Synthesis of oxazinyl analogues of fosmidomycin using RCM methodology

Article abstract of DOI:10.1055/s-2007-992373

Fosmidomycin is a promising antimalarial compound with a novel mode of action, the inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase, a key enzyme in the biosynthesis of isoprenoids through the nonmevalonate pathway. This paper describes the synthesis of a series of analogues in which the hydroxamate moiety is incorporated in a ring structure, leaving the complexation with the enzyme up to the oxygen lone pairs instead of the free hydroxyl group. The antimalarial activities of the different analogues are currently under investigation. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2007-992373

LETTER
3183
Synthesis of Oxazinyl Analogues of Fosmidomycin Using RCM Methodology
Synthesis of
O
a
xazinyl
A
n
r
alogues
aof Fosmidoh
mycin Van der Jeught,1 Christian V. Stevens,* Nicolai Dieltiens
Research Group SynBioC, Department of Organic Chemistry, Faculty of Bioscience Engineering, Ghent University,
Coupure Links 653, 9000 Ghent, Belgium
Fax +32(9)2646243; E-mail: Chris.Stevens@ugent.be
Received 20 September 2007
carried out to synthesize more bioavailable and more ac-
Abstract: Fosmidomycin is a promising antimalarial compound
with a novel mode of action, the inhibition of 1-deoxy-D-xylulose
5-phosphate reductoisomerase, a key enzyme in the biosynthesis of
isoprenoids through the nonmevalonate pathway. This paper de-
tive derivatives. The nature of the investigations was also
influenced by the elucidation of the crystallographic
structure of DXR.8 Important structural elements for anti-
scribes the synthesis of a series of analogues in which the hydrox- malarial activity are the phosphonic acid moiety, the hy-
amate moiety is incorporated in a ring structure, leaving the
droxamic acid group and the three-carbon spacer between
complexation with the enzyme up to the oxygen lone pairs instead
both functionalities. A lot of work concerned the modifi-
of the free hydroxyl group. The antimalarial activities of the differ-
cation of the phosphonate moiety, into ester prodrugs,9–11
ent analogues are currently under investigation.
carboxylic acids12 or phosphates.13 The hydroxamate
Key words: fosmidomycin, oxazinyl analogues, 1-deoxy-D-xylu-
group has also been frequently altered into benzoxazolo-
lose 5-phosphate reductoisomerase, ring-closing metathesis, heter-
ne, benzoxazolethione, oxalopyridinone, hydroxamic acid
ogeneous ruthenium catalysis
or other functionalities.14–16 Variations of the propyl spac-
er are rather scarce. Different conformationally restricted
derivatives were already investigated in order to gain new
According to the WHO, between 300 million and 500 mil-
insights regarding the structure–activity relationships.17–19
lion clinical cases of malaria occur every year and the dis-
ease is estimated to kill one to three million people
Furthermore, the synthesis and evaluation of some a-aryl-
substituted analogues as superior inhibitors compared to
annually. Almost all of the fatal cases of malaria in hu-
fosmidomycin, revealed the probability of an extra cavity
mans are due to the parasite Plasmodium falciparum, the
in the DXR enzyme at this a-position.19,20 Another ap-
causative agent of Malaria tropica. The disease is still
proach that has not yet been investigated is the incorpora-
tion of the hydroxamate moiety in a closed ring system.
gaining ground as the parasite’s resistance to drugs and
the parasite-carrying mosquito’s resistance to insecticides
The oxygen atom is hereby no longer present as a free hy-
increase.2 This highlights the urgent requirement for safe
droxyl group, but still possesses two lone pairs to complex
and effective antimalarials with new modes of action.
