Targeting an Aromatic Hotspot in Plasmodium falciparum 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase with β-Arylpropyl Analogues of Fosmidomycin

Article abstract of DOI:10.1002/cmdc.201600249

Blocking the 2-C-methyl-d-erythrithol-4-phosphate pathway for isoprenoid biosynthesis offers new ways to inhibit the growth of Plasmodium spp. Fosmidomycin [(3-(N-hydroxyformamido)propyl)phosphonic acid, 1] and its acetyl homologue FR-900098 [(3-(N-hydroxyacetamido)propyl)phosphonic acid, 2] potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this biosynthetic pathway. Arylpropyl substituents were introduced at the β-position of the hydroxamate analogue of 2 to study changes in lipophilicity, as well as electronic and steric properties. The potency of several new compounds on the P. falciparum enzyme approaches that of 1 and 2. Activities against the enzyme and parasite correlate well, supporting the mode of action. Seven X-ray structures show that all of the new arylpropyl substituents displace a key tryptophan residue of the active-site flap, which had made favorable interactions with 1 and 2. Plasticity of the flap allows substituents to be accommodated in many ways; in most cases, the flap is largely disordered. Compounds can be separated into two classes based on whether the substituent on the aromatic ring is at the meta or para position. Generally, meta-substituted compounds are better inhibitors, and in both classes, smaller size is linked to better potency.

Full text of DOI:10.1002/cmdc.201600249

DOI: 10.1002/cmdc.201600249
Full Papers
Targeting an Aromatic Hotspot in Plasmodium falciparum
1-Deoxy-d-xylulose-5-phosphate Reductoisomerase with b-
Arylpropyl Analogues of Fosmidomycin
Sanjeewani Sooriyaarachchi+,[a] Renꢀ Chofor+,[b] Martijn D. P. Risseeuw,[b] Terese Bergfors,[a]
Jenny Pouyez,[c] Cynthia S. Dowd,[d] Louis Maes,[e] Johan Wouters,[c] T. Alwyn Jones,[a]
Serge Van Calenbergh+,*[b] and Sherry L. Mowbray+*[a]
Blocking the 2-C-methyl-d-erythrithol-4-phosphate pathway for
isoprenoid biosynthesis offers new ways to inhibit the growth
of Plasmodium spp. Fosmidomycin [(3-(N-hydroxyformamido)-
propyl)phosphonic acid, 1] and its acetyl homologue FR-
900098 [(3-(N-hydroxyacetamido)propyl)phosphonic acid, 2]
potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoiso-
merase (Dxr), a key enzyme in this biosynthetic pathway. Aryl-
propyl substituents were introduced at the b-position of the
hydroxamate analogue of 2 to study changes in lipophilicity,
as well as electronic and steric properties. The potency of sev-
eral new compounds on the P. falciparum enzyme approaches
that of 1 and 2. Activities against the enzyme and parasite cor-
relate well, supporting the mode of action. Seven X-ray struc-
tures show that all of the new arylpropyl substituents displace
a key tryptophan residue of the active-site flap, which had
made favorable interactions with 1 and 2. Plasticity of the flap
allows substituents to be accommodated in many ways; in
most cases, the flap is largely disordered. Compounds can be
separated into two classes based on whether the substituent
on the aromatic ring is at the meta or para position. Generally,
meta-substituted compounds are better inhibitors, and in both
classes, smaller size is linked to better potency.
Introduction
Despite major efforts to decrease its incidence in the last
decade, malaria remains one of the leading causes of death
from a single infectious agent. The disease, caused mostly by
Plasmodium falciparum, was responsible for an estimated
438000 deaths in 2015.[1] Significant gains in recent years are
being undermined by mounting resistance of the parasite to
currently available drugs, so there is an urgent need for new
chemical entities acting on new targets.
After Jomaa and co-workers demonstrated that Plasmodia
synthesize isoprenoids via the non-mevalonate or 2-C-methyl-
d-erythritol 4-phosphate (MEP) pathway,[2] while humans
obtain these essential molecules via the orthogonal mevalo-
nate pathway,[3,4] blocking the MEP pathway became an attrac-
tive strategy to stem the proliferation of this and other patho-
gens.[5] 1-Deoxy-d-xylulose-5-phosphate reductoisomerase
(Dxr; EC 1.1.1.267) catalyzes the first committed step in the
MEP pathway, that is, the transformation of 1-deoxy-d-xylulose
5-phosphate (DOXP) to MEP.[6,7] The natural antibiotic fosmido-
mycin (1, Figure 1), a potent inhibitor of Dxr,[8,9] has been clini-
cally evaluated for the treatment of malaria, alone and in com-
bination therapy, but unfavorable pharmacokinetic properties
and low intestinal absorption have prevented it from reaching
the market.[10–12] The acetyl homologue of fosmidomycin, FR-
900098 (2), has been reported to be twice as potent against
[a] Dr. S. Sooriyaarachchi,+ T. Bergfors, Dr. T. A. Jones, Dr. S. L. Mowbray+
Science for Life Laboratory, Department of Cell and Molecular Biology,
Uppsala University, Biomedical Center, Box 596, 751 24 Uppsala (Sweden)
E-mail: sherry.mowbray@icm.uu.se
[b] R. Chofor,+ M. D. P. Risseeuw, Dr. S. Van Calenbergh+
Laboratory for Medicinal Chemistry (FFW), Gent University,
Ottergemsesteenweg 460, 9000 Gent (Belgium)
E-mail: serge.vancalenbergh@ugent.be
[c] Dr. J. Pouyez, Dr. J. Wouters
Department of Chemistry, University of Namur, Rue de Bruxelles 61, 5000
Namur (Belgium)
[d] Dr. C. S. Dowd
Department of Chemistry, George Washington University, Washington, DC
20052 (USA)
[e] Dr. L. Maes
Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University
of Antwerp, Universiteitsplein 1, 2610 Antwerp (Belgium)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600249.
Figure 1. Conversion catalyzed by Dxr and two inhibitors of this enzyme.
ChemMedChem 2016, 11, 1 – 14
1
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
P. falciparum in vitro, and against P. vinckei in a mouse malaria
model.[2] Extensive medicinal chemistry efforts seeking new an-
timalarial agents have yielded various analogues of fosmido-
mycin, the subject of multiple reviews.[13–15] The phosphonate
group and the metal-chelating reverse hydroxamate moiety
are both required for Dxr inhibitory activity. Aryl substitutions
at the a-position (with respect to the phosphonate), when at-
tached to the inhibitor backbone, have yielded some of the
most promising analogues to date.
substituents afforded potent P. falciparum Dxr (PfDxr) inhibi-
tors. These results were rationalized by crystallographic studies
of PfDxr in complex with 5d and 5e, which showed that the
phenyl rings of these compounds displace the indole ring of
the conserved Trp296 residue, and occupy its ‘usual’ position
in both active-site metal-containing[16,18,24] and metal-free struc-
tures.[17] This allows an intramolecular interaction between the
phenyl ring and the methyl group on the hydroxamic acid that
is equivalent to the intermolecular interactions observed in ter-
nary complexes with 2.[18,24] Rearrangement of the residues of
the flap results in favorable interactions between these phenyl
rings and the tryptophan residue. Importantly, both analogues
showed sub-micromolar schizontocidal activity against the
P. falciparum K1 strain, and essentially the same SAR was ob-
served as for PfDxr inhibition.
Dxr enzymes contain a strictly conserved tryptophan residue
within a flexible loop (flap) that undergoes an induced-fit con-
formational change upon fosmidomycin binding, closing over
and interacting with the bound inhibitor. This flap is consid-
ered essential for the catalytic activity of Dxr.[16–20] Murkin and
co-workers demonstrated a change in the rate-limiting step of
the Mycobacterium tuberculosis Dxr-catalyzed reaction upon al-
teration of Trp203 in the flap, thereby establishing a functional
link between this amino acid and chemical barrier crossing.[21]
Inhibition and binding studies with fosmidomycin confirmed
the importance of the flap, and the conserved tryptophan in
particular, for ligand binding. Structural evaluation of a series
of Dxr-bound compounds including 3 and 4 (Figure 2) showed
that the indole group of Trp211 in the Escherichia coli enzyme
(EcDxr) is displaced in order to accommodate the inhibitors’
pyridine/quinoline rings, which form p–p stacking or charge-
transfer interactions with the indole of the tryptophan side
chain.[22]
This follow-up study explored the influence of lipophilicity,
electronic and steric properties in variants of the phenylpropyl
side chain of 5d. We anticipated that analogues 6a–j would
retain the capacity to occupy the aromatic ‘hotspot’, while
their phenyl substituents might reinforce intra- or intermolecu-
lar interactions.
Results and Discussion
Synthesis
The synthesis of 6a–j (Scheme 1) was achieved by starting
from commercially available aryl iodides 7a–j. Sonogashira
coupling with but-3-yn-1-ol afforded the corresponding alky-
nols 8a–j, which were readily converted into 9a–j upon cata-
lytic hydrogenation. Dess–Martin oxidation to the correspond-
ing aldehydes 10a–j and subsequent Wittig olefination afford-
ed the a,b-unsaturated esters 11 a–j, which served as electro-
philes in a Michael reaction with dibenzyl methylphosphonate
to yield the 1,4-addition adducts 12a–j. Hydrolysis of the tert-
butyl ester and EDC mediated coupling with O-benzyl-N-meth-
ylhydroxylamine gave 13a–j. Finally, removal of all benzyl pro-
tecting groups by catalytic hydrogenolysis afforded the desired
analogues 6a–j.
Evaluation of function
The final compounds were tested for inhibition of recombinant
EcDxr and PfDxr using a spectrophotometric assay monitoring
the substrate-dependent oxidation of NADPH associated with
the Dxr-catalyzed reaction (Table 1).[16,23,25] None of the changes
introduced in 6a–6j improve inhibition of EcDxr relative to
5d, although 6a, 6c, 6d and 6 f are essentially equipotent to
5d. Two changes improve inhibition of PfDxr relative to 5d
(6b and 6 f), while three others give similar IC50 values (6a, 6c
and 6d). The remaining changes are associated with weaker
inhibition of PfDxr (6e, 6g–6j). Surprisingly, methyl substitu-
tion of the aromatic ring at the meta position (6b) increases
PfDxr inhibition, while it unfavorably influences EcDxr inhibi-
tion (Supporting Information (SI) Figure S1). The same may be
true for the meta-fluoro analogue 6 f.
Figure 2. Relevant Dxr inhibitors 3–5 and target b-substituted fosmidomycin
analogues 6a–j.
Recently, we assessed the ability of Dxr to accommodate
substituents in the b-position of fosmidomycin analogues
bearing a hydroxamate rather than the original reverse hydrox-
amate group.[23] We observed that direct introduction of aro-
matic rings at the b-carbon (as in 5a) afforded moderate Dxr
inhibitors. Exploration of different linkers between the b-
carbon and a phenyl ring (5b–e) suggested that a 3-carbon
linker (5d) was optimal for inhibition of EcDxr and M. tubercu-
losis Dxr, while both phenylpropyl (5d) and phenylbutyl (5e)
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
2
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
Scheme 1. Reagents and conditions: a) but-3-yn-1-ol, PdCl2(PPh3), CuI, Et3N, 1178C, 50–98%; b) H2, Pd/C, MeOH, 69–94%; c) Dess–Martin periodinane, CH2Cl2;
d) Ph3P=CHCOOtBu, toluene, 1208C, 49–67%; e) (BnO)2OPMe, nBuLi, THF, ꢀ788C, 43–71%; f) 1. TFA, CH2Cl2, 45 min, 08C!RT; 2. MeN(OBn)H, EDC, DMAP,
CH2Cl2, 18 h, RT, 44–78%; g) H2, Pd/C, MeOH, NaOH(aq), 258C, 10–15 min, quant.
None of the changes introduced in 6a–6j relative
to 5d significantly improve the IC50 values regarding
in vitro growth of the multidrug-resistant P. falcipa-
Table 1. In vitro inhibition of recombinant Dxr enzymes, as well as growth of the
P. falciparum K1 strain.
Compound
IC50 [mm]
rum K1 strain (Table 1). However, improvement of IC50
for PfDxr is well correlated with improvements in the
IC50 against the parasite (Figure 3), indicating that Dxr
is in fact the primary target of this series of com-
pounds. Only inhibitors with IC50 values better than
~0.3 mm (pIC50 greater than 6.5) had significant ef-
fects against the parasite at the highest concentra-
tion tested (64 mm).
EcDxr[a]
ND[c]
PfDxr[a]
P. fal. K1[b]
1.7ꢁ0.9[26]
0.036
(0.032–0.040)
1
2
0.03
0.045
0.4ꢁ0.2[26]
(0.02–0.05)[d] (0.041–0.049)[d]
Results against Trypanosoma brucei brucei
Squib 427, T. cruzi Tulahuen LacZ (clone C4) and
Leishmania infantum MHOM/MA(BE)/67 strains were
also negative at the highest concentrations tested
(64 mm).[29] Because these parasites do not have Dxr,
these were effectively control experiments. Cytotoxic-
ity, as assessed against MRC-5SV2 (human lung fibro-
blast) cells, was also negligible for all compounds at
this concentration.
