Synthesis and anti-parasitic activity of achiral N-benzylated phosphoramidic acid derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.103947

Synthetic pathways have been developed to access a series of N-benzylated phosphoramidic acid derivatives as novel, achiral analogues of the established Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductase (PfDXR) enzyme inhibitor, FR900098. Bioassays of the targeted compounds and their synthetic precursors have revealed minimal antimalarial activity but encouraging anti-trypanosomal activity – in one case with an IC₅₀ value of 5.4 μM against Trypanosoma brucei, the parasite responsible for Nagana (African cattle sleeping sickness). The results of relevant in silico modelling and docking studies undertaken in the design and evaluation of these compounds are discussed.

Full text of DOI:10.1016/j.bioorg.2020.103947

Journal Pre-proofs
Synthesis and Anti-parasitic Activity of Achiral N-Benzylated Phosphorami-
dic Acid Derivatives
Christiana M. Adeyemi, Anne C. Conibear, Marius K. Mutorwa, Iviwe
Nokalipa, Michelle Isaacs, Dumisani Mnkandhla, Heinrich C. Hoppe, Kevin
A. Lobb, Rosa Klein, Perry T. Kaye
PII:
S0045-2068(20)30572-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.103947
YBIOO 103947
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
16 March 2020
11 May 2020
14 May 2020
Please cite this article as: C.M. Adeyemi, A.C. Conibear, M.K. Mutorwa, I. Nokalipa, M. Isaacs, D. Mnkandhla,
H.C. Hoppe, K.A. Lobb, R. Klein, P.T. Kaye, Synthesis and Anti-parasitic Activity of Achiral N-Benzylated
Phosphoramidic Acid Derivatives, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.103947
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Synthesis and Anti-parasitic Activity of Achiral N-Benzylated
Phosphoramidic Acid Derivatives
Christiana M. Adeyemia, Anne C. Conibeara†, Marius K. Mutorwaa‡, Iviwe Nokalipaa,
Michelle Isaacsc, Dumisani Mnkandhlac, Heinrich C. Hoppeb,c, Kevin A. Lobba,c,
Rosa Kleina,c, and Perry T. Kayea,c*
aDepartment of Chemistry, bDepartment of Biochemistry and Microbiolgy and cCentre for Chemico- and
Biomedicinal Research, Rhodes University, Grahamstown, 6140, South Africa.
Abstract. Synthetic pathways have been developed to access a series of N-benzylated
phosphoramidic acid derivatives as novel, achiral analogues of the established Plasmodium
falciparum 1-deoxy-D-xylulose-5-phosphate reductase (PfDXR) enzyme inhibitor, FR900098.
Bioassays of the targeted compounds and their synthetic precursors have revealed minimal
antimalarial activity but encouraging anti-trypanosomal activity  in one case with an IC50 value
of 5.4 µM against Trypanosoma brucei, the parasite responsible for Nagana (African cattle
sleeping sickness). The results of relevant in silico modelling and docking studies undertaken in
the design and evaluation of these compounds are discussed.
Keywords: Anti-parasitic, Anti-malarial, Anti-trypanosomal, N-benzylated phosphoramidate,
Nagana Trypanosoma brucei
O
ROUTE I
P
NH2
EtO
OEt
R1
R2
O
OH
N
a
b
c
d
e
f
H
H
H
H
H
Cl
H
F
P(OEt)2
CH2OH
N
NH2
SH
Cl
Cl
F
R1
R2
O
a-h
NH2
g
h
R1
R2
F
H
ROUTE III
__________
*Corresponding author: Prof Perry Kaye. E-mail: p.kaye@ru.ac.za
†Present address: School of Biomedical Sciences, The University of Queensland, St. Lucia 4072 QLD, Brisbane,
Australia.
‡ Present address: Department of Natural and Applied Sciences, Namibia University of Science and Technology,
Namibia.
1

1. Introduction
While still unacceptably high, World Health Organization estimates of mortality levels arising
from malarial infection1 appear to have been declining in recent years. However, the emergence
of resistance to even the newest antimalarial drugs2 requires ongoing commitment to the
development of effective replacements. Our earlier research3-6 in this area has focussed on the
synthesis and evaluation of novel carboxamido alkylphosphonic acid derivatives and N-
substituted phosphoramidate esters7 (as "reverse" fosmidomycin analogues) designed to act as
inhibitors of Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductase (PfDXR). This
critical enzyme in the non-mevalonate isoprenoid biosynthetic pathway is unique to the mosquito
vector but absent in the human host, and has been validated as an antimalarial drug target.8,9
FR900098 110 is the N-acetyl derivative of the naturally occurring antibiotic, fosmidomycin 2
(Figure 1);11 both compounds mimic the PfDXR substrate and are effective PfDXR inhibitors,
but rapid in vivo clearance and recrudescence issues limit their usefulness.12,13
O
O
P
O
OH
N
O
O
O
HO
HO
H
N
P
OH
H3C
N
P
OH
OH
OH
OH
OH
1
2
3
Cl
Cl
Figure 1. Structures of known PfDXR inhibitors.
Nagana (African cattle sleeping sickness), another parasitic disease that has plagued the African
continent, is a Trypanosoma brucei (T.brucei) infection due to subspecies, such as T. brucei
brucei, carried by Tsetsi flies (Glossina). While control of this disease, which has been
responsible for major losses in animal live-stock,14 has improved dramatically, the available
drugs are limited, old and susceptible to resistance.15 Animals can also carry the subspecies,
Trypanosoma brucei gambiense, responsible for most cases of human African trypanosomiasis
(HAT).16 Although new enzyme targets are being investigated,17 to our knowledge, DXR has yet
to be identified as a target for trypanosomal therapeutic intervention.
Preliminary analysis18 of the 3-D topology of the active-site in a homology-modelled structure19
of PfDXR revealed the presence of three, adjacent, hydrophobic binding pockets. Perruchon20 and
Deng21 had located two of these pockets close to the phosphonate moiety binding pocket, and the
remarkable PfDXR inhibition activity of the alkylphosphonate derivative 3 has been attributed22
to the occupation of one of the empty pockets by the 3,4-dichlorophenyl group. Enantioselectivity
2

