Highly potent geminal bisphosphonates. From pamidronate disodium (Aredia) to zoledronic acid (Zometa)

Article abstract of DOI:10.1021/jm020819i

Bisphosphonates (BPs) are pyrophosphate analogues in which the oxygen in P-O-P has been replaced by a carbon, resulting in a metabolically stable P-C-P structure. Pamidronate (1b, Novartis), a second-generation BP, was the starting point for extensive SAR studies. Small changes of the structure of pamidronate lead to marked improvements of the inhibition of osteoclastic resorption potency. Alendronate (1c, MSD), with an extra methylene group in the N-alkyl chain, and olpadronate (1h, Gador), the N,N-dimethyl analogue, are about 10 times more potent than pamidronate. Extending one of the N-methyl groups of olpadronate to a pentyl substituent leads to ibandronate (1k, Roche, Boehringer-Mannheim), which is the most potent close analogue of pamidronate. Even slightly better antiresorptive potency is achieved with derivatives having a phenyl group linked via a short aliphatic tether of three to four atoms to nitrogen, the second substituent being preferentially a methyl group (e.g., 4g, 4j, 5d, or 5r). The most potent BPs are found in the series containing a heteroaromatic moiety (with at least one nitrogen atom), which is linked via a single methylene group to the geminal bisphosphonate unit. Zoledronic acid (6i), the most potent derivative, has an ED₅₀ of 0.07 mg/kg in the TPTX in vivo assay after sc administration. It not only shows by far the highest therapeutic ratio when comparing resorption inhibition with undesired inhibition of bone mineralization but also exhibits superior renal tolerability. Zoledronic acid (6i) has thus been selected for clinical development under the registered trade name Zometa. The results of the clinical trials indicate that low doses are both efficacious and safe for the treatment of tumor-induced hypercalcemia, Paget's disease of bone, osteolytic metastases, and postmenopausal osteoporosis.

Full text of DOI:10.1021/jm020819i

J . Med. Chem. 2002, 45, 3721-3738
3721
High ly P oten t Gem in a l Bisp h osp h on a tes. F r om P a m id r on a te Disod iu m (Ar ed ia )
to Zoled r on ic Acid (Zom eta )
Leo Widler,* Knut A. J aeggi, Markus Glatt, Klaus Mu¨ller, Rolf Bachmann, Michael Bisping, Anne-Ruth Born,
Reto Cortesi, Gabriela Guiglia, Heidi J eker, Re´my Klein, Ueli Ramseier, J ohann Schmid, Gerard Schreiber,
Yves Seltenmeyer, and J onathan R. Green
Novartis Pharma Research, Arthritis and Bone Metabolism Therapeutic Area, CH-4002 Basel, Switzerland
Received J anuary 14, 2002
Bisphosphonates (BPs) are pyrophosphate analogues in which the oxygen in P-O-P has been
replaced by a carbon, resulting in a metabolically stable P-C-P structure. Pamidronate (1b,
Novartis), a second-generation BP, was the starting point for extensive SAR studies. Small
changes of the structure of pamidronate lead to marked improvements of the inhibition of
osteoclastic resorption potency. Alendronate (1c, MSD), with an extra methylene group in the
N-alkyl chain, and olpadronate (1h , Gador), the N,N-dimethyl analogue, are about 10 times
more potent than pamidronate. Extending one of the N-methyl groups of olpadronate to a pentyl
substituent leads to ibandronate (1k , Roche, Boehringer-Mannheim), which is the most potent
close analogue of pamidronate. Even slightly better antiresorptive potency is achieved with
derivatives having a phenyl group linked via a short aliphatic tether of three to four atoms to
nitrogen, the second substituent being preferentially a methyl group (e.g., 4g, 4j, 5d , or 5r ).
The most potent BPs are found in the series containing a heteroaromatic moiety (with at least
one nitrogen atom), which is linked via a single methylene group to the geminal bisphosphonate
unit. Zoledronic acid (6i), the most potent derivative, has an ED50 of 0.07 mg/kg in the TPTX
in vivo assay after sc administration. It not only shows by far the highest therapeutic ratio
when comparing resorption inhibition with undesired inhibition of bone mineralization but
also exhibits superior renal tolerability. Zoledronic acid (6i) has thus been selected for clinical
development under the registered trade name Zometa. The results of the clinical trials indicate
that low doses are both efficacious and safe for the treatment of tumor-induced hypercalcemia,
Paget’s disease of bone, osteolytic metastases, and postmenopausal osteoporosis.
In tr od u ction
The first bisphosphonates (originally called diphos-
phonates) were synthesized in the 19th century.1 They
were initially used mainly as antiscaling and anticor-
rosive agents but also as complexing agents in the
textile, fertilizer, and oil industries.2 However, it was
not until the late 1960s that their potential for the
treatment of various diseases of bone mineral metabo-
lism became evident. Geminal bisphosphonates (for
simplicity usually just called bisphosphonates; BPs) are
metabolically stable analogues of the naturally occur-
ring inorganic pyrophosphate, which have been shown
by Fleisch and co-workers to impair the formation and
dissolution of calcium phosphate crystals in vitro.3,4 Like
pyrophosphate, bisphosphonates have high affinity for
bone mineral and at high doses can modulate calcifica-
tion both in vitro and in vivo. In contrast to pyrophos-
phate, BPs are also orally active, although their low
F igu r e 1. Bisphosphonic acids are stable analogues of pyro-
phosphoric acid. Clodronic and etidronic acid are the most
important first-generation BPs.
bioavailability often limits the usefulness of oral ad-
ministration. More importantly, the replacement of the
oxygen atom between the two phosphonic acid moieties
derivatives to act as tridentate ligands. The nature of
of pyrophosphate by a carbon atom opened up the
R2 is key to the optimization of BPs as potent inhibitors
possibility of attaching side chains (Figure 1). Prefer-
of osteoclastic bone resorption. The first-generation BPs
ably, R1 is a hydroxyl group, which increases the affinity
had either a single atom or a simple alkyl side chain at
for calcium even further owing to the ability of such
R2 (e.g., clodronic and etidronic acid, Figure 1) and were
relatively weak inhibitors of bone resorption. Antire-
sorptive potency was already markedly increased in
pamidronate (1b; see Table 1), a second-generation
* To whom correspondence should be adddressed. Phone: +41 61
696 57 86. Fax: +41 61 696 60 71. E-mail: leo.widler@
pharma.novartis.com.
10.1021/jm020819i CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/19/2002

3722 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Widler et al.
Ta ble 1. Dependence of Antiresorptive Potency of Pamidronate
Analogues on N Substituents and on the Distance to the
Phosphono Groups
b
compounda
R1
R2
n
ED50 (µg/kg)
F igu r e 2. Important bisphosphonic acids with heteroaromatic
substituents.
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
H
H
H
H
H
H
H
H
H
H
H
Me
1
2
3
4
5
2
2
2
2
2
2
150
61
pamidronate
alendronate
bone metastases arising from breast cancer or multiple
myeloma and for Paget’s disease of bone.20 Subse-
quently, several other N-containing BPs of increasing
potency have been developed for the treatment of both
benign and malignant bone diseases: alendronate,21
risedronate,22 ibandronate,23 and incadronate.24
8
20
60
neridronate
olpadronate
ibandronate
Ac
Me
Me
>1000
∼15
12
propyl Me
Et Et
pentyl Me
3
3
1.1
In 1986, a chemistry program was initiated at Ciba-
Geigy to find an even more potent BP as a follow-up to
pamidronate for the treatment of osteoporosis and
metastatic bone disease.25 Our own results, as well as
those of others,9,26 clearly indicate that the ability of BPs
to inhibit bone resorption is dependent on two separate
properties of the BP structure. The phosphonate groups
are essential for high affinity for bone mineral, which
is further increased in compounds with a hydroxyl group
at the R1 position (“bone hook”).8,9,27-30 The nature of
R2 determines the biological activity of the molecule and
influences the ability of the drug to interact with specific
molecular targets. Because second-generation BPs ex-
hibit physicochemical effects on mineralization similar
to those of early BPs while possessing markedly in-
creased potency as inhibitors of bone resorption, the
mechanism of antiresorptive activity cannot be derived
solely from direct effects on bone mineral.
This paper describes our efforts to improve the
potency and therapeutic window of pamidronate, the
first second-generation bisphosphonate, toward zoledron-
ic acid (Figure 2), which is so far the most potent agent
of its class. Concurrent to our own efforts, similar SAR
studies were undertaken in several laboratories. Pub-
lished data are mainly available from work conducted
at Procter & Gamble27,31,32 and Yamanouchi.33
1j
1k
a
Bisphosphonates were synthesized as free bisphosphonic acids;
the generic names given refer, however, to their salt forms. The
ED50 is the dose of compound administered sc, which results in a
50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3.
b
compound and the first BP with a basic aminoalkyl
group at R2. Most of the highly potent third-generation
BPs contain heteroaromatic rings with one or more
nitrogen atoms as components of their side chains.
There has been a great deal of speculation about the
exact mechanism of action of BPs. However, it is now
clear that cellular effects on osteoclasts are involved
rather than purely physicochemical effects on hydroxy-
apatite crystals as originally proposed.4,5 Recently it has
been found that the non-nitrogen-containing bisphos-
phonates with simple substituents (e.g., clodronate and
etidronate) can be metabolically incorporated into non-
hydrolyzable cytotoxic ATP analogues whereas the more
potent nitrogen-containing bisphosphonates appear to
inhibit the mevalonate pathway.6-10 They may thus
affect protein prenylation, thereby disrupting the in-
tracellular trafficking of key regulatory proteins and
inducing apoptosis (programmed cell death).11 Effects
of BPs on osteoclast recruitment12 as well as indirect
effects via stimulation of osteoblasts to produce an
osteoclast inhibitory factor have also been described.13-16
Ch em istr y
(a ) Bisp h osp h on ic a n d Bisp h osp h in ic Acid s.
Scheme 1 summarizes the methods used for the prepa-
ration of different classes of bisphosphonates.
The first BPs successfully used in the clinic were
etidronate and clodronate in the 1970s and 1980s. The
potency of these first-generation compounds was only
moderate, and moreover, for etidronate, the therapeutic
window between the inhibition of bone resorption and
impaired bone mineralization was rather small. The
search for more potent BPs by many pharmaceutical
companies has resulted in a number of novel, highly
potent derivatives.18,19
1-Hydroxybisphosphonate derivatives were prepared
from the corresponding carboxylic acids by treatment
with a mixture of phosphoric acid and phosphorus
trichloride, followed by quenching with water (method
A). While this reaction was usually performed in chloro-
benzene at the laboratory scale, an improved process
using methansulfonic acid as a solvent was preferred
for large-scale preparation. Amino groups present in the
substituent R were further modified as appropriate by
reductive amination (1h , 1i, 1o, 4l, 5r ; Tables 1, 3, 6,
7), acetylation (1f, Table 1), or hydrogenation (1g; Table
1, from the N-benzyl analogue). A similar approach
afforded 1-aminobisphosphonates starting from nitriles
(method B). This method was used to prepare 1m (Table
2). Bisphosphonates in which the 1-hydroxy substituent
is replaced by a hydrogen atom are accessible via
alkylation of tetraethyl methylenebisphosphonate fol-
lowed by acidic hydrolysis (method C; 1l, Table 2; 9a ,
The first member of the second-generation BPs was
pamidronate (1b, Table 1), originally synthesized by
Henkel as an additive for detergents and subsequently
licensed to Ciba-Geigy for development as a pharma-
ceutical agent (pamidronate disodium, Aredia). It was
the first BP to contain a basic nitrogen atom in its alkyl
side chain.20 Pamidronate exhibits increased potency as
an inhibitor of bone resorption and excellent tolerability,
resulting in a wide therapeutic window. It has been
extensively used clinically for the treatment of patients
with tumour-induced hypercalcaemia and osteolytic

Bisphosphonates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3723
Sch em e 1. General Methods for the Synthesis of
Sch em e 2. Synthesis of Aminomethylenebisphosphinic
Geminal Bisphosphonatesa
Acidsa
a
Reagents and conditions: (a) HC(OEt)3, HP(O)MeOEt (2
equiv); (b) 1 N HCl or TMSBr.
Sch em e 3. Synthesis of Arylalkylaminesa
a
Reagents and conditions: (a) (i) R1NH2, (ii) H2, Pd/C or
reductive amination with NaBH3CN; (b) (i) SOCl2, (ii) R1NH2; (c)
LiAlH4; (d) PBr3 or HBr; (e) R1NH2; (f) NaCN; (g) Raney Ni.
nic acids, by substitution of diethyl phosphite for ethyl
methylphosphonite (Scheme 2).
(b) Syn th esis of P r ecu r sor Am in es a n d Acid s.
Arylalkylamines that are not commercially available can
be prepared via several standard methods from car-
boxylic acids, aldehydes, or alcohols (Scheme 3). Aryl-
alkylaldehydes can easily be transformed into secondary
amines by reductive amination or by a two-step proce-
dure via imine formation followed by reduction (Scheme
3, method A). Alternatively, the required amines may
be synthesized starting with carboxylic acids, which are
converted to amides followed by reduction with lithium
aluminum hydride (Scheme 3, method B). Finally, a
third method used was the substitution of bromine in
arylalkyl bromides either directly with amines or with
sodium cyanide followed by hydrogenation over Raney
Ni to afford homologated primary amines (Scheme 3,
method C). All primary amines were monobenzylated
prior to their further transformation into carboxylic
acids as outlined below (cf. Scheme 5).
Most of the phenoxy- and phenylsulfanylalkylamines
were prepared by treatment of (substituted) phenols or
thiophenols with R,ω-dibromoalkylenes under basic
conditions. The reaction of the resulting bromides with
primary amines afforded the desired secondary amines.
Alternatively, alkylation of phenols and thiophenols
with N-phthalimidoalkyl bromides followed by basic
hydrolysis leads to primary amines (Scheme 4).
a
Reagents and conditions: (a) (i) H3PO4, PCl3, chlorobenzene
or MsOH, (ii) H2O; (b) H3PO4, PBr3; (c) NaH, THF; (d) 48% HBr
or TMSBr; (e) HC(OEt)3, HPO3Et2 (2 equiv); (f) 1 N HCl or TMSI;
(g) R′-X, NaH, THF.
Ta ble 2. Effect of the Substituent X on Antiresorptive Potency
compounda
X
ED50 (µg/kg)
b
1h
1l
olpadronate
OH
H
12
100
1m
NH2
>200
a
Bisphosphonates were synthesized as free bisphosphonic acids;
b
the generic names given refer, however, to their salt forms. The
ED50 is the dose of compound administered sc, which results in a
50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3.
9b, Table 11). Finally, aminomethylene BPs were
synthesized starting with a primary amine, which was
treated with triethyl orthoformate and 2 equiv of diethyl
phosphite. The phosphonate esters were hydrolyzed
with 1 M HCl or trimethylsilyl iodide to give 1-amino-
bisphosphonic acids (method D).
Analogues of pamidronate (1b, Table 1) have a two-
carbon linker between the nitrogen of the side chain and
the carbon between the phosphonic acid groups. The
required carboxylic acid precursors for these compounds
Aminomethylenebisphosphinic acids were prepared in
an analogous way as the corresponding bisphospospho-

