Design, synthesis and biological studies of a library of NK1-Receptor Ligands Based on a 5-arylthiosubstituted 2-amino-4,6-diaryl-3-cyano-4H-pyran core: Switch from antagonist to agonist effect by chemical modification

Article abstract of DOI:10.1016/j.ejmech.2017.06.056

A library of 5-arylthiosubstituted 2-amino-4,6-diaryl-3-cyano-4H-pyrans has been synthesized as a new family of non-peptide NK1 receptor ligands by a one-pot cascade process. Their biological effects via interaction with the NK1 receptor were experimentally determined as percentage of inhibition (for antagonists) and percentage of activation (for agonists), compared to the substance P (SP) effect, in IPone assay. A set of these amino compounds was found to inhibit the action of SP, and therefore can be considered as a new family of SP-antagonists. Interestingly, the acylation of the 2-amino position causes a switch from antagonist to agonist activity. The 5-phenylsulfonyl-2-amino derivative 17 showed the highest antagonist activity, while the 5-p-tolylsulfenyl-2-trifluoroacetamide derivative 20R showed the highest agonist effect. As expected, in the case of the 5-sulfinylderivatives, there was an enantiomeric discrimination in favor of one of the two enantiomers, specifically those with (S_S,R_C) configuration. The anticancer activity studies assessed by using human A-549 lung cancer cells and MRC-5 non-malignant lung fibroblasts, revealed a statistically significant selective cytotoxic effect of some of these 2-amino-4H-pyran derivatives toward the lung cancer cells. These studies demonstrated that the newly synthesized 4H-pyran derivatives can be used as a starting point for the synthesis of novel SP-antagonists with higher anticancer activity in the future.

Full text of DOI:10.1016/j.ejmech.2017.06.056

Accepted Manuscript
Design, synthesis and biological studies of a library of NK1-Receptor Ligands Based
on a 5-arylthiosubstituted 2-amino-4,6-diaryl-3-cyano-4H-pyran core: Switch from
antagonist to agonist effect by chemical modification
Rocío Recio, Empar Vegunt-Climent, Bernard Mouillac, Hélène Orcel, Miguel López-
Lázaro, José Manuel Calderón-Montaño, Eleuterio Álvarez, Noureddine Khiar,
Inmaculada Fernández
PII:
S0223-5234(17)30510-X
10.1016/j.ejmech.2017.06.056
EJMECH 9551
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 March 2017
Revised Date: 26 May 2017
Accepted Date: 26 June 2017
Please cite this article as: Rocí. Recio, E. Vegunt-Climent, B. Mouillac, Héè. Orcel, M. López-Lázaro,
José.Manuel. Calderón-Montaño, E. Álvarez, N. Khiar, I. Fernández, Design, synthesis and biological
studies of a library of NK1-Receptor Ligands Based on a 5-arylthiosubstituted 2-amino-4,6-diaryl-3-
cyano-4H-pyran core: Switch from antagonist to agonist effect by chemical modification, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.056.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Studies of a Library of NK1-Receptor LigandsBased on a 5-
arylthiosubstituted 2-amino-4,6-diaryl-3-cyano-4H-pyran Core: Switch from Antagonist to
Agonist Effect by Chemical Modification
[a]
[a]
[b]
[b]
Rocío Recio, EmparVegunt-Climent, Bernard Mouillac, HélèneOrcel, Miguel López-
cd]
[c]
[d]
[d]
Lázaro, José Manuel Calderón-Montaño, Eleuterio Álvarez, NoureddineKhiar* and
[a]
Inmaculada Fernández*
a) Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad de
Sevilla, 41012 Sevilla, Spain.
b) Institut de GénomiqueFonctionnelle (IGF), CNRS, INSERM, Univ. Montpellier, F-34094
Montpellier, France.
c) Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla,
Spain.
d) Instituto de Investigaciones Químicas, C.S.I.C-Universidad de Sevilla, C/Américo Vespucio,
4
9, Isla de la Cartuja, 41092 Sevilla, Spain
KEYWORDS. GPCR,non-peptide NK1 receptor antagonists, non-peptide NK1 receptor
agonists, 5-arylthiosubstituted 2-amino-4,6-diaryl-3-cyano-4H-pyran.
ABSTRACT. A library of 5-arylthiosubstituted 2-amino-4,6-diaryl-3-cyano-4H-pyrans has
been synthesized as a new family of non-peptide NK1 receptor ligands by a one-pot cascade
1

ACCEPTED MANUSCRIPT
process. Their biological effects via interaction with the NK1 receptor were experimentally
determined as percentage of inhibition (for antagonists) and percentage of activation (for
agonists), compared to the substance P (SP) effect, in IPone assay. A set of these amino
compounds was found to inhibit the action of SP, and therefore can be considered as a new
family of SP-antagonists. Interestingly, the acylation of the 2-amino position causes a switch
from antagonist to agonist activity. The 5-phenylsulfonyl-2-amino derivative 17 showed the
highest antagonist activity, while the 5-p-tolylsulfenyl-2-trifluoroacetamide derivative 20R
showed the highest agonist effect. As expected, in the case of the 5-sulfinylderivatives, there
was an enantiomeric discrimination in favor of one of the two enantiomers, specifically those
with (S ,R ) configuration. The anticancer activity studies assessed by using human A-549 lung
S
C
cancer cells and MRC-5 non-malignant lung fibroblasts, revealed a statistically significant
selective cytotoxic effect of some of these 2-amino-4H-pyran derivatives toward the lung cancer
cells. These studies demonstrated that the newly synthesized 4H-pyran derivatives can be used as
a starting point for the synthesis of novel SP-antagonists with higher anticancer activity in the
future.
1
. Introduction
Substance P (SP, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH ), Figure 1, neurokinin A
2
(NKA), and neurokinin B (NKB) are all mammalian tachykinins acting as both neurotransmitters
1
and neuromodulators. These peptides exert their biochemical effects in the central nervous
system (CNS) and in peripheral tissues through their binding to G protein-coupled receptors
2
NK1, NK2, and NK3, respectively. These tachykinins are involved in pain transmission,
inflammation and smooth muscle contraction, vasodilatation, gland secretion, and activation of
2

