Synthesis of N-Substituted phosphoramidic acid esters as “reverse” fosmidomycin analogues

Article abstract of DOI:10.1016/j.tet.2019.02.003

An efficient synthetic pathway to a series of novel “reverse” fosmidomycin analogues has been developed, commencing from substituted benzylamines. In these analogues, the fosmidomycin hydroxamate moiety is reversed and the tetrahedral methylene carbon adjacent to the phosphonate moiety is replaced by a nitrogen atom bearing different benzyl groups. The resulting phosphonate esters were designed as potential antimalarial “pro-drugs”.

Full text of DOI:10.1016/j.tet.2019.02.003

Accepted Manuscript
Synthesis of N-Substituted phosphoramidic acid esters as “reverse” fosmidomycin
analogues
Christiana M. Adeyemi, Heinrich C. Hoppe, Michelle Isaacs, Rosalyn Klein, Kevin A.
Lobb, Perry T. Kaye
PII:
S0040-4020(19)30141-3
https://doi.org/10.1016/j.tet.2019.02.003
TET 30126
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 15 December 2018
Revised Date: 30 January 2019
Accepted Date: 1 February 2019
Please cite this article as: Adeyemi CM, Hoppe HC, Isaacs M, Klein R, Lobb KA, Kaye PT, Synthesis of
N-Substituted phosphoramidic acid esters as “reverse” fosmidomycin analogues, Tetrahedron (2019),
doi: https://doi.org/10.1016/j.tet.2019.02.003.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis of N-Substituted Phosphoramidic Acid Esters as
"Reverse" Fosmidomycin Analogues
Christiana M. Adeyemia, Heinrich C. Hoppeb,c, Michelle Isaacsc, Rosalyn Klein,a,c
Kevin A. Lobba,c and Perry T. Kayea,c*
aDepartment of Chemistry, b Department of Biochemistry and Microbiology and
cCentre for Chemico- and Biomedicinal Research, Rhodes University, Grahamstown,
6140, South Africa.
Abstract. An efficient synthetic pathway to a series of novel “reverse” fosmidomycin
analogues has been developed, commencing from substituted benzylamines. In these
analogues, the fosmidomycin hydroxamate moiety is reversed and the tetrahedral methylene
carbon adjacent to the phosphonate moiety is replaced by a nitrogen atom bearing different
benzyl groups. The resulting phosphonate esters were designed as potential antimalarial “pro-
drugs”.
Keywords. Reverse fosmidomycin analogues, N-benzylated phosphoramidates, synthesis,
bioassay.
__________
Corresponding author: Prof Perry Kaye.
E-mail. P.Kaye@ru.ac.za
Tel. +27 46 6037030; Fax. +27 466225109.
Dept. of Chemistry, Rhodes University,
Grahamstown, 6140, South Africa.
1

ACCEPTED MANUSCRIPT
1. Introduction
The phosphonic acid derivatives Fosmidomycin 1 and its N-acetyl analogue FR900098 are
active inhibitors of the Plasmodium falciparum 1-deoxy- -xylulose-5-phosphate reductase
D
(PfDXR) enzyme1 − an established antimalarial target.2 The therapeutic value of these
compounds is, however, limited by their rapid in vivo clearance and the recrudescence
associated with their use3,4 and attention has turned to the development of novel analogues.
Brücher et al.5 have reported the preparation of promising ligands 2 (Figure 1), which exhibit
high inhibitory potency against PfDXR. In addition to a ‘reversed’ arrangement of the
hydroxamate moiety, these compounds contain an α-phenyl substituent capable of occupying
the hydrophobic pocket adjacent to the PfDXR active-site.
Our own research has focussed on developing ligands which exploit such hydrophobic binding
opportunities6-8 and, more particularly, on phosphoramidic acid derivatives 3 in which the
hydrophobic aryl group is attached to nitrogen rather than to a tetrahedral sp3 carbon (as in
compound 2), thus obviating chirality issues. Although the phosphonic acid moiety is expected
to bind to the PfDXR phosphate binding-site, phosphonate esters have, in fact, been shown to
be effective PfDXR inhibitors.5 The use of ester derivatives as pro-drugs would be expected
not only to delay exposure of the phosphoramidic acid moiety to premature decomposition, but
also to decrease the overall polarity of the ligands, possibly mitigating the unacceptably rapid
clearance exhibited by fosmidomycin itself. Recent reports9,10 reflect the potential of aryl and
dialkyl phosphoramidate pro-drugs as hepatitis C virus inhibitors. In this communication, we
now report on our attempts to access the N-aryl compounds 3 and the eventual synthesis of a
range of N-benzylated phosphoramidate ester derivatives 4 as “reverse” fosmidomycin
analogues.
Figure 1. Progression in the design of novel fosmidomycin analogues.
2

ACCEPTED MANUSCRIPT
2. Results and Discussion
In designing the synthesis of the N-aryl phosphoramidic acid analogues (3), compound 5 was
identified as a critical intermediate and several approaches to this compound were explored
(Scheme 1). In Approach I, ethyl 3-bromopropanoate 7, obtained by acid-catalysed
esterification of 3-bromopropanoic acid 611-113 (Scheme 1), was reacted with aniline and NaH
in dry THF to furnish compound 8 which contains the desired bimethylene linking group and
the functionality needed to generate the reverse hydroxamate moiety in the target ligands (3).
However, attempts to introduce the phosphonyl moiety by reacting compound 8 with diethyl
chlorophosphate and triethylamine in DCM failed to afford the critical intermediate 5 (for
R=H), as did attempts to activate the amine nitrogen by deprotonation using sodium hydride
or BuLi. In Approach II, aniline 9 was first phosphonylated to give compound 10, but
attempts to alkylate this compound with ethyl 3-bromoethylpropanoate 7 using either NaH in
dry THF (stirring for 2 days and then refluxing for 5 h) or BuLi were similarly unsuccessful
− due, presumably, to further delocalisation of the nitrogen lone-pair electrons into the
phosphonate moiety. Finally, in a variation on Approach I, a solution of methyl acrylate 11
and aniline in toluene was refluxed overnight to afford the aza-Michael addition product
1214,15 but attempts to phosphorylate this adduct using diethyl chlorophosphate and
triethylamine also failed, even after using NaH or BuLi as the base, .
Approach I
O
OEt
OH
O
O
i
ii
OEt
HN
iii
X
Br
8
6
Br
7
O
O
O P
O
O
O P
O
NH2
v
NH
Approach II
Approach III
iv
OR
N
X
R'
5
9
10
vii
X
O
O
HN
OCH3
vi
OCH3
12
11
Scheme 1. Attempted synthesis of the intermediate 5.
Reagents and conditions:- (i) H2SO4, EtOH, reflux, overnight; (ii) aniline, NaH, dry THF; (iii)
diethyl chlorophosphate, Et3N, DCM; or NaH or BuLi, dry THF; (iv) diethyl chloro-
phosphate, NaH, dry THF; (v) 7, NaH or BuLi, dry THF; (vi) aniline, toluene, refluxed
overnight; and (vii) diethyl chlorophosphate, NaH or BuLi, dry THF.
3

ACCEPTED MANUSCRIPT
Given the difficulties encountered in accessing the critical intermediate 5, the N-benzylated
analogues 4 (Figure 1) were targeted in place of the N-aryl systems 3, and the N-benzylated
phosphoramidates (17) as the critical intermediates required to establish the necessary
molecular framework (Scheme 2). While a number of the benzylamines (15) were available
commercially, 3,4-dichlorobenzylamine 15b and 3-nitrobenzylamine 15g were obtained by
reacting the corresponding benzyl halides 13b and 14g with potassium phthalimide,16,17
followed by hydrazinolysis of the resulting N-benzylphthalimides.18,19
i,ii
iv
iii
17a-g
15a-g
16a-g
13b (X = Cl)
14g (X = Br)
v
a
b
c
d
e
f
H
3,4-Dichloro
3-Chloro
3,4-Difluoro
3-Fluoro
Furfuryl*
3-Nitro
vii
vi
g
* Replacing the
phenyl group
1
2
1
1
2
21a-g
R
= OBn; R = H
19a-g
20a-g
R
R
= OBn; R = H
18a-g
1
2
2
22a,c,f,g
R
= OH;
R
= CH3
= OH;
R
= CH3
Scheme 2. Synthesis of the N-benzylated “reverse” fosmidomycin analogues (21) and (22)
via the critical intermediates (17).
Reagents and conditions:- (i) Phthalimide, K2CO3, DMF, 100 °C; ii) Potassium phthalimide
N2H4.H2O, EtOH; iii) diethyl chlorophosphate, Et3N, DCM, rt 6 h; iv) methyl acrylate,
Bu4N+Br-, anhydr. K2CO3, toluene; v) KOH, H2O-THF, 0 °C for 1 h and rt 3 h, then 1
M-HCl;
vi) CDI, dry DCM, rt 1 h, then O-benzylhydroxylamine hydrochloride, rt 48 h or N-
o
methylhydroxylamine hydrochloride, rt 7 d; vii) Bromotrimethylsilane, dry DCM, 0 C 1 h,
then rt 24 h; viii) THF-H2O, 1 h.
Reaction of each of the benzylamine derivatives 15a-g with diethyl chlorophosphate in the
presence of triethylamine afforded the N-benzylated phosphonate esters 16a-g in good yields
(Table 1) and in sufficient purity for use in the next step.22 The addition of the N-benzylated
diethylphosphonate esters 16a-g to methyl acrylate in the presence of Bu4N+Br- and anhydr.
K2CO3 in toluene afforded the phosphonate esters 17a-g23 which were hydrolysed to the
corresponding carboxylic acids 18a-g. The synthesis of O-benzyl-protected hydroxamic acids
19a-g and the N-methylhydroxamic acids 20a-g was accomplished by treating the carboxylic
acids 18a-g with CDI, followed by O-benzylhydroxylamine or N-methylhydroxylamine,
4