0.84[23]
(0.51–1.40)
0.079
5d
6a
6b
2.7ꢁ0.[f]
(0.066–0.093)[e]
1.04
(0.6–1.8)
0.175
(0.129–0.237)
1.4ꢁ1.2
X-ray crystal structures of PfDxr in complex with
seven inhibitors
The structures of PfDxr in complex with seven of the
new b-substituted inhibitors (6a–d, 6 f, 6g, 6h) are
described in more detail in the Supporting Informa-
tion. Although all of the new compounds were en-
tered into crystallization trials, only inhibitors with
IC50 values better than ~0.5 mm (pIC50 greater than
~6.2) produced structures (Figures 3–5). The struc-
tures have been solved at resolutions in the range of
1.4–1.8 ꢂ, and refined to crystallographic R-factors of
~18% and free R-factors of 20–21%. Complete data
collection and refinement statistics are given in SI
Table S2. Each complex has been crystallized in space
group P1, with similar unit cell constants. A dimer is
found in the asymmetric unit, and a manganese ion
and an inhibitor molecule are very clear in each
active site (Figure 4a). The overall electron density is
of good quality, and complete models of the enzyme
10.40
(2.8–388.3)
0.05
(0.043–0.049)
10.9ꢁ4.5
1.18
(0.4–3.3)
0.12
(0.096–0.142)
6c
3.8ꢁ2.5
7.8ꢁ3.7
0.97
(0.62–1.5)
0.15
(0.137–0.154)
6d
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
3
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
identify the favored R-enantiomer in each complex,
as observed in our earlier work.[23] The protein struc-
tures are highly conserved; a structural superposition
of each chain onto the A chain of the 5d complex
(PDB ID: 4Y67) produces RMS values of 0.16–0.36 ꢂ
for 398–410 pairs of Ca atoms, when using a 1 ꢂ Ca-
pair cutoff. The overlapping Ca-traces show separa-
tion into A and B chain clusters at the start of two
helices in the cofactor-binding domain, probably as
a result of differences in the crystal environment. Be-
cause the electron density in the flap is often poorly
defined, the chains are not tightly clustered, but
short regions before and after the flap show separate
tight A- and B-chain clusters, again separated by
~0.5–1.0 ꢂ, probably as a result of different crystal
contacts.
Table 1. (Continued)
Compound
IC50 [mm]
EcDxr[a]
PfDxr[a]
P. fal. K1[b]
51.4ꢁ21.8
10.53
(2.4–46.2)
3.2
6e
6 f
6g
(2.83–3.63)
2.00
(1.1–3.7)
0.067
(0.057–0.078)
5.7ꢁ4.4
There is no clear correlation between strength of
inhibition and temperature factors or fit to electron
density (SI Figure S2), which is perhaps to be expect-
ed given the variable conformations observed for the
flap.
7.33
(0.9–59.6)
0.27
(0.248–0.295)
45.7ꢁ21.3
Numerous crystallographic studies have shown
that fosmidomycin analogues bind in the substrate/
product-binding site of Dxr. The phosphonate group
at one end is held firmly in place by multiple hydro-
gen-bonding interactions with protein and solvent,
and the hydroxamic acid group at the other end is
coordinated to the active-site metal ion. In ternary
complexes with 1 and 2, a well-defined flap has been
observed, with a number of highly conserved amino
acid side chains contributing to the binding
site.[16–18,24] In particular, the indole ring of Trp296
(PfDxr numbering) packs against the backbone of the
two compounds, and interacts with the methyl
group of the acetyl derivative 2.[16,18,24] In the numer-
ous structures with a-aryl analogues, the flap is
either disordered or has moved to allow the substitu-
ents to bind in a depression located between three
ordered loops and a usually disordered flap. This de-
pression is large enough to accommodate analogues
with formyl, acetyl or phenyl substituents at the hy-
droxamate group, for example.[31–33] b-aryl substitu-
ents, however, show at least two modes of bind-
ing.[23] For the 5a complex that lacks a linker, the
phenyl group is positioned ‘under’ a well-defined
flap, and the methyl group on the hydroxamic acid
interacts with the indole ring of Trp296. For the 5d
and 5e complexes, however, the linkers adopt boo-
13.91
(4.3–45.5)
0.28
(0.272–0.298)
6h
49.3ꢁ18.9
46.6ꢁ15.4
4.07
(1.5–11.2)
0.87
(0.780–0.977)
6i
1.6
6j
ND[g]
>64
(1.16–2.158)
[a] IC50 values reported for EcDxr were calculated from a single curve, whereas those
for PfDxr are based on three experiments (the confidence interval for each value is
shown in parentheses). [b] IC50 values for Plasmodium growth are the meanꢁSD of
three separate experiments performed on different dates. [c] Not determined; a value
of 0.030ꢁ0.008 was reported earlier.[27] [d] Values of 0.051 and 0.018 were reported
earlier for EcDxr and PfDxr, respectively.[28] [e] A value of 0.117ꢁ0.012 was reported
earlier.[23] [f] A value of 0.43ꢁ0.09 was reported earlier.[23] [g] Not determined; 86% ac-
tivity remained after the addition of 100 mm 6j.
are deposited at the PDB for residues 77–486 in each chain.
However, the electron density for even the main chain in some
of the flap (residues 292–298) is sometimes poorly defined; see
SI Table S3. Only in the complex with 6h, and in one molecule
of 6d, is the main-chain electron density of each flap continu-
ous at the RMS value of the relevant map. The indole ring of
Trp296 is poorly defined in six of the 14 views of the active
site. Although each compound was synthesized as a racemic
mixture, the high resolution of the structures allowed us to
merang-like conformations that, together with small changes
in the fosmidomycin backbone, allow their respective phenyl
groups to interact with the methyl group of their hydroxamic
acid. In this strikingly different way of dealing with the substi-
tution, the linker occupies the volume normally occupied by a-
aryl substitutions, while the phenyl ring is co-spatial with the
indole ring of the flap tryptophan, as seen in ternary com-
plexes.[16–18,24] The edge of the phenyl ring, in turn, interacts
with the indole ring of Trp296 in the flap. All seven of the new
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
4
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
as well as 6h, which we consider as a member of the meta
class.
The members of the meta class form a tight cluster of eight
independent structures (including both subunits of the
dimers), where the phenyl rings of the new compounds closely
overlap that observed in the 5d complex (Figure 4b). Interac-
tions of the phosphonate and hydroxamic acid groups in all
complexes are essentially identical to those of the 5d com-
plex,[23] as is the overall conformation of the fosmidomycin
backbone. All members adopt the same pose, where the sub-
stituent is directed toward the indole ring of Trp296 in the 5d
complex; none points in the other direction, toward His341.
However, all inhibitors have an effect on the positioning of the
indole ring, and on the quality of the electron density of the
flap (SI Table S3). Unsurprisingly, the introduction of a naphtha-
lene ring in 6h causes a large change in the conformation of
Trp296, which is needed to prevent clashes (Figure 4b); the
flap in this structure is well defined. Although there are no
close contacts to the indole ring, only a few atoms of one
edge of the naphthalene ring are solvent-exposed. The slightly
smaller methoxyphenyl substituent in 6d causes a different
movement of the tryptophan residue, needed to prevent close
contacts between the indole ring and the methyl group. The
flap is rather well defined in both chains, as is the density for
the indole group, but the change in conformation results in
a loss of the close contacts to the indole seen in the 5d com-
plex. The methoxyphenyl group is approximately planar, and
so occupies the same place as the corresponding portion of
the naphthalene ring of 6h (Figure 4b). The methoxy group
does not form any hydrogen-bonding interactions with the
protein, and is shielded from the solvent by residues near 360.
The introduction of the methyl and fluorine substituents in 6b
and 6 f, respectively, would result in close contacts to one
edge of the indole, if the conformation seen in the 5d com-
plex were maintained (three contacts are predicted, of 2.5 ꢂ
and ~3.1 ꢂ, in 6b and 6 f, respectively). Instead, the flap
moves, and the electron density in a three-residue segment of
the flap (residues 294–296) becomes poorly defined (SI
Table S3); the indole ring is moderately clear in only one of the
four active sites (that of the 6 f A-chain). Compounds 6b and
6 f show equal or better IC50 values than 5d, however, while
the other two meta compounds have higher values (Figure 3
and Table 1). The lack of well-defined interactions between the
best inhibitors and the indole ring of Trp296 suggests that the
interactions with the indole that are observed in the 5d com-
plex are not the most important determinants of the observed
IC50 values. However, it is striking that the larger the substitu-
ent, the higher the IC50 observed (Figure 5a). We suggest that
the most energetically favorable intramolecular phenyl/methyl
interactions are harder for inhibitors with larger substituents to
attain in the complexes, because they occur in the context of
enzyme–inhibitor interactions.
Figure 3. Plot of pIC50 against in vitro growth of the P. falciparum K1 strain
versus pIC50 against PfDxr activity for the new compounds; data for 1 and 2
are shown for reference. Compounds marked with blue boxes are those for
which X-ray structures of complexes with PfDxr are reported herein, whereas
red[18] and green[23] boxes indicate structures reported elsewhere.
Figure 4. X-ray structures of PfDxr in complex with inhibitors. The cartoon
representation of the protein backbone is color coded according to position
in the sequence, going through the rainbow from red to blue. a) Electron
density for the inhibitor 6h and selected nearby atoms, contoured at the
RMS value of the sA-weighted (2mjFo jꢀDjFc j) electron-density map[30]
(0.43 eꢂꢀ3) in light blue, as well as at 2.5 eꢂꢀ3 (gold) to show the higher
electron density near the metal ion. b) Superimposed structures of the
meta-class compounds, 6b (light brown), 6d (orange), 6 f (dark brown) and
6h (yellow), on 5d (silver gray). The well-defined flap residue, Trp296, of 6h
and 5d is observed to undergo a conformational change. c) Superimposed
structures of the para-class compounds, 6a (dark green), 6c (light green)
and 6g (cyan), on 5d (silver grey). Fluorine atoms are shown in magenta.
d) All the structures superimposed, using the same coloring scheme defined
in panels b and c.
b-substituted complexes take on the same general structure
observed in the 5d complex, but can be grouped into two
sets depending on whether they represent meta or para sub-
stitutions to the phenyl ring. We have structures for three
meta (6b, 6d, 6 f) and three para (6a, 6c, 6g) substitutions,
The members of the para group (6a, 6c, 6g) form a cluster
of six similar structures that are distinct from those of the meta
group (Figure 4c). The interactions at the phosphonate and hy-
droxamic acid moieties are essentially identical in all com-
plexes, as is the conformation of the fosmidomycin backbone.
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
5
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
tronic and steric properties versus the phenylpropyl side chain
of the earlier compound 5d. Several of the new compounds
exhibit potency on PfDxr that approaches that of 1 and 2.
There is a good correlation between activity against the
enzyme, and activity against the parasite, indicating that their
primary mode of biological action is in fact via PfDxr. Seven
new X-ray structures show that all of the new arylpropyl sub-
stituents displace the key tryptophan residue of the active-site
flap, which had made favorable interactions with the reverse
hydroxamate group of 1 and its acetyl homologue 2. The plas-
ticity of the flap allows the various compounds to be accom-
modated in many ways, and indeed in most cases, the flap is
largely disordered. However, the results can be separated into
two groups, based on whether the substituent on the aromatic
ring is meta or para. Generally, meta compounds are better in-
hibitors, and in both classes smaller substituents are associated
with better potency. The large lipophilic biphenyl and naphthyl
substituents provided poor inhibitors of Dxr, which was not
compensated for by any advantages such compounds might
provide regarding entry into the parasite cell and apicoplast,
or other factors. Future directions should include tests of addi-
tional small substituents, particularly those that could make
good interactions with one of the favored conformations of
the enzyme. However, it remains to be seen whether intestinal
absorption, pharmacokinetic properties or other properties are
improved for any of the b-arylpropyl analogues. The strength
of the present study is that it provides multiple viable com-
pounds for additional biological work, increasing the chances
of ultimate success in the effort to develop useful drugs of the
fosmidomycin class.
Figure 5. Relationship between substituent size and pIC50. The inhibitors and
residues of their respective flaps are colored on a common scale, going
through the rainbow from the best pIC50 value (red, using 1 as the endpoint)
to the poorest (blue). a) Complexes of the meta cluster: it should be remem-
bered that the 6b complex, in particular, has poor electron density for the
indole of Trp296. b) Complexes of the para cluster: note that the 6g com-
plex has no significant electron density in the flap, as well as weaker elec-
tron density in the three-carbon linker of the inhibitor.
While their phenyl rings are closely co-planar with that of the
5d complex, the rings are not so tightly clustered and each is
shifted to some degree within the binding site by virtue of the
flexible 3-carbon linker. The smallest shift from 5d is associated
with the smallest substitution, and so on: ~0.5 ꢂ for 6a,
~0.7 ꢂ for 6c and ~1.0 ꢂ for 6g. This results from steric con-
straints near Met360/Pro363 that “push” the larger substituents
away. Small differences in the torsion angles give the effect of
splaying the linker, while sliding the ring in the plane of the
phenyl ring seen in 5d (Figure 4c). The important intra-ligand
ring-methyl group interactions are, therefore, maintained in all
complexes, but with variations (Figure 4d). Again, the electron
density in each complex is poorly defined in regions of the
flap; the indole ring of Trp296 is well defined in only three
active sites; see SI Table S3. In these three complexes, Trp296
is similar to the 5d complex, and the indole ring helps to
shield the edge of the phenyl ring of the inhibitor. However,
the tryptophan is not forced out of the active site, as was ob-
served for the largest meta-group compounds. Two of the fluo-
rine atoms in 6g interact with a water molecule that is highly
conserved in the various structures (although slightly displaced
in the 6a complex), while the oxygen atom in the methoxy-
substituent of 6c accepts a hydrogen bond from the main-
chain amide nitrogen of residue 359. In general, the larger the
para-group substituent, the poorer the IC50 (Figure 5b). This is
probably due to the translation of the relevant phenyl group
from its energetically-preferred position in the 5d complex, al-
though this is compensated for in part by interactions of 6g
and 6c with structurally-conserved polar atoms. Overall, the
para-substituted ligands are poorer inhibitors than their meta
equivalents (placing 6i in the para group, as the para equiva-
lent of 6h).
Experimental Section
General: All reactions described were performed under an argon
atmosphere and at ambient temperature unless stated otherwise.
