Hydroxamic Acids Constitute a Novel Class of Autotaxin Inhibitors that Exhibit in Vivo Efficacy in a Pulmonary Fibrosis Model

Article abstract of DOI:10.1021/acs.jmedchem.8b00232

Autotaxin (ATX) catalyzes the hydrolysis of lysophosphatidylcholine (LPC) generating the lipid mediator lysophosphatidic acid (LPA). Both ATX and LPA are involved in various pathological inflammatory conditions, including fibrosis and cancer, and have attracted great interest as medicinal targets over the past decade. Thus, the development of novel potent ATX inhibitors is of great importance. We have developed a novel class of ATX inhibitors containing the zinc binding functionality of hydroxamic acid. Such novel hydroxamic acids that incorporate a non-natural δ-amino acid residue exhibit high in vitro inhibitory potency over ATX (IC₅₀ values 50-60 nM). Inhibitor 32, based on δ-norleucine, was tested for its efficacy in a mouse model of pulmonary inflammation and fibrosis induced by bleomycin and exhibited promising efficacy. The novel hydroxamic ATX inhibitors provide excellent tools for the study of the role of the enzyme and could contribute to the development of novel therapeutic agents for the treatment of fibrosis and other chronic inflammatory diseases.

Full text of DOI:10.1021/acs.jmedchem.8b00232

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Hydroxamic Acids Constitute a Novel Class of Autotaxin Inhibitors
that Exhibit in vivo Efficacy in a Pulmonary Fibrosis Model
Aikaterini Nikolaou, Ioanna Ninou, Maroula G. Kokotou, Eleanna
Kaffe, Antreas Afantitis, Vassilis Aidinis, and George Kokotos
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00232 • Publication Date (Web): 05 Apr 2018
Downloaded from http://pubs.acs.org on April 7, 2018
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Hydroxamic Acids Constitute a Novel Class of Autotaxin Inhibitors that
Exhibit in vivo Efficacy in a Pulmonary Fibrosis Model
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†
§
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Aikaterini Nikolaou, Ioanna Ninou, Maroula G. Kokotou, Eleanna Kaffe, Antreas Afantitis,
§
,†
Vassilis Aidinis, George Kokotos*
†
Laboratory of Organic Chemistry, Department of Chemistry, National and Kapodistrian
University of Athens, Panepistimiopolis, Athens 15771, Greece
§
Division of Immunology, Biomedical Sciences Research Center “Alexander Fleming”, Athens
6672, Greece
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#
NovaMechanics Ltd, Nicosia, Cyprus
KEYWORDS. δꢀamino acids, autotaxin, hydroxamic acids, idiopathic pulmonary fibrosis,
inhibitors
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ABSTRACT
Autotaxin (ATX) catalyzes the hydrolysis of lysophosphatidylcholine (LPC) generating the lipid
mediator lysophosphatidic acid (LPA). Both ATX and LPA are involved in various pathological
inflammatory conditions, including fibrosis and cancer, and have attracted a great interest as
medicinal targets over the last decade. Thus, the development of novel potent ATX inhibitors is
of great importance. We have developed a novel class of ATX inhibitors containing the zinc
binding functionality of hydroxamic acid. Such novel hydroxamic acids, that incorporate a nonꢀ
natural δꢀamino acid residue, exhibit high in vitro inhibitory potency over ATX (IC values 50ꢀ
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0 nM). Inhibitor 32, based on δꢀnorleucine, was tested for its efficacy in a mouse model of
pulmonary inflammation and fibrosis induced by bleomycin and exhibited promising
efficacy. The novel hydroxamic ATX inhibitors provide excellent tools for the study of the role
of the enzyme and could contribute to the development of novel therapeutic agents for the
treatment of fibrosis and other chronic inflammatory diseases.
INTRODUCTION
Autotaxin (ATX) is an enzyme that has attracted a great interest as a medicinal target over the
1
last decade. It was originally isolated in 1992 as an autocrine motility factor from A2058
2
melanoma cells and characterized as a 125 kDa glycoprotein. ATX, also known as ENPP2,
belongs to the sevenꢀmembered family of ectonucleotidepyrophosphatases/phosphodiesterases
(ENPPs), which are characterized by their ability to catalyze the hydrolysis of pyrophosphate or
3
phosphodiester bonds in nucleotides. It is unique among the ENPPs, because it exhibits
lysophospholipase D (lysoPLD) activity, catalyzing the hydrolysis of lysophosphatidylcholine
4
,5
(
LPC) into lysophosphatidic acid (LPA) and choline (Figure 1). The bioactive lipid mediator
LPA binds to specific Gꢀproteinꢀcoupled receptors (LPA1ꢀ6 in mammals) and activates several
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signal transduction pathways including the migration, proliferation, and survival of various cell
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,7
types. ATX is produced in various tissues and is essential for vascular development, but it has
also been involved in a variety of chronic inflammatory conditions, such as arthritis and cancer,
1
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as summarized in various review articles.
As a result, both ATX and LPA have been shown
to be involved in pathological conditions and their implications in human diseases make them
attractive targets for the discovery of novel drugs.
ATX consists of two Nꢀterminal somatomedin Bꢀlike domains (SMB1 and SMB2), a central
catalytic phosphodiesterase (PDE) domain and a Cꢀterminal nucleaseꢀlike domain. A threonine
(T210) residue in the active site located in the phosphodiesterase domain, along with two zinc
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ions, are responsible for its hydrolytic catalytic mechanism. In 2011, Nishimasu et al. presented
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the crystal structures of mouse ATX alone and in complex with LPAs, that provide the first
structural evidence for LPA production by ATX and reveal how ATX generates and delivers
GPCRꢀsignaling lipid mediators, while Hausmann et al. reported on the structural basis of
1
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substrate discrimination and integrin binding by ATX, describing how ATX promotes localized
LPA signaling.
A variety of synthetic ATX inhibitors have been developed in both academia and pharmaceutical
industry and the chemical classes of synthetic inhibitors have been summarized in various review
1,16ꢀ19
articles.
The structures of selected ATX inhibitors are depicted in Figure 2. PFꢀ8380 (1,
Figure 2) exhibited low nanomolar IC in isolated enzyme assay (1.7 nM, LPC assay and 2.8
5
0
20
nM, FSꢀ3 assay) and 101 nM in human whole blood. It caused >95% reduction of rat plasma
LPA at a dose of 30 mg/kg and it reduced inflammatory hyperalgesia, in a doseꢀresponsive
manner, with the same efficacy as 30 mg/kg naproxen. HA155 (2, Figure 2) represents an
2
1
important class of ATX inhibitors, the boronic acid derivatives. Inhibition of ATX by the
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inhibitor ONOꢀ8430506 (IC50 100 nM) has shown to delay breast tumor growth and lung
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metastasis in mice. Within last year exciting results on a number of novel inhibitors have been
described. Highly potent nonꢀcarboxylic acid ATX inhibitors (for example, compound 3, Figure
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) were reported to reduce melanoma metastasis and chemotherapeutic resistance of breast
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cancer stem cells. Miller et al reported small molecule ATX inhibitors with a discrete binding
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mode, while a structureꢀguided evolution of weak physiological allosteric inhibitors (bile
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salts) led to the design of potent competitive ATX inhibitors (for example, compound 4, Figure
26
2
) that do not interact with the catalytic site. Galapagos reported an imidazo[1,2ꢀa]pyridine
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series of ATX inhibitors, demonstrating that inhibitor GLPG1690 (5, Figure 2) was efficacious
in a bleomycin (BLM)ꢀinduced pulmonary fibrosis model in mice and in reducing extracellular
28
matrix deposition in the lung. This firstꢀinꢀclass ATX inhibitor is under evaluation in a phase 2
study in idiopathic pulmonary fibrosis (IPF) patients.
The aim of our work was to develop novel ATX inhibitors that may be find applications in vivo.
We envisaged that the hydroxamic acid functionality may target the active site of ATX and in the
present manuscript we present the synthesis of a series of novel hydroxamic acids, the evaluation
of their in vitro activity against ATX and in vivo results demonstrating that such a novel
hydroxamic acid inhibitor is active in a mouse model of pulmonary fibrosis.
RESULTS AND DISCUSSION
Design and synthesis of hydroxamic acid inhibitors. Over the past decades, the biochemistry
of hydroxamic acids has attracted considerable attention due to their pharmacological
29,30
properties.
Some of them have been advanced into human clinical trials for the treatment of
several diseases. Vorinostat, a hydroxamic acid derivative which inhibits histone deacetylases, is
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an approved drug (under the name Zolinza) for Tꢀcell lymphoma. In the case of vorinostat and
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related hydroxamic acid medicines (belinostat, panobinostat), the mechanism of action is based
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on the enzyme inhibition through binding to Zn ion. We decided to use the hydroxamic acid
functionality as the zinc binding group in our ATX inhibitors expecting interaction of
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hydroxamic acid functionality with the active site Zn of ATX.
We began our studies by synthesizing a number of hydroxamic acids incorporating the 4ꢀ
aminophenylacetic acid residue, because phosphonic acids based on this moiety have been
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proven efficient inhibitors of ATX.
The synthesis of such derivatives is presented in Scheme
1
. Carboxylic acids 6aꢀm were converted into corresponding ethyl esters 7aꢀm and subsequent
treatment with hydroxylamine and EtONa led to hydroxamic acids 8aꢀm. The amino group of
phenylacetic acid was coupled with a variety of carboxylic acids containing a medium or a long
aliphatic chain or chains bearing an aromatic ring. The synthesis of carboxylic acids 6d, 6g and
6
k is presented in Scheme 2. Starting from aldehydes 9 and 12 and after a Wittig or a Hornerꢀ
Emmons olefination reaction, respectively, the unsaturated derivatives 10 and 13 were
hydrogenated leading to carboxylic acids 6d and 6g after saponification. Carboxylic acid 15 was
coupled with ethyl 5ꢀaminovalerate and saponification led to acid 6k. All the other carboxylic
acids were either commercially available or synthesized as described in literature (see
experimental part).
Since, among them, we identified a micromolar inhibitor of ATX, we synthesized the
pentafluoromethyl ketone derivative 18 as well as the boronic acid derivative 21, in order to
compare their inhibitory abilities. Compound 18 was synthesized by conversion of 17 into the
corresponding chloride and immediate treatment with pentafluoropropionic anhydride in the
3
4
presence of pyridine (Scheme 3). Commercially available boronic acid derivative 19 was
coupled with 6d using 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide (EDCI) as the coupling
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agent. The protecting group of 20 was removed under acidic conditions producing compound 21
(Scheme 3).
