Identification of bivalent ligands with melatonin receptor agonist and fatty acid amide hydrolase (FAAH) inhibitory activity that exhibit ocular hypotensive effect in the rabbit

Article abstract of DOI:10.1021/acs.jmedchem.8b00893

Activation of melatonin receptors and inhibition of fatty acid amide hydrolase (FAAH) have both shown potential benefits for the treatment of glaucoma. To exploit the combination of these biological activities in single therapeutic agents, we designed dua

Full text of DOI:10.1021/acs.jmedchem.8b00893

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Article
Identification of bivalent ligands with melatonin receptor
agonist and fatty acid amide hydrolase (FAAH) inhibitory
activity that exhibit ocular hypotensive effect in the rabbit
Gilberto Spadoni, Annalida Bedini, Lucia Furiassi, Michele Mari, Marco Mor, Laura
Scalvini, Alessio Lodola, Andrea Ghidini, Valeria Lucini, Silvana Dugnani, Francesco
Scaglione, Daniele Piomelli, Kwang-Mook Jung, Claudiu T Supuran, Laura Lucarini,
Mariaconcetta Durante, Silvia Sgambellone, Emanuela Masini, and Silvia Rivara
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00893 • Publication Date (Web): 20 Aug 2018
Downloaded from http://pubs.acs.org on August 24, 2018
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Identification of bivalent ligands with melatonin receptor agonist and fatty acid amide
hydrolase (FAAH) inhibitory activity that exhibit ocular hypotensive effect in the rabbit
a
a
a
a
b
b
c
Gilberto Spadoni, Annalida Bedini, Lucia Furiassi, Michele Mari, Marco Mor, Laura Scalvini,
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Alessio Lodola, Andrea Ghidini, Valeria Lucini, Silvana Dugnani, Francesco Scaglione,
d,b
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e
f
Daniele Piomelli, KwangꢀMook Jung, Claudiu T. Supuran, Laura Lucarini, Mariaconcetta
f
f
f
b*
Durante, Silvia Sgambellone, Emanuela Masini, Silvia Rivara
a
Dipartimento di Scienze Biomolecolari, Università degli Studi di Urbino “Carlo Bo”, Piazza
Rinascimento 6, Iꢀ61029 Urbino, Italy.
b
Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, Parco
Area delle Scienze 27/A Iꢀ43124 Parma, Italy.
c
Dipartimento di Oncologia ed EmatoꢀOncologia, Università degli Studi di Milano, Via Vanvitelli
32, Iꢀ20129 Milano, Italy.
d
Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine,
92697, California, USA.
e
Dipartimento NEUROFARBA, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università
degli Studi di Firenze, via Ugo Shiff 6, Iꢀ50019 Sesto Fiorentino (FI), Italy.
f
Dipartimento NEUROFARBA, Sezione di Farmacologia e Tossicologia, Università degli Studi di
Firenze, Viale G. Pieraccini 6, Iꢀ50019 Firenze, Italy.
*
Corresponding author
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Abstract
Activation of melatonin receptors and inhibition of fatty acid amide hydrolase (FAAH) have both
shown potential benefits for the treatment of glaucoma. To exploit the combination of these
biological activities in single therapeutic agents, we designed dualꢀacting compounds sharing the
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pharmacophore elements required for the two targets, in search for balanced potencies as MT /MT
agonists and FAAH inhibitors. In particular, the Nꢀanilinoethylamide scaffold, previously
developed for melatonergic ligands, was decorated at meta position with a polymethylene linker
bound to an Oꢀarylcarbamate group, substituted according to known structureꢀactivity relationships
for FAAH inhibition. For the most active series, the Nꢀanilinoethylamide portion was also replaced
with the indole scaffold of melatonin. Oꢀbiphenylꢀ3ꢀylcarbamate derivatives were characterized by
remarkable and balanced activity at both targets, in the nanomolar range for compound 29. Topical
administration reduced elevated intraocular pressure in rabbits, with a longer action and improved
efficacy compared to the reference compounds melatonin and URB597.
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Introduction
Glaucoma an optic neuropathy characterized by characterized by high intraocular pressure (IOP)
and progressive death of retinal ganglion cells (RGC). Chronically elevated IOP itself is a risk
factor for the development of glaucoma and can lead to visual field impairment and loss of visual
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function as a consequence of optic nerve damage. Current firstꢀline treatment of this condition is
generally based on daily topical administration of therapeutics aimed at lowering IOP. These drugs
counteract the leading causes of high IOP, i.e., they reduce the production of aqueous humor or
promote its impaired outflow through the trabecular meshwork or the uveoscleral pathway. Widely
used compounds are βꢀadrenergic antagonists, α ꢀagonists, carbonic anhydrase inhibitors,
2
2
prostaglandin analogs and miotic parasympathetic agents. Combinations of drugs are also available
for those patients in which IOP is not properly controlled by a single agent. They can be treated
either with concomitant dosing of multiple drugs or with fixedꢀcombination formulations.
Concomitant dosing, while being safe and effective, often suffers from limited patient compliance
due to several reasons, such as inconvenience, confusion caused by multiple bottles, time required
3
to administer drops and frequency of use. Nonadherence to medical prescription reduces treatment
effectiveness and leads to disease progression and visual field loss. Fixedꢀcombination formulations
simplify the treatment regimen and offer advantages not only in terms of improvement in patient
satisfaction and adherence to treatment, but also reduce exposure to ophthalmic preservatives
4
improving tolerability, avoid washout effects and may reduce cost of treatment. On the other hand,
fixed combinations have some disadvantages related to the diminished flexibility of individualized
5
,6
patient care, being not possible to titrate the dose or split the timing of doses. Moreover,
combination of drugs into one medication could in principle lead to drug interactions, instability of
7
combined molecules and the emergence of unique adverse events. In light of these limitations, a
hybrid ligand able to interact with multiple targets involved in the regulation of IOP might offer
several advantages, related to patient compliance given the reduced number of administrations and
time required and to formulation simplification, stability and shelfꢀlife of the product.
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Physicochemical properties of hybrid ligands might also differ from those of single acting agents,
which could favor the onset and maintenance of pharmacological activity. Therefore, despite the
availability of several active principles and their combinations, the search for new IOP lowering
agents is highly active and aims at developing more potent compounds with a more persistent action
and lower side effects compared to currently available drugs.
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The neurohormone melatonin (MLT, 1, Figure 1) has significant IOPꢀlowering properties. MLT
is primarily produced by the pineal gland with highest concentrations reached at night. A number of
biological activities have been ascribed to MLT, ranging from central nervous system (CNS)ꢀ
related activities, such as coordination of circadian rhythms and neuroendocrine processes, to
peripheral effects, related, for example, to regulation of the immune system, glucose handling and
cardiovascular homeostasis. In mammals, the majority of MLT actions are driven by the activation
of the two Gꢀproteinꢀcoupled receptors named MT and MT expressed in the CNS and in the
1
2
1
0,11
periphery, which signal photoperiodic information and regulate physiological functions.
binding sites have been described for MLT, such as the MT binding site, which has been identified
with the cytosolic enzyme quinone reductase 2, nuclear transcription factors of the retinoic acid
Other
3
1
2
superfamily, and other proteins such as calmodulin and calreticulin. Moreover, MLT exerts direct
antioxidant effects at high concentrations, which can be related to different biological activities
observed after its administration. MLT is produced locally in the eye and its receptors have been
1
3
identified in several areas, such as in the retina, ciliary body, cornea, lens and sclera. Topical and
systemic administration of MLT has been shown to transiently reduce IOP in normotensive and
1
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15
16
hypertensive/glaucomatous animals, such as mice, rabbits, and monkeys, as well as in
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,17
humans.
increases expression of the α
synthesis and drainage of aqueous humor in ciliary nonpigmented epithelium. Besides the natural
MLT potentiates the IOP lowering effect exerted by the adrenergic system, as it
2
and reduce expression of the β adrenergic receptors that control the
2
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19
15
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ligand, synthetic MLT receptor agents, such as agomelatine, IIK7 and 5ꢀMCAꢀNAT, have
shown IOP lowering effects.
