Isoprenoid biosynthesis via the methylerythritol phosphate pathway: Structural variations around phosphonate anchor and spacer of fosmidomycin, a potent inhibitor of deoxyxylulose phosphate reductoisomerase

Article abstract of DOI:10.1021/jo9024732

Fosmidomycin and its analogue FR-900098 are potent inhibitors of 1-deoxy-d-xylulose 5-phosphate reducto-isomerase (DXR), the second enzyme of the MEP pathway for the biosynthesis of isoprenoids. This paper describes the synthesis of analogues of the two reverse phosphonohydroxamic acids 3 and 4, in which the length of the carbon spacer is modified, the N-methyl group of 3 is replaced by an ethyl group, and the phosphate group is replaced by potential isosteric moieties, i.e., sulfonate or carboxylate functionalities. The potential of the synthesized analogues to inhibit the E. coli DXR was evaluated.

Full text of DOI:10.1021/jo9024732

pubs.acs.org/joc
Isoprenoid Biosynthesis via the Methylerythritol Phosphate Pathway:
Structural Variations around Phosphonate Anchor and Spacer of
Fosmidomycin, a Potent Inhibitor of Deoxyxylulose
Phosphate Reductoisomerase
ꢀ
Catherine Zingle, Lionel Kuntz, Denis Tritsch, Catherine Grosdemange-Billiard,* and
Michel Rohmer*
Universiteꢀ de Strasbourg/CNRS, Strasbourg, UMR 7177, Institut Le Bel, 4 rue Blaise Pascal,
67070 Strasbourg, France
grosdemange@unistra.fr; mirohmer@unistra.fr
Received November 30, 2009
Fosmidomycin and its analogue FR-900098 are potent inhibitors of 1-deoxy-D-xylulose 5-phosphate
reducto-isomerase (DXR), the second enzyme of the MEP pathway for the biosynthesis of
isoprenoids. This paper describes the synthesis of analogues of the two reverse phosphonohydroxa-
mic acids 3 and 4, in which the length of the carbon spacer is modified, the N-methyl group of 3 is
replaced by an ethyl group, and the phosphate group is replaced by potential isosteric moieties, i.e.,
sulfonate or carboxylate functionalities. The potential of the synthesized analogues to inhibit the
E. coli DXR was evaluated.
Introduction
target for the design and development of new antimicrobial
drugs.2-4 Fosmidomycin 1 (Scheme 1), a phosphonate anti-
biotic isolated from Streptomyces lavendulae,5 inhibits 1-deoxy-
D-xylulose 5-phosphate reducto-isomerase (DXR), the second
enzyme of the MEP pathway of many Gram-negative bacteria,
some Gram-positive bacteria,6 and the malaria parasites.7
The discovery of the alternative mevalonate-independent
methylerythritol phosphate (MEP) pathway for the bio-
synthesis of isoprenoids,1 which is present in pathogens
such as the tuberculosis-causing bacterium Mycobacterium
tuberculosis and the malaria protozoan agent Plasmodium
falciparum but absent in humans, represents an attractive
˚
The resolution at 2.5 A of the three-dimensional structure
of the Escherichia coli DXR-fosmidomycin complex revealed
two binding sites in the catalytic domain: a first one for
the chelation of a divalent metal ion (Mn2þ, Mg2þ, or Co2þ
*To whom correspondence should be addressed. Phone:
þ
33
)
(0)3.68.85.13.49 and þ 33 (0)3.68.85.13.46. Fax: þ þ 33 (0)3.68.85.13.45.
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Biochem. J. 1993, 295, 517–524. (b) Schwarz, M. K. Ph.D. Thesis, Nb
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DOI: 10.1021/jo9024732
r
Published on Web 04/29/2010
J. Org. Chem. 2010, 75, 3203–3207 3203
2010 American Chemical Society

Zingle et al.
