Reducing the Flexibility of Type II Dehydroquinase for Inhibition: A Fragment-Based Approach and Molecular Dynamics Study

Article abstract of DOI:10.1002/cmdc.201700396

A multidisciplinary approach was used to identify and optimize a quinazolinedione-based ligand that would decrease the flexibility of the substrate-covering loop (catalytic loop) of the type II dehydroquinase from Helicobacter pylori. This enzyme, which i

Full text of DOI:10.1002/cmdc.201700396

Accepted Article
Title: Reducing the Flexibility of Type II Dehydroquinase Enzyme for
Inhibition - A Fragment-Based Approach and Molecular Dynamics
Simulation Study
Authors: Antonio Peón, Adrián Robles, Beatriz Blanco, Marino
Convertino, Paul Thompson, Alastair R. Hawkins, Amedeo
Caflisch, and Concepcion Gonzalez-Bello
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Reducing the Flexibility of Type II Dehydroquinase Enzyme for
Inhibition – A Fragment-Based Approach and Molecular
Dynamics Simulation Study
Antonio Peón,[a] Adrián Robles,[a] BeatrizBlanco,[a] Marino Convertino,[b,δ] Paul Thompson,[c] Alastair R.
Hawkins,[c] Amedeo Caflisch,*[b] and Concepción González-Bello*[a]
Abstract: A multidisciplinary approach was used to identify and
optimize a quinazolinedione-based ligand that would reduce the
flexibility of the substrate-covering loop (catalytic loop) of the type II
dehydroquinase from Helicobacter pylori. This enzyme, which is
essential for the survival of this bacterium, is involved in the
levels is the fact that most of the antibiotics in clinical use target
a reduced and the same type of bacterial targets and resistance
to them are well known and spread worldwide. To this end, in
recent years much research has been devoted to the
identification of new and unexploited therapeutic targets involved
in key processes for bacterial survival as well as the detailed
insight of the basis of their behavior and the identification of
compounds targeting them.
biosynthesis of the aromatic amino acids.
A computer-aided
fragment-based protocol (ALTA) was first employed to identify the
aromatic fragments able to block the interface pocket that separates
two neighbor enzyme subunits and is located at the active site
entrance. Chemical modification of its non-aromatic moiety through
an olefin cross metathesis and Seebach´s self-reproduction of
One of these interesting targets is the type II dehydroquinase (3-
dehydroquinate dehydratase, DHQ2, EC 4.2.1.10) that is the
third enzyme of the shikimic acid pathway, through which
erythrose-4-phosphate and phosphoenol pyruvate is converted
into chorismic acid. The latter is the precursor of important
aromatic metabolites such as the aromatic amino acids, folate
cofactors, ubiquinone and vitamins E and K.2 DHQ2, which is
encoded by the aroD/aroQ gene, is an essential enzyme in
Mycobacterium tuberculosis, the causative agent for
tuberculosis, and Helicobacter pylori, which which is a major
cause of gastric and duodenal ulcers and it has been classified
as class I carcinogen by the International Agency of Research
on Cancer (IARC) and World Health Organization (WHO).3,4
DHQ2 catalyzes the reversible dehydratation of 3-dehydroquinic
acid (1) to form 3-dehydroshikimic acid (3) via the enolate
intermediate 2 (Figure 1).5,6 Extensive biochemical, structural
and QM/MM studies revealed that the enzymatic reaction is
initiated by an essential aspartate that deprotonates an essential
tyrosine to afford the catalytic tyrosinate, which triggers the
process.5-7 The necessary reduction in pKa of the tyrosine is
achieved by the proximity of two conserved arginine residues
and a cation-π interaction with an essential arginine. The final
step is the acid-catalyzed elimination of the C1 hydroxyl group
that is mediated by the conserved histidine acting as a proton
donor.
chirality synthetic principle allowed the development of
a
quinazolinedione derivative that disables the catalytic loop plasticity,
which is essential for the enzyme catalytic cycle. Molecular
dynamics simulation studies revealed that the ligand would force the
catalytic loop into an inappropriate arrangement for catalysis by
strong interactions with the catalytic tyrosine and by expelling the
essential arginine out of the active site.
Introduction
Although antibiotics are one of the most successful drugs in
clinic that have saved millions of lives, many of them are
nowadays ineffective in treating infections caused by resistant
bacteria.1 This is a dramatic problem in people with a weak
immune system or undergoing surgery, cancer chemotherapy,
dialysis, etc. for which the treatment of secondary processes are
crucial. One of the factors responsible for the actual resistant
[a]
Dr. A. Peón, A. Robles, Dr. B. Blanco, Prof. C. González-Bello,
Centro Singular de Investigación en Química Biolóxica e Materiais
Moleculares (CIQUS) and Departamento de Química Orgánica,
Universidade de Santiago de Compostela,
calle Jenaro de la Fuente s/n, 15782 Santiago de Compostela,
Spain.
Two of the three catalytic residues, Tyr22 and Arg17 for H. pylori
DHQ2, are located in the substrate-covering loop (catalytic loop)
that closes the active site after substrate binding (Figure 1A). It
has been shown that the arrangement of the catalytic loop
required for catalysis has: (1) the side chains of the tyrosine and
arginine residues pointing towards the active site; and (2) a
cation-π interaction between the catalytic residues.5 We believe
that targeting the loop plasticity is a good strategy for inhibitor
E-mail: concepcion.gonzalez.bello@usc.es
Dr. M. Convertino, Prof. A. Caflisch,
Department of Biochemistry,
University of Zurich,
CH-8057 Zurich, Switzerland.
[b]
[c]
E-mail: caflisch@bioc.uzh.ch
P. Thompson, Prof. A. R. Hawkins,
design since the motion of this loop is an essential process for
5,8
Institute of Cell and Molecular Biosciences, Medical School,
University of Newcastle upon Tyne,
Catherine Cookson Building, Framlington Place, Newcastle upon
Tyne NE2 4HH, UK.
the DHQ2 catalytic cycle.
We became therefore interested in
designing ligands able of altering the flexibility of the loop by
compounds structurally different from previously reported ones,
which are mainly based on the mechanism of action.7 These
reported inhibitors are either mimics of the enol intermediate 2,
Supporting information for this article is given via a link at the end of
the document.
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compounds 45,9 or substrate analogs, compounds 610 (Figure
1D). More recently, several 3-nitrobenzylgallate-based analogs 7
have been described, which incorporate relevant aromatic
moieties previously identified by structural and biological
studies.11 Remarkably, in the crystal structure of DHQ2 in
complex with the most potent inhibitors is observed that the
aromatic moiety present in their structure block the entrance of
the essential arginine side chain into the active site and cause
an important change in the appropriate arrangement and
flexibility of the substrate-covering loop.12 These aromatic
moieties are located in the interface pocket, which separates two
neighbor subunits of the enzyme and is located at the entrance
of the active site. DHQ2 is a dodecamer formed from a tetramer
of trimers, a trimer being the minimum catalytic unit of the
enzyme.13 QSAR studies also revealed that this pocket is key for
the efficacy and specificity of inhibitors.14,15
In particular, we aimed for ligands that would either stabilize the
closed form avoiding the entrance of the substrate or the open
conformation providing a widely exposed active site. For our
goal, we took into account: (1) our previous studies on the
motion of the catalytic loop during product release pinpointed by
Molecular Dynamic (MD) simulation studies of the product
enzyme complex (Figure 1B),5 (2) the loop conformational
changes observed in the crystal structures of DHQ2 from M.
tuberculosis (Mt-DHQ2) and H. pylori (Hp-DHQ2) in complex
with some of the most potent reported inhibitors,6 and (3) the
well stablished advantages of the fragment-based approaches
as will be discussed below.16-25
Figure 1 (A) Schematic representation of the DHQ2 enzyme motion required for catalysis. Note the cation-π interaction between the essential tyrosine and
arginine side chains that are required for catalysis. Both residues must be also pointing towards the active site. Only two chains of the catalytic trimer are shown
for clarity. (B) RMSD plot for the protein backbone (Cα, C, N, and O atoms) calculated per residue in the DHQ2/3 complex obtained from MD simulations studies
of the product release (DHQ2/3 enzyme complex).5 Note how the loop undergoes a large conformational change during product release whereas no significant
changes are observed in the rest of the protein. (C) Enzymatic conversion of 3-dehydroquinic acid (1) to 3-dehydroshikimic acid (3) catalyzed by DHQ2. The
reaction proceeds via the enol intermediate 2. (D) Selected examples of DHQ2 inhibitors.9-11
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We aim to identify inhibitors containing drug-like entities that
might facilitate the entrance into the bacterium cell in which the
aromatic fragments would be located in the interface pocket to
block the active site entrance. For this purpose, fragment-based
screening is nowadays a wide accepted technique to identify
relatively simple hit-compounds that possess a high binding
affinity per heavy atom, and thus are ideal compounds for
optimization into clinical candidates with good drug-like
properties.16-25 This technique has emerged as an important
alternative to high-throughput screening,24-25 because it has the
advantage that incorporates the structural information26 of the
target to preselect the molecules that are most likely to show
binding and inhibitory activity. The available experimental
knowledge of known inhibitors is incorporated by using
pharmacophore constraints to preselect compounds for docking
and therefore reducing computation times. Computer-aided
fragment-based approaches are being increasingly proven as a
successful means of generating novel hits for drug discovery
programs.27 In this context, the Anchor-based Library Tailoring
screening Approach (ALTA), which was developed in 2008 by
Caflisch et al.,28 has proven to be very efficient for the discovery
of novel inhibitors of a variety of targets. For instance, it has
been successfully applied to the discovery of inhibitors of
tyrosine kinase erythropoietin producing human hepatocellular
carcinoma receptor B4,28-29 of the West Nile virus NS3
protease,30 cathepsin B,31 the human cyclin-dependent kinase
232 and several human bromodomains33. The essential element
of the ALTA method is the use of optimally docked small
fragments to select from a large library of compounds only those
that have one (or more) of the top-ranking fragments.
compound selection and ranking. This is carried out using
FFLD37 (Fast Flexible Ligand Docking) and CHARMM34 for the
energy minimization of the enzyme-ligand resulting complex.
