Binding modes of reverse fosmidomycin analogs toward the antimalarial target IspC

Article abstract of DOI:10.1021/jm500850y

1-Deoxy-d-xylulose 5-phosphate reductoisomerase of Plasmodium falciparum (PfIspC, PfDxr), believed to be the rate-limiting enzyme of the nonmevalonate pathway of isoprenoid biosynthesis (MEP pathway), is a clinically validated antimalarial target. The enzyme is efficiently inhibited by the natural product fosmidomycin. To gain new insights into the structure activity relationships of reverse fosmidomycin analogs, several reverse analogs of fosmidomycin were synthesized and biologically evaluated. The 4-methoxyphenyl substituted derivative 2c showed potent inhibition of PfIspC as well as of P. falciparum growth and was more than one order of magnitude more active than fosmidomycin. The binding modes of three new derivatives in complex with PfIspC, reduced nicotinamide adenine dinucleotide phosphate, and Mg²⁺ were determined by X-ray structure analysis. Notably, PfIspC selectively binds the S-enantiomers of the study compounds.

Full text of DOI:10.1021/jm500850y

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Article
Binding modes of reverse fosmidomycin
analogs towards the antimalarial target IspC
Sarah Konzuch, Tomonobu Umeda, Jana Held, Saskia Hähn, Karin Brücher, Claudia
Lienau, Christoph Behrendt, Tobias Gräwert, Adelbert Bacher, Boris Illarionov,
Markus Fischer, Benjamin Mordmüller, Nobutaka Tanaka, and Thomas Kurz
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500850y • Publication Date (Web): 25 Sep 2014
Downloaded from http://pubs.acs.org on October 9, 2014
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Binding modes of reverse fosmidomycin analogs
towards the antimalarial target IspC
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Sarah Konzuch,† Tomonobu Umeda,∥ Jana Held,# Saskia Hähn,† Karin Brücher,† Claudia Lienau,†
Christoph T. Behrendt, † Tobias Gräwert, § Adelbert Bacher, § Boris Illarionov,§ Markus Fischer,§
*,†
Benjamin Mordmüller,#, Nobutada Tanaka, ∥ and Thomas Kurz
∇
†Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universität, Universitätsstr. 1,
40225 Düsseldorf, Germany; ∥School of Pharmacy, Showa University, Tokyo 142ꢀ8555, Japan; #Institut
für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstr. 27, 72074 Tübingen, Germany;
§Hamburg School of Food Science, Universität Hamburg, Grindelallee 117, 20146 Hamburg, Germany;
and ∇Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
†
CORRESPONDING AUTHOR FOOTNOTE Institut für Pharmazeutische und Medizinische Chemie,
HeinrichꢀHeineꢀUniversität, Universitätsstr. 1, 40225 Düsseldorf, Germany. Phone: (+49)21181ꢀ14985,
Fax ꢀ13847, Eꢀmail: thomas.kurz@uniꢀduesseldorf.de.
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ABSTRACT 1ꢀDeoxyꢀ
Dꢀxylulose 5ꢀphosphate reductoisomerase of Plasmodium falciparum (PfIspC,
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PfDxr), believed to be the rateꢀlimiting enzyme of the nonꢀmevalonate pathway of isoprenoid
biosynthesis (MEP pathway), is a clinically validated antimalarial target. The enzyme is efficiently
inhibited by the natural product fosmidomycin. In order to gain new insights into the structure activity
relationships of reverse fosmidomycin analogs, several reverse analogs of fosmidomycin were
synthesized and biologically evaluated. The 4ꢀmethoxyphenyl substituted derivative 2c showed potent
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inhibition of PfIspC as well as of P. falciparum growth and was more than one order of magnitude more
active than fosmidomycin. The binding modes of three new derivatives in complex with PfIspC,
NADPH and Mg2+ were determined by Xꢀray structure analysis. Notably, PfIspC selectively binds the
Sꢀenantiomers of the study compounds.
INTRODUCTION
Despite considerable research activities, malaria remains one of the most widespread and lifeꢀ
threatening infectious disease of the world. Because of new drug combinations and improved vector
control the mortality rate has been reduced significantly since 2004, when the number of deaths peaked
at 1.8 million. However, the current situation is still alarming and global efforts are required to
accomplish the goal of malaria elimination. Despite increased funding and public awareness the WHO
reported 207 million estimated malaria cases and 627,000 estimated deaths in 2012. 1
Malaria control relies heavily on early diagnosis followed by antimalarial chemotherapy to prevent
death as well as potentially fatal complications such as severe anemia and coma. Malaria therapy is
compromised by current and emerging parasite resistance towards nearly all established antimalarials.2
In most countries with endemic malaria, fixedꢀdose artemisininꢀbased combination therapies have now
become firstꢀline treatments and parenteral artesunate has become the treatment of choice for severe
malaria. However, cases of reduced parasite sensitivity towards artemisinin derivatives have been
reported.3 Thus, antimalarial drugs with new modes of action are urgently needed.4,5
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A promising approach for the discovery of antimalarials is the inhibition of the nonꢀmevalonate
isoprenoid biosynthesis (MEP pathway).6,7 The MEP pathway is essential in the Plasmodium ssp.
causing malaria (and also in some other pathogenic protozoa and many pathogenic bacteria including
Mycobacterium tuberculosis and Pseudomonas aeruginosa), but is absent in mammalians.2,8 Hence,
drugs directed at enzymes of the nonꢀmevalonate pathway should be exempt from targetꢀrelated
toxicity.
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1ꢀDeoxyꢀ ꢀxylulose 5ꢀphosphate reductoisomerase (IspC, Dxr) catalyzes the first committed reaction
D
step of the nonꢀmevalonate pathway (Fig. 1) and has been validated as a malaria target by clinical
studies using fosmidomycin, an antibiotic from Streptomyces lavendulae. 4,9,10,11,12,13
O
P
OH
HO
HO
O
O
OH
DOXP
O
P
OH
N
HO
HO
H
IspC
O
Fosmidomycin (1)
O
HO
HO
P
HO
O
OH OH
MEP
Figure 1. Inhibition of IspC.
Although crystal structures of PfIspC in the presence and absence of inhibitors (fosmidomycin,
FR900098, an αꢀpyridylꢀsubstitited FR900098 analog and a reverse fosmidomycin βꢀthia isostere) were
recently reported, the rational design of improved fosmidomycin analogs is still difficult, because
PfIspC undergoes a complex conformational transition when binding the substrate, 1ꢀdeoxyxylulose 5ꢀ
phosphate (DOXP) or one of the inhibitors studied.14ꢀ16 Earlier studies mostly focused on EcIspC and
MtIspC crystal structures as the basis for the rational design of (Pf)IspC inhibitors.17ꢀ28
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In order to gain new insights regarding the structure activity relationships of reverse fosmidomycin
analogs, we have modified several key regions of lead structures A, B (Figure 2). Specifically, we
address the substitution pattern of the αꢀphenylꢀsubstituent (first reverse compounds with donor
substituents CH3, OCH3) (2a-f), the length (4) and the chemical functionality (βꢀoxa isosters 3a-b) of
the spacer as well as the nature of the metal ion chelating group (6, 7). Moreover we report the first
crystal structures of PfIspC in complex with reverse αꢀarylꢀsubstituted carbaꢀ and oxaꢀanalogs (2a, 2c,
3a).
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O
P
O
HO
HO
2a: R=Me, R'=Me, R''=H
2b: R=Me, R'=H, R''=H
OH
N
2c: R=OMe, R'=Me, R''=H
2d: R=OMe, R'=H, R''=H
2e: R=OMe, R'=Me, R''=OMe
2f: R=OMe, R'=H, R''=OMe
R'
R''
Carba analogs 2a-f
R
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O
HO
HO
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OH
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P
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P
N
HO
HO
OH
OH
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P
O
HO
HO
3a: R'=Me
3b: R'=H
X
OH
N
OCH3
Aryl
H/CH3
Phosphonic acid analog 7
β-Oxastere analogs 3a,b
Lead structures
A: X = CH2, B: X = O
HO
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P
O
O
P
CH3
N
HO
HO
HO
HO
N
OH
H
O
O
O
P
HO
HO
OH
N
o-Hydroxyanilide analog 6
Carba homolog 4
R'
HN
O
5a: R'=Me
5b: R'=H
Benzoylamino analogs 5a,b
Figure 2. Lead structures and target compounds.
