LIT-001, the First Nonpeptide Oxytocin Receptor Agonist that Improves Social Interaction in a Mouse Model of Autism

Article abstract of DOI:10.1021/acs.jmedchem.8b00697

Oxytocin (OT) and its receptor (OT-R) are implicated in the etiology of autism spectrum disorders (ASD), and OT-R is a potential target for therapeutic intervention. Very few nonpeptide oxytocin agonists have currently been reported. Their molecular and in vivo pharmacology remain to be clarified, and none of them has been shown to be efficient in improving social interaction in animal models relevant to ASD. In an attempt to rationalize the design of centrally active nonpeptide full agonists, we studied in a systematic way the structural determinants of the affinity and efficacy of representative ligands of the V_1a and V₂ vasopressin receptor subtypes (V_1a-R and V₂-R) and of the oxytocin receptor. Our results confirm the subtlety of the structure-affinity and structure-efficacy relationships around vasopressin/oxytocin receptor ligands and lead however to the first nonpeptide OT receptor agonist active in a mouse model of ASD after peripheral ip administration.

Full text of DOI:10.1021/acs.jmedchem.8b00697

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Article
LIT-001, the First Nonpeptide Oxytocin Receptor Agonist
that Improves Social Interaction in a Mouse Model of Autism
Marie-Celine Frantz, Lucie Pellissier, Elsa Pflimlin, Stéphanie Loison, Jorge
Gandia, Claire Marsol, Thierry Durroux, Bernard Mouillac, Jérôme Becker, Julie
Le Merrer, Christel Valencia, Pascal Villa, Dominique Bonnet, and Marcel Hibert
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00697 • Publication Date (Web): 10 Sep 2018
Downloaded from http://pubs.acs.org on September 11, 2018
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LIT-001, the First Nonpeptide Oxytocin Receptor Agonist that Improves Social
Interaction in a Mouse Model of Autism
1#
2#
1#
1#
MarieꢀCéline Frantz, Lucie P. Pellissier, Elsa Pflimlin, Stéphanie Loison, Jorge
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Gandía, Claire Marsol,
Thierry Durroux, Bernard Mouillac, Jérôme A.J. Becker, Julie
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Le Merrer, Christel Valencia, Pascal Villa, Dominique Bonnet, Marcel Hibert *
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Laboratoire d’Innovation Thérapeutique, Faculté de Pharmacie, UMR7200
CNRS/Université de Strasbourg, 74 route du Rhin, Fꢀ67412 Illkirch, France.
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Physiologie de la Reproduction et des Comportements, INRA UMRꢀ0085, CNRS UMRꢀ
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247, IFCE, Université François Rabelais de Tours, Inserm, Fꢀ37380 Nouzilly, France.
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Institut de Génomique Fonctionnelle, CNRS UMR5203, INSERM U661, Université de
Montpellier (IFR3), 141 rue de la Cardonille, Fꢀ34094 Montpellier Cedex 5, France.
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LabEx MEDALIS, Université de Strasbourg, Fꢀ67000 Strasbourg, France.
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CNRS, Université de Strasbourg, PCBIS Plateꢀforme de chimie biologique intégrative de
Strasbourg UMS 3286, Fꢀ67000 Strasbourg, France.
*
Corresponding author:
Marcel Hibert: phone, + 33 (0)3 68 85 42 32; eꢀmail, mhibert@unistra.fr
Author Contributions:
#
These authors contributed equally to this work.
KEYWORDS:
Autism, oxytocin, oxytocin receptor, nonpeptide agonist, structureꢀactivity relationship, social
interaction, G protein coupled receptor, arrestin.
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ABSTRACT:
Oxytocin (OT) and its receptor (OTꢀR) are implicated in the aetiology of autism spectrum
disorders (ASD) and OTꢀR is a potential target for therapeutic intervention. Very few
nonpeptide oxytocin agonists have currently been reported. Their molecular and in vivo
pharmacology remain to be clarified and none of them has been shown efficient in improving
social interaction in animal models relevant to ASD. In an attempt to rationalize the design of
centrally active nonpeptide full agonists, we studied in a systematic way the structural
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determinants of the affinity and efficacy of representative ligands of the V1a and V
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vasopressin receptor subtypes (V1aꢀR and V ꢀR) and of the oxytocin receptor. Our results
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confirm the subtlety of the structureꢀaffinity and structureꢀefficacy relationships around
vasopressin/oxytocin receptor ligands and lead however to the first nonpeptide OT receptor
agonist active in a mouse model of ASD after peripheral i.p. administration.
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INTRODUCTION
Autism spectrum disorders (ASD) are neurodevelopment diseases characterized by impaired
social interaction and communication together with repetitive behaviors and restricted social
interest (American Psychiatric Association, 2013). These core symptoms must be present in
the early developmental period (by 3 years of age) for the diagnosis of ASD to be made. The
prevalence of ASD is currently estimated at 1 case for 88 births, however with a very large
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diversity of the associated mental disorders. The aetiology of ASD is complex. Hundreds of
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genetic mutations have been associated with the disease, explaining a number of ASD cases.
The identification of vulnerability genes, most of them coding for proteins involved in
synaptic function, as well as environmental risk factors during pregnancy and development,
has provided important insights into the understanding of the anatomical and molecular
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ꢀ7
underpinnings of ASD, notably by giving access to valuable animal models. However, most
of the etiopathological mechanisms leading to ASD remain to be identified. The social
impact of ASD is huge. US annual direct medical, direct nonꢀmedical, and productivity costs
8
combined will be $268 billion for 2015 and $461 billion for 2025.
Up to now, no drug has been approved to treat core symptoms of ASD. Antipsychotics
(aripiprazol, risperidone) or antidepressants (fluoxetine, citalopram) are often administered to
treat some of the severe associated symptoms such as irritability and compulsive, repetitive
behaviors, respectively, with associated side effects (drowsiness, weight gain, etc.). Novartis
has developed up to Phase II clinical trial an mGluR5 inhibitor, mavoglurant, showing a very
marginal improvement in Fragile X patients diagnosed with ASD. Unfortunately, trial results
did not meet the primary endpoint of showing significant improvement in abnormal behaviors
9
compared to placebo and the development has been stopped. Similarly, arbaclofen, the R
enantiomer of baclofen, showed little effect on irritability subꢀscale and development was
10
stopped after Phase IIb studies. Balovaptan is a promising drug candidate developed by
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HoffmannꢀLa Roche and entering a Phase III clinical study in ASD adults
(https://clinicaltrials.gov/ct2/show/NCT03504917) with a FDA Breakthrough Therapy
designation. This compound is claimed to be a specific V1a receptor antagonist.
Unfortunately, no preclinical pharmacological data have been published yet to support its
mechanism of action. The effect of a diuretic, bumetanide, blocking GABAꢀgated chloride
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channels, has recently been reported by Ben Ari et al. and looks promising.
Interestingly,
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there is a clear relationship between this signaling pathway and oxytocin. Furthermore,
many studies in patients demonstrate that OT may significantly improve social abilities
13ꢀ35
related to autism spectrum.
These studies differ significantly in terms of age and gender of
participants, administration route (intravenous or intranasal), doses of OT, single or multiple
long term administration, and outcomes (repetitive behavior, comprehension of affective
speech, social ball tossing game, reading the mind in eyes, etc.) making definitive conclusions
difficult to draw. However, OT clearly appears as a promising target to alleviate social
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impairments in patients with ASD.
Surprisingly, these OT clinical trials are running while very little is known about the precise
molecular pharmacology, pharmacokinetics and pathophysiological functions of OT in
animals and in man. Understanding the distribution and functions of this neurotransmitter is
crucial for the meaningful interpretation of animal and clinical studies and to rationalize the
design of better drug candidates. For instance, no OTꢀR specific brain penetrating positron
37,38
emission tomography (PET) probe is currently available.
Furthermore, the only way to get
central activity with OT is intranasal (i.n.) administration since, as most peptides, OT is not
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orally absorbed, is rapidly metabolized after i.v. administration (few minutes) and poorly
41,42
crosses blood brain barrier except very modestly via the intranasal route.
That is far from
ideal in terms of formulation since the actual concentrations of OT being administered and
42
reaching the different brain regions are unknown. In addition, the pharmacodynamics of this
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hormone is poorly documented. Thus OT has very similar affinities for its OTꢀR and for the
vasopressin receptors V ꢀR and V ꢀR and it is not fully understood which receptor is indeed
1a
1b
43ꢀ45
associated to the different OT functions.
Furthermore, these receptors are able to activate
different signaling pathways (different G proteins, arrestin) and the relationship between these
pathways and the different physiological functions mediated by OT remains to be better
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understood.
Finally, it is very unlikely that OT could ever be marketed to treat ASD since performing full
dedicated clinical trials would be very costly while intellectual property cannot be protected.
These different elements indicate that there is a clear need to discover novel centrally active,
nonpeptide OT agonists both for fundamental research and for drug development.