issues implicit in such chiral alkylphosphonates have prompted us to explore the preparation of
non-chiral N-substituted phosphoramidate analogues7 and, in this paper, we now report: i) the
design and synthesis of a series of N-benzylated phosphoramidic acid analogues of FR900098 1;
ii) their in vitro cytotoxicity and antimalarial and anti-trypanosomal activity; and iii) the results
of in silico studies of their docking in the PfDXR active site.
2. Results and Discussion
The novel phosphoramidate derivatives (4) were designed to incorporate the structural features
illustrated in Figure 2, viz., i) a phosphoramidate moiety in which the prochiral α-methylene
group in the phosphonate ligands FR900098 1 and fosmidomycin 2 is replaced by trigonal
nitrogen, thus obviating the chirality issue; ii) an N-benzyl group to occupy a hydrophobic pocket
adjacent to the PfDXR active-site; iii) a three-atom aminoethyl (N-C-C) chain replacing the
trimethylene hydrophobic patch in FR900098 1; and iv) the N-acetyl hydroxamate moiety
present in FR900098 1, which is approximately twice as active in vitro as fosmidomycin 2. It
was recognised that the phosphoramidate esters (4) might serve as ester pro-drugs, exhibiting
better lipophilicity than the phosphoramidic acids to which they could be hydrolysed in vivo by
non-specific esterases.
Figure 2. Design features of novel series of phosphoramidate ligands 4.
The synthetic pathway (Route I) outlined in Scheme 1, adapted from reported methods for the
preparation of fosmidomycin 2 and its analogues, was used to access the phosphoramidate esters
4a-d and the corresponding phosphoramidic acids 12a-d. Thus, using a method reported by
Zwierzak et al.,23 diethyl phosphoramidate 5 was converted to the silylated derivative 6 in
essentially quantitative yield (98%). Acetalisation and benzylation steps afforded the N-
benzylated phosphoramidate acetals 8a-d, acid-catalysed hydrolysis of which afforded the
corresponding aldehydes 9a-d in excellent yields. Reductive amination of the aldehydes 9a-d
3

using O-benzylhydroxylamine furnished the O-benzyl-protected amines 10a-d. Incorporation of
the acetyl functionality, required in the FR900098 analogues, was accomplished by adapting
methodology reported by Ortmann et al.24,25 to give the O-benzyl-protected N-acetyl derivatives
11a-d. The final steps in the overall synthesis involved selective catalytic hydrogenolysis, using
10% Pd/C in methanol, to yield the hydroxamate ester derivatives 4a-h, and hydrolysis of the
esters, using TMSBr, to afford the novel FR900098 analogues 12a-d.
A Saturation Transfer Difference (STD) NMR experiment26 confirmed binding of the N-
benzylated phosphoramidic acids 12a and 12d to the then available EcDXR enzyme; the acids
were used since the ester precursors were expected to undergo hydrolysis in vivo before binding
to the enzyme. Preliminary in silico docking of the N-benzylated phosphoramidic acids 12a-d
was undertaken using then available, homology-modelled structure of PfDXR.19, 26 The ligands
were observed to adopt binding orientations similar to that of fosmidomycin 2, with the N-benzyl
moieties occupying a hydrophobic pocket adjacent to the phosphonate-binding site. In each of
the ligands 12a-d, the phosphonate moiety exhibited hydrogen-bonding interactions with the
active-site residues, Ser 199 and Gly 201, while the carbonyl group interacted with residue Ser
235. Additional interactions were observed between individual ligands 12a-d and several amino
acid residues in the active-site.
In view of the activity of the halogenated phosphonate esters reported by Haemers et al.,22 the
preparation of the initial series of phosphoramidic esters 4a-d was extended to include
halogenated derivatives. In a slight modification of the approach to the critical intermediates (8)
(Route I, Scheme 1), the reaction of diethyl phosphoramidate 5 with hexamethyldisilazane was
conducted in THF instead of benzene. However, the hemiacetal 13 was isolated repeatedly and
alternative approaches, which involved introduction of a benzylamine moiety in the first step,
were explored. Reaction of benzylamine 14a with diethyl chlorophosphate in the presence of
triethylamine (Route II) afforded the phosphoramidate derivative 15a, but attempts to react this
compound with bromoacetaldehyde diethyl acetal, in the presence of sodium hydride failed to
afford the desired intermediate 8a.
4

