Anti-virulence strategy against Brucella suis: Synthesis, biological evaluation and molecular modeling of selective histidinol dehydrogenase inhibitors

Article abstract of DOI:10.1039/c1ob05149k

In the facultative intracellular pathogen Brucella suis, histidinol dehydrogenase (HDH) activity, catalyzing the last step in histidine biosynthesis, is essential for intramacrophagic replication. The inhibition of this virulence factor by substituted ben

Full text of DOI:10.1039/c1ob05149k

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 3681
www.rsc.org/obc
PAPER
Anti-virulence Strategy against Brucella suis: Synthesis, Biological Evaluation
and Molecular Modeling of Selective Histidinol Dehydrogenase Inhibitors
Marie-Rose Abdo,a Pascale Joseph,b Je´re´mie Mortier,c Franc¸ois Turtaut,a Jean-Louis Montero,a
Bernard Masereel,c Stephan Ko¨hler*b and Jean-Yves Winum*a
Received 27th January 2011, Accepted 7th March 2011
DOI: 10.1039/c1ob05149k
In the facultative intracellular pathogen Brucella suis, histidinol dehydrogenase (HDH) activity,
catalyzing the last step in histidine biosynthesis, is essential for intramacrophagic replication. The
inhibition of this virulence factor by substituted benzylic ketones was a proof of concept that disarming
bacteria leads to inhibition of intracellular bacterial growth in macrophage infection. This work
describes the design, synthesis and evaluation of 19 new potential HDH inhibitors, using a combination
of classical approaches and docking studies. The IC50-values of these inhibitors on HDH activity were
in the nanomolar range, and several of them showed a 70–100% inhibition of Brucella growth in
minimal medium. One selected compound yielded a strong inhibitory effect on intracellular replication
of B. suis in human macrophages at concentrations as low as 5 mM, with an overall survival of
intramacrophagic bacteria reduced by a factor 103. Docking studies with two inhibitors showed a good
fitting in the catalytic pocket and also interaction with the second lipophilic pocket binding the cofactor
NAD+. Experimental data confirmed competition between inhibitors and NAD+ at this site. Hence,
these inhibitors can be considered as promising tools in the development of novel anti-virulence drugs.
eventually causing death. Treatment of human brucellosis may last
Introduction
for several months and cases of relapse may be observed, reducing
Bacterial infections represent a serious public health problem
the efficacy of the therapy. Moreover, there is no human vaccine
against brucellosis.4
worldwide. The widespread emergence of resistance and multi-
resistance to antibiotics among a growing number of bacterial
pathogens represents a major threat and necessitates the develop-
ment of new molecules with innovative mechanisms of action.1
In the past few years, the growing information on the virulence
mechanisms of various pathogens in the relationship with their
hosts led to increased interest in the potential definition of
virulence factors as novel targets for anti-infectious agents.2
In intracellular bacteria such as Brucella spp., virulence is linked
to the capacity of the pathogen to replicate inside the macrophage
host cell and to escape from the host immune system.3
The genes required for intramacrophagic replication of Brucella
are a subset of the virulence genes of the pathogen and have
been called the intramacrophagic virulome.3 Definition of the
targets for antibacterials by analysis of the intramacrophagic
virulome should limit the selective pressure to the intracellular
niche and also reduce the probability of affecting the commensal
flora. Among those virulence genes, we identified, cloned and
overexpressed hisD (BR0252) from Brucella suis, which encodes
the histidinol dehydrogenase (HDH; EC 1.1.1.23), and we purified
this metalloenzyme which catalyses the final steps in the histidine
biosynthetic pathway (Fig. 1).5,6 The essential character of his-
tidine biosynthesis for virulence has been demonstrated in other
pathogenic bacteria such as Salmonella typhimurium, Burkholderia
pseudomallei and Mycobacterium tuberculosis.7 First series of
HDH inhibitors, being substituted benzylic ketones, were designed
Brucella spp. are the etiological agents of brucellosis, the major
bacterial zoonosis worldwide. They are facultative intracellular
coccobacilli, pathogenic for a variety of mammals including
ruminants and humans. Human brucellosis may become chronic,
aInstitut des Biomole´cules Max Mousseron (IBMM), UMR 5247 CNRS-
Universite´ Montpellier I-Universite´ Montpellier II, Ecole Nationale
Supe´rieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296, Mont-
pellier Cedex, France. E-mail: jean-yves.winum@univ-montp2.fr; Tel: +33-
467147234
bCentre d’e´tudes d’agents Pathoge`nes et Biotechnologie pour la Sante´
(CPBS), UMR 5236 CNRS-Universite´ Montpellier I-Universite´ Montpel-
lier II, 1919 Route de Mende, 34293, Montpellier Cedex 5, France. E-mail:
Stephan.Kohler@cpbs.cnrs.fr
cDrug Design & Discovery Center, Department of Pharmacy, University of
Namur (FUNDP), Belgium
Fig. 1 Histidine biosynthesis steps catalyzed by histidinol dehydrogenase
HDH.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 3681–3690 | 3681
©

View Article Online
and synthesized by this group, and their potential inhibitory effect
on purified Brucella HDH was studied.5,6
Results and discussion
Chemistry
The synthetic products most active on the purified enzyme
exhibited IC50 inhibition activities in the nanomolar range. These
novel inhibitors against a virulence factor were a proof of concept
that disarming bacteria leads to the inhibition of intracellular bac-
terial growth in experimental macrophage infection experiments5,6
Therefore, HDH represents a selective and promising target
for the development of new antibacterial agents, favouring the
development of HDH inhibitors.
These encouraging findings resulted in the design and synthesis
of additional structures, by developing new HDH inhibitors
using a combination of classical approaches and docking studies.
Starting from our first lead inhibitor and using palladium-
catalyzed reactions, we developed a library of inhibitors aiming
at an increased potency within the benzylic ketone series. (Fig. 2).
Encouraged by the promising HDH inhibitory activity of 1
(IC50 = 3nM), we started a systematic structure modification
program to increase the potency and to understand the structural
requirements for the key side chain by optimizing the interactions
with the enzyme active site. To this end, three structural types of
side chains, comprising para-substitution patterns in phenyl rings
were synthesized.
We envisioned that the synthetically easiest and most flexible
approach for the introduction of the different chains would
be based on the use of palladium-catalyzed reactions such as
Suzuki and Sonogashira reactions. Preparation of the different
compounds was achieved in a two-step-synthesis starting from
the fully protected L-histidine methyl ester 2, previously described
by our group.6 By reacting the corresponding aryl or vinyl or
alkyl boronic acid under Suzuki reaction conditions, followed by a
acidic total deprotection of the resulting compounds, we obtained
the series of inhibitors 3–6, as outlined in Scheme 1.
Further elaboration of new inhibitors was also accomplished
starting from the key derivative 2, by applying a Sonogashira strat-
egy with different substituted alkynes. In this case, experimental
conditions were optimized and the best results were obtained using
a palladium catalyst and copper co-catalyst at 10 mol% in THF
in the presence of triethylamine as base. Final hydrolysis under
acidic conditions yielded the series of compounds 7 (Scheme 2).
Biological Evaluation
Enzymatic assays: HDH inhibition study. The series of com-
pounds 3–7 were tested for their inhibitory activity against purified
Fig. 2 Design of improved HDH inhibitors by optimization of the initial
hit compound 1.
Scheme 1 Synthesis of HDH inhibitors 3–6 via Suzuki coupling. Reagents and conditions: (a) R-B(OH)2, Pd(Ph3)4, Na2CO3, Toluene; (b) HCl 4 M,
dioxane.
