Linking bisphosphonates to the free amino groups in fluoroquinolones: Preparation of osteotropic prodrugs for the prevention of osteomyelitis

Article abstract of DOI:10.1021/jm801007z

Osteomyelitis is an infection located in bone and a notoriously difficult disease to manage, requiring frequent and heavy doses of systemically administered antibiotics. Targeting antibiotics to the bone after systemic administration may provide both grea

Full text of DOI:10.1021/jm801007z

J. Med. Chem. 2008, 51, 6955–6969
6955
Linking Bisphosphonates to the Free Amino Groups in Fluoroquinolones: Preparation of
Osteotropic Prodrugs for the Prevention of Osteomyelitis
Tom J. Houghton, Kelly S. E. Tanaka, Ting Kang, Evelyne Dietrich, Yanick Lafontaine, Daniel Delorme, Sandra S. Ferreira,
Frederic Viens, Francis F. Arhin, Ingrid Sarmiento, Dario Lehoux, Ibtihal Fadhil, Karine Laquerre, Jing Liu, Vale´rie Ostiguy,
Hugo Poirier, Gregory Moeck, Thomas R. Parr, Jr., and Adel Rafai Far*
Targanta Therapeutics Inc, 7170 AVenue Frederick Banting, St. Laurent, Que´bec, H4S 2A1, Canada
ReceiVed August 8, 2008
Osteomyelitis is an infection located in bone and a notoriously difficult disease to manage, requiring frequent
and heavy doses of systemically administered antibiotics. Targeting antibiotics to the bone after systemic
administration may provide both greater efficacy of treatment and less frequent administration. By taking
advantage of the affinity of the bisphosphonate group for bone mineral, we have prepared a set of 13
bisphosphonated antibacterial prodrugs based on eight different linkers tethered to the free amino functionality
on fluoroquinolone antibiotics. While all but one of the prodrugs were shown in vitro to be effective and
rapid bone binders (over 90% in 1 h), only eight of them demonstrated the capacity to significantly regenerate
the parent drug. In a rat model of the disease, a selected group of agents demonstrated their ability to
prevent osteomyelitis when used in circumstances under which the parent drug had already been cleared
and is thus inactive.
Introduction
resorption,13,14 bisphosphonates have been used to deliver small
molecule therapeutics,15-20 ligands for radioisotope imaging,21-23
and even proteins24-26 to the bone. Recently, a report has
described the use of bisphosphonates for the delivery of cipro-
floxacin to bone.27 Although the ciprofloxacin-bisphosphonate
conjugate described appeared to bind efficiently to bone, it is
predictably unable to release the ciprofloxacin moiety. This
would limit the potential of this agent for treatment of bone
infections, as it is reasonable to propose that the release of the
bioactive moiety is necessary to achieve a therapeutic outcome
and bacteria are not likely to be inhibited by an antibacterial
agent that is essentially irreversibly bound to the bone surface.
They are not known to possess the bone-resorptive activity
which would be required to absorb bisphosphonates once they
are bound on bone. The relationship between resorptive activity
and efficacy of therapeutic bisphosphonates has been demon-
strated in the case of osteoporosis treatments.28 Although the
local acidity29 brought on by the infecting organism might be
associated with some bisphosphonate release from bone, the
efficiency of such a process in providing a sufficient concentra-
tion of the antimicrobial agent is doubtful.
Osteomyelitis is defined as an inflammatory process localized
to the bone and accompanied by necrosis resulting from an
underlying microbial infection,1 primarily caused by Staphylo-
coccus aureus.2 In general, it is established as a result of damage
to the osseous matrix by trauma or surgery or may appear in
loci affected by reduced vascularization, as is the case with
diabetic and elderly patients. This is an extremely difficult form
of infection to treat with a need for surgical intervention and
amputation3 and is characteristically associated with frequent
relapses.4,5 There are no marketed antibiotics with an osteo-
myelitis label, and treatment is often a result of the preferences
and experience of the attending physicians.
A combination of factors may contribute to the difficulties
in treating the disease, such as a sheltered physiological
environment that is poorly accessible to the immune system
and to antibacterial agents and conditions favoring bacterial
cells in a quiescent state and thus more resistant to treatment.
These are challenging conditions for an antibacterial agent
to overcome, requiring high drug concentrations to be
maintained in bone over a long period of time and therefore
frequent and heavy intravenous dosing. Delivery of the
antibacterial agent to the bone where it would be released
over time has been proposed to circumvent these difficulties.
In particular, the use of beads loaded with antibiotics is being
examined for such a purpose.6-9 An inconvenience of this
approach is that these materials have to be surgically
implanted, a true hurdle with a disease for which recurrence
is common and repeat applications are often necessary.
An approach based on bisphosphonate prodrugs would present
a greater potential from this respect. Such prodrugs have been
described for the delivery of small molecules to bone, such as
diclofenac,15,16 prostaglandins,17 steroids,18 and carboxyfluore-
scein.19,20 We have therefore opted for this approach.
Fluoroquinolones are a well defined class of wholly synthetic
antibacterial agents with a very successful clinical track
record.30,31 They possess a set of desirable attributes, being
generally safe agents, active orally and parenterally, with a broad
antimicrobial spectrum that includes many frequently encoun-
tered pathogens. They are bactericidal in clinically achievable
doses, a feature that is most desirable in treating osteomyelitis,
given the sheltered environment of the microorganism, the high
levels of recurrence associated with the disease, and the potential
for regrowth. Spontaneous resistance levels to these agents are
reasonably low. They have demonstrated some efficacy in
Bisphosphonates have been useful functionalities for the
delivery of agents to bone after systemic administration.10-12
These pyrophosphate analogues possess strong affinity to the
calcium phosphate bone mineral (hydroxyapatite) and rapidly
accumulate in osseous tissues in vivo. While they do possess
therapeutic activity of their own as inhibitors of bone
* To whom correspondence should be addressed. Phone: (514) 496-6667.
Fax: (514) 332-6033. E-mail: afar@targanta.com.
10.1021/jm801007z CCC: $40.75
2008 American Chemical Society
Published on Web 10/04/2008

6956 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Houghton et al.
Scheme 1a
a Reagents and conditions: (a) DMAP, CHCl3; (b) TMSBr, CH2Cl2.
animal models of osteomyelitis.32-34 From a practical perspec-
tive, they are comparatively easily synthesized and modified.
Therefore, the preparation and evaluation of bisphosphonated
fluoroquinolone prodrugs appeared to be advantageous for the
treatment and the prevention of osteomyelitis. Past reports of
prodrug strategies focusing on the release of bioactive amine
compounds provided a catalogue of functional groups tethered
to amino groups and labile under physiological conditions. The
strategy presented in this report focused on the preparation of
bisphosphonated version of these known functional groups to
provide linkers tethering the bisphosphonate group to the
secondary amino group present in many of the clinically used
fluoroquinolones and therefore representing a useful synthetic
handle.
diethyl(ethoxyphosphinyl)methylphosphonate to 6-bromo-3,4-
dihydro-4,4-dimethylchromen-2-one furnished the parent bis-
phosphonated dihydrocoumarin 23. The ring was opened and
converted to the benzyl ester 25, which was acylated or
phosphorylated to provide, after debenzylation, acids 27a-c.
These were coupled with gatifloxacin 1b to furnish, after
deprotection of the phosphonate groups, bisphosphonated pro-
drugs 29a-c.
Recently, a ꢀ-aminoketone prodrug of ciprofloxacin has been
proposed, whereby the drug is freed by elimination.41 A similar
approach was envisaged for gatifloxacin 1b by the preparation
of prodrug 36 (Scheme 5). A sequence of alkylation of the
sodium salt of tetraethyl methylenebisphosphonate with the
protected bromopropanol 30, deprotection, and iodination
furnished iodide 33. Displacement with hydroxyphenylprope-
none provided vinyl ketone 34. The conjugate addition of
gatifloxacin onto the enone, followed by deprotection of the
phosphonate esters, provided the desired prodrug 36.
Substituted acyloxyalkyl esters and carbamates are frequently
used as prodrugs.42-44 The bisphosphonated versions of such
prodrugs were prepared using acids 37, 41, and 4245 (Scheme
6). Acids 37 and 41 were obtained through oxidation of the
parent alcohols 32 and the similarly prepared alcohol 40.
Treatment of moxifloxacin 1a and gatifloxacin 1b with chlo-
roethyl chloroformate in the presence of Proton Sponge gave
the chloroethyl carbamates 43a,b (Scheme 7). Substitution of
the chlorines by acid 42 gave, after deprotection, prodrugs 45a,b.
Treatment of 43b with acid 41 gave, after deprotection, the
gatifloxacin prodrug 47. However, treatment of 43b with acid
37 failed to give the desired product 48. Reasoning that the
weak nucleophilicity of the carboxylate and the increased steric
bulk in 37, as opposed to 41, may have favored an elimination
reaction in this case, producing the labile vinyl carbamate rather
than the expected substitution product, it was decided to attempt
the same reaction with the unsubstituted chloromethyl carbamate
49 instead. In this case, the chloride was displaced as expected
providing, after deprotection of the bisphosphonates, the gati-
floxacin prodrug 51.
Chemistry
In this process, moxifloxacin 1a, gatifloxacin 1b, and cipro-
floxacin 1c were selected as starting materials for the prodrugs.
The previously described27 ciprofloxacin-bisphosphonate con-
jugate 4c was prepared by the addition of 1c to 1,1-ethenyl-
bisphosphonate 2,35 followed by deprotection of the phosphonate
esters with TMSBr (Scheme 1). The parent moxifloxacin
conjugate 4a was assembled in the same fashion.
Fluoroquinolone allyl esters 9a,b were produced via Boc
protection of the amino groups (Scheme 2). These esters were
acylated with acyl chloride 717 to supply, after deprotection,
simple bisphosphonated amides 13a,b.
The well documented use of dioxolonylmethylamines and
carbamates for the preparation of prodrugs36,37 from amine
functionalities directed our attention to the preparation of a
bisphosphonated version for the fluoroquinolones (Scheme 3).
In this case, the adequately substituted precursor 19 was
prepared from 2-bromo-1-(4-bromophenyl)propan-1-one via
palladium catalyzed coupling of a phosphinyl phosphonate38
on cyclic carbonate 17 followed by radical bromination.
Condensation of gatifloxacin 1b with 19 and deprotection of
the prodrug provided the bisphosphonated prodrug 21.
Prodrugs based on favorable cyclizations to 4,4-dimeth-
yldihydrocoumarins have been used successfully with drugs
possessing a primary or a secondary amine functionality.39,40
Bisphosphonated versions of such prodrugs (29a-c) were
prepared for evaluation as osteotropic delivery agents for
gatifloxacin (Scheme 4). Palladium catalyzed coupling of
Results and Discussion
In Vitro Evaluation of Prodrugs. The antibacterial activities
of the prodrugs against S. aureus ATCC13709 were measured
as per CLSI guidelines (results not shown).46 The minimum

Linking Bisphosphonates to Fluoroquinolones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6957
Scheme 2a
a Reagents and conditions: (a) tert-butyl bromoacetate, NaH, DMF; (b) TFA; (c) SOCl2, CH2Cl2; (d) Boc2O, NaOH, H2O, THF; (e): allyl bromide,
K2CO3, DMF; (f) AcCl, MeOH; (g) 7, Et3N, DMAP, CH2Cl2; (h) sodium p-toluenesulfinate, Pd(PPh3)4, THF, H2O; (i) TMSBr, CH2Cl2.
Scheme 3a
a Reagents and conditions: (a) formic acid, TEA, MeCN; (b) NaOH, MeOH; (c) COCl2, PhNMe2, (CH2Cl)2; (d) diethyl(ethoxyphosphinyl)methylphosphonate,
Pd(PPh3)4, THF, H2O; (e) NBS, 1,1′-azobis(cyclohexanecarbonitrile), CCl4, ∆; (f) 1b, DMF; (g) TMSBr, CH2Cl2.
inhibitory concentrations were >4 µg/mL except for 21 (<0.12
µg/mL), 36 (0.12 µg/mL), and 51 (0.5 µg/mL). These values
are generally significantly higher than those of moxifloxacin
(0.03 µg/mL), gatifloxacin (0.06 µg/mL), and ciprofloxacin (0.12
µg/mL). Given that at least a portion of the antibacterial activity
of the prodrugs may come from the release of the parent drug

6958 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Houghton et al.
Scheme 4a
a Reagents and conditions: (a) diethyl(ethoxyphosphinyl)methylphosphonate, Pd(PPh3)4, THF, H2O; (b) KOH, MeOH; (c) KOH, MeCN, then BnBr,
DMF; (d) acid chloride, DMAP, pyridine or diethyl chlorophosphate, Et3N, THF; (e) H2, Pd/C, MeOH; (f) 1b, HBTU, DIPEA, DMF; (g) TMSBr, 2,6-
lutidine, CH2Cl2.
Scheme 5a
a Reagents and conditions: (a) NaH, THF, ∆; (b) pTsOH, MeOH; (c) I2, PPh3, imidazole, CH2Cl2; (d) 1-(4-hydroxyphenyl)prop-2-en-1-one, K2CO3,
acetone; (e) 1b, Et3N, DMAP, CH2Cl2; (f) TMSBr, CH2Cl2.
during the course of the assay, it is reasonable to assume that
the prodrugs lack significant antibacterial activity of their own.
Therefore, in vivo activity would require the bisphosphonated
prodrugs to bind to bone and release the active fluoroquinolones.
These two requirements can be evaluated in vitro to predict the
therapeutic potential of these compounds.
Since bone is essentially insoluble in aqueous media,
bisphosphonates bound to bone powder are for all practical
purposes immobilized on a solid matrix, and bound and free
bisphosphonates can therefore be easily separated by cen-
trifugation. As such, a sense of the affinity of the prodrug
for osseous tissues can be obtained by determining the amount
of prodrug bound to bone powder in phosphate buffered saline
(PBS) at 37 °C over 1 h. This was ascertained by fluorescence
measurements on the supernatant to measure the unbound
fraction (Table 1).
The release of the parent fluoroquinolone from these prodrugs
immobilized on bone powder was determined by measuring the
appearance of fluorescence in the supernatant over time. This
was done in PBS and 50% rat or human sera to evaluate the
potential for enzymatic cleavage (Table 1). In addition to
fluorescence measurements, the nature of the released entity was
also confirmed by determining its antibacterial activity, and in
all cases, this activity was identical to that of the parent
fluoroquinolone (data not shown).
The results from these in vitro assays indicate several trends.
First, these prodrugs are very efficient at binding bone powder,
being taken up at >80% and generally >90% over 1 h for the

