Design of potent bisphosphonate inhibitors of the human farnesyl pyrophosphate synthase via targeted interactions with the active site 'capping' phenyls

Article abstract of DOI:10.1016/j.bmc.2012.07.019

Nitrogen-containing bisphosphonates (N-BPs) are potent active site inhibitors of the human farnesyl pyrophosphate synthase (hFPPS) and valuable human therapeutics for the treatment of bone-related malignancies. N-BPs are also useful in combination chemotherapy for patients with breast, prostate and multiple myeloma cancers. A structure-based approach was employed in order to design inhibitors that exhibit higher lipophilicity and better occupancy for the GPP sub-pocket of hFPPS than the current therapeutic drugs. These novel analogs were designed to bind deeper into the GPP sub-pocket by displacing the side chains of the 'capping' residue Phe 113 and engaging in favorable π-interactions with the side chain of Phe112.

Full text of DOI:10.1016/j.bmc.2012.07.019

Bioorganic & Medicinal Chemistry 20 (2012) 5583–5591
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
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Design of potent bisphosphonate inhibitors of the human farnesyl
pyrophosphate synthase via targeted interactions with the active
site ‘capping’ phenyls
Joris W. De Schutter a, Joseph Shaw b, , Yih-Shyan Lin a, Youla S. Tsantrizos a,b,
⇑
a Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, Canada H3A 0B8
b Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC, Canada H3G 0B1
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitrogen-containing bisphosphonates (N-BPs) are potent active site inhibitors of the human farnesyl
pyrophosphate synthase (hFPPS) and valuable human therapeutics for the treatment of bone-related
malignancies. N-BPs are also useful in combination chemotherapy for patients with breast, prostate
and multiple myeloma cancers. A structure-based approach was employed in order to design inhibitors
that exhibit higher lipophilicity and better occupancy for the GPP sub-pocket of hFPPS than the current
therapeutic drugs. These novel analogs were designed to bind deeper into the GPP sub-pocket by displac-
Received 22 May 2012
Revised 4 July 2012
Accepted 13 July 2012
Available online 24 July 2012
Keywords:
Nitrogen-containing bisphosphonates
Farnesyl pyrophosphate synthase
ing the side chains of the ‘capping’ residue Phe 113 and engaging in favorable
chain of Phe112.
p-interactions with the side
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
pre-clinical and clinical investigations have provided evidence for
multiple mechanisms that mediate N-BP-induced antitumor ef-
Human farnesyl pyrophosphate synthase (hFPPS) occupies the
first branching point of the mevalonate pathway and catalyzes
the sequential elongation of dimethylallyl pyrophosphate (DMAPP)
to geranyl pyrophosphate (GPP) and then to farnesyl pyrophos-
phate (FPP), via sequential condensation of isopentenyl pyrophos-
phate (IPP) units. Consequently, hFPPS also controls intracellular
levels of all isoprenoid metabolites and modulates the post-trans-
lational prenylation of the Ras family of GTPases (e.g., H-Ras, K-Ras,
N-Ras) that are essential for cell survival.1 It is well established that
mutated Ras proteins are predominant in oncogenesis,1 prompting
the development of farnesyltransferase (FTase) inhibitors (FTI) as
potential therapeutics for the treatment of cancer.1 Despite the
promising in vitro and preclinical efficacy of these inhibitors, only
a small subset of patients responded to FTI therapy, leading to the
realization that in vivo inhibition of farnesylation induces geranyl-
geranylation of the mutated Ras proteins, restoring their biological
function. This redundancy mechanism suggests that upstream di-
rect inhibition of FPP biosynthesis may be more effective in reduc-
ing Ras prenylation than inhibition of the FTase enzyme.
fects, including apoptotic cell death, cell cycle disruption and cd
T cell activation.4,5 N-BP-induced inhibition of cell proliferation
and apoptosis has been reported in prostate,6 and breast cancer,7,8
multiple myeloma cells,9,10 and other types of cancers. The apopto-
tic effects of N-BPs has been correlated with the intracellular accu-
mulation of IPP (as a consequence of the hFPPS inhibition) and the
formation of an ATP derivative (ApppI),4,5 which inhibits the mito-
chondrial adenine nucleotide translocase and induces apoptosis.
Additionally, IPP is a natural antigen of
by human 2 Vd2-bearing cells (alternate nomenclature
9 Vd2).11 During tumor invasion (or infection),
d T cells
expressing V 2 Vd2 T-cell antigen receptors (TCRs) expand to high
cd T cells that is recognized
V
c
T
Vc
c
c
levels and, in some cases, they can represent the majority of circu-
lating T cells. Short hairpin RNA-mediated knockdown of hFPPS in
hematopoietic and non-hematopoietic tumor cell lines has been
shown to activate V
c2 Vd2 T cells and induce IFN-
c
secretion.12
Evidence for the stimulation of V
c
2 Vd2-bearing T cells by N-BPs
has been observed in multiple myeloma patients treated with
pamidronate (3)13 and prostate cancer patients treated with
Currently, nitrogen-containing bisphosphonates (N-BPs; Fig. 1),
such as zoledronate (1) and risedronate (2a), are the only class of
clinically validated drugs that target the human FPPS.2,3 Recent
zoledronate (1);6 in the latter case, the observed
cd T cell stimula-
tion coincided with reduction in serum prostate-specific antigen
(PSA), providing further evidence of an antitumor immune re-
sponse that is induced by N-BPs in vivo. Interestingly, although
good correlation between hFPPS inhibition and cd T cell activation
has been observed, N-BPs that target other closely related
enzymes, such as the human geranylgeranyl pyrophosphate syn-
thase (hGGPPS; e.g., compound 5) or the human undecaprenyl
⇑
Corresponding author. Tel.: +1 514 398 3638; fax: +1 514 398 3797.
E-mail address: youla.tsantrizos@mcgill.ca (Y.S. Tsantrizos).
Undergraduate research participant
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.019

5584
J. W. De Schutter et al. / Bioorg. Med. Chem. 20 (2012) 5583–5591
N
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
N
N
N
PO(OH)2
H2N
O
9
OH
R'
OH
1
2a, R' = OH
2b, R' = H
3
4
PO(OH)2
PO(OH)2
O
(HO)2OP
PO(OH)2
OH
5
6
Figure 1. Examples of known N-BPs that inhibit hFPPS, hGGPPS or E. coli UPPS.
pyrophosphate synthase (hUPPS; e.g., compound 6) do not induce
c
Preparation of analogs 10 originated from 4-carboxypyridine-1-
oxide (14) which was first converted to the methyl ester, with
MeOH under acid conditions and azeotropic dehydration
(Scheme 2). The pyridine N-oxide ester was subsequently con-
verted to the corresponding 2-aminopyridine 15 using t-BuNH2
and tosyl anhydride as the activating reagent, followed by removal
of the t-butyl group with TFA.20 Conversion of the aniline to the
bisphosphonate tetraesters 16a was achieved using previously re-
ported conditions.17 Saponification of the methyl esters 16a to the
corresponding acid (16b) was performed at 0 °C in order to prevent
partial deprotection of the tetraethyl bisphosphonate esters. Amide
bond formation was catalyzed by HATU and the final bisphosphon-
ic acids were obtained after treatment with TMSBr followed by
methanolysis and trituration to give the final N-BPs of general
structure 10 (Scheme 2).
d T cell activation.14,15
The structural features and physicochemical properties of the
current N-BP drugs (e.g., 1 and 2a) limit their therapeutic use to
bone-related diseases, due to their high affinity for bone, poor oral
bioavailability, very low cell membrane permeability and negligi-
ble distribution into non-skeletal tissues.16 Therefore, the identifi-
cation of more lipophilic, potent and selective inhibitors of hFPPS is
of considerable interest. Recently, we reported the design of novel
lead structures that bind to a larger portion of the active site cavity
of hFPPS than the current drugs 1 and 2a and are also more lipo-
philic.17 In this report, we describe our subsequent efforts in
exploring the interactions between these inhibitors and the GPP
sub-pocket of the hFPPS active site.