3724 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Widler et al.
Sch em e 6. Synthesis of Imidazol-4-ylacetic Acidsa
Sch em e 4. Synthesis of Phenoxy- and
Phenylsulfanylalkylamines (X ) O or S)a
a
Reagents and conditions: (a) NH3(l), 70 °C, ∆p; (b) HCl; (c)
SOCl2; (d) NaCN, DMSO; (e) HCl, AcOH.
a
Reagents and conditions: (a) Br(CH2)nBr, NaOH, H2O; (b)
Sch em e 7. Hydroxymethylation of Imidazole
R2NH2, under pressure; (c) Br(CH2)nNPhth, NaOMe, EtOH; (d) 1
M KOH.
Derivativesa
Ta ble 3. Influence on Antiresorptive Potency by a Methyl
Substituent R to the Amino Group
compounda
R1
R2
R
ED50 (µg/kg)
b
a
Reagents and conditions: (a) paraformaldehyde or 37% HCHO,
1b
pamidronate
olpadronate
ibandronate
H
H
Me
Me
H
H
H
Me
H
61
3.4
12
18
1.2
65
120-130 °C, ∆p; (b) 37% HCHO, 40 °C.
1n c
1h
Me
Me Me
1oc
1k
Imidazol-4-ylacetic acids were synthesized according
to the literature.34,35 Formamidine and dihydroxyac-
etone was condensed in liquid ammonia under pressure
at 70 °C to give 4-hydroxymethylimidazole in moderate
yield. By replacement of formamidine by acetamidine
or benzamidine, the corresponding 2-methyl and 2-phe-
nyl derivatives were obtained in good yield. The hy-
droxymethylimidazoles were then transformed into the
corresponding chlorides with thionyl chloride followed
by reaction with sodium cyanide/DMSO to form imida-
zolylacetonitriles, which were hydrolyzed with HCl/
acetic acid (Scheme 6).
pentyl Me
pentyl Me Me
H
1p c
a
Bisphosphonates were synthesized as free bisphosphonic acids;
the generic names given refer, however, to their salt forms. The
ED50 is the dose of compound administered sc, which results in a
50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3. c Racemate.
b
Sch em e 5. Synthesis of carboxylic acidsa
Imidazol-2-ylmethanol derivatives were prepared by
heating N-substituted imidazoles with an aqueous
formaldehyde solution to 120-130 °C under pressure
according to literature procedures36,37 (Scheme 7). By
use of the same standard organic reactions as indicated
in Scheme 6, these were homologated to the desired
acetic acid analogues with 2-imidazolyl substituents.
Two-carbon homologations using Wittig-type chemistry
to afford after hydrogenation the corresponding propi-
onic acid derivatives were achieved from the corre-
sponding formyl derivatives.
a
Reagents and conditions: (a) ethyl acrylate; (b) 4 M HCl; (c)
Hal-(CH2)n-COOEt, K2CO3, 2-butanone; (d) BrCH2COOEt,
NaOMe, MeOH.
Rather surprisingly, hydroxymethylation of 4-methyl-
imidazole at 40 °C affords the 5-hydroxymethyl deriva-
tive exclusively38 (Scheme 7).
were conveniently obtained by conjugate addition of
suitably substituted amines to ethyl acrylate (ethyl
crotonate for 1p , Table 3) and subsequent hydrolysis
(Scheme 5, method A). Analogues with longer linkers
were synthesized according to method B, Scheme 5.
Bisphosphonates with heteroaromatic side chains pref-
erably have a one-carbon atom linker. In cases where
the heteroaromatic ring is linked via an aromatic ring
nitrogen, the precursor acids were prepared by alkyla-
tion with ethyl chloro- or bromoacetate under basic
conditions followed by acidic hydrolysis (Scheme 5,
method C). Alkylation with ethyl 3-bromopropionate
afforded the homologous acids.
Resu lts a n d Discu ssion
Antiresorptive potency of all compounds synthesized
was directly assessed in vivo in thyroparathyroidecto-
mized (TPTX) rats with hypercalcemia induced by 1,-
25-dihydroxyvitamin D3.39
Since it was clear that the amino group was the key
structural element for the high potency of pamidronate
(1b , Table 1), the search for more potent derivatives
concentrated on BPs containing at least one nitrogen
in the side chain. Our SAR studies started with the
synthesis of close analogues of pamidronate. The length

Bisphosphonates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3725
Ta ble 4. Antiresorptive Potency of Bisphosphonates with a
Cyclic Amino Substiutent
Ta ble 5. Antiresorptive Potency of Bisphosphonates with an
Additional Bridge between the Amino Group and the Tether to
the Bisphosphonate Unit
a, Bisphosphonates were synthesized as free bisphosphonic
acids; the generic names given refer, however, to their salt forms.
b
The ED50 is the dose of compound administered sc, which results
in a 50% reduction of the hypercalcemia induced in TPTX rats by
1,25-dihydroxyvitamin D3. c Racemate.
onate) together. Such derivatives show similar activities
as olpadronate itself for ring sizes 5-7. The eight-
member ring derivative 2l is clearly less active (Table
4). The size of the alkyl linker chain is very important;
a chain length of 2 seems to be optimal (compare 2a
with 2b and 2c and compare 2g with 2h and 2i), in
contrast to the simple derivatives of Table 1, where
alendronate with n ) 3 is the most active analogue.
Among the BPs with an additional phenyl substituent
at the pyrrolidine (or piperidine ring), only 2e exhibits
increased potency compared to the unsubstituted ring
analogue. Piperazine derivatives (2m , 2n ) show very low
activity.
Another type of cyclic derivative is summarized in
Table 5. The high dependence of antiresorptive potency
on the optimal distance between the nitrogen atom and
the bone “hook” is again nicely demonstrated by the
series of regioisomers in the piperidine series. Rather
unexpected is the higher activity of compound 3a
compared with 3b, which has the same number of
carbon atoms as pamidronate between C1 and the
nitrogen.
Addition of (substituted) phenyl groups to one of the
alkyl substituents at the amino group has resulted in
some of the most potent BPs (4g, 4j, Table 6). The best
potency is observed for derivatives with R2 ) methyl;
ethyl seems to be less favorable (compare 4d and 4e).
Also, the corresponding secondary amines are clearly
less active in all cases investigated. There is a marked
dependency of potency on the length of the linkers
between the nitrogen and the phenyl ring; 4k and
especially 4l with five and six methylene units, respec-
tively, illustrate the rapid loss of antiresorptive potency
for long-chain analogues.
a
Bisphosphonates were synthesized as free bisphosphonic acids;
b
the generic names given refer, however, to their salt forms. The
ED50 is the dose of compound administered sc, which results in a
50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3. c EB-1053. Racemate.
d
of the tether between the methylenebisphosphonate unit
and the amino group is important. Alendronate (1c,
MSD/Gentili) with an additional methylene group is
almost 10 times more potent than pamidronate and
shows the best potency of all BPs with a primary amino
group. Longer chains clearly diminish the activity.
However, potency can also be increased by alkylation
of the primary amine best exemplified by ibandronate
(1k , Roche/Boehringer Mannheim). Acetylation on the
other hand destroys the activity, indicating that a basic
nitrogen functionality is of primary importance for high
potency of BPs.
The importance of the substituent X, which may serve
as an additional ligand and therefore may add to the
strength of the bone “hook”,8,9,27-30 is highlighted in
Table 2. Compound 1l, which lacks the hydroxy group,
is about 10-fold less active compared to 1h (olpadronate,
dimethyl pamidronate). The exchange of the hydroxy
group by a primary amino group reduces the activity
even further.
Interestingly the potency of pamidronate can be
drastically increased by the addition of a methyl group
to the 2-position of the ethylene bridge. The same
modification, however, does not improve the potency of
olpadronate and, in the case of ibandronate, leads to a
significant loss of activity (cf. Table 3).
The phenyl group may be linked via an oxygen or
sulfur atom to the N-alkyl chain (Table 7). The second
N-alkyl group R1 should again be small, with methyl
The nitrogen may be incorporated into a ring system,
e.g., formally linking the methyl groups of 1h (olpadr-

3726 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Widler et al.
Ta ble 6. Antiresorptive Potency of Arylalkyl-Substituted Derivatives of Pamidronate
a
b
Bisphosphonates were synthesized as free bisphosphonic acids; the generic names given refer, however, to their salt forms. The
ED50 is the dose of compound administered sc, which results in a 50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3. c Racemate.
Ta ble 7. Antiresorptive Potency of Phenoxyalkyl- and
Phenylsulfanylalkyl-Substitued Bisphosphonates
compounds in Table 7 have ED50 values of 1 µg/kg or
less. The exact position of the tertiary amino group is
very important, exemplified by the reduction in activity
of 5n versus 5l, which have the same number of atoms
in the alkyl side chain between C1 and the phenyl ring.
Their only difference is the position of the nitrogen
atom. Very long distances between the bisphosphonate
compounda
X
R1
Me
Me
H
Me
Me
Me
Me
Me
Et
propyl
butyl
Me
Me
Me
H
R2
m
n
ED50 (µg/kg)
b
moiety and the phenyl ring are clearly detrimental to
the activity of these derivatives (cf. 5m ), which is in
accordance with the trends shown in Table 6. Small
additional substituents on the phenyl ring have very
little influence (cf. 5b, 5e-h ).
5a
5b
5c
5d
5e
5f
5g
5h
5i
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
H
2
2
3
3
3
3
3
3
3
3
3
4
6
3
2
2
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
3
2
2
2
2
2
1.5
1.7
4-Cl
H
1.2
H
0.5
3-Me
4-F
4-Cl
4-MeO
H
1.7
0.6
1.3
The important basic nitrogen does not have to be an
aliphatic amine. As was first shown by Procter &
Gamble, it may be substituted by the nitrogen of a
heteroaromatic ring system, and it has turned out that
the most promising BPs belong to this class of com-
pounds. The work by the Procter & Gamble group
concentrated on six-membered ring systems. Their
efforts culminated in the development of risedronate
(Figure 2). At Ciba-Geigy, we focused on derivatives
with five-membered heterocycles, of which the most
attractive turned out to be the imidazole ring. There
are three different ways to attach an imidazole to the
carbon chain linker. In the series of 2-imidazolyl deriva-
tives, the influence of different substituents at the
nitrogen was investigated. The N-methyl derivative 6b
(Table 8) is about 10 times more active than the NH
analogue 6a . The larger N-benzyl group (6c), on the
other hand, decreases activity. A drop in potency is
found when the number of carbons in the linker chain
1.2
20
10
5j
5k
5l
H
H
500
4
7500
100
>200
0.7
H
5m
5n
5o
5p
5q
5r
H
H
H
S
Me
H
Me
Me
H
S
H
7
0.33
7.8
S
H
5s
S
4-Cl
a
Bisphosphonates were synthesized as free bisphosphonic acids;
the generic names given refer, however, to their salt forms. The
ED50 is the dose of compound administered sc, which results in a
50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3.
b
being slightly better than hydrogen (cf. 5c, 5d , 5i-k ).
The potency-enhancing effect of the additional methyl
group becomes much more pronounced in the sulfur
series (cf. 5o vs 5p and 5q vs 5r ). Many of the

Bisphosphonates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3727
Ta ble 8. Antiresorptive Potency of Bisphosphonates with Five-Membered Heterocyclic Aromatic Ring Systems
a
b
Bisphosphonates were synthesized as free bisphosphonic acids; the generic names given refer, however, to their salt forms. The
ED50 is the dose of compound administered sc, which results in a 50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3.
is increased to two, as shown in the 4-imidazolyl series;
6e has lost almost 2 orders of magnitude in activity
compared to 6d . A methyl and especially a phenyl
substituent at the 2-position of these imidazoles also
results in a marked drop in the ED50 value (cf. 6f, 6g).
The most active BP known to date has been obtained
by linking the imidazole ring via a nitrogen atom to the
bone “hook” (6i). Extension of the linker chain leads
again to a dramatic loss of activity (6j). Additional
methyl substituents at the different ring carbon atoms
do not greatly affect the potency of imidazolyl deriva-
tives, but in all the cases investigated so far, there is a
trend of a slight loss of activity (6h , 6k , 6l). Pyrazole
and triazole proved to be less attractive heterocycles for
BPs (6m , 6n ).
Ta ble 9. Antiresorptive Potency of Derivatives of
Aminomethanebisphosphonic Acid
If the “bioactive moiety” R1 (Figure 1) is linked via a
nitrogen atom to the bone “hook”, R2 cannot be a
hydroxyl group for stability reasons. Such BPs therefore
are only bidentate ligands for calcium and thus have a
lower affinity for bone. Nevertheless, several rather
potent derivatives have been found among these deriva-
tives. Table 9 shows some simple derivatives with
aliphatic amino substituents. Whereas small amino
groups lead to only marginally active BPs (7a ,7b, Table
9), potency improves with increasing size and lipophi-
licity of the amino group (7c, 7d ). Takeuchi et al.
(Yamanouchi) have reported extensively on secondary
amine analogues.33 Their finding that a sufficiently
bulky substituent on the secondary nitrogen atom is
necessary for potent antiresoptive activity corroborates
our own data (cf. high potency of 7e).
a
Bisphosphonates were synthesized as free bisphosphonic acids;
b
the generic names given refer, however, to their salt forms. The
ED50 is the dose of compound administered sc, which results in a
50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3. c Incadronate (Yamanouchi).
The most attractive aminomethylenebisphosphonates
are again those that have a heteroaromatic substituent.
Of particular interest are compounds that bear thiazole

3728 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Widler et al.
Ta ble 10. Antiresorptive Potency of Heteroarylaminomethanebisphosphonic Acid
a
b
Bisphosphonates were synthesized as free bisphosphonic acids; the generic names given refer, however, to their salt forms. The
ED50 is the dose of compound administered sc, which results in a 50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3.
substituents. The 2-thiazolyl derivative 8a (Table 10)
has a potency comparable to that of alendronate (1c)
or at least 10 times that of pamidronate (1b, Table 1).
Potency is strongly dependent on substituents in posi-
tions 4 and 5. A methyl group in position 4, i.e., next to
the ring nitrogen, causes a 20-fold drop in activity (8b).
Shifting the methyl group to the 5-position (8c) brings
about a slight improvement in the ED50 value compared
with that of the unsubstituted analogue. Larger sub-
stituents are also tolerated (8d -f); even the phenethyl
derivative 8h shows high potency. Only residues with
high steric bulk at the point of attachment (cf. the
isoproyl substituted derivative 8g or 8i with a directly
linked phenyl group) are considerably less potent. These
residues may prevent the important interaction of the
basic amino functionality with the putative molecular
target. Compound 8f is the most potent in the series,
equipotent to ibandronate (1i). Most of the aminometh-
ylenebisphosphonates with an imidazolyl group are
surprisingly less interesting. The best derivative 8k is
the one with an N-methyl group. Larger substituents
diminish the activity (8l, 8m ) progressively, while the
unsubstituted 8j is even more than a 100 times less
potent. The benzannulated derivatives have lost practi-
cally all activity.
Ta ble 11. Comparison of Antiresorptive Potency of
Bisphosphonic and Bisphosphinic Acids Containing a Thiazole
Linked via Different Atoms X
compounda
X
R
ED50 (µg/kg)
a
8a
9a
9b
NH
CH2
S
OH
OH
OH
Me
5
200
700
10
NH
>10000
a
Bisphosphonates were synthesized as free bisphosphonic acids;
b
the generic names given refer, however, to their salt forms. The
ED50 is the dose of compound administered sc, which results in a
50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3.
P r eclin ica l a n d Clin ica l Da ta of Zoled r on ic Acid
A selection of the most potent compounds of the
different subclasses in the TPTX assay were further
profiled in vitro in mouse calvaria cultures.39 Good
correlation was found between these data and the in
vivo TPTX antiresorptive potency (cf. Table 12 and
Figure 3). In the “Schenk model”,41 an acute in vivo
model often used to compare the inhibitory potency of
bisphosphonates on metaphyseal bone remodeling in
intact, growing rats, zoledronate had a minimal effective
dose of 0.3 µg/kg sc (equivalent to approximately 0.05
µg P/kg).42 To assess the effect of BPs on bone miner-
Substitution of the nitrogen linker atom of the thia-
zole derivative 8a by a methylene or a sulfur bridge led
to 9a and 9b. Unexpectedly, these compounds are much
less potent (Table 11). The poor potency of the bispho-
sphinic acid 10 on the other hand was expected, since
the binding affinity of phosphinic acids is much reduced
compared with that of bisphosphonates.8,28,40