Consequently, our aim was to design and develop a novel nonpeptide OTꢀR agonist active in
animal models of autism after peripheral administration. To our knowledge, no compound
with such a profile has yet been disclosed. Few peptide or peptideꢀlike OTꢀR agonists have
been described but their bioavailability is not much improved over OT and their in vivo
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efficacy in autism models has not been reported.
Two nonpeptide compounds showing
40,48
some agonist activity have been discovered by Ferring and Wyeth.
The Ferring compound
49
is in fact more efficient as V ꢀR antagonist than as OTꢀR agonist and its clinical
1a
development has been stopped. The Wyeth compound, WAYꢀ267464, has been reported as a
highꢀaffinity, potent, and selective (vs. V1a, V1b, ) agonist of the OT receptor in a given
experimental system. It is more and more often used and referenced as a selective full OTꢀR
V
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agonist. However, in a different experimental setup, this compound was found to have an 8ꢀ
fold higher affinity for the V receptor with a poor functional selectivity (2x selective for OTꢀ
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a
51
R agonism over V1aꢀR antagonism) and partial OT agonism (see below). Our own efforts to
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discover OTꢀR agonists by virtual and biological high throughput screening of diverse
libraries or by rational design of oxytocin mimics, failed. Actually, OTꢀR ligands were found
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but none of them showed agonist activity (unpublished results and reference 52). We came
back to a more classical approach in looking for a common basic pharmacophore among
ligands of vasopressin/oxytocin receptors as a starting template to develop a full OT agonist.
In our eyes, this makes sense since there is a high sequence similarity between the three
vasopressin receptor subtypes and the oxytocin receptor that all belong to the same branch of
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the GPCR family. On the other hand, vasopressin and oxytocin bind with moderate to high
affinity to these four receptors. Furthermore, Table 1 shows that compounds with similar
structures have already been characterized as high affinity ligands of one or several of these
40,48,51,54ꢀ61
four receptor subtypes.
A simple examination of these representative series of
ligands reveals that a benzoyl benzazepine pharmacophore is clearly shared by this set of
molecules showing affinity for at least one member of the vasopressin/oxytocin receptor
family. Most compounds are antagonists except VNAꢀ932 and WAYꢀ141608 that display V2
54,55
48,51
receptor agonism
and the Ferring and Wyeth OT partial agonists.
Thus this arylacyl
benzazepine pharmacophore seems appropriate to afford improved OT agonists. In addition,
compared to OT, it displays the potential advantages of structural simplicity, low molecular
weight and druglikeness.
2
We report here the binding and efficacy studies around the pharmacophore of V1a, V1b, V and
OT receptor ligands using three series of compounds with (i) variations of the side chain on
the 4ꢀ position of the simplest benzoylbenzazepine pharmacophoric nucleus (Table 2); (ii)
variations on the benzazepine fragment keeping the pyrazole side chain selected previously
(Table 3); (iii) variation of the side chain of the pyrazolobenzodiazepine fragment selected on
OT agonism potential (Table 4). This led to the first identification of a new nonpeptide OTꢀR
agonist, active in a mouse model of autism after peripheral intraperitoneal administration.
RESULTS
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Chemistry
Benzazepine derivatives. Benzazepine derivatives were prepared by coupling benzazepine 8
with a series of substituted aryl acids synthesized as indicated in Scheme 1. The acid
59
precursors 3a-b were prepared according to the method described by Kondo et al (2000). .
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Acid precursors 5a-b were prepared by Suzuki coupling according to the method of Gong et
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al (2000) optimised for microwave conditions.
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Benzazepine 8 was prepared from the corresponding oxime 7 as described (Scheme 2).
Coupling was achieved by using commercially available acid chlorides (for compounds 9-14
and 18) or starting from prepared acids 3a and 5a-b (for compounds 15-17) using thionyl
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chloride.
The reduction of the nitro group of 18 was achieved as described by Ogawa et
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al (1996). Double methylation of aniline 19 was performed as described by Kondo et al
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(
2000). Peralkylation of 20 was achieved with methyl iodide and silver perchlorate
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following a procedure described by Miller et al (1989) while acetylation of 19 led to 22. The
reference antagonist 23 was obtained by acylation of 19 with 2ꢀbiphenylcarboxylic acid as
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previously described by Matsuhisa et al (1997). Structures and yields are given in Scheme 2.
Arylꢀ1ꢀ(3ꢀMe)ꢀpyrazolyl derivatives. A series of compounds resulting from the coupling of
substituted 1ꢀ(3ꢀMe)ꢀpyrazolylꢀbenzoic acids 3a-b with cyclic amines have been prepared.
The cyclic amines have been synthesized (Scheme 3) according to existing procedures.
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Ketone 25 was prepared as described by Proctor et al (1961). We had already described the
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synthesis of benzazepinone 26. Nꢀdimethylbenzazepine 28 was prepared from ketone 25
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according to a procedure described by Ogawa et al (1996). The pyrrolobenzodiazepine 31
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was obtained following a procedure described by Artico et al (1969).
The
pyrazolobenzodiazepine 37 was prepared according to the method described by Pitt et al
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(
2004). Azepine is commercially available. The different benzazepines were coupled to the
aromatic acids as described before via acyl chloride intermediate (Scheme 4). The V
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receptor
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agonist VNAꢀ932 (42) was prepared from benzodiazepine 31. The coupling products 39a
and 39b, obtained from ketone 26, were further reduced by sodium borohydride to obtain the
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alcohols 40a and 40b. Structures and yields are given in Scheme 4.
Pyrazolobenzodiazepine derivatives. A series of amines was prepared according to Scheme 5
and Scheme 6. The first series dealt with proline analogues (Scheme 5). NꢀBocꢀproline 45
reacted with N,Nꢀdimethylamine to afford the corresponding carboxamide 46 with a 75%
yield. The carboxamide 46 was transformed into the corresponding thiocarboxamide 47 with
the Lawesson reagent in 52% yield. Finally, Boc protection was removed with
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TFA/CH Cl :1/1 to afford quantitatively the building blocks 48 and 49. A second series of
building blocks was derived from glycine (Scheme 6). Nꢀprotected glycine was first turned
into the N,Nꢀdimethyl carboxamide 50 and then into the thiocarboxamide 51 following the
same sequence as above for proline derivatives. Deprotection in acidic medium afforded the
free amino thiocarboxamide 52 and carboxamide 53 with quantitative yields. The methyl ester
of glycine, 54, was commercially available. The pyrazolobenzodiazepine 37 reacted with 4ꢀ
cyanoꢀ3ꢀmethyl benzoic acid chloride to afford compound 55 in a 70% yield (Scheme 7). The
cyano group was reduced with sodium borohydride in the presence of cobalt chloride
hexahydrate to provide the primary amine 56 with 81% yield. A urea connection was then
created with carbonyldiimidazole (CDI) between the amine 56 and the previously synthesized
amines 48, 49, 52ꢀ54 leading to compounds 57 to 61. Yields ranged from 41% to 67%
(Scheme 7).
Structure-activity relationship studies
The affinities of the different ligands for the human vasopressin receptor subtypes and the
oxytocin receptor were determined as described previously (i) on CHO cell membranes by
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competition experiments against [ H]AVP; (ii) on HEK cells using a FRET based assay.
The functional agonist and competitive antagonist properties of each ligand were determined
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for the human vasopressin and oxytocin receptor subtypes in different experimental
conditions. (i) In CHO cells stably expressing receptors, the accumulation of myoꢀinositol 1ꢀ
phosphate (V1a and OT receptors) and the accumulation of cAMP (V
2
receptor) were
determined using immunoassays based on the competition between free IP
1
or cAMP and IP ꢀ
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d2 or cAMPꢀd2 conjugate, respectively. (ii) In HEK cells stably expressing V1a and OT
receptors, the intracellular calcium flux was determined using the fluorescent dye Indo1.
