Novel Oxazolidinone-Based Peroxisome Proliferator Activated Receptor Agonists: Molecular Modeling, Synthesis, and Biological Evaluation

Article abstract of DOI:10.1021/acs.jmedchem.5b00849

(Figure Presented). A series of new peroxisome proliferator activated receptors (PPARs) chiral ligands have been designed following the accepted three-module structure comprising a polar head, linker, and hydrophobic tail. The majority of the ligands inco

Full text of DOI:10.1021/acs.jmedchem.5b00849

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Novel oxazolidinone based PPAR agonists:
molecular modeling, synthesis and biological
evaluation
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a
b,c
a
b,c
Fresno, N.; Macías-González, M.; * Torres-Zaguirre, A.; Romero-Cuevas, M.; Sanz-
a
a
b,c
b,c
a
Camacho, P.; Elguero, J.; Pavón, J.; Rodríguez de Fonseca, F.; Goya, P.; Pérez-
a,d
Fernández, R. *
a
Instituto de Química Médica, IQMꢀCSIC, Juan de la Cierva, 3, Eꢀ28006 Madrid, Spain
b
CIBER Fisiopatología de la Obesidad y Nutrición. CB06/03. Instituto de Salud Carlos III,
Madrid, Spain.
c
Unidad Gestión Clínica Endocrinología y Nutrición, Instituto de Investigación Biomédica de
Málaga (IBIMA), Universidad de Málaga. Hospitales Universitarios Regional y Virgen de la
Victoria de Málaga, Universidad de Málaga, Spain.
d
Centro de Investigaciones Biológicas, CIBꢀCSIC, Ramiro de Maeztu 9, Eꢀ28040 Madrid, Spain.
KEYWORDS
oxazolidinone, PPAR agonists, molecular modeling, metabolic syndrome, diabetes
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ABSTRACT
A series of new peroxisome proliferator activated receptors (PPAR) chiral ligands have been
designed following the accepted threeꢀmodule structure comprising polar head, linker and
hydrophobic tail. The majority of the ligands incorporate the oxazolidinone moiety as a novel
polar head, and the nature of the hydrophobic tail has also been varied. Docking studies using the
crystal structure of an agonist bound to the ligand binding domain (LBD) of the PPARα
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receptor have been performed as a tool for their design. Suitable synthetic procedures have been
developed and compounds with different stereochemistry have been prepared. Evaluation of
basal and ligand induced activity proved that several compounds showed agonist activity at the
PPARα receptor, thus validating the oxazolidinone template for PPAR activity. In addition, two
compounds 2 and 4 showed dual PPARα / PPARγ agonism, and interesting food intake
reduction in rats.
1. INTRODUCTION
The peroxisome proliferator activated receptors PPARs are nuclear receptors activated by the
binding of small lipophilic ligands either natural or synthetic. They can induce or repress genes
modulating different biochemical processes such as adipogenesis, lipid and glucose metabolism,
energy balance, and inflammation among others. Considered an important target in medicinal
chemistry, significant efforts have been devoted to search new ligands capable of treating
chronic disorders such as high levels of triglycerides, diabetes and metabolic syndrome and
chronic inflammatory bowel disease.
There are three known subtypes of PPAR receptors: alpha (α), gamma (γ) and beta (β)/delta
(δ). Whereas PPARα is expressed in tissues with a high rate of fatty acid catabolism and
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modulates lipid metabolism and inflammation, PPARγ is predominant in adipose tissue and
regulates glucose and lipid metabolism. Finally, the β/δ type is the most ubiquitous and has been
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less investigated.
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,3
Despite intense research in developing PPAR ligands, there are only few marketed drugs
targeting PPARα and PPARγ and several promising candidates have failed in clinical trials
(
Fig.1). The α agonists, represented by fibrates (e.g. Fenofibrate and Gemfibrozil) are clinically
used for the treatment of dyslipidemia. The thiazolidindiones (TZDs) or glitazones (e.g.
Pioglitazone) are considered as specific ligands for the γ receptor. They are being used for
treating hyperglycemia in patients with type 2 diabetes. Due to the considerable structural
homology between these PPAR isoforms, there are other kinds of dual ligands that bind to two or
more isoforms. The most studied compounds under this description are the glitazars from which
the Saroglitazar (S enantiomer) has been recently approved and marketed under the trade name
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Lipaglyn as novel antidiabetic agent. Nowadays, there is a growing interest in developing dual
PPARα and PPARγ agonists, since they may have fewer secondary effects compared to selective
α and γ ligands.
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Fig. 1. PPARα and PPARγ marketed drugs.
The LBD (ligand binding domain) is responsible for ligand specificity and PPAR activation. The
core of the Yꢀshaped LBD consists of a set of twelve antiparallel alpha helix (helix 1 to 12) and
threeꢀstranded antiparallel betaꢀsheet. The activation functionꢀ2 (AFꢀ2) domain located at the end
of Cꢀterminal helix 12 plays a critical role after ligand binding reducing the LBD flexibility and
providing an appropriate interface for the cofactors recruitment. A large number of crystal
structures of the PPARꢀLBD with different ligands have been reported in literature enlightening
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a wellꢀdefined view of the agonists binding mode. In some cases chirality is a crucial point
determining the PPAR binding mode and synthetic strategies to deal with these stereoselectivity
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issues have been recently reviewed. Laghezza et al. reported the 2ꢀ(aryloxy)ꢀ3ꢀphenylpropanoic
acids family with PPARα/γ dual agonist activity. The (S) and (R) enantiomers of the isopropyl
derivative were crystallized bound to the LBD of the receptor. The (S) stereoisomer behaved as a
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full and partial agonist of PPARα and γ respectively. In 2002, αꢀsubstituted phenylpropanoic
acidꢀtype PPAR agonists were reported to bind to the LBD in different conformations indicating
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multiple binding points inside the binding pocket. New PPARγ scaffolds have also been
reported following a virtual screening approach to identify PPAR chemotypes, however further
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biological studies need to be performed.
The oxazolidinone is a fiveꢀmembered heterocyclic ring with several applications as
1
0
organocatalyst and chiral auxiliary (e.g. asymmetric aldol reactions). It is considered a valuable
11, 12, 13
structural motif in medicinal chemistry.
Oxazolidinones as chiral auxiliaries have been
used by Miyachi and coworkers for the asymmetric synthesis of chiral αꢀacid derivatives as
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14, 15, 16
PPARγ and α/γ ligands.
On the other hand, in vitro metabolism studies of a PPARγ ligand
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structurally similar to the oxazolidinone ring was reported by Agrawal et al.
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In this paper we describe 14 new PPAR chiral ligands mainly based on an oxazolidinone
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motif following the threeꢀmodule structure described by Pirat et al. A structureꢀactivity
relationship study was conducted by varying the hydrophobic tail and polar head. Moreover,
compounds with different stereochemistry were synthesized in order to understand the PPAR
binding mode. Docking studies were used as a tool to design these ligands. Some of these ligands
showed interesting in vitro and in vivo pharmacological activities.
2. RESULTS AND DISCUSSION
According to the accepted model, the ligands bind through three chemical features: polar head,
linker region and hydrophobic tail (Fig. 2).
In our case, the choice of the hydrophobic tail group was determined by the docking studies
going from alkyl chains (alicyclic and cyclic) to aromatic rings trying to occupy the large
hydrophobic cavity of the binding site. Besides, the hydrophobic aromatic linker with the
appropriate length was introduced. Finally, a polar head such as the oxazolidinone ring or the
carboxylic acid should enable the hydrogen bonds in front of the AFꢀ2 helix to stabilize its
conformation. The chirality in our ligands has been introduced either on the oxazolidinone ring
(
substituent in position 4, compounds 1ꢀ10) or in the optically active (S)ꢀαꢀacid (compounds 13,
14) or in both of them (11, 12).