2. Results
2.2. Biological data and discussion
2.1. Chemistry
2.2.1. Human FPPS inhibition and SAR
Inhibition of the human recombinant enzymes FPPS (hFPPS)
and GGPPS (hGGPPS) were determined using the in vitro assays de-
scribed previously.17,21 For efficiency, all compounds were initially
The synthesis of novel N-BPs is outlined in Schemes 1 and 2.
Analogs 7 and 8 were synthesized as previously reported.17 The
synthesis of analogs 9 was initiated by the condensation of 5-
bromopyridin-3-amine (11) with triethyl orthoformate and diet-
hylphosphite at 100 °C to give the bisphosphonate tetraethyl ester
12 in moderate yield (ꢀ45% isolated yield; Scheme 1). We noted
that this condensation leads to significant decomposition if carried
out at 120 °C, whereas at 80 °C the reaction proceeds very slowly.
Suzuki cross-coupling of intermediate 12 with the boronic acids or
boronate esters of fragments shown in Table 1 provided the biaryl
intermediates 13 in variable yields (35–99% isolated yields).
Deprotection of the bisphosphonate tetraesters was achieved after
treatment with TMSBr, followed by methanolysis and trituration to
give the final N-BP inhibitors of general structure 9 (Scheme 1;
Table 1).
Condensation of intermediate 12 with benzyl potassium triflu-
oroborate, as previously described by Molander,18 provided the
tetraethyl ester precursor to analog 9c. The boronate reagent of
fragment g was prepared via cyclopropanation of the precursor vi-
nyl ether using Simmons–Smith-type conditions with TFA as an
additive.19 Details on the synthesis of all non-commercial boronate
reagents are described in the supporting information.
tested at a single concentration of 1
lM and only those analogs
exhibiting greater than 80% inhibition at 1
l
M (average of tripli-
cate determinations; standard deviation of <10%) were profiled
further to determine their IC50 values (Table 2). Representative
examples were also evaluated of their ability to inhibit the struc-
turally and functionally related enzyme hGGPPS.
Recently, we reported the co-crystal structure of analog 7f
which binds deeper into the GPP sub-pocket of the hFPPS active
site than the clinical drugs 1 and 2a.17 In the hFPPS/7f complex,
the pyridine nitrogen of the inhibitor participates in a H2O-medi-
ated hydrogen-bond interactions with the side chain carbonyl oxy-
gen of Gln 254. In contrast, the heterocyclic side chains of 1 and 2a
form bifurcated hydrogen-bonds with the amide carbonyl of Lys
200 and the side chain oxygen of Thr 201.2,3 These hydrogen-bonds
play a significant role in the potency of 1 and 2a in inhibiting
hFPPS. For example, a >280-fold loss in potency has been observed
when the pyridine side chain of 2a is replaced by a simple phenyl
ring.22 Superposition of the co-crystal structures of hFPPS/2a (PDB:
1YV5) and hFPPS/7f (PDB: 4DEM) revealed that the pyridine rings
H
H
Br
NH2
Br
N
PO(OEt)2
R
N
PO(OEt)2
PO(OEt)2
d
a
b, or c
9
PO(OEt)2
N
N
N
11
12
13
Scheme 1. Reagents and conditions: (a) HP(O)(OEt)2, CH(OEt)3, toluene, 100 °C; (b) boronic acid, Pd(PPh3)4, 2 M K2CO3, dioxane,
trifluoroborate, Pd(dppf)Cl2ÁCH2Cl2, Cs2CO3, dioxane, W 125 °C (ꢀ40 W); (d) (i) TMSBr, DCM; (ii) MeOH.
lW 125 °C (ꢀ40 W); (c) benzyl potassium
l

J. W. De Schutter et al. / Bioorg. Med. Chem. 20 (2012) 5583–5591
5585
H
CO2Me
CO2R'
CO2H
N
O
R
e,f
c,d
a, b
PO(OEt)2
PO(OH)2
N
NH2
N
N
N
O
14
PO(OEt)2
H
N
N
H
PO(OH)2
15
16a, R' = Me
16b, R' = H
10
Scheme 2. Reagents and conditions: (a) cat. H2SO4, MeOH, toluene, reflux, Dean–Stark; (b) (i) Ts2O, CHCl3, 0 °C; (ii) t-BuNH2, (iii) TFA, 70 °C; (c) HP(O)(OEt)2, CH(OEt)3,
toluene, 120 °C, overnight; (d) LiOH, THF/H2O, 0 °C; (e) RNH2, HATU, DIPEA, DMF; (f) (i) TMSBr, DCM; (ii) MeOH.
Table 1
Novel N-BP inhibitors.
of both inhibitors overlapped in their hFPPS-bound conformations
(Fig. 2). However, the pyridine nitrogen of 2a (which is at the meta-
position relative to the C-b of the bisphosphonate moiety) could
more easily engage in bifurcated hydrogen-bond interactions with
Lys 200 and Thr 201 than analog 7f. We presumed that the loss of
interactions between pyridine side chain and the Lys 200/Thr 201
residues may be (in part) the reason for the lower in vitro potency
of analogs with general structure 7 as compared to 2a (e.g., 7a is
approximately eightfold less potent than 2a, under our assay con-
ditions; Table 2). However, we observed that analogs 7b and 8b are
almost equipotent (Table 2). Although the reasons for these results
are not clearly apparent, a possible explanation may be that the
O
H
N
R
Z
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
RHN
Y
N
X
7
, X = CH2, Y = N, Z = NH, R = a-j
10, R = a-j
8, X = N, Y = CH, Z = CH2, R = a-j
9, X = N, Y = CH, Z = NH, R = a-j
H
higher acidity of a Ca-amino bisphosphonate (i.e., 7b) may com-
pensate for the loss of H-bond interactions between the side chains
of analogs 7 and Lys 200/Thr 201. Consistent with the latter
assumption, a threefold increase in potency was observed for ana-
log 9b as compared to 7b (Table 2). The overall sp2 character of the
exocyclic amine linker (via conjugation of its lone-pair of electrons
with the aromatic ring) may also contribute to the stabilization of
a
b
c
f
d
MeO
O
O
the bioactive conformation of analogs 7 and 9, in which the Ca of
the bisphosphonate moiety is nearly co-planar (<10° out of plane)
e
g
with respect to the pyridine core.
In the co-crystal structure of the hFPPS-bound 7f, the 4-isopro-
poxyphenyl side-chain is almost co-planar with the pyridine ring
and inserted between the side chains of Phe 113 and Gln 185,
H
O
N
O
HN
HN
S
O
O
h
i
j
Table 2
Inhibition data for hFPPS
Compound
hFPPS IC50 (nM)
SASAe
FISAe
2aa
7ab
7bb
7eb
7fa
11
85
150
115
28
433
427
548
562
624
638
544
542
558
618
632
661
671
663
594
570
609
262
239
238
238
238
238
239
250
251
251
251
288
317
291
281
276
281
7ga
8ba
9ba
9ca
9fa
12
164
47
ꢀ1000c
60
9ga
9ha
9ia
16
IN
IN
9ja
>1000d
IN
10ba
10da
10ea
ꢀ1000c
IN
Figure 2. Superposition of X-ray structures of N-BPs bound to the active site of
hFPPS, with the bound inhibitors and key residues shown in stick form. The carbon
backbone of 2a is highlighted in cyan, and 7f in green. Oxygen, nitrogen and
phosphorus atoms are coloured in red, blue and orange, respectively. The yellow
spheres represent the Mg2+ ions. The bifurcated H-bonds of 2a with the backbone
carbonyl of Lys 200 and the side chain hydroxyl of Thr 201 are highlighted with
dashed lines; similarly, the water (red sphere) mediated H-bond of the pyridine
nitrogen of 7f with Gln 254 is also indicated. The phenyl moiety of the 4-
isopropoxyphenyl side chain of 7f is ‘stacked’ between the side chains of Phe 113
and Gln 185.
a
Compound exhibited less than 50% inhibition in the hGGPPS enzymatic assay at
10
lM.
b
Compound was not tested in the hGGPPS enzymatic assay.
c
d
e
55–65% inhibition at 1
40% inhibition at 1 M; IN = inactive at 1
Calculated using QikProp 3.2; SASA = total solvent accessible surface area in A2
lM.
l
lM.
using a probe with 1.4 A radius; FISA = hydrophilic component of SASA.

