Brucella suis histidinol dehydrogenase: Synthesis and inhibition studies of a series of substituted benzylic ketones derived from histidine

Article abstract of DOI:10.1016/j.bmc.2007.04.027

Brucella spp. is the causative agent of brucellosis (Malta fever), which is the most widespread zoonosis worldwide. The pathogen is capable of establishing persistent infections in humans which are extremely difficult to eradicate even with antibiotic therapy. Moreover, Brucella is considered as a potential bioterrorism agent. Histidinol dehydrogenase (HDH, EC 1.1.1.23) has been shown to be essential for the intramacrophagic replication of this pathogen. It therefore constitutes an original and novel target for the development of anti-Brucella agents. In this work, we cloned and overexpressed the HDH-encoding gene from Brucella suis, purified the protein and evidenced its biological activity. We then investigated the inhibitory effects of a series of substituted benzylic ketones derived from histidine. Most of the compounds reported here inhibited B. suis HDH in the lower nanomolar range and constitute attractive candidates for the development of novel anti-Brucella agents.

Full text of DOI:10.1016/j.bmc.2007.04.027

Bioorganic & Medicinal Chemistry 15 (2007) 4427–4433
Brucella suis histidinol dehydrogenase: Synthesis and
inhibition studies of a series of substituted benzylic ketones
derived from histidine
Marie-Rose Abdo,a, Pascale Joseph,b, Rose-Anne Boigegrain,b Jean-Pierre Liautard,b
Jean-Louis Montero,a Stephan Ko¨hlerb,* and Jean-Yves Winuma,*
a
ˆ
Institut des Biomole´cules Max Mousseron (IBMM), UMR 5247 CNRS-UM1-UM2, Batiment de Recherche Max Mousseron,
Ecole Nationale Supe´rieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France
b
`
´
Centre d’Etudes d’Agents Pathogenes et Biotechnologies pour la Sante (CPBS), UMR 5236 CNRS-UM1-UM2,
Universite´ Montpellier II, cc100, Place E. Bataillon, 34095 Montpellier, France
Received 10 January 2007; accepted 17 April 2007
Available online 24 April 2007
Abstract—Brucella spp. is the causative agent of brucellosis (Malta fever), which is the most widespread zoonosis worldwide. The
pathogen is capable of establishing persistent infections in humans which are extremely difficult to eradicate even with antibiotic
therapy. Moreover, Brucella is considered as a potential bioterrorism agent. Histidinol dehydrogenase (HDH, EC 1.1.1.23) has been
shown to be essential for the intramacrophagic replication of this pathogen. It therefore constitutes an original and novel target for
the development of anti-Brucella agents. In this work, we cloned and overexpressed the HDH-encoding gene from Brucella suis,
purified the protein and evidenced its biological activity. We then investigated the inhibitory effects of a series of substituted benzylic
ketones derived from histidine. Most of the compounds reported here inhibited B. suis HDH in the lower nanomolar range and
constitute attractive candidates for the development of novel anti-Brucella agents.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
aerosol. In this context, the CDC (Center for Disease
Control) classified Brucella as a class-B bioterrorism
agent (http://www.bt.cdc.gov/agent/brucellosis/). Its
known virulence, the strongly invalidating character of
the disease and its potential ability to develop resistance
to antibiotics used in treatments make this pathogen
particularly dangerous. Moreover, a vaccine for humans
is not available, and the isolation of antibiotic-resistant
strains is easily conceivable. In the case of an accident or
a bioterrorism attack with such modified strains a clas-
sical therapy would be without effect.
Brucella species are Gram-negative cocco-bacillary
organisms, of which three are pathogenic in humans
(Brucella melitensis, B. abortus and B. suis).1 They are
facultative intracellular pathogens that can survive and
multiply within the phagocytic cells of the mammalian
hosts where they are at least partially protected from
the immune system.2,3 These bacteria are the causative
agent of brucellosis (Malta fever), the most widespread
zoonosis worldwide. They are capable of establishing
persistent infections in humans which are extremely
difficult to eradicate even with antibiotic therapy.
The large-scale analysis of the virulome of intracellular
bacteria such as Brucella is an original approach for
the identification of pathogen-restricted targets essential
for the adaptation to and multiplication within the rep-
licative niche. Indeed, the virulence of these bacteria
depends on their capacity of survival and replication
in macrophages.4,5 The major goal of our project is to
use the analysis of the virulome of B. suis to develop a
non-antibiotic, target-directed therapeutic approach.6
Amino acid biosynthesis enzymes are targets of choice,
as these pathways are absent from mammalian cells.
Brucella is considered as a potential bioterrorism agent
and was the first pathogenic organism to be weaponized
by the U.S. Military as it is highly transmissible by
Keywords: Brucella; Histidinol dehydrogenase; Enzyme; Inhibitor.
*
Corresponding authors. Tel.: +33 4 6714 7234; fax: +33 4 6714 4344;
e-mail addresses: kohler@univ-montp2.fr; winumj@univ-montp2.fr
These authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.027

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M.-R. Abdo et al. / Bioorg. Med. Chem. 15 (2007) 4427–4433
The drugs will therefore inhibit the biosynthesis of cer-
tain amino acids by Brucella, which are essential for
the pathogen within the human macrophage host cell.
As a consequence, the development of the pathogen will
be blocked specifically inside the host cell niche, with-
out, however, affecting the host itself and the commensal
flora. The targeted action of these anti-infectious agents
most likely reduces the selective pressure on the micro-
organism, resulting in reduced rates of resistance. Final-
ly, the specificity of such a therapeutic agent for a
defined species of bacteria may be useful to protect a
population against an epidemic as it may occur as a con-
sequence of a bioterrorism attack.6
Starting from the commercially available L-histidine,
we synthesized on a 10-grams-scale the fully protected
histidine compound 4 in four steps. Reaction of L-his-
tidine with thionyl chloride in methanol gave the
methyl ester 1, which was then protected by tert-
butoxycarbonyl group on the NH position by action
of di-tert-butyl dicarbonate to give compound 2.