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RESULTS
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Chemical synthesis.
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Carbaꢀanalogs and βꢀoxa isosteres. Carbaꢀanalogs (2a-d) and βꢀoxa isosteres (3a-b) of fosmidomycin
(Figure 2) were prepared according to previously published procedures (supporting information Scheme
S1).29,30 Carba analogs 2e-f were synthesized by a modified procedure starting from diethyl 3,4ꢀ
dimethoxybenzylphosphonate 8a.31 Briefly, Cꢀalkylation of 8a with 2ꢀ(2ꢀbromoethyl)ꢀ1,3ꢀdioxolane in
the presence of nꢀBuLi afforded 1,3ꢀdioxolane 9a. Hydrolysis of the 1,3ꢀdioxolane moiety of 9a by
treatment with water and Dowex 50WX8 provided aldehyde 10a,32 which was subsequently oxidized to
carboxylic acid 11a. Coupling reactions of 11a with Oꢀbenzylꢀhydroxylamine or NꢀmethylꢀOꢀ
benzylhydroxylamine furnished Oꢀbenzylꢀprotected hydroxamic acids 12a and 14a.33 Dealkylation of
diethyl phosphonates (12a, 14a) with bromotrimethylsilane provided the corresponding phosphonic
acids, which were directly converted into dibenzylphosphonates 13a and 15a using N,N’ꢀ
dicyclohexylcarbodiimide (DCC) and benzyl alcohol. Finally, catalytic hydrogenation afforded
hydroxamic acids 2e and 2f as white solids (Scheme 1).
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Scheme 1. Synthesis of target compounds 2e and 2fa
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O
P
O
O
P
O
O
P
O
P
O
EtO
EtO
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3
4
EtO
EtO
EtO
EtO
EtO
EtO
OH
H
O
a)
b)
c)
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6
OMe
OMe
OMe
OMe
7
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9
OMe
OMe
O
OMe
OMe
10a
11a
8a
9a
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O
O
O
N
O
O
BnO
BnO
HO
EtO
OBn
OH
OBn
P
P
P
N
N
HO
EtO
f)
e)
d)
OMe
O
OMe
OMe
O
2e
13a
12a
OMe
OMe
OMe
11a
O
P
O
O
P
O
P
HO
HO
EtO
EtO
BnO
BnO
OH
OBn
OBn
N
N
N
H
H
H
e)
f)
d)
OMe
OMe
OMe
OMe
OMe
OMe
15a
2f
14a
aReagents and conditions: a) nꢀBuLi, 2ꢀ(2ꢀbromoethyl)ꢀ1,3ꢀdioxolane, toluene, ꢀ78 °C, 12 h, 56 %; b)
Dowex 50WX8, acetone/H2O, rt, 24 h, 70 %; c) H2O2, SeO2, THF, 4 h, reflux, 90 %; d) isobutyl
chloroformate, NMM, BnONHR (R = H, Me), THF, ꢀ20 °C, 10 min → rt, 30 min, 50 % (12a), 65 %
(14a); e) 1. TMSBr, CH2Cl2, rt, 24 h; 2. THF/H2O, rt, 1 h, 3. DCC, BnOH, benzene, 80 °C, 4h, 18 %
(13a), 30 % (15a); f) H2, PdꢀC, MeOH, rt, 3 h, 95 % (2e), 98 % (2f).
Carba homolog 4. Alkylation of phosphonate 8b29,34 with 2ꢀ(3ꢀbromopropyl)ꢀ1,3ꢀdioxolane35 in the
presence of nꢀBuLi afforded the protected intermediate 9b. Acidic hydrolysis of the 1,3ꢀdioxolane
moiety provided aldehyde 10b, which was directly oxidized to carboxylic acid 11b. Coupling of the
crude carboxylic acid 11b with OꢀbenzylꢀNꢀmethylhydroxylamine utilizing 1,1´ꢀcarbonyldiimidazole
(CDI) as coupling agent provided Oꢀbenzylꢀprotected hydroxamic acid 12b in 74 % yield and good
purity. Cleavage of phosphonic ester 12b with bromotrimethylsilane yielded phosphonic acid 16b.
Finally, catalytic hydrogenation of crude 16b afforded the target hydroxamate 4 in 92 % yield (Scheme
2).
Scheme 2. Synthesis of compound 4a
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O
P
O
P
O
O
P
EtO
EtO
EtO
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7
8
EtO
EtO
EtO
OH
P
H
O
EtO
EtO
O
N
O
O
c)
a)
b)
11b
8b
10b
9b
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O
P
O
O
EtO
EtO
HO
HO
HO
N
P
N
P
OBn
e)
OBn
HO
OH
O
O
O
f)
d)
11b
4
12b
16b
aReagents and conditions: a) nꢀBuLi, 2ꢀ(3ꢀbromopropyl)ꢀ1,3ꢀdioxolane, toluene, ꢀ78 °C, 12 h, 77 %; b)
2 M HCl, acetone, 50 °C, 3 h, 77 %; c) SeO2, H2O2, THF, 65 °C, 4 h, 66 %; d) CDI, BnONHMe,
CH2Cl2, rt, 12 h, 85 %; e) 1. TMSBr, CH2Cl2, rt, 24 h; 2. THF/H2O, rt, 1 h, 81 % (crude yield); f) H2,
PdꢀC, MeOH, rt, 1 h, 92 %.
Benzoylamino analogs 5a-b. Treatment of αꢀaminophosphonate 2536 with benzoyl chloride and TEA
afforded benzamide 26 in 59 % yield. Conversion of 26 into hydroxamic acids 27, 28 was accomplished
by hydroxylaminolysis.37 Finally, cleavage of the diethylphosphonate moiety using
bromotrimethylsilane yielded derivatives 5a-b (Scheme 3).
Scheme 3. Synthesis of benzoylamino analogs 5aꢀba
O
P
O
O
P
O
EtO
EtO
HO
HO
OH
OH
N
N
HN
O
HN
O
b)
d)
O
P
O
EtO
EtO
27
5a
OEt
O
P
O
EtO
EtO
HN
O
a)
OEt
O
P
O
O
P
O
NH2
EtO
EtO
HO
HO
OH
OH
N
H
N
25
26
H
HN
O
HN
O
c)
d)
5b
28
aReagents and conditions: a) benzoyl chloride, TEA, CH2Cl2, rt, 12 h, 59 %; b) Nꢀ
methylhydroxylamine hydrochloride, NaOH, MeOH, 0 °C → rt, 12 h, 34 %; c) hydroxylamine
hydrochloride, NaOH, MeOH, 0 °C → rt, 30 min, 53 %; d) 1. TMSBr, CH2Cl2, rt, 24 h; 2. THF/H2O, rt,
1 h, 70 % (5a), 44 % (5b).
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oꢀHydroxyanilide 6. Carboxylic acid 2929 was converted into the corresponding acyl chloride which was
subsequently reacted with 2ꢀbenzyloxyaniline to provide anilide 30. Catalytic hydrogenation of 30
yielded the protected oꢀhydroxyanilide 31. Finally, cleavage of the phosphonic acid ester group using
bromotrimethylsilane led to target compound 6 with an oꢀhydroxyꢀanilide moiety as a potential metal
ion binding group (Scheme 4).
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Scheme 4. Synthesis of oꢀhydroxyanilide 6a
BnO
HO
O
P
O
O
P
O
O
P
O
EtO
EtO
EtO
EtO
EtO
EtO
OH
N
N
H
H
a)
b)
31
29
30
HO
O
O
HO
P
c)
HO
N
H
31
6
aReagents and conditions: a) 1. (COCl)2, DMF, toluene, rt, 1 h; 2. 2ꢀbenzyloxyaniline, pyridine,
CH2Cl2, rt, 12 h, 70 %; b) H2, PdꢀC (10 %), MeOH, 2 bar, rt, 3 h, 83 %; c) 1) TMSBr, CH2Cl2, rt, 24 h;
2) THF/H2O (~100:1), rt, 1 h, 52 %.