Evaluation of the binding free energy of multiple poses of each
compound makes use of the LIECE (Linear Interaction Energy
with Continuum Electrostatics) method.39
To discard at an early stage fragments that are unlikely to bind,
several pharmacophores constraints were used. In particular,
considering that the active site of DHQ2 has several residues
capable of recognizing bidentate groups such as carboxylates or
sulfonamides, a filtering step discarded all compounds devoid of
these functional groups. It was also considered that compounds
should have a maximum of 10 rotatable bonds, between 2 to 6
acceptor groups, ≤ 3 donor groups and a total number of
hydrogen-bond acceptor and donor ≤ 10. As a consequence, the
chemical library was reduced to 239.779 compounds from which
DAIM extracted 61.549 candidate fragments.
Here, we present the computer-aided fragment-based
identification of reversible competitive inhibitors of a recognized
target for antibiotic drug discovery, Hp-DHQ2. The synthesis of
the identified potential compounds and their inhibitory activity
are also provided. Moreover, chemical modifications on the most
active compound obtained in this study were also explored for
further optimization. We provide evidence based on Molecular
Dynamics (MD) simulation studies that our inhibitors reduce the
plasticity of the catalytic loop by blocking key residues for the
catalysis.
Figure 2 Graphical representation of the workflow of ALTA procedure.
Docking was carried out using the previously reported enzyme
structure of Hp-DHQ2 in complex with (1R,4S,5R)-1,4,5-
trihydroxy-3-[(5-methyl-1-benzothiophen-2-yl)methoxy]cyclohex-
1
2-ene-1-carboxylic acid (4a, R = (5-methyl-1-benzothiophen-2-
yl)methoxy), PDB code 2WKS).9m This crystal structure was
chosen because it has an inappropriate conformation of the
substrate-covering loop for catalysis. Thus, the aromatic moiety
in 4a expels the side chain of the essential Arg17 out of the
active site and, under this arrangement the enzyme is efficiently
inhibited. After fragment docking, the library of fragments was
reduced to 10.712 candidate fragments. Using all these
fragments as queries a subset of 30.309 compounds from the
initial library was generated. After flexible docking of each
molecule of the library using the best poses of its fragments as
anchors, the best 50 top-ranking compounds were selected
(Table S1). The first two compounds and seven of remaining
ones, which were selected based on structural considerations of
the key interactions with the enzyme, were chosen for biological
evaluation (Figure 3). Compounds 8 and 15–16 were obtained
from commercial sources and compounds 9–14 were
Results and Discussion
Fragment-based Identification of Ligands
The subset “clean-leads” of the ZINC database34 with about 1.5
million compounds was chosen for this study. The computer-
aided fragment-based approach used, ALTA, involves four
consecutive steps (Figure 2): (1) automatic decomposition of
each molecule of the chemical library into fragments using the
program DAIM (Decomposition And Identification of
Molecules);35 (2) fragment docking using SEED (Solvation
Energy for Exhaustive Docking) for selection and ranking of the
fragments;36 (3) substructure search of the ALTA-selected
fragments into the chemical library for initial selection of potential
ligands; and (4) flexible docking of each selected compound
using the best poses of its fragments as anchors for final
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synthesized using standard procedures (see
description in the Supporting Information).
a
detailed
Met92* and the carbon side-chain of Asp89* (residues from this
subunit will be marked with an asterisk). The essential Asp89*
proved to be responsible for the deprotonation of the essential
Tyr22 to afford the catalytic tyrosinate, which in turn triggers the
enzymatic process.5 Moreover, several lipophilic interactions
with the side-chain of Leu11 and Leu14 were also recognized.
The carboxylate group of the inhibitors would be located in the
C1 binding pocket by forming hydrogen-bonding interactions
with the side-chain of Asn76, the main amide chain of Leu103
and Thr104. This pocket has been proven to be crucial for
substrate recognition and enzyme catalysis.40 Thus, the
carboxylate group of the natural substrate interacts with the
active site through four strong hydrogen bonds involving the
main chain NH amide group of Leu103 and Thr104, the OH
group of the side chain of Thr104 side chain and the amide side
chain of Asn76 (Figure S1). Moreover, two hydrogen bonds with
the conserved Asn76 and His102 is the way in which the
enzyme controls the correctly positioning of the C1 hydroxyl
group for the final elimination step.
Inhibition Activity of Compounds 816
The potential DHQ2 ligands identified by ALTA-approach,
compounds 816, were assayed in the presence of 3-
dehydroquinic acid (1) for their inhibitory properties against Hp-
DHQ2. The inhibition data are summarized in Table 1. In general,
these compounds were found to be reversible competitive
inhibitors of the enzyme with Ki values in the micromolar range,
below the Km of the enzyme. The most potent inhibitor proved to
be the quinazolinedione 15 with a Ki of 19 µM. This compound
clearly stands out from the series as the next ones, specifically
compounds 9, 14 and 16, showed Ki values between 77 and 91
µM.
Table 1. Ki values for compounds 8–16 against Hp-DHQ2a
Compound
8
9
Ki (µM)b
141 ± 19
77 ± 5
10
11
12
13
14
15
16
139 ± 12
103 ± 13
159 ± 15
>300
85 ± 5
19 ± 2
91 ± 12
aAssay conditions: Tris.HCl (50 mM), pH 7.0, 25 °C.
b
Km (1) = 444 μM. Values are the mean ± SEM of (n = 3)
determinations.
Remarkably, in addition to the above mentioned interactions, the
binding modes of the most active inhibitors appear to have in
common π-π stacking and hydrogen-bonding interactions with
Tyr22. Specifically, a hydrogen-bond between the phenol group
of Tyr22 and the amide carbonyl group of ligand 9 or the
carbonyl group of the aromatic ring of ligands 1416 would take
place. Tyr22 is the essential residue that removes the pro-R
hydrogen of C2 in 1 and which appropriate orientation for
catalysis is controlled by the essential Arg17. This hydrogen-
bonding interaction with Tyr22 is absent in the weakest inhibitors
of the series, ligands 8 and 1012, (Figure S2). In addition, the
predicted binding of ligands 8 and 12 would be very different
from the rest of the tested ligands in this study, which might
account for its lower potency. However, in both cases, the
aromatic ring of the ligands would be located in the interface
pocket. Once the aromatic ring is fixed in this region, the rest of
the molecule seems to be located in the space available of the
active site by establishing hydrogen-bonding interactions with
polar residues of the active site. These results revealed that a
large number of the aromatic moieties included in the reported
ligands are well located in the interface pocket and might
therefore be good hit fragments to be considered in future
designs.
Figure 3 Selected potential inhibitors of the Hp-DHQ2 enzyme obtained by the
fragment-based approach.
Binding Mode of Compounds 816
The binding modes of the most potent identified inhibitors,
compounds 9 and 1416, revealed that the aromatic ring of the
ligands 9 and 1416 would be located in the interface pocket
(Figure 4). The aromatic moiety of compounds 9 and 1416
should have apolar interactions with the side-chain of Leu93*,
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In general: (a) ligand 14 would be the only one lacking any
interactions with the C6C5 substrate recognition center of the
enzyme involving the conserved residue His82; (b) the
interaction of the aromatic moiety with the essential Tyr22 and
the interface pocket in 16 seems to be less efficient than for
compounds 9 and 1415; and (c) compound 15 would be the
ligand that would best interact with the main pockets of the
enzyme, which might explain its higher inhibitory potency.
Moreover, considering that ligand 9 is structurally very close to
3-nitrobenzylgallate-based analogs 7, which were reported by
Abell et al.11 during the realization of the herein presented
studies, our subsequent efforts were focused on the optimization
of ligand 15.
reduce the flexibility of the substrate-covering loop by interaction
with the essential tyrosine and the interface pocket. Hence, in
order to explore the possible improvement of the inhibitory
activity obtained, the modification of the chain containing the
carboxylate group in 15 was then studied.
Four new quinazolinedione derivatives, compounds 17S, 17R,
and 1819, were designed (Figure 5A). We took into account the
binding mode of citrate in the crystal structure of Hp-
DHQ2/citrate binary complex (PDB code 2C4V, 2.5 Å) reported
by Lapthorn et al.41 This compound proved to be a competitive
inhibitor of the enzyme with a Ki value of 2.5 mM. Compounds
17S and 17R containing an acetate group instead of an
isopropyl one were designed to improve the interaction with the
conserved His82, which is involved in the recognition of the C5
hydroxyl group.
Globally, the most potent inhibitor identified in this study,
compound 15, contains two key moieties: (i) the
quinazolinedione ring and (ii) the carboxylate group. Among the
two, the aromatic ring seems to be particularly well located to
Figure 4 Selected view of the predicted binding mode of ligands: (A) 9 (yellow), (B) 14 (orange), (C) 15 (green), and (D) 16 (pink) in the Hp-DHQ2 active site. The
neighboring chain close to the active site is shown in blue and its relevant side chain residues are indicated with an asterisk. Relevant side chain residues are
shown and labeled. Hydrogen-bonding (red) and lipophilic (grey) interactions are indicated as dashed lines.