All reagents and solvents were purchased from Sigma–Aldrich
(Diegem, Belgium), Acros Organics (Geel Belgium), or TCI Europe
(Zwijndrecht, Belgium) and used as received (except THF). Tetrahy-
drofuran was dried over sodium/benzophenone. NMR solvents
were purchased from Eurisotop (Saint Aubin, France). Reactions
were monitored by TLC analysis using TLC aluminum sheets (Ma-
cherey–Nagel, Alugram Sil G/UV254). Detection was observed by
spraying with a solution of (NH4)6Mo7O24·4H2O (25 gLꢀ1) and
(NH4)4Ce(SO4)4·2H2O (10 gLꢀ1) in H2SO4 (10%) followed by charring
or immersion in an aqueous solution of KMnO7 (20 gLꢀ1) and
K2CO3 (10 gLꢀ1) or an ethanolic solution of ninhydrin (2 gLꢀ1) and
acetic acid (1% v/v) followed by charring. Silica gel column chro-
matography was performed manually using Grace Davisil 60 ꢂ
silica gel (40–63 mm) or automated using a Grace Reveleris X2
system and the corresponding flash cartridges. High-resolution
spectra were recorded with a Waters LCT Premier XE Mass spec-
1
trometer. H and 13C NMR spectra were recorded with a Varian Mer-
cury 300BB (300/75 MHz) spectrometer. Chemical shifts are given
in ppm (d) relative to tetramethylsilane (1H NMR) or the NMR sol-
vent (13C NMR) as an internal standard. In 31P NMR, signals are refer-
enced to the CDCl3 or D2O lock resonance frequency according to
IUPAC referencing, with H3PO4 set to 0.00 ppm. Coupling constants
are given in Hz. Preparative HPLC purifications were carried out
using a Laprep preparative HPLC system equipped with an Xbridge
Conclusions
In the present work, we continued our systematic study of b-
substituted hydroxamate analogues of fosmidomycin. Specifi-
cally, we explored the effects of changes in lipophilicity, elec-
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
6
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
Prep C18 column (19ꢃ250 mm, 5 mm) using a H2O/CH3CN/HCOOH
gradient solvent system. All synthesized compounds were ꢂ95%
pure as verified by LC–MS.
stirred under hydrogen atmosphere while monitoring the progress
by mass spectrometry. At completion (~10 min), the reaction mix-
ture was filtered and neutralized with NaOH (1 equiv). The reaction
mixture was then concentrated in vacuo, re-dissolved in a 1:1 (v/v)
mixture of H2O and tBuOH, frozen and lyophilized to afford the de-
sired targets compounds 6a–j as monosodium phosphonic acid
salts in quantitative yield.
General procedure I: Sonogashira coupling toward aralkynols
8a–j: To a solution of the aryl iodides 7a–j in degassed Et3N was
added PdCl2(PPh3)2, CuI and but-3-yn-1-ol. The reaction mixture
was held at reflux at 1178C for 3 h, after which it was cooled and
concentrated in vacuo. Column chromatography using a hexane/
EtOAc solvent system afforded compounds 8a–j.
tert-Butyl (E)-6-(p-tolyl)hex-2-enoate (11a): Prepared according to
general procedure III. Purification 1:1 toluene/hexane v/v; yield
59%; colorless oil. 1H NMR (300 MHz, CDCl3): dH =1.47 (brs, 9H,
tBu), 1.75 (app quint, J=8.0 Hz, 2H, -CH2-), 2.12–2.23 (m, 2H,
-CH2-), 2.30 (s, 3H, Ph-CH3), 2.58 (t, J=7.5 Hz, 2H, -CH2-), 5.74 (dt,
J=1.5 Hz, 15.6 Hz, 1H, -CH=CHCO), 6.87 (dt J=7.1 Hz, 15.6 Hz, 1H,
-CH=CHCO), 7.00–7.16 ppm (m, 4H, Ar-H); 13C NMR (75 MHz,
CDCl3): dC =20.9, 28.1, 29.7, 31.4, 34.7, 79.9, 123.2, 128.2, 128.9,
135.2, 138.6, 147.5, 170.0 ppm; HRMS (ESI): calcd for C17H25O2 [M+
H]+: 261.1849, found: 261.1856.
General procedure II: triple bond reduction: To a solution of the
alkynes 8a–j in MeOH, was added 10% Pd/C under a nitrogen at-
mosphere. Molecular hydrogen (H2) was bubbled through the mix-
ture for 30 min followed by filtration through a Whatman filter
paper path. In vacuo concentration yielded compounds 9a–j,
which were used for the next step without further purification.
General procedure III: Dess–Martin oxidation and concomitant
Wittig olefination: A solution of the starting materials (9a–j) in
CH2Cl2 and a nitrogen atmosphere was cooled to 08C. Dess–Martin
periodinane (2.0 equiv) was added, and the mixture was allowed
to warm to RT. After stirring for 3 h, TLC analysis showed a complet-
ed reaction. The reaction mixture was washed once with a 5:1 mix-
ture of NaHCO3 (sat. aq.) and Na2S2O3 (aq. 2.0m), and the aqueous
layer was extracted three times with Et2O. The combined organic
layer was washed successively with a 0.1m solution of HCl and
brine, dried over anhydrous Na2SO4 and concentrated in vacuo to
obtain the corresponding aldehydes (10a–j) which was used with-
out further purification. The aldehyde was dissolved in toluene
under nitrogen atmosphere and tert-butyl(triphenylphosphoranyli-
dene)acetate (3 equiv) was added. An overnight reflux at 1208C
was followed by cooling and in vacuo concentration. Sorption of
the crude on Celite and silica gel chromatography gave access to
the tert-butyl esters 11 a–j.
tert-Butyl (E)-6-(m-tolyl)hex-2-enoate (11b): Prepared according
to general procedure III. Purification 1:1 toluene/hexane v/v; yield
51%; colorless oil. 1H NMR (300 MHz, CDCl3): dH =1.44 (brs, 9H,
tBu), 1.76 (app quint, J=7.7 Hz, 2H, -CH2-), 2.14–2.24 (m, 2H,
-CH2-), 2.32 (s, 3H, Ph-CH3), 2.59 (t, J=7.7 Hz, 2H, -CH2-), 5.75 (dt,
J=1.6 Hz, 15.6 Hz, 1H, -CH=CHCO), 6.87 (dt J=6.9 Hz, 15.5 Hz, 1H,
-CH=CHCO), 6.93–7.02 (m, 3H Ar-H), 7.12–7.20 ppm (m, 1H, Ar-H);
13C NMR (75 MHz, CDCl3): dC =21.3, 28.1, 29.7, 31.5, 35.2, 80.0,
123.3, 125.4, 126.6, 128.2, 129.2, 137.8, 141.7, 147.5, 166.0 ppm;
HRMS (ESI): calcd for C17H25O2 [M+H]+: 261.1849, found: 261.1852.
tert-Butyl (E)-6-(4-methoxyphenyl)hex-2-enoate (11c): Prepared
according to general procedure III. Purification 1:1 toluene/hexane
1
v/v; yield 64%; white wax. H NMR (300 MHz, CDCl3): dH =1.47 (brs,
9H, tBu), 1.73 (app quint, J=7.7 Hz, 2H, -CH2-), 2.12–2.23 (m, 2H,
-CH2-), 2.32 (s, 3H, Ph-CH3), 2.57 (t, J=7.4 Hz, 2H, -CH2-), 3.76 (s,
3H, PhOCH3), 5.74 (dt, J=1.7 Hz, 15.7 Hz, 1H, -CH=CHCO), 6.82 (dt
J=6.4 Hz, 15.7 Hz, 1H, -CH=CHCO), 7.26–7.42 ppm (m, 4H, Ar-H);
13C NMR (75 MHz, CDCl3): dC =28.1, 29.8, 31.3, 34.2, 55.1, 78.3,
113.7, 123.2, 129.2, 133.7, 147.5, 157.7, 166.0 ppm; HRMS (ESI):
calcd for C17H25O3 [M+H]+: 277.1798, found: 277.1790.
General procedure IV: Michael addition of methylphosphonate
diesters to a,b-unsaturated tert-butyl esters: To a solution of di-
benzylmethyl phosphonate (2 equiv) in THF and under a nitrogen
atmosphere was added nBuLi (2 equiv) at ꢀ788C. After 30 min,
a solution of the ester was added to the reaction mixture drop-
wise. Three hours later, the reaction was shown to be complete by
TLC and was quenched with NH4Cl (sat. aq.). The aqueous layer
was extracted three times with EtOAc. Organic fractions were
pooled, washed once with brine and dried over anhydrous Na2SO4.
Column chromatography (hexane/EtOAc system) afforded the ad-
ducts 12a–j.
tert-Butyl (E)-6-(3-methoxyphenyl)hex-2-enoate (11d): Prepared
according to general procedure III. Purification 1:1 toluene/hexane
v/v; yield 65%; white viscous oil. 1H NMR (300 MHz, CDCl3): dH =
1.46 (brs, 9H, tBu), 1.76 (app quint, J=7.6 Hz, 2H, -CH2-), 2.16–2.24
(m, 2H, -CH2-), 2.57 (t, J=7.6 Hz, 2H, -CH2-), 3.72 (s, 3H, PhOCH3),
5.73 (dt, J=1.6 Hz, 15.7 Hz, 1H, -CH=CHCO), 6.87 (dt J=6.9 Hz,
15.7 Hz, 1H, -CH=CHCO), 6.92–7.04 (m, 3H Ar-H), 7.12–7.21 ppm
(m, 1H, Ar-H); 13C NMR (75 MHz, CDCl3): dC =27.9, 28.1, 29.4, 31.5,
35.8, 80.0, 123.3, 125.4, 126.6, 128.2, 129.2, 137.8, 141.7, 146.8,
165.4 ppm; HRMS (ESI): calcd for C17H25O3 [M+H]+: 277.1798,
found: 277.1799.
General procedure V: acidic cleavage of the tert-butyl ester and
protected hydroxamate formation: A 0.1m solution of the start-
ing materials (12a–j) in CH2Cl2/TFA (80:20) at 08C was stirred for
2 h, after which excess toluene was added to the reaction mixture
and concentrated in vacuo. The crude acid was re-dissolved in
CH2Cl2 (0.1m), followed by addition of EDC (1.2 equiv), DMAP
(1.2 equiv), and Et3N (2.0 equiv). O-Benzyl-N-methylhydroxylamine
TFA salt (1.2 equiv) was added as a 0.2m solution in CH2Cl2, and
the mixture was stirred overnight at RT. The reaction was subse-
quently quenched with sat. aq. NaHCO3, extracted three times with
CH2Cl2, washed with brine and dried over Na2SO4. Column chroma-
tography (CH2Cl2/MeOH system) produced the protected hydroxa-
mic acids to yield compounds 13a–j.
tert-Butyl (E)-6-(4-fluorophenyl)hex-2-enoate (11e): Prepared ac-
cording to general procedure III. Purification 1:1 toluene/hexane v/
v; yield 53%; pale-yellow oil. 1H NMR (300 MHz, CDCl3): dH =1.47
(brs, 9H, tBu), 1.66–1.82 (app quint, J=7.6 Hz, 2H, -CH2-), 2.19 (m,
2H, -CH2-), 2.60 (t, J=7.4 Hz, 2H, -CH2-), 5.75 (dt, J=1.7 Hz,
15.80 Hz, 1H, -CH=CHCO), 6.80–7.00 (m, 3H, -CH=CHCO, Ar-H),
7.06–7.15 ppm (m, 2H, Ar-H); 13C NMR (75 MHz, CDCl3): dC =28.1,
29.8, 31.3, 34.4, 80.0, 115.0 (d, 2JCꢀF =20.9 Hz), 123.4, 129.7 (d,
1
3JCꢀF =8.8 Hz), 137.3 (d, 4JCꢀF =3.6 Hz), 147.2, 161.3 (d, JCꢀF
=
General procedure VI: catalytic hydrogenolysis of benzyl protec-
tive groups: The benzyl-protected compounds 13a–j (100–
130 mg) were dissolved in MeOH (10 mL) under an inert atmos-
phere and 10% Pd/C was added. The resulting mixture was then
242.9 Hz), 166.0 ppm; HRMS (ESI): calcd for C16H22FO2 [M+H]+:
265.1598, found: 265.1597.
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
7
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
2
tert-Butyl (E)-6-(3-fluorophenyl)hex-2-enoate (11 f): Prepared ac-
cording to general procedure III. Purification 1:1 toluene/hexane v/
v; yield 49%; pale-yellow oil . H NMR (300 MHz, CDCl3): dH =1.48
dC =21.2, 28.3, 28.7, 30.1 (d, 1JCꢀP =138.6 Hz), 30.6 (d, JCꢀP
=
4.1 Hz), 31.1, 34.5 (d, 3JCꢀP =10.1 Hz), 35.6, 40.5 (d, 3JCꢀP =9.3 Hz),
66.9 (d, 2JCꢀP =6.6 Hz), 67.0 (d, 2JCꢀP =6.6 Hz), 80.2, 127.9, 127.9,
1
3
(brs, 9H, tBu), 1.78 (app quint, J=8.0 Hz, 2H, -CH2-), 2.15–2.25 (m,
2H, -CH2-), 2.63 (t, J=7.7 Hz, 2H, -CH2-), 5.75 (dt, J=1.7 Hz, 15.6 Hz,
1H, -CH=CHCO), 6.79–6.97 (m, 4H, -CH=CHCO, Ar-H), 7.18–
7.27 ppm (m, 1H, Ar-H); 13C NMR (75 MHz, CDCl3): dC =28.1, 29.4,
31.3, 34.9 (d, 4JCꢀF =1.8 Hz), 80.1, 112.7, 115.2 (d, 2JCꢀF =21.1 Hz),
123.5, 124.0 (d, 4JCꢀF =2.8 Hz), 129.7 (d, 3JCꢀF =8.2 Hz), 144.3 (d,
3JCꢀF =7.1 Hz), 147.1, 162.8 (d, 2JCꢀF =245.5 Hz), 166.0 ppm; HRMS
(ESI): calcd for C16H22FO2 [M+H]+: 265.1598, found: 265.1599.
