Hit-to-Lead Optimization of Mouse Trace Amine Associated Receptor 1 (mTAAR1) Agonists with a Diphenylmethane-Scaffold: Design, Synthesis, and Biological Study

Article abstract of DOI:10.1021/acs.jmedchem.6b01092

The trace amine-associated receptor 1 (TAAR1) is a G-protein-coupled receptors (GPCR) potently activated by a variety of molecules besides trace amines (TAs), including thyroid hormone-derivatives like 3-iodothyronamine (T1AM), catechol-O-methyltransferase products like 3-methoxytyramine, and amphetamine-related compounds. Accordingly, TAAR1 is considered a promising target for medicinal development. To gain more insights into TAAR1 physiological functions and validation of its therapeutic potential, we recently developed a new class of thyronamine-like derivatives. Among them compound SG2 showed high affinity and potent agonist activity at mouse TAAR1. In the present work, we describe design, synthesis, and SAR study of a new series of compounds (1-16) obtained by introducing specific structural changes at key points of our lead compound SG2 skeleton. Five of the newly synthesized compounds displayed mTAAR1 agonist activity higher than both SG2 and T1AM. Selected diphenylmethane analogues, namely 1 and 2, showed potent functional activity in in vitro and in vivo models.

Full text of DOI:10.1021/acs.jmedchem.6b01092

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Article
Hit-to-lead optimization of mouse Trace Amine Associated
Receptor 1 (mTAAR1) agonists with a diphenylmethane-
scaffold: Design, Synthesis, and biological study
Grazia Chiellini, Giulia Nesi, Simona Sestito, Sara Chiarugi, Massimiliano Runfola, Stefano
Espinoza, Martina Sabatini, Lorenza Bellusci, Annunziatina Laurino, Elena Cichero, Raul
R Gainetdinov, Paola Fossa, Laura Raimondi, Riccardo Zucchi, and Simona Rapposelli
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01092 • Publication Date (Web): 12 Oct 2016
Downloaded from http://pubs.acs.org on October 13, 2016
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Hit-to-lead optimization of mouse Trace Amine Associated Receptor 1 (mTAAR1) agonists
with a diphenylmethane-scaffold: Design, Synthesis, and biological study
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*
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2
Grazia Chiellini , Giulia Nesi , Simona Sestito , Sara Chiarugi , Massimiliano Runfola , Stefano
3
1
1
4
5
Espinoza , Martina Sabatini , Lorenza Bellusci , Annunziatina Laurino , Elena Cichero , Raul. R.
6
,7
5
4
1
2*
Gainetdinov , Paola Fossa , Laura Raimondi , Riccardo Zucchi , Simona Rapposelli .
1
2
Dept. of Pathology, University of Pisa, Pisa, Italy, Dept. of Pharmacy, University of Pisa, Pisa,
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Italy, Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia,
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Genova, Italy, Dept. of NEUROFARBA; Section of Pharmacology, University of Florence,
5
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Italy, Dept. of Pharmacy, University of Genoa, Genoa, Italy. Institute of Translational
7
Biomedicine, St. Petersburg State University, St. Petersburg, 199034, Russia, Skolkovo Institute of
Science and Technology (Skoltech) Skolkovo, Moscow region, 143025, Russia.
Keywords: mTAAR1 agonist; diphenylmethaneꢀscaffold; Thyronamine; T1AM; T0AM
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Abstract.
The trace amineꢀassociated receptor 1 (TAAR1) is a Gꢀproteinꢀcoupled receptors (GPCR) potently
activated by a variety of molecules besides trace amines (TAs), including thyroid hormoneꢀ
derivatives like 3ꢀiodothyronamine (T1AM), catecholꢀOꢀmethyltransferase products like 3ꢀ
methoxytyramine, and amphetamineꢀrelated compounds. Accordingly, TAAR1 is considered a
promising target for medicinal development. To gain more insights into TAAR1 physiological
functions and validation of its therapeutic potential we recently developed a new class of
thyronamineꢀlike derivatives. Among them compound SG2 showed high affinity and potent agonist
activity at mouse TAAR1. In the present work we describe design, the synthesis and SAR study of a
new series of compounds (1-16) obtained by introducing specific structural changes at key points of
our leadꢀcompound SG2 skeleton. Five of the newly synthesized compounds displayed mTAAR1
agonist activity higher than both SG2 and T1AM. Selected diphenylmethane analogs, namely 1 and
0
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, showed potent functional activity in in vitro and in vivo models.
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Introduction.
Thyronamine (TAM) is generally used to indicate a class of endogenous compounds deriving from
1
thyroid hormones through metabolic reactions such as deiodination and decarboxylation . Over the
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past decade, thyronamines have been recognized as ligands of trace amine ꢀ associated receptor 1
(TAAR1), and to date they represent a thriving research field. Currently, only two compounds that
belong to the class of TAM have been identified in vivo: the 3ꢀiodothyronamine (T1AM) and the
thyronamine (T0AM). These compounds have been postulated to derive from thyroid hormone (T4)
through deiodination and decarboxylation. T1AM is widely expressed in various tissues, such as
brain, liver, heart and blood and it seems to be the most abundant thyronamine present in
2
circulation .
Among several potential receptors, it has been proved that T1AM binds to TAAR1, a Gꢀprotein
coupled receptor (GPCR) that has been identified in specific areas of the central nervous system and
in some peripheral areas. The binding of T1AM to TAAR1 receptor leads to increase of cAMP
levels by the activation of the adenylate cyclase. Further studies also highlighted that T1AM
interact with other targets such as the biogenic amines transporters and the apoB100 protein.
Thyronamines, and in particular T1AM, produce different functional effects, although their
physiological role still remains unclear. Among the most significant effects attributed to T1AM are
a hypothermic as well as negative inotropic and chronotropic cardiac effects. Moreover, endocrine
and metabolic effects such as the modulation of insulin secretion, the inhibition of catecholamines
resorption at the neuronal level and an increased metabolism of lipids at the expense of that of
3
carbohydrates have been also described . The wide variety of functions attributed to T1AM makes
this molecule a potentially useful tool for the treatment of many diseases such as obesity,
neuropsychiatric disorders and cancer. Unfortunately, TAMs are rapidly metabolized by different
enzyme systems, such as aminoꢀoxidase (MAO, SSAO), deiodinase (DIO3), sulfotransferase
(SULT1A1 and SULT1A3), Nꢀacetyltransferase, and glucuronidase. The action of these enzymes is
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a limit to their therapeutic use. Consequently, the great therapeutic potential as well as the lack of
new useful tools to elucidate the physiological function of T1AM, have pushed medicinal chemists
to develop new synthetic analogues of T1AM.
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Recently, with the aim of increasing the number of selective ligands for TAAR1 receptor and
provide new tools to facilitate the understanding of the physiological functions of this receptor, we
reported the design and synthesis of a new class of thyronamineꢀlike compounds with a
diphenylmethane scaffold, which proved to be synthetically more accessible than endogenous
4
, 5
T1AM and T0AM . Within the small series of analogues previously synthesized, SG1 and SG2
were found to activate mouse TAAR1 receptor (mTAAR1) with a potency comparable to the
4
corresponding endogenous ligands (Figure 1). In particular, SG2, the analogue of T1AM, showed
an EC50 = 240 nM comparable to T1AM (EC50 = 189 nM). In order to expand the SAR study for
this new class of compounds, in the present work we describe the design and synthesis of
compounds 1-16 obtained by introducing specific structural changes at key points of our leadꢀ
compound SG2 skeleton. The structural optimization process of the new thyronamineꢀlike
compounds was based on molecular docking studies that highlighted the main differences in the
interaction between the endogenous ligand T1AM or the synthetic analogue SG2 with the mTAAR1
receptor binding site. The newly designed compounds were first screened in vitro for mTAAR1
activation. Five compounds (1,2,4,7 and 8) showed a higher potency than the lead molecule SG2, as
well as, the endogenous ligand T1AM, with 1 and 2 being the most potent of the new series. These
compounds were then evaluated both in vitro and in vivo to characterize their ability to modulate
plasma glucose levels.
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O
I
NH2
HO
NH2
H N
R
O
2
T1AM
mTAAR1 EC50 = 189nM
SG1 R=H
mTAAR1 EC50 = 1700nM
SG2 R=Me
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mTAAR1 EC50 = 240nM
Figure 1. Structure of T1AM and the leadꢀmolecules SG1 and SG2 with a diphenylmethane
molecular scaffold
Results.
Design of new thyronamine-like analogues.
