Novel fluorescent risedronates: Synthesis, photodynamic inactivation and imaging of Bacillus subtilis

Article abstract of DOI:10.1016/j.bmcl.2012.12.051

Novel fluorescently-labeled conjugates of risedronate were synthesized using an epoxide linker, enabling conjugation of risedronate via its pyridyl nitrogen with the aromatic succinimidyl esters. The compounds were characterized by using ¹H NMR, ¹³C NMR, ³¹P NMR, UV-vis and fluorescence emission spectroscopies. Biological activity assays showed that the conjugates 14 and 15 exhibited photodynamic inactivation of Bacillus subtilis (ATCC 6633) with 91% and 47% bacterial lethality at 10 μM upon visible light irradiation, respectively. Both 14 and 15 could be also used for fluorescence imaging of Bacillus subtilis.

Full text of DOI:10.1016/j.bmcl.2012.12.051

Bioorganic & Medicinal Chemistry Letters 23 (2013) 949–954
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel fluorescent risedronates: Synthesis, photodynamic
inactivation and imaging of Bacillus subtilis
a
a
a
a
Li-Sheng Zhou a, Ke-Wu Yang a, , Lei Feng , Jian-Min Xiao , Cheng-Cheng Liu , Yi-Lin Zhang ,
⇑
Michael W. Crowder b,
⇑
a Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University,
Xi’an 710069, PR China
b Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, OH 45056, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel fluorescently-labeled conjugates of risedronate were synthesized using an epoxide linker, enabling
conjugation of risedronate via its pyridyl nitrogen with the aromatic succinimidyl esters. The compounds
were characterized by using 1H NMR, 13C NMR, 31P NMR, UV–vis and fluorescence emission spectrosco-
pies. Biological activity assays showed that the conjugates 14 and 15 exhibited photodynamic inactiva-
Received 31 October 2012
Revised 14 December 2012
Accepted 17 December 2012
Available online 25 December 2012
tion of Bacillus subtilis (ATCC 6633) with 91% and 47% bacterial lethality at 10 lM upon visible light
irradiation, respectively. Both 14 and 15 could be also used for fluorescence imaging of Bacillus subtilis.
Keywords:
Risedronates
Ó 2013 Elsevier Ltd. All rights reserved.
Photodynamic inactivation
Bis-phosphonates
Bis-phosphonates (BPs) are widely used for the treatment of
osteoporosis, bone metastases, Paget’s disease, and skeletal
complications of malignancy and have been shown to inhibit the
precipitation of calcium phosphate and biologic calcification in
vivo.1 Nitrogen-containing bis-phosphonates (N-BPs), such as
risedronate, pamidronate, and zoledronate (Fig. 1), affect the cellu-
lar function of by inhibiting farnesyl diphosphate synthase (FPPS),
a key enzyme in the mevalonate pathway, and by decreasing pre-
nylation of essential GTP-binding proteins.2–4 N-BPs that contain a
primary amino group are 100-fold more potent inhibitors than BP’s
that lack the primary amino group.5 The most potent, anti-resorp-
tive molecules are the N-BPs with a secondary or tertiary amino
group.6 Recently, N-BPs have been used for clinical purposes.7
Risedronate (monosodium 1-hydroxy-2-[3-pyridinyl]ethyli-
dene bis-phosphonic acetate), a N-BP developed by Procter &
Gamble and Hoechst Marion Roussel in 1998, is used for the treat-
ment of various systemic metabolic bone diseases.8,9 The mecha-
nism of action of BP’s in anti-metastatic and anti-tumor activities
is not well-understood; therefore, it is highly desirable to develop
specific imaging probes to elucidate the skeletal distribution and
cellular uptake of the BP’s in bone and other tissues. Fluores-
cently-labeled N-BPs could aid in elucidating cellular distribution
(including in bone), cell absorption selectivity, and cellular uptake
of BP’s.10–13 Kashemirrov et al. first reported a fluorescent risedro-
nate, which was prepared by linking of the BP’s pyridyl nitrogen
with carboxyfluorescein.14,15
While BPs have been used for the treatment of bone diseases
and cancer,16–20 the antimicrobial activity of BP’s has not received
similar attention.21,22 With the current rise of clinical antibiotic
resistance, photodynamic therapy (PDT) could be a powerful meth-
od to combat antibiotic resistant bacterial infections.23,24 PDT is a
photochemistry-based technology that relies on the wavelength–
specific light activation of certain nontoxic photosensitizers (PSs)
to produce active molecular species that are toxic to surrounding
cellular targets.25,26 PSs have been conjugated to antibodies or pep-
tides for the treatment of bacterial infections, but the sizes of the
conjugates and the complicated coupling reactions have limited
the use of antibody- and peptide-based PS’s.27 In response to this
problem, Xing et al. reported a simple and specific conjugation of
vancomycin–porphyrins to use for fluorescence imaging and anti-
bacterial studies on vancomycin resistant Enterococcus (VRE).28 Re-
cently, our group reported fluorescently-labeled vancomycin for
photodynamic inactivation of Bacillus subtilis.29
In an effort to develop photosensitive agents for fluorescence
imaging and photodynamic inactivation of Gram-positive bacteria,
we synthesized fluorescently-labeled risedronate and related ana-
logs by cross-linking risedronate and aromatic succinimidyl esters
(SE) using 3-amino-2-hydroxypropyl as a linker. We chose couma-
rin and benzocoumarin because coumarin derivatives exhibit
broad bioactivities and good photophysical properties, and the
resulting conjugates would be small in size. In order to correlate
⇑
Corresponding authors. Tel./fax: +86 29 8830 2429 (K.-W.Y.); tel.: +1 513 529
7274 (M.W.C.).
E-mail addresses: kwyang@nwu.edu.cn (K.-W. Yang), crowdemw@muohio.edu
(M.W. Crowder).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.051