with the enzyme. Analysis of compounds of this kind
Fosmidomycin and FR-900098 (Figure 1) were proven to
could give additional information on the necessity of the
show activity against the P. falciparum parasite, both in
free OH group for activity against malaria parasites. This
vitro and in clinical studies.3,4 It was only in 1998, after
paper deals with the synthesis of such analogues, employ-
extensive research, that the mechanism of action of this
ing the ring-closing metathesis (RCM) reaction as a key
new class of antimalarial drugs was revealed. The com-
step in the reaction pathway. The use of RCM to synthe-
pounds are selective inhibitors of 1-deoxy-D-xylulose 5-
size heterocycles containing the 1,2-oxaza group has been
phosphate reductoisomerase (DXR), a key enzyme in the
given little attention. In the literature, only few reports
biosynthesis of isoprenoids through the mevalonate-inde-
have been published concerning the synthesis of 1,2-ox-
pendent 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-
azines and 1,2-oxazepines by RCM.21 And even fewer ref-
erythritol 4-phosphate (DOXP/MEP) pathway.5 This
erences mentioned the preparation of 3-oxo-1,2-oxaza
pathway is present in algae, higher plants, bacteria and the
compounds, which makes these products interesting re-
malaria parasite P. falciparum, but as it is absent in hu-
search topics.
mans and mammalians, the involved enzymes are promis-
ing targets for new drugs.
NH4
+
O–
– O
Clinical trials have shown that fosmidomycin is efficient
in the treatment of acute uncomplicated Malaria tropica.
However, the oral bioavailability of both fosmidomycin
and FR-900098 is only moderate, with a resorption rate of
approximately 30%.6 In addition, the short plasma half-
life of fosmidomycin requires a repeated administration of
relatively high dosages.4,7 Various modifications were
NH4
O
O
P
OH
N
+
P
O
HO
O
N
HO
R
R1
O
(CH2)n
R = H (fosmidomycin)
R = Me (FR-900098)
R2
1a (R1 = H, R2 = H, n = 0)
1b (R1 = H, R2 = Me, n = 0)
SYNLETT 2007, No. 20, pp 3183–3187
Advanced online publication: 21.11.2007
DOI: 10.1055/s-2007-992373; Art ID: G32307ST
x
x
.x
x
.2
0
0
7
Figure 1 Structures of fosmidomycin, FR-900098 and target com-
pounds 1a,b
© Georg Thieme Verlag Stuttgart · New York

3184
S. Van der Jeught et al.
LETTER
The preparation of the RCM precursors 9a–f started with oxime was carried out with sodium cyanoborohydride in
the formation of aldimine 3 from cinnamaldehyde (2), fol- methanol acidified with HCl, in the presence of a trace
lowed by a 1,4-addition of triethyl phosphite and subse- amount of the pH indicator bromocresol green as de-
quent hydrolysis of the imine function. These reactions scribed by Eguchi.25 Other reducing agents usually result
were already optimized by other members of our research in the cleavage of the N–O bond as a side-effect. Since the
group,22 based on a procedure reported by Teulade and hydroxylamine moiety is an important feature for antima-
Savignac.23 As such, phosphonate 4 was prepared in good larial activity, this cleavage must be avoided. With this
yield (Scheme 1).
method, reduction could be performed without side-ef-
fects and in good yields. To introduce the carbonyl func-
tion and a second double bond in the structure, a
nucleophilic addition–substitution reaction of the hydrox-
ylamine nitrogen to an acyl chloride was performed. In
two cases, the acyl chloride (8c and 8d) was not commer-
cially available as such and had to be prepared in situ from
the corresponding acid, using thionyl chloride (Table 1,
entries 9 and 10). Purification of the resulting compound
9 was necessary for two analogues (9d,e) and could be
performed by column chromatography. For the other de-
rivatives, the crude reaction products were used in the fol-
lowing reaction without further purification. This reaction
consisted of the closure of the N,O-substituted hydroxy-
lamines 9a–f and was carried out via a ring-closing met-
athesis (RCM) reaction.26 Grubbs’ second-generation
catalyst (0.05 equiv) was added to a mixture of the hy-
droxylamine 9a–f in refluxing solvent. In the case of com-
pound 9e (Table 1, entry 15), the presence of the fluorine
atoms required a higher temperature to complete the reac-
tion. Therefore, benzene (bp 80 °C) was used as solvent
instead of dichloromethane (bp 40 °C), that was used for
all the other derivatives. In all six cases the ring closure
proceeded almost quantitatively, but the problem inherent
to most metathesis reactions employing ruthenium car-
benes was the effective removal of ruthenium impurities
after completion of the reaction.
