Highly potent bisphosphonate ligands for phosphoglycerate kinase

Article abstract of DOI:10.1021/jm970839y

We have synthesized a series of novel analogs of 1,3-bisphospho-D- glyceric acid, 1,3-BPG, and evaluated their binding to phosphoglycerate kinase, PGK (EC 2.7.2.3). Nonscissile methanephosphonic acids replace the two phosphate monoesters of 1,3-BPG and lead to several stable, tight-binding mimics of this intermediate species in glycolysis. Multiple fluorine substitution for hydrogen in the α-methylene groups of the phosphonic acid 1,3-BPG analogs markedly improves their binding to PGK as determined by NMR analysis. The best ligands bind some 50-100 times more strongly than does the substrate 3-phospho-D-glyceric acid and show a requirement for pK(a)³ to be generally below 6.0, while the presence of a β-carbonyl group seems to be of secondary importance.

Full text of DOI:10.1021/jm970839y

J . Med. Chem. 1998, 41, 4439-4452
4439
High ly P oten t Bisp h osp h on a te Liga n d s for P h osp h oglycer a te Kin a se†
David L. J akeman,‡ Andrew J . Ivory,‡ Michael P. Williamson,§ and G. Michael Blackburn*,‡
Krebs Institute, Department of Chemistry, University of Sheffield, Sheffield, S3 7HF, U.K., and Department of Biochemistry
and Biotechnology, University of Sheffield, Sheffield, S10 2TN, U.K.
Received December 12, 1997
We have synthesized a series of novel analogs of 1,3-bisphospho-D-glyceric acid, 1,3-BPG,3 and
evaluated their binding to phosphoglycerate kinase, PGK (EC 2.7.2.3). Nonscissile methane-
phosphonic acids replace the two phosphate monoesters of 1,3-BPG and lead to several stable,
tight-binding mimics of this intermediate species in glycolysis. Multiple fluorine substitution
for hydrogen in the R-methylene groups of the phosphonic acid 1,3-BPG analogs markedly
improves their binding to PGK as determined by NMR analysis. The best ligands bind some
50-100 times more strongly than does the substrate 3-phospho-D-glyceric acid and show a
3
requirement for pKa to be generally below 6.0, while the presence of a â-carbonyl group seems
to be of secondary importance.
Sch em e 1. Phosphate Transfer Catalyzed by PGK
In tr od u ction
Inhibition of the glycolytic enzyme phosphoglyceric
phosphokinase, PGK, leads to the conversion of 1,3-
bisphosphoglycerate, 1,3-BPG, into 2,3-BPG catalyzed
by bisphosphoglycerate mutase. Thus, when red blood
cells pass through inadequately oxygenated tissues they
produce increased amounts of 2,3-BPG. This increase
in 2,3-BPG causes a decrease in hemoglobin affinity for
oxygen which enables more oxygen to be extracted from
blood as it passes through the tissues. Thus inhibition
of PGK is a potential means for treatment of cardio-
vascular and respiratory disorders.1
PGK is the enzyme which equilibrates phosphate
transfer between position-1 of 1,3-BPG and the γ-phos-
phate of ATP (Scheme 1).2 It is the seventh enzyme in
the glycolytic pathway and is also utilized for carbon
dioxide fixation in plants. The cofactor for the reaction
is Mg2+, and the correct substrate for phosphoryl
transfer is MgATP2-. In the absence of magnesium,
ATP4- binds to the enzyme in a very different manner.3,4
PGK has been isolated from a wide variety of sources.
All forms are monomeric enzymes with molecular mass
around 45 kDa. There is a high degree of conservation
of primary protein structure,5,6 and their modes of
catalysis are similar. On the basis of the conformity of
the enzymes, Fifis and Scopes7 advanced the hypothesis
that the tertiary structure and active site regions of
PGK are conserved. The enzyme has two distinct
domains, corresponding to the N-terminal and C-ter-
minal portions of the enzyme, with the last 10 residues
of the C-terminus packed into the N-terminal domain.6
The N-terminal domain contains a “basic patch”
which is made up of several arginine and histidine
residues and provides the proposed binding site for
3-PGA or 1,3-BPG. The C-terminal domain binds the
nucleotide in a shallow hydrophobic cleft. The distance
between these substrates as bound to the enzyme was
modeled6 as approximately 11 Å for the “open form” of
PGK, which is too distant for direct phosphoryl transfer
between the substrates.8,9 In view of this bilobal shape
of PGK, it has been proposed10,11 that the two domains
swing together about a hinge region, closing down on
substrates and excluding water, and thereby effect
catalysis. Such a domain movement to generate a
“closed form” for the enzyme would support the sequen-
tial pathway for phosphoryl transfer demonstrated for
PGK.12 Many attempts to identify this “closed form” of
PGK by X-ray analysis have failed, and so it has been
sought by many other biophysical techniques. Sedi-
mentation experiments13 and small-angle scattering
experiments14,15 have given evidence for large-scale
structural changes, but the results are not conclusive.16
More recent X-ray data have still failed to identify the
fully closed state of the enzyme17 though dramatic
bending around the hinge region has been seen in a
ternary complex of Trypanosoma brucei PGK with ADP
and 3-PGA, which brings these noncomplementary
substrates 6 Å closer together than seen before and well
aligned for catalysis.18 Paramagnetic Cr3+ probes have
shown the close proximity of the substrates in the
ternary complex,19 while the kinetics of PGK have been
recently studied using a rapid quench flow apparatus,20
confirming the earlier findings that release of 1,3-BPG
is the slow, rate-determining step for the reaction.21
An early study on PGK using substrate analogs
demonstrated that 2-hydroxy-4-phosphono-DL-butyrate
is a substrate for PGK with a Km that is pH-dependent.
† Abbreviations: 1,3-BPG, 1,3-bisphospho-D-glyceric acid; 2,3-BPG,
2,3-bisphospho-D-glyceric acid; BPM, bisphosphoglycerate mutase;
DCM, dichloromethane; EDTA, ethylenediaminetetraacetic acid; LDA,
lithium diisopropylamide; 3-PGA, 3-phospho-D-glyceric acid; PGK,
yeast phosphoglyceric phosphokinase (EC 2.7.2.3); PMSF, phenyl-
methanesulfonyl fluoride; ppb, parts per billion; TRIS, tris(hydroxy-
methyl)aminomethane; ∆δmax, maximum change in chemical shift of
His residue.
2
When the pH is below pKa for the phosphonic acid, the
* To whom correspondence should be directed. Tel: +44 1142 22
94 62. Fax: +44 1142 73 86 73. E-mail: g.m.blackburn@sheffield.ac.uk.
‡ Department of Chemistry.
2
analog reacts slowly. At pHs above pKa , when the
analog is fully ionized, the kinetics observed are much
§ Department of Molecular Biology.
more comparable to those for 3-PGA.22 Further work
10.1021/jm970839y CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/15/1998

4440 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
J akeman et al.
Sch em e 2. Michaelis-Becker Syntheses of Some
controlled addition of HCl (0.1 M) to a stirred solution
of the test sample. The titration curves were plotted
in the range 3 < pH < 11 and pKa values calculated
from the experimental data. The pH range analyzed
precluded the determination by this method of values
Simple Bisphosphonate Estersa
1
2
for pKa and pKa for the bisphosphonates but supported
evaluation of pKa values separated by g0.3 units. For
the symmetrical and unsymmetrical bisphosphonates,
3
4
values of pKa and pKa were statistically corrected as
appropriate for the number of equivalent ionizable
protons and the number of equivalent phosphonate
dianions to support quantitative comparisons with 31P
chemical shift and IC50 data.
a
Reagents: (i) sodium, toluene, ∆; (ii) Br(CH2)5Br in ether, ∆,
2 h, 66%; (iii) (BrCH2CH2)2O, 150 °C, 16 h, 86%; (iv) Br(CH2)6Br,
150 °C, 16 h, 85%.
A plot of 31P chemical shift versus pKa is shown
3,4
with small analogs of 1,3-BPG has generally failed to
identify good inhibitors for PGK.23 More recently, two
keto-bisphosphonates have been studied as inhibitors
for the enzyme,24 and these compounds have been
included in the range of bisphosphonates that we have
prepared and evaluated as inhibitors of PGK.
(Figure 1). The linear regression curve has a correlation
coefficient of 0.92, and an unweighted linear fit was
found to be the best of a wide range of curve fits
attempted.
P r otein Dissocia tion Con sta n ts. Titration of the
analogs into PGK causes chemical shift changes in the
NMR spectrum. These changes are principally to
signals previously assigned as belonging to the basic
patch and, in particular, histidines-62, -167, and -170.
Dissociation constants were obtained from curve fitting,
as shown in Figure 2. Dissociation constants stronger
than 4 µM are hard to estimate accurately because of
lack of curvature in the data. The dissociation constants
for all analogs tested along with pKa data are presented
in Table 1. The ∆δmax is the average chemical shift
change for the histidine residues. The σ values are the
standard deviation calculated from the averages. When
the shifts of histidine residues were less than 10 ppb,
the value of Kd was not calculated because the shifts
were not sufficiently precise. As a result, only one set
of data was used to determine the Kd for 2.
The measurement of dissociation constants described
here builds on previous studies (reviewed in ref 5).
However, it is worth pointing out a significant difference
in the composition of the buffer used for the binding
studies. Previous work used 100 mM acetate, which
made it difficult to compare the results with those from
enzyme inhibition studies in which the buffer typically
consisted of 30 mM triethanolamine, 50 mM KCl, 40
mM (NH4)2SO4, and 0.2 mM EDTA.38 Therefore, it was
decided to use a buffer composition as close to that used
for the enzyme inhibition studies as possible, namely
10 mM triethanolamine and 40 mM KCl. The absence
of sulfate from the NMR buffer is significant: sulfate
acts as a competitive ligand at both the nucleotide
binding site and the phosphoglycerate binding site. At
low concentrations (e40 mM) it helps to displace 3-PGA
from the binding site, and hence increases the reaction
rate, and also converts the reaction into one that obeys
Michaelis-Menten kinetics.20 At higher concentrations
it is a competitive inhibitor of PGK. Addition of sulfate
to the buffer results in weaker binding and in many
cases makes it impossible to fit a dissociation constant
satisfactorily.
In our design of 1,3-BPG analogs, we have focused
on the achievement of full ionization of the phosphonic
acids, especially as effected by the use of R-fluorophos-
phonates. Our general analysis of the rectification of
the mismatch parameters in the use of alkanephospho-
nic acids as analogs of biological phosphates for this
purpose25 has been well exemplified by results in many
laboratories for analogs of nucleotides, phosphoamino
acids, glycerol phosphates, guanosine-5′-P analogs, and
the fluorophosphonate analog of 1,6-fructose bisphos-
phate, etc.26-34 The presence of a basic patch in PGK
at the site of binding 1,3-BPG makes it a suitable
enzyme for the evaluation of the contribution of anionic
charge to ligand binding. We have prepared a wide
range of the bisphosphonates synthesized in the present
work and determined their affinities for PGK to enable
us to demonstrate the superiority of R-fluoromethylene-
phosphonic acids over methylenephosphonic acids as
1,3-BPG analogs. This has both generated lead com-
pounds as possible drug candidates for respiratory
disorders and also identified symmetrical analogs of 1,3-
BPG with micromolar affinity for PGK that are suitable
for conversion into bisubstrate analogs for further use
in structural studies.
Resu lts
Syn th esis of 1,3-BP G An a logs. Chemical syntheses
were carried out as described below following Schemes
2-7. Standard procedures were employed for the
syntheses of phosphonate and R-difluoromethylenephos-
phonate esters.28 A range of methodologies was em-
ployed, each being represented in a separate scheme.
The final steps in each route were deesterifications
performed by hydrolysis in refluxing HCl (for robust
products) or by transesterification using bromotrimeth-
ylsilane followed by solvolysis with methanol for phos-
phonates that are sensitive to hot acid conditions.35 In
the case of ester (44), silylation required the use of
iodotrimethylsilane36 which was prepared in situ.37 The
free bisphosphonic acids were converted into their
sodium salts by titration to pH 7.1 with sodium hydrox-
ide solution followed by lyophilization and used in this
form for subsequent enzyme binding studies.
Discu ssion
We have synthesized a number of analogs of 1,3-BPG
and measured their dissociation constants against PGK
using NMR chemical shifts as a simple and direct
measure. It was not possible to measure the dissocia-
p Ka Deter m in a tion s. The bisphosphonic acids were
subjected to pH titration in the range pH 11 to 3 by the

Bisphosphonate Ligands for PGK
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4441
Sch em e 3. Michaelis-Becker Syntheses of Some gem-Difluoroalkanebisphosphonate Estersa
a
Reagents: (i) LDA, THF, -78 °C; (ii) 1,3-dibromopropane, 63%; (iii) bischloromethyl ether, 16%; (iv) 1,4-dibromobutane, 13%; (v)
1,4-dibromobutane, 26%; (vi) P(OiPr)3, 150 °C, 16 h, 50%.
Sch em e 4. Synthesis of 2-Aminoethylphosphonate
Sch em e 6. Syntheses of Amides of Phosphonodifluoro-
Ester and Its Amidesa
acetic Acid Estersa
a
Reagents: (i) PtO2‚xH2O, H2, MeOH; (ii) chloroacetic anhy-
dride, Et3N, DCM; (iii) P(OiPr)3, 130 °C, 73% (3 steps).
Sch em e 5. Syntheses of 2-Amino-1,1-difluoroethyl-
phosphonate Esters and Amidesa
a
Reagents: (i) oxalyl chloride, DCM, 16 h; (ii) Et3N, DCM, 0
°C, 23, 50%, 30, 89%.
Sch em e 7. Syntheses of Some 2-Oxoalkanephosphonate
Esters and of gem-Difluoro-2-oxoalkanephosphonatesa
a
Reagents: (i) Zn, CuBr, monoglyme; (ii) NH2NH2‚H2O, p-
TsOH; (iii) (ClCH2CO)2O, Et3N, DCM; (iv) P(OEt)3, 130 °C, 11%
(4 steps).
a
Reagents: (i) P(OiPr)3, 150 °C, 16 h, 33, 75%, 32, 78%; (ii)
tion constant of 1,3-BPG itself because of its very short
half-life in aqueous solution. However, the dissociation
constant of 3-PGA, which is a product of the reaction
under physiological conditions (and whose release is
normally the rate-limiting step of the reaction catalyzed
by PGK), could be measured. Many of the analogs have
dissociation constants much stronger than that of
3-PGA and are therefore potential drug candidates for
the treatment of respiratory and cardiovascular dis-
eases.
(iPrO)2P(O)CH3, LDA, -78 °C, 34, 33%, 36, 30%; (iii) n-BuLi,
iPr2NH, -60 °C, 15-30 min, (iPrO)2P(O)CF2H, -78 °C, 30 min,
38, 60%, 40, 47%.
here cast doubt on this view and support the more
traditional view that the basic patch is indeed part of
the active site in the active conformation because the
analogs described here, which were designed as analogs
of the substrate, bind well to the enzyme in the basic
patch and have no effect on signals in the region of helix-
14. Moreover, the better analogs bind more tightly to
the enzyme than does the substrate 3-PGA. Studies by
McHarg & Littlechild38 on some of these compounds
have shown that the Ki values for these compounds
when tested as inhibitors of PGK have the same order
of magnitude as the Kd values presented here in the
same conditions and that the compounds act approxi-
It has recently been proposed39 that there are two
binding sites for 3-PGA on PGK: the first in the basic
patch, as has previously been observed, and the second
next to the N-terminus of helix-14. It is further
proposed that the basic patch site primarily represents
the site of anion activation rather than the catalytically
active substrate binding site. The results presented