Bisphosphonates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3729
Ta ble 12. Inhibition of Bone Resorption vs Mineralization in
Ta ble 13. Short-Term Renal Tolerability and Therapeutic
the Calvaria Assay
Ratio of Selected BP after Repeated Subcutaneous Injections
TPTX
calvaria
calvaria
TPTX vit D3
total
therapeutic
ratiod
b
vit D3 resorption mineralization
compounda
ED50 (µg/kg) MDHc (U)
b
ED50
inhib
inhib
IC50 (µM)
pamidronate (1b)
alendronate (1c)
61
8
1.4
1.0
0.5
13.4
18.9
52.5
17.4
23.7
112.4
17.6
1.2
6.6
19
120
84
4.5
compounda
(µg/kg) IC50 (µM)
ratio
pamidronate (1b)
alendronate (1c)
4b
4d
5d
8c
61
8
1.4
1.0
0.5
0.2
100
20
30
4
9
15
30
500
400
1500
80
225
375
4b
4d
5d
8c
0.05
0.02
0.05
0.04
0.04
0.002
1.5
0.07
zoledronic acid (6i)
790
1.5
0.07
a
Bisphosphonates were synthesized as free bisphosphonic acids;
the generic names given refer, however, to their salt forms. The
zoledronic acid (6i)
15000
b
a
Bisphosphonates were synthesized as free bisphosphonic acids;
ED50 is the dose of compound administered sc, which results in a
50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3. c Total units of malate dehydrogenase (MDH)
excreted in eight 24-h urine samples collected after initiating
treatment with the test compound. The corresponding value for
b
the generic names given refer, however, to their salt forms. The
ED50 is the dose of compound administered sc, which results in a
50% reduction of the hypercalcemia induced in TPTX rats by 1,25-
dihydroxyvitamin D3.
d
control animals injected with saline was 12.8 units. Therapeutic
ratio comparing nephrotoxic potential versus bone antiresorptive
potency, calculated by dividing the reciprocal of the ED50 value in
the TPTX assay (mg/kg) by the total urinary malate dehydrogenase
excretion.
excreted (Table 13). Once again, these data demonstrate
that zoledronic acid (6i) is far superior to the other
compounds by a wide margin. It was therefore selected
for additional preclinical and clinical studies.
Overall, in a range of pharmacological assays, zoledron-
ic acid was 100-850 times more active than pamidr-
onate as an inhibitor of osteoclastic bone resorption
(Table 14).39 Short-term treatment of rats with zoledron-
ic acid prevented the bone loss associated with estrogen
deficiency following ovariectomy45 and reduced the
systemic osteopenia occurring in rats and rabbits with
experimental arthritis.45,46 Long-term treatment of ova-
riectomized rats47 and adult monkeys48 with low doses
of zoledronic acid maintained the bone mass and
strength at levels similar to those of intact control
animals without any deleterious effects on bone archi-
tecture or mineralization. Renal and intestinal toler-
ability studies indicated that at identical doses the
tolerability profile of zoledronic acid is similar to, or
better than, that of pamidronate.43 Thus, when its high
antiresorptive potency is taken into consideration,
zoledronic acid has a much greater therapeutic ratio
than pamidronate (Table 14).
Recently, the molecular target of the nitrogen-
containing bisphosphonates has been identified as an
enzyme in the mevalonate pathway: farnesyl diphos-
phate synthase. Inhibition of this enzyme prevents the
prenylation of intracellular signaling proteins, thereby
impairing many cellular functions, which in turn in-
duces apoptosis and finally cell death. Among the
bisphosphonates, zoledronic acid is the most potent
inhibitor of farnesyl diphosphate synthase and inducer
of osteoclastic apoptosis identified to date.49,50
F igu r e 3. Comparison of in vitro (calvaria assay) and in vivo
(TPTX) data.
alization, in vitro calcium incorporation into mouse
calvaria was measured. In contrast to the resorption
inhibition potency, effects on mineralization were very
similar for most of the BPs investigated. Therefore, the
increase in antiresorptive potency is not associated with
a corresponding increase in inhibition of mineralization,
and antiresorptive potency largely determines the ratio
of mineralization inhibition versus resorption inhibition.
Table 12 clearly shows the superior properties of 6i
(zoledronic acid, Figure 2) against all other compounds
investigated.
In clinical studies, renal problems have been observed
following intravenous infusions of several bisphospho-
nates. Therefore, in the profiling of new, potent BPs,
there was clearly a need to investigate renal toler-
ability.43 Urinary excretion of the cytosolic enzyme
malate dehydrogenase (MDH) is a sensitive early marker
of renal toxicity34 and was used in a rat renal tolerability
screening assay. MDH excretion values for pamidronate
were identical to control levels, whereas those for
alendronate were slightly increased. The three com-
pounds 4d , 5d , and 6i (zoledronic acid, Figure 2) showed
a similar minor, nonsignificant trend toward elevated
MDH excretion. By contrast, 8c, which had previoulsy
been shown to be nephrotoxic in rats in vivo, produced
markedly elevated MDH levels, whereas 4b showed a
similar but less pronounced profile. To rank compounds,
a therapeutic ratio was calculated by dividing the
reciprocal of the bone antiresoptive ED50 value from the
TPTX assay in mg/kg by the total units of MDH activity
The results of the clinical trials indicate that low
doses of zoledronic acid (Zometa) are both efficacious
and safe for the treatment of tumor-induced hypercal-
cemia,51 Paget’s disease of bone,52 osteolytic metastas-
es,53 and postmenopausal osteoporosis.54
The bone mass and biochemical marker data from the
phase II osteoporosis trial54 indicate that an annual
intravenous infusion of 4 mg of zoledronic acid may be
an effective treatment for postmenopausal osteoporosis.
In the only publication to date on a large phase III trial

3730 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Widler et al.
Ta ble 14. Comparison of the Relative Potencies between Zoledronic Acid and Pamidronate in Various Efficacy and Tolerability
Models in Rats
treatment
period
zoled
(a)
pamid
(b)
ratio
(b/a)
efficacy models
inhibition of 1,25(OH)2D3-induced hypercalcemia in TPTX rats, ED50 µg/kg
increased radiographic bone density in young intact rats, ED50 µg/kg
increased cancellous bone area in young intact rats, ED50 µg/kg
increased cancellous bone calcium in young intact rats, ED50 µg/kg
increased cancellous bone hydroxyproline in young intact rats, ED50 µg/kg
renal and intestinal tolerability models
4 d
0.072
1.7
0.071
0.17
1.1
61
390
7.1
22.7
210
850
230
100
130
190
10 d
10 d
10 d
10 d
renal: increased serum urea level in rats, ED100, mg/kg
renal: increased urinary excretion of MDH in rats, NOL mg/kg
intestinal: increased mucosal permeability of perfused ileal loop in rats, NOL mM
1 h
14 d
2 h
38
>1.0
10
11
0.1
10
0.3
<0.1
1
a fasting blood sample collected on day 9. In each experiment,
the upper and lower limits of hypercalcemia (100% and 0%,
respectively) were established with control TPTX animals,
which had only received 1,25(OH)2D3 or saline. Pamidronate
was used as a positive control and caused a clear dose-
dependent inhibition of hypercalcemia, achieving 100% efficacy
at a dose of approximately 500 µg/kg and a mean ED50 value
of 61 µg/kg sc. The presented TPTX data are the means of at
least three independent experiments.
Ca lva r ia Assa y in Vitr o. The effect of various bisphos-
phonates on osteoclastic bone resorption in vitro was deter-
mined in murine calvarial cultures.39 Pairs of hemicalvaria
from neonatal mice (4-7 days old) were first preincubated for
24 h in the presence or absence of test compound so that each
treated hemicalvaria had a corresponding untreated control
from the same animal. Then, a stimulator of calcium release
(20 nM 1,25(OH)2D3) was added and the incubation was
continued for a further 72 h. At the end of the incubation
period, the calcium content of the medium and that of the
calvaria were determined to permit calculation of the amount
of calcium released from the calvaria as a percentage of the
its calcium content. Bisphosphonate treatment resulted in a
dose-dependent inhibition of calcium release, with full efficacy
at the higher concentrations. Pamidronate was again used as
the reference compound, showing a mean IC50 of 0.2 µM. The
presented calvaria data are the means of two to six indepen-
dent experiments with five to seven calvaria in each treatment
group.
A modification of the calvarial assay was used to assess the
possible interference of BPs with bone mineralization in vitro.39
Calvaria were cultured in a manner similar to that described
above but with the following changes: calcium incorporation
was stimulated by replacing the 1,25(OH)2D3 with 2 mM
calcium 1,2-glycerophosphate, and the incubation period was
reduced to 48 h, but the 24 h preincubation with bisphospho-
nate remained unchanged. The amount of calcium and inor-
ganic phosphate incorporated into the calvaria was calculated
as the percent increase relative to the initial calcium content.
Bisphosphonate treatment dose-dependently inhibited the
incorporation of calcium and inorganic phosphate into the
calvaria, yet the ratio of these two ions remained close to 1.66,
the value for hydroxyapatite.
Ren a l Toler a bility Assa y. Renal tolerability of bisphos-
phonates was investigated in rats by monitoring the urinary
excretion of the cytosolic marker enzyme malate dehydroge-
nase as a parameter of nephrotoxicity.43 Test compound was
repeatedly administered to male rats (180-230 g body weight,
three animals per treatment group) as nine subcutaneous
injections (0.01, 0.1, and 1.0 mg/kg) at 1-2 day intervals over
a 2 week period. Malate dehydrogenase activity was measured
colorimetrically in eight 24-h urine collections and calculated
as total units of excreted enzymatic activity. The presented
data are from a single experiment.
with zoledronic acid, the compound proved to be supe-
rior to pamidronate for the treatment of tumor-induced
hypercalcemia.55
Exp er im en ta l Section
Reagents and solvents were used as purchased without
further purification. Column flash chromatography was per-
formed on silica gel 60 (230-400 mesh ASTM, E. Merck).
Melting points were determined in an open capillary and are
not corrected. Thin-layer chromatography (TLC) was per-
formed on precoated silica gel plates (E. Merck, silica gel 60
F
254, 0.25 mm). Compounds were visualized by UV light or
iodine vapor. For TLC of bisphosphonates, cellulose plates
without fluorescence indicator (0.1 mm thick, E. Merck) were
used. The most useful solvent system for these very polar
compounds was a five-component mixture consisting of 30
parts of water, 30 parts of pyridine, 25 parts of n-butanol, 12
parts of 2-butanone, and 3.5 parts of formic acid. For detection
a solution of a Mo/MoO3 staining reagent56 was used. Too
heavy staining should be avoided; the spots develop best
without any heating of the plates. 1H NMR spectra were
recorded on Varian-60, Varian Gemini 200/300, and Bruker
AM 250/300/360 spectrometers. Chemical shifts of signals are
expressed in parts per million (ppm) relative to tetramethyl-
silane as internal standard or to the deuterated solvents used.
Coupling constants J are reported in hertz (Hz). For 13C NMR
spectra, a Bruker XL-300 instrument was used, and for 31P
NMR, a Varian Mercury 300 instrument was used. Elemental
analyses were within (0.4% of the calculated values unless
indicated otherwise. Generally the bisphosphonates were dried
for several hours at 80-120 °C. Even so, many of them retain
residual solvents, especially water. On prolonged exposure to
air, stable hydrates can be obtained, which however was not
done for most of the compounds synthesized. Chemical yields
were not optimized and are highly dependable on the ease of
crystallization of the crude reaction product. They range from
a few percent up to 80%, typically being around 40-60% (see
details below).
Hyp er ca lcem ia Assa y in th e TP TX Ra t. As a primary
screen to assess the effects of bisphosphonates on mineral
metabolism, the thyroparathyroidectomised (TPTX) rat with
hypercalcemia induced by 1,25-dihydroxyvitamin D3 was used.
Removal of the thyroid and parathyroid glands results in a
test animal with a low serum calcium concentration that can
be modulated by various calciotropic agents. In the routine
assay, repeated injections of 1,25(OH)2D3 were used to stimu-
late bone resorption and to produce acute hypercalcemia. A
reduction in the hypercalcemia following bisphosphonate
treatment was taken as indirect evidence for the inhibition of
osteoclastic activity. In the standard assay protocol, young
male rats (130-150 g body weight) were thyroparathyroidec-
tomized and then allowed a 4-day recovery period before a
fasting serum sample was collected. Animals with a serum
calcium concentration above 1.88 mM were discarded, the
remaining animals were randomly divided into groups of 5.
On postoperative days 5-8, 1,25(OH)2D3 was administered
(125 pmol/kg sc) together with various doses of the test
bisphosphonate (sc). Total serum calcium was determined in
P r ep a r a tion of Sta r tin g Ma ter ia ls. The following het-
erocyclic amines were prepared according to the literature:
2-amino-1-methylimidazole and 2-amino-1-benzylimidazole,57
2-amino-1-phenylimidazole,58 1,2,4-thiadiazol-5-amine,59 and
5-alkyl-substituted 2-aminothiazoles.60 4,5-Dimethylimidazole

