H-
40, H-50), 7.01 (d, 2H, H-200,600), 6.44 (s, 1H, H-3), 6.38 (s, 2H, 4-NH2),

1218
M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
5.38 (s, 2H, OCH2), 3.48 (t, 2H, chain NeCH2), 2.39 (s, 3H, 2-CH3),
1.56 (m, 2H, chain CH2), 1.24 (m, 10H, chain e(CH2)5e), 0.84 (t, 3H,
chain CH3). MS (ESI) m/z: 523 (M þ H)þ. Anal. Calcd. for
C33H38N4O2: C, 75.82; H, 7.33; N, 10.72. Found: C, 75.84; H, 7.35; N,
10.77.
497 (M þ H)þ. Anal. Calcd. for C31H36N4O2: C, 74.96; H, 7.31; N, 11.29.
Found: C, 74.95; H, 7.33; N, 11.27.
6.1.6.13. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(heptylaminomethyl)
phenoxymethyl]benzamide (7e). Yield 94%, mp 64e66 ꢃC 1H NMR
(DMSO-d6) d: 10.50 (s,1H, NH), 8.38 (s,1H, H-5), 7.64 (m, 4 H, H-8, H-7,
6.1.6.7. (E)-N-(4-amino-2-methylquinolin-6-yl)-2-[4-(nonylimino-
H-30, H-60), 7.55 (m, 2H, H-40, H-50), 7.15 (d, 2H, H-300,500), 6.85 (d, 2H,
H-200,600), 6.44 (s, 1H, H-3), 6.40 (s, 2H, 4-NH2), 5.31 (s, 2H, OCH2), 3.55
(s, 2H, 400-NCH2), 2.60 (bs, 2H, chain NeCH2), 2.40 (s, 3H, 2-CH3,), 1.23
(m, 10H, chain e(CH2)5e), 0.84 (bt, 3H, chain CH3). MS (ESI) m/z: 511
(M þ H)þ. Anal. Calcd. for C32H38N4O2: C, 75.25; H, 7.50; N, 10.98.
Found: C, 75.27; H, 7.53; N, 10.93.
methyl)phenoxymethyl]benzamide (6g). Yield 80%, mp 95e98 ꢃC
1H NMR (DMSO-d6)
d: 10.54 (s, 1H, NH), 8.38 (s, 1H, H-5), 8.20 (s,
1H, CH]N), 7.64 (m, 6 H, H-8, H-7, H-30, H-60, H-300,500), 7.52 (m,
2H, H-40, H-50), 7.01 (d, 2H, H-200,600), 6.44 (s, 1H, H-3), 6.41 (s, 2H,
4-NH2), 5.39 (s, 2H, OCH2), 3.48 (t, 2H, chain NeCH2), 2.39 (s, 3H,
2-CH3), 1.56 (m, 2H, chain CH2), 1.25 (m, 12H, chain e(CH2)6e),
0.84 (t, 3H, chain CH3). MS (ESI) m/z: 537 (M þ H)þ. Anal. Calcd.
for C34H40N4O2: C, 76.07; H, 7.52; N, 10.44. Found: C, 76.05; H,
7.48; N, 10.40.
6.1.6.14. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(octylaminomethyl)
phenoxymethyl]benzamide (7f). Yield 92%, mp 63e65 ꢃC 1H NMR
(DMSO-d6) d: 10.51 (s, 1H, NH), 8.38 (s,1H, H-5), 7.65 (m, 4 H, H-8, H-
7, H-30, H-60), 7.53 (m, 2H, H-40, H-50), 7.16 (d, 2H, H-300,500), 6.87 (d,
2H, H-200,600), 6.45 (s, 1H, H-3), 6.40 (s, 2H, 4-NH2), 5.32 (s, 2H, OCH2),
3.56 (s, 2H, 400-NCH2), 2.60 (bs, 2H, chain NeCH2), 2.40 (s, 3H, 2-
CH3,), 1.23 (m, 12H, chain e(CH2)6e), 0.84 (bt, 3H, chain CH3). MS
(ESI) m/z: 525 (M þ H)þ. Anal. Calcd. for C33H40N4O2: C, 75.53; H,
7.69; N, 10.68. Found: C, 75.56; H, 7.74; N, 10.66.