128.2, 128.3, 128.5, 128.9, 135.0, 136.4 (d, JCꢀP =6.2 Hz), 136.4 (d,
3JCꢀP =6.0 Hz), 139.1, 171.6 ppm; 31P NMR (121.5 MHz, CDCl3): dP =
33.29 ppm; HRMS (ESI): calcd for C32H42O5P [M+H]+: 537.2764,
found: 537.2778.
tert-Butyl 3-((bis(benzyloxy)phosphoryl)methyl)-6-(m-tolyl)hexa-
noate (12b): Prepared according to general procedure IV. Purifica-
tion 2:1 hexane/EtOAc v/v; yield 45%; colorless oil. 1H NMR
(300 MHz, CDCl3): dH =1.39 (brs, 9H, tBu), 1.42–1.63 (m, 4H, -CH2-),
1.78–1.98 (m, 2H, -CH2-), 2.17–2.34 (m, 5H, Ph-CH3, -CH2-), 2.35–
2.54 (m, 3H, -CH-, -CH2-), 4.88–5.08 (m, 4H, -CH2-Ph), 6.88–7.00 (m,
tert-Butyl (E)-6-(4-(trifluoromethyl)phenyl)hex-2-enoate (11g):
Prepared according to general procedure III. Purification 1:1 tolu-
1
ene/hexane v/v; yield 60%; colorless oil. H NMR (300 MHz, CDCl3):
3H, Ar-H), 7.14 (t, J=7.7 Hz, 1H, Ar-H), 7.28–7.37 ppm (m, 10H, Ar-
1
dH =1.48 (brs, 9H, tBu), 1.80 (app quint, J=7.5 Hz, 2H, -CH2-), 2.21
(m, 2H, -CH2-), 2.69 (t, J=7.8 Hz, -CH2-), 5.76 (dt, J=1.8 Hz, 15.6 Hz,
1H, -CH=CHCO), 6.86 (dt, J=6.8 Hz, 15.6 Hz, 1H, -CH=CHCO), 7.28
(d, J=9.0 Hz, 2H, Ar-H), 7.54 ppm (d, J=8.5 Hz, 2H, Ar-H); 13C NMR
(75 MHz, CDCl3): dC =28.1, 29.4, 31.3, 35.0, 80.1, 123.6, 124.3 (q,
1JCꢀF =271.9 Hz), 125.3 (q, 3JCꢀF =7.2 Hz), 128.3 (q, 2JCꢀF =32.5 Hz),
128.7, 145.9, 146.9, 165.9 ppm; HRMS (ESI): calcd for C17H22F3O2
[M+H]+: 315.1566, found: 315.1570.
H); 13C NMR (75 MHz, CDCl3): dC =21.6, 28.3, 28.6, 30.1 (d, JCꢀP
=
2
3
138.6 Hz), 30.6 (d, JCꢀP =3.9 Hz), 34.6 (d, JCꢀP =10.3 Hz), 36.1, 40.5
3
2
(d, JCꢀP =10.3 Hz), 67.3 (d, JCꢀP =6.2 Hz), 80.6, 125.6, 126.7, 128.2,
128.4, 128.6, 128.8, 128.4, 136.7 (d, 3JCꢀP =7.1 Hz), 138.0, 142.5,
171.9 ppm; 31P NMR (121.5 MHz, CDCl3): dP =33.29 ppm; HRMS
(ESI): calcd for C32H42O5P [M+H]+: 537.2764, found: 537.2786.
tert-Butyl 3-((bis(benzyloxy)phosphoryl)methyl)-6-(4-methoxy-
phenyl)hexanoate (12c): Prepared according to general proce-
dure IV. Purification 2:1 hexane/EtOAc v/v; yield 53%; off-white oil.
1H NMR (300 MHz, CDCl3): dH =1.39 (brs, 9H, tBu), 1.43–1.97 (m,
6H, -CH2-), 2.17–2.52 (m, 5H, -CH2-, -CH-), 3.77 (s, 3H, PhOCH3),
4.88–5.10 (m, 4H, -CH2-Ph), 6.79 (d, J=9.1 Hz, 2H, Ar-H), 7.02 (d,
J=9.1 Hz, 2H, Ar-H), 7.26–7.44 ppm (m, 10H, Ar-H); 13C NMR
(75 MHz, CDCl3): dC =28.0, 28.5, 29.8 (d, 1JCꢀP =138.5 Hz), 30.3 (d,
tert-Butyl (E)-6-(naphthalen-1-yl)hex-2-enoate (11h): Prepared ac-
cording to general procedure III. Purification 1:1 toluene/hexane v/
1
v; yield 67%; colorless oil. H NMR (300 MHz, CDCl3): dH =1.47 (brs,
9H, tBu), 1.90 (app quint, J=7.6 Hz, 2H, -CH2-), 2.21–2.31 (m, 2H,
-CH2-), 3.08 (t, J=7.6 Hz, -CH2-), 5.78 (dt, J=1.5 Hz, 15.6 Hz, 1H,
-CH=CHCO), 6.91 (dt, J=6.9 Hz, 15.6 Hz, 1H, -CH=CHCO), 7.26–7.53
(m, 4H, Ar-H), 7.70 (d, J=8.4 Hz, 1H, Ar-H), 7.80–8.00 ppm (m, 2H,
Ar-H); 13C NMR (75 MHz, CDCl3): dC =28.1, 28.9, 31.8, 32.4, 80.0,
123.4, 123.6, 125.4, 125.5, 125.7, 126.0, 126.7, 128.7, 131.7, 133.9,
137.8, 147.3, 166.0 ppm; HRMS (ESI): calcd for C20H25O2 [M+H]+:
297.1849, found 297.1854.
2JCꢀP =3.9 Hz), 34.2 (d, JCꢀP =10.7 Hz), 34.9, 40.2 (d, JCꢀP =9.9 Hz),
3
3
55.2, 67.0 (d, 2JCꢀP =6.1 Hz), 67.0 (d, 2JCꢀP =6.7 Hz), 80.3, 113.7,
3
127.9, 128.3, 128.5, 129.2, 134.3, 136.4 (d, JCꢀP =5.3 Hz), 136.4 (d,
3JCꢀP =5.9 Hz), 157.7, 171.6 ppm; 31P NMR (121.5 MHz, CDCl3): dP =
32.18 ppm; HRMS (ESI): calcd for C32H42O6P [M+H]+: 553.2714,
found: 553.2717.
tert-Butyl (E)-6-(naphthalen-2-yl)hex-2-enoate (11i): Prepared ac-
cording to general procedure III. Purification 1:1 toluene/hexane v/
tert-Butyl 3-((bis(benzyloxy)phosphoryl)methyl)-6-(3-methoxy-
phenyl)hexanoate (12d): Prepared according to general procedur-
e IV. Purification 2:1 hexane/EtOAc v/v; yield 47%; off-white oil.
1H NMR (300 MHz, CDCl3): dH =1.39 (brs, 9H, tBu), 1.42–1.63 (m,
4H, -CH2-), 1.76–1.93 (m, 2H, -CH2-), 2.19–2.54 (m, 5H, -CH2-, -CH-),
3.76 (s, 3H, PhOCH3), 4.87–5.08 (m, 4H, -CH2-Ph), 6.65–6.74 (m, 3H,
Ar-H), 7.16 (t, J=8.1 Hz, 1H, Ar-H), 7.28–7.36 ppm (m, 10H, Ar-H);
13C NMR (75 MHz, CDCl3): dC =28.3, 28.5, 30.2 (d, 1JCꢀP =139.8 Hz),
1
v; yield 67%; colorless oil. H NMR (300 MHz, CDCl3): dH =1.48 (brs,
9H, tBu), 1.84 (app quint, J=8.1 Hz, 2H, -CH2-), 2.21 (m, 2H, -CH2-),
2.77 (t, J=7.7 Hz, -CH2-), 5.76 (dt, J=1.6 Hz, 15.2 Hz, 1H, -CH=
CHCO), 6.89 (dt, J=7.3 Hz, 15.8 Hz, 1H, -CH=CHCO), 7.29 (dd, J=
1.8 Hz, 8.4 Hz, 1H, Ar-H), 7.36–7.47 (m, 2H, Ar-H), 7.58 (s, 1H, Ar-H),
7.71–7.81 ppm (m, 2H, Ar-H); 13C NMR (75 MHz, CDCl3): dC =28.1,
29.5, 31.4, 35.3, 80.0, 123.3, 125.1, 125.8, 126.4, 127.1, 127.3, 127.5,
127.9, 132.0, 133.5, 139.2, 147.4, 166.0 ppm; HRMS (ESI): calcd for
C20H25O2 [M+H]+: 297.1849, found: 297.1852.
2
3
3
30.6 (d, JCꢀP =4.1 Hz), 34.5 (d, JCꢀP =11.0 Hz), 36.1, 40.4 (d, JCꢀP
=
9.4 Hz), 55.3, 67.2 (d, 2JCꢀP =4.1 Hz), 67.3 (d, 2JCꢀP =4.3 Hz), 80.6,
3
111.2, 114.4, 121.0, 128.2, 128.6, 128.8, 129.5, 136.6 (d, JCꢀP
=
tert-Butyl (E)-6-([1,1’-biphenyl]-4-yl)hex-2-enoate (11j): Prepared
1.5 Hz), 136.7 (d, 3JCꢀP =6.0 Hz), 136.7 (d, 3JCꢀP =6.0 Hz), 144.1,
159.8, 171.9 ppm; 31P NMR (121.5 MHz, CDCl3): dP =33.24 ppm;
HRMS (ESI): calcd for C32H42O6P [M+H]+: 553.2714, found:
553.2717.
according to general procedure III. Purification 1:1 toluene/hexane
1
v/v; yield 61%; brown oil. H NMR (300 MHz, CDCl3): dH =1.48 (brs,
9H, tBu), 1.84 (app quint, J=7.5 Hz, 2H, -CH2-), 2.21–2.35 (m, 2H,
-CH2-), 2.65 (t, J=7.8 Hz, -CH2-), 5.75 (dt, J=1.7 Hz, 15.7 Hz, 1H,
-CH=CHCO), 6.90 (dt, J=6.8 Hz, 15.7 Hz, 1H, -CH=CHCO), 7.22 (d,
J=8.5 Hz, 2H, Ar-H), 7.27–7.67 ppm (m, 7H, Ar-H); 13C NMR
(75 MHz, CDCl3): dC =28.1, 29.6, 31.4, 34.8, 80.0, 123.4, 126.9, 127.0,
127.1, 128.7, 128.8, 138.8, 140.8, 141.0, 147.4, 166.0 ppm; HRMS
(ESI): calcd for C22H27O2 [M+H]+: 323.2006, mass not found.
tert-Butyl 3-((bis(benzyloxy)phosphoryl)methyl)-6-(4-fluorophe-
nyl)hexanoate (12e): Prepared according to general procedure IV.
Purification 2:1 hexane/EtOAc v/v; yield 43%; gold colored oil.
1H NMR (300 MHz, CDCl3): dH =1.38 (brs, 9H, tBu), 1.42–1.61 (m,
4H, -CH2-), 1.76–1.96 (m, 2H, -CH2-), 2.16–2.34 (m, 2H, -CH2-), 2.18–
2.52 (m, 3H, -CH2-, -CH-), 4.89–5.08 (m, 4H, -CH2-Ph), 6.78–6.96 (m,
2H, Ar-H), 7.03 (m, 2H, Ar-H), 7.28–7.37 ppm (m, 10H, Ar-H);
13C NMR (75 MHz, CDCl3): dC =28.0, 28.4, 29.9 (d, 1JCꢀP =138.9 Hz),
tert-Butyl 3-((bis(benzyloxy)phosphoryl)methyl)-6-(p-tolyl)hexa-
noate (12a): Prepared according to general procedure IV. Purifica-
tion 2:1 hexane/EtOAc v/v; yield 54%; colorless oil. 1H NMR
(300 MHz, CDCl3): dH =1.38 (brs, 9H, tBu), 1.42–1.62 (m, 4H, -CH2-),
1.76–1.95 (m, 2H, -CH2-), 2.19–2.32 (m, 5H, Ph-CH3, -CH2-), 2.35–
2.54 (m, 3H, -CH2-, -CH-), 4.87–5.08 (m, 4H, -CH2-Ph), 6.97–7.08 (m,
4H, Ar-H), 7.28–7.35 ppm (m, 10H, Ar-H); 13C NMR (75 MHz, CDCl3):
2
3
3
30.3 (d, JCꢀP =3.9 Hz), 34.1 (d, JCꢀP =10.2 Hz), 34.9, 40.2, (d, JCꢀP
=
2
10.2 Hz), 67.0 (m), 80.3, 114.9 (d, JCꢀF =20.4 Hz), 127.9, 128.3, 128.5,
4
129.6 (d, 3JCꢀF =7.7 Hz), 136.4 (d, 3JCꢀP =6.3 Hz), 137.8 (d, JCꢀF
=
3.3 Hz), 161.1 (d, JCꢀF =243.0 Hz), 171.6 ppm; 31P NMR (121.5 MHz,
1
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
8
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
CDCl3): dP =33.00 ppm; HRMS (ESI): calcd for C31H39FO5P [M+H]+:
541.2514, found: 541.2519.
tert-Butyl 6-([1,1’-biphenyl]-4-yl)-3-((bis(benzyloxy)phosphoryl)-
methyl)hexanoate (12j): Prepared according to general proce-
dure IV. Purification 2:1 hexane/EtOAc v/v; yield 71%; colorless oil.
1H NMR (300 MHz, CDCl3): dH =1.38 (brs, 9H, tBu), 1.42–1.99 (m,
6H, -CH2-), 2.20–2.63 (m, 5H, -CH2-, -CH-), 4.88–5.09 (m, 4H, -CH2-
Ph), 7.17 (d, J=8.2 Hz, 2H, Ar-H), 7.25–7.60 ppm (m, 17H, Ar-H);
13C NMR (75 MHz, CDCl3): dC =28.0, 28.2, 29.9 (d, 1JCꢀP =138.5 Hz),
tert-Butyl 3-((bis(benzyloxy)phosphoryl)methyl)-6-(3-fluorophe-
nyl)hexanoate (12 f): Prepared according to general procedure IV.
Purification 2:1 hexane/EtOAc v/v; yield 47%; gold colored oil.
