Aryl phosphoramidates of 5-phospho erythronohydroxamic acid, a new class of potent trypanocidal compounds

Article abstract of DOI:10.1021/jm1004754

RNAi and enzymatic studies have shown the importance of 6-phosphogluconate dehydrogenase (6-PGDH) in Trypanosoma brucei for the parasite survival and make it an attractive drug target for the development of new treatments against human African trypanosomi

Full text of DOI:10.1021/jm1004754

J. Med. Chem. 2010, 53, 6071–6078 6071
DOI: 10.1021/jm1004754
Aryl Phosphoramidates of 5-Phospho Erythronohydroxamic Acid, A New Class of Potent
Trypanocidal Compounds
Gian Filippo Ruda,† Pui Ee Wong,‡ Vincent P. Alibu,‡ Suzanne Norval,† Kevin D. Read,† Michael P. Barrett,‡ and
Ian H. Gilbert*,†
†Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Sir James Black Centre, Dundee DD1 5EH,
U.K., and ‡Division of Infection and Immunity and Wellcome Trust Centre for Molecular Parasitology, Glasgow Biomedical Research Centre,
University of Glasgow G12 8TA, U.K.
Received April 19, 2010
RNAi and enzymatic studies have shown the importance of 6-phosphogluconate dehydrogenase
(6-PGDH) in Trypanosoma brucei for the parasite survival and make it an attractive drug target for
the development of new treatments against human African trypanosomiasis. 2,3-O-Isopropylidene-
4-erythrono hydroxamate is a potent inhibitor of parasite Trypanosoma brucei 6-phosphogluconate
dehydrogenase (6-PGDH), the third enzyme of the pentose phosphate pathway. However, this
compound does not have trypanocidal activity due to its poor membrane permeability. Consequently,
we have previously reported a prodrug approach to improve the antiparasitic activity of this inhibitor by
converting the phosphate group into a less charged phosphate prodrug. The activity of prodrugs
appeared to be dependent on their stability in phosphate buffer. Here we have successfully further
extended the development of the aryl phosphoramidate prodrugs of 2,3-O-isopropylidene-4-erythrono
hydroxamate by synthesizing a small library of phosphoramidates and evaluating their biological
activity and stability in a variety of assays. Some of the compounds showed high trypanocidal activity
and good correlation of activity with their stability in fresh mouse blood.
Introduction
ference in glycolysis. Inhibition of 6-PGDH should increase
the cellular level of 6-phosphogluconate, a potent inhibitor of
glycolysis. Moreover, it will reduce the cellular pool of
NADPH, which is produced by 6-PGDH along with glu-
cose-6-phosphate dehydrogenase in the pentose phosphate
pathway. Diminished NADPH production would reduce the
parasite’s ability to withstand oxidative stress and lower its
capability to carry out reductive biosyntheses. We have dis-
covered some potent inhibitors of the 6-PGDH,3 typified by
compound 1 (Figure 1), with good selectivity toward the
T. brucei enzyme over the mammalian orthologue. Unfortu-
nately these compounds are phosphates and as such do not
readily enter cells. The lack of bioavailability has been a
general hindrance to development of phosphorylated enzyme
inhibitors as drugs due to the inability of these molecules to
cross cellular membranes and reach the desired site of action/
target at an efficacious concentration.4 To circumvent this,
phosphate-prodrugs have been developed. The design of
phosphate-prodrugs is a relatively new field5 which has been
applied in antivirals, anticancer, and signaling regulators
research.6-10 A number of phosphate-prodrugs have recently
progressed to clinical trials,11,12 which proves the applicability
of this approach. In our previous work, we reported the
synthesis and the biological evaluation of several classes of
prodrug of compound 1, compounds 2-613 (Figure 1). These
prodrugs showed antiparasitic activity in vitro against cell
culture of trypanosomes with EC50 values in the micromolar
range. We also investigated their stability in pseudophysiolo-
gical conditions measuring their half-life (phosphate buffer at
pH 7.4 and 37 °C). Yet despite some good trypanocidal
Trypanosoma brucei is a parasite belonging to the order
kinetoplastida. Two subspecies of this parasite, T. b. gam-
biense and T. b. rhodesiense, are responsible for the infection
called human African trypanosomiasis (HATa), which is one
of the most lethal neglected diseases in the developing world.
Currently, only five drugs are available to treat HAT, three of
which were developed more than 50 years ago (suramin,
pentamidine, and melarsoprol); the fourth drug, eflornithine
(D,L-R-difluoromethylornithine, DFMO) is active only
against T. b. gambiense,1 and recently a new combination,
nifurtimox/eflornithine, has been introduced.2 However,
these treatments are far from satisfactory because of issues
that include toxicity, mode of administration, and efficacy
(suramin and pentamidine are only active against the first
stage of the disease). We are therefore interested in developing
new drugs to treat HAT.