6.1.6.8. (E)-N-(4-amino-2-methylquinolin-6-yl)-2-[4-(decylimino-
methyl)phenoxymethyl]benzamide (6h). Yield 80%, mp 128e130 ꢃC
1H NMR (DMSO-d6)
d: 10.53 (s, 1H, NH), 8.37 (s, 1H, H-5), 8.19 (s, 1H,
CH]N), 7.63 (m, 6 H, H-8, H-7, H-30, H-60, H-300,500), 7.53 (m, 2H, H-
40, H-50), 7.01 (d, 2H, H-200,600), 6.43 (s, 1H, H-3), 6.40 (s, 2H, 4-NH2),
5.38 (s, 2H, OCH2), 3.49 (t, 2H, chain NeCH2), 2.39 (s, 3H, 2-CH3),
1.55 (m, 2H, chain CH2), 1.23 (m, 14H, chain e(CH2)7e), 0.85 (t, 3H,
6.1.6.15. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(nonylaminomethyl)
chain CH3). MS (ESI) m/z: 551 (M
C35H42N4O2: C, 76.32; H, 7.69; N, 10.18. Found: C, 76.33; H, 7.71; N,
10.15.
þ
H)þ. Anal. Calcd. for
phenoxymethyl]benzamide (7g). Yield 95%, mp 65e66 ꢃC 1H NMR
(DMSO-d6) d: 10.52 (s,1H, NH), 8.38 (s,1H, H-5), 7.63 (m, 4H, H-8, H-7,
H-30, H-60), 7.53 (m, 2H, H-40, H-50), 7.17 (d, 2H, H-300,500), 6.88 (d, 2H,
H-200,600), 6.43 (s,1H, H-3), 6.40 (s, 2H, 4-NH2), 5.30 (s, 2H, OCH2), 3.54
(s, 2H, 400-NCH2), 2.58 (bs, 2H, chain NeCH2), 2.39 (s, 3H, 2-CH3,), 1.22
(m, 14H, chain e(CH2)7e), 0.84 (bt, 3H, chain CH3). MS (ESI) m/z: 539
(M þ H)þ. Anal. Calcd. for C34H42N4O2: C, 75.79; H, 7.86; N, 10.40.
Found: C, 75.82; H, 7.85; N, 10.42.
6.1.6.9. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(proylaminomethyl)
phenoxymethyl]benzamide (7a). Yield 92%, mp 118e120 ꢃC 1H NMR
(DMSO-d6) d: 10.50 (s, 1H, NH), 8.38 (s,1H, H-5), 7.63 (m, 4 H, H-8, H-
7, H-30, H-60), 7.55 (m, 2H, H-40, H-50), 7.17 (d, 2H, H-300,500), 6.88 (d,
2H, H-200,600), 6.43 (s, 1H, H-3), 6.39 (s, 2H, 4-NH2), 5.32 (s, 2H, OCH2),
3.60 (s, 2H, 400-NCH2), 2.56 (t, 2H, chain NeCH2), 2.40 (s, 3H, 2-CH3,),
1.30 (m, 2H, chain CH2), 1.22 (t, 3H, chain CH3). MS (ESI) m/z: 455
(M þ H)þ. Anal. Calcd. for C28H30N4O2: C, 73.97; H, 6.66; N, 12.33.
Found: C, 73.93; H, 6.61; N, 12.31.
6.1.6.16. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(decylaminomethyl)
phenoxymethyl]benzamide (7h). Yield 97%, mp 65e67 ꢃC 1H NMR
(DMSO-d6) d: 10.89 (s, 1H, NH), 8.73 (s,1H, H-5), 8.63 (bs, 2H, 4-NH2),
7.92 (bs, 2H, H-7, H-8), 7.58 (m, 4H, H-30, H-40, H-50, H-60), 7.37 (d, 2H,
H-300,500), 6.98 (d, 2H, H-200,600), 6.60 (s, 1H, H-3), 5.36 (s, 2H, OCH2),
3.99 (s, 2H, 400-NCH2), 2.79 (bs, 2H, chain NeCH2), 2.59 (s, 3H, 2-CH3,),
1.23 (m,16H, chain e(CH2)8e), 0.85 (bt, 3H, chain CH3). MS (ESI) m/z:
553 (M þ H)þ. Anal. Calcd. for C35H44N4O2: C, 76.04; H, 8.03; N,10.14.