Selective deprotection of imidazole NH with potas-
sium carbonate afforded compound 3 in good yield.
This latter was then protected on the same position
by a methoxytrityl group to lead to compound 4 in
good yield. This key compound was then condensed
with the dianion of various substituted phenylacetic
acids obtained by action of lithium bis(trimethylsilyl-
amide). The resulting compounds were purified by
silica-gel chromatography and then deprotected with
dioxane HCl solution to afford compounds 5 as
dihydrochloride salts (Fig. 1).
Recent work in our group has shown that the enzyme
histidinol dehydrogenase (HDH, EC 1.1.1.23), encoded
by gene hisD (BR0252) in B. suis, is essential for intra-
macrophagic replication.4 L-histidinol dehydrogenase is
a NAD-linked four-electron dehydrogenase that cata-
lyzes the last two steps in the L-histidine biosynthesis,
and it is found in microorganisms such as bacteria, fun-
gi, and in plants. This enzyme is a homodimeric zinc
metalloenzyme which contains one Zn2+ ion in each
identical subunit. Although histidine biosynthesis has
been elucidated in various organisms, histidinol dehy-
drogenases have been cloned and characterized to date
from only two microorganisms, Salmonella typhimurium
and Escherichia coli.7–9
2.2. Preparation of recombinant B. suis HDH protein
The HDH-encoding gene was cloned into E. coli expres-
sion vector pET15b (Novagen) downstream the T7 pro-
moter, enabling inducible expression of the recombinant
protein. The resulting plasmid, pET15bHDH, was used
to transform E. coli BL21(DE3). HDH protein was
overproduced in E. coli BL21(DE3) as N-terminal hexa-
histidine (6 · (His))-tagged fusion protein after induc-
tion with IPTG. After cell lysis by sonication and
centrifugation, His-tagged HDH protein was detected
in the soluble fraction (data not shown). The recombi-
nant HDH was then purified to homogeneity by Co-
balt-affinity chromatography as described in Section 4.
The protein fractions of the different purification steps
were analyzed by SDS–PAGE (Fig. 2). The purified pro-
tein shows a single Coomassie-stained band at ꢀ49 kDa
on SDS–PAGE (Fig. 2A, lane 6). This molecular mass
determined by SDS–PAGE is in good agreement with
the calculated value (ꢀ48.2 kDa) deduced from the re-
combinant 6 · (His)-tagged HDH. In addition, immu-
noblotting of the elution fraction with antibodies
raised against the His-tag shows a single band, indicat-
ing that the recombinant HDH protein is produced as
stable protein in E. coli (Fig. 2). The yield of B. suis
HDH protein purification was 43 mg/L of bacterial cul-
ture. The specific activity of B. suis HDH is about
15 U mgꢁ1 of protein, equivalent to a kcat of about
Ten years ago, Dancer et al. reported that HDH is a
suitable target for the development of potential herbi-
cides.10 The approach developed by this group was to
prepare HDH inhibitors which target the lipophilic
binding pocket adjoining the active site of the enzyme.
Some of their compounds exhibited nanomolar inhibi-
tion activity against HDH cabbage enzyme.10 To date,
no other work has been published on the inhibition of
this enzyme except a computational modelling study in
2001.11
As this enzyme could also constitute an attractive tar-
get for antibacterials, further development of specific
HDH-inhibitors would be highly beneficial for obtain-
ing novel types of anti-brucellosis agents, devoid of
side effects.
In the present study, we cloned and overexpressed the
HDH-encoding gene from B. suis, and we purified the
protein. We then investigated the inhibitory effects of
a series of substituted benzylic ketones derived from
histidine on the enzyme activity. The data show that
some are effective inhibitors targeting this bacterial
HDH.
12 sꢁ1. The enzyme affinity for the substrate, Km app
histidinol is approximately 12 lM.
,
2.3. Inhibition study
Inhibition data against Brucella suis HDH with com-
pounds 5a–5o are presented in Table 1.
2. Results and discussion
2.1. Chemistry
The compounds 5a to 5o showed IC50 in the range of 3–
200 nM. The most effective compounds were the bromo-
5d and the benzyloxy-5i derivatives with IC50 of 6 and
3 nM, respectively. The most ineffective inhibitors were
compounds 5g and 5m which exhibited an IC50 of 150
and 200 nM, respectively. The other compounds have
an inhibitory effect for concentrations ranging from 10
to 40 nM.
The compounds chosen in this study belong to the same
class as the previously described cabbage HDH inhibi-
tors.10 The chemistry employed for the preparation of
the compounds reported here is shown in Scheme 1.

M.-R. Abdo et al. / Bioorg. Med. Chem. 15 (2007) 4427–4433
R1
4429
R2
N
HN
HN
H2N
SOCl2, MeOH
O
OH
N
R3
N
O
O
1 R1=R2=H R3= CH3
i
ii
2 R1=R2= Boc R3= CH3
3 R1=H R2= Boc R3= CH3
4 R1=MeOTrt R2= Boc R3= CH3
iii
i: Boc2O, NEt3, MeOH.; ii: K2CO3, MeOH.; iii:
MeOTrtCl, NEt3, MeOH.; iv: substituted phenyl
acetic acid, LHMDS, -78˚C.; v: HCl, dioxane.
iv, v
HN
H2N
N
R4
O
2HCl
5
Scheme 1. Synthesis of the inhibitors.