ACCEPTED MANUSCRIPT
3
the immune system. Moreover, NK1 receptor (NK1R) is present in brain regions involved in the
3
,4
regulation of affective behaviour and the mediation of stress anxiety and depression.
Importantly, it has also been reported that the NK1R is highly over-expressed in a large number
5
6
of aggressive tumours, particularly glioma, astrocytomas and glioblastomas, where the level of
7
expression is correlated with the degree of malignancy. Indeed, the rapid internalization of SP
8
inside the cells upon interaction with NK1R, associated with the overexpression of NK1R in
most cancer cells, allowed the development of an efficient receptor-mediated delivery system of
9
synthetic antibodies into tumour cells. The observation that the release of SP is associated with
several psychopathological processes makes NK1R a therapeutic target of great relevance.
Consequently, NK1R antagonists are potential therapeutic agents for pathologies such as
4
a
4b
migraine, rheumatoid arthritis, asthma, inflammatory bowel disease and the regulation of
4
c,d
central nervous system disorders such as Parkinson's disease, anxiety or depression, emesis, as
1
0
well as cancer treatment.
The first NK1R antagonist designs, which were based on SP structure, afforded peptide
1
1
antagonists with very low affinities and limited metabolic stability. The discovery at the
1
2
beginning of the 1990s of the first non-peptide antagonist, CP-96345, boosted the research in
this area not only in academia but also in industry with almost all important pharmaceutical
companies investing in this field with the aim to identify selective and potent NK1R
1
3
antagonists. More than two decades of immense synthetic and economic efforts have allowed
the discovery of a large number of structurally diverse NK1R antagonists, though none of them
with the desired therapeutic success. Currently, there are only two NK1R antagonist-based drugs
in the market, Aprepitant (Merck) and its water soluble injectable form,
Fosaprepitantdimeglumine (Merck), approved for prevention of chemotherapy-induced nausea
3

ACCEPTED MANUSCRIPT
1
4
and vomiting (Figure 1), and the recently launched Rolapitant (Tesaro) approved for the
prevention of delayed nausea and vomiting associated with initial and repeat courses of
1
5
emetogenic cancer chemotherapy.
HN
NH2
NH
NH2
O
O
NH2
H N
O
S
2
N
O
O
N
O
O
O
H
N
H
H
H H
N
N
N
NH2
N
N
H
N
N
H
H H
H
O
O
O
O
H N
2
O
Substance P (SP)
O
O
O
N P
N
O
N
O
N
OH
NH
F3C
N
F3C
N
O
N
H
O
N
OH
H
NH
OMe
CF3
CF3
F
F
CP-96345
Aprepitant
Fosaprepitant
R3
X
Y
F3C
O
NH
S
CN
R4
NH
O
CF3
R1
R2
O
N
H
Rolapitant
I
Figure 1. Structure of substance P (SP), thenonpeptide NK1R antagonists:CP-96345, Aprepitant,
Fosaprepitant and Rolapitant, and the 5-arylthio-2-amino-4H-pyrans (I) prepared for this study.
4

ACCEPTED MANUSCRIPT
It must be taken into account that the exact structure of the NK1R, which belongs to the
1
6
superfamily of G protein-coupled protein receptors (GPCRs), is still missing in the literature.
As integral membrane proteins, the structural study of GPCRs through X-ray diffraction analysis
1
7
remained elusive for decades. Indeed, even though there are more than 800 GPCRs, only the
1
8
17
structure of 35 of them is known. The first GPCR structure was determined in 2000 but it was
not until 2007 when the other 34 structures started to be elucidated, the crystal structure of the
1
9
ETB receptor being the most recently determined GPCR.
The design and synthesis of new non-peptide molecules with high affinity for NK1Rs, and
preferentially with a different chemical structure from known NK1R antagonists, is an important
area in modern medicinal chemistry. To achieve this goal, in addition to intuition, traditional
drug design approaches are applied, along with combinatorial chemistry and computer aided
design. The considerable number of structurally diverse nonpeptidicNK1R antagonists found,
2
0
allowed the proposition of a preliminary pharmacophore model, Figure 2. The pharmacophore
consists of at least two aromatic rings kept in a fixed orientation by various scaffolds, containing
2
1
not less than one hydrogen-bond acceptor.
5

ACCEPTED MANUSCRIPT
Figure 2. Generalized nonpeptide NK1R antagonist pharmacophore consisting of two (or more)
aromatic rings held together by various scaffolds, which contains at least one hydrogen-bond
acceptor.
The relative disposition of the aromatics in such ligands has also been widely studied and has
revealed the hypothesis for the receptor bound conformation of NK1R antagonists. In most
cases, the fixed orientation between the two aromatic groups aforementioned can be either a
2
2
23
parallel face-to-face, or perpendicular “T” or edge-on “L” arrangement.
On the other hand, it is worth noting that the discovery of non-peptide tachykinin agonists can
also be of therapeutic relevance in the treatment of some diseases, as the angiogenesis-dependent
diseases where endothelial cell proliferation is required to promote vascularization and healing of
2
4
ischemic or damaged tissues. However, in contrast to the number of non-peptide NK1 receptor
antagonists described so far, the agonists for these receptors still appears to be mostly confined to
peptide compounds. Some studies have shown that slight structural variations of certain
antagonists produce marked changes in their activities and, in some cases, it causes the intrinsic
efficacy to shift towards an agonistic activity, accompanied by only minor variations in binding
2
5
affinity. Despite being aware that this behaviour is only confined to certain compounds and the
structural requirements that define an agonist versus an antagonist are poorly understood, it is
worth considering the possibility of interconverting GPCR antagonists to agonist by simple
structural modifications.
Based on these premises, and in connection with our interest in the synthesis of optically pure
2
6
sulfinyl derivatives with biological and pharmacological significance, herein we present our
studies on the design and synthesis of 5-arylthiosubstituted 2-amino-4,6-diaryl-3-cyano-4H-
pyransas new NK1R antagonists, and the quantification of their abilities to inhibit the synthesis
6

ACCEPTED MANUSCRIPT
of inositol 1-phosphate (IP
1
), by Homogeneous Time-Resolved Fluorescence (HTRF) technology
using the IPone test. Additionally, we have evaluated the possibility of employing small
structural changes to interconvert the antagonists to agonists as part of viable GPCR drug
discovery strategy. On the other hand, we have also evaluated the capacity of these 5-arylthio-2-
amino-4,6-diaryl-3-cyano-4H-pyran derivatives to act as antitumoral agents towards cancer cell
lines such as the human lung adenocarcinoma.
2
2
. Results and discussion
.1. Design
To generate the new NK1 antagonist, we utilized a ligand-based design methodology. The basis
of the present study is the observation that despite their structural disparity, CP-96345 and
S C
compound 1(S ,R ) possess significant common features in the solid state, as shown by their X-
ray crystal structures (Figure 3). CP-96345 is a quinuclidinic compound with two stereogenic
carbon atoms, while compound 1(S ,R ) belongs to the 2-amino-4H-pyran family, and encloses a
S
C
stereogenic carbon atom vicinal to a chiral sulfoxide moiety. As illustrated in Figure 3, both
compounds have two parallel aromatic rings, stabilized by a π−π stacking interaction, and at
least one hydrogen bonding acceptor atom. More specifically, compound 1(S
S C
,R ) meets the
requirements of the advanced NK1R antagonists pharmacophore proposed by Klebe’s virtual
2
1,27
NK1R model.
7