3682 | Org. Biomol. Chem., 2011, 9, 3681–3690
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Scheme 2 Synthesis of HDH inhibitors 7 via Sonogashira coupling. Reagents and conditions: (a) Arylalkyne, Pd(Ph3)4, NEt3, CuI, PPh3, THF; (b) HCl
4 M, dioxane.
Table 1 Inhibition of B. suis histidinol dehydrogenase with compounds
3–7. The values are means of three independent assays. Variations were in
the range of 5–10% of the shown data. Nd: not determined
Table 2 % Inhibition of B. suis growth in vitro in minimal medium in the
presence of various selected inhibitors at final concentrations of 100 mM
or 50 mM, measured at 7 days post inoculation. The growth curve obtained
in untreated medium was used as reference
Compounds
IC50 (nM) on HDH (B. suis)
% Inhibition of B. suis growth in minimal
medium by HDH inhibitors at a concentration of
1
3
3a
3b
3c
3d
3e
3f
3g
3h
4
30
40
20
30
nd
70
nd
30
13
30
25
40
65
nd
3
Compounds
100 mM
50 mM
3a
3b
3c
3d
3f
3h
6b
7a
7c
7d
40
60
60
90
30
50
30
70
90
70
100
100
100
30
100
100
100
30
5
6a
6b
6c
6d
7a
7b
7c
7d
7e
30
30
of the bacterium. Inhibitors have to cross the membranes by
passive diffusion, whereas in the enzymatic assays, purified HDH
was in direct contact with the molecules. The observed growth
inhibition of live brucellae is a specific effect on HDH, as we have
previously published that competitive addition of histidine relieves
the inhibition.6b
40
65
8.5
nd
B. suis HDH protein (see experimental section). Inhibition data
are presented in Table 1. All compounds 3–7 were nanomolar
inhibitors with IC50 £ 70 nM. Most of the biphenyl compounds
(3a–3h) showed an inhibitory potency in the range of 20–70nM,
demonstrating the poor influence of the nature of R.
Compounds containing phenyl alkynyl moiety such as 7a and
7d displayed the most potent activity with an IC50 of 3 and
8.5 nM, respectively. Potent inhibition was also observed for
compounds 4, 5 and 6, with IC50 concentrations ranging from
13 to 70 nM. Collectively, these results demonstrate that subtle
structural differences in the tail of the molecules can influence
enzyme inhibition characteristics.
Inhibition of intramacrophagic replication of B. suis. Com-
pound 7a, the molecule with the lowest IC50 among the molecules
tested (Table 1) and with a 100%-inhibition of Brucella growth
in minimal medium (Table 2), was selected to test the potential
effects of this HDH-inhibitor on intracellular replication of B. suis
in human macrophages. As shown in Fig. 3, the compound had
a strong inhibitory effect on intracellular growth of Brucella
at concentrations as low as 5 mM. The overall survival of
intramacrophagic bacteria was reduced by a factor 103 over the
infection period of 24 h. The viability of the macrophages was not
affected under these conditions, as determined by using trypan
blue coloration (not shown).
These results clearly show that the HDH inhibitor reaches
the target enzyme located in the bacterial cytoplasm, following
diffusion across both eukaryotic and bacterial membranes, hence
affecting histidine biosynthesis which is essential for the growth of
Brucella at the intracellular state.
Inhibition of growth of B. suis in minimal medium. Following
the determination of the IC50 values for various molecules,
the effect of selected HDH inhibitors on growth of B. suis in
minimal medium, requ◦iring histidine biosynthesis, was studied.
After incubation at 37 C with shaking and aeration for 7 days,
molecules 3d, 3f, and especially 3h, 6b, and 7a showed a 70–
100% inhibition of Brucella growth, as compared to an untreated
control culture (Table 2). To obtain satisfactory inhibition rates,
concentration of approximately 50 mM were necessary, which
is roughly 1000-fold higher than the IC50 described above. The
most likely explanation for this observation is the existence of a
physical barrier, represented by the outer and inner membranes
Molecular modeling and docking study
No experimentally-derived structural data for the B. suis isoform
of HDH have been reported to date allowing to study the molecules
within the 3-D structure of the target enzyme. Following a
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 3681–3690 | 3683
©

View Article Online
histidine) were selected, and their positions in the alignment to
the B. suis HDH were analyzed (Fig. 4). An extremely high
identity between both cavities was highlighted, since 37 out of
40 residues were perfectly recovered, including key residues in
complex with the Zn2+ cation (Q259, H262, D360 and H419) and
residues H-bonding the histidine scaffold in the catalytic pocket
(S237, H327, E356, D360 and E414). In the sequences alignment,
two E. coli residues (T141 and F213) were identified as different
but similar to those of B.suis (S141 and Y216). Nevertheless, as
T141 is H-bonding the nitrogen atom of the amide function in the
NAD+ structure, this interaction is perfectly conserved with the
substituted serine in the B. suis HDH sequence. A P–P stacking
is also observed between the aromatic side chain of F213 and the
adenine moiety of the cofactor.
No major change is then observed in the environment of NAD+
when the phenylalanine is switched to a tryptophan, as reported in
the alignment (Fig. 4). Finally, only one residue emerged as really
different in this alignment: a leucine (L138) in E. coli which was
substituted by a tryptophan (Y128) in B. suis. This amino acid is
located between the histidine/Zn2+ cavity and the NAD+ pocket
in the 3D-structure.
Once this very high similarity between both enzymatic cavities
demonstrated, E. coli HDH can be considered as a very reliable
3D-model to investigate, by molecular modeling, the histidine
derivatives series as B. suis HDH inhibitors.
Fig.
3
Intramacrophagic survival of B. suis in untreated human
macrophage-like THP-1 cells ( ), or in cells treated with 5 mM (ꢀ) or
᭹
10 mM (᭡) of compound 7a.
BLASTP alignment,8 E. coli HDH was identified as having the
protein sequence most similar to B. suis HDH, sharing 41%
sequence identity and up to 57% sequence homology (similar
residues). As the E. coli HDH has been cocristallized with NAD+
and histidine, and its structure solved (PDB code 1KAE),9 the
reliability of a docking study of B. suis HDH inhibitors within its
E. coli isoform was considered.
The 40 E. coli HDH residues whose side chain are located
in a 6 A-volume around the co-crystallized ligands (NAD and
Structures 1 and 7a were then docked inside the enzymatic
+
cavity of HDH using the automated GOLD program.10 For each
˚
Fig. 4 Swissprot (BLASTP)8 alignment of the HDH sequences from E. coli and B. suis, performed using a compositional matrix adjustment method.
Identities = 174/421 (41%), Positives = 242/421 (57%), Gaps = 11/421 (2%). Residues from the enzymatic pocket are highlighted in green (perfect
alignment), magenta (favourable alignment) and red (unfavourable alignment).
3684 | Org. Biomol. Chem., 2011, 9, 3681–3690
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Scheme 3 2D-representation of the binding mode of inhibitor 7a.
molecule, 20 conformations were generated and the conformation
with the best GOLDSCORE was further evaluated as the most
probable docking orientation. As a result, each molecule fits very
well in the catalytic pocket adopting a similar binding orientation
to histidine, the co-crystallized ligand, and product of HDH. As
expected, the zinc cation is coordinated with four nitrogen atoms
belonging to three imidazole moieties (H262, H419, compound
7a), and with the amino group of 7a (Scheme 3). Two nitrogen
lone pairs are predicted to be very available thanks to a particular
environment where the imidazole NH hydrogen is attracted by
the negatively charged E414 side chain and the amino NH2
hydrogen’s are involved in H-bonds with D360, E356 and S237 side
chains. This Zn2+ pocket is therefore targeted by each synthesized
compound, stabilized by the same key interactions as observed for
histidine.
type of inhibitors and the cofactor (Fig. 5). This region is
hydrophobic and open to the solvent, predicting a good affinity
for this lipophilic part of the molecules. Surprisingly, the aromatic
side-chains of both nanomolar inhibitors 1 and 7a are oriented in
the same direction in the NAD+ pocket. A superimposition of 7a
and NAD+ structures allowed the demonstration that an identical
volume was occupied in the active site (Fig. 6). This result is indica-
tive of a pharmacophore structure interacting most favorably with
this region and where two axial and almost perpendicular aromatic
˚
˚
planes are separated by 3.60 A to 4.10 A. Finally, after a validation
of the closest structural data available and despite potential subtle
structural effects beyond the residue changes between Brucella
and E. coli HDH, the inhibition properties of this chemical series
against our target was rationalized. This molecular modeling study
demonstrated the high capacity of both compounds to complex
the cation Zn2+ in the hydrophilic histidine pocket, and to reach
the second lipophilic pocket where the NAD+ cofactor binds.