R4
-H
HN
HN
H2N
H2N
5a
5b 4-F
5c 4-Cl
5d 4-Br
5e 4-Ph
N
N
R4
O
O
2HCl
2HCl
5m
5f
4-CH3
5g 4-OCH3
5h 4-OPh
HN
H2N
HN
H2N
5i
5j
4-OCH2Ph
2,4-F
O
O
N
N
5k 3,4-Cl
O
2HCl
O
2HCl
5l
Perfluoro
5o
5n
Figure 1. Structures of the inhibitors.
Table 1. Inhibition of B. suis histidinol dehydrogenase with com-
pounds 5a–5o
a
b
250
150
100
75
50
37
25
20
(kDa)
250
150
100
75
50
37
1
2
3
4
5
6
Compound
HDH (Brucella suis)
a
IC50 (nM)
HDH (Cabbage)
b
IC50
5a
5b
5c
5d
5e
5f
40
15
100
—
12
6
300
40
25
20
12.5
15
40
—
15
15
5g
5h
5i
150
16
1000
—
Figure 2. (a) SDS–PAGE of 6 · (His)-tagged HDH from B. suis
expressed from pET15b-HDH in E. coli BL21(DE3). Recombinant
HDH was purified by Cobalt-affinity chromatography as described in
Section 4. Lanes 1 and 5, molecular weight marker; lane 2, soluble
fraction of whole cell lysate; lane 3, proteins from soluble fraction after
dialysis; lane 4, proteins from soluble fraction after binding to the
cobalt resin; lane 6, proteins from soluble fraction after elution from
cobalt resin. (b) Western blot analysis of pure, His-tagged HDH
protein with antibodies raised against His-tag. Pure HDH is indicated
by an arrow.
3
10
—
—
5j
5k
5l
20
70
—
—
5m
5n
5o
200
14.5
25
—
—
—
a The values are means of three independent assays. Variations were in
the range of 5–10% of the shown data.
b Ref. 10.
The following should be noted regarding data of Table
1: (i) increase in the volume of R4 substituent led in gen-
eral to more potent inhibitors. This can be observed for
example in the case of the compounds 5a and 5f; 5b and
5d or 5g and 5h. The presence of the phenoxy moiety in
5h led to a 10-fold increase of drug inhibitory properties
as compared to the corresponding methoxy-substituted
compound 5g. (ii) An interesting observation can be
made with the two naphthyl derivatives 5m and 5n.
Depending on the substitution position on the naphthyl
group, a difference in behaviour could be observed. Sub-
stitution on the position 2 of the naphthyl moiety gave a
very potent inhibitor with an IC50 of 14.5 nM. Substitu-

4430
M.-R. Abdo et al. / Bioorg. Med. Chem. 15 (2007) 4427–4433
tion on position 1 led to an important decrease of activ-
ity with an IC50 of 200 nM. This could be explained by
the alternative orientation of the bulky naphthyl moiety.
In the case of compound 5n, the naphthyl moiety could
probably better accommodate in the lipophilic pocket
near the active site than in the case of compound 5m.
In the same way, the effect of compound 5i, which was
a stronger inhibitor than 5h and which was character-
ized by a longer R-chain, may give an indication of
the distance between the active site and the lipophilic
pocket. (iii) Five of the inhibitors investigated here such
as 5a, 5c, 5d, 5e and 5g also showed selectivity against B.
suis HDH compared to cabbage HDH. Thus, the potent
HDH inhibitors 5a, 5d, 5e and 5g were between 2.5 and
6.6 times, and the inhibitor 5c (chloro-derivative) 25
times more efficient on B. suis HDH than on cabbage
HDH.
4.1.2.1. Preparation of L-histidine methyl ester dihy-
drochloride (1). Thionyl chloride (1.2 equiv) was added
dropwise to a suspension of L-histidine (1 equiv) in
anhydrous methanol (50 mL) at 0 ꢁC, and the resulting
mixture was refluxed for 16 h. The reaction mixture
was allowed to cool down to room temperature and
was concentrated. Additional methanol (50 mL) was
added and the solution was evaporated again to afford
a solid which was then triturated with ethylic ether to
give a white powder. After filtration the resulting
product was used in the next step without further
1
purification: H NMR (DMSO-d6, 400 MHz) 9.05 (d,
1H, J = 1.3 Hz), 7.51 (d, 1H, J = 1.22 Hz), 4.47 (t, 1H,
J = 7.0 Hz), 3.72 (s, 3H), 3.31 (dd, 2H, J = 1.2 Hz,
J = 7.0 Hz); MS ESI+ m/z 170.20 (M+H)+.
4.1.2.2. Preparation of N-a-(tert-butoxycarbonyl)-1-
tert-butoxycarbonyl-L-histidine methyl ester (2). To a
suspension (1 equiv) of (1) in methanol (50 mL) were
added (2 equiv) of triethylamine and (2.3 equiv) of
(Boc)2O. The reaction mixture was stirred for two hours
at room temperature and monitored with TLC until no
starting material was detected. The reaction mixture was
then extracted with ethyl acetate and washed with water.
The resulting organic layer was dried over anhydrous
magnesium sulfate and concentrated under vacuum.
The crude product was purified on silica gel (ether/hex-
3. Conclusion
For the first time, histidinol dehydrogenase protein from
Brucella suis has been isolated, purified, and its biologi-
cal activity characterized. Inhibition studies with this en-
zyme are presented here. A library of new histidine
derivatives is reported and has been assayed for the inhi-
bition of HDH. Effective inhibitors were detected with
inhibitory activity below 10 nM. Our data suggest that
HDH from Brucella suis constitutes a suitable target
for such compounds, which represent valuable candi-
dates for the potential development of novel, non-classi-
cal anti-brucellosis therapy. Moreover, the study of the
interactions of the drugs with the active site of
the HDH enzyme will give a better understanding of
the drug effect, allowing the design of new potential
drugs with improved inhibitory activity.