Benzazepine series. The binding and efficacy profiles of benzoylbenzazepines 9-23 for the
V
1a, V
2
and OT receptors are given in Table 2. Simple halogen substitution of the benzoyl
fragment in para of the carboxamide link was sufficient to retain a submicromolar affinity (9-
1). A two digit nanomolar affinity was even obtained for the iodo derivative 11 on the V1a
1
receptor (Ki = 48 nM) whereas a 396 nM affinity was obtained for the OTꢀR. Antagonist
activity was clearly observed for the V and OT receptors while a mixed agonist/antagonist
1
a
profile was obtained on the V
compounds with an increased potency compared to halogens. Hence the nꢀbutyl analogue 13
had affinity constants of 28 nM, 52 nM and 37.4 nM for the V1a, V and OT receptors,
2
receptor. The nꢀalkyl substituents in 12 and 13 afforded
2
respectively. This is quite noticeable considering the simplicity and low molecular weight of
this compound. It behaved as a relatively potent antagonist at the V and OT receptors (K
inact
1
a
=
2
421 nM and 113 nM, respectively) and a quite potent V partial agonist (EC50 = 18 nM;
intrinsic activity: 74% of maximum AVP induced response). The substitution by a phenyl
fragment (14) led to a further increase in affinity with Ki of 2.28 nM, 78 nM and 10.4 nM for
the V , V and OR receptors, respectively. Compound 14 is a potent antagonist at the three
1
a
2
receptor subtypes (Kinact = 7 nM, 132 nM and 42 nM, respectively) with a preference for the
1a site. The heterocyclic homologues 15 and 16 have similar profiles with lower affinity and
potency on all sites. Interestingly, the 3ꢀmethylꢀpyrazolo analogue 17 recovered a high
affinity at both V1a (34 nM) and OT (84 nM) receptors and a lower one (282 nM) at the V
V
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receptor. It showed high potency at all sites as a V ꢀR and OTꢀR antagonist (K
= 48 nM
1a
inact
and 112 nM, respectively) and V ꢀR full agonist (EC = 13 nM). Attempts to introduce an
2
50
amino function on the benzoyl nucleus led to clearꢀcut changes: whereas the primary amino
derivative 19 and the corresponding quaternary ammonium 21 mostly lost activity at all
receptors, the intermediate dimethyl amino compound 20 showed an 82 nM affinity for the
V subtype, a 468 nM affinity for the V subtype and no affinity for the OT receptor. As most
1
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2
other compounds, 20 is a potent V ꢀR antagonist and a V ꢀR agonist with potencies of 81 nM
1a
2
and 80 nM, respectively. Finally, in contrast to the inactive acetyl compound 22, the biphenyl
58
compound 23 was confirmed as a very potent antagonist at the three receptor subtypes with
affinities of 2.74 nM, 0.57 nM and 109 nM for the V1a, V and OT receptors, respectively and
2
a marked preference for the blockade of the V ꢀR mediated function (K
= 0.096 nM
2
inact
compared to 63 nM for V ꢀR and 171 nM for OTꢀR).
1
a
Pyrazole series. The binding and potency profiles of pyrazole derivatives 17 and 38-44 for the
and OT receptors are given in Table 3. In this series, the substitution of the aryl linker
V1a, V
2
with chlorine (compound 38) led to a selectivity reversal compared to the unsubstituted
compound 17, with a decrease in affinity for the V and OT receptors and an increase for the
1
a
2 2
V receptor. The potency profile remained unaffected with agonism for the V (EC50 = 16 nM)
and antagonism for the two other receptor subtypes. Introduction of a keto group on the
benzazepine nucleus (compounds 39a and 39b) resulted in a drastic loss of affinity and
potency on all subtypes. This effect was less marked with the corresponding hydroxyl
substitution (compound 40a and 40b), in particular with a chlorine substituent since 40b
showed moderate affinity (Ki = 100 nM) and a good agonist potency (EC50 = 28 nM) at the V
2
receptor. No agonist activity was observed for the other receptor subtypes. A similar
detrimental effect was observed with an N,Nꢀdimethylamine substitution (compounds 41a and
41b). Interestingly, this structural modification is the only one that triggered a functional
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switch of the V receptor from agonism to antagonism (41b; K
= 126 nM). Compound
2
inact
WAYꢀVNAꢀ932 (43) was prepared and tested. Overall its binding and potency profiles
60
correspond to literature data. It behaved in our hands as a moderately potent, nonspecific V1a
and OT receptor antagonist (Ki = 344 nM and 199 nM, respectively) and V receptor partial
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agonist (Ki = 50 nM; EC50 = 9.5 nM with 75% of maximal response). The introduction of the
Ferring and WAYꢀ267464 pyrazolobenzodiazepine nucleus led to the potent, nonspecific
ligand 43 (Ki = 27 nM, 29 nM and 17 nM for the V , V and OT receptors, respectively).
1a
2
2
Again, 43 was a potent V ꢀR agonist (EC50 = 8.4 nM) but remained antagonist at the two other
receptor subtypes. Finally, removal of the aromatic component of this fragment of the
molecule as in 44 led to a complete loss of activity at all receptors.
Pyrazolobenzodiazepine series. The combination of the pyrazolobenzodiazepine nucleus with
a series of homologous side chains led to compounds 57ꢀ61 whose pharmacological profiles
are reported in Table 4. In our experimental conditions, the Ferring compound had a weak
affinity for the V1a and OT receptors (Ki = 196 and 141 nM, respectively) and no activity at
the V2ꢀR at micromolar concentration. We confirmed an OTꢀR agonist behavior with an EC50
++
= 28 nM and an 84% maximal response (based on Ca release) compared to OT. However,
+
+
this compound behaved as a potent V1a receptor antagonist (Ca release) with an IC50 = 8
nM. In comparison, WAY 267464 had a better affinity at both V1a (Ki = 12.5 nM) and OT (Ki
=
2
30.5 nM) receptors whereas no activity was measured at the V receptor. This compound is
an equipotent V1aꢀR antagonist compared to the Ferring compound (EC50 = 7 nM) and a
potent OTꢀR agonist (EC50 = 3 nM). However, the maximal response obtained in our
experimental conditions is limited to 40% of the maximal response obtained with OT. Hence,
the gain in affinity is balanced by a loss in potency due to partial agonism. The compound 57
had lower affinity on all receptor subtypes with a preference for the OTꢀR (Ki = 226 nM).
Very interestingly, 57 remains an agonist with a high potency (EC50 = 55 nM) and an
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increased intrinsic activity compared to WAY 267464 (52% instead of 40% of the maximal
response). The functional selectivity of 57 versus the V ꢀR is also reversed compared to the
1
a
Ferring compound since there was a moderate (two fold) preference for the OTꢀR. A very low
affinity was detected at the V receptor (Ki = 1666 nM). The carboxamide analog 58 of the
2
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thiocarboxamide 57 had a parallel profile but was much less potent. Remarkably, the three
open chain analogues 59ꢀ61 retained some activity but switched to low affinity OTꢀR
antagonists. Despite its small size and its relative flexibility, the thiocarboxamide 60 was
actually a rather potent (IC50 = 9 nM) and specific V1a receptor antagonist based on functional
assays since it was 60 fold less potent as an OTꢀR antagonist and showed no affinity for the
2
V subtype up to 1 micromolar concentration.
Signaling profile of compound 57 in vitro
Based on previous screening data and predictive bioavailability characteristics (see below),
we focused on our best candidate, compound 57, and further studied its pharmacological
profile by assessing its effects on calcium release and βꢀarrestin2 recruitment in HEK293FT
2
cell lines transiently expressing human OT, V1a, V1b or V receptors with either aequorin
sensor or βꢀarrestin2 for calcium or BRET1 recruitment assays, respectively. This activity
was compared to those of several reference molecules: oxytocin and carbetocin as full and
biased peptide agonists, respectivelly, and WAYꢀ267464 as nonpeptide partial agonist (Figure
1
and Table 5).
As regards calcium release (Figure 1A and Table 5), oxytocin was the most potent agonist on
OTꢀR; carbetocin, WAYꢀ267464 and compound 57 had similar potencies (EC50 = 25 nM),
with compound 57 showing the best maximal effect (Emax = 96%). We compared these four
compounds on mouse OTꢀR calcium release, prior to in vivo testing in mice. Oxytocin was the
most potent agonist, and compound 57 showed better potency (EC50 = 18 nM) and maximal
effect (Emax = 95%) than carbetocin and WAYꢀ267464 (Figure S1A and Table 5). Now
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focusing on βꢀarrestin recruitment (Figure 1B and Table 5), carbetocin retained a high
potency (EC50 = 10 nM) however with a partial maximal response (Emax = 42%); WAYꢀ
267464 showed a 6ꢀfold lower potency compared to that measured for calcium release,
suggesting a potential bias in its signaling. Compound 57 retained high potency (EC50 = 62
nM) and maximal response (Emax = 77%). Thus, compound 57 was the most potent agonist
behind oxytocin to recruit βꢀarrestin. Furthermore, we tested the specificity of previous
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compounds by measuring their effects on V1a, V1b and V
activation of calcium release and βꢀarrestin recruitment (Figure 1CꢀH and Table 5).
Carbetocin poorly activated V1bꢀR and V ꢀR without effect on V1aꢀRꢀ induced calcium
release. It had no effect on βꢀarrestin recruitment mediated by V1aꢀR and V1bꢀR and a very
small effect on V ꢀR mediated arrestin release (Emax = 8% at micromolar concentration).
WAYꢀ267464 activated partially V ꢀR mediated calcium release (EC50 = 2300 nM; Emax =
3%) and had no effect on AVP induced arrestin release on the three receptor subtypes.