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Figure 5 (A) Explored chemical modifications of the carboxylate containing chain in 15. New targeted quinazolinedione derivatives 1719. (B) Synthesis of
compounds 17S, 17R and 18. Reagents and conditions. (a) N-Boc-ethylenediamine (for 21) or ethyl 4-aminobutaroate (for 22), EDC, DMAP, DMF, RT; (b)
triphosgene, DIPEA, DCM, RT; (c) TFA, DCM, 0 °C; (d) 1. LiOH, THF, RT. 2. HCl (10%). (e) 1. SOCl2, 60 °C. 2. Dimethyl L-aspartate (for 27S) or Dimethyl D-
t
aspartate (for 27R), Et3N, DCM, RT; (f) 1. LiOH. 2. Amberlite IR-120 (H+); (g) 1. LHMDS, THF, 78 °C; 2. BrCH2CO2 Bu, 23 °C; (h) 24, HATU, DIPEA, DMF, RT;
(i) HCl (4.5 M), RT.
aminoquinazolinedione 24 involved the nucleophilic substitution
of commercially available chloride 28 with lithium amide, sodium
azide or benzylamine, various solvents (DMF, MeCN, Toluene),
bases (K2CO3, DIPEA) and reaction temperatures (50100 °C).
However, all the attempts afforded mainly starting material or an
intramolecular cyclization reaction of 28. The synthesis of the
amino 24 was finally achieved in three steps from antranilic acid
(20) by first incorporation of the required chain followed by
quinazolinedione ring formation. Thus, EDC-condensation with
commercially available N-Boc-ethylenediamine followed by
intramolecular condensation of the resulting amide 21 with
triphosgene and subsequent removal of the Boc protecting
group in 23 with TFA yielded the required amine 24 as
In addition, reasoning that in the Michaelis complex the C1
hydroxyl group interacts by hydrogen bonding with the
conserved Asn76 and His102 to correctly positioning this group
in the final elimination step (Figure S1),5 the incorporation of a
tertiary hydroxyl group was also explored with compound 18.
Finally, considering that the amide linker does not seem to
interact with the enzyme active site residues, the effect of the
incorporation of a flexible carbon chain was explored with
compound 19.
Chain Optimization – Quinazolinediones derivatives
1719
(a) Synthesis  Compounds 17 and 18 were prepared by amide
coupling between the acids 26 and 30 and the amines dimethyl
L-aspartate, dimethyl D-aspartate, and 24, respectively (Figure
5B). Our initial efforts for the synthesis of the
trifluoroacetic acid salt. Following
a similar strategy, the
quinazolinedione 26 was prepared from ethyl 4-aminobutaroate
and antranilic acid (20).
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Scheme 1 (A) Explored olefin cross metathesis strategy for the synthesis of 19. (B) Synthesis of compound 19. Reagents and conditions. (a) 4-bromobutene,
Grubbs-Hoveyda 2nd catalyts, PhMe, 90 °C; (b) NaN3, DMF, 90 °C; (c) 1. Ph3P, THF, H2O, ∆. 2. 20, EDC, DMAP, RT; (d) triphosgene, Et3N, DCM, RT (e) H2, Pd/C
(10%), EtOH, RT; (f) 1. LiOH. 2. Amberlite IR-120 (H+).
On the other hand, the required acid 30 for the preparation of 18
was synthesized following Seebach´s42 self-reproduction of
chirality synthetic principle. Thus, alkylation with tert-butyl
bromoacetate of the carbanion resulting of the treatment with
lithium hexamethylsilazide of previously reported dioxolanone
acid 2943 afforded the desired compound 30. Next, amide
coupling between the acids 26 and 30 and the amines dimethyl
L-aspartate, dimethyl D-aspartate and 24, respectively, using
HATU as coupling agent and in the presence of
diisopropylethylamine gave the amides 27S, 27R and 31,
respectively. Finally, deprotection of latter compounds yielded
the required amides 17S, 17R and 18, respectively.
The synthesis of compound 19, in which the quinazolinedione
and the carboxylic acid moieties are linked through a saturated
carbon chain, proved to be more challenging. An olefin cross
metathesis (OCM) approach involving the construction of the
C4C5 carbon bond was designed (Scheme 1A). This
disconnection was chosen because it would allow the use of the
allyl derivative 34,43 which is readily prepared by alkylation with
allyl bromide of the dioxolanone acid derived from L-malic acid,
compound 35. Initial alkylation attempts revealed that the use of
less reactive electrophiles such as long chain alkyl bromides
provide quite low yields of the required products.
All the attempts of OCM between the allyl derivatives 33 and 34
revealed to be unsatisfactory due to the poor selectivity towards
the required heterodimer product. Homodimerization of 34 was
the main reaction product whereas compound 33 showed to
have very low reactivity. Fortunately, the selectivity towards the
heterodimer product was achieved following the general
guidelines reported by Grubbs et al.44 consisting in the use of
two olefins of remarkably different reactivity (Scheme 1B). Thus,
the OCM between 4-bromobutene (type I olefin) and compound
34 (type II olefin) catalyzed by second generation of Grubbs-
Hoveyda catalysts gave the cross trans olefin 36 in 71% yield.
Having incorporated the carbon long chain, the following steps
were focused in the construction of the quinazolinedione ring.
First, the bromide 36 was converted into the required amine by
nucleophilic substitution with sodium azide and subsequent
Staudinger reduction of the resulting azide 37. The EDC-
coupling of the terminal amine with antranilic acid (20) in the
presence of 4-N,N-dimethylpyridine gave the amide 38. It is
important to highlight that the presence of a trans double bond in
the carbon chain is key for the successful synthesis of the
aromatic ring. Thus, the conformational restriction induced by
this olefin avoids the intramolecular nucleophilic attack by the
resulting amine to the methyl ester. Finally, the intramolecular
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condensation of 38 with triphosgene gave the quinazolinedione
39 that was transformed into the desired compound 19 by
catalytic hydrogenation of the double bond and subsequent
hydrolysis of the resulting saturated compound 40.
100 ns of MD simulations at a temperature of 300 K (Figure 6).
These studies were carried out considering a dual protonation of
His102 based on mechanistic reasons and the three possible
protonation states of His82, i.e. dual (δ and ε) and single (δ or ε).
The best results were obtained with a dual protonation of His82
because in doing so the carboxylate group is well stabilized
nearby. Other protonation states provided significant motion on
the His82 side chain. In addition, similar results were obtained
from MD simulation studies on the Hp-DHQ2/citrate binary
complex (PDB code 2C4V). These results explain why in the
latter structure the electronegative O4 atom of one of the
carboxylate group in citrate is located at 2.5 Å of the also
electronegative NE1 atom of His82 residue.
(b) Inhibitory activity of compounds 1719 and 32 The results
of inhibition activity of compounds 1719 and 32 against Hp-
DHQ2 revealed that reducing the conformational restriction in
the carbon bearing the carboxylic group, which is involved in the
recognition in the C1 pocket, enhances the inhibitory potency
(Table 2). The most favorable effect was achieved with
compound 19 (Ki = 6.3 µM) having a flexible carbon chain. In
addition, it seems that the increased flexibility also favors extra
interactions of the incorporated tertiary hydroxyl and acetate
groups with residues Asn76, Leu103, His102 and His82, as
designed. In fact, no significant effects were observed when the
latter groups were introduced in the ligand having an amide
linker, compound 18. In order to corroborate: (1) this hypothesis;
(2) to assess the reliability of the proposed complexes and (3) to
determine the conformational changes caused in the substrate-
covering loop of Hp-DHQ2 after compound 19 binding, MD
simulations studies were conducted with the highest score
solutions obtained by docking for ligands 18 and 19.
Table 2. Ki values for compounds 1719 and 32 against Hp-
DHQ2a
Compd
15
Ki (µM)b
19 ± 2
17S
17R
18
111 ± 3
52 ± 5
18 ± 2
19
32
6.3 ± 0.2
166 ± 15
(c) MD Simulations Studies – The binding modes of ligands 18
and 19 were first generated by the program GOLD45 version 5.2
using the same enzyme structure as for the fragment docking,
i.e., PDB 2WKS9m. The highest score solution for the two Hp-
DHQ2/18 and Hp-DHQ2/19 binary complexes were subjected to
aAssay conditions: Tris.HCl (50 mM), pH 7.0, 25 °C.
Km (1) = 444 μM; bValues are the mean ± SEM of (n = 3)
determinations.
Figure 6 Detailed view of the predicted binding of ligands 19 (A, yellow) and 18 (B, green) in the active site of Hp-DHQ2 obtained by MD simulation studies. Both
poses showed correspond to snapshots after 60 ns of MD simulation. Hydrogen-bonding interactions between ligand and several residues of the active site are
shown as red dashes. (C) Comparison of the binding mode of ligands 19 and 18 in the active site of Hp-DHQ2. (D-F) Variation of the relative distance between
resides Arg117 (O atom, D), His102 (ND1 atom, E), and Leu103 (N atom, F) and NH group of the aromatic moiety (N2 and N3 atoms), the medium chain
carboxylate (O4, O7 and O1 atoms) in 19 and 18, respectively, during the whole simulation. Note how ligand 19 has stronger hydrogen bonding interactions with
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10.1002/cmdc.201700396
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the latter residues than ligand 18 and provides a closer and more organized conformation of the substrate-covering loop. Side chain residues from the neighbor
chain are shown in cyan and labeled with an asterisk.
The results clearly show that the flexible carbon chain in 19: (1)
causes a more pronounced reduction of the flexibility of the
catalytic loop than in 18 and (2) enhances the strength of the
studies revealed that favoring the geometric perfection in
anchoring the ligand at the center C1 recognition center, the
motion of the loop would be frozen by the aromatic fragment and
hydrogen bonding interactions involving residues Asn76, His102, consequently the effective inhibition of the enzyme is achieved.
