Synthesis, biological evaluation and molecular docking studies of benzyloxyacetohydroxamic acids as LpxC inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.12.057

The inhibition of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represents a promising strategy to combat infections caused by multidrug-resistant Gram-negative bacteria In order to elucidate the functional groups being impor

Full text of DOI:10.1016/j.bmc.2013.12.057

Bioorganic & Medicinal Chemistry 22 (2014) 1016–1028
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular docking studies
of benzyloxyacetohydroxamic acids as LpxC inhibitors
Marina Szermerski a, Jelena Melesina b, Kanin Wichapong b, Marius Löppenberg a, Joachim Jose a,
Wolfgang Sippl b, Ralph Holl a,
⇑
a Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität Münster, Corrensstraße 48, D-48149 Münster, Germany
b Department of Medicinal Chemistry, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibition of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represents
a promising strategy to combat infections caused by multidrug-resistant Gram-negative bacteria. In order to
elucidate the functional groups being important for the inhibition of LpxC, the structure of our previously
reported hydroxamic acid 4 should be systematically varied. Therefore, a series of benzyloxyacetohydroxamic
acids was prepared, of which the diphenylacetylene derivatives 28 (Ki = 95 nM) and 21 (Ki = 66 nM) were the
most potent inhibitors of Escherichia coli LpxC. These compounds could be synthesized in a stereoselective
manner employing a Sharpless asymmetric dihydroxylation and a Sonogashira coupling in the key steps.
The obtained structure–activity relationships could be rationalized by molecular docking studies.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 3 September 2013
Revised 21 November 2013
Accepted 18 December 2013
Available online 3 January 2014
Keywords:
LpxC inhibitors
Antibacterials
Enantioselective synthesis
Phenylethylene glycol derivatives
Molecular docking studies
1. Introduction
The first irreversible step of lipid A biosynthesis is the deacety-
lation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine
Gram-negative bacteria cause several serious diseases, such as
pneumonia or sepsis, which can be fatal without treatment.1,2
Although a number of antibiotics, addressing different bacterial
targets, are currently available to treat these infections, more and
more bacterial strains evolve which are resistant to most of the
available antibiotics, and the number of infections caused by mul-
tidrug-resistant Gram-negative bacteria is steadily increasing.3,4 At
the same time, only few novel antibacterial agents are in the
research pipeline.5 These alarming data emphasize the necessity
to develop new antibacterials, which act upon unexploited bacte-
rial targets, thereby circumventing established mechanisms of
resistance.
(1), which is catalyzed by the Zn2+-dependent enzyme LpxC
(Fig. 1). This enzyme, which shows no homology to any mamma-
lian protein, could be validated as antibacterial drug target and
different structural classes of LpxC inhibitors have been described
in the literature.6,10–17 Amongst them, the N-aroyl-
L-threonine
hydroxamic acid derivatives CHIR-090 and LPC-009 represent
potent LpxC inhibitors (Fig. 2), which show excellent antibacterial
activities against clinically important Gram-negative pathogens
including Escherichia coli and Pseudomonas aeruginosa.18
Solution as well as crystal structures of various LpxC orthologs
complexed with different LpxC inhibitors have been reported.14,18–23
They show that the enzyme consists of two domains. Each of them
The inhibition of the biosynthesis of lipid A represents a
promising strategy to combat infections caused by Gram-negative
germs.6 Lipid A is a phosphorylated, (1?6)-linked glucosamine
disaccharide, which acts as the hydrophobic membrane anchor of
lipopolysaccheride (LPS) in the outer leaflet of the outer membrane
of Gram-negative bacteria, and is essential for virulence and viabil-
ity of the cells.7 Bacteria with a defective lipid A synthesis show a
higher membrane permeability leading to an increased sensitivity
to a range of antibiotics and a decreased viability.8,9 The inhibition
of lipid A biosynthesis results in cell death.10
contains two
the overall structure the
b-sheets, the enzyme displays a ‘b–
each domain contains a unique insert. Whilst Insert I of Domain I
partially defines the boundary of the active site where the catalytic
Zn2+-ion is located, Insert II of Domain II largely forms a hydrophobic
passage, which during normal function of the enzyme hosts the fatty
acid chain of its natural substrate 1.
The diphenyldiacetylene derivative LPC-009 could be crystal-
lized with LpxC orthologs from different Gram-negative species
thus giving insight into the structural requirements for ligands
binding to these enzymes as well as into the inherent conforma-
tional variations of the individual LpxC orthologs.18
a
-helices, which are packed against a b-sheet. As in
-helices are sandwiched between the
–b sandwich’ fold. In addition,
a
a–a
⇑
Corresponding author. Tel.: +49 251 8333372; fax: +49 251 8332144.
E-mail address: hollr@uni-muenster.de (R. Holl).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.057

M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
1017
Figure 1. Deacetylation of uridine diphosphate-3-O-[(R)-3-hydroxymyristoyl]-N-
acetylglucosamine (1) catalyzed by LpxC.
The crystal structure of LPC-009 in complex with E. coli LpxC
shows important interactions between the inhibitor and the
enzyme (Fig. 3). In the active site, the catalytic Zn2+-ion is com-
plexed by the hydroxamate moiety of LPC-009. Additionally, its
threonyl group interacts with conserved residues in the active site.
Whilst the methyl group forms van der Waals contacts with the
phenyl ring of Phe192, a hydrogen bond is formed between the hy-
droxyl group and Lys239. Thr191 and the backbone amide of Cys63
form additional hydrogen bonds with the NH and the carbonyl of
the amide moiety of LPC-009, respectively. The diacetylene group
of the inhibitor penetrates through the hydrophobic tunnel. The
terminal phenyl ring of LPC-009 forms van der Waals contacts with
the hydrophobic residues of Ile198, Met195, Phe212 and Val217
outside the substrate-binding passage.
CHIR-090, a slow, tight-binding inhibitor of LpxC, showed a
similar binding mode in a solution structure of the Aquifex aeolicus
LpxC–CHIR-090 complex, with its lipophilic side chain occupying
the hydrophobic channel and the threonine hydroxamic acid
moiety binding to the active site of the enzyme.16,21 This diphenyl-
acetylene derivative kills E. coli and P. aeruginosa with an efficiency
comparable to that of ciprofloxacin and was chosen as lead
compound for the development of benzyloxyacetohydroxamic acid
derivatives 3 and 4 (Fig. 2).24,25 In our previous studies we showed
that the open chain derivative 4 is more active than the conform-
ationally constrained C-glycoside 3 in the LpxC assay as well as in
disc diffusion assays against E. coli.25 Therefore, the structure of
diol 4 was further investigated. In a deconstruction reconstruction
approach, the structural features of 4 which are important for
binding to LpxC were elucidated. In particular, attention was paid
to the length of the lipophilic side chain, the hydroxymethyl
groups as well as the stereochemistry (Fig. 2). In order to gain a
deeper understanding of the deduced structure activity relation-
ships of the synthesized compounds, docking studies were
performed.
Figure 3. Comparison between docking results of compound 21 (magenta) and the
cocrystallized inhibitor LPC-009 (3P3G, inhibitor colored cyan). The zinc ion is
shown as brown ball. Hydrogen bonds are shown as dashed line.
2. Results and discussion
2.1. Chemistry
First, benzyloxyacetohydroxamic acid derivatives bearing no
hydroxymethyl side chain were synthesized (Scheme 1). For this
purpose, ethyl bromoacetate was reacted with 4-iodobenzyl alco-
hol to give benzyl ether 5. Then a Sonogashira reaction was per-
formed to establish the lipophilic side chain. The C–C coupling
with phenylacetylene and 4-(4-ethynylbenzyl)morpholine24 gave
diphenylacetylene derivatives 6 and 7, respectively. The reaction
of esters 6 and 7 with hydroxylamine led to hydroxamates 8 and 9.
For the synthesis of the phenyl ethylene glycol derivatives, sty-
rene and 4-bromostyrene were used as starting materials
(Scheme 2). According to a literature procedure, these compounds
were subjected to a Sharpless asymmetric dihydroxylation using
AD-mix-a to obtain the enantiomerically pure (S)-configured
Figure 2. Chemical structures of N-aroyl-L-threonine hydroxamic acids CHIR-090 and LPC-009 and lead compounds 3 and 4.

1018
M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
Scheme 1. Reagents and conditions: (a) NaHMDS, THF,
D
, 8 h, 62%; (b) Pd(PPh3)4, CuI, NEt3, H3CCN, rt, 2 h, 6 92%, 7 84%; (c) H2NOHꢀHCl, NaOMe, MeOH, rt, 16 h, 8 27%, 9 16%.
Scheme 2. Reagents and conditions: (a) AD-mix-a, tBuOH/H20 (1:1), 0 °C, 16 h, 10 94%, 11 82%; (b) DIPEA, H3CCN, rt, 16 h, 12 30%, 13 36%; (c) LiHMDS, THF, D, 16 h, 14 62%,
15 73%; (d) (1) pTsOH, MeOH, rt, 16 h, (2) pTsOH, H3CCN, rt, 16 h, 16 63%, 17 50%; (e) H2NOHꢀHCl, NaOMe, MeOH, rt, 16 h, 18 36%, 19 64%.
glycols 10 and 11, respectively.26,27 Then, the primary alcohol of
the diols was protected. Several protective groups were tested
(MOM, BOM, TBDMS). With respect to protection, stability and
deprotection, the MOM protective group proved to be superior.
Therefore, diols 10 and 11 were reacted with MOM-Cl to obtain
the methoxymethyl ethers 12 and 13.28 However, only moderate
regioselectivity was observed. Whilst in case of the protection of
diol 11 the desired compound 13 was obtained in 36% yield, its reg-
ioisomer as well as the bis-protected compound were isolated in
11% and 10% yield, respectively. Etherification of the secondary
alcohols 12 and 13 with ethyl bromoacetate yielded the benzyl-
oxyacetic acid derivatives 14 and 15. Then, under acidic conditions
the MOM protective group was cleaved and the resulting primary
alcohols were subsequently transformed into lactones 16 and 17.29
In order to introduce various side chains to the lactones, Sonogash-
ira couplings were performed with the 4-bromobenzene derivative
17 (Scheme 3). However, in contrast to the reaction of the iodine-
substituted compound 5, the transformations of the bromine-
substituted compounds required somewhat harsher reaction
conditions and were performed in triethylamine as solvent under
refluxing conditions. Whilst the coupling with phenylacetylene
gave access to diphenylacetylene 20, the coupling with trimethyl-
silylacetylene yielded the acetylene derivative 22. The terminal al-
kyne 24 was obtained by cleaving the TMS-protective group of 22
with TBAF. The morpholin-4-ylmethyl substituted diphenylacety-
lene derivative 27 was synthesized via a Sonogashira coupling of
alkyne 24 with 4-(4-iodobenzyl)morpholine (26). Finally, the lac-
tones 16, 17, 20, 22, 24 and 27 were reacted with hydroxylamine
to yield the desired hydroxamic acids 18, 19, 21, 23, 25 and 28,
respectively. The corresponding (R)-configured hydroxamic acids
were obtained in the same way by using AD-mix-b in the dihydr-
oxylation step of styrene and 4-bromostyrene.
2.2. Biological evaluation
The antibacterial activity of the synthesized ben-
zyloxyacetohydroxamic acids was determined in an LpxC enzyme
assay as well as in disc diffusion tests (Table 1).
In the enzyme assay, purified E. coli LpxC and the enzyme’s nat-
ural substrate were employed. The inhibition of the enzyme could
be determined by measuring the amount of the deacetylated
product. For this purpose, the resulting primary amine 2 was
transformed into a fluorescent isoindole with phthalaldehyde and
2-mercaptoethanol.
When comparing the IC50- as well as the resultant Ki-values,
several structure–activity relationships can be deduced.
In case of the hydroxamates bearing a morpholin-4-ylmethyl-
substituted diphenylacetylene moiety, the removal of both
hydroxymethyl groups of hydroxamic acid 4, leading to 9, causes
a 4-fold decrease in inhibitory activity. However, 28, which retains
the hydroxymethyl group in benzylic position, shows a 15-fold in-
crease in inhibitory activity compared to 9 and also outperforms
the activity of lead compound 4.
The deconstruction of the lipophilic side chain of ethylene
glycol ether 28, leading to the unsubstituted phenyl derivative
18, was detrimental to the inhibitory activity. In order to observe
inhibition of E. coli LpxC, a certain minimal length of the hydropho-
bic side chain is required for the ethylene glycol ethers. Like the
unsubstituted hydroxamate 18, the bromo-, the acetylene- and
the TMS-acetylene-substituted compounds 19, 25 and 23 were