O
P
O
P
NH2
R1
R2
OEt
OH
NH2
EtO
N
EtO
x
14a,e-h
H
OEt
i
OEt
5
13
ROUTE III
ROUTE I
ROUTE II
xi
14a
xii
(for
)
O
P
O
O
NH
SiMe3
P(OEt)2
O
R1
R2
HN
N
EtO
H
OEt
16a,e-h
6
15a
ii,iii
ii,iii
xi
X
O
P(OEt)2
O
O
P
EtO
H
P(OEt)2
OEt
OEt
N
N
iv
v
N
EtO
OEt
R1
R1
R2
O
H
OEt
R2
8a-h
7
9a-h
vi
N
O
N
O
O
O
OH
N
H
Bn
Bn
N
P(OEt)2
R1
P(OEt)2
P(OEt)2
O
O
N
N
xiii
vii
H
R1
O
overnight
10a-h
17
11a-h
R2
R2
xiii
5h
R1
R2
a
b
c
d
e
f
H
H
H
H
H
Cl
H
F
O
P(OEt)2
OH
O
OH
N
CH2OH
P(OH)2
N
NH2
SH
Cl
Cl
F
F
N
N
ix
R1
R2
O
R1
R2
O
4a-d
(For
)
g
h
4a-h
12a-d
H
Scheme 1.
o
Reagents and conditions:- i) Hexamethyldisilazane (20% mol excess), benzene, 80 C, 3 h; ii)
NaH, benzene, r.t., N2, then bromoacetaldehyde diethyl acetal, TBAB (10% mol), benzene, 80
oC, 4 h iii) EtOH, reflux, 1 h.; iv) NaH, benzyl halide, THF, r.t., 24 h, N2; v) 2-M HCl, r.t., 24
h; vi) O-benzylhydroxylamine.HCl in MeOH, reflux, 5 h, then NaCNBH3, conc. HCl, r.t.; vii)
acetyl chloride, DCM, Et3N, 0 oC, 1 h, then r.t., 24 h, N2; viii) H2, Pd/C, dry MeOH; ix) TMSBr,
o
DCM, N2, 0 C, 1 h, then H2O, r.t., overnight; x) hexamethyldisilazane, dry THF, 80 °C, 3 h;
xi) diethyl chlorophosphate, Et3N, DCM, r.t., 24 h; xii) bromoacetaldehyde diethyl acetal,
anhydr. K2CO3, dry CH3CN, 24 h, 85 °C; xiii) 10% Pd/C, dry MeOH, H2.
5

In Route III, initial N-alkylation of the benzylamines 14a,e-h, using bromoacetaldehyde
diethylacetal in the presence of anhydrous K2CO3 in refluxing acetonitrile, furnished the acetals
16a,e-h in variable yield (23-61%), but subsequent reaction with diethyl chlorophosphate, in the
presence of triethylamine, gave the diethyl N-benzyl-N-(2,2-diethoxyethyl)phosphor-amidate
derivatives 8a,e-h in good to excellent yield (71-94%). From this point, the original methodology
described for Route I was essentially followed to access the phosphoramidate esters 4a,e-h.
However, purification of the O-benzylhydroxyamino compounds 10a,e-h was problematic due
to decomposition during column chromatography, and the subsequent conversion to the
acetylated derivatives 11a,e-h was successfully accomplished without prior purification of the
precursors 10a,e-h. Selective de-protection of the N-benzylated hydroxylamine moiety in the
esters 11a,e-h was effected in excellent yield (89-97%) by catalytic hydrogenation using 10%
palladium-on-carbon catalyst in dry MeOH for a limited period (5 h) at room temperature. Initial
reactions, conducted overnight and using compounds 11a,e, proved to be non-selective, resulting
in cleavage of both the O- and N-benzyl rings and affording the common product 17, which is
an aza-analogue of the diethyl phosphonate ester of FR900098 1. Careful TLC monitoring of the
hydrogenolysis finally permitted selective removal of the O-benzyl group alone, to furnish the
desired N-benzylated esters 4a,e-h. Hydrolysis of the esters 4a-d using TMSBr afforded the
corresponding FR900098 analogues 12a-d.
The N-benzylated phosphoramidate esters 4a-e and 17 and the corresponding sets of intermediates
(9), (10) and (11) were examined for: i) toxicity against HeLa cells; ii) antimalarial activity against
P. falciparum using the plasmodium lactate dehydrogenase (pLDH) assay – a general screening
expected to permit hydrolysis of ester derivatives due to the presence of esterases in the Pf cells;
iii) anti-trypanosomal activity against T.brucei. The results are summarised in Table 1, and it is
apparent that all of the compounds examined were found to be essentially non-toxic against HeLa
cells at a concentration of 20 µM. Some of the 3,4-dichlorobenzyl derivatives showed a measure
of antimalarial activity at a concentration of 20 µM, as determined by the pLDH assay of malarial
cell viability (8e 54%; 9e 85%; 10e 70%; and 11e 67%). More disappointingly, none of the
targeted esters 4a,e-h exhibited any statistically significant antimalarial (pLDH) activity. (The
earlier, positive STD NMR results with compounds 12a and 12d had been obtained using the then
available EcDXR enzyme.) Surprisingly, compound 17, which has a very similar structure to
FR900098 1, also showed no promising activity against P. falciparum parasites.
6

