Synthetic fosmidomycin analogues with altered chelating moieties do not inhibit 1-deoxy-d-xylulose 5-phosphate reductoisomerase or Plasmodium falciparum growth in vitro

Article abstract of DOI:10.3390/molecules19022571

Fourteen new fosmidomycin analogues with altered metal chelating groups were prepared and evaluated for inhibition of E. coli Dxr, M. tuberculosis Dxr and the growth of P. falciparum K1 in human erythrocytes. None of the synthesized compounds showed activ

Full text of DOI:10.3390/molecules19022571

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Molecules 2014, 19, 2571-2587; doi:10.3390/molecules19022571
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthetic Fosmidomycin Analogues with Altered Chelating
Moieties Do Not Inhibit 1-Deoxy-D-xylulose 5-phosphate
Reductoisomerase or Plasmodium falciparum Growth In Vitro
René Chofor 1, Martijn D.P. Risseeuw 1, Jenny Pouyez 2, Chinchu Johny 3, Johan Wouters 2,
Cynthia S. Dowd 4, Robin D. Couch 3 and Serge Van Calenbergh 1,*
1
Laboratory for Medicinal Chemistry, Ghent University, Harelbekestraat 72, Ghent B-9000,
Belgium; E-Mails: rene.chofor@ugent.be (R.C.); martijn.risseeuw@ugent.be (M.D.P.R.)
2
Department of Chemistry, University of Namur, UNamur, Rue de Bruxelles 61, Namur B-5000,
Belgium; E-Mails: jenny.pouyez@unamur.be (J.P.); johan.wouters@unamur.be (J.W.)
3
Department of Chemistry and Biochemistry, George Mason University, Manassas, VA 20110,
USA; E-Mails: cjohny@gmu.edu (C.J.); rcouch@gmu.edu (R.D.C.)
4
Department of Chemistry, George Washington University, Washington, DC 20052, USA;
E-Mail: cdowd@gwu.edu
* Author to whom correspondence should be addressed; E-Mail: serge.vancalenbergh@ugent.be;
Tel.: +32-926-481-24; Fax: +32-926-481-46.
Received: 6 February 2014; in revised form: 18 February 2014 / Accepted: 19 February 2014 /
Published: 24 February 2014
Abstract: Fourteen new fosmidomycin analogues with altered metal chelating groups were
prepared and evaluated for inhibition of E. coli Dxr, M. tuberculosis Dxr and the growth of
P. falciparum K1 in human erythrocytes. None of the synthesized compounds showed
activity against either enzyme or the Plasmodia. This study further underlines the
importance of the hydroxamate functionality and illustrates that identifying effective
alternative bidentate ligands for this target enzyme is challenging.
Keywords: fosmidomycin; DOXP reductoisomerase; non-mevalonate pathway; isoprenoid
biosynthesis; coordination chemistry