M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
1019
Scheme 3. Reagents and conditions: (a) phenylacetylene, Pd(PPh3)4, CuI, NEt3,
Pd(PPh3)4, CuI, NEt3,
NEt3, H3CCN, rt, 2 h, 91%; (h) H2NOHꢀHCl, NaOMe, MeOH, rt, 16 h, 25%.
D
, 16 h, 65%; (b) H2NOHꢀHCl, NaOMe, MeOH, rt, 16 h, 33%; (c) trimethylsilylacetylene,
D
, 16 h, 64%; (d) H2NOHꢀHCl, NaOMe, MeOH, rt, 16 h, 16%; (e) TBAF, THF, rt, 2 h, 31%; (f) H2NOHꢀHCl, NaOMe, MeOH, rt, 16 h, 26%; (g) Pd(PPh3)4, CuI,
Table 1
Results of the agar diffusion clearance assays, the LpxC assay and molecular docking studies (higher ChemPLP Score values indicate more favourable interactions)
Compound
Substitution pattern
Stereochemistry
Zone of inhibition [mm]
IC50
(l
M)
Ki (l
M)
ChemPLP
Score
E. coli BL21 (DE3)
E. coli D22
CHIR-090
3
4
8
9
18
ent-18
19
ent-19
25
ent-25
23
24.6 1.9
<6
9.0 0.5
10.6 0.4
16.5 0.4
<6
<6
<6
<6
<6
30.3 2.5
13.1 0.8
20.8 0.6
13.2 1.6
20.3 1.0
<6
<6
<6
<6
<6
0.058 0.002
>200
2.6 0.3
>200
10.5 2.5
>200
>200
>200
>200
>200
>200
>200
>200
0.48 0.23
31.6 6.0
0.69 0.30
198 12
0.008 0.0003
—
0.358 0.038
—
1.45 0.35
—
—
—
—
—
—
—
73.75
71.32
70.76
53.94
69.47
57.42
53.05
60.67
51.06
63.37
37.56
59.57
59.87
76.24
69.03
73.06
66.29
Morpholinomethylphenylethynyl
Morpholinomethylphenylethynyl
Phenylethynyl
Morpholinomethylphenylethynyl
H
H
Br
Br
Ethynyl
Ethynyl
Trimethylsilylethynyl
Trimethylsilylethynyl
Phenylethynyl
Phenylethynyl
Morpholinomethylphenylethynyl
Morpholinomethylphenylethynyl
(2S,3R,4S,5S)
(S,S)
(S)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
<6
<6
<6
7.5 0.3
7.2 0.2
20.5 0.2
13.0 1.7
21.2 0.6
12.3 1.6
ent-23
21
ent-21
28
7.1 0.1
9.5 0.4
9.1 0.4
13.4 0.5
8.7 0.7
—
0.066 0.032
4.4 0.8
0.095 0.042
27.3 1.7
ent-28
unable to inhibit the LpxC-catalyzed deacetylation of 1 at concen-
trations up to 200 M. In contrast, compounds 21 and 28, bearing a
diphenylacetylene moiety in their side chains, inhibit LpxC at
nanomolar concentrations. With Ki-values of 66 nM and 95 nM,
respectively, these compounds are only about 10-fold less potent
than the lead compound CHIR-090.
A comparison of the activities of the enantiomeric pairs shows,
that generally the (S)-configured phenyl ethylene glycol deriva-
tives possess higher inhibitory activities than their (R)-configured
counterparts.
In the disc diffusion assays, the synthesized compounds were
tested against E. coli BL21 (DE3) and the E. coli D22 strain, which
is more sensitive towards LpxC inhibition. CHIR-090 and DMSO
were used as positive and negative control, respectively.
When tested against E. coli D22, for the phenyl ethylene glycol
derivatives similar trends could be observed as in the enzyme as-
say. For a relatively high antibacterial activity these compounds
require a lipophilic side chain of a certain length. In case of the
diphenylacetylene derivatives, representing the most active
antibacterials in this series of compounds, the (S)-configured
hydroxamates possess a higher antibacterial activity than their
(R)-configured enantiomers.
The comparison of the morpholin-4-ylmethyl-substituted
hydroxamic acids 28, 4 and 9 reveals that, in spite of their differing
Ki values, the compounds are almost equally active against E. coli
D22. When tested against the wild type strain E. coli BL21 (DE3),
compound 9, which is the least active inhibitor of E. coli LpxC,
showed the biggest halo of inhibition on the agar plate. Most
l

1020
M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
Figure 4. Docking results obtained for a) compound 21 (magenta) in comparison with b) the stereoisomer ent-21 (green). Hydrogen bonds between LpxC and the inhibitors
are shown as blue lines and distances are given in Å.
probably, these findings are due to kinetic reasons like a higher
stability towards metabolic inactivation by the bacteria or a higher
membrane permeability of compound 9 lacking the hydroxy-
methyl groups.
Also in case of the diphenylacetylene derivatives 28 and 21, the
antibacterial activities against E. coli BL21 (DE3) do not fully match
the inhibitory activities against the enzyme. The morpholin-4-yl-
methyl-substituted hydroxamic acid 28, which is slightly less
active against purified E. coli LpxC, was shown to be significantly
more active in the disc diffusion assay. This might result from
the fact that 21 does not contain a polar functional group in its
lipophilic side chain and is therefore more likely to be trapped
within the cell membrane.
trimethylsilyl group is too large for the narrow channel and result-
ing in an altered orientation of the hydroxymethyl group (Fig. S3,
Supporting Information). As a consequence the hydrogen bond to
Cys63 is lost resulting in lower scores. For compounds 8 and 9
no hydrogen bond to Met61 or Cys63 is observed resulting in
diminished activities. Docking of compound 3 shows a favourable
score as observed for the active inhibitors. However, analyzing
the docking pose of compound 3 it is recognized that only one hy-
droxyl group of the tetrahydrofuran ring is engaged in a hydrogen
bond with the backbone of Phe192 whereas the second OH-group
is located nearby the aromatic ring of Phe212 at the same place as
the methyl group of the threonyl-fragment of LPC-009 (Fig. S4,
Supporting Information). So it seems to be unfavourable to place
a polar substituent at this region which could explain the loss of
activity of 3. However, this observation is not reflected by the
docking score.
2.3. Ligand docking studies
In order to rationalize the obtained biological data (Ki on LpxC
from E. coli) all compounds were docked into the crystal structure
of LpxC. The docking studies show that all compounds are able to
coordinate the zinc ion of LpxC with the hydroxamic acid group
and are able to mimic some of the interactions observed for
LPC-009 (Fig. 3). In general, a good agreement between the
ChemPLP docking scores and binding affinities is obtained for this
homogenous data set (Table 1). A correlation coefficient (r2)
between pKi and ChemPLP Score values of 0.81 (RMSE 0.49) is
observed and the model is able to discriminate between active
and inactive compounds with only one exception, namely
compound 3 (Fig. S1, Supporting Information).
Besides the docking scores, also the coordination to the zinc ion,
the hydrogen-bonds as well as the interaction with the cluster of
hydrophobic residues have to be considered to explain the biolog-
ical data. In case of 18, ent-18, 19, ent-19, 25, and ent-25 (Figs. S2
and S3, Supporting Information) the important interaction with the
cluster of hydrophobic residues (Ile198, Met195, Phe212 and
Val217) is missing, resulting in a decrease of the docking score
and loss of inhibitory activity. In case of 23 and ent-23, the bulky
Docking of the most-active inhibitors 21 and 28, having the
hydroxymethyl group in (S)-configuration, results in favourable
docking scores (76.24 and 73.06, respectively). The hydroxyl group
is hydrogen-bonded to the backbone of Met61 (CO) and Cys63
(NH) and is mimicking the interaction of the amide group of
LPC-009 (Fig. 4a). Another hydrogen bond is observed between
the Thr191 hydroxyl group and the ether-oxygen of 21/28. The
reduced affinity compared to LPC-009 can be explained by the
missing hydrophobic interaction with Phe192. The morphonyl-
substituent of 28 is located nearby the solvated entrance of the
tunnel and is not making significant contribution to the interaction
(similar scores for 21 and 28 are obtained) (Fig. S5, Supporting
Information). For the two (R)-configured stereoisomers (ent-21
and ent-28) the hydroxymethyl group makes only a hydrogen
bond to Cys63 whereas the hydrogen bond to Met61 is lost
(Fig. 4b). The docking scores obtained for the two enantiomers
ent-21 and ent-28 are significantly lower, explaining the reduced
activities (Fig. S2, Supporting Information). For 4, a similar orienta-
tion and docking score is obtained as observed for 21 and 28. One
hydroxyl group is involved in hydrogen bonds to Met61/Cys63

M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
1021
whereas the second hydroxyl group is not directly hydrogen-
bonded, but nearby Lys239 (4.5 Å) enabling a water-bridged inter-
action (Fig. S4, Supporting Information).
5
l
m, 250 mm/4.6 mm; solvent/isohexane/ethanol = 30:1; flow
rate: 1.0 mL/min; injection volume: 15 L; detection detection at
k = 220 nm for 30 min. Synthetic starting materials were purchased
l
from Sigma-Aldrich and ACROS ORGANICS.
3. Conclusions
4.2. Synthetic procedures
With the phenyl ethylene glycol ethers this study identified a
new class of LpxC inhibitors, which serves to broaden the current
scope for compounds acting at this enzyme. A convenient synthesis
to access these compounds in enantiomerically pure form was pre-
sented, allowing especially variations of the lipophilic side chain.
The biological evaluation identified the diphenylacetylene deriva-
tives 28 (Ki = 95 nM) and 21 (Ki = 66 nM) as the most potent inhib-
itors of the presented series of compounds. A molecular docking
study was performed to help elucidating the molecular interac-
tions, which underlie these activities. The derived model empha-
sized the importance of the long lipophilic side chain, the
hydroxymethyl group in benzylic position and the (S)-configura-
tion of the stereocenter. With their promising biological activities
hydroxamic acids 28 and 21 may serve as leads for the further
development of antibacterials targeting LpxC.
4.2.1. General synthetic procedures
4.2.1.1. Synthesis of hydroxamic acids.
Hydroxylamine
hydrochloride and a 2 M solution of sodium methoxide in metha-
nol were added to a solution of the lactone in methanol and the
mixture was stirred at ambient temperature for 16 h. Then water
was added and the mixture extracted with ethyl acetate (3ꢃ).
The combined organic layers were dried (Na2SO4), filtered and
the solvent was removed in vacuo. The residue was purified by
flash column chromatography.
4.2.2. Ethyl 2-[(4-iodobenzyl)oxy]acetate (5)
Under N2 atmosphere a 2 M solution of sodium hexamethyldi-
silazane in THF (2.67 mL, 5.34 mmol) and ethyl bromoacetate
(1.42 mL, 2.14 g, 12.8 mmol) were added to a solution of 4-iodob-
enzyl alcohol (1.0 g, 4.27 mmol) in THF (50 mL) and the mixture
was heated to reflux for 8 h. Then water was added and the mix-
ture was extracted with CH2Cl2 (3ꢃ). The combined organic layers
were dried (Na2SO4), filtered and the solvent was removed in va-
cuo. The residue was purified by flash column chromatography
(4 cm, 30 mL, cyclohexane/ethyl acetate = 9:1, Rf = 0.38) to give 5
as colorless oil (850 mg, 2.65 mmol, 62%). 1H NMR (CDCl3): d
1.29 (t, J = 7.2 Hz, 3H, OCH2CH3), 4.08 (s, 2H, OCH2CO2Et), 4.23 (q,
J = 7.2 Hz, 2H, OCH2CH3), 4.57 (s, 2H, OCH2Ar), 7.11–7.14 (m, 2H,
4. Experimental section
4.1. Chemistry, general
Unless otherwise mentioned, THF was dried with sodium/ben-
zophenone and was freshly distilled before use. Thin layer chroma-
tography (tlc): Silica gel 60 F254 plates (Merck). Flash
chromatography (fc): Silica gel 60, 40–64 lm (Macherey-Nagel);
parentheses include: diameter of the column, fraction size, eluent,
20-H4-iodophenyl, 60-H4-iodophenyl), 7.67–7.70 (m, 2H, 30-H4-iodophenyl
,
50-H4-iodophenyl); 13C NMR (CDCl3): d 14.4 (1C, OCH2CH3), 61.1 (1C,
OCH2CH3), 67.5 (1C, OCH2CO2Et), 72.8 (1C, OCH2Ar), 93.7 (1C, C-
404-iodophenyl), 130.0 (2C, C-204-iodophenyl, C-604-iodophenyl), 137.0 (1C,
C-104-iodophenyl), 137.7 (2C, C-304-iodophenyl, C-504-iodophenyl), 170.3
Rf value. Melting point (mp): Melting point apparatus SMP 3 (Stuart
Scientific), uncorrected. Optical rotation
a [deg] was determined
with a Polarimeter 341 (Perkin Elmer); path length 1 dm, wave-
length 589 nm (sodium D line); the unit of the specific rotation
(1C, CO2Et); IR (neat):
m
[cmꢂ1] = 2978, 1747, 1485, 1200, 1269,
½
a 2D0 [deg mL dmꢂ1 gꢂ1] is omitted; the concentration of the sample
ꢁ
1007, 795; HRMS (m/z): [M+H]+ calcd for C11H14IO3, 320.9982;
found, 320.9964; HPLC (method 1): tR = 23.5 min, purity 97.0%.
c [mg mLꢂ1] and the solvent used are given in brackets. 1H NMR
(400 MHz), 13C NMR (100 MHz): Mercury plus 400 spectrometer
(Varian); d in ppm related to tetramethylsilane; coupling constants
are given with 0.5 Hz resolution. Where necessary, the assignment
of the signals in the 1H NMR and 13C NMR spectra was performed
using 1H–1H and 1H–13C COSEY NMR spectra as well as NOE (nucle-
ar Overhauser effect) difference spectroscopy. IR: IR Prestige-
21(Shimadzu). HRMS: MicrOTOF-QII (Bruker). HPLC methods for
the determination of product purity: Method 1: Merck Hitachi
Equipment; UV detector: L-7400; autosampler: L-7200; pump:
L-7100; degasser: L-7614; column: LiChrospherÒ 60 RP-select B
4.2.3. Ethyl 2-{[4-(phenylethynyl)benzyl]oxy}acetate (6)
Under N2 atmosphere triethylamine (1.0 mL, 7.0 mmol), cop-
per(I) iodide (38 mg, 0.2 mmol) and tetrakis(triphenylphos-
phine)palladium(0) (116 mg, 0.1 mmol) were added to a solution
of 5 (320 mg, 1.0 mmol) in acetonitrile (25 mL). Then a solution
of phenylacetylene (0.9 mL, 8.3 mmol) in acetonitrile (10 mL) was
added dropwise over a period of 2 h. After evaporation of the sol-
vent the residue was purified by flash column chromatography
(3 cm, 20 mL, cyclohexane/ethyl acetate = 9:1, Rf = 0.36) to give 6
as colorless solid (270 mg, 0.92 mmol, 92%). Mp: 124 °C; 1H NMR
(CDCl3): d 1.30 (t, J = 7.2 Hz, 3H, OCH2CH3), 4.11 (s, 2H, OCH2CO2Et),
4.24 (q, J = 7.2 Hz, 2H, OCH2CH3), 4.65 (s, 2H, OCH2Ar), 7.32–7.38
(m, 5H, Harom.), 7.51–7.55 (m, 4H, Harom.); 13C NMR (CDCl3): d
14.4 (1C, OCH2CH3), 61.1 (1C, OCH2CH3), 67.5 (1C, OCH2CO2Et),
73.1 (1C, OCH2Ar), 89.3 (1C, C„C), 89.7 (1C, C„C), 123.0 (1C,
(5
l
m); LiCroCARTÒ 250-4 mm cartridge; flow rate: 1.00 mL/min;
L; detection at k = 210 nm for 30 min; sol-
injection volume: 5.0
l
vents: (A) water with 0.05% (V/V) trifluoroacetic acid; (B) acetoni-
trile with 0.05% (V/V) trifluoroacetic acid:gradient elution: (A%):
0–4 min: 90%, 4–29 min: gradient from 90% to 0%, 29–31 min: 0%,
31–31.5 min: gradient from 0% to 90%, 31.5–40 min: 90%. Method
2: Merck Hitachi Equipment; UV detector: L-7400; pump:
C
arom.), 123.3 (1C, Carom.), 128.0 (2C, Carom.), 128.4 (1C, Carom.),
128.5 (2C, Carom.), 131.8 (2C, Carom.), 131.9 (2C, Carom.), 137.5 (1C,
arom.), 170.4 (1C, CO2Et); IR (neat):
[cmꢂ1] = 3048, 2889, 1740,
L-6200A; column: phenomenex GeminiÒ
5
l
m C6-Phenyl 110 Å;
LC Column 250 ꢃ 4.6 mm; flow rate: 1.00 mL/min; injection
volume: 5.0 L; detection at k = 254 nm for 20 min; solvents: (A)
C
m
l
1512, 1435, 1393, 1215, 1146, 937, 833, 752, 691; HRMS (m/z):
[M+H]+ calcd for C19H19O3, 295.1329; found, 295.1353; HPLC
(method 1): tR = 25.9 min, purity 99.0%.
acetonitrile: 10 mM ammonium formate = 10:90 with 0.1% formic
acid; (B) acetonitrile: 10 mM ammonium formate = 90:10 with
0.1% formic acid; gradient elution: (A%): 0–5 min: 100%, 5–15 min:
gradient from 100% to 0%, 15–20 min: 0%, 20–22 min: gradient from
0% to 100%, 22–30 min: 100%. Chiral HPLC for the determination of
enantiomeric ratios: Method 3: equipment: pump: L-6200A; Injec-
tion: manual, Rheodyne 7725i; Diode Array Detector: L-7455; data
transfer: D-line; data acquisition: HSM-Software (all except Rheodyne
7725i: LaChrom, Merck Hitachi); column: Daicel Chiralpak IB,
4.2.4. Ethyl 2-[(4-{[4-(morpholinomethyl)phenyl]ethynyl}
benzyl)oxy]acetate (7)
Under N2 atmosphere triethylamine (0.61 mL, 4.4 mmol),
copper(I) iodide (24 mg, 0.13 mmol) and tetrakis(triphenylphos-
phine)palladium(0) (72 mg, 0.063 mmol) were added to a solution
of 5 (200 mg, 0.63 mmol) in acetonitrile (25 mL). Then a solution of