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J. W. De Schutter et al. / Bioorg. Med. Chem. 20 (2012) 5583–5591
engaging in stacking interactions (Fig. 2). In an effort to optimize
these interactions, a number of derivatives were synthesized vary-
ing the electronic character and conformation of the—R substituent
in analogs 7, 8, 9 and 10. Disruption of the co-planarity between
the side chain pyridine moiety and the—R substituent by the intro-
duction of a simple methylene linker resulted in greater than 20-
fold loss in potency (e.g., analogs 9b vs 9c; Table 2). We expected
that an amide bond would maintain the overall co-planarity of
polar linker, including an amide, a reversed amide and a sulfon-
amide, that could perhaps orient the cyclopropyl moiety more
favorably towards the aromatic side chain of Phe 112. Unfortu-
nately, all these analogs (e.g., 9h, 9i and 9j) proved to be signifi-
cantly less potent than 9g, leading to less than 50% inhibition of
hFPPS at even 1 lM concentration of inhibitor (Table 1).
Many of these analogs were also tested in our enzymatic assay
using the related human enzyme GGPPS and found to exhibit less
the side chain and engage in favorable
p
-stacking interactions with
than 50% inhibition at 10 lM concentration (Table 2). This ob-
Phe 113. However, the introduction of such a linker was equally
detrimental to the potency of these compounds (e.g., IC50 of 9b
vs 10b; Table 2). A number of other modifications were also eval-
uated, including analogs with small side chains (e.g., 10d) and all
were found to be detrimental. Based on these results, we con-
cluded that a direct aryl–aryl bond between the pyridine core
and aromatic side chain is best suited to occupy the extended lipo-
philic channel formed upon binding of inhibitor 7f to the GPP sub-
pocket. It is also possible that this lipophilic channel does not tol-
erate binding of polar groups, such as an amide moiety.
In addition to the stacking interactions of the 4-isopropoxy-
phenyl moiety of 7f with the side chains of Phe 113 and Gln 185
(Fig. 2), we also observed that the isopropyl substituent was pro-
tein-bound within Van der Waals distance from the aromatic side
chain of Phe 112. Our SAR studies also suggested favorable interac-
tions between the inhibitor side chain and Phe 112. For example,
the methoxy derivative 7e was only slightly more potent than
7b, whereas analog 7f was approximately fivefold more potent
than 7b (Table 2). Consequently, we assumed that the correspond-
ing cyclopropyl analog 7g could further increase interactions with
served selectivity is uncommon for N-BP compounds that have lar-
ger side chains, such as compound 4, which is a dual inhibitor of
hFPPS and hGGPPS (with IC50 values of 100 and 280 nM, respec-
tively)14 and compound 5 which inhibits only hGPPS (an IC50 value
of 410 nM was determined under our assay conditions).
3. Conclusion
The current N-BP drugs exhibit poor cell-membrane
permeability and bind so avidly to bone that their penetration into
non-skeletal tissues is practically negligible. The
Ca-hydrox-
ybisphosphonate moiety is the main pharmacophore anchoring
these drugs to the active site of hFPPS, via chelation of three Mg2+
ions that also interact with highly conserved aspartic acid residues
within the GPP sub-pocket of the active site cavity. In spite numer-
ous efforts from both academic and industrial research group, to
date, the identification of highly potent and selective inhibitors of
hFPPS, with low affinity for bone, and sufficiently high cell mem-
brane permeability to provide good exposure of the drug in non-
skeletal tissues, remains a huge challenge.
The active site occupancy of the enzyme-bound current drugs
(e.g., 1 and 2a) is hugely suboptimal, leaving a large portion of
the cavity unoccupied. Based on this knowledge, we focused our
efforts in designing N-BP analogs that have a more optimal shape,
size and electrostatic surface complementarity with the hFPPS ac-
tive site, lower affinity for bone and increased lipophilicity. We be-
gan our studies with the virtual screening of over 1000 pyridine-
based compounds, using docking calculations.21 However, the cor-
relation between our in silico predictions and the experimental
data was generally poor;17 this observation is not unexpected, gi-
ven the high protein plasticity of hFPPS. Nonetheless, in silico
docking provided some reassurance in guiding the design of ana-
logs with very conservative structural variations and based on
the X-ray structure of our lead compound 7f.17 For example, the
design of the cyclopropoxy derivatives 7g and 9g (IC50 values of
12 and 16 nM, respectively) were based on the co-crystal structure
of 7f (IC50 = 28 nM).
the aromatic ring of Phe 112 via CH/
p-interactions (due to the
inherent -character of the C–C bonds in the cyclopropyl ring). In-
p
deed, this minor modification improved the enzymatic potency
approximately threefold in series 7 and approximately fourfold in
series 9 (e.g., 7f vs 7g and 9f vs 9g, respectively; Table 2). Consis-
tent with our prediction, in silico docking (using GLIDE) of 7g indi-
cated that the carbon atoms of the cyclopropyl ring would bind
with a ꢀ3.4 Å distance from the aromatic ring of Phe 112, allowing
for favorable CH/p
-interaction (Fig. 3).23–26
We also explored different linkers between the phenyl side-
chain and the cyclopropyl moiety of inhibitor 9g. We noted that
in the co-crystal structure of the hFPPS/7f the isopropyl moiety is
partly solvent exposed, consequently, we decided to introduce a
In summary, as part of our lead optimization efforts, we synthe-
sized analogs that are able to induce conformational changes to the
active site cavity, enlarge the available binding space within the
GPP sub-pocket by displacing the side chains of the Phe 113 and
Gln 185 and engage in novel interactions with Phe 112. In contrast
to previously reported N-BPs that have a large side chain (e.g., 4, 5
and 6) and poor ability to inhibit hFPPS, compounds 7f, 7g and 9g
exhibit an unusually high selectivity in inhibiting hFPPS versus
hGGPPS; the latter enzyme represents the most closely related hu-
man enzyme. Furthermore, the relative hydrophobicity of our most
potent analogs 7f, 7g, 9g is significantly higher than that of the
drug risedronate (2a), as estimated by the % hydrophilic compo-
nent of the total solvent accessible surface area of each molecule
in Å2 (Table 2; calculations were carried out using QuikProp and
a probe with 1.4 Å radius). For example, the% hydrophilic compo-
nent (out of the total surface area) of 2a is approximately 61% as
compared to 38–40% for inhibitors 7f, 7g, 9g. These novel com-
pounds represent promising leads towards the identification of po-
tent and selective hFPPS inhibitors with better biopharmaceutical
properties than the current drugs.
Figure 3. Docking output structure of inhibitor 9g (using GLIDE version 5.5,
Shrödinger, LLC, New York, NY 2009; standard parameters of XP-mode were used)
in the hFPPS/7f co-crystal structure (PDB: 4DEM). The predicted bifurcated H-bond
and cyclopropyl-p interactions are indicated with dashed lines.