4442 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
J akeman et al.
evaluated by the comparison between the 3-oxapentane
4 and pentane 2 and the corresponding tetrafluoro
compounds 11 and 13. The oxa-substitution resulted
in a 2-fold and 5-fold weakening of binding to PGK in
these cases.
Th e Effect of a Ca r bon yl F u n ction a lity. To
assess the importance of recognition of the carbonyl
group in 1,3-BPG, several â-ketophosphonates were
examined. The 60-fold increase in affinity for pen-
tanonebisphosphonic acid 35 compared to pentane-
bisphosphonic acid 2 clearly established the strong
recognition of the 2-keto function in analogs of 1,3-BPG.
The effect also carries over to the butanone-
bisphosphonic acid 37, and we observe values for
dissociation constants for 35 and 37 comparable to the
Ki values reported by Byers.24 A similar enhancement
for insertion of a CdO group is seen in the correspond-
ing tetrafluoro species 44 compared to 13. Unexpect-
edly, the R,R-difluoropentanebisphosphonic acid 16
binds some 20-fold more strongly to PGK than does the
difluoropentanone 39. One possible explanation is that
such difluoroketones exist predominantly as covalent
hydrates, 2,2-dihydroxy species (as seen in their 13C and
31P NMR spectra), and this may affect their binding.
Alternatively, the gem-difluoro function may itself
provide some of the recognition element of the carbonyl
group.
F igu r e 1. Phosphorus NMR chemical shift vs phosphonate
pKa of 1,3-BPG analogs.
3,4
Am id e F u n ction a lity a s a Hyd r ogen Bon d Do-
n or . The functions explored thus far lack the hydrogen
bond donor capability of the 3-hydroxyl function of 1,3-
BPG. Some isoelectronic mimics of 1,3-BPG are the
reversed amides 45 and the related N-hydroxylamides
46 (Figure 3), the latter offering a nonchiral equivalent
to the D-glycerate 3-phosphate as well as suppressing
the possibility of enolization of the ketophosphonates
or of hydration of the fluoroketophosphonates. In the
event, attempts to prepare the N-hydroxylamides were
negated by the instability of the functionality, and we
were obliged to focus efforts exclusively on the simple
amides.
F igu r e 2. Titration data for His-167: (O) compound 13, (b)
compound 16. Solid lines represent the dissociation curves
calculated from data in Table 1.
mately as competitive inhibitors to 3-PGA but are not
competitive to ATP. This is further evidence against
binding at helix-14, since this is the binding site for the
γ-phosphate of ATP.
Only a 2-fold improvement in binding results from
incorporation of the amide functionality into analog 22
derived from pentanebisphosphonic acid 2, and it is
vastly inferior to the pentanone 35. Moreover, com-
parison of 22 with 37 suggests that the ketone func-
tionality gives better binding than the amide. Although
at first sight the weak binding of the R,R-difluoroketone
39 compared to that of the amide 24 appears to go
against this trend, the weakness of binding is probably
a consequence of the preference of 39 to exist in solution
as the diol, as discussed above. The diol is too large to
fit within the basic patch and consequently binds more
weakly because of adverse steric effects. The amide 24
is not hydrated to a diol in the same way and conse-
quently binds more tightly. A similar effect is observed
for the tetrafluoroamide 31, which is better than the
tetrafluoropentanone 44 and 35 times better than the
tetrafluoropentane 13.
From comparisons of the dissociation constants mea-
sured for different ligands, it is possible to draw conclu-
sions about the characteristics required for good binding
to PGK. These are presented below.
Effect of Ch a in Len gth in Bisp h osp h on a te Bin d -
in g to P GK. There is a clear preference for a five-atom
over a six-atom spacer between the two phosphonic
acids. 1,5-Pentanebisphosphonic acid 2 is a significantly
better inhibitor for PGK than is 1,6-hexanebisphospho-
nic acid 6, while the corresponding tetrafluoropentane
13 is some seven times better than the tetrafluorohex-
ane 18. Unexpectedly, this trend is continued for the
butanone-1,4-bisphosphonic acid 37 relative to the pen-
tanone-1,5-bisphosphonic acid 35, where there is a 10-
fold preference for the shorter chain, and also for the
corresponding 1,1-difluoro compounds 41 and 39, where
there is a 25-fold advantage for the shorter chain. These
data also support the conclusion that the optimization
of chain length in the separation of the two phosphonic
acids becomes more critical as binding becomes stronger
and that the degree of concentration of anionic charge
may be important for tight binding to PGK.
r-F lu or in a tion of th e P h osp h on a tes. R-Fluorina-
2
tion is well known to reduce pKa of a phosphonic
acid.25,27,28 If the binding of analogs of 1,3-BPG is
charge-dependent, then fluorinated analogs should show
stronger binding than their unfluorinated counterparts.
For all of the tetrafluoro analogs described here, there
To evaluate chain flexibility and polarity, the effect
of replacing a methylene group by an ether link was

Bisphosphonate Ligands for PGK
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4443
Ta ble 1. Dissociation Constants for Analogs of 1,3-BPG and 3-PGA Binding to PGK and Values of Phosphonic Acid Second
Dissociation Constants, pKa and pKa Corrected for Multiplicitya
3
4
a
3
pKa values were statistically corrected for the dissociation of a proton from two equivalent sites (∆pKa + log 2) and for return of a
3
4
proton to multiple equivalent oxyanions (∆pKa - log 2 and ∆pKa - log 2 or -log 4) as appropriate.
is a uniform trend that overwhelmingly supports this
hypothesis. Thus, 18 binds twice as well as hexanebi-
sphosphonic acid 6; 13 binds 10-fold better than pen-
tanebisphosphonic acid 2; 44 binds four times more

4444 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
J akeman et al.
data can be interpreted as describing a hyperbolic curve,
strongly suggesting that tight binding of the analogs to
PGK requires that at least one of the phosphonates is
3
doubly ionized, with pKa < 6. By contrast, a plot of
4
pKa against log[Kd] gives a random scatter (not shown)
which supports the unexpected conclusion that fourth
ionization of the bisphosphonic acid is not a prerequisite
for tight binding to PGK under the conditions of pH used
in these experiments.
F igu r e 3. General structures of amide and N-hydroxamic acid
analogs of 1,3-BPG.
strongly than pentanone 35; and 31 binds 140-times
more tightly than amide 22. Even the small diol 8 binds
strongly to PGK notwithstanding its small separation
of the two phosphonic acids, possibly as this gem diol
has many opportunities for hydrogen bonding.
This conclusion is illustrated by the example of
change in pKa in the series of analogs 22, 24, 29, and
31. The amide bisphosphonic acid 22 binds to PGK
rather weakly (Kd ) 0.67 mM). Bisfluorination at either
position adjacent to phosphorus increases binding over
100-fold (Kd ) 6 µM for 24 and 2 µM for 29). Further
fluorination to the tetrafluoroamide 31 does not improve
For the difluoro analogs, the situation is rather more
variable. Thus, the difluoropentane 16 outperforms the
tetrafluoropentane 13. The two difluoroamides 24 and
29 straddle the Kd for the tetrafluoroamide 31. Yet the
difluoroketone 39 is much inferior in binding to PGK
than the tetrafluoroketone 44. These differences may
be a consequence of different orientations of the unsym-
metrical bisphosphonates in the binding site as dis-
cussed below. But with only a single exception, all of
the difluoroketonebisphosphonic acids bind more strongly
to PGK than do the nonfluorinated counterparts. This
is most powerfully exemplified by the difluoroamide 29
where the introduction of two fluorines increases bind-
ing affinity to PGK by over 300 times. Clearly, this is
primarily a function of the state of ionization of the
phosphonic acids.
Effect of Ion ic Sta te of Bisp h osp h on a te An a logs.
While no attempt has been made to correct the analog
binding data to PGK for the state of ionization of the
phosphonic acids, it was evident from the outset that
the simple methylenephosphonic acids, e.g. 2, 6, 16, and
35, will be less than completely ionized at pH ∼7 while
the R,R′-tetrafluorophosphonic acids, e.g. 8, 13, 31, and
44, will be fully ionized under the binding conditions
used.
4
binding (Kd ) 4 µM) even though pKa falls significantly.
It is clear that some additional knowledge of the
orientation of the analogs in the “basic patch” binding
site is necessary to understand these binding events
more completely.
Such orientation of bisphosphonate analogs in this
“basic patch” has not hitherto appeared important.
However, the amide analogs, keto analogs, and 16 all
share the possibility of binding in two alternative
orientations. We initially supposed that the carbonyl
functionality would determine such orientation by bind-
ing in the same locus as the carbonyl group of 1,3-BPG.
However, the very tight binding for 29 suggests the
possibility that the choice of orientation for a bisphos-
phonate analog in the “basic patch” may be primarily
determined by a high affinity site for a phosph(on)ate
dianion and a lower affinity site for the second phosph-
(on)ate species, which is less critical of complete ioniza-
tion. We note that the nonfluorinated amide 12 binds
weakly while both its difluoromethylene analogs 24 and
29 bind strongly. This further implies that binding
affinity is determined primarily by the ionization state
of the phosph(on)ate and not by functionality within the
chain (i.e. the orientation of the amide has a much
smaller effect on binding affinity than the orientation
of the difluorophosphonate). From the data presently
available, it is impossible to conclude which orientation
exists for any particular unsymmetrical analog of 1,3-
BPG bound to PGK. Additional experimentation will
therefore be necessary to study this phenomenon.
To discern the significance of the state of ionization
on analog binding to PGK, we have explored possible
correlations of pKa with log[Kd] in a variety of ways.
First, we note that there is a good linear correlation for
pKa and pKa with the 31P NMR chemical shifts of the
corresponding phosphonate sodium salts (pH* 7.1) over
some 4 orders of magnitude (Figure 1, r ) 0.92). This
extends the correlation observed previously for a rather
limited range of halogenated methanephosphonic ac-
ids.40 Moreover, the linear fit to these data is superior
to any other simple correlation examined. It is perhaps
noteworthy that the data for 3-PGA diverge markedly
from this correlation.
3
4
3
Last, there is clear evidence that lowering of the pKa
for the bisphosphonic acids below 6 conveys little
additional benefit. In this work we have not prepared
any of the monofluorophosphonate analogs that might
support a more detailed examination of bisphosphonate
3
analogs with a pKa in the range 6-7 for the quantita-
Next, we have looked at ways of correlating pKa for
these analogs with log[Kd] for PGK binding. First, a
tive structure-activity relationship, partly for synthetic
reasons and partly to avoid the problems inherent in
working with mixtures of stereoisomers. However, the
PGK affinities for analogs 37, 41, and 44 are very
similar, and the latter two appear to derive no advan-
tage from being 10-fold more acidic through fluorination.
3
4
plot of both pKa and pKa against log[Kd] for binding to
PGK shows almost random scatter (Figure 4a, r ) 0.38).
3
4
Second, a plot of the average of pKa and pKa for each
bisphosphonate against log[Kd] for binding to PGK was
created to see whether the enzyme recognizes the state
of ionization for both phosphonates. The result gives a
poor linear correlation (Figure 4b, r ) 0.47) which
indicates that the enzyme does not recognize the state
of ionization of both phosphonates equally strongly.
Con clu sion
Several bisphosphonate analogs of 1,3-BPG have been
prepared whose affinity for PGK has been determined
by NMR analysis. The resulting Kd data have been used
to analyze the structural features of the atomic frame-
3
Third, a plot of pKa against log[Kd] shows a modest
linear correlation (Figure 4c, r ) 0.66). However, these