1022
M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
4-(4-ethynylbenzyl)morpholine (253 mg, 1.26 mmol) in acetoni-
trile (10 mL) was added dropwise over a period of 2 h. After evap-
oration of the solvent the residue was purified by flash column
chromatography (3 cm, 20 mL, cyclohexane/ethyl acetate = 2:1,
Rf = 0.20) to give 7 as colorless solid (207 mg, 0.53 mmol, 84%).
Mp: 69 °C; 1H NMR (CDCl3): d 1.30 (t, J = 7.2 Hz, 3H, OCH2CH3),
2.42–2.46 (m, 4H, NCH2CH2O), 3.50 (s, 2H, NCH2Ar), 3.70–3.73
(m, 4H, NCH2CH2O), 4.11 (s, 2H, OCH2CO2Et), 4.24 (q, J = 7.2 Hz,
2H, OCH2CH3), 4.64 (s, 2H, OCH2Ar), 7.30–7.33 (m, 2H, Harom.),
7.34–7.37 (m, 2H, Harom.), 7.46–7.50 (m, 2H, Harom.), 7.50–7.53
(m, 2H, Harom.); 13C NMR (CDCl3): d 14.4 (1C, OCH2CH3), 53.8 (2C,
NCH2CH2O), 61.1 (1C, OCH2CH3), 63.3 (1C, NCH2Ar), 67.1 (2C,
NCH2CH2O), 67.5 (1C, OCH2CO2Et), 73.1 (1C, OCH2Ar), 89.2 (1C,
C„C), 89.7 (1C, C„C), 122.1 (1C, Carom.), 123.1 (1C, Carom.), 128.0
(2C, Carom.), 129.3 (2C, Carom.), 131.7 (2C, Carom.), 131.8 (2C, Carom.),
137.4 (1C, Carom.), 138.4 (1C, Carom.), 170.4 (1C, CO2Et); IR (neat):
4-bromostyrene (7.2 mL, 55 mmol) was added and the reaction
mixture was stirred at 0 °C for 16 h. Then sodium sulfite (82.5 g)
was added and the mixture was allowed to warm to room temper-
ature and stirred for 1 h. Ethyl acetate was added to the reaction
mixture, and after separation of the layers, the aqueous phase
was further extracted with ethyl acetate (3ꢃ). The combined
organic layers were dried (Na2SO4), filtered and the solvent was re-
moved in vacuo. The crude product was purified by flash column
chromatography (8 cm, 60 mL, cyclohexane/ethyl acetate = 2:1,
Rf = 0.14) to give 11 as colorless solid (9.8 g, 45 mmol, 82%). Mp:
108 °C; ½a 2D0
ꢁ
41.9 (3.1; CH2Cl2); HPLC (method 1): tR = 12.5 min,
purity 99.6%; enantiomeric ratio (HPLC method 3): tR = 25.0 min,
(R):(S) = 1.4:98.6.
4.2.8. (R)-1-(4-Bromophenyl)ethane-1,2-diol (ent-11)
As described for the preparation of 11, 4-bromostyrene (1.3 mL,
10 mmol) was reacted with AD-mix-b (14 g) in a mixture of tert-
butyl alcohol (50 mL) and water (50 mL) to give ent-11 as colorless
m
[cmꢂ1] = 2808, 1744, 1516, 1454, 1292, 1204, 1138, 1111, 1022,
864; HRMS (m/z): [M+H]+ calcd for C24H28NO4, 394.2013; found,
394.1991; HPLC (method 1): tR = 19.2 min, purity 97.7%.
solid (1.9 g, 8.8 mmol, 88%). Mp: 108 °C; ½a D20
ꢂ45.3 (2.7; CH2Cl2);
ꢁ
HPLC (method 1): tR = 12.7 min, purity 99.5%; enantiomeric ratio
(HPLC method 3): tR = 23.6 min, (R):(S) = 98.9:1.1.
4.2.5. N-Hydroxy-2-{[4-(phenylethynyl)benzyl]oxy}acetamide
(8)
4.2.9. Spectroscopic data for 11 and ent-11
Hydroxylamine hydrochloride (76 mg, 1.1 mmol) and a 2 M
solution of sodium methoxide in methanol (0.55 mL, 1.1 mmol)
were added to a solution of 6 (130 mg, 0.44 mmol) in methanol
(30 mL) and the mixture was stirred at ambient temperature for
16 h. Then the solvent was evaporated and the residue was purified
by flash column chromatography (1 cm, 5 mL, CH2Cl2/metha-
1H NMR (D3COD): d 3.58 (dd, J = 11.3/6.8 Hz, 1H, HOCHCH2OH),
3.61 (dd, J = 11.3/5.2 Hz, 1H, HOCHCH2OH), 4.65 (dd, J = 6.8/5.2 Hz,
1H, HOCHCH2OH), 7.28–7.32 (m, 2H, 20-H4-bromophenyl
,
,
60-H4-bromophenyl),
7.46–7.50
(m,
2H,
30-H4-bromophenyl
50-H4-bromophenyl); 13C NMR (D3COD): d 68.5 (1C, HOCHCH2OH),
75.2 (1C, HOCHCH2OH), 122.1 (1C, C-404-bromophenyl), 129.4
nol = 9.5:0.5, Rf = 0.37) to give
8 as colorless solid (33 mg,
(2C, C-204-bromophenyl, C-604-bromophenyl), 132.3 (2C, C-304-bromophenyl
,
0.12 mmol, 27%). Mp: 136 °C; 1H NMR (CD3OD): d 4.01 (s, 2H,
OCH2CONHOH), 4.61 (s, 2H, OCH2Ar), 7.36–7.42 (m, 5H, Harom.),
7.50–7.53 (m, 4H, Harom.); 13C NMR (CD3OD): d 69.3 (1C, OCH2CON-
HOH), 73.9 (1C, OCH2Ar), 89.9 (1C, C„C), 90.3 (1C, C„C), 124.2 (1C,
C-504-bromophenyl), 142.8 (1C, C-104-bromophenyl); IR (neat):
m
[cmꢂ1] = 3368, 2924, 1481, 1392, 1346, 1227, 1061, 1011, 895,
826; HRMS (m/z): [M+Na]+ calcd for C8H979BrNaO2, 238.9678;
found, 238.9684.
C
arom.), 124.5 (1C, Carom.), 129.1 (2C, Carom.), 129.5 (1C, Carom.), 129.6
(2C, Carom.), 132.5 (2C, Carom.), 132.6 (2C, Carom.), 139.1 (1C, Carom.),
168.9 (1C, CONHOH); IR (neat):
[cmꢂ1] = 3248, 2909, 1636,
4.2.10. (S)-2-(Methoxymethoxy)-1-phenylethanol (12)28
m
Under N2 atmosphere N,N-diisopropylethylamine (6.4 mL, 5.0 g,
39 mmol) and chloromethyl methyl ether (2.1 mL, 2.25 g,
27.9 mmol) were added to a solution of (S)-1-phenylethane-1,2-
diol (10) (1.54 g, 11.1 mmol) in acetonitrile (100 mL) at 0 °C. After
stirring the mixture at ambient temperature for 16 h, water was
added and the mixture was extracted with ethyl acetate (3ꢃ).
The combined organic layers were dried (Na2SO4), filtered and
the solvent was removed in vacuo. The residue was purified by
flash column chromatography (5 cm, 50 mL, cyclohexane/ethyl
acetate = 8:2, Rf = 0.15) to give 12 as colorless oil (600 mg,
1512, 1485, 1439, 1358, 1281, 1099, 976, 837, 752, 687; HRMS
(m/z): [M+H]+ calcd for C17H16NO3, 282.1125; found, 282.1125;
HPLC (method 2): tR = 16.8 min, purity 99.3%.
4.2.6. N-Hydroxy-2-[(4-{[4-(morpholinomethyl)phenyl]
ethynyl}benzyl)oxy]acetamide (9)
Hydroxylamine hydrochloride (59 mg, 0.85 mmol) and a 2 M
solution of sodium methoxide in methanol (0.43 mL, 0.85 mmol)
were added to a solution of 7 (140 mg, 0.36 mmol) in methanol
(30 mL) and the mixture was stirred at ambient temperature for
16 h. Then the solvent was evaporated and the residue was purified
by flash column chromatography (1 cm, 5 mL, CH2Cl2/metha-
3.3 mmol, 30%).
tR = 12.2 min, purity 99.6%.
½
a 2D0
43.1 (0.5; CH2Cl2); HPLC (method 1):
ꢁ
nol = 9.5:0.5, Rf = 0.34) to give
9 as colorless solid (22 mg,
4.2.11. (R)-2-(Methoxymethoxy)-1-phenylethanol (ent-12)
As described for the preparation of 12, the enantiomer (R)-1-
phenylethane-1,2-diol ent-10 (792 mg, 5.73 mmol) was reacted
with N,N-diisopropylethylamine (3.32 mL, 2.59 g, 20.1 mmol) and
chloromethyl methyl ether (1.09 mL, 1.15 g, 14.3 mmol) in aceto-
nitrile (50 mL) to give ent-12 as colorless oil (450 mg, 2.47 mmol,
0.06 mmol, 16%). Mp: 113 °C; 1H NMR (CD3OD): d 2.45–2.49 (m,
4H, NCH2CH2O), 3.54 (s, 2H, NCH2Ar), 3.68–3.71 (m, 4H, NCH2CH2-
O), 4.01 (s, 2H, OCH2CONHOH), 4.61 (s, 2H, OCH2Ar), 7.35–7.42 (m,
4H, Harom.), 7.47–7.53 (m, 4H, Harom.); 13C NMR (CD3OD): d 54.6 (2C,
NCH2CH2O), 63.9 (1C, NCH2Ar), 67.7 (2C, NCH2CH2O), 69.3 (1C,
OCH2CONHOH), 73.9 (1C, OCH2Ar), 89.9 (1C, C„C), 90.2 (1C,
C„C), 123.6 (1C, Carom.), 124.2 (1C, Carom.), 129.1 (2C, Carom.),
130.7 (2C, Carom.), 132.5 (2C, Carom.), 132.6 (2C, Carom.), 139.0 (1C,
43%). ½a 2D0
ꢂ45.8 (4.8; CH2Cl2); HPLC (method 1): tR = 12.6 min,
ꢁ
purity 97.8%.
4.2.12. Spectroscopic data for 12 and ent-12
C
arom.), 139.1 (1C, Carom.), 168.9 (1C, CONHOH); IR (neat): m
1H NMR (CDCl3): d 3.06 (d, J = 2.7 Hz, 1H, OH), 3.39 (s, 3H, OCH2-
OCH3), 3.59 (dd, J = 10.6/8.6 Hz, 1H, HOCHCH2O), 3.79 (dd, J = 10.6/
3.1 Hz, 1H, HOCHCH2O), 4.69 (d, J = 6.6 Hz, 1H, OCH2OCH3), 4.72 (d,
J = 6.6 Hz, 1H, OCH2OCH3), 4.90 (dt, J = 8.6/2.8 Hz, 1H, HOCHCH2O),
7.27–7.41 (m, 5H, Harom.); 13C NMR (CDCl3): d 55.7 (1C, OCH2OCH3),
73.2 (1C, HOCHCH2O), 74.6 (1C, HOCHCH2O), 97.2 (1C, OCH2OCH3),
126.3 (2C, C-20phenyl, C-60phenyl), 128.0 (1C, C-40phenyl), 128.6 (2C,
[cmꢂ1] = 3213, 2932, 2812, 1647, 1516, 1454, 1408, 1350, 1288,
1103, 1072, 999, 868, 845, 818, 787; HRMS (m/z): [M+H]+ calcd
for C22H25N2O4, 381.1809; found, 381.1824; HPLC (method 2):
tR = 13.1 min, purity 99.7%.
4.2.7. (S)-1-(4-Bromophenyl)ethane-1,2-diol (11)
AD-mix-a (77 g) was added to a mixture of tert-butyl alcohol
C-30phenyl, C-50phenyl), 140.3 (1C, C-10phenyl); IR (neat):
m
[cmꢂ1] = 3441,
(275 mL) and water (275 mL). The mixture was cooled to 0 °C,