O
H
N
t-BuNH2
H
MgSO4, CH2Cl2
overnight, 78%
3
2
2) oxalic acid
85–90%
1) P(OEt)3,
HCOOH,
EtOH
(EtO)2(O)P
O
H
4
Scheme 1
The results of the following reactions are summarized in
Table 1. Reaction of the resulting aldehyde 4 with two dif-
ferent O-substituted hydroxylamine hydrochlorides gave
oximes 6a and 6b in 93% and 74% yields, respectively
(Scheme 2). Unfortunately, O-(2-methylallyl)hydroxy-
lamine hydrochloride (5b) was not commercially avail-
able and had to be prepared starting from 3-bromo-2-
methylpropene and hydroxylamine hydrochloride accord-
ing to Albrecht’s procedure.24 Subsequent reduction of the
+
H3N
O
R1
Cl–
R1
O
NaBH3(CN),
bromocresol
green
(EtO)2(O)P
R1
5a (R1 = H)
(EtO)2(O)P
N
(EtO)2(O)P
O
5b (R1 = Me)
O
N
H
H
H
HCl, MeOH
Et3N, MgSO4
CH2Cl2, r.t.
E/Z
6a,b
7a,b
4
R2
O
(CH2)n Cl
base,
8a (R2 = H, n = 0)
8b (R2 = Me, n = 0)
8c (R2 = CF3, n = 0)
8d (R2 = H, n = 1)
anhyd CH2Cl2,
N2, r.t., overnight
(EtO)2(O)P
R1
(EtO)2(O)P
O
N
O
N
Grubbs II catalyst,
R1
O
(CH2)n
solvent, N2, ∆
O
(CH2)n
R2
R2
9a–f
10a–f
Scheme 2
Synlett 2007, No. 20, 3183–3187 © Thieme Stuttgart · New York

LETTER
Synthesis of Oxazinyl Analogues of Fosmidomycin
3185
Table 1 Summary of Reactions in Scheme 2 and Scheme 3
Entry
Starting
Product
R1
R2
n
Base
Solvent
Reaction Yield
materials
time
15 h
15 h
1 h
(%)
1
2
4
6a
6b
H
–
–
–
–
–
0
0
0
0
0
1
0
0
0
0
0
1
0
0
Et3N
CH2Cl2
93
4
Me
H
–
Et3N
CH2Cl2
74
3
6a
7a
–
–
MeOH
98
4
6b
7b
Me
H
–
–
MeOH
1 h
92
5
7a, 8a
7a, 8b
7b, 8a
7b, 8b
7a, 8c
7a, 8d
9a
9a
H
Et3N
anhyd CH2Cl2
anhyd CH2Cl2
anhyd CH2Cl2
anhyd CH2Cl2
anhyd CH2Cl2
anhyd CH2Cl2
anhyd CH2Cl2
anhyd CH2Cl2
anhyd CH2Cl2
anhyd CH2Cl2
C6H6
24 h
24 h
24 h
24 h
24 h
24 h
6 h
90
6
9b
H
Me
H
Et3N
99
7
9c
Me
Me
H
Et3N
85
8
9d
Me
CF3
H
Et3N
69a
62a
83
9
9e
pyridine
10
11
12
13
14
15
16
17
18
9f
H
pyridine
10a
10b
10c
10d
10e
10f
1a
H
H
–
–
–
–
–
–
–
–
71a
75a
68a
56a
97
9b
H
Me
H
6 h
9c
Me
Me
H
6 h
9d
Me
CF3
H
6 h
9e
5 h
9f
H
anhyd CH2Cl2
MeCN
8 h
43a
62a
65a
10a
10b
H
H
24 h
24 h
1b
H
Me
MeCN
a Yield obtained after purification by column chromatography.
A variety of methods27 have been used to scavenge ruthe- ids were purified by column chromatography on What-
nium species, including the use of: water-soluble phos- man CF11 cellulose with a mixture of MeCN and NH4OH
phines,28a,b oxidizing agents (such as triphenylphosphine (1 N) as solvent. The useful fractions were again lyo-
oxide,28c DMSO,28c or lead tetraacetate28d), mesoporous philized. The resulting bisammonium salts 1a,b29 of the
silicates,28e polar isocyanides,28f and activated carbon.28g ring-closed phosphonates (Scheme 3) proved to be hygro-
A few of these methods (ref. 28a, 28d and 28g) were ap- scopic solids.
plied on the analogues 10a–f but in these cases, normal
Using the described procedures, six analogues of the
column chromatography gave the best results despite the
phosphonate prodrugs have been synthesized and two of
inevitable losses of product.