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H
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URB597, 2
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melatonin, 1
Figure 1. Melatonin and FAAH inhibitor URB597.
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Reduction of IOP is also a wellꢀknown effect observed in cannabis users. In fact, a functional
endocannabinoid system, which includes endogenous cannabinoids, enzymes involved in their
1 2
synthesis and deactivation and the cannabinoid receptors CB and CB , has been identified in eye
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tissues and cells, comprising retina and ciliary body. Reduction of IOP has been observed after
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administration of endogenous or exogenous cannabinoids, such as ꢀ ꢀtetrahydrocannabinol, the
main active principle of cannabis, the synthetic cannabinoid receptor ligand WIN55212ꢀ2 and the
endocannabinoid arachidonoylethanolamide (anandamide, AEA), in studies performed in animals
2
2
and humans. The IOPꢀlowering effect exerted by topical AEA was counteracted by concomitant
administration of the CB receptor antagonist SR141716A (rimonabant), supporting a role for this
receptor in the modulation of IOP. However, cannabinoid actions on IOP are also mediated by
other, CB ꢀindependent, mechanisms such as activation of the TRPV1 channel and the orphan
1
2
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1
2
4
receptor GPR55 and by arachidonic acidꢀbased COX products. Experimental findings suggest that
the endocannabinoid system participates to the control of IOP modulating both production and
outflow of aqueous humor. The serineꢀhydrolase enzyme fatty acid amide hydrolase (FAAH) is the
2
5,26
main AEAꢀdegrading enzyme.
FAAH is also responsible for the hydrolysis of several other fatty
acid ethanolamides (FAEs) which have been found in the eye and might contribute to the regulation
of IOP through specific mechanisms. Interestingly, FAAH has been described as responsible for the
regulation of diurnal variation of IOP. In fact, FAAH is expressed with a circadian rhythm and high
FAAH levels drive the production of Nꢀarachidonoyl glycine which reduces IOP by activating
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GPR18 receptor. However, despite the relevance of FAAH on IOP modulation, the effect of
FAAH inhibitors on IOP has never been described.
In this study, we report the design of novel dualꢀacting compounds that behave as MLT receptor
agonists and FAAH inhibitors, and their evaluation as IOP lowering agents. We combined the
pharmacophore elements required for activation of melatonergic receptors with those for FAAH
inhibition, exploiting the relevant structural elements of two wellꢀknown classes of compounds. In
particular, Nꢀanilinoethylamides (Figure 2) are a class of versatile melatonergic ligands in which
modulations of substituents at the aniline nitrogen and on benzene ring have allowed fine tuning of
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8,29
binding affinity, receptor subtype selectivity and intrinsic activity.
The benzene ring of the
aniline structure tolerates bulky, lipophilic alkyloxy substituents, which can also provide some
30
selectivity for the MT receptor. On the other hand, NꢀalkylꢀOꢀarylcarbamates are widely used
1
31
FAAH inhibitors which irreversibly carbamoylate the catalytic serine residue. A representative
compound from this class, URB597 (2, Figure 1), has become the reference FAAH inhibitor in
pharmacological studies investigating the physioꢀpathological roles of this enzyme and its
3
2
substrates. We devised therefore two new series of Nꢀanilinoethylamide derivatives carrying on
the aniline ring an alkoxy linker bound to a carbamoyl portion, intended to exert FAAH inhibition
(
Figure 2). In one series, an NꢀcyclohexylꢀOꢀphenylcarbamate portion was joined to the aniline Oꢀ
alkyl side chain through a second bridging oxygen atom. In a second series, the cyclohexyl
substituent of URB597 was replaced by the alkyl chain stemming from the aniline portion of
melatonergic ligands, as known structureꢀactivity relationships for OꢀarylꢀNꢀalkyl carbamates had
evidenced that a linear alkyl chain is tolerated without significantly affecting FAAH inhibitory
31
potency. The length of the alkyl spacer and the substitution patterns were previously investigated
by molecular modelling, docking the modelled compounds within their putative binding sites at the
two protein targets. While for FAAH several crystal structures are available, for MLT receptors a
1
homologyꢀbased model of the MT subtype was employed. As the second series of compounds
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resulted the most promising one at preliminary biological tests, it was further explored replacing the
Nꢀanilinoethylamide portion with the original indole structure of melatonin (Figure 2).
1 2
The compounds were tested on human MT and MT receptors to evaluate their binding affinity and
intrinsic activity, on rat FAAH for their inhibitory potency and in a rabbit model of ocular
hypertension to investigate their IOPꢀlowering potential.
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O
R1
O
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H
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(CH )
2 n
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N
CH3
N
N-anilinoethylamides
CH3
+
R
H
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N
R
O
O
O
HN
O
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(CH2)n
O
NH
Bioisosteric
replacement
NHCOCH3
NHCOCH3
(CH )
2 n
R
O
N-alkyl-O-arylcarbamates
N
CH3
N
H
Figure 2. Design of dualꢀacting compounds by combination of relevant structural features of Nꢀ
anilinoethylamide melatonergic ligands and NꢀalkylꢀOꢀarylcarbamate FAAH inhibitors.
Chemistry
Compounds 10-13 (Scheme 1) were obtained by Oꢀalkylation of (3ꢀhydroxyanilinoethyl)amides
(5a-b) with the bromoalkylphenoxy derivative 6 or 7 (which in turn were prepared by Oꢀalkylation
of 3ꢀ(benzyloxy)phenol with the proper dibromoalkane) in the presence of NaH, followed by Oꢀ
debenzylation by hydrogenolysis (10-11) and subsequent Oꢀcarbamoylation with cꢀhexylisocyanate
in the presence of Et
3
N (12-13). The key intermediates 5a-b were synthesized according to a wellꢀ
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validated route to similar compounds, involving a reductive Nꢀalkylation of 3ꢀmethoxyꢀNꢀ
methylaniline with the suitable Nꢀacylaminoacetaldehyde dimethyl acetal (3a,b) in the presence of
3
TFA/Et SiH, followed by methyl ether cleavage of intermediates 4a,b using boron tribromide.
Compounds 20a-e and 20g were prepared in a convergent fashion following the steps outlined in
Scheme 2. First, the aminoalkoxyphenyl intermediates 16a-d were synthesized by Oꢀalkylation of
the previously cited phenol derivatives 5a,b with the appropriate tertꢀbutyl (ωꢀ
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2 3
bromoalkyl)carbamate 14a-c in the presence of K CO /NaI and subsequent NꢀBocꢀdeprotection
(TFA). The activated carbonates 19d-f were then prepared by reaction of 1,1ꢀbiphenylꢀ3ꢀol
derivatives (18d-f) [commercially available (18d) or prepared by a Suzuki coupling reaction of 3ꢀ
bromophenol with the suitable phenylboronic acid 17e-f] with pꢀnitrophenyl chloroformate in the
presence of N,Nꢀdiisopropylethylamine (DIPEA). Finally, carbamates 20a-f were obtained by the
convergent coupling of the aminoalkoxyphenyl intermediates 16a-d with the appropriate 4ꢀ
nitrophenyl carbonate (19d-f). The final compound 20g was achieved by hydrogenolysis of the
benzyloxy precursor 20f.
