Synthesis, stereochemistry and SAR of a series of minodronate analogues as RGGT inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.04.063

Phosphonocarboxylate (PC) analogues of bisphosphonates are of interest due to their selective inhibition of a key enzyme in the mevalonate pathway, Rab geranylgeranyl transferase (RGGT). The dextrarotatory enantiomer of 2-hydroxy-3-(imidazo[1,2-a]pyridin-3-yl)-2-phosphonopropanoic acid (3-IPEHPC, 1) is the most potent PC-type RGGT inhibitor thus far identified. The absolute configuration of (+)-1 in the active site complex has remained unknown due to difficulties in obtaining RGGT inhibitor complex crystals suitable for X-ray diffraction analysis. However, we have now succeeded in crystallizing (-)-1 and here report its absolute configuration (AC) obtained by X-ray crystallography, thus also defining the AC of (+)-1. An Autodock Vina 1.1 computer modeling study of (+)-1 in the active site of modified RGGT binding GGPP (3DSV) identifies stereochemistry-dependent interactions that could account for the potency of (+)-1 and supports the hypothesis that this type of inhibitor binds at the TAG tunnel, inhibiting the second geranylgeranylation step. We also report a convenient ³¹P NMR method to determine enantiomeric excess of 1 and its pyridyl analogue 2, using α- and β-cyclodextrins as chiral solvating agents, and describe the synthesis of a small series of 1 α-X (X = H, F, Cl, Br; 7a-d) analogues to assess the contribution of the α-OH group to activity at enzyme and cellular levels. The IC₅₀ of 1 was 5-10× lower than 7a-d, and the LED for inhibition of Rab11 prenylation in vitro was 2-8× lower than for 7a-d. However, in a viability reduction assay with J774 cells, 1 and 7b had similar IC₅₀ values, ～10× lower than those of 7a and 7c-d.

Full text of DOI:10.1016/j.ejmech.2011.04.063

European Journal of Medicinal Chemistry 46 (2011) 4820e4826
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, stereochemistry and SAR of a series of minodronate analogues
as RGGT inhibitors
a
,
Katarzyna M. B1azewska b, Feng Ni a, Ralf Haiges a, Boris A. Kashemirova, Fraser P. Coxon c,
_
Charlotte A. Stewart c, Rudi Baron d, Michael J. Rogers c, Miguel C. Seabra d, Frank H. Ebetino e,
,
Charles E. McKenna a
*
a Department of Chemistry, University of Southern California, Los Angeles, CA 90089-0744, USA
b
ꢀ
ꢀ
Institute of Organic Chemistry, Technical Unversity of Łódz, Zeromskiego St. 116, 90-924 Łódz, Poland
c Bone & Musculoskeletal Programme, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK
d Molecular Medicine, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, UK
e Warner Chilcott Pharmaceuticals, Dundalk, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphonocarboxylate (PC) analogues of bisphosphonates are of interest due to their selective inhibition
of a key enzyme in the mevalonate pathway, Rab geranylgeranyl transferase (RGGT). The dextrarotatory
enantiomer of 2-hydroxy-3-(imidazo[1,2-a]pyridin-3-yl)-2-phosphonopropanoic acid (3-IPEHPC, 1) is
the most potent PC-type RGGT inhibitor thus far identified. The absolute configuration of (þ)-1 in the
active site complex has remained unknown due to difficulties in obtaining RGGT inhibitor complex
crystals suitable for X-ray diffraction analysis. However, we have now succeeded in crystallizing (ꢀ)-1
and here report its absolute configuration (AC) obtained by X-ray crystallography, thus also defining the
AC of (þ)-1. An Autodock Vina 1.1 computer modeling study of (þ)-1 in the active site of modified RGGT
binding GGPP (3DSV) identifies stereochemistry-dependent interactions that could account for the
potency of (þ)-1 and supports the hypothesis that this type of inhibitor binds at the TAG tunnel,
inhibiting the second geranylgeranylation step. We also report a convenient 31P NMR method to
Received 26 February 2011
Received in revised form
26 April 2011
Accepted 27 April 2011
Available online 7 May 2011
Keywords:
Phosphonocarboxylates
Bisphosphonates
RGGT inhibitors
X-ray crystallography
Absolute configuration
ee determination
determine enantiomeric excess of 1 and its pyridyl analogue 2, using
solvating agents, and describe the synthesis of a small series of 1
-X (X ¼ H, F, Cl, Br; 7aed) analogues to
assess the contribution of the -OH group to activity at enzyme and cellular levels. The IC50 of 1 was 5
a- and b-cyclodextrins as chiral
a
a
e10ꢁ lower than 7aed, and the LED for inhibition of Rab11 prenylation in vitro was 2e8ꢁ lower than for
7aed. However, in a viability reduction assay with J774 cells, 1 and 7b had similar IC50 values, w10ꢁ
lower than those of 7a and 7ced.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
protein prenylation [5] by inhibiting a downstream mevalonate
pathway enzyme, Rab geranylgeranyl transferase (RabGGTase,
2-hydroxy-3-(imidazo[1,2-a]pyridin-3-yl)-2-phosphonopropa-
noic acid (3-IPEHPC, 1) [1,2] and 2-hydroxy-2-phosphono-3-(pyr-
idin-3-yl)propanoic acid(3-PEHPC, 2) [3]arephosphonocarboxylate
(PC) analogues of two clinical heterocyclic bisphosphonate (BP)
drugs, minodronic and risedronic acids, that block prenylation by
inhibiting a mevalonate pathway enzyme, FPPS [4]. In PC analogues,
one phosphonic acid group of the BP is replaced by a carboxylic acid
RGGT), thus selectively preventing prenylation of Rab family GTPa-
ses [3,6]. Rab proteins are responsible for a broad range of intracel-
lular trafficking events and some were found to contribute to the
aggressiveness and progression of various cancers [7]. 2 has shown
anti-tumour activity in vitro [8,9] and also in an animal model [10].