To introduce chirality in the backbone of hydroxamic acid potential inhibitors, the natural amino
acid glutamic acid was used as a starting material. Dimethyl (S)ꢀglutamate 22 was acylated by a
number of carboxylic acids to afford compounds 23aꢀe (Scheme 4). Hydroxamic acid derivatives
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4aꢀe were obtained by treatment of 23aꢀe with MeONa, while compound 25a containing two
hydroxamic acid functionalities was also synthesized (Scheme 4). Compounds 28a,b were
synthesized by a similar procedure, as shown in Scheme 5, starting from dimethyl (S)ꢀ2ꢀ
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aminohexanedioate (26), which was synthesized as described in literature.
We had previously described the synthesis of nonꢀnatural optically pure δꢀamino acids.
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Starting from Bocꢀprotected δꢀnorleucine (29), we synthesized methyl ester 31, which was
converted to hydroxamic acid 32 (Scheme 6).
In vitro inhibition of ATX ꢀ Structureꢀactivity relationship studies. All synthesized
hydroxamic acid derivatives were tested for their in vitro activity on recombinant mouse ATX
32,38
using the AmplexTM Red PLD assay kit (Molecular Probes, Interchim, Montlucon, France).
LPC (16:0 and 18:0) was used as substrate and the inhibition results are presented in Tables 1ꢀ3.
The molecular weights of the new synthetic compounds as well as the ClogP values, calculated
using ChemOffice Ultra 11.00, are included in Table 1ꢀ3. Reasonable molecular weights and
lipophilicity are important for the drugꢀlikeness of an ATX inhibitor and thus, such
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physicochemical properties should be taken into consideration.
At first, hydroxamic acids based on 4ꢀaminophenylacetic acid were tested and the results are
summarized in Table 1. Compound 8b containing a long acyl chain of fourteen carbon atoms
exhibited an IC value of 1 µM. Its comparison with compound 8a containing a shorter acyl
5
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chain of ten carbon atoms (IC higher than 5 µM) clearly indicates that the inhibitor should be
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lipophilic enough. To reduce the lipophilicity and the flexibility, the long chain was replaced by
various chains incorporating a benzene ring. None of the compounds 8c, 8d and 8e presented any
appreciable inhibitory activity. Insertion of an oxygen atom at the βꢀposition to the amide
carbonyl (compound 8f) led to interesting inhibition results (IC 1 µM), while insertion of the
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oxygen atom at the other side of the benzene ring (compound 8g) led to modest inhibitory
activity (IC 4 µM).
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Compound 8h consisting of a short acyl chain carrying a benzene ring linked by an amide bond
showed a promising IC value of 1.2 µM (Table 1). Its low lipophilicity (ClogP 0.89) is an
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additional attractive property. Increase of the carbon atoms number from 4 to 6 (compound 8i)
led to decrease of IC (2 µM). Inversion of the amide bond of 8h (compound 8j) led to similar
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results (IC 1 µM). The introduction of a substituent at the para position gave the same results in
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the case of a methoxy group (compound 8l), while it had a detrimental effect in the case of a
fluorine atom (compound 8k). Finally, the 7ꢀphenylheptanoyl chain (compound 8m) led again to
promising inhibitory potency (IC50 0.7 µM). It should be noticed that using 18:0 LPC as a
substrate, instead of 16:0 LPC, we observed lower IC50 values (0.2ꢀ0.3 µM) in the case of
compounds 8b, 8f and 8j, but a higher value for 8m (2 µM).
The next step was to confirm that the hydroxamic acid functionality indeed led to better
inhibitory activities in comparison to other functionalities (Table 2). Replacement of the
hydroxamic acid functionality by a simple carboxyl group (compound 17) abolished the
inhibitory activity. A pentafluoroethyl ketone functionality (compound 18) led to IC values of 3
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µM and 0.5 µM for 16:0 LPC and 18:0 LPC, respectively. The structurally related boronic acid
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derivative 21 exhibited IC50 values of 0.7 µM and 4 µM for 16:0 LPC and 18:0 LPC,
respectively.
The above structureꢀactivity relationship studies confirmed that the hydroxamic acid
functionality may efficiently serve as a novel functionality targeting ATX. Thus, we decided to
keep the hydroxamic acid functionality and the 6ꢀoxoꢀ6ꢀ(phenylamino)hexanoyl chain of
compound 8h and to replace the 4ꢀaminophenylacetic acid residue by an amino acid residue, thus
introducing chirality into our molecules. Glutamic acid was selected, because it confers a
distance of three carbon atoms between the amino group and the side chain carboxyl group.
Gratifying, compound 24a based on γꢀhydroxamate αꢀmethyl (S)ꢀglutamate exhibited an IC50
value of 0.4 µM (Table 3). However, conversion of both carboxylic groups of glutamic acid to
hydroxamic acid groups (compound 25a) led to loss of the inhibitory activity. The inversion of
the amide bond of 24a (compound 24b) destroyed the activity, while the introduction of
substituents at the paraꢀposition (compounds 24cꢀe) had also detrimental effects.
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Increase of the distance between the hydroxamic acid functionality and the amino group of the
amino acid residue by one carbon atom proved critical and led to a potent ATX inhibitor.
Compound 28a (GK400) presented an IC value of 0.05 µM (Table 3). Replacement of the 6ꢀ
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oxoꢀ6ꢀ(phenylamino)hexanoyl chain by the 7ꢀphenylheptanoyl chain (compound 28b) destroyed
the inhibitory activity. Finally, compound 32 (GK442) based on the nonꢀnatural amino acid δꢀ
norleucine exhibited potent inhibitory activity with an IC value of 0.06 µM, confirming the
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importance of the fourꢀcarbon atoms distance between the hydroxamic acid functionality and the
amino group and that the backbone of a δꢀamino acid confers the appropriate distances between
the essential functionalities.
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Among the novel hydroxamic acids developed in this work, compound 32 presents potent in
vitro inhibition of ATX (IC 0.06 µM) and possesses favorable lipophilicity (ClogP 1.11) and
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thus was selected for in vivo studies.
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Binding mode of inhibitor 32 to ATX. Computational studies, such as molecular docking and
virtual screening, may provide new insights into the inhibitory surfaces of crystal structure of
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ATX and may contribute to the development of improved small molecule ATX inhibitors.
Since hydroxamic acids represent a novel class of ATX inhibitors, docking studies were
performed to elucidate the binding mode and interactions of the most potent inhibitor 32. For the
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docking studies, the crystal structure of ATX (PDB code: 2XRG) was employed. The
nanomolar boronicꢀtype ATX inhibitor HA155 interacts with the active site forming a reversible
covalent bond between the boron atom and the hydroxyl group of the catalytic residue Thr209 of
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1
mouse ATX, which is further stabilized by two zinc ions (Figure 3a). The hydroxamic acid
functionality of inhibitor 32 (GK442) similarly interacts with Thr209 in addition to Asn230 and
Tyr 306 (Figure 3b).
In vivo effect of inhibitor 32 on a mouse pulmonary fibrosis model. IPF is a disease
4
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characterized by progressive and irreversible scarring of the lung.
The disease is fatal, with a
median survival time between 3 and 5 years from diagnosis. The pathogenesis of IPF is not
completely understood and limited therapeutic options exist. In 2014, two drugs that slow the
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5
progression of the disease, pirfenidone, a drug with poorly understood mechanisms, and
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6
nintedanib, a tyrosine kinase inhibitor, have been formally registered. However, it is unclear
whether these drugs improve symptoms such as dyspnoea and cough, or whether their beneficial
effect on functional decline results to increased survival. Thus, new therapeutic approaches that
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substantially improve the survival time and quality of life of the IPF patients are urgently
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needed.
Recent studies indicate an increase in LPA levels in the bronchoalveolar lavage fluid (BALF) of
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IPF patients, as well as increased ATX levels in the lung. Galapagos has recently reported that
a firstꢀinꢀclass ATX inhibitor, GLPG1690 (5, Figure 2), has been efficacious in a bleomycinꢀ
28
induced pulmonary fibrosis model in mice. GLPG1690 has entered clinical trials and most
recently positive topline results with GLPG1690 in patients with IPF in the FLORA Phase 2a
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trial have been announced.
The bleomycinꢀinduced pulmonary fibrosis model in rodents is considered the most commonly
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used animal model to investigate the potential of new agents as novel therapies for IPF. One of
our most potent in vitro hydroxamic acids, inhibitor 32, was tested for its efficacy in a 14ꢀday
model of pulmonary inflammation and fibrosis induced by bleomycin. C57Bl/6, BLMꢀ
challenged mice, were intraperitoneally injected with 30 mg/kg of inhibitor 32 or vehicle, once
daily for 15 days, started on dayꢀ1 prior to bleomycin installation. The administered dose was
selected based on reported efficiencies of known ATX inhibitors. The efficacy of inhibitor 32
was evaluated based on histopathological changes in lung architecture, BALF total inflammatory
cells as well as BALF’s protein and collagen content. As shown in Figure 4A, bleomycin
administration resulted in thickening of the alveolar septae, alveolar inflammation and formation
of fibrotic masses. ATX inhibition improved lung architecture; contiguous fibrotic walls and
fewer fibrotic lesions were predominantly observed in microscopic field of HE lung tissues.
Moreover, bleomycin administration caused inflammatory cell influx (neutrophils, macrophages
and lymphocytes, etc.) in bronchoalveolar lavage fluid as compared to control group (Figure 4B),
which was decreased by 50% upon inhibitor 32 treatment. Furthermore, bleomycin lung injury
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induced an increase in pulmonary vascular permeability, which was reflected by an increase in
BALF total protein after challenge (8ꢀfold compared to control group, Figure 4C). The total
protein concentration in BALFs of BLMꢀchallenged mice that received the inhibitor 32 was
markedly reduced compared to vehicle group (Figure 4C). Another hallmark of bleomycin
injury, increased collagen production and deposition in lung and BAL fluids, was also abrogated
by inhibitor 32 (Figure 4D). In addition, BALF ATX activity reduced back to normal level after
treatment with inhibitor 32 (Figure 4E). Taken together these observations suggested that the
hydroxamic acid inhibitors have beneficial effects in the development of pulmonary fibrosis.
An LC−MS/MS method for the determination of inhibitor 32 was developed and its levels in
mice plasma were quantified. Blood was sampled 1, 3 and 6 h post dosing for the determination
of the levels of inhibitor 32 and mean plasma concentrations of 32 are shown in Figure 4F.
Following i.p. administration of inhibitor 32 at a dose of 30 mg/kg, a concentration of 70 ng/mL
was detected after 1 h, while after 3 and 6 h 35 ng/mL were present in plasma.