ACCEPTED MANUSCRIPT
respectively. In view of their tendency to decompose, selected crude phosphonate ester
intermediates (19) and (20) were subjected directly to bromotrimethyl-silane-mediated
hydrolysis to afford the corresponding phosphoramidic acids (21) and (22), NMR analysis of
which confirmed the efficacy of the protocol, albeit apparently accompanied by some
decomposition – particularly evident in the NMR spectra of compound 21f. Although the
relative instability of the phosphoramidic acid moiety has proved somewhat problematic, it should be
noted that in most of the examples examined by Maiti et al.,24 P-N hydrolytic cleavage is very slow at
70 oC and pD = 7.0; half-lives of > 8 or 15 hours at 70 oC and pD=5.0 have been cited. Moreover, we
have successfully isolated ten phosphoramidic acids, and the synthetic methodology and available
data for these compounds are provided in the experimental section and the Supporting
Information file.
Some of the phosphoramidate derivatives present as pairs of rotamers and, in the case of the
13
mono- and difluorobenzyl derivatives, the complexity of the C spectra is compounded by
13
19F-13C coupling. For example, the C NMR spectrum of diethyl N-(3,4-difluorobenzyl)-N-
[2-(methoxycarbonyl)ethyl]phosphoramidate 17d reflects the presence of major and minor
rotamers in a ratio of 56:44. The complex 13C NMR signals for the two aromatic carbons
directly attached to fluorine. Careful analysis reveals 19F-13C coupling for both carbon nuclei
2
(1JC-F ~248Hz and JC-F 13-18Hz) and the doubling of the resulting multiplets due to the
presence of rotamers.
Table 1. Percentage yields of the intermediate compounds and phosphoramidic ester products
Ar
(16)
90
(17)
57
(18)
88
(19)
78
(20)
67
Benzyl
3,4-
Dichlorophenyl
93
84
94
66
70
3-Chlorophenyl
87
95
93
92
93
77
78
69
76
83
89
71
70
85
92
83
70
72
70
78
70
55
71
82
66
3,4-
Difluorophenyl
3-Fluorophenyl
Furfuryl
3-Nitrophenyl
5

ACCEPTED MANUSCRIPT
The targeted phosphoramidic acid derivatives and the intermediates involved in their
synthesis were subjected to in vitro bioassays to determine their toxicity against HEK 293
cells as well as their antimalarial and trypanocidal potential. None of the compounds listed in
the Table 1 of the Supporting Information file exhibited significant toxicity, with the
percentage viability of HEK 293 cells being > 80% at 25 µM. Their antiparasitic activity,
however, proved to be disappointing with many of the compounds showing some, albeit
weak, antimalarial activity (residual PLDH activity > 74%) and, generally, no significant
trypanocidal activity (against T.brucei). Generally, the 3-nitrophenyl derivatives exhibited the
best antimalarial potential (ca. 75% P. falciparum viability at 20 µM), while the mono-
substituted chloro analogue 20c was the only compound shown to be active against T. brucei
(ca. 49% parasite viability at 20 µM). We accept that any observed activity could arise from
interactions of the ligands at targets other than PfDXR. However, the docking studies simply
confirm the apparent unsuitability of the ligands as PfDXR inhibitors.
In silico docking studies of phosphoramidic acids (which might be expected to be formed by
intra-cellular hydrolysis of their ester “pro-drug” precursors) as their negatively charged
mono-deprotonated acids revealed that the acid analogue 21g was the only ligand to bind in
the PfDXR active-site, with a docking conformation similar to that of fosmidomycin 1. The
phosphoramidic acid analogues of all of the other ligands exhibited a preference for binding
at a different site, through which weak allosteric effects might be exerted, although this does
not necessarily preclude the possibility of a smaller population of some of these ligands
binding in the PfDXR site.
In summary, a reliable synthetic pathway has been developed which has provided access to a
series of novel phosphoramidic ester derivatives. With few exceptions, the synthetic
compounds have shown surprisingly little, if any, antimalarial (against PLDH) or trypanocidal
(against T.brucei) activity, and in silico docking studies have revealed the likely reason for
their relative inactivity as PfDXR inhibitors.
6

ACCEPTED MANUSCRIPT
3.
Experimental
3.1.
General methods
NMR spectra were recorded on Bruker 300, 400 or 600 MHz spectrometers in CDCl3,
DMSO-d6 or CD3OD, and were calibrated using solvent signals. Melting points were
measured using a hot-stage apparatus and are uncorrected. High-resolution mass spectra
(HRMS) were recorded on a Waters API Q-TOF Ultima spectrometer (University of
Stellenbosch, Stellenbosch, South Africa). NMR spectra, bioassay protocols and results and
computer modelling methods are provided in the Supporting Information.
3.2.
Reaction of benzylamine derivatives with diethyl chlorophosphate
A mixture of benzylamine 15a (2.5 mL, 23 mmol), triethylamine (3.2 mL, 23 mmol) and
diethyl chlorophosphate (3.3 mL, 23 mmol) in DCM (15 mL) was stirred in a stoppered
reaction flask at r.t. for 6 h. The mixture was then washed sequentially with 10% HCl (2 × 20
mL) and water (2 × 20 mL) and the aqueous layers were re-extracted with DCM (2 × 25 mL).
The organic layers were combined, dried with anhydrous MgSO4 and concentrated in vacuo to
afford diethyl N-benzylphosphoramidate 16a22 as a pale-yellow oil (5.0 g, 90%); νmax/cm-1
1205 (P=O); δH/ppm (600 MHz; CDCl3) 1.27 (6H, t, J = 7.1 Hz, 2 × CH3), 3.98-4.07 (6H, m, 2
× OCH2CH3 and NCH2Ph) and 7.24-7.32 (5H, overlapping m, Ar-H); δC/ppm (150 MHz;
CDCl3) 16.2 (d, JP-C = 7.2 Hz, 2 × CH3), 45.4 (NCH2Ph), 62.5 (d, JP-C = 5.2 Hz, 2 ×
OCH2CH3), 127.4, 128.6, 129.1 and 139.7 (Ar-C).
3.2.1. Diethyl N-(3,4-dichlorobenzyl)phosphoramidate 16b as a pale-yellow oil (1.4 g,
93%) [HRMS: m/z calculated for C11H1735Cl2NO3P (MH+) 312.0323. Found 312.0320];
νmax/cm-1 1200 (P=O); δH/ppm (600 MHz; CDCl3) 1.27 (6H, t, J = 7.1 Hz, 2 × CH3), 3.96-4.05
(6H, m, 2 × OCH2CH3 and NCH2Ar), 7.15 (1H, dd, J = 2.0 and 8.2 Hz, Ar-H), 7.37 (1H, d, J =
8.2 Hz, Ar-H) and 7.44 (1H, s, Ar-H); δC/ppm (150 MHz; CDCl3) 16.3 (d, JP-C = 7.1 Hz, 2 ×
CH3), 44.3 (NCH2Ar), 62.7 (d, JP-C = 5.3 Hz, 2 × OCH2CH3), 126.7, 129.4, 130.5, 131.3,
132.6 and 140.3 (Ar-C).
3.2.2. Diethyl N-(3-chlorobenzyl)phosphoramidate 16c as a pale-yellow oil (0.86 g, 87%)
[HRMS: m/z calculated for C11H1835ClNO3P (MH+) 278.0713. Found 278.0707]; νmax/cm-1
1205 (P=O); δH/ppm (600 MHz; CDCl3) 1.21 (6H, t, J = 7.1 Hz, 2 × CH3), 3.91-3.99 (6H, m, 2
× OCH2CH3 and NCH2Ar), 7.16-7.18 (3H, overlapping m, Ar-H) and 7.30 (1H, s, Ar-H);
δC/ppm (150 MHz; CDCl3) 16.1 (d, JP-C = 7.2 Hz, 2 × CH3), 44.6 (NCH2Ar), 62.4 (d, JP-C
5.2 Hz, 2 × OCH2CH3), 125.4, 127.3, 127.4, 129.7, 134.2 and 142.1 (Ar-C).
=
3.2.3. Diethyl N-(3,4-difluorobenzyl)phosphoramidate 16d as a pale-yellow oil (1.1 g,
95%) [HRMS: m/z calculated for C11H17F2NO3P (MH+) 280.0914. Found 280.0916]; νmax/cm-1
1226 (P=O); δH/ppm (600 MHz; CDCl3) 1.30 (6H, t, J = 7.2 Hz, 2 × CH3), 3.11 (1H, br s,
NH), 4.01-4.11 (6H, m, 2 × OCH2CH3 and NCH2Ar), 7.03 (1H, d, J = 8.3 Hz, Ar-H), 7.11
(1H, dd, JH-H = 8.4 and JF-H = 18.6 Hz, Ar-H) and 7.16-7.20 (1H, m, Ar-H); δC/ppm (150
MHz; CDCl3) 16.3 (d, JP-C = 7.1 Hz, 2 × CH3), 45.0 (NCH2Ar), 62.6 (d, JP-C = 5.2 Hz, 2 ×
OCH2CH3), 114.4 (dd, JF–C = 6.5 and 21 Hz), 122.9 (d, JF–C = 2.8 Hz), 130.2 (d, JF–C = 8.2
Hz), 142.5 (t, JF–C = 6.5 Hz), 162.3 and 163.9 (Ar-C).
3.2.4. Diethyl N-(3-fluorobenzyl)phosphoramidate 16e as a pale-yellow oil (1.5 g, 93%)
[HRMS: m/z calculated for C11H18FNO3P (MH+) 262.1008. Found 262.1016]; νmax/cm-1 1208
7