J. W. De Schutter et al. / Bioorg. Med. Chem. 20 (2012) 5583–5591
5587
until complete consumption of starting material (ꢀ30 min) was
observed. TFA (10.0 ml, 131 mmol) was added to the reaction mix-
ture and the reaction was stirred at 70 °C overnight. The mixture
was diluted with water (5 mL) and DCM (10 mL) and carefully neu-
tralized with saturated NaHCO3. The aqueous layer was extracted
using DCM (4 Â 10 mL). The combined organic layers were washed
once with brine, dried over MgSO4, filtered and concentrated in va-
cuo. The residue was purified by silica gel chromatography using a
CombiFlash instrument and a solvent gradient from 100% hexanes
to 100% EtOAc and then to 25% MeOH in EtOAc (all eluents con-
tained 0.1% triethylamine) to yield the desired methyl 2-aminoiso-
nicotinate product obtained as reddish/brown crystals (153.3 mg,
51%). 1H NMR (400 MHz, CDCl3) d 8.19 (d, J = 5.2 Hz, 1H), 7.16
(dd, J = 5.3, 1.3 Hz, 1H), 7.07 (s, 1H), 4.59 (s, 2H), 3.94–3.89 (m,
3H); consistent with literature.20
4. Experimental
4.1. General procedures for characterization of compounds
All intermediate compounds were purified by normal phase
flash column chromatography on silica gel using a CombiFlash
instrument and the solvent gradient indicated. The purified bis-
phosphonate tetra ester precursors of all final inhibitors were ana-
lyzed by reverse-phase HPLC and fully characterized by 1H, 13C and
31P NMR, and MS. The homogeneity of the bisphosphonate tetra es-
ters (>90%) was confirmed by C18 reversed phase HPLC, using
Method A indicated below. After ester hydrolysis, the final bisphos-
phonic acid products were precipitated and washed with deionized
H2O, HPLC grade acetonitrile and distilled Et2O to obtain the inhib-
itors as white solids; quantitative conversion of each tetra ester to
the corresponding bisphosphonic acid was confirmed by 31P NMR.
All final products were characterized by 1H, 13C, 31P and 19F NMR
and HR-MS. Chemical shifts (d) are reported in ppm relative to
the internal deuterated solvent (1H, 13C) or external H3PO4 (d
0.00 31P), unless indicated otherwise. Low- and High-Resolution
MS spectra were recorded at the McGill University, MS facilities
using electrospray ionization (ESI ). The homogeneity of the final
inhibitors was confirmed by HPLC using Method B (indicated be-
low). Only samples with >95% homogeneity were tested in the
enzymatic assays for the determination of IC50 values. HPLC Anal-
ysis was performed using a Waters ALLIANCEÒ instrument (e2695
with 2489 UV detector and 3100 mass spectrometer).
4.3. Synthesis of bisphosphonate tetraethyl esters
4.3.1. Synthesis of methyl 2-((bis(diethoxyphosphoryl)methyl)
amino)isonicotinate (16a)
A 15 mL pressure vessel was charged with the methyl 2-amino-
isonicotinate (50 mg, 0.33 mmol), diethyl phosphite (0.25 ml,
2.0 mmol), triethyl orthoformate (0.07 ml, 0.4 mmol) and dissolved
in 1 ml toluene. The mixture was stirred at 120 °C overnight. The
solution was cooled to room temperature, diluted with ethyl ace-
tate, washed with saturated NaHCO3, brine, dried over MgSO4, fil-
tered and concentrated in vacuo. The residue was purified by silica
gel (silica was pre-washed with 0.1% NEt3 in hexanes/EtOAc 3:1)
chromatography on a CombiFlash instrument, using a solvent gra-
dient from 1:3 EtOAc/Hexanes to 100% EtOAc and then to
25%MeOH in EtOAc to give the title compound as an pale yellow
powder (106.4 mg, 74%). 1H NMR (400 MHz, CDCl3) d 8.21 (d,
J = 5.2 Hz, 1H), 7.16 (d, J = 5.3 Hz, 1H), 7.10 (s, 1H), 5.51 (td,
J = 22.1, 10.1 Hz, 1H), 5.00 (d, J = 10.1 Hz, 1H), 4.26–4.07 (m, 8H),
3.92 (s, 3H), 1.25 (td, J = 7.1, 5.1 Hz, 12H).
4.1.1. HPLC sample preparation
A precise amount of bisphosphonate inhibitor was weighed out,
suspended in Milli-Q grade water and dissolved by the addition of
1–4 equiv of a 1.030 M standardized NaOH solution. The samples
were then diluted to 1 or 2 mM concentration and 10
jected for HPLC analysis.
ll was in-
Method A: Waters Atlantis T3 C18 5
Solvent A: H2O, 0.1% formic acid
Solvent B: CH3CN, 0.1% formic acid
lm
13C NMR (75 MHz, CD3OD) d 165.58, 130.92, 122.52, 113.55,
110.87, 107.87,
d 63.47 (d, J = 17.7 Hz), 54.46, 44.49 (t,
J = 149.9 Hz), 15.90–14.76 (m).
Mobile phase: linear gradient from 95%A and 5%B to 5%A and
95%B in 13 min, then 2 min at 100% B
Flow rate: 1 mL/min
31P NMR (81 MHz, CD3OD) d 18.16 (s). MS (ES+) m/z: 461.0
[M+Na+]+ (C16H28N2NaO8P2).
Method B: Waters XSELECT CSH fluoro-phenyl, 5
Solvent A: H2O, 0.06% TFA
Solvent B: CH3CN, 0.06% TFA
lm column
4.3.2. Synthesis of tetraethyl (((5-bromopyridin-3-yl)amino)
methylene)bis (phosphonate) (12)
A 15 mL pressure vessel was charged with the 5-bromopyridin-
3-amine (250 mg, 1.44 mmol), diethyl phosphite (1.12 ml,
8.67 mmol), triethyl orthoformate (0.29 ml, 1.73 mmol) and dis-
solved in 3 ml toluene. Stirred at 80 °C for 18 h, 13P NMR showed
only 10% conversion. The reaction was stirred another 14 h at
100 °C. Cooled down and concentrated in vacuo. The residue was
purified by silica gel (silica was pre-washed with 0.1% NEt3 in hex-
anes/EtOAc 3:1) chromatography on a CombiFlash instrument,
using a solvent gradient from 1:3 EtOAc/hexanes to 100% EtOAc
and then to 25%MeOH in EtOAc. The product thus obtained was
contaminated with ꢀ10% of the mono-phosphonate analog, the
mixture was purified by reverse phase chromatography on a Com-
biFlash instrument using a solvent gradient from 10% CH3CN/water
to 100% CH3CN. The desired compound was isolated as an off-
white powder (290 mg, 44%).
Mobile phase: linear gradient from 99%A, 1%B to 100%B in
10 min, then 5 min at 100% B
4.2. Synthesis of methyl 2-aminoisonicotinate (15)
Isonicotinic acid N-oxide (3.00 g, 21.6 mmol) was dissolved in
20 ml 1:1 methanol/toluene, a catalytic amount H2SO4 was added
and the flask was equipped with a Dean Stark trap filled with toluene.
The mixture was refluxed overnight. The reaction was terminated by
cooling in an ice bath, and neutralized with satd. sodium carbonate.
The solution was extracted using (5 Â 20 ml) DCM; the organic
phases were combined, washedwithbrine, driedwith MgSO4, filtered
and concentrated invacuo. Theresidue waspurified bysilica gelchro-
matography using a CombiFlash instrument and a solvent gradient
from 100% hexanes to 100% EtOAc to give 4-(methoxycarbonyl)
pyridine 1-oxide as
a
white powder (1.46 g, 45%). 1H NMR
1H NMR (400 MHz, DMSO-d6) d 8.26 (s, 1H), 7.85 (s, 1H), 7.59 (s,
1H), 6.44 (d, J = 10.8 Hz, 1H), 4.84 (td, J = 22.8, 10.7 Hz, 1H), 4.16–
3.93 (m, 8H), 1.15 (dt, J = 20.7, 7.0 Hz, 12H).
(300 MHz, CDCl3) d 8.24–8.16 (m, 2H), 7.93–7.79 (m, 2H), 3.94 (s, 3H).