2
vasopressin receptorꢀmediated
2
2
2
4
51
WAYꢀ267464 is also known to antagonize V receptors. We thus tested whether 57 would
1
a
similarly block the effects of vasopressin on V and V receptors. We found that compound
1
a
1b
5
7 poorly antagonized vasopressin induced calcium release on V1aꢀR (IC50 = 5900 nM) and
was devoid of effect on V1bꢀR (Supporting information: Figure S1, BꢀC). However, compound
7 significantly activated V receptors as measured on both pathways. Finally, we tested the
5
2
7
2
effects of the Pfizer OTꢀR antagonist PF 3274167 on the activation of OT and V receptors
2
by compound 57. PF3274167 totally suppressed and partially reduced the effects of 57 on
2
OTꢀR and V ꢀR, respectively (Table 5; Supporting information: Figure S1, DꢀE). In
conclusion, compound 57 acts as a nonꢀbiased agonist on OTꢀR with the most potent and
specific effects among tested synthetic compounds.
Biological activity of compound 57 in vivo: effects in a mouse model of ASD (Figure 2).
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ꢀ
/ꢀ
Mice lacking the mu opioid receptor (Oprm1 ) recapitulate all core symptoms of ASD:
deficient social interaction and altered communication together with stereotyped behaviors;
they also display multiple comorbid symptoms of ASD, such as exacerbated anxiety and
73ꢀ75
increased sensitivity to seizures.
They thus demonstrate remarkable face validity as a
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mouse model of ASD. Interestingly, these animals also display alterations in the
oxytocin/vasopressin system, as evidenced by reduced number of oxytocin transcripts in the
73
76
nucleus accumbens, a key structure for social reward, and consistent increase in OTꢀR
77
binding in this same region as well as in the amygdala. Here we reasoned that stimulating
OT receptors using the promising OTꢀR agonist compound 57 should relieve social
ꢀ/ꢀ
interaction deficits in Oprm1 animals. To challenge this hypothesis, we administered an
+/+
ꢀ/ꢀ
acute intraperitoneal injection of vehicle or 57 (at 10 or 20 mg/kg) to Oprm1 or Oprm1
unfamiliar (non cagemates, same age, same sex and same treatment) mice before introducing
them by pairs in an openꢀfield for 10ꢀmin testing. Behavioral parameters were scored on
videoꢀrecordings to assess the quality of social interactions in these animals, namely the
number of nose contacts (NCs), the time spent in NC, the mean duration of NCs, the number
of following episodes and the number of grooming episodes occurring after a social contact,
73
an index of social avoidance. At the dose of 10 mg/kg (Figure 2), 57 restored the number of
NCs (genotype effect: F1,42 = 54.2, p<0.0001; dose effect: F2,42 = 9.6, p<0.001; genotype x
dose interaction: F2,42 = 16.8, p<0.0001) and time spent in NC (genotype: F1,42 = 45.6,
p<0.0001; dose: F2,42 = 19.9, p<0.0001; genotype x dose: F2,42 = 17.4, p<0.0001), the mean
duration of NC (genotype: F1,42 = 15.1, p<0.001; dose: F2,42 = 19.5, p<0.0001; genotype x
dose: F2,42 = 19.8, p<0.0001) and the number of following episodes (genotype: F1,42 = 38.9,
p<0.001; dose: F2,42 = 24.4, p<0.0001; genotype x dose: F2,42 = 4.2, p<0.05) measured in
ꢀ/ꢀ
Oprm1 mice to similar levels as measured in wildꢀtype controls. Moreover, at this dose, 57
also suppressed grooming episodes occurring after a social contact in mutants (genotype: F1,42
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=
8.5, p<0.01; dose: F2,42 = 11.3, p<0.001; genotype x dose: F2,42 = 21.4, p<0.0001). The OTꢀ
+
/+
R agonist had no effect in Oprm1 mice at this dose, except that it increased the number of
following episodes. At the dose of 20 mg/kg, compound 57 demonstrated less significant
beneficial effects on social parameters measured in mutant animals, and started to show some
detrimental effects on these parameters in wildꢀtype controls (Figure 2AꢀE). Such effects may
have resulted either from excessive OTꢀR activation or from recruitment/blockade of V or
0
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0
1
a
V1b vasopressin receptors at high doses, peripherally or centrally. To our knowledge, our
results are the first evidence that a nonpeptidic OTꢀR compound can alleviate core symptoms
in the context of ASD.
The actual presence in the mouse brain of compound 57 after a 10 mg/Kg ip administration
has been determined showing an experimental brain exposure of 399 ± 104 min.ng/g.
DISCUSSION
Our ultimate goal is the design and development of drugs improving life of patients with
autism spectrum disorders. As discussed in introduction, the oxytocin receptor appears as a
valid target since OT has already shown beneficial effects in human after i.n.
15ꢀ36
administration.
However OT is not metabolically stable, poorly penetrates the brain, even
after intranasal administration, and its development as a marketed drug to treat ASD is
hampered by intellectual property considerations. Consequently, our aim was to design and
develop a novel nonpeptide OTꢀR agonist active in animal models of autism after peripheral
administration. To our knowledge, no compound with such a profile has been disclosed yet.
From the first SAR run (Table 2), we concluded that the benzoylbenzazepine pharmacophore
is indeed relevant to generate ligands with high affinity at the vasopressin/oxytocin receptors.
Very minimal substitution such as a halogen (11), an alkyl (13), a heterocycle (17) or an
amine (20) are sufficient to afford two digit nanomolar ligands at one or several receptor
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subtypes. It was encouraging to observe that minor structural changes afforded high affinity
V ꢀR ligands with functional profiles ranging from full antagonist (23) to full agonist (17).
2
Hence, as expected, replacement of the bulky and hydrophobic aromatic biphenyl fragment of
the potent, nonspecific antagonist 23 by a smaller, more hydrophilic 3ꢀmethylꢀpyrazole
1
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fragment led to a V
2
ꢀR full agonist (compound 17). It was however disappointing to observe
that such a shift could not be obtained on the V ꢀR or OTꢀR where the compound remained
1a
antagonist, despite the very high sequence homology and structural similarity of the
vasopressin/oxytocin binding sites. This illustrates one more time the subtlety of ligandꢀ
4
9
receptor interaction and activation in this receptor family.
Since the 3ꢀmethylꢀpyrazole derivative 17 of the benzoylbenzazepine pharmacophore was the
only compound showing full agonist activity at a vasopressin/oxytocin receptor (V subtype),
2
we decided to pursue our quest for an OTꢀR agonist around the 3ꢀmethylꢀ1ꢀpyrazoloaryl
pharmacophore in replacing the benzazepine fragment by other nuclei derived from reference
ligands. The central aryl linker was bare or substituted with chlorine in position 2. The
binding and functional profiles of these hybrid molecules are reported in Table 3. Taken
together, the results confirm that it is indeed possible to modulate the affinity and selectivity
profile of vasopressin/oxytocin ligands in playing with the benzazepine component of the
ligands. Hybrid molecules with the arylꢀ1ꢀ(3ꢀmethyl)pyrazole pharmacophore remained in
most cases agonists at the V
2
ꢀR, however they did not switch to agonists neither at the V1a
receptor nor at the target OT receptor. It thus appeared from this study and from our previous
4
9
work that the only entry to OT agonism is the pyrazolobenzodiazepine synthon as in Ferring
and Wyeth WAYꢀ267464 compounds.
Very limited SAR studies were reported around the Ferring compound. In the initial
48
disclosure, its functional potency using a reporter gene assay on OTꢀR and V
2
ꢀR was
reported (EC50 = 33 nM and 850 nM, respectively). Affinity data as well as its extended
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functional profile were published later together with a troncature study and the profiling of
49
few hybrid molecules. To our knowledge, only one SAR study was published around WAYꢀ
267464, dealing with flexible analogues that turned from OTꢀR agonists to V1aꢀR
78
antagonists, confirming the high sensitivity of the system. Our own SAR study around the
pyrazolobenzodiazepine reported here (Table 4) led to the discovery of a new potent agonist,
compound 57, that shows lower molecular weight, better selectivity versus the V receptor,
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
1
a
similar EC and improved efficacy (52%) compared to Ferring compound or WAYꢀ267464.
50
However, we found difficult to conclude to a superiority of 57 based on in vitro affinity or
simple potency and intrinsic activity measurements. Indeed, in our experience, such data are
very much dependent on the experimental conditions (cells, expression vectors, stable or
transient expression, receptor density and environment, assay, experimenter, etc.). As an
illustration, we report in Table 6 binding and potency data for OT, Ferring compound, WAYꢀ
267464 and compound 57 collected from literature and from our own experiments. As can be
observed, affinity data are usually consistent but may vary significantly. For example, Ki
4
0,49,51
48
varies from 12.5ꢀ113 nM
to more than 5800 nM for the reference WAYꢀ267464. As
expected, more drastic differences are observed for potency. Hence EC50 may vary for the
same compound from 33 nM to 667 nM (Ferring compound), from 3 nM to 881 nM (WAYꢀ
267464) or from 16 nM to 55 nM (57) according to differences in systems of expression or in
quantification assays. Important variations are also observed in the achievable maximal
responses. For instance, for the Ferring compound, maximal efficacy varies from 58% to 84%
2
+
and ultimately 100% in moving from an IP1, to a Ca and to a gene reporter assay in CHO
cells, respectively, which might be understood since different levels of amplification are
2+
involved. However, in looking at the Ca signal in HEK cells, the Ferring compound behaved
mainly as a full agonist (EC50 = 111ꢀ149 nM; 88% to 100% maximal response). The reverse
trend is observed for the widelyꢀused OTꢀR ‘agonist’ WAYꢀ267464 compound which
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+
2+
displayed maximal responses varying from 87% (CHO/Ca ) to 27% (HEK/Ca ). As can be
seen from Tables 4 and 6, compound 57 behaved as a partial agonist in one series of
2
+
2+
experiment (52%; HEK/Ca ) and a full agonist (93% to 101%) in three others (HEK/Ca ;
different vectors).