Interestingly, the aromatic side-chain is oriented directly to-
wards the NAD+ pocket, predicting a competition between this
Fig. 5 Compound 7a (a) compound 1 (b) and NAD+/histidine co-crystallized (c) within the HDH enzymatic cavity; in yellow highlighted H-bonds
stabilize the molecule (picture made with PYMOL)11.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 3681–3690 | 3685
©

View Article Online
of B. suis in vitro in minimal medium, and in the macrophage
host cell. Molecular modeling and competition experiment with
NAD+ confirmed the positioning of the inhibitor inside the active
site of HDH with an orientation of the imidazole ring in the
hydrophilic histidine-binding pocket, and the other part of the
structure oriented directly towards the second lipophilic NAD+-
binding pocket. As the target HDH has been directly linked to
bacterial virulence, and with the results obtained on live extra- and
intracellular brucellae, the development of these inhibitors can be
considered as a promising step towards novel anti-virulence drugs.
Experimental Section
Chemistry
General. All reagents and solvents were of commercial quality
and used without further purification, unless otherwise specified.
All reactions were carried out under an inert nitrogen atmosphere.
TLC analyses were performed on silica gel 60 F254 plates (Merck
Art.1.05554). Spots were visualized under 254 nm UV illumi-
nation, or by ninhydrin solution spraying. Melting points were
determined on a Bu¨chi Melting Point 510 and are uncorrected.
1H and 13C NMR spectra were recorded on Bruker DRX-400
spectrometer using DMSO-d6 as solvent and tetramethylsilane
as internal standard. For 1H NMR spectra, chemical shifts
are expressed in d (ppm) downfield from tetramethylsilane, and
coupling constants (J) are expressed in Hertz. Electron Ionization
mass spectra were recorded in positive or negative mode on a
Water MicroMass ZQ.
Fig. 6 Compounds 7a and NAD+ superimposed in the HDH enzymatic
cavity (picture made with PYMOL).
Table 3 : Effect of the NAD+ concentration on inhibition of B. suis
HDH by inhibitor 7a. The values are means of three independent assays.
Variations were in the range of 5–10% of the shown data
Conversion rate
(DOD340/5min)
NAD (mM)
Inhibitor (nM)
2
2
10
10
0
3
3
15
0.51
0.22
0.48
0.16
Enzymatic assays: Competition between NAD+ and HDH
inhibitors
Preparation of tert-butyl[(1S)-3-(4-bromophenyl)-1-({1-[(4-
methoxyphenyl)(diphenyl)methyl]-1H-imidazol-4-yl}methyl)-2-ox-
opropyl]carbamate (2). A solution of the 4-bromophenylacetic
acid (3 equiv.) in dry tetrahydrofuran (10 mL) was cooled
to -78 ◦C and a solution of lithium bis(trimethylsilyl) amide
(6 equiv., 1.06 M in tetrahydrofuran) was added dropwise.
After 10 min, a solution of the protected histidine methyl ester
(previously described in ref. 6) (1 equiv.) in tetrahydrofuran was
introduced through a canula. The reaction was stirred for 16 h
under argon allowing the mixture to warm to room temperature.
The mixture was then extracted with ethyl acetate (100 ml) and
washed with water (4 ¥ 25 ml). The organic layer was dried
over anhydrous magnesium sulfate and the solvent evaporated
under reduced pressure to give yellowish viscous crude. The
residue was purified by column chromatography on silica gel
(CH2Cl2/Hexane/Acetone: 5/4/1) to give the fully protected
final product as a white powder. 1H NMR (DMSO- d6, 400 MHz)
7.21 (m, 19H), 6.69 (s, 1H), 4.30 (m, 1H), 3.77 (s, 2H), 3.76 (s,
3H), 2.84 (dd, J = 6.0 Hz, 2H), 1.34 (s, 9H); 13C (DMSO- d6, 101
MHz) 206.4, 158.5, 155.2, 142.5, 137.7, 136.3, 134.1, 134, 131.9,
130.9, 130.6, 129.0, 128.0, 127.8, 119.6,119.1, 113.2, 78.3, 73.9,
55.0, 44.1, 28.4, 28.0; MS ESI+ m/z 682 (M+H)+, 704 (M+Na)+.
In order to confirm molecular modeling studies, the potential com-
petition between NAD+ and the HDH inhibitors in the enzymatic
reaction was evaluated by varying NAD+ concentrations in the
measurement cell. Standard enzymatic kinetics were performed at
a NAD+ concentration of 2mM, being the optimal concentration
of the cofactor for HDH activity (Km = 12 mM). Influence of
various NAD+ concentrations on HDH inhibition measurement
was evaluated, based on the previously established IC50 values
of inhibitors (Table 3). In parallel, NAD+ concentrations were
maintained and inhibitor concentrations were modified. Obtained
data show that NAD+ concentration has a direct and proportional
impact on IC50 values of the HDH inhibitor 7a. The curves
1/Vi = f (1/Ci) revealed that Vmax (0.0017 M s-1) is not changing
regardless of theinhibitor concentration used, whereas variation of
the apparent Km is observed (50-fold), confirming a competition
between inhibitor and the cofactor NAD+, and supporting the
predicted orientation of the inhibitor structure in the lipophilic
NAD+-binding pocket.
Conclusions
General procedure of the Suzuki cross coupling of compound 2
with various boronic acids. A 50 ml, two-necked round bottomed
flask equipped with a magnetic stirring bar and a reflux condenser
was charged with (1 eq) of compound 2 and 5 mL of toluene under
argon atmosphere. The corresponding boronic acid (3 eq) was
dissolved in a minimal amount of ethanol, and then transferred
to the previous solution. The catalyst Pd(PPh3)4 (0.1 eq) was then
By introduction of substituted aryl or substituted alkynyl aryl tails
in the lead compound 1, some potent and selective HDH inhibitors
have been discovered.
Notable among these is compound 7a with a phenyl alkynyl
chain, possessing a strong inhibitory effect on HDH, with an
IC50 of 3 nM. This compound was also able to impair growth
3686 | Org. Biomol. Chem., 2011, 9, 3681–3690
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
added, followed by (10 eq) of an aqueous solution of Na2CO3
(1 M). The resulting mixture was stirred for twelve hours under
reflux. The reaction was monitored by mass spectroscopy. Upon
completion, the mixture was extracted with ethyl acetate (50 ml)
and washed with water (3 ¥ 15 ml). The organic layer was dried
over anhydrous magnesium sulfate and the solvent evaporated
under reduced pressure to give yellowish viscous crude. The
residue was purified by column chromatography on silica gel
(CH2Cl2/Hexane/Acetone: 5/4/1) to give the fully protected final
product, a white powder. This latter was deprotected under acidic
conditions in a solution of HCl (4 M in dioxane), the reaction was
monitored by TLC until all the starting material was consumed.
The final dichlorohydrate salt was filtrated and washed with ether.