To an ice-cooled solution of the above product (4-(methoxycar-
bonyl)pyridine 1-oxide, 300 mg, 1.96 mmol) in CHCl3 was added
tosyl anhydride (2877 mg, 8.82 mmol) and the mixture was stirred
for 5 min. Next, t-BuNH2 (1.24 ml, 11.8 mmol) was added dropwise
via syringe to the activated N-oxide while maintaining the reaction
temperature below 5 °C. The reaction was monitored by TLC and
13C NMR (126 MHz, DMSO-d6) d 145.31 (t, J = 4.2 Hz), 138.10,
135.62, 121.06, 120.55, 63.08 (dt, J = 17.4, 3.1 Hz), 47.86 (t,
J = 144.7 Hz), 16.66 (dt, J = 5.7, 2.8 Hz)
31P NMR (81 MHz, DMSO-d6) d 17.22 (s).
MS (ES+) m/z: 459.2 and 461.2 [M+H+]+ (C14H26BrN2O6P2).

5588
J. W. De Schutter et al. / Bioorg. Med. Chem. 20 (2012) 5583–5591
4.4. Examples of Suzuki coupling reactions
(m, 8H), 2.95 (td, J = 7.3, 3.8 Hz, 1H), 1.23 (dt, J = 18.1, 7.1 Hz,
12H), 0.76 (m, 2H), 0.66–0.60 (m, 2H).
A 2 mL microwave reaction vessel was charged with a magnetic
stir bar, boronic acid or boronate ester (1.5 equiv), Pd(PPh3)4
(0.1 equiv) and aryl halide (if a solid); if the aryl halide is an oil, it
was added as a solution in 1,4-dioxane via syringe. The reaction
vessel was capped with a septum, degassed and flushed with
Argon. 1,4-Dioxane was added to bring the concentration of aryl
halide to 0.1 M and the mixture was again degassed and flushed
with Argon. A solution of 2 M K2CO3 (2.5 equiv) was added and
the mixture was again degassed and flushed with Argon. The
rubber septum was replaced by a Teflon lined seal, under a flow
of Argon. The reaction was irradiated to 125 °C for 15–30 min
(ꢀ40 W), reaction progress was followed by TLC and/or HPLC. The
crude was filtered through a plug of Celite, rinsed with 10 mL 1:1
EtOAc/acetone and concentrated under vacuum. The residue was
purified by silica gel chromatography (silica was pre-washed with
0.1% Et3N in hexanes/EtOAc, 3:1 ratio) using a CombiFlash instru-
ment and a solvent gradient from 1:1 EtOAc/hexanes to 100% EtOAc
and then to 25% MeOH in EtOAc. The desired product was isolated
in yields from 30% to 80% (some examples are given below).
13C NMR (75 MHz, acetone-d6) d 167.41, 138.47, 137.24, 136.10,
135.71, 129.49, 128.89, 126.62, 125.68, 117.18,
d 62.89 (d,
J = 12.8 Hz), 48.80 (t, J = 145.5 Hz), 23.02, 15.85 (d, J = 2.0 Hz), 5.56.
31P NMR (81 MHz, acetone-d6) d 20.62 (s).
MS (ES+) m/z: 540.3 [M+H+]+ (C24H36N3O7P2).
4.4.5. Tetraethyl (((5-(4-(N-cyclopropylsulfamoyl)phenyl)
pyridin-3-yl)amino) methylene)bis(phosphonate) (13i)
Yield: 35%; colorless oil.
1H NMR (400 MHz, acetone-d6) d 8.39 (d, J = 2.7 Hz, 1H), 8.28
(d, J = 1.9 Hz, 1H), 7.96 (d, J = 8.7 Hz, 4H), 7.68 (t, J = 2.3 Hz, 1H),
5.44 (d, J = 9.5 Hz, 1H), 4.69 (td, J = 22.1, 10.4 Hz, 1H), 4.23–4.08
(m, 8H), 2.25 (m, 1H), 1.23 (dt, J = 17.4, 7.1 Hz, 12H), 0.56
(m, 4H).
13C NMR (75 MHz, acetone-d6) d 143.62, 142.39, 139.99, 137.23,
137.01, 134.60, 127.82, 127.53, 117.07, d 62.86 (d, J = 11.1 Hz),
48.78 (t, J = 146.0 Hz), 24.19, 15.85 (d, J = 1.8 Hz), 5.17.
31P NMR (81 MHz, acetone-d6) d 20.56 (s).
MS (ES+) m/z: 576.3 [M+H+]+ (C23H36N3O8P2S).
4.4.1. Tetraethyl (((5-phenylpyridin-3-yl)amino)methylene)bis
(phosphonate) (13b)
4.4.6. Tetraethyl (((5-(4-(cyclopropanecarboxamido)phenyl)
pyridin-3-yl)amino) methylene)bis(phosphonate) (13j)
Yield: 48%; as colorless oil.
Yield: 34%; as clear, colorless syrup.
1H NMR (400 MHz, DMSO-d6) d 8.26 (s, 1H), 8.11 (s, 1H), 7.68 (d,
J = 7.1 Hz, 2H), 7.57 (s, 1H), 7.46 (t, J = 7.5 Hz, 2H), 7.38 (d,
J = 7.2 Hz, 1H), 6.13 (d, J = 11.0 Hz, 1H), 4.95 (td, J = 22.9, 10.9 Hz,
1H), 4.07 (d, J = 7.0 Hz, 8H), 1.14 (dt, J = 19.3, 7.0 Hz, 11H).
13C NMR (126 MHz, CD3OD) d 136.33, 134.20, 128.69, 127.91,
126.70, 118.29, d 63.61 (d, J = 33.2 Hz), d 15.29 (d, J = 5.2 Hz).
31P NMR (81 MHz, CD3OD) d 20.32 (s).
1H NMR (400 MHz, acetone-d6) d 9.52 (s, 1H), 8.27 (d, J = 2.6 Hz,
1H), 8.20 (d, J = 1.8 Hz, 1H), 7.77 (d, J = 8.7 Hz, 2H), 7.64 (d, J = 8.7
Hz, 2H), 7.55 (s, 1H), 5.27 (d, J = 10.4 Hz, 1H), 4.64 (td, J = 22.1, 10.3
Hz, 1H), 4.22–4.06 (m, 8H), 1.83–1.73 (m, 1H), 1.22 (dt, J = 17.9, 7.1
Hz, 12H), 0.95–0.87 (m, 2H), 0.83–0.75 (m, 2H).
13C NMR (75 MHz, acetone-d6) d 171.65, 139.61, 137.04, 135.61,
132.83, 127.19, 119.34, 119.25, 116.53, d 62.78 (d, J = 12.9 Hz),
48.89 (t, J = 145.0 Hz), 15.83 (d, J = 1.7 Hz), 14.62, 6.80.
31P NMR (81 MHz, acetone-d6) d 20.60 (s).
MS (ES+) m/z: 479.13 [M+Na+]+ (C20H30N2NaO6P2).
4.4.2. Tetraethyl (((5-(4-isopropoxyphenyl)pyridin-3-yl)amino)
methylene) bis(phosphonate) (13f)
MS (ES+) m/z: 540.3 [M+H+]+ (C24H36N3O7P2).
Yield: 70%, of pale yellow syrup.
1H NMR (500 MHz, CDCl3) d 8.25 (s, 1H), 8.05 (d, J = 2.4 Hz, 1H),
7.49–7.44 (m, 2H), 7.13 (s, 1H), 7.00–6.94 (m, 2H), 4.65–4.55 (m,
1H), 4.33–4.10 (m, 9H), 1.37 (d, J = 6.1 Hz, 6H), 1.29 (dt, J = 19.0,
7.1 Hz, 12H).
4.4.7. Tetraethyl (((4-(4-cyclopropoxyphenyl)pyridin-2-yl)
amino) methylene)bis(phosphonate) (7g tetraethyl ester)
Yield: 73%; white solid.
1H NMR (300 MHz, CDCl3) d 8.09 (d, J = 5.2 Hz, 1H), 7.52 (d,
J = 8.6 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 5.2 Hz, 1H),
6.70 (s, 1H), 5.58 (td, J = 22.2, 9.5 Hz, 1H), 5.01 (d, J = 9.5 Hz, 1H),
4.14–4.21 (m, 8H), 3.90–3.57 (m, 1H), 1.20–1.28 (m, 12H), 0.79
(m, 4H).