PC derivatives have proven useful as tools for investigating Rab-
dependent biological processes [11], as well as in bone distribution
studies [12] (Fig. 1).
group, creating a chiral center at the a-carbon. In general, PCs exhibit
lower bone affinity than the BPs but retain some ability to block
RGGT is responsible for transferring two geranylgeranyl (GG)
groups to Rab proteins. There is evidence that PCs prevent only the
second GG transfer to Rab proteins, showing uncompetitive inhibi-
tion against Rab proteins and mixed-type inhibition against ger-
anylgeranyl pyrophosphate (GGPP) [1]. This differentiates them
* Corresponding author.
E-mail address: mckenna@usc.edu (C.E. McKenna).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.063

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K.M. Błazewska et al. / European Journal of Medicinal Chemistry 46 (2011) 4820e4826
4821
Table 1
Selected 31P and 13C NMR chemical shifts of 6 and 7.
Compound 6a
7b
a
-X
31P NMR
20.9
13C NMRc
44.7
(d, JCP ¼ 130.2 Hz)
31P NMR
12.9
H
F
1
11.6
96.9
10.1d
2
2
(d, JPF ¼ 83.9 Hz) (dd, 1JCF
,
1JCP ¼ 202, 159 Hz) (d, JPF ¼ 70.0 Hz)
Cl
Br
14.3
68.5
9.68
1
Fig. 1. Phosphonocarboxylic acids 3-IPEHPC (1) and 3-PEHPC (2).
(d, JCP ¼ 144 Hz)
14.8
57.8
9.68
1
(d, JCP ¼ 142 Hz)
from other RGGT inhibitors, and suggests that they target a different
part of the active site [1]. A crystal structure for the complex of an
engineered form of RGGT lacking LRR and Ig-domains with the
peptide-based inhibitor is now available [13], but information con-
cerning the second prenylation step is sparse [14], limiting rational
design of more potent inhibitors derived from phosphonocarbox-
ylates. On the basis of the crystal structure, a “TAG tunnel” motif has
been proposed to play an important role in the binding of selective
inhibitors of the second prenylation step [13], and this mechanistic
hypothesis is currently being explored [1,14].
Phosphonocarboxylates have not been systematically investi-
gated as RGGT inhibitors, but among a small array of 2 derivatives,
in which a-OH was replaced with a hydrogen or halogen substit-
uent [2,5], four proved to be RGGT inhibitors at low micromolar
concentrations, placing them among the more active RGGT inhib-
itors known so far. As 1 is significantly more potent than 2, it was
a
Spectra recorded in CDCl3.
Spectra recorded in D2O, pH w2.5.
Ca.
b
c
d
Spectra recorded in D2O, pH 12.6.
synthesis, with the three
wardly from a common precursor, the
a
-halo-analogues obtained straightfor-
a-H analogue 6a (Scheme 1).
The point of departure was aldehyde 3, which was converted
into alcohol 4 via reduction with NaBH4 in refluxing methanol.
After conversion of 4 to chloride 5 by the action of thionyl chloride,
excess SOCl2 was evaporated and reaction with the carbanion of
triethyl phosphonoacetate gave 6a, which could be used directly for
the synthesis of the desoxy free acid 7a (see below), or else halo-
genated with Selectfluor, chlorosuccinimide or bromosuccinimide
leading to
tively, in yields ranging from 18 to 93%. The
esters (6b and 6c) were purified by NaHCO3 (aq) washing and
preparative TLC. The -bromo analogue 6d could be completely
a-fluoro-,
a-chloro- and
a
-bromo esters 6bed, respec-
also of interest to examine the contribution of its
activity [15].
a-OH group to its
a
-fluoro- and -chloro
a
a
purified by washing with acetone and ethanol only after hydrolysis
into the free acid. The esters 6aed were hydrolyzed by refluxing in
concentrated HCl to give the free acids 7aed, isolated in 58e86%
yield. The esters were characterized by 31P, 1H, 13C NMR spectros-
copy and by HRMS. The free acids were characterized by 31P, 1H
NMR and elemental analysis. Selected 31P and 13C NMR chemical
shifts for the esters and free acids are presented in Table 1.
2. Synthesis
We prepared a series of 1 derivatives in which the a-hydroxyl
group was replaced by a hydrogen or halogen atom, allowing
a comparison with the 2 series to explore the role of the heterocyclic
base relative to the
a-substituent in determining potency against
RGGT. Anadvantage of the newanalogues comparedto 1 istheirfacile
Scheme 1. Reagents and conditions: (a) NaBH4, MeOH, reflux; (b) SOCl2, reflux; (c) triethyl phosphonoacetate, NaH, DMF, THF, 0ꢂ to RT; (d) 12 M HCl, reflux; (e) Selectfluor, NaH,
THF; (f) N-chlorosuccinimide, NaH, THF; (g) N-bromosuccinimide, NaH, THF.