Compounds 27-29 were obtained by condensation of 5ꢀ(aminoalkoxy)ꢀNꢀacetyltryptamines 24-26
with the (4ꢀnitrophenyl)carbonate derivative 19d as outlined in Scheme 3. The intermediate amines
2
4-25 were prepared by Oꢀalkylation of Nꢀacetylserotonin with tertꢀbutyl (6ꢀbromohexyl)carbamate
(
14b) or tertꢀbutyl (8ꢀbromooctyl)carbamate (14c) in the presence of K CO , and subsequent NꢀBoc
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deprotection with trimethylsilyl bromide. The 2ꢀbromoindole derivative 23 was obtained by
bromination of 21 with trimethylphenylammonium tribromide in THF, before submitting it to Nꢀ
Boc deprotection to give 26.
a
Scheme 1
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
NH
O
2
a
b
H3CO
OC H3
N
H
R
c
H
H
N
R
N
R
OC H3
ab,
N
N
H3CO
R
O
O
CH3
CH3
a
b
C H3
C2
3
4 ab,
5ab,
H
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O H
d
H
N
Br
C H3
HO
O Bn
O
O Bn
(
CH )
N
Br-(CH ) -Br
2 n
2
n
6n= 4
O
7
n= 6
CH
3
5a
e
HN
O
OBn
O
OH
O
O
O
O
O
O
(
CH )n
(CH )n
(CH )
2 n
2
2
g
f
N HC OC H3
N HC OC H3
N HC OC H3
N
N
N
NC O
CH312n= 4
3n= 6
CH3
10 n= 4
11n= 6
CH3
8n= 4
9n= 6
1
a
Reagents and conditions: a) (RCO)
methylaniline, Et SiH, TFA, CH Cl , rt, 20 h, yield 51ꢀ52%; c) BBr
68ꢀ76%; d) K CO , acetone, 50 °C, 24 h, yield 68ꢀ82%; e) NaH, DMF, ꢀ10 °C to rt, 16 h, yield 85ꢀ
9%; f) H (1 atm), 10% PdꢀC, EtOH/EtOAc, rt, 16 h, yield 78ꢀ92%; g) Et N, EtOH/ CH CN, rt, 18
2
O, Et
3
N, CH
2
Cl
2
, rt, 2 h, yield 90ꢀ93%; b) 3ꢀmethoxyꢀNꢀ
3
2
2
3
, CH Cl , 0 °C to rt, 20 h, yield
2
2
2
3
8
2
3
3
h, yield 65ꢀ95%.
a
Scheme 2
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7
8
9
O H
O
O
(
CH ) NHBoc
(CH ) NH2
2 n
2 n
a
b
H
H
H
N
R
N
R
N
R
N
N
N
Br(CH ) -NHBoc
2
n
O
14 a- c
O
O
CH
CH3
15 a- d
CH3
16 a- d
3
ab,
5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R1
n
R
O
a
C H3
C2
H
4
O
R'
b
H
H
H
H
6
5
O
f
c
d
C H3
C H3
8
6
6
6
6
(CH2)n NH
e
C H3
C H3
C H3
CO NH2
OC H Ph
O H
H
N
R
f
g
N
g
2
O
CH3
20 a- g
O H
O
R
O
B(OH)2
c
d
e
O
or
NO 2
R
1
O H
O
C l
1
7e ,f
O
R
1
1
O2
N
R
1
Br
1
8 d- f
19 d- f
e
f
CO NH2
OC H Ph
2
a
Reagents and conditions: a) K
6 h, yield 60ꢀ64%; c) for 18e Pd(PPh
Pd(OAc) , Na CO , H O, acetone, 35 °C, 40 min, yield 81%; e) DIPEA, CH
4%; f) Et N, CH Cl , rt, 4 h, yield 38ꢀ90%; g) H (1 atm), 10% PdꢀC, EtOH/EtOAc, rt, 20 h, yield
6%.
2
CO
3
, NaI, acetone, 60 °C, 24 h, yield 20ꢀ44%; b) TFA, CH
, Na CO , CH CN, 90 °C, 1.5 h, yield 97%; d) for 18f
CN, rt, 1 h, yield 47ꢀ
2 2
Cl , rt,
1
3
)
4
2
3
3
2
2
3
2
3
7
7
3
2
2
2
a
Scheme 3
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NHBoc
NHCOCH3
NHCOCH3
(
CH )
2 n
a
HO
O
21 R=H, n=6
22 R=H, n=8
Br(CH ) -NHBoc
2
n
b
R
23 R=Br, n=6
N
N
14b n=6
4c n=8
H
H
1
c
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
CH ) SiBr
3 3
NH2
NHCOCH3
(CH2)n
O
R
O
N
O
H
NH
CH2)n
(
2
4 R=H, n=6
25 R=H, n=8
6 R=Br, n=6
NHCOCH3
d
O
2
R
N
H
O
O
27 R=H, n=6
8 R=H, n=8
29 R=Br, n=6
2
O
NO2
19d
a
Reagents and conditions: a) K
2 3 3
CO , NaI, CH CN, reflux, 20 h, yield 47ꢀ59%; b)
(
CH ) N(Br )C H , THF, rt, 30 min, yield 59%; c) CH CN, rt, 1 h; d) Et N, CH Cl /DMF, rt, 3 h,
3
3
3
6
5
3
3
2
2
two steps (c, d) yield 83ꢀ95%.
Results and discussion
Design of new compounds
The accommodation of putative dualꢀacting agents into MLT receptor binding site and FAAH
catalytic pocket was previously evaluated by docking simulations. Due to the lack of crystal
structures of MLT receptors, docking studies were performed within a homology model of the MT
1
3
0
receptor in its active conformation previously developed and tested by us. Preceding docking
models for melatonergic ligands had proposed that substituent originating from the aromatic oxygen
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3
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5
6
7
8
9
atom (e.g., the methoxy oxygen of MLT), which is important for receptor affinity, can be
accommodated within a lipophilic channel outlined by transmembrane (TM) helices 3, 4 and 5,
open toward the extracellular space in its upper portion. The presence of some smaller amino acids
lining this channel in the MT
1
receptor subtype, compared to the corresponding residues in the MT
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
subtype, has been proposed as the structural element conferring MT
1
selectivity to ligands with
30
bulkier substituents at the oxygen atom (e.g., a phenylbutyl group). Since both of these MLT
receptor subtypes have been implicated in the ocular hypotensive activity observed for MLT
15,34
agonists,
1 2
in order to obtain compounds with similar activity at MT and MT receptors an alkyl
linker was inserted to connect the MLT receptor binding portion to the FAAH inhibiting one, with
the idea that the slim alkyl chain could be tolerated into both receptors, allowing the carbamate
portion to interact with the extracellular region of the receptor. Docking models showed that both
orientations of the carbamate portion, corresponding to the two series of compounds 12-13 and 20,
could be accommodated in the extracellular lobby, provided that at least fourꢀmethylene long
1
spacers were inserted. The binding pose into the MT receptor obtained for compound 20e, which
has a sixꢀmethylene spacer, is represented in Figure 3, where the bulkier amino acids lining the
lipophilic channel of the MT receptor are also shown. The carbamoyl fragment of 20e protrudes
2
out of the TM portion of the receptor and interacts with amino acids belonging to extracellular loop
2. The Nꢀanilinoethylamide portion undertakes the pattern of interactions already proposed for
melatonergic ligands, comprising hydrogen bonds between Tyr285 in TM7 and the amide oxygen
and between Tyr187 in TM5 and the alkoxy oxygen.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
0
Figure 3. Inducedꢀfit docking solution obtained for compound 20e into the homology model of
the MT receptor. Side chains of bulkier amino acids belonging to the MT receptor are represented
in shaded green (MT
1
2
3.29
4.57
4.61
3.29
4.57
4.61
1
2
: Gly104 , Val159 , Leu163 ; MT : Ala117 , Leu172 , Phe176 ).