M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
1023
2932, 2886, 1454, 1211, 1150, 1111, 1026, 914, 756, 698; HRMS (m/z):
(dd, J = 10.9/7.5 Hz, 1H, OCHCH2O), 3.97 (d, J = 16.2 Hz, 1H, OCH2-
CO2Et), 4.10 (d, J = 16.2 Hz, 1H, OCH2CO2Et), 4.13–4.20 (m, 2H, CO2-
CH2CH3), 4.65 (d, J = 6.5 Hz, 1H, OCH2OCH3), 4.67 (d, J = 6.5 Hz, 1H,
OCH2OCH3), 4.68 (dd, J = 7.5/4.1 Hz, 1H, OCHCH2O), 7.29–7.37 (m,
5H, Harom.); 13C NMR (CDCl3): d 14.3 (1C, CO2CH2CH3), 55.4 (1C,
OCH2OCH3), 60.9 (1C, CO2CH2CH3), 66.4 (1C, OCH2CO2Et), 71.6
(1C, OCHCH2O), 81.7 (1C, OCHCH2O), 96.7 (1C, OCH2OCH3), 127.4
[M+H]+ calcd for C10H15O3, 183.1016; found, 183.1023.
4.2.13. (S)-1-(4-Bromophenyl)-2-(methoxymethoxy)ethanol
(13)
Under N2 atmosphere N,N-diisopropylethylamine (18.7 mL,
110 mmol) and chloromethyl methyl ether (5.6 mL, 73.2 mmol)
were added to a solution of 11 (7.9 g, 36.6 mmol) in acetonitrile
(70 mL) at 0 °C. After stirring the mixture at ambient temperature
for 16 h, water was added and the mixture was extracted with
ethyl acetate (3ꢃ). The combined organic layers were dried
(Na2SO4), filtered and the solvent was removed in vacuo. The resi-
due was purified by flash column chromatography (8 cm, 60 mL,
cyclohexane/ethyl acetate = 8:2, Rf = 0.14) to give 13 as colorless
(2C, C-20phenyl, C-60phenyl), 128.5 (1C, C-40phenyl), 128.7 (2C, C-30phenyl
,
C-50phenyl), 138.1 (1C, C-10phenyl), 170.3 (1C, CO2Et); IR (neat):
m
[cmꢂ1] = 2932, 1751, 1450, 1200, 1111, 1030, 918, 760, 702; HRMS
(m/z): [M+H]+ calcd for C14H21O5, 269.1384; found, 269.1390.
4.2.19. (S)-Ethyl 2-[1-(4-bromophenyl)-2-(methoxymethoxy)
ethoxy]acetate (15)
oil (3.4 g, 13.0 mmol, 36%). ½a D20
ꢁ
40.9 (3.9; CH2Cl2); HPLC (method
Under N2 atmosphere a 1 M solution of LiHMDS in THF
(32.1 mL, 32.1 mmol) was added to a solution of 13 (5.6 g,
21.4 mmol) in THF (70 mL). Then ethyl bromoacetate (7.1 mL,
10.7 g, 64.2 mmol) was added and the mixture was heated to reflux
for 16 h. After cooling the mixture to ambient temperature, water
was added and the mixture was extracted with ethyl acetate (3ꢃ).
The combined organic layers were dried (Na2SO4), filtered and the
solvent was removed in vacuo. The residue was purified by flash
column chromatography (8 cm, 60 mL, cyclohexane/ethyl
acetate = 8:2, Rf = 0.28) to give 15 as colorless oil (5.4 mg,
1): tR = 16.0 min, purity 98.6%.
4.2.14. (R)-1-(4-Bromophenyl)-2-(methoxymethoxy)ethanol
(ent-13)
As described for the preparation of 13, the enantiomer ent-11
(1.7 g, 7.8 mmol) was reacted with N,N-diisopropylethylamine
(4.33 mL, 3.39 g, 26.2 mmol) and chloromethyl methyl ether
(1.42 mL, 1.51 g, 18.7 mmol) in acetonitrile (50 mL) to give ent-
13 as colorless oil (940 mg, 3.6 mmol, 46%). ½a D20
ꢂ34.3 (3.2; CH2-
ꢁ
15.6 mmol, 73%).½a D20
ꢁ
77.2 (3.0; CH2Cl2); HPLC (method 1):
tR = 19.9 min, purity 97.8%.
Cl2); HPLC (method 1): tR = 16.3 min, purity 99.2%.
4.2.15. Spectroscopic data for 13 and ent-13
1H NMR (CDCl3): d 3.18 (s br, 1H, OH), 3.39 (s, 3H, OCH2OCH3),
3.55 (dd, J = 10.7/8.5 Hz, 1H, HOCHCH2O), 3.77 (dd, J = 10.7/3.1 Hz,
1H, HOCHCH2O), 4.68 (d, J = 6.6 Hz, 1H, OCH2OCH3), 4.71 (d,
J = 6.6 Hz, 1H, OCH2OCH3), 4.85 (dd, J = 8.5/3.1 Hz, 1H, HOCHCH2O),
7.26–7.30 (m, 2H, 20-H4-bromophenyl, 60-H4-bromophenyl), 7.47–7.50 (m,
2H, 30-H4-bromophenyl, 50-H4-bromophenyl); 13C NMR (CDCl3): d 55.7 (1C,
OCH2OCH3), 72.6 (1C, HOCHCH2O), 74.4 (1C, HOCHCH2O), 97.3 (1C,
4.2.20. (R)-Ethyl 2-[1-(4-bromophenyl)-2-(methoxymethoxy)
ethoxy]acetate (ent-15)
As described for the preparation of 15, the enantiomer ent-13
(996 mg, 3.81 mmol) was reacted with a 1 M solution of LiHMDS
in THF (5.72 mL, 5.72 mmol) and ethyl bromoacetate (1.27 mL,
1.91 g, 11.4 mmol) to give ent-15 as colorless oil (857 mg,
2.47 mmol, 65%).½a D20
ꢂ64.7 (3.4; CH2Cl2); HPLC (method 1):
ꢁ
OCH2OCH3), 121.8 (1C, C-404-bromophenyl), 128.0 (2C, C-204-bromophenyl
C-604-bromophenyl), 131.6 (2C, C-304-bromophenyl C-504-bromophenyl),
139.4 (1C, C-104-bromophenyl); IR (neat): [cmꢂ1] = 3426, 2932,
,
tR = 20.1 min, purity 95.9%.
,
m
4.2.21. Spectroscopic data for 15 and ent-15
2886, 1485, 1400, 1211, 1150, 1111, 1069, 1030, 1011, 822; HRMS
1H NMR (D3COD): d 1.24 (t, J = 7.1 Hz, 3H, CO2CH2CH3), 3.25 (s,
3H, OCH2OCH3), 3.64 (dd, J = 10.7/4.6 Hz, 1H, OCHCH2O), 3.78 (dd,
J = 10.7/6.9 Hz, 1H, OCHCH2O), 4.01 (d, J = 16.3 Hz, 1H, OCH2CO2Et),
4.09 (d, J = 16.3 Hz, 1H, OCH2CO2Et), 4.16 (q, J = 7.1 Hz, 2H, CO2CH2
CH3), 4.59 (d, J = 6.6 Hz, 1H, OCH2OCH3), 4.62 (d, J = 6.6 Hz, 1H,
OCH2OCH3), 4.65 (dd, J = 6.9/4.6 Hz, 1H, OCHCH2O), 7.28–7.34 (m, 2H,
(m/z): [M+H]+ calcd for C10H1479BrO3, 261.0121; found, 261.0118.
4.2.16. (S)-Ethyl 2-[2-(methoxymethoxy)-1-phenylethoxy]
acetate (14)
Under N2 atmosphere a 1 M solution of LiHMDS in THF (7.5 mL,
7.5 mmol) was added to a solution of 12 (910 mg, 5.0 mmol) in THF
(50 mL). Then ethyl bromoacetate (1.1 mL, 10 mmol) was added
and the mixture was heated to refulx for 16 h. After cooling the
mixture to ambient temperature, water was added and the mixture
was extracted with ethyl acetate (3ꢃ). The combined organic lay-
ers were dried (Na2SO4), filtered and the solvent was removed in
vacuo. The residue was purified by flash column chromatography
(4 cm, 30 mL, cyclohexane/ethyl acetate = 8:2, Rf = 0.28) to give
20-H4-bromophenyl, 60-H4-bromophenyl), 7.50–7.55 (m, 2H, 30-H4-bromophenyl
,
50-H4-bromophenyl); 13C NMR (D3COD): d 14.4 (1C, CO2CH2CH3), 55.5
(1C, OCH2OCH3), 61.9 (1C, CO2CH2CH3), 67.4 (1C, OCH2CO2Et), 72.2
(1C, OCHCH2O), 82.1 (1C, OCHCH2O), 97.6 (1C, OCH2OCH3), 123.0
(1C, C-404-bromophenyl), 130.3 (2C, C-204-bromophenyl, C-604-bromophenyl),
132.6 (2C, C-304-bromophenyl, C-504-bromophenyl), 139.2 (1C, C-104-bromophenyl),
171.9 (1C, CO Et); IR (neat):
m
[cmꢂ1] = 2932, 1751, 1485, 1377, 1281,
2
1200, 1111, 1034, 918, 822; HRMS (m/z): [M+H]+ calcd for C14H20
79
14 as colorless oil (830 mg, 3.1 mmol, 62%). ½a D20
ꢁ
+63.4 (4.0; CH2-
Cl2); HPLC (method 1): tR = 17.9 min, purity 95.6%.
BrO5, 347.0489; found, 347.0496.
4.2.22. (S)-5-Phenyl-1,4-dioxan-2-one (16)29
4.2.17. (R)-Ethyl 2-[2-(methoxymethoxy)-1-phenylethoxy]
acetate (ent-14)
p-Toluenesulfonic acid monohydrate (255 mg, 1.34 mmol) was
added to a solution of 14 (720 mg, 2.68 mmol) in methanol
(50 mL) and the mixture was stirred at ambient temperature for
16 h. After TLC had indicated that all of staring material was con-
sumed, the solvent was removed in vacuo. The residue was dis-
solved in acetonitrile (50 mL), additional p-toluenesulfonic acid
monohydrate (255 mg, 1.34 mmol) was added and the mixture
was stirred at ambient temperature for 16 h. Then a saturated
aqueous solution of NaHCO3 was added and the mixture was ex-
tracted with ethyl acetate (3ꢃ). The combined organic layers were
dried (Na2SO4), filtered and the solvent was removed in vacuo. The
As described for the preparation of 14, the enantiomer ent-12
(1.37 g, 7.52 mmol) was reacted with a 2 M solution of NaHMDS
in THF (5.6 mL, 11.3 mmol) and ethyl bromoacetate (2.5 mL,
22.6 mmol) in THF (50 mL) to give ent-14 as colorless oil (1.31 g,
4.88 mmol, 65%). ½a D20
ꢂ74.4 (2.8; CH2Cl2); HPLC (method 1):
ꢁ
tR = 18.3 min, purity 93.4%.
4.2.18. Spectroscopic data for 14 and ent-14
1H NMR (CDCl3): d 1.24 (t, J = 7.2 Hz, 3H, CO2CH2CH3), 3.30 (s,
3H, OCH2OCH3), 3.70 (dd, J = 10.9/4.1 Hz, 1H, OCHCH2O), 3.84