1
In our previous work, we explored the pharmacological profile of a new series of T AM
diphenylmethane analogues, and among these compounds SG1 and SG2 were found to be a good
4
mimic of the corresponding endogenous ligands T0AM and T1AM , respectively. In addition, a
docking study was performed to investigate the main differences between T1AM and the lead
diphenylmethaneꢀanalogue SG2 on mTAAR1 receptor binding. According to this study, T1AM
appears to be highly stabilized in the binding site through the formation of two Hꢀbonds between
the protonated amino group of the ethylamine side chain and aspartic acid 102 (D102) and tyrosine
291 (Y291). Furthermore, T1AM established an additional Hꢀbond between the hydroxylꢀgroup on
the outer ring and arginine 82 (R82), while no significant amino acid interactions were observed for
the biarilꢀethereal oxygen of T1AM. Notably, the SG2 derivative appeared to share most of the
4
main mTAAR1/T1AM interactions . Interestingly, the replacement of the hydroxyl group on the
T1AM outer ring with an amino group, as in SG2, allowed the formation of a cationꢀπ interaction,
while no Hꢀbonds to the R82 backbone oxygen atom were observed. Conceivably, the oxygen atom
in the SG2ꢀinner ring side chain may induce a shift of the molecule within the binding site, thus
preventing the Hꢀbond formation with R82. In order to expand the SAR study for this new class of
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thyronamine analogues, we performed specific structural changes at key points of the SG2ꢀ
molecular scaffold. Initially, to restore the Hꢀbond with R82, the oxoꢀethylamino side chain of SG2
was replaced with an ethylamine one (1-6). Among these new compounds, analogue 2 was
synthesized to evaluate the effects induced by the recovery of the phenolic function as in T1AM.
Additionally, to gain further insights on how the pattern of substitutions modulate the SG-analogues
potency as mTAAR1 agonists, we investigated the following structure modifications: (a) the
introduction of small alkyl substituents (Me, iꢀPr) on both outerꢀ and innerꢀrings (4,6-10); (b) the
removal of the methyleneꢀbridge, producing the diarylꢀanalogues 5 and 6; (c) the introduction of a
biguanideꢀmoiety at the oxyꢀalkyl side chain (15,16) and finally (d) monoꢀ and bisꢀNꢀacetylation (3,
9-15). This last chemical modification has been suggested by a recent study conducted by Hoefig et
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al , showing that Oꢀacetyl and Nꢀacetyl derivatives of T1AM may be found in white adipose tissue
and liver endogenous cells. Notably, NꢀAcꢀT1AM was also found to be an endogenous cardioactive
6
metabolite . On the basis of these findings, the corresponding monoꢀ and bisꢀNꢀacetylated
analogues of SG1 and SG2 lead compounds (i.e. 11,12 and 13,14, respectively) as well as the new
acetylated products namely 3, 9,10 and 15, were synthetised.
Synthesis.
The derivatives 1-6 in which the oxoꢀethylamino side chain of SG2 has been replaced with the
ethylamine one have been synthesized as reported in Scheme 1. Briefly, the products 17-20
obtained by the palladium(0)ꢀcatalyzed Suzuki−Miyaura crossꢀcoupling reaction of the
4
trifluoroborate salt or boronic acid with the appropriate benzylbromide , reacted with SOCl to give
2
benzylchloride 21-24. Subsequently, the nucleophilic substitution with NaCN afforded the
4
compound 25-28 with high yields. As previously described , reduction of nitrile derivative with
LiAlH
4
in the presence of a Lewis’s acid afforded the diamine derivative 1,4-6. Then, the treatment
O/NaHCO provided the acetylanilide 3 (Scheme 1a).
of 1 with a mixture of Ac
2
3
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Scheme 1
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Reagents and conditions: a: SOCl
2 3 2 3 4 3
, CHCl , rt, 2h; b: NaCN, H O/CH CN, mw; c: LiAlH , AlCl ,
THF, reflux, 12h; d: Ac₂O/NaHCO , rt, 1h, e: H , PdꢀC, AcOH, 12h; f: NaNO , H SO , H O,
3
2
2
2
4
2
1
00°C, 1h.
Compound 2 was obtained starting from the acetonitrile derivative 25. Reduction of the nitroꢀgroup
with H in the presence of 10% PdꢀC afforded the aniline 29. The subsequent hydrolysis of the
diazonium salt obtained by reaction of 29 with NaNO /H SO afforded the final product 2 (Scheme
2
2
2
4
1
b).
The Suzuki Miyaura crossꢀcoupling reaction of the 2,6ꢀdimethylꢀ4ꢀmethoxyꢀboronic acid with the
4ꢀnitrobenzyl bromide gave 31 which was demethylated with BBr at 0°C affording to phenol 32.
3
The subsequent reaction of αꢀbromideꢀacetonitrile with 32 followed by reduction to the
corresponding diamineꢀderivative gave the superior homolog of SG2 (7) (Scheme 2).
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Scheme 2
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Reagents and conditions: a: 4ꢀNitrobenzyl bromide, K CO , PdCl , Acetone/H O, rt, 72 h; b:
2
3
2
2
3
BBr , DCM, 0°C, 1 h; c: BrCH
2
CN, DMF, Cs
2
CO
3
, rt, 30'; d: LiAlH
4
, AlCl
3
, THF, reflux, 12 h.
Derivative 8-10 substituted with an iꢀPr group in the outer ring and with a small Nꢀalkyl (8) or Nꢀ
acetylꢀgroup (9,10) were synthesized starting from the cross coupling reaction of Nꢀ(4ꢀ
(
bromomethyl)ꢀ2ꢀisopropylphenyl)acetamide (34) (see SI, Scheme S1) and the (4ꢀ(hydroxymethyl)ꢀ
ꢀmethylphenyl)fluoroboronate salt (Scheme 3). The tandem reactions with SOCl and NaCN gave
the nitrile derivative 37 with high yields. Subsequent reduction with LiAlH /AlCl afforded the
in a ratio of 1:1 or 1:2 to
2
2
4
3
product 8, which was then reacted with Ac
give 9 and 10, respectively.
2 3
O in the presence of NaHCO
As concerns the synthesis of the monoꢀ and Nꢀdiacetylated derivatives 11-14 and the biguanide
analogue of SG2 (15 and 16), the synthetic pathway as well as the procedures are reported in SI
(Scheme S2).
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SCHEME 3
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Reagents and conditions: a) PdCl₂ dppf, Cs₂CO3,
H
2
O/Diossano, 95°C, 24h; b) SOCl2, CHCl3, rt,
, rt,1h.
2h; c) NaCN, H O/CH CN, mw; d) LiAlH , AlCl , THF, reflux, 12 h; e) Ac₂O/NaHCO
2
3
4
3
3
Receptor Activation.
mTAAR1 is coupled to stimulatory G protein and thus induces cAMP production in HEK293 upon
agonist exposure. We measured the activity of the new compounds using a BRETꢀbased assay as
4, 7
previously reported by us . The standard TAAR1 agonist βꢀPEA was used as reference compound
(EC50 = 138 nM).
Initially, all 1-16 compounds were tested at a screening concentration of 10ꢁM. Then, for the
compounds that were found to be active, a doseꢀresponse curve was performed to calculate their
corresponding EC50 values (Figure 2). As shown in Table 1, five compounds, namely 1,2,4,7 and 8,
appeared to be more potent than the lead compound SG2, as well as T1AM. All of them showed to
be full agonists (Efficacy 100%). In particular, the more effective structure modifications resulted
from the replacement of oxyꢀethylamino side chain of SG2 with the ethylamino one (1) and the
concomitant replacement of the amino group of the outer ring with the hydroxyl moiety (2).
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Moreover, the addition of small alkyl groups in the inner ring of SG2 (7), as well as in the outer ring
of the Nꢀethyl analogue of 1 (8), turned out to be successful, thus obtaining new compounds with a
potency 2ꢀfold higher than the lead compound SG2.
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Table 1. Activity of the new diphenylmethane derivatives 1-6, 8ꢀ10 using BRETꢀbased assay in
mTAAR1 transfected HEK 293 cells. The EC50 values are expressed in nM.
mTAAR1
compd
X
n
R
R1
R2
Y
EC (nM)
50
1
2
3
4
5
6
8
9
NH2
1
1
1
1
0
0
1
1
1
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
NH2
119
98
OH
NH2
NHAc
NH2
H
H
NH2
3100
158
Me
H
H
NH2
NH2
NH2
H
1180
633
NH2
Me
H
H
NH2
NHEt
AcNEt
AcNEt
iꢀPr
iꢀPr
iꢀPr
NH2
102
H
NH2
440
1
0
H
NHAc
>10000
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Table 2. Activity of the new diphenylmethane derivatives 7, 11ꢀ16 using BRETꢀbased assay in
mTAAR1 transfected HEK 293 cells. The EC50 values are expressed in nM.