Synthesis of bisphosphonate 7. CꢀAlkylation of phosphonate 8e17,38 with excess of 1,2ꢀdibromoethane in
the presence of nꢀbutyllithium afforded bromide 32. MichaelisꢀArbuzov reaction of 32 with triethyl
phosphite provided phosphonic ester 33 (Scheme 5). Transesterification of 33 yielded the completely
OꢀBnꢀprotected derivative 34, which was finally deprotected by catalytic hydrogenation.
Scheme 5. Synthesis of target compound 7a
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O
P
O
P
O
P
O
P
O
P
O
P
BnO
BnO
OBn
OBn
EtO
EtO
OEt
OEt
EtO
EtO
EtO
1
2
3
4
Br
EtO
b)
c)
a)
5
6
7
32
33
8e
34
8
9
O
P
O
HO
HO
OH
OH
P
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d)
34
7
aReagents and conditions: a) nꢀBuLi, 1,2ꢀdibromoethane (4 eq), toluene, ꢀ78 °C, 12 h, 60 %; b)
triethyl phosphite, 160 °C, 1 h, 180 °C 12 h, 97 %; c) 1. TMSBr, CH2Cl2, rt, 24 h; 2. THF/H2O, rt, 1 h;
3. DCC, BnOH, benzene, 80 °C, 4 h, 16 % (3 steps); d) H2, PdꢀC, MeOH, 1 bar, rt, 3 h, 97 %.
Biological evaluation.
The study compounds were screened against IspC from P. falciparum (PfIspC), Escherichia coli
(EcIspC) and Mycobacterium tuberculosis (MtIspC) using a photometric assay that has been described
earlier.29 IC50 values were determined by nonlinear regression analysis using the program package
Dynafit.39 Curve shapes were closely similar in all cases. Typical examples are shown in Fig. 3 (top).
We also measured the growthꢀinhibitory effect of the study compounds against asexual stage P.
falciparum malaria parasites. Specifically, we used one chloroquineꢀsensitive and two multiꢀdrug
resistant P. falciparum strains of different geographic origin (3D7, Dd2 and FCR3). Parasite growth was
monitored by measuring expression of P. falciparum histidine rich protein 2 by enzymeꢀlinked
immunosorbent assay exactly as described previously.40 Reverse carba and oxa analogs (2, 3) are potent
P. falciparum growth inhibitors. Several new derivatives displayed IC50 values in the double digit
nanomolare range (Table 1). Typical examples (same compounds as in Fig. 3, top) are shown as the
bottom part of Fig. 3. It should be noted that the parasite assay curves are typically descending faster
than the cognate enzyme inhibition curves.
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60
Figure 3. Top, inhibition of IspC orthologs (PfIspC, red; EcIspC, blue; MtIspC, green). Bottom,
inhibiton of P. falciparum blood stages; left, compound 2c, right, compound 2d.).
Numerical data for all compounds studied are summarized in Table 1.
Table 1. Enzyme inhibition and in vitro antiplasmodial activity.
Ar
R‘ PfIspCa
EcIspCa
MtIspCa
Pf3D7b
PfDd2b,f
PfFCR3b, f
IC50 [µM]
n.d.
IC50 [µM] IC50 [µM] IC50 [µM] IC50 [µM] IC50 [µM]
0.16
0.22
0.23
± 0.02e
1.8
0.88d
0.21
2.6
0.81 d
0.25
2.8
1
± 0.02c
± 0.01 d
4ꢀMeꢀPh
4ꢀMeꢀPh
Me 0.01
0.29
0.38
n.d.
2a
2b
± 0.002
0.19
± 0.02
0.70
± 0.1
11
H
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± 0.01
Me 0.02
± 0.1
0.07
± 0.003
0.14
± 0.01
0.32
± 0.01
1.5
± 1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
4ꢀMeOꢀPh
4ꢀMeOꢀPh
2.0
0.10
1.7
0.30
3.6
0.57
n.d.
n.d.
n.d.
0.41
n.d.
n.d.
n.d.
n.d.
2c
± 0.002
0.08
± 0.1
15
H
2d
± 0.008
± 1.0
29
3,4ꢀMeOꢀPh Me 0.06
± 0.006
0.13
± 0.02
Me 0.05
± 0.004
2.2
0.29
6.8
1.2
2e
± 1.0
163
3,4ꢀMeOꢀPh
4ꢀMeOꢀPh
4ꢀMeOꢀPh
Ph
H
8.1
2f
3a
3b
4
15
16
17
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21
22
23
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60
± 0.1
0.45
± 0.04
17
± 34
2.8
0.31
> 50
> 50
> 50
> 50
1.1
± 2.0
352
H
n.d.
n.d.
n.d.
n.d.
± 0.1
2.1
± 1.0
4.3
± 29
429
ꢀ
± 0.1
± 0.1
11
± 29
238
NHꢀCOꢀPh
NHꢀCOꢀPh
Me 9.1
± 1.3
5a
5b
± 0
± 21
> 1000
H
> 1000
741
± 94
> 500
> 500
Ph
ꢀ
ꢀ
> 300
> 300
> 500
> 500
> 50
> 50
n.d.
n.d.
n.d.
n.d.
6
7
1ꢀNaphthyl
aEnzyme assay. Values were calculated from eight or more data points. In general two or three
independent determinations have been performed.39,41 bIn vitro assay. Values are the mean of two
c
d
e
duplicate determinations. IC50 value according to ref 16. IC50 value according to ref 15. IC50 value
f
according to ref 13. n.d., not determined. Details regarding the structure of the compounds can be
found in Figure 2.
The inhibitors under study are more active against IspC from P. falciparum than against the bacterial
orthologs (Fig. S1, Fig. 4A). Notably, the inhibition of the M. tuberculosis enzyme exceeds that of the
Plasmodium enzyme by about two orders of magnitude. This is well in line with earlier observations
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indicating that αꢀaryl analogs of fosmidomycin are more potent inhibitors for the plasmodial enzyme
than for the bacterial enzymes that have been included in comparative studies.14,30 In contrast to the
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differential activities of the study compounds, authentic fosmidomycin showed similar inhibition of the
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IspC from P. falciparum and the bacterial enzymes (Table 1, Fig. S1).
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Figure 4. Top, ortholog specificity of IspC inhibition; the ordinate reflects the IC50 ratio of eubacterial
IspCs and PfIspC; the inhibitory potency of the study compounds for PfIspC exceeds that for MtIspC by
about two orders of magnitude (blue diamonds) and that for EcIspC by close to one order of magnitude.
Bottom, activity (IC50 values) against blood stages of two different P. falciparum strains versus in vitro
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inhibition of PfIspC (IC50 values); both test strains show similar responses; the sensitivity of the isolated
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enzyme exceeds that of the growing parasite by about one order of magnitude.
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According to Fig. 4B, the IC50 values of the study compounds for the enzymatic target, PfIspC, are
lower, by approximately one order of magnitude, than the IC50 values observed in asexual blood stages.
On the other hand, due to the different curve shapes illustrated in Fig. 3, the inhibitor concentrations
required for 90 % inhibition are similar in the enzyme and parasite assays.
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The most active compounds in Table 1 (carba analogs 2a, 2c) had IC50 values in the low nanomolecular
range for the plasmodial enzyme, and exceed the inhibitory effect of authentic fosmidomycin to a
significant degree (Table 1). Extending the chain length (4) caused the inhibitory activity to drop by
several orders of magnitude. The replacement of the Me2+ binding hydroxamic acid pharmacophore by
other potentially metal ion binding groups e.g. an oꢀhydroxyanilide moiety (6) and a phosphonic acid
group (7) led to complete loss of inhibitory activity .
Crystal structure analysis.
Cocrystallization of PfIspC with NADPH, Mg2+ and reverse fosmidomycin analogs (2a, 2c, and 3a)
afforded crystals with space group P21 for 2a and 2c complexes and P3121 for the 3a complex
(diffraction to resolutions of 2.25, 1.97, and 2.35 Å (Table S1)). Structures were solved by molecular
replacement using the structure of IspC in complex with NADPH, Mg2+ and fosmidomycin (PDB
accession code 3AU9) as search model.
