Divergent strategy for the synthesis of α-aryl-substituted fosmidomycin analogues

Article abstract of DOI:10.1021/jo0700981

(Chemical Equation Presented) Fosmidomycin is the first representative of a new class of antimalarial drugs acting through inhibition of 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway for the synthesis of isoprenoids. This work describes a divergent strategy for the synthesis of a series of α-aryl-substituted fosmidomycin analogues, featuring a palladium-catalyzed Stille coupling as the key step. An α-(4-cyanophenyl)fosmidomycin analogue emerged as the most potent analogue in the present series. Its antimalarial activity clearly surpasses that of the reference compound fosmidomycin.

Full text of DOI:10.1021/jo0700981

Divergent Strategy for the Synthesis of r-Aryl-Substituted
Fosmidomycin Analogues
Vincent Devreux,†,‡ Jochen Wiesner,§ Hassan Jomaa,§ Jef Rozenski,||
Johan Van der Eycken,‡ and Serge Van Calenbergh†,*
Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent UniVersity, Belgium,
Laboratory of Organic and Bioorganic Chemistry, Department of Organic Chemistry, Faculty of Sciences,
Ghent UniVersity, Belgium, Institut fu¨r Klinische Chemie und Pathobiochemie, UniVersita¨tsklinkum Giessen
und Marburg, Germany, and Laboratory for Medicinal Chemistry, REGA Institute,
Catholic UniVersity of LeuVen, Belgium
Serge.VanCalenbergh@UGent.be
ReceiVed January 17, 2007
Fosmidomycin is the first representative of a new class of antimalarial drugs acting through inhibition of
1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase (DXR), an essential enzyme in the non-
mevalonate pathway for the synthesis of isoprenoids. This work describes a divergent strategy for the
synthesis of a series of R-aryl-substituted fosmidomycin analogues, featuring a palladium-catalyzed Stille
coupling as the key step. An R-(4-cyanophenyl)fosmidomycin analogue emerged as the most potent
analogue in the present series. Its antimalarial activity clearly surpasses that of the reference compound
fosmidomycin.
Introduction
4-phosphate (MEP) pathway for the biosynthesis of isoprenoids
and absent in humans, was identified as the molecular target
for fosmidomycin.3,4
Fosmidomycin was found to be a potent inhibitor of DOXP
reductoisomerase (DXR) of P. falciparum.4 FR900098 (2), the
acetyl congener of fosmidomycin, was shown to be ap-
proximately twice as active against P. falciparum in vitro, as
well as in a P. Vinckei mouse model.4
Because of this promising antimalarial activity, fosmidomycin
received considerable attention, and recent clinical trials con-
ducted in Gabon and Thailand confirmed the potential of
fosmidomycin as an antimalarial drug.5,6These important dis-
coveries stimulated research on the chemical synthesis of
According to the WHO, malaria still causes 1.5 to 2 million
casualties in Africa alone, among which are mainly children.
One of the major reasons for the spread of the disease is the
growing resistance of Plasmodium falciparum, the parasite
species responsible for most of the diagnosed cases, toward
almost every antimalarial drug currently available. Therefore,
the discovery of novel inhibitors targeting new biochemical
pathways is of high priority.
In 1980, Okuhura and colleagues reported the first isolation
of fosmidomycin (1), a structurally simple antibiotic from
Streptomyces laVendulae.1,2
In 1998, 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoi-
somerase, an enzyme involved in the 2C-methyl-D-erythritol
(3) Kuzuyama, T.; Shimizu, T.; Takahashi, S.; Seto, H. Tetrahedron Lett.
1998, 39, 7913-7916.
(4) Jomaa, H.; Wiesner, J.; Sanderbrand, S.; Altincicek, B.; Weidemeyer,
C.; Hintz, M.; Turbachova, I.; Eberl, M.; Zeidler, J.; Lichtenthaler, H. K.;
Soldati, D.; Beck, E. Science 1999, 285, 1573-1576.
* Corresponding author. Phone: +32 9 264 81 24. Fax + 32 9 264 81 46.
† Laboratory for Medicinal Chemistry, Ghent University.
‡ Laboratory of Organic and Bioorganic Chemistry, Ghent University.
§ Universita¨tsklinkum Giessen und Marburg.
(5) Missinou, M. A.; Borrmann, S.; Schindler, A.; Issifou, S.; Adegnika,
A. A.; Matsiegui, P. B.; Binder, R.; Lell, B.; Wiesner, J.; Baranek, T.; Jomaa,
H.; Kremsner, P. G. Lancet 2002, 360, 1941-1942.
(6) Lell, B.; Ruangweerayut, R.; Wiesner, J.; Missinou, M. A.; Schindler,
A.; Baranek, T.; Hintz, M.; Hutchinson, D.; Jomaa, H.; Kremsner, P. G.
Antimicrob. Agents Chemother. 2003, 47, 735-738.
|| Catholic University of Leuven.
(1) Okuhara, M.; Kuroda, Y.; Goto, T.; Okamoto, M.; Terano, H.;
Kohsaka, M.; Aoki, H.; Imanaka, H. J. Antibiot. 1980, 33, 13-17.
(2) Okuhara, M.; Kuroda, Y.; Goto, T.; Okamoto, M.; Terano, H.;
Kohsaka, M.; Aoki, H.; Imanaka, H. J. Antibiot. 1980, 33, 24-28.
10.1021/jo0700981 CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/12/2007
J. Org. Chem. 2007, 72, 3783-3789
3783

Devereux et al.
CHART 1. Target Fosmidomycin and FR900098 Analogues and Retrosynthetic Plan Involving Key Intermediate 6
fosmidomycin analogues. Structural changes of fosmidomycin
and FR900098 could provide essential information on the
structure-activity relationship and produce improved leads
for the treatment of malaria. Mostly, attention was focused
on the modification of the phosphonate and hydroxamate
moieties, yielding, on one hand, various phosphonate prodrugs
aimed to enhance the oral bioavailability, and on the other
hand, modifications of the retrohydroxamate moiety into a
hydroxamic acid, among others.7-11 Structural variations of the
three-carbon spacer, however, are scarce. Our group recently
reported the synthesis of conformationally restricted analogues
in which the carbon spacer was part of a cyclopropyl or
cyclopentyl ring.12,13
To date, the most promising modifications of the carbon
spacer consist of the introduction of an aryl substituent in
R-position of fosmidomycin or FR900098.14 Recently, we
identified the R-3,4-dichlorophenyl-substituted fosmidomycin
analogue which exhibits a 12 times stronger in Vitro antimalarial
activity (Dd2 and 3D7 strains of P. falciparum) compared to
fosmidomycin. Lately, Kurz and co-workers evaluated the effect
of R-substituents on pivaloyloxymethyl ester prodrugs of
fosmidomycin and FR900098.15,16 The R-methyl and phenyl
analogues proved almost equipotent and the 3,4-difluorophenyl
analogue slightly better than the FR900098 prodrug in inhibiting
growth of a 3D7 P. falciparum strain.15 Incorporation of an
arylmethyl moiety in R-position generally resulted in a marked
decrease in activity.16 Unfortunately, the strategy used to
synthesize the R-aryl-substituted analogues involved a de noVo
synthetic approach, which only permitted synthesis of a limited
number of examples. To further explore the structure-activity
relationship of this class, a strategy involving the incorporation
of the aryl moiety at a later stage in the synthesis would be
desirable.