Bisphosphonate Ligands for PGK
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4445
3
4
F igu r e 4. Correlations of analog phosphonate pKa with -log[Kd] for binding to PGK. (a) Plot of -log[Kd] vs both pKa and pKa
3
4
for all bisphosphonate analogs. (b) Plot of -log[Kd] vs the average of pKa and pKa for all bisphosphonate analogs. (c) Plot of
3
-log[Kd] vs pKa for all bisphosphonate analogs.
suppression of the residual HDO resonance was applied during
the delay. The free induction decays were zero-filled to 16 k
complex points and the resolution enhanced by Gaussian
multiplication (gb ) 0.1, lb ) -10 Hz). Chemical shifts were
determined using acetone as internal reference (2.214 ppm).
Aliquots of 1,3-BPG analog solution (1-2 µL, 10 mM, D2O, at
pH* 7.1) were added to PGK samples prepared as above.
Spectra were recorded up to an analog to enzyme concentration
ratio of about 10:1. The pH* determination was repeated after
the titration to ensure that pH effects were not involved in
shifting the signals.
The change in chemical shifts of H-2 for His-62, His-167,
and His-170 versus the volume of analog solution added to a
known volume of enzyme solution was fitted using a least
squares program written by Dr. Chris Hunter (University of
Sheffield) for Apple Macintosh computers. The same results
were obtained by using a Microsoft Excel 5.0 worksheet written
to perform a least squares solver routine analysis. Both
work linking the two phosphonate functions primarily
responsible for optimum binding of these analogs to
PGK. The most effective analogs prove to have affinities
in the micromolar range. The dominant structural
parameters are the separation of the two phosphoryl
groups by a four- or five-atom spacer while the presence
of a â-carbonyl function appears to be nonessential other
3
than through its contribution to reducing pKa in some
analogs. In ionization terms, there is a clear require-
3
ment for pKa to be not greater than 6 and strong
indication that complete ionization of the second phos-
phonate is not essential. These results enable the
identification of high affinity, symmetrical bisphospho-
nate ligands for PGK capable of being incorporated into
bisubstrate analogs for PGK in pursuit of the closed
conformation of this kinase.
methods gave the same calculated values for Kd and ∆δmax
.
Dissociation constants were not determined when the change
in chemical shift was less than 10 ppb as data from the
spectrometer were insufficiently accurate.
Ch em ica l Syn th esis. Melting points were measured on
a Koffler hot stage micromelting point apparatus and are
uncorrected. Boiling points were recorded from a Kugelro¨hr
distillation apparatus and are uncorrected. Elemental analy-
ses were determined on a Perkin-Elmer 2400 CHN elemental
Exp er im en ta l Section
1H NMR Bin d in g Stu d ies. 3-Phospho-D-glyceric acid (3-
PGA) was purchased from Sigma as the trisodium salt, and
yeast phosphoglyceric phosphokinase (EC 2.7.2.3) was pur-
chased from Boehringer Mannheim for all experiments. Typi-
cally, phosphoglycerate kinase (1 mL, 10 mg) suspension of
an ammonium sulfate precipitate) was centrifuged (30 min,
3000g) to form a white pellet. The supernatant was removed,
and the pellet resuspended in an extraction buffer (2 mL, 0.1
M TRIS, 0.1 mM EDTA, 6 mM 2-mercaptoethanol, 10 µM
PMSF, 5% ammonium sulfate) in a Centricon micro-concen-
trator with a 30 000 molecular weight cutoff. The protein was
then concentrated by centrifuge (30 min, 3000g) to 0.5 mL.
The ammonium sulfate was removed and deuterium exchange
performed by suspending the protein 4 times in a deuterated
buffer (2 mL; 40 mM KCl, 10 mM triethanolamine, in D2O at
pH 7.1). It was then centrifuged to a volume of 0.5 mL. The
protein solution was split into two, the volume of each half
was made up to 500 µL with deuterated buffer, acetone (2 µL,
10% in D2O) was added, and the samples were adjusted to pH
analyzer. Infrared spectra were recorded upon
a FT-IR
Paragon 1000 spectrometer as thin films or potassium bromide
disks.
NMR spectra of analogs were recorded on a Bruker AC-250
spectrometer with a QNP probe, unless indicated otherwise
where a Bruker BMX-400 or AMX-500 instrument was used.
Spectra were recorded in CDCl3 solution unless otherwise
indicated. Coupling constants were measured in Hertz (Hz).
Spectra are reported using the δ scale in parts per million,
and proton chemical shifts are accurate to (0.01 ppm.
Phosphorus, fluorine, and carbon chemical shifts are accurate
to (0.1 ppm. Proton spectra have been calibrated by either
external tetramethylsilane or via internal solvent reference.
Phosphorus NMR spectra were recorded on the above ma-
chines at 101, 161, or 202 MHz, respectively, and are refer-
enced downfield from external 85% H3PO4. Fluorine NMR
spectra were recorded on the Bruker AC-250 or AMX-500 at
235 and 470 MHz, respectively, and are referenced downfield
from external CFCl3. Carbon NMR spectra were recorded on
the above machines at 62.9, 101, or 126 MHz, respectively,
and are referenced to external tetramethylsilane or via
internal solvent reference. All D2O spectra were referenced
7.1 ( 0.05 for NMR use. The sample concentration was
1%
1cm
measured by UV absorbance at 280 nm (A
) 4.9 mg mL-1
and a molar mass of 45 kDa).41
One-dimensional proton spectra were recorded at 300 K on
a Bruker AMX-500 spectrometer, using a spectral width of
12 500 Hz and 8 k complex data points. Between 320 and 360
scans were run, dependent on the protein concentration.
pulse angle of 90° (8-9 µs) was used with a relaxation delay
of 0.6-1.0 s between pulses. A presaturation pulse for
A

4446 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
J akeman et al.
to external 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). Mass
spectra were obtained on a Kratos MS80 mass spectrometer
in conjunction with a DS90 data station using chemical
ionization with ammonia as the reagent gas (CI) or electron
impact (EI). Fast atom bombardment (FAB) spectra were
obtained on a Kratos MS80RF machine together with the DS90
data station. Electrospray (ES) mass spectra were recorded
on a Fisons V.G. Platform instrument.
purified by dry column flash chromatography with gradient
elution (DCM-MeOH, from 100:0 to 95:5) to yield the title
compound as a colorless mobile oil (7.5 g, 86%): bp 190-195
3
°C/0.2 mmHg; δH 1.25 (24 H, d, J HH 7.0, 8 × CH3), 2.0 (4 H,
2
3
3
ddd, J HP 18.9, J HH 7.5, 8.1, 2 × CH2P), 3.57 (4 H, dt, J HP
3
3
3
9.0, J HH 7.5, 2 × CH2CH2P), 4.62 (4 H, octet, J HH 7.5, J HP
7.5, 4 × CH); δP 26.2 (2 P, s); δC 24.0, 23.9 (8 C, s, 8 × CH3),
2
28.1 (2 C, d, J PC 140.4, 2 × PCH2) 64.9 (2 C, s, 2 × CH2),
70.1, 70.0 (4 C, s, 4 × CH); m/z (CI+) 403 ([M + H]+, 15); (EI+)
403 ([M +H]+, 50). Found: C, 46.86;H, 8.82. C16H36O7P20.5H2O
requires C, 46.71; H, 9.06. Found: M+ 403.2015. C16H37O7P2
requires M, 403.2016.
Solvents were purified as follows. Tetrahydrofuran (THF),
diethyl ether (Et2O), and 1,2-dimethoxyethane (monoglyme)
were heated under reflux over potassium and benzophenone
and distilled immediately prior to use. Toluene was refluxed
over calcium hydride and distilled. Dichloromethane (DCM)
was heated under reflux with phosphorus pentoxide and
distilled. Triethylamine, diisopropylamine, and pyridine were
each heated under reflux with potassium hydroxide and
distilled. Methanol (MeOH), ethanol (EtOH), and 2-propanol
(iPrOH) were heated under reflux over magnesium and iodine;
once the iodine color disappeared further alcohol was added,
refluxed, and finally distilled. Petroleum ether (petrol, boiling
range ) 40/60 °C) and ethyl acetate (EtOAc) were distilled
from anhydrous potassium carbonate. Hexane was used
without distillation. Triisopropyl and triethyl phosphites were
stirred over sodium for 16 h and then distilled. Diisopropyl
and diethyl phosphites were distilled under nitrogen before
use. Diisopropyl difluoromethanephosphonate, diisopropyl
phosphonodifluoroethanoic acid, and diisopropyl phosphon-
odifluoromethylzinc bromide were prepared using literature
methods42-44 except for the use of toluene as solvent for
preparation of diisopropyl difluoromethanephosphonate.
TLC Rf values were obtained using Merck DC-Alufolien 60
F 254 TLC plates. Detection was by phosphomolybdic acid
(20% w/v in EtOH) dip developed by heating with a hot air
gun. Alternatively, UV or potassium permanganate spray
were used where applicable. All primary products were
homogeneous by TLC analysis on silica with solvent systems
DCM/MeOH (95:5) and hexane/EtOAc/AcOH (54:45:1). Flash
chromatography was performed on Kieselgel 60 230-400 mesh
(Merck 9385). Dry flash chromatography was performed on
Merck 60 GF254 mesh silica.
pKa values were calculated from the pH titration profile of
the free phosphonic acids, recorded using a Radiometer TTT
80 titrator, PHM 82 standard pH meter, GK2401B combined
glass electrode, ABU 80 autoburet, REC 80 Servograph, and
SAM 90 sample station. The data were analyzed using a
multiple pKa analysis program written for Kaleidagraph on
an Apple Macintosh Quadra giving an effective resolution of
∆pKa g 0.3 with an error of (0.05.
Experimental data, where applicable, are presented in the
following order: boiling point; melting point; Rf value; pKa
values; IR spectroscopy data; hydrogen NMR values; phos-
phorus NMR values; fluorine NMR values; carbon NMR
values; mass spectroscopy data; and quantitative analysis
results.
3-Oxa p en ta n e-1,5-bisp h osp h on ic Acid (4). Ester (3) (4.0
g, 10 mmol) was refluxed for 16 h in HCl (30 mL, 6 M). The
volatiles were evaporated in vacuo and the crude bisphospho-
nic acid coevaporated with MeOH (5 × 10 mL), triturated with
Et2O (20 mL), and finally lyophilized to yield the title
compound as a white crystalline solid (2.0 g, 85%): mp 121-
123 °C (lit46 mp 98-105); pKa 6.90, pKa 8.32; νmax(KBr)/cm-1
3
4
3
3
1230 (PdO); δH (D2O) 1.75 (4 H, dt, J HH 6.3, J HP 18.8, 2 ×
3
3
PCH2), 3.47 (4 H, dt, J HH 6.3, J HP 15.6, 2 × CH 2CH2P); δP
(D2O) 27.9 (1 P, s); m/z (FAB+) 235 ([M + H]+, 100%).
Tetr a isop r op yl Hexa n e-1,6-bisp h osp h on a te (5). This
was prepared as described45 and purified by Kugelro¨hr distil-
lation to yield the title compound as a colorless oil (6.16 g,
85%): bp 220-240 °C/0.5 mmHg (lit45 bp 185 °C/0.1 mmHg);
Rf (DCM-MeOH, 95:5) 0.2; δH 1.00-1.70 (36 H, m, 6 × CH2,
8 × CH3), 4.50-4.70 (4 H, m, CH); δP 30.7 (2 P, s); m/z (EI+)
414 (M+, 50). Found: M+ 414.2300. C18H40O6P2 requires M,
414.2297.
Hexa n e-1,6-bisp h osp h on ic Acid (6). Ester (5) (1.00 g,
2.42 mmol) was refluxed for 16 h in HCl (20 mL, 6 M).
Volatiles were evaporated in vacuo, and the crude bisphos-
phonic acid was triturated with Et2O (20 mL) and dried to yield
the title compound as a white crystalline solid (0.67 g, 99%):
mp 191-193 °C, (lit45 mp 206-208 °C); pKa 7.24; pKa 8.41;
3
4
ν
max(KBr)/cm-1 1245 (PdO); δH 1.25-1.80 (12 H, m, 6 × CH2);
δP (D2O) 32.9 (2 P,s); m/z (ES+) 247 ([M + H]+, 97). Found:
C, 29.35; H, 6.46. C6H16O6P2 requires C, 29.23; H, 6.55.
Tetr a isop r op yl 1,1,3,3-Tetr a flu or o-2,2-d ih yd r oxyp r o-
p a n e-1,3-bisp h osp h on a te (7). The title compound was
prepared as described48 as a colorless liquid (1.2 g, 10%): mp
3
61-62 °C; δH 1.37 (12 H, d, J HH 7.5, 4 × CH3), 1.42 (12 H, d,
3
3
3J HH 7.5, 4 × CH3), 4.80 (4 H, octet, J HP 7.5, J HH 7.5, 4 ×
CH); δP (AMX-500) 2.7-4.7 (m); δF -118.5 to -119.5 (m); m/z
(CI+) 488 (M+, 60). Found: C, 37.95; H, 6.28. C15H30F4O8P2
requires C, 37.82; H, 6.35.
1,1,3,3-Tetr a flu or o-2,2-d ih yd r oxyp r op a n e-1,3-bisp h o-
sp h on ic Acid (8). Ester 7 (2.0 g, 4.2 mmol) was refluxed for
16 h in HCl (20 mL, 6 M). The volatiles were evaporated in
vacuo and the crude bisphosphonic acid coevaporated with
MeOH (3 × 20 mL) and dried to yield the title compound as a
3
4
white hygroscopic glass (1.15 g, 95%): pKa 4.22; pKa 5.57;
2
ν
max(KBr)/cm-1 1230 (PdO); δP (D2O) 3.4 (2 P, t, J FP 91.8); δF
2 4
Tetr a eth yl P en ta n e-1,5-bisp h osp h on a te (1). This was
prepared as described45 to give the product, purified by
distillation to yield a colorless mobile oil (22.5 g, 65%): bp 174-
176 °C/0.01 mmHg (lit45 bp 180-181 °C/0.1 mmHg); δH 1.25
(D2O) -123.1 (4 F, dt, J FP 92.0, J FP 12.1).
Tetr a isop r op yl 1,1,5,5-Tetr a flu or o-3-oxa p en ta n e-1,5-
bisp h osp h on a te (10). LDA (25 mL, 2 M, 50 mmol) was
added dropwise to a solution of diisopropyl difluoromethane-
phosphonate (10.0 g, 46 mmol) in THF (60 mL) at -60 °C and
stirred for 1 h. The anion solution was then introduced by
cannula into a solution of bis(2-chloroethyl) ether (9) (2.65 g,
23 mmol) in THF (60 mL) at -60 °C and allowed to warm to
room temperature. The reaction mixture was then poured into
HCl (200 mL, 1 M) and extracted with DCM (3 × 100 mL).
The combined organic extracts were washed with brine (100
mL), dried (Na2SO4), and filtered, and the solvent was evapo-
rated in vacuo to yield a thick brown oil. This was purified
by dry column flash chromatography with gradient elution
(DCM-MeOH, from 100:0 to 95:5) which gave a mixture of
three compounds: tetraisopropyl difluoromethylenebisphos-
phonate, the desired product, and an unknown which all
coeluted. These were separated by repeated Kugelro¨hr distil-
lation taking the fraction boiling in the range 180-200 °C/1
mmHg, to yield the title compound as a pale-yellow oil (1.8 g,
3
(12 H, t, J HH 7.5, 4 × CH3), 1.35-1.76 (10 H, m, 5 × CH2),
3.95-4.10 (8 H, m, 4 × OCH2CH3); δP 32.5 (1 P, s); m/z (EI+)
344 (M+, 9). Found: M+ 344.1513. C13H30O6P2 requires M,
344.1514.
P en ta n e-1,5-bisp h osp h on ic Acid (2). Ester (1) (4.0 g, 12
mmol) was heated under reflux with HCl (30 mL, 6 M) for 10
h. The reaction mixture was cooled to room temperature and
coevaporated with methanol (3 × 50 mL). The white solid was
dried in a vacuum desiccator over phosphorus pentoxide for
16 h to yield white crystals (2.8 g, 96%): mp 154-156 °C, (lit.45
mp 155 °C); pKa 7.33, pKa 8.76; νmax(KBr)/cm-1 1220 (PdO);
δH (D2O) 1.30-1.65 (10 H, m, 5 × CH2); δP (D2O) 32.8 (s).
Tetr a isop r op yl 3-Oxa p en ta n e-1,5-bisp h osp h on a te (3).
Triisopropyl phosphite (11.2 g, 54 mmol) was heated with bis-
(2-bromoethyl) ether (5.0 g, 22 mmol) at 150 °C for 16 h under
argon.46 The reaction mixture was allowed to cool and was
3
4