1H), 4.24 (s, 2H), 3.61 (dd, J = 4.3 Hz, J = 15.5 Hz, 1H), 3.24 (dd,
J = 8.9 Hz, J = 15.5 Hz, 1H); 13C (DMSO- d6, 101 MHz) 202.7,
143.8, 137.0, 134.2, 133.8, 130.6, 127.8, 127.3, 126.8, 126.6, 125.7
(dd, JC-F = 3.6 Hz, JC-F = 7.3 Hz), 122.9, 118.2, 56.9, 44.9, 24.2;
MS (ESI+/ESI-) m/z 374 (M+H)+, 747 (2M+H)+, 372 (M-H)-,
408 (M+Cl)-, 781 (2M+Cl)-; Anal. Calcd for C20H20Cl2F3N3O:
C, 53.82; H, 4.52; N, 9.42. Found: C, 53.78; H, 4.45; N, 9.33.
(3S)-3-Amino-1-(4¢-bromobiphenyl-4-yl)-4-(1H-imidazol-4-yl)-
butan-2-one dihydrochlorhydrate (3e). Mp. 199 ◦C (decomposi-
tion); 1H NMR (DMSO- d6, 400 MHz) 14.51 (s, 2H), 9.11 (d, J =
1.0 Hz, 1H), 8.59 (s, 3H), 7.46 (m, 9H), 4.70 (dd, J = 4.2 Hz, J =
9.1 Hz, 1H), 4.14 (s, 2H), 3.58 (dd, 1H, J = 15.47, 4.33 Hz), 3.20
(dd, J = 9.0 Hz, J = 15.4 Hz, 1H); 13C (DMSO- d6, 101 MHz)
202.8, 134.2, 133.3, 130.4, 129.8, 128.9, 128.9, 128.2, 127.1, 126.8,
126.5, 118.2, 56.9, 45.2, 24.2; MS (ESI+/ESI-) m/z 383 (M+H)+,
382 (M-H)-, 416 (M+Cl)-; Anal. Calcd for C19H20BrCl2N3O: C,
49.91; H, 4.41; N, 9.19. Found: C, 49.93; H, 4.35; N, 9.22.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-(4¢-methylbiphenyl-4-yl)-
butan-2-one dihydrochlorhydrate (3a). Mp 203 ◦C (decomposi-
tion); 1H NMR (DMSO- d6, 400 MHz) 9.13 (d, J = 1.1 Hz, 1H),
+
8.65 (s, 3H, NH3 ), 7.57 (dd, J = 8.2 Hz, J = 17.6 Hz, 4H), 7.52 (s,
1H), 7.29 (dd, J = 8.1 Hz, J = 21.8 Hz, 4H), 4.73 (dd, J = 4.1 Hz,
J = 7.9 Hz, 1H), 4.20 (s, 2H), 3.61 (dd, J = 4.4 Hz, J = 15.6 Hz, 1H),
3.24 (dd, J = 8.9 Hz, J = 15.5 Hz, 1H), 2.33 (s, 3H); 13C (DMSO-
d6, 101 MHz) 202.8, 138.6, 136.9, 136.6, 134.1, 132.2, 130.4, 129.4,
126.5, 126.3, 126.2, 118.7, 56.9, 44.9, 24.2, 20.6; MS (ESI+/ESI-)
m/z 320 (M+H)+, 639 (2M+H)+, 318 (M-H)-, 354 (M+Cl)-, 673
(2M+Cl)-; Anal. Calcd for C20H23Cl2N3O: C, 61.23; H, 5.91; N,
10.71. Found: C, 61.27; H, 5.89; N, 10.74.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-(4¢-phenoxybiphenyl-4-
yl)butan-2-one dihydrochlorhydrate (3f). Mp 198–200 ◦C; 1H
NMR (DMSO- d6, 400 MHz) 14.81 (s, 2H), 9.12 (d, J = 1.08 Hz,
1H), 8.66 (s, 3H), 7.68 (d, J = 8.76 Hz, 2H), 7.61 (d, J = 8.26 Hz,
2H), 7.52 (s, 1H), 7.42 (dd, J = 8.53, 7.47 Hz, 2H), 7.33 (d, J =
8.26 Hz, 2H), 7.17 (t, J = 7.39, 7.39 Hz, 1H), 7.11–7.04 (m, 4H),
4.74 (dd, J = 8.70, 4.53 Hz, 1H), 4.20 (s, 2H), 3.61 (dd, J = 15.46,
4.36Hz, 1H), 3.24(dd, J = 15.48, 8.86Hz, 1H); 13C(DMSO- d6, 101
MHz) 202.8, 156.4, 156.3, 137.9, 134.9, 134.1, 132.3, 130.4–128.1,
126.6, 126.2, 123.6, 118.8, 118.2, 56.9, 44.9, 24.2; MS (ESI+/ESI-)
m/z 398 (M+H)+, 795 (2M+H)+, 396 (M-H)-, 432 (M+Cl)-, 829
(2M+Cl)-; Anal. Calcd for C25H25Cl2N3O2: C, 63.83; H, 5.36; N,
8.93. Found: C, 63.88; H, 5.31; N, 8.91.
(3S)-3-Amino-1-(4¢-butylbiphenyl-4-yl)-4-(1H-imidazol-4-yl)-
butan-2-one dihydrochlorhydrate (3b). Mp 155 ◦C (decomposi-
tion); 1H NMR (DMSO- d6, 400 MHz) 9.13 (d, J = 1.3 Hz, 1H),
8.62 (m, 3H), 7.58 (dd, J = 8.3 Hz, J = 14.9 Hz, 4H), 7.52 (d, J =
0.9 Hz, 1H), 7.30 (dd, J = 8.3 Hz, J = 18.1 Hz, 4H), 4.73 (dd, J =
4.3 Hz, J = 9.0 Hz, 1H), 4.19 (s, 2H), 3.61 (dd, J = 4.4 Hz, J =
15.4 Hz, 1H), 3.23 (dd, J = 8.9 Hz, J = 15.5 Hz, 1H), 2.61 (t, J =
7.74 Hz, 2H), 1.57 (td, J = 7.5 Hz, J = 15.2 Hz, 2H), 1.32 (qd, J =
7.3 Hz, J = 14.5 Hz, 2H), 0.90 (t, J = 7.3 Hz, 3H); 13C (DMSO- d6,
101 MHz) 202.8, 141.5, 138.6, 137.1, 134.2, 132.2, 130.4, 128.8,
126.5, 126.3, 126.3, 118.3, 56.9, 44.9, 34.3, 33.0, 24.2, 21.7, 13.7;
MS (ESI+/ESI-) m/z 362 (M+H)+,723 (2M+H)+,360 (M-H)-, 396
(M+Cl)-, 757 (2M+Cl)-; Anal. Calcd for C23H29Cl2N3O: C, 63.59;
H, 6.73; N, 9.67. Found: C, 63.56; H, 6.78; N, 9.63.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-(1,1¢:4¢,1¢¢-terphenyl-4-
yl)butan-2-one dihydrochlorhydrate (3g). Mp 200 ◦C (decompo-
sition); 1H NMR (DMSO- d6, 400 MHz) 14.71 (s, 2H), 9.12 (d, J =
1.18 Hz, 1H), 8.64 (s, 3H), 7.77 (m, 4H), 7.75–7.65 (m, 4H), 7.52
(s, 1H), 7.51–7.45 (m, 2H), 7.43–7.32 (m, 3H), 4.75 (dd, J = 8.70,
4.48 Hz, 1H), 4.22 (s, 2H), 3.62 (dd, J = 15.47, 4.33 Hz, 1H), 3.24
(dd, J = 15.52, 8.89 Hz, 1H); 13C (DMSO- d6, 101 MHz) 202.8,
139.5, 139.0, 138.7, 138.1, 134.2, 132.7, 130.5, 128.9, 127.5, 127.1,
127.0, 126.6, 126.5, 126.4, 118.3, 56.9, 44.9, 24.2; MS (ESI+/ESI-)
m/z 382 (M+H)+, 763 (2M+H)+, 380 (M-H)-, 416 (M+Cl)-, 797
(2M+Cl)-; Anal. Calcd for C29H27Cl2N3O: C, 69.05; H, 5.39; N,
8.33. Found: C, 69.11; H, 5.32; N, 8.28.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-(4¢-methoxybiphenyl-4-
◦
yl)butan-2-one dihydrochlorhydrate (3c). Mp 210 C (decompo-