Linking Bisphosphonates to Fluoroquinolones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6959
Scheme 6a
of these prodrugs display relatively medium independent rates
of cleavage. From this perspective, compounds 47 and 51 are
quite interesting, as simple esters were not predictably hydro-
lyzed in the absence of esterases. It is plausible that the
bisphosphonate functionality provides an intramolecular acid
that catalyzes this process. The large rate acceleration in going
from 47 to 51 likely reflects the shortened distance between
the bisphosphonate group and the acetal and supports this
hypothesis.
The very high bone affinity of these bisphosphonates and the
wide range of different rates of regenerating the parent fluoro-
quinolones provide a valuable tool for investigating the ability
of bisphosphonates to deliver fluoroquinolones to osseous
tissues, with clear potential in the prevention and the treatment
of osteomyelitis.
In Vivo Evaluation of Prodrugs. Prodrugs 36 and 51
appreciably release their parent fluoroquinolones in vitro in a
process that does not require the participation of an enzyme.
They also demonstrate a very strong affinity for bone. Given
this attractive profile, these compounds were selected for
evaluation in vivo.
a Reagents and conditions: (a) TEMPO, NaOCl, NaOCl2, H2O; (b) NaH,
THF, ∆; (c) pTsOH, MeOH; (d) PDC, DMF.
These bisphosphonated fluoroquinolone prodrugs were tested
in a rat model for the prevention of osteomyelitis, which was
adapted from the known parent treatment models.47,48 Given
the affinity of bisphosphonates for bone as demonstrated in vitro
and given the precedents for clinically used bisphosphonates,
it can be presumed that the ability to prevent infection over
time would suggest the prodrug to be binding to osseous tissues
and releasing the parent antibacterial agent over time. Inactivity
in this model on the other hand would suggest either that the
prodrug is not binding to bone in vivo or that it is not releasing
the parent drug. In other words, it provides a useful if only
indirect means to evaluate the pharmacokinetics of the prodrugs.
Briefly, a single bolus intravenous dose of the prodrug was
administered 1-2 days before an infection of the bone is
simulated by surgically inserting a bacterial load into the tibiae,
using S. aureus (ATCC 13709) as the infectious agent. The
animals were sacrificed 24 h after the infection, and the bacterial
titer in the tibiae was determined. Moxifloxacin injected at 10
mg/kg 1 h after the establishment of infection was used as a
positive control. Indeed, at that time point, moxifloxacin clears
the bacterial load, as the bacterial infection is not yet established
and therefore represents a good means to evaluate the validity
of the experiment. At this dose, moxifloxacin is not efficacious
when used 24 h after the infection is established. It is therefore
not a comparator.
methylenebisphosphonates (4a-c, 13a,b, 36, 45a,b, 47, and 51)
and 35-76% for the phosphinylmethylphosphonates (21 and
29a,b), when the parent drugs are at best negligibly bound. In
fact, it is reasonable to assume the unbound fraction to be at
least partially the result of cleavage of the prodrug during the
time course of the assay, thereby under-representing the true
efficiency of the process. The exception is compound 29c for
which there was surprisingly no binding in vitro. It is possible
that the presence of the additional phosphate group, by increas-
ing the number of potential protonation sites, might result in a
phosphinylmethylphosphonate with a different degree of ioniza-
tion at physiological pH, and this may reduce its affinity for
hydroxyapatite.
Second, it is clear that the previously published bisphospho-
nated ciprofloxacin 4c, its moxifloxacin analogue 4a, and the
simple amides 13a,b are not able to release the parent drugs.
The dioxolone based prodrug 21 released its parent extremely
efficiently. The rate accelerations observed in sera suggest that
the cyclic carbonate moiety may be subject to the action of
esterases or that its decomposition may be favored by the
presence of nucleophilic substances in plasma.
Prodrugs 29a and 29b also appeared to efficiently release
gatifloxacin. These prodrugs are expected to undergo a stepwise
hydrolysis in which the phenolic ester is saponified, releasing
a phenolate that rapidly cyclizes to the dihydrocoumarin. In PBS,
the rates of cleavage of 29a and 29b differ by nearly an order
of magnitude, reflecting the higher lability of the acetate ester
in the former over the isobutyrate ester in the latter. On the
other hand, the levels of regeneration of gatifloxacin are
comparable in sera, a result that suggests similar rates of
saponification, perhaps as a consequence of esterase action. This
would be consistent with the ester hydrolysis being the rate
determining event, followed by a very rapid cyclization.
Compound 36 demonstrates good rates for regenerating
gatifloxacin, and other than through medium effects on the
ꢀ-elimination, the process clearly does not require the participa-
tion of biocatalysts.
This experiment was performed with the bisphosphonated
prodrug 51 at 42 mg/kg of body weight 2 days prior to infection,
and this was compared with the equivalent dose of gatifloxacin
(20 mg/kg of body weight) (Figure 1).
The data provided demonstrate the ability of 51 to prevent
the growth of bacteria when used 2 days prior to infection, under
conditions in which the parent drug gatifloxacin 1b, at the
equivalent dose, is unable to do so. This is in agreement with
the prodrug going to osseous tissues and slowly releasing the
active drug but at concentrations that are sufficiently superior
to its minimum inhibitory concentration to significantly reduce
the bacterial load in bone. In fact, in a test group of five animals,
two had completely sterile bones (data not shown).
Acyloxymethyl carbamate based prodrugs are also shown to
be able to regenerate gatifloxacin. Compounds 45a and 45b were
designed to release the parent drug via cyclization of the
glutaramic ester to the glutarimide, an intramolecular process
not requiring biocatalysis. It is therefore not surprising that both
Since 51 was very rapidly regenerating in vitro, a comparison
with prodrugs regenerating their parent drugs at slower rates is
warranted. Compounds 36, which did release gatifloxacin at an
appreciable rate, and 13a, which was not able to regenerate its

6960 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Houghton et al.
Scheme 7a
a Reagents and conditions: (a) ClCH(R)OCOCl, Proton Sponge, CHCl3 or CH2Cl2; (b) 41 or 42, NaOH, THF, H2O, then 43a or 43b, MeCN, 60 °C; (c)
TMSBr, CH2Cl2; (d) 37, KOH, MeCN, then 46 or 47, DMF; (e) TMSBr, 2,6-lutidine, CH2Cl2.
Table 1. Bone Binding and Regeneration of Parent Fluoroquinolone
from Prodrugs
% parent drug released over 24 h
compd % bound to bonea PBS 50% rat serum 50% human serum
1a
1b
1c
4a
<lodb
<lodb
<lodb
92.2
99.6
99.9
98.2
63.7
36.6
76.0
<lodb
94.7
93.9
94.3
88.4
86.4
<lodb
<lodb
<lodb
<lodb
7.5
<lodb
<lodb
<lodb
<lodb
30.0
6.2
<lodb
<lodb
<lodb
<lodb
22.8
3.7
4c
13a
13b
21
29a
29b
29c
36
45a
45b
47
5.4
0.8
6.1
3.6
ndc
ndc
ndc
Figure 1. Prophylactic effect of prodrug 51 and gatifloxacin when
injected at 42 and 20 mg/kg, respectively (mole equivalent doses), 2
days prior to the establishment of a bone infection in rats.
2.3
1.2
4.2
1.4
4.3
1.8
1.5
1.8
2.4
0.7
0.7
0.9
51
13.4
25.4
21.2
Prodrug 36 was able to significantly reduce the bacterial titer
in the bone under this exposure. In a test group of five rats, one
displayed sterile bones. This is in stark contrast with compound
13a, which did not impact the establishment of the infection.
This supports the notion that not only must the prodrug reach
a At 37 °C for 1 h. b <lod: under the limit of detection (0.01%). c nd:
not determined.
parent drug moxifloxacin, were assayed at 20.8 and 24 mg/kg
of body weight a day prior to infection (Figure 2).

Linking Bisphosphonates to Fluoroquinolones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6961
These prodrugs are being further evaluated for their ability
to prevent and treat osteomyelitis in animal models, and the
results of these investigations will be presented in due course.
Experimental Section
General. 1H NMR spectra were recorded on a Varian Unity 400
spectrometer. The reported chemical shifts (in parts per million)
are referenced using the signals assigned to the residual nondeu-
terated solvents. Mass spectral analyses were performed on an
Agilent mass spectrometer under electron spray ionization (ESI).
Reactions were monitored by TLC on silica gel gel 60 F254 (0.25
mm, Merck). Silica gel column chromatography was performed
on silica gel gel 60 (70-230 mesh). THF was dried by passage
through a column of activated alumina. All reactions were carried
out under an argon atmosphere with anhydrous solvents unless
otherwise noted. All chemicals, unless otherwise stated, were
obtained from commercial sources. All compounds were g95%
Figure 2. Prophylactic effect of prodrugs 36 and 13a when injected
at 20.8 and 24 mg/kg (mole equivalent doses), respectively, a day prior
to the establishment of a bone infection in rats.
the bone, it must release its parent drug to be efficacious and
emphasizes the importance of a prodrug strategy over simple
conjugates.
1
pure as judged by H NMR and LC/MS analyses.
All animal experiments followed the Canadian Council on
Animal Care guidelines in an accredited animal facility, and
protocols were approved by the institutional animal care and use
committee.
2,2-Bisphosphonoethyl Derivatives of Fluoroquinolones. General
Procedure for Condensation with Tetramethyl Ethenylidenebi-
sphosphonate 2. The fluoroquinolone (2 mmol) was dissolved in
dry CHCl3 (30 mL). To this solution was added tetramethyl
ethenylidenebisphosphonate 2 (0.515 g, 2.11 mmol) and a catalytic
quantity of DMAP. The reaction mixture was stirred at room
temperature for 3.5 h and then evaporated at 40 °C.
3a. The residue was taken up in EtOAc and filtered, and the
product was precipitated by the addition of hexanes. Yield: 45%.
1H NMR (400 MHz, CDCl3) δ 0.86-0.87 (m, 1H), 0.94-1.05 (m,
2H), 1.10-1.35 (m, 4H), 1.55-1.85 (m, 5H), 2.25-2.40 (m, 2H),
2.64 (tt, J ) 23.9, 6.0, 1H), 2.75-2.95 (m, 2H), 3.05-3.25 (m,
2H), 3.54 (s, 3H), 3.56-3.72 (m, 6H), 3.74-4.01 (m, 8H), 7.77
(d, J ) 14.1, 1H), 8.76 (s, 1H).
3c. The residue was triturated in boiling toluene, and the insoluble
product was collected. Yield: 44%. 1H NMR (400 MHz, CDCl3) δ
1.17-1.21 (m, 2H), 1.36-1.43 (m, 2H), 1.63 (bs, 1H), 2.67-2.84
(m, 5H), 2.95-3.07 (m, 2H), 3.35 (bs, 4H), 3.48-3.56 (m, 1H),
3.80-3.88 (m, 12 H), 7.34 (d, J ) 7.0, 1H), 8.02 (d, J ) 12.9,
1H), 8.77 (s, 1H).
This experiment demonstrates the ability of these prodrugs
to produce a sufficient concentration of the parent drug to
maintain antibacterial activity when the activity of the parent
drug has already faded.
Conclusions
The free amino position of moxifloxacin, gatifloxacin, and
ciprofloxacin represents a useful handle for tethering a bispho-
sphonate group. By assessment of their affinity for bone and
their ability to release their parent drug once bound to bone, it
is possible to predict a sense for the therapeutic potential of
these fluoroquinolone bisphosphonate conjugates. These inves-
tigations highlighted the greater affinity of methylenebisphos-
phonate derivatives for bone over the parent phosphinometh-
ylphosphonates lacking one of the acidic hydroxyls but also
emphasized the far greater affinity of these two forms of
conjugates over the negligible bone affinities of the parent drugs.
With the lack of bone affinity of compound 29c, they also
exemplified the dramatic influence the overall structure can have
over bone affinity.
Whereas simple conjugates, such as bisphosphonoethyl
(4a-c) and bisphosphonopropionyl (13a,b) derivatives, were
unable to release their parent drugs once bound to bone, this
study presents the successful adaptation of the bisphosphonate
approach to known forms of prodrugs. Thus, bisphosphonated
versions of the dioxolenone (21), 2-acyloxyhydrocinnamate
(29a-c), phenylpropanone (36), and acyloxyalkyl carbamate
(45a,b, 47, 51) prodrug strategies were presented here and
shown to efficiently regenerate their parent drugs once they are
bound to bone. The comparison between 47 and 51 also shows
that, beyond its role in delivery to bone, the bisphosphonate
moiety can itself markedly impact the ability of the prodrug to
regenerate.
General Procedure for Deprotection. TMSBr (0.58 mL, 4.39
mmol) was added in one portion to a stirring solution of the
protected bisphosphonated prodrug (0.44 mmol) in dry CH2Cl2 (10
mL), and the resulting mixture was stirred at room temperature for
16 h. The solvent was removed under reduced pressure, and solid
was dried under high vacuum for 1 h. The solid was suspended in
H2O (30 mL), and the pH was immediately adjusted to pH 7.6 by
the addition of 1 M NaOH, with concomitant dissolution of the
product.
4a. The solution was concentrated in vacuo, and the solid residue
portion was purified on a C18 Sep-Pak (H2O). Yield: 59%. 1H NMR
(400 MHz, D2O) δ.0.78-1.36 (m, 4H), 1.56-2.15 (m, 4H),
2.36-2.53 (m,1H), 2.60-2.85 (m, 1H), 3.32-3.85 (m, 7H), 3.62
(s, 3H), 4.05-4.38 (m, 4H), 7.59 (d, J ) 13.7, 1H), 8.59 (s, 1H).
4c. The product solution was washed with CHCl3 (2 × 25 mL),
filtered, and evaporated to give the product in quantitative yield.
1H NMR (400 MHz, D2O) δ 1.14 (bs, 2H), 1.32-1.38 (m, 2H),
2.35-2.50 (m, 1H), 3.36-3.90 (m, 11H), 7.66 (d, J ) 7.0, 1H),
7.93 (d, J ) 13.1, 1H), 8.51 (s, 1H).
3,3-Bisphosphonoproprionamides of Fluoroquinolones. Tetra-
ethyl 2-tert-Butoxycarbonylethylene-1,1-bisphosphonate (5). To a
solution of tetraethyl methylenebisphosphonate (3.00 g, 10.4 mmol)
in dry DMF (9 mL) was added 0.46 g of NaH (60% suspension in
mineral oil, 11.5 mmol) portionwise. The resulting slurry was stirred
for 30 min at room temperature, after which tert-butyl bromoacetate
(1.7 mL, 11.5 mmol) was added neat and at a rapid pace. The
reaction mixture was stirred for 1 h and quenched by adding 2 mL
of a saturated solution of NH4Cl. The reaction mixture was
The abilities of bisphosphonate fluoroquinolone conjugates
13a, 36, and 51 to prevent infection in a rat model of
osteomyelitis were further evaluated. Compounds 36 and 51
were shown to efficaciously and significantly prevent the
establishment of infection while the parent drug gatifloxacin
was unable to do so. The inability of conjugate 13a, which was
shown in vitro to be unable to regenerate its parent drug, to
prevent infection is significant in that it highlights the fact that
these forms of stable conjugates in practical terms irreversibly
bind the drug to bone and are therefore not therapeutically useful
in tackling osteomyelitis. This finding stresses the value of the
prodrug strategy.