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Fig. 2. PPAR chiral ligands general scheme and formulae.
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2
.1. Docking studies
Docking studies of two known PPAR agonists (GW409544, WYꢀ1463) and compounds 2, 3,
, 7, 8, 9, 10, 13, 14 were carried out by means of AutoDock v4 package. Table 1 summarizes
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the most stable conformations, the percentage of hydrogenꢀbonds with the hydroxyl group of
Try464 (PPARα) and Tyr473 (PPARγ) of the 100 best docked conformations, the main polar
interactions inside the LBD and the docking energy for the selected pose of each compound.
Table 1. Docking of compounds in the LBD of PPARα and γ.
No.
Docking
energy
Tyr 464
HB (%)
Polar
Docking
Tyr473
HB (%)
Polar
interactions energy
interactions
(kcal/mol
(PPARα)
(kcal/mol
(PPARγ)
)
)
GW409544
ꢀ18.25
31
Ser280
Tyr314
Tyr464
His440
ꢀ14.45
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Ser289
His323
Tyr473
His449
WYꢀ14643
ꢀ8.99
63
Ser280
Tyr314
Tyr464
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His440
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Tyr314
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His449
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ꢀ11.93
ꢀ11.54
ꢀ12.24
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Tyr464
His440
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Tyr464
His440
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Tyr473
His449
Tyr314
Tyrꢀ64
His440
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ꢀ13.33
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Ser280
Tyr464
His440
ꢀ13.36
Ser289
His323
Tyr473
9
ꢀ10.85
2
Tyr464
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His440
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ꢀ11.24
ꢀ11.61
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Tyr464
His440
ꢀ11.21
4
7
Tyr473
His449
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Ser280
Tyr314
Tyr464
His440
ꢀ11.08
Ser289
His323
Tyr473
His449
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ꢀ9.99
22
Ser280
Tyr314
Tyr464
His440
ꢀ9.76
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Ser289
His323
Tyr473
His449
The validation of the in silico model was performed comparing the X ray data of commercial
compound GW409544 bound to the ligand domain of PPARα (PDB:1K71) and to PPARγ
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(PDB:1K74).
According to the crystal structure, the aromatic rings of GW409544 established several
interactions with nonꢀpolar amino acids of the PPARα ꢀ LBD and PPARγ ꢀ LBD. Additionally,
GW409544 adopted a conformation within the receptor that allowed the carboxylate group to
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form hydrogen bonds with Ser280, Tyr314, His440 and Tyr464 of PPARα and Ser289, His323,
His449 and Tyr473 of PPARγ. These polar interactions and specifically the formation of
hydrogen bonds with Tyr464 (PPARα) and Tyr473 (PPARγ) typically trigger a conformational
change, notably in the activation functionꢀ2 (AFꢀ2) helix, which aids in the recruitment of coꢀ
regulatory factors to regulate gene transcription.
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For the PPARα ligand binding domain, the AutoDock suite successfully reproduced the
binding mode for GW409544, showing a root mean square deviation (RMSD) of one of the most
stable binding conformations (pose #55) of 0.7 Å in comparison with the experimental geometry,
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and the same hydrogen bond patterns (Fig. 3A). An additional reference, the ligand WYꢀ1463,
which is the control ligand used in the in vivo tests, has also been included in the docking studies
(Table 1).
The most stable docking solutions for the different ligands were analyzed, and the results
suggest the existence of different potential binding modes of each compound onto the structure
of the PPARαꢀLBD. The hydrophobic tails of each compound were assigned to the same
hydrophobic region occupied by the synthetic agonist GW409544 in the crystal structure of the
1
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PPARαꢀLBD (Fig. 3). For these solutions, the oxazolidinone (CO) and carboxylic acid polar
groups were close to the hydroxyl group of the lateral chain of Tyr464, which donated a
hydrogen bond. The binding modes were stabilized by the hydrogen bond network with: Tyr314,
Tyr464 and His440 (2, 7), Tyr 464 and His440 (3, 4, 9 and 10), Ser280, Tyr464 and His440 (8)
and with the four residues just compounds 13 and 14. The chirality induced by the substituent
attached to the oxazolidinone ring in position 4 is responsible for the orientation of the CO group
towards the polar residues. For instance, enantiomers 2 and 8 showed some differences in the
percentage of hydrogen bond with the hydroxyl group of Tyr464 and in the polar residues
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interactions. However, at this point, in the absence of crystallization data, these results must be
considered as a theoretical approximation.
A)
B)
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C)
D)
Tyr
64
4
His
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4
Ser
280
Fig. 3. Docking representation of best location/orientation binding modes. Docking
representation of best location/orientation binding modes of GW409544 pose #55 (A), 2 pose
#62 (B), 14 pose #3 (C) and 8 pose #4 (D) in balls and sticks colored by atom type. The coꢀ
crystallized conformation of GW409544 is shown in green and the protein backbone by ribbons
(
blue). The most important polar interactions (dashed green lines) and residues involved are
shown (Ser280, Tyr314, His440 and Tyr464). All agonist compounds form a hydrogen bond
with Tyr464.
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In the case of PPARγꢀLBD, docking studies were carried out with 2, 4, 8, 10, 13 and 14. The
hydrophobic tails of these compounds were assigned to the same hydrophobic region occupied
by the synthetic agonist GW409544 in the crystal structure of the PPARγꢀLBD, whereas the
polar heads of the compounds were involved in a hydrogen bond network with the following
residues (i.e., Ser289, His323, His449 and Tyr473). Docking representations of the structures of
compounds 2 and 4 onto the PPARγꢀLBD are represented in Fig. 4.
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A)
B)
C)
His323
Tyr473
Tyr 473
His323
Tyr 473
His449
His323
Ser289
His449
His449
Ser289
Ser289
Fig. 4. Docking representation of best location/orientation binding modes to PPARγꢀLBD of
GW409544 pose #3 (A), 2 pose #4 (B), 4 pose #31 (C) in balls and sticks colored by atom type.
The coꢀcrystallized conformation of GW409544 is shown in green and the protein backbone by
ribbons (blue). The most important polar interactions (dashed green lines) and residues involved
are shown (Ser289, His323, Tyr473, and His449). All agonist compounds form a hydrogen bond
with Tyr473.
2.2. Chemistry
For the preparation of compounds 1ꢀ10 we followed the synthetic strategy depicted in Scheme
1.
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Reagents: i) Boc O, Et N, dioxane/H O (2:1), r.t., 24h (89%); ii) coupling agent, HOBt, Et N,
2
3
2
3
t
RꢀNH , solvent, r.t., 12h (31ꢀ84%); iii) CH Cl /TFA (1:1), r.t., 12h (80ꢀ92%); iv) BuOK,
2
2
2
Methyl 3ꢀ(bromomethyl)benzoate, THF anh., r.t., 7h (62%); v) Al(CH ) , THF, mw, 125ºC,
3
3
2
0min (40ꢀ50%); vi) Methyl 3ꢀ(bromomethyl)benzoate, Cs CO , THF anh./CH CN anh. (1:1),
2 3 3
r.t. 6h (66%); vii) 17, Al(CH ) , THF, mw, 125ºC, 20min (80ꢀ89%).
3 3
Scheme 1. Synthesis of compounds 1ꢀ10.