1
ane: 5:5) to give a white solid: H NMR (DMSO-d6,
400 MHz) 8.11 (d, 1H, J = 1.2 Hz), 7.25 (d, 1H,
J = 0.8 Hz), 7.18 (d, 1H, J = 8.1 Hz), 4.25 (dt, 1H,
J = 5.0 Hz, J = 9.0 Hz), 3.62 (s, 3H), 2.83 (dq, 2H,
J = 6.9 Hz, J = 14.8 Hz), 1.56 (s, 9H), 1.35 (s, 9H); MS
ESI+ m/z 370.28 (M+H)+ 392.13 (M+Na)+.
4.1.2.3. Preparation of N-a-(tert-butoxycarbonyl)-L-
histidine methyl ester (3). To a solution of N-a-(tert-
butoxycarbonyl)-1-tert-butoxycarbonyl-L-histidine methyl
ester (2) (1 equiv) in 30 mL of methanol, K2CO3
(0.1 equiv) was added and the mixture was refluxed for
3 h. When all of the starting was consumed, the mixture
was extracted with ethyl acetate and washed with water.
The organic layer was then dried over magnesium sul-
fate and the solvent evaporated under reduced pressure
to afford the resulting product as a white solid. This
product was used in the next reaction without further
purification:1H NMR (DMSO-d6, 400 MHz) 7.54 (d,
1H, J = 1.2 Hz), 7.15 (d, 1H, J = 7.8 Hz), 6.79 (s, 1H),
4.23 (dt, 1H, J = 5.7 Hz, J = 7.8 Hz), 3.58 (s, 3H), 2.86
(m, 2H), 1.36 (s, 9H); MS ESI+ m/z 270.20 (M+H)+
292.16 (M+Na)+.
4. Experimental
4.1. Chemistry
4.1.1. General. All chemicals, reagents and solvents for
the synthesis of the compounds were of analytical grade,
purchased from commercial sources and used without
further purification, unless otherwise specified. TLC
analyses were performed on silica gel 60 F254 plates
(Merck Art.1.05554). Spots were visualized at 254 nm
under UV illumination, or by ninhydrin solution spray-
ing. Melting points were determined on a Buchi Melting
¨
1
Point 510 apparatus and are uncorrected. H and 13C
NMR spectra were recorded on Bruker DRX-400 spec-
¨
4.1.2.4. Preparation of N-a-(tert-butoxycarbonyl)-1-
methoxytrityl-L-histidine methyl ester (4). To a stirred
solution of N-a-(tert-butoxycarbonyl)-L-histidine methyl
ester (3) (1 equiv) in dimethylformamide, triethylamine
(1.5 equiv) and methoxytritylchloride (1.1 equiv) were
added at room temperature. The reaction was moni-
tored by TLC until the starting material was consumed.
Then the mixture was extracted with ethyl acetate and
washed with brine. The combined organic layers were
dried over anhydrous magnesium sulfate and the solvent
was evaporated under vacuum. The resulting crude was
purified by flash chromatography on silica gel (hexane:
trometer using DMSO-d6 as solvent and tetramethylsi-
lane 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
(10 eV) were recorded in positive or negative mode on
a Water MicroMass ZQ.
4.1.2. Preparation of the protected histidine methyl ester
(4). This compound was prepared in a four-step
synthesis.

M.-R. Abdo et al. / Bioorg. Med. Chem. 15 (2007) 4427–4433
4431
100% to hexane/ether: 50/50) to give the pure product as
a white powder: H NMR (DMSO-d6, 400 MHz) 7.38
1H), 9.10 (d, 1H, J = 1.3 Hz), 8.62 (s br, 2H), 7.50 (d,
1H, J = 0.9 Hz), 7.39 (d, 2H, J = 8.5 Hz), 7.27 (d, 2H,
J = 8.5 Hz), 4.70 (dd, 1H, J = 4.6 Hz, J = 8.7 Hz), 4.19
(d, 2H, J = 2.8 Hz), 3.58 (dd, 1H, J = 4.3 Hz,
J = 15.5 Hz), 3.22 (dd, 1H, J = 8.7 Hz, J = 15.5 Hz)
13C NMR (DMSO-d6, 101 MHz) 202.51, 133.99,
132.42, 131.75, 131.50, 128.08, 126.53, 118.26, 56.90,
44.58, 24.09; MS ESI+ m/z 264.27 (M+H)+. Anal.
(C13H16Cl3N3O) 46.33C, 4.85H, 12.58N.
1
(m, 5H), 7.26 (d, 1H, J = 1.4 Hz), 7.07 (m, 5H), 6.95
(m, 4H), 6.65 (s, 1H), 4.21 (dd, 1H, J = 7.6 Hz,
J = 13.7 Hz), 3.76 (s, 3H), 3.55 (s, 3H), 2.80 (dd, 2H,
J = 3.4 Hz, J = 6.6 Hz), 1.34 (s, 9H); MS ESI+ m/z
542.25 (M+H)+ 564.24 (M+Na)+.
4.1.3. General procedure for the coupling of various
substituted phenylacetic acids with the protected histidine
(4) and obtaining of the resulting dihydrochloride salts (5).
A solution of the corresponding substituted phenylacetic
acid (3 equiv) in dry tetrahydrofuran (10 mL) was
cooled to 0 ꢁC, and a solution of lithium bis(trimethylsi-
lyl)amide (6 eq, 1 M in tetrahydrofuran) was added
dropwise. After 10 min, the solution was cooled to
ꢁ78 ꢁC and the protected histidine methyl ester 4
(1 equiv) in tetrahydrofuran was introduced through a
canula. The reaction mixture was stirred for 16 h under
argon allowing the mixture to warm to room tempera-
ture. The reaction mixture was extracted with ethyl ace-
tate (100 mL) and washed with water (4· 25 mL). The
organic layer was dried over anhydrous magnesium sul-
fate and the solvent evaporated under reduced pressure
to give yellowish viscous oil.