1024
M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
residue was purified by flash column chromatography (3 cm,
m
[cmꢂ1] = 2951, 2889, 1744, 1485, 1454, 1400, 1327, 1223, 1119,
20 mL, cyclohexane/ethyl acetate = 8:2, Rf = 0.28) to give 16 as col-
1011, 895, 826, 779; HRMS (m/z): [M+H]+ calcd for C10H1079BrO3,
orless oil (300 mg, 1.68 mmol, 63%). ½a D20
ꢁ
+104.3 (0.3; CH2Cl2);
HPLC (method 1): tR = 14.3 min, purity 97.4%.
256.9808; found, 256.9806.
4.2.28. (S)-N-Hydroxy-2-(2-hydroxy-1-phenylethoxy)acetamide
(18)
4.2.23. (R)-5-Phenyl-1,4-dioxan-2-one (ent-16)
As described in the general procedure for the synthesis of
hydroxamic acids, 16 (100 mg, 0.56 mmol) was reacted with
hydroxylamine hydrochloride (117 mg, 1.68 mmol) and a 2 m
solution of sodium methoxide in methanol (0.56 mL, 1.12 mmol)
in methanol (10 mL) to give 18 as colorless solid (43 mg,
0.20 mmol, 36%). Rf = 0.42 (CH2Cl2/methanol = 9:1); mp: 114 °C;
As described for the preparation of 16, the enantiomer ent-14
(1.23 g, 4.58 mmol) was reacted with p-toluenesulfonic acid mono-
hydrate (436 mg, 2.29 mmol) in methanol (50 mL) and the crude
product of the transformation was reacted with p-toluenesulfonic
acid monohydrate (436 mg, 2.29 mmol) acetonitrile (50 mL) to
give ent-16 as colorless oil (433 mg, 2.43 mmol, 53%).½a D20
ꢂ117.4
ꢁ
½
a 2D0
+117.4 (1.3; methanol); HPLC (method 2): tR = 12.5 min, pur-
ꢁ
(2.6; CH2Cl2); HPLC (method 1): tR = 14.2 min, purity 87.3%.
ity 95.3%.
4.2.24. Spectroscopic data for 16 and ent-16
4.2.29. (R)-N-Hydroxy-2-(2-hydroxy-1-phenylethoxy)acetamide
(ent-18)
1H NMR (CDCl3): d 4.44 (dd, J = 11.5/10.1 Hz, 1H, OCHCH2O),
4.50 (d, J = 17.8 Hz, 1H, OCH2CO2), 4.51 (dd, J = 11.5/3.2 Hz, 1H,
OCHCH2O), 4.67 (d, J = 17.8 Hz, 1H, OCH2CO2), 4.86 (dd, J = 10.1/
3.2 Hz, 1H, OCHCH2O), 7.35–7.44 (m, 5H, Harom.); 13C NMR (CDCl3):
d 66.2 (1C, OCH2CO2), 73.2 (1C, OCHCH2O), 74.2 (1C, OCHCH2O),
126.3 (2C, C-20phenyl, C-60phenyl), 129.0 (2C, C-30phenyl, C-50phenyl),
129.2 (1C, C-40phenyl), 135.1 (1C, C-10phenyl), 166.6 (1C, OCH2CO2);
As described in the general procedure for the synthesis of
hydroxamic acids, ent-16 (100 mg, 0.56 mmol) was reacted with
hydroxylamine hydrochloride (78 mg, 1.12 mmol) and a 2.5 M
solution of sodium methoxide in methanol (0.67 mL, 1.68 mmol)
in methanol (30 mL) to give ent-18 as colorless solid (23 mg,
0.11 mmol, 19%). Rf = 0.42 (CH2Cl2/methanol = 9:1); mp: 114 °C;
m
[cmꢂ1] = 2951, 2882, 1744, 1454, 1408, 1323, 1223,
½
a 2D0
ity 95.2%.
ꢁ
ꢂ109.7 (1.4; methanol); HPLC (method 2): tR = 12.4 min, pur-
IR (neat):
1115, 1026, 895, 752, 698; HRMS (m/z): [M+H]+ calcd for
C10H11O3, 179.0703; found, 179.0699.
4.2.30. Spectroscopic data for 18 and ent-18
1H NMR (CD3OD): d 3.61 (dd, J = 11.9/3.4 Hz, 1H, OCHCH2O),
3.70 (dd, J = 11.9/8.3 Hz, 1H, OCHCH2O), 3.86 (d, J = 14.9 Hz, 1H,
OCH2CONHOH), 3.94 (d, J = 14.9 Hz, 1H, OCH2CONHOH), 4.46 (dd,
J = 8.3/3.4 Hz, 1H, OCHCH2O), 7.30–7.41 (m, 5H, Harom.); 13C NMR
(CD3OD): d 67.6 (1C, OCHCH2O), 68.3 (1C, OCH2CONHOH), 85.7
(1C, OCHCH2O), 128.0 (2C, C-20phenyl, C-60phenyl), 129.5 (1C, C-
40phenyl), 129.7 (2C, C-30phenyl, C-50phenyl), 139.0 (1C, C-10phenyl),
4.2.25. (S)-5-(4-Bromophenyl)-1,4-dioxan-2-one (17)
p-Toluenesulfonic acid monohydrate (1.5 g, 7.8 mmol) was
added to a solution of 15 (5.4 g, 15.6 mmol) in methanol (70 mL)
and the mixture was stirred at ambient temperature for 16 h. After
TLC had indicated that all of staring material was consumed, water
was added and the mixture was extracted with ethyl acetate (3ꢃ).
The combined organic layers were dried (Na2SO4), filtered and the
solvent was removed in vacuo. The residue was dissolved in aceto-
nitrile (70 mL), p-toluenesulfonic acid monohydrate (1.5 mg,
7.8 mmol) was added and the mixture was stirred at ambient tem-
perature for 16 h. Then water was added and the mixture was ex-
tracted with ethyl acetate (3ꢃ). The combined organic layers were
dried (Na2SO4), filtered and the solvent was removed in vacuo. The
residue was purified by flash column chromatography (8 cm,
60 mL, cyclohexane/ethyl acetate = 8:2, Rf = 0.10) to give 17 as
169.1 (1C, OCH2CONHOH); IR (neat):
m
[cmꢂ1] = 3248, 2862,
1639, 1524, 1489, 1454, 1339, 1196, 1111, 1049, 895, 756, 698;
HRMS (m/z): [M+H]+ calcd for C10H14NO4, 212.0917; found,
212.0927.
4.2.31. (S)-2-[1-(4-Bromophenyl)-2-hydroxyethoxy]-N-
hydroxyacetamide (19)
As described in the general procedure for the synthesis of
hydroxamic acids, 17 (100 mg, 0.39 mmol) was reacted with
hydroxylamine hydrochloride (54 mg, 0.78 mmol) and a 2 M solu-
tion of sodium methoxide in methanol (0.58 mL, 1.17 mmol) in
methanol (30 mL) to give 19 as colorless solid (72 mg, 0.25 mmol,
colorless solid (2.0 g, 7.8 mmol, 50%). Mp: 84 °C;½a D20
ꢁ
81.2 (3.2;
CH2Cl2); HPLC (method 1): tR = 16.6 min, purity 98.5%.
64%). Rf = 0.39 (CH2Cl2/methanol = 9:1); mp: 119 °C; ½a D20
ꢁ
95.8 (1.1;
4.2.26. (R)-5-(4-Bromophenyl)-1,4-dioxan-2-one (ent-17)
As described for the preparation of 17, the enantiomer ent-15
(401 mg, 1.16 mmol) was reacted with p-toluenesulfonic acid
monohydrate (110 mg, 0.58 mmol) in methanol (50 mL) and the
crude product of the transformation was reacted with p-toluene-
sulfonic acid monohydrate (110 mg, 0.58 mmol) acetonitrile
(50 mL) to give ent-17 as colorless solid (175 mg, 0.68 mmol,
methanol); HPLC (method 2): tR = 14.3 min, purity 88.9%.
4.2.32. (R)-2-[1-(4-Bromophenyl)-2-hydroxyethoxy]-N-
hydroxyacetamide (ent-19)
As described in the general procedure for the synthesis of
hydroxamic acids, ent-17 (100 mg, 0.39 mmol) was reacted with
hydroxylamine hydrochloride (54 mg, 0.78 mmol) and a 2 M solu-
tion of sodium methoxide in methanol (0.58 mL, 1.17 mmol) in
methanol (30 mL) to give ent-19 as colorless solid (71 mg,
0.24 mmol, 63%). Rf = 0.39 (CH2Cl2/methanol = 9:1); Mp: 119 °C;
59%).½a 2D0
ꢂ72.7 (2.6; CH2Cl2); HPLC (method 1): tR = 16.8 min, pur-
ꢁ
ity 98.6%.
4.2.27. Spectroscopic data for 17 and ent-17
1H NMR (CDCl3): d 4.38 (dd, J = 11.5/10.3 Hz, 1H, OCHCH2O),
4.48 (dd, J = 11.5/3.0 Hz, 1H, OCHCH2O), 4.49 (d, J = 17.8 Hz, 1H,
OCH2CO2), 4.66 (d, J = 17.8 Hz, 1H, OCH2CO2), 4.82 (dd, J = 10.3/
½
a 2D0
98.3%.
ꢁ
ꢂ95.7 (1.1; methanol); HPLC (method 2): tR = 14.0 min, purity
3.0 Hz, 1H, OCHCH2O),), 7.24–7.27 (m, 2H, 20-H4-bromophenyl
,
4.2.33. Spectroscopic data for 19 and ent-19
60-H4-bromophenyl), 7.52–7.56 (m, 2H, 30-H4-bromophenyl, 50-H4-bromophenyl);
1H NMR (CD3OD): d 3.61 (dd, J = 11.9/3.6 Hz, 1H, OCHCH2O),
3.67 (dd, J = 11.9/7.8 Hz, 1H, OCHCH2O), 3.88 (d, J = 14.8 Hz, 1H,
OCH2CONHOH), 3.95 (d, J = 14.8 Hz, 1H, OCH2CONHOH), 4.45 (dd,
13C NMR (CDCl3): d 66.1 (1C, OCH2CO2), 72.8 (1C, OCHCH2O), 73.6 (1C,
OCHCH2O), 123.2 (1C, C-404-bromophenyl), 127.9 (2C, C-204-bromophenyl
,
C-604-bromophenyl), 132.2 (2C, C-304-bromophenyl
,
C-504-bromophenyl),
J = 7.8/3.6 Hz, 1H, OCHCH2O), 7.26–7.30 (m, 2H, 20-H4-bromophenyl
,
134.1 (1C, C-104-bromophenyl), 166.2 (1C, OCH2CO2); IR (neat):
60-H4-bromophenyl), 7.52–7.56 (m, 2H, 30-H4-bromophenyl, 50-H4-bromophenyl);