950
L.-S. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 949–954
OH
OH
HO
O
P
O
OH
OH
P
P
OH
O
OH
H2N
N
N
O
OH
OH
OH
P
P
P
HO
O
N
OH
HO
OH
O
OH
Pamidronate
Zoledronate
Risedronate
N
OH
O
C
O
O
P
P
P
OH
OH
R1
R2
OH
OH
OH
O
O
OH
OH
O
Cl
P
O
P
P
OH
OH
OH
OH
P
Cl
OH
OH
OH
OH
OH
P
HO
P
O
OH
P
HO
O
O
OH
OH
O
Ibandronate
Bisphosphonic acid
Clodronate
Pyrophosphoric acid
Etidronate
Figure 1. Structures of bis-phosphonic acids.
bioactivity to the type of aromatic rings, we also chose benzoic acid
and salicylic acid as conjugates. These synthesized compounds
were characterized spectroscopically, and their biological activities
were tested for photodynamic inactivation, minimum inhibitory
concentrations (MIC), and fluorescence-labeling.
O
O
O
NHS
DCC
+
N OH
R-OH
N
O
R
O
Carboxyfluoresceins 2 and 4 were prepared, respectively, by
two steps using the method shown in Scheme 1. A mixture of
malonic acid and acetic anhydride in concentrated sulfuric acid
was treated with acetone to afford 2,2-dimethyl-1,3-dioxane-
O
O
O
R=
6
8
R=
R=
5
7
4,6-dione 1. Naphthaldehyde
Reimer–Tiemann reaction and b-naphthol. Compound
3
was prepared by using the
was
O
O
O
1
reacted with salicylaldehyde in H2O at 75 °C to afford coumarin-
3-carboxylic acid 2. A mixture of 1 and 3 in ethanol was treated
with pyridine to afford 3-oxo-3H-benzochromene-2-carboxylic
acid 4.
O
O
R=
OH
Scheme 2. Synthesis of aromatic succinimidyl esters.
The aromatic succinimidyl esters were prepared as shown in
Scheme 2. Two solutions of 2 in acetonitrile and 4 in anhydrous
CH2Cl2 were reacted with N-hydroxysuccinimide in the presence
of N,N0-dicyclohexylcarbodiimide (DCC) to afford coumarin-3-car-
boxylic acid succinimidyl ester 5 and 3-oxo-3H-benzochromene-
2-carboxylic acid succinimidyl ester 6, respectively. Compounds
7 and 8 were prepared with a similar method using aromatic car-
boxylic acids.
The conjugates of risedronate were synthesized according to
Scheme 3. tert-Butyl allylcarbamate 9 and tert-butyl(oxiran-2-
ylmethyl)carbamate 10 were synthesized as previously reported.14
Risedronic acid 11 was prepared by reaction of 3-pyridylacetic acid
hydrochloride with phosphorous acid and phosphorus trichloride
in chlorobenzene, and the resulting product was hydrolyzed with
HCl. Risedronic acid was coupled with 10 in alkalescence aqueous
solution to afford compound 12. The amino protecting group was
removed with trifluoroacetic acid (TFA) to afford 13, which was
coupled to aromatic succinimidyl esters 5, 6, 7, and 8, respectively,
in alkaline solution, to afford the N-aromatic risdronate derivatives
14, 15, 16, and 17.
All risdronate derivatives were characterized by using 1H NMR,
13C NMR, 31P NMR, UV–vis, and fluorescence spectroscopies and
elemental analyses. 1H NMR spectra of all final compounds showed
peaks between 6.5 and 9.0 ppm, which were attributed to phenyl
protons. 31P NMR spectra showed peaks between 16 and 18 ppm.
Compound 13 exhibited a broad, 31P peak at 17.12 ppm, and com-
pounds 14, 16, and 17 displayed singlet peaks at 17.70, 17.02, and
17.08 ppm, respectively. UV–vis spectra are showed in Figure 2.
O
COOH
O
H2O, 75°C, 3h
H
+
1)
H2SO4,
O
2
OH
(CH3CO)2O,
0 °C
O
O
O
O
HO
OH
2)(CH3)2CO,
rt, 4h
COOH
O
O
CHO
ethanoll, pyri di ne,
80°C, 2-4 h
O
OH
1
O
+
4
3
50% NaOH , et hanol,
80°C, CHCl 3
OH
Scheme 1. Synthesis of carboxyfluoresceins.