ACCEPTED MANUSCRIPT
2
8
Figure 3. A) X-Ray crystal structures of CP-96345 (left), and 5-p-tolylsulfinyl-2-amino-4H-
pyran derivative1(S ,R ) (right). B) Superposed view of CP-96345 and1(S ,R ).
S
C
S
C
2
1
NK1R Klebe’s virtual model, is a ligand-supported homology model based on the known
crystal structure of the rhodopsin receptor and on the structure of CP-96345. The quality of the
model was validated by checking its ability to accommodate additional known NK1 antagonists
from structurally diverse classes, leading to the development of an advanced pharmacophore
model. We have observed that in the modeled NK1R/CP-96345 complex, compound 1(S ,R )
S
C
could replace CP-96345 and maintain all the hydrophobic and hydrogen bond interactions
proposed for the reference antagonist with the amino acids on the active site of the receptor,
namely with glutamine 165, glutamic acid 193, histidine 197, isoleucine 204 and histidine 265
(
Figure 4).
8

ACCEPTED MANUSCRIPT
S C
Figure 4. Modeled complex of the NK1R with CP-96345 (left) and compound 1(S ,R ) (right).
Accordingly, mutation studies have shown that the most relevant interaction of the NK1R /
antagonist complex is the hydrogen bond between the terminal NH of the glutamine (Gln 165)
2
29
and a hydrogen bond acceptor atom of the antagonist. Compound 1(S ,R ) positioned in the
S
C
Klebe’s model has the sulfinyl oxygen at 2.95 Å from Gln165, thus a hydrogen bond could be
established with this amino acid. Another important interaction described for the NK1R/CP-
9
9
6345 complex is the amino-aromatic hydrogen bond between the benzhydryl group of CP-
3
0
6345 with His197 , which itself is kept in place by an aromatic-aromatic interaction with
3
1
S C
Tyr272. In the case of NK1R/1(S ,R ) complex, a similar interaction could be established
between His197 and the pyridine ring either as an amine-aromatic interaction or as a hydrogen
bond with the pyridine nitrogen that lies at 2.17 Å. Finally, an in-depth examination of the
NK1R/1(S ,R ) complex shows that the amino acids Phe247 and Tyr266 are appropriately
S
C
arranged to establish an effective interaction with the ligand1(S ,R ). Particularly relevant could
S
C
be the aromatic-amine interaction between Phe247 and the amino group at the 3 position on the
pyran ring, as well as a hydrogen bond interaction between the aforesaid amino group and the
hydroxyl group of Tyr266 (2.04 Å). In summary, the former qualitative study showed a good
9

ACCEPTED MANUSCRIPT
match between the non-peptidic antagonist CP-96345/NK1R complex and the new ligands-
NK1R interactions, and indicated that the 5-arylsulfinyl-2-amino-4H-pyrans could have NK1R
binding capacity. Therefore, taking into account these observations and the influence of the
2
1
substitution pattern of both phenyl rings in the pharmacophore on binding affinity, we designed
a batch of 5-arylthio-2-amino-4H-pyran compounds in order to evaluate their potential binding
abilities to NK1R. This library was designed to be readily chemically accessible, and the 4H-
pyran derivatives were specifically chosen in order to determine the influence of (i) the nature of
the aromatic rings at C-4 and C-6 of the pyran ring, (ii) the oxidation state of sulphur at C-5 and
the nature of its substituent, and (iii) the nature of the nitrogen function in position 2 (Table 1).
Additionally, in view of the known low binding affinity of the (2R,3S) enantiomer (distomer) of
CP-96345 for the NK1 receptor (IC = 81000 ± 12000 nM) compared to the binding affinity of
5
0
1
2
the (2S,3R) enantiomer (eutomer) (IC = 3.4 ± 0.8 nM), the influence of the stereochemistry of
5
0
the stereogenic centres, at C4 and sulfur in the case of the sulfoxides were also analyzed.
Finally, it must be pointed out that these new drug candidates were designed taking into account
the Lipinski's rules, or rules of 5, so that most of them comply with these rule of 5 guidelines
(see Table 1 in Supporting Information) and are most likely to be cell permeable and orally
3
2
bioavailable.
Table 1.The 5-arylthio-2-amino-4,6-diaryl-3-cyano-4H-pyran derivatives synthesized.
R3
X, Y= :, O
X
Y
1
R = CH NO , H,
3
,
2
2
S
CN
R = 2-Py, 2-Furyl, 3,5-(CF ) C H
2 6 3
3
3
R = 4-CH , H, 4-Cl, 4-F, 4-NO , 4-OMe, 3,5-(CF )
3 2
R4
3
2
4
R2
R1
O
N
R = H, Ac, COCF
3
H
10

ACCEPTED MANUSCRIPT
1
2
3
4
Entry
Comp.
X
O
Y
R
R
R
R
Conf. at C4
1
2
3
4
5
6
7
8
9
rac-1
rac
CH3
CH
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Furyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
rac
R
1(S
S
,R
C
)
3
1(R ,S )
O
CH3
CH3
CH3
CH3
H
S
S
C
rac-2
rac
4-CH3
4-CH3
4-CH3
4-Cl
rac
R
2(S ,R )
O
O
O
S
C
2(R ,S )
O
S
S
C
rac-3
rac
CH
3
rac
R
3(S ,R )
CH3
CH3
CH3
CH3
4-Cl
S
C
3(R ,S )
O
4-Cl
S
S
C
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
rac-4
rac
4-F
rac
R
4(S ,R )
4-F
S
C
4(R
S
,S
C
)
O
CH
3
4-F
S
rac-5
5(S ,R )
rac
CH3
CH3
CH3
CH3
4-NO2
4-NO2
4-NO2
4-OCH3
rac
R
O
O
O
S
C
5(R ,S )
O
O
S
S
C
6(S ,R )
R
S
C
6(R
S
,S
C
)
CH
3
4-OCH
3
S
7(S ,R )
CH3
CH3
3,5-(CF3)2
4-Cl
R
S
C
rac-8
rac
rac
3
,5(CF
3 2
)
2
0
rac-9
rac
rac
CH3
NO2
4-CH3
4-CH3
H
H
rac
rac
C H -
6
3
rac-10
2-Py
2-Py
2-Py
2-Py
2-Py
2
2
1
2
rac-11
rac-12
rac-13
rac-14
rac
rac
rac
rac
NO
2
4-Cl
4-NO2
H
H
H
H
H
rac
rac
rac
rac
2
2
2
3
4
5
NO2
NO2
H
4-OCH
3
11