6962 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Houghton et al.
evaporated and purified by flash chromatography on silica gel,
eluting with 5% methanol/ethyl acetate to give tetraethyl 2-tert-
butoxycarbonylethylene-1,1-bisphosphonate (6, 2.1 g, 50%) as a
clear, colorless oil. H NMR (400 MHz, CDCl3) δ 1.33 (bt, J )
7.0, 12H), 1.46 (s, 9H), 2H), 2.76 (td, J ) 16.0, 6.1, 2H), 3.07 (tt,
J ) 24.0, 6.1, 1H), 4.10-4.25 (m, 8H).
5.48 (dd, J ) 17.2, 1.5, 1H), 6.00-6.11 (m, 1H), 7.90 (d, J )
12.5, 1H), 8.59 (s, 1H).
General Procedure for Preparation of Fluoroquinolone Allyl
Esters. To a solution of N-Boc fluroquinolone allyl ester 9a,b (1.5
mmol) in dry methanol (25 mL) cooled to 0 °C was added acetyl
chloride (5.33 mL, 74.6 mmol). The resulting solution was allowed
to warm to room temperature over 1.5 h and concentrated, and the
residue was partitioned between ice cold saturated NaHCO3 and
CH2Cl2. The organics were collected, dried over Na2SO4, and
concentrated in vacuo to provide the fluoroquinolone allyl ester of
sufficient purity for the next step.
Allyl 1-Cyclopropyl-6-fluoro-1,4-dihydro-7-((4aS,7aS)-octahy-
dropyrrolo[3,4-b]pyridin-6-yl)-8-methoxy-4-oxoquinoline-3-car-
boxylate (10a). Yield: 95%. 1H NMR (400 MHz, CDCl3) δ
0.75-0.85 (m, 1H), 0.95-1.10 (m, 2H), 1.14-1.24 (m, 1H),
1.53-1.68 (m, 1H), 1.72-1.90 (m, 3H), 2.38 (bs, 1H), 2.73-2.87
(m, 1H), 3.12-3.24 (m, 1H), 3.35-3.64 (m, 3H), 3.55 (s, 3H),
3.82-4.02 (m, 3H), 4.74-4.88 (m, 2H), 5.26 (dd, J ) 10.6, 1.1,
1H), 5.46 (dd, J ) 17.2, 1.5, 1H), 5.98-6.11 (m, 1H), 7.61 (d, J
) 13.9, 1H), 8.53 (s, 1H).
Allyl 7-(3-Methylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-di-
hydro-8-methoxy-4-oxoquinoline-3-carboxylate (10b). Yield: 92%.
1H NMR (400 MHz, CDCl3) δ 0.86-1.00 (m, 2H), 1.08-1.18 (m,
5H), 2.87-2.97 (m, 1H), 3.00-3.14 (m, 3H), 3.21-3.39 (m, 3H),
3.77 (s, 3H), 3.86-3.95 (m, 1H), 4.80-4.86 (m, 2H), 5.25-5.30
(m, 1H), 5.45-5.51 (m, 1H), 6.00-6.10 (m, 1H), 7.88 (d, J )
12.5, 1H), 8.58 (s, 1H).
General Procedure for Acylation of Fluoroquinolone Allyl
Esters. To a solution of the crude fluroquinolone allyl ester 10a,b
(1.4 mmol), triethylamine (0.24 mL, 1.69 mmol), and DMAP (17
mg, 0.14 mmol) in CH2Cl2 (20 mL) cooled to 0 °C was added
dropwise a CH2Cl2 solution of crude acyl chloride 7 (1.76 mmol
in 12.5 mL). The resulting mixture was allowed to warm to room
temperature overnight, diluted with CH2Cl2, and washed with
saturated NaHCO3. The aqueous washes were back-extracted with
CH2Cl2, and the combined organics were washed with brine, dried
over MgSO4, and concentrated. The product was purified by flash
chromatography on SiO2 (5% methanol/CH2Cl2).
1
Tetraethyl 2-Carboxyethylene-1,1-bisphosphonate (6). tert-Butyl
ester 7 (2.1 g, 5.2 mmol) was stirred in TFA (12 mL) for 2.5 min
and concentrated under reduced pressure. The residue was purified
by flash chromatography (gradient elution 100% ethyl acetate to
10% methanol/ethyl acetate) to furnish tetraethyl 2-carboxyethylene-
1
1,1-bisphosphonate (6) as a white solid (1.35 g, 75%). H NMR
(400 MHz, CDCl3) δ 1.28-1.39 (m, 12H), 2.86 (td, J ) 16.1, 6.3,
2H), 3.12 (tt, J ) 24.0, 6.3, 1H), 4.13-4.26 (m, 8H).
Tetraethyl 2-Chlorocarbonylethylene-1,1-bisphosphonate (7). To
acid 6 (1.02 g, 2.95 mmol) in CH2Cl2 (15 mL) was added freshly
distilled SOCl2 (0.84 mL, 11.6 mmol). The mixture was stirred at
reflux for 3 h and concentrated to dryness to give crude tetraethyl
2-chlorocarbonylethylene-1,1-bisphosphonate (7) as a colorless oil
(quantitative), which was used immediately for the next step without
further purification. 1H NMR (400 MHz, CDCl3) δ 1.30-1.40 (m,
12H), 3.05 (tt, J ) 23.5, 6.2, 1H), 3.40 (td, J ) 14.8, 6.2, 2H),
3.12 (tt, J ) 24.0, 6.3, 1H), 4.13-4.27 (m, 8H).
General Procedure for Boc Protection of Fluoroquinolones. A
mixture of the fluoroquinolone (2 mmol), Boc2O (460 mg, 2.082
mmol), and 4.2 mL of 1 M NaOH aqueous solution in 20 mL of THF
was stirred at room temperature overnight. After removal of the organic
solvent, the residue was neutralized with a saturated aqueous am-
monium chloride solution. The mixture was extracted with ethyl acetate
(3×) and dried over anhydrous sodium sulfate. Removal of the solvent
yielded the product in sufficient purity for the next step.
7-((4aS,7aS)-1-(tert-Butoxycarbonyl)octahydropyrrolo[3,4-b]py-
ridin-6-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxo-
quinoline-3-carboxylic Acid (8a). Yield: 91%. 1H NMR (400 MHz,
CDCl3): δ 0.79-0.86 (m, 1H), 1.03-1.18 (m, 2H), 1.23-1.34 (m,
2H), 1.44-1.54 (m, 1H), 1.49 (s, 9H), 1.76-1.84 (m, 2H),
2.25-2.29 (m, 1H), 2.89 (t, J)11.8, 1H), 3.22-3.30 (m, 1H), 3.38
(bs, 1H), 3.57 (s, 3H), 3.88 (dt, J ) 2.7, 10.0, 1H), 3.96-4.01 (m,
1H), 4.07-4.12 (m, 2H), 4.79 (bs, 1H), 7.82 (d, J ) 13.7, 1H),
8.79 (s, 1H) ppm.
Allyl 7-((4aS,7aS)-1-(3,3-Bis(diethylphosphono)propionyl)oc-
tahydropyrrolo[3,4-b]pyridin-6-yl)-1-cyclopropyl-6-fluoro-1,4-di-
hydro-8-methoxy-4-oxoquinoline-3-carboxylate (11a). Yield: 74%.
1H NMR (400 MHz, CDCl3, mixture of rotamers) δ 0.72-0.81
(m, 1H), 0.93-1.10 (m, 2H), 1.15-1.26 (m, 1H), 1.28-1.38 (m,
12H), 1.43-1.64 (m, 2H), 1.78-1.90 (m, 2H), 2.18-2.36 (m, 1H),
2.75-3.10 (m, 3H), 3.13-3.29 (m, 2H), 3.34-3.66 (m, 2H), 3.55
(s, 3H, major rotamer), 3.59 (s, 3H, minor rotamer), 3.74-4.26
(m, 12H), 4.53-4.66 (m, 1H), 4.76-4.88 (m, 2H), 5.17-5.35
(overlapping doublets of doublets, 1H), 5.42-5.54 (overlapping
doublets of doublets, 1H), 5.98-6.10 (m, 1H), 7.82 (d, J ) 13.9,
1H, major rotamer), 7.84 (d, J ) 14.3, 1H, minor rotamer), 8.54
(s, 1H, major rotamer), 8.55 (s, 1H, minor rotamer).
7-(4-(tert-Butoxycarbonyl)-3-methylpiperazin-1-yl)-1-cyclopro-
pyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic Acid
1
(8b). Yield: 95%. H NMR (400 MHz, CDCl3): δ 0.94-1.04 (m,
2H), 1.19-1.26 (m, 2H), 1.33 (d, J ) 6.9, 3H), 1.50 (s, 9H),
3.23-3.37 (m, 3H), 3.44-3.51 (m, 2H), 3.73 (s, 3H), 3.95-4.03
(m, 2H), 4.36 (bs, 1H), 7.89 (d, J ) 11.4, 1H), 8.83 (s, 1H) ppm.
General Procedure for Allylation of N-Boc Fluoroquinolones.
To a solution of N-Boc fluoroquinolone 8a,b (2.0 mmol) in dry
DMF (20 mL) was added K2CO3 (332 mg, 2.4 mmol) and allyl
bromide (210 µL, 2.4 mmol). The reaction mixture was heated at
75-80 °C for 24 h and concentrated in vacuo, and the residue was
partitioned between water and ethyl acetate. The organic layer was
collected, and the aqueous layer was extracted once more with ethyl
acetate. The combined organics were washed with brine, dried
(MgSO4), and concentrated to dryness to furnish material of
sufficient purity for the subsequent step.
Allyl 7-(4-(3,3-Bis(diethylphosphono)propionyl)-3-methylpiper-
azin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxo-
1
quinoline-3-carboxylate (11b). Yield: 81%. H NMR (400 MHz,
CDCl3, mixture of rotamers) δ 0.87-0.99 (m, 2H), 1.10-1.21 (m,
2H), 1.29-1.43 (m, 15H), 2.78-3.05 (m, 2H), 3.16-3.82 (m, 6H),
3.72 (s, 3H), 3.85-3.95 (m, 1H), 4.12-4.30 (m, 9H), 4.47-4.59
(m, 0.5H), 4.80-4.92 (m, 2.5H), 5.28 (dd, J ) 10.4, 1.3, 1H),
5.45-5.55 (m, 1H), 6.00-6.12 (m, 1H), 7.91 (d, J ) 12.5, 1H),
8.59 (s, 1H).
Procedure for Allyl Ester Deprotection. To a solution of allyl
ester 11a,b (1.04 mmol) in THF (20 mL) was added Pd(PPh3)4
(24 mg, 0.02 mmol) and an aqueous solution of sodium p-
toluenesulfinate (204 mg in 2 mL, 1.14 mmol). The mixture was
stirred at room temperature for 1.25 h, concentrated, and purified
by flash chromatography on SiO2.
7-((4aS,7aS)-1-(3,3-Bis(diethylphosphono)propionyl)octahydr-
opyrrolo[3,4-b]pyridin-6-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-
methoxy-4-oxoquinoline-3-carboxylic Acid (12a). 12a was pu-
rified by flash chromatography with gradient elution of 5%
methanol/CH2Cl2 to 10% methanol/CH2Cl2. Yield: 79%. 1H NMR
Allyl 7-((4aS,7aS)-1-(tert-Butoxycarbonyl)octahydropyrrolo[3,4-
b]pyridin-6-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-
oxoquinoline-3-carboxylate (9a). Yield: 74%. 1H NMR (400 MHz,
CDCl3) δ 0.73-0.83 (m, 1H), 0.88-1.12 (m, 2H), 1.18-1.29 (m,
1H), 1.48 (s, 9H), 1.58-1.85 (m, 4H), 2.19-2.28 (m, 1H),
2.82-2.93 (m, 1H), 3.15-3.26 (m, 1H), 3.30-3.40 (m, 1H), 3.56
(s, 3H), 3.78-3.92 (m, 2H), 3.99-4.11 (m, 2H), 4.70-4.90 (m,
3H), 5.27 (dd, J ) 10.4, 1.3, 1H), 5.48 (dd, J ) 17.2, 1.5, 1H),
6.00-6.11 (m, 1H), 7.84 (d, J ) 14.3, 1H), 8.56 (s, 1H).
Allyl 7-(4-(tert-Butoxycarbonyl)-3-methylpiperazin-1-yl)-1-cy-
clopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxoquinoline-3-car-
boxylate (9b). Yield: 78%. 1H NMR (400 MHz, CDCl3) δ
0.83-0.98 (m, 2H), 1.07-1.20 (m, 2H), 1.33 (d, J ) 6.6, 1H),
1.49 (s, 9H), 3.15-3.49 (m, 5H), 3.72 (s, 3H), 3.84-3.99 (m, 2H),
4.34 (bs, 1H), 4.83 (d, J ) 5.9, 2H), 5.28 (dd, J ) 10.4, 1.3, 1H),