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mTAAR1
compd
X
n
R
R1
Y
EC (nM)
50
7
NH
2
1
1
1
1
1
1
Me
H
Me
H
NH
NH
2
2
134
11
NHAc
NHAc
NHAc
NHAc
NHAc
>10000
>10000
>10000
>10000
>10000
1
1
1
1
2
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5
H
H
NHAc
NH
NHAc
Me
Me
Me
H
2
H
H
16
NH
2
1
Me
H
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Figure 2. Doseꢀresponse curves of the most active SG2ꢀanalogues. All the compounds are full
agonists (Efficacy =100%)
Docking study.
In our previous work, we investigated T AM and a series of T AM analogues whose potency profile
1
1
4
proved to be highly related to the presence of a proper basic feature linked to an aromatic core .
Indeed, our molecular modelling studies pointed out the relevance of a key salt bridge between the
protonated amine group of SG1 and SG2 lead compounds and the highly conserved residue D102,
together with a number of π−π stacking and cationꢀπ contacts with the surrounding amino acids, as
8
, 9
we previously detected in other series of TAAR1 ligands .
These findings were supported by further homology modelling and docking studies we performed
10
within the murine and human TAAR1 and TAAR5 receptors with respect to T
1
AM ligand , and
were also in accordance with the key role played by a proper basic feature included in any TAAR1
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ligand, as reported in literature . Notably, this information was definitively validated by the
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mutagenesis data involving the D102 residue, reported by Reese .
In order to rationalize the pharmacological results and refine the computational models, in silico
docking studies on the most active compounds (1,2,7 and 8) were also performed, analysing their
interactions with the putative receptor binding site (see SI, Table S1).
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In agreement with the experimental data, our docking calculations revealed that the presence of the
ethylamine chain appears to be the most effective choice, in particular within those series of
compounds bearing a mono methylꢀsubstituted inner ring (1,2 and 8). Based on a comparison with
the prototype T1AM, the most promising newly designed analogues were overturned within the
receptor binding site, maintaining in any case the key contact with D102 which is required for
1
2
TAAR1 agonism (see SI, Figure S1) .
Notably, the most effective compounds 1 and 2 were characterized by a reversed docking mode
4
when compared to that previously described for SG2 . Briefly, we originally highlighted the key
role played by the protonated amino group of the ethyloxyamine side chain of SG2 in Hꢀbonding
with D102 and Y291, while the innerꢀphenyl group was engaged in π−π stacking with Y287. On the
contrary, 1 (and 2) displayed one Hꢀbond between the aniline portion in the outer ring (or the
hydroxyl group) and the D102 negativeꢀcharged sideꢀchain, while the protonated basic side chain
appeared to be involved in one Hꢀbond and in a saltꢀbridge with T83 and D284, respectively, as
shown in Figure 3a and 3b, respectively.
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Figure 3. Docking mode of compounds SG2 (C atom cyan) and (a) 1 (C atom deep magenta) (b) 2
(C atom; coral) (c) 7 (C atom; light green) and (d) 8 (C atom; yellow) at the murine TAAR1 binding
site. The most important residues are labelled.
The addition of a methylꢀgroup in the innerꢀring of SG2 led to a 2ꢀfold increase of potency for 7,
with an EC50 of 134nM vs 240 nM for SG2. The docking study showed that also 7 takes an
overturned positioning with respect to SG2 in the binding site of mTAAR1 (Figure 3c).
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Presumably, the presence of an additional substituent into the inner ring causes a shift of the
molecule within the binding site promoting the interaction of the aniline nitrogen atom in the outer
ring with D102 residue, while the side chain’s nitrogen atom interacts with D284.
Finally, the introduction of small alkyl groups into the outer ring of 1 as in compound 8 didn’t
prevent the interaction between the nitrogen atom of aniline with D102 residue. Indeed, the EC50
value of 8 (EC =102nM) was comparable to that of 1 (EC = 119nM).
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Modulation of hepatic glucose production.
The hyperglycemic effect observed after administration of exogenous T1AM had previously been
13ꢀ15
16
attributed only to a modulation of insulin and/or glucagone secretion
. Recently, our study
showed that in human hepatocarcinoma cells (HepG2) T1AM treatment directly stimulated glucose
production (EC50 = 0.84 ꢀM), if adequate gluconeogenetic substrates were provided. Interestingly,
the effect of T1AM on gluconeogenesis displayed a bellꢀshaped doseꢀresponse curve, suggesting a
biphasic effect of T1AM on the modulation of gluconeogenesis. In the present study, the effects
induced by two of the most potent new mTAAR1 agonists (i.e. 1 and 2) on gluconeogenesis were
assessed. Preliminarily, HepG2 cells were incubated with each compound at three different doses
(0.1, 1 and 10ꢀM). By comparison, similar experiments were performed using T1AM. As shown in
Figure 4a, incubation with 0.1 and 1 ꢀM of compound 2 induced a significant increase in glucose
production (P<0.01), while a dose of 10ꢀM induced a decreased stimulation of glucose production.
On the other hand, compound 1 showed a doseꢀdependent increase of glucose production, which
reached statistical significance only when compound 1 was used at the highest dose (10ꢀM). As
shown in Figure 4a, treatment of HepG2 cells with T1AM (0.1, 1 and 10 ꢀM) confirmed the bellꢀ
16
shaped dose–response curve observed in our previous study . To better define the dose dependence
of 1 and 2 effects on glucose production additional experiments were carried out using a wider
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range of doses for both compounds. As shown in Figure 4b the EC50 calculated for the effect on
gluconeogenesis was in the submicromolar range for 2, and in the supramicromolar range for 1.
Figure 4: (a) Glucose production in HepG2 cell cultures that were incubated for 4 h in glucose
production buffer after adding vehicle (DMSO) or compounds 1, 2 and T1AM. Results are
expressed as mean ± S.E.M. and are normalized to the total cell protein content determined in cell
lysates. (n=3 in each case). (b) Glucose production in HepG2 cell in response to increasing
concentrations of compound 1 (0.1, 1, 10 and 20 ꢀM) and 2 (0.001, 0.01, 0.1, 1 and 10 ꢀM).
Results are mean ± S.E.M. and are normalized to the total cell protein content determined in cell
lysates. (n=6 in each case).*P<0.05, **P<0.01 , ***P<0.001 by ANOVA.
0
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Modulation of plasma glucose level.
As previously reported by us thyronamineꢀlike analogues SG1 and SG2 demonstrated to be able to
increase plasma glycaemia with a potency comparable to that of the corresponding endogenous
4
thyronamine (i.e. T0AM and T1AM) . Thus, in the present work the effects induced by 1 and 2
derivatives on plasma glucose level were also assessed.
Figure 5 shows that a single low dose (4.0 ꢁg/kg, i.p.) of 1 or 2 significantly increases plasma
glycaemia in CDꢀ1 mice with a potency comparable to that previously shown by leadꢀcompound
4
SG2 .
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ꢀ
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Figure 5. Effect of 1 and 2 on plasma glycaemia (mgˑL ) in CD1ꢀmice. Plasma glucose was
measured 15 min after test compound or vehicle injection (i.p.) in blood collected from the tail
veins of 4 h starved mice (n = 8 in each group). Results are expressed as means mean±SEM.*P <
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0
.05 vs vehicle. ***P < 0.001 vs vehicle.
Conclusion
Our ongoing efforts to develop thyronamineꢀlike analogues synthetically more accessible than
endogenous thyronamines yielded a new class of potent mTAAR1 diphenylmethane ligands.
A medicinal chemistry optimization combined to docking studies were used starting from the
molecular scaffold of our recently developed SGꢀcompounds to design the new ligands. In
particular, we investigated: (i) the replacement of the oxyethylamino side chain of SG2 with an
ethylamino one (ii) the introduction of small alkyl substituents (Me, iꢀPr) on both outerꢀ and innerꢀ
rings and (iii) the recovery of the phenolic function as in T1AM. Overall these chemical
modifications resulted in more potent mTAAR1 agonists when compared to T1AM, as well as, lead
compound SG2. Taking into account the high structure similarity of compounds 1 and 2 with
T1AM and their high affinity for mTAAR1, we further investigated their ability to modulate
glucose levels using both in vitro (human HepG2 cells) and in vivo (CD1ꢀmice) models. Both
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compounds demonstrated potent functional activity. In conclusion, this paper describes for the first
time the successful outcome of a hitꢀtoꢀlead optimization process leading to the identification of
new tools to explore the physiological and pharmacological functions of TAAR1.