Table1. Results for the HeLa cell toxicity and pLDH and T.brucei assays.
HeLa cell%
viability
at 20 µM
PLDH
% activity
at 20 µM
T. brucei
% viability
at 20 µM
Ar
Compound
Phenyl
8a
8e
8f
8g
8h
9a
9e
9f
9g
109.7±4.3
101.0±2.1
98.2±5.1
119.1±4.9
80.9±10.7
113.9±0.3
91.1±2.1
114.0±6.7
95.8±13.5
103.9±11.1
114.0±9.9
101.1±4.9
115.5±0.6
93.5±11.5
96.0±13.4
101.0±4.1
98.7±0.5
115.8±6.4
98.1±6.9
90.4±0.2
105.6±5.5
91.3±8.1
119.8±8.4
89.6±5.9
99.6±11.2
117.2±0.3
0a
106.2±0.5
53.9±6.5
90.5±3.9
127.9±3.8
121.3±2.8
106.8±6.7
85.4±5.0
113.9±2.6
95.5±8.9
106.8±0.1
81.6±7.1
69.8±7.9
85.5±10.7
119.9±8.5
101.2±6.8
115.8±4.3
66.5±12.5
93.6±4.0
85.5±4.3
102.5±2.0
101.3±0.2
114.4±9.0
103.1±7.6
104.1±2.8
100.0±1.4
115.9±0.4
0b
97.1±0.4
2.6±0.3
3,4-Dichlorophenyl
3-Chlorophenyl
3,4-Difluorophenyl
3-Fluorophenyl
Phenyl
3,4-Dichlorophenyl
3-Chlorophenyl
3,4-Difluorophenyl
3-Fluorophenyl
Phenyl
3,4-Dichlorophenyl
3-Chlorophenyl
3,4-Difluorophenyl
3-Fluorophenyl
Phenyl
3,4-Dichlorophenyl
3-Chlorophenyl
3,4-Difluorophenyl
3-Fluorophenyl
-
101.7±9.4
102.8±1.1
112.0±4.7
102.9±4.4
96.2±0.8
90.0±2.7
102.2±16.7
97.4±10.3
60.7±10.1
2.1±0.1
26.5±31.2
112.2±1.6
118.1±1.9
97.2±1.0
1.2±0.3
9h
10a
10e
10f
10g
10h
11a
11e
11f
11g
11h
17
9.2±0.2
31.8±0.1
60.2±2.8
106.4±3.2
110.6±3.8
103.9±2.6
117.8±2.9
104.4±10.7
108.2±0.7
0c
Phenyl
4a
4e
4f
4g
3,4-Dichlorophenyl
3-Chlorophenyl
3,4-Difluorophenyl
3-Fluorophenyl
4h
Control
c
Positive control standards: aEmetine, bChloroquine and Pentamine.
Interestingly, a number of the compounds showed some activity against T.brucei (< ca. 60% cell
viability) at a concentration of 20 µM, with four of them exhibiting significant activity (1.2 - 9.2%
viability); IC50 values (Figure 3) were obtained for these four compounds, the 3,4-dichlorophenyl
derivatives 8e (13.9 µM), 10e (16.9 µM) and 11e (5.4 µM) and the 3-chlorophenyl analogue 11f
(13.2 µM). It is also interesting to note that three of these compounds (8e, 10e and 11e) also exhibit
the best, albeit modest, antimalarial activity as determined by pLDH viability. To our knowledge,
T.brucei does not contain a DXR enzyme, and the observed anti-trypanosomal activity is
attributed to action at an unknown target.
7

Figure 3. The inhibitory effects of selected compounds, showing IC50 values for T.brucei
inhibition.
In order to rationalize the unexpectedly disappointing antimalarial data, in silico docking studies
of the structures 12a-h (and their synthetic precursors), fosmidomycin 1 and FR900098 2 were
undertaken using the initially available, homology-modelled PfDXR structure19 and the PfDXR
structures (3AU9 and 3AUA)9 which had subsequently been determined crystallographically.
For the ligand models, both the negatively charged, mono-deprotonated phosphoramidic acid
species and the phosphoramidate esters were constructed. Interestingly, many of the compounds,
while docking within the active site of the homology-modelled PfDXR19 structure, rarely docked
within the active sites of the crystallographically determined PfDXR9 structures. The failure of
the targetted ligands to dock into the active-site of the PfDXR structures is consistent with the
observed lack of antimalarial activity.
In view of the evident discrepancies between the docking data obtained in the initial studies, using
the then available homology-modelled PfDXR19 structure, and later docking studies27 using the
subsequently determined X-ray crystallographic PfDXR structure,9 attention has been given to
exploring the relative topology of the respective active sites. Examination of the active sites of the
X-ray crystallographic PfDXR9 and the homology-modelled PfDXR19 structures (Figures 4a,b)
reveals that, while the overall volume of region I of the active site appears to remain comparable
between the two structures, the overall volumes of regions II and III in the X-ray crystallographic
PfDXR9 structure are considerably more compact. These differences presumably account for the
failure of the targeted compounds to exhibit any significant anti-malarial activity.
8