Bisphosphonates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3731
was obtained by reduction of 2-hydroxymethyl-3-methylimi-
dazole with red phosphorus in hydroiodic acid at 160 °C in a
sealed tube in analogy to a literature procedure.61
A three-step procedure afforded 4-phenylpiperidines starting
with 1-benzylpiperidin-4-one. After Grignard addition of (sub-
stituted) phenylmagnesium bromide, water was eliminated
(concentrated HCl/AcOH) followed by catalytic reduction (H2,
Pd/C) of the double bond and concurrent cleavage of the benzyl
protecting group.
Reduction of ethyl 4-nitro-3-phenylbutyrate62 led directly to
the 3-phenyl-γ-lactam, which was reduced with lithium alu-
minum hydride to give 3-phenylpyrrolidine. 3-(4-Chlorophen-
yl)pyrrolidine was prepared accordingly.
All other precursors were synthesized following the outlines
given in the chemistry section above.
Abbr evia tion s. h, hour; rt, room temperature; bp, boiling
point; mp, melting point; equiv, equivalent; iV, in vacuo; HV,
high vacuum; br, broad; dec, decomposing; BP, bisphosphonate;
sc, subcutaneous.
P r ep a r a tion of Bisp h osp h on a tes. 1-Hydroxy-2-amino-
ethylidene-1,1-bisphosphonic acid (1a ) was prepared as de-
scribed in the literature.63 Pamidronate (1b) was obtained from
Henkel. Compounds 3a and 7a -d were prepared by the Agro
Chemical Division of Ciba-Geigy, following a procedure de-
scribed in a Henkel patent.64
27.2, 24.1. 31P NMR (5% NaOD/D2O): δ 19.69. Anal. (C6H17
-
NO7P2) C, H, N, P.
1-Hydr oxy-3-dieth ylam in opr opyliden e-1,1-bisph osph o-
1
n ic Acid (1j). Yield: 56%; mp 220 °C (dec). H NMR (NaOD/
D2O, 300 MHz): δ 2.42-2.62 (m, 2H), 1.24 (t, 4H), 1.63-1.80
(m, 2H), 0.70 (t, 6H). 31P NMR (5% NaOD/D2O): δ 19.15. Anal.
(C7H19NO7P2) C, H, N, P.
1-Hyd r oxy-3-(m eth ylp en tyla m in o)p r op ylid en e-1,1-bis-
p h osp h on ic Acid (Iba n d r on a te, 1k ). Yield: 6%; mp 126-
133 °C (dec). 1H NMR (NaOD/D2O, 300 MHz): δ 2.52-2.61
(m, 2H), 2.20-2.29 (m, 2H), 2.03 (s, 3H), 1.81-1.94 (m, 2H),
1.24-1.37 (m, 2H), 1.03-1.18 (m, 4H), 0.68 (t, 3H). 31P NMR
(5% NaOD/D2O): δ 19.24. Anal. (C9H23NO7P2‚0.25H2O) C, H,
N, P.
1-Hyd r oxy-3-a m in obu tylid en e-1,1-bisp h osp h on ic Acid
(1n ). Yield: 41%; mp 145-146 °C (dec). 1H NMR (NaOD/D2O,
300 MHz): δ 3.79 (m, 1H), 2.00-2.33 (m, 2H), 1.28 (d, 3H).
31P NMR (5% NaOD/D2O): δ 19.25 (AB system). Anal. (C4H13
NO7P2) C, H, N, P.
-
1-Hydr oxy-3-m eth yl-3-(m eth ylpen tylam in o)bu tyliden e-
1,1-bisp h osp h on ic Acid (1p ). Yield: 24%; mp 135-136 °C
1
(dec). H NMR (NaOD/D2O, 200 MHz): δ 3.54-3.67 (m, 1H),
2.64-2.76 (m, 1H), 2.45-2.58 (m, 1H), 2.30 (s, 3H), 1.98-2.18
(m, 1H), 1.76 (q, 1H), 1.35-1.49 (m, 2H), 1.04-1.18 (m, 4H),
0.92 (d, 3H), 0.66 (t, 3H). 31P NMR (5% NaOD/D2O): δ 18.73
(AB system). Anal. (C10H25NO7P2‚0.5H2O) C, H, N, P. C: calcd,
35.09; found, 34.6.
The other bisphosphonates were prepared according to
Scheme 1, methods A-D.
1-Hyd r oxy-3-(p yr r olid in e-1yl)p r op ylid en e-1,1-bisp h o-
sp h on ic Acid (2a ). Yield: 44%; mp 239-242 °C (dec). 1H
NMR (NaOD/D2O, 300 MHz): δ 2.74-2.53 (m, 2H), 2.25 (br
s, 4H), 1.67-1.85 (m, 4H), 1.42 (br s, 4H). 31P NMR (5% NaOD/
D2O): δ 19.07. Anal. (C7H17NO7P2) C, H, N, P.
Meth od A: Syn th esis of 1-Hyd r oxy-1,1-bisp h osp h o-
n a tes. Gen er a l P r oced u r e for th e Syn th esis of 1-Hy-
d r oxy-1,1-bisp h osp h on a tes. One equivalent of the corre-
sponding carboxylic acid is dissolved in 3 equiv of phosphoric
acid (85%) and chlorobenzene (ca. 500 mL/mol starting mate-
rial) by heating to 100-110 °C. The hot solution is treated
dropwise with 2 equiv of phosphorus trichloride, resulting in
a vigorous exothermic reaction and gas evolution. Within 15
min to 1 h, a thick precipitate forms, which eventually prevents
stirring. Heating is continued for another 3 h without stirring.
Then the bulk of the chlorobenzene is decanted followed by
removal of the remaining solvent under reduced pressure. The
white or yellow residue is taken up in water (1000 mL/mol
substrate) and heated to reflux for approximately 1 h. If
necessary, the mixture is treated with charcoal before filtration
and concentration iV. Crystallization of the resulting slightly
yellow oil may be tedious. In most cases, it can be induced by
the careful addition of small amounts of ethanol to an aqueous
solution at room temperature. Sometimes it may be advanta-
geous to wash the crude oil first with acetone, which is
decanted after phase separation.
In an alternative workup procedure, concentrated HCl is
added to the crude reaction mixture and heating continued
for several hours until gas formation gradually subsides.
Charcoal is added, and stirring is continued for a few minutes.
After filtration, the solution is concentrated iV to leave in most
cases a syrupy mass, which is crystallized by careful addition
of water. Similar to the first workup procedure described
above, the oily crude bisphosphonate may first be washed with
acetone to facilitate crystallization.
1-Hyd r oxy-4-(p yr r olid in e-1yl)bu tylid en e-1,1-bisp h os-
p h on ic Acid (2b). Yield: 75%; mp 249-250 °C. 1H NMR
(NaOD/D2O, 300 MHz): δ 2.22 (br s, 2H), 2.21 (br t, 2H), 1.35-
1.60 (m, 8H). 31P NMR (5% NaOD/D2O): δ 19.49. Anal. (C8H19
NO7P2) C, H, N, P.
-
1-Hyd r oxy-6-(p yr r olid in e-1yl)h exylid en e-1,1-bisp h os-
p h on ic Acid (2c). Yield: 62%; mp 239-240 °C. 1H NMR
(NaOD/D2O, 300 MHz): δ 2.08-2.25 (m, 6H), 1.4-1.65 (m,
2H), 1.41 (br s, 4H), 1.14-1.32 (m, 4H), 0.88-1.03 (m, 2H).
31P NMR (5% NaOD/D2O): δ 19.70. Anal. (C10H23NO7P2) C,
H, N, P.
1-H yd r oxy-3-(3-p h en ylp yr r olid in e-1yl)p r op ylid en e-
1,1-bisp h osp h on ic Acid (2d ). Yield: 18%; mp 221 °C (dec).
1H NMR (NaOD/D2O, 300 MHz): δ 7.02-7.12 (m, 4H), 6.93-
7.02 (m, 1H), 3.06 (5-line system, 1H), 2.87 (t, 1H), 2.48-2.68
(m, 3H), 2.36-2.48 (m, 1H), 2.23 (t, 1H), 1.89-2.04 (m, 1H),
1.70-1.89 (m, 2H), 1.46-1.62 (m,1H). 31P NMR (5% NaOD/
D2O): δ 19.09. Anal. (C13H21NO7P2) C, H, N, P.
1-H yd r oxy-3-[3-(4-ch lor op h en yl)p yr r olid in e-1yl]p r o-
p ylid en e-1,1-bisp h osp h on ic Acid (2e). Yield: 20%; mp 219
°C (dec). 1H NMR (NaOD/D2O, 300 MHz): δ 7.04 (d, 2H), 6.99
(d, 2H), 3.04 (5-line system, 1H), 2.85 (t, 1H), 2.47-2.67 (m,
3H), 2.33-2.47 (m, 1H), 2.20 (t, 1H), 1.87-2.03 (m, 1H), 1.69-
1.87 (m, 2H), 1.42-1.68 (m, 1H). 31P NMR (5% NaOD/D2O):
δ 19.06. Anal. (C13H20ClNO7P2‚0.5H2O) C, H, N, P.
1-Hyd r oxy-3-(p ip er id in e-1yl)p r op ylid en e-1,1-bisp h os-
p h on ic Acid (2f). Yield: 82%; mp 234 °C (dec). 1H NMR
(NaOD/D2O, 300 MHz): δ 2.27-2.40 (m, 2H), 2.14 (br s, 4H),
1.64-2.84 (m, 2H), 1.22 (br s, 4H), 1.13 (br s, 2H). 31P NMR
(5% NaOD/D2O): δ 19.13. Anal. (C8H19NO7P2) C, H, N.
1-Hyd r oxy-3-(4-p h en ylp ip er id in e-1yl)p r op ylid en e-1,1-
bisp h osp h on ic Acid (2g). Yield: 67%; mp 243-245 °C (dec).
1H NMR (NaOD/D2O, 300 MHz): δ 6.92-7.13 (m, 5H), 2.79
(br d, 2H), 2.38-2.49 (m, 2H), 2.80 (br t, 1H), 1.70-1.95 (m,
4H), 1.53 (br d, 2H), 1.27-1.46 (m, 2H). 31P NMR (5% NaOD/
D2O): δ 19.15. Anal. (C14H23NO7P2‚H2O) C, H, N, P.
1-H yd r oxy-4-(4-p h en ylp ip er id in e-1yl)b u t ylid en e-1,1-
According to this general method, the following derivatives
were synthesized.
1-Hyd r oxy-4-a m in obu tylid en e-1,1-bisp h osp h on ic Acid
(Alen d r on a te, 1c). Yield: 18%; mp 235 °C (dec). 1H NMR
(NaOD/D2O, 300 MHz): δ 2.26 (t, 2H), 1.32-1.62 (m, 4H). 31
P
NMR (5% NaOD/D2O): δ 19.51. Anal. (C4H13NO7P2) C, H, N,
P.
1-Hydr oxy-5-am in open tyliden e-1,1-bisph osph on ic Acid
(1d ). Yield: 42%; mp 208-212 °C (dec). 1H NMR (NaOD/D2O,
300 MHz): δ 2.85 (t, 2H), 1.83-1.98 (m, 2H), 1.52-1.72 (m,
4H), 19.51. Anal. (C5H15NO7P2) C, H, N, P.
1-Hyd r oxy-6-a m in oh exylid en e-1,1-bisp h osp h on ic Acid
(Ner id r on a te, 1e). Yield: 19%; mp 247-249 °C. 1H NMR
(NaO/D2O, 300 MHz): δ 2.27 (t, 2H), 1.45-1.63 (m, 2H), 1.18-
1.30 (m, 2H), 1.14 (5-line system, 2H), 0.90-1.03 (m, 2H). 13C
NMR (NaOD/D2O): δ 75.5 (t, J C-P ) 129 Hz), 40.2, 35.0, 27.8,
1
bisp h osp h on ic Acid (2h ). Yield: 75%; mp 230 °C (dec). H
NMR (NaOD/D2O, 300 MHz): δ 6.82-7.12 (m, 5H), 2.77 (br
d, 2H), 2.28 (br t, 1H), 2.00-2.09 (br, 2H), 1.81 (t, 2H), 1.44-
1.60 (m, 6H), 1.27-1.44 (m, 2H). 31P NMR (5% NaOD/D2O):
δ 19.48. Anal. (C15H25NO7P2) C, H, N, P.