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K.M. Błazewska et al. / European Journal of Medicinal Chemistry 46 (2011) 4820e4826
4822
Fig. 2. a) Left:Structure of docked (þ)-1 in RGGT/SereCyseSereCys(GG) complex. (subunit A (green mesh), subunit B (cyan mesh), (þ)-1 and SereCyseSereCys(GG) (sphere)).
b) Right: Interactions between (þ)-1 and “TAG” tunnel. ((þ)-1 (stick), protein residue (line)). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
3. Absolute configuration of (D)-1 and its RGGT binding
interaction from modeling
heterocyclic PC compounds. Initially, we used chiral HPLC to
determine ee by separating the enantiomers chromatographically,
but due to peak tailing this method is not ideal for precise ee
assignment. The measured ee values varied, depending on the
column chirality (Fig. 4a).
We then investigated the application of cyclodextrins as chiral
solvating agents to assign the ee of phosphonocarboxylate enan-
tiomers by 31P NMR. NMR spectroscopy is one of the most
commonly used techniques for the assignment of enantiomeric
purity and absolute configuration of different classes of chiral
compounds [20e22]. Enantiomers can be differentiated either by
forming a new covalent bond with a chiral derivatizing agent (CDA),
or by the addition of a chiral solvating agent (CSA), to form a dia-
Given the importance of stereochemistry in the interaction of 1
with RGGT the absolute configuration (AC) of the more potent
enantiomer is essential information, but no crystal structure of PC
inhibitor bound to RGGT has yet been obtained. We succeeded in
obtaining a crystal of (ꢀ)-1 suitable for X-ray diffraction structural
analysis by slow evaporation of an aqueous solution of the pure
enantiomer in the presence of acetone vapour at room tempera-
ture. The crystallographic structure revealed that the (ꢀ)-1 enan-
tiomer has (R) configuration at the a-C (Fig. 3), and therefore the AC
of the more potent (þ)-1 enantiomer must be (S).
Previous work showed that PCs such as 1 and 2 give mixed-type
inhibition with GGPP when inhibiting the second geranylger-
anylation (GG) of Rab protein by RGGT, implying that 1 and 2 may
bind to a site different from the GGPP binding pocket [1]. We
virtually docked (þ)-1 into the active site of monogeranylgerany-
lated peptide-bound rat RGGT (which is 91.4% and 95.8% homolo-
gous to chain A and chain B of human RGGT, respectively) [14] using
two software packages: Autodock Vina 1.1 [16] and Dock 6.3 [17].
The results show that (þ)-1 preferentially binds to a site within the
“TAG” tunnel [13] (Fig. 2a), which is close to the site where
the mono GG-cysteine complex forms and has been proposed as
the location where the first GG-cysteine is translocated [13,18].
Interactions between (þ)-1 and the protein potentially involve one
ionic H-bond between the hydroxy group of (þ)-1 and the
carboxylate of GLU140, salt bridges between the phosphonate and
ARG70/HIS111, a salt bridge between the carboxylate of (þ)-1 and
HIS94, and immersion of the imidazo[1,2-a]pyridine heterocycle in
a hydrophobic pocket formed from TYR44, LEU96, TYR97, TYR107
and PHE145 (Fig. 2b).
stereomeric complex. Enantiomeric a-aminophosphonic acids have
been analyzed by 31P {1H} NMR [23e25]. In this work we found that
acquisition of a 31P {1H} NMR spectrum of racemic phosphono-
carboxylate mixed with the appropriate cyclodextrin in D2O solu-
tion, pH 9.5 gave two signals with baseline peak resolution
(Table 2), enabling ee determination to the accuracy and precision
of the NMR peak integration, including samples of w100% ee
(Fig. 4b). It can be noted that the average of the two chiral HPLC
determinations for ee of the sample with <100% ee ((ꢀ)-1) equals
that obtained by the NMR method (Fig. 4a and 4b, right). The
method was successful with both 1 and 2, but only with a particular
Interestingly, although
a completely different enzyme is
involved, a similar mechanism has been proposed to explain the
interactions of risedronate (Ki ¼ 0.36 nmol) and minodronate
(Ki ¼ 0.0005 nmol) with FPPS, which also has a hydrophobic pocket
constructed chiefly from aromatic amino acid residues [19].
4. NMR analysis of PC chirality aided by cyclodextrin
complexes
In view of the profound effect of 1 chirality on RGGT inhibition,
we sought a method for fast and accurate ee assignment of such
Fig. 3. Molecular structure of (ꢀ)-1 from X-ray crystallographic analysis. The
(R) configuration. Thermal ellipsoids are shown at the 50% probability level.
a-C has

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K.M. Błazewska et al. / European Journal of Medicinal Chemistry 46 (2011) 4820e4826
4823
Fig. 4. Determination of ee in resolved and partially resolved samples of 1 after preparative chiral HPLC: a) Left: chiral HPLC analysis of (þ)-1 isolated as the leading peak on a QN
chiral HPLC column on a preparative HPLC column of opposite chirality (QN-quinine, QD-quinidine). Right: same analysis of (ꢀ)-1, isolated as the trailing peak on a preparative QN
HPLC column; b) Analysis of the same sample of (þ)-1 (left) and (ꢀ)-1 (right) by 31P NMR using
b-CD (D2O, pH 9.5) as a chiral solvating agent.