L.-S. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 949–954
951
O
O
O
H
NH2
CH2Cl2
rt, 4h
MCPBA
CH2Cl2
t-BuO
N
O
+
OBu-t
t-BuO
O
N
H
OBu-t
O
10
9
OH
O
P
OH
O
OH
H3PO3, PCl3
100°C, 6h
P
O
HO
HO
N
.
N
OH
HCl
11
OH
OH
OH
O
O
O
P
P
P
OH
O
OH
OH
O
NaOH, MeOH
1:1 TFA/H2O
rt, 3h
O
SE
P
HO
HO
P
P
HO
HO
HO
HO
N
OH
N
OH
N
OH
40 , 18.5 h
°C
NaHCO3/DMF
, pH8.3, rt
OH
NH
OH
NH2
OH
NH
O
R
OBu-t
12
13
O
O
15
17
R=
R=
R=
14
16
O
O
O
O
O
O
R=
OH
Scheme 3. Synthetic route of risedronate conjugates.
Figure 2. UV–vis spectra of (1) compounds 2 and 14 and (2) compounds 4 and 15, using compound 13 as a comparison. The concentrations of 13, 14, and 15 were 50
the compounds were dissolved in H2O. Compounds 2 and 4 were dissolved 50 M in methanol.
lM, and
l
Compound 13 exhibited a single absorption peak at 265 nm, and
compound 14 exhibited three peaks at 269, 327, and 386 nm.
The UV–vis absorption peaks of compound 15 at 226, 258, and
361 nm were weaker than those of 4 at 255, 327, and 361 nm.
437 nm, respectively. The fluorescence intensity of compound 14
was 11-fold weaker than that of compound 2, and the intensity
of compound 15 was fivefold weaker than that of compound 4.
We attribute the reduced emission intensity to reduced electron
delocalization as the compound(s) are conjugated to non-rigid, pla-
nar structures.
The photodynamic, antimicrobial activities of the photosensitiz-
ers against Bacillus subtilis were assayed by a traditional surface
plating method.24,30 Compounds 2, 4, 14, and 15 were incubated
with B. subtilis separately, and the cells were exposed to white light
The weaker absorbance is attributed to the weaker n–p conjugate
effect of the amino group, in comparison to a hydroxyl group, and
the red shift is due to conjugation with a chromophoric group.
Fluorescence emission spectra of compounds 14, 15, 2, and 4
(5
lM) are shown in Figure 3. The kex/kem ratios of compounds
14, 2, 15, and 4 were 309/415, 324/416, 230/445 and 361/