sition); 1H NMR (DMSO- d6, 400 MHz) 14.82 (s, 2H), 9.13 (d, J =
1.1 Hz, 1H), 8.66 (s, 3H), 7.58 (dd, J = 8.6 Hz, J = 11.4 Hz, 4H),
7.52 (s, 1H), 7.30 (d, J = 8.3 Hz, 2H), 7.01 (d, J = 8.9 Hz, 2H), 4.73
(dd, 1H, J = 4.4 Hz, J = 8.6 Hz), 4.19 (s, 2H), 3.79 (s, 3H), 3.61
(dd, J = 4.3 Hz, J = 15.5 Hz, 1H), 3.25 (dd, J = 8.9 Hz, J = 15.4 Hz,
1H); 13C (DMSO- d6, 101 MHz) 202.9, 158.8, 138.3, 134.1, 132.1,
131.8, 130.4, 127.6, 126.5, 125.9, 118.2, 114.3, 56.9, 55.1, 44.9,
24.1; MS (ESI+/ESI-) m/z 336 (M+H)+, 671 (2M+H)+ ,334 (M-
H)-, 370 (M+Cl)-, 705 (2M+Cl)-; Anal. Calcd for C20H23Cl2N3O2:
C, 58.83; H, 5.68; N, 10.29. Found: C, 58.90; H, 5.5.62; N, 10.34.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-[4-(2-naphthyl)phenyl]
butan-2-one dihydrochlorhydrate (3h). Mp 205 ◦C (decomposi-
tion); 1H NMR (DMSO- d6, 400 MHz) 14.86 (s, 2H), 9.14 (s, 1H),
8.69 (s, 3H), 8.22 (s, 1H), 8.00 (m, 2H), 7.94 (m, 1H),7.85 (dd, J =
1.6 Hz, J = 8.6 Hz, 1H), 7.79 (d, J = 8.2 Hz, 2H),7.53 (m, 3H),
7.40 (d, J = 8.2 Hz, 2H), 4.76 (dd, J = 4.7 Hz, J = 8.7 Hz, 1H),
4.25 (s, 2H), 3.63 (dd, J = 4.2 Hz, J = 15.4 Hz, 1H), 3.27 (dd,
J = 9.1 Hz, J = 15.6 Hz, 1H); 13C (DMSO- d6, 101 MHz) 202.8,
138.4, 137.1, 134.1, 133.3, 132.8, 132.1, 130.5, 128.4–127.4, 126.8,
126.6–126.0, 125.0–124.9, 118.3, 56.9, 44.9, 24.2; MS (ESI+/ESI-)
m/z 356 (M+H)+, 713 (2M+H)+ ; 354 (M-H)-, 390 (M+Cl)-, 745
(2M+Cl)-; Anal. Calcd for C29H27Cl2N3O: C, 69.05; H, 5.39; N,
8.33. Found: C, 69.11; H, 5.38; N, 8.30.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-[4¢-(trifluoromethyl) bi-
phenyl-4-yl] butan-2-one dihydrochlorhydrate (3d). Mp. 149 ◦C
(decomposition); 1H NMR (DMSO- d6, 400 MHz) 14.78 (s, 2H),
9.12 (d, J = 1.3 Hz, 1H), 8.64 (s, 3H), 7.90 (d, J = 8.2 Hz, 2H), 7.81
(d, J = 8.3 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 1.1 Hz,
1H), 7.39 (d, J = 8.4 Hz, 2H), 4.75 (dd, J = 4.6 Hz, J = 8.9 Hz,
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 3681–3690 | 3687
©

View Article Online
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-[4-(2-phenylethyl) phe-
nyl]butan-2-one dihydrochlorhydrate (4). Mp 168–170 ◦C; 1H
NMR (DMSO- d6, 400 MHz) 9.11 (s, 1H), 8.63 (s, 3H), 7.50 (s,
1H), 7.31–7.20 (m, 4H), 7.21–7.08 (m, 5H), 4.69 (dd, J = 8.03,
4.09 Hz, 1H), 4.10 (s, 2H), 3.57 (dd, J = 15.41, 4.14 Hz, 1H),
3.21 (dd, J = 15.39, 8.89 Hz, 1H), 2.86 (s, 4H); 13C (DMSO-
d6, 101 MHz) 202.9, 141.4, 140.0, 134.1, 130.7, 129.7–128.1,
126.5, 125.7, 118.2, 56.9, 44.9, 36.9, 36.6, 24.1; MS (ESI+/ESI-)
m/z 334 (M+H)+,667 (2M+H)+ , 332 (M-H)-, 368 (M+Cl)-, 701
(2M+Cl)-; Anal. Calcd for C21H25Cl2N3O: C, 62.07; H, 6.20; N,
10.34. Found: C, 62.09; H, 6.25; N, 10.31.
d6, 101 MHz) 202.7, 141.1, 135.1, 134.1, 133.4, 130.9, 130.2, 127.2,
126.9, 126.7, 126.6, 126.5, 125.5, 122.9, 118.2, 56.9, 45.0, 24.1;
MS (ESI+/ESI-) m/z 399 (M+H)+,799 (2M+H)+, 398 (M-H)-,
434 (M+Cl)-, 833 (2M+Cl)-; Anal. Calcd for C22H22Cl2F3N3O: C,
55.94; H, 4.69; N, 8.90. Found: C, 55.98; H, 4.72; N, 8.92.
(3S)-3-Amino-1-{4-[(E)-2-biphenyl-4-ylvinyl]phenyl}-4-(1H-
◦
imidazol-4-yl)butan-2-one dihydrochlorhydrate (6d). Mp 200 C
(decomposition); 1H NMR (DMSO- d6, 400 MHz) 14.54 (s, 2H),
9.12 (d, J = 1.3 Hz, 1H), 8.59 (s, 3H), 7.70 (m, 6H), 7.60 (d, J =
8.4 Hz, 2H), 7.51 (d, J = 1.0 Hz, 1H), 7.47 (t, J = 7.6 Hz, 2H),
7.37 (t, J = 1.2 Hz, 1H), 7.31 (m, 2H), 7.27 (d, J = 8.3 Hz, 2H),
4.72 (dd, J = 4.3 Hz, J = 8.3 Hz, 1H), 4.17 (s, 2H), 3.59 (dd, J =
4.3 Hz, J = 15.3 Hz, 1H), 3.22 (dd, J = 8.9 Hz, J = 15.5 Hz,
1H); 13C (DMSO- d6, 101 MHz) 202.8, 139.6, 139.1, 136.2, 135.7,
134.3, 132.8, 130.3–126.4, 118.3, 56.9, 45.1, 24.3; MS (ESI+/ESI-)
m/z 408 (M+H)+, 815 (2M+H)+, 406 (M-H)-, 442 (M+Cl)-, 849
(2M+Cl)-; Anal. Calcd for C27H27Cl2N3O: C, 67.50; H, 5.66; N,
8.75. Found: C, 67.45; H, 5.69; N, 8.72.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-{4-[(1E)-3-phenylprop-1-
en-1-yl]phenyl}butan-2-one dihydrochlorhydrate (5). Mp 178–
◦
1
180 C; H NMR (DMSO- d6, 400 MHz) 14.71 (s, 2H), 9.11
(d, J = 1.2 Hz, 1H), 8.61 (s, 3H), 7.50 (s, 1H), 7.27 (m, 9H), 6.47
(d, 1H, J = 15.8 Hz), 6.41 (dd, 1H, J = 16.5 Hz,J = 6.1 Hz), 4.69
(dd, J = 4.8 Hz, J = 8.7 Hz, 1H), 4.12 (s, 2H), 3.57 (dd, J = 4.3 Hz,
J = 15.6 Hz, 1H), 3.52 (d, J = 5.6 Hz, 2H), 3.21 (dd, J = 8.9 Hz,
J = 15.4 Hz, 1H); 13C (DMSO- d6, 101 MHz) 202.8, 139.9, 135.6,
134.1, 132.1, 130.1, 130.0, 129.2, 128.4, 128.4, 126.5, 125.9, 125.8,
118.2, 56.9, 44.9, 38.5, 24.1; MS (ESI+/ESI-) m/z 346 (M+H)+,
691 (2M+H)+, 344 (M-H)-, 380 (M+Cl)-, 725 (2M+Cl)-; Anal.