CD in each case (Table 2). The 6-membered pyridyl ring of 2
apparently fits better into the smaller cavity of -CD, whereas the
larger imidazo[1,2-a]pyridine ring of 1 is more easily accommo-
dated in the larger cavity available with -CD. Therefore, for 1
derivatives -CD was preferentially used, and -CD for 2 deriva-
tives. The Dd values also depend on the other PC -substituent, with
0.67 ppm for the bromo analogue 7d. With the fluoro derivative 7b,
additional splitting in the 31P NMR was observed due to 19F
coupling which masked the baseline separation but the alternative
of 19F NMR for the ee determination could be applied
a
b
b
a
a
(
Dd ¼ 0.08 ppm 31P NMR and Dd ¼ 0.1 ppm 19F NMR). Compared to
b-CD, use of g-CD (increased cavity size) gave worse resolution of
values ranging from 0.03 ppm for the desoxy analogue 7a to
both 1 and 7d (with 10 eq of
g
-CD the values were Dd ¼ 0.012 ppm
and Dd ¼ 0 ppm, respectively). No interference due to residual
buffer (AcOH, TEA) from preparative HPLC separation was
encountered in the 31P NMR resolutions, which eliminated the need
to transfer the enantiomer samples into their free acid form.
Table 2
31P NMR chemical shift nonequivalence (Dd) found for enantiomers of phospho-
nocarboxylates (w2.5 mM).
-CD, 31P NMR
b
eCD, 31P NMR
Dd (ppm)
5. Structureeactivity relationship studies e role of the a-OH
substituent
Compound
(racemic)
a
Dd (ppm)
2
1
7a
7b
7c
7d
0.13a
0.062c
0.045
nd
0.061b
0.13
The desoxy and halo analogues of 1 were tested for potency as
RGGT inhibitors. All of the derivatives 7aed were at least w5 times
less potent than 1 itself (IC50) (Table 3). The analogues inhibited Rab
prenylation and reduced J774 viability, but again with up to 15-fold
lower inhibitory potency `than 1. While the RGGT inhibitory
potencies of 7bed did not differ significantly (IC50 5.56e7.5 mM),
the fluoro analogue 7b was more active in the cell viability and
prenylation assays, with potencies only 1.2e2 times less than 1.
0.03b
0.082
0.379
0.666
0.081
0.055b,c
a
10 eq a-CD.
No baseline separation.
1.25 mM PC.
b
c
Overall, the
a-hydroxyl group has a more profound effect on
inhibitor activity than it does in 2, when comparing the equivalent
analogues [5]. Moreover, in contrast to the 2 analogues [5], where
the fluoro derivative was slightly more active than 2 itself, 1
remains the most active compound in this series.
Table 3
Effect on J774 cell viability, RGGT inhibition and inhibition of Rab11 prenylation of
7aed vs. 1.
Compound
Reduction of J774
viability
(IC50/mM)
RGGT inhibition
(IC50 M)
Inhibition of
Rab11 prenylation
(LED/mM)
a
-X
/
m
6. Conclusions
1
OH
F
Cl
Br
H
0.05
0.06
0.40
>0.60
>0.60
1.24
6.11
7.5
5.56
13.36
3
6
12
25
12
Firstly, the determination of the absolute configuration of the
most potent PC inhibitor of RGGT, 1, supported by computational
results has provided useful insights into the interactions between
the inhibitor and the active site of RGGT, including an explanation
7b
7c
7d
7a

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K.M. Błazewska et al. / European Journal of Medicinal Chemistry 46 (2011) 4820e4826
4824
for the stereochemical preference of the enzyme for the (þ)-1
enantiomer. Comparison of 1 with its racemic -desoxy, -fluoro,
chloro and -bromo analogues suggests that the -hydroxy of 1
contributes to its overall potency as an RGGT inhibitor, which is also
reflected at the level of in vitro cellular prenylation and, except in
the case of 7b which exhibits similar activity, impairment of cellular
viability, providing evidence for the key role of the heterocyclic
warmed to 105 ꢂC, whereupon the temperature rose to 140 ꢂC. The
oil bath was removed until the temperature stabilized at 120 ꢂC. The
reaction mixture was heated for 45 min at 120 ꢂC and 2.5 h at 85 ꢂC,
then cooled and poured into 5% HCl (600 mL) ice-cooled and
brought to pH 9 using 20% NaOH. The resulting solution was
extracted with CH2Cl2 (1200 mL) overnight. Then the organic layer
was separated and dried over MgSO4, the solvent removed under
reduced pressure, the crude product washed with water (5 ꢁ 15 mL)
and again dried, providing the pure product 3.49 g (31%).
a
a
a-
a
a
base, the other
a-substituent, on PC potency against RGGT.
Secondly, in the presence of an appropriate cyclodextrin, 31P NMR
provides a new, more convenient and accurate method of ee
assignment of PCs such as 1 or 2, or 7aed.