4.1.3.4. (S)-3-Amino-1-(4-bromophenyl)-4-(1H-imi-
dazol-4-yl)butan-2-one dihydrochloride (5d). Mp: 190–
192 ꢁC; 1H NMR (DMSO-d6, 400 MHz) 14.84 (s br,
1H), 9.11 (d, 1H, J = 1.4 Hz), 8.64 (s br, 2H), 7.52 (m,
3H), 7.21 (m, 2H), 4.70 (dd, 1H, J = 4.6 Hz,
J = 8.8 Hz), 4.19 (d, 2H, J = 4.2 Hz), 3.58 (dd, 1H,
J = 4.3 Hz, J = 15.6 Hz), 3.23 (dd, 1H, J = 8.8 Hz,
J = 15.5 Hz); 13C NMR (DMSO-d6, 101 MHz) 202.45,
134.00, 132.84, 132.14, 131.01, 126.55, 120.04, 118.25,
56.91, 44.66, 24.10; MS ESI+ m/z 308.20 (M+H)+. Anal.
(C13H16BrCl2N3O) 40.92C, 4.35H, 11.20N.
4.1.3.5. (S)-3-Amino-4-(1H-imidazol-4-yl)-1-(4-phenyl)-
phenylbutan-2-one dihydrochloride (5e). Mp: 152–154 ꢁC;
1H NMR (DMSO-d6, 400 MHz) 14.72 (s br, 1H), 9.11
(d, 1H, J = 1.1 Hz), 8.63 (s br, 2H), 7.51 (d, 1H,
J = 0.7 Hz), 7.70–7.30 (m, 9H), 4.73 (dd, 1H,
J = 4.6 Hz, J = 8.8 Hz), 4.20 (s, 2H), 3.60 (dd, 1H,
J = 4.4 Hz, J = 15.4 Hz), 3.24 (dd, 1H, J = 8.9 Hz,
J = 15.5 Hz); 13C NMR (DMSO-d6, 101 MHz) 202.77,
139.77, 138.62, 133.98, 132.60, 130.42, 128.83, 127.28,
126.53, 126.49, 126.44, 118.26, 56.93, 44.93, 24.12; MS
ESI+ m/z 306.34 (M+H)+. Anal. (C19H21Cl2N3O)
60.35C, 5.68H, 11.09N.
The residue was purified by column chromatography on
silica gel (CH2Cl2/hexane/acetone: 4:5:1) 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), and the reaction was monitored by
TLC until all the starting material was consumed. The
final salt was filtered and washed with diethyl ether.
4.1.3.1. (S)-3-Amino-4-(1H-imidazol-4-yl)-1-phenyl-
butan-2-one dihydrochloride (5a). Mp: 149–151 ꢁC; H
1
4.1.3.6. (S)-3-Amino-4-(1H-imidazol-4-yl)-1-p-tolyl-
butan-2-one dihydrochloride (5f). Mp: 148–150 ꢁC; 1H
NMR (DMSO-d6, 400 MHz) 14.77 (s br, 1H), 9.11 (d,
1H, J = 1.1 Hz), 8.62 (s br, 2H), 7.49 (s, 1H), 7.12 (s,
4H), 4.68 (dd, 1H, J = 4.5 Hz, J = 8.8 Hz), 4.09 (s,
2H), 3.56 (dd, 1H, J = 4.4 Hz, J = 15.5 Hz), 3.20 (dd,
1H, J = 8.9 Hz, J = 15.5 Hz), 2.28 (s, 3H); 13C NMR
(DMSO-d6, 101 MHz) 202.92, 135.84, 134.09, 130.17,
129.66, 128.78, 126.55, 118.20, 56.82, 44.88, 24.17,
20.61; MS ESI+ m/z 244.32 (M+H)+. Anal.
(C14H19Cl2N3O) 53.25C, 6.16H, 13.21N.
NMR (DMSO-d6, 400 MHz) 14.80 (s br, 1H), 9.11 (d,
1H, J = 1.2 Hz), 8.62 (s br, 2H), 7.50 (d, 1H,
J = 0.5 Hz), 7.29 (m, 5H), 4.70 (dd, 1H, J = 4.6 Hz,
J = 8.8 Hz), 4.15 (s, 2H), 3.58 (dd, 1H, J = 4.4 Hz,
J = 15.5 Hz), 3.22 (dd, 1H, J = 8.9 Hz, J = 15.5 Hz); 13C
NMR (DMSO-d6, 101 MHz) 202.79, 134.33, 133.30,
129.82, 128.19, 126.78, 126.57, 118.21, 56.91, 45.24,
24.17; MS ESI+ m/z 230.19 (M+H)+ 242.28 (M+Na)+.
Anal. (C13H17Cl2N3O) 51.63C, 5.72H, 13.85N.
4.1.3.2. (S)-3-Amino-1-(4-fluorophenyl)-4-(1H-imi-
dazol-4-yl)butan-2-one dihydrochloride (5b). Mp: 146–
148 ꢁC; 1H NMR (DMSO-d6, 400 MHz) 14.72 (s br, 1H),
9.10 (d, 1H, J = 1.2 Hz), 8.62 (s br, 2H), 7.50 (d, 1H,
J = 0.5 Hz), 7.28 (dd, 2H, J = 5.6 Hz, J = 8.7 Hz), 7.16 (t,
2H, J = 8.9 Hz), 4.70 (dd, 1H, J = 4.6 Hz, J = 8.7 Hz),
4.16 (s, 2H), 3.57 (dd, 1H, J = 4.4 Hz, J = 15.5 Hz), 3.21
(dd, 1H, J = 8.9 Hz, J = 15.5 Hz). 13C NMR (DMSO-d6,
101 MHz) 202.76, 161.17 (d, JC–F = 245.3 Hz), 134.14,
131.73 (d, JC–F = 8.1 Hz), 129.50 (d, JC–F = 3.3 Hz),
126.56, 118.22, 114.93 (d, JC–F = 21.2 Hz), 56.85, 44.33,
24.17; MS ESI+ m/z 248.29 (M+H)+. Anal.