M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
1025
13C NMR (CD3OD): d 67.2 (1C, OCHCH2O), 68.4 (1C, OCH2CONHOH),
84.9 (1C, OCHCH2O), 123.2 (1C, C-404-bromophenyl), 130.0 (2C,
4.2.39. Spectroscopic data for 21 and ent-21
1H NMR (CD3OD): d 3.64 (dd, J = 11.9/3.6 Hz, 1H, OCHCH2O),
3.71 (dd, J = 11.9/7.9 Hz, 1H, OCHCH2O), 3.90 (d, J = 14.8 Hz, 1H,
OCH2CONHOH), 3.98 (d, J = 14.8 Hz, 1H, OCH2CONHOH), 4.49 (dd,
J = 7.9/3.6 Hz, 1H, OCHCH2O), 7.36–7.39 (m, 5H, Harom.), 7.49–7.55
(m, 4H, Harom.); 13C NMR (CD3OD): d 67.3 (1C, OCHCH2O), 68.5
(1C, OCH2CONHOH), 85.3 (1C, OCHCH2O), 89.7 (1C, C„C), 90.5
(1C, C„C), 124.5 (1C, Carom.), 124.7 (1C, Carom.), 128.3 (2C, Carom.),
129.5 (1C, Carom.), 129.6 (2C, Carom.), 132.5 (2C, Carom.), 132.8 (2C,
C-204-bromophenyl
,
C-604-bromophenyl), 132.8 (2C, C-304-bromophenyl
,
C-504-bromophenyl), 138.5 (1C, C-104-bromophenyl), 168.9 (1C, OCH2
CONHOH); IR (neat):
m
[cmꢂ1] = 3240, 2913, 2866, 1643, 1531,
1485, 1439, 1404, 1339, 1281, 1111, 1042, 1007, 818, 667; HRMS
(m/z): [M+H]+ calcd for C10H1379BrNO4, 290.0022; found, 290.0012.
4.2.34. (S)-5-[4-(Phenylethynyl)phenyl]-1,4-dioxan-2-one (20)
Under N2 atmosphere copper(I) iodide (11 mg, 0.06 mmol), tet-
rakis(triphenylphosphine)palladium(0) (46 mg, 0.04 mmol) and
phenylacetylene (0.30 mL, 276 mg, 2.7 mmol) were added to a
solution of 17 (500 mg, 1.94 mmol) in triethylamine (50 mL). The
mixture was heated to reflux and additional phenylacetylene
(0.30 mL, 276 mg, 2.7 mmol) was added. After stirring at reflux
for 16 h, the solvent was evaporated and the residue was purified
by flash column chromatography (3 cm, 20 mL, cyclohexane/ethyl
acetate = 8:2, Rf = 0.26) to give 20 as colorless solid (350 mg,
C
arom.), 139.5 (1C, Carom.), 168.9 (1C, OCH2CONHOH); IR (neat): m
[cmꢂ1] = 3240, 2916, 2862, 1639, 1504, 1443, 1412, 1335, 1196,
1130, 1111, 1045, 829, 752, 687; HRMS (m/z): [M+H]+ calcd for
C18H18NO4, 312.1230; found, 312.1217.
4.2.40. (S)-5-{4-[(Trimethylsilyl)ethynyl]phenyl}-1,4-dioxan-2-
one (22)
Under N2 atmosphere copper(I) iodide (16 mg, 0.08 mmol), tet-
rakis(triphenylphosphine)palladium(0) (62 mg, 0.05 mmol) and
trimethylsilylacetylene (0.6 mL, 398 mg, 4.1 mmol) were added
to a solution of 17 (700 mg, 2.7 mmol) in triethylamine (40 mL).
The mixture was heated to reflux and additional trimethylsilyl-
acetylene (0.6 mL, 398 mg, 4.1 mmol) was added. After stirring at
reflux for 16 h, the solvent was evaporated and the residue was
purified by flash column chromatography (4 cm, 30 mL, cyclohex-
ane/ethyl acetate = 9:1, Rf = 0.25) to give 22 as colorless solid
1.26 mmol, 65%). Mp: 134 °C; ½a D20
ꢁ
108.4 (0.8; CH2Cl2); HPLC
(method 1): tR = 20.2 min, purity 95.8%.
4.2.35. (R)-5-[4-(Phenylethynyl)phenyl]-1,4-dioxan-2-one (ent-
20)
As described for the preparation of 20, the enantiomer ent-17
(313 mg, 1.22 mmol) was reacted with copper(I) iodide (7 mg,
0.04 mmol), tetrakis(triphenylphosphine)palladium(0) (21 mg,
0.02 mmol) and phenylacetylene (0.20 mL, 184 mg, 1.81 mmol) in
triethylamine (50 mL) to give ent-20 as colorless solid (138 mg,
(480 mg, 1.75 mmol, 64%). Mp: 113 °C; ½a D20
ꢁ
84.4 (1.3; CH2Cl2);
HPLC (method 1): tR = 20.5 min, purity 98.9%.
4.2.41. (R)-5-{4-[(Trimethylsilyl)ethynyl]phenyl}-1,4-dioxan-2-
one (ent-22)
0.50 mmol, 41%). Mp: 134 °C; ½a D20
ꢂ94.1 (1.8; CH2Cl2); HPLC
ꢁ
(method 1): tR = 21.1 min, purity 95.1%.
As described for the preparation of 22, the enantiomer ent-17
(550 mg, 2.1 mmol) was reacted with copper(I) iodide (12 mg,
0.06 mmol), tetrakis(triphenylphosphine)palladium(0) (49 mg,
0.04 mmol) and trimethylsilylacetylene (twice 0.46 mL, 315 mg,
3.2 mmol) in triethylamine (70 mL) to give ent-22 as colorless solid
4.2.36. Spectroscopic data for 20 and ent-20
1H NMR (CDCl3): d 4.42 (dd, J = 11.5/10.3 Hz, 1H, OCHCH2O),
4.51 (d, J = 17.8 Hz, 1H, OCH2CO2), 4.52 (dd, J = 11.5/3.0 Hz, 1H,
OCHCH2O), 4.68 (d, J = 17.8 Hz, 1H, OCH2CO2), 4.87 (dd, J = 10.3/
3.0 Hz, 1H, OCHCH2O), 7.34–7.38 (m, 5H, Harom.), 7.51–7.58 (m,
4H, Harom.); 13C NMR (CDCl3): d 66.2 (1C, OCH2CO2), 72.9 (1C,
OCHCH2O), 73.9 (1C, OCHCH2O), 88.7 (1C, C„C), 90.5 (1C, C„C),
123.0 (1C, Carom.), 124.3 (1C, Carom.), 126.3 (2C, Carom.), 128.5 (2C,
(420 mg, 1.53 mmol, 72%). Mp: 113 °C; ½a D20
ꢂ84.3 (1.5; CH2Cl2);
ꢁ
HPLC (method 1): tR = 20.4 min, purity 95.4%.
4.2.42. Spectroscopic data for 22 and ent-22
1H NMR (CDCl3): d 0.25 (s, 9H, Si(CH3)3), 4.38 (dd, J = 11.5/
10.3 Hz, 1H, OCHCH2O), 4.49 (d, J = 17.8 Hz, 1H, OCH2CO2), 4.49
(dd, J = 11.5/3.0 Hz, 1H, OCHCH2O), 4.66 (d, J = 17.8 Hz, 1H, OCH2-
CO2), 4.84 (dd, J = 10.3/3.0 Hz, 1H, OCHCH2O), 7.29–7.32 (m, 2H,
Carom.), 128.7 (1C, Carom.), 131.8 (2C, Carom.), 132.2 (2C, Carom.),
135.0 (1C,
C
arom.), 166.3 (1C, OCH2CO2); IR (neat):
m
[cmꢂ1] = 2870, 1740, 1512, 1400, 1223, 1107, 1022, 891, 837,
760, 691; HRMS (m/z): [M+H]+ calcd for C18H15O3, 279.1016;
found, 279.0992.
H
arom.), 7.47–7.51 (m, 2H, Harom.); 13C NMR (CDCl3): d 0.05 (3C,
Si(CH3)3), 66.1 (1C, OCH2CO2), 72.9 (1C, OCHCH2O), 73.8 (1C,
OCHCH2O), 95.6 (1C, SiC„C), 104.3 (1C, SiC„C), 124.1 (1C, Carom.),
126.1 (2C, Carom.), 132.5 (2C, Carom.), 135.2 (1C, Carom.), 166.3 (1C,
4.2.37. (S)-N-Hydroxy-2-{2-hydroxy-1-[4-(phenylethynyl)
phenyl]ethoxy}acetamide (21)
OCH2CO2); IR (neat):
m
[cmꢂ1] = 2963, 2153, 1748, 1504, 1404,
1323, 1238, 1119, 1026, 833, 760; HRMS (m/z): [M+H]+ calcd for
As described in the general procedure for the synthesis of
hydroxamic acids, 20 (57 mg, 0.20 mmol) was reacted with
hydroxylamine hydrochloride (28 mg, 0.41 mmol) and a 2 M solu-
tion of sodium methoxide in methanol (0.31 mL, 0.61 mmol) in
methanol (30 mL) to give 21 as colorless solid (21 mg, 0.07 mmol,
C15H19O3Si, 275.1098; found, 275.1110.
4.2.43. (S)-N-Hydroxy-2-(2-hydroxy-1-{4-[(trimethylsilyl)
ethynyl]phenyl}ethoxy)acetamide (23)
33%). Rf = 0.21 (CH2Cl2/methanol = 9.5:0.5); mp: 128 °C; ½a D20
ꢁ
98.8
As described in the general procedure for the synthesis of
hydroxamic acids, 22 (120 mg, 0.44 mmol) was reacted with
hydroxylamine hydrochloride (76 mg, 1.1 mmol) and a 2 M solu-
tion of sodium methoxide in methanol (0.55 mL, 1.1 mmol) in
methanol (20 mL) to give 23 as colorless solid (22 mg, 0.07 mmol,
16%). Rf = 0.10 (CH2Cl2/methanol = 9.5:0.5); Mp: 112 °C;
+104.7 (0.7; methanol); HPLC (method 2): tR = 15.5 min, purity
99.1%.
(1.1; methanol); HPLC (method 2): tR = 16.0 min, purity 97.9%.
4.2.38. (R)-N-Hydroxy-2-{2-hydroxy-1-[4-(phenylethynyl)
phenyl]ethoxy}acetamide (ent-21)
½ ꢁ
a 2D0
As described in the general procedure for the synthesis of
hydroxamic acids, ent-20 (99 mg, 0.36 mmol) was reacted with
hydroxylamine hydrochloride (49 mg, 0.71 mmol) and a 2.5 M
solution of sodium methoxide in methanol (0.43 mL, 1.1 mmol)
methanol (30 mL) to give ent-21 as colorless solid (62 mg,
0.20 mmol, 56%). Rf = 0.21 (CH2Cl2/methanol = 9.5:0.5); mp:
4.2.44. (R)-N-Hydroxy-2-(2-hydroxy-1-{4-[(trimethylsilyl)
ethynyl]phenyl}ethoxy)acetamide (ent-23)
As described in the general procedure for the synthesis of
hydroxamic acids, ent-22 (100 mg, 0.36 mmol) was reacted with
128 °C; ½a 2D0
ꢂ99.5 (1.8; methanol); HPLC (method 2): tR = 16.1
ꢁ
min, purity 95.2%.