Experimental Section
0
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Chemistry.
General Material and Methods. Melting points were determined on a Kofler hotꢀstage apparatus
and are uncorrected. Chemical shifts (δ) are reported in parts per million downfield from
tetramethylsilane and referenced from solvent references; coupling constants J are reported in hertz.
1
13
H NMR and C NMR spectra of all compounds were obtained with a Bruker TopSpin 3.2 400
13
MHz spectrometer. C NMR spectra were fully decoupled. The following abbreviations are used:
singlet (s), doublet (d), triplet (t), double−doublet (dd), and multiplet (m). The elemental
compositions of the compounds agreed to within ±0.4% of the calculated values. Chromatographic
separation was performed on silica gel columns by flash (Kieselgel 40, 0.040−0.063 mm; Merck) or
gravity column (Kieselgel 60, 0.063−0.200 mm; Merck) chromatography. The ≥95% purity of the
tested compounds was confirmed by combustion analysis. Reactions were followed by thinꢀlayer
chromatography (TLC) on Merck aluminum silica gel (60 F254) sheets that were visualized under a
UV lamp. The microwaveꢀassisted procedures were carried out with a CEM Discover LabMate
microwave. Evaporation was performed in vacuo (rotating evaporator). Sodium sulfate was always
used as the drying agent. Commercially available chemicals were purchased from SigmaꢀAldrich.
4
4
-(4-(2-Aminoethyl)-2-methylbenzyl)aniline (1). To a suspension of LiAlH (4.80 mL, 4.80 mmol)
in THF was added dropwise a solution of AlCl (639.6 mg, 4.80 mmol) in THF (20 mL), and the
3
mixture was stirred for 5 min at r.t. A solution of 25 (142.0 mg, 0.53 mmol) in THF was added
dropwise, and the mixture was heated at reflux for 12 h. The mixture was cooled to 0°C with an ice
bath and added dropwise with water and then with 10% aqueous HCl. The mixture was extracted
with diethyl ether, and the aqueous layer was made alkaline with NaOH 2N and extracted with
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CHCl . The organic phase was separated, washed with brine, dried, filtered, and concentrated. The
3
crude was purified by conversion in the corresponding hydrochloride salt. White solid. mp: 153ꢀ
1
1
57°C. (67% yield). H NMR (CD
3
OD): δ 2.21 (s, 3H, CH
); 7.07ꢀ7.15 (m, 3H, Ar); 7.29 (d, 2H,
= 8.4 Hz, Ar) ppm. C NMR (CD OD): δ 143.44, 138.48, 138.37, 136.40, 131.89, 131.66,
131.35, 129.71, 127.54, 124.11, 41.99, 39.31, 34.09, 19.70 ppm. Anal. (C H N ·HCl) C, H, N. %
3
); 2.94 (t, 2H,
J
= 7.8 Hz; CH
2
); 3.17 (t,
2
H,
J
= 7.8 Hz; CH
2
); 4.03 (s, 2H, CH
2
J
= 8.4 Hz, Ar); 7.34
10
11
12
13
14
15
16
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13
(d, 2H,
J
3
16
20
2
Calcd: 79.96 (C); 8.39 (H); 11.66 (N). % Found: 79.88 (C); 8.55 (H); 11.42 (N).
4
-(4-(2-Aminoethyl)-2-methylbenzyl)phenol (2). Compound 2 was synthesized from 30 (109 mg,
0
.46 mmol), LiAlH (4.13 mL; 4.13 mmol) and AlCl (550.8 mg, 4.13 mmol) in THF (18 mL)
4
3
following the same procedure described above for the preparation of 1. Yellow solid. mp: 138ꢀ
1
140°C. (30% yield). H NMR (CD
3
OD): δ 2.18 (s, 3H, CH
=8.4 Hz, Ar); 6.84 (d, 2H,
OD): δ 146.29, 139.28, 137.93, 137.82, 131.69, 131.64,
31.10, 130.31, 127.21, 116.88, 54.79, 39.22, 38.30, 19.74 ppm. Anal. (C16 19NO) C, H, N. %
3
); 2.70 (t, 2H,
J
= 7.2 Hz; CH
2
); 2.87 (t,
2
6
1
H,
J
= 7.2 Hz; CH ); 3.81 (s, 2H, CH ); 6.64 (d, 2H,
J
J
=8.4 Hz, Ar);
2
2
13
.94ꢀ7.03 (m, 3H, Ar) ppm. C NMR (CD
3
H
Calcd: 79.63 (C); 7.94 (H); 5.80 (N). % Found: 79.77 (C); 7.81 (H); 5.92 (N).
N-(4-(4-(2-Aminoethyl)-2-methylbenzyl)phenyl)acetamide (3). To a solution of 1 (50 mg, 0.18
mmoli) in saturated NaHCO solution (0.06 mL) was added Ac O (0.02 mL, 0.18 mmoli). The
3
2
mixture was left stirring at r.t. for 1 h, then, the mixture was dissolved in water and extracted with
CHCl . The organic layer was dried and concentrated under reduced pressure. The crude product
3
was purified by precipitation from MeOH/Et
2
O to give 3. White solid. mp: 132ꢀ136°C. (60% yield).
); 2.92 (t, 2H, = 7.7 Hz; CH ); 3.15
= 8.4 Hz, Ar); 7.05ꢀ7.11 (m, 3H, Ar);
3
.34 (d, 2H, J = 8.4 Hz, Ar) ppm. C NMR (CD OD): δ 171.57, 139.37, 138.31, 137.77, 137.62,
1
H NMR (CD
3
OD): δ 2.10 (s, 3H, CH
3
); 2.20 (s, 3H, COCH
3
J
2
(
t, 2H, = 7.7 Hz; CH ); 3.91 (s, 2H, CH ); 7.04 (d, 2H, J
J
2
2
13
7
1
35.91, 131.71, 131.52, 129.93, 127.33, 121.33, 42.01, 39.37, 34.09, 23.77, 19.74 ppm. Anal.
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2
2
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2
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(C H N ·HCl) C, H, N. % Calcd: 79.96 (C); 8.39 (H); 11.66 (N). % Found: 79.88 (C); 8.55 (H);
1
6
20
2
1
1.42 (N).
4
-(4-(2-Aminoethyl)-2,6-dimethylbenzyl)aniline (4). Compound 4 was synthesized from 26 (109
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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4 3
mg, 0.46 mmol), LiAlH (4.13 mL; 4.13 mmol) and AlCl (550.8 mg, 4.13 mmol) in THF (18 mL)
following the same procedure described above for the preparation of 1. The crude was purified by
1
conversion in the corresponding hydrochloride salt. White solid, mp: 143ꢀ145°C. (68% yield). H
NMR (CD OD): δ 2.22 (s, 6H, CH ); 2.91 (t, 2H,
J
= 7.7 Hz; CH ); 3.18 (t, 2H,
J
= 7.7 Hz; CH2);
3
3
2
4
.11 (s, 2H, CH
2
); 7.01 (s, 2H, Ar); 7.17 (d, 2H,
J
= 8.6 Hz, Ar); 7.30 (d, 2H,
J
= 8.6 Hz, Ar) ppm.
13
C NMR (CD
4.98, 34.12, 20.23 ppm. Anal. (C17
% Found: 80.40 (C); 8.59 (H); 11.31 (N).
3
OD): δ 142.77, 138.85, 136.31, 136.14, 130.58, 129.68, 129.61, 124.13, 42.00,
3
22 2
H N
·HCl) C, H, N. % Calcd: 80.27 (C); 8.72 (H); 11.01 (N).
4'-(2-Aminoethyl)-2'-methyl-[1,1'-biphenyl]-4-amine (5). Compound 5 was synthesized from 27
109 mg, 0.46 mmol), LiAlH (4.13 mL; 4.13 mmol) and AlCl (550.8 mg, 4.13 mmol) in THF (18
(
4
3
mL) following the same procedure described above for the preparation of 1. The crude was purified
by conversion in the corresponding hydrochloride salt. White solid, mp: 165ꢀ167°C. (70% yield).
1
3 3 2
H NMR (CD OD): δ 2.25 (s, 3H, CH ); 2.99 (t, 2H, J = 7.8 Hz; CH ); 3.22 (t, 2H, J = 7.8 Hz;
1
3
CH ); 7.19 (d, 2H, J=1.2 Hz, Ar); 7.25 (s, 1H, Ar); 7.51ꢀ7.46 (m, 4H, Ar) ppm. C NMR
2
(
CD
3
OD): δ 144.11, 140.55, 137.72, 137.02, 131.99, 131.21, 130.72, 127.48, 124.08, 41.92, 34.19,
2
8
0.43 ppm. Anal. (C15 ·HCl) C, H, N. % Calcd: 79.61 (C); 8.02 (H); 12.38 (N). % Found:
18 2
H N
0.00 (C); 8.10 (H); 12.22 (N).