Found: C, 76.08; H, 8.02; N, 10.16.
6.1.6.10. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(butylaminomethyl)
phenoxymethyl]benzamide (7b). Yield 92%, mp 106e108 ꢃC 1H NMR
(DMSO-d6) d: 10.51 (s, 1H, NH), 8.38 (s,1H, H-5), 7.65 (m, 4 H, H-8, H-
7, H-30, H-60), 7.55 (m, 2H, H-40, H-50), 7.16 (d, 2H, H-300,500), 6.86 (d,
2H, H-200,600), 6.44 (s, 1H, H-3), 6.39 (s, 2H, 4-NH2), 5.35 (s, 2H, OCH2),
3.60 (s, 2H, 400-NCH2), 2.57 (t, 2H, chain NeCH2), 2.40 (s, 3H, 2-CH3,),
1.28 (m, 4H, chain (CH2)2e), 0.85 (bt, 3H, chain CH3). MS (ESI) m/z:
469 (M þ H)þ. Anal. Calcd. for C29H32N4O2: C, 74.32; H, 6.89; N,11.96.
Found: C, 74.36; H, 6.91; N, 11.95.
6.1.7. General procedure for the preparation of N-(4-amino-2-methyl-
quinolin-6-yl)-2-[4-(u-aminoalkylaminomethyl)phenoxymethyl]
benzamide (8)
Aldehyde 3 (0.3 g, 0.73 mmol), N-Fmoc-diamine.(used as
hydrobromide for 8aed preparation and as free base for 8eeh
preparation) (0.73 mmol) and diisopropylethylamine (0.3 mL) were
dissolved in methanol (10 mL), and the solution was kept under
stirring for the weekend at 25 ꢃC. The reaction was monitored by
thin layer chromatography on alumina plates, eluting with an ethyl
acetate e 5% methanol mixture. Reduction of the so obtained Schiff
bases was subsequently made with sodium borohydride (0.3 g) at
room temperature for 3 h. Finally, methanolic solution was
concentrated and the residue was treated with water and extracted
with ethyl acetate. Organic layer was evaporated affording the
expected compounds N-(4-amino-2-methylquinolin-6-yl)-2-[4-
6.1.6.11. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(pentylaminomethyl)
phenoxymethyl]benzamide (7c). Yield 94%, mp 108e110 ꢃC 1H NMR
(DMSO-d6) d: 10.50 (s,1H, NH), 8.37 (s,1H, H-5), 7.61 (m, 4 H, H-8, H-7,
H-30, H-60), 7.50 (m, 2H, H-40, H-50), 7.16 (d, 2H, H-300,500), 6.89 (d, 2H,
H-200,600), 6.43 (s, 1H, H-3), 6.40 (s, 2H, 4-NH2), 5.30 (s, 2H, OCH2), 3.58
(s, 2H, 400-NCH2), 2.58 (t, 2H, chain NeCH2), 2.39 (s, 3H, 2-CH3,), 1.21
(m, 6H, chain e(CH2)3e), 0.85 (bt, 3H, chain CH3). MS (ESI) m/z: 483
(M þ H)þ. Anal. Calcd. for C30H34N4O2: C, 74.65; H, 7.11; N, 11.61.
Found: C, 74.67; H, 7.12; N, 11.60.
6.1.6.12. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(hexylaminomethyl)
(u-Fmocaminoalkylaminomethyl)phenoxymethyl]benzamides.