The key intermediate 17 was synthesized in three steps. Starting from commercially available
ꢀaminobenzoic acid, protection of the amine with Boc anhydride provided 15. Amide bond
4
formation between the carboxylic group of 15 with several amines (Fig. 1) using EDCI or
PyBOP gave intermediate 16 in moderate yields. The tertꢀbutyl carbamate 16 was cleaved under
anhydrous acidic conditions giving 17 in good yields. The oxazolidinone motif was incorporated
after a twoꢀstep synthesis starting from the commercially available (R)ꢀ4ꢀbenzylꢀ3ꢀpropionylꢀ2ꢀ
oxazolidinone in the case of compounds (1ꢀ7) and from the (S)ꢀ4ꢀbenzyl oxazolidinone for
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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5
5
5
5
5
6
t
compounds (8ꢀ10). In the first case, the use of BuOK at room temperature triggered a crossꢀ
alkylation reaction in which the oxazolidinone anion attacked the benzyl bromide derivative as
2
1
we have recently reported, providing the oxazolidinone derivative 18 in moderate yields. In the
second case, the alkyl halide underwent a nucleophilic substitution under basic conditions giving
the (S)ꢀ4ꢀbenzyl oxazolidinone derivative 19. The final step to obtain compounds (1ꢀ10)
involved a microwaveꢀassisted amide bond formation between an ester (18) or (19) and various
amine derivatives (17) mediated by a trimethyl aluminium complex.
0
1
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
The synthesis of compounds 11ꢀ14 is summarized in Scheme 2. For these compounds, two
different hydrophobic tails were selected: the pꢀtertꢀbutylbenzyl and the butyl one. Intermediate
2
1 was synthesized in two steps starting from commercially available methyl pꢀtertꢀ
butylphenylacetate or methyl hexanoate, an amide bond formation with the two different amines
followed by the acid conversion to acyl chloride gave 21 in moderate yield.
The second fragment of this synthesis (compound 23) is obtained in two steps. First, an Evans'
asymmetric alkylation in which the (R)ꢀ4ꢀbenzylꢀ3ꢀpropionylꢀ2ꢀoxazolidinone was treated with
3
ꢀnitrobenzyl bromide under Evans' protocol (NaHMDS at ꢀ78 ºC) provided 22. Secondly, a
Palladiumꢀcatalyzed reduction of the aromatic nitro group to amine gave 23 in good yield.
Compounds 11 and 12 resulted from the amide bond formation between the acyl chloride 21
and the amine derivative 23. Then, a hydrogenolysis of the oxazolidinone ring using LiOH/H O2
2
gave the chiral acid derivatives in alpha position, compounds 13 and 14.
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Reagents: i) 4ꢀaminobenzoic acid, Al(CH ) , THF, mw, 125ºC, 20min (65ꢀ70%); ii) SOCl ,
3 3
2
THF, 80ºC, 6h; iii) NaHMDS, THF anh., 3ꢀnitrobenzyl bromide, ꢀ78ºC, 6h (70%); iv) H /PdꢀC,
2
THF/EtOAc (1:2), 21 psi, r.t., 12h (90%); v) 21, THF, r.t., 12h, (34ꢀ68%); vi) LiOH, H O , 0ºCꢀ
2
2
r.t., 6h, (62ꢀ70%).
Scheme 2. Synthesis of compounds 11ꢀ14.
2.3. Pharmacology
Luciferase reporter gene assays were performed with extracts from MCFꢀ7 cells that were
transiently transfected with a reporter construct containing four copies of the human CPTI DR1ꢀ
type RE (for PPARs) and the indicated expression vectors for PPARα, RXRα, NCoR (CoR).
Cells were treated for 16 h with 10ꢁM of different compounds: DMSO (solvent), the reference
PPARα agonist GW7647, the endogenous PPARα agonist OEA, the reference PPARγ
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Rosiglitazone, and compounds 1 to 14. MD Simulation of luciferase activity was normalized to
the basal activity of PPARαꢀRXRα−SRC1 in the presence of the solvent (DMSO). Details on the
2
2,23,24,25,26
methodology have been published previously.
0
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0
Compounds 2, 4, 5, 6, 8, 11, 12, 13 and 14 showed agonist activity at the PPARα receptor.
Among them, compounds 2 and 4 displayed moderate agonism at the PPARγ receptor so they
may be considered as interesting dual PPARα/γ agonists. These compounds also showed key
important interactions in the docking studies with both PPAR isoforms. Flexibility in the
hydrophobic tail is desired (4) over rigid hydrogenated rings (7) in accordance with the
performed docking studies. The (R) stereochemistry at the oxazolidinone ring is preferred over
the (S) one, and the oxazolidinone ring choice as polar head with a shorter linker resulted to be
slightly better than the existing one in compounds 11 and 12. On the other hand, a difference of
one order of magnitude in activity was observed between the carboxylate polar head of 14 and
the oxazolidinone ring in 12 with the same hydrophobic tail and linker. This fact correlates
perfectly with the docking studies where the (S)ꢀα acid derivative showed interactions with all
the key residues at the LBD and a high HB percentage with Tyr464. As we can observe from the
in vitro and docking results, interactions with the polar residues are important but in some cases
flexibility and length of the ligand compensate the loss of HB with the polar residues in the LBD.
This could explain why smaller ligands like (R)ꢀoxazolidinone 2 show similar in vitro results to
(S)ꢀα acid 13.
Considering the EC obtained at PPARα receptors, the data suggest an intermediate position
5
0
in between fibrates and the natural ligand OEA. However, the efficacy at the receptor is not
strictly related to in vivo efficacy. The most successful PPARα agonists used in human therapy,
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the fibrates, (i.e. fenofibrate, gemfibrozil) have reported EC50 at the PPARα receptor in the
2
7
micromolar range, more than 2ꢀfold than those of compounds 2 and 4.
Based on this assumption, for the in vivo studies we selected compounds 2 and 4 since they
behaved as dual agonists with interesting PPARα EC values and moderate affinity for PPARγ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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50
2
0,28
isoform.
In vivo pharmacology was tested using the model of feeding behavior in food
deprived animals, that provides a good screening test for PPARα agonists with efficacy in
2
9,30
animal models of obesity and metabolic disorders.
plasma lipids will be analyzed in future studies.
Long termꢀeffects such as reductions in
Table 2. Pharmacological properties of compounds 1ꢀ14.
No.
PPARα activation
PPARγ activation
EC (nM)
EC (nM)
5
0
50
OEA
152+31
6.5+1
ꢀ
ꢀ
GW7647
ꢀ
Rosiglitazone
87.0+11
ꢀ4
ꢀ4
1
2
3
4
5
6
7
8
9
>10 M
>10 M
670+157
1298+261
ꢀ4
ꢀ4
>10 M
>10 M
821+139
6623+1232
1978+213
1622+388
ꢀ4
>10 M
ꢀ
ꢀ4
ꢀ4
>10 M
>10 M
756+125
ꢀ
ꢀ
ꢀ
ꢀ4
>10 M
ꢀ4
10
>10 M
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1
1
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4
966+233
1922+408
866+188
981+476
ꢀ
ꢀ
ꢀ
ꢀ
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EC50 values calculated in the presence of the different compounds by GraphPad Prism 4.
Results are the mean ± SEM. of 3 experiments.
In vivo food intake studies
The acute administration of compound 2 induced a doseꢀdependant reduction of food intake in
adult obese male rats that had been deprived of food for 24 h. Compound 2 at the effective dose
of 3.0 mg/kg is able to reduce food intake around 50% being active up to 120 min. This effect
disappears after 240 min (Fig. 5).