7.3. 3-Hydroxymethylimidazo[1,2-a]pyridine (4) [28]
Sodium borohydride (1.61 g, 2.1 eq) was added to a solution of
aldehyde 3 (2.92 g, 0.02 mol) in 400 mL of MeOH. The homogenous
solution was refluxed for 1 h (oil bath, 75 ꢂC). The progress of the
reaction was checked either by NMR (evaporation of 1 mL of
reaction mixture, dissolution in CDCl3) or by TLC (acetone,
Rf(substrate) ¼ 0.56, Rf(product) ¼ 0.24). The reaction mixture was
cooled to RT, quenched with 30 mL of 6 N HCl, evaporated to
dryness and treated with 70 mL of saturated sodium carbonate
solution. The resulting non-homogenous mixture was evaporated
to dryness and the semi-solid residue extracted with CH2Cl2
(5 ꢁ 110 mL), and then for another 2 h with 60 mL of CH2Cl2. The
combined organic extracts were dried over Na2SO4 and evaporated
to dryness, giving 2.53 g of product (85.5%).
7. Experimental
All reagents were purchased from SigmaeAldrich. DMF was of
DrySolv quality. Phosphorus oxychloride, thionyl chloride and
triethyl phosphonoacetate were distilled before use. THF was always
freshly distilled from benzophenone-sodium. For halogenation
reactions, flasks were flame-dried. Halogenation reactions were run
under Ar. NMR spectra were measured on a Varian Mercury 400
spectrometer. Chemical shifts (
(ppm) relative to internal residual CHCl3 in CDCl3
internal residual HDO in D2O (pH w2.5,
4.68 1H), or external H3PO4
0.00, 31P). The final products (free acids, 0.5e1 mg) were dissolved
d
) are reported in parts per million
(d
7.29, 1H),
d
(d
in 0.7 mL D2O, giving pH w2.5. NMR sample solutions were heated
briefly for complete dissolution. Elemental analysis was performed
by Gallbraith Laboratories Inc., Knoxville, TN.
7.3.1. Ethyl 2-(diethoxyphosphoryl)-3-(imidazo[1,2-a]pyridin-3-yl)
propanoate (6a)
Alcohol 4 (1.48 g, 0.01 mol) was added to cold (ice bath) thionyl
chloride (40 mL, 55 eq) and refluxed for 20 min. Gaseous products
and excess SOCl2 were removed in the hood under argon, using
water aspirator and then oil vacuum pump (between the pumps
and the apparatus, a trap cooled with acetone/dry ice was
installed). Then 8 mL of DMF and NaH (0.41 g, 0.017 mol) were
added. The mixture was stirred for 0.5 h at RT. In another flask
triethyl phosphonoacetate (2.24 g, 0.01 eq) in 4 mL of THF was
added dropwise to the cooled mixture of sodium hydride (0.41 g,
0.017 mol) in THF (4 mL). It was stirred on an ice bath for 15 min and
then for 45 min at RT. Chloride 5 in DMF was added via a pipette.
After 2.5 h the reaction mixture was quenched with acetic acid
(0.1 mL) in water (25 mL), pH 6, then extracted with CH2Cl2
(4 ꢁ 50 mL). After drying over Na2SO4, the organic phase was
evaporated and the residue subjected to column chromatography
(gradient elution: AcOEt to AcOEt:acetone,1:2). The product 6a was
isolated in 15% yield, 93% purity. 1H NMR (CDCl3): 1.22 (t,
Biological assays: RGGT assays, assessment of effects on protein
prenylation and measurement of viable J774 cell number were
performed according to literature procedures [5].
Molecular docking: The ligand receptor site is a structure
obtained from RGGT in complex with monogeranylgeranylated
peptide SereCyseSereCys (GG) derived from Rab7 (PDB code:
3DSV). The coordinates of SereCyseSereCys are missing from
the structure, but the coordinates of the GG moiety are clearly
determined [14]. The protein residues and C15 moiety were kept
rigid during the dockingexperiments. Ligands were generated by
Prodrg Server [26]. Autodock Vina 1 [16] was used as the initial
docking tool to generate an inhibitor/enzyme complex using the
following settings: center_x ¼ 5, center_y ¼ 15, center_z ¼ 31,
size_x ¼ 40, size_y ¼ 40, size_z ¼ 40, exhaustiveness ¼ 20,
cpu ¼ 4, num_modes ¼ 20, energy_range ¼ 5. Then the top
ranked inhibitors given by Vina were subjected to rescoring by
the amber_score module implemented in Dock 6.3 [17] using
default settings, generating the energy-minimized final coordi-
nates of the complex for analysis.
3
3JHH ¼ 7.20, C(O)OCH2CH3, 3H), 1.39 (t, JHH ¼ 7.20, P(O)OCH2CH3,
3H), 1.40 (t, 3JHH ¼ 7.20, P(O)OCH2CH3, 3H), 3.34e3.44 (m, CHHCHP,
2H), 3.65 (ddd, 2JHH ¼ 16.0, 3JHP ¼ 12.0, 7.2, CHHCHP, 1H), 4.11e4.27
3
4
(m, OCH2, 6H), 6.88 (dt, JHH ¼ 6.4, JHH ¼ 0.8, CH, 1H), 7.20 (ddd,
3JHH ¼ 8.8, 6.4, JHH ¼ 0.8, CH, 1H), 7.46 (s, CH), 7.61e7.64 (m, CH),
4
8.08 (bd, 3JHH ¼ 6.4, CH). 31P NMR (CDCl3): 20.94. 13C NMR (CDCl3):
14.45 (s, C(O)OCH2CH3), 16.85 (s, P(O)OCH2CH3, 2C), 22.00 (s,
CH2CH), 44.70 (d, 1JCP ¼ 130.2, CHP), 62.30 (s, C(O)OCH2), 63.56 (d,
2JCP ¼ 7.2, P(O)OCH2), 63.67 (d, 2JCP ¼ 5.8, P(O)OCH2), 112.81 (s, CH),
7.1. Enantiomeric purity assignment with cyclodextrins
The appropriate phosphonocarboxylate was dissolved in D2O in
an NMR tube and the pH was adjusted to 9.5 with a few microliters
of 2.5 M NaOH (concentration of PC w2.5 mM, volume 0.6 mL).