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
As reported above, variations in measurements of in vitro affinity, potency and intrinsic
activity profiles of pharmacological probes may lead to misinterpretation of in vivo data. In
40,50
particular, WAYꢀ267464 was used as a specific nonpeptide OT agonist in several studies
while it has clearly been demonstrated more recently that some of its in vivo pharmacological
51
functions are mainly driven by its V1a antagonist activity. The correspondence between
diverse in vitro and in vivo data is not straightforward and this led us to evaluate the signaling
profile and the efficacy of one of our new compounds in animal models of the target disease.
The selection criteria of the molecule to be further tested in vitro and in vivo were a
combination of its in vitro activity and its putative ability to penetrate the brain. Some
79,80
appropriate physicochemical parameters of the best candidates were calculated
(Table 7).
Compound 57 has a molecular weight slightly lower than the reference WAYꢀ267464 which
may favor passive absorption. Its thermodynamic solubility in buffer at pH 7.4 is fair : 0.68 ±
0
.07 mM (WAYꢀ267464: 5 mM). Its calculated LogD is in the perfect range for brain
penetration (LogD = 2.84; experimental LogD = 2.0 ± 0.3 at pH = 7.4). The polar surface area
is also more favorable which leads to a LogBB value (LogBB = 0.133ꢀ
0
.0153PSA+0.1522LogP) above the target threshold (LogBB = ꢀ1) to optimize brain
8
0
penetration. On this basis, compound 57 (named LIT-001) was selected for a more detailed
signaling study (Table 5, Figure 1 and Figure S1) and for in vivo studies (Figure 2).
In vitro signaling experiments allowed us to identify compound 57 as a nonꢀbiased OTꢀR
agonist on the two main signaling pathways of this receptor, with minor antagonist effect on
V
1a and agonist effect on V1b receptors, observed at high concentrations only (Table 5 and
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Figure 1). Compound 57, however, activated V ꢀR, which is not likely to impact social
2
81,82
behavior as V R are not expressed in the central nervous system
but may cause fluid
2
ꢀ
83
retention. This agonist effect was similar to the effect of oxytocin, which was shown to be
84
safe for chronic use in patients. In comparison, the reference molecule WAYꢀ267464, in our
0
1
2
3
4
5
6
7
8
9
0
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7
8
9
0
1
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5
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9
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experimental setꢀup, had only partial effects or lower affinity to stimulate OTꢀRꢀmediated
signaling. WAYꢀ267464, as compound 57, antagonizes V ꢀR, an effect that may contribute to
1
a
8
5
facilitate social interaction. We could not evidence G proteinꢀbiased agonism of carbetocin
86
on OTꢀR, as previously shown, but such bias was very clear on V1b and V
2
receptors.
Finally, oxytocin bound and activated OTꢀR as well as V1a, V1b and V receptors, as
2
8
7
previously demonstrated. Thus, compound 57 clearly appeared as the most promising
alternative to oxytocin for therapeutic development as a potent nonpeptidic OTꢀR agonist,
with potential selectivity in the CNS after peripheral administration.
ꢀ/ꢀ
Consistent with this, 57 efficiently relieved social interaction deficits in Oprm1 mice, a
7
3
mouse model of autism, by normalizing the number and duration of nose contacts, the
frequency of following episodes and the occurrence of grooming episodes after a social
contact to wildꢀtype levels at the dose of 10 mg/kg (Figure 2). These effects were observed
following systemic (intraperitoneal) administration that led to a measured brain concentration
of compound 57 in the range of its Ki (399 min.ng/g at 10 mg/kg ip). A full pharmacokinetic
study remains to be done but these preliminary results suggest brain penetrance and
bioavailability for this OTꢀR agonist, while oxytocin is known as poorly brain penetrant
(except through i.n. route) and highly sensitive to peripheral degradation.
CONCLUSION
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An extended structureꢀactivity relationship study around vasopressin/oxytocin nonpeptide
4
9
pharmacophores further illustrated the subtlety of the activation mechanisms of the V , V ,
1a
1b
2
V and OT receptors. Minor structural changes affect significantly the affinity and selectivity
profiles of ligands in spite of the high structural similarity of the binding clefts of the
members of this GPCR subꢀgroup. In this context, finding OTꢀR nonpeptide agonists
represented a challenge. Our study led to the discovery of LIT-001 (compound 57) that
represents the first nonpeptide OT receptor agonist active in an animal model of ASD after
peripheral i.p. administration. As a proof of concept, LITꢀ001 is a useful pharmacological tool
to explore the implication of oxytocin in social attachment behaviors and it may pave the way
to the development of optimized preclinical candidates to treat some components of autism.
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
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7
8
9
0
EXPERIMENTAL SECTION
Chemistry
General methods. General methods are as described in reference 49. Purity (≥ 95% for all
compounds) was determined by analytical HPLC in the same conditions as previously
49
reported. Elementary analysis of the final compounds have been determined by the Service
Central d’Analyse of the Centre National de la Recherche Scientifique (CNRS; Vernaison), or
by the core service of microanalysis of the Institute of Chemistry – Strasbourg University.
The synthesis of precursor compounds 1ꢀ8, 28, 31, 37 is reported and available in the
Supporting Information.
General method A for the coupling of amines and benzoic acids:
To a solution of benzoic acids (1.1 eq) in anhydrous CH
drops of NMP and 1.2 equivalents of SOCl . The mixture was stirred overnight at rt and
evaporated. The residue was dissolved in anhydrous CH Cl and added dropwise at 0 °C
2 2
Cl under argon were added 2 to 3
2
2
2
under argon to a mixture of the amine (1.0 eq) and anhydrous pyridine (5.0 eq) in anhydrous
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
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CH Cl . The mixture was stirred 30 min at 0 °C and then at rt for 4 to 20 h. The mixture was
2
2
then poured into water and extracted with CH Cl . The aqueous phase was washed with 1N
2
2
2 3 2 4
HCl and saturated aqueous K CO , dried over Na SO and evaporated. The residue was
purified by chromatography on a silica gel column (heptane ꢀ AcOEt excepted AcOEt ꢀ
AcOEt/MeOH 9:1 for 41a-b).
0
1
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
1-(4-Chlorobenzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepine (9). Benzazepine 8 reacted with
4
ꢀchlorobenzoic acid according to general method A to give 9 as a white solid (yield 78%). Rf
1
= 0.4 (hept/AcOEt 7/3). MP = 84 °C. H NMR (300 MHz, CDCl
3
):
δ
1.40ꢀ1.60 (m, 1H), 1.90ꢀ
2.18 (m, 3H), 2.70ꢀ3.10 (m, 3H), 5.00 (dl app, 1H, J = 13.4 Hz), 6.62 (d, 1H, J = 7.8 Hz), 6.93
13
(
t, 1H, J = 7.2 Hz), 7.03ꢀ7.18 (m, 5H), 7.23 (d, 1H, J = 6.8 Hz). C NMR (75 MHz,
CDCl ): 26.2, 29.6, 35.0, 47.7, 127.1, 127.3, 127.9, 128.2, 129.6, 130.1, 134.6, 135.5, 139.2,
143.7, 167.9. IR (ATR):
3
δ
ꢀ1
λ
(cm ) 2940, 2918, 1644, 1592, 1576, 1488, 1434, 1398, 1376,
1
7
359, 1306, 1273, 1258, 1174, 1137, 1092, 1071, 1012, 960, 917, 852, 841, 823, 766, 751,
+
45, 724, 692, 574, 555, 540. HRMS (ES) m/z calcd for C17H16ClNO [M+Na] , 308.0813;
found, 308.0819. HPLC t = 20.7 min, purity 98.8% (254 nm).