13C NMR (126 MHz, CDCl3) d 158.08, 142.30, 138.56, 136.51,
134.75, 129.92, 128.15, 117.77, 116.22, 69.94,
d 63.58 (dd,
J = 21.7, 18.6 Hz), 49.88 (t, J = 147.7 Hz), 22.00, 16.39 (dd, J = 6.0,
2.8 Hz).
13C NMR (75 Hz, CDCl3) d 159.71, 156.54, 149.33, 147.73,
131.05, 127.86, 115.39, 112.57, 106.33, 63.26–63.48 (m, CH2),
50.95, 45.09 (t, J = 146.2 Hz, CH), 16.22–16.36 (m, CH3), 6.24
31P NMR (CDCl3) d 18.99 ppm.
31P NMR (81 MHz, CDCl3) d 14.73 (s).
MS (ES+) m/z: 515.22 [M+H+]+ (C23H37N2O7P2).
4.4.3. Tetraethyl (((5-(4-cyclopropoxyphenyl)pyridin-3-yl)amino)
methylene) bis(phosphonate) (13g)
MS (ES+) m/z: 513.21 [M+H+]+ (C23H35N2O7P2).
Yield: 99% of pale yellow oil.
1H NMR (500 MHz, CDCl3) d 8.25 (s, 1H), 8.06 (d, J = 2.4 Hz, 1H),
7.51–7.45 (m, 2H), 7.16–7.11 (m, 3H), 4.30–4.10 (m, 8H), 3.81–
3.75 (m, 1H), 1.29 (dt, J = 19.1, 7.1 Hz, 12H), 0.84–0.79 (m, 4H).
13C NMR (126 MHz, CDCl3) d 159.11, 142.30, 138.63, 136.46,
134.90, 130.55, 128.05, 117.80, 115.49, d 63.63 (d, J = 36.9 Hz),
50.91, 49.89 (t, J = 147.6 Hz), 16.85–15.63 (m), 6.22.
31P NMR (81 MHz, CDCl3) d 14.73 (s).
4.5. Synthesis general procedure for amide coupling reactions
To a mixture of 2-((bis(diethoxyphosphoryl)methyl)amino)
isonicotinic acid (1 equiv) and amine (1.50 equiv) in a minimal
amount of anhydrous DMF were added in sequence: DIPEA
(3 equiv) and HATU (1.50 equiv). The reaction was stirred at RT
overnight. The reaction mixture was diluted with EtOAc and
washed with saturated NaHCO3. The aqueous layer was back-
extracted with 4 Â 10 ml of EtOAc. All organic layers were
combined, washed with brine, dried over anhydrous MgSO4,
filtered and concentrated in vacuo. The residue was purified by
silica gel (silica was pre-washed with 0.1% NEt3 in hexanes/EtOAc
3:1) chromatography on a CombiFlash instrument, using a solvent
gradient from 1:10 EtOAc/Hexanes to 100% EtOAc and then to
20%MeOH in EtOAc to give the desired amides.
MS (ES+) m/z: 513.20 [M+H+]+ (C23H35N2O7P2).
4.4.4. Tetraethyl (((5-(3-(cyclopropylcarbamoyl)phenyl)pyridin-
3-yl)amino) methylene)bis(phosphonate) (13h)
Yield: 48%; as colorless oil.
1H NMR (400 MHz, acetone-d6) d 8.34 (s, 1H), 8.23 (s, 1H), 8.13
(s, 1H), 7.90–7.80 (m, 3H), 7.62 (s, 1H), 7.54 (t, J = 7.7 Hz, 1H), 5.39
(d, J = 10.8 Hz, 1H), 4.67 (td, J = 22.2, 10.4 Hz, 1H), 4.23–4.07

J. W. De Schutter et al. / Bioorg. Med. Chem. 20 (2012) 5583–5591
5589
4.5.1. Synthesis of 2-((bis(diethoxyphosphoryl)methyl)amino)
isonicotinic acid (16b)
and flushed with Argon. A volume of 1.09 mL 1,4-dioxane was
added to bring the concentration of the aryl bromide to 0.1 M, and
the mixture was again degassed and flushed with Argon. A solution
of 2 M Cs2CO3 (0.16 ml, 2.5 equiv) was added and the mixture was
again degassed and flushed with Argon. The rubber septum
was replaced by a Teflon lined seal, under Argon flow. The reaction
was irradiated to 125 °C for 30 min (ꢀ40 W), reaction progress was
monitored HPLC. After 30 min HPLC analysis showed ꢀ30%
conversion, the same amounts of boron reagent, catalyst and
base were added to the reaction vessel and irradiation was contin-
ued. After another 30 min HPLC analysis showed ꢀ66% conversion,
the same amounts boron reagent, catalyst and base were added to
the reaction vessel and irradiation was continued. After an addi-
tional 30 min, HPLC analysis showed complete conversion. The
crude was filtered through a plug of Celite, rinsed with 10 mL 1:1
EtOAc/acetone and concentrated in vacuo. The residue was purified
by silica gel (silica was pre-washed with 0.1% NEt3 in hexanes:EtOAc
3:1) chromatography using a CombiFlash instrument and a solvent
gradient from 1:1 EtOAc/hexanes to 100% EtOAc and then to
25% MeOH in EtOAc. The product was isolated as a purple oil
in ꢀ75% purity (40 mg). The mixture was purified by preparative
HPLC on a Waters Atlantis C18 column using a gradient from 5%
CH3CN/H2O to 95% with 0.1% formic acid. Fractions of sufficient
purity were pooled and lyophilized to dryness, isolated 28 mg
(55%) of product as a white, waxy solid.
To an ice-cooled solution of methyl 2-((bis(diethoxyphospho-
ryl)methyl)amino) isonicotinate (50.0 mg, 0.114 mmol) in 1 ml of
THF was added 1.0 M LiOH (0.28 ml, 0.285 mmol) and stirred at
0 °C for 1 h. The reaction mixture was then acidified with 1 M
HCl and evaporated to dryness under reduced pressure to obtain
the free acid. The residue was re-dissolved in EtOAc and washed
with water. The aqueous layer was back-extracted with
4 Â 10 ml EtOAc. The organic layers were combined, washed with
brine, dried over MgSO4, filtered and concentrated in vacuo to give
the title compound as a yellow solid (20.6 mg, 43%).
1H NMR (400 MHz, CD3OD) d 8.13 (d, J = 3.6 Hz, 1H), 7.27
(s, 1H), 7.11 (d, J = 4.4 Hz, 1H), 5.67 (t, J = 23.5 Hz, 1H), 4.17
(m, 8H), 1.26 (dd, J = 14.7, 7.6 Hz, 12H).
31P NMR (81 MHz, CD3OD) d 19.84 (s).
MS (ES+) m/z: 447.0 [M+Na+]+ (C15H26N2NaO8P2).
4.5.2. Tetraethyl (((4-((4-methoxyphenyl)carbamoyl)pyridin-2-
yl)amino)methylene) bis(phosphonate) (10e tetraethyl ester)
Yield: 58% of pale yellow solid.
1H NMR (400 MHz, CD3OD) d 8.19 (d, J = 5.3 Hz, 1H), 7.56
(d, J = 9.0 Hz, 2H), 7.16 (s, 1H), 7.05 (d, J = 5.1 Hz, 1H), 6.92
(d, J = 9.0 Hz, 2H), 5.70 (t, J = 23.5 Hz, 1H), 4.27–4.07 (m, 8H),
3.79 (s, 3H), 1.35–1.17 (m, 12H).