Leu103 and His82. These effects are easily visualized by
analyzing the variation of the distance between the atoms
involved in these hydrogen bonds during the 100 ns of
simulation (Figures 6E-6F and S3). Thus, for ligand 19, the loop
folds on top of the ligand so that after 20 ns of simulation a
stable hydrogen bond between the NH group of the aromatic
moiety and the backbone carbonyl of Arg17 (in the catalytic
loop) is formed with an average distance of 3.1 Å (distances are
measured between heavy atoms) (Figures 6C and 6D). In fact,
our MD studies revealed that in the presence of 19 the loop is
clearly more ordered (Figure 6C). After the folding, the complex
is very stable as no significant changes were observed for the
remaining simulation (Figure S4A). This phenomenon was not
observed for ligand 18, showing an average distance of 10.9 Å.
The Hp-DHQ2/18 complex remained unchanged during the
whole simulation (Figure S4B).
Moreover, the hydrogen bonds between the hydroxyl and
carboxylate groups in 19 with the NH groups of His102, Asn76
and Leu103 have an average distance of 3.4 Å and 3.0 Å,
respectively. For 18, they are 6.4 Å and 5.7 Å, respectively.
Therefore, the incorporation of the flexible chain in 19 seems to
enhance the binding of the carboxylate group of the inhibitor in
the C1 recognition center. The strength of hydrogen bonds
involving residue His82 are quite similar (Figure S3). For both
ligands, the terminal carboxylate group would interact by
favourable electrostatic interactions with the conserved residues
Arg109, Arg113 and His82.
This would be accomplished by π-π stacking interaction
between the fragment and the catalytic tyrosine and by
hydrogen bonding with the main amide group of the essential
arginine. In doing so, the position of both residues would be well
fixed in an inappropriate arrangement for catalysis. We also
believe that some of the fragments identified in this work might
be also useful hit fragments for future designs if the interaction
with the C1 carboxylate pocket is improved.
Experimental Section
Experimental details on the synthesis of compounds 914 are detailed in
the supporting information.
2-Amino-N-[2-(N-Boc)aminoethyl]benzamide (21)  An stirred solution
of antranilic acid (20) (100 mg, 0.73 mmol) and N-Boc-ethylenediamine
(0.17 mL, 1.09 mmol) in dry DMF (3.6 mL), under argon and at room
temperature, was treated with EDC (168 mg, 0.87 mmol) and N,N-
dimethylpyridine (4.4 mg, 0.04 mmol). The resulting mixture was stirred
at room temperature for 12 h. The reaction mixture was diluted with ethyl
acetate and water. The organic layer was separated and the aqueous
phase was extracted with ethyl acetate (×2). The combined organic
layers were dried (anh. Na2SO4), filtered and concentrated under
reduced pressure. The residue obtained was purified by flash
chromatography, eluting with (60:40) ethyl acetate/hexane, to give the
amide 21 (179 mg, 88%) as a white solid. Mp: 163164 ºC. 1H NMR (300
MHz, CDCl3) δ: 7.37 (d, J = 7.8 Hz, 1H, ArH), 7.227.14 (m, 1H, ArH),
6.98 (br s, 1H, NH), 6.63 (m, 2H, 2×ArH), 5.54 (br s, 2H, 2×NH), 5.04 (br
s, 1H, NH), 3.563.43 (m, 2H, CH2), 3.37 (m, 2H, CH2) and 1.42 (s, 9H,
C(CH3)3) ppm. 13C NMR (75 MHz, CDCl3) δ: 170.0 (C), 157.4 (C), 148.9
(C), 132.4 (CH), 127.6 (CH), 117.4 (CH), 116.7 (CH), 115.7 (C), 80.0
(C(CH3)3), 41.5 (CH2), 40.2 (CH2) and 28.5 (C(CH3)3) ppm. IR (ATR) υ:
3445 (NH), 3349 (NH), 1674 (CO) and 1620 (CO) cm1. MS (ESI) m/z:
302 (MNa+). HRMS calcd for C14H21N3O3Na (MNa+): 302.1475; found,
302.1483.
Conclusions and Final Remarks
A multidisciplinary approach has been employed here to search
for ligands able to disable the enzyme flexibility that is an
essential process for catalysis. This has been applied to the
inhibition of a recognized target for antibiotic discovery, the type
II dehydroquinase from Helicobacter pylori (Hp-DHQ2), which is
essential enzyme in this pathogenic bacterium. A computer-
aided fragment-based approach, ALTA, was first employed to
identify the aromatic fragment able to block the active site
entrance by binding to the interface pocket that separates two
neighbor enzyme subunits. The identified quinazolinedione 15
proved to be a reversible competitive of the Hp-DHQ2 with a Ki
of 19 µM, below the Km of the enzyme (444 µM). Subsequent
chemical modification of the non-aromatic moiety in 15 through
an olefin cross metathesis and Seebach´s self-reproduction of
chirality synthetic principle allowed the development of a
quinazolinedione derivative containing a flexible malate moiety,
compound 19, that seems to efficiently disables the enzyme
motion. The latter ligand proved to be more potent with a Ki of
6.3 µM. The results from our Molecular Dynamics simulation
Ethyl 4-(2-aminobenzamido)butanoate (22)  A solution of ethyl 4-
aminobutaroate (1 g, 5.96 mmol) and dry triethylamine (0.9 mL, 5.96
mmol) in dry dichloromethane (52 mL) and under inert atmosphere was
treated at room temperature with EDC (1.26 g, 6.56 mmol) and antranilic
acid (20) (2.3 g, 11.92 mmol) in three portions during 1.5 h. The reaction
mixture was washed with aqueous sodium bicarbonate. The organic
phase was separated and the aqueous layer was extracted with
dichloromethane (×2). The combined organic extracts were dried (anh.
Na2SO4), filtered and concentrated under reduced pressure. The residue
obtained was purified by flash chromatography, eluting with (95:5)
dichloromethane/ethyl acetate, to give the amide 22 (782 mg, 71%) as an
orange oil. 1H NMR (250 MHz, CDCl3) δ: 7.24 (dd, J = 7.8 and 1.5 Hz, 1H,
ArH), 7.06 (td, J = 1.5 and 7.0 Hz, 1H, ArH), 6.73 (br s, 1H, NH), 6.55 (dd,
J = 8.1 and 1.1 Hz, 1H, ArH), 6.49 (td, J = 1.5 and 8.0 Hz, 1H, ArH), 5.22
(br s, 2H, NH2), 4.00 (q, J = 7.1 Hz, 2H, OCH2), 3.30 (q, J = 6.5 Hz, 2H,
NCH2), 2.28 (t, J = 7.1 Hz, 2H, COCH2), 1.79 (quint, J = 7.0 Hz, 2H,
NCH2CH2) and 1.13 (t, J = 7.1 Hz, 3H, CH3) ppm. 13C NMR (63 MHz,
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10.1002/cmdc.201700396
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CDCl3) δ: 173.5 (C), 169.4 (C), 148.5 (C), 131.9 (CH), 127.2 (CH), 117.0
(CH), 116.3 (CH), 115.9 (C), 60.4 (OCH2), 39.0 (NCH2), 31.7 (CH2), 24.4
(CH2) and 14.0 (CH3) ppm. IR (ATR) υ: 3192 (NH), 1719 (CO) and 1625
(CO) cm1. MS (ESI) m/z: 251 (MH+). HRMS calcd for C13H19N2O3 (MH+):
251.1390; found, 251.1385.
(MNa+). HRMS calcd for C14H16N2O4Na (MNa+): 299.1002; found,
299.1013.
3-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)propanoic acid (26)  A
solution of the ester 25 (200 mg, 0.73 mmol) in dry THF (1.5 mL) at room
temperature was treated with aqueous LiOH (2.1 mL, 0.7 M). The
reaction mixture was stirred at room temperature for 2 h and then
washed with diethyl ether. The aqueous phase was acidified with HCl
(10%) and extracted with ethyl acetate (×3). The combined organic
extracts were dried (anh. Na2SO4), filtered and concentrated under
reduced pressure to give the acid 26 (158 mg, 90%) as a white solid. Mp:
247249 ºC. 1H NMR (250 MHz, CD3OD) δ: 8.03 (d, J = 7.9 Hz, 1H, ArH),
7.64 (t, J = 7.6 Hz, 1H, ArH), 7.23 (t, J = 7.6 Hz, 1H, ArH), 7.16 (d, J =
8.2 Hz, 1H, ArH), 4.09 (t, J = 6.9 Hz, 2H, NCH2), 2.39 (t, J = 7.3 Hz, 2H,
COCH2) and 1.99 (quint, J = 7.1 Hz, 2H, NCH2CH2) ppm. 13C NMR (63
MHz, CD3OD) δ: 176.6 (C), 164.3 (C), 152.3 (C), 140.7 (C), 136.1 (CH),
128.7 (CH), 123.9 (CH), 116.0 (CH), 115.4 (C), 41.1 (NCH2), 32.3 (CH2)
and 24.3 (CH2) ppm. . IR (ATR) υ: 3431 (NH+OH), 1707 (CO), 1688 (CO)
and 1650 (CO) cm1. MS (ESI) m/z: 247 (MH). HRMS calcd for
C12H11N2O4 (MH): 247.0724; found, 247.0721.