3
118.37 (s, CH), 121.44 (d, JCP ¼ 17.00, C), 123.50 (s, CH), 124.29 (s,
Then
spectrum acquired.
a-CD (30 eq) or b
-CD (5 eq) was added and the 31P NMR
CH), 132.09 (s, CH), 146.04 (s, CH), 168.68 (d, 2JCP ¼ 5.85, C]O).
7.3.2. Ethyl 2-chloro-2-(diethoxyphosphoryl)-3-(imidazo[1,2-a]
pyridin-3-yl)propanoate (6c)
7.2. Imidazo[1,2-a]pyridin-3-carbaldehyde (3) [27,28]
To a cooled mixture of NaH (34 mg, 3 eq) in THF (2.5 mL)
a solution of 6a (0.165 g, 0.47 mmol) was added dropwise, giving
a yellow solution, which after stirring for 20 min at 0 ꢂC and 30 min
at RT turned brown. It was then cooled to 0 ꢂC and N-chlor-
osuccinimide (0.126 g, 2 eq) was added in one portion. The mixture
was kept at 0 ꢂC for 1 h and then at RT overnight. On the following
To DMF (120 mL, 1.55 mol) at 2 ꢂC, freshly distilled phosphorus
oxychloride (61 mL, 0.65 mol) was slowly added. The temperature
was allowed to rise gradually to room temperature. The solution was
cooled again to 2 ꢂC and a solution of imidazo[1,2-a]-pyridine 1 (10 g,
0.085 mol) in DMF (60 mL) was added dropwise. The mixture was

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K.M. Błazewska et al. / European Journal of Medicinal Chemistry 46 (2011) 4820e4826
4825
day, 31P NMR showed completion of the reaction. The reaction was
quenched with EtOH (a few drops), diluted with 10 mL CH2Cl2 and
5 mL NaHCO3 (aq) up to pH 8e9. After extraction with CH2Cl2
(30 mL) by vortex, the organic layers were separated and dried over
sodium sulfate and then subjected to prep TLC (CH2Cl2:MeOH 16:1).
Bands were extracted with MeOH (5 ꢁ 10 mL). After the prep TLC
residual succinimide remained (1H NMR: 2.8 ppm (s); 13C NMR:
w31 and w179 ppm). Therefore, a sample of 6c (31.4 mg) was dis-
solved in 1.5 mL DCM and vortexed with NaHCO3 (aq) (1 g in 20 mL)
3 times (3 ꢁ 0.350 mL),1 min each extraction. The organic phase was
dried, giving pure product (yield 93%). Fluoro-6b (18%) and bromo-
6d (60%) analogues were synthesized similarly, using Selectfluor or
bromosuccinimide respectively, instead of chlorosuccinimide.
and re-precipitation from acetone and ethanol, 7a was obtained
(36 mg) as a colourless solid (66%). Similarly obtained were 7b
(58%), 7c (75%), 7d (86%).
7.4.1. 3-(Imidazo[1,2-a]pyridin-3-yl)-2-phosphonopropanoic acid
(7a)
1H NMR (D2O, pH 2.8): 3.31e3.45 (m, 2H), 3.57e3.66 (m, 1H),
3
7.49 (t, JHH ¼ 6.8 Hz, CH, 1H), 7.74 (s, CH, 1H), 7.85e7.94 (m, 2H),
8.63 (d, 3JHH ¼ 6.8 Hz, CH, 1H); 31P NMR (D2O, pH 2.8): 12.93. Anal.
calcd for C10H11N2O5P(H2O)0.5: C 43.02%, H 4.33%, N 10.03%. Found:
C 42.88%, H 4.00%, N 9.91%.
7.4.2. 2-Fluoro-3-(imidazo[1,2-a]pyridin-3-yl)-2-
phosphonopropanoic acid (7b)
7.3.3. Ethyl 2-(diethoxyphosphoryl)-2-fluoro-3-(imidazo[1,2-a]
pyridin-3-yl)propanoate (6b)
1H NMR (D2O, pH 2.7): 3.77e4.12 (m, 2H), 7.45 (t, 3JHH ¼ 6.8 Hz,
CH, 1H), 7.78 (s, CH, 1H), 7.84e7.92 (m, 2CH, 2H), 8.67 (d, 3JHH ¼ 6.8,
CH, 1H). 31P NMR (D2O, pH 12.3): 10.13 (d, 2JPF ¼ 70.6 Hz). 19F NMR
1H NMR (CDCl3): 1.23 (t, 3JHH ¼ 7.20, C(O)OCH2CH3, 3H), 1.39 (t,
3
2
3
3JHH ¼ 7.20, P(O)OCH2CH3, 3H), 1.40 (t, JHH ¼ 7.20, P(O)OCH2CH3,
(D2O, pH 12.3): ꢀ164.5 (dd, JPF ¼ 68.9 Hz, JFH ¼ 41.0 Hz). Anal.
calcd for C10H10FN2O5P(H2O)3.9(C2H5OH)0.01(C3H6O)0.02: C 33.63%,
H 5.03%, N 7.78%. Found: C 33.99%, H 5.43%, N 7.79%.