R
1-(4-Bromobenzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepine (10). Benzazepine 8 reacted
with 4ꢀbromobenzoic acid according to general method A to give 10 as a white solid (yield
1
79%). Rf = 0.5 (hept/AcOEt 7/3). MP = 102 °C. H NMR (300 MHz, CDCl
3
):
δ
1.40ꢀ1.60 (m,
1
7
1
H), 1.87ꢀ2.20 (m, 3H), 2.70ꢀ3.10 (m, 3H), 5.00 (dl app, 1H, J = 13.1 Hz), 6.62 (d, 1H, J =
.8 Hz), 6.93 (t, 1H, J = 7.6 Hz), 7.05 (d, 2H, J = 8.4 Hz), 7.10 (t, 1H, J = 6.8 Hz), 7.23 (d,
13
3
H, J = 7.8 Hz), 7.28 (d, 2H, J = 8.4 Hz). C NMR (75 MHz, CDCl ): δ 26.2, 29.6, 34.9,
47.7, 123.9, 127.1, 127.4, 128.2, 129.8, 130.1 130.8, 135.1, 139.2, 143.7, 168.0. IR (ATR):
λ
ꢀ1
(
cm ) 2935, 2911, 1650, 1637, 1577, 1567, 1488, 1448, 1434, 1393, 1377, 1357, 1310, 1274,
1256, 1182, 1139, 1072, 1063, 1030, 1006, 954, 853, 841, 827, 760, 739, 625, 575, 550, 535.
+
HRMS (ES) m/z calcd for C17
H
16BrNO [M+Na] , 352.0307; found, 352.0324. HPLC t
R
=
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1.1 min, purity 98.9% (254 nm).
1-(4-Iodobenzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepine (11). Benzazepine 8 reacted with
4ꢀiodobenzoic acid according to general method A to give 11 as a white solid (yield 38%). R
f
1
=
0.4 (hept/AcOEt 7/3). MP = 137 °C. H NMR (300 MHz, CDCl ):
δ
1.40ꢀ1.60 (m, 1H),
3
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
1
.87ꢀ2.18 (m, 3H), 2.70ꢀ3.10 (m, 3H), 5.00 (d, 1H, J = 13.1 Hz), 6.62 (d, 1H, J = 7.5 Hz),
.91 (d, 2H, J = 8.4 Hz), 6.93 (m, 1H), 7.10 (t, 1H, J = 7.5 Hz), 7.23 (d, 1H, J = 7.5 Hz), 7.48
6
13
(d, 2H, J = 8.4 Hz). C NMR (75 MHz, CDCl
3
):
δ
26.2, 29.5, 34.9, 47.7, 96.0, 127.1, 127.4,
ꢀ1
1
28.2, 129.8, 130.1, 135.7, 136.8, 139.2, 143.7, 168.1. IR (ATR):
λ
(cm ) 2931, 2906, 2850,
2837, 1635, 1602, 1582, 1491, 1438, 1392, 1381, 1356, 1309, 1274, 1257, 1180, 1140, 1007,
+
950, 823, 765, 743, 660, 574, 552. HRMS (ES) m/z calcd for C H INO [M+H] , 378.0349 ;
1
7
16
R
found, 378.0334. HPLC t = 21.7 min, purity 98.1% (254 nm).
1-(4-Propylbenzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepine (12). Benzazepine 8 reacted
with 4ꢀnꢀpropylbenzoic acid according to general method A to give 12 as a beige glass (yield
1
8
7
6%). R
f
= 0.3 (hept/AcOEt 8/2). MP = 36 °C. H NMR (300 MHz, CDCl
3
):
δ
0.85 (t, 3H, J =
.4 Hz), 1.43ꢀ1.64 (m, 3H), 1.87ꢀ2.16 (m, 3H), 2.48 (t, 2H, J = 7.5 Hz), 2.76 (t, 1H, J = 11.6
Hz), 2.82ꢀ2.93 (m, 1H), 3.05 (t, 1H, J = 13.5 Hz), 5.04 (dl app, 1H, J = 14.1 Hz), 6.64 (d, 1H,
J = 7.8 Hz), 6.84ꢀ6.92 (m, 1H), 6.94 (d, 2H, J = 8.1 Hz), 7.02ꢀ7.17 (m, 3H), 7.22 (d, 1H, J =
13
7
1
1
.2 Hz). C NMR (75 MHz, CDCl
3
):
δ
13.6, 24.1, 26.3, 29.7, 35.0, 37.7, 47.6, 126.8, 126.9,
ꢀ1
27.7, 128.2, 128.3, 129.9, 133.5, 139.2, 144.3, 169.1. IR (ATR):
λ
(cm ) 2927, 2854, 1634,
580, 1491, 1438, 1382, 1354, 1307, 1275, 1260, 1179, 1139, 955, 852, 808, 755, 740, 720,
+
578, 547, 485, 466, 438. HRMS (ES) m/z calcd for C20
294.1845. HPLC t = 20.3 min, purity 98.8% (254 nm).
H
23NO [M+H] , 294.1852; found,
R
1
-(4-Butylbenzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepine (13). Benzazepine 8 reacted with
4
ꢀnꢀbutylbenzoic acid according to general method A to give 13 as a beige solid (yield 83%).
1
R
f
= 0.3 (hept/AcOEt 8/2). MP = 42 °C. H NMR (300 MHz, CDCl
3
):
δ
0.87 (t, 3H, J = 7.1
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2
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Hz), 1.26 (sext, 2H, J = 7.3 Hz), 1.50 (quint, 2H, J = 7.4 Hz), 1.40ꢀ1.63 (m, 1H), 1.85ꢀ2.19
(m, 3H), 2.50 (t, 2H, J = 7.7 Hz), 2.66ꢀ2.95 (m, 2H), 3.05 (t, 1H, J = 13.3 Hz), 5.04 (d, 1H, J
=
13.4 Hz), 6.64 (d, 1H, J = 7.6 Hz), 6.82ꢀ6.95 (m, 1H), 6.94 (d, 2H, J = 8.2 Hz), 7.00ꢀ7.12
13
(
m, 1H), 7.10 (d, 2H, J = 7.8 Hz), 7.23 (dd, 1H, J = 7.4, 1.2 Hz). C NMR (50 MHz,
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
CDCl
29.9, 133.4, 139.2, 144.3, 144.5, 169.1. IR (ATR):
3
): δ 13.8, 22.2, 26.3, 29.7, 33.2, 35.0, 35.3, 47.6, 126.9, 126.9, 127.6, 128.2, 128.3,
ꢀ1
1
λ (cm ) 2956, 2925, 2854, 1632, 1611,
1580, 1491, 1452, 1435, 1383, 1355, 1307, 1275, 1261, 1179, 1139, 954, 851, 837, 767, 748,
+
7
13, 579, 549. HRMS (ES) m/z calcd for C21
HPLC t = 21.8 min, purity 98.9% (254 nm).
-(1,1'-Biphenyl-4-ylcarbonyl)-2,3,4,5-tetrahydro-1H-1-benzazepine (14). Benzazepine 8
H
25NO [M+H] , 308.2009; found, 308.1994.
R
1
reacted with 4ꢀphenylbenzoic acid according to general method A to give 14 as a white solid
1
(yield 87%). R
f
= 0.4 (hept/AcOEt 7/3). MP = 162 °C. H NMR (300 MHz, CDCl
3
):
δ
1.36ꢀ
1.63 (m, 1H), 1.77ꢀ2.20 (m, 3H), 2.60ꢀ3.20 (m, 3H), 5.06 (d, 1H, J = 13.9 Hz), 6.69 (d, 1H, J
13
=
3
7.6 Hz), 6.92 (t, 1H, J = 7.1 Hz), 7.00ꢀ7.58 (m, 11H). C NMR (75 MHz, CDCl ):
δ
26.3,
29.7, 35.0, 47.7, 126.2, 127.0, 127.1, 127.6, 128.3, 128.7, 130.0, 135.0, 139.2, 140.1, 142.1,
ꢀ1
144.1, 168.8. IR (ATR):
λ (cm ) 3061, 2939, 2924, 2914, 2881, 2852, 2837, 1626, 1615,
1575, 1557, 1488, 1454, 1441, 1434, 1402, 1385, 1355, 1310, 1274, 1258, 1139, 1110, 948,
854, 840, 775, 767, 756, 747, 735, 700, 676, 579. HRMS (ES) m/z calcd for C23H21NO
+
[
M+Na] , 350.1515; found, 350.1534. HPLC t = 22.6 min, purity 99.4% (254 nm).
R
1
-(4-Pyridin-3-ylbenzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepine (15). To a suspension of
a (232 mg, 0.987 mmol) in 6 mL of anhydrous THF under argon atmosphere were added
5
2
SOCl (0.36 mL, 4.935 mmol) and 3 drops of DMF. The mixture was stirred overnight at rt.
The liquid was evaporated, the residue was dissolved in 2 mL of anhydrous THF, evaporated
again and dried in vacuo. A solid was obtained. It was suspended with DMAP (3.0 mg, 0.024
2 2
mmol) in 6 mL of anhydrous CH Cl . A solution of 8 (120 mg, 0.815 mmol) and anhydrous
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8
9
pyridine (0.20 mL, 2.445 mmol) in 1 mL of anhydrous CH Cl was added dropwise at 0 °C
2
2
under argon atmosphere. Stirring was maintained 4 h at rt. The mixture was then diluted in 30
mL of CH Cl , washed with 10 mL of 1N HCl and 10 mL of a saturated aqueous NaHCO
The organic phase was dried over Na SO and evaporated. The residue was purified by
2
2
3
.