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1. Introduction
Yearly, up to 5 million clinical cases and a million fatalities result from malaria, an infectious
disease caused by protozoa of the Plasmodium species, with P. falciparum being responsible for the
most severe cases [1]. The heaviest caseload is suffered by pregnant women and children in
sub-Saharan Africa [2]. Unlike Plasmodia which are endemic in the tropics, Mycobacterium
tuberculosis (Mtb), the causative agent of tuberculosis afflicts one-third of the world’s population
annually, leading to about 2–3 million deaths [3]. With resistance emerging to virtually all currently
used drugs for the treatment of both diseases, new, safe, effective and low cost antimalarial and
antitubercular therapeutics are highly awaited.
The discovery that fosmidomycin (1, Figure 1) and its acetyl congener FR900098 (2), both natural
products extracted from Streptomyces species inhibit 1-deoxy-D-xylulose-5-phosphate reducto-
isomerase (Dxr), opened interesting opportunities for therapeutics [4,5]. Dxr is the second enzyme in
the non-mevalonate pathway (NMP) for isoprenoid biosynthesis, which is absent in humans, but
present in most Gram-negative and some Gram-positive bacteria (including Mtb), as well as in
apicomplexan parasites (including Plasmodia) [6,7]. Fosmidomycin inhibits the Dxr-catalyzed
conversion of 1-deoxy-D-xylulose-5-phosphate (DOXP) to 2C-methyl-D-erythritol-4-phosphate
(MEP), by mimicking the binding mode of DOXP to this enzyme [8,9]. SAR studies have indicated
the importance of fosmidomycin’s hydroxamate moiety for chelation of a divalent metal cation (M:
Mn2+ or Mg2+) present in the enzyme’s active site.
Figure 1. Analogy between DOXP and Fosmidomycin/FR900098.
M2+
O
P
OH
O
P
O
P
HO
HO
O
O
P
OH
OH
OH
OH
O
O
O
O
O
O
OH
O
OH
OH OH
O
OH
O
OH
OH
DOXP
Divalent metal-bound DOXP
2-C-methyl-erythrose-
MEP
4-phosphate
M2+
OH
N
O
P
O
N
O
P
OH
OH
O
O
HO
OH
R
R
R=H: fosmidomycin, 1
R=CH3: FR900098, 2
Due to its promising antimalarial activity, fosmidomycin received considerable attention and a
combination therapy with clindamycin confirmed its potential as an antimalarial drug, following
clinical trials conducted in Gabon and Thailand [10,11]. However, the moderate bioavailability and
short serum half-life of fosmidomycin prevented the drug combination from reaching the market.
Fosmidomycin’s phosphonate group is highly ionized at physiological pH, which is the main reason
for its low bioavailability. While this does not preclude efficient uptake in P. falciparum, other
organisms like Mtb, are not sensitive to fosmidomycin because they lack a glycerol-3-phosphate
transporter (G1pT) that is known to actively transport fosmidomycin across hydrophobic cell
membranes [12,13].

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Although the chelating ability of hydroxamates often makes them potent metalloenzyme inhibitors,
most hydroxamic acids suffer from poor oral bioavailability and significant binding to other metals
(e.g., Zn2+, Cu2+, etc.) besides Mn2+ and Mg2+ [14,15]. In addition, hydroxamic acids may be rapidly
degraded in vivo by hydrolysis, glucuronidation and sulfation and may suffer from poor
pharmacokinetic and toxicological profiles [16]. In order to circumvent the limitations associated with
the phosphonate and hydroxamate moiety of fosmidomycin, two strategies have been widely exploited
in the design of potent analogues: masking of the polar phosphonate group as prodrugs and/or
substituting the hydroxamate of fosmidomycin with an alternative Mn2+ and Mg2+ binding group. The
former strategy has been relatively well investigated [17], while the latter has been studied with less rigor.
Giessmann et al. synthesized a series of amidopropylphosphonates 3 (Figure 2), but none of these
showed detectable E. coli Dxr inhibition when tested up to 30 µM, indicating the importance of the
N-OH group for Dxr inhibition [18]. This was further proven by Woo et al. following the evaluation of
compounds 4 wherein the N-OH was replaced with N-CH3 [19]. During the synthesis of -substituted
fosmidomycin analogues, our group observed that benzyl removal from the retrohydroxamate moiety
by catalytic hydrogenation typically resulted in the formation of the desired compound, but also
significant amounts of the corresponding deoxygenated derivative, i.e., the amide, due to the
competitive side reaction of “full” reduction [20]. Deprotection of the phosphonate moiety of the latter
afforded analogues such as 5, which were moderately potent in inhibiting E. coli Dxr and capable of
inhibiting the growth of a Dd2 P. falciparum strain at submicromolar concentrations (unpublished
results). The Rohmer group demonstrated that the reverse hydroxamate counterparts of fosmidomycin
or FR900098 (6) elicit comparable inhibitory activity against E. coli Dxr as the natural products [21].
This observation was further confirmed by other groups which obtained sub-micromolar IC50 values
following evaluation of fosmidomycin analogues comprising a reverse hydroxamate moiety [22–24].
Nakamura and co-workers showed that a cis arrangement of the two oxygen atoms of the hydroxamate
group is required for effective metal chelation. Furthermore, they suggested that alternative functional
groups containing cis oxygen atoms might have comparable metal coordination ability [8]. Catechols
7a and 7b showed IC50 values of 24.8 µM and 4.5 µM, respectively, when tested for inhibition
of E. coli Dxr, indicating a preference for the 1,3,4-orientation (7b) of the catechol over the
1,2,3-orientation (7a) [25].
Figure 2. Hydroxamate-modified analogs of fosmidomycin.
R1
N
O
P
O
P
O
O
P
H
OH
OH
OH
OH
OH
OH
O
O
N
HO
N
R2
H
3: R1 = H; R2 = alkyl, arylalkyl, indole-3-alkyl,...
4: R1 = methyl; R2 = H, methyl
6
5
O
OH
P
R3
OH
O
7
O
O
O
N
OH
OH
R4
P
OH
N
N
H
HO
HO
HO
8: R4 = arylalkyl,
7a: R3 =
7b: R3 =
7c: R3 =
methyl sulfonyl,...