Bisphosphonate Ligands for PGK
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4447
16%): δH 1.30 (12 H, d, 3J HH 7.0, 4 × CH3), 1.32 (12 H, d, 2J HH
108.6, 3J HF 19.6, CF2); m/z (EI+) 351 ([M + H]+, 12, 79Br), 353
([M + H]+, 12, 81Br).
3
3
7.0, 4 × CH3), 3.97 (4 H, dt, J HH 7.0, J HF 16.0, 2 × CF2CH2),
4.80 (4 H, octet, 3J HP 7.0, 3J HH 7.0, 4 × CH); δP 4.0 (2 P, t, 2J FP
Tetr a isop r op yl 1,1-Diflu or op en ta n e-1,5-bisp h osp h o-
n a te (15). Ester 14 (4.0 g, 11 mmol) was heated at 150 °C
with triisopropyl phosphite (2.37 g, 11 mmol) under argon for
16 h. The unreacted starting materials were evaporated by
Kugelro¨hr distillation (125 °C/0.2 mmHg), and the crude
bisphosphonate was purified by dry column flash chromatog-
raphy with gradient elution DCM-MeOH, from 100:0 to 99:
1, to give the title compound as a colorless oil (2.4 g, 50%): bp
2
100.1); δF -120.0 (4 F, d, J FP 100.1); δC 23.6, 23.7, 24.0, 24.1
2
2
(8 C, s, 8 × CH3), 71.5 (2 C, dt, J PC 18.0, J FC 24.3, 2 × CH2),
1
1
73.9, 74.0 (4 C, s, 4 × CH), 117.6 (2 C, dt, J PC 211.0, J FC
262.8, 2 × CF2); m/z (EI+) 474 (M+, 1.4).
1,1,5,5-Tet r a flu or o-3-oxa p en t a n e-1,5-b isp h osp h on ic
Acid (11). Ester 10 (1.79 g, 3.8 mmol) was refluxed for 16 h
in HCl (40 mL, 6 M). The volatiles were evaporated in vacuo,
and the crude bisphosphonic acid was coevaporated with
MeOH (3 × 10 mL) before being dissolved in water (10 mL),
extracted with DCM (3 × 10 mL), and lyophilized to yield the
title compound as white solid (1.12 g, 97%): mp 50-52 °C;
3
>200 °C/0.2 mmHg (decomp.); δH 1.20 (12 H, d, J HH 7.5, 4 ×
3
4
CH3), 1.28 (12 H, dd, J HH 7.5, J HH 3.8, 4 × CH3), 1.45-1.70
(6 H, m, CH2CH2CH2P), 1.80-2.10 (2 H, m, CF2CH2), 4.50-
3
3
4.67 (2 H, m, 2 × CH), 4.73 (2 H, octet, J HH 7.5, J HP 7.5, 2 ×
2
pKa 4.72; pKa 5.62; νmax(KBr)/cm-1 1290 (PdO); δH (D2O) 3.90
3
4
CH); δP 6.1 (1 P, t, J FP 108.9, PCF2), 30.1 (1 P, s, PCH2); δF
2
3
3
3
-113.5 (2 F, dt, J FP 109.0, J HF 19.7); δC 21.0-21.8 (1 C, m,
CF2CH2CH2), 23.6, 23.7, 24.0, 24.1 (8 C, s, 8 × CH3), 26.7 (1
C, d, 1J PC 142.6, CH2P), 33.3 (1 C, dt, 2J FC 21.1, 2J PC 14.4, CF2-
CH2), 69.7, 69.8, (2 C, s, 2 × CH2P(O)OCH), 73.3, 73.4 (2 C, s,
2 × CF2P(O)OCH), 122.3 (1 C, dt, 1J FC 259.2, 1J PC 217.3, CF2);
m/z (EI+) 437 ([M + H]+, 30). Found: C, 21.63; H, 4.84.
Requires C, 22.40; H, 4.51. Found: M+ 437.2033. C17H37F2O6P2
requires M, 437.2039.
(4 H, dt, J HF 14.4, J HP 5.0, 2 × CH2CF2); δP (D2O) 3.2 (2 P, t,
3
3
2J FP 91.3); δF (D2O) -122.4 (4 F, dt, J HF 15.7, J FP 91.2, 2 ×
CH2CF2).
Tetr aisopr opyl 1,1,5,5-Tetr aflu or open tan e-1,5-bisph os-
p h on a te (12). Diisopropyl difluoromethanephosphonate (10.0
g, 46 mmol) as a solution in THF (60 mL) was added dropwise
to a stirred solution of LDA (25 mL, 50 mmol, 2 M solution in
heptane/THF) at -60 °C under argon. After 30 min, the anion
solution was introduced by cannula into a stirred solution of
1,3-dibromopropane (4.66 g, 23 mmol) in THF (50 mL) at -60
°C and brought to room temperature. The reaction mixture
was then poured into HCl (200 mL, 1 M) and extracted with
DCM (3 × 100 mL). The combined organic extracts were
washed with brine (100 mL), dried (Na2SO4), and filtered, and
the solvent was evaporated in vacuo to yield a thick brown
oil. The crude bisphosphonate was further purified by dry
column flash chromatography with gradient elution DCM-
MeOH, from 100:0 to 98:2, to give the title compound as a
1,1-Diflu or op en ta n e-1,5-bisp h osp h on ic Acid (16). Es-
ter 15 (1.10 g, 2.6 mmol) was refluxed for 16 h in HCl (20 mL,
6 M). The volatiles were evaporated in vacuo, and the crude
bisphosphonic acid was dissolved in water (10 mL) and
extracted with DCM (3 × 10 mL) and EtOAc (2 × 10 mL).
Lyophilization furnished the title compound as white solid
3
4
(0.72 g, 99%): mp 158-160 °C; pKa 5.40 (CF2PO3H), pKa 7.78
(CH2PO3H); δH (D2O) 1.25-1.65 (6 H, m, PCH2CH2CH2), 1.65-
2
1.94 (2 H, m, CF2CH2); δP (D2O) 5.8 (1 P, t, J FP 101.3, PCF2),
3
3
32.5 (1 P, s, PCH2); δC (D2O) 21.2 (1 C, dd, J PC 5.0, J PC 18.4,
2
3
PCH2CH2CH2), 21.6 (1 C, d, J PC 4.5, PCH2CH2), 25.6 (1 C, d,
yellow oil (6.8 g, 63%): δH 1.30 (12 H, d, J HH 6.3, 4 × CH3),
1J PC 134.1, PCH2), 32.5 (1 C, dt, 2J FC 21.2, 2J PC 14.4, CH2CF2),
120.0 (1 C, dt, 1J FC 256.8, 1J PC 201.6, CF2), m/z (ES+) 269 ([M
+ H]+, 100).
1.33 (12 H, d, 3J HH 6.3, 4 × CH3), 1.77-1.90 (2 H, m, CH2CH2-
3
CH2), 1.92-2.20 (4 H, m, 2 × CF2CH2), 4.77 (2 H, octet, J HH
3
2
6.3, J HP 6.3, 2 × CH); δP 5.7 (2 P, t, J FP 108.2); δF -113.8 (4
2
3
3
F, dt, J FP 108.2, J HF 19.6, 2 × CF2); δC 12.6 (1 C, t, J FC 5.4,
CH2CH2CH2), 23.6, 23.7, 24.0, 24.1 (4 C, s, 4 × CH3), 33.2 (2
Tetr a isop r op yl 1,1,6,6-Tetr a flu or oh exa n e-1,6-bisp h os-
p h on a te (17). The Kugelro¨hr distillate from the preparation
of (14) was purified by dry column flash chromatography
eluting with DCM providing a pale-yellow oil which solidified
on standing to give the title compound as a waxy white solid
(3.0 g, 13%): bp > 200 °C/0.2 mmHg (decomp.); mp 54-56 °C;
2
2
C, dt, J FC 21.2, J PC 14.6, 2 × CH2CF2P), 73.4, 73.5 (4 C, s, 4
1
1
× CH), 120.1 (2 C, dt, J FC 259.6, J PC 217.6, 2 × CF2); m/z
(EI+) 473 ([M + H]+, 30). Found: M+ 473.1845. C17H35F4O6P2
requires M, 473.1848.
3
3
δH 1.30 (12 H, d, J HH 6.3, 4 × CH3), 1.33 (12 H, d, J HH 6.3, 4
1,1,5,5-Tetr aflu or open tan e-1,5-bisph osph on ic Acid (13).
Compound 12 (3.6 g, 7.6 mmol) was refluxed for 16 h in HCl
(30 mL, 6 M). The volatiles were evaporated in vacuo, and
the crude bisphosphonic acid was coevaporated with MeOH
(5 × 10 mL) before being triturated with Et2O (20 mL) to give
a brown solid. This was taken up in EtOAc, boiled with
activated charcoal, filtered, and dried to yield the title com-
× CH3), 1.50-1.68 (4 H, m, CF2CH2CH2CH2), 1.85-2.13 (4 H,
3
3
m, 2 × CH2CF2), 4.78 (4 H, octet, J HH 6.3, J HP 6.3, 4 × CH);
2
2
3
δP 5.8 (1 P, t, J FP 109.4); δF -113.4 (4 F, dt, J FP 109.9, J HF
19.7, 2 × CF2); δC 20.4, 20.5 (2 C, s, 2 × CF2CH2CH2), 23.6,
2
2
23.7, 23.9, 24.0 (8 C, s, 8 × CH3), 33.5 (1 C, dt, J FC 20.9, J PC
14.4, 2 × CF2CH2), 73.5, 73.6 (4 C, s, 4 × CH), 120.2 (1 C, dt,
1J FC 259.3, J PC 217.9, CF2); m/z (EI+) 487 ([M + H]+, 35).
1
3
pound as an off-white solid (2.0 g, 86%): mp 142-144 °C; pKa
5.09; pKa 5.87; νmax(KBr)/cm-1 1274 (PdO); δH (D2O) 1.15-
4
Found: C, 44.56; H, 7.66. Requires C, 44.45; H, 7.46.
1.35 (2 H, m, CH2CH2CH2), 1.35-1.70 (4 H, m, 2 × CF2CH2);
1,1,6,6-Tetr a flu or oh exa n e-1,6-bisp h osp h on ic Acid (18).
Ester 17 (3.0 g, 6.2 mmol) was refluxed for 16 h in HCl (30
mL, 6 M). The volatiles were evaporated in vacuo and the
crude bisphosphonic acid was coevaporated with MeOH (5 ×
10 mL) before being triturated with Et2O (20 mL) and dried
to yield the title compound as white powder (1.78 g, 91%): mp
δP (D2O) 5.5 (2 P, t, J FP 102.4); m/z (FAB+) 305 ([M + H]+,
2
100).
Diisop r op yl 1,1-Diflu or o-5-br om op en ta n e-1-p h osp h o-
n a te (14). Diisopropyl difluoromethanephosphonate (10 g, 46
mmol) as a solution in THF (60 mL) was added to a stirred
solution of LDA (25 mL, 50 mmol, 2 M solution in heptane/
THF) at -60 °C under argon. After 30 min, the anion solution
was introduced by cannula into a stirred solution of 1,4-
dibromobutane (9.76 g, 46 mmol) in THF (50 mL) at -60 °C
and slowly brought to room temperature. The reaction
mixture was then poured into HCl (200 mL, 1 M) and extracted
with DCM (3 × 100 mL). The combined organic extracts were
washed with brine (100 mL), dried (Na2SO4), and filtered, and
the solvent was evaporated in vacuo to yield a thick brown
oil. Kugelro¨hr distillation (90-100 °C/0.2 mmHg) gave the
crude bromophosphonate which was purified by dry column
flash chromatography eluting with DCM to yield the title
compound as a colorless mobile liquid (4.2 g, 26%): δH 1.30
(12 H, d, 3J HH 6.3, 4 ×CH3), 1.55-2.18 (6 H, m, CF2CH2CH2CH2),
198-201 °C; pKa 4.95, pKa 5.55; νmax(KBr)/cm-1 1250 (PdO);
δH 1.45-1.70 (4 H, m, 2 × CH2CH2CF2), 1.80-2.15 (4 H, m, 2
3
4
2
× CF2CH2); δP (D2O) 6.0 (2 P, t, J FP 100.6); m/z (FAB+) 319
([M + H]+, 100). Found C, 21.62; H, 4.24. C6H16F4O6P2
requires C, 22.66; H, 3.80.
Dieth yl 2-Am in oeth yl-1-p h osp h on a te (19). Adams cata-
lyst (PtO2 × H2O) (0.1 g, 10 wt %) was covered with ethanol
(20 mL), and diethyl cyanomethylphosphonate (1.0 g, 5.64
mmol) was added as a solution in ethanol (40 mL) followed by
HCl (1 mL, concentrated). The reaction was stirred under
hydrogen (1 atm) until gas uptake was complete (275 mL). The
solution was filtered through Celite and the solvent evaporated
in vacuo. The resulting white crystals were dissolved in DCM
(30 mL) and washed with NaOH (30 mL, 1 M). The DCM layer
was dried (MgSO4), filtered, and evaporated in vacuo to yield
the title compound as a colorless mobile oil (1.0 g, 98%): δH
3
3
3
3.38 (2 H, t, J HH 6.8, CH2Br), 4.80 (2 H, octet, J HH 6.3, J HP
6.3, 2 × CH); δP 5.6 (1 P, t, 2J FP 108.5); δF -113.2 (2 F, dt, 2J FP