Calcd for C22H25Cl2N3O: C, 63.16; H, 6.02; N, 10.04. Found: C,
63.11; H, 5.95; N, 10.06.
General procedure of the Sonogashira cross coupling of compound
2 with various substituted alkynes:. A 50 ml, two-necked round
bottomed flask equipped with a magnetic stirring bar a rubber
septum and a reflux condenser was charged with (1 equiv.) of
compound X and 10 ml of dry THF under argon atmosphere. The
corresponding alkyne (2equiv.) was added dropwise to the stirred
solution as well as (2 equiv.) of the ligand PPh3. The catalyst
Pd(PPh3)4 (0.1 equiv.) was then added followed by (0.2 equiv.) of
the cocatalyst CuI and the triethylamine (3 equiv.). The resulting
mixture was stirred for twelve hours under reflux and the reaction
was monitored by mass spectroscopy. The reaction mixture was
extracted with dichloromethane (50 ml) and washed with water (3
¥ 15 ml). The organic layer was dried over anhydrous magnesium
sulfate and the solvent evaporated under reduced pressure to
give yellowish viscous crude. The residue was purified by column
chromatography on silica gel (toluene–ethyl acetate: 7/3) to give
the fully protected final product, a yellowish powder. This latter
was deprotected under acidic conditions in a solution of HCl
(4 M in dioxane), the reaction was monitored by TLC until all the
starting material was consumed. The final salt was filtrated and
washed with ether.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-{4-[(E)-2-phenylvinyl]
phenyl}butan-2-one dihydrochlorhydrate (6a). Mp 195 ◦C (de-
1
composition); H NMR (DMSO- d6, 400 MHz) 14.58 (s, 2H),
9.12 (d, J = 1.4 Hz, 1H), 8.61 (s, 3H), 7.59 (m, 3H), 7.51 (d, J =
1.2 Hz, 1H), 7.27 (m, 8H), 4.72 (dd, J = 4.5 Hz, J = 8.6 Hz, 1H),
4.16 (s, 2H), 3.59 (dd, J = 4.1 Hz, J = 15.3 Hz, 1H), 3.22 (dd, J =
8.9 Hz, J = 15.5 Hz, 1H); 13C (DMSO- d6, 101 MHz) 202.8, 136.9,
135.6, 134.2, 132.7, 130.2, 128.6, 128.2, 127.9, 127.6, 126.5, 126.4,
126.3, 118.2, 56.9, 45.0, 24.2; MS (ESI+/ESI-) m/z 332 (M+H)+,
663(2M+H)+, 330 (M-H)-, 366 (M+Cl)-, 697 (2M+Cl)-; Anal.
Calcd for C21H23Cl2N3O: C, 62.38; H, 5.73; N, 10.39. Found: C,
62.41; H, 5.80; N, 10.36.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-{4-[(E)-2-(4-methyl phe-
nyl)vinyl]phenyl}butan-2-one dihydrochlorhydrate (6b). Mp
200 ◦C (decomposition); 1H NMR (DMSO- d6, 400 MHz) 14.56
(s, 2H), 9.12 (d, J = 1.3 Hz, 1H), 8.61 (s, 3H), 7.54 (d, J = 8.3 Hz,
2H), 7.51 (d, J = 0.8 Hz, 1H), 7.49 (d, J = 8.2 Hz, 2H), 7.24 (d,
J = 8.3 Hz, 2H), 7.18 (m, 4H), 4.71 (dd, J = 4.5 Hz, J = 8.0 Hz,
1H), 4.15 (s, 2H), 3.59 (dd, J = 4.5 Hz, J = 15.5 Hz, 1H), 3.22 (dd,
J = 9.0 Hz, J = 15.5 Hz, 1H), 2.30 (s, 3H); 13C (DMSO- d6, 101
MHz) 202.8, 136.9, 135.8, 134.2, 132.5, 130.2, 129.2, 128.1, 126.9,
126.5, 126.3, 126.2, 118.2, 56.9, 45.0, 24.2, 20.8; MS (ESI+/ESI-)
m/z 346 (M+H)+, 691 (2M+H)+, 344 (M-H)-, 380 (M+Cl)-, 725
(2M+Cl)-; Anal. Calcd for C22H25Cl2N3O: C, 63.16; H, 6.02; N,
10.04. Found: C, 63.18; H, 6.08; N, 10.01.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-[4-(phenylethynyl) phe-
nyl]butan-2-one dihydrochlorhydrate (7a). Mp 194 ◦C
(decomposition); 1H NMR (DMSO- d6, 400 MHz) 14.69
(s, 2H), 9.11 (d, J = 1.1 Hz, 1H), 8.63 (s, 3H), 7.53 (m, 5H), 7.43
(m, 2H), 7.27 (m, 2H), 4.72 (dd, J = 4.2 Hz, J = 8.5 Hz, 1H),
4.23 (s, 2H), 3.59 (dd, J = 4.4 Hz, J = 15.4 Hz, 1H), 3.23 (dd, J =
8.9 Hz, J = 15.8 Hz, 1H); 13C (DMSO- d6, 101 MHz) 202.5, 134.2,
134.2, 132.1, 131.3, 131.2, 130.3, 128.7, 126.5, 122.2, 120.7, 118.2,
89.2, 89.1, 56.9, 45.1, 24.1; MS (ESI+/ESI-) m/z 330 (M+H)+,
659 (2M+H)+, 328 (M-H)-, 364 (M+Cl)-, 693 (2M+Cl)-; Anal.
Calcd for C21H21Cl2N3O: C, 62.69; H, 5.26; N, 10.44. Found: C,
62.73; H, 5.30; N, 10.42.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-(4-{(E)-2-[4-(trifluoro
methyl)phenyl]vinyl}phenyl) butan-2-one dihydrochlorhydrate (6c).