Results and Discussion
Here we describe a highly efficient divergent synthesis of a
series of R-aryl-substituted fosmidomycin analogues 3-5,
involving the R-tributylstannyl derivative 6 as a key intermediate
(Chart 1). The vinylic tributyltin functionality of this intermedi-
ate was subjected to a palladium(0)-catalyzed Stille coupling
with a variety of commercially available or readily synthesized
aryl iodides.
The synthesis of said key intermediate 6 started with the
conversion of the THP-protected propargyl alcohol 7 to diethyl
(3-hydroxypropynyl)phosphonate (8) in a yield of 64%.17
Subsequently, the hydroxyl group of compound 8 was displaced
by a protected hydroxylamino moiety under Mitsunobu condi-
tions. Nucleophilic substitution of the corresponding tosylate
proved unsuccessful because of 1,4-addition of the nucleophile
to the alkynyl phosphonate. Although extensive examples of
successful Mitsunobu couplings are found in literature using
carbamoyl-protected hydroxylamines,18,19 introduction of a
protected hydroxylamine moiety only succeeded using N-ben-
zyloxy-2-nitrobenzenesulfonamide.20 Indeed, the 2-nitrobenze-
nesulfonamide (or nosyl/Ns) moiety effectively enhances the
acidity of the proton on the nitrogen, whereas in the case of the
Boc-protected hydroxylamine, its acidity is not sufficient for
reaction to occur.
(7) Woo, Y. H.; Fernandes, R. P. M.; Proteau, P. J. Bioorg. Med. Chem.
2006, 14, 2375-2385.
(8) Ortmann, R.; Wiesner, J.; Reichenberg, A.; Henschker, D.; Beck,
E.; Jomaa, H.; Schlitzer, M. Arch. Pharm. 2005, 338, 305-314.
(9) Ortmann, R.; Wiesner, J.; Reichenberg, A.; Henschker, D.; Beck,
E.; Jomaa, H.; Schlitzer, M. Bioorg. Med. Chem. Lett. 2003, 13, 2163-
2166.
(10) Reichenberg, A.; Wiesner, J.; Weidemeyer, C.; Dreiseidler, E.;
Sanderbrand, S.; Altincicek, B.; Beck, E.; Schlitzer, M.; Jomaa, H. Bioorg.
Med. Chem. Lett. 2001, 11, 833-835.
(11) Kuntz, L.; Tritsch, D.; Grosdemange-Billiard, C.; Hemmerlin, A.;
Willem, A.; Bach, T. J.; Rohmer, M. Biochem. J. 2005, 386, 127-135.
(12) Devreux, V.; Wiesner, J.; Goeman, J. L.; Van der Eycken, J.; Jomaa,
H.; Van Calenbergh, S. J. Med. Chem. 2006, 49, 2656-2660.
(13) Haemers, T.; Wiesner, J.; Busson, R.; Jomaa, H.; Van Calenbergh,
S. Eur. J. Org. Chem. 2006, 3856-3863.
(14) Haemers, T.; Wiesner, J.; Van Poecke, S.; Goeman, J.; Henschker,
D.; Beck, E.; Jomaa, H.; Van Calenbergh, S. Bioorg. Med. Chem. Lett.
2006, 16, 1888-1891.
(15) Kurz, T.; Schlu¨ter, K.; Kaula, U.; Bergmann, B.; Walter, R. D.;
Geffken, D. Bioorg. Med. Chem. 2006, 14, 5121-5135.
(16) Schlu¨ter, K.; Walter, R. D.; Bergmann, B.; Kurz, T. Eur. J. Med.
Chem. 2006, 41, 1385-1397.
Subsequently, a palladium(0)-catalyzed addition of Bu3SnH
to the triple bond of compound 9 afforded 1-tributylstannyl-
propenylphosphonate 6 as a single isomer in an excellent yield
3
of 90%. The E-geometry was assigned based on the JPH
1
coupling constant in the H NMR spectrum (62.7 Hz), which
is in accordance with the large coupling constant typically found
(17) Poss, A. J.; Belter, R. K. J. Org. Chem. 1987, 52, 4810-4812.
(18) Gurjar, M. K.; Krishna, L. M.; Reddy, B. S.; Chorghade, M. S.
Synthesis 2000, 557-560.
(19) Murray, A. P.; Miller, M. J. J. Org. Chem. 2003, 68, 191-194.
(20) Reddy, P. A.; Schall, O. F.; Wheatley, J. R.; Rosik, L. O.; McClurg,
J. P.; Marshall, G. R.; Slomczynska, U. Synthesis 2001, 1086-1092.
3784 J. Org. Chem., Vol. 72, No. 10, 2007

R-Aryl-Substituted Fosmidomycin Analogues
SCHEME 1. Synthesis of r-Aryl-Substituted N-Acetylfosmidomycin Analoguesa
a Reagents and conditions: (a) (i) n-BuLi, THF, -78 °C; (ii) (EtO)2P(O)Cl; HOAc; (iii) p-TsOH, MeOH, rt; (b) NsNHOBn, DEAD, PPh3, THF, rt; (c)
Bu3SnH, Pd(PPh3)4, THF, 0 °C; (d) Ar-I, Pd2dba3·CHCl3, (2-furyl)3P, CuI, NMP, rt; (e) (i) PhSH, K2CO3, MeCN, DMSO, 70 °C; (ii) Ac2O; (f) H2, Pd/C,
Na2CO3, THF, rt; (g) BCl3, CH2Cl2, -50 °C; (h) TMSBr, MeCN, rt; C-18 RP-HPLC; (i) TMSBr, MeCN, rt; NH4OH(aq); CF11-cellulose chromatography.
for a vinylic proton trans to a phosphonate.21 Optimization of
the Stille coupling on the organotin derivative 6 afforded
compounds 10f,g in good yields using a combination of Pd2-
dba3·CHCl3, tri(2-furyl)phosphine, and anhydrous CuI in NMP.22
Under these conditions, apparently no homo-coupling of 6 was
detected and the geometry of the double bond was retained.
Removal of the nosyl group of 10f,g with thiophenol and K2-
CO3 followed by in situ acylation, however, provided com-
pounds 12f,g in rather disappointing yields.23 Apparently, the
specific conditions used for the deprotection of the nosyl group
are not fully compatible with the (substituents on the) aryl
moiety. Therefore, we investigated if steps d and e could be
interchanged. An additional advantage of moving the nosyl
deprotection step forward in the synthesis is that it enhances
the divergent character of the sequence. Thus, compound 6 was
first deprotected and in situ acylated in excellent yield.