ACCEPTED MANUSCRIPT
2
2
2
2
3
3
3
3
6
7
8
0
0
1
2
3
rac-15
rac-16
rac
rac
H
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
2-Py
H
H
H
rac
rac
rac
R
H
4-NO2
rac-17
O
O
O
H
4-NO
2
H
18(S ,R )
CH3
CH3
CH3
CH3
4-CH3
4-CH3
4-CH3
4-NO2
H
Ac
S
C
18(R ,S )
O
Ac
S
S
C
rac-19
20R
-COCF3
-COCF3
rac
R
21R
CH
3
-COCF
3
R
2
.2. Synthesis
As indicated in the retrosynthetic scheme (Scheme 1), the 5-arylthio-2-amino-4H-pyran
derivatives can be synthesized in a one pot process, by reaction of the adequate
β-
33
ketothioderivative with different arylidenemalonodinitriles.
Scheme 1. Retrosynthetic scheme of 5-arylthioderivatives of 2-amino-4H-pyrans.
2
4
The series of the 6-pyridyl substituted sulfoxides, 1-7 (X= lone pair, R =Py, R =H, Scheme 1),
was prepared in both, racemic and optically pure forms, in order to determine the influence of the
stereochemistry at sulfur and C-4 on their antagonistic effect. Both enantiomers of the
12

ACCEPTED MANUSCRIPT
sulfinylderivatives 1-7 were obtained in enantiopure pure forms, starting from enantiopure R or S
aryl methyl sulfoxides 35R or 35S, as indicated in Scheme 2. β-ketosulfoxides 22R or 22S were
prepared according to the literature, by condensation of the corresponding R or S methyl
sulfoxides and ethyl 2-pyridylcarboxylate 38 in the presence of lithium diisopropylamide
(Scheme 2).
a
Scheme 2.Synthesis of optically pure 6-pyridyl-5-p-tolylsulfinyl-4H-pyran derivatives (1-7).
a
Reagents and conditions: (a) 1.LDA (2 eq.), 2.ethyl 2-pyridylcarboxylate 38, 85%; (b)
2
arylidenemalonodinitriles 28-34, piperidine (cat.), ether, (80%-quant.); (c) Ac O, DMAP (cat.),
pyridine, 0ºC (70%)
The coupling of the arylidenemalononitriles 28-34 with the enantiopure
β-ketosulfoxides
2
2Sor22R, using equimolar quantities of both reagents in diethyl ether with catalytic piperidine
as mild base, at room temperature, led the desired compounds in good to excellent yields, as an
exclusive enantiomer (Scheme 2). The reaction takes place in an addition-cyclization process,
13

ACCEPTED MANUSCRIPT
where the first addition step is an equilibrium process and it is at this moment when the new
3
4
stereogenic center is formed. The product formed after cyclization precipitates from the reaction
medium and is isolated as a white solid by simple filtration. The nature of the solvent appears to
be very important in this reaction. In general, when other solvents, such as methanol or
methylene chloride were used, yields decreased dramatically. In these conditions some by-
products such as the non-cyclic Michael adduct were obtained, or the reaction was not
completely diastereoselective and, in some cases, the 4H-pyran derivative did not precipitate.
The obtained compounds were in general highly crystalline, and we succeeded in obtaining
single crystals of some of them for the determination of their structures (see Figure 5), which
confirmed the configurational assignment of the new stereogenic carbons.
1
(R ,S )
2(S ,R )
4(S ,R )
S C
S
C
S
C
4
(R ,S )
5(S ,R )
rac-17
S
C
S
C
14

ACCEPTED MANUSCRIPT
S C
Figure 5. ORTEP drawing of optically pure sulfinyl pyran derivatives 1(R ,S ),
2
(S ,R ),4(S ,R ), 4(R ,S ),5(S ,R ) and sulfone rac-17. Thermal ellipsoids are shown at the 50%
S C S C S C S C
probability level. The hydrogen atoms are omitted for clarity.
The X-ray analysis of the sulfinyl compounds collected in Figure 5, shows a 1,3-parallel
arrangement between the aromatic rings of the two stereogenic centers (sulfinyl sulfur and
carbon 4), which can be attributed to the existence of an electronic donor-acceptor interaction
between both aromatic rings (i.e., a π−π stacking interaction). This favored disposition of the
aromatic rings, due to the π−π stacking effects, could explain the formation of a single
compound as the most stable diastereoisomer (thermodynamic control).
Treatment of 2(S
DMAP, using pyridine as base at 0°C, yielded the corresponding sulfinyl acetamides 18(S
and 18(R ,S ), respectively, with good chemical yields and in enantiopure form, after
S
,R
C S C
) and 2(R ,S ) with acetic anhydride in the presence of catalytic amounts of
S C
,R )
S
C
chromatographic purification. The racemic (R ,S /S ,R ) sulfinyl derivatives, (rac-1-rac-16),
S
C
S
C
were prepared in a similar way by reaction of the racemic
β
-ketosulfoxides rac-22-rac-26, with
-ketosulfone 27, obtained by
the Michael acceptors 28-33(Scheme 1). The reaction of the
β
oxidation of the racemic sulfoxiderac-25, with the Michael acceptor 32, under the same mild
experimental conditions, gave the sulfonyl pyran derivative rac-17 (Scheme 3).
a
Scheme 3.Synthesis of the 5-sulfonyl-4H-pyran derivative rac-17 .
15

ACCEPTED MANUSCRIPT
a
Reagents and conditions: (a) m-CPBA, chloroform, 0ºC (96%); (b p-
nitrobenzylidenemalononitrile32, piperidine (cat.), ethanol, (35%)
The influence of the nature of the solvent again proved to be crucial for the synthesis of the
sulfonyl derivative, ethanol being the best solvent in this case, but the yield was only moderate.
Finally, various racemic and enantiopure sulfenylderivatives were prepared by reduction of the
corresponding sulfoxides, by treatment with trifluoroacetic anhydride and sodium iodide in
acetone at -40°C. As expected, the trifluoroacetylation of the amino group took place
concomitantly with the reduction of the sulfinyl group (Scheme 4).
a
Scheme 4.Synthesis of 4-aryl-5-p-tolylsulfenyl-2-trifluoroacetamide derivatives .
a
Reagents and conditions: (a) 1. NaI, acetone, 2. Tf O, -40ºC (60-75%)
2
2
.3. Antagonist and agonist effect evaluation.
16