◦
1
Mp 186 C (decomposition); H NMR (DMSO- d6, 400 MHz)
14.56 (s, 2H), 9.13 (s, 1H), 8.62 (s, 3H), 7.82 (d, J = 8.1 Hz, 2H),
7.72 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 0.6 Hz,
1H), 7.43 (d, J = 16.6 Hz, 1H), 7.35 (d, J = 16.4 Hz, 1H), 7.28 (d,
J = 8.1 Hz, 2H), 4.72 (s 1H), 4.18 (s, 2H), 3.60 (dd, J = 4.6 Hz, J =
15.8 Hz, 1H), 3.22 (dd, J = 9.2 Hz, J = 15.5 Hz, 1H); 13C (DMSO-
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-{4-[(4-methylphenyl) eth-
ynyl]phenyl}butan-2-one dihydrochlorhydrate (7b). Mp 181 ◦C
(decomposition); 1H NMR (DMSO- d6, 400 MHz) 14.53 (s, 2H),
9.13 (s, 1H), 8.64 (s, 3H), 7.46 (m, 5H), 7.26 (d, 4H), 4.72 (s large,
1H), 4.22 (s, 2H), 3.59 (dd, J = 4.5 Hz, J = 15.4 Hz, 1H), 3.23
(dd, J = 8. 9 Hz, J = 15.7 Hz, 1H), 2.33 (s, 3H); 13C (DMSO- d6,
3688 | Org. Biomol. Chem., 2011, 9, 3681–3690
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
101 MHz) 202.6, 138.5, 134.1, 134.0, 131.2, 131.1, 130.3, 129.3,
126.5, 120.9, 119.2, 118.3, 89.4, 88.5, 56.9, 45.1, 24.1, 20.9; MS
(ESI+/ESI-) m/z 344 (M+H)+, 687 (2M+H)+; 342 (M-H)-, 378
(M+Cl)-, 722 (2M+Cl)-; Anal. Calcd for C22H23Cl2N3O: C, 63.47;
H, 5.57; N, 10.09. Found: C, 63.42; H, 5.61; N, 10.11.
producing 1 mmol of NADH per min in the reaction. To perform
IC50 determinations of the different inhibitors, the latter were
added at various concentratio◦ns, ranging from 1 to 200 nM, and
preincubated for 5 min at 30 C with the enzyme solution prior
to the initiation of the reaction. The enzyme concentration in the
assay system was 4.5 10-8 M.
(3S)-3-Amino-1-{4-[(4-tert-butylphenyl)ethynyl]phenyl}-4-(1H-
imidazol-4-yl)butan-2-one dihydrochlorhydrate (7c). Mp 197 ◦C
(decomposition); 1H NMR (DMSO- d6, 400 MHz) 14.49 (s, 2H),
9.11 (d, J = 1.3 Hz, 1H), 8.59 (s, 3H), 7.48 (m, 7H), 7.29 (d, J =
8.4 Hz, 2H), 4.72 (dd, J = 5.0 Hz, J = 8.8 Hz, 1H), 4.21 (s, 2H),
3.58 (dd, J = 4.5 Hz, J = 15.5 Hz, 1H), 3.21 (dd, J = 8.9 Hz,
J = 15.5 Hz, 1H), 1.29 (s, 9H); 13C (DMSO- d6, 101 MHz) 202.5,
151.5, 134.3, 134.0, 131.1, 131.0, 130.3, 126.5, 125.5, 120.9, 119.3,
118.3, 89.4, 88.6, 56.9, 45.1, 34.5, 30.8, 24.2; MS (ESI+/ESI-)
m/z 386 (M+H)+, 771 (2M+H)+, 384 (M-H)-, 420 (M+Cl)-, 805
(2M+Cl)-; Anal. Calcd for C25H29Cl2N3O: C, 65.50; H, 6.38; N,
9.17. Found: C, 65.55; H, 6.35; N, 9.21.
Bacteriology and infection
Brucella culture in minimal medium in the presence of HDH
inhibitors. B. suis was grown in Gerhardt’s minimal medium13 at
37 ◦C to an optical density (OD 600 nm) of 0.7. The culture was then
diluted 1 : 100 in fresh minimal medium in the absence or presence
of HDH inhibitor 7a at 50 mM, respectively. Bacterial growth at
37 ◦C and under shaking was followed over a period of 7 days by
OD 600 nm measurements.
Macrophage infection by Brucella in the presence of HDH in-
hibitors. Experiments were performed as described previously.14
Briefly, differentiated human THP-1 macrophage-like cells were
infected with early-stationary-phase B. suis for 45 min at a
multiplicity of infection (MOI) of 20 bacteria per cell. Cells were
washed twice with PBS and reincubated in RPMI 1640 with 10%
fetal calf serum and gentamicin at 30 mg mL-1 for at least 1 h
at 37 ◦C and 5% CO2 to kill extracellular bacteria. At 90 min.
post infection, cells of the appropriate culture wells were washed
twice with PBS and lysed in 0.2% Triton X-100. The number
of intracellular living bacteria was determined by plating serial
dilutions on TS agar plates and incubation at 37 ◦C for 3 days.
Half of the wells infected for lyses points and enumeration at 4, 7,
and 24 h were then incubated in fresh medium in the presence of the
HDH inhibitor 7a at concentrations of 5 or 10 mM, respectively,
the other half of the wells contained only fresh RPMI 1640 with
10% fetal calf serum and gentamicin at 30 mg ml-1. Experiments
were performed three times in triplicate.
(3S)-3-Amino-4-(1H-imidazol-4-yl)-1-{4-[(4-pentylphenyl)-
ethynyl]phenyl}butan-2-one dihydrochlorhydrate (7d). Mp 184 ◦C
(decomposition); 1H NMR (DMSO- d6, 400 MHz) 14.55 (s, 2H),
9.12 (d, J = 1.3 Hz, 1H), 8.62 (s, 3H), 7.50 (m, 5H), 7.26 (m, 4H),
4.72 (dd, J = 4.3 Hz, J = 8.2 Hz, 1H), 4.22 (s, 2H), 3.59 (dd, J =
4.5 Hz, J = 15.5 Hz, 1H), 3.22 (dd, 1H, J = 9.0 Hz, J = 15.5 Hz),
2.59 (t, J = 7.5 Hz, 2H), 1.57 (td, J = 7.5 Hz, J = 14.9 Hz, 2H), 1.27
(m, 4H), 0.85 (t, J = 7.0 Hz, 3H); 13C (DMSO- d6, 101 MHz) 202.5,
143.3, 134.2, 134.0, 131.2, 131.1, 130.3, 128.6, 126.5, 120.9, 119.4,
118.3, 89.4, 88.5, 56.9, 45.1, 34.9, 30.759, 30.3, 24.1, 21.8, 13.8;
MS (ESI+/ESI-) m/z 400 (M+H)+,799 (2M+H)+, 398 (M-H)-,434
(M+Cl)-, 833 (2M+Cl)-; Anal. Calcd for C26H31Cl2N3O: C, 66.10;
H, 6.61; N, 8.89. Found: C, 66.15; H, 6.68; N, 8.82.
(3S)-3-Amino-1-[4-(biphenyl-4-ylethynyl)phenyl]-4-(1H-imida-
zol-4-yl)butan-2-one dihydrochlorhydrate (7e). Mp 196 ◦C (de-
compositon); 1H NMR (DMSO- d6, 400 MHz) 14.56 (s, 2H), 9.12
(d, J = 1.3 Hz, 1H), 8.61 (s, 3H), 7.73 (m, 4H), 7.64 (m, 2H), 7.56
(m, 2H), 7.52 (d, J = 1.0 Hz, 1H), 7.49 (m, 2H), 7.40 (m, 1H),
7.31 (m, 2H), 4.73 (dd, J = 4.3 Hz, J = 8.3 Hz, 1H), 4.23 (s, 2H),
3.60 (dd, J = 4.3 Hz, J = 15.7 Hz, 1H), 3.22 (dd, J = 8.9 Hz,
J = 15.6 Hz, 1H); 13C (DMSO- d6, 101 MHz) 202.5, 140.2, 139.0,
134.2, 134.2, 131.9, 131.2, 130.3,129.0, 127.9, 126.9, 126.6, 126.5,
121.2, 120.7, 118.3, 89.9, 89.2, 57.0, 45.1, 24.2; MS (ESI+/ESI-)
m/z 406 (M+H)+, 811 (2M+H)+ , 404 (M-H)-, 440 (M+Cl)-,845
(2M+Cl)-; Anal. Calcd for C27H25Cl2N3O: C, 67.78; H, 5.27; N,
8.78. Found: C, 67.72; H, 5.31; N, 8.72.
Acknowledgements
This work was supported by a grant from the German Sanita¨tsamt
der Bundeswehr, No. M SAB1 5A002, and by ANR grant ANR-
09-MIEN-001 (to SK and J-Y W).