1H NMR (400 MHz, DMSO-d6) d 8.08 (s, 1H), 7.72 (s, 1H),
7.30–7.09 (m, 6H), 5.99 (d, J = 10.6 Hz, 1H), 4.63 (td, J = 22.9,
10.8 Hz, 1H), 4.13–3.86 (m, 8H), 3.79 (s, 2H), 1.09 (dt, J = 23.7,
7.0 Hz, 12H).
13C NMR (75 MHz, CD3OD) d 165.58, 157.14, 156.98, 147.50,
144.16, 130.92, 122.52, 113.55, 110.87, 107.87,
d
63.47
(d, J = 17.6 Hz), 54.46, 44.50 (t, J = 150.2 Hz), 15.24 (dd, J = 6.2,
3.1 Hz). 31P NMR (81 MHz, CD3OD) d 19.34 (s).
13C NMR (126 MHz, DMSO-d6) d 143.65, 141.34, 138.65, 136.65,
135.05, 128.99, 128.79, 126.44, 119.18, d 62.98 (d, J = 21.6 Hz),
48.10 (t, J = 144.6 Hz), 38.74, 16.62, 16.60.
MS (ES+) m/z: 530.36 [M+H+]+ (C22H34N3O8P2).
31P NMR (81 MHz, DMSO-d6) d 20.180 (s).
4.5.3. Tetraethyl (((4-(isopropylcarbamoyl)pyridin-2-yl)amino)
methylene) bis(phosphonate) (10d tetraethyl ester)
Yield: 54%, of beige solid.
MS (ES+) m/z: 471.33 [M+H+]+ (C21H33N2O6P2).
1H NMR (300 MHz, CD3OD) d 8.13 (d, J = 5.3 Hz, 1H), 7.05
(s, 1H), 6.94 (d, J = 5.2 Hz, 1H), 5.67 (t, J = 23.5 Hz, 1H), 4.27–4.06
(m, 8H), 3.30 (m, 7H), 1.32–1.17 (m, 12H).
4.7. Standard procedures for the hydrolysis of the
bisphosphonate tetraethyl esters to the diphosphonic acids
13C NMR (75 MHz, CD3OD) d 166.51, 157.07, 147.33, 143.93,
110.76, 107.73, d 63.45 (d, J = 18.2 Hz), 44.47 (t, J = 150.1 Hz),
41.80, 20.99, 15.25 (dd, J = 6.0, 3.0 Hz).
A solution of the tetraethyl bisphosphonate ester (1 equiv) in
CH2Cl2 was cooled to 0 °C and trimethylsilyl bromide (15 equiv)
was added via syringe. The reaction mixture was stirred at room
temperature for 5 days; the completion of conversion was moni-
tored by 31P NMR. The mixture was then concentrated in vacuo,
diluted with HPLC grade MeOH (ꢀ5 mL), the solvent was evapo-
rated to dryness and this step was repeated four times. The or-
ganic solvents were evaporated under vacuum, the residue was
suspended in 0.5 mL MeOH, excess water (ꢀ5 mL Milli-Q grade)
was added to induce full precipitation of the final product. The
amorphous powder was collected by filtration, washed with de-
ionized water, twice with HPLC-grade CH3CN, twice with distilled
Et2O or toluene and dried under vacuum to give the final com-
pound as a solid.
31P NMR (81 MHz, CD3OD) d 19.73 (s).
MS (ES+) m/z: 466.34 [M+H+]+ (C18H34N3O7P2).
4.5.4. Tetraethyl (((4-(phenylcarbamoyl)pyridin-2-yl)amino)
methylene) bis(phosphonate) (10b tetraethyl ester)
Yield: 35%, of pale brown oil.
1H NMR (400 MHz, CD3OD) d 8.19 (t, J = 5.0 Hz, 1H), 7.66
(t, J = 8.3 Hz, 2H), 7.40–7.32 (m, 2H), 7.19–7.13 (m, 2H), 7.07
(d, J = 5.3 Hz, 1H), 5.71 (t, J = 23.5 Hz, 1H), 4.25–4.13 (m, 8H),
1.27 (t, J = 7.1 Hz, 12H).
13C NMR (75 MHz, CD3OD) d 165.44, 157.01, 147.36, 144.02,
130.78, 122.39, 113.41, 110.73, 107.73, d 63.33 (d, J = 17.7 Hz),
44.36 (t, J = 149.9 Hz), 16.89–13.49 (m).
4.7.1. (((4-(4-Cyclopropoxyphenyl)pyridin-2-yl)amino)methylene)
diphosphonic acid (7g)
31P NMR (81 MHz, CD3OD) d 19.74 (s).
MS (ES+) m/z: 500.35 [M+H+]+ (C21H32N3O7P2).
Yield: 93%, as white solid.
1H NMR (500 MHz, D2O) 7.75 (dd, J = 5.8, 0.6 Hz, 1H), 7.63–7.54
(m, 2H), 7.09–7.01 (m, 2H), 6.82 (d, J = 1.0 Hz, 1H), 6.74 (dd, J = 5.8,
1.6 Hz, 1H), 3.87 (t, J = 19.2 Hz, 1H), 3.77 (hept, J = 1.8 Hz, 1H),
0.67–0.71 (m, 2H), 0.58–0.61 (m, 2H).
4.6. Tetraethyl (((5-benzylpyridin-3-yl)amino)methylene)
bis(phosphonate) (9c tetraethyl ester)
13C NMR (125 Hz, D2O) d 159.16, 157.84, 149.99, 144.55, 130.81,
128.26, 115.46, 110.28, 105.75, 51.77 (t, J = 122.5 Hz), 51.30, 5.42
31P NMR (81 MHz, D2O) d14.04.
A
2 mL microwave reaction vessel was charged with a
magnetic stir bar, tetraethyl (((5-bromopyridin-3-yl)amino)methy-
lene)bis(phosphonate) (50.0 mg, 0.11 mmol), potassium benzyltriflu-
oroborate (53.9 mg, 0.27 mmol, 2.5 equiv) and Pd(dppf)Cl2ÁCH2Cl2
(8.0 mg, 0.01 mmol, 0.1 equiv); capped with a septum, degassed
HRMS: calcd 399.05110 for (C15H17N2O7P2), found (m/z)
399.05093 [MÀH]À.

5590
J. W. De Schutter et al. / Bioorg. Med. Chem. 20 (2012) 5583–5591
4.7.2. (((5-Phenylpyridin-3-yl)amino)methylene)diphosphonic
acid (9b)
HRMS: calcd 426.0620 for (C16H18N3O7P2), found m/z
426.06280 [MÀH]À.
Isolated yield: 64%, as a white powder.
1H NMR (500 MHz, D2O) d 8.05 (dd, J = 6.0, 2.2 Hz, 2H), 7.80–
7.75 (m, 2H), 7.57 (t, J = 7.6 Hz, 2H), 7.52–7.47 (m, 1H), 7.46 (s,
1H), 3.80 (t, J = 19.0 Hz, 2H).
4.7.7. (((5-(4-(N-Cyclopropylsulfamoyl)phenyl)pyridin-3-yl)
amino)methylene) diphosphonic acid (9i)
Yield: 85%, pale yellow powder.
13C NMR (75 MHz, D2O) d 137.83, 136.75, 134.14, 133.73,
1H NMR (400 MHz, D2O) d 7.97–7.89 (m, 2H), 7.82 (q, J = 8.5 Hz,
4H), 7.32 (s, 1H), 3.62 (t, J = 19.0 Hz, 1H), 2.21–2.12 (m, 1H), 0.45–
0.38 (m, 2H), 0.32–0.25 (m, 2H).
133.53, 128.98, 128.06, 127.04, 117.68; the Ca was difficult to ob-
serve due to coupling with 31P, however, a 1H–13C correlation (d 1H
3.80 to 13C d 52.25) was clearly observed in the HSQC spectrum
(see supplementary data).
13C NMR (75 MHz, D2O) d 145.36, 142.82, 137.57, 135.31,
134.66, 134.09, 127.94, 127.44, 117.69, 52.52, 24.23, 4.72.
HSQC 1H–13C: 1H d 3.62 correlates with 13C d 53.52.
31P NMR (81 MHz, D2O) d 15.38 (s).
31P NMR (81 MHz, D2O) d 14.97 (s).
HRMS m/z: calcd 343.0249 for (C12H13N2O6P2), found m/z
343.0251 [MÀH+]À.