1H NMR (300 MHz, CDCl3): dH =1.33–1.40 (brs, 9H, tBu), 1.41–1.61
(m, 4H, -CH2-), 1.72–1.97 (m, 2H, -CH2-), 2.18–2.33 (2H, m, -CH2-),
2.36–2.55 (m, 3H, -CH2-, -CH-), 4.89–5.08 (m, 4H, CH2-Ph), 6.76–6.90
(m, 3H, Ar-H), 7.19 (td, 1H, J=6.08 Hz, 13.96 Hz, Ar-H), 7.29–
2
3
3
30.3 (d, JCꢀP =3.9 Hz), 34.2 (d, JCꢀP =10.9 Hz), 35.4, 40.2 (d, JCꢀP
=
9.3 Hz), 67.0 (d, 2JCꢀP =6.6 Hz), 67.1 (d, 2JCꢀP =6.4 Hz), 80.3, 126.9,
3
127.0, 127.9, 128.0, 128.3, 128.5, 128.6, 128.7, 136.3 (d, JCꢀP
=
7.39 ppm (m, 10H, Ar-H); 13C NMR (75 MHz, CDCl3): dC =28.2, 28.3,
6.1 Hz), 136.4 (d, 3JCꢀP =6.1 Hz), 138.6, 141.0, 141.3, 171.6 ppm;
31P NMR (121.5 MHz, CDCl3): dP =31.83 ppm; HRMS (ESI): calcd for
C37H44O5P [M+H]+: 599.2921, found: 599.2928.
3
30.2 (d, 1JCꢀP =139.9 Hz), 30.5 (d, 2JCꢀP =4.9 Hz), 34.4 (d, JCꢀP
=
3
2
10.8 Hz), 35.7, 40.5 (d, JCꢀP =9.8 Hz), 66.9 (d, JCꢀP =6.6 Hz), 67.0 (d,
2
2JCꢀP =6.4 Hz), 80.6, 112.8 (d, 2JCꢀF =21.0 Hz), 115.4 (d, JCꢀF
=
20.7 Hz), 124.2 (d, 4JCꢀF =3.1 Hz), 128.2, 128.6, 128.8, 129.9 (d,
3JCꢀF =8.3 Hz), 136.3 (d, 3JCꢀP =6.3 Hz), 136.4 (d, 3JCꢀP =6.4 Hz),
145.1(d, 3JCꢀF =7.3 Hz), 162.8 (d, 1JCꢀF =246.5 Hz), 171.8 ppm;
31P NMR (121.5 MHz, CDCl3): dP =33.16 ppm; HRMS (ESI): calcd for
C31H39FO5P [M+H]+: 541.2514, found: 541.2515.
Dibenzyl (2-(2-((benzyloxy)(methyl)amino)-2-oxoethyl)-5-(p-tol-
yl)pentyl)phosphonate (13a): Prepared according to general pro-
cedure V. Purification 3:1 hexane/acetone v/v; yield 71%; colorless
oil. 1H NMR (300 MHz, CDCl3): dH =1.34–1.58 (m, 4H, -CH2-), 1.72–
2.05 (m, 3H, -CH2-, -CH-), 2.30 (s, 3H, Ph-CH3), 2.37–2.65 (m, 4H,
-CH2-), 3.13 (s, 3H, N-CH3), 4.72 (s, 2H, NOCH2Ph), 4.86–5.06 (m, 4H,
-POCH2Ph), 6.98 (d, J=8.1 Hz, 2H, Ar-H), 7.05 (d, J=8.1 Hz, 2H, Ar-
H), 7.28–7.36 ppm (m, 15H, Ar-H); 13C NMR (75 MHz, CDCl3): dC =
19.2, 21.0, 28.7, 29.6 (d, 1JCꢀP =139.3 Hz), 29.6 (d, 2JCꢀP =5.1 Hz),
33.5, 34.6 (d, 3JCꢀP =10.2 Hz), 35.4, 36.6 (d, 3JCꢀP =9.2 Hz), 67.1 (d,
2JCꢀP =6.3 Hz), 67.5 (d, 2JCꢀP =6.1 Hz), 76.1, 127.9, 127.9, 128.2,
128.3, 128.5, 128.6, 128.9, 128.9, 129.3, 134.5, 135.0, 136.5 (m),
139.3, 173.8 ppm; 31P NMR (121.5 MHz, CDCl3): dP =33.55 ppm;
HRMS (ESI): calcd for C36H43NO5P [M+H]+: 600.2873, found:
600.2903.
tert-Butyl 3-((bis(benzyloxy)phosphoryl)methyl)-6-(4-(trifluoro-
methyl)phenyl)hexanoate (12g): Prepared according to general
procedure IV. Purification 2:1 hexane/EtOAc v/v; yield 62% color-
less oil. 1H NMR (300 MHz, CDCl3): dH =1.37 (brs, 9H, tBu), 1.41–
1.64 (m, 4H, -CH2-), 1.72–1.96 (m, 2H, -CH2-), 2.18–2.61 (m, 5H,
-CH2-, -CH-), 4.90–5.09 (m, 4H, -CH2-Ph), 7.19 (d, J=8.0 Hz, 2H, Ar-
H), 7.28–7.36 (m, 10H, Ar-H), 7.49 ppm (d, J=8.0 Hz, 2H, Ar-H);
13C NMR (75 MHz, CDCl3): dC =28.3, 28.4, 30.2 (d, 1JCꢀP =138.2 Hz),
2
3
3
30.6 (d, JCꢀP =5.4 Hz), 34.4 (d, JCꢀP =10.8 Hz), 35.9, 40.6 (d, JCꢀP
=
2
2
9.2 Hz), 67.4 (d, JCꢀP =6.7 Hz), 67.5 (d, JCꢀP =6.6 Hz), 80.8, 124.6 (q,
1JCꢀF =271.5 Hz), 125.5 (q, 3JCꢀF =3.8 Hz), 128.3, 128.5 (q, JCꢀF
=
Dibenzyl (2-(2-((benzyloxy)(methyl)amino)-2-oxoethyl)-5-(m-tol-
yl)pentyl)phosphonate (13b): Prepared according to general pro-
cedure V. Purification 98:2 dichloromethane/MeOH v/v; yield 72%;
colorless oil. 1H NMR (300 MHz, CDCl3): dH =1.37–1.59 (m, 4H,
-CH2-), 1.74–2.04 (m, 2H, -CH2-), 2.30 (s, 3H, Ph-CH3), 2.37–2.65 (m,
5H, -CH2-, -CH-), 3.12 (s, 3H, N-CH3), 4.72 (s, 2H, NOCH2Ph), 4.89–
5.05 (m, 4H, -POCH2Ph), 6.86–7.00 (m, 3H, Ar-H), 7.13 (t, J=7.4 Hz,
2
27.1 Hz), 128.7, 128.9, 129.0, 136.7 (d, 3JCꢀP =6.0 Hz), 146.7,
171.9 ppm; 31P NMR (121.5 MHz, CDCl3): dP =31.86 ppm; HRMS
(ESI): calcd for C32H39F3O5P [M+H]+: 591.2482, found: 591.2487.
tert-Butyl 3-((bis(benzyloxy)phosphoryl)methyl)-6-(naphthalen-1-
yl)hexanoate (12h): Prepared according to general procedure IV.
1
Purification 2:1 hexane/EtOAc v/v; yield 55%; colorless oil. H NMR
1H, Ar-H), 7.27–7.36 ppm (m, 15H, Ar-H); 13C NMR (75 MHz, CDCl3):
(300 MHz, CDCl3): dH =1.37 (brs, 9H, tBu), 1.45–1.75 (m, 4H, -CH2-),
1.77–1.94 (m, 2H, -CH2-), 2.20–2.49 (m, 3H, -CH2-, -CH-), 2.98 (t, J=
7.1 Hz, 2H, -CH2-), 4.86–5.09 (m, 4H, -CH2-Ph), 7.21–7.39 (m, 12H,
Ar-H), 7.41–7.51 (m, 2H, Ar-H), 7.68 (d, J=8.4 Hz, 1H, Ar-H), 7.80–
1
dC =21.3, 28.5, 28.6, 28.6 (d, 3JCꢀP =8.8 Hz), 29.5 (d, JCꢀP
=
2
3
138.8 Hz), 29.6 (d, JCꢀP =4.9 Hz), 34.6 (d, JCꢀP =10.8 Hz), 35.7, 66.9
(m), 76.0, 125.3, 126.3, 127.8, 128.1, 128.2, 128.5, 128.6, 128.8,
129.1, 129.2, 136.4 (m), 137.6, 142.3. 171.9 ppm; 31P NMR
(121.5 MHz, CDCl3): dP =33.54 ppm; HRMS (ESI): calcd for
C36H43NO5P [M+H]+: 600.2873, found: 600.2883.
7.86 (m, 1H, Ar-H), 7.93–7.99 ppm (m, 1H, Ar-H); 13C NMR (75 MHz,
2
CDCl3): dC =27.8, 28.2, 30.1 (d, 1JCꢀP =138.8 Hz), 30.5 (d, JCꢀP
=
3
3
4.5 Hz), 33.1, 34.8 (d, JCꢀP =10.9 Hz), 40.3 (d, JCꢀP =10.0 Hz), 66.9
(d, 2JCꢀP =6.7 Hz), 7.2 (d, 2JCꢀP =6.4 Hz), 80.5, 123.9, 125.5, 125.6,
125.84, 126.0, 126.7, 128.1, 128.5, 128.7, 128.9, 131.9, 134.0, 135.3
(d, 3JCꢀP =6.4 Hz), 135.7 (d, 3JCꢀP =6.1 Hz), 138.5, 171.7 ppm;
31P NMR (121.5 MHz, CDCl3): dP =33.23 ppm; HRMS (ESI): calcd for
C35H42O5P [M+H]+: 573.2764, found: 573.2761.
Dibenzyl (2-(2-((benzyloxy)(methyl)amino)-2-oxoethyl)-5-(4-me-
thoxyphenyl)pentyl)phosphonate (13c): Prepared according to
general procedure V. Purification 3:1 hexane/acetone v/v; yield
1
57%; colorless oil. H NMR (300 MHz, CDCl3): dH =1.34–1.59 (m, 4H,
-CH2-), 1.73–2.04 (m, 2H, -CH2-), 2.26–2.65 (m, 5H, -CH2-, -CH-), 3.13
(s, 3H, N-CH3), 3.75 (s, 3H, PhOCH3), 4.72 (s, 2H, -NOCH2Ph), 4.88–
5.07 (m, 4H, -POCH2Ph), 6.78 (d, J=9.6 Hz, 2H, Ar-H), 7.00 (d, J=
9.6 Hz, 2H, Ar-H), 7.27–7.39 ppm (m, 15H, Ar-H); 13C NMR (75 MHz,
tert-Butyl 3-((bis(benzyloxy)phosphoryl)methyl)-6-(naphthalen-2-
yl)hexanoate (12i): Prepared according to general procedure IV.
1
Purification 2:1 hexane/EtOAc v/v; yield 49%; colorless oil. H NMR
1
CDCl3): dC =29.1, 29.9 (d, 2JCꢀP =4.3 Hz), 30.0, 30.1 (d, JCꢀP
=
(300 MHz, CDCl3): dH =1.39 (brs, 9H, tBu), 1.44–1.96 (m, 6H, -CH2-),
2.21–2.53 (m, 3H, -CH2-, -CH-), 2.71 (t, J=7.2 Hz, 2H, -CH2-), 4.91–
5.10 (m, 4H, -CH2-Ph), 7.25–7.49 (m, 13H, Ar-H), 7.56 (s, 1H, Ar-H),
7.73–7.83 ppm (m, 3H, Ar-H); 13C NMR (75 MHz, CDCl3): dC =28.0,
28.1, 29.9 (d, 1JCꢀP =139.1 Hz), 30.3 (d, 2JCꢀP =4.5 Hz), 34.2 (d,
3
3
136.9 Hz), 34.8 (d, JCꢀP =9.8 Hz), 35.1, 36.9 (d, JCꢀP =9.2 Hz), 55.5,
67.2 (d, 2JCꢀP =6.7 Hz), 67.3 (d, 2JCꢀP =6.1 Hz), 76.3, 113.9, 128.2,
3
128.5, 128.8, 128.9, 129.1, 129.5, 129.6, 134.7, 134.8 (d, JCꢀP
=
5.5 Hz), 136.7, 157.9, 173.5 ppm; 31P NMR (121.5 MHz, CDCl3): dP =
32.35 ppm; HRMS (ESI): calcd for C36H43NO6P [M+H]+: 616.2823,
found: 616.2830.
3
2
3JCꢀP =10.5 Hz), 35.9, 40.2 (d, JCꢀP =9.8 Hz), 66.9 (d, JCꢀP =5.8 Hz),
67.00 (d, 2JCꢀP =6.9 Hz), 80.3, 125.0, 125.8, 126.3, 127.2, 127.3,
2
127.5, 127.8, 127.9, 128.3, 128.5, 131.9, 133.5, 136.4 (d, JCꢀP
=
Dibenzyl (2-(2-((benzyloxy)(methyl)amino)-2-oxoethyl)-5-(3-me-
thoxyphenyl)pentyl)phosphonate (13d): Prepared according to
general procedure V. Purification 5:1 dichloromethane/EtOAc v/v;
yield 51%; colorless oil. 1H NMR (300 MHz, CDCl3): dH =1.35–1.61
6.2 Hz), 139.7, 171.6 ppm; 31P NMR (121.5 MHz, CDCl3): dP =
32.13 ppm; HRMS (ESI): calcd for C35H42O5P [M+H]+: 573.2764,
found: 573.2772.