Fitting of dualꢀacting compounds into the active site of FAAH was also evaluated. Compounds
belonging to the NꢀcyclohexylꢀOꢀphenylcarbamate series, exemplified by compound 13 in Figure 4
(left), place their cyclohexyl ring in the acyl chain binding (ACB) channel, consistent with its
3
5
location in the crystal structure of FAAH carbamoylated by URB597. The long substituent at
carbamate oxygen atom occupies the cytoplasmic access (CA) channel taking polar interactions
with Lys263 and Gln273. The lower FAAHꢀinhibitory potency observed for this series (see below)
was attributed to possible repulsive interaction of the anilinoethylamide fragment with the dimer
interface of FAAH which could be detrimental for compound affinity.
The second class of compounds (20aꢀd) generally gave docking solutions placing the melatonergic
portion within the ACB channel, which looked more tolerant to structural alternatives. For this
reason, and because preliminary data indicated this class as more promising than the first one,
structural variants were introduced while conserving the Oꢀbiphenylꢀ3ꢀylꢀcarbamate scaffold. In
particular, small polar groups were introduced at the 3' position of the biphenyl nucleus (20e, 20g),
trying to improve FAAH inhibitory potency and the length of the spacer was increased to eight
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4
5
6
7
8
9
methylenes (20c) to exploit the steric tolerance of the ACB channel of FAAH. At the melatonergic
portion, the acetylamino group was replaced by a propionylamino one (20b) and the Nꢀ
anilinoethylamide scaffold by the Nꢀindolylethylamide one of MLT to favour interactions at the
melatomergic receptors. A representation of the docking solution obtained for the bulkiest inhibitor
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
29 is provided in Figure 4 (right). The carbamate portion occupies the catalytic site and its NH
group is hydrogenꢀbonded to the carbonyl oxygen of Met191. The carbamate oxygen is stabilized
into the oxyanion hole, interacting with the backbone NH groups of Ile238 and Gly239. The
biphenyl portion is accommodated into the CA channel, while the long substituent at carbamate
nitrogen extends inside the ACB channel. The bulky 2ꢀbromolindole terminal portion of 29 has
room enough to be properly accommodated: while the acetylamide takes a hydrogen bond with
Thr488, the bromine atom fits into a lipophilic pocket lined by amino acids Ala377, Leu380,
Phe381 and Phe432.
Figure 4. Compounds 13 (left) and 29 (right) docked into FAAH substrate binding site (PDB ID:
1MT5). Gray ribbons and surfaces refer to the monomer of FAAH which includes the catalytic site
were inhibitors were docked; orange ribbons and surfaces refer to the other FAAH monomer
associated with the first one. Docked inhibitors are represented with yellow (13) or green (29)
carbons.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In vitro potency and structure-activity relationships
Binding affinity and intrinsic activity at MT and MT receptors and FAAH inhibitory potency
1
2
obtained for the newly synthesized compounds are reported in Table 1.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Binding Affinity and Intrinsic Activity of Newly Synthesized Compounds at Human
Melatonin Receptors MT and MT and Inhibition Potency (IC ) of Rat FAAH Activity.
1
2
50
NHCOCH
3
NHCOCH
3
NHCOCH
3
O
(
CH
2
)
n
(CH
2
)
n
2 n
(CH )
HO
O
O
N
N
O
O
O
N
H
2
N
O
N
H
CH
3
CH
3
CH
3
10-11
12-13
16a,d
NHCOR1
NHCOCH3
O
H
R
(CH
2
)n
O
N
O
O
N
O
N
2 n
(CH )
H
R
CH
3
O
N
H
20a-e, 20g
27-29
Cmpd
MLT receptors
rFAAH
1
n
R
R
hMT
1
hMT
2
IC50 (nM)
a
b
a
b
p
Ki ± SD
IA ± SD
p
Ki ± SD
IA ± SD
r
r
c
±
SEM
1
d
9
.69 ± 0.09 1.00 ± 0.01 9.54 ±0.06 1.00 ± 0.03
nd
MLT
2
e
URB5
nd
nd
nd
nd
4.6 ± 1.6
9
7
10
4
6
4
6
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
6.73 ± 0.08 0.27 ± 0.07 6.22 ± 0.07 0.18 ± 0.09
7.23 ± 0.11 0.62 ± 0.14 6.48 ± 0.04 0.57 ± 0.08
7.46 ± 0.10 0.07 ± 0.07 7.27 ± 0.01 0.05 ±0.05
7.56 ± 0.09 0.71 ± 0.04 6.53 ± 0.06 0.71 ± 0.05
nd
1
1
1
1
2
3
nd
184±3
197±6
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16a
16d
4
6
4
6
8
6
ꢀ
ꢀ
ꢀ
5.51 ± 0.08 0.32 ± 0.09 4.89 ± 0.50 0.35 ± 0.04
6.35 ± 0.03 0.34 ± 0.05 6.40 ± 0.04 0.08 ± 0.01
nd
nd
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢀ
2
0a
0b
H
H
H
H
CH
3
7.89 ± 0.03 0.64 ±0.07 7.35 ± 0.03 0.39 ± 0.07 1.45±0.01
7.79 ± 0.04 0.77 ± 0.08 7.72 ± 0.02 0.88 ± 0.02 3.07±0.08
7.50 ± 0.01 0.85 ± 0.08 7.49 ± 0.02 0.96 ± 0.03 0.63±0.04
7.67 ± 0.01 0.70 ± 0.01 7.48 ± 0.02 0.75 ± 0.03 1.34±0.03
7.41 ± 0.03 0.79 ± 0.06 7.81 ± 0.05 1.01 ± 0.02 0.43±0.01
2
2 3
CH CH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20c
CH
CH
CH
CH
ꢀ
3
3
3
3
20d
20e
6 CONH
2
20g
6
6
8
6
OH
H
7.53 ± 0.02 0.89 ±0.08
7.57 ±0.01 0.94 ± 0.02 0.36±0.01
2
7
8
8.22 ± 0.01 0.73 ±0.08 8.34 ± 0.09 1.02 ± 0.02 2.38±0.16
8.31 ± 0.70 0.73 ± 0.08 8.20 ± 0.02 0.53 ± 0.02 4.00±0.10
9.11 ± 0.10 0.73 ± 0.08 8.77 ± 0.03 0.97 ± 0.02 0.85±0.01
2
H
ꢀ
29
Br
ꢀ
a
p
Ki values were calculated from IC values, obtained from competition curves by the method of
50
3
6
b
Cheng and Prusoff, and are the mean of at least three determinations performed in duplicate. The
relative intrinsic activity values were obtained by dividing the maximum analogueꢀinduced Gꢀ
c
protein activation by that of MLT. Measurements were performed in triplicate. IC50 values are the
d
e
mean of at least two determinations. nd: not determined. Ref. 32.
NꢀCyclohexylcarbamic acid Oꢀphenyl esters 12 and 13 showed only moderate binding affinity at
1 2
human MT and MT receptors, about one hundred times lower than that of MLT. These
compounds, as well as all the other agents in Table 1, showed no receptor subtype selectivity,
consistent with our design strategy. Compounds 10 and 11, which could be produced in vivo from
hydrolysis of carbamates 12 and 13, showed reduced binding affinity, that may be attributed to
lower lipophilicity of their chains. In fact, the detrimental role played by hydrophilic substituents on
the side chain stemming from the metaꢀoxygen of Nꢀanilinoethylamide derivatives has already been
37
observed. Compounds 12 and 13 also showed poor potency on FAAH. Compared to the biphenyl
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
substituent of compound 2 the longer phenoxyalkyl chain significantly impacted on the ability to
inhibit enzyme activity.