We have been working on inhibitors of 6-phosphogluco-
nate dehydrogenase (6-PGDH) as a potential way to treat
HAT. 6-PGDH is involved in the third step of the pentose
phosphate pathway and is believed to be a good drug target
for the chemotherapy of HAT.3 The bloodstream form of the
parasite relies exclusively on glycolysis as a source of energy;
therefore, the parasite should be exquisitely sensitive to inter-
*To whom correspondence should be addressed. Phone: þ44 1382
386 240. Fax: þ44 1382 386 373. E-mail: i.h.gilbert@dundee.ac.uk
a Abbreviations: 6-PGDH, 6-phosphogluconate dehydrogenase; HAT,
human African trypanosomiasis; PHLM, pooled human liver microsomal
assay.
r
2010 American Chemical Society
Published on Web 07/28/2010
pubs.acs.org/jmc

6072 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Ruda et al.
Figure 1. Lead compound 1 and the corresponding phosphate prodrugs synthesized.
Figure 2. Mechanism of cleavage of phosphoramidates as proposed by McGuigan et al.14
activity, most of the phosphate masking groups showed low
stability and very short half-life in phosphate buffer, with the
exception of the aryl phosphoramidate 4, which had both
reasonable stability and good activity. We therefore took the
phosphoramidate 4 and worked on novel chemistry to probe
stability in phosphate buffer in fresh whole blood and with
hepatic microsomes and related this to trypanocidal activity.
Work by McGuigan and co-workers has shown that it is
possible to optimize the aryl phosphoramidate stability by
combining variations of the amino acid side chain, the amino
acid ester, and the aryl group. The proposed mechanism of
cleavage for aryl phosphoramidate suggested by Mcguigan
et al.14 is shown in Figure 2. The cleavage can follow two main
pathways. The first route requires the action of a carboxyl
esterase, which hydrolyses the amino acid ester to form the
free acid carboxylate (compound A). The oxygen of the
carboxylate then attacks the phosphorus atom and displaces
the aryl ester to form a 5-membered intermediate B, which
then rearranges, forming compound D. The final step is
proposed to be catalyzed by a “phosphoramidase” enzyme
that produces the final product E. The second pathway, which
can be predominant in the presence of electron-withdrawing
groups on the phenyl ester, follows a simple chemical hydro-
lysis with releaseof the aromatic ester andthen proceedsto the
final product by enzymatic cleavage of the amino acid group.
Consequently, optimization of the chemical and enzymatic
stability of the aryl phosphoramidates can be achieved by
alterations to the amino acid side chain, the amino acid ester,
and the substituent on the aryl ester group, as illustrated in
Figure 3. To increase the stability of the prodrugs we pursued
the following modifications:
• Increasing the steric bulk of the amino acid side chain
(aimed at reducing interaction with esterases thus slow-
ing down the formation of intermediate A).
• Increasing the steric bulk of the ester (aimed at reducing
interaction with esterases and also increasing the chemical

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6073
Scheme 1a
a (a) DIAD, PPh3, N-hydroxyphthalimide, DCM; (b) Me-NHNH2, EtOH, reflux; (c) AlMe3, DCM.
may be achieved is not clear, particularly the specific activa-
tion of the prodrug within the parasite, as we have only
minimal information relating to metabolic capabilities of
blood and the parasites. However, by developing compounds
whose conversion from prodrug to drug in blood is delayed
will improve the prospects of those compounds entering the
parasites and achieving bioconversion within the target cell.
Therefore we focused our efforts on increasing the stability of
prodrugs in blood on the assumption that prodrug activation
would occur in parasites at an enhanced rate (Figure 4).
To investigate the stability of the prodrugs, compounds
were incubated in buffer at pH 7.4 and 2.0 in order to
investigate the chemical hydrolysis, and in mouse blood to
determinestabilityinthe presenceof esterases. Finally someof
them were evaluated in a pooled human liver microsomal
assay (PHLM) to determine metabolic stability. The data for
these experiments is shown in Tables 1 and 2. The stability
data was compared to the activity of compounds against
bloodstream form T. b. brucei BS427.
Figure 3. Planned modifications on the aryl-phosphoramidates.
stability of the esters, also slowing down the formation
of intermediate A).