ACCEPTED MANUSCRIPT
The biological activity of the compounds was checked by their ability to inhibit or activate
3
5
NK1R using the IPone assay which by Homogeneous Time Resolved Fluorescence(HTRF)
technology quantifies the inositol IP accumulated inside the cell. Accumulation of IP is an
1
1
indicator of NK1R activation, so that agonist ligands of NK1R cause an increase in IP levels in
1
the absence of SP while, at the contrary, antagonist ligands produce a decrease in these levels in
the presence of SP. Table 2 summarizes the results obtained with this test for the 2-amino-4H-
pyran library, using SP and CP-96345 as agonist and antagonist controls respectively. As
negative control, cells were also stimulated with DMSO in the absence of the SP and ligands
(Base, Table 2).
Table2. Inactivation of IP accumulation (pmoles) in NK1 receptor transfected CHO cells by 2-
1
-5
-7
amino-4H-pyrans (5x10 M) and CP-96345 (10 M) in the presence of SP (8nM).
Mean (pmoles
b, c
b, d
Mean % b
b, e
Entry Compound
a,b
±SD
pvalue
±SD
IP
1
)
inhibition
1
2
3
4
SP
Base
35.91
6.52 (5.51)
5.29
4.61
.47
--
--
--
--
--
2
<0.0001
<0.0001
(1.24)
CP-96345
rac-1
1.52
85.26
2.44
4
.91
12.57)
.38
8.87)
<0.0001
(0.0178)
<0.0001
(0.0028)
<0.0001
(0.0374)
<0.0001
(0.0082)
<0.0001
(0.0072)
0.0124
24.45
(35.02)
51.14
(41.98)
20.88
(28.81)
32.20
(32.77)
50.79
(36.97)
23.74
(37.45)
15.81
6.84
(20.21)
5.43
(14.26)
1.34
(18.29)
8.79
(12.78)
2.79
(16.16)
7.31
(16.64)
4.36
27.13 (23.34)
(
3
5
6
7
8
9
1(S ,R )
17.55 (20.84)
28.41 (25.57)
24.35 (24.14)
17.67 (22.63)
28.04 (22.46)
S
C
(
0
.85
1(R
S
,S
C
)
(
11.38)
7
.06
rac-2
2(S ,R )
(9.18)
1
.68
S
C
(
(
(
11.60)
5
.25
2(R ,S )
S
C
10.35)
.71
(0.008)
0.2017
(0.2937)
2
1
0
1
rac-3
30.23 (30.9)
28.38
12.14)
(13.95)
(19.52)
1
3(S ,R )
3.46
<0.0001
20.98
5.56
S
C
17

ACCEPTED MANUSCRIPT
(30.03)
(8.13)
1
1
1
1
2
3
4
5
3(R ,S )
33.83
35.01
28.37
31.89
5.78
11.89
0.87
0.149
<0.0001
<0.0001
0.1493
5.80
2.51
9,29
19.1
0.12
6.11
S
C
rac-4
4(S ,R )
20.99
S
C
4(R
S
,S
C
)
4.39
11.20
28.88
(30.04)
49.67
(43.29)
20.63
(18.50)
5
.07
5.06)
.85
6.36)
.65
5.10)
.40
9.10)
.72
3.94)
3.52
<0.0001
(0.0126)
<0.0001
(0.0013)
<0.0001
(0.1863)
0.0002
(0.0058)
<0.0001
(0.6833)
0.0038
3.53
(8.13)
2.57
(10.22)
10.65
(20.22)
2.25
(12.67)
13.53
(6.34)
21.73
(8.13)
1
1
1
1
2
2
6
7
8
9
0
1
rac-5
5(S ,R )
25.54 (25.12)
18.07 (20.37)
28.5 (20.27)
20.15 (23.52)
25.82 (37.50)
25.64 (25.14)
(
(
(
(
(
1
S
C
7
5(R ,S )
S
C
1
43.89
(34.51)
6(S
S
,R
C
)
9
6(R ,S )
28.11(-4.43)
S
C
1
28.61
(30.04)
7(S ,R )
S
C
(10.85)
(0.0289)
2
2
2
2
2
3
4
5
rac-8
rac-9
31.41
31.78
22.22
24.36
3.68
0.1441
0.0034
<0.0001
0.0013
12.52
11.51
38.12
32.18
5.92
5.43
3.63
4.51
3.38
2.26
2.81
rac-10
rac-11
0
.58
<0.0001
(0.0166)
56.08
(35.58)
0.93
(18.97)
2
6
rac-12
15.77 (23.49)
(
11.80)
2
2
7
8
rac-13
rac-14
15.57
31.92
0.68
<0.0001
0.1738
56.63
11.10
1.10
2.69
1.67
6
(
3
.12
7.83)
.43
2.28)
.97
7.75)
.01
8.59)
.63
11.64)
.41
0.0345
(0.013)
19.77
(31.97)
45.89
(33.47)
68.51
(45.52)
47.22
(33.32)
37.08
6.97
(12.59)
5.51
(4.50)
2.46
(9.65)
4.19
(11.96)
6.45
2
3
3
3
3
9
0
1
2
3
rac-15
rac-16
rac-17
28.81 (24.43)
19.43 (23.89)
11.31 (19.56)
18.95 (23.94)
22.60 (28.24)
<0.0001
(0.0175)
<0.0001
(0.0002)
0.0006
(0.0064)
<0.0001
(0.1126)
<0.0001
(0.6112)
(
(
(
1
3
18(S
S
,R
C
)
)
4
18(R
S
,S
C
(
(21.36)
(18.72)
5.50
(4.34)
4
34
35
36
rac-19
20R
22.24 (34.56)
27.36
38.07 (3.77)
23.82
(3.82)
1.72
0.0087
2.77
2
.26
<0.0001
(0.5727)
3.15
(16.86)
21R
22.68 (33.42)
36.84 (6.93)
(
10.49)
18

ACCEPTED MANUSCRIPT
a
b
The IP
1
accumulation was measured as described in the experimental section. Corresponding
-5
c
values for 10 M concentration in brackets. Data are means ±SD of IP
illustrated from a representative experiment performed at least three times. For each compound,
a statistical analysis between control and treated cells was done. Data are means ±SD of %
1
concentration (pmol) and
d
e
inactivation and illustrated from a representative experiment performed at least three times.
As shown in table 2, most of the new ligands act by inhibiting the action of SP, and thus can be
considered as a new family of SP-antagonists. Analysing these results in depth,some conclusions
on the structure-activity relationship can be drawn. Regarding the sulfinyl derivatives, those with
a p-nitrophenyl substituent at sulfur (rac-10, rac-11, rac-12, rac-13) present a slightly better
inhibitory effect of SP than those with phenyl or p-tolyl substituents (table 2, compare entries
2
4vs7, 25vs10, 26vs16and30,and27vs4and29). On the other hand, the substitution of the aromatic
ring at the C4 position has a great influence in activity. According to Klebe’s model (Figure
2
1
4
), this ring will be in proximity to Ile204 and His265 and their interaction was proposed to be
of hydrophobic nature. However, the results obtained with our derivatives can be interpreted
based on a new hydrogen bond interaction with His265 since compounds rac-5, 5(S , R ),rac-
S
C
1
2and rac-16,with a p-nitrophenyl group at this position, have some of the highest antagonist
effects. On the contrary, halogenated derivatives rac-3, 3(R , S ), 3(S , R ),rac-4, 4(R , S ), 4(S
), and rac-11showed a decreased inhibitory activity due to the lack of this proposed
interaction.
S
C
S
C
S
C
S
,
R
C
In relation to the derivatization of the amino group at C-2 in the pyran ring as an amide,
compounds 18(S ,R ) and 18(R ,S )show a similar antagonist activity compared to their
S
C
S
C
unprotected derivatives 2(S ,R ) and 2(R ,S ). Thioethers rac-19, 20R, 21R, which also present
S
C
S
C
an amide at this position, also present comparable activities to their unprotected sulfoxide
derivatives [rac-2, 5(S ,R ) and1(S ,R ) respectively]. This fact could indicate that the
S
C
S
C
19