4
'-(2-Aminoethyl)-2',6'-dimethyl-[1,1'-biphenyl]-4-amine (6). Compound 6 was synthesized from 28
(109 mg, 0.46 mmol), LiAlH (4.13 mL; 4.13 mmol) and AlCl (550.8 mg, 4.13 mmol) in THF (18
4
3
mL) following the same procedure described above for the preparation of 1. The crude was purified
by conversion to the corresponding hydrochloride salt. White solid, mp: 171ꢀ173°C. (73% yield).
1
3 3 2
H NMR (CD OD): δ 2.00 (s, 3H, CH ); 2.95 (t, 2H, J = 7.8 Hz; CH ); 3.20 (t, 2H, J = 7.8 Hz;
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1
3
CH ); 7.07 (s, 2H, Ar); 7.30 (d, 2H,
J
= 8.4 Hz, Ar); 7.53 (d, 2H,
J = 8.4 Hz, Ar) ppm. C NMR
2
(
CD OD): δ 143.35, 140.41, 137.50, 137.27, 132.17, 130.72, 128.97, 124.52, 41.98, 34.21, 20.84
3
20 2
ppm. Anal. (C16H N ·HCl) C, H, N. % Calcd: 79.96 (C); 8.39 (H); 11.66 (N). % Found: 79.90 (C);
8
.49 (H); 11.71 (N).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
-(4-(2-Aminoethoxy)-2,6-dimethylbenzyl)aniline (7). Compound 7 was synthesized from 33 (186.2
(6.87 mL, 6.87 mmol) and AlCl (915.6 mg, 6.87 mmol) in THF (25 mL)
mg, 0.66 mmol), LiAlH
4
3
following the same procedure described above for the preparation of 1. The crude was purified by
1
conversion to the corresponding hydrochloride salt. White solid, mp: 156ꢀ158°C. (75% yield). H
NMR (CD
5.0 Hz; CH
NMR (CD OD): δ 156.50, 141.82, 138.32, 129.24, 129.09, 128.16, 122.72, 114.04, 63.74, 39.07,
3.21, 19.10 ppm. Anal. (C H N O·HCl) C, H, N. % Calcd: 75.52 (C); 8.20 (H); 10.36 (N). %
3
OD): δ 2.20 (s, 6H, CH
3
); 3.37 (t, 2H,
J
= 5.0 Hz; CH
3
2
); 4.07 (s, 2H, CH ); 4.23 (t, 2H, J
1
3
=
2
); 6.76 (s, 2H, Ar); 7.15 (d, 2H,
J
= 8.4 Hz, Ar); 7.30 (d, 2H,
J
= 8.4 Hz, Ar) ppm. C
3
3
17
22
2
Found: 75.60 (C); 8.07 (H); 10.40 (N).
4
-(4-(2-Aminoethyl)-2-methylbenzyl)-N-ethyl-2-isopropylaniline (8). Compound 8 was synthesized
from 37 (130.8 mg, 0.41 mmol), LiAlH (1.84 mL, 1.84 mmol) and AlCl (244.8 mg, 1.84 mmol) in
4
3
THF (17 mL) following the same procedure described above for the preparation of 1. The crude
was purified by conversion in the corresponding hydrochloride salt. Brown solid, mp: 148ꢀ150°C.
1
(67% yield). H NMR (CD
3
OD): δ 1.28 (d, 6H,
); 2.93 (t, 2H, =7.8 Hz, CH ); 3.05ꢀ3.12 (m, 1H, CH); 3.16 (t, 2H,
); 3.43 (q, 2H, = 7.2 Hz, CH ); 4.04 (s, 2H, CH ); 7.06ꢀ7.14 (m, 4H, Ar); 7.32 (d, 1H,
Hz, Ar); 7.36 (d, 1H, = 1.6 Hz, Ar) ppm. C NMR (CD OD): δ 144.42, 143.51, 138.47, 138.36,
36.41, 131.87, 131.56, 131.00, 129.52, 128.88, 127.54, 124.53, 49.90, 41.97, 39.45, 34.11, 28.70,
4.43, 19.72, 11.38 ppm. Anal. (C21 ·HCl) C, H, N. % Calcd: 81.24 (C); 9.74 (H); 9.02 (N). %
J
= 6.8 Hz, CH
3
); 1.37 (t, 3H,
J
= 7.2 Hz, CH
= 7.8 Hz,
=8.4
3
);
2
.22 (s, 3H, CH
3
J
2
J
CH
2
J
2
2
J
1
3
J
3
1
2
30 2
H N
Found: 81.36 (C); 9.65 (H); 9.11 (N).
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2
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N-(4-(4-(2-Aminoethyl)-2-methylbenzyl)-2-isopropylphenyl)-N-ethylacetamide (9). Compound 9
was synthesized from 8 (45.6 mg, 0.15 mmol), saturated NaHCO solution (0.05 mL) and Ac O
3
2
(0.01 mL, 0.15 mmol) following the same procedure described above for the preparation of 3. The
crude solid was collected by filtration and purified by conversion to the corresponding
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
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
hydrochloride salt. Yellow solid. (30% yield). H NMR (CD
3
OD): δ 1.08ꢀ1.20 (m, 9H, CH
3
,
CH CH ); 1.73 (s, 3H, COCH ); 2.24 (s, 3H, CH ); 2.90ꢀ2.96 (m, 3H, CH , CH); 3.02ꢀ3.07 (m, 1H,
3
2
3
3
2
CH ); 3.17 (t, 2H,
J
= 7.4 Hz, CH ); 4.02 (s, 2H, CH ); 4.11ꢀ4.18 (m, 1H, CH ); 6.97ꢀ7.05 (m, 2H,
2
2
2
2
13
Ar); 7.07ꢀ7.17 (m, 3H, Ar); 7.27 (s, 1H, Ar) ppm. C NMR (CD
3
OD): δ 173.03, 146.93, 143.12,
1
38.91, 138.71, 138.36, 136.21, 131.81, 131.53, 130.08, 128.87, 128.30, 127.47, 45.29, 42.01,
39.67, 34.10, 28.83, 24.48, 24.13, 22.59, 19.77, 12.99 ppm. Anal. (C23 O·HCl) C, H, N. %
Calcd: 78.36 (C); 9.15 (H); 7.95 (N). % Found: 78.45 (C); 9.02 (H); 7.84 (N).
32 2
H N
N-(4-(4-(2-Acetamidoethyl)-2-methylbenzyl)-2-isopropylphenyl)-N-ethylacetamide (10). Compound
0 was synthesized from 8 (45.6 mg, 0.15 mmol), saturated NaHCO solution (0.05 mL) and Ac
0.02 mL, 0.30 mmol) following the same procedure described above for the preparation of 3. The
1
3
2
O
(
reaction mixture was evaporated and the crude dissolved in AcOEt and washed twice with water.
1
The evaporation of the organic collected phases gave final product as a white oil. (20% yield). H
NMR (CD
3
OD): δ 1.10ꢀ1.20 (m, 9H, CH
.21 (s, 3H, CH ); 2.74 (t, 2H, = 7.4 Hz, CH2); 2.90ꢀ2.96 (m, 1H, CH); 3.01ꢀ3.09 (m, 1H, CH
.37 (t, 2H, = 7.4 Hz, CH ); 4.00 (s, 2H, CH ); 4.11ꢀ4.20 (m, 1H, CH
= 1.2 Hz, Ar) ppm. C NMR (CD OD): δ 173.20, 173.08, 146.89, 143.36, 138.82,
38.68, 137.88, 137.74, 131.86, 131.13, 130.06, 128.84, 128.28, 127.48, 45.34, 42.13, 39.71, 36.09,
3
, CH
3
CH
2
); 1.73 (s, 3H, COCH
3
); 1.90 (s, 3H, COCH
3
);
2
3
3
J
2
);
J
2
2
2
); 6.99ꢀ7.09 (m, 5H, Ar);
1
3
7.25 (d, 1H,
J
3
1
2
8.86, 24.50, 24.16, 22.60, 22.52, 19.78, 13.00 ppm. Anal. (C25
H
34
N
2
O
2
) C, H, N. % Calcd: 76.10
(C); 8.69 (H); 7.10 (N). % Found: 76.25 (C); 8.53 (H); 7.21 (N).