• Introduction of electron donating groups on the phenyl
ring, aimed at destabilizing the anion generated during
the hydrolysis step, thus making the aryl phenolate a
poorer leaving group and increasing the stability against
chemical hydrolysis. This should slow down conversion
of A to B (mechanism 1) or formation of C (mechanism 2).
Trypanocidal Activity and Chemical Stability in Phosphate
Buffer at pH 7.4. Compounds were assessed for activity
against T. brucei. Most of the compounds showed submi-
cromolar EC50 values; four compounds were particularly
potent: 15g, EC50 90 nM; 15l, EC50 40 nM; 15p, EC50 50 nM;
15q, EC50 8 nM.
Chemistry
The phosphoramidates were prepared according to pre-
viously reported methodology. The 2,4-dimethoxybenzyl
erythrono hydroxamate was obtained in three steps from
2,4-dimethoxy benzyl alcohol and 2,3-O-isopropylidene ery-
throno lactone (Scheme 1). The product 10 was crystallized in
40% yield from EtOAc/hexane.13
The phosphorochloridates 12a-e were synthesized from
the corresponding substituted phenols, phosphorus oxychloride
and the corresponding amino acid ester hydrochloride
(Scheme 2). The phosphochloridates were then coupled with
the protected erythronohydroxamate 10 following conditions
developed by McGuigan et al.7 The intermediates 14a-q were
obtained in moderate to good yields.
The chemical stability effects of the different substituents
can be analyzed by comparing compounds where there are
stepwise changes. First, we consider the effect of changing
the aryl substituent. In general, electron donating groups
increase the buffer (chemical) stability of the compounds. In
analyzing and comparing the data for compounds 15a (4-
NO2), 15c (no substituent), 15h (4-Me), 15m (4-MeO), and
15o (2,3-diMe) (where the amino acid moiety is alanine
methyl ester), it is evident that the introduction of electron-
donating groups improves the buffer stability at pH 7.4 (half-
life increases from 0.85 h for 15a up to 30 h for compound
15o), and this is translated, in the same manner, into a higher
in vitro activity with compound 15o (IC50 0.27 μM) being the
most active in vitro for the alanine methyl ester series. There
are several exceptions. The 4-methoxy derivative 15m de-
monstrated neither increased buffer stability nor better in
vitro activity compared to the analogue 15c. This data
follows a similar trend to that of McGuigan et al.,15 who
also reported that the 4-methoxyphenyl phosphoramidate of
the nucleoside d4T was slightly less stable at pH 7.4 than the
corresponding phenyl analogue. Also for some reason, com-
pounds with valine as the amino acid and esterified as an
ethyl ester (15f) showed a small decrease in stability on
substituting the phenyl ring with either the 4-methyl (15l)
or 2,3-dimethyl (15p) substitutents. 2,3-Dimethylphenyl es-
ters and 4-methylphenyl esters showed comparable activity
and stability (see pairs 15l-15p and 15h-15o), and they are
The final step of the synthesis was the cleavage of the
protecting group from the hydroxamate moiety by trifluoro-
acetic acid in dichloromethane. The small library was obtained
in reasonable yields and in good purity as mixtures of diaste-
reoisomers due to chirality of the phosphorus atom.
Biological Stability
The prodrug approach for the treatment of a parasitic
disease such as HAT is challenging because of the complexity
of the system (host þ parasite) and the particular requirements
that the prodrug must fulfill in order to achieve the desired
effect. In the case of Trypanosoma brucei, the ideal prodrug
must have a good stability in the bloodstream of the mamma-
lian host and should be quickly converted into the active form
(drug) only inside the parasite (Figure 4). Once converted to
the active form, the charged compound should not be able to
diffuse back out of the parasite. Whether this ideal situation

6074 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Ruda et al.
Scheme 2a
a (a) POCl3, TEA, Et2O; (b) amino acid ester hydrochloride, TEA, DCM, rt; (c) NMI, DCM, (2,4-dimethoxybenzyl)-O-erythronoydroxic alcohol;
(d) TFA, DCM, rt 15 min.
Figure 4. Schematic representation of selective delivery of prodrugs into trypanosomes (MG: masking group).
generally more active than the corresponding phenyl, 4-metho-
xyphenyl and 4-nitrophenylesters.