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CONCLUSION
In conclusion, we describe a novel class of ATX inhibitors based on the hydroxamic acid
functionality. Structureꢀactivity relationship studies led to identification of hydroxamic acid
derivatives of δꢀamino acids presenting inhibitory potency over ATX at the nanomolar range and
highlighting the importance of the fourꢀcarbon atoms distance between the hydroxamic acid
functionality and the amino group. Inhibitor 32, a hydroxamic acid derivative based on δꢀ
norleucine, presented an IC value of 60 nM and exhibited promising efficacy in a mouse model
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of pulmonary inflammation and fibrosis induced by bleomycin. The low lipophilicity of inhibitor
2 (ClogP 1.11) together with the fact that the hydroxamic acid functionality is present in
3
marketed drugs make it an attractive candidate for further studies. The novel hydroxamic ATX
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inhibitors may be excellent tools for the study of the role of the enzyme in cells and in animals
and may contribute to the development of novel medicinal agents for the treatment of
inflammatory diseases.
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EXPERIMENTAL SECTION
General. Chromatographic purification of products was accomplished using Merck Silica Gel 60
(70ꢀ230 or 230ꢀ400 mesh). Thinꢀlayer chromatography (TLC) was performed on Silica Gel 60
F254 aluminum plates. TLC pots were visualized with UV light and/or phosphomolybdic acid in
1
EtOH. Melting points were determined using a Büchi 530 apparatus and were uncorrected. H
1
3
and C NMR spectra were recorded on a Varian Mercury (200 MHz and 50 MHz respectively)
in CDCl , CD OD and DMSOꢀd6. Chemical shifts are given in ppm, and coupling constants (J)
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3
in Hz. Peak multiplicities are described as follows: s, singlet, d, doublet, t, triplet and m,
multiplet. Electron spray ionization (ESI) mass spectra were recorded on a Finnigan, Surveyor
MSQ Plus spectrometer. HRMS spectra were recorded on a Bruker Maxis Impact QTOF
Spectrometer. Dichloromethane was dried by standard procedures and stored over molecular
sieves. All other solvents and chemicals were reagent grade and used without further
purification. The purity of all compounds subjected to biological tests was determined by
analytical HPLC, and was found to be ≥95%. HPLC analyses were carried out on a Shimadzu
LCꢀ2010AHT system and an ODS Hypersil (250 x 4.6 mm, 5 ꢁm) analytical column, using
H O/acetonitrile 20/80 v/v, at a flow rate of 1.0 mL/min.
2
Synthesis. Carboxylic acids 6aꢀc, 6m and 4ꢀaminophenylboronic acid pinacol ester 19 are
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53
54
54
commercially available. Carboxylic acids 6e,
6f,
6h, 6i, 6j,
6l, ethyl 4ꢀ
5
5
56
57
aminophenylacetate.HCl, ethyl 5ꢀaminovaleracetate.HCl, diethyl (S)ꢀglutamate.HCl 22 and
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dimethyl (S)ꢀ2ꢀaminohexanedioate 26 were synthesized as described elsewhere and their
analytical data are in accordance with literature.
A. General procedure for saponification of esters. A solution of the ester (1.00 mmol) in 1,4ꢀ
dioxane (2 mL) was treated with NaOH 1N (1.5 equiv, 1.5 mL, 1.50 mmol) at room temperature
overnight. The solvent was removed in vacuo, H O (10 mL) was added and extracted with Et O
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2
(
10 mL). Then the aqueous phase was acidified with 1N HCl and extracted with Et O (3 x 10
2
mL). The solvent was removed in vacuo to afford the acid.
B. General procedure for coupling. To a stirred solution of the amine (1.0 mmol) in anhydrous
CH Cl (10 mL), Et N (2.2 equiv, 0.31 mL, 2.2 mmol in case of hydrochloride salt of the amine
2
2
3
or 1.1 equiv, 0.15 mL, 1.1 mmol in case of free amine), EDCI (1.2 equiv, 230 mg, 1.2 mmol) and
o
the appropriate acid (1.2 equiv, 1.2 mmol) were added at 0 C. HOBt (1.0 equiv, 1.0 mmol, 140
mg) was added if there existed an asymmetric centre. The reaction was stirred overnight at room
temperature under argon and then washed with H O. The product was obtained after drying the
2
organic phase over anhydrous Na SO , evaporation of the solvent under reduced pressure and
2
4
purification by column chromatography.
C. General procedure for the synthesis of hydroxamic acids. To a stirred solution of esters
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aꢀm and 31 (1.0 mmol) in EtOH (4 mL), which was cooled at 0 C, NH OH.HCl (10.0 equiv,
2
6
95 mg, 10.0 mmol) and a solution of 21% w/w EtONa (20.0 equiv, 1.36 g, 20.0 mmol) in EtOH
(
7.25 mL) were added. The solution was stirred at room temperature for 2ꢀ24 hours under argon
and quenched by adding HCl 6N. The pH was adjusted 8 by the addition of 1N NaOH and the
organic solvent was evaporated under reduced pressure. Chloroform was added and the product,
which remained insoluble, was collected on a filter and washed consecutively with chloroform,
methanol and diethyl ether.
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D. General procedure for the hydrogenation of esters. A mixture of the unsaturated ester 10
or 13 (1.00 mmol) in MeOH (10 mL) and 10% palladium on activated carbon (27 mg) was
hydrogenated for 12 h. After filtration through a pad of celite, the solvent was removed in vacuo
to give the saturated compound.
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E. General procedure for the synthesis of hydroxamic acids. To a stirred solution of esters
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3aꢀe, and 27a,b (1.0 mmol) in MeOH (4 mL), which was cooled at 0 C, NH OH.HCl (10.0
2
equiv, 695 mg, 10.0 mmol) and a solution of 25% w/v MeONa (20.0 equiv, 1.08 g, 20.0 mmol)
in MeOH (4.2 mL) were added. The solution was stirred at room temperature for 48ꢀ72 hours
under argon and quenched by adding HCl 6N.The pH was adjusted 8 by the addition of 1N
NaOH and the organic solvent was evaporated in vacuo. The residue was extracted with CHCl3
(
3 x 10 mL) and the organic layer was dried over anhydrous Na SO . The solvent was
2 4
evaporated and the crude product was purified by flash column chromatography on silica gel
eluting with methanol/chloroform 1:9.
Compounds 6d, 6g and 6k were synthesized from compounds 11, 14 and 16 by procedure A.
ο
1
3
ꢀ(4ꢀOctylphenyl)propanoic acid (6d). Yield 99%; white solid; mp: 44ꢀ46 C; Η NMR (200
MHz, CDCl ): δ 7.30ꢀ7.00 (m, 4H), 2.93 (t, J = 8.0 Hz, 2H), 2.80ꢀ2.50 (m, 4H), 1.80ꢀ1.50 (m,
3
1
3
2
1
2
H), 1.50ꢀ1.10 (m, 10H), 0.88 (t, J = 7.0 Hz, 3H); C NMR (200 MHz, CDCl ): δ 179.4, 141.0,
3
37.2, 128.5, 128.1, 35.7, 35.5, 31.9, 31.5, 30.2, 29.5, 29.4, 29.3, 22.7, 14.1; MS (ESI) m/z (%):
ꢀ
61.22 [(MꢀH) , 100].
ο
1
5ꢀ(4ꢀButoxyphenyl)pentanoic acid (6g). Yield 78%; white solid; mp: 74ꢀ76 C; Η NMR (200
MHz, CDCl ) δ 7.06 (d, J = 8.0 Hz, 2H), 6.80 (d, J = 8.0 Hz, 2H), 3.92 (t, J = 6.0 Hz, 2H), 2.55
3
1
3
(
t, J = 8.0 Hz, 2H), 2.35 (t, J = 8.0 Hz, 2H), 2.00ꢀ1.40 (m, 8H), 0.95 (t, J = 8.0 Hz, 3H); C
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NMR (200 MHz, CDCl ) δ 179.3, 157.3, 133.9, 129.2, 114.3, 67.7, 34.6, 33.8, 31.4, 31.0, 24.2,
3
ꢀ
1
9.3, 13.9; MS (ESI) m/z (%) 249.22 [(MꢀH) , 100].
ο
6ꢀ((4ꢀFluorophenyl)amino)ꢀ6ꢀoxohexanoic acid (6k). Yield 85%; pink solid; mp: 110ꢀ112 C;
1
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1
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1
1
2
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2
2
2
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ΗNMR (200 MHz, CDCl ) δ 7.90ꢀ7.60 (m, 2H), 7.37 (br s, 1H), 7.10ꢀ6.80 (m, 2H), 3.50ꢀ3.10
3
13
(
m, 2H), 2.40ꢀ2.00 (m, 2H), 1.80ꢀ1.40 (m, 4H); CNMR (200 MHz, CDCl ) δ 176.5, 167.3,
3
1
64.4 (d, J = 249.5 Hz, CꢀF), 130.2 (d, J = 3.0 Hz), 129.2 (d, J = 10.0 Hz), 115.2 (d, J = 21.5
1
9
Hz), 39.4, 33.3, 28.4, 21.8; F NMR (200 MHz, CDCl ) δ ꢀ108.9 (F); MS (ESI) m/z (%) 238.19
3
ꢀ
[
(MꢀH) , 100].
Compounds 7aꢀm were synthesized from compounds 6aꢀm by procedure B.
Ethyl 2ꢀ(4ꢀdecanamidophenyl)acetate (7a). Eluent ethyl acetate/petroleum ether (40ꢀ60 C)
o
ο
1
3
0:70. Yield 85%; white solid; mp 81ꢀ83 C; Η NMR (200 MHz, CDCl ): δ 7.45 (d, J = 8.0 Hz,
3
2
H), 7.36 (s, 1H), 7.20 (d, J = 8.0 Hz, 2H), 4.13 (q, J = 7.0 Hz, 2H), 3.55 (s, 2H), 2.32 (t, J = 8.0
1
3
Hz, 2H), 1.80ꢀ1.50 (m, 2H), 1.50ꢀ1.10 (m, 15H), 0.86 (t, J = 7.0 Hz, 3H); C NMR (200 MHz,
CDCl ): δ 171.7, 171.6, 137.0, 129.7, 119.9, 60.9, 40.8, 37.7, 31.8, 29.4, 29.3, 29.2, 25.6, 22.6,
3
ꢀ
1
4.1; MS (ESI) m/z (%): 332.35 [(MꢀH) , 100].
Ethyl 2ꢀ(4ꢀtetradecanamidophenyl)acetate (7b). Eluent ethyl acetate/petroleum ether (40ꢀ60
o
ο
1
C) 30:70. Yield 55%; white solid; mp 88ꢀ90 C; Η NMR (200 MHz, CDCl ): δ 7.46 (d, J = 8.0
3
Hz, 2H), 7.24 (s, 1H), 7.19 (d, J = 8.0 Hz, 2H), 4.13 (q, J = 6.0 Hz, 2H), 3.56 (s, 2H), 2.33 (t, J
13
=
8.0 Hz, 2H), 1.80ꢀ1.50 (m, 2H), 1.40ꢀ1.10 (m, 23H), 0.87 (t, J = 6.0 Hz, 3H); C NMR (200
MHz, CDCl ): δ 171.7, 171.4, 138.0, 136.9, 129.8, 119.8, 60.9, 40.8, 37.8, 31.9, 29.6, 29.5, 29.4,
3
+
2
9.3, 25.6, 22.7, 14.1; MS (ESI) m/z (%): 390.40 [(M+H) , 100].