3732 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Widler et al.
1-Hyd r oxy-6-(4-p h en ylp ip er id in e-1-yl)h exylid en e-1,1-
bisp h osp h on ic Acid (2i). Yield: 35%; mp 236-237 °C (dec).
1H NMR (NaOD/D2O, 300 MHz): δ 6.91-7.13 (m, 5H), 2.74
(d, 2H), 2.30 (br t, 1H), 2.02-2.13 (m, 2H), 1.81 (t, 2H), 1.46-
1.66 (m, 4H), 1.39 (t, 2H), 1.16-1.33 (m,4 H), 0.90-1.05 (m,2
H). 31P NMR (5% NaOD/D2O): δ 19.70. Anal. (C17H29NO7P2‚
0.25H2O) C, H, N, P.
1-Hyd r oxy-3-[4-(3-flu or op h en yl)p ip er id in e-1yl]p r op y-
lid en e-1,1-bisp h osp h on ic Acid (2j). Yield: 11%; mp 247-
248 °C (dec). 1H NMR (NaOD/D2O, 300 MHz): δ 6.98-7.10
(m, 1H), 6.62-6.86 (m, 3H), 2.77 (d, 2H), 2.41 (br s, 2H), 2.31
(br t, 1H), 1.70-1.95 (m, 4H), 1.63 (d, 2H), 1.24-1.42 (m, 2H).
31P NMR (5% NaOD/D2O): δ 19.13. Anal. (C14H22FNO7P2‚
0.5H2O) C, H, N, P.
1-H yd r oxy-3-[m et h yl-(3-p h en ylp r op yl)a m in o]p r op y-
lid en e-1,1-bisp h osp h on ic Acid (4d ). Yield: 55%; mp 176
°C (dec). 1H NMR (D2O, 300 MHz): δ 7.00-7.20 (m, 5H), 2.48-
2.59 (m, 2H), 2.44 (t, 2H), 2.20-2.30 (m, 2H), 2.00 (s, 3H),
1.77-1.94 (m, 2H), 1.55-1.68 (m, 2H). 31P NMR (5% NaOD/
D2O): δ 19.00. Anal. (C13H23NO7P2‚H2O) C, H, N, P.
1-H yd r oxy-4-[et h yl-(3-p h en ylp r op yl)a m in o]p r op yli-
d en e-1,1-bisp h osp h on ic Acid (4e). Yield: 25%; mp 195-
197 °C (dec). 1H NMR (NaOD/D2O, 250 MHz): δ 6.88-7.08
(m, 5 H), 2.44-2.53 (m, 2H), 2.14-2.33 (m, 6H), 1.63-1.84
(m, 2H), 1.39-1.56 (m, 2H), 0.67 (t, 3H). 31P NMR (5% NaOD/
D2O): δ 19.03. Anal. (C14H25NO7P2) C, H, N, P.
1-Hydr oxy-3-[m eth yl-(3-tolylpr opyl)am in o]pr opyliden e-
1,1-bisp h osp h on ic Acid (4f). Yield: 34%; mp 193-195 °C
1
(dec). H NMR (NaOD/D2O, 250 MHz): δ 6.94-7.04 (m, 1H),
1-Hyd r oxy-3-(a zep a n -1yl)p r op ylid en e-1,1-bisp h osp h o-
n ic Acid (2k ). Yield: 32%; mp 230-232 °C. 1H NMR (NaOD/
D2O, 300 MHz): δ 2.45-2.65 (m, 2H), 2.32-2.40 (m, 4H),
1.66-1.84 (m, 2H), 1.20-1.40 (m, 8 H). 31P NMR (5% NaOD/
D2O): δ 19.15. Anal. (C9H21NO7P2) C, H, N, P.
6.79-6.91 (m, 3H), 2.38-2.42 (m, 2H), 2.31 (t, 2H), 2.11-2.24
(m, 2H), 2.05 (s, 3H), 1.94 (s, 3H), 1.66-1.88 (m, 2H), 1.42-
1.62 (m, 2H). 31P NMR (5% NaOD/D2O): δ 19.00. Anal. (C14H25
NO7P2) C, H, N, P.
-
1-Hyd r oxy-3-[(3-(4-ch lor op h en yl)p r op yl)m eth yla m in o]-
p r op ylid en e-1,1-bisp h osp h on ic Acid (4g). Yield: 21%; mp
162-166 °C (dec). 1H NMR (NaOD/D2O, 250 MHz): δ 7.04 (d,
2H), 6.95 (d, 2H), 2.38-2.50 (m, 2H), 2.30 (t, 2H), 2.08-2.19
(m,2 H), 1.89 (s, 3H), 1.65-1.88 (m,2 H), 1.40-1.60 (m, 2H).
31P NMR (5% NaOD/D2O): δ 19.02. Anal. (C13H22ClNO7P2‚
H2O) C, H, N. C: calcd, 37.20; found, 36.60.
1-Hyd r oxy-3-[(1-m eth yl-3-p h en ylp r op yl)a m in o]p r op y-
lid en e-1,1-bisp h osp h on ic Acid (4h ). Yield: 28%; mp 142-
150 °C (dec). 1H NMR (NaOD/D2O, 250 MHz): δ 6.97-7.18
(m, 5H), 2.57-2.76 (m, 2H), 2.32-2.57 (m, 3H), 1.70-1.90 (m,
2H), 1.52-1.70 (m, 1H), 1.26-1.44 (m, 1H), 0.89 (d, 3H). 31P
NMR (5% NaOD/D2O): δ 19.05. Anal. (C13H23NO7P2) C, H, N,
P.
1-Hyd r oxy-3-[(4-p h en ylb u t yl)a m in o]p r op ylid en e-1,1-
bisp h osp h on ic Acid (4i). Yield: 70%; mp 191-193 °C (dec).
1H NMR (NaOD/D2O, 250 MHz): δ 6.94-7.14 (m, 5H), 2.50-
2.62 (m, 2H), 2.27-2.40 (m,4 H), 1.66-1.87 (m, 2H), 1.30-
1.46 (m, 2H), 1.14-1.30 (m, 2H). 31P NMR (5% NaOD/D2O):
δ 18.99. Anal. (C13H23NO7P2) C, H, N, P.
1-H yd r oxy-3-[m et h yl-(4-p h en ylb u t yl)a m in o]p r op yli-
d en e-1,1-bisp h osp h on ic Acid (4j). Yield: 30%; mp 128-132
°C (dec). 1H NMR (NaOD/D2O, 250 MHz): δ 6.91-7.12 (m,
5H), 2.28-2.49 (m, 4H), 2.09-2.18 (m, 2H), 1.88 (s, 3H), 1.65-
1.88 (m, 2H), 1.12-1.40 (m, 4H). 31P NMR (5% NaOD/D2O):
δ 19.01. Anal. (C14H25NO7P2) C, H, N, P. P: calcd, 16.25; found,
15.3.
1-H yd r oxy-4-(a zoca n -1yl)b u t ylid en e-1,1-b isp h osp h o-
n ic Acid (2l). Yield: 81%; mp 248-249 °C. H NMR (NaOD/
D2O, 300 MHz): δ 2.34-2.45 (m, 2H), 2.04-2.14 (m,2 H),
1.37-1.55 (m, 4H), 1.17-1.37 (m, 10H). 31P NMR (5% NaOD/
D2O): δ 19.51. Anal. (C11H25NO7P2) C, H, N, P.
1
1-Hyd r oxy-3-(4-m eth ylp ip er a zin -1yl)p r op ylid en e-1,1-
1
bisp h osp h on ic Acid (2m ). Yield: 50%; mp 340 °C (dec). H
NMR (NaOD/D2O, 300 MHz): δ 1.8-2. 9 (br, 8H, piperazine),
2.44-2.55 (m, 2H), 1.99 (s, 3H), 1.75-1.95 (m, 2H). 31P NMR
(5% NaOD/D2O): δ 18.92. For combustion analysis, this
compound was crystallized in the form of its Na3 salt by the
addition of dilute sodium hydroxide solution. The solid contains
a small amount of phosphoric acid plus 2 mol of crystal water.
Anal. (C8H17N2O7P2Na3‚2H2O‚0.15H3PO4) C, H, N, Na, P.
1-Hyd r oxy-3-(4-p h en ylp ip er a zin -1yl)p r op ylid en e-1,1-
1
bisp h osp h on ic Acid (2n ). Yield: 17%; mp 234 °C (dec). H
NMR (NaOD/D2O, 300 MHz): δ 7.08 (t, 2H), 6.82 (d, 2H), 6.75
(t, 1H), 2.72-2.97 (br s, 4H), 2.32-2.56 (m, 6H), 1.68-1.90
(m, 2H). 31P NMR (5% NaOD/D2O): δ 19.05. Anal. (C13H22
N2O7P2) C, H, N.
-
1-Hyd r oxy-1-(p ip er id in -3-yl)m eth ylid en ebisp h osp h o-
n ic Acid (3b). Yield: 52%; mp 244-246 °C. 1H NMR (NaOD/
D2O, 300 MHz): δ 2.90 (d, 1H), 2.56 (d, 1H), 2.48 (t, 1H), 2.14
(td, 1H), 1.65-1.80 (m, 2H), 1.30-1.48 (m,2 H), 0.95-1.15 (m,1
H). 31P NMR (5% NaOD/D2O): δ 18.81. Anal. (C6H15NO7P2)
C, H, N, P.
1-Hyd r oxy-1-(p ip er id in -4-yl)m eth ylid en ebisp h osp h o-
n ic Acid (3c). Yield: 47%; mp 227-230 °C. 1H NMR (NaOD/
D2O, 300 MHz): δ 2.68 (d, 2H), 2.12 (t, 2H), 1.62-1.80 (m,
1H), 1.59 (d, 2H), 1.30-1.47 (m, 2H). 31P NMR (5% NaOD/
D2O): δ 18.97. Anal. (C6H15NO7P2‚0.25H2O) C, H, N, P.
1-Hyd r oxy-1-(a zetid in -3-yl)m eth ylid en ebisp h osp h on -
ic Acid (3d ). Yield: 16%; mp 242-243 °C (dec). 1H NMR
(NaOD/D2O, 250 MHz): δ 3.65-3.80 (m, 2 H), 3.15-3.30 (m,
3 H). 13C NMR (NaOD/D2O, XL-300): δ 76.3 (t, J C-P ) 133
Hz), 49.1 (t, J C-P ) 10 Hz), 40.4. 31P NMR (5% NaOD/D2O): δ
18.09. Anal. (C4H11NO7P2) C, H, N, P.
1-Hyd r oxy-3-p h en eth yla m in op r op ylid en e-1,1-bisp h o-
sp h on ic Acid (4a ). Yield: 50%; mp 205-206 °C (dec). 1H
NMR (NaOD/D2O, 250 MHz): δ 6.97-7.14 (m, 5 H), 2.46-
2.64 (m, 6H), 1.60-1.82 (m, 2H). 31P NMR (5% NaOD/D2O):
δ 19.02. Anal. (C11H19NO7P2) C, H, N, P.
1-Hyd r oxy-3-[(5-p h en ylp en tyl)a m in o]p r op ylid en e-1,1-
bisp h osp h on ic Acid (4k ). Yield: 30%; mp 124-127 °C (dec).
1H NMR (NaOD/D2O, 250 MHz): δ 6.93-7.13 (m, 5H), 2.40-
2.49 (m, 2H), 2.35 (t, 2H), 2.06-2.16 (m, 2 H), 1.91 (s, 3H),
1.64-1.90 (m, 2H), 1.34 (5-line system, 2 H), 1.15-1.27 (m,
2H), 0.93-1.08 (m, 2H). 31P NMR (5% NaOD/D2O): δ 19.00.
Anal. (C14H25NO7P2) C, H, N, P.
1-H yd r oxy-3-[m et h yl-(2-p h en oxyet h yl)a m in o]p r op y-
lid en e-1,1-bisp h osp h on ic Acid (5a ). Yield: 63%; mp 125-
1
130 °C (dec). H NMR (NaOD/D2O, 250 MHz): δ 7.06 (t, 2 H),
6.70-6.77 (m, 3 H), 3.86 (t, 2 H), 2.42-2.56 (m, 4 H), 1.97 (s,
3 H), 1.64-1.86 (m, 2 H). 31P NMR (5% NaOD/D2O): δ 19.00.
Anal. (C12H21NO8P2‚0.5H2O) C, H, N, P.
1-Hyd r oxy-3-[m eth yl-(2-(4-ch lor op h en oxy)eth yl)a m i-
n o]p r op ylid en e-1,1-bisp h osp h on ic Acid (5b). Yield: 14%;
mp 216-218 °C (dec). 1H NMR (NaOD/D2O, 250 MHz): δ 7.36
(d, 2H), 7.01 (d, 2H), 4.18 (t, 2H), 2.80-2.90 (m, 4H), 2.32 (s,
3H), 2.04-2.20 (br m, 2H). 31P NMR (5% NaOD/D2O): δ 18.99.
Anal. (C12H20ClNO8P2‚0.5H2O) C, H, N, P.
1-Hyd r oxy-3-(m eth ylp h en eth yla m in o)p r op ylid en e-1,1-
bisp h osp h on ic Acid (4b). Yield: 48%; mp 187-188 °C (dec).
1H NMR (NaOD/D2O, 250 MHz): δ 6.97-7.14 (m, 5H), 2.45-
2.60 (m, 4H), 2.53-2.45 (m, 2H), 2.01 (s, 3H), 1.68-1.90 (m,
2H). 31P NMR (5% NaOD/D2O): δ 19.02. Anal. (C12H21NO7P2‚
0.25H2O) C, H, N, P.
1-H yd r oxy-3-[(3-p h en oxyp r op yl)a m in o]p r op ylid en e-
1,1-bisp h osp h on ic Acid (5c). Yield: 23%; mp 148-155 °C
1
(dec). H NMR (NaOD/D2O, 250 MHz): δ 7.05 (t, 2H), 6.64-
6.76 (m, 3H), 3.78 (t, 2H), 2.52 (br t, 2H), 2.38 (t, 2 H), 1.50-
1.80 (m, 4H). 31P NMR (5% NaOD/D2O): δ 19.03. Anal.
(C12H21NO8P2) C, H, N, P.
1-H yd r oxy-3-(3-p h en ylp r op yla m in o)p r op ylid en e-1,1-
bisp h osp h on ic Acid (4c). Yield: 37%; mp 219 °C (dec). H
NMR (NaOD/D2O, 300 MHz): δ 6.91-7.09 (m, 5H), 2.46-2.56
(m, 2H), 2.35 (t, 2H), 2.27 (t, 2H), 1.62-1.80 (m, 2H), 1.47 (5-
line system, 2H). 31P NMR (5% NaOD/D2O): δ 19.17. Anal.
(C12H21NO7P2‚H2O) C, H, N, P.
1
1-Hyd r oxy-3-[m eth yl-(3-p h en oxyp r op yl)a m in o]p r op y-
lid en e-1,1-bisp h osp h on ic Acid (5d ). Yield: 72%; mp 198-
1
200 °C (dec). H NMR (NaOD/D2O, 300 MHz): δ 7.36 (t, 2H),