The importance of amino acid side chain can be seen by
analyzing the pairs 15c-15e, 15h-15j, 15i-15l, 15d-15f,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6075
Table 1. Biological Activity and Stability for Phosphoramidates
compd
R1
R2
R3
t1/2 pH 7.4 h
t1/2 pH 2 h
t1/2 mouse blood (min)
PHLM (mL/min/g)
EC50 (μM)
15aa
15b
15ca
15d
15e
15f
4-NO2
H
Me
H
Me
Me
Me
Et
0.85
43.8
11.4
6.95
0.78
0.36
0.24
0.09
0.31
0.18
0.15
0.04
3.79
0.11
0.27
0.05
0.008
<15
<15
<15
45
H
Me
Me
i-Pr
i-Pr
i-Pr
Me
Me
i-Pr
i-Pr
Me
i-Pr
Me
i-Pr
i-Pr
5
29
21
61
68
30
34
45
25
H
H
Me
Et
<0.5
<0.5
5.0
H
120
120
15g
15h
15i
H
t-Bu
Me
Et
28
4-Me
4-Me
4-Me
4-Me
4-OMe
4-OMe
2,3-Me
2,3-Me
2,3-Me
<15
15j
15l
Me
Et
28
15
8.5
45
<0.5
<0.5
<0.5
15m
15n
15o
15p
15q
Me
Et
Me
Et
30
48
10
60
>480
<0.5
10
t-Bu
>24
a Compounds 15a and 15c have been reported previously.13
Table 2. Biological Properties for the Most Potent Trypanocidal Compounds
1
>330
15l
15g
0.09
120
30
15p
0.05
60
15q
0.008
480
24
EC50 (μM)
T1/2 mouse blood (min)
0.04
45
T
1/2 ph 7.4 (h)
25
<0.5
2.4
48
<0.5
2.9
PHLM Cli mL/min/g
clogD
5.0
2.6
10
3.6
and the three phenyl esters 15b, 15c, and 15e. Changes from
glycine to alanine or alanine to valine increased the buffer
stability in every case, with the exception of 15i (Ala)-15l
(Val); in this latter case, the compounds had reasonably
comparable buffer stability with half-lives of 34 and 25 h,
respectively. The less hindered amino acid glycine 15d
showed much lower buffer stability, which made it difficult
to measure the half-life (data not reported in the table). The
poor stability for this aryl phosphoramidate was also ob-
served during its synthesis when the coupling of the phos-
phochoridate 13b with the erythronohydroxamate 10
produced the prodrug 14b with one of the lowest yields
(12%), due to a degree of decomposition that occurred
during the purification. With all of these compounds, the
increase in buffer stability also corresponded to an increase
in activity against the parasites, with valine being better than
alanine which is in turn better than glycine.
and also 15p (Et) and 15q (t-Bu) (EC50 0.050 and 0.008 μM,
respectively). A relationshipbetweenthe buffer stability at pH
7.4 and antiparasitic activityislessobvious, althoughthereare
fewer data points. The tert-butyl esters have similar half-lives
to the ethyl analogues (see pair 15p (Et) and 15q (t-Bu) and
pair 15f (Et) and 15g (t-Bu)). Comparison of the ethyl esters
15d, 15i, and 15l with the corresponding methyl ester analo-
gues 15c and 15h and 15j shows how the ethyl ester improves
both the trypanocidal activity and the buffer stability.
Overall, there is a general correlation between buffer stability
and activity of the compounds, with those compounds showing
longer half-life in phosphate buffer also the most active against
the parasite (Figure 5). Thus moving from compound 15a,
which has a half-life of about 50 min, to 15q, which has a half-
life of >24 h, the potency changes from 44 to 0.008 μM, an
increase in activity of more than 5000 times. The relationship
between in vitro antiparasitic data and buffer stability is almost
certainly complicated by the presence of esterases (see below)
which have a role in removal of the ester.
The other point of variation is with the amino acid ester
(R3). In terms of the antiparasitic activity, the tert-butyl
esters have more potent EC50 values than the ethyl and
methyl ester as shown by compounds 15e (Me), 15f (Et),
and 15g (t-Bu) (EC50 0.36, 0.24, and 0.09 μM, respectively)
We also looked at the buffer (chemical) stability of several
compounds at pH 2, to see if the compounds were likely to
survive in the stomach. The compounds showed a small

6076 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Ruda et al.
assayed had good metabolic stability (<0.5 mL/min/g).
However, the tert-butyl esters appeared to be very susceptible
to metabolism. This may be due to the extra liphophilicity of
this group. Liability of the tert-butyl derivatives to hepatic
metabolism may mitigate against their utility as drug candi-
dates in spite of their other stability characteristics being
favorable.