Subsequent Stille coupling on the organotin compound 11 using
the above-mentioned conditions led to compounds 12a-h in
good to excellent yields. The method proves highly compatible
with a wide range of diverse functionalities, including hetere-
oaromatic rings (12g,h). The current methodology requires the
use of aryl iodides, since aryl bromides only induced homo-
coupling of 6. 4-Iodobenzamide, required for preparing 12c, is
not commercially available, but was readily synthesized from
4-iodobenzonitrile.
Reduction of the R,â-double bond, combined with removal
of the benzyl protecting group on compounds 12a-f, was
accomplished using palladium on carbon (10% Pd/C) and
anhydrous Na2CO3 in dry THF under slightly positive atmo-
spheric H2-pressure. Reaction times varied from 3 to 14 h to
afford compounds 13a-d,i,j in moderate to excellent yields.
During this hydrogenation step little or no deoxygenation of
the hydroxamic acid was observed, in contrast to previously
reported N-O bond cleavage during catalytic hydrogenation.13
In case of the conversion of compound 12b, short reaction times
were necessary to prevent reduction of the nitrile substituent
on the phenyl ring. Reduction of the nitro group in compounds
12e,f was unavoidable, resulting in amino-compounds 13i,j.
Furthermore, reduction of the 2-thienyl (12g) and 3-thienyl (12h)
compounds was successfully accomplished only when using
more than 1 equiv of Pd/C. Presumably the sulfur atom in the
thiophene ring is poisoning the catalyst. Remarkably, simulta-
neous removal of the benzyl protecting group was not observed
for these thienyl compounds, yielding the O-protected saturated
compounds 14g,h. Subsequent deprotection of the benzyl
protecting group with BCl3 afforded compounds 13g,h in good
to excellent yields. Finally, the phosphonate ester groups of the
(21) Xiong, Z. C.; Huang, X. J. Chem. Res. 2003, 372-373.
(22) Sparks, S. M.; Gutierrez, A. J.; Shea, K. J. J. Org. Chem. 2003, 68,
5274-5285.
(23) Maligres, P. E.; See, M. M.; Askin, D.; Reider, P. J. Tetrahedron
Lett. 1997, 38, 5253-5256.
J. Org. Chem, Vol. 72, No. 10, 2007 3785

Devereux et al.
is Hammett σ+-controlled (4-CN > 4-SO2NH2 > 4-CF3 >
4-NH2). A similar trend was observed for the previously reported
R-phenyl-substituted analogues.14 The weak inhibitory activity
of the p-carbamoylphenyl analogue 4c of p-carbamoylphenyl
analogue 4c illustrates that the activity pattern is not exclusively
Hammett σ+-controlled. Also, incorporation of a 2- or 3-thienyl
moiety in analogues (4g,h) negatively affects the inhibitory
potency.
TABLE 1. Inhibition of Recombinant E. coli DXR by Analogues
3-5
compound
Ar
R
R
IC50 (µM)
3a
4b
4c
3d
4g
4h
3i
(4-CF3)Ph
(4-CN)Ph
(4-CONH2)Ph
(4-SO2NH2)Ph
2-thienyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
0.360
0.119
2.84
0.318
0.603
0.483
1.45
3.60
0.054
0.056
NH4
NH4
H
NH4
NH4
H
H
NH4
Na
3-thienyl
In previous studies, the R-aryl-substituted N-formyl-fosmi-
domycin analogues were generally superior DXR inhibitors as
compared to their N-acetyl counterparts. Hence, we decided to
synthesize the N-formyl congener of 4b, the most potent
N-acetyl analogue in the present study. Toward the synthesis
of 5 the above-mentioned strategy proved equally useful
upon substituting in situ N-acetylation for N-formylation, using
(3-NH2)Ph
(4-NH2)Ph
(4-CN)Ph
H
3j
5
fosmidomycin (1)
H
R-aryl FR900098 analogues 13a-d,g,j were cleaved with
TMSBr. Two different methods for purification of the final
phosphonic acid compounds were used. The first, more tradi-
tional method involves removal of the solvent, addition of water,
lyophilization of the solution, and purification of the solids via
reversed phase chromatography. This method was used for the
purification of compounds 3a,d,i,j but suffers from some serious
drawbacks. First, the resulting phosphonic acids are only
sparingly soluble in water or MeOH. Second, the C-18 reversed
phase column used for purification strongly absorbs the phos-
phonic acids 3a,d,i,j, resulting in a low recovery. This pro-
nounced retention was not observed for more polar fosmido-
mycin analogues,12 hence it could be correlated to the affinity
of the aryl moiety for the stationary phase of the HPLC column.
Third, the C-18 HPLC column used only allows for a maximum
loading of 30 mg of crude material each time, which is very
time-consuming for the purification of larger amounts of crude
analogues. Therefore, a second methodology was investigated,
comprising removal of the solvent and addition of water
followed by dropwise addition of a 5% aqueous NH4OH solution
until pH 8-9 was reached. Subsequently, the solution was
lyophilized and the product was purified via column chroma-
tography using Whatman CF11-cellulose.24 LC-MS analysis of
the crude solids before chromatographic purification only
showed NH4Br (resulting from a reaction of TMSBr with NH4-
OH) as a contaminant. The CF11-cellulose proved very effective
in separating this contaminant from compounds 4b,c,g,h, which
were isolated in high yields due to the low affinity for the
stationary phase. Additionally the resulting bis-ammonium salts
of the R-aryl phosphonates showed excellent solubility in water.
Because of the difficulties associated with the handling of
P. falciparum DXR, we investigated the ability of the synthe-
sized R-aryl-substituted FR900098 analogues 3a,d,i,j and
4b,c,g,h to inhibit the highly homologous recombinant E. coli
DXR. The conversion of DOXP to MEP by the enzyme was
determined in an assay based on the NADPH dependency of
the reaction.25 With respect to the slow, tight-binding properties
of fosmidomycin and probably most of its derivatives the
inhibitors were preincubated with the enzyme and NADPH
before the reaction was started by the addition of DOXP.7,11,26
The results are summarized in Table 1. Compared to fosmido-
mycin, all synthesized analogues were found to be weaker
inhibitors of E. coli DXR. Analysis of the order of DXR
inhibitory potency (4b > 3d > 3a > 3j) suggests that activity
a
mixture of HCOOH and 1,1′-carbonyldiimidazole.13
(Scheme 2).