ACCEPTED MANUSCRIPT
(P=O); δH/ppm (400 MHz; CDCl3) 1.30 (6H, t, J = 7.2 Hz, 2 × CH3), 3.10 (1H, br s, NH),
4.01-4.10 (6H, m, 2 × OCH2CH3 and NCH2Ar), 6.95 (1H, t, JF–H = 8.8 Hz, Ar-H), 7.08 (2H,
m, Ar-H) and 7.29 (1H, m, Ar-H); δC/ppm (150 MHz; CDCl3) 16.2 (d, JP-C = 7.1 Hz, 2 × CH3),
44.4 (NCH2Ar), 62.6 (d, JP-C = 5.4 Hz, 2 × OCH2CH3), 116.3 (d, JF–C = 17.5 Hz), 117.3 (d, JF–
= 17.1 Hz), 123.1 (dd, JF–C = 3.6 and 6.3 Hz), 124.5, 142.6 and 162.9 (d, JF–C = 240.0 Hz)
C
(Ar-C).
3.2.5. Diethyl N-(3-nitrobenzyl)phosphoramidate 16g as pale-yellow oil (3.5 g, 93%)
[HRMS: m/z calculated for C11H18N2O5P (MH+) 289.0953. Found 289.0949]; νmax/cm-1 1207
(P=O); δH/ppm (600 MHz; CDCl3) 1.07 (6H, t, J = 7.4 Hz, 2 × CH3), 3.78 (4H, m, 2 ×
OCH2CH3), 4.01 (2H, d, JP-H = 12.6 Hz, NCH2Ar), 7.33 (1H, t, J = 7.9 Hz, Ar-H), 7.55 (1H, d,
J = 7.6 Hz, Ar-H), 7.89 (1H, d, J = 8.1 Hz, Ar-H) and 8.07 (1H, s, Ar-H); δC/ppm (150 MHz;
CDCl3) 15.7 (d, JP-C = 7.1 Hz, 2 × CH3), 44.0 (NCH2Ar), 62.2 (d, JP-C = 5.2 Hz, 2 ×
OCH2CH3), 122.8, 129.1, 133.3, 135.5, 142.3 and 147.9 (Ar-C).
3.3.
Aza-Michael addition reactions of diethyl N-benzylphosphoramidate esters 16a-g
To a solution of diethyl N-benzylphosphoramidate 16a (1.6 g, 6.5 mmol) in toluene (20 mL)
was added anhydrous K2CO3 (1.8 g, 13 mmol), tetrabutylammonium bromide (0.21g, 0.65
mmol) and methyl acrylate (0.71 mL, 7.8 mmol). The reaction mixture was refluxed for 8 h at
120 ºC, and allowed to cool to r.t. Benzene (10 mL) and water (20 mL) were added and the
stirred mixture was stirred for 15 min., and then washed with cold water. The organic layer
was dried with anhydrous MgSO4 and concentrated in vacuo. The crude product was purified
by flash chromatography [on silica gel; elution with EtOAc-hexane (6:4)] to afford diethyl N-
benzyl-N-[2-(methoxycarbonyl)ethyl]phosphoramidate 17a as a yellow oil (1.5 g, 57%)
[HRMS: m/z calculated for C15H25NO5P (MH+) 330.1470. Found 330.1475]; νmax/cm-1 1734
(C=O) and 1243 (P=O); δH/ppm (600 MHz; CDCl3) 1.31 (6H, t, J = 7.5 Hz, 2 × CH3), 2.49
(2H, t, J = 7.3 Hz, CH2CO), 3.23 (2H, m, NCH2), 3.62 (3H, s, OCH3), 4.01-4.02 (4H, 2 × m, 2
× OCH2CH3), 4.20 (2H, d, JP-H = 9.6 Hz, NCH2Ph) and 7.26-7.32 (5H, overlapping m, Ar-H);
δC/ppm (150 MHz; CDCl3) 16.2 (d, JP-C = 7.2 Hz, 2 × CH3), 33.3 (CH2CO), 41.3 (d, JP-C = 4.5
Hz, NCH2), 49.9 (d, JP-C = 4.9 Hz, NCH2Ph), 51.7 (OCH3), 62.5 (d, JP-C = 5.7 Hz, 2 ×
OCH2CH3), 127.5, 128.3, 128.5 and 137.8 (Ar-C) and 172.2 (C=O).
3.3.1. Diethyl N-(3,4-dichlorobenzyl)-N-[2-(methoxycarbonyl)ethyl]phosphoramidate 17b
as a yellow oil (1.5 g, 84%) [HRMS: m/z calculated for C15H2335Cl2NO5P (MH+) 398.0691.
Found 398.0687]; νmax/cm-1 1736 (C=O) and 1243 (P=O); δH/ppm (600 MHz; CDCl3) 1.28
(6H, t, J = 7.3 Hz, 2 × CH3), 2.46 (2H, t, J = 7.3 Hz, CH2CO), 3.17 (2H, m, NCH2), 3.60 (3H,
s, OCH3), 3.97-4.01 (4H, 2 × m, 2 × OCH2CH3), 4.13 (2H, d, JP-H = 10.0 Hz, NCH2Ar), 7.14
(1H, d, J = 8.3 Hz, Ar-H), 7.35 (1H, d, J = 8.2 Hz, Ar-H) and 7.41 (1H, s, Ar-H); δC/ppm (150
MHz; CDCl3) 16.2 (d, JP-C = 7.2 Hz, 2 × CH3), 33.4 (CH2CO), 41.5 (d, JP-C = 4.3 Hz, NCH2),
49.1 (d, JP-C = 5.0 Hz, NCH2Ar), 51.8 (OCH3), 62.7 (d, JP-C = 5.7 Hz, 2 × OCH2CH3), 127.5,
130.0, 130.5, 131.5, 132.6 and 138.5 (Ar-C) and 172.1 (C=O).
3.3.2. Diethyl N-(3-chlorobenzyl)-N-[2-(methoxycarbonyl)ethyl]phosphoramidate 17c as a
pale-yellow oil (0.70 g, 77%) [HRMS: m/z calculated for C15H2435ClNO5P (MH+) 364.1081.
Found 364.1070]; νmax/cm-1 1736 (C=O) and 1244 (P=O); δH/ppm (600 MHz; CDCl3) 1.31
(6H, t, J = 7.2 Hz, 2 × CH3), 2.49 (2H, m, CH2CO), 3.22 (2H, m, NCH2), 3.62 (3H, s, OCH3),
4.01-4.07 (4H, m, 2 × OCH2CH3), 4.18 (2H, d, JP-C = 10.0 Hz, NCH2Ar), 7.20-7.28 (3H,
overlapping m, Ar-H) and 7.33 (1H, s, Ar-H); δC/ppm (150 MHz; CDCl3) 16.2 (d, JP-C = 7.2
Hz, 2 × CH3), 33.4 (CH2CO), 41.5 (d, JP-C = 4.3 Hz, NCH2), 49.6 (d, JP-C = 5.1 Hz, NCH2Ar),
8