Ethyl 2ꢀ(4ꢀ(4ꢀoctylbenzamido)phenyl)acetate (7c). Eluent ethyl acetate/petroleum ether (40ꢀ60
o
ο
1
C) 40:60. Yield 41%; white solid; mp 114ꢀ116 C; Η NMR (200 MHz, CDCl ): δ 7.85 (s, 1H),
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.76 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.26 (m, 4H), 4.13 (d, J = 7.0 Hz, 2H), 3.58
(s, 2H), 2.65 (t, J = 8.0 Hz, 2H,), 1.80ꢀ1.50 (m, 2H), 1.40ꢀ1.00 (m, 10H), 1.23 (t, J = 7.0 Hz,
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3
3H), 0.83 (t, J = 8.0 Hz, 3H); C NMR (200 MHz, CDCl ): δ 171.6, 165.7, 147.3, 137.0, 132.2,
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30.1, 129.8, 128.8, 127.0, 120.2, 60.9, 40.8, 35.8, 32.8, 31.8, 31.2, 29.4, 29.2, 22.6, 14.2, 14.1;
ꢀ
MS (ESI) m/z (%): 394.28 [(MꢀH) , 100].
Ethyl
2ꢀ(4ꢀ(3ꢀ(4ꢀoctylphenyl)propanamido)phenyl)acetate
(7d).
Eluent
ethyl
o
ο
1
acetate/petroleum ether (40ꢀ60 C) 40:60. Yield 61%; white solid; mp 99ꢀ101 C; Η NMR (200
MHz, CDCl ): δ 8.00 (s, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.20ꢀ7.00 (m, 6H), 4.12 (q, J = 8.0 Hz,
3
2
H), 3.54 (s, 2H), 2.97 (t, J = 8.0 Hz, 2H), 2.58 (q, J = 8.0 Hz, 4H), 1.70ꢀ1.40 (m, 2H), 1.40ꢀ
1
3
1.20 (m, 10H), 1.23 (t, J = 8.0 Hz, 3H), 0.89 (t, J = 8.0 Hz, 3H,); C NMR (200 MHz, CDCl ):
3
δ 171.8, 170.8, 140.7, 137.7, 136.9, 129.5, 128.4, 128.1, 120.1, 60.8, 40.6, 39.1, 35.4, 31.8, 31.5,
ꢀ
3
1.0, 29.4, 29.3, 29.1, 22.5, 14.0; MS (ESI) m/z (%): 422.36 [(MꢀH) , 100].
Ethyl
2ꢀ(4ꢀ(5ꢀ([1,1'ꢀbiphenyl]ꢀ4ꢀyl)pentanamido)phenyl)acetate (7e).
Eluent
ethyl
o
ο
1
acetate/petroleum ether (40ꢀ60 C) 40:60. Yield 85%; white solid; mp 136ꢀ138 C; Η NMR (200
MHz, CDCl ): δ 7.70 (s,1H), 7.69ꢀ7.10 (m, 13H), 4.13 (q, J = 8.0 Hz, 2H), 3.55 (s, 2H), 2.66 (t,
3
13
J = 8.0 Hz, 2H),2.34 (t, J = 8.0 Hz, 2H), 1.90ꢀ1.50 (m, 4H), 1.24 (t, J = 8.0 Hz, 3H); C NMR
(200 MHz, CDCl ): δ 171.7, 171.3, 141.1, 140.9, 138.6, 136.9, 129.6, 128.7, 128.6, 126.9, 126.8,
3
+
1
19.9, 60.8, 40.7, 37.3, 35.2, 30.9, 25.2, 14.1; MS (ESI) m/z (%): 416.15 [(M+H) , 100].
Ethyl 2ꢀ(4ꢀ(2ꢀ(4ꢀoctylphenoxy)acetamido)phenyl)acetate (7f). Eluent ethyl acetate/petroleum
o
ο
1
ether (40ꢀ60 C) 40:60. Yield 40%; white solid; mp 80ꢀ82 C; Η NMR (200 MHz, CDCl ): δ
3
8
.27 (s, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 2H), 6.88 (d,
J = 8.0 Hz, 2H), 4.56 (s, 2H), 4.13 (q, J = 8.0 Hz, 2H), 3.57 (s, 2H), 2.55 (t, J = 6.0 Hz, 2H),
13
1.70ꢀ1.50 (m, 2H), 1.50ꢀ1.10 (m, 10H), 1.23 (t, J = 8.0 Hz, 3H), 0.86 (t, J = 8.0 Hz, 3H); C
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NMR (200 MHz, CDCl ): δ 171.5, 166.4, 155.0, 137.0, 135.8, 130.6, 129.9, 129.6, 120.2, 114.6,
3
ꢀ
6
7.8, 60.9, 40.8, 35.0, 31.8, 31.6, 29.4, 29.2, 22.6, 14.2, 14.1; MS (ESI) m/z (%): 424.35 [(MꢀH)
, 100].
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Ethyl
2ꢀ(4ꢀ(5ꢀ(4ꢀbutoxyphenyl)pentanamido)phenyl)acetate
(7g).
Eluent
ethyl
o
ο
1
acetate/petroleum ether (40ꢀ60 C) 20:80. Yield 49%; white solid, mp: 132ꢀ134 C; Η NMR
(
200 MHz, CDCl ): δ 7.43 (d, J = 8.0 Hz, 2H), 7.40ꢀ7.10 (m, 2H), 7.06 (d, J = 8.0 Hz, 2H), 6.80
3
(d, J = 8.0 Hz, 2H), 6.69 (d, J = 8.0 Hz, 1H), 4.12 (q, J = 8.0 Hz, 2H), 3.91 (t, J = 6.0 Hz, 2H),
3
.55 (s, 2H), 2.57 (t, J = 8.0 Hz, 2H), 2.33 (t, J = 8.0 Hz, 2H), 1.90ꢀ1.30 (m, 8H), 1.23 (t, J = 8.0
1
3
Hz, 3H), 0.95 (t, J = 8.0 Hz, 3H); C NMR (200 MHz, CDCl ): δ 171.6, 171.1, 157.3, 136.8,
3
133.9, 129.8, 129.2, 119.8, 114.3, 67.6, 60.9, 40.8, 37.6, 34.7, 31.2, 29.7, 25.2, 19.3, 14.2, 13.9;
+
MS (ESI) m/z (%): 429.25 [(M+NH ) , 90].
4
Ethyl
2ꢀ(4ꢀ(6ꢀoxoꢀ6ꢀ(phenylamino)hexanamido)phenyl)acetate
(7h).
Eluent
ethyl
o
ο
1
acetate/petroleum ether (40ꢀ60 C) 40:60. Yield 99%; white solid; mp 160ꢀ162 C; Η NMR (200
MHz, CDCl ): δ 7.91 (s, 1H), 7.82 (s, 1H), 7.70ꢀ7.40 (m, 4H), 7.40ꢀ7.00 (m, 5H), 4.12 (q, J =
3
13
6
.0 Hz, 2H), 3.55 (s, 2H), 2.60ꢀ2.20 (m, 4H), 1.90ꢀ1.50 (m, 4H), 1.22 (t, J = 6.0 Hz, 3H); C
NMR (200 MHz, CDCl ): δ 172.4, 171.0, 166.4, 138.0, 133.3, 129.6, 128.7, 124.0, 119.9, 119.8,
3
+
1
14.8, 60.9, 49.7, 48.9, 40.7, 36.6, 24.9, 14.0; MS (ESI) m/z (%): 383.07 [(M+H) , 100].
Ethyl
2ꢀ(4ꢀ(8ꢀoxoꢀ8ꢀ(phenylamino)octanamido)phenyl)acetate
(7i).
Eluent
ethyl
o
ο
1
acetate/petroleum ether (40ꢀ60 C) 30:70. Yield 71%; white solid; mp 140ꢀ142 C; Η NMR (200
MHz, CDCl ): δ 8.00ꢀ7.80 (m, 2H), 7.60ꢀ7.40 (m, 4H), 7.40ꢀ7.00 (m, 5H), 4.12 (q, J = 8.0 Hz,
3
2
H), 3.55 (s, 2H), 2.31 (m, 4H), 1.90ꢀ1.50 (m, 4H), 1.50ꢀ1.30 (m, 4H), 1.23 (t, J = 8.0 Hz, 3H);
1
3
C NMR (200 MHz, CDCl ): δ 172.6, 172.1, 168.8, 138.1, 137.2, 129.5, 128.6, 123.9, 119.9,
3
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19.8, 117.3, 60.9, 49.8, 48.1, 40.6, 36.8, 35.6, 28.2, 25.1, 13.9; MS (ESI) m/z (%): 411.31
+
[
(M+H) , 100].
Ethyl 2ꢀ(4ꢀ(5ꢀbenzamidopentanamido)phenyl)acetate (7j). Eluent ethyl acetate/petroleum
o
ο
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
ether (40ꢀ60 C) 40:60. Yield 52%; yellowish solid; mp 148ꢀ150 C; Η NMR (200 MHz,
CDCl ): δ 8.43 (s, 1H), 7.78 (d, J = 8.0 Hz, 2H), 7.60ꢀ7.30 (m, 5H), 7.16 (d, J = 8.0 Hz, 2H),
3
6
.92 (t, J = 6.0 Hz, 1H), 4.11 (q, J = 8.0 Hz, 2H), 3.54 (s, 2H), 3.43 (q, J = 6.0 Hz, 2H), 2.37 (t,
1
3
J = 6.0 Hz, 2H), 1.80ꢀ1.40 (m, 4H), 1.22 (t, J = 8.0 Hz, 3H); C NMR (200 MHz, CDCl ): δ
3
1
2
71.8, 171.8, 168.0, 137.3, 134.3, 131.4, 129.6, 129.5, 128.5, 126.9, 119.8, 60.9, 40.7, 39.0, 36.5,
+
8.8, 22.5, 14.1; MS (ESI) m/z (%): 383.19 [(M+H) , 100].
Ethyl 2ꢀ(4ꢀ(5ꢀ(4ꢀfluorobenzamido)pentanamido)phenyl)acetate (7k).