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
9
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
(m, 4H, -CH2-), 1.72–2.07 (m, 2H, -CH2-), 2.27–2.67 (m, 5H, -CH2-,
-CH-), 3.12 (s, 3H, N-CH3), 3.75 (s, 3H, PhOCH3), 4.72 (s, 2H,
-NOCH2Ph), 4.87–5.07 (m, 4H, -POCH2Ph), 6.64–6.74 (m, 3H, Ar-H),
7.16 (t, J=7.9 Hz, 1H, Ar-H), 7.26–7.37 ppm (m, 15H, Ar-H);
(m, 6H, -CH2-), 2.29–2.64 (m, 3H, -CH2-, -CH-), 2.90–3.00 (m, 2H,
-CH2-), 3.12 (s, 3H, N-CH3), 4.68 (s, 2H, -NOCH2Ph), 4.88–5.05 (m,
4H, -POCH2Ph), 7.19–7.40 (m, 17H, Ar-H), 7.43–7.50 (m, 2H, Ar-H),
7.66–7.72 (m, 1H, Ar-H), 7.80–7.86 (m, 1H, Ar-H), 7.93–7.99 ppm
2
1
13C NMR (75 MHz, CDCl3): dC =28.4, 29.5 (d, JCꢀP =4.5 Hz), 29.6 (d,
(m, 1H, Ar-H); 13C NMR (75 MHz, CDCl3): dC =28.1, 29.9 (d, JCꢀP
=
3
3
2
3
1JCꢀP =138.4 Hz), 29.7, 34.5 (d, JCꢀP =10.3 Hz), 35.8, 36.6 (d, JCꢀP
=
138.4 Hz), 29.9 (d, JCꢀP =4.9 Hz), 33.2, 35.2 (d, JCꢀP =9.8 Hz), 36.9
9.0 Hz), 55.0, 66.8 (d, 2JCꢀP =6.7 Hz), 66.9 (d, 2JCꢀP =6.6 Hz), 76.0,
(d, 3JCꢀP =8.5 Hz), 67.2 (d, 2JCꢀP =6.7 Hz), 67.3 (d, 2JCꢀP =6.7 Hz),
110.9, 114.0, 120.7, 127.8, 128.2, 128.4, 128.6, 128.8, 129.1, 129.2,
76.3, 124.1, 125.6, 125.8, 125.9, 126.1, 126.7, 128.2, 128.5, 128.8,
3
3
3
134.5, 136.4 (d, JCꢀP =6.1 Hz), 136.4 (d, JCꢀP =6.1 Hz), 144.0, 159.5,
173.7 ppm; 31P NMR (121.5 MHz, CDCl3): dP =32.31 ppm; HRMS
(ESI): calcd for C36H43NO6P [M+H]+: 616.2823, found: 616.2831.
128.9, 129.0, 129.1, 129.5, 132.0, 134.1, 134.8, 136.7 (d, JCꢀP
=
6.4 Hz), 136.7 (d, 1JCꢀP =6.1 Hz), 138.8, 168.0 ppm; 31P NMR
(121.5 MHz, CDCl3): dP =33.49 ppm; HRMS (ESI): calcd for
C39H43NO5P [M+H]+: 636.2873, found: 636.2880.
Dibenzyl (2-(2-((benzyloxy)(methyl)amino)-2-oxoethyl)-5-(4-fluo-
rophenyl)pentyl)phosphonate (13e): Prepared according to gen-
eral procedure V. Purification 3:1 hexane/acetone v/v; yield 71%;
colorless oil. 1H NMR (300 MHz, CDCl3): dH =1.36–1.56 (m, 4H,
-CH2-), 1.71–2.02 (m, 2H, -CH2-), 2.23–2.50 (m, 5H, -CH2-, -CH-), 3.13
(s, 3H, N-CH3), 4.73 (s, 2H, -NOCH2Ph), 4.87–5.09 (m, 4H,
-POCH2Ph), 6.85–7.06 (m, 4H, Ar-H), 7.27–7.39 ppm (m, 15H, Ar-H);
Dibenzyl (2-(2-((benzyloxy)(methyl)amino)-2-oxoethyl)-5-(naph-
thalen-2-yl)pentyl)phosphonate (13i): Prepared according to gen-
eral procedure V. Purification 3:1 hexane/acetone v/v; yield 78%;
colorless oil. 1H NMR (300 MHz, CDCl3): dH =1.37–2.02 (m, 6H,
-CH2-), 2.26–2.75 (m, 5H, -CH-, -CH2-), 3.11 (s, 1H, N-CH3), 4.71 (s,
2H, -NOCH2Ph), 4.85–5.08 (m, 4H, -POCH2Ph), 7.21–7.34 (m, 16H,
2
13C NMR (75 MHz, CDCl3): dC =28.6, 29.6 (d, JCꢀP =4.3 Hz), 29.7 (d,
Ar-H), 7.36–7.47 (m, 2H, Ar-H), 7.53 (s, 1H, Ar-H), 7.70–7.81 ppm
3
2
3JCꢀP =10.0 Hz), 34.4 (d, 3JCꢀP =10.0 Hz), 34.9, 36.7 (d, JCꢀP
=
(m, 3H, Ar-H); 13C NMR (75 MHz, CDCl3): dC =28.7, 29.9 (d, JCꢀP
=
10.0 Hz), 37.0, 66.9 (d, 2JCꢀP =6.6 Hz), 67.0 (d, 2JCꢀP =6.3 Hz), 76.1,
3.9 Hz), 30.0 (d, JCꢀP =138.8 Hz), 32.9, 34.8 (d, JCꢀP =10.4 Hz), 36.2,
36.9 (d, JCꢀP =9.1 Hz), 67.2 (d, JCꢀP =6.6 Hz), 67.3 (d, JCꢀP =6.0 Hz),
1
3
114.9 (d, 2JCꢀF =21.8 Hz), 127.8 (d, 3JCꢀF =9.8 Hz), 127.9, 128.3, 128.6,
3
2
2
3
4
128.9, 129.2, 129.5, 134.5, 136.4 (d, JCꢀP =6.7 Hz), 137.9 (d, JCꢀF
=
76.3, 125.3, 126.0, 126.6, 127.5, 127.6, 127.8, 128.0, 128.2, 128.5,
4.2 Hz), 161.2 (d, 1JCꢀF =242.42 Hz), 173.4 ppm; 31P NMR
(121.5 MHz, CDCl3): dP =32.11 ppm; HRMS (ESI): calcd for
C35H40FNO5P [M+H]+: 604.2623, found: 604.2657.
128.8, 128.9, 129.1, 129.5, 132.2, 133.8, 134.8, 136.7 (d, JCꢀP
=
2
6.6 Hz), 136.8 (d, 2JCꢀP =6.2 Hz), 140.1, 170.4 ppm; 31P NMR
(121.5 MHz, CDCl3): dP =33.49 ppm; HRMS (ESI): calcd for
C39H43NO5P [M+H]+: 636.2873, found: 636.2880.
Dibenzyl (2-(2-((benzyloxy)(methyl)amino)-2-oxoethyl)-5-(3-fluo-
rophenyl)pentyl)phosphonate (13 f): Prepared according to gen-
eral procedure V. Purification 3:1 hexane/acetone v/v; yield 69%;
colorless oil. 1H NMR (300 MHz, CDCl3): dH =1.32–1.58 (m, 4H,
-CH2-), 1.69–2.03 (m, 2H, -CH2-), 2.25–2.53 (m, 5H, -CH2-, -CH-), 3.13
(s, 3H, N-CH3), 4.73 (s, 2H, -NOCH2Ph), 4.86–5.09 (m, 4H,
-POCH2Ph), 6.73–7.92 (m, 3H, Ar-H), 7.14–7.38 ppm (m, 16H, Ar-H);
Dibenzyl (5-([1,1’-biphenyl]-4-yl)-2-(2-((benzyloxy)(methyl)ami-
no)-2-oxoethyl)pentyl)phosphonate (13j): Prepared according to
general procedure V. Purification 97:3 dichloromethane/EtOAc v/v;
yield 68%; colorless oil. 1H NMR (300 MHz, CDCl3): dH =1.38–1.64
(m, 4H, -CH2-), 1.75–2.04 (m, 2H, -CH2-), 2.28–2.66 (m, 5H, -CH2-,
-CH-), 3.13 (s, 3H, N-CH3), 4.72 (s, 2H, NOCH2Ph), 4.89–5.07 (m, 4H,
-POCH2Ph), 7.11–7.19 (m, 3H, Ar-H), 7.25–7.60 ppm (m, 21H, Ar-H);
13C NMR (75 MHz, CDCl3): dC =28.5, 29.6 (d, 2JCꢀP =4.7 Hz), 30.3,
29.6 (d, 1JCꢀP =138.2 Hz), 34.5 (d, 3JCꢀP =10.2 Hz), 35.4, 36.6 (d,
2
13C NMR (75 MHz, CDCl3): dC =28.2, 29.6 (d, JCꢀP =4.1 Hz), 29.7 (d,
3
3
1JCꢀP =137.6 Hz), 31.6, 34.4 (d, JCꢀP =9.83 Hz), 35.5, 36.7 (d, JCꢀP
=
2
2
9.1 Hz), 66.9 (d, JCꢀP =6.7 Hz), 67.0 (d, JCꢀP =6.0 Hz), 76.1, 112.5 (d,
2JCꢀF =22.0 Hz), 115.1 (d, 2JCꢀF =22.0 Hz), 124.0 (d, 4JCꢀF =3.1 Hz),
127.9, 128.3, 128.5, 128.6, 128.9, 129.3, 129.5 (d, 3JCꢀF =8.6 Hz),
134.5, 136.4 (d, 3JCꢀP =6.1 Hz), 144.9 (d, 3JCꢀF =7.7 Hz), 162.8 (d,
1JCꢀF =244.7 Hz), 173.25 ppm; 31P NMR (121.5 MHz, CDCl3): dP =
33.43 ppm; HRMS (ESI): calcd for C35H40FNO5P [M+H]+: 604.2623,
found: 604.2656.
2
2
3JCꢀP =10.2 Hz), 66.9 (d, JCꢀP =6.1 Hz), 70.0 (d, JCꢀP =6.6 Hz), 76.0,
126.9, 127.9, 128.2, 128.5, 128.6, 128.7, 128.8, 128.9, 129.2, 134.5,
136.4 (d, 3JCꢀP =6.4 Hz), 138.5, 141.0, 141.5, 172.6 ppm; 31P NMR
(121.5 MHz, CDCl3): dP =32.33 ppm; HRMS (ESI): calcd for
C41H45NO5P [M+H]+: 662.3030, found: 662.3039.
Sodium hydrogen (2-(2-(hydroxy(methyl)amino)-2-oxoethyl)-5-
(p-tolyl)pentyl)phosphonate (6a): White powder. Prepared from
compound 13a (150 mg, 0.25 mmol) according to general proce-
Dibenzyl (2-(2-((benzyloxy)(methyl)amino)-2-oxoethyl)-5-(4-(tri-
fluoromethyl)phenyl)pentyl)phosphonate (13g): Prepared ac-
cording to general procedure V. Purification 3:1 hexane/acetone v/
1
dure VI; quantitative yield. H NMR (300 MHz, D2O): dH =1.29–1.78
1
v; yield 55%; colorless oil. H NMR (300 MHz, CDCl3): dH =1.34–1.59
(m, 6H, -CH2-), 1.94–2.41 (m, 4H, -CH-, Ph-CH3), 2.45–2.70 (m, 4H,
-CH2-), 3.01 (s, 5/6 of N-CH3), 3.23 (s, 1/6 of N-CH3), 6.89–7.23 ppm
(m, 4H, Ar-H); 13C NMR (75 MHz, D2O): dC =20.0, 27.6, 30.5 (d,
2JCꢀP =4.3 Hz), 31.6 (d, 1JCꢀP =130.7 Hz), 34.1 (d, 3JCꢀP =12.7 Hz),
34.5, 37.0, 39.2 (d, 1JCꢀP =7.1 Hz), 128.4, 128.9, 135.4, 139.8,
177.8 ppm; 31P NMR (121.5 MHz, D2O): dP =25.97 ppm; HRMS (ESI):
calcd for C15H23NO5P [MꢀH]ꢀ: 328.1319, found: 328.1320.
(m, 4H, -CH2-), 1.70–2.05 (m, 2H, -CH2-), 2.25–2.64 (m, 5H, -CH2-,
-CH-), 3.12 (s, 3H, N-CH3), 4.72 (s, 2H, -NOCH2Ph), 4.87–5.08 (m, 4H,
-POCH2Ph), 7.17 (d, J=8.2 Hz, 2H, Ar-H), 7.32 (m, 15H, Ar-H),
7.48 ppm (d, J=8.2 Hz, 2H, Ar-H); 13C NMR (75 MHz, CDCl3): dC =
2
1
28.2, 29.5 (d, JCꢀP =3.6 Hz), 29.7 (d, JCꢀP =138.2 Hz), 32.8, 34.4 (d,
3JCꢀP =10.1 Hz), 35.5, 36.7 (d, JCꢀP =9.5 Hz), 66.9 (d, JCꢀP =6.4 Hz),
67.0 (d, 2JCꢀP =6.6 Hz), 76.1, 124.5 (q, 1JCꢀF =272.7 Hz), 125.1 (q,
4JCꢀF =3.8 Hz), 127.9, 128.2 (q, 2JCꢀF =22.1 Hz), 128.3, 128.5, 128.6,
128.9, 129.2, 134.5, 136.4 (d, 3JCꢀP =6.3 Hz), 146.4, 173.8 ppm;
31P NMR (121.5 MHz, CDCl3): dP =33.43 ppm; HRMS (ESI): calcd for
C36H40F3NO5P [M+H]+: 654.2591, found: 654.2601.