(C13H16Cl2FN3O) 48.72C, 5.14H, 13.06N.
4.1.3.7. (S)-3-Amino-4-(1H-imidazol-4-yl)-1-(4-meth-
oxyphenyl)butan-2-one dihydrochloride (5g). Mp: 171–
173 ꢁC; 1H NMR (DMSO-d6, 400 MHz) 14.73 (s br,
2H), 9.12 (d, 1H, J = 1.3 Hz), 8.63 (s br, 2H), 7.49 (d,
1H, J = 1.0 Hz), 7.15 (m, 2H), 6.88 (m, 2H), 4.68 (dd,
1H, J = 4.5 Hz, J = 8.7 Hz), 4.06 (s, 2H), 3.73 (s, 3H),
3.56 (dd, 1H, J = 4.4 Hz, J = 15.4 Hz), 3.21 (dd, 1H,
J = 8.9 Hz, J = 15.5 Hz); 13C NMR (DMSO-d6,
101 MHz) 203.03, 158.13, 134.14, 130.79, 126.54,
125.02, 118.17, 113.65, 56.72, 54.95, 44.37, 24.18; MS
ESI+ m/z 260.31 (M+H)+. Anal. (C14H19Cl2N3O2)
50.52C, 5.79H, 12.63N.
4.1.3.3. (S)-3-Amino-1-(4-chlorophenyl)-4-(1H-imi-
dazol-4-yl)butan-2-one dihydrochloride (5c). Mp: 186–
188 ꢁC; 1H NMR (DMSO-d6, 400 MHz) 14.73 (s br,
4.1.3.8. (S)-3-Amino-4-(1H-imidazol-4-yl)-1-(4-phen-
oxyphenyl)butan-2-one dihydrochloride (5h). Mp: 165–
167 ꢁC; 1H NMR (DMSO-d6, 400 MHz) 14.80 (s br,

4432
M.-R. Abdo et al. / Bioorg. Med. Chem. 15 (2007) 4427–4433
1H), 9.11 (d, 1H, J = 1.2 Hz), 8.66 (s br, 2H), 7.51 (m,
1H), 7.39 (m, 2H), 7.25 (m, 2H), 7.13 (m, 1H), 6.98
(m, 4H), 4.71 (dd, 1H, J = 4.6 Hz, J = 8.8 Hz), 4.15 (s,
2H), 3.58 (dd, 1H, J = 4.4 Hz, J = 15.5 Hz), 3.23 (dd,
1H, J = 8.8 Hz, J = 15.5 Hz); 13C NMR (DMSO-d6,
101 MHz) 202.85, 156.61, 155.45, 134.08, 131.44,
129.97, 128.37, 126.57, 123.33, 118.44, 118.39, 118.22,
56.85, 44.47, 24.14; MS ESI+ m/z 262.24 (M+H)+,
643.38 (2M+H)+. Anal. (C19H21Cl2N3O2) 57.83C,
5.42H, 10.54N.
1H, J = 0.9 Hz), 4.86 (dd, 1H, J = 4.7 Hz, J = 9.1 Hz),
4.42 (dd, 2H, J = 18.8 Hz, J = 40.2 Hz), 3.59 (dd, 1H,
J = 4.4 Hz, J = 15.4 Hz), 3.29 (dd, 1H, J = 9.1 Hz,
J = 15.5 Hz); 13C NMR (DMSO-d6, 101 MHz) 199.60,
134.21, 126.49, 118.15, 56.74, 33.68, 24.32; MS ESI+
m/z 320.24 (M+H)+. Anal. (C13H12Cl2F5N3O) 39.95C,
3.25H, 10.58N.
4.1.3.13. (S)-3-Amino-4-(1H-imidazol-4-yl)-1-(naph-
thalen-1-yl)butan-2-one dihydrochloride (5m). Mp: 184–
186 ꢁC; 1H NMR (DMSO-d6, 400 MHz) 14.78 (s br,
1H), 9.13 (d, 1H, J = 1.32 Hz) 8.70 (s br, 2H), 7.91 (m,
3H), 7.56 (d, 1H, J = 1.2 Hz), 7.49 (m, 4H), 4.91 (dd,
1H, J = 4.8 Hz, J = 8.7 Hz), 4.67 (q, 2H, J = 18.2 Hz),
3.72 (dd, 1H, J = 4.3 Hz, J = 15.5 Hz), 3.34 (dd, 1H,
J = 8.6 Hz, J = 15.3 Hz); 13C NMR (DMSO-d6,
101 MHz) 202.70, 134.17, 133.23, 131.93, 130.22,
128.36, 128.29, 127.60, 126.75, 126.01, 125.66, 125.38,
124.53, 118.22, 56.89, 43.34, 24.47; MS ESI+ m/z
280.32 (M+H)+. Anal. (C17H19Cl2N3O) 57.83C, 5.58H,
11.98N.