1026
M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
hydroxylamine hydrochloride (76 mg, 1.1 mmol) and a 2 M solu-
tion of sodium methoxide in methanol (0.36 mL, 0.73 mmol) in
methanol (10 mL) to give ent-23 as colorless solid (86 mg,
0.28 mmol, 77%). Rf = 0.10 (CH2Cl2/methanol = 9.5:0.5); mp:
½
a 2D0
126.4 (2.7; methanol); HPLC (method 2): tR = 13.5 min, pur-
ity 95.0%.
ꢁ
4.2.50. (R)-2-[1-(4-Ethynylphenyl)-2-hydroxyethoxy]-N-
hydroxyacetamide (ent-25)
112 °C; ½a 2D0
ꢂ99.4 (1.4; methanol); HPLC (method 2): tR = 16.2
ꢁ
min, purity 96.9%.
As described in the general procedure for the synthesis of
hydroxamic acids, ent-24 (43 mg, 0.21 mmol) was reacted with
hydroxylamine hydrochloride (44 mg, 0.64 mmol) and a 2 M solution
of sodium methoxide in methanol (0.21 mL, 0.43 mmol) in methanol
(5 mL) to give ent-25 as colorless solid (34 mg, 0.14 mmol, 68%).
4.2.45. Spectroscopic data for 23 and ent-23
1H NMR (CD3OD):
d 0.23 (s, 9H, Si(CH3)3), 3.58–3.71
(m, 2H, OCHCH2OH), 3.87 (d, J = 14.8 Hz, 1H, OCH2CONHOH),
3.95 (d, J = 14.8 Hz, 1H, OCH2CONHOH), 4.44–4.49 (m, 1H,
Rf = 0.47 (CH2Cl2/methanol = 9:1); mp: 124 °C; ½a D20
ꢂ118.2 (2.1;
ꢁ
OCHCH2OH), 7.30–7.36 (m, 2H, 20-H4-((trimethylsilyl)ethynyl)phenyl
,
methanol); HPLC (method 2): tR = 14.6 min, purity 98.3%.
60-H4-((trimethylsilyl)ethynyl)phenyl),
7.41–7.46
(m,
2H,
30-
H4-((trimethylsilyl)ethynyl)phenyl
,
50-H4-((trimethylsilyl)ethynyl)phenyl); 13C-NMR
4.2.51. Spectroscopic data for 25 and ent-25
(CD3OD): d -0.1 (3C, Si(CH3)3), 67.3 (1C, OCHCH2OH), 68.5 (1C, OCH2
CONHOH), 85.3 (1C, OCHCH2OH), 94.8 (1C, SiC„C), 105.9
(1C, SiC„C), 124.5 (1C, C-404-((trimethylsilyl)ethynyl)phenyl), 128.1 (2C,
1H NMR (CD3OD): d 3.50 (s, 1H, C„CH), 3.62 (dd, J = 11.8/3.3 Hz,
1H, OCHCH2OH), 3.68 (dd, J = 11.8/7.8 Hz, 1H, OCHCH2OH), 3.88 (d,
J = 14.7 Hz, 1H, OCH2CONHOH), 3.95 (d, J = 14.7 Hz, 1H, OCH2CON-
HOH), 4.48 (dd, J = 7.8/3.3 Hz, 1H, OCHCH2OH), 7.31–7.37 (m, 2H,
C-204-((trimethylsilyl)ethynyl)phenyl
(2C,
C-304-((trimethylsilyl)ethynyl)phenyl
139.8 (1C, C-104-((trimethylsilyl)ethynyl)phenyl), 168.9 (1C, OCH2CONHOH);
,
C-604-((trimethylsilyl)ethynyl)phenyl), 133.1
,
C-504-((trimethylsilyl)ethynyl)phenyl),
20-H4-ethynylphenyl, 60-H4-ethynylphenyl), 7.45–7.50 (m, 2H, 30-H4-ethynylphenyl
,
50-H4-ethynylphenyl); 13C NMR (CD3OD): d 67.3 (1C, OCHCH2OH), 68.5
(1C, OCH2CONHOH), 79.1 (1C, C„CH), 84.0 (1C, C„CH), 85.2 (1C,
IR (neat):
m
[cmꢂ1] = 3210, 2959, 2901, 2156, 1663, 1501, 1408,
1335, 1250, 1119, 1049, 837, 756, 648; HRMS (m/z): [M+H]+ calcd
OCHCH2O), 123.9 (1C, C-404-ethynylphenyl), 128.2 (2C, C-204-ethynylphenyl
,
for C15H22NO4Si, 308.1313; found, 308.1316.
C-604-ethynylphenyl), 133.3 (2C, C-304-ethynylphenyl C-504-ethynylphenyl),
,
139.9 (1C, C-104-ethynylphenyl), 168.9 (1C, OCH2CONHOH); IR (neat):
m
4.2.46. (S)-5-(4-Ethynylphenyl)-1,4-dioxan-2-one (24)
[cmꢂ1] = 3271, 2909, 2361, 1643, 1497, 1443, 1393, 1339, 1281,
Tetrabutylammonium fluoride trihydrate (662 mg, 2.1 mmol)
was added to a solution of 22 (480 mg, 1.75 mmol) in THF
(30 mL). The mixture was stirred at ambient temperature for 2 h.
Then water was added and the mixture was extracted with ethyl
acetate (3ꢃ). The combined organic layers were dried (Na2SO4), fil-
tered and the solvent was removed in vacuo. The residue was puri-
fied by flash column chromatography (3 cm, 20 mL, cyclohexane/
ethyl acetate = 8:2, Rf = 0.23) to give 24 as colorless solid
1123, 1034, 895, 837, 683; HRMS (m/z): [M+H]+ calcd for
C
12H14NO4, 236.0917; found, 236.0907.
4.2.52. (S)-5-(4-{[4-(Morpholinomethyl)phenyl]ethynyl}phenyl)
-1,4-dioxan-2-one (27)
Under N2 atmosphere triethylamine (0.5 mL, 3.8 mmol), cop-
per(I) iodide (21 mg, 0.11 mmol) and tetrakis(triphenylphos-
phine)palladium(0) (62 mg, 0.054 mmol) were added to
a
(110 mg, 0.54 mmol, 31%). Mp: 120 °C; ½a D20
ꢁ
+97.0 (0.7; CH2Cl2);
solution of 4-(4-iodobenzyl)morpholine (26, 330 mg, 1.09 mmol)
in acetonitrile (30 mL). Then a solution of 24 (110 mg, 0.54 mmol)
in acetonitrile (10 mL) was added dropwise over a period of 2 h.
After evaporation of the solvent the residue was purified by flash
column chromatography (3 cm, 20 mL, cyclohexane/ethyl ace-
tate = 1:2, Rf = 0.27) to give 27 as colorless solid (186 mg,
HPLC (method 1): tR = 15.2 min, purity 96.1%.
4.2.47. (R)-5-(4-Ethynylphenyl)-1,4-dioxan-2-one (ent-24)
As described for the preparation of 24, the enantiomer ent-22
(420 mg, 1.53 mmol) was reacted with tetrabutylammonium fluo-
ride trihydrate (579 mg, 1.84 mmol) in THF (50 mL) to give ent-24
0.49 mmol, 91%). Mp: 143 °C; ½a D20
ꢁ
70.0 (0.9; CH2Cl2); HPLC (meth-
as colorless solid (70 mg, 0.35 mmol, 23%). Mp: 120 °C; ½a D20
ꢁ
ꢂ90.9
od 1): tR = 15.4 min, purity 95.0%.
(1.5; CH2Cl2); HPLC (method 1): tR = 15.4 min, purity 98.8%.
4.2.53. (R)-5-(4-{[4-(Morpholinomethyl)phenyl]ethynyl}
phenyl)-1,4-dioxan-2-one (ent-27)
4.2.48. Spectroscopic data for 24 and ent-24
1H NMR (CDCl3): d 3.11 (s, 1H, C„CH), 4.39 (dd, J = 11.5/
10.3 Hz, 1H, OCHCH2O), 4.49 (d, J = 17.8 Hz, 1H, OCH2CO2), 4.49
(dd, J = 11.5/3.0 Hz, 1H, OCHCH2O), 4.66 (d, J = 17.8 Hz, 1H,
OCH2CO2), 4.86 (dd, J = 10.3/3.0 Hz, 1H, OCHCH2O), 7.32–7.35
(m, 2H, 20-H4-ethynylphenyl, 60-H4-ethynylphenyl), 7.51–7.54 (m, 2H,
As described for the preparation of 27, the enantiomer ent-24 (50 mg,
0.25 mmol) in acetonitrile (10 mL) was reacted with triethylamine
(0.36 mL, 2.6 mmol), copper(I) iodide (4.3 mg, 0.02 mmol), tetrakis
(triphenylphosphine)palladium(0) (17 mg, 0.01 mmol) and 26
(225 mg, 0.75 mmol) in acetonitrile (30 mL) to give ent-27 as
30-H4-ethynylphenyl
,
50-H4-ethynylphenyl); 13C NMR (CDCl3):
d
66.1
colorless solid (80 mg, 0.21 mmol, 86%). Mp: 143 °C; ½a D20
ꢂ71.3
ꢁ
(1C, OCH2CO2), 72.9 (1C, OCHCH2O), 73.8 (1C, OCHCH2O), 78.3
(1.6; CH2Cl2); HPLC (method 1): tR = 15.5 min, purity 98.9%.
(1C, C„CH), 83.0 (1C, C„CH), 123.1 (1C, C-404-ethynylphenyl), 126.2
(2C, C-204-ethynylphenyl, C-604-ethynylphenyl), 132.7 (2C, C-304-ethynylphenyl
,
4.2.54. Spectroscopic data for 27 and ent-27
C-50
(neat):
), 135.7 (1C, C-104-ethynylphenyl), 166.2 (1C, OCH2CO2); IR
1H NMR (CD3OD): d 2.43–2.49 (m, 4H, NCH2CH2O), 3.53–3.56
(m, 2H, NCH2Ar), 3.59–3.77 (m, 6H, NCH2CH2O, OCHCH2O), 4.00–
4.07 (m, 1H, OCH2CO2), 4.08–4.14 (m, 1H, OCH2CO2), 4.52–4.57
(m, 1H, OCHCH2O), 7.34–7.40 (m, 4H, Harom.), 7.45–7.54 (m, 4H,
4-ethynylphenyl
m
[cmꢂ1] = 3248, 2878, 2357, 1736, 1504, 1458, 1404, 1323,
1231, 1107, 1026, 895, 837; HRMS (m/z): [M+H]+ calcd for
C12H11O3, 203.0703; found, 203.0726.
H
arom.); 13C NMR (CD3OD): d 54.6 (2C, NCH2CH2O), 63.9 (1C, NCH2-
4.2.49. (S)-2-[1-(4-Ethynylphenyl)-2-hydroxyethoxy]-N-
hydroxyacetamide (25)
Ar), 67.1 (1C, OCH2CO2), 67.4 (1C, OCHCH2O), 67.8 (2C, NCH2CH2O),
84.7 (1C, OCHCH2O), 89.9 (1C, C„C), 90.3 (1C, C„C), 123.5 (1C,
As described in the general procedure for the synthesis of
hydroxamic acids, 24 (40 mg, 0.2 mmol) was reacted with
hydroxylamine hydrochloride (42 mg, 0.6 mmol) and a 2 M solu-
tion of sodium methoxide in methanol (0.3 mL, 0.6 mmol) in
methanol (20 mL) to give 25 as colorless solid (12 mg,
0.05 mmol, 26%). Rf = 0.47 (CH2Cl2/methanol = 9:1); mp: 124 °C;
C
arom.), 124.5 (1C, Carom.), 128.5 (2C, Carom.), 130.8 (2C, Carom.),
132.5 (2C, Carom.), 132.7 (2C, Carom.), 139.1 (1C, Carom.), 140.0 (1C,
arom.), 172.9 (1C, OCH2CO2); IR (neat):
[cmꢂ1] = 2808, 1728,
C
m
1516, 1450, 1404, 1350, 1323, 1227, 1111, 1007, 895, 868, 829;
HRMS (m/z): [M+H]+ calcd for C23H24NO4, 378.1700; found,
378.1700.