ACCEPTED MANUSCRIPT
51.8 (OCH3), 62.7 (d, JP-C = 5.8 Hz, 2 × OCH2CH3), 126.3, 127.7, 128.2, 129.8, 134.5 and
140.2 (Ar-C) and 172.2 (C=O).
3.3.3. Diethyl N-(3,4-difluorobenzyl)-N-[2-(methoxycarbonyl)ethyl]phosphoramidate 17d
as a yellow oil (0.92 g, 78%) [HRMS: m/z calculated for C15H23F2NO5P (MH+) 366.1282.
Found 366.1286]; νmax/cm-1 1737 (C=O) and 1240 (P=O); δH/ppm (400 MHz; CDCl3) 1.33
(6H, t, J = 7.3 Hz, 2 × CH3), 2.50 (2H, m, CH2CO), 3.24 (2H, m, NCH2), 3.65 (3H, s, OCH3),
4.03-4.06 (4H, m, 2 × OCH2CH3), 4.18 (2H, d, JP-H = 10.2 Hz, NCH2Ar) and 7.06-7.18 (3H,
overlapping signals, Ar-H); δC/ppm (150 MHz; CDCl3) 16.1 (m, 2 × CH3), 33.3 (CH2CO),
41.3 (d, JP-C = 4.2 Hz, NCH2), 49.2 (d, JP-C = 5.2 Hz, NCH2Ar), 51.7 (OCH3), 62.4 (m, 2 ×
OCH2CH3), 116.2 and 117.2 (two rotameric d, JF–C = 17.6 Hz), 117.1 (t, JF–C = 16.5 Hz), 123.1
and 124.1 (two rotameric dd, JF–C = 6.6 and 3.3 Hz), 135.1 and 137.1 (two rotameric m, CF),
149.4 and 149.7 (two rotameric dd, JF-C = 247.6 and 13.2 Hz), and 150.2 and 150.3 (two
rotameric dd, JF-C = 248.7 and 18.7/17.6 Hz ) (Ar-C) and 172.1 (C=O).
3.3.4. Diethyl N-(3-fluorobenzyl)-N-[2-(methoxycarbonyl)ethyl]phosphoramidate 17e as a
yellow oil (1.3 g, 69%) [HRMS: m/z calculated for C15H24FNO5P (MH+) 348.1376. Found
348.1372]; νmax/cm-1 1738 (C=O) and 1235 (P=O); δH/ppm (600 MHz; CDCl3) 1.28 (6H, 2 ×
unresolved m, 2 × CH3), 2.46 (2H, t, J = 7.5 Hz, CH2CO), 3.20 (2H, m, NCH2), 3.59 (3H, s,
OCH3), 3.99-4.02 (4H, m, 2 × OCH2CH3), 4.17 (2H, d, JP-H = 9.8 Hz, NCH2Ar), 6.90 (1H, m,
Ar-H), 7.04 (2H, m, Ar-H) and 7.23 (1H, m, Ar-H); δC/ppm (150 MHz; CDCl3) 16.1 (d, JP-C
7.3 Hz, 2 × CH3), 33.3 (CH2CO), 41.4 (d, JP-C = 4.0 Hz, NCH2), 49.5 (d, JP-C = 5.0 Hz,
NCH2Ar), 51.6 (OCH3), 62.4 (m, 2 × OCH2CH3), 114.0 and 114.4 (two rotameric d, JF–C
=
=
20.9 Hz), 114.1 and 114.9 (two rotameric d, JF–C = 22.0 Hz), 122.7 and 123.6 (two rotameric
d, JF–C = 3.3 Hz), 129.9 (dd, JF–C = 12.1 and 8.8 Hz), 140.7 and 142.6 (two rotameric m, CF)
and 162.9 and 163.0 (two rotameric d, JF–C = 245.4/246.5 Hz) (Ar-C) and 172.1 (C=O).
3.3.5. Diethyl N-furfuryl-N-[2-(methoxycarbonyl)ethyl]phosphoramidate 17f as a yellow
oil (3.1 g, 76%) [HRMS: m/z calculated for C13H23NO6P (MH+) 320.1263. Found 320.1260];
νmax/cm-1 1735 (C=O) and 1244 (P=O); δH/ppm (600 MHz; CDCl3) 1.27 (6H, t, J = 7.5 Hz, 2 ×
CH3), 2.48 (2H, t, J = 6.2 Hz, CH2CO), 3.26 (2H, m, NCH2), 3.63 (3H, s, OCH3), 3.95-4.04
(4H, 2 × m, 2 × OCH2CH3), 4.16 (2H, d, JP-H = 9.7 Hz, NCH2Ar), 6.24 (1H, m, ArH), 6.29
(1H, m, Ar-H) and 7.34 (1H, d, J = 1.6 Hz, Ar-H); δC/ppm (150 MHz; CDCl3) 16.2 (d, JP-C
=
7.3 Hz, 2 × CH3), 33.5 (CH2CO), 41.9 (d, JP-C = 4.4 Hz, NCH2), 43.0 (d, JP-C = 5.7 Hz,
NCH2Ar), 51.8 (OCH3), 62.4 (d, JP-C = 5.4 Hz, 2 × OCH2CH3), 108.8, 110.4, 142.4 and 151.6
(Ar-C) and 172.3 (C=O).
3.3.6. Diethyl N-[2-(methoxycarbonyl)ethyl]-N-(3-nitrobenzyl)phosphoramidate 17g as a
yellow oil (3.7 g, 83%) as a yellow oil [HRMS: m/z calculated for C15H24N2O7P (MH+)
375.1321. Found 375.1328]; νmax/cm-1 1734 (C=O) and 1244 (P=O); δH/ppm (600 MHz;
CDCl3) 1.25 (6H, t, J = 7.2 Hz, 2 × CH3), 2.45 (2H, t, J = 7.5 Hz, CH2CO), 3.15 (2H, m,
NCH2), 3.54 (3H, s, OCH3), 3.97-4.03 (4H, m, 2 × OCH2CH3), 4.26 (2H, d, JP-H = 10.5 Hz,
NCH2Ar), 7.44 (1H, t, J = 7.8 Hz, Ar-H), 7.61 (1H, d, J = 6.6 Hz, Ar-H), 8.03 (1H, d, J = 6.3
Hz, Ar-H) and 8.16 (1H, s, Ar-H); δC/ppm (150 MHz; CDCl3) 16.0 (d, JP-C = 7.1 Hz, 2 × CH3),
33.3 (CH2CO), 41.6 (d, JP-C = 4.0 Hz, NCH2), 49.4 (d, JP-C = 5.0 Hz, NCH2Ar), 51.7 (OCH3),
62.7 (d, JP-C = 5.3 Hz, 2 × OCH2CH3), 122.4, 122.7, 129.5, 134.1, 140.4 and 148.3 (Ar-C) and
171.9 (C=O).
3.4.
Hydrolysis of the methyl esters 17a-g.
A solution of diethyl N-benzyl-N-[2-(methoxycarbonyl)ethyl]phosphoramidate 17a (1.1 g, 3.3
mmol) in THF (10 mL) was diluted with water (50 mL), and then cooled to 0 °C. A solution of
9