In the enzyme assay (Table 1), compound 5 was ap-
proximately twofold more active than its acetyl congener 4b
and equally active to fosmidomycin (1). More importantly, an
in vitro antimalarial assay using intraerythrocytic stages of the
P. falciparum D2d strain, proved that the activities of 4b (IC50
) 0.27 µM) and 5 (IC50 ) 0.27 µM) surpass the activity of
fosmidomycin (IC50 ) 1.1 µM) and even FR900098 (IC50
0.49 µM) to inhibit parasite growth.
)
In conclusion, a highly accessible, divergent procedure for
the preparation of R-aryl-substituted fosmidomycin analogues
3-5 with varying substituents was developed using a palladium-
(0)-catalyzed Stille coupling as a key step. The required
organotin precursor 6 was prepared regio- and stereoselectively
in four steps from protected propargyl alcohol 7, using a Pd-
(0)-catalyzed addition of tributyltin hydride as a key step. Two
compounds (4b and 5), both possessing an R-4-cyanophenyl
group, exhibited a promising in Vitro inhibitory activity for
recombinant E. coli DXR comparable to that of fosmidomycin
and were even found to show a higher activity than fosmido-
mycin in an in Vitro test against P. falciparum D2d strain.
Experimental Section
Diethyl 3-Hydroxy-propynylphosphonate (8). A 1.6 M n-BuLi
in hexanes (47.6 mL, 71.3 mmol) was added dropwise to a stirred
solution of tetrahydro-2-(2-propynyloxy)-2H-pyran (7, 10 g, 71.3
mmol) in dry THF (100 mL) at -78 °C. After stirring for 30 min
at the same temperature, a solution of diethyl chlorophosphate (11.4
mL, 78.5 mmol) in dry THF (100 mL) was added dropwise, after
which the mixture was stirred for another 30 min at -78 °C. The
reaction was quenched with saturated aqueous NH4Cl (100 mL),
and the aqueous layer was extracted three times with Et2O (100
mL). The combined organic layers were dried on anhydrous MgSO4
and filtered, and the solvents were removed under reduced pressure.
The residual oil was dissolved in MeOH (200 mL), p-TsOH (1.36
g, 7.13 mmol) was added, and the reaction mixture was stirred
overnight at rt. The solvent was removed under reduced pressure,
and the residual oil was purified via column chromatography
(pentane/acetone: 3/2) yielding 8.75 g of a colorless oil (64%). Rf
0.30 (pentane/acetone: 3/2); 1H NMR (300.13 MHz, CDCl3) δ 1.36
(6H, td, J ) 7.1 and 0.6 Hz), 2.89 (1H, br s), 4.16 (4H, dq, J ) 8.3
and 7.0 Hz), 4.35 (2H, d, J ) 3.7 Hz); 13C NMR (75.47 MHz,
CDCl3) δ 16.0 (CH3, 3JPC ) 7.1 Hz), 50.6 (OCH2, 3JPC ) 4.4 Hz),
63.5 (OCH2, 2JPC ) 5.5 Hz), 76.3 (≡C-P, 1JPC ) 298.6 Hz), 100.0
(24) Woodside, A. B.; Zheng, H.; Poulter, C. D. Org. Synth. 1988, 66,
211-219.
2
(≡C, JPC ) 50.5 Hz); 31P NMR (121.50 MHz, CDCl3) δ -6.7;
(25) Kuzuyama, T.; Takahashi, S.; Watanabe, H.; Seto, H. Tetrahedron
Lett. 1998, 39, 4509-4512.
ESMS m/z 193 [M + H]+.
Diethyl 3-[N-Benzyloxy,N-(2-nitrobenzenesulfonyl)amino]-
propynylphosphonate (9). To a stirred solution of alcohol 8 (6.58
(26) Koppisch, A. T.; Fox, D. T.; Blagg, B. S. J.; Poulter, C. D.
Biochemistry 2002, 41, 236-243.
3786 J. Org. Chem., Vol. 72, No. 10, 2007

R-Aryl-Substituted Fosmidomycin Analogues
SCHEME 2. Synthesis of r-Aryl-Substituted Fosmidomycin Analogue 5a
a Reagents and conditions: (a) (i) PhSH, K2CO3, MeCN, DMSO, 70 °C; (ii) 1,1′-carbonyldiimidazole, HCOOH, 0 °C; (b) 4-iodobenzonitrile, Pd2dba3·CHCl3,
(2-furyl)3P, CuI, NMP, rt; (c) H2, Pd/C, Na2CO3, THF, rt; (d) TMSBr, MeCN, rt; NH4OH(aq); CF11-cellulose chromatography.
g, 34.2 mmol), N-benzyloxy,N-(2-nitrobenzene)sulfonamide (11.5
g, 37.3 mmol), and PPh3 (9.8 g, 37.3 mmol) in dry THF (180 mL)
was added a solution of DEAD (6.91 mL 85%, 37.3 mmol) in dry
THF (90 mL) over 1 h at rt. The mixture was stirred for 20 h at
room temperature. The solvent was removed under reduced
pressure, and the residual oil was purified via column chromatog-
raphy (column 1: pentane/CH2Cl2/acetone: 2/7/1, column 2: Et2O
f CH2Cl2/acetone: 1/1), yielding 11.7 g of a thick amber oil (71%).
stirred for 1 h at rt. A solution of 6 (216 mg, 0.280 mmol) and
1-iodo-4-nitrobenzene (62 mg, 0.249 mmol) in anhydrous NMP (5
mL) was added dropwise over a 15 min period. After the addition
of anhydrous CuI (46 mg, 0.280 mmol), the reaction mixture was
stirred overnight protected from light. EtOAc (100 mL) was added,
and the organic layer was washed three times with 5% NH4OH
(10 mL) and three times with saturated aqueous NaCl (10 mL).