3
2
Sodium hydrogen (2-(2-(hydroxy(methyl)amino)-2-oxoethyl)-5-
(m-tolyl)pentyl)phosphonate (6b): White powder. Prepared from
compound 13b (150 mg, 0.25 mmol) according to general proce-
1
dure VI; quantitative yield. H NMR (300 MHz, D2O): dH =1.22–1.69
(m, 6H, -CH2-), 1.99–2.23 (m, 1H, -CH-), 2.28 (s, 3H, Ph-CH3), 2.49–
2.68 (m, 4H, -CH2-), 3.18 (s, 5/6 of N-CH3), 3.35 (s, 1/6 of N-CH3),
7.02–7.15 (m, 3H, Ar-H), 7.23 ppm (app t, J=7.4 Hz, 1H, Ar-H);
Dibenzyl (2-(2-((benzyloxy)(methyl)amino)-2-oxoethyl)-5-(naph-
thalen-1-yl)pentyl)phosphonate (13h): Prepared according to
general procedure V. Purification 98:2 dichloromethane/MeOH v/v;
yield 44%; colorless oil. 1H NMR (300 MHz, CDCl3): dH =1.50–2.04
2
13C NMR (75 MHz, D2O): dC =20.5, 28.4, 32.2 (d, JCꢀP =4.1 Hz), 33.4
(d, 1JCꢀP =130.7 Hz), 35.3, 35.4 (d, 3JCꢀP =9.8 Hz), 36.2, 36.3 (d,
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
10
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
3JCꢀP =5.9 Hz), 125.8, 126.5, 128.7, 129.4, 138.7, 143.8, 174.4 ppm;
31P NMR (121.5 MHz, D2O): rotamers at dP =22.14 and 22.25 ppm;
HRMS (ESI): calcd for C15H23NO5P [MꢀH]ꢀ: 328.1319, found:
328.1318.
H), 7.61 ppm (d, J=7.9 Hz, 2H, Ar-H); 13C NMR (75 MHz, D2O): dC =
2
1
27.5, 29.5, 31.5 (d, JCꢀP =4.1 Hz), 32.8 (d, JCꢀP =130.7 Hz), 34.5 (d,
1
3JCꢀP =11.0 Hz), 34.9, 35.8, 36.3 (d, 3JCꢀP =6.8 Hz), 124.4 (q, JCꢀF
=
3
270.5 Hz), 125.0 (q, JCꢀF =4.1 Hz), 128.9 (app s), 147.6, 174.9 ppm;
31P NMR (121.5 MHz, D2O): dP =21.49 ppm; HRMS (ESI): calcd for
C15H20F3NO5P [MꢀH]ꢀ: 382.1037, found: 382.1039.
Sodium hydrogen (2-(2-(hydroxy(methyl)amino)-2-oxoethyl)-5-
(4-methoxyphenyl)pentyl)phosphonate (6c): White powder. Pre-
pared from compound 13c (125 mg, 0.20 mmol) according to gen-
Sodium hydrogen (2-(2-(hydroxy(methyl)amino)-2-oxoethyl)-5-
(naphthalen-1-yl)pentyl)phosphonate (6h): White powder. Pre-
pared from compound 13h (150 mg, 0.24 mmol) according to gen-
1
eral procedure VI; quantitative yield. H NMR (300 MHz, D2O): dH =
1.23–1.66 (m, 6H, -CH2-), 2.01–2.26 (m, 1H, -CH-), 2.47–2.67 (m, 4H,
1
-CH2-), 3.17 (s, 5/6 of N-CH3), 3.34 (s, 1/6 of N-CH3), 3.78 (s, 3H,
eral procedure VI; quantitative yield. H NMR (300 MHz, D2O): dH =
PhOCH3), 6.91 (m, 2H, Ar-H), 7.21 ppm (m, 2H, Ar-H); 13C NMR
1.38–1.84 (m, 6H, -CH2-), 2.02–2.27 (m, 1H, -CH-), 2.46–2.65 (m, 2H,
-CH2-), 3.07 (t, J=7.6 Hz, 2H, -CH2-), 3.13 (s, 5/6 of N-CH3), 3.24 (s,
1/6 of N-CH3), 7.39–7.62 (m, 4H, Ar-H), 7.78 (dd, J=2.4 Hz, 7.4 Hz,
1
(75 MHz, D2O): dC =28.3, 32.0 (d, 2JCꢀP =4.0 Hz), 33.3 (d, JCꢀP
=
3
3
129.9 Hz), 34.4, 35.1 (d, JCꢀP =10.7 Hz), 36.1, 36.4 (d, JCꢀP =6.2 Hz),
55.6, 114.1, 129.9, 136.2, 156.9, 175.0 ppm; 31P NMR (121.5 MHz,
D2O): dP =22.48 ppm; HRMS (ESI): calcd for C15H23NO6P [MꢀH]ꢀ:
344.1268, found: 344.1269.
1H, Ar-H), 7.92 (dd, J=2.4 Hz, 8.1 Hz, 1H, Ar-H), 8.18 ppm (d, J=
2
8.1 Hz, 1H, Ar-H); 13C NMR (75 MHz, D2O): dC =27.8, 32.1 (d, JCꢀP
=
1
3
4.5 Hz), 32.7, 33.8 (d, JCꢀP =129.3 Hz), 34.5, 35.7 (d, JCꢀP =10.8 Hz),
3
36.4 (d, JCꢀP =7.6 Hz), 36.6, 124.4, 126.1, 126.2, 126.3, 126.4, 128.8,
Sodium hydrogen (2-(2-(hydroxy(methyl)amino)-2-oxoethyl)-5-
(3-methoxyphenyl)pentyl)phosphonate (6d): White powder. Pre-
pared from compound 13d (150 mg, 0.24 mmol) according to gen-
131.6, 133.7, 139.7, 172.7 ppm; 31P NMR (121.5 MHz, D2O): rotamers
at dP =21.94, 22.18 ppm; HRMS (ESI): calcd for C18H23NO5P [MꢀH]ꢀ:
364.1319, found: 364.1315.
1
eral procedure VI; quantitative yield. H NMR (300 MHz, D2O): dH =
1.24–1.69 (m, 6H, -CH2-), 2.02–2.26 (m, 1H, -CH-), 2.51–2.66 (m, 4H,
-CH2-), 3.17 (s, 5/6 of N-CH3), 3.34 (s, 1/6 of N-CH3), 3.79 (s, 3H, Ph-
OCH3), 6.77–6.93 (m, 3H, Ar-H), 7.26 ppm (app t, J=7.9 Hz, 1H, Ar-
Sodium hydrogen (2-(2-(hydroxy(methyl)amino)-2-oxoethyl)-5-
(naphthalen-2-yl)pentyl)phosphonate (6i): White powder. Pre-
pared from compound 13i (150 mg, 0.24 mmol) according to gen-
2
1
H); 13C NMR (75 MHz, D2O): dC =28.0, 31.8 (d, JCꢀP =4.1 Hz), 33.1 (d,
eral procedure VI; quantitative yield. H NMR (300 MHz, D2O): dH =
3
3
1JCꢀP =130.2 Hz), 35.1 (d, JCꢀP =10.6 Hz), 35.4, 36.1, 36.5 (d, JCꢀP
=
1.23–1.80 (m, 6H, -CH2-), 2.01–2.29 (m, 1H, -CH-), 2.46–2.65 (m, 2H,
-CH2-), 2.78 (t, J=7.8 Hz, 2H, -CH2-), 3.12 (s, 5/6 of N-CH3), 3.30 (s,
1/6 of N-CH3), 7.44–7.55 (m, 3H, Ar-H), 7.77 (s, 1H, Ar-H), 7.84–
7.92 ppm (m, 3H, Ar-H); 13C NMR (75 MHz, D2O): dC =29.8, 31.8 (d,
2JCꢀP =3.7 Hz), 34.1 (d, 1JCꢀP =130.8 Hz), 35.0 (d, 3JCꢀP =10.9 Hz),
7.3 Hz), 55.4, 111.5, 114.2, 121.7, 129.8, 145.5, 159.0, 175.2 ppm;
31P NMR (121.5 MHz, D2O): dP =21.72 ppm; HRMS (ESI): calcd for
C15H23NO6P [MꢀH]ꢀ: 344.1268, found: 344.1269.
Sodium hydrogen (5-(4-fluorophenyl)-2-(2-(hydroxy(methyl)ami-
no)-2-oxoethyl)pentyl)phosphonate (6e): White powder. Prepared
from compound 13e (100 mg, 0.17 mmol) according to general
3
35.8, 36.5 (d, JCꢀP =8.0 Hz), 37.3, 125.5, 126.3, 126.4, 127.5, 127.7,
127.9, 128.1, 131.7, 133.5, 141.6, 169.7 ppm; 31P NMR (121.5 MHz,
D2O): dP =22.48 ppm; HRMS (ESI): calcd for C18H23NO5P [MꢀH]ꢀ:
364.1319, found: 364.1315.
1
procedure VI; quantitative yield. H NMR (300 MHz, D2O): dH =1.14–
1.68 (m, 6H, -CH2-), 1.92–2.20 (m, 1H, -CH-), 2.42–2.66 (m, 4H, P-
CH2-, CH2-CON-), 3.14 (s, 5/6 of N-CH3), 3.27 (s, 1/6 of N-CH3), 6.97–
7.09 (m, 2H, Ar-H), 7.20–7.33 ppm (m, 2H, Ar-H); 13C NMR (75 MHz,
D2O): dC =28.0, 31.4 (d, 2JCꢀP =4.5 Hz), 33.9 (d, 1JCꢀP =139.3 Hz),
34.6 (d, 3JCꢀP =8.1 Hz), 34.7, 36.3 (d, 3JCꢀP =8.3 Hz), 37.1, 114.7 (d,
2JCꢀF =21.2 Hz), 129.9 (d, 3JCꢀF =8.3 Hz), 139.2 (d, 4JCꢀF =3.7 Hz),
160.7 (d, 1JCꢀF =239.1 Hz), 169.0 ppm; 31P NMR (121.5 MHz, D2O):
rotamers at dP =21.40, 21.50 ppm; HRMS (ESI): calcd for
C14H21FNO5P [MꢀH]ꢀ: 332.1069, found: 332.1088.
Sodium hydrogen (5-([1,1’-biphenyl]-4-yl)-2-(2-(hydroxy(meth-
yl)amino)-2-oxoethyl)pentyl)phosphonate (6j): White powder.
Prepared from compound 13j (200 mg, 0.30 mmol) according to
general procedure VI; quantitative yield. 1H NMR (300 MHz, D2O):
dH =1.23–1.73 (m, 6H, -CH2-), 1.98–2.24 (m, 1H, -CH-), 2.42–2.70 (m,
4H, -CH2-), 3.14 (s, 5/6 of N-CH3), 3.30 (s, 1/6 of N-CH3), 7.35–7.53
(m, 5H, Ar-H), 7.58–7.71 ppm (m, 4H, Ar-H); 13C NMR (75 MHz,
D2O): dC =27.9, 31.5 (d, 2JCꢀP =4.1 Hz), 33.0 (d, 1JCꢀP =130.1 Hz),
34.5 (d, 2JCꢀP =10.1 Hz), 35.0, 36.3 (d, 3JCꢀP =8.38 Hz), 37.1, 126.7,
126.8, 127.4, 129.0, 129.3, 137.8, 140.4, 143.1, 169.2 ppm; 31P NMR
(121.5 MHz, D2O): rotamers at dP =21.37, 21.54 ppm; HRMS (ESI):
calcd for C20H25NO5P [MꢀH]ꢀ: 390.1476, found: 390.1479.
Sodium hydrogen (5-(3-fluorophenyl)-2-(2-(hydroxy(methyl)ami-
no)-2-oxoethyl)pentyl)phosphonate (6 f): White powder. Prepared
from compound 13 f (130 mg, 0.22 mmol) according to general
1
procedure VI; quantitative yield. H NMR (300 MHz, D2O): dH =1.23–
1.68 (m, 6H, -CH2-), 2.02–2.23 (m, 1H, -CH-), 2.50–2.69 (m, 4H,
-CH2-), 3.17 (s, 5/6 of N-CH3), 3.35 (s, 1/6 of N-CH3), 6.88–7.10 (m,
3H, Ar-H), 7.25–7.34 ppm (m,1H, Ar-H); 13C NMR (75 MHz, D2O):
X-ray crystallography: Protein was produced as described earli-
er.[23] The water-soluble ligands (6a, 6b, 6c, 6d, 6 f, 6g, 6h) were
incubated (1 mm final concentration) with the protein solution
(0.3 mm in 20 mm Tris·HCl, pH 7.8, 200–300 mm NaCl, 5% (v/v)
glycerol, 2 mm dithiothreitol and 1 mm MnCl2) for 10–15 min at
208C before the co-crystallization experiments were set up in 2-
well MRC plates (Molecular Dimensions, UK) with a Mosquito robot
(TTP Labtech, UK). Reservoir solutions consisted of 40 mL, and the
sitting droplets contained equal volumes (100 nL each) of the pro-
tein–ligand mixture and reservoir solution. Previous screening[23]
had shown the Morpheus screen[34] was highly effective for this
protein, and crystals (0.1ꢃ0.1ꢃ0.1 mm) appeared in 1–3 days at
208C under multiple conditions (Supporting Information Table S1).
Crystals were harvested without further cryoprotection, and
plunged into liquid nitrogen for transport to the relevant synchro-
tron beamline.
dC =27.9, 31.9 (d, 2JCꢀP =4.1 Hz), 33.1 (d, 1JCꢀP =129.1 Hz), 35.0 (d,
2
3JCꢀP =8.2 Hz), 35.1, 36.1, 36.4 (d, 3JCꢀP =7.0 Hz), 112.5 (d, JCꢀF
=
2
4
23.32 Hz), 115.3 (d, JCꢀF =21.9 Hz), 124.6 (d, JCꢀF =2.7 Hz), 130.1 (d,
3JCꢀF =8.7 Hz), 146.2 (d, 3JCꢀF =8.7 Hz), 162.9 (d, 1JCꢀF =247.1 Hz),
175.0 ppm; 31P NMR (121.5 MHz, D2O): dP =22.75 ppm; HRMS (ESI):
calcd for C14H20FNO5P [MꢀH]ꢀ: 332.1069, found: 332.1067.