4.1.3.9. (S)-3-Amino-1-(4-(benzyloxy)phenyl)-4-(1H-
imidazol-4-yl)butan-2-one dihydrochloride (5i). Mp:
1
164–166 ꢁC; H NMR (DMSO-d6, 400 MHz) 14.75 (s
br, 1H), 9.11 (d, J = 1.36 Hz, 1H), 8.6 (s br, 2H), 7.49
(d, J = 1.25 Hz, 1H), 7.47-7.28 (m, 5H), 7.16 (d,
J = 8.78 Hz, 2H), 6.97 (d, J = 8.79 Hz, 2H), 5.09 (s,
2H), 4.68 (dd, J = 8.82, 4.63 Hz, 1H), 4.06 (s, 2H),
3.56 (dd, J = 15.58, 4.20 Hz, 1H), 3.20 (dd, J = 15.54,
8.86 Hz, 1H); 13C NMR (DMSO-d6, 101 MHz) 203.06,
157.22, 137.04, 134.10, 130.84, 128.34, 127.72, 127.57,
126.54, 125.35, 118.20, 114.54, 69.07, 56.75, 44.39,
24.16; MS ESI+ m/z 336.27 (M+H)+. Anal.
(C20H23Cl2N3O2) 58.80C, 5.72H, 10.21N.
4.1.3.14. (S)-3-Amino-4-(1H-imidazol-4-yl)-1-(naph-
thalen-2-yl)butan-2-one dihydrochloride (5n). Mp: 187–
189 ꢁC; 1H NMR (DMSO-d6, 400 MHz) 14.88 (s br,
1H), 9.14 (d, J = 1.27 Hz, 1H), 8.70 (s br, 2H), 7.86
(m, 4H), 7.54 (s, 1H), 7.46 (m, 3H), 4.78 (dd, J = 8.78,
4.59 Hz, 1H), 4.35 (s, 2H), 3.64 (dd, J = 15.50,
4.35 Hz, 1H), 3.28 (dd, J = 15.45, 8.87 Hz, 1H); 13C
NMR (DMSO-d6, 101 MHz) 202.94, 134.08, 132.82,
131.86, 131.10, 128.28, 128.20, 127.56, 127.45, 127.35,
126.57, 126.09, 125.73, 118.27, 56.94, 45.48, 24.20; MS
ESI+ m/z 280.32 (M+H)+. Anal. (C17H19Cl2N3O)
57.72C, 5.63H, 11.82N.
4.1.3.10. (S)-3-Amino-1-(2,4-difluorophenyl)-4-(1H-
imidazol-4-yl)butan-2-one dihydrochloride (5j). Mp:
1
195–197 ꢁC; H NMR (DMSO-d6, 400 MHz) 14.85 (s
br, 1H), 9.13 (d, 1H, J = 1.3 Hz), 8.72 (s br, 2H), 7.53
(d, 1H, J = 0.7 Hz), 7.41 (dt, 1H, J = 6.8 Hz,
J = 8.6 Hz), 7.23 (dt, 1H, J = 2.6 Hz, J = 9.8 Hz),
7.07(d dt, 1H, J = 0.9 Hz, J = 2.6 Hz, J = 8.5 Hz), 4.76
(dd, 1H, J = 4.6 Hz, J = 8.8 Hz), 4.24 (dd, 2H,
J = 18.5 Hz, J = 54.9 Hz), 3.60 (dd, 1H, J = 4.4 Hz,
J = 15.5 Hz), 3.28 (dd, 1H, J = 8.9 Hz, J = 15.5 Hz);
13C NMR (DMSO-d6, 101 MHz) 201.47, 162.23
(dd, JC-F = 11.9 Hz, JC–F = 98.1 Hz), 159.79 (dd, 1C,
JC–F = 11.8 Hz, JC–F = 100.7 Hz), 134.00, 133.18 (dd,
1C, JC–F = 6.2 Hz, JC–F = 9.7 Hz), 126.49, 118.22,
117.02 (dd, 1C, JC–F = 3.8 Hz, JC–F = 16.6 Hz), 111.23
4.1.3.15. (S)-3-Amino-1-(benzo[d][1,3]dioxo-6-yl)-4-
(1H-imidazol-4- yl)butane-2-one dihydrochloride (5o).
Mp: 159–161 ꢁC; 1H NMR (DMSO-d6, 400 MHz) 14.83
(s br, 1H), 9.11 (d, J = 1.23 Hz, 1H), 8.64 (s br, 2H), 7.50
(s, 1H), 6.85 (d, J = 7.91 Hz, 1H), 6.83 (d, J = 1.62 Hz,
1H), 6.70 (dd, J = 7.93, 1.68 Hz, 1H), 5.99 (s, 2H),
4.66 (dd, J = 8.69, 4.58 Hz, 1H), 4.06 (s, 2H), 3.55 (dd,
J = 15.50, 4.38 Hz, 1H), 3.21 (dd, J = 15.45, 8.79 Hz,
1H); 13C NMR (DMSO-d6, 101 MHz) 202.92, 147.02,
146.04, 134.05, 126.82, 126.52, 122.93, 118.23, 110.16,
108.01, 100.79, 56.71, 44.88, 24.12; MS ESI+ m/z
274.18 (M+H)+. Anal. (C14H17Cl2N3O3) 48.45C,
5.01H, 12.26N.
(dd, JC–F = 3.6 Hz, JC-F = 21.1 Hz), 103.53 (t, JC-F
=
25.9 Hz), 56.74, 39.43, 24.24; MS ESI+ m/z 266.30
(M+H)+280.33 (M+Na)+. Anal. (C13H15Cl2F2N3O)
46.25C, 4.58H, 12.36N.