1 2
Oꢀbiphenylcarbamates 20a-e, 20g showed binding affinities at MT and MT receptors similar or
slightly higher than the previous Nꢀcyclohexylcarbamates 12 and 13. The length of the alkyl spacer
did not influence binding affinity and similar potencies were observed for compounds 20a and 20d,
and for the longest compound 20c. However, chain lengthening was correlated with an increase in
intrinsic activity, with compound 20c behaving as an agonist. Also the replacement of the
acetylamino group of 20d with a propionylamino one (20b) produced the expected slight increase in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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11,29
binding affinity and intrinsic activity, consistent with SAR profile of melatonergic ligands.
These compounds showed remarkable ability to inhibit FAAH activity, with a subnanomolar IC50
value observed for compound 20c. Substituents known to be accommodated in FAAH active site
3
2
were inserted in meta position of the distal phenyl ring. As expected, the aminocarbonyl and
hydroxyl derivatives 20e and 20g showed improved potencies compared to the unsubstituted 20d.
These substituents were tolerated at MLT receptors and led to an increase of intrinsic activity at
1 2
both receptor subtypes. MT and MT binding affinity of the amino derivatives 16a and 16d,
formally deriving from carbamate hydrolysis was also tested. Consistently with the previously cited
SAR for Nꢀanilinoethylamide ligands, no significant affinity could be measured for these basic and
hydrophilic derivatives. Therefore activity of Oꢀbiphenyl carbamates at MLT receptors has to be
ascribed to the compounds per se, and not to metabolites deriving from hydrolysis reactions
performed by FAAH or other hydrolases.
OꢀBiphenylcarbamates demonstrated that it is possible to fuse the structural elements required for
FAAH inhibition and for MLT receptor binding and activation in the same molecule. However,
activity at the two targets of compounds 20a-e and 20g appeared rather unbalanced as FAAH
inhibitory potency is about 10ꢀ100 times higher than binding affinity at MLT receptors. To increase
MT
1 2
and MT binding affinity the Nꢀanilinoethylamide portion was replaced by the Nꢀ
indolylethylamide scaffold of MLT. This bioisosteric replacement allowed to increase MLT
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receptor binding affinity of about one order of magnitude as observed for compounds 27 and 28,
with maintenance of FAAH inhibitory potency in the nanomolar range. Moreover, introduction of a
bromine atom in position 2 of the indole ring in compound 29 allowed a further increase of MT
1
38
and MT
2
binding affinity, consistent with SAR for indole derivatives. The bromine atom slightly
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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9
0
improved FAAH inhibitory potency leading to a subnanomolar IC50 value, consistent with our
hypothesis that this substituent can be favorably positioned in the lipophilic acyl chain binding
pocket of FAAH (see previous modelling paragraph). Compound 29 represents therefore a potent
dualꢀacting melatonergic agonist and FAAH inhibitor with balanced potency at the two targets.
Ocular pressure lowering activity
The ability to reduce IOP of compounds belonging to the two most active series, 20c,e,g carrying an
Nꢀanilinoethylamide portion and 27ꢀ29, with an Nꢀindolylethylamide one, was evaluated in vivo in
an animal model of glaucoma. Their behavior was compared with that of compounds 1 and 2 and of
the clinically used IOP lowering agent dorzolamide (30). 50 ꢁL of a solution of the test compounds
at 1 mM concentration were topically administered into the lower conjunctival pocket to male New
Zealand albino rabbits with elevated IOP, induced by the injection of 0.1 mL of sterile hypertonic
saline solution (5% in distilled water) into the vitreous body. Reference compound 30 was tested at
1% concentration (w/v) of dorzolamide hydrochloride. IOP was measured 60, 120 and 240 min
after pressure stabilization following hypertonic saline injection.
Efficacy of compounds was monitored after 60 and 120 min, as after 4 hours IOP was returned to
basal, preꢀinjection values (Figure 5). Compound 1 had a prompt effect, visible at 60 min which
was maintained also at 120 min, even if with lower efficacy. A similar behavior was observed for
the reference FAAH inhibitor 2. Reduction of IOP by cannabinoid receptor ligands, both
endocannabinoids and synthetic compounds, had been widely described, but this is the first time
that efficacy as an IOP lowering agent is reported for a compound which acts indirectly, by
increasing the local availability of anandamide and other fatty acid ethanolamides. Coꢀ
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6
administration of compounds 1 and 2, both at 1mM concentration, produced the same effect as the
administration of single agents at 60 min. However, the IOP reduction was maintained at 120 min,
while single agents 1 and 2 lost part of their efficacy at this time. The carbonic anhydrase inhibitor
30 had a prompt effect, similar to that of compounds 1 and 2 at 60 min, which is maintained at 120
0
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min as well. The Nꢀanilinoethylamide derivatives 20c, 20e and 20g behaved similarly, showing
limited effect after 60 min, which significantly increased at 120 min, exceeding those measured for
compounds 1 and 2. In the Nꢀindolylethylamide series (27ꢀ29) compounds 27 and 28 were
relatively active at 60 min, with limited activity at longer times. On the other hand, compound 29
had significantly improved efficacy at 120 min, in analogy with compounds from the 20 series. In
fact, it provided the highest IOP reduction at 120 min which is also partly maintained at 240 min.
Figure 5. Drop of intraocular pressure (ꢁ IOP, mm Hg) versus time (min) in hypertonic salineꢀ
induced ocular hypertension in New Zealand male albino rabbits measured for compounds 1
(
melatonin), 2 (URB597), the combination of compounds 1 and 2, the reference inhibitor 30
dorzolamide) and dualꢀacting inhibitors 20c, 20e, 20g, 27, 28 and 29, tested at 1 mM concentration
(
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(
compound 30 tested at 1% (w/v) concentration). Histograms represent ꢁ IOP change, i.e., the mean
difference (n= 4 for each treatment group) in ꢁ IOP between the eye treated with vehicle and the
contralateral one, treated with the drug. Positive values mean that drugꢀtreated eyes have lower IOP
than vehicleꢀtreated ones. Error bars represent s.e.m. of mean IOP value (n=4). Tabular data of ꢁ
IOP can be found in Supplementary Table S3. Data are analyzed with 2way Anova followed by
Bonferroni multiple comparison test. p<0.05 was used to identify statistical significance. * p<0.05:
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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7
8
9
0
1
, 2 and 30 vs vehicle at 60 min; 20c vs vehicle at 120 min; 29 vs 2 at 120 min. ** p< 0.01: 30 vs
vehicle at 120 min; 29 vs 1 at 120'. *** p<0.001: 20e and 29 vs vehicle at 120 min.
Discussion and conclusions
Novel compounds targeting two physiological systems involved in the control of IOP were devised.
Exploitation of SAR for melatonin receptor agonists and carbamate FAAH inhibitors led to dualꢀ
acting agents with remarkable and balanced activity at both targets. In the rabbit model of high IOP
compounds were more effective at longer times (120 min) compared to reference singleꢀacting
agents 1, 2 and the most potent derivatives 20e and 29 were more effective than the therapeutic
agent 30. This behavior might be due either to the synergistic effect at the two targets or to the
higher lipophilicity of the dualꢀacting compounds, which could assure a longer residence time at the
site of action. Combination of compounds 1 and 2 gave the same effect as single agents at 60 min,
thus showing no additivity, but this effect was maintained at longer times (120 min). Thus, only the
combination of the two gave a time profile similar to that of dorzolamide (30). As for dualꢀacting
compounds, the Nꢀanilinoethylamide 20e and the Nꢀindolylethylamide 29 gave the best IOP
lowering effect at 120 min, comparable or better than dorzolamide (30). Comparing in vivo with in
vitro activity, the ability of dualꢀacting compounds to reduce IOP at longer times seems to be
strictly related to their potency as FAAH inhibitors. In fact, the compounds most active at 120 min
(20c, 20e, 20g and 29) showed subꢀnM IC50 values on FAAH, while compounds 27 and 28 had
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6
lower FAAH inhibitory potency. On the other hand, compound 29 having the highest efficacy at
longer times (120 and 240 min) is endowed with high potency on both targets. Therefore, the
combination of high melatonergic activity with potent inhibition of FAAH appears as a requisite to
obtain a significant and persistent IOP lowering effect, even if a role for physicochemical
properties, such as compound lipophilicity, cannot be ruled out.