The organic layer was dried over anhydrous MgSO4 and filtered,
and the solvents were removed under reduced pressure. The residual
oil was purified by column chromatography (pentane/CH2Cl2/
acetone: 3/1/1) yielding 98 mg of a pale yellow oil (58%). Rf 0.48
(pentane/acetone: 1/1); 1H NMR (300.13 MHz, CDCl3) δ 1.16 (6H,
t, J ) 7.0 Hz), 3.92 (2H, ddq, J ) 10.4, 8.4 and 7.1 Hz), 4.01 (2H,
ddq, J ) 10.3, 8.1 and 7.0 Hz), 4.47 (2H, dd, J ) 5.7 and 3.3 Hz),
5.01 (2H, s), 6.05 (1H, dt, J ) 46.0 and 6.0 Hz), 7.16-7.33 (7H,
m), 7.61 (1H, dd, J ) 7.7 and 1.4 Hz), 7.72 (1H, app dt, J ) 7.7
and 1.4 Hz), 7.79 (1H, app dt, J ) 7.7 and 1.5 Hz), 8.1 (1H, dd,
J ) 8.0 and 1.4 Hz), 8.14 (2H, d, J ) 8.4 Hz); 13C NMR (75.47
1
Rf 0.40 (pentane/CH2Cl2/acetone: 2/7/1); H NMR (300.13 MHz,
CDCl3) δ 1.29 (6H, dt, J ) 7.1 and 0.6 Hz), 3.99-4.06 (4H, m),
4.18 (2H, d, J ) 3.9 Hz), 5.15 (2H, s), 7.36-7.45 (5H, m), 7.56
(1H, dd, J ) 7.8 and 1.2 Hz), 7.67 (1H, app dt, J ) 7.7 and 1.3
Hz), 7.78 (1H, app dt, J ) 7.7 and 1.5 Hz), 8.04 (1H, dd, J ) 7.9
4
and 1.3 Hz); 13C NMR (75.47 MHz, CDCl3) δ 16.0 (CH3, JPC
)
7.2 Hz), 43.1 (NCH2, 3JPC ) 5.5 Hz), 63.3 (OCH2, 3JPC ) 5.5 Hz),
1
2
76.1 (≡C-P, JPC ) 293.7 Hz), 80.6 (OCH2), 91.9 (≡C, JPC
)
51.0 Hz), 123.9 (dCH), 126.O (dC), 128.7 (dCH), 129.2 (dCH),
129.9 (dCH), 131.4 (dCH), 133.2 (dCH), 134.2 (dC), 135.6 (d
CH), 149.5 (dC); 31P NMR (121.50 MHz, CDCl3) δ 8.2; Exact
mass (ESI-MS): calculated for C20H23N2O8PS [M + H]+ 483.0991;
found 483.0965.
3
3
MHz, CDCl3) δ 16.1 (CH3, JPC ) 6.6 Hz), 53.1 (NCH2, JPC
)
5.4 Hz), 62.3 (OCH2, 3JPC ) 6.0 Hz), 79.6 (OCH2), 123.4 (dCH),
123.9 (dCH), 126.0 (dC), 128.5 (dCH), 128.8 (dCH, 3JPC ) 1.1
Hz), 129.0 (dCH), 130.1 (dCH), 131.3 (dCH), 132.6 (dC-P,
1JPC ) 176.2 Hz), 132.7 (dCH), 134.8 (dC) 135.2 (dCH), 145.3
Diethyl (E)-3-[N-(Benzyloxy),N-(2-nitrobenzenesulfonyl)-
amino]-1-(tributylstannanyl)-propenylphosphonate (6). To a
cooled (0 °C) solution of 9 (10.4 g, 21.6 mmol) and Pd(PPh3)4
(474 mg, 0.430 mmol) in dry THF (100 mL) was added Bu3SnH
(6.28 g, 21.6 mmol) over a period of 40 min. The reaction mixture
was stirred at 0 °C for 1 h and then allowed to warm to room
temperature in 2 h. The solvent was removed and the crude oil
was purified by column chromatography (pentane/acetone: 3/1),
yielding 15 g of an amber oil (90%). Rf 0.31 (pentane/acetone: 3/1);
1H NMR (300.13 MHz, CDCl3) δ 0.87 (9H, t, J ) 7.2 Hz), 0.93-
0.98 (6H, m), 1.19 (6H, t, J ) 7.0 Hz), 1.26-1.35 (6H, m), 1.42-
1.52 (6H, m), 3.88-3.95 (4H, m), 4.39-4.41 (2H, m), 5.06 (2H,
s), 6.53 (1H, dt, J ) 62.7 and 5.5 Hz), 7.33-7.35 (5H, m), 7.54
(1H, d, J ) 7.7 Hz), 7.62 (1H, app t, J ) 7.6 Hz), 7.71 (1H, app
t, J ) 7.5 Hz), 8.04 (1H, d, J ) 8.0 Hz); 13C NMR (75.47 MHz,
CDCl3) δ 10.8 (CH2), 13.6 (CH3), 27.3 (CH2), 28.8 (CH2), 55.2
(NCH2, 3JPC ) 13.8 Hz), 61.1 (OCH2, 3JPC ) 5.5 Hz), 80.2 (OCH2),
123.8 (dCH), 126.6 (dC), 128.5 (dCH), 128.9 (dCH), 129.6 (d
CH), 131.1 (dCH), 132.6 (dCH), 136.2 (dC), 136.4 (dC-P, 1JPC
) 247.6 Hz), 149.8 (dC), 152.1 (dCH, 2JPC ) 1.7 Hz); 31P NMR
(121.50 MHz, CDCl3) δ 21.4; Exact mass (ESI-MS): calculated
for C32H51N2O8PSSn [M(120Sn) + H]+ 797.2024; found 797.1999.
Diethyl 3-[N-Benzyloxy,N-(2-nitrobenzenesulfonyl)amino]-1-
(4-nitrophenyl)-propenylphosphonate (10f). (2-Furyl)3P (12 mg,
0.051 mmol) was added to a solution of Pd2dba3·CHCl3 (6 mg,
0.0065 mmol) in anhydrous NMP (2 mL), and the mixture was
2
2
(dC, JPC ) 10.2 Hz), 145.8 (dCH, JPC ) 8.2 Hz), 147.3 (dC,
5JPC ) 1.1 Hz), 149.9 (dC); 31P NMR (121.50 MHz, CDCl3) δ
13.2; Exact mass (ESI-MS): calculated for C26H28N3O10PS [M +
H]+ 606.1311; found 606.1301.
Diethyl (E)-3-[N-Acetyl,N-(benzyloxy)amino)-1-(tributylstan-
nyl)-propenylphosphonate (11). To a stirred solution of 6 (15 g,
19.4 mmol) and anhydrous K2CO3 (10.8 g, 77.8 mmol) in MeCN
(500 mL) containing dry DMSO (10 mL) was added PhSH (6 mL,
58.3 mmol) over a 30 min-period at 70 °C. After stirring for 1 h at
the same temperature, Ac2O (18.3 mL, 195 mmol) was added and
the reaction was stirred for another 30 min. After cooling to room
temperature, the reaction was quenched with saturated NaHCO3.
The aqueous layer was extracted with CH2Cl2; the combined organic
fractions were dried over anhydrous MgSO4, filtered, and concen-
trated under vacuum. The residual oil was purified via column
chromatography (pentane/CH2Cl2/acetone: 2/1/1) yielding 11 g of
1
a yellow oil (90%). Rf 0.35 (pentane/CH2Cl2/acetone: 3/1/1); H
NMR (300.13 MHz, CDCl3) δ 0.85 (9H, t, J ) 7.3 Hz), 0.94-
0.99 (6H, m), 1.22-1.34 (12H, m), 1.41-1.51 (6H, m), 2.07 (3H,
s), 3.94-4.11 (4H, m), 4.86 (2H, s), 4.89-4.99 (2H, m), 6.48 (1H,
dt, J ) 63.5 and 5.9 Hz); 13C NMR (75.47 MHz, CDCl3) δ 10.6
3
(CH2), 13.6 (CH3), 16.4 (CH3, JPC ) 6.6 Hz), 20.5 (CH3), 27.3
(CH2), 28.7 (CH2), 57.1 (NCH2), 61.1 (OCH2, 2JPC ) 5.5 Hz), 76.0
(OCH2), 128.6 (dCH), 128.9 (dCH), 129.5 (dCH), 134.4 (dC),
J. Org. Chem, Vol. 72, No. 10, 2007 3787

Devereux et al.