JOCArticle
SCHEME 1. 1-Deoxy-D-xylulose 5-Phosphate Reducto-Isomerase (DXR) and Its Inhibitors (1-4)
by the two oxygen atoms of the hydroxamate moiety of the
antibiotic, and a second one where the negatively charged
phosphonate group is anchored by several hydrogen bonds in
a specific positively charged pocket. These two functional
groups involved in the antibiotic binding are connected by a
spacer of three methylene groups.8 Fosmidomycin 1 and its
analogue, FR-900098 2 (Scheme 1), are effective antimalarial
agents with good tolerability and rapid onset of action, but late
recrudescence and fast clearance preclude their use alone
in a single drug treatment.9 In the case of Mycobacterium
tuberculosis, the inefficiency of fosmidomycin to inhibit its
growth was due to a lack of uptake.10
yielding, on the one hand, phosphate,11 carboxylate,12 sulfo-
nate,13 sulfone,13 or sulfamate11-13 analogues and various
phosphonate diester prodrugs14 and on the other hand
rigidified carbon spacers with a cyclopropyl15or a cyclopen-
tyl ring,16a spacers with an aryl substituent in the R position
of the phosphonate,16 or spacers including oxa groups.17 We
previously reported the synthesis of the two reverse phos-
phonohydroxamic acids 3 and 4 (Scheme 1), which revealed
as potent inhibitors of the E. coli DXR the natural anti-
biotics 1 and 2. The N-methylated derivative 4 is much more
effective than the non-N-methylated compound 3 and is
nearly as efficient as fosmidomycin in enzyme assays.18
The same observation has been reported for FR-900098 2,
an analogue of fosmidomycin bearing an acetyl group in
place of the formyl group.7,8
Although X-ray crystal structures of DXR in complex
with fosmidomycin have been solved, the rational design of
new potent inhibitors by docking methods is not obvious.19
We present here the synthesis and the biological activity of
analogues of the two phosphonohydroxamic acids 3 and 4.
On the one hand, the influence of the length of the carbon
spacer and the replacement of the N-methyl group of 3 by an
ethyl group on the inhibitory activity was investigated. On
the other hand the phosphonate anchor of 3 and 4 was
replaced by other acidic bioisosters such as a carboxylate,
which displays a planar geometry, or a sulfonate sharing with
the phosphonate a pyramidal geometry around the central
atom.20
Investigations are presently oriented toward the design of
fosmidomycin analogues with improved pharmacological
properties. Attention was mostly focused on modifications
of the phosphonate group and the three-carbon spacer,
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Rohdich, F. J. Biol. Chem. 2003, 278, 18401–18407. (b) Mac Sweeney, A.;
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Matsiegui, P.-B.; Oyakhirome, S.; Mawili-Mboumba, D. P.; Missinou,
M. A.; Kun, J. F. J.; Jomaa, H.; Kremsner, P. G. J. Infect. Dis. 2004, 190,
1534–1540.
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Scherman, H.; Crick, D. C. J. Bacteriol. 2005, 187, 8395–8402. (b) Brown, A.
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Sanderbrand, S.; Altincicek, B.; Beck, E.; Schlitzer, M.; Jomaa, H. Bioorg.
Syntheses of C3 and C5 Analogues of Hydroxamates 3 and
4, and of the N-Ethyl C4 Analogue. The hydroxamates 11a,
11b, 14a, 14b, and 15 were obtained by using the same
strategy as that utilized for the syntheses of the C4 derivatives
3 and 4 previously described (Scheme 2).18
Synthesis of Carboxylate Isosters 19a and 19b. The synthe-
ses of the carboxylic acid 19a and of its N-methylated
derivative 19b are shown in Scheme 3. The first step was
the introduction of the hydroxamic acid group. Treatment of
€
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Van Calenbergh, S. J. Med. Chem. 2006, 49, 2656–2660. (d) Ortmann, R.;
Wiesner, J.; Reichenberg, A.; Henschker, D.; Beck, E.; Jomaa, H.; Schlitzer,
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Walter, R. D. Aust. J. Chem. 2007, 60, 154–158. (g) Kurz, T.; Behrendt, C.;
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Jomaa, H.; Schlitzer, M. Arch. Pharm. 2005, 338, 305–314.
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S. Eur. J. Org. Chem. 2006, 17, 3856–3863. (b) Haemers, T..; Wiesner, J.;
Van Poecke, S.; Goeman, J.; Henschker, D.; Beck, E.; Jomaa, H.; Van
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V.; Wiesner, J.; Jomaa, H.; Van der Eycken, J.; Van Calenbergh, S. Bioorg.