4
N-(4-(4-(2-Aminoethoxy)benzyl)phenyl)acetamide (11). Compound 11 was synthesized from SG1
(49.7 mg, 0.20 mmol), saturated NaHCO solution (0.07 mL) and Ac
3
2
O (0.03 mL, 0.20 mmoli)
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9
following the same procedure described above for the preparation of 3. The crude product was
1
purified by precipitation from MeOH/Et O to give 11. White solid, mp: 132ꢀ136°C. (60% yield). H
2
NMR (CD
H, = 5.2 Hz; CH
Ar) ppm. C NMR (CD
115.70, 65.29, 41.30, 40.39, 23.76 ppm. Anal. (C H N ·HCl) C, H, N. % Calcd: 71.81 (C); 7.09
3
OD): δ 2.10 (s, 3H, COCH
3
); 3.34 (t, 2H,
J
= 5.2 Hz; CH
2
); 3.87 (s, 2H, CH
2
); 4.19 (t,
2
J
2
); 6.92 (d, 2H, = 8.8 Hz, Ar); 7.10ꢀ7.14 (m, 4H, Ar); 7.42 (d, 2H,
J
J = 8.4 Hz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
3
OD): δ 171.59, 157.83, 138.90, 137.82, 136.02, 130.93, 130.03, 121.40,
1
7
20
2
(H); 9.85 (N). % Found: 71.88 (C); 7.15 (H); 9.92 (N).
N-(2-(4-(4-Acetamidobenzyl)phenoxy)ethyl)acetamide (12). Compound 12 was synthesized from
SG1 (47.0 mg, 0.19 mmol), saturated NaHCO solution (0.14 mL) and Ac O (0.04 mL, 0.38 mmol)
3
2
following the same procedure described above for the preparation of 3. The crude product was
purified by precipitation from MeOH/Acetone to give 12. White solid, mp: 152ꢀ153°C. (60% yield).
1
H NMR (DMSO): δ 2.00 (s, 3H, COCH ); 2.08 (s, 3H, COCH ); 3.34ꢀ3.38 (m, 2H, CH ); 3.79 (s,
3
3
2
2
H, CH
2
); 3.91 (t, 2H,
J
= 5.6 Hz; CH
2
); 6.84 (d, 2H, J = 8.8 Hz, Ar); 7.08ꢀ7.10 (m, 4H, Ar); 7.45
1
3
(d, 2H,
J
= 8.8 Hz, Ar); 8.06 (br t, NH
2 3
); 9.83 (br s, NH) ppm. C NMR (CD OD): δ 173.59,
1
71.54, 158.58, 139.08, 137.78, 135.20, 130.82, 130.06, 121.39, 115.59, 67.53, 41.32, 40.26, 23.70,
22.46 ppm.. Anal. (C H N O ·HCl) C, H, N. % Calcd: 69.92 (C); 6.79 (H); 8.58 (N). % Found:
19
22
2
3
6
9.88 (C); 6.67 (H); 8.61 (N).
N-(4-(4-(2-Aminoethoxy)-2-methylbenzyl)phenyl)acetamide (13). Compound 13 was synthesized
4
from SG2 (50.0 mg, 0.19 mmol), saturated NaHCO
3
solution (0.07 mL) and Ac
2
O (0.02 mL, 0.19
mmoli) following the same procedure described above for the preparation of 3. The crude product
was purified by precipitation from MeOH/Et
2
O to give 13. White solid, mp: 132ꢀ136°C. (60%
= 5.0 Hz; CH2);
= 2.4, 8.4 Hz, Ar); 6.83 (d, 1H,
= 8.4 Hz, Ar); 7.41 (d, 2H, = 8.4 Hz, Ar)
1
yield). H NMR (CD OD): δ 2.10 (s, 3H, CH ); 2.19 (s, 3H, COCH ); 3.34 (t, 2H,
J
3
3
3
3
.89 (s, 2H, CH
2
); 4.20 (t, 2H,
J
= 5.0 Hz; CH
2
); 6.77 (dd, 1H,
J
J =
2
.4 Hz, Ar); 7.03 (d, 2H,
J
= 8.4 Hz, Ar); 7.06 (d, 1H,
J
J
13
ppm. C NMR (CD
3
OD): δ 171.58, 158.00, 139.19, 138.03, 137.67, 133.61, 132.00, 129.79,
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121.32, 117.73, 112.83, 65.18, 40.41, 38.93, 23.79, 19.96 ppm. Anal. (C H N O ·HCl) C, H, N.
1
8
22
2
2
%
Calcd: 72.46 (C); 7.43 (H); 9.39 (N). % Found: 72.58 (C); 7.55 (H); 9.42 (N).
N-(2-(4-(4-Acetamidobenzyl)-3-methylphenoxy)ethyl)acetamide (14). Compound 14 was
synthesized from SG2 (50.0 mg, 0.19 mmol), saturated NaHCO solution (0.14 mL) and Ac
0.04 mL, 0.38 mmoli) following the same procedure described above for the preparation of 3. The
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
O
(
2
crude product was purified by precipitation from MeOH/Et O to give 14. White solid, mp: 139ꢀ
1
141°C. (56% yield). H NMR (CD OD): δ 1.95 (s, 3H, CH ); 2.09 (s, 3H, COCH ); 2.16 (s, 3H,
3
3
3
COCH
H,
Hz, Ar) ppm. C NMR (CD
131.94, 129.83, 121.32, 117.66, 112.72, 67.42, 40.30, 38.98, 23.70, 22.47, 19.96 ppm. Anal.
3
); 3.53 (t, 2H,
J
= 5.6 Hz; CH
2
); 3.87 (s, 2H, CH
2
); 4.00 (t, 2H,
J
= 5.6 Hz; CH
2
); 6.70 (dd,
= 8.4
OD): δ 173.60, 171.53, 158.78, 139.01, 138.27, 137.66, 132.85,
1
J = 2.4, 8.4 Hz, Ar); 6.74 (d, 1H, J = 2.4 Hz, Ar); 6.99ꢀ7.03 (m, 3H, Ar); 7.41 (d, 2H, J
13
3
(
C H N O ·HCl) C, H, N. % Calcd: 70.56 (C); 7.11 (H); 8.23 (N). % Found: 70.43 (C); 7.05 (H);
20 24 2 3
8
.22 (N).
N-(4-(4-(2-(3-Carbamimidoylguanidino)ethoxy)-2-methylbenzyl)phenyl)acetamide (15). A mixture
of 13 (32.3 mg, 0.16 mmol) and dicyandiamide (13.5 mg, 0.16 mmol) was heated to 160 °C for 100
min. The reaction mixture initially melts, then reꢀsolidifies. The reaction was cooled to room
temperature. The crude product was purified by crystallization from EtOH/Et O. Brown oil. (52%
2
1
yield). H NMR (CD
3
OD): δ 2.09 (s, 3H, CH
); 4.05 (t, 2H, = 5.2 Hz; CH
Ar); 7.41 (d, 2H, = 8.0 Hz, Ar) ppm. C NMR (CD
139.04, 138.20, 137.63, 133.00, 131.94, 129.80, 121.34, 117.63, 112.73, 67.55, 42.15, 38.94, 23.74,
9.97 ppm. Anal. (C H N O ) C, H, N. % Calcd: 62.81 (C); 6.85 (H); 21.97 (N). % Found: 62.95
3
); 2.15 (s, 3H, COCH
); 6.65ꢀ6.77 (m, 3H, Ar); 7.01 (d, 2H,
OD): δ 171.60, 162.12, 161.01, 158.56,
3
); 3.60 (t, 2H,
J
= 5.2 Hz; CH
2
);
3
.86 (s, 2H, CH
2
J
2
J
= 8.0 Hz,
13
J
3
1
2
0
26
6
2
(C); 6.73 (H); 21.76 (N).
4
-(4-(2-(3-Carbamimidoylguanidino)ethoxy)-2-methylbenzyl)aniline (16). To a solution of the
amide 15 (0.57 mmol) in MeOH was added dropwise an aqueous solution of HCl 1N (0.2 mL); the
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resulting solution was refluxed for 2h, then, after cooling, the solvent was evaporated. The residue
1
was purified by precipitation from EtOH/Et O. Brown oil. (60% yield). H NMR (CD OD): δ 2.17
2
3
(
s, 3H, CH
dd, 1H, = 2.6, 8.2, Ar); 6.83 (d, 1H,
.6 Hz, Ar), 7.32 (d, 2H, = 8.6 Hz, Ar) ppm. Anal. (C18
7.11 (H); 24.69 (N). % Found: 63.72 (C); 7.05 (H); 24.53 (N).