phenoxymethyl]benzamide (7d). Yield 96%, mp 107e109 ꢃC 1H NMR
Cleavage of Fmoc protecting group was achieved by a mixture of
20% piperidine/DMF (0.5 mL) at room temperature for 3 h. The
resulting suspension was diluted with methanol, filtered and
concentrated in vacuo. The residue was added with ethyl ether and
shaken overnight. The ethereal phase containing the side-product
fluorenylmethylpiperidine derived from the deprotection, was
(DMSO-d6) d: 10.51 (s, 1H, NH), 8.38 (s, 1H, H-5), 7.64 (m, 4 H, H-8, H-
7, H-30, H-60), 7.52 (m, 2H, H-40, H-50), 7.21 (d, 2H, H-300,500), 6.91 (d,
2H, H-200,600), 6.44 (s, 1H, H-3), 6.41 (s, 2H, 4-NH2), 5.31 (s, 2H, OCH2),
3.67 (s, 2H, 400-NCH2), 2.58 (bs, 2H, chain NeCH2), 2.40 (s, 3H, 2-CH3,),
1.22 (m, 8H, chain e(CH2)4e), 0.84 (bt, 3H, chain CH3). MS (ESI) m/z:

M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
1219
removed away by decantation and the residue rinsed thoroughly or
crystallized from ethyl ether.
4H, chain b,
b0eCH2), 1.22 (bm, 8H, chain central (CH2)4). MS (ESI) m/
z: 540 (M þ H)þ. Anal. Calcd. for C33H41N5O2: C, 73.43; H, 7.66; N,
12.98. Found: C, 73.40; H, 7.61; N, 12.93.
6.1.7.1. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(3-aminopropylamino-
1
methyl)phenoxymethyl]benzamide (8a). Yield 30%, mp 110e112 ꢃC H
6.1.7.7. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(9-aminononylamino-
NMR (DMSO-d6)
d
: 10.50 (s, 1H, NH), 8.37 (s, 1H, H-5), 7.64 (m, 4 H, H-
methyl)phenoxymethyl]benzamide (8g). Yield 25%, mp 88e90 ꢃC 1H
8, H-7, H-30, H-60), 7.51 (m, 2H, H-40, H-50), 7.14 (d, 2H, H-300,500), 6.87 (d,
2H, H-200,600), 6.44 (s, 1H, H-3), 6.39 (s, 2H, 4-NH2), 5.31 (s, 2H, OCH2),
3.54 (s, 2H, 400-NCH2), 3.30 (b, 2H, chain NH2), 2.49 (under DMSO, t, 2H,
NMR (DMSO-d6) d: 10.51 (s, 1H, NH), 8.38 (s, 1H, H-5), 7.64 (m, 4 H, H-
8, H-7, H-30, H-60), 7.52 (m, 2H, H-40, H-50), 7.18 (d, 2H, H-300,500), 6.90
(d, 2H, H-200,600), 6.43 (s, 1H, H-3), 6.40 (s, 2H, 4-NH2), 5.31 (s, 2H,
OCH2), 3.54 (s, 2H, 400-NCH2), 3.34 (b, 2H, chain NH2), 2.42 (partially
chain
1.09 (m, 2H, chain
a
-NCH2), 2.39 (s, 3H, 2-CH3,), 1.47 (m, 2H, chain a0eCH2NH2),
b
-CH2). MS (ESI) m/z: 470 (M þ H)þ. Anal. Calcd. for
under DMSO, m, 4H, chain
a
,
a
0ꢄNCH2), 2.39 (s, 3H, 2-CH3,), 1.32 (m,
C28H31N5O2: C, 71.60; H, 6.66; N, 14.92. Found: C, 71.58; H, 6.67; N,
14.93.
12H, chain central (CH2)6), 1.09 (bm, 2H, chain central CH2). MS (ESI)