ACCEPTED MANUSCRIPT
interaction of the NH
2
group with the Tyr 266 residue is not extremely relevant for the antagonist
activity.
When the pyridine ring at C-6 was substituted by other aromatic rings, such as furyl or bis-3,5-
trifluoromethyl)phenyl, (compounds rac-8 and rac-9,respectively), there was a significant
(
decrease on the antagonist activity. This fact can be explained as the consequence of the
suppression of the previously proposed stabilizing interaction between the nitrogen and the
His197 residue for pyridine derivatives. In the case of phenyl derivative rac-9, together with the
3
significant increase in the steric volume due to the presence of both CF groups in meta
positions, the strongly electron-withdrawing effect of these substituents significantly decreases
the electronic density of the aromatic ring, thus disfavouring the proposed amino-aromatic
interaction between the His197 and the aromatic ring. In the case of the furyl derivative rac-8, it
is also important to consider the low solubility of the furan derivative in the cell media, so that it
cannot be used at higher concentrations.
One of the salient features of the results summarized in table 2 is the different antagonist activity
showed by the different enantiomer partners. In this sense, all the 5-arylsulfinyl-2-amino-4H-
pyranswith (S
S C S C
,R ) absolute configuration are more active than their (R ,S )enantiomers. This
result indicates that NK1R exercises a chiral discrimination toward the 5-arylsulfinyl-2-amino-
1
2
4
H-pyran family, as it was previously described for the reference CP-96345.
Finally, it is worth mentioning that the sulfone derivative rac-17 (IC = 200±10
µ
M) was the
5
0
most active antagonist, with a 68.5% of inhibition of production of IP1. This compound
-5
presented a notable inhibitory effect at concentrations below 5x10 M (see table 2). It is
interesting to note that this increased activity of rac-17 may be explained considering the virtual
model of NK1R (Figure 6), where the presence of the sulfonylic oxygen allows the appearance
20

ACCEPTED MANUSCRIPT
of an additional stabilizing interaction by a hydrogen bond with the terminal amino group of the
amide of Gln165, located at a distance of 3.10 Å. Further, the terminal hydroxyl group of Glu193
is at the right distance to form a hydrogen bond with the other oxygen of the sulfone (2.45 Å),
instead of the electrostatic interaction proposed for the other derivatives.
Figure 6. Representation of 5-phenylsulfonyl-2-amino-4H-pyranrac-17in NK1 receptor binding
site.
Another interesting result was found when the IPone test was carried out in the absence of SP, in
order to determine the agonist activity of the compounds. In this sense, taking into account that
structural modifications in close proximity to an oxygen or a nitrogen is likely to affect
2
5
functional activity of a GPCR ligand, we decided to evaluate the possible interconvertion of
our NK1R antagonists into agonists by focusing on modifying the nitrogen at C2. Thus, it was
observed that just by acylating the amine in position 2 of the pyran ring, compounds
1
8(S ,R ),18(R ,S ), rac-19,20R, and 21R were able to exert a change in the basal activity of the
S C S C
cell, activating NK1R (Figure 7). Notably, up to now, only one non-peptide NK1R agonist has
21

ACCEPTED MANUSCRIPT
3
6
been reported and was demonstrated to be of therapeutic relevance in angiogenesis-dependent
diseases.
1
Figure 7. Activation of IP accumulation (pmoles) in NK1 receptor transfected CHO cells by
-5
-7
acylated 2-amino-4H-pyrans (5x10 M, right) and CP-96345 (10 M) in the absence of SP. The
IP accumulation was measured as described in the experimental section. Data are means ±SD of
1
IP concentration (pmol) and illustrated from a representative experiment performed at least
1
three times. For each nonpeptide compound, a statistical analysis between control and treated
cells was done: ** p<0.01 and *** p<0.001.
As depicted in table 2 and Figure7, all these compounds display both antagonistic and agonistic
effects. In the presence of the fully agonist SP, they act as antagonists by blocking access to the
receptor, but on their own they act as agonists. These compounds are, thus, partial agonists.
3
7
Many studies have been published on the benefits of partial agonists, and some current common
3
7c
drugs have been classified as partial agonists, such as buspirone and aripiprazol. Their clinical
use is being increased since they can activate receptors to give a desired submaximal response
22

ACCEPTED MANUSCRIPT
when inadequate amounts of the endogenous ligand are present, or they can reduce the
overstimulation of receptors when excess amounts of the endogenous ligand are
3
8
present. Moreover, the use of partial agonists often avoids the development of adverse effects,
such as desensitization, adaptation, tolerance and dependence, that are usually associated with
3
8
overstimulation of the receptors by full agonists.
The analysis of the structure-agonist activity relationships showed that thioethers (rac-19, 20R
and 21R) were more active than the corresponding sulfoxides [18(S ,R ) and18(R ,S )], with
trifluoroacetamides rac19 and 20R being active even at low concentrations (10 M) (see
supporting information). Compound 20R(EC = 350±22 M) was the best ligand with agonist
S
C
S
C
-5
µ
5
0
activity (Figure7), presenting a 62.34 % of activation (see table 2, SI). As in the case of the most
potent antagonists, this compound also contains a p-nitrophenyl group at position 4 of the pyran
ring, which can be responsible for a higher affinity towards the receptor. This confirms that the
2
interaction of the NH group with Tyr 266 and Phe 247 residues must be relevant for the
antagonist activity (Figure 4).
2
.4. Antitumoral activity evaluation.
Hitherto, all previous studies indicate that NK-1 receptor antagonists selectively inhibit tumour
10a, 39
cells proliferation, as well as exerting antiangiogenic and antimetastatic activities.
The
overexpression of NK-1 receptors in a wide variety of tumors opens the potential of NK-1
receptor antagonist as a specific treatment against cancer cells, decreasing considerably the side-
4
0
effects of the treatment. Moreover, their action has been shown to be dose-dependent, with the
6
b, 40, 41
therapeutic effect being at micromolar range.
Thus, the antitumoral capacity of
compounds 1(S ,R ), 1(R ,S ), 6(S ,R ), 6(R ,S ), rac-12, rac-17, 18(S ,R ) and rac-19 towards
S
C
S
C
S
C
S
C
S
C
23