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Zingle et al.
SCHEME 2. Syntheses of C3 (11a, 14a) and C5 (11b, 14b) Analogues of Hydroxamates 3 and 4, and of the N-ethyl C4 Analogue (15)
JOCArticle
SCHEME 3. Synthesis of Carboxylate Isosters 19a and 19b
the commercially available monomethylglutarate 16 with
O-benzylhydroxylamine in the presence of 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride in THF
gave the compound 17a in 53% yield. The N-methylation
of the O-benzylhydroxamate 17a was achieved by using
DBU, a softer base than sodium hydride, which led to
undesired byproduct. The N-methylated O-benzylhydroxa-
mate 17b was obtained in 58% yield. Hydrolysis of the
methyl ester was again performed in THF with lithium
hydroxide dissolved in a minimum amount of water.
This provided the carboxylic acid 18a (78% yield) and the
N-methylated analogue 18b (73% yield).
Removal of the benzyl group by catalytic hydrogenolysis
with palladium over activated charcoal at room temperature
and atmospheric pressure afforded the desired carboxylic
acids 19a and 19b in a quantitative yield.
Synthesis of the Sulfonate Isosters 25a and 25b. The synthe-
sis of sulfonate 25a and of its N-methylated analogue 25b
started with the commercially available monomethylsucci-
nate 20 (Scheme 4). The hydroxamate was introduced as
described above, leading to 21 (56% yield). Reduction of
ester 21 with NaBH4-LiCl gave alcohol 22 (73% yield).
A Mitsunobu-type reaction21 of alcohol 22 with thioacetic
acid proceeded in 84% yield to the thioacetate 23a. The N-
methylation of the thioacetate 23a was accomplished by
using methyl iodide in the presence of NaH to provide the
expected compound 23b. The oxidation of the thioacetate
23a and of the N-methylated thioacetate 23b with peroxytri-
fluoroacetic acid afforded the sulfonic acids 24a and 24b in
98% and 93% yield, respectively.22 The benzyl group of 24a
and 24b was removed by catalytic hydrogenolysis with
palladium over charcoal under atmospheric pressure and
at room temperature leading in quantitative yield and
requiring no further purification to the sulfonic acid 25a
and to its N-methylated 25b.
Most NMR spectra presented complicated signal patterns.
N-Substituted and N-and O-substituted hydroxamic acids are
present as equilibrium mixtures of Z and E conformers due to
the restricted rotation around the C-N bond.23 The relative
(22) (a) Volante, R. P. Tetrahedron Lett. 1981, 22, 3119–3122. (b) Corey,
E. J.; Cimprich, K. A. Tetrahedron Lett. 1992, 33, 4099–4102.
(23) Brown, D. A.; Glass, W. K.; Mageswaran, R.; Girmay, B. Magn.
Reson. Chem. 1988, 26, 970–973.
(21) Garcia, B.; Ibeas, S.; Munoz, A.; Leal, J. M. Inorg. Chem. 2003, 42,
5434–5441.
J. Org. Chem. Vol. 75, No. 10, 2010 3205

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JOCArticle
SCHEME 4. Synthesis of Sulfonate 25a and 25b
TABLE 1. Inhibition of Recombinant E. coli DXR by Derivatives
11-15, 19, and 25
stabilities of such Z and E conformers are influenced by the
structural and steric factors of the substituents on the hy-
droxamate moiety as well as by the nature of the solvent.