m/z: 554 (M þ H)þ. Anal. Calcd. for C34H43N5O2: C, 73.73; H, 7.83; N,
12.65. Found: C, 73.75; H, 7.86; N, 12.63.
6.1.7.2. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(4-aminobutylamino-
methyl)phenoxymethyl]benzamide (8b). Yield 25%, mp 103e105 ꢃC 1H
6.1.7.8. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(10-aminodecylamino-
NMR (DMSO-d6)
d
: 10.45 (s, 1H, NH), 8.35 (s, 1H, H-5), 7.63 (m, 4 H, H-
methyl)phenoxymethyl]benzamide (8h). Yield 25%, mp 90e92 ꢃC 1H
8, H-7, H-30, H-60), 7.51 (m, 2H, H-40, H-50), 7.17 (d, 2H, H-300,500), 6.88
(d, 2H, H-200,600), 6.44 (s, 1H, H-3), 6.32 (s, 2H, 4-NH2), 5.31 (s, 2H,
OCH2), 3.56 (s, 2H, 400-NCH2), 3.27 (b, 2H, chain NH2), 2.42 (partially
NMR (DMSO-d6) d: 10.54 (s, 1H, NH), 8.39 (s, 1H, H-5), 7.64 (m, 4 H, H-
8, H-7, H-30, H-60), 7.51 (m, 2H, H-40, H-50), 7.20 (d, 2H, H-300,500), 6.91 (d,
2H, H-200,600), 6.42 (s, 1H, H-3), 6.39 (s, 2H, 4-NH2), 5.32 (s, 2H, OCH2),
3.55 (s, 2H, 400-NCH2), 3.32 (b, 2H, chain NH2), 2.42 (partially under
under DMSO, m, 4H, chain a,
a0eNCH2), 2.39 (s, 3H, 2-CH3,), 1.38 (m,
4H, chain
b,
b0eCH2). MS (ESI) m/z: 484 (M þ H)þ. Anal. Calcd. For
DMSO, m, 4H, chain
a,a
0ꢄNCH2), 2.40 (s, 3H, 2-CH3,), 1.23 (m, 16H,
C29H33N5O2: C, 72.01; H, 6.88; N, 14.49. Found: C, 72.06; H, 6.86; N,
14.44.
chain central (CH2)8). MS (ESI) m/z: 568 (M þ H)þ. Anal. Calcd. for
C35H45N5O2: C, 74.03; H, 7.99; N, 12.34. Found: C, 74.01; H, 7.97; N,
12.33.
6.1.7.3. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(5-aminopentylamino-
methyl)phenoxymethyl]benzamide (8c). Yield 25%, mp 102e104 ꢃC 1H
6.1.8. Methyl 2-[4-(trifluoroacetylaminomethyl)phenoxy]
methylbenzoate (9)
NMR (DMSO-d6) d: 10.51 (s,1H, NH), 8.38 (s,1H, H-5), 7.64 (m, 4 H, H-
8, H-7, H-30, H-60), 7.52 (m, 2H, H-40, H-50), 7.18 (d, 2H, H-300,500), 6.90
(d, 2H, H-200,600), 6.43 (s, 1H, H-3), 6.40 (s, 2H, 4-NH2), 5.31 (s, 2H,
OCH2), 3.54 (s, 2H, 400-NCH2), 3.34 (b, 2H, chain NH2), 2.42 (partially
A mixture of 2,2,2-trifluoro-N-[(4-hydroxyphenyl)methyl]acet-
amide (3.3 g, 15 mmol), methyl 2-(bromomethyl)benzoate (3.4 g,
15 mmol) and anhydrous K2CO3 (10.4 g, 75 mmol) in anhydrous
DMF (50 mL) was kept under stirring at 30 ꢃC for 4 h, then the
mixture was filtered and evaporated in vacuo. The residue was then
washed with water and dried under vacuum to obtain 9 (4.7 g,
under DMSO, m, 4H, chain
a
,
a
0ꢄNCH2), 2.39 (s, 3H, 2-CH3,), 1.32 (m,
4H, chain b, g-CH2). MS(ESI) m/z:
b0eCH2),1.09(bm, 2H, chain central
498 (M þ H)þ. Anal. Calcd. for C30H35N5O2: C, 72.39; H, 7.09; N,14.08.
Found: C, 72.35; H, 7.04; N, 14.05.
85%); mp 98e100 ꢃC; 1H NMR (DMSO-d6)
d: 9.92 (s, 1H, NH), 7.89
(d, 1H, H-6), 7.63 (sharp m, 2H, H-3,4), 7.48 (t, 1H, H-5), 7.18 (d, 2H,
H-30,50), 6.97 (d, 2H, H-20,60), 5.39 (s, 2H, OCH2), 4.30 (d, 2H, NCH2),
3.80 (s, 3H, OCH3). MS (ESI) m/z: 368 (M þ H)þ. Anal. Calcd. for
C18H16F3NO4: C, 58.84; H, 4.39; N, 3.81. Found: C, 58.77; H, 4.28; N,
3.85.