ACCEPTED MANUSCRIPT
dil. aq. KOH (12 mL) was added dropwise followed by stirring at 0 °C for 1 h, and then at r.t.
for 3 h. The reaction mixture was acidified to pH = 2 by the dropwise addition of 1M-HCl and
the solution was then saturated with NaCl and extracted with EtOAc (2 × 20 mL). The organic
solutions were combined, dried with anhydrous MgSO4, filtered and evaporated in vacuo to
afford diethyl N-benzyl-N-(2-carboxyethyl)phosphoramidate 18a as a yellow oil (0.90 g, 88%)
[HRMS: m/z calculated for C14H23NO5P (MH+) 316.1314. Found 316.1319]; νmax/cm-1 3530-
3250 (br, OH), 1723 (C=O) and 1235 (P=O); δH/ppm (600 MHz; CDCl3) 1.29 (6H, t, J = 7.2
Hz, 2 × CH3), 2.48 (2H, m, CH2CO), 3.20 (2H, m, NCH2), 3.97-4.10 (4H, 2 × m, 2 ×
OCH2CH3), 4.19 (2H, d, JP-H = 9.8 Hz, NCH2Ph) and 7.25-7.30 (5H, overlapping m, Ar-H);
δC/ppm (150 MHz; CDCl3) 16.2 (d, JP-C = 7.4 Hz, 2 × CH3), 33.3 (CH2CO), 41.2 (d, JP-C = 4.6
Hz, NCH2), 49.8 (d, JP-C = 4.9 Hz, NCH2Ph), 62.8 (d, JP-C = 5.8 Hz, 2 × OCH2CH3), 127.6,
128.3, 128.6 and 137.6 (Ar-C) and 175.0 (C=O).
3.4.1. Diethyl N-(2-carboxyethyl)-N-(3,4-dichlorobenzyl)phosphoramidate 18b as a yellow
oil (1.3 g, 94%) [HRMS: m/z calculated for C14H2135Cl2NO5P (MH+) 384.0534. Found
384.0531]; νmax/cm-1 3526-3216 (br, OH), 1724 (C=O) and 1233 (P=O); δH/ppm (600 MHz;
CDCl3) 1.29 (6H, t, J = 7.1 Hz, 2 × CH3), 2.48 (2H, t, J = 7.2 Hz, CH2CO), 3.18 (2H, m,
NCH2), 3.99-4.09 (4H, m, 2 × OCH2CH3), 4.15 (2H, d, JP-H = 10.5 Hz, NCH2Ar), 7.15 (1H, d,
J = 8.2 Hz, Ar-H), 7.37 (1H, d, J = 8.2 Hz, Ar-H) and 7.42 (1H, s, Ar-H); δC/ppm (150 MHz;
CDCl3) 16.2 (d, JP-C = 7.2 Hz, 2 × CH3), 33.5 (CH2CO), 41.4 (d, JP-C = 4.4 Hz, NCH2), 49.0
(d, JP-C = 5.1 Hz, NCH2Ar), 63.1 (d, JP-C = 5.8 Hz, 2 × OCH2CH3), 127.6, 130.1, 130.5, 131.6,
132.7 and 138.3 (Ar-C) and 174.9 (C=O).
3.4.2. Diethyl N-(2-carboxyethyl)-N-(3-chlorobenzyl)phosphoramidate 18c as a yellow oil
(1.2 g, 89%) [HRMS: m/z calculated for C14H2235ClNO5P (MH+) 350.0924. Found 350.0912];
3550-3230 (br, OH), 1726 (C=O) and 1230 (P=O); δH/ppm (600 MHz; CDCl3) 1.32 (6H, t, J =
7.3 Hz, 2 × CH3), 2.52 (2H, m, CH2CO), 3.24 (2H, m, NCH2), 4.03-4.11 (4H, 2 × m, 2 ×
OCH2CH3), 4.19 (2H, d, JP-H = 10.1 Hz, NCH2Ar), 7.20 (1H, m, Ar-H), 7.25 (2H, m, Ar-H)
and 7.34 (1H, d, J = 6.4 Hz, Ar-H); δC/ppm (150 MHz; CDCl3) 16.3 (d, JP-C = 7.2 Hz, 2 ×
CH3), 33.5 (CH2CO), 41.5 (d, JP-C = 4.5 Hz, NCH2), 49.6 (d, JP-C = 5.0 Hz, NCH2Ar), 63.0 (d,
JP-C = 5.8 Hz, 2 × OCH2CH3), 125.4, 126.3, 127.8, 128.3, 129.9 and 140.0 (Ar-C) and 175.0
(C=O).
3.4.3. Diethyl N-(2-carboxyethyl)-N-(3,4-difluorobenzyl)phosphoramidate 18d as a yellow
oil (0.60 g, 71%) [HRMS: m/z calculated for C14H21F2NO5P (MH+) 352.1125. Found
352.1126]; νmax/cm-1 3545-3228 (br, OH), 1734 (C=O) and 1229 (P=O); δH/ppm (600 MHz;
CDCl3) 1.31 (6H, t, J = 7.1 Hz, 2 × CH3), 2.50 (2H, t, J = 7.5 Hz, CH2CO), 3.20 (2H, m,
NCH2), 4.02-4.07 (4H, 2 × m, 2 × OCH2CH3), 4.16 (2H, d, JP-H = 10.2 Hz, NCH2Ar), 7.03
(1H, m Ar-H), 7.09 (1H, m, Ar-H) and 7.19 (1H, m, Ar-H); δC/ppm (150 MHz; CDCl3) 16.1
and 16.2 (2 × CH3), 33.4 (CH2CO), 41.3 (d, JP-C = 4.6 Hz, NCH2), 49.2 (d, JP-C = 5.4 Hz,
NCH2Ar), 62.75 and 62.82 (2 × d, JP-C = 5.5 Hz, 2 × OCH2CH3), 116.2 and 117.1 (two
rotameric d, JF–C = 17.6 Hz), 117.2 (dd, JF–C = 17.5 and 4.5 Hz), 123.1 and 124.1 (two
rotameric dd, JF–C = 6.3/5.5 and 4.1/3.3 Hz), 135.0 and 136.7 (two rotameric m, CF), 148.7-
151.3 (series of m, CF) (Ar-C) and 174.7 (C=O).
3.4.4. Diethyl N-(2-carboxyethyl)-N-(3-fluorobenzyl)phosphoramidate 18e as a yellow oil
(0.68 g, 70%) [HRMS: m/z calculated for C14H22FNO5P (MH+) 334.1220. Found 334.1216];
νmax/cm-1 3535-3219 (br, OH), 1724 (C=O) and 1232 (P=O); δH/ppm (600 MHz; CDCl3) 1.31
(6H, t, J = 7.1 Hz, 2 × CH3), 2.50 (2H, t, J = 7.5 Hz, CH2CO), 3.22 (2H, m, NCH2), 4.00-4.08
(4H, m, 2 × OCH2CH3), 4.21 (2H, d, JP-H = 10.1 Hz, NCH2Ar), 6.95 (1H, t, J = 7.5 Hz, Ar-H),
7.04-7.09 (2H, m, Ar-H) and 7.28 (1H, m, Ar-H).
10

ACCEPTED MANUSCRIPT
3.4.5. Diethyl N-(2-carboxyethyl)-N-furfurylphosphoramidate 18f as a brown oil (0.98 g,
85%) [HRMS: m/z calculated for C12H21NO6P (MH+) 306.1106. Found 306.1110]; νmax/cm-1
3561-3236 (br, OH), 1730 (C=O) and 1230 (P=O); δH/ppm (600 MHz; CDCl3) 1.29 (6H, t, J =
7.1 Hz, 2 × CH3), 2.50 (2H, m, CH2CO), 3.29 (2H, m, NCH2), 3.98-4.07 (4H, 2 × m, 2 ×
OCH2CH3), 4.16 (2H, d, JP-H = 9.7 Hz, NCH2Ar), 6.26 (1H, d, J = 3.0 Hz, Ar-H), 6.31 (1H,
m, Ar-H) and 7.35 (1H, d, J = 1.0 Hz, Ar-H); δC/ppm (150 MHz; CDCl3) 16.2 (d, JP-C = 7.4
Hz, 2 × CH3), 33.7 (CH2CO), 41.9 (d, JP-C = 4.8 Hz, NCH2), 43.0 (d, JP-C = 5.6 Hz, NCH2Ar),
62.8 (d, JP-C = 5.5 Hz, 2 × OCH2CH3), 109.0, 110.5, 142.5 and 151.4 (Ar-C) and 175.3 (C=O).
3.4.6. Diethyl N-(2-carboxyethyl)-N-(3-nitrobenzyl)phosphoramidate 18g as a yellow oil
(3.0 g, 92%) [HRMS: m/z calculated for C14H22N2O7P (MH+) 361.1165. Found 361.1163];
νmax/cm-1 3535-3218 (br, OH), 1734 (C=O) and 1235 (P=O); δH/ppm (300 MHz; CDCl3) 1.30
(6H, t, J = 7.1 Hz, 2 × CH3), 2.50 (2H, m, CH2CO), 3.22 (2H, m, NCH2), 4.05-4.10 (4H, m, 2
× OCH2CH3), 4.31 (2H, d, JP-H = 10.7 Hz, NCH2Ar), 7.47 (1H, t, J = 7.9 Hz, Ar-H), 7.65 (1H,
d, J = 7.6 Hz, Ar-H), 8.09 (1H, d, J = 7.0 Hz, Ar-H) and 8.20 (1H, s, Ar-H); δC/ppm (75 MHz;
CDCl3) 16.1 (d, JP-C = 7.1 Hz, 2 × CH3), 33.5 (CH2CO), 41.7 (d, JP-C = 4.3 Hz, NCH2), 49.6
(d, JP-C = 5.1 Hz, NCH2Ar), 63.2 (d, JP-C = 5.9 Hz, 2 × OCH2CH3), 122.6, 123.0, 129.6, 134.2,
140.4 and 148.5 (Ar-C) and 174.8 (C=O).
3.5.
Conversion of the carboxylic acids 18 to hydroxamic acid derivatives 19 and 20
3.5.1. Reaction of carboxylic acids 18 with O-benzylhydroxylamine hydrochloride
To a solution of diethyl N-benzyl-N-(2-carboxyethyl)phosphoramidate 18a (0.83 g, 2.6 mmol)
in dry DCM (10 mL) was added CDI (0.47 g, 2.9 mmol) in portions and the solution was
stirred at r.t. for 1 h. O-Benzylhydroxylamine hydrochloride (0.42 g, 2.6 mmol) was added and
the resulting mixture was stirred for 48 h. The solvent was evaporated in vacuo and the residue
was dissolved in EtOAc. The organic solution was washed sequentially with 10% aq. citric
acid and saturated aq. NaHCO3, dried with anhydr. MgSO4 and filtered, and the solvent was
evaporated in vacuo to afford diethyl N-benzyl-N-[2-(N-benzyloxycarbamoyl)ethyl]phos-
phoramidate 19a as a pale-yellow oil (0.86 g, 78%) [HRMS: m/z calculated for C21H30N2O5P
(MH+) 421.1892. Found 421.1904]; νmax/cm-1 1667 (C=O) and 1227 (P=O); δH/ppm (600
MHz; CDCl3) 1.29 (6H, t, J = 7.3 Hz, 2 × CH3), 2.28 (2H, m, CH2CO), 3.22 (2H, m,
NCH2CH2), 3.97-4.05 (4H, 2 × m, 2 × OCH2CH3), 4.13 (2H, d, JP-H = 9.7 Hz, NCH2Ph), 4.88
(2H, s, OCH2Ph) and 7.31-7.37 (10H, m, Ar-H); δC/ppm (150 MHz; CDCl3) 16.3 (d, JP-C = 7.1
Hz, 2 × CH3), 33.5 (CH2CO), 42.3 (NCH2CH2), 50.2 (d, JP-C= 3.2 Hz, NCH2Ph), 62.9 (d, JP-C
= 5.3 Hz, 2 × OCH2CH3), 78.1 (OCH2Ph), 127.4, 127.7, 128.4, 128.6, 129.1, 135.7, 137.5 and
139.7 (Ar-C) and 169.0 (C=O).
3.5.1.1. Diethyl N-[2-(N-benzyloxycarbamoyl)ethyl]-N-(3,4-dichlorobenzyl)phosphorami-
date 19b as a yellow oil (1.0 g, 66%) [HRMS: m/z calculated for C21H2835Cl2N2O5P (MH+)
489.1113. Found 489.1107]; νmax/cm-1 1667 (C=O) and 1229 (P=O); δH/ppm (600 MHz;
CDCl3) 1.27 (6H, t, J = 7.2 Hz, 2 × CH3), 2.26 (2H, m, CH2CO), 3.13 (2H, m, NCH2CH2),
3.94-3.99 (4H, m, 2 × OCH2CH3), 4.05 (2H, d, JP-H = 10.5 Hz, NCH2Ar), 4.82 (2H, s,
OCH2Ph), 7.13 (1H, d, J = 7.4 Hz, Ar-H), 7.26-7.31 (6H, overlapping m, Ar-H), 7.41 (1H, s,
Ar-H) and 10.43 (1H, br s, NH); δC/ppm (150 MHz; CDCl3) 16.0 (d, JP-C = 7.1 Hz, 2 × CH3),
32.9 (CH2CO), 42.0 (NCH2CH2), 48.8 (NCH2Ar), 62.7 (d, JP-C = 5.3 Hz, 2 × OCH2CH3) 77.8
(OCH2Ph), 126.6, 127.7, 128.4, 128.3, 129.2, 130.0, 131.3, 132.3, 135.4 and 138.0 (Ar-C) and
168.6 (C=O).
11