Fig 5. Feeding inhibition induced by acute administration of compound 2. Time course of the
inhibition of food intake induced by 2 at doses of 0.3, 1.0 and 3.0 mg/kg. The compound is
active up to 120 min after administration (0'ꢀ60', P<0.05; 60'ꢀ120', P<0.05). Abscissa represents
time (min) while the ordinate axis represents the cumulative food intake (mg/kg).
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The acute administration of compound 4 induced a doseꢀdependant reduction of food intake at
the effective doses of 1.0 and 3.0 mg/kg (Fig. 6). This compound remained active 240 min after
dosing and surprisingly it continued exerting its effect 24 h later, an effect only observed before
10
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31
in nanoformulated oral oleoylethanolamide. Therefore, compound 4 is an interesting candidate
for chronic administration studies (Fig. 7).
Fig 6. Feeding inhibition induced by acute administration of compound 4. Time course of the
inhibition of food intake induced by 4 at doses of 0.3, 1.0 and 3.0 mg/kg. The compound is
active up to 240 min after administration at 1.0 mg/kg (0'ꢀ240', P<0.01) and at 3.0 mg/kg (0'ꢀ30',
P<0.0001; 30'ꢀ120', P<0.001; 120'ꢀ140', P<0.01). Abscissa represents time (min) while the
ordinate axis represents the cumulative food intake (mg/kg).
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0
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0
Vehicle
4
4
(0.3 mg/kg)
(1.0 mg/kg)
4 (3.0 mg/kg)
*
**
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Fig 7. Feeding inhibition results 24 h after compound 4 administration. The compound 4 at 1.0
mg/kg (P<0,01) remained active over 24 h after dosing in a doseꢀdependent manner. Abscissa
represents time (24 h) while the ordinate axis represents the cumulative food intake (mg/kg).
In summary, based on molecular modeling and docking studies we have prepared novel PPAR
ligands incorporating an oxazolidinone ring. Pharmacological assays, both in vitro and in vivo of
these chiral PPAR ligands have shown that the oxazolidinone motif is an interesting structural
feature for PPAR activity. The easy way to synthetically reach these compounds and to perform
structural modifications make them interesting leads for further development. Two of them
(
compounds 2 and 4) can be considered as dual wellꢀbalanced PPARα/γ ligands. Furthermore,
they have shown interesting in vivo properties that make them attractive candidates for further
research in the treatment of metabolic syndrome.
3
. EXPERIMENTAL SECTION
Chemistry. General methods
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All chemicals were purchased from commercial suppliers and used without further
purification. TLC: precoated silicaꢀgel 60 254 plates (Merck), detection by UV light (254 nm).
Flashꢀcolumn Chromatography (FC): Kieselgel 60 (230–400 mesh; Merck). Melting points (mp)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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60
1
were determined in open capillaries with a Gallenkamp capillary meltingꢀpoints apparatus. H
1
3
and C NMR spectra were recorded on Bruker Advance 300 spectrometer operating at 300.13
MHz and 75.47 MHz respectively, in CDCl or DMSOꢀd as solvents. Chemical shifts are
3
6
reported in ppm on the δ scale. In the case of multiplets, the signals are reported as intervals.
Signals were abbreviated as s, singlet; d, doublet; t, triplet; and m, multiplet. Coupling constants
are expressed in hertz. Hydrogenation reactions were carried out in a Shaker type hydrogenation
apparatus (Parr). Elemental analysis was determined with a LECO Elemental Analyzer CHNSꢀ
9
32. LCꢀMS analyses were performed using an Alliance 2695 (Waters) with a diode array
UV/Vis detector Waters 2996 and interfaced to a Micromass ZQ mass spectrometer. Analyses
were performed using reversed phase HPLC silica based columns: column Bridge C18 3.5 mm.
(
2.1 x 100 mm). Using an injection volume of 3 mL, a flow rate of 0.25 mL/min and gradient
elution (15 to 95 % over 10 min) of acetonitrile in water. Acetonitrile contains 0.08% v/v formic
acid and water contains 0.1% v/v formic acid. Analyses were monitored at 254 nm wavelength.
TM
Flash chromatography was performed using a Biotage Isolera flash purification system.
General Procedure for the Synthesis of 1-10. To a solution of 18 or 19 (1 Eq) and the
corresponding amines (2 Eq) in dry THF, it was added Al(CH ) in heptane 2.0 M (2 Eq). The
3
3
mixture was heated at 125 ºC for 35 min in a microwave reactor. The reaction mixture was
poured into an erlenmeyer, cooled in an ice bath and 1 N HCl aqueous solution was slowly added
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2
2
2
2
2
2
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4
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4
4
4
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5
5
5
5
5
5
5
6
until the effervescence ended. The mixture was taken to an extraction funnel and Et O was
2
added. The residue was purified by flash chromatography.
(R)-3-[(4-benzyl-2-oxooxazolidin-3-yl)methyl]-N-[4-(benzylcarbamoyl)phenyl]benzamide
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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(
1). Compound 18 (0.06 g, 0.18 mmol), compound 17a (0.08 g, 0.36 mmol), Al (CH ) in
3
3 3
heptane 2.0 M (0.2 mL, 0.36 mmol) in THF (10 mL) were heated at 125 ºC for 35 min in a
microwave reactor. The residue was purified: Biotage KPꢀSil 25g, EtOAc/Hexane from (5:95) up
1
to (10:90) yielding a white solid (0.04 g, 40 %). M.p. 120 ºC. H NMR (300 MHz, DMSOꢀd ) δ
6
(
ppm) 10.47 (s, 1H, NH), 8.95 (t, J = 6.0 Hz, 1H, NHCH ), 8.04 – 7.78 (m, 6H, HꢀAr), 7.64 –
2
7.48 (m, 4H, HꢀAr), 7.45 – 7.13 (m, 8H, HꢀAr), 4.68 (m, 1H), 4.47 (d, J = 6.0 Hz, 2H, NHCH2),
4.41ꢀ4.31 (m, 1H), 4.22 (m, 1H), 4.08 – 3.99 (m, 1H), 4.03 – 3.83 (m, 1H), 3.05 (dd, J = 13.5,
ab
13
J = 4.2 Hz, 1H), 2.75 (dd, J = 13.5, J = 8.2 Hz, 1H). C NMR (75 MHz, DMSOꢀd ) δ (ppm)
a4
ba
b4
6
1
65.6 (CO), 165.5 (CO), 158.6 (C), 139.8, 137.3, 136.1, 135.1, 135.5, 131.0, 129.2 (2C), 128.7,
28.5 (2C), 128.2 (3C), 127.9 (2C), 127.2 (3C), 126.8, 126.7 (2C), 119.5 (2C), 66.3 (C), 55.3
1
(
C), 45.1 (C), 42.5 (CH ), 37.2 (C). HPLC/MS: gradient elution (40 to 95 % over 10 min) of
2
+
acetonitrile in water, retention time 4.61 min [M+H] = 520. Elemental analysis calcd. for
C H N O : C, 73.97; H, 5.63; N, 8.09; found C, 73.88; H, 5.66; N, 8.08.
32
29
3
4
(
R)-3-[(4-benzyl-2-oxooxazolidin-3-yl)methyl]-N-[4-((4-(tert-
butyl)benzyl)carbamoyl)phenyl]benzamide (2). Compound 18 (0.06 g, 0.18 mmol), compound
7b (0.08 g, 0.36 mmol), Al(CH ) in heptane 2.0 M (0.2 mL, 0.36 mmol) in THF (10 mL) were
1
3
3
heated at 125 ºC for 35 min in a microwave reactor. The residue was purified: Biotage KPꢀSil
25g, EtOAc/Hexane from (5:95) up to (10:90) yielding a white solid (0.05 g, 44 %). M.p. 110 ºC.