4448 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
J akeman et al.
3
1.3 (6H, t, J HH7.5, 2 × CH3), 1.55 (2H, bs, CH2NH2), 1.9 (2H,
gradient of DCM-MeOH (from 100:0 to 85:15). This gave the
title compound as a clear oil (11.2 g, 50%): Rf (DCM-MeOH,
2
3
3
3
dt, J HP 18.8, J HH 7.5, PCH2), 2.8 (2H, dt, J HP 15.8, J HH 7.5,
3
3
3
CH2NH2), 4.05 (4H, qn, J HH 7.5, J HP 7.5, 2 × CH3CH2); δP
30.8 (1 P, s). HCl salt analysis: Found: C, 31.89; H, 7.98; N,
6.51. C6H17ClNO3P0.5H2O requires C, 31.82; H, 7.57; N,
6.12%.
95:5) 0.55; δH 1.30 (6 H, t, J HH 7.5, 2 × CH2CH3), 1.35 (12 H,
3
3
3
d, J HH 7.5, 4 × CHCH3), 2.00 (2 H, dt, J HP 18.0, J HH 7.5,
CH2P), 3.60 (2 H, dq, 3J HP 18.0, 3J HH 7.5, NHCH2CH2P), 4.00-
4.20 (4 H, m, 2 × CH2CH3), 4.85 (2 H, octet, 3J HP 7.5, 3J HH 7.5,
2
2 × CH), 7.37 (1 H, bt, NH); δP 1.9 (1 P, t, J FP 96.2, CF2P),
Diet h yl 1-Ch lor o-2-oxo-3-a za p en t a n e-5-p h osp h on a t e
(20). Triethylamine (8.36 mL, 60 mmol) was added dropwise
to a solution of chloroacetic anhydride (6.30 g, 36 mmol) and
19 (6.51 g, 30 mmol, as hydrochloride salt) in DCM (150 mL,
degassed) at 0 °C under argon. After 4 h the reaction mixture
was poured into dilute NaOH (200 mL, 1 M) and extracted
with DCM (2 × 100 mL). The combined organic layers were
washed with HCl (2 × 50 mL, 1 M), dried (Na2SO4), filtered,
and evaporated in vacuo to yield the title compound as an
orange oil (3.00 g, 40%): Rf (DCM-MeOH, 95:5) 0.36; δH 0.95
2
29.0 (1 P, s, PCH2); δF -118.1 (2 F, d, J FP 96.0, CF2P); m/z
(EI+) 423 (M+, 1). Found: C, 39.39; H, 7.1; N, 3.34. C14H29F2-
NO7P2 requires C, 39.72; H, 6.9; N, 3.31. Found: M+ 423.1387.
C14H29F2NO7P2 requires M, 423.1386.
1,1-Diflu or o-2-oxo-3-azapen tan e-1,5-bisph osph on ic Acid
(24). Bromotrimethylsilane (2.5 mL, 19 mmol) was added
dropwise to a solution of ester 23 (1.10 g, 2.6 mmol) in DCM
(25 mL) under argon. After 48 h, 31P NMR indicated the
2
reaction had reached completion, -14.7 (1 P, t, PCF2CO, J FP
3
3
3
(6 H, t, J HH 6.3, 2 × CH3), 1.65 (2 H, dt, J HH 6.5, J HP 18.8,
99.7), 12.5 (1 P, s, PCH2CH2). The volatiles were evaporated
in vacuo, and the resulting gum was solvolyzed by MeOH (3
× 15 mL). The bisphosphonic acid was then dissolved in water
(15 mL) and titrated to pH 7.1 with NaOH (1 M). Extraction
with DCM (2 × 50 mL) and EtOAc (2 × 50 mL) followed by
lyophilization furnished the title compound as a white solid
3
3
CH2P), 3.17 (2 H, dq, J HH 6.5, J HP 18.0, CH2CH2P), 3.63 (2
H, s, CH2Cl), 3.60-3.73 (4 H, m, 2 × CH2CH3), 7.23 (1 H, bt,
NH); δP 29.4 (1 P, s); δC 16.2, 16.3 (2 C, s, 2 × CH3), 25.2 (1 C,
1
d, J PC 140.1, CH2P), 33.7 (1 C, s, CH2NH), 42.4 (1 C, s, CH2-
Cl), 61.7, 61.8 (2 C, s, 2 × CH2CH3), 166.0 (1 C, s, CO); m/z
(EI+) 257 (M+, 15, 35Cl), 259 (M+, 5, 37Cl). The acidic
washings were evaporated in vacuo to yield diethyl 2-amino-
ethyl-1-phosphonate (19) hydrochloride (3.9 g).
3
4
(1.38 g, 90%): pKa 4.71 (PCF2); pKa 7.14 (PCH2); νmax(KBr
disk)/cm-1 1670 (CdO), 1240 (PdO); δH (D2O) 1.90-2.07 (2 H,
2
m, J HP 17.5, PCH2), 3.40-3.60 (2 H, m, PCH2CH2); δP (D2O)
1.5 (1 P, t, 2J FP 88.0, PCF2), 23.9 (1 P, s, PCH2); δF (D2O) -120.0
P 1-Diisop r op yl P 5-Dieth yl 2-Oxo-3-a za p en ta n e-1,5-bis-
p h osp h on a te (21). Ester (20) (2.54 g, 10 mmol) was heated
for 16 h at 130 °C with triisopropyl phosphite (4.9 mL, 20
mmol) under argon. Volatiles were evaporated under reduced
pressure (40 °C/1 mmHg), and the crude bisphosphonate (3.2
g) was purified by dry column flash chromatography with
gradient elution DCM-MeOH, from 100:0 to 50:50, to yield
the title compound as a yellow oil (2.97 g, 77%); Rf (DCM-
MeOH, 95:5) 0.56; δH 1.00-1.20 (18 H, m, 6 × CH3), 1.75 (2
2
1
(2 F, d, J FP 88.0, CF2); δC (D2O) 26.8 (1 C, d, J PC 132, CH2-
1
1
PO), 34.6 (1 C, s, CH2CH2PO), 113.9 (1 C, dt, J PC 182, J FC
2
2
268, CF2), 165.4 (1 C, dt, J PC 14, J FC 25, CO); m/z (FAB+)
372 ([M + 4Na + H]+, 6), 350 ([M + 3Na + H]+, 13), 328 ([M
+ 2Na + H]+, 5); (ES+) 371 ([M + 4Na]+, 25), 350 ([M + 3Na]+,
50), 328 ([M + 2Na]+, 56), 306 ([M + Na]+, 56).
Diisop r op yl 1,1-Diflu or o-2-N-p h t h a lim id oet h a n e-1-
p h osp h on a te (25). Diisopropyl phosphonodifluoromethyl
zinc bromide was prepared using diisopropyl difluorobro-
momethanephosphonate (10 g, 34 mmol). After addition of
CuBr (0.1 g, 0.7 mmol), bromomethylphthalimide (8.4 g, 35
mmol) was added as a powder at room temperature and stirred
for 3 d. The reaction mixture was then poured into water (100
mL) and extracted with DCM (3 × 60 mL). The organic layer
was dried (Na2SO4), filtered, and evaporated in vacuo to yield
a yellow oil. The crude product was purified by dry column
flash chromatography with gradient elution of DCM-petrol
(from 7:3 to 1:0) to yield the title compound as a soft white
3
3
2
H, dt, J HH 6.3, J HP 18.8, CH2CH2P), 2.57 (2 H, d, J HP 21.9,
COCH2P), 3.75-3.40 (4 H, m, 2 × CH2CH3), 4.35-4.55 (2 H,
m, 2 × CH), 7.22 (1 H, bt, NH); δP 20.8 (1 P, s, PCH2CO), 29.4
(1 P, s, PCH2CH2); m/z (EI+) 387 (M+, 22). Found: M+
387.1576. C14H31NO7P2 requires M, 387.1572.
2-Oxo-3-a za p en ta n e-1,5-bisp h osp h on ic Acid (22). Bro-
motrimethylsilane (1.12 mL, 8.4 mmol) was added dropwise
to a solution of ester (21) (467 mg, 1.2 mmol) in DCM (20 mL)
under argon. After 24 h, 31P NMR indicated the reaction had
reached completion; 7.0 (1 P, s, PCH2CO) and 14.0 (1 P, s,
PCH2CH2). The volatiles were evaporated in vacuo and the
resulting gum solvolyzed with MeOH (3 × 15 mL). The
bisphosphonic acid was dissolved in water (15 mL) and titrated
to pH* 7.1 with NaOH (1 M). Extraction with DCM (2 × 50
mL) and EtOAc (2 × 50 mL) followed by lyophilization
furnished the title compound as a hygroscopic glass (312 mg,
3
solid (6.38 g, 50%): mp 44-46 °C; δH 1.37 (12 H, d, J HH 6.5,
2
2
4 × CH3), 4.25 (2 H, dt, J HP 3.0, J HF 16.5, CH2), 4.87 (2 H,
octet, 2J HH 6.5, 2J HP 6.5, 2 × CH), 7.77 (4 H, m, ArH); δP 3.4 (1
3
3
P, t, J FP 100.0); δF -116.3 (2 F, d, J FP 100.0); δC 23.6, 23.7,
24.1, 24.2 (4 C, s, 4 × CH3), 39.7 (1 C, dt, 2J FC, 2J FC 22.8, CH2-
1
CF2), 74.4, 74.5 (2 C, s, 2 × CH), 116.9 (1 C, dt, J PC 212.3,
1J FC 264.2, CH2CF2), 123.6, 131.9, 134.3 (6 C, s, 6 × ArC), 167.2
(2 C, s, 2 × CO); m/z (EI+) 375 (M, 0.1); (CI+) 375 (M, 25).
89%); νmax(KBr)/cm-1 1635 (CdO), 1240 (PdO); pKa 5.62
3
4
(PCH2COCH2PO3H); pKa 6.60 (CH2PO3H); δH (D2O) 1.65-1.83
2
(2 H, m, PCH2CH2), 2.55 (2 H, d, J HP 18.8, PCH2O), 3.35 (2
Diisop r op yl 1,1-Diflu or o-2-a m in oet h a n e-1-p h osp h o-
n a te (26). Hydrazine hydrate (1.63 g, 51 mmol) was added
dropwise to a solution of 25 (6.38 g, 17 mmol) in DCM (30 mL).
After 1 h, water (30 mL) and p-toluenesulfonic acid (until pH
3) were added. The two phases were filtered and then
separated, and the organic layer was extracted with water (3
× 30 mL). The aqueous fractions were combined, basified with
NaOH (until pH 10), extracted with DCM (3 × 30 mL), dried
(Na2SO4), and evaporated in vacuo to yield a colorless oil. The
product was further purified by Kugelro¨hr distillation to give
a colorless oil (2.92 g, 70%): bp 120-125 °C/0.2 mmHg; δH
1.27 (6 H, d, 3J HH 6.3, 2 × CH3), 1.42 (2 H, bs, NH2), 1.32 (6 H,
H, q, 3J HH 6.7, 3J HP 6.7, PCH2CH2); δP (D2O) 13.0 (1 P, s, PCH2-
CO), 20.7 (1 P, s, PCH2CH2); δC 28.3 (1 C, d, PCH2CH2), 35.7
1
(1 C, s, PCH2CH2), 36.6 (1 C, d, J PC 114.5, PCH2O), 117.1 (1
C, s, CO); m/z (ES-) 290 ([M + 2Na - H]+, 5); 268 ([M + Na
- H]+, 78), 246 ([M - H]+, 100).
P 1-Diisop r op yl P 5-Diet h yl 1,1-Diflu or o-2-oxo-3-a za -
p en ta n e-1,5-bisp h osp h on a te (23). Oxalyl chloride (20.7 g,
165 mmol, freshly distilled) was syringed slowly into a solution
of diisopropyl phosphonodifluoroethanoic acid (13.9 g, 54
mmol) in DCM (50 mL) under nitrogen at 0 °C. The reaction
mixture was stirred at room temperature for 16 h. The
volatiles were evaporated under reduced pressure (0.2 mmHg).
Compound 19 (11.4 g, 53 mmol, as its HCl salt) was added at
-20 °C (ice/NaCl) followed by dropwise addition of triethyl-
amine (10.9 g, 108 mmol). The reaction was stirred 5 h, and
the volatiles were evaporated in vacuo. The residue was
dissolved in Et2O (40 mL, dry) and filtered, and the organic
layer was washed with citric acid (100 mL, 20%), NaHCO3 (100
mL, 1 M), and brine (100 mL) and then dried (Na2SO4). The
solvent was evaporated in vacuo, and the crude product (17.9
g) was purified by dry column flash chromatography with a
2
2
2
d, J HH 6.3, 2 × CH3), 3.12 (2 H, dt, J HP 7.0, J HF 16.5, CH2),
4.80 (2 H, octet, 3J HH 6.3, 3J HP 6.3, 2 × CH); δP 5.6 (1 P, t, 3J FP
2
2
106.3); δF -120.7 (2 F, dt, J FP 106.3, J HF 17.0, CF2); δC 23.7,
2
2
23.8, 24.1, 24.1 (4 C, s, 4 × CH3) 45.3 (1 C, dt, J FC 23.3, J PC
1
16.5, CH2CF2), 73.6, 73.7 (2 C, s, 2 × CH), 118.7 (1 C, dt, J PC
210.0, J FC 260.0, CH2CF2); m/z (EI+) 245 (M+, 1).
1
Diisop r op yl 1,1-Diflu or o-3-a za -4-oxo-5-ch lor op en ta n e-
1-p h osp h on a te (27). Triethylamine (450 µL, 3.2 mmol) was
added dropwise to a solution of chloroacetic anhydride (720
mg, 3.2 mmol) and 26 (790 mg, 3.2 mmol) in DCM (10 mL,