III
(a)
I
III
(b)
II
I
Figure 4. Different orientations (a and b) of the overlay of the of the homology-modelled PfDXR
active-site19 (yellow) and the active-site (blue) in the crystallographically determined PfDXR
structure (3AU9).9 In each case, the cofactor is present on the right and is coloured according to
atom type.
3. Conclusions.
The N-benzylated phosphoramidate esters 4a-h have been successfully synthesised as FR900098
ester analogues, using either a seven-step or a somewhat improved, six-step sequence. In a
further step, selective hydrolysis of four of these esters has provided access to the corresponding
phosphoramidic acids 12a-d. While STD NMR experiments on two of these acids had supported
their capacity to bind to the EcDXR enzyme, the esters 4a,e-h exhibited no statistically
significant activity in pLDH assays. In contrast with the in silico docking data obtained using the
homology-modelled PfDXR enzyme structure,19 the data obtained using the
9

crystallographically-determined PfDXR structure9 indicated preferential binding of the acid
analogues at a site slightly removed from the active site. These discrepancies are attributed to
unexpected differences in the topologies of the active-sites. The observed activity of some of
the synthetic intermediates against T. brucei, however, suggests their potential as lead
compounds in the development of novel anti-trypanosomal agents.
4. Experimental
4.1.
General methods
NMR spectra were typically recorded on Bruker 300, 400 or 600 MHz spectrometers in CDCl3
and calibrated using solvent signals [δH: 7.26 ppm for residual CHCl3. δC: 77.0 ppm (CDCl3)].
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).
The known compound, diethyl N-(2,2-diethoxyethyl)phosphoramidate 723 was isolated as a
yellow oil (4.69 g, 87%). The benzyl halides, 3-(bromomethyl)aniline and 3-mercaptobenzyl
bromide, were prepared, sequentially, from 3-aminobenzyl alcohol.General synthetic procedures
and experimental data for representative compounds are reported below. Experimental data for
the remaining synthesised compounds, NMR spectra for all new compounds, in silico docking
protocols (and the resulting binding affinities) and the bioassay results are provided in the
Supporting Information file. Bioassay protocols have been reported previously.5
4.2. The general procedure for the N-benzylation of the diethyl acetal 7 to access the
intermediates 8a-d (via Route I) is illustrated by the following example. To a stirred solution
of diethyl N-(2,2-diethoxyethyl)phosphoramidate 7 (1.00 g, 3.71mmol) in dry THF (20 mL)
under N2 was added NaH (60% dispersion in mineral oil; 0.20 g, 7.4 mmol) in small portions to
permit controlled evolution of hydrogen. Benzyl bromide (0.44 mL, 3.7mmol) in dry THF (5
mL) was then added and the resulting solution was stirred for at room temperature for ca. 24
hours. The solvent was evaporated in vacuo and the residue extracted with EtOAc (2 x 25 mL).
The organic phase was washed sequentially with satd. aq. NaHCO3 (2 × 50 mL), water (2 x 50
mL) and brine (2 × 50 mL). The aqueous washings were extracted with EtOAc (2 x 25 mL) and
the combined organic layers were dried (anhydr. MgSO4). The solvent was removed in vacuo
and the residue chromatographed [on silica gel; elution with hexane-EtOAc (4:6)] to yield diethyl
N-benzyl-N-(2,2-diethoxyethyl)phosphoramidate 8a as a yellow oil (0.60 g, 71%); (Found: C,
56.93; H, 8.49; N, 3.84%. C17H30NO5P requires C, 56.81; H, 8.41; N, 3.90%); max/cm-1 1232
(P=O); δH/ppm (600 MHz; CDCl3) 1.20 (6H, t, J = 7.0 Hz, 2 × CH3), 1.31 [6H, t, J = 7.1 Hz,
PO(OCH2CH3)2], 3.00 (2H, dd, JH-H = 5.4 and JP-H = 11.2 Hz, NCH2), 3.48 (2H, m, 2 ×
OCHaCH3), 3.67 (2H, m, 2 × OCHbCH3), 4.07 [4H, m, PO(OCH2CH3)2], 4.33 (2H, d, JP-H = 10.5
Hz, NCH2Ph), 4.59 (1H, t, J = 5.4 Hz, CH), 7.25 (1H, d, J = 7.4 Hz, Ar-H), 7.31 (2H, t, J = 7.5
Hz, Ar-H) and 7.35 (2H, d, J = 7.5 Hz, Ar-H); δC/ppm (150 MHz; CDCl3) 15.5 (2 × CH3), 16.3
[d, JP-C = 7.4 Hz, PO(OCH2CH3)2], 47.4 (d, JP-C = 4.6 Hz, NCH2), 50.6 (d, JP-C = 4.6 Hz,
NCH2Ph), 62.4 [d, JP-C = 5.6 Hz, PO(OCH2CH3)2], 62.9 (2 × OCH2CH3), 102.8 (CH), 127.4,
128.5, 128.8 and 138.2 (Ar-C).
10