M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
1027
4.2.55. (S)-N-Hydroxy-2-[2-hydroxy-1-(4-{[4-(morpholinomethyl)
phenyl]ethynyl}phenyl)ethoxy]acetamide (28)
and desalted as above using MWCO columns. The final step of pro-
tein purification was performed with a pre-packed size exclusion
chromatography column containing 300 mL of Superdex 200 (GE
Healthcare). LpxCC63A emerged in a peak after 200 mL of elution
buffer. The purified LpxC was concentrated with MWCO columns
As described in the general procedure for the synthesis of
hydroxamic acids, 27 (100 mg, 0.26 mmol) was reacted with
hydroxylamine hydrochloride (46 mg, 0.66 mmol) and a 2 M solu-
tion of sodium methoxide in methanol (0.33 mL, 0.66 mmol) in
methanol (20 mL) to give 28 as colorless solid (27 mg, 0.066 mmol,
and stored in 100 lL aliquots at 80 °C in Bis/Tris buffer 50 mM,
pH 6.0, containing 150 mM NaCl. The presence of the enzyme dur-
ing the purification progress was confirmed by sodium dodecyl
sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) with Coo-
massie brilliant blue staining. The purified LpxC had a purity above
25%). Rf = 0.34 (CH2Cl2/methanol = 9:1); mp: 129 °C; ½a D20
ꢁ
86.4 (7.1;
methanol); HPLC (method 2): tR = 12.0 min, purity 97.7%.
4.2.56. (R)-N-Hydroxy-2-[2-hydroxy-1-(4-{[4-(morpholinomethyl)
phenyl]ethynyl}phenyl)ethoxy]acetamide (ent-28)
95% according to SDS–PAGE, and a concentration of 500
according to the Bradford assay (Bio-Rad protein assay).
l
g ꢃ mLꢂ1
As described in the general procedure for the synthesis of
hydroxamic acids, ent-27 (74 mg, 0.2 mmol) was reacted with
hydroxylamine hydrochloride (35 mg, 0.5 mmol) and a 2 M solu-
tion of sodium methoxide in methanol (0.25 mL, 0.5 mmol) in
methanol (20 mL) to give ent-28 as colorless solid (30 mg,
0.073 mmol, 37%). Rf = 0.34 (CH2Cl2/methanol = 9:1); mp: 129 °C;
4.3.3. LpxC assay
A fluorescence-based microplate assay for LpxC activity was
performed as described by Clements et al.10 The wells in a black
non-binding, 96 wells fluorescence microplate (Greiner Bio One,
Frickenhausen) were filled with 97
pholinoethanesulfonic acid buffer (pH 6.0) containing 25
lL of a 40 mM sodium mor-
½
a 2D0
ꢁ
ꢂ82.4 (1.4; methanol); HPLC (method 2): tR = 11.3 min, purity
lM
96.3%.
UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine, 80
lM
dithiothreitol and 0.02% Brij 35. Inhibitors were dissolved in DMSO
and assayed over a range starting from 0.2 nM up to 200 M. The
microplate was incubated for 30 min at 37 °C in a plate shaker.
4.2.57. Spectroscopic data for 28 and ent-28
l
1H NMR (CD3OD): d 2.44–2.50 (m, 4H, NCH2CH2O), 3.53–3.56
(m, 2H, NCH2Ar), 3.61–3.73 (m, 6H, NCH2CH2O, OCHCH2OH),
3.86–3.92 (m, 1H, OCH2CONHOH), 3.94–4.00 (m, 1H, OCH2CON-
HOH), 4.47–4.51 (m, 1H, OCHCH2OH), 7.35–7.39 (m, 4H, Harom.),
7.46–7.55 (m, 4H, Harom.); 13C NMR (CD3OD): d 54.6 (2C, NCH2CH2-
O), 63.9 (1C, NCH2Ar), 67.3 (1C, OCHCH2OH), 67.7 (2C, NCH2CH2O),
68.5 (1C, OCH2CONHOH), 85.3 (1C, OCHCH2OH), 89.8 (1C, C„C),
90.4 (1C, C„C), 123.5 (1C, Carom.), 124.7 (1C, Carom.), 128.3 (2C,
After addition of 500 ng purified LpxC, the biochemical reaction
was stopped by adding 40
lL of 0.625 m sodium hydroxide, further
incubation for 15 min and neutralization by adding 40
l
L of
0.625 M acetic acid. The deacetylated product UDP-3-O-[(R)-3-
hydroxymyristoyl]glucosamine was converted into a fluorescing
isoindole by adding 120 lL of 250 nM o-phthaldialdehyde-2-
mercaptoethanol in 0.1 M borax34 and detected by a Mithras plate
reader (Berthold, Bad Wildbad) at 340 nm excitation and 460 nm
emission wavelengths. The calculation of the IC50 values was
performed with the aid of the software GraphPadPrism, which
were then converted into Ki values using the Cheng-Prusoff equa-
tion. The Ki and IC50 values are given as mean value SD from three
independent experiments. The KM value was determined using the
Carom.), 130.8 (2C, Carom.), 132.5 (2C, Carom.), 132.8 (2C, Carom.),
139.1 (1C, Carom.), 139.5 (1C, Carom.), 168.9 (1C, OCH2CONHOH); IR
(neat):
m
[cmꢂ1] = 3225, 2855, 2812, 1662, 1516, 1454, 1412,
1335, 1292, 1111, 1049, 1007, 864, 829; HRMS (m/z): [M+H]+ calcd
for C23H27N2O5, 411.1914; found, 411.1941.
4.3. Biological evaluation
Lineweaver–Burk plot and was found to be 4 lM being comparable
to the data in literature.35 To validate the test system, the IC50
value of CHIR-090 was measured and was found to be comparable
to the one in the literature.
4.3.1. Agar diffusion clearance assay
The antibacterial activity of the synthesized inhibitors was
determined by agar disc diffusion clearance assays. Liquid cultures
of E. coli BL21 (DE3) and the antibiotic resistant strain E. coli D2230
4.4. Computational methods
were grown overnight in LB broth31 at 37 °C, 200 rpm. 150
l
L of
overnight cell suspension was spread evenly onto LB agar petri
dishes. 15 L of each compound (10 mM in DMSO) was applied
Several crystal structures of LpxC from Escherichia coli, contain-
ing ligands structurally similar to the ones reported in the current
l
paper, were taken from the Protein Data Bank36 (PDB ID: 3P3G18
,
onto circular filter paper (Ø 6 mm, thickness 0.75 mm, Carl Roth).
Pure DMSO, serving as a negative and CHIR-090,16 serving as a po-
sitive control were also spotted. The petri dishes were incubated
overnight at 37 °C and the diameter of the zone of growth inhibi-
tion was measured for each compound. The measured diameters
are given as mean value SD from three independent experiments.
3PS1, 3PS2 and 3PS319).14,18–23 The crystal structures were first ta-
ken to evaluate the accuracy of the used docking and scoring
methods.
Protein models were prepared with Schrödinger’s Protein Prep-
aration Wizard (PPW).37 All solvent particles were removed except
of two conserved water molecules located near the Zn2+ ion.
Hydrogen atoms were added and subsequently an automated opti-
mization of the hydrogen bond network was performed. In this
procedure the hydroxyl and thiol groups, water molecules, amide
groups of asparagines and glutamines, and the imidazole ring in
histidines are reoriented. Also the protonation state of the struc-
tures was predicted with the PROPKA tool at pH 7.0. Both charged
and uncharged states of His265 were considered for the docking
study. Eight models were generated, which differed in hydration
of the binding pocket and His265 protonation state. Finally every
protein model was subjected to restrained energy minimization
using the force field OPLS2005 (RMSD of the atom displacement
for terminating the minimization 0.3 Å).
4.3.2. Protein purfication
The plasmid for the expression of LpxCC63A (pETEcLpxCC63A)
was kindly provided by Carol Fierke.32 The C63A mutation lowers
the undesired influence of Zn2+-concentration on enzymatic activ-
ity. The purification of LpxC was performed essentially as previ-
ously described.33 Weak anion exchange was performed with a
column containing 30 mL diethylaminoethylcellulose (DEAE)-
Sepharose fast flow media (GE Healthcare). Eluted fractions con-
taining the desired enzyme were concentrated and desalted with
molecular weight cut-off (MWCO) spin columns (10 kDa, PALL
Corporation). Strong anion exchange was then performed with a
column containing 30 mL of quaternary ammonium-sepharose
(Q-Sepharose) fast flow media (GE Healthcare). The fractions con-
taining LpxC (peak elution at 18.6 mS ꢃ cmꢂ1) were concentrated
Ligands were prepared in MOE38 (version 2012.10, Chemical
Computing Group, Montreal, Canada). Conformational search for
all structures has been carried out using the Low Mode MD

1028
M. Szermerski et al. / Bioorg. Med. Chem. 22 (2014) 1016–1028
10. Clements, J. M.; Coignard, F.; Johnson, I.; Chandler, S.; Palan, S.; Waller, A.;
Wijkmans, J.; Hunter, M. G. Antimicrob. Agents Chemother. 2002, 46, 1793.
11. Faruk, M. U.; Vitharana, D.; Reddy, P. A.; Daubaras, D. L.; McNicholas, P.; Orth,
P.; Black, T.; Arshad, S. M. Bioorg. Med. Chem. Lett. 2011, 21, 1155.
12. Li, X.; Uchiyama, T.; Raetz, C. R. H.; Hindsgaul, O. Org. Lett. 2003, 5, 539.
13. Montgomery, J. I.; Brown, M. F.; Reilly, U.; Price, L. M.; Abramite, J. A.; Arcari, J.;
Barham, R.; Che, Y.; Chen, J. M.; Chung, S. W.; Collantes, E. M.; Desbonnet, C.;
Doroski, M.; Doty, J.; Engtrakul, J. J.; Harris, T. M.; Huband, M.; Knafels, J. D.;
Leach, K. L.; Liu, S.; Marfat, A.; McAllister, L.; McElroy, E.; Menard, C. A.; Mitton-
Fry, M.; Mullins, L.; Noe, M. C.; O’Donnell, J.; Oliver, R.; Penzien, J.; Plummer,
M.; Shanmugasundaram, V.; Thoma, C.; Tomaras, A. P.; Uccello, D. P.; Vaz, A.;
Wishka, D. G. J. Med. Chem. 2012, 55, 1662.
14. Brown, M. F.; Reilly, U.; Abramite, J. A.; Arcari, J. T.; Oliver, R.; Barham, R. A.;
Che, Y.; Chen, J. M.; Collantes, E. M.; Chung, S. W.; Desbonnet, C.; Doty, J.;
Doroski, M.; Engtrakul, J. J.; Harris, T. M.; Huband, M.; Knafels, J. D.; Leach, K. L.;
Liu, S.; Marfat, A.; Marra, A.; McElroy, E.; Melnick, M.; Menard, C. A.;
Montgomery, J. I.; Mullins, L.; Noe, M. C.; O’Donnell, J.; Penzien, J.; Plummer,
M. S.; Price, L. M.; Shanmugasundaram, V.; Thoma, C.; Uccello, D. P.; Warmus, J.
S.; Wishka, D. G. J. Med. Chem. 2012, 55, 914.
sampling with a minimum RMSD between the conformations of
1 Å. This procedure was applied to obtain realistic conformations
for the flexible ligands including the ring systems.
Molecular docking studies were performed on a Linux cluster
using the Glide software (Schrödinger Inc, New York, USA).37 Con-
straints to the Zn2+ ion were set and the molecular docking was
performed in standard precision mode (SP). Glide Score has been
used to rank the docking poses. Post-docking filtering was done
using the Python script distance_to_smarts (Revision 3.5, Schrö-
dinger). Only those docking poses were selected, where the dis-
tance between both oxygen atoms of the hydroxamic acid of the
ligand and the Zn2+ ion inside the binding pocket of the protein
was below 2.7 Å. This value was chosen based on inspection of
the available X-ray structures of LpxC. Finally, rescoring of the
filtered top-ranked docking poses with GOLD version 5.2 (CCDC)39
was carried out. The piecewise linear potential implemented in the
GOLD program (ChemPLP)40 was used as scoring function to
calculate the final scores of the compounds.
15. Warmus, J. S.; Quinn, C. L.; Taylor, C.; Murphy, S. T.; Johnson, T. A.; Limberakis,
C.; Ortwine, D.; Bronstein, J.; Pagano, P.; Knafels, J. D.; Lightle, S.; Mochalkin, I.;
Brideau, R.; Podoll, T. Bioorg. Med. Chem. Lett. 2012, 22, 2536.
16. McClerren, A. L.; Endsley, S.; Bowman, J. L.; Andersen, N. H.; Guan, Z.; Rudolph,
J.; Raetz, C. R. H. Biochemistry 2005, 44, 16574.
As far as all the considered LpxC crystal structures were very
similar to each other (RMSD of the backbone atoms between
0.15 and 0.21 Å), only one crystal structure reported by Lee et al.
in 201118 (PDB ID: 3P3G) was used to dock the inhibitors under
study to save computational time.
17. Barb, A. W.; Leavy, T. M.; Robins, L. I.; Guan, Z.; Six, D. A.; Zhou, P.; Bertozzi, C.
R.; Raetz, C. R. H. Biochemistry 2009, 48, 3068.
18. Lee, C.; Liang, X.; Chen, X.; Zeng, D.; Joo, S.; Chung, H.; Barb, A. W.; Swanson, S.
M.; Nicholas, R. A.; Li, Y.; Toone, E. J.; Raetz, C. R. H.; Zhou, P. Chem. Biol.
(Cambridge, MA, U.S.) 2011, 18, 38.
19. Liang, X.; Lee, C.; Chen, X.; Chung, H. S.; Zeng, D.; Raetz, C. R. H.; Li, Y.; Zhou, P.;
Toone, E. J. Bioorg. Med. Chem. 2011, 19, 852.
Re-docking of ligands from 3P3G, 3PS1, 3PS2 and 3PS3 (Fig. S7,
Supporting Information) showed, as summarized in Figure S6 and
Table S1 (Supporting information), that top-ranked docking poses
give RMSD values lower than 1.0 Å indicating that the docking
program is able to correctly reproduce the X-ray structure of the
LpxC-inhibitor complexes. Visual inspecting of the docking results
indicates that the hydroxamic acid is coordinating the zinc ion and
making hydrogen bonds with Glu78, Thr191 and His265. The thre-
onine group is engaged in hydrogen bonds with Cys63 (backbone),
Thr191, Lys239 and van der Waals interaction with Phe192. The
hydrophobic diacetylene and the terminal phenyl are interacting
with a cluster of hydrophobic residues, including Leu18, Ile198,
Met195, Phe212 and Val217.
20. Whittington, D. A.; Rusche, K. M.; Shin, H.; Fierke, C. A.; Christianson, D. W.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 8146.
21. Barb, A. W.; Jiang, L.; Raetz, C. R. H.; Zhou, P. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 18433. S18433/1-S18433/6.
22. Mochalkin, I.; Knafels, J. D.; Lightle, S. Protein Sci. 2008, 17, 450.
23. Shin, H.; Gennadios, H. A.; Whittington, D. A.; Christianson, D. W. Bioorg. Med.
Chem. 2007, 15, 2617.
24. Oddo, A.; Holl, R. Carbohydr. Res. 2012, 359, 59.
25. Loeppenberg, M.; Mueller, H.; Pulina, C.; Oddo, A.; Teese, M.; Jose, J.; Holl, R.
Org. Biomol. Chem. 2013, 11, 6056.
26. Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G. A.; Hartung, J.; Jeong, K.
S.; Kwong, H. L.; Morikawa, K.; Wang, Z. M., et al J. Org. Chem. 1992, 57, 2768.
27. Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem. Rev. (Washington, DC)
1994, 94, 2483.
28. The preparation of compound 12 by kinetic resolution has been described in
the literature: Ema, T.; Nakano, Y.; Yoshida, D.; Kamata, S.; Sakai, T. Org. Biomol.
Chem. 2012, 10, 6299.
29.
A synthesis of compound 16 starting from (S)-mandelic acid has been
mentioned in the literature: Andrus, M. B.; Sekhar, B. B. V. S.; Meredith, E. L.;
Dalley, N. K. Org. Lett. 2000, 2, 3035.
Supplementary data
30. Normark, S.; Boman, H. G.; Matsson, E. J. Bacteriol. 1969, 97, 1334.
31. Bertani, G. J. Bacteriol. 1951, 62, 293.
32. Hernick, M.; Gattis, S. G.; Penner-Hahn, J. E.; Fierke, C. A. Biochemistry 2010, 49,
2246.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.057.
33. Jackman, J. E.; Fierke, C. A.; Tumey, L. N.; Pirrung, M.; Uchiyama, T.; Tahir, S. H.;
Hindsgaul, O.; Raetz, C. R. H. J. Biol. Chem. 2000, 275, 11002.
34. Roth, M. Anal. Chem. 1971, 43, 880.
35. Barb, A. W.; McClerren, A. L.; Snehelatha, K.; Reynolds, C. M.; Zhou, P.; Raetz, C.
R. H. Biochemistry 2007, 46, 3793.
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