HRMS: calcd 462.0290 for (C15H18N3O8P2S), found m/z
462.03099 [MÀH]À.
4.7.3. (((5-benzylpyridin-3-yl)amino)methylene)diphosphonic
acid (9c)
4.7.8. (((5-(4-(Cyclopropanecarboxamido)phenyl)pyridin-3-yl)
amino)methylene) diphosphonic acid (9j)
Yield: 79%, white powder.
1H NMR (500 MHz, D2O) d 7.86 (d, J = 2.5 Hz, 1H), 7.65 (s, 1H),
7.39–7.30 (m, 3H), 7.26 (t, J = 6.7 Hz, 1H), 7.07 (s, 1H), 3.92 (s,
2H), 3.65 (t, J = 19.0 Hz, 1H).13C NMR (126 MHz, D2O) d 145.17,
140.97, 137.71, 136.16, 132.55, 128.74, 128.69, 126.34, 120.10, d
52.10 (t, J = 123.5 Hz), 38.23.
Yield: 94%, white powder.
1H NMR (300 MHz, D2O) d 7.99 (s, 2H), 7.68 (d, J = 8.5 Hz, 2H),
7.52 (d, J = 8.5 Hz, 2H), 7.40 (s, 1H), 3.76 (t, J = 19.1 Hz, 1H), 1.76
(m, 1H), 0.93 (m, 4H).13C NMR (75 MHz, D2O) d 175.95, 145.24,
137.14, 136.05, 134.12, 134.05, 133.56, 127.55, 121.51, 117.43, d
52.30 (t, J = 124.2 Hz), 14.67, 7.32.
HSQC 1H–13C: d 1H 3.65 correlates with 13C d 52.10.
31P NMR (81 MHz, D2O) d 15.06 (s).
31P NMR (81 MHz, D2O) d 15.39 (s).
HRMS: calcd 357.0405 for (C13H15N2O6P2), found m/z
357.04149 [MÀH]À.
HRMS: calcd 426.0620 for (C16H18N3O7P2), found m/z
426.06265 [MÀH]À.
4.7.4. (((5-(4-isopropoxyphenyl)pyridin-3-yl)amino)methylene)
diphosphonic acid (9f)
4.7.9. (((4-(Phenylcarbamoyl)pyridin-2-yl)amino)methylene)
diphosphonic acid (10b)
Isolated yield: 71%, as white powder.
1H NMR (500 MHz, D2O) d 7.97 (d, J = 2.7 Hz, 1H), 7.96 (d,
J = 1.8 Hz, 1H), 7.71–7.66 (m, 2H), 7.38–7.35 (m, 1H), 7.15–7.10
(m, 2H), 4.75 (m, obscured by solvent signal, 1H), 3.74 (t,
J = 19.1 Hz, 1H), 1.35 (d, J = 6.1 Hz, 6H).13C NMR (126 MHz, D2O)
d 156.77, 145.36, 136.20, 133.72, 133.32, 131.07, 128.40, 117.19,
116.80, 71.67, 52.49, 20.99.
Yield: 49%, fine white crystals.
1H NMR (400 MHz, D2O) d 7.93 (d, J = 5.4 Hz, 1H), 7.46 (dd,
J = 8.5, 1.1 Hz, 2H), 7.38 (dd, J = 10.7, 5.2 Hz, 2H), 7.21 (t,
J = 7.4 Hz, 1H), 6.91 (s, 1H), 6.75 (d, J = 5.4 Hz, 1H); central methy-
lene obscured by solvent signal.
13C NMR (75 MHz, D2O) d 183.70, 168.57, 158.69, 147.68,
143.62, 136.47, 129.10, 125.97, 122.65, 109.11.
31P NMR (81 MHz, D2O) d 14.91.
HSQC 1H–13C: 1H d 3.74 correlates with 13C d 52.49.
31P NMR (81 MHz, D2O) d 15.05 (s).
HRMS m/z: calcd 401.0667 for (C15H19N2O7P2), found m/z
401.06784 [MÀH+]À.
HRMS: calcd 386.0307 for (C13H14N3O7P2), found (m/z)
386.03035 [MÀH]À.
4.7.5. (((5-(4-cyclopropoxyphenyl)pyridin-3-yl)amino)methylene)
diphosphonic acid (9g)
4.7.10. (((4-(Isopropylcarbamoyl)pyridin-2-yl)amino)methylene)
diphosphonic acid (10d)
27.9 mg, 63%, pale yellow powder.
Yield: 50%, of pale yellow solid.
1H NMR (500 MHz, D2O)
d 7.83 (d, J = 3.2 Hz, 2H), 7.56
1H NMR (400 MHz, D2O) d 7.90 (d, J = 5.2 Hz, 1H), 6.74 (s, 1H),
6.65 (d, J = 5.1 Hz, 1H), 4.03 (dt, J = 13.3, 6.6 Hz, 1H), 1.16 (d,
J = 6.6 Hz, 6H); central methylene obscured by solvent signal.
13C NMR (75 MHz, D2O) d 183.70, 168.91, 158.64, 147.43,
143.80, 108.90, 42.34, 21.09.
(d, J = 8.8 Hz, 2H), 7.23 (s, 1H), 7.12 (d, J = 8.8 Hz, 2H), 3.87–3.73
(m, 1H), 3.60 (t, J = 19.1 Hz, 1H), 0.77–0.57 (m, 4H).
13C NMR (126 MHz, D2O) d 158.13, 145.34, 136.24, 133.79,
133.31, 131.12, 128.26, 117.24, 115.51, d 52.47 (t, J = 125.3 Hz),
51.26, 5.43.
31P NMR (81 MHz, D2O) d 14.93.
31P NMR (81 MHz, D2O) d 15.06 (s).
HRMS: calcd 352.0463 for (C10H16N3O7P2), found (m/z)
HRMS: calcd 399.0511 for (C15H17N2O7P2), found m/z
352.04775 [MÀH]À.
399.05192 [MÀH]À.
4.7.11. (((4-((4-Methoxyphenyl)carbamoyl)pyridin-2-yl)amino)
methylene) diphosphonic acid (10e)
4.7.6. (((5-(3-(Cyclopropylcarbamoyl)phenyl)pyridin-3-yl)amino)
methylene) diphosphonic acid (9h)
Yield: 86%, fine yellow solid.
Yield: 97%, of a white powder.
1H NMR (400 MHz, D2O) d 7.91 (d, J = 5.2 Hz, 1H), 7.35 (d,
J = 9.0 Hz, 2H), 6.93 (d, J = 9.0 Hz, 2H), 6.88 (s, 1H), 6.73 (d,
J = 5.3 Hz, 1H), 3.73 (s, 3H); central methylene obscured by solvent.
13C NMR (75 MHz, D2O) d 183.70, 168.59, 158.83, 156.72,
147.70, 143.48, 129.73, 124.57, 114.34, 108.87, 55.41.
31P NMR (81 MHz, D2O) d 14.92.
1H NMR (400 MHz, D2O) d 8.02 (m, 3H), 7.91 (d, J = 8.1 Hz, 1H),
7.77 (d, J = 7.8 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.42 (s, 1H), 3.76 (t,
J = 19.0 Hz, 1H), 2.84–2.76 (m, 1H), 0.90–0.83 (m, 2H), 0.73–0.67
(m, 2H).
13C NMR (75 MHz, D2O) d 172.49, 145.34, 138.29, 136.00,
134.24, 134.10, 134.07, 130.60, 129.29, 126.59, 125.48, 117.54,
22.54, 5.44.
HRMS: calcd 416.0413 for (C14H16N3O8P2), found m/z
416.04136 [MÀH]À.
31P NMR (81 MHz, D2O) d 15.36 (s).

J. W. De Schutter et al. / Bioorg. Med. Chem. 20 (2012) 5583–5591
5591
Carpten, J.; Trent, J.; Hahn, W. C.; Garraway, L. A.; Meyerson, M.; Lander, E. S.;
Getz, G.; Golub, T. R. Nature 2011, 471, 467.
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