2
4
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
chromatography on a silica gel column (heptꢀhept/AcOEt 4/6). Recrystallization from EtOH
1
afforded white crystals (130 mg; yield 49%). R = 0.3 (hept/AcOEt 25/75). MP = 128 °C. H
f
3
NMR (300 MHz, CDCl ): δ 1.44ꢀ1.63 (m, 1H), 1.88ꢀ2.20 (m, 3H), 2.72ꢀ2.98 (m, 2H), 3.08 (t,
1H, J = 13.0 Hz), 5.05 (d, 1H, J = 13.2 Hz), 6.68 (d, 1H, J = 7.8 Hz), 6.93 (t, 1H, J = 7.4 Hz),
7.10 (t, 1H, J = 7.4 Hz), 7.20ꢀ7.40 (m, 2H), 7.30 (d, 2H, J = 8.1 Hz), 7.38 (d, 2H, J = 8.4 Hz),
13
7
.79 (d, 1H, J = 7.8 Hz), 8.57 (d, 1H, J = 3.3 Hz), 8.76 (s, 1H). C NMR (50 MHz, CDCl
3
):
δ
26.3, 29.6, 35.0, 47.7, 123.5, 126.3, 127.1, 127.3, 128.3, 129.0, 130.1, 134.2, 135.6, 136.0,
ꢀ1
138.7, 139.3, 143.9, 148.2, 148.8, 168.4. IR (ATR):
λ
(cm ) 3052, 3033, 3004, 2930, 2855,
1
7
3
1
632, 1576, 1495, 1435, 1393, 1358, 1315, 1278, 1259, 1143, 1003, 951, 854, 810, 761, 741,
+
20 2
15, 600, 583, 549, 409. HRMS (ES) m/z calcd for C22H N O [M+H] , 329.1648; found,
29.1651. HPLC t = 14.4 min, purity 99.3% (254 nm).
R
-(4-Pyrimidin-2-ylbenzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepine (16). To a suspension
Cl /THF 1/1 under
(0.18 mL, 2.463 mmol) and 3 drops of DMF. The
of 5b (164 mg, 0.821 mmol) in 8 mL of an anhydrous mixture of CH
argon atmosphere were added SOCl
2
2
2
mixture was stirred overnight at rt. Solvents were evaporated, the residue was disolved in 2
mL of anhydrous THF, evaporated and then dried in vacuo. The solid that was obtained was
mixed with DMAP (2.5 mg, 0.020 mmol) in 5 mL of anhydrous CH
benzazepine 8 (100 mg, 0.679 mmol) and anhydrous pyridine (0.16 mL, 2.040 mmol) in 1 mL
of anhydrous CH Cl was added dropwise to this suspension, at 0 °C under argon atmosphere.
The mixture was stirred overnight at rt. It was then diluted with 20 mL of CH Cl , washed
with 10 mL of 1N HCl and 10 mL of a saturated aqueous solution of NaHCO . The organic
2 2
Cl . A solution of
2
2
2
2
3
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1
1
2
2
2
2
2
2
2
2
2
2
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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
phase was then collected, dried on Na SO and evaporated. The residue was purified on a
2
4
chromatography on a silica gel column (heptꢀhept/AcOEt 6/4). Recrystallization from EtOH
f
affords transparent crystals (158 mg; yield 70%). R = 0.6 (hept/AcOEt 25/75). MP = 173 °C.
1
H NMR (200 MHz, CDCl3):
t, 1H, J = 12.9 Hz), 5.05 (d app, 1H, J = 13.4 Hz), 6.66 (d, 1H, J = 7.6 Hz), 6.87 (t, 1H, J =
.5 Hz), 7.05 (t, 1H, J = 7.4 Hz), 7.11ꢀ7.36 (m, 2H), 7.31 (d, 2H, J = 8.2 Hz), 8.23 (d, 2H, J =
δ 1.40ꢀ1.72 (m, 1H), 1.87ꢀ2.20 (m, 3H), 2.68ꢀ2.98 (m, 2H), 3.09
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
(
7
13
8.4 Hz), 8.76 (d, 2H, J = 4.6 Hz). C NMR (50 MHz, CDCl
3
):
δ 26.4 29.6, 35.0, 47.7, 119.3,
127.0, 127.3, 127.4, 128.3, 130.0, 138.3, 138.4, 139.3, 143.8, 157.2, 164.0, 168.8. IR (ATR):
ꢀ1
λ
(cm ) 3066, 3050, 2922, 2882, 2853, 1628, 1575, 1566, 1556, 1494, 1417, 1404, 1385,
1
5
356, 1313, 1279, 1260, 1142, 1018, 953, 866, 815, 804, 778, 761, 752, 744, 656, 641, 627,
+
82, 550, 476. HRMS (ES) m/z calcd for C21
= 20.2 min, purity 99.9% (254 nm).
-[4-(3-Methyl-1H-pyrazol-1-yl)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzazepine
H
19
N
3
O [M+H] , 330.1601; found, 330.1596.
HPLC t
R
1
(17).
Benzazepine 8 reacted with the acid 3a according to general method A to give 17 as
transparent crystals after recrystallization from AcOEt (yield 88%). R = 0.4 (hept/AcOEt 5/5).
f
1
MP = 124 °C. H NMR (200 MHz, CDCl
3
):
δ
1.40ꢀ1.65 (m, 1H), 1.80ꢀ2.20 (m, 3H), 2.32 (s,
3H), 2.65ꢀ3.20 (m, 3H), 5.03 (d, 1H, J = 13.2 Hz), 6.20 (d, 1H, J = 2.2 Hz), 6.64 (d, 1H, J =
7
2
3
1
.8 Hz), 6.89 (t, 1H, J = 7.6 Hz), 7.06 (td, 1H, J = 7.6, 1.0 Hz), 7.15ꢀ7.35 (m, 3H), 7.43 (d,
13
H, J = 8.6 Hz), 7.74 (d, 1H, J = 2.4 Hz). C NMR (75 MHz, CDCl
3
):
δ
13.6, 26.2, 29.5,
4.9, 47.6, 107.9, 117.3, 127.0, 127.2, 127.2, 128.2, 129.5, 129.9, 133.4, 139.1, 140.4, 143.9,
ꢀ1
50.9, 168.1. IR (KBr):
λ (cm ) 3054, 2933, 2854, 1638, 1611, 1579, 1542, 1491, 1452,
1439, 1426, 1398, 1384, 1360, 1309, 1275, 1258, 1226, 1177, 1140, 1047, 958, 945, 837, 772,
+
7
58, 577. HRMS (ES) m/z calcd for C H N O [M+H] , 332.1757; found, 332.1746. HPLC
21
21
3
t = 18.9 min, purity 99.2% (254 nm). Elemental analysis calculated (%) for C H N O: C
R
21 21
3
76.11, H 6.39, N 12.68; found: C 75.92, H 6.66, N 12.68.
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1
-(4-Nitrobenzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepine (18). A mixture of benzazepine 8
(258 mg, 1.75 mmol) and anhydrous pyridine (0.70 mL, 8.76 mmol) in 3 mL of anhydrous
CH Cl was added dropwise to a solution of 4ꢀnitrobenzoyl chloride (390 mg, 2.10 mmol) in
0 mL of anhydrous CH Cl under argon atmosphere, at 0 °C. The mixture was stirred for 45
2
2
1
2
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
min at 0 °C and one night at rt. The products were poured into 10 mL of water and extracted
twice with 30 mL of CH Cl . The organic phase was washed with 20 mL of 0.5N HCl and 20
2
2
mL of water, dried over Na SO and evaporated. The residue was purified by chromatography
2
4
on a silica gel column (hept/AcOEt 7/3ꢀ6/4) affording white crystals (360 mg; yield 69%). R
f
1
=
0.3 (hept/AcOEt 7/3). MP = 141 °C. H NMR (200 MHz, CDCl ):
δ
1.40ꢀ1.70 (m, 1H),
3
1
.85ꢀ2.25 (m, 3H), 2.70ꢀ3.15 (m, 3H), 4.99 (d, 1H, J = 13.4 Hz), 6.60 (d, 1H, J = 7.6 Hz),
.91 (td, 1H, J = 7.7, 1.7 Hz), 7.11 (td, 1H, J = 7.5, 1.2 Hz), 7.25 (d, 1H), 7.35 (d, 2H, J = 9.0
6
1
3
Hz), 8.01 (d, 2H, J = 9.0 Hz). C NMR (75 MHz, CDCl
3
): δ 26.3, 29.5, 35.0, 47.8, 123.0,
ꢀ1
127.2, 127.9, 128.2, 128.8, 130.4, 139.4, 142.5, 142.9, 147.9, 168.4. IR (KBr):
λ
(cm ) 3102,
3
7
1
074, 2939, 2916, 2849, 1642, 1598, 1513, 1491, 1440, 1412, 1344, 1316, 1271, 861, 765,
35.