4.3. The general procedure for the N-phosphonation of the diethyl acetals (16) to access the
intermediates 8a,e-h (via Route III) is illustrated by the following example. N-benzyl-2,2-
diethoxyethanamine 16a (3.79 g, 17.0 mmol) was dissolved DCM 20 mL, triethylamine (2.37
mL, 17.0 mmol) and diethyl chlorophosphate (2.45 mL, 17.0 mmol) was added to the reaction
mixture and allowed to stir at r.t for 24 h. The mixture was washed with 10% HCl and then with
water (2 × 20 mL). The organic layer was dried with anhydr. MgSO4, filtered, and solvent was
removed in vacuo to afford diethyl N-benzyl-N-(2,2-diethoxyethyl)phosphoramidate 8a as a
yellow oil (5.3 g, 87%).
4.4. The general procedure for the hydrolysis of the diethyl acetals (8) to afford the diethyl
N-benzyl-N-(2-oxoethyl)phosphoramidates 9a-h is illustrated by the following example.
Diethyl N-benzyl-N-(2,2-diethoxyethyl)phosphoramidate 8a (5.2 g, 14 mmol) was dissolved in
45 mL of 2M HCl and stirred at rt for 24 h. The resulting mixture was dissolved in CHCl3 and
washed with water (3 × 20 mL), the water layer was re-extracted with CHCl3and the combined
organic layer was washed with NaHCO3 (3 × 20 mL), brine (3 × 20 mL), dried with anhydr.
MgSO4 and concentration in vacuo afforded diethyl N-benzyl-N-(2-oxoethyl)phosphoramidate
9a as a yellow oil (1.9 g, 45%) [HRMS: m/z calculated for C13H21NO4P (MH+) 286.1208. Found.
286.1218]; max/cm-1 1695 (C=O) and 1230 (P=O); δH/ppm (600 MHz; CDCl3) 1.37 [6H, t, J =
7.0 Hz, PO(OCH2CH3)2], 3.77 (2H, d, JP-H = 11.6 Hz, NCH2), 4.16 [4H, m, PO(OCH2CH3)2],
4.28 (2H, d, JP-H= 8.0 Hz, NCH2Ph), 7.29-7.35 (5H, overlapping m, Ar-H) and 9.46 (1H, s,
CHO); δC/ppm (150 MHz; CDCl3) 16.3 [d, JP-C = 7.2 Hz, PO(OCH2CH3)2], 51.1 (d, JP-C = 3.8
Hz, NCH2), 55.4 (d, JP-C = 4.7 Hz, NCH2Ph), 63.0 [d, JP-C = 5.5 Hz, PO(OCH2CH3)2], 128.1,
128.8, 128.9 and 136.7 (Ar-C) and 199.9 (CHO).
4.5. The general procedure for reductive amination of the aldehydes (9) to afford the N-
benzyloxy derivatives 10a-h is illustrated by the following example. To a stirred solution of
diethyl N-benzyl-N-(2-oxoethyl)phosphoramidate 9a (1.8 g, 6.48 mmol), in MeOH was added
O-benzylhydroxylamine hydrochloride (1.35 g, 8.42 mmol). The solution was refluxed for 5 h
and sodium cyanoborohydride (1.22 g, 19.4 mmol), conc. HCl (1.7 mL) was added dropwise and
the mixture was stirred at r.t. for 1.5 h. Sodium cyanoborohydride (0.41 g, 0.48 mmol) was again
added and the reaction mixture was stirred for 1h. The solvent was concentrated in vacuo, the
residue was dissolved in MeOH (30 mL) and treated with ice water (20 mL) and the pH adjusted
to 10 using aq. KOH solution. The solution was extracted using DCM (3 × 25 mL) and the
combined organic layers were washed with saturated NaHCO3 (50 mL) and brine (50 mL) and
dried with anhydr. MgSO4. The solvent was evaporated in vacuo and the crude product was
purified by flash chromatography [on silica gel; elution with EtOAc-hexane (7:3)] to yield
diethyl N-benzyl-N-[2-(benzyloxyamino)ethyl]phosphoramidate 10a as a yellow oil (0.54 g,
21%) [HRMS: m/z calculated for C20H30N2O4P (MH+) 393.1943. Found 393.1946]; max/cm-1
3408 (NH) and 1237 (P=O); δH/ppm (600 MHz; CDCl3) 1.30 [6H, m, PO(OCH2CH3)2], 3.06
(4H, m, NCH2 and CH2NH), 4.05 [4H, m, PO(OCH2CH3)2], 4.10 (2H, d, JP-H= 9.7 Hz, NCH2Ph),
4.63 (2H, s, OCH2Ph) and 7.26-7.31 (10H, overlapping m, Ar-H); δC/ppm (150 MHz; CDCl3)
16.3 [d, JP-C = 7.1 Hz, PO(OCH2CH3)2], 43.0 (d, JP-C = 4.0 Hz, NCH2), 50.2 (d, JP-C = 5.0 Hz,
CH2NH), 53.9 (NCH2Ph), 62.6 [d, JP-C = 5.7 Hz, PO(CH2CH3)2], 76.2 (OCH2Ph), 127.5, 127.9,
128.3, 128.4, 128.5, 128.6, 138.0 and 138.1 (Ar-C).
4.6. The general procedure for the acetylation of the N-benzyloxy derivatives (10) to afford
the amine derivatives 11a-h is illustrated by the following example. Acetyl chloride (0.24 mL,
3.4 mmol) was added dropwise into the chilled solution of diethyl N-benzyl-N-[2-
(benzyloxyamino)ethyl]phosphoramidate 10a (0.66 g, 1.7 mmol) and triethylamine (0.36 mL,
11