135.1 (dC-P, 1JPC ) 133.4 Hz), 155.0 (dCH), 172.1 (N-CdO);
Exact mass (ESI-MS): calculated for C28H50NO5PSn [M(120Sn) +
H]+ 632.2527; found 632.2528.
76.2 (OCH2, major), 76.4 (OCH2, minor), 124.8 (dCH, 5JPC ) 2.8
4
Hz), 126.9 (dCH,3JPC ) 3.3 Hz), 127.0 (dCH, JPC ) 8.2 Hz),
128.7 (dCH), 128.9 (dCH), 129.2 (dCH), 134.4 (dCH), 137.4
2
(dC, JPC ) 8.8 Hz), 171.2 (N-CdO); 31P NMR (121.50 MHz,
Diethyl (Z)-3-[N-Acetyl,N-(benzyloxy)amino]-1-(4-trifluorom-
ethylphenyl)-propenylphosphonate (12a). (2-Furyl)3P (27.2 mg,
0.116 mmol) was added to a solution of Pd2dba3·CHCl3 (13.6 mg,
0.0149 mmol) in anhydrous NMP (3 mL), and the mixture was
stirred at rt for 1 h. A solution of 11 (400 mg, 0.634 mmol) and
1-iodo-4-trifluoromethylbenzene (154 mg, 0.566 mmol) in anhy-
drous NMP (5 mL) was added over a period of 15 min. After the
addition of anhydrous CuI (104 mg, 0.634 mmol), the reaction
mixture was stirred overnight protected from light. EtOAc (200
mL) was added and the organic layer was washed three times with
5% NH4OH (24 mL) and three times with saturated aqueous NaCl
(24 mL). The organic layer was dried over anhydrous MgSO4,
filtered, and concentrated under vacuum. The residual oil was
purified by column chromatography (pentane/acetone: 55/45) to
yield 228 mg of a pale yellow oil (88%). Rf 0.43 (pentane/acetone:
CDCl3) δ 25.9; Exact mass (ESI-MS): calculated for C20H28NO5-
PS [M + H]+ 426.1504; found 426.1488.
Diethyl 3-[N-Acetyl,N-(hydroxy)amino]-1-(thien-2-yl)-propy-
lphosphonate (13g). A 1 M solution of BCl3 in hexanes (1.6 mL,
1.60 mmol) was added dropwise to a stirred solution of 14g (169
mg, 0.397 mmol) in dry CH2Cl2 (7 mL) at -50 °C. After stirring
for 30 min at -50 °C, the reaction was quenched with saturated
NaHCO3 (10 mL) and the reaction mixture was allowed to warm
to rt. The aqueous layer was extracted with CH2Cl2, dried over
anhydrous MgSO4, and filtered, and the solvents were removed
under reduced pressure. The residual oil was purified by column
chromatography (CH2Cl2/MeOH: 94/6) to give the title compound
as a thick gray oil (69%). Rf 0.19 (CH2Cl2/Acetone: 1/1); 1H NMR
(300.13 MHz, CDCl3) δ 1.15-1.34 (6H, m), 2.14 (3H, s) 2.17-
2.60 (2H, m), 3.46 (1H, dt, J ) 22.8 and 5.3 Hz), 3.61-4.12 (6H,
m), 6.95-7.02 (2H, m), 7.20-7.22 (1H, m), 9.42 (1H, br s); 13C
1
3/2); H NMR (300.13 MHz, CDCl3) δ 1.24 (6H, t, J ) 7.1 Hz),
2.09 (3H, s), 3.98-4.18 (4H, m), 4.91 (2H, s), 5.06 (2H, dd, J )
5.8 and 2.9 Hz), 6.46 (1H, dt, J ) 47.1 and 6.4 Hz), 7.35-7.41
(5H, m), 7.43 (2H, d, J ) 8.1 Hz), 7.56 (2H, d, J ) 8.4 Hz); 13C
3
NMR (75.47 MHz, CDCl3) δ 16.3 (CH3, JPC ) 5.4 Hz), 20.6
3
NMR (75.47 MHz, CDCl3) δ 16.2 (CH3, JPC ) 6.6 Hz), 20.5
2
2
(CH3), 29.6 (CH2, major, JPC ) 3.3 Hz), 29.7 (CH2, minor, JPC
2
(CH3), 45.3 (NCH2), 62.2 (OCH2, JPC ) 5.5 Hz), 76.4 (OCH2),
1
1
3
) 3.3 Hz), 37.1 (P-CH, JPC )142.2 Hz), 46.3 (NCH2), 62.9
123.9 (CF3, q, JFC ) 286.5 Hz), 128.5 (dCH, q, JFC ) 3.4 Hz),
2
2
128.7 (dCH), 129.0 (dCH), 129.4 (dCH), 129.8 (dC, dq, 2JFC
)
(OCH2, JPC ) 6.0 Hz), 63.4 (OCH2, JPC ) 7.7 Hz), 124.9 (d
CH), 127.0 (dCH, 4JPC ) 7.5 Hz), 127.2 (dCH), 138.1 (dC), 172.5
(N-CdO); 31P NMR (121.50 MHz, CDCl3) δ 25.6, 28.0 (minor,-
major); Exact mass (ESI-MS): calculated for C13H22NO5PS [M +
Na]+ 358.0854; found 358.0842.
5
1
32.0 Hz, JPC ) 1.1 Hz), 132.9 (dC-P, JPC ) 196.5 Hz), 134.0
(dC), 142.5 (dC, 2JPC ) 11.5 Hz), 147.4 (dCH, 2JPC ) 10.5 Hz),
172.6 (N-CdO); 31P NMR (121.50 MHz, CDCl3) δ 14.3; ESMS
m/z 486 [M + H]+.
Diethyl 3-[N-Acetyl,N-(hydroxy)amino]-1-(4-trifluorometh-
ylphenyl)-propylphosphonate (13a). To a solution of 12a (266
mg, 0.547 mmol) in dry THF (8.5 mL) were added anhydrous Na2-
CO3 (174 mg, 1.64 mmol) and Pd/C (133 mg, 10%). The suspension
was hydrogenated at room temperature and at a slightly positive
atmospheric pressure for 4 h. The catalyst was removed by filtration
through celite, which was washed with portions of CH2Cl2. The
filtrate was evaporated under reduced pressure and the residual oil
was purified by column chromatography (CH2Cl2/MeOH: 94/6)
3-[N-Acetyl,N-(hydroxy)amino]-1-(4-trifluoromethylphenyl)-
propylphosphonic Acid (3a). To a solution of 13a (150 mg, 0.377
mmol) in dry MeCN (3.8 mL) was added dropwise TMSBr (497
µL, 3.77 mmol) at rt, and the mixture was stirred for 2 days at rt.