J.M. would like to thanks Dr Raphael Frederick for very fruitful
discussions and Pr Johan Wouters for providing access to his
molecular modeling platform.
References
Enzyme assays
1 D. J. Payne, M. N. Gwynn, D. J. Holmes and D. L. Pompliano, Drugs
for bad bugs: confronting the challenges of antibacterial discovery.,
Nat. Rev. Drug Discovery, 2006, 6, 29–40.
2 (a) S. Escaich, Antivirulence as a new antibacterial approach for
chemotherapy., Curr. Opin. Chem. Biol., 2008, 12, 400–408; (b) L.
Cegelski, G. R. Marshall, G. R. Eldridge and S. J. Hultgren, The
biology and future prospects of antivirulence therapies., Nat. Rev.
Microbiol., 2008, 6, 17–27; (c) D. A. Rasko and V. Sperandio, Anti-
virulence strategies to combat bacteria-mediated disease., Nat. Rev.
Drug Discovery, 2010, 9, 117–128.
3 (a) S. Ko¨hler, V. Foulongne, S. Ouahrani-Bettache, G. Bourg, J. Teyssier,
M. Ramuz and J-P. Liautard, The analysis of the intramacrophagic
virulome of Brucella suis deciphers the environment encountered by
the pathogen inside the macrophage host cell., Proc. Natl. Acad. Sci.
U. S. A., 2002, 99, 15711–15716; (b) S. Ko¨hler, S. Michaux-Charachon,
F. Porte, M. Ramuz and J-P. Liautard, What is the nature of the
Cloning, overexpression of the HDH-encoding gene from B. suis,
and purification of the enzyme were performed as previously
described by our group.6 The activity and specificity of HDH
were measured by monitoring the reduction of NAD+ to NADH
directly at 340 nM (eM = 6200 M-1 cm-1) as previously described.12
The enzymatic activity was studied at 30 ◦C in the presence of
0.5 mM histidinol, 5 mM NAD+ and 0.5 mM MnCl2 in 50 mM
sodium glycine buffer at pH 9.2. For kinetic studies, experiments
were carried out with 150 mM sodium glycine (pH 9.2) and 2 mM
NAD+. The Km for the substrate was determined by varying the
concentration of histidinol from 10 to 50 mM and was found
to be 12 mM. Activity (1 U) is defined as the amount of HDH
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 3681–3690 | 3689
©

View Article Online
replicative niche of a stealthy bug named Brucella?, Trends Microbiol.,
2003, 11, 215–219.
are avirulent., Proc. Natl. Acad. Sci. U. S. A., 1986, 83, 5189–5193; (b) S.
Pilatz, K. Breitbach, N. Hein, B. Fehlhaber, J. Schulze, B. Brenneke,
L. Eberl and I. Steinmetz, Identification of Burkholderia pseudomallei
genes required for the intracellular life cycle and in vivo virulence.,
Infect. Immun., 2006, 74, 3576–3586; (c) T. Parish, Starvation survival
response of Mycobacterium tuberculosis., J. Bacteriol., 2003, 185, 6702–
6706.
8 S. F. Altschul, T. L. Madden, A. A. Schaffer, J. H. Zhang, Z. Zhang,
W. Miller and D. J. Lipman, Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs., Nucleic Acids Res.,
1997, 25, 3389–3402.
9 J. A. Barbosa, J. Sivaraman, Y. Li, R. Larocque, A. Matte, J. D.
Schrag and M. Cygler, Mechanism of action and NAD+-binding mode
revealed by the crystal structure of L-histidinol dehydrogenase., Proc.
Natl. Acad. Sci. U. S. A., 2002, 99, 1859–1864.
10 G. Jones, P. Willett, R. C. Glen, A. R. Leach and R. Taylor, Develop-
ment and validation of a genetic algorithm for flexible docking., J. Mol.
Biol., 1997, 267, 727–748.
11 W. L. Delano, The PyMOL Molecular Graphics System on World Wide
Web. http://pymol.sourceforge.net/(accessed June 21 2010).
12 J. C. Loper and E. Adams, Purification and Properties of Histidinol
Dehydrogenase from Salmonella typhimurium., J. Biol. Chem., 1965,
240, 788–795.
13 M. Dozot, R. A. Boigegrain, R. M. Delrue, R. Hallez, S. Ouahrani-
Bettache, I. Danese, J. J. Letesson, X. De Bolle and S. Ko¨hler, The
stringent response mediator Rsh is required for Brucella melitensis and
Brucella suis virulence, and for expression of the type IV secretion
system virB., Cell. Microbiol., 2006, 8, 1791–1802.
4 (a) I. Lopes-Goni and I. Moriyon, Brucella: Molecular and Cellular
BiologyHorizon Bioscience, 2004: 1–432; (b) H. Guerra, The brucellae
and their success as pathogens., Crit. Rev. Microbiol., 2007, 33, 325–331;
(c) M. P. Franco, M. Mulder, R. H. Gilman and H. L. Smits, Human
brucellosis., Lancet Infect. Dis., 2007, 7, 775–786; (d) G. Pappas, P.
Papadimitriou, N. Akritidis, L. Christou and E. V. Tsianos, The new
global map of human brucellosis., Lancet Infect. Dis., 2006, 6, 91–99.
5 (a) C. T. Supuran, J-Y. Winum, Introduction to zinc enzymes as
drug targets. In Drug Design of Zinc-Enzyme Inhibitors: Functional,
Structural, and Disease Applications, C. T. SupuranJ. Y. Winum ed.,
Wiley, Hoboken, 2009, pp. 3–12; (b) J-Y. Winum, S. Ko¨hler, A.
Scozzafava, J-L. Montero and C. T. Supuran, Targeting bacterial
metalloenzymes: A new strategy for the development of anti-infective
agents., Anti-Infect. Agents Med. Chem., 2008, 7, 169–179.
6 (a) M. R. Abdo, P. Joseph, R. A. Boigegrain, J-P. Liautard, J-L. Mon-
tero, S. Ko¨hler and J-Y. Winum, Brucella suis histidinol dehydrogenase:
synthesis and inhibition studies of a series of substituted benzylic
ketones derived from histidine., Bioorg. Med. Chem., 2007, 15, 4427–
4433; (b) P. Joseph, M. R. Abdo, R. A. Boigegrain, J-L. Montero, J-Y.
Winum and S. Ko¨hler, Targeting of the Brucella suis virulence factor
histidinol dehydrogenase by histidinol analogues results in inhibition of
intramacrophagic multiplication of the pathogen., Antimicrob. Agents
Chemother., 2007, 51, 3752–3755; (c) M. R. Abdo, P. Joseph, R. A.
Boigegrain, J-P. Montero, S. Ko¨hler and J-Y. Winum, Brucella suis his-
tidinol dehydrogenase: synthesis and inhibition studies of substituted
N-L-histidinylphenylsulfonyl hydrazide., J. Enzyme Inhib. Med. Chem.,
2008, 23, 357–361.
14 S. Burkhardt, M. P. Jime´nez de Bagu¨e´s, J-P. Liautard and S. Ko¨hler,
Analysis of the behavior of eryC mutants of Brucella suis attenuated in
macrophages., Infect. Immun., 2005, 73, 6782–6790.
7 (a) P. I. Fields, R. V. Swanson, C. G. Haidaris and F. Heffron, Mutants
of Salmonella typhimurium that cannot survive within the macrophage
3690 | Org. Biomol. Chem., 2011, 9, 3681–3690
This journal is
The Royal Society of Chemistry 2011
©