Eluent ethyl
o
ο
1
acetate/petroleum ether (40ꢀ60 C) 40:60. Yield 53%; white solid; mp 146ꢀ148 C; Η NMR (200
MHz, CDCl ): δ 8.74 (s, 1H), 7.90ꢀ7.70 (m, 2H), 7.47 (d, J = 10.0 Hz, 2H), 7.32 (t, J = 5.0 Hz,
3
1H), 7.12 (d, J = 8.0 Hz, 2H), 6.98 (t, J = 8.0 Hz, 2H), 4.09 (q, J = 8.0 Hz, 2H), 3.52 (s, 2H),
3
.34 (q, J = 7.0 Hz, 2H), 2.31 (t, J = 7.0 Hz, 2H), 1.80ꢀ1.40 (m, 4H), 1.20 (t, J = 8.0 Hz, 3H);
13
C NMR (200 MHz, CDCl ): δ 171.9, 166.9, 164.7, 164.5 (d, J = 250.5 Hz, CꢀF), 137.3, 130.8
3
(d, J = 3 Hz, arom), 129.6, 129.5, 129.3 (d, J = 10 Hz, arom), 119.9, 115.3 (d, J = 22 Hz, arom),
1
9
6
0.9, 40.6, 39.3), 36.4, 28.7, 22.5, 14.0; F NMR (200 MHz, CDCl ): δ ꢀ108.8 (F); MS (ESI)
3
+
m/z (%): 401.21 [(M+H) , 100].
Ethyl 2ꢀ(4ꢀ(5ꢀ(4ꢀmethoxybenzamido)pentanamido)phenyl)acetate (7l). Eluent ethyl
o
ο
1
acetate/petroleum ether (40ꢀ60 C) 40:60. Yield 45%; white solid; mp 167ꢀ169 C; Η NMR (200
MHz, CDCl ): δ 8.62 (s, 1H), 7.90ꢀ7.60 (m, 2H), 7.60ꢀ7.40 (m, 2H), 7.20ꢀ7.00 (m, 2H), 6.95ꢀ
3
6
.90 (m, 1H), 6.90ꢀ6.70 (m, 2H), 4.10 (q, J = 8.0 Hz, 2H), 3.79 (s, 3H), 3.53 (s, 2H), 3.50ꢀ3.20
1
3
(m, 2H), 2.50ꢀ2.20 (m, 2H), 1.80ꢀ1.30 (m, 4H), 1.21 (t, J = 8.0 Hz, 3H); C NMR (200 MHz,
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CDCl ): δ 171.9, 167.5, 162.1, 155.2, 137.4, 129.6, 129.4, 128.8, 126.6, 119.9, 113.6, 60.9, 55.3,
3
+
4
0.7, 39.0, 36.5, 28.9, 22.6, 14.1; MS (ESI) m/z (%): 413.12 [(M+H) , 100].
Ethyl 2ꢀ(4ꢀ(7ꢀphenylheptanamido)phenyl)acetate (7m). Eluent ethyl acetate/petroleum ether
o
ο
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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5
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5
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5
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0
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2
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0
1
2
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0
1
2
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2
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8
9
0
(
40ꢀ60 C) 40:60. Yield 64%; White solid; mp 80ꢀ82 C; Η NMR (200 MHz, CDCl ): δ 8.30 (s,
3
1
H), 7.48 (d, J = 8.0 Hz, 2H), 7.40ꢀ7.00 (m, 7H), 4.14 (q, J = 6.0 Hz, 2H), 3.55 (s, 2H), 2.58 (t, J
8.0 Hz, 2H), 2.30 (t, J = 8.0 Hz, 2H), 1.90ꢀ1.50 (m, 4H), 1.50ꢀ1.20 (m, 4H), 1.24 (t, J = 6.0
=
1
3
Hz, 3H); C NMR (200 MHz, CDCl ): δ 171.8, 171.7, 142.4, 137.1, 129.4, 129.3, 128.1, 128.0,
3
1
25.4, 119.9, 60.7, 40.5, 37.2, 35.6, 31.1, 28.9, 28.8, 25.4, 14.0; MS (ESI) m/z (%): 366.26 [(Mꢀ
ꢀ
H) , 100].
Compounds 8aꢀm were synthesized from compounds 7aꢀm by procedure C.
Nꢀ(4ꢀ(2ꢀ(Hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)decanamide (8a). Yield 75%; white solid; mp
ο
1
1
87ꢀ189 C; Η NMR (200 MHz, DMSO): δ 10.63 (s, 1H), 9.83 (s, 1H), 8.83 (s, 1H), 7.49 (d, J
= 8.0 Hz, 2H), 7.15 (d, J = 8.0 Hz, 2H), 3.20 (s, 2H), 2.27 (t, J = 8.0 Hz, 2H), 1.80ꢀ1.40 (m,
1
3
2
H), 1.40ꢀ1.00 (m, 12H), 0.85 (t, J = 7.0 Hz, 3H); C NMR (200 MHz, DMSO): δ 171.2, 167.1,
1
37.8, 130.5, 129.1, 119.0, 38.8, 36.4, 31.4, 29.0, 28.9, 28.8, 25.2, 22.2, 14.0; HRMS (ESI): m/z
ꢀ
ꢀ
calculated for C H N O – H [M – H ]: 319.2027. Found: 319.2029.
18 28
2
3
Nꢀ(4ꢀ(2ꢀ(Hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)tetradecanamide (8b). Yield 67%; offꢀwhite
ο
1
solid; mp 112ꢀ114 C; Η NMR (200 MHz, DMSO): δ 10.74 (s, 1H), 9.90 (s, 1H), 8.94 (s, 1H),
.47 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 3.20 (s, 2H), 2.25 (t, J = 8.0 Hz, 2H), 1.70ꢀ
7
1
3
1.40 (m, 2H), 1.40ꢀ1.00 (m, 20H), 0.82 (t, J = 6.0 Hz, 3H); C NMR (200 MHz, CD OD): δ
3
1
69.3, 159.6, 132.4, 130.7, 129.9, 121.2, 41.4, 40.2, 37.8, 34.8, 33.7, 33.1, 30.8, 30.7, 30.5, 27.0,
ꢀ
ꢀ
2
4.1, 23.8, 22.7, 14.5; HRMS (ESI): m/z calculated for C H N O – H [M – H ]: 375.2653.
22 36 2 3
Found: 375.2675.
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Nꢀ(4ꢀ(2ꢀ(Hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)ꢀ4ꢀoctylbenzamide (8c). Yield 66%; white solid;
ο
1
mp 240ꢀ242 C; Η NMR (200 MHz, DMSO): δ 10.67 (s, 1H), 10.13 (s, 1H), 8.86 (s, 1H), 7.84
(d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
1
.24 (s, 2H), 2.62 (t, J = 8.0 Hz, 2H), 1.80ꢀ1.40 (m, 2H), 1.40ꢀ1.00 (m, 10H), 0.83 (t, J = 8.0 Hz,
13
H); C NMR (200 MHz, DMSO): δ 167.4, 165.5, 146.4, 137.8, 132.4, 131.3, 129.2, 128.4,
27.8, 120.4, 35.1, 31.4, 30.9, 28.9, 28.8, 22.2, 14.1; HRMS (ESI): m/z calculated for
ꢀ
ꢀ
C H N O – H [M – H ]: 381.2184. Found: 381.2185.
23
30
2
3
Nꢀ(4ꢀ(2ꢀ(Hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)ꢀ3ꢀ(4ꢀoctylphenyl)propanamide (8d). Yield
ο
1
7
3%; white solid; mp 195ꢀ197 C; Η NMR (200 MHz, DMSO): δ 10.67 (s, 1H), 9.90 (s, 1H),
8.88 (s, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.30ꢀ6.90 (m, 6H), 3.21 (s, 2H), 2.85 (t, J = 6.0 Hz, 2H),
13
2
.57 (t, J = 6.0 Hz, 4H), 1.70ꢀ1.40 (m, 2H), 1.40ꢀ1.00 (m, 10H), 0.84 (t, J = 6.0 Hz, 3H); C
NMR (200 MHz, DMSO): δ 170.6, 167.5, 140.1, 138.5, 137.8, 130.8, 129.3, 128.4, 128.3, 119.2,
ꢀ
35.0, 31.5, 31.2, 30.7, 29.0, 28.9, 22.3, 14.2; HRMS (ESI): m/z calculated for C H N O – H
2
5
34
2
3
ꢀ
[
M – H ]: 408.2497. Found: 409.2497.
5
5
1
2
1
ꢀ([1,1'ꢀBiphenyl]ꢀ4ꢀyl)ꢀNꢀ(4ꢀ(2ꢀ(hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)pentanamide (8e). Yield
ο
1
2%; offꢀwhite solid; mp: 181ꢀ183 C; Η NMR (200 MHz, DMSO): δ 10.67 (s, 1H), 9.87 (s,
H), 8.83 (s, 1H), 7.80ꢀ7.20 (m, 8H), 7.20ꢀ7.00 (m, 5H), 3.55 (s, 2H), 2.64 (t, J = 8.0 Hz, 2H),
13
.32 (t, J = 8.0 Hz, 2H), 1.70ꢀ1.40 (m, 4H); C NMR (200 MHz, DMSO): δ 170.9, 169.5, 156.2,
54.3, 139.4, 135.8, 128.6, 126.9, 125.1, 124.5, 117.0, 34.3, 34.2, 32.6, 28.7, 25.3; HRMS (ESI):
ꢀ
ꢀ
m/z calculated for C H N O – H [M – H ]: 401.1871. Found: 401.1870.
2
5
26
2
3
NꢀHydroxyꢀ2ꢀ(4ꢀ(2ꢀ(4ꢀoctylphenoxy)acetamido)phenyl)acetamide (8f). Yield 55%; white
ο
1
solid; mp 182ꢀ184 C; Η NMR (200 MHz, DMSO): δ 10.68 (s, 1H), 9.97 (s, 1H), 8.81 (s, 1H),
7.70ꢀ7.40 (m, 2H), 7.40ꢀ7.00 (m, 4H), 7.00ꢀ6.80 (m, 2H), 4.63 (s, 2H), 3.25 (s, 2H), 2.50 (t, J =
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13
6
.0 Hz, 2H), 1.70ꢀ1.40 (m, 2H), 1.40ꢀ1.00 (m, 10H), 0.85 (t, J = 6.0 Hz, 3H); C NMR (200
MHz, DMSO): δ 167.4, 166.8, 156.0, 136.9, 135.2, 131.5, 129.3, 119.8, 114.6, 67.4, 34.4, 31.4,
ꢀ
ꢀ
29.0, 28.8, 22.3, 14.0; HRMS (ESI): m/z calculated for C H N O – H [M – H ]: 411.2289.
2
4
32
2
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Found: 411.2289.
5
4
1
ꢀ(4ꢀButoxyphenyl)ꢀNꢀ(4ꢀ(2ꢀ(hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)pentanamide (8g). Yield
ο
1
6%; offꢀwhite solid; mp 198ꢀ200 C; Η NMR (200 MHz, DMSO): δ 10.62 (s, 1H), 9.83 (s,
H), 8.81 (s, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.30ꢀ6.90 (m, 4H), 6.81 (d, J = 6.0 Hz, 2H), 3.90 (t, J
=
6.0 Hz, 2H), 3.20 (s, 2H), 2.29 (t, J = 8.0 Hz, 2H), 1.80ꢀ1.20 (m, 10H), 0.91 (t, J = 8.0 Hz,
1
3
3
H); C NMR (200 MHz, DMSO): δ 171.1, 167.2, 156.8, 137.8, 133.8, 130.5, 129.1, 119.0,
ꢀ
114.2, 67.0, 36.3, 34.1, 30.9, 24.8, 18.8, 13.7; HRMS (ESI): m/z calculated for C H N O – H
2
3
30
2
4
ꢀ
[
M – H ]: 397.2133. Found: 397.2126.