6.1.7.4. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(6-aminohexylamino-
methyl)phenoxymethyl]benzamide (8d). Yield 32%, mp 90e92 ꢃC 1H
NMR (DMSO-d6) d: 10.53 (s,1H, NH), 8.40 (s,1H, H-5), 7.64 (m, 4 H, H-8,
H-7, H-30, H-60), 7.53 (m, 2H, H-40, H-50), 7.20 (d, 2H, H-300,500), 6.90 (d,
2H, H-200,600), 6.45 (s, 1H, H-3), 6.40 (s, 2H, 4-NH2), 5.32 (s, 2H, OCH2),
3.56 (s, 2H, 400-NCH2), 3.34 (b, 2H, chain NH2), 2.42 (partially under
6.1.9. 2-[4-(Aminomethyl)phenoxy]methylbenzoic acid (10)
DMSO, m, 4H, chain
a
,
a
0ꢄNCH2), 2.39 (s, 3H, 2-CH3,),1.38 (m, 4H, chain
A solution of 9 (3.2 g, 8.7 mmol) in tetrahydrofuran (10 mL),
methanol (10 mL) and 16% aqueous NaOH (6.5 mL) was heated at
90 ꢃC under stirring for 3 h, then evaporated in vacuo to remove the
organic solvents. The aqueous residue was then diluted with ice
water, then 1M HCl was added dropwise until to pH 3e4, and the
resulting white precipitate collected by filtration, washed with
water and dried in vacuo at 50 ꢃC to obtain 10 (1.4 g, 62%), mp
b
,
b
0ꢄCH2), 1.24 (bm, 4H, chain central
g,
g
0ꢄCH2). MS (ESI) m/z: 512
(M þ H)þ. Anal. Calcd. for C31H37N5O2: C, 72.76; H, 7.29; N, 13.69.
Found: C, 72.78; H, 7.26; N, 13.68.
6.1.7.5. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(7-aminoheptylamino-
methyl)phenoxymethyl]benzamide (8e). Yield 28%, mp 82e84 ꢃC 1H
NMR (DMSO-d6)
d
: 10.54 (s,1H, NH), 8.39 (s,1H, H-5), 7.63 (m, 4 H, H-8,
255e258 ꢃC 1H NMR (DMSO-d6)
d: 8.13 (bs, 2H, NH2), 7.92 (d, 1H, H-
H-7, H-30,H-60), 7.54 (m, 2H, H-40,H-50), 7.19 (d, 2H, H-300,500), 6.89 (d, 2H,
H-200,600), 6.43 (s,1H, H-3), 6.40 (s, 2H, 4-NH2), 5.34 (s, 2H, OCH2), 3.58 (s,
2H, 400-NCH2), 3.34 (b, 2H, chain NH2), 2.42 (partially under DMSO, m,
6), 7.58 (m, 2H, H-3 and H-4), 7.38 (m, 3H, H-5, H-30,50), 7.00 (d, 2H,
H-20,60), 5.46 (s, 2H, OCH2), 3.94 (d, 2H, NCH2). MS (ESI) m/z: 258
(M þ H)þ. Anal. Calcd. for C15H15NO3: C, 70.01; H, 5.88; N, 5.45.
Found: C, 70.16; H, 5.97; N, 5.53.
4H, chain
a,a
0ꢄNCH2), 2.39 (s, 3H, 2-CH3,),1.37 (m, 4H, chain
b
,
b
0ꢄCH2),
1.21 (bm, 6H, chain central (CH2)3 ). MS (ESI) m/z: 526 (M þ H)þ. Anal.
Calcd. for C32H39N5O2: C, 73.10; H, 7.48; N,13.33. Found: C, 73.12;H, 7.46;
N, 13.32.