3-[2-(N-Boc)aminoethyl]quinazoline-2,4(1H,3H)-dione (23)

A
suspension of amide 21 (89 mg, 0.32 mmol) and triphosgene (47 mg,
0.16 mmol) in dry dichloromethane (1.6 mL), under argon and at room
temperature, was treated with DIPEA (0.1 mL, 0.64 mmol) and the
resultant solution was stirred for 1 h. The reaction mixture was diluted
with dichloromethane and saturated aqueous solution of NaHCO3. The
aqueous layer was separated and the organic phase was washed with
water. The organic extract was dried (anh. Na2SO4), filtered and
concentrated under reduced pressure. The residue obtained was purified
by flash chromatography, eluting with (75:25) ethyl acetate/hexane, to
give the quinazolinedione 23 (58 mg, 60%) as a white solid. Mp: 204205
1
ºC. H NMR (300 MHz, DMSO-d6) δ: 11.35 (br s, 1H, NH), 7.92 (dd, J =
7.9 and 1.1 Hz, 1H, ArH), 7.657.60 (m, 1H, ArH), 7.207.14 (m, 2H,
2×ArH), 6.82 (t, J = 5.8 Hz, 1H, NH), 3.97 (t, J = 5.8 Hz, 2H, CH2), 3.19 (t,
J = 5.8 Hz, 2H, CH2) and 1.28 (s, 9H, C(CH3)3) ppm. 13C NMR (63 MHz,
DMSO-d6) δ: 162.2 (C), 155.8 (C), 150.4 (C), 139.6 (C), 134.8 (CH),
127.4 (CH), 122.3 (CH), 115.0 (CH), 114.0 (C), 77.5 (C(CH3)3), 40.2
(CH2), 37.7 (CH2) and 28.2 (C(CH3)3) ppm. IR (ATR) υ: 3372 (NH), 1703
(CO), 1671 (CO) and 1648 (CO) cm1. MS (ESI) m/z: 328 (MNa+). HRMS
calcd for C15H19N3O4Na (MNa+): 328.1268; found, 328.1262.
Diester 27S  A solution of the acid 26 (80 mg, 0.3 mmol) in tionyl
chloride (5 mL) was heated under reflux for 30 min. After cooling to room
temperature, the solvent was removed under reduced pressure and the
crude residue was dissolved in dry dichloromethane (2 mL) and treated
with dry trimethylamine (80 µL, 0.6 mmol) and dimethyl L-aspartate (60
mg, 0.3 mmol). The resulting mixture was stirred at room temperature for
2 h and then washed with saturated NaHCO3 (×3) and brine (×3). The
organic extract was dried (anh. Na2SO4), filtered and concentrated under
reduced pressure. The resulting residue was purified by flash
chromatography, eluting with ethyl acetate, to give the diester 27S (78
3-(2-Aminoethyl)quinazoline-2,4(1H,3H)-dione trifluoroacetic acid
salt (24)  A suspension of protected amine 23 (50 mg, 0.16 mmol) in
dichloromethane (1.3 mL) at 0 ºC was treated with TFA (1.3 mL) and the
resultant solution was stirred for 3 h. The reaction mixture was diluted
with dichloromethane and water. The organic phase was separated and
the aqueous phase was washed with dichloromethane. The aqueous
extract was lyophilised to give the amine trifluoroacetic acid salt 24 (50
mg, 99%) as a white solid. Mp: 253255 ºC. 1H NMR (300 MHz, CD3OD)
δ: 8.03 (dd, J = 7.9 and 1.0 Hz, 1H, ArH), 7.677.62 (m, 1H, ArH),
7.207.25 (m, 1H, ArH), 7.17 (d, J = 8.2 Hz, 1H, ArH), 4.32 (t, J = 5.5 Hz,
2H, CH2) and 3.26 (t, J = 5.5 Hz, 2H, CH2) ppm. 13C NMR (63 MHz,
CD3OD) δ: 165.3 (C), 153.1 (C), 141.4 (C), 137.0 (CH), 129.3 (CH),
124.6 (CH), 116.7 (CH), 115.9 (C), 40.4 (CH2) and 39.9 (CH2) ppm. IR
(film) υ: 3014 (NH), 1720 (CO), 1687 (CO) and 1654 (CO) cm1. MS
(ESI) m/z: 204 (MH). HRMS calcd for C10H10N3O2 (MH): 204.0779;
found, 204.0782.
mg, 66%) as a white foam.
= +27.0º (c1.1, CHCl3). 1H NMR (500
MHz, CDCl3) δ: 10.01 (s, 1H, NH), 8.09 (dd, J = 0.7 and J = 7.9 Hz, 1H,
ArH), 7.60 (td, J = 1.4 and J = 8.2 Hz, 1H, ArH), 7.21 (t, J = 7.5 Hz, 1H,
ArH), 7.13 (d, J = 8.1 Hz, 1H, ArH), 7.01 (br d, J = 8.5 Hz, 1H, NH), 4.89
(dt, J = 4.6 and J = 8.1 Hz, 1H, CH), 4.16 (m, 2H, NCH2), 3.73 (s, 3H,
OCH3), 3.68 (s, 3H, OCH3), 3.02 (dd, J = 4.7 and J = 17.1 Hz, 1H,
CHHCO2Me), 2.87 (dd, J = 4.6 and J = 17.1 Hz, 1H, CHHCO2Me), 2.36
(m, 2H, CH2CO) and 2.10 (quint, J = 7.0 Hz, 2H, CH2CH2CH2) ppm. 13C
NMR (63 MHz, CDCl3) δ: 172.2 (C), 171.4 (C), 171.2 (C), 162.5 (C),
151.7 (C), 138.6 (C), 134.9 (CH), 128.1 (CH), 123.1 (CH), 115.0 (CH),
114.2 (C), 52.6 (OCH3), 51.9 (OCH3), 48.4 (CH), 40.0 (NCH2), 36.0 (CH2),
33.4 (CH2) and 23.9 (CH2) ppm. IR (ATR) υ: 3283 (NH), 1722 (CO) and
1645 (CO) cm1. MS (ESI): 392 (MH+). HRMS calcd for C18H22N3O7
(MH+): 392.1452; found, 392.1452.
Ethyl 3-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)propanoate (25) 
A solution of the amine 22 (50 mg, 0.26 mmol) and triethylamine (72 µL,
0.52 mmol) in dry dichloromethane (1.3 mL), under inert atmosphere and
at room temperature, was treated with a solution of triphosgene in dry
dichloromethane (0.3 mL, 0.16 mmol, 0.2 M). The resulting solution was
stirred for 2 h and then washed with HCl (10%, ×3). The aqueous phase
was extracted with dichloromethane (×3). The combined organic extracts
were dried (anh. Na2SO4), filtered and concentrated under reduced
pressure. The residue obtained was purified by flash chromatography,
eluting with (40:10) diethyl ether/hexane, to give the quinazolinedione 25
(51 mg, 71%) as a yellow solid. Mp: 270271 ºC. 1H NMR (250 MHz,
CDCl3) δ: 10.85 (br s, 1H, NH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 7.61 (td, J =
1.3 and 8.2Hz, 1H, ArH), 7.20 (m, 2H, 2×ArH), 4.12 (t, J = 6.8 Hz, 2H,
NCH2), 4.05 (q, J = 7.1 Hz, 2H, OCH2), 2.43 (t, J = 7.5 Hz, 2H, COCH2),
2.08 (quint, J = 7.1 Hz, 2H, NCH2CH2) and 1.21 (t, J = 7.1 Hz, 3H, CH3)
ppm. 13C NMR (63 MHz, CDCl3) δ: 172.8 (C), 162.3 (C), 152.2 (C), 138.5
(C), 134.9 (CH), 128.1 (CH), 123.2 (CH), 115.1 (CH), 114.4 (C), 60.3
(OCH2), 40.1 (NCH2), 31.7 (CH2), 23.1 (CH2) and 14.0 (CH3) ppm. IR
(ATR) υ: 2955 (NH), 1729 (CO) and 1620 (CO) cm1. MS (ESI) m/z: 299
Diester 27R  The compound was prepared as its enantiomer 27S using
the acid 26 (60 mg, 0.2 mmol), tionyl chloride (3.5 mL), dry
dichloromethane (1.6 mL), dry trimethylamine (67 µL, 0.4 mmol) and
dimethyl D-aspartate (48 mg, 0.4 mmol). Yield = 59 mg (63%, white
foam). The compound has the same NMR data as its enantiomer 27S.
= 25.0º (c1.1, CHCl3). MS (ESI): 392 (MNa+). HRMS calcd for
C18H21N3O7Na (MNa+): 414.1272; found, 414.1268.
Diacid 17S  A stirred solution of diester 27S (30 mg, 0.1 mmol) in THF
(1 mL) was treated with LiOH (0.3 mL, 0.6 mmol, 2.5 M). The resulting
mixture was stirred at room temperature for 2 h. The reaction mixture
was diluted with water and THF was concentrated under reduced
pressure. The aqueous solution was washed with ethyl acetate (3). The
aqueous phase was treated with Amberlite IR-120 (H+) until pH 6.0 and
filtered. The filtrate and the washings were freeze-dried to afford
compound 17S (22 mg, 79%) as a white solid. Mp: 165.3–165.7 ºC.
= +10.2º (c0.8, H2O). 1H NMR (300 MHz, D2O) δ: 7.83 (d, J = 8.0 Hz, 1H,
ArH), 7.64 (t, J = 7.2 Hz, 1H, ArH), 7.23 (t, J = 7.4 Hz, 1H, ArH), 7.06 (d,
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10.1002/cmdc.201700396
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J = 8.2 Hz, 1H, ArH), 4.61 (t, J = 5.9 Hz, 1H, CH), 3.93 (t, J = 7.0 Hz, 2H,
NCH2), 2.85 (d, J = 6.1 Hz, 2H, CH2CO2H), 2.39 (t, J = 7.3 Hz, 2H,
CH2CO) and 1.95 (quint, J = 7.1 Hz, 2H, CH2CH2CH2) ppm. 13C NMR (63
MHz, D2O) δ: 177.8 (C), 177.2 (C), 177.1 (C), 166.6 (C), 154.1 (C), 140.9
(C), 138.2 (CH), 129.6 (CH), 126.1 (CH), 117.6 (CH), 115.9 (C), 52.0
(CH), 42.8 (CH2), 38.5 (CH2), 35.3 (CH2) and 25.5 (CH2) ppm. IR (ATR)
υ: 3249 (OH+NH), 1704 (CO), 1644 (CO) and 1622 (CO) cm1. MS (ESI):
362 (M–H). HRMS calcd for C16H16N3O7 (M–H): 362.0994; found,
362.0993.