2.5 mmol) in dry DCM (10 mL). The reaction mixture was allowed to stir at 0 °C for 1 h, and
then at r.t for 24 h. The solvent was removed in vacuo and the residue was dissolved in diethyl
ether (15 mL). The solution was washed sequentially with aq. K2CO3 solution, 0.5M HCl and
water. The organic solution was dried with anhydr MgSO4, the solvent was concentrated to yield
diethyl N-benzyl-N-{2-[N-(benzyloxy)acetamido]ethyl}-phosphoramidate 11a as a yellow oil
(0.23 g, 83%) [HRMS: m/z calculated for C22H32N2O5P (MH+) 435.2049. Found. 435.2046]
(Found: C, 60.88; H, 7.23; N, 6.52%. C22H31N2O5P requires C, 60.82; H, 7.19; N, 6.45%);

max/cm-1 1683 (C=O) and 1246 (P=O); δH/ppm (600 MHz; CDCl3) 1.30 [6H, m,
PO(OCH2CH3)2], 2.04 (3H, s, CH3CO), 3.14 (2H, m, NCH2), 3.71 (2H, m, CH2NHCO), 4.04
[4H, m, PO(OCH2CH3)2], 4.23 (2H, d, JP-H = 9.1 Hz, NCH2Ph), 4.75 (2H, s, OCH2Ph) and 7.27-
7.36 (10H, overlapping m, Ar-H); δC/ppm (150 MHz; CDCl3) 16.3 [d, JP-C = 7.2 Hz,
PO(OCH2CH3)2], 20.5 (CH3CO), 42.0 (NCH2), 43.6 (CH2NCO), 50.0 (NCH2Ph), 62.6 [d, JP-C
=
5.7 Hz, PO(OCH2CH3)2], 76.4 (OCH2Ph), 127.6, 128.6, 128.7, 128.8, 129.1, 129.3, 134.4 and
137.9 (Ar-C) and 172.4 (C=O).
4.7. The general procedure for the deprotection of the acetylated N-benzyloxy derivatives
(11) to afford the phosphoramidate esters (4a-h) is illustrated by the following example.
A solution of diethyl N-benzyl-{2-[N-benzyloxy)acetamido]ethyl}phosphoramidate 11a (0.12 g,
0.28 mmol) in dry MeOH (1 mL) was added to a solution of Pd/C (10%, 0.28 g) in dry MeOH
(2 mL) saturated with hydrogen and the mixture was stirred at r.t. for 5 h. The reaction mixture
was filtered through a celite pad and the filtrate was evaporated to yield diethyl N-benzyl-N-{2-
(N-hydroxyacetamido)ethyl}phosphoramidate 4a as a yellow oil (0.090 g, 91%) [HRMS: m/z
calculated for C15H26N2O5P (MH+) 345.1579. Found. 345.1584]; max/cm-1 3160 (OH) and 1634
(C=O); δH/ppm (600 MHz; CDCl3) 1.34 [6H, t, J = 7.1 Hz, PO(OCH2CH3)2], 2.18 (3H, s,
CH3CO), 3.19 (2H, m, NCH2), 3.68 (2H, m, CH2NCO), 4.05 [4H, m, PO(OCH2CH3)2], 4.12 (2H,
d, JP-H= 9.3 Hz, NCH2Ph), 7.31 (3H, m, Ar-H) and 7.35 (2H, m, Ar-H); δC/ppm (150 MHz;
CDCl3) 16.2 [d, JP-C = 7.3 Hz, PO(OCH2CH3)2], 20.9 (CH3CO), 41.0 (d, JP-C = 5.2 Hz, NCH2),
43.3 (CH2NCO), 49.1 (d, JP-C = 3.6 Hz, NCH2Ph), 63.3 [d, JP-C = 6.1 Hz, PO(OCH2CH3)2], 127.9,
128.5, 128.8 and 136.8 (Ar-C) and 172.9 (C=O).
4.8. General procedure for hydrolysis of the phosphoramidate esters (4a-d) to afford the
phosphoramidic acids 12a-d is illustrated by the following example. Trimethylsilyl bromide
(0.15 mL, 1.0 mmol) was added dropwise to diethyl N-benzyl-2-(N-hydroxyacetamido)-
ethylphosphoramidate 4a (0.12 g, 0.34 mmol) in DCM (5 mL) under N2 at 0 oC and the mixture
was stirred for 1 hour. The mixture was allowed to warm to room temperature, water was added
(1 mL) and the resulting mixture was stirred overnight. After completion, the solvent was
removed in vacuo and the residue chromatographed [preparative layer chromatography; elution
with hexane-EtOAc-MeOH (1:1:1)] to yield N-benzyl-2-(N-hydroxyacetamido)-
ethylphosphoramidic acid 12a as a colourless oil (85 mg, 87%); (Found: C, 45.76; H, 6.01; N,
9.79%. C11H17N2O5P requires C, 45.84; H, 5.94; N, 9.72%); max/cm-1 3245 (OH), 1653 (C=O)
and 1228 (P=O); δH/ppm (400 MHz; DMSO-d6) 2.09 (3H, s, CH3CO), 2.79 (2H, m, NCH2), 3.34
(2H, t, J = 6.4 Hz, CH2NCO), 3.83 (2H, s, NCH2Ph), 5.25 (1H, s, NOH), 7.29 - 7.37 (5H, m, Ar-
H) and 8.15 (2H, s, 2 x OH); δC/ppm (100 MHz; DMSO-d6) 20.7 (CH3CO), 42.6 (d, JP–C = 4.7
Hz, NCH2), 52.3 (d, JP–C = 16.9 Hz, CH2NCO), 67.2 (NCH2Ph), 126.4, 128.6, 129.4 and 139.4
(Ar-C) and 171.5 (C=O).
4.9. The general procedure for the preparation of the N-benzyl-2,2-diethoxyethanamine
derivatives 16a,e-h is illustrated by the following example. Bromoacetaldehyde diethyl acetal
(4.2 mL, 28 mmol) was added to a solution of benzylamine 9a (3.0 mL, 28 mmol) and anhydrous
K2CO3 (3.9 g, 28 mmol) in dry acetonitrile (15 mL) and the mixture was refluxed for 24 h. The
12