Conclusions
Compound 1 is a potent and selective inhibitor of T. brucei
6-PGDH; however, it is inactive against the parasite, presum-
ably due to the presence of the phosphate group. By making
prodrugs through masking of the phosphate group, it is possi-
ble to prepare compounds that have activity against the
parasite and the activityof theseprodrugs appears to correlate
to their buffer stability. In particular, the aryl phosphorami-
date prodrugs appeared to have the best balance of stability
and activity. In this article, we describe the optimization of
these prodrugs and their evaluation. By variation of the ester,
the R-amino substituent, and the substituent on the phenyl
ester on the phosphate prodrug, it is possible to fine-tune
the buffer and blood stability of the compounds. Thus, for
the carboxylic ester, increasing the bulk of the ester increases the
buffer and blood stability (presumably chemical and esterase
stability); for the R-aminosubstituent, increasing the bulkalso
increases the buffer and blood stability. For the phenyl
substituent, electron-donating substituents increase the buffer
(chemical) stability without revealing consistent effects on
blood stability. The prodrugs with the greatest buffer and
blood stability thus have a tert-butyl ester, an isopropyl
substituent at the R-position, and a 2,3-dimethyl substituent
on the phenyl ring. By these modifications, we have improved
trypanocidal activity of the compounds from 6.9 to 0.008 μM
and the stability in mouse blood from less than 15 min to
greater than 480 min.
The biological data show how important the chemical
stability in phosphate buffer at pH 7.4 is for the in vitro trypa-
nocidal activity of these compounds, thus we can possibly
assume that in the Alamar Blue assay the main decomposition
pathway of the prodrugs in the assay media is via chemical
hydrolysis.
Even though we have not proved that the mode of action of
these compounds is through inhibition of the enzyme 6-PGDH,
we have shown the aryl phosphoramidate prodrugs derived
from the inhibitor 1 have a higher antitrypanosomal activity
compared to parent compound. thus phosphoramidates show
correlation between the trypanocidal activity and the stability
in pseudophysiological conditions, with EC50 values over
5000-fold lower than compound 1. However, some microsomal
(metabolic) instability was observed for the tert-butyl-valinyl
phosphoramidates (15g and 15q), indicating thesecompounds
are probably susceptible to hepatic clearance.
Figure 5. Correlation between EC50 and half-life of the prodrugs in
phosphate buffer pH 7.4.
reduction (approximately 2-fold) in stability at more acidic
conditions, which should not preclude them being used
orally.
Mouse Blood Stability. The stability in fresh mouse blood
was investigated for key compounds. This would give an
indication of stability to esterases found in the blood. Hydro-
lysis of the prodrug by esterases is probably a key step in the
conversion of the prodrugs into the active species (Figure 2).
Many of the compounds showed a very short half-life,
probably due to hydrolysis of the amino acid ester by various
esterases in the blood, leading to loss of the masking groups.
Looking at series 15e-g and 15p-q indicates that as the
bulk of the ester increases, so does blood stability. The order
of blood stability appears to be tert-butyl > ethyl > methyl.
We hypothesize that this is the order of stability of the esters
to esterases. Similarly, an increase in bulk at the R-position
appears to increase the blood stability, as can be seen by
comparison of 15c (R2 = Me) and 15e (R2 = i-Pr) and 15d
(R2 = Me) and 15f (R2 = i-Pr). Comparison of 15c and 15h
and of 15f, 15l, and 15p indicates lack of correlation between
blood stability and the substituent on the phenyl ring. There-
fore, compounds with bulky esters or R-substituents are more
stable in blood, which is presumably a measure of increased
stability to esterases. However, the substituent on the aromatic
ring has a much smaller effect on blood (esterase) stability.
Again, this is consistent with the mechanism shown in Figure 2.
It is interesting to compare our experience with that
reported in the literature for prodrugs of the nucleotides.15,16
In the case of the nucleotide prodrugs, the stability of the
prodrug moiety, at least in human plasma, appears much
greater than we have observed here. Possibly this is due to
some intramolecular general acid/base catalysis in our sys-
tem causing a more rapid hydrolysis of the compounds. In
the case of the nucleotide prodrugs, there also was different
correlation between substituents on the prodrugs and anti-
viral activity compared to what we have seen here. According
to McGuigan and co-workers, in the case of phosphorami-
dates of d4T, the higher esterase stability was not correlated
to higher potency of the compounds. The reported data
showed that the t-butyl ester phosphoramidate of d4T was
not degraded after 21 h of incubation in the presence of
PLE.16 In our case, however, t-butyl esters are converted to
potent trypanocidal compounds in a reasonable time frame
(2-8 h).
This article shows that it is possible to improve these com-
pounds by optimizing the prodrug moiety. Further optimiza-
tion will requireretaining the good buffer andblood (chemical
and esterase) stability of the prodrug moiety while optimizing
key developability properties of the molecule (such as solubi-
lity and metabolic stability).