ACCEPTED MANUSCRIPT
3.5.1.2. Diethyl
N-[2-(N-benzyloxycarbamoyl)ethyl]-N-(3-chlorobenzyl)phosphoramidate
19c as a yellow oil (0.60 g, 83%) [HRMS: m/z calculated for C21H2935ClN2O5P (MH+)
455.1503. Found 455.1498]; νmax/cm-1 1658 (C=O) and 1214 (P=O); δH/ppm (300 MHz;
CDCl3) 1.31 (6H, t, J = 7.2 Hz, 2 × CH3), 2.32 (2H, m, CH2CO), 3.24 (2H, m, NCH2CH2),
4.02-4.13 (6H, m, 2 × OCH2CH3 and NCH2Ar), 4.87 (2H, s, OCH2Ph), 7.18-7.37 (9H,
overlapping m, Ar-H) and 9.10 (1H, br s, NH); δC/ppm (75 MHz; CDCl3) 16.2 (d, JP-C = 7.2
Hz, 2 × CH3), 33.2 (CH2CO), 44.7 (NCH2CH2), 49.6 (NCH2Ar), 62.5 and 62.8 (2 ×d, 2 ×
OCH2CH3), 78.0 (OCH2Ph), 125.5, 126.5, 127.5, 127.7, 128.3, 129.1, 129.8, 134.4, 135.6 and
139.8 (Ar-C) and 168.7 (C=O).
3.5.1.3. Diethyl
N-[2-(N-benzyloxycarbamoyl)ethyl]-N-(3,4-difluorobenzyl)phosphorami-
date 19d as a yellow oil (0.53 g, 70%) [HRMS: m/z calculated for C21H28F2N2O5P (MH+)
457.1704. Found 457.1702]; νmax/cm-1 1672 (C=O) and 1233 (P=O); δH/ppm (600 MHz;
CDCl3) 1.27 (6H, t, J = 7.3 Hz, 2 × CH3), 2.28 (2H, m, CH2CO), 3.18 (2H, m, NCH2CH2),
3.96-4.08 (6H, m, 2 × OCH2CH3 and NCH2Ar), 4.87 (2H, s, OCH2Ph) and 7.02-7.36 (8H,
series of m, Ar-H); δC/ppm (150 MHz; CDCl3) 16.2 (d, JP-C = 7.0 Hz, 2 × CH3), 33.4
(CH2CO), 44.4 (NCH2CH2), 49.4 (NCH2Ar), 62.6 (d, JP-C = 5.4 Hz, 2 × OCH2CH3), 78.1
(OCH2Ph), 116.4 (d, JF-C = 17.5 Hz), 117.3 (m), 123.2 (m), 128.6, 128.7, 129.1, 135.6, 137.0
(d, JF–C = 211.5 Hz), 149.4 (dd, JF–C = 13.6 and 244.5 Hz) and 150.9 (ddd, JF–C = 6.8, 9.1 and
247.5 Hz) (Ar-C) and 168.9 (C=O).
3.5.1.4. Diethyl
N-[2-(N-benzyloxycarbamoyl)ethyl]-N-(3-fluorobenzyl)phosphoramidate
19e as a yellow oil (0.37 g, 72%) [HRMS: m/z calculated for C21H29FN2O5P (MH+) 439.1798.
Found 439.1791]; νmax/cm-1 1674 (C=O) and 1231 (P=O); δH/ppm (600 MHz; CDCl3) 1.33
(6H, t, J = 7.3 Hz, 2 × CH3), 2.33 (2H, m, CH2CO), 3.26 (2H, m, NCH2CH2), 4.03-4.16 (6H,
m, 2 × OCH2CH3 and NCH2Ar), 4.92 (2H, s, OCH2Ph), 6.98 (1H, br s, NH), 7.07-7.41 (9H,
series of m, Ar-H) and 7.29-7.41 (4H, overlapping m, Ar-H); δC/ppm (150 MHz; CDCl3) 16.3
(d, JP-C = 7.0 Hz, 2 × CH3), 33.6 (CH2CO), 45.0 (NCH2CH2), 49.9 (NCH2Ar), 62.7 (d, JP-C
5.4 Hz, 2 × OCH2CH3), 78.2 (OCH2Ph), 114.4 (dd, JF–C = 8.7 and 20.7 Hz), 115.1 (d, JF–C
=
=
21.6 Hz), 122.9 (d, JF–C = 2.4 Hz), 124.0, 128.7 (m), 129.2, 130.2 (d, JF–C = 8.0 Hz), 135.6,
142.5 and 163.1 (d, JF–C = 246.5 Hz) (Ar-C) and 169.0 (C=O).
3.5.1.5. Diethyl N-[2-(N-benzyloxycarbamoyl)ethyl]-N-furfurylphosphoramidate 19f as a
yellow oil (0.47 g, 70%) [HRMS: m/z calculated for C19H28N2O6P (MH+) 411.1685. Found
411.1674]; νmax/cm-1 1667 (C=O) and 1226 (P=O); δH/ppm (600 MHz; CDCl3) 1.27 (6H, t, J =
7.1 Hz, 2 × CH3), 2.27 (2H, m, CH2CO), 3.28 (2H, m, NCH2CH2), 3.95-4.02 (4H, 2 × m, 2 ×
OCH2CH3), 4.08 (2H, d, JP-H = 9.5 Hz, NCH2Ar), 4.88 (2H, s, OCH2Ph), 6.26 (1H, d, J = 2.3
Hz, Ar-H), 6.29 (1H, t, J = 2.4 Hz, Ar-H), 7.33-7.39 (6H, overlapping m, Ar-H) and 9.66 (1H,
br d, NH); δC/ppm (150 MHz; CDCl3) 16.2 (d, JP-C = 7.3 Hz, 2 × CH3), 33.7 (CH2CO), 42.8
(NCH2CH2), 43.2 (NCH2Ar), 62.7 (d, JP-C = 5.5 Hz, 2 × OCH2CH3), 78.1 (OCH2Ph), 109.1,
110.5, 127.6, 128.7, 129.2, 135.7, 142.5 and 151.1 (Ar-C) and 168.9 (C=O).
3.5.1.6. Diethyl N-[2-(N-benzyloxycarbamoyl)ethyl]-N-(3-nitrobenzyl)phosphoramidate 19g
as a yellow oil (1.9 g, 78%) [HRMS: m/z calculated for C21H29N3O7P (MH+) 466.1743. Found
466.1754]; νmax/cm-1 1668 (C=O) and 1229 (P=O); δH/ppm (600 MHz; CDCl3) 1.30 (6H, t, J =
7.3 Hz, 2 × CH3), 2.33 (2H, m, CH2CO), 3.21 (2H, m, NCH2CH2), 4.01-4.04 (4H, m, 2 ×
OCH2CH3), 4.25 (2H, d, JP-H = 10.2 Hz, NCH2Ar), 4.87 (2H, s, OCH2Ph), 7.31-7.36 (5H,
overlapping m, Ar-H), 7.49 (1H, m, Ar-H), 7.66 (1H, m, Ar-H), 8.11 (1H, m, Ar-H). 8.24 (1H,
m, Ar-H) and 10.0 (1H, s, NH); δC/ppm (150 MHz; CDCl3) 16.2 (d, JP-C = 7.0 Hz, 2 × CH3),
33.3 (CH2CO), 42.4 (NCH2CH2), 49.5 (NCH2Ar), 63.1 (d, JP-C = 5.7 Hz, 2 × OCH2CH3), 78.0
(OCH2Ph), 122.2, 122.6, 123.0, 128.5, 129.0, 129.6, 134.4, 135.5, 140.1 and 148.4 (Ar-C) and
168.7 (C=O).
12