The solvents were removed under reduced pressure, and the
remaining traces of TMSBr were removed under high vacuum (0.05
mbar). The residual oil was dissolved in 2 mL of distilled water
and lyophilized. The resulting solid was purified via reversed phase
HPLC using a 5 mM NH4OAc solution for 5 min followed by a
gradient elution of 5 mM NH4OAc solution to MeCN in 15 min.
The appropriate fractions were lyophilized to give 23 mg of the
phosphonic acid as a colorless hygroscopic solid (23%). 1H NMR
(300.13 MHz, D2O) δ 1.75 (3H, minor, s), 2.01 (3H, major, s),
2.25-2.61 (2H, m), 3.00-3.18 (1H, m), 3.43-3.67 (2H, m), 7.46-
7.64 (2H, m), 7.66-7.84 (2H, m); 19.3 (CH3), 26.7 (CH3, major),
1
yielding a colorless oil (85%). Rf 0.29 (CH2Cl2/acetone: 1/1); H
NMR (300.13 MHz, CDCl3) δ 1.19 (3H, t, J ) 7.1 Hz), 1.23 (3H,
t, J ) 7.1 Hz), 2.12 (3H, s), 2.14-2.61 (2H, m), 3.18 (1H, dt, J )
23.1 and 6.1 Hz), 3.36 (1H, dt, J ) 14.2 and 4.8 Hz), 3.44-4.02
(5H, m), 7.40 (2H, d, J ) 7.7 Hz), 7.57 (2H, d, J ) 7.9 Hz), 9.44
(1H, br s); 13C NMR (75.47 MHz, CDCl3) δ 16.2 (CH3, 3JPC ) 6.0
2
Hz), 20.5 (CH3), 27.9 (CH2, JPC ) 2.2 Hz), 41.0 (P-CH, minor,
1
1JPC ) 139.9 Hz), 41.8 (P-CH, major, JPC ) 137.2 Hz), 46.2
1
26.9 (CH3, minor), 43.5 (P-CH, minor, JPC ) 131.1 Hz), 44.2-
3
3
(NCH2, major, JPC ) 10.4 Hz), 46.5 (NCH2, minor, JPC ) 15.9
1
3
(P-CH, major, JPC ) 130.0 Hz), 46.3 (NCH2, JPC ) 17.6 Hz),
2
2
Hz), 62.8 (OCH2, JPC ) 7.2 Hz), 63.2 (OCH2, JPC ) 7.7 Hz),
121.3 (CF3, q, 1JFC ) 273.2 Hz), 125.5 (dCH), 129.5 (dCH, 3JPC
) 6.6 Hz), 129.6 (dC, dq, 2JFC ) 33.2 Hz, 5JPC ) 2.2 Hz), 140.8
1
121.9 (CF3, q, JFC ) 271.1 Hz) 125.3 (dCH, major), 125.6 (d
CH, minor), 128.1 (dC, dq, 2JFC ) 31.8 Hz, 5JPC ) 3.3 Hz), 129.6
(dCH, 3JPC ) 5.5 Hz), 142.8 (dC, minor), 143.0 (dC, major, 2JPC
(dC, JPC ) 7.7 Hz), 172.5 (N-CdO); 31P NMR (121.50 MHz,
2
) 7.2 Hz), 173.7 (N-CdO, major)? 173.8 (N-CdO, minor); 31
P
CDCl3) δ 26.6, 28.7 (minor, major); Exact mass (ESI-MS):
calculated for C16H23F3NO5P [M + H]+ 398.1344; found 398.1331.
Diethyl 3-[N-Acetyl,N-(benzyloxy)amino]-1-(thien-2-yl)-pro-
pylphosphonate (14g). The title compound was prepared according
to the procedure described for 13a, but using 1.2 eq of 10% Pd/C,
resulting in a thick gray oil (50%). Rf 0.44 (pentane/acetone: 1/1);
1H NMR (300.13 MHz, CDCl3) 1.15 (3H, t, J ) 7.0 Hz), 1.26
(3H, t, J ) 7.2 Hz), 2.03 (3H, s), 2.12-2.26 (1H, m), 2.40-2.54
(1H, m), 3.39 (1H, ddd, J ) 22.9, 11.3 and 3.7 Hz), 3.52 (1H,
ddd, J ) 14.3, 8.5 and 4.9 Hz), 3.66-3.73 (1H, m), 3.77-4.12
(4H, m), 4.71 (1H, br s), 4.81 (1H, br s), 6.95-7.00 (2H, m), 7.20-
7.23 (1H, m), 7.29-7.27 (5H, m); 13C NMR (75.47 MHz, CDCl3)
δ 16.3 (CH3, 3JPC ) 6.0 Hz), 16.4 (CH3, 3JPC ) 6.0 Hz), 20.5 (CH3),
28.5 (CH2, 2JPC ) 1.7 Hz), 33.6 (P-CH, minor, 1JPC ) 139.8 Hz),
37.5 (P-CH, major, 1JPC ) 143.8 Hz), 43.7 (NCH2), 61.5 (OCH2,
NMR (121.50 MHz, D2O) δ 21.5; Exact mass (ESI-MS): calculated
for C12H15F3NO5P [M - H]- 340.0562; found 340.0564.
Bisammonium 3-[N-Acetyl,N-(hydroxy)amino]-1-(4-cyanophe-
nyl)-propylphosphonate (4b). To a solution of 13b (130 mg, 0.367
mmol) in dry MeCN (3.7 mL) was added dropwise TMSBr (484
µL, 3.67 mmol) at rt, and the mixture was stirred for 24 h at rt.