Bisphosphonates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3733
6.99-7.06 (m, 3H), 4.15 (t, 2H), 3.24 (br t, 2H), 3.10-3.20 (m,
2H), 2.61 (s, 3H), 2.10-2.33 (m, 4H). 31P NMR (5% NaOD/
D2O): δ 19.00. Anal. (C13H23NO8P2) C, H, N, P.
116 °C. 1H NMR (NaOD/D2O, 200 MHz): δ 7.21-7.44 (m, 5H),
3.08 (t, 2H), 2.70-2.88 (m, 4H), 1.83-2.09 (m, 2H). 31P NMR
(5% NaOD/D2O): δ 19.01. Anal. (C11H19NO7P2S‚0.25H2O) C,
H, N, P, S.
1-Hydr oxy-3-[m eth yl-(3-(3-m eth ylph en oxy)pr opyl)am i-
n o]p r op ylid en e-1,1-bisp h osp h on ic Acid (5e). Yield: 27%;
mp 152-158 °C. H NMR (NaOD/D2O, 300 MHz): δ 7.27 (t,
1H), 6.82-6.85 (m, 3H), 4.12 (t,2 H), 2.83-2.90 (m, 2H), 2.64-
2.72 (m, 2H), 2.35 (s, 3H), 2.32 (s, 3H), 2.06-2.21 (m, 2H),
1.96-2.06 (m, 2H). 31P NMR (5% NaOD/D2O): δ 19.00. Anal.
(C14H25NO8P2) C, H, N, P.
1-Hyd r oxy-3-[m et h yl-(2-p h en ylsu lfa n ylet h yl)a m in o]-
p r op ylid en e-1,1-bisp h osp h on ic Acid (5p ). Yield: 30%; mp
170-172 °C (dec). 1H NMR (NaOD/D2O, 300 MHz): δ 7.48 (d,
2H), 7.39 (t, 2H), 7.30 (t, 1H), 3.18-3.27 (m, 2H), 2.95-3.08
(m, 4H), 2.51 (s, 3H), 2.17 (7-line system, 2H). 31P NMR (5%
NaOD/D2O): δ 18.97. Anal. (C12H21NO7P2S) C, H, N, P, S.
1
1-Hyd r oxy-3-[m eth yl-(3-(4-flu or op h en oxy)p r op yl)a m i-
n o]p r op ylid en e-1,1-bisp h osp h on ic Acid (5f). Yield: 35%;
mp 165-172 °C. H NMR (NaOD/D2O, 300 MHz): δ 7.12 (t,
2H), 6.96-7.05 (m, 2H), 4.10 (t, 2H), 2.83-2.90 (m, 2H), 2.64-
2.73 (m, 2H), 2.31 (s, 3H), 2.07-2.17 (m, 2H), 1.95-2.07 (m,2
H). 31P NMR (5% NaOD/D2O): δ 19.00. Anal. (C13H22FNO8P2‚
0.5H2O) C, H, N, F, P.
1-Hyd r oxy-3-[(3-p h en ylsu lfa n ylp r op yl)a m in o]p r op yli-
d en e-1,1-bisp h osp h on ic Acid (5q). Yield: 63%; mp 154-
156 °C. 1H NMR (NaOD/D2O, 300 MHz): δ 7.32-7.36 (m, 4H),
7.23-7.30 (m, 1H), 3.03 (t, 2H), 2.96 (t, 2H), 2.78 (t, 2H), 2.07
(7-line system, 2H), 1.83 (5-line system, 2H). 31P NMR (5%
NaOD/D2O): δ 19.02. Anal. (C12H21NO7P2S) C, H, N, P, S.
1-H yd r oxy-3-[m et h yl-(2-(4-ch lor op h en ylsu lfa n ylet h -
yl))a m in o]p r op ylid en e-1,1-bisp h osp h on ic Acid (5s).
Yield: 31%; mp 125-130 °C. 1H NMR (NaOD/D2O, 300
MHz): δ 7.07 (s, 4H), 2.67 (t, 2H), 2.34-2.46 (m, 2H), 2.21 (t,
2H), 1.84 (s, 3H), 1.64-1.80 (m, 2H), 1.48 (5-line system, 2H).
31P NMR (5% NaOD/D2O): δ 19.11 Anal. (C13H22ClNO7P2S)
C, H, Cl, N, P, S.
1
1-Hyd r oxy-3-[m eth yl-(3-(4-ch lor op h en oxy)p r op yl)a m i-
n o]p r op ylid en e-1,1-bisp h osp h on ic Acid (5g). Yield: 71%;
1
mp 155-162 °C. H NMR (NaOD/D2O, 300 MHz): δ 7.00 (d,
2H), 6.62 (d, 2H), 3.72 (t, 2H), 2.33-2.46 (m, 2H), 2.16-2.27
(m, 2H), 1.88 (s, 3H), 1.50-1.80 (m, 4H). 31P NMR (5% NaOD/
D2O): δ 19.00. Anal. (C13H21ClNO8P2) C, H, N, Cl, P.
1-Hyd r oxy-3-[m et h yl-(3-(4-m et h oxyp h en oxy)p r op yl)-
a m in o]p r op ylid en e-1,1-bisp h osp h on ic Acid (5h ). Yield:
23%; mp 127-133 °C. 1H NMR (NaOD/D2O, 300 MHz): δ 6.78
(br s, 4H), 3.86 (t, 2H), 3.69 (s, 3H), 2.62-2.62 (m, 2H), 2.33-
2.46 (m,2 H), 2.03 (s, 3H), 1.69-1.98 (m, 4H). 31P NMR (5%
NaOD/D2O): δ 19.00. Anal. (C14H25NO9P2‚0.5H2O) C, H, N,
P.
1-Hydr oxy-2-(im ida zol-2-yl)eth yliden e-1,1-bisp h osp h o-
n ic Acid (6a ). Yield: 30%; mp 235 °C (dec). 1H NMR (NaOD/
D2O, 300 MHz): δ 6.66 (s, 2H), 3.09 (t, J H-P ) 13 Hz, 2H). 31
P
NMR (5% NaOD/D2O): δ 18.78. Anal. (C5H10N2O7P2‚0.5H2O)
C, H, N, P. P: calcd, 22.04; found, 22.5.
1-Hydr oxy-2-(1-m eth ylim idazol-2-yl)eth yliden e-1,1-bis-
p h osp h on ic Acid (6b). Yield: 46%; mp 261 °C (dec). 1H NMR
(NaOD/D2O, 300 MHz): δ 6.66 (s, 1H), 6.57 (s, 1H), 3.41 (s,
3H), 2.99 (t, J H-P ) 11 Hz, 2H). 31P NMR (5% NaOD/D2O): δ
17.96. Anal. (C6H12N2O7P2‚H2O) C, H, N, P.
1-Hyd r oxy-2-(1-ben zylim id a zol-2-yl)eth ylid en e-1,1-bis-
p h osp h on ic Acid (6c). Yield: 52%; mp 171 °C (dec). 1H NMR
(NaOD/D2O, 250 MHz): δ 7.3-7.5 (m, 5H), 7.24 (br s, 1H),
7.19 (br s, 1H), 5.43 (s, 2H), 3.52 (t, J H-P ) 12 Hz, 2H). 31P
NMR (5% NaOD/D2O): δ 17.81.
1-Hydr oxy-2-(im ida zol-4-yl)eth yliden e-1,1-bisp h osp h o-
n ic Acid (6d ). Yield: 41%; mp 238-240 °C (dec). 1H NMR
(NaOD/D2O, 250 MHz): δ 7.49 (s, 1H), 6.70 (s, 1H), 3.16 (t,
J H-P ) 12 Hz, 2H). 31P NMR (5% NaOD/D2O): δ 18.98. Anal.
(C5H10N2O7P2) C, H, N, P.
1-H yd r oxy-3-[et h yl-(3-p h en oxyp r op yl)a m in o]p r op y-
lid en e-1,1-bisp h osp h on ic Acid (5i). Yield: 61%; mp 195-
1
197 °C. H NMR (NaOD/D2O, 300 MHz): δ 7.07 (t, 2H), 6.74
(t, 1H), 6.71 (d, 2H), 3.78 (t, 2H), 2.46-2.57 (m, 2H), 2.30-
2.40 (m, 2H), 2.27 (q, 2H), 1.59-1.84 (m, 4H), 0.71 (t, 3H). 31
P
NMR (5% NaOD/D2O): δ 19.16. Anal. (C14H25NO8P2) C, H, N,
P.
1-Hyd r oxy-3-[p r op yl-(3-p h en oxyp r op yl)a m in o]p r op y-
lid en e-1,1-bisp h osp h on ic Acid (5j). Yield: 50%; mp 197-
1
199 °C. H NMR (NaOD/D2O, 300 MHz): δ 7.06 (t, 2H), 6.73
(t, 1H), 6.70 (d, 2H), 3.77 (t, 2H), 2.46-2.58 (m,2 H), 2.30-
2.40 (m,2 H), 2.08-2.19 (m, 2H), 1.58-1.84 (m, 4H), 1.16 (6-
line system, 2H), 0.52 (t, 3H). 31P NMR (5% NaOD/D2O): δ
19.16. Anal. (C15H27NO8P2‚0.5H2O) C, H, N, P.
1-H yd r oxy-3-(im id a zol-4-yl)p r op ylid en e-1,1-b isp h os-
p h on ic Acid (6e). Yield: 38%; mp 249-252 °C (dec). 1H NMR
(NaOD/D2O, 300 MHz): δ 7.25 (s, 1H), 6.53 (s, 1H), 2.52-2.63
1-H yd r oxy-3-[b u t yl-(3-p h en oxyp r op yl)a m in o]p r op y-
lid en e-1,1-bisp h osp h on ic Acid (5k ). Yield: 45%; mp 181-
1
183 °C (dec). H NMR (NaOD/D2O, 300 MHz): δ 7.07 (t, 2H),
(m, 2H), 1.77-1.95 (m, 2H). 31P NMR (5% NaOD/D2O):
19.31. Anal. (C6H12N2O7P2‚0.75H2O) C, H, N, P.
δ
6.74 (t, 1H), 6.71 (d, 2H), 3.79 (t, 2H), 2.46-2.57 (m,2 H), 2.30-
2.40 (m, 2H), 2.13-2.21 (m, 2H), 1.58-1.83 (m, 4H), 1.05-
1.19 (m, 2H), 0.94 (6-line system, 2H), 0.55 (t, 3H). 31P NMR
(5% NaOD/D2O): δ 19.16. Anal. (C16H29NO8P2) C, H, N, P.
1-H yd r oxy-3-[m et h yl-(4-p h en oxyb u t yl)a m in o]p r op y-
lid en e-1,1-bisp h osp h on ic Acid (5l). Yield: 45%; mp 153-
1-Hydr oxy-2-(2-m eth ylim idazol-4-yl)eth yliden e-1,1-bis-
p h osp h on ic Acid (6f). Yield: 43%; mp 261-262 °C (dec). 1H
NMR (NaOD/D2O, 250 MHz): δ 6.38 (s, 1H), 2.96 (t, J H-P
)
12 Hz, 2H), 2.01 (s, 3H). 31P NMR (5% NaOD/D2O): δ 19.02.
Anal. (C6H12N2O7P2‚0.5H2O) C, H, N.
1
156 °C (dec). H NMR (NaOD/D2O, 250 MHz): δ 7.15 (t, 2H),
1-Hydr oxy-2-(2-ph en ylim ida zol-4-yl)eth ylid en e-1,1-bis-
p h osp h on ic Acid (6g). Yield: 30%; mp 223-224 °C (dec).
1H NMR (NaOD/D2O, 250 MHz): δ 7.45 (d, 2H), 7.10 (t, 2H),
7.02 (t, 1H), 6.58 (s, 1H), 3.05 (t, J H-P ) 12 Hz, 2H). 31P NMR
(5% NaOD/D2O): δ 19.12. Anal. (C11H14N2O7P2‚1.5H2O) C, H,
N.
1-Hydr oxy-2-(5-m eth ylim idazol-4-yl)eth yliden e-1,1-bis-
p h osp h on ic Acid (6h ). Yield: 51%; mp 217-218 °C. 1H NMR
(NaOD/D2O, 250 MHz): δ 7.26 (s, 1H), 2.99 (t, J H-P ) 12 Hz,
2H), 1.88 (s, 3H). 31P NMR (5% NaOD/D2O): δ 19.12. Anal.
(C6H12N2O7P2‚0.25H2O) C, H, N, P.
1-Hyd r oxy-2-(im id a zol-1-yl-a m in o)eth ylid en e-1,1-bis-
p h osp h on ic Acid (6i). Yield: 67%; mp 242-244 °C (dec). 1H
NMR (NaOD/D2O, 300 MHz): δ 7.80 (s, 1H), 7.30 (s, 1H), 6.95
(s, 1H), 4.50 (t, J H-P ) 10 Hz, 2H). 31P NMR (5% NaOD/D2O):
δ 16.18. Anal. (C5H10N2O7P2‚H2O) C, H, N, P.
1-Hyd r oxy-3-(im id a zol-1-yl-a m in o)p r op ylid en e-1,1-bis-
p h osp h on ic Acid (6j). Yield: 63%; mp 246-247 °C (dec). 1H
NMR (NaOD/D2O, 250 MHz): δ 7.50 (s, 1H), 7.02 (s, 1H), 6.66
(s, 1H), 4.08-4.18 (m, 2H), 2.00-2.20 (m, 2H). 31P NMR (5%
6.76-6.88 (m, 3H), 3.85 (t, 2H), 2.42-2.55 (m, 2H), 2.16-2.27
(br t, 2H), 1.96 (s, 3H), 1.70-1.90 (m, 2H), 1.30-1.60 (m, 4H).
31P NMR (5% NaOD/D2O): δ 19.01. Anal. (C14H25NO8P2) C,
H, N, P. P: calcd, 15.59; found, 14.9.
1-H yd r oxy-3-[m et h yl-(6-p h en oxyh exyl)a m in o]p r op y-
lid en e-1,1-bisp h osp h on ic Acid (5m ). Yield: 55%; mp 183-
1
187 °C (dec). H NMR (NaOD/D2O, 300 MHz): δ 7.07 (t, 2H),
6.74 (t, 1H), 6.71 (d, 2H), 3.77 (t, 2H), 2.34-2.44 (m, 2H), 2.04-
2.14 (m, 2H), 1.87 (s, 3H), 1.64-1.84 (m, 2H), 1.46 (5-line
system, 3H), 1.09-1.26 (m, 4H), 0.97-1.09 (m, 2H). 31P NMR
(5% NaOD/D2O): δ 19.14. Anal. (C16H29NO8P2) C, H, N, P.
1-Hyd r oxy-3-[m eth yl-(3-p h en oxyp r op yl)a m in o]bu tyl-
id en e-1,1-bisp h osp h on ic Acid (5n ). Yield: 72%; mp 221-
223 °C. 1H NMR (NaOD/D2O, 300 MHz): δ 7.37 (t, 2H), 6.99-
7.08 (m, 3H), 4.11 (t, 2H), 2.72-2.80 (m, 2H), 2.55-2.63 (m,
2H), 2.38 (s, 3H), 1.96-2.09 (m, 2H), 1.76-1.93 (m, 4H). 31P
NMR (5% NaOD/D2O): δ 19.37. Anal. (C14H25NO8P2‚0.5H2O)
C, H, N, P.
1-H yd r oxy-3-[(2-p h en ylsu lfa n ylet h yl)a m in o]p r op yli-
d en e-1,1-bisp h osp h on ic Acid (5o). Yield: 71%; mp 108-