these analogues were deprotected to form their bisammo-
Although some reports have been published on ester pro- nium salts. These two final compounds and two of the
drugs of fosmidomycin analogues (vide infra), the biolog-
(EtO)2(O)P
ically active compounds are the phosphonic acids. A
O
commonly used method for the deprotection of a phos-
N
R1
phonate group into a phosphonic acid function is the reac-
tion with TMSBr in dichloromethane under a nitrogen
atmosphere, followed by addition of water. This proce-
O
(CH2)n
R2
10a,b
NH4
dure was used for analogues 10a, 10b and 10f with five
equivalents of TMSBr, but it never led to the envisaged
+
1) TMSBr, MeCN, r.t.
2) NH4OH(aq)
3) cellulose
O–
NH4
O
+
P
– O
result. Finally, the phosphonate ester groups of 10a,b
could be cleaved according to the methodology reported
by Devreux et al.20 Addition of ten equivalents of TMSBr
to a solution of phosphonate 10a,b in anhydrous acetoni-
trile at ambient temperature was followed by the addition
of a NH4OH solution and lyophilization. The residual sol-
O
chromatography
N
R1
O
(CH2)n
R2
1a,b
Scheme 3
Synlett 2007, No. 20, 3183–3187 © Thieme Stuttgart · New York

3186
S. Van der Jeught et al.
LETTER
(17) Devreux, V.; Wiesner, J.; Goeman, J. L.; Van der Eycken, J.;
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339.
phosphonates were tested for inhibition of recombinant
Escherichia coli DXR. The results will be disclosed in due
course.
In conclusion, a synthetic procedure for the preparation of
oxazinyl analogues of fosmidomycin was developed start-
ing from cinnamaldehyde and the key reaction in this
method consisted of an RCM reaction. Although the hy-
droxyl group is incorporated in the ring structure, the ox-
ygen lone pairs still comprise the potential to perform the
complexation with the enzyme.
(19) Haemers, T.; Wiesner, J.; Busson, R.; Jomaa, H.; Van
Calenbergh, S. Eur. J. Org. Chem. 2006, 3856.
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Acknowledgment
Sarah Van der Jeught wishes to thank the Fund for Scientific Rese-
arch, Flanders (FWO Vlaanderen) for the financial support.
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at reflux temperature and Grubbs’ second-generation
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ruthenium catalyst, yielding 10b (0.70 g, 1.90 mmol, 75%).
1H NMR (300 MHz, CDCl3): d = 1.08 (t, J = 7.0 Hz, 3 H,
OEt), 1.29 (t, J = 7.0 Hz, 3 H, OEt), 1.88 (q, J = 1.7 Hz, 3 H,
CMe), 2.22–2.35 (m, 1 H, CHAHBCHP), 2.40–2.54 (m, 1 H,
CHAHBCHP), 3.11 (ddd, J = 3.1, 11.2, 23.1 Hz, 1 H, CHP),
3.46 (ddd, J = 6.7, 6.8, 14.0 Hz, 1 H, CHAHBN), 3.59 (ddd,
J = 7.2, 7.2, 14.0 Hz, 1 H, CHAHBN), 3.66–4.11 (m, 4 H, 2 ×
OEt), 4.35 (ddq, J = 1.7, 3.5, 15.4 Hz, 1 H, OCHAHBCH=C),
4.44 (ddq, J = 1.7, 3.5, 15.4 Hz, 1 H, OCHAHBCH=C), 6.35
(tq, J = 1.7, 3.5 Hz, 1 H, OCH2CH=C), 7.23–7.37 (m, 5 H,
CHarom). 13C NMR (75 MHz, CDCl3, ref. CDCl3): d = 15.70
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(CMe), 16.27, 16.35, 16.45, 16.54 (2 × OEt), 27.78 (JC–P
=
2.3 Hz, CH2CHP), 42.33 (JC–P = 138.5 Hz, CHP), 45.14
(JC–P = 18.5 Hz, CH2N), 61.97 (JC–P = 6.9 Hz, OEt), 62.81
(JC–P = 6.9 Hz, OEt), 67.54 (OCH2CH=C), 77.13 (CDCl3),
127.44 (JC–P = 2.3 Hz, CHarom), 128.67 (JC–P = 2.3 Hz, 2 ×
CHarom), 129.40 (JC–P = 6.9 Hz, 2 × CHarom), 129.67 (CMe),
132.92 (OCH2CH=C), 135.48 (JC–P = 6.9 Hz, Cq,arom), 166.46
(CO). 31P NMR (109 MHz, CDCl3): d = 28.92. IR (NaCl):
1674, 1635 (C=O, C=C), 1242 (P=O), 1058, 1028 (PO)
cm–1. MS (ESI, +ve mode, %): m/z = 368.3 (100) [M + H+].