Linking Bisphosphonates to Fluoroquinolones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6963
(400 MHz, CDCl3, mixture of rotamers) δ 0.76-0.85 (m, 1H),
1.02-1.19 (m, 2H), 1.25-1.40 (m, 13H), 1.46-1.67 (m, 2H),
1.80-1.93 (m, 2H), 2.33-2.40 (m, 1H), 2.72-3.10 (m, 3H),
3.12-3.36 (m, 2H), 3.40-3.65 (m, 1H), 3.56 (s, 3H, major
rotamer), 3.60 (s, 3H, minor rotamer), 3.78-4.28 (m, 11H),
4.57-4.70 (m, 1H), 5.20-5.30 (m, 1H), 7.81 (d, J ) 13.9, 1H,
major rotamer), 7.84 (d, J ) 13.6, 1H, minor rotamer), 8.78 (s,
1H, major rotamer), 8.79 (s, 1H, minor rotamer).
7-(4-(3,3-Bis(diethylphosphono)propionyl)-3-methylpiperazin-1-
yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxoquinoline-
3-carboxylic Acid (12b). 12b was purified by flash chromatography
with 5% methanol/CHCl3. Yield: 67%. 1H NMR (400 MHz, CDCl3,
mixture of rotamers) δ 0.93-1.06 (m, 2H), 1.16-1.28 (m, 2H),
1.30-1.40 (m, 15H), 2.81-3.05 (m, 2H), 3.18-3.84 (m, 6H), 3.73
(s, 3H), 3.97-4.05 (m, 1H), 4.12-4.28 (m, 9H), 4.49-4.61 (m,
0.5H), 4.84-4.94 (m, 0.5H), 7.91 (d, J ) 12.5, 1H), 8.83 (s, 1H).
General Procedure for Deprotection to Amides 13a,b. To a
solution of protected fluoroquinolone bisphosphonate amide 12a,b
(0.82 mmol) in CH2Cl2 (40 mL) was added TMSBr (1.1 mL, 8.2
mmol). The reaction mixture was stirred for 38 h, the volatiles were
removed under reduced pressure, and the solid was dried under
high vacuum for 1 h. The solid was suspended in H2O (80 mL),
and the pH was immediately adjusted to pH 7 by the addition of 1
M NaOH, with concomitant dissolution of the product. The product
solution was concentrated and purified by reverse-phase chroma-
tography on a Waters C18 Sep-Pak cartridge.
) 7.0, 3H), 3.7 (bs, 1H), 5.11 (bq, J ) 6.9, 1H), 7.65 (d, J ) 8.5,
2H), 7.79 (d, J ) 8.5, 2H).
4-(4-Bromophenyl)-5-methyl-1,3-dioxol-2-one (17). A solution of
16 in 1,2-dichloroethane (DCE, 60 mL) was cooled in an ice bath
followed by the addition of 20% phosgene (23.5 mL, 40.1 mmol)
in toluene. After the mixture was stirred for 15 min, a solution of
N,N-dimethylaniline (4.0 mL, 79 mmol) in DCE (10 mL) was added
dropwise over a period of 1 h at the same temperature. The ice
bath was removed, and the mixture was heated to 70 °C for 20 h.
The solution was diluted with CH2Cl2, washed with water, 10%
aqueous HCl, water, brine, and then dried over Na2SO4 and filtered.
After removal of the solvent the product was recrystallized from
EtOAc/hexanes to furnish 17 (6.07 g, 63%) as a pale-green solid:
1H NMR (400 MHz, CDCl3) δ 2.36 (s, 3H), 7.33 (d, J ) 8.7, 2H),
7.58 (d, J ) 8.7, 2H).
Ethyl (Diethylphosphonomethyl)(4-(5-methyl-2-oxo-1,3-dioxol-
4-yl)phenyl)phosphinate (18). A mixture of 17 (0.323 g, 1.27
mmol), diethyl(ethoxyphosphinyl)methylphosphonate (0.325 g, 1.33
mmol), TEA (0.530 mL, 3.80 mmol), and Pd(PPh3)4 (0.146 g, 0.127
mmol) in acetonitrile (3 mL) was heated 90 °C for 3 h. The solvent
was removed at reduced pressure and the product purified by silica
gel flash column chromatography (0-6% MeOH in CH2Cl2),
1
resulting in 18 (0.368 g, 70%) as a yellow solid: H NMR (400
MHz, CDCl3) δ 1.15-1.23 (m, 3H), 1.27-1.36 (m, 6H), 2.42 (s,
3H), 2.54-2.72 (m, 2H), 3.93-4.21 (m, 6H), 7.58 (dd, J ) 2.8,
8.5, 2H), 7.94 (dd, J ) 8.3, 12.2, 2H). 31P (162 MHz, CDCl3) δ
17.41-17.54 (m, 1P), 30.86-31.08 (m, 1P).
7-((4aS,7aS)-1-(3,3-Bisphosphonopropionyl)octahydropyrrolo[3,4-
b]pyridin-6-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-
oxoquinoline-3-carboxylic Acid (13a). 13a was purified by reverse-
phase chromatography with gradient elution, 100% water to 25%
methanol/water. Yield: 32% based on tetrasodium salt of product. 1H
NMR (400 MHz, D2O, mixture of rotamers) δ 0.73-0.83 (m, 1H),
0.95-1.16 (m, 2H), 1.17-1.28 (m, 1H), 1.46-1.73 (m, 2H), 1.76-1.89
(m, 2H), 2.30-2.62 (m, 2H), 2.67-4.48 (m, 8H), 3.60 (s, 3H),
4.87-4.98 (m, 0.43H), 5.08-5.18 (m, 0.57H), 7.64 (d, J ) 14.3, 1H),
8.47 (s, 1H). 19F (376 MHz, D2O) δ -96.88 to 96.73 (m, 1F). 31P
(162 MHz, D2O) δ 19.94-20.26 (m, 2P). MS: (MH+) 618.1.
7-(4-(3,3-Bisphosphonopropionyl)-3-methylpiperazin-1-yl)-1-cy-
clopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxoquinoline-3-car-
boxylic Acid (13b). 13b was purified by two reverse-phase
chromatographies, eluting with water. Yield: 34% based on
tetrasodium salt of product. 1H NMR (400 MHz, D2O, mixture of
rotamers) δ 0.88-1.04 (m, 2H), 1.06-1.22 (m, 2H), 1.38 (d, J )
7.0, 1.7H), 1.51 (d, J ) 6.6, 1.3H), 2.39-2.62 (m, 1H), 2.78-3.08
(m, 2H), 3.27-3.48 (m, 4H), 3.77 (s, 3H), 3.98-4.78 (m, 3H),
7.75 (d, J ) 12.8, 1H), 8.53 (s, 1H). 19F (376 MHz, D2O) δ -95.77
to 95.63 (m, 1F). 31P (162 MHz, D2O) δ 19.76-20.16 (m, 2P).
MS: (M - H) 590.0.
Ethyl (Diethylphosphonomethyl)(4-(5-(bromomethyl)-2-oxo-1,3-
dioxol-4-yl)phenyl)phosphinate (19). A mixture of 18 (1.79 g, 4.28
mmol), NBS (0.761 g, 4.28 mmol), and 1,1′-azobis(cyclohexan-
ecarbonitrile) (0.11 g, 0.43 mmol) in CCl4 was heated to reflux
under a strong visible light for 4 h, at which time all the starting
material had been consumed as was evident by 1H NMR. The
solvent was removed at aspirator pressure and the crude product
was purified by silica gel flash column chromatography (0-5%
MeOH in CH2Cl2) to furnish 19 (1.26 g, 60%) as a yellow oil: 1H
NMR (400 MHz, CDCl3) δ 1.22 (t, J ) 7.1, 3H), 1.31 (t, J ) 7.1,
3H), 1.35 (t, J ) 7.1, 3H), 2.64 (ddd, J ) 2.8, 18.0, 20.4, 2H),
3.95-4.23 (m, 6H), 4.44 (s, 2H), 7.66 (dd, J ) 2.9, 8.4, 2H), 8.02
(dd, J ) 8.4, 12.2, 2H). 31P (162 MHz, CDCl3) δ 19.63 (d, J )
5.6, 1P), 32.93 (d, J ) 5.6, 1P).
1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-4-
((2-oxo-5-(4-(O-ethyl(diethylphosphonomethyl)phosphonoyl)phenyl)-
1,3-dioxol-4-yl)methyl)piperazin-1-yl)-4-oxoquinoline-3-carboxyl-
ic Acid (20). A solution of gatifloxacin 1b and 19 in DMF was
stirred at room temperature for 16 h. The reaction was quenched
by the addition of saturated aqueous NH4Cl, and the product was
extracted with ethyl acetate. The organic layer was washed with
brine and dried over Na2SO4, filtered, and concentrated at reduced
pressure. The crude product was purified by silica gel HPFC (10%
MeOH in EtOAc and then 5% MeOH in CH2Cl2) to give 20 (44
Bisphosphonated Dioxolenone Prodrug of Gatifloxacin. 1-(4-
Bromophenyl)-1-oxopropan-2-yl Formate (15). A solution of formic
acid (1.6 mL, 43 mmol) in acetonitrile (20 mL) was cooled in an
ice bath followed by the sequential dropwise addition of TEA (6.0
mL, 43 mmol) and then 2,4′-dibromopropriophenone (10.0 g, 34.2
mmol) in 10 mL of THF/acetonitrile (1:1). The resulting solution
was stirred while warming to room temperature over 18 h. The
resulting colorless precipitate was filtered off, and the organics were
removed at reduced pressure. The residue was redissolved in EtOAc,
refiltered, and concentrated to give 15 as a yellow oil that was used
1
mg, 28%) as a pale-yellow solid: H NMR (400 MHz, CDCl3) δ
0.97-1.03 (m, 2H), 1.17-1.38 (m, 14H), 2.61-2.70 (m, 3H), 2.79
(bs, 1H), 2.97 (bd, J ) 3.0, 1H), 3.16 (bt, J ) 9.2, 1H), 3.39-3.47
(m, 3H), 3.58 (d, J ) 14.7, 1H), 3.77 (s, 3H), 3.99-4.23 (m, 8H),
7.79 (dd, J ) 2.1, 8.3, 2H), 7.89 (d, J ) 7.9, 1H), 8.00 (dd, J )
8.3, 11.4, 2H), 8.82 (s, 1H).
1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-4-
((2-oxo-5-(4-(phosphonomethylphosphinoyl)phenyl)-1,3-dioxol-4-
yl)methyl)piperazin-1-yl)-4-oxoquinoline-3-carboxylic Acid (21).
TMSBr (0.175 mL, 1.33 mmol) was added to a stirring solution of 20
(70 mg, 0.088 mmol) in CH2Cl2 (4 mL). The resulting solution was
stirred at room temperature for 15 h, and then the solvent was removed
at reduced pressure. The solid was suspended in 30 mM triethylam-
monium bicarbonate buffer (2 mL), and then the pH was adjusted to
approximately 6 by the addition of triethylamine. The solution was
then subjected to two consecutive C18 silica gel chromatographies
(5-50% CH3CN in 30 mM triethylammonium bicarbonate, then
5-50% CH3CN in water) to give 21 (20 mg, 32%) as the monotri-
ethylammonium salt: 1H NMR (400 MHz, D2O) δ 1.03-1.08 (m, 2H),
1.23 (d, J ) 7.6, 2H), 1.36 (d, J ) 5.8, 2H), 2.33 (d, J ) 18.2, 2H),
1
without further purification: H NMR (400 MHz, CDCl3) δ 1.56
(d, J ) 7.0, 3H), 6.02 (q, J ) 7.0, 1H), 7.63 (d, J ) 8.7, 2H), 7.80
(d, J ) 8.7, 2H), 8.11 (s, 1H).
1-(4-Bromophenyl)-2-hydroxypropan-1-one (16). Crude 15 was
dissolved in MeOH (100 mL). Then 1 M NaOH (1.5 mL) was added
and the resulting solution was stirred for 18 h. Approximately half
of the methanol was removed at reduced pressure. The reaction
was quenched by the addition of saturated aqueous NH4Cl, and
the product was extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, and concentrated to a yellow
residue that was purified by flash silica gel chromatography
(10-50% EtOAc in hexanes), resulting in 16 as a yellow oil (5.27
g, 68% over two steps): 1H NMR (400 MHz, CDCl3) δ 1.44 (d, J