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In search for lipophilic fosmidomycin analogues, Andaloussi et al. resynthesized 7b alongside other
hydroxamate-modified compounds with a bulky heteroaryl moiety such as 7c. Tests conducted with
these compounds revealed that steric constraint in the vicinity of the Dxr active site was deleterious to
inhibitory potency [26]. Other attempts to substitute the hydroxamate group of fosmidomycin with
similar sterically demanding alternatives led to the conclusion that the Dxr active site is very narrow
around the metal cation [27,28]. Nevertheless, the Dowd group recently observed a more efficient
coordination of the metal cation by amide- versus O-linked substituents on the retrohydroxamate of
fosmidomycin [29]. They highlighted the importance of having an aromatic group in the inhibitor
while also suggesting that an alkyl chain between the retrohydroxamate and the aryl group may be
preferable for accessing an alternate binding location.
This paper aims to more systematically investigate the possibilities of replacing the retrohydroxamate
group of fosmidomycin with effective alternative bidentate ligands. Amide derivatives represented by
the general structure 8 were prepared and evaluated. We envisaged a contribution to chelation by
ortho-substituents on the amide-linked aromatic ring. Compounds with a NH moiety between carbonyl
and sulfonyl groups are very acidic (pKa ~ 2). At physiological pH, the presence of a negative charge
at this position would be expected to improve the interaction with the active-site metal ion [30].
Therefore, we included one analogue with a methylsulfonyl group in the ortho position of the phenyl
ring (compound 8h), as well as a (non-aromatic) sulfamate (compound 8m). In order to ascertain the
influence of electronic factors on chelation, aromatic substituents with various electronic properties
were selected.
2. Results and Discussion
2.1. Synthesis
The synthesis of the amide derivatives 8a–i, m–q is outlined in Scheme 1. Carboxylic acid 9 was
readily prepared starting from commercially available ethyl 4-bromo-butyrate and dibenzyl phosphite
as previously described by Kuntz et al. [21]. Anticipation that the cyano substituent on aniline 11q
would be susceptible to hydrogenation later in the synthesis necessitated the use of the diethyl
protected phosphonate 10, obtained from saponification of commercially available triethyl
4-phosphonobutyrate, for reaction with this aniline. With the exception of anilines 11i and 11l, all
other anilines used were commercially available. Synthesis of 11i (Scheme 2) started from 2-nitro-aniline
which was easily converted to the NH-Boc protected form as described by McNeil and Kelly [31].
Subsequent N,N-dimethylation, followed by Boc removal afforded the aniline. Compound 11l was
prepared from 2,6-dihydroxyaniline according to a literature procedure [32].
Anilines are often poor nucleophiles, thus carboxylic acids 9 and 10 were first converted to their
respective acid chlorides by treatment with oxalyl chloride before subsequent nucleophilic substitution
of 11a–m, 11q to generate a small library of the protected amides 12a–m, and 13q in moderate yields.
1
The H-NMR spectrum of 12c displays two peaks at 2.17 ppm and 2.21 ppm for the 2,6-dimethyl
protons corresponding to the E and Z amide rotamers in a 5/1 ratio. Hydrolysis of the tertiary butyl
ester group of 12j with TFA (20% in dichloromethane) further converted this intermediate to 12n.
Using benzyl protection for both the phosphonate and the aryl substituent (12k and 12l) allowed a mild