Bisphosphonate Ligands for PGK
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4449
CF2CO), -118.4 (2 F, dt, 2J FP 99.4, 3J HF 17.0, CF2CH2); δC 23.4,
degassed) at 0 °C under argon. After 2 h, the reaction mixture
was poured into dilute aqueous NaOH (20 mL, 1 M) and
extracted with DCM (2 × 10 mL). The combined organic layers
were washed with HCl (2 × 5 mL, 1 M), dried (Na2SO4),
filtered, and evaporated in vacuo to yield the title compound
as a white solid (400 mg, 39%): mp 58-60 °C; Rf (DCM-
2
2
23.6, 24.0 (8 C, s, 8 × CH3), 41.6 (1 C, dt, J FC 24.0, J PC 19.1,
CH2), 74.4, 74.5, 75.0, 75.1 (4 C, s, 4 × CH), 109.1 (1 C, dt,
1J FC 271.6, 1J PC 203.1, CF2CH2), 114.2 (1 C, dt, 1J FC 262.9, 1J PC
2
2
212.8, CF2CO), 159.3 (1 C, dt, J FC 25.4, J PC 17.8, CO); m/z
(EI+) 488 ([M + H]+, 25). Found: M+ 488.1590. C16H32F4-
NO7P2 requires M, 488.1582.
3
MeOH, 95:5) 0.30; δH 1.34 (6 H, d, J HH 6.3, 2 × CH3), 1.35 (6
3
3
3
H, d, J HH 6.3, 2 × CH3), 3.17 (2 H, dq, J HH 6.5, J HP 18.0,
1,1,5,5-Tetr aflu or o-2-oxo-3-azapen tan e-1,5-bisph osph o-
n ic Acid (31). Bromotrimethylsilane (1.9 mL, 14 mmol) was
added dropwise to a solution of ester 30 (1.0 g, 2.0 mmol) in
DCM (15 mL) under argon. After 24 h, the volatiles were
evaporated in vacuo, and the brown residue was taken up in
EtOAc (20 mL) and extracted with water (3 × 50 mL). The
combined water extracts were evaporated in vacuo and dis-
solved in MeOH (10 mL). Cyclohexylamine (407 mg, 4.0 mmol)
was added with swirling followed by the dropwise addition of
acetone to precipitate the cyclohexylammonium salt which was
filtered and dried to give the bis-cyclohexylammonium salt of
the title compound as a white crystalline solid (340 mg, 33%):
3
3
3
CH2CH2P), 3.82 (2 H, tt, J HF 15.6, J HH 6.3, J HP 6.3, CH2-
3
3
NH), 4.03 (2 H, s, CH2Cl), 4.82 (2 H, octet, J HH 6.3, J HP 6.5,
2
2 × CH), 7.24 (1 H, bt, NH); δP 3.6 (1 P, t, J FP 102.3); m/z
(EI+) 321 (M+, 7, 35Cl), 323 (M+, 3, 37Cl). Found: C, 37.57; H,
6.04; N, 4.23. Requires C, 37.34; H, 5.95; N, 4.35. Found: M+
321.0708. C10H19ClF2NO4P requires M, 321.0710.
P 1-Diisop r op yl P 5-Diet h yl 1,1-Diflu or o-3-a za -4-oxo-
p en ta n e-1,5-bisp h osp h on a te (28). Ester 27 (400 mg, 1.2
mmol) was heated for 16 h at 130 °C with triethyl phosphite
(425 µL, 2.5 mmol) under argon. Volatiles were removed in
vacuo (100 °C/20 mmHg, 5 h), and the crude bisphosphonate
was purified by dry column flash chromatography with gradi-
ent elution of DCM-MeOH, 100:0 to 50:50, to yield the title
compound as a yellow oil (412 mg, 81%): Rf (DCM-MeOH,
3
4
mp 167-169 °C; pKa 4.32 (COCF2PO3H); pKa 5.37 (CF2PO3H);
δH 1.00-2.10 (20 H, m, 10 × CH2(CHA)), 3.05-3.25 (2 H, m,
3
2
2 × CH), 3.90 (2 H, t, J HF 15.6, CH2CF2); δP 1.16 (1 P, t, J FP
2
3
87.1, PCF2CO), 3.3 (1 P, t, J FP 89.4, PCF2CH2); δF -121.1 (2
95:5) 0.20; δH 1.25 (6 H, t, J HH 6.3, 2 × CH2CH3), 1.30 (12 H,
2
2
3
3
2
F, d, J FP 87.2, CF2CO), -120.4 (2 F, dt, J FP 89.4, J HF 17.5,
CF2CH2); δC 23.2, 23.7, 29.7 (10 C, s, 10 × CH2(CHA)), 40.6 (1
d, J HH 6.3, 4 × CH3), 2.58 (2 H, d, J HP 18.8, COCH2P), 3.85
3
3
3
(2 H, tt, J HH 6.3, J HP 6.3, J HF 16.9, CH2CF2P) 4.10 (4 H, qn,
3J HP 6.3, 3J HH 6.3, 2 × CH2CH3), 4.80 (2 H, octet, 3J HP 6.3, 3J HH
2
2
C, dd, J FC 21.8, J PC 20.3, CH2), 49.7 (2 C, s, 2 × CH), 113.4
1
1
1
2
(1 C, dt, J FC 268.6, J PC 181.4, CF2CH2), 114.2 (1 C, dt, J FC
6.3, 2 × CH), 7.17 (1 H, bt, NH); δP 4.1 (1 P, t, J FP 102.3,
1
2
2
PCF2CH2), 22.5 (1 P, s, PCH2CO); δF -118.6 (2 F, dt, 3J HF 17.2,
260.3, J PC 192.2, CF2CO), 165.4 (1 C, dt, J FC 25.2, J PC 14.4,
CO); m/z (ES-) 318 ([M - H]+, 14).
2J FP 102.4, CF2); δC 16.2, 16.3, (2 C, s, 2 × CH2CH3), 23.6, 23.7,
1
24.0, 24.1 (4 C, s, 4 × CH), 35.1 (1 C, d, J PC 131.3, CH2P),
Eth yl Diisop r op yl 3-P h osp h on op r op ion a te (32). Ethyl
3-bromopropionate (20.0 g, 96 mmol) was heated with triiso-
propyl phosphite (17.4 g, 96 mmol) at 120 °C for 30 h under
argon. The crude phosphonate was purified by Kugelro¨hr
distillation to give the product as a colorless mobile oil (20.0
41.6 (1 C, 2J FC 23.0, 2J PC 19.0, CH2CF2), 62.7, 62.8 (2 C, s, 2 ×
CH2CH3), 74.1, 74.2 (2 C, s, 2 × CH), 117.3 (1 C, dt, 1J FC 261.8,
2
1J PC 213.1, CF2), 164.0 (1 C, d, J PC 3.9, CO); m/z (EI+) 423
(M+, 16). Found: M+ 423.1387. C14H29F2NO7P2 requires M,
423.1388.
g, 78%): bp 180-190 °C/2 mmHg;24 max(liquid film)/cm-1 1738
ν
(CdO), 1245 (PdO), 1000 (P-O-iPr); δH 1.25 (3 H, t, 3J HH 6.8,
1,1-Diflu or o-4-oxo-3-azapen tan e-1,5-bisph osph on ic Acid
(29). Bromotrimethylsilane (850 µL, 6.5 mmol) was added
dropwise to a solution of ester 28 (388 mg, 0.9 mmol) in DCM
(10 mL) under argon. After 72 h, additional bromotrimeth-
ylsilane (400 µL, 3.1 mmol) was syringed into the reaction,
and the reaction was warmed to 37 °C. After 5 h the volatiles
were evaporated in vacuo, and the resulting gum was solvo-
lyzed by MeOH (3 × 15 mL). The bisphosphonic acid was
dissolved in water (10 mL) and titrated to pH 7.1 with NaOH
(1 M). Extraction with DCM (2 × 40 mL) and EtOAc (3 × 40
mL) followed by lyophilization furnished the title compound
3
CH2CH3), 1.30 (6 H, d, J HH 6.3, 2 × CH3), 1.90-2.17 (2 H, m,
3
CH2P), 2.48-2.60 (2 H, m, CH2CH2P), 4.12 (2 H, q, J HH 6.8,
CH2CH3), 4.45-4.75 (2 H, m, 2 × CH); δP 28.4 (1 P, s).
Eth yl Diisop r op yl 4-P h osp h on obu tyr a te (33). Ethyl
4-bromobutyrate (20.0 g, 103 mmol) was heated with triiso-
propyl phosphite (18.8 g, 104 mmol) at 120 °C for 30 h under
argon. The crude phosphonate was purified by Kugelro¨hr
distillation to give the title compound as a colorless mobile oil
(20.4 g, 75%): bp 180-200 °C/2 mmHg;24 νmax (liquid film)/
cm-1 1739 (CdO), 1244 (PdO), 1011 (P-O-iPr); δH 1.20 (3 H,
3
3
3
4
t, J HH 6.8, CH2CH3), 1.25 (6 H, d, J HH 6.3, 2 × CH3), 1.58-
as a white solid (300 mg, 98%): pKa 4.82 (CF2PO3H); pKa
2
1.75 (2 H, m, CH2P), 1.75-1.95 (2 H, m, CH2CH2P), 2.83 (2 H,
6.70 (CH2PO3H); δH (D2O) 2.68 (2 H, d, J HP 18.8, COCH2P),
3
3
2
t, CH2CO2), 4.05 (2 H, q, J HH 6.8, CH2CH3), 4.53-4.70 (2 H,
3.80 (2 H, t, J HF 18.1, CH2CF2P); δP 4.6 (1 P, s, J FP 79.8,
3
m, 2 × CH); δP 29.5 (1 P, s).
PCF2), 13.2 (1 P, s, PCH2CO); δF -119.8 (2 F, dt, J HF 17.8,
2J FP 80.3, CF2); m/z (ES+) 372 ([M + 4Na + H]+, 65); 350 ([M
+ 3Na + H]+, 80).
Tetr aisopr opyl 2-Oxopen tan e-1,5-bisph osph on ate (34).
LDA (30 mL, 60 mmol, 2 M solution in heptane/THF) was
added to a solution of diisopropyl methanephosphonate (11.0
g, 61 mmol) in THF (50 mL) and stirred for 1 h at -75 °C
under argon. Ethyl diisopropyl 4-phosphonobutyrate (33) (15.7
g, 56 mmol) was added dropwise to the anion solution at -75
°C under argon and maintained at low temperature for 1 h.
The reaction mixture was brought to room temperature, then
poured into water (150 mL), acidified with HCl (50 mL, 1 M),
and extracted with DCM (3 × 125 mL). The combined organic
extracts were dried (Na2SO4) and filtered, and the solvent
evaporated in vacuo to yield a brown gum. The crude
bisphosphonate was initially purified by Kugelro¨hr distillation
(bp 220-230 °C/1 mmHg) and isolated by dry column flash
chromatography, eluting with a gradient of DCM-MeOH
(from 100:0 to 95:5), to yield the title compound24 as a colorless
oil (7.65 g, 33%): δH 1.25 (12 H, d, 3J HH 6.3, 4 × CH3), 1.28 (12
Tetr aisopr opyl 1,1,5,5-Tetr aflu or o-2-oxo-3-azapen tan e-
1,5-bisp h osp h on a te (30). Oxalyl chloride (2.18 g, 17 mmol,
freshly distilled) was added dropwise to diisopropyl phospho-
nodifluoroethanoic acid (2.1 g, 8.0 mmol) in DCM (25 mL)
under an atmosphere of argon and stirred for 16 h. The
volatiles were evaporated in vacuo, and the crude acid chloride
was dissolved in a solution of ester 26 (1.9 g, 7.7 mmol) in
DCM (25 mL). The reaction mixture was cooled to 0 °C, and
triethylamine (0.79 g, 7.8 mmol) was slowly added dropwise
and stirred for 10 min. The reaction mixture was evaporated
in vacuo, the residue dissolved in ether (30 mL) and filtered,
and the ether evaporated. The crude amide was dissolved in
DCM (50 mL), washed with HCl (50 mL, 1 M), NaOH (50 mL,
1 M), and brine (50 mL), and then dried (Na2SO4). The
solution was filtered and the solvent evaporated in vacuo to
give the title compound as a pale-yellow liquid which solidified
on standing at 0 °C (3.5 g, 89%): mp 18-20 °C; bp >190 °C/
3
H, d, J HH 6.3, 4 × CH3), 1.55-1.90 (4 H, m, PCH2CH2), 2.70
3
2
(2 H, t, J HH 6.3, CH2COCH2P), 2.95 (2 H, d, J HP 25.0, PCH2-
CO), 4.50-4.74 (4 H, m, 4 × CH); δP 18.1 (1 P, s, PCH2CH2),
29.6 (1 P, s, PCH2CO); m/z (EI+) 414 (M+, 15). Found: M+
414.1936. C17H36O7P2 requires M, 414.1937.
3
0.2 mmHg (decomp.); δH 1.30 (12 H, d, J HH 6.2, 4 × CH3),
3
3
3
1.32 (12 H, d, J HH 6.2, 4 × CH3), 3.87 (2 H, tt, J HF 15.6, J HP
6.3, 3J HH 6.3, CH2CF2), 4.80 (4 H, octet, 3J HH 6.2, 3J HP 6.2, 4 ×
2
CH), 7.15 (1 H, bt, NH); δP 3.9 (1 P, t, J FP 99.4, PCF2CH2),
2-Oxop en ta n e-1,5-bisp h osp h on ic Acid (35). Bromotri-
2
2
1.8 (1 P, t, J FP 96.8, PCF2CO); δF -118.2 (2 F, d, J FP 96.8,
methylsilane (1.4 mL, 11 mmol) was added dropwise to a