ACCEPTED MANUSCRIPT
A-549 and MRC-5 cell lines, was evaluated. In both cases MTT assay and anticancerous activity
allowed the determination of the corresponding IC .Again, the activity of CP-96345 antagonist
50
was used as a reference. Moreover, lactic acid was used as a reference to study selective toxicity,
and the well known anti-cancer drug cisplatin as positive control. Cytotoxicity vs concentration
line was drawn for each compound (see SI) allowing, thus, the determination of IC for healthy
5
0
and cancerous cells.
Table 3. IC50 values of different ligands for A549 y MRC-5 cell lines.
a
IC ±SD (
µ
M)
5
0
d
Entry
Compound
CP-96345
Lactic acid
Cisplatin
1(S ,R )
A-549
MRC-5
pvalue
0.410
0.181
0.083
0.184
0.050
0.003
---
1
2
3
4
5
6
7
8
9
46.83 ± 19.88
57.90 ± 18.03
b
b
26.60 ± 3.52
23.93 ± 2.04
11.67 ±7.10
172.22 ± 2.68
195.64 ± 4.02
156.42 ± 4.20
507.84 ± 22.08
115.71 ±85.66
132.38 ± 30.10
424.68 ± 95.35
429.66 ± 59.54
---
S
C
C
1(R
S
,S
)
6(S ,R )
S
C
6(R ,S )
S
C
18(S ,R )
107.68 ± 23.04 136.19 ± 32.43
76.47 ± 12.92 130.17 ± 12.71
0.232
0.013
0.196
0.811
S
C
rac-19
rac-12
rac-17
1
1
0
1
536.18 ± 54.38 824.57 ± 313.89
148.29 ± 25.73 140.36 ± 41.28
a
b
TheIC was calculated as described in the experimental section. Corresponding values for
5
0
c
IC50±SD (
µ
M). Data are means ±SD of IC50 and illustrated from a representative experiment
d
performed at least three times. For each compound, a statistical analysis between A-549 and
MRC-5 treated cells was done.
24

ACCEPTED MANUSCRIPT
In contrast to the behavior of the anticancerous cisplatin, which showed an important cancerous
selectivity (entry 3, table 3), no selectivity was observed for the CP-96345 antagonist used as
reference (entry 1, table 3).
For our derivatives, the highest anticancerous activities were obtained with the amide
derivatives 18(S ,R ) and rac-19 ( entries 8 and 9, table 3), highlighting a certain selectivity for
S
C
thioether rac-19 (entry 9, table 3).
In the case of enantiopure sulfoxides (entries 4-7, table 3), an enantiomeric discrimination was
observed, both in activity and selectivity. Thus, sulfoxide 1(S
its enantiomer 1(R ,S ) showed a significant selective cytotoxicity against lung cancerous cells
entries 4 and 5, table 3). At concentrations of 1(S ,R ) higher than its IC50, the cell death for the
S C
,R ) presented no selectivity, but
S
C
(
S
C
cancerous line reached up to 80% compared to less than 30% for healthy cells (figure 5, SI).
Both enantiomeric sulfoxides, 6(S ,R ) and 6(R ,S ), showed high selectivities against lung
S
C
S
C
cancer cells (entries 6 and 7, table 3). Thus at 320
µ
S C
M concentration of 6(S ,R ), the cell viability
of healthy cells was 70% compared to 7% for the A549 cancerous cell line (figure 6, SI).
Interestingly, its enantiomer 6(R ,S ) showed a high IC value for the cancerous cells (507.84
S
C
50
µ
M, entry 7, table 3), and no IC value could be obtained for the MRC-5 cell line, since at
50
3
10 µM concentration of 6(R ,S ), when the cell media is saturated in this sulfoxide, only 30% of
S C
healthy cells inhibition was observed compared to the 100% inhibition of cancerous cells (figure
, SI). These results showed therefore an excellent cancerous selectivity for this enantiomer
figure 8).
6
(
25

ACCEPTED MANUSCRIPT
MRC-5
A549
Figure 8. A) Inverted microscope images of human A-549 lung cancer cells and MRC-5 non-
S C
malignant lung fibroblasts untreated (control) and incubated with derivative 6(R ,S ) (1mM))
3
. Conclusions
In conclusion, a small library of new NK1R ligands with the 5-arylthiosubstituted 2-amino-4H-
pyran structure has been developed. Sulfonyl derivative rac-17 has proven to inhibit up to 84%
of SP activity in a micromolar range and it represents the lead compound for the design of new
potent antagonists. Moreover, a new family of non-peptide partial agonists has also been
described by the slight modification of the antagonist structure, from the 2-amino to the 2-amide
derivative. This is in concordance with an important role of the two new interactions between the
2
NH group and the Phe247 and Tyr 266 residues, proposed in the case of antagonists. The
26

ACCEPTED MANUSCRIPT
anticancer activity studies assessed by using human A-549 lung cancer cells and MRC-5 non-
malignant lung fibroblasts, revealed a statistically significant high selective cytotoxic effect of
some of these 2-amino-4H-pyran derivatives toward the lung cancer cells. These promising
results could be the adequate starting point for the development of new and more potent NK1
antagonists as anticancerous leads.
4
. Experimental section
4
.1. Chemistry. General Chemistry Methods.
All reactions were conducted under an atmosphere of dry argon using oven-dried glassware and
freshly distilled and dried solvents. TLC was performed on Silica Gel GF254 (Merck) with
detection by charring with phosphomolybdic acid/EtOH. For flash chromatography, silica Gel
(Merck 230-400 mesh) was used. Chromatographic columns were eluted with positive air
pressure and eluents are given as volume to volume ratios (v/v). NMR spectra were recorded
1
1
with a Bruker Avance DRX500 ( H, 500 MHz), and Bruker AMX500 ( H, 500 MHz)
spectrometers. Chemical shifts are reported in ppm, and coupling constants are reported in Hz.
Routine spectra were referenced to the residual proton or carbon signals of the solvent. High
Resolution mass spectra (HRMS) were recorded in “Centro de Investigación, Tecnología e
Innovación de la Universidad de Sevilla” with a Kratos MS-80RFA 241-MC apparatus. Optical
rotations were determined with a Perkin-Elmer 341 polarimeter. Elemental analyses were
measured in a LECO TruSpec® CHNS-932 apparatus. Melting points were measured in
STUART SMP3 apparatus in open end capillary tubes.
4
4
.1.1. Synthesis of 5-arylthioderivatives of 2-amino-3-cyano-4,6-diaryl-4H-pyrans.
.1.1.1. NK-1 antagonists. General Procedure
27