0
1
2
3
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8
9
0
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9
0
These compounds represent a potential alternative therapeutic approach for the treatment of ocular
hypertension and might offer a promising option for the treatment of glaucoma in light of their
potential neuroprotective effect. In fact, the endocannabinoid system is involved in cell survival
3
9
mechanisms of RGC and endocannabinoids and FAAH inhibitors have been described as able to
minimize the retinal damage and reduce RGC loss in a model of high intraocular pressureꢀinduced
40
ischemia. On the other hand, the neuroprotective activity of melatonin is widely known and its
9
protective role has been demonstrated also in ocular tissues. A neuroprotective role has been
described also for synthetic melatonin receptor ligands. In addition, reduction of RGC death may
positively modulate pineal melatonin production that could improve circadian rhythmꢀrelated
functions, thus ameliorating conditions often present in glaucomatous patients such as sleep
4
1
disorders and depression.
Experimental section
General procedures
Melting points were determined on a Buchi Bꢀ540 capillary melting point apparatus and are
1
13
uncorrected. H NMR and C NMR spectra were recorded on a Bruker AVANCE 400 instrument,
using CDCl as solvent unless stated otherwise. Chemical shifts (δ scale) are reported in parts per
million (ppm) relative to the central peak of the solvent. Coupling constants (
3
J
) are given in hertz
+
(
Hz). ESI MS spectra were taken on a Waters Micromass ZQ instrument; molecular ions [M+1] or
ꢀ
[
Mꢀ1] are given. Highꢀresolution mass spectroscopy was performed on a Micromass QꢀToF Micro
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mass spectrometer (Micromass, Manchester, UK) using an ESI source. The purity of all tested
compounds, determined by LCꢀMS using a Waters HPLC/UV/MS system (separation module
Alliance HT2795, Photo Diode Array Detector 2996, mass detector Micromass ZQ; software:
MassLynx 4.1), was greater than 95% (see Supporting Information). Column chromatography
purifications were performed under “flash” conditions using Merck 230−400 mesh silica gel.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
4
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5
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0
1
2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Analytical thinꢀlayer chromatography (TLC) was carried out on Merck silica gel 60 F254 plates. tert
ꢀ
Butyl (4ꢀbromobutyl)carbamate, tertꢀbutyl (6ꢀbromohexyl)carbamate, [1,1’ꢀbiphenyl]ꢀ3ꢀol, (3ꢀ
carbamoylphenyl)boronic acid, [3ꢀ(benzyloxy)phenyl]boronic acid, 3ꢀ(benzyloxy)phenol, 2,2ꢀ
dimethoxyethanamine and
Nꢀacetylserotonin were purchased from commercial suppliers and used
without further purification.
General Procedure for the synthesis of
propionic anhydride (12 mmol) and Et N (1.65 mL, 12 mmol) were added to a solution of 2,2ꢀ
dimethoxyethanamine (1.08 mL, 10 mmol) in dry CH Cl (16 mL) and the resulting mixture was
N-(2,2-Dimethoxyethyl)acylamines 3a,b. Acetic or
3
2
2
stirred at room temperature for 2 h. The mixture was neutralized with a saturated solution of
NaHCO and extracted with CH Cl . The combined organic phases were washed with brine, dried
3
2
2
(
Na SO ), filtered and concentrated in vacuo affording a crude residue that was purified by silica
2 4
gel column chromatography.
N-(2,2-Dimethoxyethyl)acetamide (3a). Flash chromatography (CH
2 2
Cl ꢀMeOH 98:2 as eluent).
33
Oil; 90% yield. Chemical physical data were identical to those previously reported.
2 2
N-(2,2-Dimethoxyethyl)propionamide (3b). Flash chromatography (CH Cl ꢀEtOAc 95:5 as
1
eluent). Oil; 93% yield. H NMR (CDCl
3
) δ: 1.16 (t, 3H,
J
= 7.5), 2.23 (q, 2H,
J
= 7.5), 3.40 (s,
+
6H), 3.39ꢀ3.44 (m, 2H), 4.38 (t, 1H,
J
= 5.0), 5.70 (brs, 1H). ESI MS (m/z): 184 [M+Na] .
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6
General Procedure for the synthesis of (anilinoethyl)amido derivatives 4a,b. Trifluoroacetic
acid (2.2 mL, 29 mmol) and triethylsilane (0.9 mL, 5.6 mmol) were added to a solution of 3ꢀ
2 2
methoxyꢀNꢀmethylaniline (0.3 mL, 2.3 mmol) and the suitable acetal 3a,b (3.1 mmol) in CH Cl (5
mL), and the resulting mixture was stirred at room temperature for 20 h under a nitrogen
atmosphere. After cooling to 0 °C, the mixture was carefully neutralized with a saturated aqueous
solution of NaHCO and the aqueous layer was extracted with CH Cl . The combined organic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
2
phases were washed with brine, dried over Na SO and concentrated under reduce pressure to give a
2
4
crude residue that was purified by column chromatography.
N-{2-[(3-Methoxyphenyl)methylamino]ethyl}acetamide (4a). Purification by silica gel flash
chromatography (EtOAc as eluent) and crystallization. White solid (diethyl ether/petroleum ether),
33
51% yield. Chemical physical data were identical to those previously reported.
N-{2-[(3-Methoxyphenyl)methylamino]ethyl}propionamide (4b). Purification by silica gel
1
chromatography (EtOAcꢀcyclohexane 7:3 as eluent). Oil, 52% yield. H NMR (CDCl
3
) δ: 1.12 (t,
3
6
1
H,
J = 7.5), 2.16 (q, 2H, J = 7.5), 2.95 (s, 3H), 3.46ꢀ3.47 (m, 4H), 3.80 (s, 3H), 5.70 (brs, 1H),
13
.31ꢀ6.33 (m, 2H), 6.42 (dd, 1H,
J
= 2.0 and 8.0), 7.16 (dd, 1H, J1
=
J2 = 8.5). C NMR (CDCl ) δ:
3
74.0, 160.9, 150.6, 130.0, 105.8, 102.1, 99.3, 55.2, 52.0, 38.6, 37.2, 29.6, 9.7. ESI MS (m/z): 237
+
[
M+H] .
General Procedure for the synthesis of (3-hydroxyanilino)ethylamido derivatives 5a,b. A 1M
solution of BBr in CH Cl (2.5 mL, 2.5 mmol) diluted with dry CH Cl (9 mL) was added
3
2
2
2
2
dropwise to an iceꢀcooled solution of the suitable amide 4a,b (1.23 mmol) in dry CH
2 2
Cl (9 mL)
and the resulting mixture was stirred at room temperature for 20 h. After neutralization with a 2N
aqueous solution of Na
2 3 2 2
CO , the mixture was extracted with CH Cl , and the combined organic
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phases were dried (Na SO ) and concentrated under reduced pressure to give a crude residue that
2
4
was purified by column chromatography.