Sodium hydrogen (2-(2-(hydroxy(methyl)amino)-2-oxoethyl)-5-
(4-(trifluoromethyl)phenyl)pentyl)phosphonate
(6g):
White
powder. Prepared from compound 13g (150 mg, 0.25 mmol) ac-
cording to general procedure VI; quantitative yield. 1H NMR
(300 MHz, D2O): dH =1.27–1.71 (m, 6H, -CH2-), 2.00–2.26 (m, 1H,
-CH-), 2.50–2.61 (m, 2H, -CH2-), 2.66 (t, J=7.4 Hz, 2H, -CH2-), 3.16 (s,
5/6 of N-CH3), 3.34 (s, 1/6 of N-CH3), 3.79 7.40 (d, J=7.9 Hz, 2H, Ar-
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
11
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
Diffraction data were collected at 100 K at the European Synchro-
tron Radiation Facility (ESRF, Grenoble, France) or at Diamond,
Oxford, England (Supporting Information Table S2). All crystals had
the symmetry of the triclinic space group P1, and could be classi-
fied into the same one of the two related groups of P1 cells dis-
cussed earlier.[23] Diffraction images were processed and scaled
with XDS[35] and SCALA[36] respectively, using the CCP4 package.[37]
Rigid-body refinement was used for initial placement of the struc-
tures, to maintain a similar position relative to that observed earli-
er. Structures were then subjected to alternating rounds of recipro-
cal space refinement with REFMAC5[38] and manual rebuilding with
O.[39] Solvent was added using the water tools in O. The ligands
and respective stereochemical restraints were built and generated
with the qds tools in O. Refinement restraints were then generated
from the fitted models by REFMAC5, and manually edited as
needed. Hydroxamate groups were restrained to planarity, and
metal-coordination target distances were taken from Harding.[40]
Complete data collection and refinement statistics are included in
Supporting Information Table S2.
versity. S.V.C. received support from FWO Flanders. R.C. received
a doctoral scholarship from the Special Research Fund of the
University of Gent. We thank An Matheeussen for running the in
vitro antiparasite evaluation, and Izet Karalic for technical assis-
tance.
Keywords: antibiotics
·
antiprotozoal
agents
·
oxidoreductases
relationships
·
structural biology
·
structure–activity
[1] World Malaria Report 2015, World Health Organization: http://
www.who.int/malaria/publications/world-malaria-report-2015/report/en/
(ISBN: 978 924 156 5158).
[2] H. Jomaa, J. Wiesner, S. Sanderbrand, B. Altincicek, C. Weidemeyer, M.
Hintz, I. Turbachova, M. Eberl, J. Zeidler, H. K. Lichtenthaler, D. Soldati, E.
Beck, Science 1999, 285, 1573–1576.
[3] B. M. Lange, T. Rujan, W. Martin, R. Croteau, Proc. Natl. Acad. Sci. USA
2000, 97, 13172–13177.
[4] T. Kuzuyama, Biosci. Biotechnol. Biochem. 2002, 66, 1619–1627.
[5] F. Rohdich, A. Bacher, W. Eisenreich, Biochem. Soc. Trans. 2005, 33, 785–
791.
[6] S. Takahashi, T. Kuzuyama, H. Watanabe, H. A. Seto, Proc. Natl. Acad. Sci.
USA 1998, 95, 9879–9884.
[7] M. Rohmer, Nat. Prod. Rep. 1999, 16, 565–574.
[8] T. Kuzuyama, T. Shimizu, S. Takahashi, H. Seto, Tetrahedron Lett. 1998,
39, 7913–7916.
[9] J. Zeidler, J. Schwender, C. Mꢄller, J. Wiesner, C. Weidemeyer, E. Beck, H.
Jomaa, H. K. Lichtenthaler, Z. Naturforsch. 1998, 53c, 980–986.
[10] T. Murakawa, H. Sakamoto, S. Fukada, T. Konishi, M. Nishida, Antimicrob.
Agents Chemother. 1982, 21, 224–230.
[11] S. Borrmann, I. Lundgren, S. Oyakhirome, B. Impouma, P. B. Matsiegui,
A. A. Adegnika, S. Issifou, J. F. J. Kun, D. Hutchinson, J. Wiesner, H.
Jomaa, P. G. Kremsner, Antimicrob. Agents Chemother. 2006, 50, 2713–
2718.
[12] M. Lanaspa, C. Moraleda, S. Machevo, R. Gonzꢅlez, B. Serrano, E. Macete,
P. Cisterꢆ, A. Mayor, D. Hutchinson, P. G. Kremsner, P. Alonso, C. Menꢀn-
dez, Q. Bassata, Antimicrob. Agents Chemother. 2012, 56, 2923–2928.
[13] E. R. Jackson, C. S. Dowd, Curr. Top. Med. Chem. 2012, 12, 706–728.
[14] I. Hale, P. M. O’Neill, N. G. Berry, A. Odom, R. Sharma, MedChemComm
2012, 3, 418–433.
[15] T. Masini, A. K. H. Hirsch, J. Med. Chem. 2014, 57, 9740–9763.
[16] L. M. Henriksson, T. Unge, J. Carlsson, J. ꢂqvist, S. L. Mowbray, T. A.
Jones, J. Biol. Chem. 2007, 282, 19905–19916.
Briefly, the structures of the seven new b-substituted enzyme–in-
hibitor complexes (6a, 6b, 6c, 6d, 6 f, 6g, 6h) were solved at reso-
lutions of 1.55, 1.8, 1.7, 1.7, 1.7, 1.6 and 1.4 ꢂ, respectively, and re-
fined to crystallographic R-factors of 18.6, 17.7, 18.2, 16.9, 18.2,
18.0 and 18.7% (Rfree values are 20.5, 20.4, 21.1, 20.3, 20.8 and
20.7%, respectively). Each complex has a dimer in the asymmetric
unit, with a manganese ion and ligand in each active site. Al-
though the new compounds were synthesized as racemic mixtures,
the high resolution of the study (e.g., Figure 4a) allowed us to
define the respective enantiomer of each ligand. The overall elec-
tron density is of high quality, and complete models of the
enzyme are deposited for residues 77–486 in each chain. Density
for residues in the active-site flap is discussed in the main text, and
described further in Supporting Information Table S3. Structural
comparisons were made with the lsq commands in O with close-
pair Ca cutoffs (1.0 ꢂ Ca–Ca separations).[41] Figures were created
in O, using secondary structure assignments from the yasspa algo-
rithm, and rendered in Molray.[42] Structures of the various com-
plexes were deposited at the RCSB Protein Data Bank as follows:
6a (5JMW), 6b (5JO0), 6c (5JBI), 6d (5JC1), 6 f (5JMP), 6g (5JNL)
and 6h (5JAZ). Electron density for each entry is available at the
Uppsala Electron Density Server.[43]
[17] A. Mac Sweeney, R. Lange, R. P. M. Fernandes, H. Schulz, G. E. Dale, A.
Douangamath, P. J. Proteau, C. Oefner, J. Mol. Biol. 2005, 345, 115–127.
[18] T. Umeda, N. Tanaka, Y. Kusakabe, M. Nakanishi, Y. Kitade, K. T. Naka-
mura, Sci. Rep. 2011, 1, 9.
[19] J. Xue, J. Diao, G. Cai, L. Deng, B. Zheng, Y. Yao, Y. Song, ACS Med.
Chem. Lett. 2013, 4, 278–282.
Supporting Information: Additional experimental details are pre-
sented, including comparisons of pIC50 for EcDxr and PfDxr, com-
parisons of pIC50 with crystallographic temperature factors and fit
to electron density, crystallization conditions used in the reported
X-ray structures, statistics for data collection and refinement, and
summary of electron density in residues of the flap.
[20] T. Masini, B. S. Kroezen, A. K. Hirsch, Drug Discovery Today 2013, 18,
1256–1262.
Abbreviations: Bn: benzyl, DOXP: 1-deoxy-d-xylulose 5-phosphate,
DMAP: 4-(dimethylamino)pyridine, Dxr: 1-deoxy-d-xylulose-5-phos-
phate reductoisomerase, EcDxr: Escherichia coli Dxr, EDC: N-(3-di-
methylaminopropyl)-N’-ethylcarbodiimide, MEP: 2-C-methyl-d-er-
ythritol-3-phosphate, PfDxr: Plasmodium falciparum Dxr, RMS: root
mean square, RT: room temperature, TFA: trifluoroacetic acid, THF:
tetrahydrofuran.
[21] S. A. Kholodar, G. Tombline, J. Liu, Z. Tan, C. L. Allen, A. M. Gulick, A. S.
Murkin, Biochemistry 2014, 53, 3423–3431.
[22] L. Deng, K. Endo, M. Kato, G. Cheng, S. Yajima, Y. Song, Med. Chem.
Lett. 2011, 2, 165–170.
[23] R. Chofor, S. Sooriyaarachchi, M. D. P. Risseeuw, T. Bergfors, J. Pouyez, C.
Johny, A. Haymond, A. Everaert, C. S. Dowd, L. Maes, T. Coenye, A. Alex,
R. D. Couch, T. A. Jones, J. Wouters, S. L. Mowbray, S. Van Calenbergh, J.
Med. Chem. 2015, 58, 2988–3001
[24] C. Bjçrkelid, T. Bergfors, T. Unge, S. L. Mowbray, T. A. Jones, Acta Crystal-
logr. Sect. D 2012, 68, 134–143.
[25] S. Jawaid, H. Seidle, W. Zhou, H. Abdirahman, M. Abadeer, J. H. Hix,
M. L. van Hoek, R. D. Couch, PLOS ONE 2009, 4, e8288.
[26] T. Verbrugghen, P. Cos, L. Maes, S. Van Calenbergh, J. Med. Chem. 2010,
53, 5342–5346.
[27] T. Haemers, J. Wiesner, S. Van Poecke, J. Goeman, D. Henschker, E. Beck,
H. Jomaa, S. Van Calenbergh, Bioorg. Med. Chem. Lett. 2006, 16, 1888–
1891.
Acknowledgements
We acknowledge funding from the European Community’s Sev-
enth Framework Programme (FP7/2007–2013) under BioStruct-X
(grant agreement no. 283570). T.A.J. and S.L.M. were supported
by the Swedish Research Council (VR), FORMAS, and Uppsala Uni-
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
12
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
[28] D. Giessmann, P. Heidler, T. Haemers, S. Van Calenbergh, A. Reichenberg,
J. Jomaa, C. Weidemeyer, S. Sanderbrand, J. Wiesner, A. Link, Chem. Bio-
diversity 2008, 5, 643–656.
[29] P. Cos, A. J. Vlietinck, D. Van den Berghe, L. Maes, J. Ethnopharmacol.
2006, 106, 290–302.
[30] R. J. Read, Acta Crystallogr. Sect. A 1986, 42, 140–149.
[31] L. Deng, J. Diao, P. Chen, V. Pujari, Y. Yao, G. Cheng, D. C. Crick, B. V. V.
Prasad, Y. Song, J. Med. Chem. 2011, 54, 4721–4734.
[37] M. D. Winn, C. C. Ballard, K. D. Cowtan, E. J. Dodson, P. Emsley, P. R.
Evans, R. M. Keegan, E. B. Krissinel, A. G. Leslie, A. McCoy, S. J. McNicho-
las, G. N. Murshudov, N. S. Pannu, E. A. Potterton, H. R. Powell, R. J.
Read, A. Vagin, K. S. Wilson, Acta Crystallogr. Sect. D 2011, 67, 235–242.
[38] G. N. Murshudov, A. A. Vagin, E. J. Dodson, Acta Crystallogr. Sect. D 1997,
53, 240–255.
[39] T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, Acta Crystallogr.
Sect. A 1991, 47, 110–119.
[32] M. Andaloussi, L. M. Henriksson, A. Wieckowska, M. Lindh, C. Bjorkelid,
A. M. Larsson, S. Suresh, H. Iyer, B. R. Srinivasa, T. Bergfors, T. Unge, S. L.
Mowbray, M. Larhed, T. A. Jones, A. Karlen, J. Med. Chem. 2011, 54,
4964–4976.
[33] A. M. Jansson, A. Wieckowska, C. Bjorkelid, S. Yahiaoui, S. Sooriyaarach-
chi, M. Lindh, T. Bergfors, S. Dharavath, M. Desroses, S. Suresh, M. Anda-
loussi, R. Nikhil, S. Sreevalli, B. R. Srinivasa, M. Larhed, T. A. Jones, A.
Karlꢀn, S. L. Mowbray, J. Med. Chem. 2013, 56, 6190–6199.
[34] F. Gorrec, J. Appl. Crystallogr. 2009, 42, 1035–1042.
[35] W. Kabsch, Acta Crystallogr. Sect. D 2010, 66, 125–132.
[36] P. Evans, Acta Crystallogr. Sect. D 2006, 62, 72–82.
[40] M. Harding, Acta Crystallogr. Sect. D 2006, 62, 678–682.
[41] G. J. Kleywegt, T. A. Jones, Methods in Enzymology, Vol. 277, Academic
Press, San Diego, 1997, 525–545.
[42] M. Harris, T. A. Jones, Acta Crystallogr. Sect. D 2001, 57, 1201–1203.
[43] G. J. Kleywegt, M. R. Harris, J. Y. Zou, T. C. Taylor, A. Wahlby, T. A. Jones,
Acta Crystallogr. Sect. D 2004, 60, 2240–2249.
Received: May 13, 2016
Revised: July 9, 2016
Published online on && &&, 0000
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
13
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

FULL PAPERS
S. Sooriyaarachchi, R. Chofor,
M. D. P. Risseeuw, T. Bergfors, J. Pouyez,
C. S. Dowd, L. Maes, J. Wouters,
T. A. Jones, S. Van Calenbergh,*
S. L. Mowbray*
Meta is better: X-ray structures show
that arylpropyl substituents on b-phe-
nylpropyl hydroxamate analogues of
fosmidomycin displace a key tryptophan
residue of the active site flap of Dxr,
which then becomes largely disordered.
For both meta- and para-substituted
compounds, smaller substituents are as-
sociated with better potency; meta-sub-
stituted compounds such as 6 f are usu-
ally better inhibitors, similar to the un-
substituted lead compound 5d.
&& – &&
Targeting an Aromatic Hotspot in
Plasmodium falciparum 1-Deoxy-d-
xylulose-5-phosphate
Reductoisomerase with b-Arylpropyl
Analogues of Fosmidomycin
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
14
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!