6.1.10. 2-[4-(Trifluoroacetylaminomethyl)phenoxy]methylbenzoic
acid (11)
To a suspension of 10 (0.65 g, 2.5 mmol) in pyridine (2 mL) at
15 ꢃC, N-ethyldiisopropylamine (0.41 mL, 2.5 mmol) was added,
then a solution of trifluoroacetic anhydride (0.35 mL, 2.5 mmol) in
CH2Cl2 (1 mL) was added dropwise under cooling and stirring. The
stirring was continued overnight at room temperature, then ice
water was added, followed to 1M HCl which was added dropwise to
pH about 2. The resulting precipitate was collected by filtration,
washed with water and dried in vacuo in an oven at 50 ꢃC to give 11
6.1.7.6. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(8-aminooctylamino-
methyl)phenoxymethyl]benzamide (8f). Yield 26%, mp 95e97 ꢃC 1H
NMR (DMSO-d6) d: 10.55 (s,1H, NH), 8.38 (s,1H, H-5), 7.64 (m, 4 H, H-
8, H-7, H-30, H-60), 7.53 (m, 2H, H-40, H-50), 7.19 (d, 2H, H-300,500), 6.87
(d, 2H, H-200,600), 6.43 (s, 1H, H-3), 6.40 (s, 2H, 4-NH2), 5.36 (s, 2H,
OCH2), 3.57 (s, 2H, 400-NCH2), 3.34 (b, 2H, chain NH2), 2.42 (partially
under DMSO, m, 4H, chain
a,a
0ꢄNCH2), 2.39 (s, 3H, 2-CH3,), 1.39 (m,

1220
M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
(0.36 g, 40%); mp 190e191 ꢃC dec.; 1H NMR (DMSO-d6)
d
: 9.94 (m,
imine polymer (PESI) (Filtermate 196; Packard Instruments, Mer-
iden, CT). Filters were washed three times with 1 mL of ice-cold
50 mM TriseHCl pH 7.4 and allowed to dry for 30 min at 37 ꢃC. The
plates were counted in a Top Count (Packard Instruments) after the
1H, NH), 7.62 (m, 4H, H-3, H-4, H-5, H-6), 7.21 (d, 2H, H-30,50), 6.99
(d, 2H, H-20,60), 5.44 (s, 2H, OCH2), 4.31 (d, 2H, NCH2). MS (ESI) m/z:
354 (M þ H)þ. Anal. Calcd. for C17H14F3NO4: C, 57.78; H, 4.00; N,
3.97. Found: C, 57.72; H, 3.89; N, 3.86.
addition (50 ml) of Microscint 20 (Packard) to each well.
IC50 values were obtained by fitting the competition curves
6.1.11. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(trifluoroacetylamino-
methyl)phenoxymethyl]benzamide (12)
according to a 4-parameter logistic model (ALLFIT program) [20].
A suspension of 11 (0.60 g,1.7 mmol) in oxalyl chloride (0.75 mL)
and anhydrous toluene (20 mL) was stirred overnight at room
temperature. The resulting clear solution was then evaporated in
vacuo, and the raw acyl chloride so obtained was dissolved in
CH2Cl2 (3 mL). The solution so obtained was slowly added to
a solution 4,6-diamino-2-methylquinoline (0.30 g, 1.7 mmol) in
pyridine (7.5 mL) at 0e5 ꢃC under stirring. The mixture was kept
overnight at 35 ꢃC under stirring, then evaporated in vacuo. The
solid residue suspended in water, collected by filtration, washed
with water and then crystallized from ethyl acetate. Yield 0.45 g
6.3.2. General procedure for d, k, m receptors binding assay
The binding affinities for human
d
,
k
and
m receptors were
determined with [3H]-naltrindole for
d
receptor and with [3H]-
diprenorphine for
suspension of membranes (5e15
expressing receptors [18] and receptors (Perkin Elmer Life
Sciences) and of CHO cells expressing receptors (Perkin Elmer Life
k and m receptors [17,18].
A
mg) of HEK-293 cells
d
k
m
Sciences) was incubated with the radioligand (30,000e50,000 cpm)
at room temperature for 2 h in a total volume of 1 mL containing
50 mM Hepes-Tris pH 7.4, 100 mM NaCl, 0.1 mg/mL bacitracin, 0.1%
(w/v) BSA and increasing concentrations of compounds to be tested.
Samples were incubated for 90 min at room temperature, filtered
onto GF/B glass fiber filtering microplates (Filtermate 196; Packard
Instruments, Meriden, CT) and washed three times with 1 mL of ice-
cold 50 mM TriseHCl pH 7.4 prior to scintillation counting on
a Packard Top Count.