3
); 3.79 (t, 2H,
J
= 4.8 Hz; CH
2
); 3.99 (s, 2H, CH
2
); 4.21 (t, 2H,
J
2
= 4.8 Hz; CH ); 6.79
(
J
J
= 2.6 Hz, Ar); 7.08 (d, 1H,
J = 8.2 Hz, Ar), 7.26 (d, 2H, J =
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
J
24 6
H N
O) C, H, N. % Calcd: 63.51 (C);
4-(Chloromethyl)-2-methyl-1-(4-nitrobenzyl)benzene (21). To a solution of alcohol 17 (96.0mg,
.35 mmoli) in CHCl at 0°C was added SOCl (3.50 mmol; 0.25 mL). The reaction mixture was
stirred for 2 h at room temperature, then, the solvent was evaporated. The residue was dissolved in
O and alkalized with NaOH 1N; and the aqueous layer was extracted with DCM. The organic
0
3
2
H
2
phase was dried and the solvent was evaporated to give the corresponding crude 21, which was used
1
without further purification. Yellow oil. (80% yield). H NMR (CDCl ): δ 2.22 (s, 3H, CH ); 4.08
3
3
(
s, 2H, CH
2
); 4.57 (s, 2H, CH
=7.4 Hz, Ar); 8.13 (d, 2H, = 7.4 Hz, Ar) ppm. Anal. (C15
.12 (H); 5.08 (N). % Found: 65.52 (C); 5.03 (H); 5.24 (N).
2
); 7.09 (d, 1H,
J
= 7.6 Hz, Ar); 7.19ꢀ7.24 (m, 2H, Ar); 7.26 (d, 2H,
J
J
H14ClNO
2
) C, H, N. % Calcd: 65.34 (C);
5
5-(Chloromethyl)-1,3-dimethyl-2-(4-nitrobenzyl)benzene (22). Compound 22 was synthesized from
18 (224 mg, 0.87 mmol) and SOCl (0.63 mL, 8.70 mmol) in CHCl (25 mL) following the same
2
3
procedure described above for the preparation of 21. The crude was used without further
1
purification. White oil. (76% yield). H NMR (CDCl
3
): δ 2.22 (s, 6H, CH
3
2
); 4.14 (s, 2H, CH ); 4.56
(
s, 2H, CH
2
); 7.13 (s, 2H, Ar); 7.15 (d, 2H,
J
= 8.5 Hz Ar); 8.11 (d, 2H,
J
= 8.5 Hz Ar) ppm. Anal.
(C16
2
H16ClNO ) C, H, N. % Calcd: 66.32 (C); 5.57 (H); 4.83 (N). % Found: 66.49 (C); 5.59 (H);
5
.11 (N).
4
-(Chloromethyl)-2-methyl-4'-nitro-1,1'-biphenyl (23). Compound 23 was synthesized from 19 (125
(0.37 mL, 5.10 mmol) in CHCl (25 mL) following the same procedure
mg, 0.51 mmol) and SOCl
2
3
described above for the preparation of 21. The crude was used without further purification. Yellow
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2
2
2
2
2
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4
4
4
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5
5
5
5
5
5
5
6
1
oil. (83% yield). H NMR (CDCl ): δ 2.27 (s, 3H, CH ); 4.61 (s, 2H, CH ); 7.21 (d, 2H,
J
= 7.8 Hz,
=8.8 Hz, Ar); 8.29 (d, 2H, = 8.8
2
12ClNO ) C, H, N. % Calcd: 64.25 (C); 4.62 (H); 5.35 (N). % Found:
3
3
2
Ar); 7.31 (d, 1H, J=7.8 Hz, Ar); 7.34 (s, 1H, Ar); 7.48 (d, 2H,
Hz Ar) ppm. Anal. (C14
J
J
H
6
3.91 (C); 4.68 (H); 5.40 (N).
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
4
-(Chloromethyl)-2,6-dimethyl-4'-nitro-1,1'-biphenyl (24). Compound 24 was synthesized from 20
(0.38 mL, 5.20 mmol) in CHCl (25 mL) following the same
(
134 mg, 0.52 mmol) and SOCl
2
3
procedure described above for the preparation of 21. The crude was used without further
1
purification. White oil. (81% yield). H NMR (CDCl
3
): δ 2.02 (s, 6H, CH
= 8.8 Hz Ar) ppm. Anal. (C15
H, N. % Calcd: 65.34 (C); 5.12 (H); 5.08 (N). % Found: 65.40 (C); 5.59 (H); 5.31 (N).
3
); 4.58 (s, 2H, CH
2
); 7.17
) C,
(
s, 2H, Ar); 7.33 (d, 2H,
J
= 8.8 Hz Ar); 8.31 (d, 2H,
J
H14ClNO
2
2
-(4-(4-Aminobenzyl)-3-methylphenyl)acetonitrile (25). To a solution of chloro compound 21 (78.0
mg; 0.46 mmol) in CH CN (0.62 mL) was added NaCN (45.1 mg; 0.92 mmol) in H O (0.21 mL).
3
2
The mixture was submitted to microwave irradiation (150W, 100°C, 20 min). After cooling the
solution was extracted with DCM. Organic phase was dried and evaporated to dryness. Yellow oil.
1
(
86% yield). H NMR (CDCl
3
): δ 2.22 (s, 3H, CH
= 8.6 Hz, Ar); 8.13 (d, 2H,
H, N. % Calcd: 72.16 (C); 5.30 (H); 10.52 (N). % Found: 72.34 (C); 5.02 (H); 10.65 (N).
3
); 3.71 (s, 2H, CH ); 7.09ꢀ7.18
2
); 4.07 (s, 2H, CH
2
(m, 3H, Ar); 7.25 (d, 2H,
J
J
= 8.6 Hz, Ar) ppm. Anal. (C16H N O ) C,
14 2 2
2
-(3,5-Dimethyl-4-(4-nitrobenzyl)phenyl)acetonitrile (26). Compound 26 was synthesized from 22
(
193 mg, 0.70 mmol) and NaCN (68.5 mg, 1.40 mmol) in CH CN/H O (0.94 mL/0.32 mL)
3
2
following the same procedure described above for the preparation of 25. The crude was used
1
without further purification. White oil. (70% yield). H NMR (CDCl ): δ 2.22 (s, 6H, CH ); 3.70 (s,
3
3
2H, CH ); 4.13 (s, 2H, CH ); 7.06 (s, 2H, Ar); 7.14 (d, 2H,
J
= 8.6 Hz Ar); 8.11 (d, 2H,
J
= 8.6 Hz
2
2
Ar) ppm. Anal. (C17
H
16
N
2
O
2
) C, H, N. % Calcd: 72.84 (C); 5.75 (H); 9.99 (N). % Found: 72.60 (C);
5
.60 (H); 10.02 (N).
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Journal of Medicinal Chemistry
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2-(2-Methyl-4'-nitro-[1,1'-biphenyl]-4-yl)acetonitrile (27). Compound 27 was synthesized from 23
(
95.0 mg, 0.61 mmol) and NaCN (59.8 mg, 1.22 mmol) in CH CN/H O (0.82 mL/0.30 mL)
3 2
following the same procedure described above for the preparation of 25. The crude was used
1
without further purification. Yellow oil. (86% yield). H NMR (CDCl
3
): δ 2.28 (s, 3H, CH
= 7.0 Hz Ar) ppm.
Anal. (C H N O ) C, H, N. % Calcd: 71.42 (C); 4.79 (H); 11.10 (N). % Found: 71.41 (C); 4.80
3
); 3.78
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
2
s, 2H, CH ); 7.21ꢀ7.31 (m, 3H, Ar); 7.47 (d, 2H, J = 7.0 Hz, Ar); 8.29 (d, 2H, J
15
12
2
2
(H); 11.15 (N).
2
-(2,6-Dimethyl-4'-nitro-[1,1'-biphenyl]-4-yl)acetonitrile (28). Compound 28 was synthesized from
2
4 (101 mg, 0.36 mmol) and NaCN (35.3 mg, 0.72 mmol) in CH CN/H O (0.49 mL/0.16 mL)
3
2
following the same procedure described above for the preparation of 25. The crude was used
1
without further purification. Pale yellow oil. (81% yield). H NMR (CDCl
3
): δ 2.02 (s, 6H, CH
= 8.6 Hz Ar) ppm.
) C, H, N. % Calcd: 72.16 (C); 5.30 (H); 10.52 (N). % Found: 72.40 (C); 5.59
3
);
3
.73 (s, 2H, CH ); 7.10 (s, 2H, Ar); 7.32 (d, 2H, J = 8.6 Hz Ar); 8.31 (d, 2H, J
2
Anal. (C16
H
14
N
2
O
2
(H); 10.31 (N).