The asymmetric units of crystals of 2a and 2c complexes comprise two IspC subunits, whereas that of
the 3a-complex comprises one subunit. The subunit in the asymmetric unit of the crystal containing 3a
is related by a crystallographic 2ꢀfold axis to form a homodimer. The structural differences between two
subunits in the asymmetric unit of 2a and 2c complexes are described in the “Structure determination”
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section of the Supporting Information. For simplicity, the following description refers primarily to
subunit B of the 2c complex.
Page 14 of 33
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The active site structure of the quaternary complexes reported in this paper show significant differences
from those of previously published quaternary (fosmidomycinꢀ or FR900098ꢀcontaining) complexes of
PfIspC,15 although tertiary (threeꢀdomain structure) and quaternary (homo dimer) structures are
conserved (Fig. 5a). The 2c molecule is located in the active site cavity (Figure 5a).
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The αꢀaryl substituent has van der Waals contacts with active site residues; specifically, the side chains
of Ser270, Cys338, and Pro358 are well ordered, whereas Trp296 and Met298 showed higher
temperature factors (Figure 5b). In addition, an intraꢀmolecular interaction is observed between the Nꢀ
methyl group and the aromatic ring of 2c. The Nꢀmethyl group has a contact with the side chain of
Met360. A comparison of the binding mode of 2c with that of fosmidomycin reveals that in a closed
conformation, as observed with Trp296 in the fosmidomycin complex, Trp296 would crash with the αꢀ
aryl substituent of 2c (Figure 5c). In fact, the flexible loop region (residues 291ꢀ299) in the 2c complex
adopts a relatively open conformation. As compared with the fosmidomycin complex, the flexible loop
of the 2c complex is rather disordered (poorer electron density and higher Bꢀfactors). Therefore, the
cause of the tight binding of 2c to PfIspC appears to be different from that of fosmidomycin to PfIspC,
but similar to that of the 3,4ꢀdifluorophenyl analog17 to EcIspC. Recently, we have reported the crystal
structure of PfIspC in complex with an αꢀphenyl substituted reverse thia analog of fosmidomycin.14 The
molecular interaction between 2c and PfIspC is similar to that between the thia analog and PfIspC,
however, significant conformational differences are observed for the flexible loop region.
The phosphonate group of 2c forms a tight hydrogenꢀbond network with the main chain NH and side
chain OH of Ser270, the side chain of Asn311, two water molecules, and the side chain of Ser306
(Figure 6a). In the fosmidomycin:IspC complex, the side chain of His293 is involved in the hydrogenꢀ
bond network instead of Ser306 (Figures 5c and 6b).
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The present crystal structures are the first examples of quaternary complexes of IspC with reverse carba
and oxa analogs. The reverseꢀhydroxamate group coordinates an Mg2+ ion that is bound by residues
Asp231, Glu233, and Glu315. Thus, the Mg2+ ion has distorted trigonal bipyramidal geometry. This
metal coordination geometry is consistent with that of the hydroxamate group of fosmidomycin
observed in the previously published quaternary complexes of PfIspC,14,15 but different from that of the
reverseꢀhydroxamate group observed in the ternary complex of E. coli IspC crystallized in the absence
of NADPH.17 The octahedral sixfold metal ion coordination observed in the ternary complex of
EcIspC (two inhibitor atoms, three protein ligands, and a water molecule) is unlikely to occur in the
quaternary complex of PfIspC because the water molecule would crash with the nicotinamide ring of
NADPH. Comparing a possible hydrogenꢀbond network of the 2c complex and the fosmidomycin (or
FR900098) complex (Figures 6a and 6b), the metal coordination abilities and hydrogenꢀbond network
with surrounding residues are essentially equivalent. This is consistent with the similar inhibitory
activities of fosmidomycin and a reverseꢀhydroxamate analog reported by Kuntz.42
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To examine differences in inhibitorꢀinduced conformation changes dependent on the type of inhibitors,
the crystal structures of PfIspC complexed with 2a (αꢀPhꢀCH3), 2c (αꢀPhꢀOCH3), and fosmidomycin
were compared (Figure 5d). It is interesting that the degree of loop closure is related to the size of the
bound inhibitor. This knowledge concerning the inducedꢀfit mechanism of PfIspC will be useful for
designing new inhibitors. In addition, the flexible loop of 2a complex is also rather disordered as
compared with that of the fosmidomycin complex. Thus, the primary factor of the tight binding of 2a
and 2c to PfIspC as compared with that of fosmidomycin which lacks the aryl group would be van der
Waals interactions between the αꢀaryl group and the core of the active site rather than the flexible loop
(Figures 6a and 6b).
In the present crystal structure analyses, we also determined a PfIspCꢀNADPHꢀMg2+-3a quaternary
complex. The 3a complex is quite similar to the 2c complex. The current structureꢀactivity relationship
data (Table 1) show that βꢀoxa compounds have somewhat lower inhibitory activities than their carba
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counterparts. This is explained by structural comparison of the 2c complex with the fosmidomycin
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complex (Figure 5c). Due to the presence of a bulky substituent at the αꢀposition, the methylene spacer
of 2c adopts a different conformation as compared with that of fosmidomycin. The methylene spacer of
3a adopts a conformation that is equivalent with that of 2c. In the fosmidomycin complex, the βꢀ
position of the carbon spacer is accommodated in a hydrophilic environment where it interacts with the
side chains of Glu233, Lys312 and a water molecule, whereas the βꢀposition of inhibitors 2c and 3a is
accommodated in a hydrophobic environment and interacts with the Cβ of Ser306 and the Cε of Met298.
Therefore, the βꢀoxygen of αꢀsubstituted oxa derivatives may have somewhat unfavorable interactions
with the active site as compared with their carba counterparts.
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Figure 5. Crystal structure analyses of PfIspC. (a) Overall structure of the 2cꢀcontaining quaternary
complex of PfIspC. One subunit is colored by domains: the NADPHꢀbinding, catalytic, linker, and Cꢀ
terminal domains are depicted in blue, green, yellow, and red, respectively. The other subunit is colored
cyan. The bound 2c and NADPH molecules are shown as sticks. The bound Mg2+ ions are shown as
spheres. (b) Closeꢀup view of the active site with bound 2c. The side chains surrounding the αꢀaryl
substituent are shown and the carbon atoms are colored by their Bꢀfactors: cyan (15~25 Å2), green
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(25~35 Å2), and yellow (35~45 Å2). Note that the side chains belonging to the flexible loop, Trp296 and
Met298, show higher Bꢀfactors. (c) A structural comparison of the binding mode of 2c (grey) and that of
fosmidomycin (green) (PDB code 3AU9) in the active site of PfIspC. The βꢀpositions of the methylene
spacer of the bound inhibitors are marked by arrowheads. The bound inhibitor molecules are shown as
ballꢀandꢀstick models. (d) A comparison of the inducedꢀfit movements of the flexible loop (residues
291ꢀ299) of PfIspC. The 2aꢀ, 2cꢀ, and fosmidomycinꢀbound quaternary complexes of PfIspC are shown
in blue, red, and green, respectively.
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Figure 6. Schematic overview of the interactions of inhibitors in the active site of PfIspC. Metal
coordination (2.0~2.1 Å) and possible hydrogen bonds (2.7~3.1 Å) are shown as solid and dashed lines,
respectively. Intraꢀ and intermolecular van der Waals contacts are shown as thin and thick gray arcs,
respectively. (a) 2c complex. Residues uniquely involved in direct interactions with the bound inhibitor
in the 2c complex are shown in blue. (b) Fosmidomycin (R = H) / FR900098 (R=CH3) complex.
Residues uniquely involved in direct interactions with the bound inhibitor in the fosmidomycin /
FR900098 complex are shown in green.
DISCUSSION
Using IspC orthologs from the protozoon, P. falciparum, and two eubacteria, we found that the shapes
of the inhibition curves are all closely similar (cf. Fig. 3). There is no sign whatsoever of positive or
negative cooperativity.