2
3
3
3H), 3.77 (ddd, JHH ¼ 16.00, JFH ¼ 12.4, JPH ¼ 6.8, CHHCHP, 1H),
2
3
3
3.93 (ddd, JHH ¼ 16.0, JFH ¼ 37.2, JPH ¼ 6.0, CHHCHP 1H),
3
4
4.22e4.35 (m, OCH2, 6H), 6.86 (dt, JHH ¼ 7.2, JHH ¼ 0.8, CH, 1H),
7.21 (ddd, 3JHH ¼ 9.2, 7.2, 4JHH ¼ 1.2, CH, 1H), 7.54 (s, CH), 7.62 (dd,
7.4.3. 2-Chloro-3-(imidazo[1,2-a]pyridin-3-yl)-2-
phosphonopropanoic acid (7c)
3JHH ¼ 9.2, JHH ¼ 0.8, CH), 8.17 (dd, JHH ¼ 7.2, JHH ¼ 1.2, CH). 19
F
4
3
4
NMR (CDCl3): ꢀ176.31 (bdd, JPF ¼ 85.1, JFH ¼ 35.8). 31P NMR
1H NMR (D2O, pH 2.6): 3.81 (dd, 3JHP ¼ 6.4, 2JHH ¼ 16.0 Hz, CH2P,
2
3
(CDCl3): 11.56 (d, 2JPF ¼ 83.9). 13 C NMR: 14.64 (s, C(O)OCH2), 17.04,
1H), 4.24 (dd, JHP ¼ 4.8, JHH ¼ 16.0 Hz, CH2P, 1H), 7.46 (dt,
3JHH ¼ 6.8 Hz, 4JHH ¼ 1.6, CH,1H), 7.85e7.94 (m, 2CH, 2H), 7.87 (s, CH,
3
2
2
17.10 (2s, P(O)(OCH2CH3)2, 2C), 29.00 (d, JCP ¼ 21.1, CH2C), 63.46
2
2
3
(s, C(O)OCH2), 65.16 (d, JCP ¼ 6.7, P(O)OCH2), 65.51 (d, JCP ¼ 5.2,
1H), 8.81 (d, JHH ¼ 6.8 Hz, CH, 1H). 31P NMR (D2O, pH 2.6): 9.68.
1
P(O)OCH2), 96.90 (dd, JCP/CF ¼ 202.0, 159.4, FCP), 112.9 (s, CH),
Anal. calcd for C10H10ClN2O5P(H2O)1.7: C 35.83%, H 4.03%, N 8.35%.
Found: C 35.85%, H 3.74%, N 7.98%.
117.1 (d, J ¼ 10.2, C), 118.5 (s, CH), 124.5, 124.8 (s, CH, CH), 134.7 (s,
CH), 147.0 (s, CH), 167.0 (d, 2J ¼ 18.6, C]O). Isolated by preparative
TLC (acetone:MeOH 15:1) with 18% yield.
7.4.4. 2-Bromo-3-(imidazo[1,2-a]pyridin-3-yl)-2-
phosphonopropanoic acid (7d)
7.3.4. Ethyl 2-chloro-2-(diethoxyphosphoryl)-3-(imidazo[1,2-a]
1H NMR (D2O, pH 2.5): 3.86 (dd, 3JHP ¼ 6.8, 2JHH ¼ 16.4 Hz, CH2P,
3
2
pyridin-3-yl)propanoate (6c)
1H), 4.28 (dd, JHP ¼ 5.2, JHH ¼ 16.4 Hz, CH2P, 1H), 7.45 (t,
3JHH ¼ 7.2 Hz, CH, 1H), 7.84e7.92 (m, 2CH, 2H), 7.90 (s, CH, 1H), 8.81
(d, 3JHH ¼ 6.8 Hz, CH, 1H). 31P NMR (D2O, pH 2.5): 9.68. Anal. calcd
for C10H10BrN2O5P(H2O)1.5: C 31.94%, H 3.48%, N 7.45%. Found: C
32.08%, H 3.30%, N 7.22%.
3
1H NMR (CDCl3): 1.30 (t, JHH ¼ 7.2, C(O)OCH2CH3, 3H), 1.41 (t,
3JHP ¼ 7.2, P(O)OCH2CH3, 3H), 1.42 (t, 3JHP ¼ 7.2, P(O)OCH2CH3, 3H),
2
3
2
3.79 (dd, JHH ¼ 16.0, JHP ¼ 8.8, CHHC, 1H), 4.20 (dd, JHH ¼ 16.0,
3JHP ¼ 6.0, CHHCH, 1H), 4.23e4.44 (m, 3OCH2CH3, 6H), 6.87 (bt,
3JHH ¼ 7.2, CH, 1H), 7.22 (bdd, JHH ¼ 9.6, 7.2, CH, 1H), 7.62 (s, CH,
3
3
3
1H), 7.65 (bd, JHH ¼ 9.6, CH, 1H), 8.29 (bd, JHH ¼ 7.2, CH, 1H). 31P
NMR: 14.29. 13C NMR: 14.32 (s, C(O)OCH2CH3), 16.86 (s,
P(O)(OCH2CH3)2, 2C), 30.23 (s, CH23CP), 64.04 (s, C(O)OCH2), 65.66
7.5. Crystallization of (ꢀ)-1
Fractions of (ꢀ)-1 collected in polypropylene test tubes [2] after
two prep. chiral HPLC purifications were evaporated to dryness and
converted into the free acid form by treatment with Dowex
50 W ꢁ 80e200 cation exchange resin (Hþ form). The fractions
were collected in polypropylene test tubes, evaporated to dryness,
and the yield determined by UV [2].