1
H NMR (300 MHz, CDCl ) δ (ppm) 8.91 (s, 1H, NH), 7.83ꢀ7.80 (m, 1H, HꢀAr), 7.78 – 7.63
3
(m, 5H, HꢀAr), 7.44 – 7.15 (m, 9H, HꢀAr), 7.07 – 6.96 (m, 2H, HꢀAr), 6.66 (t, J = 5.7 Hz, 1H,
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NHCH ), 4.74 ꢀ 4.65 (m, 1H), 4.57 (d, J = 5.4 Hz, 2H, NHCH ), 4.21 – 4.07 (m, 2H), 4.00 – 3.90
2
2
(
m, 1H), 3.87 – 3.76 (m, 1H), 3.04 (dd, J = 13.6, J = 4.8 Hz, 1H), 2.63 (dd, J = 13.6, J = 8.5
ab a4 ba b4
1
3
Hz, 1H), 1.30 (s, 9H, 3CH ). C NMR (75 MHz, CDCl ) δ (ppm) 166.8 (CO), 165.8 (CO),
3
3
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
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53
54
55
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57
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1
58.6, 150.5, 141.2, 139.7, 136.5, 135.2, 131.2, 129.8, 129.2, 128.9 (2C), 128.9 (2C), 127.9 (2C),
127.7 (2C), 127.2, 127.1, 126.6, 125.6 (2C), 119.9 (2C), 67.1 (C), 55.8 (C), 46.0 (C), 43.8 (CH2),
8.6 (C), 34.5 (C(CH ) ), 31.3 (3CH ). HPLC/MS: gradient elution (40 to 95 % over 10.0 min)
3
3
3
3
+
of acetonitrile in water, retention time 8.83 min [M+H] = 576. Elemental analysis calcd. for
C H N O : C, 75.11; H, 6.48; N, 7.30; found C, 75.00; H, 6.75; N, 7.70.
36
37
3
4
(R)-3-[(4-benzyl-2-oxooxazolidin-3-yl)methyl]-N-[4-((4-
(trifluoromethyl)benzyl)carbamoyl)phenyl]benzamide (3). Compound 18 (0.1 g, 0.31 mmol),
compound 17c (0.18 g, 0.61 mmol), Al(CH ) in heptane 2.0 M (0.3 mL, 0.61 mmol) in THF (10
3
3
mL) were heated at 125 ºC for 35 min in a microwave reactor. The residue was purified: Biotage
KPꢀSil 25g, EtOAc/Hexane from (5:95) up to (10:90) yielding a white solid (0.09 g, 50 %). M.p.
1
1
14 ºC. H NMR (300 MHz, DMSOꢀd ) δ (ppm) 10.49 (s, 1H, NH), 9.07 (t, J = 6.1 Hz, 1H,
6
CH NH), 7.98 – 7.77 (m, 6H, HꢀAr), 7.69 (m, 2H, HꢀAr), 7.53 (m, 5H, HꢀAr), 7.35 – 7.10 (m,
2
4
1
8
1
H, HꢀAr), 4.68 (d, J = 15.6 Hz, 1H) 4.55 (d, J = 5.8 Hz, 2H, CH NH), 4.41 (m, 1H), 4.22 (m,
2
H), 4.04 (m, 1H), 3.92 (m, 1H), 3.05 (dd, J = 13.5, J = 4.2 Hz, 1H), 2.75 (dd, J = 13.6, J =
ab
a4
ba
b4
1
3
.1 Hz, 1H). C NMR (75 MHz, DMSO) δ (ppm) 165.8 (CO), 165.7 (CO), 157.7 (C), 144.7,
41.9, 139.2, 137.3, 136.1, 135.1, 131.0, 129.2 (2C), 129.0, 128.7, 128.5 (3C), 128.0 (2C), 127.9
(3C), 127.85, 127.6, 126.8, 126.7, 125.2, 119.5, 66.3 (C), 55.3 (C), 45.1 (C), 42.3 (NHCH ), 37.2
2
(C). HPLC/MS: gradient elution (40 to 95 % over 10.0 min) of acetonitrile in water, retention
+
time 10.2 min [M+H] = 588. Elemental analysis calcd. for C H F N O : C, 67.45; H, 4.80; N,
33 28
3
3
4
7
.15; found C, 67.69; H, 5.00; N, 7.14.
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(
R)-3-[(4-benzyl-2-oxooxazolidin-3-yl)methyl]-N-[4-(pentylcarbamoyl)phenyl]benzamide
(
4). Compound 18 (0.10 g, 0.31 mmol), compound 17d (0.13 g, 0.61 mmol), Al(CH ) in heptane
3 3
2
.0 M (0.31 mL, 0.61 mmol) in THF (10 mL) were heated at 125 ºC for 35 min in a microwave
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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0
1
2
3
4
5
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7
8
9
0
1
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3
4
5
6
7
8
9
0
reactor. The crude was purified: Biotage KPꢀSil 25g, EtOAc/Hexane from (5:95) up to (10:90)
1
yielding a white solid (0.07 g, 45 %). M.p. 140 ºC. H NMR (300 MHz, DMSOꢀd ) δ (ppm)
6
1
0.46 (s, 1H, NH), 8.35 (t, J = 5.6 Hz, 1H, CH NH), 7.91 (m, 1H, HꢀAr), 7.86 (bs, 5HꢀAr), 7.58
2
–
7.51 (m, 2H, HꢀAr), 7.32 – 7.17 (m, 5H, HꢀAr), 4.69 (m, 1H), 4.42 (m, 1H), 4.23 (m, 1H), 4.06
(
m, 1H), 4.00 – 3.89 (m, 1H), 3.25 (m, 2H, NHCH ), 3.06 (dd, J = 13.6, J = 4.2 Hz, 1H), 2.77
2 ab a4
(
dd, J = 13.6, J =8.1 Hz, 1H), 1.52 (m, 2H), 1.42 – 1.18 (m, 4H), 0.85 (t, J= 6.8 Hz, 3H, CH ).
b
a
b
4
3
1
3
C NMR (75 MHz, DMSOꢀd ) δ (ppm) 166.0 (CO), 165.9 (CO), 158.1, 141.9, 137.6, 136.5,
6
1
35.5, 131.4, 130.1, 129.6 (2C), 129.1, 128.9 (2C), 128.2 (2C), 127.6, 127.2, 127.1, 119.8 (2C),
6
6.7 (C), 55.7 (C), 45.5 (C), 40.7 (CH ), 37.6 (C), 29.2 (CH ), 29.1 (CH ), 22.3 (CH ), 14.3
2
2
2
2
(
CH ). HPLC/MS: gradient elution (40 to 95 % over 10.0 min) of acetonitrile in water, retention
3
+
time 5.09 min [M+H] = 500. Elemental analysis calcd. for C H N O : C, 72.12; H, 6.66; N,
3
0
33
3
4
8.41; found C, 72.40; H, 6.83; N, 8.68.