Experimental Section
Stability in Pooled Human Liver Microsomes (PHLM).
Compounds were also investigated for their metabolic stability,
using human liver microsomes (Table 2). Most compounds
Parasite Testing. T. b. brucei (s427), wild-type cells were
cultured to the optimum density of (1-2) ꢀ 106 cells mL-1 in
HMI-9 supplemented with 10% fetal calf serum (FCS) under

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6077
environmental conditions of 37 °C and 5% CO2. Solutions of
test compounds were prepared in culture media at stock con-
centration of 200 μM and diluted serially (1:2) across the
96-well, flat-bottom solid white plates to give a total of 11 decreas-
ing concentrations (100 μL well-1). The last well of each series
was left blank, i.e., “drug-free” (negative control). Cells were
prepared at the concentration of 4 ꢀ 104 cells mL-1 and added to
each of the respective compound series (100 μL well-1). Plates
were incubated at 37 °C/5% CO2 for 48 h prior to the addition of
Alamar Blue solution (20 μL well-1, 0.49 mM in 1X PBS, pH
7.4) followed by a further 24 h incubation. Assay end points
were measured fluorimetrically with the fluorescence spectro-
meter (FluoStar, BMG LabTech, Germany) and Optima pro-
gram set at λexcitation of 544 nm and λemission of 590 nm. Data was
analyzed using Prism 5.0 software to obtain EC50 values. Exp-
eriment was performed in duplicate and repeated three times.
Microsomal Stability. Each test compound (0.5 μM) was incu-
bated with pooled human liver microsomes (Tebu-Bio, UK;
0.5 mg/mL 50 mM potassium phosphate buffer, pH7.4) and the
reaction started with addition of excess NADPH (8 mg/mL
50 mM potassium phosphate buffer, pH7.4). Immediately, at
time zero, then at 3, 6, 9, 15, and 30 min, an aliquot (50 μL) of the
incubation mixture was removed and mixed with acetonitrile
(100 μL) to stop the reaction. Internal standard was added to all
samples, the samples centrifuged to sediment precipitated pro-
tein, and the plates were then sealed prior to UPLCMSMS
analysis using a Quattro Premier XE (Waters).
suspension was left to warm to room temperature and stirred for
12 h. The triethylamine salt was filtered off, and the filtrate was
concentrated under reduced pressure to an oil, which was used
for the next step without further purification.
General Procedure B: Synthesis of Phosphorochloridates. The
appropriate phosphorodichloridate (1.2 mol) and the amino
acid esters hydrochloride (1 mol) were stirred in dry DCM with
at -78 °C. TEA was added dropwise with a syringe. The mixture
was stirred for 15 min at -78 °C and then warmed to room
temperature. The reaction was monitored by 31P NMR, and
when the analysis showed the completed disappearance of the
starting material, the reaction was concentrated under reduced
pressure. The residue was taken in Et2O, and the precipitated
triethylamine salt was filtered off. The filtrate was concentrated
in vacuo and used for the next step without further purification.
General Procedure C: Synthesis of Phosphoramidates. The
protected (2,4dimethoxybenzyl)erythrono hydroxamate (1 mol)
was stirred with the appropriate phosphorochloridate phos-
phoramidate (1.2-2.1 mol) in DCM (10 mol) and cooled to
-78 °C under argon atmosphere. N-Methyl imidazole (4 mol)
was added dropwise with a syringe to the solution. The mixture
was stirred for 15 min at -78 °C and then was left warm to room
temperature and stirred for 4-12 h. The reaction was monitored
by 31P NMR and quenched with MeOH (1 mL). The organic
phase was washed with 0.5 M HCl (3 ꢀ 10 mL), dried over
MgSO4, and concentrated under reduced pressure. The crude oil
was purified by column chromatography with 100% chloroform f
4% MeOH/chloroform.
General Procedure D: Cleavage of the 2,4-Dimethoxybenzyl
Group with TFA. The protected hydroxamate was stirred in
DCM at room temperature, 2% of TFA was added with syringe.
The reaction was stirred at room temperature until the complete
disappearance of the starting material was observed by TLC
(5% MeOH/DCM) 15-20 min. The white suspension was diluted
with Et2O, and the white precipitate was filtered off. The filtrate
was concentrated under reduced pressure and purified by chro-
matography eluting the silica with 100% DCM f 4% MeOH/
DCM.