1
6
N ꢀ(4ꢀ(2ꢀ(Hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)ꢀN ꢀphenyladipamide (8h). Yield 70%; offꢀ
ο
1
white solid; mp 253ꢀ255 C; Η NMR (200 MHz, DMSO): δ 10.20 (s, 1H), 10.16 (s, 1H), 8.49
(
s, 1H), 7.80ꢀ7.40 (m, 4H), 7.40ꢀ7.10 (m, 5H), 7.10ꢀ6.90 (m, 1H), 3.23 (s, 2H), 2.40ꢀ2.20 (m,
13
4
1
H), 1.80ꢀ1.30 (m, 4H); C NMR (200 MHz, DMSO): δ 171.4, 171.2, 167.1, 139.5, 137.9,
30.7, 129.2, 128.7, 123.0, 119.1, 119.0, 36.2, 30.8, 24.9; HRMS (ESI): m/z calculated for
ꢀ
ꢀ
C H N O – H [M – H ]: 368.1616. Found: 368.1615.
20
23
3
4
1
8
N ꢀ(4ꢀ(2ꢀ(Hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)ꢀN ꢀphenyloctanediamide (8i). Yield 96%;
ο
1
white solid; mp 195ꢀ197 C; Η NMR (200 MHz, DMSO): δ 10.64 (s, 1H), 9.84 (s, 2H), 8.83 (s,
1H), 7.70ꢀ7.40 (m, 4H), 7.40ꢀ7.10 (m, 4H), 7.10ꢀ6.90 (m, 1H), 3.22 (s, 2H), 2.40ꢀ2.10 (m, 4H),
13
1
1
.80ꢀ1.40 (m, 4H), 1.40ꢀ1.10 (m, 4H); C NMR (200 MHz, DMSO): δ 171.3, 171.1, 167.2,
39.4, 137.8, 130.5, 129.1, 128.7, 123.0, 119.1, 119.0, 38.8, 36.4, 28.6, 25.1; HRMS (ESI): m/z
ꢀ
ꢀ
calculated for C H N O – H [M – H ]: 396.1929. Found: 396.1932.
2
2
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Nꢀ(5ꢀ((4ꢀ(2ꢀ(Hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)amino)ꢀ5ꢀoxopentyl)benzamide (8j). Yield
ο
1
5
9%; offꢀwhite solid; mp 179ꢀ181 C; Η NMR (200 MHz, DMSO): δ 10.68 (s, 1H), 9.90 (s,
1H), 8.87 (s, 1H), 8.51 (t, J = 6.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.60ꢀ7.30 (m, 5H), 7.14 (d, J
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
=
8.0 Hz, 2H), 3.27 (t, J = 6.0 Hz, 2H), 3.20 (s, 2H), 2.31 (t, J = 6.0 Hz, 2H), 1.80ꢀ1.40 (m, 4H);
13
C NMR (200 MHz, DMSO): δ 171.4, 167.5, 166.4, 137.9, 134.8, 131.3, 130.7, 129.3, 128.5,
ꢀ
1
–
4
27.3, 119.2, 40.2, 39.8, 36.7, 29.4, 23.4; HRMS (ESI): m/z calculated for C H N O – H [M
20 23
3
4
ꢀ
H ]: 368.1616. Found: 368.1615.
ꢀFluoroꢀNꢀ(5ꢀ((4ꢀ(2ꢀ(hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)amino)ꢀ5ꢀoxopentyl)benzamide
ο
1
(
8k). Yield 88%; white solid; mp 269ꢀ271 C; Η NMR (200 MHz, DMSO): δ 9.89 (s, 2H), 8.56
(s, 2H), 7.91 (t, J = 7.0 Hz, 2H), 7.47 (d, J = 7.0 Hz, 2H), 7.40ꢀ7.20 (m, 2H), 7.14 (d, J = 8.0
13
Hz, 2H), 3.40ꢀ3.20 (m, 2H), 3.14 (s, 2H), 2.40ꢀ2.10 (m, 2H), 1.80ꢀ1.40 (m, 4H); C NMR (200
MHz, DMSO): δ 171.0, 166.3, 165.1, 164.8 (d, J = 240.0 Hz, CꢀF), 137.5, 131.1 (d, J = 3.0 Hz,
arom), 129.9, 129.2 (d, J = 8.0 Hz, arom), 129.1, 118.8, 115.2 (d, J = 21.0 Hz, arom), 39.2, 38.9,
1
9
3
6.1, 28.9, 22.8; F NMR (200 MHz, DMSO): δ ꢀ110.3 (F); HRMS (ESI): m/z calculated for
ꢀ
ꢀ
C H FN O – H [M – H ]: 386.1522. Found: 386.17522.
20
22
3
4
Nꢀ(5ꢀ((4ꢀ(2ꢀ(Hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)amino)ꢀ5ꢀoxopentyl)ꢀ4ꢀmethoxybenzamide
ο
1
(
8l). Yield 99%; white solid; mp 216ꢀ218 C; Η NMR (200 MHz, DMSO): δ 10.60 (s, 1H),
.85 (s, 1H), 8.81 (s, 1H), 8.34 (s, 1H), 7.81 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.14
d, J = 8.0 Hz, 2H), 6.97 (d, J = 8.0 Hz, 2H), 3.79 (s, 3H), 3.50ꢀ3.30 (m, 2H), 3.20 (s, 2H), 2.40ꢀ
9
(
13
2.10 (m, 2H), 1.80ꢀ1.30 (m, 4H); C NMR (200 MHz, DMSO): δ 171.1, 167.2, 165.6, 161.5,
37.8, 130.6, 129.2, 129.0, 126.9, 119.0, 113.5, 55.4, 38.9, 38.3, 36.1, 29.0, 22.8; HRMS (ESI):
1
ꢀ
ꢀ
m/z calculated for C H N O – H [M – H ]: 398.1721. Found: 398.1724.
2
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Nꢀ(4ꢀ(2ꢀ(Hydroxyamino)ꢀ2ꢀoxoethyl)phenyl)ꢀ7ꢀphenylheptanamide (8m). Yield 67%; offꢀ
ο
1
white solid; mp 176ꢀ178 C; Η NMR (200 MHz, DMSO): δ 10.64 (s, 1H), 9.85 (s, 1H), 8.84 (s,
1H), 7.51 (d, J = 8.0 Hz, 2H), 7.40ꢀ6.90 (m, 7H), 3.22 (s, 2H), 2.56 (t, J = 8.0 Hz, 2H), 2.27 (t, J
1
3
1
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2
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2
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
=
8.0 Hz, 2H), 1.70ꢀ1.50 (m, 4H), 1.40ꢀ1.00 (m, 4H); C NMR (200 MHz, DMSO): δ 171.2,
1
67.1, 142.3, 137.9, 130.6, 129.2, 128.3, 125.7, 119.0, 36.4, 35.2, 31.0, 28.5, 25.2; HRMS (ESI):
ꢀ
ꢀ
m/z calculated for C H N O – H [M – H ]: 353.1871. Found: 353.1872.
21 26
2
3
Methyl (E)ꢀ3ꢀ(4ꢀoctylphenyl)acrylate (10). A solution of aldehyde 9 (800 mg, 3.66 mmol) and
Ph P=CHCOOCH (1.5 equiv, 1.84 g, 5.50 mmol) in dry THF (5 mL) was refluxed under argon
3
3
overnight. The reaction mixture was then cooled to room temperature and the solvent evaporated
under reduced pressure. Then Et O (5 mL)was added, the mixture was filtered through a pad of
2
silica, and the solvent was removed in vacuo to afford the pure product. Yield 69% (0.69 g, 2.51
1
mmol); colourless oil; Η NMR (200 MHz, CDCl ): δ 7.68 (d, J =17.0 Hz, 1H), 7.43 (d, J = 8.0
3
Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.40 (d, J =17.0 Hz, 1H), 3.79 (s, 3H), 2.61 (t, J = 8.0 Hz,
13
2
H), 1.80ꢀ1.50 (m, 2H), 1.50ꢀ1.00 (m, 10H), 0.87 (t, J = 7.0 Hz, 3H); C NMR (200 MHz,
CDCl ): δ 167.6, 145.7, 144.9, 131.8, 128.9, 128.0, 116.6, 51.6, 35.8, 31.8, 31.2, 29.4, 29.2, 22.6,
3
+
2
1.6, 14.1; MS (ESI) m/z (%): 292.16 [(M+NH ) , 10].
4
Methyl 3ꢀ(4ꢀoctylphenyl)propanoate (11). Compound 11 was synthesized from compound 10
ο
1
by procedure D. Yield 88%; white solid; mp 97ꢀ99 C; ΗNMR (200 MHz, CDCl ): δ 7.30ꢀ7.00
3
(m, 4H), 3.67 (s, 3H), 2.93 (t, J = 8.0 Hz, 2H), 2.70ꢀ2.50 (m, 4H), 1.80ꢀ1.50 (m, 2H), 1.50ꢀ1.10
1
3
(m, 10H), 0.89 (t, J = 8.0 Hz, 3H); CNMR (200 MHz, CDCl ): δ 173.4, 140.8, 137.5, 128.4,
3
1
28.0, 51.5, 35.7, 35.5, 31.8, 31.5, 30.5, 29.4, 29.3, 29.2, 22.6, 14.1; MS (ESI) m/z (%): 294.41
+
[
(M+NH ) , 100].
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Ethyl (2E,4E)ꢀ5ꢀ(4ꢀbutoxyphenyl)pentaꢀ2,4ꢀdienoate (13). A suspension of aldehyde 12 (1.00
g, 5.61 mmol), triethyl 4ꢀphosphonocrotonate (1.5 equiv, 1.91 g, 8.42 mmol), lithium hydroxide
(1.5 equiv, 0.35 g, 8.42 mmol) and molecular sieves (beads, 4ꢀ8 mesh, 8.40 g, 1.50 g/mmol
aldehyde) in dry tetrahydrofuran (56 mL) was refluxed under argon for 24 h. The reaction
mixture was then cooled to room temperature, filtered through a thin pad of celite and the solvent
was evaporated under reduced pressure. The residual oil was purified by column
0
1
2
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4
5
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8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
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7
8
9
0
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8
9
0
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2
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5
6
7
8
9
0
o
chromatography on silica gel eluting with ether/petroleum ether (40ꢀ60 C) 20:80. Yield 68%
ο
1
(
1.57 g, 5.71 mmol); white solid; mp: 98ꢀ100 C; Η NMR (200 MHz, CDCl ) δ 7.50ꢀ7.30 (m,
3
3
H), 6.96ꢀ6.60 (m, 4H), 5.92 (d, J = 17.0 Hz, 1H), 4.22 (q, J = 8.0 Hz, 2H), 3.97 (t, J = 7.0 Hz,
2H), 1.90ꢀ1.60 (m, 2H), 1.60ꢀ1.40 (m, 2H), 1.30 (t, J = 8.0 Hz, 3H), 0.97 (t, J = 8.0 Hz, 3H);
13
CNMR (200 MHz, CDCl ) δ 167.3, 160.0, 145.0, 140.2, 132.0, 128.6, 123.9, 119.9, 114.7,
3
+
6
7.7, 60.2, 31.2, 19.2, 14.3, 13.8; MS (ESI) m/z (%) 275.25 [(M+H) , 100].