N
-{2-[(3-Hydroxyphenyl)methylamino]ethyl}acetamide (5a). Purification by silica gel flash
chromatography (CH Cl ꢀMeOH 98:2 as eluent). Oil, 76% yield. Chemical physical data were
identical to those previously reported.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
3
0
N-{2-[(3-Hydroxyphenyl)methylamino]ethyl}propionamide (5b). Purification by silica gel
1
chromatography (EtOAcꢀcyclohexane 7:3 as eluent). Oil, 68% yield. H NMR (CDCl
3
) δ: 1.13 (t,
3H,
J
= 7.5), 2.18 (q, 2H,
J
= 7.5), 2.94 (s, 3H), 3.45ꢀ3.47 (m, 4H), 5.71 (brs, 1H), 6.01 (brs, 1H),
+
6.25 (d, 1H,
J
= 7.5), 6.32ꢀ6.36 (m, 2H), 7.09 (dd, 1H, J1 = J2 = 8.0). ESI MS (m/z): 223 [M+H] .
2 3
General Procedure for the synthesis of bromoalkylphenoxy derivatives 6 and 7. K CO (0.31 g,
2.25 mmol) and the suitable dibromoalkane (2.25 mmol) were added to a solution of 3ꢀ
(benzyloxy)phenol (0.3 g, 1.5 mmol) in dry acetone (4 mL), and the resulting mixture was stirred at
50°C for 24 h. After addition of water the mixture was extracted with EtOAc and the combined
organic phases were dried (Na SO ). After removing the solvent by distillation under reduced
2
4
pressure, the crude residue was purified by silica gel column chromatography (cyclohexaneꢀEtOAc
5:5 as eluent).
9
1-(Benzyloxy)-3-(4-bromobutyloxy)benzene (6). Oil, 68% yield. Analytical data were in
42
agreement to those previously reported.
1
1
-(Benzyloxy)-3-(6-bromohexyloxy)benzene (7). Oil, 82% yield. H NMR (CDCl
3
) δ: 1.49ꢀ1.56
(
m, 4H), 1.81ꢀ1.92 (m, 4H), 3.45 (t, 2H, = 6.5 Hz), 3.96 (t, 2H, = 6.5 Hz), 5.07 (s, 2H), 6.58ꢀ
J
J
13
6.62 (m, 3H), 7.19 (dd, 1H, J1
=
J2 = 8.0 Hz), 7.28ꢀ7.45 (m, 5H); C NMR (CDCl ) δ: 160.3, 160.0,
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
6
1
37.0, 129.9, 128.6, 128.0, 127.5, 107.1, 106.9, 101.8, 70.0, 67.7, 33.9, 32.7, 29.1, 27.9, 25.3. ESI
+
MS (m/z): 363ꢀ365 [M+H] .
General Procedure for the synthesis of derivatives 8 and 9. NaH (60% in mineral oil, 0.026 g,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.65 mmol) was added to a solution of 5a (0.13 g, 0.63 mmol) in dry DMF (1 mL) and the resulting
mixture was stirred at ꢀ10 °C for 10 min under a nitrogen atmosphere. Then a solution of the
suitable bromoalkylphenoxy derivative 6 or 7 (0.65 mmol) in dry DMF (1.5 mL) was added and the
resulting mixture was stirred at room temperature for 16 h. After addition of water the mixture was
2 4
extracted with EtOAc, the combined organic phases were dried (Na SO ), filtered and concentrated
under reduced pressure to afford a crude product that was purified by silica gel column
chromatography (EtOAc as eluent).
N
-[2-({3-[4-(3-Benzyloxy)phenoxy]butoxy}phenyl)methylaminoethyl]acetamide (8). Oil, 89%
1
yield. H NMR (CDCl
3
) δ: 1.94 (s, 3H), 1.94ꢀ1.98 (m, 4H), 2.94 (s, 3H), 3.45ꢀ3.47 (m, 4H), 3.98ꢀ
4.09 (m, 4H), 5.05 (s, 2H), 5.61 (brs, 1H), 6.27ꢀ6.43 (m, 3H), 6.51ꢀ6.60 (m, 3H), 7.10ꢀ7.23 (m, 2H),
+
7.32ꢀ7.46 (m, 5H). ESI MS (m/z): 463 [M+H] .
N
-{2-([3-[6-(3-Benzyloxy)phenoxy)hexyloxy]phenyl(methylamino)ethyl}acetamide (9). Oil,
1
8
5% yield. H NMR (CDCl
3
) δ: 1.50ꢀ1.57 (m, 4H), 1.75ꢀ1.90 (m, 4H), 1.94 (s, 3H), 2.93 (s, 3H),
3.44ꢀ3.49 (m, 4H), 3.92ꢀ4.00 (m, 4H), 5.05 (s, 2H), 5.59 (brs, 1H), 6.26ꢀ6.40 (m, 3H), 6.50ꢀ6.59 (m,
+
3H), 7.10ꢀ7.22 (m, 2H), 7.32ꢀ7.46 (m, 5H). ESI MS (m/z): 491 [M+H] .
General Procedure for the synthesis of 3-hydroxyphenoxy derivatives 10 and 11. A solution of
the suitable benzyloxy derivative 8 or 9 (0.63 mmol) in EtOH (5 mL) and EtOAc (5 mL) was
hydrogenated (1 atm) at room temperature in the presence of 10% PdꢀC (0.05 g) for 16 h. The
catalyst was removed by filtration on Celite and the filtrate was concentrated under reduced
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pressure to afford a crude residue that was purified by silica gel column chromatography (EtOAc as
eluent) and crystallization.
N-{2-((3-(4-(3-Hydroxyphenoxy)butoxy)phenyl)(methylamino)ethyl}acetamide (10). White
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1
1
1
1
1
1
1
1
1
2
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0
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solid, mp 113ꢀ4 °C (EtOAcꢀpetroleum ether), 92% yield. H NMR (CDCl
3
) δ: 1.95 (s, 3H), 1.97 (m,
4H), 2.94 (s, 3H), 3.46ꢀ3.50 (m, 4H), 4.02ꢀ4.06 (m, 4H), 5.75 (brs, 1H), 6.33ꢀ6.49 (m, 6H), 7.00
13
(
brs, 1H), 7.09ꢀ7.18 (m, 2H). C NMR (100 MHz, CDCl ) δ: 171.1, 160.2, 157.5, 151.0, 130.0,
3
107.9, 106.7, 105.4, 102.5, 101.8, 99.7, 67.7, 51.8, 38.3, 37.5, 29.7, 26.4, 26.1, 23.1. ESI MS (m/z):
+
373 [M+H]+. HRMS (ESI): m/z calculated for C21
H
29
N
2
O
4
, [M + H] 373.2127. Found: 373.2154.
N-(2-((3-(6-(3-Hydroxyphenoxy)hexyloxy)phenyl)(methylamino)ethyl)acetamide (11). White
1
solid, mp 108ꢀ9 °C; (EtOAcꢀpetroleum ether), 78% yield. H NMR (CDCl ) δ: 1.53ꢀ1.59 (m, 4H),
3
1.78ꢀ1.85 (m, 4H), 1.95 (s, 3H), 2.94 (s, 3H), 3.45ꢀ3.52 (m, 4H), 3.93ꢀ4.01 (m, 4H), 5.80 (brs, 1H),
13
6.31ꢀ6.48 (m, 6H), 6.72 (brs, 1H), 7.07ꢀ7.19 (m, 2H). C NMR (100 MHz, CDCl
3
) δ: 170.8, 160.4,
160.3, 157.2, 130.0, 107.8, 106.6, 105.5, 102.5, 102.1, 99.9, 67.7, 67.6, 51.9, 38.1, 37.5, 29.1, 28.9,
+
+
2
4
5.8, 23.2. ESI MS (m/z): 401 [M+H] . HRMS (ESI): m/z calculated for C H N O , [M + H]
23 33 2 4
01.2440. Found: 401. 2417.
General Procedure for the synthesis of cyclohexyl carbamates 12 and 13. Cyclohexyl
isocyanate (62 µl, 0.48 mmol) and Et N (5 µL) were added to a solution of the appropriate phenol
3
10 or 11 (0.21 mmol) in absolute EtOH (0.6 mL) and dry CH CN (0.6 mL), and the resulting
3
mixture was stirred at room temperature for 18 h under a nitrogen atmosphere. After removing the
solvent by distillation under reduced pressure, the residue was purified by silica gel column
chromatography (EtOAcꢀcyclohexane 7:3 as eluent) and crystallization.