ACCEPTED MANUSCRIPT
3.5.2. Reaction of carboxylic acids 18a-g with N-methylhydroxylamine hydrochloride
N-Methylhydroxylamine hydrochloride (0.017 g, 0.20 mmol) was added to a mixture of
diethyl N-benzyl-N-(2-carboxyethyl)phosphoramidate 18a (0.13 g, 0.20 mmol) and CDI
(0.039 g, 0.24 mmol) in dry DCM (5 mL), and the mixture was left to stir at r.t. for 7 days. The
solvent was evaporated in vacuo and the residue dissolved in EtOAc. The organic solution was
sequentially washed with 10% aq. citric acid and saturated aq. NaHCO3, dried with anhydrous
MgSO4 and filtered. The solvent was evaporated in vacuo and the crude product was flash
chromatographed [on silica gel; elution with EtOAc-hexane (6:4)] to afford diethyl N-benzyl-
N-[2-(N-hydroxy-N-methylcarbamoyl)ethyl]phosphoramidate 20a as a pale- yellow oil (0.093
g, 67%) [HRMS: m/z calculated for C15H26N2O5P (MH+) 345.1579. Found 345.1577]; νmax/cm-
1
3325 (OH), 1634 (C=O) and 1227 (P=O); δH/ppm (300 MHz; CDCl3) 1.2-1.3 (6H, m, 2 ×
CH3), 2.28 and 2.65 (2H, m, CH2CO), 3.17-3.24 (5H, overlapping m, NCH3 and NCH2CH2),
3.98 (4H, m, 2 × OCH2CH3), 4.13 (2H, m, NCH2Ph) and 7.20-7.28 (5H, overlapping m, Ar-
H); δC/ppm (75 MHz; CDCl3) 16.3 (d, JP-C = 7.2 Hz, 2 × CH3), 29.8 (CH2CO), 33.7 (NCH3),
36.2 (d, JP-C = 4.8 Hz, NCH2CH2), 41.5 (d, JP-C = 5.1 Hz, NCH2Ph), 62.7 (d, JP-C = 6.0 Hz, 2 ×
OCH2CH3), 127.4, 128.4, 128.7 and 137.8 (Ar-C) and 174.4 (C=O).
3.5.2.1. Diethyl N-(3,4-dichlorobenzyl)-N-[2-(N-hydroxy-N-methylcarbamoyl)ethyl]phos-
phoramidate 20b as a brown oil (0.11 g, 70%) [HRMS: m/z calculated for C15H2435Cl2N2O5P
(MH+) 413.0800. Found 413.0797]; νmax/cm-1 3334 (OH), 1638 (C=O) and 1229 (P=O);
δH/ppm (300 MHz; CDCl3) 1.25-1.27 (6H, m, 2 × CH3), 2.3 and 2.6 (2H, m, CH2CO), 3.17-
3.27 (5H, overlapping m, NCH3 and NCH2CH2), 3.95-4.10 (6H, m, 2 × OCH2CH3 and
NCH2Ar), 7.12 (1H, d, J = 10.2 Hz, Ar-H) and 7.33-7.41 (2H, overlapping m, Ar-H); δC/ppm
(75 MHz; CDCl3) 16.3 (d, JP-C = 6.9 Hz, 2 × CH3), 29.8 (CH2CO), 33.6 (NCH3), 36.2 (d, JP-C
=
4.6 Hz, NCH2CH2), 41.1 (d, JP-C = 5.3 Hz, NCH2Ar), 63.1 (d, JP-C = 5.9 Hz, 2 × OCH2CH3),
127.6, 127.9, 130.2, 130.6, 131.8 and 137.5 (Ar-C) and 171.2 (C=O).
3.5.2.2. Diethyl N-(3-chlorobenzyl)-N-[2-(N-hydroxy-N-methylcarbamoyl)ethyl]phospho-
ramidate 20c as a yellow oil (0.09 g, 70%) [HRMS: m/z calculated for C15H2535ClN2O5P
(MH+) 379.1190. Found 379.1189]; νmax/cm-1 3329 (OH), 1642 (C=O) and 1231 (P=O);
δH/ppm (300 MHz; CDCl3) 1.2-1.3 (6H, overlapping m, 2 × CH3), 2.65 (2H, m, CH2CO),
3.17-3.28 (5H, overlapping m, NCH3 and NCH2CH2), 3.95-4.20 (6H, overlapping m, 2 ×
OCH2CH3 and NCH2Ar), 7.15-7.20 (3H, m, Ar-H) and 7.28 (1H, d, J = 7.9 Hz, Ar-H); δC/ppm
(75 MHz; CDCl3) 16.2 (d, JP-C = 6.9 Hz, 2 × CH3), 29.8 (CH2CO), 33.6 (NCH3), 36.1 (d, JP-C
= 4.9 Hz, NCH2CH2), 41.1 (d, JP-C= 5.0 Hz, NCH2Ar), 62.7 (d, JP-C = 5.4 Hz, 2 × OCH2CH3),
126.7, 127.6, 127.9, 128.6, 129.9 and 134.6 (Ar-C) and 171.2 (C=O).
3.5.2.3. Diethyl
N-(3,4-difluorobenzyl)-N-[2-(N-hydroxy-N-methylcarbamoyl)ethyl]phos-
phoramidate 20d as a pale-yellow oil (0.09 g, 55%) [HRMS: m/z calculated for
C15H24F2N2O5P (MH+) 381.1391. Found 381.1397]; νmax/cm-1 3351 (OH), 1626 (C=O) and
1227 (P=O); δH/ppm (300 MHz; CDCl3) 1.2-1.3 (6H, m, 2 × CH3), 2.69 (2H, m, CH2CO),
3.22-3.31 (5H, overlapping m, NCH3 and NCH2CH2), 3.39-4.07 (4H, m, 2 × OCH2CH3), 4.14
(2H, d, JP-H = 10.3 Hz, NCH2Ar) and 7.03-7.18 (3H, overlapping m, Ar-H); δC/ppm (75 MHz;
CDCl3) 16.3 (d, JP-C = 7.0 Hz, 2 × CH3), 29.8 (CH2CO), 33.6 (NCH3), 36.1 (d, JP-C = 4.9 Hz,
NCH2CH2), 40.9 (d, JP-C = 5.2 Hz, NCH2Ar), 62.7 (d, JP-C = 5.4 Hz, 2 × OCH2CH3), 116.3 and
117.2 (2 rotameric d, JP-C = 18.6/17.0 Hz ), 123.1 and 124.5 (two rotameric dd, JF-C = 6.0 and
3.7 Hz), 134.1 and 136.8 (two rotameric m), 147.9-152.1 (series of m, CF) (Ar-C) and 171.0
(C=O).
3.5.2.4. Diethyl N-(3-fluorobenzyl)-N-[2-(N-hydroxy-N-methylcarbamoyl)ethyl]phosphora-
midate 20e as a pale-yellow oil (0.10 g, 71%) [HRMS: m/z calculated for C15H25FN2O5P
13