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Whereas the natural product fosmidomycin inhibits IspC orthologs from P. falciparum, E. coli and M.
tuberculosis with similar potency, our data confirm earlier observations that αꢀaryl derivatives are
significantly more potent for the Plasmodium enzyme than for the eubacterial orthologs. With PfIspC,
the most potent αꢀaryl derivatives that have been described have IC50 values in the low nanomolar range.
In terms of their IC50, the most potent αꢀaryl derivatives exceed the inhibitory potential of the natural
compound, fosmidomycin, by more than an order of magnitude. A potential explanation for the higher
inhibitory activities of αꢀaryl derivatives against PfIspC is that the difference in structural flexibilities of
IspC molecules, i.e. difference in interꢀdomain and activeꢀsite loop flexibilities, may affect the
inhibitory activities of bulky inhibitors. Another explanation is that the side chain of Cys338 in PfIspC
would have a favorable van der Waals contact with the αꢀaryl group of the inhibitors (Figure 5b) as
compared with structurally corresponding serine residue in EcIspC and MtIspC.
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The IC50 values observed with the Plasmodium enzyme and with multiplying asexual P. falciparum
blood stages are remarkably well correlated; the IC50 values observed for the parasite blood stage are
typically one to two orders larger than the IC50 values observed with PfIspC in vitro. Since the inhibitors
must pass through numerous membrane barriers (the erythrocyte membrane, the parasitophorous
vacuole membrane, the cell membrane of the parasite and the four membranes of the apicoplast) in
order to reach their enzyme target in the living parasite, this may not be particularly surprising.
Importantly, however, the inhibition curves observed with the isolated enzyme and the growing parasite
are remarkably different in so far as the latter are much steeper. Whereas the IC50 values for the isolated
enzyme and the growing parasite are offset by one to two units on the log scale, the difference of
inhibitory efficacy between enzyme assay and parasite assay essentially vanishes at higher inhibitor
concentrations, as a result of the different curve shapes. We can only speculate with regard to the causes
of the curve shapes. However, there is no reason to expect a simple parallelism between IspC inhibition
and Plasmodium proliferation. Quite possibly, growth retardation may only become relevant when the
enzyme activity is depressed below a certain threshold value; such a situation could translate into a steep
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slope for the parasite growth curve. Of course, under the aspect of drug development, the minimal
inhibitory concentration is far more relevant than the IC50, and the steeper shape of the parasite growth
curves should turn out in favor of a potential therapeutic application of modified fosmidomycin
derivatives.
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Recently, we could show that PfIspC has a high degree of enantioselectivity for an αꢀarylated βꢀthia
analog of fosmidomycin (inhibition by the Sꢀenantiomer is at least three orders of magnitude stronger as
compared with the Rꢀenantiomer). Pure enantiomers of the compounds reported in this paper have
unfortunately not been obtained as yet. However, the Sꢀconfiguration of the bound inhibitors is the
preferred interpretation in this study (Fig. S2) in agreement with previous studies for αꢀaryl substituted
fosmidomycin derivatives.14,17,26,27 The clear omit map at 1.97 Å resolution for 2c (Fig. S2(b)) does not
look like a mixture of both enantiomers. The Xꢀray structure data strongly suggest that the Sꢀ
enantiomers of the compounds in this study bind preferentially to the active site of PfIspC. This will
hopefully translate into an increase of apparent inhibitory power of the study compounds, once the
preparation of pure enantiomers is achieved.
CONCLUSION
We report on kinetic and crystallographic proof for the mechanism of action of a new series of reverse
analogs of fosmidomycin. By modifying different key regions of the lead structure we provided new
insights into the structureꢀactivity relationships of reverse fosmidomycin derivatives. The 4ꢀmethoxyꢀ
phenyl substituted derivative 2c showed potent inhibition of PfIspC as well as of P. falciparum growth
and exceeds the inhibitor activity of fosmidomycin by more than one order of magnitude more.
Crystallographic studies also demonstrated that PfIspC binds selectively the Sꢀenantiomers of αꢀaryl
substituted carba and oxa analogs of fosmidomycin. Our results provide helpful information for the
design of novel IspC inhibitors.
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EXPERIMENTAL SECTION
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All solvents and chemicals were used as purchased without further purification. The progress of all
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reactions was monitored on Merck precoated silica gel plates (with fluorescence indicator UV254) using
ethyl acetate / nꢀhexane as solvent system. Column chromatography was performed with Fluka silica gel
60 (230ꢀ400 mesh ASTM) with the solvent mixtures specified in the corresponding experiment. Spots
were visualized by irradiation with ultraviolet light (254 nm). Melting points (mp) were taken in open
capillaries using a Mettler FP 5 meltingꢀpoint apparatus or a Stuart melting point apparatus SMP11 and
are uncorrected. Proton (1H) and carbon (13C) NMR spectra were recorded using Bruker Avance 500
(500.13 MHz for 1H; 125.76 MHz for 13C) or Bruker Avance 600 (600.22 MHz for 1H; 150.93 MHz for
13C) spectrometers using [D6]DMSO and CDCl3 as solvents. Chemical shifts are given in parts per
million (ppm, δ relative to residual solvent peak for 1H and 13C or to external tetramethylsilane).
Elemental analysis was performed with a Perkin Elmer PE 2400 CHN elemental analyser or a vario
MICRO cube elemental analyser (Elementar Analysensysteme GmbH, Hanau, Germany). If necessary,
the purity was determined by high performance liquid chromatography (HPLC). HPLC was performed
in analogy to a previously reported procedure.43 The Instrument was an Elite LaChrom system (Hitachi
Lꢀ2130 pump and Lꢀ2400 UVꢀdetector) or a Varian ProStar HPLC System (Varian ProStar 210 pump,
Varian ProStar 320 UVꢀdetector and Varian ProStar 410 autosampler). The column was a Phenomenex
Luna Cꢀ18(2) 5 ꢀm particle (250 mm × 4.6 mm), supported by Phenomenex Security Guard Cartridge
Kit C18 (4.0 mm × 3.0 mm). Purity of all final compounds was 95 % or higher, except compound 6
(purity 92.2 %).
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Experimental Data for Compounds. Experimental data are listed below for selected compounds
9aꢀ15a, 2e and 2f.
General procedure for the synthesis of dioxolanes (9a-d).29 At a temperature of ꢀ78 °C, a 1.6 M
solution of nꢀbutyllithium in nꢀhexane (1.1 eq, 44 mmol, 27.5 mL) was slowly added to a solution of the
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respective diethyl arylmethylphosphonate 8a-d (1 eq, 40 mmol) in dry toluene (50 mL) via a syringe
under positive pressure of dry nitrogen. After stirring for 1 h, 2ꢀ(2ꢀbromoethyl)ꢀ1,3ꢀdioxolane or 2ꢀ(3ꢀ
bromopropyl)ꢀ1,3ꢀdioxolane (1 eq, 40 mmol) was added in one portion. The reaction mixture was
allowed to warm up to room temperature and stirred overnight. Ethyl acetate (50 mL) was added and the
solution was washed three times with an aqueous solution of NH4Cl (10 %, 50 mL). The organic layer
was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude oil was purified by
column chromatography on silica gel using ethyl acetate / nꢀhexane (1:1) as the eluent to give
compounds 9a-d as colorless oils.