Ethyl 5ꢀ(4ꢀbutoxyphenyl)pentanoate (14). Compound 14 was synthesized from compound 13
1
by procedure D. Yield 84%; colorless oil; ΗNMR (200 MHz, CDCl ) δ 7.07 (d, J = 8.0 Hz, 2H),
3
6
.81 (d, J = 8.0 Hz, 2H), 4.12 (q, J = 8.0 Hz, 2H), 3.93 (t, J = 6.0 Hz, 2H), 2.56 (t, J = 6.0 Hz,
H), 2.31 (t, J = 8.0 Hz, 2H), 1.90ꢀ1.40 (m, 8H), 1.24 (t, J = 8.0 Hz, 3H),0.97 (t, J = 8.0 Hz, 3H);
2
1
3
CNMR (200 MHz, CDCl ) δ 173.7, 157.2, 134.0, 129.1, 128.6, 114.5, 114.3, 67.6, 60.2, 34.6,
3
+
3
4.2, 31.3, 31.1, 24.5, 19.2, 14.2, 13.8; MS (ESI) m/z (%) 296.24 [(M+NH ) , 100].
4
Ethyl 5ꢀ(4ꢀfluorobenzamido)pentanoate (16). Compound 16 was synthesized from compound
o
15 by procedure B. Eluent ethyl acetate/petroleum ether (40ꢀ60 C) 40:60. Yield 89%; offꢀwhite
ο
1
solid; mp: 74ꢀ76 C; ΗNMR (200 MHz, CDCl ) δ 7.90ꢀ7.40 (m, 3H), 6.90ꢀ6.50 (m, 2H), 3.75
3
(q, J = 8.0 Hz, 2H), 3.30ꢀ2.90 (m, 2H), 2.10ꢀ1.80 (m, 2H), 1.80ꢀ1.60 (m, 2H), 1.50ꢀ1.20 (m, 2H),
1
3
1.22 (t, J = 6.0 Hz, 3H); CNMR (200 MHz, CDCl ) δ 172.6, 166.3, 163.8 (d, J = 249.5 Hz, Cꢀ
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F), 130.3 (d, J = 3.0 Hz), 128.9 (d, J = 10.0 Hz), 114.4 (d, J = 21.5 Hz), 59.5, 38.9, 33.0, 28.2,
19
+
2
1.6, 13.4; FNMR (200 MHz, CDCl ) δ ꢀ109.9 (F); MS (ESI) m/z (%) 268.11 [(M+H) , 100].
3
2ꢀ(4ꢀ(6ꢀΟxoꢀ6ꢀ(phenylamino)hexanamido)phenyl)acetic acid (17). Compound 17 was
ο
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
synthesized from compound 7d by procedure A. Yield 98%; white solid; mp 215ꢀ217 C; Η
NMR (200 MHz, CD OD): δ 7.47 (m, 4H), 7.40ꢀ7.10 (m, 4H), 7.10ꢀ6.90 (m, 1H), 3.51 (s, 2H),
3
1
3
2
1
.50ꢀ2.10 (m, 4H), 1.90ꢀ1.60 (m, 4H); C NMR (200 MHz, CD OD): δ 177.0, 174.3,
3
64.9,138.4, 132.0, 130.7, 129.7, 125.1, 121.2, 119.8, 113.1, 41.1, 37.6, 30.9, 26.5, 22.1; HRMS
ꢀ
ꢀ
(
ESI): m/z calculated for C H N O – H [M – H ]: 353.1507. Found: 353.1508.
20 22 2 4
1
6
N ꢀ(4ꢀ(3,3,4,4,4ꢀPentafluoroꢀ2ꢀoxobutyl)phenyl)ꢀN ꢀphenyladipamide (18). Oxalyl chloride
(3.0 equiv, 0.93 mL 2 M solution, 1.86 mmol) and N,Nꢀdimethylformamide (25 µL) were added
to a solution of carboxylic acid 17 (220 mg, 0.62 mmol) in dry CH Cl (25 mL). After 2 h
2
2
stirring the solvent and excess reagent were evaporated under reduced pressure and the residue
was dissolved in dry dichloromethane (6 mL). Pyridine (8.0 equiv, 0.4 mL, 4.96 mmol) and
pentafluoropropionic anhydride (6.0 equiv, 0.73 mL, 3.72 mmol) were added dropwise to this
o
o
solution at 0 C consecutively. After 2.5 h at 0 C, ice was added and the mixture was stirred for
0 min. The two phases were separated and the organic phase was washed with brine and dried
Na SO ). The solvent was evaporated under reduced pressure, and the residual oil was purified
3
(
2
4
by flash column chromatography on silica gel eluting with methanol/chloroform 5:95.Yield 21%
ο
1
(
32 mg, 0.07 mmol); yellow solid; mp 184ꢀ186 C; Η NMR (200 MHz, CDCl ): δ 8.00ꢀ7.00 (m,
3
13
9H), 3.47 (s, 2H), 2.80ꢀ2.10 (m, 4H), 2.10ꢀ1.50 (m, 4H); C NMR (200 MHz, CDCl ): δ 196.2
3
(t, J = 26.1 Hz, COC F ), 172.6, 171.6, 130.7, 129.4, 128.6, 128.3, 123.9, 119.8, 119.6, 117.8
2 5
(
qt, J = 284.9 Hz, J = 34.1 Hz, CF ), 113.3, 106.9 (tq, J = 265.6 Hz, J = 37.9 Hz, CF ), 36.4,
1
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5
5
5
5
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6
19
3
0.6, 29.5, 24.9, 22.5; F NMR (CDCl ): δ ꢀ83.4 (CF ), ꢀ121.9 (CF ); HRMS (ESI): m/z
3 3 2
ꢀ
ꢀ
calculated for C H F N O – H [M – H ]: 455.1400. Found: 455.1406.
2
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21
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N ꢀPhenylꢀN ꢀ(4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)adipamide
(20).
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8
9
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1
2
3
4
5
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7
8
9
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Compound 20 was synthesized from compounds 6d and 19 by procedure B. Eluent ethyl
o
ο
1
acetate/petroleum ether (40ꢀ60 C) 40:60. Yield 60%; offꢀwhite solid; mp 211ꢀ213 C; Η NMR
(
200 MHz, DMSO): δ 10.05 (s, 1H), 9.91 (s, 1H), 7.70ꢀ7.50 (m, 6H), 7.28 (t, J = 8.0 Hz, 2H),
1
3
7
.02 (t, J = 8.0 Hz, 1H), 2.40ꢀ2.10 (m, 4H), 1.80ꢀ1.40 (m, 4H), 1.27 (s, 12H); C NMR (200
MHz, DMSO): δ 171.6, 171.3, 142.3, 139.4, 135.5, 128.8, 119.2, 118.2, 83.6, 38.3, 36.4, 25.1,
+
2
4.8, 14.4; MS (ESI) m/z (%): 423.31 [(M+H) , 100].
(4ꢀ(6ꢀOxoꢀ6ꢀ(phenylamino)hexanamido)phenyl)boronic acid (21). To a stirred solution of the
ester 20 (35 mg, 0.08 mmol) in THF (2.5 mL) an aqueous solution of HCl 1N (2.5 mL) was
added and the mixture was left under stirring for 24 hours. After evaporation of the solvent under
reduced pressure, CH Cl (10 mL) was added and washed with H O (10 mL) and brine (10 mL).
2
2
2
The product remained insoluble and was obtained after filtration and washing with CH Cl .Yield
2
2
ο
1
7
1% (20 mg, 0.059 mmol); white solid; mp 218ꢀ220 C; Η NMR (200 MHz, DMSO): δ 9.92 (d,
J = 8.0 Hz, 2H), 7.93 (s, 2H), 7.69 (d, J = 8.0 Hz, 2H), 7.65ꢀ7.40 (m, 4H), 7.40ꢀ7.10 (m, 2H),
1
3
7
.10ꢀ6.90 (m, 1H), 2.50ꢀ2.20 (m, 4H), 1.80ꢀ1.50 (m, 4H); C NMR (200 MHz, DMSO): δ 171.3,
1
71.2, 140.9, 139.3, 134.8, 128.6, 123.0, 119.2, 117.9, 36.4, 25.0; HRMS (ESI): m/z calculated
ꢀ
ꢀ
for C H BN O – H [M – H ]: 339.1522. Found: 339.1518.
1
8
21
2
4
Compounds 23aꢀe were synthesized from compound 22 by procedure B.
Dimethyl
(S)ꢀ(6ꢀoxoꢀ6ꢀ(phenylamino)hexanoyl)glutamate
(23a).
Eluent
ethyl
o
ο
20
D
acetate/petroleum ether (40ꢀ60 C) 40:60. Yield 99%; white solid; mp 116ꢀ118 C; [α]
=
1
+11.1 (c 1, CHCl ); Η NMR (200 MHz, CDCl ): δ 8.41 (s, 1H), 7.53 (d, J = 6.0 Hz, 2H), 7.26
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(
t, J = 6.0 Hz, 2H), 7.04 (t, J = 6.0 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 4.56 (t, J = 8.0 Hz, 1H),
1
3
3
.69 (s, 3H), 3.63 (s, 3H), 2.80ꢀ2.00 (m, 8H), 1.80ꢀ1.50 (m, 4H); C NMR (200 MHz, CDCl ): δ
3
173.3, 173.2, 172.4, 171.5, 138.2, 128.8, 123.9, 119.7, 52.5, 51.9, 51.6, 36.8, 35.5, 30.1, 26.7,
+
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4.8, 24.5; MS (ESI) m/z (%): 379.21 [(M+H) , 100].