3734 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Widler et al.
NaOD/D2O): δ 18.35. Anal. (C6H12N2O7P2) C, H, N, P. P: calcd,
21.65; found, 22.5.
7.08 (d, 1H), 3.15 (td, 2H, J H-P ) 15 Hz), 1.97 (tt, 1H, J H-P )
21 Hz). 31P NMR (5% NaOD/D2O): δ 19.88. Anal. (C5H9-
NO6P2S) C, H, N, P, S.
1-Hydr oxy-2-(2-m eth ylim idazol-1-yl)eth yliden e-1,1-bis-
p h osp h on ic Acid (6k ). Yield: 2.3%; mp 245-246 °C (dec).
1H NMR (NaOD/D2O, 250 MHz): δ 6.97 (s, 1H), 6.41 (s, 1H),
4.06 (t, J H-P ) 10 Hz, 2H), 2.04 (s, 3H). 31P NMR (5% NaOD/
D2O): δ 16.40. Anal. (C6H12N2O7P2) C, H, N, P. P: calcd, 21.65;
found, 21.0.
(Th ia zol-2-ylsu lfa n yl)m et h ylen eb isp h osp h on ic Acid
(9b). Tetraethyl methylenebisphosphonate (12.2 g, 42.6 mmol)
was dissolved in 100 mL of anhydrous THF, cooled to - 20
°C, and deprotonated under argon with sodium hydride (4.22
g, ca. 55% dispersion in mineral oil). After the mixture was
stirred for 5 min at this temperature, the temperature was
raised to 0 °C for another 1/2 h. After the mixture was cooled
to -30 °C, a solution of 9.00 g (38.76 mmol) of di-(2-thiazolyl)-
disulfide67 in 50 mL of THF was added dropwise within 10
min. A slightly yellow suspension formed. After being stirred
for 1 h at room temperature, the mixture was filtered and the
filtrate was concentrated. The residue was taken up in 1 N
HCl, and the resulting solution was adjusted to pH 2-3. The
aqueous layer was extracted with several portions of ethyl
acetate, which were combined and washed with a small
amount of aqueous HCl, followed by a wash with saturated
sodium carbonate solution (pH 9-10). Drying and concentra-
tion iV afforded 13.6 g of crude product, which was chromato-
graphed (ethyl acetate/acetone 4:1). Yield: 10.2 g (65%). 13C
NMR (NaOD/D2O, 300 MHz): δ 161.7, 142.0, 119.8, 63.8-64.1
(m), 38.4 (t, J C-P ) 137 Hz), 16.3. Anal. (C12H23NO6P2S2) C,
H, N, P, S.
An amount of 4.03 g (10.0 mmol) of the tetraester prepared
in the previous step was heated to reflux in 30 mL of
concentrated HCl overnight. After cooling to room tempera-
ture, the mixture was diluted with 20 mL of water and filtered.
After complete removal of the water, the solid residue was
twice dissolved in freshwater and concentrated again to
remove traces of Hl. Recrystallization from 20 mL of hot EtOH
and washing with acetone gave colorless crystals, which were
dried under HV. Yield: 2.64 g (91%), mp 255-258 °C. 1H NMR
(NaOD/D2O, 300 MHz): δ 7.29 (d, 1H), 7.09 (d, 1H), 3.10 (t,
J H-P ) 18 Hz, 1H). 31P NMR (5% NaOD/D2O): δ 14.20. Anal.
(C4H7NO6P2S2) C, H, N, P, S.
1-H yd r oxy-2-(4,5-d im et h ylim id a zol-1-yl)et h ylid en e-
1,1-bisp h osp h on ic Acid (6l). Yield: 30%; mp 251-252 °C
1
(dec). H NMR (NaOD/D2O, 250 MHz): δ 7.56 (s, 1H), 4.17 (t,
J H-P )10 Hz, 2H), 1.92 (s, 3H), 1.81 (s, 3H). 31P NMR (5%
NaOD/D2O): δ 16.55. Anal. (C7H14N2O7P2) C, H, N, P.
1-Hyd r oxy-2-(p yr r a zol-1-yl)eth ylid en e-1,1-bisp h osp h o-
n ic Acid (6m ). Yield: 23%; mp 234 °C (dec). 1H NMR (NaOD/
D2O, 300 MHz): δ 7.55 (s, 1H), 7.22 (s, 1H), 7.22 (s. 1H), 6.00
(s, 1H), 4.36 (t, J H-P ) 9 Hz, 2H). 31P NMR (5% NaOD/D2O):
δ 16.15. Anal. (C5H8N2O7P2‚2H2O) C, H, N, P.
1-Hyd r oxy-2-([1,2,4]tr ia zol-1-yl)eth ylid en e-1,1-bisp h o-
sp h on ic Acid (6n ). Yield: 32%; mp 255 °C (dec). 1H NMR
(NaOD/D2O, 300 MHz): δ 8.23 (s, 1H), 7.66 (s, 1H), 4.39 (t,
J H-P ) 9 Hz, 2H). 31P NMR (5% NaOD/D2O): δ 16.61. Anal.
(C4H9N3O7P2) C, H, N, P.
Meth od B: Syn th esis of 1-Am in o-1,1-bisp h osp h on a tes.
1-Am in o-3-(d im eth yla m in o)p r op ylid en e-1,1-bisp h osp h o-
n ic Acid (1m ). 1m was synthesized as indicated in method B
of Scheme 1, according to a literature procedure,65 in 56%
yield: mp 275-278 °C (dec). 1H NMR (NaOD/D2O, 300 MHz):
δ 2.22-2.33 (m,2 H), 1.89 (s, 6H), 1.63-1.72 (m, 2H). 31P NMR
(5% NaOD/D2O): δ 21.48. Anal. (C5H16N2O6P2‚0.25H2O) C, H,
N, P.
Meth od C: Syn th esis of 1-Am in oa lk yl-1,1-bisp h osp h o-
n a tes. 3-(Dim eth yla m in o)p r op ylid en e]-1,1-bisp h osp h o-
n ic Acid (1l). A suspension of 2.18 g (50 mmol) of sodium
hydride (ca. 55%), which had been washed with several
portions of petroleum ether to remove the mineral oil, in 45
mL of THF was treated at 0 °C dropwise with 14.4 g (50 mmol)
of tetraethyl methylenebisphosphonate. In an exothermic
reaction, a gray-yellow solution formed, which was stirred for
1.5 h at 0 °C. Meanwhile, 5.9 g (55 mmol) of 2-chloroethyldim-
ethylamine hydrochloride was partitioned between diethyl
ether and 6 N NaOH. The organic layer was concentrated to
give the free base. This was taken up in 15 mL of THF and
added dropwise to the reaction mixture, maintaining 0 °C. The
mixture was allowed to reach room temperature, and stirring
was continued overnight. Because there was still some starting
material left, another equivalent of the alkylating agent was
added and the reaction mixture was kept for 2 weeks at room
temperature. At this point, only little starting material
remained. The reaction mixture was filtered, and the filtrate
was concentrated under reduced pressure. Purification by flash
chromatography (toluene/ethanol initially 4:1, then 7:3 con-
taining 1% of aqueous ammonia solution) afforded 3.5 g (20%
yield) of tetraethyl 3-(dimethylamino)propylidene]bisphos-
Meth od D: Syn th esis of 1-Alk yl- a n d Ar yla m in o-1,1-
bisp h osp on a tes. (5-Meth ylth ia zol-2-yl)a m in iom eth ylen e-
bisp h osp h on ic Acid (8c). In a flask fitted with a distillation
head, a mixture of 114.2 g (1.00 mol) of 2-amino-5-methylthia-
zole,50 200 mL (1.20 mol) of triethyl orthoformate, and 260 mL
(2.02 mol) of diethyl phosphite was heated to 120-125 °C.
Within 3 h, 155 mL (about 90% of the calculated amount) of
EtOH was distilled off. The residual 437 g of a viscous yellow
oil was submitted to hydrolysis in the next step without further
purification. 1H NMR (CDCl3, 60 MHz): δ 6.85 (s, 1H), 5.05
(t, J H-P ) 22 Hz, 1H), 4.32 (m, 8H), 2.37 (s, 3H), 1.39 (td, 12H).
The crude tetraester (437 g) obtained in the previous step
was heated to reflux in 4 L of 1 N HCl for 24 h. The resulting
slightly yellow solution was filtered and then diluted with 1.25
L of acetone. The product crystallized in the form of a very
fine precipitate. After the mixture was stirred under reflux
for 1 h, the white solid could be filtered and washed with 1.25
L of hot acetone. Yield (after drying under HV at 120 °C):
1
phonate (bp 120 °C, 30 mbar). H NMR (CDCl3, 250 MHz): δ
1
4.12 (m, 8H), 2.61 (tt, 1H), 2.50 (t, 2H), 2.22 (s, 6H), 1.91-
2.17 (m, 2H), 1.30 (t, 12H).
194.5 g (67%); mp 225 °C. H NMR (NaOD/D2O, 300 MHz): δ
6.31 (s, 1H), 3.13 (t, J H-P ) 20 Hz, 1H), 1.90 (s, 3H). 31P NMR
(5% NaOD/D2O): δ 15.13. Anal. (C5H10N2O6P2‚0.25H2O) C, H,
N, P, S.
A mixture of 3.6 g (10.0 mmol) of tetraethyl 3-(dimethyl-
amino)propylidenebisphosphonate in 30 mL of 48% HBr solu-
tion was heated to reflux overnight. After cooling to room
temperature, the mixture was then diluted with water and
charcoal was added. Filtration and concentration gave a
yellow-brown oil, which was thoroughly dried under HV. The
residue was treated with 10 mL of MeOH and then with the
same amount of acetone to induce crystallization. After drying
several hours at 80 °C under HV, the mixture yielded 1.7 g
(51% yield) of 3-(dimethylamino)propylidene-1,1-bisphosphonic
acid in form of the hydrobromide: mp 197-200 °C (dec). 1H
NMR (NaOD/D2O, 300 MHz): δ 2.15-2.27 (m, 2H), 1.89 (s,
6H), 1.45-1.7 (m, 2H), 1.15 (tt, 1H). 31P NMR (5% NaOD/
D2O): δ 21.48. Anal. (C5H15NO6P2‚HBr) C, H, N, P, Br.
2-(Th iazol-2-yl)eth yliden e-1,1-bisph osph on ic Acid (9a).
9a was synthesized starting with 2-chloromethylthiazole:66 mp
(Ben zylim id a zol-2-yl)a m in om et h ylen eb isp h osp h on -
ic Acid (8o). A solution of 5.02 g (8.75 mmol) of tetraethyl
(1-tosylbenzylimidazol-2-yl)aminomethylenebisphosphonate in
60 mL of 1 N HCl was heated for 100 °C for 16 h. Because
some partially hydrolyzed phoshonate was still visible by TLC,
the temperature was increased to 120 °C for another 4 days.
The white solid that had formed was filtered off and washed
with a small amount of water. The crude product was taken
up in a mixture of water and 1% acetone. The insoluble part
was removed by filtration, and the filtrate was concentrated.
The residue was recrystallized from water, followed by wash-
ing with hot water and acetone to give 1.16 g (43%) of product;
mp 300 °C (dec). 1H NMR (NaOD/D2O, 300 MHz): δ 6.72-
6.81 (m, 2H), 6.46-6.52 (m, 2H), 3.51 (t, J H-P ) 20 Hz, 1H).
Anal. (C8H11N3O6P2‚1.5H2O) C, H, N.
1
259 °C (dec). H NMR (NaOD/D2O, 300 MHz): δ 7.32 (d, 1H),

Bisphosphonates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3735
In analogy to the examples described above under method
D, the following aminomethylenebisphosphonic acids were
prepared (yields given are those over two steps).
(s, 1H), 6.41 (s, 1H), 3.60 (t, J H-P ) 20 Hz, 1H). 13C NMR
(NaOD/D2O): δ 151.1, 137.1, 130.8 (2 carbons), 128.7, 126.0
(2 carbons), 123.7, 117.1, 54.4 (t, J P-C ) 126 Hz). 31P NMR
(5% NaOD/D2O): δ 15.95. Anal. (C10H13N3O6P2) C, H, N, P.
(Ben zth iazol-2-yl)am in om eth ylen ebisph osph on ic Acid
Cycloh ep tyla m in om eth ylen ebisp h osp h on ic Acid (7e).
1
Yield: 33%; mp 250-251 °C (dec). H NMR (NaOD/D2O, 360
1
MHz): δ 3.72-3.80 (m, 1H), 3.14 (t, J H-P ) 16 Hz, 1H), 2.08-
2.19 (m, 2H), 1.63-1.77 (m, 4H), 1.45-1.63 (m, 6H). 31P NMR
(5% NaOD/D2O): δ 15.47. Anal. (C8H19NO6P2) C, H, N, P.
(Th iazol-2-yl)am in om eth ylen ebisph osph on ic Acid (8a).
Yield: 19%; mp 292 °C (dec). 1H NMR (NaOD/D2O, 300
MHz): δ 6.63 (d, 1H), 6.17 (d, 1H), 3.12 (t, J H-P ) 18 Hz, 1H).
(8n ). Yield: 59%; mp 290 °C (dec). H NMR (NaOD/D2O, 300
MHz): δ 7.38 (d, 1H), 7.09 (d, 1H), 7.03 (t, 1H), 6.81 (t, 1H),
ca. 3.5 (br, 1H). 13C NMR (NaOD/D2O): δ 152.2.0, 130.3, 127.0,
122.3, 122.2, 117.7. P-C-P not visible. 31P NMR (5% NaOD/
D2O): δ 14.44. Anal. (C8H10N2O6P2S‚0.25H2O) C, H, N, P, S.
Tr a n sfor m a tion Rea ction s of Bisp h osp h on a tes. N-
Meth yla tion : 1-Hyd r oxy-3-[m eth yl-(6-p h en ylh exyl)a m i-
n o]p r op ylid en ebisp h osp on ic Acid (4l). A mixture of 1.58
g (4.0 mmol) of 1-hydroxy-3-[(6-phenylhexyl)amino]propy-
lidene-1,1-bisphosphonic acid (prepared according to method
A), 3.6 mL of 85% formic acid, and 0.9 mL of formaldehyde
solution (37% soluble in water, 12 mmol) was heated under
reflux for 17 h. The residue remaining after concentration iV
was treated with EtOH to yield, after drying under HV at 110
°C for 4 h, the product in the form of a white solid. Yield: 70%;
13C NMR (NaOD/D2O): δ 174.0, 138.4, 106.3, 59.4 (t, J C-P
)
126 Hz). 31P NMR (5% NaOD/D2O): δ 14.94. Anal. (C4H8N2-
O6P2S) C, H, N, P, S.
(4-Met h ylt h ia zol-2-yl)a m in om et h ylen eb isp h osp h on -
1
ic Acid (8b). Yield: 11%; mp 294 °C (dec). H NMR (NaOD/
D2O, 300 MHz): δ 5.80 (s, 1H), 3.09 (t, J H-P ) 19 Hz, 1H),
1.81 (s, 3H). 13C NMR (NaOD/D2O): δ 173.6, 148.2, 59.2 (t,
J C-P ) 126 Hz), 17.1. 31P NMR (5% NaOD/D2O): δ 14.90. Anal.
(C5H10N2O6P2S) C, H, N, S. P: calcd, 21.50; found, 20.89.
(5-E t h ylt h ia zol-2-yl)a m in om et h ylen eb isp h osp h on ic
Acid (8d ). Yield: 50%; mp 243 °C (dec). 1H NMR (NaOD/D2O,
300 MHz): δ 6.70 (s, 1H), 3.49 (t, J H-P ) 18 Hz, 1H), 2.64 (q,
2H), 1.20 (t, 3H). 31P NMR (5% NaOD/D2O): δ 15.11. Anal.
(C6H12N2O6P2S‚0.5H2O) C, H, N, P, S.
mp 149-152 °C (dec). 1H NMR (NaOD/D2O, 250 MHz):
δ
7.00-7.18 (m, 5H), 2.36-2.52 (m, 4H), 2.08-2.20 (m, 2H), 1.96
(s, 3H), 1.70-1.85 (m, 2H), 1.30-1.46 (m, 2H), 1.16-1.29 (m,
2H), 1.08-1.13 (m,4 H). 31P NMR (5% NaOD/D2O): δ 19.14.
Anal. (C16H29NO7P2) C, H, N, P.
(5-P r op ylt h ia zol-2-yl)a m in om et h ylen eb isp h osp h on -
ic Acid (8e). Yield: 52%; mp 275 °C (dec). H NMR (NaOD/
In an analogous way, the following N-methylated bisphos-
phonates were synthesized:
1
D2O, 300 MHz): δ 6.85 (s, 1H), 3.82 (t, J H-P ) 19 Hz, 1H),
2.62 (t, 2H), 1.61 (6-line system, 2H), 0.93 (t, 3H). 31P NMR
(5% NaOD/D2O): δ 15.14. Anal. (C7H14N2O6P2S‚0.5H2O) C, H,
N, P, S.
(5-Bu t ylt h ia zol-2-yl)a m in om et h ylen eb isp h osp h on ic
Acid (8f). Yield: 43%; mp 267 °C (dec). 1H NMR (NaOD/D2O,
300 MHz): δ 6.67 (s, 1H), 3.50 (t, J H-P ) 19 Hz, 1H), 2.62 (t,
2H), 2.55 (5-line system, 2H), 2.35 (6-line system, 2H), 0.90
(t, 3H). 31P NMR (5% NaOD/D2O): δ 15.14. Anal. (C8H16N2-
O6P2S) C, H, N, P, S.
1-Hyd r oxy-3-(d im eth yla m in o)p r op ylid en e-1,1-bisp h o-
sp h on ic Acid (1h , Olp a d r on a te). Starting with 1-hydroxy-
3-aminopropylidenebisphosponic acid (pamidronic acid), the
title compound was prepared by double methylation in 92%
yield; mp 210-212 °C (dec).1H NMR (D2O, 250 MHz): δ 3.26
(t, 2H), 2.70 (s, 6H), 2.19 (7-line system, 2H). 31P NMR (5%
NaOD/D2O): δ 18.96. Anal. (C5H15NO7P2) C, H, N, P.
1-H yd r oxy-3-(m e t h ylp r op yla m in o)p r op ylid e n e b is-
p h osp on ic Acid (1i). This compound was obtained by me-
thylation of 1-hydroxy-3-[N-propylamino]propylidenebispho-
sphonate (prepared in 67% yield following the general procedure
of method A, starting with 3-(N-propylamino)propanoic acid):
(5-Isop r op ylth ia zol-2-yl)a m in om eth ylen ebisp h osp h o-
1
n ic a cid (8g). Yield: 46%; mp 255 °C (dec). H NMR (NaOD/
1
D2O, 300 MHz): δ 6.72 (s, 1H), 3.50 (t, J H-P ) 18 Hz, 1H),
2.98 (7-line system, 1H), 1.23 (d, 6H). 31P NMR (5% NaOD/
D2O): δ 15.11. Anal. (C7H14N2O6P2S) C, H, N. P: calcd, 19.59;
found, 18.77. S: calcd, 10.14; found, 9.22.
28% yield; mp 140-145 °C. H NMR (NaOD/D2O, 250 MHz):
δ 2.30-2.41 (m, 2H), 1.95-2.05 (m, 2H), 1.83 (s, 3H), 1.60-
1.67 (m, 2H), 1.11 (6-line system, 2H), 0.51 (t, 3H). Anal.
(C7H19NO7P2) C, H, N, P. P: calcd, 21.27; found, 20.7.
1-Hyd r oxy-3-(d im eth yla m in o)bu tylid en e-1,1-bisp h os-
p h on ic Acid (1o). Yield: 47%; mp 229-230 °C (dec). 1H NMR
(NaOD/D2O, 300 MHz): δ 3.82-3.95 (m, 1H), 2.70 (br s, 6H),
2.16-2.38 (m, 1H), 1.95-2.12 (m, 1H), 1.20 (d, 3H). 31P NMR
(5% NaOD/D2O): δ 18.73. Anal. (C6H17NO7P2) C, H, N, P.
1-H yd r oxy-3-[m et h yl-(4-p h en ylb u t yl)a m in o]p r op yli-
d en e-1,1-bisp h osp h on ic Acid (4j). Yield: 30%; mp 128-132
°C (dec). 1H NMR (NaOD/D2O, 250 MHz): δ 6.91-7.12 (m,
5H), 2.28-2.49 (m, 4H), 2.09-2.18 (m, 2H), 1.88 (s, 3H), 1.65-
1.88 (m, 2H), 1.12-1.40 (m, 4H). 31P NMR (5% NaOD/D2O):
δ 19.01. Anal. (C14H25NO7P2) C, H, N, P. P: calcd, 16.25; found,
15.3.
(5-P h en eth ylth ia zol-2-yl)a m in om eth ylen ebisp h osp h o-
n ic Acid (8h ). Yield: 15%; mp 293 °C (dec). 1H NMR (NaOD/
D2O, 300 MHz): δ 7.23-7.42 (m, 5H), 6.61 (s, 1H), 3.49 (t,
J H-P ) 18 Hz, 1H), 2.84-3.00 (m, 4H). 31P NMR (5% NaOD/
D2O): δ 15.10. Anal. (C12H16N2O6P2S) C, H, N, P, S.
(5-P h en ylt h ia zol-2-yl)a m in om et h ylen eb isp h osp h on -
ic Acid (8i). Yield: 3%; mp 298-302 °C (dec). 1H NMR (NaOD/
D2O, 300 MHz): δ 7.36 (d, 2H), 6.92-7.12 (m, 3H), 6.44 (s,
1H), 3.04 (t, J H-P ) 19 Hz, 1H). Anal. (C10H12N2O6P2S‚
0.25H2O) C, H, N, P, S.
(Im idazol-2-yl)am in om eth ylen ebisph osph on ic Acid (8j).
8j was prepared as described in the general method for 8c,
using 2-amino-1-benzylimidazole as the starting material.
Then the benzyl group was cleaved by hydrogenolysis over 5%
Pd/C, followed by P ester cleavage with HCl as usual. Yield
(over three steps): 5%; mp 297 °C (dec). 1H NMR (NaOD/D2O,
360 MHz): δ 6.42 (br s, 2H), 3.24 (t, J H-P ) 18 Hz, 1H).
(1-Met h ylim id a zol-2-yl)a m in om et h ylen eb isp h osp h o-
n ic Acid (8k ). Yield: 5%; mp 323-326 °C (dec). 1H NMR
(NaOD/D2O, 300 MHz): δ 6.21 (d, 1H), 6.15 (d, 1H), 3.45 (t,
J H-P ) 20 Hz, 1H), 3.01 (s, 3H). 31P NMR (5% NaOD/D2O): δ
16.17.
(1-Ben zylim id a zol-2-yl)a m in om et h ylen eb isp h osp h o-
n ic Acid (8l). Yield: 3%; mp 261-265 °C (dec). 1H NMR
(NaOD/D2O, 360 MHz): δ 7.36-7.48 (m, 5H), 6.61 (d, 1H), 6.57
(d, 1H), 5.03 (s, 2H), 3.91 (t, J H-P ) 20 Hz, 1H). 31P NMR (5%
NaOD/D2O): δ 16.26. Anal. (C11H15N3O6P2‚1.33HCl‚0.25H2O)
C, N, P. H: calcd, 4.22; found, 3.77.
(1-P h en ylim id a zol-2-yl)a m in om et h ylen eb isp h osp h o-
n ic Acid (8m ). Yield: 25%; mp 275 °C (dec). 1H NMR (NaOD/
D2O, 300 MHz): δ 7.28 (d, 4H), 7.16 (6-line system, 1H), 6.52
1-Hyd r oxy-3-[(m eth yl-3-p h en ylsu lfa n ylp r op yl)a m in o]-
p r op ylid en e-1,1-bisp h osp h on ic Acid (5r ). A suspension of
3.08 g (8.0 mmol) of 1-hydroxy-3-[(3-phenylsulfanylpropyl)-
amino]propylidene-1,1-bisphosphonic acid (5q) in 90 mL of
acetonitrile was treated dropwise at room temperature with
16 mL of 1 N NaOH under vigorous stirring. After complete
addition, stirring was continued for another 15 min at 30 °C.
To the resulting white suspension, 3.0 mL of formaldehyde
solution (37% soluble in water, 40 mmol) was added, followed
by 0.8 g (12.8 mmol) of sodium cyanoborohydride after an
additional 5 min. The temperature rose to approximately 30
°C, and the pH increased from 7 to 9. The pH was adjusted to
7 with a small amount of acetic acid. After sitting several days
at room temperature, the white suspension was filtered and
the white solid was washed with EtOH. Recrystallization from
water/EtOH gave 2.7 g (63%) of the bisphosphonate in the form
of its disodium salt; mp 245-247 °C (dec). 1H NMR (NaOD/
D2O, 300 MHz): δ 7.25-7.50 (m, 5H), 3.15-3.40 (m, br, 4H),
3.05 (t, 2H), 2.73 (s, 3H), 2.29 (7-line system, 2H), 2.01 (5-line