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Diethyl 1-(3,4-dimethoxyphenyl)-3-(1,3-dioxolan-2-yl)propylphosphonate (9a). Colorless oil (13.05
g, 84 %); 1H NMR (500.13 MHz, [D6]DMSO): δ = 1.05 (t, J = 7.0 Hz, 3H, CH2CH3), 1.21 (t, J = 7.0
Hz, 3H, CH2CH3), 1.32ꢀ1.39 (m, 1H, CH2), 1.43ꢀ1.50 (m, 1H, CH2), 1.78ꢀ1.88 (m, 1H, CH2), 1.96ꢀ2.05
(m, 1H, CH2), 3.09 (ddd, J = 21.8, 11.4, 4.0 Hz, 1H, PCH), 3.68ꢀ3.76 (m, 9H, OCH3, CHOOCH2), 3.80ꢀ
3.87 (m, 3H, CH2CH3, CHOOCH2), 3.92ꢀ4.01 (m, 2H, CH2CH3), 4.74 (t, J = 4.6 Hz, 1H, CHOO), 6.81ꢀ
6.83 (m, 1H), 6.89ꢀ6.91 (m, 2H) ppm; 13C NMR (125.76 MHz, [D6]DMSO): δ = 16.0 (d, 3JCꢀP = 5.5 Hz,
CH2CH3), 16.2 (d, 3JCꢀP = 5.5 Hz, CH2CH3), 24.0 (CH2CH2), 31.1 (d, 2JCꢀP = 14.7 Hz, CHCH2), 41.9 (d,
1JCꢀP = 136.8 Hz, PCH), 55.3 (OCH3), 55.4 (OCH3), 61.0 (d, 2JCꢀP = 7.1 Hz, CH2CH3), 61.5 (d, 2JCꢀP
7.0 Hz, CH2CH3), 64.1 (CHOOCH2), 64.2 (CHOOCH2), 103.0 (CHOO), 111.6, 112.9 (d, 3JCꢀP = 6.4
=
Hz), 121.1 (d, 2JCꢀP = 7.4 Hz), 128.3 (d, 3JCꢀP = 6.6 Hz), 147.7, 148.3 ppm; anal. calcd. for C18H29O7P: C
55.66, H 7.53, found: C 55.29, H 7.89.
Diethyl 1-(3,4-dimethoxyphenyl)-4-oxobutylphosphonate (10a).32 Dowex 50WX8 (3mL) was added
to a solution of dioxolane 9a (14.5 mmol, 5.63 g) in water/acetone (10:1, 5 mL), . After stirring at room
temperature for 48 h, the solution was filtered. The filtrate was concentrated, diluted with saturated
aqueous NaCl solution and extracted three times with ethyl acetate (100 mL). The organic layer was
dried over Na2SO4 and concentrated under reduced pressure. Column chromatography on silica gel
using ethyl acetate gave compound 10a as a colorless oil (3.25 g, 65 %); 1H NMR (500.13 MHz,
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[D6]DMSO): δ = 1.05 (t, J = 7.0 Hz, 3H, CH2CH3), 1.22 (t, J = 7.0 Hz, 3H, CH2CH3), 1.95ꢀ2.05 (m, 1H,
CH2), 2.15ꢀ2.23 (m, 1H, CH2), 2.25ꢀ2.39 (m, 2H, CH2), 3.09 (ddd, J = 21.8, 11.3, 4.2 Hz, 1H, PCH),
3.72 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.80ꢀ3.90 (m, 2H, CH2CH3), 3.93ꢀ4.03 (m, 2H, CH2CH3),
6.79ꢀ6.82 (m, 1H), 6.88ꢀ6.91 (m, 2H), 9.55 (s, 1H, CHO); 13C NMR (125.76 MHz, [D6]DMSO): δ =
16.1 (d, 3JCꢀP = 5.6 Hz, CH2CH3), 16.2 (d, 3JCꢀP = 5.5 Hz, CH2CH3), 22.3 (CH2CH2), 40.9 (d, 2JCꢀP = 14.8
Hz, CHCH2), 41.5 (d, 1JCꢀP = 137.0 Hz, PCH), 55.3 (OCH3), 55.4 (OCH3), 61.2 (d, 2JCꢀP = 7.2 Hz,
CH2CH3), 61.6 (d, 2JCꢀP = 7.0 Hz, CH2CH3), 111.6, 112.7 (d, 3JCꢀP = 6.3 Hz), 121.2 (d, 2JCꢀP = 7.3 Hz),
127.8 (d, 3JCꢀP = 6.6 Hz), 147.8, 148.4, 202.6 (CHO); anal. calcd. for C16H25O6P: C 55.81, H 7.32,
found: C 55.79, H 7.39.
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General procedure for the synthesis of carboxylic acids (11a-d).29 To a solution of the respective
aldehyde 10a-d (1 eq, 5 mmol) in THF (7.5 mL), H2O2 (1.1 mL of a 30 % solution) and SeO2 (0.5 eq,
2.5 mmol, 0.28 g) were added. The solution was refluxed for 4 h and the reaction mixture was
concentrated under reduced pressure. Ethyl acetate (50 mL) was added to the remaining oil and the
resulting solution was washed three times with 1 M hydrochloric acid (10 mL). After drying over
Na2SO4, the organic solvent was removed under reduced pressure. The crude products 11c-d were
treated with diethyl ether (20 mL) and stored at 7 °C to give compounds 11c-d as white solids. Pure
compounds 11a and 11b were obtained without further purification.
4-(Diethoxyphosphoryl)-4-(3,4-dimethoxyphenyl)butanoic acid (11a). Yellow resin (1.58 g, 88 %);
1H NMR (500.13 MHz, [D6]DMSO): δ = 1.06 (t, J = 7.0 Hz, 3H, CH2CH3), 1.22 (t, J = 7.0 Hz, 3H,
CH2CH3), 1.91ꢀ2.01 (m, 1H, CH2), 2.06ꢀ2.10 (m, 2H, CH2), 2.13ꢀ2.19 (m, 1H, CH2), 3.10 (ddd, J =
21.8, 11.1, 4.0 Hz, 1H, PCH), 3.73 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.78ꢀ3.89 (m, 1H, CH2CH3),
3.93ꢀ4.07 (m, 3H, CH2CH3), 6.80ꢀ6.81 (m, 1H), 6.87 (s, 1H), 6.91 (d, J = 8.3 Hz, 1H), 12.10 (s, 1H,
COOH); 13C NMR (125.76 MHz, [D6]DMSO): δ = 16.0 (d, 3JCꢀP = 5.5 Hz, CH2CH3), 16.2 (d, 3JCꢀP = 5.5
Hz, CH2CH3), 25.0 (CH2CH2), 41.3 (d, 2JCꢀP = 15.4 Hz, CHCH2), 41.5 (d, 1JCꢀP = 137.7 Hz, PCH), 55.3
(OCH3), 55.4 (OCH3), 61.1 (d, 2JCꢀP = 7.1 Hz, CH2CH3), 61.6 (d, 2JCꢀP = 6.8 Hz, CH2CH3), 111.6, 112.7
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(d, 3JCꢀP = 6.3 Hz), 121.2 (d, 2JCꢀP = 7.3 Hz), 127.9 (d, 3JCꢀP = 6.7 Hz), 147.8, 148.4, 173.6 (COOH);
HPLC analysis: retention time = 1.84 min; peak area: 93.3 %. Eluent A: 5 mM NH4OAc solution; eluent
B: CH3CN, isocratic (1:1) over 20 min at a flow rate of 1 mL minꢀ1.
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General procedure for the synthesis of O-Bn-protected hydroxamic acids (12a, 14a).33 To a solution
of the carboxylic acid 11a (1 eq, 5 mmol, 1.80 g) in dry THF (50 mL) Nꢀmethylmorpholin (NMM) (1.1
eq, 5.5 mmol, 506 mg) was added. The solution was cooled down to ꢀ20 °C and isobutyl chloroformate
(1.1 eq, 5.5 mmol, 751 mg) was added. After 10 min, the appropriate hydroxylamine (1 eq, 5 mmol)
was added dropwise. The reaction mixture was warmed up to room temperature overnight. The
precipitated NMMꢀhydrochloride was filtered off and the filtrate was concentrated under reduced
pressure. The residue was dissolved in H2O / ethyl acetate and the aqueous layer was extracted three
times with ethyl acetate (50 mL). The organic layers were combined and washed twice with a saturated
aqueous solution of NaHCO3 (10 mL), dried over Na2SO4, filtered and concentrated under reduced
pressure. The crude product was purified by column chromatography on silica gel using ethyl
acetate/nꢀhexane (1:1) as the eluent to give compounds 12a and 14a as yellow oils.
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Diethyl 4-[benzyloxy(methyl)amino]-1-(3,4-dimethoxyphenyl)-4-oxobutylphosphonate (12a).