3
(d, JPC ¼ 7.1, P(O)OCH2), 66.10 (d, JPC ¼ 7.0, P(O)OCH2), 68.50 (d,
1JCP ¼ 144.1, CP), 112.4 (s, CH), 118.2 (s, CH, C, 2C), 124.5 (s, 2CH),
134.8 (s, CH), 146.2 (s, CH), 166.6 (s, 166.6, C]O). Isolated by
preparative TLC (DCM:MeOH 16:1).
7.3.5. Ethyl 2-bromo-2-(diethoxyphosphoryl)-3-(imidazo[1,2-a]
pyridin-3-yl)propanoate (6d)
The resulting (ꢀ)-1 (1.3 mg) was dissolved in water (160
mL), pH
2.5. The vial was left open in a closed jar containing acetone at RT. On
the same day, a precipitate was observed. After 2d, the system was
left open for complete evaporation and after an additional 4 d two
crystals were harvested and subjected to X-ray diffraction analysis.
1H NMR (CDCl3): 1.32 (t, 3JHH ¼ 7.2, P(O)(OCH2CH3)2, 6H), 1.36 (t,
3
2
3JHH ¼ 7.20, C(O)OCH2CH3, 3H), 3.91 (dd, JHP ¼ 16.0, JHH ¼ 12.4,
CHHCP, 1H), 4.16e4.44 (m, 3OCH2CH3, CHHC, 7H), 6.95 (bt,
3JHH ¼ 7.0, CH, 1H), 7.28e7.32 (m, CH, 1H), 7.69 (bd, 3JHH ¼ 8.8, CH,
1H), 8.39 (d, 3JHH ¼ 7.0, CH). 31P NMR: 14.78. 13C NMR: 13.79 (s, C(O)
OCH2CH3), 16.22 (d, 3JCP ¼ 7.95, P(O)OCH2CH3), 16.27 (d, 3JCP ¼ 7.95,
P(O)OCH2CH3), 29.67 (s, CH2CP), 57.80 (d, 1JCP ¼ 141.6, CP), 63.55 (s,
C(O)OCH2), 64.67 (d, 2JCP ¼ 6.6, P(O)OCH2), 65.69 (d, 2JCP ¼ 5.9, P(O)
OCH2), 112.00 (s, CH), 117.45 (s, CH), 118.78 (d, 3JCP ¼ 10.7, C), 124.26
(s, 2CH), 134.07 (s, CH), 145.36 (s, CH), 166.29 (s, C]O). Partially
purified by preparative TLC (DCM:MeOH 16:1).
7.6. Crystal structure determination for (ꢀ)-1
A
specimen of C10H15N2O8P approximate dimensions
0.22 mm ꢁ 0.21 mm ꢁ 0.09 mm, was used for the X-ray crystal-
lographic analysis. The X-ray intensity data were measured on
a Bruker SMART APEX CCD system equipped with a graphite
monochromator and a Mo Ka sealed tube (
l
¼ 0.71073 Å) using
SMART V 5.625 (Bruker AXS, 2001). A total of 1850 frames with an
exposure time of 10 s were collected. The frames were integrated
with the SAINT V 6.22 program package (Bruker AXS, 2001).
The integration of the data using a triclinic unit cell yielded a total
of 5732 reflections, of which 4674 were independent
7.4. General method for hydrolysis of triesters
The ester 6a (70.6 mg, 0.20 mmol) was dissolved in concen-
trated HCl (3 mL) and refluxed for 6 h. After evaporation to dryness

_
K.M. Błazewska et al. / European Journal of Medicinal Chemistry 46 (2011) 4820e4826
4826
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(completeness ¼ 95.4%, Rint ¼ 1.42%). The final cell constants were
a ¼ 8.1581(11) Å, b ¼ 9.0992(12) Å, c ¼ 9.3617(12) Å,
a
¼
105.929(2)ꢂ,
b
¼ 97.568(2)ꢂ, and
g
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mission coefficients (based on crystal size) are 0.791 and 0.965.The
structure was solved by direct methods and refined using the
Bruker SHELXTL 5.10 Software Package (Bruker AXS, 2000), using
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refinement on F2 with 431 variables converged at R1 ¼ 3.7%, for the
observed data and wR2 ¼ 9.36% for all data.
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CCDC813786. Copies of the data can be obtained, free of charge, on
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Acknowledgement
This work was supported by Procter & Gamble Pharmaceuticals,
Inc. and Warner Chilcott Pharmaceuticals. K.M.B. was a 2007e2009
USC WiSE Fellow.
Appendix. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.04.063.
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