6964 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Houghton et al.
3.05-3.25 (m, 2H), 3.32-3.44 (m, 2H), 3.55-3.70 (m, 3H), 3.81 (s,
3H), 4.23-4.33 (m, 2H), 4.58 (d, J ) 14.8, 1H), 7.74-7.77 (m, 3H),
7.93 (dd, J ) 8.3, 11.1, 2H), 8.92 (s, 1H). 31P (162 MHz, D2O) δ
12.82 (s, 1P), 29.37 (s, 1P). LCMS: 87.4% (254 nm), 87.1% (220
nm), 88.5% (290 nm). MS: (MH+) 708.2.
2H), 4.06-4.18 (m, 4H), 4.95 (s, 2H), 7.14-7.20 (m, 3H),
7.28-7.34 (m, 3H), 7.73 (ddd, J ) 1.9, 8.2,11.8, 1H), 7.89 (dd, J
) 1.9, 13.3, 1H). 31P (162 MHz, CDCl3) δ 20.20 (d, J ) 9.9, 1P),
33.88 (d, J ) 9.9, 1P).
Benzyl 3-(2-(2,2-Dimethylacetoxy)-5-(ethoxy(diethylphospho-
nomethyl)phosphinoyl)phenyl)-3-methylbutanoate (26b). 26bwas
purified by silica gel chromatography using 0-10% MeOH in
CH2Cl2 as the eluent. Yield: 78%. 1H NMR (400 MHz, CDCl3) δ
1.18-1.22 (m, 6H), 1.29-1.33 (m, 9H), 1.46 (s, 3H), 1.49 (s, 3H),
2.58 (ddd, J ) 6.9, 17.5, 23.5, 2H), 2.79-2.90 (m, 3H), 3.96-4.19
(m, 6H), 4.96 (s, 2H), 7.09 (dd, J ) 3.4, 8.3, 1H), 7.18-7.21 (m,
2H), 7.28-7.32 (m, 3H), 7.72 (ddd, J ) 1.9, 8.3, 11.8, 1H), 7.88
(dd, J ) 1.9, 13.3, 1H). 31P (162 MHz, CDCl3) δ 20.28 (d, J )
9.8, 1P), 34.04 (d, J ) 9.8, 1P).
Preparation of 2′-Acyloxyhydrocinnamylamides of Gatiflo-
xacin 14a-d. 6-(Ethoxy(diethylphosphonomethyl)phosphinoyl)-
3,4-dihydro-4,4-dimethylchromen-2-one (23). A mixture of 6-bromo-
4,4-dimethylchroman-2-one(3.5g,9.7mmol),diethyl(ethoxyphosphin-
yl)methylphosphonate (1.7 g, 9.7 mmol), triethylamine (4.1 mL,
29 mmol), and Pd(PPh3)4 (0.56 g, 0.48 mmol) in acetonitrile (20
mL) was heated to 100 °C for 18 h. The reaction mixture was cooled
and diluted with acetonitrile (50 mL) followed by washing with
aqueous HCl (10%), water, and saturated aqueous NaCl. The
organic phase was dried over Na2SO4, filtered, and concentrated.
The crude product was purified by silica gel chromatography
(0-10% MeOH in CH2Cl2) on a Biotage flash chromatography
Benzyl 3-(2-(Diethylphosphoryloxy)-5-(ethoxy(diethylphospho-
nomethyl)phosphinoyl)phenyl)-3-methylbutanoate (26c). Diethyl
chlorophosphate (283 µL, 1.98 mmol) was added dropwise to a
stirring solution of 25 (695 mg, 1.32 mmol) and triethylamine (368
µL, 2.64 mmol) in THF. The resulting mixture was stirred for 24 h
at room temperature followed by dilution with EtOAc (80 mL).
The organics were washed with aqueous HCl (5%), water, and
saturated aqueous NaCl, followed by drying over Na2SO4. The crude
product was purified by silica gel chromatography (0-20% MeOH
in EtOAc) on a Biotage flash chromatography system, resulting in
1
system, resulting in 23 as pale-yellow oil (3.0 g, 73%): H NMR
(400 MHz, CDCl3) δ 1.21 (t, J ) 7.2, 3H), 1.30-1.37 (m, 6H),
1.40 (s, 6H), 2.61 (dd, J ) 1.7, 17.2, 20.7, 2H), 2.66 (s, 2H),
3.95-4.08 (m, 2H), 4.11-4.21 (m, 4H), 7.16 (dd, J ) 3.1, 8.3,
2H), 7.73 (dd, J ) 3.1, 8.3, 2H). 31P (162 MHz, CDCl3) δ 20.07
(d, J ) 7.7, 1P), 33.74 (d, J ) 7.7, 1P).
3-(2-Hydroxy-5-(ethoxy(diethylphosphonomethyl)phosphinoyl)-
phenyl)-3-methylbutanoic Acid (24). A solution of 23 (0.99 g, 2.4
mmol) and KOH (0.095 g, 2.4 mmol) in MeOH was stirred at room
temperature for 2 h. The solvent was removed under reduced
pressure. The product was resuspended in water, the pH was
adjusted to 4 by the addition of HCl, and the product was extracted
with CH2Cl2. The organics were dried over Na2SO4, filtered, and
concentrated, resulting in 24 as a pale-yellow oil (1.1 g, 105%),
which was used without purification. 1H NMR (400 MHz, CDCl3)
δ 1.26 (t, J ) 7.2, 3H), 1.29-1.37 (m, 6H), 1.45 (s, 3H), 1.48 (s,
3H), 2.63 (dd, J ) 17.7, 20.9, 2H), 2.93 (AB q, J ) 14.2, 2H),
4.00-4.20 (m, 6H), 6.74 (bs, 1H), 7.56 (ddd, J ) 1.6, 8.5, 12.2,
2H), 7.63 (d, J ) 13.3, 1H).
Benzyl 3-(2-Hydroxy-5-(ethoxy(diethylphosphonomethyl)ph-
osphinoyl)phenyl)-3-methylbutanoate (25). An aqueous KOH solu-
tion (0.14 g, 2.5 mmol) was added to a stirring solution of 24 (1.1
g, 2.5 mmol) in acetonitrile (5 mL). After 10 min the solvent was
evaporated under reduced pressure and the residue was dried under
vacuum for 1 h. The pale-yellow solid was resuspended in DMF
(10 mL) followed by the addition of benzyl bromide (330 µL, 2.8
equiv). The resulting solution was stirred at room temperature for
2 h. The mixture was diluted with EtOAC (80 mL) and washed
with H2O and saturated aqueous NaCl, followed by drying over
Na2SO4. The crude product was purified by silica gel chromatog-
raphy (0-10% MeOH in CH2Cl2) on a Biotage flash chromatog-
raphy system, resulting in 25 as a pale-yellow liquid (0.64 g, 48%):
1H NMR (400 MHz, CDCl3) δ 1.23 (t, J ) 7.1, 3H), 1.26 (t, J )
7.1, 3H), 1.30 (t, J ) 7.1, 3H), 1.45 (s, 3H), 1.49 (s, 3H), 2.60 (dd,
J ) 17.4, 20.9, 2H), 3.00 (AB q, J ) 14.0, 2H), 3.78-3.88 (m,
2H), 3.99-4.15 (m, 4H), 4.93 (s, 2H), 6.75-6.78 (m, 1H), 7.14
(dd, J ) 2.0, 7.5, 2H), 7.25-7.31 (m, 3H), 7.58 (ddd, J ) 1.4, 8.0,
11.9, 1H), 7.64 (d, J ) 13.4, 1H). 31P (162 MHz, CDCl3) δ 21.04
(d, J ) 4.6, 1P), 36.00 (d, J ) 4.6, 1P).
General Procedure for the Acylation of 25. The acyl chloride
(1.56 mmol) was added dropwise to a stirred solution of 25 (825
mg, 1.56 mmol) and DMAP (catalyst) in pyridine (10 mL) in an
ice bath (26a) or at room temperature (26b,c). The resulting solution
was stirred for 2 h at the same temperature followed by dilution
with EtOAc (80 mL). The organics were washed with aqueous HCl
(5%), water, and saturated aqueous NaCl, then dried over Na2SO4.
The crude product was purified by silica gel chromatography on a
Biotage flash chromatography system.
Benzyl 3-(2-Acetoxy-5-(ethoxy(diethylphosphonomethyl)phos-
phinoyl)phenyl)-3-methylbutanoate (26a). 26awas purified by silica
gel chromatography using 0-10% MeOH in CH2Cl2 as the eluent.
Yield: 75%. 1H NMR (400 MHz, CDCl3) δ 1.20 (t, J ) 7.1, 3H),
1.30 (t, J ) 7.1, 6H), 1.49 (s, 3H), 2.33 (s, 3H), 2.56 (ddd, J )
6.8, 17.3, 23.4, 2H), 2.84 (AB q, J ) 14.4, 2H), 3.95-4.04 (m,
1
26d as a pale-yellow liquid (390 mg, 45%): H NMR (400 MHz,
CDCl3) δ 1.21 (t, J ) 7.0, 3H), 1.24-1.36 (m, 12H), 1.51 (s, 3H),
1.53 (s, 3H), 2.50-2.61 (m, 2H), 2.94 (AB q, J ) 14.1, 2H),
3.86-4.24 (m, 10H), 4.93 (s, 2H), 7.13-7.17 (m, 2H), 7.26-7.29
(m, 3H), 7.59 (dd, J ) 2.9, 8.3, 1H), 7.71 (t, J ) 9.8, 1H), 7.83 (d,
J ) 13.1, 1H).
General Procedure for Debenzylation of Esters 26a-c. Com-
pounds 26a-c (0.87 mmol) were dissolved in MeOH (20 mL) and
hydrogenated over Pd/C (10%, 250 mg) under H2 (1 atm) for 2 h.
The catalyst was filtered off and the volatiles were removed in
vacuo, resulting in the acid product.
3-(2-Acetoxy-5-(ethoxy(diethylphosphonomethyl)phosphinoyl)-
phenyl)-3-methylbutanoic Acid (27a). Yield: 98%. 1H NMR (400
MHz, CDCl3) δ 1.21 (dt, J ) 0.4, 7.1, 3H), 1.28 (dt, J ) 0.4, 7.1,
3H), 1.31 (t, J ) 7.1, 3H), 1.50 (s, 3H), 1.53 (s, 3H), 2.37 (s, 3H),
2.62 (ddd, J ) 5.4, 18.4, 22.4, 2H), 2.74 (AB q, J ) 13.9, 2H),
3.89-4.16 (m, 6H), 7.17 (dd, J ) 3.6, 8.2, 1H), 7.69 (ddd, J )
1.9, 8.2, 11.9, 1H), 7.86 (dd, J ) 1.9, 13.7, 1H). 31P (162 MHz,
CDCl3) δ 20.17 (d, J ) 5.0, 1P), 34.51 (d, J ) 5.0, 1P).
3-(2-(2,2-Dimethylacetoxy)-5-(ethoxy(diethylphosphonometh-
yl)phosphinoyl)phenyl)-3-methylbutanoic Acid (27b). Yield: 88%.
1H NMR (400 MHz, CDCl3) δ 1.21 (t, J ) 7.1, 3H), 1.29 (t, J )
7.3, 3H), 1.31 (t, J ) 7.0, 3H), 1.36 (d, J ) 7.0, 6H), 1.50 (s, 3H),
1.52 (s, 3H), 2.63 (ddd, J ) 3.9, 17.2, 22.2, 2H), 2.76 (AB q, J )
14.0, 2H), 2.86 (septet, J ) 7.1, 1H), 3.91-4.16 (m, 6H), 7.10
(dd, J ) 3.4, 8.2, 1H), 7.69 (ddd, J ) 1.8, 8.2, 11.7, 1H), 7.86 (dd,
J ) 1.8, 13.6, 1H).
3-(2-(Diethylphosphoryloxy)-5-(ethoxy(diethylphosphonometh-
yl)phosphinoyl)phenyl)-3-methylbutanoic Acid (27c). Yield: 98%.
1H NMR (400 MHz, CDCl3) δ 1.19-1.39 (m, 15H), 1.57 (s, 3H),
1.59 (s, 3H), 2.62 (bt, J ) 19.4, 2H), 2.80 (AB q, J ) 13.9, 2H),
3.90-4.17 (m, 6H), 4.22-4.32 (m, 4H), 7.57 (dd, J ) 3.4, 8.4,
1H), 7.69 (ddd, J ) 1.7, 8.4, 11.8, 1H), 7.81 (bd, J ) 13.5, 1H).
General Procedure for Acylation of Gatifloxacin with 27a-c.
HBTU (317 mg, 0.836 mmol) was added in one portion to a solution
of 27a-c (0.836 mmol), gatifloxacin 1b (310 mg, 0.84 mmol), and
diisopropylethylamine (291 µL, 1.67 mmol) in DMF (5 mL) cooled
in an ice bath. The resulting mixture was stirred and left to come
to room temperature on its own overnight. The reaction mixture
was diluted with EtOAc (100 mL) and washed with aqueous HCl
(10%), water, and saturated aqueous NaCl. The organics were dried
over Na2SO4 and concentrated in vacuo.
7-(4-(3-(2-Acetoxy-5-(ethoxy(diethylphosphonomethyl)phosphin-
oyl)phenyl)-3-methylbutanoyl)-3-methylpiperazin-1-yl)-1-cyclopro-
pyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic Acid
(28a). The residue was purified by silica gel chromatography
(0-10% MeOH in CH2Cl2) on a Biotage flash chromatography