3736 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Widler et al.
system, 2H). 31P NMR (5% NaOD/D2O): δ 19.11. Anal. (C13H21
NO7P2SNa2‚5H2O) C, H, N, P, S.
-
(5) Fleisch, H. Bisphosphonates: Mechanisms of action. Endocr.
Rev. 1998, 19, 80-100.
(6) Ciosek, C. P.; Magnin, D. R.; Harrity, T. W.; Logan, J . V. Dickson,
J . K.; Gordon, E. M.; Hamilton, K. A.; J olibois, K. G.; Kunselman,
L. K.; Lawrence, R. M.; Mookhtiar, K. A.; Rich, L. C.; Slusarchyk,
D. A.; Sulsky, R. B.; Biller, S. A. Lipophilic 1,1-bisphosphonates
are potent squalene synthase inhibitors and orally active
cholesterol lowering agents in vivo. J . Biol. Chem. 1993, 268,
24832-24837.
N-Acetyla tion : 1-Hyd r oxy-3-a cetyla m in op r op ylid en e-
1,1-bisp h osp h on ic Acid (1f). After cooling to -5 °C, a
solution of 3.5 g (15 mmol) of 1-hydroxy-3-aminopropylidene-
1,1-bisphosphonic acid (pamidronic acid) in 120 mL of 0.5 M
NaOH was treated within 1 h with 1.7 mL (18 mmol) of
acetanhydride with vigorous stirring. Stirring was continued
for another 4 h at 0 °C followed by slow warming to room
temperature. Concentration iV yielded a colorless oil, which
started to crystallize after treatment with a 2-propanol/acetone
mixture. Recrystallization from a small amount of water
afforded 3.9 g (76%) of product in the form of its 2.75 sodium
salt containing also 0.5 equiv of water (after drying overnight
at 100 °C under HV); mp 300 °C (dec). 1H NMR (D2O, 100
MHz): δ 3.40 (t, 2H), 1.85-2.30 (m, 2H), 1.92 (s, 3H). 31P NMR
(5% NaOD/D2O): δ 18.93. Anal. (C5H10.25NO8P2Na2.75‚0.5H2O)
C, H, N, Na, P.
(7) Luckman, S. P.; Hughes, D. E.; Coxon, F. P.; Graham, R.;
Russell, G.; Rogers, M. J . Nitrogen-containing bisphosphonates
inhibit the mevalonate pathway and prevent post-translational
prenylation of GTP-binding proteins, including Ras. J . Bone
Miner. Res. 1998, 13, 581-589.
(8) Rogers, M. J .; Frith, J . C.; Luckman, S. P.; Coxon, F. P.; Benford,
H. L.; Mo¨nkko¨nen, J .; Auriola, S.; Chilton, K. M.; Russell, R. G.
G. Molecular mechanisms of action of bisphosphonates. Bone
1999, 24 (Suppl.), 73S-79S.
(9) Russell, R. G. G.; Rogers, M. J .; Frith, J . C.; Luckman, S. P.;
Coxon, F. P.; Benford, H. L.; Croucher, P. I.; Shipman, C.;
Fleisch, H. A. The pharmacology of bisphosphonates and new
insights into their mechanisms of action. J . Bone Miner. Res.
1999, 14 (Suppl. 2), 53-65. Russell, R. G. G.; Rogers, M. J .
Bisphosphonates: From the laboratory to the clinic and back
again. Bone 1999, 25, 97-106. Russell, R. G. G.; Croucher, P.
I.; Rogers, M. J . Bisphosphonates: pharmacology, mechanism
of action and clinical uses. Osteoporosis Int. 1999, Suppl. 2, S66-
S80. Russell, R. G. G. The bisphosphonate odyssey. A J ourney
from chemistry to the clinic. Phosphorus, Sulfur Silicon Relat.
Elem. 1999, 144-146, 793-820.
(10) Breuil, V. Mechanisms of action of bisphosphonates. Rev. Rheum.
(Engl. Ed.) 1999, 66, 339-343.
(11) Rogers, M. J .; Watts, D. G.; Russell, R. G. G. Overview of
bisphosphonates. Cancer 1997, 80, 1652-1669.
(12) Hughes, D. E.; MacDonald, B. R.; Russell, R. G. G.; Gowen, M.
Inhibition of osteoclast-like cell formation by bisphosphonates
in long-term cultures of human bone marrow. J . Clin. Invest.
1989, 83, 1930-1935.
(13) Nishikawa, M.; Akatsu, T.; Katayama, Y.; Yasutomo, Y.; Kado,
S.; Kugai, N.; Yamamoto, M.; Nagata, N. Bisphosponates act
on osteoblastic cells and inhibit osteoclast formation in mouse
marrow cultures. Bone 1996, 18, 9-14.
(14) Sahni, M.; Guenther, H.; Fleisch, H.; Collin, P.; Martin, T. J .
Bisphosphonates act on rat bone resorption through the media-
tion of osteoblasts. J . Clin. Invest. 1993, 91, 2004-2011.
(15) Yu, X.; Scholler, J .; Foged, N. T. Interaction between effects of
parathyroid hormone and bisphosphonates on regulation of
osteoclast activity by the osteoblast-like cell line UMR-106. Bone
1996, 19, 339-345.
(16) Vitte´, C.; Fleisch, H.; Guenther, H. L. Bisphosphonates induce
osteoblasts to secrete an inhibitor of osteoclast-mediated resorp-
tion. Endocrinology 1996, 137, 2324-2333.
(17) Sietsema, W. K.; Ebetino, F. H. Bisphosphonates in development
for metabolic bone disease. Expert Opin. Invest. Drugs 1994, 3,
1255-1276.
(18) Patel, S. Drugs in development for the treatment of metabolic
bone disease. Expert Opin. Invest. Drugs 1996, 5, 429-445.
(19) Reitsma, P. H.; Bijvoet, O. L. M.; Verlinden-Ooms, H.; van der
Wee-Pals, L. J . A. Kinetic studies of bone and mineral metabo-
lism during treatment with (3-amino-2-hydroxy-propylidene)-
1,1-bisphosphonate (APD) in rats. Calcif. Tissue Int. 1980, 32,
145-157.
N-Deb en zyla t ion : 1-Hyd r oxy-3-m et h yla m in op r op yli-
d en e-1,1-bisp h osp h on ic Acid (1g). A mixture of 25.0 g (ca.
90%, 66 mmol) of 1-hydroxy-3-(benzylmethylamino)propy-
lidene-1,1-bisphosphonic acid (prepared according to the gen-
eral procedure of method A, starting with benzylmethylami-
nopropionic acid) in 500 mL of water/methanol 7:3 was
hydrogenated over Pearlman’s catalyst (2.5 g). After 1 day at
room temperature, more catalyst was added (1 g), and once
again after 1 more day (1.2 g). The mixture was filtered after
a total of 5 days (total time). The catalyst had to be extracted
several times with water (50 °C). The product could be
crystallized from 10% MeOH/water. Yield: 12.9 g (78%) after
1
drying at 80 °C under HV; mp 148 °C (dec). H NMR (NaOD/
D2O, 300 MHz): δ 2.49 (t, 2H), 1.98 (s, 3H), 1.6-1.8 (m, 2H).
31P NMR (5% NaOD/D2O): δ 19.13. Anal. (C4H13NO7P2‚H2O)
C, H, N, P.
P r ep a r a tion of Bisp h osp h in a tes. (Th ia zol-2-yl)a m in o-
m eth ylen e-1,1-bis(m eth ylp h osp h in ic Acid ) (10). In a flask
fitted with a distillation head, a mixture of 2.00 g (20 mmol)
of 2-aminothiazole,50 4.0 mL (24 mmol) of triethyl orthofor-
mate, and 4.37 g (40.4 mmol) of methylphosphinic acid methyl
ester was heated to 120-125 °C. Within 24 h, 2.8 mL (about
80% of the calculated amount) of EtOH was distilled off. The
residual 7.0 g of a viscous yellow oil was purified by flash
chromatography (10% MeOH/ethyl acetate). Yield: 3.00 g
(46%) as a mixture of diastereomers; mp 111-113 °C. Anal.
(C10H20N2O4P2S) C, H, N, P, S.
The diester (1.63 g, 5.00 mmol) obtained in the previous step
was heated to reflux in 20 mL of 1 N HCl for 6 h. The resulting
clear and slightly yellow solution was diluted with 40 mL of
acetone. Within 15 min, the product crystallized in the form
of a white precipitate. It was filtered and washed sequentially
with acetone/water 3:1, twice with acetone, and finally with
petroleum ether. Yield (after drying under HV): 1.02 g (76%);
1
mp 270 °C (dec). H NMR (NaOD/D2O, 300 MHz): δ 6.65 (d,
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in the management of osteolytic bone metastases, tumour-
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Aging 1998, 12, 149-168.
1H), 6.25 (d, 1H), 3.53 (t, J H-P ) 16 Hz, 1H), 0.95 (A6XX′ spin
system, 6H). 31P NMR (5% NaOD/D2O): δ 35.49. Anal.
(C6H12N2O4P2S‚0.25H2O) C, H, N, P, S.
(21) Yates, A. J .; Rodan, G. A. Alendronate and osteoporosis. Drug
Discovery Today 1998, 3, 69-78. Sharpe, M.; Noble, S.; Spencer,
C. M. Alendronate: An update of its use in osteoporosis. Drugs
2001, 61, 999-1039.
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