Tentative assignments of the NMR signals are proposed in
the Supporting Informations. They were made according to
NMR studiesand ab initio calculations on similar compounds
reported in the literature.24-29
Inhibition Tests on the Escherichia coli Deoxyxylulose
5-Phosphate Reductoisomerase. The compounds were tested
for inhibition against the recombinant E. coli DXR as
previously described.18 The IC50 values were determined
with and without preincubation of the compounds with the
enzyme (Table 1). Fosmidomycin and FR900098 were in-
cluded as reference compounds. Slow tight-binding inhibi-
tors such as fosmidomycin30 can thus be ascertained because
the preincubation leads to a significant decrease of the IC50
values (8- to 10-fold). On the one hand shortening the length
of the molecule (as in 11a and 11b) led to a dramatic decrease
of the affinity of DXR for these compounds. They probably
bind only to one site, either to the divalent cation or to the
phosphate-binding site. On the other hand, with compounds
14a and 14b having an additional methylene group, the
decrease was less important, about 2-fold as compared to
the IC50 values found for inhibitors 3 and 4. The slow-
binding mechanism was well pronounced insofar as the
preincubation of the enzyme with the two compounds 14a
and 14b resulted in a significant decrease of the IC50 values.
Although more flexible and prone to distortions, the longer
three methylene spacer apparently prevents compounds 14a
and 14b from efficiently binding to the active site.18
IC50 (μM)
without pre-
incubation
with pre-
incubation
compd
X
R
n
Fosmidomycin 1
FR 900098 2
H
Me
H
2
2
2
2
1
1
3
3
2
2
2
2
2
0.25
NDb
1
0.032a
0.032c
0.17a
0.049a
1000
19
3
4
PO(OH)2
PO(OH)2 Me
PO(OH)2
PO(OH)2 Me
PO(OH)2
PO(OH)2 Me
PO(OH)2 Et
CO2H
CO2H
SO3H
SO3H
0.5
11a
11b
14a
14b
15
19a
19b
25a
25b
H
1000
77
2.8
H
0.27
0.11
6.5
0.9
8.1
H
Me
H
770
270
1000
564
720
25
1000
48
Me
aReference 18. bND = not determined. cReference 13.
between this methyl group and the Trp212 indole group of
the DXR active site may explain the increased affinity for
FR-900098 2 over fosmidomycin 1. By comparing the IC50
values of compounds 4 and 15 in which the methyl group is
replaced by an ethyl group, a severe decrease of the affinity for
DXR was noted for derivative 15. Preincubation of the enzyme
with the inhibitor did not improve the inhibition potency.
Apparently the bulky ethyl group prevents the modification of
the enzyme conformation leading to the formation of a tighter
enzyme/inhibitor complex observed in the case of a slow-
binding mechanism.
Generally, all N-methyl compounds (noted b) were more
effective inhibitors than the nonmethylated compounds
(noted a). As suggested by the three-dimensional structure of
the DXR-fosmidomycin complex, a hydrophobic interaction
(24) Brown, D. A.; Coogan, R. A.; Fitzpatrick, N. J.; Glass, W. K.;
ꢀ
The replacement of a phosphonate group by other acidic
groups such as a carboxylate or a sulfonate has been widely
studied for the comparative evaluation of their bio-
isosteric relationships. Considering the non-N-methylated
compounds 19a and 25a, with respectively a carboxylate and
a sulfonate instead of a phosphonate group (compound 3),
the inhibition was drastically decreased (about 4000- to
6000-fold). Moreover the preincubation had no effect.
With compounds 19b and 25b the inhibition, although less
effective than the corresponding phosphonate derivative 4
(500- to 1000-fold), was more efficient and showed a
Abukshima, D. E.; Shiels, L.; Ahlgren, M.; Smolander, K.; Pakkanen, T. T.;
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3206 J. Org. Chem. Vol. 75, No. 10, 2010

Zingle et al.
JOCArticle
slow-binding behavior. As DXR has a rather low affinity for
DXP, its natural substrate (KM =100-300 μM), the tight
complex between DXR and compounds 1, 2, or 4 may
essentially be ascribed to the chelation of the divalent cation
by the reverse hydroxamate or the hydroxamate group of the
inhibitors and to the induced conformation change after
their binding to the active site. Clearly, this kind of complex
did not take place with compounds 19a and 25a. At best, as in
the case of compound 11a, the enzyme recognized one of the
two binding sites. In contrast, the interactions between DXR
and compounds 19b and 25b were first weak, but the con-
formation change in the active site, probably induced by the
supplementary methyl group, led to a significant tightening
of the inhibition complex.
and hydrogen bond donors display a preference for gauche
orientation, while those involving a sulfonate prefer an
eclipsed geometry.32 So subtle differences in hydrogen bond-
ing may exist between the sulfonate and the phosphonate
derivatives with the enzyme. As for carboxylate derivatives,
the consequences would be an unsuitable positioning of the
hydroxamate group with regard to the divalent cation and
a dramatic decrease of the DXR inhibition.