Linking Bisphosphonates to Fluoroquinolones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6965
system. Yield: 34%. 1H NMR (400 MHz, CDCl3) δ 0.92-0.94
(m, 2H), 1.12-1.27 (m, 14H), 1.43 (s, 3H), 1.46 (s, 3H), 2.31 (s,
3H), 2.53-5.63 (m, 1H), 2.74-2.85 (m, 3H), 3.00-3.40 (m, 5H),
3.65 (s, 3H), 3.88-4.08 (m, 6H), 4.27 (bs, 1H), 4.66 (bs, 1H), 7.07
(dd, J ) 3.2, 8.1, 1H), 7.60-7.65 (m, 1H), 7.75 (d, J ) 12.0, 1H),
7.82 (dd, J ) 5.2, 8.1, 1H), 8.72 (s, 1H). 31P (162 MHz, CDCl3)
δ 20.07 (d, J ) 8.7, 1P), 33.90 (d, J ) 8.7, 1P).
7-(4-(3-(2-(2,2-Dimethylacetoxy)-5-(ethoxy(diethylphosphonom-
ethyl)phosphinoyl)phenyl)-3-methylbutanoyl)-3-methylpiperazin-
1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxoquinoline-
3-carboxylic Acid (28b). The residue was purified by silica gel
chromatography (0-5% MeOH in EtOAc) on a Biotage flash
chromatography system. Yield: 34%. 1H NMR (400 MHz, CDCl3)
δ 0.97-1.00 (m, 2H), 1.18-1.36 (m, 14H), 1.38 (d, J ) 7.1, 6H),
1.54 (s, 3H), 1.56 (s, 3H), 2.63 (ddd, J ) 5.6, 17.2, 22.0, 2H),
2.85-2.92 (m, 3H), 3.13 (bs, 1H), 3.27-3.51 (m, 5H), 3.71 (s,
3H), 3.98-4.19 (m, 6H), 4.43 (bs, 1H), 4.83 (bs, 1H), 7.08 (dd, J
) 3.5, 8.1, 1H), 7.67-7.74 (m, 1H), 7.89 (d, J ) 12.1, 1H), 7.95
(dd, J ) 1.7, 13.7, 1H), 8.83 (s, 1H). 31P (162 MHz, CDCl3) δ
20.1. (d, J ) 9.0, 1P), 34.32 (d, J ) 9.0, 1P).
7-(4-(3-(2-(Diethylphosphoryloxy)-5-(ethoxy(diethylphospho-
nomethyl)phosphinoyl)phenyl)-3-methylbutanoyl)-3-methylpiper-
azin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxo-
quinoline-3-carboxylic Acid (28c). The material was used in the
next step without further purification.
General Procedure for Deprotection of Phosphonate Esters
28a-c. TMSBr (355 µL, 2.69 mmol) was added to a stirring
solution of 28a-c (150 mg, 0.179 mmol) and 2,6-lutidine (416
µL, 3.59 mmol) in CH2Cl2 (5 mL). The resulting yellow solution
was stirred at room temperature for 24 h, and then the solvent was
removed at reduced pressure. The yellow solid was resuspended
in water (30 mM triethylamine/carbonate buffer for 28d), and the
solution was adjusted to approximately pH 6.5 by the addition of
1 M NaOH. The solution was then subjected to reverse-phase
chromatography (0-60% CH3CN in water) on a Biotage flash
chromatography system to give the product.
Tetraethyl 4-(2-Tetrahydro-2H-pyranyloxy)butylene-1,1-bispho-
sphonate (31). To a suspension of NaH (60% suspension in mineral
oil, 900 mg, 22.0 mmol) in dry THF (20 mL) was added dropwise
tetraethyl methylenebisphosphonate (6.46 g, 22.4 mmol). The
resulting clear solution was stirred 15 min at room temperature,
after which 2-(3-bromopropoxy)tetrahydro-2H-pyran (5.05 g, 22.6
mmol) was added dropwise. The reaction mixture was heated to
reflux for 6 h, diluted with CH2Cl2 (75 mL), washed with brine (2
× 50 mL), dried (MgSO4), and concentrated in vacuo. The product
was used as such in the following step.
Tetraethyl 4-Hydroxybutylene-1,1-bisphosphonate (32). To a
stirred solution of the crude product 31 (max 22.4 mmol) in MeOH
(40 mL) was added Amberlite IR-120 (0.6 g). The reaction mixture
was heated to 50 °C for 4 h, filtered, and concentrated in vacuo.
The crude product was purified by flash chromatography on silica
gel with gradient elution from 5-10% methanol/ethyl acetate to
give 32 (2.67 g, 34% from tetraethyl methylenebisphosphonate).
1H NMR (400 MHz, CDCl3) δ 1.34 (t, J ) 7.1 Hz, 12H), 1.81
(quint, J ) 6.5 Hz, 2H), 1.99-2.13 (m, 2H), 2.37 (tt, J ) 24.4, 5.6
Hz, 1H), 2.51 (t, J ) 5.9 Hz, 2H), 3.66 (q, J ) 5.9 Hz, 2H),
4.13-4.22 (m, 8H).
Tetraethyl 4-Iodobutylene-1,1-bisphosphonate (33). To a solution
of 32 (1.52 g, 4.39 mmol) in CH2Cl2 (50 mL) were added triph-
enylphosphine (1.32 g, 5.033 mmol) and imidazole (0.45 g, 6.61
mmol). The reaction mixture was cooled to 0 °C, before the addition
of iodine (1.22 g, 4.81 mmol) in small portions. The mixture was then
removed from the cooling bath, stirred for 2 h, diluted with hexanes
(100 mL), and filtered. The filter cake was washed with further hexanes
(2 × 30 mL), and the combined filtrates were concentrated in vacuo.
The residue was purified by flash chromatography on silica gel with
gradient elution from 0 to 10% methanol/ethyl acetate to give pure 33
1
(1.6 g, 80%). H NMR (400 MHz, CDCl3) δ 1.32-1.38 (m, 12H),
1.95-2.15 (m, 4H), 2.28 (tt, J ) 24.1, 6.1, 1H), 3.18 (t, J ) 6.6, 2H),
4.12-4.24 (m, 8H).
1-(4-(4,4-Bis(diethylphosphono)butoxy)phenyl)prop-2-en-1-
one (34). A mixture of 4′-hydroxyacetophenone (2.70 g, 19.9 mmol),
paraformaldehyde (2.68 g, 89.3 mmol), and N-methylanilinium
trifluoroacetate (6.51 g, 29.4 mmol) in THF (20 mL) was refluxed
for 3 h. The mixture was cooled and added to diethyl ether (200
mL), rinsing the flask with further diethyl ether (100 mL). The
product solution was decanted from the red gum and filtered.
Evaporation gave crude 1-(4-hydroxyphenyl)prop-2-en-1-one (2.0
7-(4-(3-(2-Acetoxy-5-((phosphonomethyl)phosphonoyl)phenyl)-
3-methylbutanoyl)-3-methylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-
1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic Acid (29a).
Yield: 60% as the mono 2,6-lutidine salt. 1H NMR (400 MHz, D2O)
δ 0.99-1.09 (m, 2H), 1.16-1.29 (m, 5H), 1.51 (s, 6H), 2.46 (s,
3H), 2.70 (s, 6H), 2.98-3.10 (m,2H), 3.16-3.54 (m, 4H), 3.71 (s,
3H), 3.83 (d, J ) 12.7, 1H), 4.17 (d, J ) 12.9, 1H), 4.21-4.27 (m,
1H), 4.56 (bs, 1H), 7.20 (dd, J ) 2.9, 8.0, 1H), 7.56 (dd, J ) 3.7,
12.2, 1H), 7.62 (d, J ) 8.0, 2H), 7.71 (bd, J ) 9.2, 1H), 7.93 (dd,
J ) 3.8, 12.2, 1H), 8.26 (t, J ) 8.0, 1H), 8.89 (s, 1H). MS: (MH-)
750.1.
1
g, 68%), which was used immediately in the next step. H NMR
(400 MHz, CDCl3) δ 5.69 (bs, 1H), 5.92 (dd, J ) 10.4, 1.7, 1H),
6.44 (dd, J ) 17.0, 1.7, 1H), 6.93 (d, J ) 8.8, 2H), 7.18 (dd, J )
17.2, 10.6, 1H), 7.93 (d, J ) 8.8, 2H). A mixture of iodide 33 (3.1
g, 6.8 mmol), 1-(4-hydroxyphenyl)prop-2-en-1-one (1.21 g, 8.17
mmol), and K2CO3 (1.033 g, 7.47 mmol) in acetone (75 mL) was
heated to reflux for 6.5 h. The mixture was cooled, filtered, and
concentrated in vacuo. The residue was redissolved in CH2Cl2 (170
mL), filtered through Celite, and concentrated to give crude 34 (3.2
7-(4-(3-(2-(2,2-Dimethylacetoxy)-5-((phosphonomethyl)phospho-
noyl)phenyl)-3-methylbutanoyl)-3-methylpiperazin-1-yl)-1-cyclo-
propyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carbox-
1
ylic Acid (29b). Yield: 16% as the disodium salt. H NMR (400
MHz, D2O) δ 0.99-1.09 (m, 2H), 1.21 (d, J ) 7.3, 3H), 1.33-1.44
(m, 2H), 1.38 (d, J ) 7.0, 6H), 1.51 (s, 6H), 2.29 (AB q, J ) 16.4,
2H), 2.93-3.10 (m, 3H), 3.14-3.41 (m, 2H), 3.48-3.56 (m, 2H),
3.71 (s, 3H), 3.85 (d, J ) 13.1, 1H), 4.18 (d, J ) 13.1, 1H), 4.25
(septet,, J ) 3.6, 1H), 4.58 (bs, 1H), 7.10-7.15 (m, 1H), 7.65 (d,
J ) 12.1, 1H), 7.70 (t, J ) 9.4, 1H), 7.93 (bd, J ) 12.5, 1H), 8.89
(s, 1H). MS: (MH-) 778.1.
1
g, 99%), which was used directly in the next step. H NMR (400
MHz, CDCl3) δ 1.28-1.39 (m, 12H), 1.89-2.25 (m, 4H),
2.26-2.48 (m, 1H), 4.05 (t, J ) 5.7, 2H), 4.12-4.26 (m, 8H), 5.87
(dd, J ) 10.6, 1.8, 1H), 6.42 (dd, J ) 16.9, 1.8, 1H), 6.93 (d, J )
8.8, 2H), 7.17 (dd, J ) 17.0, 10.4, 1H), 7.95 (d, J ) 8.8, 2H),
1-Cyclopropyl-6-fluoro-1,4-dihydro-7-(4-(3-(4-(4,4-bis(dieth-
ylphosphono)butoxy)phenyl)-3-oxopropyl)-3-methylpiperazin-1-yl)-
8-methoxy-4-oxoquinoline-3-carboxylic Acid (35). A mixture of
crude enone 34 (3.2 g, 6.7 mmol), gatifloxacin 1b (3.07 g, 8.18 mmol),
DMAP (200 mg, 1.64 mmol), and triethylamine (1.4 mL, 10.0 mmol)
in CH2Cl2 (200 mL) was stirred at room temperature for 20 h. The
mixture was concentrated in vacuo, followed by flash chromatography
(gradient elution, 5% methanol/CH2Cl2 to 10% methanol/CH2Cl2) to
give 35 (3.6 g, 63%). 1H NMR (400 MHz, CDCl3) δ 0.94-1.04 (m,
2H), 1.12-1.28 (m, 5H), 1.30-1.37 (m, 12H), 2.06-2.24 (m, 4H),
2.27-2.45 (m, 1H), 2.55-3.50 (m, 10H), 3.74 (s, 3H), 3.97-4.08
(m, 3H), 4.13-4.24 (m, 8H), 6.92 (d, J ) 8.8, 2H), 7.86 (d, J )
12.1,1H), 7.94 (d, J ) 8.8, 2H), 8.80 (s, 1H).
7-(4-(3-(2-Phosphoryloxy-5-((phosphonomethyl)phospho-
noyl)phenyl)-3-methylbutanoyl)-3-methylpiperazin-1-yl)-1-cyclo-
propyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carbox-
1
ylic Acid (29c). Yield: 31% as the di-2,6-lutidine salt. H NMR
(400 MHz, D2O) δ 0.97-1.09 (m, 2H), 1.26-1.36 (m, 5H), 1.56
(s, 6H), 2.34 (t, J ) 16.7, 2H), 2.69 (s, 12H), 2.91-3.10 (m, 2H),
3.16-3.55 (m, 3H), 3.68 (s, 3H), 3.90 (d, J ) 13.5, 1H), 4.14 (d,
J ) 13.5, 1H), 4.22-4.27 (m, 1H), 4.32 (bs, 1H), 4.57 (bs, 1H),
7.49 (bd, J ) 8.5, 1H), 7.61 (d, J ) 8.1, 4H), 7.64-7.66 (m, 1H),
7.73-7.81 (m, 2H), 8.25 (t, J ) 8.1, 2H), 8.94 (s, 1H). 19F NMR
(376 MHz, D2O): δ -119.15 (d, J ) 12.8). MS: (MH+) 790.2.
Preparation of the Bisphosphonated ꢀ-Aminoketone Prodrug
of Gatifloxacin.