R)-3-[(4-benzyl-2-oxooxazolidin-3-yl)methyl]-N-[4-
(
(
dodecylcarbamoyl)phenyl]benzamide (5). Compound 18 (0.06 g, 0.18 mmol), compound 17e
(
0.11 g, 0.36 mmol), Al(CH ) in heptane 2.0 M (0.18 mL, 0.36 mmol) in THF (10 mL) were
3
3
heated at 125 ºC for 35 min in a microwave reactor. The organic layers were dried and the
solvent was evaporated under reduced pressure and the crude was purified: Biotage KPꢀSil 25g,
1
EtOAc/Hexane from (5:95) up to (10:90) yielding a white solid (0.05 g, 45 %). M.p. 115 ºC. H
NMR (300 MHz, CDCl ) δ (ppm) 8.69 – 8.56 (m, 1H, NH), 7.94 – 7.64 (m, 5H, HꢀAr), 7.53 –
3
7.19 (m, 4H, HꢀAr), 7.13 – 7.00 (m, 3H, HꢀAr), 6.34 – 6.14 (m, 1H, HꢀAr), 4.81 (m, 1H), 4.30 –
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.13 (m, 1H), 4.02 (m, 1H), 3.94 – 3.79 (m, 1H), 3.45 ꢀ 3.30 (m, 1H), 3.06 (dd, J = 13.6, J =
ab
a4
.8 Hz, 1H), 2.66 (dd, J = 13.6, J = 8.6 Hz, 1H), 1.60 – 1.55 (m, 2H, CH ), 1.46 – 1.15 (m,
ba
b4
2
13
8H, 9CH ), 0.87 (t, J = 6.7 Hz, 3H, CH ). C NMR (75 MHz, CDCl ) δ (ppm) 166.9 (CO),
2
3
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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31
32
33
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36
37
38
39
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65.6 (CO), 158.6 (C), 140.7, 139.7, 136.7, 135.4, 135.3, 131.4, 130.5, 129.3, 129.0 (2C), 127.8
(2C), 127.3, 127.0, 126.6, 119.9 (2C), 116.7, 67.2 (C), 55.7 (C), 46.1 (C), 40.2 (CH ), 38.7 (C),
2
3
1.9 (CH ), 29.6 (4CH ), 29.3 (3CH ), 27.0 (CH ), 22.7 (CH ), 14.1 (CH ). HPLC/MS: gradient
2 2 2 2 2 3
+
elution (40 to 95 % over 10.0 min) of acetonitrile in water, retention time 9.93 min [M+H] =
599. Elemental analysis calcd. for C H N O : C, 74.34; H, 7.93; N, 7.03; found C, 74.20; H,
37 47 3 4
7
.50; N, 7.04.
(R)-3-[(4-benzyl-2-oxooxazolidin-3-yl)methyl]-N-[4
(cyclopropyl)carbamoyl)phenyl]benzamide (6). Compound 18 (0.18 g, 0.56 mmol),
compound 17f (0.18 g, 0.67 mmol), Al(CH ) in heptane 2.0 M (1.11 mL, 2.24 mmol) in THF
(
3 3
(10 mL) were heated at 125 ºC for 35 min in a microwave reactor. The residue was purified:
1
Biotage KPꢀSil 25g, EtOAc/Hexane (4:6) yielding a white solid (0.05 g, 2 %). H NMR (300
MHz, MeOD) δ (ppm) 7.90 – 7.88 (m, 1H), 7.84 (bs, 2H), 7.79 – 7.77 (m, 2H), 7.55 – 7.51 (m,
4
4
2
1
H), 7.30 – 7.26 (m, 2H), 7ꢀ18 – 7.16 (m, 2H), 4.81 (d, J= 4.8 Hz, 1H), 4.36 (d, J= 4.4 Hz 1H),
.30 – 4.26 (m, 1H), 4.15 – 4.12 (m, 1H), 4.06 – 4.02 (m, 1H), 3.11 (dd, J= 13.7, 5.1 Hz, 1H),
1
3
.87 – 2.80 (m, 2H), 0.83 – 0.79 (m, 2H), 0.66 – 0.63 (m, 2H). C NMR (75 MHz, MeOD) δ
69.7 (C), 167.2 (C), 159.5 (C), 141.7 (C), 137 (C), 135.9 (C), 135.3 (C), 131.1 (2CH), 129.5
(
C), 128.9 (2CH), 128.8 (2CH), 128.4 (2CH), 126.9 (2CH), 126.7 (CH), 119.8 (2CH), 67.2
CH ), 55.9 (CH), 45.5 (CH ), 37.8 (CH ), 22.6 (CH), 5.14 (2CH ).
(
2
2
2
2
(R)-3-[(4-benzyl-2-oxooxazolidin-3-yl)methyl]-N-[4-
((cyclohexylmethyl)carbamoyl)phenyl]benzamide (7). Compound 18 (0.06 g, 0.18 mmol),
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compound 17g (0.08 g, 0.36 mmol), Al(CH ) in heptane 2.0 M (0.18 mL, 0.36 mmol) in THF
3
3
(10 mL) were heated at 125 ºC for 35 min in a microwave reactor. The residue was purified:
Biotage KPꢀSil 25g, EtOAc/Hexane from (5:95) up to (10:90) yielding a white solid (0.04 g, 42
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
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
%
). M.p. 130 ºC. H NMR (300 MHz, CDCl ) δ (ppm) 9.08 (s, 1H, NH), 7.85 (m, 1H, HꢀAr),
3
7.78 – 7.61 (m, 5H, HꢀAr), 7.45 – 7.16 (m, 5H, HꢀAr), 7.09 – 6.96 (m, 2H, HꢀAr), 6.50 (t, J = 6.0
Hz, 1H, CH NH), 4.73 (m, 1H), 4.21 – 4.06 (m, 2H), 4.00 (m, 1H), 3.93 – 3.79 (m, 1H), 3.25 (m,
2
2H, CH NH), 3.03 (dd, J = 13.6, J = 4.9 Hz, 1H), 2.63 (dd, J = 13.6, J = 8.6 Hz, 1H), 1.82 –
2 ab a4 ba b4
1
3
1
.52 (m, 6H), 1.32 – 1.10 (m, 4H), 0.93 (m, 1H). C NMR (75 MHz, CDCl ) δ (ppm) 166.9
3
(CO), 165.5 (CO), 158.5 (C), 140.8, 136.8, 135.4, 135.3, 131.4, 130.5, 129.3, 129.0, 127.9 (3C),
127.3 (2C), 127.0, 126.6, 119.8, 119.7 (2C), 67.2 (C), 55.7 (C), 46.3 (CH NH), 46.1, 38.8 (C),
2
38.0 (CH), 30.9 (2CH ), 26.4 (CH ), 25.8 (2CH ). HPLC/MS: gradient elution (40 to 95 % over
2 2 2
+
1
0.0 min) of acetonitrile in water, retention time 5.71 min [M+H] = 527. Elemental analysis
calcd. for C H N O : C, 73.12; H, 6.71; N, 7.99; found C, 73.44; H, 6.58; N, 7.52.