4450 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
J akeman et al.
3
solution of ester 34 (640 mg, 1.6 mmol) in DCM (20 mL) under
argon. After 24 h, 31P NMR indicated the reaction had reached
completion; δP 2.0 (1 P, s, PCH2CO) and 20.0 (1 P, s, PCH2-
CH2). The volatiles were evaporated in vacuo, and the
resulting gum was solvolyzed by MeOH (3 × 15 mL). The
bisphosphonic acid was then dissolved in water (15 mL) and
titrated to pH 7.1 with NaOH (1 M). Extraction with DCM (2
× 50 mL) and EtOAc (2 × 50 mL) followed by lyophilization
furnished the title compound24 as a hygroscopic white solid
(P-O-iPr); δH 1.25 (12 H, t, J HH 6.3, 4 × CH3), 1.33 (6 H, d,
3J HH 6.3, 2 × CH3), 1.34 (6 H, d, 3J HH 6.3, 2 × CH3), 1.55-1.75
(2 H, m, CH2P), 1.75-1.95 (2 H, m, CH2CH2P), 2.83 (2 H, t,
2J HP 15.0, CH2CO), 4.63-4.78 (2 H, m, 2 × CH), 4.81 (2 H,
octet, 3J HH 6.3, 3J HP 6.3, 2 × CH); δP 1.6 (1 P, t, 2J FP 97.0, PCF2),
2
29.2 (1 P, s, PCH2); δF -119.4 (2 F, d, J FP 96.6, PCF2); m/z
(EI+) 450 (M+, 3). Found: C, 45.36; H, 7.61. C17H34F2O7P2
requires C, 45.34; H, 7.61. Found: M+ 450.1747. C17H34F2O7P2
requires M, 450.1750.
3
4
(400 mg, 89%): pKa 6.39 (CH2COPO3H); pKa 7.65 (CH2PO3H);
δH (D2O, AMX-500) 1.27-1.40 (2 H, m, PCH2CH2), 1.50-1.63
(2 H, m, PCH2CH2), 2.60 (2 H, t, 3J HH 8.2, PCH2CH2CH2), 2.78
1,1-Diflu or o-2-oxopen tan e-1,5-bisph osph on ic Acid (39).
Bromotrimethylsilane (1.0 mL, 7.8 mmol) was added dropwise
to a solution of ester 38 (504 mg, 1.1 mmol) in DCM (2 mL)
under argon. After 1 week, 31P NMR indicated the reaction
2
(2 H, d, J HP 21.8, PCH2CO); δP (D2O) 11.0 (1 P, s, PCH2CO),
2
24.8 (1 P, s, PCH2CH2); δC (D2O) 18.2 (1 C, s, COCH2CH2),
had reached completion; δP -15.4 (1 P, t, J FP 97.1, PCF2CO)
1
2
27.7 (1 C, d, J PC 132.1, PCH2CH2), 44.2 (1 C, d, J PC 12.6,
and 21.3 (1 P, s, PCH2CH2). The volatiles were evaporated in
vacuo, and the resulting gum was triturated with MeOH (5 ×
15 mL). The bisphosphonic acid was then dissolved in water
(10 mL) and titrated to pH 7.1 with NaOH (1 M). Extraction
with DCM (2 × 20 mL) and EtOAc (20 mL) followed by
lyophilization furnished the title compound as a white solid
1
COCH2CH2), 47.3 (1 C, d, J PC 104.5, PCH2CO), 210.1 (1 C, d,
2J PC 16.2, PCH2CO).
Tetr a isop r op yl 2-Oxobu ta n e-1,4-bisp h osp h on a te (36).
LDA (30 mL, 60 mmol, 2 M solution in heptane/THF) was
added to a solution of diisopropyl methanephosphonate (11.0
g, 61 mmol) in THF (50 mL) and stirred for 1 h at -75 °C
under argon. Compound 32 (15.0 g, 56 mmol) was added
dropwise to the anion solution at -75 °C under argon and
stirred for 1 h. The reaction mixture was allowed to warm to
room temperature, then poured into water (150 mL), acidified
with HCl (50 mL, 1 M), and extracted with DCM (3 × 125
mL). The combined organic extracts were dried (Na2SO4) and
filtered, and the solvent evaporated in vacuo to yield a brown
paste. The crude bisphosphonate was initially purified by
Kugelro¨hr distillation (220 °C/1 mmHg) and isolated by dry
column flash chromatography, eluting with a gradient of
DCM-MeOH (from 100:0 to 95:5) to yield the title compound24
3
4
(358 mg, 98%): pKa 4.82 (CF2PO3H); pKa 7.90 (CH2PO3H);
δH (D2O) 1.45-1.65 (2 H, m, CH2P), 1.65-1.83 (2 H, m, CH2-
CH2CO), 2.93 (2 H, q, 3J HH 6.3, 3J HP 6.3, CH2CO); (D2O, BMX-
400) 1.40-1.50 (2 H, m, CH2P), 1.60-1.69 (2 H, m, CH2CH2-
3
3
CO), 2.90 (2 H, q, J HH 6.3, J HP 6.3, CH2CO); δP (D2O) 2.6 (1
2
2
P, t, J FP 76.5, PCF2, 89%); 5.5 (1 P, t, J FP 80.8, PCF2, 11%);
24.5 (1 P, s, PCH2, 89%); 25.2 (1 P, s, PCH2 11%); δF (D2O)
-115.9 (2 F, d, 2J FP 76.4, PCF2, 89%), -122.2 (2 F, d, 2J FP 80.5,
PCF2, 11%); δC (D2O, 400 MHz) 16.2 (1 C, s, PCH2CH2), 26.5
1
(1 C, d, J PC 132.9, PCH2CH2), 37.2-38.2 (1 C, m, CH2CO),
1
95.5-96.5 (1 C, m, PCF2C(OH)2), 117.5 (1 C, dt, J PC 158.3,
1J FC 270.3, PCF2), 206.0 (1 C, dt, J PC 12.2, J FC 23.2, PCF2-
CO). Found: M+ 326.958. C5H9F4O7P2Na2 requires M,
326.9587.
2
2
3
as a colorless oil (6.72 g, 30%): δH 1.28 (12 H, d, J HH 6.3, 4 ×
3
CH3), 1.32 (12 H, d, J HH 3.8, 4 × CH3), 1.85-2.05 (2 H, m,
2
PCH2CH2), 2.80-2.90 (2 H, m, PCH2CH2), 3.05 (2 H, d, J HP
Tetr a isop r op yl 1,1-Diflu or o-2-oxobu ta n e-1,4-bisp h os-
p h on a te (40). n-Butyllithium (7.8 mL, 8.3 mmol, 1.06 M
solution in hexane) was added dropwise to diisopropylamine
(0.84 g, 8.3 mmol) in THF (10 mL) at -70 °C under argon.
After 15 min, diisopropyl difluoromethanephosphonate (1.79
g, 8.3 mmol) as a solution in THF (5 mL) at -20 °C was
transferred by cannula into the solution of LDA at - 80 °C.
After 15 min, 32 (10.0 g, 36 mmol) as a solution in THF (5
mL) at -20 °C was introduced by cannula into the reaction
mixture which was maintained at -80 °C for 3 h, and then
allowed to warm to room temperature before being poured into
cold ammonium chloride (100 mL, saturated) and extracted
with EtOAc (3 × 100 mL). The combined organic extracts were
dried (Na2SO4) and filtered and the solvent evaporated in
vacuo to give an orange oil (2.7 g). The crude bisphosphonate
was purified by flash chromatography with petrol-EtOAc-
acetic acid, 54:45:1 as eluant, to yield the title compound as a
colorless oil (1.54 g, 47%): Rf (petrol/EtOAc/acetic acid, 54:45:
1) 0.30; bp > 200 °C/1 mmHg (decomp.); νmax(liquid film)/cm-1
1734 (CdO), 1248 (PdO), 1005 (P-O-iPr); δH 1.25 (12 H, t,
3J HH 6.3, 4 × CH3), 1.32 (6 H, d, 3J HH 6.3, 2 × CH3), 1.35 (6 H,
25.0, PCH2CO), 4.55-4.80 (4 H, m, 4 × CH); δP 17.8 (1 P, s,
PCH2CH2), 29.4 (1 P, s, PCH2CO); m/z (EI+) 400 (M+, 26).
Found: M+ 400.1780. C16H34O7P2 requires M, 400.1776.
2-Oxob u t a n e-1,4-b isp h osp h on ic Acid (37). Bromotri-
methylsilane (1.5 mL, 12 mmol) was added dropwise to a
solution of ester 36 (664 mg, 1.7 mmol) in DCM (20 mL) under
argon. After 24 h, 31P NMR indicated the reaction had reached
completion; δP 3.2 (1 P, s, PCH2CO) and 20.0 (1 P, s, PCH2-
CH2). The volatiles were evaporated in vacuo, and the
resulting gum was solvolyzed by MeOH (3 × 15 mL). The
bisphosphonic acid was then dissolved in water (15 mL) and
titrated to pH 7.1 with NaOH (1 M). Extraction with DCM (2
× 50 mL) and EtOAc (2 × 50 mL) followed by lyophilization
furnished the title compound24 as a hygroscopic white solid
3
4
(400 mg, 87%): pKa 5.60 (CH2COPO3H), pKa , 6.89 (CH2-
PO3H); δH (D2O) 1.50-1.80 (2 H, m, PCH2CH2), 2.70-2.93 (2
2
H, m, PCH2CH2), 3.12, (2 H, d, J HP 20.0, PCH2CO); δP (D2O)
11.1 (1 P, s, PCH2CO), 23.2 (1 P, s, PCH2CH2); δC (D2O) 22.7
1
(1 C, d, J PC 132.1, PCH2CH2), 38.3 (1 C, s, COCH2CH2), 48.0
1
2
(1 C, d, J PC 103.0, PCH2CO), 212.7 (1 C, d, J PC 15.5, CO).
3
d, J HH 6.3, 2 × CH3), 1.83-2.03 (2 H, m, CH2P), 3.00 (2 H, q,
Tetr a isop r op yl 1,1-Diflu or o-2-oxop en ta n e-1,5-bisp h o-
sp h on a te (38). n-Butyllithium (37.0 mL, 39 mmol, 1.06 M
solution in hexane) was added dropwise to diisopropylamine
(3.97 g, 39 mmol) in THF (20 mL) at -70 °C under argon. After
40 min diisopropyl difluoromethanephosphonate (8.48 g, 39
mmol) as a solution in THF (20 mL) at -20 °C was introduced
by cannula into the solution of LDA cooled to - 80 °C. After
45 min, ester 33 (10.0 g, 36 mmol) as a solution in THF (20
mL) at -20 °C was added by cannula, and the reaction mixture
was maintained at -80 °C for 3 h and then allowed to warm
to room temperature before being poured into cold ammonium
chloride (200 mL, saturated) and extracted with EtOAc (3 ×
200 mL). The combined organic extracts were dried (Na2SO4)
and filtered, and the solvent evaporated in vacuo to give a red
oil (12.3 g). The crude bisphosphonate was purified by dry
column flash chromatography with petrol-EtOAc, 80:20 as
eluant, to furnish the title compound as a colorless oil (9.47 g,
60%): Rf (petrol/EtOAc, 80:20) 0.26; bp > 200 °C/1 mmHg
(decomp.); νmax(liquid film)/cm-1 1744 (CdO), 1273 (PdO), 1007
3
3J HH 6.3, J HP 6.3, CH2CO), 4.54-4.73 (2 H, m, 2 × CH), 4.82
3
3
2
(2 H, octet, J HH 6.3, J HP 6.3, 2 × CH); δP 1.5 (1 P, t, J FP
2
97.0, PCF2), 28.1 (1 P, s, PCH2); δF -119.1 (2 F, d, J FP 97.6,
PCF2); m/z (EI+) 436 (M+, 0.3). Found: M+ 436.1591.
C16H32F2O7P2 requires M, 436.1590.
1,1-Diflu or o-2-oxobu ta n e-1,4-bisp h osp h on ic Acid (41).
Bromotrimethylsilane (1.1 mL, 8.1 mmol) was added dropwise
to a solution of ester 40 (509 mg, 1.2 mmol) in DCM (2 mL)
under argon. After 1 week, 31P NMR indicated the reaction
2
had reached completion; δP -15.7 (1 P, t, J FP 96.3, PCF2CO)
and 19.8 (1 P, s, PCH2CH2). Volatiles were evaporated in
vacuo, and the resulting gum was solvolyzed with MeOH (5 ×
15 mL). The bisphosphonic acid (348 mg, 1.2 mmol) was then
dissolved in water (10 mL) and titrated to pH 7.1 with NaOH
(1 M). Extraction with DCM (2 × 20 mL) and EtOAc (20 mL)
followed by lyophilization afforded the title compound as a
3
4
white solid (365 mg, 98%): pKa 4.77 (CF2PO3H); pKa 6.37
(CH2PO3H); δH (D2O) 1.60-1.85 (2 H, m, CH2P), 3.05 (2 H, q,