ACCEPTED MANUSCRIPT
(MH+) 363.1485. Found 363.1479]; νmax/cm-1 3334 (OH), 1638 (C=O) and 1229 (P=O);
δH/ppm (300 MHz; CDCl3) 1.2-1.3 (6H, t, 2 × CH3), 2.70 (2H, t, J = 7.1 Hz, CH2CO), 3.23-
3.34 (5H, overlapping m, NCH3 and NCH2CH2), 4.01-4.09 (4H, m, 2 × OCH2CH3), 4.18 (2H,
d, JP-H = 10.2 Hz, NCH2Ar), 6.85-6.94 (1H, m, Ar-H), 6.98-7.05 (2H, m, Ar-H) and 7.19-7.27
(1H, m, Ar-H); δC/ppm (75 MHz; CDCl3) 16.3 (d, JP-C = 7.0 Hz, 2 × CH3), 33.7 (CH2CO),
36.1 (NCH3), 40.9 (d, JP-C = 4.9 Hz, NCH2CH2), 44.9 (d, JP-C = 5.0 Hz, NCH2Ar), 62.7 (d, JP-C
= 5.4 Hz, 2 × OCH2CH3), 114.2 and 114.6 (two rotameric d, JF-C = 20.7/20.9 Hz), 114.2 and
115.3 (two rotameric d, JF-C = 21.6 Hz), 122.7 and 124.0 (two rotameric d, JF–C = 2.6/3.4 Hz),
130.1 (d, JF–C = 8.2 Hz), 139.6 and 142.3 (two rotameric m, CF), and 162.96 and 163.02 (two
rotameric d, JF–C = 246.2 Hz) (Ar-C) and 171.2 (C=O).
3.5.2.5. Diethyl N-furfuryl-N-[2-(N-hydroxy-N-methylcarbamoyl)ethyl]phosphoramidate
20f as a yellow oil (0.11 g, 82%) [HRMS: m/z calculated for C13H24N2O6P (MH+) 335.1372.
Found 335.1367]; νmax/cm-1 3334 (OH), 1632 (C=O) and 1232 (P=O); δH/ppm (300 MHz;
CDCl3) 1.2-1.3 (6H, m, 2 × CH3), 2.73 (2H, m, CH2CO), 3.21-3.37 (5H, overlapping signals,
NCH3 and NCH2CH2), 3.97-4.15 (6H, overlapping m, 2 × OCH2CH3 and NCH2Ar), 6.28-6.31
(2H, m, Ar-H) and 7.36 (1H,br s, Ar-H); δC/ppm (75 MHz; CDCl3) 16.2 (d, JP-C = 7.3 Hz, 2 ×
CH3), 29.8 (CH2CO), 33.4 (NCH3), 36.1 (d, JP-C = 5.0 Hz, NCH2CH2), 41.8 (d, JP-C = 5.2 Hz,
NCH2Ar), 62.8 (d, JP-C = 5.6 Hz, 2 × OCH2CH3), 107.8 and 108.2 (rotameric), 109.4, 141.4,
149.8 and 150.4 (rotameric) (Ar-C) and 171.5 (C=O).
3.5.2.6. Diethyl N-[2-(N-hydroxy-N-methylcarbamoyl)ethyl]-N-(3-nitrobenzyl)phosphora-
midate 20g as a pale-yellow oil (0.12 g, 66%) [HRMS: m/z calculated for C15H25N3O7P (MH+)
390.1430. Found 390.1439]; νmax/cm-1 3329 (OH), 1636 (C=O) and 1235 (P=O); δH/ppm (300
MHz; CDCl3) 1.29-1.34 (6H, m, 2 × CH3), 2.69 (2H, m, CH2CO), 3.19-3.29 (5H, overlapping
signals, NCH3 and NCH2CH2), 3.99-4.09 (4H, m, 2 × OCH2CH3), 4.28 (2H, d, JP-H = 10.1 Hz,
NCH2Ar), 7.49 (1H, t, J = 7.9 Hz, Ar-H), 7.66 (1H, d, J = 7.6 Hz, Ar-H), 8.11 (1H, d, J = 8.1
Hz, Ar-H) and 8.23 (1H, m, Ar-H); δC/ppm (75 MHz; CDCl3) 16.1 (d, JP-C = 7.1 Hz, 2 × CH3),
33.0 (CH2CO), 36.0 (NCH3), 41.3 (d, JP-C = 4.9 Hz, NCH2CH2), 48.7 (d, JP-C = 5.1 Hz,
NCH2Ar), 63.3 (d, JP-C = 5.5 Hz, 2 × OCH2CH3), 122.6, 123.1, 129.5, 134.4, 139.6 and 148.5
(Ar-C) and 171.2 (C=O).
3.6.
The synthesis of the phosphoramidic acid derivatives (21) and (22).
The general procedure is illustrated by the following example. Bromotrimethylsilane (0.86
o
mL, 6.4 mmol) was added to a chilled (0 C), stirred solution of the diethyl ester 19g (0.60 g,
1.3 mmol) in dry DCM (5 mL). After 1 h, the solution was allowed to warm to r.t and stirred
for an additional 24 h. The solvent was removed under reduced pressure and the residue was
dissolved in THF (3 mL) and H2O (0.04 mL). The resulting solution was stirred for 1 h and
then concentrated in vacuo; the residue was triturated with EtOAc at -10 °C to afford the crude
N-[2-(N-benzyloxycarbamoyl)ethyl]-N-(3-nitrobenzyl)phosphoramidic acid 21g as a cream
solid (0.42 g, 81%), m.p. 158-160 °C; νmax/cm-1 1664 (C=O); δH/ppm (600 MHz; DMSO-d6)
2.46 (2H, t, J = 7.1 Hz, CH2CO), 3.17 (2H, t, J = 7.2 Hz, NCH2CH2), 4.34 (2H, s, NCH2Ar),
4.80 (2H, s, OCH2Ph), 7.36-7.39 (5H, overlapping m, Ar-H), 7.76 (1H, t, J = 8.0 Hz, Ar-H),
7.98 (1H, d, J = 7.5 Hz, Ar-H), 8.29 (1H, d, J = 7.8 Hz, Ar-H) and 8.47 (1H, s, Ar-H); δC/ppm
(150 MHz; DMSO-d6) 28.5 (CH2CO), 42.6 (NCH2CH2), 49.2 (NCH2Ar), 77.1 (OCH2Ph),
123.6, 124.0, 125.1, 128.4, 128.9, 130.4, 134.2, 136.1, 137.1 and 147.8 (Ar-C) and 166.0
(C=O).
14

ACCEPTED MANUSCRIPT
Analytical data supporting hydrolysis of phosphoramidate ester precursors to the crude
corresponding acids (21a-f and 22a,c,f,g) are provided in the Supporting Information file.
Acknowledgements
The authors thank Rhodes University and the South African Medical Research Council
(SAMRC) for generous financial support. This research project was funded by the South
African Medical Research Council (MRC) with funds from National Treasury under its
Economic Competitiveness and Support Package.
4. References
1. Tomonobu, U., Nobutatada, T., Yoshion, K., Masayuki, N., Yukio K. and Kazuo, T.N.
Scientific report; Molecular Basis of Fosmidomycin., 2011, Pp. 1-8.
2. Singh, N., Cheve, G., Avery, M.A. and McCurdy, C.R. Curr. Pharm. Des., 2007, 13,
1161-1177.
3. Murakawa, T., Sakamoto, H., Fukada, S., Konishi, T. and Nishida, M. Antimicrob.
Agents Chemother., 1982, 21, 224-230.
4. Beytia, E.D. and Porter, J.W. Annu. Rev. Biochem., 1976, 45, 113-142.
5. Brücher, K., Gräwert, T., Konzuch, S., Held, J., Lienau, C., Behrendt, C., Illarionov, B.,
Maes, L., Bacher, A., Wittlin, S., Mordmüller, B., Fischer, M. and Kurz, T. J. Med.
Chem., 2015, 58, 2025-2035.
6. Adeyemi, C.M. Studies towards the Development of Novel Antimalarial Agents, M.Sc.
Thesis, Rhodes University, Grahamstown, 2014.
7. Adeyemi, C.M., Faridoon, Isaacs, M., Mnkandhla, D., Hoppe, H.C., Krause, R. and
Kaye, P.T. Bioorg. Med. Chem. 2016, 24, 6131-6138
8. Adeyemi, C.M., Klein, R., Isaacs, M., Mnkandhla, D., Hoppe, H.C., Krause, R. and
Kaye, P.T. Tetrahedron, 2017, 73, 1661-1667.
9. Maiti, M., Maiti M., Rozenski, J., De Jonghea, S. and Piet Herdewijn, P. Org. Biomol.
Chem., 2015, 13, 5158.
10. Mayes, B.A., Arumugasamy, J., Baloglu, E., Bauer, D., Becker, A., Chaudhuri, N.,
Latham, G.M., Li, J., Mathieu, S., McGarry, F.P., Rosinovsky, E., Stewart, A., Trochet,
C., Wang, J. and Adel Moussa, A. Organic Process Research & Development, 2014 18,
717-724.
15

ACCEPTED MANUSCRIPT
11. http://www.chemguide.co.uk/organicprops/alcohols/esterification.html (22/03/2017).
12. Ahmed, K., Anver, B.S.G. Narender, R.C., Ganesh, K., Joveeta, J.G., Bharath, K.,
Uppula, P.G. and Narahari, S. Med. Chem. Res., 2014, 23, 2080-2092.
13. Laxmi, V.P. and Narender, R. Asian J. Chem., 2015, 27, 3605-3608.
14. Horniakova, J., Komura, K., Osaki, H., Kubota, Y. and Sugi, Y. Catal. Lett., 2005, 102,
191-196.
15. Komura, K., Hongo, R., Tsutsui, J. and Sugi, Y. Catal. Lett., 2009, 128, 203-209.
16. Haitao, C., Liping, Z., Ying, X., Qiongli, Q. and Hongding, T. J. Inorg. Organomet.
Polym., 2014, 24, 780-785.
17. Juan, L., Changmei, C., Xinge, Z., Qingfei, L., Ping, Y., Yiming, W. and Guoan, L. Eur.
J. of Med. Chem., 2010, 45, 1731-1738.
18. Yu-Xiu, L., Deng-Guo, W., Ye-Rong, Z., Shao-Hua, L., Yong-Lin, Z., Qi-Qi, Z., Bao-Li,
C., Yong-Hong, L., Hai-Bin, S., Ying, L., Yong, W., Run-Qiu, H. and Qing-Min, W. J.
Agric. Food Chem., 2008, 56, 204-212.
19. Arvind, K.Y. and Lal Dhar, S.Y. RSC Adv., 2014, 4, 34764-34767.
20. Kuliszewska, E., Hanbauer, M. and Hammerschmidt, F. Chem. Eur. J., 2008, 14, 8603-
8614.
21. Kishore, K.G.D., David, S., Lokesh, G.L. and Natarajan, A. Tetrahedron Lett., 2006, 47,
6281-6284.
22. Hammerschmidt, F. and Hanbauer, M. J. Org. Chem., 2000, 65, 6121-6131.
23. Osowska-Pacewicka, K., Zawadzki, S., Zwierzak, A. Phosphorus, Sulfur and Silicon,
1993, 82, 49-54.
24. Maiti, M., Michielssens, S., Dyubankova, S., Maiti, M., Lescrinier, E., Ceulemans, A.
and Herdewijn, P. Chem. Eur. J., 2012, 18, 857–868.
16