Dimethyl (S)ꢀ(5ꢀbenzamidopentanoyl)glutamate (23b). Eluent ethyl acetate/petroleum ether
o
ο
20
1
(40ꢀ60 C) 40:60. Yield 99%; offꢀwhite solid; mp 38ꢀ40 C; [α] = +9.8 (c 1, CHCl ); Η NMR
D 3
(200 MHz, CDCl ): δ 7.75 (d, J = 7.0 Hz, 2H), 7.50ꢀ7.30 (m, 3H), 7.02 (s, 1H), 6.81 (s, 1H),
3
13
4
.60ꢀ4.40 (m, 1H), 3.64 (s, 3H), 3.58 (s, 3H), 2.60ꢀ1.80 (m, 8H), 1.80ꢀ1.40 (m, 4H); C NMR
(200 MHz, CDCl ): δ 173.2, 172.3, 170.5, 167.6, 134.4, 131.2, 128.3, 126.9, 52.3, 51.7, 51.5,
3
+
39.0, 35.2, 30.0, 28.5, 26.8, 22.3; MS (ESI) m/z (%): 379.31 [(M+H) , 100].
Dimethyl (S)ꢀ(6ꢀ((4ꢀfluorophenyl)amino)ꢀ6ꢀoxohexanoyl)glutamate (23c). Eluent ethyl
o
ο
20
D
acetate/petroleum ether (40ꢀ60 C) 40:60. Yield 94%; pink solid; mp 99ꢀ101 C; [α] = +9.6 (c
1
1, CHCl ); Η NMR (200 MHz, CDCl ): δ 8.49 (s, 1H), 7.60ꢀ7.30 (m, 2H) 7.10ꢀ6.80 (m, 2H),
3
3
6
.98 (s, 1H), 4.70ꢀ4.40 (m, 1H), 3.65 (s, 3H), 3.60 (s, 3H), 2.70ꢀ1.80 (m, 8H), 1.80ꢀ1.40 (m, 4H);
13
C NMR (200 MHz, CDCl ): δ 173.3, 173.2, 172.3, 171.5, 165.6 (d, J = 257.0 Hz, CꢀF), 134.6
3
(d, J = 3.0 Hz, arom), 121.9 (d, J = 8.0 Hz, arom), 115.5 (d, J = 22.5 Hz, arom), 52.4, 51.8,
19
5
1.7, 36.6, 35.5, 30.0, 26.7, 24.8, 24.5; F NMR (200 MHz, CDCl ): δ ꢀ119.0 (F); MS (ESI) m/z
3
ꢀ
(%): 395.13 [(MꢀH) , 100].
Dimethyl (S)ꢀ(6ꢀ((4ꢀmethoxyphenyl)amino)ꢀ6ꢀoxohexanoyl)glutamate (23d). Eluent ethyl
o
ο
20
D
acetate/petroleum ether (40ꢀ60 C) 40:60. Yield 70%; pink solid; mp 127ꢀ129 C; [α] = +9.5
1
(c 1, CHCl ); Η NMR (200 MHz, CDCl ): δ 8.74 (s, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.22 (d, J =
3
3
8
.0 Hz, 1H), 6.65 (d, J = 8.0 Hz, 2H), 4.50ꢀ4.30 (m, 1H), 3.60 (s, 3H), 3.55 (s, 3H), 3.50 (s, 3H),
1
3
2.50ꢀ1.80 (m, 8H), 1.70ꢀ1.40 (m, 4H); C NMR (200 MHz, CDCl ): δ 173.2, 172.9, 172.1,
3
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Journal of Medicinal Chemistry
Page 28 of 67
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71.3, 155.6, 131.2, 121.5, 113.5, 55.0, 52.0, 51.4, 51.3, 36.2, 35.2, 29.8, 26.3, 24.7, 24.6; MS
+
(ESI) m/z (%): 431.21 [(M+Na) , 100].
Dimethyl (S)ꢀ(6ꢀ((4ꢀethoxyphenyl)amino)ꢀ6ꢀoxohexanoyl)glutamate (23e). Eluent ethyl
o
ο
20
D
0
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0
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0
acetate/petroleum ether (40ꢀ60 C) 40:60. Yield 99%; pink solid; mp 122ꢀ124 C; [α] = +9.6
1
(c 1, CHCl ); Η NMR (200 MHz, CDCl ): δ 8.48 (s, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.14 (s, 1H),
3
3
6
2
1
2
.77 (d, J = 8.0 Hz, 2H), 4.60ꢀ4.40 (m, 1H), 3.94 (q, J = 8.0 Hz, 2H), 3.67 (s, 3H), 3.61 (s, 3H),
1
3
.50ꢀ1.80 (m, 8H), 1.80ꢀ1.50 (m, 4H), 1.33 (t, J = 8.0 Hz, 3H); C NMR (200 MHz, CDCl ): δ
3
73.8, 173.4, 172.4, 171.8, 155.3, 131.3, 121.6, 114.5, 63.6, 52.4, 51.6, 50.3, 36.6, 35.4, 30.0,
+
6.7, 24.9, 24.6, 14.7; MS (ESI) m/z (%): 423.27 [(M+H) , 100].
Compounds 24aꢀe were synthesized from compounds 23aꢀe by procedure E.
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2
Methyl (S)ꢀN ꢀhydroxyꢀN ꢀ(6ꢀoxoꢀ6ꢀ(phenylamino)hexanoyl)glutaminate (24a). Yield 20%;
ο
20
D
1
offꢀwhite solid; mp 118ꢀ120 C; [α] = ꢀ13.0 (c 0.6, MeOH); Η NMR (200 MHz, CD OD): δ
3
7.43 (d, J = 8.0 Hz, 2H), 7.17 (t, J = 8.0 Hz, 2H), 6.97 (t, J = 8.0 Hz, 1H), 4.30ꢀ4.00 (m, 1H),
1
3
3
.52 (s, 3H), 2.50ꢀ1.70 (m, 8H), 1.70ꢀ1.40 (m, 4H); C NMR (200 MHz, CD OD): δ 175.8,
3
1
74.7, 174.2, 170.6, 139.9, 129.8, 125.1, 121.1, 52.2, 51.5, 37.6, 36.4, 30.9, 28.3, 27.3, 26.4;
ꢀ
ꢀ
HRMS (ESI): m/z calculated for C H N O – H [M – H ]: 378.1671. Found: 378.1677.
18 25
3
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Methyl (S)ꢀN ꢀ(5ꢀbenzamidopentanoyl)ꢀN ꢀhydroxyglutaminate (24b). Yield 18%; offꢀwhite
ο
20
D
1
solid; mp 139ꢀ141 C; [α] = ꢀ14.0 (c 0.5, MeOH); Η NMR (200 MHz, CD OD): δ 7.77 (d, J
3
=
8.0 Hz, 2H), 7.60ꢀ7.30 (m, 3H), 4.30ꢀ4.10 (m, 1H), 3.60 (s, 3H), 2.50ꢀ1.80 (m, 8H), 1.80ꢀ1.40
1
3
(m, 4H); C NMR (200 MHz, CD OD): δ 175.9, 174.7, 170.6, 170.3, 135.8, 132.6, 129.5, 128.3,
3
ꢀ
5
2.2, 51.5, 40.5, 36.2, 31.0, 29.9, 28.3, 24.2; HRMS (ESI): m/z calculated for C H N O – H
18 25 3 6
ꢀ
[
M – H ]: 378.1671. Found: 378.1670.
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Journal of Medicinal Chemistry
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Methyl (S)ꢀN ꢀ(6ꢀ((4ꢀfluorophenyl)amino)ꢀ6ꢀoxohexanoyl)ꢀN ꢀhydroxyglutaminate (24c).
ο
20
1
Yield 19%; offꢀwhite solid; mp 147ꢀ149 C; [α]D = ꢀ18.0 (c 1, MeOH); Η NMR (200 MHz,
CD OD): δ 7.60ꢀ7.40 (m, 2H) 6.99 (t, J = 8.0 Hz, 2H), 4.26 (t, J = 8.0 Hz, 1H), 3.61 (s, 3H),
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0
2
1
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.60ꢀ1.80 (m, 8H), 1.80ꢀ1.40 (m, 4H); C NMR (200 MHz, CD OD): δ 175.8, 174.7, 174.1,
3
70.6, 169.7 (d, J = 241.5 Hz, CꢀF), 136.1 (d, J = 3.0 Hz, arom), 123.0 (d, J = 8.0 Hz, arom),
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ꢀ
ꢀ
CD OD): δ ꢀ121.1 (F); HRMS (ESI): m/z calculated for C H FN O – H [M – H ]: 396.1576.
3
18 24
3
6
Found: 396.1573.
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Methyl (S)ꢀN ꢀhydroxyꢀN ꢀ(6ꢀ((4ꢀmethoxyphenyl)amino)ꢀ6ꢀoxohexanoyl)glutaminate (24d).
ο
20
1
Yield 16%; offꢀwhite solid; mp 157ꢀ159 C; [α]D = ꢀ17.6 (c 0.5, MeOH); Η NMR (200 MHz,
CD OD): δ 7.40 (d, J = 8.0 Hz, 2H), 6.82 (d, J = 8.0 Hz, 2H), 4.30ꢀ4.10 (m, 1H), 3.72 (s, 3H),
3
1
3
3
.61 (s, 3H), 2.50ꢀ1.80 (m, 8H), 1.80ꢀ1.50 (m, 4H); C NMR (200 MHz, CD OD): δ 175.8,
3
174.7, 174.0, 170.6, 157.8, 132.8, 123.1, 114.9, 55.8, 52.2, 51.5, 37.4, 36.4, 31.0, 28.3, 26.5,
ꢀ
ꢀ
2
6.4; HRMS (ESI): m/z calculated for C H N O – H [M – H ]: 408.1776. Found: 408.1789.
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ο
20
1
Yield 17%; white solid; mp: 146ꢀ148 C; [α]D = ꢀ17.8 (c 0.4, MeOH); Η NMR (200 MHz,
CD OD): δ 7.38 (d, J = 8.0 Hz, 2H), 6.80 (d, J = 8.0 Hz, 2H), 4.30ꢀ4.10 (m, 1H), 3.96 (q, J =
3
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8
.0 Hz, 2H), 3.61 (s, 3H), 2.50ꢀ1.80 (m, 8H), 1.80ꢀ1.50 (m, 4H), 1.32 (t, J = 8.0 Hz, 3H); C
NMR (200 MHz, CD OD): δ 175.9, 174.7, 174.0, 170.6, 157.1, 132.7, 123.0, 115.5, 64.6, 52.2,
3
ꢀ
51.5, 37.4, 36.4, 31.0, 28.3, 26.5, 26.4, 15.2; HRMS (ESI): m/z calculated for C H N O – H
2
0
29
3
7
ꢀ
[
M – H ]: 422.1933. Found: 422.1908.
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(
S)ꢀN ꢀ(1,5ꢀBis(hydroxyamino)ꢀ1,5ꢀdioxopentanꢀ2ꢀyl)ꢀN ꢀphenyladipamide (25a). To
a
o
stirred solution of ester 23a (168 mg, 0.44 mmol) in EtOH (1.78 mL), which was cooled at 0 C,
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