6966 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Houghton et al.
1-Cyclopropyl-6-fluoro-1,4-dihydro-7-(4-(3-(4-(4,4-bisphospho-
nobutoxy)phenyl)-3-oxopropyl)-3-methylpiperazin-1-yl)-8-methoxy-
4-oxoquinoline-3-carboxylic Acid (36). To a solution of 35 (3.6 g,
4.3 mmol) in CH2Cl2 (150 mL) was added TMSBr (5.6 mL, 42
mmol). The reaction mixture was stirred for 27 h, the volatiles were
removed under reduced pressure, and the solid was dried under
high vacuum for 1 h. The solid was suspended in H2O (800 mL),
and the pH was immediately adjusted to pH 8 by the addition of 1
M KOH, with the concomitant slow dissolution of the product. The
solution was concentrated in vacuo at 30 °C and purified by reverse-
phase chromatography (gradient elution, 100% water to 30%
methanol/water). The pure product 36 was obtained as a white fluffy
solid (1.26 g, 33% recovery based on tetrapotassium salt of product).
1H NMR (400 MHz, D2O) δ 0.88-1.02 (m, 2H), 1.08-1.22 (m,
2H), 1.17 (d, J ) 6.2, 3H), 1.77-2.14 (m, 5H), 2.72-3.30 (m,
10H), 3.79 (s, 3H), 4.06-4.14 (m, 1H), 4.21 (t, J ) 6.4, 2H), 7.14
(d, J ) 8.8, 2H), 7.73 (d, J ) 12.8,1H), 8.05 (d, J ) 8.8, 2H), 8.52
(s, 1H). 19F (376 MHz, D2O) δ -122.44 (d, J ) 12.0, 1F). 31P
(162 MHz, D2O) δ 20.88 (s, 2P). MS: (MH+) 740.2.
eluent to afford 40 as a colorless oil (1.2 g, 50% over two steps).
1H NMR (400 MHz, CDCl3) δ 1.33-1.36 (m, 24H), 1.54-1.61
(m, 2H), 1.65-1.72 (m, 2H), 1.84-1.98 (m, 2H), 2.15 (tt, J )
24.1, 6.1, 1H), 2.28 (t, J ) 5.7, 1H), 3.66 (q, J ) 6.1, 2H),
4.72-4.82 (m, 4H).
Tetraisopropyl 5-Carboxypentylene-1,1-bisphosphonate (41).
Compound 40(365.5 mg, 0.9083 mmol) and pyridinium dichromate
(1.22 g, 3.18 mmol) were dissolved in 3 mL of N,N-dimethylfor-
mamide and stirred at room temperature overnight. After the
reaction was complete as monitored by TLC, the mixture was
diluted with water and extracted with EtOAc (3×), dried over
sodium sulfate, and concentrated in vacuo. Flash chromatography
on silica gel with 19:1 EtOAc/acetic acid afforded 41 as a colorless
1
oil (246.8 mg, 65%). H NMR (400 MHz, CDCl3) δ 1.29-1.35
(m, 24H), 1.90-1.99 (m, 4H), 2.18 (tt, J ) 24.4, 5.5, 1H), 2.34 (t,
J ) 6.8, 2H), 4.73-4.82 (m, 4H).
General Procedure for the Acylation of Fluoroquinolones
with Chloroethylchloroformate. 1-Chloroethyl chloroformate (0.27
mL, 2.478 mmol) was added to a solution of fluoroquinolone (2.477
mmol) and 547.9 mg (2.557 mmol) of Proton Sponge in 25 mL of
chloroform. The clear yellow solution was stirred at room temper-
ature for 5 h before being washed with water (3×) and dried over
anhydrous sodium sulfate. Removal of the solvent yielded the
desired product. In the cases where Proton Sponge was still present
after the water wash, the crude product was passed through a short
silica gel column with the elution of 19:1 dichloromethane/
methanol.
7-((4aS,7aS)-1-((1-Chloroethoxy)carbonyl)octahydropyrrolo[3,4-
b]pyridin-6-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-
oxoquinoline-3-carboxylic Acid (43a). Yield: 98%. 1H NMR (400
MHz, CDCl3) δ 0.78-0.86 (m, 1H), 1.03-1.17 (m, 2H), 1.25-1.35
(m, 1H), 1.47-1.64 (m, 1H), 1.78-1.90 (m, 5H), 2.32 (bs, 1H),
2.95-3.05 (m, 1H), 3.24-3.64 (m, 2H), 3.58 (s, 3H), 3.86-4.03
(m, 2H), 4.04-4.22 (m, 2H), 4.70-4.98 (m, 1H), 6.63 (q, J ) 5.9,
1H), 7.82 (dd, J ) 1.6, 13.7, 1H), 8.79 (s, 1H).
Preparation of Acyloxymethyl Carbamate Prodrugs. Tetra-
ethyl 3-Carboxypropylene-1,1-bisphosphonate (37). To a solution
of alcohol 32 (12.7 g, 36.7 mmol) in 200 mL of MeCN and 200
mL of a phosphate buffer solution made by mixing equal volumes
of 0.67 M Na2HPO4 and 0.67 M NaH2PO4 solutions at 35 °C was
added a catalytic amount of TEMPO (430 mg, 2.75 mmol). The
reaction flask, maintained at 35 °C, was fitted with two addition
funnels. One was filled with a solution of NaClO2 (8.3 g, 91.7
mmol) in 75 mL of H2O. The other one was filled with a solution
of household bleach (5.25%, 25 mL) in 250 mL of H2O. About 1/5
1
of the NaClO2 solution was added, followed by about /5 of the
bleach solution to initiate the reaction. The remainder of both
solutions was added dropwise, simultaneously, with a rate adjusted
so that both additions finished concurrently. The reaction mixture
was stirred at 35 °C for 4 h, then at room temperature for 18 h. It
was diluted with 300 mL of H2O, and the pH of the solution was
adjusted to 8.0 by adding 1 M NaOH. The resulting solution was
cooled to 0 °C, and a cold solution of Na2SO3 (6.1 wt %, 185 mL)
was added slowly. The mixture was stirred at 0 °C for 30 min,
after which a portion of Et2O was added. After being stirred
vigorously, the mixture was poured into an extraction funnel and
the Et2O layer was separated and discarded. The aqueous layer was
acidified to pH 3.4 with concentrated HCl and extracted 3× with
CHCl3/i-PrOH mixture (4:1). The combined organic layers were
dried over MgSO4, filtered, and concentrated to dryness, yielding
37 as a pale-yellow oil (12.9 g, 98%), which could be used without
7-(4-((1-Chloroethoxy)carbonyl)-3-methylpiperazin-1-yl)-1-cy-
clopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxoquinoline-3-car-
1
boxylic Acid (43b). Yield: 98%. H NMR (400 MHz, CDCl3): δ
0.94-1.04 (m, 2H), 1.21-1.26 (m, 2H), 1.40 (d, J)7.1, 3H), 1.85
(d, J ) 5.9, 3H), 3.31-3.34 (m, 2H), 3.41-3.53 (m, 4H), 3.74 (s,
3H), 3.98-4.07 (m, 2H), 4.45 (bs, 1H), 6.65 (dq, J)1.8, 5.7, 1H),
7.92 (d, J ) 12.0, 1H), 8.84 (s, 1H).
7-(4-(Chloromethoxycarbonyl)-3-methylpiperazin-1-yl)-1-cyclo-
propyl-6-fluoro-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxy-
lic Acid (49). A suspension of gatifloxacin 1b (8.65 g, 22.9 mmol)
and Proton Sponge (4.90 g, 22.9 mmol) in anhydrous CH2Cl2 (100
mL) was cooled in an ice bath followed by the dropwise addition
of chloroformic acid chloromethyl ester (2.03 mL, 22.9 mmol). The
resulting mixture was stirred at the same temperature for 4 h. The
reaction mixture was diluted by the addition of CH2Cl2 (300 mL),
washed with cold aqueous HCl (5%) and saturated NaCl, and dried
over anhydrous sodium sulfate. The organics were removed under
reduced pressure to give 47 as a yellow solid that was used without
purification (10.2 g, 95%): 1H NMR (400 MHz, CDCl3) δ
0.94-1.04 (m, 2H), 1.17-1.27 (m, 2H), 1.40 (d, J)6.7, 3H),
3.28-3.53 (m, 5H), 3.73 (s, 3H), 3.98-4.10 (m, 2H), 4.45 (bs,
1H), 5.85 (m, 2H), 7.92 (d, J ) 12.3, 1H), 8.83 (s, 1H).
General Procedure for the Reaction of Fluoroquinolone Chlo-
roethyl Carbamates with Bisphosphonated Acids. The carboxylic
acid (1.45 mmol) was dissolved in 4 mL of THF, and 1.45 mL
(1.45 mmol) of 1 N sodium hydroxide aqueous solution was added.
The mixture was stirred at room temperature for 4 h, and the organic
solvent was removed. The residual water was removed either by
applying high vacuum overnight or by freeze-drying. A mixture of
this sodium salt and a fluoroquinolone chloroethyl carbamate (1.460
mmol) in 7 mL of anhydrous acetonitrile was heated in a 60 °C oil
bath for 2 days. After cooling to room temperature, the reaction
mixture was filtered through a pad of Celite. The filtrate was
concentrated and purified by chromatography through a Waters C18
Sep-Pak cartridge (35 cm3) with gradient elution from neat water
to 2:1 water/methanol to 1:2 water/methanol to methanol.
1
further purification. H NMR (400 MHz, CDCl3) δ 1.34 (t, J )
7.0 Hz, 12H), 2.18-2.28 (m, 2H), 2.60 (tt, J ) 23.9, 6.5 Hz, 1H),
2.69 (t, J ) 7.3 Hz, 2H), 4.14-4.23 (m, 8H).
Tetraisopropyl 5-(2-Tetrahydro-2H-pyranyloxy)pentylene-1,1-
bisphosphonate (39). To a suspension of sodium hydride (60%,
342.5 mg, 8.563 mmol) in 15 mL of THF was carefully added
tetraisopropyl methylenebisphosphonate (2.80 mL, 8.61 mmol), and
the resultant pale-yellow clear solution was stirred at room
temperature for 30 min. Then neat compound 38 (2.0194 g, 8.516
mmol) was introduced by pipet plus 5 mL of THF rinse. The
mixture was refluxed for 8 h and allowed to cool to room
temperature before quenching with saturated NH4Cl. The mixture
was extracted with ethyl acetate (3×) and dried over sodium sulfate
and concentrated in vacuo. Flash chromatography with 10:1 EtOAc/
MeOH as eluent provided an inseparable mixture of the desired
product 39 and unreacted tetraisopropyl methylenebisphosphonate,
and the mixture was used directly in the next step. Selected 1H
NMR signals (400 MHz, CDCl3) δ 1.48-2.02 (m, 12H), 2.14 (tt,
J ) 24.2, 5.9, 1H), 3.36-3.42 (m, 1H), 3.46-3.52 (m, 1H),
3.71-3.77 (m, 1H), 3.83-3.89 (m, 1H), 4.57-4.58 (m, 1H).
Tetraisopropyl 5-Hydroxypentylene-1,1-bisphosphonate (40).
The mixture of 39 from the flash chromatography in the previous
step was dissolved in 4 mL of MeOH, and 24.5 mg (0.127 mmol)
of p-toluenesulfonic acid monohydrate was added. After being
stirred overnight at room temperature, the mixture was concentrated
and subjected to flash chromatography with 12:1 EtOAc/MeOH as