XLfit (IDBS, UK) was used to calculate the exponential
decay and consequently the rate constant (k) from the ratio of
peak area of test compound to internal standard at each time
point. The rate of intrinsic clearance (CLi) of each compound
was then calculated using the following calculation:
CLi ðmL=min=g liverÞ ¼ k ꢀ V ꢀ microsomal protein yield
where V (mL/mg protein) is the incubation volume/mg protein
added and microsomal protein yield is taken as 52.5 mg protein/g
liver
Verapamil was used as a positive control to confirm accep-
table assay performance.
Examples of three final compounds are given below. Details
of the remainder of the compounds is found in the Supporting
Information.
General Methods. 1H NMR, 13C NMR, 31P NMR, and 2D-
NMR spectra were recorded either on a Bruker Avance DPX
300 spectrometer or on a Bruker Avance DPX 500 spectrometer.
Chemical shifts (δ) are expressed in ppm. Signal splitting
patterns are described as singlet (s), broad singlet (bs), doublet
(d), triplet (t), quartet (q), multiplet (m), or combination thereof.
LCMS analyses were performed with an Agilent HPLC 1100
(Waters XBridge Column) and diode array detector in series
with a Bruker MicroTof mass spectrometer. Compounds were
eluted using either method A (methanol, methanol/water (95:5)
or water/acetonitrile (1:1) þ 0.1% formic acid as the mobile
phase on Phenomenex Gemini Column) or with method B
(water/acetonitrile 95:5 to 5:95 on a Water Xbridge column).
High-resolution electrospray measurements were performed on
Bruker MicroTof mass spectrometer.
2,3-O-Isopropylidene Erythronocarboxamide-4-[phenyl(meth-
oxyglycinyl)phosphoramidate] (15b). The title compound was
synthesized following the general procedure D from compound
15b (41 mg, 0.07 mmol), TFA (40 μL), and DCM (5 mL).
1
Slightly orange hygroscopic foam, 22 mg (73%). NMR (500
MHz, CDCl3) δ: 1.27 (s, 3H, 1 of C(CH3)2), 1.43 (d, 3H, J = 5.34
Hz, C(CH3)2), 3.64 (d, 3H, J = 4.70 Hz, OCH3), 3.68-3.80 (m,
2H, NHCH2), 4.08-4.25 (m, 2H, CH2OP), 4.49-4.52 (m, 1H,
CHCH2OP), 4.66 (d, 1H, J = 7.47 Hz, C(O)CH), 7.06-7.09 (m,
1H, ArH), 7.12-7.14 (m, 2H, ArH), 7.21-7.25 (m, 2H, ArH).
13C NMR (125 MHz, CDCl3) δ: 24.50, 24.60 (C(CH3)2), 26.35,
26.67 (C(CH3)2), 42.80, 42.86 (NHCH2), 52.41, 52.46 (OCH3),
65.04, 65.08, 65.24, 65.28 (CH2OP), 74.78, 75.27 (CHCH2OP),
75.76, 75.84 (C(O)CH), 110.63, 110.64 (C(CH3)2), 120.16,
120.20, 120.25 (ArCH), 125.02, 125.08 (ArCH), 129.69, 129.70
(ArCH), 150.54, 150.60 (ArC-OP), 165.76, 165.17 (HNC(O)),
171.44, 171.50, 171.60 (CO2Me). 31P NMR (121 MHz, CDCl3)
δ: 3.68, 3.50. LCMS (ESþ): m/z 419.12 ([M þ H]þ, 100%); m/z
436.14 ([M þ NH4]þ, 20%). Rt 3.0 min, (purity >99% by UV).
HRMS: Found 419.1223; C16H24N2O9P [M þ Hþ] requires
419.1214.
2,3-O-Isopropylidene Erythronocarboxamide-4-[phenyl(etho-
xyalaninyl) phosphoramidate] (15d). Compound 14d (200 mg,
0.33 mmol) was deprotected according to the general procedure
D using TFA (89 μL) and DCM (9 mL). Orange foam, 93 mg
(62%). The compound contains traces of TFA. 1H NMR (500 MHz,
CDCl3) δ: 1.16-1.28 (m, 9H, CH2CH3 þ CHCH3 þ 1 of
C(CH3)2), 1.43, 1.42 (2s, 3H, 1 of C(CH3), 3.86-4.04 (m, 2H,
CHCH3 þ OH), 4.09 (q, 2H, J = 6.9 Hz, OCH2CH3), 4.12-4.19
Thin layer chromatography (TLC) was carried out on Merck
silica gel 60 F254 plates using UV light and/or PMA or KMnO4
for visualization. TLC data are given as the Rf value with the
corresponding eluent system specified in brackets. Column
chromatography was performed using Fluka silica gel 60. All
reactions were carried out under dry and inert conditions (Ar
atmosphere) unless otherwise stated. Reactions using micro-
wave irradiation were carried in a Biotage InitiatorTM micro-
wave.