Chromatography (PE–EtOAc, 1:4): Rf = 0.18.
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Synlett 2007, No. 20, 3183–3187 © Thieme Stuttgart · New York

LETTER
Holliday, A. E.; Crudden, C. M. Org. Lett. 2006, 8, 2663.
Synthesis of Oxazinyl Analogues of Fosmidomycin
3187
desired fractions were again lyophilized, yielding 1b as a
pale brown hygroscopic solid (0.31 g, 0.88 mmol, 65%). 1H
NMR (300 MHz, CDCl3): d = 1.72 (d, J = 1.7 Hz, 3 H, CMe),
2.14–2.42 (m, 2 H, CH2CHP), 2.93 (ddd, J = 3.0, 11.8, 22.0
Hz, 1 H, CHP), 3.50 (t, J = 6.5 Hz, 2 H, CH2N), 4.31 (ddd,
J = 1.9, 3.6, 15.7 Hz, 1 H, OCHAHBCH=C), 4.44 (ddq, J =
1.9, 3.4, 15.7 Hz, 1 H, OCHAHBCH=C), 4.79 (D2O), 6.45
(tq, J = 1.7, 1.7 Hz, 1 H, OCH2CH=C), 7.19–7.35 (m, 5 H,
5 × CHarom). 13C NMR (75 MHz, CDCl3, ref. MeCN): d =
1.47 (ref. MeCN), 15.20 (CMe), 28.13 (JC–P = 2.3 Hz,
CH2CHP), 44.79 (JC–P = 129.2 Hz, CHP), 45.94 (JC–P = 17.3
Hz, CH2N), 68.24 (OCH2CH=C), 119.69 (ref. MeCN),
127.17 (JC–P = 2.3 Hz, CHarom), 128.36 (CMe), 129.00
(JC–P = 2.3 Hz, 2 × CHarom), 129.69 (JC–P = 6.9 Hz, 2 ×
CHarom), 135.26 (OCH2CH=C), 139.14 (JC–P = 6.9 Hz,
Cq,arom), 166.73 (CO). 31P NMR (109 MHz, CDCl3): d =
22.91. IR (NaCl): 1668 (C=O, C=C), 1033 (PO) cm–1. MS
(ESI, –ve mode, %): m/z = 310.3 (100) [M – +NH4 – NH3].
Chromatography (MeCN–1 M NH4OH, 5:1): Rf = 0.16.
(f) Galan, B. R.; Kalbarczyk, K. P.; Szczepankiewicz, S.;
Keister, J. B.; Diver, S. T. Org. Lett. 2007, 9, 1203.
(g) Cho, J. H.; Kim, B. M. Org. Lett. 2003, 5, 531.
(29) Preparation of Diammonium 1-Phenyl-3-(4-methyl-3-
oxo-3,6-dihydro-2H-1,2-oxazin-2-yl)propylphos-
phonate (1b): To a solution of 10b (0.50 g, 1.36 mmol) in
anhyd MeCN at r.t. was added dropwise TMSBr (13.6
mmol) and this mixture was stirred at r.t. for 24 h. The
solvents and traces of TMSBr were removed under reduced
pressure and under high vacuum. The residual oil was
dissolved in H2O and the pH was adjusted with a 5% NH4OH
solution to pH 8–9. Lyophilization of the solution resulted in
a solid and purification was performed by column
chromatography on Whatman CF11 cellulose. The solvent
used was MeCN–aq 1 M NH4OH (5:1) and the fractions
were analyzed on cellulose TLC with the use of UV light to
visualize the spots after dipping the plates in a pinacryptol
yellow solution (0.1% in H2O) and drying with hot air. The
Synlett 2007, No. 20, 3183–3187 © Thieme Stuttgart · New York

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