The solvents were removed under reduced pressure, and the traces
of TMSBr were removed under high vacuum (0.05 mbar). The
residual oil was dissolved in 2 mL of Type I water, and the pH of
the mixture was adjusted to 8-9 with a 5% NH4OH solution. The
solution was lyophilized, and the residual solid was purified by
Whatman CF11 cellulose column chromatography (MeCN/NH4-
OH (aq, 1 M): 3/1). The fractions were assayed using cellulose TLC,
and the spots were visualized under UV-light (365 nm) after dipping
in a pinacryptol yellow solution (0.1% in H2O) and drying the plate
2
2JPC ) 6.6 Hz, major), 61.6 (OCH2, JPC ) 6.6 Hz, minor), 62.3
(OCH2, 2JPC ) 7.1 Hz, major), 62.9 (OCH2, 2JPC ) 7.1 Hz, minor),
3788 J. Org. Chem., Vol. 72, No. 10, 2007

R-Aryl-Substituted Fosmidomycin Analogues
under a stream of hot air.27 The appropriate fractions were
lyophilized, yielding 79 mg of a pale amber hygroscopic solid
(66%). Rf 0.33 (cellulose TLC, MeCN/NH4OH(aq,1M): 3/1); 1H NMR
(300.13 MHz, D2O) δ 1.64 (3H, minor, s), 1.90 (3H, major, s),
2.12-2.49 (2H, m), 2.94 (1H, ddd, J ) 22.0, 12.1 and 2.8 Hz),
3.35 (1H, app dt, J ) 14.4 and 5.7 Hz), 3.50 (1H, ddd, J ) 14.4,
8.4 and 6.1 Hz), 7.39 (2H, dd, J ) 8.3 and 1.7 Hz), 7.65 (2H, d,
J ) 8.2 Hz); 13C NMR (75.47 MHz, D2O) δ 19.1 (CH3, major),
19.3 (CH3, minor), 26.5 (CH2, major), 26.7 (CH2, minor), 44.0
(P-CH, minor, 1JPC ) 126.8 Hz), 44.5 (P-CH, major, 1JPC ) 127.4
Hz), 46.1 (NCH2, major, 3JPC ) 17.1 Hz), 49.6 (NCH2, minor, 3JPC
t, J ) 7.2 Hz), 0.95-1.00 (6H, m), 1.22-1.34 (12H, m), 1.41-
1.57 (6H, m), 3.95-4.06 (6H, m), 4.71 (2H, minor, s), 4.87 (4H,
major, m), 6.34-6.55 (1H, minor, m), 6.71 (1H, major, dt, J )
64.2 and 5.8 Hz), 7.27-7.35 (5H, m), 8.16 (1H, br s); 13C NMR
(75.47 MHz, CDCl3) δ 10.6 (CH2, minor), 10.7 (CH2, major), 13.6
3
(CH3, minor), 13.6 (CH3, major), 16.3 (CH3, minor, JPC ) 6.6
Hz); 16.4 (CH3, major, 3JPC )7.2 Hz), 27.2 (CH2, minor) 27.3 (CH2,
major), 28.7 (CH2 minor), 28.8 (CH2, major), 45.7 (NCH2, major,
3JPC ) 15.4 Hz), 54.0 (NCH2, minor, 3JPC ) 13.2 Hz), 61.0 (OCH2,
2
2
minor, JPC ) 5.5 Hz), 61.1 (OCH2, major, JPC ) 5.5 Hz), 76.1
(OCH2, minor), 77.0 (OCH2, major), 127.8 (dCH, minor), 128.2
(dCH, minor), 128.3 (dCH, minor), 128.6 (dCH, major), 129.0
5
) 17.0 Hz), 108.8 (dC, JPC ) 3.3 Hz), 120.0 (CtN), 129.8
(dCH, 3JPC ) 6.1 Hz), 132.3 (dCH, major, 4JPC ) 2.2 Hz), 132.6
(dCH, major), 129.5 (dCH, major), 133.9 (dC-P, JPC ) 133.4
1
4
2
(dCH, minor, JPC ) 1.6 Hz), 145.1 (dC, JPC ) 7.2 Hz), 173.6
(N-CdO); 31P NMR (121.50 MHz, D2O) δ 20.0, 20.5 (minor,
major) Exact mass (ESI-MS): calculated for C12H21N4O5P [M -
2(NH4) + H]- 297.0640; found 297.0645.
Hz), 137.7 (dC), 153.6 (dCH, minor), 157.8 (dCH, major), 162.6
(N-CdO); 31P NMR (121.50 MHz, CDCl3) δ 21.7, 22.4 (minor,
major); Exact mass (ESI-MS): calculated for C27H48NO5PSn
[M(120Sn) + H]+ 618.2370; found 618.2373.
DOXP Reductoisomerase Inhibition Assay. The assay was
performed in a reaction mixture containing 100 mM Tris HCl (pH
7.5), 0.2% bovine serum albumin, 1 mM MnCl2, 1 mM DOXP,
0.3 mM NADPH, and 1 µg/ml recombinant DOXP reductoi-
somerase of E. coli. The mixture without substrate was preincubated
with a serial dilution of the test compounds on a 96-well plate at
30 °C for 5 min. The compounds had been dissolved in 100 mM
Tris HCl (pH 7.5) and prediluted to the 10-fold final concentrations
on a separate plate. The reaction was started by the addition of
DOXP. The decrease of absorption was monitored at 340 nm.
Diethyl (E)-3-[N-(Benzyloxy),N-formylamino)-1-(tributylstan-
nyl)-propenylphosphonate (15). To a stirred solution of 6 (3.04
g, 3.93 mmol) and anhydrous K2CO3 (728 mg, 5.23 mmol) in
MeCN (100 mL), containing dry DMSO (2 mL), was added PhSH
(404 µL, 3.93 mmol) over a 30 min period at 70 °C, and the reaction
mixture was stirred overnight at the same temperature. The reaction
mixture was then cooled to 0 °C, and the formylating solution
[prepared in a separate flask by adding HCOOH (99%, 163 µL,
4.32 mmol) dropwise to a solution of 1,1′-carbonyldiimidazole (702
mg, 4.32 mmol) in dry CH2Cl2 (6.4 mL) at 0 °C for and stirring
for 1 h] was added dropwise at 0 °C. The reaction mixture was
stirred for 1 h at 0 °C. The reaction was quenched with saturated
NaHCO3, and the aqueous layer was extracted with CH2Cl2. The
combined organic fractions were dried over anhydrous MgSO4 and
filtered, and the solvents were removed under reduced pressure.
The residual oil was purified by column chromatography (pentane/
CH2Cl2/acetone: 4/1/1) to give 1.42 g of the title compound as a
yellow oil (59%). Rf 0.43 (hexane/acetone: 7/3); 1H NMR (300.13
MHz, CDCl3) δ 0.86 (6H, minor, t, J ) 7.3 Hz), 0.87 (6H, major,
Acknowledgment. This study was supported by grants from
the European Commission (Antimal, LSHP-CT-2005-018834)
and INTAS (03-51-4077). We thank Dajana Henschker for
excellent technical assistance in performing the biological
assays.
Supporting Information Available: Experimental details (in-
1
cluding H, 13C, 31P NMR, MS data) for intermediates and final
products (3d,i,j, 4c,g,h, 5, 10g, 12b-h, 13b-d,h-j, 14h, 16, and
17), as well as full APT spectra of all new compounds. This material
is available free of charge via Internet at http://pubs.acs.org.
(27) Jork, H.; Funk, W.; Fischer, W.; Wimmer, H. Thin-Layer Chro-
matography: Reagents and Detection Methods. Vol. 1a. Physical and
Chemical Detection Methods: Fundamentals, Reagents I; Wiley-VCH:
Weinheim, 1990.
JO0700981
J. Org. Chem, Vol. 72, No. 10, 2007 3789