3
2
35
3
4
(
S)-3-[(4-benzyl-2-oxooxazolidin-3-yl)methyl]-N-[4-((4-(tert-
butyl)benzyl)carbamoyl)phenyl]benzamide (8). Compound 19 (0.20 g, 0.62 mmol), compound
7b (0.21 g, 0.74 mmol), Al(CH ) in heptane 2.0 M (1.24 mL, 2.48 mmol) in THF (10 mL)
1
3
3
were heated at 125 ºC for 20 min in a microwave reactor. The residue was purified: Biotage KPꢀ
1
Sil 25g, EtOAc/Hexane (8:2) yielding a white solid (0.02 g, 4 %). M.p. 171 ºC. H NMR (300
MHz, CDCl ) δ (ppm) 8.32 (s, 1H, H), 7.89 – 7.71 (m, 6H), 7.48 – 7.22 (m, 9H, H), 7.06 (d, J=
3
7.53 Hz, 2H), 6.44 (s, 1H), 4.81 (d, J= 15.28 Hz, 1H), 4.61 (s, 2H), 4.23 – 4.15 (m, 2H, H), 4.04
(dd, J= 5.78, 8.91 Hz, 1H), 3.89 (m, 1H), 3.08 (dd, J= 4.69, 13.47 Hz, 1H), 2.68 (dd, J= 8.42,
1
3
1
3.47 Hz, 1H), 2.17 (s, 9H). C NMR (MHz, CDCl ) δ 166.7, 165.4, 158.5, 150.7, 140.9, 136.8,
3
135.3, 135.0, 131.5, 129.4, 129.0, 128.1, 127.8, 127.3, 127.0, 126.6, 125.7, 119.8, 67.2, 55.7,
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6.1, 43.9, 38.8, 34.5, 31.3. HPLC/MS: gradient elution 30ꢀ95% in 10 min, retention time=7.66,
+
m/z [M+H] = 576. Elemental analysis calcd. for C H N O : C, 75.11; H, 6.48; N, 7.30; found
36 37
3
4
C, 75.10; H, 6.29; N, 7.18.
S)-3-[(4-benzyl-2-oxooxazolidin-3-yl)methyl]-N-[4-((4-
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
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46
47
48
49
50
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53
54
55
56
57
58
59
60
(
(trifluoromethyl)benzyl)carbamoyl)phenyl]benzamide (9). Compound 19 (0.20 g, 0.62
mmol), compound 17c (0.22 g, 0.74 mmol), Al(CH ) in heptane 2.0 M (1.24 mL, 2.48 mmol) in
3
3
THF (10 mL) were heated at 125 ºC for 35 min in a microwave reactor. The residue was
purified: Biotage KPꢀSil 25g, EtOAc/Hexane from (5:95) up to (10:90) yielding a white solid
1
(
0.32 g, 87 %). M.p. 176 ºC. H NMR (300 MHz, MeOD) δ (ppm) 7.93ꢀ7.78 (m, 4H), 7.65ꢀ7.50
(m, 7H), 7.27ꢀ7.15 (m, 6H), 4.75 (d, J= 15.32 Hz, 1H), 4.61 (d, J= 5.84 Hz, 2H), 4.57ꢀ4.50 (m,
1
2
1
H), 4.48ꢀ4.32 (m, 1H), 4.26ꢀ4.15 (m, 1H), 4.10ꢀ4.02 (m, 1H), 3.08 (dd, J= 4.88, 13.53 Hz, 1H),
1
3
.81 (dd, J= 7.78, 13.35 Hz, 1H). C NMR (75 MHz, MeOD) δ (ppm) 169.6 (CO), 168.6 (CO),
60.9, 145.0, 143.3, 138.4, 137.3, 136.6, 133.7, 132.5, 130.8, 130.3, 130.2, 129.8, 129.2, 127.1
(c, J= 30.31 Hz), 126.8, 126.4 (c, J= 9.17 Hz), 126.1 (d, J= 214.1 Hz), 124.7, 121.3, 68.6, 57.3,
46.8, 44.0, 39.2. HPLC/MS: gradient elution (30 to 95 % over 10 min), retention time 6.84 min
+
[
M+H] = 588.
(
S)-3-[(4-benzyl-2-oxooxazolidin-3-yl)methyl]-N-[4-(pentylcarbamoyl)phenyl]benzamide
10). Compound 19 (0.20 g, 0.62 mmol), compound 17d (0.15 g, 0.74 mmol), Al(CH ) in
(
3
3
heptane 2.0 M (1.24 mL, 2.48 mmol) in THF (10 mL) were heated at 125 ºC for 10 min in a
microwave reactor. The crude was purified: Biotage KPꢀSil 25g, EtOAc/Hexane from (5:95) up
1
to (10:90) yielding a white solid (0.25 g, 80 %). M.p. 267 ºC. H NMR (300 MHz, DMSOꢀd ) δ
6
(ppm) 8.36 (bs, 1H, NH), 7.92ꢀ7.82 (m, 3H), 7.57ꢀ7.53 (m, 4H), 7.31ꢀ7.19 (m, 6H), 4.69 (d,
J=15.61 Hz, 1H), 4.42 (d, J= 15.54 Hz, 1H), 4.26ꢀ4.20 (m, 1H), 4.08ꢀ4.03 (m, 1H), 3.98ꢀ3.89 (m,
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
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4
4
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1
H), 3.36ꢀ3.32 (m, 2H), 3.05 (dd, J= 3.78, 13.32 Hz, 1H), 2.75 (dd, J= 8.12, 13.43 Hz, 1H),
1
3
1.56ꢀ1.47 (m, 2H), 1.32ꢀ1.26 (m, 4H), 0.87 (t, J= 6.46 Hz, 3H). C NMR (75 MHz, DMSOꢀd ) δ
6
(ppm) 165.6 (CO), 165.5 (CO), 157.7, 141.5, 137.3, 136.1, 135.1, 131.0, 129.7, 129.3, 128.7,
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
128.5, 127.8, 126.8, 126.7, 119.5, 66.3, 55.3, 45.1, 38.7, 37.2, 28.9, 28.7, 21.9, 13.9. HPLC/MS:
+
gradient elution (30 to 95 % over 10 min), retention time 6.30 min [M+H] = 500.
N-[3-((S)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-2-methyl-3-oxopropyl)phenyl)-4-(2-(4-
(tert-butyl)phenyl)acetamido]benzamide (11).
To a solution of 23 (0.16 g, 0.56 mmol) in dry THF was added 21a in dry THF (10 mL) under
nitrogen atmosphere and stirred for 12 h. The reaction mixture was treated with 1 N HCl aqueous
solution (10 mL) and extracted with AcOEt (20 mL). The organic layer was washed with
saturated aqueous NH Cl and dried over MgSO . The residue was purified: Biotage KPꢀSil 25g,
4
4
EtOAc/Hexane from (5:95) up to (10:90) yielding a white solid (0.24 g, 68 %). M.p. 130 ºC.
2
0
1
[
α]D = ꢀ16.9. H NMR (300 MHz, CDCl ) δ (ppm) 7.75 (m, 2H, HꢀAr), 7.68 – 7.60 (m, 1H, Hꢀ
3
Ar), 7.54 (m, 2H, HꢀAr), 7.44 (m, 3H, HꢀAr), 7.32 – 7.20 (m, 6H, HꢀAr), 7.13 – 7.00 (m, 3H, Hꢀ
Ar), 4.66 (m, 1H, CH), 4.23 – 4.00 (m, 3H, CH /CH), 3.74 (s, 2H, CH CO), 3.25 – 3.06 (m, 2H),
2
2
1
3
2.63 (m, 2H), 1.34 (s, 9H, 3CH ), 1.18 (d, J = 6.6 Hz, 3H, CH ). C NMR (75 MHz, CDCl ) δ
3 3 3
(ppm) 176.4 (CO), 176.3 (CO), 169.5 (CO), 153.1 (Cꢀ2), 150.9, 140.8, 140.2, 138.0, 135.1,
130.8, 129.4 (2C), 129.2 (2C), 129.1, 128.9 (2C), 128.1 (2C), 127.3, 126.3 (2C), 125.5, 120.8,
1
19.3 (2C), 118.4, 66.0 (C), 55.2 (C), 44.5 (CH ), 39.7 (C), 39.6 (C), 37.8 (C), 34.6 (C(CH )),
2 3
+
31.3 (3CH ), 16.5 (CH ). HPLC/MS: retention time 8.78 min [M+H] = 632. Elemental analysis
3
3
calcd. for C H N O : C, 74.14; H, 6.54; N, 6.65; found C, 73.89; H, 6.82; N, 6.41.