The purity/identity of compounds was determined by a
combination of NMR (1H, 13C, 31P), LCMS, and HRMS. The
purity of all compounds was determined by LCMS.
General Procedure A: Synthesis of Phosphorodichloridates. A
solution of phosphorus oxychloride (1 mol) and the appropriate
phenol (1 mol) in dry diethyl ether was cooled at -78 °C,
followed by the addition of triethyl amine (1 mol). The white

6078 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Ruda et al.
(m, 2H, CH2OP), 4.32 (bs, 0.5H, PNH), 7.08 (q, 1H, J = 7.4 Hz,
ArH), 7.10-7.15 (m, 2H, ArH), 7.22-7.26 (m, 2H, ArH), 8.89,
9.20 (2bs, 1H, ONH). 13C NMR (125 MHz, CDCl3) δ: 14.07,
14.10 (CH2CH3), 20.82, 20.95 (CHCH3), 24.35, 24.52, 26.25,
26.64 (C(CH3)2), 50.01, 50.29 (CHCH3), 61.82 (OCH2CH3),
64.84 (CH2OP), 75.40, 75.75 (CHCH2OP), 75.84, 76.08 (C(O)-
CHCH), 110.46, 110.54 (C(CH3)2), 120.15, 120.33, 120.37, (ArCH),
125.02, 125.08 (ArCH), 129.73 (ArCH), 150.49, 150.52 (ArC-
OP), 165.12 (HNC(O)), 173.49, 173.72 (CO2Et). 31P NMR (121
MHz, CDCl3) δ: 2.56, 2.30. LCMS (ESþ): m/z 447.18 ([M þ
H]þ, 100%). Rt 3.4 min, (purity 97.6% UV). HRMS: Found
447.1532; C18H28N2O9P [M þ Hþ] requires 447.1527.
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2,3-O-Isopropylidene Eerythronocarboxamide-4-[phenyl(meth-
oxyvanlinyl)phosphoramidate] (15e). The title compound was
synthesized following the general procedure D from 14e (191
mg, 0.31 mmol), TFA (40 μL), and CM (5 mL). Orange foam, 62
mg (43%). 1H NMR (500 MHz, CDCl3) δ: 0.76-0.85 (m, 6H,
CH(CH3)2), 1.29-(d, 3H, J = 3.0 Hz, 1 of C(CH3)2), 1.44 (s, 3H,
1 of C(CH3)2), 1.95-2.02 (m, 1H, CH(CH3)2), 3.64 (d, 3H,
J = 10.6 Hz, OCH3), 3.85-3.87 (m, 1H, NHCH), 3.85-3.87 (d,
1H, J = 11.2 Hz, PNH), 4.14 (t, 1H, J = 4.3 Hz, CHHOP),
4.18-4.35 (m, 1H, CHHOP), 4.49-4.53 (m, 1H, CHCH2OP),
4.69 (dd, 1H, J1 = 3.0 Hz, J2 = 7.5 Hz, C(O)CHCH), 7.06-7.10
(m, 1H, ArH), 7.14-7.16 (m, 2H, ArH), 7.22-7.27 (m, 2H,
ArH), 8.85 (bs, 1H, ONH). 13C NMR (125 MHz, CDCl3) δ:
17.12, 18.93 (CH(CH3)2), 24.49, 26.63 (C(CH3)2), 31.97, 32.02
(CH(CH3)2), 52.43 (CO2CH3), 59.94 (PNHCH), 64.84, 64.89
(CH2OP), 75.49, 75.80 (CHCH2OP), 75.89 (C(O)CHCH),
110.51 (C(CH3)2), 120.30, 120.34 (ArCH), 125.00 (ArCH),
129.64 (ArCH), 150.68, 150.74 (ArC-OP), 166.10 (HNC(O)),
174.54, 174.57 (CO2Me). 31P NMR (121 MHz, CDCl3) δ: 3.49,
3.32. LCMS (ESþ): m/z 461.18 ([M þ H]þ, 100%). Rt 3.1 min,
(purity 92.5% by UV). HRMS: Found 461.1680; C19H30N2O9P
[M þ Hþ] requires 461.1683.
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acid, an inhibitor of 6-phosphogluconate dehydrogenase. Chem-
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Acknowledgment. We acknowledge the Wellcome Trust
(075277 and 083481) for financial support.
Supporting Information Available: Experimental procedures
and spectral data for all intermediates. This material is available
free of charge via the Internet at http://pubs.acs.org.
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