952
L.-S. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 949–954
Figure 3. Fluorescence spectra of (A) compounds 2 and 14 and (B) compounds 4 and 15. Concentrations of compounds 14 and 15 were 5
were 5 M in methanol. Compounds 14, 15, 2, and 4 were excited with 309, 230, 324 and 361 nm light, respectively.
lM in H2O, and compounds 2 and 4
l
irradiation (400–800 nm, 350 mW/cm2) and spread on Luria-
Bertani (LB) agar plates. B. subtilis cells, which had been treated
with the photosensitizers but not irradiated with light, were used
as a control. The antimicrobial activity was evaluated by counting
the number of colony forming units (CFU) on the LB agar plates.
The number of colony forming units on photosensitizer-treated
plates that were irradiated and on photosensitizer-treated plates
that were not irradiated was divided by the CFU’s on plates that
were not treated with photosensitizers nor irradiated. Survival
fractions were determined, and the percentage of dead bacteria
was obtained indirectly. Figure 4 shows the percentage of B. subtilis
cells that died as a function of different photosensitizer concentra-
tions. After irradiation, the percentage of dead bacteria was 91%
and 47% when cells were exposed to 10 lM of 14 or 15, respec-
tively. Compounds 2 and 4 exhibited no obvious photodynamic
inactivation on the tested bacterial strain. We hypothesize that
the photodynamic effect exhibited by 14 and 15 originates from
singlet oxygen (1O2) that is generated through the photosensitiza-
tion process and that singlet oxygen is responsible for the damage
of cell walls and membrane structures.30 Fluorescein-containing
compounds have been shown to produce 1O2, which can irrevers-
ibly damage living cells.31 While compounds 2 and 4 probably also
produce singlet oxygen, compounds 14 and 15 exhibit a higher
binding affinity to cell walls (see fluorescence microscopic results
below). These previous results along with the results presented
herein suggest that the photodynamic inactivation by the risedro-
nate derivatives against B. subtilis is due to singlet oxygen
toxicity.32,33
Compounds 14, 15, 16, and 17 were also evaluated for MIC val-
ues with B. subtilis using the method of the macrodilution (tube)
broth according to the Clinical and Laboratory Standards Institute
(CLSI).34 The results indicated that all four compounds (without
photoactivation) exhibited low antimicrobial activity to the bacte-
ria with MIC values >128 lg/mL. Photoactivation resulted in irradi-
ated compounds 14 and 15 exhibiting significant antimicrobial
activity (Fig. 4).
Since compounds 14, 15, 16, and 17 fluoresce, cells that were
treated with these compounds were imaged on a confocal fluores-
cent microscope. The bacterial strains were incubated with the
photosensitizer at 37 °C for 30 min in phosphate-buffered saline
(PBS) buffer. As shown in Figure 5, both 14 and 15 showed obvious
fluorescence in Gram-positive bacteria (B. subtilis). This result is
attributed to the high affinity of bis-phosphonates to bacteria.
Novel fluorescently-labeled risedronate and related analogues
were synthesized by conjugating risedronate with aromatic succ-
inimidyl esters using 3-amino-2-hydroxypropyl as a ‘linker’. The
conjugates of risedronate were characterized by 1H NMR, 13C
NMR, 31P NMR, UV–vis, and fluorescence emission spectroscopies
and by elemental analysis (see Supplementary data). Biological
activity assays found that the fluorescently-labeled risedronates
14 and 15 showed photodynamic inactivation against B. subtilis
Figure 4. Photodynamic inactivation of N-aromatic risedronate 14, 15 and
carboxyfluorescein 2, 4 against Bacillus subtilis. The white-light dose was 63 J/cm2
(exposure for 3 min at a rate of 350 mW/cm2). The concentrations of 2, 4, 14, and 15
with 91% and 47% lethality at 10
lM, respectively, while none of
were 0–10
lM, and bacteria were treated with compounds 14 or 15 with no light
the N-aromatic risdronate derivatives exhibited low MIC values
illumination as control groups.

L.-S. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 949–954
953
Figure 5. Confocal microscopic (left) and differential interference contrast (right) images of incubated B. subtilis with (A) compound 14 and (B) compound 15.
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Acknowledgments
We are grateful for assistance in the photodynamic studies from
Dr. Dan Sun at the Laboratory of Photonics and Photon Technique
in Northwest University. Also we thank professor Jia-Yun Liu at the
Clinical Laboratory at Xijing Hospital, Fourth Military Medical Uni-
versity for the tested bacteria. This work was supported by the Na-
tional Natural Science Foundation of China (20972127, 21272186),
Doctoral Foundation of China (200806970005), National Science
Foundation of Shaanxi Province (2009JM2002), Key Fund for Inter-
national Cooperation of Shaanxi Province (2010KW-16) and Sha-
anxi Provincial Bureau of Foreign Experts Fund (SLZ2008013),
and from the National Science Foundation (CHE402191 to M.W.C.).
15. McKenna, C. E.; Kashemirov, B. A.; Bala, J. L. F. Synthesis of Drug Conjugates via
Reaction with Epoxide-Containing Linkers In Patent, U., Ed.; University of
Southern California: United States, 2008.
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Supplementary data
Supplementary data (detailed synthesis protocols and methods)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2012.12.051.
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