Ester 32  A stirred solution of ketal 31 (40 mg, 0.08 mmol) in water (0.3
mL) was treated with LiOH (0.7 mL, 0.08 mmol, 0.11 M). The resulting
mixture was stirred at room temperature for 4 h. The reaction mixture
was diluted with ethyl acetate and water. The organic layer was
separated and the aqueous phase was washed with ethyl acetate (×2).
The aqueous phase was treated with Amberlite IR-120 (H+) until pH 6.0
and filtered. The filtrate and the washings were freeze-dried to afford
compound 32 (30 mg, 88%) as a white solid. Mp: 170 ºC (dec.).
=
0.6° (c 0.9, CH3CN). 1H NMR (300 MHz, D2O) δ: 7.74 (d, J = 7.9 Hz, 1H,
ArH), 7.52 (t, J = 7.8 Hz, 1H, ArH), 7.11 (t, J = 7.7 Hz, 1H, ArH), 6.96 (d,
J = 8.2 Hz, 1H, ArH), 3.94 (t, J = 5.7 Hz, 2H, NCH2), 3.62 (t, J = 5.7 Hz,
2H, NCH2), 2.70 (t, J = 15.4 Hz, 1H, CHH), 2.51 (d, J = 14.5 Hz, 1H,
CHH), 2.41 (d, J = 15.4 Hz, 1H, CHH), 2.37 (d, J = 14.5 Hz, 1H, CHH)
and 1.25 (s, 9H, C(CH3)3) ppm. 13C NMR (63 MHz, D2O) δ: 172.5 (C),
171.8 (C), 165.1 (C), 152.5 (C), 139.2 (C), 136.5 (CH), 127.9 (CH), 124.3
(CH), 116.7 (C), 115.9 (CH), 114.1 (C), 83.7 (C), 74.8 (C), 45.6 (CH2),
44.8 (CH2), 40.6 (CH2), 37.7 (CH2) and 27.7 (C(CH3)3) ppm. IR (ATR) υ:
Diacid 17R  The compound was prepared as its enantiomer 17R using
the diester 27R (70 mg, 0.17 mmol), THF (1.8 mL), and LiOH (0.34 mL,
2.5 M). Yield = 50 mg (79%, white solid). The compound has the same
NMR data as its enantiomer 17S.
= 10.1º (c0.8, H2O). MS (ESI):
362 (M–H). HRMS calcd for C16H16N3O7 (M–H): 362.0994; found,
362.0994.
3440, 3125 and 3355 (OH+NH), 1729, 1710, 1668 and 1652 (CO) cm1
.
Acid 30  A solution of 2-((2R,4R)-2-(tert-butyl)-5-oxo-1,3-dioxolan-4-
yl)acetic acid (29)40 (300 mg, 1.48 mmol) in dry THF (14.8 mL), under
argon and at 78 ºC, was treated with LHMDS (2.97 mL, 2.97 mmol, ca
1M in THF). After 30 min, a solution of tert-butyl bromoacetate (0.33 mL,
2.22 mmol) in dry THF (3 mL) was added. The reaction mixture was
allowed to warm up to 23 ºC and it was stirred at this temperature for
another 10 h. HCl (1 M) was added and the reaction mixture was
extracted with ethyl acetate (×3). All the organic extracts were dried (anh.
Na2SO4), filtered and concentrated under reduced pressure. Purification
MS (ESI) m/z: 434 (MH). HRMS calcd for C20H24N3O8 (MH): 434.1569;
found, 434.1566.
Acid 18  A stirred solution of ester 32 (10 mg, 0.02 mmol) in water (0.11
mL) was treated with aqueous HCl (0.08 mL, 0.37 mmol, 4.5 M). The
resulting mixture was stirred at room temperature for 6 h. The reaction
mixture was diluted with ethyl acetate and water. The organic layer was
separated and the aqueous phase was washed with ethyl acetate (×2).
The aqueous phase was freeze-dried to afford the acid 18 (7 mg, 78%)
by
dichloromethane/methanol/trimethylamine, gave the ester 30 (164 mg,
35%) as a white solid.
= +18° (c 1.7, CH3OH). 1H NMR (250 MHz,
flash
chromatography,
eluting
with
(91:8:1)
as a white solid. Mp: 151152 ºC.
= +1.6° (c 0.8, H2O). 1H NMR
(500 MHz, D2O) δ: 7.83 (d, J = 8.0 Hz, 1H, ArH), 7.56 (t, J = 7.7 Hz, 1H,
ArH), 7.15 (t, J = 7.7 Hz, 1H, ArH), 7.05 (d, J = 8.3 Hz, 1H, ArH), 3.99 (m,
2H, NCH2), 3.443.32 (m, 2H, NCH2), 2.77 (t, J = 16.1 Hz, 1H, CHH),
2.52 (d, J = 15.3 Hz, 2H, CHH+CHH) and 1.25 (d, J = 14.6 Hz, 1H, CHH)
ppm. 13C NMR (100 MHz, D2O) δ: 176.4 (C), 173.3 (C), 171.1 (C), 164.3
(C), 151.7 (C), 138.4 (C), 135.7 (CH), 127.1 (CH), 123.6 (CH), 115.1
(CH), 113.3 (C), 73.3 (C), 44.4 (CH2), 42.6 (CH2), 39.9 (CH2) and 36.9
(CH2) ppm. IR (ATR) υ: 3264, 3095, 2917 and 2850 (OH+NH), 1704,
1644 and 1622 (CO) cm1. MS (ESI) m/z: 378 (MH). HRMS calcd for
C16H16N3O8 (MH): 378.0943; found, 378.0942.
CDCl3) δ: 5.30 (s, 1H, CH), 3.03 (d, J = 16.1 Hz, 1H, CHH), 2.95 (d, J =
15.6 Hz, 1H, CHH), 2.87 (d, J = 16.1 Hz, 1H, CHH), 2.85 (d, J = 15.6 Hz,
1H, CHH), 1.16 (s, 9H, C(CH3)3) and 0.96 (s, 9H, C(CH3)3) ppm. 13C
NMR (63 MHz, CDCl3) δ: 174.0 (C), 173.1 (C), 168.3 (C), 110.0 (CH),
82.5 (C), 77.5 (C), 41.3 (CH2), 40.6 (CH2), 34.6 (C(CH3)3), 28.1 (C(CH3)3)
and 23.7 (C(CH3)3) ppm. IR (KBr) υ: 3009 (OH), 1806 (CO), 1724 (CO)
and 1708 (CO) cm1. MS (ESI) m/z: 315 (M–H). HRMS calcd for
C15H23O7 (M–H): 315.1449; found, 315.1456.
Amide 31  A stirred solution of acid 30 (16 mg, 0.05 mmol) and amine
24 (24 mg, 0.12 mmol) in dry DMF (3.6 mL), under argon and at room
temperature, was treated with HATU (36 mg, 0.09 mmol) and DIPEA (8.5
µL, 0.05 mmol). The resulting mixture was stirred at room temperature for
1 h. The reaction mixture was diluted with ethyl acetate and water. The
organic layer was separated and the aqueous phase was extracted with
ethyl acetate (×2). The combined organic layers were dried (anh.
Na2SO4), filtered and concentrated under reduced pressure. The residue
obtained was purified by flash chromatography, eluting with (75:25) ethyl
acetate/hexane, to give the amide 31 (15 mg, 60%) as a white solid. Mp:
3-allylquinazoline-2,4(1H,3H)-dione (33)  A suspension of N-allyl-2-
aminobenzamide (50 mg, 0.26 mmol), which was prepared as indicated
below, in dry dichloromethane (1.3 mL), under inert atmosphere and at
room temperature, was treated with triphosgene (38 mg, 0.13 mmol) and
DIPEA (0.1 mL, 0.052 mmol). The resulting solution was stirred for 1 h
and then diluted with dichloromethane and saturated solution of NaHCO3.
The aqueous phase was separated and the organic layer was washed
with water. The organic extract was dried (anh. Na2SO4), filtered and
concentrated under reduced pressure. The residue obtained was purified
by flash chromatography, eluting with (75:25) ethyl acetate/hexane, to
give the quinazolinedione 33 (35 mg, 61%) as a yellow solid. Mp:
221224 ºC. 1H NMR (300 MHz, CDCl3) δ: 10.82 (br s, 1H, NH), 8.26 (d,
J = 7.9 Hz, 1H, ArH), 7.61 (t, J= 8.1 Hz, 1H, ArH), 7.23 (d, J = 7.9 Hz, 1H,
ArH), 7.17 (t, J = 8.1 Hz, 1H, ArH), 5.955.82 (m, 1H CH=CH2),
5.125.01 (m, 2H, CH=CH2), 4.19 (t, J= 7.1 Hz, 2H, CH2) and 2.51 (q, J =
7.1 Hz, 2H, CH2) ppm. 13C NMR (63 MHz, CDCl3) δ: 162.3 (C), 152.4 (C),
138.7 (C), 134.9 (C), 134.8 (CH), 128.3 (C), 123.3 (C), 117.1 (C), 115.1
(C), 114.6 (CH2), 40.2 (CH2) and 32.9 (CH2) ppm. IR (ATR) υ: 3183 (NH),
1714 (CO) and 1629 (CO) cm1. MS (ESI) m/z: 217 (MH+). HRMS calcd
for C12H13N2O2 (MH+): 217.0972; found, 217.0969.