4.1.3.11. (S)-3-Amino-1-(3,4-dichlorophenyl)-4-(1H-
imidazol-4-yl)butan-2-one dihydrochloride (5k). Mp:
1
193–195 ꢁC; H NMR (DMSO-d6, 400 MHz) 14.91 (s
br, 1H), 9.24 (d, 1H, J = 1.3 Hz), 8.80 (s br, 2H), 7.73
(d, 1H, J = 8.2 Hz), 7.69 (d, 1H, J = 2.0 Hz), 7.65 (d,
1H, J = 0.7 Hz), 7.39 (dd, 1H, J = 2.0 Hz, J = 8.3 Hz),
4.84 (dd, 1H, J = 4.6 Hz, J = 8.6 Hz), 4.38 (d, 2H,
J = 5.1 Hz), 3.71 (dd, 1H, J = 4.4 Hz, J = 15.5 Hz),
3.36 (dd, 1H, J = 8.8 Hz, J = 15.6 Hz); 13C NMR
(DMSO-d6, 101 MHz) 202.18, 134.63, 134.00, 131.86,
130.58, 130.42, 130.20, 129.50, 126.49, 118.32, 56.91,
44.25, 24.04; MS ESI+ m/z 298.21 (M+H)+. Anal.
(C13H15Cl4N3O) 42.16C, 4.27H, 11.22N.
4.2. Biology
4.2.1. Cloning and overexpression of the HDH-encoding
gene from B. suis, and purification of the enzyme. The
HDH-encoding gene, BR0252, was specifically amplified
by PCR using B. suis 1330 chromosomal DNA as tem-
plate and OPJ5-foward primer (50-GCGGGCATATG
GTCACAACGCTCAGACAGACCG-30) and OPJ6-re-
verse primer (50-GCGCGGGATCCTCATAGGTTCA
GACGAATGGCGACG-30) which contain BamHI
and NdeI recognition sequences (underlined), respec-
tively. The PCR products were digested with BamHI
and NdeI and ligated to BamHI- and NdeI-digested
pET15b (Novagen) prior to introduction into E. coli
4.1.3.12. (S)-3-Amino-4-(1H-imidazol-4-yl)-1-(perflu-
orophenyl)butan-2-one dihydrochloride (5l). Mp: 193–
195 ꢁC; 1H NMR (DMSO-d6, 400 MHz) 14.74 (s br,
1H), 9.12 (d, 1H, J = 1.3 Hz), 8.77 (s br, 2H), 7.52 (d,

M.-R. Abdo et al. / Bioorg. Med. Chem. 15 (2007) 4427–4433
4433
strain DH5a. The integrity of the cloned gene was veri-
fied by sequencing, using primers OPJ5 and OPJ6 de-
scribed above. The construct pET15bHDH was then
transformed into E. coli strain BL21(DE3) for produc-
tion of the 6 · (His)-HDH fusion protein.
6200 Mꢁ1 cmꢁ1) as previously described.13 The enzy-
matic 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 so-
dium glycine (pH 9.2) and 2 mM NAD+. The Km for
the substrate was determined by varying the concentra-
tion of histidinol from 10 to 50 lM. Activity (1 U) is de-
fined as the amount of HDH producing 1 lmol of
NADH per min in the reaction. To perform IC50 deter-
minations of the different inhibitors, the latter were
added at various concentrations, 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 · 10ꢁ11 M.
Escherichia
coli
BL21(DE3)
cells
harbouring
pET15bHDH were grown at 37 ꢁC in one litre of Lur-
ia-Bertani medium supplemented with 50 lg/mL ampi-
cillin. When the culture reached an optical density at
600 nm (OD600) of approximately 0.6, expression of
6 · (His)-HDH protein was induced by the addition of
isopropyl-thio-b-D-galactoside (IPTG) to a final concen-
tration of 1 mM and growth was continued for 5 h. Cells
were then harvested by centrifugation at 3500 rpm at
4 ꢁC for 20 min and broken by sonication in buffer A
(200 mM KCl, 50 mM Tris–Cl [pH 7.5], 10% glycerol,
0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluo-
ride, 2 lM pepstatin A) supplemented with 0.5 mM
dithiothreitol and 0.2 mM disodium EDTA. All subse-
quent steps were performed at 4 ꢁC. After centrifugation
(13,000 rpm, 20 min), the soluble extract was treated
with streptomycin sulfate12 to remove ribosomes and
nucleic acids. The suspension was then centrifuged at
13,000 rpm for 15 min, and the supernatants were dia-
lysed against 2 L of sonication buffer A for 1 h. The dia-
lysed lysates were mixed with Talon Co+-affinity resin
(Clontech) that had been equilibrated with buffer I
(20 mM Tris–HCl [pH 8.0], 5 mM b-mercaptoethanol,
10% glycerol, 1 mM phenylmethylsulfonyl fluoride,
2 lM pepstatin A and 0.1% Nonidet P-40) supple-
mented with 150 mM KCl. The resin and bound His-
tagged protein were collected by centrifugation at
2500 rpm for 10 min and washed with buffer I contain-
ing 500 mM KCl and 10 mM imidazole. The 6 · (His)-
HDH protein was eluted with buffer I containing
125 mM KCl and 75 mM imidazole without Nonidet
P-40. Elution fractions were free of detectable contami-
nating proteins as determined by Coomassie blue stain-
ing of sodium dodecyl sulfate (SDS)–polyacrylamide
gels. The fractions containing the His-tagged proteins
(estimated purity, >95%) were pooled and stored in elu-
tion buffer supplemented with 40% (v/v) glycerol at
ꢁ20 ꢁC. Protein concentration was determined with
the bicinchoninic acid reagent (Pierce) using bovine ser-
um albumin as a standard.
Acknowledgment
This work was supported by a grant from the German
Sanita¨tsamt der Bundeswehr, No. M SAB1 5A002.
References and notes
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4.2.2. Enzyme assays. The activity and specificity of
HDH were measured by monitoring the reduction
of NAD+ to NADH directly at 340 nM (eM
=
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