Linking Bisphosphonates to Fluoroquinolones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6967
Mixed Acetal 44a. From acid 42 and carbamate 43a. Yield: 43%.
1H NMR (400 MHz, CDCl3) δ 0.78-0.86 (m, 1H), 1.04-1.20 (m,
2H), 1.28-1.36 (m, 12H), 1.46-1.64 (m, 4H), 1.74-2.03 (m, 5H),
2.26-2.44 (m, 4H), 2.90-3.02 (m, 1H), 3.22-3.50 (m, 3H), 3.58
(s, 3H), 3.84-4.03 (m, 3H), 4.03-4.28 (m, 8H), 4.64-4.94 (bs,
1H), 5.02 (dt, J ) 10.0, 21.9, 1H), 6.15 (d, J ) 10.2, 1H), 6.84 (q,
J ) 4.7, 1H), 7.82 (d, J ) 13.9, 1H), 8.79 (s, 1H).
Mixed Acetal 44b. From acid 42 and carbamate 43b. Yield: 43%.
1H NMR (400 MHz, CDCl3) δ 0.96-1.04 (m, 2H), 1.16-1.29 (m,
2H), 1.34 (t, J ) 7.1, 12H), 1.52 (d, J ) 5.5, 3H), 1.69 (bs, 3H),
1.99 (quint, J ) 7.0, 2H), 2.35 (t, J ) 7.2, 2H), 2.42 (t, J ) 7.2,
2H), 3.22-3.53 (m, 5H), 3.74 (s, 3H), 3.98-4.04 (m, 2H),
4.14-4.24 (m, 8H), 4.36-4.44 (m, 1H), 5.03 (dt, J ) 10.2, 21.7,
1H), 6.18-6.21 (m, 1H), 6.86 (q, J ) 5.5, 1H), 7.91 (d, J ) 12.1,
1H), 8.83 (s, 1H).
Mixed Acetal 46. From acid 41 and carbamate 43b. Yield: 74%.
1H NMR (400 MHz, CDCl3) δ 0.88-1.04 (m, 2H), 1.14-1.25 (m,
2H), 1.28-1.42 (m, 24H), 1.51 (d, J)5.5, 3H), 1.61 (bs, 3H),
1.86-2.00 (m, 4H), 2.13 (tt, J ) 4.7, 24.3, 1H), 2.35 (t, J ) 6.0,
2H), 3.22-3.53 (m, 5H), 3.73 (s, 3H), 3.96-4.04 (m, 2H),
4.34-4.44 (m, 1H), 4.78 (septet, J ) 6.1, 1H), 6.86 (q, J ) 5.3,
1H), 7.90 (d, J ) 12.2, 1H), 8.83 (s, 1H).
4.34 (bs, 1H), 6.79 (q, J ) 5.5, 1H), 7.73 (d, J ) 12.7, 1H), 8.51
(s, 1H). 31P NMR (162 MHz, D2O) δ 14.27. 19F NMR (376 MHz,
D2O) δ -122.32 (bs). MS (m/e): 751 (M + H).
Mixed Acetal 47. Yield: 30%. 1H NMR (400 MHz, D2O) δ
0.88-1.02 (m, 2H), 1.07-1.20 (m, 2H), 1.37 (t, J ) 7.3, 3H), 1.55
(d, J)5.5, 3H), 1.72-1.85 (m, 5H), 2.48 (t, J ) 6.4, 2H), 3.25-3.34
(m, 2H), 3.43-3.52 (m, 4H), 3.75 (s, 3H), 3.93 (bs, 1H), 4.10
(septet,, J ) 3.5, 1H), 4.36 (bs, 1H), 6.80 (dq, J ) 0.8, 5.5, 1H),
7.73 (d, J ) 12.7, 1H), 8.52 (s, 1H). 31P NMR (162 MHz, D2O) δ
-20.87. 19F NMR (376 MHz, D2O) δ -122.32 (bs). MS (m/e):
708 (M + H).
Mixed Acetal 51. A solution of crude 53 (3.25 g, 4.11 mmol)
and 2,6-lutidine (9.53 mL, 82.1 mmol) in CH2Cl2 (30 mL) was
cooled in an ice-bath followed by the dropwise addition of TMSBr
(8.13 mL, 61.6 mmol). The resulting yellow solution was stirred
while warming to room temperature over 24 h. The solvent and
excess lutidine were then removed under reduced pressure. The
residue was resuspended in H2O and purified by C18 reverse phase
chromatography (0-60% CH3CN in water) on a Biotage flash
chromatography system. The CH3CN was removed under reduced
pressure and the water was removed by lyophization to give the
1
pale-yellow mono 2,6-lutidine salt of 54 (1.58 g, 49%): H NMR
(400 MHz, D2O) δ 0.88-1.11 (m, 2H), 1.20-1.31 (m, 2H), 1.36
(d, J ) 6.9, 3H), 2.04-2.22 (m, 3H), 1.79 (t, J ) 7.7, 2H), 2.71 (s,
6H), 3.28-3.55 (m, 5H), 3.75 (s, 3H), 3.97 (bd, J ) 12.0, 1H),
4.20-4.26 (m, 1H), 4.38 (bs, 1H), 5.82 (s, 2H), 7.41 (d, J ) 12.8,
Mixed Acetal 50. Acid 37 (3.70 g, 10.3 mmol) was dissolved in
CH3CN (20 mL), followed by the addition of KOH (0.634 g, 11.3
mmol) in H2O. The resulting solution was stirred for 5 min and
then concentrated under reduced pressure. This sodium salt was
dissolved in DMF (25 mL) followed by the addition of carbamate
49 (2.09 g, 4.47 mmol). The resulting solution was stirred at room
temperature for 3 h and then quenched by the addition of ice-cold
H2O (150 mL). The product was extracted with EtOAc (3 × 100
mL), and the combined organics were washed with water and brine
and then dried over Na2SO4. After the drying agent was filtered
off, the organics were removed under reduced pressure, resulting
in 50 as yellow solid that was used without purification (3.3 g,
93%): 1H NMR (400 MHz, CDCl3) δ 0.92-1.04 (m, 2H),
1.17-1.27 (m, 2H), 1.34 (t, J ) 6.9, 12H), 1.39 (d, J)10.5, 3H),
1.95 (bs, 2H), 2.19-2.33 (m, 2H), 2.47 (tt, J ) 7.5, 31.1, 1H),
2.77 (t, J ) 7.6, 2H), 3.28-3.52 (m, 5H), 3.73 (s, 3H), 3.98-4.23
(m, 8H), 4.42 (bs, 1H), 5.87 (m, 2H), 7.90 (d, J ) 11.9, 1H), 8.83
(s, 1H).
1H), 7.62 (d, J ) 8.2, 1H), 8.26 (t, J ) 7.7, 2H), 8.84 (s, 1H). 31
P
NMR (162 MHz, D2O) δ -20.94 (s, 1P). 19F NMR (376 MHz,
D2O) δ -119.05 (d, J ) 10.5, 1F). LCMS: -98.8% (254 nm),
98.8% (220 nm), 98.9% (320 nm). MS: (MH+) 680.2.
Biology. Determination of Minimum Inhibitory Concentra-
tions against S. aureus ATCC13709. The antimicrobial activity of
the compounds were tested against S. aureus strain ATCC 13709.
Minimum inhibitory concentration (MIC) testing was performed
by the microdilution method according to the guidelines set by the
Clinical and Laboratory Standards Institute (formerly the National
Committee for Clinical Laboratory Standards).46
Determination of Levels of Bone Binding in Vitro. An individual
compound was dissolved in PBS and added, at a final concentration
of 1 mg/mL, to a slurry of bone meal powder (Now Foods,
Bloomingdale, IL) in PBS at 10 mg/mL. The suspension of drug/
prodrug in bone meal powder was incubated at 37 °C for 1 h to
allow for binding and centrifuged at 13 000 rpm for 2 min before
recovering the supernatant. The bone meal powder pellet was then
washed three times with 1 mL of PBS. All supernatants were saved
and assessed for fluoroquinolone content by fluorescence measure-
ments at excitation/emission wavelengths of 280/465 nm. The
amount of fluoroquinolone was determined from standard curves
generated for each experiment. Amount of drug/prodrug bound to
bone powder was deduced from the difference between the input
amount (typically 1 mg) and the amount recovered in the
supernatants after binding. In control binding experiments, >99%
of input parent (nonbisphosphonated) drug was recovered in the
supernatant.
Regeneration of Parent Drug from Bone-Bound Prodrug.
Washed bone powder-bound prodrugs from the previous section
were resuspended in 400 µL of PBS or in 400 µL of 50% (v/v in
PBS) human or rat serum. The suspension was incubated for 24 h
at 37 °C and centrifuged at 13 000 rpm for 2 min, and the
supernatant was recovered. Methanol (5× volume relative to
supernatant) was added to each supernatant, and the mixture was
vortexed on a floor model vortex for 15 min to extract freed
fluoroquinolone. The mixture was then centrifuged at 10 000 rpm
for 15 min to pellet the insoluble material. The supernatant
containing the extracted fluoroquinolone was recovered and evapo-
rated to dryness in a speed vac. The dried pellets were resuspended
in PBS, and the amount of fluoroquinolone was determined by
fluorescence measurements as described previously. The percentage
of drug regenerated was deduced from the difference between
amount of bound prodrug and the amount of regenerated drug. The
identity of regenerated drug was deduced by determination of
General Procedure for Bisphosphonate Deprotection. To a
solution of bisphosphonated tetraester (0.63 mmol) in 5 mL of
CH2Cl2 was added 0.83 mL (6.29 mmol) of bromotrimethylsilane.
After being stirred at room temperature for 6 h, the mixture was
concentrated and the residue was kept at high vacuum for at least
30 min. The resulting material was dissolved in dilute sodium
hydroxide solution (e2 equiv of NaOH; this process was fairly
time-consuming, and ultrasound sonication was needed from time
to time to maintain the solution) prior to the careful adjustment of
pH to 7.20 with 1 N sodium hydroxide solution. The aqueous
solution obtained was subjected to a Waters C18 Sep-Pak cartridge
(20 cm3) with gradient elution from neat water to 10:1 water/
methanol. All fractions containing the desired product were
immediately combined and frozen in an acetone/dry ice cold bath.
The solvents were removed by freeze-drying, and the material
obtained was washed with CH2Cl2.
1
Mixed Acetal 45a. Yield: 18%. H NMR (400 MHz, D2O) δ
0.75-0.83 (m, 1H), 0.96-1.04 (m, 1H), 1.04-1.13 (m, 1H),
1.18-1.27 (m, 1H), 1.46-1.60 (m, 2H), 1.52 (d, J ) 5.5, 3H),
1.73-1.86 (m, 2H), 1.93 (quint, J ) 7.6, 2H), 2.28-2.40 (m, 1H),
2.38 (t, J ) 7.1, 2H), 2.49 (t, J ) 7.2, 2H), 2.98-3.12 (m, 1H),
3.30 (d, J ) 9.4, 1H), 3.43-3.53 (m, 1H), 3.59 (s, 3H), 3.90-4.10
(m, 4H), 4.24 (t, J ) 19.4, 1H), 6.77 (q, J ) 5.5, 1H), 7.64 (d, J
) 14.5, 1H), 8.46 (s, 1H) ppm. 31P NMR (162 MHz, D2O) δ 14.23
ppm. 19F NMR (376 MHz, D2O) δ -123.52 (bs) ppm. MS (m/e):
777 (M + H).
1
Mixed Acetal 45b. Yield: 30%. H NMR (400 MHz, D2O) δ
0.89-1.00 (m, 2H), 1.08-1.17 (m, 2H), 1.37 (t, J ) 7.2, 3H), 1.53
(d, J ) 5.5, 3H), 1.93 (quint, J ) 7.2, 2H), 2.38 (t, J ) 7.6, 2H),
2.50 (t, J ) 7.0, 2H), 3.24-3.34 (m, 2H), 3.42-3.50 (m, 3H), 3.75
(s, 3H), 3.93 (bs, 1H), 4.06-4.12 (m, 1H), 4.22 (t, J ) 19.0, 1H),

6968 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Houghton et al.
(6) Nelson, C. L.; McLaren, S. G.; Skinner, R. A.; Smeltzer, M. S.;
Thomas, J. R.; Olsen, K. M. The Treatment of Experimental
Osteomyelitis by Surgical Debridement and the Implantation of
Calcium Sulfate Tobramycin Pellets. J. Orthop. Res. 2002, 20, 643–
647.
the minimum inhibitory concentration (as per the guidelines of the
Clinical and Laboratory Standards Institute, section M7 A7), which
always matched those of parent drugs but not those of prodrugs.
Prophylactic Use of Prodrugs in a Rat Model of Bone Infec-
tion. S. aureus ATCC 13709 cells were grown overnight at 37 °C
in brain heart infusion broth (BHIB). Cells were subcultured into
fresh BHIB and incubated for 4-5 h at 37 °C. The cells were
washed twice with phosphate-buffered saline (PBS) and resus-
pended in BHIB supplemented with 10% (vol/vol) fetal bovine
serum at a density of approximately 1010 colony forming units
(CFU)/mL (based upon turbidimetry). The suspension was ali-
quoted, and a portion was used to check the CFU count by serial
dilution plating. The culture was stored frozen (-80 °C) and was
used without subculture. For use as an inoculum the culture was
thawed, diluted in PBS, and kept in an ice bath until it was used.
Animals were injected with the drugs intravenously and, after
the indicated duration, infected as described47,48 to generate the
bone infection. Female CD rats (age, 57-70 days; n ) 5/group;
Charles River, St-Constant, Canada) were anesthetized by isofluo-
rane before and during the surgery. Following complete induction
of anesthesia, the rat was placed ventral side up and hair was shaved
from the surgical site. The skin over the leg was cleaned and
disinfected (proviodine-ethanol 70%). A longitudinal incision
below the knee joint was made in the sagital plane. The incision
was made over the bone below the “knee joint” (tibia head or
condyle) but not completely extending to the ankle. A high speed
drill fitted with a 2 mm bulb bit was used to drill a hole into the
medullar cavity of the tibia. Rats were injected intratibially with
0.05 mL of 5% sodium morrhuate (sclerosing agent) and then with
0.05 mL of S. aureus suspension (about 5 × 105 CFU/rat). The
hole was sealed by applying a small amount of dry dental cement
that immediately absorbs fluids and adheres to the site. The wound
was closed using three metal skin clips. Moxifloxacin 3 (as a
positive control) was injected once at 10 mg/kg intravenously 1 h
postinfection in saline, while the test compounds (prepared in 0.9%
saline) were injected as a single intravenous bolus dose at the
indicated time points prior to the infection.
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Infected rats were sacrificed by CO2 asphyxiation 24 h postin-
fection to monitor the bacterial CFU count. Infected tibiae were
removed, dissected free of soft tissue, and weighed. The bones were
ground using a metal ball mill, resuspended in 5 mL of 0.9% NaCl,
serially diluted, and processed for quantitative cultures. Treatment
efficacy was measured in terms of the logarithm of the amount of
viable bacteria (log CFU per gram of bone). The results obtained
for each group of rats were evaluated by calculating the mean
log CFU and standard deviation. The limit of detection was
2 log CFU/g of bone. Statistical comparisons of viable bacterial
counts for the different treated and untreated groups were performed
with Dunnett’s multiple-comparison test. Differences were con-
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treated infected animals to the untreated infected ones.
Supporting Information Available: Chromatographic methods,
HPLC purities, and LC/MS traces for compounds 4a, 13a,b, 20,
29a-c, 36, 45a,b, 47, and 51. This material is available free of
charge via the Internet at http://pubs.acs.org.
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