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3
-(4-{3-[(2-Acetamidoethyl)methylamino]phenoxy}butyloxy)phenylcyclohexylcarbamate (12).
1
White solid, mp 115ꢀ6 °C; (EtOAcꢀpetroleum ether), 65% yield. H NMR (CDCl ) δ:1.14ꢀ1.50 (m,
3
6H), 1.58ꢀ1.80 (m, 4H), 1.93 (s, 3H), 1.92ꢀ2.05 (m, 4H), 2.94 (s, 3H), 3.44ꢀ3.47 (m, 4H), 3.54ꢀ3.58
(m, 1H), 4.00ꢀ4.06 (m, 4H), 4.98 (brd, 1H), 5.72 (brs, 1H), 6.30ꢀ6.40 (m, 3H), 6.70ꢀ6.74 (m, 3H),
0
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8
9
0
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9
0
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9
0
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8
9
0
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8
9
0
13
7.10ꢀ7.23 (m, 2H). C NMR (100 MHz, CDCl
3
) δ: 170.4, 160.2, 159.8, 153.5, 152.0, 150.9, 130.0,
129.6, 113.7, 111.6, 108.1, 105.5, 102.3, 99.7, 67.6, 67.3, 51.7, 50.1, 38.4, 37.3, 33.2, 26.0, 25.4,
+
+
24.7, 23.2. ESI MS (m/z): 498 [M+H] . HRMS (ESI): m/z calculated for C H N O , [M + H]
28 40 3 5
498.2968. Found: 498.2961.
3-(6-{3-[(2-Acetamidoethyl)methylamino]phenoxy}hexyloxy)phenylcyclohexylcarbamate (13).
1
White solid, mp 99ꢀ101°C; (EtOAcꢀpetroleum ether), 95% yield. H NMR (CDCl ) δ:1.17ꢀ1.46 (m,
3
6H), 1.48ꢀ1.65 (m, 4H), 1.72ꢀ2.04 (m, 8H), 1.94 (s, 3H), 2.94 (s, 3H), 3.45ꢀ3.47 (m, 4H), 3.54ꢀ3.59
(m, 1H), 3.92ꢀ4.01 (m, 4H), 4.95 (brd, 1H), 5.66 (brs, 1H), 6.31ꢀ6.40 (m, 3H), 6.69ꢀ6.75 (m, 3H),
1
3
3
7.10ꢀ7.24 (m, 2H). C NMR (100 MHz, CDCl ) δ: 170.4, 160.4, 159.9, 153.5, 152.0, 150.9, 130.0,
129.5, 113.6, 111.6, 108.1, 105.4, 102.3, 99.8, 67.9, 67.7, 51.8, 50.1, 38.4, 37.3, 33.2, 29.3, 29.1,
+
25.9, 25.8, 25.4, 24.7, 23.2. ESI MS (m/z): 526 [M+H] . HRMS (ESI): m/z calculated for
+
C H N O , [M + H] 526.3281. Found: 526.3251.
30
44
3
5
tert-Butyl (
ω-bromoalkyl)carbamates (14a-c). tertꢀButyl (8ꢀbromooctyl)carbamate (14c) was
prepared according to literature procedure and chemical physical data were identical with those
4
3
previously reported.
tertꢀButyl (4ꢀbromobutyl)carbamate (14a) and tertꢀbutyl (6ꢀ
bromohexyl)carbamate (14b) are commercially available.
General Procedure for the synthesis of N-Boc-aminoalkoxyphenyl derivatives 15a-d. The
2 3
suitable tertꢀbutyl (ωꢀbromoalkyl)carbamate 14a-c (1.50 mmol), K CO (0.315 g, 2.25 mmol) and a
catalytic amount of NaI were added to a solution of the appropriate (3ꢀhydroxyanilino)ethylamido
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derivative 5a or 5b (1.50 mmol) in dry acetone (8 mL), and the resulting mixture was stirred at 60
°C for 24 h under a nitrogen atmosphere. After water addition addition the mixture was extracted
2 4
with EtOAc, the combined organic phases were dried (Na SO ) and the solvent was removed by
distillation under reduced pressure to afford a crude residue that was purified by column
chromatography.
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tert-Butyl {4-[3-((2-acetamidoethyl)methylamino)phenoxy]butyl}carbamate (15a). This product
was obtained following the above general procedure by reacting 5a with 14a. Purification by silica
1
gel chromatography (EtOAc as eluent). Oil, 37% yield. H NMR (CDCl
3
) δ: 1.45 (s, 9H), 1.65ꢀ1.71
(m, 2H), 1.77ꢀ1.83 (m, 2H), 1.94 (s, 3H), 2.94 (s, 3H), 3.17ꢀ3.21 (m, 2H), 3.44ꢀ3.48 (m, 4H), 3.98
(t, 2H,
J
= 6.5), 4.66 (brs, 1H), 5.71 (brs, 1H), 6.28ꢀ6.38 (m, 3H), 7.13 (dd, 1H, J1 = J2 = 8.0). ESI
+
MS (m/z): 380 [M+H] .
tert-Butyl {4-[3-((2-propionamidoethyl)methylamino)phenoxy]butyl}carbamate (15b). This
product was obtained following the above general procedure by reacting 5b with 14b. Purification
1
by silica gel chromatography (EtOAcꢀcyclohexane 7:3 as eluent). Oil, 20% yield. H NMR (CDCl )
3
δ: 1.12 (t, 3H,
J
= 7.5), 1.45 (s, 9H), 1.38ꢀ1.52 (m, 6H), 1.74ꢀ1.81 (m, 2H), 2.16 (q, 2H,
= 6.5), 4.55 (brs, 1H), 5.65 (brs,
8.0). C NMR (100 MHz, CDCl ) δ: 174.0, 160.3,
J = 7.5),
2.94 (s, 3H), 3.10ꢀ3.15 (m, 2H), 3.45ꢀ3.49 (m, 4H), 3.94 (t, 2H, J
1
3
1H), 6.29ꢀ6.38 (m, 3H), 7.13 (dd, 1H,
J
1
=
J
2
=
3
156.0, 130.0, 105.5, 102.2, 99.7, 67.6, 51.8, 40.5, 38.4, 37.2, 30.0, 29.6, 29.2, 28.4, 26.5, 25.8, 9.7.
+
ESI MS (m/z): 422 [M+H] .
tert-Butyl {8-[3-((2-acetamidoethyl)methylamino)phenoxy]octyl}carbamate (15c). This product
was obtained following the above general procedure by reacting 5a with 14c. Purification by silica
1
gel chromatography (EtOAc as eluent). Oil, 37% yield. H NMR (CDCl
3
) δ: 1.22ꢀ1.37 (m, 6H),
1.43 (s, 9H), 1.39ꢀ1.48 (m, 4H), 1.70ꢀ1.77 (m, 2H), 1.93 (s, 3H), 2.92 (s, 3H), 3.05ꢀ3.13 (m, 2H),
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