ACCEPTED MANUSCRIPT
To a solution of the corresponding β-keto thioderivative (0.38 mmol, 1 eq.) and the appropriate
Michael acceptor (0.76 mmol, 2 eq.) in ether or methanol (5-20 mL), a catalytic amount of
piperidine is added as base. After stirring overnight, the final product is isolated as a solid by
simple filtration, followed by washing with ether. In general, all the products are obtained in
good purity, and can be recrystallized from ethanol.
rac-2-Amino-3-cyano-4-phenyl-6-(2-pyridyl)-5-p-tolylsulfinyl-4H-pyran,rac-1
Prepared following the general procedure from the racemic (p-tolylsulfinyl)methyl 2-pyridyl
ketone 22S (98.5 mg, 0.38 mmol) and benzylidenemalononitrile 28 (117.17 mg, 0.76 mmol) in
ether (5mL). The product rac-1 is obtained as a white solid (144.72 mg, 0.35 mmol) in 92%
1
yield. M.p.: 227-228 ºC. H NMR (500 MHz, DMSO-d )
δ
: 8.72 (d, J = 4.6 Hz, 1H), 8.11 (td, J
6
=
1.6 and 7.6 Hz, 1H), 8.07 (d, J = 7.8 Hz,1H), 7.60 (t, J = 6.0 Hz,1H), 7.45 (d, J = 8.2 Hz,2H),
7
.25 (s, 2H), 6.93 (m, 3H), 6.87 (d, J = 8.1 Hz, 2H), 6.76 (d, J = 6.3 Hz, 2H), 4.66 (s,1H), 2.13
1
3
(
s,3H) ppm. C NMR (125.7 MHz, DMSO-d
6
) δ: 160.2, 149.5, 148.7, 148.1, 143.2, 139.5,
1
39.4, 137.7, 128.6, 128.0, 126.8, 126.1, 125.6, 125.2, 123.6, 122.7, 119.6, 59.2, 32.2, 20.6 ppm.
+
HRMS Calc. for C H N O S: [M+H] 414.1276, found 414.1253 (-5.6 ppm).Anal. Calc. for
2
4
20
3
2
C H N O S:C, 69.71; H, 4.63; N, 10.16; S, 7.75. Found. C, 68.75; H, 4.63; N, 9.99; S, 7.88.
24
19
3
2
(
R ,S )-2-Amino-3-cyano-4-phenyl-6-(2-pyridyl)-5-p-tolylsulfinyl-4H-pyran,1(R ,S )
S
C
S
C
Prepared following the general procedure from the (S)-(p-tolylsulfinyl)methyl 2-pyridyl ketone
2S (98.5 mg, 0.38 mmol) and benzylidenemalononitrile 28 (117.2 mg, 0.76 mmol) in ether
5mL). The product 1(R ,S ) is obtained as a white solid (146.13 mg, 0.35 mmol) in 93% yield.
The spectroscopic data are similar to those of the rac-1. M.p.: 213-215 ºC. HRMS Calc. for
2
(
S
C
+
20
C H N O S: [M+H] 414.1276, found 414.1272 (-1.0 ppm).[
α
]
D: +198.6 (c 0.37, chloroform)
24
20
3
2
(
S ,R )-2-Amino-3-cyano-4-phenyl-6-(2-pyridyl)-5-p-tolylsulfinyl-4H-pyran, (S ,R )
1
S
C
S
C
28

ACCEPTED MANUSCRIPT
Prepared following the general procedure from the (R)-(p-tolylsulfinyl)methyl 2-pyridyl ketone
2
2R (98.5 mg, 0.38 mmol) and benzylidenemalononitrile 28 (117.2 mg, 0.76 mmol) in ether
(
5mL). The product 1(S ,R ) is obtained as a white solid (141.41 mg, 0.34 mmol) in 90% yield.
S
C
The spectroscopic data are similar to those of the 1(R ,S ) enantiomer. M.p.: 214-215 ºC. HRMS
S
C
+
20
Calc. for C H N O SNa: [M+Na] 436.1096, found 436.1096 (0.1 ppm).[
α]
D: -203.2 (c 0.37,
2
4
19
3
2
chloroform)
rac-2-Amino-3-cyano-4-p-tolyl-6-(2-pyridyl)-5-p-tolylsulfinyl-4H-pyran,rac-2.
Prepared following the general procedure from the racemic (p-tolylsulfinyl)methyl 2-pyridyl
ketone rac-22 (98.5 mg, 0.38 mmol) and p-methylbenzylidenemalononitrile 29 (127.83 mg, 0.76
mmol) in ether (8 mL). The product rac-2 is obtained as a yellow solid (161 mg, 0.38 mmol) in
1
quantitative yield. M.p.: 218-219 ºC. H NMR (500MHz, DMSO-d )
δ 8,69 (d, J = 4.7 Hz, 1H),
6
8
1
6
.09 (td, J = 1.6 and 7.7 Hz, 1H), 8.04 (d, J = 7.9 Hz, 1H), 7.58 (ddd, J = 1.3, 4.9 and 7.3 Hz,
H), 7.41 (d, J = 8.2 Hz, 2H), 7.20 (s, 2H), 6.86 (d, J = 8.1 Hz, 2H), 6.70 (d, J = 7.8 Hz, 2H),
1
3
.61 (d, J = 8.0 Hz, 2H), 4.61 (s, 1H), 2.15 (s, 3H), 2.10 (s, 3H) ppm. C NMR (125.7 MHz,
: 160.0, 149.1, 148.7, 148.2, 140.2, 139.6, 139.3, 137.6, 135.4, 128.5, 128.4, 126.8,
25.5, 125.2, 123.7, 122.6, 119.6, 72.1, 59.1, 48.7, 31.9, 26.8, 20.6, 20.5 ppm. HRMS Calc. for
6
DMSO-d ) δ
1
+
C H N O S: [M] 427.1354, found 427.1381 (6.2 ppm).Anal. Calc. for C H N O S: C, 70.24;
25
21
3
2
25 21
3
2
H, 4.95; N, 9.83; S, 7.50. Found. C, 69.78; H, 4.93; N, 9.71; S, 8.02
R ,S )-2-Amino-3-cyano-4-p-tolyl-6-(2-pyridyl)-5-p-tolylsulfinyl-4H-pyran,2(R ,S ).
S C
(
S
C
Prepared following the general procedure from the (S)-(p-tolylsulfinyl)methyl 2-pyridyl ketone
2S (98.5 mg, 0.38 mmol) and p-methylbenzylidenemalononitrile 29 (127.83 mg, 0.76 mmol) in
2
ether (8 mL). The product 1 is obtained as a yellow solid (160 mg, 0.38 mmol) in quantitative
yield. The spectroscopic data are similar to those of the rac-2. M.p.: 186-187 ºC. HRMS Calc.
29