Yellow oil (1.53 g, 64 %); 1H NMR (600.22 MHz, [D6]DMSO): δ = 1.05 (t, J = 7.0 Hz, 3H, CH2CH3),
1.21 (t, J = 7.0 Hz, 3H, CH2CH3), 1.90ꢀ1.97 (m, 1H, CH2), 2.13ꢀ2.28 (m, 3H, CH2), 3.10ꢀ3.14 (m, 4H,
PCH, NCH3), 3.71ꢀ3.77 (m, 7H, OCH3, CH2CH3), 3.80ꢀ3.86 (m, 1H, CH2CH3), 3.94ꢀ4.01 (m, 2H,
CH2CH3), 4.66 (dd, J = 22.5, 9.9 Hz, 2H, PhCH2), 6.82 (d, J = 8.2 Hz,1H), 6.88 (s, 1H), 6.92 (d, J = 8.2
Hz), 7.17ꢀ7.18 (m, 2H), 7.30ꢀ7.36 (m, 3H) ppm; 13C NMR (150.93 MHz, [D6]DMSO): δ = 16.0 (d, 3JCꢀP
= 5.5 Hz, CH2CH3), 16.2 (d, 3JCꢀP = 5.5 Hz, CH2CH3), 24.6 (CH2CH2), 29.2 (d, 2JCꢀP = 16.2 Hz,
CHCH2), 32.6 (NCH3), 41.7 (d, 1JCꢀP = 137.2 Hz, PCH), 55.3 (OCH3), 55.4 (OCH3), 61.1 (d, 2JCꢀP = 7.0
Hz, CH2CH3), 61.5 (d, 2JCꢀP = 6.9 Hz, CH2CH3), 75.1 (PhCH2), 111.6, 112.8 (d, 3JCꢀP = 6.4 Hz), 121.2
(d, 2JCꢀP = 7.4 Hz), 128.1 (d, 3JCꢀP = 6.7 Hz), 128.3, 128.6, 129.3, 147.8 (d, 5JCꢀP = 2.3 Hz), 148.4 (d, 4JCꢀP
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= 1.7 Hz), 173.0 (C=O) ppm; anal. calcd. for C24H34NO7P: C 60.12, H 7.15, N 2.92, found: C 60.37, H
7.31, N 2.65.
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Diethyl 4-(benzyloxyamino)-1-(3,4-dimethoxyphenyl)-4-oxobutylphosphonate (14a). Yellow oil
(1.07 g, 46 %); 1H NMR (500.13 MHz, [D6]DMSO): δ = 1.05 (t, J = 7.0 Hz, 3H, CH2CH3), 1.22 (t, J =
7.0 Hz, 3H, CH2CH3), 1.82 (t, J = 7.5 Hz, 2H, CH2), 1.91ꢀ1.99 (m, 1H,CH2), 2.12ꢀ2.21 (m, 1H, CH2),
3.05 (ddd, J = 21.8, 11.3, 3.5 Hz, 1H, PCH), 3.72 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.79ꢀ3.87 (m, 2H,
CH2CH3), 3.93ꢀ4.00 (m, 2H, CH2CH3), 4.74 (s, 2H, PhCH2), 6.78 (d, J = 8.2 Hz, 1H), 6.84 (s, 1H), 6.89
(d, J = 8.3 Hz, 1H), 10.51 (s, 0.1H, Eꢀisomer, OH), 10.87 (s, 0.9H, Zꢀisomer, OH) ppm; 13C NMR
(125.76 MHz, [D6]DMSO): δ = 16.1 (d, 3JCꢀP = 5.6 Hz, CH2CH3), 16.2 (d, 3JCꢀP = 5.4 Hz, CH2CH3),
25.3 (CH2CH2), 29.9 (CHCH2), 41.7 (d, 1JCꢀP = 136.7 Hz, PCH), 55.3 (OCH3), 55.4 (OCH3), 61.2 (d,
2JCꢀP = 6.9 Hz, CH2CH3), 61.6 (d, 2JCꢀP = 6.7 Hz, CH2CH3), 76.6 (PhCH2), 111.5, 112.7 (d, 3JCꢀP = 6.3
Hz), 121.2 (d, 3JCꢀP = 7.0 Hz), 127.8 (d, 2JCꢀP = 6.7 Hz), 128.1, 128.2, 128.6, 136.0, 147.7 (d, 5JCꢀP = 2.8
Hz), 148.3, 168.5 (C=O) ppm; anal. calcd. for C23H32NO7P: C 59.35, H 6.93, N 3.01, found: C 59.08, H
7.22, N 2.82.
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General procedure for the synthesis of tri-O-benzyl protected phosphonohydroxamic acids
(15a,c,d, 13a, 24, 34).29 To a solution of the respective phosphonic acid diethyl ester (14a,c,d, 12a, 23,
33) (1 eq, 3 mmol) in dry CH2Cl2 (10 mL), trimethylsilyl bromide (5 eq, 15 mmol, 1.99 mL in case of
compounds 15a,c,d, 13a, 24; 10 eq, 30 mmol, 3.98 mL in case of compound 34) was added at 0 °C.
After 1 h, the solution was allowed to warm up to room temperature and stirred for 23 h. The solvent
was removed under reduced pressure. The residue was dissolved in THF (10 mL) and treated with water
(0.1 mL). After 30 min, the solvent was evaporated and the residue was dried under reduced pressure
overnight. The residue was dissolved in benzene (20 mL). 2ꢀBenzylꢀ1,3ꢀdicyclohexylꢀisourea (2 eq,
6 mmol, 1.89 g in case of compounds 15a,c,d, 13a, 24; 4 eq, 12 mmol, 3.78 g in case of compound 34)
was added. The mixture was refluxed for 6 h. After cooling to room temperature, ethyl acetate (30 mL)
was added, solid 1,3ꢀdicyclohexylꢀurea was filtered off and the solvent was removed under reduced
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pressure. Column chromatography on silica gel using ethyl acetate provided compounds 15a,c,d, 13a,
24 and 34.
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Dibenzyl 4-[benzyloxy(methyl)amino]-1-(3,4-dimethoxyphenyl)-4-oxobutylphosphonate (13a).
Yellow oil (550 mg; 30 %); 1H NMR (500.13 MHz, [D6]DMSO): δ = 1.99ꢀ2.03 (m, 1H, CH2), 2.20ꢀ
2.28 (m, 3H, CH2), 3.09 (s, 3H, NCH3), 3.26ꢀ3.32 (m, 1H, PCH), 3.61 (s, 3H, OCH3), 3.73 (s, 3H,
OCH3), 4.64 (dd, J = 24.6, 9.9 Hz, 2H, PhCH2), 4.76 (dd, J = 12.0, 7.8 Hz, 1H, PhCH2), 4.85 (dd, J =
12.0, 6.8 Hz, 1H, PhCH2), 4.96 (dd, J = 12.0, 7.1 Hz, 1H, PhCH2), 5.02 (dd, J = 12.0, 8.5 Hz, 1H,
PhCH2), 6.83ꢀ6.84 (m, 1H), 6.87 (s, 1H), 6.91 (d, J = 8.3 Hz), 7.17ꢀ7.18 (m, 4H), 7.29ꢀ7.37 (m, 11H)
ppm; 13C NMR (125.76 MHz, [D6]DMSO): δ = 24.4 (CH2CH2), 29.2 (d, 2JCꢀP = 16.4 Hz, CHCH2), 32.6
(NCH3), 41.8 (d, 1JCꢀP = 136.3 Hz, PCH), 55.2 (OCH3), 55.4 (OCH3), 66.5 (d, 2JCꢀP = 6.9 Hz, CH2CH3),
66.8 (d, 2JCꢀP = 6.5 Hz, CH2CH3), 75.1 (PhCH2), 111.7, 112.8 (d, 3JCꢀP = 6.2 Hz), 121.2 (d, 3JCꢀP = 7.4
Hz), 127.4, 127.6, 127.7 (d, 2JCꢀP = 7.2 Hz), 127.9, 128.0, 128.2, 128.2, 128.3, 128.6, 129.3, 134.3,
136.5 (d, 3JCꢀP = 6.1 Hz), 136.6 (d, 3JCꢀP = 6.4 Hz), 147.9 (d, 5JCꢀP = 2.3 Hz), 148.5 (d, 4JCꢀP = 1.5 Hz),
172.9 (C=O) ppm; anal. calcd. for C34H38NO7P: C 67.65, H 6.35, N 2.32, found: C 67.43, H 6.20, N
2.28.