-(4-Aminobenzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepine (19). Few mg of 10% Pd/C were
added to compound 18 (356 mg, 1.20 mmol) in 10 mL EtOH and 10 mL AcOEt and the
suspension was stirred under H (20 psi) at rt for 5 h. The mixture was then filtered on celite
and the filtrate was evaporated. The residue was taken up in 50 mL of CH Cl and washed
with 40 mL of water. The organic phase was dried over Na SO and evaporated. Compound
2
2
2
2
4
f
19 was obtained as a white powder (326 mg; quantitative yield). R = 0.3 (hept/AcOEt 5/5).
1
MP = 162 °C. H NMR (300 MHz, CDCl ):
δ 1.49 (m, 1H), 1.70ꢀ2.20 (m, 3H), 2.50ꢀ3.10 (m,
3
3H), 3.15ꢀ4.00 (bs, 1H), 5.02 (m, 1H), 6.38 (d, 2H, J = 8.1 Hz), 6.67 (d, 1H, J = 7.8 Hz), 6.93
(t, 1H, J = 7.6 Hz), 7.02 (d, 2H, J = 8.1 Hz), 7.07 (t, 1H, J = 7.5 Hz), 7.22 (d, 1H, J = 7.5 Hz).
13
3
C NMR (75 MHz, CDCl ): δ 26.2, 29.7, 34.9, 47.6, 113.7, 125.8, 126.7, 126.9, 128.2, 129.9,
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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
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5
5
5
5
5
5
5
5
5
5
6
ꢀ
1
130.4, 139.0, 144.8, 147.5, 168.9. IR (KBr):
λ
(cm ) 3447, 3351, 2931, 2838, 1617, 1383,
+
1
308, 1273, 1170, 764, 503. HRMS (ES) m/z calcd for C18
67.1477. HPLC t
20NO: C 76.66, H 6.81, N 10.52; found: C 76.50, H 6.98, N 10.29.
N,N,N-Trimethyl-4-(2,3,4,5-tetrahydro-1H-1-benzazepin-1-ylcarbonyl)benzenaminium
H
20NO [M+H] , 267.1492; found,
2
R
= 17.8 min, purity 99.2% (254 nm). Elemental analysis calculated (%) for
18
C H
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
(
20). A solution of 19 (400 mg, 1.50 mmol), MeI (0.20 mL, 3.00 mmol) and K CO (311 mg,
2 3
2 2
2.25 mmol) in 8 mL of CH Cl was warmed at 70 °C under argon atmosphere during 6 h.
After return to rt, the mixture was poured into 50 mL of water and extracted with 80 mL of
AcOEt/toluene. The organic phase was washed with 25 mL of water, dried over Na SO and
2
4
f
evaporated. Recrystallization from MeOH afforded white crystals (293 mg, yield 66%). R =
1
0
.6 (hept/AcOEt 5/5). MP = 152 °C. H NMR (300 MHz, CDCl
3
):
δ
1.45 (m, 1H), 1.75ꢀ2.20
(m, 3H), 2.50ꢀ3.20 (m, 9H), 4.90ꢀ5.30 (m, 1H), 6.34 (d, 2H, J = 7.9 Hz), 6.66 (m, 1H), 6.89
13
(m, 1H), 7.00ꢀ7.40 (m, 4H). C NMR (75 MHz, CDCl ):
δ
25.9, 29.5, 34.6, 39.6, 47.3, 110.0,
3
ꢀ1
1
3
22.3, 126.3, 126.6, 127.8, 129.6, 130.1, 138.6, 144.8, 150.7, 168.6. IR (KBr):
λ
(cm ) 3094,
007, 2933, 2845, 1612, 1524, 1484, 1437, 1369, 1302, 1194, 1169, 762, 500. HRMS (ES)
+
m/z calcd for C19
H
22
N
2
O [M+H] , 295.1805; found, 295.1798. HPLC t
R
= 21.1 min, purity
98.8% (254 nm). Elemental analysis calculated (%) for C19
found: C 77.69, H 7.75, N 9.62.
22 2
H N O: C 77.52, H 7.53, N 9.52;
N,N,N-Trimethyl-4-(2,3,4,5-tetrahydro-1H-1-benzazepin-1-ylcarbonyl)benzenaminium,
Trifluoroacetate (21). To a solution of 20 (80 mg, 0.273 mmol) in 10 mL of anhydrous
CH
.300 mmol). The mixture was stirred for 2 h at rt in the dark, then filtered and the solvent
was evaporated. The residue was purified by semiꢀpreparative HPLC (H O/0.1% TFA) for 5
2 2 4
Cl under argon atmosphere were added AgClO (62 mg, 0.300 mmol) then MeI (19 µL,
0
2
min, then from 0 to 100% of MeCN/0.1% TFA in H O/0.1% TFA from 5 to 30 min, at a flow
2
rate of 10 mL/min. After lyophilisation, compound 21 was obtained as a hygroscopic white
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powder (49 mg; yield 42%). R = 0.2 (CH Cl /MeOH 8/2). MP = 60 °C. H NMR (300 MHz,
f
2
2
CDCl
2.9 Hz), 3.75 (s, 9H), 4.95 (d, 1H, J = 12.9 Hz), 6.60 (d, 1H, J = 7.5 Hz), 6.92 (t, 1H, J = 7.6
Hz), 7.11 (t, 1H, J = 7.4 Hz, H ), 7.24 (d, 1H, J = 7.2 Hz, H ), 7.37 (d, 2H, J = 8.7 Hz, H15a,b),
.70 (d, 2H, J = 9.0 Hz). C NMR (75 MHz, CDCl ): 26.2, 29.5, 34.8, 47.9, 57.0, 119.4,
127.2, 128.0, 128.1, 130.2, 130.4, 138.8, 139.3, 142.8, 147.3, 160.9, 161.2, 166.5. IR (ATR):
3
): δ 1.53 (m, 1H), 1.90ꢀ2.20 (m, 3H), 2.35 (bs, 2H), 2.72ꢀ2.92 (m, 2H), 3.03 (t, 1H, J =
1
9
8
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
13
7
3
δ
ꢀ1
λ
(cm ) 3417, 3042, 2930, 2854, 1683, 1633, 1579, 1492, 1455, 1442, 1414, 1396, 1358,
1
314, 1277, 1198, 1168, 1146, 1118, 1071, 1016, 956, 940, 847, 824, 800, 761, 749, 717, 598.
+
HRMS (ES) m/z calcd for C20
H
25
N
2
O.C
F
2 3
2
O .H
2
O [M] , 309.1961; found, 309.1961.
Elemental analysis calculated (%) for C H N O.C F O .H O: C 59.99, H 6.18, N 6.36;
20
25
2
2
3
2
2
found: C 59.21, H 6.56, N 6.18.
N-[4-(2,3,4,5-Tetrahydro-1H-1-benzazepin-1-ylcarbonyl)phenyl]acetamide
solution of 19 (60 mg, 0.225 mmol), Ac O (25 µL, 0.248 mmol) and DIEA (60 µL, 0.338
mmol) in 1.5 mL of anhydrous CH Cl was stirred for 4 h at rt. The mixture was then diluted
with 10 mL of CH Cl and the organic phase was washed with 5 mL of water, 5 mL of 10%
(22).
A
2
2
2
2
2
citric acid and 5 mL of saturated aqueous Na
with 40 mL of CH Cl . The organic phases were gathered, dried over Na
The residue was purified by chromatography on silica gel column (hept/AcOEt 5/5ꢀ1/9).
2
CO
3
. The aqueous phases were extracted again
2
2
2
SO and evaporated.
4
f
Compound 22 was obtained as a white powder (68 mg; yield 98%). R = 0.5 (hept/AcOEt
1
1
3
7
/9). MP = 168 °C. H NMR (300 MHz, CDCl
3
):
δ
1.51 (m, 1H, 1.75ꢀ2.00 (m, 3H), 2.09 (s,
H), 2.65ꢀ3.10 (m, 3H), 5.00 (d, 1H, J = 13.5 Hz), 6.61 (d, 1H, J = 7.5 Hz), 6.89 (t, 1H, J =
1
3
.2 Hz), 7.00ꢀ7.15 (m, 3H), 7.21 (d, 1H, J = 7.2 Hz), 7.20ꢀ7.40 (m, 2H), 7.72 (bs, 1H). C
NMR (50 MHz, CDCl
3
):
δ
24.6, 26.2, 29.6, 34.9, 47.7, 118.3, 127.0, 127.2, 128.2, 129.2,
ꢀ1
1
1
30.0, 131.4, 139.1, 139.3, 144.0, 168.5, 168.6. IR (KBr):
λ
(cm ) 3289, 3036, 2925, 2861,
+
691, 1620, 1529, 1406, 1308, 1262, 503. HRMS (ES) m/z calcd for C19
H
20
N
2
O
2
[M+H] ,
28
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