3
9
41
3
5
N-[3-((S)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-2-methyl-3-oxopropyl)phenyl]-4-
hexanamidobenzamide (12). To a solution of 23 (0.39 g, 1.65 mmol) in dry THF was added
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7
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1b in dry THF (10 mL) under nitrogen atmosphere and stirred for 12 h. The reaction mixture
was treated with 1 N HCl aqueous solution (10 mL) and extracted with AcOEt (20 mL). The
organic layer was washed with saturated aqueous NH Cl and dried over MgSO . The residue was
4
4
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
purified: Biotage KPꢀSil 25g, EtOAc/Hexane from (5:95) up to (10:90) yielding a white solid
20
1
(0.21 g, 34 %). M.p. 141 ºC. [α]D = ꢀ32.2. H NMR (300 MHz, CDCl ) δ (ppm) 8.13 – 8.10 (bs,
3
1
H, NH), 7.86 – 7.81 (bs, 1H, NH), 7.76 – 7.70 (m, 2H, HꢀAr), 7.67 – 7.54 (m, 3H, HꢀAr), 7.47
7.37 (m, 2H, HꢀAr), 7.24 – 7.15 (m, 4H, HꢀAr), 7.11 – 7.00 (m, 2H, HꢀAr), 4.67 – 4.57 (m,
–
1H), 4.22 – 4.01 (m, 3H), 3.14 – 3.06 (m, 2H), 2.62 – 2.50 (m, 2H), 2.36 (t, J= 7.6 Hz, 2H,
CH CO), 1.70 – 1.59 (m, 2H), 1.37 – 1.23 (m, 4H), 1.16 (d, J= 6.7 Hz, 3H, CH CH), 0.97 – 0.79
2
3
1
3
(
t, J= 6.6 Hz, 3H, CH ). C NMR (75 MHz, CDCl ) δ (ppm) 176.3 (CO), 172.1 (CO), 165.3
3
3
(CO), 153.2, 141.3, 140.1, 138.1, 135.1, 129.9, 129.3 (2C), 129.0, 128.9 (2C), 128.2 (2C), 127.3
2C), 125.5, 121.0, 119.3, 118.6, 66.0, 55.2, 39.7, 39.6, 37.8, 37.7 (CH CO), 31.4 (CH ), 25.2
(
2
2
+
(CH ), 22.4 (CH ), 16.5 (CH CH), 13.9 (CH ). HPLC/MS: retention time 7.70 min [M+H] =
2
2
3
3
556. Elemental analysis calcd. for C H N O : C, 71.33; H, 6.71; N, 7.56; found C, 71.18 ; H,
33 37 3 5
6
.42 ; N, 7.31.
(S)-3-[3-(4-(2-(4-(tert-butyl)phenyl)acetamido)benzamido)phenyl]-2-methylpropanoic
acid (13).
To a solution of 11 (0.14 g, 0.22 mmol) in THF/H O (3:1) (30 mL) at 0 ºC were added 30 %
2
H O aqueous solution (0.1 mL, 0.88 mmol) and LiOH (0.01 g, 0.44 mmol) in H O (2 mL). The
2
2
2
solution was stirred for 2 h at r.t. The reaction mixture was treated with 1 N HCl aqueous
solution (1 mL) and extracted with AcOEt (2x 20 mL). The organic layers were washed with
brine and dried over MgSO . The residue was purified: Biotage KPꢀSil 25g, Ethanol/CH Cl (0ꢀ6
4
2
2
20
1
%
) yielding a white solid (0.07 g, 70 %). M.p 180.9 ºC. [α]D = 11.1. H NMR (300 MHz,
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Journal of Medicinal Chemistry
Page 30 of 51
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DMSOꢀd ) δ (ppm) 12.16 (s, 1H, COOH), 10.40 (s, 1H, NH), 10.04 (s, 1H, NH), 7.92 (d, J = 8.7
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Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H), 7.64 – 7.57 (m, 2H, HꢀAr), 7.34 (m, 2H, HꢀAr), 7.26 (m, 3H,
HꢀAr), 6.91 (m, 1H, HꢀAr), 3.62 (s, 2H, CH CO), 2.89 (m, 1H,), 2.66 – 2.54 (m, 2H), 1.26 (s,
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H, 3CH ), 1.05 (d, J = 6.5 Hz, 3H, CH ). C NMR (75 MHz, DMSOꢀd ) δ (ppm) 176.7 (CO),
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3
6
169.7 (CO), 164.7 (CO), 148.9 , 142.1, 139.9, 139.1, 132.6, 130.3, 129.1, 128.8 (2C), 128.5
(
2C), 128.3, 125.1 (2C), 123.9, 120.8, 118.2 (2C) , 42.9 (CH CO), 40.6, 38.7, 34.1 (C(CH ) ),
2 3 3
3
1.1 (3CH ), 16.7 (CH ). HPLC/MS: gradient elution (40 to 95 % over 10.0 min) of acetonitrile
3 3
+
in water, retention time 6.72 min [M+H] = 473. Elemental analysis calcd for C H N O : C,
2
9
32
2
4
73.7; H, 6.83; N, 5.93; found C, 74.00; H, 7.00; N, 5.70.
(S)-3-[3-(4-hexanamidobenzamido)phenyl]-2-methylpropanoic acid (14). To a solution of
2 (0.10 g, 0.18 mmol) in THF/H O (3:1) (30 mL) at 0 ºC were added 30 % H O aqueous
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2
2
2
solution (0.1 mL, 0.72 mmol) and LiOH (0.008 g, 0.36 mmol) in H O (2 mL). The solution was
2
stirred for 2 h at r.t. The reaction mixture was treated with 1 N HCl aqueous solution (1 mL) and
was extracted with AcOEt (2x 20 mL). The organic layers were washed with brine and dried
over MgSO . The residue was purified: Biotage KPꢀSil 25g, CH OH/CH Cl (0ꢀ6 %) yielding a
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3
2
2
20
1
white solid (0.09 g, 62 %). M.p 200 ºC. [α]D = 12.9. H NMR (300 MHz, DMSOꢀd ) δ (ppm)
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1
7
3
2.18 (s, 1H, COOH), 10.13 (s, 1H, NHCO), 10.03 (s, 1H, NHCO), 7.91 (d, J = 8.7 Hz, 2H),
.71 (d, J = 8.5 Hz, 2H), 7.68 – 7.53 (m, 2H), 7.23 (m, 1H), 6.91 (m, 1H), 2.90 (m, 1H), 2.61 (m,
H), 2.33 (t, J = 7.4 Hz, 2H, CH CO), 1.58 (m, 2H, CH ), 1.28 (m, 4H, 2CH ), 1.05 (d, J = 6.2
2
2
2
13
Hz, 3H, CH CH), 0.87 (t, J = 6.6 Hz, 3H, CH ). C NMR (75 MHz, DMSOꢀd ) δ (ppm) 176.7
3
3
6
(CO), 171.7 (CO), 164.7 (CO), 142.2, 139.9, 139.1, 128.7, 128.5 (2C), 128.2, 124.0, 120.7,
18.2, 118.1 (2C), 40.6 (CH), 38.6, 36.4 (CH CO), 30.9 (CH ), 24.6 (CH ), 21.9 (CH ), 16.7
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