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Dibenzyl 4-(benzyloxyamino)-1-(3,4-dimethoxyphenyl)-4-oxobutylphosphonate (15a). Yellow oil
(248 mg; 14 %); 1H NMR (500.13 MHz, [D6]DMSO): δ = 1.83ꢀ1.86 (m, 2H, CH2), 1.97ꢀ2.06 (m, 1H,
CH2), 2.24ꢀ2.28 (m, 1H, CH2), 3.23 (ddd, J = 22.0, 11.4, 3.2 Hz, 1H, PCH), 3.61 (s, 3H, OCH3), 3.73 (s,
3H, OCH3), 4.73ꢀ4.77 (m, 3H, PhCH2), 4.83ꢀ4.87 (m, 1H, PhCH2), 4.94ꢀ5.04 (m, 2H, PhCH2), 6.80 (d, J
= 8.2 Hz, 1H), 6.83 (s, 1H), 6.89 (d, J = 8.2 Hz, 1H), 7.17ꢀ7.19 (m, 2H), 7.30ꢀ7.37 (m, 12H), 10.51 (s,
0.1H, Eꢀisomer, OH), 10.87 (s, 0.9H, Zꢀisomer, OH) ppm; 13C NMR (125.76 MHz, [D6]DMSO): δ =
5.2 (CH2CH2), 29.8 (d, 2JCꢀP = 16.0 Hz, CHCH2), 41.8 (d, 1JCꢀP = 136.1 Hz, PCH), 55.2 (OCH3), 55.4
(OCH3), 66.5 (d, 2JCꢀP = 6.7 Hz, CH2CH3), 66.8 (d, 2JCꢀP = 6.8 Hz, CH2CH3), 76.6 (PhCH2), 111.6, 112.7
(d, 3JCꢀP = 6.3 Hz), 121.3 (d, 3JCꢀP = 7.3 Hz), 127.4, 127.5, 127.6, 128.0, 128.1, 128.2, 128.2, 128.4,
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128.6, 136.0, 136.5 (d, 3JCꢀP = 6.4 Hz), 136.6 (d, 3JCꢀP = 6.0 Hz), 147.9 (d, 5JCꢀP = 2.5 Hz), 148.4, 168.47
(C=O) ppm; anal. calcd. for C33H36NO7P: C 67.22, H 6.15, N 2.38, found: C 67.47, H 6.27, N 2.36.
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General procedure for the synthesis of carboxylic acid 20, hydroxamic acid 22, o-hydroxyanilide
31, bisphosphonate 7 and target compounds 2a-f, 3b and 4.29 To a solution of the appropriate OꢀBnꢀ
protected hydroxamic acid (1 mmol) in freshly distilled methanol (20 mL), PdꢀC catalyst (10 %, 40 mg)
was added. The mixture was hydrogenated for 1 h (in case of compounds 2a, 2c, 4, 20, 22, 31) and 3 h
(in case of compounds 2b, 2dꢀf, 3b, 7). The catalyst was removed by filtration and the solvent was
evaporated under reduced pressure. Pure target compounds 2b and 2d-f were obtained as white solids
after addition of ethyl acetate. Recrystallization in ethyl acetate gave compound 31 as colorless crystals.
Pure compounds 2a, 2c, 3b, 4, 7, 20 and 22 were obtained without further purification.
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1-(3,4-Dimethoxyphenyl)-4-[hydroxy(methyl)amino]-4-oxobutylphosphonic acid (2e). White solid
(290 mg, 87 %) mp: 70 °C; 1H NMR (500.13 MHz, [D6]DMSO): δ = 1.89ꢀ1.99 (m, 1H, CH2), 2.19 (s,
3H, CH2), 2.77ꢀ2.84 (m, 1H, PCH), 3.03 (s, 3H, NCH3), 3.71 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 6.74ꢀ
6.76 (m, 1H), 6.85ꢀ6.87 (m, 2H), 9.68 (br s, 1H, OH) ppm; 13C NMR (125.76 MHz, [D6]DMSO): δ =
25.3 (CH2CH2), 30.1 (d, 2JCꢀP = 15.2 Hz, CHCH2), 35.6 (NCH3), 44.1 (d, 1JCꢀP = 136.8 Hz, PCH), 55.3
(OCH3), 55.45 (OCH3), 111.6, 112.8 (d, 3JCꢀP = 5.6 Hz), 121.3 (d, 2JCꢀP = 7.2 Hz), 130.5 (d, 3JCꢀP = 6.5
Hz), 147.3, 148.3, 172.7 (C=O) ppm; anal. calcd. for C13H20NO7P: C 46.85, H 6.05, N 4.20, found:
C 46.61, H 6.05, N 4.06.
1-(3,4-Dimethoxyphenyl)-4-(hydroxyamino)-4-oxobutylphosphonic acid (2f). White solid (313 mg,
98 %), mp: 75 °C; 1H NMR (500.13 MHz, [D6]DMSO): δ = 1.73ꢀ1.93 (m, 3H, CH2), 2.20ꢀ2.28 (m, 1H,
CH2), 2.75 (ddd, J = 14.9, 10.9, 3.5 Hz, 1H, PCH), 3.72 (s, 6H, OCH3), 6.75ꢀ6.76 (m, 1H), 6.84ꢀ6.87
(m, 2H), 8.66 (br s, 1H, OH), 9.71 (s, 0.1H, Eꢀisomer, NH), 10.27 (s, 0.9H, Zꢀisomer, NH) ppm; 13C
NMR (125.76 MHz, [D6]DMSO): δ = 26.4 (CH2CH2), 30.6 (d, 2JCꢀP = 15.2 Hz, CHCH2), 44.2 (d, 1JCꢀP
134.6 Hz, PCH), 55.3 (OCH3), 55.4 (OCH3), 111.6, 112.8 (d, 3JCꢀP = 6.2 Hz), 121.1 (d, 3JCꢀP = 6.7 Hz),
=
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130.5 (d, 2JCꢀP = 6.4 Hz), 147.3 (OCH3), 148.2 (OCH3), 168.8 (C=O) ppm; HPLC analysis: retention
time = 1.54 min; peak area: 97.5 %. Eluent A: 5 mM NH4OAc solution; eluent B: CH3CN, isocratic
(1:1) over 20 min at a flow rate of 1.5 mL minꢀ1.
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ASSOCIATED CONTENT
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Supporting Information. Experimental procedures, analytical data, enzyme assays, biological
evaluation of in vitro antiplasmodial activity, and experimental data regarding crystallization and
structure determination. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (+49)21181ꢀ14984. Fax: (+49)22181ꢀ13847. Eꢀmail: Thomas.kurz@uniꢀduesseldorf.de.
ACKNOWLEDGMENT
Nobutada Tanaka was supported in part by a grant from the Takeda Science Foundation, the Naito
Foundation, and GrantꢀinꢀAid for Scientific Research (C) from the MEXT of Japan.
We are very grateful for the support by the HansꢀFischerꢀGesellschaft and the Deutsche
Forschungsgemeinschaft, grant GR 3866/1ꢀ1.
ABBREVIATIONS
nꢀBuLi, nꢀbutyllithium; CDI, 1,1`ꢀcarbonyldiimidazol; DCC, N,N`ꢀdicyclohexylcarbodiimide; DMF,
N,Nꢀdimethylformamide; DMSO, dimethyl sulfoxide; DOXP, 1ꢀdeoxyꢀ
D
ꢀxylulose 5ꢀphosphate; Dxr
ꢀerythritol 4ꢀphosphate;
(IspC), 1ꢀdeoxyꢀ ꢀxylulose 5ꢀphosphate reductoisomerase; MEP, 2Cꢀmethylꢀ
D
D
NADPH, nicotinamide adenine dinucleotide phosphate; NMM, Nꢀmethymorpholine; PdꢀC, palladium
on activated carbon; rt, room temperature; TEA, triethylamine; THF, tetrahydrofurane; TMSBr,
bromotrimethylsilane; WHO, World Health Organization.
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