(52%); mp 164e165 ꢃC. 1H NMR (DMSO-d6)
d: 10.91 (s, 1H, benza-
mide NH),10.00 (t,1H, acetamide NH), 8.74 (bs, 3H, H-5 and 4-NH2),
7.99 (dd, 1H, H-7), 7.65 (m, 5H, H-8, H-30, H-40, H-50, H-60), 7.16 (d,
2H, H-300,500), 6.93 (d, 2H, H-200,600), 6.64 (s, 1H, H-3), 5.34 (s, 2H,
OCH2), 4.28 (d, 2H, 400-CH2), 2.61 (s, 3H, 2-CH3). MS (ESI) m/z: 509
(M þ H)þ. Anal. Calcd. for C27H23F3N4O3: C, 63.76; H, 4.56; N, 11.02.
Found: C, 63.65; H, 4.72; N, 10.98.
IC50 values were obtained by fitting the competition curves
according to a 4-parameter logistic model (ALLFIT program) [20].
6.1.12. N-(4-amino-2-methylquinolin-6-yl)-2-[4-(aminomethyl)
phenoxymethyl]benzamide (13)
6.3.3. GTP
The 35S GTP
mixture containing 50 mM Hepes-Tris pH 7.4, 1 mM Dithiothreitol
(DTT),100 mM NaCl,10 mM MgSO4, 0.1 nM 35S GTP
S (Perkin Elmer
Life Sciences), 300 nM GDP (or concentrations varying between
0.1 nM and 100 M) and 1e2 g of membrane proteins, with or
g
S binding
A solution of 12 (0.20 g, 0.4 mmol) in saturated methanolic
ammonia (4 mL) was kept overnight at room temperature; the
solvent was then removed in vacuo and the residue suspended in
diethyl ether, collected by filtration, washed with diethyl ether and
then crystallized from ethyl acetate with some drop of methanol.
gS binding was determined in a 1 mL reaction
g
m
m
Yield 0.15 g (92%), mp 213e215 ꢃC 1H NMR (DMSO-d6)
d: 10.53 (s,
without compounds to be tested [16].
1H, NH), 8.39 (s, 1H, H-5), 7.65 (sharp m, 5H, H-8, H-7, H-40, H-50, H-
60), 7.53 (d, 1H, H-30), 7.20 (d, 2H, H-300,500), 6.90 (d, 2H, H-200,600),
6.45 (s,1H, H-3), 6.42 (bs, 2H, 4-NH2), 5.33 (s, 2H, OCH2), 3.62 (s, 2H,
400-CH2), 2.40 (s, 3H, 2-CH3). MS (ESI) m/z: 413 (M þ H)þ. Anal.
Calcd. for C25H24N4O2: C, 72.78; H, 5.87; N, 13.59. Found: C, 72.84;
H, 6.04; N, 13.50.
Acknowledgments
We wish to thank Dr. A. Alvino (Department of Chemistry,
University “La Sapienza” e Roma) for mass spectra and Mr. A.
Puccio for technical assistance.
6.2. Molecular Modeling
References
Minimum energy conformations of the compounds were
generated by Molecular Modeling Software Chem-X (Chemical
Design, Ltd.) and used to measure intramolecular distances. At the
lowest energy the molecules showed a stretched molecular shape.
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6.3.1. General procedure for NOP Receptor binding assay in
membrane preparations
Enriched plasma membranes from transfected cells were
prepared by differential centrifugations and stored at ꢄ80 ꢃC
(protein concentration 2e4 mg/mL) until used. The binding of 125I-
Tyr14-nociceptin (Perkin Elmer Life Sciences) was measured in 1 mL
reaction mixture containing 50 mM Hepes-Tris pH 7.4, 10 mM
MgSO4, 10
mM leupeptin, 10
m
M bestatin, 0.1 mg/mL bacitracin, 0.1%
(w/v) bovine serum albumin (BSA), and 3
mg of membrane proteins
from HEK-293 cells transfected with the NOP receptor as previously
described [16]. The concentration of radiotracer was maintained
constant at 50,000e100,000 cpm in the presence of increasing
concentrations of compounds to be tested. Reactions lasted 90 min
at room temperature and were terminated by rapid filtration onto
GF/B glass fiber filtering microplates pre-treated with 0.1% ethylene

M.R. Del Giudice et al. / European Journal of Medicinal Chemistry 46 (2011) 1207e1221
1221
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