203205 ºC.
= +6.7º (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3) δ:
10.3 (br s, 1H, NH), 8.05 (dd, J = 8.0 and 1.0 Hz, 1H, ArH), 7.68 (m, 1H,
ArH), 7.16 (m, 2H, 2×ArH), 6.94 (t, J = 5.0 Hz, 1H, NH), 5.26 (s, 1H, CH),
4.27 (t, J = 5.5 Hz, 2H, CH2), 3.753.55 (m, 2H, CH2), 2.99 (t, J = 16.6 Hz,
1H, CHH), 2.99 (d, J = 16.6 Hz, 1H, CHH), 2.89 (d, J = 16.6 Hz, 1H,
CHH), 2.67 (d, J = 14.7 Hz, 1H, CHH), 2.60 (d, J = 14.7 Hz, 1H, CHH),
1.41 (s, 9H, C(CH3)3) and 0.89 (s, 9H, C(CH3)3) ppm. 13C NMR (75 MHz,
CDCl3) δ: 173.8 (C), 168.9 (C), 167.8 (C), 163.2 (C), 151.9 (C), 138.9 (C),
135.4 (CH), 128.4 (CH), 123.5 (CH), 115.5 (CH), 114.3 (C), 110.2 (CH),
82.3 (C(CH3)3), 78.0 (C(CH3)3) 44.2 (CH2), 40.9 (CH2), 40.0 (CH2), 39.3
(CH2), 34.5 (C), 28.1 (C(CH3)3) and 23.7 (C(CH3)3) ppm. IR (ATR) υ:
3512 (OH), 3362 (NH), 1793 (CO), 1724 (CO) and 1659 (CO) cm1. MS
(ESI) m/z: 526 (MNa+). HRMS calcd for C25H33N3O8Na (MNa+):
526.2160; found, 526.2168.
N-allyl-2-aminobenzamide  An stirred solution of antranilic acid (30)
(100 mg, 0.73 mmol) in dry DMF (3.6 mL), under argon and at room
temperature, was treated with EDC (168 mg, 0.87 mmol), N,N-
dimethylpyridine (4.8 mg, 0.04 mmol), DIPEA (0.25 mL, 0.15 mmol) and
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10.1002/cmdc.201700396
ChemMedChem
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3-butenylamine (120 mg, 0.11 mmol). The resulting mixture was stirred at
room temperature for 1 h. The reaction mixture was diluted with ethyl
acetate and water. The organic layer was separated and the aqueous
phase was extracted with ethyl acetate (×2). The combined organic
layers were dried (anh. Na2SO4), filtered and concentrated under
reduced pressure. The residue obtained was purified by flash
chromatography, eluting with (20:800) ethyl acetate/hexane, to give N-
allyl-2-aminobenzamide (105 mg, 70%) as a white solid. Mp: 181−184 ºC.
1H NMR (300 MHz, CDCl3) : 7.28−7.25 (d, J = 7.8 Hz, 1H, ArH),
7.19−7.13 (t, J = 7.7 Hz, 1H, ArH), 6.64 (d, J = 8.8 Hz, 1H, ArH), 6.59 (t,
J = 8.2 Hz, 1H, ArH), 6.27 (br s, 1H, NH), 5.86−5.72 (m, 1H, CH=CH2),
5.48 (br s, 2H, NH2), 5.15−5.07 (m, 2H, HC=CH2), 3.44 (q, J = 6.4 Hz, 2H,
CH2) and 2.32 (q, J = 6.7 Hz, 2H, CH2) ppm. 13C NMR (63 MHz, CDCl3)
δ: 169.2 (C), 148.6 (C), 135.3 (C), 132.1 (CH), 127.1 (CH), 117.3 (CH),
117.2 (CH), 116.6 (CH2), 116.3 (C), 38.5 (CH) and 33.7 (CH2) ppm. IR
(ATR) υ: 3295 (NH) and 1616 (CO) cm1. MS (ESI) m/z: 191 (MH+).
HRMS calcd for C11H15N2O (MH+): 191.1179; found, 191.1178.
mixture was dissolved under inert atmosphere in dry dichloromethane
(2.5 mL) and then treated with EDC (340 mg, 1.7 mmol), DMAP (53 mg,
0.4 mmol) and anthranilic acid (20) (130 mg, 0.4 mmol). The resulting
mixture was stirred at room temperature for 1 h and then diluted with
dichloromethate and saturated sodium bicarbonate. The aqueous layers
were extracted with dichloromethane (3). The combined organic
extracts were washed with water, dried (anh. Na2SO4), filtered and
concentrated under reduced pressure. The resulting residue was purified
by flash chromatography, eluting with (4:1) diethyl ether/ethyl acetate, to
give the amide 38 (105 mg, 74%) as a white foam.
= –5.8º (c1.0,
CHCl3). 1H NMR (250 MHz, CDCl3) δ: 7.35 (d, J = 8.0 Hz, 1H, ArH), 7.17
(t, J = 7.7 Hz, 1H, ArH), 6.67 (d, J = 8.1 Hz, 1H, ArH), 6.60 (t, J = 7.3 Hz,
1H, ArH), 6.44 (br s, 1H, NH), 5.66–5.40 (m, 3H, NH+CH=CH), 5.18 (s,
1H, CH), 3.63 (s, 3H, OCH3), 3.573.30 (m, 2H, NHCH2), 2.73 (s, 2H,
CH2CO2), 2.50 (d, J = 6.8 Hz, 2H, NCH2CH2) 2.32 (q, J = 6.6 Hz, 2H,
HC=CHCH2), and 0.92 (s, 9H, 3×CH3) ppm. 13C NMR (63 MHz, CDCl3) δ:
173.2 (C), 169.2 (C), 168.5 (C), 148.2 (C), 133.8 (CH), 132.0 (CH), 127.1
(CH), 124.0 (CH), 117.2 (CH), 116.7 (CH), 116.0 (C), 107.9 (CH), 80.0
(C), 51.9 (OCH3), 39.2 (CH2), 38.5 (CH2), 36.6 (CH2), 34.1 (C(CH3)3),
32.6 (CH2) and 23.4 (3×CH3) ppm. IR (ATR) υ: 3168 (NH), 3096 (NH),
1773 (CO) and 1738 (CO) cm1. MS (ESI) m/z: 419 (MH+). HRMS calcd
for C22H31N2O6 (MH+): 419.2177; found, 419.2180.
Methyl (2S,4S)-2-[(E)-4-(5-bromo-2-pentenyl)-2-(tert-butyl)-5-oxo-1,3-
dioxolan-4-yl)]acetate (36)  A solution of alkene 3443 (160 mg, 0.62
mmol) and 4-bromobutene (63 μL, 0.62 mmol) in dry toluene (3 mL)
under inert atmosphere was treated with second generation Grubbs-
Hoveyda catalyst (78 μg, 0.12 mmol) and heated at 90 ºC for 16 h. After
cooling to room temperature, the reaction mixture was filtered through
Celite. The filtrate and the washings were concentrated under reduced
pressure and the resulting brown oil was purified by flash
chromatography, eluting with (50:50) diethyl ether/hexane, to yield the
Quinazolinedione 39  A solution of the aniline 38 (40 mg, 0.1mmol)
and triethylamine (27 µL, 0.2 mmol) in dry dichloromethane (0.5 mL),
under inert atmosphere and at room temperature, was treated with a
solution of triphosgene (18 mg, 0.06 mmol) in dry dichloromethane (0.3
mL). The reaction mixture was stirred at room temperature for 2 h and
then washed with HCl (10%, 3). The combined aqueous layers were
extracted with dichloromethane (3). The combined organic extracts
were washed with water, dried (anh. Na2SO4), filtered and concentrated
under reduced pressure. The resulting residue was purified by flash
chromatography, eluting with (4:1) diethyl ether/ethyl acetate, to afford
bromide 36 (185 mg, 77%) as a yellow oil.
= +2.2º (c1.0, CHCl3). 1H
NMR (250 MHz, CDCl3) δ: 5.625.50 (m, 2H, CH=CH), 5.19 (s, 1H, CH),
3.63 (s, 3H, OCH3), 3.37 (t, J = 6.6 Hz, 2H, BrCH2), 2.78 (s, 2H, CH2CO2),
2.61–2.48 (m, 4H, CH2CH=CHCH2), and 0.90 (s, 9H, 3×CH3) ppm. 13
C
NMR (63 MHz, CD3OD) δ: 173.5 (C), 168.6 (C), 133.2 (CH), 124.8 (CH),
108.1 (CH), 79.9 (C), 51.9 (OCH3), 39.7 (CH2), 37.0 (CH2), 35.5 (CH2),
34.1 (C(CH3)3), 32.1 (CH2) and 23.4 (3×CH3) ppm. IR (ATR) υ: 1795
(CO) and 1743 (CO) cm1. MS (ESI) m/z: 385 and 387 (MNa+). HRMS
calcd for C15H23Br79O5Na (MNa+): 385.0621; found, 385.0621.
the quinazolinedione 39 (31 mg, 70%) as a white foam.