Bisphosphonate Ligands for PGK
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4451
m, 10 × CH2(CHA)), 2.20-2.50 (2 H, m, CH2CF2), 3.00-3.25
3J HH 6.3, 3J HP 6.3, CH2CO); δP (D2O) 1.9 (1 P, t, 2J FP 80.0, PCF2,
2
2
85%); 5.5 (1 P, t, J FP 80.0, PCF2, 15%); 23.7 (1 P, s, PCH2,
(2 H, m, 2 × CH2); δP (D2O) 0.9 (1 P, t, J FP 85.8, PCF2CO,
2
2
85%); 25.7 (1 P, s, PCH2 15%); m/z (ES-) 311 ([M + 2Na -
H]+, 15); 289 ([M + Na - H]+, 100); 267 ([M - H]+, 100).
Found: M+ 467.1902. C16H35F2N2O7P2 requires M, 467.1889.
60%), 3.8 (1 P, t, J FP 90.1, PCF2CO, 40%), 5.0 (1 P, t, J FP
2
95.6, PCF2CH2, 57%), 5.4 (1 P, t, J FP 97.0, PCF2CO, 43%); δC
(D2O, 400 MHz) 23.2 (2 C, s, 2 × CHA C-4), 23.7 (4 C, s, 2 ×
CHA C-3,5), 25.1-26.2 (2 C, m, PCF2CH2CH2), 29.7 (4 C, s, 2
× CHA C-2,6), 49.7 (2 C, s, 2 × CHA C-1), 94.3-94.5 (1 C, m,
PCF2C(OH)2), 111.8-125.5 (2 C, m, PCF2), 201.5-202.5 (1 C,
m, PCF2CO); m/z (ES-) 317 ([M - H]+, 33); (ES+) 100 (CHA+,
100). Found: M+ 517.1825. C17H35F4N2O7P2 requires M,
517.1856.
Eth yl Diisop r op yl 4,4-Diflu or o-4-p h osp h on obu tyr a te
(42). n-Butyllithium (17.5 mL, 18.6 mmol, 1.06 M solution in
hexane) was added dropwise to diisopropylamine (1.87 g, 18.6
mmol) in THF (30 mL) at -70 °C under argon. After 15 min
the solution was cooled to - 80 °C. Diisopropyl difluorometha-
nephosphonate (4.0 g, 18.5 mmol) as a solution in THF (5 mL)
at -20 °C was transferred by cannula into the LDA. After 15
min, ethyl acrylate (1.20 g, 12 mmol) as a solution in THF (5
mL) at -20 °C was introduced by cannula into the reaction
mixture. The reaction mixture was maintained at -80 °C for
3 h and then allowed to warm to room temperature, poured
into cold aqueous ammonium chloride (100 mL, saturated),
and extracted with EtOAc (1 × 100 mL). The combined
organic extracts were dried (Na2SO4) and filtered, and solvent
evaporated in vacuo to give an orange oil (3.7 g). The crude
phosphonate was purified by dry column flash chromatography
with gradient elution of DCM-MeOH (from 0:100 to 2:98) as
a solvent to yield the title compound as a colorless oil (800
Ack n ow led gm en t. These studies were supported by
the BBSRC with a research studentship (to D.L.J .) and
Grant GR/H/32780. We thank Mr. P. Ashton, Birming-
ham University, for assistance with HRMS measure-
ments. The Krebs Institute is a BBSRC Biomolecular
Sciences Center.
Refer en ces
(1) Voet, D.; Voet, J . G. Biochemistry, 1st ed.; J ohn Wiley & Sons:
New York, 1990; Vol. 1.
(2) Scopes, R. K. 3-Phosphoglycerate Kinase. In The Enzymes, 3rd
ed.; Boyer, P. D., Ed., Academic Press: New York, 1973; Vol VIII,
Chapter 10.
3
mg, 21%): Rf (DCM-MeOH, 95:5) 0.34; δH 1.15 (3 H, t, J HH
3
6.3, CH2CH3), 1.27 (6 H, d, J HH 6.3, 2 × CH3), 1.32 (6 H, d,
3J HH 6.3, 2 × CH3), 2.16-2.56 (4 H, m, CH2CH2CO), 4.04 (2
3
3
3
(3) Fairbrother, W. J .; Graham, H. C.; Williams, R. J . P. The Roles
of ATP4- and Mg2+ in Control Steps of Phosphoglycerate Kinase.
Eur. J . Biochem. 1990, 190, 407-414.
H, q, J HH 6.3, CH2CH3), 4.75 (2 H, octet, J HH 6.3, J HP 6.3, 2
2
× CH); δP 5.2 (1 P, t, J FP 107.2, PCF2); δF -114.3 (2 F, dt,
3
2J FP 107.4, J HF 18.8, PCF2); m/z (EI+) 316 (M+, 0.4).
(4) Graham, H. C.; Williams, R. J . P. The Roles of ADP2- and Mg2+
in Control Steps of Phosphoglycerate Kinase. Eur. J . Biochem.
1991, 197, 81-91.
Tetr a isop r op yl 1,1,5,5-Tetr a flu or o-2-oxo-p en ta n e-1,5-
bisp h osp h on a te (43). n-Butyllithium (2.3 mL, 2.4 mmol,
1.06 M solution in hexane) was added dropwise to diisopro-
pylamine (245 mg, 2.4 mmol) in THF (6 mL) at -70 °C under
argon. After 20 min the solution of LDA was cooled to -80
°C, and diisopropyl difluoromethanephosphonate (525 mg, 2.4
mmol) in THF (5 mL) at -20 °C was transferred into the
reaction mixture by cannula. Ester 42 (700 mg, 2.21 mmol)
as a solution in THF (10 mL) at -20 °C was added by cannula
to the reaction mixture after 15 min, and the reaction mixture
was maintained at -80 °C for 4 h and then allowed to warm
to room temperature, poured into cold aqueous ammonium
chloride (30 mL, sat.), and extracted with EtOAc (3 × 30 mL).
The combined organic extracts were dried (Na2SO4) and
filtered, and the solvent was evaporated in vacuo to give an
orange oil (864 mg). The crude bisphosphonate was purified
three times by flash chromatography with petrol/EtOAc (twice
at 70:30, and once at 80:20 ratio) as eluant to furnish the title
compound as a colorless oil (312 g, 29%): Rf (petrol-EtOAc-
acetic acid, 50:49:1) 0.50; bp > 200 °C/1 mmHg (decomp.); νmax
(liquid film)/cm-1 1747 (CdO), 1268 (PdO), 1001 (P-O-iPr);
δH 1.27-1.46 (24 H, m, 8 × CH3), 2.25-2.55 (2 H, m, CF2-
(5) J oa˜o, H. C.; Williams, R. J . P. The Anatomy of a Kinase and the
Control of Phosphoryl Transfer. Eur. J . Biochem. 1993, 216,
1-18.
(6) Watson, H. C.; Walker, N. P. C.; Shaw, P. J .; Bryant, T. N.;
Wendell, P. L.; Fothergill, L. A.; Perkins, R. E.; Conroy, S. C.;
Dobson, M. J .; Tuite, M. F.; Kingsman, A. J .; Kingsman, S. M.
Sequence and Structure of Yeast Phosphoglycerate Kinase.
EMBO J . 1982, 1, 1635-1640.
(7) Fifis, T.; Scopes, R. K. Purification of 3-Phosphoglycerate Kinase
from Diverse Sources by Affinity Chromatography. Biochem. J .
1978, 175, 311-319.
(8) J ohnson, P. E.; Abbott, S. J .; Orr, G. A.; Semeriva, M.; Knowles,
J . R. Phosphoryl Enzyme of Phosphoglycerate Kinase. Biochem-
istry 1976, 15, 2893-2901.
(9) Mildvan, A. S. Conformation and Arrangement of Substrates at
Active Sites of ATP-Utilizing Enzymes. Philos. Trans. R. Soc.
London B. 1981, 293, 65-74.
(10) Banks, R. D.; Blake, C. C. F.; Evans, P. R.; Haser, R.; Rice, D.
W.; Hardy, G. W.; Merrett, M.; Phillips, A. W. Sequence,
Structure, and Activity for Phosphoglycerate Kinase - a Possible
Hinge-Bending Enzyme. Nature (London) 1979, 279, 773-777.
(11) Wilson, H.; Williams, R. J . P.; Littlechild, J . A.; Watson, H. C.
NMR Analysis of the Interdomaine Region of Yeast Phospho-
glycerate Kinase. Eur. J . Biochem. 1988, 170, 529-538.
(12) Webb, M.; Trentham, D. Analysis of Chiral Inorganic {16O,17O,18O]-
Thiophosphate and the Stereochemistry of the 3-Phosphoglyc-
erate Kinase Reaction. J . Biol. Chem. 1980, 255, 1775-1778.
(13) Mas, M.; Resplandor, Z. E. Structure-Function Relationships
in 3-Phosphoglycerate Kinase - the Role of the Carboxy-
Terminal Peptide. Proteins: Struct. Funct. Gen. 1988, 4, 56-
62.
3
3
CH2), 3.07 (2 H, t, J HH 6.3, CH2CO), 4.85 (4 H, octet, J HH
3
2
6.3, J HP 6.3, 4 × CH); δP 1.5 (1 P, t, J FP 97.2, PCF2CO), 5.1
2
2
(1 P, t, J FP 106.8, PCF2CH2); δF -113.9 (2 F, d, J FP 106.8,
2
PCF2CH2), -119.3 (2 F, d, J FP 98.1, PCF2CO); m/z (CI+) 487
([M + H]+, 100). Found: M+ 487.1638. C17H33F4NO7P2
requires M, 487.1636.
(14) Pickover, C. A.; McKay, D. B.; Engelman, D. M.; Steitz, T. A.
Substrate Binding Closes the Cleft Between the Domains of
Phosphoglycerate Kinase. J . Biol. Chem. 1979, 254, 11323-
11329.
(15) Sinev, M. A.; Razgulyaev, O. I.; Vas, M.; Timchenko, A. A.;
Ptitsyn, O. B. Correlation Between Enzyme Activity and Hinge-
Bending Domain Displacement in 3-Phosphoglycerate Kinase.
Eur. J . Biochem. 1989, 180, 61-66.
(16) Roustan, C.; Fattoum, A.; J eanneau, R.; Pradel, L. Yeast
3-Phosphoglycerate Kinase: Sulfate and Substrate Binding.
Their Effects on the Conformational State of the Enzyme.
Biochemistry 1980, 19, 5168-5175.
(17) Davies, G. J .; Gamblin, S. J .; Littlechild, J . A.; Dauter, Z.; Wilson,
K. S.; Watson, H. C. Structure of the ADP Complex of the
3-Phosphoglycerate Kinase from Bacillus stearothermophilus at
1.65 Å. Acta Crystallogr. D 1994, 50, 202-209.
1,1,5,5-Tet r a flu or o-2-oxop en t a n e-1,5-b isp h osp h on ic
Acid (44). Ester 43 (110 mg, 225 µmol) was dissolved in dry
acetonitrile-d3 (1.125 mL). Chlorotrimethylsilane (209 µL, 1.6
mmol) was syringed into the solution under argon followed
by solid sodium iodide (236 mg, 1.6 mmol). The reaction was
monitored by 31P NMR, and after 8 h the reaction was
complete; δP -14.7 (1 P, bt, PCF2CO) and -10.5 (1 P, bt, PCF2-
CH2). The reaction mixture was filtered through Celite, and
the volatiles were evaporated in vacuo. Extraction with MeOH
(2 × 20 mL) gave the bisphosphonic acid which was taken up
into MeOH (2 mL), and cyclohexylamine (45 mg, 450 µmol)
was added. Dropwise addition of acetone (15 mL) with rapid
swirling gave the cyclohexylammonium salt (65 mg) as an off-
white solid. This was taken up in MeOH and reprecipitated
with acetone to yield the bis-cyclohexylammonium salt of the
title compound as a white crystalline solid (40 mg, 34%): mp
(18) Bernstein, B. E.; Michels, P. A. M.; Hol, W. G. J . Synergistic
Effects of Substrate-Induced Conformational Changes in Phos-
phoglycerate Kinase Activation. Nature (London) 1997, 385,
275-278.
3
4
210-212 °C; pKa 4.47; pKa 5.10; δH (D2O) 1.00-2.20 (20 H,

4452 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
J akeman et al.
(19) Gregory, J . D.; Serpersu, E. H. Arrangement of Substrates at
the Active-Site of Yeast Phosphoglycerate Kinase - Effect of
Sulfate Ion. J . Biol. Chem. 1993, 268, 3880-3888.
B. H.; Sawyer, T. K. The Role of 4-Phosphonodifluoromethyl-
and 4-Phosphono-Phenylalanine in the Selectivity and Cellular
Uptake of SH2 Domain Ligands. Bioorg. Med. Chem. Lett. 1997,
7, 1909-1914.
(20) Schmidt, P. P.; Travers, F.; Barman, T. Transient and Equilib-
rium Kinetic Studies on Yeast 3-Phosphoglycerate kinase -
Evidence that an Intermediate Containing 1,3-Bisphosphoglyc-
erate Accumulates in the Steady-State. Biochemistry 1995, 34,
824-832.
(34) Wrobel, J .; Dietrich, A. Preparation of L-(Phosphonodifluoro-
methyl)phenylalanine Derivatives as Nonhydrolyzable Mimetics
of O-Phosphotyrosine. Tetrahedron Lett. 1993, 34, 3543-3546.
(35) McKenna, C. E.; Higa, M. R.; Cheung, N. H.; McKenna, M. The
Facile Dealkylation of Phosphonic Acids Dialkyl Esters by
Bromotrimethylsilane. Tetrahedron Lett. 1977, 739-743.
(36) Blackburn G. M.; Ingleson, D. Specific Dealkylation of Phos-
phonate Esters Using Iodotrimethylsilane. J . Chem. Soc., Chem.
Commun. 1978, 870-871.
(37) Olah, G. A.; Husain, A.; Gupta, B. J . B.; Narang, S. C. Trichloro-
(methyl)silane-Sodium Iodide, a New Regioselective Reagent for
the Cleavage of Ethers. Angew. Chem., Int. Ed. Engl. 1981, 20,
690-691.
(38) McHarg, J .; Littlechild, J . A. Studies with Inhibitors of the
Glycolytic Enzyme Phosphoglycerate Kinase for Potential Treat-
ment of Cardiovascular and Respiratory Disorders. J . Pharm.
Pharmacol. 1996, 48, 201-205.
(39) McPhillips, T. M.; Hsu, B. T.; Sherman, M. A.; Mas, M. T.; Rees,
D. C. Structure of the R65Q Mutant of Yeast 3-Phosphoglycerate
kinase Complexed with Mg-AMPPNP and 3-Phospho-D-glycer-
ate. Biochemistry 1996, 35, 4118-4127.
(40) Blackburn, G. M.; Taylor, G. E.; Tattershall, R. H.; Thatcher,
G. R. J .; McLennan, A. G. Phosphonate Analogs of Biological
Phosphates. In Biophosphates and Their Analogs; Bruzic, K. S.,
Stec, W. J ., Eds.; Elsevier Science Publishers: Amsterdam, 1987;
pp 451-464.
(41) Krietch, W. K. G.; Bu¨cher, T. 3-Phosphoglycerate Kinase from
Rabbit Skeletal Muscle and Yeast. Eur. J . Biochem. 1970, 17,
568-580.
(42) Burton, D. J .; Ishihara, T.; Maruta, M. A Useful Zinc Reagent
for the Preparation of 2-Oxo-1,1-difluoroalkylphosphonates.
Chem. Lett. 1982, 755-758.
(43) Burton, D. J .; Sprague, L. G. Preparation of Difluorophospho-
noacetic Acid and its Derivatives. J . Org. Chem. 1988, 53, 1523-
1527.
(21) Scopes, R. K. The Steady-State kinetics of Yeast Phosphoglyc-
erate Kinase. Anomalous Kinetic Plots and the Effect of Salts
on Activity. Eur. J . Biochem. 1978, 85, 503-516.
(22) Orr, G. A.; Knowles, J . A. Interactions of the Phosphonate Analog
of 3-Phosphoglycerate with Phosphoglycerate Kinase. J . Bio-
chem. J . 1974, 141, 721-723.
(23) Vas, M. Modeling of Substrate Binding to 3-Phosphoglycerate
Kinase with Analogs of 3-Phosphoglycerate. Eur. J . Biochem.
1990, 194, 639-645.
(24) Li, Y., & Byers, L. D. Phosphonate Inhibitors of Glyceraldehyde
3-Phosphate Dehydrogenase and Phosphoglycerate Kinase. Bio-
chim. Biophys. Acta 1993, 1164, 17-21.
(25) Blackburn, G. M. Phosphonates as Analogs of Biological Phos-
phates. Chem. Ind. (London) 1981, 134-138.
(26) Berkowitz, D. B.; Shen, Q.; Maeng, J .-H. Synthesis of the R,R-
difluoroalkylphosphonate analog of phosphoserine. Tetrahedron
Lett. 1994, 35, 6445-6448.
(27) Blackburn, G. M.; Eckstein, F.; Kent, D. E.; Perre´e, T. D. Isopolar
vs. Isosteric Phosphonate Analogs of Nucleotides. Nucleosides
Nucleotides 1985, 4, 165-167.
(28) Blackburn, G. M.; Perre´e, T. D.; Rashid, A.; Bisbal, C.; Lebleu,
B. Isosteric and isopolar Analogs of Nucleotides. Chem. Scr.
1986, 26, 21-24. Blackburn, G. M.; Parratt, M. J . Synthesis of
R-Flurovinyl Phosphonates. J . Chem. Soc., Perkin Trans. 1 1986,
1417-1424. Blackburn, G. M.; Parratt, M. J . The Synthesis of
R-Fluoroalkylphosphonates. J . Chem. Soc., Perkin Trans. 1 1986,
1425-1430.
(29) Burke, T. R.; Smyth, M. S.; Nomizu, M.; Otaka, A.; Roller, P. R.
Preparation of fluoro-4-(phosphonomethyl)-D,L-phenylalanine
and Hydroxy-4-phosphonomethyl-D,L-phenylalanine Suitably
Protected for Solid-Phase Synthesis of Peptides Containing
Hydrolytically Stable Analogs of O-Phosphotyrosine. J . Org.
Chem. 1993, 58, 1336-1340.
(30) Halazy, S.; Ehrhard, A.; Danzin, C. 9-(Difluorophosphonoalkyl)-
guanines as a New Class of Multisubstrate Analog Inhibitors of
Purine Nucleoside Phosphorylase. J . Am. Chem. Soc. 1991, 113,
315-317.
(31) Humphries, R. G.; Tomlinson W.; Ingall, A. H.; Cage, P. A.; Leff,
P. FPL-66096 - A Novel, Highly Potent, and Selective Antago-
nist at Human Platelet P-2T Purinoceptors. Brit. J . Pharmacol.
1994, 113, 1057-1063.
(32) Nieschalk, J .; O’Hagan, D. Monofluorophosphonates as Phos-
phate Mimics in Bioorganic Chemistry - a Comparative Study
of CH2-Phosphonate and CF2-Phosphonate Analogs of sn-
Glycerol 3-Phosphate as Substrates for sn-Glycerol-3-Phosphate
Dehydrogenase. J . Chem. Soc., Chem. Commun. 1995, 719-720.
(33) Stankovic, C. J .; Surendran, N.; Lunney, E. A.; Plummer, M.
S.; Para, K. S.; Shahripour, S.; Fergus, J . H.; Marks, J . S.;
Herrera, R.; Hubbell, S. E.; Humblet, C.; Saltiel, A. R.; Stewart,
(44) Soborovskii, Z.; Baina, N. F. Dichlorodifluoromethane as
a
Difluoromethylating Agent: (I) Reaction of Dichlorodifluo-
romethane with Sodium Alcoholates and Mercaptides. J . Gen.
Chem. USSR 1959, 29, 1115-1117.
(45) Moedritzer, K.; Irani, R. R. Direct Synthesis of R-Aminometh-
ylphosphonic Acids - Mannich-Type Reactions with Orthophos-
phorous Acid. J . Inorg. Nucl. Chem. 1961, 22, 297-304.
(46) Maier, L.; Crutchfield, M. M. Organic Phosphorus Compounds
70. Preparation and Properties of New Phosphorus-Containing
Chelating Agents for Calcium and Magnesium Ions. Phosphorus
Sulfur 1978, 5, 45-51.
(47) Blackburn, G. M.; J akeman, D. L.; Ivory, A. J .; Ha¨gele, G.
Theoretical Analysis of the Complex NMR Spectra of Esters of
1,1,3,3-Tetrafluoro-2,2-dihydroxypropane-1,3-bisphosphonic Acid.
J . Chem. Soc., Perkin Trans. 1, manuscript in preparation.
J M970839Y