2
-(4-(4-Aminobenzyl)-3-methylphenyl)acetonitrile (29). A solution of 25 (136 mg, 0.5 mmoli) in
AcOH (0.5mL) was hydrogenated in the presence of 10% PdꢀC (28.3 mg), for 12 h. Then the
catalyst was filtered off, and the solvent was removed to dryness to give a crude product that was
1
used in the subsequent step without any further purification. Yellow oil. (80% yield). H NMR
(
CDCl
.88 (d, 2H,
(C); 6.82 (H); 11.85 (N). % Found: 81.45 (C); 6.73 (H); 11.98 (N).
3
) δ: 2.24 (s, 3H, CH ); 6.61 (d, 2H,
3
); 3.68 (s, 2H, CH
2
); 3.85 (s, 2H, CH
2
J = 8.4 Hz, Ar);
6
J
=8.4 Hz, Ar); 7.06ꢀ7.12 (m, 3H, Ar) ppm. Anal. (C16H N ) C, H, N. % Calcd: 81.32
16 2
2-(4-(4-Hydroxybenzyl)-3-methylphenyl)acetonitrile (30). To a mixture of aniline derivative 29
63.0 mg, 0.26 mmoli) in H O was added dropwise with H SO4conc (0.06 mL) and the mixture was
stirred at room temperature for 20min. Then a solution NaNO (17.9 mg, 0.26 mmoli) in H O (0.19
(
2
2
2
2
ml) was added dropwise to the reaction mixture. The resulting solution was stirred for 1 h at 100°C.
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1
2
2
2
2
2
2
2
2
2
2
3
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5
5
5
6
The mixture was cooled to room temperature, and the residue diluted with AcOEt and washed with
brine. The collected organic layers were dried and evaporated to give compound 30 that was
1
directly used in the next step. Yellow oil. (60% yield). H NMR (CDCl
3
) δ: 2.22 (s, 3H, CH
3
); 3.84
2 2
(s, 2H, CH ); 4.06 (s. 2H, CH ); 7.14ꢀ7.19 (m, 3H, Ar); 7.27ꢀ7.33 (m, 4H, Ar) ppm. Anal.
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
16
(C H15NO) C, H, N. % Calcd: 80.98 (C); 6.37 (H); 5.90 (N). % Found: 80.72 (C); 6.51 (H); 5.84
(N).
5-Methoxy-1,3-dimethyl-2-(4-nitrobenzyl)benzene (31). To
dimethylphenylboronic acid (473 mg, 2.63 mmol) in acetone/H
nitrogen flux, ꢀnitrobenzyl bromide (569 mg, 2.63 mmol), K
catalytic amount of PdCl . The resulting mixture was stirred at r.t. for 62 h in a sealed vial. The
crude mixture was evaporated and then diluted with water and extracted with Et O. The organic
a
solution of 4ꢀmethoxyꢀ2,6ꢀ
2
O 1:1 (4 mL) was added, under
p
2 3
CO (1.24 g, 6.58 mmol) and a
2
2
phase was dried over sodium sulfate and concentrated under vacuum. The crude residue was
purified by column chromatography Petroleum Ether /AcOEt (99:1), affording 31. Yellow oil. (49%
1
yield). H NMR (CDCl
3
): δ 2.19 (s, 6H, CH
= 8.8 Hz, Ar); 8.09 (d, 2H,
Calcd: 70.83 (C); 6.32 (H); 5.16 (N). % Found: 71.01 (C); 6.12 (H); 5.00 (N).
3
); 3.80 (s, 3H, OCH ); 6.65 (s, 2H,
3
); 4.08 (s, 2H, CH
2
Ar); 7.16 (d, 2H,
J
J
= 8.8 Hz, Ar) ppm. Anal. (C16H17NO ) C, H, N. %
3
3,5-Dimethyl-4-(4-nitrobenzyl)phenol (32). A solution of 31 (0.39 mmol) in anhydrous DCM
1.5 mL) was cooled to −78 °C and treated dropwise with a solution of BBr in DCM (3.94 mL,
.24 mmol); the resulting solution was stirred at the same temperature for 5 min and at 0 °C for 1 h.
(
3
1
The mixture was then diluted with water and extracted with DCM. Organic phase was dried and
1
evaporated to give the product 32. Pale yellow oil. (64% yield). H NMR (CDCl
3
): δ 2.16 (s, 6H,
CH ); 4.06 (s, 2H, CH ); 6.59 (s, 2H, Ar) 7.16 (d, 2H,
J
=8.8 Hz, Ar); 8.10 (d, 2H, J=8.8 Hz, Ar)
3
2
ppm. Anal. (C15
H
15NO
3
) C, H, N. % Calcd: 70.02 (C); 5.88 (H); 5.44 (N). % Found: 70.00 (C); 5.91
(H); 5.63 (N).
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2-(3,5-Dimethyl-4-(4-nitrobenzyl)phenoxy)acetonitrile (33). To a mixture of cesium carbonate (725
mg, 2.22 mmol) and phenol derivative 32 (0.44 mmol) in 50 mL of DMF was added BrCH CN
2
(0.03 mL, 0.44 mmol). The reaction mixture was stirred for 30 min at r.t., poured into 100mL of
cold HCl 1N and extracted with AcOEt. The organic phase was dried and evaporated to give the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
product 33. White oil. (98% yield). H NMR (CDCl
3
): δ 2.22 (s, 6H, CH3); 4.10 (s, 2H, CH
= 8.8, Ar); 8.11 (d, 2H, J=8.8 Hz, Ar) ppm. Anal.
C H N O ) C, H, N. % Calcd: 68.91 (C); 5.44 (H); 9.45 (N). % Found: 68.65 (C); 5.52 (H); 9.65
2
); 4.77
(s, 2H, CH CN); 6.73 (s, 2H, Ar); 7.15 (d, 2H,
J
2
(
(
1
7
16
2
3
N)
N-(4-(4-(Hydroxymethyl)-2-methylbenzyl)-2-isopropylphenyl)acetamide (35). To a solution of
trifluoro(4ꢀ(hydroxymethyl)ꢀ2ꢀmethylphenyl)borane salt (509 mg; 2.38 mmol) in dioxane/H O 9:1
(4 mL) was added, under nitrogen atmosphere, Nꢀ(4ꢀ(bromomethyl)ꢀ2ꢀisopropylphenyl)acetamide
4 (513 mg; 2.38 mmol), cesium carbonate (2.30 g; 7.1 mmol) and PdCl dppf (34.8 mg; 0.05
2
3
2
mmol). The resulting mixture was stirred at 95°C for 24 h in a sealed vial. The crude mixture was
evaporated and then diluted with water and extracted with DCM. The organic phase was dried over
sodium sulfate and the solvent removed. The crude product was chromatographed on a silica gel
1
column, eluting with Petroleum Ether/AcOEt (9:1). Brown oil. (50% yield). H NMR (CDCl ): δ δ
3
1
.20 (d, 6H,
.94 (s, 2H, CH
= 8.0 Hz, Ar); 7.17 (s, 1H, Ar); 7.47 (d, 1H,
% Calcd: 77.14 (C); 8.09 (H); 4.50 (N). % Found: 77.39 (C); 8.21 (H); 4.64 (N).
J
= 6.8 Hz, CH
3
); 2.19 (s, 3H, CH
3
); 2.26 (s, 3H, CH
= 1.6, 8.0 Hz, Ar), 7.05ꢀ7.09 (m, 2H, Ar); 7.13
= 8.0 Hz) ppm. Anal. (C20 ) C, H, N.
3
CO); 2.96ꢀ3.03 (m, 1H, CH);
3
2
); 4.65 (s, 2H, CH
2
); 6.90 (dd, 1H,
J
(d, 1H,
J
J
H25NO
2
N-(4-(4-Cchloromethyl)-2-methylbenzyl)-2-isopropylphenyl)acetamide (36). Compound 36 was
synthesized from 35 (208 mg, 0.67 mmol) and SOCl (0.49 mL, 6.70 mmol) in CHCl (25 mL)
2
3
following the same procedure described above for the preparation of 21. The crude was used
1
without any further purification. Brown oil. (70% yield). H NMR (CDCl
3
): δ 1.20 (d, 6H,
J
= 6.8
3 3 3 2
Hz, CH ); 2.20 (s, 3H, CH ); 2.26 (s, 3H, CH CO); 2.96ꢀ3.03 (m, 1H, CH); 3.93 (s, 2H, CH
); 4.57
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