= +5.7º
(c1.25, CHCl3). 1H NMR (300 MHz, CDCl3) δ: 9.85 (br s, 1H, NH), 8.11 (d,
J = 7.9 Hz, 1H, ArH), 7.60 (td, J = 1.5 and 7.4 Hz, 1H, ArH), 7.22 (td, J =
0.9 and 8.0 Hz, 1H, ArH), 7.10 (d, J = 8.0 Hz, 1H, ArH), 5.71 (m, 1H,
CH=CH), 5.44 (m, 1H, CH=CH), 5.11 (s, 1H, CH), 4.15 (t, J = 7.0 Hz, 2H,
NCH2), 3.63 (s, 3H, OCH3), 2.76 (s, 2H, CH2CO2), 2.50–2.41 (m, 4H,
CH2CH=CHCH2) and 0.87 (s, 9H, 3CH3) ppm. 13C NMR (63 MHz,
CDCl3) δ: 173.5 (C), 168.8 (C), 162.2 (C), 151.8 (C), 138.5 (C), 134.9 (C),
133.0 (CH), 128.2 (CH), 124.0 (CH), 123.2 (CH), 115.0 (CH), 114.3 (C),
107.9 (CH), 79.9 (C), 51.8 (OCH3), 39.9 (CH2), 39.3 (CH2), 36.7 (CH2),
34.0 (C(CH3)3), 31.1 (CH2) and 23.4 (3CH3) ppm. IR (ATR) υ: 2952 (NH),
1795 (CO), 1748 (CO), 1717 (CO) and 1659 (CO) cm1. MS (ESI) m/z:
445 (MH+). HRMS calcd for C23H29N2O7 (MH+): 445.1969; found,
445.1975.
Methyl (2S,4S)-4-[(E)-4-(5-azido-2-pentenyl)]-2-(tert-butyl)-5-oxo-1,3-
dioxolan-4-yl)acetate (37)  A solution of bromide 36 (60 mg, 0.15
mmol), sodium azide (40 mg, 0.62 mmol) in dry DMF (4 mL) under inert
atmosphere was heated at 90 ºC for 2 h. After cooling to room
temperature, the reaction mixture was diluted with ethyl acetate and
water (1:4). The organic layer was separated and the aqueous layer was
extracted with ethyl acetate (×3). The combined organic extracts were
washed with water, dried (anh. Na2SO4), filtered and concentrated under
reduced pressure. The resulting residue was purified by flash
chromatography, eluting with (50:50) diethyl ether/hexane, to afford the
azide 37 (39 mg, 74%) as a colorless oil.
= +10.4º (c1.0, CHCl3). 1H
NMR (250 MHz, CDCl3) δ: 5.585.51 (m, 2H, HC=CH), 5.18 (s, 1H, CH),
3.63 (s, 3H, OCH3), 3.29 (t, J = 6.6 Hz, 2H, N3CH2), 2.77 (s, 2H,
CH2CO2), 2.50–2.48 (m, 2H, CH2CH), 2.34–2.27 (q, J = 6.2 Hz, 2H,
CHCH2) and 0.91 (s, 9H, 3×CH3) ppm. 13C NMR (63 MHz, CDCl3) δ:
173.4 (C), 168.6 (C), 132.4 (CH), 124.8 (CH), 108.2 (CH), 79.9 (C), 51.9
(OCH3), 50.5 (CH2), 39.6 (CH2), 37.0 (CH2), 34.1 (C(CH3)3), 32.0 (CH2)
and 23.4 (3×CH3) ppm. IR (ATR) υ: 2095 (N3), 1798 and 1746 (CO) cm1.
MS (ESI) m/z: 348 (MNa+). HRMS calcd for C15H23N3O5Na (MNa+):
348.1530; found, 348.1530.
Quinazolinedione 40  A solution of the alkene 39 (45 mg, 0.1 mmol) in
ethanol (1 mL) under hydrogen atmosphere was added via canula to a
suspension of Pd-C (5 mg, 10%) in ethanol (0.5 mL) under hydrogen
atmosphere. The resulting suspension was stirred at room temperature
for 12 h. Hydrogen was removed under vacuo and the suspension was
filtered through a plug of Celite. The filtrate and the washings were
concentrated under reduced pressure. The resulting residue was purified
by flash chromatography, eluting with (4:1) diethyl ether/ethyl acetate, to
afford the saturated quinazolinedione 40 (38 mg, 84%) as a white foam.
1
= +7.3º (c1.25, CHCl3). H NMR (250 MHz, CDCl3) δ: 10.20 (s, 1H,
NH), 8.12 (dd, J = 8.0 and 1.0 Hz, 1H, ArH), 7.63 (td, J = 8.5 and 1.4 Hz,
1H, ArH), 7.24 (t, J = 7.9 Hz, 1H, ArH), 7.13 (d, J = 8.1 Hz, 1H, ArH),
5.16 (s, 1H, CH), 4.08 (t, J = 7.2 Hz, 2H, NCH2), 3.66 (s, 3H, OCH3), 2.82
(s, 2H, CH2CO2), 1.86–1.71 (m, 4H, 2CH2) and 0.93 (s, 9H, 3CH3) ppm.
13C NMR (63 MHz, CD3OD) δ: 173.6 (C), 168.5 (C), 162.0 (C), 151.6 (C),
138.1 (C), 134.7 (CH), 128.0 (CH), 123.1 (CH), 114.6 (CH), 114.2 (C),
Methyl
(2S,4S)-4-[(E)-5-(2-aminobenzamido)-2-pentenyl]-2-(tert-
butyl)-5-oxo-1,3-dioxolan-4-ylacetate (38)  A solution of azide 37 (170
mg, 0.6 mmol) in tetrahydrofuran (15 mL) was treated with
triphenylphosphine (180 mg, 0.6 mmol) and distilled water (0.15 mL) and
heated under reflux for 12 h. After cooling to room temperature, the
solvent was removed under reduced pressure. The resulting crude
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10.1002/cmdc.201700396
ChemMedChem
FULL PAPER
107.8 (CH), 79.8 (C), 51.6 (OCH3), 40.3 (CH2), 39.2 (CH2), 33.9 (CH2),
33.2 (C(CH3)3), 27.2 (CH2), 26.5 (CH2), 23.2 (3CH3) and 22.6 (CH2)
ppm. IR (ATR) υ: 2952 (NH), 1790 (CO), 1743 (CO), 1711 (CO) and
1649 (CO) cm1. MS (ESI) m/z: 447 (MH+). HRMS calcd for C22H31N2O7
(MH+): 447.2126; found, 447.2120.
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Acid 19  A solution of the diester 40 (35 mg, 0.08 mmol) in THF (0.8
mL) was treated with aqueous LiOH (4.8 mL, 0.48 mmol, 0.1 M) and
stirred at room temperature for 6 h. The reaction mixture was diluted with
ethyl acetate and MilliQ water. The organic layer was separated and the
aqueous extract was washed with ethyl acetate (×3). The aqueous layer
was treated with Amberlite IR-120 (H+) until pH 6. The filtrate and the
washings were lyophilized to give the acid 19 (22 mg, 76%) as a white
[9]
solid.
= –14.0º (c1.0, H2O). Mp: 177.9–178.1 ºC. 1H NMR (500 MHz,
D2O) δ: 8.10 (d, J = 8.0 Hz, 1H, ArH), 7.83 (td, J = 7.9 Hz and 1.3 Hz, 1H,
ArH), 7.42 (t, J =7.7 Hz, 1H, ArH), 7.31 (t, J = 8.2, 1H, ArH), 4.07 (t, J =
7.3 Hz, 2H, NCH2), 3.15 (d, J = 16.3 Hz, 1H, CHH), 2.86 (d, J = 16.3 Hz,
1H, CHH), 1.94–1.81 (m, 2H, CH2), 1.75 (q, J = 7.7 Hz, 2H, CH2), 1.68–
1.56 (m, 1H, CHH), 1.48 (q, J =7.6 Hz, 2H, CH2) and 1.44–1.37 (m, 1H,
CHH) ppm. 13C NMR (63 MHz, D2O) δ: 178.4 (C), 174.5 (C), 164.6 (C),
152.1 (C), 138.8 (C), 136.0 (CH), 127.5 (CH), 123.9 (CH), 115.5 (CH),
113.9 (C), 75.8 (C), 43.5 (CH2), 41.2 (CH2), 38.8 (CH2), 26.9 (CH2), 26.3
(CH2) and 22.5 (CH2) ppm. IR (ATR) υ: 3325 (OH) and 1582 (CO) cm1.
MS (ESI) m/z: 363 (M–H). HRMS calcd for C17H19N2O7 (M–H): 363.1198;
found, 363.1198.
Acknowledgements
Acknowledgements Text. Financial support from the Spanish
Ministry of Economy and Competiveness (SAF2013-42899-R
and SAF2016-75638-R), the Consellería de Cultura, Educación
e Ordenación Universitaria (Centro singular de investigación de
Galicia accreditation 2016-2019, ED431G/09) and the European
Regional Development Fund (ERDF) is gratefully acknowledged.
AP, AR and BB thank the Spanish Ministry of Science and
Innovation for their respective FPU and FPI fellowships. We are
also grateful to the Centro de Supercomputación de Galicia
(CESGA) for use of the Finis Terrae II supercomputer.
Keywords: Enzyme Motion • Inhibitors • Fragment-based •
Molecular Dynamics Simulations • Antibiotics
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Entry for the Table of Contents
FULL PAPER
Antonio Peón, Adrián Robles, Beatriz
Blanco, Marino Convertino, Paul
Thompson, Alastair R. Hawkins,
Amedeo Caflisch,* and Concepción
González-Bello*
Page No. – Page No.
A multidisciplinary approach to identify and optimize a quinazolinedione-based
ligand that would reduce the flexibility of the substrate-covering loop (catalytic loop)
of the type II dehydroquinase from Helicobacter pylori is presented. This enzyme is
essential for the survival of this pathogenic bacterium and is involved in the
biosynthesis of the aromatic amino acids.
Reducing the Flexibility of Type II
Dehydroquinase Enzyme for
Inhibition – A Fragment-Based
Approach and Molecular Dynamics
Simulation Study
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