mixture was diluted with EtOAc (20 mL) and washed sequentially with water (3 × 25 mL) and
dried with anhydr MgSO4, filtered and concentrated in vacuo. The crude material was purified
by flash chromatography [on silica gel; elution with EtOAc-hexane (6:4)] to afford N-benzyl-
2,2-diethoxyethanamine 16a30 as a yellow oil (3.8 g, 61%); max/cm-1 3399 (NH); δH/ppm (600
MHz; CDCl3) 1.20 (6H, t, J = 7.1 Hz, 2 × CH3), 2.75 (2H, d, J = 5.6 Hz, NCH2), 3.53 (2H, m, 2
× OCHaCH3), 3.68 (2H, m, 2 × OCHbCH3), 3.81 (2H, s, NCH2Ph), 4.62 (1H, t, J = 5.6 Hz, CH),
7.25 (1H, m, Ar-H) and 7.32-7.33 (4H, overlapping m, Ar-H); δC/ppm (150 MHz; CDCl3) 15.6
(2 × CH3), 51.7 (NCH2), 54.0 (NCH2Ph), 62.5 (2 × OCH2CH3), 102.3 (CH), 127.1, 128.3, 128.5
and 140.3 (Ar-C).
4.10. Diethyl N-{2-(N-hydroxyacetamido)ethyl}phosphoramidate 17
A solution of diethyl N-benzyl-{2-[N-benzyloxy)acetamido]ethyl}phosphoramidate 14a (0.12 g,
0.28 mmol) in dry MeOH (1 mL) was added to a solution of Pd/C (10%, 0.28 g) in dry MeOH
(2 mL) saturated with hydrogen and the mixture was stirred overnight at r.t. The reaction mixture
was filtered through celite pad and the filtrate was evaporated to yield diethyl N-{2-(N-
hydroxyacetamido)ethyl}phosphoramidate 17 as a pale-yellow oil (0.063 g, 90%) [HRMS: m/z
calculated for C8H20N2O5P (MH+) 255.1110. Found. 255.1103]; max/cm-1 3222 (OH), 1623
(C=O) and 1209 (P=O); δH/ppm (600 MHz; CDCl3) 1.28 [6H, m, PO(OCH2CH3)2], 2.15 (3H, s,
CH3CO), 3.18 (2H, m, NCH2), 3.67 (2H, m, CH2NCO), 3.84 (1H, s, NH), 3.99 [4H, m,
PO(OCH2CH3)2] and 9.70 (1H, s, OH); δC/ppm (150 MHz; CDCl3) 16.2 [d, JP-C = 7.1 Hz,
PO(OCH2CH3)2], 20.7 (CH3CO), 37.9 (NCH2), 48.3 (CH2NCO), 63.1 [d, JP-C = 5.7 Hz,
PO(OCH2CH3)2] and 173.2 (C=O).
Acknowledgements
The authors thank Rhodes University and the South African Medical Research Council
(SAMRC) for generous financial support and the SAMRC for a bursary (to C.M.A.). This
research project was funded by the South African Medical Research Council (SAMRC) with
funds from National Treasury under its Economic Competitiveness and Support Package. A.C.C.
was supported by a Beit Trust scholarship.
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O
P
ROUTE I
NH2
EtO
OEt
R1
R2
O
OH
N
a
b
c
d
e
f
H
H
H
H
H
Cl
H
F
P(OEt)2
CH2OH
N
NH2
SH
Cl
Cl
F
R1
R2
O
a-h
NH2
g
h
R1
R2
F
H
ROUTE III
15

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
16

Highlights
1. Design and successful development of synthetic pathways to novel phosphoramidic acid
derivatives.
2. Bioassay data reveals the anti-trypanosomal potential of some of the compounds.
3. Detailed in silico modelling and docking studies have been undertaken to explore the topology
of the actives site in an initial homology-modelled PfDXR enzyme strycture and in a
subsequent, crystallographically determined PfDXR structure, the results of which account for
the unexpected lack of antimalarial activity.
17