In trying to understand the biosteric relationships among
carboxylic, sulfonic, and phosphonic acid groups, it has been
shown that a selective molecular recognition of the phospho-
nate group is achieved by high polar binding sites as in the
DXR enzyme, whereas, strikingly, the binding sites of
sulfonates display a relatively low polarity. In addition,
the negative pKa value of a sulfonic acid, which results in a
constant loss of the acidic proton in any protein environ-
ment, could explain the loss of specificity of the sulfonate
analogues for the active site of the DXR enzyme and the low
IC50 values. For instance, the inhibition potency of
carboxylate and sulfonate derivatives of 9-(3,3-dimethyl-
5-phosphonopentyl)guanine was tested on purine nucleo-
side phosphorylase. The sulfonate compound binds about
one-fourth as tightly as the phosphonate. The interaction of
the carboxylate derivative was even much weaker (180-
fold). The carboxylate group turns away from the center
of the phosphate-binding site forming hydrogen bonds with
other amino acids.33
In the case of phosphonate derivatives, the two acidic
groups can be involved in electrostatic interactions and
hydrogen bonds. The binding mode of fosmidomycin 1 to
the E. coli DXR revealed from the X-ray structure seemed to
show that hydrogen bond formation is privileged over
electrostatic interactions. Indeed, the phosphonate moiety
is embedded in a network of hydrogen bonds with the
phosphonate oxygen atoms acting as hydrogen acceptors
and the side chains of polar amino acids such as Ser186,
Ser222, Asn227, and Lys228 as hydrogen bond donors.8a
Our results show that in the case of DXR a carboxylate or a
sulfonate are not suitable surrogates of a phosphonate. The
sulfonate analogue of FR900098 displayed an IC50 value of
23 μM and is approximately 500-fold less active than the
corresponding phosphonate derivative, FR900098.12 The
reason is probably that the latter interacts in the phosphate
recognition site of DXR via specific hydrogen bonds and not
electrostatic interactions. Indeed, only two hydrogen bonds
can be generated with a carboxylate group while a phospho-
nate group is able to be involved in three hydrogen bonds.20
Structurally these two functional groups are different: a
phosphonate has a pyramidal structure, and a carboxylate
a planar one. If the carboxylate binds to the phosphonate
recognition site, it would accommodate differently, maybe
forming other hydrogen bonds. Consequently, the hydro-
xamate group would become unable to chelate the divalent
cation present in the active site leading to a considerable
decrease of the inhibition potency of the carboxylate analo-
gue. The explanation for the relative failure of the sulfonate
derivatives to inhibit the DXR is less obvious. The dibasic
phosphonate group and the monobasic sulfonate share a
considerable structural similarity. Both adopt a pyramidal
geometry around the phosphorus and the sulfur atom. The
reason seemed to be the hydrogen bonding capacity of the
sulfonate and the phosphonate groups with the DXR. A
crystallographic database survey of geometrical features of
the hydrogen bonding interactions revealed that a sulfonic
group tends to form longer interactions than carboxylic and
phosphonic groups.31 Interactions between phosphonate
Acknowledgment. The authors are indebted to Jean-
ꢀ
Daniel Sauer and Maurice Coppe for running the NMR
spectra and Helene Nierengarten, Nathalie Zorn, and
ꢀ ꢁ
ꢁ
Romain Carriere for measuring the mass spectra. This work
was supported by a grant from the “Agence Nationale de la
Recherche” (Grant Nb ANR-06-BLAN-0291-01).
Supporting Information Available: General Methods and
1
Experimental Section; H, 13C, and 31P NMR spectra for
1
compounds 8-15; H, 13C spectra for compounds 17-25;
HSQC and HMBC spectra for compounds 24 and 25;
NOESY spectra for compounds 24b and 25b; and protocol
for enzyme assays. This material is available free of charge
via the Internet at http://pubs.acs.org.
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